The History of Neuroscience in Autobiography

VOLUME 12

The History of Neuroscience in Autobiography

VOLUME 12

Edited by Thomas D. Albright
and Larry R. Squire

Society for Neuroscience

Society for Neuroscience

The Society for Neuroscience publishes works that advance
the understanding of the brain and nervous system.

Copyright © 2022 by the Society for Neuroscience

Published by the Society for Neuroscience

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Washington, DC 20005

www.sfn.org

All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise without the prior permission of the Society for Neuroscience.

Library of Congress Control Number: 96070950

978-0-916110-11-6

Printed in the United States of America

Contents

Previous Contributors

Preface to Volume 1

Preface to Volume 12

Richard A. Andersen

Corey S. Goodman

Tomas Hökfelt

Darcy B. Kelley

George F. Koob

Stephen G. Lisberger

Gail Mandel

Kelsey Catherine Martin

Kevan A. C. Martin

Pasko Rakic

Joshua Richard Sanes

Wolfram Schultz

David L. Sparks

Nicholas C. Spitzer

Index

Previous Contributors

Volume 1

Denise Albe-Fessard

Julius Axelrod

Peter O. Bishop

Theodore H. Bullock

Irving T. Diamond

Robert Galambos

Viktor Hamburger

Sir Alan L. Hodgkin

David H. Hubel

Herbert H. Jasper

Sir Bernard Katz

Seymour S. Kety

Benjamin Libet

Louis Sokoloff

James M. Sprague

Curt von Euler

John Z. Young

Volume 2

Lloyd Beidler

Arvid Carlsson

Donald Griffin

Roger Guillemin

Ray W. Guillery

Masao Ito

Martin Larrabee

Jerry Lettvin

Paul MacLean

Brenda Milner

Karl Pribram

Eugene Roberts

Gunther Stent

Volume 3

Morris H. Aprison

Brian B. Boycott

Vernon B. Brooks

Pierre Buser

Hsiang-Tung Chang

Claudio A. Cuello

Robert W. Doty

Bernice Grafstein

Ainsley Iggo

Jennifer Lund

Edith and Patrick McGeer

Edward R. Perl

Donald B. Tower

Patrick D. Wall

Wally Welker

Volume 4

Per Andersen

Mary Bartlett Bunge

Jan Bures

Jean Pierre G. Changeux

William Maxwell (Max) Cowan

John E. Dowling

Oleh Hornykiewicz

Andrew F. Huxley

JacSue Kehoe

Edward A. Kravitz

James L. McGaugh

Randolf Menzel

Mircea Steriade

Richard F. Thompson

Volume 5

Samuel H. Barondes

Joseph E. Bogen

Alan Cowey

David R. Curtis

Ennio De Renzi Wilfrid Rall

Mark R. Rosenzweig

Arnold Bernard Scheibel

Gerald Westheimer

Lynn T. Landmesser

Rodolfo R. Llinás

Alan Peters

Martin Raff

John S. Edwards

Mitchell Glickstein

Carlton C. Hunt

Volume 6

Bernard W. Agranoff

Emilio Bizzi

Marian Cleeves Diamond

Charles G. Gross

Richard Held

Leslie L. Iversen

Masakazu Konishi

Lawrence Kruger

Susan E. Leeman

Vernon B. Mountcastle

Shigetada Nakanishi

Solomon H. Snyder

Nobuo Suga

Hans Thoenen

Volume 7

Floyd E. Bloom

Joaquín Fuster

Michael S. Gazzaniga

Bertil Hille

Ivan Izquierdo

Edward Jones

Krešimir Krnjević

Nicole M. Le Douarin

Terje Lømo

Michael M. Merzenich

John Wilson Moore

Robert Y. Moore

Michael I. Posner

Peter H. Schiller

Gordon M. Shepherd

Robert H. Wurtz

Volume 8

Huda Akil

Philippe Ascher

Giovanni Berlucchi

Lily Jan

Yuh-Nung Jan

Bruce Sherman McEwen

Frederick A. Miles

Peter M. Milner

Fernando Nottebohm

Tomaso A. Poggio

Marcus E. Raichle, Sr.

Pablo Rudomin

Anne Treisman

Stanley J. Watson, Jr.

Anne Buckingham Young

Volume 9

David H. Cohen

Yadin Dudai

Gerald D. Fischbach

Sten Grillner

Jon Howard Kaas

Eric Kandel

Nancy Kopell

Margaret Stratford Livingstone

Giacomo Rizzolatti

Trevor W. Robbins

Wolf Singer

David C. Van Essen

Volume 10

Ben A. Barres

Anders Björklund

Thomas J. Carew

Dennis W. Choi

Joseph T. Coyle

Kjell Fuxe

Michael E. Goldberg

Mary Elizabeth Hatten

Steven E. Hyman

Eve Marder

Roger A. Nicoll

S. Murray Sherman

Douglas G. Stuart

Volume 11

Barry J. Everitt

Tamás F. Freund

Uta Frith

Mary B. Kennedy

Eric I. Knudsen

Pierre J. Magistretti

Yasushi Miyashita

Dale Purves

Larry R. Squire

Michael Paul Stryker

Larry W. Swanson

Richard W. Tsien

Preface to Volume 1

Before the Alfred P. Sloan Foundation series of books began to appear in 1979, the scientific autobiography was a largely unfamiliar genre. One recalls Cajal’s extraordinary Recollections of My Life, translated in English in 1937 and the little gem of autobiography written by Charles Darwin for his grandchildren in 1876. One supposes that this form of scientific writing is scarce because busy scientists would rather continue to work on scientific problems than to indulge in a retrospective exercise using a writing style that is usually outside their scope of experience. Yet, regardless of the nature of one’s own investigative works, the scientific enterprise describes a community of activity and thought in which all scientists share. Indeed, an understanding of the scientific enterprise should in the end be accessible to anyone because it is essentially a human endeavor, full of intensity, purpose, and drama that are universal to human experience.

While writing a full autobiographical text is a formidable undertaking, preparing an autobiographical chapter, which could appear with others in a volume, is perhaps less daunting work and is a project that senior scientists might even find tempting. Indeed, a venture of this kind within the discipline of psychology began in 1930 and is now in eight volumes (A History of Psychology in Autobiography). So it was that during my term as president of the Society for Neuroscience in 1993 to 1994, I developed the idea of collecting autobiographies from senior neuroscientists, who at this period in the history of our discipline are in fact pioneers of neuroscience. Neuroscience is quintessentially interdisciplinary, and careers in neuroscience come from several different cultures, including biology, psychology, and medicine. Accounts of scientific lives in neuroscience hold the promise of being informative and interesting, and they could be a source of inspiration to students. Moreover, personal narratives provide for scientists and nonscientists alike an insight into the nature of scientific work that is simply not available in ordinary scientific writing.

This volume does have a forerunner in neuroscience. In 1975, MIT Press published The Neurosciences: Paths of Discovery, a collection of 30 chapters in commemoration of F. O. Schmitt’s 70th birthday, edited by F. Worden, J. Swazey, and G. Adelman. The contributing neuroscientists, all leaders of their discipline, described the paths of discovery that they had followed in carrying on their work. While writing in the style of the conventional review article, some authors did include a good amount of anecdote, opinion, and personal reflection. A second, similar volume, The Neurosciences: Paths of Discovery II, edited by F. Samson and G. Adelman, appeared in 1992.

In any case, neuroscience writing that is deliberately and primarily autobiographical has not been collected before. This project, The History of Neuroscience in Autobiography, is the first major publishing venture by the Society for Neuroscience after The Journal of Neuroscience. The book project was prepared with the active cooperation of the Committee on the History of Neuroscience, which serves as an editorial board for the project. The first chairperson of the committee was Edward (Ted) Jones; its members were Albert Aguayo, Ted Melnechuk, Gordon Shepherd, and Ken Tyler. This group compiled the names and carried out the deliberations that led to the first round of invitations. In 1995 Larry Swanson succeeded Ted Jones as chair of the committee, and as we go to press with Volume 1 the committee members are Albert Aguayo, Bernice Grafstein, Ted Melnechuk, Dale Purves, and Gordon Shepherd.

In the inaugural volume of the series, we are delighted to be able to present together 17 personal narratives by some of the true pioneers of modern neuroscience. The group includes four Nobel Laureates and 11 members or foreign associates of the National Academy of Sciences, USA. The contributors did their scientific work in the United States, Canada, England, Australia, France, and Sweden. It is difficult to imagine a finer group of scientists with which to inaugurate our autobiographical series. The autobiographical chapters that appear here are printed essentially as submitted by the authors, with only light technical editing. Accordingly, the chapters are the personal perspectives and viewpoints of the authors and do not reflect material or opinion from the Society for Neuroscience.

Preparation of this volume depended critically on the staff of the book’s publisher, the Society for Neuroscience. The correspondence, technical editing, cover design, printing, and marketing have all been coordinated by the Society’s central office, under the superb direction of Diane M. Sullenberger. I thank her and her assistants Stacie M. Lemick (publishing manager) and Danielle L. Culp (desktop publisher) for their dedicated and skillful work on this project, which was carried out in the midst of demands brought by the first in-house years of the Society’s Journal of Neuroscience. I also thank my dear friend Nancy Beang (executive director of the Society for Neuroscience) who from the beginning gave her full enthusiasm to this project.

Larry R. Squire
Del Mar, California
September 1996

Preface to Volume 12

In this twelfth volume of The History of Neuroscience in Autobiography, we are pleased to present 14 personal narratives by leaders in our field of science.

Larry Squire founded this autobiographical series 28 years ago during his tenure as president of the Society for Neuroscience. From the beginning, the Society leadership understood the importance of documenting the explosive modern growth of a field that brings innovation to medical practice and resonates with a public eager to understand the workings of the brain. Larry’s key insight was that the historical record need not simply reflect the timeline of discoveries; there is also value in recording the “activity and thought in which all scientists share,” for science itself “is essentially a human endeavor, full of intensity, purpose, and drama that are universal to human experience.”

Collectively, the chapters contained in this series do exactly that: They provide a firsthand chronicle of a bold venture in biomedical research, as it has played out in the late 20th and early 21st centuries. Individually, they are treasures, for they are cast in the irresistible and uniquely human form of expression: the story. As such, they not only connect the dots along paths of scientific discovery but also convey the human elements behind those connections, the rich drama and comedy of interpersonal relationships, the sudden flashes of insight, the folly of hubris and the pain of missed opportunities, the beauty of new ideas, and the raw excitement of discovery. These stories are populated by imposing and quirky characters, magnified by the lenses of memory and attention. There are tales of brilliance, fantasy, risk, sacrifice, triumph, and camaraderie, but the theme that binds every one of the chapters in this series is success. Thus, not only do these chapters entertain in the way that only good stories can, but they also offer appealing models and nuanced lessons for success in the human side of science for the next generation of vanguards in our field.

Tom Albright joined Larry as coeditor beginning with Volume 9. With the publication of Volume 12, the library of chapters now numbers 169. Many more great stories are in the making, of course, as the field of neuroscience bounds ahead, and we look forward to curating them in these pages.

Beginning with Volume 8 in the series, the Society for Neuroscience assumed full responsibility for this project. Volume 12 was prepared with the advice of the Society’s Publications Committee, which serves as editorial board for the project. With input from this committee, from the Council of the Society for Neuroscience, and many others, names were compiled for Volume 12. Production was overseen by Kelly Newton, who serves as the Society’s director of scientific publications.

Autobiographical chapters contained in Volume 12 are freely available from the SfN website, along with the 155 chapters that appeared in the first eleven volumes of the series. From the SfN homepage, chapters can be accessed either via the “Publications/History of Neuroscience Autobiographical Chapters” menu tabs or via the “About/History of Neuroscience” menu tabs. They can also be accessed directly at https://www.sfn.org/about/history-of-neuroscience/autobiographical-chapters. Hardbound copies of Volume 12 are available for purchase via inquiry to jn@sfn.org.

We hope readers will find Volume 12 as interesting and useful as the earlier volumes.

Thomas D. Albright and Larry R. Squire
Del Mar, California
April 2022

Richard A. Andersen

BORN:

New Kensington, Pennsylvania

October 27, 1950

EDUCATION:

University of California, Davis, B.S. Biochemistry (1973)

University of California, San Francisco, PhD Physiology (1979)

Johns Hopkins Medical School, Postdoctoral Fellow (1981)

APPOINTMENTS:

Assistant Professor, Salk Institute (1981–1986)

Adjunct Assistant Professor, University of California, San Diego (1982–1987)

Associate Professor, Salk Institute (1986–1987)

Associate Professor, Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology (1987–1990)

Professor, Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology (1990–1993)

James G. Boswell Professor of Neuroscience, Division of Biology and Biological Engineering, California Institute of Technology (1993–present)

T & C Chen Brain-machine Interface Center Leadership Chair, California Institute of Technology (2017–present)

HONORS AND AWARDS (SELECTED):

Sloan Foundation Fellow (1982)

McKnight Foundation Scholars Award (1983)

Spencer Award, Columbia University (1994)

Fellow, the American Association for the Advancement of Science (2000)

Fellow, the American Academy of Arts and Sciences (2002)

Visiting Professor, College de France (2005)

Member, National Academy of Sciences (2005)

Member, National Academy of Medicine (2007)

A Recipient of UCSF’s 150th Anniversary Alumni Award (2015)

Richard Andersen’s research uses neurophysiological, psychophysical, neuroanatomical, and computational methods to understand the processing of sensory and motor signals in the cerebral cortex. These studies have included examining the senses of sight, hearing, balance and touch, the neural mechanisms of action, and the development of neural prosthetics. He discovered gain fields, the method the brain uses to transform signals between spatial representations. He also discovered neural signals of intention in the posterior parietal cortex, proving that they are not sensory in nature but rather reflect the planning of the actor. He applied this discovery of intention signals to advance research in brain-machine interfaces, showing that paralyzed patients’ intentions can be decoded from brain activity to control assistive devices, such as robotic limbs and computers. He used intracortical electrical stimulation of somatosensory cortex to show that natural sensations of touch and body position can be restored to patients with cervical spinal cord lesions that have left them otherwise insensate to somatosensory stimulation below the lesion.

Richard A. Andersen

Family Background

I was born in New Kensington, Pennsylvania, in 1950. My mother Norma was born in Queens, New York, and my father John in Brooklyn, New York. The name Andersen comes from my grandfather on my father’s side who was Danish. My grandmother on my father’s side was from Germany. John was a first-generation American. My mother’s parents were American and their parents came from Germany, making my mother a second-generation American. My brother Cliff was born in 1954 in Buffalo, New York.

My father joined the U.S. Army at the beginning of World War II and was in officer training, coincidently here in Pasadena where I now work. He and several soldiers contracted polio from the swimming pool at Pasadena City College, literally across the street from Caltech. His case was severe and he spent many months in a hospital. Although he recovered, his lower legs were weakened, and later in life, he suffered from post-polio syndrome, which greatly hampered his mobility. Interestingly, I am now studying ways to assist people with paralysis. My mother was his primary caregiver, with me as backup, so I have a firsthand appreciation of the suffering from loss of mobility.

My parents met through family friends in New York City. My mother was a secretary in Manhattan and my father was an engineering student at Brooklyn Polytechnic (now the NYU Tandon School of Engineering).

My father designed and oversaw the building of chemical plants, mostly for Kaiser Chemical. As a result, our family moved quite a bit, including Buffalo, New York; Columbus, Ohio; Walnut Creek, California; Baton Rouge, Louisiana; and Moraga, California. He enjoyed talking about different chemical processes and engineering, and so I was getting exposed to the world of science at an early age. In high school, I enjoyed doing science projects, including building a fuel cell and studying pollutants in the San Francisco Bay. So it was natural that when I went to college at the University of California, Davis (UC Davis), that I was a chemical engineering major for the first two years, which was a good choice for getting a quantitative education. My brother Cliff took a different career route and became a musician, mostly playing jazz guitar.

In college, I was naturally interested, as many students are, in one’s own psychology. At the time, psychology had the biggest classes and was a very popular major. But my leanings were more toward understanding the biological basis of brain function. At the time, there was not a major for neuroscience, so I pieced together a degree in biochemistry that would prepare me best for studying the brain. I was particularly lucky in my last two years to be hired to do research in Robert Scobey’s lab. Scobey studied the processing of visual information by retinal ganglion cells, and I participated in single neuron recording experiments in the retinas of cats, examining the response of these cells to small displacements of visual stimuli.

I also enjoyed nonacademic pursuits in college; maybe too much—but it was, after all, the late 1960s and early 1970s, which were socially dynamic times. I started a student newspaper and was a disc jockey at the college radio station, hosting a jazz and blues radio show. One of my friends went to work at a radio station in the Bay Area, and he hoped I would join him. DJ or scientist? I did go to San Francisco, but chose the latter career path.

Graduate School

After receiving my B.S. from UC Davis I entered the Physiology Graduate Program at UC San Francisco (UCSF). Again there was no formal neuroscience or neurobiology program, but UCSF had a number of young and now famous neuroscientists on the faculty at that time.

During my first year at UCSF, I worked in the lab of Ben Libet (see Volume 1). He was collaborating with a neurosurgeon on the timing of the awareness of sensation in patients. Later Libet did a groundbreaking experiment in which he found that the awareness of movement intentions came after the movement plans had already been formed. He interpreted the results as showing that the sense of free choice was not causal to action planning. He wanted me to work on his other main topic, which was neural transmission in the superior cervical ganglion by way of second messengers. Although these were interesting experiments, I decided to transfer to another lab, that of Michael Merzenich (see Volume 7).

Merzenich came from the Hopkins school of neurophysiologists, being a graduate student with Vernon Mountcastle (see Volume 6). At Hopkins, Merzenich studied the somatosensory system and, as postdoctoral fellow with Jerzy Rose from the University of Wisconsin, subsequently studied the auditory system. Merzenich was one of the primary developers of the cochlear prosthetic. Seeing the neural plasticity in humans who learned to interpret the rudimentary artificial signals from the prostheses, led him to become intensely interested in brain plasticity. He next performed famous experiments with Jon Kaas and colleagues on the plasticity of cortical somatosensory maps after deafferentation from peripheral nerve lesions. Merzenich was a tremendous and inspirational mentor who emphasized studying big ideas.

Thesis Research

In Merzenich’s lab, I studied the anatomy of the auditory system in cat, detailing the topography of the cortical-cortical, cortico-thalamic, and cortico-collicular connections. From these experiments, I found two parallel systems. Highly tonotopically organized cortical fields were connected to corresponding tonotopic regions of the thalamus, whereas another, nontonotopically organized cortical area was connected to more complex and multimodal areas of the thalamus. These two pathways, which I called “tonotopic” and “diffuse,” suggest that the former system is very much like visual cortex with its strong topography, and the latter is highly integrative (Andersen, Knight, and Merzenich 1980).

Perhaps the most remarkable finding from my thesis research was that all cortico-cortical and thalamocortical pathways are highly reciprocal. We were using new anatomical tracers, one that was transported retrogradely and another anterogradely. By mixing the anterograde and retrograde tracers and making punctate cortical injections, a technique developed by one of my fellow graduate students Steve Colwell, the resulting labeling showed nearly identical patterns of projection for both tracers. This result indicated a precise reciprocity of connections between cortical areas and between the cortex and thalamus. Although other track tracing techniques had by that time shown that these connections were reciprocal, the newer tracers that we employed, horseradish peroxidase and tritiated amino acids, provided the first detailed demonstration of the spatial precision of this reciprocity. These results were similar to Colwell’s earlier findings in the rabbit visual cortico-thalamic system. They bear out the notion that all cortico-cortical and cortico-thalamic connections are precisely reciprocal, which to my knowledge is a rule to which subsequent research has not found an exception.

First Interest in Posterior Parietal Cortex

UCSF, which at the time was located only at the hospital on Parnassus Avenue, had a tremendous medical library. It was there that I would read books and papers by famous neurologists like Critchley, Luria, Geschwind, Holmes, and Balint. I found the deficits from posterior parietal lesions particularly fascinating. For instance, Balint’s syndrome, first described by the Hungarian neurologist Rezso Balint in 1909, results from bilateral lesions of the posterior parietal cortex (PPC). These subjects have difficulty making voluntary eye movements, misreach for objects in the peripheral visual fields, and cannot perceive multiple objects simultaneously. Another example deficit is neglect, which results from unilateral lesions, particularly of the right hemisphere of PPC. Patients with neglect are not blind but have an attentional deficit in which they are seemingly unaware of visual space contralateral to the lesion. I had the opportunity to go on rounds at a hospital while a graduate student and observed patients with PPC lesions. I was struck by how similar these patients’ deficits were to those described in the literature.

Postdoctoral Study

Around the time of my graduation, a few labs were using the new technique of single neuron recording in behaving non-human primates (NHPs). Luck would have it that one of the labs was Vernon Mountcastle’s, and that Mountcastle was examining higher cortical functions in the PPC of NHPs. Following Merzenich’s advice, I contacted Mountcastle and he accepted me for postdoctoral study. Mountcastle was a giant in the field, having discovered the cortical column, a shaft of neurons arranged vertically within cortex with similar response properties. These columns are the basic building blocks of much of cortex. Mountcastle was the world leader in quantifying the relation of neural activity in the somatosensory system to perception. I had heard stories that he was a tough taskmaster and, since I was a bit of a night owl, Merzenich thought the discipline of working in Mountcastle’s lab would be good for me. However, on my arrival in Baltimore I found Mountcastle to be an amazingly devoted, inspiring, and exacting teacher. He had gathered at Hopkins top researchers in NHP research, including faculty Apostolos Georgopoulos, Mahlon DeLong, and Gian Poggio. Postdoctoral fellows beside myself in Mountcastle’s lab were John Kalaska, Roberto Caminiti, and Brad Motter, who all went on to stellar careers in systems neuroscience. Around the time of the move I married Carol Ahern, an audiologist. We met at a seminar on the cochlear hair cells of the inner ear at UCSF. We moved together to Johns Hopkins in Baltimore, where she entered the master’s program in audiology at Hopkins.

Mountcastle was interested in how perception leads to action along cortical pathways. This led him to the PPC because it is the major association cortical area bridging between somatosensory cortex and premotor cortex. His experiments on this sensory-to-motor transformation required recording from neurons while animals experienced perceptual goals and directed actions toward these goals. With the help of Ed Evarts from NIH, who was recording from the motor cortex of behaving NHPs, Mountcastle retooled his lab to record from PPC in behaving NHPs. What he found has provided the foundation for research in PPC to this day. In landmark papers in 1975 and 1977, Mountcastle and his colleagues described neural responses to specific actions and high-order sensory phenomena. Numerous cells were selective to the particular voluntary oculomotor behaviors of saccades, smooth pursuit, and fixation. Others responded to reach or hand manipulation of objects. High-level visual sensory properties included modulation by attention, and responses to global motion patterns that are indicative of flow fields during locomotion. Mountcastle proposed a command hypothesis in which PPC integrated sensory information and emitted command signals for action, which were elaborated by downfield motor areas. In 1978, Michael Goldberg (see Volume 10) and colleagues from NIH published a paper in NHPs in PPC. They replicated many of the findings of Mountcastle, but they proposed a different interpretation in which the observed changes in PPC activity were better explained by visual attention. It is not too surprising, in retrospect, that an area involved in visuomotor transformations would have both high-level motor and sensory properties.

Gain Fields

My main research at Hopkins involved the discovery of “gain fields.” The PPC has long been implicated in spatial awareness, as evidenced by spatial-perceptual deficits in humans with lesion to this area. Among the spatial deficits, people fail to be aware of visual stimuli in the contralateral space, show spatial memory defects, are aware of only half an attended object, and have difficulty in navigation. From these observations, I reasoned that the PPC would have visual receptive fields anchored to the outside world. In healthy subjects, this hypothesized anchoring could account, for instance, for the world appearing to be stable in spite of the many eye movements we make, which constantly change the images on the retinas. Likewise, a coordinate transformation from retinotopic receptive fields in early visual areas is required for observers to make visually guided body movements. An example is that observers can reach to an object accurately, independent of gaze direction.

What Mountcastle and I found was that the visual receptive fields in PPC remained retinotopic—that is, anchored to the retina. But we also found that many of the visually responsive neurons are also modulated by the direction the eyes are looking in the orbits of the skull—that is, they convey eye position signals (Andersen and Mountcastle 1983). Moreover, while some neurons carried only eye position or retinal signals, it was most common that individual neurons carried both signals. This convergence of different eye position and retinal signals on individual neurons in PPC could conceivably represent locations with respect to at least the head, invariant of eye position. Mountcastle felt rather that the systematic modulation of the gain of the visual response by eye position had a role in attention, not coordinate transformations, and so my first paper on gain fields had essentially no discussion.

Faculty Years: Parallel Paths in Research

Over my past 40 years of research, and still going, I followed a few selective lines of research. I was a faculty member at three institutions, first the Salk Institute, then MIT, and then Caltech. I will follow the paths of research through these great institutions in chronological order, but my major research interests spanned these sites. From my postdoc days, I continued my interest in spatial representations and coordinate transformations. A second interest was, and is, the encoding of intent in the posterior parietal cortex. And a third is studying structure from motion processing in the pathway leading from primary visual cortex into the PPC.

Along the way, I transitioned from primarily using the behaving NHP paradigm to performing clinical studies in humans (although we are still doing NHP studies). This path was made possible by applying what we learned about the PPC in NHPs to translate to neuroprosthetics for tetraplegic participants. We use brain-machine interfaces (BMIs) which record brain activity from electrodes implanted in the brain and translate the signals to control external devices such as robotic limbs or computers. BMIs typically have used motor cortex activity for control. We refer to our approach as cognitive neuroprosthetics because we implant the electrodes in PPC, a high-level/cognitive brain area, and we record the intent of the individual. As one might predict from association cortex, recording in PPC enables access to a plethora of different signals, from intended movements of all parts of the body to high-level cognitive signals, such as those related to semantics and memory decisions. This has been a thrilling ride—that is, to do basic research in NHPs and then to translate that research directly in clinical studies. Moreover, the work in humans has led to new scientific discoveries that contribute to new prosthetic applications.

Needless to say, BMIs allowed us to sail into rather uncharted territory, as we had never seen the human cortex functioning at such fine temporal and spatial scales. And, unlike animal studies in which inner mental processes are at best inferred, we can ask the participants how they did a task, what they felt with microstimulation, or what they imagined introspectively. This new clinical direction has left me at this point in my career excited every day about what new results we will find and how they will contribute to helping individuals with paralysis.

The Salk Institute

In some ways, my disagreement with Mountcastle about gain fields left the field of coordinate transformations open to me, and I continued studying this topic when I moved to my first faculty position as an assistant professor at the Salk Institute in 1981. Mountcastle felt it would be a good move for me to go to the Salk. I could concentrate on research without teaching or administrative duties, as the Salk was small and almost entirely research oriented. Mountcastle told me his first few years as a faculty member at Hopkins were similar in that he had few teaching and administrative responsibilities. Carol and I had our son Michael at the Johns Hopkins hospital and three months later we moved to San Diego.

At the Salk Institute, I was within the division directed by Max Cowan, a well-known neuroanatomist (see Volume 4). The process of setting up my behaving monkey lab and collecting the first neural data would take some time. So, to get an early start, I took advantage of working in Cowan’s state-of-the-art neuroanatomy lab to begin examining the neural connectivity of the PPC. In Mountcastle’s papers, he had found a clumping of response properties for saccades, pursuit, and reaching. He speculated that a columnar organization in PPC was made up of mixed saccade, pursuit, and reach columns.

I reasoned that the best way to visualize the saccade columns in PPC was to make tracer injections into the frontal eye field (FEF). The FEF is a cortical region in the frontal lobe specialized in producing voluntary saccades, and a presumed target of saccade neurons in PPC. Instead of finding columns in PPC, the major reciprocal connections with FEF were mostly with a single cortical area within the lateral bank of the intraparietal sulcus, which we named the lateral intraparietal area (LIP) (Andersen, Asanuma, and Cowan 1985). LIP has become popular for the study of early eye movement processing, including movement planning, attention, and decision making. Other columns postulated by Mountcastle in PPC also turned out to be cortical areas, including the medial superior temporal area for pursuit eye movements and the parietal reach region for reaching.

With Jim Gnadt, a postdoctoral fellow at the time and now a program director at the National Institute of Neurological Disorders and Stroke (NINDS), we studied the saccade properties of LIP neurons. We found that if (1) a saccade target was briefly flashed in the visual response field of an LIP neuron, and (2) the monkey had to withhold a saccade to that target location for a delay period, the neuron remained active throughout the delay period. To show that this persistent activity was not a memory signal for the location of the visual target, we also used a task in which the flashed saccade target (the cue) was delivered outside the neuron’s response field. The monkey was then instructed to make a saccade that brought the cued location into the response field. In this case, the delay period activity emerged when the previously cued saccade target location was now in the response field of the neuron. Jim and I reasoned that the activity represented the intended movement of the animal, and not the memory of the location of the target (Gnadt and Andersen 1988).

My early description of gain fields with Mountcastle was limited to only a few eye positions and retinal visual stimulus locations. With postdoctoral fellows Ralph Siegel and Greg Essick, we continued the examination of gain fields in PPC with much more thorough mappings of the visual receptive fields at different eye positions. These new findings reinforced our earlier interpretation that the response fields were strongly retinotopic, and the modulation by eye position could be best modeled as the product of an eye position gain factor on a retinotopic visual receptive field. Likewise, these new results strengthened the interpretation that the eye position–dependent modulation produced a tuning for locations in head-centered space (Andersen, Essick, and Siegel 1985).

As mentioned, a strategy I used to jump-start my lab at the Salk was to do anatomical experiments, which were sure to render quick results, while building up a recording lab for behaving monkeys. Another strategy was to pick two major lines of research as a precaution in the event that one did not succeed. The first, outlined earlier, was to study the visual-motor properties of PPC neurons. The second was to study motion perception. The motion pathway had already been well established anatomically as proceeding from layer 4b neurons in the primary visual cortex (V1), to the middle temporal area (MT), to the medial superior temporal area (MST) in PPC. In the end, both lines of research were successful and certainly kept me busy.

The three-dimensional (3D) structure of objects can be perceived from two-dimensional (2D) motions of random moving dots that are arranged as if they were dots painted on a rotating object projected onto a 2D screen. A nice feature of this stimulus is that it is ambiguous, and sometimes the object is seen to rotate in one direction, but then it flips to appear to be rotating in the opposite direction. This stimulus can be made unambiguous by adding disparity to the dots. Thus, I started investigating structure-from-motion (SFM) along the motion pathway in NHPs. Beginning at the Salk with Ralph Siegel, we showed in psychophysical experiments that monkeys perceive SFM and that both monkeys and humans integrate spatial and temporal parameters of SFM similarly (Siegel and Andersen 1988).

A major influence on me during my time at the Salk Institute was Francis Crick. I had read James Watson’s book, The Double Helix, which begins with the famous line “I have never seen Francis Crick in a modest mood.” It was thus with some initial trepidation that soon after I arrived at the Salk, Francis walked into my office, extended his hand, and introduced himself. To my delight, he became a supportive and brilliant mentor.

Crick had taken an interest in consciousness as the other major question in biology from the one he had already tackled successfully: what is the genetic code? To this end, Crick; V. S. Ramachandran, a vision scientist from UC San Diego (UCSD); and Gordon Shaw, a physicist from UC Irvine (UCI), organized the Helmholtz Club. There were about 20 members from around southern California. Besides those of us from San Diego, regulars included Stan Schein, Joaquin Fuster (see Volume 7), John Shlag, and Madeleine Schlag-Rey from UC Los Angeles (UCLA) and John Allman and David Van Essen (see Volume 9) from Caltech. Every month or so, we would have a meeting at the UCI faculty club in which two renowned speakers from around the world gave extended talks with much interaction followed by dinner at a nearby restaurant. These were lively meetings. I recall that Richard Gregory, a famed British psychophysicist, barely got past his first slide before there was a vigorous, long debate with Francis. Of course, this was a bit of an exception from most presentations, but the long format provided a tremendous avenue for presenting research in depth with lively discussion.

One nascent field that was emerging from these meetings, from neural network researchers at UCSD, and from artificial intelligence (AI) researchers at MIT, was computational neuroscience. This move toward more theoretical neuroscience helped me greatly to formulate my own research with more of a theoretical perspective.

I was particularly fortunate that right across the street from the Salk, at UCSD, there was the Parallel Distributed Processing group, a collection of mathematical psychologists, including David Rumelhart, Geoffrey Hinton, and James McClelland, who were advancing connectionist models of cognition. In 1986, this group developed backpropagation, a method for training multilayered neural networks by propagating error back through the networks. In 1988 David Zipser, a member of this UCSD group, and I published a paper in Nature, which was one of the first uses of neural networks to model a brain computation and to compare the network to neural data (Zipser and Andersen 1988). We trained a three-layer neural network that received retinotopic targets and eye position signals as inputs and, as outputs, produced receptive fields that coded the targets in head coordinates. After training the network with backpropagation, we found that the middle layer units developed gain fields, with retinotopic visual fields modulated by eye position very similar to the recorded neurons in PPC. Thus, the network model demonstrated that gain fields could be a method of computation for making coordinate transformations.

San Diego was a great environment for me and my family. Our daughter Kristen was born there. The Salk Institute was a great place to focus on basic research. We lived in Encinitas, a beautiful beach town just north of San Diego. I even learned to surf when I was there. In part through Crick and the Helmholtz Club, I also got to know several of the faculty in the Brain and Cognitive Sciences Department at MIT. MIT was the center for AI. At the time, AI referred to more algorithmic/symbolic theories of brain function. Many neuroscientists in my generation were deeply influenced by the posthumously published book Vision by David Marr, one of the luminaries in the MIT AI group. His book emphasized three levels for understanding the visual system: computation, algorithm, and hardware. The MIT faculty in Brain and Cognitive Sciences was an impressive group of neurophysiologists, cognitive scientists, and AI researchers. I was honored to receive a job offer from them, and it was an opportunity that was too great to pass up. So in 1987, we left the West Coast behind and moved to Boston. My family and I settled outside of Boston in Wellesley.

MIT

At the Salk, I had the only behaving monkey lab. At MIT, there were two giants of behaving monkey research. Emilio Bizzi was one of the founders of the field of motor control, studying how the motor system controls the trajectories of the limbs. He was also the chair of the department and was very supportive, patient, and a great leader of the faculty (see Volume 6). Peter Schiller was an eminent visual neurophysiologist who has made many seminal findings of the cortical processes of vision and attention (see Volume 7). Ann Graybiel was the world expert on the anatomy and functioning of the basal ganglia, and Sue Corkin was a renowned neuropsychologist. I mentioned the AI component of the department that included the giants in the field, among them Tomaso Poggio (see Volume 8) and Simon Ullman. It was a most fertile ground for the study of systems neuroscience.

Structure from Motion

MIT was a perfect place to study SFM. Faculty members Shimon Ullman and Ellen Hildreth were world experts on the topic. At the time, there was a running challenge in the literature to see what algorithms could determine whether a rotating object was rigid or not from viewing the fewest dots and the fewest frames.

We chose the topic of how the perception of surfaces is produced from SFM. When viewing a few dots on the surface of an otherwise invisible cylinder, one gets the clear impression that there is a surface between the dots. With postdoctoral fellow Masud Husain and graduate student Stefan Treue, we used random dot patterns that had limited lifetimes before they were repositioned to another location on the surface of the object defined by motion. We found that humans would integrate the information from such displays spatially and temporally across several point lifetimes, providing strong evidence that the brain constructs perceived surfaces from transient velocity information to produce more global surfaces. In fact, points could be left out of a quarter of an object’s surface and the interpolation process would still fill in a perceived surface. Ellen Hildreth, collaborating with us, showed that a computational model of surface interpolation could account for all of the aspects of SFM perception that we had found.

The SFM displays we used had no disparity depth cues, being presented as moving dots on a flat screen. Yet, even viewed with just one eye, the motion fields produced vivid percepts of a 3D object. How is it possible for the nervous system to separate these directions into separate surfaces, since each patch of the display contains motions moving in different directions? Particularly problematic is the mechanism of motion perception itself, which is a result of inhibitory interactions between neurons. The question has even broader implications about how we can perceive transparency. For instance, we can make out both the reflection on a window and the scene behind it. We postulated that there would be two stages in the visual pathway. The early stage would measure local motions. The later stage would integrate the motion signals from the earlier stage more globally to construct the perception of surfaces.

Ning Qian, a postdoc in the lab, had the brilliant idea of presenting exactly balanced motion signals at every sample location on the surface. This idea was motivated by the observation that counter-phase sine-wave gratings, which are mathematically equivalent to two sine wave gratings drifting in opposite directions, produce no perceived motion but rather static standing wave oscillations. We paired each moving dot with another dot moving in the opposite direction. This display destroys the perception of moving transparent surfaces, and rather appears like a field of flickering points.

Our recording experiments in areas V1 and MT of NHPs explained the phenomena of transparent surface perception. V1 neurons measured local motions with their small receptive fields, and did not reliably distinguish between the paired, perceptually nontransparent stimuli and the unpaired, perceptually transparent stimuli. In contrast, the directionally selective MT neurons, with their large receptive fields, responded better to the nonpaired stimuli than to the paired stimuli. Ted Adelson is a computational neuroscientist at MIT who introduced the concept of motion energy to explain neural interactions that produce direction selectivity. With Ted, we built a computational model and tested it with the same stimuli we used in the NHP experiments. The first stage models V1, which measures local motions and does not distinguish between transparent and nontransparent motions. The second stage models MT, in which motion energies of opposite direction but the same spatial frequency and disparity contents suppress one another. Like the MT neural responses, this second stage distinguishes between transparent and nontransparent motions. These results were published in three companion papers in the Journal of Neuroscience, which beautifully explained transparent motion perception based on psychophysical measurements and neural recording data, and conceptualized the process with a computational model (Qian, Andersen, and Adelson 1994a, 1994b; Qian and Andersen 1994).

Optic Flow

Another important function of motion is determining the direction of locomotion for navigation. The next stage in the V1-MT pathway is the dorsal division of the medial superior temporal area (MSTd). This area has large, bilateral receptive fields that are selective for patterns of motion that one sees when moving through the environment. Neurons were described that were sensitive to expansion/contraction (like a field of stars while moving through space in a science fiction movie), rotation, and translation. These different motion patterns could be decomposed by the nervous system into three channels (expansion/contraction, translation, and rotation) for analyzing the optic flow field. A possible advantage of such a decomposition is that translation motion due to eye movements could be subtracted from expansion components due to locomotion to determine the focus of expansion, which would correspond to the direction of heading.

Mike Graziano, Bob Snowden, and I tested this decomposition hypothesis by constructing a 2D spiral motion space, which had on the horizontal axis clockwise/counterclockwise rotation, and expansion/contraction on the vertical axis. A neuron tuned to 45 degrees in this space, for instance, would be selective to a clockwise outward spiral. We found many MSTd neurons that were tuned to spirals, indicating that a simple three channel decomposition is not correct, and rather there is a continuum of motion pattern selective neurons. We also found that the tuning was invariant for patterns over these large receptive fields. Thus, single neurons cannot determine accurately the direction of heading from expansion and a course coding at the level of populations of neurons would have to account for the precision of heading direction from optical flow patterns (Graziano, Andersen, and Snowden 1994). In a subsequent experiment with Bard Geesaman, we showed that the tuning of MSTd neurons to complex motions was invariant for the type of stimuli, whether they were moving dot patterns, solid squares, or a figure/ground display (Geesaman and Andersen 1996). Thus, MSTd neurons would appear to be involved in both the analysis of object motion as well as optic flow from observer translation.

Saccades and LIP

After having discovered LIP at the Salk Institute, two postdoctoral fellows in my lab at MIT, Shabtai Barash and Leonardo Fogassi, and graduate student Martyn Bracewell, began looking at LIP response properties in detail. LIP could be differentiated from adjoining area 7a of PPC by saccade timing. We found that the saccadic responses of most LIP neurons began just before saccades, whereas responses in 7a were mostly postsaccadic. The response fields in LIP were well defined, and their visual, delay, and saccade responses were spatially overlapping. Peter Their, who was doing a sabbatical in the lab, showed that microstimulation of LIP produced saccades, although requiring higher currents than in the frontal eye field in the frontal lobe. In experiments later carried out at Caltech, we further showed that pharmacological inactivation of LIP produced deficits in the accuracy and dynamics of saccades, particularly for saccades to remembered locations and primarily in the visual field contralateral to the inactivation. The combination of evidence from neurophysiology, neuroanatomical connectivity, microstimulation, and inactivation led us to conclude that LIP is a posterior eye field for saccades in parietal cortex, much like the frontal eye field is for saccades in the frontal lobe.

Gain Fields

While at MIT, we also extended research on gain fields. Previously, we had shown gain fields in area 7a of PPC, but in further study, we found them to exist in LIP as well. Large shifts in gaze combine eye and head movements. Interestingly, we discovered that neurons in LIP had both eye and head gain fields and that these gain fields were aligned (Brotchie et al. 1995). Thus, the gain fields in LIP are, in fact, modulating visual responses by the gaze direction with respect to the body.

We also extended our theoretical studies of gain fields. We found that microstimulation of different locations in LIP produced saccades in different directions toward the contralateral field. However, the initial eye position fixation modulated the amplitude of the saccades, similar to simulations of microstimulation of middle-layer units in the neural network models of PPC. As a serendipitous control, we found that microstimulation of cortex in the intraparietal sulcus just ventral to LIP produced complicated and often head-centered goal-directed saccades when varying eye position. These stimulation results were consistent with retinotopic-eye position modulated neurons in LIP, which could converge onto the more ventral location in the intraparietal sulcus to code goals in head-centered coordinates.

A common criticism of neural network modeling of brain processes at the time was that backpropgation learning is not biologically plausible. With Pietro Mazzoni, a graduate student in the lab, and Michael Jordan, a faculty member in our department at MIT, we showed that a biologically plausible reinforcement model produced gain fields when training neural networks to convert stimulus locations from retinal to head centered, and with approximately the same number of training cycles as with backpropogation (Mazzoni, Andersen, and Jordan 1991). Thus, our networks were consistent with long-term/skill learning with biologically realistic constraints.

Caltech

When I was up for tenure at MIT, which I received, I was also offered positions at Rockefeller University and the California Institute of Technology. My wife Carol was a fourth-generation Californian on one side of her family and fifth on the other. MIT was an excellent academic environment for me, and New York City is in my roots, but for Carol, the desire to get back to California was very strong. Needless to say, Caltech and MIT have numerous similarities, particularly in my areas of interest of the visual system and computational neuroscience. Given these excellent opportunities it was not an easy decision, but family won out and we moved to California in 1993. My son was in sixth grade at the time and my daughter was in third. The transition for them was pretty smooth. My son and his wife are now graphic designers living in Brooklyn, back near where my parents grew up, and my daughter is a pediatrician in Redondo Beach, just south of Los Angeles.

An advantage of moving is that one can design a new lab fitted to exploring new research directions. In my case, I wanted to add vestibular and auditory stimuli to the arsenal of sensations we could study. This opportunity was particularly important because the PPC is an association cortical area, and these senses, along with somatosensation and vision, all converge on this multimodal area (Andersen et al. 1997).

Caltech: Monkey Days

Intention

Intention and attention are intertwined. It was argued by some that the delay activity we saw in LIP was a correlate of attention. In fact, when one plans to look somewhere, visual attention is also directed to that location. But attention is a sensory process. If you plan to reach to a location, then attention is likewise directed to that location. But the plans, to saccade or reach, are very different since the action involves separate effectors. In experiments with Larry Snyder and Aaron Batista, we reasoned that LIP would be more active for planned eye movements than for planned reaches if it was coding intent, but would show similar activities if it was encoding spatial attention (Snyder, Batista, and Andersen 1997). In fact, we found the former. Serendipitously, we also found that regions within the intraparietal sulcus on the medial bank showed the opposite pattern of activity, being selective for reaches but not saccades. We termed this area the parietal reach region (PRR), which included the medial intraparietal area and area V6. Another area in the intraparietal sulcus that is anterior to LIP is the anterior intraparietal area (AIP). Hideo Sakata and colleagues found that single neurons in AIP coded entirely different grasps. These findings of eye movement, reach movement, and grasp movement fields in the intraparietal sulcus led Chris Buneo and me to propose that there is a map of intentions within PPC (Andersen and Buneo 2002).

In further experiments, we showed that PPC can plan simultaneously a sequence of reaches. Also, if NHPs do not know early in a trial whether a reach or a saccade will be required to obtain a target, they will plan movements to both; in other words, PPC can form intended movement plans that can be scrubbed later.

Gain Fields and Multimodal Integration

In the move to Caltech, we installed an anechoic chamber for auditory experiments, which has no reflections of sound off the walls of the chamber and is ideal for sound localization. Another addition was a 3D vestibular chair that could move the NHPs around all axes in three dimensions and record eye position at the same time. Included in our arsenal of new paradigms was training monkeys to rotate their heads on their bodies without changing gaze direction.

Not surprisingly, given that PPC is an association area that integrates different sensory modalities, we found that PPC neuronal activity was affected by vision, audition, and vestibular inputs, but in very specific ways. When passively stimulated with auditory and visual stimuli, in which the stimuli had no behavioral significance, LIP neurons respond only to visual stimuli. Thus, in terms of passive stimulation, LIP would be considered a visual and not an auditory sensory area. This preferred selectivity for visual stimulation is not surprising given its role in eye movements. However, graduate student Jennifer Linden and postdoctoral fellow Alexander Grunewald found that when saccading to sounds in space, LIP is also activated by the auditory stimulus. Auditory targets are initially encoded in head coordinates in early stages of the auditory system, given that locations of sounds in space are derived from intra-aural cues of phase and intensity at the two ears. However, the auditory stimuli in LIP were represented toward eye coordinates, although many were intermediate between eye and head coordinates. Thus, the auditory target representation is shifted toward a common coordinate frame with visual stimuli in LIP for making saccades (Cohen and Andersen 2002). Eye position modulated the auditory responses, suggesting that gain fields also account for the coordinate transformation of auditory space from head to eye coordinates.

Another way that gain fields, and presumably coordinate transformations, are handled in PPC came from studying how head position signals are represented in LIP and area 7a. By using various combinations of head position with respect to the body and world using a vestibular chair, we found that neck proprioceptive signals produced gain fields in LIP, whereas vestibular signals also accounted for gain fields in area 7a (Snyder et al. 1998). Thus, LIP, using the sense of the position of the head on the body, could code gaze location (eye plus head position) with respect to the body. Area 7a projects to the presubiculum of the hippocampal formation and, using the vestibular sense of the position in the world, could code visual stimuli in the world (so-called allocentric coordinates). This result is consistent with the finding of place and grid cells, which code in allocentric coordinates, in the hippocampal complex.

The Parietal Reach Region

The natural coordinate frame for coding a visually guided reach is for the visual target to be in the coordinates of the hand—that is, the movement vector from the hand to the target. But surprisingly, we found that PRR codes reaches, but in eye coordinates, not in hand coordinates (Batista et al. 1999)! Again gain fields were present in PRR, and they included modulations for gaze and limb position. Thus, the signals were all there for converting visual targets to hand coordinates further along in the sensorimotor pathway for reaching.

Gain fields for eye position have been measured by a number of groups since our initial findings in PPC and in a large number of cortical areas. These findings suggest that it is a general method for making coordinate transformations. However, to sample even two variables, for instance, eye and target position, in two dimensions (horizontal and vertical) in different combinations, requires a large number of trials. This experimental version of the “curse of dimensionality” is even greater if a third variable, hand position, is included. Chris Buneo came up with a novel idea, which was to use a matrix design for plotting pairs of the three variables (gaze, target, and hand position) in three matrices, and then to calculate the gradients of the matrices for the firing rate changes. This technique could distinguish gain fields from coordinate frames. For example, when plotting the firing rate of a neuron as a function of horizontal limb position and horizontal retinal stimulus location, the activity will vary only as a function of limb position for a gain field. For a visual stimulus in hand coordinates, the activity will vary for both hand position and retinal location to maintain selectivity for a location with respect to the hand. With this technique, we reaffirmed that PRR codes visual targets in retinal coordinates, but moving forward in the sensorimotor pathway to area 5d in PPC, anterior to PRR, we found a transition to representing the target in hand coordinates (Buneo et al. 2002). Interestingly, we also found that the coordinate representations in area 5d were dynamic. In the task, initially only cues of where to look and where to initially place the hand were present. In that case, the neurons coded only the hand position relative to the gaze direction (hand-eye coordinates). When the target for the reach appeared, the reference frame switched to coding the hand relative to the target (Bremner 2014). In our subsequent PPC recordings in humans, we have found similar coding of imagined hand movements to visual targets to be in hand coordinates, suggesting that the PPC recordings in humans are further downstream, like area 5 of the NHPs. We use imagined hand position because the subjects are tetraplegic and cannot actually move their limbs. We also found that the dynamics in the coordinate frames depend on the tasks. If the human subjects are making eye movements, the PPC neurons code the target in retinal coordinates, and if they are imagining hand movements, the same population of neurons code the target in (imagined) hand coordinates.

Perhaps the most direct evidence suggesting that the gain fields are important for coordinate transformations comes from the inactivation of PRR in NHPs. Although the representation of targets in PRR are in retinal coordinates, modulated by eye position, we found that inactivation of PRR produced one of the hallmarks of Balint’s syndrome, optic ataxia, which is the misreaching to visual targets. These reach inaccuracies were not due to a general sensory effect, since saccade deficits were not found. Thus, the inactivation of PRR interferes with the spatial transformation of the retinotopic locations of stimuli to hand coordinates for spatially accurate reaches. In other words, PRR plays a critical role in coordinate transformations (Hwang et al. 2012).

It is remarkable that so much of the cortex is affected by eye position. What is the source of this signal? It could be derived from proprioceptors in the extraocular muscles. Alternatively, it could be derived from an efference copy of the command to move the eyes. One study suggested that the gain fields in LIP are too slow to follow the rapid number of eye movements we make, whereas another found that their dynamics in LIP match the psychophysics of perceived stability of the world across eye movements. Using statistical modeling, we found that there is even a prediction in the population response of LIP neurons of where the new position of the eyes will be prior to an eye movement (Graf and Andersen 2014). Because proprioceptive and efference copy signals are prominent in cortex, it is likely that both contribute to gain fields.

Internal Models

In studying the dynamics of reach movement-induced activity in PPC of NHPs, we found that the responses were too fast (occurring before reaches) to be sensory feedback, and too slow to be the command signal to move the limbs, since PPC activity began after M1 activity. This finding we interpreted as consistent with an internal model of the state of the limb (Mulliken, Musallam, and Andersen 2008). Internal models are popular in the motor control literature, as they can take out the lags introduced in the nervous system due to brain processing. These internal models are updated by sensory inputs as a consequence of movement as well as monitoring movements with internal copies of the commands (efference copies) sent out by motor cortex. But in PPC, they are also likely to be more general and provide a model of the position of the body in the world. This idea of PPC being a site for an internal model has been proposed by neuropsychologists studying deficits in the PPC, in which patients are slow or unable to correct movements. Interestingly, in our human experiments, we found that receptive fields for reach can be changed even when the tetraplegic subjects, who cannot move their limbs, imagine their hands at different locations in space. Presumably, imagination is able to access the internal models.

Free Choice

A large body of research focuses on decision making in NHPs. Generally, animals choose between alternatives that vary the amount, probability, or type of reward. In orbital frontal cortex, decisions are made in selecting between potential types of rewards. In PPC, decisions are made between actions. Alex Gail at Gottingen showed that potential action plans are represented in PPC for two potential targets, followed by a decision favoring one plan. This result is consistent with what we found with decisions between effectors. In our study with He Cui, we found that a potential plan for reach is made in PRR and for a saccade in LIP, and when the animal is cued to make a free choice between saccading and reaching, one plan remains and the other disappears (Cui and Andersen 2007). This action choice mechanism, and preplanning, affords rapid decisions and actions over short timescales between competing alternatives.

We asked whether PPC is actually part of the circuit involved in decision making, or whether it just reflects decisions made elsewhere. The effector selectivity of PRR and LIP allowed us to address this question. NHPs were given the choice to move to two targets, one in each visual field, to obtain equal reward for either choice. Postdoctoral fellows Vasileios Christopoulos, James Bonaiuto, and Igor Kagan showed that PRR inactivation biased the animals to make more ipsilesional choices for reaches, but not saccades. The reverse was found for LIP, biasing saccades more than reaches. The dorsal pulvinar is a major thalamic input to PPC. Postdoctoral fellows Melanie Wilke and Igor Kagan found that inactivation of the pulvinar produced a bias for ipsilesional saccade choices when saccade targets were simultaneously presented in the two visual hemifields. If, however, rewards were increased for the contralesional saccades, this bias was largely erased. Thus, the animal could see the target in the contralateral visual field, but it became less appealing and thus was chosen less often. Increase in the salience of the contralesional target could not explain the magnitude of the reward effect, because increasing the contrast of the contralesional target produced much less modulation of the decision bias than increasing the reward.

A major deficit of PPC lesions, particularly to the right hemisphere, is neglect in which patients fail to be aware of stimuli located in the contralateral space. What has been thought previously to be a milder form of neglect is extinction. For extinction, subjects can see stimuli in the contralesional visual field. However, when a competing stimulus is simultaneously presented to the ipsilateral space, the patients report only the ipsilesional stimulus. Our experiments with changing reward between competing saccade targets after pulvinar inactivation suggest that an element of extinction may be a deficit in decision-making circuitry. These inactivation data explain why patients with PPC lesions tend to explore the contralesional space with eye movements less often.

If PPC is involved in action decisions, how do other areas in the frontal lobe coordinate the action plan with PPC? Using simultaneous recordings from PRR and dorsal premotor cortex (PMd), we found that correlations between these two areas are stronger during free choices than during instructed movements. This finding suggests that there is a subpopulation of neurons in the frontal and parietal cortex that orchestrates activity between these areas during free choice (Pesaran, Nelson, and Andersen 2008).

Visual Motion

At Caltech, we made great strides in understanding the neural mechanisms for SFM perception and optic flow navigation. These studies were made in behaving NHPs with single neuron recordings from MT and MSTd. Much of this research was headed by a brilliant postdoctoral fellow, David Bradley.

Structure-from-Motion

At MIT we had found that V1 neurons measured local motions for transparent two-surface random dot patterns and that the responses of MT neurons to the transparent stimuli were suppressed by the two surfaces moving simultaneously in the preferred and antipreferred directions, compared with neuronal responses to single surfaces moving in the preferred direction. Neurons in MT are also selective for binocular disparity. Natural transparent stimuli usually would contain other surface cues, such as depth from binocular disparity. When we presented the two surfaces at different depths using disparity cues, we found that the inhibition occurs mainly for transparent stimuli at the same depth, which is consistent with the idea that this inhibition exists to reduce spurious motion signals (noise). Thus, transparent surfaces are represented independently in MT when binocular disparity cues are present to disambiguate the depth ordering of surfaces (Bradley, Qian, and Andersen 1995).

In a follow-up study, we used the illusion of a rotating transparent cylinder composed of random dots. This display is perceptually bistable, much like a Necker cube and will appear to flip front and back surfaces, and thus, the direction of the perceived rotation of the cylinder. We trained NHPs to indicate the surface order they perceived, even though the visual stimulus was always the same. Many neurons in MT exhibited changes in activity that coincided with the reversals in perception, indicating that MT has a basic role in SFM perception (Bradley, Chang, and Andersen 1998). In a subsequent study, it was found that MT neurons were much more affected by the surface order of these perceived 3D stimuli than were V1 neurons. These results suggest that V1 is not directly involved in SFM perception but MT is.

Optic Flow and the Direction of Heading

We had studied the global motion fields in spiral space at MIT. The expanding and contracting stimuli have an important role in the perception of direction of locomotion. For instance, while walking or driving, the motion that is generated on the retina, for moving forward, is an expansion (contraction if you are moving backward), and the focus of expansion indicates the direction of heading when the eyes are still. However, when the eyes also move, as happens when moving in one direction but fixating on an object to the side using smooth pursuit eye movements, this introduces an additional translation motion on the retinas, shifting the focus of expansion. Marty Banks and colleagues at UC Berkeley showed that humans are still able to perceive the correct direction of heading and this correct percept requires eye movement information. Marty and I were at a meeting together and discussed trying his paradigm while recording from area MSTd, which has expansion selective neurons.

We simulated locomotion with random dot fields. We measured the tuning of MSTd neurons for the location of the focus of expansion and found that this tuning shifted toward correctly registering the heading direction during pursuit eye movements. This tuning shift is possible only if MSTd neurons receive either visual or motor signals about pursuit. We eliminated the visual signal hypothesis by showing that the same neurons did not exhibit a compensatory tuning shift when the display simulated the retinal stimulus that occurs during pursuit, but with the eyes still (Bradley et al. 1996). Thus, MSTd neurons compensate for the motor signals that drive eye movements and register the true heading direction. Further experiments led by Krishna Shenoy, a postdoctoral fellow in the lab, found that this was the case when gaze was shifted by vestibular signals during whole body rotations. These results indicate that the compensation is a result of gaze rotation, whether it is the eyes moving in the head (pursuit) or the head rotating in the world (vestibular-ocular reflex). In another study he found that the amount of compensation was correlated with the speed of pursuit. Brian Lee, a graduate student, found there was also a retinal component of adjustment, which was apparent when the rate of visual expansion was changed, simulating different speeds of locomotion in the environment.

My studies on visual motion, spanning my faculty positions at the Salk, MIT, and Caltech, addressed fundamental questions about SFM perception and heading perception. Personally I felt I was ready to pivot to explore a new topic. A new direction was catching my interest: to explore brain-machine interfaces in NHPs and later in human clinical trials.

Brain-Machine Interfaces

As I was investigating the sensorimotor transformations in cortex, I began to think about how this knowledge might be used clinically. One straightforward application would be to design neural prosthetics that would help people suffering from paralysis. A colleague at Harvard, Marge Livingstone (see Volume 9), told me I was already talking about this when I was still at MIT, so at least 27 years ago.

The technology used for neural prosthetics is referred to as brain-machine interfaces (BMIs) or brain-computer interfaces (BCIs). A BMI is a device that interfaces a machine with the brain. It can write in or read out information. Write-in BMIs generally use electrical stimulation to modulate brain activity. Examples are already available clinically, including cochlear implants for restoring hearing, deep brain stimulators for movement disorders, and cortical stimulators to disrupt epileptic seizures.

Read-out BMIs are still in development. The challenge of read-out BMIs is to record signals from the brain and decode them with mathematical algorithms to produce control signals for operating external devices, such as computers and robotics. One important use of such a read-out system is the development of neural prosthetics systems to assist patients with severe paralysis. Thus, a person who was paralyzed from neurological injury or disease affecting parts of the motor system could still generate signals from less affected parts of the brain to control external, assistive devices. Examples of patients that can benefit from a BMI are those suffering from spinal cord lesion, stroke, anterolateral sclerosis, multiple sclerosis, or Duchenne muscular dystrophy.

This field of read-out BMIs was established before neurophysiologists like myself, using single neuron recordings in NHPs, became involved. The signals for driving the BMIs were recorded primarily from electroencephalogram (EEG) recordings. Subjects could train their EEGs to control external devices like the movement of a cursor on a computer screen. An advantage of EEG is that it is noninvasive. However, a disadvantage is the spatial resolution is very low, averaging brain signals from centimeters of cortex. To overcome this spatial resolution limitation, ideally one would record the activity of single neurons. Importantly, not only would one record from single neurons, but also a number of single neurons simultaneously. Thus, populations of single neurons are recorded all at once. Arrays of microelectrodes have been developed over the past two decades that allow this simultaneous recording from populations of single neurons. The array that we use, the Utah array, was originally developed by Richard Normann at the University of Utah and refined by two companies, Cyberkenetics and later Blackrock Neurotech. It is a small four- by four-millimeter flat surface with 100 protruding microelectrodes that looks a bit like a bed of nails. Each of these arrays typically records from 100– to 200 neurons, generally more than one array is implanted in each subject, and the recordings are viable in humans for about five years.

Brain Control in Monkeys

We began using brain signals to control computers in earnest at Caltech in the late 1990s. This idea of single-neuron population BMIs had occurred independently at a few NHP neurophysiology labs, including Miguel Nicolelis at Duke, John Donoghue at Brown, and Andy Schwartz at Arizona State University and later the University of Pittsburgh. These labs concentrated on primary motor cortex with the exception of Nicolelis who studied several cortical areas. I had been working on the high-level intent signal in PPC and so had concentrated on making a more cognitive neuroprosthetic—that is, one that specified the intended goal of a movement. These first experiments were in NHPs and were necessary to establish the success of brain control with single-neuron population recording in animal studies before human clinical studies. These animal studies were followed by studies in humans with severe paralysis by the Donoghue lab, the Schwartz lab, and our lab.

Our first paper on cognitive neural prosthetics with NHPs was published in Science in 2004 (Musallam et al. 2004). In this study, implants were made in PPC and the dorsal premotor cortex, another high-level pre-motor area. The study focused on decoding the intended goals of a movement instead of the trajectory to a goal, which was the approach of other NHP BMI studies. Decoding is a necessary step between the raw neural data and the control of assistive devices. Training data are collected at the beginning of an experiment that determines the correlation between patterns of neural activity and motor outcomes. The training data teach the decoder to subsequently predict the intended motor outcome from patterns of activity generated by single trials to drive the prosthetic device.

We found that determining goals could be accomplished very quickly, within 200 ms, and much quicker than the time that would be required for guiding the trajectories. Performance improved over a period of months suggesting that learning was at play. The desirability of the goals also could be decoded by indicating the value of each trial, which was cued at the target location. The value manipulations were amount, probability, and type (juice or water) of the reward. These cognitive variables reflecting the desirability of goals are factored into decision making. This finding of desirability indicated that very high-level signals related to the state of the NHP could be decoded. Conceivably these high-level signals can be used in clinical studies for assessing the motivation and preferences of the patient. They also suggest that implants, particularly those introduced in centers more involved with emotion and mood, could be used in clinical studies of neuropsychiatric disorders, such as depression, and could provide feedback to guide neuromodulation with, for instance, electrical stimulation.

Next, graduate students Markus Hauschild and Grant Mulliken showed that a trajectory signal also could be decoded from PPC. The trajectories could be decoded for targets in 2D and even in 3D, where NHPs moved a cursor to locations in different depths combined with horizontal and vertical movements. Graduate students Bijan Pesaran, John Pezaris, and Maneesh Sahani; postdoctoral fellows Hans Scherberger and Murray Jarvis; and colleague Partha Mitra showed that local field potentials (LFPs) are prominent and predictive of behavioral state in PPC (Pesaran et al. 2002; Scherberger, Jarvis, and Andersen 2005). Postdoctoral fellow EunJung Hwang showed that LFPs recorded from the microelectrodes also could be used for brain control. Adding LFP information to the spiking information improved performance. LFPs average neural activity over larger volumes of cortex, resulting in more redundant information being recorded across electrodes. Spike recordings reflect activity in much more localized volumes of cortex, resulting in much more independent information recorded across electrodes and thus leading to better decoding performance. However, an important feature of the LFPs is that they can be recorded from most microelectrodes in an array, whereas spikes are more limited in the number of electrodes that are close enough to single neurons to record spikes. Also, over time, the spiking signals fade. LFP signals still remain and thus can be used to extend the time implants are viable.

While we were doing these NHP studies to confirm the feasibility of implanting PPC in humans, we were also in the process of preparing for the human studies. We built a multidisciplinary team of scientists and physicians from Caltech, the Keck Medical School of the University of Southern California, the Geffen Medical School at UCLA, the Rancho Los Amigos National Rehabilitation Center, and the Casa Colina Hospital and Centers for Health Care. We worked with regulatory bodies, including the U.S. Food and Drug Administration, and the institutional review boards at Caltech and at our collaborators’ institutions to receive the go-ahead. It took about 15 years of NHP studies, building the infrastructure, and obtaining approvals before we began our first human clinical trials.

Caltech: Human Days

Neuroprosthetics Studies in Humans

Participants in neuroprosthetic studies are true heroes. At the time of my writing this autobiography, there have been only about 20 participants who have entered this type of clinical trial among the groups doing this research worldwide. The participants are akin to astronauts who have landed on the moon; an elite group with tremendous bravery and resolve to enter this new world of brain-controlled neuroprosthetics. They volunteer selflessly, as there is no direct medical benefit for them personally in enrolling in the study; only the knowledge that their groundbreaking work is contributing to medical advances that someday will benefit a future generation of people suffering from severe paralysis.

Our first participant was Erik Sorto. He was the victim of a gunshot that severed his spinal cord when he was 21 years old, rendering him tetraplegic. When Sorto entered the study he had already been paralyzed for 10 years. The implant surgery took place at the Keck Medical School at the University of Southern California in April 2013. Two microelectrode arrays were placed in the PPC. The surgeons were Charles Liu and Brian Lee, and the surgery went flawlessly. Going back to the planetary space analogy, my colleagues at the Jet Propulsion Laboratory (JPL) in the Pasadena area refer to the seven minutes of terror when a Mars rover enters the atmosphere of Mars before it lands. For us, it was two weeks of terror after the implantation, during the healing period, before Sorto had his first session. We had every reason to believe that the implant would work based on our NHP studies. However, the monkeys were healthy and Sorto had been paralyzed for 10 years. Would the motor-related signals in PPC still be there after 10 years of disuse? No one had ever implanted human PPC with microelectrode arrays. We used functional magnetic resonance imaging preoperatively to help guide us to locations in PPC that were active when Sorto imagined a reach or a grasp. But this technique is indirect and monitors increases in blood flow when a brain area is active. Would we record single neurons in PPC of Sorto, and would he be able to modulate them with his thoughts when controlling a robotic limb or a computer cursor?

Our team was led by Spencer Kellis, a senior scientist at Caltech. During the first session, we isolated only a few neurons. This is not uncommon as yields of neurons become much larger over the first month. Also we had to adjust the unit isolation on the channels with the data collection software. In the next session, something amazing happened. As more neurons came into view, Sorto was asked to try to control their firing rates. Some of the neurons were activated when he imagined rotating his paralyzed wrist. We used the firing rates of these neurons to control the wrist of a robotic arm. He was able to use his thought to rotate the robot hand to gesture shaking the hand of a graduate student. This result was amazing. First, it showed that, even after 10 years of disuse, the intrinsic circuits for motor control were still intact. Second, it required no practice on Sorto’s part; the brain control worked right out of the box. Third, Erik was ecstatic. This was the first time in 10 years that he had been able to move a limb and with a social gesture.

We ask participants at the beginning of the study what they hoped to achieve. In Erik’s case, he wanted to be able to drink a beer on his own. A year into the study, he achieved this goal by controlling a robotic limb. The first time he accomplished this feat, there were shouts of joy from Erik, staff, and lab members present. Our second subject, Nancy Smith, was a high school teacher who had played the piano before a car accident six years earlier that left her tetraplegic. The implant surgery was performed by Nader Pouratian at UCLA, and the research team was led by Tyson Aflalo, a senior scientist at Caltech. Surprisingly, we found that she had a motor representation of all 10 individual fingers in PPC and was able to play a virtual keyboard piano with imagined finger strokes of one of her hands.

We have now had five participants over the past eight years, Erik Sorto, Nancy Smith, DL, FG, and JJ. In every case, it leaves me breathless to see them control their physical environment with just their thoughts. For me, it has been a high point in my career, being able to do both the basic research of studying neuroscience, and then being able to apply what we learn directly to the clinic. These studies with humans reveal new scientific findings that are then used to further advance the clinical aspects of the study. The team is truly interdisciplinary. No single person can do this kind of project, and it requires physicians, scientists, engineers, and computational neuroscientists. The patients are truly members of the team. Unlike conventional clinical studies that involve large numbers of people for very short periods of time, these studies involve single individuals over extensive time periods. Each person does two to three sessions per week lasting about four hours per session. The total length of time a person is in a clinical trial is about five years.

As mentioned earlier, when we obtain consent from our participants, we indicate that they will not directly benefit from the study. However, I have found that this type of study is tremendously beneficial to them, and to us, in ways that became apparent only over the course of the studies. People with paralysis often do not leave their home environment and are depressed. The clinical studies enable them to have a job, get out of the house, meet interesting people, and have a purpose. Some come to engage more in outside interests, including giving lectures and being interviewed by media. From our team’s side, we have direct contact with members of the population we want to help, being able to better understand their needs. We get to know their families and, before the Covid pandemic, we would have holiday and birthday parties with the participants, their family members and caregivers, and our team of scientists and physicians. It is a truly unique experience for all of us.

In 2015, we published our first human study with Sorto in Science (Aflalo et al. 2015). This first-ever use of PPC single-neuron population recordings for prosthetic control showed that indeed PPC provides high-level movement-related activity through motor imagery. Intended goals of movement could be decoded very rapidly, within 200 ms, similar to our findings in NHPs. Also the trajectories could be decoded. Moreover, goal signals were effector specific, and separate neurons encoded the contralateral limb or ipsilateral limb, referenced to the implanted hemisphere. Thus, this was the first demonstration of a cognitive neural prosthetic in humans.

Our Science paper received considerable press. A couple, Tianqiao Chen and Chrissy Luo, saw a news story on our findings on television in Singapore. They had founded a company, Shanda in 1999, which became the biggest online entertainment developer in China. They subsequently founded the Tianqiao and Chrissy Chen Institute for philanthropy targeting brain science. Tianqiao visited me at Caltech, and a month later, both he and Chrissy visited again. Their interests in our research and in Caltech led to their making a gift of $115 million in 2016. This was the largest gift given to Caltech by a donor that was not an alumnus or previously connected or known to the Caltech administration. The gift provided $50 million toward the construction of the Chen Neuroscience Research Building and the remainder to support the Tianqiao and Chrissy Chen Institute for Neuroscience at Caltech. This new neuroscience institute included several endowed centers. One of these is the T&C Chen Brain-Machine Interface Center, for which I am currently the director.

“It Was the Best of Times, It Was the Worst of Times.”

This Charles Dickens quote (A Tale of Two Cities) captures the next few years of my life. Research was going fabulously, and building up the BMI Center was crucial in sustaining our BMI research with humans and to also building interest in BMI research by fellow Caltech faculty through seed grants from the BMI Center (Andersen 2019). But in early 2017, my wife Carol was diagnosed with an incurable disease. That year, I became her caregiver until her passing at home with our family in January of 2018. I lost my lifelong love and emotional foundation. Not long after her passing came the pandemic, still with us two years later as I write these words. My fantastic children, lab, and colleagues have helped me to weather these tragedies and also to keep my research moving forward.

Partially Mixed Selectivity

We were pleased that even after years of spinal cord injury the intent of the subjects could be recorded from PPC. As we explored the activity of the populations of recorded neurons, we found that a large number of other cognitive variables were present and decodable. These included mental strategy (imagined versus attempted movement), finger movements, decisions involving memory recall, hand shaping for grasp, observed actions of others, and action verbs, such as “grasp” or “drop.” From a few hundred neurons, we could decode much of what a person intends to do. How is this possible?

Recall that the gain fields we found in NHPs mixed gaze position–tuned activity with retinotopically tuned visual stimulus positions. In the Zipser-Andersen model, we showed that as few as nine neurons could represent all of visual space with respect to the head. Recent animal studies from other labs have shown a mixing of variables. For example, recordings from prefrontal cortical neurons in NHPs showed a mixing of two types of memory task and different visual objects. The key is that one neuron can respond to more than one variable and can be involved in more than one circuit. This type of coding was coined randomly mixed selectivity. However, in our studies, we found that, although there is mixing, there is also structure in the statistics of the representation. We referred to this as partially mixed selectivity.

We studied these statistical structures formally in two experiments. In the first, we examined the representation of task strategy (imagine/attempt), side of the body (left/right), and effector (hand/shoulder). Graduate student Carey Zhang, along with Tyson Aflalo, found that whereas the effectors were randomly mixed on PPC neurons, the strategy and body side were correlated (Zhang et al. 2017). Thus, if a neuron responded to an imagined right hand movement, it was more likely to respond also to an imagined left hand movement. One could not predict, however, if that same neuron would respond to an imagined shoulder movement. This structure makes functional sense since, for instance, something learned with one hand is transferable to the other hand. However, the mechanics of the shoulder and hand are very different, and thus it is useful to have their activities more separated in the neural population.

A second experiment led by Tyson Aflalo and colleague Guy Orban examined selectivity for observed actions and action verbs. Participants viewed videos of five actions of another person manipulating objects. These included drag, drop, grasp, push, and rotate. Each action was shown from different views of the person performing the action. Besides the action videos, some trials showed only the text of the action verbs and the participants were asked to read the text silently. It was again found that all of these variables were mixed in the population, but there were correlations of the videos showing the same action from different points of view and even reading the verb for the same action (Aflalo et al. 2020).

Partially mixed selectivity may explain why so many roles have been attributed to NHP LIP. As mentioned, we had found the area and ascribed it to have a role in saccades. Subsequent studies emphasized its role in decision making, attention, and categorization. In NHP experiments, one selects an area of the cortex to study, usually based on preliminary data or prior literature suggesting its function. To train the NHPs to perform a behavioral task takes months, and collecting data is also very time consuming with the NHPs performing many thousands of trials. Not surprisingly, researchers find what they are looking for and then try to reconcile their results to other functions ascribed to the same brain area by different labs using different tasks. With humans, you can ask them to perform a task, so there is almost no training involved. Data collection is from array recordings so each daily yield is a large data set. With language, you can instruct many different types of tasks. With humans, you can also obtain their verbal reports of sensations for stimulation studies and mental strategies for behavioral tasks.

Although areas of the PPC show a great diversity of action variables recorded from single small patches, I still believe that there are core local functional differences within PPC. We have seen differences between human area 5, which appears to be reach selective, and AIP, which appears to be more involved in shaping the hand during grasp. The inactivation studies in NHPs produced a specific reach deficit for PRR, and a saccade deficit for LIP. I believe that what we record from an implant in PPC are the core local computation at the implant site as well as the information coming into the area from the many other brain areas that project into the local region. This idea emphasizes that each node is a part of a network.

Stimulation

The people with tetraplegia from spinal cord injury not only are unable to move their limbs, but also are unable to feel them since the main touch and body position signals travel through the spinal cord to the brain. This deficit is problematic for two reasons. One is a sense of embodiment. When Nancy Smith imagined moving the avatar’s fingers to play a virtual piano, she did not receive touch feedback when she struck the piano keys. Second, somatosensation is necessary for manual dexterity. Under visual feedback our participants are able to move a robot limb and grasp an object using visual feedback for guidance. However, once the robot hand has grasped an object, somatosensation is necessary for manually manipulating the object.

We and others have used one method to evoke somatosensation by delivering current through electrodes imbedded in a grid (referred to as electrocorticography or ECoG) that sits on the surface of the primary somatosensory cortex. The arrays are placed under the dura but do not penetrate the cortex, and the electrodes themselves can be small with diameters around two millimeters. Even so, currents to evoke sensation are high, in the order of milliamps, and the sensations are unnatural and have been reported as tingling or electric. On the positive side, the grids can be large and cover extensive areas of the brain, and the somatotopic map in S1 can be determined by stimulating different electrodes in the grid. Thus, this method can provide touch sensation at localized regions of a robotic hand and may improve dexterity.

Intracortical microstimulation can be used to stimulate sensation through the microelectrodes in the microelectrode arrays. Because the tips are in the cortex rather than on the surface, much smaller currents can be delivered to evoke a sensation. Rather than stimulating large numbers of neurons as in the case of ECoG, only a small number of neurons are activated around the tips of the electrodes. Using this technique, the laboratory of Rob Gaunt at the University of Pittsburgh implanted the hand region of S1, and the participant reported natural-like feelings of pressure. Our team, led by Michelle Armenta Salas, Luke Bashford, and Spencer Kellis, implanted the arm region of one of our participants and for the first time, he reported natural touch sensations, such as tap, squeeze, and vibration (Armenta Salas et al. 2018). Also for the first time, we also found proprioception, in which stimulation of some sites produced the sense of the arm moving. Interestingly, as we increased the current delivered, we could change the sensation elicited from a touch sensation to a proprioceptive sensation for many of the electrodes, providing preliminary evidence that the sensations can be manipulated by changing the properties of the electrical stimulus.

Spatial Representation

Using the relative position task and analysis we previously used in NHPs, we have been exploring the representation of space in humans. In our electrode placements, we see that the coordinate transformations are often largely complete, for instance, an imagined reach is coded in hand coordinates. One reason for this difference, I suspect, is that our human participant implants are recording from locations more anterior in PPC than in the NHPs. We began seeing hand coordinate frame neurons in area 5 of NHPs. Another interesting feature we saw in area 5 of NHPs, and now in PPC of humans, is a switching of neural representations. In area 5, when the initial hand and eye position were obtained in the task, the neurons coded the relative position of the eye and hand. When a reach cue was provided, the representation shifted to coding the location of the target relative to the hand. In humans, we have found a similar contextual shifting of coordinate frames. When the target is for a saccade, the coding is the gaze relative to the target (eye coordinates). When the target is for a reach, the coding in the population shifts coordinates to coding the target with respect to the hand (hand coordinates). We also are finding not only egocentric spatial representations but also allocentric representations. An egocentric representation is where an object is with respect to a part of the body. An allocentric representation is where objects in the environment are placed with respect to each other. Interestingly, both egocentric and allocentric deficits in spatial perception are seen with PPC injury in humans.

Although many of our spatial representation experiments are currently ongoing, graduate student Matiar Jafari and Tyson Aflalo led a study, published recently, in which we looked at reach activation and coordinate frames in the population of S1 neurons (Jafari et al. 2020). There are two remarkable findings from this study. The first is that even though the patient is paralyzed, we designed a task in which he imagined different initial hand positions as well as imagined reaching to the target. In this task, there is a visual cue phase, a delay phase when motor planning occurs, and then an action phase in which a reach is imagined. The first finding was that during the visual cue and motor planning phases, which produce robust activation in PPC neurons, there was no activity in S1. Thus, vision and planning activity are not variables that are represented in S1 but are present in a comparable task in PPC. Even if there is mixed selectivity in S1, it does not share the same variables as PPC. Second, during the imagined reach the activity was in hand coordinates, the appropriate coordinates one would expect for an actual reach.

Internal Models and Imagination

A third interesting feature of the S1 recording results was the activation of S1 for imagined reach. Reach activity has been recorded in S1 of NHPs before an actual reach, although the coordinates were not determined. The fact that the reach activation in human S1 is in hand coordinates, and there was no activity in the cue or planning phase, suggests that the source of the reach activation is computed in another brain area and then sent to S1. This findings suggests that the activation is an efference copy and a component of an internal model.

As mentioned, the timing of reach responses in NHP PPC suggested that they were efference copies for updating an internal model in the sensorimotor system. With our human participants, we have been able to examine task strategies in which the patient imagines moving, attempts to move (for paralyzed parts of the body), or actually moves (for nonparalyzed body parts). It is rather amazing how much of the brain is activated by imagination. For example, S1 and PPC neurons can be activated for touch or imagined touch. This extends not only to the part of the body of the imagined touch, but also to the quality of the touch, such as a squeeze or a tap. The results from the coordinate frame studies suggest that PPC keeps a representation of the world and the relation of the body in the world. Injury to PPC further supports its role in this internal model framework, since these patients have great difficulty adjusting and correcting their movements to changes in the environment.

Awareness of Intent

Harkening back to the Libet experiment, we designed a study in which participants were free to choose when to move, or both what to move and when to move. They were asked to retrospectively report the time they felt the urge to move. Similar to Libet’s findings, and subsequent findings by others, PPC activity rises several hundred milliseconds before subjects report the initial urge to move. Moreover, our BMIs can decode the subject’s intended movement before they are aware of it, and they report that a brain-controlled action often occurred before they intended it. To provide better embodiment, we found that we could design decoding algorithms that predict when the subject would be aware of the intent in order to synchronize intention awareness with the movement output of the neuroprosthetic. In the bigger picture, these results indicate that a lot of movement computation is occurring implicitly or preconsciously in PPC.

Stability and Learning

There is an extensive literature documenting changes in cortical representations of the body in somatosensory and motor cortex; in fact, many of the seminal studies were made by my mentor Mike Merzenich. Perhaps less studied is the functional organization that remains intact after injury. The study of BMIs in tetraplegics has provided a unique opportunity to determine remaining function. BMI studies from our group and others have found considerable preserved structure even years after the initial injury. The motor cortex is active and this activity can be immediately harnessed in brain-control tasks. Microstimulation of S1 produces the same somatotopy of sensation one would expect in a healthy individual, and the machinery for natural perception is still intact. Mixed selectivity, which is found in association cortex of laboratory animals, is also present in PPC of the human participants.

Learning, in which the control of the prosthetic improves over months, has been observed in most studies. Very fast learning, on the scale of one day, often involves cognitive strategies using existing intrinsic patterns of activity within an area. For instance, postdoctoral fellows EunJung Hwang and Sofia Sakellaridi have led studies that show a rotation of targets will lead to re-aiming, documented in NHPs and humans (Hwang et al. 2013; Sakellaridi et al. 2019). In the case of humans, they can tell you what cognitive strategy they are using. Studying long-term learning is currently more problematic because of the difficulty in recording from the same neurons over many days with the current recording technologies. The long-term learning that has been observed is in the domain of the cortical areas implanted—in other words, a sharpening of the skill of the area. Further work on plasticity with BMIs is needed. But at least in viewing what we know today, it is important to select areas of the brain that naturally function in the domain of the desired BMI output. For instance, one would likely be more successful to implant language cortex for decoding language than to implant visual cortex for language decoding.

Functional Ultrasound

No doubt there will be improvement in materials and electronics as BMIs move to the clinic. The gold standard for BMIs is to record from single-neuron populations. But to record the extracellular electric field of a neuron requires the microelectrode to be within a few tens of microns from the neuron. Thus, at least within the foreseeable future, this sort of spatial resolution will always require an invasive brain implant. As the technology becomes refined, this invasiveness may not be a major stumbling block as current neurotechnologies like cochlear implants and deep brain stimulators are invasive and yet readily available from the clinic.

Another avenue is to produce minimally invasive technologies. One such technology we have been working on is functional ultrasound. This technique detects changes in blood flow with the activation of brain areas. It has a very high spatial resolution, in the order of a 100 microns, but it is slow as it is limited temporally by the hemodynamic response. In a collaboration with Mikhail Shapiro’s lab at Caltech and Mickael Tanter’s lab in Paris, postdoctoral fellow Sumner Norman and graduate student Whitney Griggs have shown that functional ultrasound can be used in NHPs for single-trial decoding of the direction of movement and by the effector used (Norman et al. 2021). This technique could be used as a minimally invasive BMI in humans by replacing a small bit of skull with an acoustically transparent window. It would not require surgery to the dura or penetration into brain tissue. In addition to BMI applications, this technology may be useful for decoding the state of the brain. It has a very wide field of view and could detect epileptic seizure locations as well as changes in more global brain activity that accompany neuropsychiatric diseases.

Final Thoughts

I have had extremely good fortune in my life and career. I have had the privilege to work with the brightest and best students and colleagues from around the world. I was fortunate to be at the beginning of what is now the huge and evolving field of neuroscience and to be trained by some of the giants in the field. I again find myself at the beginning stages of a new and expanding field: BMIs. I thank my family who have supported me all these years and for the great joy they have brought me. I thank the many brilliant students, postdoctoral fellows, and staff who studied with me. Many I have mentioned in the text and all have contributed tremendously to the research coming out of the lab. The following table lists our lab members and the decade in which they graduated or are currently in the lab for the decade beginning 2020. Finally, I am still doing research, which seems to me to be at a level comparable to early in my career, and it certainly bringing me the same exhilaration of adventure.

Lab Members

Postdoctoral Fellows

Graduate Students

Research Support

1980s

1980s

Chiko Asanuma
Greg Essick
Ralph Siegel
Peter Brotchie
Gene Blatt
Jim Gnadt
Masud Husain
Roger Erickson
Leonardo Fogassi
Peter Thier (faculty on sabbatical)

Martyn Bracewell
Michael Graziano

1990s

1990s

1990s

Robert Snowden
Shabtai Barash
Ning Qian
Larry Snyder
Sohaib Kureshi
David Bradley
Jim Crowell
Jing Xing
Philip Sabes

Stefan Treue
Bard Geesaman
Pietro Mazzoni
Brigette Stricanne
Ray Li
Maneesh Sahani
Jennifer Linden

Gail Robertson
Catherine Cooper
Betty Gillikin Grieve
Viktor Shcherbatyuk
(to present)
Cierina Reyes Marks

2000s

2000s

2000s

Jyl Boline
Yale Cohen
Kenneth Grieve
Alex Gruenwald
Krishna Shenoy
Murray Jarvis
David Dubowitz
Brian Corneil
Hans Scherberger
Shau-Ming Wu
Rodrigo Quian-Quiroga
Jason Connolly
Chris Buneo
Bijan Pesaran
Bradley Greger
Alexander Gail
Sam Musallam
Boris Breznen
Marina Brozovich
Daniel Rizzuto
Axel Lindner
Elizabeth Torres
He Cui

John Pezaris
Aaron Batista
Kyle Bernheim
Daniella Meeker
Matthew Nelson
Brian Lee
Michael Campos
Daniel Baldauf
Asha Iyer
Grant Mulliken
Hillary Glidden
Rajan Bhattacharyya

Kelsie Pejsa (to present)
Tessa Yao

2010s

2010s

Melanie Wilke
Claudia Wilimzig
Igor Kagan
Carlos Pedreira
Xoana Troncoso
EunJung Hwang
James Bonaiuto
Lindsay Bremner
Markus Hauschild
Arnulf Graf
Christian Klaes
Michelle Armenta Salas
Vasileios Christopoulos
Dan Kramer
Spencer Kellis

Boris Revechkis
Carey Zhang
Matiar Jafari
HyeongChan Jo

2020s (current lab members)

2020s (current lab members)

Tyson A.alo (member of professional staff)
Luke Bashford
David Bjanes
Jorge Gamez
Sumner Norman
So.a Sakellaridi
Xinyun Zou
Liang She

Srinivas Chivukula
Whitney Griggs
Charles Guan
Kelly Kadlec
Isabelle Rosenthal
Sarah Wandelt

Selected Bibliography

Aflalo, T., S. Kellis, C. Klaes, B. Lee, Y. Shi, K. Pejsa, K. Shanfield, S. Hayes-Jackson, M. Aisen, C. Heck, C. Liu, and R. A. Andersen. 2015. “Decoding motor imagery from the posterior parietal cortex of a tetraplegic human,” Science, 348: 906–10.

Aflalo, T., C. Y. Zhang, E.R. Rosario, N. Pouratian, G. A. Orban, and R. A. Andersen. 2020. “A shared neural substrate for action verbs and observed actions in human posterior parietal cortex,” Science Advances, 6.

Andersen, R. 2019. “The intention machine,” Scientific American, 320: 25–29.

Andersen, R. A., C. Asanuma, and W. M. Cowan. 1985. “Callosal and prefrontal associational projecting cell populations in area 7A of the macaque monkey: a study using retrogradely transported fluorescent dyes,” J Comp Neurol, 232: 443–55.

Andersen, R. A., and C. A. Buneo. 2002. “Intentional maps in posterior parietal cortex,” Annual Review of Neuroscience 25: 189–220.

Andersen, R. A., P. L. Knight, and M. M. Merzenich. 1980. “The thalamocortical and corticothalamic connections of AI, AII, and the anterior auditory field (AAF) in the cat: evidence for two largely segregated systems of connections,” J Comp Neurol, 194: 663–701.

Andersen, R. A., and V. B. Mountcastle. 1983. “The influence of the angle of gaze upon the excitability of the light-sensitive neurons of the posterior parietal cortex,” J Neurosci, 3: 532–48.

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Corey S. Goodman

BORN:

Chicago, Illinois

June 29, 1951

EDUCATION:

Stanford University, B.S. (1972)

University of California, Berkeley, PhD (1977)

University of California, San Diego, Helen Hay Whitney Postdoc Fellow (1979)

APPOINTMENTS:

Assistant to Associate Professor of Biology, Stanford University (1979–1982; 1982–1987)

Professor of Neurobiology and Genetics, University of California, Berkeley (1987–2005)

Investigator, Howard Hughes Medical Institute (1988–2001)

Head of Neurobiology, Dept. of Molecular & Cell Biology, U.C. Berkeley (1992–1999)

Director, Helen Wills Neuroscience Institute, U.C. Berkeley (1999–2001)

President and CEO, Renovis (2001–2007)

Adjunct Professor of Neuroscience, U.C. Berkeley (2005–2007, 2015–present)

President, Biotherapeutics and Bioinnovation Center, Pfizer (2007–2009)

Managing Partner, venBio Partners (2010–present)

HONORS AND AWARDS (SELECTED):

Alan T. Waterman Award, National Science Board (1983)

Elected Member, American Academy of Arts and Sciences (1993)

Elected Member, National Academy of Sciences (1995)

Fondation IPSEN Prize (with Marc Tessier-Lavigne and Friedrich Bonhoeffer, 1996)

J. Allyn Taylor International Prize (with Tom Jessell, 1996)

Canada Gairdner International Award (1997)

Wakeman Award (with Tessier-Lavigne, 1998)

Elected Member, American Philosophical Society (1999)

March-of-Dimes Prize (with Jessell, 2001)

Reeve-Irvine Medal (with Tessier-Lavigne, 2006)

Dawson Genetics Prize (2011)

Gruber Neuroscience Prize (with Tessier-Lavigne and Bonhoeffer, 2020)

Corey Goodman helped decipher the logic and molecular mechanisms of axon guidance. He and his colleagues used genetic analysis in Drosophila to discover how growth cones navigate intermediate targets to reach their final target. The orchestration of these guidance decisions involves attraction and repulsion, switching from one to the other, and uses an array of signals and receptors. They conducted the first large-scale genetic screen for axon guidance mutants, looking for mutants in which too few or too many axons cross the midline, and then conducted a second screen, looking for mutants in which motor axons do not go to the right muscles. Among their discoveries were Slit and Robos and their regulator Commissureless, as well as Semaphorins and Plexins, Side and Beats, and other guidance and signaling molecules. They then conducted a third screen, looking for mutants in which neuromuscular synapses were too big or too small. Goodman’s work showed that the molecules and mechanisms of axon guidance are highly conserved across the animal kingdom. Goodman moved from academia to biotechnology to help turn great science into impactful medicine. He cofounded eight biotechnology companies and led one of them as chief executive from a private to public company. He was president of Pfizer’s Biotherapeutics and Bioinnovation Center. Today, he is managing partner of venBio, a venture capital firm he cofounded. venBio already has five U.S. Food and Drug Administration–approved drugs on the market, saving and improving lives.

Corey S. Goodman

Background

I’ve lived my life in chapters. Although I spent three decades as a developmental neurobiologist, I left academia two decades ago to devote myself to translating great science into impactful medicine in the world of biotechnology and drug discovery. Given that history, I was touched when asked to write this autobiography for the History of Neuroscience series. This invitation recognizes the contributions from my lab from the late 1970s to the early 2000s.

When we began studying axon guidance, it was an open frontier. Over those three decades, we helped decipher the logic and molecular mechanisms of axon guidance. We used genetic analysis in Drosophila to discover how growth cones navigate a series of intermediate targets to reach their final target. The orchestration of these guidance decisions involves attraction and repulsion, switching from one to the other, and uses an array of signals and receptors. We conducted the first large-scale genetic screen for axon guidance mutants, looking for mutants in which too few or too many axons cross the midline, and then a second screen, looking for mutants in which motor axons either do not enter muscle domains or go to the wrong muscles. Among our discoveries were Slit and Robos and their regulator Commissureless, as well as Semaphorins and Plexins, Side and Beats, and other guidance and downstream signaling molecules. We then conducted a third screen, looking for mutants in which neuromuscular synapses were too big or too small, or absent all together.

That was an exciting period of discovery and one I shared with the extraordinary graduate students, postdocs, and staff in my lab, whose enthusiasm and creative abilities made it all possible. I’ll mention many by name here, but due to space limitations, I can’t possibly do justice to all of them and their contributions. Nevertheless, I hope they all know that I am deeply thankful for what they added to the stories I tell here, and to the culture they helped create.

I feel as if my personal journey has been much like that of a growth cone, navigating a series of choice points, each decision building upon the previous one, with no dead reckoning or master plan from the outset, but rather moving on to the next challenge, influenced by what came before. I’ve followed my passions and instincts, looking for the next frontier. I think what has been consistent in the chapters of my life has been the desire to learn and expand my horizons, discover things no one has ever seen before, take risk, and use my core as a scientist to contribute to knowledge, health, society, and policy.

In this essay, I’ll recount stories and influences throughout my journey. I hope it helps students realize there is no right or wrong path to success. Sometimes the combination of interests, instincts, and intuition, along with the influences around you, lead to creative and impactful outcomes. Some may say I was lucky. Of course I was. As Louis Pasteur said, “Chance favors the prepared mind.” Sensing when to seize the moment is a key to success. Science and music have been inexorably intertwined throughout my life. My musical explorations taught me to learn from history, improvise and create, explore what I don’t know, and take risks and expose myself to failure—important lessons for how to be a creative scientist.

Family

I was born in Chicago on June 29, 1951. My parents, Florence and Arnold Goodman, were Ashkenazi Jews. My father was born in Baltimore in 1919; he was an only child. My mother was born in Chicago in 1922; I have been close with her sister Myra (12 years younger) throughout my life. My parents grew up during the Great Depression and met before my father went off to the navy during World War II. He became a lieutenant on the destroyer USS Hilary P. Jones. After he came back, they had two children—my brother Michael (born 1947) and me.

In my early childhood, my parents lived in a poor, heavily Jewish, neighborhood on the near-northside of Chicago, in a one-bedroom apartment in the back of the same building where my maternal grandparents lived. When my parents retired, they moved to Florida and lived in a small one-bedroom condo they inherited from my mother’s parents. They lived on their social security checks and had little money. When my first biotech company became successful in the late 1990s, my wife Marcia and I were able to help buy them a nicer home in Boca Raton and supplement their income until they died. It felt good to make their final years a little bit more comfortable. My father, who had been a heavy smoker, died from lung cancer in 2004 at the age of 84. My mother died from a multitude of health issues in 2008 at the age of 85.

Both sides of my family came from Eastern Europe, and both had modest means in the United States. My mother’s parents, Jean and Bill Friedman, were born in 1902 in Chicago to parents who immigrated from Kiev, Ukraine. In Ukraine, Jean’s family had been professors and teachers, whereas Bill’s had been rabbis for many generations. Their respective families lost everything when they fled to America. Jean and Bill were both orphaned at the age of 15. Bill was one of nine siblings, all taking care of each other and living on the streets. Jean was one of three sisters. Jean and Bill married when they were 16. Having grown up on the streets, Bill did what he needed to survive. In his 20s, he earned money by working gambling tables and running liquor trucks during Prohibition, quite a change from his rabbi forbearers in Ukraine. By the time I knew him, he was in his 50s, and those days were behind him.

My father’s parents, Frieda and Morris, were born in northeastern Poland in villages that were wiped out in the Holocaust. Around World War I, they both immigrated to the United States from Riga, Latvia. They met on the boat coming over and got married in the United States. At Ellis Island, my grandfather’s name was changed from Yagodnik to Goodman. Morris never learned English, although Frieda did. He worked in a Jewish butcher shop. He enjoyed watching baseball games and playing pinochle and other games with his Jewish buddies, all in Yiddish. They were poor and lived in a tiny apartment. Most of what I know about Jewish cooking comes from Frieda—her matzo ball soup, gefilte fish, borscht, and poppy seed cookies were delicious.

After the war, my father went back to school and got a degree in optometry. Something happened to his optometry practice when I was young; his partner left amid some sort of financial scandal, and my father closed the business. I never knew what happened. Meanwhile, my mother was an artist and had started an art business that was growing. My father joined my mother in her business, which imported and made paintings for furniture showrooms. We moved to a small two-bedroom, one-bath house they rented in Evanston, north of Chicago, where I went to kindergarten. We then moved to Skokie and lived in a modest three-bedroom, one-bath house, a move they made for the education of their two boys. We moved into the Niles Township school district, where I went to junior high and high school.

In the old country, my mother’s family was highly educated, but in the United States, my immediate family was not, except for my Great Aunt Isabel, my maternal grandmother’s younger sister. Isabel was born in 1905 and got a PhD at the University of Chicago in anthropology. She spent years living with Native American tribes along the west coast of Canada and became an expert in their culture. She taught anthropology at the University of Washington. For decades, she lived with the beatnik writer Piro Caro. As I was growing up, my family would tell me disapproving stories of my Aunt Isabel’s bohemian lifestyle, about her leaving academia, living in a Beat Generation commune in California, singing and playing guitar, and living on a houseboat in Sausalito. When I heard these stories, I longed to meet her.

When I began college at Stanford in 1968, I finally met Isabel and Piro. At the time, Isabel lived in San Francisco. Piro had become the leader of the houseboat community in Sausalito and lived on a converted ferryboat called the City of San Rafael. He would come over to Isabel’s house, and we would have dinner together. Piro was often on the evening news as the community leader during the Houseboat Wars of the mid-1970s. He fought for the rights of his community and saved the houseboat community. He was arrested for leading protests and gave eloquent testimony in court rooms and city council rooms. Piro died in his mid-80s while I was on faculty at Stanford and Isabel in her late 80s after I moved back to Berkeley. I admired their passion and intellect and identified with their lives.

My brother Michael and I were close when we were growing up. He was three years ahead of me in high school. He too was good at math, science, and music. He went to the University of Chicago as a physics major. He switched major to psychology and never graduated. By the early 1970s, he became a disciple of Maharishi Mahesh Yogi. He moved to the Maharishi’s ashram and research facility in Weggis, Switzerland.

In the summer of 1975, while a graduate student at Berkeley, I visited him in Weggis. I was given a tour of the Maharishi’s neurophysiology lab. With my understanding of neuroscience, I could see that their equipment had not been calibrated and likely had never been used. They told me about results that they claimed confirmed what the Maharishi had predicted. They couldn’t answer my questions and passed me from one person to another like a hot potato. In the middle of the night while I was sleeping, the Maharishi’s guards came into my room and kicked me out of the compound onto the streets of Weggis. They told me my presence was disrupting the meditation of the Maharishi’s disciples. Evidently, they didn’t like having a real scientist in their midst. Michael walked in and thanked me for coming to visit him, but his demeanor was flat and without affect. He has never spoken about it since.

When he returned to the US years later, Michael became involved with Maharishi International University in Iowa, and then moved to Florida to take care of our mother. He works as a relationship therapist, spiritual guide, vedic teacher, and leader of drum circles. He calls himself the Relationship Doctor. With him, there is a fine line between reality and delusion. On Facebook and LinkedIn, he claims to have a PhD from University of Chicago, as well as other degrees, when in truth, he does not, and in fact never graduated from college. After decades of trying to connect as adults, we are estranged.

Childhood

While I was growing up, my parents and grandparents impressed on me repeatedly that education was the key to success and to a life better than theirs. Once they saw my aptitude in math and science, they naturally assumed that I would become a “real doctor.” And they expected that I would marry a Jewish woman. I let them down in both regards.

In the summer of 1957, we moved to Skokie where I went to Devonshire School. Skokie in those days was a white middle-class suburb of Chicago and had a large Jewish population. My schools were largely white and 35–40 percent Jewish. My high school class of 500 or so had very little diversity—one Asian, no African Americans, and a few Hispanics.

Science and music were becoming my major interests. I also enjoyed sports. I was outgoing and had many friends in my neighborhood, some of whom I remain friends with today. We played lots of baseball, football, and basketball.

My interest in music began when I was very young. My parents noticed me sitting at the piano at my maternal grandparent’s house, playing melodies by ear. During grammar school, they bought a piano, and got me classical piano lessons. My parents were big fans of big band and swing jazz music. By the time I entered junior high school, they noticed that I was playing jazz and blues by ear, and so they found me a jazz piano teacher.

At Devonshire School, I excelled at math and science. I was often far ahead of the class and started to become bored. My school counselor convinced my parents to allow me to skip from the middle of fifth to the middle of sixth grade.

My parents were not religious, and neither am I. They wanted me to be proud of my Jewish heritage and sent me to Sabbath School for a few years to learn our history and culture. I was one of the few Jewish kids in my school who did not have a bar mitzvah. My parents didn’t have much money, but in the summer of 1962 when I was 11, they needed to go to Europe to meet with artists and buy and commission paintings, which was part of their business. They gave me the choice: I could have a bar mitzvah or accompany them on a trip to Europe. I chose the trip. We spent over four weeks in Europe; visiting the great cities as well as small villages in France, Italy, and Spain, where my parents met with artists and my brother and I wandered on our own around the countryside.

That trip was my first exposure to other cultures. Throughout my life, I’ve traveled to more than 90 countries. In my student days, I backpacked throughout Europe. I traveled extensively as a scientist. Marcia and I have been to many far-flung corners of the planet, snorkeling the coral reefs of Raja Ampat, birding the rain forests of Borneo, watching polar bears above the Arctic Circle, exploring the Amazon by riverboat, trekking to watch mountain gorillas in Uganda, and listening to monks chant in Himalayan Buddhist monasteries. My love of travel was born on that trip when I was 11 and never waned.

I went to Old Orchard junior high school from 1962 to 1964. I continued to excel at math and science. I was lazy when it came to foreign languages which became the bane of my education. I loved music, playing timpani in my junior-high orchestra and in a local community orchestra. I also played piano in a rock band and continued jazz piano lessons.

High School

Skokie was growing in the 1960s, as more families moved to the suburbs for the schools. The school district’s third high school, Niles North, opened in 1964, and I was in the first freshman class to enter. It was a modern high school in terms of facilities and design. I enjoyed my high school years. More than 55 years later, I still have several close friends from those days.

In high school, I excelled in the advanced placement courses in calculus, biology, and chemistry. My high school had lab space for independent science projects, and throughout high school, I did my own projects. I entered several in the state science fair and won awards. My junior year project on paramecium genetics won the highest honor at the state science fair and won the 1966–1967 Ford Future Scientists of America Award from the Ford Foundation. During the summer of 1967, after my junior year, I attended a National Science Foundation (NSF)–sponsored summer research program at the Jackson Laboratory in Bar Harbor, Maine, where I studied mouse genetics. I was beginning to get a taste of real science. I came back after that summer to serve as president of the Illinois Junior Academy of Sciences.

I also found time to do other things in high school. I served on our class council for several years and chaired the homecoming committee. I enjoyed public speaking and debating and won the Illinois high school debate competition in my sophomore year. I also played piano in my high school jazz band.

The Summer of Love

There was another side of me that most of my classmates didn’t know about, and that worried my parents: my growing musical interests. I played in a rock band that gave local dance concerts. We listened to the British invasion of blues rock, and in 1967–1968, to the psychedelic rock coming out of San Francisco. I would venture into Chicago to hear blues concerts by Otis Spann, Muddy Waters, and others. I was getting interested in playing Chicago blues and New Orleans rhythm and blues, influences that persist in my piano style today. One evening I traveled to the southside to see Ray Charles. The audience was almost entirely African American. Everyone, including me, knew every one of his songs, and we were all on our feet, singing, swaying, and clapping. It felt like a revival meeting. For days to come, I wanted to play rhythm and blues like Ray.

I was also beginning to experiment with psychedelic drugs. I had my first acid trips during my senior year. Only one high school friend knew because she and I tripped together. I was starting to explore consciousness and eastern philosophy and mind and behavior, but at school, I kept that a secret, preferring to be known as a science and math nerd and class leader.

During my summer (1967) at the Jackson Laboratory in Maine, I met a girl in our NSF program named Annette Melville who lived in San Francisco and was planning to attend Stanford. We listened to a lot of San Francisco rock music. I used money I had saved to buy a ticket to fly to San Francisco during spring break of my senior year, ostensibly to visit Stanford, but really to visit Annette and explore San Francisco. The summer of 1967 was the Summer of Love. Haight-Ashbury was exploding with rock music.

Back home, my parents, grandparents, and school counselor all wanted me to go to Harvard or Yale since those schools had more name recognition in their Jewish community, and a lot more Jewish women for me to meet and marry. My grandma Frieda warned me: “You’ll never meet a nice Jewish woman at Stanford.” I didn’t care. I accepted the offer from Stanford.

How could my family afford Stanford or Harvard or Yale? My parents had little money, but they also had not, for reasons I never understood, encouraged me to apply for financial aid. I was rescued by a family that owned a hometown pharmaceutical company. In those days, the G.D. Searle family gave two scholarships each year across the three Niles High Schools to the top two students planning to major in biology, chemistry, or biochemistry. Fortunately, I won one of the Searle Scholarships, which allowed me to attend Stanford, all expenses paid.

The spring and summer of 1968, right before I went off to college, were politically charged, and I got more involved in antiwar protests and campaigning. Martin Luther King was assassinated in April, and Bobby Kennedy in June. When I had to register for the draft, I declared myself a conscientious objector. I thought the war was immoral. That summer, a friend and I campaigned for liberal-candidate Eugene McCarthy in conservative, Ku Klux Klan–supporting areas of northern Indiana. One man greeted us with a shotgun and told us to get the hell off his property. The Democratic National Convention took place in Chicago that August, and thousands of Vietnam War protesters, including me, took to the streets. We chanted “the whole world is watching.” A few days later, I left for California.

Undergraduate Years at Stanford

I started at Stanford in September 1968. I lived in Toyon Hall and ate at El Tigre eating club. My Searle Scholarship covered my tuition and room and board, but I still needed spending money and funds to fly back to Chicago, and so I served meals, cleaned tables, and washed dishes at El Tigre. I placed out of calculus and chemistry, and started with more advanced math classes, the biology core, and organic chemistry. To fulfill my breadth requirements, I took courses in philosophy and Eastern religion.

“First Imitate, Then Innovate”

I also took a performing arts course during which I learned a key piece of wisdom that I have repeated countless times to students in my lab. Michael Bloomfield, one of the greatest guitarists of our time, gave a guest lecture. He showed us on an acoustic guitar how he had developed his style. First, he copied other’s licks and only once he had perfected what others before him had done, did he begin to develop his own style. He said that every great artist, whether a musician, dancer, or painter, would first master what had come previously before embarking on creating new styles. “First imitate, then innovate,” he told us. I realized this principle applies to science as well.

The summer after my freshman year (1969) was the last time I went home for more than a visit. I worked as a research assistant at a hospital in Chicago, an experience that convinced me I was more interested in basic research than medical research. But by far the most memorable experience of that summer was my trip with my brother to the Woodstock rock festival. We arrived during the first performance on Friday by Richie Havens and stayed to the last performance by Jimi Hendrix on Monday morning. It was an amazing experience, full of music, people, and drugs.

My sophomore year I moved into the Manzanita trailer park at Stanford. Now long gone, the trailers were temporary overflow housing for students until more dorms were built. I became manager of El Tigre eating club, a job I kept for several years. It was an easy way to earn money and get free meals.

My science classes all went well in autumn and winter. Then that spring (1970), Nixon invaded Cambodia, and our campus, like many others, erupted in protests in what was called the Cambodia Spring of 1970. Four students were shot during a Kent State protest. The Stanford campus was nearly shut down and many classes were disrupted. My only class on campus was molecular biology, which went on. I spent a lot of time off campus that spring, working as a research assistant for one of Hal Mooney’s graduate students. It was a great hands-on education in evolutionary biology and ecology.

Music vs. Science

At the beginning of my sophomore year, a friend played me a new album: Miles Davis’s In a Silent Way. The music was beautiful, spiritual, ethereal. It was the birth of electric jazz with a heavy use of keyboards. I was captivated by the music and began to toy with the idea of becoming a professional musician.

Later that year came a profound week for me, which drove me to a key life decision. I heard Josh Lederberg, a Nobel laureate and chair of genetics at Stanford, give a seminar on his research. I sat in the back of the lecture hall and had the audacity to think: “I can do that.” That same week, I heard my favorite jazz pianist, Bill Evans (who had also played with Miles Davis years earlier on Kind of Blue) give a concert, and I had the clarity to think: “I can’t do that.” Could I have done that? I will never know. But my decision was clear: I could do science as a profession and music as a hobby, but not vice versa. I knew I wanted to be a scientist. I just didn’t know what kind.

Kicking Sigourney Out of the Band

Music continued to be a major interest, and I frequently drove up to San Francisco with my friends to see rock concerts at Fillmore, Winterland, and the Family Dog. My sophomore and junior years, I played keyboard in a rock band I cofounded, Spody Odie, named after a phrase from a Robert “R.” Crumb comic. We played lots of our own songs with crazy lyrics and fun arrangements. I wrote several, including “Hard Hat Blues,” with our lead guitarist, a political satire on Richard Nixon’s Hard Hat Riot of 1970. Everyone in the band was talented. We were known for long guitar and keyboard solos, and a great rhythm section. We briefly had a female singer who went on to be a famous actress: Sigourney Weaver. She had real stage presence, which was something the rest of us lacked. The problem was that Sigourney envisioned us as her backup band, featuring her singing, with only short instrumental solos, whereas we wanted to do our own songs with long solos. At a rehearsal one Saturday morning, she stopped our lead guitarist in the middle of a brilliant solo and laid down the law: no more long solos. No one said a word. I looked at my bandmates, read their eyes, looked at her, and pointed to the door.

My bandmates, Craig Okino, Roger Davis, Steve Grushkin, and Rick Ries, and I were close friends. We lived together, played music together, and partied together. I was scientist by day and musician by night, and I thoroughly enjoyed my double life. I studied hard and worked long hours in the lab during the day. But I had my other life at night and on weekends. In my junior year, we recorded a demo tape, with the hope of getting a recording contract. But while some of the guys wanted to go professional, I had made my decision to be a scientist and Rick wanted to go to medical school. We broke up but have stayed close friends over the decades since college.

The others have played in professional bands over the years. In October 1973, while I was a graduate student at Berkeley, Steve, Craig, and I went to see the original Wailers on their first California tour. Within a few months, Steve moved to Jamaica, where he lived for many years, and became a well-known reggae bass player. Craig played for years in Pagan Babies, a worldbeat band in Honolulu. Steve (aka Herb Daily) continues to play in reggae bands, while Roger plays in a band called Tropic Sōl. For decades, Spody Odie had reunions. We would rent a rehearsal studio in the Bay Area, come together from all over the country, and spend a day playing music, telling stories, eating and drinking, reminiscing about kicking Sigourney out of the band, and enjoying our lifelong friendship.

My Introduction to Fruit Flies

Early in my junior year, I heard a lecture on biological clocks by Colin Pittendrigh and found it a fascinating model for studying the physiological control of behavior. For winter and spring quarters, I worked with one of Pitt’s graduate students. I mastered the use of his “bang boxes,” used to study the physiology of the eclosion rhythm (the emergence from the pupal case) in Drosophila. That was my first experience with Drosophila, and I enjoyed it.

Students in Pitt’s lab told me about what Ron Konopka was doing in Seymour Benzer’s lab at CalTech, using Pitt’s bang boxes to do a genetic screen for eclosion-rhythm mutants. I thought Ron’s genetic approach would crack the field wide open, and he did, with the discovery of the per mutants. It was my introduction from afar to Seymour Benzer and his genetic approach to neurobiology and behavior. I came to greatly admire Seymour. Years later, he and I visited each other’s labs, talked science, and enjoyed some fabulous meals together.

My Mentor for Life

My junior year is when I discovered my calling in science. In the winter quarter of 1971, I took Don Kennedy’s class, Physiological Basis of Behavior. Kennedy was the best teacher I ever had; every one of his lectures was an epic story. He was entertaining and captivating. At the end of each lecture, you closed your notebook, and felt you had learned something profound. Neuroscience brought together all my scientific interests, using biology, chemistry, and biophysics to understand how we behave, think, and perceive.

From Kennedy’s list of term-paper topics, I picked what became my focus for decades to come: the formation of nerve connections. I spent long days and nights in a small alcove in the biology library. Most of the key research at the time was focused on regeneration of retinotectal connections in fish and amphibians. I read papers by Roger Sperry, Marcus Jacobson, and others. By 1971, there were a lot of naysayers about Sperry’s chemoaffinity hypothesis and a lot of seemingly conflicting experimental results. I did not see a knockout punch and thought Sperry was right. I titled my paper “Specification of Neuronal Connections” and concluded that Sperry’s notion of molecular specificity and, in particular, gradients controlling neuronal connections was “plausible but lacks supporting evidence.” Little did I know that I would spend the next three decades obtaining that evidence.

Kennedy had us come into his lab to pick up our term papers from a table outside his office. He just happened to walk out of his office as I was looking at his comments on my paper, and he said, “So you’re Corey.” I was amazed he knew my name. He told me that my analysis of the formation of neuronal connections was the best he had ever read. He put his arm around me and walked me around his lab, introducing me to some of his graduate students and postdocs and telling them that this was the undergraduate student whose term paper he had read to them at their group meeting earlier that week. I was shocked. Professor Kennedy, who for the rest of my life would be Don, made me feel special. He asked me if I wanted to do my honor’s thesis in his lab. I was overwhelmed. Of course, I said yes. The next quarter, he had me take Biology 253, his intensive graduate neurophysiology lab course.

I tried to keep my double life as scientist and musician a secret from Don and others in his lab. I worried they wouldn’t take me seriously if they knew I performed in a rock band. Then one morning at his weekly group meeting, Don told me that he and some others from the lab had heard me perform at a concert the previous Saturday night. I had no idea they had been in the audience. He smiled and said that in addition to being a very promising young scientist, I was a damn good keyboard player. I was relieved. I no longer had to hide my other life.

While most of Don’s lab worked on crayfish, I did my own project, using the salamander for limb-transplant experiments to test the specificity of regenerating motoneurons for specific muscles. I worked hard and had the support of many of his terrific students and postdocs. I learned a lot from them about how to do science.

Don became the major mentor of my scientific career. While I was a graduate student at Berkeley, I would frequently go to Stanford to use Don’s microscopes, interact with his lab team, and discuss my research with Don. In some respects, Don knew more about my graduate thesis than did my adviser back in Berkeley. At every step of my career, I turned to him for advice. To this day, I have several of his books that he gave me, a cherished gift to remember him by. Don lived his life in chapters, and in that regard, he was a great role model for me. He went from neuroscience professor and inspirational teacher, to head of the U.S. Food and Drug Administration (FDA) under President Carter, to president of Stanford, to editor of Science magazine. Don remained a dear friend throughout my career until his death from Covid in 2020.

What a Long, Strange Trip It’s Been

In the spring of my junior year (1967), while living in the hills of Los Altos, I experienced a convergence of neuroscience and psychedelic drugs. I had learned about the maps in the visual system and had read about how different aspects—form, color, movement—are encoded in different regions of visual cortex and are kept aligned by interconnections between the various maps, so that we perceive the world as a single visual picture. One Saturday afternoon, I dropped acid and took a walk up the hill behind our house. I picked up a long blade of grass and waved it back and forth in front of my face. As I enjoyed the visual sensation I was creating, I realized what I was seeing: the different cortical areas had come unglued. Form, color, and movement were all on their own and no longer aligned. I marveled at how the interconnections of different regions of the cortex that normally hold all of this together in a single perception of visual space had been uncoupled by LSD, so that the output of each region was perceived independently. The experience further cemented my desire to be a neuroscientist.

During my senior year, my girlfriend LaDene Otsuki and I lived with seven friends, two dogs, and three cats, in a six-bedroom house on Richard Court in Mountain View. It was communal living in which we cooked and shared together. LaDene and I lived in the garage with my keyboards. Two of my band mates, Craig and Steve, lived with us. The nine of us, with various partners, developed a very tight bond. We continue to have Richard Court reunions. Music, drugs, politics, and communal living were key influences on how we grew during our undergrad years.

I was taking classes and spending most of my daylight hours doing research in Don’s lab. I filed my senior honors thesis, and Don wrote a letter for my graduate-school and fellowship applications. I was awarded an NSF Predoctoral Fellowship. I applied to many graduate programs and was accepted by all of them. LaDene’s aspiration was to become a world-class classical pianist and she found a great teacher near Berkeley. Don thought Berkeley was one of many good choices for me. Thus, just as I had picked Stanford because of Annette, so I picked Berkeley because of LaDene.

Graduate School at Berkeley

LaDene and I moved to Berkeley in August of 1972. We rented a three-bedroom house and found other roommates. I bicycled to campus. My NSF fellowship paid $300 per month. We had little money but still managed to enjoy the diversity of food and culture in Berkeley. LaDene and I bought an old A.B. Chase upright piano for $150 at Salvation Army. It was a remarkably good piano, given how cheap it was. LaDene worked at the law school while studying piano with her teacher. The pieces I heard her practice—certain Beethoven sonatas, Chopin etudes, and Scarlatti sonatas—became permanently etched in my brain.

We went to plenty of rock and jazz concerts, but, given LaDene’s passion, we also went to every classical piano recital in the Bay Area. A few years later, when we moved into a bigger six-bedroom house in Berkeley, LaDene bought a grand piano, quit her job, and focused all her energy on piano. Sitting next to her grand piano was my Fender Rhodes electric keyboard. The house was often filled with music. Our housemates were a diverse group, including my college band guitarist Steve before he moved to Jamaica, an English graduate student from our Stanford gang, a law student, and a scientist whom I had met in Don’s lab.

Music continued to play a large role in my life. My old rock-musician friends would occasionally get together. I met a terrific jazz bass player and jazz guitarist. The three of us would get together and play jazz standards and free-form improvisations. I developed my jazz-piano style during those years and learned a lot from them.

My first year of graduate school was boring. It felt like my undergraduate days all over again, but without the research I had been doing in Don’s lab at Stanford. The coursework was material I felt I could learn on my own. I was anxious to get into the lab. I was required to take the graduate neurophysiology lab course, but it felt redundant, because I had already taken Don’s graduate course at Stanford.

To entertain myself, I surprised some faculty by taking the undergraduate Natural History of Vertebrates two-quarter course in winter and spring. Berkeley was renowned for having one of the very best vertebrate-biology museums in the world. I thought that while I was waiting for the purgatory of my first year to be over, I would at least learn something interesting—the natural history of birds, herps, and mammals. That course had a big impact on me. I became an avid birder. Later I got my wife Marcia hooked, and we both love heading off to rain forests to watch birds and monkeys. We’ve been birding in Trinidad, Belize, Costa Rica, Panama, the Amazon via Ecuador and Peru, many parts of Africa, Borneo, Sri Lanka, and the Arctic.

By the end of spring quarter, I had decided to work in the lab of Hugh Rowell. He had two terrific scientists: a graduate student just finishing up, Carol Mason, and a postdoc, Michael (Mick) O’Shea. Carol graduated, I bonded with Mick, and Hugh promised to let me do whatever I wanted. The lab was joined by another postdoc, Bill Heitler, and a neuroscientist on sabbatical, Keir Pearson. I had the opportunity to apprentice and collaborate with Mick, Bill and Keir. Although Hugh was going through a divorce and largely absent, the lab environment was quite stimulating. And Don Kennedy remained a valuable mentor.

Before starting in Hugh’s lab, I spent the summer of 1973 at Friday Harbor Laboratory, University of Washington’s marine biology station in the San Juan Islands. I learned a lot more marine biology and invertebrate development and evolution. This too had a big impact on me. I had spent the summer after my junior year of college at Stanford’s Hopkins Station. My two summers at marine biology stations turned me into a naturalist and marine biologist. Marcia shares that interest as well, having spent a summer while she was an undergraduate at the Fairleigh Dickinson marine biology station on St. Croix. Our common interest in marine biology has lasted for decades, taking us to remote coral reefs in Raja Ampat, Komodo, and Palau. We also spend a lot of time on the Big Island of Hawaii, where today we have a home just a short walk from one of the island’s best coral reefs

One of my teachers at Friday Harbor was Fu-Shiang Chia from University of Alberta. He came from a very poor family in China, left home at a young age, traveled around China for several years as an apprentice to a palm reader, and then escaped to Taiwan where he was educated. Fu-Shiang was a talented palm reader, so I asked him to read mine. Looking at the palm of my hand, he predicted my life. He said I would live a long life, with one major health scare that I would survive, that I would become famous, and at the peak of my career, I would change professions and succeed again. I asked him if he always told people good things. He laughed and answered no, that in his village, if you were good at something, you were expected to keep at it. He had just told me my future, he said with a twinkle, and it was up to me how I interpreted it. Obviously, what he told me about my career (not my health) resonated with my thinking. Remarkably, concerning my health, he was right as well.

Isogenic Grasshoppers

My thesis was titled “Identified Neurons and Behavior in Isogenic Locusts.” How I got there is an interesting story of serendipity of a conversation one day with a student from Uganda.

Hugh had been educated at Cambridge University in England. He had also done research and taught in Uganda before coming to Berkeley. When I joined, his lab was studying the identified neurons that control the visually induced jump reflex in the grasshopper (or locust). His postdoc Mick O’Shea had discovered the descending contralateral movement detector (DCMD) interneuron, with its large axon, that connected the brain and visual system to the motoneurons and jump circuitry on the other side of the thoracic segments. As I looked at Mick’s data, I noticed that DCMD did not have the same branches in every animal. You could recognize it by its general shape and position of its cell body and axon, but the pattern of its axon branches varied.

To start my thesis, I wondered if I could study the constancy and variability of identified neurons, both in their anatomy and physiology, and then study the role of nature and nurture in the development of their anatomy and physiology. To what extent is the variability inherited? Might this be the substrate of evolutionary selection?

I wanted to find a group of identified neurons to study. After studying the grasshopper’s neuroanatomy, I focused on the ocelli, the three small eyes that insects have in addition to their large compound eyes. For any single ocellus, I found a way to stain a group of seven large, identified interneurons. I discovered that I could simply hold the grasshopper down, stick a pin into the ocellus and mash up the interneuron endings, inject a drop of cobalt solution into the ocellus, cover the hole, and wait a day. Presto. Seven beautiful, cobalt-stained ocellar neurons every time. This method was reliable enough to do large numbers.

In 1974, I published a paper on the constancy and variability of the ocellar interneurons. There was overall constancy in the general shape of each neuron, but variation in the branching patterns, including cases in which axons extended into regions where they normally were not found, and cases in which extra neurons occurred, which appeared to be duplicates of normally occurring cells. In 1976, I published a paper in Science showing that the same principles of constancy and variability pertained to a group of 61 small interneurons.

I was starting to get a hunch that some of the variability in identified neurons had a genetic basis. Certain kinds of variability clustered in different clutches (i.e., offspring from the same female). I learned that some insects could reproduce parthenogenically (i.e., without fertilization), leading to isogenic (genetically identical) offspring. One day, I was talking with Bill and said I wished I could breed grasshoppers parthenogenically. Just at that moment, Ochong Okelo, a graduate student from Uganda who Hugh sponsored, walked into the lab, heard my question, and said: “Corey, but you can.” Ochung taught me that if you crowd the females and let them see and smell males but not copulate, they will ultimately lay parthenogenic eggs and produce all female offspring.

I determined that parthenogenesis in grasshoppers led to homozygous, diploid females, and isogenicity. Having shown that I could produce clones of isogenic female grasshoppers, I started making many different clones and observing their behavior, neuroanatomy, and synaptic physiology. The first thing I noticed was that I was getting the same kinds of variability I had seen in the breeding population, but it was clustered in specific clones.

In 1977, I published another paper in Science entitled: “Neuron Duplications and Deletions in Locust Clones and Clutches.” I showed that duplications and deletions of identified neurons can occur with a high degree of genetic control and specificity, as shown by examining the ocellar interneurons from different parthenogenetic clones of isogenic animals.

Perhaps the most interesting results from my thesis came from my examination of genetic variability in the morphology of an identified neuron. I focused on one ocellar interneuron, L5, on each side of the brain. I looked at this neuron in 11 different isogenic clones. In most clones, the morphology was normal. But clone 2 had a high percentage of animals with axon abnormalities of L5. The other ocellar interneurons were normal.

The axonal abnormalities were not random but rather showed a pattern. In clone 2, L5’s axon would sometimes either grow more laterally on its way to its normal posterior arborization, or it would send an extra branch laterally. In both cases, it would have an extra arborization in a specific lateral neuropil region. It would also sometimes grow further posterior than normal and have yet a different extra arborization in a specific posterior neuropil in the brain. Finally, the axon sometimes grew across the midline to the other side, and when it did, it ultimately terminated and arborized in the correct region on the other side or additionally in one of the extra neuropil regions that it arborized in on its own side in clone 2.

These data led to interesting insights about normal development. The results eliminated certain models for axonal branching and termination. They were not determined by distance or time. Moreover, the signal for branching in an area was not directional but rather induced branching and termination irrespective of the direction from which the axon approached the area. Finally, the signals looked to be the same on both sides of the brain. I hypothesized a multi-epicenter model in which gradients of the same morphogen (or chemoattractant) are used in different regions of the neuropil, but a given axon is normally only exposed to one or a few of those regions.

I got my PhD in five years and published twelve papers, two of them in Science. My research received a lot of attention, including invitations to speak at scientific meetings. But, as I was finishing up my thesis, I became self-critical of where it was going. I was looking at axon trajectories and numbers of neurons in the adult after genetic manipulations and speculating about what happened in the embryo. I wanted to understand the cellular and molecular mechanisms that generate neural specificity during embryonic development.

Stepping Stones

I needed to find an organism in which I could study and manipulate embryonic development of individual neurons. I started looking throughout the animal kingdom, reading papers, trying to find the perfect preparation. And then came another chance occurrence. Mike Bate, who became a collaborator for years and a friend for life, visited Berkeley in 1976 and gave a seminar on two key papers he either had or was just about to publish in 1976 from his postdoctoral work in Australia. His first seminal paper in 1976 described his discovery of pioneer neurons that navigate along stepping stones in the grasshopper embryo limb bud, supporting what Ross Harrison had proposed in 1910. His second paper was on a map of neuroblasts in the grasshopper embryo. Both studies came from his analysis of fixed and sectioned material. Mike was in Berkeley on his way to be an independent scientist with Friedrich Bonhoeffer in Tubingen, Germany, to continue his studies on the grasshopper embryo.

Mike and I spent the evening talking about how to study the development of the nervous system. We fast became friends and realized we had similar goals. We discussed someday moving into Drosophila and using the power of genetics, but for now, we would learn the cell biology of neuronal development in the grasshopper embryo. We realized we had to be able to do this in living embryos, not just fixed and sectioned material.

The next morning, I collected some grasshopper eggs of different ages. I popped them open, figured out how to dissect and hold them down as flat fillets, and looked at the living grasshopper embryos in a dissecting microscope and then in a compound microscope. I realized grasshoppers had a large, transparent embryo. I could see the developing axon pathways, neuroblasts, and neurons, and in some cases, it looked like I could visualize individual axons in the living grasshopper embryo.

Shortly thereafter, I applied for and was awarded a Helen Hay Whitney Postdoctoral Fellowship to study neuronal development in grasshopper embryos. My original plan was to go to Cambridge, England, to join the lab of Malcolm Burrows. But then a friend introduced me to Nick Spitzer’s work at University of California, San Diego. Nick was using Nomarski interference contrast optics to visualize and record from developing Rohon-Beard neurons on the dorsal surface of the living Xenopus spinal cord. I arranged to visit Nick and give a seminar about my thesis work. I asked him if I could come to his lab, and together we could work on the development of identified neurons in the grasshopper embryo. Nick said yes. I wrote to the Helen Hay Whitney folks for permission to take their fellowship to Nick’s lab, and they agreed. That led to an incredible time as a postdoc in San Diego. Mike came over several times and worked with Nick and me.

Postdoc Years in San Diego

I moved from Berkeley to San Diego in the spring of 1979 and stayed for two years. I moved into a house just a few blocks from Windansea Beach in La Jolla. I made a lot of new friends, some of whom I’ve stayed close to over these years. My devotion to music slowed down as science became my main focus. I had a piano and played often. I met another jazz pianist who introduced me to a jazz music-theory teacher, and for most of my time in San Diego, I took weekly lessons from him. For a while, I played piano behind a jazz singer who sounded like Billie Holiday. But I didn’t have the circle of musician friends like I did in the Bay Area, and I didn’t have the time.

Dancing with My Star

In my second year (1978–1979), I went to a party for the new first-year graduate students in biology. There I met Marcia Barinaga, my future wife. She was beautiful, bright, fun, playful, and captivating. I was hooked. We had lunch together a few days later.

A week later, some friends asked if I was interested in joining them to take country-swing dance lessons at one of the local cowboy bars. I enjoyed country music and the occasional beer but was a little ambivalent about dance lessons, that is, until I looked down the list and saw Marcia’s name on it with no partner listed next to her. I suddenly decided that I had always wanted to take country-swing lessons. She and I did in fact become dance partners, and great ones at that (we still go out swing dancing), and clearly, from my perspective, we developed something special. I couldn’t figure out why we weren’t progressing beyond dancing together, until a mutual friend told me that Marcia lived with her boyfriend, but he didn’t like dancing. For my final year in San Diego, I had a different girlfriend, but continued to go out dancing with friends, including Marcia.

To finish the story of how Marcia and I finally got together, fast forward to the first few years when I was on faculty at Stanford. I had occasionally seen Marcia on visits to San Diego. Marcia was working on her thesis at the Salk Institute in Ron Evans’ lab. Then in the summer of 1982, I heard from mutual friends that Marcia had broken up with her boyfriend. An amazing coincidence occurred. Work in my lab was going very well. I was being quite selective in agreeing to give only a few seminars and scheduling dates a year or more in advance. I got a call from Max Cowan at the Salk, asking me to come down and give a seminar. I surprised him by asking, “how soon?” He said there had been a cancellation the following month, and I agreed. I wrote to Marcia and told her I was coming down to give a Thursday seminar, and suggested we get together that Friday and take a walk on the beach. A walk became dinner, and dinner became the weekend. We commuted for more than a year. Marcia moved up to live with me in the summer of 1984. We were married on December 8, 1984, the same date that appears on her PhD diploma. She likes to joke that she got her PhD and Mrs. on the same day.

We were married with family and a few close friends in Florida near where my parents lived, and then we had a big party at Stanford at Richard Scheller’s home. Of course, just as my PhD didn’t fulfill my family’s dream that I would become a real doctor, neither did marrying Marcia, who is of Basque and Polish heritage, fulfill their dream that I would marry a Jewish woman. There was a little tension with my family, but they had raised me to be independent. My allegiance was with Marcia. One of my cousins had a traditional Jewish wedding the next year (with their rabbi and cantor playing key roles), and during the festivities, my father—after a few drinks—said to Marcia and me, with my mother nodding in agreement, “Now this is a real wedding!” Fortunately, as years went on, my parents came to love Marcia and realized that I was lucky to have found her.

Grasshopper Embryos

In San Diego, Nick had arranged for a small room in the building where I could raise grasshoppers and grow the wheat to feed them. I brought grasshoppers, and some of my cages, down from Berkeley, and we were up and running in no time. We did experiments together. Soon I was doing experiments day and night, and as Nick had other commitments, he would join me when he could and stayed in daily touch with our data.

As Mike Bate had described, each thoracic segment has a plate of 31 ventral neuroblasts on each side of the midline. We could see them and their offspring in the living embryo. We could also see axons and even growth cones. Even more clear and distinct was the single dorsal unpaired median (DUM) neuroblast at the posterior end of each segment and dorsal to the plates of ventral neuroblasts. The DUM neuroblast gives rise to a set of neuronal progeny that are clearly distinguishable. We could visualize the DUM neuroblast and its progeny, and identify some of them individually, by removing the embryo from the egg, desheathing the dorsal surface, and viewing the dorsal surface under a water immersion lens with Nomarski optics.

Nick and I published seven papers from our two years together. We initially focused on the differentiation of a single identified DUM neuron, called DUMETi, followed it from neuroblast to neuron, recorded from it, looked at its electrical coupling and uncoupling, examined its sensitivity to neurotransmitters from its cell body to growth cone, and injected dye and followed its morphological development. We published that work, mapping the temporal and spatial differentiation of a single identified neuron, in a feature article in Nature in 1979.

At the same time, we collaborated with Mick O’Shea, by this time at USC, and Dick McCaman, at City of Hope, two experts on these neurons in the adult, to study the biochemical differentiation of the neurons in correlation with their morphology and other properties. The four of us published this work in Science in 1979. Nick and I published two papers on the development of electrical properties of these neurons, and another with Keir Pearson (Edmonton) on the morphological and electrical properties in the progeny from a single neuroblast.

I invited my good friend Mike Bate to come over. He slept on my couch for a month and worked with me in the lab. He became a pro at injecting and recording from embryonic neurons. Mike discovered a second class of neuronal precursor cells along the midline of each segment in the grasshopper embryo, the midline precursors (MPs), that each divide once to generate two progeny. While Mike was working with us, and after he went back to Germany, we worked together on the differentiation of the H neuron, which arises from MP3. Mike, Nick, and I published two papers in 1981 on the work we did together. Mike and I continued musing about whether neuronal development in Drosophila was similar to grasshopper, and whether we could use the power of genetics to study neuronal development. That would have to wait a few years.

The body of work Nick and I did together launched the field, and launched my career. We were the first to watch identified neurons develop from neuroblast to differentiated neuron, to map the extension of growth cones from outgrowth to synapse formation, and to map the coordinated development of electrical excitability and neurotransmitter receptors. I am indebted to Nick for giving me the opportunity to develop the grasshopper embryo in his lab. When it came to jobs, I didn’t surprise anyone by accepting an offer from Stanford, my alma mater. After two fabulous years in San Diego, I moved back to the Bay Area in the summer of 1979 to start my own lab.

Back on the Farm

That summer, I returned to Stanford to fulfill my dream of running my own lab, launching my academic career as a professor and lab director, which spanned the next 25 years, from 1979 to 1987 at Stanford, and from 1987 to 2005 at U.C. Berkeley. We were in the right place at the right time to discover some of the key cellular rules and molecular mechanisms of axon guidance. Over those 25 years of rapid discovery, I was joined by 25 graduate students and 52 postdocs. It was an amazing team and an incredible journey. Together we published 212 papers with an h-index of 132. Many brilliant careers were launched from that group. Two of my graduate students, Chris Doe and Denise Montell, and one of my postdocs, Alex Kolodkin, are already elected members of the U.S. National Academy of Sciences (NAS), and others will no doubt join them. Another graduate student, Hailan Hu, won the 2022 L’Oreal-UNESCO International Prize for Women in Science.

When I first arrived, I was given a windowless space in the basement of Herrin labs, next to the boiler room. Rats scurried in the ceilings overhead. Don Kennedy called us the troglodytes. But we had great times there. Early on, I was joined by some terrific postdocs: Jon Raper, Paul Taghert, Mike Bastiani, John Kuwada, and Andy Harris. Kathryn Kotrla and Susannah Chang were early grad students. Robert Ho was an undergrad. Eldon Ball came from Australia on sabbatical. Together, we troglodytes did a lot of exciting science in that basement.

I will always be indebted to the McKnight Foundation for giving me a Scholars Award in my first year. That funding was essential for launching my lab. My interview with the review committee was unsettling. I expected they would ask me why I was working on something so simple. But Sam Barondes took over and asked the opposite: why was I working on something so complicated, and how did I ever hope to unravel the molecular mechanisms of axon guidance? Sam was getting at the core of whether we could do genetics of axon guidance in Drosophila. His questions were worrisome because I didn’t yet have all the answers. I was convinced I had blown the interview, but I got the award, and Sam and I became lifelong friends.

The Chemoaffinity Hypothesis

The human brain contains trillions of neurons, each of which can make hundreds to thousands of synaptic connections with specific targets. Every human brain, while marvelously unique, is constructed based upon a common wiring diagram. This problem is extraordinarily complex. Millions of neurons located in one region in the developing brain form connections with the correct subset of target neurons in another region by growing processes over long distances and bypassing incorrect targets along the way. What molecules and mechanisms control brain wiring? Where in the genome is the instruction manual for this complex wiring diagram?

The problem had been defined more than a century ago by Santiago Ramón y Cajal, who hypothesized that growth cones are lured to their targets in the developing nervous system by chemical factors released by the target cells, a concept called chemotropism. In the 1930s and 1940s, Paul Weiss claimed to have disproved chemotactic guidance and instead proposed the resonance hypothesis, suggesting that axons grow at random and later prune inappropriate connections, maintaining those that are functionally correct.

Roger Sperry, who had been a student of Weiss, was convinced his former advisor was wrong. In 1963, Sperry proposed the “chemoaffinity hypothesis,” based on his studies on the regeneration of retinal nerve fibers into the tectum of the frog brain. During the 1970s, Sperry’s hypothesis came under considerable criticism, as the field became mired in debate over whether neural specificity existed, and if so, to what degree and using what mechanisms. It was in this historic context that I began my quest to understand the molecular basis of neural specificity

The G Growth Cone

The most impactful of our early discoveries was the work of two postdocs, Jon Raper and Mike Bastiani, published in full in 1983 and 1984, on the exquisite specificity of the G growth cone for the two P axons in the grasshopper embryo. In 1979, Jon and I picked the G neuron as the ideal growth cone to follow and manipulate, because it was accessible and easy to identify, and we knew it in the adult from Keir Pearson’s work. G’s growth cone first extends toward the midline, and then it leaves the midline and continues laterally on the other side. Finally, it turns anterior along a specific longitudinal bundle of axons that it had ignored on its own side. We reasoned that if we could discover the cellular and molecular mechanisms for that set of early growth cone choices toward, across, and away from the midline, and the choice of a specific pathway on the other side, we would understand much of the cellular and molecular logic of axon guidance.

Jon and I used microelectrodes to inject dye into the G neuron and then into the axons upon which it was extending. We were joined by Mike Bastiani who did electron microscope reconstructions of the dye-filled G growth cone and filopodial contacts at various stages along the choice of its specific longitudinal pathway. We discovered that at this stage, there were around 150 axons organized into around 20 bundles of axons, called axon fascicles, each in specific locations. The filopodia from the G growth cone encountered most if not all of them, but it always picked a bundle of four axons, that we called the A/P fascicle, and of those four axons, always grew along the surface of the two P axons, and not the two A axons. This was a remarkable degree of specificity.

To test the exquisite specificity of the G growth cone for the two P axons, Jon selectively ablated the P axons, the A axons, and other neurons, and followed the growth of the G growth cone. The results were striking. When the two P axons were ablated, the G growth cone stalled and stayed in that region of neuropil and did not extend on any other axons. No other ablation had this effect. Clearly, the G growth cone is determined to follow the two P axons and no others after crossing the midline, even though it ignores the same P axons on its own side. These results gave rise to the labeled-pathways hypothesis, and the notion that affinities can switch upon crossing the midline.

A few years later, Mike, along with Chris Doe and Sascha du Lac, showed the same degree of specificity of two other identified neurons, aCC and pCC, for different axonal and glial pathways. Chris used laser ablations to create elegant temporal delay experiments and showed that even with a 5 percent temporal delay in the development of an individual neuron, that its growth cone still recognizes the same pathway.

Although the detailed series of Raper and Bastiani papers weren’t published until 1983 and 1984, by 1982 we had already published some preliminary symposium papers on these findings. Around the same time, Robert Ho, Paul Taghert, and Mike published papers on pioneer neurons and peripheral pathways and filopodial contacts and dye coupling. And Robert and Eldon Ball discovered muscle pioneers that erect a scaffold for developing muscles and guide motoneuron growth cones. Things were moving fast.

Our results on the G growth cone got a lot of attention at scientific meetings. It was clear we were heading toward a genetic analysis of axon guidance in Drosophila. Harvard offered me an endowed professorship in 1982. In response to that and other offers, Stanford tenured me in the spring of 1982. To convince me to stay, the Biology Department committed to hiring a neurobiologist of my choice and to give both of us adjoining labs on the newly renovated first floor of Herrin labs. The Goodman cave denizens were offered the opportunity to move upstairs into fresh air and sunshine. We felt like the prisoners as they emerge from the dungeon to sing their chorus in Beethoven’s opera Fidelio.

For my new colleague, I picked a postdoc from Eric Kandel’s and Richard Axel’s labs named Richard Scheller. Besides being a superb scientist, Richard was a kindred spirit, having grown up in the Midwest, played bass in a rock band, and lived a life very similar to my own. My lab moved upstairs, and Richard moved next door, beginning a fabulous five-plus-year period of productivity and a lifelong friendship. We learned a lot as we pushed each other to take on big questions, usually while playing nerf basketball in his office. We attracted some incredibly talented postdocs and graduate students into the dynamic environment of our adjoining labs.

In 1983, I was honored to receive the annual Alan T. Waterman Award (an award Richard won in 1989), given each year by the National Science Board to a single outstanding young scientist in any field of science or engineering under the age of 40. The award ceremony was at the U.S. State Department. In addition to Marcia, I was joined by my parents and my Aunt Myra and Uncle Leo. That was an important moment for my family. My parents finally came to appreciate my success and abandoned their belief that I should have become a real doctor.

From Grasshopper to Drosophila

Mike Bate and I were anxious to move our studies from grasshopper to Drosophila. In the winter of 1983/1984, I spent a week working with Mike in Cambridge, England. Just before I arrived, Mike had figured out how to dissect the fruit fly embryo as a fillet, much like we did with grasshopper embryos, permitting visualization of the dorsal surface of the embryonic nerve cord. He was anxious to show me but hadn’t yet identified our old friends, the identified neurons from grasshopper. I looked through the Nomarski microscope and saw the developing nerve cord with two cell bodies looking just like aCC and pCC. I stuck microelectrodes into them and filled them with dye. We were thrilled to see that it was indeed aCC and pCC. Over the next few days, we filled a bunch of our old friends with dye. It was exciting working together. It was clear that the fruit fly embryonic central nervous system (CNS) was highly homologous to the much larger grasshopper embryo.

When I got back to Stanford, Mike and I enlisted the help of John Thomas and Mike Bastiani in my lab to compare grasshopper, moth (Manduca), and fruit fly embryonic nervous systems by filling identified neurons and doing electron microscopy on their growth cones. We showed that the G growth cone in Drosophila, just as in grasshopper, crossed the midline and turned anterior along the two P axons in the A/P fascicle. John, Mike Bastiani, Mike Bate, and I published a feature article in Nature in 1984 entitled: “From Grasshopper to Drosophila: A Common Plan for Neuronal Development.” On both sides of the pond, we embarked upon a genetic analysis, my lab focusing on axon guidance and Mike’s on muscle and neuromuscular development.

Questioning the Relevance of Our Model System

By 1984, we believed our data showed Sperry was right. We now needed to identify the guidance molecules in Drosophila that generate the remarkable degree of specificity we had discovered in grasshopper. But not everyone shared our enthusiasm. Gerry Edelman was not going to let grasshopper experiments get in the way of what he coveted—a second Nobel Prize for his discovery of the neural cell adhesion molecule (NCAM). He was convinced that NCAM and one other CAM were sufficient, along with activity-dependent competition, to wire up the nervous system. He called this “modulation theory” and said it refuted Sperry’s chemoaffinity hypothesis. In a 1983 article in Science, he wrote: “The accumulated data on . . . CAM’s favor modulation theories rather than strict chemoaffinity theories of cell-cell recognition.”

Most young scientists in their early 30s don’t have to face a relentless onslaught from an aggressive Nobel Laureate seeking to discredit their work, but I did in the mid-1980s. Edelman attacked me at scientific meetings with arrogance and showmanship, barraging me with questions without letting me answer, telling jokes and stories to distract the audience, and trying to fluster me. But I wasn’t intimidated.

Gerry next convened a group of four senior developmental neurobiologists for a meeting at Rockefeller. Not one of them was a molecular biologist or geneticist, but they initiated an inquisition of my work and the work of scientists working in parallel on the nematode. The leader of Edelman’s team appeared to be Dale Purves. Dale had told me in private that he believed our work was irrelevant for mammals. Dale’s notion was akin to Paul Weiss’s resonance theory from the 1930s, but with the addition of nerve growth factor (NGF)-like growth factors and NCAM. This seemed absurd. “When exactly, during evolution,” I asked Dale, “did the mechanisms that were used to construct complex nervous systems in simpler organisms get abandoned and replaced by random growth and competition?”

In the winter of 1984/1985, I was summoned to fly to New York to meet with Edelman and his four senior elders. I wasn’t surprised to see Dale Purves, Pasko Rakic, and Steve Easter. But I was dismayed to see Nick Spitzer, my former postdoc adviser, as a member of Edelman’s group. Dale and Steve proclaimed that I was either dead wrong in how I interpreted our experiments, or I was studying an evolutionary dead-end that had nothing to do with the mammalian nervous system. This criticism reminded me of anonymous reviews we had received for the Raper and Bastiani papers, saying we were either wrong or studying something irrelevant.

Thus, I wasn’t surprised when their paper downplaying our work, entitled “The Changing View of Neural Specificity,” was published as an article in Science in 1985. They wrote: “recognition is probably a relatively weak force in the generation of connections.” They marginalized our work by writing that “a more rigid plan may prevail in some invertebrates.” They cited Edelman’s claim that modulation of two CAMs explained nervous system wiring.

From my perspective, it was their paper, not our model system, that was irrelevant. The specificity of the G growth cone for the two P axons (out of 150 or so axons within filopodial grasp) was impossible to explain using Edelman’s model. If axon guidance were simply based on an adhesive hierarchy of two molecules, then when the P axons were ablated, G would have picked another pathway, but it didn’t. As their paper was published, John Kuwada in my lab was in the process of showing the same degree of neuronal specificity in the fish embryonic spinal cord, with results that were virtually identical to what we had shown in the grasshopper embryo. John published his work in Science a year later in 1986.

The Easter, Purves, Rakic, and Spitzer 1985 paper is interesting from a historical perspective. They and others held onto these antispecificity notions until the discoveries in the late 1980s and 1990s became overwhelming in revealing the molecules and mechanisms that control guidance and their conservation across phylogeny.

Labeled Pathways

During the mid-1980s, a group of superb postdocs joined my lab, including John Thomas, Steve Crews, Kai Zinn, Peter Snow, Allan Harrelson, and Tom Elkins. Kai and Steve brought a whole new level of molecular biology to the lab, Peter brought protein biochemistry, and John and Tom brought Drosophila genetics. Many of them went on to make major discoveries and have highly successful careers. Several superb graduate students also joined my lab around the same time, including Chris Doe, Denise Montell, Nipam Patel, and others (of that trio—two today are elected members of the NAS, two have been Howard Hughes Medical Institute [HHMI] investigators, and one runs Woods Hole Institute). Nipam got his PhD and stayed as a postdoc, opening the field of evolutionary developmental biology.

We took several parallel approaches. In the grasshopper, we went looking for surface molecules on subsets of axon pathways, with the hope of cloning them in Drosophila. In Drosophila, we began to use genetics to dissect the developing nervous system. In separate studies, we began to explore the determination of cell fate from neuroblast to neuron.

We isolated large numbers of grasshopper embryonic nervous systems, made membrane preparations from them, generated monoclonal antibodies against those preparations, and screened the antibodies on embryonic nervous systems, looking for antigens expressed on subsets of axon pathways. Mike, Nipam, and Allan made and screened the antibodies; Peter isolated and microsequenced the proteins; and Kai Zinn made grasshopper embryonic nervous system cDNA libraries and cloned the genes.

Based upon our monoclonal antibody screens, we initially isolated and cloned the genes encoding three proteins that are expressed on subsets of axon pathways in the grasshopper embryo: Fasciclin I, Fasciclin II, and Fasciclin III. All three can function as homophilic cell-adhesion molecules. Fasciclin II and III are members of the immunoglobulin superfamily, whereas Fasciclin I defined a new family of homophilic cell-adhesion molecules. A year or two later, we cloned another CAM on a larger subset of axon pathways, called Neuroglian. Fasciclin II is related to mammalian NCAM, whereas Neuroglian is related to mammalian L1.

None of these proteins is expressed on the entirety of a given neuron’s axons and dendrites. Rather, all three labels are regionally expressed on segments of axons that bundle (or fasciculate) together. For example, some axons express Fasciclin I on their surface as they extend across the midline, but when they turn anteriorly or posteriorly in a longitudinal pathway, they turn off Fasciclin I and turn on Fasciclin II (Cell 1987).

Alan Harrelson and I published a paper on Fasciclin II (Science 1988) entitled: “Growth Cone Guidance in Insects: Fasciclin II Is a Member of the Immunoglobulin Superfamily.” We had already learned that the cellular cues that guide neuronal growth cones toward their targets appear to be highly conserved in such diverse organisms as insects and vertebrates. Our data suggested that the molecular mechanisms underlying these events may be equally conserved.

John Thomas and Steve Crews began our studies on the midline in the Drosophila embryo by cloning the single-minded gene that controls the fate of midline cells (Cell 1988a,b). Denise Montell began studies of the role of extracellular matrix in Drosophila neuronal development (Cell 1988).

Chris Doe studied the role of cell interactions in the determination of neuroblasts and their neuronal progeny in grasshopper and Drosophila (Developmental Biology 1985a,b). His cell-ablation experiments were key to understanding the cell interactions controlling neuronal fate and lineage. Chris was first to show that segmentation genes (e.g., fushi tarazu and even-skipped) have a second pattern of expression during neurogenesis where they control neural fate (Science 1988; Nature 1988). In his lab at University of Oregon, Chris has continued his elegant studies on the mechanisms controlling neural fate in Drosophila.

In 1982 and 1984, Paul Patterson invited me to lecture in the two-week Cold Spring Harbor (CSH) developmental neurobiology course he taught with Dale Purves. The students were terrific, and I enjoyed teaching with Paul. Dale often challenged my interpretations and the relevance of our work. It was these discussions that foreshadowed his authorship of the 1985 Science paper. In 1985, Paul called me and said he and Dale were splitting up and asked me to teach the course with him. I accepted. Paul and I taught the CSH course together in 1986, 1988, and 1990. In 1988, one of our students was a postdoc with Tom Jessell named Mary Hynes. During the weekend break in the middle of the course, her boyfriend, also a postdoc in Tom’s lab, came out to stay with her, and he and I enjoyed talking about axon guidance and became lifelong friends. That is how I first met Marc Tessier-Lavigne.

Stanford was the perfect place for me in my early years. The biology department kept my teaching load and committee assignments low. They gave me, and later Richard and me together, a supportive environment in which to focus on our research. We thrived. I also developed a wonderful lifelong friendship with Carla Shatz in the Neurobiology Department. I arrived after her, and no surprise, given our interests, we became friends and colleagues.

Back to Berkeley

Marcia and I moved to Berkeley in the summer of 1987. The lab followed a few months later. I have been asked why I left Stanford to move back to Berkeley. The reasons were both personal and professional. On the personal side, it was a good move for Marcia, who had just finished a science journalism program and was launching her career as a science writer for Nature and then Science magazine. Berkeley was an exciting environment for a budding science journalist.

The move also helped us with our housing situation. My parents had no money to help us. I had little savings and we lived on my faculty salary as Marcia was earning very little writing for Nature. At Stanford, I had bought a little two-bedroom, one-bathroom house in Menlo Park one block from the freeway with the help of Stanford’s COIN (coinvestment) program. That initial program was flawed and provided little long-term hope that we would ever sufficiently grow our equity and come to own our own home. Berkeley offered me meaningful mortgage assistance that made it possible for us to own our own home.

On the professional side, at Stanford, Richard’s and my labs were bursting at the seams, and we both needed more space and more funding for the ambitious research programs and goals we were embarking on. I needed more space for fly genetics and more benches for molecular biology. The HHMI wanted to support both of us, but in those days, the university tightly controlled who got nominated, and at Stanford, those nominations were under the control of the medical school. One day, Max Cowan, chief scientific officer (CSO) of HHMI, came to see the two of us. He told us that the Stanford medical school refused to nominate us so long as we were in the biology department. Biology had pushed, but to no avail. Our choice was simple, he said: either we needed to move to a medical school department (which is what Richard did) or move to another university (which is what I did). Berkeley assured me that they would nominate me for HHMI, and Max assured me he would approve the appointment. Berkeley offered me more space than I had at Stanford, in a new building, and adjoining the lab of Gerry Rubin, a superb Drosophila geneticist and developmental biologist. It was clear I would have everything I needed to carry out my ambitious research goals at Berkeley.

I enjoy leading and building teams. At Stanford, I had no immediate path to a leadership position. Berkeley offered me the opportunity to help build neuroscience. Dan Koshland hoped I would want to become head of the Neurobiology Division of the newly formed Department of Molecular and Cellular Biology (MCB) and to work on unifying neuroscience across campus. With this opportunity, I moved to Berkeley in 1987.

A few years later, in 1992, I recruited Carla to join me at Berkeley. She stayed until 2000 when she moved back to her alma mater Harvard to become chair of the Neurobiology Department. While we were together at Berkeley, with the help of Vice Chancellor Carol Christ, Carla and I cofounded the Helen Wills Neuroscience Institute. And of course, we taught, collaborated, and wrote together, including our key 1993 Cell/Neuron review.

We were grateful that the Cellular and Molecular Neuroscience Section of the NAS helped elect both of us in the same year (1995). It was an honor to sign the NAS book with Carla.

With HHMI funding at Berkeley, I was able to recruit fabulous staff to support me and advance our science. I searched nationwide for a top electron microscopist and found Rick Fetter. Hardly a project took place that Rick did not touch. From 1989 until 2001 when I departed for biotech and Rick moved to University of California, San Francisco (UCSF), he was coauthor on 20 papers. I also had a terrific executive admin in Inez Drixelius, and a superb lab manager in Beth Blankemeier, who also made all our monoclonal antibodies. Together, Inez and Beth kept the lab running, protected me, and allowed me to focus on the science. I couldn’t have done it without them.

The Many Faces of Fasciclin II

In 1988, shortly after arriving at Berkeley, Alan Harrelson and I showed that Fasciclin II functions in axon guidance and selective fasciculation in the grasshopper embryo (Science 1988). Gabi Grenningloh and Jay Rehm cloned the gene encoding Fasciclin II in Drosophila, generated mutations in it, and along with Dave Lin and Rick, examined the genetics of Fasciclin II (Cell 1991). Dave Lin, with Gabi, Rick, and Casey Kopczynski, showed that both loss- and gain-of-function genetics altered growth cone guidance and patterns of selective fasciculation, confirming the role of Fasciclin II as a recognition molecule expressed on a subset of axon pathways (Neuron 1994a,b).

We also discovered that Fasciclin II is localized to developing neuromuscular synapses. Grae Davis and Christoph Schuster used Fas II expression to begin our studies on the development of synapses. What began with Fas II led us to wonderful discoveries of retrograde signals and other mechanisms. They showed that Fas II controls synaptic stabilization and growth as well as presynaptic plasticity (Neuron 1996a,b). They went on to show that target-derived Fas II regulates synapse formation as well (Neuron 1997). In the process of these experiments, we discovered the homeostasis of synaptic transmission (Journal of Neuroscience 1996). Grae, along with Aaron DiAntonio and Sophie Petersen, discovered evidence for a retrograde signal that regulates presynaptic transmitter release (Neuron & Nature 1998). Karen Zito, a joint graduate student with Udi Isacoff, discovered the molecular mechanisms that cluster Fas II and the Shaker potassium channel at the neuromuscular synapse (Neuron 1997). Grae, Christoph, Aaron, and Karen launched their own labs at UCSF, Heidelberg, Washington University, and University of California, Davis.

Searching for Semaphores

Whereas most of the surface molecules we discovered that are expressed on subsets of axon pathways (e.g., Fasciclin I, II, and III, and Neuroglian) can function as homophilic cell adhesion molecules, one (Fasciclin IV) does not, as shown by Alex Kolodkin and David Matthes (Neuron 1992). In collaboration with Tim O’Connor in David Bentley’s lab, Alex and David showed that Fas IV is also expressed on circumferential bands of epithelial cells in developing limb buds, where it functions to help guide the growth cones of pioneer neurons (the ones originally discovered by Mike Bate). When cloned in 1992, Fas IV was revealed to be a transmembrane protein that showed no homology with any protein in available databases.

We went looking for a Drosophila homologue, but couldn’t find one, probably because these insects diverged some 300 million years ago, and because Fas IV was a pioneer protein, we didn’t know which sequences might be conserved across species. To get to Drosophila, Alex and David crawled across insect phylogeny, first isolating Fas IV-like sequences from a beetle and then a moth. These sequences were used to refine their primers and ultimately allowed them to clone related genes in Drosophila. We discovered a gene family that we named the Semaphorins, with transmembrane Fas IV in the grasshopper (and Drosophila) as the founder renamed Sema I. We also discovered a second Semaphorin in Drosophila that encodes a secreted protein we called Sema II. We generated mutations in the semaII gene and showed they lead to behavioral defects and death. In the process, Alex and David discovered an important new family of transmembrane and secreted axon guidance molecules (Cell 1993), something Alex has continued to study in his own lab at Johns Hopkins.

All our insect Semaphorins share a highly conserved extracellular domain of around 500 amino acids, with certain highly conserved sequences. Based on these conserved sequences from grasshopper to Drosophila, we used primers to isolate a related human gene, Sema III. While our work was being prepared for publication, Jon Raper and colleagues at University of Pennsylvania published another member of the family from the chick, called Collapsin, based on its ability to promote the collapse of sensory growth cones in cell culture. Our analysis suggested that human Sema III was the homologue of chick Collapsin. We discovered Semaphorin sequences in two poxviruses, suggesting a potential role for Semaphorins in blunting the host inflammatory and immune response against viral infection. Today, we know that Drosophila has 5 Semaphorins organized into 3 classes while human has 21 Semaphorins organized into 7 classes.

Two years later, in 1995, we published two papers showing the function of Semaphorins in Drosophila and mouse. In the first paper (Cell 1995), David and Alex showed that Sema II can function as a selective target-derived signal that inhibits the formation of specific synaptic terminal arbors. In the second paper (Neuron 1995), in collaboration with Carla’s and Marc’s labs, we presented evidence in the mouse showing that Sema III, a diffusible guidance molecule expressed by ventral spinal-cord cells, inhibits NGF-responsive sensory axons that normally terminate dorsally but that has little effect on NT-3-responsive sensory axons, which terminate ventrally. These studies showed that Semaphorins in both Drosophila and mammals can function as selective chemorepellents to pattern motor and sensory projections.

Over the next few years, we began to elucidate the receptors for the Semaphorins. In 1997, Zhigang He in Marc’s lab discovered that Neuropilin is a receptor for Semaphorin III; Alex Kolodkin in his lab at Johns Hopkins made the same discovery. Alain Chedotal in my lab collaborated with Hong Chen and Zhigang in Marc’s lab to show that Neuropilin 2 is a receptor for Semaphorins Sema E and Sema IV but not Sema III.

In that same year (1997), I was contacted by Melanie Spriggs at Immunex in Seattle. She had followed up on our discovery in 1993 of secreted Semaphorin sequences in poxviruses and our hypothesis that they functioned as immune repellents. She used the vaccinia A39R Semaphorin domain to expression-clone an immune receptor called VESPR. She said that VESPR is related to a protein identified in Xenopus called Plexin. Fujisawa’s lab in Kyoto had generated monoclonal antibodies against optic tectum in Xenopus and had published in 1987 on two different epitopes on different layers of neurons in the optic tectum. They cloned the genes that encoded these proteins in Xenopus and mouse and published them in 1995 and 1996. One of these proteins, Neuropilin-1, was a Sema III receptor. It seemed a remarkable coincidence that the other protein, Plexin-1, might also be a Semaphorin receptor.

Melanie’s insight led to a wonderful collaboration between her lab, Marc’s lab, and Meg Winberg in my lab. In 1996, the lab of Paolo Comoglio in Torino, Italy, had published on a family of transmembrane proteins in the mouse that were homologous to Plexin-1. Luca Tamagnone from Paolo’s lab had contacted me, I believe after Melanie had, and asked if we had considered the possibility that Plexins might be Semaphorin receptors. They had less direct evidence than did Melanie, and all these years later, I can’t remember what it was, but Luca was on the same track. They had cloned the entire family of Plexins in mouse and human. I brought Luca, Melanie, Marc, and Meg together under the tent—we shared data and worked together.

The multilab collaboration led to two Cell papers, the first in 1998 and the second in 1999. In the first, Meg showed that Drosophila has two Plexins and that Plexin A is a neuronal receptor for class I Semaphorins, by both binding studies and genetics. Meg did beautiful genetic analysis of Plexin A and Sema 1 to show that Plexin A controls motor and CNS axon guidance. Interestingly, upon further sequence analysis, it became clear that Plexins also contain complete Semaphorin domains. Whereas Drosophila has neither a Neuropilin homologue nor class III Semaphorins, it does have Plexins.

In the second paper, the collaboration enlarged to include Mu-ming Poo’s lab and additional postdocs in Paolo’s and Marc’s labs, and Alain Chedotal from my lab. In that paper, we showed that Plexins are a large family of receptors—at least nine in human—that appear to bind to most classes of human Semaphorins, including being coreceptors along with Neuropilins for class 3 Semaphorins. Moreover, it looked as if signaling could go both ways.

Alain got involved in a series of collaborations with different laboratories in 1998 and 1999 elucidating the function of several novel Semaphorins in mammals. In 2001 (Neuron), with Denise Montell (in her own lab at Johns Hopkins), Meg showed that the transmembrane protein Off-track associates with Plexins and functions downstream of Semaphorin signaling. That same year (Neuron 2001), Hailan Hu in my lab showed that Plexin B mediates axon guidance in Drosophila by inhibiting Rac and enhancing RhoA.

Different Semaphorins can be attractive vs. repulsive for the same growth cone. Alex Kolodkin (Johns Hopkins) and Tim O’Connor (Vancouver) used the pioneer growth cones in the limb bud of the grasshopper embryo, the place where we first observed a guidance function for Sema 1a (Fas IV; Neuron 1992), to show that Sema 1a is an attractant and Sema 2a is a repellent for those growth cones (Development 1999).

Hailan Hu in my lab collaborated with Tanja Godenschwege in Rod Murphey’s lab (Nature Neuroscience 2002). In the adult giant fiber (GF) system of Drosophila, they showed that transmembrane Sema 1a is involved in synapse formation as well as axon guidance. Genetic analysis showed that Sema 1a is involved in assembly of a central synapse and that it is required both pre- and postsynaptically, suggesting that Sema 1a is part of a bidirectional attractive signaling system that leads to the formation of the GF synapse.

To Cross or Not to Cross

After crossing the midline, growth cones change their behavior, turn, and selectively fasciculate with specific axon pathways on the contralateral side of the nervous system. In contrast, before crossing the midline, these growth cones ignore the same pathways on their own (ipsilateral) side of the nervous system. Axon guidance changes as the growth cone navigates from one intermediate target to another.

In 1987, Mike Bastiani, Allan Harrelson, and Peter Snow showed just this pattern of expression of Fasciclin I (on subsets of commissural axons) and Fasciclin II (on subsets of longitudinal axons), with individual neurons expressing Fasciclin I on the commissural segment and then switching Fasciclin I OFF and Fasciclin II ON as they turned onto a longitudinal pathway (Cell 1987).

Given our discovery of the key role of the midline, in the mid- to late 1980s, first at Stanford and then at Berkeley, we began to focus on the genetic analysis of the midline in Drosophila and the role of midline glia in axon guidance. Steve Crews and John Thomas cloned the single-minded gene that controls the development of midline cells (Cell 1988a,b). We developed markers for midline glia and longitudinal glia. Roger Jacobs elucidated the scaffold and role of the midline and longitudinal glia in axon guidance (Journal of Neuroscience 1989a,b). Roger collaborated with Jonathan Rothberg in Spyros Artavanis-Tsakonas’s lab to study the role of the slit gene in midline development (Genes and Development 1990). Christian Klämbt and Roger used genetic analysis to show the steps and cellular interactions involved in axon guidance toward and away from the midline (Cell 1991). Together these discoveries set the stage for what came next—our first large-scale genetic screen for genes that control axon guidance at the midline.

The Power of Genetics

A few years after arriving at Berkeley, around 1990, we embarked on the first large-scale genetic screen to dissect the problem of axon guidance. We modeled our screen after what Christiane Nusslein-Volhard and Eric Wieschaus had accomplished in Drosophila using large-scale genetic screens to collect mutations in genes affecting the pattern of bristles on the outside of the embryo (Nature 1980) and used this collection of mutants to deduce the genetic logic of pattern formation, for which they won the Nobel Prize in 1995.

We applied this same approach for the first time to the inside of the embryo. We had a monoclonal antibody, BP102, that stains all axon pathways and used it to visualize the axon commissures in the whole embryo. We collected mutations in genes affecting the pattern of axon commissures in the developing embryo and used this collection of mutants to deduce the genetic logic of midline guidance (Neuron 1993). This first screen was started by Mark Seeger who was joined by Guy Tear. Much of the screen was done by a talented technician, Dolors Ferres-Marco. Dolors went on to get her PhD and today has her own lab in Alicante, Spain. Mark and Guy went on to have their own labs at The Ohio State University and King’s College London.

We used a high rate of mutagenesis to produce many independent mutations on each chromosome. We anatomically screened a total of 13,529 mutagenized lines: 6,211 mutant lines on the second, 5,197 on the third, and 2,121 on the X chromosome. We identified 263 mutant lines that showed interesting CNS axon pathway defects (180 on the second, 70 on the third, and 13 on the X). Since the average mutagenized chromosome carried two to three independent lethal mutations (plus additional viable mutations), the actual number of mutations screened was far greater than the 13,529 lines examined and appeared to have covered a large fraction of the genome. Our previous studies had revealed a handful of genes that have easily detectable abnormalities in CNS axon pathways, and we used those genes as controls. For example, we collected six mutants in the single-minded gene on the third chromosome. Of the 263 mutant lines collected, around 140 led to axon commissures that were either partially or completely absent, or fuzzy, fused, or thicker.

Shortly after launching the midline axon guidance screen, David Van Vactor began a second large-scale genetic screen, collecting mutations on the second chromosome in genes affecting the pattern of motor axon pathways in the periphery to deduce the genetic logic of motor axon guidance (described below). Davie was joined by Helen Sink, Doug Fambrough, and Rosalie Tsoo. They used a monoclonal antibody against Fasciclin II, which stained motor axons in the periphery. Both screens were published in 1993, the midline screen in Neuron and the motoneuron screen in Cell. Helen passed away in 2021 after being hit by a motor vehicle, the same fate as happened years earlier to Tom Elkins and Peter Snow. Davie went on to become a professor at Harvard, Doug the CEO of biotech company Dicerna, and Helen (before her death) became a highly acclaimed high-school science teacher in Harlem.

My job for both screens was to keep our team focused and not let them get discouraged. Outside the lab, these screens, which took several years to conduct, were met with skepticism of whether the approach would bear fruit, and if so, whether the genes discovered in Drosophila would be relevant to mammals. I had taken the risk of committing a lot of lab resources to both screens. Our goal was to collect all the genes that gave mutant phenotypes in midline or motor axon guidance. I enjoyed my daily visits to the fly room where Dolors would show me what mutants she had found, and how they were falling into a small number of discrete complementation groups (what became robo, comm, and slit).

A Roundabout Discovery

The midline guidance screen by Mark, Guy, and Dolors (Neuron 1993) had a simple logic: to search for mutants in which too many (roundabout) or too few (commissureless) axons cross the midline, or axons enter but fail to leave the midline (slit). We had collected 8 mutants in robo, 2 in comm, and 13 in slit, proportional to the size of the three target genes. Those were the three stars of the screen—the three mutants with the most dramatic and penetrant phenotypes.

In wild-type embryos, around 90 percent of the CNS axons first extend toward and across the midline before turning anterior or posterior on the contralateral side, while around 10 percent never cross the midline and extend anterior or posterior on their own side. In comm mutants, none of the axons cross the midline. It is a complete split-brain mutation with no commissures. In robo mutants, all of the axons cross the midline, and many cross and recross multiple times. In slit mutants, all axons extend toward the midline, but never leave, as all longitudinal axons coalesce along the midline. These were our starting points for unraveling midline axon guidance.

As published a few years later, we also collected mutant alleles in the Netrin receptor frazzled (the DCC/UNC-40 homologue) and published this in collaboration with the Jan lab (Cell 1996). Its phenotype was partial and less dramatic than comm. But one gene was dramatically missing from our midline screen. We had not collected a single mutation in the Netrin gene. We couldn’t imagine it wasn’t present since it existed in nematodes (UNC-6) and mammals. Kevin Mitchell and Barry Dickson sorted this out. In collaboration with Marc’s lab, they discovered that Drosophila has two tandem Netrin genes with redundant midline function but with divergent functions in peripheral guidance (Neuron 1996). They had to delete both genes to see a mutant phenotype with partially missing or thinner commissures; expressing either of the two genes at the midline in the double-mutant restored midline function.

The big insight into the function of these genes, and the logic of midline axon guidance, came from Tom Kidd in the summer of 1997. Tom had been working on the genetics of these three genes (robo, comm, slit), using loss-of-function (LOF) and gain-of-function (GOF) genetics in different combinations and strengths of expression. In the middle of June, Marcia and I were on safari in Zimbabwe, our first of many trips to Africa. The day I returned to the lab, Tom sat me down at the microscope and said he had something to show me. I came to learn that he had some of these results before I left for Africa, but he made everyone in the lab promise not to tell me, so he could surprise me with a more complete story when I got back.

Tom showed me what I guessed was a comm LOF mutant with no axons crossing the midline. No, he said, it was a robo strong (double dose) GOF. With all axons expressing high levels of Robo, none cross the midline. If so, then perhaps Comm normally regulates Robo. Then he showed me what looked like a robo LOF mutant. No, he said, it was a comm weak (single dose) GOF. That result further suggested that Comm regulates Robo. Finally, he showed me what looked like a slit LOF. No, he said, this was a comm strong (three dose) GOF.

As best as we could predict from genetic analysis, Tom had shown that Comm functions to down-regulate the Robo receptor, and Slit was likely to be the ligand for the Robo receptor. Moreover, since the comm mutant phenotype was much stronger than the robo phenotype, it followed that there must be additional Robo receptors for Slit. We just had to clone the genes and sort out all the interactions. We also wanted to jump to mammals. I called Marc and proposed that the two labs collaborate on the mammalian molecules and mechanisms.

Tom, Guy, Kevin, and Rick, in collaboration with Katja Brose in Marc’s lab, showed that Robo encodes a receptor that defines a novel subfamily of immunoglobulin superfamily proteins that is highly conserved from Drosophila to mammals (Cell 1998). There are three Robos in Drosophila, four in human, and one (Sax-3) in nematode. Using monoclonal antibodies against Robo, we showed that for axons that project across the midline, Robo is not expressed on their commissural axons but is expressed highly on their longitudinal axons. This regional expression is similar to what we had seen earlier with Fas II expression. On axons that project ipsilaterally, Robo is expressed on their surface from the outset. Tom and Guy published the robo and comm dosage-sensitive and complementary genetic interactions (Neuron 1998).

Marc’s and my groups worked on Slit-Robo together. Kuan Hong Wang in Marc’s lab discovered a truncated Slit protein in mammals that functions as a positive regulator of sensory axon elongation and branching. Tom in my lab discovered that Slit is the repulsive midline ligand for the Robo receptor. We knew about Slit-Robo in summer of 1997, but had a lot of work to do, and Marc and I wanted to publish complete stories. In March 1999, we published three papers in Cell on Slits and Robos in Drosophila and mammals. Others jumped on the story and tried to scoop us after hearing Tom’s job talks in spring 1998, but we were first, and colleagues knew it. History has appropriately given us the credit for the Slit-Robo discovery.

In our series of three papers (Cell 1999a,b,c), Tom and Kim Bland showed that Slit is the midline repellent for the Robo receptor in Drosophila. Katja (with Tom, Kim, and others) showed that Slit proteins bind Robo receptors and have an evolutionarily conserved role in repulsive axon guidance from Drosophila to mammals. Kuan Hong and Katja showed that an N-terminal fragment of Slit2 is a positive regulator of axon elongation and branching in mammals.

A few years later, in studies focused on migrating mesodermal cells in the Drosophila embryo, Sunita Kramer in my lab (with Tom and Julie Simpson) showed that Slit can function as both an attractant and a repellent and that individual cells can switch from repulsion to attraction at different points in their migration (Science 2001). Tom and colleagues in his own lab at University of Nevada, Reno, brought clarity to this issue by showing that Slit is proteolytically cleaved, which converts a repulsive cue into a positive cue (Development 2020).

Robo is also proteolytically cleaved to regulate its function. From our genetic screen for midline mutants also came mutations in the Kuzbanian gene that encodes a metalloprotease. Doug Fambrough in my lab, in collaboration with Gerry Rubin’s lab, showed Kuzbanian regulates axon guidance (Proceedings of the National Academy of Sciences 1996). Mutations in kuzbanian resemble mutations in robo. My former postdocs Pablo Labrador and Greg Bashaw at University of Pennsylvania went on to show that the Kuzbanian protease activities Robo signaling by cleaving Robo (Development 2010).

My final graduate student, Hua Long, switched to Marc’s lab when I moved to biotech. She showed that ventral midline cells in the embryonic spinal cord express all three mammalian Slit homologs. When all three genes are deleted, many commissural axons fail to leave the midline, whereas others recross it, demonstrating a key role for Slit-Robo signaling in midline commissural axon guidance in vertebrates just as in Drosophila (Neuron 2004).

The same midline glia cells in Drosophila secrete NetA, NetB, and Slit, the attractive and repulsive midline guidance cues. How do growth cones navigate to the midline and then away from it? Commissureless seemed critical to this switch.

Sorting out Robo

Guy Tear and Mark Seeger cloned the commissureless (comm) gene (Neuron 1996), but its sequence didn’t teach us much. It was a novel transmembrane protein, and nothing immediately popped out as either another family member in Drosophila or a homologue in mammals. Genetic analysis by Tom and Guy taught us that Comm downregulates Robo (Neuron 1998) and led to the insight that Slit was the repulsive ligand for Robo (Cell 1999), but our initial analysis of Comm itself led to conflicting ideas about how the protein functions.

Some of these issues were resolved shortly after I moved to biotech, and many of those insights came from former Goodman lab members. Guy Tear and colleagues, in his lab in London, showed that Comm recruits a specific ubiquitin ligase (Nedd4) to control cell surface levels of Robo (Neuron 2002). Barry Dickson and colleagues, in his lab in Vienna, showed that Comm sorts Robo to the endosome (vs. the cell surface) and targets Robo for degradation, allowing growth cones to cross the midline.

For neurons that extend across the midline, comm is ON until they cross, keeping Robo off the surface. As soon as they cross, comm turns OFF, letting Robo go to the cell surface and repelling them from the midline. For neurons that extend on their own side, comm is OFF from the outset, letting Robo go to the cell surface and stopping those growth cones from heading towards or crossing the midline. It is still unclear what turns comm ON and OFF.

Finally, do mammals have Comm or Comm-like functions for sorting Robo receptors? In 2017, Tom Kidd and colleagues in Reno showed that mammalian PRRG4 shares certain structural motifs with Comm and is a functional homologue of Comm, recruiting human Robo1 from the cell surface (PLoS 2017). In 2019, Greg Bashaw and colleagues at Penn showed that mammalian Ndfip proteins bind to and recruit Robo receptors to late endosomes, and recruit Nedd4-family E3 ubiquitin ligases to trigger Robo degradation, thus defining a conserved Robo1 intracellular sorting mechanism between flies and mammals to avoid premature responsiveness to Slit (Cell Reports 2019).

Robos Rock

In robo mutants, axons cross and recross the midline, whereas in slit mutants, they grow to the midline but never leave it. In comm GOF experiments, a lower dose of Comm leads to a robo-like phenotype, whereas a higher dose of Comm leads to a slit-like phenotype. Taken together, these results suggest that there are additional Robos, that they too respond to Slit, and that they too are downregulated by Comm. In the late 1990s, Julie Simpson, along with Tom Kidd and Kim Bland, discovered two additional Robo receptors in Drosophila: Robo2 and Robo3 (Neuron 2000). When overexpressed at high levels, either Robo1 or Robo2 can generate comm-like phenotypes. Moreover, the robo/robo2 double mutant is largely identical to slit. Julie began to see that Robo and Robo2 are not identical in function. Robo2 can inhibit Robo at the midline. Greg Bashaw, in his lab at Penn, elucidated this difference in 2015 by showing that Robo2 acts in trans to inhibit Slit-Robo repulsion in commissural axons (eLife 2015).

Julie then made a profound discovery with the help of Kim and Rick. She discovered that Robos can function in short-range and long-range guidance, presumably based on a gradient of Slit emanating from the midline, and that a combinatorial code of Robo receptors controls lateral position (Cell 2000). Barry Dickson and colleagues, in his lab in Vienna, discovered the same Robo code and published back-to-back papers with us (Cell 2000). Robo is expressed on all longitudinal axons, Robo 3 is expressed on intermediate and lateral longitudinal axons, and Robo2 is expressed on lateral axons. Misexpressing different Robos on specific embryonic neurons sends their longitudinal axons to specific M-L positions within the nerve cord.

The discovery of the Robo code finally solved, at a first approximation, the question that arose from the original Raper and Bastiani papers in 1984. The G growth cone showed a remarkable specificity for the 2 P axons vs. 150 or so other axons in the longitudinal tracks. Is there a single unique molecule that labels the P axons or is there some sort of combinatorial code? We had found surface labels on subsets of pathways, such as Fasciclin II, but they are not expressed on single bundles of axons, but rather on subsets of pathways in different regions. Fas II, for example, is on four bundles of 18 median, 8 ventro-medial, 10 intermediate, and 8 lateral axons with a total of 44 Fas II-positive axons out of approximately 150 longitudinal axons.

The answer, as Julie’s work revealed, is that there is a code, combining long-range regional cues (the Slit gradient and Robo code) with local attractive and repulsive cues (such as Fas II). She showed, for example, that the Fas II-positive dMP2 and vMP2 axons extend in the medial Fas II-positive axon bundle. Those two axons normally express Robo but not Robo2. When she expressed Robo2 in those two neurons, they then extended in either the intermediate or lateral Fas II-positive bundles. In other words, precise lateral location is determined by a combination of the Robo code and local cues, in this case Fas II. The Robo code on its own generates a coarse topography of projections. The refinement of topography is provided by discrete local cues. Neither on its own is sufficient to generate the precision of axon pathways.

Thus, in 2000, we discovered that the M-L axis is patterned by Slit and Robos. In 2003, John Thomas and colleagues at the Salk Institute showed that the A-P axis is patterned by a Wnt and Wnt receptors. In 2009, Mike Bate and colleagues in Cambridge showed that the D-V axis is patterned by Semaphorins and Plexins. Axon patterning is achieved by a series of M-L, D-V, and A-P gradients and receptors, combined with local cues.

Swimming Downstream

We also started to elucidate the signal transduction machinery downstream of Slit-Robo signaling. This was primarily the work of Greg Bashaw and Hailan Hu in the lab. First, Greg showed that attraction vs. repulsion is encoded in the cytoplasmic domain of the receptors (Cell 1999). With Tom and Tony Pawson’s lab in Toronto, Greg then showed that Abl and Enabled play opposing roles downstream of the Robo receptor (Cell 2000). Greg and Hailan went on to identify a novel RhoGEF that promotes Rho-dependent axon attraction at the midline that overcomes Robo repulsion (Journal of Cell Biology 2001). Finally, Hailan, with Greg, Pablo Labrador, and others, went on to show that a cross GTPase-activating protein (CrossGAP) links the Robo receptor to Rac to regulate midline repulsion (Proceedings of the National Academy of Sciences 2005). Greg has gone on in his own lab at Penn to elucidate the signaling machinery downstream of Slit-Robo. Hailan went on to explore the cellular and molecular mechanisms underlying social behaviors and psychiatric diseases. Today, she is director of the Center for Neuroscience at Zhejiang University in China.

Going for a Walkabout

In each abdominal hemisegment of the Drosophila embryo, an array of 30 muscle fibers is innervated by 38 motoneurons in a highly stereotyped and cell-specific fashion. To begin to elucidate the molecular basis of neural specificity in this system, we conducted the first genetic screen for mutations affecting neuromuscular specificity. Led by David (Davie) Van Vactor, and joined by Helen Sink, Doug Fambrough, and Rosalie Tsoo (Cell 1993), this screen also had its own simple logic: to search for mutants in neuromuscular connectivity in which either pathway (beaten path, stranded, and short stop) or target (walkabout and clueless) recognition is perturbed. The initial screen was on the second chromosome. Our data suggested that around 10 genes on the second chromosome (about 40 percent of the genome) control specific aspects of motoneuron pathfinding or target recognition. Helen went on to finish the motoneuron screen on the third chromosome in 1994 (published by Helen and Davie as an abstract), which led to the discovery by Helen of sidestep (Cell 2001).

Davie initially focused his efforts on walkabout (wako) with its fascinating mutant phenotype. In wako mutants, motoneuron growth cones reached their potential target regions, but then do not recognize the surfaces of the appropriate muscle targets. Instead, the growth cones wander around, contacting other neighboring muscle fibers. Davie spent nearly two years taking his own “walkabout” to positionally clone wako. He was excited by its interesting muscle expression pattern. But that elation led to disappointment when he sequenced wako. It encodes a chaperonin component that controls protein folding. Finding the muscle substrate protein(s) that accounted for the targeting defects in wako would be a tough slog.

Doug Fambrough cloned beaten path (beat) (see the next section). Davie then focused on another mutant on the second chromosome with a beat-like phenotype. He had better luck on this one. With the help of Hong Wan, Davie went on to show that this beat-like phenotype was caused by mutations in transmembrane tyrosine phosphatase DLAR. He published his findings with Haruo Saito and colleagues at Harvard who were taking a reverse genetics approach to DLAR (Cell 1996). In Dlar mutant embryos, specific motor axons bypass their normal target region. While we were working on DLAR, Kai Zinn and his colleagues at Caltech showed that two other receptor tyrosine phosphatases (DPTP69D and DPTP99A) were expressed on motor axons and that mutations in these genes led to similar kinds of motoneuron pathfinding errors in different but overlapping subsets of motoneurons. We and Kai published back-to-back papers in Cell (1996) on the role of transmembrane tyrosine phosphatases in motor axon pathfinding.

Davie, in collaboration with Kai, went on to show that Profilin and the Abl tyrosine kinase function to control motor axon guidance (Neuron 1999), a complement to their work on tyrosine phosphatases. Davie has continued in his own lab at Harvard to use genetic analysis to study the formation and maintenance of synaptic connections.

The Beat Generation

A wonderful story in motor axon guidance emerged from Davie’s (Cell 1993) and then Helen and Davie’s (unpublished) large-scale genetic screens. The keys to the start of this story were two genes (beaten path and sidestep) that, when mutated, showed similar phenotypes in which motor axons fail to defasciculate and leave the main motor nerve to grow into and innervate their specific muscle domains. The story began with the identification of the beaten path (beat) gene by Doug Fambrough (Cell 1996), followed by Helen’s identification of the sidestep (side) gene (Cell 2001). In a third paper from the lab during this period (Development 2001), Teg Pipes discovered a family of beat-like genes in Drosophila. These three papers, and in particular Helen’s and Teg’s as I was leaving academia, started a cascade of discoveries on motor axon guidance in the labs of three former postdocs: Hermann Aberle, Pablo Labrador, and Kai Zinn.

Doug showed that the Beat protein was a novel protein of the Ig superfamily expressed by motoneurons during axon outgrowth. In the absence of Beat, many motor axons fail to defasciculate and leave the major motor nerves at specific choice points. Helen showed that Side is also a novel member of the Ig superfamily that is expressed on both specific embryonic muscles and other peripheral cells and tissues. Using genetic analysis, Helen reasoned that Side appeared to function as a substrate attractant for motor axons.

Teg identified 14 beat-like genes in Drosophila. Many Beats appear to be expressed by subsets of neurons, including motoneurons. Mutations in other Beat-family members led to more subtle guidance phenotypes that observed for beat itself. From a historical perspective, fortunately at least one of the Beat-family members (Beat or as it is now called, Beat-Ia) has a strong mutant phenotype on its own (with no genetic redundancy) such that it showed up in Davie’s original genetic screen. The project was picked up by several former postdocs.

Herman Aberle and his colleagues in Muenster, Germany (Genes and Development 2009) showed that Side and Beat bind and directly interact, with Side as the pathway label for motor axons and Beat as the motoneuron receptor for Side. He showed that motor axons recognize and follow Side-expressing cell surfaces to their target region. Misexpression of Side in side mutants strongly attracts motor axons to ectopic sites. Over the next decade (Cell 2013; eLife 2017), Kai Zinn and colleagues, in collaboration with Chris Garcia’s lab and Pablo Labrador, used new analytical methods to show that the 14 Beat-family members and 8 Side-family members in Drosophila bind to one another with specific affinities and form a family of axon guidance ligand-receptor pairs. All of the Side-family members (with one exception) are expressed on specific peripheral tissues traversed by motor and sensory axons in the periphery where they function together in axon guidance.

The Yin and Yang of Target Recognition

When Meg Winberg was a postdoc in my lab, she became best known for leading the discovery of Plexins as the receptors for Semaphorins (Cell 1998, 1999). But my favorite paper of Meg’s came from a different study in which she sought to understand the logic of target recognition. Several molecules had been discovered in my lab that are expressed by subsets of embryonic muscle targets (e.g., Netrin B, Fas III, Connectin), but each on their own had only weak mutant targeting phenotypes. She wanted to better understand the logic of how targeting was accomplished. She picked NetB for her test.

Meg used genetic analysis (loss-of-function and over-expression and misexpression gain-of-function) to reveal the complementary and combinatorial functions of Netrins, Semaphorins, and IgCAMs in controlling target recognition by motoneurons in Drosophila (Cell 1998). Fas II and Sema II are expressed by all embryonic muscles, where Fas II promotes, and Sema II inhibits, promiscuous synaptogenesis. NetB is expressed by a subset of muscles where it attracts some axons and repels others. However, growth cones in this system apparently do not rely solely on single molecular labels on individual targets. Growth cones assess the relative balance of attractive and repulsive forces and select their targets based on the complementary and combinatorial input of multiple cues. By shifting the levels of guidance cues up or down, she could alter target selection. For example, NetB functions as a target-recognition molecule guiding specific motoneurons to recognize a subset of muscles, but it does not function alone in specifying a specific muscle target. Targeting is based on the balance of attractive and repulsive forces on any given target in relationship to neighboring cells, a model very similar to how we had come to view axon guidance by the late 1990s.

Wishful Thinking

The synapse folks in my lab were feeling a bit envious of the midline and motoneuron teams that were doing large-scale genetic screens using specific antibodies to stain either axon commissures or motor axons, respectively, in whole embryos. They had a hankering—call it wishful thinking—to do a similar anatomical screen for mutants that altered the formation and growth of a specific glutamatergic synapse: the neuromuscular junction (NMJ).

We wanted to understand what signals regulate synaptic structure and function. We already knew from earlier studies in the lab by Grae Davis and Christoph Schuster that different levels of the cell adhesion molecule Fasciclin II would lead to smaller or larger synapses (Neuron 1996a,b,c, 1997). We also learned from studies by Sophie Petersen, Aaron DiAntonio, and Grae, using genetic analysis of glutamate receptors that an unknown retrograde homeostatic signal regulates transmitter release at this synapse (Neuron 1997, 1998; Journal of Neuroscience 1999), something Grae has pursued in his own lab at UCSF.

Hong Wan, a graduate student in the lab, took the first crack at this problem by conducting a pilot genetic screen for synapse mutants by screening a collection of walking defect mutants on the X chromosome, and in so doing, she discovered Highwire (Neuron 2000). I had heard Roland Strauss (Würzburg, Germany) talk about his collection of 230 mutant lines on the X chromosome with various degrees of behavioral walking defects in adult flies. We reasoned that some of those mutations might involve defects in wiring or synaptic growth and function. Hong used antibodies to label the larval NMJ in Roland’s mutants.

One mutant line, highwire (hiw) has a dramatic synaptic phenotype: synapses have greatly expanded branching with a large increase in the number of boutons. Hong showed that Highwire encodes an E3 ubiquitin ligase and appears to function as a negative regulator of synaptic growth. Hong collaborated with Aaron DiAntonio, a postdoc in the lab, and this began a beautiful set of studies that Aaron continued in my lab (Nature 2001), and then in his own lab at Washington University, on mechanisms controlling synaptic growth and function. For example, while in my lab, Aaron used genetic analysis to show that Highwire interacts with Fat Facets (FAF), a deubiquitinating protein, to regulate synaptic sprouting. The hiw loss-of-function was the same as the faf gain-of-function as both led to synaptic overgrowth.

But how could we take Hong’s pilot screen and Aaron’s subsequent genetic analysis and turn them into a large-scale genetic screen? Fortunately, Karen Zito, a joint graduate student with Udi Isacoff, provided the perfect tool (Neuron 1999). Karen had generated transgenic fruit flies that express a novel green fluorescent membrane protein (CD8-GFP-Shaker fusion protein) at the postsynaptic specialization of muscles only, allowing for repeated noninvasive confocal imaging of synapses through the translucent live, developing Drosophila larvae. Karen used her new tool to watch living synapses grow. She made novel discoveries about how new synaptic boutons insert between existing ones or add at the ends of existing strings of boutons.

As soon as Dorit Parnas and I saw Karen’s florescent synapses in the living larvae, we knew we could begin a genetic screen of these synapses by viewing living synapses in thousands of mutant lines of larvae under the confocal microscope (Neuron 2001). Dorit was joined by Hermann Aberle. So began our large-scale genetic analysis of synapse formation and growth. This screen had its own simple logic: to search for mutants in which the NMJ was either too big or too small (or absent all together). Dorit and Hermann screened 3,000 mutant lines on the second chromosome (Neuron 2001) and 4,973 lines on the third chromosome (Neuron 2002) and collected 19 complementation groups on the second and 17 complementation groups on the third chromosome that altered synaptic growth and structure.

In 1999, Brian McCabe and Pejmun Haghighi joined the lab, and along with Hermann and Rick Fetter, the analysis of these mutants took off. They discovered that bone morphogenetic protein (BMP) retrograde signaling from the postsynaptic muscle to the presynaptic motoneuron terminal controls synaptic growth. The initial genetic inroad from the screen came from the discovery of mutations in the wishful thinking (wit) gene that led to small synapses and decreased levels of synaptic Fas II. Mike O’Connor (Minnesota) had cloned the gene. Hermann mapped and identified our wit mutants, which showed the highly penetrant synaptic phenotype. It was a surprise to find a neuron-specific Type II BMP receptor. We showed that Wit is expressed by motoneurons. Wit appears to function as a presynaptic receptor that regulates synaptic size.

In the end, some of the other components in the pathway were discovered from our screen, and others by collecting mutants and collaborating with other labs that had been working on BMP signaling. The team showed that the receptor subunits and downstream signaling components are expressed in the presynaptic motoneurons. Brian, along with Mike O’Connor and colleagues, showed that the BMP homolog Gbb (encoded by the glass bottom boat gene) is the signal secreted by postsynaptic muscle cells (Neuron 2003). Pejmun and Brian went on to show that Highwire regulates synaptic size by binding the Smad protein Medea (Med) and thereby regulating the presynaptic BMP signaling cascade. Brian, Pejmun, and Hermann went on to further elucidate this pathway in their own labs.

Thus, the large-scale genetic screen for genes controlling synaptic size led to the ligand (a BMP) and receptors (Type I and II BMP receptors) and downstream signaling components (Smads) involved in the retrograde control of synaptic size and showed how Highwire interacts with this pathway. It was gratifying a decade later to see Ralf Schneggenburger and colleagues (Lausanne) show that retrograde BMP signaling controls synaptic growth at a mammalian synapse (Nature Neuroscience 2013), in this case at the calyx of Held auditory relay synapse.

The Likeness of Being

During the late 1970s and early 1980s, we watched and manipulated growth cones in the developing embryo. We discovered remarkable specificity. What came as a surprise was the amount of pushback we got during the 1980s. The common thread of those arguments was that mammals don’t show the kind of specificity we had seen in insects.

As a result, during the 1990s while I was at Berkeley, as we and others were discovering the molecules and mechanisms of guidance and target recognition, and realized they were conserved across phylogeny, I had the opportunity to write three reviews (the first with Carla Shatz, the second alone, and the third with Marc Tessier-Lavigne) to provide overviews and historical context, and to lay to rest some of the lingering questions and controversies from the 1980s. More than two decades later, it is gratifying to see that these three reviews remain historically relevant, largely correct, and highly cited.

While my lab was working on the earlier activity-independent stages of brain wiring involving axon guidance and initial target recognition, Carla’s had been working on the later activity-dependent stages that control the refinement and precision of connections. Carla and I had been teaching Developmental Neurobiology together at Stanford and Berkeley, and at CSH with Paul Patterson, and had come to see the 1980s argument of chemoaffinity vs. competition as off the mark. It wasn’t either–or. Rather, it was both–and. We viewed the development of wiring as using both mechanisms in tandem and in a coordinated fashion to control brain wiring. Carla and I wrote a review titled “Developmental Mechanisms That Generate Precise Patterns of Neuronal Connectivity” (Cell/Neuron 1993). We resolved the issue about Sperry and chemoaffinity by arguing that he was right, as Friedrich Bonhoeffer showed, but erred by expecting chemoaffinity to do everything.

I wrote the second review a year later (Cell 1994). It was entitled “The Likeness of Being: Phylogenetically Conserved Molecular Mechanisms of Growth Cone Guidance.” It was written as the first wave of guidance molecules were cloned and shown to be conserved from invertebrates to mammals. In 1992, we published on what became the first Semaphorin, called Fasciclin IV in the grasshopper. In 1993, we published the cloning of the Semaphorin gene family from insects to mammals at the same time as Jon Raper published the cloning of Collapsin (a Semaphorin) in the chick. Marc’s lab published the cloning of Netrin in mammals, which is homologous to UNC-6 in the nematode. The handwriting was on the wall: there was a clear likeness of being when it came to the molecules and mechanisms of axon guidance.

I felt two questions had been answered. First, do two cell adhesion molecules on their own control axon guidance? The answer was no: a variety of attractants and repellents control axon guidance. Second, how different are vertebrates from invertebrates? Here too, the answer was clear. Axon guidance molecules are highly conserved across phylogeny.

The field progressed quickly in the mid-1990s. Within a few years, Marc and I thought it was time to write a major review (Science 1996) on the “Molecular Biology of Axon Guidance.” We concluded: “Growth cones appear to be guided by at least four different mechanisms: contact attraction, chemoattraction, contact repulsion, and chemorepulsion. Evidence is accumulating that these mechanisms . . . are mediated by mechanistically and evolutionarily conserved ligand-receptor systems.” By this time, we already knew about the Netrins and their receptors, Semaphorins, IgCAMs, and other cell surface and extracellular matrix molecules.

Within a few years, those conclusions were solidified by the cloning of the Semaphorin receptors (Plexins and Neuropilins), the Ephrins and their receptors, and Slits and their Robo receptors. By the end of the 1990s, we and others had discovered the big four families of guidance molecules—Netrins, Semaphorins, Slits, and Ephrins—and their receptors. And we had come to understand a great deal about the logic and mechanisms of axon guidance.

Over the years, Marc and I won several awards together. It was an honor for both of us to share the 1996 IPSEN Prize and the 2020 Gruber Prize in Neuroscience with our friend Friedrich Bonhoeffer. Another friend had a big impact on my scientific career—Tom Jessell. It was an honor to win two awards with Tom: the 1996 Taylor Prize and the 2001 March-of-Dimes Prize. I lost two of my favorite scientists when Tom passed away in 2019 and when Friedrich died from Covid in 2021, just a few months after he, Marc, and I celebrated the Gruber Prize together.

A Grand Slam for Berkeley Neuroscience

When I got to Berkeley in the fall of 1987, Berkeley was undergoing a massive reorganization of the life sciences to bring its antiquated set of small departments into the modern era, led by Dan Koshland. By 1989, 13 small biology departments had been replaced by MCB and the Department of Integrative Biology (IB). The Division of Neurobiology was in MCB. Until this time, neurobiology had grown within a variety of small departments, all supported by a Graduate Group in Neurobiology that had little power on campus. I had been recruited in part to help build molecular and cellular neurobiology within MCB, and eventually to help unite all of neuroscience.

I served as head of the Division of Neurobiology from 1992 to 1999. With the encouragement of Gerry Rubin, I also had a secondary appointment in the Division of Genetics so that I could have a voice in those faculty recruitments as well. I recruited Carla Shatz (now Director, BioX, Stanford) to come join me in 1992, and Carla and I together recruited John Ngai (now Director of the National Institutes of Health [NIH] BRAIN Initiative), Udi Isacoff (now an elected member of the NAS and director of the Helen Wills Neuroscience Institute), Yang Dan (now also an elected member of the NAS and an HHMI investigator), Mu-ming Poo (now director of the Shanghai-based Institute of Neuroscience), Tito Serafini (now chief strategy officer and cofounder of Atreca), and Rich Kramer. We helped Psychology recruit Jack Gallant, Frederic Theunissen, Bob Knight, and Mark D’Esposito; Optometry recruit John Flannery; and Biochemical Engineering recruit David Schaffer. Overall, Neuroscience did very well during the 1990s as strong appointments were made across campus.

The reorganization of biology had brought the molecular and cellular side of neuroscience into MCB but had not provided a united home that also included the systems and cognitive side of the field. The Psychology Department emerged as the de facto center for that side, but there were debates within Psychology as to whether cognitive neuroscience was or was not really psychology. Clearly, the campus-wide neuroscience community needed to be unified, with a single voice and strong leadership, and with its own FTEs and graduate program. That was what the campus had recruited me to do.

Fortunately, the campus had visionary leadership in Chancellor Chang-Lin Tien and Vice Chancellor Carol Christ. We would not have achieved the unification of neuroscience without their strong support. Starting in the mid-1990s, Carla and I, with the support of the neuroscience community, began to lay the groundwork for an interactive program stretching from genes and molecules to behavior and cognition. We thought this was critical since neuroscience transcends the boundaries that define traditional academic departments.

MCB and Psychology were worried that what we really wanted was to create a new Neuroscience Department that might take away many of their faculty and resources. Their concern was reinforced by an outside review that Carla and I had encouraged the chancellor to commission. The review committee included Zack Hall, Larry Squire, and Torsten Wiesel. On October 3, 1995, they concluded: “We think the formation of a Department of Neuroscience would be an important, vital step for neuroscience and for Berkeley.”

That recommendation by the review committee enabled us to offer a less-radical compromise that we believed would receive broad support: a strong institute with degree-granting ability and its own FTEs, whose faculty would retain appointments in existing departments. An independent department, we reasoned, was too big a step and could come later. MCB readily embraced our proposal. Psychology did not, doubting that Carla and I, as faculty in MCB, would truly work to enhance cognitive neuroscience. It took them a while to realize we were sincere. Fortunately, the younger generation, and in particular Rich Ivry, who later became chair of Psychology, appreciated the importance of our proposal for the campus and his department. Rich persuaded Psychology to embrace our proposal, joined our executive committee, and was a tremendous help in guiding the institute. Although winning over Psychology was our first major hurdle, there were still many rivers to cross—in particular, we had to persuade the Academic Senate.

We received the support of Chancellor Chang-Lin Tien. But we would never have created the Helen Wills Neuroscience Institute were it not for Vice Chancellor Carol Christ (today Berkeley’s chancellor). We made presentations in front of countless committees of the Academic Senate. We never fully knew what power some of them had over our proposal. Their worries concerned resources—both faculty appointments (FTEs) and money for staff and graduate students. Very few institutes on campus had the FTEs and resources we were seeking. In my experience, academics often tend to view their world as a zero-sum game, reasoning that if someone else gets something, you get less. That view lacked the entrepreneurial perspective that you could grow new resources if you have a bold vision. And what better goal to have than to understand the brain by harnessing Berkeley’s breadth from the social to the biological to the physical sciences.

With the help of Vice Chancellor Christ, Carla and I finally succeeded. In 1996, the Academic Senate approved, and in 1997, Chancellor Tien officially signed into creation, the Center for Neuroscience, later renamed the Neuroscience Institute, with broad powers and resources in terms of faculty, students, and staff. Carla initially became director of the center/institute, and I remained head of the Division of Neurobiology in MCB, but in reality, we made every decision together. A few years later, as she was leaving Cal to become chair of neurobiology at Harvard, I became director of the institute. The institute integrated the campus neuroscience community, helped recruit new faculty in a variety of departments, and oversaw the Neuroscience PhD Program.

While we were convincing the campus to create the Neuroscience Institute, we knew that one of our best powers of persuasion would be our ability to raise new resources. We were trying to raise money for our graduate program, help recruit new faculty, and build special core facilities. We also wanted to build a brain-imaging center with a state-of-the-art research fMRI. Moreover, we wanted to help the campus raise money for a new building with a greatly expanded animal facility so that we could expand cognitive neuroscience. We were ultimately successful in all these goals. But we never would have gotten started had it not been for Mike Desler in the development office, whose goal was to build a new basketball pavilion.

One day in early 1995, Mike came to my office and said he wanted to help neuroscience. He had befriended a famous Cal alumna, Helen Wills, one of the greatest women’s tennis players of all time. Helen won Wimbledon eight times, captured 31 Grand Slam titles, and once won 180 consecutive matches. She grew up in the Bay Area, went to Cal, and was already a tennis star while a student. By the time Mike had become her friend, she was living in a convalescent home in Carmel. Mike had tried to convince Helen to leave her estate to athletics. But Helen told Mike that he could raise his money elsewhere. Rather, in her late 80s, she wondered why some of her friends had lost their cognitive ability. Mike knew what Carla and I were trying to do for neuroscience, and so he offered to introduce us to Helen.

Carla and I went down to Carmel together in 1995 to visit Helen in her nursing home, and I visited her again some months later. Her body was weakening, her hearing was failing, but her mind was sharp. She wanted to understand how the brain works, and why some people lose their memory and cognitive abilities whereas other don’t. We told her about our proposed institute. She loved the idea, engaged with us, and asked good questions.

Ultimately, Helen Wills offered to leave her estate to our Neuroscience Institute that didn’t even exist yet. By doing so, she helped create the institute by calming worries about funding. We discussed how she could have the biggest impact. On October 20, 1995, she bequeathed $10.5 million to endow the Neuroscience Graduate Program and to support the growth of neuroscience at Cal through support of new faculty and special facilities. With Vice Chancellor Christ’s support, Helen pledged her estate to an Institute that wouldn’t be officially approved for another two years (1997). At the age of 92, on January 1, 1998, Helen Wills died in her convalescent home. We renamed the center the Helen Wills Neuroscience Institute.

In 2000, we persuaded Henry H. (Sam) Wheeler Jr., a philanthropist who loved innovative science and cutting-edge technology, to give $5 million to help launch the Wheeler Brain Imaging Center to support advanced brain imaging technologies. Sam later gave the Center another $2.5 million gift. Years earlier, when we promised Psychology that we would strengthen their enterprise and help provide resources, we meant what we said, and in that spirit, ultimately all areas of neuroscience have prospered.

Today, more than two decades after Carla and I co-founded the Neuroscience Institute, the campus is on the verge of forming a Department of Neuroscience. That was always our dream, but as we realized back in the 1990s, it required evolution, not revolution. The establishment of a Department of Neuroscience was what several outside review committees had proposed, the first in 1995 chaired by Zach Hall and the second in 2019 chaired by Tom Carew, and soon it will be a reality. I continue to be a huge supporter of the Neuroscience Institute. Udi Isacoff is director and has taken it to the next level, helping to build the Weill Neurohub, a groundbreaking partnership to cure disease. I have an adjunct-professor appointment in the institute and love interacting with students. And given the financial success of the more recent chapters of my life, Marcia and I today are major donors to help facilitate the recruitment of new faculty and graduate students.

Flies Are Just Little People with Wings

Starting in the late 1980s, I wanted to learn more about biotechnology and the private sector. I accepted offers to consult for various companies, to help them, and also to learn more about drug discovery and development. In the late 1980s, I joined the scientific advisory boards of Athena Neurosciences in the Bay Area (later acquired by Elan) and Teijin Limited in Japan.

In 1985, my good friend Spyros Artavanis-Tsakonas and his colleagues at Yale cloned the Notch gene in Drosophila. In 1987, Spyros cloned the human Notch gene and filed a patent on it, following that with additional patents on Notch, its ligands, and its applications and utility for human disease. When I heard that Spyros had patented human Notch and its therapeutic applications for human disease, I asked my lab neighbor Gerry Rubin: “Is Spyros nuts?” The answer of course is yes (which is why we love him). But the relevant question was to probe into what Spyros was thinking. To our knowledge, he was the first person to clone the homologue of a Drosophila development gene in humans and to patent it. Within a few minutes, the realization hit both of us. Spyros wasn’t nuts. Mutations in human Notch are associated with lymphoblastic leukemia. Aberrant Notch signaling is probably involved in other cancers. We began to think about all the ways in which reagents against Notch might be used as human therapeutics. And we began to consider ways in which Gerry’s work on the Ras pathway, and ours on Semaphorins and other pathways, might be relevant for human health.

In 1991, Spyros asked me if I would join him and Gerry Rubin to form what became Exelixis, which means evolution in Greek, to use fly genetics to dissect human disease pathways. Stelios Papadopoulos, a scientist turned banker, summarized that concept when, after listening to Spyros, Gerry, and me prattle on about the relevance of our discoveries, said, “What you are telling me is that flies are just little people with wings.” Stelios joined us, and with his help, we raised venture capital and opened our lab doors in January 1995. Our focus was the Notch and Ras pathways. We recruited great people. We brought in Marc Tessier-Lavigne as scientific adviser in addition to the three scientific founders. During our early years, I was the spokesperson for Exelixis at biotech meetings. Stelios predicted that of the three of us, I would be the first to leave academia to move into the private sector, and he was right. I had caught the bug. There was no zero-sum game, and no constraints. In the private sector, if you have a vision and articulate specific goals, you can raise the resources you need to realize that vision.

The early years of Exelixis were a learning experience for me. I was surrounded by great teachers and learned how to build and lead a company. I felt like I got the equivalent of an MBA, and degrees in corporate and patent law. Our business plan was to apply functional genomics in model organisms (fruit flies, nematodes, and zebrafish) to identify pathways and biological targets that could be exploited for human health. By 2000, we transitioned to focus on drug discovery and development. We acquired and built a chemical library with millions of compounds. Goldman Sachs helped take Exelixis public on April 10, 2000. As of the writing of this biography, Exelixis remains an independent public company with several FDA-approved cancer drugs, some of which are against downstream targets in the Ras pathway. I learned a tremendous amount about building biotech companies from my Exelixis experience.

Exelixis gave Marcia and me some financial freedom and provided us with the means to help support my parents. We sold our stock over the next few years and used the proceeds to help buy a home for my parents in Florida, purchase the ranch that would be our future home, and begin a financial nest egg. It gave us the freedom to take risk. Without that freedom, I doubt Marcia and I would have felt comfortable with me leaving a tenured academic position, as I did for my next chapter.

Leaving the Ivory Tower

In 1999, Marc Tessier-Lavigne, his former postdoc Tito Serafini, who was at the time an assistant professor at Berkeley and head of the campus Functional Genomics Laboratory, and I were cooking up an idea for a new biotech company focused on neurological diseases. We pitched our idea—a start-up called Renovis—to our friend and senior adviser Ed Penhoet, who had been a biochemistry professor at Berkeley, then was cofounder and CEO of Chiron, and had returned to Berkeley as dean of the School of Public Health. Ed was enthusiastic. He gave us space and provided seed funding. We raised our first institutional financing in spring of 2000. We obtained space at Children’s Hospital in Oakland and then in South San Francisco. Tito closed his Berkeley lab and joined the company full time as CSO. We recruited some terrific scientists. Marc and I went by often and helped oversee Renovis.

In winter–spring of 2001, Renovis was searching for its CEO. If we went with an outside candidate, we knew it would soon become their company and Marc and I would lose control. Ed asked me to do it and expressed confidence that I was a natural for it. Of course, that was what I had heard several years earlier from Stelios. I had always imagined my life in chapters. In June 2001, I was turning 50. If I was ever going to make a jump into the private sector, this seemed like the right time.

Going Over to the Bright Side

One Sunday afternoon in March–April 2001, I got a phone call from my friend Richard Scheller. He was thinking of leaving Stanford to become senior vice president of research at Genentech. He asked me what I thought about the idea. He confessed that several senior advisers were telling him not to do it and said he felt some academics were unforgiving and prejudiced against colleagues who left to work in the private sector. I told him to go for it. He could have a big impact on human health by applying his 30 years of scientific experience to medical problems. Richard made the leap and has had a highly impactful career in biotech.

A month or so later, in May 2001, Richard got a reciprocal call from me. I told him I was thinking about leaving Berkeley to become CEO of Renovis. I asked him what he thought about the idea. He told me I should do it, for the same reasons I had told him. He cautioned me that senior advisers would tell me not to do it, just as they had him. Although I had many details to sort out concerning my lab, taking care of my students and postdocs, and arranging for a leave of absence, that weekend Marcia and I both knew I was going to do it.

When I announced that I was taking a leave of absence from Berkeley to become CEO of Renovis, I got interviewed by many in the media. When asked by the San Francisco Chronicle why I was switching, I answered: “This time I decided I didn’t want to hand over the car keys to somebody else.” Was I going to continue cofounding biotech companies, remain in academia, and repeatedly hand the keys over to someone else? Or was I going to take the responsibility? I love to lead. I’m not afraid of risk. Now felt like the right time. My decision to leave academia was unusual in 2001 (although it would have been less so today) and surprised many colleagues. Some told me that I was burning bridges regarding future awards. Others told me that I was setting a bad example by going over to the “dark side.” Others told me they were envious and wished they had the chutzpah to do it.

Academics love calling the private sector the dark side as if it is all about money whereas academia is not. That is a false dichotomy. Neither side is what it is made out to be. Richard and I moved to biotech to turn our scientific experience into developing medicines. Both of us have had an impact on saving lives and improving human health. I call that the bright side—just as much as is basic research. Fundraising is as key to universities and individual labs as it is to biotechs. Some academics claim innocence and purity while constantly seeking ways to make money via consulting or founder’s equity. In academia, I raised money in the evening. In biotech, I raise money in the light of day. Both pursuits require the resources necessary to fuel science. And both are essential for the development of technologies to improve human health.

From Gross National Happiness to IPO

I started as CEO of Renovis in summer of 2001. For the first few years, I led a double life, building Renovis while also overseeing the final years for the graduate students and postdocs finishing up their work in my lab at Berkeley. I was fortunate that Beth Blankemeier continued to work part time as manager of my lab at Berkeley while also working part time at Renovis; her double life helped make my double life possible (after Renovis she moved to Genentech; in 2015, she sadly passed away from cancer).

The university was gracious in negotiating my leave of absence. Vice Chancellor Paul Gray granted me a four-year leave and asked me to continue to help Berkeley raise money in the biomedical sciences and to keep an eye on the neuroscience program. He hoped I would go off to industry for a few years and then return to take on a leadership position at Berkeley.

In founding Renovis, Marc, Tito, and I proposed to build a biotech company based upon Tito’s single-cell genomic profiling, and bacterial artificial chromosome (BAC)-mediated transgenesis, which had been pioneered by Nat Heintz at Rockefeller. We planned to identify novel, druggable targets for neurological and psychiatric diseases, and use them to develop drug candidates and take these drug candidates into the clinic.

We raised our first institutional round of funding in July 2000. The second round was raised in May 2001. I joined as CEO in September 2001. In January 2002, Ed and I got together on the Berkeley campus for lunch to talk about what to do with Renovis. The platform was taking longer than anticipated to build out—Tito’s and Nat’s technologies both needed technical improvements and more time to move from academia to industry. Faced with this reality, we reasoned that we needed to get a drug candidate sooner than the platform would produce one. We had a good valuation, great investors, an A+ team, and plenty of money. We decided the best path was to license, buy, or acquire a lead drug. In summer of 2002 we found what we were looking for, a biotech company called Centaur that was in financial trouble but had a potentially fabulous drug candidate—Cerovive (NXY-059)—that was partnered with AstraZeneca (AZ) for ischemic stroke.

AZ had finished its Phase II trial of Cerovive but given the expense of conducting two large Phase III trials, AZ paused the clinical development and asked for further primate experiments. Centaur couldn’t raise money because investors thought AZ’s request for more animal data was an indication that they were balking on taking Cerovive forward.

I brought in a top stroke expert, Wade Smith from UCSF, who helped me analyze the animal and clinical data. We became convinced that the primate data would be positive (they were) and that AZ would decide to take Cerovive into two Phase III trials (they did). We acquired Cerovive and their other chemical libraries and drug candidates. With AZ’s decision to move forward, Cerovive gave Renovis a clear path to an initial public offering (IPO) by having a clinical asset partnered with pharma.

The Centaur acquisition allowed us to build out our chemistry and drug discovery and development capabilities as well as our clinical team. We licensed another drug candidate from Jon Levine’s lab at UCSF, a kappa opioid agonist and low-dose opioid antagonist for postoperative pain that was designed to replace morphine. Jon already had data from hundreds of patients at UCSF. We began early clinical trials on his drug combination. Over the next year, we established collaborations with Pfizer and Genentech, and took a third drug candidate we had made internally, our TRPV1 antagonist against the capsaicin receptor, into the clinic for pain with Pfizer. In summer 2003 we raised a our final financing round as a private company preparing for its initial public offering (IPO).

In fall 2003 we prepared for our IPO. By middle of November, our documents were filed with the U.S. Securities and Exchange Commission (SEC). We had lined up Goldman Sachs to take us public. We would wait until mid-January to begin our IPO roadshow. The next one to two months was a quiet period. Marcia and I decided to use that period to go on an adventure to Cambodia and Bhutan. We started in Phnom Penh, visiting a program we had been funding that helped girls get a high school education. We visited the schools, met many girls we had been supporting, and their families. It was deeply moving.

We then flew to Siem Reap, our second time visiting Angkor Wat, Bayon, Preah Kahn, Ta Prohm, and other ancient temples. Beyond being tourists, our mission was to spend time at the Angkor Hospital for Children, run by Friends Without a Border, a nonprofit we had been supporting ever since we met its founder, photographer Kenro Izu, and were inspired by his vision to help Cambodian children.

Days earlier, I had been at Renovis, trying to help design new drugs for Western patients. Now Marcia and I were in the developing world helping with much more basic medical needs—saving children’s lives with clean water, antibiotics, intravenous fluids, generic drugs, and clean operating and anesthesia equipment. What a contrast in human needs. We heard stories of everyday “miracles” from Cambodian doctors and nurses. This hospital, as well as a more recent one that we helped Friends Without a Border build in Luang Prabang, Laos, have become lifelong missions for us.

We next traveled to Bhutan. We wanted to experience a Himalayan Buddhist kingdom. Our two weeks in Bhutan were more inspirational than we anticipated. The king of Bhutan has focused his country on gross national happiness. People were deeply Buddhist and happy. We visited many temples. With pack horses and guides, we trekked up into the Himalayas to stay at the village next to the Ngang Lhakhang temple. The village was welcoming as we became their guests to watch their Buddhist Tshechu festival with dancing, costumes, music, and food.

One day we hiked up a mountain to the Tiger’s Nest monastery. It was a long and exhilarating hike through fields of prayer flags. We noticed lots of trash along the trail and decided to clean up the holy site, picking up the trash and collecting it in a bag as we descended the mountain. Partway down, we passed an entourage of monks in maroon robes. At the end of the entourage was a lama wearing a special hat. Our guide told us this was a very famous lama, known in Bhutan as the Thai lama. The lama saw our bag of trash and gave us a wink and a thumbs-up. He directed his monks to bless us. They poured holy water into our cupped hands, from which we sipped a bit, and then poured the rest over our heads. It was an auspicious moment. The monks brought out wrist-strings made of saffron and maroon yarn. The lama tied a holy string onto my wrist, but not being allowed to touch a woman, dropped another in Marcia’s hand, and I tied it onto her wrist. We felt blessed. I wore that holy string until it fell off months later.

A few weeks after returning from Bhutan, in mid-January 2004, with the help of Goldman Sachs, we did our two-week IPO roadshow, meeting potential investors across Europe and throughout the United States. We ended up in New York to raise the funds needed to make new drugs to cure disease. Just a few weeks earlier, I had been in Bhutan in a Buddhist culture focused on gross national happiness. Now I was meeting with individuals who handled billions of dollars, with their focus on making money. Money vs. happiness and the contrast of Western vs. Buddhist culture. My holy string symbolized my internal conflict. During our IPO roadshow, so as not to spook investors, I taped the string above my wrist so it would not show. We completed our IPO on February 4, 2004. At our celebration dinner that evening, with a table full of bankers in suits, I rolled up my sleeve, took off the tape, and showed them all the holy string from the Thai lama that had blessed me throughout our IPO. Taking Renovis public and being a public CEO was a great learning experience, but one I personally only cared to do once.

Cerovive was the first drug candidate to fulfill the STAIR criteria established by the Stroke Therapy Academic Industry Roundtable in 1999. AZ conducted two large Phase III trials for Cerovive. The first trial, SAINT I, reported out in May 2005 with positive results. We were almost to the finish line with the first neuroprotective drug to limit brain damage after a stroke. This was potentially huge for human health. On the strength of the results of that first Phase III trial, AZ began building worldwide manufacturing facilities for Cerovive.

All that remained was the conclusion of the second trial, SAINT II, due to report out in October 2006. The results of that trial were a shock. Although directionally supportive of SAINT I, the second trial failed to reach statistical significance. We worked with our Stanford statistician to try to determine if there was a difference in the patient population in terms of ethnicity, stroke location, severity, or timing that determined the two different statistical outcomes, but nothing could be found. Given the enormous cost of another stroke trial, AZ decided, and we concurred, not to do another Phase III trial.

Our programs with TRPV1, P2X3, and P2X7 continued moving forward. By spring 2007, several companies approached Renovis about a possible acquisition for those programs. My decision to sell Renovis was a painful one, but the right one. Without Cerovive, Renovis would have required a large influx of funds and several years to grow as an independent company. To try to push on would have been the wrong decision for the company and its shareholders. In summer 2007, Evotec negotiated the acquisition of Renovis with me, which was signed and announced in mid-September 2007. One of Evotec’s conditions was that I join the Evotec Board of Directors which I did for three years from 2008 to 2010, the last two as vice chair. The Renovis P2X3 program, acquired by Evotec, was later licensed to Bayer. Bayer announced positive Ph IIa data for chronic cough in July 2019 and positive Ph IIb data in August 2021.

Too Big to Innovate

In spring and summer 2007, while negotiating with Evotec’s CEO for their acquisition of Renovis, I was considering my next chapter. I was regularly contacted by universities and research institutes to inquire if I might be interested in a position as president or provost. Marcia and I discussed these positions, and decided they weren’t right for me or for us. I was offered a partner position at a large private equity and venture capital firm, but as I explored that position, I became concerned about their internal politics. I was willing to take my time to find the right position. I wanted to do something transformational and impactful for human health.

Then in early July 2007, I was contacted by recruiters and asked if I might be interested in becoming head of research and development (R&D) at Pfizer. I had several times before been offered high-level positions in pharma or large biotech, and had turned them down, but this one sounded intriguing. I answered that I would like to learn more. A week or so later, Pfizer CEO Jeff Kindler flew out to meet with me in San Francisco. He offered me the opportunity to become head of R&D at the world’s largest pharmaceutical company and to change the future of pharma R&D. I think he was surprised when, a few weeks later, I turned him down.

Pfizer’s research organization in 2007 seemed unproductive to me when I considered how much money they spend each year, and how few new drugs resulted. Pfizer was no Genentech when it came to discovering and developing new drugs. Rather, most of Pfizer’s new drugs came from acquisitions, and most of their drugs in 2007 were small molecules with few biologics. If I became head of R&D, I would want to make massive cultural and structural changes that would require firing people and closing sites. I would want a larger focus on biologics. I suspected I would face internal resistance. This was not how I wanted to spend my energy, and so I said no.

A week or two later, Jeff came back with a more interesting offer. He proposed a two-headed system reporting to him. He agreed that the current research organization, called PGRD (Pfizer Global R&D), focused mostly on small molecules. He proposed to promote his internal candidate to be head of PGRD. I would join his Executive Leadership Team to build out a separate biotherapeutics unit focused on everything that wasn’t small molecules—namely, antibodies, proteins, peptides, nucleic acids, vaccines, gene therapy, and cell therapy. He asked me to build what he called Pfizer’s Genentech. My core site would be Rinat, a spin-off of Genentech in South San Francisco that Pfizer had acquired.

Building something new, starting with Rinat, sounded more exciting than having to deconstruct something large and cumbersome before building a new organization. Jeff wanted my unit to be in California. He wanted me to make Pfizer more innovative and nimbler. I could remain living in the Bay Area (where Marcia and I were building our ranch) and come to New York one week per month. In September, I accepted the offer to join Pfizer as president of a new division called the Biotherapeutics and Bioinnovation Center (BBC). I joined as a member of Pfizer’s Executive Leadership Team reporting to the CEO. I started on October 4, 2007.

I proposed a new model, starting with Rinat, which would lower clinical attrition and cost, and increase speed of new medicines. The goal was to make Pfizer a top-tier biotherapeutics company, and in so doing, to develop a new model for the rest of Pfizer’s R&D. I would build Pfizer’s biotherapeutics capability by a combination of building internally and small biotech acquisitions. My BBC model was to create a federation of small biotech-like R&D units in major academic hubs. Each unit would have fewer than 150 scientists, be led by a world-class CSO, take drug candidates to human proof-of-concept (typically Phase II clinical trials), and then hand them off to Pfizer’s business units for Phase III trials, approval, and sales.

I established nimble decision-making and an entrepreneurial culture, and proposed biotech-like equity incentives to recruit and retain top people. We started with antibodies and various alternative scaffolds, and then continued with RNAi and stem cells. We used human genetics and system biology to validate targets. I learned a tremendous amount about the pharmaceutical world. My leadership team and our scientists were superb. I formed many lifelong friendships.

In summer 2008, Jeff was negotiating the acquisition of Wyeth. Given the sorry state of Pfizer’s clinical pipeline, it made sense. Jeff told me he wanted this acquisition to be different from Pfizer’s prior acquisitions of Warner-Lambert and Pharmacia. He wanted to use the acquisition to reorganize R&D to become more productive and innovative. He asked me to produce a white paper on how we might achieve that goal, combining the best of both companies to build something new rather than simply shutting Wyeth’s sites and leaving Pfizer’s sites intact. I sent my white paper to the executive team. Within 24 hours, most of the team members said they fully supported my proposal. Leadership at PGRD, however, didn’t like my proposal because it would shake up their world. Succumbing to this pressure, Jeff squashed any discussion of better ways to integrate Wyeth, and my white paper was buried. Jeff brought in Boston Consulting Group, who worked with PGRD to make the acquisition of Wyeth just like the acquisitions that came before. Productive Wyeth sites were shut down in favor of problematic Pfizer sites. Same as it ever was. I didn’t want to be part of that and so concluded it was time for me to go.

Transforming Great Science into Impactful Medicine

After leaving Pfizer, I reimmersed myself into the innovation ecosystem. I pursued two parallel paths. The first was new company formation. From the summer of 2009 and into 2010, I cofounded and helped launch several biotech companies, including Second Genome, Kodiak (originally called Oligasis), and others; continued to serve as vice chair of the Evotec Board; and joined several additional boards, including as chair of iPierian.

The second path was venture capital. I was getting tired of having to pitch my ideas to venture capitalists. Perhaps the best solution was to become one of them, as I had come close to doing in 2007 before I joined Pfizer. If I wanted to have more say at the board level, then I needed to have more control over the purse strings. The solution, I felt, was to find some like-minded colleagues and together start our own biotech venture capital (VC) firm.

We launched venBio Partners in 2010 and raised our first fund of $179 million in September 2011. My partner Rob Adelman had been an undergraduate at Berkeley and helped launch a biotech with Bruce Ames while still an undergrad. He got his medical degree at Yale, became a practicing surgeon, and, after cofounding several more biotechs, joined another venture group. Rob and I work well together and found we had similar philosophies about building biotech companies and recruiting talent. For the first few years, we were joined by Laura Deming, a brilliant young Thiel Fellow.

We recruited a great science-based team with company-building and venture capital experience, including Aaron Royston, MD, MBA; Rich Gaster, MD, PhD; Yvonne Yamanaka, PhD; and Jaume Pons, PhD. We raised our second fund ($340 million) in 2015, our third ($394 million) in 2019, and our fourth ($550 million) in 2021. Today, our investors are mostly family offices and university endowments. While we provide our investors with top-tier financial returns, which is why they keep coming back, I am most proud of the fact that in 10 years, we already have five FDA-approved drugs on the market for seven indications, with many more coming along in the clinic. I am fulfilling my dream of turning great science into impactful medicine.

Two of my most rewarding experiences at venBio have been companies I cofounded: Labrys and ALX Oncology. While at Pfizer, I was a champion for a program Jaume Pons started when he made an antibody against the 37-amino-acid calcitonin gene-related peptide (CGRP) for treatment of chronic migraine. Pfizer decided that it was not interested in pursuing a migraine drug. After I left, Pfizer decided to out-license the CGRP antibody. They opened a competition among leading VC groups. Most other VCs did not submit proposals because key opinion leaders (KOLs) in migraine told them the CGRP antibody would not work. Rather, the KOLs believed that a CGRP antagonist would have to cross the blood-brain barrier (BBB) to work, something an antibody would not do. I believed the KOLs were wrong. There was evidence suggesting that release of CGRP from trigeminal sensory endings onto smooth muscle around the blood vessels leading into the brain likely causes migraine, suggesting that this approach would directly address the underlying cause without crossing the BBB.

Convinced that the CGRP antibody would work, I won the competition. I founded Labrys Biologics in 2012. We licensed the CGRP antibody from Pfizer. It took longer than I anticipated to build the investor syndicate to join venBio because so many VCs had been convinced by KOLs that a CGRP antibody would not work. I recruited the management team and chaired the board. In 2013 and 2014, we conducted Phase I clinical studies and then two Phase II clinical trials. The antibody was given as a subcutaneous injection. The clinical data were strikingly positive: with very few side effects, 25 percent of the treated patients stopped getting migraines, and the rest had reduced frequency and intensity of migraines.

Julia, the daughter of a good friend, had been a chronic migraine sufferer since she was in her late teens. She had multiple migraine days every week. I convinced her to enroll in our Phase II trial. The trial had three arms (two active and one placebo), so she had a two-thirds chance of getting active drug. About four months after she started the trial, I saw Julia at a party. She threw her arms around me and said her life was transformed; she had not had a migraine since the day she got her first shot. I was moved to tears.

In 2014, based on our Phase II trials, Teva acquired Labrys. My investors made nearly 25 times their investment. It was even a bigger win for patients. Teva completed two Phase III trials in 2017. Papers on the Phase II and Phase III trials were published in the New England Journal of Medicine The drug, called Ajovy, was approved by the FDA in 2018, the European Union in 2019, and the United Kingdom in 2020.

Second on my “most rewarding” list is ALX Oncology, an immuno-oncology company. My interest in immuno-oncology goes back to my days at Berkeley in the 1990s when I was head of Neurobiology and Jim Allison was head of Immunology. He was discovering the stop signals in the immune system, what are called checkpoint inhibitors, while we were discovering stop signals in the nervous system, what are called repellents. Jim made the first discovery of a stop signal for adaptive immunity: CTLA-4. Scientists subsequently discovered the major checkpoint inhibitor for T cells: PD-L1 on cancer cells and their receptor PD-1 on T cells. Successful tumors—those that thrive and metastasize—express stop signals on their surface to inhibit immune cells from attacking them. These discoveries led Jim and Tasuku Honjo to win the Nobel Prize in 2018.

In subsequent years, it was discovered that there are checkpoint inhibitors for myeloid cells in innate immunity. Many successful tumors express CD47 on their surface, which inhibits innate immunity by binding to its receptor, SIRP, on the surface of macrophages and dendritic cells. CD47 functions as a “don’t eat me” signal to macrophages and to stop dendritic cells from activating T cells. In 2013, Chris Garcia and colleagues at Stanford published a paper in Science showing that if you block CD47 with a biologic agent that lacks an Fc domain (and thus does not recruit macrophages), that you potentiate the efficacy of other anti-cancer antibodies without causing cytopenias, as are found with antibodies against CD47 with functional Fc domains.

Jaume Pons had taken over much of my responsibilities at Pfizer when I resigned in 2009. He was head of Pfizer/Rinat, and oversaw protein engineering, immuno-oncology, and oncology biologics. From 2011–2014, Jaume and I discussed lots of ideas for starting a new biotech together. In late spring 2014, we decided to use Chris Garcia’s paper as our starting point by blocking CD47 using a biologic with a “dead” Fc.

We licensed Chris’s intellectual property (IP) from Stanford, and Jaume, Chris, and I cofounded Alexo (now called ALX Oncology) in March 2015. Jaume resigned from Pfizer/Rinat to join as CEO. At the same time, I recruited Hong Wan to leave Pfizer and join as CSO. Hong had been one of my star graduate students at Berkeley (she discovered Highwire). She had been a leader in translational medicine for neurological diseases at Wyeth, and at Pfizer/Rinat became head of preclinical development, investigational new drug applications (INDs), and early clinical biomarkers for immuno-oncology.

We made our unique biologic against CD47 in 2015 and did the necessary preclinical development in 2016. We dosed our first patient in 2017, and since then have shown that ALX148 is safe and efficacious in hematological and solid tumors. We took the company public in July 2020. Today, ALXO is a successful public company with many ongoing Phase II clinical trials in a variety of cancer indications and drug combinations. It has been a success story from academic paper to company-formation to protein-engineering to positive human clinical trials.

At venBio, we recently raised our fourth fund as I turned 70. Some people ask how many more funds I will participate in, and how many more biotech companies I plan to cofound. The answer is simple—I intend to keep doing this until I get bored or lose my ability. I love what I do. I get to work with great people, build terrific teams, and create innovative medicines.

When Scientist Became Patient

I can’t write my life history without addressing a profound moment when I came face-to-face with my own mortality and felt first-hand the impact of the work I’ve been involved with on people’s lives. In September 2014, as I was working on cofounding ALX, I became an immuno-oncology patient. On September 5, after returning home from a party celebrating the acquisition of Labrys by Teva, at age 63, I was diagnosed with metastatic melanoma.

In September 1991, when I was age 40 and a Berkeley professor, Marcia noticed an irregular pigmented lesion on my right leg and said it looked like a melanoma. She was right. It was on the back of my right thigh where I had some terrible sunburns as a child. I went to Richard Sagebiel, head of the Melanoma Center at UCSF. Sagebiel thought it was an early-stage melanoma and had it surgically removed.

The pathology report confirmed I had malignant melanoma of Clark’s Level II – having already grown vertically from the epidermis into the papillary dermis. Sagebiel said I was lucky. Clark’s Level II was considered curable by excision. For decades, I believed my melanoma was gone. Fourteen years later in 2005, I watched my good friend and neuroscientist Larry Katz die of metastatic melanoma, realizing that could have been me.

Then 23 years later, in late August 2014 when I was age 63, I felt an enlarged lymph node in my right groin. I had a needle biopsy at UCSF. One day later, on September 3, the first anti-PD-1 antibody, Merck’s Keytruda (Pembrolizumab), was approved by the FDA for metastatic melanoma. On September 5, I received the report from my biopsy: it was a recurrence of my melanoma in what was likely the sentinel lymph node.

I spent the weekend reading the literature and talking to my friends Chuck Baum, Jim Allison, and Roger Perlmutter, who were experts in immuno-oncology. Jim introduced me to Toni Ribas, an immuno-oncology expert at UCLA. I arranged to see Adil Daud on Monday, Sagebiel’s successor and head of UCSF’s Melanoma Center. I learned from Adil that, since 1991, clinicians had learned that 5–10% or so of the time, melanomas at Clark’s Level II have already gone metastatic. Evidently mine had.

I went back to my 1991 path report and found that my melanoma had a “lymphocytic host response” which is probably why I am still alive. The report said I had a moderate number of tumor-infiltrating lymphocytes (TILs) invading my melanoma when it was excised, which indicated my melanoma was being recognized by my immune system. The experts told me that I was an outlier—they had never seen a reappearance that many years after the primary. This suggested my T cells had been killing the cancer cells in a battle waged largely in my groin lymph nodes. Why hadn’t they finished the job? They were most likely stopped by checkpoint inhibitors such as PD-L1 on the cancer cells or pesky regulatory T cells (called Tregs), or both. Perhaps more recently a cancer cell mutated and started expressing higher levels of PD-L1 or some other gene, or my T cells became exhausted – whatever the cause, after a 23-year standoff, the cancer cells had overcome my immune response and I had two enlarged lymph nodes.

Had my melanoma re-appeared 5, 10, or 15 years after my primary lesion, I likely wouldn’t be writing this autobiography. I was incredibly lucky to be diagnosed with metastatic melanoma two days after Keytruda was approved. When I saw Adil, we were entering a new frontier – trying to figure the best path forward. A PET-CT scan revealed no macroscopic cancer anywhere except in two adjoining lymph nodes in my groin. However, it was hard to imagine that some cancer cells hadn’t taken a walkabout to other distant sites in my body.

I had my first Keytruda infusion on September 23, 2014 and continued infusions every three weeks for the next 15 months. Scans over the first few months showed that I was a partial responder. My affected lymph nodes got smaller, and the radioactive glucose signal weaker, but it didn’t go away. We suspected that Tregs were stopping my T cells from finishing the job. Adil had shown that the T cells in my lymph node were high in both PD-1 and CTLA-4. We didn’t want to give me systemic anti-CTLA-4 because it is known to cause autoimmune disease and my mother had Crohn’s disease, but we knew that Ron Levy at Stanford had shown in animal studies that local intra-tumoral injection of anti-CTLA-4, in combination with systemic anti-PD-1, was very potent.

I reached out to Ron, and we forged a UCSF-Stanford collaboration. On March 19, 2015, I had the first of two intra-lymph node injections of anti-CLTA-4 by Sunil Reddy and Ron Levy at Stanford. I was the first human patient in which Ron tried this approach. I had a second injection a month later on April 10. Sunil took immune cells by needle biopsy for cell sorting. The most striking finding was a dramatic increase in NK cells in my lymph node. A year later, in July 2016, a friend told me about a talk Ron Levy had given in which he mentioned the results from his first human patient with local anti-CTLA-4 injection and systemic anti-PD-1. It was a surreal moment. I stayed silent, but I knew—that first patient was me.

On May 8, 2015, I had a partial dissection of my groin lymph nodes at UCSF. Pathology on the other 6 dissected lymph nodes showed two with a small number of cancer cells and four with no cancer cells. I stayed on anti-PD-1 for another 6 months or so until Adil and I decided to stop treatment. Throughout the rest of 2015 and much of 2016, my quarterly scans were clean. Then a year later, in November 2016, I felt a small nodule on my right thigh. A PET-MRI scan showed it was my melanoma in a small focal lesion.

Since I had the BRAF mutation (found in up to 60 percent of melanomas), Adil and I decided that it was time to give me BRAF/MEK inhibitor combination, a targeted therapy first approved in 2013. In patients with high tumor load, they reduce the cancer, but escapers with mutations can break away from the treatment. We reasoned that I had such a small tumor load – this small focal lesion on my right thigh and perhaps some other cells but nothing macroscopically in my body—that the BRAF/MEK combo would likely kill off the remaining cancer cells. I started a year of BRAF/MEK in December 2016. Within 1-2 weeks of beginning therapy, the bump on my right leg disappeared.

November 2016 was the last time a scan showed any evidence of melanoma cells in my body. Beginning in February 2017, and annually for the past five years, my scans have been clean. Our best guess is that I am now cancer free. Regardless of what the future brings, I’ve had a fabulous 30 years since this began. No surprise, I’ve become quite an expert at the interface of the immune system and oncology. Perhaps my personal experience gives me a little extra drive to make sure that my oncology companies pick the right drug candidates and do the right clinical trials. For me, the impact of this field on patients is not abstract. I’ve experienced it first-hand.

Public Service

I have always been drawn to the interface between science and public policy, an area of growing importance, given the increasingly important role science and technology play in our lives, at the same time as the scientific literacy of so many politicians and citizens seems to be declining.

I became involved in public service in the late 1980s. I chaired the McKnight Neuroscience Scholars Award Committee and later became president of the McKnight Neuroscience Fund. I served on other review committees for universities, institutes, and disease foundations.

On the Berkeley campus, I cofounded the Biology Scholars Program (BSP) in 1992 with John Matsui and Caroline Kane. Our goal was to provide mentoring and an inclusive study environment and community for students from low-income, first-to-college, or black and indigenous people of color (BIPOC) backgrounds. The success of the program is clear: similar students not in BSP are four times more likely to leave biology.

The word must have gotten out that I took public service seriously. The day after I was elected to the NAS in 1995, a staffer invited me to become a member of the Board on Biology of the National Research Council (the public policy arm of the NAS). I joined the board, whose name was changed in 1997 to the Board on Life Sciences. I had a strong voice and worked to help drive decisions. In 2001, NAS President Bruce Alberts asked me to chair the Board on Life Sciences, which I did from 2001 to 2006.

We soon found ourselves in the national spotlight. Early in his presidency, George W. Bush stated his opposition to using human embryonic stem cells for research. Questions were raised about whether government funding should be used for stem-cell research. Members of the U.S. Senate asked us to convene a panel of experts to propose policy for the use of human embryonic stem cells in medicine and to help chart a path forward. We convened the panel and released two reports: “Stem Cells and the Future of Regenerative Medicine” and “Guidelines for Human Embryonic Stem Cell Research.”

In addition to embryonic stem cells, we were asked to address human cloning. In 1996, Ian Wilmut and colleagues at the Roslin Institute in Scotland reported on the cloning of Dolly the sheep. Questions were raised as to whether humans could—and should—be cloned using similar methods. The genie was out of the bottle. In 2002, Brigitte Boisselier, a Raelian and director of the biotech Clonaid, claimed to have cloned a human baby in Florida (likely a publicity stunt). Congress asked us to investigate whether that claim was true, and whether the scientific community and ethicists thought humans should be cloned. We published a report in 2002 on “Scientific and Medical Aspects of Human Reproductive Cloning.”

We also made an assessment of extraterrestrial life in the universe, reviewed concerns about animal biotechnology, examined the efficacy and safety of dietary supplements, proposed how to transform undergraduate biology education, studied how to counter agricultural bioterrorism, proposed policy on the responsibilities of authorship in the life sciences in terms of sharing of data and materials, did a number of commissioned studies on how to counter bioterrorism, oversaw a study on the reorganization of NIH, addressed the nation’s environmental challenges, proposed confinement methods to deal with genetically engineered organisms, reported on how to measure waterborne pathogens, and assessed the safety of genetically engineered foods. Over those five years, we accomplished a lot for the public good.

A few months after my tenure on the Board on Life Sciences ended in 2006, I began working with the California Council on Science and Technology (CCST), which serves the same role for California as the National Research Council (NRC) does for the federal government. In 2006, I was asked to chair CCST’s Innovation Task Force and was asked to consider the implications of the NRC’s 2005 study “Rising Above the Gathering Storm” from the perspective of the California economy and innovation network. The thesis was that the United States was losing its competitiveness and preeminence in science, technology, and innovation. Congress requested a report from the NRC on what to do about it. Shortly after the NRC study was released, California’s governor Arnold Schwarzenegger requested a parallel study from CCST focused on the California economy—which is the sixth largest economy in the world and based heavily on science and technology—and what actions state policymakers could take to create high-quality jobs in all areas of innovation. We published our report in 2007.

Shortly after our task force released its report in 2007, I was asked to join the CCST as a member. Seven years later in 2014, I was asked to chair CCST which I did for a three-year term from 2014 to 2017. Much of what the governor and legislature asked us to study concerned energy and water. For example, the California Natural Resources Agency asked us to assess the pros and cons concerning advanced well stimulation technologies in California, one major form of which is called hydraulic fracturing or “fracking.” We released our report in 2015.

Life Balance

In April 2000, Exelixis had gone public, and six months later, for the first time, Marcia and I were selling stock and in better financial shape than ever before. We increased our support of my parents. We gave more money away to causes we felt deeply about, such as the girl’s education program and children’s hospital in Cambodia. We also started discussing buying land in West Marin on which we could build a home, have a garden, and Marcia could have a horse. We were thinking perhaps 5–10 acres.

Then we fell in love with a large ranch that was 823 acres of rolling hills, deep ravines with creeks and riparian habitats, and beautiful views of Tomales Bay, the Point Reyes peninsula, and the Pacific Ocean. In recent years, it had been used only for cattle grazing and had no buildings, no roads, and no infrastructure. It was relatively affordable because the previous owner had sold off the development rights to the Marin Agricultural Land Trust (MALT), but still it was a big commitment for us. But they aren’t making any more California coast and this was a once-in-a-lifetime opportunity. In June 2001, we bought the ranch.

That purchase changed our lives. Marcia comes from a ranching family. Her father was Basque and grew up on a remote sheep ranch in southern Idaho. He went off to college and became an engineer for General Electric but stayed close to his Idaho ranching family. Marcia was very close with her father and her Idaho cousins.

Sheep ranching is in Marcia’s DNA, so it came as no surprise when she and her father told me their plan to convert our ranch from purely cattle grazing to sheep. Marcia subsequently decided, following in the tradition of some of her relatives back in the Basque region of Spain, that she would make a Basque-style sheep-milk cheese.

We built the infrastructure (roads, power, water, septic, fences), two barns, a creamery, and a home for our ranch manager, and Marcia began her sheep dairy and cheese business in 2009. We completed and moved into our beautiful rustic home in 2010. Today, we also have a chicken coop, a large greenhouse, and outdoor garden boxes, and Marcia has a horse that she rides many days a week. All the grazing on our ranch is low-impact and rotational. The pastures are certified organic. Part of our ranch is leased to a friend and fellow rancher for his organic grass-finished beef that is sold locally.

Seeking more balance in her life, Marcia retired from cheesemaking in 2016, after receiving one of the highest honors in American cheesemaking—first place in her category in the American Cheese Society awards. We still raise lambs for restaurants and individuals. Marcia has transitioned her flock from dairy sheep to fiber breeds (Romney, Corriedale, and Cormo) that produce delicious lambs and high-quality wool for knitters and spinners. We eat exceptionally well, feasting on our own lamb and pork, and enjoying the vegetables from our greenhouse and eggs from our chickens (see www.barinagaranch.com).

I increasingly work from the ranch. We have a home in San Francisco, and I spend a few days a week there, going into my venBio office, and work the rest of the time from the ranch. Our ranch house has a music studio, which is home for Nina (named after Nina Simone), my 106-year-old Steinway grand piano built in 1916. Playing piano is still a big part of my life.

Epilogue

In this essay, I’ve tried to convey the sense of fun and excitement I’ve experienced at every stage of my career. At each stage, I’ve tried to use my gifts as a scientist to innovate, whether it was in answering fundamental questions in basic science or applying great science to impactful medicine, and as a leader and communicator to inspire others. At my core, I am a scientist. Every chapter has been rewarding to me on its own and each has enriched my knowledge and given me a broader perspective. I certainly no longer have the math skills I did when I was a student. But what I’ve gained over these decades is the wisdom that comes from having viewed biomedical science from such a variety of different vantage points.

Mentorship played a major role in my development. My major mentor, Don Kennedy let me know he believed in me and gave me the confidence to aim high in my aspirations. I can only hope that throughout my career, I have had the same impact on some of the young scientists who I have had the pleasure of mentoring.

When I look back on my academic years, my lab was family. I was blessed with having such terrific and dedicated students, postdocs, and staff. Many of us established lifelong friendships, and I have enjoyed watching their careers blossom. Thus far, we’ve gotten together twice for reunions, once in June 2001 as I was leaving academia for biotech, and again in August 2014. I’ve enjoyed learning about their science and catching up on their lives. Some of them have blown my mind with their accomplishments. This was a passionate and talented group to whom I will always be grateful for how they enriched my life.

Tomas Hökfelt

BORN:

Stockholm, Sweden

June 29, 1940

EDUCATION:

Karolinska Institutet Stockholm, Medicine Candidate (1962)

Karolinska Institutet Stockholm, PhD (1971)

Fogarty Fellow, National Institutes of Health, Bethesda, MD (1981/1982)

APPOINTMENTS:

Docent, Histology, Karolinska Institutet (1969–1979)

Professor of Histology and Cell Biology, Karolinsk Institutet (1979–2016)

Associated member/member of the Nobel Committee for Physiology or Medicine (1981–1991, 1994–1996)

HONORS AND AWARDS:

Associate of Neuroscience Research Program (1977–1989)

Foreign Associate, National Academy of Sciences USA (1984)

Member of Swedish Royal Academy of Sciences (1985)

Member of Academia Europaea (1989)

Member, Accademia Nazionale Dei Lincei (1993)

Member, American Academy of Arts and Sciences (1996)

Member, Royal Danish Academy of Sciences and Letters (1996)

Foreign Member, Chinese Academy of Sciences (2000)

Honorary Member, Hungarian Academy of Sciences (2004)

Foreign Associate, Institute of Medicine/National Academy of Medicine USA (2006)

Foreign Member, The Norwegian Academy of Science and Letters (2021)

Doctor honoris causa at several universities

Hilda and Alfred Eriksson’s Prize, Royal Academy of Sciences, together with K. Fuxe (1979)

The Artois-Baillet Latour Health Prize, together with V. Mutt (1987)

The Bristol-Myers Award for Distinguished Achievement in Neuroscience Research, together with W. Nauta and T. P. S. Powell) (1988)

Anders Jahre’s Prize (1990)

The Eli Lilly Preclinical Research Award (1991)

Grand Medaille, Academie des Sciences (Paris) (2007)

Golden Kraepelin Medal (Munich) (2012)

The Arvid Carlsson Prize (2022)

Tomas Hökfelt is a histochemist with a focus on chemical signaling in the nervous and endocrine systems using a variety of approaches: the formaldehyde fluorescence (Falck-Hillarp) method for monoamines, autoradiography for amino acids, immunohistochemistry, and in situ hybridization. These techniques have been complemented with electrophysiology and standard molecular biology. He has explored the localization and role of neuropeptides and their coexistence with classic transmitters with special interest in pain and mood behavior, as well as neurodegeneration and systems important for control of food intake. Over many years, he studied rodents but has more recently analyzed human postmortem brains, also from depressed patients. A goal has been to search for novel strategies to treat human diseases.

Tomas Hökfelt

Prologue

The reason for me being invited to write this article can be spelled Nils-Åke Hillarp. He was recruited from Lund to our Histology Department at Karolinska Institutet (KI) in 1962, passed away in 1965, just 49 years old, but still changed my life and that of several others, as will be told. Of course, many other people have been important. This article is dedicated to all of the wonderful and talented PhD students (more than 20), postdoctoral fellows and guest scientists (altogether about 75), mentors, worldwide colleagues, and, last but not least, technicians and secretaries, with whom I have collaborated for nearly six decades. During some periods, my research group included more than a dozen members, often thanks to U.S. National Institutes of Health (NIH) and Canadian Medical Research Council (MRC) grants to support U.S. and Canadian postdocs. In later years, Chinese PhD students and postdoc fellows played a particularly important role. Through the years, many of the ideas and projects in the laboratory sprung out of my collaborators’ minds (motivation is strong when working on own ideas!). Also, antibodies have been the scientific oxygen in my professional life, and many of them have been generously supplied by colleagues around the globe. I will first go through my life and research chronologically, the first part describing the period before my Fogarty scholarship at NIH, Bethesda, USA, the second part covering research after returning to Sweden. I will present both interesting (to me) results as well as some interesting errors. The last part deals with special topics.

My Family and Early Life

My mother Sonja was born in Stockholm; her father died early. Her mother Olga was a wonderful (grand-)mother who supported herself and her two children by running a small cab company; she also had a garden with delicious raspberries. There was neither time nor financial resources for higher education. My father Paul’s family had a small farm in Småland, a poor region from where many Swedes emigrated to the United States. My father was born, however, in southern Lapland (Lapponia), high up in the North in the small village of Vinliden. The reason was that my grandfather, Gustav Karlsson, had become a missionary in Lapland, a region even poorer than Småland. During my military service in the North of Sweden in 1960, my father visited me, and we made a tour, including Vinliden. His birth house was still there but uninhabitable. We met an old lady who remembered the Karlsson family, and we were invited for coffee and buns. A very moving event.

The family moved back to their farm, called Hökargården (the origin of my family name), in Landsbro in Småland. My grandfather regularly alone went back to Lapland for further missions, whereby his return to the farm resulted in my father getting five siblings. As the eldest child, my father became responsible for the farm and only completed four years in school. As a young man, he moved to Stockholm and worked as an errand boy in his uncle’s trading company. In 1937 he married my mother. I was born in 1940, my brother Stefan in 1944, and my second brother Paul (Jr.) in 1953.

Choosing a career in business with several job changes and mostly living abroad, Stefan and Paul always asked at our fairly rare meetings what I was doing. Their somewhat compassionate reaction to my attempts to explain was always, “so just the same.” I attended school in Stockholm for six years. My real interest was sports—ice hockey, swimming, basketball, and more—but these activities stopped after our family moved to Germany. Here I started to play golf in 1955. Two years later, I was down to a 7 handicap. But then I hit a caddie: from a high tee, my 150 m shot made a perfect curve and the boll dropped right on the poor boy’s head (concussion but fast recovery), as close as I ever got to a hole-in-one. (An interesting example of the impact of “free fall”; a second one will follow.) After that, I never played a decent round of golf. A tough but I guess fair punishment. This said, I played many rounds of golf with my father up till his too early passing away.

Still, I had some use of my swimming: during my military service, I won the regiment championship in three disciplines. The lieutenant under whom I served was initially very much against my participation in this event. After my “victories,” he suddenly became amicable. My grandson Gustav, who was initially coached by my son Patrik, has taken over the competitive swimming baton. Gustav now holds some Swedish records in backstroke, thanks to a generous four-year stipend from George Washington University, Washington, D.C., where he greatly improved his skills and was appointed team captain of the GWU swimming team. In all fairness, my granddaughter Hedda is also great. She has studied digital cultures at Lund University and now works as a social media coordinator in a company manufacturing electric boats.

While raising a family in Stockholm, my father was employed by the Swedish National Association of Farmers (SNAF), trading agricultural products. In the early 1950s, he was offered a job by a large international company, Bunge and Born, based in Buenos Aires, as head of their Scandinavian and German operations. This precipitated our family’s move to Germany. After 10 years the family moved back to Sweden, with my father now serving as the CEO of SNAF (well done considering only four years of school).

Frankfurt am Main and Hamburg

In the autumn of 1953, our family landed in Frankfurt. This meant a dramatic change in lifestyle and economic resources; and a transfer from a clean and orderly Sweden, not harmed by war, to a Frankfurt badly damaged by bombs. Because I did not speak German, I was placed into a private school in a small village 10 miles outside of Frankfurt, with hens and ducks in the street, a bakery (wonderful bread!) in one corner of the inner yard, and a dung hill at the opposite corner.

My mother had always been ambitious on my behalf. Before moving to Germany, she insisted that I learn the German irregular verbs; so I studied and she examined. The first exam in the class was actually irregular German verbs. It turned out that German kids also had difficulties with them. Thanks to my mother, I wrote the best test, even though I could not speak a single sentence in German. Half a year later, I moved to a school in the city, Goethe Real Gymnasium. There was a shortage of teachers and lecture halls, and half the pupils occupied the rooms during the morning and the other half in the afternoon. A sick teacher meant cancelled lectures and free time. By that point, I was pretty fluent in German. To improve my language skills, my parents allowed my brother Stefan and me to go to the movies two or three times a week (foreign movies were dubbed into German). So I have heard them all—John Wayne, Richard Widmark, Cary Grant, and many more—“speaking German.”

In 1955, my father’s company moved to Hamburg, and five very pleasant years followed, after which I finished up with abitur in 1960. I very much enjoyed and appreciated attending the Ernst-Schlee-Gymnasium, a fine school with great teachers and class mates. Several teachers had served in the war with different types of experiences. Our Latin teacher “survived” because he was sitting in the trenches, by heart reciting the works of the great Latin poets. Another teacher, in physics, had a badly hurt leg, but he stays in my mind for another reason: his demonstration of gravity from the roof of the school, with a stopwatch in his right hand and a stone in the other became a memorable event: while pressing the stone, he dropped the watch (my second example of “free fall”).

Every second year, the class went for a week to a school home on the island of Föhr in the North Sea. We wandered over the wet land exposed to ebb and flow of the tides, a wonderful and unique experience. A special event was the personal washing procedure, which took place twice during the visit. We pupils stood in a long queue in front of a single outdoor shower with the gymnastics/sports teacher holding a stopwatch in his hand: shower for 60 seconds, then soaping, followed by another 60 seconds of rinsing. Very efficient, and cost effective. What we suspected was confirmed just a couple of years ago when visiting my old classmates in Hamburg: our teacher had been an SS officer during the war.

Return to Sweden

After abitur in Hamburg, I went back to Sweden for university studies. The move from Sweden in 1953 had been somewhat of a nightmare, losing my comerades, no more ice hockey, new language and much more, but I now realized that the seven years in Germany had indeed widened my horizons. I had experienced a people that had been hit hard by war and now was rebuilding their country. Travelling with my family I also early on got to know many other European countries and their customs and practices. Indeed, these years shaped my life.

The grading system for university access was different in Germany and Sweden, and my total points were not enough to get into medical school. So my mother (again) and I summarized my German curriculum and sent that to the University Chancellor in Sweden for evaluation. My first choice was to study medicine. One influence behind my choice was my uncle Bernt Hökfelt, who has been a bit of a hero for me. Bernt had a small sports car, which I always included, along with my uncle, in my evening prayer. In contrast to my father, Bernt had been able to go through all school classes, and after matriculation he was admitted to KI. Bernt made his thesis on noradrenaline (NA), adrenal gland, and more with Ulf von Euler in the Department of Physiology as supervisor; he then transferred to Karolinska Hospital. Here, Rolf Luft had created an excellent Department of Endocrinology and been appointed the first professor in endocrinology in Scandinavia. Together with Nils-Åke Hillarp my uncle published, in 1953, results that would end up in textbooks: there are separate adrenaline and NA cells in the adrenal medulla. My uncle later moved to Malmö to become the first professor of endocrinology at Lund University.

With the uncertainty of the value of my German degree in mind, I had also applied for other academic fields upon my return to Sweden, including at the Royal Technical High School (KTH). On a Monday in August 1960, I had an appointment with the professor in Technical Physics. When I opened the daily newspaper, I found my name on the list of students accepted to KI. I immediately cancelled my appointment at KTH, which was a big relief. In my official examination book, it is written that I had been admitted by a personal decision of the university chancellor. Thus, the German school curriculum was of a high quality, and the summary had been useful.

Karolinska Institutet: Medical Studies

In 1960, after three summer months of military service, I entered KI to start the class in anatomy/histology. The first examination was in histology. I was so nervous that the examiner, Professor Lars Gyllensten, asked me to take a walk and come back in 10 minutes. Already also an established novel writer, Lars Gyllensten had started has literary career with a prank: Gyllensten wrote together with Torgny Greitz, later chair of radiology at the Karolinska Hospital, under pseudonyms, a small, positively reviewed pamphlet. When exposed as a joke, the blame was initially on the critics, but the future would prove them right: Gyllensten became one of Sweden’s most famous authors and permanent secretary of the Swedish Academy, the body that awards the Nobel Prize in Literature, and later was named chair of the Nobel Foundation. In 1971, he stepped down as professor of histology at KI, a position that I eight years later successfully applied for.

In 1966, I had completed all medical courses, except one: oto-rhino-laryngology. I also had worked as a deputy physician for a couple of months in a mental hospital. In parallel, I had started modest experiments in the Histology Department and was involved in teaching. And, importantly, I had married Lil, and soon Patrik (1964) and Paula (1967) were born. This was economically a difficult time, and the very generous support provided by my father was essential for my possibility to early form a family and to pursue a research career.

Nils-Åke Hillarp

Hillarp was appointed professor in the Department of Histology at KI in 1962. He came from Lund University, where he had made several seminal contributions to neuroscience and endocrinology (see Hökfelt, 2010). A more detailed article on Hillarp has been written by Annica Dahlström (1996). Hillarp’s last project before moving to Stockholm was the development of a novel method: the formaldehyde fluorescence method (FAM), also known as the Falck-Hillarp technique, allowing demonstration of the monoamine transmitters dopamine (DA), NA, and, less efficiently, serotonin (5-hydoxytryptamine, 5-HT) in brain and peripheral tissues in a microscope—a revolution in neuroscience. Hillarp worked out the method in Lund together with his younger colleague and former student Bengt Falck, and with a team around Arvid Carlsson in Gothenburg (Falck et al., 1962); and they published the first study with the title “Cellular localization of brain monoamines” (Carlsson et al., 1992). A new field, “chemical neuroanatomy” was born.

The Amine Group and PhD Theses: Exciting Times

It is a special privilege for a young scientist to be lucky and work in a lab, where everybody feels that new knowledge is continuously generated, and to see things no one else has seen before. That is what happened in 1962 to the 10 students Hillarp recruited after his arrival at KI, the first ones being Kjell Fuxe and Annica Dahlström, followed by Torbjörn Malmfors (passed away 2022), Karl-Axel Norberg (passed away 2021), Gösta Jonsson (passed away 2019), Bertil Hamberger, Tomas Hökfelt, Lars Olson, Charlotte Sachs and, the last, Urban Ungerstedt. We all defended our thesis, but sadly we did so after Hillarp has passed away because of a malignant melanoma. Six of Hillarp’s initial 10 students advanced to become full professors. This was achieved despite limited resources, because Hillarp gave us great projects and excellent guidance, because of our hard work, and because the 10 of us stuck together and helped each other by forming the Amine Group (AG). The premature departure of our leader and supervisor, however, allowed us to flourish independently from early on.

When setting up FAM at KI, it was recognized that diffusion of the amines in the tissue was a critical issue. To that end, the samples were freeze-dried and reacted with para-formaldehyde (PFA) vapors. Moreover, the humidity of the PFA powder was monitored, and all steps up to embedding in paraffin were carried out in a climate-controlled room, a protocol developed by Bertil Hamberger, Torbjörn Malmfors, and Charlotte Sachs and reported in a one-page, key paper in 1964.

My own project under Hillarp’s supervision was to localize the monoamine transmitters at the ultrastructural level—that is, in the electron microscope (EM)—with special focus on the brain. I was given this task, because I had trained with Professor Ove Nilsson, an excellent electron microscopist. I defended my thesis in 1968, but was saddened that Hillarp never knew of my success at confirming the results he had predicted. (Hillarp always wrote protocols for his students, including number of animals, drugs, doses, times, and expected results, and the results he had proposed were always correct.) Being the last student joining Hillarp, his illness left him with very little time to spend on me. Also sadly, several years after Hillarp’s passing away I was told by another AG member that he during the daily coffee pause had looked around and asked for me. When noting my absence, he said “well, Tomas is apparently not that interested in research.” I was too busy perfusing rats in the basement and had no time to attend the coffee pause. So my advice to young scientists is this: don’t miss the coffee hour with your supervisor.

The background for my thesis project was that Keith Richardson, as well as Eduardo de Robertis and Amanda Pelligrino De Iraldi and several more had demonstrated that peripheral sympathetic, noradrenergic nerve terminals contain a special type of synaptic vesicles (SVs) (diameter about 500 Å), which have a reserpine-sensitive, electron-dense core, thus in all probability representing NA. In addition, there were large dense core vesicles (LDCVs) (diameter about 1,000 Å) that, especially after glutaraldehyde exposure, had a dense core of variable opaqueness. It had been suggested that these LDCVs may represent the amine storage sites in the anterior hypothalamus—that is, the brain (Pellegrino de Iraldi et al., 1963). Hillarp, however, was convinced that the bulk of monoamines in the brain was in the small SVs. Both Dr. De Robertis and Amanda visited the AG very early. I remember Dr. De Robertis sitting in the middle as we formed a circle around him, competing to impress him with our data, as he took notes. Amanda was the first ever foreign visitor who stayed for a longer time with the AG. We were so young and greatly impressed by this elegant and somewhat exotic lady from a far-away country.

SVs with a dense core, however, had not been demonstrated in the brain and, surprisingly, not in sympathetic nerves in rat iris. I worked hard with different fixatives (including 0.5% potassium permanganate, KMnO₄). I even asked Professor von Euler for advice, and he suggested that alkaline pH may retain the amine in the vesicles. So I used alkaline phosphate buffer and, indeed, it worked in the rat. My short paper on rat iris was just published in 1966, when everything changed for me. Keith Richardson in a short Nature paper showed that high (3%) concentrations of KMnO₄ allowed demonstration of SVs with a dense core in the rat iris (Richardson, 1966). This confirmed the idea that the amine rapidly diffuses out of the vesicle and that a super-fast/-strong fixation is needed. I immediately repeated Richardson’s results on the rat iris and expanded the analysis to the entire sympathetic neuron, and the brain.

The main results from analyses of KMnO₄ fixed tissues were a series of mostly single-authored papers published from 1966 to 1969 reporting on the following: (1) the dense core both in SVs and LDCVs reflects stored NA, based on in vivo and in vitro experiments conducted together with Gösta Jonsson, our methods expert (1966); (2) the dense core in the SVs in nerve endings in the rat iris disappears after preganglionic electrical stimulation combined with inhibition of tyrosine hydroxylase (TH) synthesis (and of course after reserpine) (1967), and here help from Torbjörn Malmfors and his technician was critical; (3) the first ultrastructural histochemical demonstration of a transmitter (monoamine) in classic SVs (and LDCVs) in the brain, in nerve terminals, and cell bodies in the locus coeruleus (LC) (Hökfelt, 1967) (previous evidence was based on subcellular fractionation studies by de Robertis’s and Victor Whittaker’s groups); (4) the presence of noradrenergic SVs in the cell body, dendrites, and axons of sympathetic neurons (i.e., in all parts of the neuron), providing morphological support, for example, for dendritic NA release (1969) (unfortunately, however, I did not suggest that mechanism); (5) the ultrastructural identification of DA nerve terminals in the striatum and that they represent 12–15% of all nerve endings (Hökfelt, 1968)—I incubated thin slices with alpha-methyl-NA, a compound that is resistant to breakdown, to increase intravesicular amine levels and thus facilitating detection, which was the slice technique I learned from Bertil Hamberger; (6) a lesion of the ascending nigro-striatal DA bundle reduced the number of nerve endings with SVs with a dense core in the striatum to a few percent, confirming their dopaminergic nature; together with Urban Ungerstedt (1969); (7) the presence of monoaminergic type 1 synapses in the hypothalamus, but not in association with DA nerve endings in the striatum, which served as evidence for nonsynaptic striatal DA release (Hökfelt, 1968; Torbjörn Malmfors scrutinized this big manuscript), and further, in the hypothalamus (periventricular zone) about 4% were monoaminergic and about 5% in the suprachiasmatic nucleus (mainly serotonin) (Hökfelt, 1968); and (8) the nerve endings of the tubero-infundibular DA (TIDA) neurons in the external layer of the median eminence (ME) abut the portal vessels, suggesting release into the blood (1967, cf. below). I also may add a paper not included in my thesis showing that a single nigral DA neuron can give rise to some 500,000 nerve endings in the striatum, an enormous divergence (Andén et al., 1966). This was later beautifully shown by Wakoto Matsuda, Takeshi Kaneko, and colleagues who in 2009, using a virus tracer, could visualize the entire axonal arborization of a single nigro-striatal DA neuron.

Taken together, my work showed that the monoamine synapses in most regions represent only a small population of all nerve endings in the brain, as also reported in parallel autoradiographic studies by George Aghajanian, Floyd Bloom, and others. I was able to fulfill the main task that Hillarp had given to me, that is, to demonstrate the ultrastructural localization of monoamines in the brain. Nils-Åke Hillarp not being around anymore, I had the privilege to visit Arvid Carlsson in Gothenburg to discuss my results and get advice. Moreover, I would not have been able to carry out my thesis work without Waldtraut Hiort, one of the many technicians at the Histology Department at the time. She had extensive experience in all details and steps involved in EM.

Studies Parallel to My Thesis Work

Selected Studies with FAM

Along with my first PhD student, Åke Ljungdahl I made some modifications of FAM, using the first ever Vibratome and formalin perfusion. This approach resulted in beautiful micrographs without the disturbing cracks seen in freeze-dried tissue after processing for FAM (Hökfelt and Ljungdahl, 1972). Thus, it is possible to retain amines at their storage sites after perfusion even with liquid formalin. This finding was appreciated by Olavi Eränkö in Helsinki, a pioneer in histochemistry, who actually before Falck and Hillarp had shown that liquid formalin fixation results in fluorescent NA cells in the adrenal medulla. How the Vibratome method was later combined with the glyoxylic acid (GA) reaction, a new method developed in Lund, has been described by Anders Björklund (see Volume 10). With this modified method, we for the first time visualized DA fibers in limbic cortical areas, including dense fiber patches in the entorhinal cortex (Hökfelt et al., 1974), confirming the pioneering biochemical work of Ann Marie Thierry, Jacques Glowinski, and colleagues, who showed, surprisingly, a cortical DA innervation (1973).

Early Neuroendocrine and Pharmacological Studies

Parallel to these studies, I started to work with Kjell Fuxe focusing on neuroendocrine and pharmacological experiments with FAM. To work with Kjell was for me a learning experience, like a pre-postdoctoral period with Kjell as supervisor, before having finished my thesis work.

The neuroendocrine studies focused on the role of monoamines in the control of hormone secretion from the anterior pituitary, research pioneered by Charles Sawyer and colleagues in the United States. Kjell had in 1963 reported an intense fluorescence in the external layer of the ME, the first evidence for involvement of DA in the brain control of pituitary hormones. Kjell suggested that I should help him in this project, and the first joint, short paper on these TIDA neurons was published in 1966.

Using semiquantitative fluorescence combined with TH synthesis inhibition, we monitored the rate of DA depletion in the ME as an indirect measure of activity and turnover of DA, using different models and drugs (estrous cycle, ovariectomy, hormones, DA agonists, and more) (Fuxe et al., 1967). Our results strongly suggested that DA inhibits the release of luteinizing hormone (LH), yet others had an opposite view. Thus, both at meetings and in reviews of our papers, we felt resistance (e.g., one reviewer said: “what’s the evidence that this green stuff in the median eminence is dopamine?”). But Kjell was fearless and argued against our famous opponents. Eventually Kjell’s PhD student Anders Löfström, in 1977, reported objective quantification of the intensity using microfluorimetry and could show that, in fact, DA turnover in the lateral palisade zone of the ME is low only at proestrus (i.e., preceding and allowing ovulation), confirming our subjective evaluations. Further support was given by Nira Ben-Jonathan, John Porter, and colleagues, who in 1977 reported that portal plasma DA levels were at the lowest levels during proestrus (i.e., less inhibition).

We speculated that DA released from nerve terminals in the ME, through axo-axonic contacts, inhibits luteinizing hormone-releasing hormone (LHRH). Interestingly, our immunohistochemical studies, several years later, in 1975, showed a distinct overlap between TH-positive(+) and LHRH⁺ nerve endings in the external layer of the ME.

I also proposed an alternative hypothesis involving structural changes: DA may expand the surface area occupied by tanycytes along the portal vessels, in this way preventing LHRH nerve endings from releasing their hormone (Hökfelt, 1973). This was indirectly supported by Björn Meister’s demonstration of the DA- and cyclic adenosine-3’:5’-monophospate (cAMP)-regulated phosphoprotein (DARPP-32) in tanycytes in close relation to both TH⁺ and LHRH⁺ nerve endings (Meister et al., 1988). DARPP-32 was discovered in the Paul Greengard laboratory and is known to be expressed in DAceptive neurons that possess D1 receptors. Taken together, we may have been correct in the view that DA inhibits LH secretion.

In 1968, J. H. van Maanen and Peter Smelik provided evidence that DA may also inhibit prolactin release, and in 1974, Robert MacLeod and Joyce Lehmeyer were able to provide final evidence for the view that DA, in fact and surprisingly, is the prolactin inhibitory factor (PIF) and thus directly acts on the lactotrophs. This was a major discovery that “cleared the air.” So PIF is the only principal anterior pituitary “controller” that is not a peptide. In addition, as mentioned earlier, my own demonstration of DA terminals directly abutting the portal vessels in the external layer supported that view. Kjell and I also pursued this issue with our CA-synthesis-inhibition approach and reported in 1969 that prolactin administration increases TIDA neuron activity during, for example, pregnancy and lactation, but, without going into detail, this time we barked up the wrong tree in terms of site of action. Thus, as we suggested, DA is a coordinator inhibiting both prolactin and LH release, but by different mechanisms and at different levels (pituitary vs. ME). My previous PhD student Christian Broberger has together with his postdoctoral fellows recently shown that the frequency of TIDA neuron oscillations is important for prolactin secretion.

Our neuroendocrine research took a new turn when Barry Everitt from Cambridge joined, introducing us to novel models, like sexual behavior, and adding important tools and perspectives to our work. Barry would come back as guest professor a second time and remain a collaborator (see the section “The Hypothalamic/Neuroendocrine Theme”). Barry became master of Downing College, Cambridge; a fellow of the Royal Society and Honorary Doctor at KI; and in 2020 and 2021, the first ever non–U.S. citizen president of SfN.

The collaboration with Kjell resulted in a long series of papers on the neuroendocrine role of hypothalamic monoamines and several reviews. At international meetings, often in Italy and sometimes organized by Luciano Martini and colleagues, we had the privilege to meet some of the pioneers in neuroendocrinology, from the United States, Charles H. “Tom” Sawyer, William “Fran” Ganong, Samuel “Don” McCann (of course, I never dared to address them by their nickname) and, from Europe, Geoffrey Harris, Claude Kordon, János Szentágothai, Béla Flerkó, Béla Halász, and many more. Later, after the discovery of the releasing factors by Roger Guillemin’s and Andrew Schally’s teams, my exploration of the neuroendocrine hypothalamus would continue (see the section “Neuropeptides and Coexisting Messenger Molecules”).

An exciting event for Kjell and me was Dr. Guillemin’s visit at KI shortly before the final discovery that thyrotropin-releasing hormone (TRH) is “just” a tripeptide. Dr. Guillemin, knowing that TRH is a small molecule, contacted Kjell and said he had seen his data on the DA system in the ME. He suspected that TRH could in fact be DA. We told him we could easily test that. So he came all the way to KI with a small tube containing TRH, and we put a drop on an object slide and reacted it with PFA vapors. Upon inspection in the fluorescence microscope, the droplet remained nonfluorescent. Thus, TRH was not DA.

During my career, I have made several blunders. One happened in relation to the Fifth International Congress of Endocrinology in Hamburg in 1976. I was invited to be a member of the Program Committee, representing the Nordic countries. It was a big honor, and I was certainly the youngest committee member by far. I contacted dozens of colleagues in the five Nordic countries, asking for names of potential speakers and collected an impressive list of names. I arrived at the hotel and presented myself to the concierge and told him about the committee meeting. But he couldn’t find my name. Finally, he returned, excusing himself and saying “die Herren sind schon gestern angekommen” (i.e., “the gentlemen [of the committee] already arrived yesterday”). I was one day late! I checked my plane ticket—yes, I should have traveled the day before, but the check-in also had overlooked the wrong date. When I entered the meeting room, I was met with disparaging looks, and I heard a senior German professor saying, “he could at least have excused himself.” Principally, the whole program had been decided the day before, without considering my proposals. When I returned to my room on the 10th floor of the hotel, I seriously considered jumping out of the window.

One more, less sad story, is also related to neuroendocrinology. Umberto Scapagnini studied medicine at the University of Catania, Sicily, and did postdocs in the United States, among other places in San Francisco, working with Dr. Ganong on the monoaminergic control of the stress axis. I met him at several conferences. After returning to Italy, he became full professor at the University of Catania and later moved into politics eventually becoming the mayor of Catania. In 2003, I was invited to give a lecture at that university. A young female scientist was appointed to take me on a tour through the city. We arrived at City Hall with a statue of an elephant outside, the symbol of the city. I mentioned to my young colleague that I actually know your mayor. The girl looked skeptical and said, “so let’s go and visit him.” I thought it was not a good idea, and we walked toward a big avenue. We heard music from a distance that grew stronger. A parade with many people was approaching headed by a big, live elephant and Umberto Scapagnini. Still surprised that I did what I did: I took some steps into the street and said, “Hi, Umberto.” He looked at me and gave me a big smile and warmly said, as only Italians can, “Tomas, Tomas, come here, join me.” So I ended up heading the parade through Catania, arm-in-arm with Umberto and after the elephant. To be honest, I felt a bit satisfied having provided convincing evidence that I did indeed know the mayor.

In a different set of pharmacological experiments, along with the Gothenburg group (Nils-Erik Andén, Hans Corrodi and colleagues), Kjell and I and other AG members analyzed the effect of several psychoactive drugs on the turnover of the monoamines using the TH synthesis inhibition approach. This strategy was based on Arvid Carlsson and Margit Lindquist’s 1963 pioneering paper strongly suggesting that chlorpromazine is a DA receptor blocker. The Gothenburg group used biochemistry, and we used the histochemistry protocol to look at the monoamine systems in the fluorescence microscope. We concluded, for example, that apomorphine activates receptors for DA (1967) and clonidine receptors for NA (1970), that LSD stimulates serotonin receptors (1970) and that ergot alkaloids stimulate DA receptors (1973). This latter finding would turn out to have implications for the treatment of Parkinson’s disease.

I defended my PhD thesis in December 1968. So what to do after that? I had planned to complete my medical studies, but the prospect to remain at the department did not seem good, even if Kjell Fuxe had been appointed professor in 1968. Thus, at least one AG member had a permanent position. The selection process for Kjell’s professorship was a drama. In the first round, Nils Ringerts, an eminent cell biologist and later secretary of the Nobel Committee, was listed first and Kjell second by the faculty committee. However, after Kjell’s formal complaint, the faculty changed the order. My aim was to become, like my Uncle Bernt, a clinical endocrinologist. However, I had also applied to attend a neuroscience course in Boulder, Colorado.

The Neuroscience Research Program: The Second Study Program

In 1962, leading U.S. scientists joined forces to promote interactions among researchers working on the nervous system, and they established the Neuroscience Research Program (NRP). It was sponsored by MIT and initially based in Boston and led by Dr. Francis O. Schmitt of Harvard University. One may say that this initiative represented the foundation of a new discipline: modern neuroscience. In 1969, the NRP announced the second Intensive Study Program in Boulder, Colorado. I applied and, to my surprise, was accepted. I arrived in Boulder on July 20, 1969, and together with some of the faculty and other students, watched the first landing of man on the Moon. An exciting start for my first visit to the United States. It was, of course, an unbelievable experience listening to wonderful lectures from scientists that I knew only by name (and some not at all). This firework of (neuro)science at its best made me finally choose basic research. That same year, I applied for a research position at the Swedish Medical Research Council and was successful, thus making it possible for me to pursue a career in research.

I would later, once more, be surprised by the NRP. In the mid-1970s, I was invited to become an associate of the NRP, which meant annual visits to Boston (and after 1981 to New York and Rockefeller University when Gerald Edelman took over as scientific chair) with the benefit of listening to brilliant scientists as well as to attend so-called work sessions organized on specific hot topics that were then summarized and published in the NRP Bulletin. In fact, I got the privilege of co-organizing such a session, together with Geoffrey Burnstock, in November 1978. Also participating were Michael Gershon, Leslie Iversen, Hans Kosterlitz, Joseph Szurszewski, Frederic Worden, and NRP Staff coordinator Key Dismukes. The topic was “nonadrenergic, noncholinergic autonomic transmission mechanisms,” which also included my presentation of neuropeptides and their coexistence with classic transmitter. The proceedings were swiftly published in the NRP Bulletin in 1979.

Amino Acid Neurotransmitters

In 1966, I attended the first major symposium of my life, “Structure and Function of Inhibitory Neuronal Mechanisms,” a Wenner-Gren Center International Symposium in Stockholm. Virtually all famous scientists in the field attended, and at that meeting glycine as transmitter was born, “delivered”, as it were, by Robert Werman and Morris Aprison. I was baffled by the tough critique expressed by some participants. This lecture and much discussion on GABA primed me for amino acid transmitters. At that time, however, there was no reliable histochemical technique available to demonstrate, in the microscope, which neurons utilize such transmitters.

The finding, in my thesis, that only about 15% of all nerve endings in the striatum were dopaminergic was surprising to me. To be honest, looking in the fluorescence microscope with FAM, it seemed that there were only DA fibers. This raised my interest in the identity of the remaining 85%. Leslie Iversen had published, in 1968, two papers with Michael Neal and Sol Snyder on uptake of tritiated GABA in synaptosomes. So why not try autoradiography, as already carried out with tritiated monoamines by several groups?

Åke Ljungdahl and I started autoradiographic studies after incubation of brain slices with ³H-GABA and direct isotope injections into the brain and spinal cord. At least half a dozen experiments failed over the first half year, but fortunately we followed Thomas Edison’s adage that “Our greatest weakness lies in giving up. The most certain way to succeed is always to try just one more time” (Thomas A. Edison). So, we decided on one more, final experiment (without having read Edison). Thanks God, this one was successful, and we could report the labeling of (the expected) GABA neurons in cerebellum, both Golgi type neurons in the granular layer, and basket cells in the molecular layer (Hökfelt and Ljungdahl, 1970). Parallel studies using a similar approach yielded corresponding results in both retina (Bernt Ehinger) and cerebral cortex (Floyd Bloom and Leslie Iversen). We also obtained positive results in the spinal cord by injecting either tritiated GABA, glycine or glutamate, although the latter mainly accumulated in glial cells.

In 1970, I was surprisingly invited to present these data at a high-profile electrophysiological meeting in Basel organized by Leo Hösli. All of the big names were there. I had prepared a dozen slides and was very nervous, and got even more so when the chair, David Curtis, announced my lecture saying, “you have two minutes.” I presented 10 slides but am not sure that the electrophysiologists understood anything, except perhaps John Eccles who worked with János Szentágothai and was interested and knowledgeable in anatomy. Floyd Bloom and Leslie Iversen understood, of course. Floyd presented, at the same meeting, the first data on the effect of cAMP on Purkinje cells. John Eccles, acting as chair (and a hero of mine), gave some positive comments on Floyd’s talk but still recommended not take these results “too seriously.”

Floyd Bloom and Leslie Iversen are both pioneers in neuroscience. They were close to us thematically, but we really never collaborated on a specific topic. Leslie, like us, moved early into substance P research. His focus was on the receptors, which he continued after assuming a position as director of a newly established Merck, Sharp, and Dohme (MSD) Research Centre in Terlings Park, United Kingdom. My wife Lil and I had the privilege to be invited to the festive inauguration of the center. The MSD big shots were flying in from the United States (flying the last leg in two parallel helicopters, in case one were to crash, I think someone told me). Our own flight was complicated by a last-minute strike in Sweden, making it necessary for us to fly from Copenhagen at 10 am. Our plan was to drive for five-plus hours from Stockholm to Halmstad, from where my father would take us to the Copenhagen Airport. Unfortunately, we overslept the alarm clock and woke up at 2 am instead of midnight. This time, we made the 500 km journey in 3.5 hours and arrived, thanks to my father, just in time to catch the plane to London.

Eventually, Åke and I decided to give up mapping GABA neurons with autoradiography. One reason was that Eugene Roberts had started to purify the GABA synthesizing enzyme, glutamic acid decarboxylase (GAD), for immunohistochemistry (IHC); and in 1973–1975, Roberts, Chung Wu, and their collaborators published pioneering papers on the distribution of GABA neurons with this method. This was in 1983 followed up by Jon Storm-Mathisen, Ole Petter Ottersen, and colleagues using antibodies to the transmitters GABA, glycine, and glutamate themselves. More recently, antibodies to the vesicular transporters for GABA and glutamate (VGLUT1-3), generated by several groups, have emerged as valuable tools for studying glutamatergic neurons in the microscope.

The Immunohistochemical Era (1969–present day)

In the present day (2020–2021), there are hardly any physical gatherings, only virtual video meetings, because of the Covid-19 pandemic. I keep wondering what my life would have been in a video-only world with no physical meetings? In fact, my scientific life changed because of the (live) Fourth International Congress on Pharmacology, which was held July 14–18, 1969, in Basel. This was the first time that I, together with Kjell Fuxe, met Menek Goldstein, professor at New York University School of Medicine. He told us that he had purified enzymes in the catecholamine (CA) synthesis pathway and made antibodies. He asked if we would be interested in a collaboration using the immunofluorescence method. Menek mentioned a then freshly published paper by Laurie Geffen, Bruce Livett, and Robert Rush (Geffen et al., 1969), reporting staining of chromaffin cells in the adrenal medulla and sympathetic neurons with antibodies to dopamine ß-hydroxylase (DBH). We gratefully accepted Menek’s offer, and I started immediately to set up the method with help from excellent technicians (including first Ann-Cathrin Swenson/Radesäter, and later Annie Nygårds, Katarina Åman, and others). Also, I soon realized that fluorescein-isothiocyanate (FITC)-labeled antibodies were commercially available in Stockholm, thanks to the work carried out at the Department of Microbiology headed by Professor Astrid Fagraeus. I got help and advice from her younger colleagues Peter Biberfeld and his wife Gunnel.

The Immunofluorescence Method/Immunohistochemistry

The immunofluorescence method already had been introduced by Albert Coons and collaborators in 1942 and was widely used in the field of microbiology and later also by the pioneer in protein tracing, Richard Nairn. The insight of a possible broader applicability started decades later with the purification of DBH by Stanley Friedman and Seymour Kaufman in 1965 and the generation of anti-DBH antibodies by James Gibb, Sydney Spector, and Sidney Udenfriend in 1967, followed by the previously mentioned paper by Geffen and collaborators. Why this delay of some three decades? One reason was perhaps the lack of suitable (pure) immunogens, so the purification of DBH was indeed a pioneering effort.

I have tried to find out how the Australian group started the immunofluorescence project and have been told the following story by Bruce Levitt and Robert Rush (and they have graciously allowed me to include their story in this article): Bruce had begun dating a girl named Barbara. He was aware that her father was head of pathology at Monash, but was not familiar with his research. At the time, pathology announced an introductory course on IHC. Bruce suggested to Laurie Geffen that they should both enroll, but Laurie replied, “No Bruce, you enroll!”

The next weekend, Barbara’s father appeared in the living room and asked Bruce, “What are you working on?”

Bruce answered, “purifying DBH together with Robert Rush in the Geffen/Austin laboratories in Physiology/Biochemistry to raise anti-DBH antibodies.”

After a thorough interrogation, Bruce getting more and more impressed and curious, the father suggested that Bruce should use their anti-DBH antibodies to localize the enzyme with the immunofluorescence method. Bruce agreed and informed him that he had already enrolled in the course to be run by pathology. Barbara’s father happened to be Richard Nairn, the noted pioneer in immunofluorescence protein tracing. So, choosing the right girlfriend seems important.

IHC meant an unprecedented advance in our ability to study the cellular and subcellular localization of proteins; and even smaller molecules, like peptides and even very small molecules like 5-HT, the latter pioneered by Harry Steinbusch, the late Albert Verhofstad, and Henk Joosten in the Netherlands (Steinbusch et al., 1978). There were at least two questions: Can one really make specific antibodies to such small molecules, and are 5-HT/amino acids retained in the tissue after perfusion with liquids? Albert had previously spent some time in our lab looking at the adrenal gland, his favorite tissue, with our antibodies to the CA-synthesizing enzymes. Not being familiar with the brain, he and Harry sent 5-HT antibodies to me in 1978 and asked for my opinion. Having seen the weak, and rapidly fading 5-HT-induced fluorescence in neurons visualized with FAM, I could not believe what I saw in the microscope: It was absolutely wonderful, a highlight in my “microscopy life.” Clearly, this was the way to visualize 5-HT neurons.

With “immunocytochemistry” as key word 733,134 hits appeared in PubMed on March 4, 2021, and 737,903 on May 27, 2021 (i.e., more than 50 publications per day). A useful method, to say the least.

Methodological Improvements

The initial publication by Geffen et al. (1969) was followed in 1970 by papers from Hartman and Udenfriend and our group. These studies suffered from suboptimal quality of illustrations, the main reason being the mild fixation used (acetone, chloroform, methanol, alcohol) to retain immunogenicity (Fuxe et al., 1971). Having worked with fixatives and transcardial perfusion for my electron microscopic studies, I started to test all possible fixatives and perfusion. The conclusion was that “ordinary formalin” gave excellent results without compromising immunogenicity (Goldstein et al., 1972; Hökfelt et al., 1973b). The 1972 immunohistochemical mapping of NA neurons by Boyd Hartman, Doris Zide, and Sidney Udenfriend using DBH antibodies also introduced an important piece to the puzzle: the addition of Triton X-100 to the diluted antibody not only “reduced nonspecific binding” but also enhanced antibody penetration, so that not only cell bodies but also terminals and dendritic processes could be visualized (Hökfelt et al., 1973a). The introduction of monoclonal antibodies to the field in 1979, pioneered by Claudio Cuello, represented a novel tool, also useful for double-labeling experiments. Subsequent method modifications—for example, using peroxidase labeling—were reported by Paul Nakane and Barry Pierce in 1966 and by Ludwig Sternberger and colleagues in 1970, as well as the tyramide signaling amplification method by Joe Adams in 1992. The latter offered higher sensitivity and allowed use of up to tenfold higher dilutions of the antisera. Some old “not-so-good” antisera became useful. Today, genetically modified mice expressing a certain tagged protein are often used for identifying neuronal (and glial) subpopulations.

Specificity Controls

Specificity of antibody labeling became more critical when expanding the method to fields outside microbiology. While studies on the CA-synthesizing enzymes did not present problems, this became an issue with the neuropeptides, that is, small molecules that had to be ligated to larger molecules like bovine serum albumin before immunization. Thus, the rabbit may generate antibodies also to the carrier protein. And it would turn out that there are families of structurally similar peptides, opening up for cross-reactivity. We acknowledged this in an early paper on substance P (Hökfelt et al., 1975b) by using the term substance P-like immunoreactivity, as suggested by my coauthor Göran Nilsson. This was good advice because, for example, several more tachykinins were subsequently identified. However, the real specificity problems arose when turning to the G protein-coupled receptors (GPCRs), where antibodies very often show false positives.

In those early days, specificity controls were based on testing cross-reactivity with different known peptides and, especially, preadsorption of the antibody with the immunogen. We noted that this was not enough, and more recently the introduction of peptide/receptor knock-out (KO) mice offers superior specificity controls. As does the possibility to compare with results from in situ hybridization (ISH) (localization of mRNA transcripts), especially if double-labeling could be achieved on the same section (IHC+ISH). Several colleagues have published on this problem.

In-House Problems (Our Errors)

In spite of our insight specificity being a problem, we (and others) have made some serious mistakes. For example, antibodies to the C-terminal part of cholecystokinin (CCK) cross-react with the calcitonin gene-related peptide (CGRP): these peptides share a C-terminally amidated phenylalanine, and a glycine in the 5th last amino acid position. Not much similarity, but still enough for cross-reactivity. Fortunately, we detected the error, and in a 1986 paper with Gong Ju as first author, we corrected our mistake.

Unfortunately, a second case was more serious: we reported in PNAS in 1983 that corticotropin-releasing factor (CRF) and peptide histidine-isoleucine (PHI) coexist in rat hypothalamus, allowing for some interesting speculations on functionality. In 1986, I received for review a manuscript from Neuroendocrinology with the title: “Colocalization of PHI- and CRF-immunoreactivity in neurons of the rat hypothalamus: a surprising artefact” by Fred Berkenbosch and colleagues. This was a shock, but eventually we managed to present an explanation (Hökfelt et al., 1987a). Coauthor Jan Fahrenkrug showed that the antiserum we used and believed to be specific for the N-terminal portion of PHI, in fact contained a subpopulation of C-terminally directed antibodies that also reacted with rat CRF: rat CRF and PHI share a common C-terminally amidated isoleucine. So only one amino acid is identical and was sufficient to cause cross-reactivity. Why did we not spot the problem in our control adsorption experiments? The answer: because we used for adsorption the ovine peptide that does not have a C-terminal leucine, and because at that time, only the ovine CRF peptide was available—the sequence of rat CRF was reported by Wylie Vale and co-authors six months after our 1983 publication. So, staining was not blocked with ovine CRF suggesting specificity. Had we waited until the rat CRF sequence was known, we would have avoided this serious mistake. As always, timing is important.

Remapping the Monoamine Systems: The Adrenaline Neurons

Using Menek Goldstein’s antibodies to the ‘monoamine enzymes’, we published a series of papers essentially confirming the Dahlström-Fuxe map of monoamine localization in the rat brain. (Dahlström and Fuxe, 1964). A member of the AG, passing by me sitting at the microscope looking at immunostained CA neurons, remarked “why are you doing this, everything has already been discovered?” But it turned out there was more to see. Of particular importance (for us) was the discovery of a third, albeit small, CA system based on the antibodies to phenyletholamine N-methyltransferase (PNMT), which allowed identification of two adrenaline (epinephrine) neuron groups, C1 and C2, in the medulla oblongata (Hökfelt et al., 1974).

In 1980, the Australian scientists Peter Howe, John Chalmers, and colleagues described a third adrenaline group, C3, and showed, with double FAM-immuno labeling, that the adrenaline cells do not show formaldehyde-induced fluorescence. In 1985, George Foster, a postdoctoral fellow from the United Kingdom, reported interesting differences during brain development: PNMT appeared before TH. We summarized the main findings of our work on the CA systems in a review article in 1984 in a special issue of Science on neuroscience edited by Solomon H. Snyder. Sol is a brilliant scientist who has made many fundamental discoveries (one of them would have been enough to make a great career), and who over decades has been very supportive, always generous, and interested.

The function of adrenaline system was difficult to define, but involvement in blood pressure control was explored by Kjell Fuxe and colleagues, and several other groups. In 1974, we suggested that C1 neurons represent a vasodepressor system, whereas other groups (Donald Reis, John Chalmers, Alan Sved, and Patrice Guyenet) provided evidence for a vasopressor system, involved in the baroreflex. It is now clear that our interpretation was wrong as discussed in Hökfelt (2010). In general terms, roles of the C1 adrenaline neurons include involvement in pain, hypoxia, blood loss, and more, and these neurons have fittingly been called the body’s “emergency medical technicians” (Gyenet et al., 2013).

Two major papers on the CA enzymes, the “crowning” of our work with Menek’s antibodies, were published in volume 2 of the Handbook of Chemical Anatomy series: Classical Transmitters in the CNS, Part I. This series was initiated by Anders Björklund after discussions with Elsevier. Anders then invited me to join as coeditor. I was delighted to accept, and we started with the first volume in 1983, the last one, number 21 on DA, was published in 2005. The series ran for two decades. Anders and I are deeply thankful to all colleagues who contributed with excellent chapters to the 21 volumes and to those who served as coeditors (Michael Kuhar, Larry Swanson, Christer Owman, Floris Wouterlood, Anthony van den Pol, Masaya Tohyama, Floyd Bloom, Rémi Quirion, Harry Steinbusch, Jan de Vente, Steven Vincent, Ole Petter Ottersen, Jon Storm-Mathisen, Leszek Kaczmarek, Harold Robertson, Stephen Dunnett, and Marina Bentivoglio). Volume 1 was on methods, and we were both grateful and proud when Professor Walle Nauta accepted to write the foreword.

The second volume, in 1984, then focused on the CA systems in the rat brain and included, for example, a detailed map of the DA system by Anders and Olle Lindvall, a comprehensive comparison of all four enzymes with focus on the adrenaline neurons (Hökfelt et al., 1984a), a complete map of the TH-positive (+) systems showing coronal sections from about 180 levels (Hökfelt et al., 1984b). For the latter study, I traveled to Lund with stained sections to work with Anders, a memorable and exciting experience. All photos (half or full coronal rat brain sections) were taken by Ragnar Mårtensson, a medical hospital photographer in Lund. Inspired by Anders, Ragnar had built a new microscope variant with a scanning condenser that allowed for seamless photos of half a rat brain section (instead of merging some 50 single photos to a montage). This method is also described in the same volume. This was a remarkable progress for that time. We were amazed and delighted with the outcome.

To work with Anders was a treat. As a PhD student, Anders had revived brain research in the Histology Department in Lund. After having made a thesis with a methodological angle, Anders then carried out, mainly with Olle Lindvall, outstanding histochemical studies on, especially, the DA systems in the rat brain, and thereafter became a pioneer in brain transplantation research, again together with Olle—the rest is history.

I also immensely enjoyed working with Menek Goldstein. He had, after being sheltered against the Holocaust in Poland, escaped to Switzerland, where he received his PhD in Bern in 1955. He then moved to the United States and worked at New York University School of Medicine from 1957 onward. Menek was a sweet and considerate human being. When Kjell and I visited the United States, Menek took care of us. For example, we attended the CA meeting in wonderful Asilomar in 1996. Menek arrived a day earlier and reserved the best room of the hotel facility for Kjell and me, a big room with an open fireplace. (We understood that Arvid Carlsson did not live in a similarly elegant room.) A lunch at the crowded deli on Second Avenue was imperative when visiting New York. With Menek as principal investigator (PI), I twice received support from NIH, which made much of our collaboration possible. Menek was awarded a doctor honoris causa at KI. He mostly lived alone and, in 1997, was found dead, sitting in the chair in front of his computer with an NIH application on the screen.

Neuropeptides and Coexisting Messenger Molecules (1974–present day)

Neuropeptides are the most diverse signaling system in the nervous system with more than 100 peptides and at least twice as many receptors. The immunohistochemical studies on the CA neurons might perhaps not have revealed so many new things, but this technique was our inroad to the peptidergic systems. At this point, Kjell Fuxe and I took different paths. That occurred quite naturally, also because Kjell had been joined by a brilliant Italian guest scientist, Luigi Agnati, who would become Kjell’s close collaborator for decades to come.

All of my PhD students and most postdocs worked on neuropeptides, with major foci being peripheral neurons, especially dorsal root ganglia, brain, and coexistence of neuropeptides and classic neurotransmitters. These systems offered a wealth of new information, simply because hardly anything was known about their anatomical or/cellular localization. For example, immunohistochemical analysis of the parvocellular hypothalamic hormones expressing the releasing and inhibitory factors discovered by Roger Guillemin’s and Andrew Schally’s teams, provided surprising information. They were, as expected, found in nerve endings in the external layer of the ME but, surprisingly, also in many other brain regions. For example, in 1975, we detected TRH in nerve terminals throughout the brainstem, and as caudally as around spinal motoneurons. Could they originate from the hypothalamic TRH neuronal cell bodies? They did not. We showed with Olle Johannsson and other colleagues in 1981 that TRH in the spinal ventral horn is produced by medullary neurons projecting to the spinal cord having 5-HT as the main principal transmitter. This wide brain distribution of the peptides was demonstrated, in parallel, using highly sensitive radioimmunoassays (RIA) (Michael Brownstein, Miklós Palkovits, Seymour Reichlin, Keith Porter, and associates). TRH was also found in peripheral tissues, such as pancreas and gastrointestinal tract, as shown by John Morley in 1979; in ß-cells as shown by Ivor Jackson and Ronald Lechan as well as Hitoshi Kawano and colleagues, both in 1984; and in enteroendocrine cells and nerves in the intestine, as shown by our Japanese postdoc Yoshihiro Tsuruo in a 1988 article.

Altogether these findings heralded what now appears to be a rule: neuropeptides are cogwheels in many large machineries. The same neuropeptide is used in many parts of the nervous and other systems. The functional effects of peptides then depend on the type of GPCRs and G proteins involved. The name given to a peptide following its discovery has in some cases turned out to be a “misnomer,” because it may reflect the tissue or organ from which the neuropeptide was first isolated, or the very first function associated with the peptide. Viktor Mutt, a pioneer in peptide research at KI and another hero of mine as well as of many more colleagues, discovered dozens of peptides. He recognized this problem and started to give “his” peptides neutral names—for example, galanin was named after its first (glycine) and last (alanine) amino acid.

Dorsal Root Ganglion Neurons and Pain

This project eventually would encompass a large number of collaborators. In the early phase, Zsuzsanna Wiesenfeld-Hallin and her team, including Xiao-Jun Xu, carried out the physiological experiments. Zsuzsanna was already an established and skillful pain physiologist using the flexor reflex as a pain model. I was fortunate and grateful to work with her over several decades, given that our collaboration gave meaning to our histochemical results.

Robert Elde was my second guest scientist, from the University of Minneapolis. We joined forces to study somatostatin (SST) in dorsal root ganglion (DRG) neurons, and he also raised antibodies to enkephalin. He later returned for a second period and made me aware of confocal microscopy, among many other things, and helped us to acquire perhaps the first microscope of this type in Sweden. Bob served as dean at the University of Minneapolis and was elected doctor honoris causa at KI.

Jan Lundberg had already started PhD studies with Annica Dahlström in Gothenburg. After a lecture in Gothenburg, Jan approached me and asked if he could join us. His arrival transformed our lab. He approached the work on peripheral systems with an enormous amount of energy and competence. It is an interesting question, where our group would have been, if I had not given that live lecture in Gothenburg. Jan had the gift of doing the right thing at the right time. His wife Ingeborg was expecting their first child, when the Swedish annual moose hunt started. Jan is from Värmland, a county where hunting is top priority, so this was really a difficult situation: attend birth or hunt? After much agony, Jan went back to Värmland, shot a moose, and returned home just in time to witness the birth of his daughter.

Strategic as always, half-way through his thesis work, Jan transferred to the Pharmacology Department to work with Anders Änggård and his cat salivary gland model, an ideal preparation to monitor the stimulus-induced release of coexisting neuropeptides. There, he early on became professor and successfully supervised some 20 PhD students over 15 years (approximately the same number as I have had over 40 years). He then joined the Astra drug company, eventually becoming Astra/AstraZeneca’s head of worldwide research, a position that he later assumed at Eli Lilly in Indianapolis. Jan and I share coauthorship of more than 100 papers (but only he owns a Ferrari).

Antibodies to peptides had been generated for RIAs, and many colleagues were prepared to share these reagents. The first antibodies that we tested were raised against SST and substance P. SST was discovered in 1973 in Roger Guillemin’s laboratory as the growth hormone release-inhibiting factor, but we received the antibody in 1974 from Dr. Akira Arimura, a close coworker of Dr. Andrew Schally, thanks to support by Rolf Luft.

The substance P antibody was a different story. It was raised at KI by Göran Nilsson working in Börje Uvnäs’s laboratory. The project was initiated after Susan Leeman, Michael Chang and Hugh Niall identified, in 1970/1971, substance P as an undecapeptide (11 amino acids), 40 years after its discovery by Ulf von Euler and John Gaddum in 1931, a true breakthrough. Bengt Pernow, who defended his thesis on substance P in 1953 with Ulf von Euler as supervisor, realized the opportunity. He and Börje Uvnäs, decided to send Göran to Rosalyn Yalow’s laboratory to learn RIA and how to generate antibodies. Yalow had together with Solomon Berson developed RIA in the 1950s, and she received the Nobel Prize for this achievement in 1977 (Berson passed away in 1972), together with Guillemin and Schally.

Completely ignorant of this background, I had a meeting with Göran in 1974, when he told me about the antibodies that he had generated for RIA. Would I be interested in testing them for IHC? Guess if I was! This was a very fortunate offer, but peptide antibodies turned out to be more complicated than those directed against the CA enzymes. Göran had immunized 10 rabbits that were boosted four times. I analyzed serum from all rabbits and from all blood drawn. The immune response of the rabbits was indeed variable: some rabbits did not respond at all, there was one winner and some were useful; and the strongest staining was mostly seen with the blood drawn after the third and fourth boost. I right away understood that differences in antibody quality are a fundamental factor and realized that there always may be a better antibody around the corner.

Substance P had been associated with sensory neurons and pain already in the 1950s, first by Fred Lembeck of the University of Graz in Austria. Thus, I first applied the antibody to sections of the spinal cord. I got help from Jan-Olof Kellerth, a student of Ragnar Granit, with surgery and perfusion of the cat spinal cord. The result was spectacular: the superficial layers were shining like a semi-solar eclipse outlining the superficial layers, suggesting that peptide was present in primary sensory afferents. A second highlight in my “fluorescence microscopy life.” In agreement, fibers were also found in the skin and in cell bodies in the sensory ganglia, that is, as expected from sensory neurons. Importantly, the substance P⁺ cell bodies constituted only some 15–20% of all neurons and were always of small size, both in cat and rat, suggesting a relation to pain sensation (Hökfelt et al., 1975b). A parallel paper was submitted to Brain Research, but having never worked on the spinal cord, I placed the ventral horn upward in the figures. The founder and editor in chief of this journal, Professor Konrad Akert of the Brain Institute in Zürich, politely asked if I could consider turning the photos 180 degrees with the dorsal horn appropriately oriented? And so I did, and suddenly I became a “spinal cord scientist.”

Together with Bob Elde, we demonstrated in 1976 that SST is also present in small DRG neurons, but in a population different from substance P. We were surprised to find that SST, an inhibitory transmitter, is expressed in DRG neurons at all. Were these neurons inhibiting pain? To help address this question, Bob generated antibodies to enkephalin using a peptide supplied by Lars Terenius. This was the start of my long and fruitful collaboration with Lars, a pioneer in the opiate field. The enkephalins are endogenous ligands for morphine receptors and were discovered by John Hughes, Hans Kosterlitz, and coworkers as first reported in a Nature paper in 1975 (for history, see the chapter by Huda Akil and Stanley J. Watson, Jr. in Volume 8). That Nature paper evoked enormous interest (and hope)—hundreds of labs started projects based on this discovery, including our group. With these antibodies, we could demonstrate the cellular localization of enkephalins. These peptides were not present in DRG neurons, but in spinal dorsal horn interneurons, and in many brain regions (Elde et al., 1976). In 1977, we reported that spinal enkephalin⁺ interneurons intermingled with substance P afferents, providing a morphological basis for endogenous pain defense. This result fitted nicely with the parallel demonstration by Thomas (Tom) Jessell and his coworkers that morphine/enkephalin inhibits substance P release. In fact, Leslie Iversen’s group in Cambridge along with Tom, Claudio Cuello, Piers Emson, and others were important early players in the substance P field, showing, among other things, for the first time that substance P is also present in human DRG neurons.

We explored many aspects of substance P expression. Stephen Brimijoin, a senior and eminent scientist based at the Mayo Clinic, wished to come to study axonal transport of substance P in human nerves, which he would arrange to provide. Long before Steve’s planned arrival in Sweden, I traveled to a meeting in the United States. So we decided that Steve should send the nerve(s) to the conference center. And, indeed, I received a thermos with ice and the tissue sample in a bottle. To ensure the tissue quality, I took the thermos to the hotel kitchen each day and asked the staff to fill it with ice. Once I returned to Sweden, I opened the thermos, still with ice, but no bottle. In the kitchen they had thrown out the bottle with all ice and added new ice. What an embarrassment! Things got much worse when Stephen arrived and I confessed to the mishap. He then pulled up his trousers and showed the scar caused by the removal of a piece from his own sural nerve.

Our work on peptides in DRG neurons was the beginning of our efforts to phenotype these neurons. We showed that they are not only of different sizes but also heterogeneous with regards to chemistry. Several years later, in 1982 and 1983, Susan Amara, Geoffrey Rosenfeld, and colleagues made a remarkable discovery: the calcitonin gene generates, by splicing, a second tissue-specific peptide only expressed in the nervous system: CGRP. This turned out to be the most abundant and arguably the most important peptide in DRG neurons. We demonstrated that CGRP is coexpressed with substance P and sensitizes a substance P-induced pain reaction (Wiesenfeld-Hallin et al., 1984), possibly via inhibition of a peptidase breakdown of substance P, as suggested in a 1985 paper together with Pierre Le Greves and LarsTerenius. The 1984 paper, published in a low-impact factor journal, is one of my most interesting publications: intrathecal injection of substance P alone at the lumbar level caused scratching for a few minutes (assumed to be a pain response), but CGRP alone did not induce any effect at all. Combining the two peptides, however, resulted in scratching for up to 30 minutes, a dramatic potentiation—and the first direct evidence that CGRP is associated with pain. More recent research found unexpected and important roles of the CGRP system in several peripheral systems, and the U.S. Food and Drug Administration (FDA) approved CGRP antibodies for the treatment of migraine, a major breakthrough both for patients and peptide research alike. This major advancement was reviewed by the two main players, Lars Edvinsson and Peter Goadsby, in 2019. Of note, Lars is a Swedish colleague working at Lund University.

Our work on neuropeptides in sensory neurons and dorsal horn had obvious clinical implications. Looming—then and now—over research on this topic was the issue of the generally inefficacious treatment of, especially, neuropathic pain. There had “always” been morphine, the gold standard, but with serious side effects and lower efficacy for neuropathic pain. Might the discovery of substance P and/or enkephalin(s) lead to new pain treatments? In 1975, James Henry and colleagues had shown that substance P excites spinal dorsal horn neurons supporting a role as a pain transmitter. Thus, a substance P antagonist could very well be a “pain killer.” Hopes were high, until Pfizer finally succeeded in developing a small-molecule substance P antagonist that passed the blood-brain barrier: it worked in rats but not in humans, a big disappointment. My personal explanation is that the DRG neurons are not only releasing substance P, but also CGRP and the excitatory amino acid glutamate (and even further transmitters). So, maybe it is not enough to just block substance P (neurokinin) receptors?

The Endocrine, Autonomic, and Gut Nervous Systems, and Coexistence

The autonomic nervous system is classically divided into two parts: sympathetic and para-sympathetic neurons using, respectively, NA and acetylcholine as neurotransmitter. In the mid-1970s, I started a collaboration with (the late) Lars-Gösta Elfvin, an anatomist who had trained with Fritiof Sjöstrand. This collaboration took me back from the sensory to the autonomic nervous system, which I had studied in my thesis. Elfvin’s work focused on autonomic ganglia and included cat and guinea pig, The original question was whether these ganglia have adrenaline (PNMT⁺) neurons (they did not). While working on this project, I did some unplanned experiments by applying peptide antisera on some sections of guinea pig sympathetic ganglia. It turned out that this species is wonderful for IHC—the antibodies cross-reacted and all stainings were much stronger than in rat. This work involved several PhD students, including Marianne Schultzberg, Jan Lundberg, Carl-Johan Dalsgaard, and the late Björn Lindh (the latter two were Elfvin’s PhD students). Marianne Schultzberg was a PhD student with Tamas Bartfai at Stockholm University, and Tamas contacted me about her when he was leaving for a postdoc with Paul Greengard at Yale in 1976. As described next, Marianne got a new project and completed her thesis in 1980.

In the endocrine system, we, in 1974/1975, found SST in cells of the islands of Langerhans, thus a new pancreatic hormone, and we also found SST is parafollicular cells in the thyroid gland and in endocrine cells in the stomach mucosa, as well as in nerves in lamina propria and the myenteric plexus (Hökfelt et al., 1975a). Similar results on the Langerhans islets were reported by Stephen Bloom/Julia Polak, Anthony Pearse, Lelio Orci, Paul Goldsmith/William Ganong, and collaborators. In 1979–1980 articles, in the adrenal medulla, we reported expression of the pentapeptide enkephalin in the chromaffin cells, also found in the human adrenal medulla—that is, a new putative hormone or regulator had been identified in addition to NA and adrenaline.

We then started to phenotype the autonomic nervous system and found, like in the DRGs of the sensory system, a distinct heterogeneity regarding chemical phenotype: (1) presence of SST in NAergic ganglion cells, the first example of coexistence of a peptide with a classical transmitter (Hökfelt et al., 1977); (2) in the same year, sensory substance P fibers in sympathetic ganglia, establishing a connection between sensory and autonomic functions; (3) in 1978, a preganglionic enkephalin innervation in sympathetic ganglia); and (4) subpopulations of nerve terminals/neurons in prevertebral ganglia defined by neuropeptides neuropeptide tyrosine (NPY, SST, and vasoactive intestinal polypeptide [VIP] as reviewed by Elfvin et al. [1993]).

A start of a major project was the observation by Jan Lundberg and colleagues that VIP is expressed in cholinergic neurons in cat sympathetic ganglia (1979), followed by functional data showing how acetylcholine and VIP cooperate in producing vasodilation (Lundberg et al., 1980). In a series of papers, we also defined the subcellular localization of neuropeptides vs. classic transmitter in these systems and provided strong evidence that peptide storage is exclusive to LDCVs (see Fried et al., 1985). Interestingly, as shown in my thesis, the LDCVs can store monoamines. But could they also store amino acid transmitters? Immunoelectron microscopy on the dorsal horn by Adalberto Merighi, a champion in ultrastructural IHC, working with Julia Polak and Dionysia Theodosis reported that GABA (1989) and glutamate (1991) can be detected only in the SVs (vs. CGRP only in LDCVs). If true, a distinct difference exists between classic transmitters like amino acids and monoamines, the latter being stored both in SVs and LDCVs (my thesis work).

Regarding the functional roles of coexistence, Jan and colleagues in 1982 presented evidence that NPY in sympathetic neurons primarily inhibits the release of NA—that is, it exerts a presynaptic effect (Lundberg et al., 1982). Jan summarized much of this work in a comprehensive, single-author article published in Pharmacological Reviews in 1996. In 1976, Geoffrey Burnstock wrote a visionary paper raising the transmitter coexistence and cotransmission issue, although his own work mainly focused on purines and classical neurotransmitters. Lily Yeh Jan and Yuh Nung Jan explored in 1979 cotransmission in elegant studies on bull frog ganglia, revealing involvement of an LHRH-like peptide. To be honest, transmitter coexistence had already been shown in studies on nonvertebrates (but not involving neuropeptides), for example, in 1974 by Michael Brownstein working in the Axelrod laboratory on Aplysia. Mike was one of Julius Axelrod’s many brilliant postdocs (also see the section “National Institute of Mental Health”)

Finally, Marianne Schultzberg went on to describe in detail the peptidergic innervation of the gut (Schultzberg et al., 1980), in parallel with Frank Sundler and associates in Lund and with Marcello Costa and John Furness and collaborators at Flinders in Australia, the latter complementing their beautiful histochemistry with elegant electrophysiological analyses.

The Peptidergic Brain and More Transmitter Coexistence

While these studies of the autonomic nervous system showed important principles, a main interest for me was the brain. After remapping the CA neurons, we generated complete rat brain mappings of three neuropeptides: substance P in 1978, first author Åke Ljungdahl (parallel to Claudio Cuello and Ichiro Kanazawa); SST in 1984, Olle Johansson first author (but James Finley, Peter Petruzs, and colleagues already had published an SST map in 1981); and galanin in 1986, Tor Melander first author (parallel to Gerhard Skofitsch and David Jacobowitz). We also published two neuropeptide receptor maps: the NPY Y1 receptor in 2002, with Jutta Kopp as first author (parallel to Janice Urban and colleagues) and NPY Y2 in 2006 with Davor Stanic as first author. We also paid special attention to the hypothalamus, a brain region that, as noted earlier, has been central to our efforts in the neuroendocrine field.

The substance P map is the first detailed map of a brain peptide, which I made together with Åke Ljungdahl, already mentioned and a clever young person with whom it was a treat to work. Åke went on to clinical work, becoming an expert in Parkinson’s disease. He sadly succumbed to lung cancer in 2003. We learned the brain together, sitting side by side at two microscopes with the König and Klippel Rat Brain Atlas in between. A short report of the substance P map was published in Science in 1975 (Hökfelt et al., 1975b), but it took us three more years to publish the full 82-page paper in Neuroscience (Ljungdahl et al., 1978). Something important happened in between: in the 1975, we reported only a few substance P⁺ cell bodies and wondered from where all the dense fiber plexuses originated? Could they come from sensory ganglia, similar to the belief held at one point that brain NA may originate from peripheral sympathetic ganglia present in nerves following the blood vessels?

The substance P cell body issue was resolved by intraventricular (i.vt.) application of colchicine, and in the 1978 paper, we observed more than 30 substance P⁺ cell groups in different brain regions, explaining the extensive fiber networks in most parts of the brain. In the same year, the first substance P pathway in the brain was reported by Claudio Cuello and colleagues. How did colchicine reveal substance P cell bodies? Annica Dahlström and George Kreutzberg of the Max Planck Institute in Munich had in 1968/1969 shown that this “microtuble-destroyer” blocks axonal transport of NA (Dahlström) and acetylcholine esterase (AChE) (Kreutzberg). Thus, the centrifugal transport of peptides produced in the cell bodies was inhibited, leading to accumulation in the soma. In fact, colchicine i.vt. became a standard for peptide mapping studies. But already in 1973, Julien Barry and colleagues in France used colchicine to detect hypothalamic LHRH neurons.

The use of the colchicine approach deserves discussion, since the method has been so widely used. Do the cells really produce or utilize the same peptide under normal conditions, or does colchicine induce synthesis of novel peptides? Is the detection dependent on only the inhibition of axonal transport? ISH could later contribute to this discussion: we can almost always see the peptide transcript in cells (without colchicine), where the peptide itself only appears after colchicine treatment. There are exceptions: DRG neurons are fairly unique in that colchicine is not needed to show peptides in cell bodies with IHC. There are also cases in which colchicine changes both peptide/enzyme and transcript levels, as shown by Roser Cortes and colleagues in 1990.

After the discovery of NA-SST coexistence in sympathetic neurons, we wondered if this was a unique situation and therefore looked for more examples. The presence of multiple substance P neurons in the medullary raphe nuclei and adjacent regions was strongly reminiscent of the 5-HT neurons described by Dahlström and Fuxe in 1964. Having tested the splendid Harry Steinbusch-Albert Verhofstad 5-HT antibody I asked if we could use their antibody. They agreed but I would first have to ask their chair, Professor Rudolf Nieuwenhuys, a pioneer in the field of comparative neuroanatomy. So, I traveled to Nijmegen and, after a lengthy discussion, I got permission to use the antibody. We reported in Neuroscience in 1978 that many medullary descending 5-HT neurons coexpress substance P. In the same year, this coexistence was also published by Victoria Chan-Palay, Gösta Jonsson, and Sanford Palay. In 1980, together with Lana Skirboll and Jens Rehfeld, we reported that many mesencephalic DA neurons coexpress cholecystokinin (CCK), findings that were subsequently followed up by Kim Seroogy, a postdoctoral fellow from the United States, who also discovered additional coexisting peptides in DA neurons. Thus, we became convinced that peptide-classical transmitter coexistence is real and, in fact, may be a rule. I was invited to write a review for Nature (Hökfelt et al., 1980). Not yet having realized the importance of this journal, I had to be reminded twice by the Nature editor before we prepared and submitted the manuscript.

We are often asked to present a list of our most important publications. This 1980 Nature paper would be very high on such a list, because it summarizes the early phase of neuropeptide research and peptide-classical transmitter coexistence, including Jan Lundberg’s functional and, in any case for us, fundamental studies. Notably, it made our group known also locally at KI. I had just been appointed professor, and now the senior and famous colleagues took notice of us and got an idea of what we were doing. I had never believed that a Nature paper could be so important. I therefore contacted Nature, 10 years later, and asked if I could write a 10-year perspective. In fact, I right away submitted a fairly complete draft, finishing up by saying (something like) “if monoamines were the transmitters of 1960s and GABA of the 1970s, we hoped that the 1980s would be the decade of the peptides, but then glutamate appeared and grasped the attention.” After several weeks the editor responded (again something like): “If now glutamate is so important, why don’t you write a review on that transmitter?” Fortunately, Eric Kandel, who insisted I should be sole author, showed mercy and supported an improved version (Bob Elde helped, too) for publication in Neuron (Hökfelt, 1991).

National Institute of Mental Health (1981–1982)

The 1970s and 1980s had been very productive, but the group had been small. My first student, Åke Ljungdahl, had defended his thesis in 1980, and I had two more ongoing PhD students. Also, Lana Skirboll, a postdoc from Yale, had made important contributions to establish the DA-CCK coexistence, findings that she followed up with electrophysiological studies published in 1981. She also established methods to combine IHC and retrograde tracing using fluorescent dyes (1983) introduced by Hans Kuypers and Marina Bentivoglio. Lana returned to the United States and joined NIH to run a lab but soon moved into science policy, eventually becoming director of, first, the Office of Science Policy of the National Institute of Mental Health (NIMH) and, then, of the NIH under the NIH director Harold Varmus, managing a range of important policy issues. Under NIH Director Elias Zerhouni, she helped to create the NIH Roadmap for Medical Research. She then moved to the private sector, to Sanofi. Lana and her husband Leonard were great hosts during our time at NIH.

At KI, Steven Vincent from the University of British Columbia (UBC) had arrived in 1981 for postdoctoral studies. During his stay Steve reported a wealth of interesting data, on peripheral and central dynorphin pathways (1982); the coexistence of two peptides in human cortex (1982); glutamate decarboxylase (GAD) in pancreatic ß-cells (1983); and, together with Lana Skirboll, in Science, a GABAergic hypothalamic projection to the neocortex (1983). Steve brought with him an NADPH-diaphorase staining protocol, which he called the “magic stain,” showing beautiful neurons in cortex. Later, after returning to UBC, Steve reported, in the Proceedings of the National Academy of Sciences in 1991 with Hope and colleagues, that this staining in fact showed nitric oxide synthase (NOS), the enzyme that had just been discovered by Ted Dawson, Sol Snyder, and colleagues.

In 1980, I received an invitation to visit the NIH in Bethesda, Maryland, as a Fogarty Scholar, and I felt it would be good for me to be exposed to a different research environment. The Fogarty International Center was established by the U.S. Congress in 1968 and named after Congressman John E. Fogarty, who during his almost three decades in Congress “was a champion for NIH and for the value of research.”

I had never done postdoc studies, and there was no tradition in the AG to undertake a postdoc challenge abroad. I was at this point 41 years old, a bit late for “postdoctoral training.” My host at NIMH was Michael Brownstein, who is incredibly bright and was chief of the Molecular Brain Laboratory, previously headed by Julius Axelrod, the 1977 Nobel laureate. The invitation came as a big surprise, but I did accept. We leased our house in Stockholm and arrived in Bethesda in September 1981. My technician, Gun Norell, had traveled to Bethesda before us, so that when I arrived the laboratory on the 10th floor of Building 10 was ready and outfitted with a cryostat and a Swedish flag in the window of the door. Thus, everyone knew where the Swedes were.

The first thing we had to do was buy a car. After signing the contract for an old, but good-looking Mercury and walking out of the door, I saw over my shoulder how several colleagues patted my salesman on the shoulder and congratulated him. It wasn’t long before I had to bring the car to the repair shop, and I understood why.

My goal at the NIH was to work with Mike Brownstein to set up what was, at that time, a novel histochemical technique, ISH, to map the transcripts for various neurotransmitter-related molecules. In 1981, only a single study related to messenger molecules had been published (by Roland Pochet, Jean Pasteels, and colleagues). As noted, my lab and Mike and his collaborators at NIH had in parallel been mapping the distribution of many transmitters including neuropeptides, we with IHC, and they using highly sensitive biochemical techniques and the elegant punch method developed by Miklós Palkovits. So now we were going to go for the transcripts. At that time, however, it was deemed necessary to have a plasmid for the molecule you wanted to investigate. There were only a few such plasmids available, one being for the enkephalin precursor, at Mount Sinai in New York. As time passed, however, it became obvious that we would not have access to any plasmid, and our original plans evaporated.

In spite of this setback, I was very busy setting up IHC in Bethesda. I started a collaboration with Lou Sokoloff who, together with Seymour Kety, had made a major contribution with the introduction of the 2-deoxy-glucose (2-DG) technique to monitor activity in brain sections, and whose work also created a basis for positron emission tomography (PET). One unresolved question raised by his work was as follows: where is the 2-DG localized at the cellular level? Cutting frozen tissue in a cryostat may cause diffusion/redistribution. With Carolyn Smith, Gun Norell, Lou Sokoloff, and colleagues, we worked out an autoradiographic method showing the cellular localization of 2-DG (1983). This method also could be combined with IHC (1984). Even if these papers have left few traces, it was an honor to collaborate with Lou Sokoloff, a wonderful and generous person, and a pioneer in neuroscience.

We Fogarty Scholars had simple offices on the top floor of the Stone House, which was certainly fine with me. In fact, it was a tremendous courtesy in view of the general shortage of space at the NIH. Mike Brownstein’s own office in Building 10 was a platform a meter above the stereotaxic instrument, and Mike had to climb up using a small ladder. This elevated floor was so close to the roof that Mike could not use a normal chair but had installed an old car chair without legs that allowed him to sit and work in his “office.” Dr. Axelrod still came in and had a desk in one corner. I felt it would have been good for some of the PhD students at KI to see this, particularly those who were not happy until they had their own office.

During my time at NIMH, there was a second Swedish Fogarty Fellow, Professor Lars Svennerholm from Gothenburg University, a neurologist and pioneer in the field of gangliosides. Lars was not happy with his office. He felt that a Fogarty Scholar should be housed in one of the nice rooms on the second floor. He approached several of the NIH directors and, in fact, he succeeded: after several months, the Fogarty Scholars moved down from the attic. But this was too late for Lars, he already had moved back to Gothenburg. To my biggest surprise, I got a wonderful office with two open fireplaces designated, I guess, for Lars.

Overall, I am forever grateful for the invitation to visit NIH as a Fogarty Scholar. The weekly presentations in the Stone House by heads of the various NIH branches were a treat, and the exposure to this unique, biggest-in-the-world research organization strongly influenced my professional life and way of thinking. I like to see my research life as before (small team) and after (eventually large group) NIH.

The visit to the United States and Bethesda was also a big adventure for the family. Our children, Patrik and Paula, were exposed to a very different school system. They liked it and performed well, even though the U.S. history curriculum was very complex, and Paula struggled with studying French from English. Patrik learned to enjoy football. When back in Sweden he, together with some friends, started a team (Danderyd’s Mean Machines) and eventually the first ever American football league in Sweden (still ongoing). Lil drove the kids to school. One day they were stopped by a big, impressive motorbike police officer outside the school: the car lacked the tax badge (I had it in my pocket and was, as pretty often, in Sweden). Paula was scared to death, also because Lil had just swept a coat over her nightgown, and Paula wondered if she really could enter the school building under these circumstances. But the officer was polite and understanding. Patrik got his driver’s license one year earlier than would have been possible in Sweden. Around Christmas we drove, with the Mercury, to New York (yes, the Mercury still made it, even if one window could not be fully closed) and embarked on a flight for a one-week vacation in the Bahamas—a highlight for the family.

Return to Sweden

We returned to Sweden in late spring 1982. It did not start well. I was told that I would not get any salary for the coming three summer months. This was because I had not worked during the preceding nine months. Since the summer months in those days were, in principle, holidays for academics, the summer salaries had to be “earned” during the previous nine months. Bengt Pernow, at that time president at KI, stepped in and solved the problem. But worse was to come. Paula, our daughter, had had several infections in Washington. Back home in Sweden, we spent some summer days on the southern West coast visiting my parents. Despite wonderful weather, Paula did not want to join us on the beach. I had to go back to NIH for one more month, and I needed to find out what was wrong before departing. In the late afternoon the day before departure to Washington, D.C., we went to the local hospital. After a blood test, the doctor told us that it did not look good, but he was not sure. Only a specialist at the university hospital in Lund/Malmö could give a correct diagnosis. I drove the 100 miles and dropped the sample in a box around midnight. Early the next morning, we learned that Paula had acute leukemia, and our lives changed. The trip to Washington was canceled and a horrible period started with initial visits to Karolinska Hospital. Was a transplantation possible? The first test said no, but after retesting it changed to a yes. The chemotherapy was efficient, and we were told that the relapse risk was 85%. We were recommended, and chose, transplantation. This initially was successful, but then a graft vs. host reaction set in with an enormous force, bleeding, and unbearable pain that ensued for weeks. In spite of the extensive and committed work from doctors and nurses, Paula passed away on March 31, the day before her 16th birthday. We wondered, and still do, whether we did the right thing to go for transplantation. We were deeply impressed and grateful for the heroic efforts of the doctors and nurses at KI Huddinge Hospital, and we were touched and truly comforted by the many letters of condolences from colleagues around the world. However, the loss of our daughter (sister) will always be on our mind, and there is not a single day when Lil and I do not think about her. We were comforted by having our son Patrik who later married Ulrika, and they gave us the two already mentioned grandchildren Gustav and Hedda.

Return to Research at KI

Even though my return to Sweden had started with a tragedy for our family, the coming two post-NIH decades saw an upswing of research. Tremendously talented and hard-working Swedish PhD students and postdocs from abroad joined my lab in increasing numbers, creating an exciting and (almost always) collegial and interactive atmosphere, just like in the Hillarp days. Together we generated a wealth of interesting results and published many and, hopefully, some rather important papers.

The first major project after my return centered on galanin in the brain, a peptide discovered in 1983 by Kazuhiko Tatemoto, Viktor Mutt, and colleagues at KI. One of the co-authors on the original paper was Åke Rökaeus in the Department of Pharmacology. He had continued the tradition of Göran Nilsson and raised antibodies against galanin, and now offered them to me for immunohistochemical analysis. I made a preliminary mapping, and we published a short paper with Åke Rökaeus as first author in 1984.

At this point Tor Melander came to my lab from the United States with experience in experimental brain research obtained in Joseph Well’s lab at the University of Vermont. Immediately after my return from my NIH period, the lab was fairly deserted. My daughter had just passed away, and so the mood was low. Tor’s arrival was a boost to my mood, and he wanted to continue the 2-DG studies I had begun at NIMH with Louis Sokoloff. However, eventually and fortunately, Tor agreed to do the full galanin mapping, and he did it virtually by himself (1986), in parallel with Skoftisch and Jacobowitz at NIMH (1985). Our main interest from Tor’s work was the coexistence of galanin with classical transmitters in many systems. What caught our special attention was the galanin expression in NAergic neurons in the LC and serotonergic neurons in the dorsal raphe nucleus (DRN) (Melander et al., 1986) as well as in cholinergic forebrain neurons (1985).

Many excellent researchers contributed to our studies on galanin in the brain, including Tamas Bartfai, a truly brilliant colleague with extensive experience from both academia and big pharma. After working at Roche in Basel, he was appointed professor in biochemistry at KI, but he decided to move to the Scripps Research Institute in La Jolla as chair of neuropharmacology. He and Ulo Langel became important players in the galanin field and created the first galanin receptor ligands that were used all over the world. Tamas and colleagues also made important contributions to the work on sensory neurons and pain, as will be described later. Tamas has throughout been a most valued adviser and inspiring colleague. My collaboration with Tamas has resulted in more than 40 publications.

Anders Björklund happened to visit our lab in 1984, and Tor Melander and I showed him some of Tor’s galanin stainings. “These are cholinergic forebrain neurons,” Anders said. I had never worked in this region of the brain, so this was new and exciting information for both Tor and me, especially since these neurons had been associated with Alzheimer’s disease (AD). Together with Tamas and Gilberto Fisone, a PhD student in Tamas’s lab and today chair of our department at KI, we showed that galanin inhibits acetylcholine (ACh) release in the rat (1987) and monkey (1991) (ventral) hippocampus. Marcelo Villar, a postdoc from Buenos Aires, took on an incredible amount of experimental work ranging from studies of peptide localization in DRG neurons and hypothalamus, and using diverse methods including hypophysectomy and brain lesions. After Marcelo returned to Buenos Aires, he soon became rector of the Austral University. He and Roser Cortes, from Barcelona, found that the cholinergic forebrain neurons, like the DRG neurons upregulate galanin synthesis after axonal lesions (Cortes et al., 1990). We hypothesized that in AD, degeneration starts in some nerve terminals (axonal/synaptic damage) initiating galanin upregulation and release from intact terminals, inhibiting acetylcholine (ACh) release and aggravating the disease. We proposed that a galanin antagonist may initially in the disease process have therapeutic effects by counteracting reduction of ACh levels.

This work on AD was continued by other groups, with studies of human brain tissue, in particular by Victoria Chan-Palay and by Elliott Mufson and their colleagues. They could not detect galanin in cholinergic forebrain neurons, which, without colchicine, was also the case in rats. (No one has used colchicine with the human brain.) However, and interestingly, Victoria discovered in 1988 a galaninergic hyperinnervation of cholinergic forebrain neurons. This was confirmed by Mufson’s group, who suggested that galanin exerts trophic effects. We felt that we were at a dead end, and Tamas Bartfai suggested that we should leave AD and focus on depression, in view of galanin’s coexistence in NA and 5-HT neurons. Kjell Fuxe, Luigi Agnati, and Sven Ove Ögren were already working on this hypothesis. Later, Sven Ove, an expert in animal behavior and pharmacology with a past as a successful drug developer at Astra/AstraZeneca, and I extensively collaborated on the galanin project.

Our focus for functional studies thus was directed toward galanin in LC and DRN. It was obvious that an electrophysiological approach might reveal interesting results. Eventually and thanks to Vincent Pieribone, a U.S. postdoc, electrophysiology was introduced in our laboratory and, in 1995, we were able to publish our first paper on LC (Pieribone et al., 1995). Sten Grillner, an outstanding electrophysiologist who headed the Nobel Institute of Neurophysiology at KI was the expert coauthor. What a big moment for our lab this was. When writing the manuscript, however, we discovered that the groups of Albert Dresse and Peter Illes had already shown that galanin inhibits the spontaneous firing of LC neurons. Our electrophysiological studies of galanin were then led by Zhi-Qing David Xu, my second Chinese PhD student, who defended his thesis on this project in 1997. In brief, using receptor selective ligands obtained from colleagues at AstraZeneca, we proposed involvement of inhibitory GalR1 autoreceptors activated by galanin released from dendrites in the LC. The work to support this hypothesis was carried out by Xiasong Ma and David (2001) and by Evelyne Vila-Porcile, Marc Landry, and colleagues (2009). Somato-dendritic release of peptides is an accepted concept thanks to the extensive work by Mike Ludwig, Gareth Length, and colleagues on oxytocin and vasopressin in the magnocellular nuclei in the hypothalamus. In 1998, David showed a similar effect of galanin on DRN 5-HT neurons. David stayed with me for almost two decades before returning to Beijing. We still collaborate.

Even if our focus was on GalR1, there was also evidence for involvement of GalR2 in depression-like behavior. Thus, Xiaoying Lu, Tamas Bartfai, and colleagues provided evidence that GalR2 mediates antidepressant effects (Lu et al., 2005). Some years later, we could together with Sven Ove Ögren confirm this view in a rat model using swim stress and ISH (Kuteeva et al., 2008). Taken together and overall, the two galanin receptors appear to have opposite effects, GalR1 being pro- and GalR2 being antidepressive. These results from animal experiments were the basis for our later studies of the human brain.

Clearly, it would be critical to understand the localization of the galanin receptors. Though many antibodies have been generated against these receptors, it remains unclear to this day whether any one of them is specific, even if some generate “convincing” stainings. The general specificity of GPCR antibodies has been tested by Martin Michel (Amsterdam) and of galanin receptor antibodies by Tamas Bartfai (Scripps) and Barbara Kofler (Salzburg) and their collaborators. False positive labeling occurred with many antibodies examined at that time.

We also have been working on NPY which, like galanin, is coexpressed in the LC as described in some detail by Vicky Holets, a U.S. postdoc (Holets et al., 1988). NPY was also discovered in the Mutt laboratory, in 1982. The NPY1 and NPY2 receptors are particularly abundant. In this case, we have been more successful with the receptor antibodies. The NPYR1 mapping by PhD student Jutta Kopp was a heroic enterprise. She first mapped the Y1 receptor using our in-house antibody and basically had finished the work. At this point, I received antibodies from John Walsh and Helen Wong at UCLA, where John had established an NIH-supported antibody core facility. They generously sent me many antibodies, among others a dozen raised against NPYR1. Two of these (here called #1 and #2) were particularly interesting. Scanning revealed that #1 and #2 showed extensive, but completely different staining patterns; #1 showed the few systems that Jutta had already discovered, but in addition the whole brain was “covered” by fluorescence, an enormous difference. So we decided to work with #1 and Jutta now had to redo everything. After exceptionally hard work, she produced a likely complete and absolutely wonderful map (Kopp et al., 2002).

Before manuscript submission there had been some highly relevant questions that made me very nervous: First, was the staining specific? To address this question, Jutta studied an NPYR1-KO mouse generously supplied by Thierry Pedrazzini in Lausanne. Second, is the NPYR1 distribution in mouse similar to what Jutta had seen in rat? Thus, Jutta had to make a fairly detailed pilot study of the mouse brain, and yes, the staining patterns for NPYR1 were similar in mouse and rat; and none of the #1-induced staining could be seen in the KO mouse. However, the wide staining with antibody #2 remained in the Y1 KO mouse—strongly suggesting a lack of specificity, false positive staining. Had we studied only antibody #2, without checking a KO mouse, we might have reported the wrong map. Jutta was then able to publish an 89-page paper in Neuroscience and defend her thesis.

Neuroscience is a journal that was started in the mid 1980s, with Professor David Smith of University of Oxford as founding editor and chief editor for many years to come. We published many of our most important papers in his journal. David, a brilliant scientist, was always helpful and constructive, and he generously allowed publication of Jutta’s very long Y1R paper, as well as our already mentioned and similarly long substance P mapping paper, and even foldouts for some micrograph montages.

The In Situ Hybridization Era (ongoing)

If IHC was our main tool from 1970 and onward, ISH became a second basic, complementary method some 15 years later. After my failure at NIMH it was Martin Schalling who took on the challenge to establish ISH in our group. He joined my group, even if he had to support himself by working as a psychiatrist. He managed to introduce a TH insert into a vector in the lab of Rolf Ohlsson in Umeå and proceeded to collect the data that resulted in our first ISH paper (Schalling et al., 1986). However, Martin realized that the method needed significant improvements and decided to make a tour in the United States, visiting a series of labs, including Michael Brownstein/Scott Young (who at that time had started up the oligo approach = no plasmid needed) at NIMH in Bethesda. Martin returned and developed a new protocol based on the discussions he had with Scott Young and others. The first papers using this protocol were published 1987 and 1988 (e.g., Schalling et al., 1988), whereby the help by the late Håkan Persson and his student Anders Ericsson was most valuable. Subsequently, Åke Dagerlind and Stefan Brené came on board to help develop and publish protocols in 1996.

Martin defended his thesis in 2002 and succeeded in obtaining a competitive European Molecular Biology Organization (EMBO) postdoctoral fellowship to work in David Housman’s laboratory at MIT, where he completely shifted gears and became a geneticist. After returning to Sweden, he established a large group working on the genetics of psychiatric disorders. Taken together, Martin and his coworkers’ contributions were essential for the survival and productivity of our lab—in the years to come, the ISH method was to be used in many studies. For the past year or so we have been using the RNAscope method, and we have complemented ISH with quantitative polymerase chain reaction (qPCR).

Continuation of the Studies of DRG Neurons and Pain

The failure of a substance P antagonist in treating pain in humans made us look for alternatives, and the two novel peptides, galanin and NPY, seemed interesting to us. Galanin is normally visible in only a few neurons in DRGs under normal conditions, as first shown by the Polak/Bloom and Skofitsch/Jacobowitz labs in 1986. Subsequently, we observed a dramatic upregulation (100-fold) of galanin, mainly in small/midsize DRG neurons after peripheral nerve injury (Hökfelt et al., 1987b). Of note, Shehab and Atkinson already in 1986 demonstrated increased expression of a peptide, VIP, in DRG neurons after nerve injury. After the dramatic upregulation of galanin, again published in a low-impact factor journal, I became convinced that galanin research should be a priority. Regarding NPY, this peptide shows even lower expression in normal DRGs but is also strongly upregulated after axotomy, mainly in large DRG neurons, as first demonstrated by Satoshi Wakisaka, Keith Kajander, and Gary Bennet in 1981.

In our continuing fruitful collaboration with Zsuzsanna Wiesenfeld-Hallin, the physiological experiments now focused mainly on galanin and NPY. Tamas Bartfai got rapidly involved in this work. Most collaborators were, however, guest scientists. Marcelo Villar extended the original finding by elegant studies reporting quantitative results on galanin and other peptide levels. The RIA analyses were carried out by Elvar Theodorsson and showed that, contrasting galanin, CGRP and substance P levels are decreased after nerve injury (Villar et al., 1989). Marc Landry, an excellent histochemist from Bordeaux, provided great methodological upgrades to our lab. When I asked Marc why he chose our lab for postdoctoral studies, he said “I listened to a lecture of yours in Paris and really liked your tie.” Val Verge, from Canada, an elegant lady, who introduced elegant and for us novel quantitative methods, also complemented our pain research with aspects on growth factors. Andrew Bean, from the United States, contributed to the development of methods as well as to cloning and work on growth factors. Jacquie Corness, a PhD student from Canada, took on the task to explore molecular mechanisms underlying the nerve injury-induced upregulation of galanin. With help from Peter Burbach, Utrecht University, she demonstrated an involvement of leukemia inhibitory factor (LIF) and NGF (1996). Thus, Jacquie followed up the pioneering studies of LIF and nerve injury by Yi Sun, Story Landis, and Richard Zigmond, which they had published in 1993. Nora Kerekes focused her PhD work on growth factors and DRG neurons using DRG cell cultures and electrophysiology. Importantly, together with Francoise Mennicken and Dajan O’Donnell from AstraZeneca Montreal and Russell Hill, Nora showed that a large proportion (about 75%) of DRG neurons express GalR2 and that galanin, through this receptor, increases excitability (Kerekes et al., 2003).

During this period, many researchers in our lab were Chinese. Ju Gong Ju was my first Chinese guest scientist. Expelled from the university due to the cultural revolution, he was now back as professor in Xi’an eager to catch up in research. LKB, a Swedish company, asked if I was willing to host Ju Gong. When I heard of his comparatively old age, I expressed doubt (today I feel ashamed, thinking of my own age when I write this). Fortunately, LKB convinced me, and Gong Ju, a lovely man, arrived in 1986. He became a real asset, carrying out all experimental work himself, and writing the manuscript, the first one published in 1987. Gong Ju also liked singing, and he even learned some Swedish songs.

Xu Zhang (Chang) and his wife Lan Bao followed. Chang was a student of Gong Ju and had previously learned EM from Björn Afzelius, another Swedish pioneer, at Stockholm University. (I was responsible for a PhD course in EM at KI for several years. The highlight of the course was a lecture by Björn and Arvid Maunsbach, professor of anatomy and another student of Fritof Sjöstrand, dealing with published errors in ultrastructural research. The students and I loved their presentation.) So Chang came to me to study peptides in DRG neurons at the ultrastructural level. He produced a massive amount of data, with the help of his wife, Lan Bao—her contribution was initially unbeknownst to me. His results provided evidence that galanin was upregulated in CGRP neurons, alike replacing CGRP in the LDCVs, and he outlined the peptidergic secretory pathway in DRG neurons in a beautiful 1995 article, and much more. Chang completed his thesis in 1994. We have more than 60 publications together. Chang and Bao returned to China, first to Ju Gong and the Fourth Military Medical University in Xi’an. Chang then received one of seven positions at the Chinese Academy of Sciences (CAS) in Shanghai aimed at recruiting young successful Chinese scientists working abroad, a project masterminded by Jian-Ping Wu and Mu-Ming Poo. (I was proud that someone from our laboratory was selected.) In Shanghai, both Chang and Lan Boa embarked on successful careers, Chang being elected academician of the CAS at a young age.

Other Chinese collaborators were, as mentioned, Zhi-Qing David Xu, who also worked on galanin in DRGs. Using ISH, he studied galanin during development and found prenatal expression not only in DRGs but also in the ear, eye, nose, and, surprisingly, during bone formation, suggesting a developmental role for this peptide (Xu et al., 1996). Ru-Rong Ji studied growth factors and neuropeptides after inflammation and the effect of antisense nucleotides on peptides and pain behavior. He then went on to work with Clifford Woolf at Harvard and is now professor at Duke and a leading pain researcher. Tiejun Sten Shi completed his thesis on regulation of galanin and NPY in different mononeuropathic pain models, including a quantitative analysis based on stereology of the effect of nerve injury on the actual number of DRG neurons in mice. Mice, in contrast to rats, exhibit a fairly pronounced and rapid loss of neurons (Shi et al., 2001). Sten served as the lead person for our pain research program for a long time. Mingdong Zhang joined later to work on calcium-binding proteins, and more. Hong-Xiang Ruby Liu was, like Ru-Rong Ji, a postdoctoral fellow from the laboratory of the legendary Professor Ji-Sheng Han in Beijing, well known for his fundamental studies on acupuncture.

The work by Marc Landry and Hong-Xiang then gave us the insight that two galanin receptors are involved: GalR1 on glutamatergic spinal interneurons is antinociceptive (Landry et al., 2006). By contrast, GalR2 on DRG neurons is pronociceptive (Liu et al., 2001). These findings suggest that pain alleviation could be achieved by galanin, or better a selective GalR1 agonist, given intrathecally (or by a compound passing the blood-brain-barrier), or alternatively by a GalR2 antagonist that is administered peripherally.

The main conclusion from our extensive work on galanin and pain is that we have defined a new endogenous antinociceptive system that protects against neuropathic pain, which is intrinsic to a population of DRG neurons. This system is activated by nerve injury and involves GalR1 and upregulation of, among other molecules, inhibitory galanin (and downregulation of the excitatory peptides substance P and CGRP in the same neuron subpopulation). Interestingly, after nerve injury, galanin has an initial pronociceptive phase that involves GalR2 and is part of the warning system. So the effect of galanin is biphasic, via two galanin receptors. We speculated that when the protective mechanism(s) is fatigued, neuropathic/chronic pain will arise. A second endogenous antinociceptive system had previously been recognized: dorsal horn interneurons expressing, for example, the enkephalins (opioid peptides) (their receptors also being targets for morphine). This system is activated by inflammatory pain as shown, for example, by Ronald Dubner and Mary Ann Ruda (1992) and Eberhard Weihe and colleagues (1994). Inflammatory pain upregulates expression of substance P and CGRP in DRG neurons.

Having often stressed the importance of studying human tissues, long after submission of this article, I got a major shock: Tavares-Ferreira, Price, and colleagues reported in a single-cell transcriptomic study that the only galanin receptor they could detect in human DRGs was GalR3 (Tavares-Ferreira et al., 2022). So I will have to rethink my view on how to treat pain in humans based on galanin receptors. My hope is that also in the human spinal cord GalR1 is present in dorsal horn interneurons, as in rat.

In animal experiments, intrathecal administration of galanin/GalR1 agonists does reduce pain, in accord with our hypothesis. However, peripheral injection of GalR2 antagonists did not show pain relief (unpublished work by Tiejun Sten Shi and Mingdong Zhang). We have tried to explain this as follows: GalR2 stimulation results in activation of phospholipase Cß (PLCß), which increases intracellular Ca²⁺, resulting in increased glutamate release and pain. After blockade of GalR2 alone, pain persists, possibly because DRG neurons also express several other receptor types, that are parallelly upregulated after nerve injury and equally activate PLCß. One example is cholecystokinin B (CCKB) receptors, as shown by Xu Zhang and colleagues in 1993. To reduce pain, it therefore would be necessary to block GalR2 and several other receptors at the same time. An alternative could be to directly inhibit PLCß, that is, the common intracellular target for these receptors, including GalR2. Preliminary results published by Shi and colleagues in 2008 (PNAS) suggest that this may be a plausible idea.

Parallel analysis of the involvement of the NPY system in pain has revealed many similarities with galanin, as reviewed by Pablo Brumovsky (Brumovsky et al., 2007) and more recently by Tyler Nelson and Brad Taylor, in 2021. Detailed information on localization of NPY-Y1 and -Y2 receptors in DRGs and spinal cord was first reported by Xu Zhang and later by Pablo Brumovsky and colleagues, in 1999 and 2005, respectively.

At this point, an important question was whether these galaninergic mechanisms operate in humans. Zhang Xu showed upregulation of galanin in monkey DRGs after nerve injury as well as regulation of other peptides (Zhang et al., 1993). Thanks to Zsuzsanna Wiesenfelt-Hallin’s contact with two Canadian colleagues (Jonathan Dostrovsky and Andres Lozano, Toronto) and their generosity, we got access to human DRGs (unlesioned [donated] or brachial plexus injury). Thankfully, Marc Landry and colleagues showed robust galanin expression in normal human DRGs, and possibly an increase of expression after brachial plexus injury, reported in 2003. Taken together, these results suggested that similar galaninergic mechanisms may operate in rodents and primates/humans, which supported our conviction that the galanin system is an important target for developing drugs for the treatment of pain. A big relief.

The Hypothalamic/Neuroendocrine Theme

Neuroendocrinology has its roots in neurosecretion—that is, the release of messenger molecules from neurons into blood, and therefore hormones. Pioneered by great scientists like Ernst and Berta Scharrer, Wolfgang Bargmann, and many more, early studies were focused on the magnocellular hypothalamic neurons in the paraventricular (PVN) and supraoptic (SON) nuclei. The classic book by Geoffrey Harris entitled Neural Control of the Pituitary Gland, published in 1955, may represent the start of modern neuroendocrinology. This was followed by the work of Roger Guillemin and Andrew Schally and their colleagues reporting the structure of several such “controlling” molecules—that is, the releasing and inhibitory hormones.

The fact that the hormones discovered by Guillemin and Schally’s teams were peptides made it possible to study them by IHC. Thus, we initiated a second wave of neuroendocrinology at KI, in which my PhD students together with guest scientists made important contributions. Björn Meister focused on the many peptides in neurons in the arcuate nucleus, partly working with Barry Everitt, and together they in 1986 published a comprehensive review on this tiny nucleus. Sandra Ceccatelli studied nitric oxide synthase, growth factors, and peptides in the PVN, partly together with Björn and Marcelo Villar. Sandra, after a short postdoc period with Donald Pfaff at Rockefeller, came back to KI to join Sten Orrenius in the Department of Environmental Medicine. She later returned to our Neuroscience Department to become our first female chair.

In 1995, Martin Schalling imported a new mouse line from the Jackson Laboratories to KI: the anx/anx mouse that lost weight and survived for only three weeks or so. He generously shared this “anorectic” project with us, initiating research related to food intake. This resulted not only in Christian Broberger’s thesis focusing on the arcuate nucleus (Broberger et al., 1997) and the lateral hypothalamus, but also two further theses with Martin as main supervisor for Ida Nilsson and Jeanette Johansen who explored possible mechanisms underlying the anx/anx phenotype.

Christian took a postdoctoral position with David McCormick at Yale and learned electrophysiology, a method that he then successfully combined with histochemistry, resulting in a novel understanding of synaptic signaling in the arcuate nucleus and publications in high-profile journals (and a professorship at Stockholm University).

For over three decades, our lab has had a fruitful collaboration with Laura Calza’s team in Bologna, working on a palette of projects mainly associated with hormones, in particular the thyroid axis, and neuropeptides, especially galanin, both at the hypothalamic and spinal level, as well as using tissue cultures. Laura’s lab has many skills that we lack, and it has been a privilege to interact with her and her team.

We have had a long-standing collaboration with the Endocrinology Department headed by Rolf Luft, who initiated our work on SST. With Anna-Lena Hulting, we have analyzed pituitary tumors, and the effect of leukotrienes on the release of pituitary hormones (in collaboration with Bengt Samuelsson).

In 2016, Tibor Harkany moved to our department to work on hypothalamus and neuropeptides. Tibor’s efforts represent a wonderful continuation of the neuroendocrine tradition at KI, using, for example, single-cell analysis of hypothalamic neurons (Romanov et al., 2017), perhaps one could say a third wave, now based on modern molecular–biological methods. Tibor is a stellar scientist with an extraordinarily wide competence and great ability to win big grants. His move was an enormous boost for the Neuroscience Department, and for me personally, in terms of intellectual (and economic) support, including recruiting (and paying for) PhD and postdoc students (Joanne Bakker, Martino Caramia) for our group. In 2015, Tibor accepted a generous offer to move to the Medical University of Vienna, retaining 20% involvement at KI.

Human Postmortem Tissues (2010–present day)

Having carried out animal experiments for many decades and trying to relate our results to illnesses like pain and mood disorders, I every now and then contemplated the relevance of animal experiments. So some 10 years ago I started a project with the question: how similar is the chemical makeup of rodent vs. human nervous tissues? Are results from animal studies relevant when embarking on the (very costly) development of drugs for human clinical treatment? I approached Miklós (Miki) Palkovits on this topic. Miki had established the Human Brain Bank in Budapest, and I have long enjoyed a professional relationship with him, since my time at NIMH. I know Miki to be a master of brain anatomy and brain dissection, and a true scholar.

Together with postdoc Erwan LeMaitre and colleague Rochellys Diaz-Heijtz and, later, PhD student Swapnali Barde, we embarked on the analysis of galanin and its three receptors primarily in two human brain regions: LC and the DRN, using ISH and qPCR (Le Maitre et al., 2013). Importantly, these are big tissue samples where the NA and 5-HT neurons represent only a small population of all neurons. We found strong galanin expression in NAergic LC neurons (like in rat and mouse), and in the DRN region, but not in 5-HT neurons (as in rat, but not mouse). Regarding receptor distributions, we need more exactly defined, microdissected regions or even single-cells studies to gain a clear picture of their localization (under way).

The next project concerned five human male and female brain areas from controls and subjects who had committed suicide (Barde et al., 2016). The samples were obtained from Naguib Mechawar, who heads the brain bank at the Douglas Hospital (McGill) in Montreal: LC, DRN, the medullary raphe region, and two prefrontal cortex (PFC) regions were studied using RIA, qPCR, and bisulfite pyrosequencing (for DNA methylation). The results provided unique information on the distribution of the transcript and peptide, and on differences in their expression patterns in depressed vs. control brains. For example, transcript levels for galanin and GalR3 were higher in depressed DRN and LC than in controls, and in all cases, these increases were paralleled by a decrease in methylation (i.e., evidence for epigenetic mechanisms).

We had, together with a dozen research groups, obtained a European grant (acronym NEWMOOD) to explore various aspects of depression, headed by the leading psychiatrist William (Bill) Deakin of University of Manchester, and with Hungarian colleagues, including György Bagdy and Gabriella Juhász. Here a candidate gene study revealed that variants in genes for galanin and its three receptors confer increased risk of depression and anxiety in people who experienced childhood adversity or recent negative life events (Juhasz et al., 2014). Together, the two studies suggest that the galanin system may be involved in major depression disorder.

With Mathias Uhlén we published an RNAseq study in Science (2020) comparing the mouse, pig, and human brains with Evelina Sjöstedt as first author. Most recently, we have analyzed, with RNAseq and RNAscope, 17 regions of the human prefrontal cortex with a focus on neuropeptide systems, including galanin, where Wen Zhong, a biostatistician, has made a terrific contribution (submitted). By integrating RNA-seq, ISH, and single-cell data we could propose, for example, microcircuits involving galanin and galanin receptors in glutamtergic pyramidal neurons. Mathias is a great scientist and entrepreneur alike, and the Swedish scientist who by far has received the biggest grants in Sweden, by the Knut and Alice Wallenberg Foundation. With part of this money, Mathias funded the Science for Life Laboratory, a unique resource and core facility for Swedish research now housing some 800 scientists offering support, for example, for RNAseq studies and genetic analyses.

Taken together, results from our lab and others, and from both animal and our human studies, have provided a coherent, hypothetical view of the involvement of galanin in mood regulation: Galanin synthesized in LC neurons is present in noradrenergic nerve terminals in cortex but also locally in their dendrites. Stress leads to increased firing of LC neurons and upregulation of galanin synthesis. If firing becomes excessive, galanin will be released from dendrites and activate inhibitory GalR3/GalR1 autoreceptors to prevent overexcitation. We and others have proposed that this is part of the resilience machinery (Weinschenker and Holmes, 2016; Hökfelt et al., 2018). Exhaustion of this protective mechanism may result in depression. Interestingly, like in the pain models, the two galanin receptors may have opposite effects.

The just-mentioned analysis of human PFC suggests that galanin also plays a role in the anterior cingulate cortex, in this case coexisting as an antagonist in glutamatergic pyramidal neurons. Here, galanin may surveil and suppress the excitability in this circuit. This is interesting in relation to the recent successful treatment of depression with ketamine, a noncompetitive NMDA antagonist. Thus, John Krystal and colleagues (Krystal et al., 2019) have proposed “a paradigm shift for depression research and treatment”—that is, from the old monoamine hypothesis to involvement of glutamatergic mechanisms. Maybe we can maintain an interest in both?

Some Reflections on Progress in the Neuropeptide Field

The discovery of an increasing number of neuropeptides accelerating from the 1960s raised interest and hope in this new type of signaling molecules, amplified by the discovery of corresponding receptors. Eventually, it was realized that the peptides and their receptors represent the most diverse of all transmitter systems (>100). Important insights include the fact that peptides coexist with classic transmitters and thus are auxiliary messengers, which are primarily released when neurons fire at high rates or burst fire. This explains the early observation that peptide KO mice do not necessarily present a phenotype different from wild types. Findings suggest that neuropeptides are essential only under certain circumstances, such as stress, injury, seizure, and addiction (Hökfelt, 1991). From this growing body of evidence, thoughts about developing new therapeutic strategies emerged. These included, for example, the use of substance P antagonists for treatment of pain, and use of antagonists to CRH for relief of stress. Although CRH receptor antagonists that pass the blood-brain barrier have been developed, they, and many antagonists at other receptors, often elicit serious side effects, like liver toxicity. Merck’s failure with a neurokinin 1 antagonist for treatment of depression was a major setback for the whole field. But there are some successes with, for example, CGRP antibodies/antagonists to treat migraine, orexin/hypopcretin antagonists for insomnia, and substance P antagonists for chemotherapy-induced nausea. Our own hope for the treatment of pain and depression based on galaninergic mechanisms has not materialized. But we have not given up. Nonetheless, the development of pharmacological tools for influencing peptide signaling still has limitations compared with the monoamine systems, in which each metabolic and signaling step is known, offering a multitude of targets.

Other Projects

We have over three or so decades also pursued several other lines of research, as listed next.

Neuropeptides in Glia

It had long been known that neuropeptides are expressed in glia. Our own work with Laura Calza, Ruud Ubink, and Norbert Halasz focused on galanin and NPY, and we have reviewed this topic (Ubink et al., 2003).

Basal Ganglia

Together with Mario Herrera-Marschitz (Mario is a wonderful scientist without initial resistance; “let’s do it” and we did) and Urban Ungerstedt, using Urban’s pioneering microdialysis method, we focused on peptides in basal ganglia, involving many postdocs: Marie-Noëlle Castel, Patrizia Morino, Rafael Rodriguez-Puertas, Zhi-Bing You, and Sonia Gomez-Urquijo.

Adrenergic Receptors

Anthony Nicholas, a postdoc from the United States, initiated a major study on adrenergic receptors based on ISH. Together with Vincent Pieribone, he explored the distribution of alpha-1, alpha-2, beta-1, and beta-2 adrenergic receptor mRNAs in the rat brain. We have summarized the results in a review (Nicholas et al., 1996).

Neurodegeneration

Thanks to the generosity of U.S. and Swiss colleagues, including Dr. Stanley Prusiner of the University of California, San Fransisco, we had the possibility to analyze several models of neurodegeneration, including Alzheimer and prion mouse models, with this work showing dramatic changes in peptide expression and carried out by my PhD student Margarita Diez (e.g., Diez et al., 2000).

SNAP-25 and the Metabolic Syndrome

Christina Bark working as a postdoc with Michael Wilson discovered two isoforms of SNAP-25: SNAP 25a and -b. After returning to Sweden, she created a unique mouse model in which SNAP-25b had been replaced by SNAP-25a. This SNAP-25b-deficient mouse present a distinct metabolic disease phenotype, especially after Western diet, including serious liver pathology (Valladolid-Acebes et al., 2015). Following up using three-dimensional imaging, Csaba Adori has been able to describe dramatic changes in the sympathetic liver innervation both in this mouse model and human fat liver (Adori et al., 2021).

Autoantibodies

Autoantibodies increasingly have been implicated in various diseases. Searching for such antibodies, we applied sera from human subjects with a particular disease onto sections of a formalin-fixed rat brain, hoping to find binding to discrete structures (neurons/glia) in specific brain regions, results that may give disease-related clues. Sergui Fetissov used sera from patients with anorexia and bulimia nervosa and found labeling of arcuate neurons expressed in food-intake-controlling alpha-melanocyte-stimulating hormone⁺ neurons (Fetissov et al., 2002). Serguei in 2009 reported on serum from patients with the autoimmune polyglandular syndrome type 1, where one serum (with anti-AADC autoantibodies) stained nigral DA neurons even at a dilution of 1:1,000,000 (in collaboration with Olle Kämpe). Together with Henning Vaeröy, a Norwegian forensic psychiatrist, Serguei found that sera from violently aggressive subjects harbor antibodies reactive to adrenocorticotropic hormone (2018).

In collaboration with the Finnish sleep researcher Markku Partinen, we studied sera from narcoleptic patients. Peter Bergman and Csaba Adori showed that several sera from such patients reacted with hypothalamic melanin-concentrating hormone⁺ and POMC⁺ neurons, and together with Hungarian scientists a possible role of the immunogen in the fine regulation of sleep was reported (Bergman et al., 2014). Finally, together with Krister Kristensson, Marina Bentivoglio, and others, my PhD student Mingdong Zhang analyzed viral invasion of the brain and could show that the H1N1 virus is transported to nuclei involved in sleep regulation and causes narcoleptic-like sleep (Tesoriero et al., 2016).

Kidney Research

Ulla Kopp is Swedish and married to Gerald Dibona, both eminent kidney physiologists working at the University of Iowa. Combining her experimental kidney model with IHC, Ulla explored among other things, the role of peptides and prostaglandins in renal nerve activity. I was surprised about the many similarities with our work on DRGs and pain. As a bonus, Ulla was a super-skilled organizer who, like a general, oversaw our move to new labs.

Committees

The Nobel Committee

In 1981, I was elected into the Nobel Committee (NC; today 18 members). The NC handles the many (today up to 800) proposals and meets with the Nobel Assembly (final vote, 50 members) to discuss the results of the crucial, extensive evaluations of the top candidates, followed by the Nobel Prize announcement the first week in October and a ceremony on December 10. The deliberations carried out by these two Nobel bodies are supported by the secretary, who also is a member of the NC, and during most of my period it was Jan Lindsten (1979–1990).

Already my first year on the NC was exciting, the prize for neuroscience was awarded to David Hubel, Torsten Wiesel, and Roger Sperry. The prize awarded to Köhler and Milstein for monoclonal antibodies in 1983 was important for its potential clinical relevance, and it also provided new reagents for our immunohistochemical work, as said. However, who could anticipate that such antibodies would revolutionize biomedicine, including the treatment of breast cancer and rheumatoid arthritis, and more? In fact, according to a 2021 report by Asher Mullard in Nature Reviews: Drug Discovery, the 100th monoclonal antibody product was approved by the U.S. Food and Drug Administration (FDA) in 2021, and in 2019, 9 of the top 20 therapeutics by sales were antibodies, with cumulative earnings of US$75 billion. This prize certainly was awarded to scientists who have “conferred the greatest benefit on mankind” (Alfred Nobel’s testimony).

In 1986, the prize was awarded to Rita Levi-Montalcini for the discovery of nerve growth factor (NGF) and to Stanley Cohen for the discovery of epidermal growth factor (EGF). Even if well-received, some colleagues felt that one scientist had been left out: Viktor Hamburger. Why was Hamburger not included? There are many factors that contribute to deciding on an award. Foremost among them being reviews by specialists in the field, but also the nominations, including how many and by whom. As stated by Professor Bengt Pernow, previous chair of the NC and KI president: “We had been discussing the field of growth factors for around 5 years before Levi-Montalcini’s prize and asked five or six scientists to review the field for us as a normal part of the evaluation” (Williams, 1995). Apparently these reviews had not favored Hamburger. Why not? Perhaps Hamburger received fewer, if any, nominations than Levi-Montalcini? If so, this will remain a secret until 2037, when the archives including the 1986 Nobel Prizes can be accessed (after application).

Other exciting events for us neuroscientists were during my time in the Nobel assembly (ending 2006) the Nobel Prizes in Physiology and Medicine given to Erwin Neher and Bert Sakmann in 1991 (the introduction of the patch clamp technique), to Stanley Prusiner in 1997 (the discovery of prions), to Arvid Carlsson, Paul Greengard, and Eric Kandel in 2000 (discoveries concerning chemical signaling in the nervous system), and to Richard Axel and Linda Buck in 2004 (the discoveries of odorant receptors and the organization of the olfactory system).

The IBRO Dargut and Milena-Kemali Foundation for Research in Neuroscience

The IBRO Dargut and Milena-Kemali Foundation was established in 1996 by Dargut Kemali, a professor of psychiatry in Naples, to honor the memory of his wife Milena Kemali. Milena was an anatomist/histochemist, and I gave her advice. On one visit to Naples, she drove me from the airport respecting traffic rules (which is not always the case in Naples), but after leaving the city she drove against three red lights. I couldn’t help but ask why? The answer was (something like) this: “These red lights are not needed but were put up by a relative to the mayor, a clear case of nepotism.” I had the privilege, together with Marina Bentivoglio, to assist with setting up the Dargut and Milena-Kemali Foundation. The Foundation, now taken over by IBRO, supports a course and awards a prize every other year with a lecture presented at the Federation of European Neuroscience Societies (FENS) Forum.

The Gruber Prize Selection Committee

In 2004, the Peter and Patricia Gruber Foundation established a substantial prize in neuroscience. Sol Snyder invited me to become a member of the first selection advisory board, and the first prize was presented to Seymour Benzer at the SfN meeting in San Diego the same year. It was a special honor to be part of this group, who met in the sitting room of Peter and Patricia Gruber’s private flat on the west side of Central Park in New York City. To get to know the Grubers, a wonderful and modest couple with a strong philanthropic engagement, was inspiring. The Prize Lecture continues to be an important highlight at the annual SfN meeting.

The Brain Prize Selection Committee (2010–2012)

The Brain Prize is the brainchild of Nils Axelsen, former department chair and scientific director of Statens Serum Institute, Denmark, and at that time vice chair of the board of directors of the Lundbeck Foundation. Having been asked to explore the possibility of creating a major international prize in neuroscience, Nils evaluated many award-giving foundations and suggested a prize in the field of basic and clinical brain research, with the name The Brain Prize. Nils proposed that this prize should (i) be awarded each year to one or more scientists after evaluation of nominated candidates by a selection committee; (ii) stimulate European and particularly Danish neuroscience; and (iii) be supported by an independent foundation, with Nils as chair of the board of directors. Anders Björklund and I assisted with recruiting the first selection committee.

The original emphasis was on European scientists and research carried out in Europe, but collaborating researchers from, for example, the United States also could be included. In this spirit, two U.S. colleagues were on the first selection committee (Huda Akil and Fred Gage), and Colin Blakemore acted as chair, and I as vice chair. This was a wonderful committee. I was happy to see three Hungarian scientists receive the first prize (György Buszáki, Tamás Freund, and Péter Somogyi). The third prize went to six scientists behind optogenetics. Because there was no strict limitation on the number of awardees, we could honor the several scientists behind this important methodological advancement. Now the prize is available to scientists all around the world, and the award is 10 million DKK (around US$1.6 million). It is recognized by the scientific community as one of the world’s major scientific awards.

International Alliance for Translational Neuroscience

In 2012, the International Alliance for Translational Neuroscience (IATN) was founded in Beijing, a vision of Xiaomin Wang, professor at the Capital Medical University (CMU), assisted by Zhi-Qing David Xu, my previous PhD student. This organization eventually encompassed eight universities around the world. I had the honor of helping prepare the founding documents. Over five years, scientists from these universities met and discussed important issues in the field of basic and clinical neuroscience. A highlight was the Xianshan Science Conference 506th session, International Forum on Brain Disorders, in Beijing, in 2014. I was invited to many unique events in China, with Zhi-Qing David Xu often being a wonderful cohost. These included a visit to an illuminated Great Wall on a dark late evening and the opera “Impression West Lake” performed on the lake and directed by Yimou Zhang (who also choreographed the opening ceremony at the 2008 Beijing Olympic games). Both were unforgettable experiences. It was a privilege to be involved in this exciting enterprise and interact with Chinese colleagues.

The Dagens Nyheter Affair (1995)

Toward the end of this article, the worst experience in my professional career, an event that made me seriously think of quitting, shall be told. Early summer 1995, I got a call from a journalist, BGA, from Dagens Nyheter, the largest morning newspaper in Sweden. BGA asked me, surprisingly, about the situation with my U.S. grants. I guessed he was alluding to an NIH grant with Menek Goldstein as PI, and I responded “fine.” Thinking more about it, I remembered that I had received mail from NIH referencing an “audit.” Not knowing what to do, I had left the letter on my secretary’s desk for a couple of months. At that time, my secretary helped several other groups in the department, and it occurred to me that someone might have seen the mail and noted the word “audit” and contacted the journalist.

At the end of the summer, BGA called me again and said he wanted to go through my correspondence. KI is a governmental institution and all correspondence is open to the public. In the follow-up interview, I slowly realized that it was not simply a matter of my U.S. grant: BGA suspected that I had been bribed by the Italian pharmaceutical company Fidia to promote the selection of Rita Levi-Montalcini for the Nobel Prize, which she, as said, received in 1986. He actually said that he was aiming for the prestigious annual Prize for Investigative Journalists and that he wanted to put an end to all hush-hush associated with the Nobel Prize.

BGA mainly based his accusation on a Fidia check deposited to my account in a U.S. bank in the amount of US$5,000. I still had that account from may stay at NIH 1981/1982 and had received this money from my coorganizer Erminio Costa for the organization of a symposium on “Neuropeptide-Catecholamine Interactions” at the Fifth International Catecholamine Symposium, held in Gothenburg, Sweden, in June 1983. Costa had raised the money from Fidia, and it was to be used to pay travel costs for eight speakers. Instead of changing the check into SEK (Swedish krona) and paying the speakers in cash (SEK), I deposited it in my U.S. account and wrote a check to the speakers (most from the United States). I could present, to the Dagens Nyheter’s editor (and BGA), written confirmation by all of the participants that they had received travel reimbursement (altogether US$4,600). The rest I used for my travel to Gothenburg.

Fidia was a small Italian pharmaceutical company, producing and selling GM1, a ganglioside. Fidia gave grants to work on the mechanism of action of GM1 (I did not receive a grant) and supported high-profile meetings in the neuroscience area. Fidia also awarded a modest prize (US$2,000), that several distinguished colleagues had received, including me. Fidia both paid travel and lodging at the meetings but almost always required a manuscript for the proceedings book. I accepted several invitations and sometimes even my wife was invited.

Some time after the interview, I understood that the story was soon going to be published. At this point, I just asked BGA for one favor: please tell me a day in advance of publication so that I could warn my elderly mother. I had been invited to a meeting in Amsterdam to give a talk on Sunday in the Dutch Academy of Sciences, and I told BGA that I would be in my office on Saturday until around 1 p.m. before leaving for the airport. I did not hear anything and flew to Amsterdam and gave my talk Sunday morning. After that, I called Stockholm and then got to know that Dagens Nyheter had published what was going to be the first of four one- to two-page articles with me as target. An hour later, I came down with a terrible pain attack—a kidney stone. After treatment at a hospital, I could return to the meeting. But even if that acute pain was gone, the coming months, even years, would turn out to cause chronic pain and frustration.

Completely inexperienced, I did not know how to handle the situation but was advised to keep quiet. Sten Grillner and Nils Ringertz, chair and secretary of the NC, respectively, wrote a response, in which they explained the principles of the work in the NC and Nobel Assembly. What helped me overcome the trauma were the wonderful and thoughtful letters from many colleagues, from all around the world, who expressed support and even indignation on my behalf. Things soon calmed down, but I of course was heavily afflicted and incapacitated, pondering whether I could proceed with my research career. What did the colleagues really think, the relatives of my Swedish collaborators, and the public in any case? (I did receive some anonymous mails suggesting that I should be ashamed.)

Decisive support came from a small note in Science, one of the most prestigious journals, by Nigel Williams (1995), for me one of the most important articles ever published. Nigel interviewed eminent and knowledgeable scientists, by phone, and asked what they thought about the serious accusations in the Dagens Nyheter articles. In fact, my case had been published in newspapers around the world. One important voice was Professor Bengt Pernow, as said, a member of the NC, who was quoted as saying, “We had been discussing the field of growth factors for around 5 years before Levi-Montalcini’s prize and asked five or six scientists to review the field for us as a normal part of the evaluation, but Hökfelt was not one of them” (Williams, 1995). This, along with the fact that the Nobel Prize was given for “growth factors” (and not nerve growth factors) and that the laudation in the Concert Hall was presented by Kerstin Hall, professor of endocrinology at KI, certainly confirmed that I did not play a major role in the decision of the Nobel Prize (I, of course, voted for the prize). Previously, the secretary of the NC had not allowed me to reveal that I was not one of the reviewers, but now Bengt Pernow had made this public. As said, these reviews are extensive and in depth, up to 30 pages or so, and are the basis for the work by the NC and the decision by the Nobel Assembly, so I felt “cleared” from the accusations. The editor in chief of Dagens Nyheter, later published some retractions: “There were no bribes” and “The laureate is not questioned” but still “We stand by the basic message . . . and I haven’t regretted we published the articles” (Williams, 1995). One may wonder, what the “basic message” really was, or even what it was intended to be. I felt great gratitude to Nigel Williams. I have today made considerable efforts to get in contact with him, to express my appreciation, but so far I have failed.

Shortly after the note in Science, I was contacted by a journalist working for an influential investigative Swedish television program that was produced by a media star, Robert Aschberg. They had read the note in Science and realized that I had been wrongly accused of taking a bribe, and they wanted me to confront BGA in their program. It felt good, of course, to get a chance to publicly clear my name, and I was tempted to accept. But knowing myself, I thought that I would probably mess things up. So I said no thanks. I also considered contacting the Swedish “press ombudsman.” In fact, I spent a lot of time writing a long and detailed response to all accusations, but never submitted.

Traveling and Meetings

Meetings have always been an important and fun part of my life. Therefore, visits to the United States and other countries have been important. I have always been grateful for invitations and for the warm hospitality of my many hosts, too many to mention here. During one period, I often visited Japan and would like to thank some wonderful Japanese colleagues for many memorable meetings: Professors Yutaka Sano, Yasuhiko Ibata, and Nobutaka Hirokawa. I have already mentioned some special Chinese colleagues. Of the many meetings I attended over five decades, some, for various reasons, remain special to me and will briefly be mentioned here.

Nerve Cells, Transmitters, and Compartments Seminar

This seminar took place at the Pontifical Academy on October 9–14, 1978. The symposium was held in the Vatican Gardens, and I was humbled by the list of the brilliant participating scientists, whom I had never met but only read about. Several topics were discussed, ranging from growth factors through amino acid transmitters, opioid peptides, cotransmission, and sympathetic ganglia, to the brain-mind problem.

This seminar was somewhat overshadowed by excitement over the possibility that the participants would have the opportunity to meet the Pope, Paulus VI, who had been in office since 1963, for 15 years. Unfortunately, the pope passed away on August 6, 1978, so that chance faded away. Surprisingly, a new pope, Johannes Paulus I, was elected on August 28, again raising the hope of a personal encounter. Unbelievably, Johannes Paulus I passed away on September 28, just a few days before the symposium was to begin. Our seminar took place, but with the Vatican in mourning and with certain restrictions. When departing from the Vatican we, the participants, saw the white smoke raising over the conclave of the Cardinals. The Cardinals had elected Johannes Paulus II who remained in office until 2005, that is, for more than a quarter of a century. The timing of my once-in-a-lifetime opportunity to meet a pope had been exceptionally unfortunate, indeed.

Central Regulation of the Endocrine System

Already in 1979, Kjell Fuxe and I had the great honor to organize a Nobel Symposium (#42) together with and thanks to Professor Rolf Luft. The meeting was attended by leaders in the field, three Nobel laureates (Axelrod, Schally, and von Euler) and three to be named: Martin Rodbell talked about early evidence for G-proteins, findings heralding the Nobel Prize that he would share with Alfred Gilman in 1994. Paul Greengard talked about protein phosphorylation; and Arvid Carlsson, surprisingly, talked about neuropeptides and their effect on gross behavior. But he would soon return to DA and other classical transmitters; and, as said, share the Prize with Greengard and Eric Kandel in 2000. This was a fantastic meeting and an honor to meet all these brilliant scientists at such an early stage in my career.

Coexistence of Neuronal Messengers: A New Principle in Chemical Transmission

I had the privilege to organize, together with Kjell Fuxe and Bengt Pernow, this symposium in Stockholm in 1985. It was supported by the Wallenberg Foundation for International Cooperation in Science, Astra Pharmaceuticals, and the Nobel Foundation. The meeting was attended by many outstanding colleagues. It was very exciting and a great honor, as well as a true highlight of my professional life.

Symposium Commemorating the 90th Anniversary of the Nobel Prize

The Nobel Foundation decided to celebrate the 90th anniversary of the Nobel Prize with pomp and circumstances. All previous laureates were invited (and more than 140 accepted) and four parallel, one-day symposia were held on December 7, 1991. One was on neuroscience, and Kjell Fuxe, Gunnar Grant, Sten Grillner, Lars Olson, and myself had the honorable assignment to organize the program. The 1991 Awardees, Erwin Neher and Bert Sakmann, served as chairs. Two participating laureates shared the 90th birthday with the Nobel Prize: Linus Pauling and Charles Huggins—impressive, to say the least. Two speakers were brilliant recruitments to the neuroscience field, Leon Cooper (from physics) and Gerald Edelman (from chemistry), together with many old heroes. It was a great day.

Finally, What About Grants?

Today, it is obvious that grants are important, and they must be big grants. This was not always so. Governmental and institutional support in the form of position, PhD students, technicians, and secretary represented a reliable basis for research. Very importantly, during the 1980s and 1990s, many foreign guest scientists came to our lab with their own support and salary, for example, from NIH, the Canadian MRC, and Chinese bodies. I also want to gratefully acknowledge several further sources, some unexpected. In Sweden, the (Medical) Research council has supported me uninterruptedly from 1969 to this day (more than 50 years). Another source was the Wallenberg Foundations, after the Research Council, the largest donors to Swedish research. Their support to the research community here in Sweden cannot be overestimated. I also was lucky to obtain NIH grants, as co-PI, one in the 1970s focused on substance P with my Swedish mentor Bengt Pernow as PI, and later grants for the immunohistochemical analysis of the monoamine neurons, with Menek Goldstein of NYU as PI. I was grateful, impressed, and surprised by the U.S. generosity to support researchers in Sweden.

Even more surprising was another U.S. grant: It started, without any application, with a message from Dr. Perry Molinoff formerly at the University of Pennsylvania; at that time, he was the head of neuroscience discovery at Bristol-Myers Squibb (BMS). He informed me that I was being considered for an unrestricted neuroscience grant. However, before making the award, he and a group from BMS wanted to make a site visit to KI. They suggested a date and planned to visit me in the morning. I realized that the group was too large for me to pick them up by my car at the hotel. So I gave them, I thought, exact instructions for how to get to me. I was standing, dressed in white shirt and tie, in the window of our building starting 8.00 a.m., looking over the campus and waiting for their arrival. It was raining, and with every minute I got more and more nervous, and saw the money slowly fading away in the grey sky. Finally, around 10 a.m. a group crossed the campus; they had been left at the main entrance that is East on the campus (our building is West), with umbrellas and carrying/dragging their luggage. Now I was definitely sure about the failure. But it ended well. After a short presentation, they left for Arlanda airport, and the company jet that took them to Copenhagen. Some time later, I learned that I would have US$100,000 for each of the next five years. It was a big sum, in the range of my MRC grant. This meant a lot. Thanks Perry, and thanks BMS.

I had 10 years earlier very good reasons to be extremely grateful to BMS or, more specifically, to their Prize Committee: I was awarded the first BMS Award for Distinguished Achievement in Neuroscience Research together with Drs. Walle Nauta and Thomas Powell. It was an incredible honor for me to share the award with these giants in neuroanatomy. Incredible also was what happened when my wife and I were traveling to attend the prize ceremony at noon in The Pierre Hotel in New York. I had since long agreed to give three lectures at College de France in Paris, and the last one was at 5 p.m. the day before the prize ceremony in New York. This seemed like an impossible situation, until BMS came back and told me that my wife and I could fly with the Concorde from Paris to New York. The supersonic plane would only take three-and-a-half hours and would land at JFK at 11 a.m. on the day of the Ceremony. We arrived by public transport in good time at Charles de Gaulle (together with the passengers traveling in Bentleys and similar luxury cars) and boarded. After having been seated for 20 minutes or so we were told to disembark—there was a problem with a vault on the aircraft. I am always the nervous type, but this was too much. Heart racing: what if we arrived too late and BMS had paid these terribly expensive tickets? Eventually the problem was fixed and Air France guaranteed arrival at JFK at 11:45 am, so we decided to fly. I was completely exhausted and slept the whole trip, and did not note the breaking of the sound barrier nor enjoyed the caviar and champagne (but Lil did). A waiting driver took us to New York City and, at exactly 12:30 p.m., we ran into The Pierre, convinced that half the ceremony lunch had passed. However, we had forgotten that the lunch was preceded by a 30-minute cocktail session. We joined the last guests on their way to the lunch, and slipped unnoticed into our chairs. No one seemed to have missed us. An unbelievable relief, after unbelievable stress and an unbelievable journey in an unbelievable plane.

Concluding Remarks

I consider myself an explorer, some would say stamp collector, providing information on the localization of chemicals in the nervous system, information to be used for functional studies. But to see, with one’s own eyes in the microscope, what no one else has seen before has given me an enormous joy, even if just a twinkle in the universe. Much work has focused on neuropeptides. Four of them, galanin, NPY, CCK, and VIP, were discovered at KI, in the Viktor Mutt Laboratory, and it has been an honor to follow up Viktor’s groundbreaking work.

I have been awarded several prestigious honors and have always felt a bit embarrassed. But these experiences have stimulated me to work harder to show that I perhaps was worthy such distinctions. My school period in Germany made me familiar with its history, including Otto von Bismarck, the mighty chancellor who united Germany. He once said (among many other quotes): “A statesman . . . must await until he hears the steps of God sounding through events, then leap up and grasp the hem of His garment.” By replacing some revered words, this has characterized much of my career: I have listened to and learned from many inspiring and inventive colleagues and collaborators, including more than 20 PhD students and some 75 postdocs, and then I have put all my energy into every project. Things have sometimes gone wrong, but I believe that our results have mostly been correct. Nine of my PhD students became full professors (Marianne Schultzberg, Martin Schalling, Björn Meister, Jan Lundberg, Sandra Ceccatelli, Christian Broberger, and Nora Kerekes; and, in China, Zhang Xu, a member of the Chinese Academy of Sciences, and Zhi-qing David Xu.

And it all began with one person, Nils-Åke Hillarp. It is hard to overestimate the importance of a single person for advances in biomedicine (in Hillarp’s case, achieved in three years). Times have changed, as has the process of conducting science and publishing results. In the old days, with our simple techniques, it could be that a postdoc left after two years with 20 papers. Today, especially in the good labs, postdocs may leave with one publication after four years or more. Hopefully, it is clear that I, during my six decades in research, have been supported by many to whom I owe sincere thanks, but here I would like to express my gratitude to Solomon Snyder, Eric Kandel, Jean-Pierre Changeux, and Huda Zoghbi for support, advice and taking interest in our work over decades; to Paul Greengard for support also in personal matters; to Tamas Bartfai for collaboration and friendship; to Anders Björklund, Robert Elde, and Tibor Harkany for reading and providing valuable input to the draft; and, finally, to Tom Albright for extensive editing. I appreciate and am honored by the invitation from Drs. Larry R. Squire and Tom Albright to contribute to their unique series The History of Neuroscience in Autobiography. It is and will remain the account of neuroscience research carried out over the past seven decades or so. Finally, none of the things described in this article could have been achieved without my wife Lil.

Selected Bibliography

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Darcy B. Kelley

BORN:

New York City, New York

November 29, 1948

EDUCATION:

Barnard College, New York, NY, BS (1970)

The Rockefeller University, New York, NY, PhD (1975)

APPOINTMENTS:

Assistant Professor, The Rockefeller University (1977–1978)

Assistant Professor, Princeton University (1978–1982)

Associate Professor, Biological Sciences, Columbia University (1983–1987)

Professor, Columbia University, New York, NY (1983–present)

Course Director, Neural Systems and Behavior, Marine Biological Laboratory (1985–1989)

Founding Co-Director Doctoral Program in Neurobiology and Behavior (1995)

Howard Hughes Medical Institute Professor (2002–present)

Harold Weintraub Professor, Columbia University (2010–present)

Director, Doctoral Program in Neurobiology and Behavior (2021–present)

HONORS AND AWARDS (SELECTED):

National Science Foundation Graduate Research Fellowship (1970–1973)

Alfred P. Sloan Foundation Research Fellowship in Neuroscience (1978–1981)

Javits Neuroscience Investigator Award, National Institutes of Health (1988–1995)

Fellow, American Association for the Advancement of Science (1989–present)

Forbes Medal, the Grass Foundation (2003)

CSAB Award, Center for the Integrative Study of Animal Behavior IU (2009)

Fellow, International Society for Neuroethology (2014–present)

Member, American Academy of Arts and Sciences (2017 – present)

Sinauer Associates Distinguished Scientist Lecture, University of Massachusetts, Amherst (2018)

Darcy Kelley’s research has elucidated the broad range of biological mechanisms—from hormones to genomics—that shape innate vocal communication. She developed Xenopus as a biological model for developmental, cellular, and molecular explorations of sexually differentiated behaviors. She and her laboratory members identified mechanisms through which gonadotropins, androgens, and estrogens shape auditory processing and vocal pattern generation. Together they developed powerful ex vivo approaches to identify evolutionarily conserved components of vocal neural circuits from forebrain to hindbrain. Kelley and former trainees—now tenured faculty members in the United States and abroad—collaborate in developing new approaches for identifying how neural circuits for vertebrate social communication evolve. Her current research foci include genetic mechanisms that support the divergence of vocal communication during speciation and the biology of neurodevelopmental disorders of social communication. She has served as trustee of the Marine Biological Laboratory, the Grass Foundation, and the Association of American Colleges and Universities.

Darcy Brisbane Kelley

Family

I grew up in New York City. My mother’s family (Brisbane) was from upstate New York and my father’s (Kelley) was from New England. My mother Elinor was the youngest of six, and her father Arthur died on Christmas day when she was 11. My grandmother, Phoebe Cary, was ill throughout my childhood; I did not know my maternal grandparents. I was, however, very close to my paternal grandmother, Harriet, whom I named “Gaga.” Gaga grew up in North Attleboro, Massachusetts, but was a frequent visitor to her aunts in Brooklyn and loved New York City. After she had broken off several engagements, my great-grandmother Maud (“Mother”) Tweedy ordered Gaga to accept her most recent offer: from my grandfather, Solon Kelley II, a New York City stockbroker. When he insisted on moving the family to Connecticut, Gaga left him and supported herself by working at Macy’s. Her sons (my father and his younger brother Ned) had to live with Mother Tweedy until they were dramatically rescued in the midst of a snowstorm by Gaga and her new husband, Freddy Wildman. Mother Tweedy was so terrifying that whenever we had to visit, my mother and I would hide inside a car in her garage.

My first year in graduate school, grandfather Solon came to visit. Over a luncheon (I figured it was now or never), I asked him what had happened with Gaga. Grandpa said that he had followed Harriet to Paris (she had to go to France to get a divorce). When he caught up with her on a bridge over the Seine, Harriet took off her wedding ring and threw it into the river. He only saw her again once, at a party. Grandpa was ascending the up staircase and Gaga descending the down; she winked at him and went on. Before I headed back to Rockefeller, Grandpa asked me whether I was going to be a “bluestocking”: an old-fashioned term for a scholarly woman who never marries. I had to agree with Gaga; Freddy was much more fun.

My father (Solon III) grew up with Gaga and Freddy in New York City. He was educated at the University of Virginia before leaving to join the Army Air Corps (later the Air Force) at the beginning of World War II. He was a decorated fighter pilot, served in the China-India-Burma and European theaters and was released with the rank of major. My parents met after the war, married in 1947, and started their family: me (the oldest), my sister Harriet (the artist Mandy Staffa), and my brother Soso (Solon IV, and the last). I do not know why they were all named after a lawgiver in ancient Greece. My father never discussed his war service but continued to fly, piloting us to Martha’s Vineyard to vacation with my cousins Allaire and Cary; my mother was very close to her sister Alice. My father died of bladder cancer at 51 during my first year at Rockefeller. He was the last living member of his squadron; they all died of the same disease. Air Force and Navy pilots continue to die of bladder cancer to this day.

My mother Elinor also grew up in New York City; she graduated from Bennington College in Vermont. Bennington attracted outstanding faculty during the war. W. H. Auden delivered a series of lectures that contributed to my mother’s desire to be a writer. Her thesis on “The Brothers K” (Dostoyevsky) was supervised by Peter Drucker, professor of politics and philosophy at that time (and later a management guru). Solon joined his stepfather, his brother Ned, and his half brother Freddy Jr. to form Wildman and Sons, a wine importing firm. Freddy Sr. served as both a lieutenant with the Second Division in France in World War I and then as an Air Force Colonel in World War II. According to family lore, after World War II ended, “Big” Freddy somehow secured a car and drove—from one notable French vineyard to another—lining up the wines that Wildman and Sons would later import. Big Freddy was a man of considerable charm: a “grand seigneur.” My grandmother loved to travel, and their partnership was strong.

The Wildman and Sons trips through Europe were a frequent part of our childhood and usually included my mother, who had spent a year in Versailles as a child and so was fluent in French. After one long separation, we children pretended not to recognize our parents. This move did not have the desired effect. We did, however, get to tag along once for a summer vacation in France at Pyla sur la Mer where I learned to play couteau (a child’s version of mumbley peg) on the beach. Stony Beach in Woods Hole, where I later spent summers with my own family when at the Marine Biological Laboratory (MBL), always brings back memories of that beach in France. If you have read the opening of Lewis Thomas’s Lives of a Cell, you might see the resemblance, which is one reason why I have always been so happy at the MBL.

Throughout my childhood, we lived off of East End Avenue. Before the East River Drive was built, our apartment building fronted directly on the river, and tenants could dock their boats. The bottom part of the building was later converted to fallout shelters that my friends and I would explore on rainy weekends; they were stocked with supplies during the Cuban missile crisis while John F. Kennedy was president. Rockefeller also had subterranean domains with a series of tunnels. While exploring them with my fellow graduate students, we once found one that led to a cowshed, erected for a cell biologist studying the pancreas, if memory serves.

Education

When the time came for first grade, my mother sent me to the Chapin School because the headmistress, Miss Stevenson, had said she would watch out for me as I crossed East End Avenue. I was a moody child, and Chapin was a good choice. My education led me to believe, despite growing up in the 1950s, that women could be powerful and erudite leaders. Chapin was run entirely by women; the only men were talented musicians (Robert Schrade and Charles Walker). Miss Stevenson succeeded Miss Chapin and in turn transferred power to Mrs. Mildred Berendsen, a most formidable leader, whom we called Milly Bear. Mrs. Berendsen’s subject was history, and seniors had the privilege of taking it in her office. My other teachers, equally demanding, were kind.

I developed a passion for science beginning at age 11. My inspiration was a renowned, French–American child psychiatrist: Dr. J. Louise Despert. I met her because she had written a book for parents, Children of Divorce. One evening in 1959, our parents invited us to sit down and then announced that they were getting a divorce. My brother and I reacted to this unexpected news by developing hysterical paralysis and my sister looped the cord of the venetian blinds in our shared bedroom around her neck. Our mother had to bribe us with treats to go talk to Dr. Despert every week, but eventually we felt better; she was a skilled clinician. As I recovered, we’d discuss the science books I took out each week from the library on my walk to her office. Dr. Despert remained a lifelong friend of our family, inscribing another book—The Inner Lives of Children—to my son Alex when he was born in 1975. Until redirected as a teenager, I wanted also to be a doctor and a child psychiatrist.

Despert was also a distinguished and prolific academic psychiatrist, widely recognized as an expert in childhood schizophrenia. In the Ackerly oral history project on “Pioneers of the Child Guidance Movement” (William Gardner Collection, Kornhauser Health Sciences Library Historical Collections, University of Louisville), Despert recounts leaving her medical studies in Paris to become a nurse during World War I. She moved to New York in 1920 and then completed her studies at Barnard and NYU Medical School, paid for—as she told my mother—by a grateful patient from her days as a nurse. In her 1971 paper reflecting on infantile autism, Despert describes the link between aversion to cuddling in infancy and complete lack of sexual drive in adulthood and asks, “Is there not something lacking, some hormone perhaps, an ingredient which interferes with the early affective development by its very absence?”

My newfound interest in science was tolerated graciously, although my English teacher—Judith Phelps—said that I gesticulated too much to ever be a doctor. I learned to think and to write, however imperfectly, over the course of my 12 years at Chapin and to deeply respect educators. Mathematics was not my strong suit except for geometry, but I did advance in calculus thanks to a brilliant teacher, Ruth Hutter. I ran into Mme Meller, my French teacher, on the bus some years ago, and she complained about the ascendance of computer “languages.” French was very handy though during our later fieldwork in Gabon. I keep Chapin’s motto—Fortiter et Recte—in mind; it helps during tricky situations at Columbia.

“Here’s the thing,” as we say today. In the 1950s, women worked when they had to support themselves and their families. Only some jobs were available to them, but these included teaching, not just as a job but as a profession, and a powerful one. As Columbia students, Bridges and Sturtevant decided to join Thomas Hunt Morgan’s lab after taking introductory biology from him—the only time Morgan ever taught it—and not after hearing a research talk. They liked the cut of his jib.

I began my own teaching by tutoring college classmates in Biology I in the dinner line at Grinnell College, gave a lecture on bats in Biology II at Barnard, and was teaching assistant in a neuroscience course at the New School while a first-year graduate student at Rockefeller. Today, many of my Columbia Medical School colleagues believe that teaching interferes with research: why teach when you could be in the lab? However, learning about something new to teach it can pave the way for new research directions. When I moved to Columbia in 1983, I began teaching developmental and systems neuroscience to undergraduates. A few years ago, I added viral vectors to my lecture on tracing neural circuits, and so I had to learn virology. Then I joined forces with other amphibian research labs to develop viral vectors for frogs and newts. Next, came Covid. Ironically the question of how this virus jumped from bats to humans—changing its host specificity—is basically the same problem as figuring out how viral vectors that transduce mouse neurons can be biologically reengineered to figure out fascinating questions in newt and frog brains: regeneration of adult brains, evolution of cortex, and genetic control of circuits for innate behaviors.

The 1960s

I was a teenager in the 1960s: Selma and Martin Luther King Jr. marches and protests. My father was conservative; he hated my brother’s long hair, and we fought bitterly about the Vietnam War. My mother, though, was a “total liberal.” The father of my classmate, Christina Berlin, was a newspaperman and took his daughter with him to meet the Beatles. Everyone in my class was sick with envy. With my close friend and classmate, the painter Adele Alsop, and some musical buddies, we’d roam across the NYC boroughs to play folk music. Our banjo player, Joseph Pennock, introduced us to Flatt and Scruggs backstage at Carnegie Hall. I tutored kids, supported the Student Nonviolent Coordinating Committee, and watched the assassination of JFK on black-and-white television.

In 1957, the Soviet Union launched Sputnik, creating consternation in the United States; clearly, we had fallen behind the U.S.S.R. The National Science Foundation (NSF, only just founded in 1948) was moved to sponsor summer science programs for high school students. By then, I had become interested in biology, and endocrinology specifically, through reading Nobel Laureates in Physiology and Medicine, especially the part on Banting and Best’s discovery of insulin. In 1965, as a high school junior, I applied to two NSF-supported summer science institutes: one at Mt. Holyoke on chemistry and another at Grinnell on the biological basis of behavior. I didn’t know that behavior had a biological basis. Luckily, as chemistry is not my thing, I chose Grinnell and, having passed the required psychological test, traveled to the cornfields of Iowa.

Neil Kent’s NSF Behavior Science Institute at Grinnell College aimed to prepare high school students with scientific potential to major in psychology as undergraduates. The test (of religious beliefs) weeded out applicants that might have a problem with an assigned text: Walden II, B. F. Skinner’s Utopian novel. D. W. Tyler taught us to operantly condition “rats” in simulated experiments and use statistics to analyze the results. The Rutgers ethologist, Danny Lehrman, traveled to Iowa to talk to us. He did a great impression of bow-cooing in ring doves. The experimental psychologists Ogden Lindsley and Nate Azrin described using operant conditioning to shape behaviors of autistic children. Arnie Kriegstein, now a stem cell neuroscientist and former Columbia colleague, was in my Grinnell cohort, as was the psychologist, Michael Domjan. And I did end up majoring in experimental psychology because neuroscience and behavior majors did not yet exist. The upshot was that I decided to matriculate at Grinnell because I could do research as an undergraduate. I withdrew my application to Radcliffe, and even tried to skip my last semester at Chapin to start college early. Mrs. Berendsen talked me out of it; among other arguments, I was school president. My yearbook motto was “Dux femina facti” (from Vergil’s Aeneid, chapter 1; roughly “The leader of the exploit a woman”).

It is hard to imagine now how different the 1950s and 1960s were, especially for women. You can get some flavor for the times by watching “The Marvelous Mrs. Meisel,” although it wasn’t as much fun in real life. In 1966 in Iowa, women students had “parietals” and had to be back in the dorms by 10 p.m. on weekdays and 11 p.m. on weekends. We had bed checks; the men were still outside serenading the women who were leaning out of the dorm windows. Girls got pregnant and dropped out of college to marry, so parietals and bed checks clearly were not doing the job. I gave evening talks on birth control in the lounges. Coeducation had other curious features that I had not encountered at Chapin. Girls, even the most brilliant ones, almost never spoke up in class. This experience, and a book, Sex and Internal Secretions, awoke a life-long interest in the biology of sex differences.

Barnard College

My father’s illness worsened, and in 1968, I returned to New York, transferring to Barnard College as a second semester sophomore. Columbia did not admit women until after I joined their faculty in 1983. I was a psychology major, in part because my NSF summer institute allowed me to skip introductory psychology. However, while at Grinnell, I had also read Don Griffin’s book (Listening in the Dark) on bat echolocation and was entranced by the ability to paint a picture of the world in sound. I devoured everything I could read about bats. I wanted to find out how echolocation developed in baby bats, using the classic paradigm for discerning innate behaviors: social isolation in infancy. Barnard’s introductory biology course (the one I gave the bat lecture in) was taught by David Ehrenfeld (now a noted conservationist at Rutgers). Ehrenfeld, who studied sea turtle navigation, called Don Griffin (see Volume 2), who had been recruited to Rockefeller from Harvard, and arranged for us to meet.

When I walked into Griffin’s lab in Smith Hall, in 1969, a group was clustered around a tape recorder. Roger Payne had just returned from recording whale songs; beautiful, mysterious, very low-frequency sounds. Griffin suggested that I contact Marta Nottebohm, the wife of my future postdoctoral adviser, Fernando (see Volume 8), as she had hand-reared baby bats during one of their field expeditions. Richard Penney, stationed at the NY Zoological Society, knew of a colony of Eptesicus fuscus (big brown bats) in Purdy Station, northwest of New York City. We met at the Institute for Research on Animal Behavior at the Bronx Zoo, Penny drove me and Marta to the barn to get the bats, and we returned with three. Marta gave me tips on feeding them (tiny baby bottle) and also on getting the baby bats back to New York City safely. This trip later produced the infamous “bats in the bra in the subway” story that I gave yearly when I lectured to first-year Columbia students taking “Frontiers of Science.” From sound spectrograms made in Griffin’s lab, I could see baby bat ultrasonic calls shortening daily, supporting the idea that the capacity for producing the short chirps of echolocation sounds was innate.

Don Griffin also introduced me to Jim Simmons, a psychology grad student of Glen Wever’s at Princeton. I graduated from Barnard a semester early and traveled to Europe with Simmon’s group to collect the horseshoe bat, Rhinolophus ferrumequinum, at a field station in the mountains between France and Spain. Our guide, the subterranean insect expert, Michel Bouillon, took us to private caves where we wiggled though tunnels to see prehistoric cave drawings illuminated by smelly acetylene headlamps. We returned to Paris by train with the bats and stayed with our host, Rene Guy Busnel; and I went to my first scientific meeting. Rhinolophus has a two-part echolocation call: constant frequency followed by a frequency modulated sweep. Later work by Nobuo Suga’s lab (see Volume 6) at Washington University showed that each component is represented in separate areas of auditory cortex. Neurons are tuned to distance-dependent echo delays and amplitudes relative to the bats own ultrasonic sound pulses.

In the 1970s, when I was graduate student at Rockefeller (see the next section), lunch was served at a long table in a room looking over the East River. If a student settled next to Don Griffin, he or she would be recruited for one of his projects: going aloft in a hot air balloon to record the vocalizations of migrating birds or using a “bat detector” to study bats emerging from under the roof tiles of the field station in Millbrook, New York. Don’s wife, Jocelyn Crane, was an authority on fiddler crabs and our lab at Columbia inherited Joycelyn’s tanks when she later turned to the study of art. The Griffins took me to dinner once at restaurant in the Hotel des Artistes below Jocelyn’s apartment. They first retired to Princeton, New Jersey, and I house sat for them, luckily not when an ancient toaster set off a fire. Don and Joycelyn then moved to Massachusetts. Don kept a small sailboat and used it to work on the terminal buzz of Myotis lucifigus (the little brown bat) until he died. His admirable curiosity is well captured by the biographical sketch for National Academy of Sciences (NAS) by my Princeton University colleague, the late Charlie Gross (see Volume 6). I miss them both.

The Rockefeller University

Laboratory rotations, I believe, are as much about learning what you don’t enjoy in research as what you do. My first laboratory rotation at Rockefeller was in Bruce McEwen’s lab (see Volume 8). Bruce was a great mentor; not just when I was a student but throughout my career. He died unexpectedly last year (early 2020) to my great sorrow. My rotation project was characterizing corticosteroid receptor expression in the brains of baby hamsters in response to stress. After injection with tritiated steroid and then removing and homogenizing the brains, cellular components (membranes, cytoplasm, nuclei) were separated by high-speed centrifugation and the nuclear fraction was eluted from gel columns in the cold room. Bruce didn’t have a fraction collector, so this meant moving scintillation vials by hand in sequence to receive the correct number of drops so that radioactivity could be measured in a counter after adding scintillation fluid.

I had nightmares about the baby hamsters. I was sure the centrifuge would blow up,. It was freezing in the cold room, and, more crucially, the data were all numbers on a paper strip. Where was the brain; where was the behavior? I did, however, learn enough steroid biochemistry to run studies when I had my own lab. I edited a volume of papers (Bruce put me up for the job) and presented my work in his lab at the first meeting of the SfN. The abstract (which I had forgotten) was followed 50 years later by an invitation from Journal of Neuroscience to review our research in an issue celebrating that first meeting. I greatly enjoyed writing this with many former trainees and current collaborators (Kelley et al. 2020).

Next, to try out electrophysiology, I examined the neural circuitry underlying the female rat lordosis reflex in Don Pfaff’s lab. The idea was to determine whether neurons in the central gray (PAG) were responsive to the sensory stimuli that induced lordosis: touch to the perineum. Surgery took the entire day, the recordings took the entire night, and I am an early morning person. Don then suggested a possible project that would fit my interests in sex determination and development. Emil Witschi had discovered that rearing Xenopus tadpoles in estradiol resulted in frogs that were 100% female. Backcrossing these frogs (treated as embryos) to untreated males resulted in sex ratios that revealed a zz (male), zw (female) primary sex determination system (i.e., some phenotypic females produced entirely male offspring). The experimental system seemed to be a promising one to determine whether there were any genetic contributions to sex differences in behavior or whether gonadal steroids (androgens and estrogens) were the sole determinants. This same question motivated the development of the “four core genotypes” in mice studied later by my fellow Grinnellian and Rockefeller graduate, Art Arnold, at the University of California, Los Angeles.

During my first year at Rockefeller I found out that many of my fellow RU graduate students (including Art) were going to Africa for a course on field studies of animal behavior. I asked to join them but they hadn’t counted on including a psychologist. So, I applied to the Organization for Tropical Studies for a course on “Habitat exploitation and diversity; an ecological approach with vertebrates.” My final rotation, during the summer of 1971, was thus field biology in Costa Rica. The course was organized by Roy McDiarmid (University of Florida) and Robert Rickliefs (University of Pennsylvania), a distinguished amphibian ecologist and ornithologist, respectively; visiting faculty included Jiro Kikawa from Queensland and Gordon Orians from Seattle. Among my fellow students were Sandy Vehrencamp, Frank Bonnacorso, Donna Howell, Marty Crump, Jim DeWeese, Ron Orenstein, and Dennis Turner. We started with Howler monkeys (and bats with harems for me) and then moved to the Osa Peninsula. Some highlights were mist netting in dry streambeds and finding the bottom panel full of vampire bats in the morning, the Jesus Christ lizard (Basiliscus basiliscus) running on top of a stream, gold Ponerine ants with painful bites, the huge Vampyrum spectrum that Donna caught flying around the mess hall, and the bat sea cave. Our course spanned the transition between wet and dry seasons, and when we returned to Guanacaste at the end of the course, my tree with the harem bat (Saccopteryx bilineata) was bat free. The advantages of laboratory research were clear to me. In my files, I find feedback on my lack of preparation in advanced theoretical ecology from Gordan Orians. Absolutely true; I was there mainly for the bats.

When I returned to Rockefeller, I started to explore the hormonal basis of reproductive behaviors in Xenopus laevis. Figuring out what was known about Xenopus required hours in the library stacks making notes on cards; lots to be said for Google Scholar and personal computers. However, I discovered the eccentric Sir Lancelot Hogben, the psychoanalytically inspired studies of William Russell on Xenopus sex behaviors and John Hutchison’s thesis research at the University of Capetown (UCT) on male clasping. I decided to start by determining whether the zz genotype contributed to the clasping “reflex” of males.

Mikamo, a scientist in Emil Witschi’s group, then at the Population Council, gave me advice on rearing frogs. I started by replicating the estradiol effect with tadpoles. I’d induce females to lay eggs and males to fertilize them while clasping using human chorionic gonadotropin (the basis of the first pregnancy test). I set up the tanks, went away for the weekend and they had all died by Monday. It was the water (it is pretty much always the water with Xenopus). I next tried to feminize males with estradiol; they died too (too much estradiol is toxic to the liver). But when I ovariectomized females and implanted them with pellets of testosterone, they clasped other females. Clearly their zw genotype did not prevent them from exhibiting androgen-stimulated, male-typical sex behaviors. I turned from behavioral genetics to endocrinology and set about describing the effects of gonadal steroids on the behaviors of adults and mapping the distribution of androgen and estrogen receptors in their brains with Joan Morrell, now at Rutgers, but then a new postdoc in Don Pfaff’s lab.

It was not until 2008 that the genetic underpinnings (i.e., DMW) of the X. laevis sex determination pathway were identified. How estrogen exposure during a short developmental window (tadpole stages 50–52) produces ovaries in genetic males, and whether or not estrogen plays an endogenous role in producing females, is not clear to this day. During this critical period, the developing gonad expresses ESR1 (estrogen receptor α) but not aromatase (the enzyme that converts testosterone or T to estradiol or E). However, the gonad is not the only possible source of estrogenic compounds in tadpoles, nor is estradiol the only estrogen. In 1995, before DMW was identified, Lydia Kang in our lab examined steroid synthesis in tadpole gonads and interrenal (frog adrenals) glands. Androgens (T and dihydrotestosterone or DHT) are detectable as early as tadpole stage 47. Δ5-3β-Dihydroxy steroid dehydrogenase, an essential enzyme for generating Δ5 steroids, is present in both interrenals and gonads at stage 47. Both DHT and T bind to the androgen receptor, but DHT metabolites, 3α and 3β diol, do not; the latter is a high-affinity ligand for ER2 (estrogen receptor β). My guess is that circulating 3βdiol from the interrenals binds to ER2 in the developing gonad and facilitates ovarian determination.

The genetic mechanisms responsible for primary sex determination vary across the Xenopus phylogeny; even within populations of one species (e.g., X. tropicalis; Roco et al. 2015). In contrast to the roles of estrogens and androgens, strongly conserved across vertebrates, primary sex determination is remarkably labile. All that matters is reliably producing offspring with ovaries and others with testes, although some genes (such as DMRT1) are particularly handy for the process.

Rockefeller University: Neural Control of Vocal Behavior

I defended my PhD in 1975. My papers (three research reports and a review) were already published. Don Griffin, who was on my committee, gently suggested that vocal behaviors might be more interesting than clasping. Art and Fred had just published a paper illustrating the pronounced sexual dimorphism of vocal control nuclei in the canary and zebra finch forebrain, so this experimental system seemed like a good one for a postdoc.

The goal of my postdoctoral research was to establish the connectivity of vocal control circuitry in the canary forebrain, starting with the thalamo-recipient nucleus, Field L, using two newly developed methods: anterograde tracing with tritiated amino acids and retrograde tracing with horseradish peroxidase (HRP). Bernice Grafstein (see Volume 3) and McEwen had just shown that radiolabeled amino acids are incorporated into proteins, transported from the cell body to axon terminals, released, and taken up by postsynaptic neurons. Jennifer and Matthew LeVail had shown that HRP is taken up by presynaptic endings and transported to the cell body. I determined how auditory input from a primary forebrain auditory nucleus (Field L) gains access to vocal motor circuitry (HVc and its target in ventral forebrain, RA). Some months into the project I asked to see the brain sections (Fink-Heimer staining to reveal degenerating terminals) that Teg Stokes and Tiana Leonard had prepared, outlining the location of Field L after lesions in the thalamic auditory nucleus, ovoidalis. To my horror I discovered that months of work had been based upon a single brain, which, however, turned out to have had a perfectly placed lesion. Tracing results appeared in two papers identifying new regions (Nif, Uva) in the song circuit. Coordinating what is heard with what is uttered, is one theme of our current research in Xenopus.

I had a marvelous time in grad school and as a postdoc. Lily Conrad, a very close friend to this day, joined Don Pfaff’s lab in 1972 after undergrad research in Gary Lynch’s lab at University of California, Irvine. Don describes Lily in one of his books as someone who could have been “the next Cajal” had she not gone off to med school. It was Harvard Medical School. Lily went into emergency medicine at the beginning of this specialty; her paper on cougar attacks is a classic. We’d hang out with the physics grad students, go disco dancing, take ballet and modern dance lessons, and run around at SfN meetings together.

Once I started looking for a job, about the middle of my second year as a postdoc, I’d share what I’d figured out with other friends, such as Tom Rainbow in the McEwen lab. I applied to two places, was invited to give a talk at both and chose the Princeton Psychology Department. I started the process of job negotiation, designing a lab, and writing a National Institutes of Health (NIH) grant while awaiting the birth of our daughter Danielle. Later, when I moved to Columbia, Tom and I collaborated on steroid receptor biochemistry; we’d go out to play video games (PacMan, Centipede) during incubations. Tom taught me how to use my first personal computer, an Atari. One day at Columbia, in front of the mailboxes, my fellow faculty member, Steve Schuetze, asked me if I had heard the “news from Princeton.” Tom had been killed jumping onto a train from Princeton to Philly (he was a faculty member at Penn). Steve, also a close friend, was himself killed by a truck during a bicycle trip to celebrate his promotion and tenure. “When sorrows come, they come not single spies, but in battalions” (Hamlet).

Mentors and Role Models

Becoming a faculty mentor is quite a lot like becoming a parent—or laboratory rotations—in the sense that it is as much about what you did not like—as a child or a trainee—as what you did. Today, when choices are wider, students can use the mentoring reputation and record of the advisor in addition to the science itself. At Rockefeller, when I was a graduate student and postdoctoral fellow, there were no women in tenure-track faculty ranks in my fields of interest. Don Pfaff, my PhD advisor, was a good model of a careful, methodical, productive, and thorough scientist. Fred Nottebohm, my postdoc advisor, was also very productive with an intellectual style characterized by imagination and metaphor.

No women faculty did not mean no women mentors. Christiana (Tiana) Leonard, for example, was a member of Carl Pfaffmann’s group. Her MIT PhD advisor was Walle Nauta, a gifted neuroanatomist, and she trained me and Lily in histology and neuroanatomy. Tiana was also a major contributor to the early mapping of bird song system and subsequently had a distinguished career at the University of Florida. Bernice Grafstein, still an active professor at Weill-Cornell Medical School, was another mentor; she continued to teach developmental neurobiology to us even after she moved across the street to Cornell and is responsible for my enduring interest in the subject. I reconnected with her when I became a term trustee of the Grass Foundation. While Tiana and Bernice were clearly gifted researchers and role models, it did not appear, at that time, that Rockefeller would be the right environment for a young woman faculty member. Fortunately, time has wrought changes at Rockefeller, not just for women but for scientists from diverse backgrounds.

Princeton: Establishing a Model for Vocal Communication

When I interviewed for the job in psychology at Princeton, Mark Konishi (see Volume 6), who I’d also thought of for a postdoc along with Harvey Karten, then at Stony Brook), was on his way to CalTech. I asked him what he thought about the place, and he said that it was a good first job, and so it was. When I started, as is somehow usually the case, my lab wasn’t ready, so I commuted to teach and kept doing research at Rockefeller. I’d been hired, I surmised, to teach neuroanatomy and I prepared by sitting in on the Cornell Medical School course and making a slide set of the rat brain (mice had not yet ascended). I’d ferry frogs to and from New York City, usually by train. At PJ Clarke’s restaurant one night, I met my mother for a dinner during which the people at the next table all stood up and fled. I figured it was a gigantic cockroach, but when I got home, two frogs were missing. I dashed back and scrabbled through the sawdust on the floor until the clean-up crew told me they were safe in a tub in the basement.

At Princeton, I also taught one lab course. My “students” were Dan Sanes, Terry Sejnowski, John Paton, and Marilyn Yodlowski. Louis Sokoloff’s (see Volume 1) 2-deoxyglucose method had just come out (basically a precursor to the immediate early gene approach but based on trapping labeled metabolites of glucose in active neurons,) so I figured we’d try that out. Terry, then a postdoc in Alan Gelperin’s lab, used the technique in a mollusk (Limax) and the rest of us applied it to Xenopus. The course resulted in four papers, including mine in Science (1980) and Terry’s in Nature (1980). The first was a help in getting my second job. The second paper was also consequential, although perhaps not just for the science.

I was at a Gordon Conference that summer and arranged to meet Terry (who had just taken the neurobiology course at the MBL and was on his way to Steve Kuffler’s lab at Harvard) in Woods Hole to revise the paper. I drove to Woods Hole and my strong ties to the MBL began (Kelley 2017). While writing Miranda Robertson (editor at the time; Dear Madam, so pompous of us), Terry and I decided to send in a possible cover picture of a 2DG-labeled Limax neuron. This same picture later appeared on the cover of a book on how life on Earth had been seeded by aliens. Don’t ask; I have absolutely no idea. Anyway, Terry got Harvard to invite me for a seminar. and I met Alison Doupe, an extraordinary and creative bird song neuroscientist, then in Paul Patterson’s lab. Alison was a wonderfully generous person and was also very funny. Mark Konishi (her postdoc advisor) once sent her in his stead to Paris to give a talk as she was fluent in French. Alison practiced the talk with her Parisian uncle and his partner. Their major comment was her Canadian accent; not something easily revised for a talk the next day. Alison and her lab were responsible for our understanding that two mysterious forebrain songbird nuclei, lMAN and area X, are homologs of the mammalian basal ganglia, leading to the idea that injecting variability into programs is an important circuit function for motor learning.

Why did I go back to Xenopus after my bird song postdoc? Mostly because I wanted to understand male- and female-specific innate vocal communication at the cellular and molecular levels. Also, birds are fragile, short-lived, and have small clutches; Xenopus is robust and long-lived and a single mating produces hundreds of tadpoles. Because Xenopus are nocturnal, totally aquatic throughout life and prefer murky ponds, sound production and reception are essential for social communication. The songs themselves produce acoustical “proxies” for analysis of motor programs. Birds instead are diurnal and thus also use visual cues in social behavior. Their vocal organ is an evolutionary novelty, not an adaptation of the ancestral vocal organ shared by frogs and mammals: the larynx. Male canaries and zebra finches learn their songs, making them one of the few model systems for vocal learning, but I was more interested in sex differences in innate behaviors. Another consideration was independence. Nottebohm and Konishi had established a powerful model system for learned vocal behaviors; there was no reason I couldn’t do the same for innate vocal behaviors, although I still maintain close ties to the bird song community.

At Princeton, our lab began to establish Xenopus as a model system for understanding vocal communication and its sexual differentiation. I wrapped up my thesis work by characterizing female sexual behavior, mapping DHT binding in the central nervous system (CNS), and (with Sol Erulkar’s lab at Penn) showing that androgen controls the excitability of clasping motor neurons in the spinal cord. Dan Wetzel, our first graduate student (now a researcher at IBM), characterized male advertisement calling and its endocrine control, including an unexpected role for gonadotropin. Using the 2DG method, combined with steroid autoradiography, I showed that both auditory and vocal nuclei in X. laevis accumulate gonadal steroids. Martha Constantine-Paton, a frog developmental neurobiologist, then married to John Paton, was in the Princeton Biology Department. I had first met her as a graduate student in Bob Capranica’s lab, swapping eye and ear anlagen in tadpoles. I planned to explore the developmental origins of sex differences in the vocal system; Martha helped me learn classic embryological transplantation methods at Princeton. On the train between New York City and Princeton, I’d spend the morning planning my lectures and the evenings marking up Current Contents for reprints.

Moving to Columbia

When I first visited the MBL (summer of 1980), Fred was teaching in the Neural Systems and Behavior course, as was Eduardo Macagno from Columbia. Ed had joined Cyrus Levinthal to convert Columbia’s Zoology and Botany Departments into a single department: Biological Sciences. John Hildebrand had been recruited from Harvard. He shared my interests in developmental neurobiology as did Steve Schuetze, who had just arrived from Gerry Fischbach’s lab (see Volume 9). Cy offered me a job, but I told him that I saw no point in starting over again but would move if Columbia offered me tenure. The result (after the usual to and fro, with some complications of departmental politics) was that I moved to Columbia in January of 1983 as a tenured faculty member, six and a half years after I was awarded the PhD. I thus was never subject to the excruciating experiences of even the most accomplished junior faculty today and reduced my commute from 4 hours to 1.5 hours daily. I was also shielded from possible adverse consequences of being an outspoken person and never had to worry about antagonizing someone who might participate in the tenure decision.

As I reviewed my Princeton correspondence just before the move (there were secretaries who typed and made carbon copies, not emails), I remember the fall of 1981 as a busy and happy time with exchanges of paper reprints between our lab and many others. Marty Chalfie was also moving to Columbia from Sidney Brenner’s lab and offered me a place to stay before that year’s Neuroethology Gordon Research Conference in the United Kingdom. In 2008, I traveled to Stockholm when Marty was awarded the Nobel Prize and Martha Constantine-Paton, now at MIT and married to Bob Horvitz, was a fellow guest. I interviewed John and Martha’s son Joe for Columbia’s Neurobiology and Behavior (NB&B) graduate program without realizing that I’d published papers with both of his parents. Joe now directs the Champalimaud in Lisbon, and I advise their graduate program. Science is social.

When I joined Columbia’s Biology Department, they had admitted a graduate student interested in our lab: David Sassoon. David (now at University of California, San Francisco, and the Salpetriere) pioneered our studies of sexually differentiated laryngeal development and its hormonal control and identified the juvenile muscle satellite cell (discovered by Alex Mauro at Rockefeller) as an androgen-responsive myogenic stem cell. Dennis Gorlick, our first postdoc, and Dan Wetzel also moved from Princeton to Columbia with me. Dan and Ursula Haerter (now a labor lawyer) mapped the Xenopus vocal circuit. Patty Hannigan (now Dr. Lotitio and in medical practice) continued her Princeton undergraduate thesis at Columbia, defining sensitive periods for masculinization of vocal behaviors.

I started teaching in NB&B and would take the entire lab to Woods Hole for the summer, including Rob Raguso (now at Cornell), a family friend of Patty’s on his way to start college at Yale. Andy Bass, a PhD with Glenn Northcutt (now also at Cornell), joined us in teaching NB&B as did Neil Segil, a former lab member, now at USC. Russ Fernald (Stanford) set up a unit on the diurnal rhythm of photoreceptors in monkfish, collecting them with students in the morning, teaching the dissections after lectures, and cooking them for dinner. I was so happy that our children could be in Woods Hole during the summers, at Stony Beach, the Children’s Science school, riding, swimming, playing tennis, and collecting insects. My MD/PhD husband Richard took the physiology course one summer and stood by to resuscitate us if needed when the collecting boat came up with a huge electric eel for Andy and Neil to dissect.

In 1982, I was also in conversation with Martha Tobias, a PhD student of Roy Ritzmann’s at Case Western, to join the lab as a postdoc. John Hildebrand was directing the neurobiology course and Martha, who had stayed on at the MBL to do research after the NS&B course was over, asked who at Columbia was working in neural development. We hadn’t published anything yet, but John knew what was in the works and Martha joined us, rising through the ranks to senior research scientist.

Having Martha as a research partner was a boon; she brought with her not only a strong interest in behavior and development but also remarkable skills in electrophysiology. Until her (much-lamented) retirement, we ran the lab together. Martha was responsible for many early research findings, including the isolated vocal organ (larynx) that “sings” ex vivo and the role of estrogen in the strength of the vocal neuromuscular synapse. Although she did not share my interests in molecular biology and genetics, Martha became keenly interested in vocal evolution after Joe Thornton (now at Chicago) and Ben Evans (now at McMaster) joined us as graduate students and published two seminal papers on the evolution of vocal signaling in 2011 and 2014. Also in 1995, after Apartheid ended, Martha moved her family (the artists Richard and Luca Chiriani) to South Africa to start fieldwork on Xenopus (see the section “Field Research in Africa”). We later went to Gabon, advised by Carl Hopkins at Cornell, who was studying electric fish and had noticed Xenopus for sale in a market north of Libreville. The songs of the species we collected in Gabon (X. mellotropicalis) were included in Martha’s 2011 paper, using the robust molecular and morphological phylogeny that Ben Evans and his lab at McMaster had established.

Sexual Differentiation

In the 1970s, our understanding of the basis for male- and female-specific (“sexually dimorphic”) behaviors was based on a pivotal study in guinea pigs (Phoenix et al. 1959). The authors concluded that androgens act early in development to organize sexually differentiated behaviors and later in adulthood to activate behaviors. The questions we addressed in Xenopus in the 1980s were as follows: (1) How are these developmentally segregated effects exerted at the molecular and cellular levels? (2) What muscles and neural circuits are responsible? (3) How does the action of hormones sculpt and energize the neural circuits underlying sexually differentiated vocal behaviors in Xenopus? and (4) What are these circuits?

Answering these questions required characterizing the vocal repertoires of the sexes as well as their endocrine control, identifying sexually differentiated features that underlie the neuromuscular control of vocal behaviors and determining how these diverge developmentally in males and females. We discovered the responsible sensory and motor components, the neural circuitry that connects them as well as how each is shaped developmentally in the sexes and affected by hormones. For example, David Sassoon discovered that the greater number of laryngeal muscle fibers in males is due to androgen-stimulated proliferation of a specific class of muscle stem cells. Neil Segil and David discovered the extraordinarily high levels of androgen binding in the developing larynx. John Roberts (now at Westminster College) discovered that thyroxine exposure during metamorphosis is required for testosterone-directed laryngeal masculinization (androgen competence). In the nervous system, I used the Golgi method to characterize the sexually differentiated dendritic arbors of adult vocal motor neurons and Jane Dennison from Australia used EM to find out how and when vocal motor neuron numbers diverged in the sexes. I also carried out my first molecular biology experiments in 1985 to identify tissue-specific expression of mRNAs in Xenopus tadpoles with fellow faculty Mark and Eva Rastl-Dworkin.

The 1980s were a prime period for developmental neurobiology. We learned, for example, that in Xenopus androgens can regulate ontogenetic cell death to sculpt sex differences in laryngeal motor neuron numbers. As an undergraduate, Jeremy Kay (now at Duke) identified the dying laryngeal motor neurons. The complex crosstalk between these neurons, their muscle targets, and androgens was clarified by Melanie Marin (now at Mt. Sinai) and Martha. Chris Edwards determined that prolactin closes the sensitive period for androgen action on laryngeal motor neurons and their muscle targets. Chris went on to postdoc with Gary Rose and discovered the fascinating neurons in the frog inferior colliculus that count sound pulses; he is now in medical practice.

In short, sexual differentiation of the effectors for male-specific vocal patterns is due to orchestrated action of hormones that determine timing of cell proliferation, differentiation, and cell death. In many ways, Xenopus—with its great experimental advantages for developmental biology and striking sex differences in vocal behaviors and their neural underpinnings as well as sensitivity to the same hormones that direct sexual differentiation across vertebrates—has been a powerful model system for understanding the hormonal control of sex differences during development.

Vox ex Vivo: The Isolated Brain and Larynx as Engines for Discovery

At Columbia, we also began to explore how the brain and vocal organs generate vocalizations in males and females. In the summer of 1984, Martha and I stayed on at the MBL after the neural systems course ended to try to coax isolated brains to “sing”—that is, to generate patterns of activity recorded from the laryngeal nerve as it exits the brain that match patterns of male- and female-specific songs (known as “fictive” songs). When these attempts failed, Martha turned to the larynx, stimulating the laryngeal motor nerve as it enters the brain posteriorly. If the nerve was stimulated with the pattern of a male’s song, the attached larynx generated actual sound pulses that matched those produced by a frog singing under water, including the sound frequencies that make up each sound: vox ex vivo. As a postdoc, Ayako Yamaguchi (now at Utah) recorded from the laryngeal nerve of singing male and female frogs and showed that nerve compound action potential (CAP) patterns matched male- and female-specific actual vocal patterns. CAP shapes in males (but not females) closely resemble individual motor neuron action potentials, suggesting that male laryngeal motor neuron firing is closely coordinated. Martha then used the isolated larynx to discover (using a classic quantal analysis) that laryngeal motor neuron synapses (NMJs) in females are “strong”: each nerve action potential releases enough acetylcholine for the muscle fiber to depolarize and produce an action potential, whereas male synapses are “weak,” requiring multiple action potentials for muscle fiber contraction, a sex difference I have always relished. We then went on to show that the synapse starts out during development as weak in both sexes and strengthens in females during exposure to estrogen during puberty. Exactly how this happens at the molecular level is still not known, though Kwok Hang Wu (now at Yiling Pharmaceutical) in his postdoctoral research, uncovered several estrogen nuclear receptor variants that are candidate participants in this process. Together these studies revealed that, in X. laevis, male- and female-specific vocal patterns are generated by the brain and that the male- and female-specific spectral features of vocalizations are due to the larynx. This separation of functions turned out to be critical for our current genomic approaches.

Refining the medium bathing the frog brain and continuously oxygenating this “frog juice” allowed us to use the ex vivo preparations to understand the neural circuitry that generates song patterns. With Blair Simpson (an NS&B student who joined our lab briefly after 1984 NS&B; now professor of psychiatry at Columbia), we used the isolated brain to map the locations of glottal and laryngeal motor neurons by applying the fluorescent dye lucifer yellow to each of the nerve rootlets that form the N.IX-X bundle as it exits the brain case. She then double-labeled laryngeal motor neurons by injecting HRP into laryngeal muscles, revealing sexual dimorphisms in number, location, and size of vocal motor neurons. Later, an undergraduate at Columbia, Catherine Brahic (now environment editor at the Economist), confirmed and extended our previous in vivo studies using HRP-WGA in vivo. She used anterograde and retrograde tracing in the ex vivo brain and discovered the involvement of the dorsal raphe nucleus in hindbrain vocal circuitry. All of the connections Catherine described are present in both male and female brains (though more robust in males) but must function differently in response to the sex-specific hormonal environment.

The motor neurons that drive sound production are located at the caudal end of nucleus ambiguus (NA) in the hindbrain. As a graduate student, Erik Zornik (now at Reed) injected fluorescent tracers into NA and its major afferent, the parabrachial nucleus (PB) in ex vivo brains, establishing the interneuron classes that account for the ipsi- and contra-lateral connection in the hindbrain vocal circuit. Ayako Yamaguchi (now at the University of Utah and Erik’s postdoc advisor) followed up on this observation when she established her lab at Boston University, showing that 13 weeks of androgen treatment can completely masculinize vocalizations of adult females. With Heather Rhodes (now at Dennison), Ayako found that when an isolated Xenopus brain is exposed to serotonin, fictive male- and female-specific “songs” can be recorded from the laryngeal nerve as it exits the hindbrain. This effect of exogenous serotonin reflects the endogenous action of serotonergic neurons in the raphe on the vocal circuit described by Catherine Brahic.

One challenge in going from hormonal control of behavior to underlying mechanisms is determining which hormone targets (sensory, motor, CNS) are responsible for endocrine actions. The ex vivo approach allowed us to define the effects of hormones on multiple components of the vocal circuit. As a graduate student in our lab, Erik Zornik used the ex vivo brain to establish that PB, formerly known as DTAM), provides powerful, monosynaptic, excitatory input to laryngeal motor neurons in caudal NA while inhibiting glottal motor neurons in rostral NA. This circuit motif is essential for vocal production in Xenopus because the closed glottis prevents fluids in the buccal cavity from entering the space above the arytenoid disks, which (as I showed later with Coen Elemans at Southern Denmark University or SDU) prevents disk separation from attaining the velocity required for sound production.

The nuclei of the hindbrain are highly conserved across extant vertebrates, from frogs to mammals. The parabrachial complex in mammals includes neurons that gate the expiratory to inspiratory transition. Air-powered sound production requires an open glottis. Maintaining the ability to produce courtship vocalizations during the evolutionary transition from land back to water in Xenopus thus required a switch in PB neuron connectivity from excitatory to inhibitory. What drives the activity of PB neurons? In her PhD thesis, Irene Ballagh (now at the University of British Columbia) used microstimulation and dye-tracing in ex vivo male and female brains to establish reciprocal, functional connectivity between PB and the central nucleus of the amygdala (CeA). Fictive sex-specific songs can be elicited ex vivo by microstimulation of the CeA in both sexes. In mammals (mice, bats, and rhesus monkeys) the CeA also responds to, and is active during, the production of vocal signals.,

The ex vivo brain also revealed novel endocrine contributions to the Xenopus song circuit. Dan Wetzel’s PhD thesis had shown that male advertisement calling relies on gonadal androgen and that a pituitary hormone, gonadotropin (luteinizing hormone, LH) synergizes with androgen to enable robust male calling behavior. As a postdoc, Eun-Jin Yang (now at GlaxoSmithKline) and graduate student Brian Nasipak (now at Alnyam Pharmaceuticals) discovered that gonadotropin, acting through the LH receptor, acts directly on the CNS to increase calling. LH application to the ex vivo Xenopus brain increases expression of its target, egr-1, even in the absence of neural transmission. Gonadotropin thus may play a novel integrative role in modulating both reproductive physiology (modulating circulating androgen or estrogen levels) and behaviors

Field Research in Africa

Xenopus are species of the family Pipidae, native to Africa. Pipids are Paleobatrachian frogs that migrated from what is now Europe into Gondwanaland (then the conjoined continents of Africa and South America) approximately 160 million years ago. Other Pipids are found in South and Central America and all are aquatic. The Pipids survived the mass extinction event at the Cretaceous-Paleocene boundary that wiped out many terrestrial frog species, leading to the rapid speciation of the more recently evolved Neobatrchia species threatened today by global climate change and habitat loss. I suspect that when we humans are long gone from this planet (via migration to other worlds and/or extinction), if Earth still supports its animal life forms, Xenopus will be among them.

To get at the actual biology of our lab findings in Xenopus, we traveled to Africa. When Apartheid ended in 1995, Martha and her family moved to Capetown in South Africa to study the species’ vocal communication. Luca enrolled in a Rudolf Steiner school where he never had to wear shoes, Richard painted, and Martha was hosted by Mike Picker at UCT, who also had hosted Ben Evans’s research on hybrid zones between X. gilli and X. laevis a few years before. Martha’s family settled into a house in Rondebosch that (crucially) had a swimming pool and into life in immediately post-Apartheid South Africa, with its multiple curious aspects that I also experienced when I visited.

Usually, research in neuroethology starts from behavior and then moves to neural circuits. In our case, however, this direction was reversed. A male X. laevis’ advertisement call contains alternating rapid and slow trains of sound pulses (fast trill and slow trill). Fast trill is amplitude modulated: the loudness of each sound pulse increases progressively within each fast trill segment. Male-specific facilitation at the laryngeal neuromuscular synapses (Martha) and progressive recruitment of motor neurons (described by Ayako Yamaguchi during her postdoc) account for the progressive loudness of sound pulses during fast trill. Female laryngeal synapses do not facilitate. Martha used quantal analysis to confirm that the locus of this sex difference was pre- rather than postsynaptic. The male larynx can only produce sound pulses because each successive action potential increases the amount of acetylcholine released from the synapse.

In the lab, females are more attracted to intensity-modulated male songs than to calls without this feature. Modulating the intensity of a behavior used during social interactions is a common motif in ethology (animal behavior). I wondered whether, for example, this modulation helped the female find the singing male underwater when she was just about to extrude her eggs. You might ask, why did we have to go to Africa to find this out? We had tried phonotaxis (attraction to sound) behavioral experiments in the lab using Jocelyn Crane’s crab tanks. A rotating graduate student spent an entire summer broadcasting calls that varied in amplitude modulation to sexually receptive females from an underwater loudspeaker. He discovered that a fiberglass tank created acoustic “nulls” as the female swam toward the male (the reflected sound waves cancelled out the frog’s actual song) and so returned to medical school. We eventually built a large trapezoidal tank that had better acoustics for recording male songs across species. Recently, Ursula Kwong-Brown and Young Mi Kwon in our lab found that a large glass aquarium works best because the sound energy radiates into the air outside the tank in response to sound pulse impact and echoes are greatly diminished. However, our “field research” study was in 1995 not in 2019 to 2022.

The swimming pool in Rondebosch (termed the “artificial pond” in our 1998 PNAS paper) turned out to have excellent acoustics. Martha’s approach was to slow down the male’s ability to locate the female by confining her to the end of the pool, where the steps were, with a plywood barrier. A funnel trap inserted into a hole in the middle of the barrier let the male enter the female’s chamber but not exit. However, wet plywood is acoustically transparent. The male could not find the hole and began swimming rapidly back and forth along the length of the barrier, producing advertisement calls. The sexually receptive female responded to the vocalizing male with a call we had never heard before that Martha named “rapping” (it sounds like a Geiger counter or machine-gun fire). The male then called back with an ardent version of advertisement calling, the answer call, creating underwater duets. Males do not habituate to rapping; it is an acoustic aphrodisiac. In contrast, Taffeta Elliot (now at New Mexico Tech) showed in her PhD thesis that female ticking suppresses male calling.

In ponds that contained hundreds of sexually mature males, Martha would record only one calling male. Back in the lab, she determined that this observation reflects male vocal dominance. Pair-wise testing revealed that dominance hierarchies last for up to two weeks. Groups of terrestrial male frogs form calling choruses. Females can locate males because in air call intensity diminishes rapidly with distance. In water, however, sounds can be heard over very long distances. Male-male vocal suppression avoids underwater cacophony that would hinder the female’s ability to locate a calling male. Testing pairs of males revealed that male-male vocal dominance is long-lasting (at least 45 minutes). Taffeta, however, found that the suppression of male calling by the female unreceptive call (ticking) is transitory. Males habituate after about 15 minutes and go back to calling. So, the socially appropriate response of a male-to-female rapping is to switch from advertisement to answer calling. The appropriate response to male ticking is transient vocal suppression, while the response to advertisement calling should be prolonged vocal suppression. These observations later enabled Ian Hall to determine, using lesions, that the extended amygdala (CeA/BNST) is essential for a male’s ability to produce socially appropriate vocal responses to ticking vs. rapping calls from females.

Back in the lab, Sandya Viswanathan during her undergraduate (USC) summer research helped us replicate and extend the Rondebosch results. Because the mechanism of sound production in Xenopus is internal (rapid separation of the arytenoid disks producing vibration of the whole body), visual inspection doesn’t tell you which frog in a pair is calling. However, the frequencies that make up sound pulses in males and females differ, allowing male and female contributions to the duets to be readily distinguished. For male-male pairs, we used an insensitive hydrophone (a waterproofed hearing aid) to distinguish caller from listener. Vocal responses can also be reliably evoked by underwater call broadcasts. Taffeta Elliott presented males with broadcasts of female calls in which the interval between sound pulses varied from short (rapping) to long (ticking). From the male’s response (transient vocal suppression or answer calling), she determined that the interpulse interval border between ticking and rapping is graded rather than categorical. Playbacks also allowed us to disentangle the respective roles of spectral (pitch) and temporal (rhythm) features in vocal communication. As a postdoc, Clementine Vignal (now at the Sorbonne) then determined that males use the frequency content (spectral features) of sound pulses to identify the sex of the caller and the rate of sound pulses (temporal features) to distinguish attractive from repulsive female calls.

Neural Circuits for Vocal Communication

Understanding the neural link between production and perception of vocal signals requires knowing where auditory information intersects with pathways that control vocal patterns. Chris Edwards mapped the hindbrain and midbrain components of the ascending auditory pathway and Taffeta (coadvised by Jakob Christensen-Dalsgaard at SDU) recorded from the Xenopus inferior colliculus, determining that neurons in the laminar nucleus are rate tuned. At the time, we had not yet discovered the phylogenetic significance of the two dominant frequencies (dyads) that comprise male sound pulses in Xenopus, so the spectral representation of dyads from the auditory midbrain to the forebrain remains to be explored.

Many years earlier, Aubrey Gorbman had speculated that a region of the forebrain, the ventral striatum (VS), might receive auditory information. When Catherine Brahic was in our lab, she placed fluororuby into the parabrachial nucleus in the ex vivo brain and showed that VS and PB are reciprocally connected. Studies of transcription factor expression by Spanish neuroanatomists (Morona and Gonzalez, 2013) then revealed that the VS is homologous to the central nucleus of the mammalian amygdala (CeA). When Ian Hall (now at Benedictine) lesioned the CeA in vivo, males lost their ability to distinguish information on sex and reproductive state conveyed by calls. Males responded to rapping with vocal suppression (the appropriate response to a dominant male) rather than with the answer call (the appropriate response to an ovipositing female). How this acoustic information is represented within the CeA remains to be discovered. However, the highly evolutionarily conserved CeA is clearly a key hub for recognizing social cues conveyed by conspecific sounds (across species, including mice, bats, and nonhuman primates). Ian and Irene’s experimental results form the basis for our current explorations in Xenopus of human neurodevelopmental disorders that affect social interactions.

Evolution of Vocal Circuits

Anyone exploring the biology of courtship, sex differences in behavior, neural circuits for innate behaviors, and related subjects will, sooner or later, be drawn to Darwin’s ideas on sexual selection. I started reading The Descent of Man on my train trips back from Princeton, perhaps influenced by colleagues in biology: Bob May and the Grants (Rosemary and Peter). Darwin’s convictions about the intellectual differences between men and women were irritating but provoked the idea that the expression of receptors responsive to sex-specific patterns of hormone exposure could be an important underlying mechanism for sex differences.

The evolution of neural circuits for producing and responding to innate (species-specific) vocalizations is a subject of our current research. Ursula Kwong-Brown, then a Columbia College Music and Neuroscience major, working with Martha, made a key discovery. Looking up from the bench one day while the underwater songs of males from various species were being played, Ursula asked: “Why is that frog singing a perfect fourth?” You can listen to what she heard online (Kelley and Kwong-Brown 2019).

Ursula and Martha discovered that the two dominant frequencies (DFs; i.e., dyads) in the sound pulses of male advertisement calls in each Xenopus “subgroup” (A, M, and L) form a musical interval: a perfect fourth (A), an octave (M), or a major/minor third (L). The exception is X. laevis, the species we had studied for many years, in which the DF2 (higher) to DF1 (lower) ratio is 1.14 instead of 1.22. This difference in sound dyad ratios between species that can hybridize (produce fertile progeny) now allows us to identify genetic loci associated with species differences in laryngeal sound production.

Ursula, Martha, and I then traveled to the University of Massachusetts, Amherst, to visit Sheila Patek (now at Duke), an expert in animal sound production mechanisms. Sheila pointed out that acoustic properties of Xenopus sound pulses could simply be an artifact of recording in glass tanks. For example, the snaps of the snapping shrimp in her lab weren’t “snappy” when recorded in the field (the great barrier reef in Australia); they were “thuddy.” We knew from Martha’s research that the sound frequencies in a pulse recorded from a male X. laevis in “the dish” (vox ex vivo) were the same as recordings in tanks (vox in vivo). I had just heard a talk by Ron Hoy about Damian Elias’ studies of the courtship “songs” of spiders: web vibrations that could be analyzed by shining light from a laser at webs and measuring reflected wavelength’s Doppler shifts. The returning reflection is higher when the web moves toward the laser source and lower when the web moves away. This is the same idea as the Doppler shift used by echolocating bats to determine whether the flying insect is going away from or coming toward them. I called Ron and he suggested contacting Damian. We called Damian and he hopped on a plane from Berkeley bringing his field laser with him.

The laser and sound recordings from a frog singing in a glass tank matched the acoustic recordings in vivo and in vitro across species. In fact, all parts of a frog’s body vibrate as a dyad when he sings; Ursula could record dyads from a single toe. These observations were key to figuring out (with Coen Elemans at SDU) how Xenopus produce their songs underwater without using vibration of laryngeal membranes powered by expiration. Air-powered vocal production is the mechanism we humans use and is one shared with current terrestrial frogs as well as ancestral Xenopus before they ventured back from land to water. Laryngeal vibration that produces two sound frequencies is an evolutionary adaptation that enabled Xenopus to continue to use vocal communication during courtship underwater.

Genetic Underpinnings of Innate Songs

While I was at Princeton, Christine (“Janni“) Nusslein-Volhard gave a lecture on the genetic control of development in fruit flies. When we talked, she revealed that she had wanted to determine the genetic underpinnings of fly innate behaviors but switched to development as more tractable. Later, at a Genes and Behavior Gordon Conference, I heard Kerry Shaw (Cornell) describe her investigations of courtship songs in Hawaiian crickets using a quantitative trait loci (QTL) approach, eventually revealing a close linkage between the courtship signals of males and the acoustic preferences of females.

Shaw’s studies illustrate one of the major challenges for understanding the evolution of vocal communication in courtship. How, as species and their courtship songs diverge, does the perception of that song diverge in concert? While we usually think of males doing the singing and females the listening, conspecific males also interact vocally (e.g., choruses); how do they recognize their vocal partners or adversaries? What about female duets? Martha showed that X. laevis females are not vocal when together, although they do compete for a male when gravid. I suspect that in species in which access to males poses a challenge to reproductive success, we might find females that also compete vocally.

When I met David Kingsley (development and evolution of spines in sticklebacks), he pointed out that the major challenge in using QTLs to identify genomic loci associated with a particular trait is quantitatively phenotyping that trait. However, Martha’s evolutionary studies of males and female Xenopus vocalizations had done just that, establishing temporal and spectral characterizations of vocal signals and their variation across the Xenopus phylogeny. So, with advice from Ben Evans, I began to think about how we might approach the genetic basis for species differences in Xenopus song production and perception.

One catalyst was Charlotte Barkan’s PhD thesis in our lab, which explored characteristics of male songs in two related species: X. laevis from South Africa and X. petersii from Congo. Hybrids between the two species produce intermediate vocal patterns in males. When Andres Bendesky joined the Columbia faculty, I heard him describe his postdoctoral research with Young Mi Kwon in Hopi Hoeckstra’s lab at Harvard. They used F2 hybrids between two species of Peromyscus (biparental and uniparental) to identify genomic loci associated with male and female parental behaviors. For this challenging project, Andres developed quantitative measures of parenting in both sexes. Using a hidden Markov model and low coverage sequencing, he correlated parenting styles and maternal or paternal species-specific genomic loci to identify candidate genes associated with these innate behavioral traits. Another catalyst was Yun Ding and David Stern’s (Janelia) 2016 Nature paper identifying an intronic retroelement in a calcium-activated potassium channel gene associated with interspecific variation in the frequency of sine song in flies. The “power of Drosophila genetics” (yes, I have species envy) pointed to a genetic change affecting sine song frequency. While female preference actually relies on the pulse part of the male’s biphasic songs rather than the sine part, this paper provided important proof of principle that the genetic approach to understanding the divergence of vocal behavior during speciation could work.

Xenopus has some real advantages for dissecting the genetic bases for innate differences in vocal communication. Studies in ex vivo preparations (the isolated larynx and brain) clearly revealed that the spectral properties of male songs are inherent to the vocal organ, while the temporal properties are inherent to the brain. This is not the case for other vocalizing species, such as terrestrial frogs, birds, and mammals, in which air movement and vibrating membranes power sound production, and both are driven by CNS motor neurons. Spectral features in birds/primates also require coordination with the syrinx/vocal tract or larynx/vocal tract and thus also rely on the CNS. In contrast, frequency information in Xenopus sound pulses is entirely due to the properties of its vocal instrument, the larynx. Temporal information in calls is almost entirely due to the hindbrain vocal pattern generator. In both sexes, laryngeal motor neurons and muscles must contract sufficiently rapidly to separate the arytenoid discs and cause the larynx (and the whole body) to vibrate, generating sound pulses underwater.

Martha had shown in her 2011 paper that the calls of both X. petersii and X. laevis are biphasic: periods of fast trill alternate with slow trill. By recording from the vocal nerve in the fictively singing brain of male X. laevis, together with recording from the PB in the rostral hindbrain, Ayako’s laboratory found that a distinctive local field potential (LFP) accompanies fast trill. Bob Schmidt had also shown much earlier that this LFP could be recorded from the PB in terrestrial frogs; thus, PB is a candidate ancestral character for anuran vocal production. In her thesis research (coadvised by Erik Zornik), Charlotte Barkan found a PB LFP in X. petersii with a duration corresponding to fast trill. The PB includes two types of rhythmically active neurons: fast trill neurons (FTNs) and early vocal neurons (EVNs). When synaptically isolated and stimulated with NMDA, the membrane potential of FTNs (but not EVNs) oscillates at the species-specific duration and rhythm. FTNs are thus strong cellular candidates for species-specific genetic differences that drive innate song patterns. An extensive pharmacological survey has not yet revealed specific cellular or molecular candidates for the species-specificity of FTN membrane oscillations. Our current QTL approach focusing on temporal aspects of advertisement calls should help to reveal candidate molecular mechanisms for heritable song patterns (CNS) as well as dyad harmonics (vocal organ).

X. laevis and X. petersii male songs alternate between fast and slow trills. D. melanogaster male songs are also biphasic; they alternate between pulse and sine song. So, when Emilie Perez and Avelyne Villain joined our lab as postdocs after their PhDs with Clementine Vignal, we asked Christa Baker and Mala Murthy at Princeton to collaborate in developing ways to analyze the quantitative variations in call features that we expected to find in X. laevis/X. petersii hybrids. Emilie, Avelyne, and Christa developed analytical methods that demonstrated variability in the spectral and temporal characteristics of F1 male hybrid songs. Andres and I recruited Young Mi Kwon back to the United States from Cambridge in the United Kingdom, and she then developed an automated pipeline for acoustic analyses for the many hundreds of male F2 hybrids required for the QTL analysis. Stay tuned, but we are cautiously optimistic that genomic loci associated with species-specific temporal features will include genes that contribute to innate vocal rhythms. Similarly, biomechanical and/or morphological features of the larynx also should be associated with the genetic divergence responsible for species-specific dyad ratios.

What about matching production to perception? While in our lab as a postdoc, Clementine Vignal had shown that Xenopus can recognize sex-specific spectral features of songs. Does auditory recognition of spectral or temporal vocal features coevolve with speciation? When Ian Hall joined our lab as a postdoc, Martha and Ursula had started developing auditory evoked potentials (AEPs) to look at auditory sensitivity to spectral features with help from Bob Dooling’s lab and Catherine Carr at the University of Maryland. Ian Hall then used AEPs to show that the auditory sensitivity of females was greatest, across several Xenopus species, for the spectral features of their own species’ male songs. Shifting either the higher frequency (DF2) or the lower frequency (DF1) in a sound pulse by a few hundred Hz abolishes this preferential sensitivity. This acoustic advantage is present in females but not in males and depends on gonadal steroids (testosterone is the major circulating hormone of females). AEPs most likely reflect activity in the acoustic ganglion and perhaps the dorsal acoustic medulla. I had shown that neurons in the X. laevis acoustic ganglion accumulate DHT (an androgenic precursor to 3β-diol), providing a possible explanation for hormonal regulation of auditory sensitivity. Emilie Perez then used AEPs to examine the auditory sensitivity of female F1 hybrids and found them to be most sensitive to the frequencies in male hybrid sound pulses compared with either parental species. One goal of our current studies in F2 X. laevis/X. petersii hybrids is to determine whether this sensitivity confers a behavioral advantage for females in locating a genetically compatible male.

I’ve glossed over the major experimental disadvantage of Xenopus for QTL analyses: the long time to reach sexual maturity and adult song production. Although careful attention to rearing can accelerate development, male X. laevis typically take at least 6 months (and females a year) before exhibiting an adult repertoire of behaviors. So, while a single mating can generate the large numbers of animals needed for statistical analyses of QTL data, we have had to wait for the F2s to grow up. Ironically, the 2020 Covid research pause was an advantage. Before the pause, we collected promising preliminary QTL data on male song spectral features. We are now analyzing temporal F2 hybrid vocal features in both sexes and their association with genomic fragments of maternal (petersii) or paternal (laevis) origin. When I first described this project to Mala Murthy, she commented that it is exceptionally ambitious. My suspicion is that she meant: “for someone whose experimental animals might outlive her.” I do have 35 year-old Xenopus in our colony. “If not now, when?” I replied.

Behavioral Genetics

One of the most contentious aspects of research in behavioral genetics is the idea that there are “genes for a behavior.” Many human behaviors are learned (e.g., language), and the idea that the individual’s genetic background might make a major contribution to their behavioral repertoire is controversial, to put it mildly. We do accept, however, that many human disorders are strongly influenced by an individual’s genome. For example, mutations in the BRACA1 and 2 genes have strong associations with breast cancer. Women with these genetic variants can choose to have prophylactic mastectomies. Some communities require testing for Tay-Sachs disease before a couple’s first date, identifying the risk that falling in love would have for future children. The brain is also an organ and subject to genetic variation, especially during development. Genetic variants that shape neural circuits have the potential to profoundly influence behaviors, including social behaviors and cognition, for better or for worse, and sometimes for both. For example, an individual’s genome might contribute to an intellectual disability or speech alterations or instead to enhanced sociality or creativity. Bipolar disorder (manic depression)—the most heritable of the neuropsychiatric disorders—is characterized by periodic alterations in mood but also by epochs of heightened creativity, as Kay Jamieson has so vividly described in her books.

After my initial exposure to autism (now termed autism spectrum disorder or ASD) as a high school student at the NSF summer science institute in Iowa, I was introduced again to behavioral genetics and neuropsychiatry when Ed Kravitz (see Volume 4) asked me to join the scientific advisory board of the Hereditary Disease Foundation (HDF), which is where I met Anne Young (see Volume 8). Founded by Milton Wexler and his daughter Nancy, the HDF is focused on Huntington’s disease (HD) but with the underlying premise that many neuropsychiatric diseases treated by Milton in his practice also were subject to inherited genetic variants.

My first meeting was in Santa Monica. The HD gene (Huntingin) had not yet been identified. The session opened with the presentation of a patient and was followed by a brainstorming session on how to identify the gene and ultimately understand how its known effects, the death of medium spiny neurons in the striatum, contribute to motor phenotypes (chorea) as well as to emotional and cognitive phenotypes. This simultaneously fascinating and catastrophic disease was ultimately traced to the Huntingtin gene and its expanded trinucleotide repeats; some progress in treatment has begun. My experience with the HDF, and that early exposure in Iowa to Nate Azrin and Ogden Lindsley’s attempts to use operant conditioning to shape the behaviors of autistic children, contributed to my current interests in the biology of neuropsychiatric disorders described next. The other contributing motivation came from realizing that my grandmother’s long illness was the result of severe bipolar disorder that must have genetic antecedents, although what these are, and how they relate to her psychosis, are not yet clear.

My thinking about genetics and behavior was also influenced by a workshop on the “Neuroscience Workforce” at the National Institute of Medicine, attended by representatives of “big Pharma.” The research programs at leading companies had switched from neural diseases, such as Alzheimer’s, to cancer because the biology was much better understood. Most neuropsychiatric disorders have a characteristic developmental trajectory. How can neuroscientists understand the basic biology of complex, genetically influenced behavioral variants and their severity?

While I was growing up, autism (see Family Pictures by Sue Miller), was viewed as the result of maternal behaviors: the cold mother. While behavioral therapies could be effective, particularly for self-harm, the psychoanalytic viewpoint prevailed for many years. Today, efforts by individual researchers and foundations (e.g., Simons) have enabled the identification of rare, highly penetrant genetic variants associated with ASD. Figuring out how these genes contribute to disease is a focus of many laboratories using mouse models and organoids.

The problem as I see it, however, is the basic biology. Autism is a neurodevelopmental disorder sometimes associated with intellectual disability and sometimes with specialized intellectual abilities (or both). A common feature across ASD is difficulty in social interactions. Would it be possible to understand the biological consequences of neuropsychiatric disorders and their associated genetic variants in a model system in which neural development is accessible, in which social behaviors and their development are well-characterized and quantifiable, and for which underlying neural circuits have been identified? That would be Xenopus.

Autistic Frogs?

We currently are gearing up to determine if knockouts of rare, highly penetrant ASD genes in Xenopus produce behavioral phenotypes, and if these can be rescued by exposure to estrogen during development. The inspiration for this project comes directly from recent work by Helen Willsey on X. tropicalis (Willsey et al., 2021) in which CRISPR knockouts of rare, highly penetrant ASD candidate genes in one cell of the two-cell-stage embryo produce either a larger or smaller forebrain in tadpoles. These morphological phenotypes can be rescued by drugs in the estrogen signaling pathway. Was Despert’s 1971 paper prescient?

I concede that frogs are not great models for the intellectual disabilities that often accompany early onset neuropsychiatric disorders. However, we have characterized the behavioral repertoire of both sexes in Xenopus during courtship and mating, and vocal components are readily quantified. From Gurdon’s early work growing tadpoles from eggs with nuclei from somatic cells to the present, Xenopus has been a powerful model for developmental biology. Mouse models currently dominate explorations of human developmental disorders, despite the inconvenience of interuterine development, in part because of major investment by the NIH in creating transgenic mouse lines and multiple experimental tools (e.g., CRISPR, viral vectors). However, as more and more genetic variants associated with human disease are identified, and as these experimental tools are applied to Xenopus, this animal model has emerged as a powerful biological system for understanding the basic biology of genetically influenced human disorders. We are simply extending this approach to innate social behaviors and their underlying neural circuitry.

Large stretches of the human genome are devoid of gene ontology (annotation linking genotype to phenotype). Families faced with a variant identified as contributing to the illness of their child, a variant whose “function” is not known, can recognize (through studies in animal models) that the variant is a “real thing.” It has a biological basis rather than resulting from their parenting styles or maternal factors. The social behavior network in the brain is one of the most evolutionarily conserved circuits in vertebrates. For example, the central nucleus of the amygdala—known to contribute to the production and perception of vocal signals during social behaviors from bats to monkeys—is essential for Xenopus males to distinguish the “voice” (male or female) of a singing frog and respond with socially appropriate vocalization (Hall, Ballagh, and Kelley 2013). The ability to make this distinction is required for reproductive success, plays a role in speciation and must be strongly selected for over evolutionary time scales. Autistic frogs? Maybe not, but genes and circuitry and behavioral alterations, probably yes. We will see.

Educating Scientists

Undergraduate Education

At Princeton, my first teaching assignment was developmental psychology. This was not a course I had ever taken. When I asked: “Why me?” I was told: “Well, you have children, don’t you?” I asked Marc Bornstein—who had taught this course the previous year but then moved to NYU—what I should cover. He said that it didn’t really matter so long as I gave one lecture on Freud and one on Piaget. I followed his advice; the remaining lectures were on the development of behaviors in species other than our own. Incorporating Piaget and Freud was easier than I imagined, in part because both had started out as biologists: Piaget studying the shells of mollusks and Freud search for the elusive reproductive organs of eels. As Piaget said: “I am convinced that there is no sort of boundary between the living and the mental or between the biological and the psychological. From the moment an organism takes account of a previous experience and adapts to a new situation, that very much resembles psychology” (Bringuier, 1969). I covered bird song as a model for children’s language development, as well as the seminal studies of Patricia Kuhl and the near-at-hand developing speech of our own children. My graduate teaching at Princeton was focused on neuroanatomy, a foundational skill for neuroscientists.

When I arrived at Columbia, John Hildebrand was teaching the cellular and molecular neuroscience course and I began teaching a complementary developmental and systems neuroscience course (always including bat echolocation) that I continue to teach to this day. I alternate with Rafa Yuste, who I recruited to Columbia during my brief stint as chair of biological sciences. In 1995, with Don Hood in psychology, I also created the neuroscience and behavior major that has had broad appeal for Columbia undergraduates.

Columbia College is distinguished by its Core curriculum: “The central intellectual mission of the Core is to provide all students with wide-ranging perspectives on significant ideas and achievements in literature, philosophy, history, music, art, and science” (Kelley, 2010). The Core originated in 1919, but science was not added to its mission until 2004. With David Helfand in astronomy and fellow science faculty members, I helped to create a new core course, Frontiers of Science (FroSci). FroSci started as a discussion in the Committee on Science Education about what students generally took to “satisfy” the science requirement, then two courses in one discipline for depth and one course in another for breadth. The problem as we saw it was that, as a general rule, only science majors had any contact with modern science. We proposed a one-semester course for all entering students, comprising both life and physical sciences, that would be taken by all first-year Columbia College students. The original idea was that faculty members would deliver one lecture, but Brian Greene pointed out that presenting a narrative really required three lectures. I was enthusiastic and so David and I went from one science department to another, promoting the idea and recruiting a small band of fellow scientists of like opinions. The inaugural Howard Hughes Medical Institute (HHMI) Professors program supported my efforts in FroSci. Today, convening yearly with my fellow, distinguished science educators and researchers (many of whom are members of the NAS) has been incredibly helpful, particularly in discovering new approaches (such as “intelligent tutors”) to help level the playing field for a talented entering class with diverse preparation in science. FroSci now includes three weekly lectures from effective science communicators among the research faculty. Student skills are honed by weekly small seminars led by postdoctoral Columbia Science Fellows dedicated to teaching.

Of every hurdle that I have faced, FroSci was the most difficult. We were strongly backed by the then deans of the college (Austin Quigley and Kathryn Yatrakis). The faculty were skeptical, however, most students were hostile, and the ratings were abysmal. Strenuous efforts were initiated by a new dean and other faculty to come up with a replacement course that would be more popular. However, everyone who was interested enough to teach science to first-year students was already busy with other initiatives or teaching in FroSci. Then in 2007, David went to a new college in Canada, Quest University (eventually as its president), leaving me to hold the fort, luckily with Dr. Ivana Hughes, a chemist and gifted educator. We struggled on, and David returned in 2015. Finally, the course emerged triumphant during the pandemic. FroSci was exceptionally well prepared for new learning styles, and thus has inched, bit by bit, year after year, into favor with Columbia students. As Churchill said (1941 at Harrow): “Never ever, ever give up.”

Graduate Education

In 1983, there was no Neuroscience Department at Columbia. Neuroscience PhD trainees at the Medical School received their PhDs from physiology, with many mentors housed in the Center for Neurobiology and Behavior that Eric Kandel (see Volume 9) had set up when he moved to Columbia from NYU. Trainees at Columbia’s Arts and Sciences campus received PhDs from biological sciences or psychology. I had, by then, a strong interest in developmental neurobiology and taught a graduate course on this subject with Jane Dodd and Tom Jessell. I also took an Ion Channels course from Steve Siegelbaum and John Koester. I pictured ion channels as tiny entities with innate behaviors.

Eventually, I talked John Koester into starting a new PhD program with me: NB&B. We began with a class of three: Sally Till, Sandya Viswanathan, and Jill Willdonger. Sandya had a productive career in documentary films and is now in medical practice. Sally is at the University of Edinburgh studying autism and Jill is at the University of California, San Diego. Our NB&B graduate program currently attracts 600–700 applicants annually for a typical entering class of 12 students. I continue to teach all NB&B students in their first years. After John Koester retired, I led NB&B with Carol Mason and Ken Miller, who founded our program in theory with Larry Abbott. Carol now chairs intercampus neuroscience efforts at Columbia. Ken and I were joined by Wes Grueber and Ai Yamamoto at Columbia’s Medical School. Aniruddha Das (primate visual system) leads our diversity, equity, and inclusion (DEI) initiatives.

Diversity, Equity, and Inclusion

Science runs on talent. Nurturing careers—from undergraduates to faculty members—has been a central focus for me. Many women undergraduates have carried out research in our laboratory and authored research papers, beginning with Patty Hannigan at Princeton and, more recently, Ursula Kwong-Brown, now a composer (PhD, University of California, Berkeley), with whom I continue to collaborate. I fought for many years to bring more women faculty into the biological sciences. At first, the issue was advancing these recruits through tenure and then the issue was retaining them in the face of outside offers. We had much better luck recruiting senior women scientists. When Oliver Hobert moved from the medical school to biological sciences, recruiting young women faculty members shifted into high gear. Three extraordinary new women faculty (Maria Tosches, Erin Barnhardt, and Laura Duvall) joined biological sciences just as the pandemic began. Our department and the Zuckerman Institute also had the good fortune to attract Ishmail Abdus-Saboor from Penn. Marty Chalfie in biological sciences now leads our DEI efforts. In 2020, a group of undergraduates in our lab founded the Research and Diversity (RnD) initiative to bring undergraduate scientists into conversations with researchers from underrepresented groups. NB&B graduate student have also just created a program, CAN, to coach college students from under-represented groups with the graduate application process. Fingers crossed for all of these efforts as well.

Coda

I write this essay during the time of Covid from Baltimore, the home of my beloved second husband, Kenneth Frank. I met Kenny on Amtrak in the café car 16 years ago when he commented on my screen saver (blue-footed boobies) and asked if I had been to the Galapagos. Between us, we have four children and eight grandchildren, each one talented and beautiful. Separation from them during the pandemic has been hard. When I finally saw my grandson, he wept for the loss of a year without his friends. This spring and early summer, the 17-year cicadas emerged in Baltimore. The old oak trees were festooned with their husks. They hid under leaves from the cold rain and decorated the tires of the car. Kenny described them as “kind and curious,” although not everyone agreed.

In February and March of 2020 many friends and even our young graduate students fell ill. We had no Covid tests for a long time, but we all knew. All recovered but many, many, others did not. The still-ongoing pandemic has been the most remarkable period, so far, that I have lived through; such a tricky virus. Who is most at risk, how to treat the infection and its consequences, and how to protect the world preoccupied us then and now. I virtually attended webinars across the city, the United States, and the world. We saw researchers pool resources; experienced highly collaborative science and were encouraged when its application flourished. However, we also learned that superb science is not enough to prevent illness: people, and above all policymakers in government, must believe in and act on the science.

However, in the emerging “variants” of Sars-CoV-2, we can see, in real time, how organisms evolve, including the contributions of small genetic changes to their reproduction and survival. We also have come to a greater understanding of human biology, particularly the damage that can be inflicted by our own immune system. Laboratory research, except for Covid, was paused and scientists put aside their projects to try to help out by, for example, developing home tests for Covid that regular people can afford. Ivana Hughes and David Helfand pivoted to incorporate Covid into FroSci.

Some years ago, my last FroSci lecture on the evolution of language concluded with the “tangled bank” quote from Darwin’s Origin of Species:

It is interesting to contemplate an entangled bank, clothed with many plants of many kinds, with birds singing on the bushes, with various insects flitting about, and with worms crawling through the damp earth, and to reflect that these elaborately constructed forms, so different from each other, and dependent on each other in so complex a manner, have all been produced by laws acting around us. These laws, taken in the largest sense, being Growth with Reproduction; Inheritance which is almost implied by reproduction; Variability from the indirect and direct action of the external conditions of life, and from use and disuse; a Ratio of Increase so high to lead to a Struggle for Life, and as a consequence to Natural Selection, entailing Divergence of Character and the Extinction of less-improved forms. Thus, from the war of nature, from famine and death, the most exalted object which we are capable of conceiving, namely, the production of the higher animals, directly follows. There is grandeur in this view of life, with its several powers, having been originally breathed into a few forms or into one; and that, whilst this planet has gone cycling on according to the fixed law of gravity, from so simple a beginning endless forms most beautiful and most wonderful have been, and are being, evolved (Darwin, 1872).

Afterward, a student approached me to say that this view of the world seems utterly bleak to him. It does not seem bleak to me. I find this view of life wondrous. Understanding the little, red-eyed cicada, tucked underground as a larva for 17 years, feeding off the sap of trees, answering the mysterious signal to bore upwards, crawl up the trees or telephone poles, sing, mate, and then die does not take away the joy of unravelling their mystery. I do admit that periodic cicadas are not perhaps—as Andres Bendesky pointed out to me—a great model species for the lab, but they are an inspiration. The virus that now governs our lives (and deaths) is also an example of evolution. It is tempting to confer intent (such a tricky virus) but really all that is in play is selection for the ability to survive and replicate.

The evolution of neural circuits that have enabled our own species to survive this period are only one example of the “endless forms most beautiful and most wonderful” of nervous systems on our planet. We will need these and more to continue to survive the havoc we’ve unleashed on the planet lest it goes on cycling without us.

From Baltimore and New York City, November 2021

This memoir is dedicated to the memory of my lifelong friend, Adele LeBourgeois Alsop, who was, unbeknownst to almost all of us, bravely dying as I wrote this.

Selected Bibliography

Bringuier, JC (1969). Conversations with Jean Piaget (translated by Basia Gulati). University of Chicago Press.

Darwin, C (1872). On the origin of species by means of natural selection, 6th Edition. London: John Murray.

Hall IC, Ballagh IH, Kelley DB (2013). The Xenopus amygdala mediates socially appropriate vocal communication signals. J Neurosci 33(36), 14534–14548. https://doi.org/10.1523/JNEUROSCI.1190-13.2013

Kelley DB (2010). Science for all in a core curriculum: Frontiers of Science. Science and the Educated American: A Core Component of Liberal Education. Meinwald J and Hildebrand JG, Eds, 218–227.

Kelley D (2017). History of the Marine Biology Laboratory. YouTube. https://www.youtube.com/watch?v=tGD_8TlR-RA

Kelley DB, Ballagh IH, Barkan CL, Bendesky A, Elliott TM, Evans BJ, Hall IC, Kwon YM, Kwong-Brown U, Leininger EC, Perez EC, Rhodes HJ, Villain A, Yamaguchi A, Zornik E (2020). Generation, coordination, and evolution of neural circuits for vocal communication. J Neurosci 40(1), 11–36. https://doi.org/10.1523/JNEUROSCI.0736-19.2019

Kelley D, Kwong-Brown U (2019). Frogs singing submerged. The Naked Scientist. https://www.thenakedscientists.com/articles/interviews/frogs-singing-submerged

Morona R, González A (2013). Pattern of calbindin-D28k and calretinin immunoreactivity in the brain of Xenopus laevis during embryonic and larval development. Journal of Comparative Neurology, 521(1), 79–108.

Phoenix CH, Goy RW, Gerall AA, Young WC (1959). Organizing action of prenatally administered testosterone propionate on the tissues mediating mating behavior in the female guinea pig. Endocrinology 65(3), 369–382. https://doi.org/10.1210/endo-65-3-369

Roco AS, Olmstead AW, Degitz SJ, Amano T, Zimmerman LB, Bullejos M (2015). Coexistence of Y, W, and Z sex chromosomes in Xenopus tropicalis. PNAS 112(34), E4752–E4761. https://doi.org/10.1073/pnas.1505291112

Willsey HR, Exner CRT, Xu Y, Everitt A, Sun N, Wang B, Dea J, Schmunk G, Zaltsman Y, Teerikorpi N, Kim A, Anderson AS, Shin D, Seyler M, Nowakowski TJ, Harland RM, Willsey AJ, State MW (2021). Parallel in vivo analysis of large-effect autism genes implicates cortical neurogenesis and estrogen in risk and resilience. Neuron 109(5), 788–804. https://doi.org/10.1016/j.neuron.2021.01.002

George F. Koob

BORN:

Okinawa, Japan

August 29, 1947

EDUCATION:

Pennsylvania State University, BS, Zoology (1969)

Johns Hopkins University, PhD, Behavioral Physiology (1972)

APPOINTMENTS:

Predoctoral Fellow, Johns Hopkins University, Department of Environmental Medicine (1969–1972)

Staff Scientist, Department of Neurophysiology, Walter Reed Army Institute of Research (1972–1975)

Postdoctoral Fellow with Dr. Susan D. Iversen, University of Cambridge, Department of Experimental Psychology, Dr. Leslie Iversen, Medical Research Council, Neurochemical Pharmacology Unit (1975–1977)

Staff Scientist, Arthur Vining Davis Center for Behavioral Neurobiology, The Salk Institute for Biological Studies (1977–1983)

Assistant, Associate, Full Adjunct Professor, Departments of Psychology, Psychiatry, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego (1980–2014)

Associate Member (with tenure), Division of Preclinical Neuroscience and Endocrinology, The Scripps Research Institute (1983–1989)

Professor, Department of Neuropharmacology, The Scripps Research Institute (1990–2006)

Director, Alcohol Research Center, The Scripps Research Institute (1995–2014)

Acting Chair, Department of Neuropharmacology, The Scripps Research Institute (2000–2002)

Professor and Chair, Committee on the Neurobiology of Addictive Disorders, The Scripps Research Institute (2006–2014)

Director, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health (2014–present)

Senior Investigator and Section Chief, Neurobiology of Addiction Section, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health (2014–present)

HONORS AND AWARDS (SELECTED):

Daniel H. Efron Award, Excellence in Research in Neuropsychopharmacology, American College of Neuropsychopharmacology (1991)

Highly Cited Researcher, Institute for Scientific Information/Web of Science Group, Clarivate Analytics (2001, 2018, 2019, 2021)

Distinguished Investigator Award, Research Society on Alcoholism (2002)

Mark Keller Award, National Institute on Alcohol Abuse and Alcoholism (2004)

Honorary Doctorate of Science, Pennsylvania State University (2009)

Marlatt Mentorship Award, Research Society on Alcoholism (2012)

Julius Axelrod Mentorship Award, American College of Neuropsychopharmacology (2012)

Docteur Honoris Causa, Universite Bordeaux Segalen (2013)

IPSEN Foundation Neuroplasticity Award (2014)

Distinguished Scientist Award, American Society of Addiction Medicine (2015)

Chevalier de la Legion d’Honneur in recognition of outstanding contributions to Franco-American scientific relations (2016)

National Academy of Medicine (2017)

Jellinek Memorial Award, Research Society on Alcoholism (2018)

George Koob has made major contributions to our understanding of the neurobiology of addiction. He has guided nationwide efforts to address the scourge of alcohol use disorder by directing the National Institute on Alcohol Abuse and Alcoholism. His early research focused on the neurobiology of stress and drug reward and then evolved into understanding neuroadaptations of the brain stress and reward systems in the progression to compulsive-like drug seeking. He hypothesized that hyperkatifeia is driven by allostatic changes of loss of reward and gain of stress neuroadaptations to drive and perpetuate the cycle of addiction through negative reinforcement. These advances provide a bridge to a key motivational component of the real-world misery of addiction and have implications for how humans experience negative emotional states in general.

George F. Koob

I was born in a Quonset hut in Okinawa, Japan, on August 29, 1947, the son of Robert Anthony Koob from East Rutherford, New Jersey, and Theodora Johanna Koob (née Foth) from Jersey City, New Jersey. I was the oldest of four children. Kathy, my younger sister, tragically died of non-Hodgkin’s lymphoma at the age of 46. My mother and father met in Carlstadt, New Jersey, and married in 1942. My father was subsequently drafted into the U.S. Army to serve in World War II, and he participated in the invasion of Okinawa as a warrant officer in the 305th Ordinance Battalion. He won a commission as second lieutenant after the cessation of hostilities with Japan. He went on to obtain a bachelor’s degree and master’s degree in business administration at Babson Institute and ultimately pursued a career as a regular Army officer. After 32 years, he retired from the Army as a full colonel at Fort Campbell, Kentucky, where he served as comptroller for the base that houses the 101st Airborne Division. My mother was a teacher, professor of English, and writer. My mother earned her PhD in linguistics in 1946 from New York University and joined my father on Okinawa in 1946. There, she founded the U.S. Dependent school system on Okinawa, which remains to this day, and served as its first principal. Theodora subsequently wrote and published eight children’s books and was recruited as a professor of English at Shippensburg State College in Shippensburg, Pennsylvania, in 1964. She retired in 1980. Throughout her career, she taught at the grade school, junior high school, senior high school, and university levels.

The Early Years

I grew up as an Army brat and had a rich intellectual but socially tumultuous upbringing as we moved every one to three years, from Okinawa to Aberdeen, Maryland (my first grade); Fort Bragg, North Carolina (second grade); Natick, Massachusetts (third grade); Angouleme, France (fourth to sixth grades); Augusta, Georgia (seventh and eighth grades); and finally Chambersburg, Pennsylvania (ninth to twelfth grade). I had two younger brothers (Joe and Steve) and a younger sister (Kathy). We were always challenged by our parents to pursue academic careers. There was never any doubt that each of us would go to college and that I would go on to pursue an advanced degree. Joe became a college professor of music, motivational speaker, and writer. Steve became a conservator archaeologist and curator of glass at the Corning Glass Museum. Kathy became a textile conservator. Despite moving every one to three years throughout my youth, there were many constants, mostly within our family unit as we regularly gained and lost childhood friends because of my father’s frequent Army reassignments. My mother insisted on music lessons and the pursuit of hobbies for each of us. Mine was collecting butterflies (which was not, alas, an ecologically appropriate choice). My father was a dedicated Boy Scout leader who won every award they conferred—he was most proud of his Silver Beaver Award. My brothers and I all joined the Boy Scouts and earned the rank of Eagle Scout. Looking back, the wonderful experiences of Boy Scout camps, a time when we really connected with our father, contributed to my later career in physiology and avocation of gardening (more on that later). Another plus of my being an Army brat was the extraordinary and wonderful opportunities to travel and see new places. My mother took us everywhere in France, with the American military base near Angouleme as the launching point. From the castles of the Loire to real caves of Lascaux to opera in Bordeaux, I was imprinted early on as a devout Francophile and even developed an ear for the French language, having to learn the catechism in French for my second French First Communion.

In high school, I had a particularly charismatic biology teacher, Louise Grove, who was also my Latin teacher. I became enthralled with both subjects. Salient during this time was teaching the discovery of the DNA structure by Watson and Crick and the beginning of lay books on ethology, such as Territorial Imperative by Robert Ardrey. I was equally fascinated by the classics and actually was accepted as a classics major at Pennsylvania State University but changed at the last minute to zoology. My brother, Steve Koob, became the classics major and went on to a brilliant career as a conservator/archeologist.

Pennsylvania State University, 1965–1969

My studies at Pennsylvania State University during the first year were at the Mont Alto campus, which was 10 miles from our house in Chambersburg. This afforded a more gradual transition to the large campus at State College and allayed some of the costs. I remember a beautiful wooded campus, dedicated teachers, and a harrowing drive in some very large snow storms. I enjoyed such subjects as botany and zoology but struggled with calculus. Probably most important was that I was able to skip the regular first English courses at Penn State and took the advanced English course, which was taught by a charismatic teacher who challenged students with literature from Dostoevsky to Shakespeare, honing our ability to think critically and integrate concepts. I aced the final with a Blue Book essay on how love transcended as a key element across all of what we had read that term (more on my writing in my graduate years below).

The move to the Penn State main campus for my sophomore year was a culture shock of sorts, given all the opportunities to meet new people and make new friends. But alas, I eventually settled in to studying. During that second year, I met members of the Alpha Zeta fraternity, which was an honorary fraternity with origins in agriculture but included many members with aspirations for related careers in zoology, veterinary medicine, landscape architecture, and even medicine. I pledged the fraternity and was elected. We had a fraternity house in the center of campus, right next to Recreation Hall. One had to maintain a 3.0 (B) academic record, and we were a dry house (no alcohol). Here, I made lifelong friends and acquired a yearning for gardening that evolved into my current main avocation of growing exotic fruit trees in Southern California.

My move to the main campus of Penn State allowed my choice of a zoology major to flourish. Here, my undergraduate advisor was the famous ecologist David E. Davis. He guided me to courses in physiology and psychology, which dovetailed nicely with my early interests in ethology. I looked to join the primate work of a Penn State professor who divided his time between the University of Georgia and Penn State, but the timing was off and he was at the University of Georgia. One of my psychology professors, Michael Warren, sent me to Howard S. Hoffman, who was studying the power of imprinting to shape operant conditioning. He had baby ducklings imprinted to a milk bottle that moved back and forth on a train track (Hoffman et al., 1972). Needless to say, I was fascinated. Howard Hoffman immediately assigned me to reading Ferster and Skinner (1957) and to working with Ivan Divac, two events that presaged my ultimate career pursuit of bridging behavior and the brain. I also worked through the summers doing paid jobs at Penn State, cleaning rabbit cages and helping study wild rats. One series of events that I noted retrospectively was that I excelled in all courses on physiology at Penn State while my fraternity brothers often struggled with them. There was never any doubt that I would apply to graduate school and pursue a career in academia, something likely related to animal behavior.

But there was a cloud on the horizon. The Vietnam conflict became fully engaged, and the draft was activated in the United States. I drew a low draft number and was clearly facing the draft upon graduation. Then “The Colonel” (my father) intervened. In the spring of my second year at Penn State, I received a phone call one afternoon from the Colonel who directed ROTC at Penn State. Unbeknownst to me, my father had enrolled me in ROTC, and I was to report to training the next day. However, I quickly figured out that there was a program that allowed one to delay active duty after being commissioned in the officer core to attend graduate school, making ROTC training much more palatable. To qualify, I ultimately had to graduate one term early from Penn State in the spring of 1969 and was commissioned as a second lieutenant in the U.S. Army Signal Corps. Meanwhile, I had applied to five graduate schools to pursue studies in ethology. I was called for an interview for the Pathobiology Program in the School of Public Health at Johns Hopkins University in Baltimore, Maryland. Unfortunately, they had no slots available on their training grant, so they negotiated with the Department of Environmental Medicine for a slot on their National Institutes of Health Heart and Lung training grant. I poignantly remember my interview with the chair of Environmental Medicine, Richard Riley, who was questioning my transcript and pointed out a few C grades intermixed with mostly A and B grades. He specifically asked about my C grades in map reading from my ROTC courses. I answered that ROTC had not been my highest priority for study, to which he smiled and said he was against the war and I would be welcomed to his department.

Johns Hopkins University, Graduate School, 1969–1972

My graduate career in the Department of Environmental Medicine began in April 1969. I graduated with my PhD in June 1972. I worked continuously throughout that period, including all summers, to meet the Army’s time requirement of how long active duty could be delayed by attending graduate school. Upon joining the Johns Hopkins program, I immediately did a rotation with Charles Southwick, exploring mouse behavior and doing ecological field work on the Chesapeake Bay, and in a course with Edwin Gould, I trained bats to run down a runway to obtain mealworms. At the same time, I was assigned to Stephen Weinstein and Zoltan Annau, who had a program that explored the effects of hypoxia on motivated behavior, including brain stimulation reward. Zoltan Annau had trained with Jim Olds, the codiscoverer of brain stimulation reward with Peter Milner (Olds and Milner, 1954). We embarked on measuring brain stimulation reward concomitantly with food and water intake over long periods to account for the long-term effects experienced by humans who move to high altitude. This study led to my first eureka! moment and my first published scientific paper (Koob et al., 1970). I define a eureka! moment as one of those results in an experiment that was unpredicted and leads to a completely different conceptual direction. Here, we found that rats that were exposed to 10% oxygen (equivalent to about 18,000 feet above sea level) exhibited a profound and prolonged increase in lever pressing for brain stimulation reward that we assumed reflected a euphoric-like response, particularly given that orally administered amphetamine at a large dose produced an identical effect. Both hypoxia and amphetamine also reduced responding for food and water. I went on to show that this effect was not mediated by peripheral sympathetic activation, including a heroic adrenal demedullation surgical procedure, and hypothesized it was attributable to central nervous system monoamine dysregulation, including my first catecholamine assay (Koob and Annau, 1973, 1974). During this period, Stephen Weinstein left academia for business, and Zoltan Annau became my PhD adviser. I learned much about basic physiology and behavior from Zoltan and much about the excitement of new findings that science provides. During this period, my interest in ethology faded, and my interest in what was to be the new field of the neuroscience of emotion and motivation blossomed. Zoltan also stimulated my future career not only with erudite guidance of my thesis project but also with his strong encouragement to interact with world-class scientists at Johns Hopkins. Zoltan was also rigorous about experimental design, statistics, and writing. When my first scientific paper came back from him covered in red ink, he, a Hungarian by birth, remarked that “if I did not know better, one might think that English was not your first language.” Ultimately, my first paper (revised) was read by Philip Bard, who had a wonderful story about the paradoxical effects of opioids in cats. My first event recorder was borrowed from Curt Richter, deemed by many as the father of physiological psychology. I often saw Curt Richter, Anna Baetjer, Philip Bard, and Vernon Mountcastle, all towering figures in their fields, in the lunchroom at the top of the Johns Hopkins School of Public Health. I took psychiatry at the Johns Hopkins Medical School. I so enjoyed the section by Joe Brady that I took his graduate course on behaviorism, and he ultimately became the chair of my thesis committee. I took Solomon (Sol) Snyder’s neuropharmacology course, and he ultimately advised us on our amphetamine brain stimulation studies. Both Joe and Sol became my career-long guardian angels, opening doors and advising me throughout my career. All of them—Zoltan, Joe, and Sol—planted the seeds of a career that involved understanding the neurochemical/neurocircuitry of how the brain mediates motivation and emotions.

Indeed, my early interactions with Joe Brady illustrate this motivational imprinting. I remember one day after his demonstration of operant conditioning to the medical school psychiatry course, where he trained chickens to walk off the end of a large table, I queried him by asking, “Dr. Brady, how hungry were those chickens?” His response: “Koob, damn hungry.” This interaction contributed to my lifelong interest in motivation and what drives behavior.

However, as my PhD neared completion, and thus the completion of my delay of my active duty in the U.S. Army, the dark cloud that hung over my generation had not dissipated. I was commissioned as a second lieutenant in the Army Signal Corps, a combat branch. My goal, at the advice of Joe Brady among others, was to fulfill my service obligation in the Medical Service Corps at the Walter Reed Army Institute of Research (WRAIR) in Washington, D.C. To accomplish this, I had to be moved from the Signal Corps to the Medical Service Corps, a task for which I enlisted the aid of three Army Colonels: Joe Brady (who introduced me to the Division of Neuropsychiatry at WRAIR), Harry Holloway (director of the Division of Neuropsychiatry at WRAIR), and my father (who followed my paperwork). By the summer of 1972, I received my orders to report to Fort Sam Houston in San Antonio, Texas, for officer basic training in the Medical Service Corps. I joined WRAIR in the fall of 1972.

Walter Reed Army Institute of Research, 1972–1975

Now a captain in the Medical Service Corps, I was assigned as a staff scientist to the Department of Neurophysiology at WRAIR where my director was Herb Spector. However, I quickly began to work also with Jim Meyerhoff, who had studied with Sol Snyder at Johns Hopkins. I was very keen to pursue studies of the role of key brain catecholamine circuits in reward using the brain stimulation reward technique that was developed in my thesis work. A highly influential series of papers published by Tim Crow and colleagues (Anlezark et al., 1973; Crow, 1973) generated great interest that I conveyed to Jim Meyerhoff and two other colleagues and collaborators, Jean Balcom (Kant) and Rufus Sessions. At WRAIR, I was able to set up a bank of operant chambers, procure a PDP-8 computer, and take a course in Maynard, Massachusetts, on how to program the computer. We generated a number of salient findings, including showing that heroin facilitated brain stimulation reward (Koob et al., 1975), charting the projections of dopamine neurons in the ventral tegmental area (Koob et al., 1975), and finding that the locus coeruleus seemed more involved in stress-like responses than reward and learning (Sessions et al., 1976). The intellectually rich environment at WRAIR provided an equally important direction in my career. Here, I worked as a teaching assistant with Jim Petras in the human neuroanatomy course at WRAIR. Michie Vane, who had worked with Walle Nauta, taught me numerous neuroanatomical staining procedures, including Nauta-Gygax silver staining. Jim Petras introduced me to Harvey Karten, Sven Ebbesen, Walle Nauta, Lennart Heimer, and Floyd Bloom. My introduction to Floyd Bloom was especially notable. I had been so interested in the locus coeruleus that I was keen to know the afferent inputs to the locus coeruleus. I pestered Jim Petras to introduce me to Floyd. I had heard him give a seminar on his elegant work on norepinephrine in the hippocampus. I thought that if anyone knew afferents to the locus coeruleus, then it would be him. When Jim Petras called Floyd at his National Institute of Mental Health laboratory at St. Elizabeth’s Hospital in Washington, D.C., Floyd immediately wanted to know who was asking and then asked Jim to put me on the phone. I introduced myself as George Koob, “K-O-O-B, book spelled backwards,” something my father always said. Floyd laughed for what seemed like minutes and that was the beginning of a lifelong mentorship and friendship. He invited me to all their research seminars at St. Elizabeth’s Hospital, and I visited them regularly. What I found out later was that he was looking for someone in behavioral neuroscience to join their group. These collaborations and interactions left me with a thirst to receive further training in functional neuroscience. I had thought of working in Paris with Jacques Glowinski, but Sol Snyder instead suggested a postdoctoral stage at the University of Cambridge, England, a position for which I was accepted. Just before leaving for Cambridge, at one of the last visits to Floyd Bloom’s group, he asked me what my plans were upon my return to the United States from Cambridge. I remarked that I had no plans as of yet. He said he would like me to join their group, either in D.C. or possibly at The Salk Institute in La Jolla, California.

University of Cambridge, 1975–1977

For me, my postdoctoral stage at the University of Cambridge was magical. Where else could one live in Grantchester and on the way to work walk past the cows in the fens, through the gardens of a 700-plus-year-old college (Pembroke), and past the Eagle Pub where Watson and Crick declared that they had found the secret of life. In the summer of 1975, Susan (Sue) Iversen had offered me a position as a postdoctoral fellow in her group in the Department of Experimental Psychology at the University of Cambridge. Shortly thereafter, I won an R21 grant from the National Institute of Mental Health to pursue postdoctoral studies in Sue’s laboratory. I combined both sources of funds and was able to stay in Cambridge from 1975 to 1977. A fellowship offer had later come from the University of Bordeaux, but I decided to go to Cambridge. My French connection would flourish later. My time at Cambridge was probably my most formative from the perspective of shaping the framework for my life’s work to understand the neurochemical neurocircuitry of motivation. The openness, generosity, and hospitality of Sue and her husband, Leslie, as well as my friends and collaborators at Cambridge, including Trevor Robbins, Barbara Sahakian, Paul Fray, Piers Emson, and Claudio Cuello, created a standard to which I have aspired ever since. More important, the thirst for knowledge and the framework of trying to understand how the brain worked via neurochemical neurocircuits were enduring legacies imparted on me particularly by Sue and Les Iversen.

Upon my arrival at Cambridge, my first charge was to work with Les, who directed the Medical Research Council research unit, called the Neurochemical Pharmacology Unit (NCPU) at Cambridge, to improve the catecholamine assay and move it to psychology. Just before Christmas 1975, we successfully improved the assay using paper chromatography. My participation in this effort earned me a high table dinner for Candelmas Feast with Les at Trinity College, a wonderful evening never to be forgotten. With Sue, I investigated the functional significance of norepinephrine and dopamine in motivation in a series of studies that used intracerebral administration of the catecholamine neurotoxin 6-hydroxydopamine and dopamine receptor antagonists. In parallel, I collaborated with many students and faculty in psychology and the NCPU, many of whom became lifelong friends. My work with Sue Iversen resulted in a paper on how stress-induced eating sustains learning (also with Paul Fray; Koob et al., 1976) and a key study that showed that the destruction of dopamine projections produced persistent and pronounced deficits in intracranial self-stimulation from ipsilateral electrodes in both lateral hypothalamus and locus coeruleus groups but only transient effects on self-stimulation from contralateral electrodes at these sites (Koob et al., 1978a). Sue had insisted that I do some teaching in the context of tutorials. Both Sue and Trevor Robbins, a demonstrator at Cambridge (equivalent to the U.S. academic ranking of assistant professor), patiently taught me basic principles of general psychology so that I could be an effective tutor. In turn, I taught all of what I knew about brain dissection and surgical techniques. Ultimately, my collaboration with Trevor emerged into an enduring, lifelong, and intellectually fruitful friendship. I like to think that our brains fed off each other in our early attempts to understand the role of the mesolimbic dopamine system in motivated behavior. The arguments ranged from reward to conditioned reinforcement to incentive motivation, resulting in a series of papers that anticipated our future pursuits. We showed that nucleus accumbens dopamine depletion decreased activation that was associated with the anticipation of food in hungry rats (Koob et al., 1978b) and later blocked schedule-induced polydipsia (Robbins and Koob, 1980). Later, Trevor introduced me to Barry Everitt, and another lifelong friendship developed. With Trevor and Barry, we first outlined the neurocircuitry of addiction in the context of dysfunctional domains in a chapter for the textbook (Koob et al., 2008) where I assiduously wedged in the circuitry of the negative emotional state of drug withdrawal (more on that to come). Trevor, Barry, and I subsequently had many theoretical discussions that helped shape my view of addiction and significantly broadened my perspectives on addiction.

As my time in Cambridge neared 18 months, Sue Iversen brought me into her office. She wondered if I had been thinking of what I would do when my funds for working with her ran out. She noted that she was seeing a significant number of positions opening in the United States and asked if Floyd had written to me yet. He had not. But that week, after my meeting with Sue, a blue airmail letter arrived from Floyd, offering me a position as a research associate, then staff scientist, in the Arthur Vining Davis Center for Behavioral Neurobiology at The Salk Institute. That July, I arrived in La Jolla and joined the Bloom lab.

The Salk Institute, 1977–1983

My arrival was heralded by my colleagues from the University of Cambridge, including Sue and Les Iversen, who then visited every summer for several years after that. Their presence eased my transition into a completely new world of outstanding biologists and neuroscientists. I immediately set up a single operant chamber and began establishing an operant conflict test with the goal of studying the intoxicating effects of alcohol as Floyd challenged us to participate in winning an Alcohol Research Center (ARC) grant to study the neurobiology of alcohol. There was very little known about the neurobiology of alcohol in 1977. In fact, a prevailing hypothesis at that time was that alcohol acted mainly like an anesthetic that interfered with membrane dynamics. I poignantly remember the site visit for the ARC grant, chaired by Joe Brady from Johns Hopkins University. After his bear hug of a greeting, he announced he would see me at my second thesis defense, alluding to the fact that he had chaired my PhD thesis defense. The site visit went well, and the study of the neurobiology of alcohol has paralleled my studies of the neurobiology of addiction in general. Both of these pursuits further opened windows on my search to understand the neurobiology of motivation.

Two separate tracks of research preoccupied me in these early years at Salk. First, the arrival of David Roberts as a postdoctoral fellow from the University of British Columbia in Canada introduced us to intravenous drug self-administration. Here, we replicated a key finding from Dave’s PhD thesis that removal of the dopamine projection to the nucleus accumbens with the neurotoxin 6-hydroxydopamine produced a pronounced decrease in intravenous cocaine self-administration and produced an extinction-like pattern of behavior (Roberts et al., 1980). David Roberts worked with both me and Floyd, as did many of my earlier postdoctoral fellows. This shared mentoring structure with Floyd Bloom endured while I was at Salk and beyond. Notably, this type of shared mentoring reversed as my career grew, with fellows primarily trained by junior faculty also asking to be comentored by me via collaboration. The drug addiction and motivation work continued with results showing convergence of the role of dopamine and opioid mechanisms for opioid- and psychostimulant-induced locomotor activation and reward in the nucleus accumbens (Pettit et al., 1984). At the same time, we mounted significant refutation of the hypothesis of Roy Wise that dopamine was the reward neurotransmitter (Ettenberg et al., 1982). In a series of studies, we argued that dopamine was key to psychostimulant reward and played a major role in incentive motivation (i.e., more of a functional role in the activation of behavior anticipating reward; Koob, 1982; Koob, 1992). This debate raged for years and to some extent presaged the concept of incentive salience, which probably best explains the functional role of the dopamine system (Robinson and Berridge, 1993). A vignette from this period amply illustrates the argument. Roy Wise had given the Bloom laboratory a talk on his dopamine reward hypothesis, and I had countered with a rebuttal. The final word came from Floyd when he said, “I do not know, Roy. If you ever followed a Parkinson’s patient into the bathroom to take a piss, you know they are motivated.” Roy and I finally buried the hatchet in our shared commentary in Neuropsychopharmacology in 2014 (Wise and Koob, 2014).

At the same time and in parallel, we began to explore measures of alcohol intoxication to ultimately study the neuropharmacological/neurocircuitry mechanisms that mediate alcohol reward. Early on, we adopted an incremental shock conflict test (Pollard and Howard, 1979) that reliably proved sensitive to alcohol. In a series of studies in collaboration with Karen Thatcher-Britton, professor in the Department of Psychiatry at the University of California, San Diego (UCSD), we identified an anxiety-like role for corticotropin-releasing factor (CRF; Thatcher-Britton and Koob, 1986), proconflict effects of a benzodiazepine receptor inverse agonist that was being touted as an “alcohol antagonist” (Britton et al., 1988), and powerful anticonflict effects of neuropeptide Y (NPY; Heilig et al., 1992). These studies ultimately led to our parallel efforts to examine the neural substrates that mediate alcohol self-administration in rodents. Here, we adopted a sweet solution fadeout procedure from Hank Samson (Samson, 1986) to initiate drinking at levels that generated reliable blood alcohol levels (Koob and Weiss, 1990). Indeed, our CRF and NPY work and the animal model development of Karen Thatcher-Britton, Markus Heilig, Emilio Merlo-Pich, Friedbert Weiss, and Amanda Roberts, among many others, provided the foundation for the ultimate transition to studying neuroadaptations that drive excessive drug taking.

Another research program we developed at The Salk Institute came from Floyd’s becoming very interested in the functional roles of neuropeptides in the brain. We began work on β-endorphin, showing that when it was injected in the ventral tegmental area, it produced robust locomotor activation (with Luis Stinus), presumably by activating the mesolimbic dopamine system (Stinus et al., 1980). At that time, Floyd wondered about studies from David De Wied and colleagues in The Netherlands that showed dramatic behavioral effects of vasopressin and vasopressin-like peptides in facilitating memory (De Wied, 1976; Walter et al., 1978). At the same time, a confluence of factors converged to move neuropeptide studies into high gear. Norman White joined us on sabbatical from McGill University and prompted us to utilize established animal models of memory in our studies of the effects of peripherally administered neuropeptides. Soon afterward, a watershed moment in my career arrived. Michel Le Moal from the University of Bordeaux came to work with us in the summer of 1978. He had regularly joined the laboratory of Jim Olds at Caltech in Pasadena, California, for his annual summer visit to a U.S. laboratory. In the summer of 1976, however, Jim had passed away, and Michel asked Floyd if he could join the Bloom laboratory at Salk for the summer to work with me. This was the beginning of another lifelong collaboration and friendship that has endured for more than 40 years. Michel Le Moal became my foremost experimental and conceptual collaborator.

The ensuing Koob-French connection then became one of the most enduring and pleasurable experiences of my research career. I learned to speak French fluently by living with my French colleagues in their homes, specifically Herve Simon early on and Michel Le Moal when he bought a townhouse near the medical center. Herve and his wife, Marianne, not only taught me to speak French but also taught me to cook, and I still have my cherished copy of Cuisine San Souci (Montigny and Kahn, 1977) though its cover is torn. Luis Stinus and Robert Dantzer became dear friends and collaborators as well, introducing me to new exciting dimensions of science and additional myriad elements of French culture. I actually gave several science lectures exclusively in French, including a plenary talk at the French Society for Neuroscience. Ultimately, I mentored 13 French postdoctoral fellows and seven French visiting scientists in my laboratories at Salk and The Scripps Research Institute (TSRI). I am very proud of winning an honorary doctorate from the University of Bordeaux and the French Legion of Honor, but considering Michel Le Moal as my “French brother” and the enduring friendships and wonderful immersion in the rich French culture are what I value most from my French connection.

After our first successful summer exploring the functional role of neuropeptides, Michel invited me to Bordeaux, thus beginning an annual spring/summer exchange. I worked in Bordeaux with Michel’s group at Institut National de la Sante et de la Recherche Medicale (INSERM) and Centre National de la Recherche Scientifique (CNRS) units in April to May, and Michel worked with us at Salk (and later at Scripps) each summer for six weeks, every year from 1978 until 2005 when administrative responsibilities precluded long visits for both of us. We won support for this exchange for nine years from the National Science Foundation and CNRS. Finally, the return of Roger Guillemin to Salk and the move of the Wylie Vale group to the temporary building space between the Salk main building and the Glider Port near campus, which housed my first laboratory, provided us with an unending source of contact, discussions, novel neuropeptides, and collaborations. What evolved were again long-term collaborations and personal friendships with Catherine Rivier, Jean Rivier, and Wylie Vale that were key to our studies on the function of brain stress systems and that provided cultural delights. Wylie became a never-ending source of non-science-related book reading recommendations, and Jean and I went on frequent fruit tree foraging expeditions to expand our burgeoning gardening avocations.

On the neuropeptide front, Michel and I successfully replicated the prolongation of extinction in rats that is produced by subcutaneous vasopressin administration and went on to show that we could reverse such effects with doses of a vasopressin antagonist that also reversed vasopressin-induced blood pressure activation (Le Moal et al., 1981). This was followed by numerous studies and collaborations with the Bordeaux group, with the conclusion that the neuropeptide vasopressin has both an interoceptive arousal function in the periphery and a parallel arousal effect in the brain, independent of the periphery (Le Moal et al., 1984; Lebrun et al., 1985; Le Moal et al., 1987). Later, this role of vasopressin came to be understood as part of brain stress systems, with significant data suggesting that vasopressin 1b receptor antagonists have antistress/anxiolytic-like effects in animal models (Griebel et al., 2002).

In 1981, a second eureka! moment occurred. Wylie Vale, Catherine Rivier, Jean Rivier, and Jocham Speiss at Salk sequenced the 41-amino-acid CRF, the long-awaited neuropeptide that drove the release of adrenocorticotropic hormone and subsequent adrenal steroid response to arousal and stress (Vale et al., 1981). Marvin Brown had already seen preliminary evidence of a neurotropic action of intraventricular CRF administration producing sympathetic activation (Brown et al., 1982). With Wyle’s urging, Michel Le Moal and I designed an experiment to test the hypothesis that CRF had a neurotropic action in the brain to facilitate behavioral responses to stress. We injected CRF at three doses intracerebroventricularly and observed behavior and locomotor activity in well-habituated rats. We observed dose-dependent activation, even with climbing on the walls of the wire mesh test cages. We also observed some very peculiar behavior, expressed as forepaw treading and tremor-like behavior. I called Wylie and told him the rats were “levitating,” and he came over to watch. Later, in a more stressful open-field environment, the rats exhibited a dose-dependent increase in sensitivity to the inhibitory effects of the open field (Sutton et al., 1982). We later learned that at high doses, CRF could produce electroencephalographic activation that is associated with seizures. Altogether, these findings supported the hypothesis that CRF plays a neurotropic role in the brain to facilitate behavioral responses to stress.

Thus, my time at The Salk Institute began my two-track independent research programs, one exploring the neurobiological substrates of motivation and drug reward and the other exploring the functional role of endogenous neuropeptides. These research programs interacted but never fully merged until later, in what I consider a third eureka! moment after we moved to TSRI. During this period, I was joined by a number of postdoctoral fellows under my tutelage, including Aaron Ettenberg, Franco Vaccarino, Marianne Amalric, and Christine LeBrun (the latter two from France). My first PhD student, Neal Swerdlow, also joined, who was a walk-on from the UCSD Medical School.¹ We explored the neural and neuropharmacological substrates for the reinforcing effects of psychostimulants, opioids, and alcohol, with a focus on dopamine and opioid peptides converging on the nucleus accumbens.

The Scripps Research Institute, The First Half, 1983–1997

In 1983, Floyd Bloom decided to move his laboratory to The Scripps Clinic and Research Foundation (renamed later TSRI), forming the Division of Preclinical Neuroscience and Endocrinology in the Department of Basic and Clinical Research, then chaired by Ernie Beutler. Floyd was particularly intrigued by the burgeoning field of molecular biology and its potential application for discovering previously unknown neurotransmitters/neuromodulators and their receptors and functions in the brain. His studies began with Greg Sutcliff at TSRI, and this approach ultimately resulted in a number of discoveries, including isoforms of myelin-associated glycoprotein (Lai et al., 1987) and the neuropeptide hypocretin (de Lecea et al., 1998). I was invited to join Floyd’s lab in this move and was offered a position as associate member (equivalent to associate professor) with tenure, which I readily accepted.

Joining TSRI also marked the point at which both my independent addiction and stress research programs began to merge. Accomplishments that moved us toward the conceptual merge were studies of output neurocircuitry that mediates psychostimulant and opioid reward (Hubner and Koob, 1990; Robledo and Koob, 1993), the establishment of a role for dopamine and opioid peptides in the nucleus accumbens in alcohol reward (Rassnick et al., 1992; Heyser et al., 1999), and confirmation that CRF had a neurotropic function in the central nervous system to mediate behavioral responses to stress and that CRF receptor antagonists block behavioral responses to stressors (Tazi et al., 1987; Swerdlow et al., 1989; Heinrichs et al., 1992).

The ultimate merging of these two research tracks came from five breakthroughs that comprised several eureka! moments. The first centered on studies that showed that motivational aspects of drug withdrawal (anxiety-like and aversive-like effects of drug withdrawal) could be reversed by CRF receptor antagonists. The second was an unexpected finding, in which the nucleus accumbens was implicated in the motivational effects of opioid withdrawal (Koob et al., 1989). The third was conceptualizing neuroadaptations that are associated with addiction, which was published in Science in 1988 (Koob and Bloom, 1988). The fourth was stimulated by my teaching an undergraduate course at UCSD, when I recognized a common pattern of behavioral dysregulation across all addictions. The fifth came from maturation of my collaboration with Michel Le Moal to the theoretical level, where he introduced me to the concept of allostasis.

For the first major breakthrough, in a clever use of microdialysis and radioimmunoassay, in which the perfusion medium contained anti-CRF serum, Emilio Merlo-Pich, a Fogarty fellow, was able to measure CRF in the central nucleus of the amygdala (CeA) in freely moving animals. He showed a dramatic increase in extracellular CRF in the CeA during alcohol withdrawal (Merlo-Pich et al., 1995). Similar results were found by others in the bed nucleus of the stria terminalis (BSNT; Olive et al., 2002). Extracellular CRF also increased in the CeA during precipitated withdrawal from chronic nicotine (George et al., 2007), withdrawal from binge cocaine self-administration (Richter and Weiss, 1999), and withdrawal from opioids (Weiss et al., 2001) and cannabinoids (Rodriguez de Fonseca et al., 1997). Equally compelling, we were able to reverse the anxiogenic-like effects and aversive effects of alcohol, opioids, Δ⁹-tetrahydrocannabinol (THC), and nicotine withdrawal with systemic, intracerebroventricular, and intracerebral administration of CRF receptor antagonists (Rassnick et al., 1993; Baldwin et al., 1991; Grieder et al., 2014; Rodriguez de Fonseca et al., 1997).

The second major breakthrough came from the results of an experiment in which we sought to identify potential targets in the brain that mediate motivational aspects of opioid withdrawal with Tamara Wall, an honors student from UCSD, and Floyd Bloom (Koob et al., 1989). The literature had suggested that somatic signs of precipitated opioid withdrawal in rats, such as wet dog shakes and ptosis, were mediated by opioid receptors in such regions as the medial thalamus and periaqueductal gray. However, when we used an operant measure of malaise associated with precipitated opioid withdrawal, the suppression of lever pressing for food pellets and increased sensitivity to the response-disruptive effects of an intracerebrally injected opioid receptor antagonist showed that opioid receptors in the region of the nucleus accumbens were far and away the most sensitive site relevant to the medial thalamus and periaqueductal gray. Thus, I realized that the nucleus accumbens, which was known to be important for the positive reinforcing effects of opioids, also may be responsible for the response-disruptive, aversive stimulus properties of opioid withdrawal. We argued that “euphoria” and “dysphoria” that are induced by opioids may reflect opponent motivational processes that operate at the cellular level within the nucleus accumbens. The specter of opponent process theory penetrated my thinking and moved my research from studying how drugs produce their acute positive reinforcing effects to how drug addiction is driven by negative reinforcement.

For the third breakthrough in 1988, Floyd Bloom was invited to write a review on addiction for Science, and he asked me to do it with him. The paper (Koob and Bloom, 1988) was one of the first attempts to attach neurocircuit changes to adaptations that are associated with repeated drug seeking and taking. In a sense, we applied what we knew about the neurobiology of repeated exposure to drugs of addiction to the opponent process theory of Richard Solomon (Solomon and Corbit, 1974; Solomon, 1980), which we took one step further. We hypothesized that tolerance and dependence resulted from two types of neuroadaptations: within-system neuroadaptations and between-system neuroadaptations. In the paper, we established a framework that has held up over time and provided a heuristic framework to identify key elements of the addictive process:

In a within-systems opposing process, the primary cellular response element to the drug would itself adapt to neutralize the drug’s effects; persistence of the opposing effects after the drug disappears would produce the withdrawal response …

In the between-systems opposing process, a different cellular system and separable molecular apparatus would be triggered by the changes in the primary drug response neurons and would produce the adaptation and tolerance. (Koob and Bloom, 1988, 720)

The fourth breakthrough was initiated early upon my arrival to San Diego in 1977. I had met and was befriended by J. Anthony “Tony” Deutsch, a professor of psychology at UCSD. Somewhere around 1979–1980, Tony offered to let me help him teach undergraduates. I ultimately began teaching a course he taught called Drugs, Addiction, and Mental Disorders. Tony was an endless source of intellectual discussion about various topics related to motivation. Perhaps ultimately the most rewarding for me, he introduced me to exotic fruit gardening in San Diego County by supplying me with everything from figs to cherimoyas to tropical guavas. This evolved into a significant avocation, where we currently care for more than 150 varieties of fruit trees and have fruit year-round on our property in Rancho Santa Fe, California.

I enjoyed teaching so much that I created two undergraduate courses: Drugs and Behavior and Impulse Control Disorders. I also created the graduate-level psychopharmacology course. My teaching had two other major benefits to my career: (1) enabling me to explain the meaning of what I did in research with regard to translation to the human condition, and (2) allowing UCSD undergraduate students to join our laboratory for independent study and honor research thesis projects. Almost all the research assistants we hired over my tenure at TSRI were former students with independent research projects. However, it was with the creation in 1997 of my course Impulse Control Disorders, which used as a background framework the principles of self-regulation outlined in the book Losing Control (Baumeister et al., 1994), that I began to see a common pattern of phenotypes that we collectively term “addiction,” whether an addiction to drugs or a nondrug addiction (or “process addiction”; e.g., gambling). I realized that addiction inevitably involves a repeated three-stage cycle: binge/intoxication, withdrawal/negative affect, and preoccupation/anticipation.

The fifth breakthrough again came through an invitation from Floyd Bloom, then Editor-in-Chief of Science, that Michel Le Moal and I write a review on addiction entitled, “Drug Abuse: Hedonic Homeostatic Dysregulation” (Koob and Le Moal, 1997). Here, for the first time, all our thinking about addiction converged, and we characterized addiction as a chronically relapsing disorder, characterized by loss of control and compulsive drug seeking within a heuristic framework comprising a three-stage addiction cycle: binge/intoxication, withdrawal/negative affect, and preoccupation/anticipation. We integrated the addiction cycle with elements of the diagnostic criteria for substance dependence from the Diagnostic and Statistical Manual of Mental Disorders, 4th edition (American Psychiatric Association, 1994), with the elements of self-regulation failure and with hypothesized neurochemical mechanisms (Koob and Le Moal, 1997). My participation in an episode of the documentary series directed by Bill Moyers, The Hijacked Brain (season 1, episode 2, Addiction: Close to Home, 1998), where I explained my perspective on addiction to a lay audience, also helped me consolidate my thinking on the hedonic dysregulation that was caused by addiction. During this period from 1983 to 1997, a large number of outstanding postdoctoral fellows (38) and visiting scientists (12) had passed through the Koob lab.²

The Scripps Research Institute, The Second Half, 1998–2014

Our role and integration at TSRI grew during this period, and ultimately Floyd formed the Department of Neuropharmacology in 1990. In 1990, I was promoted to professor in the Department of Neuropharmacology. From 2002 to 2004, I served as acting chair of the Department of Neuropharmacology. Then in 2006, to consolidate and coordinate our work on the neurobiology of addiction and at the suggestion of the president of TSRI, Richard Lerner, we formed the department-level Committee on the Neurobiology of Addictive Disorders. I then served as a professor and the Chair until leaving to become director of the National Institute on Alcohol Abuse and Alcoholism (NIAAA) in 2014.

At this point, approximately midway through my research career, my initial research programs on drug reward, neuropeptides, and stress converged, and all of my energy began to focus on what we termed the negative emotional side (or “dark side”) of addiction and how such negative emotional states drive negative reinforcement to contribute to the compulsive drug-seeking and drug-taking characteristics of addiction. More eureka! moments awaited, which were now directed at the role of stress and negative emotional states in addiction.

My next eureka! moment occurred when Serge Ahmed, my postdoctoral fellow from Bordeaux, decided to test the hypothesis that extended access to cocaine would prolong extinction to cocaine. Rats were divided into two groups: one with access to intravenous cocaine self-administration for 1 hour (short-access group) and one with access to intravenous cocaine self-administration for 6 hours (long-access group). An escalation in intake was observed only in the long-access group with the increase in intake observed during not only during the first hour of the session, but also sustained over the entire session and an upward shift of the dose-effect function. When the animals were allowed access to different doses of cocaine, both the long- and short-access animals titrated their cocaine intake, but long-access rats consistently self-administered almost twice as much cocaine at any dose tested, suggesting a decrease in reward or increase in hedonic threshold in the escalated animals (Ahmed and Koob, 1998). Such an escalation in intake was then observed by us not only for cocaine but also for methamphetamine, heroin, and nicotine and in alcohol-dependent animals (Kitamura et al., 2006; Chen et al., 2006; Cohen et al., 2012; Roberts et al., 2000). Confirming the hypothesized set point change was the observation that intracranial self-stimulation thresholds increased in parallel with the escalation of cocaine (Ahmed et al., 2002), methamphetamine (Jang et al., 2013), and heroin (Kenny et al., 2006). While no animal model fully replicates the panoply of symptoms that are associated with drug addiction in humans, animals with extended access to drug, in effect, meet most of the criteria associated with addiction, including tolerance, withdrawal, substance taken in larger amounts and over a longer period than intended, and elements of compulsive-like drug seeking, thus providing a powerful means of examining the neuroadaptations driving compulsive-like drug seeking (Edwards and Koob, 2013; George et al., 2014).

As such, extended access to drugs of abuse and dependence-induced alcohol drinking have provided significant insight into the role of stress-related neurotransmitter neuromodulators in driving compulsive drug seeking. For alcohol, intracerebroventricular administration of a CRF₁/CRF₂ receptor antagonist blocked the increase in alcohol self-administration during acute withdrawal from alcohol vapor-induced dependence in rats, but CRF receptor antagonists had no effect on alcohol self-administration in nondependent animals (Valdez et al., 2002). When administered directly into the CeA, the same CRF receptor antagonist blocked the increase in alcohol self-administration in dependent rats (Funk et al., 2006). These data suggest an important role for CRF, primarily within the CeA, in mediating the increase in self-administration that is associated with dependence. We also found that CRF receptor antagonists, injected intracerebroventricularly or systemically, blocked potentiated anxiety-like responses to stressors during protracted abstinence (Breese et al., 2005; Valdez et al., 2003) and the increase in alcohol self-administration that is associated with protracted abstinence (Valdez et al., 2002; Chu et al., 2007), similar to results reported by others (Sommer et al., 2008; Gehlert et al., 2007).

Critical to these studies was another of my long-standing collaborations with my colleagues at the University of Bordeaux. Luis Stinus had established a robust conditioned place aversion paradigm using three chambers and a central triangular area. Luis and I, with Gery Schulteis (my postdoctoral fellow), then went on to show that this paradigm produced robust conditioned place aversion to very low doses of naloxone (4–8 μg/kg) injected subcutaneously in opioid-dependent rats (Schulteis et al., 1994). We went on to show that such place aversion lasted for weeks once established (Stinus et al., 2000) and was sensitive to blockade by CRF receptor antagonists (Stinus et al., 2005). In a study of the suppression of operant responding as an index of withdrawal malaise, naloxone precipitated an aversive response after a single morphine injection (Schulteis et al., 1997). These studies together pointed to long-lasting conditioned responses to neuroadaptation in hedonic systems (aversive responses) that occurred early in the addiction process, much earlier than had been previously thought.

CRF₁ receptor small-molecule receptor antagonists also selectively reduced the increase in alcohol self-administration associated with withdrawal (Funk et al., 2007). CRF receptor antagonists also blocked the increase in intake that was associated with extended access to the intravenous self-administration of cocaine (Specio et al., 2008), nicotine (George et al., 2007), heroin (Greenwell et al., 2009; Park et al., 2015), and food (Cottone et al., 2009).

During this period, around 1999–2000, one of my postdoctoral fellows, Noelle Gracy, took a position with Academic Press/Elsevier, and she commissioned Michel Le Moal and me to write a book on the neuroscience of addiction, which was finally published as the Neurobiology of Addiction in 2006 (Koob and Le Moal, 2006). Then, in 2014, in collaboration with Michael (Mike) Arends (who began working with me as an undergraduate research student in 1994 and became instrumental to my publishing efforts ever since), we wrote a college-level textbook called Drugs, Addiction, and the Brain (Koob et al., 2014). Because of the enormity of research generated on the neurobiology of addiction in the 10 years since publication of Neurobiology of Addiction in 2006, we embarked on writing an update, which blossomed into a series of five volumes. As of this writing, three of these volumes are published—Neurobiology of Addiction: Volume 1. Introduction to Addiction (Koob et al., 2019), Neurobiology of Addiction: Volume 2. Psychostimulants (Koob et al., 2020a), and Neurobiology of Addiction: Volume 3. Alcohol (Koob et al., 2021)—and the next two volumes (Opioids and Nicotine/Cannabis) are well underway.

A key to success in any endeavor requires skills from many quarters. Since 1994, Mike Arends has served as my own editor in chief, polishing my writing and correcting all errors since that time (more than 600 peer-reviewed articles and book chapters). He also took on managing editor roles for two journals for which I served as Editor-in-Chief: Pharmacology, Biochemistry and Behavior and Journal of Addiction Medicine. Mike has also aided me tremendously since the mid-1990s with salient literature searches that are integral to my writing, to the point that I included him as a coauthor on books I have written with Michel Le Moal, including Drugs, Addiction, and the Brain (Koob et al., 2014) and the Neurobiology of Addiction series. With his invaluable help, I am afforded the time to focus on writing, thinking, and synthesizing. He continues to be instrumental and to make substantive contributions to my success.

The research results and our conceptual evolution generated over this period led Michel and me to refine our thinking about a more prominent role for neuroadaptations in brain stress systems as one progresses through the stages of the addiction cycle. Here, we added to our conceptualization of the hedonic dysregulation associated with drug addiction by invoking the contribution of an anti-reward system and the role of negative reinforcement (Koob and Le Moal, 2008). A further part of this thesis was that a key part of addiction involves long-term, persistent dysregulation of the activity of neural circuits that mediate anti-reward motivational systems that derive from two sources: a decrease in the function of brain reward systems that normally mediate natural rewards (within-system neuroadaptations) and recruitment of brain stress systems (between-system neuroadaptations), both of which drive aversive states (Koob and Le Moal, 2008). We argued that such anti-reward neuroadaptations represent the opponent process state that is hypothesized to be triggered by the excessive activation of brain reward systems that forms a general feature of biological systems (Solomon, 1980). The development of the aversive emotional state that drives the negative reinforcement of addiction was conceptualized as the “dark side” of addiction (Koob and Le Moal, 2005). Thus, drug seeking in the binge/intoxication stage was hypothesized to largely involve positive reinforcement. As hedonic allostasis develops, an additional source of motivation, negative reinforcement, is recruited, in which the individual engages in compulsive drug seeking and drug taking to relieve withdrawal symptoms (Koob and Le Moal, 2008).

In 2010, Nora Volkow, director of the National Institute on Drug Abuse (NIDA), and I were asked by the journal Neuropsychopharmacology Reviews to review the literature on the neurocircuitry of addiction. Here, we bridged what was known about the neurocircuitry of addiction from both preclinical data from animal models of addiction and neuroimaging data from human studies to the three-stage cycle framework and provided a heuristic basis for studying the neurobiology of addiction (Koob and Volkow, 2010).

Around this time and in a different context with two anesthesiologists, Joe Shurman at Scripps Memorial Hospital and Howard Gutstein at MD Anderson Cancer Center, we attempted to explore why some individuals who take prescription opioids progress to opioid addiction (Shurman et al., 2010). Our hypothesis was that opioid misuse in the context of pain management produces hypersensitivity to emotional distress. Our thesis was that opioid withdrawal consisted of a major negative emotional state that is characterized not just by physical symptoms and physical pain but also by negative emotional symptoms that are associated with opioid withdrawal and alcohol and drug withdrawal in general, including chronic irritability, physical pain, emotional pain, malaise, dysphoria, alexithymia, and the loss of motivation for natural rewards. These symptoms could be engaged if a subject took opioids at levels above those needed to address acute pain (Shurman et al., 2010). In this context, I decided to create my own word—hyperkatifeia, defined as the increased intensity of the constellation of negative emotional/motivational symptoms and signs observed during withdrawal from drugs of abuse (hypersensitive negative emotional state)—to parallel the word hyperalgesia, which described a hypersensitive pain state. Both hyperkatifeia and hyperalgesia were hypothesized to be generated by opioid withdrawal (Shurman et al., 2010).

Thus, the compulsive drug seeking that is associated with addiction is hypothesized to derive from multiple sources of motivational dysregulation that occur in three functional domains that reflect the three stages of the addiction cycle: incentive salience/pathological habits in the binge/intoxication stage, negative emotional states in the withdrawal/negative affect stage, and executive function deficits in the preoccupation/anticipation stage (Everitt and Wolf, 2002; Koob and Volkow, 2010). These three domains and stages are hypothesized to be mediated by three major neurocircuitry elements: basal ganglia, extended amygdala, and prefrontal cortex, respectively. The hyperkatifeia that drives negative reinforcement is hypothesized to derive from the loss of function of key neurochemical circuits within the brain reward system in the basal ganglia and gain of function within the brain stress systems (CRF, dynorphin, norepinephrine, hypocretin, vasopressin, glucocorticoids, and neuroimmune factors) in the extended amygdala. Compelling evidence exists to argue that hyperkatifeia that is triggered by acute excessive drug intake is sensitized during the development of compulsive alcohol taking with repeated withdrawal, persists into protracted withdrawal, and contributes to the development and persistence of the compulsive loss of control of drug seeking. Again, all this work was facilitated by a large number of outstanding postdoctoral fellows (34) and visiting scientists (8) who had passed through the Koob lab from 1998 to 2014.³

On a personal note, from 1994–2014, I raised my son, Cameron Michel Koob, who went on to Franklin and Marshall College, graduated and worked in politics helping with the Democratic National Committee and a number of political candidates, including Tom Steyer’s campaign for president. He is currently a Georgetown University law school student, aspiring to a career in corporate law with a possible focus on the biomedical domain.

At this point, my career had made a full circle. I had returned to the original findings of my PhD thesis that hypoxic stress facilitated reward. I reflected on the relationship between reward and stress and two towering figures from the field of psychology who influenced my early career, Horsley Gantt from the Pavlovian tradition and Joseph Brady from the operant tradition (Gantt, 1942; Brady, 1956; Koob, 2017). Their observations that conflict and aversive stimuli can result in emotional-like responses imprinted me in how aversive stimuli can motivate behavior in the form of negative reinforcement and how this process forms the basis for a major part of the pathophysiology of addiction. Thus, as observed by Louis Pasteur, in a sense, my PhD training supported the argument, “In the fields of observation, chance favors only the prepared mind” (Louis Pasteur lecture, University of Lille, December 7, 1854).

So how did my graduate studies at Johns Hopkins with Zoltan Annau prepare me to go from the positive reinforcement studies in the first half of my career to negative reinforcement studies in the second half of my career? In my PhD thesis studies, we identified a role for catecholamines in how mild to moderate stress can facilitate brain reward systems. Sol Snyder introduced me to how drugs of abuse profoundly activate brain reward systems. Perhaps as part of the rich tradition of the study of reinforcement, from both classical and operant conditioning perspectives, Horsley Gantt and Joseph Brady laid the conceptual groundwork for motivation and emotion that resonated with me and prepared me to recognize that the converse could also be true: excess reward can engage homeostatic mechanisms to regulate motivation. However, when Michel Le Moal and I embraced Richard Solomon’s opponent process theory, with an overlay of allostasis, the neurocircuitry of the emotional states that were described by Gantt and Brady were unveiled.

During this period, I took on some significant administrative responsibilities. Floyd Bloom became editor in chief of Science in 1995, and I replaced him as director of the NIAAA-funded Alcohol Research Center at TSRI from 1995 to 2014, carried through four 5-year renewals. In 2001, I successfully won an NIAAA consortium grant, Integrative Neuroscience Initiative on Alcoholism (INIA), and served as consortium coordinator from 2001 until 2014, successfully renewing it twice. In 2006, I formed the Committee on the Neurobiology of Addictive Disorders at TSRI. During my tenure as director of the TSRI ARC, we began to focus on the neurobiology of alcohol use disorder (AUD) at multiple levels of analysis, from molecular to cellular to neuropharmacology to neurocircuitry. In parallel with our changing conceptual framework, we moved from studies on the acute intoxicating effects of alcohol to the neuroadaptations associated with the development of dependence. A significant advance was developing an animal model of dependence-induced drinking using a chronic intermittent ethanol exposure procedure. Another major advance was to focus molecular and cellular studies on brain circuits that are relevant to the engagement of excessive drinking in dependence. We also began examining the role of not only GABA and glutamate neurotransmission but also their interaction with neuropeptides in the extended amygdala in the negative emotional state and stress associated with alcohol dependence. Finally, while directing the TSRI ARC, I developed the concept of the “Center at Large” to expand the ARC to be a resource for alcohol research across the whole San Diego community to recruit new young investigators and disseminate alcohol-related information in community outreach.

Some of the original goals of the INIA consortium were to first identify gene targets that are produced by excessive drinking in animal models, nominated by expression assays or other methods, and then to confirm gene targets using transgenic, knockout, inducible knockout, site-specific knockout, RNAi, in situ hybridization, in vivo electrophysiology and imaging, and next-generation sequencing. Another goal was to attract new and innovative investigators to the field of alcohol research by recruiting individuals for U01 grants and Pilot Projects and making informatics-integrated datasets accessible, searchable, and interactive with other databases for all scientists who are interested in alcoholism research. When I handed the reins of INIA over to R. Adron Harris at the University of Texas, Austin, in 2014 to then become director of NIAAA, the consortium had evolved even further. New goals included the identification of druggable targets that would be the most promising for medications development for the treatment of AUD.

Significant progress was made on all the goals of INIA and centered on the refinement and standardization of animal models of excessive drinking, identification of new gene targets for excessive drinking, and implementing novel approaches for functional validation. Extensive gene expression studies using gene arrays were completed on genetic and behavioral models of excessive drinking. Three major functional gene pathways were identified, providing novel gene targets: neuroinflammation, ion channels, and second messenger systems. A key result was the finding of a significant number of genes that are related to neuroimmune function that are activated in a transcriptome meta-analysis across INIA (Mulligan et al., 2006). Subsequent studies supported a key role for neuroimmune function with chronic alcohol exposure, ultimately leading to this part of INIA being termed INIA-Neuroimmune (Blednov et al., 2011). Other major contributions of INIA to the field included the development, refinement, and standardization of excessive drinking animal models so that they reliably produce blood alcohol levels greater than 100 mg% and can be widely used in interactive studies across INIA, including the development of an animal model of binge-like drinking in mice (drinking in the dark; Rhodes et al., 2005). Novel approaches to functional validation that were introduced within INIA were the use of next-generation transcriptome sequencing and the design and production of viral vectors for RNAi knockdown and the development of knockout mice and rats. Additionally, numerous outstanding neuroscientists were recruited to the alcohol field, thereby broadening and diversifying the scientific base. Thus, using NIAAA’s INIA consortium approach, the discovery of neuroimmune and other alcohol targets based on cross-species genomic, cellular, and behavioral neuroadaptations across different laboratories has systematically allowed the identification of conserved signaling pathways that are associated with AUD (Harris and Koob, 2017). This approach to neuroscience has provided the field with rigorous replicable measures for translation to the human condition and a heuristic framework advancing treatment strategies.

However, my interest in translational research had earlier roots with another conceptual breakthrough that originated with Barbara Mason at the University of Miami. She was the principal investigator on the U.S. multicenter clinical trial launched by Lipha Pharmaceuticals for the ultimate approval of acamprosate for the treatment of AUD. I was recruited to the team to help coordinate preclinical evidence of the mechanism of action of acamprosate from our own studies and those across the world (De Witte et al., 2005). Through these interactions, I came to learn about innovative human laboratory strategies for translation that were initiated by Barbara (Mason et al., 2008). She showed the efficacy of nalmefene, an opioid receptor antagonist, in treating alcoholism in a four-year double-blind study (Mason et al., 1999). She also directed the pivotal U.S. study for the approval of acamprosate for the treatment of AUD (Mason et al., 2006). She had developed a highly innovative and pioneering human laboratory paradigm for the development of medications for AUD using cue reactivity under conditions of different emotional settings in alcohol-dependent subjects (Mason et al., 2008). Her studies have shown that the response to drugs in the human laboratory paradigm is predictive of efficacy in clinical trials (Mason et al., 2009; Mason et al., 2014). Given our extensive studies identifying targets for the development of medications for AUD, Barbara and I decided to join forces and won support for the funding of a joint grant in 1999 on medication development in protracted abstinence in alcoholism, which was renewed several times until I left for NIAAA in 2014. Barbara validated her human model of craving with existing medications, and then we “translated” a number of novel compounds from the preclinical model of excessive drinking during withdrawal during chronic intermittent alcohol exposure in rodents to the Mason human laboratory study paradigm. Successful compounds from this work included gabapentin (Mason et al., 2014) and mifepristone (Vendruscolo et al., 2015). Her human laboratory approach is now being widely adopted in the field and should facilitate the development of medications for all drug addictions. Perhaps more importantly, this collaboration led to a conceptual breakthrough, what we called the Rosetta Stone approach, in which we hypothesized that existing pharmacotherapies for addiction could be used to validate and improve animal and human laboratory models to identify viable new treatment candidates (Koob et al., 2009). Barbara not only provided me with a conceptual framework for the movement of my career to translational neuroscience—she also helped me understand the clinical syndrome of addiction and the suffering such patients experience. During this period, I began a series of lectures at a local private treatment facility, Casa Palmera, in Del Mar, California, founded and run by Lee Johnson. With Barbara’s help, I was able to translate my brain science to patients, helping them understand that their conditions were treatable and that recovery was possible. My collaborations with Barbara also contributed to the clinical “top-down” impetus for the emphasis on negative emotional states and stress as driving forces of addiction and a key neglected opportunity for treatment targets (Koob and Mason, 2016).

At the same time, Barbara became my muse, intellectual sounding board, best friend, and soul mate. Floyd Bloom recruited Barbara to TSRI as the Pearson Family Professor in 2007, and our relationship flourished, and we fell in love. We have lived together since 2013, and we married in Fiji on February 14, 2018. Barbara is currently professor at Scripps Research, The Pearson Family Professor, Director of the Pearson Center for Alcoholism and Addiction Research, director of the TSRI Alcohol Research Center (the same one founded by Floyd Bloom in 1977), and adjunct professor in psychiatry at UCSD.

Director of the National Institute on Alcohol Abuse and Alcoholism, 2014–present

When T. K. Li retired as director of NIAAA, a five-year debate ensued to merge NIAAA with NIDA while acting director, Ken Warren, guided NIAAA. Ultimately, this merger failed, largely because of a lack of enthusiasm of all parties and the enormous effort and disruption such a merger would cause in the field. As a result, in 2013, the National Institutes of Health (NIH) began a search to recruit a new director of NIAAA. I was urged to apply by many of my colleagues and friends, notably by Nora Volkow, director of NIDA, with whom I had been collaborating in writing and scientific discussion for many years. I was offered the position. After much discussion, I realized that this was a prime opportunity to move the field forward and possibly move the field in directions that in my opinion were long neglected. Notable among these were the lack of emphasis on alcohol as a drug of addiction when considering its enormous burden on society, the lack of a heuristic framework for studying the role of motivational withdrawal (hyperkatifeia) in driving AUD and substance use disorder in general, the conceptual gap between clinical studies and preclinical work on which my collaboration with Barbara Mason had shone a light, and the lack of translation of evidence-based information about alcohol to the public.

During my tenure as director of NIAAA, I can list many accomplishments, including the current organization of the institute; its direction; its major mission of generating evidence-based information about the etiology of AUD, including its diagnosis, prevention, and treatment; and the dissemination of this information to the public.

For the organization of NIAAA, I developed and implemented a vision for the institute that effectively included providing a positive environment for the exchange of ideas by including my leadership team and entire staff in decisions. I streamlined the office of the director and shared all decision making with my deputy (Patricia Powell), executive officer (Vicki Buckley), and Associate Director for Basic Research (Bridget Williams-Simmons), which because of each of our unique skills and backgrounds, strengthened our approach to leadership. We also welcomed regular input from my intramural scientific director (George Kunos) and clinical director (David Goldman) and division directors (Antonio Noronha, Ralgh Hingson, Kathy Jung, and Raye Litten), and senior adviser to the director (Aaron White). These individuals and many others became what for me is our NIAAA family, and all that follows was the result of our efforts together as a team.

In the Intramural Research Program, I dissolved the large laboratory structure and employed an academic structure in which each investigator directed their own independent laboratory. I also complied with the overall NIH guidance to have the clinical director report directly to me. As a result, our outstanding NIAAA Intramural Research Program has become an incubator for innovative basic, translational, and clinical alcohol research and training.

Given that alcohol misuse contributes to more than 200 diseases and disorders, I made collaborations across NIH institutes/centers and throughout the federal government a priority. NIAAA maintains a very active Collaboration on Research on Addiction (CRAN) with NIDA and the National Cancer Institute (NCI). One of the most notable accomplishments of CRAN was the development and continued success of the Adolescent Brain Cognitive Development (ABCD) study, which is following the development of more than 10,000 children from nine to ten years old to adulthood, conducting every-other-year brain imaging and yearly neuropsychological testing and other environmental measures (Volkow et al., 2018). The ABCD study benefited significantly from the National Consortium on Alcohol and Neurodevelopment in Adolescence study launched by NIAAA in 2012. The ABCD data are available to researchers through the National Institute of Mental Health data archive and will provide a rich set of data on normal adolescent development of inestimable value for future research. NIAAA staff remain actively involved in planning and cosponsoring programs with other NIH institutes that are associated with pain, fetal alcohol spectrum disorder, child development, basic neuroscience (the BRAIN initiative), liver disease, cancer, and SARS-CoV-2.

As NIH sought to increase diversity and inclusion in the workforce and support research on health disparities, I rallied NIAAA staff at multiple levels to address these issues in meaningful ways with actionable steps, including an emphasis on hiring women to close the male–female gap in senior positions. I also strongly supported NIAAA’s Intramural Research Program in recruiting highly talented underrepresented minority investigators into tenure-track positions. I have led efforts to improve the culture and expand the health disparities portfolio, and I established an NIAAA Advisory Council working group on mentoring underrepresented minorities. I also continued to foster the next generation of alcohol researchers by promoting training through career development awards, strongly supporting early-stage investigators through funding and outreach, and developing multiple avenues for mentorship.

Throughout my tenure at NIAAA, I have developed not only a conceptual neurobiological framework to guide studies of the neurobiology of AUD that has proven to have invaluable heuristic utility but also a research translation framework for NIAAA to convey its evidence-based research to the real world. This research translation framework has resulted in a number of direct web-based programs to inform the public about alcohol misuse and key conceptual framework papers and reviews on such topics as the brain disorder model of addiction (Volkow and Koob, 2015); development of an Addictions Neuroclinical Assessment Framework (Kwako et al., 2016); microcircuitry analysis of the neurobiology of addiction (Koob and Volkow, 2016); a review of the state of knowledge of the molecular site of action for the initiation of alcohol effects on the brain (Cui and Koob, 2017); validation of the three domains of dysfunction that correspond to the three-stage cycle of addiction (Kwako et al., 2019); a major review on alcohol and sleep (Koob and Colrain, 2020); a commentary on the interaction between alcohol misuse, hyperkatifeia, the SARS-CoV-2 pandemic, and deaths of despair (Koob et al., 2020b); and a review with both the NIDA and National Institute of Mental Health directors on choosing appropriate language to reduce stigma in the fields of addiction and mental health in general (Volkow et al., 2021).

With regard to informing the public, I supported and initiated major efforts to increase awareness of the consequences of alcohol misuse, including AUD, to all facets of the community. I fully supported and helped with the rollout of College Alcohol Intervention Matrix (College AIM), an evidence-based menu of prevention options for college and university students. Started before my arrival at NIAAA, College AIM rates both individual and community prevention programs by effectiveness and cost. Under my tenure, it has been distributed to every college and university in the United States. In addition, in 2016, NIAAA collaborated with the television network HBO to produce a landmark documentary called Risky Drinking, which charts the spectrum of AUD across four human vignettes in the context of AUD as a brain disorder and in the context of treatment options.

I then initiated the NIAAA Alcohol Treatment Navigator, a clinical core resource, efforts to operationally define recovery, efforts to develop a consensus definition of fetal alcohol spectrum disorder, efforts to facilitate concurrent treatment of AUD with alcohol-associated liver disease, and efforts to compile a prevention core resource for adolescents. For example, the Alcohol Treatment Navigator provides key information about how AUD is a spectrum disorder with a corresponding range of treatments and two locators whereby one can enter a zip code and find a treatment provider in that zip code area. Subsequently, we added a portal to assist healthcare providers in making effective referrals for treatment.

I also initiated and led the development of a Healthcare Professional’s Core Resource released in 2022 that provides key information to healthcare providers, including physicians, pharmacists, psychologists, and nurse practitioners, about alcohol and treatment options. Input from clinicians, researchers, and other stakeholders has produced an accurate, extensive resource for what all healthcare providers who interface with patients need to know about alcohol’s effects on the human body across the lifespan. An outreach plan to maximize the impact of this critical resource is underway. Our outreach efforts through our website, Rethinking Drinking, College Aim, and the NIAAA Navigator have been updated to meet the challenges of the COVID-19 pandemic and new advances.

We also have made significant strides engaging clinicians and other healthcare providers to factor in a patient’s alcohol consumption in their overall healthcare. My efforts to engage specialists in liver disease through clinical researchers and the American Association of Liver Disease is also transforming healthcare by increasing the concurrent treatment of alcohol-associated liver disease and AUD. I also shifted priorities in the NIAAA portfolio toward research on the older adult population and women in response to epidemiological studies that show an increase in alcohol use, binge drinking, and consequences in these subpopulations. Collectively, these efforts highlight our efforts at understanding the changing needs of the U.S. population and ability to nimbly shift priorities as necessary to maximize program efficiency and results.

Finally, under my leadership, NIAAA expanded its research portfolio and maintained a robust, high-quality research program despite the challenges of SARS-CoV-2, which necessitated adapting to new research priorities and opportunities. Although considerable staff time was devoted to pandemic-related activities, I ensured that priorities to synergize research efforts and disseminate information to the public advanced with vigor. Examples include integrating pain, opioid use disorder, and sleep measures with AUD in clinical studies; supporting pharmacotherapy clinical trials for alcohol-related liver disease and AUD; finalizing the Healthcare Professional’s Core Resource; implementing a definition of AUD recovery for research; initiating a registry to follow patients who received AUD treatment; boosting research on fetal alcohol spectrum disorder prevention and treatment; and increasing focus on reducing stigma. Of all of our tangible efforts at NIAAA during my tenure, I am most proud of the Risky Drinking documentary, College Aim, Rethinking Drinking, the NIAAA Treatment Navigator, development of an operational definition of recovery, and the Healthcare Professional’s Core Resource.

National Institute on Drug Abuse, Intramural Research Program, 2014–present

Upon arriving at NIH in 2014, I was allowed to continue supervising my research group at TSRI for two years while transitioning all my trainees to their next career goals. At the same time, I won an appointment as a tenured senior investigator in the NIDA Intramural Research Program where I established a basic research laboratory called the Neurobiology of Addiction Section in the Integrative Neuroscience Research Branch at the Bayview Center in Baltimore. I immediately recruited Leandro Vendruscolo, a senior postdoctoral fellow from my laboratory at TSRI, to coordinate my laboratory at NIDA. Brooke Schmeichel, who had just begun her fellowship with me at TSRI, also made the transition to NIDA. I then began a new group that consists of three to four postdoctoral fellows at any given time, up to six post-baccalaureate fellows, and currently two PhD students. My role gradually is evolving into what I call a “Le Pere Koob” role (“the father Koob” role), in which I help direct the overall program of the laboratory, participate in all the experimental designs, and help guide postdoctoral fellows toward K99 grant awards or faculty positions and postbaccalaureate fellows toward graduate or medical school.⁴

Some of the main accomplishments include the continued development of new animal models of “endophenotypes” that are associated with addiction, with a focus on negative emotional states that are associated with the withdrawal and negative affect stage of the addiction cycle, including the vapor-induced escalation of drug intake in rats (Vendruscolo et al., 2018), escalation of intravenous self-administration in mice (Towers et al., 2019), vapor escalation in mice (Moussawi et al., 2020), and pain measures (Marchette et al., 2021). We also identified and focused on exploring the neurocircuitry that is mediated by three neurochemical systems that are involved in the negative emotional state that drives the withdrawal and negative affect stage, including CRF, glucocorticoids, and mineralocorticoids (de Guglielmo et al., 2019; Vendruscolo et al., 2015; Aoun et al., 2018); dynorphin/κ-opioid systems (Marchette et al., 2021); and oxytocin (Tunstall et al., 2019).

To extend our neurocircuitry approach to protracted withdrawal, with implications for the etiology of craving and relapse, functional magnetic resonance imaging (fMRI) was applied in rats to identify amygdala and hypothalamic neurocircuits that were activated by stimuli that were previously paired with heroin withdrawal. This activation was related to the degree of heroin dependence, supporting the significance of conditioned negative affect in sustaining compulsive-like heroin seeking and taking (Carmack et al., 2019). These results were consistent with earlier observations in collaboration with Barry Everitt and Gery Schulteis, in which conditioned withdrawal (conditioned suppression of responding on a fixed-ratio schedule for food) could be blocked by lesions of the basolateral amygdala (Schulteis et al., 2000). In a follow-up imaging study, a putative rat salience network was identified using modularity analysis on resting-state fMRI data from rats to parcellate the rat insula into functional subdivisions, and mouse tract tracing data from the Allen Brain Atlas was used to confirm the network’s underlying structural connectivity. From the data from animals in the Carmack et al. (2019) study, a salience network, which consisted primarily of the ventral anterior insula and anterior cingulate cortex, responded to conditioned cues and showed greater functional connectivity to the default mode network during conditioned heroin withdrawal (Tsai et al., 2020). These results suggest that the insula and salience network may serve as markers of highly salient environmental stimuli, particularly conditioned stimuli paired with heroin withdrawal.

During the SARS-CoV-2 pandemic shutdown in 2020–2021, my collaborators and I wrote several reviews that have refined a conceptual framework that focuses on the role of negative emotional states in the withdrawal/negative affect stage of the addiction cycle. Noteworthy reviews included alcohol tolerance (Elvig et al., 2021); allostasis theory in opioid tolerance (Ballantyne and Koob, 2021); cues conditioned to withdrawal and negative reinforcement (Pantazis et al., 2021); opponent process, hyperkatifeia, negative reinforcement, and opioid addiction (Koob, 2020; Koob, 2021); using neuroimaging for the assessment of AUD (Voon et al., 2020); tolerance to opioids and allostasis theory (Ballantyne and Koob, 2021); and neurocircuitry of the comorbid posttraumatic stress disorder-opioid use disorder state (Upadhyay et al., 2021). To date, I have mentored or comentored 13 PhD students, 84 postdoctoral fellows, and 11 K99 grant awardees.

Summary

My journey in research has always been predicated on trying to understand the neurobiological mechanisms of the hedonic components of motivation: reward and anti-reward. After spending the first half of my career understanding how we feel good, I have spent the remainder of my career trying to understand how we feel bad. My studies on the neurobiology of stress and the neurobiology of addiction have come full circle and have provided windows on how the brain processes such emotions and how such processes go awry. I would like to think that our efforts have contributed to considering hyperkatifeia as a driving force of negative reinforcement in addiction and have provided insights into how we feel and express negative emotions in general. I have been blessed with wonderful mentors, students, postdoctoral fellows, collaborators, and staff, all of whom have enriched my journey, and I want to thank them all. Perhaps most poignantly as my career winds down, I am grateful for the opportunities I have had at NIAAA and in my writing to disseminate evidence-based science, particularly evidence-based neuroscience, to the public. Hopefully, our efforts in this regard will be our legacy of helping humankind in its never-ending quest for hedonic homeostasis.

Selected Bibliography

Ahmed SH, Kenny PJ, Koob GF, Markou A (2002) Neurobiological evidence for hedonic allostasis associated with escalating cocaine use. Nat Neurosci 5: 625–6.

Ahmed SH, Koob GF (1998) Transition from moderate to excessive drug intake: change in hedonic set point. Science 282: 298–300.

American Psychiatric Association (1994) Diagnostic and Statistical Manual of Mental Disorders, 4th edition. American Psychiatric Press, Washington DC.

Anlezark GM, Crow TJ, Greenway AP (1973) Impaired learning and decreased cortical norepinephrine after bilateral locus coeruleus lesions. Science 181: 682–4.

Aoun EG, Jimenez VA, Vendruscolo LF, Walter NAR, Barbier E, Ferrulli A, Haass-Koffler CL, Darakjian P, Lee MR, Addolorato G, Heilig M, Hitzemann R, Koob GF, Grant KA, Leggio L (2018) A relationship between the aldosterone-mineralocorticoid receptor pathway and alcohol drinking: preliminary translational findings across rats, monkeys and humans. Mol Psychiatry 23: 1466–73.

Ballantyne JC, Koob GF (2021) Allostasis theory in opioid tolerance. Pain 162: 2315–9.

Baldwin HA, Rassnick S, Rivier J, Koob GF, Britton KT (1991) CRF antagonist reverses the "anxiogenic" response to ethanol withdrawal in the rat. Psychopharmacology 103: 227–32.

Baumeister RF, Heatherton TF, Tice DM (1994) Losing Control: How and Why People Fail at Self-Regulation. Academic Press, San Diego.

Blednov YA, Ponomarev I, Geil C, Bergeson S, Koob GF, Harris RA (2011) Neuroimmune regulation of alcohol consumption: behavioral validation of genes obtained from genomic studies. Addict Biol 17: 108–20.

Brady JV (1956) Assessment of drug effects on emotional behavior. Science 123: 1033–4.

Breese GR, Overstreet DH, Knapp DJ, Navarro M (2005) Prior multiple ethanol withdrawals enhance stress-induced anxiety-like behavior: inhibition by CRF1- and benzodiazepine-receptor antagonists and a 5-HT1a-receptor agonist. Neuropsychopharmacology 30: 1662–9.

Britton KT, Lee G, Koob GF (1988) Corticotropin releasing factor and amphetamine exaggerate partial agonist properties of benzodiazepine antagonist Ro 15-1788 in the conflict test. Psychopharmacology 94: 306–11.

Brown MR, Fisher LA, Rivier J, Spiess J, Rivier C, Vale W (1982) Corticotropin-releasing factor: effects on the sympathetic nervous system and oxygen consumption. Life Sci 30: 207–10.

Carmack SA, Keeley RJ, Vendruscolo JCM, Lowery-Gionta EG, Lu H, Koob GF, Stein EA, Vendruscolo LF (2019) Heroin addiction engages negative emotional learning brain circuits in rats. J Clin Invest 130: 2480–4.

Chen SA, O’Dell L, Hoefer M, Greenwell TN, Zorrilla EP, Koob GF (2006) Unlimited access to heroin self-administration: independent motivational markers of opiate dependence. Neuropsychopharmacology 31: 2692–707 [corrigedum: 31:2802].

Chu K, Koob GF, Cole M, Zorrilla EP, Roberts AJ (2007) Dependence-induced increases in ethanol self-administration in mice are blocked by the CRF1 receptor antagonist antalarmin and by CRF1 receptor knockout. Pharmacol Biochem Behav 86: 813–21.

Cohen A, Koob GF, George O (2012) Robust escalation of nicotine intake with extended access to nicotine self-administration and intermittent periods of abstinence. Neuropsychopharmacology 37: 2153–60.

Cottone P, Sabino V, Roberto M, Bajo M, Pockros L, Frihauf JB, Fekete EM, Steardo L, Rice KC, Grigoriadis DE, Conti B, Koob GF, Zorrilla EP (2009) CRF system recruitment mediates dark side of compulsive eating. Proc Natl Acad Sci USA 106: 20016–20.

Crow TJ (1973) Catecholamine-containing neurones and electrical self-stimulation: 2. A theoretical interpretation and some psychiatric implications. Psychol Med 3: 66–73.

Cui C, Koob GF (2017) Titrating tipsy targets: the neurobiology of low-dose alcohol. Trends Pharmacol Sci 38: 556–68.

de Guglielmo G, Kallupi M, Pomrenze M, Crawford E, Simpson S, Schweitzer P, Koob GF, Messing RO, George O (2019) Inactivation of a CRF-dependent amygdalofugal pathway reverses addiction-like behaviors in alcohol-dependent rats. Nat Commun 10: 1238.

de Lecea L, Kilduff TS, Peyron C, Gao X, Foye PE, Danielson PE, Fukuhara C, Battenberg EL, Gautvik VT, Bartlett FS 2nd, Frankel WN, van den Pol AN, Bloom FE, Gautvik KM, Sutcliffe JG (1998) The hypocretins: hypothalamus-specific peptides with neuroexcitatory activity. Proc Natl Acad Sci USA 95: 322–7.

De Wied D (1976) Behavioral effects of intraventricularly administered vasopressin and vasopressin fragments. Life Sci 46: 27–9.

de Witte P, Littleton J, Parot P, Koob G (2005) Neuroprotective and abstinence-promoting effects of acamprosate: elucidating the mechanism of action. CNS Drugs 19: 517–37.

Edwards S, Koob GF (2013) Escalation of drug self-administration as a hallmark of persistent addiction liability. Behav Pharmacol 24: 356–62.

Ettenberg A, Pettit HO, Bloom FE, Koob GF (1982) Heroin and cocaine intravenous self-administration in rats: mediation by separate neural systems. Psychopharmacology 78: 204–9.

Elvig SK, McGinn A, Smith C, Arends MA, Koob GF, Vendruscolo LF (2021) Tolerance to alcohol: a critical yet understudied factor in alcohol addiction. Pharmacol Biochem Behav 204: 173155.

Everitt BJ, Wolf ME (2002) Psychomotor stimulant addiction: a neural systems perspective. J Neurosci 22: 3312–20 [erratum: 22(16): 1a].

Ferster CB, Skinner BF (1957) Schedules of Reinforcement. Appleton-Century-Crofts, New York.

Funk CK, O’Dell LE, Crawford EF, Koob GF (2006) Corticotropin-releasing factor within the central nucleus of the amygdala mediates enhanced ethanol self-administration in withdrawn, ethanol-dependent rats. J Neurosci 26: 11324–32.

Funk CK, Zorrilla EP, Lee MJ, Rice KC, Koob GF (2007) Corticotropin-releasing factor 1 antagonists selectively reduce ethanol self-administration in ethanol-dependent rats. Biol Psychiatry 61: 78–86.

Gantt WH (1942) The origin and development of nervous disturbances experimentally produced. Am J Psychiatry 98: 475–81.

Gehlert DR, Cippitelli A, Thorsell A, Le AD, Hipskind PA, Hamdouchi C, Lu J, Hembre EJ, Cramer J, Song M, McKinzie D, Morin M, Ciccocioppo R, Heilig M (2007) 3-(4-Chloro-2-morpholin-4-yl-thiazol-5-yl)-8-(1-ethylpropyl)-2,6-dimethyl-imidazo[1,2-b]pyridazine: a novel brain-penetrant, orally available corticotropin-releasing factor receptor 1 antagonist with efficacy in animal models of alcoholism. J Neurosci 27: 2718–26.

George O, Ghozland S, Azar MR, Cottone P, Zorrilla EP, Parsons LH, O’Dell LE, Richardson HN, Koob GF (2007) CRF-CRF₁ system activation mediates withdrawal-induced increases in nicotine self-administration in nicotine-dependent rats. Proc Natl Acad Sci USA 104: 17198–203.

George O, Koob GF, Vendruscolo LF (2014) Negative reinforcement via motivational withdrawal is the driving force behind the transition to addiction. Psychopharmacology 231: 3911e7.

Greenwell TN, Funk CK, Cottone P, Richardson HN, Chen SA, Rice K, Zorrilla EP, Koob GF (2009) Corticotropin-releasing factor-1 receptor antagonists decrease heroin self-administration in long-, but not short-access rats. Addict Biol 14: 130–43.

Griebel G, Simiand J, Serradeil-Le Gal C, Wagnon J, Pascal M, Scatton B, Maffrand JP, Soubrie P (2002) Anxiolytic- and antidepressant-like effects of the non-peptide vasopressin V1b receptor antagonist, SSR149415, suggest an innovative approach for the treatment of stress-related disorders. Proc Natl Acad Sci USA 99: 6370–5.

Grieder TE, Herman MA, Contet C, Tan LA, Vargas-Perez H, Cohen A, Chwalek M, Maal-Bared G, Freiling J, Schlosburg JE, Clarke L, Crawford E, Koebel P, Repunte-Cononigo V, Sanna PP, Tapper AR, Roberto M, Kieffer BL, Sawchenko PE, Koob GF, van der Kooy D, George O (2014) VTA CRF neurons mediate the aversive effects of nicotine withdrawal and promote intake escalation. Nat Neurosci 17: 1751–8.

Harris RA, Koob GF (2017) The future is now: a 2020 view of alcoholism research. Neuropharmacology 122: 1–2.

Heilig M, McLeod S, Koob GK [sic], Britton KT (1992) Anxiolytic-like effect of neuropeptide Y (NPY), but not other peptides in an operant conflict test. Regul Pept 41: 61–9.

Heinrichs SC, Cole BJ, Merlo-Pich E, Menzaghi F, Koob GF, Hauger RL (1992) Endogenous corticotropin-releasing factor modulates feeding induced by neuropeptide Y or a tail-pinch stressor. Peptides 13: 879–4.

Heyser CJ, Roberts AJ, Schulteis G, Koob GF (1999) Central administration of an opiate antagonist decreases oral ethanol self-administration in rats. Alcohol Clin Exp Res 23: 1468–76.

Hoffman HS, Ratner AM, Eiserer LA (1972) Role of visual imprinting in the emergence of specific filial attachments in ducklings. J Comp Physiol Psychol 81: 399–409.

Hubner CB, Koob GF (1990) The ventral pallidum plays a role in mediating cocaine and heroin self-administration in the rat. Brain Res 508: 20–9.

Jang CG, Whitfield T, Schulteis G, Koob GF, Wee S (2013) A dysphoric-like state during early withdrawal from extended access to methamphetamine self-administration in rats. Psychopharmacology 225: 753–63.

Kenny PJ, Chen SA, Kitamura O, Markou A, Koob GF (2006) Conditioned withdrawal drives heroin consumption and decreases reward sensitivity. J Neurosci 26: 5894–900.

Kitamura O, Wee S, Specio SE, Koob GF, Pulvirenti L (2006) Escalation of methamphetamine self-administration in rats: a dose-effect function. Psychopharmacology 186: 48–53.

Koob GF (1982) The dopamine anhedonia hypothesis: a pharmacological phrenology. Behav Brain Sci 5: 63–4.

Koob GF (1992) Dopamine, addiction and reward. Semin Neurosci 4: 139–48.

Koob GF (2017) The dark side of addiction: the Horsley Gantt to Joseph Brady connection. J Nerv Ment Dis 205: 270–2.

Koob GF (2020) Neurobiology of opioid addiction: opponent process, hyperkatifeia and negative reinforcement. Biol Psychiatry 87: 44–53.

Koob GF (2021) Drug addiction: hyperkatifeia/negative reinforcement as a framework for medications development. Pharmacol Rev 73: 163–201.

Koob GF, Annau Z (1973) Effect of hypoxia on hypothalamic mechanisms. Am J Physiol 224: 1403–8.

Koob GF, Annau Z (1974) Behavioral and neurochemical alterations induced by hypoxia in rats. Am J Physiol 227: 73–8.

Koob GF, Annau Z, Blake WD Jr, Weinstein SA (1970) Hypothalamic self-stimulation, eating, and drinking: the effects of hypoxia. Commun Behav Biol A 5: 205–10.

Koob GF, Arends MA, Le Moal M (2014) Drugs, Addiction, and the Brain. Academic Press, San Diego.

Koob GF, Arends MA, McCracken M, Le Moal M (2019) Neurobiology of Addiction: Volume 1. Introduction to Addiction. Elsevier, New York.

Koob GF, Arends MA, McCracken M, Le Moal M (2020a) Neurobiology of Addiction: Volume 2. Psychostimulants. Elsevier, New York.

Koob GF, Arends MA, McCracken M, Le Moal M (2021) Neurobiology of Addiction: Volume 3. Alcohol. Elsevier, New York.

Koob GF, Balcom GJ, Meyerhoff JL (1975) Dopamine and norepinephrine levels in the nucleus accumbens, olfactory tubercle, and corpus striatum following lesions in the ventral tegmental area. Brain Res 94: 45–55.

Koob GF, Bloom FE (1988) Cellular and molecular mechanisms of drug dependence. Science 242: 715–23.

Koob GF, Colrain IM (2020) Alcohol use disorder and sleep disturbances: a feed-forward allostatic framework. Neuropsychopharmacology 45: 141–65.

Koob GF, Everitt BJ, Robbins TW (2008) Reward, motivation, and addiction. In: Squire LG, Berg D, Bloom FE, Du Lac S, Ghosh A, Spitzer N (eds) Fundamental Neuroscience, 3rd edition. Academic Press, Amsterdam, pp. 987–1016.

Koob GF, Fray PJ, Iversen SD (1976) Tail-pinch stimulation: sufficient motivation for learning. Science 194: 637–9.

Koob GF, Fray PJ, Iversen SD (1978a) Self-stimulation at the lateral hypothalamus and locus coeruleus after specific unilateral lesions of the dopamine system. Brain Res 146: 123–40.

Koob GF, Le Moal M (1997) Drug abuse: hedonic homeostatic dysregulation. Science 278: 52–8.

Koob GF, Le Moal M (2005) Plasticity of reward neurocircuitry and the “dark side” of drug addiction. Nat Neurosci 8: 1442–4.

Koob GF, Le Moal M (2006) Neurobiology of Addiction. Academic Press, London.

Koob GF, Le Moal M (2008) Addiction and the brain antireward system. Annu Rev Psychol 59: 29–53.

Koob GF, Lloyd GK, Mason BJ (2009) Development of pharmacotherapies for drug addiction: a Rosetta Stone approach. Nat Rev Drug Discov 8: 500–15.

Koob GF, Mason BJ (2016) Existing and future drugs for the treatment of the dark side of addiction. Annu Rev Pharmacol Toxicol 56: 299–322.

Koob GF, Powell P, White A (2020b) Addiction as a coping response: hyperkatifeia, deaths of despair, and COVID-19. Am J Psychiatry 177: 1031–7.

Koob GF, Riley SJ, Smith SC, Robbins TW (1978b) Effects of 6-hydroxydopamine lesions of the nucleus accumbens septi and olfactory tubercle on feeding, locomotor activity and amphetamine anorexia in the rat. J Comp Physiol Psychol 92: 917–27.

Koob GF, Volkow ND (2010) Neurocircuitry of addiction. Neuropsychopharmacol Rev 35: 217–38 [erratum: 35:1051].

Koob GF, Volkow ND (2016) Neurobiology of addiction: a neurocircuitry analysis. Lancet Psychiatry 3: 760–73.

Koob GF, Wall TL, Bloom FE (1989) Nucleus accumbens as a substrate for the aversive stimulus effects of opiate withdrawal. Psychopharmacology 98: 530–4.

Koob GF, Weiss F (1990) Pharmacology of drug self-administration. Alcohol 7: 193–7.

Kwako LE, Momenan R, Litten RZ, Koob GF, Goldman D (2016) Addictions neuroclinical assessment: a neuroscience-based framework for addictive disorders. Biol Psychiatry 80: 179–89.

Kwako LE, Schwandt ML, Ramchandani VA, Diazgranados N, Koob GF, Volkow ND, Blanco C, Goldman D (2019) Neurofunctional domains derived from deep behavioral phenotyping in alcohol use disorder. Am J Psychiatry 176: 744–53.

Lai C, Brow MA, Nave KA, Noronha AB, Quarles RH, Bloom FE, Milner RJ, Sutcliffe JG (1987) Two forms of 1B236/myelin-associated glycoprotein, a cell adhesion molecule for postnatal neural development, are produced by alternative splicing. Proc Natl Acad Sci USA 84: 4337–41.

Lebrun C, Le Moal M, Koob GF, Bloom FE (1985) Vasopressin pressor antagonist injected centrally reverses behavioral effects of peripheral injection of vasopressin, but only at doses that reverse increase in blood pressure. Regul Pept 11: 173–81.

Le Moal M, Dantzer R, Michaud B, Koob GF (1987) Centrally injected arginine vasopressin (AVP) facilitates social memory in rats. Neurosci Lett 77: 353–9.

Le Moal M, Dantzer R, Mormede P, Baduel A, Lebrun C, Ettenberg A, van der Kooy D, Wenger J, Deyo S, Koob GF, Bloom FE (1984) Behavioral effects of peripheral administration of arginine vasopressin: a review of our search for a mode of action and a hypothesis. Psychoneuroendocrinology 9: 319–41.

Le Moal M, Koob GF, Koda LY, Bloom FE, Manning M, Sawyer WH, Rivier J (1981) Vasopressor receptor antagonist prevents behavioural effects of vasopressin. Nature 291: 491–3.

Marchette RCN, Gregory-Flores A, Tunstall BJ, Carlson ER, Jackson S, Sulima A, Rice KC, Koob GF, Vendruscolo LF (2021) κ-Opioid receptor antagonism reverses heroin withdrawal-induced hyperalgesia in male and female rats. Neurobiol Stress 14: 100325.

Mason BJ, Goodman AM, Chabac S, Lehert P (2006) Effect of oral acamprosate on abstinence in patients with alcohol dependence in a double-blind, placebo-controlled trial: the role of patient motivation. J Psychiatr Res 40: 383–93.

Mason BJ, Light JM, Escher T, Drobes DJ (2008) Effect of positive and negative affective stimuli and beverage cues on measures of craving in non treatment-seeking alcoholics. Psychopharmacology 200: 141–50.

Mason BJ, Light JM, Williams LD, Drobes DJ (2009) Proof-of-concept human laboratory study for protracted abstinence in alcohol dependence: effects of gabapentin. Addict Biol 14: 73–83.

Mason BJ, Quello S, Goodell V, Shadan F, Kyle M, Begovic A (2014) Gabapentin treatment for alcohol dependence: a randomized clinical trial. JAMA Intern Med 174: 70–7.

Mason BJ, Salvato FR, Williams LD, Ritvo EC, Cutler RB (1999) A double-blind, placebo-controlled study of oral nalmefene for alcohol dependence. Arch Gen Psychiatry 56: 719–24.

Pantazis CB, Gonzalez LA, Tunstall BJ, Carmack SA, Koob GF, Vendruscolo LF (2021) Cues conditioned to withdrawal and negative reinforcement: neglected but key motivational elements driving opioid addiction. Sci Adv 7: eabf0364.

Merlo-Pich E, Lorang M, Yeganeh M, Rodriguez de Fonseca F, Raber J, Koob GF, Weiss F (1995) Increase of extracellular corticotropin-releasing factor-like immunoreactivity levels in the amygdala of awake rats during restraint stress and ethanol withdrawal as measured by microdialysis. J Neurosci 15: 5439–47.

Montigny R, Kahn O (1977) Cuisine Sans Souci. Le-Livre, Sablons.

Moussawi K, Ortiz MM, Gantz SC, Tunstall BJ, Marchette RCN, Bonci A, Koob GF, Vendruscolo LF (2020) Fentanyl vapor self-administration model in mice to study opioid addiction. Sci Adv 6: eabc0413.

Mulligan MK, Ponomarev I, Hitzemann RJ, Belknap JK, Tabakoff B, Harris RA, Crabbe JC, Blednov YA, Grahame NJ, Phillips TJ, Finn DA, Hoffman PL, Iyer VR, Koob GF, Bergeson SE (2006) Toward understanding the genetics of alcohol drinking through transcriptome meta-analysis. Proc Natl Acad Sci USA 103: 6368–73.

Olds J, Milner P (1954) Positive reinforcement produced by electrical stimulation of septal area and other regions of rat brain. J Comp Physiol Psychol 47: 419–27.

Olive MF, Koenig HN, Nannini MA, Hodge CW (2002) Elevated extracellular CRF levels in the bed nucleus of the stria terminalis during ethanol withdrawal and reduction by subsequent ethanol intake. Pharmacol Biochem Behav 72: 213–20.

Park PE, Schlosburg JE, Vendruscolo LF, Schulteis G, Edwards S, Koob GF (2015) Chronic CRF₁ receptor blockade reduces heroin intake escalation and dependence-induced hyperalgesia. Addict Biol 20: 275–84.

Pettit HO, Ettenberg A, Bloom FE, Koob GF (1984) Destruction of dopamine in the nucleus accumbens selectively attenuates cocaine but not heroin self-administration in rats. Psychopharmacology 84: 167–73.

Pollard GT, Howard JL (1979) The Geller-Seifter conflict paradigm with incremental shock. Psychopharmacology 62: 117–21.

Rassnick S, Heinrichs SC, Britton KT, Koob GF (1993) Microinjection of a corticotropin-releasing factor antagonist into the central nucleus of the amygdala reverses anxiogenic-like effects of ethanol withdrawal. Brain Res 605: 25–32.

Rassnick S, Pulvirenti L, Koob GF (1992) Oral ethanol self-administration in rats is reduced by the administration of dopamine and glutamate receptor antagonists into the nucleus accumbens. Psychopharmacology 109: 92–8.

Rhodes JS, Best K, Belknap JK, Finn DA, Crabbe JC (2005) Evaluation of a simple model of ethanol drinking to intoxication in C57BL/6J mice. Physiol Behav 84: 53–63.

Richter RM, Weiss F (1999) In vivo CRF release in rat amygdala is increased during cocaine withdrawal in self-administering rats. Synapse 32: 254–61.

Roberts DCS, Koob GF, Klonoff P, Fibiger HC (1980) Extinction and recovery of cocaine self-administration following 6-hydroxydopamine lesions of the nucleus accumbens. Pharmacol Biochem Behav 12: 781–7.

Roberts AJ, Heyser CJ, Cole M, Griffin P, Koob GF (2000) Excessive ethanol drinking following a history of dependence: animal model of allostasis. Neuropsychopharmacology 22: 581–94.

Robbins TW, Koob GF (1980) Selective disruption of displacement behaviour by lesions of the mesolimbic dopamine system. Nature 285: 409–12.

Robinson TE, Berridge KC (1993) The neural basis of drug craving: an incentive-sensitization theory of addiction. Brain Res Rev 18: 247–91.

Robledo P, Koob GF (1993) Two discrete nucleus accumbens projection areas differentially mediate cocaine self-administration in the rat. Behav Brain Res 55: 159–66.

Rodriguez de Fonseca F, Carrera MRA, Navarro M, Koob GF, Weiss F (1997) Activation of corticotropin-releasing factor in the limbic system during cannabinoid withdrawal. Science 276: 2050–4.

Samson HH (1986) Initiation of ethanol reinforcement using a sucrose-substitution procedure in food- and water-sated rats. Alcohol Clin Exp Res 10: 436–42.

Schulteis G, Ahmed SH, Morse AC, Koob GF, Everitt BJ (2000) Conditioning and opiate withdrawal: the amygdala links neutral stimuli with the agony of overcoming drug addiction. Nature 405: 1013–4.

Schulteis G, Heyser CJ, Koob GF (1997) Opiate withdrawal signs precipitated by naloxone following a single exposure to morphine: potentiation with a second morphine treatment. Psychopharmacology 129: 56–65.

Schulteis G, Markou A, Gold LH, Stinus L, Koob GF (1994) Relative sensitivity to naloxone of multiple indices of opiate withdrawal: a quantitative dose-response analysis. J Pharmacol Exp Ther 271: 1391–8.

Sessions GR, Kant GJ, Koob GF (1976) Locus coeruleus lesions and learning in the rat. Physiol Behav 17: 853–9.

Shurman J, Koob GF, Gutstein HB (2010) Opioids, pain, the brain, and hyperkatifeia: a framework for the rational use of opioids for pain. Pain Med 11: 1092–8.

Solomon RL (1980) The opponent-process theory of acquired motivation: the costs of pleasure and the benefits of pain. Am Psychologist 35: 691–712.

Solomon RL, Corbit JD (1974) An opponent-process theory of motivation: 1. Temporal dynamics of affect. Psychol Rev 81: 119–45.

Sommer WH, Rimondini R, Hansson AC, Hipskind PA, Gehlert DR, Barr CS, Heilig MA (2008) Upregulation of voluntary alcohol intake, behavioral sensitivity to stress, and amygdala Crhr1 expression following a history of dependence. Biol Psychiatry 63: 139–45.

Specio SE, Wee S, O’Dell LE, Boutrel B, Zorrilla EP, Koob GF (2008) CRF₁ receptor antagonists attenuate escalated cocaine self-administration in rats. Psychopharmacology 196: 473–82.

Stinus L, Koob GF, Ling N, Bloom FE, Le Moal M (1980) Locomotor activation induced by infusion of endorphins into the ventral tegmental area: evidence for opiate-dopamine interactions. Proc Natl Acad Sci USA 77: 2323–7.

Stinus L, Caille S, Koob GF (2000) Opiate withdrawal-induced place aversion lasts for up to 16 weeks. Psychopharmacology 149: 115–20.

Stinus L, Cador M, Zorrilla EP, Koob GF (2005) Buprenorphine and a CRF₁ antagonist block the acquisition of opiate withdrawal-induced conditioned place aversion in rats. Neuropsychopharmacology 30: 90–8.

Sutton RE, Koob GF, Le Moal M, Rivier J, Vale W (1982) Corticotropin releasing factor produces behavioural activation in rats. Nature 297: 331–3.

Swerdlow NR, Britton KT, Koob GF (1989) Potentiation of acoustic startle by corticotropin-releasing factor (CRF) and by fear are both reversed by α-helical CRF (9-41). Neuropsychopharmacology 2: 285–92.

Towers EB, Tunstall BJ, McCracken MM, Vendruscolo LF, Koob GF (2019) Male and female mice develop escalation of heroin intake and dependence following extended access. Neuropharmacology 151: 189–94.

Tazi A, Dantzer R, Le Moal M, Rivier J, Vale W, Koob GF (1987) Corticotropin-releasing factor antagonist blocks stress-induced fighting in rats. Regul Pept 18: 37–42.

Thatcher-Britton K, Koob GF (1986) Alcohol reverses the proconflict effect of corticotropin-releasing factor. Regul Pept 16: 315–20.

Tsai PJ, Keeley RJ, Carmack SA, Vendruscolo JCM, Lu H, Gu H, Vendruscolo LF, Koob GF, Lin CP, Stein EA, Yang Y (2020) Converging structural and functional evidence for a rat salience network. Biol Psychiatry 88: 867–78.

Tunstall BJ, Kirson D, Zallar LJ, McConnell SA, Vendruscolo JCM, Ho CP, Oleata CS, Khom S, Manning M, Lee MR, Leggio L, Koob GF, Roberto M, Vendruscolo LF (2019) Oxytocin blocks enhanced motivation for alcohol in alcohol dependence and blocks alcohol effects on GABAergic transmission in the central amygdala. PLoS Biol 17: e20066421.

Upadhyay J, Verrico CD, Cay M, Kodel S, Yammine L, Koob GF, Schreiber R (2021) Neurocircuitry basis of the OUD-PTSD comorbid state: conceptual analyses using a dimensional framework. Lancet Psychiatry, in press.

Valdez GR, Roberts AJ, Chan K, Davis H, Brennan M, Zorrilla EP, Koob GF (2002) Increased ethanol self-administration and anxiety-like behavior during acute withdrawal and protracted abstinence: regulation by corticotropin-releasing factor. Alcohol Clin Exp Res 26: 1494–501.

Valdez GR, Zorrilla EP, Roberts AJ, Koob GF (2003) Antagonism of corticotropin-releasing factor attenuates the enhanced responsiveness to stress observed during protracted ethanol abstinence. Alcohol 29: 55–60.

Vale W, Spiess J, Rivier C, Rivier J (1981) Characterization of a 41-residue ovine hypothalamic peptide that stimulates the secretion of corticotropin and β-endorphin. Science 213: 1394–7.

Vendruscolo LF, Estey D, Goodell V, Macshane LG, Logrip ML, Schlosburg JE, McGinn MA, Zamora-Martinez ER, Belanoff JK, Hunt HJ, Sanna PP, George O, Koob GF, Edwards S, Mason BJ (2015) Glucocorticoid receptor antagonism decreases alcohol seeking in alcohol-dependent individuals. J Clin Invest 125: 3193–7.

Vendruscolo JCM, Tunstall BJ, Carmack SA, Schmeichel BE, Lowery-Gionta E, Cole M, George O, Vandewater S, Taffe M, Koob GF, Vendruscolo LF (2018) Compulsive-like sufentanil vapor self-administration in the rat. Neuropsychopharmacology 43: 801–9.

Volkow ND, Gordon JA, Koob GF (2021) Choosing appropriate language to reduce the stigma around mental illness and substance use disorders. Neuropsychopharmacology, in press.

Volkow ND, Koob GF (2015) Brain disease model of addiction: why is it so controversial? Lancet Psychiatry 2: 677–9.

Volkow ND, Koob GF, Croyle RT, Bianchi DW, Gordon JA, Koroshetz WJ, Perez-Stable E, Riley WT, Bloch MH, Conway K, Deeds BG, Dowling GJ, Grant S, Howlett KD, Matochik JA, Morgan GD, Murray MM, Noronha A, Spong Y, Wargo EM, Warren KR, Weiss SRB (2018) The conception of the ABCD Study: from substance use to a broad NIH collaboration. Dev Cogn Neurosci 32: 4–7.

Voon V, Grodin E, Mandali A, Morris L, Weidacker K, Kwako L, Goldman D, Koob GF, Momenan R (2020) Addictions NeuroImaging Assessment (ANIA): towards an integrative framework for alcohol use disorder. Neurosci Biobehav Rev 113: 492–506.

Walter R, Van Ree JM, De Wied D (1978) Modification of conditioned behavior of rats by neurohypophyseal hormones and analogues. Proc Natl Acad Sci USA 75: 2493–2496.

Weiss F, Ciccocioppo R, Parsons LH, Katner S, Liu X, Zorrilla EP, Valdez GR, Ben-Shahar O, Angeletti S, Richter RR (2001) Compulsive drug-seeking behavior and relapse: neuroadaptation, stress, and conditioning factors. In: Quinones-Jenab V (ed) The Biological Basis of Cocaine Addiction (series title: Annals of the New York Academy of Sciences, vol. 937). New York Academy of Sciences, New York, pp. 1–26.

Wise RA, Koob GF (2014) The development and maintenance of drug addiction. Neuropsychopharmacology 39: 254–62.

¹(1977–1983) Predoctoral fellow: Neal Swerdlow. Postdoctoral fellows: David C.S. Roberts, Aaron Ettenberg, Jeffrey Schwartz, Derek van der Kooy. Visiting Scientists: Norman White, Michel Le Moal, Trevor Robbins, Charles Ksir.

²(1984–1997) Predoctoral fellows: Lisa H. Gold, Athina Markou, Stefanie Rassnick, S. Barak Caine, Brian A. Baldo, Shelly S. Watkins, Darrel Macey. Postdoctoral fellows: Franco J. Vaccarino, Tom Blasco, Marianne Amalric, Christine Lebrun, Matthew B. Weinger, Abdeloughhab Tazi, Carol Hubner, Friedbert Weiss, Belinda J. Cole, Luigi Pulvirenti, Helen Baldwin, Rafael Maldonado, S. Stevens Negus, Markus Heilig, Stephen C. Heinrichs, Patricia Robledo, Emilio Merlo-Pich, Athina Markou, Frederique Menzaghi, Michelle D. Brot, Gery Schulteis, Loren H. Parsons, Petri Hyytia, Carmen Maldonado-Vlaar, Mark P. Epping-Jordan, Ana Maria Basso, Amanda J. Roberts, Francoise Dellu-Hagedorn, Charles J. Heyser, Yvette Akwa, Mariarosa Spina, Amanda D. Smith, John R. Walker, Michael R. Weed, K. Noelle Gracy, Serge H. Ahmed, Vincent David, Angelo Contarino. Visiting Scientists: Luis Stinus, Martine Cador, Joe Martinez, Keith Matthews, Luigi Pulvirenti, Angela Roberts, S. Mechiel Korte, Barry Everitt, Fernando Rodriguez de Fonseca, Bo Soderpalm, Regina Richter, Victor A. Molina.

³(1998–2013) Predoctoral fellows: Glenn Valdez, Paula Park, Cara Buck. Postdoctoral fellows: Monique Vallee, Cristina Orsini, Daniel Lin, Emanuela Izzo, Andrew Morse, Ahmed Alomary, Joseph Lewis, Marc Azar, Eric Zorrilla, Benjamin Boutrel, Scott Chen, Heather Richardson, Cindy Funk, Sandy Ghozland, Brendan Walker, Tom Greenwell, Valentina Sabino, Pietro Cottone, Sunmee Wee, Chitra Mandyam, Nicholas Gilpin, Laura Orio, Dong Ji, Olivier George, Kaushik Misra, Candice Contet, Scott Edwards, Leandro Vendruscolo, Timothy Whitfield, Ami Cohen, Joel Schlosburg, Carrie Wade, Brooke Schmeichel, Michel Verheij. K99 awardees: Valentina Sabino, Pietro Cottone, Nicholas Gilpin, Sunmee Wee, Scott Edwards, Marian Logrip, Joel Schlosburg. Visiting Scientists: Miguel Navarro, Dale L. Birkle, Koki Inoue, Antoine Tabarin, Serge Ahmed, Osamu Kitamura, Olivier Halfon, Olivier Deschaux.

⁴(2014–2021) Predoctoral fellows: Kay Nisbett, Rani Richardson. Postdoctoral fellows: Stephanie Carmack, Mandy McCracken, Brendan Tunstall, Emily Lowery-Gionta, Renata Marchette, Caroline Pantazis, M. Adrienne McGinn, Nadia Said. K99 awardees: Emily Lowery-Gionta, Brooke Schmeichel, Brendan Tunstall, Khaled Moussawi.

Stephen G. Lisberger

BORN:

New York City

October 28, 1949

EDUCATION:

Cornell University, Ithaca NY, BA (1971)

University of Washington, Seattle WA, PhD (1976)

APPOINTMENTS:

Postdoctoral Fellow, University of Munich (1977)

Postdoctoral Fellow, National Institute of Mental Health, NIH (1977–1981)

Assistant Professor of Physiology, University of California San Francisco (1981–1986)

Associate Professor of Physiology, University of California San Francisco (1986–1989)

Professor of Physiology, University of California San Francisco (1989–2011)

Investigator, Howard Hughes Medical Institute (1997–2015)

Professor of Neurobiology, Duke University (2012–present)

George Barth Geller Distinguished Professor, Duke University (2012–present)

HONORS AND AWARDS (SELECTED):

Elected Member, National Academy of Sciences (2022)

Elected Fellow, American Association for the Advancement of Science (2021)

David A. Robinson Lecture, Johns Hopkins University (2014)

Cesar Fernandez Memorial Lecture, University of Chicago (2013)

Bernice Grafstein Prize for Mentoring Women in Neuroscience, Society for Neuroscience (2011)

Elected member, American Academy of Arts and Sciences (2008)

Distinction in Teaching Award, First-Year Medical Students (1998)

McKnight Foundation Development Award (1988–1991)

Young Investigator Award, Society for Neuroscience (1986)

The Academic Senate UCSF, “Distinction in Teaching Award” (1986)

McKnight Foundation Scholar Award (1981–1984)

Alfred P. Sloan Fellow (1981–1983)

Steve Lisberger has been a pioneer in understanding the neural circuit basis for motor control and motor learning using eye movements as a model system in awake, behaving non-human primates. Trained in mathematics and computer science, he turned to neuroscience as a graduate student. Throughout his 50-year career, he has used as tools single-unit electrophysiology, clever target motion paradigms, quantitative analysis of eye movement behavior, and computational modeling. He made important discoveries about how the output from the cerebellar cortex controls movement and about its interaction with the vestibulo-ocular reflex (VOR). His analysis of the neural circuit basis for motor learning in the VOR revealed three parallel VOR pathways with adaptive plasticity present in the vestibular inputs to the cerebellar cortex and to the “cerebellar nucleus” neurons in the vestibular nucleus. The second half of his research career expanded to the analysis of visual guidance of smooth pursuit eye movements. He evaluated how the population response for visual motion is decoded from the extrastriate visual cortex and characterized the neural circuit basis for the decoder as one pathway that estimates the speed and direction of physical target motion and one that evaluates motion reliability and uses it to set the strength of signal transmission from the visual system to the motor system. Most recently, he used motor learning in pursuit eye movements to elucidate the operating principles of a learning neural circuit in the cerebellar cortex.

Stephen G. Lisberger

As I write this, I am completing the first half-century of my career as a neuroscientist. I do not know where the time has gone. It has been great fun even when it wasn’t fun. The highlight of my career has been the people I have met and known or even known well. I also am proud of my accomplishments in advancing knowledge. People think of me as an experimental neuroscientist. But I am a computational neuroscientist who does his own experiments and summarizes his model with computer simulations or equations. I enjoy the process of science almost as much as the thrill of discovery. I also enjoy the success of others and I have devoted much energy to scientific leadership and mentoring.

For the first 20 years of my career, I was a neuroscientist almost single-mindedly. But I had a “work/life balance” even before that became something we talked about. I view my life as running along three parallel paths—“science,” “family,” and “fun”—with the width of each path widening and narrowing dynamically in relation to the competing opportunities and demands of the moment. Of course, “science” and “family” are fun, and it is possible to think about science while pursuing fun activities. Indeed, some of my best ideas in the past 10 years came to me while I was on my bike on the backroads of Chapel Hill. One of my favorite books read during my first trip to Europe in the summer of 1968 was A Moveable Feast by Ernest Hemingway—I see science as a moveable feast. This has made it possible for me to enjoy science even during the Covid-19 pandemic. I can do science happily at my kitchen table and over video calls, even if I prefer strongly to do it with people in a group setting.

Growing Up, School, and University

Born in New York City on October 28, 1949, I grew up first in the suburbs in North Stamford, Connecticut, and then as sort of a faculty brat in Ithaca, New York, home of Cornell University. Before too long, I had four younger sisters, and then my father passed away in 1961. I was a solitary child with only a few, but close, friends, and I spent a great deal of my time in my room. I built model rockets that I left my room to fly at an abandoned airstrip next to Cayuga Lake. I became intrigued with transistors and designed (but did not build) a digital computer circuit on a very big sheet of paper. I listened to rock-and-roll in the 1960s on a wonderful old AM radio with tubes that allowed me to receive WLS (Chicago) or WABC (Cousin Brucie in New York) at night. My mother had enough money for us to live comfortably, but I had a pathetic allowance and took a paper route for the Ithaca Journal and mowed lawns for pocket change. I could not be bothered with homework and seldom attended to it until late, frequently turning off my lights until I heard my mother go to her room and then doing homework under the covers with a flashlight.

My first important mentor was Mr. Howitt who taught math in grades seven to nine. It was the start of the “modern math” era, and I took to it right away. He entered me in math contests and took me to various nearby cities on Saturdays to compete. I excelled, and it was one of the only positive influences on my confidence in junior high school. It was a perfect time for me to have a confidence builder, just as I was grappling with my father’s untimely death. I remember that my high school calculus teacher, Mrs. Hockett, told me that I could be a theoretical mathematician if I wanted. College math, especially n-dimensional vector spaces and multivariate calculus, disabused me of any such illusions.

My mother arranged for me to enroll at Phillips Andover Academy starting in 10th grade, thinking I needed some male influence given that I was growing up with four sisters and a mother. It was the single most important influence on me academically in my entire life. But I hated it because of the academic and social pressure cooker created by putting a large number of very smart teenage boys together for full-time living and studying. I returned to Ithaca High School after one year plus one week. At Andover, I completed two years of high-school math in one year, allowing me learn computer programming instead of taking precalculus when I returned to Ithaca High School in 11th grade. Here, I found my second critical mentor, Mr. Holman, who invited me to the Cornell Computer lab every Saturday morning while he “operated” Cornell’s CDC 1604 computer by running the machine that read punch cards for each job. I rode my bike there every Saturday morning and stayed in the computer room from 8:00 a.m. until noon. I was intrigued by the huge Fortran programs run by the various graduate students, who seemed serious and mysterious to me. Computers and computer programming became major forces in my life before almost anyone knew about them. It was transformational. Andover also taught me how to take notes, how to organize my time, how to deal with the stress of public speaking, and how to recover self-esteem after harsh criticism, all skills that have served me well as a neuroscientist. But the biggest win of the year at Andover was that it allowed me to avoid high-school biology, a huge relief because I was too squeamish to dissect a frog.

As a kid, the family path was poorly developed. The fun path was a challenge for me. I loved sports, but I was small and weak and was challenged to compete with my peers—at least until my senior year of high school when I was number two on the tennis team, an experience that was exhilarating because of the enthusiasm, support, and sense of humor of the coach, Mr. Cowan. The neighborhood kids and I played basketball or whiffle ball or street hockey in the neighborhood almost every day after school, and we were a “team” even though our families and our aspirations were utterly different. I loved ice hockey, and in those days, the ponds froze in winter and we played outside. I learned to skate when we lived in Stamford, Connecticut, almost as early as I learned to walk. I played intramural hockey in high school but didn’t blossom as a hockey player until graduate school. As a child, I went to public skating sessions at Lynah Rink on the Cornell campus, and we rooted passionately for Cornell’s hockey team. In a way, I was a college kid on the Cornell campus as soon as I was allowed to ride my bike to campus (seventh grade or so), so I had a very college-oriented youth. I think that is why it has been meaningful for me to return to a full-featured college campus at Duke, where I value both my medical school workplace and the broader academic campus environment.

I stayed in Ithaca for college at Cornell University. I lived in the dorms my first year, a fraternity my second and third years, and a house by Cayuga Lake as a senior. I planned to major in physics, but that plan was dashed in the first semester of my sophomore year when I could not get up for the required course for majors at 8:00 a.m. on Tuesday, Thursday, and Saturday. I note that my daughter Emika proved much tougher than I and completed a physics major at Cornell in 2016, just 45 years behind me. I changed to economics which I enjoyed but found to be “too soft.” During the economics phase, I had the amazing experience of taking a seminar in the “Economics of Regulation” from Alfred E. Kahn who went on to deregulate the airlines as head of the Civil Aeronautics Board. Turnabout is fair play—in the course, he was a strident regulator. I ended up as a math major, mainly because Cornell did not have a computer science major in those days. One of the great features of Cornell was that star faculty taught undergraduate courses. I audited the Introduction to Biology Course taught by Bill Keeton (he wrote the book), I took Shakespeare from Arthur Mizener, one of the preeminent Shakespeare scholars of his era, and “Mill, Marx, and Nietzsche” from Werner Danhauser, again the preeminent Nietzsche scholar of the time.

After my sophomore year, two important accidents occurred. First, my year of computer programming in 11th grade gave me the background to receive the top grade in a large graduate-level computer science course as a freshman. The professor, Dick Conway, called me afterward and offered me a job in what I think of as the first dot-com, Compuvisor. I worked with him, Howard Morgan, and Bill Maxwell to develop a programming language called ASAP, which was the predecessor to VisiCalc, Lotus 123, and Excel. I almost became a computer professional—there is no telling where I would be today but for the second accident. At the start of the summer when I was a rising junior, Amy Pruitt (my then-girlfriend’s roommate) gave me a book called Neurophysiology, a Primer by Charles F. (Chuck) Stevens. I read it and my reaction was: “OMG, I had no idea you could study how the brain works, that’s what I want to do.” I continued to work for Compuvisor, but I immediately started taking neurobiology courses. At Cornell, that was mainly psychology, for example, with the greats J. J. and Eleanor Gibson, or a graduate course in animal behavior with Tom Eisner, Steve Emlen, Jeff Camhi, and Bill Keeton. I applied to graduate school in neuroscience and matriculated to the Department of Physiology and Biophysics (PBio) at the University of Washington (UW, pronounced Udub) in Seattle. My Uncle Dick, a banker, was correct when he told me that “Seattle is depressed,” but it was a fantastic place to live and study, and I enjoyed a terrific environment at UW. I owe a great debt to the faculty leaders of PBio, Harry “Pat” Patton, Bertil Hille, Thelma (Temy) Kennedy, Wayne Crill, and Orville Smith, for the environment they created and the way they supported their students.

Initiation to Science

The second person I met after I arrived at UW was Chuck Stevens. He and I both worked at the lab almost every evening and his lab was right next to the bullpen for first-year graduate students. We talked frequently and started a friendship that has lasted many years. Like me, Chuck was a theorist who did his own experiments and summarized them with equations. We had a great deal in common. When I told him years later that his book had turned me from computers to neuroscience, his flattering response was “that alone makes it worthwhile to have written the book.”

It is frightening how little I knew about science and neuroscience when I started graduate school. I had never read a scientific paper, never been trained to think in terms of experimental questions and answers, and never learned much of anything about neurons or brains. All I knew was how to program computers and a little bit of abstract mathematics. In the fall quarter of my first year, we had a course taught by Chuck Stevens. It was at the same time the most frightening episode of my entire education and the beginning of me as a scientist. Chuck would come into class at 9:00 a.m., put his thermos of coffee on the table at the front of the room, pour himself a cup, and say “give me a few minutes.” He was deciding the structure of the next 50 minutes of class and what he was going to say. Once the caffeine started to work, he would draw his lecture about cell membranes and the proteins within them (e.g., Na-K pumps) on the blackboard. Now and then he would stop and say, “Steve, what experiment would you do to test this.” Fortunately, Stephen J. Smith, now of the Allen Institute, sat behind me in class and would always pick me up by giving a cogent answer. I simply was not ready to think in terms of experiments about the cell biology of neurons—or about much else, for that matter.

I stopped looking at Twitter some time ago because I grew tired of hearing the narrative that “graduate school sucks.” Maybe I had a charmed five years in graduate school, but I think we need to figure out how everyone can have the same charmed graduate career. For me, a major contributor to my happiness in graduate school was the first person I met at UW, Albert Fuchs, who was assigned to me as my first-year adviser and became my thesis adviser. I found the eye movement system that Albert investigated appealing in its combination of simplicity, accessibility of the essential neural circuits, and natural amenability to quantitative analysis and the thought processes of an engineer. Albert told me, “We already know how the final motor machinery in the brainstem works and what the sensory inputs look like in the visual cortex—all we need to do is figure out what happens across the 2 or 3 synapses that connect them.” We are still working on that!

I started graduate school in 1971 when the theory of cerebellar learning had just achieved prominence. During the previous 10 years, experiments by Masao Ito, Sir John Eccles, and others had brilliantly elaborated the circuitry of the cerebellum and the signs of the synapses within the canonical circuit. Seminal papers by David Marr (1969) and James Albus (1971) posited that inputs on the climbing fiber caused changes in the strength of synapses from parallel fibers to Purkinje cells. Ito (1972) gave functional context to the theory. In addition, the collaboration of Bob Baker, Wolfgang Precht, and Rodolfo Llinas (1972) had just discovered that Purkinje cells in a part of the cerebellar cortex called the “flocculus” inhibit neurons in the vestibular nucleus and that the vestibular nucleus neurons are interneurons in the three-neuron vestibulo-ocular reflex (VOR) arc. The VOR uses inputs from the vestibular apparatus that signal head turns to drive an eye movement that compensates for head turns. It keeps the eyes stable in space as we move about. Errors in keeping the eye stable in space cause “retinal slip,” the motion of the visual scene across the retina.

Ito (1972) postulated, and Kyoji Maekawa and Jerry Simpson (1972) demonstrated, that the climbing fiber input to the flocculus responds to visual inputs. Over the mossy fiber-parallel fiber pathway, the flocculus receives vestibular inputs as a side loop of the brainstem VOR pathways. Ito’s theory was simple: if the VOR makes an error, the visual scene slips across the retina. Climbing fibers in the flocculus signal the error. Plasticity modifies the strength of the vestibular parallel fiber inputs. Changes in simple-spike firing of Purkinje cells correct the VOR through their direct projection to the brainstem VOR pathway.

For me, this all came together at a small meeting at the Good Samaritan Research Institute in Portland, Oregon, in the spring of 1972. The flocculus was the anatomical substrate for taking advantage of the accessibility of eye movements to study cerebellar learning. I was intrigued.

Joe Kimm and Albert Fuchs had been recording single axons in the vestibular nerve of monkeys, and Albert told me that they had recorded lots of fiber activity related to eye movements in the structure that lies just above the vestibular nerve. I put two and two together and realized that they were recording mossy fiber inputs to the flocculus. I petitioned Albert to allow me to try to find Purkinje cells in the flocculus. The first 20 electrode penetrations were frustrating. It was easy to find the part of the cerebellum with mossy fibers related to eye movements. But when I isolated Purkinje cells, identified by the characteristic infrequent popping of the climbing fiber, their simple-spike activity (caused by mossy fiber inputs) was unmodulated during the VOR.

Then, ah ha! Serendipity struck! I found a Purkinje cell that showed strong modulation of simple-spike firing rate. Why? During experiments, the monkey faced an eight-inch radius dome that had seven push-buttons with LEDs in them spaced along the equator of the dome. The monkey’s job was to push the lighted button in exchange for applesauce, something he did only after looking at the button. That allowed us to calibrate the electro-oculogram electrodes that we used to monitor eye position and to get the monkey to look where we wanted him to. The dome was attached to the monkey’s chair and moved exactly with him when we turned the chair to evoke a VOR. I had been recording from Purkinje cells while rotating the monkey back and forth in the dark to study them during the VOR, but on this day, I accidentally left the room lights on. Instead of making a VOR in the dark, the monkey was preventing his VOR by tracking the button in front of him as it moved exactly with him. I quickly corrected this “error” by placing the monkey in the dark and the modulation of Purkinje cell firing went away. This was the rule, not the exception. Purkinje cell simple-spike firing is modulated strongly when the animal cancels his VOR by tracking a target that moves exactly with him. Firing is weakly or not-at-all modulated during the VOR in the dark. I published this finding in my first paper (Lisberger and Fuchs, 1974) as a short communication in Brain Research, a prestigious venue in those days.

In parallel, my future postdoc mentor Fred Miles and I went on to discover that Purkinje cell simple-spike firing is also modulated strongly when the monkey tracks a smoothly moving target with his head stationary (Miles and Fuller, 1975; Lisberger and Fuchs, 1978a). We both realized that the responses during cancellation of the VOR arise in the vestibular system and are related to head velocity; the responses during tracking with the head stationary are a corollary discharge of the signal related to eye velocity going to motoneurons. The head and eye velocity inputs cancel during the VOR because eye and head velocity are equal and opposite. David A. Robinson recognized that Purkinje cell output is related to eye velocity relative to the stationary world (also known as “gaze velocity”) and named these horizontal gaze velocity Purkinje cells (HGVPs). Fred Miles pointed out to me that Purkinje cells form an internal model of what happens in the orbit where head and eye velocity sum to create the desired eye velocity relative to the world. Knowing that the Purkinje cells were two synapses from the motoneurons, it was easy to develop a story of the functional roles for each aspect of their discharge. During the VOR in the dark, the brainstem has it all figured out, and the cerebellum leaves well-enough alone. If the VOR isn’t appropriate for the motion of the target, then the cerebellum engages.

In my data, the climbing fiber responses occurred out of phase with simple-spike firing during tracking of a moving target, with or without head rotation. Fred Miles did not observe modulation of climbing fiber responses, probably because he used a much lower set of eye and head speeds than I did. I submitted a paper on the modulation of climbing fiber activity, but it was rejected by Experimental Brain Research, and the reviews were so outrageous that Albert and I decided not to try to fix it. My first PhD student, Leeland Stone, revisited the climbing fiber responses as part of his PhD thesis (Stone and Lisberger, 1990). Climbing fiber firing still occurred out of phase with simple-spike firing. Lee went a step farther and used an elegant analysis to provide evidence that the climbing fibers were being driven by retinal slip. He computed averages of retinal slip aligned on the (rare) occurrence of climbing fiber responses and demonstrated that a brief retinal slip of a few degrees per second (deg/s) preceded climbing fiber responses by 100 ms, a typical visual delay. He found no evidence that climbing fibers caused any eye movement.

There is much more to the graduate school story, but the executive summary is that I received my PhD in 1976 and published the two papers that are still my most cited nonreview papers (Lisberger and Fuchs, 1978a, 1978b). I am deeply indebted to a few people who made a huge difference in my graduate career. Albert Fuchs was an awesome mentor who walked the line between guidance and independence perfectly. Mike King and Craig Evinger were close friends and coconspirators and together we developed a great lab group. I was inspired by Erich Luschei, who taught me how to program the Digibits and all about quantitative control of behavior. It was a work-hard-play-hard paradigm that I think originated for me at Cornell, itself a work-hard-play-hard school. We had a great time outside of the lab, and there were many times when the “have fun” path was wide and the science path very narrow. I took the Mountaineers’ climbing course and we scaled multiple peaks and hiked many gorgeous trails in Washington State. Most memorable was climbing Mt. St. Helens before it blew up and taking several backpacking trips into the Enchantment Lakes. I played on the UW hockey club, one year centering for Craig Beveridge who had a tryout with the Vancouver Canucks and Jim Wellington who started for the University of Vermont in Division III. Every good pass turned into an assist. We practiced a couple of times a week and played in the Northwest Amateur Hockey League. It was the pinnacle of my ice hockey career. I started exercising regularly in February of 1972 and became an avid runner. Sad that my joints no longer allow me to run, but this was my main form of exercise for 30 years. Graduate school was a time when I blossomed personally and intellectually. I strive to create the same positive experience for all PhD students.

In Search of the Modifiable Synapse or “The Fastest Boys at the NIH”

Postdoc is said to be the best time of a scientist’s career. For me it was really good, but maybe not better than my graduate school experience. Again, I was blessed with a fabulous mentor in Fred Miles. Fred had taken on the very large task of chasing the modifiable synapse (pronounced s-eye-napse, please, in Fred’s English) for motor learning in the VOR. Before I arrived in 1977, he had pretty much found Ito’s hypothesis for VOR learning to be wanting (Miles et al., 1980a), and he had documented that VOR learning had zero effect on the responses of fibers in the vestibular nerve (Miles et al., 1980b).

Fred and I had a pretty simplistic view of how to find the site of the modifiable synapse in those days, and we set out to do a simple experiment. If the site of learning wasn’t in the cerebellar cortex or the vestibular nerve, then we reasoned it must be in the brainstem. So, my first project as a postdoc was to record as many neurons in the vestibular nuclei as I could, both before and after motor learning in the VOR. Long, tedious recording sessions led to a negative result (Lisberger and Miles, 1980). We found no evidence that changes in the size of the VOR had any effect on the responses of neurons that were related principally to head turns and not to eye movements. One of the reviewers of the paper reported that “the authors have taken a seemingly rich and interesting subject and converted it into a boring and vacuous effort.” Ouch. And, 10 years later I discovered that we threw out the baby with the bathwater: we encountered but had not recorded from the right neurons—namely, the neurons that did respond in relation to eye movements. Ed Keller and Wolfgang Precht (1979) had recorded from them and discovered that the key neurons disappeared from the sample when learning had rendered the amplitude of the VOR really small, but they didn’t do the right experiments to identify them either.

I learned a ton from Fred about how to do science, how to think about science, and how to write about science. He introduced me to “plasticity.” We invented a strategy for learning about how brain circuits work through strategic and quantitative analysis of behavior. By recording the effect of VOR learning on an arcane visual-motor reflex called the “optokinetic response,” we predicted which neural circuits would or would not contain sites of learning (Lisberger et al., 1981). We developed the concept that the VOR could “learn by doing,” and the theory that signals related to motor activity were sufficient to cause learning. We theorized in a paper in Annual Review of Neuroscience (Miles and Lisberger, 1981) that the output from the flocculus was exactly the “doing” signal that was needed to guide learning in the vestibular nucleus. We tested the hypothesis in futility by coordinating electrical stimulation in the bilateral flocculi with vestibular rotation in the dark. Our stimulation probably was too crude and unnatural, but 32 years later, Jennifer Raymond’s lab validated the theory with an elegant application of optogenetics in mice (Nguyen-Vu et al., 2013). Fred and I also invented the idea that we should use a transient vestibular stimulus rather than sinusoidal rotation so that we could study the latency and temporal evolution of behavioral and neural responses rather than merely the steady-state. We did not do much with it because, well, our vestibular stimulator was wholly unsuited to the task. I leveraged the transient stimulus to huge advantage later. But it is important to know who invented the approach. I just used it. Fred was burned out on the VOR and on single-unit recordings in monkeys, and I took the project away to make it my own.

My experience at NIH was terrific. It was a work-hard-play-hard environment, just like graduate school and college. I had very good colleagues in Ed Evarts’s Laboratory of Neurophysiology, and we had a great community of neuroscience postdocs across the NIH campus. We had fun together, too. The RIFs (named after an impending reduction-in-force at NIH) developed into the best men’s softball team at the NIH and won the intramural championship. Our running relay team—myself, Von Jennings, Jerome Sanes, Miles Herkenham, and Ed Evarts—finished second in the first annual “Director’s Cup,” a five by half-mile relay on the NIH campus. I still think of us as the “fastest boys at the NIH” because the team that won was from the Food and Drug Administration, technically not the NIH. During my four years at NIH, I had a close relationship, including daily lunch, with Bob Wurtz’s lab on the floor below us. While I was at NIH, Bob was given his own lab, the Laboratory of Sensorimotor Research, and I guess I might have stayed on there. But I wanted to return to my roots as an academic following the engram that developed during my critical period when I grew up with the benefits of Cornell’s campus.

San Francisco and the Rise of UCSF

I accepted a faculty position at the University of California, San Francisco (UCSF) in 1981 in the Department of Physiology and Division of Neurobiology. It was special for me to return to my father’s 1916 birthplace and to be in the same city as my paternal grandmother, who was 96 years old when I arrived at UCSF.

My bosses at UCSF were Fran Ganong and Zach Hall. The initial renovation of my lab provided me with an office for myself, an office for a couple of lab members, a closet for training monkeys, and a single combination of a rig-room and behavior room for monkey experiments. Monkeys lived nearby. We wheeled them through a public corridor in covered primate chairs. Between 1981 and 1994, I published 24 papers that were completed in that 600-square-foot space, including my major series of papers that elucidated the neural circuit basis for learning in the VOR. When UCSF was on the Parnassus Campus, no one had a lot of space and we had a highly collaborative, interactive community. A lesson for the future?

UCSF had a great and growing neuroscience community. I received fantastic support and mentoring from Zach Hall, Michael Stryker, and Allan Basbaum. The community was small at the outset but grew quickly, especially in the 1990s when we created the W.M. Keck Foundation Center for Integrative Neuroscience to pull our systems neuroscientists into a single space. Key hires included Allison Doupe, Ken Miller, and many others into the systems neuroscience community and Marc Tessier Lavigne and Cori Bargmann in the Department of Anatomy. The Keck Center’s renovated space opened in 1994 and shortly thereafter we became one of the first five Sloan Centers for Theoretical Neurobiology. At the same time as UCSF was becoming the national leader in all things molecular and cellular, including neuroscience, the Keck Center and Sloan Center put us on the map in questions of how the brain works when it is working. By 2000, UCSF was among the best neuroscience groups in the country. Lots of places had great faculty and great students. But UCSF also had great community. This was Zach Hall’s vision and accomplishment. I try to emulate him in my current position as chair of neurobiology at Duke.

In USCF’s systems neuroscience, I saw a huge pivot in the 1990s. The principal investigators (PIs) in the Keck Center were quantitative, but our science was not leveraging neural theory or neural computation. With the funds from the Sloan Center, we began to hire postdocs who had just gotten their degrees in a quantitative science, mostly physics. Many of them succeeded in our environment, remarkably without being remade to be like “us.” More important, I saw a qualitative change in how each lab approached its research, and I think it elevated our science hugely. Bill Bialek was a regular visitor and spent six months in residence as part of the Sloan Center. His presence made a big difference to our Sloan postdocs, the faculty, and me. He and I struck up an amazing collaboration, I learned a huge amount from him, and he changed how my lab approached science.

My personal life also pivoted in the 1990s. Bill Newsome introduced me to a postdoc in his lab, Chieko Murasugi. We married in 1992, and our children Emika and Theodore were born in 1994 and 1997. Chieko was a neuroscientist who worked on eye movements, having received her PhD with Ian Howard at York University in Canada. She dropped science like a hot potato shortly after we married in favor of art, her real love, and became an abstract painter. The 1990s were intense with all the scientific developments and the family. We purchased our first dream house seven blocks from UCSF, which allowed me to walk to work and prevented a long commute so that I could be home for breakfast and dinner. In due course, we raised the roof and built a third floor onto the house to create a gorgeous studio for Chieko and an office for me. The studio became a bedroom for Emika when she started high school on the premise that it would give her a place to spread her wings a bit at home and keep her off the streets. As far as I know, it worked.

After Emika was born in 1994, the 1990s and the first decade of the 21st century were crazy with a constant balancing of work and life. Somehow, we survived. The kids had tons of activities. Both became stellar musicians (long story), great friends to me, and people very different from each other. We had many memorable family vacations, mostly based on miles accumulated from my work travels. My favorite anecdote from the whole time was from an exchange I had with one of Theodore’s little league coaches. On a Sunday morning, I dropped him at the field on Treasure Island in the middle of San Francisco Bay an hour before the first pitch and told the coach, “I’ll be back in time for the game, I am going back to the City to do some work in a café.” I returned at game time and the coach asked me, “Did you finish your work?” Of course, in science we never finish our work, nor was it ever in my mind that I would “finish my work.” I laughed to myself. I still wonder if I ever will “finish my work” and how I will know that I have finished.

Cracking the VOR Circuit and Learning in Monkeys

The pinnacle of my contribution to neuroscience was my series of 1994 papers on the neural circuit basis for motor learning in the VOR, published back-to-back-to-back-to-back in the Journal of Neurophysiology with me as the first author of all four papers (Lisberger, 1994; Lisberger et al., 1994a, 1994b, 1994c). I set out to find the neural circuit basis for VOR learning when I moved to UCSF and started my own lab. It was very much my work, of course with some help from trainees and staff, but I was the intellectual force behind it. Those papers have stood the test of time, although there have been additions, subtractions, and adjustments to the story.

I concluded that there are two sites of learning in the circuit for the VOR. One site is in the vestibular nucleus at the synapses of vestibular inputs onto neurons that I called floccular target neurons (FTNs) because they are inhibited at monosynaptic latencies after single-shock stimulation in the flocculus. The other site is in the vestibular inputs to Purkinje cells in the cerebellar cortex, presumably at the parallel fiber to Purkinje cell synapse. Both sites are needed to allow a circuit model of the VOR to reproduce neural firing patterns and eye speeds during the VOR before and after VOR learning. But there is nuance to my conclusion, nuance that has been swept under the rug, misunderstood, or ignored. Direct measurements of the vestibular sensitivity of Purkinje cells show that learning in the cerebellar cortex is in the “wrong” direction—that is, the direction opposite to that needed in Ito’s theory to cause VOR learning. My data agreed with Fred Miles’s earlier data (Miles et al., 1980b). My computational models suggest that the learning in the cerebellar cortex rebalances the brainstem and cerebellar pathways and allows the system to remain stable at its new settings, instead of causing learning.

One of my lab’s mantras is that “the truth is complicated.” My science doesn’t simplify a story just to make it accessible or palatable. For example, the previous paragraph summarizes the VOR story in 200 words, but the four papers in 1994 occupied nearly 90 pages of the Journal of Neurophysiology, with a total of 63 figures and 178 panels. My full contribution to the neural circuit basis for VOR learning appears in about 22 original papers. Even an essay of the scope of this one cannot capture all the important detail. My strength and weakness as a scientist are the same: I value that detail. So, my strategy in this essay is to outline the key insights that drove the research, rather than trying to explain all the details.

The first insight that drove my research on VOR learning was the need to identify the neurons that received monosynaptic inhibition from the flocculus. Fred Miles and I had successfully implanted Peter Rhodes’s bipolar concentric stimulating electrodes in the flocculus, so I decided to do this again and now record from the brainstem and search for neurons that were inhibited by single shocks. We lowered the stimulating electrode toward the correct stereotaxic coordinates with the monkey in his chair and awake and cemented the electrode in a location where stimulation evoked ipsiversive smooth eye movements. Single-shock stimulation caused monosynaptic inhibition of a distinctive group of neurons that we called FTNs. Their responses during the VOR underwent profound changes in association with learning, changes that far exaggerated the changes in the VOR itself.

The second insight was that the tradition in the field of using sinusoidal oscillation of the monkey as a vestibular stimulus was depriving us of critical information about timing. So, we improved the strategy that Fred Miles and I had tried to deploy of using a transient pulse of head motion as a stimulus. Our stimulus never was perfect, but this strategy quickly revealed that the earliest part of the VOR response, the first 5 ms, was unmodified by learning while the modified part of the response happened later (Lisberger, 1984). For our stimulus, VOR latency was 14 ms and the latency of the modified pathways was 19 ms. We concluded that parallel brainstem pathways were unmodified vs. modified. Our recordings (Lisberger et al., 1994b) later established that the unmodified pathways use a brainstem interneuron that responds with very short latencies called the PVP by David A. Robinson for position-vestibular-pause. The modified pathways use the FTNs as the brainstem interneuron. FTNs respond during the VOR with latencies that are compatible with the modified component of the eye movement response. The flocculus inhibited only the modified pathway—or perhaps more accurately, the FTNs were subject to learning and became different from the PVPs because they received inputs from floccular Purkinje cells.

The third insight came from a wonderful collaboration with Terry Sejnowski. In the late 1980s and early 1990s, Terry had invented “NETtalk,” an artificial neural network that learned iteratively to pronounce written English text by matching its own output to phonetic transcriptions. He was a guru, perhaps “the” guru, of three-layered neural networks that learned by back propagation (backprop). Backprop was a mathematically derived learning rule that used the chain rule (I remembered this from my math major) to propagate errors in network output backwards to the weights between model units. It changed the weights in a direction that would reduce error the next time. It was like having a screwdriver that could turn each weight in the network up and down a little bit and retain the direction of change that reduced output error. I had great visits to the Salk during our collaboration. I would fly down in the early evening, and Terry and I would have an animated dinner; frequently I stayed at his house. We would think and talk the next day, I would attend tea at 3:00 p.m. with Terry’s lab, including Francis Crick and Patricia Churchland, and then rush to the airport for the short flight back to San Francisco. When I read anything written by Terry, Francis, or Pat, I can hear them each saying it in their own distinctive way. Pat went on to join my scientific family as both of her children received their PhDs from my lab.

Terry and I started from Barak Perlmutter’s elaboration of backprop into recurrent networks. We implemented it to work on signals that varied as a function of time on a scale of milliseconds and used it to explore how a network with the architecture of the VOR circuit would compute. We were interested in whether the sites of learning could be anywhere in the network, or if the circuit architecture or the behavioral tasks it had to solve would constrain the sites of learning. The sites of learning were narrowly constrained if we forced the network to learn a modified VOR under conditions in which it had to both generate a VOR and track moving targets with the head still or turning. Learning had to occur in parallel in the vestibular inputs to the model network’s FTNs and floccular Purkinje cells and the learning on Purkinje cells had to involve changes in time constants as well as the amplitudes of neural signals. Terry’s brilliance at assembling a story led to a short paper (Lisberger and Sejnowski, 1992a), but our most important discoveries were relegated to a technical report from UCSD (Lisberger and Sejnowski, 1992b). The latter guided a generative learning neural circuit model that I published in the fourth of my papers in 1994.

I have learned over the years that my greatest ability is to conceive and navigate a process that solves big problems. My contributions to understanding learning in the VOR relied very heavily on my management of process. The key inventions—electrical stimulation of the flocculus, transient vestibular stimuli, and the backprop model—all were the results of two minds working together, mine with either Fred’s or Terry’s. Throughout my career, major developments in my own research started through collaborations with amazing senior scientists and were brought to fruition by my persistence and management of the research process.

By 2021, much has been added to the VOR learning story. In monkeys, Jennifer Raymond and I verified that the learning mechanisms in the VOR “know” about the 100 ms difference between the latency of the vestibular signals subject to learning and the latency of the visual signals that instruct learning (Raymond and Lisberger, 1998). Jennifer’s lab demonstrated that long-term depression of parallel fiber to Purkinje cell synapses in the mouse flocculus is tuned for this time difference, placing the burden of temporal-credit-assignment on the cellular mechanisms of plasticity (Suvrathan et al., 2106). Multiple labs have developed VOR learning preparations in mice and the story has become more granular. For example, different neural sites and mechanisms are responsible for learning that increases vs. decreases the size of the VOR. Jason Christie and Sascha du Lac have made important contributions. Chris deZeeuw provided a wealth of evidence for many details of cerebellar learning. Chris and I became especially good friends recently, and I learned much from him as coauthors with Jennifer Raymond of a recent Perspective in Nature Neuroscience (de Zeeuw et al., 2021). The three of us truly contributed equally to the paper, and I am the middle and corresponding author.

One tragic omission from the thinking about VOR learning in mice is the failure to include the eye movement input to the flocculus in interpreting any of the data. I hope that will change. Other niggling issues remain. First, my recordings (and Fred Miles’s) were made after weeks of continuous exposure to conditions that cause VOR learning. The learning demands on the cerebellar cortex could be completely different after 10 minutes vs. weeks of learning. The Ito theory might be correct for the first 10 minutes of learning—just not after 10 days. Second, the terminology “flocculus” has proved to be tricky. In the monkey, the strictest use of terminology notes that the flocculus is continuous with the ventral paraflocculus. Most of the knowledge from monkeys comes from recordings in the ventral paraflocculus, which is very small in other species. With the help of neurosurgeon Luc Jasmin, we demonstrated that lesions of the more caudal flocculus had small effects on either pursuit eye movements or VOR learning. Lesions that extended more anteriorly to include more ventral paraflocculus had much larger effects on both (Rambold et al., 2002). We think that the evolution of pursuit eye movements expanded the ventral paraflocculus to join the flocculus in controlling eye movement. We use the terminology “floccular complex” to encompass both.

Sadly, few if any labs are studying motor learning in the VOR in monkeys now. I think there is still much to do in monkeys, even using conventional techniques, and eventually by deploying modern molecular approaches as well. The story deserves to be completed, including taking advantage of the approaches that are possible in both mice and monkeys to constrain the final models.

Visual Control of Movement

When I started my independent career at UCSF, I knew that the VOR learning project was risky and difficult. I would need some low-hanging fruit for my trainees and for papers for my tenure dossier. I already had published my first paper on smooth pursuit eye movements (Lisberger et al., 1981), so I chose the neural basis for pursuit as a parallel project that seemed likely to yield more papers sooner. My first NIH grant was entirely on pursuit, although it included identifying FTNs by electrical stimulation in the flocculus. It was funded for three years starting on the day I arrived at UCSF.

Again, I start with a succinct summary of what I learned about how a moving target leads to accurate smooth eye movements. The middle temporal visual area of the extrastriate cortex (MT) represents the image motion (defined as target motion across the potentially moving retina) that drives pursuit. Sensory signals proceed from MT to the pontine nuclei in a pathway that estimates the speed and direction of image motion by finding the preferred speed and direction of the most active MT neurons. Sensory signals from MT also pass through the parietal cortex to the smooth eye movement region of the frontal eye fields (FEFSEM). FEFSEM implements a reliability-weighted combination of sensory data and priors based on past experience. FEFSEM sets how much credence should be given to the estimates of physical image motion by adjusting the strength or “gain” of visual-motion transmission. The gain mechanism prevents visual sensory signals from having unfettered access to the motor system. Signals from FEFSEM and MT converge in the pons and pass to the floccular complex of the cerebellum and the oculomotor vermis. There, pontine inputs integrate with positive feedback of an eye velocity corollary discharge and create Purkinje cell simple-spike output that inhibits last-order interneurons in the vestibular nucleus and drives motor output.

Several key insights were critical. The first insight occurred in a collaboration with Bill Newsome and Bob Wurtz while I was at the NIH. In 1978 or 1979, a big NIH-wide systems neuroscience journal club discussed a paper on MT neurons. Bill, Bob, and I thought that MT might provide the visual input for pursuit, and we decided to ask how lesions of MT affected pursuit. We knew that MT was organized retinotopically, so we would have to study pursuit by placing the moving target in the lesioned part of the visual field. We appropriated the “step-ramp” target motion invented by Rashbass (1961). The monkey fixated at straight-ahead, and we controlled retinal location by stepping the target to a specific retinal location as it started to move. We measured the first 100 ms of the eye movement response before any eye movements could change the retinal location, speed, or direction of the stimulus. The initiation of tracking in the “open-loop interval” was a response to a visual stimulus that we controlled with the step and the ramp. This step-ramp target motion moved us away from analysis of steady-state behavior for sinusoidal target motions and into a new era in studying smooth eye movements. Bob, Bill, and I discovered that surgical lesions of MT create a visual scotoma for the initiation of pursuit without any effect on the ability to generate the motor behavior for visual field locations outside of the scotoma. We had some concerns about the surgical lesions, and so we decided to publish only some fundamental discoveries about visual control of pursuit from the prelesion data. Sadly, an anonymous (sort of) reviewer prevented us from publishing the paper. Later, Bob and Bill replicated the lesions results with ibotenic acid lesions of MT and came up with better-controlled data (Newsome et al., 1985). My first independent publication was a cleaner version of the fundamental discoveries about normal pursuit that I think of as kicking off the modern analysis of smooth pursuit eye movements (Lisberger and Westbrook, 1985). It was great to collaborate with Bill, who also became a terrific friend. He brought tons of energy and optimism to the table, not to mention considerable scientific insight. I only regret that we have not copublished except as the 7th and 20th authors on a paper by Mark Churchland that used data from both of our labs with no extra effort from either of us (Churchland et al., 2010).

The second insight came from analysis of an arcane feature of pursuit. Once a monkey initiates pursuit, eye velocity oscillates around target velocity at a frequency as high as 6 Hz. We were pretty sure that the oscillations were a symptom of a high-gain, closed-loop feedback control system, but why can the same subject not track sinusoidal target oscillation at 6 Hz? We discovered that the explanation is what we now call “gain control.” Visual motion has very weak access to the motor system when the subject is fixating a stationary target and much stronger access when the monkey is tracking a moving target (Goldreich et al., 1992; Schwartz and Lisberger, 1994). Then, Masaki Tanaka took a page from the book of Newsome. Bill’s lab found that stimulation in area MT could change what a monkey told you he had seen (Salzman et al., 1990). Masaki found that stimulation in FEFSEM could change what the pursuit system said it had seen and make it respond much more strongly to a given target motion (Tanaka and Lisberger, 2001). We concluded that the output from FEFSEM doesn’t drive eye motion, but rather modulates how strongly sensory inputs drive eye motion. This is still true 20 years later and is a major feature of how we understand the neural control of pursuit.

The third insight was courtesy of my terrific friend and long-time collaborator Tony Movshon. I participated in the Cold Spring Harbor Course on Vision in 1988, and on the way back to New York City in his car, Tony explained to me that Rich Krauzlis’s and my ideas about representations of both image acceleration and image velocity in MT (Krauzlis and Lisberger, 1989) had to be wrong and could be disproved in a few simple recording experiments. We agreed to collaborate. I spent many months at NYU for the next 10 years, we recorded from MT of anesthetized monkeys, and I decoded image acceleration but not image deceleration from MT. Sadly, Tony was correct. Our behavioral experiments and models of pursuit required image deceleration to keep eye speed from overshooting the target during pursuit initiation, a problem I still haven’t solved. But my time with Tony was a scientific awakening. Tony thinks deeply about perception, motor behavior, theories of sensory processing, sensory neural responses, and much more. He pushed me intellectually. Our experimental designs went way beyond anything I had ever imagined. Tony had me program motion energy models. Tony also happens to be more knowledgeable about classical music, and almost everything else, than anyone I know. We share huge enthusiasm for air travel and its idiosyncrasies, although Tony’s travels make me look like a homebody, even during my 15 consecutive years of flying more than 100,000 miles each year on American Airlines. We remain best of personal and scientific friends.

The fourth insight resulted from a collaboration with Bill Bialek that grew out of UCSF’s Sloan Center for Theoretical Neurobiology. During one of his visits to our Center, Bill came to my office door and asked: “This system you study, pursuit eye movements—how variable is it?”

I responded: “It is fairly reliable but still pretty variable.”

To which Bill replied: “I think we could learn something if we could put a number on that.”

The next day, I collected hundreds of eye movement responses to the same seven target motions. Bill took the data home and a year later he came back, said he understand the variation, and backed that up with an equation he wrote on my whiteboard. The equation said that more than 90 percent of the trial-by-trial variation in the initiation of pursuit could be understood as small errors in estimating the direction, speed, and time of target motion. Leslie Osborne picked this up, collected much more data, and concluded that sensory noise was the primary cause of motor variation (Osborne et al., 2005). We no longer think her conclusion is correct, but the data stand, and Leslie’s conclusion was half-wrong for innocent reasons.

A chance conversation, a single experiment, and astute analysis by Bill Bialek transformed my research program and put variation on my map as a part of the neural code that mattered and as a tool for analyzing systems. It marked the end of averaging the responses to 16 repetitions of the same stimulus and declaring victory. Suddenly there was a good reason to repeat the same stimulus 100, 200, or even 500 times and analyze trial-by-trial statistics. Javier Medina discovered impressive trial-by-trial correlations between the responses of individual Purkinje cells and eye speed at the initiation of pursuit (Medina and Lisberger, 2007). Sonja Hohl recorded shocking trial-by-trial correlations between the responses of individual neurons in MT and eye speed at the initiation of pursuit (Hohl et al., 2013). Before she did her experiments, Sonja described them at the Cold Spring Harbor Course on Vision. She reported to me that Tony Movshon and Eero Simoncelli told her the experiments “wouldn’t work.” Tony had once told me that our ideas about the sensory origin of motor variation had to be wrong because “there are so many neurons in MT that the sensory noise will average out.” True, if the neural responses vary independently. But we understand “neuron-behavior” correlations as unavoidable consequences of the “noise correlations” between neurons with similar sensory or motor preferences in a given part of the brain (Huang and Lisberger, 2009).

Seth Egger rediscovered recently (Egger and Lisberger, 2022) something that David Schoppik and Kathy Nagel had started to explore (Schoppik et al., 2008), namely that neuron-behavior correlations provide powerful constraints on models of the pursuit system. Seth also demonstrated that part of the trial-by-trial variation in pursuit comes from noise in the gain of visual-motor transmission, while part comes from correlated sensory noise as Leslie Osborne had concluded. As is always the case in science, we weren’t alone in analyzing neural and motor variation. It had started with Harris and Wolpert’s (1998) report of signal-dependent “motor” noise but proceeded in parallel in many labs. It is unknowable and immaterial who was first—we participated in an “age of variation” in systems neuroscience and it opened our eyes. And it was a big focus of the Keck Center at UCSF, led to our NIMH Conte Center entitled “Variation as a Neural Code,” and infiltrated the thinking and research of many of us.

I remain steadfastly loyal to pursuit eye movements as a model system. We can quantify the sensory input and motor output and, if we measure the eye movements, we know how neurons in the motor system are firing. It gives us profound interpretational power and my aspiration is to take advantage of that. My goal is to develop a circuit model of pursuit that (1) has the architecture of the pursuit circuit; (2) operates on a millisecond timescale throughout a two-second initiation and steady-state of pursuit; (3) has model neurons with mean, variance, and neuron-behavior correlations that mimic those recorded at different nodes of the pursuit circuit; (4) reproduces behavioral and neural responses to many target forms, including those with degraded motion reliability; and (5) operates autonomously based on the feedback structure of a closed-loop system that eliminates its sensory input within 200 ms but still produces persistent motor output. Maybe then I will have “finished” my work.

Cerebellar Learning, Redux

I thought I had declared victory over cerebellar learning in the 1990s, but Javier Medina had other ideas. He came to work with me as a postdoc in 2001 and reshaped our nascent efforts in studying motor learning in smooth pursuit eye movements. In collaboration with Megan Carey, he developed a paradigm for eliciting reliable motor learning in the direction of pursuit eye movements (Medina et al., 2005). Learning trials started with target motion in a fixed “pursuit” direction and then, 250 ms later, a component in an orthogonal, “learning” direction. Learning is expressed in pursuit as an eye movement that takes the eye in the learning direction at a time that anticipates the instruction. Further, direction learning in pursuit has the same time selectivity as Mike Mauk had found in classical conditioning of the eyelid response (Perrett et al., 1993). By comparison with learning in the VOR, pursuit learning occurs quickly. It was possible to record from floccular Purkinje cells during baseline pursuit before learning and then throughout the substantial learning caused by a block of 100 repetitions of the same learning trial. Javier demonstrated that instructive changes in target direction caused climbing fiber spikes on about 40 percent of trials and that 100 learning trials caused a well-timed suppression of simple-spike activity of about 20 spikes/s. Correlative for sure, but the direction, reliability, and timing of the responses were nicely in line with the Ito hypothesis of cerebellar learning in the eye movement motor system and, remarkably, involved the same Purkinje cells that participate in VOR learning (Medina and Lisberger, 2008).

Our research on pursuit learning led David Herzfeld to a statement of four principles of operation of a learning neural circuit (Herzfeld et al., 2020).

Principle 1: The earliest learning, even after a single instruction, is fast and easily forgotten and occurs at the parallel fiber to Purkinje cell synapse in the cerebellar cortex, driven by inputs on climbing fibers.

Principle 2: Early learning in the cerebellar cortex transfers to later, slower learning in the cerebellar nucleus, instructed by the learned simple-spike responses in Purkinje cells.

Principle 3: Neural inputs to each site of learning need to signal the context at the time of the instruction to be subjected to learning. For pursuit learning, those inputs have different functional relationships to eye movements in the cerebellar cortex vs. the cerebellar nucleus.

Principle 4: Feedback from Purkinje cells through the cerebellar nucleus to the inferior olive modulates the transmission of errors via the climbing fibers and limits the amount of learning that is possible in the cerebellar cortex. Learning must be transferred out of the cerebellar cortex to consolidate and persist, leaving the cerebellar cortex in a nimble learning state most of the time.

A single critical insight jump-started and drove our discoveries about pursuit learning. Javier came to me about noon one day as we were preparing a paper on neural learning in floccular Purkinje cells and said: “Steve, there is a problem.” Pushed to elaborate, he explained that most of the neural and behavioral learning occurred over the first 30 learning trials, when there were at most 12 climbing fiber responses to the instruction. He went on to predict that “there is no way 12 climbing fiber responses could cause a 20 spike/s depression of simple-spike firing. The cerebellar learning theory must be wrong.”

I don’t know how I had this idea. I said to Javier: “This isn’t a problem, it is an opportunity. If 12 climbing fiber inputs are causing a 20 spike/s depression, then each climbing fiber should cause a 2 spike/s depression and you should be able to measure that.” I suggested that he break the sequence of trials into pairs, separate the pairs according to whether or not the first trial included a climbing fiber response to the instruction, and ask whether the simple-spike firing showed a small depression on the second trial if (and only if) there was a climbing fiber response on the first trial. Javier came back late that afternoon and said: “You are completely crazy, but I will do the analysis.” It worked. A single climbing fiber input is linked tightly to a well-timed depression of simple-spike firing on the next trial. I remained a little concerned that our discovery was “too good to be true” and I predicted correctly that the reviewers of the paper would tell us to do it again. Fortunately, one reviewer also suggested a way to “do it again” with the data we already had and didn’t require us to redo the experiments on more monkeys. Still, it was a great relief when Jennifer Raymond’s lab found the same single-trial effects in data recorded during motor learning in the VOR (Kimpo et al., 2014), and David Herzfeld found related effects in data recorded by Robi Soetedjo and Yoshiko Kojima in the oculomotor vermis during saccadic adaptation (Herzfeld et al., 2018). If I had to name my lab’s single most important discovery, this would be it.

Javier’s discovery of single-trial plasticity of simple-spike responses was based on a tiny database. The next step was to develop a paradigm that would allow us to investigate single-trial learning and plasticity in depth. Yan Yang invented the “random direction paradigm,” in which the instruction was randomized between two directions along a learning axis so that there was no long-term learning (Yang and Lisberger, 2010). Now, we could present 400 instructions while recording from a single Purkinje cell, meaning that we might get as many as 80 climbing fiber responses for each recording. Yan obtained beautiful data. She verified single-trial plasticity of simple-spike responses linked to climbing fiber inputs. She solidified that single-trial learning in eye movements was larger if there had been a climbing fiber response on the previous trial in the Purkinje cell under study (Yang and Lisberger, 2013). She added that single-trial learning in eye movements and plasticity in cerebellar output both scaled with the duration of the calcium-related events caused by climbing fiber spikes (Yang and Lisberger, 2014). Not only did this make me feel more confident of climbing-fiber driven single trial plasticity and learning, but it also implied that on any given trial, climbing fiber events occurred either on most Purkinje cells or on a few.

Since Yan’s key papers in 2013–2014, the lab continues to explore cerebellar learning in pursuit eye movements. We learned that pursuit learning evolves over multiple timescales (Hall et al., 2018). We think that different time courses arise at different learning sites. We obtained preliminary evidence that consolidation over longer learning blocks causes learning to be transferred out of the cerebellar cortex, presumably to the cerebellar nucleus. We have started to employ approaches with multicontact probes to assess neural correlates of plasticity other than at the parallel-fiber to Purkinje cell synapse. My goal, in collaboration with Court Hull, Javier Medina, and Nicolas Brunel, is to construct a biologically motivated circuit model of the cerebellum that includes all neuron types, transforms the known mossy-fiber inputs into the known Purkinje cell output, and learns autonomously in a way that mimics biology, complete with realistic first- and second-order statistics of neural responses. Then I wish to fit that model into the larger model of the pursuit circuit outlined in the previous section.

The Cerebellum: Back to the Future

My CV lists 152 “original” publications. Only about 33 of these are on the cerebellum. The small number belies how central the cerebellum has been in my career. It is my favorite brain structure. In my earliest days as a student, I was influenced profoundly by the papers, the passion, and the personal attention I received from greats like Masao Ito, Rodolfo Llinas, Bob Baker, and even Sir John Eccles. I had the enormous honor of knowing these individuals personally, and others who influenced the cerebellar field profoundly such as Jerry Simpson, Tom Thach, Peter Strick (senior to me but still active), David A. Robinson, Geoffrey Melvill Jones, and of course my graduate and postdoctoral mentors, Albert Fuchs and Fred Miles.

I have watched the field of cerebellar research go through multiple evolutions. In 1972, cerebellar learning was one of the most exciting concepts of neuroscience and The Cerebellum as a Neuronal Machine had been published only recently (Eccles et al., 1967). But then, it got complicated and, perhaps more important, we did not have any access to methods to establish causation. We knew that the parallel-fiber to Purkinje cell synapse was subject to long-term depression caused by climbing fiber inputs (Ito and Kano, 1982), but does that happen in real life? Neuroscience was correlative then, and we needed to establish causation. In the meantime, other aspects of neuroscience moved to the forefront: modern anatomical methods, neural networks, gene-targeting knock-outs, functional imaging, brain machine interfaces, and the neurophysiology of “cognition,” to name a few. Cerebellum was reduced to a smallish field that risked imploding through self-criticism. We did not know it, but we were waiting for technology to loop back to us in the 21st century and enable answers to the kinds of questions we had in the 1980s.

Thanks to the efforts of a handful of cerebellar neuroscientists younger than I, the field is back on its feet and is flourishing. The Gordon Research Conference on the Cerebellum started in 2011, was an immediate success, and has grown better and better over the years. Kamran Khodakhah invited me to present the inaugural Keynote Lecture in 2011, bestowing on me one of the greatest honors of my career. Roy Sillito started the Cerebellum Social at the Annual Meeting of the Society for Neuroscience (SfN) and it was an immediate hit. Attendance was great and people wanted to be there rather than at the companion socials. We had several informal meetings of cerebellar neuroscientists to talk about how we could support the growth and success of our field while maintaining scientific standards. Those meetings created a unity of purpose. Now, modern molecular approaches are integrated into cerebellar research and there are many labs run by young cerebellar scientists. I am proud that one of my trainees, Megan Carey, has assumed complementary leadership roles in nurturing and expanding cerebellar neuroscience. The terrific Grass Lecture of another trainee, Jennifer Raymond, shone a spotlight on our field at the 2019 Annual Meeting of the SfN. The future looks bright for cerebellar research.

Eye Movements as a Window on the Brain

I chose to research eye movements because I believe that we can use eye movements as a model system to understand general properties of how the brain works. Eye movements afford many advantages. We know the sensory stimuli that drive eye movements—head turns (VOR), eccentric target positions (saccades), and target motion (pursuit)—and we can deliver those stimuli precisely and accurately. We can measure eye movements quantitatively with magnetic search coils and increasingly with video technology. I refined our surgical procedures for implanting eye coils to the point at which coils seldom need to be replaced (thanks to Howard Egger of NYU for showing me the surgery and Creig Hoyt of UCSF for teaching me how to suture halfway through the sclera). The final motor machinery for eye movements is in the brainstem (rather than the spinal cord) where it is accessible to microelectrodes in behaving monkeys. If we know the kinematics of eye movement, we can use known equations to describe the time-varying firing rates of the extraocular motoneurons and of many brainstem and cerebellar neurons. The depth of knowledge about the connections and activity in the final motor machinery for eye movements affords interpretational power for recordings of neural activity elsewhere in the brain. The field, including my lab, has realized that potential to some degree. We have illuminated general principles of brain function in sensory decoding, cerebellar learning, Bayesian-like sensory-motor behavior, and mechanisms that create persistent activity.

In sensory decoding, we sat on the shoulders of two seminal contributions (Groh, 2001; Salinas and Abbott, 1994). In Jennifer Groh’s framework, the population response in area MT is a “place code” in the sense that the speed and direction of target motion is represented by which neurons are most active. In contrast, the signals that drive smooth eye movement are a “rate code.” In the cerebellum, firing rate encodes eye velocity and direction is represented roughly along the horizontal and vertical axes. We deployed vector averaging calculations to show how to transform the sensory code into motor commands. We made it work, while also realizing the importance of the amplitude of neural responses, as pointed out by Krekelberg et al. (2006). Recently, however, we had an epiphany. The sensory-motor decoder for pursuit eye movements isn’t an equation. It is a set of parallel neural circuits that implement the strategies proposed by theorists but in very brain-like ways. We now think of the sensory-motor decoder as a question of how the brain implements equations. Our extensive data on the first- and second-order statistics of neural responses in multiple nodes of the pursuit system provide constraints on a circuit model of decoding. Similar logic should work for other sensory-motor systems, and I look forward to seeing it applied to decode the output of the dynamical system framework (Shenoy et al., 2013).

The entire cerebellum has the same architecture, so I used to think that all parts worked and learned according to what we knew about the floccular complex and eye movements. I think research on eye movements, including ours, has revealed principles that generalize. But the task for the rest of the cerebellum is more complicated than it is for its eye movement regions. An important concept for control of limb movements has been that the cerebellum learns “internal models.” I think of the processing in the floccular complex in terms of an internal model. Purkinje cells sum signals related to eye velocity with respect to the head and head velocity with respect to the world. This coordinate transformation—from eye to head coordinates—computes an internal model of the orbit and represents eye velocity with respect to the world (also known as gaze velocity). This internal model seems almost trivial compared with those needed for arm movement, and I wonder whether the concept generalizes. My belief about generalization of our findings on cerebellar learning is undermined further by recent discoveries of climbing fiber signals that seem more appropriate for reinforcement learning than for traditional cerebellar error-correcting learning (Heffley and Hull, 2019). Even in classical conditioning of the eyelid response, climbing fiber responses shift back to the conditioned stimulus instead of the unconditioned stimulus after full conditioning (Ohmae and Medina, 2015). Perhaps these complications will prove to be layers on top of the simple principles enunciated in research on eye movements. Perhaps the floccular complex, where climbing fiber responses signal movement errors and also are modulated by reward size (Larry et al., 2019), will provide insight.

“Bayesian inference” is shorthand for a computation that the brain performs routinely, namely a reliability-weighted combination of current sensory data with adaptable priors (or expectations) based on context and past experience. We demonstrated that pursuit obeys Bayesian inference in the sense that it is based on the relative reliabilities of context and sensory data (Yang et al., 2013; Darlington et al., 2017). We controlled context by providing a blend of target motions in blocks that were mainly slow (2 deg/s) or mainly fast (20 deg/s) but always included a few probe trials at the fixed speed of 10 deg/s. We controlled motion reliability by adjusting the contrast of the moving target. When tested with a given probe target speed, the strength of the initiation of pursuit is biased toward the speed of most of the target motions in the block. The bias is greater when the contrast, that is the motion reliability, of the sensory stimulus is lower. Our data implied that the prior would be implemented by control of the strength of visual-motor transmission, linking to our demonstration of control of visual-motor gain as a key component of the pursuit circuit. Tim Darlington used this paradigm to demonstrate the neural computations that implement Bayesian inference for pursuit in FEFSEM (Darlington et al., 2018). He discovered a ramp of preparatory firing during fixation even before the target started to move. The amplitude of the ramp depended on whether most of the target motions in a block were slow or fast. Thus, the preparatory activity represented the prior. The activity in FEFSEM during pursuit initiation encodes the maximum a posteriori estimate of target speed that appears to be computed by combining visual motion inputs with the prior represented in the preparatory activity. A recurrent local circuit model with balanced excitatory and inhibitory model neurons reproduced all the features of the representation in FEFSEM, and even adapted autonomously as did the monkey’s pursuit behavior when presented with mostly fast vs. mostly slow target motions. FEFSEM may be an example of how Bayesian behavior can be implemented with neural circuits and may be a potential template for other systems in which behavior results from a reliability-weighted combination of sensory data and priors based on past experience.

An autobiographical essay by Steve Lisberger would not be complete without some self-criticism. When I started my career in 1971, we viewed eye movements as a “window on the brain” that would reveal general properties of brain circuits. Sometime between then and now, that vision went off the rails. I think some of us still have that vision. Also, eye movements have proven invaluable to probe decisions, attention, learning, and other higher brain functions. But I think the field lost its way. Instead of my vision of using eye movements to discover general brain properties, most of the field descended into discussion and analysis of intricacies using jargon that left most neuroscientists out of the discussion. Research on the control of eye movements turned inward and became increasingly arcane and inaccessible to the rest of the field. I wonder whether I could have exerted leadership that would have prevented this inward turn. For sure, I aligned myself with good friends who had a much broader view of systems neuroscience—people like Tony Movshon, Bill Newsome, Michael Stryker, Carla Shatz, Terry Sejnowski, Bill Bialek, and Dora Angelaki, to name a few. I think the eye movement field saw me as a “social climber” rather than as a leader, and I didn’t do much to change that view and explain to them what I saw as the rightful destiny of eye movement research. I regret that eye movements have failed—not entirely but to some degree—to lead neuroscience by revealing general principles of how the brain works. I hold myself partly responsible.

Howard Hughes Medical Institute

It was a huge luxury to be an investigator of the Howard Hughes Medical Institute (HHMI) from 1997 until 2015. That said, I always believed that at least 3,200 scientists were as qualified to be HHMI investigators as the 320 of us who were lucky enough to be selected. I celebrated two renewals before my nonrenewal cast me into one of the most distinguished clubs in the field, the club of former HHMI investigators. I was worried about getting back into the NIH system and it was a struggle to do so—my main grant from the National Eye Institute went unfunded as an A0 and an A1 and then again as a new A0 before being funded as a second A1.

In 1997, HHMI made a big step into systems neuroscience by adding Bill Newsome, John Maunsell, Tom Albright, and myself as investigators. The Science Meetings we were required to attend every year were a real highpoint, especially as the cadre of systems neuroscientist expanded and lots of molecular and cellular neurobiologists started to study brain circuits. We were in a Science Meeting at HHMI Headquarters in Chevy Chase, Maryland, on September 11, 2001, when the Twin Towers were brought down. It was dramatic and unsettling. I remember our collective shock and concern for our New York colleagues’ friends and family. I remember the stunning silence in the skies as we sat outside on the evening of September 11. I remember our feeble attempt at conducting an impromptu scientific meeting that same evening. I was supposed to be the next speaker when Tom Cech interrupted to tell us there had been a terrorist attack in New York and the formal meeting would be suspended. The talk I gave that evening was terrible—it would not have been a good thing if I had given the same talk at the real Science Meeting.

HHMI came at a tipping point in my career. I had published my papers on motor learning in the VOR in 1994 and the lab was small. Before 1997, I had been an independent investigator for 16 years and only 3 PhD students had joined the lab. It was a transition point for postdocs: everyone who was in the lab in 1997 was gone by 1998. Before HHMI, I had done much (but not all) of my own research. In my first 10 years as an HHMI investigator, 11 postdocs and 9 PhD students joined the lab. They brought new energy and new ideas and my research thrived. The lab was bigger than ever, reaching 12 scientists plus me at one point. It was stimulating and productive. My trainees changed my research program dramatically during that time. I do not know whether that would have happened if I had not been an HHMI investigator. Fortunately, we cannot “rerun the tape” (attribution to Stephen Jay Gould), and I do not care to know.

Leadership

During the Covid-19 pandemic when we mostly worked from home and kept our distance from each other, my view was that we were doing research but not doing science. Our trainees were coming to the lab to do their experiments, but there was no interaction, no chance insights over discussions at the coffee machine. We were doing what we had promised our granting agencies, but we were not delivering added value. Leadership is about creating a culture in which research turns into science. Of course, it also is about creating a culture of excellence, professionalism, inclusiveness, integrity, and support for everyone, and I have strived to succeed at the whole package.

I guess people saw me as a leader. Or maybe they merely realized that I am not good at saying “no.” In 1990, my colleagues asked me to be the director of the W. M. Keck Foundation Center for Integrative Neuroscience, founded with a generous gift from the Keck Foundation to bring our systems neuroscientists together by remodeling 10,000 square feet of space on the eighth floor of the Health Sciences East (HSE) tower. In the Keck Center, we had the crazy idea that labs didn’t have to be silos. We divided the 105- by 105-square-foot space diagonally into a triangle for dry labs and a triangle for animal labs, the latter connected to the vivarium by a back door. We created six or seven bullpens for our lab members and required that each bullpen have major representation from at least two labs. We clustered the rooms where each lab did their wet research but didn’t partition the labs. It worked. People talked to each other, collaborations sprang up between labs, and it was a vital, active, fun place to do science.

The Keck Center had a big impact on neuroscience at UCSF and its creation was temporally correlated with UCSF’s final rise to the top of the field. The concepts behind the Keck Center weren’t new to UCSF. It was simply UCSF’s concepts instantiated in a group that was interested in how the brain worked while it was working. But the Keck Center was an example of how establishment of an appropriate physical structure can have a huge impact on science. We hired brilliant assistant professors who embraced the idea that great colleagues and collaboration were more important than space and money—Allison Doupe, Ken Miller, Philip (Flip) Sabes, Loren Frank, and Michael Brainard in the early days and others later. We developed the Sloan Center for Theoretical Neurobiology and recruited quantitative postdocs who changed how we did our science.

I don’t take credit for most of what we did—in many ways, I was the person who developed and managed the process to implement the brilliant ideas of my colleagues. The original Keck Center included myself, Mike Merzenich, Christoph Schreiner, Roger Nicoll, Howard Fields, Allan Basbaum, Michael Stryker, Allison Doupe, and Ken Miller. Zach Hall, one of my main leadership mentors, was very much the “man behind the curtain.” His advice, support, and efforts made a huge difference. I learned a ton from him. Between the founding grant from the Keck Foundation, generous funding from the Sloan Foundation, some philanthropy, 10-years of Program Project support from National Institute of Neurological Disorders and Stroke (NINDS), and 5-years of a Conte Center from the National Institutes of Mental Health, we had terrific financial support. But we were more than well-funded. We had a community that made a difference, and we used our funding to move science and the culture of science forward qualitatively.

It was difficult for people to imagine why I would leave UCSF and San Francisco. Indeed, up to the day I announced my decision, I do not think any of my colleagues believed that I would leave. Yet many of us moved to other positions. The place thrived even as people who seemed like senior leaders and glue departed, validating the notion that it is something in the overall culture that makes UCSF special. For me, however, there also was a glass ceiling. I had leadership aspirations, and they never were going to be realized at UCSF. I had concerns about how strongly the Department of Physiology was going to support neuroscience, especially systems neuroscience. And personally, San Francisco had gone from an easy wonderful place to live in 1981 to an expensive and crowded place to live that presented many challenges for everyday life. For complicated and personal reasons, the last five years at UCSF were not very happy years for me in my job. So, I was ripe to be plucked. We were in Eilat, Israel, for the night before crossing the border to Jordan to visit the amazing archeological site at Petra when Nicole Calakos Emailed me to ask if I would throw my hat in the ring to become chair of neurobiology at Duke. We discussed it as a family over dinner at a Brazilian steakhouse, the family was unanimously on board, and, 11 months later, Dean Nancy Andrews offered me the job.

UCSF and the Keck Center had something special that I tried to create anew at Duke. I accepted the job in June 2011 and started in early September 2011. For nine months, I commuted between San Francisco and Durham, making two to three weeklong trips to Durham each month. I had an apartment near the Duke campus and, after a while, a car that I barely needed. I devoted 100 percent time and effort to getting things rolling at Duke. I started with a sense of urgency because I saw that it was necessary. Duke Neurobiology had enjoyed glory days in the 1990s and at the start of the 2000s under the stewardship of Dale Purves, but because of forces outside of his control, Jim McNamara had faced huge obstacles as Dale’s successor. By the time I got there, the carriage was sitting by the side of a bumpy road with the wheels totally off. Larry Katz had passed away (a tipping point), many senior members of the group had left, and morale was low. Of the faculty in the Department of Neurobiology at the time I was recruited, only five remain today. Twelve, including me, are new.

My main leadership goals at Duke were to establish a culture of “us” rather than “me” and to hire great faculty who would embrace that culture. I saw myself as using leadership to change how science is done, not simply to make the department financially successful. I recognized during my recruitment that many young faculty across the campus were going to be star neuroscientists. I saw my role as getting them all to the table. Yes, I was given generous resources (do I detect a theme here: HHMI, Keck/Sloan, now Duke?), and my goal was to use those resources to change how science is done at Duke Neurobiology. I am pleased with how it has turned out. Duke Neurobiology now includes about 50 faculty across 17 departments in the Schools of Medicine, Engineering, and Arts and Sciences. But more important, we have an inclusive culture in which the faculty collaborate and interact. Also, remembering the impact the Sloan Center for Theoretical Neurobiology had at UCSF, I have been intentional about bringing theory and computation into the community. Again, I see a qualitative change in the way that many purely experimental labs are doing their science.

I never shied away from a leadership role. Spurred on by a postdoc colleague from the NIH days, David Friedman, I helped start an ad hoc Committee on Scientific Literacy for the SfN. Our committee played a big role in starting the SfN’s initiatives in this area by organizing a symposium at the annual meeting of a high school science teachers society. I do not recall the details, but I think our ad hoc committee gradually evolved into the current standing Public Education and Communication Committee. I was elected treasurer of the SfN in 2012 and as treasurer I chaired the Finance Committee and served on the Audit Committee, the Investment Committee, and Council. I agreed to run for president of SfN once and lost to Rick Huganir. I had a vision for what I would have done if elected, but in the end, I merely was immunized against running again. I served as the chair of the Sensory-Motor Integration (SMI) study section and I have cochaired numerous study sections for the BRAIN Initiative. As a study section chair, I try to make a contribution to neuroscience by making sure that the review process is fair and unbiased, and that the applicants receive narrative feedback that matches their numerical scores so that they have actionable criticisms.

I was a high-level editor for two journals. For the Journal of Neuroscience, I served as section editor and senior editor for 10 years. I led the initiative to take the manuscript handling and editorial processing online for the journal and was very closely involved in the design of the manuscript handling system we adopted. Given how we take this and much more for granted now, it is shocking to recall that I had to work against very considerable resistance. Once he was persuaded of its virtues, Marty Saggese, then the new executive director of the SfN, became a major ally in this effort and a very good colleague and friend for years since then. I took one year off from being an editor after my term ended at the Journal of Neuroscience and then became the associate editor for the International Brain Research Organization’s (IBRO’s) flagship journal Neuroscience, working under Ole Petter Ottersen for four years. Finally, I was the chief editor of Neuroscience for six years. Editorial service to Neuroscience is an important community service because the profits from Neuroscience provided IBRO’s entire revenue stream in those days.

Mentoring

My contribution to science has been multiplied by a commitment to mentoring. In my own laboratory, I have mentored 15 PhD students and 29 postdocs. Of these, 32 remained active in scientific research (including 5 who are still in my lab) and most of the others have high-level positions in the for-profit or not-for-profit worlds. Many have gone on to have distinguished careers. Of my 44 mentees, 16 were women, and I received the Bernice Grafstein Prize from the SfN in 2011 to recognize my successes in mentoring women.

I devoted considerable effort to mentoring of junior faculty. At UCSF, I was a primary mentor for Allison Doupe (my late, great friend and coconspirator), Ken Miller, Michael Brainard, Philip (Flip) Sabes, Loren Frank, Eddie Chang, Vikas Sohal, and others. Three of them have gone on to become HHMI investigators and Eddie won the Blavatnik Prize for Young Scientists. Of course, the success of these scientists is a testimony to their brilliance, but I like to think that my guidance helped to channel that brilliance. At Duke, I have mentored Rebecca Yang, Anne West, Lindsey Glickfeld, Kevin Franks, Court Hull, Jeremy Kay, Greg Field, Eva Naumann, Anita Disney, Kafui Dzirasa, John Pearson, Nicole Calakos, Mike Tadross, Tim Dunn, Greg Cogan, Jeff Beck, Cagla Eroglu, and others. Many of these faculty are not in my department and are not among my direct reports, but I am a mentor-at-large and I offer free advice to anyone.

One of my major mentoring successes has been guidance in scientific thinking for Duke Neurobiology graduate students. I run the Neurobiology Study Section in a course called “Grant Writing,” but I view the course as training in how to think about their own project in depth (for the first time for most of them) with a draft of a National Research Service Award (NRSA) application as a by-product. The study section has been enormously successful and rewarding for me. I now have had the chance to guide each of the students in Duke Neurobiology in their science and their approach to science early in their career. They appreciate my style (tough love) and the excellent and honest guidance I give them, and so I serve on many PhD thesis committees and I provide informal mentoring for many of them. My office door is open and they seem to be comfortable coming to talk to me.

Perhaps my greatest success as a mentor was to help Richard O’Brien, chair of neurology at Duke, with the development of a successful application for an Alzheimer’s disease Research Center (ADRC). Once again, my role was process. I didn’t know much about Alzheimer’s disease then, but I learned from Rich and I channeled his brilliance on how an ADRC should look at Duke into a process that ultimately identified (1) ideal leadership in Heather Whitson; (2) a fantastic and forward-looking set of cores; (3) a collaboration with our neighbor nine miles south in Chapel Hill, the University of North Carolina; and (4) a first-time application that was funded in 2021. My guidance and mentoring were necessary (but not sufficient) for this success. Credit goes to Rich’s brilliance about Alzheimer’s disease and to Heather’s awesome organizational skills.

Animal Rights

The most difficult part of my career was 28 years of harassment by so-called animal rights groups. Fred Miles once told me that he would have quit science if subjected to the pressures I had experienced. I agree with him that it was incredibly tough. It wasn’t just the episodic harassment, horrible publicity, demonstrations in front of UCSF, and fear for the safety of myself and my family. It was also the knowledge that even when it was quiet, the other shoe could drop without a moment’s notice. I am and always have been resilient. Perhaps I was prepared for dealing with this kind of situation by the uncertainty I faced as a child as to when my stressed mother, as a widow at age 36 with 5 kids under 12, would explode in my face. I persevered.

It all started in about April 1983 when a well-known TV personality declared on Animal Rights Day in Davis, California, that my two monkeys, Beau and Captain, were being deprived of food and water for “some stupid experiment.” The story broke all over the news media. UCSF’s Institutional Animal Care and Use Committee (IACUC) summoned me to their meeting the next day. On the basis of what they had read in the news media, the IACUC was prepared to suspend my protocol. But Mary Dallman, a senior neuroendocrinologist in my department and an animal user pointed out that I had done nothing wrong. It wasn’t my fault that an animal activist had broken into UCSF’s animal facility, awakened my monkeys in the middle of the night, and taken photographs of groggy monkeys in unflattering light. At that moment, I joined a select club of neuroscientists who would experience animal activism, in some cases much more severely that I would.

We learned a lot from that first episode. Zach Hall’s brilliant intuitions, personal counsel, and undying support made a big difference. We learned about the modus operandi of the animal activists, we learned that Richard Nixon’s policy of stonewalling was a good one, we learned that I as the accused should never represent myself, and we learned that time almost always would heal all wounds. My good friend Rick Van Sluyters was a target across the bay at UC Berkeley and we coordinated and consoled each other with humor not fit for repeating.

I once sat next to an airline pilot on a cross-country flight and I asked him what it was like to fly a commercial airliner. His response: “hours and hours of utter boredom punctuated by moments of abject terror.” My next 25 years after 1983 were similar. Plenty of time to do my science, punctuated by moments of frightening terrorist actions against me by the animal activist groups. Over the years, there were two public hearings on my research at the meetings of the San Francisco Board of Supervisors. Louis Reichardt led a large group of neuroscientists to pack the room for the first meeting and successfully overwhelmed a poorly prepared presentation by the animal activists. For the second meeting, my lab was bigger and more militant, and Megan Carey agreed to testify. When she answered that she had never seen any of the atrocities that were alleged, they asked her whether she had ever been to my “secret lab”! Megan still asks to see my secret lab. On a third occasion, around 2003, the Board of Supervisors was asked again to investigate. Gavin Newsom, now governor of California but then the chair of the Board of Supervisors (I think) threw out the first pitch at “opening day” for the San Francisco Little League. While my six-year-old son Theodore was getting a hot dog with the rest of his team, I approached the pitcher’s mound and introduced myself to Gavin as “the guy at UCSF with the monkeys.” I was all prepared with an elevator speech, but Gavin just said: “Oh, yes, we know about that. You have nothing to worry about.”

I found it unsettling when the animal activists glued the locks on the front door at our home and then crowed about it in their blogs and on their websites. It was frightening that they knew who we were and where we were. As soon as the locksmith left the house, I walked the seven blocks to UCSF and straight to the office of the executive vice chancellor of UCSF, Eugene Washington. I burst through his assistant’s office saying that I did not care what Gene is doing, I needed to talk to him. Gene immediately authorized 24-hour protection at the house and a security appraisal. The security professionals made quite a few recommendations, and UCSF had them done. Gene’s responsiveness made a big difference to me and the family. At that time, I also made appointments to visit the head of school at both children’s schools to educate them about the animal activist movement and how it was affecting us and to tell them what sorts of (very low probability) events they should be alert to. Those were not easy meetings.

Along with the negative publicity and overt attacks from outside, I also experienced challenges from legitimate regulators, namely the UCSF veterinarians and the USDA inspector. Whenever the USDA inspector visited, he went straight to my protocol looking for something marginal or wrong. We were diligent about animal care and animal welfare, thanks very much to my staff, Karen MacLeod, Elizabeth Montgomery, and Stefanie Tokiyama. He didn’t find much and I didn’t get into more trouble, but there always was the concern. My problems with our USDA inspector ended at an Annual Meeting of the Society for Neuroscience. I spoke on the Animal Panel about my experiences and about all the refinements we had made in my research on monkeys, including our decision to place the monkeys in sanctuaries when we were finished using them for research, rather than euthanizing them. Our USDA inspector was at that talk. He came up to me afterwards, introduced himself, and thanked me for my informative presentation. That was the end of my issues with him. I think it has been constructive to speak openly about what I experienced with animal activism and it is cathartic. I do not condone the activists’ methods, but I also think we have come a long way since 1983 in ensuring the welfare of experimental animals in our labs.

Then and Now

Things have changed. In the 1970s, eye movements were the hottest model system on the planet and everyone wanted to record the electrical activity of single neurons in the brains of behaving monkeys. Now, eye movements have become a niche system. Monkey research is a tiny corner of neuroscience and is not expanding. New techniques have exploded serially into the field, taken over much of neuroscience research, and then retracted into their rightful place in the neuroscientist’s tool kit. In the 1970s, it was single-unit recording in awake behaving monkeys. In the 1980s, it was tract tracing with horseradish peroxidase. In the 1990s, it was computational modeling and parallel distributed processing. In the 2000s, it was gene targeting knockouts and fMRI. In the 2010s, it was optogenetics and other molecular approaches. What is next?

At my first SfN meeting in San Diego in 1973, there probably were fewer than 2,000 attendees and it was very personal. I remember meeting Steve Kuffler in the hotel pool. Now, there are 30,000 attendees, and it can feel quite impersonal and difficult to connect. When I started my first faculty job in 1981, I activated my first R01 on the day I arrived. That was the rule; not the exception. Now, we counsel new faculty to wait a couple of years to apply for their first R01 and to live off their startup funds. It never occurred to me that my first grant would not be funded. Now I and everyone else worries about whether their first, next, or last grant will be funded. In the 1970s, a paper in the Journal of Neurophysiology was the pinnacle of one’s career. Many of my most important papers were published in JNP. Of the 152 papers on my CV, 22 appeared in what we think of as prestige journals and 101 in either the Journal of Neurophysiology or the Journal of Neuroscience. Now we value a paper by where it is published as much as by what it contributed. I don’t like this change, but I understand that my trainees perceive a need for high-profile publications.

I understand that there always will be “then and now” and that those of us who were around for “then” will lament the changes that created “now.” Still, science used to be smaller, personal, and supportive. Now it has become too big, quite impersonal, and cutthroat. I would like to see us return to the good old days to some degree. Science needs to be about advancing knowledge and improving our world, not about self-promotion. To go back to “then,” the incentives will need to change.

People

Near the end of her life, my mother once said to me: “I have had a great life, except for the deaths of close family members.” In her case, that included two husbands, one long-term boyfriend, a daughter, a sister, a brother-in-law, both parents, and both parents-in-law. I feel the same way about my career as a neuroscientist. I have had a charmed career, except for the deaths of close colleagues. Roy Steinberg was a fantastic scientist and a regular Saturday morning coffee companion at the Tassajara bakery in Cole Valley, San Francisco. Carol Basbaum was not a neuroscientist, but Allan Basbaum was a close colleague, friend, and adviser, and I admire how both of them dealt with her life and death. Allison Doupe was a treasured friend and colleague, and a coconspirator in the design and construction of the Sandler Neuroscience Building (originally “19A”) at Mission Bay. I remember sitting on a bench outside the New Orleans Convention Center when I was recruiting Allison to UCSF, hearing her vision for how birdsong would conquer the world of neuroscience. She had it right, and she turned a lot of her vision into reality. I miss her terribly.

Science is full of great people and I have had the honor of knowing and working with some of them. The correlation between greatness as a person and stardom as a scientist is zero (or maybe negative). Some people are brilliant in life, some are brilliant in science, and a few are brilliant in both. I cannot even begin to mention the scientists (and nonscientists) who have been role models for me in some aspect of my life.

Chieko, Emika, and Theodore (also called Theo) have been great supporters and advisers. Chieko received a PhD from York University in experimental psychology with Ian Howard, so she understands the pressures and challenges of a life in science. She was always incredibly supportive of my need to work in all unscheduled moments and of the critical importance of travel for science. I’ve been equally supportive of her art career. I used travel to work intensely and I used travel to write, freeing up time to be with the family on weekends and evenings. I recall sending her an email one day in a February or March that said, “My grant got funded, let’s celebrate!”

She responded, “You wrote a grant?”

And I replied: “Yes, on airplanes last summer.”

Emika once said to me: “Dad, I want to do neuroscience, like you do, but I need goals that are months away, not 50 years away.” She gets science and as someone who goes all in on everything she does, she understands why I had to do the same thing. But she also appreciated that I would get up at 4:00 a.m. on Saturday to take her to a crew regatta and then would freeze by the water for the 30 seconds when I could see her boat come in. Theodore is like me in that he only does stuff that is relevant to him. He is the athlete I always wanted to be, and we have enjoyed many afternoons together at the ballpark—watching the Giants at AT&T Park or just working on his fastball at Grattan playground. Theodore is acutely aware of what is going on around him, much like his dad. He also knows as much about the past 50 years of music as anyone I know and always amazes me with the breadth of his knowledge about many things. We’ve had many special vacations as a family—most of them enabled by the miles I accumulated in my travels. And we have been healthy. I’ve been lucky.

Finally, just like everyone else in neuroscience, my science has been done by my students and postdocs. I succeeded because I sat on their shoulders. I was not able to mention all of them in the narrative, so I list them here without commentary in approximate chronological order. Almost all of them are stars in their chosen walks of life, and I am proud of them as my scientific children.

Students: Leland Stone, Edward Morris, Richard Krauzlis, Maninder Kahlon, Nicholas Priebe, Mark Churchland, Justin Gardner, Anne Churchland, Megan Carey, David Schoppik, Sonja Hohl, John O’Leary, Jennifer Li, Ramanujan Raghavan, Timothy Darlington, Stuart Behling.

Postdocs: Lawrence Tychsen, Keith Grasse, Dianne Broussard, Helen Bronte-Stewart, Sascha Du Lac, Vincent Ferrera, Jennifer Raymond, Gal Cohen, Holger Rambold, Masaki Tanaka, I-han Chou, Ram Ramachandran, Leslie Osborne, Javier Medina, Siobhan Garbutt, Hilary Heuer, Jin Yang, Xin Huang, Alex Roitman, Yan Yang, Yu-Qiong Niu, Joonyeol Lee, Kris Chaisanguanthum, Mati Joshua, Matthew Phillips, J. Patrick Mayo, Nathan Hall, David Herzfeld, Seth Egger.

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Gail Mandel

BORN:

Atlanta, Georgia

June 29, 1950

EDUCATION:

University of California, Los Angeles, BS (1972)

University of California, Los Angeles, PhD (1977)

University of California, Los Angeles, Molecular Biology Institute, Postdoctoral (1977–1978)

University of California, San Diego, Department of Biology, Postdoctoral (1979–1981)

APPOINTMENTS:

Instructor, Department of Pathology, Harvard Medical School, Boston, MA (1982–1984)

Assistant Prof. of Molecular Medicine, Tufts-New England Medical Ctr, Boston, MA (1984–1987)

Associate Prof. of Molecular Medicine, Tufts-New England Medical Ctr, Boston, MA (1987–1989)

Associate Prof. of Neurobiology and Behavior, SUNY at Stony Brook (1989–1993)

Professor, Department of Neurobiology and Behavior, SUNY at Stony Brook (1993–2002)

Investigator, Howard Hughes Medical Institute (1997–2016)

Distinguished Professor, Dept of Neurobiology and Behavior, SUNY at Stony Brook (2002–2006)

Senior Scientist, Vollum Institute, Oregon Health and Science University, Portland, OR (2006–present)

HONORS AND AWARDS:

American Cancer Society Scholar, Harvard Medical School (1982–1984)

National Science Foundation Faculty Award for Women Scientists and Engineers (1992–1996)

Director, Program Project Grant - Institute for Cell and Developmental Biology (1996–2001)

McKnight Investigator Award in Neurosciences (1997–1999)

Jacob J. Javits Neuroscience Investigator Award, NINDS, NIH (1997–2004)

Investigator, Howard Hughes Medical Institute (1997–2016)

Chancellor’s Award Honoring Research in Science, Engineering and Medicine (2001)

Chancellor’s Award as First Time Patent Recipient (2002)

Fellow of the American Association for the Advancement of Science (2003–present)

Member, American Academy of Arts and Sciences (2007–present)

Member, National Academy of Sciences (2008–present)

Discovery Award, OHSU Medical Research Foundation (2011)

NIH Director’s Transformative Research Award (2013–2019)

Councilor, Society for Neuroscience (2017–2022)

Gail Mandel was instrumental in launching the nascent field of molecular neurobiology through research, teaching, and mentoring. She was among the first to clone and functionally express ion channels with a principal focus on the voltage-dependent sodium channel. Her research led to identification of the genetic elements that control neuronal-specific expression and, ultimately, mechanisms that underlie the development of the nervous system. Contrary to expectations, her identification of the transcription factor, REST, showed that the acquisition of a terminally differentiated neuronal phenotype was not due to activators but rather to lack of this repressor. REST has been shown subsequently to be a master regulator for a wide cast of genes, many of which underlie human neuropathologies, including diseases external to the nervous system. She recently applied her background on gene repression to study of the transcriptional repressor, MeCP2, and its role in human diseases, showing that RETT is a disease involving both neuronal and glial dysfunction, thereby developing therapeutic approaches toward a cure.

Gail Mandel

I am a first-generation American. My mother, Judith Scheiner, was born in Berlin to Russian parents. She attended middle school in Paris and was living there at the outbreak of World War II. She, along with her parents, attempted to escape to the United States (her older sister was married to a British citizen and they had already immigrated to the United States). While her parents had Russian passports, my mother had a German passport, making it all but impossible to immigrate directly, because German Jews were flocking to enter the United States at the same time. I have the paperwork tracing her successful efforts to obtain safe passage, eventually gaining entrance through Portugal and Cuba, arriving finally in New York City in 1939. My father, Herman Mandel, the youngest of three boys, immigrated with his family from Poland when he was a toddler. Interestingly, both parents became naturalized citizens well after their immigration to the United States, with my mother filing in 1953. Dad’s naturalization papers are dated 1943 as he filed to join the U.S. Army infantry in World War II. He was captured in France and was a prisoner of war until the end of the war. As he said, they woke up one morning to find all the Nazis gone and the Americans arriving. Neither parent spoke much of their struggles, but it clearly shaped their lives, as they surrounded themselves with friends who had shared similar experiences and had liberal values. This included a lot of bridge and chess, which was used as a metric for the worthiness of my boyfriends.

I was born in Atlanta, Georgia, in 1950, while my father was attending Georgia Tech. After nine years or so, we were a family of five. We moved around, first from Georgia to Lynchburg, Virginia. My mother, a talented pianist and dancer, attended Randolph-Macon Woman’s College in Lynchburg, Virginia, where I had occasion as a young girl to see her perform as a ballerina in college performances. We spent several years in Glens Falls, New York, before settling in 1959 in idyllic Southern California. My dad was an engineer at Atomics International, my mother taught piano and then became a social worker, and my life centered on Malibu and Muscle Beach in Santa Monica. The beach life paid off with my first publication, featuring my friends and me in a Life magazine photo-op “California Girls Spangle the Beach.” This lifestyle was turned upside down when, in my junior year of high school, my father was commissioned as part of a team to help research coolants in a nuclear power plant in Pinawa, a small town in Manitoba, Canada. Pinawa is a very cold place, positioned in the midst of boreal forest, beautiful lakes, and spectacular wildlife. There were activities to enjoy, like skating and canoeing the frigid waters with friends, but no beaches, I turned to studies for the first time. My efforts landed me in the top 10 of my graduating class of 12. Still, my standardized test scores were competitive enough to be awarded a scholarship to the University of Colorado, Boulder, where I majored in journalism. I joined the Tri-Delt sorority and was having a blast when the power plant downsized in Canada, and with it, my scholarship. I moved back to the San Fernando Valley in Los Angeles with my parents and reconnected with many of my old friends. I scraped together enough money to buy a used, fire engine red Chevrolet Malibu super-sport convertible to cruise the valley and beaches, always with the top down (car that is). I was once again living the Southern California 1960s lifestyle.

My sophomore year in college was spent at what is now Cal State Northridge, where I continued to major in journalism, although an interest in biology was taking root. To investigate this new topic more thoroughly, I applied to the Eugene and Ruth Roberts summer student academy at the City of Hope Research Center in Pasadena. I didn’t know it then, but this was to be the beginning of a career in science shaped by a succession of wonderful mentors with creativity in their science, alongside huge personalities that included an equally large sense of humor. As I chronicle here, they showed me that research could be both challenging and fun, and that being a good human being was, as it is in life, a necessary component for a fulfilling career.

I was accepted into the City of Hope research program for summer 1970 and assigned to the neurochemist, Bernard Haber. Bernie had an ebullient personality. We clicked right away. My project was to test acetylcholine and GABA as “putative” neurotransmitters in chick brain. While this seems like what must have been a foregone conclusion even at that time, we were very conservative in those days before stating any finding as fact. It was my first introduction to bench science with state-of-the-art techniques, such as cell fractionation and sucrose gradients. I became friends with another neurochemist, Richard Hammerschlag. Richard did beautiful work, described to me as nature offering hints to answers for questions he didn’t ask. As a graduate student with Susan Leeman, Richard set out to purify corticotropin-releasing factor (CRF) from extracts of bovine hypothalami, which led not to CRF, as planned, but to the identification of substance P and neurotensin. Then, as a faculty researcher at City of Hope, he established an in vitro axonal transport system as a new approach to identify the elusive sensory neurotransmitter, only to be led to the early steps in the transport system itself. In addition to talking about science, Richard and I spent leisure time discussing the complexities of the Vietnam War and composing limericks. The latter groomed me for limerick compositions that were called upon as a postdoc with Bill Wickner at University of California, Los Angeles (UCLA) and much later as a member of the legendary NIH Physiology Study section. The panel’s cynical sense of humor belied an ability to provide incisive, critical, but fair reviews of their peers. I benefited from their collective wisdom, and limericks were subject to the same level of scrutiny as the grant proposals.

UCLA, 1970–1972

Despite my increasing interest in biology, I still clung to journalism when I transferred to UCLA in fall of my junior year. For one of my writing classes, I interviewed celebrities, for which there was no shortage in Westwood. This led me to coffee with comedians Gene Wilder, Phil Silvers (as Sargent Bilko), Carl Reiner, and Mel Brooks, as well as an in-depth interview with Jay Silverheels (Tonto in the Lone Ranger television show). My interest in biology took center stage, however, after I met UCLA graduate student, Paul Brehm, a transplant from Ferguson, Missouri. Paul was living in a small studio apartment in Beverly Hills and was having difficulty adjusting to the extroverted LA lifestyle. He made me laugh with his perfect irreverent imitations of certain UCLA faculty, and I was more than happy to help along these lines. Paul was studying bioluminescence with Jim Morin in the zoology department. Jim, along with his mentor, Woody Hastings at Harvard, was actually the first to identify and name green fluorescent protein (GFP; Morin & Hastings, 1971). They correctly surmised that GFP was an energy acceptor for the calcium-activated photoprotein, shifting light emission from blue to green. Paul was using GFP to identify and record electrophysiological responses from bioluminescent cells in the hydrozoan coelenterate where GFP was first discovered. The summer of 1971 would find us separated, with Paul at the Marine Biological Lab in Woods Hole, where Morin was an instructor in the now extinct Experimental Invertebrate Zoology Course. As for me, I was up for trying another summer with Bernie Haber. However, he had just moved his lab to the University of Texas Medical Branch, so it was summer in Galveston for me. At least there was a beach. I finalized my studies from the previous summer and presented an abstract at the annual Neurochemistry meetings, my first real scientific publication.

During the next year at UCLA, Paul and I grew close, and we moved into a small apartment at 1024 Hill Street, just blocks away from Santa Monica beach. We would walk the Venice boardwalk and eat at Al’s Kitchen on the Santa Monica Pier, still home to the hippodrome that houses the merry-go-round in the movie The Sting. Paul and I enjoyed the same music, food, backpacking, and exploring Southern California. We would ride our bikes to and from work down Pico Boulevard into Westwood and life was great. We both grew up loving animals and just kept accumulating them without regard for the impracticalities. We started with three parakeets that lived on our pole lamp and flew wild in our small apartment and then added two cockatiels. They controlled the air space. Next, we rescued two chinchillas that were about to be sacrificed in a research lab at UCLA. Those animals were lucky there was no Institutional Animal Care and Use Committee (IACUC) in those days. We quickly learned that they were male and female as they pumped out offspring at a nonstop pace. They controlled the ground space along with a desert packrat who nested under our refrigerator. When I was missing shiny jewelry, I knew where to find it.

During my senior year at UCLA, at the encouragement of Paul, I took all of the invertebrate courses offered, including the graduate seminars. The graduate students with faculty Jim Morin and Len Muscatine were a tight group, but I was deemed acceptable company. We made regular trips to collect invertebrates, looking especially for species that were bioluminescent. We would take clam guns to the Morro Bay mud flats and core drill for bioluminescent worms. For intertidal zones, we would carry squirt guns loaded with concentrated potassium chloride to hunt down new bioluminescent species (virtually all bioluminescent animals emit light when depolarized). Class trips included marine stations at Bodega Bay, Santa Catalina, and Puerto Penasco, Mexico. Paul introduced me to the field book Between Pacific Tides; Sea of Cortez, which was a field guide and the story of Ed Ricketts, a marine biologist, and author John Steinbeck, coadventurer with Ricketts. This guide colored our frequent visits to Pacific Biomarine, a collecting service headed by chemist Dr. Rim Fay who was a modern-time Ed Ricketts.

A memorable non-marine course for me was a botany course for non-majors led by Mildred Mathias, for whom the UCLA botanical garden is dedicated. With her class of about 12 students, I keyed out plants in and around LA on our field trips. Mildred walked the botanical gardens with all of us trying to keep up with her. She enthusiastically grilled us on different genus and species names for exotic plants based on our dichotomous key. I was pretty good at this, but always in the shadow of Evelyn Zwicker (now Maisel) who had a photographic memory. Evelyn became a medical doctor and talented artist. Both of us remember our time at UCLA with great warmth and were drawn back together as I researched the past for this biography.

UCLA, 1972–1978

Nearing the end of my senior year, I was looking into the prospect of graduate school. In seeking advice about applying to the UCLA Zoology program, I received conflicting recommendations, including one by a senior faculty member that I train instead for a career in dental hygiene. Somehow the liberal thinking, which then focused primarily on marijuana and protesting the Vietnam War, had not percolated down to the level of tackling gender bias in careers. This bias would take a much longer time to take center stage.

Between 1972 and 1973, two major events happened. First, in 1972, I entered graduate school in the Molecular Biology Institute (MBI) at UCLA, directed by Nobel laureate Paul Boyer. The field of molecular biology was in its infancy, which excited me, and they had hired junior rising stars that included Tom Kornberg, Mike Grunstein, Bill Wickner, and Judith Lengyel. I joined the lab of Bill Clark, by that time an MBI associate professor, working in the exciting and expanding area of immunology. Second, in 1973, Paul and I were married. At that time, it was difficult to find a rabbi who would perform a union between a Jewess and non-Jew, even in Los Angeles. At the request from my parents, we did manage to find one and tried, somewhat unsuccessfully, to keep the event low-key. Our fellow graduate students and friends were kept in the dark. The following day we set out with classmates on a collecting trip to Jade Cove. When information was leaked about our wedding, they decorated our Chevrolet Blazer, and we suffered through a weekend of jokes and pranks.

In the summer of 1974, I spent time at the Woods Hole Marine Biological Lab (MBL). Paul had returned to work with Woody Hastings to investigate the biochemistry of brittlestar bioluminescence. This would be my first experience at the MBL and one that had a major impact on my future. I was intrigued. I took advantage of the courses and seminars and was witness to the unparalleled intensity of the MBL. Much later, I would return to the MBL as an instructor in the neurobiology course, directed by Arthur Karlin, teaching the first molecular neurobiology techniques in the biochemistry section. This experience also introduced me to the unique community in Woods Hole, which prompted us to buy a cottage there and that now serves as our east-coast residence when we are not in Oregon.

My continued interest in invertebrate biology carried on in the background of mammalian immunology studies. Back in the lab, I had been working on an ambitious project aimed at identifying the T-cell receptor, using anti-idiotypic antibodies, but after three years of effort, I ultimately failed. The technology for identifying the T-cell receptor was just not there yet. Indeed, it was another nine years before cDNA cloning resolved the predicted primary structure of the T-cell receptor. By the summer of 1975, I was midstream into my second thesis project. I spent the final two years on this project, which involved electron spin resonance to measure membrane fluidity. The idea under test was whether changes in membrane fluidity in tumor cells affected their cytolysis by antibody plus complement or by killer T cells. We published a methods paper on the approach for altering membrane lipids and, in a related paper, I ruled out fluidity as an underlying mechanism for cytolysis. However, membrane fluidity did affect patching of H-2 surface antigens, suggesting involvement in other immune processes. Although not the outcome I had envisioned, the results were still a contribution in the early days when the roles for membrane fluidity were poorly understood. I published my findings in the Journal of Immunology (Mandel et al., 1978; Mandel & Clark, 1978), a top journal focused, like most others at the time, on just the quality of the science. My PhD experience was also teaching me an important lesson; science operates on the premise of testing, not proving, an idea.

I was learning this lesson while Paul and I were living on different coasts. Paul graduated with his PhD in 1975, just in time to be called into active duty into the Army Chemical Corps. He was located at Aberdeen Proving Grounds in Maryland and the assignment was for an indeterminate amount of time. I was left on my own to finish up my thesis work at UCLA. When the Vietnam War ended abruptly, Paul was given notice of release from active duty so he was free to look for a postdoc. Opportunities were scarce, even for those of us willing to work for little pay, just for the chance to continue a career in research. He wanted to stay at UCLA while I finished my thesis work. By chance, a spot opened up in Roger Eckert’s lab. My fellow graduate student and close friend, Laurinda (Rindy) Jaffe and I wasted no time in browbeating Roger into submission. In an inebriated state, Roger called Paul in Maryland and made him an offer. Roger made the right decision. In the three years in his lab, Paul would discover calcium-dependent inactivation of calcium channels, thereby completely changing the direction of the lab. By this time, we had acquired three rescued dogs along with our other animals, forcing us to move to the San Fernando Valley where we rented a house with a fenced yard.

As my thesis project drew to a close in 1977, I started to look for a postdoc position at UCLA because Paul was still with Eckert. I happened to run into Bill Wickner on the MBI stairway, and we started talking. At conversation’s end, I was to be his first postdoc. Once again, I had chosen wisely. Like my experience in the Clark lab, Bill was a hands-on mentor, a lab rat that is still doing research in his own lab. Bill came into the lab every day in a jolly mood singing the lyrics of some lame song he had in his head. “Jeremiah Was a Bullfrog” from Joy to the World by Three Dog Night was one of his favorites. Bill also had a gift for word play that often turned up in limerick form, something I had taken to well over the years. The Wickner lab was small, with Craig Zwizinski, Pam Silver, and a technician, Teresa Killick. Science-wise, a hot topic in the field was the emerging appreciation that proteins spanned membranes and not simply adhered to the bilayer formed by lipids. Bill is a bold scientist. Not only did I have an opportunity to learn biochemistry from first principles in his lab, just as important, Bill was a role model for applying these fundamentals to risky experiments with potential for new discovery. One question at the base of much controversy was the question of how transmembrane proteins, made up of charged amino acids, could insert into the lipid interior of the plasma membrane and be stable in the face of the hydrophobic environment. In the case of secreted and transmembrane proteins, answers were emerging from the Blobel lab at Rockefeller. Small noncharged sequences on secreted and membrane proteins, “signal peptides” would guide them during translation into the membrane and subsequently be cleaved, a discovery that earned Blobel the Nobel Prize. The labs of Wickner at UCLA and Masayori Inouye at Stony Brook had proposed different models. In the Wickner model, which I pursued using a viral coat protein that is synthesized as a soluble protein, the heart of the idea was that transmembrane proteins, if properly folded to hide charged residues and aided by the “leader sequence,” could self-assemble into a membrane. Bill named our model the “Membrane-Triggered Folding Hypothesis” (Wickner et al., 1978; Ito et al., 1979; Mandel & Wickner, 1979; Wickner et al., 1980). In the Inouye “loop model,” the idea was that the signal sequence could associate with the membrane in a way (looping in) that would allow self-assembly followed by an energetically favorable environment allowing the protein to traverse the membrane by itself during translation (Inouye & Halegoua, 1980). I loved being involved directly in the excitement of being part of a competitive, discovery-driven field.

As both Paul and I reflect on the years in graduate school at UCLA, we recognize that it was a period where we really could enjoy ourselves with little responsibilities while living on a shoestring budget. Graduate stipends were rare, and we relied on salary as teaching assistants. Both Paul and I were teaching assistants in a variety of high-enrollment courses that generally involved three full days of lab exercises each week. It paid very little but was enough to live on. There were no credit cards and everything was paid by check. We were terrible at money management and routinely bounced checks. That would be our signal that we were out of money. This was a lifestyle that would come back to haunt us when we applied for our first home mortgage years later. Credit scores were other things we knew nothing about. As for science, we both had experienced initial thesis projects that failed and had found new projects with the help of our thesis advisors. We were never discouraged by failure, nor did we focus on the uncertainties of the future, which were as prevalent then as they are today. We were just focused on learning as much as we could.

Science was our first priority, so we spent long hours in the lab, typically every day of the week. However, we did a have life-work balance with quality time spent outside the lab. We scheduled that time around our experiments. Paul and I would often backpack with friends deep into the California wilderness. We hiked Sequoia, Mammoth Lakes, Yosemite, Kennedy Meadows, and Kings Canyon, to name some of our favorites. We always brought along the dogs, and we would be somewhere in wilderness for a week or so. Those backpacks were heavy from food and gear, and there was no room for a tent, just a sleeping bag. Every trip was an adventure. Hiking back into Sawtooth Pass, at 12,000 feet in January was a risk, with no provisions for a possible snowstorm and no means of communication other than a mirror. I can’t imagine what we were thinking. We would sign a register declaring we were in the wilderness somewhere should we turn up missing. We would drink water along the way in streams and fill our canteens with little concern about water purity. Over the many years of backpacking, Paul was a notorious prankster. We would normally gather with our friends at our place to distribute weight among the backpacks. There were no freeze-dried foods so we would take jars of peanut butter, crackers, fruit, pasta, and tomato sauce, all of which were pretty heavy. When no one was looking, Paul would load the backpacks with additional heavy surprises, such as bricks and, once, an antique steam iron. Often, we would backpack with our close friends, Jane and Bill Koenig. Bill was a medical student and Janey was a talented fellow graduate student in the genetics program at UCLA. She would go on to a productive postdoc with Kazuo Ikeda at the City of Hope working with the now famous Drosophila mutant, Shibere. At the time, we had a 1972 Chevrolet Blazer with four-wheel drive. We used that truck for field trips to the Puerto Penasco Marine lab in Mexico and to tropical La Paz, located at the tip of Baja. The latter trip preceded the construction of a road, so it was very risky to make that thousand-mile trip, not knowing where to obtain gas and drinking water. Fortunately, we had a fellow lab mate of Paul with us, marine biologist Jon Kastendiek, who was conducting research there. We would follow the beach much of the way down, camping on the beach and snorkeling in the tropical waters.

In total, our years at UCLA were truly “golden,” as Alan Grinnell, faculty leader of the neurophysiology group, described this time period. Alan was one of the people who encouraged me to pursue a career in research and who also helped with my tennis game. Many a night was spent playing Ping-Pong in front of the Grinnell and Eckert labs, followed by free food from the medical school cafeteria. Alan was a very competitive Ping-Pong player and he was very proud of his bloopers that dribbled over the net for a point. We would call him on that, accusing him of cheating. He maintained an active research program split between synaptic competition and bat echolocation that took him all over the world. He still has probably the foremost private collection of pre-Columbian artifacts in the country, so truly a Renaissance scholar, always fun to talk to. Despite our diverse research interests, the graduate students during this period remained a large cohesive group. We would work late into the night, taking breaks for Ping-Pong and hall Frisbee. When one of us would make a breakthrough in our work, the students and postdocs would celebrate together with a bunny hop down the hall chanting “publishable results—YEAH!” Funny, those breakthroughs always seemed to happen late at night. Also, in those days, major revisions were not the norm, so we were forever optimistic of seeing our results in print.

I am a loyal Bruin. I am a member of the external scientific advisory board of the UCLA Broad Stem Cell center, directed for many years by the talented Owen Witte who hired and nurtured a terrific group of scientists. Over the years, the advisory board meetings have provided me the opportunity to hear presentations of interesting science, to reconnect with faculty, and to once again experience the hickory burger, fries, and banana cream pie from the Apple Pan in Los Angeles, which thankfully survived the pandemic.

UCSD, 1978–1981

In late 1978, my time at UCLA ended and I moved to the University of California, San Diego (UCSD) to join Paul, who had begun a second postdoc at the Salk Institute. We bought a house at 11822 Jonny Lane in Mira Mesa, a San Diego development built originally as naval housing. Thousands of homes that came in a few slightly different models dotted the landscape. We managed to score a special one, at the end of a cul-de-sac offering a panoramic view of a canyon, complete with coyotes, hawks, rattlesnakes, and tarantulas. This was a house we figured we could afford, which turned out not to be the case. Our misplaced belief stemmed from a one-time postdoctoral stipend from the Muscular Dystrophy Association to Paul that was more money than we had ever seen. We took all $16,000 and invested it in a money market that was making insane interest. Being the shrewd money managers that we were, we forgot that we had mortgage payments that amounted to more than my income. Realizing our mistake, we forfeited the money market and paid a huge penalty, but it really didn’t faze us. We had learned a lesson in money management and were happy with what we had.

For my choice of a second postdoc mentor, I had asked Harvey Herschman, one of my thesis committee members, to recommend someone whose research he admired and would complement my training interest. He suggested Melvin Simon. Mel was yet another timely choice. He was working in a new field of gene translocation, guided by his gifted senior postdoc, Mike Silverman, and star graduate student, Janine Zieg. I was lucky to join this group. I first worked with a postdoc in the lab, Alan Boyd, to continue studying integral membrane proteins, this time focused on proteins required for bacterial motility (Boyd et al., 1982). Alan is a Brit, and between experiments we competed in crossword puzzles. I contributed to a Science paper on the evolution of phase variation, a gene inversion event causing a loop to form in the DNA that regulates the type of flagellum expressed by Salmonella (Simon et al., 1980; Silverman et al., 1979, 1981). This project allowed me to do some of the first DNA sequencing chemistry. It is a tribute to Zieg, Silverman, and Simon, who were able to identify the sequence of the invertible loop of DNA, the precursor to modern day three-dimensional (3D) chromatin looping, with merely conventional genetics and Maxam and Gilbert sequencing (Zieg et al., 1977). Russell Doolittle at UCSD was also a coauthor on the Science paper. I did some chemical syntheses in his lab, although for the life of me I cannot remember why. Russ was a scholar and competitive athlete. He gave me a copy of his favorite book, The Decline and Fall of the Roman Empire, by Gibbon, which I still have today. Russ regularly ran in the Boston Marathon, and he invited Paul and me to his final marathon dinner in Boston. Over dinner, he disclosed that he was also a regular participant in the debate circle with creationists. He recounted his most recent debate with proponents of intelligent design, which in simple terms reflects antievolutionary thought. He said he had always enjoyed this sparring, but this time, he was bushwhacked by a hostile audience and an unannounced television filming. Unprepared, it became clear that this event greatly influenced his decision to leave the debate stage, which coincided with ending his marathon participation. Russ was a person of great intellect and presentation abilities, so for this to happen to him has been a constant reminder to me to be well prepared for every speaking opportunity (although Chuck Stevens, a renowned neuroscientist and great speaker, once told me that 90 percent was just showing up, helping to settle my nerves before a talk).

While at UCSD, I would reunite with Rindy Jaffe, who would continue to explore the voltage-dependent block to polyspermy, a fundamental discovery she had made as a graduate student. She still pursues this question, applying rapidly evolving optical approaches to creative preparations she develops. It was also a time we would meet another lifelong friend, Simon Halegoua. Paul was a postdoc in the newly formed Molecular Neurobiology group at the Salk Institute. He mentioned that he had befriended a fellow postdoc in the group, a quintessential New Yorker with a quick wit who was as irreverent as Paul. When he told me the name, I recognized it as an author of the loop hypothesis for membrane insertion. I was intrigued and anxious to compare notes, so Paul introduced us. We quickly became fast friends and shared the Jewish high holy holidays together, a tradition that remains. Paul’s experience in the Molecular Neurobiology group would also play an unexpected role in directing my future work on the voltage-dependent sodium channel. His decision to do a physiology postdoc with Yoshi Kidokoro in the Neurobiology group had placed him squarely in the middle of a group seeking to clone an ion channel. The group, led by Steve Heinemann and his research colleague, Marc Ballivet, was in hot pursuit to obtain the first primary sequence for a subunit of the nicotinic acetylcholine receptor. This effort was among the first to apply molecular cloning techniques to ion channels and the community of ion channel biophysicists waited anxiously in the wings. My training in DNA sequencing with Mel Simon would prove to be important preparation for my eventual entry into this field.

In 1981, Paul accepted a faculty job at Tufts Medical School in Boston to join Kathy Dunlap, a fellow colleague from the Eckert lab. He missed the electrifying atmosphere of the Salk but was excited to be with Kathy and have his own lab. As well as having no one to talk to about ion channel cloning, he was one of only two single-channel “patch clamp” physiologists in Boston, David Clapham being the other. As was our custom, I stayed behind a bit to finish up my second postdoc with Mel Simon. During this 3,000-mile separation, I lived with Rindy Jaffe and Carol Vandenberg in a small beach house, since washed away I think, where I could enjoy walking the beach and seeing beautiful sunsets before heading East. Rindy, Paul, and I were to form an enduring friendship. We still have lively discussions about science, and more recently, about interesting recipes. This friendship is easy to maintain because Rindy, and her husband Mark Terasaki, also an excellent cell biologist (and pianist), own a cottage a block away from ours in Woods Hole.

Harvard Medical School, 1981–1983

To join Paul in Boston, I accepted an instructor position in Tom Benjamin’s lab in the Pathology Department at Harvard Medical School. My motivation for applying to his lab was research on polyoma virus proteins, and I thought it would be a good entrée back into eukaryotic biology. I had intended on trying to figure out how middle T antigen inserted into the host membrane, returning to my previous interests, but somehow that never came to pass. Instead, I participated in biochemistry experiments related to phosphorylation of proteins, all new to me. Although this was not my most productive time publication-wise, I owe Tom a debt of gratitude for his support, and I was fortunate to meet and work with some excellent postdocs in his group—Gordon Carmichael, Brian Schaffhausen, and Kurt Ballmer-Hofer. Both Gordon and Brian have a terrific sense of humor and a very quick wit, increasing entertainment at the lab bench. Part of my distraction around this time in Tom’s lab was that Paul and I had our first child. Josh Mandel-Brehm was born in 1983. Once again, our lives were turned financially upside down. Seemingly incapable of any financial planning we had bought a house, 68 Adella Avenue in West Newton, another one we learned we could not afford. The interest rates were at 17.5 percent, a historical high, and our combined salaries were only slightly higher than the mortgage payments, forcing us to borrow more money. Our long hours in the lab were under siege with daycare adding to our commuting time and financial debt. I was feeling the pinch trying to balance science with my new life as a mother. However, a fortuitous career-changing event was about to happen.

Tufts New England Medical Center, 1983–1990

I had been thinking about projects that would bring me closer to the emerging field of neurobiology when, serendipitously, one evening, Paul spoke highly about a new faculty member at Tufts New England Medical Center who had presented a seminar on cloning neuropeptides in the Physiology Department. Neuropeptides would take me closer to my long-term interests as they played an essential, receptor-mediated role in the nervous system. The faculty member was Richard Goodman, an MD/PhD in the field of endocrinology. Dick trained with Joel Habener at Massachusetts General Hospital in Boston and was interested in cloning the cDNAs for somatostatin, vasoactive intestinal peptide, thyroid releasing hormone, and bombesin, among others. By chance, we ran into Dick and his wife, Eve, at a movie theater, and by the end of the discussion, Dick had graciously offered me a faculty appointment. I seized this wonderful scientific opportunity. It was a move that also would mitigate the time-consuming and stressful cross-town commuting between Harvard and Tufts. I joined a large group that included Marc Montminy, Andy Leiter, Malcolm Low, Ron Lechan, Philip Stork, Thom Segerson, Steve Fink, Kevin Sevarino, and Stephanie Lee.

The years between 1984 and 1989 with Dick represented an exciting time as the lab was bursting with projects (e.g., Montminy et al., 1986; Lechan et al., 1986; Leiter et al., 1987; Cooperman et al., 1987; Fink et al., 1988; Sevarino et al., 1989; Maue et al., 1990). Cloning of neuropeptides was so successful that the lab turned toward cloning neuropeptide receptors. I was anxious to steer efforts more toward cloning of ion channels. I had the skill-set and, influenced by Paul, it was clear that cloning ion channels was an exciting new field. The major driving force was led by biophysicists who had developed models for gating that made specific structural predictions that awaited testing. The 1984 Excitable Membranes Gordon Conference sealed the deal and pointed me toward the voltage-dependent sodium channel. Traditionally focused exclusively on ion channel biophysics, Chuck Stevens had carved out a big chunk of the presentations to promote ion channel cloning and his new molecular neurobiology group at Yale. At the meeting, rumors were circulating that Shosaku Numa in Japan had already resolved the primary sequence for a voltage-dependent sodium channel, but no details were available. Oocyte expression from synthetic RNA recently had been developed so it was conceivable he had functionally expressed the first voltage-dependent ion channel. This idea really lit a fire under competitors. The ion channel biophysicists were not pulling for anyone in particular; they just wanted ion channel clones for expression, mutagenesis, and testing structural models based on physiology. It had been 30 years since Hodgkin and Huxley showed the features of the sodium channel responsible for an action potential. Since that time, some of the greatest biophysicists had generated models that could account for the voltage dependence of activation, inactivation, and selective permeability. The problem with Numa winning the race was that he was not interested in sharing when it came to his clones. With few exceptions, Numa refused to distribute his clones, even after they were published. One exception was his willingness to share the clones for the nicotinic acetylcholine receptors with the Sakmann group in Germany. Another exception was sharing the clones encoding the dihydropyridine receptor with Kurt Beam at Colorado State University (I suspect Numa had lost interest when he realized that it was a nonconducting channel). Kurt applied his strengths in biophysics and cell biology to unravel the signaling pathway between muscle depolarization and release of intracellular calcium using the cDNA clones.

Still, the dihydropyridine receptor was not on the path to delineating the pore mechanisms in voltage-dependent ion channels. Thus, using techniques developed in tandem with Dick, I set out to clone the cDNA encoding the mammalian voltage-dependent sodium channel. I say “the” channel because we had no idea about the extent of sodium channel diversity at that time. The project was launched in our lab when Paul convinced Bob Barchi (University of Pennsylvania) to collaborate with us using his antibodies to screen a lambda gt10 muscle expression library for the sodium channel alpha subunit peptide. In the midst of our muscle cloning effort, the rumors were confirmed with Numa publishing the primary sequence of the eel sodium channel (Noda et al., 1984). Once again, however, the eel sodium channel turned out to be nonfunctional when expressed in heterologous cells, which remains a mystery today. We altered our strategy in light of Numa’s paper and decided to use the eel cDNA to cross species to clone the mammalian sodium channel cDNA. This experiment carried the basic assumption that mammalian and fish sodium channels were sufficiently homologous, so it remained a bit risky. Dick and I purchased an electric eel from Florida and had it flown into Boston Logan Airport. It arrived the day before Thanksgiving. We picked up the rather large box at the airport and brought it with us on the subway during rush hour. We had no problem finding a seat because the box was stamped officially in large letters, DANGER, HIGH VOLTAGE. We commissioned a marine biologist at the Boston Aquarium to help us extract the electric organ, a modified electroplax muscle. He showed up in heavy rubber boots and gloves as we gathered around in curiosity. The extraction was successful and we made a cDNA library from the eel electric organ RNA. We used this reagent to identify a large sodium channel clone from our mammalian brain cDNA library. We surmised, correctly, that Numa was already well on the way to cloning mammalian sodium channels using the same strategy as ours. Once again, Numa won the race, publishing the full-length sequence of three rat brain sodium channel isoforms. This time the channels were functional when expressed in Xenopus oocytes. There was, however, some pause for concern as there was abnormally slow inactivation compared with sodium currents recorded in vivo, a feature also seen when expressed in mammalian cell lines. I return to this curiosity later.

In addition to Numa, Dick and I were competing with several other formidable labs in the United States, including the Lester-Davidson team at Cal Tech, which also succeeded in functional expression of a full-length brain-type sodium channel (Auld et al., 1988). Catterall, Pongs, Yuh Nung, and Lily Jan also were involved in cloning ion channel cDNAs in brain. Dick and I decided to change strategy again and return to cloning the skeletal muscle sodium channel. However, there was concern that we would end up recloning the same sodium channels identified in brain, so we performed the first sodium channel molecular RNAse protection analyses to test whether the rat brain sodium channels also were expressed in muscle. They were not (Cooperman et al., 1987)! Therefore, with Jim Trimmer and Bill Agnew at Yale, we closed in on cloning and expressing the first cDNA encoding a non-neuronal voltage-dependent sodium channel (Trimmer et al., 1989). One of the most important and unexpected revelations from our work was the discovery that the muscle sodium channel sequence was quite divergent from the brain sequences. Distinctions in tetrodotoxin (TTX) sensitivity of sodium channels were known, but this was not enough information to have drawn conclusions about protein structure. One very important contribution of our cloning effort was the first full-length cDNA clone for a voltage-dependent ion channel that would be freely available, for the first time, to all interested scientists, without stipulations of collaboration. The sharing of such an important reagent began to reap benefits for the biophysicists and provide new mechanistic insights into congenital diseases that had been entirely unexplored. One of the first studies revealed that mutations in this channel (SkM1) turned out to be the basis of hyperkalemic periodic paralysis (Cannon et al., 1995). The biophysical community would soon learn that structural diversity within the sodium channel gene family was even greater than we had imagined. At last count, there are 10 distinct mammalian sodium channel subtypes in different excitable tissues, many expressed in tissue-specific fashion. I was to investigate the tissue-specificity more intensively later.

This period of ion channel gene discovery was an exciting time in my career. In addition to mammalian cloning efforts, there was a collaborative project with Larry Salkoff at Washington University. Around 1985, Paul ran into Larry at a Federation of American Societies for Experimental Biology (FASEB) meeting, and Larry was very anxious to apply ion channel cloning to his research. Larry had been the first to identify Shaker as a potassium channel defect and clearly understood the potential of cloning the cDNA. Paul directed him to me and working together we obtained the first genomic sequence for a fly sodium channel (Salkoff et al., 1987). In another important project, Paul’s lab and mine were working together to look at sodium channel induction in a neuronal rat model, pheochromocytoma (PC12) cells. This cell line was used to investigate the signaling pathways by which nerve growth factor (NGF) led to a neuronal-like phenotype, including the ability to generate a TTX-sensitive action potential. Our objective was to be the first to link expression of a specific sodium channel isoform with native sodium current using a combination of patch clamp of the sodium current and molecular biology. Beyond identifying this link, our results would provide a direct comparison of functional properties of native and heterologously expressed sodium channels. Using RNA protection analyses, combined with whole-cell patch clamp analysis, we assigned induction of excitability, after long-term NGF treatment, to induction of the brain type II sodium channel (Nav1.2) (Mandel et al., 1988). Strikingly, the functional properties of this sodium current were markedly dissimilar to that reported by oocyte expression studies on brain type II alpha subunit. Moreover, the fast inactivation observed for PC12 sodium current resembled that of traditional in vivo inactivation widely reported among different species and tissue types, which was distinct from the slow inactivation of Nav1.2 sodium channel alpha subunits expressed in the oocytes. This result put to rest the idea that the slowly inactivating sodium channel currents observed in oocytes from expressed RNA reflected function in certain native neurons, previously overlooked by electrophysiologists. Instead, the results supported the idea of the existence of an auxiliary subunit that was present in native tissue, such as the sodium channel beta subunits identified biochemically by the Catterall and Barchi labs. This proposition was later tested and proven through coexpression with a beta subunit newly cloned by others.

Paul and I submitted the PC12 sodium channel findings at the same time that Larry and I submitted the Drosophila sodium channel findings, both as reports to Science. Both papers were accepted for publication to appear in 1987, and we were told that they would likely appear in the same issue. We were excited to also learn that the Drosophila channel paper had been upgraded to an article. However, shortly thereafter, Paul received a call from an editor at Science (when journals still relied on the telephone for both good and bad news) saying that they decided to unaccept the PC12 paper. The editor told him that sodium channel papers, in particular, were no longer a priority and “were being sent to the circular file.” Paul challenged him, pointing out that he had just upgraded a sodium channel paper from a report to an article. The editor asked how he knew about this, and Paul told him to check the authorship on the two papers. Clearly, there was more going on than the editor wanted to share. I think this was the instance that led to our referring to manuscripts as “under rejection” as opposed to “under review” when our work was submitted to one of the high-profile journals. In the end, the article with Salkoff was published in the same 1987 issue of Science as the sequence of the fly shaker K channel from the Jan labs. This was not the end, however, for the odyssey of the PC12 paper. Paul asked one of the leading ion channel biophysicists, Susumu Hagiwara, our UCLA colleague, to communicate the manuscript to Proceedings of the National Academy of Sciences (PNAS). He reviewed it and had some suggestions that were easily incorporated. Hagi called back, embarrassed after checking with PNAS, to say that he unwittingly had already given away his last direct communication slot. Hagi convinced a fellow NAS colleague at UCLA, George Bartholomew, a physiological ecologist, to communicate the paper (Mandel et al., 1988). After the work was published, Hagi confessed that he was in hot water with PNAS for transferring the paper. This was an impetus, among others, for NAS to adopt a policy for members that restricted direct communications of papers to people who are experts in the field covered by the paper.

Another ion channel cloning project that originally involved the joint Brehm/Mandel/Goodman labs was cloning cDNA that encoded the rat epsilon subunit of the pentameric muscle acetylcholine receptor (AChR). This project was initiated shortly after Numa had identified bovine epsilon as a new AChR subunit. Epsilon was to add another member to the already four subunits (alpha, beta, delta, and gamma) known to form the AChR. The burning question became whether developmental changes in AChR conductance and gating seen in mammals and frogs could be accounted for by epsilon. The prevailing view was that the changes were due to post-translational phosphorylation of the receptor. Paul’s findings with Yoshi, however, found that the changes required transcription and were consistent with the kinetics of receptor turnover and replacement. We pushed hard to obtain a rat epsilon cDNA for expression for testing, as Numa had not yet published functional properties of receptors containing the new subunit. Once again, however, by sharing his clone with the Sakmann lab, we would lose the race. Oocyte expression by this group showed that substitution of the gamma subunit by epsilon recapitulated the developmental changes in conductance and kinetics. Still, this set into motion our efforts to obtain the entire complement of Xenopus subunits for investigating receptor function during synapse development using the advantages offered by spinal neuron/muscle cocultures. This long-term molecular/physiology collaboration between Paul and me on AChRs would last decades, transitioning later to zebrafish.

On the home front, our daughter, Caleigh Mandel-Brehm, had been born in 1985. Now, with two children in tow, and Paul spending each summer with Kathy Dunlap at the MBL, we were, once again, stretched thin. It was worth it, however, as the MBL project was remarkably productive and fascinating, involving GFP and bioluminescence. Kathy and Paul were following up on Paul’s first failed thesis project, which sought to identify the source of calcium for bioluminescence using GFP as a cell marker. Using the newly developed patch clamp technology, Kathy and Paul would be the first to discover a role played by gap junctions in chemical signaling between cells. Along with Stephen Smith from Stanford and a talented Chilean biophysicist David Naranjo, they were also the first to use bioluminescence to image calcium changes in vivo. This led to the discovery of intercellular propagating calcium waves, a new kind of cellular communication and a new role for gap junctions. Given the excitement of these discoveries, there was every reason to push forward with the summer separation.

Our lives would be simplified by an opportunity for me at the MBL. Arthur Karlin was the new director of the MBL neurobiology course and he invited me to join as an instructor in 1987. Summers were much more relaxed and enjoyable with the whole family at Woods Hole. As one of only a handful of ion channel molecular biologists, I was responsible for introducing molecular neurobiology to the course. Each summer we would craft a molecular biology project for the students. As one example, we set out to clone the sodium channel cDNA from the rough-skinned salamander Taricha, a species that produced TTX. The idea was to see whether the sodium channels in the salamander were resistant to the TTX, as first reported for puffer fish (Kidokoro et al., 1974). If true, was the TTX resistance due to an amino acid alteration in the primary structure? It was productive research-wise for me as well, as Rindy Jaffe and I collaborated on a paper that appeared in Science, showing that cortical contraction in Xenopus oocytes could be induced by Ach following injection of RNA coding for the M1 muscarinic acetylcholine receptor (Kline et al., 1988).

Back at Tufts, our commute was steadily taking its toll and impacted negatively on my time in the lab. We tried every combination of car and rapid transit, but there was nothing rapid about the rapid transit in Boston, which included the MBTA, the bus, and the light rail. We eventually turned to driving around downtown trying to find a parking spot. We would trade off days during the week for going home while the other stayed late. Evenings involved two dinners, an early one for the kids and a much later one for us. We flipped for the weekends, with the winner going to the lab for a few hours of uninterrupted thinking and benchwork. Finally, after years of applying for a Tufts parking spot, I was approved. Still, when it came time to pick up the kids from daycare one of us would drive as in “It’s a Mad-Mad-Mad-Mad World,” trying to avoid the embarrassment of being the last parent to pick up their child. To this day, I see parents rushing to their cars at the end of the day in what I imagine is a fear of being late to daycare. In the final two years of Boston, we made just enough to hire a daytime nanny who would come to our house. Finally, we could take advantage of every minute in the lab and still have quality time with our kids.

In 1988, I was about to enter the most exciting time in my career, where all prior experience would come together to place me in a unique position. My background and interests were not in structure-function of ion channels or biophysics, and I was not going to follow a path of mutagenesis and functional studies. Instead, my interests were in achieving a basic understanding how the neuronal phenotype was acquired, focusing on the genetic components of the sodium channel gene that led to its neuronal-specific expression. Because the sodium channel is central to commerce throughout the nervous system, I reasoned that if I could understand what regulated sodium channel gene expression, it would lead me to more general mechanisms underlying formation of the nervous system. Indeed, the sodium channel work was going to lead me directly to the discovery of a fascinating transcription factor, the RE1 Silencing Transcription Factor (REST). The project had its roots in a set of experiments we did to isolate and clone an upstream regulatory region for the sodium channel gene, containing the promoter. We showed that this relatively small genomic fragment of about 1,200 base pairs programmed expression of a bacterial reporter gene in the PC12 neuronal cell model. Together with Bob Maue, Susan Kraner, and Dick Goodman, we designed simple deletional analyses of this genomic region in both PC12 and muscle cell lines. Unexpectedly, deleting a sodium channel gene regulatory sequence resulted in robust ectopic expression in the muscle cells with little change in the PC12 neurons. The simplest interpretation of this result was that the regulatory region was acting as a repressor sequence in the muscle cells, and not as an activator in the neuronal PC12 cells (Maue et al., 1990). This discovery flew in the face of current dogma, holding that positively acting transcription factors were responsible for conferring tissue-specific expression. At the same time, David Anderson’s group at Cal Tech published a similar story line for the neuronal SCG10 gene (Mori et al., 1990). The SCG10 gene encoded a neuronal-specific phospho-protein of unknown function, which David had identified in a screen for neuronal genes as a postdoc with Richard Axel. David and I were to do this two-step tango for the next several years as we each followed up on our initial discoveries.

Dick’s and my scientific interests continued to move in different directions. His principal interests remained centered on cloning neuropeptide receptors, whereas mine were on ion channel regulation. Still, we worked together closely and toward the end of my time at Tufts, we recruited Paul to help with the neuropeptide receptor cloning, as he was routinely using oocyte expression. Several of the endocrine fellows (Tom Segerson, Eliot Spindel, and Kevin Sevarino) wanted to use RNA expression in oocytes to clone the neuropeptide receptors. Paul instructed all of them in oocyte expression and two microelectrode recordings, capitalizing on the common pathways involving intracellular calcium release used by these neuropeptides. The oscillating membrane currents would provide a faithful readout of expression and working through pools of RNA candidates ultimately would lead to identification of the single desired cDNA. The receptors included those corresponding to many of the previously cloned neuropeptides

When 1989 rolled around, I was showing signs of fatigue, reflecting the mounting demands on both scientific and personal time. Paul and I realized we could not continue making what amounted to entry-level salaries with the high cost of living in Boston. A visit by Jim Patrick, of the Salk Molecular Neurobiology group, would open our eyes to other opportunities. Jim knew Paul as a postdoc in that group and Jim was establishing the Department of Neuroscience at Baylor College of Medicine. He wanted us both to join and help guide development and Baylor rolled out the red carpet. Their offer stretched well beyond our imagination but, although greatly tempted, we ultimately turned down this opportunity in favor of Stony Brook University in New York. Our decision was driven by several factors that centered on a wonderful New York public school system for Josh and Caleigh and its seamless connection to high-quality daycare, minutes from campus. Another plus was the proximity to Woods Hole, making continued summer research at the MBL feasible and convenient. What clinched our decision, however, was our close friend and colleague, Simon Halegoua, who was working with the new chair of Neurobiology and Behavior at Stony Brook, Lorne Mendell, to recruit a person who could bring molecular neurobiology to the department. I was excited to work with Simon to develop molecular neurobiology at a place already strong in cellular neurophysiology. As for Tufts, it was about to lose its core molecular neurobiology contingency in 1989, as Dick took Segerson, Low, Stork, and Roger Cone with him when he assumed the directorship of the Vollum Institute in Portland, Oregon. As he headed west, I would head further east with postdoc Susan Kraner, to Long Island.

Stony Brook, 1990–2007

With our move to Stony Brook University, we could finally afford a house that provided for all our needs. We bought a contemporary house at 7 Emmet Way that was located on two wooded acres. This would be the house that the kids would grow up in and the one that they would turn into the ultimate party house, much to our chagrin. The public school, only minutes away, offered great after school daycare, which included help with homework, social fun time, and putting on plays until 5 p.m. We were able to better balance family life with life in the lab. My lab was located next door to Simon Halegoua’s on the fifth floor of the Life Sciences Building, and Paul was in the basement with fellow physiologist Gary Matthews. As per our tradition, Paul moved first and I sent Susan Kraner to set up the lab. Unlike Tufts, there was a graduate program and both Paul and I each accepted our first graduate students after 10 years of being faculty. Drew Chong was my first student and was exceptionally talented. He would play a major role in shaping the future direction of the lab.

In the summer of 1991, I was an instructor in the Neurobiology course at the Stanford University Hopkins Marine Station. Fellow instructors were my friends Bill Gilly and Rock Levinson. Both of them were highly entertaining and made for a great summer back on the west coast. Rock would later play a major part in my studies on sodium channel expression by providing years of experience with immunohistochemistry and antibody production. This course took on particular personal importance for two reasons. First, Hurricane Bob struck Long Island while I was away. Paul had no electricity or water for days, with our two young kids and our elderly dog, Shadow, who sadly died during the storm. Evidently, Paul tried for days to contact me, but there were no cell phones then and we were used to going long stretches without communication. I never heard the end of this one. Second, I made strong connections with two exuberant students in the course: Juan Jose Toledo-Aral, (Juanjo) from Seville, Spain, and Maurice Kernan, a fly geneticist with an interest in mechanoreceptors. Both ended up at Stony Brook, Juanjo as my postdoc, and Maurice as a faculty colleague. Juanjo would join me to explore the mechanisms underlying sodium channel expression in PC12 cells. This would set the stage for a three-way collaboration among Paul, Simon, and myself. My group at that time was brimming with talent and, with spirited Juanjo, afterwork hours seemed like one big party at our house. Juanjo would organize the rest of the lab to spend many an evening at our house where he made authentic Sangria cocktails and taught us to dance the Merengue. My technician, David Kennedy, was a cartoonist and immortalized many of our after-hours parties as shown in the following illustration. Another talented postdoc, Yasushi Okamura, would also join in the fun both inside and outside the lab. Before joining my lab, Yasushi had been an MD/PhD student in the lab of an exceptionally creative scientist, Kuni Takahashi, at Tokyo Medical School. Yasushi led a project on proto-chordates in my lab and identified a contact-mediated induction mechanism of sodium channel expression at the gastrula stage (Okamura et al., 1994). In his independent lab, Yasushi later discovered the voltage sensitive phosphatase, which has revealed new functions for voltage sensors. With the core of Kraner, Chong, Okamura, and Juanjo, I was well positioned to take on new research challenges.

Thus, 1992 was a critical year for my group. Together, with Susan Kraner, Drew Chong, and Huey-Jen Tsay in the lab, we published the second part of the REST story (Kraner et al., 1992). Here, we identified Repressor Element 1 (RE1) as a 23 base pair sequence in the sodium channel promoter region. We showed that this sequence recruited a protein-binding complex only in terminally differentiated non-neuronal cell types such as fibroblasts. Furthermore, we devised a “repressor trap” by transfecting many copies of the RE1 sequence into cells, thereby preventing the complex from binding to the RE1. This trap resulted in derepression of the promoter in non-neuronal cells. The same year, in the same issue of Neuron, David’s group published a similar sequence finding for SCG10 (Mori et al., 1992). And away we go.

The next step was obvious to both of our groups. We set out to identify the repressor protein that bound to the RE1/NRSE sequences. I recall being very nervous because David’s group seemed like it was also moving into sodium channel regulation, and I was at a disadvantage having just established my lab at Stony Brook. Moreover, our son and daughter were getting to ages where they needed more time with us. We seemed to be failing at establishing the simple structured family life that was expected. No sit-down dinners together, no restaurants or movies, and no traditional family vacations. The NY schools piled on the homework with the expectation that the parents would have to play an integral part, competing with one another to help with take-home science projects, dioramas, and writing projects. It seemed as if a project was always in play, so the table saw was churning away and there was a chest of art supplies. Fast-drying plaster was a lifesaver. It was getting harder to trade off with each other for weekend days in the lab as both kids became social butterflies and freely offered our house for the frequent sleepovers that invariably turned into all night parties. Then there was soccer, starting with modest competition and ascending to travel teams, select teams, and eventually school teams. I would take one and Paul would take the other to the games and tournaments. Mix in school plays, clubs, orchestra, wrestling, baseball, lacrosse, and basketball, and we had no free time left. We entertained all of these activities through grade school, junior high, and high school. Paul finally had too much with commuting between Stony Brook and Woods Hole. He ended his very productive stint with Kathy Dunlap at the MBL in 1993 and turned in their National Science Foundation grant. We needed a reprieve and found it in overnight Camp Half Moon for kids. Located in the Berkshires, the camp would take both Caleigh and Josh for eight consecutive weeks. Thankfully, cell phones had not been invented, and they were allowed only to write letters that, not surprisingly, didn’t come that often. Both children went to this camp for the next six summers, providing them with new friends and adventures and freeing us to once again to live like postdocs.

As 1995 rolled around, I learned of an impending Science paper from the Anderson lab announcing the identification of neural restrictive silencing factor (NRSF), which mediated repression from the NRSE site. We were close to identification of the binding factor ourselves, using a one-hybrid screening approach, and to lose at this point was unimaginable. Encouraged by Simon, I phoned Ben Lewin, then editor at Cell, and he said he would reserve a place in the next issue if we could show function with a full-length cDNA for REST. The entire lab pitched in for an intense stretch of 24/7 experiments. Susan Kraner had already left for a faculty position in Kentucky, but we drew her back in to run a critical experiment to show biochemical evidence for the binding of REST to the RE1 sequence in vitro. Late one night Drew Chong phoned me at home and said, “Gail, it worked”! The full-length REST cDNA in the PC12 cells repressed the endogenous sodium channel gene promoter present on an accompanying mini-gene. We had REST in time for publication in Cell. In the end, both my lab and David’s lab prevailed (Chong et al., 1995; Schoenherr & Anderson, 1995). Together, we had discovered the basis for neural specificity, and it was due not to activation but to repression. If you wanted to be anything but a neuron, you needed to suppress neuronal genes with REST. I figured we had won in the name game with REST but, in fact, it now turns out to be difficult to use REST as a key word in a literature search. Google REST and you come up with any word that contains the letters r e s t. On the flip side, REST has provided an endless source of humorous seminar titles like “Despite Jet Lag, Stress and Grant Deadlines, Adults Get Plenty of REST” (thanks to Gary Westbrook); “The Rise and Fall of REST: Creating the Nervous System”; “Repression in the Nervous System; How to Quiet Your Nerves”; and “Advice to Neurons, Lose the REST.”

It was during this same period that Simon, Paul, and I began our three-way collaboration on NGF-induced sodium channel excitability in PC12 cells. Simon had a long history with oncogenic signaling underlying growth-factor actions, beginning from his postdoc at the Salk Institute. He was an expert with signaling pathways in PC12 cells, while I had the molecular expertise with sodium channels, and Paul had the electrophysiological skills. We knew from previous molecular analysis that the Nav1.2 sodium channel was highly inducible by NGF in PC12 cells (Mandel et al., 1988). However, quite unexpectedly, Simon and I identified a second sodium channel type that was also NGF-induced. As this channel type was expressed specifically in peripheral neurons, we named it peripheral nerve sodium channel type 1, PN1 (D’Arcangelo, 1993), later changed to Nav1.7. At the time of publication in 1993, we were able to pin down the induction of Nav1.2 to a cAMP-dependent pathway. Juanjo Toledo Aral joined in on the hunt for NGF mechanisms that would account for PN1 induction and scored an immediate hit. He found that a brief exposure to NGF, as short as one minute, resulted in long-lasting induction of PN1 expression (Toledo-Aral, et al., 1995). The physiology would go on to show that the sodium current induction was as high as that from long-term NGF action, but short-term exposure was selective to PN1. It worked through an immediate-early pathway that converged with an interferon-gamma signaling pathway. Simon, Juanjo, and I enlisted Rock Levinson to perform immunohistochemistry with a pan neuronal sodium channel antibody. Consistent with our initial predictions of this being a peripheral-type sodium channel, we found it was present in a subset of neurons in cultured dorsal root ganglia (DRG) neurons. Based on the distribution, we proposed that it had a specific function in nociceptors, rendering it central to pain sensation (Toledo-Aral et al., 1997). This idea was largely dismissed based on its TTX sensitivity, but when families were identified subsequently as unable to sense pain, molecular analysis assigned the pathophysiology to PN1. It was gratifying that our idea was eventually validated in humans.

In 1997, Max Cowan, then vice president of the Howard Hughes Medical Institute (HHMI), announced that I was selected as an HHMI investigator. Simon and I had already relocated to the basement of the Life Sciences building to form a new Center, joining Gary Matthews and Paul. We designed newly renovated labs from classrooms and organized the labs like a giant maze with common core facilities. There were many interconnected rooms, with people going in and out of different labs all day, and I have to say this arrangement and the personalities represented the most interactive group of scientists I have encountered in my career. Jim Trimmer, my collaborator and friend from the original sodium channel cloning days, also joined us in the basement Center from the biochemistry department. Jim built a big lab that was churning out antibodies for ion channels and dissecting the targeting elements for neurons. In addition to our individual grants, I was directing a program project from the NIH awarded to our group. I was fortunate to have Karen Warren as my HHMI administrative assistant. She would also keep all five of our labs on the rails as well. Her equally talented mother, Pat Leavy, would later join the group as my Society for Neuroscience (SfN) editorial assistant while I was on the editorial board. In short, the years in the basement would be special for all of us, but when offered luxurious labs in a brand-new building at Stony Brook, we moved. Unfortunately, it would never be the same. We were split between just floors 3 and 5, but it was enough to have a negative impact on our group interactions. Jim Trimmer would leave for a new position at UC Davis, and our NIH program project eventually terminated because of my desire to shed administrative duties. Publications continued, but we would never feel the excitement that was generated by the basement group.

The boost in funding provided by the HHMI appointment allowed me to expand the lab. New members adapted rapidly, evolving molecular approaches toward understanding how REST functioned as a repressor, as these were the early days of studying repressor complexes. We were able to make important contributions to our general understanding as well as to the understanding of nervous system development. For example, our findings pointed to REST recruitment to the chromatin of two distinct corepressors, one of which was completely novel. Postdoc Julia Grimes showed that the amino terminal repressor domain of REST recruited the already identified general corepressor, Sin3, to neuronal gene chromatin (Grimes et al., 2000). Three other postdocs in the lab, Elena Battaglioli, Maria Estella Andres, and Pepe Tapia-Ramirez, identified a corepressor that mediated repression from the carboxyl terminal REST repressor domain (Tapia-Ramirez et al., 1997; Andres et al., 1999). That corepressor we named CoREST, and it turns out to be one of the major corepressors in cells of all types, even in the blood lineage, as one of my later graduate students Huilan Yao went on to show (Yao et al., 2014). Further interrogation of CoREST function directly linked REST repression to the chromatin modifier, histone deacetylase, thereby indicating a prime mechanism for its corepressor function (Ballas et al., 2001; You et al., 2001). A problem that had us stumped for a while involved the evolution of the REST repression mechanism. We turned to the fly model with the idea that we could make faster progress using the genetic advantages offered by this system. We were to learn that while CoREST is present from yeast to humans, REST evolved with the vertebrates. Based on her fly work (Dallman et al., 2004), my postdoc, Julia Dallman, showed that a tram-track protein, with no homology to REST, could still serve the same repressor function in neurodevelopment by coopting CoREST. This idea, of nonhomologous gene replacement for REST in invertebrates, has since been supported by studies in other laboratories.

I was also fortunate to recruit Nurit Ballas to my group. Nurit had published beautiful molecular work on plants, and she and I initially thought it would be fun to look at repressor function in plants. Ultimately, however, Nurit switched to mammalian molecular biology in the nervous system. This was an excellent decision on her part. Exploiting her earlier biochemistry skills, Nurit demonstrated, using mouse embryonic stem cells, that REST repressed neuronal genes not just in non-neuronal fibroblasts, but also in pluripotent stem cells and dividing neural progenitors. Using this ideal cell culture system, she went on to become the first person to illuminate how the REST repressor complex controlled chromatin status during the different stages of neuronal differentiation, including the differentiation to mature neurons (Ballas et al., 2005; Ballas & Mandel, 2005). For example, Nurit found that REST had to depart neuronal gene chromatin in neural progenitors before terminal neuronal differentiation, and that when REST left the chromatin, its loss caused the disengagement of the entire repressor complex, specifically primary corepressors, CoREST and histone deacetylase 2 (Ballas et al., 2001; Ballas & Mandel, 2005; Ballas et al., 2005). Furthermore, persistent expression of REST during early development delayed neuronal differentiation in vivo (Mandel et al., 2011). Taken together, these studies provided a prototype for the epigenetic function of repressors in the developing central nervous system. I still have students and postdocs new to the lab refer to these early papers to measure REST function in the context of differentiating cells. Nurit and I also initiated studies of the cellular basis of the neurological disease Rett syndrome, caused by sporadic mutations in the gene encoding the transcriptional repressor, Methyl CpG binding protein 2 (MeCP2). We were the first to provide experimental support for the idea that glia, as well as neurons, were affected in this neurodevelopmental disorder (Ballas et al., 2009). This idea was met with some skepticism initially, but after my move to Vollum, we strengthened the proposal, in collaboration with Nurit, through in vivo studies (Lioy et al., 2011). Nurit continues elegant studies elucidating the role of human astrocytes in Rett syndrome in her independent lab at Stony Brook.

In addition to revealing more about repressor function, REST was poised to make another major contribution. In a chance meeting with John Dunn (Brookhaven National Laboratory in Long Island) around 2003, I became aware of his recently developed Genome Signature Tag method for profiling genomic DNA (Dunn et al., 2002). John was a very creative scientist who openly shared his ideas with those around him. He had used this technique for genomic profiling of the Yersinia pestis genome, and I asked him about adapting his method for identifying REST binding sites genome-wide. John agreed it was a reasonable idea, and we agreed to work together on the experimental design and analysis. We recruited a talented bioinformatics colleague of John’s at Brookhaven, Sean McCorkle, who had worked on the Yersinia project. We invited my previous collaborator, Dick Goodman, to participate in the project, surmising that it would be beneficial to use two mammalian transcription factors with completely different binding site motifs. Although now separated by 3,000 miles, we worked together again, with Dick working on CREB at the Vollum with his group, and my group working on REST at Stony Brook, with open communication among us. Graduate student Stephanie Otto led the effort in our group. Our collective efforts were successful (Impey et al., 2004; Otto et al., 2007). For us, the results represented a major contribution toward understanding REST function. We showed, unequivocally, that REST occupied, and presumably repressed, thousands of neuronal genes. For the first time, we were seeing the entire scope of REST-dependent regulation. We proposed that “neural-ness” was actually a default pathway in non-neuronal cells, which was prevented by the REST repressor mechanism. Remarkably, our REST paper was published the same year that the Wold group at Cal Tech used REST as the proof of concept for high-throughput chromatin immunoprecipitation technology (ChIP seq; Johnson et al., 2007). Although our two groups used different species and different non-neuronal cell types for the genome-wide studies, and different approaches for the binding analyses, both studies converged on the same REST binding site motifs on thousands of neuronal genes. Years earlier, David Anderson and I had predicted that vast numbers of neuronal genes would share the REST binding site, simply using our eyes to pick out the REST binding sites in the neuronal sodium channel and SCG10 genes, so it was a rewarding denouement to our first findings.

Simon Halegoua was not the only colleague at Stony Brook who heavily influenced my research. Joe Fetcho was an engine driving both Paul and me to collaborate on zebrafish physiology and genetics. Joe had abandoned goldfish, turning to zebrafish as a model for circuitry analyses. Around 1997, he invited Michael Granato for a seminar. Granato introduced his cool motility mutants that he was using to identify central circuits involved in swimming. Paul and I thought that it was more likely that the phenotypes instead reflected defects in the final motoneuron pathway. Indeed, our intuition was correct and the mutants would pave the way to identifying key players in synaptic transmission and human congenital myasthenic syndrome. The work would involve synaptic physiology and zebrafish behavioral analysis performed in the Brehm lab and molecular analysis in my lab. One of the first mutants we explored was intriguing. The twitch once line was unable to mount swimming due to fatigue setting in after the first or second tail bend. Paul’s postdoc, Fumi Ono, found that the acetylcholine receptors were present in the muscle membrane but in the wrong location, being distributed and not clustered at the synapse. Rapsyn was a protein known to be involved in AchR clustering and, by sheer coincidence, a postdoc in my lab, Shinichi Higashijima, had created a rapsyn-GFP transgenic zebrafish line in Japan before coming to my lab. On a hunch, Fumi crossed the rapsyn-GFP transgenic line with twitch once. When in-crossed, all of the fluorescent fish were able to mount normal swimming and the receptor clustering phenotype was rescued. We had our first bona fide hit without having to do positional cloning, which was tedious at that time. There was more to come. Fumi noticed that the fatigue associated with the mutant phenotype was similar to a very common phenotype in humans afflicted with a variety of myasthenic syndromes. When he administered cholinesterase inhibitors used to treat humans, he observed greatly improved swimming in the mutant line. On this basis, he proposed that mutations in rapsyn might represent a currently unidentified form of myasthenic syndrome. Our announcement at the 2001 SfN meeting spawned the discovery of human rapsyn deficiency (Ono et al., 2001; Ono et al., 2002). The stage was now set for our collaborative exploration into zebrafish motility mutants as a platform for identifying and studying myasthenic syndromes in humans.

My lab members who participated in the Mandel/Brehm collaboration included Julia Dallman, Michelle Gleason, and Becky Mongeon. Through combined patch clamp, cloning, and imaging, our group assigned functional defects to mutations that represent human myasthenic syndromes. A mutation in muscle calcium ATPase in the accordion line informed us about contractile defects in Brody disease (Gleason et al., 2004). A mutation in the pore of the AchR in the twister line provided key insights as to pharmacologic treatment of slow channel syndrome (Walogorsky et al., 2012), as did the bajan line, a mutation in choline acetyltransferase responsible for episodic apnea (Wang et al., 2008). Other important studies involved mutations of the glial glycine transporter (Shocked line; Mongeon et al., 2008) and Eaton Lambert syndrome, a mutation in the P/Q calcium channel (tb204a line; Wen et al., 2013). In addition to resolving these neuromuscular phenotypes, we made other fundamental findings together. Led by Hua Wen in Paul’s lab, we were the first to identify synaptotagmin 7 as the calcium sensor mediating asynchronous transmitter release (Wen et al., 2010). There were also rewarding collaborations with Joe Fetcho’s group on zebrafish that were among the first to track activity in spinal circuits using in vivo imaging (Gleason et al., 2003; Higashijima et al., 2003; Armisen et al., 2007). My collaborations with Paul and Joe on zebrafish have been one of the truly fun epochs in my career, persisting over a 20-year span.

Meanwhile, back at home, our kids were growing up and becoming competitive New Yorkers, much to the trepidation of a Midwest father and West Coast mother. Pressure was on in every aspect of their lives, from sports to parties to academics and to the uncertainties about which college would admit them. That last concern was driven more by the parents than the kids as competition spread through Long Island like wildfire. Both kids were captains of their school soccer teams, and Josh was on a select travel team as well. Caleigh was traveling to Europe to compete. Josh was also captain of his wrestling team, and Caleigh was captain of the charter female golf team at her school. We managed to squeeze in a bit of cultural heritage in the form of Hebrew school. They never complained, but they clearly had a healthy skepticism of many teachings, like why miracles only happened in Biblical times and whether these people really existed. Speaking frankly, the kids stuck it out for one reason: the Bar and Bat Mitzvah parties following graduation from Hebrew School. On Long Island, these parties take on mammoth proportions, rivaling weddings. When Josh held his party at the Stony Brook Yacht club, the drapes mysteriously caught on fire and was on the fringe of becoming out of control. The Commodore informed us that for the first time in their 100-year history, the Yacht Club would not be returning a security deposit. Not to be outdone, Caleigh held her Bat Mitzvah at the historical Stony Brook Three Village Inn and restaurant, forcing closure because of noise and smoke cannons that the DJ was firing into the crowd.

All in all, we had managed to make career choices that would balance opportunities for the kids, while still providing a good opportunity for each of us to pursue research. Of course, the kids continued to get into trouble in college, but at least they were a thousand miles away and for the most part it was “good trouble.” Caleigh protested for human rights of the homeless in the unforgiving winters of Madison, Wisconsin, and Josh formed the charitable Linus Foundation with members of his fraternity. They devised an idea that involved raucous fundraising parties as a means to provide sporting equipment for poor intercity kids. They both made it through; Caleigh with a degree in zoology from the University of Wisconsin and Josh with a degree in neuroscience from Washington University. Caleigh took advantage of a wonderful opportunity to be a technician in the lab of Huda Zoghbi at Baylor College of Medicine. This would be her gateway into graduate school at Harvard Neurobiology, under the mentorship of friend and colleague, Mike Greenberg. Josh accepted a position at Hydra Bioscience in Boston, founded by David Clapham. Although Josh started off doing patch clamp screening for drug compounds, he decided he liked the business end better and was off to the University of Michigan to earn an MBA. The kids were set for the time being and we could, for the first time in many years, think more selfishly, opening the last chapter in our careers.

Vollum Institute, 2006

In what Dick Goodman refers to as his seventeen-year recruitment, Paul and I returned to the west coast to join the faculty at the Vollum Institute in Portland, Oregon. The Department of Neurobiology and Behavior at Stony Brook had become very senior and our leaving would provide them with the opportunity to recruit new junior faculty. Our star colleague and close friend, Joe Fetcho, who introduced us to zebrafish, had already left for Cornell University in Ithaca, so our lunch party was down to Simon, Paul, and me. Our decision meant we would be leaving Simon, our close friend and scientific colleague, but the west coast was calling. I had been eager to return to the west coast, but finding jobs for two very senior faculty, neither of whom was interested in being a departmental chair, had greatly narrowed the possibilities.

After being hired at the Vollum, we bought a charming house in Portland that had been part of the Lewis and Clark Centennial Exposition in 1905. The house was moved by mule from the original site of the Exposition along the Willamette River to its final resting place at 1717 NW 33rd Avenue. We found the small city to be very lively, blending rural and urban lifestyles, good music, and good food. Still missing the northeast, however, we eventually purchased and renovated a 1907 house at 10 Orchard Street, in the heart of Woods Hole, very close to the MBL. We needed sanctuary from Portlanders and their obsession to be unique, which is tantamount to their doing the opposite of whatever New York and California were doing. Oregon had dramatic (but not warm) beaches, mountains, and wineries to explore, and our wonderful dog, Blue, benefited from the lack of leash laws at the beach. She loved chasing birds and playing in the waves. All things considered, we have enjoyed Portland and doing research in the Vollum.

I was able to move my HHMI lab to the Vollum, which certainly helped in the transition. There was space available for Paul and me to have labs across from each other, as we had enjoyed at Stony Brook. Also, as in Stony Brook, when students and postdocs in my lab needed imaging or physiology technology, they migrated into Paul’s lab, and when Paul’s group needed molecular biology, they had dedicated benches in my lab. Thus, we continued to be able to collaborate on projects, for example, using zebrafish, but we also continued to pursue our individual research interests. Happily, Hua Wen, Paul’s extremely talented long-term research professor and the mainstay of zebrafish physiology and molecular biology in his lab, decided to move with us to the Vollum. Even with a skeleton crew, the move was very good for me as well. I was elected into the American Academy of Arts and Sciences and, with Dick’s support, also was elected into the National Academy of Sciences. With the move to the Vollum, I took the opportunity to move my research into new projects and technologies, which are still unfolding.

Our original discovery of REST opened up areas beyond what we had imagined in 1995. Because of its regulation of thousands of neuronal genes, REST became a widely studied transcriptional repressor in the central nervous system (CNS), both during neurodevelopment and in the adult. We continued our work on REST during early neurogenesis in mice and found that REST not only repressed expression of neuronal genes until cell cycle exit, as predicted, but also protected the genome from DNA damage during the cell cycle (Nechiporuk et al., 2016). Premature deletion of REST in mice resulted in DNA breaks during the cell cycle that led ultimately to depletion of the progenitor pool and microcephaly in the adult. This result added more support, from a different perspective, to previous work by others of a link between REST and neurogenesis related to human microcephaly (Yang et al., 2012). We also identified sequences in REST that regulated its stability, with consequences on axonal integrity in peripheral neurons, as well as a signaling pathway that was important in transcriptional regulation at REST-regulated promoters (Cargnin et al. 2014; Nesti et al., 2014).

Most recently, in an unexpected turn of events, we found that by studying mice exclusively we had missed an important function in REST that was specific to primates. REST levels in mouse brain decrease during aging, while in human brain they begin to increase during aging. This difference coincides with a completely distinct set of target genes in human hippocampus that represent genes encoding inflammatory and immune pathways rather than the stereotypical “synaptic” genes (McGann et al., 2021). It had been reported that REST regulates human longevity and that REST levels are lower in Alzheimer’s disease. Our findings support the idea of a new role for REST in aging, and the results have opened the doors to new functions for REST in protecting human brain health. Finally, we have identified newly evolved motifs in the primate REST protein, absent in lower mammals, which we hypothesize play important roles in the expansion of the numbers of neural progenitors in the cortex in primates. In addition to providing a model for basic mechanisms of transcriptional repression, REST dys-regulation recently has been implicated in studies by other investigators in diverse abnormal CNS pathologies, including epilepsy, cancer and hearing loss.

We continue to focus on gene repression in our studies of MeCP2, and its function in different CNS cell types. Because MECP2 is on the X chromosome, females are mosaic for loss of MeCP2 function due to dosage compensation in mammals. A clever physiology study, led by graduate student Benjamin Rakela, comentored by both Paul and me, exploited a female Rett mouse model generated in Adrian Bird’s lab (University of Edinburgh). In this model, wild-type cells in the brain can be distinguished by their GFP fluorescence from cells lacking MeCP2 expression due to mutations. In cortical slice recordings using this Rett mouse model, and by testing every combination of wild-type and MeCP2 mutant astrocytes and neurons, Benjamin found a perturbation in synaptic communication that was manifest as decreased astrocyte: neuronal excitatory signaling (Rakela et al., 2018). This central role played by astrocytes confirmed a proposal we had made earlier using cre-lox technology and the astrocyte effects on mouse Rett-like behaviors (Lioy et al., 2011). We also pursued high-resolution microscopy experiments, stimulated by the creativity of Stephen Smith. We adapted his technique of array tomography, created specifically to investigate synapses, to instead investigate architectural changes in chromatin. One of the advantages of this technique, which uses extremely thin sections, is that cells within the same tissue section are treated identically for direct side-by-side direct comparisons. Furthermore, multiple rounds of immuno-labeling can be performed. A research assistant professor in the lab, Mike Linhoff, exploited this technology, again using the female Rett mouse model in which only the wild-type neurons are GFP labeled. Mike found robust changes in the 3D-chromatin structure in the mutant MeCP2-deficient neurons in the hippocampus of these female mice. The chromatin in mutant neurons was more compact, which would be predicted to inhibit gene activation (Linhoff et al., 2015). We propose that the chromatin structural changes might make the mutant neurons less able to adapt normally to stimuli within a circuit. While this proposal awaits confirmation, independent studies by others have confirmed the chromatin structural changes that we first reported.

Because no single gene target has been identified to explain Rett syndrome, in our most recent endeavor, we have bridged basic science and translational medicine by designing experiments to repair MeCP2 mutations at the level of RNA, using a targeted RNA editing approach (Sinnamon et al., 2017; Sinnamon et al., 2020). Using this approach, John Sinnamon, a research assistant professor, led a team to show the first efficient reversal of a Rett patient mutation in nondividing cells (neurons) in culture, as well as the first effective editing of the same patient mutation across different hippocampal neuronal types in mice (Sinnamon et al., 2020). Studies of the consequences of Rett patient mutation reversal by targeted RNA editing, on Rett-like behaviors in mice, are ongoing and promising.

Reflections on My Career

I did not know from the beginning what I wanted to do as a profession. I cannot say I always wanted to peer into a microscope, although I did have an innate curiosity about how things worked. My future direction decision came late in college, fueled by the scientists around me. From them I was to learn what it was to make a discovery, a revelation that changes the way we think about a biological process or problem. Most of science is spent trying to piece together a puzzle, but those of you who have been the very first to solve a puzzle know the thrill. To the many extraordinary colleagues who helped guide and inspire me to set the bar high, I owe my heart-felt gratitude. I have always enjoyed being in the lab, sharing pivotal moments in a project with my students and postdocs. Let discovery serve as their ambition. In closing, I have also been lucky to be part of a wonderful and interesting family, especially our two children, who are in science in one way or the other; their spouses; and our lively and inquisitive grandchildren, all of whom are the primary reason for writing this autobiography.

Selected Bibliography

Andres ME, Burger C, Peral-Rubio MJ, Battaglioli E, Anderson ME, Grimes J, Dallman J, Ballas N, Mandel G. (1999) CoREST: a functional corepressor required for regulation of neural-specific gene expression. Proceedings of the National Academy of Sciences of the United States of America 96(17): 9873–8.

Armisen R, Gleason MR, Fetcho JR, Mandel G. (2007) Tracking tagged molecules in single neurons in intact zebrafish. CSH Protocols 2007: pdb prot4780.

Auld VJ, Goldin AL, Krafte DS, Marshall J, Dunn JM, Catterall WA, Lester HA, Davidson N, Dunn RJ. (1988) A rat brain Na+ channel alpha subunit with novel gating properties. Neuron 1(6): 449–61.

Ballas N, Battaglioli E, Atouf F, Andres ME, Chenoweth J, Anderson ME, Burger C, Moniwa M, Davie JR, Bowers WJ, Federoff HJ, Rose DW, Rosenfeld MG, Brehm P, Mandel G. (2001) Regulation of neuronal traits by a novel transcriptional complex. Neuron 31(3): 353–65.

Ballas N, Grunseich C, Lu DD, Speh JC, Mandel G. (2005) REST and its corepressors mediate plasticity of neuronal gene chromatin throughout neurogenesis. Cell 121(4): 645–57.

Ballas N, Lioy DT, Grunseich C, Mandel G. (2009) Non-cell autonomous influence of MeCP2-deficient glia on neuronal dendritic morphology. Nature Neuroscience 12(3): 311–7.

Ballas N, Mandel G. (2005) The many faces of REST oversee epigenetic programming of neuronal genes. Current Opinion in Neurobiology 15(5): 500–6.

Boyd A, Mandel G, Simon MI. (1982) Integral membrane proteins required for bacterial motility and chemotaxis. Symposia of the Society for Experimental Biology 35: 123–37.

Cannon SC, Hayward LJ, Beech J, Brown RH, Jr. (1995) Sodium channel inactivation is impaired in equine hyperkalemic periodic paralysis. Journal of Neurophysiology 73(5): 1892–9.

Cargnin F, Nechiporuk T, Mullendorff K, Stumpo DJ, Blackshear PJ, Ballas N, Mandel G. (2014) An RNA binding protein promotes axonal integrity in peripheral neurons by destabilizing REST. Journal of Neuroscience 34(50): 16650–61.

Chong JA, Tapia-Ramirez J, Kim S, Toledo-Aral JJ, Zheng Y, Boutros MC, Altshuller YM, Frohman MA, Kraner SD, Mandel G. (1995) REST: a mammalian silencer protein that restricts sodium channel gene expression to neurons. Cell 80(6): 949–57.

Cooperman SS, Grubman SA, Barchi RL, Goodman RH, Mandel G. (1987) Modulation of sodium-channel mRNA levels in rat skeletal muscle. Proceedings of the National Academy of Sciences of the United States of America 84(23): 8721–5.

D’Arcangelo G, Paradiso K, Shepherd D, Brehm P, Halegoua S, Mandel G. (1993) Neuronal growth factor regulation of two different sodium channel types through distinct signal transduction pathways. Journal of Cell Biology 122(4): 915–21.

Dallman JE, Allopenna J, Bassett A, Travers A, Mandel G. (2004) A conserved role but different partners for the transcriptional corepressor CoREST in fly and mammalian nervous system formation. Journal of Neuroscience 24(32): 7186–93.

Dunn JJ, McCorkle SR, Praissman LA, Hind G, Van Der Lelie D, Bahou WF, Gnatenko DV, Krause MK. (2002) Genomic signature tags (GSTs): a system for profiling genomic DNA. Genome Research 12(11): 1756–65.

Fink JS, Verhave M, Kasper S, Tsukada T, Mandel G, Goodman RH. (1988) The CGTCA sequence motif is essential for biological activity of the vasoactive intestinal peptide gene cAMP-regulated enhancer. Proceedings of the National Academy of Sciences of the United States of America 85(18): 6662–6.

Gleason MR, Armisen R, Verdecia MA, Sirotkin H, Brehm P, Mandel G. (2004) A mutation in serca underlies motility dysfunction in accordion zebrafish. Developmental Biology 276(2): 441–51.

Gleason MR, Higashijima S, Dallman J, Liu K, Mandel G, Fetcho JR. (2003) Translocation of CaM kinase II to synaptic sites in vivo. Nature Neuroscience 6(3): 217–8.

Grimes JA, Nielsen SJ, Battaglioli E, Miska EA, Speh JC, Berry DL, Atouf F, Holdener BC, Mandel G, Kouzarides T. (2000) The co-repressor mSin3A is a functional component of the REST-CoREST repressor complex. Journal of Biological Chemistry 275(13): 9461–7.

Higashijima S, Masino MA, Mandel G, Fetcho JR. (2003) Imaging neuronal activity during zebrafish behavior with a genetically encoded calcium indicator. Journal of Neurophysiology 90(6): 3986–97.

Impey S, McCorkle SR, Cha-Molstad H, Dwyer JM, Yochum GS, Boss JM, McWeeney S, Dunn JJ, Mandel G, Goodman RH. (2004) Defining the CREB regulon: a genome-wide analysis of transcription factor regulatory regions. Cell 119(7): 1041–5.

Inouye M, Halegoua S. (1980) Secretion and membrane localization of proteins in Escherichia coli. CRC Critical Review Biochemistry 7(4): 339–71.

Ito K, Mandel G, Wickner W. (1979) Soluble precursor of an integral membrane protein: synthesis of procoat protein in Escherichia coli infected with bacteriophage M13. Proceedings of the National Academy of Sciences of the United States of America 76(3): 1199–203.

Johnson DS, Mortazavi A, Myers RM, Wold B. (2007) Genome-wide mapping of in vivo protein-DNA interactions. Science 316(5830): 1497–502.

Kidokoro Y, Grinnell AD, Eaton DC. (1974) Tetrodotoxin sensitivity of muscle action potentials in pufferfishes and related fishes. Journal of Comparative Physiology 89:59–72.

Kline D, Simoncini L, Mandel G, Maue RA, Kado RT, Jaffe LA. (1988) Fertilization events induced by neurotransmitters after injection of mRNA in Xenopus eggs. Science 241(4864): 464–7.

Kraner SD, Chong JA, Tsay HJ, Mandel G. (1992) Silencing the type II sodium channel gene: a model for neural-specific gene regulation. Neuron 9(1): 37–44.

Lechan RM, Wu P, Jackson IM, Wolf H, Cooperman S, Mandel G, Goodman RH. (1986) Thyrotropin-releasing hormone precursor: characterization in rat brain. Science 231(4734): 159–61.

Leiter AB, Toder A, Wolfe HJ, Taylor IL, Cooperman S, Mandel G, Goodman RH. (1987) Peptide YY. Structure of the precursor and expression in exocrine pancreas. Journal of Biological Chemistry 262(27): 12984–8.

Linhoff MW, Garg SK, Mandel G. (2015) A high-resolution imaging approach to investigate chromatin architecture in complex tissues. Cell 163(1): 246–55.

Lioy DT, Garg SK, Monaghan CE, Raber J, Foust KD, Kaspar BK, Hirrlinger PG, Kirchhoff F, Bissonnette JM, Ballas N, Mandel G. (2011) A role for glia in the progression of Rett’s syndrome. Nature 475(7357): 497–500.

Mandel G, Clark W. (1978) Functional properties of EL-4 tumor cells with lipid-altered membranes. Journal of Immunology 120(5): 1637–43.

Mandel G, Cooperman SS, Maue RA, Goodman RH, Brehm P. (1988) Selective induction of brain type II Na+ channels by nerve growth factor. Proceedings of the National Academy of Sciences of the United States of America 85(3): 924–8.

Mandel G, Fiondella CG, Covey MV, Lu DD, Loturco JJ, Ballas N. (2011) Repressor element 1 silencing transcription factor (REST) controls radial migration and temporal neuronal specification during neocortical development. Proceedings of the National Academy of Sciences of the United States of America 108(40): 16789–94.

Mandel G, Shimizu S, Gill R, Clark W. (1978) Alteration of the fatty acid composition of membrane phospholipids in mouse lymphoid cells. Journal of Immunology 120(5): 1631–6.

Mandel G, Wickner W. (1979) Translational and post-translational cleavage of M13 procoat protein: extracts of both the cytoplasmic and outer membranes of Escherichia coli contain leader peptidase activity. Proceedings of the National Academy of Sciences of the United States of America 76(1): 236–40.

Maue RA, Kraner SD, Goodman RH, Mandel G. (1990) Neuron-specific expression of the rat brain type II sodium channel gene is directed by upstream regulatory elements. Neuron 4(2): 223–31.

McGann JC, Spinner MA, Garg SK, Mullendorff KA, Woltjer RL, Mandel G. (2021) The genome-wide binding profile for human RE1 silencing transcription factor unveils a unique genetic circuitry in hippocampus. Journal of Neuroscience 41(31): 6582–95.

Mongeon R, Gleason MR, Masino MA, Fetcho JR, Mandel G, Brehm P, Dallman JE. (2008) Synaptic homeostasis in a zebrafish glial glycine transporter mutant. Journal of Neurophysiology 100(4): 1716–23.

Montminy MR, Sevarino KA, Wagner JA, Mandel G, Goodman RH. (1986) Identification of a cyclic-AMP-responsive element within the rat somatostatin gene. Proceedings of the National Academy of Sciences of the United States of America 83(18): 6682–6.

Mori N, Schoenherr C, Vandenbergh DJ, Anderson DJ. (1992) A common silencer element in the SCG10 and type II Na+ channel genes binds a factor present in nonneuronal cells but not in neuronal cells. Neuron 9(1): 45–54.

Mori N, Stein R, Sigmund O, Anderson DJ. (1990) A cell type-preferred silencer element that controls the neural-specific expression of the SCG10 gene. Neuron 4(4): 583–94.

Morin JG, Hastings JW. (1971) Energy transfer in a bioluminescent system. Journal of Cellular Physiology 77(3): 313–8.

Nechiporuk T, McGann J, Mullendorff K, Hsieh J, Wurst W, Floss T, Mandel G. (2016) The REST remodeling complex protects genomic integrity during embryonic neurogenesis. eLife 5: e09584.

Nesti E, Corson GM, McCleskey M, Oyer JA, Mandel G. (2014) C-terminal domain small phosphatase 1 and MAP kinase reciprocally control REST stability and neuronal differentiation. Proceedings of the National Academy of Sciences of the United States of America 111(37): E3929–36.

Noda M, Shimizu S, Tanabe T, Takai T, Kayano T, Ikeda T, Takahashi H, Nakayama H, Kanaoka Y, Minamino N, et al. (1984) Primary structure of Electrophorus electricus sodium channel deduced from cDNA sequence. Nature 312(5990): 121–7.

Okamura Y, Ono F, Okagaki R, Chong JA, Mandel G. (1994) Neural expression of a sodium channel gene requires cell-specific interactions. Neuron 13(4): 937–48.

Ono F, Higashijima S, Shcherbatko A, Mandel G, Brehm P. (2001) A mutation in zebrafish rapsyn disrupts ACh receptor clustering and promotes synaptic depression. Society for Neuroscience Abstract 27:918.1.

Ono F, Shcherbatko A, Higashijima S, Mandel G, Brehm P. (2002) The zebrafish motility mutant twitch once reveals new roles for rapsyn in synaptic function. Journal of Neuroscience 22(15): 6491–98.

Otto SJ, McCorkle SR, Hover J, Conaco C, Han JJ, Impey S, Yochum GS, Dunn JJ, Goodman RH, Mandel G. (2007) A new binding motif for the transcriptional repressor REST uncovers large gene networks devoted to neuronal functions. Journal of Neuroscience 27(25): 6729–39.

Rakela B, Brehm P, Mandel G. (2018) Astrocytic modulation of excitatory synaptic signaling in a mouse model of Rett syndrome. eLife 7: e31629.

Salkoff L, Butler A, Wei A, Scavarda N, Giffen K, Ifune C, Goodman R, Mandel G. (1987) Genomic organization and deduced amino acid sequence of a putative sodium channel gene in Drosophila. Science 237(4816): 744–9.

Schoenherr CJ, Anderson DJ. (1995) The neuron-restrictive silencer factor (NRSF): a coordinate repressor of multiple neuron-specific genes. Science 267(5202): 1360–3.

Sevarino KA, Stork P, Ventimiglia R, Mandel G, Goodman RH. (1989) Amino-terminal sequences of prosomatostatin direct intracellular targeting but not processing specificity. Cell 57(1): 11–9.

Silverman M, Zieg J, Hilmen M, Simon M. (1979) Phase variation in Salmonella: genetic analysis of a recombinational switch. Proceedings of the National Academy of Sciences of the United States of America 76(1): 391–5.

Silverman M, Zieg J, Mandel G, Simon M. (1981) Analysis of the functional components of the phase variation system. Cold Spring Harbor Symposia on Quantitative Biology 45(Pt 1): 17–26.

Simon M, Zieg J, Silverman M, Mandel G, Doolittle R. (1980) Phase variation: evolution of a controlling element. Science 209(4463): 1370–4.

Sinnamon JR, Kim SY, Corson GM, Song Z, Nakai H, Adelman JP, Mandel G. (2017) Site-directed RNA repair of endogenous Mecp2 RNA in neurons. Proceedings of the National Academy of Sciences of the United States of America 114(44): E9395–E402.

Sinnamon JR, Kim SY, Fisk JR, Song Z, Nakai H, Jeng S, McWeeney SK, Mandel G. (2020) In vivo repair of a protein underlying a neurological disorder by programmable RNA editing. Cell Repair 32(2): 107878.

Tapia-Ramirez J, Eggen BJ, Peral-Rubio MJ, Toledo-Aral JJ, Mandel G (1997) A single zinc finger motif in the silencing factor REST represses the neural-specific type II sodium channel promoter. Proceedings of the National Academy of Sciences of the United States of America 94(18): 1177–82.

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Kelsey Catherine Martin

BORN:

Seattle, Washington

June 3, 1958

EDUCATION:

Harvard University, AB (1979)

Yale University, PhD (1991)

Yale University, MD (1992)

Columbia University, Postdoctoral (1999)

APPOINTMENTS:

Assistant Professor, Department of Psychiatry and Biobehavioral Sciences, Department of Biological Chemistry, UCLA (1999–2006)

Associate Professor, Department of Psychiatry and Biobehavioral Sciences, Department of Biological Chemistry, UCLA (2006–2010)

Professor, Department of Psychiatry and Biobehavioral Sciences, Department of Biological Chemistry, UCLA (2010–present)

Chair, Department of Biological Chemistry, UCLA (2010–2015)

Interim Dean, David Geffen School of Medicine at UCLA (2015–2016)

Dean, David Geffen School of Medicine at UCLA (2016–2021)

Director, Simons Foundation Autism Research Institute (SFARI) and Neuroscience Collaborations, Simons Foundation, New York, NY (2021–present)

HONORS AND AWARDS (SELECTED):

Jordi Foch-Pi Award, American Society of Neurochemistry (2001)

Daniel X. Freedman Award, National Alliance for Research on Schizophrenia and Depression (NARSAD) (2001)

Brain Research Institute, UCLA, H. W. Magoun Award (2011)

UCLA Society for Postdoctoral Fellows, Postdoctoral Mentorship Award (2012)

Society for Neuroscience Presidential Lecture (2014)

Senior Fellow, Janelia Research Campus, HHMI (2015–2020)

Election to the American Academy of Arts and Sciences (2016)

Election to the National Academy of Medicine (2016)

Wilber Lucius Cross Medal, Yale University School of Arts and Sciences (2018)

Eric R. Kandel Award, Columbia University (2019)

Alpha Omega Alpha Medical Honors Society (2020)

Kelsey C. Martin’s research focuses on the cell and molecular biology of long-term synaptic plasticity and memory, asking specifically how experience alters gene expression within neurons to change neuronal connectivity in the brain. She demonstrated that long-lasting, transcription-dependent plasticity can occur at specific synapses made by a single neuron and went on to elucidate a role for mRNA localization and local translation during synapse-specific forms of plasticity. Together, her studies have revealed the importance of both transcriptional and post-transcriptional mechanisms of gene regulation during long-term plasticity and memory. Her laboratory identified pathways and molecules that travel from stimulated synapses to the nucleus to impact transcription, and mechanisms and molecules that regulate the localization, stability and translation of mRNAs at synapses during synapse formation and long-term synaptic plasticity. In addition to her research, she has been dedicated to advancing biomedical research and academic medicine, including serving as dean of the David Geffen School of Medicine at UCLA and as Director of the Simons Foundation Autism Research Initiative and Neuroscience Collaborations.

Kelsey Catherine Martin

I am writing this autobiographical essay as I move from UCLA and Los Angeles to the Simons Foundation in New York—a ripe time for self-reflection. I’ve spent my scientific career studying how experience changes brain connectivity to store long-term memories, and as I pack up my family’s home of 22 years to start a new chapter, I find myself flooded by the meaning and power of those memories, memories that in many ways feel more lasting than any physical object or material reality. At the same time, as I navigate a new city, job, home, and colleagues, I am profoundly aware of how much the external environment affects my neural circuits. I have a palpable sense that my brain is undergoing new programs of gene expression and forming new synaptic connections.

Childhood

I was born in Seattle, Washington, the second of four children and the only girl in the family. My father, George M. Martin, is a physician-scientist who has spent his career studying the biology of aging. Although he retired from the University of Washington at age 75, he remains active and NIH-funded to this day, at age 94, often joking that his goal is to “die funded.” My dad is and has always been entirely consumed by science and his research—it’s what brings a sparkle to his eyes. He grew up in Queens, the son of a New York City police officer who changed his name from Cohen to Martin because of the anti-Semitism he faced on the police force. My dad joined the Navy at age 17, and when World War II ended, he spent a year at Cooper Union College in New York before moving across the country to work on the railroad and play trumpet in a jazz band in Alaska. He moved to Seattle to finish his premedical classes before enrolling in the medical school class of 1952 at the University of Washington.

My dad met my mother, Julaine Martin, during one of his pre–medical school chemistry courses. As my mom described it, she was charmed by his near-sightedness and his high-water trousers. My mom was the oldest of three, born in Nebraska before her family lost their farm in the Dust Bowl and moved to rural Washington. She won a full scholarship to Mount Holyoke College, but her family could not afford her trip across the country; they also did not support the idea of women going so far away to attend college. She became a laboratory technician to ensure employment, but her true love was always art, and especially folk art. My parents had a very traditional marriage, and my mom stopped working once my brother was born to become a full-time extraordinary homemaker. When I was in high school, my mom went back to get a master’s degree in art history at the University of Washington and then found incredible joy managing several art galleries in Seattle.

My older brother Peter was born two years before me; my younger brother Tommy just a year after me, and my youngest brother Andrew was born when I was six years old. My mom loved to travel and made sure that my dad took every sabbatical year he could. When I was three, we spent a year in Glasgow, Scotland, and when I was ten my family spent a year in Paris, France. True believers in the value of international experiences, my parents sent me back to Paris to spend a year living with a French family when I was 14 years old, and then, when I was 16 years old, they sent me to live with a family in Mexico City. While going to a French-speaking school as a ten-year-old was scary, I have incredibly sweet memories of traveling with my family throughout Europe. My mom took us to visit museums in Paris every weekend, where I have a vivid memory of standing, awestruck, in front of Monet’s Water Lilies in the Orangerie museum. My dad, a true pathologist, would stop at every cemetery we drove by in our Volvo station wagon and have us read all the tombstones to determine if an epidemic had struck the town. In these travels, I learned to speak French and Spanish fluently, which I am sure was made possible by immersion before what Steven Pinker and colleagues have described as the closure of a critical period for second language acquisition (Hartshorne et al., 2018). I can’t help but think this helped me see the world through different lenses growing up.

In addition to our travels, my parents made our Seattle home an international environment, constantly hosting students from around the world. When I was 11, Anper, a 13-year-old boy from Truk Atoll, now called Chuck Lagoon, lived with us for a year while his adoption with another family was being finalized. I remember picking Anper up at SeaTac airport, where he was terrified to get on the escalator—a contraption he had never seen or heard of. When Anper arrived at our house, he removed all the houseplants and put them in our yard, having a very different sense of “indoor” and “outdoor.” Within days, our neighbors complained because Anper was killing birds with a slingshot, with remarkable precision. He got kicked out of our local public middle school because he was constantly getting in fights with other boys, and always winning those fights. I’ll add that he picked these fights because he thought that anyone with skin darker than his was “evil,” and so he targeted dark-skinned black students—an interesting lesson about racism for me as an 11-year-old.

Given all of my family’s travels, I never attended the same school for more than two or three years. I also ended up skipping grades, graduating from high school at age 16. I attended public schools in Seattle, and, as a teenager, fell head over heels in love with literature and especially with F. Scott Fitzgerald. To this day, reading The Great Gatsby, and its last line, “So we beat on, boats against the current, borne back ceaselessly into the past,” fills me with a sense of wonder. My love for Fitzgerald made me intent on attending his alma mater, Princeton. I applied early and was rejected. My public high school teacher told me, and my parents, that I was aiming way too high. I’d received a “D” in French III (with no acknowledgment that the “D” was earned my first semester as a 14-year-old in an 11th-grade class in one of the top public schools in Paris). While I was crushed at the time, this was also a great lesson for me about the arbitrariness of some institutional decisions. I graduated early from high school, in December, and flew to Mexico to work in the lab of one of my dad’s former postdoctoral fellows and to take courses at the Universidad Autónoma de Mexico.

College

I returned from Mexico to start as a freshman, in the Harvard-Radcliffe class of 1979. I lived in Weld Hall in Harvard Square, with four roommates, and, not knowing what to expect, was the last to show up and thus ended up with the bottom bunk of the one double room in the suite. This was the first of many examples of being a somewhat “clueless” Harvard undergrad. I had never heard of Advanced Placement, which was not available at my high school, had never heard of Andover or Philips Exeter, and did not understand the fashion of tartan kilt skirts.

I was not particularly interested in science when I started college. Despite having grown up with a father who so deeply loved science, I found science to be somewhat soulless and heartless. I have strong childhood memories of lying in bed, telling my dad that I was afraid of death, and especially of my parents’ deaths. To comfort me, he gave me a genetics lecture, excited to tell me how he and my mom would live on in my genes and in the genes of my children. I now find this to be deeply comforting, having children of my own. At the time, however, it was too abstract to satisfy any of my younger existential fears and feelings and added to my feelings that science failed to answer the questions I was most curious about.

Nonetheless, I started college thinking I might go to medical school, mostly out of an interest in global health. This was a short-lived plan. I found myself turned off by the competitive culture I encountered in the premedical science courses I took. In contrast, I loved reading and painting, was captivated by my literature courses, and loved spending time in the painting studio I was assigned in the Carpenter Center for the Arts. In my sophomore year, I chose, very happily, to major in English and American literature and language and to take as many courses as I could in visual and environmental studies.

One of the most memorable courses I took was a poetry seminar on Emily Dickinson, Robert Frost, and Wallace Stevens. Three decades later, when my daughter was a junior in high school, her English teacher told me that he loved teaching poetry because it made his students slow down to read and think. I continue to feel that time stands still when I read words that so perfectly capture complex states, feeling, and experiences—from Emily Stevenson’s “Hope is the thing with feathers - / That perches in the soul” to Wallace Steven’s “…For the listener, who listens in the snow, / And, nothing himself, beholds / Nothing that is not there and the nothing that is” (The Snow Man).

I wrote my senior honors thesis comparing Thomas Carlyle’s 1836 British novel Sartor Resartus to Herman Melville’s 1851 American novel Moby Dick. Although seemingly unrelated narratives, I saw them as two distinct responses to understanding “truth” at a time when the certainty of theism, the belief in God as the divine creator and ruler of the universe, was increasingly questioned by European and American philosophers and scientists. While Carlyle responded to this doubt by proclaiming that meaning and truth could only be found in manmade fashions and institutions (Sartor Resartus meaning “The Tailor Retailored”), Melville viewed the pursuit of meaning and truth—the white whale—through more open, porous fishing nets. In exalting the importance of clothing as a container for meaning, the protagonist of Sartor Resartus, Professor Teufelsdröckh, proclaims, “Matter exists only spiritually, and to represent some Idea, and body it forth. Hence Clothes, as despicable as we think them, are so unspeakably significant. Clothes, from the King’s mantle downwards, are emblematic, not of want only, but of a manifold cunning Victory over Want” (Carlyle, Sartor Resartus, p. 56). In contrast, Ishmael, the protagonist of Moby Dick, in considering the white whale, asks, “Is it that by its indefiniteness it shadows forth the heartless voids and immensities of the universe, and thus stabs us from behind with the thought of annihilation, when beholding the white depths of the milky way?” (Moby Dick, p. 136). Decades later, I can’t help but think of Carlyle and Melville as scientists—one who likes to tell a coherent, simple, hypothesis-confirming story, and the other “released from the shackles of hypothesis-driven science” (Lehmann, 2018).

Peace Corps

I graduated from Harvard in 1979. At the time I was living with my then-boyfriend, Mark Barad, in a communal home in Allston, Massachusetts. We spent many dinners with our roommates discussing the ways in which western imperialism contributed to injustices around the globe. After six months of a series of temporary jobs, Mark and I decided we wanted to join the Peace Corps to fight western imperialism. Not wanting to teach English, I capitalized on my French-speaking skills and asked that we be assigned to work in public health, agriculture, or sanitation—arguing that we needed less language training and thus could learn new fields of expertise. We were assigned to a maternal and child public health position in Zaire—now the Democratic Republic of Congo. Told that we had to be married to be assigned to the same site, we wed in February 1980, when I was 21 years “young” in my childhood home in Seattle, Washington.

Mark and I were assigned to a public health position in the Kasai Oriental rural zone of Zaire, in the middle of the country, in the Kasai Oriental district, in a village called Bibanga, home of a former U.S. Southern Presbyterian mission hospital. A village of 1,000, Bibanga Hospital served a rural zone of 30,000 and was reachable only by foot, or across unmaintained dirt roads by four-wheel drive. The hospital was an old brick building, with no electricity, open wards, a center courtyard with washed disposable gloves hanging to dry from clothes lines, an operating room with a generator for electricity during surgeries, one doctor (Dr. Katambwe), a small pharmacy, and long queues of patients lining up each day to be seen.

We were charged with setting up a program for malnourished children, those with kwashiorkor (protein malnutrition) and marasmus (severe, total energy malnutrition). We quickly learned that mothers who brought their malnourished children to Bibanga left many other children behind at home, who subsequently became malnourished because the moms were the ones who did all the farming and ensured that their families were fed. Moreover, children who were malnourished enough to be brought to the hospital usually did not survive more than a day or two. This was the first time I’d ever seen anyone die, and it was a harsh introduction to the tragedy of healthcare disparities.

The amazing “bible” of international health, Where There Is No Doctor, A Village Health Care Handbook (1977) by David Werner, inspired us to reevaluate and change directions by developing a preventive health program for maternal and child healthcare workers. We organized a program for villages across the rural zone served by the Bibanga Hospital, submitted grants to international development agencies, and wrote healthcare manuals in French and in the local Bantu language Tshiluba (titled Mdimu Mbupita Buanga—a Tshiluba proverb meaning “cleverness is better than medicine,” the equivalent of “an ounce of prevention is worth a pound of cure,”) along with a poster-size visual aid book I illustrated. With funding from Oxfam, the Canadian Embassy, UNICEF and others, we had our books and visual aids printed at a printing press in Kananga, Zaire; bought a motorcycle for travel between villages; and obtained a large supply of polio, MMR, and DPT vaccines, along with a large kerosene freezer and set of cold boxes. We then started intense community organizing: traveling from village to village to meet with village elders to identify midwives and childcare workers to spend a month attending our maternal and child health “school” in Bibanga; recruiting a small public health outreach and education team; identifying lodging and hiring a “cook” for the school; and establishing a cold-chain for the vaccines, including connecting with village bar owners to use their kerosene refrigerators to store vaccines alongside cold beer.

After the first month-long training session, we initiated a series of monthly rounds to meet with the trained village midwives and childcare workers and to host village gatherings for child-weighing (hanging a baby scale from a tree branch), health education (topics ranging from nutrition to family planning to water sanitation), and immunization. The latter was the most effective intervention by far. Childhood mortality in 1980’s Zaire was high, with more than 20% of children dying before their fifth birthday, primarily from infectious diseases—many of which could be prevented by vaccination. Each fall, during a period of time called the “month of the flying termites,” our rural zone was hit with a measles epidemic, causing significant morbidity and mortality. After vaccinating 30,000 individuals within the rural zone, the “month of the flying termites” passed without any measles. This was a conversion experience for me, making me acutely and profoundly aware of the power of science to save lives.

Living in a mud hut with no electricity, I spent most evenings reading novels by kerosene lantern. Pre-cell phone and internet, all communication with our families was by regular (albeit extremely irregular) mail. As soon as my dad heard of my “conversion,” he started sending me science books—the first edition of Ben Lewin’s Genes, and biographies of John Enders and Jonas Salk. As much as I loved literature, I remember feeling that my knowledge of literature left me feeling empty-handed and in need of more practical, helpful expertise. At that point, I became intent on a career in medicine or public health.

Our project was going so well that we decided to stay on for a third year. Three months into that year, however, I got very sick. With my immune system weakened from having lost 30 pounds (largely because so little food was regularly available), two bouts of malaria (treated effectively with chloroquine), and more than a handful of infectious diarrheal diseases, within 24 hours of getting a splinter in my foot, I spiked a fever of 104°C and my foot swelled up unrecognizably. The next morning Mark used a shortwave radio to call the nearest Peace Corps office in the town of Mbuji-Mayi (translation, “goat water”), the Peace Corps officer drove up in a four-wheel drive Land Rover, drove me back to Mbuji-Mayi, and loaded me on the back of a cargo plane headed to Kinshasa. This was the only flight to Kinshasa for a week, and had I missed it, I would not have survived. In Kinshasa, after a week of IV penicillin, although my fever broke, the top of my foot had turned black and I was medically evacuated to the U.S. Army Hospital in Frankfurt, Germany. In Frankfurt, an Army doctor put a probe in one end of my foot and out the other, without my feeling a thing. Within an hour, I was given a diagnosis of necrotizing fasciitis, sent to surgery, and told that I might wake up without a foot. This is another vivid memory for me—shivering and terrified, lying on the gurney with cold IV fluids running into my arm.

I woke up with my foot but with a large area on the top of my foot missing from surgical debridement. After three weeks in Frankfurt, I was airlifted with other U.S. Army patients to Walter Reed Hospital in Washington, D.C., and from there to Harborview Hospital in Seattle, where I had a skin graft and then spent a month recovering at my parents’ home. To this day, I have a scar in the shape of Africa on my right foot.

Medical and Graduate School

I moved back to Cambridge, MA to find a job and take the rest of my premed courses. I worked part time in the Harvard Institute for International Development, working with Richard A. Cash, who had directed the first clinical trials for oral rehydration therapy in Bangladesh, helping write a book about a public health program he had just completed in Mali. Immersed in my work with Cash and intent on a career in medicine, I found myself wonderfully liberated from any awareness of or anxiety about premed competition, and completely enjoyed my premed classes.

While applying to medical school, I worked for a year as a research technician in the lab of pediatrician and virologist George Miller at Yale University. I was hired to study the transmission of HIV to and from children—a question that would inform the critical public health questions of whether children with AIDS should go to public schools or be placed in foster families. Although the first part of the project was true public health work—identifying children with AIDS and all their contacts, and then obtaining blood samples from them—the second part involved being in the lab and doing immunoblots to see who was HIV positive. This was when I realized how much I enjoyed bench work. I remember, for example, reading an early paper about the benefits of milk as a blocking agent for Western blotting and being delighted when I developed a blot that had crystal clear, sharp bands without any dark splotches on the film. I also attended the weekly Miller lab meetings and learned more about the other projects in the group. The lab was focused on the question of how Epstein Barr virus (EBV) herpesvirus switches between latency and replication. One of the truly exciting discoveries made while I was in the lab was the identification of a viral gene (ZEBRA) that triggered activation of latent EBV (Countryman and Miller, 1985). I fell in love with so many aspects of research in the Miller lab, from the medical significance of the questions and discoveries to George Miller’s clear devotion to mentorship, to the collective and communal problem solving, the satisfying, sleuthful aspects of experimental science, and the pleasure of hands-on experimentation. With encouragement from George, I applied to and was accepted into the Yale Medical Scientist Training Program (MSTP).

After my first two preclinical years, I chose to do my PhD training in the lab of Ari Helenius, a cell biologist and biochemist who was a pioneer in the use of viruses to study membrane trafficking and protein folding in cells. Thinking that I wanted to work in international health or infectious diseases, and being drawn to cell biology, the Helenius lab was the perfect fit for me. It was a medium-size lab, with four graduate students and four postdoctoral fellows, and Ari was deeply involved as a mentor, meeting with me for hours on end to discuss every aspect of my experiments. Our lab was next door to Ira Mellman’s lab, and we had joint Helenius-Mellman lab meetings and many joint lab parties. Ari had moved from Finland, and these parties always included singing the traditional drinking song “Helan Går” with shots of Acquavit, followed by theatrical skits directed by Ari and Ira, and ending with time in the sauna, imported from Finland to Ari’s backyard in Madison, Connecticut.

My PhD thesis project focused on the transport of influenza ribonucleoproteins (RNPs) into and out of the nucleus of infected epithelial cells. I spent the first part of my PhD using biochemical fractionation to study this process, which was a complete failure because the fractionated nuclei were either contaminated with cytoskeleton (using mild fractionation protocols) or broken open (using harsh fractionation protocols). I thus embarked on what I thought of as “single-cell biochemistry,” leveraging the newly available technology of laser scanning confocal microscopy to image the location of viral proteins at various time points after infection and under various experimental manipulations (Martin and Helenius, 1991a). Influenza virus was known to enter cells by receptor-mediated endocytosis, with penetration into the cytosol occurring by low pH-activated fusion between the membrane of the virus and the membrane of the late endosome. I discovered that the low pH of the late endosome triggered dissociation of the viral matrix protein M1 from the viral ribonucleoprotein particle (vRNP), exposing nuclear localization signals (NLSs) on the viral nucleoprotein NP (which bound the viral RNA to form the vRNP), resulting in the rapid, active nuclear import of the vRNP (Martin and Helenius, 1991b). I further found that the antiviral drug amantadine blocked the dissociation of M1 from the RNP, thereby preventing nuclear import. Next, I asked why vRNPs enter the nucleus at the start of infection, but are exported from the nucleus at the end of infection to form new viral particles that bud out from the plasma membrane—that is, what triggered the switch between disassembly and assembly of influenza viral nucleocapsids and between nuclear import and export of vRNPs? I found that M1 was synthesized late in infection, diffused into the nucleus, where, under neutral pH conditions, it bound the vRNP and escorted them out of the nucleus through the nuclear pores. These findings demonstrated that control of the assembly/disassembly of influenza vRNPs, and the directionality of nuclear transport of the viral genome, depend on Ml and its low pH-sensitive association with vRNPs. I have incredibly happy memories of graduate school. I loved being in the lab, spending time with my labmates, and doing experiments (especially microscopy experiments), and I had the fortune and elation of making discoveries that held clear significance to human health. I also loved the freedom I experienced as a graduate student—I was in the lab into the wee hours of the morning, but I also swam nearly every day, and took dance classes in New York once or twice a week.

The Yale MSTP had a very flexible reentry into clinical training from the PhD. I did clerkships in surgical subspecialties, internal medicine, and ob-gyn before returning to the lab to complete my last experiments and defend my PhD, which I did while I was eight months’ pregnant with my son Ben. A woman faculty member in a basic science department told me that that was the end of my career. My male internal medicine attending pulled me aside to ask if I had ascites, and the dean of student affairs counseled me that having a child was the financial equivalent of buying a new car each year and driving it off a cliff. Quite to the contrary, I found that having children not only provided the greatest joy of my life, but also provided critical perspective and made me much more focused and efficient in the lab.

Motherhood did, however, have a different impact on my clinical training. I had terrible morning sickness during my first trimester, when I was doing my first internal medicine clerkship, and I developed a very strong “odor aversion” conditioning to the smell of hospital medicine wards, such that to this day I feel slightly nauseous when I am on clinical wards (the irony of this was not lost on me when I became dean of a medical school). Being a third-year medical student was especially challenging as the mother of an infant. Ben’s father was an intern, working upward of 100 hours a week, and we had no family nearby to help and could not afford a full-time nanny. Instead, Ben started daycare at Yale Medical School when he was six weeks old, and while Yale was ahead of its time in offering on-site, subsidized daycare, I struggled with the limited hours and found myself in a near-impossible predicament every time Ben was sick. As dedicated as I was to patient care, I disliked what I perceived as the rigid hierarchy and lack of flexibility of the clinical setting.

As a student, I experienced cultural and philosophical differences between learning in the lab and on the wards: in the lab, I was encouraged to always ask questions and to always try to “disprove my hypothesis,” whereas on the wards, I was evaluated less on my curiosity and more on my fund of knowledge and my ability to succinctly and quickly articulate and act on that knowledge. I now appreciate the rationale for this approach to teaching medicine, but I also think something important is lost by not adopting the open-mindedness that Zen master Shunryu Suzuki calls the “beginner’s mind” (Suzuki, 1970). This has become clearer to me through the years, as I recognize that truly masterful clinicians are often those who practice an open-minded approach to differential diagnosis.

Shortly after defending my PhD, I did my psychiatry clerkship and was struck by how little was understood about the pathophysiology of psychiatric illness. This was in stark contrast to my experience in other clerkships, where therapeutic interventions were usually based on a relatively in-depth understanding of the biology of the organ and the disease process. It became clear to me that psychiatry was of critical public health importance and was in desperate need of strong scientific research. Finally, similar to my interest in literature and art, I was drawn to psychiatry because it is so central to human identity and behavior and to what it means to be human. My cell biological training as a graduate student made neurons, with their incredible morphological polarization and compartmentalization, seductively fascinating. Exploring intracellular trafficking in neurons felt like entering a gold mine to me. All of this propelled me to pursue postdoctoral training in neuroscience instead of following the traditional path of residency training, with the thought that I could always go back to clinical training.

Postdoctoral Training

I considered a number of labs for my postdoc training, and ended up in just the right place for someone with interests in psychiatry and in molecular cell biology—working with Eric Kandel on experience-dependent brain plasticity. While I was primarily drawn to the research in Eric’s lab, four other qualities of Eric as a mentor stood out: his wonderful sense of humor, his incredibly synthetic intellect, his clinical background and interest in psychiatry, and, finally, his apparent delight in learning that I had a child and clear communication that he did not see this as a hindrance to my productivity, but rather as an added richness to my life.

The Kandel lab was very different from the Helenius lab. Every variable of every experiment in the Helenius was understood and controlled for—for example, information was available about the epitopes recognized by every antibody, the temperature at which each antibody should be used for each assay, and great detail was available about every cell line we used. As much as Eric Kandel pioneered a “simple system” approach amenable to studying the cell biology of learning and memory using Aplysia sensory-motor synapses in culture, it became immediately apparent to me that neuroscience was much messier than the cell biology I had been trained in. I remember my reaction to experiments being done by my labmates in which they dissected Aplysia ganglia and incubated them with serotonin at room temperature, with the aim of identifying changes in gene expression occurring in sensory neurons. Coming from the Helenius lab, I worried about the variability of room temperature in an aging New York building, about the other cell types in the ganglion, and about the impact of the injury of dissection. These concerns nearly paralyzed me—and in fact led me to think I’d made a mistake. I visited Mu-Ming Poo, who had been at Yale and had recently moved to Columbia, to ask for his advice. He urged me stay—telling me that it would be an amazing education to spend time in two outstanding labs that were so different. I was not fully convinced and applied midyear to a residency program in pathology at Columbia. After being accepted into the residency, I made an appointment to meet with Eric Kandel to tell him of my change in plans. I remember my nervousness waiting outside his office, and then his loud and emphatic “NOOOO” and his very convincing suggestion that I give the lab another six months.

I am so glad I stayed. As an experimentalist, I have a propensity for carefully controlled biological reductionism, coupled with a reticence to tackle big, messy, important questions. I also have learned, however, that the “experimental control” of biological reductionism can create a fallacy about biological truths—for example, influenza infections in humans are undoubtedly more complex than the satisfying solutions I identified in a synchronized tissue culture cell line, and the mechanisms underlying learning and memory are certainly more complex than the plasticity in a dish that I have studied throughout much of my career. Eric’s bold and fearless curiosity were a perfect (albeit on occasion frustrating) antidote to my more cautious tendencies in science.

When I joined the Kandel lab in 1992, the lab was focused on identifying and characterizing the signaling pathways and gene regulatory switches required to consolidate short- to long-term facilitation—candidates for what we thought were master molecular switches for long-term memory formation. This line of inquiry had roots in a longer history elucidating a requirement for new gene expression during long-term but not short-term memory. In the early to mid-1960s and 1970s, a number of investigators, including Louis Flexner (Flexner et al., 1962), Bernie Agranoff (Agranoff et al., 1966), Larry Squire and Sam Barondes (Squire and Barondes, 1973), showed that inhibitors of protein and RNA synthesis blocked long-term memory but not short-term memory formation. In the mid-1980s, Eric Kandel and colleagues demonstrated that inhibition of RNA and protein synthesis in Aplysia sensory neurons blocks serotonin-induced long-term facilitation of sensory-motor neurons (Montarolo et al., 1986). In 1990, taking a cue from the beautiful work from Seymour Benzer and colleagues demonstrating a role for the cAMP signaling pathway in long-term memory in Drosophila melanogaster (e.g., Dudai et al., 1976), Pramod Dash in the Kandel lab showed that the consolidation of short- to long-term plasticity at Aplysia sensory-motor synapses required a cAMP-dependent transcription factor, the cyclic AMP response element binding protein (CREB). He did this by injecting CRE oligonucleotides into the sensory neuron, blocking long- but not short-term facilitation of the sensory-motor synapse (Dash et al., 1990).

Soon after I joined the lab, in 1994, Cristina Alberini cloned the Aplysia CCAAT enhancer-binding protein (C/EBP) and demonstrated that it was an immediate early gene (IEG) required for long- but not short-term synaptic plasticity (Alberini et al., 1994). There was tremendous excitement about this discovery as the first transcriptional “master switch” for long-term memory. Dusan Bartsch in the lab then looked upstream to learn what regulated expression of C/EBP, and using a yeast two-hybrid system, cloned Aplysia CREB2 (Bartsch et al., 1995). He went on to show that CREB2 functioned as a transcriptional repressor during long-term facilitation of Aplysia sensory-motor synapses, and, as such, served as a distinct type of master regulator. A few years later, Dusan Bartsch went on to clone and characterize Aplysia CREB1 and showed that one of three isoforms encoded by the CREB1 gene, CREB1a, functioned as a transcriptional activator that was necessary and, following phosphorylation, was sufficient for long-term facilitation of Aplysia sensory-motor synapses (Bartsch et al., 1998). These discoveries generated a true sense of excitement and optimism in the lab that we had “cracked” the code for long-term memory formation. In retrospect, these discoveries were more like reaching a mountain peak that, once the clouds cleared, revealed a landscape of yet higher peaks across the horizon.

I brought a cell-biological bent to the study of long-term memory. I was fascinated by two cell biological questions that emerged from the finding that long-term plasticity and memory required new gene expression. Neurons are spectacularly polarized cells, extending processes that extend great distances from the cell body. The requirement for new transcription immediately raised the question of how signals are transported the long distances from stimulated synapses to the nucleus. The second, perhaps more profound, question emerged from the remarkable compartmentalization of neurons, with each neuron forming up to thousands of synaptic compartments. The finding that long-term plasticity required transcription in the nucleus raised the question of whether the cellular unit of plasticity is the cell and its nucleus, or the synapse. If the synapse, what are the mechanisms that allow the products of gene expression made in a shared nucleus to alter the strength of some synapses but not others within a single neuron?

My first independent project in the Kandel lab addressed the first question. Another postdoctoral fellow in the lab, Dan Michael, was studying the potential phosphorylation and activation of Aplysia CREB2 and C/EBP by extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) (Michael et al., 1998). Dan told me about the graded response of MAPK to extracellular signals in PC12 cells, where epidermal growth factor (EGF) causes transient MAPK activation and cell proliferation, whereas nerve growth factor (NGF) causes sustained MAPK activation and nuclear translocation and results in neuronal differentiation. I immediately wondered if MAPK might respond differentially to a single pulse of serotonin (which produces short-term facilitation of Aplysia sensory-motor synapses) and to five spaced pulses of serotonin (which produces long-term facilitation). I took a single cell approach, which the giant neurons of Aplysia are so perfect for, and found that MAPK underwent a graded response to serotonin stimulation and that inhibition of MAPK blocked long-term but not short-term facilitation of sensory-motor synapses. I found that plasticity-inducing stimuli also triggered nuclear translocation of MAPK in CA1 pyramidal neurons in rodent hippocampus, suggesting that MAPK might be a common player in multiple forms of learning-related plasticity (Martin et al., 1997a). Together, these findings provided a mechanism by which synaptic stimulation, through sustained activation of MAPK and translocation to the nucleus, could activate gene transcription to convert short-term to long-term plasticity. The finding that the nuclear translocation of MAPK was graded supported a combinatorial model for transcription-dependent plasticity—in which multiple signaling pathways respond in a graded manner to reach a combinatorial threshold to convert short-term to long-term plasticity.

Aplysia neurons were ideal for single cell biology and electrophysiology, with the sensory cell body measuring 50 microns in diameter, the motor neuron about 100 microns in diameter, and the individual synaptic compartment a few microns in diameter and hence easily resolvable by light microscopy, but they are difficult to culture! It took me several months to learn how to use glass electrodes to identify and pull Aplysia neurons out of their protease-treated ganglia, hooking, but not puncturing, the cell soma and carefully pulling the neuronal cell body out along with a long neuronal process and transferring it to a culture dish without damaging the neuron. Once the neurons were in the culture dish, I used glass electrodes to create eddies in the culture medium to lay the neuron flat on the surface of the coverslip in the dish, ensuring that the process extended away from the soma. Finally, I used the electrodes to gently manipulate the position of the neurons, manually arranging the geometry of the coculture such that the sensory neuron process contacted the motor neuron process, but the cell bodies were separated from one another. It took me an entire day, and great manual dexterity and patience, to culture anywhere from 8 to 10 culture dishes each containing a single sensory-motor coculture.

As occasionally mind-numbing as it was to culture Aplysia neurons, it taught me the importance of being involved in every aspect of an experiment (something I sometimes think about in a world in which so much science involves and benefits from large team collaborations—underscoring the need for outstanding team communication to make sure that important experimental insights don’t fall through the cracks). The fact that I made the cultures myself was necessary for a “eureka” moment that opened the door to studying whether and how transcription-dependent plasticity could occur at specific synapses made by a single neuron. I was on a pumpkin-picking preschool field trip with my two-year-old daughter Maya (born during my second year as a postdoc), sitting on a bale of hay on the back of a truck, when it dawned on me that some of the sensory neurons that I isolated had bifurcated processes. If I cultured these such that one process made synapses with one motor neuron, and the other with another spatially separated motor neuron, I could test whether serotonin application to one branch triggered facilitation only at that branch or at both branches of the sensory neuron. While the former would demonstrate synapse-specific plasticity, the latter would be consistent with cell-wide plasticity. I couldn’t wait to get back to the lab to try this out.

I used a perfusion micropipette to deliver five spaced applications, and the next day, to my enormous delight, the strength at the serotonin-treated branch had increased, with no change in synaptic strength at the untreated branch. I went on to show that this branch-specific form of long-term facilitation required transcription in the sensory neuron and involved growth of new synaptic connections exclusively at the serotonin-treated branch (Martin et al., 1997b). In exploring what might underlie this branch-specific long-term facilitation, I asked whether it involved local translation of synaptically localized mRNAs. This question was inspired by work of others, including Oswald Steward, who had identified polyribosomes at the base of spines in hippocampal neurons (Steward and Levy, 1982), and Erin Schuman, who had recently demonstrated a role for local translation in dendrites during brain derived neurotrophic factor (BDNF)-induced plasticity of hippocampal synapses (Kang and Schuman, 1996). I found that local perfusion of membrane-permeant translational inhibitors and injection of membrane-impermeant translational inhibitors into the sensory but not the motor neuron blocked branch-specific long-term facilitation, consistent with translation of localized mRNAs in the sensory neuron process being required for synapse-specific plasticity. Another advantage of Aplysia neurons is that neuronal processes will survive for days after the cell soma has been removed, allowing one to monitor translation in isolated processes independently of contributions from cell body translation and protein transport. I leveraged this advantage using metabolic labeling to demonstrate that isolated sensory neuronal processes were capable of protein synthesis and that this translation was upregulated by serotonin. Together, these studies demonstrated at the level of a single cell that long-lasting plasticity could occur within specific synapses made by a given neuron and that this synapse-specific plasticity required the translation of localized mRNAs.

The simple bifurcated culture system, while technically challenging to prepare, opened the door to many more questions about compartmentalized gene expression and plasticity in neurons. In collaboration with another postdoc in the lab, Andrea Casadio, I showed that a synaptic “tag” was required for persistent long-term synaptic plasticity: application of five pulses of serotonin to the sensory cell body produced long-term facilitation at both branches of a bifurcated sensory neuron, but that facilitation, while present at 24 hours, did not persist to 48 hours and was not associated with the growth of new synapses (Casadio et al., 1999). If, however, a single application of serotonin was applied at the site of contact onto the motor neuron, that was sufficient to generate persistent, branch-specific plasticity that lasted 48 hours and that was accompanied by the branch-specific growth of new synapses. We interpreted these findings as indicating that the single synaptic application of serotonin was sufficient to create a synaptic tag that could capture the products of gene expression induced by the five spaced applications of serotonin to generate long-lasting synaptic plasticity. Varying the time between the synaptic single application of serotonin and the somatic spaced applications of serotonin, we estimated that the synaptic tag persisted between 4 and 5 hours, whereas the products of gene expression induced by somatic stimulation persisted between 1 and 4 hours. Together, these studies provided insights into the temporal and spatial orchestration of signaling and gene expression within a single neuron that gives rise to the persistence of activity-dependent synaptic plasticity. Unlike the initial gestalt when I joined the Kandel lab that there might be a “master switch” for long-term plasticity and memory, these discoveries underlined the complexity and breadth of gene regulatory mechanisms—transcriptional and post-transcriptional—by which neuronal activity could drive long-lasting changes in synaptic efficacy.

While most of my work in the Kandel lab was done using the Aplysia system, I collaborated with many colleagues in the lab to study plasticity in rodent hippocampus (Son et al., 1996; Patterson et al., 1996; Winder et al., 1999; Huang et al., 2000). Compared with Aplysia, rodents offered several advantages. Genetically modified mice offered the possibility to study the link between genes and plasticity and behavior. Additionally, defined genetic strains of mice were readily available as well as genetic tools and antibodies. In contrast, Aplysia are largely wild, genetically heterogeneous animals—with breeding pairs caught in the Pacific and then bred through one generation in a facility at University of Miami. When harvested in the wild, animals are selected based solely on their size. Depending on the time of the year, the Aplysia we received were more or less healthy. It was rare to find breeding pairs in August, creating a scarcity of animals from Miami; in the winter, the FedEx packages sat in the cold at the JFK tarmac, and in the summer, they sat in the heat. Neither condition made for healthy animals. I decided that when starting my own lab, I would use both systems.

UCLA

I was in the Kandel lab for nearly seven years. I was just about ready to stay in New York when a Yale MD-PhD friend, Dan Geschwind, wrote and encouraged me to apply to UCLA, where he had just finished his neurology residency. I had never been in Southern California and had a somewhat negative view of the region from having grown up in Seattle and lived in New York. Our visit was illuminating in ways that completely changed my perception. A defining moment was participating in the Friday morning Learning and Memory Journal Club, an incredibly interactive journal club that had started a few years earlier and that continues to this day, attended by faculty, staff, and trainees interested in the biology of learning and memory. I was also struck by how many neuroscientists I knew had recently joined the UCLA faculty—Tom Otis, Alcino Silva, Tom O’Dell, Felix Schweizer, Dean Buonomano, and Dan Geschwind—and by the open interactions between the faculty, staff, and trainees. Finally, I realized it would be a good thing for my independence as a scientist to have 3,000 miles between me and my postdoctoral mentor.

We moved to Los Angeles in July 1999, moving into a house just a mile away from campus. I was fortunate to arrive with an NIH R01 in hand, which I had written toward the end of my postdoc, and a Burroughs Wellcome Career Award. Within a few months of joining UCLA, I also joined the Department of Biological Chemistry, a basic science department that was much smaller than the Psychiatry Department, which ended up becoming my true intellectual home on the campus.

I was intent on pursuing two questions in my own lab. First, I wanted to identify the mRNAs that localized to neuronal processes, convinced that this would provide a useful tool for studying how local translation contributes to learning-related synaptic plasticity. Second, I wanted to understand the cell biological mechanisms by which signals were relayed from stimulated synapses to the nucleus. To address the first question, I undertook two approaches. First, working with my first MD-PhD student, Robert Moccia, we prepared cultures of isolated Aplysia sensory neurons, cut off all their cell bodies, isolated RNA from the processes, and generated cDNA libraries to identify the population of localized mRNAs. We identified hundreds of transcripts in the library, indicating that a large repertoire of mRNAs was present in neuronal processes, where their local translation could generate an array of changes in synaptic structure and function (Moccia et al., 2003). Among the mRNAs we identified, several encoded ribosomal proteins, and we confirmed the localization of these transcripts by in situ hybridization. At the time, this seemed like a heretical finding, because the textbook definition of ribosome biogenesis was that it occurred exclusively in the nucleus. We proposed, in collaboration with Joachim Frank, that there might be translation of ribosomal proteins followed by an exchange of preexisting proteins on localized ribosomes. In subsequent years, other studies identified ribosomal protein mRNAs in distal neuronal processes, and more recent work on ribosome heterogeneity and specialization (Genuth and Barna, 2018) has revealed significant heterogeneity and specialization of ribosomes, supporting the possibility that local translation of ribosomal proteins could result in a specialized population of ribosomes at the synapse that translate specific localized mRNAs. I raise this here because I remember at the time vacillating between a deep sense of doubt about the finding (“it must be contamination”) and a thrilling sense of excitement about the possible implications of the finding. The trick was waiting for technologies to be available to rigorously test this idea—technologies that are just now, well over a decade later, coming on line.

With another graduate student, Michael Poon, we isolated the processes of cultured hippocampal neurons by culturing them on filters that had 3-micron pores, so that the cell bodies stayed on the top surface, but some of the dendrites and axons penetrated through and were mechanically separated from the cell bodies. We generated a cDNA library from these isolated processes and again identified more than a hundred dendritically localized transcripts (Poon et al., 2006). As we had found in Aplysia, many of these encoded components of the translational machinery, again consistent with the idea that one function of local translation was to enhance the translational capacity of the synapse.

To study the mechanisms by which synaptically localized signals traveled back to the soma and into the nucleus, I asked whether the active nuclear import machinery played a role in the process. While neurons are specialized for rapid signaling between compartments using electrochemical signaling and calcium waves, the finding that MAPK and other molecules underwent stimulus-induced nuclear import indicated that mechanisms must exist for the transport of soluble molecules from the synapse to the nucleus. This type of nuclear signaling pathway could provide a distinct temporal regulation of stimulus-induced transcription, and also, unlike electrochemical signaling, which is cell-wide, might provide spatial information about the site of stimulation within the neuron. I decided to focus on the classical nuclear import pathway, which consists of a family of nuclear import proteins called importins that recognize NLSs in karyophilic proteins and transport these proteins into the nucleus. Together with a graduate student, Kim Thompson, and in collaboration with my colleague Tom O’Dell—who had been a postdoc in the Kandel lab when I arrived, and became a wonderful collaborator throughout my time at UCLA—we found that importins localized to distal processes of Aplysia neurons and to dendrites of rodent hippocampal neurons. We further found that long-term facilitation of Aplysia sensory-motor neurons and long-term potentiation of rodent hippocampal neurons triggered the nuclear translocation of importins (Thompson et al., 2004). What excited me most about these studies was the possibility of using the importin transport proteins as bait to pull out novel synaptically localized signaling molecules whose activity-dependent nuclear import contributed to the consolidation of short- to long-term plasticity.

Despite multiple efforts, however, this line of investigation never yielded fruit. Instead, a postdoctoral fellow, Toh-Hean Ch’ng, made advances by focusing on a candidate transcriptional coregulator, the CREB Regulated Transcription Coactivator 1 (CRTC1), whose activity in non-neuronal cells was known to be regulated by stimulus-induced nucleocytoplasmic trafficking. His work showing stimulus-induced nuclear transport of CRTC1 from synapse to nucleus in rodent hippocampal neurons provided new insights into the role of phosphorylation–dephosphosphorylation cascades. It also showed that neuromodulatory inputs that regulated cAMP concentrations could regulate the duration of stimulus-induced transcription required for transcription-dependent plasticity (Ch’ng et al., 2012, 2015). Moreover, Toh Hean’s studies of CRTC1 inspired many other ongoing projects in the lab, led by other talented students and postdocs, including Jennifer Achiro, Sylvia Neumann, Martina DeSalvo, and Shivan Bonanno. They would go on to ask a series of important questions: does differential phosphorylation and dephosphorylation of CRTC1 serve as a phospho-code that couples specific types of stimuli to specific transcriptional programs? Are the transcriptional programs recruited by stimuli that elicit long-term potentiation distinct from those that elicit long-term depression? What is the profile of genes regulated by CRTC1? How does catecholaminergic stimulation, which increases intracellular cAMP, affect the transcriptional program elicited by glutamatergic activity and by stimulus-induced CRTC1 nuclear translocation?

Over a decade after our study on importin-mediated synaptonuclear import, Wendy Herbst, a talented graduate student in the lab, undertook the challenge of identifying novel synaptonuclear signaling molecules that might play a role in plasticity. In addition to the role of these signaling molecules in regulating activity-dependent transcription, we recognized that they also might play a role in synaptic tagging during synapse-specific plasticity, such that loss of a particular synaptonuclear signaling molecule from the synapse could tag that synapse for synapse-specific plasticity. Wendy Herbst used importins as bait to immunoprecipitate cargo signaling molecules from the synapse. Although this approach identified a number of potentially interesting cargoes, she was unable to detect robust synapse to nucleus import during plasticity. She then decided to switch her focus from identifying synaptically localized karyophilic proteins to identifying activity-dependent changes in the population of proteins present in the nucleus. Just as I had done as a graduate student in studying the nuclear import of influenza vRNPs, Wendy started by fractionating nuclei from stimulated and unstimulated neurons, but found—despite following published reports claiming pure, intact nuclei—that the nuclei were either contaminated with cytoskeleton or were leaky. She then turned to using the proximity ligation assay pioneered by Alice Ting, expressing an engineered ascorbate peroxidase enzyme with two NLSs that localized the enzyme to the nucleus of cultured rodent forebrain neurons, where it could biotinylate neighboring proteins when biotin-phenol and hydrogen peroxidase were added (Hung et al., 2016). We collaborated with the neighboring proteomics lab of James Wohlshlegel to use mass spectrometry to identify differences in the nuclear proteome of silenced and stimulated rat forebrain neurons. In following up on one of the candidate proteins, Programmed Cell Death Protein 4 (PDCD40), whose nuclear concentration was decreased by glutamatergic stimulation, Wendy identified a novel mechanism for altering the subcellular concentration of PDCD4. This involved stimulus-induced, PKC-mediated nuclear proteolysis of PDCD4 (Herbst et al., 2021). While PDCD4 has been shown to function as a translational regulator in cancer cells, Wendy’s findings also supported a role for PDCD4 in transcriptional regulation, and using RNA-sequencing, suggested a specific role in transcription of genes encoding synaptic proteins. Together, Wendy’s studies elucidated the power of the proximity ligation methodology in studying the proteome of specific cell types and subcellular compartments during plasticity. Her findings also underscored the wide array of cell biological mechanisms by which activity can regulate gene expression during plasticity—in this case through activity-dependent nuclear degradation of a transcriptional regulator.

My lab continued to work in the Aplysia sensory-motor culture system until 2015. The system has many ideal attributes for cell biological studies of plasticity. In addition to the large size of the neurons, one can study sensory neurons in the presence or absence of chemical synapses (isolated sensory neurons do not form chemical synapses; sensory neurons paired with follower motor neurons form glutamatergic synapses). Taking advantage of this, graduate student Vlasta Lyles showed that a sensory neuron specific mRNA, sensorin, was diffusely distributed throughout the processes of isolated sensory neurons, but concentrated at glutamatergic synapses in sensory neurons paired with follower motor neurons. She further showed that reduction of sensorin mRNA but not protein was sufficient to block synapse formation with the motor neuron, indicating that local translation of sensorin mRNA at the synapse was required for synapse formation.

A postdoc in the lab, Ohtan Wang, took advantage of the simplicity of the Aplysia sensory-motor system to visualize in real time local translation at stimulated synapses (Wang et al., 2009). To do this, she developed a reporter fusing the 5’ and 3’ untranslated regions of sensorin to the photoconvertible protein Dendra2. Expressing this in neurons, she was able to ultraviolet-photoconvert the Dendra2 protein from green to red, and then monitor new translation of the reporter by the appearance of new green signal. To ensure that the new translation was occurring in the neuronal process, and that it did not reflect transport of newly synthesized protein from the soma, Ohtan removed the cell body and then locally perfused serotonin onto specific sites of contact between the sensory neurite and the follower motor neuron. The results were stunning, with translation visualized in real time only at stimulated synapses. Ohtan went on to show that this translation was stimulus specific (occurring only during long-term facilitation and not long-term depression) and that it required the presence of a chemical synapse (i.e., was not observed in isolated sensory neurons).

A few years later, Sangmok Kim, another graduate student in the lab, cultured a bifurcated sensory motor neuron with a follower motor neuron, with which it formed synapses, and a nonfollower motor neuron, with which it formed adhesive interactions but no chemical synapses. He found that RNAs localized throughout the arbor of the sensory neurons, but that local translation occurred only at the site of synapse formation (Kim and Martin, 2015). When he paired a bifurcated sensory neuron with two follower motor neurons and locally perfused serotonin to induce branch specific long-term facilitation, he found that RNAs—including transcriptionally induced mRNAs—were delivered throughout the sensory arbor, but that translation was restricted to the site of stimulation. In investigating the mechanisms underlying this local translation, Sangmok found that it required trans-synaptic netrin/DCC signaling between the sensory and motor neuron. What was most exciting about these findings was that they revealed a decoupling between transcriptional and translational regulation in neurons, with stimuli triggering transcription of mRNAs that are delivered throughout the neurons, and local cues independently regulating the translation of these mRNAs. Decentralizing the control of gene expression to individual synapses allows all the branches of a neuron to be in a state of readiness to respond to local stimuli by changing their local proteome to alter synaptic structure and function.

As incredibly satisfying as it was to use Aplysia cultures to explore these cell biological questions, there were limitations to the system. While other fields had developed powerful shared databases for model organisms (e.g., C. elegans or D. melanogaster), this never occurred in the Aplysia field. As one example, although there were efforts to sequence the Aplysia genome, these took a long time to become publicly available. In part, this was because of technical difficulties in sequencing the genome, but in part, I believe it was also because the Aplysia research community did not have a history of developing shared resources. The fact that Aplysia could not be genetically manipulated also was a limitation. The final challenge arose from climate change. Being in Los Angeles was ideal for working on Aplysia because there are wild populations off the California coast. I hired a scuba diver who collected for me, but over the years it became more and more difficult for our diver to find healthy animals, which I can’t help but attribute to rising Pacific Ocean temperatures.

At the same time, the tools available in mouse and rat made cell biological analyses more and more tractable. Patrick Chen in the lab used RNA sequencing and translating ribosome affinity purification-sequencing (TRAP-seq) to identify changes in transcription and translation following induction of long-term potentiation of mouse hippocampal CA3 to CA1 synapses (Chen et al., 2017). Mariana Fontes collaborated with the lab of Bin Tian to study alternative cleavage and polyadenylation of mRNAs after induction of hippocampal long-term potentiation (Fontes et al., 2017). Ji-Ann Lee, a postdoc in my lab, used knock-down and rescue approaches, along with RNA sequencing and crosslinking-immunoprecipitation and RNAseq (CLIP-seq), to identify cytoplasmic targets of the RNA binding protein RbFox1. He found that these were enriched in genes involved in cortical development and autism (Lee et al., 2016). He further identified the mechanisms by which RbFox1 regulated the stability and translation of its mRNA targets in the cytoplasm. These and other studies in the lab made it clear that newly developed tools and technologies would enable powerful cell biological studies in mouse systems. For me, this was a striking example of the importance of tool and technology developments in neuroscience and of the importance of sharing those tools and technologies with the broader scientific community.

Looking back over the past 30 years, I’ve watched the field of memory research move from excitement about the molecular and genetic tools that felt so enabling when I started as a postdoc in the Kandel lab to excitement about what could be learned about brain and behavior from advances in human genetics—including through next-generation -omics approaches to excitement about enabling circuit-level tools such as large-scale, cellular-level recording of neural activity and optogenetics, to advances in quantifying behavior and advances in brain imaging, to the application of new computational and mathematical tools to understanding brain function. As exciting and innovative as each new approach is, there is often an overly exuberant faith that each new shiny method will be the “breakthrough” to understanding how the brain “really” works, with the field then moving like an amoeba toward that promising strategy. For me, I see the neuroscientific progress as meta-incorporation of all these levels of analyses, a “stitching together” of multiple ways of seeing and understanding the brain.

At the same time, I recognize I’m a cell biologist at heart. I find most satisfaction thinking about the cells in the brain, the molecules that move within those cells, and the interactions between cells in the brain. I initially believed there would be simple, elegant engrams for memory (CamKII alpha, CREB) or that there would be a single “synaptic tag,” but I have grown to realize how much more elegantly complex and dynamic cell networks are in the brain. I fell in love with the gorgeous images of neurons in Ramon y Cajal’s golgi stains, inspired to understand the mechanisms of intracellular transport within these spectacularly polarized cells. As I’ve learned more about glial cells and extracellular matrix, exosomes that can transfer genetic materials between cells, and liquid-liquid phase partitioning within cells and subcellular compartments, I’ve become more and more interested in the local neighborhoods of synapses—inside and outside of the neuronal membrane. I’ve even found myself wondering if Cajal’s “neuron doctrine,” and its ascendancy over Golgi’s “reticular network” theory created a framework that focused too narrowly on individual, membrane-bound neurons, rather than on cell-cell interactions and cell-extracellular matrix interactions in the brain (Poo et al., 2016).

Family and Love

Many changes occurred in my family life after moving to UCLA. In 2004, my then-husband Mark announced that he was moving out, just about a month after my mother was diagnosed with a glioblastoma. I was heartbroken and struggled to look forward with any hope or joy, other than by focusing on taking care of Ben, then 12, and Maya, then 9. There were some funny moments, but only in retrospect—including our dog killing a skunk under the house who sprayed into the heating vents, leaving an aroma that haunted our house and all our clothes for months (a half a year later, I was asked by a woman in an elevator what perfume I was wearing). I really never thought I would be happy again.

Six months after Mark had left, a colleague of mine, Joel Braslow—an MD-PhD psychiatrist at UCLA with a PhD in the history of science, dropped by our house. Joel had been divorced about a year earlier, and his two sons, Seth and Sam, went to the same elementary school as Ben and Maya. Joel invited us all over for dinner, and then took Ben and Sam on a hiking trip. Over a period of months, we spent more time together and fell in love. Joel supported me through the death of my mother in 2005 and, with patience and humor, helped me through the challenges of being a single, working mom. In 2006, we were married in our home in Los Angeles. While having a blended family was not always easy (I remember Ben throwing all of Joel’s things into our swimming pool), it has ended up being one of the happiest surprises of my life. My oldest stepson, Seth, lives in China, where he teaches English and is planning to marry a woman he met while he was at Changsha University (which Seth chose to attend because it was where Chairman Mao studied). My youngest stepson, Sam, is a reporter who now works for the Beverly Hills Courier. Over the years, we have developed a strong sense of family with Seth, Ben, Sam, and Maya and their significant others, with my nephew T. J. (my brother Tommy’s son, who lives in LA) and his significant other, and with my father after he moved in with us. These are my beloveds, and being with them is my home. Looking back to how devastated I felt in 2004, the happiness I now feel in my marriage and family life is a lesson about the unknown silver linings of change. I see the world differently now, have learned so much from Joel (including, not incidentally, about social sciences, mental illness and health, psychiatry, and public mental health delivery), and feel in every way that I always have a partner and friend—someone I not only love but also like and admire and trust.

I have to add something about the absolute pleasures of being a mom. For me, it has been so much more meaningful than what my dad taught me about the comfort of genetic transmission. It’s been awe-inspiring to watch my children grow and become the individuals they are. Ben was always a mathematics and puzzle lover. He was taken with Latin in middle school and attended Junior Classical League annual meetings; he loved math and competed in math competitions across California; he played chess as a five- and six-year-old (and my heart melted when he learned to shake hands with his opponents regardless of whether he had won or lost); he received an award for math and science and for “beauty and kindness of spirit” when he graduated from middle school; he loved Pokemon, Magic Cards, and Dungeons and Dragons; throughout his childhood, he read voraciously and would laugh out loud in the middle of books; and he loved, and still loves, food and cooking. Ben went to Stanford for college and studied chemistry, and then went to UCSF where he got his PhD in biophysics in the lab of Jamie Fraser, where I thought he was the happiest graduate student I had ever met (which says something remarkable about Jamie Fraser). He is now a postdoc with Danielle Grotjahn at the Scripps Research Institute in La Jolla, using cryo-electron tomography to study mitochondrial dynamics. He loves every aspect of science, excels at computational analyses, and amazes me with his resilience to all the hard stuff in science—the rejections, the experiments that just don’t work, the competition. Somehow his scientific curiosity transcends everything else. He is also stupendously happy in his relationship with Rachel, whom he met as a freshman at Stanford and with whom he’s managed to build a life that balances work and play.

Maya spent much of her childhood in ballet. She started taking ballet when she was five, and by the end of high school was spending 10 to 20 hours a week at her ballet studio. Watching her dance was a thing of beauty—she exuded a radiance that felt contagiously joyful. She spent many summers at ballet camps, and followed the Royal Academy of Dance curriculum up through the Solo Seal competition. After high school, she went to Columbia University in New York in part so that she could continue to dance. In addition to dance, Maya was always deeply engaged in her studies. At Columbia, she majored in history and wrote her honors thesis on the emergence of eugenics in early 20th-century Britain. She graduated summa cum laude and phi beta kappa, and returned to Los Angeles to work part time in a human genetics lab and part time in her old dance studio teaching ballet. Under the mentorship of Deborah Krakow, she studied the genetics of musculoskeletal dysplasias, and identified and characterized a mutant leading to a novel bent bone dysplasia. She also loved spending time at the Little People of America annual convention, learning about the sociology and clinical care of individuals with short stature. Ultimately, she decided to go to medical school and is currently a second-year medical student. Like Ben, Maya strikes me as one of the happiest medical students I have ever met, convincing me that she found the absolutely right “career” for her. She lives with her boyfriend of six years, Aaron, and they too have managed to build a life that balances work and play, including weekly tennis sessions and going to the local bar with their friends on “trivia” nights.

While I am not genetically related to Seth or Sam, and was not a part of their lives until they were 16 and 11 years old, respectively, I’ve been equally amazed watching who they have become, and feel equally inspired by the goodness of who they are. Seth may be one of the kindest persons I’ve ever met—I’m not sure he has a mean bone in his body. He is perfectly fluent in Mandarin, and feels a true sense of belonging in China. Sam has become a fierce, articulate journalist and compelling writer, committed to reporting on “the truth,” and is unabashed in his pursuit of important stories. He is brilliantly imaginative and funny.

Academic and Community Service

I recognized early on as a faculty member that in addition to being a scientist, a part of me was an activist who wanted to be engaged in my community—likely the part that led me to join the Peace Corps. Soon after I became a UCLA faculty member in 1999, I decided to learn about the MD-PhD program because it had been so important to me. I learned that only 20% of UCLA MSTP students were women (unlike the 50% in my class at Yale) and when I asked why, was told that women students got pregnant and dropped out. That was enough to motivate me to join the admissions committee, become the associate director, and then, in 2005, with my colleague Steve Smale, take on the codirectorship of the UCLA-Caltech MSTP. This provided a fantastic exposure to many aspects of the university, including its relationship to Caltech. We were able to recruit amazing students, significantly increase the representation of women and underrepresented minorities, renew the NIH grant, develop mentoring programs, and, working with my husband Joel, start a new social science track in the MSTP. I learned a lot about university administration and finances, about mentors and mentoring, about the stresses on students, and the qualities necessary to succeed in science and medicine.

In 2010, my colleagues in biological chemistry asked if I would take on the chairpersonship of the department. After having been led by Elizabeth Neufeld for 20 years, the department turned to a rotating chairpersonship. Larry Zipursky, my close colleague and mentor throughout my time at UCLA, took on a three-year term, followed by Michael Grunstein. I found a cherished scientific home in the department and in 2006 moved my lab to the new biological chemistry building, where my lab shared a large open lab space with stem cell biologist Kathrin Plath, developmental biologist Gerry Weinmaster, and yeast geneticist and cell biologist Greg Payne. Our offices were adjacent to one another, and our trainees all worked together in common space. Colleagues with deep expertise in gene expression—Siavash Kurdistani, Michael Carey, Reid Johnson, and James Wohlschlegel—were just across the hall. The proximity of these colleagues had a profound impact on my research program. The department had just over 20 faculty, all deeply committed to research and education on basic molecular biological mechanisms, with particular strengths in gene regulation, chromatin biology, and structural biology. I was especially taken with the departmental academic promotion meetings: in psychiatry, the focus was on numbers of papers and amount of grant dollars, whereas in biological chemistry, the evaluation began with a discussion of the questions the faculty member under review was asking, discoveries he or she had made, teaching he or she did, and then moved on to numbers of papers and grants. This order of things was what mattered to me in academic research, and so when I was asked to be chair, I accepted.

As chair, I found that I enjoyed supporting the department faculty, recruiting and mentoring faculty, facilitating collaborative projects, and setting up enabling shared technologies and equipment. I also enjoyed working with the other basic science department chairs to advance graduate education and basic science in the medical school. I became the chair of the basic science chairs, and was invited by the dean of the medical school, Gene Washington, to join his research executive committee. I was deeply involved in strategic planning that led to the design of a unified biomedical graduate program in life sciences across the medical school and college as well as in the design and development of interdepartmental research “themes” in the medical school; cofounded a campus-wide effort now called the Depression Grand Challenge that supports scholarship across disciplines to understand, prevent, and treat depression; and joined a chancellor’s task force aimed at unifying the many disparate neuroscience units on campus (I came to view neuroscience at UCLA as a dilute solution with many gems, in great need of nucleating agents for crystallization). I was energized by all these activities and was optimistic that working with my colleagues we could enable great discoveries and breakthroughs.

In 2015, Dean Washington announced that he was leaving to take a position at Duke. The chancellor asked me if I would serve as executive vice dean, working with John Mazziotta, a neurologist-scientist, serving as vice chancellor for health and dean. Being a dean in a medical school was certainly not a position I had ever considered, but, just as I had felt about serving as chair of biological chemistry, I felt so committed to my colleagues, so optimistic about what could be done at UCLA, and so inspired by the potential of biomedical research to improve human health and well-being that I was convinced it was the right thing to do and I said yes. Within a few months, the UCLA chancellor and provost decided to split the role of vice chancellor for UCLA health and dean of the David Geffen School of Medicine (DGSOM) into two separate roles, and asked me to become the interim dean. Again, even though being a dean had never been a blip on the radar screen of my future, I felt incredibly excited about what I could do to advocate for biomedical research and education.

As dean, I became painfully aware that academic medicine is at critical crossroads—facing an existential threat in balancing the research, education, and community engagement missions of a public medical school with the business of a healthcare system. Over the last half-century, medical schools have become increasingly dependent on clinical revenue, which has shifted priorities and decision-making toward generating profit over the research, educational, and community-service missions. For example, in 1965, 40% and 6% of U.S. medical school revenue came from federal research funding and from clinical/hospital revenue, respectively, whereas in 2015, the revenue sources had changed dramatically, to only 14% from federal research funding and 61% from clinical and hospital revenue (Education Advisory Board). By the time I left UCLA, close to 60% of the entire UCLA campus (not just the medical school) revenue derived from the clinical enterprise. As a faculty member, I was unaware of this dramatic change in funding of academic medical centers, much less the implications of such a shift. As a dean, however, I saw the extent to which the logic of business practices affected the fundamental values of academic medicine as the engine for basic research, education, and community engagement. Moreover, the value of basic research, of education, and of community engagement are often long-term propositions, and as such, are not easily compatible with a healthcare business that needs to make margins over the short term and needs to compete in the market-driven business of U.S. health care. This has led to a debate in academic medicine about “margin versus mission” and to the frequent statement of “no margin, no mission.”

Ironically, as the values of academic medicine are threatened by a market-driven healthcare system, biomedical research has been undergoing a revolution, with new and enabling technologies and discoveries that have been transforming medicine. The opportunities for synergy between clinical medicine and biomedical research have become increasingly extraordinary; compared with when I began my research and clinical training, the opportunities for bidirectional exchanges of knowledge have blossomed with advances in human genetics, biomedical informatics, electronic health records (EHRs), clinical imaging, and more. UCLA was and is perfectly poised to exploit this synergy, with its top-ranked hospital and clinics sharing a single, walkable campus with the entire UCLA research enterprise. As a public medical school in one of the most diverse cities in the country, the DGSOM was and is ideally positioned to address the increasing imbalance in U.S. healthcare funding that underlies the tragic health and healthcare disparities our nation faces. I was deeply motivated to address these challenges.

I felt a longing for my work to have a more immediate social impact and was excited to help drive forward a range of fields, from immunology to cancer biology to metabolism and social medicine and beyond. And I was eager to help change the face of science and medicine—it was clear to me that both science and medicine had been limited by the demographics of the “club” of academic medicine. All of this inspired me to take on the role of interim dean, and then, following a national search, of dean.

I spent six years in the dean’s office at the DGSOM, from 2015 to 2021. I look back on this time as being perhaps the steepest learning curve of my life. A school of 3,000 faculty, 6,000 staff, and 3,000 trainees (including medical students, residents and fellows, graduate students, and postdoctoral fellows), with a budget of nearly $2.5 billion, each day presented new and unexpected (and often unimaginable) challenges. I worked with an amazing team in the dean’s office, from whom I learned so much about organizational management, finances, employment law, leadership, academic medicine, communication and marketing, philanthropy, change management, and more. While our work often felt Sisyphean, with imperceptible impact day to day or week to week, the changes we made over longer time frames were incredibly satisfying. I will only briefly outline four areas here—precision medicine/genomics, neuroscience, culture, and COVID-19 and social injustice.

To leverage the strengths in human genetics and genomics at UCLA as well as the diverse clinical population served by UCLA and more broadly by all five University of California medical centers (all of whom shared a common EHR data warehouse of nearly 16 million patients), we established an Institute for Precision Health (IPH), with Dan Geschwind as director. Dan recruited a remarkable deputy director, Dr. Clara Lajonchere. Within a few years, IPH developed a program to recruit patients for genetic sequencing from across the UCLA Health System, started the California Center for Rare Diseases at UCLA, founded a center for digital healthcare technologies, started an induced pluripotent stem cell (iPSC) core, and spearheaded multiple collaborations across the campus and city. With support from IPH, we started a Master’s Program in Genetic Counseling in 2020, with a specific focus on increasing diversity in genetic counseling. In 2017, I partnered with the dean of the UCLA Samueli School of Engineering to create a new cross-school Department of Computational Medicine committed to transforming patient care by leveraging recent advances in artificial intelligence and genomics. Under the leadership of computer scientist and geneticist Eleazar Eskin, this department immediately had an impact on bringing data scientists and clinicians together—for example, to use artificial intelligence to build a preoperative predictor to identify patients scheduled for elective surgery who are at high risk for complications. Together, these efforts all demonstrated the transformative impact of connecting research and scholarship—in this case in genetics, genomics, and data sciences—with the clinical enterprise.

Before becoming dean, I led what was called the “neuroscience research theme” at UCLA, an effort to drive synergy across the basic and clinical neurosciences. As dean, I was able to move a group of neuroscientists to shared, newly renovated lab space in the medical school, which provided an exciting concentration of outstanding neuroscientists working on neural circuits. More ambitiously and less successfully, I set out to integrate the clinical neurosciences—psychiatry, neurology, and neurosurgery, with the basic neurosciences. UCLA benefits from having tremendous resources in a state neuropsychiatric institute now called the Semel Institute for Neuroscience and Human Behavior. The Semel Institute is directed by the chair of psychiatry, and out of a deep belief that neurosciences are enriched by inclusion of many disciplines, from basic neurobiology to social sciences to clinical neuroscience, I sought to split this leadership into two. I reasoned that while the chair of psychiatry needed to be a board-certified psychiatrist, the director of the Semel Institute needed to be a neuroscientist, who could be a neurologist, a neurosurgeon, a neurobiologist, a social scientist, or a neurobiologist. I also believed that there was a conflict in having the director of an interdisciplinary institute of neuroscience also serve as chair of a single department. The timing for this change, however, was not right and it (and I) ran into great opposition from the leadership of the Department of Psychiatry and the Semel Institute. This was a real lesson for me in the need to listen to all voices in a community early on, to understand their views, and to gain trust. It was also a lesson in what often feels like a glacial pace of change in academic organizations.

One of the most surprisingly satisfying initiatives during my time as dean was the establishment of what we called our “Cultural North Star.” I realized early on that there was not a unifying sense of mission within the school—clinicians did not understand scientists, staff and faculty and trainees were guided by three different sets of expectations, and the community had grown so large that rather than having a cohesive sense of community, we were a patchwork of smaller and often isolated enclaves. I was also surprised by how much time (and resources) were taken up by resolution of end-stage conflicts, and hoped that articulating an explicit set of cultural values would help to generate a framework or playbook to guide decisions, interactions, and behavior. After a year-long audit of the school’s culture, in partnership with Emily Rose and Emilie Marcus, we developed the Cultural North Star as a means of unifying our efforts to advance science and medicine—with three pillars, Do What’s Right, Make Things Better, and Be Kind, and 12 supporting statements (e.g., “we listen to diverse voices,” “we are grounded in ethics and data,” “we embrace failures as opportunities to learn”). As a scientist, I had not put much thought into organizational culture and harbored some skepticism. To my surprise, this turned into one of the most impactful efforts I undertook with my team because it provided the necessary context for self-reflection and open discussion, catalyzing change in the school’s historic power structures. At the same time, it enhanced the school’s collaborative efforts and our sense of community and purpose.

Serving as dean during the COVID-19 pandemic was nothing less than inspiring and humbling. In early spring 2020, the school rapidly pivoted from in-person research and education, to virtual and remote—quickly developing innovative approaches to teaching and communicating. Faculty, staff, and trainees came together to build and distribute personal protective equipment for healthcare workers, and a remarkable group of faculty developed a novel large-scale, inexpensive technology for COVID-19 testing, called SwabSeq, which is now being used across our campuses as well as by other universities. Our clinical trials infrastructure enabled deployment of multiple clinical trials in record time, and we collaborated with institutions across the country as a center for therapeutic trials and for vaccine testing. The philanthropic community was extremely generous, and we developed a structure for evaluating and rapidly distributing these funds for research on COVID-19, including processes for prioritizing precious patient samples for this research. In many ways, COVID-19 showed me what was possible—what the research community could do if it came together to use our expertise to address a shared crisis.

The COVID-19 pandemic shone a stark light on health disparities in Los Angeles, as it did across the nation and the globe. Similarly, the murder of George Floyd in May 2020 forced a long overdue reckoning with social and racial injustices within our school. We established an Anti-Racism Roadmap, led by Executive Vice Dean and Vice Dean for Education, Clarence Braddock, to cocreate with the DGSOM community a path to advancing racial justice, equity, diversity, and inclusion within our institution. Hosting a series of zoom dialogues, with hundreds of DGSOM members, we addressed issues including data transparency, the minority tax, accountability and reporting, and beyond. These discussions led to the development of publicly available databases on faculty, staff, and trainee demographics across the school, a clear portal for reporting incidents of mistreatment or discrimination, new policies and goals for recruitment and retention, and more. The Anti-Racism Roadmap gained an unstoppable momentum, one that made it clear to me that the world was changing in a way that—while it would not happen overnight—would continue to move forward.

The “Me Too” movement also has helped to remake the culture in medical school during the six years I was in the dean’s office. A new line was drawn in the sand making sexually harassing behaviors that plague the work place, including academic medicine, no longer acceptable. Along with our long-needed reckoning with racism reckoning, academic medicine is confronting uncomfortable truths that have been too long ignored. As dean, I tried to create institutional structures that hopefully would allow for the sustained examination and alleviation of the often-corrosive relationships of power and domination within our medical school as well as those social forces that create health and social inequities. To this end, for example, I established a new interdepartmental research initiative in the school called “Health Equity and Translational Social Sciences,” recruiting outstanding physicians and social scientists to understand and address health disparities, creating a translational social medicine to affect health in a manner analogous to the translation of biomedical research to health. This effort had the unexpected and powerful impact of changing the way the more biologically oriented research programs viewed their own work, as they began to think about the impact of their studies on equity.

Simons Foundation

Sitting in my office one day in the midst of the COVID-19 pandemic, I received a call from Huda Zoghbi asking if I would interview to become the head of the Simons Foundation Autism Research Initiative (SFARI). I remember the sense of excitement I felt after our discussion, recognizing the potential to advance our understanding of mind, brain, and behavior in a significant way. As dean, I deeply missed thinking about neuroscience. While I kept my lab going, thanks in large part to the amazing scientists in my group, I missed having time to think about science in a substantive way. I found it frustrating that while the opportunities for making transformative discoveries in neuroscience (and in other fields) were so astounding, I was spending so much of my time simply fighting for the resources to support this research. I often felt like a combination of Sisyphus and Don Quixote—deeply believing in the power of biomedical research and academic medicine and fighting for that against many currents and tides. And meeting with everyone at the Simons Foundation and at SFARI made it clear to me that I would be joining a team with a truly shared mission of advancing neuroscience, making a difference in the lives of autistic individuals and their families, making a difference in the way we do science, and making a difference in how the power of science is understood. I also saw this as a way to have more time for my own science, with my lab continuing at UCLA. And, personally, it felt like the right time for a change for Joel and for me—we were ready for a new adventure—and I missed New York!

As I look back over the years, I recognize that the path I have taken has been a combination of purpose and chance. I see the thread and the focus that links literature and public health and neuroscience and academic administration and the Simons Foundation, but I also recognize that I’ve not necessarily followed a traditional path, but rather have moved in different directions at many points in my life. I think back to the metaphors of clothing in Carlyle’s Sartor Resartus and the fishing net in Melville’s Moby Dick, and recognize that I’ve built my life around the latter. Rather than define my life by a fixed and tailored roadmap, I’ve been open to the often unanticipated opportunities I’ve encountered along the way. As I get older, I also recognize how much we all live in worlds that we believe are as permanent and natural as the physical world (the world of English literature, the world of medicine, the world of science, the world of international development), but instead that are all simply human constructions. While the worlds we inhabit give meaning to our lives, they have always struck me as arbitrary and relative. The joy that I have experienced in discovering biological truths, in poetry and art that captures beauty and truth, in working with others to create or improve a community, and the immeasurable love I feel for the beloveds in my life provide a more transcendent, and less relative, meaning that fills me with a sense of great fortune and gratitude.

From the perspective of a neuroscientist studying the biology of memory, I am also aware that memory is dynamic. The story I would tell 5, 10, 15 years from now would likely be changed as a result of future experiences. This narrative is my autobiography that is bounded by and given its specific context-dependent meaning at this particular moment in time. But just as I imagine my brain is undergoing new programs of gene expression and forming new synaptic connections as I begin a new adventure with the Simons Foundation, I suspect that with new experiences and new synaptic connections, my narrative will be given new meanings as my brain transforms the dynamic and complex ebb and flow of information between genes, proteins, cells, synapses, circuits, thoughts, feelings, and experiences into a single coherent story.

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Thompson, KR, Otis, KO, Chen, DY, Zhao, Y, O’Dell, TJ, Martin, KC. (2004) Synapse to nucleus signaling during long-term synaptic plasticity; a role for the classical active nuclear import pathway. Neuron 44: 997–1009.

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Kevan A. C. Martin

BORN:

Cape Town, South Africa

May 22, 1952

EDUCATION:

University of Cape Town, BSc (1973)

University of Cape Town, BSc Honors (1974)

University of Cape Town, MSc (1975)

Wolfson College, Oxford, DPhil (1978)

APPOINTMENTS:

Weir Junior Research Fellowship, University College, Oxford (1979)

Junior Dean, University College, Oxford (1979)

Salverson Junior Research Fellowship, University College, Oxford (1982)

E. P. Abrahams Cephalosporin Junior Research Fellowship, Linacre College, Oxford (1984)

Ovenstone Senior Research Fellow, University College, Oxford (1985)

Senior Scientist, Medical Research Council Unit, Oxford (1985)

Foreign Associate of the Royal Society of South Africa (1990)

Old Members Trust Senior Research Fellow, University College Oxford (1990)

Henry Head Research Fellow of the Royal Society London (1990)

Senior Treasurer of the Amalgamated Clubs, University College, Oxford (1993)

Double Professor of Systems Neurophysiology and Director of the Institute of Neuroinformatics, University of Zurich and Swiss Federal Institute of Technology (ETH; 1995–2019)

Kevan A. C. Martin forged an interdisciplinary, multilevel approach to analyze and describe the structures and functions of the local and long-distance circuits in the neocortex in multiple species. His work has been highly influential in establishing general principles of structure, function, and computation in neocortical circuits. With Rodney Douglas, he founded the Institute of Neuroinformatics in Zurich, whose mission is to discover key principles by which brains work and implement these in artificial systems that interact intelligently with the real world. His experimental research is characterized by detailed quantitative analyses of the physiology and morphology of identified neurons and their synaptic connections and synthesizing these results in computational models that can be realized in software and in novel analogue ‘very large scale integrated’ (VLSI) silicon circuits. One key result of this synthesis was a “Canonical Circuit” for neocortex that, while originating from studies of the cat’s visual cortex, provides an operational description of local cortical processing in species from mouse to man. Formulated at a mesopic scale, the Canonical Circuit forms the critical bridge between the microscopic synaptic and single neuron levels and the macroscopic levels of systems and behavior.

Kevan A. C. Martin

Nature or Nurture?

Any autobiography immediately raises in its author questions of free will and determinism. To what extent is their history simply a random walk determined by chance encounters, and to what extent is their history tightly constrained by circumstances? Perhaps one’s personal history can be read either as a pinball’s path of chance encounters and lucky breaks, or a predetermined journey along a well-constrained path. A neuroscientist’s knowledge of both genetic inheritance and neuroplasticity assures us that we can never free ourselves of the influences that shape us; indeed, our own biology ensures that we can never have a truly original thought. Having spent most of my academic life studying central processing in the visual system, what significance do you give to the fact that my earliest years were spent at a blind school, or that my father founded a school for children with cerebral palsy?

With a long look in the rearview mirror, I see that structure is an abiding interest of mine. Was this because my paternal grandmother, whom I never knew, had left us boxes of microscope slides she had prepared for her biology students? There is evidently a strong aesthetic element too, for magnified views of nature I find beguiling. And then there is the pull of understanding how things might be built and work. Was this a consequence of being around my father who was forever repairing cars, building boats, and recycling and repurposing what my mother called “junk,” and why I still ride (and maintain) his 1936 Ariel Square Four motorcycle? “Nothing knowingly thrown away” could have been his motto; hence, the fascinating museum of flotsam and jetsam I grew up with, which included my grandmother’s histology collection. What looms largest in the mirror are the people I encountered and worked with, but two most especially: David Whitteridge, who introduced me to the visual cortex and with whom I shared a lab and office for 17 years until his death, and Rodney Douglas, my scientific partner on a voyage of discovery that is now in its fourth decade. The longevity of these scientific relationships is a pertinent fact, as is the presence of a penumbra of colleagues who have accompanied me for long periods of my scientific life. But the rearview mirror also reveals that my greatest joy is being with people who know a lot more than I do about all kinds of things.

Out of Africa

I was born on the southern tip of Africa, the middle of three sons. We lived in a bungalow in the grounds of The Athlone School for the Blind, a school for black children, which was surrounded by indigenous bush on the Cape Flats, a sandy plain between Table Mountain and the Helderberg Mountains. My father had been appointed vice principal at the ripe age of 23, more on the basis of his three degrees than on any expertise in special education. He majored in physics for his BSc, English for his BA, and education for his BEd—a graduate degree. He had entered the University of Cape Town (UCT) at age 16, propelled by his parents, both of whom were émigrés from the East End of London. His mother had earned a BSc in biology from Bedford College, which was founded in 1849 as the first higher education college for women in the United Kingdom and became one of the University of London’s colleges in 1900. He was musically gifted and immensely practical, no challenge seemed to faze him. Years later I came across a pupil’s braille essay about the visit of Helen Keller to the school in 1951, which my father had read and transcribed between the lines in his neat longhand. One of my earliest memories is of blind children on a terrace, weaving baskets from long canes and grasses. The school still exists, and its chapel records that my grandfather was a benefactor.

In 1954, my father was invited to found “The Cape School for Cerebral Palsied Children.” My mother, who read avidly and had the most extraordinary memory for names and faces, which she retained well into her 90s, took care of her sons’ schoolwork while my father set up the new school in a Victorian villa in a suburb of Cape Town. There were no African models for the school, but from the outset, my father understood that these children would need not just teachers with special skills, but the support of a multidisciplinary team including parents; physio-, speech, and occupational therapists; psychologists; social workers; and nurses and doctors. This holistic care meant that children with brain damage, mainly due to birth trauma and many wheelchair-bound, could now be educated at a school, a hugely positive step for both pupils and parents. It was successful and a bespoke new school complex was built and named “Vista Nova.” As young children, we met children with conditions that are never seen now, mainly because of the huge improvements in perinatal care and attention to genetic disorders, like phenylketonuria. Athetoid, ataxic, and spastic cerebral palsy were common. Before ventricular shunts were used, we saw hydrocephalic children with huge heads, and before seatbelts were compulsory, we saw the consequences of head injuries from car accidents.

Despite my mother’s best efforts, I was an indifferent pupil, but, like her, I was an avid reader. My academic progress was not helped by being at an eminent school for white boys. The school’s main interest seemed to be in its sporting achievements—rugby, field hockey, cricket, athletics, swimming, and tennis—not academic excellence. I developed a preference for gymnastics, which was a minor sport, so I trained at a local club. Throughout my high school years, I represented the Cape Province at the National Championships, and in my final year of school, I represented South Africa as a junior. This competence attracted an invitation for me to join a flying trapeze troupe to tour Europe. It was tempting, but at age 17, it seemed premature to make such a flying leap.

National service was compulsory for all white men, so January 1970 found me starting basic training as a rifleman in the 1st South African Infantry Battalion, which had its camp outside the semi-desert town of Oudtshoorn, famous for its ostrich farms and the Cango Caves. All South Africans were officially segregated by race, but whites were further divided by language and culture. All the teaching at my high school was in the English medium, so although Afrikaans, the language of the ruling Nationalist Party, was a compulsory subject, I could not speak it, nor then had any interest in doing so, for Afrikaners were generally regarded by English-speaking whites as nationalistic, unsophisticated, and racist. For a year, I lived in an Afrikaner community and heard (and spoke) little else than Afrikaans. To escape the discipline of the army, I would disappear from camp on Friday evening and hitchhike hundreds of miles, returning late Sunday evening, hoping my absence hadn’t been noticed by those in charge. The motorists who gave me a lift were invariably rural Afrikaners, who warmed when hearing my improving Afrikaans, and this resulted in many enlightening conversations and more often than not, they would invite me back to their homes. In the face of their spontaneous kindness and generosity, it was impossible for me to sustain my English-speaker’s negative caricature of Afrikaners.

Undergraduate Times

Discharged from the army, I joined a bank, but apart from learning the joys of double bookkeeping, it became rapidly clear that banks embodied the same divisions of rank as the army, so an escape to “higher education” at UCT beckoned. But what to study? Some years after establishing Vista Nova, my father had completed a psychology major by correspondence. I found his textbooks fascinating reading, so psychology went on the list of my major subjects along with logic and metaphysics as well as physiology. As first-year preparatory subjects, I read chemistry 1, physics 1, and zoology 1.

The first semester of zoology was taught by the head of department, Professor John H. Day, a marine biologist and invertebrate specialist. He took us on a tour of the animal kingdom, and despite complete ignorance of biology, and Latin, I was instantly entranced by his lectures. I couldn’t believe how much was known about obscure (to me a least) animals with Latin names. Why would anyone want to measure the pH of the fluid in the nephron of an Amazonian beetle, for example? Similarly, psychology 1, which initially seemed to be dominated by lectures on learning theory and statistics, was no less gripping. At my secondary school, we had done precious little practical work, so the afternoon practical classes were exciting. There was one exception: I discovered from the physics practical notebooks of my father, which he had kept, that ours were pretty much identical to those he had worked through 30-plus years earlier, so this greatly dented my enthusiasm for experimental physics. Nonetheless, after just a month at UCT, the uncomfortable question arose: how was it possible that my secondary school teachers, all of whom had university degrees, had not been able to convey one jot or iota of the intellectual delights I was experiencing?

With four lectures each morning and four afternoons of lab practicals, there was little opportunity for any outside activities, but I squeezed in flamenco guitar lessons with the Cape Town doyen, Pablo Navarro. As I progressed, I joined Pablo to accompany student flamenco dancers at Hazel Acosta’s studio in Cape Town. Academically, I hung on by my fingertips and no one was more surprised than I was when I managed to pass all my end-of-first-year exams.

I came from a frugal family and despite my brief encounter with banking, I never wanted to be in debt, so cash flow was always a problem. During the long vacation at the end of my first year, I managed to get a job with Jeffares and Green, a civil engineering firm that specialized in road design. As rank junior (temporary) I was assigned with the vital task of interpolating levels from the field notebooks that arrived back from the land surveyors, which I did with the help of a Facit mechanical calculator. There was a large pile of notebooks and my three months were spent working in the corner of the office of one of the design engineers, so I learned a lot about road design by osmosis. The highlight of the day, however, was hearing the tea trolley rattling slowly down the corridor to our door.

For similar cash-strapped reasons, during the second year of psychology, I worked as a “demonstrator” (i.e., technical assistant) in practical classes for the fresher-year psychologists. One of my co-demonstrators and practical partner was Tim Jenkin, who later joined the banned African National Congress and built letter bombs for scattering leaflets. He was arrested and sentenced to imprisonment in Pretoria jail, from which he escaped by fashioning wooden keys to open all the doors. His autobiography was made into the film: Escape from Pretoria. The university campus was a hotbed of antigovernment protests, in which many of us participated, but it was also infiltrated with informers, so detention without trial of students was commonplace.

My second year saw my entry to logic and metaphysics. Dr. Barney Keaney had just been appointed as the senior lecturer, and he had devised a compendious two-year course, which began with Greek philosophy and logic and ended with the linguistic turn of the 20th century. It was a complete contrast to the work I was busy with on the science side of the campus. The criticism might be levelled that such a whirlwind ride through centuries of logic and philosophy was far too superficial for a major university course and that it would have been better to concentrate on one or two philosophic schools of thought. On the contrary, what the course did in effect was to provide me with a series of hand- and toe-holds up an otherwise blank cliff face that I could then use to climb whatever route I chose. It has worked as well as intended.

Physiology 1 was taught at the medical school as part of the preclinical years for the medical students. The head of department was a ruddy Scotsman, Archibald Sloan, who chain-smoked, but whose research area was exercise physiology. The most memorable lectures, however, were the laconic deliveries of Professor Leon Isaacson, a renal physiologist who had made fundamental measurements of the electrophysiology of the proximal tubule in Maurice Burgs’s lab at NIH, where the preparation of the perfused isolated proximal tubule was first developed. Leon gave the most research-based lectures in the course, yet as a practicing consultant at the neighboring Groote Schuur hospital, his clinical experience was immense, so the medical students benefited hugely by getting his firsthand accounts of diseases of the kidney. Unlike most of the other lecturers, if I managed to generate one page of notes during one of Leon’s lectures, it was a lot.

The physiology practical classes seemed to have been devised in the late-19th century, and apart from the histology practical, many of them used equally vintage apparatus, gleaming of brass. Spirometer recordings of respiration, or recordings of frog muscle twitches, were made using clockwork-driven smoked-drum kymographs. We made our own smoked paper using a paraffin burner, and then varnished the results before we smudged them, cutting out the relevant piece to mount in our lab notebooks. Although the practicals and the apparatus we used were antiquated, having a mechanical rather than electronic connection between organ and recording was instructive, and the necessity of adding significant details like date, time-base, and signal calibration to the smoked drum paper, made us more aware of the nature of the signals we were recording and the limitations and potential artifacts imposed by the methods of recording.

Psychology 2 demanded volumes of reading and essay writing. The problem of accessing the original literature was solved by the campus central library making multiple photocopies of papers or book chapters, which we could then reserve for an hour at a time. This was a cheap and efficient system and gave us good practice in reading comprehensively and note-taking. The contrast to the modern era could not be more profound: on most students’ desks (and usually the surrounds of the printer) one now finds large piles of PDFs, which seem to act more as a comfort blanket than a desk-top reference library. Seeing these piles always reminds me of the story Sydney Brenner told of when he was director of the Molecular Biology Laboratory and wanted to photocopy an article after-hours. A graduate student had commandeered the sole photocopier and was copying multiple articles from a large pile of journals. Watching this for some minutes, Sydney could stand it no more and interrupted the student (who apparently had no idea to whom he was talking):

“Why don’t you try neuroxing the articles instead of xeroxing them?”

“What is neuroxing?”, the student asked, innocently.

“Well,” said Sydney, “you open the journal and you scan the pages one after another with your eyes.”

The ingenuity of psychology’s system of short loans of critical readings also meant that the lecturers could include on our reading list otherwise-obscure or hard-to-obtain articles. In this way, I read Sigmund Freud’s investigations of the histology of the nervous system where his embryonic ideas about excitation and inhibition began to be formed, and discovered German physiological psychologists like Willem Wundt, Hermann von Helmholtz, and Hermann Ebbinghaus through translations. It was the beginning of my long relationship with libraries, now a lost pleasure for most. Our reading is now corrupted by the predictive capitalism of websites with their siren call—“if you like that, you’ll like this”—whereas in a library, there is always the serendipity of a book title catching your eye, or accidentally getting absorbed in a journal article whose topic is at oblique angles to the one you were intending to read. While we have acquired instant access to the world’s libraries, the traps set by clickbait are too tempting, and so the accidental encounters are greatly diminished if not lost.

During my second year I met Fred Roux, a PhD student in the Department of Civil Engineering who was studying the properties of concrete at elevated temperatures to see whether it could be used for the secondary containment of nuclear reactors. He was funded by the Atomic Energy Commission and sought an assistant to help him with data analyses, and who better than I to calculate Young’s modulus, plot stress-strain diagrams, and make the coffee? Fred lodged with the Hofmeyr family, and through Billy and Ursula Hofmeyr, I got to meet some of the leading artists and musicians in South Africa and, inter alia, learn about fine wines—something that never appeared on our Baptist table. Now with an additional income from Fred, I could contemplate buying small artworks on paper and began to frequent the gallery of Joe Wolpe, whose fine eye, prodigious memory, and a gentle humor was a delight. The first painting I ever bought was from another friend of the Hofmeyr’s, Paul du Toit, the former pupil of Jean Welz, an Austrian émigré artist who happened to live around the corner from us and later drew my portrait. After Paul du Toit’s early death, I wrote his biography, which was beautifully produced by the Fernwood Press.

The Department of Civil Engineering became my second home, for next door to Fred’s concrete blocks, ovens, and testing machines was the Water Resources Laboratory, run by the formidable Professor Gerrit van Rooyen Marais. He needed someone to make figures for his reports and publications. I made myself available and was introduced to the “Leroy Lettering Instrument,” Rotring pens, and many variants of stencils and French curves. Professor Marais showed me how to use a form of graphical statistics called probability paper, which I later adopted for analyzing data for my final year psychology thesis on time perception.

Without the medical students, my final-year physiology course had shrunk to a dozen or so students. The course material was top-heavy with biochemistry that required many metabolic pathways to be learned, which I found a chore, but the practicals were more interesting, having moved into the electronic age. We were taught how to use an oscilloscope, how to make pipettes to record intracellularly from the isolated heart, how to do calculations with an analogue computer, and how to measure sodium transport across frog’s skin using an Ussing chamber. We also had to give two seminars on set topics in front of the faculty. I got the short straw as my first seminar was on sodium transport. I set off boldly, but was brought up short by Leon Isaacson when I cited an experiment in which they had used red blood cell ghosts: “What are red blood cell ghosts?” Leon inquired. I had to confess I had not the ghost of an idea and fell silent in acute embarrassment while Leon explained what they were. It was the best lesson ever to never stand up in front of an audience without thorough preparation and understanding of what I was talking about.

As final-year psychology students, we had a series of lectures from the head of department, Professor W. D. Radloff, for the first time. He would bring into the class a box file of reprints, which he would rifle through, pull out one that caught his attention, and give us a brief synopsis of its contents, and then go on to find another. One morning he pulled out a review published in Nature in 1972, with title “The Visual System and Neuronal Specificity” by R. M. Gaze and M. J. Keating. It was a critical review of Roger Sperry’s “chemoaffinity hypothesis,” and it concluded with the statement that there was much confusion in the field, not helped by ill-defined terms and ambiguity of the meanings of descriptions like “order,” “randomness,” and “specificity.” Scientifically and philosophically it sounded intriguing, and the paper stuck in my mind. Little did I then know that five years later I would be sitting in a small room in Oxford defending my DPhil thesis against a sustained critique by my external examiner, the same Mike Keating.

Engineering and Electrodes

In the long vacation Professor Marais gave me a job, and so I was introduced to the kinetics of waste-water treatment in the activated sludge system. The lab was a large temperature-controlled space with rows of benches for chemical testing, side rooms with chemicals, ovens and fine balances, a cold room for storing raw sewage brought in a tanker from a local plant, and a mezzanine of small offices for the master’s and PhD students, each of whom ran a lab-scale treatment plant. Taliep Lakay, the multitalented master-technician, who knew how to do everything, as well as the washing-up, taught me the battery of chemical tests used. He had pinned up a gallery of quotes he overheard from the professor: “Think until your head gets hurt,” “Write it over and over until you get it right.” Stalking the lab, chewing on an unlit pipe, was Professor Marais himself, who was merciless with any student he thought was not paying full attention to their experiment, like not maintaining their pumps, properly adjusting the aeration levels, or cleaning the settling tanks. A red rag was for the student to announce they couldn’t be there on an evening or weekend because they were seeing their significant other or family. I soon realized that his insistence on undivided attention to the experiment in progress had a point, because the treatment plants took some weeks to reach steady-state and any shutdown meant time was lost in getting them back to steady-state. I later distilled this notion into the mantra for my own students during experiments: “constant vigilance.” One day I had finished all my analyses early and was heading for the door. Professor Marais spotted me and called me back:

“Where are you going?”

“I’ve finished all my analyses for today,” said I.

“Well go back and look at your apparatus and think harder about your experiment—if you walk out of the door you will not be thinking about your experiment at all!”

It was no nine-to-five environment: Professor Marais was first in and last to leave and expected us to follow his example.

I wanted to do a fourth honors year in ethics and physiology. Unfortunately, Archie Sloan insisted that the honors program in physiology was so demanding that there was no possibility of my completing a double honors—this despite the fact that I had just managed to graduate with a BSc degree in three major subjects. For my project, I decided to record from neurons in the preoptic area of the hypothalamus, which was an area of interest to my supervisor, the endocrinologist, Cyril Beardwood. The problem was that no-one knew how to make single-unit unit recordings. Fast-forward three months and after many failed attempts to record with insulated insect pins, I discovered an abstract by David Hubel (see Volume 1) with his recipe for a varnished tungsten electrode. Another month went by before I figured out how to straighten a coil of tungsten wire, etch it, and insulate it. Eventually, I recorded a fair number of stable single units. The real lesson learned, however, was never to reinvent the wheel—if someone already knew how to do something, go and learn it from them.

In the Civil Engineering Department, my career as a draughtsman flourished. The head of department, Professor John D. Martin, was writing a theoretical work for MIT Press, titled Plasticity, and he needed someone to draw the figures. He would give me a sheaf of pencil sketches of what he wanted, and it was my job to turn them into an annotated pen-and-ink drawing of publication quality. I never got to see the published tome until decades later and the wonder was that I had found the time to do so many drawings.

Sir Bryan Matthews—inventor of the oscillograph, differential amplifier, and other physiological instruments, and rare collaborator of E. D. Adrian—visited South Africa in 1974 as part of a Royal Society-British Council educational tour. His lecture in the Physiology Department included a demonstration of the activity of a muscle endplate, which he recorded from himself with an electrode fashioned from a hypodermic needle. Seeing an action potential firing under conscious control made a huge impact on me. In the lab, he tried to show us honors students how to record from the optic tectum of a frog, but our pipettes were inadequate. The most significant part of his visit, however, was giving me advice about which neurophysiologist I might approach in England about the possibility of doing a doctorate. He suggested I write to Professor Charles Phillips, a distinguished motor cortex physiologist in Oxford. I duly posted off a hand-written blue aerogramme and waited. Some weeks later I had a reply: Professor Phillips had recently been appointed head of the Department of Human Anatomy following the untimely death of Geoffrey Harris, discoverer of the portal system of the hypothalamus, which delivers releasing factors to the anterior pituitary. Although he was not in a position to take on a student, he had asked around the department and had found that someone called Tim Horder might be interested. In those pre-internet days, there was not much I could discover in the library about what research topic Tim actually pursued. Still, I wrote to him explaining that I was particularly interested in the work of Gaze and Keating on the retinotectal system and Sperry’s theory of chemospecificity. It was a bull’s-eye, for Mike Gaze had been Tim’s PhD supervisor at Edinburgh University. A slow exchange of blue aerogrammes followed about my science, my unusual academic trajectory, funding, and choice of college. Tim typed all his letters, but was obviously not a touch typist as the letters bounced up and down and there were many xxxx crossings-out.

Mastering Wastewater

With the increasing likelihood that I could go to Oxford, whose term started in October, I needed to save some money to supplement the small bursary I had been awarded from UCT for postgraduate study. I asked Professor Marais if he had a lab job for me for nine months after I finished my BSc (Honors) in December. He made the unexpected suggestion that I enroll for a master’s degree in civil engineering. Thus, in addition to the long days running my experiments on the biological removal of phosphorus from wastewater, which was a new topic in the lab, I found myself sitting with card-carrying civil engineers in night courses in aquatic chemistry, kinetic modeling, and plant design. I already knew how do the chemical analyses and maintain the reactors, so I was quickly up and running, but the theory was a longer haul and I needed a lot of handholding from one of the PhD students, George Ekama. Professor Marais’s kinetic equations showed that only a small amount of phosphorus was removed by the practice of routinely extracting a fixed fraction of the liquor in order to maintain a constant “sludge age.” The fraction of phosphorus taken up by the active organisms could be significantly increased, however, by subjecting the sludge to a short anoxic phase, which could evoke something called “luxury uptake” of phosphorus—that is, uptake more than is needed for building cells. I realized that Professor Marais’s kinetic equations predicted that under luxury uptake significant amounts of phosphorus would be removed if the sludge age was kept sufficiently short. When I took my reasoning to him, I expected the worst, but he saw the significance immediately, and we devised a series of experiments to test the quantitative predictions.

Six months later, and a lot of intensive work, a good fit of experimental data to the theoretical curve had emerged. It was my first experience of having a significant insight and testing it experimentally—hugely encouraging. By then I was reasonably experienced at writing up experiments and, unlike Professor Marais, who was an Afrikaner, my mother tongue was English. I gave him the first draft of my research report. Some days later, he stalked in to the lab and gave me my typescript back without any annotations or corrections, and simply said, “write it over.” I was livid, but I had forgotten that he was an avid reader of English prose and poetry, and that the essayist, Lord Macaulay was one of his favorite authors. Indeed, Professor Marais wrote succinct and limpid prose, and no one could set out the derivation of an equation more elegantly than he. Some 15 drafts later, we called it quits. My study was preliminary, and there were years of theory and experiments by others ahead, but what my work had done was to embed the problem of phosphorus removal in the familiar kinetic language of the lab so that what could be predicted and what had to be measured was defined. Much later, I discovered the Zen concept of Shoshin (the beginner’s mind), in which there are many possibilities, unlike the expert’s mind, in which there are few. A beginner’s mind certainly applied to mine. The rare opportunity I had been given was to plant the seed for what became a major research area of the lab.

The Dreaming Spires

I wrote my theory exams, submitted my thesis, and left for Oxford in September 1975 with one suitcase, my guitar, and clutching a blue aerogramme from Tim Horder saying he had accepted me as a DPhil student. Tim’s unconventional typing style made for an interesting conversation with the U.K. immigration officer, who reluctantly let me in, but with the injunction to register immediately at the Oxford police station. Out of the train window, I caught my first glimpse of the dreaming spires of Oxford, which was to be my home for the next two decades.

Tim had secured a place for me at a new graduate college, Wolfson College. Its founding principal, Sir Isaiah Berlin, had decreed that it would not follow the traditional college hierarchy of Junior (undergraduate), Middle (graduate), and Senior (Fellow) Common Rooms and it would have no “high table” for Fellows in the dining hall, and would be mixed, with families in residence. This was an ideal choice for me as I had not the faintest clue of the subtle mores of Oxbridge college life. Even then, the competitive nature of the intellectual conversations in the Wolfson common room was like nothing I had ever experienced, and it had a strange psychological consequence: after about six months I realized that people were finishing my sentences, so tentative had I become about uttering anything. But, understanding that most of these graduates had been training in this clever repartee for years, I consciously remedied my speech impediment. What was most exciting was to go to lectures by people whose work I had studied, particularly the philosophers, like Gilbert Ryle and Peter Strawson, and to repair my deficient knowledge of classic films through the Oxford University Film Club. At the end of the year, good news arrived from Cape Town: I had been awarded an MSc in civil engineering cum laude. I felt an imposter, but because I was on another track, no card-carrying engineer need be too offended.

My work pattern also had to adapt to Oxford life. In the first weeks, I would arrive too early at the Department of Human Anatomy and have to wait for someone to unlock the front door. Unlike Professor Marais, Tim did not stalk the lab or berate his students. Instead, he would appear at random intervals, have a quick chat, and then rapidly disappear again to the library or to his heavy tutorial load in Jesus College. He was a decided iconoclast, which made for interesting conversations on embryology and pattern formation, about which I knew nothing. Tim spent one afternoon initiating me into the techniques of anesthetizing a goldfish, preparing it for recording from the optic tectum, and mapping multiunit receptive fields using an Aimark perimeter he had loaned from the University Laboratory of Physiology where he had been a Demonstrator. Being able to wave a wand around in the visual field and find the small area where the neurons responded is a wonder that has never left me. The electrodes and amplifiers Tim used were decidedly primitive, even compared to what I had used in Cape Town, so as soon as I acquired a license from the Home Office to do animal experiments on my own, I asked Professor Phillips if he had any more modern equipment to spare, whereupon he lent me one of his valve cathode followers, powered by a 110-volt battery, and some Tektronix valve amplifiers. Not quite solid-state technology, but nonetheless, it all worked flawlessly. I also secured three glass-insulated tungsten electrodes from Alan Ainsworth, Colin Blakemore’s technician in Cambridge. They were top-of-the-range and I managed to make them last the entire two years of my DPhil experiments. I had two projects, the first was to extend Mike Gaze’s work on the plasticity of retinotectal connections, the second was to explore further the phenomena first observed by Mike Keating that binocular receptive fields on the optic tectum of frogs remained congruent throughout post-metamorphic development despite large shifts in the relative positions of the two eyes.

Figure 1.    Preparing to map the optic tectum of an anesthetized goldfish using an Aimark perimeter. Photo credit: Stuart M. Bunt.

Anatomical Matters

As I was in the Department of Human Anatomy, I thought it would be a good opportunity to learn some human neuroanatomy, so I asked the Reader who ran the class, Tom Powell, if I could watch the medical students’ dissections.

“Oh,” he said. “I’m short of a Demonstrator, would you be interested?” Of course, I would! Every week, I took my Cunningham’s Manual of Practical Anatomy and sat down with a fixed brain and learned how to do the week’s dissection. Having crammed the necessary knowledge, I thought I was sufficiently well-prepared for the class, but I had not anticipated the cleverness of the Oxford medical student, who needed no dissection guide, just a gesture in the direction of which part of the brain to cut into very small pieces. Ten minutes into a three-hour class, I would be called over to a group clustered at a dissection table, given a handful of brain fragments and interrogated:

“Where is the fifth nerve nucleus?”

“Show us the corticobulbar tract.”

I would then have to reconstruct the three-dimensional (3D) jigsaw, not always possible, and resection the assemblage along the textbook dissection planes hoping there was still something left to see. I had not the confidence to tick them off, or the 3D knowledge to identify structures from odd planes of section, so it was all acutely embarrassing. I did learn neuroanatomy, however.

Tom Powell had a reputation for not suffering fools gladly, but he made an exception in my case and allowed me to use his histology facilities for my anatomical studies. His technician, Ron Brooks, taught me the newly introduced method of horseradish peroxidase (HRP) for tracing fibers from tectum to retina and to use tritiated proline to trace projections from retina to tectum. Powell’s group always took their “elevenses” in the lab, so while they drank tea, I cut and reacted sections and had free tutorials on the work they were doing on the macaque brain. Tom Powell is best known for his studies with Vernon Mountcastle on the columnar organization of the somatosensory cortex in the cat and monkey and of promoting the idea of the uniformity of the neocortex, but he made significant contributions to all the major systems of the brain. He was mentor to a stellar list of medically qualified doctoral students and postdoctoral colleagues, one of whom was Ted Jones—certainly the most scholarly and erudite neuroscientist I ever met. Many years later, I started some physiology experiments with Ted at UC Irvine, but the first Gulf War burst upon the TV screens, and we found it too distracting to continue.

In Tim’s lab, I was analyzing the phenomenon of “compression” in the retinotectal system of goldfish. The entire visual field of one eye is represented on the optic tectum and Mike Gaze and colleagues had shown that following removal of half the tectum, a complete retinal representation would reform on the remaining half, whether or not the optic nerve was cut. If the nerve was left intact, they occasionally found that the original projection did not compress and that the regenerating projection then formed an orderly map on the same piece of tectum, with the result that one location in the tectum received input from two quite different locations of the retina. I discovered how to induce “duplication” reliably and then found that, in a minority of cases, the duplicate projection was reversed in polarity compared with the remaining intact projection. This had never been seen and it led to another series of experiments to show that the ordering across the mediolateral axis could similarly be reversed reliably. I presented preliminary results on the duplication and polarity reversal at a meeting of the Society for Experimental Biology. The raw data seemed too complicated to show in a short talk, so I made schematics of the essential results, which may have sufficed for the non-cognoscenti, but unfortunately for me, Mike Gaze was in the audience. His comment was cutting: “Might have been interesting, but I needed to see the data.” Point taken!

On a visit to Mike Keating and Mike Gaze at the Medical Research Council’s National Institute of Medical Research at Mill Hill, I met the theorist David Willshaw, who was doing a master’s degree with Mike Gaze. David had already produced some important results from models of pattern formation with Martin Prestige in Edinburgh and later with Christoph von der Malsburg in Göttingen. Our paths were to cross many times over the next decades. I also met Chris Kennard, a medical doctor who was doing his PhD with Mike Keating, and later, as a distinguished neurologist, gave me critical support when my father was diagnosed with an acoustic neuroma.

Reading and Writing

Tim’s favorite medium of publication was the Proceedings of the Physiological Society, which were the abstract of talks given at the Physiological Society meetings and refereed by those present. The author order on Physiological Society publications was then alphabetical. Philosophically and morally, this seemed right to me and it removed all the jostling and elbowing for positions in the list of authors. All publications from my lab followed this alphabetical ordering, regardless of journal. We had interesting discussions over the years about this policy and its alternatives, but there was never great resistance to it. It was impossible in our team-style of working to define exactly who first thought what and who did what, as many journals now insist. The present absurdity of identifying several “first authors” and a similar slew of “senior authors,” with the “also-helped” authors sandwiched between, was avoided. I made it a practice not to include my name on an experimental paper simply because I had suggested the experiment, but had not made any significant practical contribution.

Tim had been invited to take part in a Society for Experimental Biology symposium, and the lectures were to be published in a book. He asked me to help with his chapter. I agreed, but what I had not properly understood was that he had no intention of publishing his lecture. Instead, Tim intended to review every publication ever published on fiber ordering in the vertebrate visual system. I spent countless hours in the Radcliffe Science library and the Bodleian library reading obscure journals, as well as sifting through mountains of reprints left by Sir LeGros Clarke in the department’s library. The pithy title of our chapter said it all: “Morphogenetics as an Alternative to Chemospecificity in the Formation of Nerve Connections: A Review of the Literature before 1978, Concerning the Control of Growth of Regenerating Optic Nerve Fibres to Specific Locations in the Optic Tectum and a New Interpretation Based on Contact Guidance.” Professor Marais would have been envious of the number of drafts it went through, but at least it gave me a comprehensive knowledge of the literature and gave grateful retinotectal researchers easy access to obscure observations. Perhaps this experience is the source of my frequent despair at the lack of scholarship evident in so many contemporary publications, even reviews. Our claim that the ordering of fibers emerging from the retina mattered to their final destination prompted a number of prominent investigators—David Hubel and Otto Creutzfeldt included—to investigate the retinotopic ordering of fibers in the cat’s optic nerve and tract. For me, it was a close encounter with an iconoclastic and perfectionist mind.

One benefit of my new knowledge was that Tim now let me tutor some of his students. I had little idea of what an Oxford tutorial was, but Tim explained that it involved the student reading set texts, writing an essay, and then discussing it, or “reading it,” during an hour-long one-on-one tutorial. Again, I had not reckoned with the cleverness of Oxford students, for one of my first tutees was someone called Mike Fischer, who had already graduated in physics and was taking a second bachelor’s degree in physiology. To describe our tutorials as “lively” is an understatement, for as experienced tutee, Mike easily anticipated all the twists and turns of my critiques of his essays and taught me more than I gave him, but it was a great experience for me.

In my third year I became ‘Captain of Men’s Boats’ at Wolfson College and captain of the Oxford University Men’s Gymnastics Team. The rowing, coxing, and coaching commitments meant that six days a week my early mornings were spent on the river—and there was that thesis to write.

The Waynflete Professor

It was time to think of where I was heading next. I wanted to continue research on the visual system, but I knew I did not want to continue working on retinotectal development and plasticity, which now needed a much more cellular-molecular approach to determine the mechanisms underlying the phenomena I had been observing. A solution was close at hand: Tim had heard that the Waynflete Professor of Physiology and head of the University Laboratory of Physiology was looking for someone to replace his postdoc, Peter Clarke, who was leaving to take up a position at the University of Lausanne. I put up my hand and was invited for an interview. It was memorable. In his vast office, I was introduced to Professor David Whitteridge, who sat in an armchair, with stockinged and sandaled feet, and proceeded with an interrogation. In addition to Tim’s recommendation, I had three pluses to offer: I was from a former British Colony, I knew a huge amount about a subject dear to his heart (retinotopic maps), and as a boy, I had made a crystal radio and knew what a “cat’s whisker” was and did. I got the gig. We worked together until his death 17 years later. He was the most gracious scientist I ever met, and although I wrote three different obituaries when he died, I still felt I had not given an adequate account of his life.

David Whitteridge was universally called “DW” or “Prof,” even by his wife Gweneth, who was a medical historian and an expert on William Harvey, discoverer of the circulation of the blood. Prof was a product of Oxford’s Medical School and had been Sir Charles Sherrington’s last Demonstrator. He did his BSc (now MSc) with Jack Eccles on transmission through the ciliary ganglion, work he published in the Journal of Physiology in 1937. For a meeting of the Physiological Society, he renovated and demonstrated some of Sherrington’s original apparatus and later wrote a Trends in Neuroscience article about it. I served as a subject for an experiment using Sherrington’s original flicker-fusion apparatus, which Prof had resurrected. Prof was at the frontline when Jack Eccles and Henry Dale were locking horns about the nature of synaptic transmission. According to Prof, who was then an undergraduate, Sherrington told them he thought that Dale was right and synaptic transmission was chemical, not electrical, as Eccles supposed. They rushed to tell Eccles, who riposted, “The Old Man hasn’t seen my latest results.” By the time Prof introduced me to his erstwhile supervisor, Eccles was an old man himself, and a grand one. I had been hoping to discuss his work with Karl Popper on consciousness, but had to content myself with listening.

During World War II, Prof did research on paraplegics at Stoke Mandeville Hospital with Sir Ludwig Guttman, who pioneered paraplegic sports, which eventually became the hugely successful Paralympic Games. Together, they discovered autonomic dysreflexia, which affected the control systems for blood pressure and temperature. After the Bhopal disaster in 1984, when due to human error, poisonous gas escaped from a chemical plant, he resumed his collaboration with Autor Singh Paintal in Delhi to study the mechanism of phosgene gas poisoning, which they had last investigated in the 1950s in Edinburgh.

Although Prof was reputed to have a “high IQ and low pH,” I soon discovered that he had a wicked sense of humor, a compendious knowledge of physiology, and an unlimited fund of stories about scientists, especially Sherrington. His own papers were characterized by one or two elegant summary figures and a polyglot authorship. His mother was French, so he delighted in translating key passages of Ramón y Cajals’s Histologie de Systeme Nerveaux for us. When Peter Clarke, a devout Christian, asked Prof for advice about how he might learn French, he twinkled, “On the pillow, dear boy, on the pillow.” Peter took his advice to heart and when he arrived in Lausanne, he married Stephanie, a cognitive neuroscientist from the French-speaking part of Switzerland. Whenever Prof launched into a story with a visitor, he would often preface it with, “Kevan has probably heard this one already,” but I rarely had. His best-known work on vision was with Peter Daniel of the topographic representation of primate area 17. Over five summers, they mapped the entire cortex of different old-world species and using the metric they called the “magnification factor,” they developed a mathematical model that accurately predicted the 3D shape of the monkey’s area 17. That the physical shape of the neocortex might be determined by the constraints of a representation of a peripheral organ was a completely novel concept, and their example remains unique.

When I joined his lab, Prof was working on the development and plasticity of binocular vision in sheep, which he chose because the precocial lambs could avoid a visual cliff on their day of birth and thus were an excellent choice for exploring questions of nature or nurture. He had a long history of working with sheep, with classic studies on eye muscle spindles with Peter Daniel and Sybil Cooper. His lab was huge—and needed to be to accommodate a lifetime’s collection of antique electrophysiology equipment, along with plastic and latex rubber models of the visual cortex of cats and primate, which were frequently pulled out for impromptu tutorials. He believed that building one’s own apparatus was “good for the soul and reduced the number of reported artifacts.” I had to learn how to operate a series of “Jock Boxes,” which were bespoke interfacing instruments designed and made by W. T. S. “Jock” Austin, Prof’s erstwhile technician in Edinburgh. Calibrating blood pressure manometers and CO₂ machines was a major pre-experiment activity, as was grinding the seats of metal catheter taps using jeweler’s rouge. Prof loved doing experiments and was first at the operating table when venous and arterial cannulations and tracheostomies were needed, whistling tunelessly when things were not going smoothly. His maxim: “the only thing certain in an experiment is that long pieces of tubing will be cut in to shorter pieces” was practiced diligently. He admonished clumsy experimenters (i.e., me) who caused voltage transients by touching forceps to the stereotaxic apparatus, saying, “that would have broken a galvanometer string,” but when I innocently asked how many strings he had broken, he guffawed: “I’m not that old!”

Another new arrival in the lab was Henry Kennedy, who was taking a sabbatical year from an institute of the National Institute of Health and Medical Research (Institut national de la santé et de la recherche médicale; INSERM) at Bron, near Lyon. Henry shared with Prof the descent of a French mother and an English father. He was then collaborating with Guy Orban in Leuven and Jean Bullier in Bron, both of whom had learned their trade in Peter Bishop’s (see Volume 1) department in Australia and had adopted Geoff Henry’s methods and nomenclature for mapping and classifying cortical receptive fields. Our introduction got off to a bad start when Henry, an ardent devotee of traditional English pubs, suggested we meet in the King’s Arm to discuss what we might do together. I replied that I never went to pubs. He has never forgotten the rebuff.

Ungulate Investigations

Prof had recently published a Nature paper with Peter Clarke and V. S. Ramachandran, reporting that neurons with binocular receptive field disparities were present in newborn lambs. Henry and I decided to do a more in-depth analysis of the development of receptive fields in sheep. We found that while most neurons were orientation-tuned in the newborn, other features like direction selectivity and end-inhibition were absent. Of most interest were the so-called obligate binocular cells, which would respond only to binocular simulation over a narrow range of binocular disparity. These were absent in the newborn, but constituted 15 percent of the neurons we encountered in the adult. One particular neuron lives on in memory: we were recording from a 70 kilogram adult sheep and encountered a neuron I could not drive. Henry decided that by hook-or-by-crook he would map its receptive field. He lit a cigarette, sat down at the tangent screen, and over the next 40 minutes gave me a master class in hand-plotting receptive fields. The receptive field he painstakingly teased out was not only obligate binocular cell, requiring a precise binocular alignment of the eyes, but the receptive field was tiny, strongly end-inhibited, and completely directional. We had encountered nothing remotely like it in the newborn, whose circuits seemed to lack all adult mechanisms for facilitating or inhibiting a response.

We also defined the critical period for ocular dominance plasticity by monocular deprivation and explored various methods for reversing the shift in dominance. Fergus Campbell at that time was promoting a therapy to reverse amblyopia in children, which involved exposing the amblyopic eye to rotating gratings. Ramachandran persuaded Prof to try it on monocularly deprived lambs and Fergus came to see one of the experiments. Prof enthusiastically explained the experimental setup as Henry and I were mapping a receptive field. We always measured blood pressure with an arterial catheter, which occasionally blocked. Seeing the telltale loss of a pulse amplitude on the blood pressure meter, Prof picked up a large syringe, thinking it was filled with saline, and injected the contents into the artery. The syringe was filled with air. The blood pressure dropped to zero and the cell stopped responding. Henry and I looked balefully at an embarrassed Prof. Then before our eyes, the blood pressure slowly started increasing until it was back to normal, and the cell responded again. Prof smiled triumphantly at Fergus as if he had intended this demonstration of the robustness of the experimental sheep all along.

My first opportunity to attend an international meeting came at the end of my first year as a postdoc. The meeting, “The Developmental Neurobiology of Vision,” was organized by Ralph Freeman and Wolf Singer (see Volume 9) and took place in Crete in 1978. It was a unique opportunity for me as a neophyte to meet the gods of vision. The retinotectal system was well-represented with luminaries like Steve Easter, Pamela Johns, Barbara Finlay, and Scott Fraser, but for me, it was a golden opportunity to put names to faces and talk to many visual system experts, including Horace Barlow, David Hubel, Pasko Rakic (see Volume 12), and Colin Blakemore. In retrospect, the most significant person for my future was Murray Sherman (see Volume 10), who had begun using HRP to label intracellularly physiologically-identified neurons in the thalamus. Alan Brown in Edinburgh was using the same technique to great effect in mapping sensory afferents and motoneurons in the cat’s spinal cord, work that had greatly impressed Prof and me.

Retiring Research

Two years into my postdoc, Prof retired. We had discussed our options, and because Prof was keen to continue research, we decided to link up structure and function in the visual cortex using the intracellular-HRP technique. His aphorism, “physiology is anatomy plus thought,” was to take a new turn. In the tradition of Oxford, the retiring head moved out of his department. Prof’s colleague at Magdalen College, Larry Weiskrantz, offered him space in his Department of Experimental Psychology. After the cavernous space of the Waynflete Professor’s office, we now shared a tiny office with no view, but it was on the same corridor as Larry, Alan Cowey (see Volume 5), Dick Passingham, and Edmund Rolls. A framed photograph of Dorothy Hodgkin, the crystallographer, reminded us she had worked on the same corridor. I had studied Jerome Bruner’s and Michael Argyle’s work in Cape Town, and here they were in the flesh. Under the same roof was the Zoology Department, where Richard Dawkins unselfishly showed me his simulations of artificial life on an early desktop computer. Experimental Psychology had no common room, so the department members gathered in a large communal hallway at elevenses to buy tea and donuts at a serving hatch. Larry was always present for informal discussions—it felt like a very democratic and open system—and the most convenient way to get business done with Larry.

The Medical Research Council (MRC) gave us a one-year grant to see if we could get the new technique up and running. We were now on probation, and I urgently needed to learn how to do intracellular recording in vivo. Alan Brown’s technique was specialized for the spinal cord, so not readily adapted to neocortex. While we were mulling this over, a bolt from the blue arrived in the form of a Nature paper from Charles Gilbert and Torsten Wiesel entitled, “Morphology and Intracortical Projections of Functionally Characterised Neurons in the Cat Visual Cortex.” It was not much different from the title of the grant application we had just submitted to the MRC. Prof immediately wrote to Torsten, whom he knew, to ask if his postdoc could visit his lab to learn the technique. A polite letter of refusal came back, citing time limitations and raising the issue of competition. This left Murray Sherman, then in the middle of a move from the University of Virginia to The State University of New York at Stony Brook. I wrote to him and he generously said, “yes.”

This was my first visit to the United States, and I flew to San Francisco to visit Ken Brown (Torsten Wiesel’s erstwhile colleague) and Dale Flaming, to see the pullers and bevellers they had developed for their intracellular retinal work. I also visited Harvard Neurobiology, where there was much going on of interest to me besides Charles and Torsten’s work, which remained behind closed doors. While there I gave one of their “brown bag” lunchtime talks on our sheep work. After my talk, a small man came up to me and asked many questions—indeed, apart from Prof and David Attenborough, I never met someone so interested in the sheep’s visual system. Only later I discovered I had been talking to Steve Kuffler. David Ferster and Simon LeVay had been labeling thalamic afferents extracellularly with HRP so I had a chance to see their spectacular histology. Jonathon Horton was using HRP to look at fiber-ordering in the visual pathway, which was still a keen interest of mine. Then, wandering down the corridor was David Hubel holding up to the light a cytochrome oxidase-stained tangential section of macaque V1 and showing me the leopard spots that were visible to the naked eye. The discovery of the cytochrome blob system by Margaret Wong-Riley became a major research avenue for David, and led to a series of papers with Marge Livingstone (see Volume 9) on the physiology and anatomy of the cytochrome system, and a series of psychophysical experiments in which they tried to tie their discoveries of multiple parallel pathways to behavior. My own thoughts about their ambitious multilevel investigation were expressed in a Trends in Neuroscience article: “From Enzymes to Perception: A Bridge too Far?” The main subjects responded with an enthusiastic letter; Semir Zeki was less happy with it, as he felt they had stolen his thunder and needed no more column inches from me. Much later Geoffrey North asked me to write David Hubel’s obituary for Current Biology, but despite being allowed extra pages, I still felt I had only given the sparest details of his extraordinarily rich contribution to science.

Juniors and Seniors

Oxford colleges have a system of competitive Junior Research Fellowships (JRFs). I applied for the Weir JRF at University College. I submitted my DPhil thesis and made it onto the short list. At the interview, I was ushered into a wood-paneled room lined with Fellows, with the Master, Lord Goodman, presiding from one corner. My examiner was Julian Jack, the renowned biophysicist and coauthor with Denis Noble and Dick Tsien (see Volume 11) of the classic, “Electric Current Flow in Excitable Cells.” I imagined we would reprise my DPhil viva encounter with Mike Keating, but no, Julian decided to launch into a full-frontal attack on the ideas contained in the chapter on morphogenetics that I had coauthored with Tim. Deciding I had nothing to lose, I parried and thrusted back as best I could. I guess our performance was entertaining enough for the Fellows to elect me. At my first lunch in the Senior Common Room, a small elderly man limped up to ask if I would take on the job as the College’s Junior Dean. My inquirer, I later discovered, was the Senior Fellow, David Cox, one of the great mountaineers of the interwar years and founder-member of the Oxford Night Climbers, who scaled college and town buildings under cover of darkness. His climbing career had sadly been cut short by polio. I said, “yes,” having no idea of what a Junior Dean did. He introduced me to the Dean, David Bell, a volcanologist, who explained that I was now responsible for the discipline and welfare of the students. Since University College was known as “the Pub on the High Street,” this was not welcome news. Fortunately for me, however, the combination of the sensitive hand of David Bell and the fact that the College had just admitted women undergraduates for the first time since its founding in 1249, which had a remarkably pacifying effect, made my apprenticeship as Junior Dean relatively incident-free.

The Senior Common Room seemed to be filled with philosophers. Peter Strawson, whose papers I had studied in my Logic and Metaphysics course, was an Emeritus Fellow, Gareth Evans would not let any loose thinking go unpunished, John Finnis was a Reader in legal philosophy, Ronald Dworkin held the chair of jurisprudence and was a relentless debater. In my first week, I found myself defending animal experiments to another eminent legal philosopher, Herbert Hart, who challenged me with the view of the utilitarian, Jeremy Bentham: “The question is not, can they reason? nor, can they talk? but, can they suffer?” I was getting a fast-track education in ethics. Through Herbert Hart, I met other giants like Sir Isiah Berlin and John Rawls, as well as Herbert’s graduate student, Nicola Lacey, who later wrote a sensitive biography of Herbert. Dan Cunningham was the other medical Fellow along with Julian Jack. He was a respiratory physiologist who had the distinction of having had Roger Bannister and Edmund Hilary on his treadmill.

The Oxford Colleges rank their Fellows in order of seniority, with the longest serving fellows at the top and the recently appointed fellows at the bottom. This ranking determined which Fellow presided at dinner and said the grace. One vacation I found myself to be the sole Fellow at dinner, where unbeknownst to me, Stephen Hawking, an alumnus, had also signed in. Saying the Latin grace with Hawking listening was memorable. He was then still speaking with his own voice and fortunately on that occasion he was dining with colleagues who helped translate. Our later conversations were easier, if slower, when he used his famous speech synthesizer.

Experiments in Psychology

Murray Sherman’s new lab in Stony Brook was being set up by Mike Friedlander, with help from Rick Lin, Jim Wilson, and Larry Stanford. Mike took me through the methods they were using for their lateral geniculate nucleus (LGN)—both the intracellular methods and the methods for classifying thalamic receptive fields. I began a long collaboration with Mike on development and plasticity of thalamic afferents when he moved to his first faculty job at the University of Alabama at Birmingham. Murray, too, was not only a generous host, but influential in my own thinking, particularly about parallel processing pathways in the visual system. His serial electron microscope (EM) reconstructions of the retinal afferents and their targets in the LGN were groundbreaking and also an encouragement for us to attempt a similar approach for cortical circuits.

Figure 2.    David Whitteridge (“Prof”) and his postdoc in the Department of Experimental Psychology, Oxford. Photo credit: Rodney Douglas.

Prof and I set up our lab in a tiny room next to the goods lift in the animal house on level F. This seemed a particularly inhospitable location for intracellular recording, so we improvised an isolated table by floating a solid steel lathe bed on pneumatic truck shock absorbers, which we inflated with a bicycle pump. It served for decades. I pulled pipettes on the classroom pipette puller back in physiology and beveled them using the poor-man’s beveler involving a thick slurry of alumina powder, a method developed by Tony Spindler for recording heart muscle in Denis Noble’s lab. Marianne Dawkins in the next-door Department of Zoology, used pigeons for behavioral studies, so Prof decided we should start by intracellular recordings of the tiny cells in the pigeon’s superior colliculus. So it was that our first HRP-labeled neuron was a pigeon’s tectal neuron. We soon graduated to our familiar adult sheep, which were brought in from the university’s farm and temporarily housed on the roof. Stabilizing the brain was a formidable issue, but it was in the sheep’s V1 that we obtained our first well-labeled pyramidal neurons.

We had passed the MRC’s audition and wrote a three-year grant to explore the circuits of V1, but in the cat, not the sheep. Sherrington, Eccles, Kuffler, Phillips, Hubel, and Wiesel and many others had ensured that the cat had made the most significant contribution to neurophysiology of any species. Using another species for our study, like the sheep or mouse, inevitably meant the overhead of much derivative research. By selecting cat V1, the dangers of generalizing from highly specialized cortical areas, like rodent barrel cortex or primate area 17, could be avoided. Additionally, I wanted to add Jean Bullier and Geoff Henry’s method of using electrical stimulation to identify the X- and Y-type inputs to visual cortex and the ordinal position of the cells in the cortex. Although Prof sniffed that electrical stimulation was something he’d left behind in the 1930s, he was persuaded, neither of us dreaming how significant the technique was to be.

We began with two experiments per week, with me doing all the preparation and post-processing in-between. Fortunately, we now had funding for a technician and we advertised the position. On our short-list was a Yorkshireman who had studied social anthropology at Oxford Polytechnic and, as employment for social anthropologists was proving elusive, he had taken a job as a forester in the local Wytham Wood. After our interviews, Prof expressed his strong preference for the forester, saying, “Well if he can cut trees into sections, he can surely cut brains into sections.” With this inescapable logic John Anderson joined us in the small office and took over the post-processing in the histology section of the Department of Human Anatomy. What we had not uncovered in our interview was that in addition to fishing, which requires infinite patience, John also painted landscapes—he was an artist; much later on, he took to sculpturing in clay, and we worked together in the same pottery studio. I had been doing all the reconstructions of the neurons, and showed John what the main structure of neurons were and how to use the microscope and drawing tube. When he found a missed collateral from the axon of one of the cells I had drawn, I knew Prof had made the right choice. John’s painstakingly meticulous work was an essential contribution to our research over the next four decades. His first efforts were displayed to the public in our comprehensive account of the structure and function of spiny neurons in the cat, published in the Journal of Physiology. His pen-and-ink drawings have appeared in touring science museum exhibits. His cumulative contribution to our understanding of the long-distance cortical circuits in primate cortex is unmatched and he more than deserved the belated reward of MSc and PhD degrees from the University of Zurich.

John’s many hours at the microscope was taking its toll, however, and he began complaining of various discomforts and chronic pains, so some physiotherapy was clearly indicated. I knew of the F. M. Alexander Technique, a method admired by Sherrington, which teaches awareness of one’s body in order to move more efficiently and to improve poor posture, so acquiring a better “use of self,” the term Alexander coined. I suggested to John he try a few lessons. He did, and it worked. In fact, it worked so well that both of us subsequently trained as teachers—John with Don Weed and me with David Gorman, both mavericks in the field. David Gorman handwrote and illustrated a brilliantly informative functional anatomy book, The Body Moveable, which became a ready reference. I later had the opportunity to work with two teachers who were trained by F. M. Alexander himself, Marjorie Barlow and Elizabeth Walker, and I invited Michael Frederick, the founder of the Alexander Technique International Congresses, to be our teacher-in-residence for a fortnight in exchange for organizing a series of plenary neuroscience talks for one of the Congresses.

Prof and I had soon discovered that there were no “low-hanging fruit” to be had in the form of simple structure-function correlations, despite hints in that direction from the Harvard investigators. Nonetheless, a far deeper analysis of the cortical circuits was now possible, not least because the HRP-filling revealed the neurons in an isolated splendor of detail that was breathtaking even to those familiar with Golgi-stained material. We now sought a collaborator to identify the targets of our labeled neurons, using electron microscopy (EM). Tom Powell demurred, believing the effort involved in looking at the axons using EM was too great. We then met Peter Somogyi, a protégé of Hungarian anatomist János Szentágothai, coauthor of The Cerebellum as a Neuronal Machine together with Jack Eccles and Masao Ito (see Volume 2). Peter was working on the basal ganglia with David Smith and Paul Bolam, but was eager to collaborate on cortex, particularly if the cells were smooth (inhibitory), so we began with cortical basket cells. I journeyed up to Alan Brown’s lab in Edinburgh to reconstruct our best example using their new computer-aided microscope, a device known as the Joyce-Loeb Magiscan. State-of-art it may have been, but it was exceedingly laborious and it could not stitch the transverse sections together or easily correct for the 80 percent shrinkage in the z-dimension of the air-dried sections. It did give us, however, the first 3D picture of the large basket cell’s dendrites and axonal arbor. The EM revealed that its major targets were dendrites and spines, not cell bodies as Ramón y Cajal had supposed with his nids pericellulaire, which Prof translated as “pericellular nests.” It was a convincing demonstration that a combination of light-microscope and EM offered potentially huge gains and it became our standard procedure in the long march to understand the structural basis of cortical function.

Pub Proceedings

Leon Isaacson had been in touch, because one of his medical colleagues at UCT wanted to use his sabbatical to study the brain rather than the kidney, and was I interested? I said, “yes,” and on a visit to Cape Town, Leon invited me to the “Foresters Arms” to meet Rodney Douglas—one of my rare forays into a pub! My first encounter with Rodney sparked, and indeed, I cannot recall us ever having a dull conversation. He immediately invited me to go sailing on the South Atlantic Ocean in the yacht that he and his father had built in their back garden. In the UCT lab, we initiated a project with Lauriston Kellaway to map the visual cortex of the indigenous rock hyrax (Procavia capensis, locally known as a “Dassie”), whose closest relative is the elephant. We made some successful experiments, but quickly ran out of adult animals when the local villagers in the area where the “Dassies” were caught, discovered that we had (inadvertently) provided them with a cache of fresh “bush meat.”

Although by reputation a brilliant diagnostician, computers were Rodney’s forte: in a shed on the Medical School campus he had set up the revolutionary Data General Eclipse minicomputer, which sported a generous 64 kilobytes of RAM. On arriving for his sabbatical in Oxford, he immediately embarked on a program of computerizing our physiological stimulus and data acquisition and building a microscope system for 3D reconstructions. Brian Baker, the electronics guru in Experimental Psychology, designed and built all the interfaces. My erstwhile tutorial “student,” Mike Fischer, had founded Research Machines to build some of the earliest desktop computers for schools in the United Kingdom. He was hugely supportive and provided us not only with his RML 380Z, but also a series of programmers.

Figure 3.    Mapping the visual cortex of Procavia capensis in the Department of Physiology at UCT with Rodney Douglas (left) and Lauriston Kellaway (middle). Photo credit: L. Kellaway.

The success of the collaboration with Peter Somogyi and his students, Tamás Freund (see Volume 11) and Zoltán Kisvárday, had not gone unnoticed and MRC decided to form a Unit to study the circuits of the neocortex and basal ganglia, headed by David Smith who was head of the Department of Pharmacology, which became our temporary home while a new MRC annex was built. We moved out of our happy home in Experimental Psychology to a tiny lab/office in pharmacology. There was no room for John and his microscope and drawing board, so he had to camp in the passageway. Yet we started to expand. Charmaine Nelson came as an EM technician, Neil Berman arrived from Cape Town as a PhD student, Paul Gabbott came from the Open University as a postdoc, and we had sabbatical visitors like Christovam Diniz from Belem, Brazil; Colette Dehay, from Henry Kennedy’s INSERM lab; and Clay Reid from Bob Shapley’s lab at New York University. Larger than life in every way, Danie Botha arrived from Cape Town and the elegant 300 lines of Pascal he wrote for the 3D reconstruction system we called TRAKA served us well for decades. Rodney wrote a companion program, TRAKEM, to reconstruct serial EM sections, and this was expanded greatly by Albert Cardona and it had a second life as a key tool for connectomics.

Figure 4.    John C. Anderson in his passageway drafting room, Department of Pharmacology, Oxford. Photo credit: Kevan A. C. Martin.

John was tasked with making highly accurate TRAKA reconstructions of the dendritic trees of different types of cells. These seven cells, labeled J (for John) 1 to 7, became key datasets for biophysical modeling. Ironically, the first papers that used them were not ours, but from the labs of Terry Sejnowski and Christof Koch, so we were never cited. A layer 5 pyramidal cell, “J5,” had a particularly stellar career, which was reviewed in a paper by Bartlett Mel entitled “J5 at Sweet 16.” J-cells still keep popping up in various publications, with variable attributions.

Julian Jack was the principal player in another piece of theater that took place at one of the Oxford meetings of the Physiological Society. To a packed lecture hall, Rodney presented modeling work using J5 to estimate the amplitude of a unitary excitatory postsynaptic potential (epsp). The session was chaired by Peter Matthews, doyen of muscle spindles and son of Sir Bryan. There were a few straightforward questions after Rodney’s 10-minute presentation and then Julian asked where we had got the conductance values from, as he doubted them. “From your book,” replied Rodney, which evoked a ripple of amusement from the audience. Their reaction was unfortunate because Julian, embarrassed, now had to save face and tore in. Peter Matthews tried to moderate, but the 5 minutes allotted for questions quickly stretched to 15 as the Irresistible Force encountered the Immoveable Object. Since publication was dependent on the audience approving it, it was evident at one point that if Peter Matthews took it to a vote, we would lose. Prof, silent in the back row, left it to me to suggest modifications to the text. Julian eventually realized he might have over-reached, and backed off. We offered our compromise text and our Proceedings was voted through. That evening at the Society dinner, the Secretary reported on the day’s meeting, noting that over years he had witnessed a regrettable and progressive decline in the quality of the debates in the sessions, but today he was glad to report that the old ways were not entirely lost. Reading our published Proceedings again today, our estimate turned out to be well in the range of those seen experimentally.

I reviewed our progress for an issue of the Quarterly Journal of Experimental Physiology. It was clear that our understanding of the machinery underlying cortical computation was still primitive and that analyses of receptive field properties and back-of-envelope models had long ago reached their limits. At the other end of the spectrum, most computational models were far removed from providing any explanation of the neural machinery we were seeing. David Marr’s three levels of analysis—theory, algorithm, implementation—is still widely used as a throat-clearer in talks as it seems to many the logical route to discovery, but as Marr himself explained, an algorithm could be implemented in many different ways, so they offer no ready insights into how brain circuits are built or perform their operations. Whenever Tony Movshon was in my audience, he would tease me with his aphorism: “anatomy tells you what might be, physiology tells you what is,” but the history of neuroscience begs to differ. Structure endures, physiological knowledge is highly technique- and concept-dependent (e.g., Eccles vs. Dale, or Hodgkin vs. Wald). Ramón y Cajal is still frequently cited, whereas his contemporary Sherrington is virtually forgotten. We were pursuing the “principles and technology of neural engineering,” as Horace Barlow put it, and the yawning gap between the macrolevel theories and microlevel experimental data now begged for a bridging mesopic level of analysis to link a circuit level description to both the fine structure and the overall operation of the cortical circuit—and ultimately behavior.

Canonical Concerns

To probe the whole cortical circuit, Rodney, Prof and I took an alternative “engineering” approach and recorded the intracellular impulse response of identified cells to a brief electrical pulse delivered by stimulating electrodes placed in the optic tracts. To the intracellular electrode we glued an extracellular “piggy-back” multibarreled pipette so that we could deliver agonists and antagonist of the type a and b receptors for the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). By labeling the cells, we had exact information as to their morphology and laminar position. We found distinct differences in the time course of the inhibitory responses of superficial and deep layer pyramidal cells. Rodney developed a rate model to predict how the GABAa and GABAb receptors shaped the excitatory pulse response. This combination of experiment and simulation revealed three main features: the thalamic input was small, it was amplified by recurrent excitation, and the positive feedback of recurrent excitation was balanced by recurrent inhibition. It was clear from their elaborate local axons that pyramidal cells must connect mainly to other pyramidal cells, so the recurrent excitation was expected. But having grown up with Hubel and Wiesel’s textbook models of “simple” and “complex” cells, we had not appreciated that the thalamic input was functionally so weak, and by identifying a role for recurrent excitation in amplifying the thalamic input we introduced a completely novel mechanism. That excitation and inhibition would wax and wane together seemed paradoxical, but in the presence of strong recurrent excitation, inhibition acted as a governor.

With Rodney’s stimulus presentation and data-collection system now in full swing, we were doing two long experiments each week. For light relief, Rodney and I took it in turns to cook elaborate dinners for a dozen people every Saturday. Danie Botha was a fixture and took us on a tour through the winelands of Bordeaux with his highly researched purchases. On a visit to Oxford, David Hubel was a guest at one of these dinners and forever after reminded me of “the fish”—a whole salmon baked in puff pastry that we served up on that occasion. These delights and distractions were all to end with the return of Rodney to Cape Town to take up a senior lectureship in the Department of Physiology at UCT.

I went to Cape Town to write up our findings on the cat’s cortex for Terry Sejnowski’s new journal Neural Computation. Our paper was modestly titled, “A Canonical Circuit for Neocortex,” taking a cue from Tom Powell’s concept of the uniformity of neocortex. It was a significant synthesis, for our canonical circuit not only gave an immediate logic for components that Prof and I had identified, but it was also a short step to “canonical computations,” which we proposed were composed of computational “primitives,” like linear gain, nonlinear gain control, soft winner-take-all, and signal-restoration, all functions later realized in various models and in neuromorphic circuits. Although our term “canonical circuit” rapidly become common currency, it took longer to show that it could seamlessly link synapses to behavior. The cat’s cortex was the ideal bridge between mouse and primates, and the canonical circuit even played a key role in Karl Friston’s theory of dynamic causal modeling for human functional imaging.

Silicon Alley

After two years at UCT, Rodney decided to return to the MRC Unit as a non-tenured scientist. We had moved into an annex of the new Department of Pharmacology and had more space. John Anderson, at least, was no longer camping in a corridor. We had begun a collaboration with Christof Koch, who was then at the California Institute of Technology (Caltech). Rodney went to Pasadena for some months to work with Christof’s students and while there he met Carver Mead, who had created critical tools for designing very large scale integrated (VLSI) circuits. Carver had also taught a course with Richard Feynman and John Hopfield on neural computation and had the insight that by using transistors in subthreshold mode, he could mimic the physics of nerve membranes and began designing analogue rather than digital VLSI circuits. With one of the few women at Caltech, the biology graduate Misha Mahowald, he created the first silicon retina in analogue VLSI (aVLSI), which was featured in a cover article in Scientific American. Rodney decided to see if an action potential could be realized in an aVLSI circuit and set to work with Misha, with Carver looking over their shoulders. They succeeded in designing and fabricating the Hodgkin-Huxley action potential in aVLSI, and their advance was rewarded with a Nature letter: “A Silicon Neuron,” with a cover illustration of the layout.

Soon after their publication, I was invited to give a talk at King’s College, Cambridge as part of a conference on “The Neuron as a Computational Unit” organized by Richard Durbin, Chris Miall, and Graeme Mitchison. Rodney and Misha came along, so at the end of my talk, I mentioned their silicon neuron. At the tea interval, Andrew Huxley (see Volume 4) came up to me and asked about the silicon neuron.

I beckoned Misha over and introduced her, “Misha, this is Andrew Huxley, he is interested to know more about your silicon neuron.”

As happens, she missed the name and launched straight in with: “Do you know anything about the action potential?”

Without missing a beat, Huxley twinkled, “A little.”

By coincidence, I was then sharing a house in Oxford with Alan Hodgkin’s daughter, Deborah, so I heard many stories of the legendary pair, who set a mark for computational neuroscience that has never been surpassed, although, as Alan Hodgkin reminisces in Chance and Design, he saw their model as ultimately a failure, because the mechanisms of the voltage gates within the channels remained beyond their grasp.

Prof, Misha, Rodney, and I went to London to present our work at one of the Royal Society’s evening soirées. Prof had been elected a fellow in 1953, and I was soon to be the Royal Society’s Henry Head Research Fellow. The soirées were invitation only for the great and the good, and the science on display was a cross-section of cutting-edge research assembled for their Summer Science Exhibition. In true British tradition, we were in formal evening garb. Prof seemed to know everyone and introduced us to eminences like Paul Fatt and Miriam Rothschild. For Misha it was a voyage into antiquity, but she gamely demonstrated her silicon chips to the assembled elite.

Figure 5 (left to right):   Prof Whitteridge, Kevan A. C. Martin, Misha Mahowald, and Rodney Douglas at the Royal Society Soiree. Photo credit: Janet Whitteridge.

Misha had now arrived in Oxford as a postdoc. She was passionately convinced that her form of neuromorphic engineering could lead to the solution of biological problems and that in turn biology could point her to engineering solutions. Her mission was to use the machine as a means to understand the brain. For her science, was a dynamic network, not a process of constructing an edifice. Her influence is strongly evident in a paper we wrote for Science magazine on recurrent circuits, in which we used the electronic circuit metaphor to explain aspects of recurrent excitation and inhibition. We summarized some of the wider ideas we had been discussing in a short article, “Neuroinformatics as Explanatory Neuroscience.” Often in the evenings, Misha would appear at my desk with a question, “what do you think about . . . ?” This typically would be the start of hours-long discussions that explored in depth some aspect of biological computation she had been pondering. I came very much to value her probing questions, her ability to synthesize, and her deep and original ways of thinking. It was an acute shock and long-lasting loss when some years later Rodney phoned on a late December evening to break the devastating news that Misha had taken her own life.

Synaptic Mapping

Our main experimental work was to test predictions of the canonical circuit. We began by mapping the synapses formed by the different inputs to the thalamorecipient layer 4, where the “simple cells” of Hubel and Wiesel lived. Such quantitative mappings were to become a theme of our research, but on our first pass, we found that thalamic synapses in cat and monkey V1 formed less than 10 percent of the excitatory synapses in their target layer 4, as the canonical model predicted. We also found a strong recurrent connection between layer 4 cells, but the surprise was the input from layer 6 pyramidal cells, which formed nearly half of the complement of excitatory synapses in layer 4. Highly unusually for pyramidal cells, they formed most of their synapses with dendritic shafts, not spines. This quantitative synaptic map raised the intriguing question of whether the different anatomical inputs had different physiological properties. I took advantage of the College lunchtimes to discuss with Julian Jack how best to tackle this question experimentally.

Julian and his team had used the method of “minimal-stimulation” to study synaptic plasticity in slices of hippocampus, and I thought we might adopt the method to screen for the presence of the different classes of synaptic inputs to layer 4 of cat V1. I carried out the experiments in Julian’s lab with his PhD student Kristina Tarczy-Hornoch, postdoc Neil Bannister, and Julian’s research assistant, Ken Stratford. For reasons never fully explained, Kristina felt the need to slice the cat’s cortex while barefooted, but it worked: from the first experiment, when we evoked very different types of epsps in layer 4. Strikingly, one type we encountered was large in amplitude, had virtually no variance, and showed strong paired-pulse depression. We followed up the minimal-stimulation survey with paired intracellular recordings and convinced ourselves, and our referees, that the large amplitude potential was the thalamic afferent input—and characterized the excitatory inputs from layer 4 spiny stellate cells and layer 6 pyramidal cells.

The Worst of Times

Our knowledge of the canonical circuit and its operations now made it possible to embark on a program to build a “silicon cortex.” Mike Fischer generously supported this, with his programmer Adrian Whatley coming on board to develop the software interfaces. It was a hugely ambitious undertaking, but driven with great energy. Then one day the sky fell in. Rodney arrived with a letter from the MRC Unit directors, informing him that his five-year contract would not be renewed. Unfortunately, the directors, David Smith and his deputy, Peter Somogyi, had neglected to mention their decision to me beforehand. The timing was particularly unfortunate, for Rodney’s daughter was being treated for a serious illness at a specialist pediatric cancer unit in Oxford, and without a work permit, he would have to leave the country, with dire consequences for his daughter’s life. No amount of rational argument would persuade the directors to extend his contract, for even a year. Relations became so sour that I also was shown the door, which is how I found myself without a lab, sharing a small office in Experimental Psychology, but this time not with Prof, but with J. Z. Young (see Volume 1), the eminent anatomist who discovered the squid’s giant axon.

The White Knight who rode to our rescue was the Gatsby Charitable Foundation in the form of Roger Freedman. He had been a regular visitor to our lab because the settlor, David Sainsbury, was keen to invest in computational neuroscience. Roger rapidly negotiated a salary and space for Misha and Rodney at Imperial College, London. Then, out of the blue, an emissary arrived from the Swiss Federal Institute of Technology (Eidgenössische Technische Hochschule, ETH) in Zurich, who had decided to team up with the cantonal University of Zurich (UZH) to create a joint Institut für Neuroinformatik. The emissary, Klaus Hepp, was a theoretical particle physicist turned theoretical systems neuroscientist. The Brain Research Institute in Zürich was then headed by Michel Cuenod, who had been urged by Eberhard Fetz to establish computational neuroscience in Switzerland. Klaus and Michel were intrigued by our combination of experiment, theory, computation, and engineering and we were invited to Zurich for interviews, discovering on arrival that a source in Oxford had been briefing against us. The Zurich interest galvanized the Gatsby, and they offered us generous funding to create a computational neuroscience unit, an initiative that eventually became the Gatsby Computational Neuroscience Unit under Geoff Hinton, who steered it strongly in the direction of machine-learning rather than neuroscience. It was clear our days in Oxford were over: Prof had mapped his last receptive field and was no longer with us, and at our erstwhile directors’ instigation, the MRC was suing me for the heirloom equipment that Prof and I had brought into the Unit; fortunately sanity eventually prevailed at the MRC Head Office, and they dropped the case. It was the right time to make a clean break and move to Zurich.

The Institute of Neuroinformatics

Rodney and I arrived in Zurich in late 1995 to find ourselves proud double professors and directors of a to-be double institute, our positions and institute equally funded by the ETH and the UZH. But we had no instruction manual, no “guide for dummies,” and all official business was in German, which neither of us yet read or spoke. Our ignorance and naïvety now seems risible, for what followed was years of trial-and-error learning, with rather too much error for comfort. There were no models for double institutes or double professors like us—we were Versuchskaninchen—guinea pigs. The UZH and ETH differed in every way, including academic and administrative structures, salary scales, pension schemes, subjects, semester dates, and lecture timetables. Nothing was in synch. ETH professors were appointed by the President himself (always a him) and were lords of their fiefdoms. But what institute professors chose to locate themselves in or what other professors they associated with was largely irrelevant to the President. Years later, this came back to bite us when we read in the Friday evening edition of the local free newspaper that the President had decided, unilaterally, that the Institute of Neuroinformatics (INI) was to be transferred from our happy home in the Department of Physics (the Department of Einstein and Pauli), to a newly created health sciences department, and that double professorships, equally funded by the ETH and UZH, were now history. The level playing field we had worked so hard to create between ETH and UZH within INI had tilted, and we no longer had any control over the appointment of new ETH professors in INI. It was a top-down decision quite at odds with the admirable system of direct democracy and consensus government that the Swiss justifiably regard with pride.

The tradition of German science was that the Professor was the top of a hierarchy of Oberassistents, Assistents, and technische Mitarbeiter (technicians), all devoted to the professor’s research. We intended to use our Oberassistent positions to create independent groups, and finance from our own budget their lab set-up, a PhD student, and some running costs, with the agreement that they would seek external grant funding to continue. For professors to give away their resources was seen as wildly eccentric, but it worked very well and year-on-year about half of the Institute’s funding was from external sources—highly atypical for Swiss Institutes, which run mainly on internal funding.

We packed into a temporary space in the center of Zurich while a building was being planned and built for the INI and the Brain Research Institute on a former farm on the outskirts of Zurich. The institute they had designed for us was in the form of the standard German-style “container,” a long rectangular plan with two long corridors flanking a windowless central spine containing communal services, with laboratory-cum-offices lining the outside walls. From visits to similar buildings in Germany, I knew that one could gaze down the long empty corridors all day and never see another soul, as everyone worked behind closed doors. It wouldn’t do. We had to prise biologists, engineers, psychologists, computer scientists, physicists, applied mathematicians, and other disciplines out of their silos and get them to learn each other’s argot and ways of thinking, which meant taking down every wall possible to provide large spaces where we could mix all these cultures, maximizing osmosis. White boards were to line the corridor walls, encouraging spontaneous discussions. To emphasize in the architecture our flat organizational structure there were to be no private offices, not even for the directors. And then there was the matter of a kitchen . . .

Thankfully, the architects responded creatively and deconstructed our space. Rodney and I positioned our desks as far as possible from the administrative offices, sharing one end of the “Big Room,” with its long refectory table, sofas inviting conversation, piano, journals, jungle of plants, tie-dyed wall hangings, circus equipment, and adjacent (small) kitchen. One unintended consequence of taking away the walls of the silos was the emergence of a grassroots’ network of expertise. The students rapidly discovered who among them was the go-to person for a particular corner of knowledge, and so they created organically a self-perpetuating network of competences. The endless flow of visitors, from schools to lay public, gave them many opportunities to rehearse simple and short explanations of their research.

The animal house was in the building and included large outside enclosures filled with an endlessly changing landscape of ladders, ropes, boxes, and trees, to which the cats and monkeys had free access. Visitors were always astonished to discover our “zoo.” The animal caregivers were exceptionally devoted and could call any cat to them by name. I made it a rule that all the PhD students working with these animals should spend time with them, for this produced calmness on both sides. One student, Franziska Sägesser, was our champion cat whisperer and held long conversations with the cats in Swiss German, which they certainly understood better than I. Experiments with large animals present large challenges, and it typically took two or three of us several hours of concentrated and well-rehearsed preparation before we could begin recording, even longer if optical imaging was involved. After training the students to do the experiments, I always assisted them (but never quite finessed the tuneless whistle that Prof emitted when things were not going smoothly) and in the old tradition, I always helped with the endgame.

Soon after we had moved into our new Bau 55 we became embroiled in a public controversy. In Switzerland, some fraction of the cost of public building was devoted to Kunst am Bau (“art within architecture”). The building occupants had no say over the choice of artists, so one day we found Thomas Hirschhorn—renowned for his Wegwerf Kunst (“disposable art”)—building a large carboard “Kiosk” in our entrance hall. Eight were planned, each wallpapered inside and out with photocopies of a particular artist or writer’s work, starting with one dedicated to Robert Walser. Inside were books by, and videos about, the artist. After six months the Kiosk was torn down and a new one was erected, all at a cost of SFr 400000. We protested at the waste, but found ourselves vilified in the press as the “philistine professors.” We quickly cottoned-on that we were now part of the exhibit and shut-up. For the vernissage of the final Kiosk, we all arrived dressed up in carboard costumes, to Hirschhorn’s amused surprise. It was a wonderful piece of Dada, quite fitting for a Zurich art event.

Persons

We kept busy recruiting. One key position was the system manager. Dave Lawrence, Terry Sejnowski’s former System Manager had been a key component of the NSF Telluride Neuromorphic Engineering Workshop, which had been initiated by the group around Carver Mead, including Rodney. Rodney invited Dave to come to Zurich, and he was eventually persuaded, “I’ll just come for 6 months to set up the network.” He is still there now, as the INI manager. We were hugely impressed with the quality of the administrative staff in Switzerland for everyone we interviewed had formal training and seemed to speak at least four languages fluently. Kathrin Hofacker was one early arrival who became the senior administrator, ably supported by staff that included a former dancer from Balanchine’s New York City Ballet, Simone Schumacher. This dependable core of competence created the critical interfaces we needed with our two central administrations. Marie-Claude Hepp-Reymond, a motor cortex physiologist, retired from the Brian Research Institute and, along with Dan Kiper, became an indefatigable promoter of our outreach activities.

Another innovation was a weekly plenary meeting, for the whole institute—mandatory attendance for everyone year-round—one of our few diktats. The first part was the “business meeting,” where anything could be raised and discussed, followed by one or two presentations by graduate students on their work in progress, chaired by the students themselves. Various embellishments were added, like “Swiss German for beginners,” “word of the week,” or “puzzle of the week,” which built the esprit de corps.

Swiss women had (finally) won the vote, but school hours were still designed for stay-at-home mothers and childcare was complicated and expensive, so when a pregnant postdoc arrived in our first year to work with Peter König, we immediately made the policy decision that mothers could be with their children in the INI and bring them to any meeting. We converted one room into a crèche, but infants could be found cradled under their mother’s desks. Surprisingly, the students were unphased by this unusual situation and became adept supplementary caregivers. The benefits were that the parents were less stressed, their work was less interrupted, and the children enjoyed being in the INI. Indeed, we could quickly identify which children had been raised in the INI—they were the quieter, socialized ones who just got on with entertaining themselves. Visiting children, by contrast, were noisy and excitable, scribbling all over the boards lining the corridor walls and being generally disruptive.

After a year in Zurich, we were ready for a formal opening of the INI and invited the great and the good and asked Terry Sejnowski to give the inaugural lecture. I worked hard with my German tutor, Verena Müller, to give the vote of thanks in German. We had arrived.

ADA, the Intelligent Space

Through Rodney and Misha’s connections with Carver Mead’s alumni, we rapidly built up a formidable critical mass in neuromorphic engineering. Tobi Delbrück, Shih-Chii Liu, Giacomo Indiveri, and Jörg Kramer (a Swiss) arrived and rapidly attracted students to their innovative theoretical and practical courses. It then made sense to create a Europe-based neuromorphic engineering workshop, like Telluride, which brought together the same constellation of skills we had in the INI. Thus, the Capo Caccia Workshop was conceived. Located on the remote coast of Sardinia and lasting a fortnight, it quickly became the international annual meeting place for a cross-section of brain-interested scientists, engineers, entrepreneurs, and students. Another unintended consequence of removing the silo walls in INI and giving our students international exposure at gatherings like Telluride and Capo Caccia was that employees saw our engineering graduates as having an added-value over their peers because they knew something about brains, and similarly, our biology graduates were viewed as having the added-value of exposure to the neuromorphic engineering and neural computation that their peers lacked. About half our PhD graduates are recruited by industry, some founding start-ups with projects originating in INI.

We pulled some of these strands together in an INI-wide project to make an “intelligent space,” which would sense, learn, and act in response to its occupants. The catalyst for this was an invitation to propose an exhibit for the Swiss Expo. We submitted a proposal for “ADA, the Intelligent Space,” which was named after Ada Lovelace, who wrote the first computer program for Charles Babbage’s analytical engine. ADA was intended to evoke wider discussions about two premises: first, that brains continuously construct their own interpretation of the world, and second, that intelligent technologies of the future will share this property with the brain. This was a mammoth undertaking with a very hard deadline, so it was driven with great urgency by Rodney, Tobi Delbrück, and Paul Verschure, a computational psychologist who had joined us from Gerry Edelman’s Institute, along with multiple students and associated scenographers, lighting designers, musicians, and even H. R. Giger of Alien fame, who contributed artworks, most notably his “Biomechanoid” metal cubes, one of which ended up as a frieze in the INI’s entrance hall. After multiple iterations and stress testing, ADA appeared on a platform in Lake Neuchâtel, where she operated with virtually no downtime for six months and was experienced by half a million visitors. It was the only exhibit from a German Swiss university and was a convincing demonstration of the power of harnessing multiple disciplines in a common cause. Its influence is still felt: a decade later, Michael Arbib used ADA as the case study to discuss the implications of neuromorphic engineering for the future of architecture. The dislocation caused by half our students and senior staff working in Neuchâtel for extended times made an added challenge for me to keep the home fires burning in our fledgling institute.

Canonical Advances

Rodney and I wanted to elaborate our canonical circuit. A mathematician, Tom Binzegger, one of our first PhD students, had worked with John Anderson to create a database of cat V1 neurons reconstructed in 3D using TRAKA. We then applied “Peters Rule,” a term coined by Valentino Braitenberg after Alan Peters proposed that cells connect in proportion to the boutons and dendrites that the different cell types contribute to a given volume of neuropil. We also took into account known cases of specificity, like the chandelier cell connection to the pyramidal cell’s axon. From this, we synthesized a quantitative map of the circuit of V1 of the cat, which was soon used by other groups to make supercomputer simulations and by us to explore the dynamical aspects of the pulse response. Getting Tom to finish the project, however, did require the patience of a saint, because just as we were nearing a version of a typescript I thought we could submit, Tom would pop up with another idea of how he could extend, cross-check, fine-tune, or rearrange the results, which usually meant another extensive redraft. But far better to work with someone who keeps worrying the data than with one who jumps ahead to a conclusion too easily won.

The quantitative “Binzegger circuit” provided the ideal opportunity for a proof-of-principle exercise to explore the universality of the canonical circuit by (virtually) translocating a piece of cat primary visual cortex into the prefrontal cortex of the primate to test if it could do something useful. Klaus Hepp proposed we use saccadic generation by the frontal eye field area (FEF) as the behavioral read-out, and he enthusiastically piled high the desk of our student Jakob Heinzle with reprints of every electrophysiological study of FEF in behaving monkeys he could find. Our spiking neuron model behaved excellently and predicted some novel properties discovered later in experimental studies in behaving monkeys. Buoyed by this success, we then applied the model to explain saccadic eye movements during reading, for which there was a large experimental literature. My one-and-only paper in Psychological Reviews was the delightful outcome.

Widening Horizons

Smaller internal meetings that required the directors’ presence began to proliferate, so for efficiencies sake, we scheduled them all on the same day. Wednesdays began with my breakfast meeting around the refectory table to which everyone was invited, and ended at 5 p.m. with the whole INI gathering. Breakfast was the occasion for wide-ranging discussions about questions of science and of being a scientist. Students who saw me active in the lab, were surprised to learn that most of my contemporaries had become managers, fundraisers, and conference-circuiteers around the age of 35 and were seldom active again in the lab—quite the opposite of Prof’s generation, who died with their boots on. But out-of-lab activities were necessary skills, as were refereeing papers, writing grants, choosing and mentoring students, preparing for committee meetings, and public engagement. How to learn these skills was an important topic that we discussed and practiced. The Nobel Prize awards were a perennial breakfast topic, to which I usually offered the opinion that prizes in science were like awarding a gold medal for a winning relay team only to the member who actually crossed the finish line, and then heaping yet more honors on that individual for “winning” the same race.

All the students found giving talks particularly stressful, even when faced with a friendly critical audience. To give an overly polished, overly sunny, TED-style talk seemed their ambition, but such presentations clearly lacked an authentic voice, so as an antidote I decided that we would practice some of the exercises I had learned in many of the theater and music workshops I had participated in over the years while researching for a book on, “the performer’s brain.” The exercises helped the students to remain grounded and authentic, themselves, even when faced with the powerful and distracting stimulus of an audience. One such exercise was playing “pass-the-story,” in which one person in the group would begin an improvised story and then point to the person they wanted to continue it. The difficulty of not knowing whether you were next, and then having to extemporize on the spot, was not too different from having to think on your feet when faced with random questions from an audience, but the exercise often proved paralyzing. To help them further, I organized workshops for the INI members with outside teachers. One memorable weekend was with the late Kristin Linklater, author of Freeing the Natural Voice, who at end of her workshop, gave us the gift of a 15-minute voice warm-up that she had distilled for attendees of the World Economic Forum at Davos. I still use it.

One breakfast exercise that turned out to be unexpectedly moving was when I asked everyone to draw on the whiteboard the story of the work they did. It was an exercise that they clearly thought about a lot, because week-on-week one of them would produce a sketch that captured in a very creative way the essence of what they did, as well as revealing to the rest of us unexpected insights as to how they saw their own work.

Figure 6.    Rodney Douglas (in fancy dress) and Kevan A. C. Martin at INI’s tenth birthday party at Zurich’s Frauenbad. Photo credit: Simone Schumacher.

I also invited people I knew with interesting life stories for conversations with students in our Big Room. The musicians ranged from David James, the counter-tenor of the Hilliard Ensemble; to David Earl, a concert pianist-composer with whom I had collaborated on a song-cycle, Island Owl; to Suggs, the singer-songwriter for the hit Ska band Madness. Matthieu Ricard, the Buddhist monk who has spent more time meditating in a brain scanner than most, came to discuss his book Happiness, and Rod McKinnon entranced his young audience with tales of crystals of potassium channels. All of these visitors had unusual trajectories, and their stories helped inspire and expand the horizons of the students. In house, we also grew a team of qualified teachers of language, music, meditation, tango, yoga, and the Alexander Technique. Were these activities good for anything in an academic institute? At one level, these were simply offers of positive relaxation and a change of gear for a very head-centric people. At a deeper level, these activities joined mind and body—the psychophysical unity of self as Karl Popper and F. M. Alexander termed it—and were an effective, lived experience that reminded them that our brains are not us.

As a student in Oxford, I had made a sponsored parachute jump to raise money for a clinic in Khayelitsha, one of the informal settlements that had grown up on the Cape Flats. This experience led skydiving to be added to my list of sports. I was a member of a number of teams, most notably with “4-Brain,” led by a well-known Swiss skydiver, Mafalda Fent. In competition, a “four-way” skydiving team is scored by how many different formations of a predetermined sequence the four skydivers can complete in 35 seconds of free fall. Bemused passers-by in the lobby of INI would see us clad in multicolored jumpsuits, sliding around on trolleys—“dirt diving”—as we rehearsed the mechanics of the transitions between successive formations. We competed in a number of Swiss National Championships and won medals, but with the introduction of vertical wind-tunnels, the sport has become exceedingly technical, and now only professionals have any chance for medals in the open category.

One of the delights of being on the UZH campus was bumping into Rüdiger Wehner, the sometime director of the Institute of Zoology, whose research on mechanisms of navigation in the desert ant Cataglyphis ranks among the classics of neuroethology. Rudiger was always brief on casual chat, and long on telling me about his latest discoveries. He seemed to know everybody, and invited luminaries like E. O. Wilson to Zurich. We shared an interest in the history of neuroscience and ethology and from time to time he sent me the draft of something he had just written for comment. He did not need my help—his book Desert Navigator, shows what a fine writer and photographer he is.

Student Life

When I began teaching neuroinformatics to non-English speakers, I adopted a lecture-demonstration, “volunteers-please?” approach and, contra the fashion, used no PowerPoint, supplied no handouts, and delivered only old-fashioned “chalk-talks.” I assured the students that I would ask nothing in the exam that was not in my lecture, which seemed to concentrate their minds wonderfully, and they even began practicing note-taking and neuroxing, for which atavistic strategy the ETH Students’ Association awarded me their Golden Owl for excellence in teaching!

The quality of the master’s students was a revelation. The first MSc projects I supervised were those of Sylvia Schröder and Antonia Drinnenberg, who set a very high bar for their successors. Sylvia went on to do her PhD with me and introduced natural stimuli to our studies of cat V1. An informal seminar I began on the foundational literature of neuroscience, became a set course for our in-house MSc degree in neural systems and computation, which was initiated and directed by our new professor, Richard Hahnloser, a former PhD student of Rodney and Klaus Hepp. My first assistant, Georg Keller, devised an experiment to accompany each week’s set of historic papers. One example was his working replica of the Hodgkin-Keynes mechanical model of the potassium channel, something that had inspired Rod McKinnon in his structural studies. The attendees soon understood the meaning of the word “tralaticious” when we read Roy and Sherrington’s 1890 paper, which had spiked in citations after the discovery of the BOLD signal. I had written “A Brief History of the Feature Detector,” which highlighted Horace Barlow’s early work, and the seminar gave me an opportunity to introduce some of his fellow travelers in the legendary Ratio Club, the hub for British cyberneticists. The 1950s was an exceptionally fertile era that generated new ways of thinking about the nervous system in a quantifiable way. The interchange between engineers, physiologists, psychologists, mathematicians, and computer designers in the Ratio Club was hugely productive and something we tried to emulate on a daily basis in INI. The famous photograph of the Ratio Club, taken in May 1952 outside Peterhouse College, Cambridge, shows Horace (at the time a PhD student of E. D. Adrian) sitting on the ground in the front row holding a glass of wine. Cross-legged at the other end of the row is Alan Turing. I once asked Horace why he was the only one in the photograph holding a glass. He replied, straight-faced, “It must be because I’m a slow drinker.”

Idan Segev, whom I’d not met, invited me to a conference at the Hebrew University in Jerusalem. It was the beginning of a friendship and a long collaboration. He had worked at National Institute of Mental Health with Wilfred Rall and is surely the inheritor of Rall’s mantle. As an advocate for the biophysical modeling of neurons he is unsurpassed, always clear and passionate, full of insights. Mike Shadlen, a jazz guitarist of note and sometime graduate of “Uncle” Bill Newsome’s Monkey Business, was at the same conference. He had hired a car and offered to take us on a day trip to the Mediterranean. I suggested we try swimming in every sea and, Red Sea excepted, we did, but it involved driving along the West Bank, from the Dead Sea up to the Sea of Galilee, something Mike had been strongly advised against. It was a fascinating, if disturbing journey, now quite impossible. On a later occasion, Idan took Tobias Bonhoeffer and me to Petra along the opposite bank of the Jordan river.

Tobias taught a summer school at the Gulbenkian Institute in Lisbon, and while there, advised a young biology student to apply to me to do a PhD. So a tassel-haired, bearded and braces-clad Portuguese, Nuno Maçarico da Costa, arrived in the lab, staying long enough to get a PhD and start his own junior group before moving on to lead a connectomics project at the Allen Brain Institute. By the time he completed his 10,000-hour apprenticeship in INI, he was the master of his trade. He was extraordinarily generous with his time and knowledge and helped me train another generation of experimentalists, including Georg Keller’s younger brother Andi, who introduced the strangely silent world of two-photon imaging into our cat studies (making me grateful for the inestimable boon of Lord Adrian’s insight to play the voltage transients of neurons over a loudspeaker). Nuno’s own PhD project was the impossible dream: to discover how synaptic inputs from the thalamus relate to the functional ocular dominance of their target cells in layer 4. There was almost no technique we did not deploy in an attempt to winkle out the answer, including optical imaging, tract tracing, immunochemistry, intracellular recording, and an inordinate amount of correlated light and EM. His project had the unintended and significant consequences of not only introducing new technologies to the lab, but also of driving the skills of key people to new levels. For his dream, though, we had to admit defeat. Nonetheless, the spin-offs were significant and his quantitative map of the thalamic input to spiny cells in cat V1 was used in a biophysical model of the simple cell with Idan Segev and Yoav Banitt, which alerted us to the importance of synchrony and stochastic resonance.

Lines of Communication

John Anderson and I had been working on the quantitative ultrastructure of the long-distance connections between visual areas in the macaque. With Kathy Rockland, we described the anterograde pattern of synapses for the V1 projection to MT, which resembled the magnocellular thalamic projection to layer 4 in V1. John and I subsequently found that, while every projection we studied had a unique laminar pattern of innervation, all contributed only a tiny fraction of the synapses in their target layer. This matched well the quantitative results obtained by Henry Kennedy using retrograde tracers to label the cells of origin. How could such tiny numbers of synapses be effective? It was not by making large synapses, for when we reconstructed in 3D the postsynaptic densities, we found that their sizes typically ranged over at least two orders of magnitude. We were particularly intrigued by the presence of so-called perforated synapses, which took on horseshoe-like shapes, which we had seen previously for the thalamocortical synapses in cat and monkey. This prompted a recurring thought: how could a single vesicle of neurotransmitter ever saturate all the postsynaptic receptors at a such synapses, as distinguished investigators like Julian Jack and Bert Sakmann assured us it did?

Primate Matters and Moonshots

For her PhD, Isabelle Spühler had mapped the pattern of dopamine boutons in macaque frontopolar cortex. She then teamed up with Andreas Hauri, a student of Rodney’s, who had co-developed the Cx3D, a program for simulating millions of cells interacting with each other both biochemically and physically. They produced an insightful paper that used the structural data to model the nonsynaptic release and receptor-binding of dopamine. Their study, along with our FEF modeling papers, made me even more curious to explore the circuits of prefrontal cortex. Thus, when I applied to the Zurich Animal Commission for a renewal of my animal license, I proposed making a quantitative analysis of the local circuits in prefrontal cortex. Another group leader, Dan Kiper applied at the same time to continue his behavioral studies on visual acuity. Zurich is the only one of the 26 Swiss Catons where a minority of members could block, through a mechanism called a Rekurs. a license being granted. Three positions on the commission were reserved for animal welfare organizations, so this mechanism was highly likely to be used. The chair of the Zurich Animal Commission was Klaus Pieter Rippe, a philosopher who wore another hat as president of the Swiss Ethics Committee of Non-human Biotechnology, which had been commissioned by the Zurich Animal Commission to assess the ethics of using marmosets as a model for depression. They took this particular case as a remit to consider all Swiss primate experiments. Their deliberations led to both our license applications being subject to a Rekurs. Dan and I engaged in a wearying legal battle to have it overturned, but eventually the Swiss High Court declared that fundamental research should not be carried out on primates—only research that had a direct outcome for human health and welfare. It is not difficult to point out the flaws in their reasoning, which if universally applied, would actually have devastating consequences for human health and welfare. Ours was far from an isolated instance, as similar cases were repeated across Europe.

Our planned work was stopped in its tracks, but research involving monkeys did continue in INI with Hans-Jörg Scherberger, who was studying the cortical representation of grasp and precision grip. Later, after another lengthy legal process and a very supportive UZH vice president of research, Danny Wyler, Valerio Mante was granted a license to study decision-making and coding in monkeys. The Swiss Ethics Committee meanwhile had assigned themselves the task of advising the Swiss Federation on “the dignity of plants” (sic), where they came to the conclusion that decapitation of wild flowers at the roadside without rational reason is “morally impermissible.” Even Jeremy Bentham would demur.

An important source of funding for the INI was the European Union’s (EU’s) Future and Emerging Technologies (FET) program, which funded consortia of labs across Europe. Because of its portfolio of skills, INI members had been very successful in obtaining FET grants. All this was to end, however, when the EU decided to fund just a few flagship projects. At the front of the queue was a former UCT student of Rodney’s, Henry Markram, who had started the Blue Brain Project at ETH’s sister institution EPFL (École Polytechnique Fédérale Lausanne). Neuroscience is a cottage industry, but Henry’s proposal for an FET flagship project was presented as neuroscience’s moonshot: to simulate the human brain within a decade, and by large-scale simulations, find cures for neurological diseases, thus eliminating any further need for animal experiments. Rodney and I were convinced the extravagant claims made for the Human Brain Project (HBP) were bogus and that it would be a tragic waste of Swiss academic funding. Because no new money was involved, a flagship project would also divert much-needed funds away from many EU neuroscience projects for at least a decade and signal the end the very successful FET program. Henry’s public relations machine swung into action and he convinced the Swiss politicians, the president of EPFL, and the ETH Board to back his bid. We did point out the absurdity and hubris of HBP’s claims in person and in print, but to our surprise, many eminent scientists climbed aboard and the HBP flagship was funded to the tune of 1 billion Euros over 10 years. Now after a decade, the failure of the HBP to realize its ambition to simulate a human brain, or publish any groundbreaking insights from their simulations, is plain for all to see.

Daisies and Bouquets

The clustered lateral connections made by pyramidal cell axons, which we called a “daisy,” had long intrigued us. These originally were discovered by Kathy Rockland and Jennifer Lund (see Volume 3) and had been assigned many roles, including forming an inhibitory surround for the receptive field and for linking domains that had the same orientation preference—the so-called “like-to-like” connectivity. In an epic series of experiments led by Elisha Rüsch and German Köstinger that required intrinsic imaging, intracellular labeling, and correlated light and electron microscopic reconstructions, we overturned most of the previous speculations, not the least of which showed convincingly that the daisies did not create the surrounds of receptive field and did not exclusively connect like-to-like orientation domains. Our new interpretation was that daisies provided a structure for local context-dependent processing, something for which we had supporting evidence from our previous physiological experiments with Cyrille Girardin, Anita Schmidt, and Sylvia Schröder.

Rodney and I decided to mark the 20th birthday of INI by thanking all our supporters. The Hilliard Ensemble was tempted out of retirement to give us a concert in the majestic Semper Aula of the ETH main building. It was a moment to savor and enjoy; to remember the friends we’d made, and lost; to give credit to all the people who helped us build our collective enterprise; and for me to say a prayer of thanks to Leon Isaacson for introducing me to Rodney. We had been roped together on the same mountain for four decades, ascending in tandem by the best routes we could find, leading, following, endlessly debating and creating science and environments for science.

Quantum and Solace

The question of the relation of synaptic structure to epsp amplitude reared its head whenever we did correlated LM and EM, but to get a definitive answer required high-risk experiments, so I was reluctant to offer it ever as a PhD project. An MSc student, Gregor Schuhknecht, who had been studying thalamic synapses in the mouse motor cortex with Rita Bopp, an EM expert, expressed a strong desire to try. Knowing it was now or never, I sent him to Hamburg to take an advanced patch-recording course with Ora Ohana. Ora had done her PhD with Bert Sakmann before setting up her own group in INI, so she was the perfect tutor. I tapped Ken Stratford, then busy with ecology research in Namibia, to look over Gregor’s shoulder at the quantal analyses, and with German Köstinger and Simone Holler taking on the structural side, we now had a once-in-a-generation A team, and they played a blinder. We established that there was a linear relationship between the amplitude of the epsp and the area of the postsynaptic density, and more significantly, we provided definitive evidence that individual synapses had multiple transmitter release sites, thus overturning decades of authoritative assertions that neocortical synapses possess only a single release site. The shades of gray EM structure could now be colored with functions beyond a simple plus or minus. The project epitomized INI’s ethos of collective endeavor. Another door had opened, beckoning us to explore further the nature of cortical synapses and the circuit functions they enable.

Charles Sherrington, who made the link from synapses to behavior surer than anyone, gave us the indelible image of the “enchanted loom,” by whose means we weave our dreams into realities: how favored are we who can explore its secrets?

Pasko Rakic

BORN:

Ruma, Former Yugoslavia

May 15, 1933

EDUCATION:

University of Belgrade, MD (1960)

University of Belgrade, PhD (1969)

APPOINTMENTS:

Resident in Neurosurgery, University of Belgrade Faculty of Medicine (1961–1962)

Clinical and Research Fellow in Neurosurgery, Harvard Medical School, Boston (1962–1966)

Assistant Professor, Developmental Biology and Genetics, Biological Institute, Belgrade (1967–1969)

Assistant to Associate Professor of Neuropathology, Harvard Medical School, Boston (1969–1978)

Professor and Chair of Neurobiology, Yale University School of Medicine (1978–2015)

Director, Kavli Institute for Neuroscience, Yale University (2004–2015)

Dorys McConnell Duberg Chair in Neuroscience, Yale University (1978–present)

HONORS AND AWARDS (SELECTED):

Member, National Academy of Sciences, United States (1985)

Member, Serbian Academy of Arts and Sciences (foreign member) (1985)

Karl Spencer Lashley Award, American Philosophical Society (1986)

Francois I Medal, College de France (1986)

Member, Croatian Academy of Arts and Sciences (foreign member) (1990)

Fyssen International Science Prize, Paris (1992)

Member, American Academy of Arts and Sciences, United States (1994)

President, Society for Neuroscience (1996–1997)

The National Academy of Medicine, United States (1999)

Bristol-Myers Squibb Award for Distinguished Achievement in Neuroscience (2002)

Kavli Neuroscience Prize (2008)

Member, Norwegian Academy of Science and Letters (foreign member) (2008)

Krieg Lifetime Achievement Award shared with Paul Allen (2010)

Child Mind Institute Award, New York City (2014)

Member, Royal Society London, United Kingdom (foreign member) (2016)

The Charles Branch Brain Health Award (2018)

Member, Royal Academy of Medicine, Spain (foreign member) (2018)

Connecticut Medal of Science (2019)

Sterling Fellow, Yale University (2020)

Pasko Rakic is recognized for his discoveries of the molecular and cellular principles of neuronal generation, migration, and differentiation that underlie the development and evolution of the brain, particularly the cerebral cortex. His pioneering studies of cortical neurogenesis in rodents, nonhuman primates, and humans led him to propose the radial unit and related protomap hypothesis of the development and evolution of the cerebral cortex, which explain how three-dimensional laminar, columnar, and areal organization of the cortex is built from a two-dimensional layer of neural stem cells in distant proliferative zones. Rakic then identified genes involved in specific developmental events and provided quantitative data for the overproduction and selective elimination of axons, synapses, and neurotransmitter receptors in the primate brain before they decline to adult levels during prolonged primate adolescence.

Pasko Rakic

I admit that it took me a long time to finally decide to write this autobiography. I had not responded to numerous kind invitations by Larry Squire, more recently joined by Thomas Albright. The idea behind the book series entitled The History of Neuroscience in Autobiography was not to write or document dry scientific facts that can be found in the original research papers, but rather to provide personal recollections and stories about your professional and private life and how it all happened, who inspired your choices and actions, and who were your role models. In addition, it should contain some interesting and unusual anecdotes. So, it is an arduous task, particularly if one has a long history, and I am probably one of the oldest neuroscientists who is writing this type of autobiography. Therefore, I was wondering who would be interested to read it when many of my colleagues that participated in some of these events are no longer with us? It is also rather difficult to make statements in personal terms, like “I made some significant discoveries,” without looking self-serving. I was also reluctant to spend the time necessary for writing an autobiography at the expense of my still very active enterprise. I finally did it now, because of my solitary disposition in my home as a result of COVID-19, which allowed me to indulge in reminiscence and to devote sufficient time to complete this difficult task.

Origin and Early Years

I was born in Ruma, a small town in Vojvodina, which was at that time a province in the Kingdom of Yugoslavia. Although this autonomous province is now part of the Republic of Serbia, members of my family were immigrants with multi-ethnic roots. My father, Toma Rakić, the son of Anton Rakić, was Croatian born in Istria, a peninsula at the Adriatic Sea, which at the time was part of Italy. My paternal grandmother, Maria Cukon, was also born in Istria and of mixed Croatian, Italian, and Albanian lineage. In 1924, to escape from rising fascism in Italy, my father emigrated to the newly formed South Slavic country named Yugoslavia. There, he met my mother, Juliana, who was born in Dubrovnik on the Dalmatian Coast of the Adriatic Sea. Her father, Miloš Todorić, was Serbian and her mother, Pepa Kuzma, was of Croatian and Slovakian ancestry. At the inauguration of the Kavli Prize in Oslo, when asked my origin, I explained that it is a rather complex issue. So, the narrator of the ceremony, Jon Storm-Mathisen said, “Maybe the best information would be to tell us where your ancestors were born?” I replied, “When my grandparents were born, the peninsula of Istria was part of the Austro-Hungarian Empire, in 1918 it became Italy, in 1946 it became Yugoslavia, and in 1991 it became Croatia. Therefore, they lived in four countries without moving from the same house” (figure 1).

My father had innate intelligence and was particularly adept at working with numbers. In his new country, he attended a financial school in Novi Sad and subsequently earned a job as the district director of the Internal Revenue Service. At that time in Yugoslavia, in order to prevent the temptation of extending favors to local friends, district directors, and of course members of their families, were required to move from one town to another about every three years. It was emotionally difficult for me as a young boy to leave friends behind and then repeatedly try to make new friends in new places. Thus, later in my life, I related more to the people than to the places and never felt that I fully belonged anywhere. I was born in Yugoslavia, a country that no longer exists, and have lived the longest and made my professional career in the United States, which is a multi-ethnic society.

Figure 1.   House of my ancestors in Istria Peninsula at the Adriatic Sea.

Apart from being devoted to raising two children, my mother was an avid reader and collected books that ranged from classical French, Russian, and American literature, like Honoré de Balzac, Fyodor Dostoevsky, and Sinclair Lewis, to the psychology classics, like Sigmund Freud and Alfred Adler. Reading books or even items in an encyclopedia during my childhood was the only escape from the cruel and senseless World War II that deeply affected our country. During German occupation, I saw people killed in the street or relatives disappearing overnight from my life without any trace. Some local people cooperated or participated in the quisling governments created by German occupiers, and three different national factions, Croatian fascists “Ustaše,” Serbian nationalists “ˇCetnici,” and Marxist league of “Communists” killed my grandfather, one uncle, and a cousin during the war. Hence, I could say that I was a real Yugoslavian, as I was suffering equally at the hands of extremists from all sides.

For a while, I was seriously devoted to chess, memorizing entire movements made by the World Champions. At the age of 14, I was beating my classmate Vladimir Sokolov, who eventually became a world-class International Chess Federation master. Unlike Vladimir who persisted, however, I stopped playing chess at the age of 15, because I felt that it was too time-consuming and did not produce or explain anything. The exposure to chess at that critical developmental age, however, had a long-lasting positive effect on my thinking and selecting between various possibilities in the future. My mother had unrealistic expectations for me and insisted that I take strict classical piano music lessons. I did not have much enthusiasm or talent for the piano, and instead I mastered playing the accordion. I was sufficiently proficient to play in the school’s orchestra at weekly dance parties. However, I did not like this arrangement, as while I was playing, my friends were dancing with nice girls. Another, more enjoyable pastime was painting watercolors and drawing graphics, as well as creating wooden and metal models of trucks, tanks, and airplanes, which were not available as toys during the war. I was also quite athletic and was winning most of the 100-meter dash races and my class represented our school at the national competition.

Although I was raised as a Catholic, my parents were not religious and went to church only occasionally to attend Midnight Mass on the night of Christmas Eve. Perhaps motivated by resentment of the dominating atheistic communism in postwar Yugoslavia, I tried to find answers to some basic questions of life in religion. Also, Catholics were advocating forgiveness and were against killings. Thus, on my own, I decided to become an altar boy at the age of 8 and began to get up at 6:30 a.m. every morning and walk several miles to church to serve at the mass. There I would join the priest and listen to the long ceremony, which was at this time entirely in Latin language. I did not fully understand all the words but learned the entire mass by heart and even now remember some long passages. However, over time I found myself debating biblical explanations of the origin of the universe at meetings with my fellow altar boys, as well as with the priest. For example, I was upset to learn that Giordano Bruno was burned and Galileo Galilei was tried by the Roman Inquisition because of their views of the universe that were contrary to religious beliefs at the time. By the age of 14, I asked, “How could people who thought that the earth is flat teach us anything about the origin of the universe?” After I made this statement, our priest told me to leave and stop attending the group meetings. Thus, at that early age I realized that scientific facts and strict religious beliefs could be in conflict; and, intuitively, I have preferred to err on the side of science. Many years later my friend Michael Bennett, a member of the National Academy of Sciences (NAS) and smart guy, told me, “Gentleman never talk about religion,” and I said, “If one aspect of being a gentleman is to be afraid to say what they think, then I do not have this particular aspect.” However, I do think that most religions have the basic intent to do some good and that the believers in the afterlife are lucky, and I still like to attend Midnight Mass for nostalgic music and sentimental reasons.

I obtained high school education in the town of Sremska Mitrovica, which even after the Communist takeover, continued education in the Austro-Hungarian-style gymnasium (equivalent to high school and junior college in the United States). The gymnasium had a strong emphasis on classics, history, philosophy, and literature, but also on algebra, geometry, calculus, and the natural sciences of biology and physics. I also had courses in Latin, French, and Russian languages, but unfortunately English was not recommended as a choice because it was used in the countries with a “rotten capitalist system.” My three-years-older sister Vera attended the same school and after completion entered university and received a diploma in higher mathematics, which at the time was rather unusual for a woman. I was a top student in the gymnasium class and was elected to be a leader of the debating Literary Club, which met monthly. I enjoyed vigorous discussions with both fellow students and professors. However, I was not a conformist and often took a position against the prevailing views, established dogmas, and authorities, a practice which I continued later in my life, sometimes to my detriment.

Higher Education

There was never any doubt in my mind that I should continue to seek the highest educational level possible, especially since education in socialistic Yugoslavia was free as long as one had good grades. Initially, I was interested in pursuing a career in art or architecture as they offered some level of freedom of expression, even in the socialistic society. However, my father thought that even art was controlled by communists and dominated by “socialistic realism” and convinced me to apply to medical school with the statement that, “If things do not work as well as you wish, it is better to be a mediocre doctor than a mediocre artist.” This advice was, perhaps, influenced by his experience of losing his life savings after the Socialists came to power in 1946 and expropriated all his holdings in the banks. He also told me at this time the popular proverb, “that knowledge is the only thing that cannot be taken from you.” Thus, I enrolled in Medical School at the University of Belgrade to hopefully learn some useful information.

Belgrade was, at that time, the capital of Yugoslavia, a country with a multi-ethnic population of about 20 million. It was a cosmopolitan city, with excellent classical music, opera house, and several repertory theaters that I regularly attended and enjoyed. I also kept in touch with art by writing reviews for the major Belgrade newspapers about the avant-garde theater shows. I was also publishing reflective and satirical poetry. In addition, I earned some money by drawing cartoons and caricatures for the local satirical journals. Some of my friends suggested that I abandon medical school and become a professional writer and cartoonist, because “anybody can finish medical school, but only a few have talent for writing poetry and to create original cartoons.” However, my writing and drawing activity did not prevent me from getting top grade at the Medical School.

As the medical student with the best record in the class, I won a summer scholarship to Finland and spent two summer months in the Department of Gynecology at the Helsinki Medical School. I liked Finnish people and their customs, including hot nudist saunas at the coast of a cold lake, which I attended with my professor, his wife, and daughter. I was particularly impressed with Finnish women, whom I thought were nice looking, and I had several dates with Miss Helsinki, Karina Tapiovaara. This was my first trip abroad, and I also used this opportunity to visit several capitals of other Western European countries, including Vienna, Amsterdam, and Stockholm. I began to appreciate the value of freedom and expression of rights to have different views, which was being so strongly suppressed by the socialistic regimes.

After returning to Yugoslavia, during a regular histology course at the Medical School, which I also taught as a Student Aid, I was exposed to a book with Ramon y Cajal’s drawings. This book opened to me a universe of unbelievable complexity and beauty, formed by the black silhouettes of nerve cells, as well as Cajal’s imaginative thinking about how they may have evolved and function. This exposure to the “neuronal forest,” perhaps in addition to the compelling problems of neuropsychiatric diseases, drew my interest to the brain. Many years later, I was asked by Javier DeFelipe from the Cajal Institute in Madrid to write a foreword to his book entitled Cajal, Neuronal Forest: Science and Art, which I gladly did (DeFelipe, 2019; Rakic, 2019).

Residency in Neurosurgery and Fellowship to Harvard Medical School

Following graduation from medical school, I entered into an internship at the University Hospital and then enrolled in the residency program in neurosurgery. The training in neurosurgery was rigorous, but I was a bit disappointed as it did not meet my expectation as an intellectual endeavor. I also did not like that my daily activity did not depend on me, but rather on the strict schedule, which started very early in the morning and was prepared by the head nurse. There was no choice or creativity and most of the operated patients died almost at the same rate as the unoperated. Nevertheless, according to my professors, I was quite skillful with my hands, and on my way to becoming a successful neurosurgeon with a secured university faculty position. However, during the second year of my residency, I met Hannibal Hamlin, a neurosurgeon from Boston (United States) who was visiting the Neurosurgery Department at the University of Belgrade. We went to dinner, and he liked very much our discussion about the brain and urged me to apply for an international fellowship to train at Harvard Medical School. He assured me that my rather poor English would not be an obstacle and that I would be able to learn. So, I applied and obtained a Clinical and Research Fellowship to be spent in the Department of Neurosurgery at Massachusetts General Hospital in Boston, which was chaired by William Sweet.

After arriving in Boston in the fall of 1962, I had my first Thanksgiving Dinner at Hamlin’s home at 270 Benefit Street in Providence, Rhode Island. I had not realized that he was a descendent of the senator from Maine, Hannibal Hamlin, who was the 15th vice president during Abraham Lincoln’s term. Some other guests at the dinner were descendants of the former U.S. president Van Buren and vice president Rockefeller. I went to the dinner in a tailormade black suit with a white shirt and red tie that I brought from Yugoslavia to impress my new colleagues. To my surprise, the other guests at the dinner were dressed very casually in old, wrinkled shirts, cheap jerseys, and jeans. I learned my lesson; poor people in the United States at that time dressed up, and rich people tried to hide their wealth by wearing cheap clothing. These days both rich and poor try to dress as unattractively as possible. In the backyard of the house and before Thanksgiving turkey dinner, I played my first (American) football game with the other guests and spoiled my beautiful white shirt.

During my fellowship at Harvard Medical School, I had an additional assignment to perform some research on neuroanatomy of the human brain in the Warren Anatomical Museum, which was part of the Department of Neurology and Neuropathology. There, I was lucky to meet the world-famous neuropathologist, Paul Yakovlev. He was originally trained with the legendary Ivan Pavlov in his native St. Petersburg, and after the October Revolution, he escaped from the new Soviet Union and immigrated to France and worked in Paris with another famous neurologist, Joseph Babinski. Paul, a European-style intellectual, introduced me to the challenges and joys of studying the development of the human brain. He also instilled in me the idea that understanding of the human brain will not come from the study of single cells or identification of specific signaling molecules that we share with other creatures, but from unraveling the pattern of neuronal circuitry that is exclusively human and that underlies aspects of our unique mental capacity, such as language and abstract thinking. He also believed, like Albert Einstein, that the understanding of the meaning of a finding is as important as the finding itself. My original two-year fellowship transformed into a four-year stay in Boston, during which time I immersed myself in analyzing the neuronal organization of the human brain in Yakovlev’s collection. Paul was an erudite who loved history and introduced me to the classical literature on human brain development. He also showed me that research is fun, and when asked when he would stop working, Paul would reply, “I never did work!”

In addition to assisting neurosurgery operations and following patients’ postoperative states, I was interested in neurology and regularly attended Tuesday’s “Brain Cutting Sessions” at Massachusetts General Hospital, conducted by Raymond Adams, as well as Saturday’s weekly conferences held by Norman Geschwind and Derek Danny-Brown at the Boston City Hospital. Nevertheless, because of my disillusion with neurosurgery, I devoted most of my time to learning about advances made in basic neuroscience research. I was particularly interested to probe fundamental questions, including exploration of the molecular and cellular mechanisms that enable initially uncommitted progenitor cells (now renamed stem cells) in the embryo to generate neurons that migrate to their proper areal and laminar destinations and form the mature human cerebrum, the most complex organ in the known universe.

Visit to the Moscow Brain Institute

Following my exposure to the human brain at Harvard, and my experience in research on the developing human brain for my doctoral dissertation after I returned to Belgrade, I was offered and accepted a two-month exchange program to visit the Moscow Brain Research Institute of the Soviet Academy of Sciences. There in 1967, I met several of the prominent Russian scientists, like Semen Sarkisov, Gregory Poliakov, and Tatiana Leontowich. They showed me at the microscope sections of Lenin’s brain that were cut into serial tissue sections and stained by Cécile and Oskar Vogt. I was looking at the neurons in Lenin’s auditory cortex and thought that these cells created speeches that influenced millions of people and changed the history of the entire world. During that time, I also went to St. Petersburg (then called Leningrad) to visit the Sechenov Institute of Evolutionary Physiology and Biochemistry.

I spent New Year’s Eve 1968 at the home of Andrey Polenov, whose predecessors were on the ship Aurora, which fired guns at the onset of the October Revolution. It happened that during the time of my visit, the Soviet army invaded Czechoslovakia. My host toasted with a glass of slivovitz, which I had given him as a gift, with the words, “For the friendship without tanks.” However, a KGB agent was also present at the dinner and reported that conversation. Nothing happened to Polenov, but the KGB suspected that I might be an American or one of Tito’s secret agent. Josip Broz Tito indeed came in defense of Czech leader Alexander Dubcek, and all airplane flights to Yugoslavia were cancelled. Therefore, I could not leave the Soviet Union and instead had become a candidate for deportation to a camp in Siberia. For days I was detained in Moscow’s Hotel Ukraina (at Soviet expense) and was very scared. The person who was assigned to watch me was a nice young lady, who told me openly that I may be imprisoned in Siberia because I was suspected of being an American or Yugoslavian spy. She suggested that we walk out of the hotel to talk more freely, and I was happy to oblige. After hearing my recital of the Russian love poems by Fyodor Tyutchev, which I knew by heart, she was very impressed and began to like me. She did not believe that someone who is in love with subtle Russian romantic poetry, could hate Russia, and would be a spy. She also seemed to like me and was trying to help. Finally, one night at 2 o’clock after midnight, KGB agents picked me up and drove me to the Sheremetyevo Airport, which was at that hour closed for passenger traffic. They embarked me on a military cargo plane, which was loaded with arms destined for Uganda (Africa). The Soviets made a deal that if Yugoslavia allowed their plane to stop at Belgrade Airport to refuel, in return, they will dispose of me. Indeed, I was left after midnight at the Belgrade Airport, which was closed for the regular passengers. At that time, I was very frightened and decided never to visit Russia again, and so far, I have kept that promise, even though over the years I have been invited to several interesting meetings.

Changing from Medical to Basic Science and Enrolling in Graduate Program

After a productive experience in Boston (United States) and Moscow (Soviet Union), I returned to Belgrade (Yugoslavia) and decided not to rejoin the residency in the Harvard Neurosurgery Department. I thought that I could contribute more by doing basic research than by caring for individual patients, given that so much was unknown about causes and pathogenesis of congenital neuropsychiatric disorders. I also wanted to escape the routine duties in clinical medicine, especially because so little could be done for patients with neurological disorders or brain trauma without more knowledge about the brain. However, I recognized that to become a good basic scientist, I needed to learn more about genetics and molecular and cellular biology. Therefore, I decided to obtain a graduate degree in the Belgrade University program of Developmental Biology and Genetics. For my thesis dissertation, I decided that the question of how a complex organ like the human brain, which mediates thought and creativity, and develops from a single fertilized cell, is the most important question in medicine that probably could be answered by modern methods and creative thinking.

As part of my research on my doctoral thesis at Belgrade University, I used a recently available radioactive marker for DNA replication, (H³-thymidine- [³HdT]), to label the last cell divisions in viable tissue slice preparations of postmortem embryonic human cerebrum. I had smuggled a small container with ³HdT on my return from my previous visit to Boston. I can admit this unlawful act now because the statute of limitation has passed. However, I knew that ³HdT has such weak radioactivity that it could not penetrate even a sheet of paper and could not endanger me or anyone else on the plane. This sample of radioactive chemical enabled me to perform experiments on human slice preparations to identify neural stem cells, which continue to divide “supravitally” (after death) in the tissue culture medium. I created the title “Supravital DNA Synthesis,” but because the initial observations and ideas in my dissertation were written in Serbo-Croatian language, Sidman helped me to make translation to proper English. Thus, his name was added to the authorship of the paper, which was published in The Journal of Neuropathology and Experimental Neurology (Rakic and Sidman, 1968). In the 18th century the classical anatomist, Wilhelm His, suggested that postmitotic neurons may migrate based on the examination of the histological preparations from embryonic human cerebrum. However, my work was the first direct experimental evidence that showed that in the several regions of the human brain that I examined, including the large and highly convoluted cerebral cortex, neurons are not generated locally. Rather, they actively migrate from the place of their origin in the proliferative centers near the cerebral ventricle to their final positions. In my doctoral thesis, I described that heavily radioactive nuclei, indicating cell division, are concentrated in the two distinct transient proliferative cellular layers situated near fluid filled cerebral ventricles. Based on these findings in my doctoral dissertation, I subsequently termed these transient cellular compartments ventricular (VZ) and subventricular (SVZ) zones. I also observed that heavily labeled cells but are rare or absent in the superjacent intermediate zone (IZ) and cortical plate (CP) below the pial surface.

Emigration to United States

On the basis of these fundamental discoveries and formulation of new concepts, I was offered a faculty position as an assistant professor in the Department of Neurology and Neuropathology at Harvard University in Boston, which was chaired by Raymond D. Adams. I did not have a problem obtaining a permit to leave Yugoslavia, because Marshall Tito was trying to create “communism with a human face” and was allowing emigration to the West, as he pointed out: “If people want voluntarily to be exploited by capitalists let them go.” Indeed, many including me wanted to leave. However, the U.S. Embassy in Belgrade presented some obstacles because at the time they had a strict quota for immigration from the Socialistic Republic of Yugoslavia, and I was told that I must wait at least two years before I could get a proper legal U.S. visa. Again, I was lucky that at that time (during Richard Nixon’s administration) the U.S. secretary of state was Elliot Richardson, the brother of the Massachusetts General Hospital neuropathologist Edward Pierson Richardson, Jr., who enthusiastically supported my appointment. Elliot Richardson contacted the U.S. Embassy in Belgrade, which then called me on the very same day and asked me to come to their office to obtain my U.S. visa.

At that time, people leaving Communist Yugoslavia were allowed to take with them no more than $20 in cash. On the first leg of the flight from Belgrade to Paris, I ordered a Coca Cola to taste it. I did not like it, but it did not cost me anything. So, on the flight from Paris to Boston, I ordered a martini, about which I knew from watching Humphrey Bogart drinking it with Ingrid Bergman in the movie Casablanca. However, it cost me $3, so that I immigrated to the United States with a total possession of only $17 and no relatives and no friends. Indeed, I left a unilateral, leftist Marxist, egalitarian society in which all are equally poor, to come to the American libertarian system, which at that time, emphasized individualistic competitive meritocracy and allowed opportunity to all. People usually say that I am highly competitive and that, therefore, America was great for me. It is interesting that today, many of the people who benefited from that American meritocracy now attack it as being inhumane and preach the Marxist ideology that I, and many others who escaped, left behind. However, I liked the capitalistic system of the United States, which at that time provided me with an opportunity to succeed according to my abilities.

Faculty Position at Harvard Medical School

I loved the opportunities at Harvard Medical School, which I considered to be unlimited. Because of my knowledge of human brain anatomy, I was asked to be the main lecturer in the neuroscience course, and in spite of my heavy accent, I was voted by medical students as the most popular teacher. In addition, before I received sufficient grant support for my research, I began translation of my doctoral thesis into English with the help of Richard Sidman, who was an excellent English writer. Part of this task was a description of my drawings of the developmental stages of the fetal human cerebral wall, based on the Golgi silver impregnation method. These findings led me to suggest new terminology for the basic cellular compartments in the developing cerebrum.

At that time Sidman was a member of the Boulder Committee for embryonic terminology, formed by the American Anatomical Association, and he presented my original drawings and my concepts and basic terminology at their meeting in Colorado. The members of the committee thought that this was very useful information and adopted my terms for the transient embryonic zones (VZ, SVZ, and IZ) as universal new terminology for the developing vertebrate nervous system. To make it clear that I created these terms, I was advised by my colleagues to sign my schematic drawing, which was reproduced in the Boulder Committee Report published in the Anatomical Record (1970) as evidence of the original source of the concepts and terms (see review of this subject in Bystron et al., 2008). Only one additional significant transient cellular compartment, the subplate zone (SPZ), which was subsequently discovered by Ivica Kostovic in 1974, was added to the nomenclature (see details in Kostovic and Rakic, 1990). In addition, my postdoctoral fellow Donald Schmechel used the Golgi method to examine in more detail the macaque outer half of the SVZ, at the border to IZ. This zone generates some neurons destined for the superficial cortical layers, but produces mostly glial cells, which in primates outnumber neurons (Schmechel and Rakic, 1979a). Some of the the radial glial cells even transiently stop dividing to serve as a stable cytoskeleton of the expanding CP (Schmechel and Rakic, 1979b). This subdivision of the SVZ was later confirmed by immunohistochemistry and named the outer subventricular zone (oSVZ) by Smart et al. (2002). The production of glial cells in this zone continues after birth and contributes substantially to the formation of secondary and tertiary gyri in primates, including humans (Rash et al., 2019; Arellano et all., 2021).

I was lucky that when I entered the field there was an influx of new methods in genetics, molecular and cell biology, electron microscopy, immunohistochemistry, and tracing techniques to detect the pattern of neuronal connectivity. I had an opportunity to meet MIT professor Francis O. Schmitt who created the Neuroscience Research Program (NRP) which was the first society in the word with this name. I was invited to the regular meetings to serve as “scribe,” which provided me an opportunity to meet and discuss issues with the best known intellectuals and researchers, including Nobel Prize laureates, such as Francis Crick, James Watson, John Eccles, Severo Ochoa, Jerry Edelman, Marshall Nirenberg, and even mathematician Ilya Prigogine. For example, Francis Crick was originally an expert in X-ray diffraction methods, but he recognized the value and importance of neuroanatomy (e.g., Crick and Jones, 1993). He made a serious effort to learn neuroanatomy from the world experts, like MIT professor Walle Nauta (also a member of the NRP). During dinner and continuing into late hours passing midnight, we would spend hours discussing the cellular and developmental mechanism of the visual system formation as well as radial unit hypothesis (RUH). Francis Crick thought that a physical diagram, similar to the Double Helix in genetics, also was needed for the developmental neurosciences and that my Radial Unit model was the most appropriate candidate for this. I was still young and honored that he found it worthwhile to spend so much time with me discussing various conceptual issues and valued and liked some of my ideas. I also benefited from the new advances and changing concepts made by my colleagues in developmental biology, too many to be listed here. Thus, I decided to build upon my initial discovery and to focus on the kinetics of progenitor cell proliferation, now called stem cells, and the mechanisms of neuronal migration. I also contributed to NRP publications (e.g., Rakic, 1975b; 1980). The fact that the different neuronal classes must be produced at precise times and sequentially migrate to distant prespecified locations fascinated me to the extent that I was willing to devote, if necessary, the rest of my life to decipher how it works at the cellular and molecular level.

Initially, I used spontaneous mutation in inbred strains of mice as a model system to study how genes affect neuronal connectivity. Today the role of gene mutations on the synaptic pattern is being hotly pursued in Drosophila and Nematode. However, the first description of changes in synaptic organization due to single gene deficits was not obtained from a simple model organism but, rather, was based on electron microscopic examination of neurological mutant mice. In collaboration with Verne Caviness and Richard Sidman, the first evidence of abnormal synaptic contacts due to single gene mutation was obtained in mice named weaver and reeler (e.g., Rakic and Sidman, 1973; Caviness and Rakic, l978). These initial studies that were carried out in mice with spontaneous mutations opened new fields for exploration (reviewed in Rakic and Caviness, 1995). I became interested in the cells that were considered non-neuronal or glial but serve as predecessors for all neurons. These cells, which can be well stained by Golgi silver impregnation method, were described by classical neuroanatomists and were referred to as “Epithelial cells,” “Radial cells,” “Fetal ependymal cells,” “Spongioblasts,” “Tanycytes,” and “Faserglia.” I proposed the term radial glia cell (RGC) that over time became universally adopted (Rakic, 1972).

The next logical question was how the neurons, after their last mitotic division, find the way to their proper positions in the increasingly distant and highly convoluted primate cerebrum. With the application of serial electron microscopic sections, combined with DNA labeling and immunohistochemistry, I discovered that migrating neurons find their way to the appropriate areal, laminar, and columnar positions within the cortex by following scaffolding formed by the elongated shafts of RGCs, a concept that became known as “glia guided neuronal migration” and the concept to which I devoted numerous studies (Rakic, 1971; 1972; 1973; 1978; 1981b). This was before the era of PowerPoint, so during my lectures, I was often trying to explain this extraordinary phenomenon by making drawings on a nearby blackboard (figure 2). With advances made in computer technology, I started to use computerized animations to illustrate this dynamic process based on my initial India ink drawings). On the basis of these discoveries, I proposed the RUH of cortical development and evolution, which provides insight into how the complex, three-dimensional (3D) organization of the brain is built from an initially two-dimensional layer of dividing neural stem cells (e.g., Rakic, 1988b; 1995). This model, despite some initial opposition to it, is now generally accepted and confirmed experimentally in my laboratory, as well as in many other laboratories (see review in Jones and Rakic, 2010). Recently, it was supported by a multi-authored paper in Science (Grasby et al., 2020), which “identified 199 significant loci and found significant enrichment for loci influencing total surface area within regulatory elements that are active during prenatal cortical development, supporting the radial unit hypothesis.” As some of my colleagues say, “the 367 authors of this paper cannot be wrong!”

Figure 2.   Pasko lecturing by drawing the concepts or equations on the nearby board.

Migration of neurons occurs not only during the development of the cerebral cortex but also in many other regions of the large primate brain, and particularly dramatic migration occurs in the developing cerebellar cortex (Rakic, 1971). My first graduate student at Harvard, Richard Nowakowski, who joined this project, showed that neuronal migration along RGC glial fibers occurs also in the hippocampus (Nowakowski and Rakic, 1979; 1981). However, the transient population of RGCs is most remarkable in the large primate fetal cerebral wall where they span the full distance between ventricular and pial surfaces. It is obviously especially important for the development of the even larger human brain in which many neurological and neuropsychiatric disorders display neuronal dysplasia (e.g., Rakic, 1988a). At that time, minicomputers for three-dimensional reconstruction from electron microscopic images were not yet available. With the help of a friend and excellent neuroanatomist, Larry Stensaas from the University of Utah, we manually outlined images of migrating neurons and adjacent RGC fibers from the serial electron microscopic sections of the fetal monkey cerebral wall. I brought these images to the office of the NASA Space Division in Lowell, Massachusetts. They had an IBM 360/75 computer that was used to make the outer structure of the Apollo space vehicle that was sent to the Moon. It enabled us to rotate images to examine the relationship between migrating neurons and RGC fibers. This computer had a much larger capacity than we needed. However, at that time, NASA was not busy and they agreed to help us free of any charge. We published the paper on computer-aided 3D reconstruction and quantitative analysis migrating neurons and RGC relationships based on serial electron-microscopic montages of fetal monkey brain in Nature (Rakic et al., 1974).

As a next step, I initiated a comprehensive autoradiographic study of the specific DNA replication marker ³H-thymidine in nonhuman primates to determine the precise time of neuronal origin, routes of their migration, and settling pattern. I selected the Old World primate—rhesus macaque (Macaca mulatta)—because it possessed a similar cortical organization to that of humans. This project, which was supported by a large grant from the U.S. National Institutes of Health, provided the most comprehensive data and sequence of neuronal origin in any species at the time. I recognized that to obtain this amount of data from a large and slowly developing primate with state-of-the-art methods was extremely expensive. Indeed, the initial message from the NIH Study Section was that for this amount of money they could give five grants for research on small and fast developing species. My reply was: “You still will not know how this occurs in primates!” and they gave me the grant with a high priority score.

Among many instructive and biomedically relevant findings from this series of studies, we discovered that the genesis of neurons occurs at a strict areal and limited temporal schedule (Rakic, 1973). For example, in the cerebral cortex in the macaque monkey, both the beginning and ending of neurogenesis occur before birth and each cytoarchitectonic area has a different time window (Rakic, 1974; 1975b). This study induced the idea that determination of neuronal fate occurs at the time of the last cell division. See the section “Human-Specific Features” for more about this idea and why it is an evolutionary advantage, or even a necessity, for a species like humans, to keep accumulated knowledge for many decades in neurons that should not divide for the duration of their life.

These findings led to the proposed elaboration of the RUH with the protomap hypothesis, as a related model of how different regions of the cerebral cortex evolve during development and evolution to acquire most of their specialized molecular, anatomical, and functional properties through genetic programs intrinsic to the neuronal stem cells during their last division in the VZ and SVZ (Rakic, 1972; 1975b; 1988b; 1990). I was happy that, in spite of initial opposition to this concept by the researchers who overemphasize the role of the environment, the radial unit and protomap hypotheses became generally accepted as basic principles and have received support from numerous laboratories (e.g., Arcaro et al., 2020; Grasby et al., 2020). Furthermore, recent experimental studies, using genetically altered mice, viral gene transfer for tracing cell lineages, and the in utero electroporation method, confirmed the validity of my models (e.g., Miyashita-Lin et al., 1999; Cholfin and Rubenstein, 2007). Contemplating the meaning of these findings, I admired the fact that Charles Darwin had a vision and the insight to propose the theory of evolution based on only crude observations available at the time. Our findings on molecular and cellular development of the mouse, monkey, and human brains clearly expose a progression that could have only occurred during evolution by random mutations of genes and their regulatory elements in our common ancestors.

Today, the notion that connectivity in the developing central nervous system is initially more widespread than in adults is commonplace. However, it was not a popular notion at the time when I discovered that in the primate embryo, the central projections from the two eyes are intermixed in their first brain target, the lateral geniculate nucleus, before segregating into six eye-specific layers, and this occurs before birth. I was inspired by the research by David Hubel and Torsten Wiesel and their students Marge Livingstone and Simon LeVay, who shared with me their exciting findings about the organization of ocular dominance in cats and macaques (LeVay et al., 1975; Hubel et al., 1975). By manipulating visual connections in the embryo, I obtained the first direct evidence that in the nonhuman primate visual system competitive interactions between axons starts before birth. Thus, segregation of ocular dominance columns begins before animals are exposed to vision. In addition, we found that prenatal eye nucleation prevents this process (Rakic, 1976b). This paper was published before the famous Nobel winning report on ocular dominance segregation in primates (Hubel et al., 1977) and the findings were subsequently confirmed by additional studies (e.g., Rakic, 1977; 1981a; Meissirel et al., 1997; Kuljis and Rakic, 1990). Specific neuronal classes of areas 17 and 18 are generated independently at different times (Rakic, 1976a) and as subsequently shown are divided into independent subareas before and without visual input (Rakic, 1991b; Bourgeois and Rakic, 1996; Arcaro et al., 2020). Our use of a strict, time-consuming, and tedious quantitative analyses of developing human and primate brain sections processed with Golgi and electron microscopic methods paid off. We discovered that neurons began to form connections even before they arrive at their final positions. For example, callosal neurons send their axons to the opposite hemisphere while still migrating across fetal cerebral wall to the CP (Schwartz et al., 1991) and thalamo-cortical axons make transient connections with the SPZ neurons (Kostovic and Rakic, 1990; Rakic, 1991a).

At about that time Nada Zecevic, who received her MD degree in Belgrade, attended my course on developmental neurobiology when I was there and decided to change her career from clinical neurology to basic neuroscience research on the developing human brain. She obtained a U.S visa and joined my laboratory in Boston, and we collaborated on many papers, even after she obtained a faculty position at the University of Connecticut (e.g., Zecevic et al., 1989; Zecevic and Rakic, 1991; 2001; Radonjic et al., 2014). Toward the end of my stay in Boston, Carla Shatz, who received PhD training with David Hubel and Thorsten Wiesel, decided to join my lab as a postdoctoral fellow. Initially, she continued to work on the development of the efferent connections in the primate visual system (Shatz and Rakic, 1981). However, she was very fascinated by research on the transient embryonic SPZ conducted by Ivica Kostovic, who was working in my lab at the same time (e.g., see review in Kostovic and Rakic, 1990). When I decided to leave Harvard and move to Yale, Carla did not want to transfer with me from Boston to New Haven and, therefore, did not use the full term of the fellowship. In addition, she received an attractive offer to take a faculty position and open her own lab at Stanford University. However, she decided to continue work on the SPZ in developing cats, rather than monkeys, “because they are less expensive.” She did that in spite of my advice to continue work on macaques. I was pleased that 40 years later, during dinner at a restaurant in Amsterdam following a meeting at the Brain Institute, Carla admitted that my advice was probably correct and that few scientists these days work on developing cats, which anyway have a quite different organization of the visual system compared with primates. However, she became one of the most successful neuroscientists in the world, eventually best known for her excellent work on neuronal plasticity and neuroimmunology.

Horrific Incident at Boston Fenway

I enjoyed living in Boston, a cosmopolitan city with several top-level universities, excellent symphony orchestra, and international restaurants. When I was discussing with my friends my negotiations to possibly move to Yale, they told me, “Why should you leave a big and nice city like Boston and go to crime-ridden New Haven?” Then, I explained what happened to me in the nice downtown city of Boston in 1975. After picking up Ivica Kostovic from Logan Airport, who had come from Yugoslavia to join my lab, I was driving him to Harvard in my Volvo sedan. I stopped at the red light at Fenway, near the Boston Museum of Fine Arts, and suddenly a man opened the door, entered the car, and placed a gun on the back of my head shouting, “Drive, or I will kill you!” I was so stunned that I stalled the car and felt the gun directly touching the back of my head while the intruder continued to verbally threaten to kill me. I shouted back, “Wait a minute, I am on your side, but this is a standard shift car, and I am trying to start running it again.” Indeed, I did, but as I was driving, I suddenly saw several police cars with many policemen around, who started shooting at my car. My car’s windows, including the front windshield, disintegrated into small glass pieces, like cubes of sugar. The next several seconds were the worst and scariest in my life. My heart stopped beating and I was wondering why the police shot at my car, maybe Ivica was some kind of criminal drug dealer” I did not know that it was possible, but in a few seconds, I reviewed all my life and was wondering why I ever came to this country. Then I saw some blood on the floor of the car and decided to open the door and jump out, letting Ivica and the gunman to continue moving with flat tires and without the driver until it hit the cars parked on the street side. One policeman grabbed Kostovic from the car and used the knee to neck maneuver to hold him to the ground. Then one other policeman shouted, “These are not the guys.”

What happened was a remarkable coincidence. Police were parked there because the Irish prime minister was giving a talk in the Museum of Fine Arts, and there was some suspicion that the Irish National Liberation Army was planning to assassinate him. While stationed there, the police received a telephone message that “Three dangerous armed men committed a robbery on Newbury Street are driving in a foreign car toward Fenway.” It turned out that on the way they stopped and dispersed, and one of the robbers commandeered my car. Thus, police thought that we were the three armed men who wanted to kill the Irish prime minister and started shooting at us. This was big news, and this is the only time when Ivica and I were featured on the front page of the Boston Globe newspaper (figure 3).

Figure 3.   Incidence at Fenway Park in Boston.

However, this incident in Boston had another interesting consequence. After the former Yugoslavia divided into several smaller but independent states, for a short time Ivica Kostovic was vice president of Croatia. I was joking with my other postdocs, who were unsuccessfully looking for positions: “If you cannot make it in science, you can always become vice presidents of a country.” In 1997, in this capacity as vice president, Kostovic met the transitional administrator of the United Nations agency UNTAES, Jacques Klein, who did not as yet want to remove sanctions against this new country because, “some cities, like Vukovar in Slavonia Province of Croatia, were still not safe.” Then Ivica told him his story about being kidnapped in Boston by an armed robber, and Klein recognized that there are places in the United States that are as dangerous as in Croatia, and he removed the sanctions and opened the process of peaceful reintegration of this region into Croatia.

Move to Yale University

In 1978, I was recruited to Yale School of Medicine by famed cell biologist and Nobel Laureate, George E. Palade, to become the Dorys McConnell Duberg Professor of Neuroscience and the founding chair of the Section of Neuroanatomy. I had a New Year’s Eve dinner at Palade’s home with his wife Marilyn Farquhar, and they convinced me that my dedication to basic science will be appreciated at Yale Medical School. At that time, Yale was ranked among the top universities in the world, and I decided to accept this position. However, my decision was influenced by the possibility to be joined by my wife-to-be, Patricia Goldman. Unlike some other universities that did not like recruitment of couples to the same department, Palade told me that Yale would not mind this and said, “We can get two for the prize of one.”

When we met, Patricia was already a highly successful neuroscientist at the National Institute of Mental Health and was recruited to Yale at my suggestion, where we would get married. She also decided to retain her already recognized last name and add a hyphen to take mine and become Goldman-Rakic. We were working in different subfields of neuroscience, so we did not publish many papers together, but we were each other’s constructive critics, regularly commenting on each other’s manuscripts before submission. We were also organizing multidisciplinary meetings and applied for Center and Program Project grants. Our common denominator in science was our interest in the biological basis of the highest brain cognitive functions that make us human. Thus, together we founded a new journal Cerebral Cortex, now a well-established and high-impact journal, which became a force in this research area. We were devoted to fostering the new multidisciplinary approach through various functions at the Society for Neuroscience for which we both served as presidents (figure 4). Over the years, Pat helped me build and transform the Section of Neuroanatomy into a thriving, modern Department of Neurobiology. She was focused on the organization, physiology, and pathology of the prefrontal association cortex in nonhuman primates and was so successful that many people think that probably only her premature death prevented her from receiving the Nobel Prize (e.g., see Arnsten and Rakic, 2022). Together, Pat and I attracted the Kavli Institute for Neuroscience to Yale, which was awarded only after her premature death. Fred Kavli, who visited us at Yale and enjoyed red vintage wine at dinner in our house, stated that “the Kavli Institute at Yale should focus on the research of the cerebral cortex, the only organ which can make connection between the smallest particles (nanoscience) and the largest—universe (astrophysics).” The dean’s adviser at that time suggested that Fred also meet the director of Interdepartmental Neuroscience Program and chairs of Neurology and Psychiatry, but he refused: “Students are supported by tuition, and disease by NIH, I want to understand biological bases of human cognitive capacities via cerebral cortex.” Fred Kavli told me that he visited Yale University only because Pat and I worked on the cerebral cortex. He would probably turn in his grave if he learned that his wishes were totally ignored after I stepped down as director. I learned my lesson, and when I gave over $6 millions to Yale, I requested that my gift be spent while I was still alive because if given as an endowment, Yale administrators possibly would not respect the donor’s wishes.

Figure 4.   Pat and Pasko at the meeting together.

The move to Yale was also an opportunity to select some new direction for my research. With the support of the Ford Foundation, Yale was able to establish a timed breeding colony of nonhuman primate, macaque monkeys, providing an opportunity to study the development of some primate-specific features. I resisted the temptation to use some of the new and popular experimental methods that can be performed only in simple animal models to focus on what is possible to do in primates, including human. I became convinced that understanding the human cerebral cortex is the most important subject in science at the basic, medical, as well as philosophical level. I defended this position when I was invited to participate in the 2014 Science Forum in NYC. For this occasion, the famous actor and panelist Alan Alda assembled a distinguished panel of scientists that included several Nobel and Kavli Laureates in various disciplines ranging from cancer research and neuroscience to nanotechnology and astrophysics. To start the Forum, Alan gave us a probing question, “What is the single most important question in science? Please do not list several, I want only a single subject.” I dared to argue that the single most important subject is “An understanding of the human cerebral cortex.” I met with some resistance from the Forum participants, “What do you mean? How about origin of the universe?” argued one astrophysicist, citing a new Nobel Prize winning theory of cosmic inflation. To that I replied, “Which organ was used to make this theory?” By the reaction of the audience and from the panel’s subsequent responses, it seemed that I won that argument because without cortex there is no astrophysics. However, I must admit that it is possible that, as mice are incapable of understanding the equation E = mc², the human brain may not be capable of solving the problem of the origin of the universe.

To focus on the understanding of the development and evolution of the human brain seems obviously a worthwhile and positive endeavor, but surprisingly I learned that it also had some negative consequences. When I presented lectures stating that one probably cannot adequately model human language disorders in rodents, many in the audience were not only unhappy but some were even negative. This response was quite disappointing to me because, although I worked mostly on human and nonhuman primates, I greatly valued research on inbred strains of mice and used them as a model system to understand some basic principles of neuronal interactions in the brain (e.g., Rakic and Sidman, 1973; Caviness and Rakic, 1978). In the book entitled Mouse Brain Development (Goffinet and Rakic, 2000), I declared that “mouse is perhaps the best model system to study genetic brain disorders.” However, I also recognized that despite the superficial similarity, cerebral cortex in mice and primates, including humans, are significantly different. Thus, I organized some meetings with participants who use different model systems that range from nematode and fruit fly to carnivores and primates (e.g., Easter et al., 1985). Finally, I decided to study the development of the brain simultaneously in three mammalian species—mouse, macaque monkey, and human, to learn from the similarities as well as from the differences that occurred during the process of mammalian brain evolution. This was not commonly done in the same laboratory, but I was hoping to obtain specific anatomical and molecular comparative data relevant to the evolutionary and developmental basis of uniquely human mental capacities. Therefore, in collaboration with scientists in other fields, I applied transgenic technology to delete or manipulate mammalian homologs of several Nematode, Drosophila, and rodent genes (e.g., BMPs, Caspase–9, Cx43, CPP32, FGFs, JIP1, Jnk1–3, MeCP2, MEKK4, METTL7B, Numb, Notch, WNTs, and too many others to be listed) involved in programmed cell death, migration, and differentiation as well as creation and maintenance of neuronal connections in mammals. The results showed that cortical development and evolution do not depend on a single gene or regulatory element, but rather on a combination of many (e.g., Kwan et al., 2012; Franjic et al., 2022). We uncovered similarities between species but also some human-specific genetic pathways that also were supported by the work of Christopher Walsh (Huang et al, 2020).

My decision to perform research on nonhuman primates was not without some other consequences and was not easy to defend, even to some of my close relatives and in-laws. My justification was that our animals are treated humanely and are not exposed to any pain or procedures that doctors do not perform on their patients. I was once confronted in the front of Sterling Hall at the Yale Medical School by a protestor from the People for the Ethical Treatment of Animals (PETA), holding an offensive poster that asked, “Why do research on animals”? I replied, “If your son or mother are sick with a disease that can be cured only by the antibodies made in an animal, would you allow it, or let them die?” He gave the usual answer that it is a hypothetical situation, but in such a case, he would allow use of the animal. So, I said, “You care about your family more than the families of others!” Protestors did not return with the offensive posters.

One of the major goals was to perform electron microscopic quantitative analysis of synaptogenesis in nonhuman primates to explore in greater detail Peter Huttenlocker’s finding on human postmortem tissue that cortical synapses are initially overproduced before declining to the adult level. On my invitation, Jean-Pierre Bourgeois decided to leave a job at the Pasteur Institute in Paris to spend several years working on this project in my lab. He performed detailed quantitative analysis first in the macaque visual cortex and found enormous overproduction of synapses (Rakic et al., 1986, 1994; Bourgeois et al., 1989). In addition, time-consuming 3D quantitative analyses showed that during the period of maturation before puberty, monkeys lose about 2,500 synapses every second in the visual cortex alone and a total of about 30,000 in all cortical areas every second during the day and night in these three years. This looked unbelievably large, so even my wife Patricia said that it might be nonsense. So, for the first time in my career, I asked the editor in chief of the Journal of Neuroscience (at that time Dale Purves) to assign my paper to the hardest reviewers possible because I did not want to be a fool. He apparently did, but I only know that one of them was Francis Crick, who acknowledged his identity and who accepted the paper (Bourgeois and Rakic, 1993).

At about the same time, my first graduate student at Yale, Anthony LaMantia, found that axons of the corpus callosum in macaque monkey are 60 percent overproduced, before decreasing to the adult level (LaMantia and Rakic, 1990). We then found that similar decline occurs in anterior commissure (LaMantia and Rakic, 1994) as well as in other large axonal tracts (e.g., Rakic and Riley, 1983a, 1983b). We also discovered that a population of GABAergic neurons in the SPZ, form transient synapses, is also eliminated (Meinecke et al., 1992). These discoveries led me to propose the selective elimination hypothesis as a mechanism for tuning synaptic connections by interaction with the environment during the period of most intense learning. My colleague and friend Jean-Pierre Changeux found that similar overproduction and elimination occurs during development of neuromuscular junction, but he called it “selective stabilization.” We were joking during dinner in a nice Paris restaurant that it is actually the same phenomenon that we termed differently. When we looked at the glass of wine on the table, I said that it is half empty and he said that it is half full, and we are both right. These days selective elimination or stabilization is commonly called “pruning” (e.g., Keshavan et al., 1994). We also discovered that major neurotransmitter receptors are similarly overproduced (Lidow et al., 1989, 1991). Only recently we found that although childhood and adolescence does represent a period of the highest decline in dendritic spine number, surpassing the adult values for two- to three-fold, overproduction and developmental remodeling of cortical circuitries continues throughout the third decade of life, before stabilizing to the adult level (Petanjek et al., 2011).

In collaboration with Pat Levitt, who came to my laboratory after working with Bob Moore at the University of California, San Diego, we documented using immunocytochemistry at the light and electron microscopy levels, the early separation of neuronal and glial cell lineages (Levitt and Rakic, 1980; Levitt et al., 1981). This work has modified traditional ideas about phenotypic neuronal specification, suggesting that the basic phenotype of cortical cells is specified close to the time of a cell’s last division. Many other members of my lab, including Tarik Haydar, Matt Sarkisian, and Joshua Breunig, applied the most advanced molecular methods to study lineage transition from RGCs to Intermediate Neuronal Stem Cells during development and maintenance of the embryonic cerebrum (e.g., Haydar et al., 1999, 2000, 2003; Breunig et al., 2007b; Liu et al., 2008)—a subject that now also is studied in many other laboratories. With another postdoctoral fellow Robert Williams, we created and patented a 3D counting method for numbering cells in serially sectioned brain tissue (Williams and Rakic, 1988). He was also engaged in creating experimentally novel cytoarchitectonic areas within the visual cortex in developing macaque cerebrum (Rakic et al., 1991). This was an important finding, because reduction of the area V1 did not cause expansion of V2, but rather acquired a totally different (novel) features, which support the protomap hypothesis. Comparing monkey data on timing of the emergence of selected developmental landmarks indicated that the same early development occurs in humans. By necessity, we were working on the development of new methods and modification of existing methods (e.g., Rakic and Goldman-Rakic, 1985; Williams and Rakic, 1988; Cameron and Rakic, 1994; Kornack et al., 1995; Ang et al., 2003; Miska et al., 2004; Micali et la., 2020; Morozov et al., 2020).

I was lucky to work with a highly talented PhD student, Chia-Yi (Alex) Kuan, who emigrated with an MD degree from his native Taiwan and was admitted to the Yale neuroscience graduate program. He was crucial for forming my collaboration with Richard Flavell, chair of the Department of Immunobiology at Yale, who was the world leader in creating single-gene mutation in mice. In this collaboration, Kuan obtained evidence of the enormous importance for the role of programmed cell death/apoptosis in normal brain development as well as in pathogenesis of many brain malformations (Kuida et al., 1996; Young et al., 1997; Kuan et al., 1999, 2000). Alex was imaginative and productive, and we published about a dozen papers on this subject that became highly cited in several fields of biology. Alex obtained a faculty position and has established a successful laboratory, and our collaborations has continued.

At about the same time, Nenad Sestan, who also had already received an MD degree from the University of Zagreb in Croatia, came to my lab to obtain a PhD at Yale. He was both an original thinker and technical maverick. As part of the research on his doctoral dissertation, he rotated through the lab of a Drosophila specialist Spyros Artavanis-Tsakonas and established a collaboration between his and my labs. Through this effort, Nenad discovered the role of Notch gene signaling in neuronal differentiation and neurite outgrowth, which were published in two Science papers within the same year (Sestan et al., 1999; Qi et al., 1999). Although he has received many opportunities and offers to take attractive and lucrative jobs elsewhere, he remains at Yale, where he established his own large laboratory. He advanced quickly and at a relatively young age became the Cushing Endowed Professor in the Department of Neuroscience, with joint appointments in Genetics, Psychiatry, and Comparative Medicine. Nenad is presently considered to be of the most successful developmental neuroscientists, has been elected to the National Academy of Medicine (NAM), and has received many prestigious prizes. His recent study in Nature on reviving the brain of a dead pig gained international recognition from 355 different news outlets making it one of the most recognized scientific breakthroughs of the year and placed Nenad on the list of “ten people who mattered in science.”

It is interesting that three people of Croatian origin (P. Rakic, I. Kostovic, and N. Sestan) end up exploring evolution, development, and congenital disorders of the human cerebral cortex. Ivica Kostovic became the founder and director of the Croatian Brain Institute, which is devoted to research of the human brain, so our laboratories have continued to collaborate (e.g., Duque et al., 2016). Among the few neuroscientists who share the enthusiasm and recognize the necessity to explore human differences are Christopher Walsh at Harvard (e.g., Huang et al., 2020) and Javier DeFelipe at the Cajal Institute (e.g., Eyal et al., 2016), although John Rubenstein (e.g., Raju et al., 2018) and Arturo Alvarez-Buylla (e.g., Sorrells et al., 2018) have also made important contributions. Without such research, we would still be ignorant about basic molecular and cellular events that underly cortical expansion and elaboration in humans that enables creation of our civilization.

One feature that is most prominent in the human brain is elaborate and deep secondary and tertiary cerebral convolutions. With Brian Rash, who joined my lab as a postdoctoral fellow, we provided new insight into the cellular mechanisms of gyrification. At that time, the mechanisms underlying gyral development became controversial, with some disputing the classical Van Essen model of the role of white matter connections and suggesting instead that cortical gyri occur because of an increase in neurogenesis of the superficial layers neurons in the underlying hotspots of the oSVZ. However, many studies have shown that cortical neurogenesis and settling of neurons into the cortical plate in primates, including humans, is finished before gyral development and that the oSVZ produces mainly oligodendrocytes and astrocytes during the period of gyrification (Rash et al., 2019; Arellano et al., 2021). More specifically, we showed that in the developing macaque cerebrum, secondary and tertiary gyri form after the completion of neurogenesis, driven primarily by the rapid growth of intracortical neuropil and subjacent axonal fascicles in the white matter influenced by intracortical neurotrophin signaling through brain-derived neurotrophic factor (BDNF) (Rash et al., 2019).

I recognized that a multidimensional and dynamic process of brain development cannot be fully explained by the study of a single gene or single molecule and that we need to understand underlying cell-to-cell interactions. Over the years, my laboratory has identified several membrane polypeptides as well as voltage- and ligand-activated ion channels on the surface of migrating neurons and radial glial cells. These factors contribute jointly to cell orientation and recognition of migratory pathways through differentially distributed cell adhesion molecules that lie between the surfaces of migrating neurons and adjacent RGC fibers. These same factors also regulate the rate of nuclear movement in migrating neurons by controlling the dynamics of cytoskeletal proteins (Rakic 1972, 1978; Komuro and Rakic, 1992, 1993, 1996; Breunig et al., 2007b; Torii et al., 2009; Rash et al., 2016). About that time, Eva Anton, who graduated from Duke University, joined my lab because of his belief that the molecular mechanisms of neuronal migration in the central nervous system have a good chance to be solved by modern neurobiological methods. Indeed, Eva used the most advanced methods for studying cell movement in the cerebral cortex in vitro. He explored the functional significance of two novel molecules that recently had been identified on the radial glial cell surface membrane. He also generated an antibody to a new type of molecule that may serve as a signal for cessation of cell migration at the interface between the cortical plate and marginal zone of the embryonic cerebrum (Anton et al., 1996, 1997, 1999). I recognized that this work needed diverse knowledge, and together with another talented graduate student of mine, Joshua Breunig, we explored diverse molecules and cell phenotypes involved in this complex process. So far, we have identified that at least 20 diverse molecules, some of which were initially discovered in invertebrates, control specific phases and components of neuronal stem cell proliferation and migration in the cerebrum. These include the mode of neuronal proliferation, phenotype determination, establishment of polarity, detachment from the local substrate, and rate of nuclear and somal translocation to the proper areas, layers, and columns of the cerebral cortex (e.g., Breunig et al., 2007b, 2008, 2010).

Although we were quick to adopt the newest and the most advanced methods, we also continued to use the older techniques, such as electron microscopy, in combination with immunocytochemistry and autoradiography. I collaborated with Pat’s group (e.g., Schwartz et al., 1991) but was also lucky to have in my lab two excellent electron microscopists, one who emigrated from Japan, Hitoshi Komuro, and one who emigrated from Russia, Yury Morozov. They were research scientists in my lab and were engaged in many projects, including national and international collaborations on the various topics that required ultrastructural, subcellular localizations of specific molecules (e.g., Komuro and Rakic, 1992, 1993; Berghuis et al., 2007; Breunig et al., 2008; Dominquez et al., 2013, Datta et al., 2020; Galvin et al., 2020; Morozov et al., 2020).

We were also very active in research on the effect of genetic and environmental factors on brain development. By manipulating the speed and pattern of neuronal migration using various genetic (e.g., reelin, Notch, Lis1, Neuropeptide-Y,TCF4, Connexin 43, Numb), environmental (e.g., x-rays, ultrasound), and chemical factors (e.g. alcohol, antidepressants, cannabis, and opioids), we discovered the hidden abnormalities of neuronal positioning and small changes in synaptic pattern (e.g., Levitt et al., 1984; Kuljis and Rakic 1989; Lidow et al., 1989; Schull et al., 1986; Qi et al., 1999; Sestan et al., 1999; Ouimet et al., 1992; Kuida et al., 1996; Algan and Rakic, 1997; Anton et al., 1999; Li et al., 2003, 2019; Ang et al., 2006; Breunig et al., 2007a; Selemon et al., 2005, 2009, 2013; Rash et al., 2018; Morozov and Rakic 2009; Liu et al., 2012) that cannot be discerned by routine examination of the postmortem human brain (e.g., in the so-called idiopathic cortical digenesis). These findings opened a new window into understanding pathogenesis of congenital brain malformations, which I termed “neurodislocation syndrome.” This concept provided insight into how individuals with seemingly “normal” brains display major neurological and neuropsychiatric conditions. Specific molecules and cellular mechanisms that place neurons into proper position proved to be universal and have spawned studies of surface mediated interactions in other brain structures that are now carried out by developmental neurobiologists all over the world.

I was also lucky that Kazue Hashimoto-Torii and her husband Masaaki Torii, who received their graduate degrees in Japan, joined my lab. They discovered the relationship between Reelin and Notch signaling pathways and how this cooperation regulates neuronal cell migration from the proliferative centers to the embryonic cerebral cortex (e.g., Hashimoto-Torii, 2008). They also discovered the role of ATP and gap junctions in interkinetic and multipolar phase of neuronal migration (Liu et al., 2008, 2010). They left my laboratory at Yale after obtaining faculty positions at George Washington University, but our collaborations have continued, resulting in several manuscripts, including one on the development of a novel fluorescence reporter system in mice (Hashimoto-Torii, 2014). These heat shock proteins and factors may be potentially very useful as the means to detect prospective disorders before the appearance of visible symptoms and before the pathological effect becomes irreversible.

Role of Genes vs. Environment

As evident from my bibliography, I was initially fascinated by the effect of the environment on brain development, which was popularly known as “plasticity.” However, the more I learned about brain development, through my own work as well as from the large literature, including papers being evaluated in the journal Cerebral Cortex, the more I became aware of evidence indicating that the basic human characteristics, including temperament; talent for singing, arts, or sports; and basic intelligence, are inherited and thus, depend on the combinations of genes. It was Seymour Benzer, during our dinner at Caltech where they tried to recruit me, who finally convinced me that genes are primary. He repeated to me several times during our dinner in Pasadena (California): “Genes are very, very stubborn.” Indeed, I learned that there are no two human beings that are created equal because of genetic variance. Even identical twins are different. This eternal debate about the roles of genes and the environment led to the one that was held at Oxford University Library in the United Kingdom between Colin Blakemore and me, with the presence of some other notable developmental neuroscientists like Zoltan Molnar (figure 5).

Colin asked, “OK. You, Pasko, believe in the crucial role of genes, but what is your estimation, 75 percent genes–25 percent environment?”

I replied, “100 percent genes.”

He was upset: “Do you mean that environment does not play a role?”

I answered, “Yes, environment is also 100 percent.”

Colin, “So, you want it both ways, do you want to be president of USA?”

Figure 5.   Debate at the Oxford Library in the United Kingdom.

To which I answered, “No, I will disappoint you. I cannot be president. I was not born in USA and have the birth certificate to prove it.” I also cited Niles Bohr that, “Opposites are not contradictory but complementary.”

Furthermore, the initial question was not correct, it should have been, “What is primary and what is secondary?”

I said, “The answer is of course genes because without the brain created by genes, the environment would have nothing to influence.”

I was dedicated to exploring the development and evolution of the entire cerebral cortex, including all areas since, for example, even primary visual cortex in rodents and primates is dramatically different. Therefore, we explored species-specific differences in cortical organization. However, perhaps in part due to Pat’s interest, I was perusing peculiarities of the association areas of the prefrontal cortex. She had evidence that rodents do not have the types of connections like the primate prefrontal cortex with its input from the structures like part of the pulvinar, which does not even exist in rodents. After one of her talks at University of California, Los Angeles, she was asked by the founding father of neuroethology, Ted Bullock, “These are nice neurophysiological studies, but they are expensive to perform in monkeys, so why don’t you do it in mice?”

Pat replied, “Would you use mouse as a model system to study the development of wings?”

Ted said, “No, I would not. Mice do not have wings.”

Pat won the argument with her reply, “Mice do not have prefrontal cortex.” So even now, many years later, I am collaborating with Pat’s former postdoctoral fellow Amy Arnsten, now the Kent Professor of Neuroscience and Psychology at Yale, on the cellular and molecular organization of the prefrontal cortex in macaque monkey (e.g., Datta et al., 2020, 2021; Galvin et al., 2020). My justification for this extraordinary effort is that it is not possible to model the structure in an organism that does not have it.

Among various chemical and physical environmental factors influencing prenatal brain development, we also examined the effect of exposure to ultrasound waves (USW) on the developing brain in pregnant mice. We found that it has an effect in developing mice (Ang et al., 2006) and therefore advised that, as is done for the X-ray, which is harmful, USW also should be used only when medically indicated. However, because this test was done in quickly developing small mice, there was a question of how much effect USW have on the large, slowly developing brain in humans, which also have much thicker abdominal and uterine walls. Proper testing in nonhuman primates, such as the macaque monkey, is very time-consuming and extremely expensive, and I was informed that NIH would not consider this as an additional research grant for me since I already had two. I was discussing this issue with Nobel Laureate Salvador Luria from MIT, who was very interested in this issue and he said, “You should not worry, I am on the board of the Rockefeller Foundation and you should submit it there.” To my and his surprise, Sheldon Segal, a distinguished reproductive biologist, who served as the director for Population Sciences at the Rockefeller, rejected my grant application on September 4, 1983, with the statement: “It is difficult to understand what significance we could learn from a few monkeys that has not already been revealed by literally thousands of human fetuses . . . overall, negative about the need for study.” Both Salvador Luria and I were stunned by this reply since this was like saying, “What can we learn from studying the effect of smoking in animals that we cannot learn from millions of healthy and smiling over 70-year-old individuals.” However, I was persistent and eventually received funds from the NIH for the USW project, but indeed it took a lot of time to obtain the proper number of USW exposed and controls in slowly developing monkeys and to wait for their postnatal maturation to examine and perform statically significant analysis of effects on developmental cellular events. At the time of this writing, we are completing this prolonged double-blind study, which revealed the same small, but statistically significant effect of USW on migration of cortical neurons in the primate telencephalon (Duque and Rakic, work in progress).

Human-Specific Features

By studying simultaneously development and organization in three mammalian species, we recognized that there are human-specific features. One of the most unusual is the existence of a stream of migrating cells from the ganglionic eminence of the human fetal telencephalon to the thalamic nucleus pulvinar in the diencephalon (Rakic and Sidman, 1969). I initially described this structure in my doctoral thesis and named it corpus gangliothalamicus. It is a relatively large cellular stream clearly visible using basic histologic stains, but we added new methods to determine that they are indeed postmitotic migrating prospective GABAergic neurons (Letinic et al., 2001). This structure so far could not be found in the developing brain of any other species, including rodents, carnivores, and nonhuman primates. Another feature that seems to be present only in primates, including human, is that a subclass of GABAergic neurons originate in the proliferative zones of the dorsal telencephalon (Letinic et al., 2002; Radonjic et al., 2014). Some aspects of the RGC proliferation are also different (e.g., Kornack and Rakic 1998) and some of the genes and regulatory elements are human specific (see review by Molnar et al., 2019; also see the newest data by Franjic et al., 2022). Another type of species-specific features are large quantitative differences. For example, primates have much more interstitial neurons in the subcortical white matter than in the lateral geniculate nucleus (LGN) (Kostovic and Rakic, 1980). Yet, there are hundreds of physiological studies on the LGN and none on the interstitial neurons. I was compelled to quote Georg Hegel: “quantity changes lead to quality changes.” This issue of species-specific differences deserves our attention because there is no question that some features of the cortical neuronal organization in primates are different than in any other mammalian species (e.g., Reilly et al., 2015; Duque et al., 2016; see also reviews in Rakic 2003, 2009; Clowry et al., 2010; Kwan et al., 2012; Cotney et al., 2013; Reilly et al., 2015; Molnar et al., 2019; Silver et al., 2019; Geschwind and Rakic, 2013; Arnsten and Rakic, 2022).

Our initial analysis of time of cell origin using DNA replication markers revealed that the neurons serving the most precious mental functions, such as the cerebral cortex in primates, including humans, have neurogenesis limited to the developmental period, and postmitotic neurons last the entire life span and are irreplaceable (Rakic, 1974; Rakic and Nowakowski, 1981). These findings led me to suggest that the stability and longevity of the neuronal populations in the adult primate, including the adult human brain in general and cerebral neocortex in particular, may be an evolutionary adaptation for the retention of learned and stored information over the prolonged life span of the individual (Rakic, 1985). In contrast, fish and amphibians continue neurogenesis during their entire life, and the salamander can regenerate an entire spinal cord, but mammals are drastically reduced in this capacity. I was wondering why this seemingly useful capacity would be systematically reduced during evolution. This steady decrease in adult neurogenesis during evolution sems to have occurred at the expense of the capacity for regeneration and natural turnover of neurons that exists and is prominent in many lower vertebrates. This subject was not within my main research interests, but I noticed that some scientists, as well as the popular press, initially strongly contested my conclusions. This subject came in focus when Elizabeth Gould published in Science paper claiming that thousands of new neurons are added daily to the prefrontal cortex of the adult macaque monkey (Gould et al., 1999). This was a stunning claim, and Eric Kandel commented in the New York Times (October 15, 1999), that “this idea was perfectly possible, given how little was known about the brain system for ultimate long-term memory storage.” I said to Eric at one of our many dinners: “If this is true, you should return your Nobel Prize since you established that memory is preserved in the pattern of synapses, and newly generated neurons in human would need more that 8 months to form a first synaptic contact.” In the subsequent article in The New Yorker (July 1, 2001), I was portrayed as a dogmatic, male chauvinist who attacked a talented young woman, Elizabeth Gould from Princeton, who made a huge discovery and destroyed the old dogma. Pat cried when she read this article, but I decided not to reply to this uninformed and offensive article. Instead, I offered to visit Princeton to examine the original slides, and if such cells indeed existed, I would accept it. They refused! However, I am happy that many other labs, including the Gould lab, could not replicate her findings. My original statement of the limit of the primate cortex also has been confirmed by different methods, including carbon–14 birth dating of neurons in the adult human cerebral cortex (Bhardwaj et al., 2006). Most important, Alvarez-Buylla has shown that it is nonexistent or negligible in adult human hippocampus (Sorrells et al., 2018). I was gratified that this finding and its message against unrealistic hopes has helped to redirect neural stem cell research, from how to replace degenerating or injured neurons, to how to preserve them. A postdoc in my lab, David Kornack, became interested in this subject of adult neurogenesis and confirmed my original findings (e.g., Eckenhoff and Rakic, 1988; Rakic and Kornack, 1993; Kornack and Rakic, 2001; Rakic 2002b). After getting a faculty position at the University of Rochester, he tried to continue doing research in support of the concept that the lack of adult neurogenesis may be an evolutionary advantage. However, at many study sections for scientific peer review, he encountered difficulties obtaining grants on this subject because of the opposition of the true believers in human adult neurogenesis.

The idea of obtaining new neurons at old age is apparently very attractive and popular among nonspecialists and lay people. For example, I attended the Yale fundraising dinner, and Ted Turner, founder of CNN, and his wife Jane Fonda, were sitting next to me.

After learning that I am a neuroscientist, her first question was: “Is it possible that I can get some new neurons?”

She did not like my reply that “it is unlikely and would actually be counterproductive.”

I was also a participant of the famous debate at the satellite meeting on the topic of adult neurogenesis where two presidents of the Society for Neuroscience, Fred (Rusty) Gage and I, tried to be presidential and paraphrase statements made by former U.S. presidents.

Rusty paraphrased President George Bush and claimed that I said, “Read my lips: No new neurons.”

I replied by paraphrasing President John Kennedy, “Ask not what new neurons can do for you; ask what you can do for your old neurons.”

Rusty is a good friend and an excellent neuroscientist with whom I have several papers (e.g., Brennand et al., 2015), but in the research area of adult neurogenesis, he like many others, believe that although small, it may have huge importance and that stimulating its increase may help many neuropsychiatric disorders. Indeed, an enormous amount of money has been spent on research on how supposedly new adult neurons can prevent and heal aging disorders. Many, notably Arvid Carlsson, a Nobel laureate from Göteborg, strongly complained that this field was highly supported at the expense of funding research on how to prevent the decline of neurons or heal existing neurons. I was surprised and disappointed that so many scientists are still using inadequate methods in this field even though there is clear evidence of the significant technical and conceptual problems (e.g., Arellano et al., 1999; Kuan et al., 2004; Breunig et al., 2007a; Duque and Spector, 2019; Duque at al., 2021; Franjic et al., 2022).

Another contentious argument that I was involved in is my criticism of the implicit assumption by numerous publications that neural structure for human language is evolutionarily related to bird singing abilities and that human cerebral cortex is an expansion of the mouse cortex with the addition of a few association areas. I argued that a bird’s song does not have genetic, anatomic, or functional substrates in common with the human language, which involves abstract thinking as advocated by Fernando Nottebohm. Human language is basically a cortical function, the structure that birds do not have. In addition, in my opinion, the Foxp2 was inappropriately called the “language gene” because it is also expressed in the rodent spinal cord. Furthermore, there is no presently living species that should be classified as lower or higher because, for example, birds are as advanced in their phylogenetic tree as humans are in theirs.

When I was at a meeting on evolution at the Royal Society in England, Fernando asked me, “How do you know, without doing more research, that birds do not have language?”

I replied, “I know; if they had language, they would tell me!”

I argued that the work on bird singing is an excellent contribution to understanding the path of their evolution, but they are not our ancestors. It was not a new and original idea because it is well established that presently living animals evolved from extinct common ancestors, as so clearly and eloquently formulated by Charles Darwin. Unfortunately, it is often a forgotten truth that many young neuroscientists, and even members of the NAS, seem not to know or choose to ignore. This is the reason why in my lectures I emphasize the old truth that all presently living species are equally evolved from a long extinct ancestor, which we cannot examine. I also make the point that rodents, like mice, are closer to humans than carnivores, like ferrets and dogs, who were separated from the primate phylogenetic tree 7 million years before mice. At a large meeting on the development of the cerebral cortex in Greece, attended by more than 250 researchers, I asked the audience to raise their hand if they knew that rodents are evolutionarily much closer to human than carnivores. Only a few hands went up, including one by evolutionary biologist Leah Krubitzer who supported my statement. I was stunned by the lack of knowledge of this basic information by the researchers who work on the cerebral cortex in mice. However, I do not blame them. It is the fault of their mentors who ask them to learn complex techniques and perform time-consuming experiments and do not teach them some basic facts and the history of the subject. I had many useful and animated discussions at some meetings, but my main reward was the positive messages that I received from participating students, who thanked me for pointing out this simple truth. They were indoctrinated in their courses that humans have evolved from the simpler animals that are used as the model systems, rather than, as suggested by Darwin, we derived from a common ancestor, which we will never be able to examine.

Because studies on brain development in the valuable and endangered primate species probably will never be repeated, my concern was how to preserve this material to be useful for the present as well as future generations. Over many decades, the late Patricia Goldman-Rakic and I accumulated large collections of stained and unstained slides, electron microscopy blocks, and frozen tissue that can be utilized with the application of new staining and molecular methods. I urged younger members of the Yale faculty, Alvaro Duque and Lynn Selemon, to create the Macaque Brain Resource, designed to utilize the available material for new studies on the brain of nonhuman primates without sacrificing additional animals (e.g., Duque et al., 2019). I believe so much in this project that I have personally donated more than $6 million for its creation and maintenance. In addition, Duque and Selemon applied and were awarded an NIH grant to support the Macaque Brain Resource. It recently became an Independent Center at Yale University School of Medicine. The unstained sections and EM block of the tissue from developing monkey brain are available to scientists from around the world who visit the Macaque Brain Resource Center at Yale. Scientists may process material with new antibodies and molecular and cellular markers. Access to this resource is available by arranged visits or through remote access of digitized images of slides via website access (https://medicine.yale.edu/neuroscience/macbrain/).

Administrative Duties

I served for 37 years as department chair, the longest length of time of any chair in the history of Yale University. My chairmanship spanned seven Medical School deans, starting with Bob Berliner, and five presidents of the university starting with Bartlett Giamatti. As one of my friends said, “If you survived seven deans, you must be doing something wrong.” Some of the deans did not like my nonconformist, straightforward, and direct style. However, they may have kept me as a chair for so long because our department was never in the red, and our members had very large grants, including several big Center and Program Projects. We never received adequate space and the department was relatively small. Nevertheless, in 1996, it was ranked number one in the world with only 10 faculty members (five females and five males). The equal ratio was not planned that way, and I did not perform an “affirmative action” search. I just selected the best, and it turned out to be about 50–50. For example, I recruited and promoted Susan Hockfield to her tenure and eventual provost position before she left Yale to become the first woman president of MIT. This was a friendly departure since I provided a positive recommendation and subsequently was a frequent guest at her dinners and stayed at her MIT residence on the Charles River in Cambridge, Massachusetts. Other notable members, who unfortunately left Yale, were Mriganka Sur, Pat Levitt, Anna Wang Roe, David McCormick, and Patricia-Godman-Rakic, who left under worse circumstances. I also recruited Michael Schwartz and Michael Crair, who become dean’s advisers for education and research. Despite the internationally recognized success of the Neurobiology Department at Yale, our space was kept at a minimum because some of the deans did not appreciate the value of basic brain research and instead favored genetics. However, I took advantage of this fact and remembered the advice from the famous neurophysiologist Otto Creutzfeldt who said, “Chairman should never allow a department to become big.” Indeed, I did not mind that the department was small as this gave me a chance to devote more time to my research. Our department was also a good example of diversity and my group at Yale is like the United Nations. Remarkably, 12 out of 12 recent members were born in 12 different counties! That also was not planned—again, I tried to select the best.

After stepping down as chair, I retain a full-time faculty position. This allows me to spend even more time on research so that the past few years have become even more productive. Our research remains unique as recognized by many study sections. For example, I recently received a large 10-year MERIT award from the NIH to support an active lab of about eight members with different scientific backgrounds. We are presently as productive as ever.

Traveling and Lecturing to Transfer Knowledge to the Next Generation

During all these years I have hoped that my research will be useful to other scientists at both a conceptual and a clinical level. To this end, I have given lectures at numerous meetings and visited institutions in more than 35 countries on five continents. I enjoyed traveling and frequently have visited some of the world’s most magnificent cities (Paris 21 times, Stockholm 17 times, Madrid 15 times, London 14 times). I have close relationships with many French scientists, including Jean-Pierre Changeux, Jacques Glowinski, and Michael Imber, and was invited several times to spend weeks in the residence at Rue de l’Universite and to give biweekly lectures at the local institutions. I am also on the board of Oxford University in the United Kingdom.

As a recipient of a Selby Fellowship in the Australian Academy of Science, I was required to visit seven cities on this continent, spending at least two days at each city to give two lectures, one for the general public and one for the scientists. When asked how I did that tedious task, I said, “To the general public, I gave the most detailed and complex lecture,” and so the attendees said, “He is very smart, I did not understand anything.” And for the specialists, I gave a very simple general lecture, so that even scientists could understand. I also have had many visits to Israel because Pat was on the Advisory Council of the Weizmann Institute in Rehovot. During some of those visits, we had the privilege and honor to be driven by Yadin Dudai in his car to see some of the most precious places in this part of the world, with his personal explanations about their historical significance. I also had great experiences visiting China several times, including the last one as a member of the U.S.-China Scientific Exchange Committee chaired by Torsten Wiesel. After the main meeting in Beijing and as a guest of the Chinese Government, my present wife, Sandra, and I visited some of the most treasured historical monuments in the history of the world, including the Great Wall of China and Terracotta Warriors in Xi’an City.

I considered that the most effective way to propagate your ideas is to participate in multidisciplinary international meetings. I attended far too many meetings to list each one, and so I will select one that was most unusual and memorable. I was a participant at several meetings of the Pontifical Academy in the Vatican that were held in the very same building in which Galileo was tried. The other notable participants include Rita Levi-Montalcini, John Eccles, Patricia Goldman-Rakic, János Szentágothai, and Vernon Mountcastle. At one of these meetings, Sir John Eccles, who later in his life became a dualist and started to believe in independent Worlds of Religion and Science, presented his idea that the World of God commutates with the World of Brain through the Radial Units of Pasko Rakic. He discussed the RUH and related issues in several of his many books (e.g., Eccles, 1989). To Sir John’s dismay, Vernon Mountcastle said plainly, “This is nonsense.” This was an additional nail in the coffin of the relationship between these two great neuroscientists. I was embarrassed by the whole situation and tried to play innocent and said that I did not understand these issues and arguments. Sir John accepted my excuse, “Pasko is honest, he admits that he does not understand it.” The local permanent members of the Pontifical Academy and the Pope himself actually accepted the concept of evolution with the idea that seven days in the Bible is only symbolic and that each “day” may last millions of years.

It seems rather unlikely that someone born in Ruma (not Roma), a small provincial town in Yugoslavia, would later in life entertain for dinner in my private dining room about 15 Nobel Prize winners. These were not group gatherings but rather individual dinners with a lot of discussions over a nice glass (or two) of wine. In fact, I often served Grgich Hills’ chardonnay from Napa Valley, the same wine that was served in the White House during the dinner meeting of Ronald Regan and Mikhail Gorbachev when they were talking about “breaking down” the wall. It is interesting that Miljenko Grgich was born on the Adriatic coast and after graduating from the University of Zagreb, he emigrated to the United States to become a winemaker in Napa Valley, which eventually obtained a Prize in Judgment of Paris as the best white wine and placed American wine at the top of the world. Discussions with Nobel Laureates were always animated. They seemed to like the wine, and I was happy that they really listened and appreciated my opinion.

My discoveries, concepts, and graphic illustrations of the developmental events underlying brain structure and function have been well received and highly cited. I have published more than 350 scientific papers that according Google Scholar are reaching the mark of 90,000 citations with h-factor of 147. Some scientists do not appreciate morphometric data, but many foundations and institutions are interested in the hard data when selecting prize winners. I had a lunch long ago in Boston with the founder of the Institute for Scientific Information, Eugene Garfield, and told him that many scientists and administrators think that the number of citations does not necessarily correlate with the quality of the contribution. For example, Elizabeth Gould has thousands of citations of papers that have been proven wrong. So, Eugene replied: “Citation index does have one high correlation: those who have less citations like it less.” It is difficult to find a contemporary review article or textbook in neuroscience that does not refer to my contributions, although I complain that sometimes they forget to mention my name. However, Francis Crick once told me, “When they describe your discoveries as common knowledge and do not cite your name, that means that you really made it.”

Initially, I worked mostly alone and performed most of the experiments with help from technicians, and it may be considered unusual that on three of the five most cited papers, I am the sole author! However, over the years, as neuroscience research became a multidisciplinary activity, I have enjoyed the benefits of highly productive collaborations with my talented and dedicated students, postdoctoral fellows, and colleagues. Modern neuroscience research requires experts with different backgrounds, and without these interactions, at both technical and intellectual levels, this work simply could not be possible. The debates with my students and postdocs at regular lab meetings or individually were essential for my creative thinking and balance of my views. I was not a strict adviser, but rather I tried to learn from them. I liked controversies, and when my student won an argument, this would make two people happy: my student and myself. I have lived during the time of globalization of science; and over the years, I have had more than 50 graduate and postgraduate students from more than 20 countries, most of which became highly successful neuroscientists and, presently, seven are chairing a department or an institute. I consider my students as my children.

During my lectures, I often return to my roots and try to entertain the audience with my drawings and anecdotes (figure 1). I was illustrating my findings and conceptual models by personal drawings using pen and India ink. My four-dimensional (three dimensions of space plus time) drawing of the development of cerebral cortex is reproduced in medical textbooks around the world. Carl Hugo Lagercrantz, professor of Pediatrics at the Karolinska Institute in Stockholm, in his book Infant Brain Development (Springer, 1996), translated to several languages, cites my paper on RUH, including the illustration of the four-dimensional (space and tine) model of neuronal migration to the cerebral cortex. Interestingly, on page 10, he also reproduced one of my cartoons on the problem of how relying on a single method, like gene expression, can be misinterpreted. Apart from my talks, which were full of factual details as well as broad conceptual generalizations, I became known as an authentic and original reviewer, as indicated by some of the titles in the reputable journals: “A Small Step for the Cell—A Giant Leap for Mankind: A hypothesis of Neocortical Expansion during Evolution” (Trends in Neurosciences, 1995), “Young Neurons for Old Brains?” (Nature Neuroscience, 1998), “The Importance of Being Well Placed and Having the Right Connections” (Annals of the New York Academy of Sciences, 1999), “Discriminating Migrations” (Nature, 1999), “Illegal Immigrations” (Neuron, 2000), “Neurocreationalism: Making New Cortical Maps” (Science, 2001), “Neuroscience: Immigration Denied” (Nature, 2004), and “No More Neurons for You” (Science, 2006). However, I was also engaged in editing several comprehensive reference books that have a large and diverse audience (e.g., Rakic and Singer, 1988; Gazzaniga et al., 2009; Singer et al., 2019; Rubenstein and Rakic, 2020).

Over the years, I was rewarded for my scientific achievements by election to memberships of academies and foundations, including the U.S. triad (the National Academy of Sciences, NAS; the American Academy of Arts and Sciences; AAAS, the National Academy of Medicine, NAM) as well as the Royal Society (London) and the Spanish and Norwegian Royal Academies. For many years, I was also on advisory boards of institutions like RIKEN (Tokyo, Japan), Dahlem Conferences (Berlin, Germany), the National Alliance for Research on Schizophrenia and Depression (NARSAD; now called Brain and Behavior Research Foundation) (New York City), the DANA Foundation (New York City), and the Max-Planck Institute for Developmental Biology (Tubingen, Germany). At these remarkable organizations and institutions, I had opportunities to interact with intellectuals like Masao Ito, Otto Creutzfeldt, Karl-Friedrich Bonhoeffer, and Christiane Nüsslein-Volhard, and I learned a lot from them. I was also offered the opportunity to become president of IBRO, which included use of an apartment in the center of Paris, but I did not accept it, because I was afraid that it would take time away from my research activity. I also did not accept the invitation to the White House to celebrate Obama’s Brain Initiative because I did not like that it was created at the expense of other fields and without increasing the overall NIH budget. When some of my colleagues said, “How can you refuse the honor to be one of those who were invited to the White House?” I replied, “Yes, it is indeed an honor to be invited, but it is an even bigger honor to be the one who has the courage to decline it.”

However, I did support and contribute to the negotiation and lobbying about another White House initiative by George H. W. Bush’s administration called “Decade of the Brain”(1990–1999), which was also implemented with only a modest overall NIH budget. I had an opportunity to visit Congress several times and have had lunch with influential politicians like senator Mark Hatfield, chair of the Appropriations Committee of the U.S. Congress (figure 6). I tried to persuade him to make a more substantial increase during a very nice conversation about the prospect of the Decade of the Brain. Unfortunately, the amount of money dedicated to this endeavor was less than we expected. As pointed out by Dominick Purpura, former chair of Neuroscience and dean of the Albert Einstein Medical School in New York, none of the 10 proposed goals were actually achieved. One of the goals was a promise to find a cure for Christopher Reeve’s quadriplegia. However, I recognized that if Chris, whom I met, had fallen from a horse in Belgrade in 1962, when I was a resident in neurosurgery, he would give them the definition of this term that was popular in Yugoslavia, “A pessimist is an optimist with information!”

I suppose because of my activity on behalf of neuroscience, I was elected and accepted the position to serve as the 26th president of the Society of Neuroscience (1995–1996). This job was indeed very time-consuming, but I take pride that during my presidency, we had successful negotiations to obtain support for the Decade of the Brain. In addition, during my presidency and with Publication Committee members Solomon Snyder and Peter Strick, we succeeded to make the Journal of Neuroscience available online in spite of the strong opposition of some Council members who were worried irrationally that the cost would eventually result in an increase in annual dues for students. I am also one of only two members of the SfN who attended all 49 annual meetings. Larry Swanson being the only other one with a similar record. The 50th in-person meeting was canceled because of COVID-19. In addition, I also served on many of the Society’s committees. Looking back, I spent almost a full year in hotels attending SfN meetings.

Figure 6.   Lunch at U.S. Capital, with Director of the Appropriation Committee Mark Hartfield.

Over the past 50 years, I cannot help but notice a dramatic change in the evaluation of science and neuroscientists. Of course, enormous progress has been made in the use of the most sophisticated technology and accumulation of a huge number of new facts. However, one of the problems is that students are not taught about the history and philosophy of science so that some of them think that all this started after the introduction of the internet. In addition, in the past, the significance of the contributions was the main or only question that mattered. As I pointed out in my History of Neuroscience Lecture, these days, major questions about scientists are their geographic location, gender, ethnic origin, and skin color. Unfortunately, presently these are the dominant issues at many universities, foundations, and learned societies. In this respect, I must admit that the communists were more open-minded and rational, and when they declared that religion is not important, they never asked the applicant’s religion. If it is not important, why ask? The communists were also better educated and understood the philosophy of equality and were aware that there could be different opinions. Another difference is that they had a sense of humor, and even Marshall Tito laughed at the jokes about himself.

Private Life

My private life has been closely related to my professional life, and it has been eventful but in general very happy. Before I emigrated to United States, I married a Serbian woman, Ljiljana Lekić, who was a student at the University of Belgrade. After our wedding, we emigrated to the United States, and she initially became a technician in our new department at Harvard Medical School. After about two years, she entered a PhD program, and her mentor and thesis adviser and was my friend and collaborator Richard Sidman. I was stunned to learn that during that time they started a sexual affair because he was much older than she was, but also he seemed to have a very nice family with two children. However, after learning of this relationship, we divorced, and they married. In those days, there was not the institutional disapproval of faculty–student relationships that there is today. In retrospect, I was actually very lucky that my first marriage ended in divorce relatively quickly, which gave me an opportunity to marry again and spend the next 25 wonderful years with Patricia Goldman-Rakic. She was my beloved wife, devoted friend, and great supporter. She was also a very trustworthy and honest person, and I appreciated these qualities. We had a wonderful marriage. Then, while I was visiting Japan in 2003, she died after being struck by a car while crossing a street near our home in New Haven.

Pat’s death was devastating to me to the extent that I needed psychotherapy sessions because I felt survivor guilt, with an irrational feeling that if I had not gone to Japan to give the talk, this tragedy would not have happened. I also thought that I would never again have a romantic relationship with any woman or marry again. However, contrary to my expectations, I was very lucky again to meet another truly wonderful woman in New Haven, Sandra Biller. She also lost her husband under tragic circumstances, and we understood each other. In addition, this is a different relationship because she is a psychotherapist and not a basic scientist, and we do not work in the same place. However, we discovered that we have much in common and we wed, not just to console each other, but also to support each other psychologically (figure 7). She is an extraordinary person with whom I share many interests, such as the love of art. After seeing some of my research papers and my drawings on gene structure, she was inspired to make a sculpture “DNA of Love” that is displayed in my home (figure 8). We also enjoy discussions about the meaning of life and have learned how to age together. She retained her nice house in the woods at the edge of the Quinnipiac National Park, and I kept one in downtown New Haven. We have a large library of over more than 3,000 books, mostly on brain subjects, including some classics, such as Wilhelm His (1889), Ludwig Edinger (1895), Korbinian Brodmann (1925), and Santiago Ramon y Cajal (1895), that are considered as relatively rare antiques.

Outside of science and art, I am also interested in national and international world politics (as entertainment). Sandra and I were actually invited to the White House after I received the Kavli Award since U.S. presidents invite to a reception only Nobel, Kavli, and Pulitzer prize winners. In the Oval Office, G. W. Bush looked at Sandra and said, “Do you know that I am the only president of the United States who was born in Connecticut—and, did you know, they did not vote for me! At any rate, you have a wonderful smile!” She replied, “Pity I did not know that you are so charming; otherwise, I would have voted for you!”

Figure 7.   Sandra and Pasko at the 2008 reception for the Kavli Prize in Oslo (Norway).

Figure 8.   DNA of love.

I am an avid competitive tennis player who runs after every ball like it is the Wimbledon Final. I played regularly with many famous neuroscientists like Jack Cooper and Alan Wagner until they passed away. However, even these days, I continue to play regularly and enjoy beating younger players, including my students. This, besides the genes and two glasses of wine a day, is the reason that I enjoy excellent health and thus far have not spent a single night in the hospital.

Concluding Remarks

Looking back on my long scientific career, I recognize how fortunate I have been and how small the chances were that someone born in Ruma (not Roma!) could achieve this much in this highly competitive field. I also recognize that one must have luck. Here is one remarkable example. One evening in the summer of 1962 at about 6:30 p.m., after finishing with the assistance of the removal of a neuroglial tumor in the neurosurgery operating room, I received a call from my professor that a neurosurgeon from Harvard, Hannibal Hamlin, was visiting Belgrade Medical School. Because his host was not free that evening, I was asked whether I would join him for dinner this evening in one of Belgrade’s best restaurants. I said I would be happy to, but only if I can cancel a 7:00 p.m. date with my girlfriend at downtown square Terazije. So, I called her, and after five long rings she replied, out of breath, and said that she was already at the stairs when she heard the telephone ringing and ran back to her apartment to answer the call. She agreed to cancel our date and let me go to dinner at such a fine restaurant. If she was one step more down the stairs and had not answered the telephone, I would not have gone to the dinner and would not have met Hamlin, who suggested and helped me apply for a Fulbright Scholarship in the United States. This is how life depends on luck and unrelated occasions, but as one multiple Open Golf Champion said when it was reported that he won with a lucky shot, “Yes, I was lucky the last seven times.”

At the end, if I have to select a single sentence of advice that had a crucial influence on me, it would be the words of my high school teacher who said, “Always ask how and why.”

Selected Bibliography

Algan O, Rakic P. 1997 Radiation-induced area- and lamina-specific deletion of neurons in the primate visual cortex. J. Comp. Neurol. 381: 335–352.

Ang ESBC, Jr, Haydar TF, Gluncic V, Rakic P. 2003 Four-dimensional migratory coordinates of GABAergic interneurons in the developing mouse cortex. J. Neurosci. 23: 5805–5815.

Ang ESBC, Jr. Gluncic V, Duque A, Rakic P. 2006 Prenatal exposure to ultrasound waves impacts neuronal migration in mice. Proc. Nat. Acad. Sci. (USA) 103: 12564–12568.

Anton ES, Cameron RS, Rakic P. 1996 Role of neuron-glial junctional proteins in the maintenance and termination of neuronal migration across the embryonic cerebral wall. J. Neurosci. 16: 2283–2293.

Anton ES, Marchionni MA, Lee K-F, Rakic P. 1997 Role of GGF/ neuregulin signaling in interactions between migrating neurons and radial glia in the developing cerebral cortex. Development 124: 3501–3510.

Anton ES, Kreidberg J, Rakic P. 1999 Distinct functions of a3 and av integrin receptors in neuronal migration and laminar organization of the cerebral cortex. Neuron 22: 227–289.

Arcaro MJ, Mautz T, Livingstone MS. 2020 Anatomical correlates of face patches in macaque inferotemporal cortex. Proc. Natl. Acad. Sci. (USA) 117: 32667–32678.

Arellano JI, Harding B, Thomas JL (2018) Adult human hippocampus: no new neurons in sight. Cereb. Cortex 28: 2479–2481.

Arellano JI, Morozov YM, Micali N, Rakic P. 2021 Radial Glial Cells: New Views on Old Questions. Neurochem. Research 46: 2512–2524.

Arnsten AFT, Rakic P. 2022 Patricia S. Goldman-Rakic (1937–2003) A biographical memoir. National Academy of Science. Proc. Natl. Acad. Sci. (USA). www.nasonline.org.memoirs.

Berghuis P, Rajnicek AM, Morozov YM, Ross RA, Mulder J, Monory K, Marsicano G, Matteoli M, Canty A, Yanagawa Y, Rakic P, Lutz B, Mackie K. Harkany T. 2007 Hardwiring the brain: endocannabinoids control axon guidance. Science 316: 1212–1216.

Bhardwaj RD, Curtis MA, Spalding KL, Buchholz BA, Fink D, Bjork-Eriksson T, Nordborg C, Gage FH, Druid H, Eriksson PS, Frisen J (2006) Neocortical neurogenesis in humans is restricted to development. Proc. Natl. Acad. Sci. U S A 103:12564–12568.

Bourgeois, J-P, Jastreboff P, Rakic P. 1989 Synaptogenesis in the visual cortex of normal and preterm monkeys: Evidence for intrinsic regulation of synaptic overproduction. Proc. Nat. Acad. Sci. (USA) 86: 4297–4301.

Bourgeois J-P, Rakic P. 1993 Changing of synaptic density in the primary visual cortex of the rhesus monkey from fetal to adult stage. J. Neurosci. 13: 2801–2820.

Bourgeois J-P, Rakic P. 1996 Synaptoarchitecture of the occipital cortex in macaque monkey devoid of retinal input from early embryonic stages. Euro. J. Neurosci. 8: 942–950.

Brainard MS, Doupe AJ. 2013 Translating Birdsong: Songbirds as a model for basic and applied medical research. Annu Rev Neurosci. 36: 489–517.

Brennand KJ, Savas JN, Kim Y, Tran N, Simone A, Hashimoto-Torii K, Beaumont KG, Kim HJ, Topol A, Ladran I, Abdelrahim M, Matikainen-Ankney B, Chao Sp-h, Mrksich M, Rakic P, Fang G, Zhang B, Yates JR, Gage FH. 2015 Phenotypic differences in hiPSC NPCs derived from patients with schizophrenia. Mol. Psych. 20: 361–368.

Breunig JJ, Arellano JI, Macklis JD, Rakic P. 2007a Everything that glitters isn’t gold: a critical review of analysis of postnatal neural stem cells. Cell Stem Cell 1: 612–627.

Breunig JJ, Silbereis J, Vaccarino FM, Šestan N, Rakic P. 2007b Notch regulates cell fate and dendrite morphology of newborn neurons in the postnatal dentate gyrus. Proc. Nat. Acad. Sci. (USA) 104: 20558–20563.

Breunig JJ, Sarkisian MR, Arellano JI, Morozov YM, AE, Sojitra S, Wang B, Flavell RA, Rakic P, Town T. 2008 Primary cilia regulate hippocampal neurogenesis by mediating sonic hedgehog signaling. Proc. Nat. Acad. Sci. (USA) 105: 13127–13132.

Breunig JJ, Arellano JI, Rakic P. 2010 Cilia in the brain: going with the flow. Nature Neurosci. 13:654–655.

Bystron I, Blakemore C, Rakic P. 2008 Development of human cerebral cortex. Boulder Committee revisited. Nature Review Neurosci. 9: 110–122.

Cameron RS, Rakic P. 1991 Glial cell lineage in the cerebral cortex: Review and synthesis. Glia 4: 124–137.

Cameron S, Rakic P. 1994 Identification of membrane proteins that comprise the plasmalemmal junction between migrating neurons and radial glial cells. J. Neurosci. 14: 3139–3155.

Caviness VS, Jr, Rakic P. l978 Mechanisms of cortical development: a view from mutations in mice. Ann. Rev. Neurosci. l: 297–326.

Cholfin JA, Rubenstein JR. 2007 Genetic regulation of prefrontal cortex development and function. Novartis Foundation Symposium 288: 165–173.

Clowry G, Molnár Z, Rakic P. 2010 Renewed focus on the developing human neocortex. J. Anat. 207: 276–288.

Cotney J, Leng J, Jun Yin J, Reilly SK, DeMare LE, Deena Emera D, Ayoub AE, Rakic P, Noonan, JP. 2013 The evolution of lineage-specific regulatory activities in the human embryonic limb. Cell 154:185–196.

Crick F, Jones E. 1993 Backwardness of human neuroanatomy. Nature 561: 109–110.

Datta D, Leslie SN, Morozov YM, Duque A, Rakic P, van Dyck CH, Nairn AC, Arnsten AFT. 2020 Classical complement cascade initiating C1q protein within neurons in the aged rhesus macaque dorsolateral prefrontal cortex. J. Neuroinflammation 17: 1–15.

Datta D, Leslie SN, Wang M, Morozov YM, Yang S, Mentone S, Zeiss C, Duque A, Rakic P, Horvath TL, van Dyck CH, Narin, AC, Arnsten ATF. 2021 Age‐related calcium dysregulation linked with tau pathology and impaired cognition in non‐human primates. Alzhemer’s Dementia. 17: 920–932.

DeFelipe J. 2019 Cajal’s Neuronal Forest. Science and Art. 484 pages, Oxford University Press. Oxford UK.

Dominguez MH, Ayoub AE, Rakic P. 2013 POU-III Transcription Factors (Brn1, Brn2, and Oct6) Influence neurogenesis, molecular identity, and migratory destination of upper-layer cells of the cerebral cortex. Cereb. Cortex 23: 2632–2643.

Duque A, Arellano JI, Rakic P 2021 An assessment of the existence of adult neurogenesis in humans and value of its rodent models for neuropsychiatric diseases. Mol. Psych., online ahead of print. doi:10.1038/s41380-021-01314-8

Duque A, Krsnik Z, Kostovic I, Rakic P. 2016 Secondary expansion of the transient subplate zone in the developing cerebrum of human and nonhuman primates. Proc. Natl. Acad. Sci. (USA) 113: 9892–9897.

Duque A, Spector R. 2019 A balanced evaluation of the evidence for adult neurogenesis in humans: Implications for neuropsychiatric disorders. Brain Struc. Funct. 24: 2281–2295.

Easter SS, Jr, Purves D, Rakic P, Spitzer NC. 1985 The changing view of neural specificity. Science 230: 507–511.

Eccles JC, 1989 Evolution of the Brain: Creation of Self. Routledge, London, 282 pages.

Eckenhoff ME, Rakic P. 1988 Nature and fate of proliferative cells in the hippocampal dentate gyrus during the life span of the rhesus monkey. J. Neurosci. 8: 2729–2747.

Eyal et al., 2016 Unique membrane properties and enhanced signal processing in human neocortical neurons. eLife 5: e16553.

Franjic D, Skarica M, Ma S, Tebbenkamp ATN, Arellano JI, Choi J, Xu C, Li Q, Morozov YM, Andrijevic D, Vrselja Z, Spajic A, Santpere G, Li M, Liu Y, Spurrier J, Zhang L, Gudelj I, Rapan L, Takahashi H, Huttner A, Fan R, Strittmatter SM, Sousa AMM, Rakic P, Sestan N. 2022 Transcriptomic taxonomy and neurogenic trajectories of adult human, macaque, and pig hippocampal and entorhinal cells. Neuron, 110: 452–469.

Galvin VC, Yang ST, Paspalas CD, Yang Y, Jin EL, Datta D, Morozov YM, Lightbourne TC, Lowet AS, Rakic P, Arnsten AFT, Wang M. 2020 Muscarinic M1 receptors modulate working memory performance and activity via KCNQ potassium channels in primate prefrontal cortex. Neuron 106: 649–661.

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Joshua Richard Sanes

BORN:

Buffalo, New York

September 5, 1949

EDUCATION:

Yale College, New Haven, CT, Scholar of the House, BA (l970)

Harvard University, Boston, MA, Neurobiology, PhD (1976)

APPOINTMENTS:

Assistant, Associate, Full and Alumni Endowed Professor, Washington University School of Medicine, St. Louis, MO (1980–2004)

Sabbatical, Institut Pasteur, Paris, France (1985–1986)

Wiersma Visiting Professor of Neurobiology, California Institute of Technology (1993)

Sabbatical, University of Marseille, Luminy, France (1999)

Jeff Tarr Professor of Molecular and Cellular Biology, Harvard (2004–present)

Paul J. Finnegan Director, Center for Brain Science, Harvard (2004–2020)

Visiting Fellow, Trinity College, University of Cambridge (2008)

Visiting Scientist, Biogen (2013–present)

HONORS AND AWARDS (SELECTED):

Councilor, Society for Neuroscience (1990–1994)

Elected Fellow, American Association for the Advancement of Science (1992)

Stephen Kuffler Lecture, Harvard Medical School (1996)

National Advisory, Council, NINDS, NIH (1999–2003)

Alden Spencer Award, Columbia University (2000)

Elected member, National Academy of Sciences USA (2002; section chair, 2014–2017)

Visiting Professorship, College de France, Paris (2004)

Elected member, American Academy of Arts and Sciences (2006)

Grass Lecture, Society for Neuroscience (2008)

Bishop Lecture, Washington University (2010)

Javits Neuroscience Investigator Awards NIH (1989, 2005, and 2012)

Harvey Lecture, New York (2012)

Member, NIH Advisory Committee on BRAIN Initiative (2013–2014)

Vernon Mountcastle Lecture, Johns Hopkins University (2017)

Gruber Prize in Neuroscience, Society for Neuroscience (2017)

Perl-UNC Neuroscience Prize (2019)

Maxwell Cowan Award in Developmental Neuroscience (2019)

Scolnick Prize in Neuroscience (2020)

Honorary Doctoral Degree, Hebrew University of Jerusalem (2022)

Joshua Sanes and his colleagues have studied the formation of synapses, which transmit information between nerve cells. For many years, they used the neuromuscular junction to identify intercellular signals that regulate formation and maturation of this model synapse. In the course of this work, they pioneered new ways to mark and manipulate neurons and the synapses they form. Subsequently, they turned to the retina to identify molecules underlying the specificity of synapse formation, which is responsible for assembling the complex circuits that underlie information processing. Most recently, they have used high-throughput transcriptomic methods to generate cell atlases of the retina in mice, humans, and other vertebrates and have used them to analyze the development, resilience, dysfunction, and evolution of retinal cell types.

Joshua Richard Sanes

Introduction

I have had a wonderful life so far: loving parents and grandparents, a long happy marriage, terrific children, good health, financial stability, the respect of my peers, and many friends in the neuro-verse. However, none of this would be of particular interest to the likely readers of this chapter. There have also been some bumps along the road, but they are likewise of modest general interest. So this will be a scientific autobiography—appropriate for the venue and appropriate to me, since my primary self-identification for more than a half century has been as a neurobiologist.

I view the core of my work as being about synaptic specificity—how axons choose particular targets (cellular specificity) and, in some cases, particular parts of a target cell (subcellular specificity) on which to form connections. My lab has undertaken three major efforts directed at this goal. The first used the neuromuscular junction (NMJ) to study subcellular specificity and, as the work unfolded, synaptogenesis in general. The second used the retina to study cellular specificity. The most recent applied high-throughput transcriptomic methods to retina to see if we could comprehensively classify and characterize the full cast of characters. The efforts overlapped, occupying roughly 1976–2010, 1995–2018, and 2014–present, respectively. Rather than taking a strictly chronological approach, I’ll describe them in turn. To avoid abandoning chronology altogether, I’ll intersperse these sections with ones on other projects and activities, and precede them with brief comments on my family and childhood.

Childhood

When I was a child, my parents seemed remarkably different from each other, but I now see astonishing similarities between them. First, both were children of immigrants from Eastern Europe—Russia/Poland (it kept switching back and forth) for my paternal grandfather, Romania for my paternal grandmother, and Lithuania for both maternal grandparents. Second, after my grandparents passed through several cities—Patten (Maine), Rochester (New York), Chicago, and Philadelphia—they ended up in Buffalo, where my parents grew up in a lower-middle-class predominantly Jewish neighborhood just a few blocks from each other. In fact, they were casual friends, although my father was three years older than my mother and they traveled in different social circles. Third, both of my parents had older sisters who died in childhood from conditions that would not be lethal today—Down’s syndrome and appendicitis. This left them both as only children in melancholy families. Fourth, both were very smart and—especially my father—fiercely intellectual. Fifth, both grew up with thwarted ambitions. Shortly after my mother, social and scholastic, enrolled in college as a premedical student, her father suffered financial reverses and she was forced to drop out. My father, too rebellious and politically active to finish high school, wanted to be writer, but was never able to make that his profession.

As a consequence, both ended up leaving Buffalo during World War II. My father was in the Army, serving as a radio operator in New Guinea. My mother moved to Utah with a friend, and then on to San Diego. There she worked in an airline control tower, bought a Piper Cub, learned to fly it, and volunteered in the Civil Air Patrol, flying up and down the coast looking for Japanese submarines. She didn’t find any.

After the war, both returned to Buffalo, reunited, married, and moved to New York, where my father tried to write fiction, my mother worked as a copy editor for left-wing magazines, and both ran with a crowd of New York intellectuals many of whom remained lifelong friends. When my mom found out that she was pregnant with what became me, they moved back to Buffalo. My father joined his father in the latter’s automotive parts store. In the optimistic postwar years, his idea was that he would soon make his fortune, leave daily operations to subordinates, and return to writing. Sadly, although he was able to support the family, the riches did not materialize. He remained at the store for many years, eventually became a salesperson for a college textbook company, and finally retired. At that point, he was finally happy and spent a decade making wonderful collages and enjoying his grandchildren (two of mine and two of my brother’s) until he was taken by a glioblastoma in 1995.

My mother stayed home for around 15 years after I was born, volunteering in all manner of activities in Williamsville, the suburb of Buffalo to which we moved when I was 3. Then, ambition bubbling to the surface, she returned to college, earned bachelor’s and master’s degrees in audiology and spent 20 years working as a speech therapist before retiring. She lived to the age of 97, full of energy and opinions until the last few years of her life.

I went to elementary, middle, and high school in Williamsville. Although children of parents with high aspirations are now often sent to private schools, that wasn’t the case then, and in fact the Williamsville schools were excellent. When I was in second grade, the teacher suggested to my parents that I be skipped to third grade. After much thought, they consented. In retrospect it was a mistake. I was already small, young for my grade, and physically and socially awkward, all qualities accentuated by skipping. Nonetheless, I generally enjoyed school, had a modest number of friends, participated in a full range of nonathletic extracurricular activities, and spent happy Saturdays and summers helping out at my father’s and grandfather’s store.

My dad was interested in psychology and psychoanalysis, and among the many books in the house were some collections of case histories jazzed up for general audiences by their therapist-authors—for example, The Fifty-Minute Hour (Lindner, 1955). I began reading them in junior high school. These books, discussions of psychology at home, and the novels that I was devouring awoke in me a fascination with mental illness. I even wrote a high school term paper about schizophrenia, in which I described biochemical changes that had been reported to occur in the brain of people with these afflictions (all of which later turned out to be irreproducible). In this way I began transitioning from a literary sort (contributor to the school literary magazine, editor of the school newspaper, and so on) to one with an interest in something that didn’t have a name then but that we would now call neurobiology.

Becoming a scientist

High School: Guthrie

Robert (Bob) Guthrie was a microbiologist who grew up poor in the Ozarks, found his way to the University of Minnesota where he earned a PhD and an MD, and then bounced around for several years (NIH, University of Kansas, Staten Island Public Health Hospital, Sloan Kettering) before moving to Williamsville and landing at the University of Buffalo. His research was on the susceptibility of bacteria to purine-based cancer chemotherapeutic agents. When his second child (of six) was born with a severe intellectual disability, he began to think about using his expertise to find ways to diagnose these disabilities.

In early work, he had isolated E. coli mutants that required purine for growth (Guthrie, 1949). In a prescient (and awkward) sentence in the introduction of the report, he noted that “the nature of the purine requirement of this organism appeared to be such as to warrant further investigation of its use for assay of natural materials for purine content.” I suspect he was thinking of this when he tackled phenylketonuria (PKU), in which mutations of a gene that turns phenylalanine into tyrosine leads to accumulation of high levels of phenylalanine and its metabolites in serum, with devastating effects on brain development. There was already evidence that a low-phenylalanine diet could attenuate symptoms if begun in infancy, but there was no way to screen infants for the defect. Guthrie and Susi (1963) grew B. subtilis in the presence of an inhibitor of phenylalanine synthesis. They spotted a few drops of blood from a newborn on filter paper, and placed a small punch on the plate. Bacterial grew only if the blood contained high levels of phenylalanine, with the diameter of the growth ring roughly proportional to the phenylalanine concentration. Guthrie then turned into an advocate, lobbying state legislatures to mandate the test for all newborns. He was eventually successful and now all 50 states require the “Guthrie test.”

Another passion of Guthrie’s was engaging students in life science research at an early age. To this end, he obtained a National Science Foundation grant and hired a Williamsville High School biology teacher (Mr. Stanford) to run a program. Somehow, Mr. Stanford found and recruited me. The idea was to have us find amino acid auxotrophs (bacterial mutants that would grow only in the presence of specific amino acids) that could be used to generate similar tests for other inborn errors of metabolism. It was a perfect project for young students: technically simple, easily scalable, and requiring no fancy equipment, yet having a valuable goal. I joined the lab, and began working both summers and weekends during the school year.

It was an amazing experience. I was able to do things that would be forbidden today: design, perform, and interpret experiments with minimal oversight; run an autoclave all by myself; and even watch an autopsy on a stillborn infant (for which my medical student mentor was severely chastised). It also introduced me to college students who helped me envision a world beyond Williamsville. And I even did something useful, generating a leucine auxotroph that was later used (sadly without attribution) in a test for Argininosuccinic Aciduria (Murphey et al., 1972). It was called JP3 for Josh and Paula, a student in the program on whom I had an unrequited crush.

College: Greengard

I enrolled at Yale in 1966, the first year in which public school students were admitted in equal numbers to prep school students. Enthused by my experience with Guthrie, my initial idea was to major in biochemistry and find a way to apply biology to brain disorders. However, I quickly realized that one year of math and one year of physics were enough to last me for a lifetime. I then cycled rapidly through several other majors before settling on psychology. I delighted in the stories, especially in abnormal psychology. However, naïve though I was, I felt that there had to be a way to get beyond description to some sort of explanation.

The opportunity arose through a course called Physiological Psychology; it would now be called Introductory Neurobiology. A friend (Bruce Krueger, now a neurobiologist at the University of Maryland) and I were enthralled and approached the professor for advice on how we could do research in this area as seniors. After a few intermediate steps, we ended up in the office of Paul Greengard. Paul had just arrived at Yale at the age of 43, feeling that he had not lived up to his potential and had one last chance to succeed. In fact, he was already quite successful, but that’s not how he saw it. At any rate, he had the big idea that mechanisms by which hormones act on target organs—formation of second messengers and stimulation of protein phosphorylation—might also be used by slow or “modulatory” neurotransmitters at synapses. This is obvious in retrospect but was daring at the time and viewed by many as outlandish. Undeterred, he attacked it with remarkable focus, energy, and creativity. In a series of papers published between 1969 and 1975, he made most of the key discoveries for which he later won the Nobel Prize, showing that slow synaptic transmission is mediated in large part by effects of neurotransmitters on cyclic nucleotides via cyclic nucleotide-dependent phosphorylation of synapse-associated proteins. At the time, I thought this was just what happened in top-tier labs; only later did I realize how unusual it was. (I describe this time in an obituary I wrote; Sanes, 2019.)

Paul invited Bruce and me to join for senior theses. I hatched an ambitious (and unrealistic) plan to unravel the mysteries of the “pink spot,” a component of urine visualized on chromatograms, that appeared to distinguish schizophrenics from neurotypicals (Bourdillon et al., 1965). The pink spot had been identified as the dopamine metabolite 3,4-dimethoxyphenylethylamine (DMPEA); dopamine abnormalities had been implicated in the pathogenesis of schizophrenia; and Paul was interested in how dopamine activated adenylate cyclase. So the idea was to find out if those schizophrenics who did and didn’t excrete DMPEA formed diagnostically distinct groups (under the supervision of a psychology professor, Ed Zigler) and then find out how DMPEA affected dopamine-sensitive neurons and enzymes. I proposed this plan to a program called “Scholar of the House,” which absolved students of course requirements so they could devote the full year to research. To my surprise, I was admitted to the program.

Neither prong worked as planned, but both worked. The collaborator in New York who was going to test patient urine for DMPEA became seriously ill and left her position. However, Zigler arranged for me to visit Bellevue Hospital and go through the records of some 40 schizophrenics to test his hypothesis that developmental level (estimated from indicators such as occupation, level of education, and employment history) was related to symptoms he had characterized as reflecting turning away from the self, turning away from others, or turning against others. Our results supported the hypothesis (Sanes and Zigler, 1970). In Paul’s lab, Bruce and I isolated cyclic adenosine monophosphate (cyclic AMP)-dependent kinases from a variety of bovine organs (which we collected from an abattoir) and asked whether they exhibited tissue specific features—which they did (Kuo et al., 1970). In addition, I followed up one difference, that protein kinases from cerebellum were activated by cyclic guanosine monophosphate (cyclic GMP) nearly as well as by cyclic AMP. By fractionation, I obtained the first evidence for a cyclic GMP-dependent protein kinase. More sophisticated work by others was used in the eventual publication, but an abstract reporting the initial finding is the only abstract I list in my CV (Kuo et al., 1969).

Paul was brilliant, funny, lovable, and loving. His lab was still small, and I had much more direct interactions with him than would have been possible a decade later, when the group was an order of magnitude larger. My experience in his lab gave me not only a taste of exciting science, and a lifelong mentor and friend, but also the credentials to apply to graduate school in neuroscience. Bruce stayed in Paul’s lab for his PhD, and I was strongly tempted to do the same, but in the end decided to seek out a new adventure.

Graduate School: John Hildebrand

I applied to the newly formed Departmenr of Neurobiology at Harvard Medical School. They rejected me, but I was accepted into the Department of Biochemistry under the auspices of a separate neuroscience program run by Richard Sidman, who had pioneered the use of mouse mutants (all naturally occurring at that point) to address issues in neural development. I had little to do with the Sidman group, and instead did rotations with Fred Goldberg, who was beginning the studies of protein turnover that subsequently made him the leading figure in this area; and with Larry Okun, an independent fellow working on methods to culture and record from neurons. Larry assigned me the task of testing whether neurons, which are postmitotic, could be provoked to undergo “unscheduled” DNA synthesis following DNA damage. It was exciting to find out that they could (Sanes and Okun, 1972). Larry was one of the smartest and kindest people I ever met, and every minute I spent with him was pure pleasure. In fact, he was so smart that he had been allowed to attend seminars in the Neurobiology Department and sometimes invited me to accompany him. This was a rare privilege, a point I’ll come back to shortly.

The rotations were fun, and coursework was enlightening—including a course in molecular biology from Mario Capecchi. (Mario’s stay at Harvard was short-lived, in part because he had the audacity to seek mutants in mammalian cells rather than in bacteria or phage as all self-respecting geneticists were supposed to do. After he won a Nobel Prize, Harvard may have regretted letting him go.) Nonetheless I still wanted to be a neurobiologist, and signed up for a rigorous nine-week-long summer course at the Marine Biological Laboratory in Woods Hole, then taught by John Dowling (see Volume 4), Michael Bennett, Rudolfo Llinas, and others. Just as I was leaving, though, the two Neurobiology Department graduate students who had been admitted instead of me left the program and, needing to fill their training grant slots, they admitted me.

The Department of Neurobiology was, at that time, a remarkable place. Founded only a few years earlier, it was chaired by Steve Kuffler and had, as senior faculty, Ed Furshpan, David Hubel (see Volume 1), Ed Kravitz (see Volume 4), David Potter, and Torsten Wiesel. There were also a few junior faculty members, Zach Hall, Jack McMahan, John Nicholls, and Ann Stuart at the time I started, joined by John Hildebrand, Story Landis, and Paul Patterson by the time I finished. The students and postdocs during that time were a cast of all-stars, including Eric Frank, Charles Gilbert, Jim Hudspeth, Mary Kennedy (see Volume 11), Simon Levay, Louis Reichardt, Carla Shatz, Mike Stryker (see Volume 11), David Van Essen (see Volume 9), and King Wai Yau. The faculty were well aware of their excellence, which had both unpleasant and pleasant consequence. On the downside, the atmosphere was unabashedly elitist, with very few outsiders meriting respect. Work done outside the department was often treated dismissively: only when a result had been replicated in The Department (it was always called The Department) could it be trusted. Seminars, which occurred several times a week at lunch, were not publicized and interlopers were made to feel unwelcome. Indeed, as I mentioned, Larry Okun’s admittance, and Gerry Fischbach’s later, were rare exceptions. When I joined a lab in The Department, I was asked to renounce membership in Sidman’s program and to take the full suite of neurobiology courses. Another consequence of this high self-regard was that all new faculty until around 1990 were chosen from the ranks of departmental students and postdoctoral fellows.

On the positive side, once one was on the inside, the atmosphere was marvelous. Hierarchical distinctions were minimal. The whole Department, including students, postdocs, faculty, and staff had lunch together every day. A set of four semester courses was unparalleled—ones on axon and synaptic physiology taught by Potter and Furshpan, one on neurochemistry by Kravitz and Hall, and one on systems neuroscience by Hubel and Wiesel. Courses were held in the evening, and each week one student gave a lecture on an assigned topic following extensive rehearsal with an instructor (no teaching assistants). As the instructors were all excellent lecturers, we learned presentation skills along with neurobiology. There was hardly any pressure to publish—we were all so sure of our status that there was little need for external validation. Even the junior faculty were spared much of the anxiety that usually accompanies that status. The department had six rather young full professors, and it was clear that no additional tenured appointments would be made for a long time. So the assistant professors could eschew competition (mostly), follow their passion, recruit better students than they would likely get elsewhere, and be assured that the Harvardian glow would earn them a tenured professorship when they had to leave.

My aim was to join Ed Kravitz’ lab, collaborate with Paul Greengard, and use Ed’s model system, the lobster, to learn more about the role of cyclic nucleotides in synaptic transmission. I would use a leg muscle, which is innervated by both a GABAergic inhibitory neuron (as Kravitz and Potter had demonstrated) and an excitatory glutamatergic neuron. In one set of experiments, I would stimulate one axon or the other, then freeze the muscles and take them back to New Haven to be assayed for cyclic AMP and cyclic GMP. In a parallel approach, I would record from muscle fibers, apply reagents known to mimic or inhibit cyclic AMP or cyclic GMP pathways, and ask how they affected inhibitory or excitatory synaptic potentials.

It was a great idea that failed. I was in over my head and not getting the support an unskilled graduate student needed. In retrospect I suspect that Kravitz was torn between two strong beliefs. One was the egalitarian ethos that compelled him to support a student’s desires whatever his own feelings. The other was his skepticism about roles of cyclic nucleotides in neural function. His view—admittedly shared by many neuroscientists at the time—was that the role of cyclic AMP in the brain had already been discovered: it was to modulate metabolism, especially glycolysis, just as it did in other tissues. Cyclic AMP levels were high in brain because it needed a lot of energy. End of story. It all came to a head when I discovered that an exciting result was artifactual, stemming from changes in perfusion flow rate on membrane potential and resistance. When I sheepishly confessed this to Ed he said, oh yes, he had known all about that for years. I was crushed by what I viewed as his lack of concern and realized I needed to get out.

I was torn between finding another advisor and leaving the program. Two conversations with Ed Furshpan helped me recover. In the first, he stood in the stairwell with me for hours after our evening class, and—mostly by listening sympathetically—talked me down off the ledge. In the second, he recommended looking at the arthropod antenna, a rich and pure (neuronally speaking) source of sensory neurons. As luck would have it, John Hildebrand, a postdoctoral fellow with Kravitz, was about to ascend to an assistant professorship and had decided to adopt the holometabolous (undergoes a complete metamorphosis) moth Manduca sexta as his model system, so he could address developmental questions. We agreed that development of sensory neurons in the Manduca antenna would be a good starting point for his lab and a plausible path to a PhD for me.

The next few years were happy and productive (which for me are nearly synonymous). Each week I would bicycle to Bio Labs in Cambridge and pick up a small vial of Manduca eggs from a lab that had a breeding colony. I’d bring them back to our lab, lay them on cubes of food that I mixed up, and then transfer them to larger and larger plastic dishes as they passed through their larval (caterpillar) instars, before eventually molting to form pupae. During this metamorphosis, the cellular buds (imaginal discs) that will become appendages evert from inside the larva and become sandwiched between the body proper and the hard protective pupal case. Then, over the next 20 days, a simple epithelial tube develops into a gorgeous antenna, revealed in all its glory when the moth ecloses from the pupa, dries its wings, and flies off.

Sensory organs called sensilla arise by mitosis of epithelial cells; the supporting cells form the cuticular protrusions of various many forms, depending on what modality the neurons are destined to sense (mechanical, olfactory and so on); and the neurons extend axons toward the brain and dendrites into the cuticular sheaths. My job was to follow the process in as much detail and with as many methods as I could muster. I mapped out the development of the antenna proper, using light and scanning electron microscopy; followed the genesis of sensilla using birth-dating methods (tritiated thymidine autoradiography), and light and transmission electron microscopy; confirmed that the neurons were cholinergic (as had been proposed) and followed the accumulation of the transmitter synthetic and degradative enzymes; and documented the development of responses to odorants using a bulk recording method called the electroantennogram (Sanes and Hildebrand 1975, 1976a, 1976b, 1976c; Schweitzer et al., 1976). And finally, I compared neuronal development in unmanipulated pupae with those in which the brain was excised at the onset of pupation (Sanes et al., 1976). (It turns out that pupae don’t need brains to turn into moths.)

Throughout this project, John Hildebrand was an ideal mentor for an ambitious, insecure, and somewhat obnoxious young scientist. He provided me with great independence, gentle guidance, and unwavering support. In return, as his only student or postdoc for the first few years, I was able to help him get his lab off the ground more quickly than would have been possible otherwise. And the detailed description of sensory neuronal development, although short on major surprises, provided a solid foundation for the beautiful work on olfactory behavior that his lab produced over the following decades. I also ended up getting great help from Jack McMahan, whose lab was down the hall. His expertise (histology) complemented John’s (biochemistry), and I sought his advice almost daily as I learned light and electron microscopy—technical help and, as important, how to interpret and not overinterpret what I saw. He had recently begun to work on the NMJ, and our many conversations ignited my interest in that system.

As the project on Manduca wrapped up, I began to look for a lab in which to pursue postdoctoral studies. I contacted several eminences and made a decision. But before starting, I took some time off.

Interludes: Teaching and Policy

Zambia

My freshman advisor, Frank Ballance, spent several years in Uganda and Tanzania in a precursor of the Peace Corps before returning to Yale Law School. After he graduated, he returned to Africa, helping set up the economic ministry in Lusaka, capital of the newly independent Zambia (previously Northern Rhodesia). We stayed in touch and, to my delight, he offered to host my roommate and me during the summer of 1968, after our sophomore year.

It was my first trip abroad, and replete with adventures both en route and there. Frank picked us up in Johannesburg and we visited Soweto (the infamous center of oppression under and resistance to apartheid), then drove South to Lesotho, back North through Botswana and Rhodesia (now Zimbabwe) to Lusaka, passing through game parks along the way.

Once in Lusaka, I started the job that Frank had found for me in the University of Zambia Medical School. It had been founded in 1965 and hadn’t yet graduated a class. I worked in the Department of Biochemistry, which was run by a British expatriate named David Mobbs. Actually, he didn’t run the department, he was the department, so I was able to take on responsibilities that would have been unimaginable under other circumstances—especially since I had not gotten beyond high school biology. I planned, tested, and oversaw the lab sessions for the students. It was a great confidence builder and also my first experience of teaching.

Office of Technology Assessment

I spent most of 1973 cutting sections of Manduca antennae and examining them in the microscope. This coincided with the Senate investigation of the break-in of the Democratic National Committee headquarters by a cadre of Republican operatives, working under the direction of the White House—the so-called Watergate hearings. The hearings were broadcast live and I listened to every word as I cut, stained, looked, and photographed. I was obsessed by the shenanigans and the characters—Sam Ervin, the committee chair (“I’m just a simple country lawyer”); Howard Baker, the ranking Republican (“What did the President know and when did he know it?”); the turncoat Howard Dean (“there was a cancer growing on the presidency”); Rose Mary Woods (the 18-minute gap in the Oval Office tape); the Plumbers; and so much more. I wondered if there might be a way to see some of this stuff up close.

Following his time in Zambia, Frank Balance had returned to the United States and become a staff member for Senator Jacob Javits. He offered to arrange a few interviews, and I ended up taking a position with the Office of Technology Assessment (OTA), a congressional agency that had been set up to provide guidance on scientific and technological issues when called upon by senators, representatives, or committees. It has been established in 1974 with an emphasis on energy policy and was now (late 1975) launching a health policy program. Although I knew nothing whatsoever about health policy, I was an attractive candidate for two reasons: I had a shiny new PhD from Haahvahd and I promised to leave in a year.

While there, I worked on two reports. One was on the value of basic research—scarcely a controversial topic, but useful to the Health Subcommittee of the Senate Labor and Public Welfare Committee. The other, more substantive, was requested by the Senate Finance Committee. They wanted to know about the then-new computed axial tomography (CAT) scanner. Because of cost controls in hospitals, physician groups had taken to buying scanners, setting them up in offices near hospitals, and billing insurance for scans. The issue was whether it was sufficiently useful that Medicare should provide reimbursement. As always, if a procedure was approved, price controls were minimal—what has been called a healthcare system with capitalist supply and socialist demand. But we were asked only about safety and efficacy. We were skeptical initially but as we conducted interviews, read the literature, and attended radiology conferences, we concluded that it had numerous advantages over previous technologies (e.g., pneumoencephalography and arteriography).

The benefits of the year for me were numerous. Most important, I met my wife Susan there. She was also spending a transitional year—in her case between a stint in the Peace Corps and a program in public health. Most immediately, I satisfied my desire to be a voyeur in the federal government, observing the types of people who had fascinated me during the Watergate hearings. A highlight was briefing Senator Edward Kennedy about CAT scanners. I earnestly explained the pros and cons. He listened politely for a few minutes, then interrupted: “Thanks. But what I want to know is who’s for it and who’s against it.” I also learned a bit about science and healthcare policy—an interest I have maintained and nurtured, most recently through membership on the National Academies’ Committee on Science, Technology, and Law.

OTA also provided me with the opportunity to test my commitment to a life in academic neuroscience research. I had enjoyed the lab enormously but still had a sliver of doubt, and I had entertained thoughts of alternatives such as science journalism. What I found was that I loved the style of policy research but was far less interested in its substance than in learning how the brain worked.

Regarding style, the appeal was irresistible. For example, one day, I called Donald Fredrickson, then director of the NIH, to see if my boss and I could get his views. Had I called six months earlier, I’m sure his assistant’s assistant would not have taken my call. Now it was, “I’m so sorry Dr. Sanes, he is completely booked today, but would be happy to meet any time tomorrow that’s convenient for you.” It’s easy to get used to that sort of deference! However, I found myself spending weekends in the library of the George Washington University Medical School, reading journals, taking notes, and thinking about experiments. When September came, I was ready to return to the lab.

The Neuromuscular Junction

Basal Lamina

During the last year of my thesis work, Jack McMahan told me about a remarkable observation he had made. He had been visualizing NMJs in live frog muscles using Nomarski optics and trying to record from them. On occasion, he accidently injured a muscle fiber with his electrode. What he saw was that the cell itself retracted, but the nerve terminal persisted on what turned out to be the basal lamina that ensheathes each muscle fiber and runs through the synaptic cleft. We called these sheaths “ghosts.” Jack’s observation revealed what should have been obvious but wasn’t: the nerve terminal adheres to the basal lamina, which it contacts directly, rather than the underlying plasma membrane. More intriguing, it made us wonder whether motor axons could be made to reinnervate ghosts following denervation. It had been known since the work of Tello (Cajal’s student) that regenerating motor axons selectively reinnervate original synaptic sites, even though those sites occupy less than 1 percent of the muscle fiber surface, a striking example of subcellular specificity in synapse formation. If regenerating axons reinnervated original synaptic sites on ghosts, one would know where to look for the recognition molecules responsible.

I was eager to take this on—it combined my initial interest in synapses with my newer interest in neural development. Before leaving for OTA, I arranged a joint postdoctoral fellowship, in which I would work with Jack to test the idea that regenerating axons would reinnervate initial synaptic sites on basal lamina “ghosts” and, in parallel, work with Zach Hall, another faculty member in the department, to probe the molecular composition of the basal lamina. I had some hesitation about staying in the same department rather than moving to a new environment, but I couldn’t imagine anything more interesting than this project. And in fact, Zach Hall had moved to the University of California, San Francisco (UCSF) by the time I got to work, so the two parts of the project ended up being sequential rather than simultaneous, and I did get a chance to experience a different academic environment in a different part of the country.

Over the next year, I worked with another postdoctoral fellow in Jack’s lab, Larry Marshall, to test the basal lamina hypothesis. The experiment was conceptually simple. We used a thin flat frog muscle, the cutaneous pectoris, directly beneath the skin of the chest. The muscle fibers ran rostrocaudally and the nerve from lateral to medial along the middle of the muscle, with axons leaving the nerve to deposit a single NMJ on each muscle fiber in a narrow “end-plate band.” We would anesthetize a frog, incise the skin to expose the muscle, and cut out “windows” on either side of the nerve, leaving a “bridge” of innervated muscle fibers. If the bridge was wider than 1.5 mm, the muscle fibers would seal off and survive, but if it was narrower, they would degenerate and be phagocytized, leaving a ghost town behind. Resident myoblasts called satellite cells would then divide to generate new myotubes, so in many cases, we x-irradiated the chest (covering the head and legs with strips of lead) to prevent myoblast proliferation and thereby leave the ghosts more or less empty. When we damaged the muscle, we also crushed the nerve lateral to the muscle. The distal axons degenerated but the nerve sheaths persisted, and guided regeneration back to the muscle. The questions then were (1) could we recognize original synaptic sites on ghosts after the axons and muscle fibers had degenerated, and (2) would regenerated axons reach and preferentially reinnervate these sites?

We recognized the original sites in two ways. One was with a histochemical stain for acetylcholinesterase, which had long been used as a marker for NMJs. (An ancillary benefit was that we demonstrated that acetylcholinesterase was attached to or part of the basal lamina, the first documented association of an enzyme with basal lamina in any tissue; McMahan et al., 1978.) The second was by electron microscopy. In normal muscle, junctional folds invaginate the postsynaptic membrane directly beneath nerve terminals, and struts of basal lamina project into the folds. These struts survived loss of the muscle fiber and its plasma membrane.

By both criteria, reinnervation of original synaptic sites was as precise in the absence of muscle fibers as it was in their presence. We then looked further at the axonal segments that contacted synaptic basal lamina. In normal adult muscle, synaptic vesicles accumulate in nerve terminals, and cluster at active zones that lie directly opposite the mouths of junctional folds. This apposition was reestablished in regenerated axons on ghosts, even including the formation of active zones at sites on the presynaptic membrane that abutted the struts of basal lamina—in other words, where junctional folds had been. Together these results established that at least some of the factors responsible for directing subcellular specificity and presynaptic differentiation, long assumed to be membrane components, were instead stably associated with the basal lamina of the synaptic cleft (Sanes et al., 1976).

The next step was to find those factors—far easier said than done at a time when neither monoclonal antibodies nor recombinant DNA methods had been invented. I moved to Zach Hall’s lab, now at UCSF, to get started. With Zach’s guidance I tried several approaches. Attempts to isolate synaptic portions of the basal lamina failed completely—no surprise, because it comprised about 0.1 percent of the basal lamina sheath and less than 0.01 percent of muscle extracellular matrix. What did work was to generate antisera from matrices that could be isolated, and then find antibodies within the sera that selectively immunostained synaptic basal lamina. In the end, we found evidence for four distinct synaptic basal lamina antigens—acetylcholinesterase and three others (Sanes and Hall, 1979). These antibodies could then be used to seek bioactive synaptic components.

At this point, early 1979, it was time to look for an independent position. I was very happy in Zach’s lab—he remains a good friend—but I was increasingly eager to strike out on my own. Luckily, the work with Jack and Zach had made me fairly marketable, so several opportunities opened up. The two I ended up deciding between were Stanford and Washington University in St. Louis (Wash U). The allure of California was great, as was the possibility of rejoining Jack. Conversely, there were some drawbacks. One was laid out by the chair of the Stanford Neurobiology Department, Eric Shooter. In a shockingly candid conversation, for which I remain grateful, he said that we would offer me an assistant professorship, but I should realize that if I continued to work on the NMJ, much of the credit for anything I did would accrue to Jack. The other factor was Susan’s desire to move Eastward, closer to family (Massachusetts) and farther from what seemed to be a wave of insanity in the Bay Area. In early November 1978, nearly 1,000 cult members who had moved from San Francisco to Jonestown, Guyana, committed mass suicide. Just a few weeks later, Harvey Milk, a city supervisor and gay icon, and George Moscone, the mayor, were assassinated by a disgruntled supervisor.

Luckily, Wash U was attractive in many ways. It was already, and still is, unusually collaborative and collegial. Dale Purves (see Volume 11), a former collaborator of Jack McMahan’s at Harvard, took it upon himself to recruit me to the Department of Physiology, which he had joined a few years earlier. He instilled confidence as did the chair, Cuy Hunt (see Volume 5), who had already built successful departments at Yale and Utah and had the perfect light touch. There was also a long and glorious tradition of neuroscience—among some 20 Wash U Nobel laureates were Joseph Erlanger and Herbert Gasser, who characterized sensory nerves, and Rita Levi-Montalcini and Stanley Cohen, who discovered nerve growth factor (NGF). At the time I was recruited, chairs of three medical school departments were neuroscientists—Cuy in physiology, Luis Glaser in biochemistry, and Max Cowan (see Volume 4) (who left before I arrived) in anatomy, as well as Viktor Hamburger (see Volume 1), who had stepped down as chair of biology but remained a powerful presence. Indeed, although I never grew to love St. Louis, I never ceased to love Wash U.

The first order of business at Wash U was to learn more about the basal lamina. My first postdoctoral fellow, Arlene Chiu, generated monoclonal antibodies that defined three groups of basal lamina components: those concentrated at synaptic sites, those excluded from synaptic sites, and those present throughout the basal lamina (Sanes, 1982; Sanes and Chiu, 1983). Arlene also obtained initial evidence that one synaptic component, recognized by an antiserum and a few monoclonal antibodies (C1 and C4; C for Chiu), was a protein of about 190 kD that was stably associated with basal lamina but could be solubilized by denaturing and reducing agents.

Dale Hunter, a postdoctoral fellow who joined the lab in 1985, took the next steps. He noted that C1 and C4 stained not only synaptic basal lamina but also glomerular basal lamina in kidney, a vastly richer source. He used glomeruli to generate another set of monoclonal antibodies (D5 and D7 for Dale) that he used to purify the protein and show that neurons were adherent to it. Most important, we collaborated with John Merlie—about whom more later—to isolate cDNAs encoding the antigen, using expression cloning in bacteria. Following several rounds of screening (in each case using a recovered partial cDNA to identify clones that extended further), we ended up with the sequence of a protein that was clearly related to laminin (Hunter et al., 1989a). This was interesting for a few reasons. First, laminin was a known component of basal laminae. It was a heterotrimer of three subunits, initially called A, B1, and B2 and subsequently renamed α1, β1 and γ1. Our protein, which we called synaptic laminin or s-laminin (pronounced slaminin) expanded the laminin gene family; it was later renamed (to my distress) β2. We and others later showed that there are some 5α, 3β, and 3γ chains that can assemble in multiple αβγ trimers. Second, and more relevant here, the initial α1/β1/γ1 laminin had already been shown to support neuronal adhesion and promote neurite outgrowth. It made sense that a laminin variant could act in a related but distinct way. Indeed, Dale and Brenda Porter, an MD-PhD student, showed that s-laminin bore a small adhesive domain that was selective for motor neurons and that could act to inhibit the growth that classical laminin promoted—in other words, while classical laminin made axons grow, s-laminin made them stop growing and start differentiating (Hunter et al., 1989b; Porter et al., 1995).

The discovery that there were multiple laminins with a special form in synaptic basal lamina raised the possibility that the same was true for other basal lamina components—collagens IV, heparan sulfate proteoglycans, and entactins/nidogens. We tested this idea over the next several years and found that it was correct. In fact, basal laminae are much more variable than had previously been thought, with differences among cells in a tissue, transitions in isoform composition during development, and compensatory changes in mutant mice lacking specific isoforms (e.g., Sanes et al., 1990; Miner and Sanes, 1994, 1996; Miner et al., 1995, 1997; Patton et al., 1997; Fox et al., 2008). Thus, although our focus remained on the NMJ, the analysis, and the conclusions we drew from it, extended to many tissues and organs. In particular, several of the mutant mice we generated (including lamb2 mutants) had lethal glomerular (kidney) defects (Noakes et al., 1995b; Miner and Sanes, 1996) leading Jeff Miner, who had led much of this work, to abandon neuroscience in favor of nephrology; he now leads the nephrology research division at Wash U.

Collaboration with John Merlie

Our entrée to the synaptic cleft was through studies of reinnervation following injury. The second order of business at Wash U (once biochemical analysis was underway) was to look at development: how the basal lamina formed, how its components were regulated by neuromuscular interactions, and what roles (if any) they played in synapse formation and maturation. We documented the series of steps by which the basal lamina formed (Chiu and Sanes, 1984) and collaborated with John Lawrence (a new assistant professor in pharmacology) to show that activity and nerve-derived factors regulate basal lamina deposition in cultured muscle (Sanes and Lawrence, 1983; Sanes et al., 1984). Functional studies, however, required a different approach. A life-changing collaboration with John Merlie made that possible.

By the time John was recruited to Wash U in 1982, he was already a major contributor to studies of the nicotinic acetylcholine receptor (AChR), the neurotransmitter receptor concentrated in the postsynaptic membrane of the NMJ. He worked with pioneers in the area, Jean-Pierre Changeux at Institut Pasteur in Paris and Steve Heinemann and Jon Lindstrom at the Salk Institute in La Jolla, before moving to a faculty position at the University of Pittsburgh, where he began applying recombinant DNA methods to the problem. He was soon tenured there, but Phil Needleman, the chair of pharmacology, persuaded him to move without tenure, and trust that it would come soon—which it did.

John and I had our first long talk in 1983 on a plane taking us to Cold Spring Harbor for their annual symposium, at which we were both speaking. It was on molecular neurobiology that year and served as a sort of coming out party for what was then an emerging field. We quickly realized that our interests were similar (synapse formation at the NMJ) but slightly different (presynaptic vs. postsynaptic) and that our distinct skill sets would allow us to do experiments together that neither of us could do alone. Our first foray was to show that denervation supersensitivity, the appearance of AChRs in extrasynaptic membrane following denervation or paralysis, was preceded by, and presumably due to, an increase in AChR subunit mRNA (Merlie et al., 1984). What I remember most vividly about this study was that after the experiments were complete, we had to hold the manuscript for a few months until we obtained 80 base pairs of sequence to verify that the cloned cDNA probe indeed corresponded to the AChRa subunit. What a contrast to experiments I’ll describe below in which more than 10 billion base pairs of sequence are delivered to our hard discs overnight—a rate increase of more than 10⁹-fold.

In our next study, we asked whether there might be transcriptional contributions to the concentration of AChRs in the postsynaptic membrane of normal adult muscle. I used microscissors to dissect end plate-rich and -free portions of diaphragm—not unlike what I had done to make bridges of basal lamina ghosts eight years earlier, except in this case from dozens of mice per experiment. John then generated RNA from each sample and probed it on Northern blots with cDNAs encoding two AChR subunits. In both cases, levels of mRNA were several-fold higher in the synaptic sample than in the extrasynaptic sample (Merlie and Sanes, 1985). Because the synapse comprised only a small fraction of the nominally synaptic sample, we considered that perhaps the true difference was much greater. Diaphragm muscle fibers contain about 500 nuclei of which about five lie directly beneath the postsynaptic membrane. Thus, only 3 percent of the nuclei in the synaptic third were actually synaptic, so even if they transcribed AChR genes at 100x the rate of extrasynaptic nuclei, the difference between sample would only be a few-fold. We therefore speculated that synaptic nuclei were transcriptionally specialized, implying that the nerve acted not only to determine where AChRs accumulated but also where they were synthesized. Later we generated transgenic mice in which regulatory elements of AChR subunit genes drove expression of a nuclear-localized reporter and showed that indeed transcription was largely confined to synaptic nuclei (Sanes et al., 1991; Gundersen et al., 1993). We also used in situ hybridization (which had failed in 1985) to show that several other components of the postsynaptic membrane are synthesized locally (Moscoso et al., 1995). Later still, Masashi Kishi, a postdoctoral fellow, used microarrays to identify mRNAs concentrated in synapse-rich samples, identifying novel components of the postsynaptic membrane (Kishi et al., 2005a). Thus, muscles, like some neurons, harness local protein synthesis to help sculpt the postsynaptic membrane, but by different mechanisms—localized transcription in the multinucleated muscle fiber and selective RNA transport to dendrites in neurons.

As this work was proceeding, John had become proficient in the generation of transgenic mice. In this method, developed by Richard Palmiter and Ralph Brinster in the early 1980s, DNA is injected into the pronucleus of a fertilized mouse egg, which is then implanted in the oviduct of a pseudopregnant mother—much as is now done with people. The transgene integrates at random into a chromosome and essentially becomes part of the genome. John initially used the method to explore regulatory elements of AChR genes in bulk assays (Merlie and Kornhauser, 1989) and soon, as noted earlier, with reporters that could be visualized (Sanes et al., 1991). Around 1990, he decided to take the brave next step of importing knock-out technology, in which endogenous genes could be mutated—the method that Mario Capecchi coinvented after leaving Harvard, and for which he shared a Nobel Prize. The first reports on generation of such mice appeared in 1989, and it was immediately clear that the method would be transformative: one could, for the first time, choose which gene to mutate and, soon, what mutation to make (knock-in mice). This was not even possible in Drosophila! It was, however, very difficult. Among the many steps, perhaps the most challenging were learning to cultivate embryonic stem cells without having them differentiate or get contaminated, and generating vectors at a time when neither PCR nor genome sequence was available.

Two postdoctoral fellows—Peter Noakes in my lab and Medha Gautam in John’s—rose to the challenge. We decided to mutate three genes—s-laminin (lamb2); rapsyn, an AChR-associated scaffolding protein that John had cloned and studied; and agrin, a nerve-derived basal lamina-associated organizer of postsynaptic differentiation that Jack McMahan and colleagues had discovered. They initially used the “bridge” assay that had taught us about subcellular specificity of reinnervation, but in this case, they allowed myotubes to regenerate in persistently denervated basal lamina “ghosts.” Steve Burden in Jack’s lab found that AChRs clustered in the new myotubes directly beneath original synaptic sites, showing that basal lamina could direct both pre- and postsynaptic differentiation (Burden et al., 1979). Based on this finding, they purified the aggregating factor, and eventually both McMahon’s group and Scheller’s, also at Stanford, cloned it (McMahan, 1990). Along the way, we established a collaboration with Yancopoulos and colleagues at Regeneron, who had isolated muscle-specific receptor tyrosine kinase (MuSK), which turned out to be a critical subunit of the agrin receptor. We believed that together these might comprise the core components of a neuromuscular synaptogenesis program.

Progress was slow. I threw most of my NIH funding behind the effort, as did John. Eventually we applied for a program project grant from NIH to allow us to proceed. We had precious few “preliminary results,” but I wrote that if our application was turned down, this new technology would be available only to investigators in “Highly Huge Medical Institutes.” Whether for this reason or another, we were funded.

Perhaps most disheartening was agrin. Vectors had to be made from the genomic clones that could be recovered, and we used one in which we could delete a set of exons in the 5’ (N-terminal) portion. The mutant mice were horribly normal. It turned out that the deletion was in a region encoding multiple copies of an apparently dispensable domain (called a follistatin repeat), and the mice were able to make a protein that lacked these exons but was nonetheless functional. We therefore went back and deleted a few more exons—and got the same result. Finally, in what would surely be the last try, we succeeded in inactivating the gene. In quick succession, we obtained laminin β2, rapsyn, and agrin mutants, and the Yancopoulos group obtained MuSK mutants, which we subsequently analyzed together. All were lethal—agrin and MuSK at birth, rapsyn shortly thereafter and laminin β2 a few weeks later—demonstrating, in combination with other observations, that they were essential for neuromuscular synaptogenesis (Noakes et al., 1995a; Gautam et al., 1995, 1996; DeChiara et al., 1996; Apel et al., 1997; Zhou et al., 1999). Our bet had paid off.

With this success we began thinking about next steps—more genes, more analyses, and forays into the central nervous system. Then disaster struck. John and I usually got together on Saturday mornings to discuss progress and make plans. On Saturday May 27, 1995, we were particularly excited, because we were going to dissect a litter of agrin knockout mice and image NMJs. As I prepared to drive into the lab, the phone rang. John had suffered a heart attack and died almost immediately. In a moment, I lost a dear friend and my closest collaborator.

It was a horrible time. In the few months after John’s death, I lost my father to a glioblastoma, one of my best friends from college to AIDS, and my grandmother to the ravages of old age. My wife became extremely ill from a large abdominal infection and its sequelae. And overnight the size of my group doubled, as I stepped in to mentor John’s students, fellows, and research assistants, and to supervise the transgenic facility that he had recently established. It also marked the end of my having time to do any experiments with my own hands.

Formation and Maturation of the Neuromuscular Junction

Time—and the unsurprising depression that followed—passed, and both scientific and family life recommenced. Particularly important was the strong support from Wash U, which allowed John’s lab to remain open until we could find new homes for his group members. A few joined my group, a few completed their theses and moved on, a few got other positions, and so on. One senior research assistant, Mia Wallace, took over the transgenic facility, which survived my year as the weekend staff member; she eventually built an institute-wide facility that she supervises to this day. Eventually, we returned to the genetic analysis of neuromuscular synaptogenesis that John had begun. Over the years, we generated (or, in some cases, obtained) mutants in more than 40 genes and analyzed their roles in the formation, maturation, and maintenance of this synapse. I believe this remains the most complete genetic analysis of development for any synapse, and it laid the foundation for studies of central synapses by numerous other groups.

I’ll mention three sets of studies from this long series here. Most related to the questions I initially set out to answer was a detailed analysis of muscle-derived factors that organize presynaptic differentiation. We analyzed roles of several laminin subunits, showing distinct roles for the β2, α4, and α5 chains. In brief, β2 appears to promote maturation of nerve terminals (Noakes et al., 1995a; Fox et al., 2007), in part by binding to calcium channels in the presynaptic membrane (Nishimune et al., 2004); it also actively excludes glial (Schwann) cells from the synaptic cleft (Patton et al., 1998). α4 is responsible for the precise apposition of active zones with junctional folds (Patton et al., 2001), and α5 has autocrine functions in postsynaptic maturation (Nishimune et al., 2008).

We also sought roles for neural cell adhesion molecule (NCAM), Tenascin-c, fibroblast growth factor 5 (FGF5), and ColQ, the collagenous subunit of acetylcholinesterase. However, despite discovering many interesting features of their localization and regulation, we obtained no evidence for major roles in synaptogenesis (Covault and Sanes, 1985; Covault et al., 1986; Sanes et al., 1986b; Gatchalian et al., 1989; Moscoso et al., 1998; Feng et al., 1999). This was disappointing but illustrates a great advantage of the genetic approach: one can test candidates nominated based on, for example, expression patterns or bioactivity in cell culture and then triage poor candidates to focus on the critical ones. Eventually, Hisashi Umemori, later joined by Mike Fox, took a more direct, unbiased approach. Hisashi devised a bioassay based on clustering of synaptic vesicles within cultured primary neurons and then used it to monitor purification of active material from brain and Torpedo electric organ. This approach yielded three sets of molecules: a group of FGFs (7, 10, and 22), a signal regulatory protein (SIRPα), and cleaved terminal domains of several collagen subunits (Umemori et al., 2004; Fox et al., 2007; Umemori and Sanes, 2008). Hisashi and Mike have continued to pursue roles of FGFs, SIRP, and collagens in their own labs (e.g., Terauchi et al., 2010; Toth et al., 2013; Su et al., 2012).

A second set of studies following up the analysis of postsynaptic differentiation was initiated with Merlie. We used targeted mutagenesis to selectively delete several alternatively spliced exons in agrin whose existence had been noted but whose function remained unclear. In this way, we found that one short exon near the C-terminus was essential for its AChR-aggregating activity (consistent with previous work in tissue culture), whereas several other segments were dispensable (Burgess et al., 1999, 2000). Surprisingly, a synapse-specific carbohydrate that we had identified much earlier (Sanes and Cheney, 1982) also affected AChR clustering (Martin et al., 1995). Of several components of the postsynaptic membrane and cytoskeleton that we studied, the most interesting were members of the dystrophin-associated glycoprotein complex (DGC) that Kevin Campbell had isolated in his studies of Duchenne muscular dystrophy, caused by loss of dystrophin. We followed up on hints that—just as had been the case for laminins and collagens—the DGC differed between synaptic and extrasynaptic membrane, with different members of gene families and/or different alternatively spliced isoforms in the two regions. An autosomal homologue of dystrophin, called utrophin, is concentrated beneath the postsynaptic membrane. Loss of either utrophin or dystrophin had only a modest effect in mice—surprisingly, given the lethality of dystrophin loss in humans. What we found was that in mice, utrophin compensates for loss of dystrophin, with double mutants faithfully recapitulating virtually all aspects of the Duchenne phenotype (Grady et al., 1997). These double mutants have since been used in preclinical tests of potential therapies in mice. As for other synaptic DGC components, they appear to be dispensable for initial formation of the postsynaptic membrane but are crucial for its postnatal maturation (e.g., Grady et al., 2000, 2003).

Third, we used genetic methods to explore roles of electrical activity in synapse formation and maturation. Our reagent was a mutant lacking choline acetyltransferase (ChAT), the enzyme that synthesized the neurotransmitter acetylcholine; in the absence of ChAT, neuromuscular transmission was abolished, and the mice died at birth (Misgeld et al., 2002). With this assurance, we performed two experiments. In one, we studied the role of activity in synapse elimination. In early postnatal life, individual muscle fibers are innervated by multiple axons, and then all but one withdraw, resulting in the adult pattern of single innervation. It was abundantly clear that elimination was driven by activity, but it was less clear whether it was the total or relative activity of the inputs that mattered. By inactivating some or most inputs (using a conditional allele), we showed that the more active axon won the competition; when both (or all) axons were inactive, multiple inputs persisted (Buffelli et al., 2003).

The second experiment helped resolve a paradox that had arisen with respect to how pre- and postsynaptic specialization come to be apposed to each other. The dogma was that axons grow into the center of a muscle and secrete agrin to generate postsynaptic specializations in the central end-plate band. However, we and others found that the endplate band formed when agrin—or even innervation—was missing (Yang et al., 2001; Lin et al., 2001), and in some cases, axons grew toward preexisting postsynaptic specialization (Flanagan-Steet et al., 2005). By studying double mutants lacking both agrin and choline acetyltransferase, we found that synapses form in the absence of agrin provided that acetylcholine is also absent. Our conclusion from these and other experiments was that activity destabilized postsynaptic specializations unless agrin is present; that is, a major role of agrin is to give the “correct” postsynaptic structure immunity from the dispersing effect of activity (Misgeld et al., 2005). I found this conclusion especially pleasing because it was completely consistent with essentially all previous results of many groups—but provided a strikingly new interpretation (Kummer et al., 2006).

Imaging

With the initial aim of visualizing NMJs, Guoping Feng generated a large variety of transgenic lines that expressed various fluorescent proteins (XFPs) in motoneurons. This is fairly routine today, but at that time (around 1997), there were many fluorescent proteins and many regulatory elements that needed to be tested in many combinations. The result was a set of XFP lines expressing green, yellow, blue, or red fluorescent proteins in a variety of patterns—some, for example, in all motoneurons and others in just one or two per muscle (Feng et al., 2000b). Most also lit up other neuronal populations, and have been used in literally hundreds of studies by dozens of laboratories; Guoping’s paper has now been cited more than 2,000 times. Later, Jean Livet, a remarkably creative postdoc, took this approach a step further, inventing the “Brainbow” method that enables marking neurons in a range of dozens to hundreds of colors (Livet et al., 2007; Cai et al., 2013). Although frankly less useful than the XFP lines, they generated far more beautiful micrographs, which have now appeared in museums throughout the world.

Jeff Lichtman, an expert in imaging methods, helped in the initial characterization of the XFP lines and cosupervised development of the Brainbow method. Jeff was a postdoctoral fellow with Dale Purves, left Wash U for a short postdoctoral fellowship, and then returned to join the faculty shortly after I arrived. We quickly became friends and eventually collaborated on a variety of studies using the XFP lines to investigate neuromuscular development, regeneration, disease, and aging (e.g., Nguyen et al., 2002; Schaefer et al., 2005; Valdez et al., 2010). Jeff had (has) a deep suspicion of studies with molecular goals or that use manipulations to investigate mechanisms, so our points of view couldn’t be more different. Although he failed to change my mind in hundreds of conversations over some three decades, his influence on my thinking was profound. We also combined our contrasting views productively to comment on issues that spanned these areas (Sanes and Lichtman, 1999; Lichtman and Sanes, 2008).

Interlude: Lineage

I had long wanted to spend time in France, and even began arranging a postdoctoral fellowship with Jean-Pierre Changeux (see Volume 4) before being seduced by the basal lamina. An opportunity arose in 1985, thanks to a junior faculty grant called an Established Investigatorship of the American Heart Association, which encouraged recipients to spent protracted time in another lab. Medical schools in general don’t have clear sabbatical policies, but I argued successfully that as long as they weren’t paying my salary they shouldn’t tell me where to sit. Changeux was still the obvious choice, but I wanted something less related to my current work. I asked John Merlie, who had loved his years in Paris, for advice, and he suggested I contact Francois Jacob. I did so, and arranged an interview.

Jacob was courtly and kind when I visited, and said he’d be willing to host me. I found an apartment—a former chamber de bonne (maid’s quarters) atop an apartment building a few blocks from the Pasteur. It was wonderful—recently renovated with a clear view of the Eiffel Tower from one window, on a quiet side street, and just one block from a Saturday farmer’s market. In August, Susan and I moved in.

By 1985, although he continued to preside over a unit that filled a floor of “Batiment Monod,” Jacob was rather disengaged from science. At our first meeting, he suggested I visit the junior faculty arrayed across the floor, each with a few rooms, a few students, and a few technicians, choose one and let his secretary know my decision. Over the next year, he took me to lunch at a nearby café every few months, where we had omelets and frites, and made awkward conversation, but we seldom talked about science. I later found out that he was writing his wonderful autobiography, La Statue Interieure (The Statue Within) at the time; much of it described his childhood, life in the French Resistance, and the war wound that dashed his hope of becoming a surgeon—so it’s easy to imagine that he was lost in that world.

After some wandering, I joined the group of Jean-Francois Nicolas and began working with John Rubenstein. Jean-Francois had worked on teratoma cells (an early version of embryonic stem cells) as a PhD student with Jacob and then spent a few years as a postdoctoral fellow at Stanford before returning to Pasteur to apply molecular methods to early embryogenesis. John had earned an MD and PhD at Stanford and then joined Jean-Francois as a postdoctoral fellow; he is now an eminent neuroscientist at UCSF. John and I quickly became good friends and have remained close ever since.

At the time, Jean-Francois and John were trying to generate transgenic mice by infection of early embryos with a recombinant retrovirus containing the bacterial beta-galactosidase (lacZ) gene—which Jacob and Monod had used long before in their discovery of the operon. I spent a few weeks trying to demonstrate lacZ activity in transgenic embryos, but it soon became clear that the method was doomed to failure—because, we now know, the viral regulatory sequences are silenced by methylation early in embryogenesis. Seeking to find a better use for the virus, Jean-Francois came up with the idea of using it to trace cell lineage at later embryonic stages. We would infect single cells in postimplantation embryos, the retroviral genome would be integrated into the host genome, and we would be able to track progeny over many generations, based on their expression of lacZ. John was heading back to California, but I thought it was a wonderful idea and took it on.

The first step was to devise a histochemical stain for lacZ. A substrate that generated a blue product, X-gal, had long been used to stain bacterial colonies, but it took weeks of fiddling to find ways to immobilize the product as soon as it was generated, so that it would provide cellular resolution and survive tissue processing. Next, I used cultured cells to show that infection had no adverse effects on proliferation, that the regulatory elements in the virus were active in a wide variety of cell types, and, most important, that expression persisted over many cell divisions, indelibly marking the progeny of the infected cell. Once that all worked, I injected minute amounts of virus into early mouse embryos, waited several days, then looked for groups of lacZ-positive cells—putative clones. I was able to recover many cell clusters in yolk sac and skin, accumulate evidence that the clusters were indeed clones and, by comparing clones at various intervals following infection, reconstruct cell lineages (Sanes et al., 1986). As far as I know, this provided the first way to trace postimplantation cell lineages in a mammal, enabling a rigorous comparison of actual cell fate with the range of potentials a cell could exhibit in culture or after transplantation. Clearly, the combination of information about fate and potential was far more revealing than either alone.

In these first experiments, I had little success infecting cells in the brain. Once I got home, I was eager to try again. I decided to use the chick embryo, because it was much easier to expose and target the neural tube when the creature was surrounded by an egg rather than a mother. In fact, Marla Luskin, an extremely talented neuroanatomist, soon found ways to study the mouse cortex (Luskin et al., 1988), but it was much more difficult, so we largely stuck with chick.

The viral vectors I had used in France did not infect chick cells, but we were able to adapt (repackage) them. We also collaborated with John Majors, who had studied avian tumor viruses with Mike Bishop and Harold Varmus at UCSF before joining the Wash U faculty. He was extremely smart and absolutely adored cloning, and soon generated several new vectors. We initially used the optic tectum, because it is large, prominent, and has a very orderly arrangement of cells and laminae. Grace Gray, a graduate student and two postdoctoral fellows, Joel Glover and Deni Galileo, showed that single tectal progenitors could give rise to nearly all major tectal neuronal classes as well as glia (Gray et al., 1988; Galileo at al., 1990). Connie Cepko, who had devised essentially the same method at the same time, reached a similar conclusion in rodent retina (Turner et al., 1990). Tracing the distribution of cells in the clones over time also helped us understand the migratory paths that neurons take on their way from their birthplace in the ventricular zone to their final laminar destinations (Gray and Sanes, 1991). In the course of this work, we obtained some insight into the then-elusive radial glial cells, making the case that they are in fact the tectal stem cells (Gray and Sanes, 1992)—which was later shown to be the case in mammalian cortex as well (Fishell and Kriegstein, 2003). We also applied the method to spinal cord (Leber et al., 1990) and sensory ganglia (Frank and Sanes, 1991).

By 1990, I began to phase out this work. One problem was that it was fundamentally unrelated to the project on neuromuscular synaptogenesis, and I found it unpleasant to have two subgroups in the lab with little interest in each other’s work and little possibility of working together. In addition, it was becoming clear that further progress would require moving beyond description to elucidate the transcriptional programs that underlie fate choices—a path that Connie Cepko (then a competitor, now a friend and colleague) has followed with great success. That was not a prospect I found too attractive, especially when the collaborative work with Merlie was so exciting and fun. I remember telling a colleague about our work on lineage around that time. He said, “that’s interesting, but I think of you more as a synapse guy.” He was right.

Synaptic Specificity

My core interest has been in how neurons make the right connection—synaptic specificity. Studies on the basal lamina began with a focus on subcellular specificity. Once I got to Wash U, along with pursuing that theme, I was eager to move on to the cellular specificity that accounts for the stereotypy of neural circuits.

Dale Purves provided an entrée. He had been using physiological methods to find the cellular basis for a result that J. N. Langley (in my opinion the second greatest neurobiologist of all time, after Cajal) had obtained long before (Langley, 1895). Langley studied the innervation of peripheral targets of sympathetic ganglia that were supplied by preganglionic neurons from different spinal levels. He found that neurons innervated from rostral or caudal spinal segments preferentially innervated rostral or caudal targets, respectively. He then severed the preganglionic nerve and waited for regeneration. What he found was that this segmental preference was reestablished, leading him to propose a molecular explanation that predated Roger Sperry’s chemoaffinity hypothesis by more than 50 years. Purves confirmed Langley’s result at a single cell level and then, with Wes Thomson and Joe Yip, performed an experiment that I view as his best ever (although I know he will disagree violently). They transplanted ganglia from various segmental levels to the bed of the superior ganglion, and showed that ganglia from rostral or caudal levels were preferentially innervated by preganglionic axons running through rostral or caudal ventral roots, respectively (Purves et al., 1981).

Langley had also shown that autonomic preganglionic axons could innervate skeletal muscle fibers, so we wondered if such connections would also exhibit positional selectivity. Don Wigston, who had been a postdoctoral fellow with both Dale and Viktor Hamburger, joined the lab to test this idea. He transplanted intercostal muscles from different segmental levels to a common site and sutured in the preganglionic trunk. He then dissected the muscle in contiguity with ventral roots (an arduous procedure that he taught me so we could both do experiments) and recorded from muscle fibers to determine the segmental origin of their inputs. As was the case for ganglia, rostral and caudal muscles were preferentially innervated by axons from rostal and caudal levels, respectively (Wigston and Sanes, 1982, 1985), suggesting the existence of a body-wide program of positional information. Mike Laskowski, a faculty member at St. Louis University who came for a sabbatical, later showed that the mapping function was a bona fide part of neuromuscular development, not some peculiarity of the cross-innervation paradigm: in muscles innervated from multiple spinal segments, the rostrocaudal axis of the motor pool was systematically mapped onto the rostrocaudal axis of the muscle, and the map was reestablished following denervation and reinnervation (Laskowski and Sanes, 1987, 1988).

How to go forward? A serendipitous observation provided a way. Maria Donoghue, a graduate student in Nadia Rosenthal’s laboratory at Boston University, had characterized regulatory elements from a myosin gene that Merlie used to generate transgenic mice (Rosenthal et al., 1989). Nadia asked if Maria could visit our labs to characterize patterns of transgene expression. Maria and I began assaying many different muscles and found, to our surprise, that levels of reporter varied among muscles in several independently derived transgenic lines, forming a 100-fold gradient in which a muscle’s position in the rostrocaudal axis was correlated with its level of reporter expression (Donoghue et al., 1991). Although we never fully understood the basis of this peculiar pattern, it provided a useful marker: by generating muscle cultures and even immortalized cell lines from the transgenic mice, we could show that the muscles bore a cell-autonomous, heritable memory of their rostro-caudal position that was maintained by transgene methylation (Donoghue et al., 1992a, 1992b).

The implication was that this positional memory might drive expression of a recognition molecule that accounted for positionally selective innervation. Maria therefore used a subtractive hybridization approach to look for genes differentially expressed between rostral and caudal muscles and muscle cell lines, and came up with a great candidate. Disappointingly, while she was cloning her way through a ridiculously long untranslated region to reach the protein-coding sequence, the same gene, ephrinA5, was discovered and implicated in retinotectal mapping—also along the rostrocaudal axis—by the Bonhoeffer and Flanagan groups (Cheng et al., 1995; Drescher et al., 1995). On the bright side, the parallel spurred our efforts to implicate ephrins in the positional selective innervation of muscle. In favor of that idea, we found that recombinant ephrinA5 inhibited outgrowth from cultured motoneurons in a position-dependent manner and that manipulation of ephrin A5 in mice (by knockout or transgenic overexpression) degraded the topographic motor maps (Donoghue et al., 1996; Feng et al., 2000a).

This series of studies was deeply gratifying, but I was increasingly concerned that the effects we saw were quantitative rather than qualitative—highly significant statistically but scarcely overwhelming. The reason may be that once a motor axon is guided to the appropriate target muscle it doesn’t care all that much which muscle fiber it innervates. Specificity matters more in the brain, where highly stereotyped patterns of connectivity underlie neural circuitry. So, we had to study the brain itself.

Retina

Studies in Chick Embryos

The first steps in this project were taken by Masa Yamagata, a postdoctoral fellow with perhaps the greatest range of skills and most astonishing work ethic of anyone who has ever been in my lab. After a discouraging attempt to find more “neuromuscular specificity” genes, we decided to tackle the retinotectal projection in the chick embryo. There were two reasons. First, it was familiar territory from the studies on neuronal lineage and migration described above. Second, it had long been a favorite preparation for studies of axon guidance, culminating in the discoveries of ephrins and eph kinases, so a lot was known about the projection. Our idea was to look at laminar specificity—the phenomenon whereby once the axons of retinal ganglion cells (RGCs, the sole output neurons of the retina) arrive at their proper position along the dorsoventral and rostrocaudal axes, they dive into the neuropil to form synapses in specific tectal laminae. Masa began with a comprehensive inventory of the expression of “known” candidate adhesion molecules (Yamagata et al., 1995) and then adapted a “cryoculture” system that we had devised in studies on the NMJ (Covault et al., 1987). In this method, neurons are cultured on cryostat sections of a target tissue, and the trajectories of their axons assessed. Masa found that outgrowth, branching, and arborization patterns of retinal axons on tectal slices were lamina-selective, indicating the existence of localized cues that guide retinal axons (Yamagata and Sanes, 1995). We made modest progress over the next years in identifying a few of these cues (Inoue and Sanes, 1997).

Masa returned to Japan in 1995, and established a group at an institute in Okazaki. However, despite substantial success, he was unhappy in that position, so I suggested he return to Wash U for a respite while he figured out what to do next. He did return, took up where he had left off, and ended up staying through 2020. He began by looking for recognition molecules that might explain how different types of RGCs terminate in different tectal laminae, where they synapse on different target cells. To this end we contacted Catherine Dulac, who had invented a method for generating cDNA libraries from single neurons and differentially screening them to isolate cell type–specific genes—in her case, genes encoding vomeronasal receptors (Dulac and Axel, 1995). Catherine agreed to help, provided we promised to follow her protocol exactly—if we deviated and ran into problems, she would cut us off. I was in complete agreement with this restriction, having learned from Zach Hall that when trying a new method, one must begin by slavishly following the established protocol before trying to get clever and improve matters. I have preached the gospel of “slavish following” many times over the years—with mixed success.

Masa generated single cell libraries from RGCs that differed in size and surface properties and then used differential screening to find genes selectively expressed by one or another of the cells. He ended up with two closely related genes that encoded large transmembrane molecules with features that placed them in the “immunoglobulin superfamily,” a group known to include numerous recognition and adhesion molecules. We called them Sidekick-1 and -2 based on very weak homology to a Drosophila gene. With Josh Weiner, Masa showed that they were expressed by nonoverlapping subsets of RGCs (perhaps 20 percent each) as well as by subsets of retinal interneurons, that the Sidekick proteins were concentrated at synaptic sites, and that they both mediated homophilic adhesion—that is, beads coated with recombinant Sidekick-1 adhered to other Sidekick-1 bearing beads but not to Sidekick-2 bearing beads and vice versa (Yamagata et al., 2002). These results suggested that the Sidekicks could indeed promote differential connectivity of RGC subsets with synaptic partners.

When Masa and Josh Weiner began looking at the morphology of the RGCs that expressed each Sidekick, we had a surprise. In the retina, interneurons called bipolar and amacrine cells project to a synaptic layer called the inner plexiform layer (IPL), where they synapse on dendrites of RGCs. The IPL can be divided into numerous sublaminae, with each interneuronal and RGC type arborizing on only one or a few of the sublaminae. This laminar specificity is akin to the laminar specificity we had been studying in tectum. What Masa and Josh found was that processes of both interneurons and RGCs that expressed Sidekick-1 projected primarily to the same sublamina, and Sidekick-2 positive cells projected to a different sublamina. We realized at once that we should be looking at specificity in the retina itself rather than in the tectum, and soon found that overexpression or depletion of Sidekicks led to redistribution of arbors in predictable ways (Yamagata et al., 2002; Yamagata and Sanes, 2008). Masa also went on to show similar patterns of expression and function for several related molecules, called Dscams and Contactins, together leading us to posit the existence of an “immunoglobulin superfamily code” for laminar specificity (Yamagata and Sanes, 2008, 2012).

Studies in Mice

The retina has many advantages for studies of neural circuit assembly. It is accessible because it is outside of the skull. It comes with its own lens for viewing in vivo. Its cells and synapses are arranged in neat layers and thus are easy to identify. It is a complete circuit with a single controllable input (light), a single output (the optic nerve), and few if any long-range inputs. Best of all, we actually know what it does, which cannot be said for many other regions. Yet it is a bona fide part of the brain and about as complicated as other parts of the brain in terms of cellular and synaptic diversity. In a sense, it is for synaptic specificity what the NMJ was for synaptogenesis per se: a model that has all the key molecular and cellular features of, say, the cortex, but can be studied in far more satisfying detail. So, I quickly fell in love with the retina and have studied it almost exclusively over the past 15 years.

On the other hand, the chick retina does have some drawbacks: little functional information is available about it, we were not going to be able to study post-hatching stages, and it is refractory to the sort of genetic analysis that had proved to be so powerful in studies of the NMJ. The obvious solution was to transfer the retinal project from chicks to mice. Two developments made this possible. First, In-Jung Kim came to the lab as a postdoctoral fellow and undertook a heroic screen of more than 100 immunoglobulin superfamily molecules to find ones expressed in subsets of mouse RGCs. Second, once I moved (back) to Harvard in 2004, I established a collaboration with one of the leading scholars of retinal circuitry, Markus Meister.

My path back to Harvard began in 2000. Around that time, I realized that I was coming to a “now or never” point. Either I would move in the next few years or I’d soon become less marketable (I was already 51) and would likely finish my career at Wash U I loved Wash U and loved science but remained less than enamored of St. Louis and wanted another professional challenge. The obvious solution was to do more or less the same thing somewhere else. Luckily, Harvard came calling. Neurobiology at Harvard had long been centered at the Medical School. In 2000, the dean at the time, Jeremy Knowles, called a meeting to discuss the idea of establishing a new center for neuroscience on the Cambridge (undergraduate) campus. I was part of a group invited to discuss the idea and became excited by the prospect. Over the next year, the idea of launching a center crystallized and the prospect of leading it was enticing—but Harvard had other ideas and I began to explore other options. A few years later, their exploration complete (or failed), I was offered the job and took it.

There is a long story to be told about the Center for Brain Science, but I won’t tell it here. In brief, we decided to focus on trying to find out how neural circuits underlie behavior. This area was far from my own, but I was convinced it would be the most productive over the next few decades—and also one that would allow us to harness strengths in social and physical sciences that could distinguish us from medical school–based groups. We would be open to any model system (mice, fish, flies, worms…), and method (physiology, anatomy, computation, behavior…) as long as the investigator shared that overarching goal. We also realized that the tools needed to achieve the goal were insufficient, and we supported efforts to invent new and better methods. At the outset, progress was rapid but slowed down for a few reasons. First, Larry Summers, our visionary president and a major force in promoting life sciences on the Cambridge campus, put his foot in his mouth one too many times and was dismissed; his successor was far less of an advocate. Second, 2008 happened, and resources became scarce. And by the time the ship had righted itself, we were no longer (as a friendly dean explained it) the Shiny New Thing; there were now Newer New Things. Nonetheless, over a period of 10 years, we recruited a superb group of neuroscientists (e.g., Takao Hensch, Jeff Lichtman, Bence Olveczky, and Nao Uchida), who joined and helped coalesce several already on campus (e.g., Catherine Dulac, Markus Meister, and Venki Murthy), and also urged tool builders in the physical science (e.g., Adam Cohen, Xiaowei Zhuang, and Charles Lieber) to turn their talents to inventing tools for neuroscience. By the time I stepped down in 2020, the center was well established as one of two key nodes of neuroscience at Harvard, the other being the Department of Neurobiology at the Medical School.

But back to the retina. As soon as I got to Harvard, In-Jung Kim began collaborating with Yifeng Zhang, a postdoctoral fellow in Markus Meister’s lab. Having completed her screen of immunoglobulin superfamily genes, In-Jung had begun generating transgenic lines to mark RGC subsets with fluorescent proteins. Yifeng learned to target fluorescent RGCs in these lines for patch clamp recording and elucidated the visual features to which they were most sensitive. This allowed them to characterize RGC types by a combination of molecular, physiological, and structural features. They discovered several novel types, including J-RGCs and W3B RGCs, and found novel distinctions among RGCs responsive to motion in different directions (Kim et al., 2008, 2010; Kay et al., 2011a; Zhang et al., 2012). Perhaps most striking were J-RGCs, with absolutely gorgeous asymmetric dendrites that “pointed” in the direction of the motion to which they responded best; In-Jung’s images now decorate a few textbooks. And in a fortuitous turn of events, the W3B RGCs turned out to express Sidekick 2; Arjun Krishnaswamy and Masa were later able to show how Sidekick shaped the response properties of this RGC type (Krishnaswamy et al., 2015).

We also dug into the cadherin superfamily, which accounts, along with immunoglobulin superfamily, for the majority of known neuronal adhesion molecules. Looking at the assembly of just the circuits that tell the brain about motion in specific directions, we found roles for six different cadherins (Duan et al., 2014, 2018). We extended the study to transcription factors that came to our attention because their genes were expressed by intriguing subsets of cells—for example, Fezf1, FoxP1, FoxP2, NeuroD6, Tbr1, and Satb1 (Kay et al., 2011b; Rousso et al., 2016; Peng et al., 2017, 2020; Liu et al., 2018)—discovering additional RGC subclasses (e.g., F-RGCs) and showing roles in circuit assembly for several of them. It was particularly satisfying that we could assess the effects of loss- and gain-of-function genetic manipulations by electrophysiological as well as morphological criteria, providing a link between circuit assembly and circuit function.

A few of these genes led us to an aspect of organization that we hadn’t been looking for—the arrangement of cells and processes along the surface of the retina, or what we called lateral specificity as opposed to laminar specificity. Retinal cell types are nonrandomly arrayed along the retinal surface, a so-called mosaic arrangement that ensures coverage of all parts of the visual field by a full complement of feature detectors. Jeremy Kay isolated several retinal cell types by FACS from our GFP lines and profiled them by microarrays (this was before RNAseq was widely available). Among the genes he studied were MEGF10 and 11, two transmembrane proteins selectively expressed by a set of amacrine neurons called “starbursts” that play a key role in direction selectivity. He found that MEGF10 and its homologue, MEGF11, regulate the mosaic arrangement of starbursts, the first molecules implicated in this process for any cell type (Kay et al., 2012). Starbursts also figured prominently in studies of a remarkable clade of more than 50 tightly linked genes within the cadherin superfamily, the clustered protocadherins, which we had begun analyzing many years earlier (Wang et al., 2002). Julie Lefebvre and Dimitar Kostadinov found that they act to endow starbursts with individual identities, thereby preventing dendrites of a single starburst from synapsing with other dendrites of the same starburst (self-avoidance) while allowing them to synapse on dendrites of other, nominally identical, starbursts (self-/non-self-discrimination) (Lefebvre et al., 2012; Kostadinov and Sanes, 2015; Ing-Esteves et al., 2018).

A Retinal Cell Atlas

Most of the cell types we studied in retina were those for which we had transgenic lines that allowed us to mark, isolate, and manipulate them. In short, we were largely restricted to cells that we already knew something about. It was clear that a full account would require a more comprehensive method for cataloguing and characterizing all retinal cell types. Over the years, based on Masa’s success with Sidekicks, we had attempted transcriptomic profiling of single mouse RGCs, but the results were too noisy and the numbers too small to be useful. Our opportunity to expand the search came through a project begun with Aviv Regev at the Broad Institute and Alex Schier in the Center for Brain Science. Under the auspices of the BRAIN Initiative, the NIH had allocated funds for development of new “neurotechnologies,” and we were awarded a large grant to support several groups trying to develop methods for more efficient transcriptomic characterization of large numbers of cells. The idea was that we would use the retina, for which so much ground truth was available, to validate and optimize the methods. (I had served on the planning committee for the BRAIN Initiative, but we had disbanded before grant applications were solicited, and so there was no conflict.)

The clear winner was a method called “Drop-seq,” invented by Evan Macosko in Steve McCarroll’s lab (Macosko et al., 2015). In this method (and two other versions invented nearly simultaneously), a microfluidic device captures single cells (or, more recently, single nuclei) in nanoliter-size droplets along with a small bead that captures the mRNA. The mRNA from thousands of cells is then converted to DNA and amplified in a single tube, yielding 100- to 1,000-fold savings in time and money compared with previous laborious one-cell-at-a-time methods. An elaborate barcoding system ensures that transcripts from a single cell can be grouped after sequencing, and computational methods are then used to divide the cells into clusters or putative cell types.

The method and its variants are so powerful and informative that they have been the main focus of the lab’s work for the past six years. In a first set of experiments, Evan used Drop-seq to profile some 45,000 mouse retinal cells, which then were divided into 39 clusters. By inspecting genes selectively expressed in each cluster, and relating them to what we already knew about the retina, we could see that they all made sense and made up a low-resolution retinal cell atlas—low in the sense that many clusters contained cells of several closely related types. We then went on to enrich the most heterogeneous classes so we could obtain enough cells to finish the job, eventually ending up with what we believe to be a nearly complete atlas of 130 neuronal types plus another 10–15 non-neuronal types (Shekhar et al., 2016; Tran et al., 2019; Yan et al., 2020a). This impressive number, along with subsequent data from other brain regions, supported the assertion I’d been making for many years with limited evidence: that although the retina is easier to study than other brain areas, it is no less complex. Importantly, we checked whether types defined molecularly correspond to types defined by criteria that most neurobiologists care about—structure and function. Fortunately, at least for retina, they do.

With the atlas in hand, we have spent the past few years using it to ask biologically interesting questions. They include: How do newborn RGCs diversify into the 45 distinct types present in the adult (Shekhar et al., 2022)? Do different RGC types vary in their resilience or vulnerability to injury, and can we use those difference to improve the resilience of vulnerable types (Tran et al., 2019; Jacobi et al., 2022)? What are the cell types in the retinas of humans and nonhuman primates, and how do they differ between the peripheral retina and the fovea, which mediates our high-acuity vision (Peng et al., 2019; Yan et al., 2020b)? Can we use atlases from multiple species to learn something about the evolution and conservation of cell types (e.g., Yamagata et al., 2021; Kölsch et al., 2021). Answering these questions will take longer than my lab will be open, but fortunately, several of the key figures in these studies now have their own groups—Yirong Peng at UCLA, Karthik Shekhar at UC Berkeley, and Nick Tran at Baylor—where they each are taking this work in exciting new directions.

Sabbaticals

I am always surprised that so few tenured faculty members take sabbaticals. I have had six, each a valuable experience scientifically and an exciting adventure personally. I described the first, at Institut Pasteur, previously. Here are the others:

Cal Tech: 1993

For the first few years of our collaboration, John Merlie supervised all experiments that involved recombinant DNA, but as time passed, most of the students and postdocs in my lab incorporated molecular methods into their projects. This started to get embarrassing for me, because I had no practical experience in any of these techniques. I therefore arranged to spend several months at Cal Tech learning molecular biology, under the auspices of a Wiersma Professorship, which provided our family with a house near campus. I wanted to find a lab directed by someone senior enough to be a wise mentor but junior enough to be in town most of the time. Kai Zinn, a fly geneticist who had worked with Tom Maniatis and Corey Goodman, fulfilled both criteria—and turned out to be a patient teacher as well.

By 1993, several genes encoding homophilic neural recognition molecules (i.e., ones that bind to their own kind) important for neural connectivity had been identified, cloned, and sequenced in both flies and mice. Orthologs had been postulated, but their sequence similarity was decidedly unimpressive. My idea was to generate recombinant proteins from both species and ask whether they bound to each other. If they did, it would not only be interesting but also provide a new way to find mammalian cell recognition molecules.

With Kai’s close guidance, I set out to clone extracellular binding domains, insert them into expression vectors, purify recombinant proteins, and assess binding by a phage display assay. This all worked, but I ran out of time. New Year’s came, it was time to return to St. Louis, and attempts to interest students in my lab in continuing the work failed. I still think these would be experiments worth doing, although their value declined once genome sequence became available. Nonetheless, the sabbatical was a success in that I came back with enough hands-on experience to have some modest credibility in directing molecular projects in my own lab.

University of the Mediterranean at Luminy: 1999.

I met Chris Henderson during my sabbatical at the Institut Pasteur. He had been a postdoctoral fellow with Jean-Pierre Changeux, then stayed on briefly as a junior faculty member before moving south to Montpelier and then east to Luminy, a campus set close to the Mediterranean in a particularly beautiful part of Provence. Chris’s interest in trophic support of motoneurons and mechanisms underlying motoneuron diseases (especially amyotrophic lateral sclerosis) overlapped mine in neuromuscular development, so we saw each other at various meetings over the years and remained friends. In thinking about a next sabbatical, the idea of learning his methods for purifying, culturing, and studying motoneurons was appealing. The location was not an insignificant factor.

The project I chose was to see if I could generate cell lines from embryonic mouse motoneurons, with the aim of eventually being able to study those from the mutants we had generated. Today, the obvious strategy would be to edit the genome of stem cells and then direct their differentiation into motoneurons, but that wasn’t an option at the time, so I used a method of hybridizing embryonic motoneurons to immortalized lines. I achieved some success, and we made use of a few of the lines later, although the development of these better methods soon made mine obsolete. It was a happy time, though: I learned a lot, had the first chance to work with my own hands since Merlie’s death, and absolutely loved Provence—especially the charming, small village of Cassis where we (and Chris) lived. We’ve gone back to that area for vacations every few years since.

Cambridge University: 2008

By 2008, the lab was largely devoted to studies of circuit formation in retina. I was in an embarrassing position not dissimilar to the one that had motivated my sabbatical at Cal Tech: I was directing a program on a topic about which I had inadequate knowledge. When the possibility arose of spending a term in Cambridge as a Visiting Fellow Commoner at Trinity College, I decided to use it as an opportunity learn about the retina. I was loosely affiliated with the conjoined laboratories of Bill Harris and Christine Holt, both of whom were leaders in the area—Bill primarily using zebrafish to study retinal lineage and Christine primarily using Xenopus to study retinotectal pathfinding. I attended their lab meetings each week but, unlike my stay at Cal Tech, I did not work in the lab. Instead, I visited with local experts (e.g., Leon Lagnado and Horace Barlow) and immersed myself in the relevant literature. As a way to enforce some discipline, I took on authorship of two substantial reviews, one with Masa Yamagata on synaptic specificity (Sanes and Yamagata, 2009) and the other with Larry Zipursky, comparing the visual systems of insects and vertebrates (Sanes and Zipursky, 2010).

Biogen: 2013

My next sabbatical was spent at Biogen, one of the world’s oldest and largest biotech companies. Biogen is less than a half mile from our home in Cambridge, but it was in many ways a different world. Spyros Artavanis-Tsakonas, an eminent Drosophila geneticist with a long-time involvement in the world of biotechnology, had been recruited to be chief scientific officer a few years earlier. Biogen had a distinguished history: two of its founders, Phil Sharp and Walter Gilbert, were Nobel laureates, and another founder, Charles Weissmann, cloned interferon-alpha, initiating the company’s long dominance in therapies for multiple sclerosis. However, the tradition of basic research (called “early discovery research” in biotech) had waned over the years, and Spyros’s mission was to revive it. To this end, he tried to hire scientists from academia in a variety of capacities. I had become friends with Spyros, an extraordinarily gregarious individual, on an NIH study section many years earlier, so I was one of the many he called. Learning that I was not interested in moving from academia, he invited me to test the waters by spending a sabbatical semester there. It was an intriguing idea.

I began by proposing a program to use the retina as a model for testing mechanisms, targets, and therapeutics for neurological diseases. There was a lot of enthusiasm for the plan at first, but Biogen is a business, and soon their interest moved from using the retina for research to finding therapies for retinal diseases. Since then, I have served as a consultant helping to get that program launched. This has involved developing strategic plans, reviewing smaller biotech companies that are eager to partner with or be acquired by a larger entity, and helping to hire people who actually knew what they were doing. Most recently, because of an administrative reorganization, I ended up as interim head of ophthalmology research—an appointment that was meant to last a few months but, thanks to COVID, ended up lasting over a year.

It has been both a fascinating and a frustrating experience. I had minimal previous contact with the worlds of biotech or clinical medicine, so there was a lot to learn and I enjoyed learning it. I am disappointed, however, that Biogen slowly abandoned Spyros’s goal (and that of the then-CEO George Scangos) of inventing new therapies, and instead retreated to a strategy based largely on buying ideas, reagents, methods, and drugs from other companies. In short, there has been nothing to make me second-guess my decision to stick with academia.

New York University Medical Center: 2017

Steve Burden, who I mentioned earlier, had remained a good friend for some 40 years. Through him, I got to know his partner, Ruth Lehmann, who studies germ and stem cell development in Drosophila. Steve and Ruth had moved from MIT to the Skirball Institute at NYU (now, thanks to vigorous fundraising, the NYU Skirball Institute in the NYU Grossman School of Medicine at NYU Langone Health Center) many years earlier, as directors of their neuroscience and developmental biology programs, respectively; Ruth later became director of the entire Skirball Institute as well as chair of cell biology.

Steve and Ruth arranged for Susan and me to spend the fall of 2017 at NYU, and Ruth even managed to get us a studio apartment in a building on the health sciences campus. It ended up being a semester of very long weekends: we’d take the train from Boston to New York on Thursday evening, and return to Boston of Tuesday evening. Thus, I could spend Wednesday and Thursday in my lab at Harvard (with Susan spending those days at the legal services center where she was then working), work from New York the other three weekdays, and explore the city on the weekends.

My main neuroscience-related activity in New York was to revise chapters for the next edition of Principles of Neural Science. The first edition of this textbook was written by Eric Kandel (see Volume 9) and James Schwartz in 1980. Tom Jessell, who joined the team for the third edition, asked me to coauthor the section on neural development for the fourth edition, which appeared in 2000. This was a massive task, because the field had advanced radically over the preceding decade, and we undertook to completely reimagine and rewrite the section. We wrote what was essentially a short book inside a long book. In fact, we contacted the publisher to ask whether they would consider letting us expand the section modestly (dividing each jam-packed somewhat telegraphic chapter into two slightly more discursive ones) and turn it into a stand-alone volume. They refused. By the time of the fifth edition (2012), Tom had taken on leadership responsibilities at Columbia and what had been a shared burden fell mostly on me. For the sixth edition, Tom faced serious health and other problems, so I ended up doing it entirely myself. By the time the edition finally appeared in 2021, Tom had passed away. With his death, that of Schwartz much earlier, that of the artistic director, Sarah Mack, in 2020, and the decreased involvement of Eric Kandel, my guess is that the sixth edition will be the last.

Family

The reader may wonder whether I abandoned my family during these adventures. In fact, things worked out well. Susan knew she was pregnant before we went to Paris and enjoyed the opportunity to slow down and appreciate the process. Her employer in St. Louis (Planned Parenthood) gave her a leave of absence; given their name, how could they not? Jesse, our son, was born in April and we were able to appreciate his first few months and host his grandparents before returning to “real life.”

Seven years later, Susan was able to use our semester in Cal Tech as a pretext to extricate herself from an administrative job (not at Planned Parenthood) that she had outgrown and accept a new position. Jesse, now joined by Milla, were 7 and 5. We enrolled them in the local school, in which most of the students were working class and foreign born. Their exposure to people they would not have seen in their excellent but liberal/yuppie St. Louis school was a valuable experience.

By the time we went to Cassis, Susan had decided to go to law school. She studied hard, aced the LSATs, and was admitted. Time in Cassis gave her a chance to take a breather, get ready for the rigorous three years ahead, and leave her current position sooner than would have been politic otherwise. As to school, we found out what our children would need to do to return without penalty and made sure they did it. We had asked them to try out the local schools but gave them the option to stay home if things didn’t work out. In fact, both thrived, made friends, became fairly fluent in French, and came home with the desire to visit again, which both have done.

Final Thoughts

Mentors and Collaborators

A majority of the authors in this history series owe much of their success to the students and fellows who worked in their laboratories and actually carried out the experiments for which their mentors got the credit. I am certainly in that category. I’ve tried to acknowledge their contributions to the work I described, and I remain proud by proxy of their subsequent achievements. It would be impolitic to name those to whom I felt closest or who went on to greatest glory—but they know who they are.

What I’ve also tried to do is emphasize the importance of mentors and collaborators, perhaps less generally acknowledged. I had mentors every step of the way: Robert Guthrie in high school, Paul Greengard in college, John Hildebrand in graduate school, Jack McMahan and Zach Hall during postdoctoral study, and Dale Purves when I was a junior faculty member. I don’t know where I’d be without their wisdom, guidance, and forbearance—but I’m sure I would not be writing this chapter.

Likewise, as important as students to me (heretical though it may seem) were true collaborators: faculty in other laboratories. Here, I’m thinking of those with whom there was a relationship that extended beyond a single project or paper: Ann Marie Craig, John Lawrence, Jeff Lichtman, John Majors, John Merlie, Alan Pearlman, and Rachel Wong at Wash U; Zhigang He, Markus Meister, Aviv Regev, and Larry Zipursky since moving to Harvard. Of these, I’d single out John Merlie, Jeff Lichtman, and Markus Meister as having particularly profound effects on the direction of our work.

Style

It is the height of hubris to talk about a scientific style, but as I look back, I see that we have in most cases tried to answer a question rather than exploit a specific method. In service of answers, I was perfectly happy to play histologist to Merlie the molecular biologist, molecular biologist to Lichtman the histologist, and geneticist to Meister the physiologist. The advantage of this approach is obvious, but it does have disadvantages. We never became true experts in any single method, nor did we have simple projects that would allow undergraduates or beginning graduate students to “turn the crank.” We were never able to knock at the gates of Hershey Heaven, the vision of scientific joy enunciated by the pioneer molecular biologist, Alfred Hershey: “to have one experiment that works and keep doing it all the time.” And I do think failure is more common with this approach than with a methods-driven one—see how many times I’ve used that word or its relatives.

A few months ago, I heard a related idea put forward by Ardem Patapoutian, who won the Nobel Prize in 2021. At the end of a (virtual) seminar I attended, he had a single “advice for young scientists” slide. It showed a Venn diagram in which one circle labeled “big questions” overlapped only slightly with another circle labeled “enabling technology.” He advised ambitious neuroscientists to focus their attention on the overlap. This captured my view perfectly. There are some questions I would love to have addressed, such as what causes schizophrenia or what is consciousness—but there was no way to approach them in a way I thought was rigorous. On the other side, there were no end of questions we could have answered with the methods we had—but I couldn’t make myself care about many of them. I tried to stick to the overlap.

Next Step

Some of my friends in the world of science wanted to work until their last day on earth—notably John Merlie and Paul Greengard, both of whom got their wish. Not me. Neurobiology research has always been the most fascinating activity I could think of, but never the only one. I believe that for most scientists, it’s a good idea to get out of the way at some point, making room for younger people with more energy, more passion, new technological expertise, and better ideas. When I began telling friends several years ago that I planned to phase out my lab by my early seventies, the invariable response was, but what will you do? The answer is, I don’t know. Having viewed myself primarily as a neurobiologist for more than a half-century, I’m curious to find out what it feels like to not be a neurobiologist. This is scary to be sure, but quite exciting.

Selected Bibliography

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Gray GE, Sanes JR. 1992. Lineage of radial glia in the chicken optic tectum. Development 114: 271–83.

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Hunter DD, Shah V, Merlie JP, Sanes JR. 1989b. A laminin-like adhesive protein concentrated in the synaptic cleft of the neuromuscular junction. Nature 338: 229–34.

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Jacobi A, Tran NM, Yan W, Benhar I, Tian F, Schaffer R, He Z, Sanes JR. 2022. Overlapping transcriptional programs promote survival and axonal regeneration of injured retinal ganglion cells. Neuron, in press.

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Kay JN, Voinescu PE, Chu MW, Sanes JR. 2011b. Neurod6 expression defines new retinal amacrine cell subtypes and regulates their fate. Nat Neurosci 14: 965–72.

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Kim IJ, Zhang Y, Yamagata M, Meister M, Sanes JR. 2008. Molecular identification of a retinal cell type that responds to upward motion. Nature 452: 478–82.

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Kishi M, Pan YA, Crump JG, Sanes JR. 2005b. Mammalian SAD kinases are required for neuronal polarization. Science 307: 929–32.

Kölsch Y, Hahn J, Sappington A, Stemmer M, Fernandes AM, et al. 2021. Molecular classification of zebrafish retinal ganglion cells links genes to cell types to behavior. Neuron 109: 645–62.e9.

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Misgeld T, Kummer TT, Lichtman JW, Sanes JR. 2005. Agrin promotes synaptic differentiation by counteracting an inhibitory effect of neurotransmitter. Proc Natl Acad Sci USA 102: 11088–93.

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Moscoso LM, Merlie JP, Sanes JR. 1995. N-CAM, 43K-rapsyn, and S-laminin mRNAs are concentrated at synaptic sites in muscle fibers. Mol Cell Neurosci 6: 80–9.

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Wolfram Schultz

BORN:

Meissen, Germany

August 27, 1944

EDUCATION:

Dr. Med. University of Heidelberg, Germany, 1973

Habilitation (PhD) in Neurophysiology, University Fribourg, Switzerland, 1981

APPOINTMENTS:

Postdoc, MPI Biophysical Chemistry, Göttingen, Germany (1973)

Postdoc, State University of New York at Buffalo, United States (1975)

Postdoc, Karolinska Institute, Stockholm, Sweden (1976)

Lecturer–Professor Neurophysiology, University Fribourg, Switzerland (1977–2001)

Wellcome Trust Principal Res Fellow, University Cambridge, United Kingdom (2001–present)

Professor of Neuroscience, University Cambridge, United Kingdom (2002–present)

Visiting Professor, Tamagawa University, Japan (2002)

Fellow, Churchill College, Cambridge, United Kingdom (2004–present)

Visiting Associate, California Institute of Technology, United States (2004–present)

Visiting Professor, Tohoku University, Sendai, Japan (2018)

HONORS AND AWARDS (SELECTED):

Ellermann Prize, Switzerland (1984)

Brooks International Lecture, Department of Neurobiology, Harvard University (1993)

Theodore-Ott-Prize, Switzerland (shared) (1997)

Golden Brain Award (2002)

Pfizer Lecture, Society of Neuroscience, San Diego (2004)

Ipsen Prize for Neuronal Plasticity (shared) (2005)

Fellow, Royal Society, United Kingdom (2009)

European Journal of Neuroscience Award—FENS Award (2010)

Kavli Presidential Lecture, Society of Neuroeconomics (2010)

Zülch Prize, Germany (shared) (2013)

Brenda Milner Lecture, Montreal Neurological Institute (2014)

Qi Zhen Global Lecture, Zhejiang Univ, Hangzhou, China (2014)

EBBS/Behavioral Research Prize Lecture, FENS Forum, Copenhagen (2016)

Plenary Lecture, 39th Annual Meeting, Japan Neuroscience Society, Yokohama (2016)

Brain Prize (shared) (2017)

Caltech Chen Distinguished Lecture (2017)

Erlanger Distinguished Lecture, San Diego (2018)

Volker Henn Lecture, University of Zürich (2018)

Gruber Prize (shared) (2018)

Waelsch Lecture, Columbia University (2018)

Karl Spencer Lashley Award (2019)

AV Hill Lecture, Cambridge, United Kingdom (2021)

Wolfram Schultz has combined behavioral and neurobiological techniques to investigate the neuronal mechanisms of reward processing and economic decision-making. He has used behavioral concepts from animal learning theory and economic decision theories to study individual neurons and global activity of the dopamine system, striatum, orbitofrontal cortex, and amygdala. He and his group discovered the dopamine reward prediction error signal that indicates how much a reward is better or worse than predicted. They also described other reward, risk, and decision signals in the striatum, frontal cortex, and amygdala.

Wolfram Schultz

Reward is in my genes. My maternal great-grandfather Carl A. Beegen demonstrated this fact by running a wine shop and spirit distillery in Meissen, Germany, many decades before I was born there. The genes were passed on to his son, my maternal grandfather, who took over the booze shop. He initially emigrated to the United States on the occasion of the 1904 World Fair in St. Louis. But his mother could not accept the absence of her beloved son and lured him back by claiming a serious health scare. When that health scare turned out to be a fake, my grandfather became unhappy. But by now he had met a woman with whom he ultimately fathered 12 children, only six of whom survived their first year, and one of whom became the mother of my younger brother and me. But my grandfather’s new wife would in no way emigrate. So, given the fake health scare, my grandfather coerced his father into selling him the shop for the money he had made in St. Louis. He then built a flourishing business, importing wine by the barrel on ships and river barges from Bordeaux all the way to Meissen, which borders the Elbe River. He also distilled spirits in the basement of the shop for his own brands, all legal. He was very social, had a small tasting corner in his shop, and offered free wine to the many unemployed workers in 1920s and 1930s Germany. My paternal grandfather was slightly different. He was a colonel and also into reward but not the liquid variety. He married a much younger woman who also happened to be the daughter of a Royal Saxonian Major General whose hunting ground was next to that of Frederick Augustus III, King of Saxony (i.e., before the monarchy was toppled in 1918). The two noble gentlemen occasionally shot bucks on the other’s range and then moved them slightly over into their own territory. With his young wife, the colonel fathered half a dozen children most of whom survived beyond childhood. One of them was my father, Robert.

Ultimately, I was born in 1944, also in Meissen. At the time, my father had followed the footsteps of his military pedigree and was captain of the infantry and was mostly stationed in Russia during the Second World War. He was caught by the Russians at the end of the war in Germany, transported back to Russia, interned in Siberia, and sent back home in 1946 because his abdominal scar from a skin transplant for a lost finger was mistaken as a potentially troubling stomach problem. He joined his wife Herta, my mother, said hello to me, and fathered my brother who was born in 1947 and is now a retired physics professor and former head of a large metal physics research laboratory at the University of Dresden. My father had been trained before the war as an accountant with Siemens, the big electro company. I inherited his attention to detail and perseveration, which served me well in my later laboratory life. My mother, encouraged by her socially responsible, wine- and booze-selling father, was one of the few female students of German, English, and history at the Universities of Leipzig and Vienna; there she occasionally heard about gender discrimination but was overall happy. She became a high school teacher before and during the war but afterward was a housewife to raise my brother and me. She was surely a lost talent, and very supportive, but staying home for a married woman with children but without servants was necessary at a time when cars, supermarkets, central heating, washing machines, and refrigerators did not exist or were unaffordable.

My parents were fed up with having another dictatorship and with the poor educational prospects of their non-proletarian offspring in communist Eastern Germany and moved to Western Germany. So, in October 1950, I was thrown over a two-meter-high fence into West Germany at a moment when the armed fence patrol had reliably passed on their hourly rounds. There was mention of border guards having shot dead another refugee a couple days earlier, but we were not deterred. We made it without problems, like thousands of others, took a train to a small town in northern Bavaria (Bad Neustadt) and settled where my father had been hired by his former boss in his former company, Siemens.

Getting Started

Early Learning

I was neither a great pupil nor a proficient high school student. The stuff was just too much routine and boring, and my performance was borderline. My mother, as a former high school teacher, was shocked but nevertheless installed in me a desire for academic work. When I was in danger of not surviving the final high school exam required for university, she persuaded me to think further and continue the boring school. To address my weakest point, she arranged for instructions in Latin from a private and expensive teacher. Her leverage was my interest in becoming an automotive engineer, a popular job in car-crazy Germany, which at the time required a university education and thus a high school diploma, and for that I needed a pass in Latin. At the final exam, after eight years of studying the language, the examiners let me succeed after I had promised to not become a Latin teacher; they were sympathetic but did not want to compromise their teaching reputation. I soon lost interest in car engineering, but I am eternally grateful to my mother for having kept me on a path that kept all education options open; she had no easy job. While my father often expressed shock about my undisciplined behavior, he was liberal enough to let me explore the world instead of doing boring homework (he himself had not been a great high school student, so that is also in my genes).

Indeed, the German high school at the time was fantastic, because it ended at noon or shortly thereafter. If you could escape homework without completely blowing school, you could live a decent life full of self-driven activity and exploration. I found plenty of attractions on the sports grounds, where I learned persistence in the face of failure: I tried very hard, for several years, to run the 100-meter dash faster than 12.5 seconds but never succeeded. My biggest sports achievement was to take second place in the 200-meter breaststroke in a regional swimming championship, which eliminated another potential career, that of sports teacher. But the benefit was that I became resilient and enduring. Learning to do something for its own sake, and trying hard, irrespective of expected glory, helped me throughout my life to undertake seemingly useless tasks that at some point might pay off.

In the winter, I was attracted to metal constructions after we had found an abandoned and incomplete but large Märklin metal construction set. I constantly was constructing something, including a huge tower with blinking lights that were visible through the window from the street. On visits to my grandparents, who had remained in Eastern Germany, my grandfather bought me and my brother model trains that, unfortunately, were of the poor quality of the time and hardly ever worked (we never had money to buy the more reliable Märklin trains). They required constant mechanical and electric adjustments, which taught me mechanics, electricity and, maybe most important, to never give up. Ultimately, I succeeded in constructing an operation of several trains running automatically in parallel and alternatingly (when they worked).

I never got rid of my obsession with exploration and novelty-seeking, and I kept avoiding repetitive tasks with prescribed procedures, unnecessary detail, and slow progress. I was good in math, which was easy, but I lost interest because of its repetitive and procedural nature. When my math grades declined, I worked on math for a few days, and my mother was worried about my monomaniac insistence; I lost interest again when my marks had recovered. Except for sports, basically all other disciplines bored me. Despite all this jittering, I could focus very well, with great persistence, when I liked tasks that were interesting and captivating, like the eternal tinkering with the never-working model trains, and I would not stop until I was content with the outcome. I understood later that the combination of these behaviors may inadvertently expose one to rare events and lead to novel experiences. It can be formalized as exploration and positive skewness seeking. The epsilon-greedy exploration model of reinforcement learning of Sutton and Barto (1989) might apply; rather than always seeking the best-known option, a small percentage (epsilon) of choices are made randomly, which occasionally leads to unpredicted and unknown outcomes that are much better than the best-known option and are worth the many failures. Anyway, that was my not-so-great school life until I ran into the idea to do a year of high school exchange.

A Healthy Interruption

After the war, various organizations brought German high school students for a year to the United States. For me, this was another opportunity to experience the unknown and nonrepetitive. I was selected by Youth for Understanding, for which Ulrich Irmer, the later German and European parliamentarian, did the screening in the American Cultural Mission (Amerika Haus) in Munich and in his own, slightly bohemian student flat. Thus, I left Europe in the summer of 1962 on a four-engine Super Constellation propeller plane from the Flying Tiger charter line (reportedly owned by the CIA). We flew from Amsterdam for almost 24 hours to Detroit, with intermediate landings in Ireland and Newfoundland. I entered a welcoming family of a gas station owner (the father) and special education teacher (the mother) in Mayville in the “thumb” of Michigan. The father was very practical and had built, with his own hands, six or seven private houses, his own included, and the mother with her patience and knowledge was very reassuring. This was real life; a small village with a main street that was deserted during the day because everybody was working, a run during the morning school break to the dime store for sweets, work at the Mobile gas station on Wednesday afternoons and all day Saturday, and the oldest daughter married to a dairy farmer (what a nice guy, actually both of them). A number of lakes nearby allowed me to practice water skiing in the summer and to go ice skating and watch motorcycle races on ice in the winter. I also enjoyed American football and track, although with my usual moderate success in sports.

The U.S. experience provided quite a contrast to postwar Germany with its painful recovery from misleading demagogic warfare and industry-level murder. But what amazed me most, and would become the most lasting influence, was the unpresumptive attitude and openness. By attending the local Methodist church with my host family, while not being religious at all, I met the preacher, Reverend Bill Lutz, and struck a great friendship. We discussed the state of the world on many Sunday evenings, starting with his sermon and widening into the history of the United States and the benefits of democracy. It was just the right moment, when everybody was optimistic, everything seemed possible if you had enough initiative, and no political murders had yet taken place. Reverend Lutz’s broad intellect and profound knowledge, in the middle of the countryside, was impressive. At the end of the school year, he suggested that I take a trip through the United States. He picked up the phone a few times, called up fellow Methodist and Presbyterian preachers throughout the west and south of the country, and arranged a tour for me through Wyoming, Oklahoma, California, Arizona, Texas, and Louisiana, using a Continental Trailways bus ticket one of my German uncles had donated. Afterward, I went back to my old high school in Germany and finished the last year in 1964 with new motivation and moderate success.

Another Healthy Interruption

With my broad and unfocussed interests, I had no idea about a future profession. The fresh high school diploma and my mother’s guidance toward academia both suggested that I should attend university. But what discipline? The decision was not imminent because of mandatory military service (which was only abandoned many years later by Angela Merkel’s government). The standard duration was 18 months, but I received a regular salary for doing 24 months that could supplement my future government studentship. The military produced another welcome delay for my professional indecision. I passed a comprehensive entrance exam with more points than anyone else had scored in the past several years and then was thoroughly interviewed by a suspicious psychologist. I could only explain that my performance might be due to my extracurricular interests, which convinced him that I was not nuts. Thus, I went for 24 months to the Air Force. Rather than flying, I served in the ground troops because of the short duration and my less-than-perfect eyesight. I was stationed in the Netherlands (on a NATO base), Munich, northern Germany and Bavaria again, regions I had not known before. I found that the boring military routine removed the idiosyncratic routines, unconscious prejudices, and reduced motivation that I had acquired in high school. The biggest benefit of all was that I met a lovely, and ultimately irresistible, girl in a club in Munich in 1966; she had cancelled a midsummer party in the Bavarian mountains because of rain and thus had run unintentionally into me in that club. Gerda was to become my wife a few years later.

University Studies

My interest in careers spanned from sports teacher (which is nice because of the exercise in fresh air and swimming pools but not realistic given my mediocre sports performance) through foreign diplomatic service (compatible with my desired international living but incompatible with my lack of patience in the face of malfunction) to law (more serious and promising, as I had done an excellent job in the law course at the German Air Force Officer’s Training School). I also annoyed my parents and everyone else by proposing to become a politician, which was a true joke, almost surreal, and incompatible with my dislike for slow progress. I sometimes shudder when thinking how these options could have gone wrong. At the end, I decided on medicine, as some relatives were medical doctors, and as I wanted a job with a large spectrum of possibilities. I found biology interesting and wanted to help people; however, I was not quite so sure, as I had been public enemy number one for my high school biology teacher, who was back from an American two-year postwar de-Nazification camp and still showed remnant behaviors. And of course, medical school required excellent school grades. But luck does exist. On one of my last trips home from the military, I had bought a newspaper and read that the University of Hamburg was crediting former soldiers one half mark for every half year of service, because the military had helped the city of Hamburg a few years earlier to recover from a surge of the North Sea and a massive flooding from the Elbe River. With my two years of service, I seemed to be eligible, and that opportunity finally made the decision for me. I was accepted by Hamburg University on the second reserve list two weeks before the term started in the fall of 1966.

The two-year military service had left my brain drained. I was even more bored in the military than in high school and was longing for something truly useful and engaging to do.

Thus, at Hamburg University, I soaked up, frantically, like a dry sponge, any knowledge and ideas I could get hold of. Others talked about parties and girls, but I was already busy with Gerda in the early stages of becoming her future husband. I attended every possible and impossible lecture in medicine, and some in psychology and philosophy, in particular the small ones with good interactions with the lecturer. I just could not get enough. I went to Carl-Friedrich von Weizsäcker’s lectures on Friday evening at the end of a busy student week. He read Kant’s Critique of Pure Reason in an overflowing auditorium of 2,000 students sitting everywhere. This was a cultural, and partly morally redeeming, event; Weizsäcker had been a nuclear scientist who had reportedly delayed his work on nuclear fission to avoid building an atomic bomb for Hitler. After the war, he had become a philosopher. His Kant lectures taught me to question dogmatic beliefs and the limits of knowledge as a prelude to Horkheimer and Adorno’s critical theory, which I encountered later in Heidelberg.

I found the fundamental biology in undergraduate medicine absolutely wonderful. It was so easy to understand. Almost everything makes perfect sense, falls into place naturally, and works the way it should, as if it had been tuned to optimal function. I lived in a nice student housing and had many discussions that helped us bring everything down to the essentials, including physics that I had forgotten from high school and, most interestingly, physiology that was just beautiful if you could boil it down to the essential laws. As Einstein says, “if you cannot explain it in simple words, you have not understood it well enough.” My teachers and fellow students had understood it. Then my “career” in neuroscience began in 1968 when I asked Professor Duncker about working in his neurophysiology laboratory. He immediately suggested that I do my medical thesis, which is like a master’s for medics but conveys the title of medical doctor and allows one to do research without a further doctorate. While exploring my possibilities, I also had tried a lab stunt in the biochemistry department of the natural sciences faculty, but medical students were considered inferior to natural science students, and the lecturer told me to come back when I had done my formal lecture course (which turned out to be super simple and all written out clearly in an excellent textbook). Thus, I became a neurophysiologist and not a molecular biologist.

After my medical undergraduate exam, I wanted to move to a less sober environment and went to cozy Heidelberg. It was the only university, apart from Hamburg, that would accept my high school grades for clinical medical studies. The city is absolutely delightful, a classic center of poetry, literature, and philosophy, like the German Romanticists around Hölderlin. There is the philosopher’s path on the mountain overlooking the Neckar River, a pretty old town (infested by tourists but beautiful on rainy days when nobody is around), and a classic university that later turned out to be partnered with Cambridge on the basis of its many similarities. Besides my clinical medicine, I enrolled in undergraduate mathematics because I figured that the brain is so complex that math could provide reductionist formalizations, and I attended a few select philosophy lectures in the old town. There I met Harald Pilot who was a senior philosophy student and who served as a tutor for younger students like me. His simple and profound explanations of the philosophy of science and critical theory, which continued informally in the pub after his evening sessions, left a profound desire to search for unifying concepts underlying the complexity of nature. Without him and the Heidelberg philosophy environment, I probably would not have used the formal theories of economics for 20 years in my neurophysiology research in Cambridge. I also learned from these philosophy seminars how to run a contemplative discussion that mixes formal issues with exploration and allows one to pursue unexplored arguments and to make mistakes, which can be quite fruitful. These experiences became very helpful for the interactions in my later research group and prevented me from running sterile report-to-the professor meetings. I was also somewhat engaged in the political student activities of the time where we read volume 1 of Karl Marx’s Capital, learned to doubt the unmerited authority of people and concepts, and used critical theory to search for the big picture, to get to the bottom of problems rather than simply understanding and superficially fixing them, and to question and reflect on the usefulness, ethics, and wider implications of our actions, all of which helped me avoid many unnecessary steps in life and research. Studying medicine in Heidelberg was also a cultural enterprise; I was attracted by the small evening lectures of Professor Tellenbach who showed us the relationship between psychiatric disease, culture, and history. It was a truly great time! Of course, things go well when one is in resonance with the world: I did the best undergraduate medical exam in Hamburg and the second best in Heidelberg out of several hundred students; I never realized the value of it until I saw applicants to my lab boasting about such marks. In the end, I did a practical year in which I spent several months in neurosurgery and neurology and found the textbook by the renowned neuropsychologist Klaus Poeck to be very stimulating and beautifully written. I wrote long and detailed patient reports after hours of examination and was close to becoming a neurologist and neuropsychologist myself. But I wanted to try neurophysiology first.

Postdoc Life

Without much connection to real science, I asked fellow Heidelberg students with lab experience for advice and was pointed to Otto Creutzfeldt, the son of the neurologist who defined Creutzfeldt-Jakob disease. Otto Creutzfeldt had done pioneering intracellular recordings in the visual cortex at a Max-Planck Institute in Munich and was in the process of moving to a new MPI in Göttingen. During a brief visit with him in Munich, he asked how many years I had taken to study full medicine and undergraduate math (I took only the minimum time required for medicine and did math in parallel), and he accepted me as a postdoc in his new department. I was amazed by his neuroscience perspective and his international horizon. He had a whole Max-Planck department in a new building overlooking the city and a dozen groups working on all forms of sensory processing—very up to date. I worked with Christian Hellweg between 1973 and 1975 on intracellular (whole cell) recordings in the cat somatosensory cortex and natural mechanical vibrissae stimulation. To learn such a sophisticated technique, I pestered colleagues in the lab until it worked and am much indebted to Klaus Albus and Uli Kuhnt for their tolerance of this impatient newcomer. In the course, Sir John Eccles visited and was looking for a postdoc for his lab in Buffalo. Creutzfeldt suggested me, we talked, and off we went to Buffalo with Gerda and our one-year-old son Johannes in the snowy spring of 1975.

In Eccles’ lab in Buffalo, I worked with Gary Allen on connections between the cortex and the cerebellum in anesthetized monkeys, using electrical stimulation of the motor cortex and single-neuron recordings in the dentate nucleus of the cerebellum. This was connectionist work very similar to current experiments that use optogenetic stimulation, except that we had no idea about the transmitter identity of the stimulated cortical and recorded cerebellar neurons. But the results showed that the anatomically demonstrated pathway from cortex via pons and inferior olive was functional. Gary had bought a primate chair for trying out experiments on awake behaving monkeys but never got around to using them because he became a Christian missionary. The monkey chair together with the ensuing discussions stimulated my interest in this elegant work. Watching single neurons in real time while a monkey performs in a controlled behavioral task seemed the most direct and precise way to watch the brain in action. I did a further experiment on the cerebellum with Erwin Montgomery in which we stimulated the cerebellar nuclei and investigated the resulting muscle contractions in anesthetized monkeys. We discovered an output pathway from the dentate nucleus (which was a part of the neocerebellum and primarily linked to the cortex) that bypassed the cortex and went through the brainstem, suggesting an evolutionary ancient cerebellar output to behavior. Eccles closed his lab during this period, and I needed to find another postdoctoral opportunity before starting my own research. Having experienced a wonderful introduction into classical neurophysiology, I was attracted to the new field of neuropharmacology that defined brain function on the basis of neurotransmitters instead of electrical brain activity. I contacted Urban Ungerstedt in Stockholm, who obtained a fellowship for me to introduce neurophysiology into his lab, and that is how I got acquainted with the dopamine system.

We arrived with Gerda, Johannes, and our few weeks’ old son Thomas on a sunny winter morning in 1976 in beautiful and serene Stockholm. I conducted a few straightforward neurophysiology experiments in Urban’s lab that would complement his pioneering histological and behavioral work on rats. The neurophysiology of dopamine neurons was just starting to be explored, and its sophistication did not compare to that of the well-developed motor control field. I recorded from striatal neurons in rats whose dopamine system had been lesioned at different time points. The striatal neurons showed elevated activity a few days after the lesion but no longer a year later. The literature was very limited, and I was able to follow the complete literature on dopamine electrophysiology, anatomy, and a few extra things, like neuronal plasticity and adaptation. This seems unbelievable now when interesting paper after interesting paper appears each week, and it is quite impossible to keep up with fields outside one’s own perspective. In any case, Urban and his Stockholm lab gave me the opportunity to get fully acquainted with the state of the dopamine field at the time and thus provided me with an excellent start for one of my major scientific avenues.

The Dopamine Reward Signal

My Plans

With my Stockholm introduction into neurophysiology based on neurotransmitters rather than anatomy, I wanted to start my own work and moved in 1977 into a junior faculty position at the university in the lovely city of Fribourg in Switzerland. From my medical studies, boosted by the short stunt in neurology, I was familiar with Parkinson’s disease that arises from the degeneration of midbrain dopamine neurons. From my postdoctoral stay with Gary Allen in Eccles’ Buffalo laboratory I was keen to enter motor neurophysiology and monitor single neurons while monkeys performed well-controlled movements. This research interest had been boosted by Eccles’ almost daily lunch discussions about Free Will in the cafeteria of the Amherst campus at Buffalo, where he emphasized the importance of studying self-initiated movements in which free will might be expressed in an objectively measurable way. With the cortex-centered view of neuroscience at the time, Eccles’ focus was not on dopamine neurons but on the supplementary motor area (SMA) where he thought such neuronal activity should first arise, and Ben Libet’s readiness potential in humans supported this notion (1978). But deficits in spontaneously initiating movements also constituted a substantial symptom of Parkinson’s disease. To address these aspects in combination, I thought to first establish recordings from single dopamine neurons in awake behaving monkeys and to see how their activity might be related to the Parkinson’s deficits; then I thought to take a stab at the SMA to find activity during spontaneous movement initiation.

However, trying to link dopamine signals to specific behaviors was not without risk. Sure, the function of dopamine neurons in movement was much less known than that of motor cortex, cerebellum, and much of the basal ganglia, and any data in this respect would be interesting. But Parkinson’s patients got better by administration of dopamine drugs that cannot replicate subsecond neuronal changes. So many knowledgeable people reasoned that dopamine was simply a chemical that was necessary to prevent Parkinsonian symptoms but might not show a relationship to specific behaviors. It might be a necessary ingredient of the brain’s chemistry, something like a “rain on the brain” or a “fuel” that was necessary for other brain structures to control behavior, a neuromodulator that does nothing on its own and only enables other processes.

Building My Own Lab

To get started in Fribourg, I visited Uli Büttner and Volker Henn at the University Neurology Clinic in Zurich in 1977. They studied oculomotor nuclei and explained to me in one day how to record from individual neurons in the midbrain of monkeys. We are still using some of their techniques today, and I am eternally grateful for their help and was much honored to be invited to present the Volker-Henn-Lecture in 2018 in Zurich. I went back to Fribourg and started my new monkey lab in an empty room with little money but with the help of two young workshop members named André Gaillard and Edouard Regli. They were absolutely wonderful, ingenious, super precise, and supportive people who liked to create something interesting. To celebrate the new lab, I designed a little demonstration of the work on reward I was planning to do. I put a bottle of brandy into the liquid-dispensing bottle, programmed the liquid valve to just fill just one small brandy glass with a single key touch, and off we went to a very happy (human) party that became the talk of the place for many years to come.

Searching for a Dopamine-Specific Neuronal Discharge

From my teenage mechanical and electrical tinkering, I had gained plenty of hands-on experience, including resilience from repeated failure. As good commercial metal microelectrodes were unavailable at the time, I learned to make my own glass-insulated tungsten recording electrodes under the microscope, which was a feast on quiet evenings in the lab. These electrodes turned out to be crucial for recording from dopamine neurons; the impedance-reducing platinization of their tips was important for reducing the background noise that made neuronal discharges more recognizable. There are certain things in life that you have to do yourself, and electrodes with micrometer precision tips are good examples. Tungsten is the English word for a hard metal whose chemical element name is Wolfram (W, atomic number 74, melting point 3,422 C); some of my friends call me Tungsten (as did a Swiss databank website of scientific talents). So, I thought I might have a native relationship to tungsten electrodes and thus have used them for all 40 years of my work in neurophysiology.

While working in Stockholm, I had run into the work of George Aghajanian in the 1970s who had reported that rat midbrain dopamine neurons discharge extracellularly recorded action potentials with a distinct waveform (Guyenet & Aghajanian 1978). I tried this while still in Stockholm and confirmed on the first recording day the wonderful positive-negative-positive waveform with a little break in the upgoing flank and the characteristic blop-blop-blop in the loudspeaker. Without optogenetic identification at the time, the waveform would be crucial to identify dopamine neurons in awake monkeys in which we needed to test antidromic stimulation or systemic pharmacological injections.

To become familiar with dopamine recordings in monkeys, I implanted a recording chamber that allowed me repeated electrophysiological testing under anesthesia without worrying about the animal’s behavior. The first challenge came when locating dopamine neurons at 30 millimeters below the cortical surface. Stereotaxic coordinates in macaque monkeys are notoriously inaccurate, which we overcame by locating the face and head area of the ventromedial somatosensory thalamus that are positioned above the lateral region of dopamine neurons. The thalamic neurons had fantastic strong and precise responses to the touch of single facial hairs or a single tooth or the gingiva; the responses were a beauty for any electrophysiologist and a reason to call in departmental colleagues to illustrate what dry textbooks claimed. From then on, on every recoding day, we would pass our electrode through the thalamus before going for lateral dopamine neurons below.

After several months of weekly recording in the anesthetized monkey, I had developed the electrodes I needed to confirm that I could see in the recordings all the essential characteristics: the characteristic waveform with a systematic polarity change associated with electrode movement, the blop-blop-blop sound, a reduction of impulse rate by systemic apomorphine, as is typical for dopamine neurons, and identification of striatum-projecting dopamine neurons by antidromic responses. Then I tested the same monkey sitting awake in a monkey chair and confirmed the striking electrophysiological characteristics. Now I was ready to record from neurons that used the neurotransmitter dopamine during well-controlled behavioral tasks in macaque monkeys, taking advantage of their exquisitely controllable behavior. While I always remained anxious about the identity of our dopamine neurons, recent optogenetic identification of monkey dopamine neurons revealed the same waveforms (Stauffer et al. 2016). My moment of gratitude came on a psychopharmacology conference in 2007 in Boca Raton, Florida, where I happened to sit opposite George Aghajanian in the hotel bus and could tell him how much of a game-changer his papers on identified dopamine recordings had been for me.

I was working on my own in Fribourg and had all the time in the world to explore what dopamine neurons were doing during behavior. Nobody else was doing similar recordings, so there was no pressure or competition, and hardly anybody took notice of my work at conferences. I benefited from the low-level but permanent faculty position and the modest but pretty reliable funding from the Swiss National Science Foundation. Great, leisurely moments with lots of time for imagination and trying things out, a pleasant and ultimately very productive period.

Surprise 1a: Phasic Movement-Related Dopamine Activity

Before going any further, the first tests of dopamine neurons in the awake monkey had to address the potentially discouraging issue of “rain on the brain,” according to which there might not be any phasic, subsecond changes in dopamine neurons related to behavior. Even before perfecting my electrodes, I designed an experiment based on the known Parkinsonian motor and cognitive deficits. Together with my first graduate student, André Ruffieux, who later became a respected neurologist in Fribourg, we trained monkeys in a then-state-of-the-art delayed go/no-go task that included many sensory, movement, and cognitive processes appropriate for wide screening of behavioral relationships. Indeed, variable fractions of dopamine neurons showed diverse, excitatory, and inhibitory changes with reaching or mouth movements, during reward reception, and even during the whole task (Schultz et al. 1983). Thus, dopamine neurons were more than “rain on the brain,” and we might even have found a positive correlate with Parkinsonian deficits. We immediately submitted a paper to Science that was, just as immediately, rejected. This was fortunate, as Mahlon DeLong, a pioneer and authority on the motor neurophysiology of the primate basal ganglia, and a friend, reported an absence of dopamine changes in his more controlled and concise arm movement task (DeLong et al. 1983). Their result fit well with the “rain on the brain” notion of dopamine function. How embarrassing: The first findings on movement relationships in a novice laboratory were not reproducible in a world-class laboratory!

Being nevertheless convinced of the validity of our dopamine movement relationships, the only explanation we had was that Mahlon’s study used much more constrained arm movements, whereas our monkeys performed a large arm reaching movement that engaged more than 35 hand, arm, and shoulder muscles plus several muscles on the back, as our widespread muscle recordings had shown. We had already cautiously interpreted our data as reflecting behavioral activation rather than motor control, and that still seems to be the best explanation. We then used the muscle recordings to refine our tasks and to reduce muscle activity, and now we rarely found movement-related dopamine changes. We also tested a wider variety of movement situations, including eye movements and, separately, more sluggish arm movements following the offset of a visual stimulus rather than its onset (Schultz & Romo 1990). None of these tests showed any dopamine activation. Now we had what Mahlon had seen: Dopamine neurons, despite their crucial role in Parkinson’s akinesia, are not activated during well-controlled movements. Still, the irreproducibility of our earlier movement activation remained a sensitive issue.

Then Karl Deisseroth, Ed Boyden, and their colleagues developed optogenetics around 2010. They used rodents because of their suitability for molecular biology, but rodents are not monkeys and have more difficulty controlling their difficult-to-monitor movements. Also, rodents are often tested during whole body movements in boxes, or during locomotion in T-mazes, or when running on treadmills or wheels, all of which engage hundreds of muscles and sensory receptors in the skin, joints, and muscles and lead to substantial behavioral activation. Although these might be straightforward tasks for behaviorists, they are confusing for behavioral neurophysiologists. Even seemingly simple licking movements can be accompanied by uninstructed movements and unintended neuronal activity, as Anne Churchland’s laboratory at Cold Spring Harbor showed (Musall et al. 2019). When testing neurons in rodents, with or without optogenetics, an early voltametric study (Stuber et al. 2005)—and later several high-profile neurophysiological studies—reported time and again dopamine activations during these movements (Berke 2018; Engelhard et al. 2019). This was a relief: Finally, someone confirmed our movement-related activity, which we ourselves had difficulty doing when studying better controlled movements. Dopamine neurons would still not code individual muscle contractions or movements but would get all excited when the animal became active. Could this be what was causing Parkinsonian hypokinesia: not specific motor control but rather a lack of behavioral activation by dopamine neurons?

Surprise 1b: Phasic Dopamine Stimulus Response

The movement-related changes were statistically significant but were nevertheless quite modest, resulting rarely in more than 20–50 percent increase of activity. I just was not convinced that this was all. Maybe the task in our study, and also in Mahlon’s study, contained too many stimuli and movements. At a conference in 1978 in Denmark, I expressed my worries to Susan Iversen who suggested a more natural, scaled-down task to get to the bottom of things. Instead of the stimuli from the previous task, I used a new custom-made food box whose front door (40 by 40 millimeters) would open rapidly upward through an experimenter-operated release cable (400 degrees/second); then the animal released a touch-sensitive resting key, reached into the food box, retrieved a raisin or a morsel of apple, brought it to its mouth, and ate it—all quite intuitive and natural. Infrared photo beams detected the door opening and the animal’s hand entering the box.

On my first dopamine recordings with this task, something happened that neither Mahlon nor we had ever seen before: Less than 100 milliseconds after the food box door opened, dopamine neurons showed a sharp, phasic, stereotyped increase in activity that lasted less than 200 milliseconds (Schultz 1986b). The increase consisted of just a few extra dopamine impulses that were sharply time-locked to the precise time of the door opening. Well more than half of the dopamine neurons in the substantia nigra with all their fancy electrophysiological characteristics showed this response, and so did more than 90 percent of the dopamine neurons located in the ventral tegmental area. The response of only a few extra discharges was not much in itself, and became an object of occasional ridicule, but the high proportion of dopamine neurons responding with similar latency and duration made the response a substantial population signal that was likely to get noticed by postsynaptic neurons. The response occurred with the opening of the food box and not when the animal received the reward. The response could reflect many things: general alert, salience, and attention; the sensory sight of the opening door and the food and/or the door opening sound; the initiation of an arm and hand movement into the box; or maybe the prediction of obtaining a reward inside the box. We had much more work to do before putting a name on it.

Nevertheless, the response was a surprise: The simplified task with limited stimuli had yielded a clear, phasic, rapid, consistent, and substantial subsecond signal in most dopamine neurons. Now I had two separate phasic dopamine changes: the rather sluggish and heterogeneous changes with motor activation during various task epochs seen before, and now the sharper, faster, stronger, stereotyped, and more substantial response to the food box stimulus. I drew a scheme that separated the two responses (Schultz 1986b), which despite many refinements and better specifications stood the test of time (Engelhard et al. 2019). These two dopamine changes contradicted the notion of unmodulated dopamine activity, including Mahlon’s lack of task modulation: Dopamine neurons were more than stupid “rain on the brain.” Sometimes when I woke up in the early hours of morning, I wondered whether I could convince the community about the existence of a subsecond dopamine signal.

Surprise 2: Reward!

Then Ranulfo Romo joined the laboratory in 1985, and we took up the idea of spontaneous movements, which were better related to Parkinsonian deficits. We covered the food box with a shield and allowed the monkey to reach into the open box from underneath to fetch a piece of apple at a time of its choosing and without a triggering stimulus. Ranulfo was great in patiently training the capricious monkeys. Indeed, some dopamine neurons showed changes with these self-initiated movements (Romo & Schultz 1990), which was a nice confirmation of the movement activity observed earlier (and which also removed the lump in my stomach caused by Mahlon’s negative results). Still, when we opened the food box door in control trials, many more of the same dopamine neurons showed the familiar, much sharper response.

With Ranulfo’s enthusiasm, we asked whether this phasic response to the food box door opening might reflect more than simple alertness or stimulus-driven attention, as someone suggested politely. To start, I went home and asked my wife. After my lengthy description, Gerda suggested a German word for incentive (Anreiz): As the food box opening allowed the animal to capture a piece of food, the neuronal response might simply reflect the incentive for getting a reward. Based on Gerda’s common-sense suggestion, we looked at Bindra’s description of “incentive motivation” (1968), which combines “incentive” (to do something) with “motivation” (the reason for doing something, like getting a reward). Another possible term is “incentive salience,” which captures the alert and attention function expressed in our earlier minimal-assumption interpretation; we later identified an initial attentional component of the phasic dopamine response (Schultz 2016). As it turned out, both “incentive” interpretations were superseded by our findings on reward unpredictability and reward prediction error (RPE) coding described below in sections “Surprise 4: Reward Unpredictability” and “From Reward Unpredictability to RPE.”

To address the issue of “incentive,” we wanted to see what happened when the animal encountered the food that provided the incentive. So, we stuck the food onto a touch-sensitive wire that told us precisely when the animal touched the food. As the shield prevented the animal from seeing into the box, any dopamine response could not be linked to seeing the food, and there was no stimulus that triggered the movement and could elicit a difficult-to-interpret dopamine response. Now, Eureka, the dopamine neurons responded to the touch of the food (Romo & Schultz 1990)! This was not a somatosensory touch response: On occasional test trials without food, the response was gone, or replaced by depressed activity, even though the animal touched the wire and moved its hand in the empty box (into which it could not see). Thus, there was a sharp dopamine signal that was linked to the reward and not to visual or somatosensory stimulation nor to hand movement.

This reward response was surprise 2 (after the phasic dopamine changes) and a reward for us that needed to be properly acknowledged at the end of a recording morning. We had bought a bottle of rum from a nearby supermarket and fetched a coke from the machine in the basement; we drank a bit coke off the top of the bottle, replaced it with rum, and drank a Cuba Libre to the discovery of a dopamine reward signal. As rum is lighter than coke, the two did not mix, and we drank almost pure rum, which inevitably finished the morning session in good spirits.

New data bring new problems. At the time, the definition of reward function was too unclear for precise neurophysiology. I was told about reinforcement (which includes punishment) and operant or goal-directed behavior (which might be too narrow). For the “man on the street,” reward is a bonus for having done something special (also too narrow). There is also happiness (maybe the essence but hopeless to test in animals). All this was inadequate for the millisecond precision afforded by electrophysiology. The same Volker Henn whose laboratory members showed me their neurophysiology recording techniques in awake monkeys emphasized the importance of reducing the complex notion of reward to measurable variables. We had to generate not only our own microelectrodes but also the concepts for understanding the data.

It was first Roy Wise who helped a lot. I met Roy during a summer conference in 1989 in the Sardinian sea resort of Capo Boi. While the wind blew softly through the open shutters of the lunch place, we kept discussing, nonstop and repeatedly, the definitions of reward functions and their suitability for controlled testing of fast neuronal signals. It was unbelievable how his sharp mind combined with common sense dissected these inscrutable notions and came up with straightforward suggestions. We became good friends and had many more such discussion, including one during a conference organized by Ranulfo Romo in 2010 in Mexico DF. While visiting the different buildings around the great pyramid of Teotihuacan, we intermittently mixed Aztec culture with neuronal reward signals.

Back in Fribourg, I put together our own definition—rewards have three global functions: learning (as Pavlov has it), approach behavior (straightforward and intuitive) along with value for economic decision-making (requiring further concepts), and happiness (unavoidable but untestable in animals). These definitions, with many ramifications and sophistications, have proven to be very helpful to guide our work through the years.

But there were also words of caution. We had occasional visitors working on motor control whose expertise I valued highly. I told them that I wanted to test reward processing by neurons. But I was very clearly told, repeatedly, by prominent people in the field, that reward is exactly what everybody tries to factor out because it messes up their data interpretation. True, larger rewards shorten reaction times and speed up movements, so you want to hold reward constant to keep perfect control over the movement. Fortunately, I did not listen.

But a dopamine reward function is nothing new. A researcher at the Imperial College of Tropical Agriculture in Trinidad had shown that 3,3-dihydroxyphenylethylamine, now referred to as dopamine, is involved in keeping the banana healthy (Griffiths 1959). If dopamine is oxidized, the banana gets brown, and when the enzymatic reaction is prevented and dopamine remains detectable, the banana stays nice and yellow. One might call it the “Rotten Banana Model of Dopamine Reward Function.” Thus, all we were doing beyond this discovery was to associate the known dopamine reward function with neurons.

Surprise 3: Reward Prediction

While it was nice to see a response to the reward, the response to the opening of the food box still required interpretation. We found that the same dopamine neurons that responded to the food touch during spontaneous movements also responded to the door opening (Romo & Schultz 1990). Maybe the door opening response was related to the reward the animal would find in the box? That was all fine, but something was funny about these responses: The food touch response was gone when the door opening triggered the animal’s reaching into the box. How can that be a neuronal reward signal when the neuron fails to respond to reward in some situations? Maybe the dopamine response to the reward had simply been transferred to the door opening, as if the neuron had done enough by responding to the door opening and no longer cared for the reward itself? It seemed most likely that the door opening was a reward predictor, in which case we would have surprise 3 (after the phasic dopamine changes and the reward response). To substantiate this assumption, we needed better controlled tests.

This is where Tomas Ljungberg came in. Tomas had done a PhD with Urban during my postdoctoral stay, at which time we had many discussions about the lack of knowledge about neurophysiological dopamine signals. He came to Fribourg 1987 with his experience in animal conditioning, which was the occasion to address the reward and prediction issues. Tomas wanted to understand stuff and was not content with our simple and intuitive tests that had been crucial for the initial detection of phasic dopamine signals. He suggested that we use concepts from animal learning theory and try a decent conditioning experiment in which we had control over the animal’s behavior and could monitor the learning progress. We would use an arbitrary, intrinsically neutral stimulus and associate it with reward to make it a reward predictor.

We replaced the food box with a light-emitting diode and a lever, and the food with apple juice whose quantity was perfectly controlled by a computer-controlled electromagnetic valve that delivered drops from a spout in front of the animal’s mouth. Thus, like the more intuitive food box opening, the small light-emitting diode would become a conditioned stimulus that predicted the juice and elicited the reaching, and the animal received the juice for touching the lever. Sure enough, most dopamine neurons began responding to the light shortly after it had been presented together with the juice, around the same time the animal began to touch the lever that produced the reward. In comparison tests, the same dopamine neurons responded to the food box opening, and both responses were neither time-locked to the reaching movement nor to the muscle activity in the arm, shoulder, and back that we monitored via implanted and inserted electrodes (Ljungberg et al. 1992). How nice to reproduce our own data! Thus, our previous dopamine responses were not specific for the food box opening. Dopamine neurons did not seem to care about the kind of reward the stimulus predicted—food morsels or liquid drops.

But what about the response to the reward that we saw with Ranulfo when the animal touched the food during spontaneous movements without preceding stimuli? I was brooding over this question during a quiet weekend and asked Tomas on Monday morning whether he had ever referenced the dopamine responses to the moment of juice delivery. He came back a couple hours later with a bright smile on his face: Indeed, at the beginning of conditioning, dopamine neurons did respond to the juice reward. But with advancing learning trials, the reward response disappeared and reappeared at the stimulus that had come to predict the reward and elicit the movement (Schultz et al. 1993). The process resembled the response transfer we had seen with Ranulfo between the touch of a food morsel without a preceding stimulus and the stimulus of the food box opening (Romo & Schultz 1990). Now we had a response that engaged most dopamine neurons and transferred from reward to reward-prediction stimuli.

These experiments told us also something fundamental about reward processing. The response to the food morsel inside the food box (without any predictive door opening stimulus) occurred outside any learning episode. Maybe the reward response did not depend on an explicit learning process? Here, Jacques Mirenowicz came in who had joined us from Paris for a PhD. He was reading philosophy texts while supervising a monkey’s task training (which is admittedly boring) and thus had a more reflective attitude. He figured that the simplest reward test would be a drop of juice reward delivered outside any task or learning episode, out of the blue. Sure enough, dopamine neurons responded strongly to such a “free reward” (Mirenowicz & Schultz 1994). The reward may well serve to condition all stimuli present at that moment, but the test was remote from any learning. It looked like the dopamine reward response reflected a more fundamental process still obscure to us.

The response transfer from reward to reward-predicting stimuli occurs not only in the described operant tasks but also during Pavlovian conditioning (Waelti et al. 2001). Of course, reward predictors are Pavlovian conditioned, not operantly conditioned. Any stimulus in an operant procedure becomes inadvertently a Pavlovian conditioned reward predictor through its association with the reinforcer, as long as the animal is awake and attentive.

Together these results were funny in several respects. Dopamine responses do not categorically distinguish between liquid and food reward nor between primary and higher-order rewards. The response transfer occurs also between reward-predicting stimuli, and it is not restricted to learning. And the response occurs with artificial visual and auditory stimuli and with more natural food box opening when they predict reward. Thus, the dopamine response looks like a basic and fundamental reward signal. Our later economic approach would tell us that this is exactly what one would expect from a general reward value signal, which we then specified as coding formal economic utility (Stauffer et al. 2014).

Surprise 4: Reward Unpredictability

The response transfer was a funny thing, but we had no idea how to approach it. I remember a late morning in the summer of 1989 when Tomas Ljungberg and I were getting tired of searching for another dopamine neuron after several hours of recording and we started telling each other jokes. They were pretty good, but they were distracting to the point that we missed mistakes the monkey made. Of course, you don’t get reward for making mistakes, and monkeys are no exception. When the predicted juice drop failed to occur because of a mistake, the dopamine neurons did not simply stop responding. Rather, they shut down their activity completely for a couple hundred milliseconds, time after time, exactly at the moment at which the reward would have occurred had the animal not made the mistake. We saw the dopamine depression clearly on the monitor of our computer, which was programmed to display, among other things, behavioral errors.

Well, the animal should not control the experiment, so we stopped chatting and occasionally withheld the juice deliberately even when the animal performed perfectly well. We saw the same brief dopamine depression when the reward would have occurred had we not interfered (Ljungberg et al. 1991). And there was no depression on those trials on which we did not omit the reward and gave it to the animal as predicted (which were most trials). Instead of going home for family lunch, we continued recording and found time and again the same depression with reward omission in all further dopamine neurons we recorded before stopping for the day. Thus, while we were joking, we had found that dopamine neurons were depressed when a predicted reward was omitted. The depression reminded us of an earlier result with Ranulfo in which dopamine neurons were activated when the animal touched food inside the food box into which it could not see; when the food box turned out to be empty, dopamine activity was depressed (Romo & Schultz 1990). The experiment had been too uncontrolled to give the result much thought, but it had presaged the depression in the better controlled task and now made perfect sense. We later saw the same dopamine depression during well-controlled learning when the animal failed to get the reward because of making the wrong choice (Hollerman & Schultz 1998).

A dopamine depression with reward omission contrasts with intuition and neurophysiology: Neurons do not respond to absent events. Something else was going on, and its persistent and reproducible nature made us suspicious. A reward that is predicted by a stimulus and then does not occur is more than just an absent event. There was not nothing but something—that is, there was a stimulus, which had predicted a reward, and then the predicted reward failed to occur because of the animal’s mistake or because a mean experimenter had blocked the reward tube. So, we were dealing with a violation of reward prediction, not simply reward absence.

A response that reflects violation of reward prediction indicates a relationship to unpredictability, which could explain all dopamine responses. Reward unpredictability was surprise 4, the biggest and most exotic of all surprises (Mirenowicz & Schultz 1994). And it explained all dopamine reward responses: the activation by unpredicted reward with learning, food touch and free delivery, the absence of response to predicted rewards, and the depression with omission of predicted reward. These results pretty much nailed unpredictability as a condition for the dopamine reward signal. The question was then, what function would a neuronal response have that reflects reward unpredictability? What behavioral or neuronal process might involve reward unpredictability?

From Reward Unpredictability to RPE

It was time to formalize these results to integrate them into existing theories of reward and decision-making. The solution came during my short sabbatical with Tony Dickinson in 1993 in Cambridge. We had sharp discussions about reward function every day in the coffee room of the Psychology Department, which were occasionally interrupted by a low-flying crow that contributed an occasional croak and more solid stuff. I asked Tony what reward function might involve reward unpredictability. Tony had just written his book on animal learning theory and was the right person at the right time (Dickinson 1980). He opened John Pearce’s undergraduate textbook on animal learning (Pearce 1987), which stated on half a page the requirement of reinforcer unpredictability for associative conditioning as the basis of Rescorla and Wagner’s reinforcement learning theory (1972). Amazing: This was exactly the way dopamine neurons code rewards! These gentlemen must have had a secret look at dopamine signals. What we labeled unpredictability is often referred to as prediction error and constitutes the basis of Rescorla and Wagner’s associability term.

From my student days, I was familiar with control theory and the role of errors in correct performance. The only neuronal error signal known at the time was related to movements and involved the climbing fibers of the cerebellum (Gilbert & Thach 1977), as hypothesized by David Marr (1969). But error signals with reward? Why would you get a reward for having committed an error, as Roy Wise asked wisely? I was surely worried about generalizing the error concept to reward learning and its potential neuronal signals. As an experimentalist, I am closer to data than others and feel a responsibility to not mislead the field by engaging in overly enthusiastic interpretations. Others have called my attitude as struggling and not understanding my own data, but that is a small price to pay for being cautious; fancy interpretations can be falsified on the slightest occasion.

An error can be defined more broadly than just a behavioral mistake; an error is the discrepancy between what happens and what was predicted, like a positive or negative surprise, irrespective of requiring an action. This is like when your pub runs out of your favorite ale: You don’t care about what exactly went wrong or who misjudged the barrel or forgot to refill it. You just sense the difference against the prediction and are disappointed. With such a definition, the use of the error concept by accepted reinforcement theories would allow us to understand the dopamine reward signal by using fundamental error concepts, and possibly contribute a neuronal foundation to animal learning theory.

While we had done well with intuition and ad hoc definitions, better scrutiny of a dopamine RPE signal would require more formal tests. Tony suggested the blocking paradigm of Kamin that tests the requirement of prediction errors in learning (Kamin 1969). We presented an initial stimulus that fully predicts a reward and added another stimulus without changing the reward; the reward did not change with the added stimulus and hence would fail to elicit a prediction error. Then we tested whether the added stimulus would be learned. Indeed, the monkey’s licking demonstrated learning of the initial stimulus but not of the added stimulus. In parallel, dopamine neurons responded to the initial stimulus (that had been learned) but not to the added stimulus (that had not been learned). And the dopamine neurons were excited by a reward that was delivered unexpectedly after the added, nonpredictive stimulus (Waelti et al. 2001). Thus, dopamine responses followed the prediction error assumptions of the blocking paradigm: An RPE is necessary for learning, and the dopamine neurons signal that prediction error. Subsequently, Tricia Janak’s group demonstrated behavioral learning when dopamine neurons were optogenetically stimulated at the moment at which a fully predicted reward elicited no response (Steinberg et al. 2013). Thus, the blocking paradigm nails the notion that dopamine responses code an RPE, and that their response drives learning.

While these dopamine responses conform with the Rescorla-Wagner learning model, our relentless playing around revealed another interesting property that put the icing on the cake. The animals tested by Tomas Ljungberg had been trained in our spatial delayed response task, such that an initial stimulus instructs the animal to touch a left or right lever when a movement-triggering stimulus occurs and that movement-triggering stimulus then predicts the reward. In fact, both stimuli predict reward. Monkeys learn such a task backward: first the movement-triggering stimulus, and then the earlier instruction stimulus. With learning, the dopamine response occurs initially to the reward, then gets lost and transfers to the triggering stimulus, and then gets lost again and transfers to the instruction stimulus, which is now the earliest reward predictor (Schultz et al. 1993). Thus, dopamine responses transfer from reward to reward-predicting stimuli and then to the earliest reward predictor. All these responses depend on unpredictability, not only of the reward but also of the reward predictors. RPEs occur not only with rewards but also with reward predictors: Each stimulus elicits a prediction error relative to the prediction from the immediately preceding stimulus.

Reward-predicting stimuli elicit approach behavior and learning, like primary rewards, and thus constitute higher-order rewards. In fact, primary rewards are often difficult to identify and to distinguish from higher-order rewards. A juice drop at the mouth may be formally called a primary reward, but it predicts the liquid acting on sensory receptors in the mouth and reducing the salt concentration in the blood, both of which may be genuine, real “primary” rewards (of course, the juice taste on the tongue itself may also be a genuine reward). Thus, the liquid drop at the mouth may be primarily a reward predictor or higher-order reward, and the prediction error it elicits is a higher-order RPE. Hence, dopamine neurons detect prediction errors for both primary and higher-order rewards relative to the reward prediction at that moment.

Our finding about reward unpredictability seemed to relate well to RPE as the crucial variable of the Rescorla-Wagner learning model (Mirenowicz & Schultz 1994). But our additional observation that dopamine neurons signal both primary and higher-order RPEs matched a more advanced and efficient reinforcement model. In 1996, Read Montague, Peter Dayan and Terry Sejnowski reported the similarity between dopamine responses and temporal difference (TD) learning (Montague et al. 1996). TD learning was developed in 1981 by Rich Sutton and Andy Barto at Amherst. Their mentor Harry Klopf had been intrigued by the Bellman equation that describes how reward can be maximized by optimizing the pursuit of immediate reward together with all future predicted rewards. Learning through prediction errors at both primary and higher-order rewards can be more accurate as compared with considering only the final reward. Specifically, the single RPE of the Rescorla-Wagner model (reward minus prediction) is replaced in TD learning by (the current reward together with the temporally discounted sum of all future rewards) minus (the current prediction). Thus, our dopamine responses in the delay task complied with the more efficient TD reinforcement learning formalism by coding RPEs with both primary and higher-order rewards.

Signals like the dopamine prediction error response serve as effective teaching signals; artificial models using dopamine-like TD signals, together with other machine learning algorithms, acquire performance of backgammon and Go games at world-champion level (Tesauro 1994; Silver et al. 2017). The fact that these implementations differ vastly from the brain’s architecture emphasizes the importance of neuronal signals that may dominate over any particular architecture implementing the reinforcement algorithm (Purves 2019). The architecture in biological brains seems to represent an implementation that has emerged from evolution but that may not be crucial for reinforcement learning.

Simple Schemes That Convey Complicated Concepts

I was planning to attend the 1995 German Neurobiology Meeting in Göttingen where I would meet Rich Sutton. Rich had been the first graduate student of Andy Barto and developed TD learning together with him, and TD learning has since then become a mainstay in machine learning and artificial intelligence. I thought he might be interested to hear about a corresponding neuronal mechanism. I was frequently visiting my mother in Germany, which required long train rides from Switzerland with many hours to fill. I took essentials from our studies and played around with a graph until I got it the way I wanted. Rich loved it and told me that I was holding some genuine truth in my hands. I used the graph later for a review in the Journal of Neurophysiology to which Peter Strick had invited me (Schultz 1998). I also had the graph at hand when Science invited Read Montague, Peter Dayan, and me to write a review about the similarity between neuronal responses to RPEs, which had never been heard of in neuroscience, and artificial algorithms that were designed from scratch based mainly on insight and which were highly efficient for learning (Schultz et al. 1997). It goes like this:

An unpredicted reward elicits a dopamine activation, like a free reward, or the touch of a food morsel without a preceding stimulus, or a drop of liquid before and during conditioning.

Fully predicted rewards at exactly the predicted time elicit no dopamine response, as shown in established tasks or at the end of learning.

Omission of predicted reward elicits a dopamine depression, as shown with behavioral mistakes or the withholding of predicted reward.

Dopamine neurons respond also to unexpected reward-predicting stimuli (which are higher-order rewards). While a neuronal response to a reward-predicting stimulus may not in itself reflect a prediction error and might be compatible with Rescorla-Wagner learning, the disappearance of that response when the stimulus is predicted by another, earlier stimulus indicates higher-order RPE coding compatible with the TD formalism.

Multiple Dopamine Response Components

The dopamine RPE response and its correspondence to sophisticated artificial reinforcement models differed from all traditional hypotheses of dopamine function. While the data were accepted by the community amazingly rapidly, dissenting voices were heard. There were indications of attention and punishment activating dopamine neurons that needed to be taken care of. True, we had ourselves seen dopamine activations to novel or known neutral control stimuli, and there were reports about responses to tail pinch, but we put them aside because we lacked solid interpretations. The solution came after a few extra experiments of our own (Mirenowicz & Schultz 1996; Kobayashi & Schultz 2014) and a couple of insightful studies by Masamichi Sakagami at Tamagawa University near Tokyo (Nomoto et al. 2010) and by Christopher Fiorillo at Stanford after he had left our lab (Fiorillo et al. 2013). Looking at these data together, we realized that dopamine neurons do what many neurons in other cortical and subcortical brain regions do: They show an unselective early activation to all kinds of stimuli before the animal and its neurons identify and evaluate the stimulus (Schultz 2016). The early response to unrewarded stimuli grows with the attention the stimulus generates, which would also explain the activation by aversive stimuli. The early response increases also when the stimulus resembles a rewarded stimulus or simply when rewards occur in the same environment. A few tens to a couple hundred milliseconds later, the true dopamine value signal emerges that is not explained by attention or punishment. Only recent experiments in rodents have revealed an anatomically separate subgroup of dopamine neurons that are activated by specific liquids that are also aversive, but without coding aversive prediction errors. These responses are in themselves interesting but leave the RPE response as the main (“canonical”) phasic dopamine signal (Menegas et al. 2018). It might be that the early dopamine response component serves to alert the animal about a possible reward in the environment. The animal can prepare a rapid reaction but cancel the action if the object turns out a few hundred milliseconds later to be nothing of interest. The capacity to react quickly, before the second component identifies the object’s nature, would provide an advantage over competitors in the long run of evolutionary selection.

From Stereotypy to Free Will

As soon as we saw the first strong phasic dopamine responses after having found the much smaller and rather boring movement-related changes, we were stunned by the observation that these phasic responses occurred only with rewards and reward-predicting stimuli (when unpredicted) and with no other stimuli. Also, almost all responses were excitatory, whereas depressions of activity occurred almost only with reward omission. Thus, as reward-predicting stimuli are higher-order rewards and are treated similarly to primary rewards by TD models, the strong phasic dopamine responses (distinct from the smaller movement-related changes) were stereotyped for the kind of event that elicited them (reward) and for the kind of response (excitation with unpredicted reward, depression with omitted reward). This simplicity was a huge contrast to the vast response differences between neurons and task events in other brain structures that we had read about and that we saw in the neighbouring nondopamine neurons of substantia nigra pars reticulata while searching for dopamine neurons (Schultz 1986a). Thus, before going any further, we needed to make sure we were not missing anything. Thus, we recorded neurons in the striatum and SMA in the same monkeys and tasks and found time and again responses to all kinds of events and a mix of fast and slow excitations and inhibitions. The contrast to dopamine responses could hardly have been more impressive.

Our recording in striatum and SMA allowed us to catch up with the question of Free Will that remained from my time in Eccles’ lab in Buffalo. Thus, we set out to test spontaneous, internally driven, “freely willed” movements. We recorded not only in the SMA but also in the striatum, which receives dopamine axons and is an important hub in cortico-basal ganglia loops with the SMA. At the time, SMA and striatum were both relatively unexplored, and any experiment would be exciting and result in novel data. The SMA receives strong transsynaptic inputs from the basal ganglia, and the striatum receives strong monosynaptic input from the frontal cortex, including SMA. Fuster (1973) and Kubota and Niki (1973) described prefrontal activity lasting several seconds before stimulus-driven movements. We wondered whether such activity might also exist when the animal itself decides to make a movement rather than in reaction to an imperative stimulus. Ranulfo trained monkeys patiently to self-initiate arm movements into our covered food box in a reproducible manner. We then recorded from about 20 arm, shoulder and back muscles to monitor, and control for, hidden muscle activity.

Our caution and self-scrutinizing paid off double. First, neurons in SMA and striatum showed large responses variations, both between neurons and between task events (Schultz et al. 1993); no comparison to the rather stereotyped dopamine responses. Second, subsets of SMA and striatal neurons increased their activity gradually for one to three seconds before self-initiated movements; the increased activity terminated abruptly as soon as the animal started the movement (Schultz & Romo 1992; Romo & Schultz 1992). Many of these neurons failed to show such sustained activity during the preparation of movements triggered by external stimuli and thus indeed may have been involved in the internal generation of movement. I was very proud of this internally generated activity and wanted to follow it up later. We were touching the neuronal basis of intention-in-action (Searle 1983) on the road to Free Will. But the discovery of the dopamine RPE signal took all my attention, and we never went back to these ideas, which was quite a shame actually.

Given the huge diversity of cortical and striatal neurophysiology signals that others and we have seen, the question remains in what respect the much simpler and more stereotyped dopamine responses are homogeneous. People looking at the molecular, cellular, release and connection characteristics of dopamine neurons are emphasizing their heterogeneity, so the word homogeneous requires specification. Many dopamine responses are not homogeneous, including the slower movement relationships and the specific aversive non-RPE responses. But ‘homogeneous’ would apply to the events that trigger the phasic, two-component RPE response, which are both primary and higher-order rewards (Schultz 1998; Eshel et al. 2016). But while the RPE response is homogeneous in terms of triggering events, it varies quite a bit among individual neurons. For example, we found time and again that dopamine neurons in medial parts of the midbrain (ventral tegmental area, VTA) respond more frequently and strongly to RPEs than more lateral neurons (substantia nigra pars compacta). A recent and more quantitative study made this point clearer by describing optimistic dopamine neurons whose responses scale well with positive RPEs and hardly change with negative RPEs, whereas responses of ‘pessimistic’ dopamine neurons don’t care much about positive RPEs but scale well with negative RPEs (Dabney et al. 2019; Lovet at al. 2020). Only the population activity of these neurons would code RPEs adequately.

Thus, we had seriously worried about missing something important when we had noticed the homogeneity of the reward triggering events for dopamine neurons. But addressing that worry resulted in our foray into the Free Will problem that together with consciousness still constitutes a main challenge for neuroscience.

What Does Dopamine Do?

I often hear this question when walking the corridors of conferences. Many years ago, a prominent Parkinson researcher asked me, rather incredulously, how a dopamine reward signal could account for Parkinsonian movement deficits. He obviously assumed that a dopamine signal should correspond to Parkinsonian deficits, and the RPE signal hardly did so.

There is a wide spectrum of motor and cognitive impairments in diseases like Parkinsonism and schizophrenia and in experimental pharmacological and lesion tests on humans and animals. Although interference with dopamine neurotransmission induces reward learning deficits in humans and animals (Zweifel et al. 2009), many deficits cannot be easily explained by phasic RPE signaling, including the Parkinsonian movement deficits. This is the issue: Although it is reasonable to infer a natural function from the deficits that occur when the system is compromised, the RPE signal simply won’t explain the many motor and cognitive deficits. I have only one general, and not necessarily satisfying, answer for these deficits that comes back to the notion of “rain on the brain”: Dopamine is a chemical that is released in a slowly changing or even tonic manner and serves as a fuel that enables other neurons to do their job; a true neuromodulator without a sophisticated function on its own. Of course, this tonic function is only one of the many dopamine functions (Schultz 2007).

But the confusion about dopamine function goes deeper and persists even when only considering phasic dopamine changes. I have been talking for more than 30 years about dopamine RPE responses and have sidelined the far less interesting slower and lower dopamine changes with behavioral activation, but others are now rediscovering these earlier reported changes. As electrophysiologists, we study subsecond neuronal signals, and an initial sharp and short reward response is not the same thing as a slower and weaker change during behavioral activation. But the two dopamine changes might have a common function, like motivation, that combines reward detection with the behavioral activation that is necessary to obtain the reward. The two dopamine signals may even have a common RPE function (Kim et al. 2020), which might suit the lower time resolution of complex rodent behavior and some new optical recording methods.

Thus, to answer the question: dopamine does have multiple behavioral relationships at different time scales (Schultz 2007): (1) the fast, two-component signal coding RPEs, the phasic changes with behavioral activation and movements (that might overlap with the RPE function); and (2) the slow or even tonic enabling function inferred from compromised dopamine neurotransmission like Parkinson’s. In any event, dopamine neurons contradict the widespread assumption that one brain system has one function.

Leaving Fribourg

I will always be grateful for two assets this Swiss city provided me with: the possibility to discover the dopamine RPE signal in a well-organized environment with good grant support, and the safety, structuredness, and coziness of a pretty city between mountains and lakes that allowed us to bring up a family with three children in a joyful manner, including our daughter Carolina who was born in 1980. But I had become infected with, or spoiled by, a certain academic atmosphere as a postdoc in Creutzfeldt’s, Eccles’, and Ungerstedt’s laboratories. I encountered such an atmosphere in Cambridge during the 1993 stay with Tony Dickinson and Nick Mackintosh, and I was longing to have it again. I expressed my interest to Tony and some of the people I had met in Cambridge, including Trevor Robbins and Barry Everitt. At one point, Martin Johnson as head of the Anatomy Department heard about my interest and suggested that I look into a research professorship from the Wellcome Trust that would bring my laboratories to Cambridge. I applied to the Wellcome Trust and wrote a Home Office animal license application. When both were granted, my move to Cambridge was sealed for a start in October 2001.

I admit that a number of people were stunned by my easy movement from one country to another. Some people even thought I wanted to avoid Germany, but I had not the slightest such intention. It was a job and academic offer I could not refuse, nor get anywhere else at the time. I don’t take national allegiance too narrowly. If anything, I am European and would be comfortable to live in any western European country (as well as in the United States). Hence, moving to England for me was more like moving from one U.S. state to another, which Americans are accustomed to doing. The disrespect for local boundaries was reinforced by the excellent travel connections within Europe by Eurostar and the budget airlines that brought travel costs down and made visits with our adult children in Germany, Switzerland, and Britain frequent and routine events. One of my sons living in Geneva even took a job at King’s College London and travels there every week for term lectures and meetings, sometimes on the same day going back and forth. It is so nice and easy to benefit from the sophisticated culture of Europe!

Cambridge Work

Neuroeconomics: A New Research Direction

At one of Terry Sejnowski’s summer conferences in the 1990s in Woods Hole, I was not only charmed by this wonderful Cape Cod harbor village, but I was also impressed by a talk from Paul Glimcher who suggested investigating reward processing with formalisms of economic theory. He emphasized particularly the subjective nature of reward value, which in economics is formalized in various versions of utility theory. I had encountered utility in small lectures on game theory in Heidelberg, with many informal discussions with the lecturer. So, Paul’s presentation rang a bell to an initiated but otherwise naïve mind.

Applying economic theory to neuronal reward studies is not trivial: the amazing similarity between the dopamine RPE signal and the basic TD algorithms provides a biological foundation for efficient learning machines. As the algorithms are superb and should be further developed and exploited, why bother with biological details such as reward subjectivity, as some of my machine learning colleagues argue? Sure, the Bellman equation as a basis of TD learning already takes subjective reward value implicitly into account when describing reward maximization, so everything should be settled. But biological organisms are not silicon machines, and we need to know whether reward neurons indeed code subjective value as assumed by the Bellman equation. Thus, before going any further in the parallelism between dopamine responses and TD learning, we needed to know whether the dopamine signal represents reward value in a subjective but formal metric. Furthermore, utility coding might also explain how dopamine neurons deal with risk, which is crucial as rewards are, by default, uncertain. And how would the dopamine signal be involved in choice? This is where economic theory comes in. So, I started building my next experimental designs on the basic concepts of economic decision theory. The move to Cambridge offered the possibility to incorporate this new direction into the next 20 years of work even before neuroeconomics became a field.

My entry into neuroeconomics was aided by almost yearly visits to Caltech, which began with initial seminar invitations from John Allman around the year 2004 and were hosted by Colin Camerer and Ralph Adolphs. So, during most of my Cambridge time, Gerda and I spent about two months per year in Pasadena. This was not only sunny California but also provided easy personal access to world leaders in neuroscience and economics. I gave lectures and seminars, participated in a large NIH center grant, visited also with Richard Andersen and John O’Doherty, and learned economics from Colin Camerer, Charlie Plott, David Grether, Antonio Rangel, and Federico Echenique, which amounted almost to a private economics PhD. I was also impressed by Caltech’s attitude toward monkey neurophysiology. As a result, Richard Andersen’s monkey neurophysiology flourished, and its extension to human brain-machine interface (Aflalo et al. 2015) attracted a large donation from Tianqiao and Crissy Chen for a huge new neuroscience institute (with a Zen Garden in the basement).

The Dopamine Utility Prediction Error Signal

The economic value of a reward depends on its amount and the probability with which it occurs. A bigger reward has more value than a smaller one and a more frequent reward has more value than a less frequent one, provided satiety is ruled out. To understand whether dopamine neurons might code reward value thus defined, Christopher Fiorillo and Philippe Tobler tested rewards of different amounts occurring with various probabilities. Sure enough, most dopamine neurons increased their activity monotonically with both parameters (Fiorillo et al. 2003; Tobler et al. 2005). We then used the reinforcement learning framework to follow the intuitive, frequentist approach in probability theory that derives probability from the frequency of past events. Indeed, the frequency of past rewards translates into a neuronal code for reward probability (Lak et al. 2016). Thus, dopamine neurons code reward amount and probability as the two most basic economic value parameters. Furthermore, dopamine neurons are sensitive to both liquid and food rewards (and to higher-order visual, somatosensory, and auditory rewards) and code their value in a common currency (Lak et al. 2014), rather than being interested only in particular rewards. These value properties made dopamine reward signals amenable to a neuroeconomics approach.

To address subjective dopamine reward value coding in a simple manner, we used a traditional test called temporal discounting: reward delays decrease the subjective value of rewards without changing their physical properties. When monkeys choose the same reward at different delays, they prefer the early reward; their choices indicate an exponential value drop (Kobayashi & Schultz 2008), exactly as theory would have it. Sure enough, the dopamine responses to delay-predicting stimuli decrease in a similar manner, indicating sensitivity to subjective reward value. The evidence of subjectivity was restricted to temporal discounting, however, and a more general assumption of subjective value coding would require at least one other situation. Risk presented such a possibility.

Risk concerns both loss and uncertainty and comes in at least two forms. When trusting a surgeon with your life, you probably prefer one with a lower failure rate to one with a higher failure rate, everything else being equal. Here risk emphasizes loss while also dealing with uncertainty; it is identical to negative (probabilistic) value, and thus basically the opposite to (probabilistic) reward. By contrast, economic risk emphasizes uncertainty while also dealing with loss. When shopping for a car, you probably prefer one that works more or less well, and thus is only little risky, to one that works sometimes really well and on other days really poorly and thus is quite risky. You never quite know whether the riskier car works, and you incur a big loss when it doesn’t. Thus, economic risk varies with the difference between outcomes, with larger difference being riskier. It is captured by the higher statistical “moments” of probability distributions, primarily variance, skewness, and kurtosis. We implemented the simplest and unequivocally defined economic risk, the so-called mean-preserving spread in which either one or the other of two equally probable rewards occurs; both gambles have the same mean reward amount but different variance (Rothschild & Stiglitz 1970). Such abstract academic definitions may have real-world bearing: Stiglitz later became the chief economist of the World Bank and won the Nobel Prize. Monkeys often prefer the riskier to the less risky gamble despite their identical mean as long as the reward distribution is not skewed. Dopamine responses to cues predicting gambles are stronger with the preferred riskier gamble and reflect its higher value (Stauffer et al. 2014). Thus, as with temporal discounting, dopamine neurons code subjective reward value with risk, rather than with physical value.

The probability and subjective value coding of dopamine neurons provided enough arguments for trying utility functions that represent subjective value in a mathematically coherent way. Bernoulli claimed 280 years ago (1738) that agents value rewards nonlinearly, based on the observation that extra rewards add gradually less subjective value (diminishing marginal utility, represented by concave utility). Such functions have the property of all mathematical functions and can predict events that were not used for their construction, like choices. Von Neumann and Morgenstern’s (VNM) utility axioms (1944) conceptualize choice under risk and allow to estimate utility functions with cardinal numeric properties (immune to change in mean and variance), which is mathematically appropriate for neuronal response functions that are intrinsically cardinal.

We estimated utility functions iteratively from psychophysically estimated certainty equivalents in choices between a variable safe reward and a set gamble with two equiprobable rewards. I designed the test during one boring flight to the United States and inadvertently reinvented the fractile, chaining procedure described by Caraco (1980); so we were in business. We fitted splines to the certainty equivalents and obtained an S-shaped utility function that was convex with small rewards (0.1–0.5 milliliters, indicating risk-seeking), linear with larger rewards (risk neutrality), and slightly concave with larger rewards (1.0–1.3 milliliters, indicating risk avoidance). We also estimated risky and riskless utility functions from various direct parametric fits. The utility functions were similar between the different models but varied between individual monkeys. Although VNM utility is a first step toward a fundamental, well-defined decision variable, there are limits to its general applicability, which are addressed in Kahneman and Tversky’s prospect theory (1979) by adding probability weighting, reference dependency, and gain-loss slope differences. Now we had an experimentally viable method for assessing subjective value in a general mathematically defined way, which could be used for testing utility coding in dopamine neurons.

We did three tests: First, increasing amounts of unpredicted reward (eliciting a positive RPE) induced a nonlinear dopamine response increase that resembled nicely the curved utility function. That was encouraging but not a very controlled result. Second, in gambles with two outcomes, the larger reward elicited a positive RPE, and the smaller reward elicited a negative RPE. The RPEs were identical in gambles with identical spread, even if the gambles had different means. When the gambles were positioned at different parts of the nonlinear utility function, the identical RPEs became varying utility prediction errors (UPE): The same RPE became a small UPE where the utility slope was flat, and a big UPE where the slope was steep. Indeed, the dopamine response to the large gamble reward was small when the gamble was placed on the flat part of the utility function and large when the gamble was placed on the steep part (the typical low background activity allows only a small difficult-to-quantify depression induced by the small gamble reward). That result basically nailed utility coding by dopamine neurons and was confirmed by the third out-of-sample test. Being a mathematical function, utility predicts choice: A convex utility function predicts preference for a riskier compared to a less risky gamble, even with the same physical mean amount of the two gambles. The monkeys know the theory and follow that prediction, and their dopamine response as well: the neuronal response is stronger to the stimulus predicting the preferred riskier gamble compared to another stimulus predicting the less risky gamble. These three results suggest that dopamine neurons code economic utility and thus follow the necessary assumptions of a biologically relevant reward signal. Such dopamine signals could indeed be useful for updating decision variable coding in postsynaptic neurons. Now, Bernoulli’s suggestion from 280 years ago had a firm biological basis: Utility exists in the hardware of the brain and can be measured, which is much better than inferring it only from observable choice.

Then the real question arises: Would such a dopamine signal have behavioral consequences? The answer is yes. Artificial electric and optogenetic dopamine excitation and inhibition direct animals toward reward and away from nonrewards, respectively (Olds & Milner 1954; Corbett & Wise 1980; Tsai et al. 2009). Bill Stauffer in our laboratory confirmed and dissected the effects in monkeys. Optogenetic dopamine stimulation enhances dopamine responses to reward-predicting stimuli compatible with reinforcement learning and directs behavioral choice toward the stimulus associated with the stimulation (Stauffer et al. 2016). Thus, dopamine excitation follows the definition of reward function, generating learning and approach behavior. Dopamine inhibition has the opposite effect, generating unlearning and avoidance. As there are no real liquid or food rewards in these laboratory stimulation experiments, what the animals seek is a dopamine excitation, rather than a better real liquid or food reward that does not exist. Likewise, animals avoid dopamine inhibition, rather than a worse real reward that does not exist in the lab. Thus, when animals visibly seek and avoid real rewards outside the lab, the rewards are only the means to obtain the desired dopamine excitation and to avoid the hated dopamine inhibition. Or, when I encounter a better-than-predicted reward, it generates a positive RPE and dopamine excitation; when I go after that reward, I do it because I want to experience that dopamine excitation. Likewise for worse-than-predicted reward: I hate dopamine inhibitions and therefore will avoid the worse reward. Thus, when we go to the pub, we only seem to want drinks and food and see friends, but what we really seek is the dopamine excitation elicited by the prediction and experience of drinks, food, and friends.

And there is one more point (Schultz 2021): When we succeed in getting a dopamine excitation, the excitation increases the reward prediction in the next trial through reinforcement learning. But the increased prediction requires better reward for eliciting the same dopamine prediction error excitation the next time; to obtain that same dopamine excitation, we now need to seek an even better reward. When we obtain that better reward and the desired dopamine excitation it elicits, it increases the prediction again, and the iteration continues and drives us toward better and better rewards. Such a reward maximization process is beneficial for the individual decision maker, but it also makes us restless as we become dissatisfied with what we have; and it drives us continuously to ever better rewards. Little dopamine devils in the brain!

Reward Neurons Galore

The Striatum

Our recordings in nondopamine brain structures that served as controls for the rather homogeneous dopamine reward signal brought us to the striatum. We quickly realized a wider engagement beyond its traditional function in movement. Using a go/no-go task in which a particular stimulus required the animal to withhold a movement, we saw activity in many striatal neurons during the no-go period before the reward. It looked like we had found a movement inhibition signal, which had never been seen before. But then we used control go trials in which the reward was delayed by a similar duration between the movement and the reward and found a similar activity as in no-go trials. So, no movement inhibition activity, but maybe reward expectation? We played with the duration of the waiting period before the reward, and the striatal neurons continued to show the persistent activity before the reward, even with an 80-second delay, way beyond the sampling duration of our computer data collection program. The activity only went down when the animal lost patience and moved his arm a bit and then resumed for the rest of the 80 seconds when he settled down again. This reward expectation activity was just one of many forms of reward, stimulus, and movement processing at various task events in the striatum (Apicella et al. 1992; Hollerman et al. 1998). By contrast, neurons in the ventral striatum, also called nucleus accumbens, showed less diversity and more relationship to reward (Apicella et al. 1991). Thus, striatum neurons were at the crossroads between movement and reward; this was maybe the region in which the reward signals of dopamine neurons met the local reward and movement signals required for economic choice.

The recordings in the striatum allowed for a true extension of our reward work, beyond comparison with dopamine neurons. We were particularly interested in social comparisons of reward. When Raymundo Báez-Mendoza joined the lab from Mexico thanks to a Max-Planck master program, he set out to test cooperative choices of two monkeys in the popular prisoner’s dilemma. To provide well-controlled test conditions, we set up cameras by which each monkey watched the well-separated partner monkey. With all that technology, we slowly realized that this artificial lab situation was nowhere near the natural world of monkeys and that we might not trust the data we were to collect. We also contacted Chris Harris from our economics department, who suggested we use a dictator game as a scaled-down social test with less complexity. One monkey would simply decide how much of available reward he would get and how much the other monkey would get. A good social neuronal reward signal should make a distinction between which of the two animals gets the reward. We confirmed that our monkeys in the lab distinguished between their own and the conspecific’s reward and knew who acted. We found that most striatal neurons coded the monkey’s own reward rather than the reward for the other monkey. Importantly, the responses depended on which monkey performed the action that led to the own reward; different striatal neurons coded the reward for the recorded monkey when his own action or the other monkey’s action led to that reward, thus distinguishing the actor whose movement led to that reward (Báez-Mendoza et al. 2013). The actor-specific responses disappeared in some neurons when the other monkey was replaced by a computer, demonstrating specificity for biological agents. Thus, in one of the first social neurophysiology reward studies on monkeys, we had found a reward signal in striatal neurons that identified the social agent responsible for the monkey’s own reward.

Upstairs, the Orbitofrontal Cortex Serves Economic Choice

The frontal cortex is another major brain structure whose activity we wanted to compare with the dopamine reward signal, and the orbitofrontal cortex (OFC) was at that time the neurophysiologically least known frontal area. Edmund Rolls in Oxford had done pioneering reward studies (Thorpe et al. 1983), but there was more to be done when Leon Tremblay joined the lab from Canada via Paris. Starting with the task that had demonstrated sustained reward expectation signals in the striatum, we found similar reward expectation activity in OFC neurons, as well as responses to reward-predicting stimuli and rewards, but the signals were less frequent and less strong than in the striatum (Tremblay & Schultz 2000). With Leon’s usual imagination, he tried out something that originated more from a gut feeling than a theoretical concept. He wondered whether adaptation might occur in OFC neurons. He was guided by ideas from Horace Barlow’s and Simon Laughlin’s earlier work on retina adaptation that makes processing within the neuron’s limited coding range more efficient. He first established the monkey’s choice preference between two rewards. Then he asked whether the same OFC neuron might show different responses to the same reward when he changed only the alternative reward. Indeed, the neuronal response followed the animal’s preference: When the animal preferred a piece of apple to a piece of cereal, the neuron responded more to apple, but when the animal preferred a raisin to the same piece of apple, the neuron lost most of its response to the apple and instead responded well to the raisin (Tremblay & Schultz 1999). Although this neuronal change seemed compatible with adaptation to the current reward distribution (which changed between the two experimental steps), the responses also followed the definition of preference as the probability of choosing a reward from a given set of rewards. Formal economic preference coding would bring OFC signals into the domain of economic choice variables, which became an active research field after Camillo Padoa-Schioppa and John Assad’s ingenious experiments on common currency reward value coding in OFC neurons (Padoa-Schioppa & Assad 2006). But the adaptation issue stuck: When Shunsuke Kobayashi joined us from Tokyo, he thought that neuronal adaptation should not only occur relative to the mean value but also relative to the width (statistical variance) of the reward distribution. Indeed, some OFC neurons show steeper reward-response slopes with narrower distributions, and thus better reward discrimination; the response slopes were flatter with wider distributions, indicating better use of the neuron’s limited coding range (Kobayashi et al. 2010). Thus, besides the issue of reward preferences, OFC neurons adapt to the first two statistical moments of reward probability distributions as indication of efficient processing.

As the adapting OFC responses demonstrated sensitivity to reward statistics, we remembered variance as a definition of economic risk. We had seen a slow signal in dopamine neurons that increased with variance, but because of its slow time course, we thought it might serve the dopamine teaching function rather than economic decisions (Fiorillo et al. 2003). Now we wondered whether OFC neurons might carry an explicit risk signal whose latency and duration would be short enough to serve economic decisions and change monotonically with variance. We used Rothschild and Stiglitz’s mean-preserving spread and asked whether some OFC neurons show stronger responses to stimuli indicating higher risk, as defined by a larger spread between the top and bottom rewards of binary gambles. Indeed, a population of OFC neurons showed stronger responses with larger risk, but without coding reward value itself (O’Neill & Schultz 2010). Apparently, distinct OFC neurons coded reward value and risk, both of which are important for choice under risk.

The work on nondopamine reward structures encouraged us to investigate reward functions beyond reinforcement learning. There is the old question of how rewards that typically have several dimensions, and thus are vectors, could be coded as single-dimensional (scalar) neuronal signals. An apple has taste and amount, but how could the lower or higher activity of a reward neuron represent the combined value from the two components? The issue might be approached by looking at revealed preference theory, which describes how an integrated subjective value results from different reward components. For best experimental control, we would test choices between two “bundles” that each have the same two distinct rewards, like apple juice and grape juice, which we could set experimentally to specific amounts. When options with different compositions are chosen equally frequently, they are assumed to have equal subjective value. To participate in the choice between such options, individual neurons should show similar responses to these options despite their different composition, and more preferred bundles should elicit stronger OFC responses even if one bundle component was smaller than in a nonpreferred option. This question stimulated Alex Pastor-Bernier, who had joined us from Montreal and set out with great enthusiasm to test bundles with two juice components on monkey OFC neurons. To get familiar with the concepts and develop a feasible behavioral task, Alex first did an extensive behavioral study in which Charlie Plott at Caltech provided encouragement and the necessary economic background (Pastor-Bernier et al. 2017). I had met Charlie many years ago as a founder of experimental economics (now more fashionably called behavioral economics) during one of my early visits to Caltech and had benefited from his vast practical knowledge and lucid explanations. Based on the well-tested behavior, Alex went on to record from OFC neurons and identified neuronal signals that integrated the values from all bundle components combined (Pastor-Bernier et al. 2019). The neurons showed similar responses to bundles that were similarly preferred by the monkey (choice indifference), while showing larger responses to more preferred bundles (confirming the neurons’ sensitivity). The neuronal responses remained strongest with the preferred bundle even when a third bundle was added to the option set, thus complying with Arrow’s weak axiom of revealed preference and the independence of irrelevant alternatives (IIA) as a requirement for utility maximizing choice. Interestingly, humans often violate the IIA. This contrast does not necessarily indicate that monkeys are more rational than humans but may reflect the fact that monkeys have experienced tens of thousands of such tests and thus don’t get fooled, whereas humans are tested only a few times and get it wrong more easily. As a solid confirmation, a support vector machine classifier correctly predicted behavioral choices from neuronal responses. With this result, the single-dimensional OFC reward signal seems to be involved in choices of typically multidimensional rewards.

The bundle design provided us also with a chance for testing selective reward satiety. Earlier, nonbundle tests had failed because satiety for one reward destroys the monkey’s appetite for all rewards. By contrast, the finely controlled reward adjustments afforded by the bundle design preserves the animal’s motivation. Indeed, the choice indifference curves show graphically the value reduction of the sated bundle reward in a beautiful, intuitively understandable pattern, and the OFC responses follow the differential change (Pastor-Bernier et al. 2021). Thus, the concepts of economic theory had allowed us for the first time in neurophysiology to identify reward-specific satiety in a controlled manner.

An Open Mine: The Amygdala

While the reward signals in striatum neurons provided a stark contrast to the dopamine reward responses, our experience with OFC neurons went well beyond comparison with dopamine responses and showed us signals for an economic decision machine. But there was one more brain structure in which we suspected major reward signals. As judged by current reviews, the amygdala is primarily involved in aversive processing, because fear is lost after lesions and neurons respond well to fear-inducing stimuli. But we were not convinced. When Istvan Hernadi joined the lab in 2002, he irreverentially designed, together with Ken Tsutsui, a sophisticated reward and economic choice schedule in which the monkey chose between saving a reward with interest for future consumption and immediately consuming the already saved reward. Sure enough, amygdala neurons responded well to reward and reward-predicting stimuli, and their activity transitioned from reward value to coding the actual choice. Amygdala activity could even predict whether the monkey would save more or cash in the saved reward. In the meantime, Dan Salzman convincingly demonstrated amygdala reward responses in a straightforward Pavlovian task (Paton et al. 2006), which discouraged our immediate publication. Then Fabian Grabenhorst joined the lab and wanted to apply his data analysis skills from his Oxford PhD with Edmund Rolls. His analysis demonstrated an amygdala involvement in multiple aspects of sophisticated reward processing and economic choice, thus advancing Dan Salzman’s amygdala reward signals toward economic choice (Grabenhorst et al. 2012; Hernadi et al. 2015; Grabenhorst et al. 2016).

After Istvan had left for his native Hungary, Maria Bermudez came from Spain and tested more basic theoretical concepts of reward processing, such as reward contingency. A stimulus is only learned as a reward predictor when the reward occurs more frequently in the presence of the stimulus compared with its absence. Simple stimulus-reward pairing does not explain learning (although it is often claimed so); even a stimulus that is paired with a reward does not become conditioned as a reward predictor when the reward occurs as frequently without the stimulus as with the stimulus, because such a stimulus does not contain distinct reward information. The crucial function of reinforcer contingency was unknown to Pavlov and was first demonstrated with aversive stimuli by Robert Rescorla, but it had never been shown before with neurons. Indeed, Maria’s amygdala responses to reward-predicting stimuli disappeared when the reward occurred as frequently without the stimulus as with the stimulus, thus demonstrating a neuronal correlate for crucial contingency (Bermudez & Schultz 2010). Further experiments by Maria demonstrated adaptive coding of reward amounts and sensitivity to reward timing as typical features of reward neurons, which encouraged further consideration of the amygdala as a key reward structure.

But our curiosity did not stop here. Lesion studies of the amygdala have indicated a social function. By the time Fabian was done publishing Istvan’s data, he had accumulated a full data set from his new experiments. We had designed a task in which the monkey learns the value of probabilistic rewards from repeated choice and observes a partner monkey sitting opposite (Grabenhorst et al. 2019). Based on his own experience, the monkey learns the reward-predicting stimuli of the other monkey and might understand and simulate his choices. Besides coding their own reward, amygdala neurons learned the value of the reward for the other monkey. A support vector machine classifier developed by Fabian and Arek Stasiak showed that the monkey’s own amygdala responses predicted the partner monkey’s choices. Thus, while the sophisticated reward signals in the amygdala extended well beyond its traditional role in fear, amygdala neurons are even engaged in social reward processing.

A Look Back

Would I become a scientist again? Definitely, if one could make it a paying profession. It is never guaranteed that the stuff one imagines and designs works. It often looks more like wonder. Yes, there is a gut feeling of what may work and what may not work, but gut feelings can be wrong. Distractions are there, and they can compromise scientific productivity. I believe in the notion that 90 percent of our activities are governed by unconscious processing. You are focusing on a text, and when you wake up in the morning you suddenly have the exact formulation on which you were laboring. Your brain did that for you. But if you flood your brain with tons of unnecessary things, the brain will faithfully advance on these unnecessary things, and the interesting stuff gets left behind. So, your first thoughts in the morning tell you what your mind works on. Even better, you can preload your brain with your problem before sleep, and the brain works it out and tells you the solution in the morning. Thus, your brain’s night job does what you are being paid to do during the day and which brings in the big money that your dopamine neurons seek.

Would I become a neuroscientist again? That is more difficult to answer: what would I want to investigate? Many basic processes are known by now, published in hundreds of thousands of papers. There is of course one big and most important question that has stubbornly escaped understanding and might have the importance of Watson and Crick’s double helix: consciousness. It would be fantastic to contribute, but one needs to generate ingenious experimental plans and lots of concepts. The other direction is human neuroscience. The new methods for investigating the human brain in action are fantastic and have the added advantage of addressing clinical questions, which is very moral and justified when it comes to the responsible use of research money, often provided by the taxpayer. And of course, there will always be a new method, like optogenetics currently, which is very exciting. But then, what do you want to discover with the new method that is not already at least partly known? Would this mean filling important gaps, which can be satisfying and attract big grants, or can one, maybe inadvertently, make a surprising discovery? These are the reasons why so many people are interested in new methods that open new research avenues.

Why did my work work? Well, factors were surely luck, persistence, sweat, diligence, stubbornness, and irreverence toward established career goals. I started with a temporary and inferior faculty position in Fribourg, but it became quickly permanent and allowed me to apply for rather easy, relatively small grants with good advice (notably from Dr. Winkler at the Swiss National Science Foundation). This unambitious position was no way to make a career, but I could advance as slowly as necessary to understand what I was doing and work gradually toward a good grasp of the data rather than following publication pressure.

What could have gone wrong? Everything. There is no way to predict a surprising discovery. But my head of department was impressed when a departmental colleague working on blood vessels told him that by experimenting with dopamine neurons, I might be doing something of potential importance. But that was not the point: what mattered was that I could do what I felt was interesting. I did not focus on immediately obvious research questions, and I even had difficulties understanding them; I rather felt that we scientists should generate new problems, in the sense of identifying a new paradigm worth the effort, and then go for it when it seemed to work.

Did people understand what I was doing? It is difficult to say, but initially not many, and I felt sometimes outside of mainstream discussions at conferences. While some people did not sign on, others appreciated the cautious and tedious progress and encouraged me. Discovery at any level does not come for free and is never certain. There is always frustration when trying to understand what one is doing and seeing. The struggle is not always apparent when looking at the result, in particular, when it finally, after lots of effort, becomes simple and straightforward: everything is clear, so why did it take so long to figure it out? It took us 20 years to unravel a particularly telling reward signal, and another 20 years to characterize its biological usefulness, but at the beginning even the term of reward needed a better definition, which is not the case when working on the visual system, for example. People gave us credit for such risky undertaking. In addition, there are the difficulties of experiments with capricious monkeys, supervised by capricious humans, when the animal won’t perform the task, and I still don’t know whether this is the right test. These experiences teach me something, and I feel the progress—tedious, slow, and never sure, but ultimately a lot of fun and very rewarding.

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Mirenowicz J, Schultz W. Preferential activation of midbrain dopamine neurons by appetitive rather than aversive stimuli. Nature 379: 449–451, 1996.

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David L. Sparks

BORN:

Guntersville, Alabama

December 22, 1937

EDUCATION:

University of Alabama, BA (1959)

University of Alabama, MA (1962)

University of Alabama, PhD (1963)

APPOINTMENTS:

Postdoctoral Research Fellow, University of Mississippi Medical School (1963–1965)

Instructor in Psychiatry, University of Alabama in Birmingham (UAB) (1965–1967)

Assistant Professor of Psychiatry, UAB (1967–1969)

Associate Professor and Chair of Psychology, UAB (1969–1972)

Professor and Chair of Psychology, UAB (1972–1973)

Professor of Psychology, UAB (1973–1981)

Professor of Physiology and Biophysics, UAB (1981–1989)

Senior Research Scientist, Neurobiology Research Center, UAB (1987–1989)

Trustee Professor of Behavioral Neuroscience, University of Pennsylvania (1989–1997)

Professor, Department of Neuroscience, Baylor College of Medicine (1997–2004)

HONORS AND AWARDS (SELECTED):

University Scholar, University of Alabama in Birmingham (1976–1981)

Recipient of Minerva Foundation Golden Brain Award (1987)

Fellow, American Association for the Advancement of Science (1990)

Sprague Lecture, Institute of Neurological Sciences, University of Pennsylvania (1996)

Recipient of an NIH Merit Award: (5 R37 EY01189) (1987–1997)

Elliot Stellar Lecture, University of Pennsylvania (2001)

The Sparks lab used the primate superior colliculus (SC) as a model for studying how sensory signals are transformed into commands for the generation of movements. The lab discovered cells in the SC with high-frequency bursts of activity tightly coupled to the onset of saccades (the quick, high-velocity movements of the eyes), which are involved in the initiation of saccades. The accuracy of saccades results from a combination of the movement tendencies produced by the entire active population of coarsely tuned cells rather than the discharge of a small number of finely tuned cells. The saccadic system does not localize visual targets based solely on information about the region of the retina activated, but also uses information about the current positions of the eyes in the orbits to localize the target in a more abstract spatial frame of reference. The visual, auditory, and somatosensory signals observed in the SC specify the movement required to direct gaze to the location of a stimulus, not the location of the stimulus in external space. Information about the change in eye position used for remapping sensory signals into motor coordinates is still available after extraocular muscle proprioceptive signals are eliminated by surgical sectioning of the afferent pathways. Corollary discharge, a copy of a motor command, must have provided the eye-position information instead. The motor map in the SC represents a command for a change in gaze, not separate eye, or head commands. Correlations between the activity of neurons above the level of the motoneurons and the parameters of the eye movement may be misleading when more than one oculomotor subsystem is active. The collicular signals involved in specifying the direction and amplitude of a movement are separable from those initiating the action.

David L. Sparks

Overview

The manuscript for this autobiography was due November 1, 2021—two months shy of the 17th anniversary of my retirement date. In those 17 years, I have enjoyed extended time with family, and I communed with nature while sailing in French Polynesia, the Strait of Georgia, and along sections of the U.S. coast of the Gulf of Mexico. I renewed my interest in nature photography and photographed birds and other animals on all seven continents. As I learned while writing this article, I also forgot many of the details about the research done in my lab during the 40 years before retirement!

In this autobiography I focus on telling the story of how my interest in neuroscience developed and how I adopted a motor, rather than perceptual, perspective on the processing of sensory signals. The story has a somewhat unusual trajectory. It is a testament to the impact that textbooks and published papers can have on the interests and actions of a reader. I still marvel at the chance encounters that led to, and facilitated, a research career trying to understand how sensory signals are translated into commands for the control of movements.

To allow for grouping of experiments relating to particular topics, sketches of research projects are separated from descriptions of the different faculty positions I held and of the extraprofessional pursuits at each place of employment. Students, postdoctoral fellows, and colleagues are introduced after descriptions of their research projects.

Family Background

I was born in Guntersville, Alabama in 1937, the eldest of the two sons of Lee and Ruth (Mooney) Sparks. During my early childhood, we lived in Alabama, Tennessee, Georgia, Kentucky, and North Carolina while my father worked on many of the dams that the Tennessee Valley Authority (TVA) built on the Tennessee River to provide power, improve navigation, and control periodic flooding. The TVA was part of President Roosevelt’s New Deal designed to help the United States recover from the Great Depression.

Both of my parents were born in Alabama. My mother graduated from high school, but my father dropped out of school after the ninth grade. He was a skilled operator of heavy equipment but progressed to the roles of foreman and superintendent on construction projects during the earthmoving phase. Later, when I was in high school and college, he worked on the construction of a power plant in Kentucky; turnpikes and interstate highways in Ohio, Michigan, Illinois, Indiana, and Pennsylvania; the Lewis Smith Dam in north Alabama; Keystone Dam near Tulsa, Oklahoma; and the nuclear test site in Nevada.

Between 1937 and 1954, when I graduated from high school, my best estimate is that we lived in 18 different towns or cities in seven different states. I do not remember where we lived when I started the first grade, but I was permitted to start at age five. I skipped the fifth grade. The material to be covered in the fifth grade in Florida was mostly a repeat of what I had studied the year before in three different public schools in Georgia, a system that had only 11 grades at that time. I regretted graduating from high school at age 16 because I could have had two more years to play high school basketball, my favorite sport. Needless to say, the peripatetic nature of my father’s work resulted in a very spotty educational background. Continuous compensation was required.

I adjusted to the frequent moves by creating my own cognitive world, a space that did not need to be left behind when we changed locations. Happiness, aspirations, feelings of success or failure, and other psychological states occurred within that world and were not dependent on input from individuals who would not make the move with me. I developed a sense of independence that allowed me to follow the path paved with chance encounters that led to a research career in the field of neuroscience.

My father enjoyed fishing and hunting. Fish, quail, and other game were a significant part of our diet. My mother was a homemaker. She managed to keep track of our possessions during all our moves. She strongly encouraged us to be good students and pinched pennies to purchase a World Book Encyclopedia and several volumes of Will and Ariel Durant’s The Story of Civilization and to pay for our piano lessons.

Memories of most of the places we lived when I was a child are sparse. My most vivid childhood memories are from 1948 and 1949 when my father worked on a construction project in Saudi Arabia. My mother, my brother, and I stayed in the United States and lived in a small house close to the home of my maternal grandparents on their small 80-acre farm in northern Alabama. There was a barn for storing hay and maintaining livestock, a potato house, a chicken house, and a smokehouse for preserving the salt-cured or smoked meat of the hogs that were slaughtered after the onset of cold weather. A large garden and orchard provided enough vegetables and fruit for consumption during the growing season and for canning, which preserved them for the remainder of the year. Neither house had running water the first year we lived there. Electricity had only recently become available. A pump was added to the well during the second year, and water lines were run to the small house where we lived. I no longer had to carry buckets of water from the well at my grandparents’ house to the house where we lived.

I learned about farm life by helping with the farming activities of my two uncles and my grandparents when I was not in school on weekends, holidays, during the summer, and the annual two-week break from school, a break that allowed students to assist in harvesting crops. In northern Alabama, this was primarily cotton-picking time. I was 11 and 12 years of age during this period. My uncles and grandfather taught me how to manipulate the combination plow and hopper filled with corn seed. As the plow was pulled by the mule (Betsy), seeds dropped into the furrow and were covered by the rear of the planter. The difficult parts for a young boy were making sure the seeds were dispensed in a straight row and getting the planter turned around and headed in the opposite direction at the end of the row. Betsy was very experienced and made the learning process less arduous by being tolerant of the novice that was following behind.

Cotton seeds were spaced such that when they germinated and sprouted, there was a continuous row of plants. When the plants reached a certain height, it was time to “chop” the cotton. This was done manually. A hoe was used to thin the plants so that the space between them would accommodate the mature plants with their branches and bolls. In the process, weeds were removed, and the remaining plants were propped up with a mound of dirt. As I was learning to do this, one of my uncles would be working an adjacent row while checking my work and correcting my errors. But after a couple of days, I could “carry my own row.”

A long sack made of heavy cloth and reinforced on the bottom was used for picking cotton. The shoulder strap allowed it to be dragged along the ground while using both hands to remove the cotton from the bolls. Initially, one of my uncles would be on an adjacent row checking to make sure I didn’t leave too much cotton in the boll and didn’t put too much trash in the sack until I was once again trusted to carry my own row. The most cotton I was able to pick in one day was 150 pounds.

When the first wagon was full of cotton and ready to go to the cotton gin, my grandfather told me to get aboard. He drove the horses the seven miles to the cotton gin on the outskirts of Decatur, Alabama. After the wagon was unloaded and he received his payment, my grandfather took me to the Blue Bird Cafe and bought us ice cream cones. As we sat at a small table in the cafe, he pulled out his book and totaled how much I had earned that year ($3 a day for chopping cotton, planting corn, etc., $3 for each 100 pounds of cotton picked). I don’t remember the total, but, psychologically, I was wealthy. Developmentally, these interactions with my grandfather were important. He reinforced hard work and emphasized the importance of education by giving me problems, such as converting feet and yards into acres, furlongs, and hectares. When not doing farm work, I roamed the woods and fields with my brother and with Ralph and Ray, nonidentical twins about my age, who lived a few miles from our house. We swam in the local creeks and fished in all the ponds located on the surrounding farms. These were the years when I learned to love being outdoors, a passion that carried over to later years when, at various times, activities such as camping, fishing, hiking sections of the Appalachian Trail, sailing, and nature photography became my way of accessing and enjoying nature.

My brother, Gary, earned a bachelor’s degree in architecture from Oklahoma State University (OSU) in 1966. He worked for architectural firms in Tulsa after his service in the U.S. Army until he started his own firm in 1986. His most noted architectural projects were the expansion, renovation, and preservation of the historically valued sporting facilities on the OSU campus. In 1995, there were rumors that the once-famed, but now outdated, Gallagher-Iba Arena constructed in 1938 would be replaced by a new off-campus arena. The existing arena was the home to more NCAA Championships than any other facility in America. It was named for Edward Gallagher, the university’s first wrestling coach whose teams won 11 NCAA team championships, and the basketball coach, “Hank” Iba, whose teams won national championships in 1945 and 1946. Gary thought it would be a tragedy to destroy the arena with its rich history and the special 1-1/4 inch white maple basketball court. He devised a plan by which the historic building would be preserved and renovated, and seating capacity increased from 6,000 to 13,600 by constructing a new seating bowl and roof structure over the existing arena while continuing to have events during the two-year construction process. He convinced the decision makers that this could be done, and his firm was awarded the contract.

College and Graduate School (1954–1963)

Upon graduating from high school in 1954, encouraged by my parents (devout Southern Baptists), I attended Tennessee Temple University, a four-year private Christian university located in Chattanooga, Tennessee, for two years. Finding the course offerings very limited compared with what I saw in the course catalogs of other universities, I transferred to the University of Alabama in Tuscaloosa. My parents were Alabama residents at that time, and the lower cost of tuition for in-state residents was a determining factor in the choice of an alternative college. My parents assisted with tuition expenses, but I covered much of the cost of attending college with my savings, earned each summer while working on the construction jobs my father acquired for me. The first summer, I worked as a manual laborer, but the next summer I worked as a mechanic helper. Oil changes, lubrication, and minor repairs of the large pieces of equipment occurred during lunch breaks and in the interval between the two 10-hour shifts. While working as a mechanic helper, I learned to operate some of the heavy equipment. In subsequent summers, I worked as an operator of Caterpillar Dozers and the Caterpillar DW21 Scraper. When operating the heavy equipment, I earned a little more than $3 per hour and usually worked 50–60 hours per week.

As I sampled the broader range of courses available at the University of Alabama, I gravitated to those offered by the Department of Psychology. Three of the psychology courses (Experimental Psychology, Physiological Psychology, History and Systems of Psychology) and a course in the Philosophy Department that included a section on the philosophy of science were the ones that most intrigued me. The textbooks in the psychology courses I enjoyed were Experimental Psychology (1949) by Benton J. Underwood; Physiological Psychology (1950) by Clifford Thomas Morgan and Eliot Stellar; A History of Experimental Psychology (1950) by E. G. Boring; and Experimental Psychology (1954) by Robert S. Woodworth and Harold Schlosberg. I graduated with a BA in psychology in 1959.

In his Notes for a Life Story, S. S. Stevens wrote “Any man’s life builds on a succession of accidents and that among the chance encounters, there are some that take effect while others do not.” Several of the chance encounters that affected either my choice of a career or facilitated my research efforts are described in this autobiography. The first occurred in my senior year of college. History and Systems of Psychology, a required course for an undergraduate major, was being removed from the undergraduate listing and becoming one of the core courses of the graduate program. In this transition year, most members of the class were graduate students.

Perhaps that motivated me because I was receiving good grades on the exams. One day the professor, George Passey, stopped me as I was leaving the classroom and asked if I had considered graduate school. I told him I had not. He then asked what I would do if he promised me an assistantship that would pay tuition and provide some funds for living expenses. I told him I would go to graduate school.

I entered graduate school the following fall as a relatively undifferentiated student. I knew my interest was in experimental, rather than clinical, psychology but I did not have a particular area of experimental psychology in mind. The seminar offered by Dempsey Pennington, a visiting professor, was the chance encounter that was decisive in my choice of a career path. The Organization of Behavior by D. O. Hebb and Science and Human Behavior by B. F. Skinner were among the books and readings selected for discussion. Interestingly, my decision to pursue a career studying the biological bases of behavior was stimulated by Skinner’s book, not Hebb’s.

I recognized the importance of Hebb’s proposed mechanism for increasing synaptic efficacy and his attempt to bridge the gap between neurophysiology and psychology. But, as a first-year graduate student, I was not excited by it. I was unable to envision ways of testing his theoretical formulations experimentally using the methods then available to an investigator.

In contrast, I had very strong negative reactions to many of the statements in Skinner’s book. He argued that psychology should engage exclusively in research that would eliminate the type of experimentation that had aroused my interest in the field. According to Skinner, placing an emphasis on physiological variables obscured attending to external ones, the variables he thought should receive priority. Skinner recognized a “causal chain” with three links: an operation performed on the organism, an inner condition, and a kind of behavior. Skinner did not deny the existence of internal states. “The objection to inner states is not that they do not exist, but that they are not relevant in a functional analysis.” Skinner maintained that it was not possible to account for behavior while considering only inner conditions, and that if it was always necessary to go back to variables outside the organism, it would be possible to “avoid many tiresome and exhausting digressions” by simply studying behavior as a function of the external variables.

The strength of my negative reactions to Skinner’s assertions was informative. I had not recognized that I felt so strongly about the need to learn about the internal events associated with behaviors. I began thinking about how to counter Skinner’s arguments in favor of pursuing a strictly behavioral approach to experimentation. I had “conversations” with Woodworth and Schlosberg by rereading their chapters on vision. Suppose, I asked, we had avoided the “many tiresome and exhausting digressions” of learning about the types of receptors in the retina, the anatomical distribution of rods and cones, and their differential sensitivity to light levels. Would an accurate estimate of the visual sensitivity of the human visual system be available? Not knowing that the receptors were not evenly distributed, those trying to obtain an estimate would not know to control the fixation point of the subject. Lacking information about the sensitivity of the different receptors to various wavelengths, an arbitrary range of wavelengths might have been used for the test stimulus. After a few of these “conversations,” I realized that, for me, whether or not my ruminations countered any of Skinner’s arguments, I was very much interested in the anatomical and physiological aspects of sensory systems and how they related to the results of psychophysical studies. I was fascinated by how biological information informed the design of behavioral experiments, and the ways that behavior could be used to test hypotheses about underlying biological mechanisms. I was attuned to Woodworth and Schlosberg’s point of view: “Research at all levels is important in giving us a full account of vision — no single level is enough. The brilliance, hue, and saturation of a color sensation depend not only on the stimulus but also, to a marked degree, upon the receiving apparatus and on its condition.”

These reactions suggested that the biological bases of behavior could be the direction I should pursue as a career. I began spending hours in the library. I discovered the Journal of Comparative and Physiological Psychology and the Journal of Neurophysiology. I found papers from Vernon Mountcastle’s lab and resonated with his effort to quantify neural events at successive stages of the somatic afferent central projections, his expectation of finding lawful transformation between peripheral sensory and central neural events, and his hope of learning “which quantitative aspects of the neural response tally with psychophysical measurements” (Mountcastle et al. 1963, Journal of Neurophysiology, 26: 807–834). Now, instead of just being interested in some area of experimental psychology, I had a goal. I needed to find a way to become involved in studying the three-dimensional correlations between the physical properties of the stimulus, the psychophysical report of the subject, and the intervening neural events. Although there were excellent experimental psychologists on the faculty, none were studying the biological bases of behavior. But I had a lot to learn and needed to address the inadequacies in my knowledge base. I changed my graduate minor to biology and took courses in mammalian anatomy, histology, and genetics and continued my voyages to the library.

A third chance encounter was with a second-year graduate student, Bob Travis. He had worked in Jim Olds’s lab at the University of Michigan as a research assistant for several years after his undergraduate degree. He had a small office in the abandoned army barracks building that was being used to house the animals used in experiments conducted by faculty members and students and oversaw the animals. Bob and I had frequent conversations. One day I mentioned reading an interesting article in Science about a phenomenon called spreading cortical depression (SD), a depression of brain activity reflected in changed EEG patterns following application of electrical, mechanical, or chemical stimuli to the cortical surface. The paper used SD produced by the application of potassium chloride (KCl) to the cortex as a functional unilateral ablation technique because SD is restricted to a single hemisphere. Bob then told me about the experiment he had been involved in when Jan Bures visited the Olds lab. They studied the effects of SD on the rate of electrical self-stimulation in rat. After further discussion of the article, we designed an experiment that evaluated the effect of prior training under varying degrees of SD on the subsequent performance of the nondepressed organism. We built an avoidance box, obtained 25 percent KCl from a local drugstore, and constructed a trephine of the appropriate size to place an opening in the skull that would allow a two-millimeter-by-two-millimeter piece of filter paper soaked in the KCl solution to be placed on the surface of a rat’s dura. This was done by welding a section of a fine-toothed hacksaw blade around a small-diameter welding rod. The relays, photoelectric switches, and electronic timing units necessary to automatically schedule the experimental events were available in the lab.

Not attuned to the fact that we should let someone on the faculty know what we were doing, we conducted the experiment, wrote up our results, and submitted a manuscript to the Journal of Comparative and Physiological Psychology. After a few months we received the two reviews. The first sentence of the first review read “the title is a meaningless word salad,” and the comments went downhill from there. The cover letter from Eliot Stellar, the editor of the journal and one of the authors of my undergraduate text in Physiological Psychology, stated that despite the harshness of the criticisms, he believed that if we performed the yoked control suggested by one of the reviewers that the results were interesting and worthy of publication. We did the additional experiments and asked one of the faculty members for assistance in preparing the manuscript, and this became my first original scientific publication (Travis and Sparks 1963).

Many years later, I met Eliot Stellar when interviewing for a position at Penn. Eliot was provost. Of course, he had no memory of his role in the publication of the SD paper when I told him this story 27 years later. Eliot died while I was still working at Penn. In 2001, after I had moved to the Department of Neuroscience at the Baylor College of Medicine, I was honored to accept an invitation to give the Stellar Lecture at Penn.

The textbook for the Sensation and Perception course taught by George Passey was The Human Senses (1953) by F. A. Geldard. Too large to be required as a textbook, multiple copies of the Handbook of Experimental Psychology (1951) by S. S. Stevens were available in the departmental library. The content of this course reinforced my decision to focus on the biological bases of behavior.

Even though no faculty member was involved in research related to the biological bases of behavior, Bob Travis and I were permitted to conduct research related to our interests for our dissertations. Bob did a series of SD experiments for his thesis, and I studied how radio-frequency lesions in several limbic-system structures affect active and passive avoidance behavior.

The next chance encounter was highly significant for me. The event was the first meeting of the lab component of an upper-level undergraduate course in genetics that I was taking as part of my graduate minor. As students arrived, they were told to pair up as lab partners. The even number of students in the room when I arrived had already formed pairs. For a brief period, I was the only student without a lab partner. A few minutes later, Betty Ellis arrived, and we became lab partners. Our lab exercises involved selecting fruit flies with certain physical features, allowing them to mate, and cataloging the characteristics of the offspring through as many generations as possible. Coffee after class transitioned into study dates, which evolved into a state in which we were considering marriage.

Betty obtained her BS degree before I received my PhD. She graduated in January 1963 and accepted a research assistant position at Southern Research Institute in Birmingham. After Betty began working in Birmingham, I learned that my application for a National Science Foundation (NSF) Postdoctoral Fellowship in the Department of Neurosurgery at the University of Mississippi Medical School in Jackson, Mississippi, had been approved. We needed to decide if we were staying together. The wedding was in Birmingham on the last day of August 1963. The day after the marriage, we drove to Jackson in the 1958 black Chevrolet Impala convertible I had won in a poker game.

Postdoctoral Fellowship (1963–1965)

Betty began work as a research assistant in the lab of Dr. Charles Randall, the chair of the Microbiology Department at the University of Mississippi Medical School. My goal during the two years of the NSF Postdoctoral Fellowship was to increase my knowledge about the nervous system and to learn different research methods. I took courses in neuroanatomy, histology, and medical physiology and volunteered as an assistant in the neuroanatomy labs the second year. I began research projects in the lab of Irwin Powell in the Department of Anatomy. I learned several methods of anatomical tracing that were being used in his lab, and we published a paper on the interaction of evoked potentials in the anterior thalamus of the cat.

Don Foshee (joint appointment in neurosurgery and in physiology and biophysics) and I published a paper describing experiments in which we recorded EEG activity from a variety of subcortical sites to determine whether seizure activity accompanied electrical self-stimulation of hypothalamic structures producing high self-stimulation response rates.

I observed, but did not participate in, the intracellular recordings from rabbit retina performed in the laboratory of Fred Sias. Fred also taught a basic electronics course for graduate students, which I attended. That course was followed by another course about the use of digital logic components in lab instrumentation. The knowledge I acquired from the two courses was invaluable when setting up a lab at the University of Alabama in Birmingham (UAB).

The Neurosurgery Department had a small library adjacent to the departmental office, containing a wonderful assortment of books and journals. I had 24-hour access to the library and spent a lot of nighttime hours there. In my spare time, I taught a physiological psychology course at nearby Millsaps College, a private liberal arts college in Jackson.

In 1964, I was a participant in a special six-week Summer Institute on Behavioral Genetics at the University of California, Berkeley, organized by Gerald McClearn and James McGaugh. In addition to what I learned about behavioral genetics, I was able to sample the knowledge and research experience of participants from other universities and assess where I stood in terms of preparation for a career in behavioral neuroscience. Participants had access to the university’s library. I compared the books on reserve for various courses in biology and psychology with the ones I had read and found several, especially ones related to neuroethology, of which I was unaware.

Betty and I enjoyed being tourists on the weekends while in Berkeley. We checked out Carmel, Pebble Beach, Cannery Row, and the tide pools off Monterey where Doc, the main character of John Steinbeck’s novel, spent so many hours. One highlight of our tourist activity was freezing in Candlestick Park during a night game between the Dodgers and Giants that lasted four hours and three minutes. Koufax pitched for nine innings; Gaylord Perry for five. Willie Mays got one hit and had one stolen base. The Giants won in 13 innings.

Support from the NSF Postdoctoral Fellowship was for only two years. I needed to find a job. Bob Travis had acquired a position in the Department of Psychiatry in the Medical College at UAB, and there was a possibility that I could join him there. During the interview with Jim Sussex, the chair of the Department of Psychiatry, I learned that S. Richardson Hill, the new dean of the Medical College, had begun evaluating the clinical programs based on research productivity as well as clinical service. Sussex said he looked at the credentials of his faculty and saw that he would need to recruit additional members of the department to generate significant research output. Bob Travis had been added to the faculty two years earlier. Sussex said he was now in the position of having one lovebird. I was offered a position as a second lovebird. I accepted the offer. We moved to Birmingham in June 1965 and lived there for 24 years.

University of Alabama in Birmingham (1965–1989)

Overview of Faculty and Research Positions

Both Betty and I were employees of UAB, and each of our positions changed several times. She worked for Drs. Wayne and Sarah Finley in the Laboratory of Medical Genetics from 1965 until 1970. She taught biology labs part-time at Samford University (a private university in Homewood, Alabama) from 1973 to 1978 to be home when school hours were over for the boys (more about them later). In 1981 and 1982, Betty worked in the lab of Dr. Jimmy Neill, chair, Physiology and Biophysics at UAB. She noticed that biology had changed so much that her degree (1963) was out of date. To compensate, she entered the graduate program in biology and received a MS degree in 1983. She then began working for Dr. Ken Zuckerman, deputy director of the Hematology–Oncology Division of the Department of Medicine. From 1985 until 1989, Betty worked in the laboratory of Dr. John Whittaker, chair of the Department of Neurology.

My initial appointment at UAB was in the Division of Behavioral Science in the Department of Psychiatry, first as an instructor and then as assistant professor. This was a full-time research position, but I did teach the Physiological Psychology course in the Psychology Department in the newly established College of General Studies. George Passey, the professor who offered me the assistantship that enabled me to enter graduate school, was now head of the social sciences division of the College. UAB, located in the largest city in the state, became an autonomous component of the University of Alabama system in 1969 and I moved to the Department of Psychology as chair and associate professor. It was obvious to me that growth in enrollment would be rapid and that graduate programs would emerge as the faculty grew in size and areas of expertise. Accepting the position as chair allowed me to influence the direction of growth, to recruit faculty with a strong interest in research and teaching, and to make sure the administration provided space and facilities for their research programs. I resigned as chair in 1973 to have more time for research after a proposal for a graduate degree in psychology had been submitted. I remained in the department as a professor until 1981, when I switched back to the medical school component of UAB as Professor of Physiology and Biophysics. While at UAB I was an active participant in two interdisciplinary groups: visual sciences and neuroscience.

Extraprofessional Pursuits While at UAB

Betty was a member of groups that raised funds to support blind and disabled individuals and to support the Birmingham Symphony. She completed the classes and voluntary work required to become a Master Gardener. In the spring, Betty and I would usually go to Sarasota for the annual meeting of the Association for Research in Vision and Ophthalmology. Bob Travis and I walked the Appalachian Trail from Davenport Gap to Clingman’s Dome each year for several years.

Our three sons, Steve, Greg, and Scott were born in 1965, 1967, and 1971, respectively. The family spent many summer vacations at Perdido Bay (Pirates Cove) near the pass to the Gulf of Mexico close to the Alabama/Florida border. The boys and I would leave to fish in the gulf in our Boston Whaler boat before sunrise. After fishing, we would go sailing, water skiing, and shrimping. We enjoyed the time at the coast and made an offer on a unit in a planned extension of a condo complex. The building permit for the extension was not granted, and we bought a 27-foot sailboat instead.

In the fall, we would often camp for a week in the Smoky Mountains, enjoying the autumn colors, hiking to the waterfalls, and watching the black bears overturn the garbage cans in the Cades Cove campground. Camping trips over the years included trips to Tuxpan (Mexico), Rocky Mountains, Yellowstone, Tetons, Jasper, and Banff National Parks (Canada), the Oregon coast, and the Florida Keys.

When I was still the lab’s computer programmer and the same computer was used for developing programs, training animals, collecting data, and analyzing data, I would often go back to the lab after spending a little time with the family at dinner. Betty was encouraging me to spend more time with the boys. So, in 1978, I bought an Apple II computer, thinking I would be involved with the boys while they were learning to use it. I brought the computer home and the boys gathered around as we set it up and started working through the instruction manual. At some point, I became tired and told the boys we could continue tomorrow. That was the end of my spending time with them with the Apple II computer. By the next morning, they knew much more about how the Apple II worked than I would ever know.

Greg (11 years old at the time) and his friends Archie Cobbs and Mark Russinovich became Apple II experts. While they were high school students, Greg had a summer job programming for people in the vision research community, and Archie programmed for someone in the chemistry department. Archie and Mark both wrote articles for the Apple II Nibble magazine.

Greg graduated from the University of Texas in Austin with a major in computer science. Currently, he is a principal software engineer for Dell EMC. Archie got a BA in mathematics at Princeton, and both an MA in mathematics and a PhD in computer science at the University of California, Berkeley. He is founder and CEO of various software companies specializing in medical applications and has several patents for Java methodologies.

Mark Russinovich also achieved much success and is chief technical official for Microsoft Azure. According to Wikipedia, he “earned his BS degree in computer engineering from Carnegie Mellon University, MS degree in computer engineering from Rensselaer Polytechnic Institute, and PhD in computer engineering from Carnegie Mellon. He was introduced to computers when his friend’s father got an Apple II in the 1970s. He was able to reverse engineer its ROM and write programs for it.”

University of Pennsylvania (1989–1997)

In 1989, I accepted a position as a trustee professor of behavioral neuroscience in the Department of Psychology. There, I was able to interact with the faculty of the Mahoney Institute of Neurological Sciences and the students in the Neuroscience Graduate Group. I enjoyed team-teaching courses in the neuroscience group and the interactions with students in the psychology and neuroscience programs. Jack Nachmias, who studied small eye movements early in his career, participated in our lab journal club sessions.

When we arrived in Philadelphia, Betty was the occupant of an empty nest. Our sons were all in college. She did not continue working because back problems prevented her from standing for long periods. She arrived in Philadelphia knowing no one, but she adjusted. When we moved to Houston eight years later, Betty was a graduate of a two-year art appreciation course at the Barnes Museum, and a three-year program of horticulture at the Barnes Arboretum in Merion, Pennsylvania. She was on the Nomenclature Committee for the Philadelphia Flower Show; chair of the Botany-Horticulture committee of the Herb Society of America, Philadelphia Unit; and chair of the Long-Range Planning Committee and head of the Salvia/Tender Perennial Special Interest Group of the Philadelphia unit of the Hardy Plant Society. She worked as a volunteer in the Experimental Horticultural Section at Longwood Gardens. Articles about the garden she constructed in our back yard appeared in two Sunday editions of the Philadelphia Inquirer.

The Move

The rumor that the only reason I accepted the job at Penn was so I could sail our (now 30-foot) boat around the Florida Keys and up the east coast to the Chesapeake was not true. But moving the boat was fun. It was a (mostly) family affair. Gary (my brother), Jess (a fishing/sailing friend), Greg (middle son) and I sailed the boat from Mobile Bay to Fort Myers. Steve and Scott (our other sons), Greg, Jess, and I spent a week fishing, snorkeling, and sailing from Fort Myers to Fort Lauderdale. Jess, Greg, and I continued sailing up the east coast until the time allocated for the first stage of moving the boat expired. We left the boat at a marina in Oriental, North Carolina. After the lab equipment was transferred to Penn, Betty and I found a slip at a marina on the eastern shore of the Chesapeake and then sailed the boat from Oriental, North Caroline, to the Chesapeake.

Extraprofessional Pursuits While at Penn

We enjoyed sailing on the Chesapeake and discovered some wonderful anchorages. I also explored the coast of Maine during the summer months. I chartered sailboats in Maine for a couple of weeks for two summers and sailed my boat from the Chesapeake to Maine and back three times.

Betty enjoyed gardening, working in the Philadelphia Flower Show, and visiting gardens in New England with members of the Hardy Plant Society. She and friends obtained U.S. Department of Agriculture permits and made two trips to England to visit gardens and nurseries and returned with lots of new items for their gardens.

Baylor College of Medicine (1997–2004)

After presenting a seminar to the Division of Neuroscience, Baylor College of Medicine in 1996, I called to check on Betty. She asked whether they offered me a job. To my surprise, she became excited when I said yes. With all her gardening friends in Philadelphia and her activities in the gardening societies and art museums, I did not think she would want to leave Philadelphia. Her desire to return to the South was well hidden from me.

Nikos Logothetis and John Maunsell had set up animal surgery, histology, and animal facilities in space close to their labs. Nikos, who had been at Baylor since 1990, had just accepted a position at the Max Planck Institute for Biological Cybernetics. All Nikos’s lab equipment would stay at Baylor. I could move in and begin doing experiments immediately because Nikos and I were doing similar experiments. Major attractions included the rapidity with which I could resume research after a move, the reduction in teaching and administrative responsibilities, and the proximity to the labs of other faculty and their associates engaged in outstanding research in systems neuroscience. During my time at Baylor, my lab adjoined the labs of John Maunsell, Mike Crair, and Fabrizio Gabbiani. Joint journal club meetings were held weekly.

Extraprofessional Pursuits While at Baylor

Betty and I enjoyed the classical music programs available in Houston. Rice University, near Baylor College of Medicine, has an outstanding chamber music series. While we were in Houston, Betty and I attended concerts by the Academy of St. Martin in the Fields and the Pacifica, Emerson, Julliard, American, Shanghai, and Tokyo String Quartets. Another highlight for me was hearing, for the first time, a live performance of Verdi’s Requiem by the Houston Symphony and Chorus. Using contacts in the Hardy Plant Society, Betty was able to connect with gardeners in the Houston area and very quickly became an active member of several groups. In 2000, Betty began dividing her time between the house we had purchased in Pensacola, Florida, as an eventual retirement home, and our apartment in Houston. An only child, she needed to supervise the medical care of her parents in Mobile, Alabama. I moved the sailboat (now 32 foot) to Pensacola, and we both traveled between Houston and Pensacola frequently. Betty’s gardening activities switched to Florida.

Research: Acquiring Equipment, Developing Methods, and Exploratory Data

I arrived at UAB in 1965, eager to begin projects related to the goal I had set in graduate school—exploring the three-dimensional correlations between the physical properties of an external stimulus, the psychophysical report of the subject, and the intervening neural events. I wanted to study these relationships in awake, behaving animals. This seemed ambitious but I was optimistic. Wall, Freeman, and Major had recorded from dorsal horn cells in freely moving rats. O’Keefe and Bouma recorded the activity of amygdala cells in freely moving cats. Hubel had successfully recorded single-unit activity in the lateral geniculate body in unrestrained cats and Jasper, Ricci, and Doane reported studies of single-cell activity during avoidance conditioning in monkeys. I never lost sight of my goal, but, in retrospect, it was seven years before I made any serious progress. In those seven years, we migrated from bulky relay modules to more compact digital logic modules to laboratory computers for controlling experiments.

An issue I had not resolved was which sensory system to study. The visual system was my first choice, but a major concern was whether the small receptive fields of the cells in most of the visual areas that had been studied could be studied in an alert animal. Even small movements of the eyes could cause the image of a stationary spot to move on the retina over an area larger than the visual receptive fields that had been reported. I planned to explore the properties of visually responsive neurons in the superior colliculus (SC), an area that lesion experiments (Sprague and Meikle 1965, Experimental Neurology, 11, 115–146) had implicated in visual functions that might not demand small receptive fields.

I worked with Bob Travis on his studies of the neural correlates of discriminative conditioning while setting up a separate area for conducting my own experiments. While discussing the data Bob had obtained recording evoked potentials during conditioning paradigms, we decided to work together to develop a chronic single-unit recording setup. Bob began making microelectrodes. I built a cathode-follower high-impedance probe. Within a couple of days, we were successfully recording extracellular action potentials in anesthetized rats. Next, we needed a way to control the position of an electrode in the brain of the squirrel monkey subjects Bob was studying in his experiments. UAB had a well-equipped machine shop staffed with excellent personnel. With their assistance, we designed and built an 80-threads-per-inch screw-driven electrode carrier (Sparks and Travis 1967). One complete turn of the screw advanced the electrode approximately 300 microns. The electrode carrier, machined for a slip fit with the support rods, was internally threaded so that rotation of the screw advanced the electrode carrier without rotating the electrode.

By 1968, Bob and I were joint authors on six papers that involved recording the activity of single neurons in extrapyramidal and other subcortical areas. These were exploratory studies, for there was little to guide us concerning how individual neurons would respond to the behavioral variables we were manipulating. Mostly unknown to us, several other labs had also become actively engaged in experiments recording the activity of individual neurons in alert, behaving animals. Evarts published a description of his technique for recording the activity of subcortical neurons in moving animals, and Wurtz and colleagues had a series of papers describing single-cell activity in striate cortex in awake monkeys.

The International Congress of Physiological Sciences was held in Washington, D.C., in 1968. Bob and I went to the meeting and arranged to visit the labs of Evarts and Wurtz at the National Institutes of Health (NIH). Evarts very graciously gave us a set of drawings of the microdrive they were using with rhesus monkeys. After observing the rhesus monkeys performing behavioral tasks in the Evarts and Wurtz labs, Bob and I decided to switch from squirrel monkeys to rhesus monkeys.

In 1968, we had noticed unit activity closely related to stereotyped head, neck, and eye movements in the reticular formation, but we could not document it because we were not measuring eye or head movements in those experiments. We followed up those observations in experiments using rhesus monkeys and the Evarts microdrive, and we determined that the firing patterns of some reticular formation neurons was related to horizontal eye movements (Sparks and Travis 1971).

A number of important chronic single unit recording papers related to the neural control of eye movements appeared in the early 1970s. Fuchs and Luschei recorded the activity of neurons in the abducens nucleus during horizontal eye movement made by alert monkeys. Schiller described the activity of single cells in the oculomotor and abducens nuclei during eye movements of unanesthetized monkeys. Robinson reported on the activity of oculomotor cells in monkeys, and Bizzi and Schiller recorded single-unit activity in the frontal eye fields of unanesthetized monkeys during eye and head movements. Luschei and Fuchs reported on the activity of brainstem neurons during eye movements of alert monkeys. Six papers related to the visual, attentional, and motor properties of neurons in the monkey SC were published by Schiller and Koerner, Schiller and Stryker, Wurtz and Goldberg, and Goldberg and Wurtz. Fuchs and Robinson (1966, Journal of Applied Physiology, 21: 1068–1070) published a paper describing a new high-resolution method of recording the horizontal and vertical movement of the eyes in rhesus monkeys. I contacted David Robinson and went to Baltimore to learn more about the technique. Alex Skavenski was finishing his postdoctoral training in Robinson’s lab and had made extensive notes about construction of the field coils and electronic circuits for generating the magnetic fields. He generously gave me a copy of his notes, and I used them to replicate Robinson’s setup in Birmingham.

In 1967, UAB received a NIH grant to purchase a large central computer system for online acquisition and processing of data from biophysical experiments. Small laboratory computers installed in a few labs were directly connected over long coaxial cables to the large central computer. One of the laboratory computers (a Digital Equipment Corporation [DEC] PDP-8/I with 8 k of 12-bit memory, a Teletype, and an AXO8 interface box with eight channels of a/d conversion [12 bit], two channels d/a conversion [10 bit], eight digital inputs, and four digital output lines) was installed in our lab. A few days before his putative start date, the person we had hired to program the PDP-8/I informed us that he had accepted a position in California and was no longer in Alabama. I used the DEC Introduction to Programming book to learn the assembly language instructions and became the lab programmer. A free one-week assembly-language programming course came with the lab computer. After I had written code that met our most immediate needs, I signed up for the course offered in California rather than one on the east coast and modified our Dodge van so it would accommodate our camping gear, Betty, me, and two small boys. We camped in the Rocky Mountains, Yellowstone, Tetons, Jasper, and Banff National Parks in Canada, and along the Oregon coast on our way to Palo Alto.

In 1972, Bob Travis stopped conducting experiments in the lab to focus on clinical research. I inherited his electrically shielded, sound-attenuated, walk-in room with a rhesus monkey restraining chair and computer-controlled HP 1310A large-screen oscilloscope for presentation of targets for visual tracking tasks. I had completed winding the coils and connecting the circuitry used to produce the magnetic fields. The equipment necessary for detecting and recording movements of the eyes was installed and working. The PDP-8/I had been programmed to enable direct experimental control of the monkey’s eye position and velocity, along with the measurement of attendant neuronal activity. The system (1) generated and controlled the position and velocity of a visual tracking target presented on an oscilloscope; (2) generated a serial pulse code of target parameters, which was recorded on analog tape; (3) monitored horizontal and vertical eye position; (4) compared horizontal and vertical eye position with target position; (5) delivered reinforcement for appropriate tracking of the visual target; (6) provided online displays of eye positions, target position, and spike activity; and (7) permitted modification of target parameters based on the response properties of each neuron studied. Several offline data-analysis programs also had been written.

I now had the tools to pursue my goal of studying the three-dimensional relationships between stimulus input, psychophysical report, and the intervening neural events. But the scientific environment had changed. Some questions I had about the SC were already answered by the experiments done in the labs of Schiller and Wurtz. The grant application I submitted in September 1972 to study the role of pontine and other brainstem neurons in the control of saccadic and pursuit eye movements did not include experiments relating neural activity to sensory input. The need to obtain funds to keep the lab going was prepotent, and I made a vow to myself to add the sensory component later. The application to the National Eye Institute for funds to support the lab for three years was approved.

Many anxious minutes spread over more than three decades would have been avoided if I could have looked into the future and discovered that this grant would receive continuous funding and still be active when I retired at the end of the year in 2004. If this sounds as if obtaining funds to support research was smooth sledding, it did not seem that way at the time. Initially, I requested three years of additional support on each renewal application. After each submission, there was a long period of uncertainty and anxiety about the outcome of the review process. Nine years later, I began requesting five years of additional funding, and the periods of uncertainty and anxiety were not as frequent. The renewal application requesting funding from 1987 to 1992 was ranked 1 out of 62 applications recommended for approval by the VIS B study section and was selected for a NIH Merit (Method to Extend Research in Time) Award from 1987 to 1997.

Evolution of Ideas Concerning the Role of the SC in the Control of Movements

Recordings from the SC: A Signal for Saccade Initiation

Shortly after beginning to record the activity of neurons in the pontine reticular formation, I deviated from the proposed research plan. The optimal orientation of the microdrive for reaching the pontine target cells was such that the electrodes passed through the SC before reaching the pons. I could not resist observing for myself the important findings that had then been recently reported by Wurtz and Goldberg (1971, Science, 171: 82–84; 1972, Journal of Neurophysiology, 35: 575–586; 587–596) and by Schiller and Koerner (1971, Journal of Neurophysiology, 34: 920–936). Also, I had questions about the activity of collicular neurons, which were not answered in those papers. This diversion was supposed to be short, but it was not.

I was not yet convinced that the SC played a critical role in either the initiation of saccades or the precise specification of the direction and amplitude of a movement. My reasons for skepticism were these: the coarse tuning of the movement fields (the range of saccade directions and the amplitudes associated with movement-related activity of a neuron), and the failure of earlier papers to report a tight coupling between saccade-related activity in collicular neurons and the onset of the associated saccade. I thought that if the SC participates in the initiation of saccades, it should be possible to isolate a subpopulation of neurons in which the onset of neuronal activity is tightly coupled to saccade onset. Furthermore, in a situation in which a visual stimulus sometimes elicits a saccade and sometimes fails to do so, the occurrence of the neuronal activity should be highly correlated with saccade occurrence. Computer-generated plots of instantaneous spike frequency (the reciprocal of interspike intervals, ISIs) provided a better view (Sparks and Sides 1974) of the temporal pattern of spike activity on each trial than did the raster plots and cumulative histograms used in earlier papers. These plots revealed saccade-related burst neurons (SRBNs) that generated an initial low-frequency component, followed by a pulse of activity beginning approximately 20 milliseconds (ms) before saccade onset (Sparks 1978).

The probability of saccade occurrence was manipulated by varying target duration in a way that when two successive dots (A and B) were presented, the monkey would sometimes make a saccade to position A (near the center of the movement field of the cell) and then a second saccade (out of the movement field of the cell) to position B. On other trials, with the same duration of target A, only a single saccade (not in the movement field of the cell) to target B was observed.

The probability of a spike burst occurring in SRBNs was perfectly correlated with the occurrence of a saccade into the movement field of the cell. A spike burst always occurred if such a saccade was produced. Some buildup of spike activity might be observed but a discrete high-frequency pulse of activity never occurred when the animal canceled the saccade or produced a saccade that was not in the movement field of the cell (Sparks 1978). I speculated that the discharge of SRBNs served as a trigger input to the pontine and midbrain circuitry that generates the horizontal and vertical signals required for a saccade.

Saccade Commands: Organized in Nonretinal Coordinates

Preliminary studies of the functional organization of the SC were interpreted as supporting early models of the saccadic system in which a signal of retinal error (RE), the distance and direction of the target image from the fovea, was relayed directly to the oculomotor system, which generated a command to correct for RE. Saccades were assumed to be ballistic because the trajectory of the saccade was determined at saccade onset. The site of neural activity in the retinotopic map of the superficial layers of the SC indicates the position of a visual target relative to the fovea. Schiller and Koerner 1971, Journal of Neurophysiology, 34: 920–936) hypothesized that the cells in the superficial layers activated underlying regions of the SC containing neurons that discharge before saccades. Because the motor map of saccadic eye movements is aligned with the retinotopic map (Robinson 1972, Vision Research, 12: 1795–1808), the ensuing saccade would direct the foveal projection toward the region of the visual field containing the target. Later models assumed that visual targets for saccadic eye movements are localized with respect to the head or body and not with respect to the retina.

The results of experiments Larry Mays and I conducted strongly supported the view that saccade targets are localized in a non-retinocentric frame of reference (Mays and Sparks 1980b, 1981; Sparks and Mays 1983b). On a typical trial, the fixation target was extinguished, and an eccentric target was illuminated for 50–100 ms. The monkey usually looked to the position of the target with a latency of 160–200 ms. Randomly, on 30 percent of the trials, after the target was extinguished, but before the animal could begin a saccade, the eyes were driven to another position in the orbit by electrical stimulation of the SC. Animals compensated for the stimulation-induced perturbation by looking to the approximate position of where the target had appeared. This happened regardless of the position of the target and regardless of the direction and amplitude of the saccade required to compensate for the stimulation-induced movement. Visual background cues could not be used as an external frame of reference for localizing the targets because, except for the fixation target and the briefly flashed target, the task was performed in total darkness. Also, the target was extinguished before the stimulation-induced saccade and could not provide a visual update of the target position.

Retinocentric models predict that the animal will produce a saccade with a predetermined distance and direction—a saccade that will direct gaze to a point in space that differs from the target location by an amount equal to the direction and amplitude of the stimulation-induced saccade. The result predicted by retinocentric models was never observed. In spatial models, RE signals will be combined with information about the change in eye position produced by collicular stimulation. They predict that the animal will look to the actual position of the target in space. This was the result we observed.

The eye movement that compensated for the change in eye position produced by stimulation was not a passive, low-velocity movement to an orbital position of mechanical equilibrium established by a tonic pattern of motoneuron activation specified by the visual target. It was a saccade. A new saccade command, based on stored information about the location of the retinal image and information about the new position of the eyes, had been issued.

Larry Mays and I are joint authors on 17 publications. The chance encounter that was the basis of our collaboration happened in 1976 while recruiting Joan Lorden to the Department of Psychology. I learned that her husband, Larry Mays, had a PhD and was also looking for a research/ teaching position. Larry had recorded unit activity from hippocampus in awake rats in his dissertation research, and he had measured eye movements using the magnetic search coil while working with Jim McElligott at Temple University. I secured a postdoctoral position on a training grant for Larry, and Joan and Larry came to UAB. Joan is now provost and vice chancellor for Academic Affairs at the University of North Carolina (UNC) at Charlotte. Larry is professor and chair, Department of Bioinformatics and Genomics, UNC at Charlotte.

Activity of SC Neurons Preceding Compensatory Saccades

John Porter and I did follow-up experiments to determine whether neurons in the SC discharging before visually triggered saccades also discharge before saccades compensating for stimulation-induced perturbations in eye position (Sparks and Porter 1983). Target coordinates were arranged so that the vector of the compensatory movement was in the movement field of the cell under investigation. Forty-nine of the 50 cells studied discharged before compensatory saccades in their movement field. The only neuron that did not was shown to require prior activation by a visual stimulus within its movement field. This finding provided additional support for the hypothesis that the SC contains a spatial map of motor error (ME), the difference between current eye position and desired eye position.

Population Coding of Motor Commands

Spatially, the movement fields of most SC neurons are large and coarsely tuned (Sparks 1975). Each neuron fires before a broad range of saccades. Thus, a large population of neurons is active before each saccade. The discharge of individual SC neurons is ambiguous with respect to saccade direction or amplitude because identical discharges precede many saccades with different directions and amplitudes (Sparks and Mays 1980). It is the location of active neurons within the topographical map of movement fields, not their frequency of firing, that must specify saccade direction and amplitude.

How are the signals needed to precisely control the direction and amplitude of a saccade extracted from the activity of this large population of coarsely tuned cells? We tested predictions of a population coding scheme in which each member of the active population contributes to the movement, and the exact trajectory of a saccade is determined by the average or sum of the population response (McIlwain 1975, Journal of Neurophysiology, 38: 219–230; Sparks et al. 1976).

An insulated, silver-coated glass pipette designed by Malpeli and Schiller (1979, Journal of Neuroscience Methods, 1: 143–151) was used to record extracellular unit activity, for microstimulation, and for pressure injection of various agents into the SC. If, as we proposed, the direction and amplitude of a saccade is the result of a weighted average of each neuron’s vector contribution, deactivation of a small region of collicular neurons may produce saccades with amplitudes that are either too large or too small. Which one occurs depends on the location of the deactivated neurons within the population of cells active before and during a saccade. For example, if the inactivated cells are “voting for” relatively small amplitude movements, the amplitude of the saccade specified by the remaining active neurons would be larger than the one specified by the full complement of cells. A smaller movement would occur if a different movement (involving a different subset of SC cells) was made in which the same inactivated cells were ones contributing to relatively large amplitude movements.

As predicted by the vector-averaging hypothesis, a systematic pattern of errors in direction and amplitude was observed (Lee et al. 1988; Sparks et al. 1990). Movements to targets requiring a saccade smaller in amplitude than the “best saccade” (the movement produced by passing small-current pulses through the recording/injection probe) were too small; saccades to targets requiring a movement larger than the “best saccade” were too large. The direction and amplitude of movements similar to the “best saccade” were not altered noticeably after the injection. But the velocity of visually guided movements corresponding to the “best saccade” was slower, and the duration was prolonged so that the amplitude of the movement was about the same. The findings of our experiment were important for first demonstrating that saccadic accuracy results from the movement tendencies produced by the entire active population rather than by the discharge of a small number of finely tuned cells. A “News and Views” article written by Terry Sejnowski (1988, Nature, 332: 308) highlighted the findings of the experiment in the issue of Nature in which our paper appeared.

Choongkil Lee did his PhD research under the supervision of Joseph Malpeli at the University of Illinois and had experience with the Malpeli and Schiller technique for reversible activation or inactivation of small regions of brain tissue. Choongkil is now professor emeritus, Department of Psychology, Seoul National University.

Bill Rohrer obtained his PhD from the University of Notre Dame. Bill worked with Choongkil on the population coding experiment and conducted experiments using express saccades that increased our knowledge about the motor commands found in the SC. He is now a senior professor of Biology and Allied Health Technology, Union County College, Cranford, New Jersey.

Influences of the Site, Duration, and Level of Collicular Activity

Early papers reported that the properties of movements evoked by SC stimulation in monkeys were not influenced by varying stimulation parameters. This led to the conclusion that, within the SC, information relating to the properties of a saccade are encoded solely by the spatial distribution of activity and that neither the level nor the duration of activity plays a role in determining the properties of the movement. However, in various nonprimate species, varying the parameters of microstimulation has large effects on stimulation-evoked orienting movements. Incidental observations in recent (at that time) primate studies were not consistent with the hypothesis that the activity of SC neurons plays no role in the control of saccade velocity or duration. It seemed prudent to check the validity of these conclusions before launching experiments based on the assumption that they were correct.

Thus, we repeated the early primate microstimulation experiments but systematically varied the duration and frequency of the stimulation trains over a wider range than used in the early experiments (Stanford et al. 1996). In contrast to the results of the earlier studies that varied current strength but not train duration, we found that both the site of stimulation and the parameters of stimulation are important factors in determining the properties of stimulation-induced eye movements.

As early studies reported, the site of stimulation within the collicular motor map determined the largest stimulation-induced movement that we could generate. But varying the duration and frequency of the stimulation train produced dramatic effects. For any stimulation site, movement amplitude depends on the duration of the stimulation train: As train duration increases, movement amplitude increases monotonically until it reaches the site-specific limit. Within the range over which amplitude can be modulated, the end of the movement is tightly linked to the offset of the stimulation train. The frequency of stimulation influences the peak velocity of an evoked movement: Higher frequencies produce movements with higher velocities. The parameters of collicular stimulation can be adjusted to provide independent control over saccade amplitude and saccade velocity. Movements that have comparable amplitudes, but different dynamic characteristics can be produced by varying train duration and frequency. Collicular activity must be sustained throughout the execution of the movement if the site-specific maximal amplitude is to be achieved, and the peak velocity of a saccade is determined, at least in part, by the level of collicular activity.

Terry Stanford received his PhD from the University of Connecticut Health Center in 1989. He developed a method for studying the responses of neurons in the inferior colliculus in the unanesthetized rabbit to auditory stimuli in the lab of Shigeyuki Kuwada and decided to spend some time in my lab to learn more about chronic single unit recording techniques. Terry is now professor, Neurobiology and Anatomy, Wake Forest School of Medicine.

Movement-Related Activity in the SC: A Command for a Change in Gaze

Microstimulation of the optic tectum/SC in several nonprimate species produces coordinated movements of the eyes, head, pinnae, vibrissae, and body. Early experiments in which the parameters of stimulation were not varied systematically failed to observe coordinated movements of the eyes and head following microstimulation of the SC in monkeys. But coordinated eye–head gaze shifts were evoked by electrical stimulation of the SC in rhesus monkeys in a preliminary report by Seagraves and Goldberg (The Head–Neck Sensory Motor System, eds. Berthoz, Graf, and Vidal, 1992, 292–295). For his dissertation research, Ed Freedman performed three experiments related to this unresolved issue. The first provided a comprehensive description of the visually guided gaze shifts that occur when the head is completely unrestrained. The second involved a reexamination of the effects of microstimulation of different regions of the SC, and the third examined the pattern of activity of individual neurons before and during the execution of large visually guided gaze shifts involving combined eye–head movements.

Several general rules about the coordination of the eyes and head during large rapid visually guided gaze shifts emerged from the first experiment (Freedman and Sparks 1997a, 2000). These general rules were used as the basis for comparison with movements evoked during microstimulation of the primate SC, and to develop a strategy for determining whether the activity of individual cells in the SC was correlated with the overall change in gaze or with the eye and/or head components of the gaze shift.

The site and parameters of microstimulation of the SC of rhesus monkeys was varied systematically when the head was completely unrestrained (Freedman et al. 1996). Collicular stimulation produces high-velocity, combined eye–head gaze shifts that are remarkably similar to naturally occurring visually guided gaze shifts of comparable amplitude and direction. The amplitude and velocity of stimulation-induced gaze shifts depend on the parameters (frequency, current level, and duration of the stimulation train) of stimulation and the site of stimulation. Increasing the duration of the stimulation train systematically increases the amplitude of evoked gaze shifts until a site-specific maximal amplitude is reached. The frequency of stimulation affects the velocity of evoked gaze shifts: Higher frequencies result in higher peak velocities.

As is true for visually guided gaze shifts, the head contribution to stimulation-induced gaze shifts depends on the position of the eyes relative to the head at the onset of stimulation. When the eyes are deviated in the direction of the ensuing gaze shift, the head contribution increases and the latency to head movement onset decreases. The head contribution to stimulation-induced gaze shifts also depends on the direction of the gaze shift. The head contribution decreases as the gaze shift becomes more vertical.

Gaze shifts with relatively constant amplitudes and directions occur when stimulating a particular site using constant stimulation parameters. But these gaze shifts of similar directions and amplitudes are accomplished with many combinations of eye and head components, depending on the initial positions of the eyes in the orbits and the direction of the gaze shift. These data are inconsistent with the hypothesis that the SC generates separate eye and head displacement commands. Instead, the findings indicate that a unitary signal of desired gaze displacement is derived from the locus of collicular activity. This signal is separated into eye and head components below the level of the SC. But the level of collicular activity can influence the velocity of gaze shifts without affecting the gaze displacement signal.

The activity of single neurons in the intermediate and deep layers of the monkey SC was recorded during combined eye–head gaze shifts (Freedman and Sparks 1997b). The cells were studied in conditions in which (1) the amplitude and direction of gaze movements was relatively constant, but the eye and head components varied over a wide range; and (2) either the eye or head contribution was fixed but the direction and amplitude of gaze changed over a large range. This allowed the activity of collicular neurons to be recorded under conditions in which gaze and the eye and head contributions to gaze were dissociable.

For all the cells for which these analyses could be performed, motor-related activity was best correlated with the amplitude and direction of the gaze shift and was only weakly correlated with eye or head components of gaze. Gaze shifts having similar amplitudes and directions were associated with similar motor-related bursts. These findings supported the hypothesis that a desired gaze-displacement signal is derived from the locus of collicular activity and is decomposed into separate eye and head signals downstream from the SC.

I hired Ed Freedman as a research assistant because of his previous research experience at Brown University and Cornell. Later, he was accepted into the graduate program in neuroscience and did his dissertation research in my lab. Ed received the 1997 Lindsley Prize in Behavioral Neuroscience awarded by the Grass Foundation and the Society for Neuroscience. He also won the Flexner Award for outstanding dissertation research in the Institute of Neurological Sciences at Penn. Ed spent two years as a postdoctoral fellow in the lab of Albert Fuchs at the University of Washington in Seattle and then accepted a position in the Department of Neuroscience, University of Rochester Medical Center, where he is currently an associate professor.

Evolution of Ideas Concerning the Coordinates of Sensory Signals in the SC

Visual Maps in the Primate SC: What Is Represented?

Visually responsive neurons in the superficial layers of the SC are activated by stimulation of a particular region of the retina. They respond only to the appearance of a visual stimulus in their receptive field, and the location of the receptive field shifts with each change in gaze. Thus, the site of neuronal activity in the retinotopic map of the superficial layers represents a map of RE, the distance and direction of the image from the fovea (Goldberg and Wurtz 1972, Journal of Neurophysiology, 35: 542–559; Mays and Sparks 1980a). The deeper layers of SC contain visually responsive cells with activity that represents ME, the difference between current eye position and desired eye position. We labeled these as quasi-visual or QV cells. They discharge whenever a saccade with a particular direction and amplitude is appropriate, regardless of whether the movement becomes appropriate because of the onset of a visual stimulus or because of an eye movement occurring after the disappearance of the target (Mays and Sparks 1980a).

Monkeys were trained to make saccades in response to a single target in one half of the visual field or to two brief targets usually in the other hemifield. On single-saccade trials, after subjects maintained fixation of a target (O) at the center of the oscilloscope screen for a variable period, target C appeared in the receptive or movement field of the neuron under observation. On double-saccade trials, the offset of the center fixation target (O) was followed by successive presentations of targets A and B, neither of which were in the neuron’s receptive or movement field. The total duration of targets A and B was less than the reaction time of the monkey. Nevertheless, we programmed the computer to make reward contingent upon a saccade to position A followed by a saccade to position B. A critical feature of this paradigm is that a saccade from position A to position B has the same amplitude and angle as a saccade from the fixation point to C. Only the origins of the movements differ.

On the single-saccade trials, when the animal is maintaining a single fixation position, QV cells appear to be visually responsive, to have visual receptive fields and to respond to targets in their receptive field whether or not a saccade is made toward the target. But on double-saccade trials requiring a change in fixation, QV cells begin to fire only after the eye has reached position A, and they continue to fire until after the saccade from A to B. The cells firing in this case are not those whose receptive field contained the original target B, but the cells whose receptive field would contain target B if the target were flashed again in the same spatial location after the eyes reached position A. Unlike neurons in the superficial layers, which are activated if, and only if, a particular region of the retina is stimulated, QV cells are activated by stimulation of any region of the retina if, after a subsequent movement of the eye, a saccade with a particular trajectory is required to look to the target. No component of the discharge of the QV cells appeared to be motor in the usual sense. Instead, the activity of QV cells reflects eye-position error (the difference between actual and desired eye position) and holds this information in spatial register until a saccade occurs or is canceled.

Note that the presumed linkage, implied in earlier versions of the foveation hypothesis, between the superficial layers (receiving direct retinal inputs) and the deeper layers of the SC is not necessary for the activation of neurons with saccade-related activity. In this experiment, the discharge of overlying visual cells was neither necessary nor sufficient to activate most cells with saccade-related activity.

Auditory Maps in the Primate SC: What Is Represented?

Experiments studying saccades to auditory targets also indicated that the responses of sensory cells in the deeper layers of the SC are encoded in motor, rather than sensory, coordinates (Jay and Sparks 1984, 1987a, 1987b, 1990). Monkeys trained to look to either visual or auditory targets in a completely darkened room were placed with their heads fixed in the center of a semicircular track. Movement of a speaker (with a light-emitting diode attached) along the track and rotation of the track allowed targets to be presented at most locations on an imaginary sphere surrounding the animal. Three fixation lights separated by 24 degrees were placed along the horizontal meridian. At the beginning of each trial, one of the three fixation lights was randomly activated. After a variable interval, an auditory (broad-band noise burst) or visual target was presented, and the animal was required to look to the target location to receive a liquid reward. A delayed saccade task was used to separate, temporally, sensory and motor activity.

The major objective of the experiment was to plot the receptive fields of sound-sensitive cells in the SC of alert monkeys while varying the direction of visual fixation. If the receptive fields of auditory neurons in the SC are based on interaural cues and are organized in head-centered coordinates, the direction of fixation would have no effect. But, if the response of auditory neurons is organized in ME coordinates, then the response should depend on both speaker position and fixation direction. We found that the position of the eye in the orbit had a distinct effect upon the response of sound-sensitive cells in the SC. When the magnitude of the neural response was plotted as a function of the stimulus location in space, the receptive fields of the neurons shifted with the position of the eye in the orbit. But when the magnitude of the neural response was plotted as a function of the direction and amplitude of the movement required to look to the auditory target, the plots obtained with the different fixation positions were closely aligned. Thus, the discharge of sound-sensitive neurons in the SC is not determined solely by the position of the auditory stimulus in space, but rather depends upon ME, the trajectory of the saccade required to look to the target.

We also monitored the activity of cells that generated a burst of activity before saccades to visual targets while monkeys made saccadic eye movements to auditory targets. Most neurons generating bursts before saccades to visual targets also burst before saccades to auditory targets. Visual and auditory signals, originally encoded in retinal and head-centered coordinates, respectively, have undergone a transformation that allows them to share a common efferent pathway for the generation of saccadic eye movements.

The sound delivery system we used in these experiments was constructed by the technical staff at Cal Tech. It was a duplicate of the system used by Knudsen and Konishi (1979, Journal of Comparative Physiology, 133: 13–21) in their studies of auditory localization by the barn owl. John Pettigrew (1984, Nature, 309: 307–308) wrote a “News and Views” article in Nature about the use of the hoop in the “revelations” of Knudsen and Konishi finding a computational map of auditory space in the brain of the barn owl and the “surprising” finding that the coordinate frame of the map of sound space in the monkey SC shifts when the monkey’s eyes change positions.

Sometime in the early 1980s, I received a letter from Albert Fuchs inquiring about the possibility of transferring a graduate student, Martha Jay, from the University of Washington to UAB. Martha’s husband had accepted a faculty position in the Department of English at the University of Alabama in Tuscaloosa. Martha was in the MD/PhD program at the University of Washington and her thesis for the MS degree involved studies of the auditory system. The timing was perfect. I had just completed installing the sound delivery system described previously. After she received her PhD, Martha’s husband accepted faculty positions in South Carolina and then Wisconsin. Martha received her MD from the University of South Carolina School of Medicine and did her residency in Ophthalmology at Northwestern University. She was the first eye surgeon in the Milwaukee area to perform laser refractive surgery and remains at the forefront of the field.

Somatosensory Maps in the Primate SC: What Is Represented?

The saccadic eye movements directing gaze to the source of a tactile stimulus cannot be based solely on information about the region of the body surface that is activated. Information about the current direction of gaze also must be considered. Jennifer Groh’s dissertation research at the University of Pennsylvania addressed this issue. Her thesis combined behavioral, electrophysiological, and computational methods. The computational component demonstrated that the required sensorimotor transformations could be performed using conventional neural circuits and neurons with typical biophysical properties (Groh and Sparks 1992).

Animals (with the head fixed) were trained to look to the source of visual or somatosensory stimuli (Groh and Sparks 1996a, 1996b, 1996c). Behavioral data were used to compare the accuracy and other characteristics of saccades to visual targets with those of saccades directed to vibratory stimuli. Somatosensory saccades were less accurate than visual saccades, and the peak velocities of somatosensory saccades were lower than those of visual saccades of the same amplitude.

Primary objectives of neurophysiological experiments were to determine whether cellular responses to somatosensory stimuli are affected by the positions of the eyes in the orbits and to determine whether cells that discharged before saccades to visual targets also discharged before saccades to somatosensory stimuli.

All but one of the 86 cells with saccade-related activity studied discharged before saccades to both visual and somatosensory targets. The remaining cell was active only for visual saccades. This result indicates that visual and somatosensory signals, originally encoded in quite different coordinate frameworks, have been translated into a common frame of reference and are sharing premotor neural elements.

Nearly all cells responding to somatosensory stimuli also responded to visual targets. Eye position significantly affected the responses to both somatosensory and visual stimuli in a similar manner. The cells responded to visual and somatosensory targets located at approximately the same direction in space with respect to the eyes. The activity of these cells exhibited both sensory and motor qualities. The discharge was more closely linked in time to stimulus onset than to the movement. Nonetheless, the activity of collicular cells was related to the change in eye position necessary to bring the target onto the fovea, rather than the location of the stimulus on the body surface. Somatosensory signals in the SC, like auditory ones, appear to be encoding the metrics of a movement to a stimulus rather than the location of that stimulus in space.

Jennifer Groh learned about my research in a graduate course at the University of Michigan, and she had heard rumors that I was moving to Penn, where she had also been accepted into their graduate program after she applied the year before. She wanted to know if I moved to Penn, would it be possible for her to transfer and to do her dissertation research in my lab. Jennifer flew to Birmingham to discuss a possible transfer before actually doing so. She recently told me I met her at the gate in the airport holding up a copy of the Journal of Neurophysiology so she would recognize me. After receiving a PhD from Penn, Jennifer spent three years as a postdoctoral fellow in Bill Newsome’s lab at Stanford University. She is currently professor in the Departments of Psychology and Neuroscience, Neurobiology, and the Center for Cognitive Neuroscience at Duke University.

The Importance of the Findings of the Experiments Conducted by Jay, Groh, and Mays and Sparks

The research of most sensory neurophysiologists is devoted to understanding how neural representations of external and internal events are related to cognitive factors, especially perception. But sensory processing is also required to initiate and guide action. Indeed, perception and cognition have no adaptive value in the absence of action. Action is usually directed to the spatial location of objects, but the localization of a stimulus in space requires complex neural computations. Visual stimuli activate receptors in specific regions of the retina, but there is not a one-to-one correspondence between which region of the retina is activated and the location of the visual object in space. Which region of the retina is activated by a stationary visual stimulus depends on the positions of the eyes in the orbits, the position of the head, and the position of the body.

Information about the region of retinal activation must be combined with information about eye, head, and body position to compute the location of the visual stimulus. It is not possible to know the location of a visual stimulus in space by processing only information that arrives over the optic nerve. The localization of somatosensory and auditory stimuli also requires complex neural computations. The raw sensory data must be combined with information about the positions of the eyes in the orbits, the position of the head on the body, and other sources of information to compute the location of the target and the trajectory of a movement to the target. Results of experiments by Mays and Sparks (1980a), Jay and Sparks (1984, 1987b), and Groh and Sparks (1996c) provided early examples of cases in which sensory stimuli were coding information in a motor frame of reference. They also provided examples of the types of experimental manipulations required to demonstrate coding in motor coordinates.

Extraocular Muscle Proprioception: Essential for the Spatial Localization of Saccade Targets?

Localizing visual stimuli in spatial coordinates requires that retinal and eye-position information be combined. The two possible sources of the eye-position signal are extraocular muscle afferents and a neural copy of the command to move the eye. Bart Guthrie, John Porter, and I (Guthrie et al. 1983) investigated the role of extraocular muscle afferents and, by exclusion, the contribution of a copy of the central motor command to the eye-position signal used in target localization.

We did anatomical studies to determine whether, in the monkey, extraocular muscle afferents reach the central nervous system exclusively through the ophthalmic branch of the trigeminal nerve, as they do in the cat. Injections of horseradish peroxidase into individual extraocular muscles of monkeys, labeled pseudounipolar neurons, localized only within the ipsilateral semilunar ganglion (Porter et al. 1983). Intracranial transection of the ophthalmic nerve before muscle injection eliminated all labeling within the ganglion. Thus, the extraocular muscles can be selectively deafferented to examine the role of proprioception in the control of eye movements.

Bart Guthrie developed a surgical procedure for reliably sectioning the ophthalmic branch of the trigeminal nerve. After surgical elimination of extraocular muscle proprioception, we tested animals on a stimulate/compensate task (Mays and Sparks 1980b; Sparks and Mays 1983b), a task requiring rapid and accurate feedback about eye position. If extraocular muscle proprioception is an essential source of eye-position information, without it, the oculomotor system would not be capable of generating an accurate compensatory saccade. In contrast, if corollary discharge is used as an accurate eye-position signal, the animals would continue to compensate in the absence of extraocular muscle proprioception.

After initial testing of animals on the stimulate/compensate task, the ophthalmic nerves were transected bilaterally at their junction with the trigeminal ganglion, eliminating extraocular muscle proprioception. Complete transection was determined postoperatively by loss of the corneal blink reflex and confirmed postmortem by the absence of labeling of trigeminal ganglion sensory neurons after injection of horseradish peroxidase into the extraocular muscles. Postoperatively, animals were still able to compensate for stimulation-induced perturbations in eye position and look to the approximate position of the targets in space (Guthrie et al. 1983). This is possible only if information about the stimulation-induced eye movement is still available. This information could not be provided by retinal stimulation. Experiments were conducted in total darkness, and the visual target was not present during or after the stimulation-induced saccade. Because transection of the ophthalmic nerves eliminated extraocular muscle proprioception, the eye-position information must have been provided by a centrally generated corollary discharge.

Bart Guthrie was in the MD/PhD program at UAB, had finished medical school and a one-year internship, and was working to finish his PhD before beginning a residency in the Neurosurgery Department at the Mayo Clinic in Minnesota. After completing his neurosurgery training at the Mayo Clinic and a fellowship at Stanford University, Bart was on the faculty at George Washington University. He returned to UAB in 1993 and currently serves as the director of Computer-Assisted Neurosurgery (CANS) at UAB.

John Porter received his PhD in Anatomy from the Medical College of Virginia in 1980. He worked with Robert Spencer studying the morphology and innervation of extraocular muscle fiber types and the destination of extraocular muscle afferents. After a successful research career, John left his position at the Case Western Reserve University School of Medicine to become program director, Neuromuscular Disease at the National Institute of Neurological Disorders and Stroke.

The overlap of Bart’s and John’s time in the lab was fortuitous. John’s anatomical expertise allowed us to perform the experiments needed to see whether the anatomical data about the location of cell bodies of extraocular muscle afferents in cats could be extended to primates.

Single-Cell Signals

Gradual Development of Signals Specifying Saccade Direction and Amplitude

Stimulation of a discrete region of SC produces a sequence of neural events that specifies the horizontal and vertical amplitude of a saccade (specification) and provides the signals necessary to initiate that movement (initiation). As discussed earlier, the high-frequency burst of SRBNs that precedes saccade onset by 18–20 ms is a collicular signal probably involved in the initiation of saccades. What is the evidence that other types of collicular signals are involved in the specification of saccade direction and amplitude?

While testing the ability of rhesus monkeys to compensate for perturbations in eye position produced by microstimulation of the paramedian pontine reticular formation (PPRF; Sparks et al. 1987), we accidentally discovered that pontine stimulation sometimes triggered, prematurely, a component of the impending saccade to a visual target. The speed of the movements observed after stimulation could be used to distinguish (1) pontine stimulation-evoked low-velocity movements from (2) saccades made to acquire the visual target. This premature saccade was considered a component of the impending movement to the visual target because both its direction (up or down) and amplitude depended on target position.

The amplitude of the prematurely triggered saccade increased as the time of stimulation approached the point at which a normal, visually triggered saccade would have occurred. This suggests that, for normal visually directed saccades, during the presaccadic interval, the input to the saccadic generator circuits gradually develops over at least a 100-ms period. We accidentally discovered that pontine stimulation could be used to sample, at various intervals, the status of this latent signal.

Glimcher and Sparks (1993) tested the effects of low-frequency stimulation, subthreshold for movement initiation, on spontaneous and visually guided saccades. Subthreshold stimuli biased the direction and amplitude of spontaneous and visually guided eye movements in the direction and amplitude of movements evoked by suprathreshold stimulation of the same collicular location without affecting the time at which the movements were initiated. Movement specification is separable from initiation.

The low-frequency signals of (1) the QV cells that encode ME (Mays and Sparks 1980) and (2) those that become active when a target is selected as the goal of an impending saccade (Glimcher and Sparks 1992) are signals that could be involved in the specification of saccade direction and amplitude.

Paul Glimcher received his PhD in neuroscience at the University of Pennsylvania in 1989. His dissertation research was directed by Randy Gallistel. Paul was interested in the neural bases of attention and the processes involved in selecting targets as goals for action. He pursued this line of research after accepting his initial faculty position as an assistant professor in neural science at New York University in 1994. He is now the Julius Silver Professor of Neural Science and professor of neuroscience and physiology at New York University.

Single-Cell Signals: Sometimes Seductive Saboteurs

We encountered two situations in which, for neurons above the level of the motoneurons, correlations between the saccade-related activity of neurons and the properties of the movement are misleading. The first case occurs when recording the activity of excitatory burst neurons (EBNs) in the pontine reticular formation during coordinated eye–head movements. In animals with the head in a fixed position, the number of spikes in the saccade-related burst is highly correlated with saccade amplitude, and the peak velocity of the movement is tightly coupled to the peak frequency of the burst. EBNs have monosynaptic connections with motoneurons innervating extraocular muscles. Early papers viewed the activity of EBNs as a command for a saccadic eye movement. Later experiments found that during coordinated eye–head movements, the spike count of EBNs was better correlated with gaze amplitude than with the amplitude of the eye component (Ling et al. 1999, Journal of Neurophysiology, 82: 2808–2811). An explanation for this discrepancy in findings is presented below.

Neeraj Gandhi and I tried to resolve this discrepancy by stimulating the omnipause neurons (OPNs) before and during a gaze shift (Gandhi and Sparks 2007). OPNs, a discrete collection of cells found along the midline of the PPRF, discharge tonically during fixation and are silent during saccades. They inhibit, monosynaptically, the EBNs and, thus, gate the activity of cells presumably generating a command for an eye movement. OPNs are clustered together in a small nucleus that contains only OPNs, and it is possible to selectively activate OPNs with electrical stimulation. When this is done in animals with the head restrained, OPN stimulation interrupts ongoing saccades for the duration of the stimulation train. The saccade is resumed when the stimulation train ends.

If the EBNs are generating a command only for moving the eyes, then OPN stimulation should perturb only movements of the eyes. If, however, as the correlation between the number of spikes in the EBN burst and gaze amplitude implies, the output of EBNs is sent to both eye and neck motoneurons, the inhibition of EBN activity produced by stimulation of the OPNs should affect both the eye and the head components of the gaze shift.

Stimulation of the OPNs synced to the onset of large, combined eye–head movements interrupted the ongoing gaze shift but not the movement of the head. The interruption of gaze occurred because the saccadic component of the eye movement was halted. Gaze was constant during the interruption period because the eyes counterrotated and completely compensated for the continuing movement of the head. The gaze shift was resumed shortly after the offset of the stimulation train.

We concluded that stimulation of the OPN region gates the neural pathway involved in the production of the saccadic eye component of the gaze shift. The EBN signal is not necessary for the initiation of the head component of a gaze shift or for the continued execution of an ongoing head movement. Having a separate gate for the onset of saccadic movements allows eye and head movements to begin at different times. Also, the separate gate can prevent saccadic disruptions of smooth pursuit eye movements tracking a target moving in a single-depth plane or disruptions of vergence eye movements when tracking a target that is moving through different depth planes. Inhibition of saccade initiation also occurs when the vestibulo-ocular reflex (VOR) allows fixation of a stationary target to be maintained by a counterrotation of the eyes during movements of the body and/or the head.

Why, then, in head-unrestrained animals, is the number of spikes generated by pontine burst neurons more highly correlated with the amplitude of the gaze shift than it is with the amplitude of the eye movement? My suggestion (Sparks 1999) was that during combined eye–head gaze shifts, the eye movements that are executed are not the movements requested by the EBNs. Because the head was moving and the VOR was active and producing a counterrotation of the eyes, the executed eye movements had amplitudes smaller than the movements that would have occurred if the head had not been moving.

In experiments with the head restrained, measures of executed eye movements provide accurate estimates of the saccadic commands transmitted to the extraocular motoneurons. The estimates are accurate because the motoneurons receive no additional inputs from other oculomotor subsystems (e.g., vergence, pursuit, vestibular) during the time when the saccade command is being implemented. Under these conditions, the amplitude of the movement is tightly correlated with the number of spikes in the saccade-related burst of pontine EBNs, and the peak velocity of the movement is tightly coupled to the peak frequency of the burst. However, when the motoneurons are receiving inputs from parallel oculomotor subsystems while a saccade is being generated, measures of the amplitude, direction, or velocity of the executed saccade provide unreliable and potentially misleading estimates of the saccadic command sent to the motoneurons. In the absence of precise information about other influences on motoneuron activity, correlations of the activity of neurons above the level of motoneurons with various movement parameters cannot be interpreted accurately when more than one oculomotor subsystem is active. I concluded (Sparks 1999) that “advances in understanding the neuronal bases of eye–head coordination and saccadic–vergence interactions will be impeded until we find a way to cope with this neural uncertainty problem—namely, the problem of not being able to determine whether or not the eye movement that was executed is the one that was requested by the subsystem under investigation.” See Sparks and Gandhi (2003) for a longer discussion of this issue.

The second case we encountered in which correlations between the saccade-related activity of neurons and the properties of the movement are misleading happens when saccades are made to the remembered location of a target. “Remembered saccades” are characterized by an “upward bias,”—that is, overshooting upward targets and undershooting downward targets (White et al. 1994). We recorded the movement fields of the same SRBN for movements to visual and remembered targets (Stanford and Sparks 1994). In each case, we observed an upward shift of the movement field to remembered targets relative to that observed for movements to visual targets. The most parsimonious explanation for this result is that the signal responsible for the observed upward bias is added at a site downstream to the SC. Because of the dissociation between SC commands and the movements that are observed on memory trials, saccades to remembered targets cannot be used to study the relationship between neuronal activity and the metrics or velocity of the executed movement. Perhaps it is more appropriate to talk about a “collicular request” rather than a “collicular command.”

Janis White was the first author of the first of two papers in the volume of Vision Research describing our findings about saccades to remembered target locations. Janis has OD and PhD degrees and is currently director of the Low Vision Service of the East Orange VA Medical Center in New Jersey. She did a postdoc in the lab after she finished her PhD in physiological optics at the University of Houston, College of Optometry.

Neeraj Gandhi did his dissertation under the supervision of Ed Keller and obtained his PhD in bioengineering in 1997 from the bioengineering graduate group offered jointly by the University of California, San Francisco, and the University of California, Berkeley. He is now professor and graduate program director, Department of Bioengineering at the University of Pittsburgh.

The Reproducibility of Motor Command Signals Carried by Individual Neurons

The responses of visual neurons are highly reproducible when the same time-varying luminance patterns are presented repeatedly (e.g., Reinagel and Reid, 2002, Journal of Neuroscience, 22: 6837–6841). Xintian Hu and I wanted to perform the motor equivalent of these sensory experiments by measuring the reliability of motor commands for saccadic eye movements generated by single premotor neurons.

We recorded the activity of the EBNs in the PPRF while subjects made repeated saccades from the same initial fixation target to the same eccentric target. Although considerable variability in the amplitude, duration, and velocity of movements was observed, software was used to identify subsets of movements with similar amplitudes and velocity profiles.

We (Hu et al. 2007) found that the ISIs of the high-frequency component of the bursts are very similar during movements having similar amplitudes and velocity profiles. Except for jitter in the duration of the first interval and variability in the last few intervals of the burst, most of the ISIs are similar, at a submillisecond timescale, from movement to movement. This is true despite the facts that (1) the movements selected for having similar amplitudes and velocity profiles occurred over a relatively long period and (2) movements of other directions and amplitudes and velocities were interspersed. Thus, each time the cell’s burst has a particular temporal pattern, a saccade with a particular velocity profile will occur. Such a relationship between the activity of a single pontine burst cell and the velocity profile of a subset of movements would not be observed if (1) other members of the large active population of pontine burst cells had quite different temporal profiles of burst activity or (2) if, in general, the burst profiles were heterogeneous and uncorrelated. Thus, these findings indicate that variability in the discharge of a single pontine burst cell is not independent of, but, instead, must be strongly correlated with the activity of other active pontine cells generating saccade-related bursts.

Xintian Hu graduated from the University of Science and Technology of China in 1988. He received his PhD degree from Princeton University in 2000 performing his dissertation research in the lab of Charles Gross. In 2005, he was awarded the One Hundred Persons Project of the Chinese Academy of Sciences and took his current position at the Kunming Institute of Zoology as a full professor.

Other Research

By focusing on experiments related to the format of the motor commands and the frame of reference of sensory signals found in the SC, I have neglected research conducted in the lab on other topics and the individuals performing those experiments. To partially compensate, the neglected individuals, their current positions, if known, and the papers they contributed to are listed here.

Additional Collaborators

Terry Hickey is now retired after resigning as provost of the University of Central Florida in 2010. Terry and I were co–principal investigators on a National Eye Institute grant to study the effects of short-term monocular visual deprivation on oculomotor function.

Mike Gurski was a postdoc in Terry’s lab.

Jon Nelson is currently professor of physical therapy at College of Saint Scholastica in Duluth, Minnesota.

Rosemarie “Rosi” Hartwich-Young (deceased) worked for Alcon Laboratories in Fort Worth, Texas, until retirement. Rosi wrote the anatomy section of our chapter in the book The Neurobiology of Saccadic Eye Movements, edited by Wurtz and Goldberg.

Laurel Carney received her bachelor’s degree in electrical engineering from MIT and her master’s and doctoral degrees, also in electrical engineering, from the University of Wisconsin at Madison. She published several papers related to auditory physiology with Tom Yin while at Madison. While a postdoctoral fellow in my lab, Laurel and Terry Stanford devised a behavioral paradigm that revealed a gradual build-up of the signals specifying the amplitude of a saccade to a single visual goal in human subjects. Laurel is currently the Marylou Ingram Professor of Biomedical Engineering and professor of neuroscience at the University of Rochester.

Ellen Barton is now teaching in the Oklahoma State University system. Ellen entered the neuroscience graduate program at Penn after receiving an MSEE degree from Rensselaer Polytechnic Institute.

Jamie Nichols did a postdoctoral fellowship in Bill Newsome’s lab at Stanford and later decided not to pursue a career in neuroscience.

Rimas Kalesnykas is the first author of our review of the role of the SC in the control of saccadic eye movement which appeared in The Neuroscientist. Yihong Zhang did his dissertation research in Larry Mays’s lab at UAB. He worked in my lab at Penn for a short while before I moved to Houston.

Longteng Chen continued the experiments he began in my lab when he moved to the Department of Otolaryngology at the University of Texas Medical Branch in Galveston. He was examining the effects of stimulation of the frontal eye fields and supplementary eye fields on movements of the eyes and head.

Laurent Goffart is currently a member of the Institut de Neurosciences de la Timone in Marseille, France. In my lab, he was able to delineate the time in which activity in the caudal fastigial nucleus can modify the trajectory of a saccade.

Mark Walton is currently a senior scientist at the University of Washington Seattle. He received his PhD in behavioral neuroscience working with Larry Mays. His time in the lab was shortened by my retirement.

Kathy Pearson became the lab programmer in 1980, and, except for three or four years when she was employed elsewhere, she was an integral part of the laboratory until I retired in 2004. For part of the time when the lab was at Penn, Kathy lived in Virginia and worked remotely. When I accepted the position at the Baylor College of Medicine, Kathy stayed in Virginia but moved, virtually, from Philadelphia to Houston.

Papers by the Additional Collaborators

Barton EJ, Nelson JS, Gandhi NJ, Sparks DL (2003) Effects of partial lidocaine inactivation of the paramedian pontine reticular formation on saccades of macaques. J Neurophysiol. 90: 372–386.

Barton E, Sparks DL (2001) Saccades to remembered targets exhibit enhanced orbital position effects. Vision Res. 41: 2393–2406.

Chen LL, Goffart L, Sparks DL (2001) A simple method for constructing microinjectrodes for reversible inactivation in head-unrestrained, behaving monkeys. J Neurosc Methods. 107: 81–85.

Goffart L, Chen LL, Sparks DL (2003) Saccade dysmetria during functional perturbation of the caudal fastigial nucleus in the monkey. Ann NY Acad Sci. 1004: 220–228.

Goffart L, Chen LL, Sparks DL (2004) Deficits in saccades and fixation during muscimol inactivation of the caudal fastigial nucleus in the rhesus monkey. J Neurophysiol. 92: 3351–3367.

Hartwich-Young R, Nelson JS, Sparks DL (1990) The perihypoglossal projection to the superior colliculus in the rhesus monkey. Vis Neurosc. 4: 29–42.

Kalesnykas RP, Sparks DL (1996) The primate superior colliculus and the control of saccadic eye movements. The Neuroscientist. 2: 284–292.

May PJ, Hartwich-Young R, Nelson J, Sparks DL, Porter JD (1990) Cerebellotectal pathways in the macaque: Implications for collicular generation of saccades. Neuroscience. 36: 305–324.

Nichols J, Sparks DL (1995) Nonstationary properties of the saccadic system: new constraints on models of saccadic control. J Neurophysiol. 73: 431–435.

Nichols J, Sparks DL (1996a) Component stretching during oblique stimulation-evoked saccades: the role of the superior colliculus. J Neurophysiol. 76: 582–600.

Nichols J, Sparks DL (1996b) Independent feedback control of horizontal and vertical amplitude during oblique saccades evoked by electrical stimulation of the superior colliculus. J Neurophysiol. 76: 4080–4093.

Sparks DL, Barton, EJ, Gandhi, NJ, Nelson, J (2002) Studies of the role of the paramedian pontine reticular formation (PPRF) in the control of head-restrained and head-unrestrained gaze shifts. Ann NY Acad Sci. 956: 85–98.

Sparks DL, Freedman EG, Chen, LL, Gandhi NJ, (2001) Cortical and subcortical contributions to coordinated eye and head movements. Vision Res. 41: 3295–3305.

Sparks D, Barton EJ (1993) Neural control of saccadic eye movements. Curr Opin Neurobiol. 3: 966–972.

Sparks DL, Gurski MR, Mays LE, Hickey TL (1986) Effects of long-term monocular deprivation upon oculomotor function in the rhesus monkey. In Keller EL, Zee DS, eds. Adaptive Processes in Visual and Oculomotor Systems. Oxford: Pergamon Press, 91–200.

Sparks DL, Hartwich-Young R (1989) The deeper layers of the superior colliculus. In Wurtz RH, Goldberg ME, eds. Reviews of Oculomotor Research: The Neurobiology of Saccadic Eye Movements. Amsterdam: Elsevier, 213–255.

Sparks DL, Mays LE, Gurski MR, Hickey TL (1986) Long-term and short-term monocular deprivation in the rhesus monkey: Effects on visual fields and optokinetic nystagmus. J Neurosci. 6: 1771–1795.

Sparks D, Nelson J (1987) Sensory and motor maps in the mammalian superior colliculus. TINS. 10: 312–317.

Sparks DL, Rohrer B, Zhang Y (2000) The role of the superior colliculus in saccade initiation: A study of express saccades and the gap effect. Vision Res. 40: 2763–2777

Stanford TR, Carney LH, Sparks DL (1990) The amplitude of visually guided saccades is specified gradually in humans. Soc Neurosci Abstr. 16: 372.19.

Walton MG, Sparks DL, Gandhi NJ (2005) Simulations of saccade curvature by models that place superior colliculus upstream from the local feedback loop. J Neurophysiol. 93: 2354–2358.

Committees, Editorial Boards, Review Articles

I felt an obligation to contribute to the rapidly growing community of neuroscientists and the organizations that supported and published my research, including the following:

Member, Bio-Psychology Study Section, NIH

Chair, Bio-Psychology Study Section, NIH

Member, Program Committee, Society for Neuroscience

Member, National Advisory Eye Council, National Eye Institute

Member, National Task Force on the NIH Strategic Plan

Ad-Hoc Member, Board of Scientific Counselors, National Eye Institute

Motor Commission, International Union of Physiological Sciences

Board of Trustees for the Society for the Neural Control of Movement

Board of Scientific Advisors, Regional Primate Research Center, University of Washington

Editorial Board: Behavioural Brain Research, Reviews of Oculomotor Research, Journal of Neuroscience, Vision Research, Visual Neuroscience, Behavioral Neuroscience, Journal of Vision, Journal of Neurophysiology

In addition, Ron Harris-Warrick and I were occasional section editors of the motor control issue of Current Opinions in Neurobiology. Several review articles, not cited in previous sections of this document, are listed in the Selected Bibliography.

Retirement (December 31, 2004–present)

After I announced my retirement, Jennifer Groh and Neeraj Gandhi organized my Roast at a combined meeting of the Vision and Oculomotor social groups at the 2004 meeting of the Society for Neuroscience in San Diego. Jennifer chaired the session and did the initial searing before introducing the main Roasters: Kathleen Cullen, Bill Newsome, and Ed Keller. I felt well done when I gave a short, humorous (hopefully), and intellectually light response. At the end, there was a long standing ovation. I was most appreciative.

Xintian Hu’s experiments were still in progress when I retired. The National Eye Institute approved an arrangement in which John Maunsell became PI on my active grant (funding had been approved through July 31, 2007) and supervised Xintian’s research until he finished collecting data.

Xintian had a position awaiting him at the Kunming Institute of Zoology in China. The Institute purchased the equipment in my lab and would have it shipped to China after Xintian finished his current project. I spent a month in China in 2006 and five weeks in 2007 working with Xintian on the analysis of data he had collected after I retired and preparing the manuscript (Hu et al. 2007).

I was invited to give the keynote address at a Gordon Research Conference (Oculomotor System Biology) in Maine in 2005. Betty went to the meeting so she could visit with participants who had worked in my lab. The last scientific meeting I attended was the 2007 Oculomotor Gordon Conference. Xintian and I had a poster displaying the results of the experiments concerning the reliability of motor-command signals.

Florida was battered by four major hurricanes within a six-week period during the 2004 hurricane season. Hurricane Ivan made a direct hit on Pensacola four months before I retired in December 2004. My boat (En Passant) survived with no damage. All the marinas in the area suffered serious damage and the cost of slip rentals increased significantly after the marinas were rebuilt. Insurance for boats the size of mine was difficult to obtain in Florida and became almost prohibitively expensive. The cost–enjoyment ratio was not working for me. I sold En Passant and nature photography became my primary retirement hobby.

Nature photography, especially bird photography, combines birdwatching, being outdoors, photography, travel, messing about with computers, and learning new skills and concepts—all things I enjoy. Much of my time in retirement has been devoted to this activity. My goal has been to capture, in an unobtrusive manner, the beauty and uniqueness of birds, and some of the characteristic behaviors displayed in their natural habitat. I have self-published several photo books to preserve images that I think were successful in achieving this goal in a format that will be durable and accessible to family and friends when I am no longer around.

Since the Gordon Conference in 2007, almost all my in-person contact with neuroscientists has been related to sailing or photography. I helped Ken Britten and Sandra Aamodt sail their new (for them) boat from the Straits of San Juan de Fuca to San Francisco in 2006. Ken, Sandra, and Paul Gamlin came to Pensacola, and we spent a few days sailing in the nearby bays and sounds in 2007. In 2010, I spent a few days photographing the California wildflowers with Bill Newsome and his son David. In 2011, I met Bill Newsome and Brie Linkenhoker in New Mexico, and we photographed the migrating sandhill cranes and snow geese at the Bosque del Apache National Wildlife Preserve. In 2018, Bill Newsome and I went on a South Georgia–Falklands Expedition led by four well-known nature photographers.

In Pensacola, Betty continued her gardening activities, enjoyed the monthly meetings of a literary study group, and spent a lot of time doing genealogical research. She spent two weeks in Kunming in 2006 while Xintian and I were analyzing the data collected in Houston. In 2007, Betty, Sadie (one of our granddaughters), and I met Xintian in Beijing. Xintian was our guide to many of his favorite places in the area for a week. Then Betty and Sadie returned to the United States. Xintian and I went to Kunming. I stayed for four weeks while we finished the data analysis and preparation of the manuscript that we submitted for publication.

We sold our house in Pensacola in 2015 and bought a condo in Colorado to be near our sons and grandkids. Initially, we spent half our time in Colorado and half in Florida, but we moved to Colorado full time in 2017.

During the COVID-19 lockdown, I have been occupied by putting together photo books for family and friends, compiling a book about Betty after her sudden and unexpected death in 2019, and preparing this manuscript.

Acknowledgments

I was fortunate to have been born at a time and in a place in which it was possible to spend most of a lifetime pursuing one’s curiosity. The 12-year-old boy following Betsy (the mule) and a plow could not have imagined the research career he would have because of the many chance encounters described here. Nor could he imagine that in retirement he would visit places such as St. Paul Island in the middle of the Bering Sea to photograph the colonies of seabirds nesting on the cliffs.

The era in which my research was performed was an exciting one. It was a time when many technological advances altered dramatically what could be accomplished in the laboratory. Especially important, this was a time when the activity of neurons involved in the control of movements could be observed in moving animals and not just in anesthetized or paralyzed preparations.In the oculomotor realm, researchers in many different countries were contributing to the rapid increase in knowledge about the vestibular system, the extraocular muscles, and the anatomical areas that provided inputs to the motoneuron pools, as well as the biophysical and neurochemical attributes of the neurons in the neural circuits involved in oculomotor control. International meetings devoted to the neural control of eye movements began to be organized. The meetings facilitated the assimilation of the new information and provided opportunities for individual interactions with researchers doing similar research.

I am grateful for the generosity of researchers who shared the methods and techniques they developed when performing related research. These were essential for many of the experiments described here. The research would not have happened without the financial support provided by several sources. Over the years, I was PI on three different grants funded by the National Eye Institute. I was also recipient of a McKnight Senior Investigator Award and a Sloan Foundation grant in computational neuroscience.

I thank the students, technicians, programmers, postdocs, and colleagues who worked in the lab or made other contributions to the experiments for the effort exerted during all phases of the experiments. The intellectual stimulation occurring during our weekly journal club meetings was one of the most rewarding aspects of my work. Most of the concepts considered in this review were discussed extensively during the meetings, and it would be difficult to know the actual source of some of the ideas.

I have a close-knit and supportive family. I am especially indebted to my wife, Betty. She understood and supported my passion for my work and my hobbies, as I understood and supported her passion for her undertakings. The move to Colorado to be near family in retirement was a decision we should have made earlier. The family gatherings have been wonderful and watching our two youngest grandchildren develop is something we missed with the older ones because of geographical distance.

I thank Jennifer Groh for helpful comments on an early version of this document and Shari Hatch-Gately for detecting numerous formatting errors and making other useful suggestions.

Selected Bibliography

Freedman EG, Sparks DL (1997a) Eye–head coordination during head-unrestrained gaze shifts in rhesus monkeys. J Neurophysiol. 77: 2328–2348.

Freedman EG, Sparks DL (1997b) Activity of cells in the deeper layers of the superior colliculus of rhesus monkey: Evidence for a gaze displacement command. J Neurophysiol. 78: 1669–1690.

Freedman EG, Sparks DL (2000) Coordination of the eyes and head: Movement kinematics. Exp Brain Res. 131: 22–32.

Freedman EG, Stanford TR, Sparks DL (1996) Combined eye–head gaze shifts produced by electrical stimulation of the superior colliculus in rhesus monkeys. J Neurophysiol. 76: 927–952.

Gandhi NJ, Sparks DL (2003) Changing views of the role of superior colliculus in the control of gaze. In Chalupa LM, Werner J, eds. The Visual Neurosciences. Cambridge: MIT Press, 1449–1465.

Gandhi NJ, Sparks DL (2007) Dissociation of eye and head components of gaze shifts by stimulation of the omnipause neuron region. J Neurophysiol. 98: 360–373.

Glimcher P, Sparks DL (1992) Movement selection in advance of action: saccade-related bursters of the superior colliculus. Nature. 355: 542–545.

Glimcher P, Sparks DL (1993) The effects of low frequency stimulation of the superior colliculus on spontaneous and visually guided saccades. J Neurophysiol. 69: 953–964.

Groh JM, Sparks DL (1992) Two models for transforming auditory signals from head-centered to eye-centered coordinates. Biol Cybernetics. 67: 291–302.

Groh JM, Sparks DL (1996a) Saccades to somatosensory targets. I. Behavioral characteristics. J Neurophysiol. 75: 412–427.

Groh JM, Sparks DL (1996b) Saccades to somatosensory targets. II. Motor convergence in primate superior colliculus. J Neurophysiol. 75: 428–438.

Groh JM, Sparks DL (1996c) Saccades to somatosensory targets. III. Eye-position-dependent somatosensory activity in primate superior colliculus. J Neurophysiol. 75: 439–453.

Guthrie BL, Porter JD, Sparks DL (1983) Corollary discharge provides accurate eye-position information to the oculomotor system. Science. 221: 1193–1195.

Hu X, Jang H, Gu C, Li C, Sparks DL (2007) The reliability of oculomotor command signals carried by individual neurons. Proc Natl Acad Sci USA. 104(19): 8137–8142.

Isa T, Sparks DL (2006) Microcircuit of the superior colliculus: A neuronal machine that determines timing and endpoint of saccadic eye movements. In Grillner S, Graybiel AM, eds. Microcircuits: The Interface between Neurons and Global Brain Function. Cambridge: MIT Press, 5–34.

Jay MF, Sparks DL (1984) Auditory receptive fields in primate superior colliculus shift with changes in eye position. Nature. 309: 345–347.

Jay MF, Sparks DL (1987a) Sensorimotor integration in the primate superior colliculus: I. Motor convergence. J Neurophysiol. 57: 22–34.

Jay MF, Sparks DL (1987b) Sensorimotor integration in the primate superior colliculus: II. Coordinates of auditory signals. J Neurophysiol. 57: 35–55.

Jay MF, Sparks DL (1990) Localization of auditory and visual targets for the initiation of saccadic eye movements. In Berkley M, Stebbins W, eds. Comparative Perception. Vol. I: Basic Mechanisms. New York: Wiley and Sons, 351–374.

Lee C, Rohrer WH, Sparks DL (1988) Population coding of saccadic eye movements by neurons in the superior colliculus. Nature. 332, 357–360.

Mays LE, Sparks DL (1980a) Dissociation of visual and saccade-related responses in superior colliculus. J Neurophysiol. 43: 207–232.

Mays LE, Sparks DL (1980b) Saccades are spatially, not retinocentrically, coded. Science. 208: 1163–1165.

Mays LE, Sparks DL (1981) The localization of saccade targets using a combination of retinal and eye-position information. In: Progress in Oculomotor Research. Fuchs A, Becker W, eds. New York: Elsevier, 39–47.

Porter JD, Guthrie BL, Sparks DL (1983) Innervation of monkey extraocular muscles: Localization of sensory and motor neurons by retrograde transport of horseradish peroxidase. J Comp Neurol. 218: 208–219.

Sparks DL (1975) Response properties of eye movement-related neurons in the monkey superior colliculus. Brain Res. 90: 147–152.

Sparks DL (1978) Functional properties of neurons in the monkey superior colliculus: Coupling of neuronal activity and saccade onset. Brain Res. 156: 1–16.

Sparks DL (1986) Translation of sensory signals into commands for the control of saccadic eye movements: Role of primate superior colliculus. Physiological Reviews. 66: 118–171.

Sparks DL (1988a) Neural cartography: Sensory and motor maps in the superior colliculus. Br Beh Evol. 31: 49–56.

Sparks DL (1988b) Saccadic command signals in the superior colliculus: Implications for sensorimotor transformations. Canad J Physiol Pharm. 66: 527–531.

Sparks DL (1989) Neural commands for the control of saccadic eye movements: Required transformations of signals observed in the superior colliculus. In Ito M, ed. Neural Programming, Taniguchi Symposia on Brain Sciences No. 12. Tokyo: Japan Scientific Societies Press, 125–136.

Sparks DL (1991a) Sensorimotor integration in the primate superior colliculus. Seminars in the Neurosciences. 3: 39–50.

Sparks DL (1991b) The neural control of orienting eye and head movements. In Humphrey DR, Freund H-J, eds. Motor Control: Concepts and Issues. Dahlem Konferenzen. Chichester: Wiley and Sons, 263–275.

Sparks DL (1999) Conceptual issues related to the role of the superior colliculus in the control of gaze. Curr Opin Neurobiol. 9: 698–707.

Sparks DL (2002) The brainstem control of saccadic eye movements. Nat Rev Neurosc. 3: 952–964.

Sparks DL (2004) Commands for coordinated eye and head movements in the primate superior colliculus. In Hall WC, Moschovakis AK, eds. The Superior Colliculus: New Approaches for Studying Sensorimotor Integration. Boca Raton: CRC Press, 303–318.

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Sparks DL, Mays LE, Porter JD (1987) Eye movements induced by pontine stimulation: Interaction with visually-triggered saccades. J Neurophysiol. 58: 300–318.

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Sparks DL, Travis RP (1971) Firing patterns of reticular formation neurons during horizontal eye movements. Brain Res. 33: 477–481.

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Nicholas C. Spitzer

BORN:

New York, New York

November 8, 1942

EDUCATION:

Harvard College, BA (Biology, 1964)

Harvard Medical School (1964–1966)

Harvard University, PhD (Neurobiology, 1969)

APPOINTMENTS:

Fellow, Harvard Medical School (1969–1970)

Fellow, University College London (1970–1972)

Assistant to Distinguished Professor, University of California San Diego (1972–present)

Chair, Department of Biology (1988–1990); Co-Chair (1992–1993)

Chair, Academic Senate (1994–1995)

Founding Chair, Neurobiology Department, Division of Biological Sciences (1999–2003)

Founding Co-Director, Director, Kavli Institute for Brain and Mind (2004–2020)

Founding Editor-in-Chief, BrainFacts.org (2011–2014)

HONORS AND AWARDS (SELECTED):

Alexander Forbes Lectures, Grass Foundation, MBL (1998)

Trustee, The Grass Foundation (1998–2002)

Elected Councilor, Society for Neuroscience (2000–2004)

Elected Fellow, American Association for the Advancement of Science (2001)

Elected, American Academy of Arts and Sciences (2002)

Wiersma Visiting Professor of Neuroscience, California Institute of Technology (2006)

Elected, National Academy of Sciences, USA (2013)

Atkinson Family Endowed Chair (2016–present)

Kuffler Lectures, University of California, San Diego (2017)

Karl Spencer Lashley Award (2022)

Nick Spitzer carried out a sustained line of research originating in studies of the development of neuronal excitability. This led to the discovery, in the embryonic amphibian nervous system, of calcium-dependent action potentials that generate transient elevations of intracellular calcium before the action potentials achieve classical sodium-dependence. He found that changes in this calcium-dependent electrical activity cause neurotransmitter switching, which is associated with matching changes in postsynaptic receptors. He subsequently discovered that changes in electrical activity also drive transmitter switching and receptor matching in the adult mammalian brain. Transmitter switching often switches the sign of the synapse and leads to changes in behavior that can be detrimental or beneficial. These findings identified a novel form of plasticity, distinct from changes in synaptic weight and synapse number, and overturned the long-held view that the choice of transmitter is irreversibly specified by cell identity at an early stage of development.

Nicholas C. Spitzer

Parents

I was born into an academic family with high expectations of achievement, shortly after the United States entered World War II. In addition to the chromosomes, there was a culture of accomplishment. I am grateful for both, but have always wondered how my career would have turned out with a different starting point. Finis origine pendet—the end hangs on the beginning—so I shall start with a description of the family into which I arrived.

My father, Lyman Spitzer Jr., was an astrophysicist who in 1946, when I was four, published a report for the Rand Corporation pointing out the benefits of space astronomy and the value of an orbiting telescope. He would go on to develop suborbital telescopes and promote the realization of the Hubble Space Telescope. These and other achievements were later recognized in many ways, including the National Medal of Science, the Crafoord Prize, and posthumously when the last of the four great orbiting telescopes was named the Spitzer Space Telescope. At the time I was born, he was working in the Empire State Building on development of sonar to enhance submarine detection for the Navy. He was younger than his two sisters and the older of two sons of a Toledo Ohio businessman who ran a paper box factory. He had won laurels in boarding school at Andover Academy and in college at Yale, received his PhD at Princeton, and was on leave from his faculty position at Yale. At the age of 33, he was appointed professor and chair of the Department of Astronomy at Princeton and director of the University Observatory.

My mother, Doreen Canaday Spitzer, was the archeologist daughter of a Toledo industrialist at the Willys Overland Company who was known as the father of the Jeep. She attended boarding school at Dongan Hall on Long Island and college at Bryn Mawr, her major in archeology inspired by her mother’s devotion to the classics. After graduation, she spent three years as a student at the American School for Classical Studies at Athens, working on excavations in Corinth, Greece. This launched a lifelong love of Greek language, culture, and the arts. She later returned to Greece many times and served as a trustee of the school for 18 years. She took all four of her children and later 10 of her 13 grandchildren on individual trips “to the old country.” Being the eldest, I had the privilege of being the first. My mother was also a sparkling hostess at dinner parties in Princeton for a steady stream of visiting astronomers. In early years, my sisters and I watched them come and go but ate supper by ourselves in the kitchen. In later years, we were welcomed at the dining room table, fascinated by the conversations.

Lyman and Doreen met each other at an early age, as the two families knew each other socially in Toledo. They were mutually attracted and enjoyed dancing together but decided to pursue their different objectives for a while and stay in touch. They met again in Switzerland when the Spitzers invited the Canadays to visit them at the Chateau Gumligen bei Bern. Doreen and Lyman played tennis, recited poetry, danced, and climbed the Stockhorn. Their wedding in 1940 led to a happy and mutually supportive marriage of 57 years, as Doreen recounted in her memoir, As Long As Ye Both Shall Live.

My father studied interstellar matter and star formation from both theoretical and observational perspectives. He was known for an unusual ability to visualize physical processes, examining them in his mind and then checking conclusions mathematically. My mother delved into the origins of the culture she loved so much. My parents’ interests in these evolving and changing processes likely contributed to my later interest in the development of the nervous system.

Childhood

In connection with my father’s war work, our family moved to Washington, D.C., where my sister Dionis was born in 1945. We subsequently moved back to live in New Haven, Connecticut, before moving to Princeton in 1947. The Observatory on Prospect Avenue adjacent to the campus was connected to a three-story Victorian house with stairs equipped with bannisters that we loved to straddle and slide down. My sisters Sarah Lutetia and Lydia were born in 1950 and 1954, and Dionis and I graduated to bedrooms on the third floor of the house. We enjoyed climbing on the peaked, slate-tiled roof with its wide gutters and in the towering maple and beech trees in the yard.

My father installed a chairlift in the yard on a rope that ran from a low point on one tree to a high point on another; we took turns pulling each other up with a tow rope, letting go the rope and watching the rider come careening down in high glee. He also dug a small swimming pool in the yard that was later enlarged in excavation by the faculty and graduate students of the department in a single afternoon at one of the annual Observatory picnics. My mother built a playhouse nearby from the large shipping crate in which our grand piano arrived. She also converted a back room of the house into a playroom with a curtain that fostered impromptu theatrical performances. She enjoyed gardening and taught me how to separate bulblets and plant out daffodil and tulip bulbs.

The house was situated on the same street as the University eating clubs and saw a steady flow of undergraduates along the sidewalks on which we roller skated. When there was significant snowfall in winter (and snow days with no school), we went skiing on nearby country slopes, climbing up for each descent. When our neighbor Don Griffin brought out his horse and sleigh on Prospect Avenue, we rode in the sleigh or skijored behind. Both my parents loved to ski and from an early age took us on week-long ski vacations from school every year at Lake Placid, New York, or Stowe, Vermont. I continue to enjoy both downhill and cross-country skiing with family and friends. I have faint memories of my attendance at Miss Mason’s Nursery School and Miss Fine’s School, followed by stronger recollections of the Nassau Street grade school that was a short walk from home. My parents were ardent Francophiles (my father took all his sabbatical leaves in Paris), so my mother arranged for a series of French undergraduates to provide brief afternoon tutorials for me and my sisters in conjugating avoir and être and learning a modest vocabulary. These were followed by a demonstrably French dinner at which we all had to speak French. After dinner she played the piano and we gathered round to sing French songs.

When my father took his position at Princeton, he had arranged for a six-month leave of absence every other year to work at Caltech where he could make observations with the large telescopes on Mt. Wilson and have opportunities for collaboration. Some years we took a train to Chicago and another one on to Los Angeles, a three-day trip through spectacular western scenery. Other years we took the train to Chicago and drove the rest of the way, camping out and visiting National Parks. We lived in Pasadena in a series of rented houses near Caltech and were enrolled in school. We got out on hikes in the San Gabriel Mountains along old trolley lines. We stayed in the Kapteyn Cottage when my father was observing with the 100-inch telescope that Hubble had used to make observations of galaxies beyond our own and obtain evidence for an expanding universe. My father’s war work had led to friendship with Roger Revelle at the Scripps Institution of Oceanography; we visited the Revelles in La Jolla and our families went camping together in the desert. The palm trees, bougainvillea, and sagebrush of southern California combined with camping and hiking trips in the Sierra Nevada mountains and in Yosemite, Sequoia, and Kings Canyon National Parks produced many warm memories. Years later, these experiences predisposed me to be enthusiastic about a faculty position at the University of California, San Diego (UC San Diego).

My father greatly enjoyed planning family trips, as had his father before him. One of these saw us sailing on the Isle de France ocean liner from New York to Le Havre in 1955. Dionis and I were introduced to the many sights of Paris and then traveled to Zermatt in Switzerland with our parents for an introduction to serious climbing with guides. We enjoyed climbing peaks on both rock and snow, but were too young to join our parents on their ascent of the Matterhorn. When my father afterward participated in a meeting of the International Astronomical Society in Dublin, Ireland, my mother, Dionis, and I cycled around the countryside exploring and climbing in the ruins of old castles. My two younger sisters stayed at home with a good family friend.

After completing fifth grade at the Nassau Street school, I progressed to the Quarry Street school for the sixth through eighth grades. I did well in all subjects and founded a student newspaper (Teen Topics). However, my behavior in school was rambunctious, and I was sent to the principal’s office on more than one occasion for disruptive behavior in class. I was caught shoplifting in Woolworth’s store and entering locked university buildings through open windows—activities that reflected my teenage testing of limits—what could I get away with? My parents sensed that I was not being sufficiently challenged academically and enrolled me at Andover Academy for high school. It was a perfect move.

Education

My four years at Andover were transformative. Academic demands were challenging and I learned how to focus my efforts. I enjoyed Mr. Maynard’s Math 2x-4x, which covered three years of math in two years, and Dr. Hammond’s Science Honors course. With Dr. Gillingham, I learned Latin and read Caesar, Virgil, Horace, and Ovid. Monsieur Gilbert taught me French by la méthode direct (he never spoke in English). Emory Basford was a stern but forgiving task master in English, and I remember memorizing long soliloquies in Shakespeare’s Richard II (popularly referred to as Dick the Deuce). I played club-level sports, rowing crew or running cross-country in the fall, skiing in the winter, and playing lacrosse in the spring. I enjoyed my theatrical roles as King Claudius in Hamlet and the sinister Jonathan in Arsenic and Old Lace. I have always been grateful for the breadth and rigor of my Andover education, which prepared me to enter Harvard in 1960 as a sophomore with advanced standing.

I declared my major in physics and enrolled in math and physics courses that I soon realized were over my head. I also spent a great deal of time dating a classmate at Radcliffe whom I had met the previous summer in the Experiment in International Living in France. I was encouraged to try out for the fencing team and the épée became my weapon. I enjoyed the regular workouts and the strategies for achieving a touch against opponents, going on to become team captain my senior year. However, my grades were so poor at the end of my first year that I received a letter from the dean suggesting that I might like to take a year’s leave of absence. I spent the summer in school at UC Berkeley, taking a course in English literature and another in Italian, with time off to go climbing in Yosemite. My second year at Harvard, I built on my facility with languages and pendulumed my major from physics to Slavic and linguistics, taking intensive courses in these subjects.

Fortunately, I also enrolled that year in George Wald’s new introductory biology course, Natural Sciences 5. Wald was a Nobel laureate for his discovery of the role of vitamin A in forming the three color pigments and showing that color blindness is caused when one of the pigments is absent. He was a compelling lecturer, holding the entire class in rapt attention, starting with prebiotic synthesis at the beginning of the semester and concluding with animal behavior at the end. I found his lectures on the nervous system inspiring, and learned later that he had persuaded half a dozen other students that year to take up careers as neuroscientists. I switched my major to biology and approached John Dowling (Volume 4), who had worked with Wald and was now a member of the faculty, to ask if I could work in his lab.

I was delighted when John took me on in the fall of 1962. He had just demonstrated the existence of rapid dark adaptation in the albino rat that did not have a photochemical basis—an apparently neural process. He suggested that I study the horseshoe crab, Limulus, to see if it had a similar component to the visual adaptation of its compound eyes. If it did, the simplicity of neural processes beyond the receptors would raise the possibility that rapid dark adaptation is a property of the receptor itself. His enthusiasm was contagious. I drove to Woods Hole, brought animals back to the Bio Labs on Divinity Avenue, and maintained them in seawater in a cold room. I spent many happy hours recording from single nerve fibers dissected from the optic nerve bundle of young crabs. For work the following summer, I had a National Science Foundation (NSF) fellowship of $600 that was just sufficient to rent a room in Cambridge and support my steady diet of whole wheat bread and peanut butter. I supplemented this occasionally with bullfrog legs from Wald’s freezer where, after removing the frogs’ retinas, he had stored them for his later consumption. Fortunately, I had graduated and left the lab by the time Wald returned, but I learned later that he was greatly annoyed by the loss.

My results were compatible with the existence of a neural component to dark adaptation. I loved this research. I found electrophysiology intensely satisfying: One asks the question and the data become available immediately. Change an experimental parameter, new data appear. This experience launched my career. Discussions with John both in his lab and during picnics on the Boston North Shore directly shaped the next phase of my education. He had gone to medical school at Harvard and thought that he had been more broadly educated than he would have been in graduate school, where prompt specialization and narrowing of focus is emphasized. I found this persuasive and matriculated in the fall of 1964 at the Harvard Medical School.

The entering class was told that a substantial fraction of everything we would be taught would prove incorrect, but the identity of that fraction was unknown so that it was essential to learn all of it. In the first two years, the rapid pace included gross anatomy, biochemistry, and physiology, as well as parasitology and pathology courses, all requiring hours of study. The latter courses introduced me to subjects with which I was wholly unfamiliar and contributed to my later interest in scientific problems with clinical relevance. The block of lectures on the nervous system were particularly good and especially interesting for their clarity and rigor. They were presented by faculty in Stephen Kuffler’s neurobiology unit, at that time a component of Otto Krayer’s Pharmacology Department. In the second year, we were given white coats and introduced to taking histories on hospital wards. This tipped the scale for me, as I soon realized that there is a routine and even repetitive quality to medicine because patients often have the same disorders. I was sufficiently self-aware to recognize that continuity of this experience would not be sustainable.

With the prospect of two more years of study focused increasingly on clinical aspects of medicine, I gave my college senior honors thesis to Steve Kuffler in support of my application to become a graduate student in the neurobiology unit. I was very glad to be accepted and supported by a predoctoral fellowship from the NIH. The extraordinary faculty included David Potter, Ed Furshpan, Ed Kravitz (Volume 4), David Hubel (Volume 1), and Torsten Wiesel. Jim Hudspeth, David Van Essen (Volume 9) and Eric Frank joined me in this class of graduate students. Demanding seminar courses taught by the faculty, based on original papers, were intensely stimulating. David Potter became my adviser and got me started with a cathode follower amplifier, making intracellular recordings with sharp electrodes and replicating published findings. As postdoctoral fellows, Dave and Ed Furshpan had discovered the electrical synapse in the crayfish nervous system, and they were pursuing their joint interest in electrical coupling among electrically inexcitable cells. Dave suggested the rat seminiferous tubule as an experimental preparation because of the potential role of somatic supporting cells in coordinating synchrony of meiosis by large numbers of germinal cells. I was able to be quite independent, and I became interested in a similar arrangement in the anther of the lily bud, which could be conveniently investigated once a year in the spring. Short-range interactions between cellular elements might be afforded by gap junctions in the seminiferous epithelium and by plasmodesmata in the anther, directly connecting the interior of one cell to that of another. In both of these preparations, my recordings of electrical coupling and observation of the spread of intracellular tracers demonstrated interaction between cell types not previously known to be joined by appropriate membrane junctions. However, I did not obtain evidence for a role for these connections in controlling or metabolically supporting synchronous meiosis. I received my PhD in 1969 and published my first paper on the results from the anther of the lily (Spitzer, 1970). My unpublished results of coupling in the rat seminiferous tubules later received published support in the form of electron microscopic images of gap junctions (Pelletier & Byers, 1992, Microscopy Research Technique, 20, 3–33).

Steve’s neurobiology unit had become the Neurobiology Department by the time I received my PhD. Steve ran it like a family, warmly engaged in everyone’s work and life. He led by example, scientifically and personally. At seminars in the lunchroom, he illustrated how one could be penetratingly critical and also exhibit an extraordinary sense of humor, sometimes simultaneously. His influence on members of the department in those times is captured in a wonderful monograph (STEVE, Remembrances of Stephen W. Kuffler; McMahan, 1990).

Postdoctoral Years and Future Planning

Jack McMahan was working with Steve and pioneering the use of Nomarski interference contrast optics for visualization of neurons in situ. I approached him to ask if I could do a brief postdoc with him. I had spent the summer between my two years in medical school in the lab of J. David Robertson, learning electron microscopy (EM). Robertson’s technician, Janet Lamborghini, taught me how to cut thin sections and capture exquisite EM images. The experience not only gave me a deep appreciation of the power and beauty of anatomy, but also introduced me to my future wife (married in 1967). My studies with Jack enhanced my appreciation of the importance of anatomy when we developed the frog cutaneous pectoris muscle as a preparation for direct visualization of single nerve terminals for physiological studies.

Steve was a good friend of Bernard Katz (Volume 1) from their time together in Sydney, and there was an academic pipeline bidirectionally between Steve’s Department and Katz’s Biophysics Unit at University College London. I was impressed by the way Bernard Katz and Ricardo Miledi thought about solving scientific problems and asked Steve if I might pursue further postdoctoral work there. In response to my letter of inquiry to Katz, I soon received a letter of welcome in return. The next two years were exhilarating. Dale Purves (Volume 11), Bert Sakmann, Bill Betz, Jean Rosenthal, Terje Lomo (Volume 7), and Bob Zucker contributed to the lively discussions of research (or cricket) in the tearoom at 11 a.m. and 4 p.m., in which Katz and Miledi occasionally participated. I worked under the supervision of Ricardo, studying the effect of botulinum toxin on synaptic transmission at the amphibian neuromuscular junction and collaborating with Mollie Holman during her sabbatical visit from Monash University. Ricardo was an extraordinarily creative investigator and I watched him closely in order to absorb as much of his talent as possible. Living in London on a NATO NSF postdoctoral fellowship enabled enjoyment of West End theater, music on the South Bank, side trips to notable sites in the countryside and travels in Europe.

As my second year in the Biophysics Unit moved along, I started to think about what to do next. I believed that the careful choice of the problem on which to begin work in my own lab would be essential and could define the arc of my career. It was critical to select a problem in which I had a deep interest so that I would work tirelessly to address it. I wanted to go in untraveled directions and pick a problem unidentified by others or that had received little attention. A less crowded field would provide more opportunities for significant discovery, and it would be important to embrace risk. I wanted to select the largest problem on which I thought I could make serious headway in a reasonable time. It would also be useful to pick a problem that appeared to be fundamental and thus of broad significance. I grew up intellectually in the heyday of molecular biology in the 1950s and 1960s. Fundamental questions about DNA structure and replication, coding of information in DNA, and operation of the Central Dogma had been answered. These discoveries were inspiring, and I was looking for similarly foundational questions to address.

Neuronal development was endlessly fascinating to me although I had had no practical experience in the field. The interface of electrophysiology with developmental biology was relatively unexplored. I boiled down my choice to either the development of electrical excitability or the development of specific neuronal connections. Both action potentials and brain wiring are crucial to brain function. Studies of axon pathfinding and connectivity were already underway by Michael Gaze, Marcus Jacobson, and others. Much work focused on development of retinotectal connections that Roger Sperry’s incisive studies had drawn to attention. However, the use of genetically tractable models for developmental studies was in its infancy. Ed Furshpan and Dave Potter had led a graduate seminar discussing the famous five Hodgkin (Volume 1) and Huxley (Volume 4) 1952 papers in the Journal of Physiology. Like many others, I was captivated by their findings. How did the action potential develop during embryogenesis? I wanted to find out.

In my graduate work, I had learned how to make intracellular recordings from small cells in novel preparations. My knowledge of Kuffler’s pathbreaking research made me aware of the importance of selecting the appropriate model system with which to answer a question. In my postdoctoral studies, I was sensitized to the advantages of an amphibian model. The available literature led me to focus on the primary sensory Rohon-Beard neurons of the Xenopus embryo. These neurons were first described by Freud during his early career as a neuroanatomist. Their names came from James Victor Rohon, in Germany, and John Beard, in England, who studied them later. I was to fall in love with them, as they kept yielding up secrets about neuronal development. The location of their 20-µm diameter cell bodies on the dorsal surface of the neural tube would facilitate recordings. Anne Warner gave me some embryos on which to try dissections and impalements and that went well. My first goal would be to chart the ontogeny of the neuronal action potential.

Starting My Lab at UC San Diego

I flew to the United States for a whirlwind visit to five universities and found UC San Diego particularly attractive. Twelve years earlier, Roger Revelle had recruited outstanding faculty with whom to launch the University, and its cross-disciplinary spirit and ambition were appealing. I accepted the offer and arrived in January 1972. My startup package included a laboratory in Bonner Hall, two offices, $25,000 and several used microscopes. Janet Lamborghini was my first postdoc and helped me set up the lab. After futile efforts to capture the elusive Xenopus in the storm drains along the freeways, we ordered adult animals to begin generating embryos. Sharp, high-resistance microelectrodes were pulled for physiological recordings, and fixatives and microtomes were prepared for anatomical studies.

It was exciting to make the first successful impalements of Rohon-Beard neurons in the embryos and in the premetamorphic tadpoles, although success came only once or twice a week in the beginning. To provide a thorough grounding in the membrane properties of these neurons, I began by studying their resting potential, which proved interesting in its own right. It sat at –88 mV in the presence of 3 mM external potassium chloride (KCl), like the resting potential of glia (Kuffler et al., 1966, Journal of Neurophysiology, 29, 768–787). This value was more negative than most intracellular recordings of neuronal resting potentials known at the time, but followed the prediction of the Nernst equation for a K⁺-selective membrane. Stimulation of the anterior end of the spinal cord elicited antidromic action potentials in Rohon-Beard neurons, followed by slower depolarizations. These depolarizations could be attributed to accumulation of extracellular K⁺ from the action potentials of neighboring axons, as earlier demonstrated for glia (Orkand et al., 1966, Journal of Neurophysiology, 29, 788–806). I suggested that in intact animals, these depolarizations of the membrane potential should decrease the distance to threshold so that a smaller generator potential would be effective in eliciting an action potential (Spitzer, 1976).

The Development of Excitability: Intracellular Recordings

I was particularly eager to investigate the appearance of the action potential. My recordings from Rohon-Beard neurons at the tadpole stage had shown that the action potential was brief in duration and conventional in waveform. Does it spring into existence fully formed, like Athena from the head of Zeus? Progress with the embryos was slow because dissections were often destructive and because visualization of the neurons viewed through a 40x water immersion lens was hindered by intracellular inclusions of yolk and lipid. Fortunately, Paola Baccaglini, my first graduate student, joined me in this work. We both felt a great urgency for success and worked late into most evenings. At the neurula stage, when the neural tube had just closed, no action potential was detected in response to a depolarizing current pulse. However, a hyperpolarizing current pulse yielded an inflection in the voltage trajectory at the conclusion of the pulse, suggestive of anode break excitation. Wonderfully, depolarization of Rohon-Beard neurons in embryos only a few hours older, at early tailbud stages, produced all-or-none, overshooting action potentials that ranged in duration from as long as 500 milliseconds (msec) to as short as 50 msec. The use of ion substitutions and pharmacological blockers led us to the conclusion that most of the inward current was carried by Ca²⁺. Calculation suggested that the Ca²⁺ influx could raise the intracellular Ca²⁺ concentration from 10⁻⁷ to 10⁻⁵ M, sufficient to influence later neuronal differentiation. When embryos were a few hours older, at late tailbud stages, the action potential acquired a new, rapidly rising component. This was followed by a slower component that created a shoulder on the repolarizing phase. The peak proved to be Na⁺-dependent and the shoulder was Ca²⁺-dependent. The overall duration now ranged from 20 msec to 4 msec. Then, by early tadpole stages, the action potential was 1 msec in duration and wholly Na⁺-dependent, as in the mature animal (although Ca²⁺-dependent action potentials could still be elicited when K⁺ currents were blocked). These findings demonstrated the sequence in which neuronal excitability developed in the early stages of maturation (Spitzer & Baccaglini, 1976; Baccaglini & Spitzer, 1977).

Recognizing that dissociated cell cultures would be useful for extending the analysis of differentiation, we modified procedures of others so we could track the development of neurons from cells that had been dissociated from the neural plate of embryos before the onset of their excitability. It was gratifying to discover that the somatic action potential of Rohon-Beard neurons, motor neurons, and interneurons all matured in vitro in a similar sequence and with a time course similar to what we had observed for Rohon-Beard neurons in vivo (Spitzer & Lamborghini, 1976). In addition, finding this same sequence of maturation in cultures of single neurons demonstrated that the process is cell autonomous (Henderson & Spitzer, 1986). The same sequence of maturation of action potentials, from Ca²⁺- to Na⁺-dependence, was later observed in neurons from embryos of chick and mouse (Spitzer, 1985).

Anatomical studies of the birth of Rohon-Beard neurons, their sequestration of intracellular Ca²⁺, their transmitter uptake and the death of the neurons were carried out in the following years (Lamborghini et al., 1979; Lamborghini, 1980, 1985, 1987). Following Steve Kuffler’s practice, I added my name to publications only when I had participated in the experiments, though I was wistful when three of the papers from the lab appeared in Nature. Still, I believed it was the right thing to do, and it does not seem to have hindered my career. I continued this practice for 22 years until the lab grew larger, with many projects underway simultaneously, so that I needed to leave the joys of doing the experiments to my colleagues.

In 1977, Corey Goodman (Volume 12) visited and made a persuasive case for studying the development of embryonic grasshopper neurons. He subsequently joined the lab as a postdoctoral fellow. The dorsal unpaired median (DUM) neurons of grasshoppers, like the Rohon-Beard neurons of Xenopus, were identifiable, such that the same cells could be examined from animal to animal. Simple dissections allowed both classes of neurons to be visualized and studied in vivo early during embryogenesis, and their large size expedited intracellular recordings. What aspects of functional and anatomical development of Rohon-Beard neurons were conserved in the development of DUM neurons? A grasshopper breeding colony was quickly set up in the basement of Bonner Hall. We mastered the dissections of segmental ganglia in embryos at different stages of development and investigated the anatomical, biochemical, and electrophysiological properties of DUM neurons from birth to maturation. The development of axon projections, their central arborizations and the appearance of their transmitter, octopamine, were characterized first (Goodman et al., 1979). We also examined the development of chemosensitivity to neurotransmitters, the onset of electrical excitability, and the loss of extensive electrical and dye coupling to other cells that is frequently observed during cell-specific differentiation (Goodman & Spitzer, 1979, 1981a, 1981b; Goodman et al., 1980). The earliest action potentials were elicited from neurites about the time of electrical uncoupling and were Na⁺-dependent. Action potentials appeared shortly afterward in the soma and were dependent on both Na⁺ and Ca²⁺. I was early to bed and early to rise during those years, while Corey rolled in later and worked into the evening. This arrangement provided the opportunity to carry out my experiments on Xenopus in the morning, investigating the development of chemosensitivity and electrical uncoupling of Rohon-Beard neurons. Corey and I worked together on grasshopper in the afternoon. We concluded that the developmental trajectories of Rohon-Beard and DUM neurons were similar for aspects of development known at the time. Later, we discovered another similarity. Transmitter expression in Rohon-Beard neurons starts at the stage of development when axon outgrowth begins (Root et al., 2008; Jacobson & Huang, 1985, Developmental Biology, 110, 102–113), just as we had found for DUM neurons.

After completing a two-year postdoc, Corey joined the faculty at Stanford, and I returned to full-time work with Rohon-Beard neurons. I had found they were electrically coupled before the time when Ca²⁺-dependent action potentials could be recorded. The coupling was voltage-dependent, and sustained depolarization or hyperpolarization of one neuron reversibly uncoupled that neuron from others, as previously reported for amphibian blastomeres. I was intrigued by the voltage dependence of this process. Reduction of intracellular pH eliminated the voltage dependence but left the neurons in a coupled state. When Ca²⁺-dependent action potentials appeared, the impulse in one neuron could uncouple it from others. At early stages of development, Rohon-Beard cells were coupled along and between two rows on either side of the dorsal spinal cord. These neurons became permanently uncoupled around the time that the prominent Na⁺ current appeared in the action potential, while other cells remained coupled (Spitzer, 1982). The role of voltage-dependent uncoupling was unclear, but I supposed it might serve to isolate neurons from one another, so that they could begin to develop independently.

Postdoc John Bixby and I completed the study of developing chemosensitivity in Rohon-Beard neurons, finding that responses to GABA first appeared at late tailbud stages. The responses had a reversal potential of –30 mV, which appeared to depend on Na⁺ and K⁺ instead of Cl⁻ and did not change during development, but were blocked by bicuculline or picrotoxin. Later research indicated that elevation of the intracellular concentration of Cl⁻ by a Na⁺- and K⁺-dependent Cl⁻ transport mechanism (likely NKCC1) underlies the Cl⁻-dependent depolarization by GABA receptors in these neurons (Rohrbough & Spitzer, 1996). Surprisingly, both young and mature neurons appeared to be insensitive to glutamate, acetylcholine, or five other transmitters (Bixby & Spitzer, 1982). Responses to GABA appeared at about the same time as the appearance of the Na⁺ current in the action potential and contemporaneous with electrical uncoupling. In addition, study of the appearance and development of transmitter sensitivity in cultures of Xenopus spinal neurons demonstrated the presence of two populations of cells. One population had the chemosensitivity profile of Rohon-Beard neurons, depolarized by GABA and insensitive to glutamate. The other population had the profile of motor neurons and interneurons. They were depolarized by glutamate and were hyperpolarized by GABA and glycine, with a reversal potential of –59 mV (Bixby & Spitzer, 1984a).

In the meantime, we demonstrated a requirement for RNA and protein synthesis for maturation of the action potential, using our cell culture system. Early inhibition of transcription with actinomycin D, or inhibition of protein synthesis with cycloheximide or puromycin, blocked the appearance of brief Na⁺-dependent action potentials such that they remained long in duration and Ca²⁺-dependent. Application of the inhibitors at later times in development allowed action potentials to develop normally (O’Dowd, 1983; Blair, 1983).

The Development of Excitability: Voltage Clamp Studies

The next step was to make voltage-clamp recordings of the ionic currents underlying the action potential to quantify their changing contributions during development. Patch electrodes had recently been perfected, and these seemed ideal for voltage-clamp recordings from cultured cells (Hamill et al., 1981, Pflugers Archiv, 391, 85–100). As part of her graduate training, Diane O’Dowd took a Cold Spring Harbor course to learn how to voltage clamp excitable cells and set up our first patch-clamp rig when she got back to the lab. Angie Ribera joined me for her second postdoc and became an invaluable colleague. In short order, we recorded isolated Ca²⁺currents, Na⁺ currents, and both voltage- and Ca²⁺-dependent K⁺ currents from young neurons and mature neurons. The high-voltage-activated Ca²⁺ current (ICa) showed only a small increase in the extent of inactivation during the development of excitability. However, the small Na⁺ current (INa), which was present when the action potential first appeared, increased twofold in density. Ca²⁺-dependent K⁺ current (IKc) density increased twofold, and the voltage-dependent K⁺ (delayed rectifier) current (IKv) density increased threefold and became activated almost twice as fast (O’Dowd et al., 1988). IKv was emerging as a major contributor to maturation of the action potential. Single channel recordings identified 15 and 30 pS conductance classes of voltage-dependent K⁺ channels in both young and mature neurons. We detected no changes in open probability of these channels, enabling calculation that the number of channels had increased threefold. Only the 30 pS class showed an increase in its activation rate (Harris et al., 1988). These single channel recordings showed that the changes in IKv during maturation reflected increases in the number of channels and not increases in single channel conductance.

In an elegant study, Angie Ribera discovered the period during which transcription was required for the differentiation of IKv and for the maturation of the action potential. A nine-hour application of a reversible RNA synthesis inhibitor to cultured neurons irreversibly prevented both events. Removal of the inhibitor after nine hours allowed resumption of RNA synthesis, but IKv did not appear and the action potential remained long in duration. Instead, a different K⁺ current, IKA, which normally appears later than IKv, was now expressed, albeit delayed in its appearance by the nine hours of inhibited RNA synthesis. These results demonstrated a critical period for transcription essential for IKv to appear. After the critical period, no recovery of IKv was observed (Ribera & Spitzer, 1989; 1990).

The Development of Excitability: Reconstruction of the Developing Action Potential

Voltage-clamp recordings of amphibian spinal neurons during development had revealed changes in ICa, INa, IKc, IKv, and IKA. To determine whether these changes were sufficient to produce the changes in action potential duration and ionic dependence that we had observed, Shawn Lockery and I constructed a HodgkinHuxley model of the electrical excitability of these neurons. During the day, Shawn was doing postdoctoral work with Terry Sejnowski, and I was doing experiments in the lab, so we worked in the evenings. Had we identified all the currents underlying the young and mature action potentials, or were additional currents required? Does IKv make the largest contribution? Could changes in other currents be even more important? The model showed that the equations describing the voltage-clamped currents of young and mature neurons generated action potentials that were appropriate in duration and ionic dependence for each developmental stage. Moreover, the observed changes in the currents were quantitatively sufficient to produce the changes in the action potential observed during development. The effect of the change in each current was detectable in the model. Notably, the increase in amplitude of IKv had the largest effect. These results were hugely rewarding. The model showed that the currents we had characterized could account for all our observations of action potentials during development. Furthermore, changes in action potential duration could be achieved with changes in kinetics rather than in the amplitude of IKv or with changes in amplitudes of other currents (Lockery & Spitzer, 1992). How IKv came to have such an important role in development remains a subject for future study.

The Development of Excitability: K⁺ Channels Underlying IKv

We began analyzing the expression of different K⁺ channel genes to identify the ones that underlie IKv, which had proved to be the workhorse in shortening the action potential and altering its ionic dependence. xSha2 (xKv1.2) was the first Xenopus K⁺ channel gene to be cloned. xSha2 was expressed in the nervous system at the neurula stage and encoded a delayed rectifier current when expressed in oocytes. However, it was a relatively rare transcript, suggesting that it would make only a small contribution to IKv (Ribera, 1990). Single-cell reverse transcription–polymerase chain reaction (RT-PCR) analysis of K⁺ channel gene expression following recording of IKv during its development in culture identified transcripts of xKv1.1 and xKv2.2, which encode delayed rectifier current, but neither was expressed throughout the development of IKv (Gurantz et al., 1996). We also isolated xKv.3.1, which encoded a delayed rectifier current in oocytes and was upregulated in the nervous system throughout the period in which IKv develops in vivo. xKv3.1 was expressed in 40 percent of neurons in culture, suggesting that it contributes to maturation of IKv and to shortening of the action potential (Gurantz et al., 2000). To assess its contribution directly, we used antisense cRNA to xKv3.1 to suppress expression of this gene in the spinal cord. These experiments reduced the amplitude and rate of activation of IKv and increased the duration of the action potential in 40 percent of the neurons, matching the expression of xKv3.1 that we had observed in cultured neurons (Vincent et al., 2000). These findings, along with Angie Ribera’s results from her lab at the University of Colorado, indicated that heterogeneous K⁺ channel gene expression gives rise to homogenous IKv current. It was satisfying to have an understanding of the development of the action potential at the level of expression of single K⁺ channel genes.

Intracellular Ca²⁺ Transients

We expected that the influx of Ca²⁺ during long-duration, Ca²⁺-dependent action potentials would raise intracellular levels of Ca²⁺. Janet Holliday joined the lab for her postdoc, and we began imaging intracellular Ca²⁺ with fura-2AM to study the effects of Ca² influx more directly. We first discovered that neurons exhibit a transient elevation of Ca²⁺ in response to a single Ca²⁺-dependent action potential. We also observed spontaneous, transient elevations of intracellular Ca²⁺ in culture during early development at the time when action potentials produce the greatest Ca²⁺ influx. The imaging studies further revealed that rapid Ca²⁺ elevations, both in the nucleus and in the cytosol, were primarily due to Ca²⁺-induced Ca²⁺-release from intracellular stores that was triggered by Ca²⁺ influx (Holliday & Spitzer, 1990; Holliday et al., 1991).

Xiaonan Gu wrote to me to inquire about a postdoc while he was completing his graduate work with Ken Muller. Ken recommended him with great enthusiasm and I was glad to take him on. Xiaonan was deeply insightful and extraordinarily creative. We established an exceptional partnership, working initially with cultured neurons to understand how Ca²⁺ transients are triggered, and studying the biophysical properties and function of low-voltage-activated (LVA) T-type Ca²⁺ current (ICaT). There were no significant changes in threshold, peak density, kinetics, and pharmacological sensitivity of ICaT during development. Spontaneous Ca²⁺ transients in young neurons assayed by fluo-3AM fluorescence were blocked by Ni²⁺ or amiloride at concentrations that specifically blocked ICaT. This current had the lowest threshold of activation among other inward currents in young neurons, and mathematical simulations showed that ICaT lowers the threshold of the action potential by 15 mV. We concluded that ICaT can depolarize cells and trigger action potentials and that this current is part of the cascade of events leading to spontaneous elevations of intracellular Ca²⁺ in cultured neurons at early stages of differentiation (Gu & Spitzer, 1993). Michel Desarmenien, visiting on leave from the French National Centre for Scientific Research, undertook voltage clamp analysis of embryonic neurons developing in vivo, focusing on the Ca²⁺ and K⁺ currents that play key roles in triggering and controlling the duration of action potentials (Desarmenien et al., 1993). Reassuringly, low-voltage-activated Ca²⁺ current, ICaT, IKv, and IKA all developed as previously observed in culture.

Imaging Xenopus spinal neurons with fluo-3AM both in culture and in the intact isolated spinal cord for one hour during development identified two classes of spontaneous Ca²⁺ transients, Ca²⁺ spikes and Ca²⁺ waves, often in the same neuron. Graduate student Eric Olson joined us in these studies. We found that propagated Ca²⁺ spikes had rapid, stereotyped rise and decay, were about 10 sec in duration, depended on Ca²⁺-dependent action potentials and occurred at a frequency of about three spikes per hour that decreased during development. Waves were located in growth cones, had a slow nonuniform rise and decay, and were several minutes in duration. They did not depend on Ca²⁺-dependent action potentials and occurred at frequencies of about eight waves per hour throughout development. Both spikes and waves were abolished by removal of extracellular Ca²⁺ (Gu et al., 1994). These observations raised the possibility that the two distinct signals (spikes and waves) regulate different aspects of neuronal differentiation.

Regulation of Neuronal Differentiation by Intracellular Ca²⁺ Transients

We found three distinct neuronal properties that were regulated by either Ca²⁺ waves or Ca²⁺ spikes. One property, the extent of neurite outgrowth, depended on waves. Because Ca²⁺ waves were expressed in growth cones, they were in a position to regulate the extension of neurites. We found that Ca²⁺ waves inhibited neurite outgrowth. Thus, neurites were short in the presence of extracellular Ca²⁺ when waves were present. When waves and Ca²⁺ influx were prevented, by removal of extracellular Ca²⁺, neurites were long. Depletion of intracellular Ca²⁺ stores during development, even in the presence of extracellular Ca²⁺, also produced long neurites. The additional effect of these manipulations on Ca²⁺ spikes proved unrelated to the growth of neurites, because selectively blocking Ca²⁺ spikes did not affect growth (Bixby & Spitzer, 1984b; Holliday & Spitzer, 1990; Holliday et al., 1991; Gu et al., 1994). The inhibiting action of Ca²⁺ waves on neurite extension was mediated by calcineurin, a Ca²⁺-dependent phosphatase. Inhibition of this enzyme blocked the Ca²⁺-dependent reduction in rate of outgrowth, even though the frequency of waves was not affected. Moreover, constitutive expression of Xenopus calcineurin led to the generation of short neurites in the absence of Ca²⁺ waves. We were able to show that calcineurin suppressed the phosphorylation of GAP-43, thereby destabilizing actin filaments and slowing neurite extension (Lautermilch & Spitzer, 2000). It was gratifying to understand the molecular basis of Ca²⁺ wave action.

The second property, the rate of activation of IKv during development, proved to depend on Ca²⁺ spikes. Blocking voltage-dependent Ca²⁺ channels or intracellular Ca²⁺-induced Ca²⁺-release prevented the increase in activation rate of IKv. Significantly, depletion of protein kinase C in the presence of extracellular Ca²⁺ also prevented the increase, thereby identifying a molecular mechanism for the action of intracellular Ca²⁺. Stimulating the kinase in the absence of extracellular Ca²⁺ permitted an increase in the activation rate. Thus, Ca²⁺ spikes at early stages of development regulate the differentiation of the kinetics of IKv, perhaps by increased expression of the 30 pS channel described earlier, and modulate the dependence of action potentials on this current (Desarmenien & Spitzer, 1991).

The third property, neurotransmitter identity, also turned out to be regulated by Ca²⁺ spikes. We knew that the early differentiation of excitability in Xenopus spinal neurons in culture parallels their development in vivo. This encouraged us to examine the maturation of gamma-aminobutyric acid (GABA) immunoreactivity in cultured neurons, using an excellent antibody that recognized GABA. We found that GABA immunoreactivity in culture developed with the same time course previously described in vivo, and its appearance was blocked by transcriptional inhibitors. After prolonged absence of Ca²⁺-dependent action potentials, or prolonged depletion of Ca²⁺ stores, the number of GABAergic neurons was dramatically reduced. Importantly, this reduction in neuronal number occurred in the absence of cell death. The loss of GABA depended on sustained absence of Ca²⁺ influx. Janet Holliday and I, along with others, published a modest report of this finding, which would shape the direction of my scientific work from that point forward (Spitzer et al., 1988). Full accounts of these findings appeared subsequently (Holliday et al., 1991; Spitzer et al., 1993). Although it never occurred to us at the time, the reduction in number of GABAergic neurons would later prove to be associated with an equivalent increase in the number of neurons expressing a different transmitter, glutamate (Borodinsky et al., 2004). This occurred in the absence of neurogenesis. Looking back, I see that this finding of a reduced number of GABAergic neurons, when Ca²⁺ elevations were blocked, was the first hint of our subsequent discovery of neurotransmitter switching, which occurs in response to sustained changes in electrical activity.

The control of neuronal differentiation by Ca²⁺ spikes and waves suggested the principle of a developmental handshake involving reciprocal control, in which differentiation regulates ion flux and ion flux regulates differentiation (Spitzer, 1991). Later, Yehezkel Ben-Ari and I realized that there is a more general version of this principle. The development of the central nervous system requires intermediate stages of differentiation, providing functions that inﬂuence subsequent gene expression. We proposed that embryonic neuronal functions constitute a series of checkpoint signatures. Neurons failing to express these functions are delayed or developmentally arrested (Ben-Ari & Spitzer, 2010).

The difference in frequency of Ca²⁺ spikes and Ca²⁺ waves reminded me of the frequency coding of information in the peripheral nervous system (Adrian & Zotterman, 1926, Journal of Physiology, 61, 151–171). This led me to wonder whether the frequencies of spikes and waves were important for regulating expression of the three neuronal properties we were studying. Xiaonan Gu perfused cultures of Xenopus spinal neurons with Ca²⁺-free medium to eliminate spontaneous Ca²⁺ transients and used computer-controlled solenoid valves to deliver pulses of Ca²⁺ medium containing K⁺ to depolarize neurons. This procedure generated transient elevations of intracellular Ca²⁺ that mimicked spikes or waves, and these could be produced at different frequencies. Imposing different frequencies of Ca²⁺ elevation in this way demonstrated that the spontaneous wave frequency in the cultures was sufficient to regulate normal neurite extension, and the spontaneous spike frequency in the cultures was sufficient to produce normal neurotransmitter expression and current maturation. Chelation of intracellular Ca²⁺, while imposing the spontaneous frequencies of Ca²⁺ transients, demonstrated that these frequencies were also necessary for normal differentiation. These tests with different frequencies showed that the frequency of Ca²⁺ transients encoded information, like the frequency of action potentials, although the Ca²⁺ transients were 10⁴ times longer in duration and less frequent than action potentials (Gu & Spitzer, 1995).

Activity-Dependent Growth Cone Motility

To what extent were our findings about the role of growth cone Ca²⁺ waves in regulating neurite outgrowth in culture relevant to axon extension in the intact spinal cord? Lateral dissections of Xenopus embryos allowed us to observe migrating growth cones in vivo. By imaging fluo-3AM loaded neurons, postdoc Tim Gomez recorded transient elevations of [Ca²⁺]I with the same properties of growth cone waves we had seen in vitro. We found that growth cones of sensory Rohon-Beard neurons, motor neurons, and interneurons exhibited different migration velocities at different points in their trajectories in the spinal cord. For each class of neurons, the frequency of their growth cone Ca²⁺ transients was inversely proportional to the velocity of growth cone migration. Photoactivation of an intracellular Ca²⁺ chelator in slowly migrating growth cones suppressed Ca²⁺ transients and increased the rate of outgrowth. Photoactivation of intracellular caged Ca²⁺ in rapidly migrating growth cones generated Ca²⁺ transients and decreased the rate of outgrowth. At turning points, growth cones increased the frequency of Ca²⁺ transients and paused, as if sampling the environment for critical cues. We concluded that these growth cone Ca²⁺ transients provide a natural signaling mechanism that regulates axon extension and perhaps axon guidance (Gomez & Spitzer, 1999). Imaging growth cone Ca²⁺ transients in primary motor neurons of transparent zebrafish embryos with GCaMP3 also revealed differences in frequency that were correlated with the location that the growth cones had reached. High-frequency bursts of Ca²⁺ transients were associated with growth cone stalling at turning points. Suppression of Ca²⁺ transients by Kir2.1 K⁺ channel expression in single zebrafish primary motor neurons caused the neurons to make pathfinding mistakes (Plazas et al., 2013). These results raised the question of what cues were eliciting the Ca²⁺ transients.

The Origin of Spontaneous Ca²⁺ Transients in Growth Cones

Growth cone filopodia are thin sensory-motor protrusions from the palm of the growth cone that detect environmental cues and translate them into changes in motility. High-speed (8 Hz) confocal imaging of magnified regions of growth cones of cultured Xenopus spinal neurons loaded with fluo-4AM revealed brief (0.3-second duration) Ca²⁺ transients in filopodia. Their frequency, but not their amplitude or duration, depended on the identity and concentration of the environmentally relevant proteins on which the neurons were cultured. These filopodial transients occurred at a frequency of one to nine per minute in active filopodia and were rapidly propagated to the palm of the growth cone. The lifetimes of filopodia were proportional to the frequency of their filopodial transients. Higher frequencies initiated the larger Ca²⁺ transients in the palm of the growth cone that we previously had shown to suppress axon extension. Variations in the frequency of filopodial transients across the filopodia of a growth cone, when it encountered a boundary between two different substrate proteins, caused the growth cone to turn away from the side with the higher frequency. Thus, the filopodial Ca²⁺ transients, by way of a frequency code, were poised to regulate both growth cone extension and the dependence of turning on environmental cues (Gomez et al., 2001).

We also found that Ca²⁺ transients and transient elevations of cAMP were interdependent in the cell bodies of cultured Xenopus spinal neurons (Gorbunova & Spitzer, 2002). This discovery prompted investigation of the interactions between these two second messengers in the filopodia of the growth cones of these neurons. Postdoc Xavier Nicol found that Netrin-1, which attracts growth cones to the midline of the nervous system, drives a transient elevation of cAMP in filopodia, which does not depend on Ca²⁺, and briefly increases the frequency of filopodial Ca²⁺ transients. In contrast, Netrin-1 in the palm of the growth cone drives a transient Ca²⁺-dependent increase in cAMP and a sustained increase in the frequency of the Ca²⁺ transients that inhibit axon extension. Optogenetically generated cAMP transients in filopodia, but not in the palm of the growth cone, caused growth cone turning, and the angle of the turn was proportional to the frequency of imposed filopodial cAMP transients. We confirmed in vivo the relevance of these in vitro observations by spatially restricting the optogenetic stimulation of cAMP transients to filopodia. This was sufficient to steer growth cones to the midline when the Netrin-1 receptor was blocked (Nicol et al., 2011). It was clear that electrical activity, in different forms, regulates many aspects of development of the nervous system (Spitzer, 2006). Further investigation of activity-dependent growth cone motility was inviting, as it harked back to my enthusiasm for working on the wiring problem. However, our study of activity-dependent changes in neurotransmitter expression was heating up and began to consume my attention.

The Discovery of Activity-Dependent Transmitter Switching During Development

Laura Borodinsky joined the lab as a postdoctoral fellow, and we discovered transmitter switching in the developing spinal cord. Laura had the impressive ability to find the right way to do the right experiments. Previous studies had demonstrated that neurotransmitter identity is specified by intrinsic transcription factors, as Tom Jessell and his colleagues had discovered (Tanabe et al., 1998, Cell, 95, 67–80), and modified by cytokines, elevation of intracellular Ca²⁺, and neurotrophic factors, as Paul Patterson, Story Landis, and their collaborators had shown (Patterson & Chun, 1974, Proceedings of the National Academy of Sciences USA, 71, 3607–3610; Walicke & Patterson, 1981, Journal of Neuroscience, 1, 343–350; Landis and Keefe, 1983, Developmental Biology, 98, 349–372). Ed Furshpan and Dave Potter provided functional evidence for a change in transmitter within single sympathetic neurons (Furshpan et al., 1976, Proceedings of the National Academy of Sciences USA, 73, 4225–4229; Potter et al., 1986, Journal of Neuroscience, 6, 1080–1098). When we imaged fluo-4AM-loaded glutamatergic Rohon-Beard neurons, glycinergic dorsolateral and GABAergic ventral interneurons, and cholinergic ventral motoneurons in the embryonic Xenopus neural tube, we found that each class of neurons exhibited a specific pattern of spontaneous Ca²⁺ spike activity. Sustained suppression or enhancement of this activity, either by misexpression of K⁺ or Na⁺ channels or with pharmacological antagonists or agonists, altered the number of neurons expressing each of the four neurotransmitters, as identified by immunocytochemistry. Suppressing Ca²⁺ spike activity increased the number of neurons expressing glutamate and acetylcholine, the canonical excitatory transmitters, and decreased the number of neurons expressing GABA and glycine, the typical inhibitory transmitters. In contrast, enhancing Ca²⁺ spike activity had the opposite effect. It decreased the number of neurons expressing the excitatory transmitters and increased the number of neurons expressing the inhibitory transmitters (Borodinsky et al., 2004).

These findings suggested that activity-dependent regulation of neurotransmitter identity is homeostatic. Suppression of electrical activity appeared to be compensated by an increase in the number of excitatory neurons and a decrease in the number of inhibitory neurons. In a similar manner, enhancement of activity was compensated by an increase in the number of inhibitory neurons and a decrease in the number of excitatory neurons. Significantly, the total number of Rohon-Beard sensory neurons and motoneurons, for which independent markers of neuronal identity were available, was unchanged even while the number of neurons expressing glutamate and acetylcholine was altered (markers for the interneurons were not known at that time).

These results compelled us to conclude that neurons, responding to changes in activity, were changing their transmitters. Similar changes in transmitter identity were observed in culture, where we could show that the transmitter changes were functional. When we recorded spontaneous synaptic currents, we found that the proportion of neurons generating glutamatergic or cholinergic currents was indistinguishable from the proportion of neurons expressing glutamatergic or cholinergic immunoreactivity. Roles for neurogenesis or apoptosis seemed unlikely to be relevant because the number of Rohon-Beard neurons and motor neurons did not change following changes in Ca²⁺ spike activity. It was difficult to avoid the conclusion that neurotransmitter identity can be plastic in vivo, at least in the embryonic nervous system.

The Discovery of Transmitter-Receptor Matching

The discovery of transmitter switching created a quandary with respect to the postsynaptic transmitter receptors. A mismatch between the new transmitter and the old receptors would render the synapse inoperative. Fortunately, Laura Borodinsky’s examination of embryonic Xenopus striated muscle cells revealed that before innervation they normally expressed receptors for glutamate, GABA, and glycine in addition to acetylcholine. During normal differentiation and innervation of the muscle cells, acetylcholine receptor expression was sustained and the other receptors were downregulated. When the motoneuron transmitter was changed to glutamate, GABA, or glycine by altering Ca²⁺ spike activity, the appropriate cognate noncholinergic receptors were upregulated. In these conditions, glutamatergic, GABAergic, and glycinergic synaptic currents as well as nicotinic cholinergic currents could all be recorded from the muscle cells. When neurons were cocultured with muscle cells in the presence of extracellular Ca²⁺ (but not in its absence), which transmitter receptors were upregulated depended on which neurotransmitter was expressed by the neurons (Borodinsky & Spitzer, 2007). Thus, prior to innervation and in the absence of synapses, the selection of receptors seemed to depend on diffusible factors from the neurons.

We later tested the hypothesis that the diffusible factors include the neurotransmitters themselves. Using a functional assay, we found that if embryonic vertebrate skeletal muscle cells are cultured before they are innervated, then sustained application of glutamate to the cells is necessary and sufficient to upregulate ionotropic glutamate receptors from the pool of receptors that are normally expressed at low levels. Extending this result in vivo, by using implanted agarose beads impregnated with 2 mM glutamate to achieve sustained local diffusion of agonist, we saw that application of glutamate to embryonic skeletal myotomes was necessary and sufficient for upregulation of the GluN1 NMDA receptor subunit. These results suggested a mechanism by which neuronal release of a new (switched) transmitter upregulates the postsynaptic expression of appropriate, matching transmitter receptors (Hammond-Weinberger et al., 2020).

The Origin of Spontaneous Ca²⁺ Spike Activity

We now wanted to understand the cause of the spontaneous Ca²⁺ spike activity that, when suppressed or enhanced, leads to transmitter switching during development. It seemed possible that neurotransmitters could themselves regulate production of Ca²⁺ spikes. Cory Root, my creative and productive lab manager, took up this challenge (he would later return to UC San Diego as a faculty member). We found that, at the neural tube stage of development, both Rohon-Beard neurons and motoneurons coexpress glutamate and GABA. At the later tailbud stage, Rohon-Beard neurons selectively express glutamate and motoneurons selectively express acetylcholine, while interneurons selectively express GABA. Neither neurogenesis nor cell death could account for these changes. At the neural tube stage, both glutamate and GABA stimulated the generation of Ca²⁺ spikes via metabotropic receptors and activation of protein kinases A and C. Preventing the expression of GABA, or blocking GABA and glutamate receptors, lowered the frequency of Ca²⁺ spikes. This had the effect of increasing the number of neurons that express the excitatory transmitters, acetylcholine and glutamate, and of decreasing the number of neurons that express the inhibitory transmitters, GABA and glycine (Root et al., 2008). These findings answered our persisting question about the origin of spontaneous Ca²⁺ spike activity early in development. Neurotransmitters expressed early in development cause this spontaneous activity.

Environmental Stimuli Cause Transmitter Switching That Changes Behavior

At this point we understood that experimental manipulations of Ca²⁺ spike activity can regulate which neurotransmitter is expressed. But we had no idea whether transmitter expression in a neuronal population can also be specified by natural environmental stimuli. Furthermore, we did not know if transmitter switching had relevance for behavior. Davide Dulcis, who had a marvelous talent for doing ingenious and revealing experiments, joined the lab as a postdoc at this point. To address these questions, we first studied neurons in the ventral suprachiasmatic nucleus that in teleosts, amphibia, and primates regulates neuroendocrine pituitary function in response to light. In Xenopus larvae, we found that increasing or decreasing exposure to light for as little as 10 minutes regulated camouflage by clustering or dispersing the distribution of melanin in melanocytes. Skin pigmentation decreased in the light and increased in the dark. The effect of light on pigmentation was prevented by blocking D2 dopamine receptors.

Neurons in the ventral suprachiasmatic nucleus of the larvae normally exhibit Ca²⁺ spikes similar to those observed in the neural tube. We found that light, by increasing Ca²⁺ spike activity, caused the appearance of dopamine specifically in neurons that already expressed neuropeptide Y (NPY). Intriguingly, these newly appearing dopaminergic neurons already projected to the melanotrope cells that regulate melanocytes. After dopamine was expressed as a cotransmitter, the melanotrope cells expressed D2 dopamine receptors. NPY expression continued. We did not investigate whether another cotransmitter disappeared when dopamine appeared.

When the endogenous population of dopaminergic neurons was ablated, there was no change in skin pigmentation in response to a brief period of illumination. However, when these larvae were exposed to bright illumination for two hours, the newly appearing dopaminergic neurons rescued the camouflage regulation. This result demonstrated that the new dopaminergic neurons have relevance for behavior. We concluded that a natural stimulus, light, can alter the number of amine-transmitter-expressing neurons in the brain at larval stages of development and control a natural behavior, camouflage (Dulcis & Spitzer, 2008).

We later discovered another instance in which environmental stimuli drive transmitter switching and change behavior, when we studied changes in social preference of Xenopus larvae that ordinarily result from sustained exposure to kinship odorants. After normal development in the presence of siblings, sibling-raised larvae cluster in the water by swimming either toward a briefly available (10 minutes) kin odorant (sibling-conditioned water) or away from a non-kin odorant (nonsibling conditioned water). However, after sustained exposure (24 hours) to water containing kinship-signaling odorants released from nonsiblings, sibling larvae change their social preference and cluster with nonsiblings.

We found that this altered behavior was mediated by changes in the expression of dopamine and GABA in granule and periglomerular interneurons of the accessory olfactory bulb. In both granule and periglomerular cells, sustained exposure to kin odorants increased the number of interneurons expressing dopamine and decreased the number of interneurons expressing GABA. GABAergic neurons expressing the Pax6 transcription factor became dopaminergic neurons expressing Pax6 and no longer expressed GABA. In contrast, sustained exposure to non-kin odorants decreased the number of neurons expressing dopamine and increased the number of neurons expressing GABA. Blocking Na⁺-dependent action potentials or suppressing elevations of intracellular Ca²⁺ prevented these changes in transmitter expression.

We then looked for a causal connection between the newly speciﬁed transmitter and changes in kinship recognition. We ﬁrst ablated endogenous dopaminergic neurons in the accessory olfactory bulb of sibling-raised larvae. This treatment prevented attraction to kin odorants, suggesting that dopaminergic neurons in the bulb are ordinarily necessary for normal processing of the kinship odorants that elicit attraction to siblings. We next exposed these larvae to kin odorants for 24 hours to induce the expression of dopamine in neurons that initially expressed GABA. This treatment restored the attraction to kin odorants. Rescue of sibling preference suggested that the newly speciﬁed dopamine-expressing neurons were sufﬁcient to support kinship recognition (Dulcis et al., 2017).

Reserve Pool Neurons

I was fascinated by the finding that transmitter switches were associated with changes in behavior. Later work would continue to demonstrate that the switches caused the changes in behavior, as we were able to show that overriding the switches prevented the behavioral changes. However, transmitter switching during development occurred only in a subset of the neurons in a nucleus or region. Davide Dulcis and I proposed that these cells be designated reserve pool neurons. Reserve pool neurons already participate in neuronal circuits with afferent and efferent connections. In response to sustained activity, they acquire or lose or switch neurotransmitters, thus endowing circuits with a broader range of function. It was as if they had a day job and potentially a second night job and that activity could recruit them for this second job. In the case of a transmitter switch, reserve pool neurons would then give up their day job. Reserve pool neurons may be endowed with special signal transduction pathways not possessed by their nonswitching neighbors (Dulcis & Spitzer, 2011). Alternatively, their synaptic connections may enable them to generate higher or lower levels of activity than their neighbors, and in that case, they may not have special signal transduction machinery. I hope to understand what gives reserve pool neurons their special status.

The Discovery of Activity-Dependent Transmitter Switching in the Adult

Up to this point, I had supposed that transmitter switching was solely a developmental form of synaptic plasticity. However, when we searched for it in the adult rat brain, we were in for a big surprise. We knew that, during development, neurons in the suprachiasmatic nucleus can change their transmitter in response to light (Dulcis & Spitzer, 2008). Working from this finding, we tested the effect of a one-week exposure to long-day, balanced-day, or short-day photoperiods on transmitter expression and behavior. Rats are nocturnal and prefer short-day (long-night) photoperiods. We found that the paraventricular nucleus and several other hypothalamic nuclei increased the number of dopaminergic neurons during short-day photoperiods and decreased the number of dopaminergic neurons during long-day photoperiods, when compared to the number resulting from balanced-day photoperiods. The number of dopaminergic neurons resulting from long- and short-day photoperiods was matched by corresponding increases and decreases in the number of neurons expressing somatostatin. Thus, after exposure to the three different photoperiods, the number of somatostatin neurons was inversely related to the number of dopaminergic neurons. There was no change in adult neurogenesis or apoptosis in any of these conditions. Individual neurons appeared to be reciprocally switching transmitter expression between dopamine and somatostatin.

Somatostatin neurons of the paraventricular nucleus, as well as dopaminergic neurons, make synapses on corticotropin-releasing factor (CRF) neurons distributed along the third ventricle. After the stressful long-day photoperiod, and along with the decrease in number of dopaminergic neurons, dopamine D2 receptors on these CRF neurons were downregulated. In contrast, after the short-day photoperiod, and in parallel with an increase in number of dopaminergic neurons, the D2 receptors were upregulated. To our surprise, the level of somatostatin receptors did not change. After the short-day photoperiod, the level of CRF in the cerebrospinal fluid was lower, presumably because its release could be inhibited by both dopamine D2 receptors and somatostatin receptors. As expected, the level of CRF was higher after the long-day photoperiod, when D2 receptors were rare, and release could be inhibited only by somatostatin receptors. These findings reassured us that the D2 dopamine-somatostatin transmitter switch was functional.

To investigate the behavioral effects of the exposure to different photoperiods, we tested rats on the elevated plus maze and in the forced swim test, widely used assays of stress. Relative to controls exposed to the balanced-day photoperiod, rats with short-day exposure exhibited less stress. They spent more time exploring the open arm of the maze and spent a longer time swimming before becoming immobile. Long-day exposure produced the opposite effects. To determine whether these behavioral changes were specifically related to changes in the number of dopaminergic neurons, we ablated these neurons in animals maintained on the balanced-day photoperiod. This procedure increased stressful behavior in the same way as exposure to long-day photoperiods. Rats spent less time exploring the open arm of the elevated plus maze and less time swimming before becoming immobile. These findings showed that reducing the number of dopaminergic neurons was sufficient to lead to stress-like behavior.

We also asked whether photoperiod-dependent, hypothalamic transmitter switching was necessary for the observed changes in behavior. We first ablated dopaminergic neurons in the hypothalamic nuclei and then exposed animals to the short-day photoperiod to recruit more neurons to become dopaminergic. The stress-like behavior that we had seen after ablation of dopaminergic neurons was now reversed. This behavioral rescue was prevented by acute, local infusion of dopamine receptor antagonists, showing that the rescue depended on the newly recruited dopaminergic neurons and not on some other mechanism (Dulcis et al., 2013). These findings made a big impression on me, suggesting a relationship to the characteristics of seasonal affective disorder, a form of clinical depression that is more common at high latitudes in association with the short-photoperiod days of winter.

We followed up this notion in a long-range collaboration with colleagues in Melbourne after discussion of the relevance of our findings to humans. Thinking of high latitudes where photoperiod durations undergo wide swings over the course of a year, we secured postmortem brains from residents of Scotland, which we obtained from the Edinburgh University Brain Bank. These individuals had died from heart disease or cancer during either the summer (long-day photoperiod) or during the winter (short-day photoperiod). We found that the density of neurons expressing dopamine and its transporter was higher in the midbrains of those who had died in the summer than in the midbrains of those who had died in the winter. This study established an association in humans between photoperiod and the number of midbrain dopaminergic neurons and suggested to us that dopaminergic neurotransmitter switching might underlie this association (Aumann et al., 2016).

Beneficial Effects of Switching

Our discovery of transmitter switching in the adult mammalian brain was astounding to me, because neuronal transmitters were considered to be fixed and unchanging in the mature nervous system. I thought that transmitter switching might have explanatory power for a whole range of instances in which there is loss or gain of function in response to experience. Evidently, transmitter switching was not a process restricted to the developing nervous system. Where else in the brain could it occur and in response to what kinds of experience? Thinking about the path forward stimulated consideration of the model system that could best address the next set of questions. We quickly turned to adult mice, recognizing the availability of genetic tools with which to pursue our studies. I wondered whether the behavioral effects of transmitter switching resulted only from aversive experience, or whether rewarding experience could induce transmitter switches as well and, if so, was the resulting behavior beneficial? Hui-quan Li came to the lab as a postdoc, and we tackled this question.

Mice love to run. When running wheels are placed outdoors, infrared video cameras have revealed that the wheels are frequently used by wild mice at night. Moreover, running wheel use in the wild matches running wheel use by laboratory mice. We found that one week of voluntary wheel running in the lab induced transmitter switching from acetylcholine to GABA in neurons of the caudal pedunculopontine nucleus, which is important for gait and balance. The switch involved a decrease in the number of neurons expressing acetylcholine and a matching increase in the number of neurons expressing GABA. We next genetically marked cholinergic neurons to identify ones that had switched their transmitter. This method permanently labeled cholinergic neurons with mRuby2 fluorescent protein, even those that lost acetylcholine as a result of running. We could then score the number of mRuby2-labeled fluorescent neurons that expressed GABA after running. The results showed unambiguously that formerly cholinergic neurons now expressed GABA. No neurogenesis or apoptosis was detected. The switch was prevented by expressing K⁺ channels in the cholinergic neurons to suppress the generation of action potentials, demonstrating that the switch was activity-dependent.

Following sustained running, mice also exhibited enhanced learning of motor skills, mastering the accelerating rotarod more rapidly and accommodating to the narrow balance beam more quickly than mice without access to a running wheel. Interestingly, we found that switching neurons project to the substantia nigra, ventral tegmental area, and the thalamus, all areas that regulate motor skill learning. Enhanced performance of the motor skills was sustained for at least two weeks, although the transmitter switch spontaneously reversed at the end of a week of rest following the one week of running. Because the behavior persisted even when the switch had reversed, this finding suggested that plasticity had occurred downstream of the switching neurons, perhaps at one of the targets of the switching neurons, such as the substantia nigra.

To determine whether transmitter switching was necessary for the beneficial effect of running on motor skill learning, we sought to override the switch in transmitter identity. First, prior to access to the running wheel, we introduced the gene encoding choline acetyltransferase into genetically marked cholinergic neurons to sustain expression of acetylcholine in neurons that otherwise would lose it after running. Now, one week of running no longer enhanced motor skill learning. In parallel experiments, we introduced a gene encoding an inhibitor of the gene encoding glutamic acid decarboxylase to prevent the appearance of GABA after running. Again, we found that one week of running no longer enhanced motor skill learning. These experiments showed that either compensating for the loss of acetylcholine, or preventing the gain of GABA, blocked the beneficial effect of sustained running. Clearly, rewarding experience stimulated transmitter switching, and the effects were beneficial (Li & Spitzer, 2020). We began to suspect that transmitter switching could be induced by many kinds of experience, particularly when they were sustained in duration.

Mechanisms of Transmitter Switching in Development: Transcription Factor Activation

I wanted to understand the cellular and molecular mechanisms of transmitter switching, as this information might make it possible to regulate transmitter switching for beneficial purposes. The preponderance of glutamate and GABA as neurotransmitters recommended study of the glutamate-to-GABA switch that we had discovered in the embryonic Xenopus spinal cord. Because Ca²⁺ spike activity initiates transmitter switching, when Alicia Güemez-Gamboa joined the lab as a postdoc, we began by investigating the mechanism by which Ca²⁺ spike activity is transduced. Graduate student Lin Xu first suppressed Ca²⁺ spikes in single neurons to determine whether glutamate-to-GABA switching is cell autonomous. Transmitter switching did not occur, suggesting that it is regulated by the level of Ca²⁺ spike activity in surrounding neurons. We then found that brain-derived neurotrophic factor (BDNF) was expressed in the spinal cord and that increasing Ca²⁺ spike activity in cultures of spinal neurons caused the release of BDNF. Activation of TrkB receptors by BDNF in the spinal cord triggered a mitogen-activated protein kinase signaling cascade, which concluded in cJun N-terminal kinase-mediated activation of the cJun transcription factor and glutamate-to-GABA switching. Introduction of BDNF, after suppression of Ca²⁺ spikes, drove the switch, increasing the number of GABAergic neurons and decreasing the number of glutamatergic neurons. Inhibiting production of BDNF or blocking activation of TrkB receptors, reversed the switch, increasing the number of glutamatergic neurons and decreasing the number of GABAergic neurons. These ﬁndings identified the initial transduction mechanism for activity-dependent transmitter switching in developing spinal neurons (Güemez-Gamboa et al., 2014).

Postdoc Kurt Marek and graduate student Lisa Kurtz led the search for gene expression regulated by this signaling cascade. Using a functional screen, we identified tlx3 (the t-cell leukemia homeobox-3 gene). We found that tlx3 expression depended on Ca²⁺ spike activity. Additionally, tlx3 was expressed in Rohon-Beard neurons, which we had shown to undergo the glutamate-to-GABA switch. Thus, this gene was expressed in the right place at the right time to regulate the switch. Knocking down tlx3 function with morpholinos produced the switch, increasing the number of GABAergic neurons in the spinal cord and decreasing the number of glutamatergic neurons. Overexpression of tlx3 had the opposite effect on the transmitter switch, increasing the number of glutamatergic neurons and decreasing the number of GABAergic neurons. These results showed that tlx3 can function as a switch that specifies the level of expression of glutamatergic and GABAergic phenotypes.

To learn how tlx3 was regulated by activity, we surveyed the promoter region of the frog, mouse, and human tlx3 genes for conserved transcription factor binding sites. This led us to identify a variant form of the cAMP response element (CRE), with high conservation, suggesting that this nucleotide sequence could be important for the regulation of tlx3 transcription. We next directly assessed the role of cJun in transmitter switching by overexpressing wild-type or dominant-negative cJun. Overexpressed cJun produced the switching that we had observed following increased Ca²⁺ spike activity, increasing the number of GABAergic neurons and decreasing the number of glutamatergic neurons, similar to the results after tlx3 knockdown. In contrast, overexpressed dominant-negative cJun caused switching in the opposite direction, decreasing the number of GABAergic neurons and increasing the number of glutamatergic neurons, similar to the results after tlx3 overexpression. We found further that Ca²⁺ spike activity signals through cJun phosphorylation and that phosphorylated cJun binds to the CRE site in the tlx3 promoter. The results supported a model in which Ca²⁺ spike activity culminates in regulation of the level of cJun phosphorylation, and phosphorylated cJun binding to the CRE site modulates transcription of tlx3 to achieve the transmitter switch. These experiments suggested a framework for understanding the mechanism of transmitter switching (Marek et al., 2010). Activity-dependent binding of a transcription factor regulates the transcription of a gene, tlx3, which controls the number of neurons that express glutamate vs. GABA.

Postdoc Michaël Demarque extended our understanding of the molecular mechanism of activity-dependent transmitter switching and related it to changes in behavior. We investigated in Xenopus larvae the conditions under which the number of serotonergic neurons changes. We found that presumptive serotonergic neurons in the developing dorsal raphe, which express the xLmx1b transcription factor but not tryptophan hydroxylase (the serotonin synthetic enzyme), generated spontaneous Ca² spike activity. Suppressing Ca²⁺ spike activity increased the number of neurons expressing the xLmx1b transcription factor, as well as tryptophan hydroxylase, whereas the number of neurons expressing the upstream Nkx2.2 transcription factor did not change. Increasing Ca²⁺ spike activity decreased the number of neurons expressing xLmx1b and tryptophan hydroxylase, again without effect on expression of Nkx2.2. Neuronal proliferation was not affected. Directly overexpressing xLmx1b also increased the number of serotonergic neurons, while suppressing xLmx1b expression reduced their number. Ca²⁺ spike activity appeared to exert its effects on the number of serotonergic neurons through the expression of xLmx1b (Demarque & Spitzer, 2010).

We now turned to the study of ﬁctive swimming, because of the well-described role of serotonin in its modulation. Fictive swimming patterns consist of periods of successive action potential bursts in ventral root recordings, called swimming episodes, which are followed by periods of inactivity. Bath application of serotonin reversibly decreased the duration of swimming episodes. Suppressing Ca²⁺ spike activity to increase the number of serotonergic neurons produced a similar decrease in swimming episode duration. We knocked down the xLmx1b transcription factor to determine if its expression was responsible for the effects of Ca²⁺ spike activity on both the number of serotonergic neurons and the duration of swimming episodes. Now the number of neurons expressing xLmx1b and tryptophan hydroxylase was reduced and swimming episodes were longer in duration. These results indicated that the appearance of more serotonergic neurons can influence neuronal activity (bursts of action potentials in ventral roots) at the network level. In the intact larvae, free-swimming episodes varied in duration as predicted from analysis of fictive swimming. Overexpressing xLmx1b decreased the duration and the distance of free swimming, and knocking down xLmx1b increased both duration and distance. Although we did not search for a switch partner that disappears with the appearance of serotonin, these results showed that, in Xenopus larvae, Ca²⁺ spikes regulate xLmx1b expression, which specifies the number of serotonergic neurons, and the serotonergic neurons alter swimming behavior (Demarque & Spitzer, 2010).

Mechanisms of Transmitter Switching in Development: microRNA Expression

Later, as we thought more about potential mechanisms regulating transmitter switching, I wondered whether coordinated regulation of the loss of one transmitter, and the gain of another, might involve the action of microRNAs (miRs). Giordano Lippi, then a postdoc with Darwin Berg, had helped us appreciate that miRs can function to suppress expression of one gene and silence the repressor of another, thus activating the second gene, which is the scenario in a transmitter switch. Accordingly, as we progressed in studying the social preference of Xenopus larvae to kin and non-kin siblings, described earlier, we profiled small RNA molecules to identify miRs in the accessory olfactory bulb that were differentially regulated by kinship exposure. We first validated the top candidates by qPCR. We then narrowed the analysis to identify those candidates that directly target the Pax6 and Bcl11b genes, which are determinants of the dopaminergic and GABAergic phenotypes. We found that increased miR-375 and miR-200b levels in nonsibling conditions could contribute both to the reduction of dopaminergic neurons by suppressing expression of the stimulatory Pax6 transcription factor and to the increase in GABAergic neurons by suppressing production of the inhibitory Bcl11b transcription factor. Application of antagonists to miR-375 and miR-200b showed that changes in the levels of the transcripts of each miR were sufficient to drive changes in the number of dopaminergic or GABAergic neurons in vivo (Dulcis et al., 2017). Although the signaling cascade leading from Ca²⁺ spike activity to miRNA expression remains to be worked out, the results raised the possibility that microRNAs have a significant role in transmitter switching.

Mechanisms of Transmitter Switching in the Adult

Up to this point, what we knew about the mechanism of neurotransmitter switching came from our work on Xenopus. To study the mechanism in the case of the adult mammalian brain, we began by analyzing the activity-dependence of the dopamine-to-somatostatin switch induced by long-day photoperiod stress. Graduate student Da Meng found that suppressing activity of dopaminergic neurons in the paraventricular nucleus of the hypothalamus (PaVN) in the adult rat prevented the loss of dopamine expression we had previously observed (Dulcis et al., 2013). Interestingly, the neurons that lost dopamine in response to the long-day photoperiod were drawn from the 50 percent of the dopaminergic neurons that normally coexpress glutamate. We noted that the CRF neurons innervated by dopaminergic neurons of the PaVN express both inhibitory dopamine D2 receptors and excitatory glutamate receptors. Accordingly, loss of D2 receptors following exposure to the long-day photoperiod could account for the increased release of CRF and the ensuing stress behaviors we had reported. We also found that suppressing activity of all PaVN excitatory glutamatergic neurons decreased the number of inhibitory PaVN dopaminergic neurons. The match of decreased excitation with decreased potential for inhibition indicated homeostatic regulation of transmitter expression in the PaVN, similar to the homeostatic regulation we had seen in the spinal cord of Xenopus larvae (Borodinsky et al., 2004). Thus, electrical activity regulates transmitter switching and homeostasis in the adult brain, as it does in the developing nervous system (Meng et al., 2018; Li & Spitzer, 2020). Additional studies will reveal the extent to which other aspects of regulation in the adult are the same as those identified for transmitter switches during development.

Current Research

Several ongoing projects in the lab are investigating the role of transmitter switching in mouse models of clinical disorders. One study addresses the mechanism underlying the generalized fear that results from footshock stress, which has been used as a model of post-traumatic stress disorder (PTSD). Following footshock, serotonergic neurons in the lateral wings of the dorsal raphe switch their cotransmitter from glutamate to GABA, as we also observe in postmortem human brain tissue of individuals with PTSD. The loss of glutamate and gain of GABA occur in the same neurons, and this transmitter switch appears to cause generalized fear. Expressing exogenous glutamate partially reduces generalized fear, and suppressing expression of GABA completely erases it. Prompt administration of fluoxetine directly after the footshock, but not later, blocks both the transmitter switch and acquisition of generalized fear (Li et al., 2021).

Another study investigates whether neurotransmitter switching in mice, driven by drugs of abuse, can account for changes in cognitive function. Repeated administration of phencyclidine or methamphetamine causes pyramidal neurons in the prelimbic cortex to express GABA and lose expression of glutamate, indicating that a neurotransmitter switch has occurred. Preventing these neurons from gaining GABA prevents the appearance of drug-induced deficits in the novel object recognition test and spontaneous alternation task, demonstrating a causal link between the transmitter switch and the cognitive deficits. Interestingly, chemogenetic stimulation of parvalbumin-positive inhibitory interneurons that innervate pyramidal neurons, either during drug exposure, or later after the transmitters have switched, suppresses pyramidal neuron hyperactivity and prevents, or reverses, the appearance of both the switch and the cognitive deficits (Pratelli & Spitzer, 2021).

A third study tests the hypothesis that transmitter switching contributes to the generation of autism spectrum disorders. Pregnant female mice are treated with either Poly Inosine:Cytosine (Poly I:C) or valproic acid (VPA) on embryonic day 12.5. In adulthood, the progeny exhibit enhanced stereotypy and deficits in social interaction. We identify a transient decrease in the number of GABAergic neurons and a matching transient increase in the number of glutamatergic neurons in the medial prefrontal cortex, at postnatal day 10, specifically in interneurons expressing parvalbumin or cholecystokinin. Genetic marking of the originally GABAergic neurons demonstrates that they acquire glutamate and confirms that the transmitter switch has occurred. The switch reverses spontaneously by one month. To test the role of the transmitter switch in the emergence of the behavioral disorders, we restore GABA expression specifically in postnatal parvalbumin and cholecystokinin neurons. This treatment prevents the gain of glutamate in these neurons and reverses altered behavior that otherwise would be observed in male mice (Godavarthi & Spitzer, 2020).

Additionally, we are continuing studies of the benefits that may be achieved through transmitter switching. One week of running induces an NPY-to-glutamate switch in neurons in the hilus of the dentate gyrus of adult mice, in addition to the acetylcholine-to-GABA switch that we found in the midbrain, as discussed earlier. The increase in the number of glutamatergic neurons induced by running matches the decrease in the number of neurons that express NPY and occurs in the absence of neurogenesis or apoptosis in the hilus. The switch is associated with improved performance of adult mice on tests of novel object recognition and contextual fear conditioning. We hypothesize that replacement of NPY with glutamate in hilar neurons reverses the inhibitory regulation that these neurons otherwise exert on the lateral entorhinal cortex-to-dentate gyrus projection. This substitution of excitation for inhibition may underlie the enhanced performance induced by one week of running (Zambetti et al., 2017).

Looking Backward and Going Forward

The road from embryonic development of electrical excitability to neurotransmitter switching in the adult brain has been exciting. There have been slow stretches and fast tracks, as well as intriguing side streets not discussed here. The trip was made possible by collaboration with wonderful colleagues (see Table 1). Our analysis of the development of electrical excitability was born out of a keen interest in learning the process by which it occurs. Curiosity-driven research once again paid off handsomely, providing the deep satisfaction of understanding the process quantitatively. Usefully, it provided a pathway to the discovery of neurotransmitter switching in the adult mammal (Spitzer, 1979, 2017).

Table 1.   Collaboration with Students and Postdocs

Students

Postdocs

Paola Baccaglini

Janet Lamborghini

Alan Willard

Corey Goodman

Matt Lee

John Bixby

David Zimmerman

Leslie Henderson

Marsha Revenaugh

Angie Ribera

Leslie Blair

Greg Harris

Amanda Iles

Janet Holliday

Diane O’Dowd

Michel Desarmenien

Rosario de Baca

Shawn Lockery

Nicolas Hussy

Beverly Clendening

Keith Allen

Xiaonan Gu

Maureen Ruchhoeft

Uwe Ernsberger

Eric Olson

Jeffrey Rohrbough

Kate Ribbeck

Devorah Gurantz

Donald Hagler

Mike Ferrari

Steve Watt

Evanna Gleason

Nate Lautermilch

Tim Gomez

Guo-li Ming

Anne Vincent

Ronald Swanger

Paul Kingston

Yuliya Gorbunova

Tim Manning

Cory Root

Ray Smith

Gabriela Montsalve

Laura Borodinsky

Sharon Sann

Brent Miller

Julia Cronin

Matt Conklin

Margaret Lin

Davide Dulcis

Elena Minakova

Kurt Marek

Estuardo Robles

Michaël Demarque

Linda Chang

Xavier Nicol

Lisa Kurtz

Paola Plazas

Sera Chung

Sheila Rosenberg

Qian Xiao

Claudia Alvarez-Baron

Lin Xu

Dena Weinberger

Norma Velázquez-Ulloa

Alicia Güemez-Gamboa

Kwan Pyo Hong

Matt Eckler

Pouya Jamshidi

Hui-quan Li

Amy Guzdar

Stefania Zambetti

Vaishnavi Rao

Swetha Godavarthi

Da Meng

Vincent Luczak

Nandu Prakash

Marta Pratelli

Christiana Stark

Kyle Jackson

Wuji Jiang

Arth Thaker

Three aspects of transmitter switching are particularly noteworthy. The first is that switching is most often observed between glutamate and GABA or vice versa. This might have been anticipated because these transmitters are abundantly expressed in nervous systems. Additionally, the switch from glutamate to GABA is metabolically simple, involving only a single enzyme. The second point is that switching between glutamate and GABA, like other switches such as acetylcholine to GABA, appears to change the sign of the synapse from excitatory to inhibitory or vice versa. Switches in synaptic sign reverse the signals to downstream neurons. The third point is that transmitter switching causes changes in behavior in all cases in which behavior has been tested. Strikingly, behavior is changed even when the switch is between two inhibitory transmitters, such as dopamine and somatostatin. Thus, the impact of transmitter switching on brain function and behavior can be expected to be substantial, both for ill and for good.

There are several issues that I hope to address in the near future. One urgent question is whether there are morphological changes in the downstream (and upstream) connectivity of the neurons that have switched their transmitter. Particularly in the adult nervous system, where the process of transmitter switching takes several days, there is opportunity for branching or retraction of nerve terminals and the formation or loss of synaptic contacts. It is also important to examine the consequences of transmitter switching for synaptic physiology. At an early stage in the study of transmitter switching, I chose to jump over the synapse and leap to behavior to determine if switching had biological significance. Now it is time to fill in this important piece of the picture. It also will be interesting to learn whether a single transmitter switch can drive switching in downstream neurons and induce multiple concatenated, serial switches. I am particularly eager to find out, in cases in which transmitter switching has adverse consequences, whether transmitter switching can be overridden or reversed by noninvasive alterations in electrical activity. The ability of transcranial magnetic stimulation to affect spiking activity of neurons, in areas of cortex measuring less than 2millimeters in diameter, may avoid problems associated with drug action on unwanted targets and reduce the cost of treatment. Overall, the prospects seem bright for manipulations of transmitter switching to treat mental illness and promote health.

Academic Life Beyond the Lab

My career got off to a strong start. Then, not long after I was promoted to professor, and had served on a variety of committees, I was elected chair of the Biology Department. Although this reduced my time in the lab, it was rewarding to solve problems for my colleagues and lead faculty recruitment and planning of student curriculum. Jennefer Collins, the superb management services officer, made the job easy. Fortunately, the position of chair is typically a three-year appointment on the UC San Diego campus, and I was soon back in the lab full time. A few years later, I had the rewarding opportunity to cochair the department with Peter Geiduschek, a brilliant molecular biologist with a magnificent, wry sense of humor. I was chair in the morning. Peter was chair in the afternoon. This was a compromise acceptable to both of us because we were each immersed in our separate research programs. We did not see one another during the day, but talked on the phone each evening to provide mutual briefings. I recall those conversations with exceptional pleasure.

My experience as a department chair led to election as chair of the Academic Senate. Now the problems to be solved were those on the whole-campus scale. I enjoyed working with the administration, and particularly with Chancellor Dick Atkinson, to realize the benefits of shared governance that is one of the founding principles of the University of California. In contrast with decisions made solely by the administration, shared governance leads to decisions that integrate the interests of all parties. This partnership has been a major contributor to the success of UC San Diego. The collaboration requires a willingness to work together that has occasionally seemed in jeopardy. Later, when the Biology Department became the Division of Biological Sciences, I was elected founding chair of the Neurobiology Section. This was the most enjoyable administrative position yet, providing leadership for a smaller group of colleagues, all of whom were deeply invested in studies of the nervous system.

Looking back, I recognize that I have enjoyed starting enterprises that have, at least so far, gone on to thrive. There are several features that seem to fascinate me. There is the opportunity to develop and shape a vision of the possible future. There is the energy that comes with starting something new. There is the tension that failure could be lurking. The opportunity to launch the Kavli Institute for Brain and Mind (KIBM) was such an enterprise. UC San Diego Chancellor Bob Dynes had sat next to Fred Kavli at a meeting of the President’s Council of Advisors on Science and Technology and had learned about the Kavli Institutes that Fred was planning to create. The institutes would be established under the auspices of the Kavli Foundation, with its substantial assets from his business success. Later, in 2003, Fred visited the campus to discuss the possibility of establishing an institute at UC San Diego. Jeffrey Elman, a professor of cognitive science well known for his contributions to the field of neural networks, and I were asked to assemble a proposal for consideration by the Kavli Foundation. We consulted with colleagues and presented a vision for an institute that would bridge disciplinary boundaries in order to understand human cognition, from the brain to the mind. The KIBM was one of the group of four institutes that Fred established first. Jeff and I greatly enjoyed working together. We established several programs, enabled by the founding endowment of $7.5 million. For eight years I was glad to play the role of codirector and then director for three years, when Jeff stepped up to become dean of the Division of Social Sciences. Rusty Gage joined me as codirector for another five years. Catalyzing new scientific research and bridging the gap between neurobiology on the one hand and cognitive science and psychology on the other, the highly successful Innovative Research Grant program annually funded 10 seed grants at $50,000 each.

Another opportunity to launch a new enterprise came from the Society for Neuroscience. I had been a member ever since my arrival at UC San Diego, serving on several committees, and in 2000 was elected to the SfN Council. I found it stimulating to be involved in planning the activities of the Society and determining its future initiatives. Particularly interesting were discussions of the benefits and drawbacks of investing in the construction of what became the Society’s office building at 1121 14th Street, NW. In 2011, I was invited to become the founding editor in chief of BrainFacts.org, the Society’s public-facing website about the nervous system that aims to inform and educate a broad audience. UC San Diego is a public university, and I had been teaching neurobiology to undergraduates for four decades. Here was a chance to reach an even larger audience. It was a splendid team effort. Early on, the Kavli and Gatsby Foundations committed support. We had an outstanding editorial board of neuroscientists, the full involvement of the Public Education and Communication Committee that I had previously chaired, and a spectacular SfN staff. There were consultants for online surveys and for focus groups to assess interest, and consultants for web design. An absorbing, collegial energy pushed us all forward. It was rewarding both then and now to see the number of hits on the website grow and to see other Foundations jumping in to provide support. Since I stepped down as editor in chief in 2014, the reach and impact of BrainFacts.org have continued to expand.

Coda

I write this in what one hopes is the closing phase of the great coronavirus pandemic of 2020–2021. It began with a lockdown and complete stoppage of all university activity and confinement to home. We came to the lab for mouse and frog colony maintenance. Experiments slowed to a crawl. My lab kept in touch with weekly Zoom journal clubs and lab meetings. Data analysis began to catch up with experimental results. Figures were prepared. We wrote and published what we hoped was a preview of things we could learn about transmitter switching in the future, thinly disguised as a review article (Li et al., 2020). Gradually, we have been able to return to a semblance of business-as-usual, working masked and in shifts to ensure adequate distancing. My colleagues were amazing, pleasantly pushing to and beyond the limits of what the campus allowed. Nonetheless, the pandemic inserted a delay in our progress at a time when our pace was accelerating.

My family has been consistently supportive of my work. Janet Lamborghini and I had two children, Julian and Hilary. Xiaoping Wang, my second wife, and I had three children, William, Addison, and Alexandra. I assisted with many homework assignments and attended many soccer games, but I am grateful to both Janet and Xiaoping for the lioness’s share of the heavy lifting of childrearing. After a visit to the Hirshhorn Museum, during a Washington, D.C., study section meeting, I built Julian a tensegrity jungle gym copy of Kenneth Snelson’s Coronation Day sculpture. At the conclusion of another study section, Julian flew to Washington and we visited the Air and Space Museum and other engaging sights. Visits to the lab provided opportunities for Hilary, Addison, and Alexandra to see frog embryos and tadpoles under a microscope when not creating artwork on the whiteboard in my office. We enjoyed hiking in the Torrey Pines State Park and along the beach, climbing Mt. Woodson and Stonewall Peak in east San Diego County, and camping in the Anza Borrego Desert. Annual ski trips to Mammoth in the winter and vacations in Vermont in the summer were keenly anticipated. My family was understanding of my absence on trips for seminars and conferences. I took them with me whenever I could.

Thirty years ago, I began solo trips to the Sierra Nevada Mountains of California to climb the high peaks. I knew I enjoyed climbing from childhood trips with my parents. Life was marching on. If not now, when? I found that long weekends of solitude above 10,000 feet, with magnificent views, provided inspiration to think creatively about my scientific objectives. One Monday in 1995, UC San Diego Vice Chancellor Bruce Darling learned at Dick Atkinson’s cabinet meeting that, although I was chair of the Academic Senate and usually in attendance, I was absent because I was in the hospital following a climbing accident. Bruce was an accomplished alpinist and had climbed all over the Sierras. As soon as I had recovered, we started rock climbing and ice climbing together. We also shared an enthusiasm for backcountry ski touring and launched an annual series of trans-Sierra ski tours, crossing east-to-west or north-to-south over the course of a week. We took friends when we could persuade them to join us, and have made 13 of these trips during the years when the snowpack permitted. We hope that the warmer winters associated with climate change will not curtail these high adventures.

Selected Bibliography

Aumann, T.D., Raabus, M., Tomas, D., Prijanto, A., Churilov, L., Spitzer, N.C. and Horne, M.K. (2016) Differences in number of midbrain dopamine neurons associated with summer and winter photoperiods in humans. PLoS One 11: e0158847.

Baccaglini, P.I. and Spitzer, N.C. (1977) Developmental changes in the inward current of the action potential of Rohon-Beard neurones. J Physiol 271: 93–117.

Ben-Ari, Y. and Spitzer, N.C. (2010) Phenotypic checkpoints regulate neuronal development. Trends Neurosci 33: 485–492.

Bixby, J.L. and Spitzer, N.C. (1982) The appearance and development of chemosensitivity in Rohon-Beard neurones of the Xenopus spinal cord. J Physiol 330: 513–536.

Bixby, J.L. and Spitzer, N.C. (1984a) The appearance and development of neurotransmitter sensitivity in Xenopus embryonic spinal neurons in vitro. J Physiol 353: 143–155.

Bixby, J.L. and Spitzer, N.C. (1984b) Early differentiation of vertebrate spinal neurons in the absence of voltage-dependent Ca⁺⁺ and Na⁺ influx. Dev Biol 106: 89–96.

Blair, L.A.C. (1983) The timing of protein synthesis required for the development of the sodium action potential in embryonic spinal neurons. J Neurosci 3: 1430–1436.

Borodinsky, L.N., Root, C.M., Cronin, J.A., Sann, S.B., Gu, X. and Spitzer, N.C. (2004) Activity-dependent homeostatic specification of transmitter expression in embryonic neurons. Nature 429: 523–530.

Borodinsky, L.N. and Spitzer, N.C. (2007) Activity-dependent neurotransmitter-receptor matching at the neuromuscular junction. Proc Natl Acad Sci USA 104: 335–340.

Demarque, M. and Spitzer, N.C. (2010) Activity-dependent expression of Lmx1b regulates specification of serotonergic neurons modulating swimming behavior. Neuron 67: 321–334.

Desarmenien, M.G., Clendening, B. and Spitzer, N.C. (1993) In vivo development of voltage-dependent ionic currents in Xenopus embryonic spinal neurons. J Neurosci 13: 2575–2581.

Desarmenien, M.G. and Spitzer, N.C. (1991) Role of calcium and protein kinase C in development of the delayed rectifier potassium current in Xenopus spinal neurons. Neuron 7: 797–805.

Dulcis, D., Jamshidi, P., Leutgeb, S. and Spitzer, N.C. (2013) Neurotransmitter switching in the adult brain regulates behavior. Science 340: 449–453.

Dulcis, D., Lippi, G., Stark, C.J., Do, L.H., Berg, D.K. and Spitzer, N.C. (2017) Neurotransmitter switching regulated by miRNAs controls changes in social preference. Neuron 95: 1319–1333.

Dulcis, D. and Spitzer, N.C. (2008) Illumination controls dopaminergic differentiation regulating behavior. Nature 456: 195–201.

Dulcis, D. and Spitzer, N.C. (2011) Reserve pool neuron transmitter respecification: novel neuroplasticity. Dev Neurobiol 72: 465–474.

Godavarthi, S. and Spitzer, N.C. (2020) Transmitter switching contributes to two environmental models of neurodevelopmental disorders. Abstract Soc Neurosci.

Gomez, T.M., Robles, E., Poo, M.-M. and Spitzer, N.C. (2001) Filopodial calcium transients promote substrate-dependent growth cone turning. Science 291: 1983–1987.

Gomez, T.M. and Spitzer, N.C. (1999) In vivo regulation of axon extension and pathfinding by growth-cone calcium transients. Nature 397: 350–355.

Goodman, C.S., O’Shea, M., McCaman, R. and Spitzer, N.C. (1979) Embryonic development of identified neurons: Temporal pattern of morphological and biochemical differentiation. Science 204: 1219–1222.

Goodman, C.S., Pearson, K.G. and Spitzer, N.C. (1980) Electrical excitability: a spectrum of properties in the progeny of a single embryonic neuroblast. Proc Natl Acad Sci USA 77: 1676–1680.

Goodman, C.S. and Spitzer, N.C. (1979) Embryonic development of identified neurones: Differentiation from neuroblast to neurone. Nature 280: 208–214.

Goodman, C.S. and Spitzer, N.C. (1981a) The mature electrical properties of identified neurones in grasshopper embryos. J Physiol 313: 369–384.

Goodman, C.S. and Spitzer, N.C. (1981b) The development of electrical properties of identified neurones in grasshopper embryos. J Physiol 313: 385–403.

Gorbunova, Y.V. and Spitzer, N.C. (2002) Dynamic interactions of cyclic AMP transients and spontaneous Ca²⁺ spikes. Nature 418: 93–96.

Gu, X., Olson, E.C. and Spitzer, N.C. (1994) Spontaneous neuronal calcium spikes and waves during early differentiation. J Neurosci 14: 6325–6335.

Gu, X. and Spitzer, N.C. (1993) Low-threshold Ca²⁺ current and its role in spontaneous elevations of intracellular Ca²⁺ in developing Xenopus neurons. J Neurosci 13: 4936–4948.

Gu, X. and Spitzer, N.C. (1995) Distinct aspects of neuronal differentiation encoded by frequency of spontaneous Ca2+ transients. Nature 375: 784–787.

Güemez-Gamboa, A., Xu, L., Meng, D. and Spitzer, N.C. (2014) Non-cell-autonomous mechanism of activity-dependent neurotransmitter switching. Neuron 82: 1004–1016.

Gurantz, D., Lautermilch, N.J., Watt, S.D. and Spitzer, N.C. (2000) Sustained upregulation in embryonic spinal neurons of a Kv3.1 potassium channel gene encoding a delayed rectifier current. J Neurobiol 42: 347–356.

Gurantz, D., Ribera, A.B. and Spitzer, N.C. (1996) Temporal regulation of Shaker- and Shab-like potassium channel gene expression in single embryonic spinal neurons during K⁺ current development. J Neurosci 16: 3287–3295.

Hammond-Weinberger, D.R., Wang, Y., Glavis-Bloom, A. and Spitzer, N.C. (2020) Mechanism for neurotransmitter-receptor matching. Proc Natl Acad Sci USA 17: 4368–4374.

Harris, G.L., Henderson, L.P. and Spitzer, N.C. (1988) Changes in densities and kinetics of delayed rectifier potassium channels during neuronal differentiation. Neuron 1: 739–750.

Henderson, L.P. and Spitzer, N.C. (1986) Autonomous early differentiation of neurons and muscle cells in single cell cultures. Dev Biol 113: 381–387.

Holliday, J., Adams, R. J., Sejnowski, T.J. and Spitzer, N.C. (1991) Calcium-induced release of calcium regulates differentiation of spinal neurons. Neuron 7:787–796.

Holliday, J. and Spitzer, N.C. (1990) Spontaneous calcium influx and its roles in differentiation of spinal neurons in culture. Dev Biol 141: 13–23.

Lamborghini, J.E. (1980) Rohon-Beard cells and other large neurons in Xenopus embryos originate during gastrulation. J Comp Neurol 189: 323–333.

Lamborghini, J.E. (1987) Disappearance of Rohon-Beard neurons from the spinal cord of larval Xenopus laevis. J Comp Neurol 264: 47–55.

Lamborghini, J.E. and Iles, A. (1985) Development of a high affinity GABA uptake system in embryonic amphibian spinal neurons. Dev Biol 112: 167–176.

Lamborghini, J.E., Revenaugh, M. and Spitzer, N.C. (1979) Ultrastructural development of Rohon-Beard neurons: Loss of intramitochondrial granules parallels loss of calcium action potentials. J Comp Neurol 183: 741–752.

Lautermilch, N.J. and Spitzer, N.C. (2000) Regulation of calcineurin by growth cone calcium waves controls neurite extension. J Neurosci 20: 315–325.

Li, H., Jiang, W., Chen, C., Cleveland, D.W. and Spitzer N.C. (2021) Role of transmitter plasticity in fear generalization. Abstract Soc Neurosci.

Li, H., Pratelli, M., Godavarthi, S., Zambetti, S., Spitzer, N.C. (2020) Decoding neurotransmitter switching: the road forward. J Neurosci 40: 4078–4079.

Li, H. and Spitzer, N.C. (2020) Exercise enhances motor skill learning by neurotransmitter switching in the adult midbrain. Nat Commun 11: 2195.

Lockery, S.R. and Spitzer, N.C. (1992) Reconstruction of action potential development from whole cell currents of differentiating spinal neurons. J Neurosci 12: 2268–2287.

Marek, K.W., Kurtz, L.M. and Spitzer, N.C. (2010) cJun integrates calcium spike activity and tlx3 expression to regulate neurotransmitter specification. Nature Neurosci 13: 944–950.

Meng, D., Li, H.Q., Deisseroth, K., Leutgeb, S. and Spitzer, N.C. (2018) Neuronal activity regulates neurotransmitter switching in the adult brain following light-induced stress. Proc Natl Acad Sci USA 115: 5064–5071.

Nicol, X., Hong, K.P. and Spitzer, N.C. (2011) Spatial and temporal second messenger codes for growth cone turning. Proc Natl Acad Sci USA 108: 13776–13781.

O’Dowd, D.K. (1983) RNA synthesis dependence of action potential development in spinal cord neurones. Nature 303: 619–621.

O’Dowd, D.K., Ribera, A.B. and Spitzer, N.C. (1988) Development of voltage dependent calcium, sodium and potassium currents in Xenopus spinal neurons. J Neurosci 8: 792–805.

Plazas, P.V., Nicol, X. and Spitzer, N.C. (2013) Activity-dependent competition regulates motor neuron axon pathfinding via PlexinA3. Proc Nat Acad Sci USA 110: 1524–1529.

Pratelli, M. and Spitzer, N.C. (2021) Manipulating brain activity to rescue drug-induced cognitive defects. Abstract Soc Neurosci.

Ribera, A.B. (1990) A potassium channel gene is expressed at neural induction. Neuron 5: 691–701.

Ribera, A.B. and Spitzer, N.C. (1989) A critical period of transcription required for differentiation of the action potential of spinal neurons. Neuron 2: 1055–1062.

Ribera, A.B. and Spitzer, N.C. (1990) Differentiation of IKA in amphibian spinal neurons. J Neurosci 10: 1886–1891.

Rohrbough, J. and Spitzer, N.C. (1996) Regulation of [Cl-]i by Na+-dependent Cl- cotransport distinguishes depolarizing from hyperpolarizing GABAA receptor-mediated responses in spinal neurons. J Neurosci 16: 82–91.

Root, C.M., Velázquez-Ulloa, N.A., Monsalve, G.C., Minakova, E. and Spitzer, N.C. (2008) Embryonically expressed GABA and glutamate drive electrical activity regulating neurotransmitter specification. J Neurosci 28: 4777–4784.

Spitzer, N.C. (1970) Low resistance connections between cells in the developing anther of the lily. J Cell Biol 45: 565–575.

Spitzer, N.C. (1976) The ionic basis of the resting potential and a slow depolarizing response in Rohon-Beard neurones of Xenopus tadpoles. J Physiol 255: 105–135.

Spitzer, N.C. (1979) Ion channels in development. Ann Rev Neurosci 2: 363–397.

Spitzer, N.C. (1982) Voltage and stage dependent uncoupling of Rohon-Beard neurones during embryonic development of Xenopus tadpoles. J Physiol 330: 145–162.

Spitzer, N.C. (1985) The control of development of neuronal excitability. In Molecular Bases of Neural Development, G. M. Edelman, W.E. Gall and W.M. Cowan, eds. Rockefeller Univ. Press, New York, pp. 67–88.

Spitzer, N.C. (1991) A developmental handshake: neuronal control of ionic currents and their control of neuronal differentiation. J Neurobiol 22: 659–673.

Spitzer, N.C. (2006) Electrical activity in early neuronal development. Nature 444: 707–712.

Spitzer, N.C. (2017) Neurotransmitter switching in the developing and adult brain. Ann Rev Neurosci 40: 1–19.

Spitzer, N.C. and Baccaglini, P.I. (1976) Development of the action potential mechanism in embryonic amphibian neurons in vivo. Brain Res 107: 610–616.

Spitzer, N.C., de Baca, R.C. and Holliday, J. (1988) Developmental acquisition of GABA-like immunoreactivity of amphibian spinal neurons differentiating in vitro. Abstract Soc Neurosci.

Spitzer, N.C., de Baca, R.C., Allen, K.A. and Holliday, J. (1993) Calcium dependence of differentiation of GABA immunoreactivity in spinal neurons. J Comp Neurol 337: 168–175.

Spitzer, N.C. and Lamborghini, J.E. (1976) The development of the action potential mechanism of amphibian neurons isolated in culture. Proc Natl Acad Sci USA 73: 1641–1645.

Vincent, A., Lautermilch, N.J. and Spitzer, N.C. (2000) Antisense suppression of a potassium channel gene demonstrates its role in maturation of the action potential. J Neurosci 20: 6087–6094.

Zambetti, S., Connors, J.O. and Spitzer, N.C. (2017) Neurotransmitter switching in the adult mouse hippocampus and changes in episodic memory. Abstract Soc Neurosci.

Index

Abdus-Saboor, I., 203

Aberle, H., 79, 81

Achiro, J., 345

Adams, R. D., 420

Adelman, R., 96

Adler, A., 413

Adolphs, R., 544

Adori, C., 152

Aflalo, T., 25, 28, 30

Aghajanian, G., 115

Agnati, L., 127, 141

Agnew, B., 303

Agranoff, B., 338

Ahern, C., 5

Ahmed, S., 223

Ainsworth, A., 375

Akert, K., 130

Akil, H., 130

Alberini, C., 338

Alberts, B., 101

Albus, K., 523

Alda, A., 431

Alexander, F. M., 387, 403

Allen, G., 524

Allison, J., 97, 99

Allman, J., 9, 544

Amalric, M., 219

Åman, K., 122

Amara, S., 131

Ames, B., 96

Andersen, R. A., 2, 544

Anderson, D., 307, 314

Anderson, J., 385, 405

Andres, M. E., 313

Andrews, N., 276

Angelaki, D., 274

Änggård, A., 129

Annau, Z., 211, 228

Anton, E., 435

Aprison, M., 120

Arbib, M., 400

Ardrey, R., 209

Arends, M., 225

Arimura, A., 129

Arnold, A., 179

Arnsten, A., 438

Artavanis-Tsakonas, S., 71, 87, 434, 499

Aschberg, R., 158

Assad, J., 550

Atkinson, D., 644

Attenborough, D., 383

Auden, W. H., 173

Austin, W. T. S., 380

Axel, R., 154

Axelrod, J., 133, 136, 137, 159

Axelsen, N., 154

Azel, R., 61

Azrin, N., 176

Babbage, C., 399

Babinski, J., 417

Baccaglini, P., 618

Baetjer, A., 212

Báez-Mendoza, R., 548

Bagdy, G., 149

Baker, B., 254, 270

Baker, H., 475

Balcom, J., 213

Balint, R., 4

Ball, E., 59

Ballagh, I., 189

Ballance, F., 474

Ballas, N., 313

Ballivet, M., 299

Ballmer-Hofer, K., 300

Balzac, H., 413

Banitt, Y., 405

Banks, M., 21

Bannister, N., 394

Barad, M., 331

Barash, Shabtai, 13

Barchi, B., 302

Bard, P., 212

Barde, S., 149

Bargmann, C., 259

Bargmnn, W., 147

Bark, C., 152

Barkan, C., 195, 196

Barlow, H., 382, 391, 404, 549

Barlow, M., 387

Barnhardt, E., 203

Barondes, S., 338

Bartfai, T., 132, 140, 141, 142

Bartholomew, G., 304

Barto, A., 537, 538

Barton, E., 597

Bartsch, D., 338

Basbaum, A., 259, 276

Basbaum, C., 282

Bashaw, G., 74, 75, 76, 77

Bashford, L., 29

Bass, A., 185

Bastiani, M., 59, 60, 70

Bate, M., 55, 57, 58, 62

Batsita, A., 15

Battaglioli, E., 313

Baum, C., 99

Beam, K., 301

Bean, A., 144

Beard, J., 617

Beardwood, C., 371

Beegan, C. A., 516

Ben-Ari, Y., 626

Bendesky, A., 195, 204

Benjamin, T., 300

Ben-Jonathan, N., 116

Bennet, G., 144

Bennett, M., 414, 471

Bentham, E. J., 406

Bentivoglio, M., 136, 152, 154

Benzer, S., 437

Berendsen, M., 174

Bergman, P., 152

Berkenbosch, F., 125

Berlin, C., 175

Berlin, I., 374, 384

Berliner, B., 443

Berman, N., 389

Bermudez, M., 552

Betz, B., 616

Beutler, E., 220

Beveridge, C., 256

Bialek, B., 259, 266, 274

Biberfeld, P., 122

Biller, S., 450

Binzegger, T., 400

Bird, A., 319

Bishop, M., 489

Bismarck, O., 161

Bixby, J., 621

Bizzi, E., 10

Björklund, A., 115, 126, 140, 155

Blakemore, C., 375, 437

Bland, K., 74

Blankemeier, B., 67, 90

Bloom, F., 121, 213, 219, 221, 231

Bloom, S., 132

Bloomfield, M., 47

Bogart, H., 421

Bohr, N., 438

Boisselier, B., 101

Bolam, P., 387

Bonaiuto, J., 19

Bonanno, S., 345

Bonhoeffer, F., 55, 83

Bonhoeffer, K.-F., 448

Bonhoeffer, T., 404

Bonnacorso, F., 179

Bopp, R., 408

Boring, E. G., 566

Bornstein, M., 200

Borodinsky, L., 628, 630

Botha, D., 389, 391

Boullon, M., 177

Boyd, A., 298

Boyden, E., 528

Boyer, P., 293

Braddock, C., 356

Bradley, D., 20

Brady, J., 212, 216, 228

Brahic, C., 188

Brainard, M., 276, 278

Braslow, J., 349

Brehm, P., 292

Brené, S., 143

Brenner, S., 368

Breunig, J., 433, 436

Brimijoin, S., 130

Brinster, R., 482

Broberger, C., 117, 148

Brodmann, K., 450

Brooks, M., 292

Brooks, R., 376

Brose, K., 74

Brown, K., 383

Brown, M., 219

Brownstein, M., 133, 136, 137, 143

Brumovsky, P., 146

Brunel, N., 270

Bruno, G., 414

Buck, L., 154

Buckley, V., 232

Bueno, C., 15

Bullier, J., 380, 386

Bullock, T., 438

Buneo, C., 17

Buonomano, D., 343

Burbach, P., 144

Burden, S., 483, 500

Bures, J., 568

Burgs, M., 368

Burnstock, G., 133

Burrows, M., 56

Bush, G. W., 101

Busnel, R. G., 177

Buszáki, G., 155

Büttner, U., 525

Calza, L., 151

Camerer, C., 544

Camhi, J., 253

Caminiti, R., 5

Campbell, F., 381

Campbell, K., 485

Capecchi, M., 471

Carew, T., 87

Carey, M., 268, 271, 280, 351

Carlsson, A., 112, 115, 119, 154, 442

Carlyle, T., 331

Carmichael, G., 300

Carter, J., 50

Casadio, A., 341

Cash, R. A., 334

Castel, M.-N., 151

Caviness, V, 423

Ceccatelli, S., 147

Chalfie, M., 185

Chalmers, J., 125, 126

Chang, M., 129

Chang, S., 59

Changeux, J.-P., 433, 445, 481, 487, 498

Chan-Palay, V., 135, 140

Chedotal, A., 70

Chen, C., 26, 544

Chen, L., 597

Chen, P., 347

Chen, T., 26

Chiu, A., 479

Ch’ng, T.-H., 344

Chong, D., 308, 310

Christ, C., 67, 85

Christensen-Dalsgaard, J., 192

Christie, J., 263

Christopoulus, V., 19

Churchill, W., 202

Churchland, A., 528

Churchland, P., 262

Clapham, D., 299, 317

Clark, B., 294

Clarke, L., 377

Clarke, P., 378

Cobbs, A., 573

Cohen, S., 153, 479

Collins, J., 643

Colwell, S., 4

Conrad, L., 181

Constantine-Paton, M., 184

Conway, D., 252

Cooper, J., 452

Cooper, L., 159

Cooper, S., 380

Corkin, S., 11

Corness, J., 144

Cortes, R., 140

Costa, E., 156

Costa, M., 133

Costa, N. M., 405

Court, R., 51

Cowan, M., 7, 66, 312

Cowey, A., 382

Cox, D., 384

Crair, M., 444, 575

Crane, J., 178, 191

Creutzfeldt, O., 378, 444, 448, 523

Crews, S., 64, 65, 71

Crick, F., 9, 262, 422, 432

Crill, W., 253

Crow, T., 213

Crump, M., 179

Cuello, C., 123, 130, 134, 214

Cukon, M., 412

Cunningham, D., 384

Curtis, D., 121

Dagerlind, Å., 143

Dahlström, A., 112, 128, 134

Dallman, J., 313, 315

Dallman, M., 279

Dalsgaard, C.-J., 132

Dan, Y., 84

Danhauser, W., 252

Daniel, P., 380

Danny-Brown, D., 418

Dantzer, R., 218

Darling, B., 646

Darlington, T., 273

Darwin, C., 426

Das, A., 202

Dash, P., 338

Davis, D. E., 210

Davis, G., 67, 80

Davis, M., 47

Davis, R., 48

Dawkins, R., 382

Dawson, T., 136

Day, J. H., 366

Dayan, P., 537

Deakin, W., 149

Dean, H., 475

DeFelipe, J., 416, 435

Dehay, C., 389

Deisseroth, K., 528

Delbrück, T., 399

DeLong, M., 5, 527

Demarque, M., 638

Deming, L., 96

de Robertis, E., 113, 114

DeSalvo, M, 345

Desarmenien, M., 624

Desler, M., 86

Despert, J. L., 174

D’Esposito, M., 84

Deutsch, J. A., 22

DeWeese, J., 179

De Wied, D., 217

deZeeuw, C., 263

DiAntonio, A., 67, 80, 81

Diaz-Heijtz, R., 149

Dibon, G., 152

Dickens, C., 27

Dickinson, T., 535, 542

Dickson, B., 73, 75, 76

Diez, M., 151

Diniz, C., 389

Dismukes, K., 120

Disney, A., 278

Divac, I., 210

Dodd, J., 202

Doe, C., 59, 61, 62, 65

Domjan, M., 176

Donoghue, J., 22

Donoghue, M., 490

Doolittle, R., 298

Dostoevsky, F., 413

Douglas, R., 364, 388

Doupe, A., 183, 259, 276, 278, 282

Dowling, J., 471, 614

Dresse, A., 141

Drinnenberg, A., 404

Drucker, P., 173

Dubner, R., 146

Dudai, Y., 445

Dulac, C., 492

du Lac, S., 61, 263, 283

Dulcis, D., 631, 633

Dunlap, K., 299, 305, 310

Dunn, J., 314

Duque, A., 443

Durant, W., 563

Durbin, R., 392

Duvall, L., 203

Dynes, B., 644

Earl, D., 402

Easter, S., 63, 382

Ebbesen, S., 213

Ebbinghaus, H., 369

Eccles, J., 121, 254, 270, 379, 422, 445, 523

Echenique, F., 544

Edelman, G., 62, 119, 400

Edelman, J., 422

Edinger, L., 450

Edison, T., 120

Edvinsson, L., 131

Edwards, C., 187, 192

Egger, D., 267

Ehrenfeld, D., 177

Einstein, A., 417

Eisner, T., 253

Ekama, G., 373

Elde, B., 130

Elde, R., 128

Elfvin, L.-G., 132

Elkins, T., 72

Elliot, T., 191

Ellis, B., 569

Elman, J., 644

Emlen, S., 253

Emson, P., 130, 214

Enders, J., 333

Eränkö, O., 115

Erlanger, J., 479

Ervin, S., 475

Essen, D. V., 472, 615

Essick, G., 8

Ettenberg, A., 219

Evans, B., 186, 195

Evans, G., 384

Evans, R., 57

Evarts, E., 5, 258

Everitt, B., 117, 147, 215, 236, 543

Fahrenkrug, J., 125

Fambrough, D., 72, 77, 78, 79

Fargraeus, A., 122

Fatt, P., 393

Feng, G., 486

Fent, M., 403

Fernald, R., 185

Ferres-Marco, D., 71

Ferster, D., 383

Fetcho, J., 315, 317

Fetissov, S., 152

Fetter, R., 67

Fetz, E., 395

Feynman, R., 392

Field, G., 278

Fields, H., 276

Fink, S., 301

Finlay, B., 382

Finley, S., 571

Fiorillo, C., 539, 544

Fischbach, G., 184, 472

Fischer, M., 378, 395

Fisone, G., 140

Fitzgerald, F. S., 329

Flaming, D., 383

Flannery, J., 84

Flavell, R., 434

Flerkó, B., 117

Flexner, L., 338

Floyd, G., 356

Fogarty, J. E., 136

Fogassi, L., 13

Fontes, M., 347

Foshee, D., 570

Fox, M., 484

Frank, E., 472, 615

Frank, J., 343

Frank, K., 203

Frank, L., 276, 278

Franks, K., 278

Fraser, J., 350

Fraser, S., 382

Fray, P., 214

Fredrickson, D., 476

Freedman, E., 585

Freedman, R., 395

Freeman, R., 381

Freud, S., 369

Freund, T., 155, 389

Friedman, B., 41

Friedman, D., 277

Friedman, S., 122

Fuchs, A., 254, 270, 586, 588

Funess, J., 133

Furshpan, E., 471, 473, 615, 617, 629

Fuster, J., 9

Fuxe, K., 112, 116, 119, 127, 141, 159

Gabbiani, F., 575

Gabbott, P., 389

Gaddum, J., 129

Gage, F., 441

Gage, R., 644

Gail, A., 18

Gaillard, A., 525

Galilei, G., 414

Galileo, D., 489

Gallant, J., 84

Gandhi, N., 593

Ganong, F., 258

Ganong, W., 117, 132

Gantt, H., 228

Gasser, H., 479

Gaster, R., 96

Gautam, M., 482

Gaze, R. M., 370, 372, 376, 617

Geesaman, B., 13

Geffen, L., 122

Geiduschek, P., 643

Geldard, F. A., 569

Georgopolous, A., 5

Gershon, M., 120

Geschwind, D., 342, 343, 354

Geschwind, N., 418

Giamatti, B., 443

Gibb, J., 122

Gibson, E., 253

Giger, H. R., 400

Gilbert, C., 382, 472

Gilbert, W., 499

Gilly, B., 308

Gilman, A., 159

Girardin, C., 407

Gleason, M., 315

Glickfeld, L., 278

Glimcher, P., 543, 593

Glover, J., 489

Glowinski, J., 115, 213, 445

Gnadt, J., 8

Goadsby, P., 131

Godenschwege, T., 70

Goffart, L., 598

Goldberg, M., 5

Goldman, D., 232

Goldman, P., 429

Goldman-Rakic, P., 443, 445, 450

Goldsmith, P., 132

Goldstein, M., 125, 127, 156, 160

Gomez, T., 627

Gomez-Urquijo, S., 151

Goodman C. S., 40, 497, 618

Goodman, D., 306, 317

Goodman, R., 300

Gorbachev, M., 446

Gorbman, A., 192

Gorman, D., 387

Gould, E., 211, 440–41, 446

Gracy, N., 225

Grafstein, B., 181, 182

Granato, M., 315

Granit, R., 130

Grant, G., 159

Gray, G., 489

Graybiel, A., 11

Graziano, M., 13

Greenberg, M., 317

Greene, B., 201

Greengard, P., 116, 132, 154, 159, 469, 472, 502

Gregory, R., 9

Greitz, T., 112

Grether, D., 544

Greves, P. L., 131

Griffin, D., 177, 181, 611

Griggs, W., 33

Grillner, S., 141, 157, 159

Grimes, J., 313

Grinnell, A., 297

Groh, J., 272, 589

Gross, C., 178

Grotjahn, D., 350

Grove, L., 209

Gruber, P., 154

Grueber, W., 202

Grunewald, A., 16

Grunstein, M., 294, 351

Grushkin, S., 48

Gu, X., 624, 626

Guillemin, R., 117, 129, 147, 218

Gurski, M., 597

Guthrie, B., 591

Guthrie, R., 468, 502

Gutstein, H., 226

Guttman, L., 79

Guyenet, P., 126

Gyleensten, L., 111

Haber, B., 291

Haerter, D., 185

Haghighi, P., 81

Hagiwara, S., 304

Hahnloser, R., 404

Halász, B., 117

Halasz, N., 151

Halegoua, S., 299, 307

Hall, I., 193, 197

Hall, K., 157

Hall, Z., 85, 87, 258, 259, 280, 477, 478

Hamberger, B., 112, 113, 114

Hamburger, V., 153, 479, 490

Hamlin, H., 417, 452

Hannigan, P., 202

Harkany, T., 148

Harrelson, A., 62, 65, 70

Harris, A., 59

Harris, B., 499

Harris, C., 549

Harris, G., 117, 372

Harris, R. A., 229

Harrison, R., 55

Hart, H., 384

Hartman, B., 123

Hartwich-Young, R., 597

Harvey, W., 379

Hashimoto-Torii, K., 437

Hastings, W., 292, 294

Hatfield, M., 448

Hauri, A., 406

Hauschild, M., 23

Havens, R., 47

Hawking, S., 384

Haydar, T., 433

Hebb, D. O., 566

Hegel, G., 440

Heilig, M., 217

Heimer, L., 213

Heinemann, S., 481

Heintz, N., 90

Heitler, B., 52

Helenius, A., 334

Helfand, D., 201, 204

Hellweg, C., 523

Helmholtz, H., 369

Hemingway, E., 250

Henderson, C., 498

Hendrix, J., 47

Henn, V., 525

Henry, G., 385

Henry, J., 131

Hepp, K., 395, 404

Hepp-Reymond, M.-C., 398

Herbst, W., 345

Herkenham, M., 258

Hernadi, I., 552

Herzfeld, D., 268

Hickey, T., 597

Higashijima, S., 315

Hildebrand, J., 184, 201, 471, 474, 502

Hildreth, E., 11

Hill, S. R., 571

Hillarp, N.-Å., 108, 111–113, 115, 139, 162

Hille, B., 253

Hills, G., 446

Hingson, R., 232

Hinton, G., 10, 395

Hirokawa, N., 158

His, W., 450

Ho, R., 59, 61

Hobert, O., 202

Hodgkin, A., 393

Hofacker, K., 398

Hoffman, H. S., 210

Hofmeyr, U., 369

Hogben, L., 179

Hökfelt, B., 111

Hökfelt, T., 108, 112

Holiday, B., 56

Holler, S., 408

Holliday, J., 624, 626

Holloway, H., 212

Honjo, T., 97

Hood, D., 201

Hopfield, J., 392

Hopkins, C., 186

Horder, T., 372, 373

Horton, J., 383

Hösli, L., 121

Housman, D., 143

Howard, I., 259, 282

Howe, P., 125

Howell, D., 179

Hoy, R., 194

Hoyt, C., 271

Hu, H., 70

Hu, X., 596

Hubel, D., 153, 378, 382, 391, 426, 471, 615

Hudspeth, J., 472, 615

Huganir, R., 277

Huggins, C., 159

Hughes, I., 201, 204

Hughes, J., 130

Hull, C., 270, 278

Hunt, C., 479

Hunter, D., 479

Husain, M., 11

Hutchinson, J., 180

Huttenlocker, P., 432

Hutter, R., 174

Hwang, E., 32

Hynes, M., 65

Ibata, Y., 158

Illes, P., 141

Imber, M., 445

Indiveri, G., 399

Iraldi, A. P. D., 113

Irmer, U., 519

Isaacson, L., 387, 408

Isacoff, U., 67, 81

Ito, M., 254, 270, 448

Iversen, L., 120, 121, 130

Iversen, S., 214, 215

Ivry, R., 85

Izu, K., 92

Jackson, I., 128

Jacob, F., 487

Jacobs, R., 71

Jacobson, M., 49, 617

Jafari, M., 30

Jaffe, L., 295

Jaffe, R., 299, 306

Jamieson, K., 198

Jan, L. Y., 133, 302

Jan, Y. N., 133

Janak, T., 536

Jarvis, M., 23

Jasmin, L., 263

Javits, J., 475

Jay, M., 588

Jennings, V., 258

Jessell, T., 65, 130, 202, 500

Ji, R.-R., 145

Johansen, J., 148

Johansson, O., 127, 134

Johns, P., 382

Johnson, L., 231

Johnson, M., 543

Johnson, R., 351

Jones, G. M., 270

Jonsson, G., 112, 114, 135

Joosten, H., 123

Jordan, M., 14

Ju, J. G., 144

Juhász, G., 149

Jung, K., 232

Kaas, J., 3

Kagan, I., 19

Kahn, A. E., 252

Kajander, K., 144

Kalesnykas, R., 597

Kandel, E., 61, 135, 154, 159, 202, 337, 441

Kane, C., 101

Kaneko, T., 115

Karlin, A., 294, 305

Karlsson, G., 108

Karten, H., 213

Kastendiek, J., 297

Katz, B., 616

Katz, L., 277

Kaufman, S., 122

Kavli, F., 430, 644

Kawano, H., 128

Kay, J., 187, 278, 495

Keaney, B., 367

Keating, M. J., 370

Keating, M., 375, 376

Keeton, B., 252

Keller, E., 257, 595

Keller, G., 405

Kellerth, J.-O., 130

Kelley, D. B., 172

Kellis, S., 25, 29

Kemali, M., 154

Kennard, C., 377

Kennedy, D., 49, 59, 104, 309

Kennedy, H., 380, 405

Kennedy, J. F., 173

Kennedy, M., 472

Kent, N., 176

Kety, S., 137

Khodakhah, K., 271

Kidd, T., 73, 75

Kidokoro, Y., 299

Kikawa, J., 179

Killick, T., 295

Kim, I.-J., 493

Kim, S., 346

Kimm, J., 254

Kindler, J., 94

King, M., 256

Kingsley, D., 195

Kiper, D., 398, 406

Klämbt, C., 71

Klein, J., 428

Klopf, H., 537

Knight, B., 84

Knowles, J., 494

Kobayashi, S., 550

Koch, C., 390, 392

Koenig, B., 297

Koester, J., 202

Kofler, B., 142

Kohl, P., 200

Kolodkin, A., 68, 69, 70

Komuro, H., 436

König, P., 398

Konishi, M., 182, 183

Konopka, R., 49

Koob, C. M., 228

Koob, G. F., 208, 213

Kopczynski, C., 67

Kopp, J., 134, 142

Kopp, U., 152

Kordon, C., 117

Kornberg, T., 294

Koshland, D., 66, 84

Kostadinov, D., 495

Kosterlitz, H., 120, 130

Köstinger, G., 408

Kostovic, I., 422, 427, 434

Kotrla, K., 59

Krakow, D., 350

Kramer, J., 399

Kramer, R., 84

Kramer, S., 74

Kraner, S., 306, 308, 310, 311

Krauzlis, R., 265

Kravitz, E., 176, 471, 615

Krayer, O., 615

Kreutzberg, G., 134

Kriegstein, A., 176

Krishnaswamy, A., 495

Kristensson, K., 152

Krubitzer, L., 443

Krueger, B., 469

Krystal, J., 149

Kuan, C.-Y., 434

Kuffler, S., 281, 383, 615, 619

Kuhnt, U., 523

Kunos, G., 232

Kurdistani, S., 351

Kuwada, J., 59

Kuwada, S., 584

Kuypers, H., 136

Kuzma, P., 412

Kwan, Y. M., 195

Kwong-Brown, U., 191, 193, 202

Labrador, P., 74, 79

Lagercrantz, C. H., 447

Lajonchere, C., 354

Lakay, T., 371

LaMantia, A., 433

Lamborghini, J., 616, 618, 645

Landis, S., 144, 471, 629

Landry, M., 141, 144, 145, 147

Langel, U., 140

Langley, J. N., 489

Laskowski, M., 490

Lawrence, D., 398

Lawrence, J., 480

Le Moal, M., 217, 218, 220, 225

Le, X., 141

LeBrun, C., 219

Lechan, R., 128, 301

Lederberg, J., 47

Lee, B., 21, 24

Lee, C., 583

Lee, J.-A., 347

Lee, S., 301

Leeman, S., 129, 291

Lefebvre, J., 495

Lehmann, R., 500

Lehmeyer, J., 117

Leiter, A., 301

Lekić, L., 449

LeMaitre, E., 149

Lembeck, F., 130

Length, G., 141

Lengyel, J., 294

Leonard, C., 182

Leonard, T., 181

Leontowich, T., 418

Lerner, R., 223

LeVail, J., 181

LeVay, S., 383, 472

Levi-Montalcini, R., 153, 156, 445, 479

Levinhal, C., 14

Levinson, R., 308, 312

Levitt, B., 122

Levitt, P., 433, 444

Levy, R., 100

Lewin, B., 311, 333

Lewis, S., 413

Li, T. K., 231

Libet, B., 3, 525

Lichtman, J., 486

Lin, D., 67

Lindh, B., 132

Lindquist, M., 119

Lindsley, O., 176

Lindsten, J., 153

Lindstrom, J., 481

Lindvall, O., 126

Linhoff, M., 319

Linklater, K., 401

Lisberger, S. G., 250, 273

Litten, R., 232

Liu, C., 24

Liu, R., 145

Liu, S.-C., 399

Livet, J., 486

Livett, B., 122

Ljungberg, T., 532, 534, 537

Ljungdahl, Å., 115, 120, 134, 136

Llinas, R., 254, 270, 471

Lockery, S., 622

Logothetis, N., 574

Lomo, T., 616

Lorden, J., 581

Low, M., 301

Ludwig, M., 141

Luft, R., 111, 148, 159

Lund, J., 407

Lundberg, J., 128, 132, 135

Luo, C., 26

Luria, S., 438

Luschei, E., 256

Luskin, M., 488

Lutz, B., 518

Lyles, V., 346

Ma, X., 141

Macagno, E., 184

Mackintosh, N., 542

MacLeod, K., 281

MacLeod, R., 117

Macosko, E., 496

Maekawa, K., 254

Mahowald, M., 392

Malmfors, T., 112, 113, 115

Mandel, G., 290

Mandel-Brehm, C., 305

Mandel-Brehm, J., 300

Maniatis, T., 497

Mante, V., 406

Marcus, E., 355

Marek, K., 636

Marin, M., 187

Markram, H., 407

Marr, D., 10, 254, 390, 536

Marshall, L., 477

Mårtensson, R., 126

Martin, G. M., 328

Martin, J. D., 371

Martin, K. A. C., 364

Martin, K. C., 328

Martini, L., 117

Marx, K., 522

Mason, B., 230, 232

Mason, C., 202

Mason, H. C., 52

Mathias, M., 293

Matsuda, W., 115

Matsui, J., 101

Matthes, D., 68

Matthews, B., 372

Matthews, G., 308, 312

Matthews, P., 390

Maue, B., 306

Mauk, M., 268

Maunsbach, A., 145

Maunsell, J., 274, 574–75

Maxwell, B., 252

May, B., 193

Mays, L., 581

Mazziotta, J., 352

Mazzoni, Pietro, 14

McCabe, B., 81

McCaman, D., 58

McCann, S., 117

McCarroll, S., 496

McCarthy, E., 46

McClearn, G., 570

McClelland, J., 10

McCorkle, S., 314

McCormick, D., 444

McDiarmid, R., 179

McEwen, B., 178

McGaugh, J., 570

McKinnon, R., 402, 404

McMahan, J., 471, 476, 479, 482, 502, 616

McNamara, J., 277

Mead, C., 399

Mechawar, N., 149

Medina, J., 267, 270

Meister, B., 116, 147

Meister, M., 493

Melander, T., 134, 140

Meller, M., 174

Mendell, L., 307

Meng, D., 639

Mennicken, F., 144

Merighi, A., 133

Merkel, A., 520

Merlie, J., 479, 481, 497

Merlo-Pich, E., 217, 220

Merrera-Marschitz, M., 151

Merzenich, M., 3, 276

Meyerhoff, J., 213

Miall, C., 392

Michael, D., 339

Michel, M., 142

Miles, F., 255–56, 257, 260, 270

Milk, H., 479

Miller, G., 334

Miller, K., 202, 259, 276, 278

Milner, P., 211

Miner, J., 480

Mirenowicz, J., 533

Mitchell, K., 73

Mitchison, G., 392

Mizener, A., 252

Mobbs, D., 475

Moccia, R., 343

Molinoff, P., 160

Mongeon, B., 315

Montague, R., 537

Montell, D., 59, 64, 65, 70

Montgomery, E., 281, 523

Montminy, M., 301

Mooe, B., 433

Mooney, H., 47

Morgan, C. T., 566

Morgan, H., 252

Morgan, T. H., 175

Morin, J., 293

Morino, P., 151

Morozov, Y., 436

Moscone, G., 479

Motter, B., 5

Mountcastle, V., 3, 5, 212, 376, 445, 567

Movshon, T., 265, 274

Moyers, B., 223

Mufson, E., 141

Mullard, A., 153

Muller, K., 624

Müller, V., 399

Mulliken, G., 23

Murasugi, C., 259

Murthy, M., 197

Muscatine, L., 293

Mutt, V., 128, 138

Nachmias, J., 573

Nagel, K., 267

Nairn, R., 122

Nakane, P., 124

Nasipak, B., 189

Naumann, E., 278

Nauta, W., 160, 182, 213, 422

Navarro, P., 367

Neal, M., 120

Neher, E., 154, 159

Neill, J., 571

Nelson, J., 597

Nelson, T., 146

Neufeld, E., 351

Neumann, S., 345

Newsom, G., 280

Newsome, B., 259, 264, 274, 590

Ngai, J., 84

Niall, H., 129

Nicholls, J., 471

Nichols, J., 597

Nicol, X., 628

Nicolas, J.-F., 487

Nicolelis, M., 22

Nicoll, R., 276

Nieuwenhuys, R., 135

Nilsson, G., 129, 138

Nilsson, I., 148

Nilsson, O., 113

Nirenberg, M., 422

Nixon, R., 47

Noakes, P., 482

Noble, D., 383, 385

Norberg, K.-A., 112

Norell, G., 136, 137

Norman, S., 33

Normann, R., 22

Noronja, A., 232

Northcutt, G., 185

Nottebohm, F., 442

Nottebohm, M., 177

Nowakowski, R., 424

Nuerohub, W., 87

Numa, S., 301

Nusmel, R. G., 177

Nusslein-Volhard, C., 71, 194, 448

Nygårds, A., 122

Ochoa, S., 422

O’Connor, M., 82

O’Connor, T., 68, 70

O’Dell, T., 343

O’Doherty, J., 544

O’Donnell, D., 144

O’Dowd, D., 621

Ögren, S. V., 141

Ohana, O., 408

Ohlsson, R., 143

Okamura, Y., 309

Okelo, O., 54

Okino, C., 48

Okun, L., 471, 472

Olds, J., 211, 217, 568

Olson, L., 112, 159

Ono, F., 315

Orban, G., 28, 380

Orci, L., 132

Orenstein, R., 179

Orians, G., 179

Orrenius, S., 147

Osborne, L., 266, 267

O’Shea, M., 52, 58

Otis, T., 343

Otsuki, LaDene, 51

Ottersen, O. P., 121, 126, 278

Otto, S., 314

Padoa-Schioppa, C., 550

Palade, G. E., 429

Palay, S., 135

Palkovits, M., 137, 148

Palmiter, R., 482

Papadopoulos, S., 88

Partinen, M., 152

Passey, G., 569, 571

Passingham, D., 382

Pasteels, J., 137

Pasteur, L., 41, 228

Pastor-Bernier, A., 551

Patek, S., 193

Patel, N., 64

Paton, J., 183

Patrick, J., 307

Patterson, P., 65, 83, 629

Patton, H., 253

Pauling, L., 159

Paulus, I. J., 158

Pavlov, I., 417

Pawson, T., 77

Payne, G., 351

Payne, R., 177

Pearce, J., 535

Pearse, A., 132

Pearson, K., 52, 60, 598

Pedrazzini, T., 142

Peng, Y., 497

Penhoet, E., 89

Penney, R., 177

Pennington, D., 566

Pennock, J., 175

Perez, E., 196, 197

Perlmutter, B., 262

Perlmutter, R., 99

Pernow, B., 138, 153, 157, 159

Persson, H., 143

Pesaran, B., 23

Peters, A., 400

Petersen, S., 80

Petras, J., 213

Pettigrew, J., 588

Pezaris, J., 23

Pfaff, D., 147, 178, 181, 182

Phelps, J., 174

Phillips, C., 372

Picker, M., 190

Pierce, B., 124

Pieribone, V., 141

Pinker, S., 329

Pipes, T., 79

Plath, K., 351

Plott, C., 544, 551

Pochet, R., 137

Poeck, K., 522

Poggio, G., 5

Polak, J., 132, 133

Poliakov, G., 418

Pons, J., 96, 98

Poo, M., 84

Poo, M.-M., 70, 84, 145, 337

Poon, M., 344

Popper, K., 379, 403

Porter, B., 480

Porter, J., 116, 581, 591, 592

Potter, D., 615, 629

Powell, P., 232

Powell, T., 160, 375, 387, 392

Precht, W., 254, 257

Prigogine, I., 422

Pruitt, A., 252

Prusiner, S., 151, 154

Puratian, N., 25

Purpura, D., 448

Purves, D., 63, 65, 479, 486, 489, 616

Qian, N., 11

Quigley, A., 201

Radloff, W. D., 370

Raguso, R., 185

Raki, T., 412

Rakic, P., 63, 382, 412

Ramachandran, V. S., 9, 381

Ramon y Cajal, S., 60, 348, 379, 387, 391, 416, 450

Randall, C., 570

Rangel, A., 544

Raper, J., 59, 60, 68, 83

Rash, B., 435

Rastl-Dworkin, E., 187

Raymond, J., 258, 263, 271

Reagan, R., 446

Reddy, S., 100

Regli, E., 525

Rehfeld, J., 135

Reichardt, L., 472

Reid, C., 389

Reiner, C., 292

Reis, D., 126

Rescorla, R., 552

Revelle, R., 612, 617

Rhodes, H., 189

Rhodes, P., 261

Ribas, T., 99

Ribera, A., 621, 622, 623

Ricard, M., 402

Richardson, E., 420

Richardson, K., 113

Richter, C., 212

Ricketts, E., 293

Rickliefs, R., 179

Ries, R., 48

Riley, R., 211

Ringerts, N., 119, 157

Rippe, K. P., 406

River, C., 218

Robbins, T., 214, 215, 543

Roberts, A., 217

Roberts, D., 216

Roberts, E., 121

Roberts, R., 291

Robertson, J. D., 616

Robertson, M., 183

Robinson, D. A., 255, 261, 270, 577

Rockland, K., 405, 407

Rodriguez-Puertas, R., 151

Roe, A. W., 444

Rohon, J. V., 617

Rohrer, B., 583

Rökaeus, Å., 138

Rolls, E., 382, 552

Romo, R., 530

Root, C., 631

Rooyen Marais, G., 369

Rose, E., 355

Rose, J., 3

Rosenfeld, G., 131

Rosenthal, J., 616

Rosenthal, N., 490

Rothschild, M., 393

Roux, F., 369

Rowell, H., 52

Royston, A., 96

Rubenstein, J., 487

Rubin, G., 66, 74, 84, 87

Ruda, M. A., 146

Ruffieux, A., 527

Rumelhart, D., 10

Rüsch, E., 407

Rush, R., 122

Russell, W., 180

Russinovich, M., 573

Ryle, G., 374

Sabes, P., 276, 278

Sachs, C., 112, 113

Saggese, M., 278

Sahakian, B., 214

Sahani, M., 23

Sainsbury, D., 395

Saito, H., 78

Sakata, H., 15

Sakmann, B., 154, 159, 616

Sala, M. A., 29

Salk, J., 333

Salkoff, L., 303

Salzman, D., 552

Samson, H., 217

Sanes, D., 183

Sanes, J. R., 466

Sanes, J., 258

Sano, Y., 158

Sarkisian, M., 433

Sarkisov, S., 418

Sassoon, D., 185, 186

Sawyer, C., 116

Scangos, G., 500

Scapagnini, U., 118

Schaffer, D., 84

Schaffhausen, B., 300

Schalling, M., 143, 147

Schally, A., 117, 127, 147

Scharrer, B., 147

Schein, S., 9

Scheller, R., 61, 89

Scherberger, H., 23

Scherberger, H.-J., 406

Schier, A., 496

Schlag-Rey, M., 9

Schlosberg, H., 566

Schmechel, D., 422

Schmeichel, B., 235

Schmidt, A., 407

Schmidt, B., 196

Schmitt, F. O., 119, 422

Schneggenburger, R., 82

Schoppik, D., 267

Schreiner, C., 276

Schröder, S., 404, 407

Schuetze, S., 182

Schuhknecht, G., 408

Schulteis, G., 225, 236

Schultz, W., 516

Schultzberg, M., 132

Schumacher, S., 398

Schuster, C., 67, 80

Schwartz, A., 22

Schwartz, M., 444

Schwarzenegger, A., 101

Schweizer, F., 343

Scobey, R., 3

Searle, G. D., 46

Seeger, M., 75

Segal, S., 438

Segerson, T., 301

Segil, N., 185, 187

Sejnowski, T., 262, 274, 390, 398, 537, 582

Seknowski, T., 183

Selemon, L., 443

Serafini, T., 84, 89

Seroogy, J., 135

Sessions, R., 213

Sestan, N., 434

Sevarino, K., 301

Shadlen, M., 404

Shapiro, M., 32

Sharp, P., 499

Shatz, C., 65, 82, 84, 274, 427, 472

Shaw, G., 9

Shaw, K., 194

Shekhar, K., 497

Shenoy, K., 21

Sherman, M., 382, 383, 385

Sherrington, C., 379, 408

Shi, T. S., 145

Shlag, J., 9

Shooter, E., 478

Shurman, J., 226

Sias, F., 570

Sidman, R., 421, 423, 450, 471

Siegel, R., 8, 9

Siegelbaum, S., 202

Sillito, R., 271

Silva, A., 343

Silver, P., 295

Silverman, M., 298

Silvers, P., 292

Simon, H., 217

Simon, M., 298

Simoncelli, E., 267

Simpson, B., 188

Simpson, J., 254, 270

Singer, W., 381

Sink, H., 72, 77

Sinnamon, J., 319

Sjöstedt, E., 149

Sjöstrand, F., 132, 145

Skavenski, A., 577

Skinner, B. F., 566

Skirboll, L., 135, 136

Sloan, A., 368

Sluyters, R. V., 280

Smale, S., 351

Smelik, P., 117

Smith, C., 137

Smith, D., 142, 387

Smith, N., 25, 29

Smith, O., 253

Smith, S., 305, 319

Smith, W., 91

Snow, P., 62, 70, 72

Snowden, B., 13

Snyder, L., 15

Snyder, S. H., 125

Snyder, S., 136, 213, 228

Sokoloff, L., 137, 140, 183

Sokolov, V., 414

Solomon, R., 221, 228

Somogyi, P., 155, 387, 395

Sorto, E., 24, 25

Southwick, C., 211

Spann, O., 45

Sparks, D. L., 562

Spector, S., 122

Speiss, J., 219

Sperry, R., 49, 60, 153, 370, 490

Spindler, T., 385

Spitzer, N. C., 610

Spriggs, M., 69

Spühler, I., 406

Squire, L., 85, 338

Stanford, T., 584

Stauffer, B., 547

Steinbeck, J., 293

Steinberg, R., 282

Steinbusch, H., 123, 126, 135

Stellar, E., 569

Stensaas, L., 424

Sternberger, L., 124

Stevens, C. F., 252, 253, 299, 301

Stevens, S. S., 566

Steward, O., 341

Stinus, L., 218, 224

Stokes, T., 181

Stone, L., 256

Stork, P., 301

Storm-Mathisen, J., 121, 126, 412

Stratford, K., 394, 408

Strauss, R., 80

Strawson, P., 374

Strick, P., 58, 270

Stryker, M., 259, 274, 276, 472

Stuart, A., 471

Suga, N., 177

Summers, L., 494

Sun, Y., 144

Sur, M., 444

Sutcliff, G., 220

Sutton, R., 537, 538

Suzuki, S., 336

Sved, A., 126

Svennerholm, L., 138

Swanson, L., 448

Swenson, A.-C., 122

Swerdlow, N., 219

Szentágothai, J., 117, 121, 445

Szurszewski, J., 120

Taghert, P., 59, 61

Takahashi, K., 309

Tamagnone, L., 69

Tanaka, M., 265

Tanter, M., 32

Tapia-Ramirez, P., 313

Tapiovaara, K., 416

Tarczy-Hornoch, K., 394

Tatemoto, K., 139

Taylor, B., 146

Tear, G., 75

Terasaki, M., 300

Tessier-Lavigne, M., 65, 82, 88–89

Thach, T., 270

Thatcher-Britton, K., 217

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Theodorsson, E., 144

Theunissen, F., 84

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Thomas, J., 62, 64, 65, 71, 77

Thomas, L., 173

Thompson, K., 344

Thomson, W., 490

Thornton, J., 186

Tien, C.-L., 85

Till, S., 202

Ting, A., 345

Tito, M., 420

Tobias, M., 185

Tobler, P., 544

Todorić, M., 412

Toit, P., 369

Tokiyama, S., 281

Toledo-Aral, J. J., 308

Tosches, M., 203

Tran, N., 497

Travis, B., 568, 569, 571, 572, 576, 578

Trimmer, J., 303, 312

Tsay, H.-J., 310

Tsien, D., 383

Tsoo, R., 72, 77

Tsuruo, Y., 128

Tsutsui, K., 552

Turner, D., 179

Turner, T., 441

Tyler, D. W., 176

Tyutchev, F., 419

Ubnik, R., 151

Udenfriend, S., 122, 123

Uhlén, M., 149

Ullman, S., 11

Umemori, H., 484

Underwood, B. J., 566

Ungerstedt, U., 113, 114, 524

Uvnäs, B., 129

Vaccarino, F., 219

Vaeröy, H., 152

Vale, W., 218–19

Van Buren, M., 417

Vandenberg, C., 299

Vane, M., 213

van Maanen, J. H., 117

Van Vactor, D., 72, 77

Varmus, H., 136, 489

Vehrencamp, S., 179

Vendruscolo, L., 235

Verge, V., 144

Verhofstad, A., 123, 135

Verschure, P., 399

Vignal, C., 192

Vila-Procile, E., 141

Villain, A., 196

Villar, M., 140, 147

Vincent, S., 136

Viswanathan, S., 192, 202

Vogt, O., 418

Volkow, N., 226, 231

von Euler, U., 111, 114, 129, 159

Wagner, A., 452

Wakisaka, S., 144

Walker, E., 387

Wall, T., 221

Wallace, M., 484

Walsh, C., 435

Walsh, J., 142

Walton, M., 598

Wan, H., 80

Wang, K. H., 74

Wang, O., 346

Wang, X., 155, 645

Warren, K., 312

Warren, M., 210

Washington, E., 280

Washington, G., 352

Waters, M., 45

Watson, J., 9, 422

Watson, S. J., Jr., 130

Weaver, S., 48

Wehner, R., 403

Weihe, E., 146

Weiner, J., 492

Weinmaster, G., 351

Weinstein, S., 211

Weiskrantz, L., 382

Weiss, F., 217

Weiss, P., 60

Weissmann, C., 499

Weizsäcker, C.-F., 521

Wen, H., 316, 318

Werman, R., 120

Werner, D., 332

West, A., 278

Wetzel, D., 184

Wever, G., 177

Wheeler, H. H., Jr., 87

White, A., 232

White, J., 595

White, N., 217

Whitson, H., 279

Whittaker, J., 571

Whitteridge, D., 378

Wickner, B., 294, 295

Wieschaus, E., 71

Wiesel, T., 85, 153, 382, 426, 445, 615

Wiesenfeld-Hallin, Z., 128, 144, 147

Wigston, D., 490

Wilder, G., 292

Wilke, M., 19

Willdonger, J., 202

Williams, N., 157

Williams, R., 433

Wills, H., 84, 86

Willsey, H., 199

Willshaw, D., 377

Wilmut, I., 101

Wilson, E. O., 403

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Winberg, M., 69, 79

Wise, R., 216, 531

Witschi, E., 179

Witschi, W., 180

Wohlschlegel, J., 351

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Wong-Riley, M., 383

Woodworth, R. S., 566

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Worden, F., 120

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Wu, J.-P., 145

Wu, K. H., 188

Wundt, W., 369

Wurtz, B., 264

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Xu, X.-J., 128

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Xu, Z.-Q. D., 141, 145, 155

Yakovlev, P., 417

Yamagata, M., 499

Yamaguchi, A., 187, 188, 190

Yamamoto, A., 202

Yamanaka, Y., 96

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Yang, Y., 269

Yatrakis, K., 201

Yau, K. W., 472

Yip, J., 490

Yodlowski, M., 183

Yogi, M. M., 43

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Young, S., 143

Zecevic, N., 426

Zerhouni, E., 136

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Zhang, M., 145, 152

Zhang, X., 146

Zhang, Y., 155, 494

Zhong, W., 149

Zide, D., 123

Zieg, J., 298

Zigler, E., 470

Zigmond, R., 144

Zinn, K., 64, 79, 497

Zipser, D., 10

Zipursky, L., 499

Zito, K., 67, 81

Zoghbi, H., 317, 357

Zornik, E., 188

Zucker, B., 616

Zuckerman, K., 571

Zwicker, E., 293

Zwizinski, C., 295