Mechanisms of Synaptic Transmission: Bridging the Gaps (1890-1990)

MECHANISMS OF SYNAPTIC TRANSMISSION

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MECHANISMS OF SYNAPTIC TRANSMISSION Bridging the Gaps

(18QO-1990) JOSEPH D. ROBINSON, MD Professor of Pharmacology Emeritus State University of New York, Syracuse

OXFORD UNIVERSITY PRESS

2001

OXJORD UNIVERSITY PRESS Oxford New York Athens Auckland Bangkok Bogota Buenos Aires Calcutta Cape Town Chennai Dar es Salaam Delhi Florence Hong Kong Istanbul Karachi Kuala Lumpur Madrid Melbourne Mexico City Mumbai Nairobi Paris Sao Paulo Shanghai Singapore Taipei Tokyo Toronto Warsaw and associated companies in Berlin Ibadan

Copyright © 2001 by Oxford University Press. Published by Oxford University Press, Inc., 198 Madison Avenue, New York, New York, 10016 http://www.oup-usa.org Oxford is a registered trademark of Oxford University Press 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 Oxford University Press. Library of Congress Cataloging-in-Publication Data Robinson, Joseph D. Mechanisms of synaptic transmission : bridging the gaps (1890-1990) / Joseph D. Robinson, p. cm. Includes bibliographical references and index. ISBN 0-19-513761-2 1. Neural transmission—Research—History—20th century. 2. Synapses—Research—History—20th century. I. Title. QP364.5 .R63 2001 573.8'09—dc21 00-053071

135798642 Printed in the United States of America on acid-free paper

for Carol, with love

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PREFACE

Our understanding of how the nervous system accomplishes its wondrous feats—sensing, thinking, willing, imagining, learning—took a decisive turn late in the nineteenth century. At that time, anatomists, embryologists, and physiologists, led by Santiago Ramon y Cajal, Wilhelm His, and Charles Sherrington, developed the Neuron Theory, specifying a nervous system composed of discrete nerve cells communicating through their synaptic contacts. But this formulation was a beginning, not an end, for it immediately raised questions that its authors and their contemporaries set about addressing. How can nerve cells assemble into the organized pathways required for reflexes and other precise neural responses? How can neural function change to exhibit learned behaviors? And, the major focus here, how can impulses pass from cell to cell at the synapses? A century later these questions continued to challenge experimental ingenuity and interpretive insight, for initial efforts to answer them exposed a cascade of further questions. If cell processes form specific junctions by growing along particular chemical gradients, how are these gradients formed, what are their identities, and how do they direct growth? If learning occurs through altering synaptic transmission, what aspect is altered, how does experience guide this alteration, and how does this alteration modify transmission? If, as the next generation argued, transmission at synaptic junctions occurs chemi-

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cally, what are the chemicals, how are they formed, how are they eliminated, how do they act, and how can these processes be modified therapeutically? This book outlines the course of such inquiries through a hundred years of scientific endeavor. It is a tale of old formulations transformed gradually or replaced abruptly, of plausible schemes supported or refuted, of incremental advances gained through the cumulative efforts of generations of participants from across the globe, and of new concepts achieved through new experimental approaches exploiting new methods. It is also a tale of integration. A plethora of disparate observations were synthesized into coherent models, uniting approaches and conclusions from anatomy, biochemistry, embryology, medicine, pharmacology, and physiology. The composite field, neuroscience, that emerged from these inquiries, however, was itself integrated within the mainstream of general cell biology. A secondary goal of this narrative is to illustrate the diversity of scientific practices, a goal that requires an inclusive account of a complex field over a substantial time span. (This approach contrasts with a common historical practice of selecting isolated cases to exemplify science, a practice that often strips the episodes from antecedents and consequences and that raises the specter of selecting data to fit a cherished hypothesis.) Nevertheless, the chosen span of a hundred years is arbitrary in the sense that final answers were nowhere apparent in 1990. The terminal'year was chosen for the practical advantage of allowing some retrospection. The demands of breadth and inclusion force this account to approximate a simple chronicle of who did what, when, where, how (and often why). It is not a biographical account, for the cast is too numerous; moreover, individuals are named often without mentioning their colleagues (the phrase "and associates" should everywhere be added by the reader), although collaborating authors are identified in the bibliography. Publication dates are included, but the adjudication of priority claims is not attempted (quests for priority have fired some of the individuals appearing in this narrative, but where disputes arise, the issues are often too complex for resolution at this scale). It is not a social history, either, although human interactions surely affected the course, and various institutions facilitated or impeded research. Cities are listed, but chiefly to illustrate the geographical extent of the investigators' activities. Whereas certain centers fostered notable discoverers—as, for example, did London, Berlin, and New York—other scientists prospered in the hinterlands, as did Cajal in Barcelona, Sherrington in Liverpool, and their successors at such distant sites as Graz, Oslo, Aberdeen, Dunedin, Taipei, and Beijing. But of the questions insufficiently examined, probably the most serious to a scientist is how: scientific advances are notably dependent on enabling techniques, and the development of new methods permits the resolution of questions long asked but unapproachable previously. This account, therefore, is intended as a framework

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to provide the scientific context for future studies that can then examine in detail—and from other perspectives—individual episodes only sketched in this account. The predominant sources here are published scientific papers. These record the experimental results and present the argued interpretations. They thus served as both the repository of current knowledge and the vehicle for communication and persuasion (formal scientific meetings and informal personal interactions also served these ends, but such channels ultimately flowed into published papers). Accounts from memoirs and biographical compilations also helped to establish the origins and courses of studies noted here. Consultations with contributors to these efforts in the form of conversations, inquiries, and formal interviews have been particularly valuable. For their kind and thoughtful assistance I am particularly indebted to George Aghajanian, Oliver Brown, Jack Cooper, Mario Delmar, Robert Furchgott, Ian Glynn, Steven Grassl, Jack Green, Frederic Holmes, Peter Holohan, Andrew Huxley, Jose Jalife, Eric Kandel, Bernard Katz, Mahlon Kriebel, Joseph Larner, Karina Meiri, Ruth Nadelhaft, Lisa Robinson, Amar Sen, Gordon Shepherd, Eric Simon, Mikulas Teich, Helen Tepperman, Jay Tepperman, Richard Veenstra, Irwin Weiner, and Richard Wojcikiewicz. The library staffs at the State University of New York in Syracuse and at the University of Virginia in Charlottesville provided willing and efficient help in finding sources, fulfilling requests, and answering vague questions. Fiona Stevens and Jeffrey House at Oxford University Press graciously provided interest, assistance, and an indulgent tolerance. Financial support from the Hendricks Fund, State University of New York, Syracuse, and from the National Science Foundation made this project possible. Finally, I am deeply grateful to my wife, Carol, for innumerable reasons. On the most mundane level, I thank her for efficiently typing all the references for this book—even retyping, without reproach, a major chunk of these after I ineptly and irretrievably erased the references. Syracuse, N.Y.

J.D.R.

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CONTENTS

1. Beginnings: Cajal and tke Neuron Tkeory (1889-1909), 1

Cajal at Berlin, 1 Background: Cells, Nerve Cells, and Nerve Impulses, 4 Proclamation of the Neuron Theory, 10 CajaFs Contributions, 11 Confirmations, Criticisms, and Responses, 18 Conclusions, 26 2. Beginnings: Snerrington ana the Synapse (1890—1913), 31 Sherrington, Reflexes, and the Synapse, 31 Background: Reflexes, 33 Sherrington s Achievements, 35 Synapses and the Reflex Arc, 37 Conclusions, 44 3. Chemical Transmission at Synapses (1895—1945), 49

Nerve Impulse Conduction and Synapse Structure, 49 Background: The Autonomic Nervous System, 50

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CONTENTS Chemical Transmission in the Autonomic Nervous System, 55 Chemical Transmission at Neuromuscular Junctions, 72 Chemical Transmission in the Central Nervous System, 75 Electrical Transmission, 76 Conclusions, 78 4. Chemical Transmission at Synapses (1945—1965), 87

Postwar Progress, 87 Identifying Chemical Transmission, 89 Visualizing Synaptic Gaps and Synaptic Vesicles, 106 Identifying Electrical Transmission, 111 Conclusions, 112 5. Identifying Neurotransmitters (1946-1976), 119

Scope and Criteria, 119 Acetylcholine, 120 Noradrenaline, 123 Dopamine, 125 Serotonin, 126 GABA, 129 Glutamate, 132 Glycine, 133 Neuropeptides: Substance P and Enkephalins, 134 Conclusions, 137 6. Ckaracterizing Receptors (1905-1983), 143

Essential Issues, 143 Drug-Receptor Interactions, 143 Receptor Classification, 152 Structure-Activity Relationships, 156 Receptor Identification and Purification, 157 Responses of Individual Receptor Molecules, 163 Conclusions, 166 7. Second Messengers (1951-1990), 171 Cyclic AMP, 171 Protein Kinases and Phosphatases, 177

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G-Proteins, 181 Ca2+, 186 Inositol-£mphosphate and Diacylglycerol, 189 Conclusions, 193 8. Receptor Structures and Receptor Families (1983 — 1990), 199 Molecular Biology and Recombinant DNA Techniques, 199 Nicotinic Cholinergic Receptors, 200 Ligand-Gated Ion Channels, 206 Adrenergic Receptors, 208 G-Protein Coupled Receptors, 210 Receptor Regulation, 212 Conclusions, 214 9. Synthesis, Storage, Transport, ana Metabolic Degradation 01 Neurotransmitters, 219

Steps in Chemical Transmission, 219 Synthesis, 219 Storage, 225 Degradation, 229 Transport ("Reuptake"), 234 Conclusions, 238 10. Neurotransmitter Release, 245 Proposals, 245 Evidence for Exocytotic Release, 249 Triggering of Release, 257 Mechanism of Release, 259 Endocytotic Retrieval of Vesicles, 262 2+

Ca -Independent Non-Exocytotic Release, 265 Conclusions, 267 11. Formation or Specific Synapses, 273 Embryonic Development of Synaptic Connections, 273 Approaches and Possible Mechanisms, 274 Early Arguments Concerning Chemotaxis (1890-1963), 276 Cell Death and Neurotrophic Factors, 280

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CONTENTS Chemical Guidance (1963-1990), 283 Growth Cone Motility, 286 Synapse Formation, 288 Conclusions, 290 12. Learning, 295

Background, 295 Chemical Representations, 297 Learning in Aplysia, 300 Learning in Drosophila, 306 Learning in Mammals: The Hippocampus and Long-Term Potentiation (LTP), 306 Conclusions, 314 13. Diseases ana Therapies, 319 Defining and Developing, 319 Parkinson s Disease, 320 Schizophrenia, 327 Depression and Manic-Depressive Illness, 337 Conclusions, 343 14. Epilogue, 349 Progress, 349 Historical Accounts and Conclusions, 350 Assumptions, 351 Approaches, 351 Goals, 352 Generalities and Exceptions, 353 Conflict Resolution, 355 Lessons, 356 References, 359 Index, 443

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1 BEGINNINGS: CAJAL AND THE NEURON THEORY (1889-1909)

Cajal at Berlin

In October 1889 Santiago Ramon y Cajal (Fig. 1-1), a new member of the German Anatomical Society, appeared in Berlin for its annual meeting, having pooled his meager resources to make a first trip in Europe beyond his native Spain.1 For the previous two years Cajal had striven tirelessly to delineate the microscopic structure of the nervous system, examining histological sections stained by the notoriously difficult Golgi technique using a Zeiss microscope presented by the provincial government in gratitude for Cajal's zealous efforts during a cholera epidemic. Now 37 and recently appointed professor of anatomy in Barcelona, Cajal was determined to present a new vision. What he saw and how he interpreted it had been published in Spanish, chiefly in a journal Cajal founded for that purpose and whose cost had drained his minimal salary.2 Recognizing how little Spanish was read by the central Europeans who dominated histology and neuroanatomy, he had recently arranged to have French translations published in German journals. But more important, he realized, would be an opportunity for meeting the scientific establishment and for demonstrating his slides to them. The consequences of his German trip3 satisfied even Cajal's driving ambition. During the initial sessions, devoted to formal lectures by the elite, Cajal was

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FIGURE 1-1. Santiago Ramon y Cajal (1852-1934; from P. Yakovlev, courtesy of P. Rakic).

too distracted by anticipation to listen. But on the day for demonstrations he eagerly set up his slides—using his Zeiss as well as a couple of requisitioned microscopes—and lectured all who ventured by in his imperfect French. (Although Cajal had learned German and English to read scientific publications, he spoke only Spanish and French.) Cajal recollected that most of those present were intent on demonstrating their own preparations. But among those drawn into Cajal s orbit4 were such luminaries—and future allies—as Wilhelm His (Leipzig), Rudolf von Kb'lliker (Wiirzberg), Gustav Retzius (Stockholm), and Wilhelm Waldeyer (Berlin). CajaFs preparations were superficially unprepossessing: multiple thick tissue sections arrayed under an uneven layer of resin on slides without coverslips.5 But viewed through the microscope—when accompanied by Cajals identification of forms and explication of methods—were clear revelations. Kolliker, the reigning authority on neuroanatomy, was so enthusiastic that he took Cajal to dinner for further discussion. Indeed, Kolliker subsequently learned Spanish in order to read Cajal s earlier papers, and he later counted among his accomplishments the discovery of Cajal. What those slides showed were silhouettes—dark red to black against a pale yellow background—of twisting threads dividing and subdividing profusely after extending from a central mass (Fig. 1-2). What those silhouettes represented to Cajal and his audience were single nerve cell bodies and their branching processes. What that interpretation centered on was a critical issue of struc-

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FIGURE 1-2. Photomicrograph of a Golgi-stained pyramidal neuron of the hippocampus. Cajal, however, relied on drawings rather than photographs. (Courtesy of Fidia Research Laboratories.)

tural and functional concern: whether the brain was composed of a reticulum of continuous, anastomosing fibers connecting everything to everything, or an assemblage of individual cells making discrete and specific contacts. Cajal's slides and arguments favored the latter view, an interpretation recently championed by His and by August Forel in Zurich. Theirs, however, was a minority stance against the prominent reticularist position of Joseph von Gerlach and Camillo Golgi. This chapter describes selected developments between this Berlin meeting and the publication twenty years later of Cajal's treatise on regeneration in the nervous system. But before considering further Cajal's images and interpretations, a brief summary of preceding events is necessary.

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MECHANISMS OF SYNAPTIC TRANSMISSION Background: Cells, Nerve Cells, ana Nerve Impulses

Cells

Formulation of the cell theory is conventionally ascribed to Matthias Schleiden in 1838, working with plants, and to Theodor Schwann in 1839, working with animals.6 Through the nineteenth century their initial pronouncements were developed and refined, so that a textbook of 1895 could state authoritatively: "all vital processes of a complex organism appear to be nothing but the highly-developed result of the individual processes of its innumerable variously functioning cells[, each] a little mass of protoplasm."7 Nevertheless, a troubling uncertainty remained: what marked, anatomically and functionally, the boundary of a cell?8 If cells were separate entities, what separated them? In plant tissues, the cell borders were demarcated by cell walls, readily visible with a microscope.9 But in animal tissues no such structures were apparent. Cell margins might represent merely the undifferentiated edge of its protoplasm, with protoplasm then understood to be the vital, organic stuff of life. Or the margins might reflect some surface modification where protoplasm abutted its nonliving environment. In fact, indications of a distinguishable cell boundary were noted in some circumstances,10 and functional evidence was accumulating. For example, Carl Nagli at midcentury inferred the presence of membranes surrounding the protoplasm from observing that pigment granules could neither enter nor escape, and subsequently Wilhelm Pfeffer attributed selective permeability to a cell membrane from observing the swelling and shrinkage of cells immersed in various media. But in 1889 nearly a decade remained before Ernest Overton would publish his prescient studies postulating a lipoidal barrier on the cell surface, and even in 1897 Max Verworn, a leading figure in German biology and an advocate of protoplasmic preeminence, dismissed the notion, claiming that "the idea that the cellmembrane is a general cell-constituent has completely disappeared."11 Correspondingly, William Halliburton s text of 1904 referred to an animal cell as "a little naked lump of living material."12 Cell Theory ana the Nervous System

The cell theory included the notion of its universal applicability. Examinations of animal tissue raised few doubts, and easily recognized nuclei served as indicators of cells even when cell margins were not obvious. But where in most tissues a packing together of polygonal forms could be seen or inferred, microscopic examinations of the nervous system presented, instead, a confusing complexity. Transected nerves revealed, under microscopic examination, numerous circular profiles, interpretable as cross sections of tubes running longitudinally. Those outlines were subsequently identified with "myelinated" fibers bearing

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a coat of lipoidal myelin. On the other hand, sections of brain and spinal cord revealed areas of white and gray matter: white matter appeared to be filled with sections of myelinated fibers, whereas gray matter contained sections of fibers without such thick coatings ("unmyelinated" fibers) together with globular bodies containing nuclei. From ancient times nerves were thought to contain hollow tubes through which animating spirits flowed, so the microscopic fibers could be likened to such structures and functions. The nucleated globules were interpretable as nerve cells, but relating those globules to the array of fibers was hindered by the profusion of interlaced processes. The decades of description and debate must here be compressed into a brief listing. In the 1830s Jan Purkinje and his students in Breslau—notably Gabriel Valentin—identified in the cerebellar cortex large corpuscles shaped like teardrops, some with branched tails (Fig. 1-3A; these subsequently were christened "Purkinje cells"). Elsewhere they identified globules with sharp outlines containing granules and a nucleus, although Valentin concluded that these globules were not continuous with the numerous fibers present.13 In 1853 Kolliker noted that "most observers . . . regard [fibers arising from cell bodies] as not always present, but rather as a secondary formation which does not exist during life"; still, he considered that such cellular fibers were "an essential constituent of the living nerves."14 In a posthumous publication of 1865 Otto Deiters in Bonn took a major step further, distinguishing between branching "protoplasmic processes" (later termed "dendrites"), which extended in variable numbers from the cell body, and the single "axis cylinder" (later termed "axon").15 Deiters's definitions were based on microscopic dissection of nerve cells with their processes attached, as well as on microscopic examination of tissue sections. Progress at midcentury was vastly facilitated by the development of better methods for fixing tissues and by the introduction of new stains that colored the constituents differentially.16 (In 1856 Rudolf Virchow in Berlin described "a kind of glue in which the nervous elements are planted," containing soft and fragile cells that he named "neuroglia";17 these subsequently were recognized as a second kind of neural cells, ones involved in nutritive and supportive roles. The emphasis here, however, is on nerve cells themselves.) In retrospect, Deiters's distinctions identify the tripartite nature of the prototypic nerve cell: dendrites, cell body, and axon. But in 1872 Gerlach, using newer staining techniques, saw the cell processes forming a continuous and interconnected reticulum, with dendrites joined and with axons extending either directly from cell bodies or from the reticulum itself. Gerlach's forceful advocacy of a protoplasmic continuum and his drawings of a pervasive network (Fig. 1-3B) reinforced earlier but less absolutist claims.18 From Italy Golgi endorsed the principle of an interconnected reticulum in a series of papers beginning in 1873, recasting the particular form somewhat.19 Golgi approached these considerations through his discovery of a revolutionary

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FIGURE 1-3. Nerve cells and fibers. A. Purkinje's 1837 drawing of the cerebellar cortex, showing the large corpuscles subsequently named Purkinje cells (one shown enlarged). B. Gerlachs 1872 drawing of processes between two cell bodies, above and below, branching and interconnecting. (Reproduced from Shepherd [1991], Figs. 2 and 8.)

black stain, his reazione nera. His guide, however, was a conviction that neural function was comprehensible as a holistic system evidenced in the total connectivity of a reticulum. Golgi's stain, as noted above, showed distinct silhouettes and allowed him to confirm Deiters s view of an axis cylinder distinguishable from other processes. And examining that axis cylinder, Golgi discovered a new characteristic, its branching into what are now termed "axon collaterals." These he felt would form a widespread reticulum of anastomosing—and therefore continuous—processes. What he actually saw, however, were merely branchings into finer and finer processes: either with no termination visible in

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the mass of images, or with terminations attributed to the fiber diameters becoming submicroscopic. Golgi's conclusion about axons forming a reticulum agreed with Gerlach's interpretation, even though Golgi's stain showed apparently free endings. On the other hand, Golgi differed with Gerlach's view of protoplasmic processes. Instead of the anastomoses that Gerlach specified, Golgi saw free endings here also. And since free endings could not participate in a continuous reticulum, Golgi imagined a quite different role for the protoplasmic processes. Seeing them ending near blood vessels or neuroglia, he proposed that the protoplasmic processes served a nutritive function. Contrasting with these views of a continuous reticulum were the proposals advanced by His and Forel of discrete, discontinuous nerve cells.20 His concluded that the nerve cell was a "genetic, nutritive, and functional entity" from studying embryological development;21 moreover, he discovered that primordial nerve cells initially developed axis cylinders and subsequently branched extensions (protoplasmic processes) to form a distinct tripartite entity. Forel followed the antithesis of development, degeneration. Augustus Waller had shown that after transecting a nerve the peripheral process degenerated within a few days. Bernhard von Gudden, with whom Forel studied, later showed that damage could extend centrally as well.22 Forel's insight was to stress the loss of a functional unit—both peripheral and central—after local injury, emphasizing by the limits of such degeneration that the functional unit was discrete and discontinuous. Nerve Impulse Conduction

Another pertinent concern is the process of nerve conduction.23 The ancients described vital spirits flowing from the brain through nerves to animate the body. In the eighteenth century the characterization of electricity as an "imponderable fluid" suggested its association with nerve impulses. At the end of that century Luigi Galvani in Bologna furthered this notion by showing that electrical discharges could stimulate muscle contractions—as had others before him. But Galvani next claimed that living tissues released electricity. He hung the isolated spinal cord plus hind legs of a frog from an iron railing, using a brass hook to impale the spinal cord: when the legs happened to touch the railing their muscles contracted. Galvani proposed a circuit for "animal electricity" from muscle to iron railing to brass hook to spinal cord to nerve to muscle; he imagined that in vivo the brain secreted electricity through nerves to muscles, where the electricity was stored.24 Alessandro Volta in Pavia challenged Galvani's interpretation, claiming that metallic contacts between the iron railing and the brass hook generated a stimulating current. Volta confirmed his proposal of "metallic electricity"—arising

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from the moist contact of dissimilar metals—by constructing a bimetallic "pile" of alternating silver and zinc discs separated by moist cardboard: the first battery.25 In the course of their debate, Galvani described a nerve-muscle unit stimulated by draping its nerve across a second muscle and its cut surface: the first muscle then contracted. This "contraction without metals" Volta also challenged, arguing that electricity arose from the junction of dissimilar tissues, just as from dissimilar metals. Nevertheless, Volta recognized that electric fish, such as the torpedo, could generate electricity.26 In the 1820s Leopoldo Nobili in Florence recorded, using a galvanometer he constructed, a "frog current" passing from legs to body of a skinned, decapitated "frog. Carlo Matteucci in Pisa extended that observation in the next decades by demonstrating a current between the cut end of a muscle and its intact surface, although he could show no such current in nerve. Matteucci also found that when strychnine was given to a frog, producing strong, persisting contractions ("tetany"), this current then decreased. Emil du Bois-Reymond in Berlin meticulously examined these phenomena, beginning in the 1840s. With improved galvanometers,-du Bois-Reymond measured a "resting current" in nerve as well as muscle; he also recorded the "negative variation"—the decrease in that resting current—during tetany. Du BoisReymond imagined that the current flow was due to an electrical potential arising from polarized "electromotive particles" oriented within nerve and muscle. At midcentury Hermann Helmholtz in Berlin calculated the rate at which impulses pass along a nerve by timing the interval between electrical stimulation of a frog nerve and contraction of the muscle it innervated. His value, roughly 30 meters/second, was far slower than electrical conduction along a wire. Julius Bernstein in Halle, a former student of du Bois-Reymond, then showed that the negative variation traveled along nerves at an equivalent velocity, furthering its association with the nerve impulse. Moreover, Bernstein reconstructed the timecourse of the negative variation by measuring its magnitude at successive intervals: current fell and rose within milliseconds. In the latter decades of the nineteenth century, Ludimar Hermann in Konigsberg, also a former student of du Bois-Reymond, vigorously attacked the notion that resting currents were present normally. He found no such current flowing over the surface of intact tissues and concluded that the currents measured by Nobili, Matteucci, and du Bois-Reymond were instead "injury currents," flowing between injured regions of nerve or muscle and an intact surface. On the other hand, Hermann characterized the negative variation as an "action current." He proposed that a self-propagating wave of electrical activity advanced by self-stimulating circuits: local currents passed from the point of excitation to adjacent regions, generating there a new action current that again

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stimulated locally, advancing thus along the nerve or muscle fiber. At the turn of the century Hermann likened this process to transmission along submarine cables, in which a central "core conductor" is separated from a conducting environment (the sea) by an insulating sheath. Hermann also rejected du BoisReymonds electromotive particles, suggesting that the action current was due instead to the formation and disappearance of ions: on stimulation, an organic electrolyte in the nerve and muscle fibers transiently formed ions having different mobilities. By contrast, Bernstein in 1902 argued for preexisting potentials at rest, based on cell membranes selectively permeable to certain ions.27 In the first half of the nineteenth century Michael Faraday founded the science of electrochemistry, in the process naming the positively charged ions ("cations") and negatively charged ions ("anions") formed from "electrolytes." In the 1880s Svante Arrhenius in Upsala argued comprehensively for the spontaneous dissociation, in solution, of electrolytes into cations and anions. Shortly thereafter, Walther Nernst in Gottingen specified the electrical potential associated with a concentration differential of ions, and before the turn of the century both he and Wilhelm Ostwald were linking ionic processes to neural electricity. In this context, Bernstein's membrane theory identified resting potentials with concentration potentials: they arose from concentration differentials across membranes that were permeable to one ionic species but not its counter-ion. For the action current, Bernstein proposed that excitation produced, transiently, a general increase in membrane permeability. This loss of selectivity would decrease the transmembrane potential, transiently; the consequent current could then propagate through the local circuits that Hermann envisaged (although in terms of transmembrane currents rather than through Hermann's release and recapture of ions within the fiber). Appearing in the same volume with Bernstein's proposal was a complementary suggestion by Overton in Wiirzburg.28 He reported that if sodium ions (Na + ) were absent from the bathing medium, muscles failed to contract when stimulated; potassium ions (K + ) could not substitute for Na + . Since muscles were known to be rich in K + and the extracellular fluid in Na + , Overton suggested that excitation was associated with an exchange of intracellular K + for extracellular Na + . Subsequent developments had by mid-twentieth century validated Bernstein's thesis of selective permeabilities underlying the resting and action potentials as well as Overton's suggestion of opposing fluxes of K + and Na + , but in the first decades of the twentieth century these formulations were not uniformly accepted. Thus, Howell's 1905 textbook included arguments for nerve impulses being chemical phenomena, repeating the analogy with "a spark along a line of gunpowder"; moreover, it ignored Bernstein and Overton, although it

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cited Albrecht Bethe's proposal for neurofilaments as core conductors.29 Keith Lucas in Cambridge, in his Croonian Lecture of 1912, cited both Bernstein and Overton, but he emphasized a proposal by Nernst relying on a series of transverse membranes; however, Lucas reformulated Nernst s proposal in terms of "a sheath membrane impermeable to certain ions."30 But in his influential 1920 textbook, William Bayliss in London devoted more attention to a scheme reminiscent of Hermann's than to Bernstein's and Overton's.31 Bayliss favored the intracellular scheme of John Macdonald in Sheffield, who imagined that axons were filled with colloidal material that trapped certain ions, notably K+.32 Stimulation, Macdonald argued, altered the colloidal state transiently, releasing K + ; K + was recaptured when the colloid reverted to its initial state. In short, at the beginning of the twentieth century no firm understanding existed of how impulses traveled along fibers or between cells—and hence no mechanistic basis for determining whether these processes were similar or different.

Proclamation or the Neuron Tneory

The clear images on Cajal's slides in Berlin and his ready advice on how better to apply the Golgi stain encouraged others to attempt with it the defining of nerve cells as well as the mapping of the nervous system. The sharp outlines—coupled with Cajal's evangelical interpretation of discontinuity—also provided a morphological dimension to the generative/degenerative studies of His and Forel, encouraging others to look and think again. At the turn of the decade, confirmations of Cajal's approach and Cajal's interpretation came from, among others, Kolliker, Retzius, Arthur van Gehuchten in Louvain, and Michael von Lenhossek in Basel. Perhaps most consequential, however, was a summarizing synthesis by Wilhelm von Waldeyer, professor of anatomy in Berlin. In a serialized review published in 1891, Waldeyer introduced the term neuron to name as a discrete whole the cell body plus all its processes; the "Neuron Theory" (or "Neuron Doctrine") explicitly enunciated this unity of parts and separateness of cells.33 Although Cajal noted with some pique that Waldeyer "did not personally investigate the problem of interneuronal connections, confining himself to making a popular review of [Cajal's] works in a German weekly and inventing the word neuron,"34 Waldeyer's contribution was significant. Not merely did he lend his personal prestige as an establishment authority, Waldeyer assembled a careful compilation of the evidence leading to his conclusion. (At that time His introduced dendrite to supplant protoplasmic process, and Kolliker replaced axis cylinder with axon. These distinguishing new names also helped to propel the Neuron Theory.)

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Cajal's Contributions Teennique

Cajal first viewed the Golgi stain in 1887, just two years before the Berlin meeting. On a trip to Madrid from Valencia, where he was then professor of anatomy, he visited Luis Samarro, a neurologist who had studied in Paris and was "trying out patiently and carefully all the new technical methods which had appeared abroad"; Samarro's sharp, discrete images inspired Cajal on his return to Valencia to try the Golgi method "on a large scale."35 Four technical and strategic aspects of Cajal's approach are significant: 1. Cajal toiled assiduously to make the staining more reliable. At first telling the method seems routine: blocks of tissue were soaked in potassium bichromate and then transferred to a solution of silver nitrate, which produced a dark red to black image of silver chromate.36 Nevertheless, staining was erratic, incomplete, and tedious. Cajal, who praised persistence as "the virtue of the less brilliant,"37 set about perfecting the method, optimizing concentrations, times, temperatures, and light exposure of the photosensitive silver salts. Through this careful and deliberate search Cajal standardized a rapid staining method as well as developed a doubleimpregnation approach, garnering success where others failed.38 Thus, Samarro had soon abandoned the technique, and although Kolliker had traveled to Pavia in 1887 to learn the technique from Golgi, Kolliker progressed significantly after being tutored by Cajal in Berlin.39 Others soon adopting the Golgi stain included von Lenhossek, Retzius, and van Gehuchten. But even after Cajal's modifications, the procedure retained a reputation for difficulty. Nevertheless, one puzzling characteristic, its staining only a tiny fraction of the cells (a few out of every hundred), was quite beneficial: the complexities of overlapping cell bodies and processes were reduced to more distinguishable images. A composite picture of the whole could then be constructed from multiple preparations, each defining different selections of images. (Subsequently, Cajal used other stains as well, borrowing Paul Ehrlich's methylene blue stain and devising new methods, such as his reduced silver nitrate technique, as will be noted below.) 2. Cajal used thick sections for microscopic examination. Since neuronal processes can be quite long, having bigger, thicker sections increased the opportunities for tracing those processes to their terminations. (The light background and paucity of images produced by the Golgi stain permitted the use of such sections, which would have been opaque with older techniques.)

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3. Cajal used tissues from embryos and young animals. The neurons in these animals are shorter (thereby increasing the likelihood that their terminations would lie within the microscopic section), more separated from one another (thereby minimizing the complexity of images), and not yet myelinated (depositions of myelin, which occur relatively late in development, hinder staining by the Golgi method). 4. Cajal examined a wide range of species. Through extensive comparisons he could then define similarities of structure and attribute these to similarities of function. Cajal's Principle 01 Dynamic Polarization

Before considering his anatomical investigations further, a crucial functional characterization should be noted: Cajal's Principle of Dynamic Polarization. Formulated as an "induction from numerous morphological facts,"40 this principle specified the direction in which nerve impulses move through neurons: the dendrites and cell body conduct toward the axon, whereas the axon conducts impulses away from the dendrites and cell body. This principle delineated functional circuits that could be discovered by identifying points of contact between adjacent neurons. It also contradicted Golgi s scheme of impulses traveling through a reticulum, where no directionality could be inferred from morphological examination and in which dendrites played no part in conducting impulses. Relating Form ana Function

Two examples of Cajal's anatomical studies can exemplify his meticulous approach and clarifying interpretations: 1. Cerebellum. When he began using the Golgi stain, Cajal turned first to the cerebellum. This structure at the base of the brain has bilateral hemispheres displaying a structural pattern evident even by cursory histological approaches. (Indeed, the strikingly regular architecture of the cerebellar cortex remains a beacon to those attempting to decipher behavioral responses in terms of neuronal connections.) Cajal's first depiction (Fig. 1-4A)—a composite reconstruction of transverse sections—shows the prominent dendrites of Purkinje cells rising in a fan toward the cerebellar surface, with the Purkinje axons running in the opposite direction. The figure also shows a series of "descending fringes" from "stellate cells" that envelop the Purkinje cell bodies (these fringes and their cells of origin Kolliker renamed "baskets" and "basket cells"). The projections of other cells are less definitively drawn, but a clearer sense of organization is apparent than in Golgi s view (Fig. 1-4B).

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FIGURE 1-4. Cerebellar cells. A. Cajal's 1888 drawing of the cerebellar cortex stained by the Golgi method, showing (A) Purkinje cell bodies and (B) axons, (C) descending fringe depicted without the Purkinje cell bodies they envelop, and (D) stellate cells with their (L) prolongations. B. Golgi s earlier drawing of the cerebellar cortex, stained by his method, showing Purkinje cells. C. Cajals 1906 diagram of the functional organization of cerebellar cortical cells, showing (A) axonal input to the cerebellum; (B) axon from Purkinje cell; (C) axon from climbing fiber that twists around dendrites of Purkinje cells (seen edge on); (a) granule cell; (b) basket cell; and (c) basket of basket cell around Purkinje cell body. D. Cajals 1909 drawing of climbing fibers and baskets around Purkinje cells, from Golgi-stained sections of two-month-old guinea pig brain, showing (A) basket cell axon; (B) basket cell; (C) climbing fiber passing over Purkinje cell body to its dendrites; (a) and (b) fibers forming "nests" around basket cells; and (c) fibers forming baskets. (A and B are from Shepherd [1991], Figs. 19 and 12; C from Ramon y Cajal [1967], Fig. 5; D from Ramon y Cajal [1995], Fig. 21.)

For his Nobel lecture of 1906, however, Cajal filled out that scheme with representative diagrams (Fig. 1-4C), showing not only the baskets covering Purkinje cell bodies but also ascending fibers (arising from cell bodies outside the cerebellum) "wrap[ping] around the ascending trunk of the Purkinje cell like creepers along the branches of a tropical tree" (here the fan of Purkinje cell dendrites is shown edge-on, in a longitudinal section).41 In addition, his diagram includes axons of "granule cells" rising toward the cerebellar surface, where they split: running longitudinally as "parallel fibers" to contact the dendrites of Purkinje and basket cells. The granule cells themselves receive axons called "mossy fibers"

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(arising from cell bodies outside the cerebellum). Less diagrammatic views were shown in CajaFs monumental atlas published in French translation in 1909: Fig. l^D illustrates again the ascending fibers and the baskets enveloping Purkinje cells. From these images could be divined cerebellar inputs coming through ascending and mossy fibers, impinging on Purkinje cells whose axons provide the sole output; modulating those interactions were intracerebellar basket and granule cells. It should be stressed again that Cajal s figures are composites. The Golgi stain reveals few cells. Only from extensive examinations of repeated preparations could the full picture be constructed. This approach demanded Cajal's essential characteristics: patience, persistence, care, and comprehension. So endowed, Cajal could see what others overlooked. For example, Cajal drew spines—perpendicular spikes—on Purkinje cell dendrites, whereas Golgi had drawn smooth dendrites (although for his 1906 Nobel lecture Golgi added spines but in an "unrealistic drawing that was clearly not copied from nature").42 2. Spinal cord. Cajal also examined the spinal cord in his early studies. Although its organization is less apparent, the anatomy, when interpreted by Cajal, illuminated the function. But before describing Cajal's images and interpretations, a brief look at human neuroanatomy may be helpful (Fig. 1-5). Nerves—bundles of conducting fibers—carry sensory information to the central nervous system (brain plus spinal cord) from the rest of the body. They also carry motor commands to effector organs in the periphery, such as muscles and glands. These nerves are paired, innervating the right and left halves of the body; 12 pairs of "cranial nerves" arise from the brain and 30 pairs of "spinal nerves" from the spinal cord. These latter, which are of interest here, run out between the vertebrae of the spinal column (8 cervical, 12 thoracic, 5 lumbar, and 5 sacral pairs) and then branch to innervate the body. Each, however, emerges from the spinal cord as two branches that quickly merge to form the nerve (Fig. 1-5B). The branch that is anterior in humans (but ventral in animals moving on four legs and called therefore the "ventral root") contains motor "efferent fibers": those carrying outgoing commands to effector organs. The branch that is posterior in humans (dorsal in quadrupedal animals and thus the "dorsal root") contains sensory "afferent fibers": those carrying incoming sensory information. Each dorsal root also contains a bulbous swelling filled with nerve cell bodies, the "dorsal root ganglion." These structural and functional relations were established in the first half of the nineteenth century due to the studies of, among others, Charles Bell in London and, more importantly, Francois Magendie in Paris; the

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FIGURE 1-5. Spinal cord and nerves. A. Longitudinal section through brain, spinal cord, and vertebrae showing spinal nerves emerging between the vertebrae. B. Cross section of the spinal cord showing dorsal roots, ventral roots, and the dorsal root ganglion. C. CajaFs drawing comparing bipolar sensory cell of a fish (above) with unipolar sensory cell of a mammal (below): (C) dendrite; (e) cell body in ganglion; (c) spinal cord, and (D) axodendritic process. (C is from Ramon y Cajal [1937], Fig. 49, courtesy of the American Philosophical Society.)

distinction in function between the two roots became known as the Law of Bell and Magendie. What happened to the fibers after they entered the spinal cord and how they functioned in "spinal reflexes" then became a major concern in the second half of that century. Golgi's view (Fig. 1-6A, as drawn by Cajal) depicted sensory afferent fibers of the dorsal roots entering the "dorsal horn" of the spinal cord gray matter. There, as axis cylinders, they gave off numerous collaterals contributing to the reticulum. The motor efferent fibers leaving through the ventral roots Golgi identified as axis cylinders of large cells in the "ven-

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tral horn" of the spinal cord gray matter ("ventral horn cells"). Since Golgi believed that protoplasmic processes served only nutritive roles, connections with the reticulum were through branching collaterals of the axis cylinders from these ventral horn cells. Thus, the conducting pathway, sensory to motor, ran: axis cylinders from the afferent fibers to reticulum to axis cylinders of the ventral horn cells via their collaterals. Not only did that pathway contrast with CajaFs Principle of Dynamic Polarization (enunciated subsequently), but the totality of its connectivities offered no clues to the subtleties of spinal reflexes then being examined. CajaFs view, by contrast, provided alternative pathways accommodating distinct functional responses, as well as exemplifying the uniform pattern of impulse conduction from axon terminal of one neuron to dendrite and/or cell body of the next. For example, Fig. 1-6B-D, from CajaFs massive treatise, distinguished three pathways for three types of responses. A. For direct, local, unilateral reflexes, Cajal showed impulses flowing from the periphery through afferent sensory neurons having their cell bodies in the dorsal root ganglia and axons terminating directly on the dendrites and cell bodies of the ventral horn neurons; the axons of these motoneurons then innervated the muscles on the same half of the body (Fig. 1-6B). The axons of the dorsal root neurons split on entering the spinal cord, however, with major branches ascending and descending. Consequently, the dorsal root ganglion cell of one segment had terminations directly on ventral horn motoneurons a few segments above and below its level of entry.

FIGURE 1-6. Cellular relationships in the spinal cord. In these cross sections the dorsal direction is to the right. A. CajaFs 1894 drawing of Golgi's view of the spinal cord; (s) is the sensory input and (m) the motor output, linked by a reticulum. B. CajaFs 1909 drawing of the cellular organization for direct, local, unilateral reflexes: (P) are the sensory endings in the skin; (G) is the dorsal root ganglion cell body; (C) is the spinal cord; (b) are the branches of the dorsal root ganglion cell axon; (d) is the ventral horn motoneuron; and (M) are nerve endings of the motoneuron axons on muscle. Arrows show the direction that impulses travel. C. Corresponding diagram for indirect, diffuse, unilateral reflexes: (A) is the dorsal root ganglion cell body; it makes contact with the cell body and dendrites of an interneuron; (B) is the ventral horn motoneuron cell body and dendrites receiving impulses from the interneuron. D. Corresponding diagram for crossed reflexes: (B) is the axon of a dorsal root ganglion cell whose axon branches within the spinal cord, making contact with an interneuron (top) that sends its axon to the opposite side of the spinal cord, where it makes contact with a ventral horn motoneuron. (A is from Ramon y Cajal [1894], Fig. 2, courtesy of the Royal Society. B, C, and D are from Ramon y Cajal [1995], Figs. 209, 210, 211.)

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B. For indirect, diffuse, unilateral reflexes, Cajal showed the dorsal root ganglion neurons terminating also—through other branches—on neurons within the spinal cord gray matter (Fig. 1-6C). These "interneurons" then sent longer ascending and descending branches that terminated on ventral horn motoneurons. Thus, by passing through interneurons, the impulses from the periphery were spread to innervate additional units. Moreover, the pathway could be modulated by other inputs affecting the interneurons. C. For crossed reflexes, Cajal showed afferent dorsal root ganglion neurons terminating also on still other interneurons; the axons of these interneurons then crossed to the opposite half of the spinal cord before terminating on ventral horn motoneurons there (Fig. 1-6D). By this pathway, stimulation on one side of the body could cause responses in the other half. Cajal's neuroanatomy included far more intricacies, identifying a half dozen cell types in the gray matter of the spinal cord and following myelinated axons through specific tracts in the white matter. Moreover, he pursued such detailed examinations throughout the nervous system.

Continuations, Criticisms, ana Responses

Within a few years of Cajal's visit to Berlin, a number of leading neuroanatomists confirmed his results with the Golgi stain and joined Waldeyer in affirming the Neuron Theory. Cajal was invited to present the Croonian Lecture to the Royal Society in 1894; that same year a synopsis of his results was published in French.43 In 1899 Lewellys Barker in Baltimore persuasively summarized the Neuron Theory in a book that swayed the English-speaking world.44 And in 1906 Cajal shared the Nobel Prize for Physiology or Medicine with Golgi.45 This was a curious juxtaposition, since their acceptance lectures are mutually contradictory. Where Cajal saw processes end, as in the baskets around Purkinje cell bodies (Fig. 1-7A), Golgi saw—amid the profusion of images—those fibers continuing onward (Fig. 1-7B). It was not just Golgi who resisted Cajal's interpretations, however. Persisting objections and criticisms can be grouped into five categories.

Fixing and Staining Artiiacts

What does a real neuron look like? Efforts to answer this question must avoid an array of potential artifacts, from optical distortions, to fixation artifacts during conversion of live cells into durable forms for sectioning, to staining arti-

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FIGURE 1-7. Alternative views of the baskets around cerebellar Purkinje cells. A. Cajal s 1909 drawing of the cerebellar cortex of a 20-day-old cat stained by the Golgi method, showing: large Purkinje cell bodies with their extensive dendritic branchings, and baskets enclosing the cell bodies (not shown) arising from horizontal axons of basket cells. B. Golgi s 1906 drawing of baskets, showing the fibers passing from the region of the Purkinje cell bodies to join the reticulum below. (A is from Ramon y Cajal [1995], Fig. 7. B is from Golgi [1967], Fig. 2.)

facts that can alter, blur, and conceal. Thus, an obvious problem in arguing for discontinuity is that the preexisting continuity may have been lost during processing. Alex Hill in Cambridge, a harsh critic of CajaFs viewpoint, complained in 1896 that Cajal "has no right to conclude that the position of structures in hardened and shrunken tissues is the position which they occupy during life."46 The threat of damage during fixation is, of course, a perennial problem in microscopy. A conventional precaution is to try alternative modes of fixation in the hope that a consistent result from multiple approaches signifies the natural state. Similar concerns about staining were raised as well, especially in the early years, when the Golgi stain alone gave such clear images. Cajal felt that the Golgi stain represented the entire cell. But "understated" preparations failed to show the processes as fully, whereas "overstained" preparations revealed further images. How could one be certain that the edge of the stain was the edge of the cell? Hill questioned whether the staining might "stop short at the edge of a favourable zone, giving an incomplete picture of the elements which it colours," and he protested that "IT DOES NOT FOLLOW THAT BECAUSE THE [AXON TERMINALS] CANNOT BE FOLLOWED [FURTHER

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THAT] THEY END."47 Moreover, since only a few of the cells present are stained, Hill questioned "what is proved by a stain which picks out one cellsystem and leaves a number of similar cells uncoloured" since the uncolored cells could be in protoplasmic continuity?48 The profusion of images also reinforced skeptics' doubts about just where— in an era when the cell membrane was doubted by some and not visible to any—cells terminate. What Cajal could do, however, was show that a quite different stain gave similar results. In 1896 he began using Ehrlichs methylene blue stain, producing the same picture of axons and dendrites terminating freely (and confirming the existence of dendritic spines as well). This corroboration was particularly significant because methylene blue did not stain merely a tiny fraction of the cells, it stained myelinated axons as well as unmyelinated ones, it differed chemically so that interactions with the cells would be through different means (as opposed, say, to adventitious deposits of silver salts), and it was applied to living tissues as a "vital stain." Still, arguments would persist as long as two uncertainties remained: what constitutes the margin of a cell, and how can that margin be identified microscopically? (Subsequent studies on the Golgi stain, using both continuous examination by light microscopy to follow the progress of staining and electron microscopy to define where the silver chromate crystals are, support CajaPs interpretations. Precipitation begins at "nucleation centers" in the cytoplasm and continues to fill the protoplasm. Unlike earlier conclusions that silver salts coated the surface, these results show that neurons are filled with the salts, which do not escape across the cell membrane.49)

Discontinuity

A crucial issue for the Neuron Theory was the meaning of and evidence for "discontinuity." The Golgi stain showed no indication of continuity. On the other hand, the Golgi stain did not show cell contacts either: the model of axon terminals making contact with dendrites and cell bodies was constructed from multiple images of various cells seen separately. The gaps that Cajal drew (e.g., Figs. 1.6B-C) were diagrammatic (and considerably larger than those later demonstrated by electron microscopy). Some investigators, moreover, continued to see continuity. Hans Held in Leipzig, using other stains, described in 1897 axon terminals ending in contact with cell bodies in the embryo but in adults forming a "delicate histological relationship between the axis cylinder and the protoplasm of the enveloped nerve cell" with "the very dense, thinnest parts of protoplasm . . . fused together."50 Cajal countered with claims that he could see no protoplasmic anastomoses; toward the end of his life he summed up his conclusions, avowing "what I have seen during fifty years of work and what any observer who is free

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from prejudice of a doctrine can easily verify, not by [relying on] this or that nerve cell, perhaps badly fixed or of an abnormal type, but instead on millions of neurons deeply stained by different methods."51 More serious to many at the time were the claims by Stefan Apathy in Naples and Albrecht Bethe in Strassburg. They described "neurofibrils"—fine filaments in the dendrites, cell bodies, and axons of gold- or toluidine blue-stained tissue—that crossed from the axon terminal of one neuron to the dendrite or cell body of another.52 Continuity, according to Apathy and Bethe, was through these fibrils. Cajal, on the other hand, could see fibrils, but not fibrils crossing from one neuron to another: "In vain I toiled in search of the external course of the delicate filaments."53 Cajal then developed a silver stain for the fibrils, and examining a range of organisms, including the leeches that Apathy studied, found not "the slightest indication that the neurofilaments pass from one cell to another."54 Nevertheless, claims for continuity through connecting neurofibrils persisted for decades. In part, the controversy lingered because the images were at the limit of resolution by light microscopy. More significant in keeping alive the controversy, however, was the unresolved functional problem of how nerve impulses pass from cell to cell. Those advocating neurofibrillar continuity solved this problem by proposing that neurofibrils were the conducting elements of the nervous system (comparisons were made to muscle fibers, which are the contractile elements of muscle). Thus, Howell's physiology textbook of 1905 likened nerve conduction to transmission by a submarine cable. In each case a conducting "central thread" was surrounded by an insulating sheath. Then, "the central threads are represented by the neurofibrils . . . and the surrounding sheath by the perifibrillar substance."55 But if the neurofibrils did not play a central role in conducting impulses from one neuron to another, then their linking one neuron to another would seem of little importance.

Impulse Conduction

The Neuron Theory proclaimed the structural individuality of cells within the nervous system, with impulses passing from neuron to neuron. Forel likened that process to interlaced limbs in a forest conveying an impetus from tree to tree.56 Less picturesquely, Cajal proposed that "current must be transmitted from one cell to another by way of contiguity or contact, as in the splicing of two telegraph wires."5' Just such an image he drew in the ascending fibers twining about Purkinje cell dendrites (Fig. 1-4C). This emphasis on functional contact, however, contradicts the diagrammatic gaps Cajal drew when emphasizing morphological discontinuity (Fig. 1-6B-D), although he did consider that electrical conduction might cross a gap "by an induction effect, as in indue-

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tion coils."58 Cajal also imagined a "granular cement, or special conducting substance" providing physical and conductive integrity at the contact zone.59 But without a clearer sense of how nerve impulses travel, Cajal s suggestions are no less vague than notions of conducting neurofilaments linking neuron to neuron. Golgi imagined the reticulum linking units together so that "one single fibre may have connections with an infinite number of nerve cells."60 Such connections affirmed Golgi's opposition to the doctrine of "cerebral localization," which delegated particular functions to particular areas of the brain.61 "The concept of so-called location of the cerebral functions . . . would not be in perfect harmony with the anatomical data[, for with the elements] conjoined by means of a diffuse network . . . it is naturally difficult to understand a rigorous functional localization."62 Thus, Golgi conceived of the diffuse reticulum as a "nerve organ" to which "every nerve element of the central nervous system contributes," concluding that he could not "abandon the idea of a unitary action of the nervous system."63 Cajal caricatured Golgi's holistic view as an "unfathomable physiological sea into which . . . pour the streams arriving from the sense organs, and from which . . . the motor . . . conductors were supposed to spring like rivers originating in motor lakes."64 Cajals harsh conclusion was that "the reticulum hypothesis, by dint of pretending to explain everything easily and simply, explains absolutely nothing."65 On the other hand, Cajals delineation of broadly branching processes—as in the ascending and descending branches of dorsal root ganglion axons (Fig. 1-6B-D)—implied generalized responses, whereas Golgi admitted a fuzzy sort of localization in "territories [whose] nerve fibres coming from, or going to, the periphery . . . have a more direct and intimate connection . . . than . . . those at some distance from them [and with] those territories slowly merg[ing] with other regions where other bundles of fibres prevail."66 Both Cajal and Golgi faced the dilemma of discrete sensory inputs causing discrete motor responses (as in direct spinal reflexes) and of other discrete sensory inputs causing complex responses (as reacting to painful stimuli by withdrawal plus strong emotions). A related issue concerned changeable responses, as must occur in learning. Matthias Duval argued that if the reticulum were fixed "it is difficult to understand how practice makes certain . . . acts that are difficult to learn (such as ... playing a musical instrument) so easy to perform."67 Instead, Duval imagined the sites of contact to be "malleable," with the junctions along a chain of neurons serving as a "series of switches."68 Nevertheless, the neurofibrillary proposal could include the notion that impulses passed along particular conducting strands within a vast reticulum and that the conductivities over specific fibrils could be altered by prior experience. Indeed, Hill elaborated a mechanism by

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which the first impulse conducted by an interneuronal fibril "creates an open road along which subsequent impulses [then] pass with ease."69

Development ana Regeneration

As noted above, His formulated one of the early arguments for the Neuron Theory from studying the embryological development of nerve cells, particularly the sequential growth of their processes: observing first the axon and then the dendrites growing out from the cell body. By the end of the nineteenth century, Cajal, using the Golgi stain, had confirmed and extended these observations, in particular describing the growth cone at the end of the developing axon.70 This point of view—that the cell body plus processes represented developmentally a single cellular unit—Cajal referred to as the "monogenist hypothesis."71 A rival view, which he termed the "polygenist hypothesis," considered nerve fibers to be formed through fusing a chain of primordial cells. Although polygenist views had been prominent before His and Cajal, their new evidence, in conjunction with analogous studies by Retzius, Lenhossek, and others, seemed compelling. Moreover, confirmation also came from a dramatic new approach. In 1907 Ross Harrison in New Haven described the growth of axons in vitro, a pioneering success in what became the field of cell and tissue culture.72 Harrison first removed a tiny fragment of a frog embryo destined to give rise to nerve fibers and placed that tissue on a cover slip in a drop of frog lymph; when the lymph clotted it trapped the tissue on the cover slip. Harrison next placed the cover slip with adhering tissue over the concavity of a microscope slide and sealed the edges. With aseptic conditions the tissue survived for a week or more, allowing him to observe continuously the development of individual processes (unlike Cajal, who had to construct successive events from studying a series of embryos killed at successive times in their development). What Harrison then saw were the naked fibers growing out from the mass of tissue into the surrounding cell-free clot: an extension of the nerve process rather than a fusion of preexisting elements. Harrison could also distinguish the terminal growth cone, which—as Cajal had inferred from a series of static images—was moving in amoeboid fashion. Despite those demonstrations of a monogenist embryological development, opponents continued their advocacy of polygenist views by focusing instead on nerve regeneration: after a nerve is cut the processes peripheral to the injury first degenerate and later regenerate. The monogenist view held that axons regenerated by "sprouting" from the central stump of the cut nerve; these sprouts then grew from the stump along the course of the degenerated peripheral portion. The rival polygenist view, as exemplified notably by Bethe,73 argued that the regenerated axon arose instead from the chain of cells surrounding its former course. (Schwann had described a series of bodies—

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subsequently named "Schwann cells"—that formed a sheath about myelinated nerve fibers. A prominent proposal from the polygenist camp was that Schwann cells gave rise to the regenerated axon.) Evidence in favor of the polygenist argument included: (1) physiological studies showing that, early in the course of regeneration, electrical stimulation peripheral to the cut caused motor response, whereas electrical stimulation central to the cut did not; (2) inability to observe microscopically at that time the continuation of fibers across the cut region; (3) observation of Schwann cells along the course of regeneration; and (4) regeneration despite mechanical blocks between the central stump and the peripheral course (a block, for example, created by folding the cut central end back and suturing it to a nearby structure far from the site it formerly innervated).74 Cajal, who found it inconceivable that regeneration would differ so fundamentally from development, attacked the morphological arguments vigorously in the 1900s/5 At that time he developed a staining procedure, the reduced silver nitrate method, that allowed him to detect processes that Bethe's approach could not. Using this stain, Cajal then described two sites of axonal regeneration: by sprouting from the cut central stump, and by sprouting from the axon more centrally, as if forming new collaterals. Extensions of these sprouts Cajal could then see crossing the cut region and invading the degenerated peripheral course. Moreover, he found that these sproutings next became naked axons, coursing before any proliferation of Schwann cells; only later did Schwann cells form a sheath and the new axons become myelinated. When Cajal imposed a porous barrier between the central stump and peripheral destination, such as blotting paper or cork, he saw new fibers growing through the barrier. When he folded back the cut end of a nerve and sutured it out of place, he still saw new fibers invading the peripheral course, but in this case they sprouted as collaterals from the nerve before its fold. The obvious conclusions were that sprouts arose more prevalently and diffusely from the central portion of the nerve than the polygenist opponents had imagined, and that these sprouts then coursed more widely in avoiding obstacles. These conclusions sprang from the use of a new staining technique coupled with Cajal's skill, effort, and determination. (Cajal did not reexamine Bethe's physiological studies stimulating peripheral and central regions, although he remarked that stimulation of uncut collaterals—arising either from different nerves or from the cut nerve central to the point where Bethe stimulated— could produce the responses attributed by Bethe to regeneration of an unconnected peripheral segment.) An unanswered question pervading these studies of development and regeneration, however, was what guided the growing fibers to their proper destinations. Cajal suggested "neurotropic" factors produced by Schwann cells and/or the end organs ultimately innervated. But the nature of those factors and the mechanisms by which they directed growth remained unidentified.

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Anomalies

Two instances of apparent anomalies may suffice to illustrate ways that Cajal met contradictions to key elements of his formulation. The first concerns the Principle of Dynamic Polarization. As originally proposed in 1891, it stipulated that impulses pass in dendrites toward the cell body and in axons away from the cell body. '6 But applying this characterization to a prominent cell type, neurons of the dorsal root ganglia (Fig. 1-5C), presented two problems. What in this neuron is a dendrite? And need the impulse pass through the cell body before entering the axon? The questions arose because these cells deviate strikingly from prototypic neurons, such as Purkinje cells. Instead of a bipolar configuration, with dendrites arising from one pole of the cell body and the axon from another, neurons of the dorsal root ganglia in mammals are unipolar: a single process extends from the periphery to its prominent branching within the spinal cord, with the cell body attached by a stalk part way along that course. Cajal resolved the issue by declaring the peripheral segment, extending outward from the cell body stalk, to be a dendrite even though it had "all the structural and morphological characters of the axis cylinder."7' That identification he justified on developmental and phylogenetic grounds. As His had shown previously, neurons of the dorsal root ganglia begin in the embryo as bipolar cells having a peripheral dendritic process and a central axon; during development, however, the peripheral segment assumes the appearance of an axon and the cell body becomes separated by a stalk, achieving the unipolar configuration. Moreover, as His had also noted previously, the dorsal root neurons of lower vertebrates, such as fish, remain bipolar in adults. Since the conduction pathway in a unipolar neuron would be more direct if it bypassed the cell body, Cajal proposed—without direct evidence—that such was the case. In 1897 he revised the Principle of Dynamic Polarization accordingly: dendrites conduct impulses toward the axon, and axons conduct impulses away from the dendrites.78 (Cajal promulgated this formulation after physiologists had shown that both axons and dendrites could, when stimulated at a point away from their termini, conduct impulses in both directions. Contradictions may be avoided and the bidirectionality accommodated, however, by postulating that dendrites contain receptive elements at their termini, so impulses would then be conducted away from those termini, whereas axons contain transmitting elements at their termini. Identifying and characterizing these two classes of elements then became a major quest, as subsequent chapters will describe.) The second instance also involves aberrant morphology, again as deviations from a prototypic form. Cajal described certain cells in the retina, called amacrine cells, that contained no dendrites; he could easily concede, in accord with his Principle, that "axonal arborizations are applied only to the surface of the cell body."79 But even though earlier investigators had described "horizontal

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cells" of the retina lacking axons, Cajal found only horizontal cells having axons, attributing contradictory accounts to failures in staining an axon that surely must be present. Nevertheless, subsequent neuroanatomists described two classes of horizontal cells, those with and those without axons; Marco Piccolino concluded that "Cajal 'saw' only axon-bearing horizontal cells because [only] these conformed well" to CajaPs formulations.80

onclusions

Cajal was born in a remote Spanish village and was uprooted during his childhood to successively larger towns as his fathers medical practice grew. His first ambition was art, a career that clashed with his father's expectations of his son. Rebellious youth faded only when Cajal discovered the satisfactions of anatomical study, although that schooling sparked continuing conflicts, as in his repudiating the vitalist views of some professors. And Cajal, a provincial socially as well as academically, failed at Madrid in his first bid for a professorship. Coming thus from a country one contemporary dismissed as "remarkable for its barrenness in original research,"81 how did Cajal reach the pinnacle of scientific greatness? He toiled relentlessly. He developed techniques and used them wisely, choosing specimens broadly and pursuing comparative, developmental, and functional correlations. He enlisted his considerable artistic talents and promoted his conclusions tirelessly. He persevered in his ambition.82 He grasped similarities astutely,83 and he believed fervently in "the unity of biological laws,"84 which assured discoverable generalities. And he concluded that biology displays a "unity of plan with infinite variety of forms,"85 allowing him to extract prototypic classes inclusive of their variants. Cajal, of course, did not labor alone. His work was extended as well as confirmed by allies, whom he courted. He developed a strong and able following in Spain, including his brother Pedro. Although an ardent nationalist himself, he was not burdened with inherited doctrines like those contemporaries from countries endowed by past accomplishments. And his work was sharpened by the criticisms of opponents, whom he did not shrink from criticizing in return. Some opposition he attributed to "the feverish thirst for novelty [and] the suggestion of fashionable theories," and he noted that some "young enthusiasts [were] as eager for reputation as they were uncritical in observation."86 Among those criticisms, however, lurked fundamental disagreements of what was seen and what was absent and therefore not seeable.87 Thus, Bethe failed to see (and denied the existence of) regenerating sprouts, and Cajal failed to see horizontal cells without axons. Conversely, Apathy saw neurofilaments con-

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necting cells (which Cajal failed to see and denied the existence of), and Cajal saw tight contacts and dismissed continuity. In retrospect, errors may be attributed to insufficient resolution by light microscopy, inadequate fixing and staining, and confounding complexity. At the time, progress was achieved through bolstering morphological uncertainties with developmental, phylogenetic, regenerative, and functional arguments. Hence, in the two decades following Cajal's mission to Berlin, a flourishing research program was established, rooted in the neuron, the fundamental morphological and functional unit of the nervous system. The image of a prototypic neuron—with receptive dendrites and a transmitting axon—left unresolved, however, crucial uncertainties about where and how cell margins end and about how nerve impulses pass along the chains of neurons making up the nervous system. Notes 1. For historical accounts—in many cases pertinent, as well, to other topics—see Cannon (1949); Clarke and Jacyna (1987); Clarke and O'Malley (1968); Finger (1994, 2000); Glynn (1999); Jacobson (1994); Jones (1994); Meyer (1971); Shepherd (1991), as well as Ramon y Cajal (1937). He and others frequently simplified his surname to Cajal, but bibliographers prefer the full surname, Ramon y Cajal. Earlier, Cajal had served as an army physician in Cuba, where he contracted malaria and tuberculosis; on his return, he was able with his back pay to purchase his first microscope. 2. The Revista Trimestral de Histologia normal y patologica. He also illustrated this journal with his own lithographs. 3. Other stops on this tour included Lyons, Geneva, Frankfurt, Gottingen, and Pavia (where he missed meeting Golgi, who was away). 4. Pi-Suner and Pi-Suner (1936) tell of Cajal dragging Kolliker by the sleeve to his demonstration. 5. Described in Sherrington's foreword to Cannon (1949). 6. For accounts of a more complex history, see Harris (1999); Hughes (1959). 7. Hertwig (1895), pp. 1, 8. 8. For historical accounts of the cell membrane see Baker (1952); Jacobs (1962); Kleinzeller (1995); Smith (1962). For more recent developments, see Robinson (1997). 9. Unfortunately, before the cell membrane of plant and animal cells was identified, the cell wall of plants was frequently termed a "membrane." 10. For example, W. Bowman in 1840 saw through the microscope—and drew—the "sarcolemma" (cell membrane plus adhering fibrous material) around muscle fibers. 11. Verworn (1899), p. 65: the English translation of the 1897 text. 12. Halliburton (1904), p. 5. 13. Kollikers interpretation of Valentin's views, quoted by Shepherd (1991), p. 34. Moreover, Purkinje in 1837 conceded that "nothing definite can be ascertained about the connection between the ganglion corpuscles and the elementary brain . . . fibers" (quoted by Clarke and O'Malley, 1968, p. 55). 14. Kolliker, quoted by Shepherd (1991), p. 34. Previously, Robert Remak had described nonmyelinated fibers originating from the nucleated globules, and others,

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including Hermann von Helmholtz, had described particular instances where fibers arose from cell bodies. 15. Shepherd (1991) attributes the term "axis cylinder" to J. F. Rosenthal in 1839. 16. Previously, unfixed tissue was often macerated in water for viewing, with consequent osmotic distortions. 17. Virchow, quoted by Clarke and O'Malley (1968), p. 86. 18. For example, Kolliker conceded in 1853 that "nerve cells may anastomose," and in 1867 he claimed that "the simplest hypothesis" favors a reticulum "linked by anastomoses" (quoted by Shepherd, 1991, pp. 36, 53, 54). 19. Golgi's initial paper of 1873 was largely overlooked; subsequent papers, beginning in 1883, published also in Italian, attracted more notice. 20. Their views were published in 1887, just as Cajal was beginning his research; however, Cajal was unaware of their work until after he published his initial papers. 21. From Hiss paper of 1877, quoted by Clarke and O'Malley (1968), p. 103. 22. After an axon is transected the peripheral process degenerates; centrally, the cell body may swell and stain abnormally, but generally it recovers and regenerates new peripheral processes. 23. For historical accounts, see Brazier (1959); Clarke and Jacyna (1987); Clarke and O'Malley (1968); Mauro (1969); Piccolino (1997); Tasaki (1959). 24. Galvani had hung the legs on the railing to study their response to an approaching thunderstorm. (He had earlier shown that nearby electrical discharges could trigger contractions.) Subsequently, Galvani placed the frog legs on an iron plate; when a brass hook through the attached spinal cord contacted the plate, the legs moved. 25. Galvani responded by using a single metal rod to connect frog muscle to spinal cord, producing contraction. Volta then claimed that the metal rod was not homogeneous—with bimetallic contacts in the rod itself. 26. Electric fish had been known since antiquity, and both Galvani and Volta studied their properties. Piccolino (1997) notes that Volta constructed his battery on the pattern of stacked units within fish electric organs, and that Volta then interpreted the electric organ as a physical entity. 27. Bernstein (1902). He did not speculate on which ions were involved, although in 1913 he proposed a variable permeability to K + . He did not cite Overton's proposal (below). Neither did Bayliss, who stated that "the only satisfactory way of explaining such electrical states is by the assumption of a membrane which is permeable to one of the ions into which an electrolyte inside the axis cylinder is dissociated, but not permeable to the oppositely charged fellow ion" (1920, p. 392). 28. Overton (1902). He could show no such dependence on extracellular Na+ for nerve, but he suggested that sufficient extracellular Na + could be trapped within the nerve sheath. 29. Howell (1905), p. 113. 30. Lucas (1912), p. 521. Nernsts argument concerned the dependence of nerve excitation on the frequency of stimulation by an alternating current. Lucas also cited a proposal for selective permeability advanced in 1890 by Ostwald. 31. Bayliss (1920). He discussed Macdonald but not Bernstein under the topic of nerve and muscle excitation, although he discussed Bernstein under the topic of cellular potentials (which did not include action potentials). 32. Macdonald (1905). 33. These are summarized and the final article translated in Shepherd (1991). 34. Ramon y Cajal (1937), p. 587; italics in original.

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35. Ibid., pp. 308, 309. Cajal contrasts this with the tendency for investigators generally to use only the methods they (or their teachers) developed. 36. Cajal also included a fixative, osmic acid. 37. Ramon y Cajal (1937), p. 309. 38. Cajal described his method in his first book, translated into French in 1894 and into English in 1990 (Ramon y Cajal, 1990). 39. For a less enthusiastic view of Cajal's contributions, see Jacobson (1994). 40. Cajal's Nobel Lecture of 1906, translated into English (Ramon y Cajal, 1967, p. 221). Cajal reported that he proposed the Principle in embryonic form in 1889, in fuller form in 1892, and in revised form in 1897 (Ramon y Cajal, 1937); van Gehuchten formulated the notion independently, and there was some rivalry about priority. 41. Quotation from Ramon y Cajal (1990), p. 34. 42. Palay and Chan-Palay (1975), p. 52. 43. Ramon y Cajal (1990): the English translation of the French edition of 1894. 44. Barker (1899). Earlier English writers, such as W. A. Turner (1893), also endorsed the Neuron Theory. 45. Golgi's other accomplishments should not be discounted. These include descriptions of two morphological classes of neurons (now called "Golgi type I" and "Golgi type II") and of a significant structure in the cytoplasm (the "golgi apparatus"). 46. Hill (1896), p. 27. Hill was also critical of Golgi's views, particularly of Golgi's relegating dendrites to nutritive functions alone. 47. Ibid., pp. 20, 25; capitals in original. 48. Ibid., p. 27. 49. Blackstad (1965); Chan-Palay and Palay (1972); Spacek (1989); Stell (1965). 50. Held, quoted in Clarke and O'Malley (1968), pp. 120, 121; italics in original. The stains Held used were hematoxylin and erythrosin-methylene blue. 51. Ramon y Cajal paper of 1933, quoted in Clarke and O'Malley (1968), p. 137; italics in original. 52. Apathy (1897); Bethe (1900). For Cajal's harsh opinion of Bethe's preparations see Ramon y Cajal (1937), pp. 519-520. 53. Ramon y Cajal (1937), p. 520. 54. Ibid., p. 563. 55. Howell (1905), p. 114. 56. Forel, quoted in Clarke and O'Malley (1968), p. 106. Forel went on to say: "Electricity gives us ... innumerable examples of similar transmissions without direct continuity." 57. Ramon y Cajal (1990), p. 161. 58. Ramon y Cajal (1937), p. 323. 59. Ramon y Cajal (1967), p. 220. 60. Golgi's Nobel lecture of 1906 (Golgi, 1967, p. 215). 61. See Finger (1994), chapters 3 and 4. 62. Golgi paper of 1883, quoted by Finger (1994), p. 53; italics in original. 63. Golgi (1967), pp. 193, 216. 64. Ramon y Cajal (1937), p. 336. 65. Ibid., p. 337. 66. Golgi (1967), pp. 215-216. 67. In afterword to Ramon y Cajal (1990), p. 192. Correspondingly, Cajal attributed "genius" to the richness of neuronal contacts (Ramon y Cajal, 1937, p. 459). 68. Ibid.

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69. Hill (1900), p. 685. 70. See Ramon y Cajal (1937), chapter 7. The growth cone was not visible with His's staining. 71. Ramon y Cajal (1991), a newly edited translation from the French edition of 1909-1911. 72. Harrison (1907); a fuller account is Harrison (1908). 73. For references to Bethe, see Ramon y Cajal (1991), p. 11. 74. Summarized in Ramon y Cajal (1991). 75. Ibid. 76. See Ramon y Cajal (1937). 77. Ibid., p. 385. 78. Ibid. 79. Ramon y Cajal (1990), p. 161. 80. Piccolino (1988). 81. Barker (1899), p. 20. 82. Cajal was not overly generous in evaluating the contributions of others; thus, he characterized His and Forel as "timidly suggesting" that nerve cells were discrete entities (Ramon y Cajal, 1937, p. 334). 83. Sherrington reported that when Cajal included a specimen from birds to illustrate pyramidal tracts of the spinal cord, he protested that birds did not have pyramidal cells; Cajal responded, "Bien; c'est la meme chose." (In Cannon, 1949, p. xiii). 84. From Cajal's autobiography, translated in Ramon y Cajal (1991), p. 31. 85. Ramon y Cajal (1937), p. 433. 86. Ramon y Cajal (1991), p. 16; (1937), p. 536. 87. In a paper of 1888, Cajal wrote that his failure to see anastomoses of neural processes "did not deny indirect anastomoses," but "having never seen them, we dismiss them from our opinion" (in Clarke and O'Malley, 1968, p. 112). An impasse can occur when one individual claims to see something that another fails to see; however, for the positive case Cajal concluded that "the repeated observation of a particular histological feature in a number of different preparations is the absolute guarantee of its reality" (Ramon y Cajal, 1990, p. 173). The seeing or not seeing by others can be weighed. Probably more beneficial in such cases is the marshalling of other lines of evidence.

2 BEGINNINGS: SHERRINGTON AND THE SYNAPSE (1890-1913)

Snerrington, Reilexes, and trie Synapse

When Santiago Ramon y Cajal journeyed to London in 1894 to deliver his Croonian Lecture, he stayed for two weeks at the house of Charles Scott Sherrington (Fig. 2-1 ).1 Sherrington, then 37, was five years younger than Cajal and quiet and reserved, whereas Cajal was voluble and dramatic. Both, however, were careful, imaginative, and tireless investigators, and Sherrington's own studies were advancing on a complementary course. Before completing his medical training at St. Thomas's Hospital in London, Sherrington had studied at Cambridge. There he came under the influence of John Newport Langley and Walter Gaskell in the Physiological Laboratory, and they instilled in him an appreciation of how biological structure underlies function. Initially, Sherrington's interests focused on cellular pathology and bacteriology. (He worked in Spain during the cholera outbreak of 1885,2 when Cajal's efforts were rewarded with a Zeiss microscope, and he studied with Rudolph Virchow and Robert Koch in Berlin.) He had also worked on the nervous system with Friedrich Goltz in Strassburg. And in 1890, following Gaskell's advice, Sherrington began to examine spinal reflexes: functional studies interpretable in terms of discrete neurons arranged in the distinct pathways that Cajal was demonstrating histologically. 31

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FIGURE 2-1. Charles Scott Sherrington (1857-1952; photograph by Louis Cobbett).

By 1894 Sherrington had been elected to the Royal Society and was a lecturer in physiology at St. Thomas's Hospital and physician-superintendant of the Brown Institute (an institute for research on animal diseases affiliated with the University of London); in 1895 he was named professor of physiology in Liverpool, where he remained until his appointment at Oxford in 1913. Some relevant aspects of Sherrington s work in London and Liverpool I will note later; first, however, I wish to celebrate the term synapse, which—after some prompting—Sherrington introduced. While preparing the seventh edition of his multivolume textbook of physiology, Michael Foster, the eminent professor of physiology in Cambridge,

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sought out Sherrington to write the section on neurophysiology. That challenge pushed Sherrington to recognize the need for a convenient name for the junction between neurons, Cajal's point of contact without continuity. Earlier Sherrington had used the term adjunction;3 he now offered syndesm, from the Greek for a bond.4 Foster, however, consulted a Greek scholar in Cambridge who suggested instead synapsis, which means the process of contacting, because it formed a better adjective. The 1897 edition of Foster's textbook then proposed, with customary diffidence: we are led to think that the tip of [an axon] is not continuous with but merely in contact with the substance of the dendrite or cell body on which it impinges. Such a special connection of one nerve cell with another might be called a synapsis.5

Shortened to synapse, that term was soon accepted widely.

Background.: Reflexes

Reflexes—involuntary, automatic, but characteristic responses to particular stimuli—represent a fundamental physiological phenomenon of major theoretical interest.6 The issue here, however, is how their study influenced contemplations of synaptic transmission. Still, a brief listing of some steps prior to Sherrington's efforts seems pertinent. Greek physicians and philosophers developed the notion of "sympathies," nonmaterial psychic principles that evoked bodily responses to perturbations locally or even distantly. But in the seventeenth century Rene Descartes extended his mechanistic formulations to include sensory impressions being "reflected," like light, back to the muscles. Descartes imagined that sensation and execution traveled through the same nerve; at that time the spinal cord was considered to be merely a collection of nerves descending from the brain. A century later Robert Whytt in Edinburgh added experimental demonstrations that reflections occurred in the spinal cord: he showed that certain responses, such as movement of the leg after local irritation, persisted in decapitated frogs; conversely, destruction of the spinal cord (by passing a needle down the spinal canal) abolished these responses.7 Whytt, moreover, described what later was termed "spinal shock," a diminished response immediately after transection of the spinal cord; thus, Whytt noted that a decapitated frog would not respond to stimulations for 15 minutes or so. In 1812 Julien Legallois in Paris described a "segmental" organization of the spinal cord. After transecting the cord at successive levels, he found that specific areas of sensation and movement were affected. Legallois concluded that

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each segment—associated with a pair of nerves emerging from the spinal cord—represented a distinct, responsive center. Beginning in the 1830s Marshall Hall in London extended Whytt's observations, establishing—and naming—the "reflex arc": an ingoing path to the central nervous system and an outgoing path to muscle. His arc thus corresponded with the recent discoveries of Charles Bell and Francois Magendie allocating sensory inputs to the spinal cord through dorsal roots and motor outputs through ventral roots. Hall also mapped responses relative to the site at which he transected the spinal cord, finding that sensory inputs may evoke responses beyond their segment. And he included responses of voluntary systems, such as skeletal musculature, as well as of involuntary and complex systems, such as sneezing and coughing. Hall distinguished reflex action from conscious volition, although he recognized that reflex responses could be modified by volition. In the latter half of the nineteenth century, attention was diverted by protracted debates over a "spinal soul," an entity granting conscious sensation to the spinal cord; principal protagonists were Eduard Pfliiger in Bonn (physiologist, pro) and Rudolf Lotze in Gottingen (philosopher, con). According to Pfliiger, the spinal "soul" was one with the conscious, volitional "soul" of the brain; after transection there were then two "souls," one on each side of the cut. More productive were the studies in 1845 by the brothers Ernst and Eduard Weber in Leipzig, who described a slowing of the heart after electrically stimulating the brain. They traced this effect to the base of the brain and then to a specific cranial nerve, the vagus, that emerges there to innervate the heart (among other structures). Soon afterward Pfliiger identified nerves that, when stimulated, slowed intestinal peristalsis; other apparently inhibitory nerves to the viscera were soon described. Nevertheless, Goltz argued in 1863 against specifically inhibitory nerves, and attempts to demonstrate inhibitory nerves to skeletal muscle were unsuccessful. In the 1860s Ivan Setchenov, a Russian physiologist visiting Claude Bernard in Paris, showed that the time delay in responses—such as the interval between presenting a noxious stimulus to a leg and that leg's movement—could be prolonged reversibly by chemical stimulation of particular regions of the frog brain. Setchenov argued for master inhibitory centers in the brain. Others, such as Moritz Schiff in Florence, found that strong stimulation at multiple sites, peripherally as well as centrally, could produce inhibition; he claimed that inhibition arose in each spinal segment. Thus, as the nineteenth century was closing, a mass of fragmentary data was interpreted through opposing hypotheses. In retrospect, these disputes reflect a host of problems, including nonuniform experimental approaches (e.g., differences in stimulus intensity, duration, and mode) and inappropriate conceptual schemes (e.g., irrelevant criteria, such as classifying reflexes into superfi-

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cial vs. deep). Moreover, vast areas of ignorance, anatomical as well as physiological, prevented sharp distinctions between rival claims.

Snerringfton's Achievements

A half century after his death in 1952, Sherrington s influence continues to pervade the field of neuroscience. Like Cajal, he remains a heroic figure, having seen further and more clearly than his contemporaries and having assimilated those glimpses into a whole, as specified in the title to his Silliman Lectures of 1904, The Integrative Action of the Nervous System? Integration, Sherrington concluded, "welds . . . together from its components . . . an animal individual."9 Although other means, such as chemicals circulating in the bloodstream, may participate in coordinating this unity, Sherrington judged the nervous system—by virtue of its rapid conductance and broad distribution—to be the principal agent. Sherringtons course to that conclusion began through analyzing motor behavior into its fundamental units. These he deemed to be the simple reflexes, dependent on particular nerve cells and their connections. The daunting complexity was thus resolved into receptors (sensing some stimulus), conducting pathways (at least two neurons, transmitting impulses to and from the central nervous system), and effectors (then responding). Sherrington, nevertheless, recognized that such simple reflexes were abstractions, for the pathways could be modified by other neural systems impinging on them. Individual reflexes were subject to hierarchical levels of control that mold complex behaviors through coordinating, suppressing, and enhancing individual reflexes. His lectures displayed this viewpoint through a wealth of experimental observations unified by broad generalizations, summarizing a decade and a half of extraordinary effort and insight. When Sherrington began studying spinal reflexes in 1890, there was, as noted above, a long history of stimulating various parts and recording the consequent responses. But the synthesis of such observations into a comprehensible whole was frustrated conceptually by the general ignorance of functional anatomy— compounded by the prevailing view of protoplasmic continuity (which specified neither the route nor the direction an impulse would travel through the reticulum)—and technically by the diversity of stimuli employed and the variability of responses elicited. Sherrington addressed both classes of problems, favored by his experience with microscopic anatomy as well as by his patience and precision. He was an early convert to the Neuron Theory, and he began his Silliman Lectures by professing that "Nowhere in physiology does the cell-theory reveal its presence more frequently in the very framework of the argument than . . .

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in the study of nervous reactions."10 And he bolstered that outlook with necessary examinations of the sensory and motor connections, studies he considered "boring" and "pedestrian" but which he pursued carefully and fruitfully.11 Thus, in 1892 he published a 150-page paper describing the source of motor fibers to the hind leg muscles of various species:12 he cut successively the ventral roots from the lumbosacral region of the spinal cord, stimulated electrically the peripheral stump of the cut root, and recorded which muscles then contracted. (As noted in chapter 1, the spinal cord is organized segmentally, with dorsal and ventral roots passing from the spinal cord between the vertebrae. These roots then join to form spinal nerves carrying both sensory and motor fibers: axodendritic processes of unipolar sensory neurons and axons of motor neurons. Moreover, the spinal nerves formed from the roots then merge into brachial and pelvic plexuses, which branch again into nerves to various muscles of the fore and hind legs. Because of these mergings and branchings, tracing fibers from muscle to spinal cord root cannot be achieved by inspection alone.) Correspondingly, in 1893 Sherrington published a 120-page paper describing the course of the sensory fibers to the hind leg:13 he cut peripheral nerves and stimulated the central stump, recording which muscles responded and thus which muscles were connected reflexively to sensory fibers in the stimulated nerve; in like manner, he traced which dorsal root was involved by stimulating its central stump. He also mapped the areas on the skin where local stimulation caused specific motor responses. Also in 1893 he identified the region of the spinal cord through which fibers passed upward toward the brain:14 after cutting specific spinal cord roots and waiting some days for the consequent degeneration, he recorded the behavioral changes in the animal and then examined microscopically the path of the degenerating fibers. Meanwhile, Sherrington was also examining reflexes. In 1892 he described initial studies on the knee jerk, demonstrating, contrary to some opinion, that it was a true reflex.15 Thus, he showed that the response required innervation through identified dorsal (sensory) and ventral (motor) roots controlling the active muscle (an extensor16 of the lower leg). In the course of these studies he also discovered that the knee jerk was exaggerated by cutting the dorsal roots supplying the antagonistic muscles, the flexors of the lower leg.17 Furthermore, he found that even after the flexors were freed from the leg (so they could exert no mechanical effect on the reflex) stimulating the nerve to the flexors could abolish the knee jerk, whereas cutting the sensory root from these flexors prevented that abolition.18 Sherrington concluded that "a stream of [sensory] impulses . . . passes up from the [flexors] and . . . in the cord exerts a depressing or restraining influence on the jerk."19 These experiments Sherrington complemented with anatomical studies demonstrating that sensory fibers ran from muscles to the spinal cord. (He cut

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the dorsal roots and, after waiting some days for degeneration to occur, showed that the nerves to the muscles then contained degenerated fibers.)20 The presence of sensory fibers from muscles was implicit in his studies of reflexes, but it was nevertheless a new discovery. It was also a crucial element for Sherrington's formulation of the neural control of posture, which depended on sense organs in the muscles and tendons reporting to the central nervous system the extent of contraction. (Muscles in the body are routinely contracted to some degree: they have a certain "tone." Maintaining posture requires balancing flexor and extensor contractions. This process is regulated unconsciously by reflex action, although it can also be consciously controlled, as in voluntarily changing posture.) From these studies Sherrington proposed the principle of "reciprocal innervation," specifying that contraction of one muscle was associated with a reflex relaxation of the antagonistic muscles synchronously—as in contraction of an extensor being accompanied by relaxation of the flexor.21 Inhibition of the antagonistic muscle was not merely a mechanical response, however, but an active process, for if a muscle were cut free and its motor nerve then stimulated, as that freed muscle shortened the antagonistic muscle still relaxed.22 Sherrington extended his studies to a variety of other reflexes, in the process using two approaches to isolate the spinal reflexes from influences of the brain: (1) in "spinal animals" the spinal cord was severed near its origin from the brain, producing, after a period of spinal shock, flaccid paralysis of the limbs; and (2} in "decerebrate animals" the cerebral hemispheres were removed, producing rigid extension of the limbs.23 In particular, he examined flexion reflexes, in which sharp, noxious stimuli to the foot caused flexion of that limb and extension of the others (as in animals with an injured foot hobbling on three legs), and extension reflexes, in which a steady, firm pressure to the foot caused extension (as in legs supporting an animal against gravity). To explain "crossed reflexes"—where stimuli to one side produce responses on the other side—he drew neural circuits (Fig. 2-2) that are reminiscent of CajaFs histological drawings of pathways in the spinal cord (Fig. 1-6D; Cajal, however, ignored inhibition). These coordinated reflexes also demonstrated that reciprocal innervation was applicable to symmetrical muscles on opposite sides of the animal as well as to antagonistic muscles of a limb; thus, when the left ear of a "decerebrate cat" was stimulated the left foreleg moved forward and the right foreleg backward, with the hind legs moving oppositely (Fig. 2-3).

Synapses ana the Rerlex Arc

In his Silliman Lectures Sherrington listed 11 ways that the conduction of impulses over reflex arcs differed from conduction by nerve trunks, empha-

FIGURE 2-2. Diagram for coordinated, crossed extension and flexion. Sherringtons 1905 drawing shows a dorsal root ganglion cell a transmitting sensory information from the skin and making synaptic contact in the ventral horn of the spinal cord on the same side (excitatory + to motoneuron d, which causes contraction of the flexor F, and inhibitory — to motoneuron e, which causes relaxation of extensor E); a also crosses to the other side of the spinal cord, there making synaptic contact in that ventral horn (excitatory to motoneuron e', which causes contraction of extensor E', and inhibitory to motoneuron 8', which causes relaxation of flexor F'). The drawing also shows a second dorsal root ganglion cell a' carrying sensory information from the flexor F and producing on the same side excitation of 8 to the flexor and inhibition of e to the extensor. Both sensory fibers also make further contacts, illustrated by the upward branches. Arrows show the direction of impulse flow. Note that Sherrington depicts dorsal root ganglion cell axons crossing to the opposite side, rather than an inhibitory interneuron crossing (see Fig. 1-6D) and that each dorsal root axon has both direct excitatory and inhibitory influences. (From Sherrington [1905], Fig. 8, courtesy of the Royal Society.)

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FIGURE 2-3. Decerebrate rigidity and evoked responses. Sherrington's 1906 drawing shows, on the left, the characteristic posture of a "decerebrate cat," with its limbs extended. (The portion of the brain removed is shown by cross hatching.) The drawing on the right shows the response to stimulating the left ear. (From Sherrington [1947], Fig. 47, courtesy of Yale University Press.)

sizing that additional processes must participate.24 His emphasis, however, was less on defining causal mechanisms for these differences than on demonstrating the functional processes present in reflex arcs. Indeed, some of these differences remained unexplainable for decades, such as the greater sensitivity of reflex arc conduction to drugs like strychnine. Here I will merely note five salient features of synapses and synaptic transmission that Sherrington addressed. Suriace 01 Separation

Sherrington deduced that "if there is not actual continuity of physical phase between [two neurons in a chain] there must be a surface of separation."25 This dividing line he imagined as a "delicate transverse membrane" that "might restrain diffusion, bank up osmotic pressure, restrict the movement of ions, accumulate electric charges, support a double electric layer, . . . alter in dif-

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ference of potential with changes in surface-tension or in shape, or intervene as a membrane between dilute solutions of electrolytes."26 Sherrington, however, had no direct evidence for such a structure or its properties. But he claimed that "in the neurone-chains . . . of vertebrates, histology on the whole furnishes evidence that a surface of separation does exist," although he provided no citations and conceded that in invertebrates "many nerve-cells are actually continuous one with another."2'

Valved Conduction

Whereas numerous studies showed that nerve trunks—both sensory and motor—conduct in either direction, impulses passed through reflex arcs in only one direction. Cajal's Principle of Dynamic Polarization specified just such a directionality in chains of neurons, and Sherrington proposed that the origin of this directionality lay in "valved conduction" at the synapse, where the hypothetical "membrane" might be "more permeable in one direction than the other."28 Again, direct evidence for a membrane having the requisite properties was not available.

Synaptic Delays

Another notable characteristic of conduction over reflex arcs was the longer time required—between applying a stimulus and recording the response— when compared with the conduction of impulses along a corresponding length of nerve. This delay Sherrington attributed to the time required for crossing the synapse. Although he admitted that the delay might "be due to the minute, branched, and more diffuse conducting elements" in the gray matter of the spinal cord, he argued that the neuron "itself is visibly a continuum from end to end."29 Moreover, Sherrington presented evidence against one means by which synaptic delays might occur through time required for closing a synaptic gap. He recorded the time between stimulus and response (in this case for the flexion reflex of a "spinal dog") when excited by two stimuli in rapid succession, the first submaximal and the second maximal. According to the hypothesis being tested, the first impulse should "set" the synapse for the second, but there was little difference (Fig. 2-4A). These delays he then compared with that from a single maximal stimulus (Fig. 2-4B); the response time was shorter than for the second of two stimuli. This result Sherrington considered "conclusive [evidence] against any major portion of the latent period being consumed at the synapse in a process which sets the synapse ready to conduct," as in proposals by Cajal for an "amoeboid movement of the protoplasm" occurring during the synaptic delay.30

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FIGURE 2-4. Effect of prior stimulation on the lag between stimulus and response. Sherrington's 1906 reproduction of myograph tracings, showing the extent of the flexion reflex of a dog's leg (on the vertical axis), traced on a smoked drum by an attached lever, as a function of time (on the horizontal axis). A. Sherrington evoked the flexion reflex first by a weak alternating current applied to the sole of the foot (s); after that response had leveled off he administered a stronger shock (s1) producing further flexion. B. Sherrington administered a stimulus (s2), equal in magnitude to s1 but without the prior stimulation. The pertinent measurement is the time interval between the shock and the onset of flexion (or its increase). This is shorter for s2 than s1, even though s1 was preceded by a "priming stimulus" s. These drawings are "negative" images of the myograph tracings, which appear as white lines against the black background of the smoked drum. (From Sherrington [1947], Fig. 4, courtesy of Yale University Press.)

With Willem Einthoven s development of the string galvanometer, it became possible to measure electrical responses in nerve and muscle with sufficient time resolution to quantitate synaptic delays. This W. A. Jolly in Edinburgh did in a straightforward fashion in 1911. First, he measured the time between applying a stimulus—either striking the patellar tendon for the knee jerk or pricking the skin for the flexion reflex—and the appearance of the response, recorded galvanometrically as the action current of the appropriate muscle, extensor or flexor, respectively.31 That measured interval, however, included times for (i) the sensory response; (ii) conduction from sense organ to spinal

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cord along the sensory nerve; (iii) conduction in the spinal cord including, ex hypothesi, synaptic delays; (iv) conduction from spinal cord to muscle along the motor nerve; and (v) the initiation of the muscle response. Times for (ii) and (iv) he evaluated by measuring the lengths of the nerves, applying the recently reported value for nerve conduction in human median nerve, 120 meters/second.32 For (i) he measured the interval between stimulation (striking the patella or pricking the skin) and the arrival of an electrical signal, recorded galvanometrically, in the sensory nerve, measured the distance along the sensory nerves between point of stimulation and point of electrical recording, and subtracted the calculated conduction time for that length of nerve from the measured interval. Similarly, for (v) he measured the interval between electrical stimulation of the motor nerve and production of the electrical response, recorded galvanometrically, in muscle, measured the distance along the motor nerve from point of stimulation to the point of recording on the muscle, and subtracted the calculated conduction time for that length of nerve from the measured interval. The synaptic delay (iii) is the difference between the overall time for the reflex and the sum of (i) + (ii) + (iv) + (v); distances in the spinal cord were so short that neuronal conduction times, if the impulse were transmitted like those in nerve, would be insignificant. Table 2-1 shows values Jolly reported for the knee jerk and flexion reflex of a "spinal cat," with synapse times of 2.1 and 4.3 milliseconds, respectively. Jolly concluded that "the knee-jerk mechanism involves one spinal synapse . . . while the flexion reflex involves two";33 compare Figures 1-6B and 1-6C of Cajal. Subliminal Stimuli and Synaptic Summation

A number of the differences between nerve and reflex conduction that Sherrington listed involved disparities between stimuli and responses, such as disTABLE 2-1. Response Times for Knee Jerk and Flexion Reflexes0 TIME (MILLISECONDS) KNEE JERK

FLEXION

Overall time from stimulus to response (T)

5.5

10.6

Sensory response time (i)

0.5

2.8

Conduction time to and from cord (ii + iv)

1.4

2.0

Motor response time (v)

1.5

1.5

Conduction time in cord (iii) = T - (i + ii + iv + v)

2.1

4.3

PROCESS

°The experiment is described in the text. (From Jolly [1911], p. 86. Used by permission of The Physiological Society.)

Skerrington and tke Synapse (1890-1913)

43

cordances in frequencies and durations. Such concerns may be exemplified by Sherringtons account of summation.34 An impulse transmitted by sensory nerves may be unable to evoke the reflex response and is therefore "subliminal." Nevertheless, a sequence of such subliminal stimuli can, if administered within a brief time, evoke the response. Analogously, a subliminal stimulus from each of two different sensory receptors, again if delivered within a given interval, can evoke a response. In these instances subliminal impulses from sensory neurons seemed to be summed together to produce the response. Since such summation did not occur with normal nerve or muscle, Sherrington localized that summation to the synapse. The means by which summation occurred was, however, not obvious. Sherrington imagined that the synapse represented a "considerable resistance to the passage of a single nerve-impulse"; a succession of impulses, on the other hand, "forced" their way through.35 In 1912 Edgar Adrian and Keith Lucas in Cambridge suggested that summation in reflex arcs occurred at—and reflected the presence of—a "region of decrement," that is, of diminished impulse conduction.36 They had showed that although a single impulse was unable to pass an experimentally-induced region of diminished conduction in a nerve (caused, for example, by local heating), a second impulse of identical magnitude could be conducted beyond the block if it followed the first impulse within a certain interval. Adrian and Lucas interpreted their results in terms of a brief period of "supernormal" excitability following the passage of any impulse, so that a second impulse arriving during the supernormal period would be magnified sufficiently to pass the block. A synapse, then, could be a "region of decrement," with the second impulse magnified to pass the block. They, however, did not address what the physical basis of this "synaptic block" might be.37

Inhibition

Inhibition was a crucial element in Sherringtons formulation of how integration is achieved: from hierarchical control manifested experimentally by spinal shock and by decerebrate rigidity, to reciprocal innervation dependent on sensory nerves from muscle, to the convergence of excitatory and inhibitory influences on the spinal motoneuron (the "final common path" to a response).38 He stressed that inhibition was an active process, not merely the absence of excitation, and was not due artifactually to experimental perturbations. (Thus, he argued that spinal shock resulted from the loss of input above the point of section rather than from the injury of sectioning, showing that a subsequent transection of the spinal cord—below the first cut—did not produce a second episode of spinal shock.39) Here, however, emphasis is on possible mechanisms for inhibition, which Sherrington localized "in all probability . . . at points of synapsis."40 He then

44

MECHANISMS OF SYNAPTIC TRANSMISSION

suggested that inhibition was "referable to a change in the conduction of the synaptic membrane causing a block in conduction."41 Nevertheless, in an earlier lecture in that series he argued against inhibition resulting from a simple blockade of synaptic conduction—against "merely arresting . . . an [excitatory] afferent channel" to the motoneuron—since cessation of the excitatory stimuli was followed by a continued, albeit brief, response (the "afterdischarge"), whereas stimulation of an inhibitory pathway halted the response immediately.42 He imagined that some process continued after the excitatory stimulus ceased, a process that was susceptible to immediate inhibition. (If the continuing response, the afterdischarge, were generated in the motoneuron, then merely blocking the synapse between that neuron and the excitatory sensory nerve would not halt the response immediately; however, if other mechanisms for the continuing response were responsible, such as continued excitation of the motoneuron through ancillary pathways, then blockade of synapses with the motoneuron would produce the observed effect.) Sherrington also dismissed four other proposals then current: (I) a shift in the metabolic balance of neurons between anabolism and catabolism, reflecting qualitative differences between excitatory and inhibitory impulses,43 a process that Alexander Forbes also criticized;44 (2) changes in binding between certain salts and proteins presumably involved in conducting nerve impulses;45 (3) "drainage" of excitation to alternative neural courses;46 and (4} the mutual annihilation of two waves—excitatory and inhibitory—that have opposing phases.47 Ultimately, Sherrington admitted "We do not yet understand the intimate nature of inhibition."48 Bayliss, in his textbook of 1920, agreed that "It cannot be said that any one of the theories suggested is a satisfactory one."49 Still, Bayliss noted that if excitation were associated with an increased permeability to certain ions, then inhibition could be associated with a decreased permeability.

Conclusions

Sherrington was a slight, spectacled man, notably courteous and gentle, but with firm opinions, sharply critical judgments, and a strong sense of rectitude. He was a published poet and discerning bibliophile, with broad interests and a formidable memory for literature as well as science. He was a tireless yet patient experimenter and a vigorous, competitive athlete. He was an entertaining raconteur50 and valued companion who cultivated ties across the globe. He was a central figure of the scientific establishment, enjoying a rising sequence of appointments from London to Liverpool to Oxford. He benefited from the blossoming of British physiology, led by Michael Foster, Walter Gaskell, John Newport Langley, Keith Lucas, Edward Schafer, Ernest Starling,

Skerrington and the Synapse (1890-1913)

45

and Augustus Waller. He garnered hosts of academic recognitions, extending to the presidency of the Royal Society and the Nobel Prize (1932). He established by precept the "Sherrington School of Physiology,"51 a style of approaches and analyses inculcated in students and colleagues (who included such notable figures in the neurosciences as R. S. Creed, Harvey Gushing, Derek DennyBrown, John Eccles, John Fulton, Ragnar Granit, E. G. T. Liddell, and Wilder Penfield). Undoubtedly, this web of associations and affiliations assisted Sherrington in securing audiences and their sympathetic hearings. Nevertheless, Sherrington's rise to preeminence—above mentors and colleagues—is readily attributable, as the published record illustrates, to effort, care, skill, thought, and imagination. Where chapter 1 dealt with sharp conflicts, this chapter relates developments: clarifications, revisions, and extensions. Although aspects of Sherrington's formulations were not without challenges, and although later decades saw striking metamorphoses of such notions as synaptic transmission and the mechanisms of inhibition, the fundamental principles that Sherrington established served as firm points of departure. Before Sherrington, descriptions of reflex responses were often fragmentary and conflicting. Then, in a patient and orderly progression, he delineated circumscribed phenomena: eliciting quantifiable responses from specific muscles by standardized electrical stimulation of identified neural pathways.52 From simple responses characterized in this fashion, Sherrington then reconstructed a coordinated, hierarchical system of identified pathways. This program he continued in Oxford until his retirement in 1935 at age 78, extending his examination of reflex modulation to the brain, localizing higher centers of control, and describing "central inhibitory states" and "central excitatory states." Although in later years he was tardy in applying newer techniques, he retained the virtues of clarity, simplicity, careful control of variables, and rigorous examination of alternatives. In the course of these endeavors, Sherrington not only introduced the term "synapse," he also attributed to this unseen entity certain functional properties: acting as a one-way valve for impulse conduction between neurons, producing a delay during that conduction, and modifying conduction to produce such effects as summation and inhibition. But identifying the physical nature of the synapse that underlay these functional properties was a task beyond Sherrington's approach. Notes

1. For biographies, see Cohen (1958), Eccles and Gibson (1979), Granit (1967), and Swazey (1969). 2. Contrary to some accounts, Eccles and Gibson (1979) state that Sherrington did not meet Cajal in Spain and that their only meeting was in London in 1894.

46

MECHANISMS OF SYNAPTIC TRANSMISSION

3. Sherrington (1893b), p. 300. 4. The account is in Fulton (1938), where it is clear that Sherrington preferred "syndesm"; see also Shepherd and Erulkar (1997) and Tansey (1997). Sherrington had a thorough classical education, and in his old age returned to reading Greek. 5. Foster (1897), p. 60. 6. For historical accounts, see Clarke and Jacyna (1987), Clarke and O'Malley (1968), Dodge (1926), Fearing (1964), Hoff and Kellaway (1952), Jeannerod (1985), and Smith (1992). 7. Stephen Hales had previously performed these experiments and told Whytt of them. 8. The lectures were published in 1906, with a second edition (differing only in a new preface) in 1947: Sherrington (1947). 9. Ibid., p. 2. 10. Ibid., p. 1. French (1970) argues that Edward Schafer's description in 1879 of discrete nerve fibers in jellyfish predisposed Sherrington to accept the Neuron Theory. 11. These quotations appear in the entry on Sherrington in The Dictionary of Scientific Biography, but without citations. 12. Sherrington (1892a). 13. Sherrington (1893a). 14. Sherrington (1893b). A further lengthy anatomical study appeared a few years later (Sherrington, 1898a). 15. Sherrington (1892b). At that time, criticisms of the knee jerk being a true reflex included comparisons of the time between stimulus and response for the knee jerk (brief) vs. other reflexes (long). Thus, Augustus Waller (1890) argued that although innervation was necessary for the knee jerk, it was really a direct muscle response; why innervation was necessary he did not explain. For a later evaluation of the time course, see W. A. Jolly (1911). 16. Extensors straighten a joint (in this case the knee), whereas flexors bend it. 17. Sherrington (1892c). 18. Sherrington (1893c).

19. Ibid., p. 563. 20. Sherrington (1894). The principle here is that nerve fibers cut from their cell bodies (here in the dorsal root ganglion) will degenerate; the motor fibers, passing through the ventral roots, will not be affected. 21. Sherrington (1897). Antagonism between flexors and extensors when a joint moves had been noticed by the ancients; Sherrington s emphasis is on the active, coordinated contraction/relaxation of these antagonistic couples. 22. Sherrington (1947). 23. Sherrington (1898b). Sherrington thought that this was a new discovery; however, a number of others had described the phenomenon, although without pursuing the functional implications. 24. Sherrington (1947), pp. 13-14. 25. Ibid., p. 16. 26. Ibid., pp. 16, 17.

27. Ibid., p. 17. 28. Sherrington (1947), p. 39. Sherrington cited William James's Law of Forward Direction, but not Cajal. 29. Ibid., p. 21. Sherrington's assumption that visual continuity implied uniform physiological mechanisms in dendrite, cell body, and axon was, however, not borne out in later studies.

Sherrington and the Synapse (1890-1913)

47

30. Ibid., pp. 23, 25. No reference is given to Cajal's views. 31. Jolly (1911). A pendulum swing initiated the stimulus and also started a photographic recording, which timed the delay until the galvanometric response. 32. Piper (1908). 33. Jolly (1911), p. 87. Since it was then known that the overall time between stimulus and response varied with the intensity of the stimulus, an essential consideration was that stimuli in both cases be equivalent. A number of other assumptions make these calculations less clearcut than they may seem at first glance; nevertheless, the general conclusion was subsequently confirmed. 34. Sherrington (1947), pp. 36-38. 35. Ibid., p. 37. 36. Adrian and Lucas (1912). 37. Adrian and Lucas were prominently involved in establishing the all-or-nothing response in nerve: that an impulse of a definite magnitude is transmitted or no impulse at all. However, these considerations did not forbid the threshold for excitation at synapses to be modifiable. 38. Sherrington (1913). It is important to distinguish between central inhibition, associated with the skeletal musculature, and peripheral inhibition, associated with the autonomic nervous system (chapter 3). 39. Sherrington (1947), pp. 242-245. 40. Ibid., p. 193. 41. Ibid., p. 194. 42. Ibid., p. 101. 43. See Gaskell (1886). 44. Forbes (1912). 45. See Macdonald (1905). 46. See McDougall (1903). 47. See Brunton (1883). 48. Sherrington (1947), p. 193. 49. Bayliss (1920), p. 426. 50. Thus, Sherrington (in Cannon, 1949, p. x) relates the following tale of Cajal's visit to his house. "He did not smoke, not even a cigarette. On being offered, inadvertently twice over, 'something to smoke,' his reply was a vigorous, 'Mais la vie moderne est une chose deja fort compliquee. Porter du tabac, des allumettes, etc., ga serait de la compliquer encore plus. Merci non!' His philosophy of life even in little things was never far to seek." 51. Denny-Brown (1957). 52. Muscle responses Sherrington recorded with a "myograph," a lever attached to the muscle that inscribed tracings on a smoked drum: a system of low friction and low inertia not surpassed until electronic recording techniques appeared. Stimulation was by "faradic" current, alternating current produced by an inductorium; however, quantitating the magnitudes of those stimuli seems to modern readers rather quaint: "The currents were usually just perceptible to the tongue tip" (Frohlich and Sherrington, 1902, p. 15).

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3 CHEMICAL TRANSMISSION AT SYNAPSES (1895-1945)

Nerve Impulse Conduction ana Synapse Structure

Significant advances in understanding synaptic transmission soon followed new observations and new interpretations.1 This chapter describes early evidence for chemical transmission in the autonomic nervous system, at neuromuscular junctions, and in the central nervous system. For narrative ease I will discuss studies at these three sites successively, even though experiments at all three sites were proceeding contemporaneously. But first I should note efforts in two related areas that were less successful in these decades. By 1915 nerve impulses were identified with waves of electrical activity; these electrical responses Julius Bernstein had attributed to changing ionic fluxes across the axon membrane, reflecting changing permeabilities of the membrane to particular ions (chapter 1). But in 1941 a reviewer still could not decide among three postulated mechanisms for impulse conduction, "all involving] a cell membrane or interface": Bernstein's theory of selective and variable permeability, analogies with oxidation electrodes that changed potential with changing oxidation/reduction states, and even vaguer formulations implicating colloidal structures ("Excitation might involve a change toward dispersal of colloids, due to the transitory production of cytolytic agents by the stimulation . . .").2 The reviewer cited recent papers by K. S. Cole demonstrating alterations in 49

50

MECHANISMS OF SYNAPTIC TRANSMISSION

membrane resistance during action potentials—consistent with Bernstein's theory—but overlooked a significant contribution by Alan Hodgkin and Andrew Huxley exploiting a novel approach that also appeared in 1939 (these will be noted further in chapter 4).3 While physiologists were concentrating on impulse conduction arising from changes in axonal membranes, a few microscopists persevered with earlier proposals that instead identified neurofibrils as the conductor of nerve impulses. This interpretation accompanied their continuing to see neurofibrils running from neuron to neuron, creating a conducting pathway "by living substance."4 Their images, however, provided no clue to the mechanisms peculiar to synapses, such as unidirectional conduction and synaptic delay. These residual affirmations of reticularist sentiments provoked Santiago Ramon y Cajal to denounce again in his old age this theme.5 Most microscopists agreed, aided by reports like that of George Bartelmez. While admitting that neuronal "elements are all at the verge of the resolving power of the microscope," Bartelmez in 1933 denied neurofibrillar continuity and maintained instead that "each of the elements of the synapse has a limiting membrane, although at the contact only one membrane can be resolved."6 David Bodian, also in Chicago, commented in 1937 that after 40 years of debate, "the morphological problem . . . will not be furthered . . . by the exclusive use of the older . . . methods."7 Following Bartelmez, Bodian concentrated on better fixation techniques and on correlating images from different staining procedures, concluding in 1942 that at synapses "only one membrane can be resolved, presumably because of the intimacy of contact of the separate neuronal limiting membranes"; the fused membrane separating two neurons he labeled a "synaptolemma."8 This structure had mechanistic implications, although not discriminatory ones. If synaptic communication were through electrical excitation of one cell by another, then tight apposition would favor it. This structure could also accommodate newer notions of synaptic transmission by chemical means: if these chemicals acted intracellularly, as was often assumed, a fused membrane would lessen the barriers to chemical transmitters passing from cell to cell.

Background: The Autonomic Nervous System

Greek anatomists described two chains of interconnected ganglia passing from the base of the brain and running beside the vertebral column.9 From these trunks nerves passed both to the spinal cord and to the viscera. In the midseventeenth century Thomas Willis in Oxford considered the ganglia to be storehouses of animal spirits. And since the chains of ganglia ran past the bases of the ribs, Willis named them the "intercostal nerves." The "sympathy of parts"—the means whereby one region affects another (such as associations

Chemical Transmission at Synapses (1895—1945)

51

between viewing dangerous sights and the heart pounding)—would follow from the apparent connections. On the other hand, the anatomical separation from brain and spinal cord would underlie the contrasts between involuntary ("visceral") and voluntary ("somatic") actions. Willis also discovered that cutting cranial nerve X, the vagus nerve (named for its wandering course from brain through neck and chest to abdomen), caused "great trembling" of the heart. In 1729 Frangois Pourfour du Petit in Paris showed that the intercostal nerves were not directly connected to the brain. Moreover, the cranial nerves—even the vagus, which passes to the viscera—were separate from the intercostal nerves. Pourfour du Petit also related specific lesions (such as cutting fibers from the anterior portion of the intercostal nerve) with specific functional changes (such as paralysis of the pupil). Shortly thereafter, Jacques-Benique Wilson in Paris renamed the intercostals the "great sympathetic nerves," in accord with their function. Another Parisian, the influential Xavier Bichat, stressed at the beginning of the nineteenth century the functional independence of the visceral and somatic systems, despite the anatomical connections that were then recognized between them: ganglia of the great sympathetic nerve were connected to nerve roots emerging from the spinal cord by two branches, or "rami," "gray rami communicantes" and "white rami communicantes." By midcentury Robert Remak in Berlin had characterized the microscopic composition of these rami: white rami contained myelinated fibers that passed centrally to the spinal roots, whereas gray rami contained fine myelinated and unmyelinated fibers that originated in sympathetic ganglia and then passed peripherally in spinal nerves as well as in sympathetic nerves to the viscera. Friedrich Henle and Rudolf Kolliker had shown that sympathetic fibers ran to muscle layers in the walls of arteries, and in 1852 Charles-Edouard BrownSequard, a peripatetic investigator then in the United States, and Claude Bernard in Paris demonstrated independently that electrical stimulation of sympathetic nerves to the face caused a local constriction of the blood vessels. A few years later Bernard reported that stimulating sympathetic nerves to the submaxillary gland constricted the blood vessels, whereas stimulating a cranial nerve innervating that region dilated them. Such antagonistic effects Walter Gaskell in Cambridge characterized anatomically as well as functionally in the 1880s.10 Gaskell had entered Trinity College intending a career in clinical medicine, but while he was an undergraduate Michael Foster arrived in Cambridge to teach physiology, and Gaskell became the first of Foster's stellar recruits to the field. After exploring the known antagonistic effects on heart rate of stimulating the vagus nerve (slowing) vs. stimulating fibers from the sympathetic trunk (accelerating), Gaskell next turned to microscopic examinations of the spinal cord roots and rami communicantes, establishing five categories of motor fibers (Fig. 3-1). (1) Nonmyelinated fibers ran from sympathetic ganglia through gray rami and then

52

Cnemical Transmission at Synapses (1895 — 1945)

53

peripherally in spinal and sympathetic nerves. (2) Myelinated fibers of large diameter—easily distinguishable after staining—ran from ventral horn cells in the spinal cord, out ventral roots, and then through spinal nerves to the voluntary muscles. (3) Far smaller myelinated fibers ran from cells in the lateral horns of the spinal cord, out ventral roots, and through white rami to sympathetic ganglia. These fibers and the white rami arose from thoracic and upper lumbar segments of the spinal cord. (4) Small myelinated fibers also arose from sacral segments of the spinal cord; however, after passing through ventral roots, these ran to the pelvic plexus of nerves, which innervated lower intestine, bladder, and reproductive organs. (5) Yet other small myelinated nerves arose in the brain, passing out in certain cranial nerves, such as the vagus, to innervate head, chest, and upper abdomen. Gaskell, therefore, identified three outflows of structurally similar small motor fibers: cranial, thoracolumbar, and sacral.11 Moreover, he identified a functional opposition between thoracolumbar and craniosacral fibers extending throughout the body (Table 3-1). John Newport Langley (Fig. 3-2) furthered these studies at the turn of the century. Langley, like Gaskell and Sherrington, had been recruited as an undergraduate by Foster, in his case from an intended career in the civil service. (Langley not only succeeded Foster as professor of physiology in Cambridge, he also founded and edited the Journal of Physiology, which for many years he owned.) Langley s approach included a careful mapping of the sympathetic chain, made possible by the use of nicotine.12 He found that nicotine initially stimulated but then blocked the transmission of nerve impulses through sympathetic ganglia. After he painted a ganglion with nicotine solutions, a characteristic response resulted that soon disappeared. Stimulating fibers leading from the spinal cord to this ganglion then evoked no responses. By contrast, stimulating fibers running/row this ganglion still produced the normal response. In that manner Langley matched each root, ramus, and ganglion with its sympathetic response.13 In the process he also provided new names, including "preganglionic fibers" and "postganglionic fibers"; the overall system he labeled the FIGURE 3-1. Anatomy of the autonomic nervous system. A. This diagrammatic view from the rear shows on the left parasympathetic fibers from the cranial and sacral regions, having long preganglionic fibers (dashed lines) and short postganglionic fibers (solid lines). On the right, sympathetic fibers from the thoracic and lumbar regions of the spinal cord have short preganglionic fibers, making synaptic contact in the chain of sympathetic ganglia, and long postganglionic fibers. In addition, some preganglionic fibers pass upward and downward within the sympathetic chain. B. Cross-section of the spinal cord showing the sympathetic preganglionic motoneurons in the lateral horns. The axons of these neurons exit by the ventral roots and pass through the white ramus communicans to the sympathetic ganglion, where they make synaptic contact with sympathetic postganglionic neurons. The axons of these postganglionic neurons can pass by the gray ramus communicans to the spinal nerve or run in a sympathetic nerve.

TABLE 3—1. Examples of Antagonistic Effects in the Autonomic Nervous System

SYMPATHETIC STIMULATION (THROUGH THORACOLUMBAR FIBERS)

PROCESS

PARASYMPATHETIC STIMULATION (THROUGH CRANIOSACRAL FIBERS)

Pupil diameter

constricts

dilates

Heart rate

increases

decreases

Bronchial muscle

relaxes

contracts

Intestinal peristalsis

decreases

increases

Bladder sphincter

contracts

relaxes

FIGURE 3-2. John Newport Langley (1852-1925). 54

Chemical Transmission at Synapses (1895—1945)

55

"autonomic nervous system," allocating "sympathetic" to the thoracolumbar outflow and applying "parasympathetic" to the craniosacral.14 The newly enunciated Neuron Theory guided the identification of these ganglionic elements. Ganglia were collections of neuronal cell bodies, plus neuronal processes entering and leaving. Preganglionic fibers were axons that made synaptic contact with ganglion cells; postganglionic fibers were axons of these ganglion cells (Fig. 3-1). Furthermore, ganglia of the sympathetic division generally lay close to the spinal cord, so preganglionic fibers were short and postganglionic fibers to innervated organs long. By contrast, ganglia of the parasympathetic division generally lay close to or even within the organ innervated, so preganglionic fibers were long and postganglionic fibers short. Finally, two further sets of clarifications are necessary. (I) Early microscopists distinguished between "striated muscle," so named for the transverse stripes then visible, and "smooth muscle," which lacked stripes. Smooth muscle is present in blood vessels and viscera; its contractions are not under voluntary control but are regulated—excited or inhibited—by the autonomic nervous system. (Here, inhibition is "peripheral," through inhibitory nerves to the muscle.) Striated muscle is present in somatic skeletal muscle subject to voluntary control. (Here, inhibition is "central," at the level of the spinal motoneuron, as Sherrington demonstrated.) Striated muscle is also present in the heart; there, however, it is controlled by the autonomic nervous system. (2) For emphasis as well as convenience, I use the term "synapse" for junctions: between neurons; between autonomic motoneurons and the glands or smooth muscles they innervate (commonly termed "neuroeffector junctions"); and between voluntary motoneurons and the skeletal striated muscles they innervate (commonly termed "neuromuscular junctions").

Chemical Transmission in the Autonomic Nervous System

Here I describe separately synaptic transmission at three sites: between sympathetic postganglionic fibers and their effector cells (smooth muscle and gland), at corresponding junctions of parasympathetic postganglionic fibers, and in autonomic ganglia between preganglionic fibers and ganglionic neurons.

Adrenaline and Postganglionic Sympathetic Nerve Endings (1895—1920)

In the winter of 1893-1894, George Oliver, a physician in Harrogate, was exploring responses of the radial artery to extracts of assorted organs, using an instrument he devised to measure its diameter.15 In the course of this survey he found that adrenal extracts, when given orally to his son, constricted the artery.16 That experiment was, among other characteristics, particularly rash,

56

MECHANISMS OF SYNAPTIC TRANSMISSION

since earlier reports noted that adrenal extracts killed dogs, rabbits, and guinea pigs.17 In any event, Oliver took his extract to London, where he persuaded the professor of physiology Edward Schafer to study its properties further. In 1895 Oliver and Schafer described remarkable increases in the blood pressure of animals given adrenal extracts intravenously, responses they attributed to constriction of the arterioles (fine arteries leading to the capillaries, which had earlier been identified as a site of blood pressure regulation).18 Since vasoconstriction occurred in isolated organs as well, they concluded that the response was "due to the direct action of the active principle . . . upon the muscular tissue of the blood vessels."19 Oliver and Schafer traced the source of their active principle to the adrenal medulla, which they considered to be a "ductless . . . gland" releasing its secretion into the bloodstream. Their extract also slowed the heart, noticeable after the rise in blood pressure; however, when both vagus nerves (parasympathetic) were cut, the extract markedly increased the heart rate and the force of contraction.20 Furthermore, the extract increased the heart rate and contractile force in isolated frog hearts (hearts freed from neural control). These dramatic effects attracted great interest, and others soon extended Oliver and Schafer's observations. In 1901 Langley confirmed a catalog of responses (such as slowing intestinal peristalsis and dilating the pupil), added some new (such as increased salivation), and stressed the parallel between administering the extract and stimulating the sympathetic division of the autonomic nervous system.21 Langley also pointed out that extracts were active even after nerves to the affected tissues were cut and had degenerated: the extract affected not nerve endings but the tissues themselves. Meanwhile, Jokichi Takamine, an independent chemist with ties to Parke, Davis & Co., developed procedures for isolating from adrenals a crystalline extract having these pharmacological properties (which he patented), named it Adrenalin (which he registered as a trademark), and calculated its empirical formula as CioHisNOa.22 (Since Parke, Davis obtained Takamines trademark for their commercial product, American pharmacologists generally use the name epinephrine, introduced by John Jacob Abel in Baltimore during his unsuccessful attempt at purification; the rest of the world more commonly uses adrenaline, which I follow here.23) In 1901 T. B. Aldrich at Parke, Davis & Co. recalculated the formula as CgHiaNOs and in 1905 suggested a structural formula containing catechol, a secondary alcohol, and a methylated amine (Fig. 3-3A).24 That year H. D. Dakin in London synthesized this compound, showing it to have the requisite pharmacological activities.25 How this chemical produced Langleys parallels, mimicking the effects of stimulating sympathetic nerves, was suggested the year before. Thomas Elliott, a research fellow in GaskelFs and Langleys Cambridge, proposed in 1904 that adrenaline was "the chemical stimulant liberated on each occasion when the

Chemical Transmission at Synapses (1895—1945)

57

FIGURE 3-3. Chemical structures of adrenergic agents and analogs.

[nerve] impulse arrives at the periphery."26 Elliott, too, noted that adrenaline did not excite sympathetic ganglia but was effective on end organs even after the nerve endings had degenerated. (Indeed, he observed that after degeneration the response was augmented, an important phenomenon later termed "denervation supersensitivity.") Elliott thus surmised that nerve endings excite their effector cells—smooth muscle or glands—by releasing adrenaline: transmission was effected chemically. In 1905 Elliott published a full paper surveying the effects of adrenal extracts and of Parke, Davis's Adrenalin on a range of tissues, concluding that each response was "of a similar character to that following excitation of the sympathetic . . . nerves."27 But Elliott did not repeat his earlier suggestion about sympathetic nerve endings releasing adrenaline onto their end organs.28 Elliott's basis for that proposal rested purely on correlating effects, and he now noticed some divergences. Although Langley in 1905 affirmed that "the nervous impulse should not pass from nerve to muscle by an electric discharge, but by the secretion of a special substance at the end of the nerve," he, too, cited divergences between administering adrenaline and sympathetic stimulation.29 Still stronger challenges to the parallels between added adrenaline and sympathetic stimulation came from Henry Dale (Fig. 3-4), yet another who followed undergraduate years in Cambridge with medical training in London prior to a scientific career. Dale, however, joined the Wellcome Physiological Research Laboratories in 1904, persuaded by a liberal offer from the parent pharmaceutical company.30 Exploring the biological properties of natural substances was then an active and profitable enterprise, and Dale was soon examining responses to extracts of ergot, a deadly fungus. In 1906 he reported that ergot extracts

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MECHANISMS OF SYNAPTIC TRANSMISSION

FIGURE 3^t. Henry Hallett Dale (1875-1968; courtesy of the Wellcome Trust Medical Photography Library).

blocked adrenaline s ability to raise blood pressure without blocking adrenaline s inhibitory actions, concluding that "probabl[yj these two sets of effects [excitatory and inhibitory] are produced by different active principles."31 In 1910 Dale documented further divergences while comparing systematically a "range of compounds which . . . simulate the effects of sympathetic nerves" (simulations he labeled "sympathomimetic" effects).32 In retrospect, three compounds among those he studied are notable, those now known as dopamine, noradrenaline, and adrenaline (Fig. 3-3). Since all contain both catechol and amine constituents, they are known collectively as catecholamines. Dale's critical observation was that the rank order of these three differed for

Chemical Transmission at Synapses (1895—1945)

59

different responses. For example, noradrenaline was most potent in raising blood pressure and dopamine least, whereas adrenaline was more potent than noradrenaline in reducing contractions of the cat uterus. Consequently, Dale asserted that Elliott's proposal "assum[ed] a stricter parallelism between the two actions than actually exists."33 Elliott subsequently completed his medical training and pursued a successful career in clinical medicine, becoming a professor of medicine in London. He left no explicit explanation of why he abandoned his proposal.34 But with the parallelism challenged by his seniors and with no further avenues of exploration apparent, he had little choice.

The Concept or Receptors

Whether or not adrenaline was released from sympathetic nerve endings, it— and many other synthetic as well as naturally occurring chemicals—affected biological systems. How could this occur? In 1905 Langley proposed that the "action of adrenalin depends upon the presence in the muscle protoplasm of some substance."35 Consequently, drugs as well as the "effective material of internal secretions [would] produce their effects by combining with the receptive substance."36 To account for inhibitory as well as excitatory responses, Langley imagined that "both inhibitory and motor substance[s] might be present [at a synapse, and thus] the effect of a nervous impulse depends upon the proportion of the two kinds of receptive substances" present.37 The following year Langley suggested that these reactive substances were "radicles of the protoplasmic molecule,"38 in accord with contemporary views of a unitary protoplasmic substance bearing distinct side chains, or radicles (or radicals), that mediated specific functions. In this sense, Langley imagined that "a special radicle is necessary for the combination with a number of chemical bodies, and that the compound formed [from the bodies and the radicle] leads to further change."39 (Chapter 6 will describe further developments of the receptor concept.) Acetylcnoline ana Postganglionic Parasympatnetic Nerve Endings

Like many who contemplated parasympathetic transmission, Walter Dixon focused on how stimulating the vagus nerve could inhibit the heart. Dixon, however, began further afield. Before moving to Cambridge, he had studied medicine in London, where William Bayliss and Edward Starling were demonstrating how the intestine produced secretin, a factor triggering the release of digestive enzymes. Bayliss and Starling proposed in 1902 that secretin was formed when acidic stomach contents reached the duodenum, "probably by hydrolysis" of a precursor in the duodenal wall, "prosecretin."40 Dixon, too,

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began investigating the secretin system, generalizing that sequence to proposals that many natural substances (as well as drugs) liberate active substances from tissues. Extrapolating to the vagal innervation of the heart, Dixon in 1906 suggested that the heart contains " 'pro-inhibitin,' which, as a result of vagus excitation, is converted into . . . Inhibitin.'"41 His brief paper in 1907 dropped these names but described an experiment: he stimulated the vagus nerve electrically for a half hour and then made an extract of the heart; when he added this extract to a second heart its beating slowed (Fig. 3-5).42 Dixon likened the response to that produced by muscarine (Fig. 3-6C) and noted that atropine (Fig. 3-6D) reversed the inhibition. (Forty years earlier Oswald Schmiedeberg in Dorpat purified muscarine from the mushroom Amanita muscaria and described its ability to inhibit the heart, like vagal stimulation. The poisonous properties of belladonna had been known for centuries, and Schmiedeberg also showed that atropine, a purified extract from the plant Atropa belladonna, blocked the effects of both muscarine and vagal stimulation. By the turn of the century these two compounds, one mimicking and the other blocking, had become identifying reagents for parasympathetic effects.) Although Dixon did not cite Elliott, he, too, suggested that the active substance was stored in the nerve ending, was released by excitation to combine with "a body in the cardiac muscle," and thereby produced inhibition.43 But Dixon did not try to identify the active substance or even extend his initial observations; he was deterred, he said later, by universal skepticism.44 Earlier, Reid Hunt in Baltimore found that administering choline (Fig. 3-6B), known to be present in adrenal extracts, caused blood pressure to fall; furthermore, this fall could be blocked by atropine.45 Other active substances,

FIGURE 3-5. Dixon s experiment showing the slowing of a recipient heart induced by an extract from a vagally stimulated donor heart. The recording shows the heart beats (vertical excursions of a lever attached to an exposed frog heart) with time (horizontal axis). At the first arrow was added an extract from the donor heart whose vagus nerve had been stimulated, showing the slowing of contractions in the recipient heart. At the second arrow atropine was added to the recipient heart, and its rate increased. (Reprinted from Dale [1934], Fig. 1, courtesy of the BMJ Publishing Group. Dale noted that the figure, previously unpublished, was from a lantern slide given to him by Dixon.)

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FIGURE 3-6. Chemical structures of cholinergic agents and analogs.

unidentified, were present in adrenal extracts, and Hunt imagined that they might be precursors to choline.46 Consequently, in 1906 he tested the acetyl ester of choline, acetylcholine (Fig. 3-6A): it was a thousand times more potent, and its effects were blocked by atropine.47 But Hunt, having no evidence that acetylcholine was present in the body, merely suggested that acetylcholine acted on "terminations of the vagus in the heart."48 Subsequently, Dale, while continuing his studies of ergot extracts, detected aberrant muscarine-like actions in a particular batch. In 1914 Arthur Ewins at the Wellcome Laboratories identified acetylcholine in this extract, and Dale set about cataloging its pharmacological properties.49 The range of actions— including slowing the heart, lowering blood pressure, speeding peristalsis, constricting the pupil—corresponded closely with those that followed stimulation of parasympathetic nerves; moreover, atropine blocked these effects. Dale emphasized that responses to added acetylcholine were evanescent and suggested that esterases in the body might split acetylcholine rapidly into acetate

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and far weaker choline (Fig. 3-6A-B). Consequently, if acetylcholine were present in animals at levels expected from its intrinsic potency, this lability ensured that "its detection [would be] impossible by known methods."50 In any case, the parallelism—as with adrenaline and sympathetic stimulation—was not perfect; for example, added acetylcholine mimicked sympathetic stimulation of sweat glands. Development of these considerations came after World War I, notably through the new experimental approaches of Otto Loewi (Fig. 3-7) in Graz. Loewi, after completing medical training in Strassburg (including a research project with Schmiedeberg), practiced medicine briefly before turning to pharmacology. His earlier studies in Marburg and Vienna ranged over carbohydrate

FIGURE 3-7. Otto Loewi (1873-1961).

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and protein metabolism as well as kidney and heart function. In 1902 he worked briefly with Starling in London and while there met Dale, with whom he developed a lifelong friendship. Loewi also met Elliott during a visit to Cambridge, but he stated that Elliott's and Dixon's pioneering papers escaped his notice.51 In any event, by 1903 he was convinced that synaptic transmission occurred through chemical means (according to the recollection of an acquaintance52), but not knowing how to demonstrate this conviction, he pursued other issues for nearly two decades. Then in 1920 at age 47: The night before Easter Sunday . . . I awoke, turned on the light, and jotted down a few notes on a tiny slip of thin paper. Then I fell asleep again. It occurred to me at six o'clock in the morning that during the night I had written down something most important, but I was unable to decipher the scrawl. The next night, at three o'clock, the idea returned. It was the design of an experiment to determine whether or not the hypothesis of chemical transmission that I had uttered seventeen years ago was correct. I got up immediately, went to the laboratory, and performed a simple experiment on a frog heart according to the nocturnal design.53

Loewi s tale of this key experiment is equally disarming: The hearts of two frogs were isolated, the first with its nerves, the second without. Both hearts were attached to ... canulas [sic] filled with a little Ringer solution.54 The vagus nerve of the first heart was stimulated for a few minutes. Then the Ringer solution that had been in the first heart during stimulation . . . was transferred to the second heart. It slowed and its beats diminished just as if its vagus had been stimulated. Similarly, when the [sympathetic] accelerator nerve was stimulated and the Ringer from this period transferred, the second heart speeded up and its beats increased. These results unequivocally proved that the nerves do not influence the heart directly but liberate from their terminals specific chemical substances which, in turn, cause the well-known modifications of the function. . . . 55

The published reports, however, were less straightforward than Loewi's recollections, and the consequent skepticism represented no unequivocal acceptance of proof. Loewi's 1921 paper described, briefly, the results of stimulating the vagus trunk of an isolated, cannulated heart (donor), and then transferring its Ringer solution to a second isolated, cannulated heart (recipient).56 With one species of frog, Rana esculenta, the force of the recipient heart's contractions decreased, diminished with time, disappeared when the perfusing Ringer solution was changed, and was blocked by atropine (Fig. 3-8A). But there was no obvious slowing of the recipient heart. With a second species, Rana temporaria, the recipient heart slowed, but no effect of atropine was reported (Fig. 3-8B). And with a toad, the force of contraction of the recipient heart instead increased,

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although the rate did not increase (Fig. 3-8C). Loewi concluded that nerve stimulation formed or released either an inhibitory substance, whose effects were blocked by atropine, or a stimulatory substance. Cautiously, Loewi named the inhibitory substance "VagusstofP' and the stimulatory substance "Acceleransstoff." The diversity of responses invited criticism, and for some the "records [were] far from convincing."57 Active chemicals could have been released artifactually when solutions were changed, for amphibian hearts are exquisitely sensitive to experimental manipulations.58 Particularly disturbing was the mix of inhibitory and stimulatory responses. In his 1921 paper Loewi noted that the vagus trunk near the heart contains not only cranial parasympathetic fibers but also sympathetic fibers from the thoracolumbar division; in his 1922 paper he pointed out that the balance between these two systems not only differed between species but also with the season of the year.59 The earlier paper reported experiments in February and March, the latter experiments from April through

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August; with summer toads the recipient heart first decreased and then increased its contractions (Fig. 3-8D). But he failed to show comparable changes in the donor frog and toad hearts when their vagal trunks were stimulated (or, better yet, to stimulate electrically only parasympathetic or only sympathetic fibers—as his retrospective summation implied).60 Loewi also showed that atropine did not block by inhibiting the release of Vagusstoff: Vagusstoff appeared in the Ringer's solution after stimulating in the presence of atropine as well.61 For a decade these experiments were criticized, and contrary results were reported.62 Nevertheless, others reproduced Loewi's results, extended the experiments to additional parasympathetic sites, and improved the technique.63 In particular, W. A. Bain in Edinburgh avoided serious shortcomings of Loewi's experiments and provided missing data (Fig. 3-9); indeed, this figure is reproduced in textbooks rather than Loewi's.64 Most persuasive, however, was Loewi's development of the issues, especially his identifications of the responsible chemicals. Although in 1922 he had referred to the inhibitory substance circumspectly as Vagusstoff,65 Loewi soon showed that these inhibitory Ringer's solutions contained choline, which he

FIGURE 3-8. Loewi's experiments on chemical transmission. A. As described in the text, the Ringers solution from a cannulated donor heart—whose vagal trunk had been stimulated electrically—was transferred to a cannulated recipient heart at the first arrow. The magnitude of the beats (vertical excursions) of the recipient heart then decreased with time (horizontal axis). At the second arrow Ringers solution was added from a donor heart whose vagal trunk had not been stimulated, and heart beats returned to their original extent. At the third arrow Ringers fluid from a stimulated donor heart was again added. And at the fourth arrow atropine was added, restoring the extent of contractions. Rana esculenta were used. B. Similar experiments with a different species of frog, Rana temporaria, showed a slowing of the recipient heart as well as a diminution of contractions after adding (at the arrow) Ringer's solution from a stimulated donor heart. No atropine was added. C. Similar experiments with a toad heart showed an increased magnitude of contractions in the recipient heart after adding (at the arrow) Ringer's solution from a donor heart whose vagal trunk had been stimulated. Experiments were done in the spring. D. Similar experiments on a toad done in the summer: adding the Ringer's solution from a stimulated donor toad first decreased and then increased the magnitude of contractions. E. In experiments similar to those in A, the Ringer's solution from a stimulated donor heart was first heated for 20 minutes at 55° C and then allowed to stand at room temperature for one-half to three hours. This "heat-inactivated" solution, added at the arrow, caused decreased contractions in the recipient heart, which declined only slowly. (The numbers on the recording refer to intervals when the chart was not moving.) After contractions returned to control levels, Ringer's solution from a stimulated donor heart was added, but this sample had been left at room temperature without prior heat inactivation and its potency had disappeared. (A, B, and C are from Loewi [1921], Figs. 1, 2, and 3; D is from Loewi [1922], Fig. 2; E is from Loewi and Navratil [1926a], Fig. 1. Courtesy of Springer-Verlag.)

FIGURE 3-9. Bain's demonstration of chemical transmission. A. The perfusion apparatus contained a reservoir of Ringer's solution (A) that could be refilled through (C) and whose hydrostatic pressure was controlled by an-overflow tube (B). Ringer's solution passed to the donor heart (F), whose vagus nerve could be stimulated electrically, by the tubes (D) and (E). The perfusion fluid next passed by (G) and (H) to the recipient heart (I). (J) and (K) were levers attached to the hearts that inscribed on a smoked drum the heart beats. B. Tracings of the heart beats from donor (D) and recipient (R) hearts were displayed against time (T). During the stimulus period (dip in the line marked S) the donor heart ceased beating, followed by the recipient heart. After electrical stimulation ceased the donor heart resumed beating, as did the recipient heart. (From Bain [1932], Figs. 1 and 2, courtesy of the Physiological Society.]

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identified by acetylating the extract to form acetylcholine.66 This conversion multiplied the extract's potency enormously, allowing him to establish its presence through biological assays ("bioassays"). Loewi calculated that insufficient choline was present in the Ringer's solutions to produce, as choline, the observed inhibition; consequently, Vagusstoff was probably some more active derivative of choline. As had Dale earlier, Loewi concluded that the evanescent effect of choline esters, such as acetylcholine, was likely due to the presence of degrading enzymes; such cholinesterases would generate from its more active ester the choline he found. Accordingly, when Loewi heated the Ringer's solution from stimulated donor hearts to destroy enzymatic activity, the inhibitory potency remained even after the solution sat at room temperature for some hours. By contrast, the inhibitory potency of unheated Ringer's solutions disappeared in that time (Fig. 3-8E).67 Physostigmine (also known as eserine), a poison purified from seeds of Physostigma venenosum, was then known to augment and prolong the effects of parasympathetic nerve stimulation. In 1926 Loewi described how physostigmine also augmented and prolonged the effects of both Vagusstoff and acetylcholine.68 And in 1930 both K. Matthes, who worked with Dale, and Loewi found cholinesterase activity in blood that could be inhibited by physostigmine.69 Thus, physostigmine's potentiation of the actions of both acetylcholine and Vagusstoff was attributable to physostigmine's ability to inhibit the cholinesterases that destroyed both acetylcholine and Vagusstoff. Adding further plausibility to the identification of Vagusstoff with acetylcholine was Dale's announcement the previous year that acetylcholine indeed existed in animal tissues. In 1929 Dale, now at the National Institute for Medical Research in Hampstead, reported the chemical identification of acetylcholine in bovine spleens.70 Measuring acetylcholine in other animal organs by chemical means was, however, not possible for several decades. Instead, pharmacologists relied on various bioassays, such as frog heart, rabbit blood pressure, and, notably, frog rectus abdominis muscle and leech dorsal muscle. The essential considerations for a valid bioassay were sensitivity to the small amounts of acetylcholine present and specificity (i.e., relative insensitivity to the multitude of other active substances that could be present in perfusates and extracts). Confirmation came from characteristic responses to blocking and potentiating agents (e.g., atropine, physostigmine). Most significant, however, was demonstrating a fixed quantitative ratio of activities for an unknown substance to activities of authentic acetylcholine at different dilutions and in different bioassay systems. In this fashion, H. C. Chang and John Gaddum, working with Dale, measured in 1933 the acetylcholine content of dozens of organs from a half dozen species.71 They also showed that no other choline ester had the requisite characteristics.72

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In 1933 Wilhelm Feldberg (Fig. 3-10) in Berlin described the definitive presence of acetylcholine in venous blood from mammalian hearts after vagal stimulation using the leech bioassay that Bruno Minz in Feldberg's laboratory developed.73 Feldberg had studied medicine in Berlin and then in 1925 journeyed to Cambridge to work with Langley; after Langleys death he worked with Dale in Hampstead before returning to Berlin in 1927. When he was dismissed by the Nazi government late in 1933, Dale took him in. Together they exploited the leech bioassay to demonstrate acetylcholine release from the stomach after vagal stimulation, providing identification at a further site and resolving a possible anomaly. (Vagal effects on the gastrointestinal tract were poorly blocked by atropine, raising the possibility that acetylcholine might not be the neurotransmitter there.)74 By the mid 1930s these studies had demonstrated that acetylcholine was present in nervous tissue, was released by nerve stimulation, and mimicked the effects of parasympathetic nerve stimulation.

FIGURE 3-10. Wilhelm S. Feldberg (1900-1993; courtesy of the Physiological Society and the Wellcome Institute for the History of Medicine).

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Adrenaline ana Postganglionic Sympathetic Nerve Endings (1920—1945)

In 1922 Loewi reported that ergot extracts blocked the effect of Acceleransstoff, the stimulating substance from toad hearts,75 just as Dale had earlier shown they blocked the effects of both adrenaline and sympathetic stimulation. In 1926 Loewi provided a further link by demonstrating that ultraviolet light inactivated Acceleransstoff, as it did adrenaline.76 A decade later he finally convinced himself that Acceleransstoff was adrenaline: perfusates from stimulated hearts, as well as extracts of these hearts themselves, showed the characteristic green fluorescence of adrenaline.77 During that interval further reports confirmed the release of adrenaline-like substances when sympathetic nerves were stimulated.78 On the other hand, stimulating sympathetic nerves to sweat glands caused secretion, but here administering acetylcholine rather than adrenaline caused secretion. Dale and Feldberg established this as a defined anomaly: acetylcholine appeared in the venous blood from a cat's foot when they stimulated sympathetic nerves to its sweat glands.79 They considered this "an exception to the generally valid rule" that postganglionic sympathetic neurons release as their neurotransmitter an adrenaline-like substance, generalizing their interpretation to other animals (including humans), in which atropine inhibited sweating.80 Consequently, Dale suggested a pharmacological classification into "cholinergic" and "adrenergic" neurons, depending on the neurotransmitter released, that could differ from the anatomical classification: We can then say that postganglionic parasympathetic fibres are predominantly, and perhaps entirely, "cholinergic," and that postganglionic sympathetic fibres are predominantly, though not entirely, "adrenergic."81

Meanwhile, Walter Cannon in Boston was pursuing a diverging path. While a student at Harvard Cannon had studied gastrointestinal motility using recently developed X-rays; as a faculty member at Harvard he recognized that emotional states could alter this motility. He then linked emotional states, adrenal hormones, and bodily functions. So in 1921—the year of Loewi's initial study— Cannon published analogous experiments: stimulating sympathetic nerves to the liver released into the bloodstream substances that increased the heart rate of cats who had had their adrenals removed and hearts denervated.82 A decade later Cannon described an increased heart rate, blood pressure, and salivary secretion after stimulating sympathetic nerves to tail hairs in cats whose adrenals were removed, hearts denervated, and spinal cords transected.83 Again, the only link between the site of stimulation and the affected organs was the bloodstream: an adrenaline-like substance had been liberated into the bloodstream even when the adrenals were absent. Cannon cautiously called the circulating material "sympathin," even as he noted obvious parallels with adrenaline (such

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as blocking of both by ergot extracts and increased sensitivity to both after denervating tissues). In 1933, however, Cannon and Arturo Rosenblueth argued that two varieties of sympathin were formed: excitatory ("sympathin E") and inhibitory ("sympathin I").84 This distinction also accounted for different responses of their test organs—cat nictitating membrane85 and uterus—after stimulating different sympathetic nerves. For example, stimulating nerves to the intestine released into the bloodstream substances causing nictitating membrane contraction and uterine relaxation (sympathins E and I), whereas stimulating sympathetic nerves to the liver caused nictitating membrane contraction but no uterine relaxation (sympathin E only). Cannon and Rosenblueth adapted Langley s notion of receptive substances to depict complexes formed in the tissues from transmitter plus excitatory receptive substance (sympathin E) and/or inhibitory receptive substance (sympathin I); in this way an adrenaline-like substance became "differentiated for positive and negative action."86 Now they added a further characteristic: neurotransmitter plus receptive substance could be released together into the bloodstream to act beyond their site of formation. Cannon and Rosenblueth defended their proposal vigorously through the next decade. Others remained critical. Sympathetic stimulation having some excitatory and some inhibitory effects was analogous to parasympathetic stimulation having correspondingly diverse effects, but no one felt that two forms of acetylcholine were required.87 Moreover, Zenon Bacq in Liege, who had collaborated with Cannon before Rosenblueth, argued that different responses to intestinal and hepatic nerve stimulation could be attributed to quantitative effects: when lesser amounts of sympathin were released the weaker inhibitory responses were then not obvious.88 Nerves might also release additional substances besides sympathin. In any case, chemical assays revealed adrenaline in brain and sympathetic nerves as well as in adrenals, although debates continued about the specificity of these analyses.89 Some investigators, including Bacq, proposed that noradrenaline (Fig. 3-3B) was a neurotransmitter, in addition to or instead of adrenaline.90 Although that issue was not resolved by 1945, it was clear that adrenaline-like substance(s) were present, were released, and mimicked the effects of sympathetic stimulation.

Acetylcnoline ana Preganglionic Autonomic Nerve Endings

While cataloging the pharmacological properties of acetylcholine in 1914, Dale distinguished two opposing sets of actions.91 (1) Administering acetylcholine to cats lowered blood pressure, slowed the heart rate, speeded peristalsis, constricted the pupils, etc.—the well-known array that muscarine elicited and

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atropine blocked. These Dale now classed as "muscarine actions" of acetylcholine (later called "muscarinic actions"). (2) But in the presence of atropine, larger doses of acetylcholine produced the opposite effects: elevated blood pressure, accelerated heart rate, retarded peristalsis, dilated pupils, etc.92 Such effects, as Langley had shown during his identification of autonomic ganglia,93 could be elicited by low doses of nicotine as well as by stimulating sympathetic preganglionic fibers. On the other hand, Langley had also shown that higher doses of nicotine blocked these effects. Dale now found that such doses of nicotine blocked responses to acetylcholine plus atropine as well, responses he labeled "nicotine actions" (later called "nicotinic actions"). These distinctions reflected anatomical as well as pharmacological differences. Muscarinic responses occurred at synapses between postganglionic parasympathetic neurons and their effector cells (smooth muscles and glands). Nicotinic effects were localized to synapses in autonomic ganglia between preganglionic fibers and postganglionic neurons of both sympathetic and parasympathetic divisions.94 The accumulating evidence for chemical transmission by postganglionic neurons suggested that chemicals might mediate synaptic transmission by preganglionic neurons as well. Dale's demonstration suggested what the chemical might be. Contradicting these parallels were differences in time courses for transmission at these loci, differences as striking as those in susceptibilities to blocking agents. Muscarinic responses to the postganglionic release of acetylcholine, according to Dale, "have a long latency, rise slowly to a maximum with repetitive stimulation of the nerve, and . . . outlast the period of such stimulation"; by contrast, nicotinic responses to preganglionic neuronal activity "had the appearance of a direct, unbroken, physical propagation [with a] transmission delay [of] at most a very few milliseconds."95 Nevertheless, Chang and Gaddum in 1933 identified acetylcholine in the sympathetic chain, placing this potential neurotransmitter at the appropriate site.96 The following year Feldberg and Gaddum found that stimulating preganglionic fibers to sympathetic ganglia released acetylcholine into the perfusion fluid.97 Feldberg then localized the acetylcholine to preganglionic fibers: he cut these preganglionic fibers, allowed the processes peripheral to the cut to degenerate, and showed that acetylcholine disappeared from the ganglia.98 Moreover, nicotine did not affect acetylcholine release into the perfusion fluid when it inhibited transmission through the ganglia; instead, it blocked the response of the postganglionic neurons to acetylcholine.99 Further support came from studies on adrenals. The adrenal medulla is related embryologically to sympathetic ganglia and their postganglionic neurons, and the nerves evoking adrenaline release from adrenal medullas correspond to preganglionic fibers. Accordingly, Feldberg and Minz found that stimulating these nerves released acetylcholine.100

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Together, these experiments demonstrated that acetylcholine was present in the terminals of preganglionic neurons, that stimulating preganglionic fibers released acetylcholine, and that added acetylcholine mimicked the effects of such stimulation. But several decades passed before a mechanistic explanation could account for the different time courses of synaptic transmission at muscarinic vs. nicotinic sites (chapter 7).

Chemical Transmission at Neuromuscular Junctions

Determining how nerve impulses induce skeletal muscles to contract was a similar problem, approached similarly. Again, new insights emerged from exploring the properties of natural toxins. Claude Bernard began by defining the site at which curare (Fig. 3-6F), an arrow poison extracted from certain South American plants,101 produced paralysis. His initial experiments, summarized in 1856,102 demonstrated that electrical stimulation of the nerves failed to evoke muscle contractions in frogs injected with curare, although direct electrical stimulation of the muscles evoked the usual response. Subsequently, Bernard showed that applying curare to a limited region of the frog (1) blocked responses to motor nerves innervating muscles within that region, but (2) did not affect responses of sensory nerves carrying impulses from that region. This distinction was consistent with curare imparing nerve-muscle interactions but not nerve conduction (at least in sensory nerves). Nevertheless, various reports over the following decades proposed a range of actions, from curare indeed affecting nerve conduction to it "paralyzing" nerve endings. With the turn of the century, however, studies using purified curare revealed no alteration in nerve action potentials.103 And in 1905 Langley described explicit experiments showing that curare acted on muscle after all, but in a quite localized fashion.104 Nicotine affected striated muscle of chickens as it did ganglia: low doses stimulated whereas high doses inhibited the response to nerve stimulation. Curare blocked this stimulation by nicotine;105 moreover, the antagonism between curare and nicotine persisted in denervated muscles. Langley concluded that "nicotine and curari [sic] do not act on the axonendings but on the muscle itself . . . by combining with the receptive substance."106 Transmission at neuromuscular junctions was therefore through chemical actions: stimuli passing by the nerve cannot affect the contractile molecule [of muscle] except by the radicle which combines with nicotine and curarif; this] seems to require that the nervous impulse should not pass from nerve to muscle by an electric discharge, but by the secretion of a special substance at the end of the nerve.1107

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This interpretation echoed not only Elliott's proposal but also a suggestion by Emil du Bois-Reymond in 1877: There must be either a stimulating secretion in the form perhaps of a thin layer of ammonia or lactic acid . . . on the outside of the contractile tissue so that violent excitation of the muscle takes place [after nerve stimulation], or the influence must be electric.108

Indeed, du Bois-Reymond then argued against electrical transmission on theoretical grounds.109 Despite du Bois-Reymond's stature as a founding father of physiology, scientific opinion in his and Langley's times favored electrical transmission. In 1888 Willy Kiihne in Heidelberg stated that "a nerve only throws a muscle into contraction by means of its current of action"; Kiihne, a pioneering microscopist, had observed what became known as the muscle "endplate," where "[n]erves end blindly in the muscles," an apposition he thought favored electrical transmission.110 And in the early decades of the twentieth century Louis and Marcelle Lapicque in Paris developed an encompassing theory of electrical transmission rooted in the relationship between the strength of stimulation required to evoke responses and the duration of that stimulation.111 The minimal voltage necessary for excitation decreased as the duration was prolonged. The Lapicques noted that plots of such strength-duration relationships appeared hyperbolic, with the strength approaching asymptotically, as the duration was prolonged, a limiting value they called the "rheobase." From these strength-duration plots they then calculated a time, the "chronaxie," equal to the duration of stimulation required when the stimulation strength was twice rheobase. The Lapicques asserted that effective transmission at synapses required "isochronicity." Elements on either side of the synapse must have the same chronaxie, and altering the chronaxie of either—producing "heterochronicity"—impeded transmission. They argued that curare blocked transmission in just this fashion: by increasing the chronaxie of muscle. Keith Lucas in Cambridge, however, obtained different results and drew different conclusions. Like the Lapicques, Lucas viewed neuromuscular transmission as an electrical process. But in 1907 he described triphasic plots of minimal current strength for excitation vs. current duration.112 These phases he attributed to different strength-duration relationships for muscle, for nerve, and for a third "substance," which he identified as the junctional material. Furthermore, he found no effect of curare on the strength-duration plots for muscle. Lucas died during the war, and for decades thereafter the Lapicques forcefully defended their observations of isochronic plots for nerve and muscle, the effects of curare on these, and the consequent significance for junctional transmission.113 But in 1930 W. A. H. Rushton in Cambridge reinvestigated Lucas's

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conclusions and addressed the Lapicques' specific criticisms (these centered on possible experimental artifacts).114 Like Lucas, Rushton found that muscle and nerve had quite different chronaxies, although he could not distinguish Lucas's third substance. He also confirmed Lucas's observation that curare did not alter the chronaxie of muscle.115 And in 1932 Harry Grundfest, then in Philadelphia, where Rushton was visiting, confirmed Rushton s results using single nerve and muscle fibers to avoid heterogeneous responses: he recorded two strength-duration curves, attributable to nerve and to muscle, with different chronaxies unaffected by curare.116 Despite such disputes, the concept of electrical transmission dominated these decades. Loewi himself proclaimed in 1934: "Personally I do not believe in a humoral mechanism existing in the case of striated muscle."117 Nevertheless, experiments in the 1920s, patterned on Loewi's demonstrations, had revealed acetylcholine-like material appearing in the perfusion media after nerves to striated muscles were stimulated.118 In 1933 Feldberg, while still in Berlin, employed his leech assay to identify the released material as acetylcholine.119 And in 1936 Feldberg, with Dale and Marthe Vogt, another refugee from Germany, showed, by selectively destroying sensory and autonomic fibers in the nerve, that acetylcholine came from motor fibers.120 Acetylcholine release began when nerve stimulation started, ceased when stimulation stopped, and occurred even in the presence of curare, when muscle contraction was prevented. These results related acetylcholine release to motor nerve activity convincingly, but causal interpretations were strongly hampered by failures to show that administering acetylcholine produced true contraction of striated muscles. Instead, early reports described "contractures": prolonged shortenings with slow onsets that were not associated with characteristic muscle action potentials.121 (With mammalian muscle acetylcholine evoked contractures only after denervation.122) In 1933, however, Feldberg obtained responses from innervated mammalian muscles by injecting acetylcholine into an artery leading to that muscle.123 Then, with Dale and G. L. Brown, an electrophysiologist Dale recruited for these studies, Feldberg reported in 1936 that "close arterial injection" not only produced responses without prior denervation, but that the responses resembled true contractions; they concluded that "acetylcholine . . . is liberated by [the] arrival of nerve impulses at the nerve ending, and destroyed by a local concentration of cholinesterase" to achieve the transient response of a true muscle "twitch."124 Brown supported this assertion with electrical recordings showing appropriate muscle action potentials.125 Large doses of acetylcholine, on the other hand, caused contractures. Fritz Buchtal and J. Lindhard in Copenhagen administered acetylcholine even more discretely, using microsyringes to deliver minute volumes to the

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endplate region where motor nerve endings terminate.126 After a single administration of acetylcholine, sufficient to produce a contraction, muscles would not respond to further acetylcholine until the first had been removed or destroyed; in this situation contractions could not be evoked by stimulating the motor nerve, either. On the other hand, single large doses of acetylcholine caused contractures. These observations finally linked motor nerve activity with acetylcholine release. An appropriate dose of acetylcholine could elicit genuine contractions, whereas large doses produced contractures. And acetylcholine's stimulation could be mimicked by nicotine and blocked by curare.

Chemical Transmission in the Central Nervous System

Aware of Loewi's experiments on Vagusstoff, Edgar Adrian in Cambridge suggested in 1924 that central inhibitory effects might be mediated by chemical transmitters similarly.127 The following year Charles Sherrington in Oxford acknowledged the same notion.128 But characterization of chemical transmission in the brain and spinal cord progressed slowly, due in large part to formidable difficulties. As Minz observed: the extreme anatomical complexity . . . with its innumerable physiologically interlocking pathways . . . excludes the possibility of analyzing drug effects on single isolated units . . . and thus eliminates a source of information to which we owe very precious knowledge . . . of neuro-muscular and ganglionic transmission.129

Moreover, attention during these decades was focussed almost exclusively on acetylcholine in light of successes at other sites (and a paucity of alternative candidates). Here I will note briefly efforts, as in studies at other loci, to show acetylcholine's presence, its release during neural activity, and its ability to mimic that activity. Chang and Gaddum, as cited above, reported in 1933 the presence of acetylcholine in the central nervous system as well as in other tissues. In 1941 F. C. Macintosh in London described an uneven content of acetylcholine among different portions of the brain and spinal cord.130 Such local variations accorded with principles of neural localization attributing specific functions to particular areas of the brain: acetylcholine might mediate only certain functions in certain locales. (What mediated other functions in other locales was a different issue.) Since many central neurons are continuously active, acetylcholine release might be expected even in the absence of experimental stimulation. In 1936 Feldberg described an unstimulated release into the cerebrospinal fluid of dogs

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given physostigmine intravenously, and these observations were subsequently confirmed.131 Demonstrating a stimulated release was more difficult. Stimulating sensory nerves generally produced no measurable increase in acetylcholine release, although in 1941 Edith Biilbring and J. H. Burn in Oxford reported that stimulating the sciatic nerve caused a release of acetylcholine from the spinal cord.132 Other modes of activating the nervous system were generally more successful. Minz found an increased release of acetylcholine after electrically stimulating the spinal cord, and Feldberg after injecting adrenaline or potassium chloride intravenously.133 But—in contrast with experiments on the autonomic nervous system and neuromuscular junctions—none of these studies could correlate a discrete release of acetylcholine with the stimulation of particular fibers evoking specific physiological responses. Attempts to show that added acetylcholine initiated particular physiological responses were equally fragmentary. In 1934 B. B. Dikshit in Edinburgh reported that injecting acetylcholine into the cerebral ventricles reproduced some effects of vagal stimulation: depressed respiration and decreased heart rate.134 Nils Emmelin and Dora Jacobsohn in Lund then described depressed respiration after injecting acetylcholine close to the hypothalamus, results mimicking electrical stimulation of this region of the brain.135 Others cataloged various inhibitory or excitatory effects of administering acetylcholine.136 But as Samson Wright in London observed in 1944, there were "striking differences in the actions [of acetylcholine] in different species, in different preparations and under different anesthetics."137 Together, these findings at most suggested a role for acetylcholine in central neurotransmission. Perhaps the strongest argument for chemical transmission were analogies with the autonomic nervous system and neuromuscular junctions.

Electrical Transmission

Opposing these formulations were continuing arguments for electrical transmission based on experimental as well as theoretical grounds and bolstered by considerable achievements in electrophysiology. Technical advances during these decades facilitated better quantification of better differentiated phenomena. In 1920 Alexander Forbes in Boston introduced the vacuum tube amplifier to neurophysiology, making detectable electrical responses previously unmeasurable, and in 1922 Herbert Gasser and Joseph Erlanger in St. Louis adapted cathode ray tubes to display signals free from distortions inherent in earlier recording devices.138 Gasser and Erlanger then found that action potentials in nerve trunks had complex wave forms, representing different fibers conducting at different velocities; various agents suppressed specific responses, allowing a classification of fibers by that sensitivity and also by conduction veloc-

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ity and fiber diameter.139 Reevaluations of the synaptic delay, calculated in light of different conduction velocities in different fibers, gave values as low as a millisecond or less.140 And in 1928 Adrian described impulses from a single neuron by cutting away all but one fiber of a nerve trunk.141 During these decades Sherrington and his collaborators extended earlier analyses of reflex action. In particular, Sherrington enunciated the concepts of "central excitatory state" and "central inhibitory state," identified as transient increases or decreases, respectively, in excitability following a stimulus.142 For example, a weak sensory stimulus to the spinal cord may fail to evoke efferent motor responses, but a second weak stimulus, by itself also subthreshold, may do so if it follows within a brief interval. The observed summation Sherrington attributed to "an enduring central excitatory process," and he suggested two possible mechanisms: that the "electrical processes of successive nerveimpulses summate," or that each impulse "produces a quantum of exciting agent, a chemical substance, which sums with other quanta formed at the same or neighboring points by other impulses."143 Prominent among physiologists advocating electrical transmission and rejecting Sherrington's second alternative was John Eccles, Sherrington's former student and collaborator.145 Eccles was an ingenious and resourceful critic of chemical transmission in ganglia, at neuromuscular junctions, and in the central nervous system, although he accepted the principle of chemical transmission at terminals of postganglionic autonomic fibers. Indeed, he and Brown in 1934 specified characteristics that pointed to a chemical link at a postganglionic parasympathetic site: long latencies between vagal nerve impulses and changes in heart rate, roughly a hundred milliseconds; prolonged durations of these responses after vagal stimulation ceased, lasting for seconds; and extensions of these durations after administering physostigmine.145 These observations fitted with a slow diffusion of acetylcholine from nerve to heart and a slow inactivation of the acetylcholine, inhibitable by physostigmine.146 Such characteristics Eccles contrasted with synaptic properties at other sites: latencies of a millisecond or less, responses lasting only milliseconds, and—he claimed—no prolongation by physostigmine.147 For these sites Eccles proposed a conduction sequence of impulse at synapse, transmitter action, "detonator response," and impulse generation in postsynaptic cell.148 The transmitter action Eccles considered to be electrical. The detonator response (so named because it set off an "explosive" postsynaptic impulse) was the resulting electrical change in the postsynaptic cell that grew in a millisecond or so, accounting for the synaptic delay, and then disappeared within a few milliseconds, consistent with the brief duration of the transmitter action. Eccles acknowledged accumulating evidence that implicated acetylcholine's action at such synapses. He, however, relegated acetylcholine to a secondary role in a dual mechanism: the primary neurotransmitter was electrical, but chemical neurotransmitters

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could be responsible for slower and longer influences, detectable as a tail to the detonator response.149 But brief latencies could occur with chemical neurotransmitters also if release sites were close to receptors, thereby minimizing diffusion time. And brief durations would follow a rapid removal of the neurotransmitters, as by enzymatic destruction. Cholinesterase activity could be measured in tissue samples, but the rate of acetylcholine hydrolysis at the synapse could not; a corresponding problem was determining whether administered physostigmine inhibited cholinesterase at the synapse fully. Eccles initially claimed that physostigmine had no effect at ganglia, but Rosenblueth in 1938 argued that Eccles had not used sufficient physostigmine; indeed, Rosenblueth found prolonged responses to elecrical stimulation with higher concentrations of physostigmine.150 On the other hand, Dale pointed out that high doses of acetylcholine could depress synaptic transmission, implying that too much physostigmine could interfere with cholinergic transmission as well as too little.151 Eccles in 1944, now returned to Australia, acknowledged that "the very prolonged transmitter action which appears [after administering physostigmine to ganglia] is due to acetylcholine," but this concession was still in the context of his dual mechanism.152 Joined by two able emigres from Europe, Bernard Katz and Stephen Kuffler, Eccles had been investigating "endplate potentials" at neuromuscular junctions, brief electrical changes in muscle elicited by nerve stimulation (see chapter 4). They agreed in 1941 that the "transient effect on the muscle membrane [might be due to] a chemical transmitter such as acetylcholine," even conceding that evidence "favour[ed] the acetylcholine theory."153 The following year they noted "indications" that endplate potentials were "set up by a chemical membrane action rather than by extrinsic currents from the motor nerve."154 But at mid-decade Eccles was proclaiming electrical transmission vigorously, drawing potential contours for model synapses that would enable intercellular excitation, citing recent experiments demonstrating excitation between experimentally apposed nerves, and accounting for unidirectional electrical transmission at synapses by the local geometries.155

Concr nclusions

Loewi and Dale shared the Nobel Prize in 1936 that rewarded their complementary achievements and careers. Loewi's scientific lineage stretched back to Schmiedeberg, acclaimed as the "father of pharmacology," and to the nineteenthcentury titans of German physiology. Loewi progressed through German and Austrian universities before a forced departure to England in 1938 and emigration to New York as war broke out. Dale, who profitably examined other topics as well (notably the actions of histamine), attained a rank in neurophar-

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macology comparable to Cajal's in neuroanatomy and Sherrington's in neurophysiology. Dale came from the Cambridge of Foster, Gaskell, Langley, Elliott, Dixon, Lukas, and Adrian, but he abandoned academe for an industrial laboratory in 1904; in 1914 he moved to a precursor of the National Institute for Medical Research. Appreciations of Loewi s contributions often center on his experiments published in 1921. Their general design, allegedly revealed in a dream, seem nowadays more trivially obvious than some subtle synthesis of protracted ruminations, conscious or not. As Dale noted in 1934, these experiments "demanded no special techniques or apparatus [and] might. . . have been made at any time during the fifteen years . . . since the idea of ... chemical transmission . . . first took shape."156 In fact, Dixon attempted quite similar studies 15 years earlier, but his extraction procedure was too harsh for labile substances like acetylcholine; Dixon's choosing extraction over perfusion probably reflected his sense that nerves abutted muscles tightly, so neurotransmitters would pass directly from neuron to muscle cell. Loewi, moreover, was fortunate in using frogs having low levels of cholinesterase activity and experimenting at colder temperatures where that activity would be still less. And Loewi was fortunate, as he acknowledged, in doing the experiment before assessing its unlikeliness: If I had carefully considered in the daytime I would undoubtedly have rejected the kind of experiment I performed. It would have seemed likely that any transmitting agent released by a nervous impulse would be in amount just sufficient to influence the effector organ. It would seem improbable that an excess that could be detected would escape into the fluid which filled the heart.157

More significantly, Dixon abandoned his approach without exploring alternatives, whereas Loewi met criticisms with reaffirmations, with a public demonstration in 1926 at the International Congress of Physiology in Stockholm, and with improved experiments. Loewi also pursued the implications of his experiments astutely. He showed that perfusates from stimulated hearts contained a choline-like substance, that cholinesterase activity was present, that physostlgmine (previously thought to act by exciting nerves) inhibited cholinesterase activity, and that physostigmine potentiated responses to both Vagusstoff and acetylcholine. These crucial developments, in the context of replications and extensions by others, secured a firm experimental basis for chemical transmission from vagus nerve to heart. Dale, with Feldberg and Feldberg's assay, confirmed the identification of Vagusstoff with acetylcholine and extended the formulation to autonomic ganglia and neuromuscular junctions. "From isolated facts of apparently moderate significance there emerged general conclusions of high value."158 Acetylcholine was present, it was released by nerve stimulation, and its administration mimicked the results of such nerve stimulation at synapses of postganglionic

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parasympathetic neurons, of preganglionic autonomic neurons, and at neuromuscular junctions. Adrenaline (or some closely similar substance) was present, was released, and mimicked stimulation of postganglionic sympathetic neurons. Analogies with these results encouraged the pursuit of acetylcholine as a neurotransmitter in the central nervous system also. This accumulated evidence convinced even those who proclaimed electrical transmission. They then compromised by asserting dual mechanisms: electrical transmission for the initial fast response and chemical transmission for the slower prolonged response that followed. Neither party to the controversy could devise—with methods then available—discriminating experiments. They could not show that electrical impulses in presynaptic neurons could induce currents in postsynaptic cells sufficient to generate new impulses (or that any induced currents were insufficient). And they could not show that neurotransmitters released at the synapse could induce the immediate, transient response seen with nerve stimulation (or that such release produced only delayed and protracted responses).

Notes 1. For historical accounts, see Bacq (1975); Cannon (1934); Clarke and O'Malley (1968); Dale (1934, 1958); Davenport (1991); Eccles (1959); Feldberg (1977); Pick (1987); Finger (2000); Gerst and Brumback (1984); Grundfest (1957a); Holmstedt (1975); Holmstedt and Liljestrand (1963); Mclntyre (1947); Sinister (1962); Thomas (1963); Whitteridge (1993). 2. Bishop (1941), pp. 1, 3. 3. Cole and Curtis (1939); Cole and Hodgkin (1939); Hodgkin and Huxley (1939). 4. Boeke (1965), p. 309, italics in original. The original edition was published in 1932. For early physiological arguments against such neurofibrillar functioning, see Langley (1901a); for general discussions, see Parker (1929) and Nonindez (1944). 5. Ramon y Cajal (1934). 6. Bartelmez and Hoerr (1933), pp. 401, 426. They also described how successive sections of a tissue could show either neurofibrillar continuity or discontinuity, depending on the fixation conditions. 7. Bodian (1937), p. 118. 8. Bodian (1942), p. 150. 9. For historical accounts, see Clarke and Jacyna (1987); Gaskell (1916); Hoff (1940); Langdon-Brown (1939); Sheehan (1936, 1941). 10. Gaskell (1886, 1889). 11. For earlier reports of similar studies by others, see Sheehan (1941). 12. Multitudes of new plants and animals, identified in voyages of discovery, were then being studied for practical as well as scientific value. Langley began, at Fosters suggestion, with pilocarpine ("jaborandi") and moved to nicotine ("pituri") when a supply was offered to him (Fletcher, 1926). 13. Langley and Dickinson (1889); Langley (1893). 14. Langley (1893, 1898, 1905). Gaskell preferred "involuntary" to "autonomic."

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15. For historical accounts, see Barcroft and Talbot (1968); Dale (1948); Oliver (1895). 16. Barcroft and Talbot (1968) point out that oral administration of adrenaline should have no effect on arteries. 17. Cited in Oliver and Schafer (1895). 18. Oliver and Schafer (1895). Preliminary accounts were published in 1894 and 1895. 19. Ibid., p. 247. 20. They interpreted the fall in heart rate as a reflex response (parasympathetic) to the rise in blood pressure. 21. Langley (1901b). This paper cites intervening studies. 22. Takamine (1901). For a historical account, see Davenport (1982). 23. Actually, Abel named his material "epinephrin." Tansey (1995) described Dale's successful struggle with the Burroughs, Wellcome hierarchy in 1906 to use adrenaline when Parke, Davis had Adrenalin as a trademark. Dale, however, reverted to adrenine in 1910. 24. Aldrich (1901, 1905). Aldrich had worked under Abel at Johns Hopkins. 25. Dakin (1905). Stolz (1904) in Germany also synthesized that structure. 26. Elliott (1904), p. xxi. 27. Elliott (1905), p. 466. Elliott thanked Langley, Gaskell, and Dixon. Notable is a citation to Kipling's The Jungle Book, concerning mongooses ruffling their fur. 28. Elliott's sole reference to his proposal is buried in a section on the manner of disappearance of adrenaline in the tissues. Here he refered to "the conjecture that [adrenaline] is concerned in the transference of a sympathetic nervous impulse, and stored to such an end in the neighbourhood of the myoneural junction." But he then dismissed this along with the preceding notions: "The evidence does not conclusively disprove any of these." Elliott (1905), p. 455. 29. Langley (1905), p. 183. Langley called attention to discrepancies reported in Langley (1901b), which compared the degree of responses to adrenaline vs. sympathetic stimulation, and in Elliott (1905), which noted opposite effects, such as pupilary constriction in dogs with adrenaline vs. dilatation with symapthetic stimulation. Langley (1906, p. 191) also stated that "some tissues are readily affected by stimulation of the sympathetic nerves, and barely at all, or only in enormous doses, by adrenalin." 30. Dale (1958). 31. Dale (1906), p. 206. He thanked Elliott for suggestions and for help with some experiments. 32. Barger and Dale (1910), p. 21. Barger synthesized the compounds and Dale tested them. 33. Ibid., p. 54. 34. Feldberg (1977) noted an attempt by Elliott in 1914 to identify a chemical agent acting at synapses on muscle and on fish electric organ, and his questioning Elliott about this in 1942, but Feldberg did not state whether he asked about adrenaline. Dale (1961), in his memoir of Elliott, noted that Langley supervised Elliotts research and had a strong aversion to speculation. Dale also pointed out Elliott's apparent renunciation in 1914 of his earlier view: "It is always a pleasure, and therefore a temptation, to accept a theory which harmonizes all the facts into a close-fitting plan. But the evidence at present does not justify us in welcoming this simplification" (p. 1395). What theory Elliott is there referring to, however, is not clear; it may be that Elliott is concerned with the notion that adrenergic nerve endings do not synthesize adrenaline but take it from the circulation. 35. Langley (1905), p. 375. Boeke (1965) suggested that Langley's receptive substances were neurofibrils.

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36. Langley (1905), p. 400. 37. Ibid., pp. 404, 412. 38. Langley (1906), p. 194. 39. Ibid., p. 181. 40. Bayliss and Starling (1902), p. 340. They also coined the term hormone for bloodborne substances acting at sites distant from their origins, like secretin. 41. Dixon (1906), p.1807. 42. Dixon (1907). The nature of Dixon s active substance is unknown, but acetylcholine is unlikely to have survived his extraction procedure. Dale (1934) suggested that it was choline. 43. Dixon (1907), p. 457. Elliott, however, thanked Dixon for help. 44. Quoted in Dixon's obituary (Gunn, 1932). Dixon and Hamil (1909, p. 335) proposed that "excitation of a nerve induces the local liberation of a hormone which causes specific activity by combining with some constituent of the end organ, muscle, or gland"; a footnote stated that experimental evidence would be presented subsequently, but this did not appear. 45. Hunt (1901). 46. However, the drop in blood pressure due to this other substance was not blocked by atropine; Dale (1934) suggested that it was not a precursor but probably histamine. 47. Hunt and Taveau (1906). They later surveyed a dozen groups of homologous compounds (Hunt and Taveau, 1909). 48. Hunt and Taveau (1906). Hunt made this suggestion at the same meeting where Dixon described inhibitory effects of heart extracts, but, as Dale pointed out, "Neither, apparently, saw any connection between the two sets of observations" (1937, p. 230). 49. Ewins (1914); Dale (1914). 50. Dale (1914), p. 189. 51. Loewi (1945a). 52. Loewi (1954). 53. Loewi (1960), p. 17. Cannon (1934) gave a slightly different account; see also Weiss and Brown (1987); Davenport (1991). Loewi's dating requires that a year passed before he reported his experiments. He cannot be merely a year off, because Easter of 1921 fell after he submitted his manuscript. Possibly both the year and the Easter date were misremembered. 54. Ringer's solutions are artificial salt solutions mimicking the composition of extracellular fluids, named from Sidney Ringer's description in 1883. 55. Loewi (1960), p. 17. 56. Loewi (1921). 57. Bacq (1975), p. 15. 58. Minz (1955, p. 12) noted that "no other pharmacological test shows so many irregularities as ... the frog's heart [preparation that Loewi used, with] grouped beats, spontaneous acceleration, spontaneous block, [and] increase and decrease of the height of contraction. . . . Nothing is easier than to stop such a heart. The smallest trace of blood serum is sufficient and one can obtain slowing or total block of the control heart simply by adding the liquid of a fresh normally beating heart without any . . . stimulation. On the other hand, when the heart is very carefully washed for a long time one can be sure to get a hypodynamic heart and it is a very embarassing fact that just those kinds of hearts give the best results in Loewi's experiments." 59. Loewi (1922). 60. In his 1921 paper Loewi showed that stimulating the vagus trunk in a toad

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increased the contractions of that toad's heart (his Fig. 3a), but this is his only portrayal of a donor heart. 61. Loewi and Navratil (1924). 62. For example, Asher and Scheinfinkel (1927). Kahn (1926) cites criticisms. 63. For example, Bain (1933); Brinkman and van Dam (1922); Engelhart (1931); Kahn (1926). 64. Bain (1932). For a textbook reproduction see Goodman and Gilman (1956). 65. In 1921 Loewi concluded that the active material was not potassium ions (K + ), as Howell had suggested (Howell and Duke, 1910). 66. Loewi cited Hunt, but he was aware that no ester of choline had yet been identified in animals. 67. Loewi and Navratil (1926a). 68. Loewi and Navratil (1926b). Earlier, Dixon and Brodie (1903) reported that physostigmine potentiated the effects of vagal stimulation on the lungs, and Anderson (1904) described antagonistic effects of atropine and physostigmine on the iris. Since Anderson showed that physostigmine was ineffective in denervated irises, Langley (1905) concluded that physostigmine acted on nerve endings. 69. Matthes (1930); Engelhart and Loewi (1930). 70. Dale and Dudley (1929). They discovered acetylcholine by accident while searching for histamine. The functional significance of high levels of acetylcholine in bovine spleens remains unknown. 71. Chang and Gaddum (1933). 72. Dale (1934, p. 838) stated that "when . . . the activity of a solution containing the [unknown] neurotransmitter is matched by the same strength of acetylcholine [in different bioassays], we can be practically certain that we are dealing with [acetylcholine] and with no other choline ester." 73. Feldberg and Krayer (1933); Minz (1932). Physostigmine was given to the donor animals and was present in the leech bioassay to prevent destruction by cholinesterases at all stages. 74. Dale and Feldberg (1934). Dale considered that atropine was ineffective against vagal stimulatation at these sites due to poor access: the nerve ending might be tightly apposed to the muscle, whereas added acetylcholine and added atropine must diffuse into this region. 75. Loewi (1922). Since Acceleransstoff was destroyed by heating, it could not be an inorganic substance like calcium ions (whose effects on the heart Loewi had studied). 76. Loewi and Navratil (1926a). 77. Loewi (1936). For specificity Loewi relied on the demonstration by Gaddum and Schild (1934) that fluorescence in the presence of alkali was due to adrenaline; Loewi repeated that "substances related to adrenaline show this reaction only in concentrations of a much higher order of magnitude" (1945, p. 806). 78. For example, Brinkman and van Dam (1922); Kiilz (1928); Finkleman (1930); Bacq (1933); Bain (1933). 79. Dale and Feldberg (1934). Cats have sweat glands in their footpads. 80. Ibid., p. 125. 81. Dale (1933), p. IIP. 82. Cannon and Uridil (1921). These experiments arose from a preceding controversy concerning emotions and adrenal secretions (Barger, 1992). 83. Newton et al. (1931); Cannon and Bacq (1931). 84. Cannon and Rosenblueth (1933).

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85. The nictitating membrane is a clear protective membrane that moves across the eye, under autonomic control. 86. Cannon and Rosenblueth (1933), p. 568. 87. See Minz (1955), p. 126. 88. Bacq (1975), p. 43. 89. For example, Shaw (1938); Raab (1943). 90. Bacq (1934); Stehle and Ellsworth (1937); Greer et al. (1938). 91. Dale (1914) 92. He noted that such sympathetic effects of administering acetylcholine were "seen best in a cat which has had the spinal cord cut in the neck and the brain destroyed" to diminish further any parasympathetic effects of acetylcholine (ibid., p. 157). 93. Langley and Dickinson (1889). 94. Most studies of nicotinic effects deal with sympathetic responses, both because atropine is often added to block postganglionic parasympathetic responses to added acetylcholine and because sympathetic ganglia are more accesible. 95. Dale (1938b), pp. 416, 417. 96. Chang and Gaddum (1933). 97. Feldberg and Gaddum (1934). They identifed acetylcholine, after perfusion with physostigmine, by six different bioassays. Their study followed a similar one reporting acetylcholine release into perfusion media of a substance that could activate ganglia (Kibjakow, 1933). However, Kibjakow did not perfuse with physostigmine, and Feldberg and Gaddum reported that they could not reproduce Kibjakows experiment "with any regularity" (p. 306). 98. Brown and Feldberg (1936b). 99. Feldberg and Vartiainen (1934). Langley (1901a) concluded that nicotine acted on postganglionic cells in ganglia since nicotine was effective after preganglionic fibers degenerated. 100. Feldberg and Minz (1933); Feldberg et al. (1934). 101. Various arrow poisons contained different mixtures of ingredients in addition to curare; moreover, three varieties of curare were initially identified by the containers in which they reached investigators: pots, gourds, and tubes. The purified active ingredient from the last of these, tubocurarine (Fig. 3-5F), has been studied most. Here I use the term curare generically. 102. Bernard (1856). A translation appears in Shuster (1962). 103. For example, Garten (1912). 104. Langley (1905). 105. Curare can also block transmission at autonomic ganglia: it is antagonistic to nicotinic actions of acetylcholine at both sites. 106. Langley (1905), pp. 411, 400. 107. Langley (1906), p. 183. 108. du Bois-Reymond (1877), as translated in Clark and O'Malley (1968), p. 241. Dale (1938a) considered this the first enunciation of chemical transmission. Langley also referred to du Bois-Reymond's formulation, although without specific citation. 109. Grundfest (1957a) summarized some criticisms of electrical transmission elaborated by du Bois-Reymond. 110. Kiihne (1888), pp. 446, 441. 111. Lapicque and Lapicque (1908); Lapicque (1909); Lapicque (1926). 112. Lucas (1907a, b, c). 113. For example, Lapicque (1931, 1934).

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114. Rushton (1930). 115. Rushton (1933). 116. Grundfest (1932). He also demonstrated that the measured chronaxie varied with type and size of electrode as well as with the positioning of electrodes along the fibers. 117. Loewi (1934), p. 232. 118. For example, Brinkman and Ruiter (1924); Shimidzu (1926). 119. Feldberg (1933b). 120. Dale et al. (1936). 121. Riesser and Neuschloss (1921). For a contemporary discussion of contractures, see Gasser (1930). 122. Frank et al. (1922). Dale and Gaddum (1930) explained an ancient observation of E.F.A. Vulpian—that stimulating autonomic nerves to skeletal muscles deprived of their motor nerves causes contractures—in terms of parasympathetic fibers to blood vessels in the muscle releasing acetylcholine, which then diffused away to stimulate the skeletal muscle. 123. Feldberg (1933a). A further problem in studying transmission at neuromuscular junctions was interference at this site by ether anesthesia (Simonart and Simonart, 1935). 124. Brown et al. (1936), p. 423. 125. Brown (1937). 126. Buchtal and Lindhard (1942). 127. Adrian (1924). 128. Sherrington (1925). 129. Minz (1955), p. 165. 130. Macintosh (1941). 131. Feldberg and Schriever (1936); Adam et al. (1938); Chute et al. (1940). Cerebrospinal fluid resembles blood plasma in composition and is secreted by the brain; it surrounds the brain and spinal cord and also fills the cerebral ventricles (see note 134). 132. Feldberg and Schriever (1936); Adam et al. (1938); Bulbring and Burn (1941). Chang et al. (1938) described acetylcholine efflux after stimulating the vagus nerve in animals given physostigmine into the cerebrospinal fluid as well as intravenously; the vagus also carries sensory fibers having terminals within the brain. 133. Minz (1936); Feldberg and Schriever (1936); Chute et al. (1940). 134. Dikshit (1934a, b). The brain contains within it several ventricles filled with cerebrospinal fluid; these connect with each other as well as with the surface, also bathed with cerebriospinal fluid (see note 131). 135. Emmelin and Jacobsohn (1945). The hypothalamus is a region of the brain that influences many body functions through the autonomic nervous system, including respiration. 136. For example, Schweitzer and Wright (1937); Bulbring and Burn (1941); McKail et al. (1941). See also Feldberg (1945). Subsequently, Kuno and Rudomin (1966) showed that the release described by Bulbring and Burn was due to impulses passing back through motoneuron axons and then via recurrent collaterals that make cholinergic synapses on Renshaw cells in the spinal cord (chapter 4). 137. Calma and Wright (1944), p. 102. 138. Forbes and Thacher (1920); Gasser and Erlanger (1922). For historical accounts, see Finger (2000); Frank (1986); Perl (1994). 139. Erlanger et al. (1924); Bishop et al. (1933).

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140. Lorrente de No (1935); Eccles and Pritchard (1935). 141. Adrian and Bronk (1928). 142. See Creed et al. (1932). 143. Ibid. pp. 44, 45. 144. Others advocating electrical transmission included Erlanger (1939); Lorrente de No (1939); and Fessard (1951). 145. Brown and Eccles (1934a, b). 146. Eccles (1937b). 147. Eccles (1937a). 148. Eccles (1937b). 149. Monnier and Bacq (1935) also proposed a dual mechanism. 150. Rosenblueth and Simeone (1938). 151. Dale (1937). 152. Eccles (1944), p. 49. 153. Eccles et al. (1941), p. 383. 154. Eccles et al. (1942), p. 211. 155. Arvanitaki (1942); Katz and Schmitt (1940); Eccles (1946). 156. Dale (1934), p. 836. 157. Loewi (1960), p. 18. 158. Holmstedt and Liljestrand (1963), p. 185.

4 CHEMICAL TRANSMISSION AT SYNAPSES (1945-1965)

Postwar Progress Scientific accomplishments surged after World War II, due in part to confidence among the victors and to the euphoria of peace, in part to the imaginative exploitation of technical capabilities developed for that conflict, and in part to pent-up desires within the scientific community to resume interrupted interests.1 Perhaps the most significant factor, however, was public enthusiasm for the promised benefits of scientific investigation that translated into a vastly increased sponsorship. The National Institutes of Health began a generous patronage that would extend beyond the United States, wisely administered as direct grants to the individual investigators who proposed the projects2 and allocated according to the informed evaluations of their peers. Through these decades this sponsorship continued to grow as achievements accumulated and as perceived needs, ranging from health care to national prestige, were publicized broadly. New funding coupled to new expertise meant new instrumentation: new types of microscopes, centrifuges, spectrometers, and electronic devices for stimulating, recording, counting, and analyzing. And with these instruments widely available, new techniques flourished, including those for separating and visualizing subcellular components, for determining molecular structures, and

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for identifying metabolic interconversions. Analytical advances—notably through chromatographic and electrophoretic separation methods and radioactive tracer techniques—increased capabilities enormously and made measurements previously impossible now routine.3 By 1945 the successive steps of "intermediary metabolism" had been largely identified; these chronicled the conversion of glucose to carbon dioxide and water (through the pathways for glycolysis, the Krebs cycle, and oxidative phosphorylation) and the trapping of liberated energy as an "energy-rich" compound, adenosine triphosphate (ATP), available for powering cellular work. Over the next two decades the participating enzymes were then characterized and localized within the cell. (For example, in 1949 Albert Lehninger in Baltimore showed that enzymes for the Krebs cycle and for oxidative phosphorylation lay within mitochondria, organelles isolated as one of four subcellular fractions by ultracentrifugation and visualized soon thereafter by electron microscopy.) Also by 1945 George Beadle and Edward Tatum in Palo Alto had completed their studies establishing the dictum of one gene/one enzyme, and Oswald Avery in New York had identified deoxyribonucleic acid (DNA) as the chemical embodiment of genetic information. Then in 1953 James Watson and Francis Crick in Cambridge proposed a double helical structure for DNA with farreaching functional implications. Within a dozen years a host of scientists deciphered the genetic code and described the enzymatic synthesis of proteins on cytoplasmic ribosomes, a synthesis guided by messenger ribonucleic acid (mRNA) carrying genetic information from DNA in the nucleus. In the early 1950s Frederick Sanger in Cambridge reported the first amino acid sequence of a protein (insulin). A decade later John Kendrew and Max Perutz in Cambridge and David Phillips in London described from X-ray crystallographic studies the three-dimensional structures of myoglobin, hemoglobin, and lysozyme. Their structural models not only showed the a-helices and j8-sheets that Linus Pauling had predicted in 1951, they also revealed the catalytic complexes of enzyme plus substrate and the conformational changes accompanying such functional interactions. Their images accorded, too, with notions—advanced in these decades by Daniel Koshland in Brookhaven and by Jacques Monod in Paris—of how substrate binding could favor catalytically competent structures and how "allosteric" modifiers could alter structure and hence regulate activity. The long-standing mystery of how muscle contracts was resolved in these decades, also, in terms of reversible associations between two major proteins of muscle, myosin and actin. In 1954 Andrew Huxley in Cambridge, UK, and Hugh Huxley in Cambridge, Mass., independently formulated sliding filament models in which interdigitating myosin and actin molecules slid past one another to effect the shortening. Subsequent elaborations depicted peptide "side arms" of myosin cyclically binding to actin, swinging to pull actin toward

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it, and then releasing actin for a further cycle: these steps were driven by ATP binding to myosin, its hydrolysis to adenosine diphosphate (ADP) and phosphate, and then release of these products. Electron micrographs also revealed cellular membranes, structures under 100 A in thickness and thus well below the resolving power of light microscopy. In 1959 J. D. Robertson in London interpreted these images, following James Danielli's proposal from the 1930s, as a bilayer of lipids sandwiched between two layers of protein. That organization, however, provided no sense of how polar substances could cross the nonpolar membrane interior. Nevertheless, studies during the 1940s demonstrated convincingly such movements of polar substances both with and against transmembrane electrochemical gradients (passive and active transport, respectively). And in 1957 J. C. Skou in Aarhus argued that a Na + - and K+-stimulated ATP-hydrolyzing enzyme (later named the "Na+/K+-ATPase") was responsible for the active transport of Na + and K + across the outer membrane of cells, serving as a Na + /K + -pump to create asymmetric distributions of these ions between cell interior and cell environment. In 1961 Robert Crane in St. Louis and Peter Mitchell in Edinburgh presented models for "secondary active transport," in which the energy stored in such transmembrane gradients could power the transport of other substances.

Identifying Chemical Transmission Impulse Conduction Along Axons

Before considering transmission across synapses further, it is important to note contemporary advances in understanding how impulses were conducted along axons.4 Recognizing the distinctions between these two classes of processes, it turned out, was a crucial requirement for further understanding of each. Two important characteristics of nerve conduction had been established earlier. First, impulses were conducted not in a decremental fashion but at a constant, undiminished intensity in a self-propagating fashion. Second, impulses were generated in an "all-or-nothing" manner: stimulus intensities below a critical value, the "threshold," produced no propagated impulses; intensities above the threshold produced identical propagated impulses.5 Through the 1930s the most prominent—but not the only—explanations for propagated impulse conduction were based on Julius Bernstein's proposal specifying a membrane that was selectively permeable to K + at rest but became transiently permeable to other ions upon excitation. The resting potential then represented a diffusion potential for K + , described at equilibrium by the Nernst equation:

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where E m is the potential across the membrane, R the gas constant, T the absolute temperature, F Faraday's constant, and [K + ] in /[K + ] out the ratio of the K + concentration in the axonal cytoplasm to that in the surrounding medium.6 (Potentials are expressed relative to the medium defined as 0 mV; with cytoplasmic K + concentrations higher than those in the medium, the interior would then be negative.) Since, according to Bernstein's formulation, the action potential reflected a transient loss of selective permeability, the membrane potential should then fall toward 0 mV. The impulse then propagated by inducing currents, flowing in local circuits, that altered the permeability of the axon membrane just ahead of the advancing action potential. Unfortunately, such proposals could not be tested quantitatively because transmembrane potentials could not be measured directly. The routine approximation involved measuring the "injury potential": one electrode was in the surrounding medium and the second on a damaged (and therefore leaky) portion of the nerve; the measuring circuit ran from the first electrode, through the intact membrane, through the axonal cytoplasm, out the damaged membrane to the second electrode, and through a voltmeter back to the first electrode. Among the deficiencies was a short circuit through the extracellular medium between the two electrodes. When J. Z. Young described to neurophysiologists in 1936 the giant axons of squid, with diameters of 0.5 to 1 mm, they soon recognized the experimental opportunities.7 In 1938 K. S. Cole, joined in Woods Hole by H. J. Curtis and Alan Hodgkin, evaluated membrane resistances during rest and after stimulation with external electrodes: resistance fell 400-fold while an action potential passed, consistent with Bernstein's formulation.8 The following year Hodgkin, joined in Plymouth by Huxley, inserted a fine glass cannula longitudinally down a squid axon through a nick in its surface; they then measured directly the transmembrane potential between an electrode in the cannula and an electrode in the bathing medium (Fig. 4-1 A).9 The resting potential was about —50 mV, somewhat less than that predicted by the Nernst equation.10 The action potential, on the other hand, overshot 0 mV and rose to about +40 mV, in sharp contradiction to Bernstein's formulation. Hodgkin and Huxley published a brief report just as the war began and a fuller description afterward, but neither accounted for the overshoot convincingly.11 By 1952, however, they had encompassed all these issues in a paragon of physiological explanation that became the foundation for all further advances in understanding axonal conduction. From precise experiments measuring transmembrane currents and voltages in the presence of varied external media, Hodgkin, Huxley, and in some important studies Bernard Katz collected the necessary data for evaluating the variables of an equivalent circuit for the axon membrane (Fig. 4-1B).12 The equation describing that circuit, the "HodgkinHuxley equation," could then reproduce quantitatively the shape and charac-

FIGURE 4-1. Nerve action potentials. A. The resting and action potentials of a squid giant axon were recorded between a cannula inserted in the axon and an electrode in the seawater bath. The vertical scale is in millivolts (bath defined as 0 mV), and the truncated sine wave at the bottom (500 Hz) indicates time. B. The equivalent circuit for squid axon membrane depicts membrane capacitance (CM), currents for Na + , K + , and other ions, L (lN a , IK, and IL), and the corresponding batteries (E) and resistances (R); resistances to Na + and K + are variable. These components were readily interpretable as biological entities, the capacitance as the insulating membrane lipid bilayer, the batteries as the ion gradients, and the variable resistors as selective channels for specific ions whose conductances were sensitive to time and voltage. (A. from Hodgkin and Huxley [1939], Fig. 2, © Macmillan Magazines Ltd., reprinted by permission. B. from Hodgkin and Huxley [1952], Fig. 1, courtesy of the Physiological Society. 91

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teristics of propagated action potentials. Equally important, the components of that circuit could be interpreted as physiological entities. Without stimulation, the transmembrane potential reflected the far higher permeability of the membrane to K + than to Na + , so the resting potential was near the K + equilibrium potential.13 With stimulation there was first a marked increase in permeability to Na + , causing the upstroke of the action potential as Na + flowed down its electrochemical gradient into the cell, passing through 0 mV and approaching the Na + equilibrium potential. (Permeability to Na + thus varied with the transmembrane potential; impulse propagation reflected an action potential triggering, through local circuits, the permeability change in adjacent regions of the membrane.) The increased permeability to Na + then ceased and the permeability to K + increased, causing the downstroke of the action potential as K + flowed down its electrochemical gradient out o/the cell. The action potential thus arose from a transient opening of membrane channels14 first for Na + and then for K + . Richard Keynes in Cambridge subsequently measured fluxes of radioactive 24Na and 42K consistent with this model.15 (With squid axons as well as the vast majority of mammalian cells, the cytoplasm contains high concentrations of K + and low concentrations of Na + , whereas the extracellular fluid contains the opposite ratio. Consequently, when their respective channels open Na + flows into the cell and K + out. The asymmetric distributions of Na + and K + that drive such flows are maintained by an energy-consuming Na+/K+-pump.16)

Intracellular Microelectroaes

Just as the higher resolving power of electron microscopes could settle controversies that light microscopes could not, so intracellular electrodes could provide new values crucially important in resolving long-standing disputes. But the intracellular electrodes that Hodgkin and Huxley used—glass cannulas 100 (Jim in diameter—were clearly unsuitable for neurons and skeletal muscle cells only a fraction of that size. The obvious solution was to use smaller electrodes, and this was accomplished by two graduate students of Ralph Gerard in Chicago.17 In 1946 Judith Graham described resting potentials of —41 to —80 mV from frog muscle cells impaled by glass electrodes she pulled from capillary tubing to tip diameters as small as 2 /Jim.18 Gilbert Ling inherited Graham's equipment and succeeded in pulling electrodes with tip diameters of 0.5 fjim or less; in 1949 Ling reported resting potentials averaging — 98 ± 6 mV.19 With such electrodes Hodgkin and W. L. Nastuk in 1950 recorded muscle action potentials having an overshoot to +30 mV from a resting potential of -90 mV.20 Evidently the cell membrane, when penetrated carefully, sealed around the electrodes, preventing short-circuiting through leaks. These electrodes were applied to mammalian neurons at this time also. But

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before relating their use in resolving the question of synaptic transmission in the central nervous system, the earlier resolution of synaptic transmission at neuromuscular junctions should be noted.

Transmission at Neuromuscular Junctions

In the nineteenth century Willy Kuhne described microscopically distinguishable regions on muscle surfaces where motor nerve fibers end in discrete, circumscribed areas. These regions became known as muscle "endplates," but their detailed relationships could not be distinguished by light microscopy.21 In 1907 John Newport Langley localized the actions of curare and nicotine to the region where nerve fibers terminated—and where the proposed reactive substances would function.22 Then in the late 1930s Hans Schaefer and Herbert Gopfert in Bonn described characteristic electrical responses at the endplate region of frog muscles, evoked by stimulating the motor nerve: transient depolarizations—decreases in the resting potential toward 0 mV.23 These they measured in the presence of curare to reduce responses to nerve stimulation (otherwise, induced muscle action potential would mask these depolarizations; with higher concentrations of curare the depolarizations could be blocked totally). Through the 1940s and beyond John Eccles (Fig. 4-2), Bernard Katz (Fig. 4-3), and Stephen Kuffler developed such studies of "endplate potentials" (e.p.p.s) to define functional properties and to reveal origins from neurotransmitter actions, working first together in Sydney and then separately across the globe. Eccles, after receiving his medical degree in Melbourne, went in 1925 as a Rhodes scholar to Oxford, where he was Sherrington's student and final collaborator. After Sherrington's retirement, Eccles moved in 1937 to Sydney, in 1944 to Otago, and in 1952 to Canberra. Katz received his medical degree in 1934 in Leipzig, next studied with A. V. Hill in London, and then emigrated to Sydney. Katz worked with Eccles from 1939 to 1942, when he joined the Australian air force; Katz returned to London in 1946, where he remains. Kuffler received his medical degree in 1937 in Vienna and left for Sydney in 1938, moving to Chicago in 1945, Baltimore in 1947, and Boston in 1959. Their joint paper of 1941 presented admirably the central properties of e.p.p.s evoked by stimulating the motor nerves to frog and cat muscles: depolarizations were local and spread decrementally, their magnitude varied without a threshold, and they could sum with another depolarization to form still larger depolarizations.24 These characteristics thus differed sharply from those of muscle action potentials, which propagated without loss of amplitude ("allor-nothing"). Eccles and associates also showed that when e.p.p.s exceeded a critical threshold they elicited muscle action potentials: e.p.p.s served as electrical stimuli to the adjacent muscle.

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FIGURE 4-2. John C. Eccles (1903-1997; courtesy of the National Library of Medicine).

The following year Kuffler described experiments with single frog muscle fibers innervated by single nerve terminals. This preparation sharpened the time resolution by reporting from a single endplate rather than a population of them, and it also minimized distortions due to electrical shunting through extraneous tissues.25 Kuffler added curare to the surrounding medium and

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FIGURE 4-3. Bernard Katz (1911-; courtesy of Bernard Katz).

could then record a progressive delay in the action potential's onset, eventually leaving only the e.p.p. when sufficient curare diffused into the region (Fig. 4—4A). Consequently, Kuffler could show individual endplate depolarizations and the minimal amplitude sufficient to initiate action potentials in regions adjacent to the endplate. As noted in chapter 3, compelling evidence for the involvement of acetylcholine was accumulating at this time, including Fritz Buchtal and J. Lindhard's demonstration in 1942 that endplate regions were 1000-fold more sensitive to acetylcholine than the remaining muscle surface, observations confirmed and extended by Kuffler in 1943.26 Nevertheless, Eccles resisted until 1948 the notion that acetylcholine alone produced e.p.p.s. But that year he reported that

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FIGURE 4-4. Muscle endplate potentials. A. Potentials at the endplate region of single frog muscle fibers were measured with extracellular electrodes before adding curare (bottom, showing the muscle action potential) and at successive times after adding curare to the bathing medium, concluding with an endplate potential alone (top). Voltage is displayed on the vertical axis (no calibration presented) and time on the horizontal axis. B. Endplate potentials of frog muscles were measured with intracellular microelectrodes in media containing low Na + concentrations (to prevent action potential formation) in the absence (above) and the presence (below) of the cholinesterase inhibitor prostigmine. Voltage is displayed on the vertical axis and time on the horizontal. (A. from Kuffler [1942], Fig. 5., courtesy of the American Physiological Society. B. from Fatt and Katz [1951], Fig. 13, courtesy of the Physiological Society.)

both phases of the e.p.p.—the fast and slowly decaying components visible with low levels of cholinesterase inhibitors such as physostigmine—"reacted similarly to every test": adding curare or acetylcholine, changing the temperature, and stimulating repetitively.27 Since he had previously acknowledged that acetylcholine was responsible for the slow component, "this precise corre-

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spondence indicates that the fast component is also due to acetylcholine."28 The following year he added to the similarities parallel responses to seven cholinesterase inhibitors.29 Katz in 1942 measured decreases in membrane resistance corresponding with the e.p.p.s and suggested that acetylcholine might act by increasing membrane permeability to ions.30 Then in 1951, using intracellular microelectrodes to impale muscle cells, he and Paul Fatt could not only record e.p.p.s clearly and the effects of cholinesterase inhibitors on them (Fig. 4-4B), they could also further pursue the question of changeable membrane permeabilities.31 Since acetylcholine caused potentials to fall nearly to 0 mV, they argued that it produced a general increase in membrane permeability at the endplate. In addition, they showed that when muscle action potentials were initiated at a distance from the endplate, they skirted the endplate as they spread across the muscle surface. Katz and Fatt concluded that the endplate membrane was inexcitable electrically and therefore "differs from the surrounding fibre surface not only in its specific sensitivity to chemical stimulants, but in its lack of sensitivity to electric currents."32 Pursuing the issue of ionic permeability, Katz and Jose del Castillo showed in 1954 that the maximal e.p.p. evoked by nerve stimulation was —10 to —20 mV, "close to the estimated free diffusion potential between fibre contents and Ringer solution," and that the depolarization correlated with large decreases in membrane resistance.33 Unlike action potential depolarizations that rise to +30 mV or more and are associated with a selective influx of Na + , depolarizations at the endplate must therefore involve other ions. A. Takeuci and N. Takeuci in Salt Lake City concluded, through systematically removing ions from the bathing media, that e.p.p. depolarizations were due to an increased permeability to both Na + and K + , but not to Cl'.34 Related experiments concerned the mode of acetylcholine release from nerve terminals. In 1952 Katz and Fatt described the "chance observation" that tiny depolarizations of frog muscle endplates appeared sporadically in the absence of nerve stimulation.35 These depolarizations originated at the endplate but were a hundredth the amplitude of evoked e.p.p.s (Fig. 4—5) and disappeared after the muscle was denervated. The magnitude of these "miniature endplate potentials" (m.e.p.p.s) varied from preparation to preparation but was constant for a given preparation; curare decreased their magnitude whereas cholinesterase inhibitors increased it. On the other hand, the frequency with which these m.e.p.p.s appeared varied a thousand-fold during observations of a given preparation; neither curare nor cholinesterase inhibitors affected the frequency. In 1956 I. A. Boyd and A. R. Martin in London reported the presence of m.e.p.p.s at cat muscle endplates and A. W. Liley in Canberra at rat muscle endplates.36 Descriptions at other neuromuscular junctions soon followed. Katz and Fatt also described the statistical distribution of the occurrence of m.e.p.p.s, which they then interpreted as independent events appearing ran-

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FIGURE 4-5. Miniature endplate potentials. Spontaneous electrical activity measured with intracellular microelectrodes, recorded (A) at the endplate region and (B) 2 mm away (upper traces in each panel). The lower traces, at higher speed and lower amplification, show the response to nerve stimulation. Voltage is displayed on the vertical axis and time on the horizontal. For the upper traces, the scale is 3.6 mV and 47 msec; for the lower, 50 mV and 2 msec. (From Fatt and Katz [1952b], Fig. 1, courtesy of the Physiological Society.)

domly in time.37 These m.e.p.p.s therefore seemed like spontaneous leaks of neurotransmitter, but with a definite relationship to the evoked release of neurotransmitter: Katz and del Castillo suggested that an e.p.p. was "built up statistically of small all-or-none units which are identical in size," so that m.e.p.p.s "could be regarded as the least unit, or the 'quantum', of end-plate response."38 Nerve action potentials apparently increased by orders of magnitude the probability of such quanta being released. Transmission in the Central Nervous System

Although Eccles conceded in the late 1940s that transmission at neuromuscular junctions was by chemical means, he remained convinced that transmission in the central nervous system was electrical. One reason was the meager evidence for acetylcholine functioning there: "it now seems probable that . . . acetylcholine . . . plays no part whatsoever in central synaptic transmission."39 Another was his success in devising a scheme for inhibition at electrical synapses, which had been an elusive goal.40 Eccles in 1947 depicted hypothetical interneurons (which he termed "Golgi cells") shunting electric currents into—and thereby depressing the excitability of—motoneurons (Fig. 4-6).

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FIGURE 4-6. Model for inhibition at electrical synapses. A motoneuron (M) receives synaptic contact from an excitatory neuron (E) and an inhibitory "Golgi" interneuron (G), which is excited by another neuron (I). Excitation by I shunts electrical currents through G to alter the excitability of M. (From Brooks and Eccles [1947], Fig. 1, © Macmillan Magazines Ltd., reprinted by permission.)

With the advent of intracellular microelectrodes, however, discriminating experiments became possible. Eccles soon set about examining synaptic transmission in the central nervous system, culminating in 1952 with a stellar paper.41 For this he chose ventral horn motoneurons of the spinal cord, which he had studied with Sherrington and which have the advantage of large cell bodies (50-70 /Am across), known sensory inputs, and identified outputs. On the other hand, they lie well below the surface and must therefore be impaled "blind." This necessitated advancing the microelectrodes slowly into the exposed spinal cord of anesthetized cats while monitoring the voltage relative to an external electrode: recorded voltages would fall abruptly to about —60 mV when electrodes penetrated cells. Eccles could then identify the impaled cell as a motoneuron by stimulating the corresponding ventral root electrically: action potentials spread in the axons from the stimulated point, both peripherally along the nerve ("orthodromically") and centrally into the spinal cord ("antidromically"). Recording an action potential indicated that the impaled cell body was continuous with a stimulated axon.42 Eccles also elicited responses in impaled motoneurons by stimulating sensory nerves that make synaptic contact with those motoneurons ("orthodromic stimulation"). Impaled motoneurons had

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resting potentials around —60 mV and action potentials with overshoots to +25 mV, like skeletal muscle and squid axons. To examine synaptic potentials Eccles stimulated excitatory sensory nerves (orthodromic stimulation) while he advanced the microelectrode. Just before he impaled a motoneuron he could record, extracellularly, small potential changes (Fig. 4-7A). But after impaling the motoneuron the potential changes (1) were 20-fold larger, (2) had the opposite sign, and (3) had slower time courses (Fig. 4-7B and C]). Moreover, presynaptic potentials recorded intracellularly in the motoneuron (i.e., the initial deflections synchronous with the stimulatory response) were no larger than presynaptic potentials recorded extracellularly, so there was occurring "no special method of injecting current across the post-synaptic membrane."43 Current flow from pre- to postsynaptic

FIGURE 4-7. Excitatory and inhibitory postsynaptic potentials. A. Potentials just outside the motoneuron were recorded extracellularly after a sensory nerve exciting the motoneuron was stimulated. Voltage is displayed on the vertical axis and time on the horizontal. (The breaks in the recording represent intervals of about 4 msec.) B. Potentials in the motoneuron were recorded with an intracellular microelectrode after a sensory nerve exciting the motoneuron was stimulated. In both B and C a downward deflection represents a depolarization. C. The trace in panel B is shown at lower amplification. D. Potentials recorded intracellularly from a motoneuron are shown after stimulation of a sensory nerve inhibiting the motoneuron (upper trace); the lower trace show potentials recorded from the sensory nerve. Here and in E the more usual convention is followed: a downward deflection represents a hyperpolarization. E. For comparison with D, potentials are shown after stimulating a sensory nerve exciting the motoneuron (as in panels B and C, but here with the conventional orientation). (From Brock et al. [1952], Fig. 9A-C [A-C] and Fig. 12 C and D (D and E; courtesy of the Physiological Society.)

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cell was insufficient to produce the measured depolarizations in the postsynaptic cell. Eccles concluded: "The problem of [electrical] continuity versus discontinuity at the synapse can now be regarded as decisively settled."44 When Eccles stimulated instead an inhibitory sensory input, the intracellular potential was hyperpolarized, that is, became more negative (Fig. 4—7D; for comparison, the results of stimulating an excitatory sensory input were also shown: Fig 4-7E). The observed hyperpolarization directly contradicted Eccles's Golgi cell model for electrical inhibition (Fig. 4-5), which required a depolarization of the motoneuron by currents shunted through the Golgi cells. Eccles conceded that "experimental evidence has falsified the Golgi-cell hypothesis . . . and left the chemical neurotransmitter hypothesis as the only likely explanation."45 Eccles referred to the electrical changes as "post-synaptic potentials" ("p.s.p.s");46 these became subdivided into "excitatory postsynaptic potentials ("e.p.s.p.s") and "inhibitory postsynaptic potentials" ("i.p.s.p.s"). The graded nature of e.p.s.p.s indicated how stimuli that were individually ineffective could add to reach the threshold necessary for generating action potentials, and how i.p.s.p.s. could sum algebraically with e.p.s.p.s to prevent action potentials. And in 1963 Katz described miniature e.p.s.p.s from frog spinal motoneurons and argued for the quantal release of neurotransmitters in the central nervous system as well as at neuromuscular junctions.47 How chemical neurotransmitters could induce such depolarizations and hyperpolarizations was less easy to study in the central nervous system than at neuromuscular junctions, where the external environment can be manipulated easily. Instead, Eccles altered the intracellular ion contents by injecting ions through one barrel of a double-barreled micropipet (the other barrel he used to measure electrical responses). Eccles concluded that e.p.s.p.s occurred like e.p.p.s: through an increased permeability that short-circuited the resting potential toward 0 mV.48 Defining ionic permeabilites to account for i.p.s.p.s was less clear-cut, but Eccles argued for increased permeabilities to K + and/or Cl~.49 In the meantime, evidence for inhibitory changes in ionic permeabilities were reported at another site. Some invertebrate muscles are directly innervated by both excitatory and inhibitory nerves (unlike mammalian skeletal muscle, which is innervated solely by excitatory nerves), and in 1958 Fatt described an increased permeability to Cl~ at crustacean neuromuscular junctions associated with hyperpolarizing e.p.p.s.50 Soon after the identification of i.p.s.p.s, Eccles described interneurons in the spinal cord whose neurotransmitter(s) hyperpolarized their target cells, publishing a model in 1954 (Fig. 4-8A) for the antidromic inhibition that Birdsey Renshaw in New York had described in the 1940s.51 Stimulating ventral roots can inhibit, for a brief interval, the firing of motoneurons whose axons run through that root. Renshaw invoked two structures to account for this inhibi-

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tion: branches of the stimulated axon that run back in the spinal cord ("recurrent collaterals") and interneurons stimulated by these collaterals. The causal pathway thus lay from (1) antidromic stimulation of motoneuron axons in the ventral root to (2) orthodromic spread along recurrent collaterals that (3) excite interneurons that then (4) inhibit the motoneurons. The time course was appropriate for a two-neuron pathway, and Renshaw recorded discharges from interneurons after antidromic stimulation of ventral roots, although he could not connect interneuron firing to motoneuron inhibition. This Eccles did, using intracellular microelectrodes to follow motoneuron potentials.52 He found that stimulating motoneurons caused their own inhibition (hyperpolarization), and he confirmed Renshaw's finding that action potentials in motoneuron axons excite interneurons, presumably through recurrent collaterals. Eccles named the interneurons "Renshaw cells" and showed, importantly, that their activation was blocked by compounds such as dihydro-/3-erythroidine, which block the effects of acetylcholine, whereas their activation was augmented by cholinesterase inhibitors. By contrast, hyperpolarization of the motoneuron was blocked by strychnine, a drug known to cause excessive motor discharges. The major conclusions were (I) motoneurons release acetylcholine at neuromuscular junctions and, by their recurrent collaterals, on Renshaw cells, exciting both; and (2) Renshaw cells inhibit motoneurons using an unknown inhibitory neurotransmitter whose actions are blocked by strychnine. Eccles stressed that acetylcholine was released from all terminals of motoneurons, a manifestation of what he termed "Dale's Principle": a neuron releases the same neurotransmitter from all its terminals.53 A second invocation of interneurons concerned inhibition that was associated with the activation of sensors in muscle. For reciprocal innervation Sherrington had depicted sensory fibers from muscle exciting motoneurons to one set of muscles while inhibiting motoneurons to the antagonistic set (Fig. 2-2). In accord with this scheme, David Lloyd in New York argued for a "direct inhibition," in which the sensory neuron acted without intervening interneurons on the motoneuron.54 But in 1956 Eccles recalculated the latency between sensory nerve activity and motoneuron inhibition and concluded that two synapses, not one, intervened.55 Moreover, he found that stimulating sensory nerves activated certain interneurons (different from Renshaw cells). This scheme (Fig. 4-8B) differs from Sherrington's proposal not merely by including interneurons but, more significantly, by restricting the influence of a neuron to either excitation or inhibition at all its terminals. But what is the excitatory neurotransmitter released by sensory neurons to activate motoneurons and interneurons? Identifying that substance—as well as the inhibitory substance released by the interneurons—remained a challenge during the following decades (see chapter 5).

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FIGURE 4-8. Proposed inhibitory circuits. A. Inhibition of a motoneuron (the large cell at the bottom) occurs by means of a recurrent collateral, branching at a node from the thick myelinated motoneuron axon, and exciting the small "Renshaw" interneuron, which in turn inhibits the motoneuron. (Redrawn from Eccles et al. (1954), Fig. 18, courtesy of the Physiological Society.) B. A sensory nerve from an extensor muscle sends one branch to excite a motoneuron to that extensor muscle, while another branch excites an inhibitory interneuron that in turn inhibits the motoneuron to the flexor muscle. (From Eccles [1961], Fig. 1, courtesy of the Royal Society. This figure had been redrawn Eccles et al. [1956], Fig. 13.)

Presynaptic Innimtion Inhibition described in the preceding section was "postsynaptic": inhibitory neurons hyperpolarized their target cells, hindering their activation by excitatory neurons. In 1957 Karl Frank in Bethesda described a different type of inhibition, which he called "presynaptic."56 Frank found that stimulating certain inhibitory nerves alone did not inhibit a motoneuron but could reduce its activation (i.e., reduce the magnitude of e.p.s.p.s in that motoneuron) evoked

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through stimulating an excitatory nerve to that motoneuron. Thus, the inhibitory nerve, when stimulated just before the excitatory nerve, diminished the motoneurons response to that excitatory nerve. Eccles confirmed these observations in 1961, describing decreases in evoked e.p.s.p.s without changes in responsiveness of the motoneuron to electrical

FIGURE 4-9. Presynaptic inhibition in the spinal cord. A sensory nerve from an extensor excites the motoneuron to that extensor (EM). Sensory nerves from a flexor and from a flexor tendon excite interneurons (white) that excite a second-order interneuron (black). This intemeuron makes synaptic contact with the sensory nerve terminal from the extensor. The black neuron inhibits presynaptically by diminishing neurotransmitter release from the terminal it contacts. Pathways exciting and inhibiting the motoneuron to the flexor (FM) are not shown. (From Eccles et al. [1962], Fig. 10, courtesy of the Physiological Society.)

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stimulation.5' These decreases in e.p.s.p.s he interpreted as due to inhibitory neurons reducing neurotransmitter release from excitatory neurons. To explain how one neuron could reduce neurotransmitter release by another, Eccles correlated the reductions with depolarizations of the presynaptic excitatory fibers, arguing that depolarizing nerve terminals diminished their release of neurotransmitters.58 Although he could not demonstrate altered ionic permeabilities to account for such presynaptic depolarization, he was able to show marked differences between pre- and postsynaptic inhibition in the cat spinal cord: strychnine blocked the latter, but picrotoxin blocked the former.59 Eccles also incorporated presynaptic inhibition into a still more complex diagram of spinal cord excitation and inhibition (Fig. 4-9). At this time Kuffler demonstrated presynaptic inhibition at crayfish neuromuscular junctions.60 Stimulating inhibitory nerves not only reduced excitatory e.p.p.s but also diminished the number of excitatory quanta: presynaptic inhibition was associated with fewer quanta of excitatory neurotransmitter being released.61 Moreover, administering y-aminobutyric acid (GABA), a known inhibitory substance, reproduced these effects (see chapter 5).

Responses at Other Sites

Studies at three other sites were influential then and were developed subsequently. 1. Neurons of the stellate ganglion in squid form "giant synapses" with the giant axons. These axo-axonal synapses are sufficiently large and accessible that microelectrodes can be advanced, guided visually (with a microscope), through the giant axons and into the presynaptic terminals of the stellate ganglion cells. In 1958 S. Hagiwara and Ichiji Tasaki in Woods Hole described synaptic potentials in a postsynaptic giant axon when the presynaptic stellate ganglion cells were stimulated.62 As they advanced the microelectrode farther, the resting potential abruptly fell to zero and synaptic potentials were replaced by far smaller deflections of the opposite polarity. As they advanced it still farther, the resting potential reappeared; there they also recorded signals from stimulating the stellate ganglion cells. Hagiwara and Tasaki concluded that the electrode recorded from three loci, and that "between the pre- and post-synaptic membrane [there is] a small 'space' where the potential is close to that of the surrounding sea water."63 Thus, they confirmed Eccles's 1952 interpretation in a system where the entities could be seen with a microscope and where potentials in each of the three compartments could be measured directly. They also confirmed that current applied locally could not flow in significant amounts from one neuron to another.

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2. Earlier, Alfred Fessard, working in Arachon, France on the electric ray Torpedo mamwrata, found that the electric organ discharged when its nerve was stimulated but that denervated electric organs could not be excited electrically.64 Then in 1942 he and Wilhelm Feldberg reported strong evidence for acetylcholine mediating the neural stimulation: acetylcholine was present in innervated electric organs, acetylcholine was released into the bath when the nerve was stimulated, the cholinesterase inhibitor physostigmine potentiated the response to nerve stimulation, and added acetylcholine mimicked the effect of nerve stimulation.65 This sensitivity to acetylcholine and insensitivity to electrical stimulation is similar to later characterizations of muscle endplates. Fish electric organs are developmentally related to muscle, and Torpedo electric organ represents a collection of endplates. 3. By contrast, the electric organ of the electric eel, Electrophorus electricus, responds to electrical stimulation even after denervation. In 1955 Harry Grundfest in New York described local, graded depolarizations after stimulation that, when sufficiently large, led to action potentials; he likened these to e.p.p.s that could trigger all-or-nothing impulses.66 (Indeed, Keynes had pointed out in 1953 that these membranes were more than muscle endplates, having, unlike cells of Torpedo electric organs, electrically-excitable domains as well.67) Carlos Chagas in Rio de Janeiro had found that curare blocked responses to nerve stimulation, and Grundfest, in collaboration with David Nachmansohn, then reported that physostigmine potentiated such responses.68 Added acetylcholine depolarized electric organ cells but did not, under their conditions, mimic nerve stimulation. Their failure to assemble the parallels for Elerctrophorus that Fessard and Feldberg had for Torpedo probably reflected Nachmansohn's insistence, despite a wealth of contradictory evidence, that acetylcholine was involved with impulse conduction along axons and not with synaptic transmission.69

Visualizing Synaptic Gaps ana Synaptic Vesicles

The physiological studies cited above required functionally asymmetric synapses with systems for liberating quanta of neurotransmitters from presynaptic elements. Anatomists had depicted terminals (or "boutons" or "endfeet") but were unable, with light microscopes, to distinguish details. Electron microscopes, with the potential for far greater resolution, were just beginning to be marketed by Siemens in Germany and RCA in the United States when World War II erupted; after the war some years were consumed in developing better optics and better fixing, staining, embedding, and sectioning procedures. Then

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several groups almost simultaneously published electron micrographs that correlated with physiological function, supplying entities for those processes. In 1954 Sanford Palay (Fig. 4-10A), while visiting George Palade in New York, described in an abstract the axo-dendritic synapses from rat cerebellum and brainstem: between the plasma membranes surrounding presynaptic and postsynaptic cells intervened a space approximately 200 A across.70 Not only was there no continuity of cytoplasm between the cells, but a significant gap separated them, a gap that came to be called the "synaptic cleft." A paper the following year centered on the Nissl substance in neuronal cytoplasm, interpreted as endoplasmic reticulum with attached granules, a subject that Palade was investigating vigorously in other cells.71 Not until 1956—after his return to New Haven—did Palay describe fully the synaptic morphology, adding more kinds of synapses at more locations in the central nervous system and including a careful rationale for identifying synapses in the limited fields and thin sections required by electron microscopy (Fig. 4-11 A).72 Meanwhile, Eduardo DeRobertis and Stanley Bennett in Seattle described gaps of 100-150 A between presynaptic and postsynaptic cells of frog sympathetic ganglia and earthworm nerve cord, again in an abstract in 1954; their full paper appeared the following year.73 On the other hand, Robertson, then in Cambridge, Mass., had noted in 1953 membranes "closely applied to one

FIGURE 4-10. A, left. Sanford L. Palay (1918-). B, right. Victor P. Whittaker (1919-). (Courtesy of Sanford Palay and Victor Whittaker.)

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FIGURE 4-11. Electron micrographs of synaptic junctions and of synaptosomes. A, on the left. An axon terminal, containing mitochiondria "and numerous synaptic vesicles" makes contact with a dendrite, with a synaptic cleft discernable between their cell membranes. At x the "intrasynaptic space is enlarged . . . permitting . . . glial fibers to pass between the terminal and the dendrite." B, on the right. An isolated synaptosome contains a mitochondrion and numerous synaptic vesicles (sv). It is surrounded by a "thin surface membrane" (tm). (A from Palay [1956], Fig. 2., reproduced by copyright permission of the Rockefeller University Press. B from Gray and Whittaker [1962], Fig. 5, courtesy of the Anatomical Society of Great Britain and Ireland.)

another" in squid synapses, but he felt that areas where the membranes seemed to be separated were "probably ... a result of alterations associated with fixation and preparation."74 Robertson's further studies on membrane structure, which culminated in his description of "unit membranes," clarified where the edges of membranes lay among the stained images, and he then joined in advocating synaptic clefts not only at neuronal synapses but also at neuromuscular junctions.' 5 Eccles in 1957 remarked on the suitability of 200 A clefts: "There are two conflicting requirements ... of the synaptic cleft: that it should be very narrow, so that the . . . transmitter is applied . . . efficiently and . . . quickly ... to the subsynaptic membrane; that it should be wide, so that the postsynaptic currents flow as freely as possible."76 In 1964 he added that a fairly wide cleft would provide "a very effective shunt preventing electrical interaction between the presynaptic and postsynaptic components of a chemically transmitting synapse."7'

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Palay identified neurofilaments in his micrographs, but while they were abundant in dendrites and cell bodies and extended into axons, the neurofilaments stopped short of the terminals.78 Neurofilaments seen by light microscopists existed, but, contrary to arguments by Stefan Apathy, Albrecht Bethe, and their successors (chapters 1 and 2), no continuity was found of neurofilaments across synaptic clefts. In 1954 Palay also reported a characteristic "agglomeration of mitochondria and small vesicles (300-500 A)" in presynaptic terminals.79 Two years later he described vesicles 200-650 A in diameter, some having a densely staining interior. He noted that these "vesicles may be considered as containing small units of a chemical transmitter, like acetylcholine," and cited Fatt s recent review on quantal release.80 In their 1954 abstract DeRobertis and Bennett also described vesicles 200-500 A in diameter, naming them "synaptic vesicles" and suggesting the following year that "[i]t is not unreasonable to speculate that active compounds . . . might . . . be associated with . . . vesicles."81 DeRobertis, too, promoted the notion that vesicles could represent quantal units of neurotransmitter, but his micrographs showed holes in the presynaptic membranes and vesicles apparently in the cleft.82 Attempts to define mechanisms for the quantal release of neurotransmitter from vesicles I will discuss in chapter 10, but here I will note initial experiments showing that acetylcholine actually was present in synaptic vesicles. These approaches continued earlier efforts at cell fractionation, begun successfully by Albert Claude in New York and culminating in George Hogeboom and Walter Schneiders four fractions obtained by centrifuging "homogenized" tissues at successively greater speeds and longer times: (1) "nuclei and debris," (2) "mitochondria," (3) "microsomes," and (4) "supernatant."83 Indeed, a major focus of Palade s research at this time was correlating biochemical and structural properties of these fractions with structures visible (by microscopy) in cells and tissues. Successful applications of such procedures to brain tissue came, however, from England. In 1958 Victor Whittaker (Fig. 4-10B), who after biochemical training in Oxford was now working with Catherine Hebb in Babraham, found that most of the acetylcholine present in homogenates of rabbit and guinea pig brains could be recovered, after such differential centrifugation, in the mitochondrial fraction.85 But when they separated that fraction further—by "discontinuous sucrose density gradient centrifugation," in which material is layered atop sucrose solutions of successively higher concentrations (thus of increasing density from top to bottom) and then centrifuged—most of the acetylcholine was in a fraction different from mitochondria.86 The following year Whittaker reported the presence of acetylcholine in a similar subfraction, which he now identified as synaptic vesicles.8' Again, he prepared a conventional mitochondrial fraction and then separated this by den-

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sity gradient centrifugation, collecting material accumulating at the interface between the layers of 0.8 and 1.2 M sucrose. This fraction contained the highest amount of acetylcholine as well as its highest "relative specific activity" (defined as the acetylcholine content of the fraction divided by the total acetylcholine content of all the fractions, relative to the protein content of that fraction divided by the total protein recovered in all the fractions).88 Electron micrographs showed few mitochondria in this fraction but large numbers of smaller particles, ranging from 200 to 3000 A in diameter, that seemed to be "simple vesicles."89 By contrast, the fraction collected at the bottom of the centrifuge tube, under 1.2 M sucrose, contained the highest specific activity of mitochondria; micrographs of this fraction showed conventional mitochondria. In 1960, however, Whittaker published a reinterpretation in collaboration with E. G. Gray, an able electron microscopist in London.90 Gray and Whittaker's micrographs of the acetylcholine-rich fraction showed "synaptic vesicles . . . enveloped within membranes forming larger particles" that they interpreted as "pinched-off nerve endings" formed during homogenization of the brain.91 These particles contained occasional small mitochondria with, in some cases, apparent fragments of the postsynaptic membrane attached. They stated that "the complete structure is identical with [nerve] endings seen in the cerebral cortex."92 A more complete report in 1962 emphasized this interpretation, depicting nerve ending particles about 10,000 A across (Fig. 4-1 IB); they now interpreted as a fixation artifact Whittaker s misidentification in 1959 of these as naked synaptic vesicles of far smaller diameter.93 In 1964 Whittaker renamed these particles "synaptosomes."94 At that time he also described a procedure for osmotically lysing their outer membrane and then separating—by density gradient centrifugation over a broader range of sucrose concentrations—seven fractions. These included synaptic vesicles, of proper size by electron microscopy and containing the highest specific activity of acetylcholine; "synaptosomal ghosts," considered to be emptied plasma membranes from nerve endings; and free mitochondria (Table 4-1). The enzymes responsible for synthesizing and destroying acetylcholine, choline acetyltransferase and cholinesterase (chapter 9), were not present at highest specific activities in the synaptic vesicle fraction. DeRobertis, who had moved to Buenos Aires, challenged in 1961 Whittaker's initial identification just as Whittaker was recanting; DeRobertis, too, described detached nerve endings filled with synaptic vesicles and some small mitochondria, surrounded by an intact membrane with patches of postsynaptic membrane attached.95 DeRobertis then published methods also based on osmotic lysis of the synaptosomes; however, his synaptic vesicle fraction had the highest specific activities not only of acetylcholine but also of cholinesterase and choline acetyltransferase,96 and its purity was in turn challenged by Whittaker.97

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TABLE 4—1. Separation of Synaptic Vesicles by Sucrose Density Gradient Centrifugation0 RELATIVE SPECIFIC ACTIVITIES IN FRACTION CHOLINE ACETYL-

CHOLINES-

SUCCINIC

ACETYLCHOLINE

TRANSFERASE

TERASE

DEHYDROGENASE

O (no structures)

0.07

2.61

0.18

0.18

D (synaptic vesicles)

2.50

0.67

0.75

0.42

E (microsomes, some synaptic vesicles)

1.00

0.25

2.50

0.50

F (synaptosome ghosts)

0.42

0.42

2.25

0.42

G (synaptosome ghosts)

0.92

0.42

1.75

0.50

H (damaged synaptosomes)

2.33

0.27

0.67

1.47

I (mitochondria, shrunken synaptosomes)

0.69

0.23

0.61

3.77

FRACTION

"The crude mitochondria! fraction prepared by differential centrifugation of brain homogenates in 0.32 M sucrose was lysed osmotically by adding water. This material was then placed in centrifuge tubes atop a series of five sucrose concentrations and centrifuged. Fraction O was material remaining at the top; D, at the interface with 0.4 M sucrose; E, at the interface between 0.4 and 0.6 M; F, between 0.6 and 0.8 M; G, between 0.8 and 1.0 M; H, between 1.0 and 1.2 M; and I, the pellet at the bottom of the centrifuge tube below 1.2 M sucrose. Fractions were characterized by electron microscopy and by measuring the relative specific activities of markers. Choline acetyltransferase is the enzyme synthesizing acetylcholine, cholinesterase the enzyme hydrolyzing it, and succinic dehydrogenase is found solely in mitochondria. Microsomes are small vesicles formed during homogenization from the endoplasmic reticulum, an interlacing series of tubules within cells. (From Whittaker et al. [1964], Tables 1 and 3. © the Biochemical Society; reproduced by permission.) Identifying Electrical Transmission The preceding studies established chemical transmission convincingly at both central and peripheral synapses. To explore further the relationships between currents in presynaptic terminals and neurotransmitter release, Edwin Furshpan and David Potter, visiting in Katz's laboratory, then examined synapses in crayfish nerve cords. There "lateral giant fibers" (presynaptic) and "giant motor fibers" (postsynaptic) are each large enough to impale with microelectrodes. To Furshpan and Potters surprise, conduction at these synapses was, as they reported in 1957, electrical yet unidirectional.98 Synaptic delays were quite

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short, perhaps 0.1 millisecond. Current passed in a graded, decremental fashion from exciting electrode in presynaptic neuron to recording electrode in postsynaptic neuron; such currents could depolarize but not hyperpolarize postsynaptic neurons. Conversely, currents from exciting electrodes in postsynaptic neurons could hyperpolarize but not depolarize presynaptic neurons. The electrical junction rectified, passing current in one direction but not the other. In 1964 Furshpan, now in Boston, described nonrectifying electrical synapses in a vertebrate brain (in goldfish, at particular synapses on giant Mauthner neurons).99 Robertson, now also in Boston, interpreted electron micrographs of these sites as showing "synaptic discs" formed from a fusion of pre- and postsynaptic membranes; tangential sections revealed characteristic hexagonal arrays.100 But in 1967 Jean-Paul Revel and Manfred Karnovsky in Boston detected 20 A gaps between the membranes, although they confirmed the hexagonal structures seen in tangential sections.101 Because of this spacing, such contacts became known as "gap junctions"; the hexagonal arrays were identified as cross sections of clustered channels spanning the intercellular gap and connecting the cytoplasms of the two cells.102 Revel and Karnovsky's studies were on mouse heart and liver. By that time such contacts had been established at numerous sites between nonexcitable cells (such as liver) as well as excitable cells (neurons, heart, smooth muscle). Through these can flow not only electric (ionic) currents but also small molecules. Gap junctions thus provide a means for rapid communication and for coordination among groups of cells of many types. In mammalian nervous systems such electrical synapses seemed in 1990 far less common than chemical synapses. For that reason, and because their manipulation therapeutically had not been achieved by the time this history closes, I will not consider electrical synapses further. Nevertheless, gap junctions play vital roles throughout the human body, and identified disorders are responsible for specific diseases.

Conclusions

Eccles dominated synaptic physiology at midcentury, first defending electrical transmission energetically and then demonstrating chemical transmission decisively. A skillful experimenter and imaginative interpreter—with significant accomplishments beyond those cited here—Eccles was justly rewarded with a Nobel Prize in 1963. (Others discussed in this chapter also so honored include Hodgkin, Huxley, Katz, and Palade.) Nevertheless, Eccles was not chosen to succeed Sherrington at Oxford. Instead, he returned to Australia in 1937 to head a small research institute in the pathology department of a Sydney hospital. Kuffler arrived as a pathologist, and Eccles diverted him to research. With

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Katz they persevered admirably, although Eccles remembered them as "three . . . huddled together in an alien world."103 The war and a change in hospital administration made research impossible. With no academic positions available in Australia, Eccles ventured alone to New Zealand, beginning at the Otago medical school in 1944. Despite a consuming teaching load he attracted new collaborators, notably L. G. Brock and Jack Coombs, and together they refuted his model of electrical conduction. In 1951 Eccles returned to Australia again, now to Canberra and the Australian National University, then being founded as a graduate research institution. There, visited by dozens of scientists from across the globe, he did what he considered his best work. Eccles, like Sherrington, was a confirmed dualist and spent his final decades pondering the mind-brain enigma.104 But the pervasive philosophical view guiding his research was, by his account, Karl Popper's.105 Eccles learned of, met, and was won over by Popper in 1944, when both were (temporarily) in New Zealand. Eccles interpreted Popper's views as the methodological imperative "to formulate clear hypotheses and then test them by rigorous experiment."106 Accordingly, Eccles viewed his microelectrode study of postsynaptic inhibition as "a clear test," since his electrical hypothesis predicted depolarization and the chemical hypothesis hyperpolarization.107 That test, however, refuted his hypothesis: "The result was repeatable, graded with stimulus strength, and indubitable."108 (Perhaps Eccles considered Popper's views also as consolation; Zenon Bacq quotes Eccles: "I can now rejoice in the falsification [refutation] of a cherished theory, because even this is a scientific success."109 Bacq also observed that "Eccles had changed mounts, the rider was as tempestuous as ever, but the horse was much better."110 And in fairness to Dale, Feldberg, et al., it is important to remember that there were good reasons for believing synaptic transmission was chemical before Eccles's experiments.) Of course, admonitions about formulating hypotheses clearly and testing them rigorously are routine scientific advice independent of Popper's writing, even if this advice is not universally heeded. But Popper's interpretation of science as conjecture and refutation accompanied his adamant rejection of inductive inference. This distinctive aspect of Popper's teaching Eccles did not adopt in practice. Eccles accepted chemical transmission at neuromuscular junctions not after refuting electrical hypotheses but after assessing the positive evidence in favor of chemical hypotheses. Instances of corroborative weight hailed by the scientific community also included independent but mutual support between physiological and anatomical studies: discovering synaptic clefts with appropriate magnitudes and identifying synaptic vesicles suitable for storing neurotransmitter quanta. Scientific research is opportunistic not only in seizing available techniques and ideas to make old problems soluble, but also in choosing to corroborate

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one theory rather than refute another. When experiments to refute either hypothesis for neuromuscular transmission were not apparent, Eccles instead sought evidence favoring one. Definitive experiments can sometimes be planned, as in Eccles's application of microelectrodes, even if the results are unanticipated. Sometimes rewarding results appear by chance, as in Katz and Fatt's discovery of m.e.p.p.s. Moreover, initial generalizations derived from observations and corroborations are, at least in such biological research, inevitably modified. Intrinsic complexities ensure that with closer scrutiny new entities and interactions will appear. And biological diversity—reflecting random mutations and contingent selections—makes exceptions likely. Generalizations are tied to specified domains, as in recognizing that some neurons communicate electrically through gap junctions while others communicate chemically across synaptic clefts. (Further qualifications may then be required, specifying how particular instances diverge from a prototypic manifestation.) But useful, instructive generalizations can result nonetheless. During these two decades new experimental techniques, notably those exploiting intracellular microelectrodes and electron microscopes, did extend the principle of chemical transmission from the autonomic nervous system to neuromuscular junctions and the central nervous system. New general phenomena were recognized, such as presynaptic inhibition, and new circuits for reciprocal control were delineated. New details were added, such as the quantal release of neurotransmitters packaged in synaptic vesicles and the identities of ionic currents responsible for membrane voltage changes. Still, many questions remained unanswered, including the identities of the neurotransmitters for sensory excitation and for central inhibition. Notes 1. For general accounts, see Fruton (1999); Judson (1979); Morange (1998); Robinson (1997). 2. See Kornberg (1997). 3. For example, fluxes occurring during steady states—when there are no net changes—cannot be measured by chemical analyses but can be followed as tracer fluxes using radioactive isotopes added initially to one of the several compartments exchanging materials. 4. For historical accounts, see Clarke and O'Malley (1968); Hodgkin (1992); Robinson (1997); Tasaki (1959). 5. Action potentials were identical for a given fiber and experimental conditions, such as temperature, ionic milieu, etc. Stimuli below threshold values produced electrical changes that were conducted decrementally. 6. The equilibrium distribution may be considered the result of K + , high in concentration within the cell, diffusing outward in the absence of accompanying negative

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charges or of an exchange for positive charges diffusing inward. The resulting electrical potential, representing the loss of positive charges within the cell, will oppose the concentration gradient favoring a net outward diffusion. The Nernst equation states the equilibrium potential and concentration ratio for such a system. 7. Young described the axons at a meeting attended by neurophysiologists (Young, 1936). 8. Cole and Curtis (1939); Cole and Hodgkin (1939). The experiments, performed the previous summer, involved measuring transverse impedance to alternating current. 9. Hodgkin and Huxley (1939). 10. The calculation requires knowing the concentration of intracellular K + , which was reported that year (Bear and Schmitt, 1939). 11. Hodgkin and Huxley (1945); their 1939 note did not attempt an explanation of the overshoot. 12. Hodgkin and Huxley (1952) and preceding papers. These studies relied crucially on an enabling new technique, the voltage clamp method (see Robinson, 1997). 13. The equilibrium potential across a membrane is that at which no net movement of the ion occurs. Above this potential net movement of the ion occurs in one direction, below it in the opposite direction. 14. The term channel was introduced by Hodgkin and Keynes (1955); pore was a common designation earlier. 15. Keynes and Lewis (1951) and preceding papers. 16. See Robinson (1997). 17. For historical accounts, see Frank (1986); Marshall (1987); Robinson (1997). 18. Graham and Gerard (1946). This represented the completion of work interrupted by the war. 19. Ling and Gerard (1949). 20. Nastuk and Hodgkin (1950). 21. Even in the 1930s some argued about the continuity of such regions with muscle cytoplasm: see Eccles and O'Connor (1939). 22. Langley (1907). 23. Gopfert and Schaefer (1938). 24. Eccles et al. (1941). These experiments, too, were made in the presence of curare. Pertinent earlier work includes Gopfert and Schaefer (1938); Eccles and O'Connor (1939); Feng (1940). Eccles (1977) stated that he was initially unaware of the work by Gopfert and Schaefer and by Feng. 25. Kuffler (1942). With external electrodes at the endplates, e.p.p.s could be measured even without curare (Fig. 4-4E). 26. Buchtal and Lindhard (1942); Kuffler (1943). 27. Eccles (1948), p. 103. 28. Ibid., p. 104. At this time Kuffler announced that Eccles now believed that "the electrical hypothesis cannot be reconciled with more recent experimental results" and that his "evidence favors [acetylcholine] as the sole mechanism" (Kuffler, 1948, p. 445). 29. Eccles and MacFarlane (1949). 30. Katz (1942). 31. Fatt and Katz (1951). 32. Ibid., p. 362. Grundfest (1957b) emphasized the characteristic electrical inexcitability. 33. del Castillo and Katz (1954b), p. 564. 34. Takeuchi and Takeuchi (1960).

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MECHANISMS OF SYNAPTIC TRANSMISSION

35. Fatt and Katz (1952b). 36. Boyd and Martin (1956); Liley (1956). 37. Fatt and Katz (1952b). See also del Castillo and Katz (1954b). 38. del Castillo and Katz (1954b), p. 560. 39. Brooks and Eccles (1947), p. 760. 40. Ibid. Earlier attempts at explaining inhibition included schemes invoking the axonal refractory period, a brief interval immediately following passage of an action potential when that axon cannot again be excited (see Adrian, 1924). 41. Brock et al. (1952). 42. The cell body must be connected directly since, according to Cajal and Sherrington, antidromic impulses cannot cross synapses. 43. Brock etal. (1952), p. 455. 44. Ibid. p. 452. 45. Ibid. p. 455. 46. Eccles (1953), p. 130. 47. Katz and Miledi (1963). Earlier, Eccles had noticed random low-amplitude electrical activity but attributed it to distant synaptic activity (Brock et al., 1952). After Fatt and Katz's initial description, quantal release of neurotransmitters was then described at several other sites, including autonomic ganglia (Nishi and Koketsu, 1960). 48. Coombs et al. (1955b). 49. Coombs et al. (1955a); Eccles et al. (1964). 50. Boistel and Fatt (1958). 51. Renshaw (1946) and preceding papers. 52. Eccles et al. (1954). 53. Ibid. See also Eccles (1976). 54. Laporte and Lloyd (1952). Eccles had considered such inhibition to be monosynaptic earlier (Bradley et al., 1953). 55. Eccles et al. (1956). 56. Frank and Fuortes (1957). The effect is still across a synapse, but the immediate target is the presynaptic terminal of the second neuron. Frank (1959) later termed the phenomenon "remote inhibition," including an alternative mechanism. 57. Eccles et al. (1961). 58. Eccles inferred the depolarization from the greater excitability of these presynaptic fibers that he also observed. In addition, he cited Hagiwara and Tasakis demonstration (1958) that neurotransmitter release declined sharply as the presynaptic terminal was depolarized. 59. Eccles (1964), Fig. 96. 60. Dudel and Kuffler (1961). 61. Similarly, Kuno (1964) described a decrease in the probability of neurotransmitter release associated with presynaptic inhibition of spinal motoneurons. 62. Hagiwara and Tasaki (1958). 63. Ibid., p. 124. 64. Auger and Fessard (1938). 65. Feldberg and Fessard (1942). They also cited Elliott's attempt to extract the chemical neurotransmitter at this site. 66. Altamirano et al. (1955a). 67. Keynes and Martins-Ferreira (1953). 68. Chagas et al. (1951); Altamirano et al. (1955b). 69. Nachmansohn (1961).

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70. Palade and Palay (1954); Palay and Palade (1954). The second of these is chiefly concerned with the Nissl substance. 71. Palay and Palade (1955). These granules were identified subsequently as ribosomes, the sites of protein synthesis. 72. Palay (1956). Palay argued from the prominence of mitochondria in presynaptic terminals, visible by light microscopy, to the identification of nerve terminals by the large numbers of mitochondria visible in electron micrographs. Recognizing synaptic vesicles then added another identifying characteristic for presynaptic terminals. Although electron microscopy showed small areas and great complexity (e.g., Fig. 4-10A), such comparisons established common identities. 73. DeRobertis and Bennett (1954, 1955). 74. Robertson (1963), pp. 221-222. 75. Robertson (1956, 1960). Birks et al. (1960) provided further detailed views of neuromuscular junctions. Palade and Palay (1954) mentioned neuromuscular junctions, but their account of an intervening space is vague. 76. Eccles (1957), p. 217. 77. Eccles (1964), p. 28. 78. Palay (1958). 79. Palade and Palay (1954), p. 355. Robertson (1956) saw circular profiles that he interpreted as tubes cut in cross section; however, Palay (1956) argued that circular profiles were apparent after sectioning at assorted angles and thus the images represented spheres not cylinders. 80. Palay (1956), p. 199. 81. DeRobertis and Bennett (1954); (1955), p. 55. 82. DeRobertis and Bennett (1955); DeRobertis (1956, 1958). 83. Hogeboom et al. (1948); Hogeboom (1955). The starting material was a homogenate, prepared by grinding tissue with a cylindrical pestle turned inside a closefitting tube. This homogenate was then placed in plastic centrifuge tubes and spun at successively higher speeds and longer times to obtain the characteristic four fractions. (Homogenates originated as biochemists' preparations to provide a uniform stock from which identical samples could be tested and analyzed.) 85. Hebb and Whittaker (1958). They modified Hogeboom and Schneider's scheme somewhat, including the homogenizing of brain tissue in 0.32 M sucrose. 86. Mitochondria were recognized biochemically by the presence of an enzymatic "marker," succinic dehydrogenase, an enzyme previously shown to be present solely in mitochondria. 87. Whittaker (1959). 88. Whittaker measured the nitrogen content, which parallels the protein content closely. 89. Whittaker (1959). p. 698. 90. Gray and Whittaker (1960). 91. Ibid., p. 35P. 92. Ibid. In contrast to earlier proposals that neurons were linked by conducting fibers, this connection of pre- and postsynaptic elements is by extracellular materials that do not participate in impulse conduction. 93. Gray and Whittaker (1962). In retrospect, it is obvious that small synaptic vesicles should not have penetrated so far into the sucrose gradient as did the nerve terminals. 94. Whittaker et al. (1964).

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95. DeRobertis et al. (1961). 96. DeRobertis et al. (1963). 97. Whittaker et al. (1964) noted differences in distribution of markers, lower relative specific activities of acetylcholine, and claimed that DeRobertis s fixation technique for electron microscopy caused "extensive disruption of the more organized structures . . . leaving clumps of synaptic vesicles and membrane fragments" (p. 301). 98. Furshpan and Potter (1957, 1959). For a historical account, see Hagland and Collett (1996b). 99. Furshpan (1964). 100. Robertson (1963). 101. Revel and Karnovsky (1967) 102. See Goodenough and Revel (1970); Makowski et al. (1977). 103. Eccles (1977), p. 5. 104. Eccles also expressed unorthodox views about "mind" influencing synaptic transmission (see Robinson, 1987). 105. Eccles (1976). Popper's seminal book of 1934 was not translated into English until much later (Popper, 1959). 106. Eccles (1976), p. 225. 107. Ibid. 108. Ibid. 109. Bacq (1975), p. 65. See also Eccles (1970), pp. 104-106. 110. Ibid.

5 IDENTIFYING NEUROTRANSMITTERS (1946-1976)

Scope and. Criteria

During the postwar decades the discovery of new neurotransmitters burgeoned unexpectedly into an expanding enterprise. Earlier studies on the autonomic nervous system had provided models for neural control through antagonistic systems using two neurotransmitters, acetylcholine and adrenaline (chapter 3). Each could be either excitatory or inhibitory, depending on the tissue, and two neurotransmitters should be sufficient for such accelerator/brake control. But despite this theoretical sufficiency, further experiments on the central nervous system revealed that neither could fill prominent excitatory and inhibitory roles (chapter 4). If other neurotransmitters participated, what were they—and how could they be identified? The criteria were obvious. The candidate neurotransmitter should be present in the presynaptic terminal, be released when the presynaptic terminal was active, and, when applied experimentally, induce faithfully the responses in the postsynaptic neuron.1 In practice, since central nervous system neurons continuously integrate diverse excitations and inhibitions, the last criterion was relaxed to demonstrating merely changes in such activity. Not surprisingly, satisfying these criteria in the central nervous system was frustrated by inherent complexities and impeded by imperfect techniques lack119

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ing adequate precision and sensitivity. Nevertheless, efforts applied diligently and imaginatively over the decades implicated substances in unanticipated numbers: dozens upon dozens of likely neurotransmitters. Here I will illustrate these quests by describing briefly some diverse courses by which nine putative neurotransmitters gained credibility. (Neurotransmitters are sometimes distinguished from "neuromodulators," the latter having a slower onset and longer duration of action and/or an indirect effect through modifying responses to other agents.2 Here, however, neurotransmitter is used inclusively for substances released by a neuron to affect nearby cells acutely.)

Acetylcholine

By 1945 acetylcholine (Fig. 3-6A) was established as the excitatory neurotransmitter at neuromuscular junctions and autonomic ganglia and as the excitatory or inhibitory neurotransmitter of postganglionic parasympathetic fibers. Defining its role in the central nervous system was far less straightforward, although satisfying the first criterion, demonstrating its presence, was soon achieved. Not only was acetylcholine present in brain and spinal cord, it was present in synaptic vesicles within nerve terminals (chapter 4). These analyses were made by bioassays, however, and to skeptical chemists such identifications lacked—however close the correlations with authentic acetylcholine across multiple systems—the assurance of molecular analyses. Measuring acetylcholine chemically was a daunting challenge since it is intrinsically labile (splitting to acetate and choline), exists among enzymes that catalyze this hydrolysis, and.is present in minute quantities.3 Finally, in 1968 Bo Holmstedt in Stockholm and Donald Jenden in Los Angeles collaborated to demonstrate the molecule in mammalian brain, combining gas chromatographic methods for separation and mass spectrometry for identification and quantitation.4 Alternative choline esters, if present, existed at far lower concentrations. The following year Jack Green in New York reported the chemical identification of acetylcholine released into the bath after he stimulated parasympathetic fibers to isolated intestine.0 Still more difficult was correlating acetylcholine release with impulse transmission across specific synapses in the brain. For autonomic ganglia and neuromuscular junctions, acetylcholine release into the surrounding bath or the venous effluent could be matched to stimulation of the presynaptic fibers. For the brain, however, release from one pathway might be commingled with, and thus masked by, release from multitudes of other pathways if the total release into bath or venous outflow were examined. More localized collections were required to distinguish particular releases from the general background release.

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This James Mitchell in Babraham achieved in the 1960s by catching released acetylcholine in small cups on the cortical surface, formed by pressing hollow plastic cylinders against the brain surface.6 Mitchell found that the spontaneous release was "roughly proportional to the electrical activity of the brain"; moreover, when he placed the cups over the sensory cortex, an increased release of acetylcholine followed stimulation of peripheral sensory nerves or of sensory pathways in the brain.7 By contrast, when he removed the cortex and placed the cups on the underlying white matter, he could detect no acetylcholine release. In subsequent studies Mitchell found that acetylcholine release from the visual cortex increased severalfold when the eyes were exposed to light or the visual pathway was stimulated electrically.8 Severing the neural pathways eliminated light-evoked release. He also measured release into cups implanted in conscious, unrestrained animals that correlated with their activity.9 Demonstrating release beneath the surface obviously required different approaches. In 1961 John Gaddum (Fig. 5-1A), then also in Babraham, described "push-pull" cannulas for perfusing deeper regions of the brain. Gaddum contributed broadly to neuropharmacology, just as he appears in numerous chapters here. Now he inserted parallel hollow needles into brain tissue

FIGURE 5-1. A, left, John Henry Gaddum (1900-1965). B, right, Ulf S. von Euler (1905-1983). (A courtesy of Wellcome Trust Medical Photography Library. B courtesy of Leo von Euler.)

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and then forced perfusion fluid through one, collecting it from the other.10 In 1964 Hugh McLennan in Vancouver applied this method to show acetylcholine release within the caudate nucleus (a cellular region deep within the brain known for its high acetylcholine content; see chapter 13); furthermore, acetylcholine release increased after he stimulated certain pathways to this nucleus.11 The previous year Mitchell had used push-pull cannulas to show that acetylcholine was released within the cortex but not from the white matter immediately beneath.12 But more discrete localizations were not possible by such techniques. The third criterion, mimicry of neurally evoked responses, was satisfied earlier.13 In 1958 David Curtis and Rosamond Eccles in Canberra applied Bernard Katz and Jose del Castillo's "microelectrophoretic" approach to spinal cord Renshaw cells.14 Curtis and Eccles used five-barreled micropipets; the central pipet was filled with concentrated NaCl to record electrical potentials, and the other four contained acetylcholine and various analogs, acetylcholine antagonists, and cholinesterase inhibitors (Fig. 5-2A). Each of these charged molecules could be released electrophoretically by passing an electric current through its particular barrel.15 Since Renshaw cells are excited by recurrent collaterals of ventral horn motoneurons (chapter 4), Curtis and Eccles identified these cells by stimulating the ventral roots. Impulses passed antidromically back toward motoneurons and then ortfoodromically along branching recurrent collaterals to excite, across the synapse, a Renshaw cell. So they advanced the fivebarreled micropipet into the ventral horn until they recorded action potentials correlating with this stimulation. At that point they could release, electrophoretically, each of the substances in the other four barrels near this Renshaw

FIGURE 5-2. Electrodes for electrical recording and for microelectrophoretic application. A. Five barreled glass microelectrodes allowed one barrel to be used for measuring electrical responses extracellularly and the surrounding barrels to release four test substances extracellularly. Each barrel was about a micron in diameter. B. Concentric glass microelectrodes contained an inner barrel for penetrating the cell, to permit transmembrane electrical measurements, and an outer barrel for releasing a test substance extracellularly.

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cell while continuing to monitor its activity. As John Eccles—with less precise localization—concluded earlier, acetylcholine excited Renshaw cells. Curtis and Eccles now showed that nicotine also excited, whereas the nicotinic antagonists curare and dihydro-/3-erythroidine blocked. Cholinesterase inhibitors potentiated responses to acetylcholine. With five-barreled micropipets both release of reagents and measurements of potential were extracellular. To measure transmembrane potentials Curtis used concentric pipets: the inner pipet penetrated to the cell interior, while the outer released reagents extracellularly (Fig. 5-2B). Shortly thereafter, Kresimir Krnjevic in Babraham applied these techniques to neurons of the cerebral cortex.16 Acetylcholine excited only 15% of the thousands of cortical neurons examined, although it excited 60% to 90% of the Betz cells (large neurons of the motor cortex). By contrast, glutamate (see below) excited all the cortical cells. And instead of the nicotinic responses of Renshaw cells, the responses of cortical neurons to acetylcholine were muscarinic: atropine blocked, but not curare or dihydro-/3-erythroidine. Moreover, responses to acetylcholine were slow in onset and prolonged after administration ceased, time courses reminiscent of muscarinic postganglionic parasympathetic responses. Others soon reported similar results at other sites in the brain, finding muscarinic responses predominantly.17 But these experiments reaffirmed the conclusion that acetylcholine played a far less pervasive role in central neurotransmission than it did in the periphery.

Noraarenaline

By 1945 adrenaline (Fig. 3-3A), too, seemed established as a neurotransmitter, in its case as the excitatory or inhibitory neurotransmitter of postganglionic sympathetic fibers. Nevertheless, uncertainties about the true identity of the sympathetic agent stretched back to the arguments between Thomas Elliott and Henry Dale (chapter 3). Walter Cannon had proposed sympathins E and I, whereas Zenon Bacq considered noradrenaline the excitatory agent and adrenaline the inhibitory. Others favored noradrenaline alone as the neurotransmitter, and in 1942 Hermann Blaschko in Oxford presented biochemical evidence for noradrenaline being formed first and adrenaline from it.18 Beginning in 1946, Ulf von Euler (Fig. 5-1B) in Stockholm marshalled persuasive evidence for noradrenaline (Fig. 3-3B) fulfilling the role of sympathetic neurotransmitter.19 After medical training in Stockholm, von Euler had worked with Dale in Hampstead (where he collaborated with Gaddum). He then went on to identify several physiologically significant substances, including prostaglandins, but his major interest in later years centered on noradrenaline— studies rewarded with a Nobel Prize in 1970. Initially, von Euler identified

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FIGURE 5-3. Catecholamine biosynthetic pathway. Tyrosine, B, a conventional amino acid, may be synthesized from another conventional amino acid, phenylalanine, A, by hydroxylation of its benzene ring. Hydroxylation of tyrosine forms dopa, C, a third amino acid but one not found in proteins (unlike phenylalanine and tyrosine). Decarboxylation of dopa forms dopamine, D, which, like the succeeding products, is a catecholamine. (Catechol consists of a benzene ring with two adjacent hydroxyls.) Hydroxylation of dopamine on its sidechain forms noradrenaline, E. Methylation of noradrenaline s amine group forms adrenaline, F.

noradrenaline by comparing responses from a broad range of bioassays, which he then supplemented with discriminating chemical analyses: the noradrenaline content of sympathetic nerves was 50-fold that of adrenaline.20 Others soon confirmed this reinterpretation.21 (In the meantime, P. Holtz in Rostock detected noradrenaline in mammalian adrenals, although at far lower levels than adrenaline.22) Dale, looking back in 1953 on his 1914 paper showing a close parallel between sympathetic stimulation and added noradrenaline, acknowledged: "I ought to have seen that noradrenaline might be the main transmitter—that Elliott's theory might be right in principle and faulty only in this detail."23 On the other hand, von Euler found adrenaline instead of noradrenaline in frog hearts.24 Otto Loewi s identification in 1936 was correct for the species he studied. In 1946 von Euler also described noradrenaline s presence in brain extracts, but he concluded that the low levels he found arose from postganglionic sym-

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pathetic fibers accompanying blood vessels in the brain.25 Marthe Vogt, now in Edinburgh, disagreed. In 1954 she reported that noradrenaline levels in brain tissue were higher in some regions (notably the hypothalamus) than in others, but there was no correlation between this uneven density and blood vessel distribution.26 Moreover, after Vogt cut the sympathetic nerves to the brain blood vessels and allowed the terminals to degenerate, she found no change in the noradrenaline content of the hypothalamus. Detailed localizations within the brain became possible with the development of microscopic techniques for identifying catecholamine-containing neurons. In the early 1960s Bengt Falck in Lund and Nils-Ake Hillarp in Goteborg found that treating fixed tissue sections with formaldehyde vapor produced yellow-green fluorescent products from noradrenaline and dopamine.27 This histochemical technique was then widely employed to define pathways using noradrenaline, investigations of immense significance in understanding the functional organization of the nervous system but lying beyond the bounds of this history. It is, however, important to add that discriminating between noradrenaline- and dopamine-containing neurons was initially based on responses to pharmacological agents, but in 1968 Falck devised a microspectrofluorometric method for distinguishing between these catecholamines that confirmed earlier assignments.28 Unlike acetylcholine, noradrenaline is not rapidly destroyed in the brain, and its metabolic products do not mix appreciably with general cellular metabolites. Consequently, it is possible to add isotopically labeled noradrenaline and follow it by means of this radioactivity. 3H-noradrenaline was used from the early 1960s for such purposes, including both light and electron microscopic investigations of cellular and subcellular distribution.29 Radioactive labeling also facilitated demonstrations that noradrenaline, like acetylcholine, was present in brain synaptosomes.30 Documenting noradrenaline release from sympathetic fibers followed soon after von Euler's identification.31 Measuring noradrenaline release from the central nervous system was technically more difficult, but by the mid-1960s that, too, was achieved.32 Curtis s initial microelectrophoretic studies on the spinal cord failed to identify neurons responding to noradrenaline.33 Nevertheless, analogous studies by others then cataloged and mapped neuronal responsiveness to noradrenaline throughout the brain.34

Dopamine

Suggestions that dopamine (Fig. 3-3C) might serve as a neurotransmitter emerged from the recognition that it was present in amounts greater than would be expected for a mere metabolic precursor to noradrenaline. Blaschko showed

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in 1956 that labeled dopa gave rise to labeled dopamine and noradrenaline, in accord with the sequential conversion: dopa —» dopamine —» noradrenaline (Fig. 5-3).35 But Blaschko, noting that some nerves contained as much dopamine as noradrenaline, proposed the next year that "dopamine has some regulatory functions of its own."36 In 1958 Arvid Carlsson in Lund found dopamine levels in brain equivalent to noradrenaline levels; he, too, proposed that such amounts "may indicate that the function of [dopamine] is not merely that of a precursor."37 A. Bertler and E. Rosengren in Lund and I. Sano and associates in Osaka then provided strong support for this proposal: they described in 1959 distributions of dopamine in the brain that differed from those of noradrenaline.38 Combining chemical analyses with fluorescence microscopic techniques, Swedish investigators then mapped dopamine-containing neurons in the brain quite distinct from noradrenaline-containing ones.39 Concurrently, Victor Whittaker in Babraham identified dopamine in brain synaptosomal fractions.40 These mappings revealed prominent concentrations of dopamine in the caudate nucleus of the basal ganglia (see chapter 13). At the same time that McLennan described the stimulated release of acetylcholine in the caudate nucleus using push-pull cannulas, he also reported dopamine release that increased when he stimulated a different pathway to the caudate.41 Floyd Bloom in Washington then showed that administering dopamine microelectrophoretically inhibited certain caudate nucleus neurons.42 These experiments identified particular neuronal pathways containing dopamine as their catecholamine, with the dopamine present in nerve endings and released during stimulation. Administered dopamine affected the activity of other neurons. But the enormous interest in dopaminergic systems that developed subsequently represented further interests: changes in dopamine levels accompanied certain diseases, and drugs effective in treating certain diseases altered dopamine metabolism, transport, and receptors (chapter 13).

Serotonin

Scattered among the mucosal cells lining the gastrointestinal tract are "enterochromaffin cells," named for their location plus their characteristic staining by chromium salts. Vittorio Erspamer, first in Pavia and then Rome, began in the 1930s efforts to identify the substances within these cells that were responsible for the staining (as well as for the characteristic fluorescence and chemical reactions).43 By 1940 he had prepared extracts that reproduced the fluorescence and color reactions; in addition, these extracts caused various smooth muscles to contract, notably the rat uterus and small intestine. From its gastrointestinal origin and amine content, Erspamer named the substance

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"enteramine," although he soon identified it in such disparate loci as mammalian spleen, octopus salivary gland, and amphibian skin. His early attempts at chemical identification were not definitive, but by 1952 he concluded, despite the name but because of observed responses, that enteramine was "a true hormone" released into the bloodstream "to regulate bloodflow through the kidney."44 Irvine Page, first in New York, then Indianapolis, and finally Cleveland, approached from different interests.45 Beginning in the 1930s, Page was searching for circulating factors that produce malignant hypertension, a severe and mysterious malady. His goal was obscured by the multitudes of substances that constrict blood vessels and hence elevate blood pressure. For decades it had been known that serum from clotted blood, when transfused into animals, elevated their blood pressures;46 to eliminate this potentially confounding process, Page attempted to identify the responsible factor in serum. At the end of the war, he hired a young chemist, Maurice Rapport, to isolate this vasoconstricting substance using the rabbit ear vein as a bioassay. By 1948 Rapport obtained crystalline material (from 900 liters of serum derived from two tons of beef blood), which they named "serotonin" from its source and action.47 The following year Rapport, now in New York, identified serotonin as 5-hydroxytryptamine (Fig. 5^4A).48 Chemical syntheses came from two pharmaceutical firms, who then made the pure synthetic material available for study,49 effectively promoting further research. Through the 1940s Page was unaware of Erspamers work on enteramine, but in 1952 Erspamer showed that enteramine also was 5-hydroxytryptamine.50 (Most of the body's 5-hydroxytryptamine is in enterochromaffin cells, which release it to alter gastrointestinal motility. Thus, Erspamers naming is justifiable. Platelets are the source of 5-hydroxytryptamine liberated during blood clotting, when it helps constrict blood vessels to facilitate hemostasis. So Page's naming is also justifiable, albeit on a quantitatively lesser scale. Many pharmacologists prefer 5-hydroxytryptamine, which has a convenient abbreviation, 5-HT. But serotonin has gained broad acceptance and hence is used here.) In 1953 Page surveyed several organs and tissues for serotonin (using clam heart bioassays) and reported equivalent concentrations in brain and kidney, but negligible amounts in nerve and muscle.51 The following year Gaddum, then in Edinburgh, described a more detailed localization within the brain (using rat uterus bioassays), noting high concentrations in the hypothalamus and midbrain, with an overall distribution like that of noradrenaline.52 Sidney Udenfriend and associates in Bethesda confirmed and extended these identifications in 1957 using chemical (fluorescence) assays.53 Other studies soon described serotonin throughout the biological universe, from bee venoms to bananas. Since serotonin, like noradrenaline, is metabolized relatively slowly and its

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FIGURE 5-4. Structures of some neurotransmitters. The structure of the opioid morphine is also shown.

metabolites do not mix with general cellular metabolites, administering radioactive serotonin allowed its subsequent localization by electron microscopy,54 as with radioactive noradrenaline. Moreover, the fluorescence microscopic techniques that formed yellow-green products with catecholamines formed yellow products with serotonin.55 Mappings by this method demonstrated that serotonin-containing cell bodies were localized in the midbrain, separate from catecholamine-containing cell bodies. Again, these methods provided detailed demonstrations of neuronal pathways and relationships essential to understanding brain function. In the early 1960s Whittaker reported serotonin's presence in synaptosomal fractions from the brain, like acetylcholine.56 Independently, Eduardo De-

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Robertis also described serotonin-containing synaptosomes prepared by density-gradient centrifugation.57 Serotonin release after neural stimulation was soon reported, despite the rapid transport of this substance into cells (chapter 9).58 Descriptions of responses to microelectrophoretically applied serotonin also followed.59 But, as with dopamine, the extraordinary interest throughout the succeeding decades sprang from serotonin's association with particular disorders and the associated pharmacological interactions (chapter 13).

GABA While comparing the chemical composition of cancerous and noncancerous tissues, Eugene Roberts in St. Louis noticed a prominent spot on paper chromatograms of normal brain tissue. This he identified in 1950 as y-aminobutyric acid (GABA; Fig. 5-4C).60 Roberts could detect GABA only in the central nervous system, where it was formed by enzymatic decarboxylation of glutamate (Fig. 5-4D), one of the conventional a-amino acids that make up proteins; GABA was then converted enzymatically to succinate, an intermediate in the Krebs cycle for cellular metabolism. Since glutamate could be formed from another intermediate in this cycle, Roberts interpreted the pathway forming and degrading GABA as a metabolic "shunt," bypassing steps in the Krebs cycle.61 Ernst Florey approached quite differently, from studying substances present in the brain that elicited inhibitory responses.62 Favorite test systems for inhibition were crustacean synapses, where inhibitory nerves, readily identifiable and accessible, participate.63 In 1956 Florey, then in Montreal, identified GABA as the active substance in his "Factor I," an extract from beef brain that inhibited crustacean synapses.64 Others soon joined in studying GABA as a possible inhibitory neurotransmitter. In 1958 Paul Fatt in London, while examining changeable ionic permeabilities at crustacean neuromuscular junctions (chapter 4), showed that GABA affected ionic conductances in postsynaptic neurons, as did stimulating inhibitory nerves.65 And Stephen Kuffler in Baltimore found that the electrical responses of crustacean synapses were similar when evoked by adding GABA or by stimulating inhibitory nerves.66 Nevertheless, Florey was having second thoughts about his identification. In the mid-1950s Florey began collaborating with McLennan, then also in Montreal; together they showed that Factor I inhibited various mammalian processes, including synaptic transmission in autonomic ganglia.67 But after Florey identified Factor I as GABA, McLennan pointed to discrepancies—activities that Factor I possessed but GABA lacked—and reported that his preparation of Factor I did not contain GABA.68 Florey agreed with the implicit assumption that Fac-

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tor I should contain a single inhibitory substance displaying the entire range of inhibitory responses, and in a review published in 1961 Florey declared that "GABA is not the inhibitory neurotransmitter although it imitates the transmitter action in many crustacean preparations."69 Florey also failed to find GABA in crustacean nerves, but Edward Kravitz and David Potter did.70 The following year, 1963, they, together with Kuffler, now also in Boston, reported that inhibitory but not excitatory nerves contained GABA.71 They pointed out that Florey's argument against GABA being the inhibitory substance could be countered by acknowledging that additional inhibitory substances might be present in tissue extracts: the amount of GABA present in extracts need not equal the total inhibitory capacity of the extract; GABA might represent only part of the inhibitory capacity while being the physiologically active substance. And in 1966 Kravitz found that stimulating inhibitory nerves released GABA in amounts proportional to that stimulation; by contrast, stimulating excitatory nerves released no GABA.72 The previous year A. and N. Takeuchi in Tokyo described microelectrophoretic administration of GABA onto crustacean muscles: GABA evoked inhibitory electrical responses when applied to neuromuscular junctions, but not elsewhere on muscle surfaces or when injected into muscles.73 Thus, GABA was present specifically in inhibitory nerves, it was released specifically from them, and its localized application to neuromuscular junctions mimicked nerve stimulation. Florey was won back to his original view.74 Building a comparable case for GABA in vertebrate nervous systems was more protracted. GABA, as Roberts initially demonstrated, was present in the central nervous system. Moreover, its uneven distribution there could reflect diverse functions, as uneven distributions of established neurotransmitters were so interpreted.75 But when Roberts examined the subcellular distribution in 1963, he found that most of the GABA was "free" in the supernatant fraction. (The largest fraction of "bound" GABA, however, was associated with synaptosomes.76) At this time Roberts was still focusing on metabolic roles for GABA, so this distribution was not crucial. Others interested in GABA as a neurotransmitter, including Whittaker, also found that most of the GABA was free, but in 1971 Leslie Iversen in Cambridge argued persuasively that in vivo GABA was largely confined to vesicles but was artifactually released during cell fractionation.77 In the spinal cord, where Charles Sherrington and John Eccles had established central inhibition decisively (chapters 2 and 4), GABA did not at first satisfy expectations. In 1959 Curtis found that microelectrophoretically applied GABA blocked excitation in the spinal cord but did not hyperpolarize the neurons; moreover, strychnine, which both Sherrington and Eccles used to block inhibition, did not alter GABAs action.78 Curtis concluded that GABA produced a nonspecific "depressant" effect, rather than the true inhibition expected of a neurotransmitter.

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Studies on the brain were more encouraging. In 1963 Krnjevic reported that applying GABA microelectrophoretically to the cerebral cortex inhibited both spontaneous activity (Fig. 5-5A) and excitation induced by applied glutamate.79 Krnjevic subsequently found that applied GABA hyperpolarized cortical neurons, producing i.p.s.p.s just as did stimulating inhibitory pathways. Moreover, responses to applied GABA or to stimulation were similarly sensitive to injected Cl~, indicating that similar changes in ionic permeability underlay each.80 Particularly persuasive were studies on the cerebellum, where earlier research established Purkinje cells in the cerebellar cortex as prominent inhibitors of neurons in subcortical nuclei and brainstem. In 1967 K. Obata in Tokyo demonstrated that microelectrophoretically applied GABA hyperpolarized these neurons.81 Obata also showed that Purkinje cells contained GABA and that stimulating the cerebellum increased GABA release into the ventricle severalfold.82 Mitchell and Iversen then collected GABA from cups placed on the cerebral cortex: electrical stimulation of inhibitory pathways increased GABA release here as well.83

FIGURE 5-5. Electrical responses to microelectrophoretic application of neurotransmitters. A. Applying GABA extracellularly blocked spontaneous activity. Electrical activity of cerebellar neurons is represented by the vertical traces displayed against time (marked in seconds below the trace), measured by one barrel of the electrode. When a 60 nA current was passed through the barrel containing GABA (horizontal line beneath the time trace), discharging GABA electrophoretically, the spontaneous activity ceased. When the application of GABA stopped, the spontaneous activity resumed. B. Applying the L-stereoisomer of glutamate (L-Glut) by a 60 nA current through one barrel of the electrode produced electrical activity in cortical neurons (vertical excursions, recorded through a separate barrel of the micropipet) that began shortly after glutamate was released and ceased abruptly when release was halted. Applying the D-stereisomer (D-Glut) produced no response. (A and B from Krnjevic and Phillis [1963a], Figs. 17 and 4, respectively, courtesy of the Physiological Society.)

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By 1968 Curtis realized that his multibarrel electrodes could damage neurons, and he redesigned their configuration. He now found that applied GABA indeed hyperpolarized spinal motoneurons.84 Strychnine, however, still did not block the effects of GABA. On the other hand, earlier studies by Florey and others demonstrated that picrotoxin, another well known convulsant derived from plants, blocked inhibition at crustacean synapses as well as the effects of Factor I and of GABA.85 Curtis surveyed an array of convulsants for a more convenient reagent, and in 1970 he reported that bicuculline, another plant product, blocked GABA's effects specifically.86 Bicuculline also blocked inhibition occurring at certain sites in the brain and spinal cord, but not at sites sensitive to strychnine. There another inhibitory neurotransmitter must act.

Glutamate

With evidence accumulating that GABA served as an inhibitory neurotransmitter in crustaceans, Jay Bobbins in New York screened dozens of other amino acids for effects on crustacean synapses. In 1958 he reported that—alone among these—glutamate (Fig. 5-4D) and aspartate excited crustacean muscles.87 Glutamate was more potent, and Robbins suggested that it might be the neurotransmitter of excitatory nerves, opposing GABA released by inhibitory nerves. Independently, Anthonie Van Harreveld in Pasadena identified glutamate as the active substance in rabbit brain extracts that excited crustacean muscles.88 And in 1964 the Takeuchis used microelectrophoretic application to localize glutamate s action to crustacean neuromuscular junctions.89 Glutamate excited even after denervation, indicating direct effects on the endplate rather than through the nerve. (Due to technical obstacles, glutamate release from excitatory nerves was not demonstrated until 1981.90) Meanwhile, Curtis was curious about structural and metabolic relationships between GABA and glutamate, and he found that microelectrophoretically applied glutamate excited spinal cord neurons.91 But Curtis judged glutamate not to be a neurotransmitter, in part because he considered antagonism between applied glutamate and applied GABA as competition for a common receptor (and he then considered GABA not to be a neurotransmitter) and in part because he found the D- and L-stereoisomers of glutamate to be equally effective.92 Perhaps he also shared the contemporary skepticism about ordinary amino acids serving as neurotransmitters, for in 1960 the likely neurotransmitters (those considered above) were all specialized derivatives. Nevertheless, even though glutamate as a conventional amino acid was a component of all cells, its distribution in the nervous system was uneven, suggesting a functional localization.93 Of particular note were glutamate concentrations in dorsal root ganglia and regions of the spinal cord where sensory dor-

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sal root neurons terminate.94 This localization suggested a role in sensory limbs of spinal cord reflex arcs, arcs where acetylcholine served in motor limbs. Initial investigations of glutamate s subcellular distribution revealed—as with GABA—that the greatest amount was free in the soluble fraction.95 In 1971, however, Solomon Snyder in Baltimore found a portion of the neuronal glutamate concentrated in synaptosomal fractions, and two years later H. F. Bradford in London described a vesicular fraction containing glutamate.96 Demonstrating a stimulated release of glutamate was hindered by the high background of "nonspecific" release, reflecting glutamate's multiple physiological roles. In 1969, however, Herbert Jasper in Montreal described glutamate release into fluid flowing over the brain surface that increased after electrical stimulation.97 In 1972 P. J. Roberts and Mitchell, now in Bristol, reported a stimulated release from the spinal cord and the following year a stimulated release into cups on the visual cortex.98 Unlike Curtis, who found both stereoisomers of glutamate equally effective on the spinal cord, Krnjevic showed in 1963 that the naturally occurring Lstereoisomer was more effective on cortical neurons (Fig. 5-5B).99 Krnjevic also found that glutamate excited spinal cord cells in regions where sensory dorsal root neurons terminate.100 By 1972 Curtis agreed that glutamate was a likely neurotransmitter in spinal cord and brain,101 exciting a far larger fraction of neurons than any other putative neurotransmitter then known. But the delineation of glutamatergic systems was hindered during this period by the lack of specific blocking agents to assist in identifying such synapses.

Glycine

Glycine (Fig. 5-4B), too, is an ordinary a-amino acid present in all cells, making its candidacy as a neurotransmitter "easy to deride."102 But in 1965 Morris Aprison in Indianapolis pointed out that glycine, measured during surveys of amino acid content, was concentrated in the spinal cord gray matter, where inhibitory interneurons and their terminals are localized.103 Two years later Aprison furthered his proposal by describing sharp decreases in glycine content after destroying the spinal cord interneurons.104 Demonstrating glycine's presence in synaptic vesicles, however, was not achieved during this period, although Snyder provided circumstantial evidence for glycine's association with synaptosomal fractions.105 Measuring a stimulated release was also challenging. In 1971 R. A. Webster in London described an outflow of labeled glycine into fluid perfusing the cat spinal cord, and this outflow increased when he stimulated peripheral nerves electrically (he first loaded the spinal cord with labeled glycine by perfusing with 14C-glycine.106) The following year Roberts and Mitchell, in experiments

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paralleling their study on glutamate release, also reported an increased outflow of labeled glycine after stimulating isolated frog spinal cords that were first loaded with 14C-glycine.107 Most convincing were studies showing that added glycine inhibited the spinal cord ventral horn motoneurons that Sherrington and Eccles had characterized. In 1967 Aprison described hyperpolarizations of these motoneurons after applying glycine microelectrophoretically, inducing i.p.s.p.s.108 That year Curtis reported similar results; he also found that strychnine blocked this response to glycine, in accord with Sherringtons and Eccles's observation that strychnine blocked inhibition in the spinal cord.109 These and subsequent studies confirmed glycine as an inhibitory neurotransmitter in the spinal cord, notably as the inhibitory neurotransmitter that Renshaw cells released onto motoneurons.110 In the brain neither added glycine nor strychnine had prominent effects, although Obata at this time showed that added glycine inhibited in a strychnine-sensitive manner at certain loci.111 By contrast, added GAB A inhibited broadly throughout the brain in a strychnine-insensitive but picrotoxin- and bicuculline-sensitive manner. Moreover, added GABA also inhibited particular pathways in the spinal cord, consistent with Eccles's earlier demonstration that presynaptic inhibition was strychnine-insensitive but picrotoxin-sensitive (chapter 4).

Neuropeptio.es: Substance P and Enkepnalins

During the postwar decades dozens of peptides emerged as acknowledged neurotransmitters, including many previously identified as gastrointestinal hormones (such as secretin). Here I will note the course to recognizing two novel peptides.

Substance P

Working in Dale's institute in Hampstead, von Euler and Gaddum sought to extend Dale's identification of acetylcholine to tissues beyond the spleen. In 1931 they described an alcoholic extract of horse brain and intestine that dilated blood vessels and contracted intestines like acetylcholine, but unlike acetylcholine acted in the presence of atropine.112 Their partial purification included a precipitation from the alcoholic extract, which they dried to a powder: they called this product "preparation P," for powder. Gaddum in 1933 named the active agent "substance P," and in 1936 von Euler, back in Sweden, reported that digestion with the proteolytic enzyme trypsin destroyed activity.113 Thus, the active agent was probably a peptide. Isolation and chemical identification were not achieved in the next several

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decades; nevertheless, further purification implied that substance P was a definite entity with actions distinguishable by certain bioassays. Again, uneven distributions—as reported by Gaddum, now in Edinburgh, Bengt Pernow in Stockholm, and F. Lembeck in Graz—suggested specific functional roles in brain and spinal cord.114 A notable concentration in dorsal roots and dorsal horns of the spinal cord suggested that substance P participated in sensory transmission. Moreover, in the early 1960s substance P was localized to synaptosomal fractions.115 Although Gaddum failed to find release from the spinal cord using push-pull cannulas, in 1968 Jane Shaw and P. W. Ramwell in Shrewsbury, Mass., reported that stimulatory drugs increased the release of substance P into cortical cups.116 And although Krnjevic found only weak responses of spinal cord neurons to microelectrophoretically applied substance P preparations, H. Caspars and P. Stern in Miinster described in 1961 an enhanced electrical activity in the cortex after topical administration.117 Chemical identification, which permitted experiments using the pure substance and measurements of it, resolved and extended old issues. In the 1960s Susan Leeman in Waltham began studying brain extracts that promoted salivary secretion. This culminated in 1970 when she (1) isolated an active 11amino acid peptide of specified composition and (2) showed its pharmacological identity to substance P.118 The next year Leeman and colleagues reported the sequence of 11 amino acids and the chemical synthesis of this peptide.119 Antibodies to the pure peptide could now be used in highly sensitive analytical procedures to confirm the distribution of substance P in the brain and spinal cord, again emphasizing a localization in dorsal root ganglion cells as well as distinct regions of the brain.120 Masonori Otsuka in Tokyo then demonstrated a stimulated release of substance P specifically121 And application of the pure substance, by perfusion or microelectrophoresis, excited cortical neurons and spinal cord ventral horn motoneurons.122 Whereas glutamate evoked rapid responses of brief duration in the motoneurons, substance P caused more prolonged responses. Subsequent studies identified substance P as a major neurotransmitter in sensory neurons mediating pain (so P stands not only for powder and peptide, but also pain). Enkepnalins

Opium, the dried exudate from seed capsules of the poppy, has been used to relieve pain and induce euphoria for millennia. Early in the nineteenth century morphine (Fig. 5-4F) and codeine were isolated from opium, and by midtwentieth century synthetic opioids123 were developed as well as specific antagonists. Precise structural requirements for morphine and its analogs to produce analgesia and euphoria (and for antagonists to block the responses) suggested that these compounds interacted with specific receptors in the body. In 1973

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three groups identified such receptors in the brain (chapter 6). The existence of specific receptors for exogenous opioids then suggested that endogenous opioids might exist. So several investigators, including Lars Terenius in Upsala, Avram Goldstein in Palo Alto, and Snyder, set about searching for substances in brain extracts that bound to their receptor preparations and whose binding could be displaced by known opioids and opioid antagonists.124 The initial success, however, was achieved through a traditional route. Hans Kosterlitz had initially studied sugar metabolism and diabetes in his native Germany and continued this research after fleeing to Aberdeen in 1934.125 After the war he turned his attention to how the autonomic nervous system affects digestion; this endeavor included examining morphine's inhibition of peristalsis,126 demonstrable on the isolated guinea pig ileum. On retiring in 1973 at age 70, Kosterlitz founded the Unit for Research on Addictive Drugs in Aberdeen, where he and a young associate, John Hughes, exploited an extremely sensitive and selective bioassay for opioids, the mouse vas deferens. Electrical stimulation of sympathetic nerves to the vas caused it to contract; morphine blocked this response, whereas opioid antagonists, such as naloxone, prevented morphine's action.127 (Hughes and Kosterlitz also demonstrated that morphine acted by preventing noradrenaline release from the excitatory nerve.) Hughes and Kosterlitz then showed that pig brain extracts contained material that blocked contractions of both the vas deferens and ileum; naloxone antagonized the extracts' effects in both bioassays.128 The active substance, named "enkephalin," had a molecular weight of roughly 1000, and its potency was destroyed by enzymes cleaving peptide bonds. In 1975 they identified two active peptides in the extract (and synthesized them): met-enkephalin (Fig. 5-4E) and leu-enkephalin; each contained five amino acids, differing in only one of these.129 The following year Snyder reported that calf brain extracts also contained met- and leu-enkephalin, whose purification he followed by assessing binding to opioid receptor preparations.130 Both described uneven distributions of enkephalins in the brain, extending beyond known pathways for pain perception and implying that enkephalins played broader functional roles.131 In 1976 Snyder also reported that enkephalins were localized in synaptosomal fractions.132 Demonstrating enkephlin release was technically difficult, in part due to free enkephalins rapid destruction by endogenous peptidases. Within five years of enkephalins discovery, however, descriptions of its release appeared.133 If exogenous opioids produce analgesia by acting on specific receptors in the brain, then endogenous opioids should also be able to relieve pain. Indeed, injecting enkephalins into brain ventricles produced analgesia.134 Reports also published in 1976 described microelectrophoretic application of enkephalins that altered the electrical activities of particular neurons in certain brain regions, effects blocked by naloxone.135 (Although the structures of met-enkephalin

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and morphine [Figs. 5-4E and F] seem at first glance dissimilar, molecular models showed that strong resemblances in three-dimensional configurations exist.136) Meanwhile, Goldstein in 1975 found larger peptides in the pituitary that had opioid activity both in bioassays and on receptor preparations.137 Within a decade two further families of endogenous opioid peptides were characterized in addition to enkephalins: endorphins and dynorphins, ranging from 16 to 31 amino acids.138

Concl onciusions

After decades of concentration on acetylcholine and adrenaline/noradrenaline, new studies shifted the focus. To the surprise of many, glutamate turned out to be the major excitatory neurotransmitter in the brain as well as the sensory neurotransmitter of dorsal root ganglion cells. And GAB A turned out to be the major inhibitory neurotransmitter in the brain, with glycine a prominent inhibitory neurotransmitter in the spinal cord (notably as the neurotransmitter of Renshaw cells). Furthermore, considerations of pathologies and therapeutics fostered an interest in many of the more newly established neurotransmitters, notably dopamine, serotonin, GABA, and enkephalin. In all cases, however, the pathways to validating these nine substances as authentic neurotransmitters began from diverse personal interests that then converged on commonly recognized steps: demonstrating presence, release, and activity (mimicking physiological responses and pharmacological sensitivities to antagonists). Nevertheless, different candidate neurotransmitters posed different conceptual and technical challenges, and viewing these quests as stereotyped sequences of experiments would undervalue the skill and ingenuity demanded. Instead, successes reflected directed efforts to develop new strategies and modify old ones, although the cast here is so large that individual contributions blur together. Moreover, the successes represent communal accomplishments. Some scientists concentrated on a few techniques applicable to several neurotransmitters, while others examined only a few neurotransmitters but these extensively. Together, their findings corrected earlier errors through adapting alternative approaches and extending efforts to further issues and concerns. These studies also illustrate the common perils of oversimplification and overgeneralization. Thus, some investigators sought the inhibitory neurotransmitter and so discarded evidence for an inhibitory neurotransmitter when the candidate failed certain specifics. But an opposite extreme, assuming a new entity for each phenomenon, would also lead to error. The effective course required a reluctance to advocate new neurotransmitters and a tentative accept-

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ance only as the weight of evidence accumulated. This evidence included not only satisfying the criteria stated above but also displaying further attributes recognized later. Important among these were identifications of receptors specific for the candidate neurotransmitter, receptors that, when occupied by these substances or their close structural analogs, evoked demonstrable biochemical and cellular responses (chapters 6 and 7). During these decades evidence for and against other substances that might serve as neurotransmitters also accumulated, but space does not allow discussion of some later accepted, such as adenosine, and others that remained in limbo, such as taurine. But it is important to recognize that the list of likely neurotransmitters continued to grow throughout the period covered in this book.

Notes

1. For a contemporary discussion, see Werman (1966). Werman also stated other criteria that seemed obvious, too, such as the presence of enzymes for neurotransmitter synthesis and degradation. The applicability of these to all neurotransmitters turned out, nonetheless, to be limited (chapter 9). 2. See Cooper et al. (1996), pp. 4-5. 3. Using tracer techniques, such as radioactive labeling, is frustrated by the rapid breakdown, with the products then being incorporated into multitudes of other cellular constituents. 4. Hammar et al. (1968). 5. Schmidt et al. (1969). They, too, used a gas chromatographic approach. 6. Mitchell (1963). Physostigmine was present in the Ringers solution in the cups; acetylcholine was measured by bioassays. Acetylcholine concentrations in blood and cerebrospinal fluid were lower, so the released acetylcholine could not simply have leaked from these sources. This approach was first described in an abstract in 1953 by Macintosh and Oborin in Montreal, but they did not pursue it. 7. Ibid., p. 114. 8. Collier and Mitchell (1966). 9. Collier and Mitchell (1967). 10. Gaddum (1961). 11. McLennan (1964). 12. Mitchell (1963). 13. Exact mimicry would be unlikely. For example, as McLennan (1970) pointed out, much of the endogenous neurotransmitter is released onto the branched dendrites, whereas acetylcholine was administered in such experiments onto the cell body, close to the recording electrode. 14. del Castillo and Katz (1955a); Curtis and Eccles (1958a, b). These studies were extended in Curtis et al. (1961). Rosamond Eccles is John Eccles's daughter. 15. The number of ions released is proportional to the amount of current passed through the pipet. To prevent leakage from the pipets before release is desired, an opposing "back current" is applied. 16. Krnjevic and Phillis (1961, 1962, 1963a, 1963b, 1963c).

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17. For example, Spehmann and Kapp (1961); Spehmann (1963); Crawford and Curtis (1966); McLennan and York (1966). 18. Blaschko (1942). 19. von Euler (1946). Von Euler also included chemical assays, but these could not discriminate between noradrenaline and adrenaline when the quantities of each were unknown (e.g., noradrenaline was stated to fluoresce more weakly than adrenaline). 20. von Euler (1948, 1949); von Euler and Hamberg (1949). 21. Bacq and Fischer (1947); Gaddum and Goodwin (1947); Holtz et al. (1947). 22. Holtz and Schumann (1948). 23. Dale (1965), p. 98 (a reprint of the 1953 edition). In 1914, however, noradrenaline was not known to exist in mammalian tissues. 24. von Euler (1946). 25. Ibid. At that time sympathetic nerves were known to accompany blood vessels, where they were importantly involved in maintaining blood pressure through activating smooth muscles that controlled vessel diameter. 26. Vogt (1954). 27. Carlsson et al. (1962); Falck (1962); Falck et al. (1962). With adrenaline the fluorescence had a slower onset and was thus distinguishable. 28. For the pharmacological procedures, see Corrodi and Jonsson (1967). Bjorklund et al. (1968) described treatment with hydrochloric acid that shifts the excitation spectra between dopamine and noradrenaline, thereby making them distinguishable. 29. Samorajski and Marks (1962); Wolfe et al. (1962). In both these studies animals were injected with 3H-noradrenaline and tissue sections then exposed to photographic emulsions: the radioactive noradrenaline caused a deposition of silver grains in these emulsions that could be correlated with the cytological images from light or electron microscopy. 30. For example, Potter and Axelrod (1963); Glowinski et al. (1966). 31. For example, Peart (1949); West (1950). 32. For example, Anden et al. (1965); Baldessarini et al. (1967). Alternatively, decreases in tissue content were reported (for example, Gunne and Reis, 1963). 33. Curtis et al. (1961). 34. For example, Bloom et al. (1963); Bradley and Wolstencroft (1962); Krnjevic and Phillis (1963d). 35. Demis et al. (1956). They incubated adrenal medullary homogenates with 14Cdopa and recovered 14C-dopamine and 14C-noradrenaline. 36. Blaschko (1957), p. 11. 37. Carlsson et al. (1958), p. 471. 38. Bertler and Rosengren (1959); Sano et al. (1959). 39. For example, Anden et al. (1964b, 1965); Hillarp et al. (1966). 40. Laverty et al. (1963). They found, however, considerable amounts of "free" dopamine, that is, dopamine in the supernatant fraction. 41. McLennan (1964). He noted that dopamine "is not normally regarded as a likely transmitter substance" (pp. 152-153). 42. Bloom et al. (1965). Dopamine excited a smaller number of caudate nucleus neurons. See also McLennan and York (1967), who, citing recent reports, now considered dopamine a likely neurotransmitter. 43. Vialli and Erspamer (1933); Erspamer (1940a, 1940b, 1940c). 44. Erspamer and Asero (1952), p. 801. Erspamer noted that far lower concentrations of enteramine were required to affect the kidney than to raise blood pressure; moreover, vessels leading to the glomeruli (the site of filtration where urine formation begins) were most sensitive (Erspamer, 1953).

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45. For autobiographical accounts, see Page (1976); Rapport (1987, 1997). 46. Blood consists of various blood cells plus liquid "plasma." After blood coagulates, the clot contains trapped cells plus the clotting proteins. The remaining liquid is "serum," which is plasma minus the clotting proteins (it also contains substances liberated from the cells during coagulation, such as serotonin). 47. Rapport (1948a, 1948b). 48. Rapport (1949). 49. Hamlin and Fischer (1951) at Abbott Laboratories; Speeter et al. (1951) at the Upjohn Co. 50. Erspamer and Asero (1952). 51. Twarog and Page (1953). 52. Amin et al. (1954). 53. Bogdanski et al. (1957). The easy assay by fluorescence spectroscopy greatly facilitated further research. 54. Aghajanian and Bloom (1967). 55. Dahlstrom and Fuxe (1964); Anden et al. (1966). Procedures to distinguish serotonin from catecholamines included pharmacological approaches and microspectrofluorescence methods (see Corrodi and Jonsson, 1967; Bjorklund et al., 1970). 56. Michaelson and Whittaker (1962, 1963). 57. DeRobertis et al. (1962); Zieher and DeRobertis (1963). DeRobertis's separation of cholinergic from serotonergic synaptosomes was, however, controversial. 58. For example, Anden et al. (1964a); Beleslin and Myers (1970). Sometimes the disappearance of serotonin and appearance of its metabolite was interpreted as release: for example, Aghajanian et al. (1967). 59. For example, Bloom et al. (1963); Curtis et al. (1961); Krnjevic and Phillis (1963a). 60. Roberts and Frankel (1950). Awapara et al. (1950) independently identified GABA in chromatograms of the brain. 61. Roberts (1956). The GABA shunt depicted a-ketoglutarate converted to glutamate, which was then decarboxylated to GABA; GABA was next oxidized to succinate, so the GABA shunt bypassed the steps in the Krebs cycle between a-ketoglutarate and succinate. 62. Florey (1954). 63. Chief among these crustacean systems are neuromuscular junctions, where separate excitatory and inhibitory nerves innervate muscle, and stretch receptors (sense organs in muscle), on which inhibitory nerves end. 64. Bazemore et al. (1956, 1957). 65. Boistel and Fatt (1958). Hagiwara et al. (1960) confirmed these observations. 66. Kuffler and Edwards (1958). Others studying these issues included Grundfest et al. (1959) and Van der Kloot and Robbins (1959). 67. Florey and McLennan (1955). 68. McLennan (1957, 1958). 69. Florey (1961), p. 513. 70. Florey and Chapman (1961); Kravitz et al. (1962). 71. Kravitz et al. (1963). Their analyses were facilitated by using a highly sensitive and specific enzymatic assay for GABA. 72. Otsuka et al. (1966). 73. Takeuchi and Takeuchi (1965). 74. Koidl and Florey (1975). 75. Baxter and Roberts (1959). See also Fahn and Cote (1968). 76. Weinstein et al. (1963).

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77. Mangan and Whittaker (1966); Neal and Iversen (1969). 78. Curtis et al. (1959); Curtis and Watkins (1960). 79. Krnjevic and Phillis (1963a). 80. Krnjevic and Schwartz (1967). 81. Obata et al. (1967). 82. Obata (1969); Obata and Takeda (1969). Obata dissected Purkinje cells from cerebella and measured the GABA content on the pooled sample. By contrast, spinal cord motoneurons contained no GABA. Earlier, Kuriyama et al. (1966) measured the GABA content of the cerebellar region that includes Purkinje cell bodies. 83. Mitchell and Srinivasan (1969); Iversen et al. (1971). 84. Curtis et al. (1968). 85. Elliott and Florey (1956); Van der Kloot and Bobbins (1959). 86. Curtis et al. (1970, 1971). Picrotoxin is not appreciably ionized at neutral pH, making electrophoretic release difficult, so a better agent was needed. 87. Bobbins (1958,1959). The status of aspartate as aneurotransmitter remains uncertain, in part because it has been studied less vigorously and in part because both it and glutamate affect many of the same receptors. 88. Van Harreveld (1959). 89. Takeuchi and Takeuchi (1964). 90. Kawagoe et al. (1981). Success was achieved by elegant analytical methods applied to release in tiny volumes. 91. Curtis et al. (1960); Curtis and Watkins (1960). 92. Since physiological interactions usually exhibit stereoselective preferences, the equivalence of D- and L-stereoisomers implied that both acted by nonspecific nonphysiological mechanisms. Subsequently—and for reasons that are not apparent—the equivalence of the two stereoisomers vanished from reports (see Krnjevic and Phillis, 1963a). 93. For example, Berl and Waelsch (1958); Johnson and Aprison (1971). 94. For example, Graham et al. (1965, 1967); Duggan and Johnston (1970). 95. For example, Mangan and Whittaker (1966). 96. Wofsey et al. (1971); DeBelleroche and Bradford (1973). 97. Jasper and Koyama (1969). 98. Boberts and Mitchell (1972); Boberts (1973). 99. Krnjevic and Phillis (1963a). 100. Galindo et al. (1967). 101. Curtis et al. (1972). 102. Aprison and Werman (1965), p. 2081. 103. Ibid. 104. Davidoff et al. (1967). They destroyed interneurons by cutting off the blood supply to the spinal cord and demonstrated the selective loss of interneurons histologically. 105. Mangan and Whittaker (1966) found glycine chiefly free in the supernatant fraction. Young and Snyder (1973) argued for glycine-containing synaptosomes by identifying a synaptosomal fraction that bound strychnine. Strychnine presumably bound to glycine receptors on posfsynaptic membrane attached in the synaptosomal fraction to presynaptic terminals, and these terminals presumably contained glycine to release onto the adjacent postsynaptic receptors. 106. Jordan and Webster (1971). This demonstration required adding an inhibitor of glycine transport to block backflow into the spinal cord. 107. Boberts and Mitchell (1972). No transport inhibitor was used. 108. Werman et al. (1967).

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109. Curtis et al. (1967). 110. See Belcher et al. (1976). 111. Obata et al. (1970). 112. von Euler and Gaddum (1931). 113. Chang and Gaddum (1933); von Euler (1936). 114. Amin et al. (1954); Pernow (1953); Lembeck (1953). 115. Kataoka (1962); Cleugh et al. (1964). 116. Gaddum and Szerb (1961); Shaw and Ramwell (1968). 117. Galindo et al. (1967); Caspars and Stern (1961). 118. Chang and Leeman (1970). 119. Chang et al. (1971); Tregear et al. (1971). 120. Takahashi et al. (1974); Hokfelt et al. (1975). 121. Otsuka and Konishi (1976). 122. Konishi and Otsuka (1974); Phillis and Limacher (1974); Henry (1976). 123. Opioid refers to all substances with morphinelike properties, replacing terms like opiate and narcotic. 124. Terenius and Wahlstrom (1975); Teschemacher et al. (1975); Pasternack et ai. (1975). 125. For autobiographical reminiscences, see Kosterlitz (1979). 126. Opioids have been used to combat diarrhea also. 127. Henderson et al. (1972). 128. Hughes (1975); Hughes et al. (1975b). 129. Hughes et al. (1975a). One of the peptides contains leucine (leu), where the other has methionine (met). 130. Simantov and Snyder (1976). 131. Hughes et al. (1975a); Simantov and Snyder (1976). With pure met-enkephalin available, antibodies could then be prepared for use in immunohistological localization (for example, Hokfelt et al., 1977). 132. Simantov et al. (1976). 133. For example, Henderson et al. (1978); Iversen et al. (1978); Glowinski (1981). 134. For example, Belluzzi et al. (1976); Biischer et al. (1976). Earlier, Liebeskind found that electrical stimulation of certain brain regions produced analgesia, consistent with a stimulated release of neurotransmitters in a pathway relieving pain (Mayer et al., 1971). 135. For example, Frederickson and Norris (1976); Hill et al. (1976). 136. For example, Smith and Griffin (1978). 137. Teschemacher et al. (1975). 138. Frederickson (1984).

6 CHARACTERIZING RECEPTORS (1905-1983)

Essential Issues

John Newport Langley in 1905 introduced the notion that nerve activity, excitatory drugs, and their antagonists all elicited cellular responses through "receptive substances," hypothetical structures that soon became known by the shorter title receptors (chapter 3).1 This and the following two chapters describe attempts to answer essential questions raised by Langley s proposal: what, chemically, are these receptors; how do neurotransmitters and drugs interact with them; and how do such interactions trigger the cellular responses? These investigations also inspired various schemes for classifying receptor families, revealing generalities about evolutionary relationships and illustrating mechanistic similarities—despite the multitudes of entities cataloged over the decades. Moreover, demonstrations that certain neuronal constituents interacted with specific receptors complemented the identifications of neurotransmitters that were proceeding by other criteria (chapter 5). Drugj—Receptor Interactions One approach to discovering how receptors function lay in examining the ways cells responded to various agents that (presumably) acted through these receptors.

143

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Interpreting Dose—Response Plots

Among such characteristics were the time courses and magnitudes of these responses. In 1909 A. V. Hill, then in Cambridge, turned his formidable analytical powers to this issue, although only briefly.2 While examining Langley s proposal quantitatively, Hill focused first on the time courses of nicotineinduced muscle contraction and of curare-induced relaxation. Rather than the time course representing merely the physical process of drug diffusion into or out of the tissue, the response times, he concluded, reflected reversible interactions, "a gradual combination of the drug with some constituent of the muscle."3 As to the magnitude, Hill expressed the contraction height in terms of such combinations between excitatory drug or "agonist," A, and receptor, R:

A + R <=> AR. But his formulation included a threshold value, T, that AR must exceed before contraction resulted; thus, the contraction magnitude was proportional to (AR - T). A. J. Clark, also educated in Cambridge, developed Hill's model of reversible associations between drug and receptor. Clark's version, however, depicted the response as proportional to the amount of this complex, without it having to exceed some threshold level. In 1926 Clark, then in London, plotted the magnitudes of frog heart and skeletal muscle contractions (response) against administered acetylcholine concentrations (dose), identifying the consequent "doseresponse plots" as rectangular hyperbolas.4 (With dose on the x-axis and response on the i/-axis, the curves approached the horizontal asymptote of maximal response at infinite dose.) For large concentration ranges it is more convenient to plot the logarithm of the dose against the response, and rectangular hyperbolas are transformed in "log dose-response plots" into sigmoidal curves (Fig. 6-1 A). Clark noted that with both tissues the dose-response data "lie along a curve which can be fitted closely by the equation [for a rectangular hyperbola of] K.x = y/(lOO — y), where x = molecular concentration of the drug, y — action produced as percentage of maximal action [i.e., 100]; K = constant" (the line in Fig. 6-1A was drawn to this formula).5 He concluded that "the simplest explanation [for this fit] is to suppose that a reversible monomolecular reaction occurs between the drug and some receptor in the cells."6 An equivalent formula expresses the ratio of drug-receptor complex, AR, to the maximal drug-receptor complex possible, ARmax, in terms of the binding equilibrium A + R <^> AR:

where A is the concentration of free agonist, R the concentration of free receptor, AR the concentration of agonist-receptor complex, and K^ the association

FIGURE 6-1. Acetylcholine concentration and muscle contraction. A. Acetylcholine, at the concentrations indicated (logarithmically) on the x-axis, was added to baths containing frog rectus abdominis muscles. The magnitude of contraction is plotted on the y-axis, expressed as a percentage of the maximal contraction. The curve is that of a rectangular hyperbola transformed in the semilogarithmic plot. B. In similar experiments acetylcholine was added in the presence of no atropine (I) or 33 /iM (II), 100 ^iM (III) or 1 mM (IV) atropine. Here, the logarithm of the normalized contraction is plotted on the y-axis. (A and B from Clark [1926a], Fig. 6, and Clark [1926b], Fig. 4, respectively, courtesy of the Physiological Society.) 145

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constant for this binding. Response, according to Clark's principles, was then linearly proportional to AR/ARmax. Although Clark cited the similarities of his curves to those for oxygen's association with hemoglobin, he did not explicitly relate his equation to binding formulas then known, such as those for Irwin Langmuir's adsorption curves or for Leonor Michaelis and Maud Menten's substrate-velocity curves for enzyme activity.7 Moreover, Clark did not explicitly identify his K as the association constant, Ka, in the binding equilibrium. Clark also described atropine's inhibition of these acetylcholine-induced contractions: with successively higher concentrations of this antagonist, the log dose-response curve was shifted further and further to the right (Fig. 6-1B).8 To describe this effect he inserted a factor in his equation, multiplying K by the ratio of acetylcholine concentration to atropine concentration.9 This modification, however, had no theoretical justification, and he acknowledged that "it only holds when atropine is present in a concentration sufficient to produce a well-marked action," that is, at high atropine concentrations.10 Indeed, Clark argued against atropine and acetylcholine competing for the same site on the grounds that added acetylcholine did not accelerate atropine's dissociation from the tissue.11 The next significant advance came from John Gaddum, who has appeared in earlier chapters. He, too, was educated in Cambridge but then worked with Henry Dale, Wilhelm Feldberg, and Ulf von Euler before beginning a series of academic moves—from Cairo to London to Edinburgh (where he succeeded Clark, who had moved there in 1927) and finally to Babraham. But it was while in London in 1937 that Gaddum revised Clark's formulation for antagonism by explicitly representing competition between agonist and antagonist for the same receptor site:

where Kj and K% are the respective association constants for agonist GI and for antagonist C% binding to the same receptor site.12 Again, the fractional response was equated to the extent of agonist binding. The Hill-Clark-Gaddum formulation stressed reversible binding to receptors governed by distinct affinities for specific substances. Since this proposal may seem trivially obvious to modern pharmacologists, it seems worth emphasizing that alternative views were also expressed. For example, others, including Gaddum, reported sigmoidal log dose-response plots before Clark did, but they had interpreted their plots differently: as agonists interacting with heterogeneous populations of units "whose susceptibility is distributed about a mean in accordance with a probability curve [so that the dose-response] curve [is] the integral of the normal distribution."13 This formulation was difficult to disprove

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before identical, pure receptors were available for study, but Clark in his influential review of 1937 disagreed on other grounds, judging the population argument "unfruitful, since, if the response to drugs is attributed to a peculiarity of living tissue, there is no means of linking up such responses with the known laws of physical theory."14 Even more dissimilar was Walther Straub's potential theory of 1907.15 He considered that drugs exerted their effects not by combining with specific receptors but through the concentration gradient of a drug from outside the cell to inside. Straub derived his model from observing that added drugs could produce large effects initially (when the gradient would be high), but that this response would often decline slowly (presumably as the drug entered the cell and the gradient dissipated). But, as Clark pointed out, many drugs do not show decreased responses over time.16 Moreover, such time courses can be explained by heterogeneous drug-receptor interactions, with receptors having different rates of association and dissociation (falling back on the population argument). Clark also attributed the decline to tolerance, a well-recognized phenomenon, albeit one then without a clear mechanistic explanation.

Intrinsic Activity, Efficacy, Partial Agonists, and Spare Receptors

The Hill-Clark-Gaddum model specified that responses were linearly proportional to the fractional occupancy of receptors by agonists, but this agreeably simple relationship was not obeyed in many cases. In some instances the discrepancies were attributable to extraneous complexities, such as metabolism of the added drugs or their slow diffusion to the sites of action; actual concentrations at the receptors would then be unknown and equilibrium between drug and receptor, on which the formulation relied, would not obtain. In other instances paradoxical responses appeared, suggesting intrinsic disagreements. For example, J. Raventos, working with Clark, reported in 1937 that a homologous series of compounds, varying in the length of the side chain bonded to an amine group, excited when the side chains were short, but their maximal responses declined with increasing chain length rather than remaining constant.17 Moreover, at still longer chain lengths the compounds inhibited. Even more puzzling were observations of K. H. Ginzel in Vienna. In 1951 he identified a member of such a homologous series that could act as either agonist or antagonist.18 Such gradations of activity E. J. Ariens in Utrecht explained in terms of "intrinsic activity." In 1954 Ariens proposed that response magnitudes did not reflect the fractional occupancy of receptors alone, but the nature of the complexes formed between agonist and receptor as well.19 Accordingly, substances having high intrinsic activity would induce strong activation when occupying their receptors and be strong agonists. Substances having lower intrinsic activ-

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ity would produce weaker activation. These weak agonists could also act as antagonists toward strong agonists by competing with them for receptor occupancy: when weak agonists occupied the receptors instead of strong agonists, they would evoke lesser responses, manifested as inhibition toward strong agonists. Ariens formalized this proposal by including a factor to represent intrinsic activity, a, so responses would be proportional to a times the fractional occupancy: a/(l + l/K^A). Two years later R. P. Stephenson in Edinburgh called attention to further discrepancies and added new hypotheses to account for drug—receptor interactions.20 Stephenson pointed out that actual dose-response plots frequently deviated from true hyperbolas. He also repeated Ariens's conclusions that a homologous series of compounds could have different maximal responses (Fig. 6-2A) and that some compounds could have both agonist and antagonist properties. But Stephenson rejected the basic tenet of Hill, Clark, Gaddum, and Ariens that responses must be linearly proportional to receptor occupancy. Instead he introduced three hypotheses: (1) A maximum effect can be produced by an agonist when occupying only a small proportion of the receptors, (2) The response is not linearly proportional to the number of receptors occupied, and (3) Different drugs may have different capacities for initiating a response.21 Stephenson named agonists that were unable to elicit maximal

FIGURE 6-2. Responses to acetylcholine homologs. A. Alkyl-trimethylammonium compounds, containing ethyl, butyl, hexyl, heptyl, octyl, nonyl, or decyl groups as the alkyl sidechains, were added—at the concentrations indicated (logarithmically) on the xaxis—to baths containing guinea pig ileum segments. The magnitude of the contractions are plotted on the y-axis as a percentage of the maximal contraction. B. Responses to the alkyl-trimethylammonium compounds calculated from the affinities and efficacies listed in Table 6—1, with response a hyperbolic function of the stimulus. (A and B from Stephenson [1956], Figs. 1 and 10, respectively, courtesy Macmillan Magazines, Ltd.)

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responses, even at infinite dose, "partial agonists," and the excess of receptors, unneeded when a full agonist acted, "spare receptors." The factor modifying maximal response he named "efficacy," e, which he multiplied times fractional occupancy (as had Ariens with intrinsic activity, a). Stephenson, however, equated this product not to response, as had Ariens, but to "stimulus," S:

Whereas S directly represented the affinity of receptor for agonist and the efficacy of agonist at receptor, response was now an unspecified function of S. Stephenson illustrated these properties with a calculated family of curves for a series of homologous compounds. These he derived from values for affinity, which increased with side chain length, and for efficacy, which first rose with side chain length and then fell (Table 6-1), and from a hyperbolic relationship of response to S. These calculated curves mimicked experimental results: Figure 6-2B vs. 6-2A. Since responses were not necessarily linear functions of S (or of fractional occupancy), Stephenson's formulation allowed dose-response plots to deviate from true hyperbolic curves. At this time Mark Nickerson in Winnipeg published results consistent with Stephenson's formulation.22 Nickerson described how diphenhydramine irreversibly blocked responses to histamine over a slow time course (tens of minutes). During the early minutes diphenhydramine appeared to be a competitive (reversible) antagonist: the maximal response to histamine was unchanged but the apparent affinity decreased. Later in the time course, however, the maximal response decreased. Nickerson interpreted these results in terms of an excess of histamine receptors: initially enough (spare) receptors were available so that a maximal response could occur even though some were blocked irreversibly, but the decreased number simulated a reduction in affinity. Later, when the number of available receptors was decreased still further, too few remained to generate a maximal response.

TABLE 6-1. Affinity and Efficacy of Chiolinergic Homologs* ETHYL

BUTYL

HEXYL

HEPTYL

OCTYL

NONYL

DECYL

Affinity

0.63

3.8

19

41

63

110

190

Efficacy

31

200

21

2.2

1.4

1.0

0.6

ALKYL GROUP:

"Affinities for the partial agonists (hexyl and longer derivatives) were estimated from the concentrations for half-maximal responses. Affinities for the shorter full agonists were estimated by extrapolating the plot for partial agonist affinity vs. chain length (Stephenson, 1956, Fig. 4). Values are for the affinity constant X 10~3. Efficacies were then calculated from these affinity constants and the magnitudes of the responses, using the concentrations for half-maximal responses. (From Stephenson [1956], Table V. Used by permission of Nature Publishing Group.)

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Stephenson considered the block by irreversible antagonists to be "surmountable" as long as sufficient receptors remained to give maximal responses, although with time, as more receptors were inactivated, the block would become "insurmountable." This phenomenon of surmountable but irreversible inhibition, Stephenson claimed, would be difficult to explain without the occurrence of spare receptors. From independent investigations of irreversible antagonists, Robert Furchgott in New York also recognized that maximal responses might be obtainable if agonists required only a tiny fraction of the total receptors present.23 Furchgott developed these considerations in parallel with Stephenson, and in 1966 he defined "intrinsic efficacy" as Stephenson's efficacy divided by the concentration of receptors present, a term emphasizing the ability of an agonist to produce a stimulus at a single receptor.24 These studies thus prompted appreciations of (1) receptor affinities for specific substances (agonists, antagonists, and partial agonists) that governed receptor occupancy, with (2) the occupied receptor responding not merely by activation or inhibition but by degree of activation or inhibition (notions of intrinsic activity and efficacy). Thus, receptor responses need not be linearly related to receptor occupancy. (3) Tissue responses, such as smooth muscle contraction, reflected the constraints of cellular capabilities as well as the sequence of steps between receptor and response. Consequently, tissues might respond nonlinearly to receptor occupancy and be unable to exceed certain maximal responses even when further receptors were occupied by strong agonists (notions of stimulus and of spare receptors). With weaker agonists (partial agonists), though, tissues might never reach that maximum, despite the full complement of receptors being occupied. Two-State Models or Receptor Activation

Neither Ariens nor Stephenson explained how intrinsic activity or efficacy might be manifested, and the link between hypothesized receptor occupancy and observed tissue response remained mysterious as the 1950s ended. The chemical nature of receptors was then also unknown, although many assumed that receptors, like enzymes, were proteins. Such assumptions sanctioned further speculations, founded on new insights into protein structures and their functional changes. By the early 1960s John Kendrew had determined with X-ray crystallography the first three-dimensional structure of a globular protein, and Max Perutz had reported the crystal structures of both oxygenated and deoxygenated hemoglobin, revealing a distinct shift in protein conformation that accompanied oxygen binding (chapter 4). Perutz's demonstration complemented recent studies indicating that some enzyme activators and inhibitors bound to regulatory sites

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distinct from catalytic sites ("allosteric" sites), thereby altering structure and thus function. In 1965 Jacques Monod in Paris, together with Jeffries Wyman and Jean-Pierre Changeux, presented a striking and influential model for allosteric control of protein conformations.25 They proposed that proteins could exist in two alternative, interconvertible forms, R and T, of which R was active and T inactive (or significantly less active); moreover, if R and T had different affinities for a ligand,26 L, its binding would shift the equilibrium between R and T:

Consequently, if L bound more tightly to R than to T, L would shift the conformational equilibrium toward R and be an activator. If L bound more tightly to T, then L would be an inhibitor. Also incorporated in their model was the possibility for cooperative binding among multiple sites: if ligand binding favored the conformation having a higher affinity for this ligand, then binding of the first ligand would promote the binding of subsequent ones (as observed with oxygen binding to hemoglobin). Such cooperativity would be represented by sigmoidal binding plots rather than hyperbolic ones. (Log binding plots would also be sigmoidal, but steeper than for noncooperative binding.) In 1967 Arthur Karlin in New York adopted this model for drug interactions with acetylcholine receptors, as did Changeux, visiting in New York, the following year.27 Others soon followed.28 Not only did the Monod- WymanChangeux model account for the apparent cooperativity of nonhyperbolic dose-response curves, it also rationalized agonists, partial agonists, antagonists, and intrinsic activity/efficacy: full agonists bound preferentially to R forms and shifted receptor molecules to these active conformations; antagonists bound preferentially to T forms and shifted receptors to inactive conformations; partial agonists bound to both, so significant amounts of both T and R forms would be present and maximal activity unachieved even with all binding sites occupied.

Desensitization

Pharmacologists in the 1930s noticed that when certain substance were added to tissues at high concentrations, they initially excited but then prevented further responses, so that subsequent additions were for some time thereafter ineffective.29 Possible explanations included presumed depletions of cellular energy stores during the initial excitation.30 But in 1957 Bernard Katz in Lon-

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don accounted for this "desensitization" with a three-state model depicting receptors: (1) in resting states capable of activation, (2) in active states, and (3) in desensitized states incapable of activation.31 Katz considered various paths among these states. Subsequent studies favored his cyclical scheme, which rotated receptors through resting to active to desensitized and back to resting states.32 Katz's model clearly harked back to Hodgkin and Huxley's 1953 model for nerve impulse conduction (chapter 4), in which Na+-channels undergo a voltage-dependent transition from closed to open states, followed by a spontaneous conversion to inactive (unexcitable) states before reverting to closed (excitable) states. Moreover, Katz's three-state model for activation and desensitization was compatible with proposals for conformational changes in receptors, just as were two-state models then also being considered.

Rate Theory

William Paton, unlike the Langley, Hill, Clark, Gaddum succession, was educated in Oxford, although he worked briefly with Dale before returning to Oxford. There in 1961 Paton published an ingenious alternative to these "occupation theories," a "rate theory" proposing that "the stimulant effect produced by a drug depends, not on the number of receptors occupied, but on their rate of occupation."33 Maximal responses required maximal numbers of interactions, so agonists should dissociate rapidly from receptors to permit further interactions. Conversely, antagonists would block by dissociating only slowly, thereby preventing agonists from interacting. Paton accumulated an impressive body of corroborative evidence from studies on cholinergic stimulation of the guinea pig ileum in the absence and presence of various inhibitors. But others were less enthusiastic, and in 1968 D. R. Waud summarized criticisms.34 These ranged from the lack of chemical precedent for such rate-dependent stimulation to the lack of similar data at other loci where they were sought, including neuromuscular junctions and adrenergic systems. Moreover, Waud provided alternative explanations for Paton's observations, couched in terms of occupancy theories and plausible assumptions that attributed Paton's time courses not to rates of interaction with receptors but to rates of access to receptors, with diffusion modified by tissue binding sites. In the face of these criticisms and the continuing development of occupation theories, enthusiasm for Paton's rate theory ebbed.

Receptor Classification

Another approach to understanding receptors was to sort them into meaning ful categories, expressing similarities and exhibiting anomalies.

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Classification by Agonist

One obvious scheme involved classifying neurotransmitter receptors by their physiological agonists: cholinergic receptors responding to acetylcholine, adrenergic receptors to noradrenaline, and so forth. With the development of multitudes of therapeutic agonists and antagonists, this criterion provided a practical ordering. But simple models in biology are frequently transformed, after closer scrutiny, into more complex ones. Dale in 1914 distinguished between two classes of responses to acetylcholine, those elicited by muscarine and by nicotine (chapter 3). This distinction evolved into the recognition of two classes of cholinergic receptors, muscarinic and nicotinic. Subsequently, Walter Cannon and Arturo Rosenblueth argued for two types of sympathin, arising through combinations with circulating receptors: excitatory sympathin-E and inhibitory sympathin-I (chapter 3). In 1948, however, Raymond Ahlquist in Augusta offered a different interpretation, proposing two types of adrenergic receptors fixed in the tissues.35 Ahlquist based this formulation on his comprehensive survey of how 22 tissues responded to equimolar doses36 of six agonists, including adrenaline, noradrenaline, and the synthetic agonist isoproterenol. In some assays isoproterenol was most active and noradrenaline least; in others adrenaline was most active and isoproterenol least (Table 6-2). So he concluded that there were "at least two distinct general types of these receptors," which he labeled a and ft.37 Ahlquist s classification was strongly supported by the discovery of antagonists that affected /3-receptors selectively. In 1965 James Black in Macclesfield described the first clinically satisfactory agent, propranolol (Inderol), a drug that affirmed the concept and added beta blacker to the common vocabulary.38 Comparisons of other adrenergic agents in other assays, however, suggested

TABLE 6-2. Relative Magnitudes of Responses to Adrenergic Agonists* RANK-ORDER OF RESPONSES WITH RESPONSE

ADRENALINE

NORADRENALINE

ISOPROTERENOL

Vasoconstriction

1

2

3

Vasodilation

2

3

1

Intestinal contraction (inhibition)

1

2

3

Uterine contraction (inhibition)

2

3

1

"Rank-order of responses in rabbits to equimolar amounts of the three adrenergic agonists are tabulated. Vasoconstriction was measured from blood pressure changes and vasodilation from coronary artery flow. (From Ahlquist [1948], Table I. Used by permission of the American Physiological Society.)

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further subdivisions. In 1967 A. M. Lands in Rensselaer identified j3i and @2 receptors. Among other responses, the former was prominent in cardiac stimulation, the latter in bronchodilation.37 The pharmaceutical industry—in which both Black and Lands worked—then developed practical (3i and /3a agonists and antagonists. For example, the fiz agonist terbutaline (Brethine) is useful in treating asthma since it dilates bronchi without stimulating the heart; on the other hand, the fii antagonist atenolol (Tenormin) is useful in treating hypertension without affecting pulmonary airflow. Then in 1974 Salomon Langer in Buenos Aires divided a-receptors into a\ and «£ after exploring different issues (see below). Many early classifications, such as Ahlquist's and Lands's, were based on agonist responses. A better discriminator would be agonist affinity, avoiding possible variations in efficacy as well as the unknown links between responses and stimuli (in Stephenson's sense). But direct measurements of agonist affinity were not practical until labeled ligands of high specificity became generally available in the 1970s. The third approach, advanced by Heinz Schild in London, focused on affinities for competitive antagonists. Binding to receptors could then be assessed without the confounding uncertainties of how responses reflected receptor occupancy. Moreover, Schild in 1947 formulated a convenient experimental protocol based on "dose ratios": determining the antagonist concentration necessary to reduce (1) the response elicited by an x-fold dose of some agonist to (2) the response of a single dose of this agonist in the absence of antagonist.40 In 1959 he devised a simple plot of such data that revealed the agonist association constant, a value that should be the same for all receptors of a given class but would likely be different for receptors of other classes.41 These approaches both certified similarities between receptors in different tissues and established distinctions between different receptor classes. Other techniques supplemented these methods, including direct measurements of agonist affinity, possible when suitably labeled ligands became available, and examinations of receptor structures revealed by molecular biological techniques (chapter 8). These approaches also culminated in the realization that multiple receptors for each neurotransmitter were the rule; for example, 15 distinct serotonin receptors were distinguished by 1994.

Classification by Cellular Localization Since many neurotransmitters produced their effects quite rapidly, the responding receptors would seem to be on or near the cell surface, as Clark pointed out in 1937.42 In 1955 Katz demonstrated just such a localization at neuromuscular junctions: acetylcholine released from micropipets near the muscle cell surface excited responses, whereas acetylcholine released into the protoplasm did not.43 Later microelectrophoretic experiments confirmed this cell

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membrane localization for many neurotransmitter receptors on many cell types (chapter 5). A different issue was where on the cell surface the receptors lay. Early models depicted the obvious localization at postsynaptic sides of synaptic clefts, where receptors would receive neurotransmitters released from presynaptic terminals, in accord with the principle of synaptic polarization. But further studies uncovered further complexities. In 1963 Charles Edwards in Minneapolis described a decrease in the number of acetylcholine quanta released at neuromuscular junctions when exogenous acetylcholine was added. He considered that this response was due to a "direct action [of acetylcholine] on the presynaptic terminals."44 The following year John Hubbard in Canberra confirmed this phenomenon, which he interpreted as "a negative feed-back on further acetylcholine release."45 Surprisingly, these observations were not pursued at that time, and the notion of regulatory presynaptic receptors was developed instead from studies on adrenergic synapses. In 1957 G. L. Brown, then in London, reported that certain adrenergic antagonists, including phenoxybenzamine, increased noradrenaline outflow from stimulated sympathetic nerve endings.46 Several explanations were advanced in the following decade, including phenoxybenzamine s interference with mechanisms for noradrenaline disposal. As Langer pointed out in 1970, another possibility "cannot be excluded," that a-receptor antagonists such as "phenoxybenzamine may increase the release of [noradrenaline]."47 Klaus Starke in Essen provided strong support for this alternative the following year, describing how a-receptor agonists reduced noradrenaline release—the converse of antagonists enhancing release.48 In 1972 Starke reexamined responses to aadrenergic antagonists and concluded that "a-receptors are involved in the regulation of the release of noradrenaline from adrenergic nerve terminals."49 Langer that year reached a similar conclusion from similar studies, invoking a "negative feedback mechanism regulating transmitter release"; in 1974 he named the presynaptic receptors a2 and the postsynaptic receptors ai.50 Langer and Starke also described differences in drug specificities between a\- and «£receptors.51 But these studies did not demonstrate directly that the regulatory receptors were indeed presynaptic, although Langer soon provided independent support, albeit also indirect: binding of a-adrenergic ligands decreased after he destroyed sympathetic nerve endings in the heart.52 (But later studies revealed a2~receptors postsynaptically as well as presynaptically.53) On the other hand, Paton showed that added noradrenaline diminished acetylcholine release from cholinergic nerve terminals, whereas E. Muschall in Mainz found the converse: muscarinic agonists diminished noradrenaline release from adrenergic nerve terminals.54 Such processes would augment antagonisms between alternative systems, allowing one neurotransmitter (say, acetylcholine) not only to direct the target organ oppositely from its rival neu-

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retransmitter (say, noradrenaline) but also to diminish the release of its rival. These presynaptic "heteroreceptors," which respond to neurotransmitters released from different nerve terminals, thus contrast with presynaptic "autoreceptors," which respond to the neurotransmitter released from their own terminal.

Classification by Function

Langley imagined two classes of receptors, one eliciting excitatory and the other inhibitory responses (chapter 3). Later studies, however, demonstrated that the same receptor could mediate excitatory or inhibitory responses depending on the cellular circumstances. A more rewarding classification considered classes of receptor mechanisms, as further studies disclosed (chapters 7 and 8).

Structure—Activity Relationships

Receptor structures could not be determined directly with techniques then available. Nevertheless, since neurotransmitters were thought to fit their receptors like keys in locks, the receptors ligands should provide a complementary image of its binding site. "Structure-activity" studies thus attempted to correlate ligand structures—accessible through contemporary chemical methods— with receptor responses. A few steps in the analyses of cholinergic ligands will exemplify such endeavors. Acetylcholine is a flexible molecule, able to assume alternative conformations in solution. Some of its analogs, however, have more restricted motions, so plausible common dimensions can be derived. This Carl Pfeiffer in Chicago reported in a pioneering study published in 1948: he calculated common critical distances between a methyl group on the quaternary nitrogen and the carbonyl and ester oxygens of various muscarinic agonists and antagonists (see Fig. 3-6).55 Pfeiffer s distances came from molecular models, but in the following decades X-ray diffraction and nuclear magnetic resonance techniques provided specific dimensions. From these data Cyrus Clothia in London differentiated between the muscarinic and nicotinic surfaces required for acetylcholine binding, which he localized in 1970 to the methyl and carbonyl sides of the molecule (Fig. 6-3A).56 Selective muscarinic agonists had the methyl side accessible but the carbonyl side blocked (Fig. 6-3B), whereas selective nicotinic agonists had the methyl side blocked but the carbonyl side accessible (Fig. 6-3C). William Beers and Edward Reich in New York inferred similar requirements from studying molecular models: in addition to common interactions through the quaternary nitrogen, muscarinic ligands interacted through the methyl group and ester

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FIGURE 6.3. Muscarinic and nicotinic conformations. A. The acetylcholine structure was derived from X-ray diffraction and nuclear magnetic resonance studies. The drawing shows methyl carbons (Cl, C2, C3) bonded to the quaternary nitrogen (N) of choline, the two methylene carbons of choline ((C4, C5), the ester oxygen (Ol), the carbonyl carbon (C6) of acetate and its carbonyl oxygen (O2), and the methyl carbon (C7) of acetate. C7 and O2 define the methyl and carbonyl sides. B. Muscarinic structures are superimposed on the acetylcholine structure: M identifies muscarine atoms; ft, acetyl/3-methylcholine; and ACTM, acetoxycyclopropyltrimethylammonium. C. Nicotinic structures are superimposed on the acetylcholine structure: DMPP identifies dimethylphenylpiperazinium atoms; LC, lactoylcholine; and a, acetyl-a-methylcholine. (A, B, and C from Chothia [1970], Figs. 1, 3, and 5, respectively. Reprinted by permission of Nature, ©Macmillan Magazines, Ltd.)

oxygen, whereas nicotinic ligands interacted through the carbonyl oxygen.5' The receptors presumably had groups that fitted these surfaces and accommodated the ligands' polarities. Neither analysis, however, accounted convincingly for relative efficacies or revealed how ligand binding triggered receptor responses.

Receptor Identification ana Purification

More direct and detailed information was needed, but to characterize receptors chemically and mechanistically they must be isolated from contaminating entities and confounding processes. Serious conceptual uncertainties and technical inabilities, however, impeded progress toward receptor purification. By 1960 neurotransmitter receptors were localized to cell membranes and thought to be, at least in part, proteins.58 The structural organization of lipids and proteins within membranes, however, was then unclear. The favored model depicted protein layers coating each surface of a lipid bilayer; not until late in the

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1960s did S. J. Singer revive mosaic models, showing "intrinsic" membrane proteins that extended across the bilayer (as would be appropriate for transporters, channels, and receptors that must mediate between extracellular and intracellular environments).59 Extracting such intrinsic proteins in their native state was also a daunting challenge. During the 1960s methods appeared for fragmenting membranes mechanically and for "solubilizing" their proteins with various detergents. The detergent-solubilized proteins could then be purified by ultracentrifugation and chromatography. As the 1970s began, a crucial new technique, electrophoresis through polyflcrylamide gels of proteins solubilized in the detergent sodium dodecylsulfate (SDS-PAGE), was being employed for separating membrane proteins according to their molecular weights. Membrane-bound enzymes could be followed through purification procedures by assaying catalytic activity at successive steps. Following receptors was less straightforward. The binding of specific ligands, such as receptor agonists or antagonists, was obscured by "nonspecific" binding to other sites as well as to irrelevant "specific" sites, including those on neurotransmitter transporters and on synthetic and degradative enzymes. Moreover, purifying a ligandbinding entity did not guarantee that this entity was the entire receptor. Not surprisingly, some commentators were pessimistic about the prospects for isolating intact receptors,60 and the piecemeal progress fostered skepticism. Still, some successes accrued; three examples may illustrate the quests. Nicotinic Cnolinergic Receptors

The first neurotransmitter receptor to be isolated was the nicotinic receptor, although early attempts foundered for want of sufficiently discriminating ligands. For example, Carlos Chagas in Rio de Janeiro tried to extract receptors from eel electric organs—a wise choice since the organs are large and richly innervated with cholinergic fibers—through their affinity for curare and gallamine (a synthetic antagonist). By 1959 Chagas had separated a fraction containing acidic mucopolysaccharides that bound radioactive derivatives of these ligands tightly.61 But critics soon pointed out that the binding measured in dilute solution in vitro was unlikely to occur in vivo, where higher salt concentrations would interrupt electrostatic binding between positively charged ligands and negatively charged acidic mucopolysaccharides.62 Furthermore, acidic mucopolysaccharides (and cholinergic ligand binding) were not confined to innervated regions, as would be expected for neurotransmitter receptors. So in 1962 Chagas modified his designation to "acceptor" sites.63 Changeux, then back in Paris, chose instead radioactive decamethonium, an antagonist having a high affinity for nicotinic receptors. With this ligand,

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Changeux in 1970 demonstrated binding to a detergent-solubilized membrane fraction from eel electric organ that could be displaced by nicotinic agonists and antagonists.64 Procedures that destroy proteins eliminated binding, so Changeux concluded that the receptor was (at least in part) protein. And, importantly, a-bungarotoxin displaced decamethonium; in 1963 Chen-Yuan Lee in Taipei had purified a-bungarotoxin from the venom of a Formosan snake, the banded krait (Bungarus multicinctus), and showed that this protein blocked nicotinic receptors specifically, not affecting acetylcholinesterase or other cholinergic processes.65 The next year, 1971, Lincoln Potter in London and Michael Raftery in Pasadena reported experiments using radioactive a-bungarotoxin to label receptors from torpedo or eel electric organs through several purification steps.66 Since the toxin-receptor binding is extremely tight, the labeling survived separations based on chromatography and ultracentrifugation. Changeux and others subsequently used a comparable toxin from cobra venom and Karlin a covalently bound affinity label.67 Significant progress in purification then came almost simultaneously from a number of investigators applying affinity chromatography.68 With this technique, solubilized but unlabeled membrane fractions were passed through chromatography columns containing a matrix to which binding ligands, such as cobra toxin or choline-like sidechains, were linked. These fixed ligands bound the solubilized nicotinic receptors, impeding their flow through the column and thereby separating them from other materials. The receptors could then be displaced from the fixed ligands and collected in highly purified form; their presence was followed by binding assays, using radioactive toxins. (Affinity chromatography had recently been used to purify acetylcholinesterase, using choline-like sidechains to bind the enzyme.69) In 1974 Karlin described four protein subunits in the purified receptor complex, separable by SDS-PAGE and named a through 5; their apparent molecular weights were, respectively, 39, 48, 58, and 64 kDa.70 Only the a-subunits bound the acetylcholine-like affinity label. And in 1978 Karlin calculated from ultracentrifugation experiments a molecular weight for the receptor complex of 250 kDa (confirming Raftery s early estimate).71 With two cholinergic binding sites per 250 kDa, Karlin specified a subunit stoichiometry of azfiyS. These approaches isolated and identified 250 kDa complexes having distinct subunits with appropriate affinities for cholinergic agonists and antagonists. But they did not show that the 250 kDa complexes constituted the functional receptor, which must also include channels to allow transmembrane passage of Na + and K + when acetylcholine binds. This concern, however, was addressed concurrently in "reconstitution" experiments. In the early 1970s Efraim Racker in Ithaca had developed procedures for incorporating detergent-solubilized intrin-

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sic membrane proteins into phospholipid bilayers forming microscopic vesicles ("liposomes").72 By 1979 several investigators, including Racker, had reported acetylcholine-stimulated Na + fluxes across liposome membranes that contained purified receptors.73 Electron microscopic studies complemented these approaches. Early micrographs showed rosettes on the surface of electric organs,74 and in 1982 Robert Stroud in San Francisco published a model, based on examinations at higher magnifications, that depicted a radial, pentameric array of subunits around a central ion-conducting channel (Fig. 6-4).75 Finally, the availability of purified receptor preparations permitted more direct studies on drug-receptor interactions, demonstrating cooperative binding of acetylcholine to the two a-subunits and responses supporting multistate models for receptor activation and desensitization.76 jS-Aarenergic Receptors

Success with other receptors was more elusive. Whereas fish electric organs provided large quantities and high densities of nicotinic receptors, no organs were so richly endowed with receptors for any other neurotransmitter. This low density not only necessitated more extensive purification from extraneous materials, it also required that labeling ligands have extremely high specific activities.77 Indeed, initial attempts with radioactive noradrenaline revealed only nonspecific binding: antagonism by other ligands did not follow their pharmacological ranking, and binding was neither saturable nor stereospecific.78

FIGURE 6.4. Model of the nicotinic receptor. The receptor is composed of (A) five subunits traversing the lipid bilayer of the membrane and enclosing a (B) central ionconducting channel. (From Kistler et al. [1982], Fig. 6, courtesy of the Biophysical Society.)

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In 1974, however, experiments using radioactive /3-adrenergic antagonists with far higher specific activities overcame these difficulties. Robert Lefkowitz in Durham labeled frog (Rana pipiens) red blood cells with alprenolol, Alexander Levitzki in Rehovot labeled turkey red blood cells with propranolol, and Gerald Aurbach in Bethesda labeled turkey red blood cells also, but with hydroxybenzylpindolol.79 Within three years /3-agonists as well as antagonists were employed successfully, with labeling of /3-receptors in neural tissues as well.80 Solubilizing /3-receptors was also difficult, but by 1976 Lefkowitz uncovered a suitable detergent for frog red blood cells membranes.81 Nevertheless, five more years were consumed in purifying the receptors. This feat was again achieved through affinity chromatography; here Lefkowitz linked alprenolol to the chromatography matrix and identified a prominent 58 kDa band after SDSPAGE.82 The purified material had the same affinity and specificity as the original membranes. The following year, 1982, he improved the preparative procedure to yield the 58 kDa band alone.83 Earlier studies had demonstrated that /3-receptors activate the enzyme adenylate cyclase (chapter 7), so reconstituting this activation would bolster confidence in the ligand-binding protein being a functional receptor. Since adenylate cyclase had not yet been purified, Lefkowitz proceeded in a less straightforward but nonetheless convincing manner. Red blood cells of another frog, Xenopus laevus, have the adenylate cyclase system but no /3-receptors (i.e., adding /3-adrenergic agonists has no effect on adenylate cyclase). So Lefkowitz incorporated the 58 kDa protein in the phospholipid bilayer of liposomes and then fused these vesicles with Xenopus red blood cells: /3-agonists now stimulated the cells' adenylate cyclase.84 Earlier, Lefkowitz showed that frog red blood cells contain /32-receptors, whereas turkey red blood cells contain /3i.85 He now isolated /3i-receptors from turkey cells using similar methods, although they found two bands by SDSPAGE with lower apparent molecular weights: 40 and 45 kDa.86 But neither f3\- nor /3a-receptors resembled nicotinic receptors. There was no evidence of subunits assembled into an oligomeric complex and no indication of an intrinsic ion channel.

Opioid Receptors

Identifications of opioid receptors garnered more attention, even though further progress was relatively modest. In fact, purification was not achieved by 1983, when this account concludes. Interest instead reflected the importance of morphinelike substances medically and socially, coupled to demonstrations soon afterward that the body makes and uses morphinelike substances (chapter 5). Investigations of opioid receptors also established general techniques

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for labeling neurotransmitter receptors in the brain, launching a "grind and bind" industry that soon identified multitudes of receptors. And these studies also attracted public notice through widely aired disputes over priority and the allocation of scientific rewards.87 Initial attempts in 1971 to tag opioid receptors, however, failed.88 Again, this resulted from using high concentrations of radioactive ligands—necessitated by the low specific activities then available—so nonspecific binding swamped any specific binding. Avram Goldstein in Palo Alto first incubated 2000 nM radioactive levorphanol (a synthetic analgesic) with brain membrane fractions, in the absence or presence of a 100-fold excess of unlabeled levorphanol or unlabeled dextrorphan (the pharmacologically inactive stereoisomer of levorphanol). Goldstein then separated membranes from unbound label in the incubation medium by centrifugation and counted the radioactivity remaining in the pellet. The excess unlabeled levorphanol should displace radioactive levorphanol at stereospecific sites, whereas dextrorphan would not compete. But by this argument only 2% of the total labeling was attributable to saturable, stereospecific sites: a fraction too small to pursue profitably.89 In 1973, however, three independent investigations, using far lower concentrations of ligands, succeeded. (1) Lars Terenius in Upsala incubated 0.6 nM radioactive dihydromorphine with brain synaptosomal membranes, in the absence or presence of the two stereoisomers of methadone (a synthetic agonist), both unlabeled. He next centrifuged the mixture, washed the pellet (unlike Goldstein), and then counted the remaining radioactivity.90 Lmethadone reduced total binding by 45%, identified with specific binding, whereas pharmacologically inactive D-methadone reduced total binding by only 10%. (2) Solomon Snyder in Baltimore, together with Candace Pert, a graduate student, incubated 5 nM radioactive naloxone (an antagonist) with homogenized brain in the absence or presence of unlabeled levorphanol or dextrorphan. They then separated tissue from medium by rapidly filtering through glass-fiber discs, washed these disks twice to remove loosely bound label, and counted the residue.91 Levorphanol reduced labeling by 70%, identified with specific binding; dextrorphan had negligible effect. Snyder and Pert also compared the abilities of various other ligands to displace radioactive naloxone, amassing a good correlation between relative affinity, so calculated, and the rank-orders of pharmacological potency, strong evidence that binding was to genuine receptors. Their filtration procedure also became a standard method for subsequent successes with other receptors. (3) Eric Simon in New York incubated 5 nM radioactive etorphine (a synthetic analgesic) with brain homogenates in the absence or presence of unlabeled levorphanol or dextrorphan, separating the tissue from medium either by centrifugation or filtration. Specific binding was 40%-60% of the total.92 Simon noted that binding was

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abolished by first incubating the tissue with proteolytic enzymes, suggesting that binding sites were (at least in part) protein. These studies gained significance from the discovery of endogenous opioids that soon followed (although, as noted in chapter 5, the initial isolation relied on traditional bioassays). And although these studies did not culminate in receptor isolation by 1983, they led to further revelations, including demonstrations of how opioid receptors were distributed within the nervous system and allocations of opioid receptors among several subclasses.93 Moreover, an intriguing difference in the interactions of receptors with agonists vs. antagonists also supported proposals for alternative receptor conformations: Na + reduced the binding of agonists but increased that of antagonists, implying that this cation induced or selected protein conformations having different specificities for these oppositely acting agents.94

Responses 01 Indiviaual Receptor Molecules

Models for neuromuscular transmission developed by Katz in the early 1950s depicted populations of receptors that responded to discrete quanta of acetylcholine: to the spontaneous release of single quanta by miniature endplate potentials and to the evoked release of multitudes of quanta by endplate potentials (chapter 4). Twenty years later Katz and Ricardo Miledi advanced this characterization a level further, identifying the responses of individual receptors to administered acetylcholine.95 Since the "statistical variation of highfrequency collisions between [acetylcholine] molecules and end-plate receptors . . . might be discernable as an increase in membrane [electrical] noise," they recorded electrical fluctuations from endplates in the presence of constant levels of acetylcholine.96 Katz and Miledi then analyzed these traces in terms of discrete electrical pulses, representing abrupt transitions between nonconducting and conducting receptor states. In 1972 they reported unitary conductance changes on the order of 10 pS lasting roughly 1 ms.97 Others, notably Charles Stevens in Seattle and David Colquhoun in Southampton, pursued the "fluctuation analysis," calculating single-channel conductances of 25 pS and mean open-channel lifetimes of 3 ms.98 But the high electrical background from extraneous regions and processes required model-dependent statistical analyses to uncover the individual events. Through a spectacular technical triumph (celebrated with a Nobel Prize in 1991), Erwin Neher and Bert Sakmann (Fig. 6-5) in Gottingen solved the problems of reducing this background and of displaying the discrete events: they devised a "patch electrode," a glass pipet with a polished tip 3-5 jjim in diameter, that they pushed tightly against the carefully cleaned muscle surface. Since

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FIGURE 6.5. A., left, Erwin Neher (1944-). B., right, Bert Sakmann (1942-). [A courtesy of E. Neher, B courtesy of B. Sakmann.)

the patch was isolated by the tight seal from electrical events elsewhere, the electrode responded prominently to ionic currents through the few channels bounded by its orifice. So, with cholinergic agonists in the electrode bathing the patch, Neher and Sakmann in 1976 recorded discrete electrical pulses (Fig. 6-6A), identifiable as multiples of a basic amplitude and interpretable as currents through channels that abruptly opened when agonist bound and later abruptly closed." They calculated single-channel conductances of 22 pS and mean open-channel lifetimes of 11 ms.100 Responses from a population of such receptors would then sum to give the composite endplate currents previously measured with intracellular microelectrodes. Neher and Sakmann improved and extended their technique, and patch electrodes soon became widely used to characterize channels throughout the biological realm.101 An alternative approach for recording responses from single receptor molecules was through reconstituting purified receptors in lipid bilayers. In this case the preferred system is a planar bilayer, formed across a tiny hole in a septum that separates two solutions. With receptors incorporated in and penetrating across this insulating bilayer, gross electrodes in the two solutions can then monitor conductance through the receptor channels. By 1980 successful reconstitutions revealed the same discrete, abrupt steps in conductance, attribute-

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FIGURE 6.6. Openings and closings of individual nicotinic receptor channels. A. Patch electrodes containing the cholinergic agonist suberyldicholine were placed tightly against the muscle fiber surface to measure transmembrane currents. Downward deflections record an increased current flow under the electrode and upward deflections a decreased flow. These deflections were interpreted as agonist-induced openings of ionconducting channels with a unitary magnitude, followed by the return of the conductance to baseline values when the channel closed. In some cases the downward deflection is twice the unitary magnitude, interpreted as two channels under the electrode being open at the same time. B. Gross electrodes were in two solutions separated by a lipid bilayer containing purified nicotinic receptors. Currents measured between the two electrodes then include ion flows through these receptors. In these tracings upward deflections record an increased current. The occasional increases in conductance were interpreted as spontaneous openings of receptor channels, producing currents of a unitary magnitude. C. More frequent conductance increases occurred after the cholinergic agonist carbamylcholine was added to the solutions. These often appeared as multiples of the unitary conductance and were interpreted as multiple channels being open at the same time. (A from Neher and Sakmann [1976], Fig. 3, reprinted by permission of Nature, ©Macmillan Magazines, Ltd. B and C from Schindler and Quast [1980], Figs. 4A and B, courtesy of H. G. Schindler.) ble to the unitary conductance of single receptor molecules (Fig. 6-6B).102 Again, adding cholinergic agonists increased the probability of channel openings (Fig. 6-6C). This method not only could check the functional capabilities of purified receptors, it also allowed the easy pharmacological manipulation of receptors through additions to the bathing solutions.

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From initial notions of a neurotransmitter acting through a cellular receptor, the studies cited here refined these concepts and extracted new generalities. The studies also uncovered complexities and elaborations, including a growing catalog of receptors. Indeed, one striking conclusion was that a single neurotransmitter could act through multiple classes of receptors, each dedicated to controlling particular cellular processes. This realization had practical implications as well, for the multiplicity afforded opportunities for directed therapy: through the design of new drugs aimed at distinct receptors modulating specific functions. At this scale little can be said about individual scientists. A succession that began once again in Cambridge at the turn of the century was soon joined by other investigators across the globe. Effective communication and sharp competition then merged individual styles into a cosmopolitan throng assimilating new insights and extending new approaches. Indeed, their practice was marked by the fruitful application of techniques devised for other inquiries—such as detergent extraction of proteins, SDS-PAGE, affinity chromatography, and the reconstitution of membrane proteins—as well as by the development of innovative new techniques for these studies—such as patch electrodes (which were then applied to studies of membrane channels at diverse loci). Initial analyses of dose-response relationships revealed the fundamental characteristic of receptor functioning: selective occupancy that initiated particular cellular changes. Further examinations of responses and broader explorations with additional agonists and antagonists complicated the models but merged the structures and functions into the mainstream of cellular biochemistry, with its protein conformational transitions and allosteric regulation. The major achievement, however, was the isolation of functional receptors and their recognition as integral membrane proteins. This feat rescued receptors from the stigma of hypothetical entities, providing chemical identities and structures that illuminated function. Purification of nicotinic receptors revealed an oligomeric, funnel-shaped transmembrane unit surrounding a central channel for conducting ionic charges. By constrast, /3-adrenergic receptors had simpler, quite different structures that—as the next chapter will relate—coupled occupancy to the activation of enzymatic systems that then amplified responses through the cell's interior. Notes 1. Langley (1905, 1906). In fact, Langley enunciated the principle in 1878: "We may . . . assume that there is some substance . . . in the nerve endings or gland cells with which both atropin and pilocarpin are capable of forming compounds. On this assump-

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tion then the atropin and pilocarpin compounds are formed according to some law of which their relative mass and chemical affinity for the substance are factors" (p. 367). From considering antiparasitic actions, Paul Ehrlich at the turn of the century also postulated that receptors mediated drug effects (see Bloch, 1994, chapter 5). 2. Hill (1909). For example, Hill described the onset of contraction as y = k(l — e~ At ), where y is the magnitude of contraction, t the time since the beginning of the contraction, and k and A constants. 3. Ibid., p. 372. 4. Clark (1926a). 5. Ibid., p. 535. 6. Ibid., p. 545. 7. Langmuir (1916); Michaelis and Menten (1913). The Michaelis-Menten analysis proposed that enzyme velocity was proportional to the concentration of an enzymesubstrate complex, ES, formed reversibly by the association of substrate, S, and enzyme, E: E + S <=> ES. Their formula for velocity, v, in terms of these parameters, of maximal velocity, Vmax, and of the constant for ES dissociation, Km, is:

Clark's equation is formally equivalent, although it is expressed in terms of the association constant, the reciprocal of the dissociation constant. In his 1937 review Clark made this comparison. 8. Clark (1926b). 9. Clark justified his evaluation by citing a previous expression of this form for oxygen vs. carbon monoxide binding to hemoglobin, as well as earlier pharmacological studies. But Clark presented no model of reversible antagonist binding to match his proposal for reversible agonist interaction. 10. Clark (1926b), p. 555. When atropine concentrations are large compared to the association constant for atropine, then Clark's formula approximates Gaddum's (1937, cited below). 11. Clark (1926b). Clark's argument is mistaken. He came to his conclusion not from analyzing competing equilibria but from reports that carbon monoxide accelerated oxygen dissociation from hemoglobin. It was subsequently realized that hemoglobin has multiple interacting sites, so that binding of carbon monoxide to one site can alter oxygen affinity at another site; such interactions would not occur on receptors having independent (noninteracting) sites. Clark repeated his argument in 1937, after Gaddum's 1937 formulation appeared. 12. Gaddum (1937). Gaddum's equation describes reversible, equilibrium binding to receptors of antagonist, I, or agonist, A : I R < = > I + R + A<=> AR, where IR represents the antagonist-receptor complex and the response is proportional to AR. This is formally equivalent to earlier expressions of competitive inhibition of enzymes, expressed in terms of dissociation constants, Km (for ES) and K, (for El):

When I is large relative to K,-, values of v/Vmax approximate those from Clark's formulation for inhibition. 13. Gaddum (1926). See also Shackell et al. (1924). 14. Clark (1937), p. 205. 15. Straub (1907). Straub imagined that antagonists, such as atropine, inhibited by

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preventing an agonist, such as acetylcholine, from entering the cell and thereby establishing a gradient. 16. Clark (1937). 17. Raventos (1937). 18. Ginzel et al. (1951). 19. Ariens (1954). 20. Stephenson (1956). 21. Ibid., p. 380. 22. Nickerson (1956). 23. Furchgott (1955). 24. Furchgott (1966). 25. Monod et al. (1965). Koshland et al. (1966) presented a somewhat different multistate model. 26. "Ligand" is a general term for a substance—atom, ion, or molecule—that binds. 27. Karlin (1967); Changeux and Podleski (1968). Changeux et al. (1967) earlier proposed a related model in terms of cooperative effects transmitted through a membrane lattice. 28. For example, Colquhoun (1973); Thron (1973); Pert and Snyder (1974). 29. For example, Barsoum and Gaddum (1935); Brown (1937). 30. For example, Cantoni and Eastman (1946). 31. Katz and Thesleff (1957). 32. For example, Rang and Ritter (1970). 33. Paton (1961), p. 23. 34. Waud (1968). 35. Ahlquist (1948). 36. Expressing dosages in molar terms allowed comparisons of the potency per molecule. 37. Ahlquist (1948), p. 595. 38. Black et al. (1965). At the time Ahlquist proposed his dichotomy, all known adrenergic antagonists affected a-receptors predominantly. 39. Lands et al. (1967a). A subsequent paper introduced the terms "/3-1" and "/3-2" (Lands et al., 1967b). 40. Schild (1947). For example, the response to an agonist dose, A, will equal that with twice the dose, 2A, in the presence of an antagonist, I, when I = 1/K, :

where Ka and K, are the association constants for agonist and antagonist binding. 41. Arunlakshana and Schild (1959). 42. Clark (1937). 43. del Castillo and Katz (1955a). 44. Ciani and Edwards (1963), p. 23. 45. Hubbard and Yokota (1964), p. 1073. 46. Brown and Gillespie (1957). 47. Langer (1970), p. 544. 48. Starke (1971). 49. Starke (1972), p. 19. 50. Enero et al. (1972), p. 672; Langer (1974). Delbarre and Schmitt (1973) suggested splitting a-receptors into ai and 0.% categories but did not indicate what those categories were.

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51. Dubocovich and Langer (1974); Starke et al. (1975). 52. Story et al. (1979). 53. See Doxey et al. (1977). 54. Paton and Vizi (1969); Loffelholz and Muscholl (1969). 55. Pfeiffer (1948). 56. Chothia (1970). He was collaborating with Peter Pauling, the son of Linus Pauling. 57. Beers and Reich (1970). Chothia and Pauling (1971) challenged a bond angle, but Beers and Reich (1971) claimed that their model still fit when the angle was corrected. 58. Nachmansohn (1953). Others proposed different compositions. For example, Wooley (1958) argued for lipoidal receptors. 59. For the development of membrane models and experimental approaches, see Robinson (1997). 60. For example, Waud (1968). 61. Chagas (1959). 62. Ehrenpreis and Kellock (1960). 63. Chagas (1962). He imagined that acceptor sites would, by binding acetylcholine, increase the local concentration of acetylcholine near its receptor. 64. Changeux et al. (1970a, 1970b). He measured binding by equilibrium dialysis: receptor fractions were confined inside dialysis sacks whose membrane pores prevented proteins from escaping but allowed smaller ligands to enter from the bathing media. Binding was calculated from the concentrations of ligands inside and outside the sacks after equilibration. This approach gave precise values, but the prolonged exposures allowed receptor desensitization. More rapid and convenient approaches used centrifugation or filtration to separate, after an initial mixing, receptor-plus-ligand from unbound ligand. 65. Chang and Lee (1963). a-bungarotoxin has a molecular weight of only 8 kDa, so its mass contributed little to that of the receptor complex. 66. Miledi et al. (1971); Raftery et al. (1971). The electric organ of the ray, Torpedo marmorata, has even higher densities of cholinergic innervation. 67. Changeux et al. (1971); Karlin and Cowburn (1973). Affinity-labeling uses a reagent containing (1} a group that binds selectively but reversibly to the protein of choice and (2) a highly reactive group that will bond irreversibly to proteins it contacts. Thus, the affinity-group selects the protein for labeling with the reactive group. 68. These included Eldefrawi and Eldefrawi (1973); Karlin and Cowburn (1973); Karlsson et al. (1972); Klett et al. (1973); Olsen et al. (1972); Schmidt and Raftery (1972). 69. Kalderon et al. (1970). Pedro Cuatrecasas, who developed this approach, also applied it in 1972 to purifying insulin receptors. 70. Weill et al. (1974). 71. Reynolds and Karlin (1978); Raftery et al. (1971). 72. For a historical sketch and references, see Robinson (1997). 73. Changeux et al. (1979); Huganir et al. (1979); Wilson and Raftery (1979). 74. For example, Nickel and Potter (1973); Cartaud et al. (1978). 75. Kistleretal. (1982). 76. For example, Heidmann and Changeux (1980); Hess et al. (1983); Neubig et al. (1982). 77. "Specific activity" refers to the radioactivity per molecule, expressed as curies/ mole; curies are measures of radioactive disintegrations per unit of time. 78. Cuatrecasas et al. (1974); Lefkowitz (1974); Maguire et al. (1974). Rank-order

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for displacing radioactive noradrenaline did not follow rank-order for antagonizing noradrenaline's effects at receptors; binding did not follow the hyperbolic "saturable" response of dose-response plots but continued without apparent asymptote; and binding of the pharmacologically inactive stereoisomer was comparable to the active one. 79. Lefkowitx et al. (1974); Levitzki et al. (1974); Aurbach et al. (1974). 80. For example, Lefkowitz and Williams (1977); Bylund and Snyder (1976). 81. Caron and Lefkowitz (1976). 82. Shorr et al. (1981). 83. Shorr et al. (1982a). 84. Cerione et al. (1983). 85. DeLean et al. (1982). 86. Shorr et al. (1982b). 87. See Cozzens (1989); Goldberg (1988); Kanigel (1986). 88. Goldstein et al. (1971). 89. Goldstein et al. reported that stereospecific labeling increased at higher ligand concentrations; as Pert and Snyder (1973a) noted, such a relationship is unexpected. Moreover, Simon et al. (1973) stated that they could not replicate Goldstein's results. Goldsteins subsequent attempts at isolation then resulted in a lipoprotein (Lowney et al., 1974), inconsistent with later findings. 90. Terenius (1973a, 1973b). 91. Pert and Snyder (1973a, 1973b). 92. Simon et al. (1973). Simon had been pursuing opioid receptors since the mid 1960s (Van Praag and Simon, 1966), prior to efforts by Terenius and Snyder. 93. Kuhar et al. (1973); Martin et al. (1976); Lord et al. (1977). 94. Pert et al. (1973); Simon et al. (1973); Simon and Groth (1975). 95. Katz and Miledi (1970). 96. Ibid., pp. 963, 962. 97. Katz and Miledi (1972). 98. Anderson and Stevens (1973); Colquhoun et al. (1975). 99. Neher and Sakmann (1976). 100. Since obtaining good electrode seals at neuromuscular junctions was difficult, they used denervated muscle, which has "extrajunctional" receptors over its surface where seals are easier. These extrajunctional receptors were previously known to have responses about threefold longer. 101. Hamill et al. (1981). See also Robinson (1997). 102. Schindler and Quast (1980); Nelson et al. (1980).

7 SECOND MESSENGERS (1951-1990)

Cyclic AMP

During the early 1950s Bernard Katz identified muscle e.p.p.s with increased membrane permeabilities to Na + and K + induced by acetylcholine, while John Eccles linked e.p.s.p.s and i.p.s.p.s of spinal cord motoneurons to increased membrane permeabilities to these and other ions (chapter 4). The notion that neurotransmitters excited or inhibited by altering specific ionic permeabilities, which had been established at these sites, seemed applicable at all sites. But the responses that Katz and Eccles studied had rapid onsets and brief durations, whereas responses at other sites were notably delayed and prolonged. Indeed, such striking differences in time courses were one cornerstone for earlier arguments against chemical transmission at neuromuscular junctions and in the central nervous system (chapter 3). Then Eccles had contrasted rapid responses at presumed electrical synapses with slow responses at acknowledged chemical synapses (as with the vagal innervation of the heart). The latter, he believed, reflected inevitable delays while released acetylcholine diffused to its receptors and while cholinesterase inactivated it. But if fast responses also relied on chemical mechanisms, indicating that chemical transmission could be quite rapid, what produced the slow onset and protracted time courses at other synapses? An unanticipated explanation for 171

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such delays and prolongations (and much more) emerged from different interests. Earl Sutherland (Fig. 7-1) defined a new mode of cellular communication—and one that operates at many synapses—through an urge to understand how hormones act. Sutherland had received his medical training in St. Louis, and after military service in World War II, he returned to work on the hormonal regulation of glucose metabolism with the eminent biochemist Carl Cori. Cori was then studying phosphorylase, a crucial en2yme in cellular metabolism that splits glycogen, a polymerized storage form of glucose, into glucose-1-phosphate subunits (Fig. 7-2A). Glucose-1-phosphate is next converted to glucose-6phosphate, which is either metabolized locally to supply cellular energy or dephosphorylated to glucose, which is released into the bloodstream for metabolism elsewhere.1 Phosphorylase, however, exists in an active form, phosphorylase a, and an inactive form, phosphorylase b (Fig. 7-2B). By 1951 two hormones, adrenaline and glucagon,2 were known to raise blood glucose levels by promoting glycogen breakdown in the liver. That year Suther-

FlGURE 7-1. Earl Wilbur Sutherland, Jr. (1915-1974; courtesy of the National Library of Medicine).

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FIGURE 7-2. Control of glycogen breakdown to glucose-1-phosphate. A. Active phosphorylase a catalyzes the phosphorolysis of glycogen, forming glucose-1-phosphate with inorganic phosphate (Pi). Glycogen synthesis is catalyzed by a distinct enzyme, glycogen synthase. B. Phosphorylase kinase catalyzes the conversion of inactive phosphorylase b to active phosphorylase a through the transfer of a phosphate from ATP to a serine of phosphorylase. C. Protein kinase A catalyzes the conversion of inactive phosphorylase kinase to active phosphorylase kinase through the transfer of a phosphate from ATP to a serine of phosphorylase kinase. cAMP stimulates protein kinase A. D. Adenylate cyclase catalyzes the conversion of ATP to cAMP and inorganic pyrophosphate (PPj); this enzyme is activated by, among other agents, glucagon and adrenaline. cAMP is hydrolyzed to AMP by phosphodiesterase (i.e., one of the ribose-phosphate bonds is hydrolyzed).

land and Cori identified phosphorylase as the rate-limiting step for glucose release.3 They also demonstrated that adrenaline and glucagon increased glucose release by stimulating the conversion of inactive phosphorylase b to active phosphorylase a.

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After Sutherland moved to Cleveland in 1953, he continued to study phosphorylase, aided over the years by a number of accomplished colleagues, including Reginald Butcher, Theodore Rail, Alan Robison, and Walter Wosilait. There he showed that activation involved phosphorylation of phosphorylase b, whereas inactivation involved dephosphorylation of phosphorylase a. Moreover, adrenaline and glucagon promoted the phosphorylation of phosphorylase b.4 (The enzyme catalyzing the phosphorylation of phosphorylase Sutherland named phosphorylase kinase. At that time kinase referred to an enzyme that activated; it also carried the connotation of phosphorylation. The latter sense prevailed, and enzymes later termed kinases cause inhibition as well as activation through phosphorylations.) For these experiments Sutherland used liver slices, very thin tissue sections that allow oxygen to diffuse easily to the interior. Such slices were considered "intact cell" preparations: although cells on the cut surfaces were damaged, those in the interior remained undisturbed and functioning. At this time hormone responses could be demonstrated with slices but not with "broken cell" preparations, such as homogenates. Indeed, some claimed that intact cells— or perhaps even groups of cells—were required for hormones to act.5 But the contemporary biochemical approach to circumventing the impenetrable complexities of whole cells was to study simplified systems, isolating and examining individual components. Sutherland's first stellar achievement was to succeed with such simplification, demonstrating in 1957 hormone responses in broken cell preparations: added adrenaline or glucagon markedly increased phosphorylase activity in liver homogenates.6 Moreover, he showed that this activation occurred in two steps with two distinct cellular components. First, a membrane fraction, separated from liver homogenates, formed a low molecular weight, heat-stable factor in the presence of the hormones. Second, although the hormones had no effect on supernatant fractions of the homogenate, adding this factor to the supernatant fractions increased their phosphorylase activity. Sutherland's ultimate triumph that year was identifying the activating factor as cyclic adenosine monophosphate (cAMP; Fig. 7-3A), an unexpected substance with a structure not previously known in biological systems.7 Independently, David Lipkin in St. Louis identified cAMP in barium hydroxide digests of ATP and then synthesized cAMP chemically.8 Sutherland proceeded to characterize the enzyme catalyzing cAMP synthesis from ATP, adenylate cyclase, and the enzyme destroying cAMP, phosphodiesterase (Figs. 7-2D and 7-3A).9 Functional models thus depicted hormones activating the membrane-bound adenylate cyclase to produce cAMP, with the elevated concentrations of cytoplasmic cAMP promoting the conversion of phosphorylase b to phosphorylase a. Cytoplasmic concentrations of cAMP then returned to basal levels (and the hormone stimulation disappeared) as the phos-

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FIGURE 7-3. ATP, GTP and some of their products. A. ATP can be hydrolyzed to ADP and Pi by various ATPases. ATP can also be converted to cAMP and PPj by adenylate cyclase (below). B. Analogously, GTP can be hydrolyzed by GTPases or converted to cGMP by guanylate cyclase.

phodiesterase destroyed cAMP. In 1965 Sutherland designated cAMP a "second messenger," since cAMP carried information from receptor to cell interior, while hormones, the "first messengers," brought information to the receptor.10 The general process became known as "signal transduction." Sutherland found adenylate cyclase activity in all organs; highest activities were in the brain, where added adrenaline also stimulated cAMP formation.11 At the same time, adrenaline's ability to elevate cAMP levels was tied to cellular responses beyond phosphorylase, for cAMP affected a wide array of cellular properties sensitive to adrenaline, including other enzymes (e.g., inhibiting glycogen synthase, the enzyme that opposes phosphorylases actions12) and processes (e.g., water flow across the amphibian bladder wall13). The cataloging of cAMP responses, however, was hindered by the inability of added cAMP to cross cell membranes and reach its intracellular sites of action. Consequently, the synthesis of a derivative that could gain entry more easily, dibutyryl-cAMP, greatly facilitated these surveys.14 Subsequent studies then revealed cAMP's participation in the whole gamut of cellular regulation, from cell membrane to nucleus. One prominent topic was adrenergic stimulation of the heart. In 1965 Sutherland showed that adrenaline s excitatory effects were preceded by a sharp rise in cAMP levels (Fig. 7-4A).15 Since /3-adrenergic agonists stimulated adenylate cyclase in the same rank order as they excited cardiac function, Sutherland concluded that adenylate cyclase mediated these adrenergic responses. And

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FIGURE 7-4. Effects of adrenaline on heart cAMP levels, contractile force, and phosphorylase activity. A. Adrenaline was added to perfused hearts, and after the times (in seconds) indicated on the x-axis, the amount of cAMP (upper panel, expressed on the y-axis as nmoles per gram of heart) was measured. Contractile force (middle panel, expressed on the y-axis as percent of the unstimulated value) was also measured after addition of adrenaline. Contractile force rose after cAMP rose. Phosphorylase activity of the heart (bottom panel, expressed on the y-axis relative to the mean value before adding adrenaline) also rose, but more slowly, peaking after the rise in contractile force. These time courses suggested that the increased contractile force was not due solely to increased phosphorylase activity. B. A model for the adrenergic receptor-adenylate cyclase complex shows a catalytic subunit (C) regulated by inhibitory a-adrenergic receptor subunits and by excitatory /3-adrenergic receptor subunits. (A from Robison et al. [1965], Fig. 2, courtesy of the American Society for Pharmacology and Experimental Therapeutics. B from Robison et al. [1967], Fig. 4, courtesy of the New York Academy of Sciences.) since a-adreneregic agonists decreased cAMP levels in some tissues and antagonized some /3-adrenergic effects, he suggested that a- and /3-receptors might be linked to adenylate cyclase in an opposing fashion (Fig. 7-4B).16 By the early 1970s further investigations demonstrated that administering dibutyryl-cAMP extracellularly or cAMP intracellularly (by microelectrophoresis) altered electrical responses that correlated with accelerated heart rate

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and increased contractile force.17 These responses, which mimicked the addition of /3-adrenergic agonists, were then linked to alterations in particular K + and Ca2+ currents. In later years the numbers of these currents (and of the membrane channels conducting them) increased dramatically, and causal explanations for adrenergic effects became correspondingly more complex. Nevertheless, a fundamental notion remained: adrenergic agonists affected cardiac function through second messenger systems that altered specific channel conductances. Sutherland also noted that muscarinic cholinergic agonists decreased cardiac cAMP levels, albeit modestly.18 Cholinergic antagonism to adrenergic stimulation could then result from cholinergic agonists opposing the rise in cAMP due to adrenergic agonists. But this explanation, too, was superseded by more complex formulations (see below). Meanwhile, it had become apparent that additional substances altered cellular levels of cAMP. These included not only other hormones but also acknowledged neurotransmitters, beginning with serotonin (initially demonstrated in liver flukes, but in 1968 in mammalian brain).19 Then in 1971 Paul Greengard in New Haven reported, first, that excitation of preganglionic fibers elevated cAMP levels in sympathetic ganglia, and, second, that dopamine, released from interneurons in the ganglia, was the stimulant to adenylate cyclase.20 The next year Greengard demonstrated dopamine-stimulated adenylate cyclase activity in the brain's basal ganglia, where dopamine plays a central role.21 Thus, dopamine receptors were coupled to cAMP production in both ganglia and brain, with cAMP apparently mediating the dopaminergic responses. And in 1974 he described the block of this dopaminergic stimulation of adenylate cyclase by antipsychotic drugs, those used to treat schizophrenia but known also to produce motor symptoms through actions on the basal ganglia (chapter 13).22 Still missing from this account, however, are the mechanisms by which cAMP induces its responses and the mechanisms by which receptors stimulate or inhibit adenylate cyclase. (A second cyclic nucleotide, cyclic guanosine monophosphate [cGMP, Fig. 7-3B], was discovered in 1967 during a survey of urinary constituents.23 Over the next two decades the properties of cGMP were scrutinized, and although significant physiological roles were discovered—notably in the chain of visual responses in retinal cells—a definite participation in synaptic transmission was not established.)

Protein Kinases and Pnospnatases

Glycogen breakdown to glucose is a primary step in liberating energy for muscle contraction, also. While Sutherland was examining the hormonal activation of phosphorylase b in liver, Edwin Krebs (Fig. 7-5A) was examining the acti-

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FIGURE 7-5. A, left, Edwin G. Krebs (1918-). B, right, Edmond H. Fischer (1920-). (A courtesy of E. G. Krebs. B courtesy of E. H. Fischer.)

vation of phosphorylase b in muscle. Krebs had initially studied medicine a year behind Sutherland at Washington University, and after military service he, too, worked with Cori in St. Louis. In 1948 Krebs moved to a faculty position in Seattle, and there eight years later he described the conversion of inactive phosphorylase b to active phosphorylase a, collaborating with Edmond Fischer (Fig. 7-5B), who had emigrated from Geneva a few years earlier.24 This study with cell-free muscle extracts was published the same year that Sutherland reported the phosphorylation of liver phosphorylase b. Krebs and Fischer, however, proceeded to show in 1959 that the phosphorylating enzyme, phosphorylase kinase, also existed in two forms, activated and inactivated.25 Incubation with ATP activated phosphorylase kinase, and adding cAMP augmented this conversion. They offered the "speculative hypothesis" that "phosphorylase . . . kinase itself exists in phosphorylated and dephosphorylated forms."26 Finding evidence for this suggestion consumed eight more years. During this time Krebs showed that administering adrenaline (which stimulates muscle phosphorylase just as it does liver phosphorylase) increased both phosphorylase activity and the level of cAMP in muscle.27 Three years later, in 1968, Krebs reported that phosphorylation of phosphorylase kinase indeed accompanied its activation; moreover, cAMP promoted this phosphorylation.28 Later

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that year he purified "phosphorylase kinase kinase" from muscle, the enzyme catalyzing the phosphorylation of phosphorylase kinase.29 Subsequently this enzyme became known as "cAMP-dependent protein kinase" or "protein kinase A" (I will use the latter name). The causal chain thus ran (Fig. 7-2): adrenaline => increased cAMP => active protein kinase A => active (phosphorylated) phosphorylase kinase => active (phosphorylated) phosphorylase a =$ glycogen breakdown. Two protein kinases, protein kinase A and phosphorylase kinase, are involved, each transferring the terminal phosphate of ATP to a protein (onto the hydroxyl of the amino acid serine, forming phosphoserine esters, as Krebs showed). Sutherland described a phosphatase cleaving the phosphate from phosphorylase and Krebs a phosphatase similarly dephosphorylating phosphorylase kinase.30 Activation could be turned off as well as on. Independently, Joseph Larner in Cleveland was examining the opposing reaction, the formation of glycogen from glucose catalyzed by glycogen synthase. As noted above, adrenaline inhibited glycogen synthase while activating phosphorylase, a prime example of what came to be called reciprocal control (the biochemical equivalent of Sherrington s reciprocal innervation). In 1963 Larner showed that glycogen synthase also existed in two forms, with their interconversion reflecting phosphorylation/dephosphorylation of this enzyme, too.31 Moreover, cAMP activated a kinase that phosphorylated glycogen synthase to produce the less active form.32 Evidently, cAMP bound to this glycogen synthase kinase to activate its phosphorylating ability; Larner invoked Monod's notion of allosteric control of protein function in describing the activation of this kinase.33 Krebs then showed that the kinases were identical: phosphorylase kinase kinase and glycogen synthase kinase were the same enzyme (protein kinase A).34 In 1968 Krebs reported that protein kinase A phosphorylated several other proteins in vitro.35 Whether it phosphorylated any of these in vivo was not established, however, and all the enzymes then known to be regulated by cAMP and protein kinases were involved with glycogen synthesis and breakdown. But the next year Lester Reed in Austin showed that phosphorylation/dephosphorylation also regulated a key enzyme of intermediary metabolism, pyruvate dehydrogenase.36 That year, 1969, Greengard proposed that "protein kinases mediate all the diverse effects" of cAMP; indeed, he found protein kinase A activity in every tissue he examined, concluding that cAMP through this kinase "may play a role in the regulation of all animal tissues."37 In 1957 P. J. Heald in London had described an increased phosphorylation of brain proteins following electrical stimulation in vitro; Heald related this protein phosphorylation to transport mechanisms necessary to restore ionic balance after stimulation.38 Instead, Greengard in 1969 demonstrated receptormediated increases not only in cAMP synthesis (see above) but also in protein

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kinase activity; furthermore, he showed that protein kinases phosphorylated particular, identifiable proteins in the brain.39 Independently, Richard Rodnight in London reported in 1970 that cAMP stimulated the phosphorylation of brain proteins and in 1973 that added noradrenaline as well as electrical stimulation increased protein phosphorylations.40 Protein phosphorylation thus might play a major role in synaptic transmission, with likely targets including receptors and the ion channels facilitating excitatory and inhibitory ionic fluxes. Accordingly, in 1977 Ivan Diamond in San Francisco and Jean-Pierre Changeux in Paris reported that nicotinic acetylcholine receptors, present in postsynaptic membranes from fish electric organs, could be phosphorylated in vitro by endogenous protein kinases.41 And in 1986 Greengard showed that phosphorylating the y- and 5-subunits of this receptor altered its functional properties (increased its rate of desensitization).42 The next year Greengard found that cAMP analogs promoted phosphorylation of these receptors in muscle cells in vivo.43 Other studies demonstrated phosphorylation of ion channels and consequent changes in their properties. For example, in 1980 Greengard described enhanced Ca2+ fluxes after he injected protein kinase A into neurons.44 Similarly, W. Trautwein in Homberg and F. Hofmann in Heidelberg found that injecting protein kinase A into heart cells enhanced Ca2+ influx, as did adding noradrenaline.45 Noradrenaline's stimula-

FIGURE 7-6. The spreading recognition that multitudes of proteins are regulated through phosphorylation/dephosphorylation. (Reprinted from Krebs [1994], ©1994, with permission from Elsevier Science.)

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tory effect on the heart was thus attributable to noradrenaline elevating protein kinase A activity through stimulation of cAMP formation; protein kinase A would then phosphorylate Ca2+ channels to promote ion fluxes that stimulate cardiac activity. Through the 1980s further evidence accumulated for protein phosphorylation throughout the organism, with such phosphorylations representing a ubiquitous means for modifying protein function. Other protein kinases were soon identified (see below), and the number of proteins known to be phosphorylated soared (Fig. 7-6). Phosphorylations could participate in these regulatory processes only if the phosphorylations were readily reversible. As cited above, Sutherland and Krebs demonstrated the enzymatic dephosphorylation of phosphorylase and phosphorylase kinase. Protein phosphatase activity in the brain was identified soon afterward.46 By 1983 four classes of protein phosphatases were established,4' and further studies revealed that these phosphatases were themselves regulated, in part by phosphorylation/dephosphorylation.

G-Proteins

Still missing from this account is the link between neurotransmitters binding to receptors and the activation of adenylate cyclase to synthesize cAMP. By 1971 neither receptors nor enzyme had been isolated, but proposals centered on complex systems with regulatory and catalytic subunits.48 Ligand-induced conformational changes in receptor subunits could induce, through contiguity, conformational changes to activate catalytic subunits, following the pattern of allosteric enzymes then being described. But evidence for an intervening entity—a mobile, amplifying entity—emerged from studies by Martin Rodbell (Fig. 7-7A) in Bethesda. Rodbell was born a decade after Sutherland, Krebs, and Fischer, with World War II interrupting his education. He received his Ph.D. in biochemistry from the University of Washington (where Krebs and Fischer were) in 1954, and in 1956 he began his career at the National Institutes of Health (NIH), studying hormonal responses. In 1971 these investigations had evolved into examining how glucagon stimulated adenylate cyclase. While measuring the binding of labeled glucagon to liver cell membranes, Rodbell found that adding nucleotides—most prominently guanosine triphosphate (GTP) and guanosine diphosphate (GDP) (Fig. 7-3B)—affected the process: as little as 50 nM GTP or GDP decreased the affinity for glucagon.49 Since GDP as well as GTP was effective, Rodbell considered that the nucleotides acted by binding to regulatory (allosteric) sites rather than by phosphorylation. In addition to these effects on binding, GTP also increased the rate of cAMP

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FIGURE 7-7. A, left, Martin Rodbell (1925-1998). B, right, Alfred Goodman Oilman (1941-). (A courtesy of the National Institutes of Health. B courtesy of A. G. Gilman.)

synthesis, and at very low substrate levels GTP was required for catalytic activity.50 Rodbell concluded that GTP "play a specific and obligatory role in the activation . . . by glucagon."51 Moreover, Rodbell showed in 1974 that this activation by GTP occurred after glucagon bound to the receptor and that an analog of GTP that was not hydrolyzed could induce a persisting activation.52 Such analogs proved to be valuable both practically, because of this long-lasting activation, and conceptually, because binding rather than phosphorylation must then be the essential process. On the other hand, GDP did not activate (in contrast to its effects on glucagon binding), and GTP's activation was transient, presumably because it was hydrolyzed to inactive GDP. Others soon found that GTP and GTP analogs activated adenylate cyclases from several sources following stimulation by several ligands.53 Efforts then focused on identifying the component to which GTP bound and on defining how the resulting complex activated adenylate cyclase. In 1976 Dan Cassel and Zvi Selinger in Jerusalem described a distinct GTPase activity in avian red blood cell membranes that was stimulated by adrenergic agonists; they argued that GTP hydrolysis was required for returning the activated system to its unstimulated state.54 Cassel and Selinger next showed that ligands

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binding to their receptors promoted the release of GDP.55 (Rodbell initially found that GDP decreased hormone binding to receptor; the necessary correlate is that hormone binding to receptor will decrease GDP binding to its sites.) Meanwhile, Thomas Pfeuffer in Wiirzberg labeled a 42 kDa peptide from avian red blood cell membranes using a reactive analog of GTP, and he also separated this peptide by affinity chromatography using GTP complexed to a matrix.56 Pfeuffer then demonstrated that when GTP bound to this peptide (which he accordingly named "G-protein"), its association with—and thus activation of—adenylate cyclase was promoted.57 A model from 1981 (Fig. 7-8A) showed a complex of receptor, G-protein (containing GDP), and adenylate cyclase. Ligands binding to the receptor facilitated the association between receptor and G-protein, with concomitant release of GDP. The G-protein then bound GTP, permitting association with and activation of adenylate cyclase. This activation ceased when the G-protein hydrolyzed GTP to GDP, causing dissociation from the adenylate cyclase. The model also indicated that the G-protein moved between receptor and adenylate cyclase.58 And implicit was the capacity for amplification: one receptor could activate multiple G-proteins, and each G-protein could activate one adenylate cyclase to form multiple cAMP molecules before the slow hydrolysis of GTP terminated this activation. Purification and further characterization of the G-proteins came prominently from Alfred Goodman Gilman (Fig. 7-7B), destined by nature, nurture, and name to be a pharmacologist: he was the son of one author of the standard textbook of pharmacology and namesake of the other.59 Gilman was born a generation after Rodbell and received his M.D. and Ph.D. degrees in 1969 from Case Western Reserve University, where Sutherland had identified cAMP. After two years at NIH, he moved to Charlottesville and was soon resolving a number of significant issues. Pertinent here is his purification of the G-proteins. Gilman approached this problem by exploiting various mutant tissue culture cells that lacked different components of the receptor/G-protein/adenylate cyclase system. Gilman could then characterize the components by reconstituting the functional system from constituents lacking certain capabilities.60 And he could use systems lacking certain capabilities (such as response to GTP) to assay for components that restored function. By 1980 Gilman isolated from mammalian liver three peptides that participated in the activation by GTP, having molecular weights of 52, 45, and 35 kDa.61 Avian red blood cells, however, contained only the 45 and 35 kDa peptides; the 52 kDa peptide was subsequently recognized as a variant of the 45 kDa unit.62 And in 1984 Lutz Birnbaumer in Houston identified a 5 kDa peptide (later corrected to 7 kDa).63 The native G-protein was thus a trimer of a (45 kDa), ft (35 kDa), and y (7 kDa) subunits. Only a, however, bore the GTP-binding site.

FIGURE 7-8. Models for G-protein actions. A. Receptor (R), G-protein (G), and adenylate cyclase (C) are in, or tethered to, the cell membrane. When an agonist binds to R, G interacts with R and exchanges its bound GDP for GTP. With GTP bound, G interacts with C, stimulating catalytic activity that continues until G hydrolyzes GTP to GDP. This hydrolysis promotes dissociation of G from C, turning off the stimulation. B. The exchange of GTP for GDP triggers a dissociation of a from /3y subunits of a heterotrimeric G-protein. Hydrolysis of GTP to GDP allows reassociation of the complex. (A from Limbird [1981], Fig. 1, reproduced by permission of the author, ©The Biochemical Society. B from Stryer [1988], Fig. 38-5, ©1988 by W. H. Freeman and Company, used with permission.)

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Parallel studies on visual receptors in the retina revealed an analogous GTPbinding protein that coupled light-sensitive rhodopsin to the enzyme hydrolyzing cGMP (cGMP phosphodiesterase). In 1981 Lubert Stryer in Palo Alto showed that this protein, which he named "transducin," was a trimer of a (39 kDa), ft (36 kDa), and y (~10 kDa) subunits.64 Subsequent studies revealed structural similarities with a, (3, and y subunits of other GTP-binding proteins, and in 1983 Gilman argued that they represented a family of structurally and functionally similar GTP-binding proteins.65 These became known as "heterotrimeric Gproteins" to distinguish them from smaller, single-subunit GTP-binding proteins that are also extremely important in cellular functioning, but in ways beyond the scope of this book. (By 1998 four major families of heterotrimeric G-proteins were recognized, with 20 a, 6 ft, and 12 y subunits identified.66) Moreover, these heterotrimeric G-proteins were soon linked not only to a host of different receptors (chapter 8) but also to another second messenger system (see below). A further complexity emerged when Gilman showed that GTP promoted the separation of the heterotrimeric G-protein into two components,67 the a and fty subunits. (A textbook depiction from 1988 is reproduced in Fig. 7-8B.) It was a that, when it contained GTP and was separated from fty, bound to and activated adenylate cyclase. (ft and y bound tightly together and normally did not separate; before the identification of y, Gilman wrote of dissociation into a and ft subunits.) The slow onset of synaptic action at /3-adrenergic receptors (and at many others) was thus attributable to chains of events including G-protein interaction with these receptors, G-protein dissociation into its subunits, binding of the a subunits to adenylate cyclase, synthesis of cAMP, activation of protein kinases, and catalysis of various protein phosphorylations—followed by the cellular responses to these phosphorylated proteins. The slow decline in synaptic activation then reflected the slow hydrolysis of GTP, breakdown of cAMP, and dephosphorylation of activated proteins. A still further complexity was added by Gilman s recognition of inhibitory as well as stimulatory G-proteins, with GI inhibiting adenylate cyclase and Gs stimulating.68 Differential sensitivities to certain bacterial toxins assisted in these identifications and distinctions: cholera toxin catalyzed the modification of Gsa, making it persistently active, whereas pertussis toxin catalyzed the modification of Gj«, rendering it inactive.69 When the inhibitory G,a subunit was isolated, it had a molecular weight of 41 kDa, although it was structurally similar to the 45 kDa Gsa subunit. Since the fty complexes seemed to be the same in Gj and Gs, the inhibitory and stimulatory capabilities were attributed to these distinct a subunits. According to this formulation, a receptor would inhibit or stimulate adenylate cyclase depending on whether it bound Gj or Gs preferentially. Sutherland's model (Fig. 7-4B) now required appropriate G-proteins as intermediaries: Gs for j8-adrenergic and Gj for ag-adrenergic receptors.70

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The initial concept of G-proteins as merely intermediaries in the production of second messenger systems had to be revised during this decade, however. Muscarinic receptors involved in the vagal slowing of the heart seemed to be coupled to Gj-proteins,71 so when acetylcholine bound to the receptors G;a would be liberated to inhibit adenylate cyclase. But in 1985 compelling evidence appeared for G-proteins interacting directly with ion channels and thereby modifying their conductances.72 Two years later Eva Neer and David Clapham in Boston showed that liberated /3y subunits interacted directly with a cardiac K + channel, opening it to cause a slowing of the heart.73 Previously, the /3y complexes had been thought to function solely through their uniting with a subunits and—through formation of inactive a/3y complexes—preventing a from binding to adenylate cyclase. Now distinctions between various /3y subunits were stressed, as these subunits appeared to act differentially on their own. Moreover, further studies in the succeeding years provided additional evidence for both a and j3y subunits binding to and affecting directly various cellular proteins beyond the second messenger systems.

Ca2+

By 1970 cyclic nucleotides were acknowledged as second messengers that carried information from surface receptors throughout the cell interior. That year Howard Rasmussen in Philadelphia proposed as a companion to these organic molecules a quite different substance, inorganic calcium ions (Ca2+), providing cells with "two interrelated intracellular messengers" acting in concert.74 Rasmussen suggested that after cAMP triggered protein phosphorylations—as shown by Krebs, Fischer, Greengard, and others—Ca2+ would then activate the phosphorylated proteins. But he admitted that Ca2+ might also serve as a signaling substance independent of cAMP.

Responses to Ca

Several roles for Ca2+ were then apparent. Longest established was the Ca2+ requirement for muscle contraction, noted in 1883 by Sydney Ringer in London.75 In 1947 Victor Heilbrunn in Philadelphia showed that injecting Ca2+ into muscle provoked contraction, and in 1952 Alexander Sandow in New York formulated a scheme for "excitation-contraction coupling" in which muscle action potentials liberated Ca2+ intracellularly, with this Ca2+ then activating the contractile proteins.76 During the 1950s and 1960s electron microscopists identified both a tubular system within muscle cells and the association of this "sarcoplasmic reticulum" with periodic imaginations of the muscle cell membranes, "transverse tubules."77 Physiologists showed that these transverse

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tubules conducted electrical signals to the cell interior,78 where Ca2+, sequestered within the sarcoplasmic reticulum,79 would be released into the cytoplasm. Meanwhile, Wilhelm Hasselbach in Heidelberg and Setsuro Ebashi in Tokyo demonstrated in the early 1960s that muscle relaxation was effected by removing Ca2+ from the cytoplasm: Ca2+ was reaccumulated within the sarcoplasmic reticulum.80 The cycle of muscle contraction and relaxation thus followed a cycle of Ca2+ release and sequestration. A second role was in secretion. As an initial step toward defining how cholinergic nerves to the adrenals evoke the secretion of adrenaline, William Douglas in New York perfused the adrenals with acetylcholine in media containing various ions. Acetylcholine, Douglas reported in 1961, caused adrenaline release only when Ca2+ was also present in the perfusion media. Moreover, the magnitude of the adrenaline release varied with the Ca2+ concentration.81 Since Katz had shown that acetylcholine acts on the exterior of muscle cells to alter permeability to Na + and K + , Douglas proposed that acetylcholine stimulated secretion similarly: "by causing calcium ions to penetrate the adrenal... cells."82 Indeed, he found a marked increase in the uptake of labeled Ca2+ when he added acetylcholine to the perfusion media.83 Douglas named the process "stimulus-secretion coupling" to stress the parallel with muscle, and he suggested that Ca2+ might participate analogously in noradrenaline secretion from sympathetic nerves. He also demonstrated a Ca2+ requirement for secretion by other glands.84 A third and related role was in neurotransmitter release, as delineated at neuromuscular junctions and ganglionic synapses by Katz and Ricardo Miledi in London. At these sites the absence of Ca2+ prevented synaptic transmission, just as it prevented adrenaline secretion. In 1965 Katz and Miledi showed that during perfusion with Ca2+-deficient media, the localized addition of Ca2+ at motorneuron terminals, delivered by micropipets, promoted acetylcholine release.85 This observation thus countered arguments that Ca2+ was required merely for impulse propagation to the nerve terminals. Rasmussen's 1970 proposal specified several additional roles for Ca2"1", including several steps in the control of metabolism; others roles soon appeared. But two salient issues loomed beyond the mere recognition that Ca2+ participated: how the local Ca2+ concentration was regulated—increased to initiate responses and reduced to terminate them—and how Ca2+ actually caused these responses. Regulation or Cytoplasmic Ca

Concentrations

Two sources for elevating cytoplasmic Ca2+ levels were illustrated in these studies, extracellular and intracellular. Katz and Miledi defined inward Ca2+ currents, attributable to Ca2+ flowing through transmembrane channels from

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extracellular fluid to cytoplasm.86 Throughout the next decades a host of Ca2+ channels—present almost ubiquitously in cells across the biological universe— were distinguished according to their electrical properties and sensitivities to various inhibitors.87 These, like the Na+ and K + channels of the Hodgkin-Huxley model for nerve action potentials (chapter 4), were voltage-gated channels: their openings and closings depended on the transmembrane potential. Consequently, the voltage-gated Ca2+ channels linked membrane potential changes to the multitudes of cellular responses regulated by cytoplasmic Ca2+ concentrations. And in 1972 A. Fleckenstein in Freiburg demonstrated that certain drugs known both to alter cardiac function and to be antagonized by higher Ca2+ concentrations acted through a blockade of particular Ca2+ channels.88 These "calcium channel blockers"—including such drugs as nifedipine (Procardia) and diltiazem (Cardizem)—became widely used in treating angina, cardiac arrhythmias, hypertension, and other disorders. On the other hand, in muscle the sarcoplasmic reticulum was an intracellular reservoir of releasable Ca2+. The corresponding structure in nonmuscle cells, the "endoplasmic reticulum," was later recognized as such a source in these cells, too (see below). For influxes of Ca2+ to alter the cytoplasmic concentration acutely, that concentration must normally be maintained at a lower level89 by Ca2+ pumps, energy-consuming active transport systems capable of extruding Ca2+ against its electrochemical gradient.90 In 1966 H. J. Schatzmann in Bern identified a cell membrane transport system that used ATP as its energy source (it was a Ca2+-ATPase).91 A few years later Harald Reuter in Bern and Mordecai Blaustein in Cambridge described a parallel transport system that exchanged extracellular Na+ for intracellular Ca2+, driven by the transmembrane electrochemical gradient for Na + . 92 As noted above, Hasselbach and Ebashi identified a system that actively transported Ca2+ from cytoplasm to sarcoplasmic reticulum. This was also a Ca2+-ATPase, related to but distinct from the cell membrane Ca2+-ATPase. The same Ca2+-ATPase was later recognized in the endoplasmic reticulum of nonmuscle cells.

Mechanisms or Action

Accounts of how Ca2+ initiated its multitudes of responses were less tidy, evolving into a catalog of mechanisms. In some instances Ca2+ acted directly, as in binding to, and thereby activating, the proteolytic enzyme calpain or various K + and Ca2+ channels that control cellular function.93 In other instances Ca24" functioned by binding to, and thereby activating, regulatory proteins, which in turn affected the responses of further systems. During this time two important examples of such regulatory proteins were characterized, troponin and calmodulin.

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Ebashi, while continuing his investigations on Ca2+ activation of muscle, separated in 1965 a substance from the contractile proteins that conferred on them sensitivity to Ca2+. He named this Ca2+-binding protein "troponin."94 Troponin itself turned out to contain three subunits, of which troponin C, with a molecular weight of 18 kDa, bound Ca2+.95 In 1973 John Collins and James Potter in Boston reported its amino acid sequence, identifying four binding sites for Ca2+.96 The route to defining the other regulatory protein, calmodulin, was less direct. For studies on how cAMP degradation is regulated, Wai Yiu Cheung in Memphis set about purifying the responsible enzyme, phosphodiesterase. But, as Cheung reported in 1970, he found sharp losses in phosphodiesterase activity during this purification, which he traced to the separation from the enzyme of an activating protein.97 Three years later Jerry Wang in Winnipeg showed that this protein bound Ca2+ and that Ca2+ was required for its activation of phosphodiesterase.98 The purified activator had a molecular weight of 19 kDa and other structural similarities to troponin. Comparisons of the amino acid sequences in 1978 confirmed the close relationship.99 That year Cheung proposed a new name, "calmodulin," to emphasize the Ca2+ dependency of its actions in modulating a growing number of systems.100 Targets included adenylate cyclase as well as phosphodiesterase, various phospholipases and protein phosphatases, and the cell membrane Ca2+ ATPase.101 Particularly significant were several protein kinases, ranging from those with narrowly specific targets—such as myosin light chain kinase, which phosphorylates a component of the muscle contractile apparatus—to the broadly active CaM kinase II (named for its activation by calmodulin). Greengard identified CaM kinase II in 1978.102 It was soon shown to regulate a number of neural processes, including systems for neurotransmitter synthesis, neurotransmitter release, and neuronal structural changes.103 Finally, three enabling techniques that played essential roles in these investigations deserve mention.104 Ca2+ ionophores, which carry Ca2+ across membrane barriers, provided a means for changing intracellular Ca2+ concentrations. Ca2+ chelating agents, which bind Ca2+ specifically and reversibly, allowed intracellular Ca2+ concentrations to be kept at desired levels. Ca2+ indicators (proteins or dyes whose emission of light is a function of ambient Ca2+ concentration) and Ca2+ electrodes reported the local Ca2+ levels.

Inositol-fr/spnospnate ana Diacylglycerol

Different concerns prompted studies culminating in the identification of two further second messengers that were unrelated structurally to any previously known. The paths to their recognition originated in Lowell Hokin and Mabel

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Hokin's search in Montreal for an acetylcholine-stimulated labeling of nucleoproteins.105 But after incubating pancreas slices with acetylcholine plus radioactive phosphate, the Hokins found instead an increased incorporation of the label in phospholipids, as they reported in 1953.106 Atropine blocked the stimulated incorporation, indicating a specific muscarinic response. This stimulated incorporation represented an increased turnover, with synthesis and degradation rising in parallel and no net change in phospholipid content. By 1955 the Hokins had identified two phospholipids responsible for the observed turnover: phosphatidylinositol (then not distinguished from phosphatidylinositol-fosphosphate; Fig. 7-9) and phosphatidic acid (phosphatidylinositol minus the inositol group, i.e., phosphorylated diacylglycerol).107 Both were minor constituents of the cell membrane. Since acetylcholine promotes the secretion of digestive en2ymes by the pancreas, they thought the stimulated turnover of these phospholipids might be part of the secretory mechanism. In 1959 the Hokins, now in Madison, turned their attention to nasal salt glands of the albatross, which secrete concentrated NaCl and thereby compensate for the seabirds' consumption of salty food and drink. The glands are stimulated by cholinergic nerves in vivo, and added acetylcholine dramatically increased the labeling of phosphatidylinositol and phosphatidic acid in slices of these glands in vitro.108 The Hokins proposed a cycle in which phosphatidic acid served as a carrier for transporting Na + from gland to exterior, ferrying Na + across intervening cell membranes. They vigorously pursued this scheme for several years, but in 1964 they found that the rate of labeling was too slow and abandoned the proposal.109 What then might be the role of the acetylcholine-stimulated turnover of phospholipids? Examples of stimulated turnover were soon recognized in nonsecretory cells,110 so turnover could not reflect solely the process of secretion. In 1964 the Hokins suggested that the cycle between phosphatidylinositol and phosphatidic acid was linked to transitions between "resting" and "stimulated" cellular states,111 but they were unable to characterize the linkage. Four years later Jack Durell in Washington showed that acetylcholine promoted the hydrolysis of phosphatidylinositol instead to diacylglycerol and inositolphosphate; he argued that the acetylcholine-stimulated hydrolysis increased membrane permeability to cations enough to depolarize cells, although he was unable to demonstrate altered permeabilites.112 In 1975 Robert Michell in Birmingham published a lengthy review scrutinizing two decades of such studies and concluding that certain receptors (including those for a-adrenergic as well as muscarinic cholinergic agonists) transmitted information to the cell interior through the hydrolysis of phosphatidylinositol.113 By this time Ca2+ had attained prominence as a second messenger, and Michell cited studies indicating an association between receptor activation and increased cytoplasmic Ca2+ levels. He concluded that the stim-

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FIGURE 7-9. Conversion of Phosphatidylinositol-fcisphosphate into two second messengers, diacyglycerol and inositol-frisphosphate. Phosphatidylinositol-Znsphosphate contains a three-carbon, three-hydroxyl backbone, glycerol. The first two hydroxyls of this alcohol are esterifed (acylated) with long chain fatty acids and the third with a phosphate group. This phosphate also forms an ester linkage with inositol, a six-carbon, sixhydroxyl cyclic alcohol. Two other hydroxyls of inositol are esterifed with phosphate groups. Phospholipase C cleaves between the third glycerol hydroxyl and the linking phosphate, liberating glycerol with two fatty acids esterified (diacylglycerol) plus inositol with three phosphates esterified (inositol-frisphosphate).

ulated hydrolysis could "raise cell-surface permeabilities to Ca2+" and thereby pass the signal on.114 Direct evidence came from an insect physiologist in Cambridge, Michael Berridge. He had been studying the secretion of saliva by blowflies, which is stimulated by serotonin, and he could measure secretion, Ca2+ influx, and phosphatidylinositol hydrolysis continuously.115 In accord with MichelPs hypothesis, Berridge in 1979 found that when serotonin stimulated salivary secretion, it increased both Ca24" entry into isolated salivary glands and phosphatidylinositol hydrolysis.116 On the other hand, removal of external Ca2+ prevented the increased secretion without affecting phosphatidylinositol hydrolysis, as would

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be expected if hydrolysis were a step between stimulation of receptor and Ca2+dependent activation of secretion. By this time phosphatidylinositol was known to exist also in a form containing two additional phosphates, phosphatidylinositol-£MSphosphate; the hydrolysis product of this compound is inositol-fmphosphate (Fig. 7-9). Although Berridge had reported in 1979 that serotonin accelerated the hydrolysis of both phosphatidylinositols, further studies on blowflies in 1983 revealed that the initial product of stimulated hydrolysis was inositol-£raphosphate, derived from phosphatidylinositol-fowphosphate (the liberated inositol-tnsphosphate was then rapidly dephosphorylated).117 The same year Berridge showed that adding inositol-fmphosphate to cell interiors evoked a release of Ca2+ from intracellular reservoirs.118 Rapid dephosphorylation of inositol-faisphosphate would then terminate this release.119 In 1988 Solomon Snyder in Baltimore identified a receptor for inositol-fraphosphate present on the endoplasmic reticulum,120 providing the link to Ca2+ release into the cytoplasm. A phospholipase that cleaves between the glycerol backbone and phosphate is designated as phospholipase C (Fig. 7-9), and in 1987 a phospholipase C specific for phosphatidylinositol-foisphosphate was characterized.121 Suggestions that G-proteins activated such phospholipases were advanced by the middle of that decade, although definitive demonstrations appeared only later.122 Overall, this pathway for signal transduction begins with receptors on the cell membrane that interact with particular G-proteins. These G-proteins activate a phospholipase C specific for phosphatidylinositol-feisphosphate, liberating inositol-fraphosphate. Inositol-£nsphosphate binds to its receptors on the endoplasmic reticulum, releasing Ca2+. Ca2+ then elicits its multitudes of effects. Inositol-fraphosphate is thus a second messenger that acts by releasing another second messenger, Ca2+. While this pathway was being examined, independent investigations revealed another route by which phosphatidylinositol-tephosphate hydrolysis generates a second messenger. Even before inositol-fraphosphate was recognized as a second messenger, Yasutomi Nishizuka in Kobe was studying a novel protein kinase in the brain.123 Moderately high concentrations of Ca2+ (50 /xM) plus membrane phospholipids activated this protein kinase.124 In 1979, however, Nishizuka found that diacylglycerol (Fig. 7-9) also activated, reducing the concentrations of Ca2+ and phospholipids then required.125 In fact, the basal levels of Ca2+ normally present in cytoplasm, <1 /xM, were sufficient in the presence of diacylglycerol. Nishizuka named the enzyme protein kinase C. By the end of the decade, it was recognized as a family of protein kinases having distinct localizations and substrate preferences.126 Among the targets were ion channels and the machinery for neurotransmitter release.127 In 1979 Nishizuka concluded that receptor-stimulated hydrolysis of phos-

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phatidylinositol-Znsphosphate yielded active diacylglycerol as well as active inositol-£mphosphate.128 Diacylglycerol thus served as a second messenger between receptor occupancy and protein kinase C activation. Its actions were then terminated by phosphorylation, which converted active diacylglycerol to inactive phosphatidic acid.129 Receptor-stimulated hydrolysis of phosphatidylinositol-fozsphosphate thus generates two second messengers. (1) Released diacylglycerol activates particular members of the protein kinase C family present locally, with consequent phosphorylation of proteins that regulate processes from cell membrane to nucleus. (2) Released inositol-£hsphosphate triggers Ca2+ efflux from stores in the endoplasmic reticulum, elevating cytoplasmic Ca2+ levels. This Ca2+ then acts directly or through binding to regulatory proteins such as calmodulin; its targets include different protein kinases with different substrate specificities.

Concr onclusions

The concept of a second messenger arose from studies on the hormonal control of glucose availability, but it matured into appreciations of general mechanisms for signal transmission within and among all cells. Receptors could trigger an increase in the cytoplasmic concentrations of second messengers through their formation (e.g., cAMP, inositol-fmphosphate, diacylglycerol) and through their entry or release (e.g., Ca 2+ ). The second messengers could then alter cellular function directly (e.g., Ca2+ affecting certain ion channels) or by activating certain enzymes (e.g., protein kinase A, CaM-kinase II, protein kinase C). Protein kinases could phosphorylate particular proteins to regulate discretely, if sometimes broadly, a range of cellular functions: cell division, growth, gene expression, biochemical syntheses and degradations, motility, permeability, excitation, and so on. In some cases G-proteins linked receptor occupancy to second messenger formation (e.g., cAMP, inositol-fnsphosphate, diacylglycerol). Moreover, G-proteins could also act directly on cellular components (e.g., ion channels). These cascades of reactions amplified responses. They also directed them, depending on the particular receptor occupied, the second messenger formed, the effector activated, and the substrates available to that effector. The functional consequences could be evanescent, reflecting the rapid removal of second messenger and dephosphorylation of proteins, or prolonged as a result of some long-lasting change initiated by the second messenger, such as the new synthesis of some protein. Defining the complexities of these systems consumed several decades and demanded notable ingenuity and insight. Indeed, Nobel Prizes were awarded to Sutherland (1971), Krebs (1992), Fischer (1992), Rodbell (1994), Gilman

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(1994), and Greengard (2000) for their achievements—as well as to Carl and Gerty Cori (1947), who laid the groundwork for the initial discoveries. Appreciating the significance of these processes required time and flexibility, also. Through the 1950s neurotransmitter responses had been depicted as depolarizations or hyperpolarizations elicited through the opening of channels specific for certain ions. The subsequent investigations then added second messenger systems, as primary effectors or as modulators of neurotransmitter actions, to the earlier mechanisms for synaptic transmission. Moreover, this distinction between receptors opening channels and receptors acting through second messengers complemented contemporary realizations that the structures of neurotransmitter receptors also fall into two major classes (chapter 8). Notes 1. At this time both the breakdown and the synthesis of glycogen were thought to be catalyzed by the same enzyme, phosphorylase. (Leloir et al., 1959, demonstrated that a different enzyme, glycogen synthase, was responsible for synthesis; this was a major conceptual advance, establishing the pattern seen frequently thereafter of synthesis and degradation following separate pathways, separately regulated.) Phosphorylase was so named because the breakdown proceeds by phosphorolysis: breaking glucose-glucose bonds by inserting a phosphate group (forming glucose-1-phosphate fragments). Only liver possesses glucose-6-phosphatase, so only liver can liberate free glucose from glycogen. 2. Hormones are regulatory substances released into the bloodstream. Adrenaline is released by the adrenals, glucagon (then known as the "hyperglycemic-glycogenolytic factor") by the pancreas. 3. Sutherland and Cori (1951). 4. Sutherland and Wosilait (1955); Rail et al. (1956). 5. See Sutherland (1972). 6. Rail et al. (1957). 7. Sutherland and Rail (1957). 8. Cook et al. (1957); Lipkin et al. (1959). Sutherland and Lipkin independently approached Leon Heppel for a reagent, and he put them in touch with each other. Cook et al.s original formula, containing two adenine nuclei, was corrected in their second paper. 9. Sutherland et al. (1962); Butcher and Sutherland (1962). Alternative names for adenylate cyclase are adenyl cyclase and adenylyl cyclase. 10. Sutherland et al. (1965). 11. Sutherland et al. (1962); Klainer et al. (1962). 12. Belocopitow (1961). 13. Handler et al. (1968). 14. Falbriard et al. (1967). 15. Robison et al. (1965). 16. Robison et al. (1967). See also Turtle and Kipnis (1967). By contrast, others at this time were looking for adrenergic receptors elsewhere. For example, Honig and Stam (1967) proposed that adrenaline acted directly on the contractile proteins of heart muscle.

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17. Yamasaki et al. (1974); Reuter (1974). See also Tsien (1974). 18. Murad et al. (1962). 19. Mansour et al. (1960); Kakiuchi and Rail (1968). 20. McAfee et al. (1971); Kebabian and Greengard (1971). 21. Kebabian et al. (1972). 22. Clement-Cormier et al. (1974). 23. Ashman et al. (1963). 24. Krebs and Fischer (1956). 25. Krebs et al. (1959). 26. Ibid., p. 2872. 27. Posner et al. (1965). 28. DeLange et al. (1968). 29. Walsh et al. (1968). 30. Sutherland and Wosilait (1955); Riley et al. (1968). 31. Friedman and Lamer (1963). 32. Rosell-Perez and Lamer (1964). 33. Huijing and Lamer (1966). 34. Solderling et al. (1970). 35. Walsh et al. (1968). 36. Linn et al. (1969). 37. Kuo and Greengard (1969), p. 1354. 38. Heald (1957, 1962). 39. Miyamoto et al. (1969); Ueda et al. (1973). 40. Weller and Rodnight (1970); Reddington et al. (1973). 41. Gordon et al. (1977); Teichberg et al. (1977). 42. Huganir et al. (1986). 43. Miles et al. (1987). 44. Kaczmarek et al. (1980); Castellucci et al. (1980). 45. Osterrieder et al. (1982). 46. For example, Weller and Rodnight (1971); Maeno and Greengard (1972). 47. Ingebritsen and Cohen (1983). 48. For example, Robison et al. (1967). 49. Rodbell et al. (1971b). 50. Rodbell et al. (1971a). In retrospect, it seems that RodbelFs ATP contained low levels of contaminating GTP. Consequently, only with low concentrations of ATP (and thus negligible adventitious GTP) did the requirement for added GTP become obligatory. 51. Ibid., p. 1877. 52. Rodbell et al. (1974); Londos et al. (1974). 53. For example, Bockaert et al. (1972); Wolff and Cook (1973). 54. Cassel and Selinger (1976). 55. Cassel and Selinger (1978). 56. Pfeuffer (1977). 57. Pfeuffer (1979). 58. By this time the reigning conception of membrane structure was the "fluidmosiac" model, which depicted proteins free to move laterally through the fluid lipid bilayer (see Robinson, 1997). Consequently, G-proteins would be able to move in the plane of the membrane from receptor to adenylate cyclase. 59. L. S. Goodman and A. Gilman's The Pharmacological Basis of Therapeutics, was first published in 1941 (and continues to the present, although with different authors).

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MECHANISMS OF SYNAPTIC TRANSMISSION

60. This reconstitution technique was previously applied to receptor and adenylate cyclase by Orly and Schramm (1976). 61. Northup et al. (1980). 62. Hanski et al. (1981); Robishaw et al. (1986). 63. Hildebrandt et al. (1984). 64. Fungetal. (1981). 65. Manning and Gilman (1983). 66. Hamm (1998). 67. Hanski et al. (1981); Northup et al. (1982). See also Pfeuffer (1979). 68. Bokoch et al. (1983, 1984). Independently, Birnbaumer obtained similar results (Hildebrandt et al. 1983). Arguments for the existence of inhibitory G-proteins existed earlier (for example, Londos et al., 1981). 69. For example, Cassel and Pfeuffer (1978); Katada and Ui (1982). 70. Arguments for G; coupling to o^-adrenergic receptors included reconstitution experiments by Cerione et al. (1986). See also Cotecchia et al. (1990). 71. Arguments for Gj coupling to muscarinic receptors included reconstitution experiments by Florio and Sternweis (1985). 72. Pfaffinger et al. (1985); Breitwieser and Szabo (1985). 73. Logothetis et al. (1987). However, these experiments were disputed for some time: see Birnbaumer et al. (1990); Schneider et al. (1997). 74. Rasmussen (1970), p. 409. 75. For historical accounts see Needham (1971); Robinson (1997). 76. Heilbrunn and Wiercinski (1947); Sandow (1952). 77. For example, Porter and Palade (1957); Franzini-Armstrong and Porter (1964); Peachey (1965). 78. For example, Huxley and Taylor (1958); Freygang et al. (1964). 79. Costantin et al. (1965); Winegrad (1965). 80. Hasselbach and Makinose (1961); Ebashi and Lipmann (1962). 81. Douglas and Rubin (1961). 82. Ibid., p. 40. 83. Douglas and Poisner (1962). 84. For example, Douglas and Poisner (1963, 1964). 85. Katz and Miledi (1965). In these and other electrophysiological studies cited here, the release of neurotransmitter was inferred from the presence of postsynaptic potentials. Earlier proposals for Ca2+ involvement include Hodgkin and Keynes (1957) and Birks and Macintosh (1957). 86. Katz and Miledi (1969). Hodgkin and Keynes (1957) identified a Ca2+ current associated with the squid action potential, but this Ca2+ current was relatively tiny. Fatt and Ginsborg (1958) showed that invertebrate muscle action potentials included a predominant Ca2+ current. 87. See Hagiwara and Byerly (1981); Bean (1989). 88. Kohlhardt et al. (1972); Spedding (1985). 89. See Hodgkin and Keynes (1957). 90. For a historical account, see Robinson (1997). 91. Schatzmann (1966). 92. Reuter asnd Seitz (1968); Blaustein and Hodgkin (1969). 93. See Melloni and Pontremoli (1989); Marty (1989). 94. Ebashi and Kodama (1965); Ebashi et al. (1967). 95. Greaser and Gergely (1971). 96. Collins et al. (1973).

Second Messengers (1951-1990)

19?

97. Cheung (1970). See also Kakiuchi and Yamazaki (1970). 98. Teo et al. (1973). 99. Dedman et al. (1978). Watterson et al. (1980) reported the complete sequence. 100. Cheung et al. (1978). 101. See Cheung (1981); Means et al. (1982). 102. Schulman and Greengard (1978); Kennedy and Greengard (1981). See also Goldenringet al. (1983). 103. See Colbran and Soderling (1990). 104. See Campbell (1983); Hille (1992). 105. For a historical account, see Robinson (1997). 106. M.R. Hokin and Hokin (1953, 1954). 107. L.E. Hokin and Hokin (1955). 108. L.E. Hokin and Hokin (1959). 109. M.R. Hokin and Hokin (1964a). 110. For example, Fisher and Mueller (1968). 111. M.R. Hokin and Hokin (1964b). 112. Durell et al. (1968, 1969). 113. Michell (1975). 114. Ibid., p. 137. 115. Berrdige showed that the rate-limiting step for Ca2+ appearance in the saliva was Ca2+ influx into the gland cells; hence the rate of apppearance—which could be monitored continuously—equaled the rate of influx. Berridge also equilibrated the gland cells with radioactive inositol, which labeled the phosphatidylinositol; hence the subsequent release of radioactive inositol—which also could be monitored continuously— reflected the hydrolysis of phosphatidylinositol. 116. Fain and Berridge (1979). 117. Berridge (1983). 118. Streb et al. (1983). 119. Berridge (1983); Storey et al. (1984). 120. Supattapone et al. (1988); Ross et al. (1989). 121. Ryu et al. (1987). See also Rhee et al. (1989). 122. For example, Cockcroft and Gomperts (1985); Smrcka et al. (1991); Taylor et al. (1991). 123. Inoue et al. (1977). 124. Takai et al. (1979a). 125. Takai et al. (1979b). 126. See Kaczmarek (1987); Nishazuka (1988). 127. For example, Tanaka et al. (1984); DeRiemer et al. (1985). 128. Takai et al. (1979b). 129. Kaibuchi et al. (1983).

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8 RECEPTOR STRUCTURES AND RECEPTOR FAMILIES (1983-1990)

Molecular Biology ana Recomiinant DNA Techniques

In 1953 James Watson and Francis Crick proposed a double-helix model for DNA, with helices linked by pairings between the complementary nucleotide bases of each strand. The elaborations and extensions that followed in subsequent decades included demonstrations of how DNA specified protein structure.1 Successive triplets of the four nucleotide bases that constitute DNA (adenine, guanine, thymine, and cytosine) designate successive amino acids of the encoded protein. To direct protein synthesis, the sequence of nucleotide base triplets is first "transcribed" into messenger RNA (mRNA) bearing the complementary2 sequence of nucleotide bases. The mRNA then migrates from nucleus to cytoplasm, where its complementary nucleotide triplets are "translated" into the sequence of amino acids.3 These triplets direct the stepwise linking of amino acids into a peptide chain, with that assembly catalyzed by ribosomal enzymes. Information thus flows from DNA to mRNA to protein. These understandings led also to the development of new techniques, including powerful methods for examining protein structure and altering it in highly specific ways. A range of restriction endonucleases were identified that cleave DNA at distinct sites specified by characteristic sequences of nucleotide bases. Various restriction endonucleases could thus generate specific fragments of 199

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MECHANISMS OF SYNAPTIC TRANSMISSION

DNA. Also identified were DNA ligases that join together free ends of DNA strands. Recombinant DNA could therefore be constructed by cutting native DNA with restriction endonucleases and assembling fragments into new strands with DNA ligases. Novel strands of DNA with desired sequences could be formed from existing fragments or by incorporating short stretches of nucleotides (oligonucleotides) synthesized chemically or enzymatically. Important tools for generating multiple copies of a particular stretch of DNA were bacterial plasmids, strands of DNA separate from the bacterial chromosome and serving as accessory chromosomes. These plasmids are replicated during bacterial division like the single bacterial chromosome. Bacterial plasmids could be isolated, their DNA cut by restriction endonucleases, pieces of new or altered DNA inserted with ligases, and the plasmid then replaced in the bacteria ("transformation"). When a single bacterium containing a plasmid with a particular strand of DNA undergoes successions of cell division, its progeny—a colony of cells grown in vitro—will contain that DNA. These progeny are identical genetically and thus are "clones"; in laboratory parlance the DNA is "cloned." This procedure provides a means for generating macroscopic quantities of a selected or constructed DNA molecule. Among the various "expression" procedures for synthesizing protein encoded by DNA, the Xenopus oocyte system is particularly relevant here. First, the DNA is transcribed into complementary mRNA. For this, tissue culture cells may be "transfected" with plasmid cDNA, which then directs a massive transcription. (In later experiments the mRNA was often synthesized by cell-free systems.) Second, the resulting mRNA is extracted from the transfected cells and injected into oocytes of the frog Xenopus laevis, which readily synthesize protein from exogenous mRNA (translation or expression). The properties of this newly synthesized protein, such as a neurotransmitter receptor that oocytes ordinarily do not express—can then be studied in the injected cell.

Nicotinic Cholinergfic Receptors

Earlier investigators isolated and purified nicotinic cholinergic receptors from fish electric organs, describing four protein subunits present as a pentameric a$y8 complex that surrounded a central ion-conducting channel (chapter 6). Specifying the chemical identity, however, requires knowledge of the sequence in which amino acids appear in this protein and their spatial relationship (i.e., the three-dimensional structure of the protein). Mechanistic understandings require, in addition, an explanation of how acetylcholine s binding to a subunits alters the overall structure and its conductivity: triggering the transition from a closed, resting configuration to an open, active one.

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Amino Acid Sequence

In 1980 Michael Raftery in Pasadena reported the amino acid sequences of the first 50 or so amino acids from each of the isolated a, /3, y, and 8 subunits.4 These he determined by stepwise cleavages of the terminal amino acid from a chain, identifying the liberated amino acid after each cycle. The identified sequences, however, represented only a tenth of the total, and that procedure could not progress significantly further. Although the cleavage efficiency at each cycle is high, even tiny errors are multiplied progressively. Frederick Sanger devised an approach for sequencing insulin in the early 1950s that used enzymatic and chemical means to cleave the peptide chain at specific points, producing various overlapping fragments short enough to be sequenced stepwise. Piecing together the individual sequences then revealed the overall sequence. Sangers method, however, was laborious. It also was not feasible with many intrinsic membrane proteins. Their transmembrane segments were highly hydrophobic (necessary for insertion within the surrounding lipid bilayer), and this hydrophobicity limited their compatibility with solvents required for separatory and analytical procedures. In 1977 Sanger in Cambridge, UK and Allan Maxam and Walter Gilbert in Cambridge, Mass., independently developed rapid methods for determining the sequence of nucleotide bases along a strand of DNA.5 The amino acid sequence can then be read from the DNA sequence, following the rules for which nucleotide triplet specifies which amino acid. These techniques greatly facilitated protein sequencing, but they required an initial identification of the particular DNA sequence encoding that protein. Shosaku Numa (Fig. 8-1A) soon applied these methods to sequencing peptide hormones, endorphins, and their precursors. Numa had received his M.D. from Kyoto in 1953, and after three years of clinical training he was awarded a Fulbright fellowship to work in biochemistry at Harvard University. After a further three years examining enzymes of lipid biosynthesis in Munich, he returned to Kyoto, where he continued with lipid biochemistry for a decade and a half. But with the advent of molecular biological approaches, Numa turned his efforts to these techniques, which he then adapted to studying nicotinic receptors. As the initial step in this venture, he reported in 1982 the amino acid sequence for the a subunit.6 First, Numa isolated the mRNA from Torpedo electric organs, which specifies all the proteins being produced by the organ. From these mRNA molecules he synthesized, enzymatically,' their complementary DNA (cDNA) sequences. The resulting cDNA fragments thus encoded all the proteins being produced in the organ. Numa next created a "cDNA library" by inserting all these cDNA fragments randomly into plasmids, which he then incorporated into bacteria so that an individual bacterium received randomly a particular

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MECHANISMS OF SYNAPTIC TRANSMISSION

FIGURE 8-1. A, left, Shosaku Numa (1927-1992). B, right, Robert]. Lefkowitz (1943-). (A courtesy of Osamu Hayashi. B courtesy of R. J. Leftowitz.)

fragment. The progeny of each transformed bacterium—its clones—would all contain plasmids bearing a certain fragment of cDNA. To find which bacterial clones contained the cDNA for the a subunit, Numa then screened the cDNA library with probes based on Raftery's partial determination of the sequence. This screening involved searching, through hybridization experiments,8 for complementary matches between the nucleotide base sequences of clone and probe. One probe represented the pentapeptide including amino acids 25-29 of the a subunit. However, more than one nucleotide triplet may encode a given amino acid (the code is "degenerate"), so Numa was forced to use 32 different DNA oligonucleotides to represent all the ways that these five amino acids could be encoded. From 200,000 bacterial clones in the library, this probe selected 57. A second probe, representing amino acids 13-18 from Raftery's partial sequence and also containing 32 alternative representations, selected 20 clones from the first 57: these clones thus contained DNA complementary to both probes. Numa then sequenced the DNA from two of these clones.9 With this information, plus Raftery's determination of the first 54 amino acids, Numa could then deduce the sequence of all 437 amino acids in the a subunit.10 Numa

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203

identified four stretches of highly hydrophobic amino acids long enough to form an a-helix that could span the membrane's lipid bilayer, and he suggested that these might represent transmembrane segments of the receptor. In 1983 Numa published sequences for the /3, y, and 8 subunits as well.11 The sequences of all four had conspicuous similarities, including four likely transmembrane segments, implying both a common evolutionary ancestry and a common structural organization. The following year Numa described the expression in Xenopus oocytes of mRNA for each of these subunits. The four were sufficient for administered acetylcholine to evoke its characteristic electrical response, demonstrating that functional receptors were now present.12 By contrast, omitting the mRNA for any one of the subunits markedly reduced the response. Electric organs are closely related to muscle, and their nicotinic receptors are physiologically and pharmacologically similar. In 1983 Numa applied these techniques to nicotinic receptors from mammalian muscle, demonstrating that their amino acid sequences were closely similar as well.13 On the other hand, neural nicotinic receptors are functionally distinguishable, and in 1986 Jim Patrick in La Jolla showed that the sequences were distinguishable also.14 Patrick used probes from the a subunit of muscle nicotinic receptors to screen a neural cDNA library under "low stringency" conditions (removing less vigorously probes loosely hybridized to the target and thereby identifying cDNA that was not fully complementary to the probe). The selected cDNA encoded a protein generally similar to the a subunit of muscle nicotinic receptors, although there were extensive differences in two regions of the sequence.

Structure ana Function

Delineating the receptor's three-dimensional structure and its functional motions were still more formidable challenges. The standard approach for determining protein structures involved X-ray crystallography. Crystallizing intrinsic membrane proteins turned out to be quite difficult, however, and the first success—providing a resolution to 3 A for a protein involved with photosynthesis—did not occur until the mid-1980s (a feat that merited a Nobel Prize).15 Unfortunately, that achievement did not point to easy methods applicable readily to other membrane proteins. Electron microscopy remained the principal tool for revealing structures. During the 1980s (and continuing through the 1990s) Nigel Unwin extended these efforts significantly, attaining resolution to 17 A by 1988.16 This success came in part from progressively improved preparations, begun in Palo Alto and continued after his return to Cambridge. Unwin used postsynaptic membranes from Torpedo electric organs that formed flattened tubes, with receptors then organized into helical arrays; these he suspended in ice films for low-

204

MECHANISMS OF SYNAPTIC TRANSMISSION

temperature electron microscopy. Unwins success was also founded on exhaustive analyses, with images reconstructed statistically from the diffraction patterns of thousands of receptor molecules. The derived representation provided convincing views of the receptor's contours, including cross-sections depicting access routes to and from the transmembrane region (Fig. 8-2A). Nevertheless, the course of the channel across the bilayer thickness could not be defined at this resolution, nor could the individual amino acids of the protein be identified. Meanwhile, Numa was providing complementary information about the receptors functional structure using other new techniques of molecular biol-

FIGURE 8—2. Structures of ligand-gated ion channels. A. Section through the nicotinic receptors of Torpedo electric organ, determined by electron microscopy/image reconstruction. The figure depicts large lobes surrounding the extracellular access route to the transmembrane channel (not resolved) as well as the smaller lobes surrounding the intracellular egress route for Na + entering the cytoplasm. (K + moves along this route in the opposite direction.) The elliptical image below the receptor was identified as an extraneous protein (see Mitra et al., 1989). B. Diagram of the three rings of negative charges flanking the transmembrane channel. A cation (circle enclosing +) is shown at the extracellular mouth of the channel. This diagram is superimposed on Toyoshima and Unwin's recontructed image. The chains of hexagons represent chains of sugars covalently bonded to extracellular regions. C. Common folding pattern for subunits of ligand-gated ion channels, showing the extracellular N-terminal and extracellular C-terminal segments, four transmembrane a-helices that span the bilayer (M2 is shaded), and the cystine loop in the N-terminal segment. (A from Toyoshima and Unwin [1988], Fig. 4, reprinted by permission of Nature. © 1988, Macmillan Magazines, Ltd. B from Hucho and Hilgenfeld [1989], Fig. 5C, courtesy of the Federation of European Biochemical Societies. C from Ortells and Lunt [1995], Fig. 1, © 1995, by permission of Elsevier Science.)

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ogy: forming "chimeras," by joining strands of DNA from different sources to synthesize composite structures, and effecting "site-directed mutagenesis," by changing the DNA coding for particular amino acids (omitting, adding, or substituting). Numa expressed the altered DNA in Xenopus oocytes to produce the altered receptors, and his collaborator in Gottingen, Bert Sakmann, then examined the conductances of single nicotinic receptors in these oocytes using patch electrodes (chapter 6). Substituting 8 subunits from mammalian receptors for the 8 subunits from fish affected receptor conductances. To find out which regions of this subunit determined the divergent properties, Numa and Sakmann replaced various regions of the fish 8 subunit with corresponding regions of the mammalian muscle 8 subunit: they formed chimeras by splicing the cDNA for the respective segments. In 1986 Numa and Sakmann reported that the rate of cation flow through the receptor channel was sensitive to the source of one of the proposed transmembrane segments in the 8 subunit (the second in the amino acid sequence and designated "M2").17 Cation flow was also sensitive to variations in the adjacent sequence linking M2 to the next putative transmembrane segment. They suggested that negatively charged amino acids in the linking segment were "located near the mouth of the channel, attracting . . . cations toward the channel."18 Two years later Numa and Sakmann described an extension of these studies, now using site-directed mutagenesis.19 They identified three clusters of negatively charged amino acids flanking M2 (in a, j3, and y subunits as well as in 5), which apparently served as "major determinants of the channel conductance," since substitution with uncharged amino acids sharply affected electrical responses.20 They located a ring of negatively charged amino acids at each end of the channel, plus an intermediate ring "positioned between [to] form a narrow . . . constriction" that would serve as a selectivity filter, restricting the passage to cations of certain diameters.21 Henry Lester in Pasadena, also using site-directed mutagenesis, found in 1988 that substituting a nonpolar amino acid (alanine) for polar serines in the M2 segments decreased channel conductance. He concluded that the serines lay within the aqueous cationconducting channel.22 A third approach used compounds that inhibit receptor conductance by (apparently) blocking the central channel. Jean-Pierre Changeux in Paris selected an inhibitor that was photoactive to label the receptor: after allowing the inhibitor to bind, he activated it with a flash of light, causing it to form covalent bonds with any portions of the receptor then adjacent to it. Changeux found that the amino acids linked to the inhibitor lay on M2 segments of a, /3, y, and 8 subunits.23 Models based on these studies depicted M2 segments of each subunit lining the narrow channel as it crossed the bilayer thickness, with access to this

206

MECHANISMS OF SYNAPTIC TRANSMISSION

channel controlled by rings of negatively charged amino acids that accepted positively charged cations (Fig. 8-2B). Models also included sites for acetylcholine on the N-terminal segment of a subunits.24 But the mechanism by which acetylcholine s binding alters the structure—and in precisely what ways— remained undetermined in 1990. Ligfana-Gatea Ion Channels

Physiological and pharmacological studies had established a group of "ligandgated ion channels," so named because the binding of neurotransmitters (ligands) opened gates to allow ion flow through receptor channels (chapter 6). This group included receptors for GABA, glycine, and glutamate as well as nicotinic receptors for acetylcholine; in 1988 one class of serotonin receptors, SHTs, was shown to function as a ligand-gated ion channel also.25 But determining whether or not this common function reflected a common structure required the newer approaches. GABA ana Glycine Receptors

In 1987 Eric Bernard in Cambridge reported the sequences for two subunits of the GABAA receptor and Heinrich Betz in Heidelberg that for one subunit of the glycine receptor.26 They, too, determined these sequences by cDNA techniques, using oligonucleotide probes based on partial amino acid analyses of purified subunits. From their similar results they now stressed the chemical and structural similarities to nicotinic receptors and to each other: they argued for the existence of a superfamily of related receptors. Indeed, there was about 50% homology27 between GABAA and glycine receptors and about 25% homology with nicotinic receptors. All subunits were of similar size and all had four hydrophobic stretches likely to be transmembrane segments (Fig. 8-2C). In fact, the homologies lay chiefly in the four putative transmembrane segments, notably in M2. All had lengthy, extracellular N-terminal domains where the binding sites for their neurotransmitters lay. These domains also included two cysteines that were 15 amino acids apart, apparently forming a loop through disulfide bonding between these cysteines., Differences among these receptors are to be expected, of course, for they bind different ligands selectively. Moreover, GABA and glycine are inhibitory neurotransmitters that promote Cl~ fluxes through their receptors, rather than the Na + and K + fluxes of excitatory nicotinic receptors. Correspondingly, the GABAA and glycine receptors had a cluster of positively charged amino acids at each end of the presumed channel,28 where nicotinic channels had negatively charged amino acids. Based on the pattern of nicotinic receptors, the GABAA and glycine receptors should consist of five subunits arranged around a central channel, although

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the precise number of subunits for the GABAA receptor was debated for many years. In any case, there appeared to be fewer kinds of subunits than for the electric organ nicotinic receptor, although the GABAA receptor had multiple varieties of each kind of its subunits.29 Glutamate Receptors

Certain glutamate receptors also function as ligand-gated ion channels, and these had been subdivided pharmacologically according to their differential sensitivities to characteristic agonists, including kainate, quisqualate, and N-methylD-aspartate (NMDA). It seemed likely, therefore, that these receptors would be structurally similar to nicotinic, GABAA, and glycine receptors. These glutamate receptors had not been purified, however, so there were no partial amino acid sequences available and thus no guides for constructing oligonucleotide probes. Michael Hollmann in La Jolla instead sequenced the kainate receptor in 1989 by a different cDNA approach: "expression cloning."30 First he formed a cDNA library from rat brain mRNA, representing all proteins synthesized in the brain and consisting of 800,000 clones. He divided this library into 18 sublibraries of 44,000 clones each, and he then transcribed the cDNA of each sublibrary in vitro, obtaining 18 pools of the corresponding mRNAs. He injected each of these pools into a separate Xenopus oocyte, testing each oocyte for the kainateinduced depolarizations that would signal the expression of functional receptors. Hollmann next subdivided the positive cDNA sublibrary successively, testing pooled mRNA from 4000 clones, then 400 clones, and finally 40 clones. At this point he tested individually the mRNA from 12 clones of that final pool, selecting clones having the longest stretches of cDNA. The mRNA from only one of these clones elicited responses to kainate, and Hollmann sequenced the corresponding cDNA, deducing an encoded protein with a molecular weight of 100 kDa. This protein was twice the size of the subunits from other known ligandgated ion channels. Moreover, there was no cysteine loop and "no significant over-all homology" with the other receptors, although Hollmann did identify four "candidates" for transmembrane segments.31 At best, it seemed that glutamate receptors were "distant cousins" of the known ligand-gated ion channels.32 Sequences of other ligand-gated glutamate receptors, including NMDA receptors,33 were determined soon afterward, and all these showed marked similarities to this kainate receptor. Structural ana Evolutionary Superramilies

Strong similarities in the sequences of nicotinic, GABAA, and glycine receptors—and among the subunits of a given receptor—implied not only a close structural and functional pattern (Table 8-1) but also a common ancestry: an

208

MECHANISMS OF SYNAPTIC TRANSMISSION TABLE 8-1. Receptor Classes

LIGAND-GATED ION CHANNELS

G-PROTEIN COUPLED RECEPTORS

nicotinic cholinergic

muscarinic cholinergic

GABAA

GABAB

glycine

a- and /3-adrenergic

5HT3

5HTi and 5HT2 dopamine

glutamate kainate quisqualate NMDA

metabotropic glutamate

evolutionary descent from a primitive precusor.34 (The 5HTs receptor was sequenced by expression cloning in 1991;35 its similarities placed it within this superfamily. The 5HTs receptor conducts cations, and it is related more closely to nicotinic receptors than to the anion-conducting GABAA and glycine receptors. By contrast, further studies in the 1990s placed the glutamate receptors in an unrelated superfamily. These studies also argued for only three transmembrane segments in glutamate receptor subunits.)

Aarenergfic Receptors Robert Lefkowitz (Fig. 8-1B) had isolated /3-adrenergic receptors and reconstituted the purified proteins to form functional receptors—capable of stimulating adenylate cyclase—as one step in his comprehensive study of adrenergic processes (chapter 6). Lefkowitz had received his M.D. from Columbia University in 1966 at age 23, and after clinical training in internal medicine, he spent two years in research at NIH before further training in cardiology at Harvard University. In 1973 he was appointed associate professor of medicine and assistant professor of biochemistry at Duke University, where he initiated an active and productive research program. Lefkowitz now obtained partial amino acid sequences from purified fizadrenergic receptors and used these sequences to construct oligonucleotide probes for screening a hamster library, identifying in 1986 the DNA that encoded a protein containing 418 amino acids with a molecular weight of 46 kDa.36 The deduced sequence contained likely sites—"consensus sequences," since these appeared at demonstrated occurrences in other proteins—for phosphorylation by protein kinases and for glycosylation (covalent attachment of

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sugars). Most notable, however, were seven stretches of hydrophobic amino acids, each long enough to form a-helices that could span the membrane's lipid bilayer. Lefkowitz pointed out that these putative transmembrane segments were reminiscent of the seven putative transmembrane segments recently deduced in the amino acid sequence of rhodopsin and the seven transmembrane a-helices visualized in bacteriorhodopsin.37 (Rhodopsin is the protein in retinal rod membranes that bears the light-sensitive pigment retinal and that couples with a G-protein to initiate its functional response to light. Bacteriorhodopsin is a retinal-containing, light-driven membrane pump that extrudes H + from certain bacteria.) Lefkowitz suggested that (3 agonists might bind to the receptor as retinal binds to rhodopsin, in both cases to activate a coupled G-protein. The next year, 1987, Lefkowitz reported the sequence of human /^-receptors, presenting a model with seven transmembrane segments (Fig. 8-3).38 He also reported the sequence for human jSi-receptors: this protein contained 477 amino acids (51 kDa) and had 54% homology with human /3£.39 The conserved features between /3\, {$%, rhodopsin, and recently sequenced muscarinic receptors (see below) included seven likely transmembrane segments containing notable sequence homologies, plus consensus sites for phosphorylation on intracellular domains and for glycosylation on extracellular domains. Lefkowitz expressed fi\- and /^-receptors in Xenopus oocytes, obtaining receptors specific for (3i and (32 agonists and antagonists.40 Next, he constructed chimeras of /3i//32-receptors and expressed these also in oocytes. The fourth transmembrane segment controlled specificity for (3\ vs. {$2 agonists, whereas the sixth and seventh segments controlled specificity for (3i vs. /3£ antagonists.41 Both /3r and /^-receptors bind Gs, which stimulates adenylate cyclase, whereas o^-receptors bind Gj, which inhibits it (chapter 7). So Lefkowitz obtained sequences for o^-receptors by similar means and then constructed a 2/Pz chimeras.42 The ability to couple with Gs lay in the third cytoplasmic loop (between transmembrane segments 5 and 6). He refined this localization using site-directed mutagenesis, implicating portions of the second intracellular loop and of the intracellular tail as well as of the third intracellular loop.43 The ai-receptors activate, through a different G-protein, a phospholipase C that splits phosphatidylinositol-tephosphate into two second messengers, inositol-fnsphosphate and diacylglycerol (chapter 7). Lefkowitz determined the sequence of this adrenergic receptor also, although he did not explore its properties at that time.44 These studies established a characteristic structure having seven transmembrane segments, with ligands binding in a pocket formed within the array of transmembrane a-helices. Other approaches also contributed, including examinations of ligand-binding sites using reactive analogs that would label adjacent amino acids.45 Intracellular loops evidently should be involved with coupling

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MECHANISMS OF SYNAPTIC TRANSMISSION

FIGURE 8-3. Amino acid sequence and folding pattern for the human /32-adrenergic receptor. The amino acid sequence is depicted in the one-letter symbols for the amino acids (A for alanine, R for arginine, N for asparagine, etc.). Black circles with white letters indicate sites where the human and hamster receptors differ. The seven transmembrane a-helices, containing hydrophic amino acids, are shown, as well as two sites for glycosylation on asparagines in the N-terminal extracellular segment. (From Kobilka et al. [1987], Fig. 3, courtesy of Robert J. Lefkowitz.)

to G-proteins, but how ligand binding activated the coupled G-protein remained undetermined.

G-Protein Coupled Receptors

In 1987 Lefkowitz proffered the "interesting speculation . . . that the seven membrane-spanning . . . feature might be common to a l l . . . membrane receptors . . . coupled to G proteins."46 The next few years justified his generaliza-

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tion and affirmed the common name for this group (alternative names include "heptahelical receptors" and "metabotropic receptors").

Muscarinic Cnolinergic, Serotonergic, ana Dopaminergic Receptors

The same year that Lefkowitz reported the first sequence for an adrenergic receptor, 1986, Numa reported the first sequence for a muscarinic cholinergic receptor.47 Muscarinic receptors had just been purified from pig brain, so Numa obtained partial amino acid sequences by chemical means, produced oligonucleotide probes based on these sequences, and then screened a pig brain cDNA library with these probes. He identified the cDNA for a 51 kDa protein containing 460 amino acids, with sequence homologies to rhodopsin and the /?2adrenergic receptor, including seven likely transmembrane segments. Expression in Xenopus oocytes produced characteristic electrical responses to administered acetylcholine that were blocked by atropine.48 At that time pharmacological approaches distinguished two major classes of muscarinic receptors, MI and MZ; the receptor classes also had different distributions in the brain. Numa identified his protein as MI based on the corresponding localization of its mRNA in the brain as well as on the binding of a diagnostic MI antagonist to oocytes expressing the cDNA. The cDNA representing this receptor did not, however, encode the sequences of certain peptide fragments present in proteolytic fragments of muscarinic receptors purified from pig brain. Numa concluded that these aberrant sequences might belong instead to M£ receptors present along with MI in his receptor preparation. Accordingly, he synthesized oligonucleotide probes corresponding to the aberrant sequences and screened cDNA libraries from brain and from heart, where M2 receptors are prominent. The identified cDNA encoded a protein of 466 amino acids (52 kDa) having sequence homology to MI and containing seven likely transmembrane segments.49 Localization of mRNA for this protein in the brain and heart indicated that it was M£. Daniel Capon in San Francisco identified the cDNA encoding this receptor independently, reporting the next year, 1987, that expression of this cDNA in tissue culture cells led to the binding of a characteristic antagonist to M£ receptors.50 Also in 1987 Tom Bonner in Bethesda and Capon independently identified two additional muscarinic receptors, MS and M^51 They used oligonucleotide probes based on conserved sequences for screening libraries under conditions of lowered stringency. The four encoded proteins that they detected—corresponding to MI through M4—had similar sequences but distinctive cytoplasmic loops; when expressed individually, the proteins bound cholinergic ligands with distinguishable affinities. (Their approach, searching for previously unknown proteins through low-stringency screening, represented a powerful exploratory mode then coming into prominence.)

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In 1988 Capon showed that these receptors were coupled to particular second messenger systems.52 He expressed each individually in tissue culture cells that otherwise lacked muscarinic receptors: administering cholinergic agonists then inhibited cAMP production in cells expressing M£ or M4 but increased phosphatidylinositol-fozsphosphate hydrolysis in cells expressing MI and Ma.53 Bonner identified a fifth subtype in 1988; it also increased phosphatidylinositol-£>isphosphate hydrolysis.54 During these years similar approaches revealed sequences for serotonin (SHTiA, 5HTic, and 5HT2) and dopamine (D£) receptors.55 These receptors, too, had sequence homologies, seven likely transmembrane segments, and responses mediated through G-protein coupled systems: SHTiA and Dg affecting cAMP production and 5HTic and 5HT2 phosphatidylinositol-tephosphate hydrolysis.

Structural ana Evolutionary Superramily

Common features—sequence homologies, seven transmembrane segments, intracellular phosphorylation and extracellular glycosylation sites, and signalling mediated through coupled G-proteins—indicated a structural and functional superfamily (Table 8-1) as well as an evolutionary one. These genetic, structural, and functional analyses thus uncovered unifying generalizations despite the plethora of neurotransmitters and their receptor subtypes. (Continued studies in the 1990s added further members to this superfamily, including GABAfi and metabotropic glutamate receptors, plus receptors for enkephalins and other peptides.)

Receptor Regulation

Earlier studies demonstrated that cells could adjust the responses of their receptors, often in a compensatory, homeostatic manner. One well-recognized process was desensitization, a diminishing response despite continued stimulation. With some receptors this desensitization represented an obligatory conformational step between open and resting stages (chapter 6). Chronic administration of agonists could, however, alter responses in more complex fashions. For example, adding /3-adrenergic agonists elevated cellular cAMP levels acutely, but the cAMP levels then plateaued or even fell despite the continued presence of agonist. Plausible explanations invoked feedback loops. In this case, /3-adrenergic agonists would elevate cAMP levels and thereby activate protein kinase A; protein kinase A would phosphorylate the receptor and thus diminish its response. When cAMP levels returned to basal levels, protein phosphatases would dephosphorylate the receptor, restoring its basal activity.

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In agreement with this scheme, Lefkowitz correlated the desensitzation of j8-adrenergic receptors with the receptors' phosphorylation by protein kinase A, and the desensitization of ai-adrenergic receptors with their phosphorylation by protein kinase C.56 Phosphorylations by protein kinases A and C, however, affect not only the receptor being activated. Other cellular constituents were phosphorylated, too, including other receptors (e.g., protein kinase A will phosphorylate nicotinic cholinergic receptors, reducing their responses as well).5' Thus chronic administration of a neurotransmitter could modify responses not only at its own receptors but also at receptors for other neurotransmitters. Contrasted with such "heterologous desensitization" was receptor-specific "homologous desensitization."58 In 1986 Lefkowitz described a kinase that phosphorylated—and inactivated—/3-adrenergic receptors preferentially.59 Moreover, this kinase phosphorylated only /3-adrenergic receptors that were actually binding agonists. Evidently, the agonist induced the necessary conformation in its receptor to make the receptor susceptible to this phosphorylation. The desensitization that followed this agonist-dependent phosphorylation, however, required the presence of another protein, /3-arrestin, which Lefkowitz identified in 1990.60 (This process paralleled one described earlier for rhodopsin. Rhodopsin kinase phosphorylates light-activated rhodopsin and thereby promotes its binding to arrestin; bound arrestin then blocks further light-activated responses. Studies continuing in the 1990s grouped these kinases into a family whose members phosphorylated various receptors specifically, including receptors for acetylcholine, dopamine, serotonin, and enkephalin.61) A second means for decreasing responses involved sequestering the receptors within the cell.62 For example, administering adrenergic agonists reduced the number of j8-receptors on the cell surface, as measured by the binding of a labeled ligand that did not penetrate the cell membrane. The sequestered receptors could be recovered in a membrane-bound fraction, separate from the fraction containing surface receptors, after density-gradient centrifugation of homogenized cells. But the total number of receptors, measured in broken cells or by using ligands that penetrate membranes, did not change. There appeared to be two distinct fates awaiting these sequestered receptors. In some cases the sequestered receptors appeared to be "resensitized" and returned to the surface.63 In other cases receptors disappeared, so that new protein synthesis was required to replenish the cellular complement.64 "Downregulation" refers to the loss of receptors, as opposed to sequestration in which the total number is unchanged. Accounts of downregulatlon during these years argued variously for receptor degradation and/or decreased synthesis.65 Both processes were plausible, for receptors, like other cell proteins, are continuously broken down and replaced. Different studies stressed one or the other of these mechanisms, depending on the cell type, receptor, and ago-

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nist studied. Although no general account was possible by 1990, interest was growing in mechanisms for altering the cellular content of mRNAs encoding these receptors. For example, direct measurements of the mRNA for /3-receptors revealed agonist-induced decreases that paralleled downregulation.66 Molecules of mRNA are also continuously broken down and replaced, so decreased mRNA levels could be due to decreased transcription or increased degradation. As the decade closed several studies favored agonist-mediated "destabilizations" of mRNA—a shortening of their functional lifetimes—without a change in transcription rates.67 "Upregulation," on the other hand, refers to a gain of receptors. Prolonged administration of receptor antagonists could increase the number of receptors,68 just as prolonged administration of agonists could decrease their number. In some instances the increase was confined to the particular receptor subtypes to which that antagonist bound.69 But broader changes could also occur, as shown by responses to cAMP. Many genes have regions in their DNA where particular proteins bind to enhance the expression of that gene. In 1986 Marc Montminy in La Jolla identified such a site for cAMP-stimulated protein synthesis. He named this region of the gene the "cAMP response element" (CRE). 70 The next year he identified a CRE binding protein (CREB), which was activated through phosphorylation by protein kinase A.71 The sequence thus ran: elevated cAMP => protein kinase A => phosphorylated CREB => activated CRE => mRNA transcription. In 1989 Lefkowitz showed that cAMP enhanced the transcription of mRNA for ^3-adrenergic receptors through such a mechanism.72 Consequently, it would seem that any agonist elevating cAMP levels might increase the synthesis of /3-receptors. By 1990 it was clear that homeostatic processes could attenuate the therapeutic responses to chronically administered agonist or antagonist drugs. Furthermore, even when the agonist or antagonist was highly specific for a given class of receptors, heterologous regulatory mechanisms could extend the drugs' actions to affect other proteins, including receptors for other neurotransmitters.

Conclusions

New techniques of molecular biology rapidly disclosed the amino acid sequences for an expanding roster of neurotransmitter receptors. These sequences identified the receptors chemically, even if they did not immediately reveal the precise mechanisms. Still, two major classes of receptor function as well as structure became apparent. (1) Oligomeric subunits surrounding an ion-conducting pore formed a ligand-gated ion channel that opened when neurotransmitters filled the binding sites. (2) Monomeric proteins with seven transmembrane seg-

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ments formed receptors that activated a coupled G-protein. With the first class, chemical signals (neurotransmitters) were converted to electrical signals. With the second, chemical signals were converted to different chemical signals: G-proteins that then interacted with various enzymes and channels to elicit both chemical and electrical responses. The reductionist program that delineated these structures and functions thus disclosed unanticipated unities characterizing these classes, despite a proliferating catalog of distinguishable receptors.73 These unities, of course, reflected evolutionary relationships. Indeed, such similarities are expected in evolutionary systems: random modifications in an ancestral protein enable new ligands to bind (as in the divergence of serotonergic and nicotinic receptors) and thereby activate a functional component, either similar (as in cation-conducting channels of serotonergic and nicotinic receptors) or modified (as in the appearance of GABAergic and glycinergic anion-conducting channels). This spate of discoveries and realizations was made possible by the development of new methods, just as the great advances in understanding metabolic syntheses and degradations in the 1940s and 1950s followed the introduction of new separatory techniques and radioisotope labeling procedures. The new methods were labor intensive, however, and one further consequence was the creation of large research groups generating multiauthored papers (one cited here had 16 authors). These new methods also provided a new research strategy. As Lefkowitz pointed out,' 4 the old paradigm directed a progression from physiological phenomena to pharmacological characterization to biochemical specification to molecular biological identification and manipulation. But a new paradigm now emerged: probes corresponding to a given protein's amino acid sequence could be used to screen DNA libraries at various degrees of stringency, uncovering genes for related but previously unknown proteins having similar functions as well as similar structures. Moreover, the growing compilations of protein sequences, structures, and functions also facilitated deductions of the corresponding structures and functions when a new sequence was determined. The recognition of conserved motifs in amino acid sequences—again, a characteristic to be expected with evolutionary changes—also aided the assignment of functions to structures. But these new understandings of neurotransmitter receptors were advanced not only by the new techniques and principles from molecular biology and biochemical genetics. Progress was assisted also by concomitant advances in understanding hormonal and sensory receptors and by the deciphering of further intracellular signalling and regulatory processes. Better understandings of receptor structure and function improved understandings of diseases as well (chapter 13), and certain pathologies could now be attributed to precise aspects of receptor structure. For example, the muscular weakness of myasthenia gravis is due to a failure in transmission at neu-

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romuscular junctions: antibodies to muscle nicotinic receptors prevent acetylcholine from causing contractions. The sites to which the disabling antibodies bind were now localized to specified regions on the extracellular N-terminal segment of nicotinic receptor a subunits/ 5 Better understandings also promised better therapies by guiding the quest for more precise interventions with fewer extraneous actions and side effects. The recognition of multitudes of distinguishable receptor subtypes encouraged this quest for specificity through the development of drugs acting on a single subtype, leaving unaffected all other receptors. But dimming this prospect were realizations of the cell's intrinsic homeostatic mechanisms. Although the initial administration of a drug could evoke a particular response, the continued use of that drug often resulted in altered responses. Studies during this decade showed that after chronic administration the compensatory changes could extend to other receptor species also.

Notes 1. For histories, see Fruton (1999); Judson (1979); Morange (1998). 2. "Complementary" refers to the structural fit that allows specific pairing between particular bases on the two strands of DNA (in the double helix) and on a strand of DNA and of RNA (in transcription): guanine with cytosine, thymine with adenine (thymine is absent from RNA, where uracil takes it place, pairing with adenine). 3. Each nucleotide triplet has a specific meaning, although all triplets do not encode an amino acid (e.g., some are "stop codons," signalling the end of a protein). 4. Raftery et al. (1980). 5. Both sequencing techniques rely on introducing base-specific breaks in the DNA. Experimental conditions are chosen to produce only one or a few breaks per chain, but with those breaks randomly distributed—in the population of chains—among the various occurrences of the particular base. With one end of the original chain labeled, the breaks then produce a family of labeled fragments, each beginning at the labeled end and extending to an occurrence of the particular base. These fragments are next separated according to length (by gel electrophoresis, which resolves fragments differring by as little as one nucleotide) and compared with the families of fragments for each of the other three bases, forming a ladder of DNA fragments from which the base sequence is read. 6. Noda et al. (1982b). 7. The enzyme "reverse transcriptase" transcribes mRNA to form cDNA. (The information flow is opposite to that of the "Central Dogma" of molecular biology, from DNA to mRNA.) 8. In hybridization experiments the two strands of the plasmid cDNA are separated and fixed to a matrix. The labeled probe is added and allowed to bind to its complementary sequence. (If the probe is identical to the encoding sequence on one strand of DNA, the probe will bind to the other, complementary DNA strand.) Labeled probe that is not complementary, and therefore does not bind tightly, is washed away vigorously ("high stringency" conditions). Labeled probe that survives such washing thus identifies the plasmid cDNA bearing the complementary sequence.

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9. Numa chose the two clones having the longest inserts and therefore the greatest likelihood of containing the entire representation of the a subunit. 10. In addition, the cDNA encoded a preceding stretch of 24 amino acids that formed a "signal sequence" of amino acids, which assists in inserting the peptide chain into the membrane and is then removed. 11. Noda et al. (1983b, 1983c). Others at that time also sequenced particular subunits by similar methods: y by Claudio et al. (1983); a by Devillers-Thiery et al. (1983). 12. Mishina et al. (1984). 13. Noda et al. (1983a) 14. Boulter et al. (1986). 15. See Robinson (1997). 16. Brisson and Unwin (1984); Toyoshima and Unwin (1990); and intervening papers. 17. Imoto et al. (1986). 18. Ibid., p. 673. 19. Imoto et al. (1988). 20. Ibid., p. 648. 21. Ibid. 22. Leonard et al. (1988). 23. Giraudat et al. (1986); Revah et al. (1990). See also Hucho et al. (1986); Charnet et al. (1990). Leonard et al. (1988) also used channel blocking agents to assess functional changes produced by site-directed mutagenesis. 24. For example, Kao and Karlin (1986). 25. Yakel and Jackson (1988). See also Derkach et al. (1989). 26. Schofield et al. (1987); Grenningloh et al. (1987). GABAA receptors were distinguishable pharmacologically from GABAfi receptors, which are not ligand-gated ion channels. 27. In contrast to an "identity" of amino acids between two sequences being compared, a "homology" refers to the presence of the identical amino acid or a "conservative substitution": an amino acid of the same size and polarity. 28. Montal (1990); Stroud et al. (1990). 29. For example, Levitan et al. (1988). See also Montal (1990); Stroud et al. (1990). 30. Hollmann et al. (1989). 31. Ibid., pp. 646, 647. 32. Stroud et al. (1990), p. 11,017. 33. Moriyoshi et al. (1991). 34. See Ortells and Lunt (1995). 35. Maricq et al. (1991). 36. Dixon et al. (1986). Initially they screened a genomic library, which contained the entire DNA content of the nucleus and thus encoded all the proteins the organism was capable of synthesizing. By contrast, a cDNA library represents only the proteins synthesized by the particular cell type at the particular time. Elliott Ross in Dallas reported the sequence of a /3-receptor from turkey red blood cells also in 1986 (Yarden et al., 1986). 37. Nathans and Hogness (1983); Henderson and Unwin (1975). 38. Kobilka et al. (1987). 39. Frielle et al. (1987). To locate a similar sequence they used the probe from /3g, hybridizing with low stringency. This encoded a then unrecognized protein; however, a probe based on the sequence of this unrecognized protein hybridized with the DNA for the j3i-receptor. (The unrecognized protein turned out to be a serotonin receptor. Fargin et al. [1988].)

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40. Frielle et al. (1987). 41. Frielle et al. (1988). 42. Kobilka et al. (1987, 1988); Regan et al. (1988). 43. O'Dowd et al. (1988). 44. Cotecchia et al. (1988). 45. For example, Dohlman et al. (1988); Wong et al. (1988). 46. Kobilka et al. (1987), p. 49. 47. Kubo et al. (1986a). 48. The electrical responses were presumably manifested through altered channel permeabilities mediated by G-protein-dependent processes. The electrical responses were readily distinguishable from those of nicotinic receptors. 49. Kubo et al. (1986b). 50. Peralta et al. (1987b). 51. Bonner et al. (1987); Peralta et al. (1987a). See also Akiba et al. (1988). The numbering of muscarinic receptors 3 and 4 differ in these papers; that in Bonner et al. prevailed. 52. Peralta et al. (1988). See also Fukuda et al. (1988). 53. Nevertheless, other studies suggested that a single receptor subtype might couple with different G-proteins (for example, Ashkenazi et al., 1987). 54. Bonner et al. (1988). 55. Fargin et al. (1988); Julius et al. (1988); Pritchett et al. (1988); Bunzow et al. (1988); Neve et al. (1989). 56. Stadel et al. (1983); Leeb-Lundberg et al. (1987). 57. Huganir et al. (1986); Miles et al. (1987). 58. Sibley et al. (1987). 59. Benovic et al. (1986, 1987). 60. Lohse et al. (1990). 61. Pitcher et al. (1998). 62. For example, Chuang and Costa (1979); Maloteaux et al. (1983); Waldo et al. (1983); DeBlasi et al. (1985). 63. For example, Feigenbaum and El-Fakahany (1985); Sibley et al. (1986). 64. For example, Mahan et al. (1985); Ray et al. (1989). 65. See Hausdorff et al. (1990); Hulme et al. (1990). 66. Hadcock et al. (1988). 67. For example, Collins et al. (1989); Hadcock et al. (1989). 68. See Creese and Sibley (1981). 69. For example, Hess et al. (1988). Nevertheless, more complex patterns of receptor changes were also reported: see McGonigle et al. (1989). 70. Montminy et al. (1986). See also Comb et al. (1986); Short et al. (1986). 71. Montminy and Bilezikjian (1987). 72. Collins et al. (1989). This represents a positive feedback loop, but Collins et al. attempted to reconcile the stimulated mRNA synthesis with an expected desensitization of the receptor. 73. Nevertheless, the ability of reductionistic approaches to disclose unities has sometimes been challenged: see Robinson (1992). 74. Lefkowitz et al. (1989). See also Gilbert (1991). 75. Tzartos et al. (1988).

9 SYNTHESIS, STORAGE, TRANSPORT, AND METABOLIC DEGRADATION OF NEUROTRANSMITTERS

Steps in Chemical Neurotransmission

With the acceptance of chemical neurotransmission came the recognition that synaptic transmission required a number of discrete processes: synthesis of the neurotransmitter, its storage, release, and interactions with receptors (and the consequences thereof), and the termination of its actions. All these steps were obviously important functionally. Each of these steps, it also became apparent, was a potential site of pathological malfunction. And each was a potential site for therapeutic intervention. Chapters 6 through 8 dealt with receptors and their associated signal transduction systems. Chapter 10 will deal with neurotransmitter release into the synaptic cleft. This chapter is concerned with synthesis, with metabolic degradation, and with two transport steps, one for storing the neurotransmitter in vesicles and one for terminating the responses to released neurotransmitter. Because of the variety and complexity of these processes, I must restrict this account to representative samplings. Synthesis

If chemical neurotransmitters are present, then some means for their synthesis must also be present, either locally or elsewhere in the body (or perhaps in 219

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the body's foodstuffs). Delineations of these biosynthetic pathways sometimes arose from, and usually became integrated with, general biochemical programs addressing cellular metabolism.

Acetylcnoline

In the mid 1930s Wilhelm Feldberg in Hampstead amassed convincing evidence for acetylcholine s participation in synaptic transmission in autonomic ganglia (chapter 3). As part of these studies, Feldberg demonstrated that acetylcholine was being synthesized during periods of prolonged stimulation: the amount of acetylcholine released during stimulation was five times the amount initially present in the ganglia.1 That same year, 1936, Juda Quastel in Cardiff described an increase in the acetylcholine content of brain slices that was markedly stimulated by adding oxygen and glucose (or certain other metabolic substrates).2 It seemed obvious that acetylcholine was formed by the condensation of acetate with choline (Fig. 3-6), but such a reaction could not be defined using whole cell preparations such as tissue slices. So in 1943 David Nachmansohn in New York used brain homogenates to form acetylcholine in the presence of the cholinesterase inhibitor physostigmine, choline, and, as a necessary energy source, ATP.3 Adding acetate was not required—presumably there was enough in the homogenate—nor was oxygen or any metabolizable substrate. Nachmansohn named the enzyme responsible for this synthesis choline acetylase, although he did not then purify it further. (Nachmansohn was studying acetylcholine not as a neurotransmitter—a role he denied it—but as a component of the mechanism underlying the conduction of nerve impulses along axons. Despite convincing evidence in later decades establishing both a different mechanism for axonal conduction and the irrelevance of acetylcholine, Nachmansohn continued to proclaim his beliefs.4) Acetylation reactions, then a topic of general biochemical interest, seemed to require a modified form of acetate, termed—for lack of a more precise identification—"active acetate." Among those studying these acetylations was Fritz Lipmann in Boston, and in 1945 he reported that acetylating homogenates contained a necessary heat-stable factor.5 The following year Lipmann referred to this factor as a coenzyme and demonstrated that it was also involved in acetylating choline.6 By 1949 Lipmann had characterized the factor chemically and named it coenzyme A (CoA), for acetylation.7 The acetate-bound form, acetylCoA, was soon recognized as an intermediate in numerous metabolic pathways, including the synthesis of fatty acids and the formation of citrate in the Krebs cycle (arising in this case from pyruvate, a product of glycolysis). A specific and widely distributed enzyme synthesized acetylCoA from acetate, Co A, and ATP. In 1951 Nachmansohn acknowledged that choline acetylase catalyzed only

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the ATP-independent condensation of acetylCoA with choline, leaving the ATPdependent formation of acetylCoA to other enzymes.8 The following year Nachmansohn described the partial purification of choline acetylase from squid head ganglia, which catalyzed the synthesis of acetylcholine from acetylCoA and choline.9 Progress in purifying choline acetylase was slow, however, but in 1986 Paul Salvaterra in Duarte used cDNA methods to deduce the amino acid sequence of a 67 kDa protein.10 The source of acetylCoA for synthesizing acetylcholine in vivo was debated for decades, with favored sources including pyruvate and citrate as well as acetate.11 The source of choline was identified as simply choline, but its cellular content could govern the rate of acetylcholine synthesis in certain circumstances. Of particular interest was the action of hemicholinium-3, a synthetic compound that produces muscular paralysis. F. C. Macintosh in Montreal surmised that this compound might affect acetylcholine synthesis, and in 1956 he showed that hemicholinium-3 competed with choline for transport into neurons.12 Subsequent studies defined a transport system sensitive to hemicholinium-3: after released acetylcholine is hydrolyzed by cholinesterase, the resulting choline is pumped across the cell membrane into the neuronal cytoplasm, where it can be reused for making new acetylcholine.13 In some disorders, such as Alzheimer's disease, acetylcholine levels are reduced; in other disorders, such as Parkinson s disease, cholinergic activity is excessive. By 1990, however, no therapeutically useful activators or inhibitors of choline acetylase were available. Since cellular levels of choline seemed to affect rates of acetylcholine synthesis, attempts were made to elevate acetylcholine levels by increasing dietary choline or its metabolic precursors. Unfortunately, this approach provided little benefit.14

Dopamine ana Noraarenaline

While studying the metabolism of amino acids, Peter Holtz in Greifswald described in 1938 the decarboxylation of dopa (Fig. 9-1) by kidney extracts.15 Holz considered this reaction to be a step between the amino acid tyrosine and its oxidized degradation products. The following year Hermann Blaschko (Fig. 9-1A) in Oxford proposed that this decarboxylation was instead a step in the biosynthesis of physiologically active catecholamines (then recognized as adrenaline and perhaps noradrenaline; see chapter 5). Blaschko, after leaving Germany for Cambridge and then Oxford, enjoyed a long career examining the syntheses and degradations of various catecholamines. He was also a convincing advocate of their functional roles. At this point he argued that decarboxylation followed hydroxylation of tyrosine to dopa in this pathway (Fig. 9-2A) since he found that decarboxylation of dopa was far faster than decarboxylation of tyrosine.16

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FIGURE 9-1. A, left, Hermann Blaschko (1900-1993). B, right, Julius Axelrod (1912-). (A courtesy of the department of pharmacology, University of Oxford. B courtesy of J. Axelrod.)

In 1950 Blaschko reported that mammalian tissues did not decarboxylate the dopa derivative having a hydroxylated side chain (dihydroxyphenylserine), so decarboxylation must precede hydroxylation of the side chain.17 The reaction sequence, he concluded, ran from tyrosine hydroxylation forming dopa, to dopa decarboxylation forming dopamine, to dopamine hydroxylation forming noradrenaline (Fig. 9-2A). When radioactively labeled dopa became available, Blaschko was able in 1955 to demonstrate this pathway: adrenal homogenates rapidly converted labeled dopa into labeled dopamine, and after prolonged incubation into labeled noradrenaline.18 Characterization of the responsible enzymes—tyrosine hydroxylase, dopa decarboxylase, and dopamine 0-hydroxylase (Fig. 9-2A)—progressed during the following decades.19 Here, however, I will include only some of the steps taken in recognizing the properties of tyrosine hydroxylase. In 1964 Sidney Udenfriend in Bethesda achieved a partial purification of tyrosine hydroxylase from adrenals, but further purification was achieved only slowly.20 Nevertheless, in 1985 Jacques Mallet in Gif-sur-Yvette determined the amino acid sequence of a 65 kDa protein by cDNA techniques; the active enzyme was a tetramer of these subunits.21

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FIGURE 9-2. Biosynthetic pathways. A. Synthesis of catecholamine can begin with either phenylalanine or tyrosme, each a common amino acid. Hydroxylation of tyrosine forms dopa, and decarboxylation of dopa forms dopamine. Noradrenergic neurons contain an additional enzyme that hydroxylates dopamine to form noradrenaline. B. Synthesis of serotonin (5-hydroxytryptamine) begins with hydroxylation of the common amino acid tryptophan, followed by decarboxylation.

Udenfriend in 1964 also argued for tyrosine hydroxylase being the controlling enzyme of the biosynthetic pathway.22 It was the rate-limiting step in the sequence (slowest in vivo), so changes in tyrosine hydroxylase activity— increases or decreases—would change catecholamine production accordingly. Earl Stadtman in Bethesda had recently stressed a principle of feedback control in biosynthetic pathways termed "end-product inhibition," whereby the final biosynthetic product inhibits the first step in the pathway.23 Correspondingly, Udenfriend showed that dopamine and noradrenaline inhibited tyrosine hydroxylase.24 Moreover, Norman Weiner in Boston found that stimulating sympathetic nerves increased tyrosine hydroxylase activity. He suggested that the drop in noradrenaline levels, due to its release during stimulation, removed noradrenaline s inhibition of the enzyme.25 Other regulatory modes soon became apparent. For example, cAMP increased tyrosine hydroxylase activity acutely, and protein kinase A phosphorylated tyrosine hydroxylase.26 Protein kinase C also phosphorylated tyrosine hydroxylase.27 But what signalled the rise in cAMP or diacylglycerol was still being debated as the 1980s closed.28 Changes in total enzyme content could occur in addition to acute regulation through feedback inhibition and enzyme phosphorylation. For example, depleting an animals stores of catecholamines with drugs or by stress (e.g., exposure

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to cold) increased the amount of tyrosine hydroxylase present, with this change following increases in mRNA for this enzyme.29 Added cAMP could also increase the cellular content of tyrosine hydroxylase, and this change, too, was associated with increased mRNA levels.30 Furthermore, the gene for tyrosine hydroxylase, like the genes for certain adrenergic receptors (chapter 8), was associated with a cAMP response element (CRE).31 The rate-limiting step in a biosynthetic pathway should be a prime target for pharmacological control as well. But no practical activators of tyrosine hydroxylase were discovered, and although an inhibitor, a-methyltyrosine, was studied, it found no significant role in therapy.

Serotonin

The biosynthetic pathway for serotonin resembles closely that for catecholamines (Fig. 9-2B), and its elucidation sprang from parallel studies. In 1954 Udenfriend described the enzymatic decarboxylation of 5-hydroxytryptophan, but he subsequently discovered, to his surprise, that this decarboxylating enzyme was identical to that for dopa.32 So in 1962 he suggested that a better name for the common enzyme was aromatic amino acid decarboxylase.33 The hydroxylating enzyme, tryptophan hydroxylase, was identified in the mid-1960s but not purified until the 1980s.34 Its amino acid sequence, obtained by cDNA techniques in 1987, revealed a close structural and evolutionary relationship to tyrosine hydroxylase and phenylalanine hydroxylase.35 All three hydroxylases commanded considerable attention because of an intriguing reaction mechanism involving oxygen, iron, and a unique cofactor, tetrahydrobiopterin.36 Tryptophan hydroxylase is the rate limiting step in the serotonin pathway, as is tyrosine hydroxylase in the catecholamine pathway. It, too, is phosphorylated,37 but, unlike tyrosine hydroxylase, it is not subject to end-product inhibition.38 No therapeutically useful activators or inhibitors were developed by 1990, but another route to manipulating serotonin synthesis was explored during these decades. The cytoplasmic concentrations of tryptophan are insufficient to saturate tryptophan hydroxylase, so increases or decreases in these levels would increase or decrease 5-hydroxytryptophan formation (and serotonin also, since 5-hydroxytryptophan is rapidly decarboxylated). Accordingly, feeding large amounts of tryptophan could raise brain levels of serotonin.39 By 1990 clinical studies with tryptophan elicited some enthusiasm.40

Enkepnalins

When John Hughes and Hans Kosterlitz identified the first opioid peptides in 1975 (chapter 5), they noted that the amino acid sequence of met-enkephalin

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was present within a known pituitary hormone, /3-lipotropin.41 That presence suggested a source. Smaller peptide hormones were then known to originate through cleavage of larger precursor proteins; the prime example was insulin, whose two chains are clipped from the larger proinsulin.42 C. H. Li in San Francisco had identified /3-lipotropin, and in 1976 he described a 31-amino acid fragment as a proteolytic product.43 This peptide was quite active at opioid receptors, it contained the met-enkephalin sequence, and he named it /3-endorphin.44 In 1979 Shosaku Numa in Kyoto obtained the sequence of proopiomelanocortin by cDNA methods. This 29 kDa protein contained the sequence of /3-lipotropin (and /3-endorphin within it) as well as the sequences of the hormones ACTH and MSH.45 Proopiomelanocortin thus seemed to be the precursor of several hormones. But although the sequence of met-enkephalin lay within the /3-endorphin segment, the sequence of leu-enkephalin was absent. Moreover, the various hormones within proopiomelanocortin were missing from areas of the brain rich in met- and leu-enkephalin.46 Opioid peptides are also present in adrenals, and Udenfriend turned to this source, identifying in 1980 proteins containing both met- and leu-enkephalin sequences.4' That same year he showed that labeled amino acids appeared in these proteins and then in enkephalins.48 Two years later Numa published the sequence of proenkephalin: a 30 kDa protein containing one leu-enkephalin segment, four met-enkephalin segments, and two segments that were slight variants of met-enkephalin.49 This ratio was close to the prevalence of the two enkephalins within the brain, adding further weight to arguments that proenkephalin was the physiological source. Numa also pointed out that the enkephalin sequences in proenkephalin were flanked by pairs of basic amino acids. Such demarcations had been established for peptide hormones within precursor proteins, where the pairs of basic amino acids served as recognition sites for specific proteases that clipped out the proper peptides. (By this time it had become apparent that a single precursor protein could give rise to different peptide products in different cells, depending on which proteases were active where.) During the 1980s (and beyond) the search continued for proteases liberating the enkephalins, although little was known about their control.50 On the other hand, the rate of precursor synthesis did respond to perturbations.51 But no therapeutic modifications of enkephalin synthesis were devised.

Storage

While Feldberg was considering acetylcholine synthesis in ganglia, he also commented on a current controversy: whether nerve impulses triggered a new synthesis of acetylcholine in the presynaptic neuron, which then interacted with

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the postsynaptic cell, or instead triggered a release of previously synthesized acetylcholine. In 1936 Feldberg acknowledged that "the theory of immediate synthesis . . . affords an easier and more direct interpretation."52 But by 1945, with acetylcholine measurements in tissues accumulating, Feldberg argued for the storage of acetylcholine.53 Storage required that acetylcholine be protected from degradation by cholinesterase. Feldberg rejected the possibility that acetylcholine was "present in a diffusible form . . . but spatially separated from . . . cholinesterase."54 So he concluded that acetylcholine was "bound in the tissue or cell granules to some constituents" at synthesis, and in such linkage was "immune to cholinesterase."55 For over a decade the focus was on neurotransmitters stored in secretory granules through their binding to proteins or lipids or even nucleotides.56 A different model arose from examinations not of nerve or brain, but of adrenals. This course began in 1953, when Blaschko and, independently, NilsAke Hillarp in Stockholm separated catecholamine-containing "granules" from adrenal homogenates.57 They used differential centrifugation, following procedures recently established for liver,58 and found 70%-90% of the tissue catecholamines in what, with liver, would be the mitochondrial fraction. Blaschko noted that this fraction contained mitochondrial enzymes but did not examine its morphology. Hillarp failed to see "conventional mitochondria" but presented electron micrographs showing large granules with diameters of 0.1-0.6 />tm; he raised the possibility that these granules were "comparable to mitochondria."59 Subsequently both Blaschko and Hillarp used sucrose density-gradient centrifugation to separate from mitochondria a pure fraction of catecholaminecontaining granules.60 By 1955 both Hillarp and Blaschko argued that the particles were bounded by a semipermeable membrane (vesicles would then be a better name than granules).61 But the prominent osmotic effect expected of catecholamines free at high concentration within a semipermeable barrier was not apparent. On the other hand, if the positively charged catecholamines were bound to constituents of the granules, this osmotic potential would be diminished. Both Hillarp and Blaschko then reported that high concentrations of negatively charged ATP were present in the granules together with catecholamines.62 Such binding might also account for the concentration differential between catecholamines in granules and in cytoplasm. In 1962 Norman Kirshner in Durham described an uptake of catecholamines into isolated adrenal granules that was markedly stimulated by added ATP.63 Kirshner cited proposals that catecholamines and ATP were "associated in a nondiffusable complex within the granules."64 He, however, argued for accumulation through active transport rather than by binding: "catecholamines . . . react with some component of the granule membrane [and] are transported across the membrane and released into the [granule] interior."65 ATP was not

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taken up with the catecholamines, and Kirshner, in accord with Hillarp s report of ATPase activity in granules,66 proposed that ATP hydrolysis provided the energy for active transport. Although binding within the granules could minimize osmotic effects, active transport would account for the concentration gradient. Reserpine, a plant alkaloid recently established as an effective drug for treating schizophrenia (chapter 13), blocked ATP-dependent uptake. This was a significant datum, since Marthe Vogt in Edinburgh had reported in 1956 that reserpine caused a loss of catecholamines from brain and adrenals in vivo.67 Reserpine-inhibitable accumulation thus became the hallmark of catecholamine storage. (Adrenals could also accumulate serotonin in vitro, and reserpine blocked serotonin uptake in adrenals as well as serotonin uptake in the brain.68) A plausible mechanism for ATP-dependent transport would feature a catecholamine-transporting ATPase, along the lines of the recently described Na+/K+-ATPase that functions as the cell membrane Na'VK^-pump.69 The Na+/K+-ATPase was the first transport ATPase to be characterized, and it established the class of what became known as primary active transport systems: these coupled metabolic energy (here, ATP) directly to transport (here, Na + from and K + into the cell). But attempts to identify an analogous catecholamine-transporting ATPase were unsuccessful. The relevant precedent came instead from formulations of an alternative mechanism, promulgated in the early 1960s by Robert Crane in St. Louis and Peter Mitchell in Edinburgh.70 Their models of secondary active transport depicted transmembrane gradients of one solute serving as energy sources for transporting another solute. In these co-transport systems the energetically downhill fluxes of one solute drove energetically uphill fluxes of the other, as in the coupled influxes of Na+ and glucose that occur across the cell membranes of the intestinal epithelia. Mitchell also incorporated this concept in his chemiosmotic hypothesis for oxidative phosphorylation, whereby metabolic oxidations established H+-gradients across mitochondrial membranes, and these gradients then drove ATP synthesis. Mitchell proposed that a class of reagents known as uncouplers of oxidative phosphorylation prevented ATP synthesis by dissipating transmembrane H+-gradients. More than a decade after Kirshner's description and Mitchell's proposal, George Radda in Oxford reported that such uncouplers also blocked ATPdriven catecholamine uptake by adrenal granules.71 Radda suggested that the granule ATPase was a H+-transport pump that established H+-gradients across granule membranes. Then catecholamine "transport could . . . occur by [a] cotransport mechanism associated with the movement [of H+]."72 Two years later, in 1977, Radda demonstrated an ATP-dependent acidification of the granule interior, consistent with an ATPase transporting H + from cytoplasm to gran-

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ule.73 Subsequent studies characterized this H+-transporting ATPase of the granule membrane, showing that it belonged to a family of ATPases closely related to the mitochondrial ATP synthase.74 In 1978 Shimon Schuldiner in Jerusalem provided the complementary observation: H+-gradients imposed across the granule membranes drove catecholamine uptake.75 The energetically downhill efflux of H + from the granule drove an energetically uphill influx of catecholamines; in Mitchell's terms, the catecholamines were carried by an antiporter. Reserpine blocked the H + dependent transport, confirming reserpines effect on this segment of the process (as expected from its competitive inhibition toward catecholamines76). Antonio Scarpa in Philadelphia then explored the relative contributions to catecholamine transport of the chemical and electrical potentials created by the membrane H+-ATPase.77 Purification of the catecholamine transporter was protracted, but in 1990 Schuldiner isolated a protein that bound reserpine and that, when reconstituted, effected transport driven by H+-gradients.'8 (The amino acid sequence of the vesicular transporter, obtained by cDNA methods and published in 1992, represented a 65 kDa protein with 12 transmembrane segments and an apparent structural/evolutionary relationship to certain transporters in bacteria.'9) Characterizing transport systems in neural tissue was more difficult. In the mid-1950s electron microscopists described synaptic vesicles in nerve terminals, and a few years later neurochemists isolated them (chapter 4). But these vesicles were far smaller than adrenergic granules (roughly 0.05 yum in diameter), could not easily be obtained in such large quantities, and were pharmacologically heterogeneous, containing a variety of different neurotransmitters. Nevertheless, with the adrenal granule as a model, similar properties were ascribed to vesicles for amino acid and amine neurotransmitters.80 Storage of peptide neurotransmitters, however, occurred by a quite different mechanism. Earlier studies on peptide hormones indicated that the protein precursors were incorporated into a vesicular system as they were synthesized by membrane-bound ribosomes. Processing of the precursors then took place in the vesicles.81 Whereas synthesis and storage of amino acid and amine neurotransmitters could occur at the nerve terminals, synthesis and storage of peptide neurotransmitters occurred at the site of protein synthesis in the cell body, with the filled storage vesicles then carried to the nerve terminals.82 The peptide-containing vesicles contained a dense core (in electron micrographs) and were larger and more variable in size than the small, clear vesicles containing amino acid and amine neurotransmitters. Studies on peptide neurotransmitters also led to the notion of "co-transmitters."83 It was possible to identify neurons containing particular peptide neurotransmitters by using specific antibodies that could then be visualized by electron microscopy.84 This approach revealed many neurons containing one or more peptide neurotransmitters (in large, dense core vesicles) together with

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an amino acid or amine neurotransmitter (in small, clear vesicles). Consequently, a neuron could release more then one neurotransmitter and thereby evoke still more complex responses in the postsynaptic cells. Altering synaptic transmission by affecting the storage of certain neurotransmitters was an obvious approach to therapy. Reserpine s ability to ameliorate the symptoms of schizophrenia centered attention on catecholamines and serotonin as participants in the pathophysiology of this disease (chapter 13). Reserpine s ability to do so through blocking transport into synaptic vesicles also suggested a particular mode of action for new drugs. Similar agents did not prove clinically effective, however, and reserpine itself fell from favor, being replaced by drugs that act instead on receptors. Degradation Cnolinesterase

The actions of a neurotransmitter could be terminated by destroying the neurotransmitter, and just such a mechanism was established for acetylcholine in its early years (chapter 3). Henry Dale in 1914 attributed the evanescence of acetylcholine s actions to enzymatic destruction. Otto Loewi in 1926 showed that frog hearts broke down Vagusstoff/acetylcholine but that physostlgmine blocked this degradation. And they and their associates in 1930 found that blood catalyzed a physostigmine-inhibitable hydrolysis of acetylcholine. In 1932 Edgar and Ellen Stedman in Edinburgh described acetylcholine hydrolysis by blood serum, attributed to "choline-esterase."85 Further examinations revealed different substrate preferences with cholinesterases from different sources. For example, Gordon Alles and Roland Hawes in Los Angeles reported in 1940 that red blood cells hydrolyzed acetylcholine faster than other choline esters, whereas serum hydrolyzed some other esters (notably butyrylcholine) faster than acetylcholine.86 Nachmansohn in 1949 renamed the former activity— present predominantly in neurons, muscle, and electric organs—acetylcholinesterase.87 (The activity in serum, glial cells, liver, and many other sites has been known by several names, including butyrylcholinesterase. Later studies revealed two distinct but related genes for these two catalytic specificities. The differences are not essential here, and for continuity I shall use the general term cholinesterase for acetylcholinesterase.) Nachmansohn identified cholinesterase activity in eel electric organs in 193988 while in France, following medical studies in Berlin and before his further emigration to the United States. In New York Nachmansohn proceeded with purifications from this source, rich in cholinesterase as well as in choline acetylase and nicotinic receptors. By 1947 he achieved a several hundredfold purification, freeing the enzyme from competing activities.89 Nachmansohn s associates continued this program through the 1960s, obtaining crystals of

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apparently pure enzyme.90 In 1986 Palmer Taylor in La Jolla reported the amino acid sequence for a 66 kDa protein from Torpedo electric organ, using cDNA techniques.91 The enzyme had no transmembrane segments but was tethered to the outside of the cell membrane through a covalently-attached lipid: cholinesterase was thus sited effectively for cleaving released acetylcholine. (In 1991 Joel Sussman and Israel Silman in Rehovot determined the threedimensional structure by X-ray crystallography.92) The reaction mechanism attracted enzymologists' attention, not only because it resembled the extensively studied mechanism of serine proteases93 but also because it approached a "perfect enzyme" in catalytic efficiency: the reaction rate was near the limit set by substrates diffusing to and by products diffusing from the active site. In the 1950s I. B. Wilson, initially collaborating with Nachmansohn, examined the enzyme s kinetic properties and proposed a model for its active site bearing an "esteratic" or catalytic domain plus a negatively charged "anionic" domain where the quaternary ammonium group of choline bound.94 Wilson also formulated a two-step reaction sequence, with an initial acetylation of the enzyme by acetylcholine, releasing choline, followed by hydrolysis of this acetylenzyme, releasing acetate.95 Certain organophosphorus compounds inactivated cholinesterase by forming nonhydrolyzable enzyme-phosphoryl adducts, corresponding to the acetylenzyme intermediate.96 Radioactive inhibitors labeled a serine of the enzyme, and Wilson in 1966 proposed that this serine was also acetylated by acetylcholine during hydrolysis.97 These organophosphorus compounds were first studied as insecticides in the 1930s, before their mode of action as cholinesterase inhibitors was recognized. Then during World War II scientists in Germany, Britain, and the United States developed organophosphorus compounds for chemical warfare.98 These also became valuable reagents for studying the serine proteases as well as cholinesterase. And they continued to be important insecticides (e.g., parathion, malathion) and potential war gases (e.g., tabun, sarin, soman). (Wilson in 1955 developed a reagent to reactivate cholinesterases blocked by these organophosphorus compounds, providing an antidote to such poisonings.99) Reversible cholinesterase inhibitors—physostigmine and various relatives both natural and synthetic—found continuing therapeutic uses, notably in treating glaucoma and myasthenia gravis. As the 1980s closed, new interest in such drugs sprang from their potential benefits in treating Alzheimer's disease. Monoamine Oxidases

The discovery of cholinesterase provided the precedent for neurotransmitter actions being terminated by enzymatic destruction. Apparently in keeping with

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this mechanism, adrenaline, whose candidacy as a neurotransmitter was being explored at the same time as acetylcholine's, was readily oxidized in vitro. Correct identification of the pertinent mode of oxidation is traceable to studies in 1928 by Mary Hare in Cambridge. She described, however, the oxidation by liver extracts of a related compound, tyramine, an oxidation accompanied by deamination.100 In 1937 Blaschko reported the oxidation of adrenaline, noradrenaline, and dopamine by extracts of brain as well as of liver, kidney, and intestine.101 He had just completed his Ph.D. studies at the University of Cambridge following medical training in Germany, and now turned to catecholamines, collaborating initially with Derek Richter, also in Cambridge. They noted the similarity to Hare's observations and attributed their results to an enzyme, amine oxidase, that catalyzed an oxidative deamination: forming an aldehyde and releasing ammonia or an amine (Fig. 9-3). In 1949 Efraim Racker in New York described an aldehyde dehydrogenase that oxidized a broad range of aldehydes to acids.102 For catecholamines the two enzymes acting sequentially would produce deaminated acid metabolites (Fig. 9-3). (In 1951 Albert Zeller in Chicago suggested

FIGURE 9-3. Metabolic degradation of noradrenaline. The pathway on the right shows the more common sequence: methylation of one ring hydroxyl to form normetanephrine, followed by oxidative deamination and further oxidation to the final methylated, deaminated acid. Either sequence can occur, however, and all products, except the reactive aldehydes, can be identified in animals.

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the name monoamine oxidase to distinguish this amine oxidase from enzymes that oxidatively deaminated diamines, such as histamine.103) Richter concluded in 1937 that the "most probable function of the amine oxidase appears to be the destruction of toxic amines . . . but it may also have a special role in ... the physiological inactivation of adrenaline."104 Both those roles were subsequently verified, but here the focus is on the latter process. Blaschko and Richter had identified the enzyme in the brain, and others subsequently identified it in sympathetic nerves.105 Still, the relative slowness of noradrenaline s and adrenaline's oxidations in vitro fostered considerable skepticism about the physiological relevance of this degradation.106 But with the advent of radioactive tracer techniques, Richard Schayer in Chicago was able to show in the early 1950s that administered adrenaline was deaminated/ demethylated in vivo. He concluded that these results "indicate a major role for amine oxidase in [adrenaline] metabolism in the intact animal."107 Bernard Brodie in Bethesda then used a recently recognized inhibitor of monoamine oxidase, iproniazid (Marsilid), to produce marked increases in noradrenaline levels in the brain.108 This finding implied that monoamine oxidase degraded noradrenaline in the absence of this inhibitor. In 1955 Udenfriend discovered that monoamine oxidase acted on serotonin also, catalyzing its oxidative deamination to an aldehyde.109 After the action of aldehyde dehydrogenase, the ultimate product would then be 5-hydroxyindoleacetic acid. Brodie found that inhibiting monoamine oxidase in vivo increased the brain levels of serotonin as well.110 Subsequent studies confirmed the degradation of both catecholamines and serotonin by monoamine oxidase in vivo, although they did not demonstrate that monoamine oxidase acted physiologically to destroy neurotransmitters released at the synapse.111 In fact, the enzyme was localized to mitochondria in 1952,112 indicating an mfracellular site of degradation. With the development of new monoamine oxidase inhibitors, different patterns of susceptibility appeared, depending on the source of monoamine oxidase and the particular substrate assayed. This diversity led to proposals in the late 1960s that two (or perhaps more) forms of monoamine oxidase existed.113 Although purification of monoamine oxidases was difficult and fraught with artifactual fragmentations, the amino acid sequences of two distinct monoamine oxidases, A and B, were obtained by cDNA methods in 1988.114 Both were oligomers with subunits of 60 and 59 kDa, respectively. Even though other mechanisms for terminating the actions of catecholamines and serotonin were recognized later (see below), interest in monoamine oxidases continued because of the therapeutic potential of drugs that inhibited these enzymes. In the mid-1950s the first effective drug for treating psychological depression, iproniazid, was discovered by chance; when its antidepressant efficacy was attributed to inhibition of monoamine oxidase, a search for further antidepressant inhibitors followed (chapter 13). These drugs were

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prescribed extensively in the 1960s, but they fell from favor when newer drugs, acting by different mechanisms and having fewer side effects, became available. The original monoamine oxidase inhibitors blocked both monoamine oxidase A and B. In the 1970s, however, deprenyl (selegilene, Eldepryl) was shown to inhibit monoamine oxidase B selectively. Rationales then appeared for using this drug in the treatment of Parkinson's disease (chapter 13). The clinical successes in the 1980s soon reinvigorated the search for clinically effective monoamine oxidase inhibitors.

Catecnol-O-Methyltransrerase

In 1957 Marvin Armstrong in Salt Lake City described a methylated compound from human urine that he identified as a metabolite of noradrenaline.115 He suggested that this substance, 3-methoxy-4-hydroxymandelic acid (Fig. 9-3), could be formed through amine oxidation followed by O-methylation. That finding turned Julius Axelrod's attention to a new route for catecholamine metabolism. Axelrod (Fig. 9-1B) had just received his Ph.D. in 1955, although he had worked for over a decade with Brodie—first in New York and then in Bethesda—as an often-independent investigator. With his doctoral degree, his own laboratory at NIH, and a background in drug metabolism, Axelrod now set about defining the pathways for catecholamine disposition. Giulio Cantoni, then also in Bethesda, had found a few years earlier that S-adenosyl methionine was the donor for some biochemical methylations.116 Accordingly, Axelrod showed in 1957 that extracts of rat liver destroyed catecholamines when incubated with S-adenosyl methionine. He then identified O-methylated metabolites (metanephrine from adrenaline and normetanephrine from noradrenaline; Fig 9-3) in vitro and in vivo, and he partially purified the responsible enzyme, catechol-O-methyltransferase.11' Subsequently, Carola Tilgmann and Nisse Kalkkinen in Helsinki purified the enzyme fully and in 1990 published the amino acid sequence determined by cDNA methods.118 Although Axelrod concluded in 1958 that O-methylation was the "principal route" for metabolism—a larger fraction was methylated than deaminated— uncertainty about the physiological function of this metabolism persisted.119 Clearly, catecholamines were usually methylated as well as deaminated before they were excreted from the body, but whether catecholamines were methylated to terminate their actions was less obvious. For example, inhibitors of catechol-O-methyltransferase could prolong the responses to administered adrenaline but not noradrenaline, and these inhibitors affected responses to nerve stimulation only modestly.120 Moreover, the bulk of the enzyme was present in the cytoplasm, as Axelrod showed initially, and this localization made a role in terminating synaptic transmission unlikely.121 By 1990 no clinically useful inhibitors of catechol-O-methyltransferase were available.122

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Enkepnalin-Cleaving Proteases

As soon as enkephalins became available for study, it was apparent that their actions in vivo were quite brief. Neural tissues rapidly destroyed activity in vitro, apparently through enzymatic destruction, and in 1978 Bernard Roques and Jean-Charles Schwartz in Paris identified a cleavage point between glycine and phenylalanine (Fig. 5-4E).123 Roques and Schwartz named the responsible enzyme "enkephalinase," and they argued for a physiological role since treating with morphine increased this enzyme s level, whereas inhibiting this enzyme produced experimental analgesia.124 Four years earlier John Kenny in Leeds had described a neutral endopeptidase125 in kidney, and in 1983 he showed that enkephalinase was the same as this widely distributed enzyme.126 Moreover, this neutral endopeptidase, a membrane-bound enzyme belonging to a large family of zinc-containing peptidases, had its active site oriented toward the extracellular milieu, appropriate for a neurotransmitter-destroying enzyme. In 1987 groups in Paris, Montreal, and San Francisco reported the amino acid sequence, defining an 85 kDa protein with one transmembrane segment.127 Early studies also revealed the cleavage of tyrosine from the N-terminus of enkephalin, reflecting the action of an aminopeptidase.128 In 1985 Kenny identified this aminopeptidase as membrane-bound aminopeptidase-N.129 This enzyme, too, belonged to the family of zinc-containing peptidases and was widely distributed throughout the body. In 1988 Ove Noren in Copenhagen reported the amino acid sequence for a 110 kDa protein with a single transmembrane segment.130 These peptidases cleaved other peptides besides enkephalin, and other peptidases could cleave enkephalin. Nevertheless, they were closely tied to enkephalin destruction and considerable interest then focused on finding inhibitors of the neutral endopeptidase and/or aminopeptidase-N. Such inhibitors could produce analgesia,131 but by 1990 no clinically useful agent was identified. Transport ("Reuptake") While extending his metabolic studies on catecholamines, Axelrod identified a different mode of inactivation, one that for many neurotransmitters was more significant physiologically. Seymour Kety, Axelrod's superior at NIH, had commissioned, for a study on schizophrenia, the custom synthesis of adrenaline labeled to high specific activity, and Axelrod obtained some of the leftover compound. He then described in 1958 the labeled metabolites in humans and the following year in cats and mice.132 In all species O-methylated metabolites appeared in the blood rapidly, followed by deaminated ones. But by using cats and mice Axelrod was also able to examine tissue contents post mortem. Sev-

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eral organs, notably heart, spleen, and adrenal, took up significant amounts of labeled adrenaline; furthermore, the loss of labeled adrenaline from these organs was protracted, with an appreciable fraction retained for hours.133 Axelrod repeated this study with labeled noradrenaline, and an even larger fraction was taken up and retained by tissues.134 This noradrenaline was in nerves, since prior denervation markedly diminished uptake.135 Moreover, autoradiography combined with electron microscopy—as well as cellular fractionation studies— showed labeled noradrenaline in synaptic vesicles.136 And although intravenously administered noradrenaline did not appear in the brain (like many polar substances, it is excluded by the "blood-brain barrier"), when labeled noradrenaline was injected into the cerebral ventricles it entered brain cells, too.137 In 1961 Axelrod showed that noradrenaline initially taken up by nerves was released when he stimulated these nerves.138 His model (Fig. 9-4A) summarized this observation, depicting both release from the nerve ending and uptake into it. Also noteworthy were the effects of drugs on noradrenaline uptake. Reserpine, cocaine, and the recently introduced antidepressant drug imipramine (Tofranil) all decreased tissue levels of labeled noradrenaline but increased blood levels. In 1960 Axelrod concluded that these drugs acted "presumably by interfering with binding" and in 1961 "by altering the binding sites at the nerve endings"; later that year he added that they could act by "preventing the entry and/or the binding."139 To distinguish between drugs blocking entry from the circulation (uptake) and drugs preventing storage in neurons (viewed as binding), Axelrod gave drugs before or after injecting labeled noradrenaline into animals.140 Cocaine and imipramine reduced tissue levels when given before but not after; Axelrod concluded that they "block the entry . . . into storage sites but do not cause . . . release."141 Reserpine, on the other hand, acted when given after as well as before.142 The experiment not only tied specific drugs to actions at particular sites, it also distinguished between uptake, soon assigned to transport at the cell membrane (see below), and storage, soon attributed to transport within vesicles (see above).143 These findings, confirmed and extended by others, established transport into nerve endings as the major means for terminating the effects of released noradrenaline.144 Since the transport system retrieved noradrenaline for reuse,145 the term "reuptake," emphasizing this recycling process, appeared in the 1960s146 and became the prominent designation. These findings also accounted for some previously unexplained phenomena. For example, denervation was known to enhance the responses to certain neurotransmitters, and such "denervation supersensitivity" was now attributable to the loss of reuptake—and hence the loss of inactivation—that followed the

FIGURE 9-4. Fate of released noradrenaline. A. Noradrenaline released from the nerve ending (the horizintal Y on the left) can react with the receptor or be O-methylated (within cells) or be lost into the circulation. In addition, as Axelrod's experiments showed, noradrenaline in the blood can be taken up by the nerve endings, which can also take up released noradrenaline. B. Brodie's scheme shows, at the top, a ouabain-inhibitable ATPase (the Na+/K+-ATPase) that pumps Na + out of and K + into the nerve ending, thereby establishing an electrochemical gradient for Na + across the membrane. This gradient then drives the influx of noradrenaline by the carrier (C, in the middle of the figure), a cotransport system (symporter). The noradrenaline that is taken up into the cytoplasm is next packaged in storage vesicles, safe from monoamine oxidase (MAO) that destroys cytoplasmic noradrenaline. The bottom of the figure indicates a Ca2+activated release of noradrenaline into the synaptic cleft. (A from Herting and Axelrod [1961], Fig. 2, reprinted by permission of Nature, © 1961, Macmillan Magazines, Ltd. B from Bogdanski and Brodie [1969], Fig. 4, courtesy of the American Society for Pharmacology and Experimental Therapeutics.)

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destruction of nerve endings. And the potentiating effect of cocaine on administered adrenaline, which Loewi had shown in 1910, was now attributable to cocaine preventing inactivation by blocking reuptake.147 In the 1960s similar studies demonstrated the reuptake into tissues and nerve endings of serotonin and GAB A, then candidate neurotransmitters.148 Later studies showed that glycine and glutamate also relied on reuptake to terminate their synaptic actions.149 But attempts to demonstrate reuptake of acetylcholine failed. This neurotransmitter, unlike the other amine and amino acid neurotransmitters, relies on metabolic degradation rather than transport. Choline, however, is transported into the nerve terminal after it is cleaved from acetylcholine in the synaptic cleft (see above). Meanwhile, experiments in vitro explored the mechanisms involved in reuptake. In 1961 Elwood Titus in Bethesda described an uptake of labeled noradrenaline into brain slices that achieved higher concentrations in the tissue than were in the medium.150 Cocaine, imipramine, and reserpine blocked uptake, as did ouabain, an inhibitor of the Na+/K+-ATPase and thus of the active transport of Na + . Five years later Leslie Iversen, then in Boston, reported that noradrenaline uptake into isolated hearts required Na + in the medium; independently Brodie reported a similar dependence for uptake into rat heart slices.151 Both cited similarities to recent studies showing that sugar and amino acid transport required extracellular Na+.152 Brodie s model in 1969 (Fig. 9-4B) depicted a primary active transport system (the ouabain-sensitive Na + /K + ATPase) that established a Na+-gradient across the cell membrane, together with a secondary active transport system, driven by this Na+-gradient, for pumping noradrenaline across the membrane.153 The energetically downhill flow of Na + into the cell drove an energetically uphill influx of noradrenaline (in Mitchell's terms, noradrenaline was carried by a symporter). Noradrenaline arriving in the cytoplasm could next be packaged into storage vesicles to prevent destruction by monoamine oxidase. (Storage, as described above but shown later, relied on another secondary active transport system, this one an antiporter driven by the H+-gradient formed by a H+-ATPase.) In 1967 R. J. Blackburn in Sandwich and S. B. Ross and A. L. Renyi in Sodertalje found that ouabain, cocaine, and imipramine inhibited serotonin uptake into brain slices.154 Brodie then showed that synaptosomes could accumulate serotonin as well as noradrenaline by Na+-dependent processes.155 Subsequent studies characterized the affinities of distinguishable cell membrane transporters for various neurotransmitters, their specific ionic requirements, and their inhibitors.156 Isolating these transporters proved difficult, but in 1990 Baruch Kanner in Jerusalem and his associates reported the amino acid sequence, obtained by cDNA methods, for the GABA transporter.15' (Sequences for dopamine, noradrenaline, and serotonin transporters followed the next year.158 These sequences identified a family of cell membrane transport proteins

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having 12 transmembrane segments but distinct from the family of vesicle transporters for these same neurotransmitters.159) A major interest driving these investigations was the clinical efficacy of reuptake inhibitors. Imipramine and its siblings—the tricyclic antidepressants (named for their chemical structure)—supplanted monoamine oxidase inhibitors as the first choice for treating depression by the mid-1960s because of their greater efficacies and lesser toxicities (chapter 13). Then in the late 1980s fluoxetine (Prozac)—the first of the "specific serotonin reuptake inhibitors" and which enjoyed still fewer side effects—supplanted in turn the tricyclic antidepressants. The popularity of these drugs resulted not only from their safety but also from their utility in treating a range of disorders: depression, obsessive-compulsive disorder, panic attacks, bulimia and eating disorders, and more.

Conclusions

By 1990 various investigators across the globe had delineated specific steps in neurotransmitter synthesis, storage, and disposition. In doing so they defined particular processes and uncovered energetic and regulatory niceties, all in accord with general mechanisms of cellular metabolism and transport. For his contributions Axelrod shared the Nobel Prize in 1970 with Bernard Katz and Ulf von Euler. This chapter illustrates some diverse courses of discovery, citing studies on acetylcholine, catecholamines, serotonin, and enkephalins. The resulting models depicted how: (1) Acetylcholine is synthesized through the condensation—catalyzed by choline acetylase—of acetylCoA, a common unit in metabolic pathways, with choline. Newly synthesized acetylcholine is next transported into storage vesicles by a H+-driven antiporter. Released acetylcholine is hydrolyzed by cholinesterase, liberating acetate plus choline (which is then transported into the cytoplasm of the presynaptic nerve ending by a Na+-driven symporter). (2) Dopamine is synthesized from the amino acid tyrosine by enzymatic hydroxylation and decarboxylation. Dopamine is next transported into storage vesicles by a H+-driven antiporter; dopamine is converted enzymatically to noradrenaline within adrenergic vesicles. The actions of released dopamine and noradrenaline are terminated through transport into the cytoplasm by a Na+-driven symporter (reuptake); there they may be reincorporated into storage vesicles for reuse. Lesser amounts of catecholamines are degraded—in neurons and glia as well as liver and kidney—by catechol-O-methyltransferase and monoamine oxidase.

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(3) Serotonin is synthesized from the amino acid tryptophan by enzymatic hydroxylation and decarboxylation. Serotonin is next transported into storage vesicles by a H+-driven antiporter. The actions of released serotonin are terminated through transport into the cytoplasm by a Na + driven symporter, where it may be reincorporated into storage vesicles for reuse. Lesser amounts are degraded by monoamine oxidase. (4) Enkephalins, like other peptide neurotransmitters, are clipped enzymatically from precursor proteins. The precursor protein is translocated within the cellular tubulo-vesicular system during its synthesis, and there processing to the final peptide occurs. Enkephalins within storage vesicles are then carried from the cell body to the nerve endings. The actions of released enkephalins are terminated through destruction by peptidases. Practical interest in these processes sprang from their sensitivities to newly discovered drugs effective against previously intractable disorders. Reserpine, which depletes stores of noradrenaline and serotonin (and dopamine, too, as shown later), relieves the symptoms of schizophrenia. However, it can also produce psychological depression. Monoamine oxidase inhibitors decrease the metabolism of noradrenaline and serotonin (and dopamine, too, as shown later) and relieve the symptoms of depression. Tricyclic antidepressants such as imipramine block the reuptake of—and thus also potentiate the effects of— noradrenaline and serotonin (to different degrees depending on the particular drug) and relieve the symptoms of depression. Clinical utility was often discovered before the biochemical mechanisms. Recognition of these mechanisms then inspired new studies on schizophrenia and depression based on catecholamine and/or serotonin malfunctionings (chapter 13). Such recognition also prompted searches for better agents acting in these manners (uncovering, for example, the specific serotonin reuptake inhibitors such as fluoxetine [Prozac]).

Notes 1. Brown and Feldberg (1936a). They measured acetylcholine release into the veins draining the ganglia, using a leech bioassay. Physostigmine was present to block acetylcholine destruction by cholinesterase. 2. Quastel et al. (1936). They, too, added physostigmine and measured acetylcholine with a bioassay. 3. Nachmansohn and Machado (1943). They used homogenates initially but later saline extracts of the homogenates; they also included fluoride to prevent depletion of ATP by irrelevant ATPases. Lipmann (1941) had recently published his magisterial review delineating the role of ATP and "energy-rich" phosphate bonds. 4. Nachmansohn (1953, 1961).

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5. Lipmann (1945). 6. Lipmann and Kaplan (1946). Nachmansohn was also pursuing a cofactor (Nachmansohn and Berman, 1946), as was Feldberg (Feldberg and Mann, 1946). 7. Kaplan and Lipmann (1949). It was Feodor Lynen in Munich, however, who described the chemical structure of acetylCoA (see Lynen, 1953). 8. Korey et al. (1951). 9. Korkes et al. (1952). 10. Itoh et al. (1986). 11. See Jope (1979). 12. Macintosh et al. (1956). 13. See Jope (1979). 14. See Johnston et al. (1992). 15. Holtz et al. (1938). 16. Blaschko (1939). See also Blaschko (1942); Holtz (1939). 17. Blaschko (1950). 18. Demis et al. (1955, 1956). Blaschko was then visiting with Arnold Welch in New Haven, following Welch's visit with Blaschko in Oxford. See also Hagen (1956); Udenfriend and Wyngaarden (1956); Kirshner (1957). 19. Dopaminergic neurons lack dopamine-/3-hydroxylase, which is present in adrenergic neurons. The adrenal contains in addition an N-methyl transferase that converts noradrenaline to adrenaline. 20. Nagatsu et al. (1964); Shiman et al. (1971); Haavik et al. (1988). 21. Grima et al. (1985). 22. See also Levitt et al. (1965). 23. Stadtman (1963). See also Bonner (1961). 24. Nagatsu et al. (1964). 25. Alousi and Weiner (1966). 26. For example, Goldstein et al. (1973); Harris et al. (1974); Joh et al. (1978); Meligeni et al. (1982). 27. For example, Albert et al. (1984); McTigue et al. (1985). 28. See Zigmond et al. (1989). 29. For example, Mueller et al. (1969); Thoenen (1970); Black et al. (1985); Faucon Biguet et al. (1986). 30. For example, Kumakura et al. (1979); Lewis et al. (1987). 31. Lewis et al. (1987). They also showed that glucocorticoids, which are released with stress, interact with the gene for tyrosine hydroxylase through a response element to which the steroid receptor binds. 32. Clark et al. (1954); Lovenberg et al. (1962). See also Sumi et al. (1990), who showed that the expressed cDNA exhibited both catalytic activities. 33. Lovenberg et al. (1962). 34. For example, Grahame-Smith (1964); Lovenberg et al. (1967); Nakata and Fujisawa (1982); Cash et al. (1985). 35. Grenett et al. (1987). 36. See Hufton et al. (1995). 37. For example, Hamon et al. (1978); Ehret et al. (1989). 38. For example, Jequier et al. (1969). 39. For example, Green et al. (1962); Wang et al. (1962). 40. For example, Boman (1988). 41. Hughes et al. (1975a).

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42. Steiner (1977). 43. Li and Chung (1976). 44. Although Simon had suggested "endorphin" as a generic name for all endogenous opioids, the name instead became attached to this family. A third family of endogenous opioids, the dynorphins, arises from a third precursor. 45. Nakanishi et al. (1979). The pituitary secretes adrenocorticotropic hormone (ACTH) to regulate adrenal release of cortical hormones and melanocyte-stimulating hormone (MSH) to control pigmentation. 46. Rossier et al. (1977); Lewis et al. (1978). 47. Lewis et al. (1980). They cleaved the presumed precursors with proteases and identified released enkephalins chromatographically and by their actions on opioid receptors. 48. Rossier et al. (1980). 49. Noda et al. (1982a). See also Gubler et al. (1982); Comb et al. (1982). 50. For example, Fricker and Snyder (1982); Hook and Eiden (1984); Seidah et al. (1990). 51. For example, Temple et al. (1990). 52. Brown and Feldberg (1936a), p. 282. 53. Feldberg (1945). 54. Ibid., p. 614. Feldberg argued that acetylcholine was still protected from cholinesterase after homogenizing the tissue (he assumed homogenization would rupture all membranes). 55. Ibid., pp. 614, 615. 56. For example, Green (I960); Green et al. (1961); Burack et al. (1961). 57. Blaschko and Welch (1953); Hillarp et al. (1953). Welch was visiting Blaschko in Oxford; shortly thereafter Blascko visited Welch in New Haven, where they studied the formation of labeled catecholamines from labeled dopa (ref. 18). 58. See Robinson (1997). 59. Hillarp et al. (1954), p. 166. 60. Blaschko et al. (1957); Hillarp (1958b). 61. Hillarp and Nilson (1954); Blaschko et al. (1955). A semipermeable membrane would permit diffusion of water but not catecholamines, allowing the catecholamines to exert osmotic effects. 62. Hillarp et al., (1955); Blaschko et al. (1956). 63. Kirshner (1962). See also Carlsson et al. (1963). 64. Ibid., p. 2311. 65. Ibid., p. 2316. 66. Hillarp (1958a). 67. Holzbauer and Vogt (1956). Earlier that year Brodie described a reserpineinduced "release" of serotonin from its stores in brain (Pletscher et al., 1956). 68. Bertler et al. (1960). See also Paasonen and Vogt (1956); Bertler (1961); Bogdanski et al. (1968); Phillips (1974). 69. For the historical background, see Robinson (1997). 70. Ibid. 71. Bashford et al. (1975). 72. Ibid., p. 155. 73. Casey et al. (1977). See also Flatmark and Ingebretsen (1977). 74. See Robinson (1997). The ATP synthase can run backwards, in this mode hydrolyzing ATP to pump H + .

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75. Schuldiner et al. (1978). See also Johnson et al. (1978); Phillips (1978). 76. Jonasson et al. (1964). See also Kanner et al. (1979). 77. Johnson et al. (1979). See also Knoth et al. (1980). The gradient is an electrochemical gradient, dependent on both the active transport pump and the passive permeabilities of the membrane. 78. Stern-Bach et al. (1990). 79. Liu et al. (1992); Erickson et al. (1992). 80. For example, Anderson et al. (1982); Naito and Ueda (1985); Kish et al. (1989). For early studies on vesicles from peripheral nerve, see von Euler and Lishajko (1963). 81. See Mains et al. (1990). 82. See DeCamilli and Jahn (1990). 83. Hokfelt et al. (1980). 84. For reports of enkephalins localized with amine neurotransmitters, see Charnay et al. (1982); Hunt and Lovick (1982); Altschuler et al. (1983). 85. Stedman et al. (1932). 86. Alles and Hawes (1940). 87. Augustinsson and Nachmansohn (1949). 88. Nachmansohn and Lederer (1939). 89. Rothenberg and Nachmansohn (1947). 90. Kremzner and Wilson (1963); Leuzinger and Baker (1967). 91. Schumacher et al. (1986). There are, however, alternative forms and sequences: the enzyme exists as an oligomer of different multiples and the mRNA is processed ("alternative splicing") to produce different sequences from the same gene. 92. Sussman et al. (1991). Their structure featured an active site serine deep within a catalytic "gorge" and adjacent to a histidine adjacent to a glutamate, forming a chargerelay system analogous to that of the serine proteases. 93. See Robinson (1997). 94. Wilson and Bergmann (1950). The crystal structure, however, showed that the "anionic site" was not composed of negatively charged amino acids but of aromatic amino acids, whose 7r-electron clouds serve the same function. 95. Wilson et al. (1950); Wilson (1951). 96. See Burgen (1949); Aldridge (1950). 97. Wilson (1966). 98. For a historical account, see Holmstedt (1963). 99. Wilson and Ginsburg (1955). See also Childs et al. (1955). 100. Hare (1928). Tyramine is the decarboxylation product of tyrosine, analogous to dopamine but having a single phenolic hydroxyl. Hare reported that her enzyme did not affect adrenaline, apparently because enzmatic oxidation of adrenaline is far slower than oxidation of tyramine, whereas the spontaneous oxidation of adrenaline is rapid. 101. Blaschko et al. (1937a, 1937b); Richter (1937). The key to their success was blocking other routes of oxidation with cyanide. For an autobiographical reminiscence, see Blaschko (1972). 102. Racker (1949). 103. Zeller (1951). This repeated a less accessible naming in German during the war (Zeller et al., 1940). 104. Richter (1937), p. 2028. 105. For example, Holtz and Westermann (1956) found monoamine oxidase activity in adrenergic nerves, while Burn and Robinson (1952) and Stromblad (1956) reported the disappearance of such activity after nerve degeneration. 106. See Kopin (1972). Richter (1940) had also expressed doubts about the func-

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tional role, and Blaschko (1952) wondered whether an active termination mechanism was necessary in light of the prolonged effects of peripheral sympathetic stimulation. 107. Schayer (1951a, 1951b); Schayer and Smiley (1953). Schayer did not demonstrate deamination directly but showed a cleavage between the methyl carbon of adrenaline and the a carbon of the side chain. 108. Spector et al. (1958); see also Pletscher (1957). These experiments were with rabbits and rats; with some other species monoamine oxidase inhibitors produced lesser effects (Kopin, 1972). 109. Sjoerdsma et al. (1955). Blaschko (1952) had reported the oxidation of serotonin by tissue extracts earlier. 110. Spector et al. (1958). 111. For example, Grout (1961) found that inhibiting monoamine oxidase in vivo did not affect cardiovascular function. See also Kopin (1972). 112. Hawkins (1952). 113. For example, Maitre (1967); Johnston (1968); Squires (1968). 114. Bach et al. (1988); Hsu et al. (1988); Ito et al. (1988). 115. Armstrong et al. (1957). 116. Cantoni (1953). 117. Axelrod (1957); Axelrod et al. (1958); Axelrod and Tomchick (1958); LaBrosse et al. (1958). 118. Tilgmann and Kalkkinen (1990); Salminen et al. (1990). 119. LaBrosse et al. (1958), p. 593. See also Kopin (1972); Guldberg and Marsden (1975). 120. Wylie et al. (1960). 121. Depending on the organism, there could be a substantial fraction of membranebound catechol-O-methyltransferase, although this enzyme, too, acted on cytoplasmic catecholamines. See Broch and Fonnum (1972); Roth (1980, 1992). 122. In the late 1990s an inhibitor of catechol-O-methyl transferase, tolcapone (Tasmar), showed promise in the treatment of Parkinson s disease. 123. Malfroy et al. (1978). See also Sullivan et al. (1978); Gorenstein and Snyder (1979). 124. Malfroy et al. (1978); Roques et al. (1980). 125. Peptidases that cleave the terminal amino acids are exopeptidases; aminopeptidases and carboxypeptidases cleave the N- and C-terminal amino acids, respectively. Endopeptidases cleave peptide bonds father within. 126. Kerr and Kenny (1974); Matsas et al. (1983). The designation refers to the optimal pH for catalysis and distinguished this endopeptidase from the well-known one that cleaves optimally at acidic pHs. 127. Devault et al. (1987); Malfroy et al. (1987). 128. Hambrook et al. (1976). 129. Matsas et al. (1985). 130. Olsen et al. (1988). 131. For example, Roques et al. (1980); de la Baune et al. (1982); Waksman et al. (1985). 132. LaBrosse et al. (1958); Axelrod et al. (1959). Axelrods methods for separating adrenaline and its metabolites were crucially important for these studies. 133. Earlier Burn (1932) had inferred an uptake of adrenaline into tissues, and subsequent investigators, measuring unlabeled adrenaline, also argued for an uptake (e.g., Raab and Humphries, 1947; Nickerson et al., 1950). 134. Whitby et al. (1961).

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135. Hertting et al. (1961a). Fluorescence techniques also revealed the uptake of unlabeled catecholamines into neurons (Hamberger et al., 1964). 136. Wolfe et al. (1962); Potter and Axelrod (1963). 137. Glowinski et al. (1961). 138. Hertting and Axelrod (1961). They gave labeled noradrenaline to animals, loading nerve endings in the spleen. They then removed the spleen and perfused it. When they stimulated the nerves to the spleen, labeled noradrenaline appeared in the perfusate. 139. Whitby et al. (1960), p. 605; Axelrod et al. (1961), p. 384; Hertting et al. (1961b), p. 152. 140. Axelrod et al. (1962). 141. Ibid., p. 297. 142. Reserpine was then thought to cause depletion of stores, an effect later attributable to enzymatic destruction of catecholamines that had been prevented by reserpine from entering the storage vesicles. 143. Iversen (1965) identified a second uptake system, designated uptake£, with lower affinity for catecholamines. This system was later localized to non-neuronal cells, where it uses quite different transporters; these can nevertheless function importantly in neurotransmitter disposal. 144. For example, Stromblad and Nickerson (1961); Iversen (1967). Moreover, Kopin et al. (1962), using perfused rat hearts, found less than half as much labeled noradrenaline metabolized as taken up into reserpine-sensitive stores. Correspondingly, Grout (1961) demonstrated that even when administered together, the inhibitors of monoamine oxidase and of catechol-O-methyltransferase altered adrenergic responses minimally. 145. See Brown (1965). 146. For example, Folkow et al. (1967). 147. Frohlich and Loewi (1910). MacMillan (1959) proposed that cocaine blocked uptake into neural stores, as did Muscholl (1960). 148. For example, Marchbanks (1966); Blackburn et al. (1967); Ross and Renyi (1967); Iversen and Neal (1968); Kuriyama et al. (1969). 149. For example, Neal and Pickles (1969); Logan and Snyder (1971). 150. Dengler et al. (1961, 1962); Dengler and Titus (1961). 151. Iversen and Kravitz (1966); Bogdanski and Brodie (1966). 152. See Robinson (1997). 153. Bogdanski and Brodie (1969). 154. Blackburn et al. (1967); Ross and Renyi (1967). 155. Bogdanski et al. (1968). 156. See Kanner and Schuldiner (1987). Although the electrochemical Na+-gradient was the crucial driving force for these transporters, many had various requirements for other ions, too. 157. Guastella et al. (1990). 158. Pacholczyk et al. (1991); Usdin et al. (1991); Hoffman et al. (1991); Kilty et al. (1991); Blakely et al. (1991); Shimada et al. (1991). 159. See Schloss et al. (1992). Glutamate transporters belong, however, to a different family (see Worrall and Williams, 1994).

10 NEUROTRANSMITTER RELEASE

Proposals

Schemes for chemical neurotransmission needed explanations also of how neurotransmitters could pass from presynaptic to postsynaptic neuron. Pertinent issues included identifications of the source from which the neurotransmitter was released, descriptions of the molecular machinery responsible for release, and characterization of the regulatory systems that controlled release. Defining the underlying mechanisms proved to be a particularly formidable task: the machinery operated rapidly and intermittently, within only minute regions of the neuron, yet depended on complex molecular arrays. By the mid-1930s arguments for neurotransmitters being stored in presynaptic neurons focused attention on how release from such stores occurred and what triggered it. G. L. Brown and Wilhelm Feldberg in Hampstead found that adding K + to the fluid perfusing sympathetic ganglia augmented the response of postganglionic fibers to stimulation of preganglionic fibers, and in 1936 they suggested that K + "might be the agent responsible for the discharge of [acetylcholine]."1 Two years later Henry Dale endorsed this suggestion, noting that the "mobilization" of K + accompanying an action potential could "release . . . acetylcholine from its depot at the nerve ending."2 In 1939, however, Brown showed that adding K + stimulated nerve fiber activity, indicating 245

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a different source for the augmented response,3 and by 1950 Alan Hodgkin and Andrew Huxley had demonstrated that during action potentials K + flowed from the cell (chapter 4). Nevertheless, Brown, Feldberg, and Dales suggestion persisted for some years. For example, a prominent pharmacology textbook stated in 1956 that K + "released during passage of the nerve impulse . . . may liberate free acetylcholine from a precursor protein complex, perhaps by cation exchange."4 On the other hand, Bernard Katz and Paul Fatt in London found that a decrease in the external Na + concentration diminished e.p.p.s at neuromuscular junctions.5 So they suggested in 1952 that the inward flow of Na + during action potentials stimulated acetylcholine release. Their model of Na+ exchanging for acetylcholine invoked a membrane carrier that could alternately transport Na + inward and acetylcholine outward across cell membranes.6 (The notion of membrane carriers ferrying polar solutes across nonpolar membrane barriers was then in vogue.7 James Danielli had developed a model for cell membranes in the 1930s that featured layers of lipid molecules flanked by surface proteins. To account for the ready diffusion into and from cells of small ions, such as K + , or small polar molecules, such as water, Danielli later invoked pores through his model membrane. In the 1940s and 1950s selective "carriers" were also included: such lipoidal carriers would form nonpolar complexes that could diffuse across the lipid barrier, transporting larger polar substances, such as sugars and amino acids. No carriers had been identified by 1952, but Katz and Fatt's suggestion met the spirit of these conjectures.) Then in 1954 Katz and Jose del Castillo described a quantal model for neurotransmitter release (chapter 4). They identified the smallest e.p.p. evoked at neuromuscular junctions—achieved by lowering the external Ca2+ concentration— with the smallest spontaneously occurring m.e.p.p.s, and they interpreted this "unit potential" as the response to one quantum of released acetylcholine. Ordinary e.p.p.s thus represented the summed responses to hundreds of such fundamental quanta. That year Eduardo DeRobertis in Seattle and Sanford Palay in New York independently described electron micrographs of nerve endings filled with numerous small vesicles (chapter 4). DeRobertis, moreover, reported that "some of these vesicles seem to perforate the presynaptic membrane, so that portions of the vesicles seem to lie in the intermembranal space [i.e., the synaptic cleft] and come into direct contact with the post-synaptic membrane."8 The obvious identification of physiologists' quanta with microscopists' vesicles (or "particles") led Katz and del Castillo in 1955 to "imagine a mechanism by which each particle loses its charge of [acetylcholine] in an all-or-none manner when it collides with, or penetrates, the membrane of the nerve terminal."9 (They also presented evidence against Katz's earlier model for Na+/acetylcholine

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exchange via a membrane carrier.) Later in 1955, however, Katz and del Castillo specified a more restricted process that became the standard model for neurotransmitter release through 1990 (and beyond): vesicles might bind to reactive sites on the membrane and there fuse with the membrane, opening a pathway to the exterior through which the vesicle contents could enter the synaptic cleft (Fig. 10-1A).10 In his Croonian Lecture of 1961, Katz specified that "We do not believe that . . . vesicles are discharged bodily from the nerve terminal."11 Soon afterward, Christian de Duve named the process of vesicles fusing with cell membranes "exocytosis"; the complementary process—pinching off invaginations of the membrane to form vesicles—he named "endocytosis".12 Accordingly, neurotransmitter release achieved through fusion of vesicle with cell membrane became known as "exocytotic release." But even if neurotransmitters were stored in synaptic vesicles, other mechanisms for release were conceivable. Indeed, some investigators claimed that vesicles dispensed their neurotransmitter into the cytoplasm of the presynaptic neuron. Release into the synaptic cleft would then occur from the cytoplasm through pores or carriers in the cell membrane. For example, Nils-Ake Hillarp in Lund reported in 1954 that after catecholamines were released from the adrenal, the storage vesicles (or "granules") remained in the cells "apparently unchanged in number"; he concluded that "secretion is not accompanied by the discharge of these granules from the cells, but rather [catecholamines are] liberated and then secreted."13 Later, he found no appreciable loss of adrenal granule proteins during catecholamine release, in accord with his contention that the entire granule was not discharged.14 Since less of the nonmembrane ("soluble") protein of the granules was lost than would occur if the entire granule contents were discharged, he also concluded that catecholamines were released first into the cytoplasm, from which they would "have little difficulty in permeating the cellular membrane."15 By contrast, the soluble proteins would be retained within the cell. Arguments for neurotransmitter release from the cytoplasm were reinforced in the 1960s by comparisons of the neurotransmitter content of isolated synaptic vesicles with the content of free, unbound neurotransmitter (chapter 4). Appreciable amounts of neurotransmitter appeared to be free in the cytoplasm, and although some of this was clearly due to losses from the vesicles that occurred during their isolation, significant amounts of cytoplasmic neurotransmitter still seemed to occur in the native cell. Moreover, studies on neurotransmitter synthesis, storage, and reuptake demonstrated that neurotransmitters passed through the cytoplasm to gain entry to the vesicles (chapter 9). Correspondingly, neurotransmitters might pass from vesicles to cytoplasm en route to the synaptic cleft. Thus, in 1966 Ulf von Euler in Stockholm referred

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to an extravesicular pool of noradrenaline that was refilled by reuptake as well as from the vesicular stores, and he identified this pool as the source of released neurotransmitter.16

Evidence for Exocytotic Release Morphological Studies

If synaptic vesicles fuse with the cell membrane to release neurotransmitters, then stimulating presynaptic neurons should deplete their complement of vesicles—unless the process of replacement keeps pace with exocytosis. Stimulating presynaptic neurons should also increase the surface area of these nerve endings through the addition of vesicle membranes—unless the process of retrieval keeps pace as well. On the other hand, attempts to outrace replacement and retrieval by stimulating at high rates for long times might reveal changes due to neural exhaustion rather than physiological release. Through the 1960s studies on various systems under various conditions generated reports that vesicle numbers did or did not change with stimulation.17 In the 1970s, however, more convincing evidence for exocytosis appeared. Thus, Suthiwan Kwanbunbumpen in Canberra described in 1970 changes in the numbers of synaptic vesicles at the "active zones" of neuromuscular junctions (regions of the presynaptic ending filled with vesicles and lying opposite postsynaptic folds of the muscle18). Kwanbunbumpen found reduced vesicle numbers if, before stimulating, he administered hemicholinium, which halts acetylcholine synthesis by blocking choline transport (chapter 9).19 Without hemicholinium, however, stimulation increased vesicle numbers. He interpreted this contrary observation as a physiological recruitment of new vesicles for the increased activity. Soon afterward several investigators found decreases

FIGURE 10-1. Proposals for exocytotic release. A. Katz and del Castillo's version, showing as dots the molecules on each membrane that interact to assure fusion of vesicle with presynaptic membrane, followed by exocytotic release. B. Heuser and Reese's cycle at neuromuscular junctions, showing the presynaptic terminal lying beneath a (striped) Schwann cell and above the (striped) muscle. On the left, synaptic vesicles move toward the membrane and fuse for exocytotic release. On the right, vesicle membrane is retrieved as coated pits and coated vesicles, enclosed within clathrin baskets and bound for intraterminal cisternae from which new synaptic vesicles bud. C. Breckenridge and Aimer's sequence of reversible and irreversible steps, showing the initial formation of a fusion pore followed by full exocytosis. (A from Fig. 5 of the report of a meeting held in 1955 [del Castillo and Katz, 1957]. B from Heuser and Reese [1973], Fig. 36, reproduced by permission of Rockefeller University Press. C from Breckenridge and Aimers [1987], Fig. 6, courtesy of Wolfhard Aimers.)

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in synaptic vesicle numbers in sympathetic ganglia following intense stimulation.20 Furthermore, J. J. Pysh and R. G. Wylie in Chicago described an increase in the nerve ending surface, too, consistent with vesicle membrane being added during exocytosis: "initial calculations indicate that the estimated quantity of vesicle membrane lost agrees with the . . . increase of membrane in axon endings."21 Also in 1972 Bruno Ceccarelli, visiting Alexander Mauro in New York, reported decreased stores of acetylcholine accompanying decreased numbers of vesicles at neuromuscular junctions.22 These changes followed moderate rates of stimulation for 6-9 hours in the absence of hemicholinium. After 4 hours of stimulation the number had not changed, but by this time the nerve endings contained the enzyme peroxidase, which he had included in the bathing medium to demonstrate endocytosis. (Peroxidase, like other proteins, cannot diffuse across membranes but can enter cells by endocytosis: it can be taken up within vesicles newly formed from the cell membrane. The presence of peroxidase is demonstrable in electron micrographs through its oxidation of added substrates to form electron-opaque deposits.) Ceccarelli interpreted these observations as exocytotic release in the first several hours being matched by an endocyiotic retrieval of vesicle membranes that maintained vesicle numbers.23 Only with prolonged stimulation did retrieval and reformation of vesicles lag behind release. John Heuser and Thomas Reese in Bethesda confirmed and extended these observations. In 1973 they described a depletion in synaptic vesicles at neuromuscular junctions after stimulating for 1 minute at high frequencies, and this depletion was balanced by an increase in the cell membrane so the total membrane content remained constant.24 After stimulating for 15 minutes, there was an even larger depletion accompanied now by the appearance of numerous membrane-bounded cisternae within the nerve ending. After allowing the nerve to rest for 15 minutes, these cisternae disappeared and new vesicles appeared. When they included peroxidase in the bathing media, this tracer first entered the cisternae and later the synaptic vesicles. When they stimulated the nerve, the tracer disappeared from the synaptic vesicles. Accordingly, Heuser and Reese proposed a scheme with neurotransmitter release via exocytosis, followed by vesicle retrieval through endocytosis and the subsequent transfer of membrane through cisternae to new vesicles (Fig. 10-1B). Heuser and Reese next applied a technique recently developed for studying intramembrane components (such as intrinsic membrane proteins), freezefracture electron microscopy. They split frozen tissues mechanically, and some of the cleavages passed between the leaflets of the membrane lipid bilayer. Electron microscopy could then reveal structures extending through the membrane interior. They found "dimples" appearing in the presynaptic membranes after nerve stimulation, which they interpreted as exocytotic channels occur-

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ring at the site of vesicle fusion.25 By conventional thin-section electron microscopy they also found profiles identifiable as vesicles fused with the membrane (Fig. 10-2). (Others had already reported such profiles of fused vesicles.26) An obvious problem was distinguishing between exocytotic and endocytotic images. Heuser and Reese argued that exocytosis occurred at the active zones, whereas endocytosis occurred beyond this region. Moreover, they identified endocytosis with "coated pits" and "coated vesicles," so named because of their fuzzy surfaces (Figs. 10-1B and 10-2).27

FIGURE 10-2. Electron micrographs of exocytosis and endocytosis at neuromuscular junctions. Figures 23 through 25 show, at the arrows, profiles of exocytotic release. Figure 26 shows, at the arrow, a coated pit undergoing transformation into a coated vesicle. (From Heuser et al. [1974], Figs. 23-26, courtesy of Kluwer Academic Publishers.)

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A further problem lay in the poor time resolution of these approaches. So Heuser and Reese developed a quick-freeze technique that fixed tissues within a few milliseconds, sufficient to resolve a single nerve impulse. This approach allowed them in 1979 to photograph nerve endings "caught in the act of exocytosis" (Fig. 10-3); moreover, they correlated the number of exocytotic figures with the quanta of transmitter discharged, equating one vesicle with one quantum.28 And in 1985 Ceccarelli, back in Milan, improved the time resolution to less than 1 millisecond: he could now show that vesicles fused with the membrane at the same time that quanta were released.29

FIGURE 10-3. Freeze-fracture electron micrographs of quick-frozen neuromuscular junctions, from Heuser et al. (1979). Figure 7 shows the array of intramembrane particles, later identified as Ca2+ channels, at the active zone of an unstimulated neuromuscular junction. Figure 8 shows an active zone after stimulation, with transmembrane openings (one within a box) considered to be "exocytotic stomata." The arrows point to regions interpreted as collapsed vesicles incorporated into the presynaptic membrane. (Reproduced by permission of Rockefeller University Press.)

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Ceccarelli, however, argued against a collapse of vesicle into the cell membrane and for a "quick direct removal of the vesicle membrane without it flattening into the [cell membrane]."30 Through the 1980s controversies persisted about the fate of the vesicle membrane: whether release required coalescence with the cell membrane or involved merely a transient opening between vesicle interior and synaptic cleft, followed by withdrawal of the emptied vesicle.

Biochemical Studies

Adrenal vesicles contain ATP as well as catecholamines, and in 1956 Hillarp described the concomitant disappearance of these compounds during stimulation.31 Since ATP was readily hydrolyzed, Hillarp suggested that ATP played a role in catecholamine release, which he imagined was first from vesicle to cytoplasm and second from cytoplasm to exterior. But in 1965 William Douglas in New York described an efflux into the perfusion fluid of ATP (and its degradation products) as well as of catecholamines.32 Moreover, he calculated that the ratio of released ATP (plus degradation products) to released catecholamines was close to the ratio present in vesicles. Douglas pointed out that the polar adenine nucleotides could not diffuse readily across lipoidal cell membranes, so release from the cytoplasm seemed unlikely (catecholamines would experience the same difficulty). Nevertheless, membrane carriers might be present to allow their exit from adrenal cells. Proteins also do not diffuse across membranes, and carriers for mediating protein transport seemed implausible. Consequently, any exit of proteins accompanying catecholamine release would support proposals for exocytosis. As noted above, Hillarp in 1956 felt that a smaller fraction of "soluble" proteins was released from vesicles than of catecholamines.33 But more specific studies on intravesicular proteins proved otherwise. In 1965 P. Banks in Sheffield and Karen Helle in Bergen collaborated to demonstrate the release of a prominent protein from within adrenal vesicles.34 This protein, subsequently purified and named chromagranin A, appeared in the perfusion fluid along with catecholamines when and only when adrenals were stimulated.35 By contrast, stimulation did not release cytoplasmic proteins. Equally compelling were studies on dopamine-j8-hydroxylase, the enzyme that converts dopamine to noradrenaline and is present within adrenal vesicles. In 1968 Norman Kirshner in Durham reported that after stimulation this enzyme, too, accompanied catecholamines into the perfusion fluid.36 Adrenergic nerves also contain chromagranin A and dopamine-/3-hydroxylase within their synaptic vesicles, and by 1971 similar studies demonstrated that adrenergic nerves, too, released these proteins in response to stimulation.37 Biochemical evidence for the fusion of vesicle with cell membrane appeared as well. For example, Regis Kelly in San Francisco reported in 1981 that after

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stimulation some components of the vesicle membrane were now detectable (with antibodies) in the presynaptic membrane.38 Electrical Studies

The quantal hypothesis arose from studying electrical depolarizations in postsynaptic cells (e.p.p.s and m.e.p.p.s). Two other modes of electrical recordings also provided continuous records of rapidly changing events, bolstering the static morphological and biochemical observations. Exocytotic fusion would increase the surface of presynaptic neurons by adding vesicle membrane to cell membrane. Among the consequences of this increased area would be an increased electrical capacitance.39 Accordingly, J. I. Gillespie in Plymouth reported in 1979 that stimulation increased the capacitance of presynaptic terminals at squid giant synapses, changes he identified with "the incorporation of synaptic vesicles into the presynaptic membranes."40 Although Gillespie could not quantitate the changes, Erwin Neher in Gottingen applied his patch electrodes (chapter 6) successfully to this task, describing in 1982 discrete steps of capacitance changes during stimulation of adrenal cells.41 These he attributed to successive exocytotic fusions of catecholamine-containing vesicles. To achieve still better resolution, Neher next studied exocytosis from mast cells (cells present throughout the body that participate in inflammatory responses and that contain large histamine-containing vesicles available for release). With these cells he could detect an initial flickering of the capacitance values that he interpreted as the reversible formation of an "aqueous pore between the secretory vesicle and [cell] membrane, before formation of the (irreversible) fused state."42 And in 1987 Wolfhard Aimers in Seattle combined patch electrode recordings with fluorescence measurements of exocytotic release from mast cells (Fig. 10-4).43 He described the flickering capacitance changes as the reversible openings of "fusion pores" linking vesicle interior to cell exterior prior to the irreversible fusion of membranes (Fig. 10-1C). Neher s and Almers's studies thus supported Heusers characterization of "narrownecked pores" in electron micrographs of mast cells, a process that began "with formation of a narrow orifice" before the ultimate fusion of vesicle with cell membrane.44 These studies provided strong evidence for exocytotic release from mast cells, but comparable studies on neurons were not technically feasible by 1990. A second realm of electrical studies used voltammetry to monitor neurotransmitter release. Katz's identification of quantal release—and subsequent electrophysiological studies—equated presynaptic release with postsynaptic depolarizations. These postsynaptic responses, however, need not represent release precisely, for they depended on the intervening ability of postsynaptic

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FIGURE 10-4. Capacitance and fluorescence studies of release from mast cell vesicles. The upper tracing shows an initial flickering of the capacitance changes (in pF) after stimulation of release. The lower trace, over the same time course, shows the fluorescence change (F), representing the release of a fluorescent compound from the vesicles, beginning after the period of flickering. (From Breckenridge and Aimers [1987], Fig. 3B, courtesy of Wolfhard Aimers.)

receptors to mirror every change in neurotransmitter level. Obviously, a better approach would be to measure the released neurotransmitter directly. For neurotransmitters that are readily oxidized, such as catecholamines and serotonin, voltammetry provided such an alternative. (In this technique current is passed through electrodes and the flow measured at various voltages. Oxidations at the electrode tip alter this current flow at characteristic voltages.) This technique had been used for over a decade to record neurotransmitter release in vivo when in 1990 Mark Wightman in Chapel Hill placed microelectrodes near adrenal cells and found current transitions that he interpreted as the "direct chemical measurement of single exocytotic events."45 Complexities ana Criticisms

By 1990 a wealth of studies provided firm support for exocytosis. Still, evidence for exocytosis was most compelling for secretory cells, such as adrenal and mast cells; for neurons evidence was strongest at neuromuscular junctions. Generalizations from these cases to all chemical synapses satisfied most investigators, but sufficient complexities were apparent to provoke continuing criticisms from an active few. First, not all neurotransmitter release was quantal. In the absence of stimulation, even acetylcholine release at neuromuscular junctions failed this criterion: in 1963 John Mitchell in Babraham measured release by bioassay and found that at rest only a small fraction was attributable to quantal release, with "a large proportion . . . derived from some other source."46 Indeed, Katz and

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Ricardo Miledi argued in 1977 that "a steady leakage . . . from the terminals [being monitored could] exceed the . . . quantal discharge by at least an order of magnitude."47 Leakage, however, did not increase during stimulation, so that quantal release then became far larger. Second, newer studies revealed less clear-cut characteristics of quanta!/ vesicular release than initial measurements at neuromuscular junctions had implied. The number of acetylcholine molecules per vesicle, calculated from brain and sympathetic ganglia preparations, was 1000-2000, whereas the number of molecules per m.e.p.p. (or quantum) was 6000-10,000; in the far larger vesicles of electric organ preparations, the number was greater than 100,000.48 These imperfect correlations between vesicle and quantum accompanied renewed uncertainty about statistical analyses of release. Katz and del Castillo calculated in 1954 that the number of quanta available for discharge was roughly 200.49 This was far smaller than the number of vesicles present in the region being studied, and in 1977 A. Wernig in Munich argued that "a quantum might be due to the simultaneous discharge of several or all the vesicles in one 'active zone.' "50 Wernig's interpretation not only provided a value comparable to Katz and del Castillo's estimate, it also fitted descriptions by Mahlon Kriebel in Syracuse of postsynaptic responses smaller than m.e.p.p.s (together with multiples of these sub-m.e.p.p.s).51 KriebePs observations raised the possibility that m.e.p.p.s—and conventional quanta—were composed of subunits: each m.e.p.p. could require the release of more than one vesicle, and one vesicle (or less) could elicit a sub-m.e.p.p. Moreover, Henri Korn in Paris and Donald Faber in Buffalo analyzed release at brain synapses as independent all-or-none processes at each active zone, which could represent the coordinate discharge from an invariant number of vesicles.52 Third, neurotransmitters seemed to be present in at least two pools, but the functions and subcellular locations of these pools were debated. On the one hand, Richard Birks and F. C. Macintosh in Montreal described the stimulated release of acetylcholine from sympathetic ganglia in 1961, interpreting their data as the sequential outflow from two intraneuronal pools.53 They suggested that the smaller, more readily releasable pool corresponded to vesicles close to the presynaptic membrane. The larger pool might then serve as a reservoir for replacing acetylcholine in emptied vesicles of the smaller pool. Indeed, studies in the late 1960s using radioactive tracers supported models depicting a readily releasable pool that was preferentially replenished. For example, Irwin Kopin in Bethesda administered a labeled precursor of noradrenaline and then compared the radioactivity in the noradrenaline released by stimulation from an adrenergic nerve to that remaining in the nerve: he concluded that "newly synthesized [noradrenaline] is selectively released."54 On the other hand, these labeling experiments did not actually identify the pool of readily releasable neurotransmitter as that in vesicles adjacent to the presynaptic membrane. And in the 1970s some—notably Maurice Israel in

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Paris, Y. Dunant in Geneva, Ladislav Tauc in Gif-sur-Yvette, and Roger Marchbanks in London—concluded that the newly synthesized and readily releasable pool was cytoplasmic, not vesicular.55 By this time the synthesis of many neurotransmitters was known to occur in the cytoplasm, and cell fractionation studies revealed significant amounts of cytoplasmic neurotransmitter. These critics noted that vesicle numbers did not always change in parallel with neurotransmitter content and that after stimulation the cytoplasmic neurotransmitter could change more—in absolute amount and in degree of labeling—than the vesicular. In 1974 Herbert Zimmermann, visiting Victor Whittaker in Cambridge, found, contrary to the report of Dunant and Israel, that after stimulating nerves to Torpedo electric organ, the acetylcholine content of the vesicles initially fell in parallel with the decline in transmission; only after prolonged stimulation were the vesicles depleted of acetylcholine.56 And in 1977 Zimmermann and Whittaker, now in Gottingen, described the acetylcholine content of vesicles that had undergone exocytosis/endocytosis. They stimulated electric organ tissue in media containing sucrose as well as a labeled precursor of acetylcholine, so that sucrose was taken up by the endocytosis that followed exocytosis. When they next isolated the vesicles by density-gradient centrifugation, those vesicles containing sucrose were denser and thus separable from vesicles that had not undergone the exocytotic/endocytotic cycle, and the denser vesicles contained manyfold higher amounts of radioactivity.57 Thus, the emptied vesicles were preferentially filled with new acetylcholine, in accord with the exocytotic model. Advocates of cytoplasmic release also had to struggle to imagine a mechanism that could discharge neurotransmitters in quantal packets. One suggestion was for release through voltage-gated channels that would open transiently to allow a given number of neurotransmitter molecules to diffuse out.58 But William Van der Kloot in Stony Brook showed that quantal size was not altered by changes in the membrane potential of the nerve ending, as would be expected if acetylcholine release were governed by such a channel.59 Alternatively, channel openings for given periods of time might produce quantal release. But Van der Kloot found that altering neurotransmitter concentrations in the cytoplasm (by increasing or decreasing the water content of the cell, concentrating or diluting cytoplasmic solutes) also failed to change quantal size.60 Nevertheless, Israel continued through the 1980s to advocate release by channels, even isolating a protein that he believed was this channel.61

Triggering or Release

In 1940 Macintosh, then in Hamptead, reported that extracellular Ca2+ was required for acetylcholine release from sympathetic ganglia.62 His observation extended earlier studies stretching back to the nineteenth century that showed

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extracellular Ca2+ was necessary for nerves to elicit muscle contractions. Others, however, advocated roles for K + and Na + in promoting neurotransmitter release, as noted above. During investigations of such cation effects, Katz and Fatt included examinations of how Ca2+ affected neuromuscular transmission, and in 1952 they uncovered a "curious effect": lowering extracellular Ca2+ "reduces the e.p.p. in definite 'quanta,'" that is, in a stepwise fashion.63 That year del Castillo reported correlations between Ca2+ concentrations and e.p.p. amplitude, suggesting that "the amount of acetylcholine released . . . is a function of the concentration of calcium ions."64 Two years later Katz and del Castillo recast such interpretations as Ca2+ increasing the quantal content of e.p.p.s, and they argued that Ca2+ acted by raising the probability of quantal release.65 Katz and Miledi then used ganglionic synapses of squid giant axons, where neuronal elements are large enough for direct electrical manipulations, to demonstrate that graded electrical depolarizations, induced presynaptically, could elicit graded responses postsynaptically as long as extracellular Ca2+ was present.66 But when they raised the transmembrane potential of the presynaptic terminals so that Ca2+ influx could no longer occur,67 there was no sign of neurotransmitter release. Douglas's work on secretory cells in the early 1960s, from which he promulgated the concept of Ca2+-dependent "stimulus-secretion coupling" (chapter 7), thus complemented the conclusions of Katz and his collaborators. Taken together, these studies promoted the notion that exocytotic release required Ca2+ and that a requirement for Ca2+ implied exocytotic release. The mechanism by which Ca2+ acted was less clear, however. Katz and collaborators first proposed an acetylcholine carrier in the membrane that must initially contain Ca2+.68 On the other hand, Hodgkin and Richard Keynes, citing the disruptive effects of Ca2+ on cytoplasmic structures, suggested in 1957 that intracellular Ca2+ might be involved "in breaking up the intracellular vesicles near the membrane . . . and releasing acetylcholine from them."69 Although it was soon clear that Ca2+ must enter the cytoplasm to act, what happened next would remain uncertain during the next 30 years. Defining the entry process progressed more rapidly. In 1967 Katz and Miledi concluded that "depolarization opens a 'gate' to calcium," and since pulses of extracellular Ca2+ were effective only at brief intervals during a stimulus, nerve activity must open such gates transiently.70 They also measured currents, attributable to Ca2+ influxes, that were associated with neurotransmitter release.71 The Hodgkin-Huxley model for nerve action potentials relied on voltagegated channels for Na + and for K + that opened in response to membrane depolarizations. Voltage-gated Ca2+ channels were identified soon thereafter. In 1985 voltage-gated Ca2+ channels were allocated among several classes, based on electrical properties and sensitivities to particular inhibitors, and the amino

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acid sequence of one class of channels was defined in 1987.72 Meanwhile, Baldomero Olivera and William Gray identified a toxin from cone snails that caused muscle paralysis, a peptide that they named w-conotoxin.73 The previous year, 1984, Lynne Kerr and Doju Yoshikami, also in Salt Lake City, had described this toxin's ability to block neurotransmitter release by preventing Ca2+ entry.74 The w-conotoxin was subsequently confirmed as a specific blocker of the pertinent class of Ca2+ channels and became a valuable reagent for studying the role of Ca2+ in neurotransmitter release as well as for identifying these channels. Miledi succeeded in 1973 in showing that Ca2+ injected into presynaptic terminals could evoke neurotransmitter release, confirming a cytoplasmic role.75 A year earlier Rodolfo Llinas in Rochester, Minnesota demonstrated, using a light-emitting Ca2+-indicator, that cytoplasmic Ca2+ levels within presynaptic terminals rose following stimulation, in accord with electrical measurements of Ca2+ currents through transmembrane channels.76 Further studies with a variety of indicators confirmed such rises, but the limited spatial and temporal resolution of these techniques were inadequate for precise characterization of the cytoplasmic Ca2+ changes. Nevertheless, calculations showed that, due to local binding, the regions of elevated Ca2+ would be tightly circumscribed after a localized influx.77 This spatial restriction was pertinent to the placement of calcium channels relative to neurotransmitter release sites. In 1974 Heuser and Reese suggested that the rows of intramembrane particles visible in freeze-fracture micrographs of synaptic active zones (Fig. 10-3) might be Ca24" channels.78 Llinas, now in New York, supported this identification in 1981 by correlating the number of such particles, the conductance of Ca2+ channels (previously determined), and the measured current flow across presynaptic terminals.79 And in 1990 Milton Charlton in Toronto used labeled w-conotoxin to demonstrate that these Ca2+ channels were present exclusively at the active zones.80 This localization would therefore allow the efficient interaction of incoming Ca2+ with synaptic vesicles arrayed at the active zones, poised—according to the exocytotic model— to discharge their neurotransmitter into the synaptic cleft.

Mechanism of Release

How does this localized Ca2+ influx trigger exocytosis and how does the exocytotic machinery work? Early explanations invoked simple solutions. John Eccless suggestion in 1959 took the form of a question: "Is it possible that vesicles are positively charged and hence are greatly accelerated . . . across a depolarized membrane?"81 In 1966, however, Banks demonstrated that adrenal vesicles bore instead net negative charges, incompatible with such an elec-

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trophoretic extrusion.82 Banks then proposed that divalent Ca2+ could link negatively charged vesicle membranes to (presumably) negatively charged cell membranes, thereby promoting fusion and exocytosis, although he did not specify how the latter events occurred. Defining the basic processes, it was clear, would require a better understanding of the components. It was also clear in 1990 that the identification of these components was still incomplete. Synapsin

The first participating protein to be identified was synapsin I, although it was discovered through another interest. While exploring cAMP-stimulated protein phosphorylations, Paul Greengard in New Haven found that synaptic membranes provided excellent substrates, and in 1973 he identified a protein in these membranes that was rapidly phosphorylated when he added cAMP.83 He named it protein I because its molecular weight, 86 kDa, placed it first among the phosphorylated proteins on his electrophoresis gels. With the purified protein, isolated in 1977,84 Greengard prepared specific antibodies for identifying its subcellular localizations. He had initially shown that protein I was present only in the nervous system; with these immunocytochemical techniques he could show in 1979 that protein I was not only concentrated in presynaptic terminals, but it lay on the surface of synaptic vesicles.85 In 1983 he renamed it synapsin I to emphasize this localization.86 Meanwhile, Greengard found that Ca2+-dependent CaM kinase as well as cAMP-dependent protein kinase A phosphorylated synapsin I, but at different points on its amino acid sequence.87 Then, collaborating with Llinas, he showed in 1985 that injecting unphosphorylated synapsin I into presynaptic terminals of squid giant synapses decreased postsynaptic responses.88 By contrast, injecting CaM kinase into the terminals increased responses. Greengard could now argue that Ca2+ influx through voltage-gated channels would facilitate neurotransmitter release through Ca2+-dependent phosphorylation of synapsin I. Earlier, Greengard suggested that synapsin I mediated interactions between synaptic vesicles and cytoskeletal proteins, such as actin and tubulin, that form a scaffolding of rods and filaments to control cell shape and to guide organelles through the cytoplasm.89 In 1987 Greengard, now in New York, showed that adding synapsin I to actin filaments in vitro caused them to coalesce into bundles, whereas phosphorylating synapsin I with CaM kinase abolished this ability.90 And in 1989 Nobutaka Hirokawa in Tokyo published electron micrographs of nerve terminals demonstrating networks of actin filaments that were linked by synapsin I to the synaptic vesicles. He, too, argued that phosphorylation of synapsin I "could release synaptic vesicles from actin filaments . . . and thus increase the mobility of synaptic vesicles to the presynaptic membrane."91 These interactions, however, seemed to be involved with recruiting vesicles to active zones rather than with fusion and exocytosis.

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Synaptotagmin, Synaptophysin, and. Synaptobrevin

Synapsin I was a peripheral membrane protein, linked to the synaptic vesicle surface but readily dissociable. In the 1980s three tightly bound integral membrane proteins were identified, and these seemed likely participants in the exocytotic process. At the beginning of this decade Louis Reichardt in San Francisco identified a 65 kDa protein present in synaptic vesicle membranes, using a collection of antibodies against synaptic membranes to identify component proteins.92 Initially known as p65, it was renamed synaptotagmin in 1991.93 Thomas Siidhof in Dallas reported the amino acid sequence in 1990, which indicated a single transmembrane segment.94 Of particular interest were sequences in synaptotagmin similar to the regulatory domain of protein kinase C, for that domain was thought to include Ca2+- and phospholipid-binding regions. As the decade closed enthusiasm was growing for synaptotagmin being part of the Ca2+sensitive trigger for exocytosis. A second integral membrane protein, synaptophysin, was identified in 1985 by Greengard as a prominent 38 kDa band on SDS-PAGE gels of synaptic vesicle proteins, and by Bertram Wiedenmann and Werner Franke in Heidelberg using antibodies against synaptic vesicle proteins to screen extracts.95 Two years later they reported the amino acid sequence, which indicated four transmembrane segments.96 They also noted analogies to the protein that forms gap junctions (chapter 4), and they suggested that oligomers of synaptophysin also might form a transmembrane pore. In 1988 the Heidelberg group reconstituted synaptophysin into artificial lipid membranes, demonstrating channel conductances.97 To form a pore from vesicle interior to synaptic cleft, however, would require alignment with another channel to cross the presynaptic membrane. Richard Scheller in Palo Alto described in 1988 a third integral membrane protein from synaptic vesicles, using antibodies against the membranes to screen proteins expressed by a cDNA library from Torpedo electric organ.98 The following year Reinhard Jahn in Martinsreid identified the corresponding protein in mammalian brain, which he named synaptobrevin because of its low molecular weight, 18 kDa.99 But the function of this protein remained unknown as the decade ended. Neurexin and SNAP-25

Two proteins associated with presynaptic membranes were also detected. A protein in black widow spider venom, a-latrotoxin, causes a massive exocytotic release of neurotransmitter even in the absence of Ca2+, and in 1985 J. Meldolesi in Milan identified a protein in synaptic membranes that bound to a-latrotoxin.100 It was subsequently included in a family of integral membrane proteins called neurexins.101 And Michael Wilson in La Jolla found a 25 kDa protein by screening a cDNA library, which in 1989 he named SNAP-25 (for

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synaptosomal associated protein, plus its molecular weight).102 SNAP-25 did not contain a likely transmembrane segment, although it was tightly bound to membranes. No more could be said about its physiological role by 1990. Vesicular Transport in Non-Neural Systems

While these studies were in progress, considerable success rewarded examinations of intracellular transport between membrane-bounded cisterns of the cellular Golgi system as well as secretory systems of various nonneural cells (notably yeast).103 These processes represented "membrane trafficking" through the budding off of vesicles from one membrane and their fusion with another: endocytosis and exocytosis. As the 1990s began interest was growing in the parallels between exocytotic fusions at diverse cellular sites.104 (During the 1990s further progress was achieved in identifying additional components of the synaptic exocytotic process, in drawing parallels with entities and processes among disparate secretory systems, and in constructing models showing how interacting entities accomplished exocytotic release. Nevertheless, no definitive model was available as the century ended.)

Enaocytotic Retrieval or Vesicles

Given the exocytotic explanation of neurotransmitter release, there remained the problem of what happens to the emptied synaptic vesicle. Models for vesicle retrieval ranged from a simple reversal of the exocytotic event to an endocytotic recapture, through coated vesicles, following the full incorporation of synaptic vesicles into presynaptic membrane (Fig. 10-1C and B). The former mechanism gained favor in the 1980s, reinforced by observations of flickering capacitance measurements that were interpreted as transient openings and closings of a fusion pore. But this flickering preceded the massive expulsion (Fig. 10-4), and little quantitative evidence was forthcoming for such reversible processes being the physiological mode of neurotransmitter release. On the other hand, studies on coated vesicle systems through the 1980s demonstrated their involvement in a broad range of cellular functions, from membrane transfer between intracellular organelles to uptake of extracellular proteins. These studies also revealed a spectacular molecular mechanism. While studying protein secretion and absorption, Keith Porter in Cambridge, Mass., turned to the uptake of yolk protein by mosquito oocytes. In 1964 he reported that this uptake correlated with the appearance of fuzzy "bristlecoated" pits, which apparently next became fuzzy bristle-coated vesicles.105 Porter suggested that this coating on the convex surfaces might serve a mechanical function in such transitions. Five years later Toku Kanaseki and Ken Kadota

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in Osaka described "baskets" around vesicles from brain: a series of regular pentagons and hexagons like the seams on a soccer ball (Fig. 10-5).106 They proposed that transformations of hexagons into pentagons caused the curvatures required to form first pits and then vesicles from the relatively planar presynaptic membrane. Barbara Pearse in Cambridge, UK, then developed an effective procedure for isolating coated vesicles from brain. The protein component of this purified preparation, analyzed by SDS-PAGE, consisted of "essentially just one protein species" having a molecular weight of 180 kDa; this she named clathrin.107 The following year, 1976, Pearse concluded from electron micrographs that these coated vesicles were bounded by 12 pentagons plus a variable number of hexagons, depending on the vesicle diameter.108 She suggested that three clathrin molecules met at each vertex of the polygons. In 1981 Ernst Ungewickell and Daniel Branton in Cambridge, Mass., dissociated the baskets into three-legged structures with pronounced knees, which they named triskelions (Fig. 10-6).109 Each triskelion contained three clathrin molecules (180 kDa) plus three smaller proteins (33—36 kDa); these became known as the clathrin heavy and light chains, respectively.110 That year Pearse depicted rearrangements of triskelions to convert hexagons to pentagons (Fig. 10-7),m but the mechanism for such transformations was still unresolved when the decade ended. Meanwhile, James Keen and Ira Pasten in Bethesda showed that clathrin baskets could be released from coated vesicles by solutions of high ionic strength, fragmenting the baskets into filaments.112 Remarkably, this transformation was reversed when they reduced the ionic strength sharply, with the filaments reassembling into empty baskets. This spontaneous reconstitution of

FIGURE 10-5. Baskets of coated vesicles, from Kanaseki and Kadota (1969). Figure 24 shows a soccer ball composed of hexagonal and pentagonal panels, while Figure 25 shows a model of a coated vesicle with similar hexagons and pentagons outlining its surface. Figure 26 is an electron micrograph of a coated vesicle from the brain, also showing outlines of hexagons and pentagons. (Reproduced by permission of Rockefeller University Press.)

FIGURE 10-6. Electron micrographs of clathrin triskelions from brain coated vesicles. (From Ungewickell and Branton [1981], Fig. 3a, reprinted by permission of Nature, ©1981 Macmillan Magazines, Ltd.)

FIGURE 10-7. Proposal for the conversion of clathrin hexagons to pentagons through "small distortions of the triskelion." (From Crowther and Pearse [1981], Fig. 4. Reproduced by permission of Rockefeller University Press.)

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the baskets did not occur at cytoplasmic ionic strengths, but Keen and Fasten identified a fraction from coated vesicles—containing a 110 kDa protein—that mediated the reconstitution under physiological conditions. They named this the basket assembly factor. Subsequent studies in the 1980s revealed additional assembly proteins with molecular weights of roughly 50 and 20 kDa; together with the 110 kDa protein, they formed complexes capable not only of controlling the size of the coated vesicles but also of selecting which areas of the membrane surface would be transformed into coated pits and vesicles.113 Moreover, the assembly protein complexes were themselves regulated by phosphorylation/dephosphorylation cycles. Before endocytotic vesicles can fuse with membranes their coats must be removed. But whereas coat assembly proceeded without an extrinsic energy source, coat removal required added ATP. In 1982 James Rothman in Palo Alto found that a cytoplasmic factor promoted dissociation of the baskets at physiological ionic strengths if ATP were present.114 ATP was hydrolyzed during the dissociation process, and Rothman then purified the "uncoating ATPase," identifying a 70 kDa cytoplasmic protein that stripped the coats away.115 In 1986 he grouped this enzyme with a family of 70 kDa proteins mediating cellular responses to stress (called heat shock proteins because of the stress first studied).116 The following year John Ellis in Coventry included these proteins in his category of molecular chaperones, proteins that assist other proteins in folding and unfolding.11' But what spurred the uncoating ATPase into action was not then apparent.

Ca

-Independent Non-Exocytotic Release

The critics of exocytotic release noted above were concerned with Ca2+dependent processes. Different issues arose with the recognition by 1970 that release could also occur in the absence of extracellular Ca2+.118 If such Ca2+independent release implied nonexocytotic mechanisms, then an obvious candidate for carrying neurotransmitters from cytoplasm to synaptic cleft would be the cell membrane reuptake transporter (chapter 9) running in reverse.119 These Na + cotransport systems are normally driven by Na4" flowing down its electrochemical gradient from outside the cell to the inside, carrying the neurotransmitter in the same direction. But if the gradients were reversed—by electrical depolarization and/or lower ratios of extracellular to intracellular Na + and/or lower ratios of extracellular to intracellular neurotransmitter—the Na + cotransport system could carry cytoplasmic Na + plus neurotransmitter out of the cell.120 Accordingly, in 1978 Carl Cotman in Irvine reported that depolarizing synaptosomal preparations with high concentrations of extracellular K + could trig-

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ger the release of GABA even in the absence of extracellular Ca2+.121 Moreover, this Ca2+-independent release appeared to come from a different intracellular pool of GABA (as reflected in the degrees of labeling) than did the Ca2+-dependent release that could also be evoked. If they first incubated the synaptosomes in Na+-free media with agents that facilitated Na+ fluxes, then the subsequent Ca2+-independent release was diminished; this was explainable as the manipulations causing a loss of intracellular Na + , which would magnify the Na + gradient from out to in. Conversely, raising intracellular Na + by facilitating influx or by hindering efflux, which would minimize the gradient, promoted Ca2+-independent GABA release.122 Nevertheless, release in the absence of extracellular Ca2+ might still be occurring through a Ca2+-dependent exocytotic process, but one sensitive to elevated levels of intracellular Na + : in 1970 David Lust and Joseph Robinson in Syracuse proposed that higher concentrations of intracellular Na + could free into the cytoplasm Ca2+ sequestered within mitochondria.123 Consequently, orthodox exocytotic release that requires a rise in intracellular Ca2+ could still be operating in the absence of extracellular Ca2+ under these circumstances. But in 1988 S. Bernath and M. J. Zigmond in Pittsburgh directly implicated the cotransport system, showing that an inhibitor of the Na + -GABA cotransporter blocked neurotransmitter release in the absence of extracellular Ca2+.124 Similar studies with other neurotransmitters also implicated Ca2+-independent release in particular instances.125 Three categories of such Ca2+-independent release were established by 1990, as illustrated below.

Physiological Release

Horizontal cells in the retina release GABA even though they do not contain synaptic vesicles, and in 1982 Eric Schwartz in Chicago described release from these cells in the absence of extracellular Ca2+.126 Subsequently, Schwartz showed that such release occurred even when the intracellular Ca2+ concentration was kept low with buffers to counteract any rise in Ca2+ contributed by intracellular reservoirs; on the other hand, GABA release was blocked by an inhibitor of the Na + cotransporter.12'

Pathological Release

Various deleterious conditions, such as anoxia, damage the brain by triggering the release of glutamate, but this release does not require extracellular Ca2+.128 Anoxia and similar perturbations also elevate cytoplasmic Na+ concentrations, due in part to the loss of active transport systems that normally extrude this cation. So in 1990 David Attwell in London proposed that this glutamate release was due to a reversal of the Na + cotransporter: experimentally elevated con-

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centrations of intracellular Na + and glutamate promoted release, whereas elevated concentrations of extracellular Na + and glutamate inhibited release.129

Pharmacological Release

Amphetamine acts in large part by promoting the release of catecholamines and serotonin, but in 1975 Julius Axelrod in Bethesda demonstrated that such release was not exocytotic.130 Electrical stimulation of adrenergic nerves released not only noradrenaline but also dopamine-/3-oxidase, an enzyme normally present within the synaptic vesicles, so that its appearance extracellularly signified exocytosis. By contrast, administering amphetamine released noradrenaline without dopamine-j3-oxidase, and this release occurred in the absence of extracellular Ca2+. In 1990 David Sulzer in New York argued that amphetamine promoted the loss of catecholamines from the synaptic vesicles, with the consequent rise in cytoplasmic levels of catecholamines then driving the Na + cotransporters in reverse to release catecholamines.131 Indeed, inhibitors of the cotransport systems diminished amphetamines ability to elicit release.132 These explanations of C a2+-independent release seemed plausible for neurotransmitters having Na + cotransport systems, such as catecholamines, serotonin, GAB A, and glutamate. But they were obviously inapplicable to acetylcholine, which has no such system. For this case Charles Edwards, visiting Frantisek Vyskocil in Prague, noted that after exocytotic release of acetylcholine, the vesicular H+/acetylcholine exchanger would be incorporated by exocytotic fusion into the presynaptic membrane.133 They suggested that these exchangers could then mediate Ca2+-independent acetylcholine release across the cell membrane until they were retrieved by endocytosis (they would then be replaced by new exchangers from vesicles that fused subsequently). Correspondingly, Vyskocil reported in 1985 that an inhibitor of the H+/acetylcholine exchanger blocked Ca2+-independent release of acetylcholine.134

Conclusions

The quantal hypothesis, advanced by Katz in the early 1950s from studies at neuromuscular junctions, attributed e.p.p.s to summations of m.e.p.p.s, each representing the release of a uniform packet of neurotransmitter. Soon afterward electron microscopists described vesicles in nerve terminals, and Katz identified his quanta with the contents of these vesicles. This elaboration transformed his quantal hypothesis to an exocytotic one: release was achieved through the fusion of neurotransmitter-containing vesicles with presynaptic membrane. Evidence supporting and extending these models accumulated in the following decades, with measurements of neurotransmitters within vesicles,

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identifications of vesicular contents released exocytotically, micrographic demonstrations of vesicle fusion and retrieval, and calculations of membrane surface changes attendant on these fusions. Other studies, again initiated by Katz and associates, tied quantal/vesicular release to an influx of Ca2+ through specific channels in presynaptic membranes. In the 1980s biochemists began identifying proteins in vesicle and presynaptic membranes that were plausible components of the exocytotic machinery. Essential entities were still unidentified as the decade closed, however, and a comprehensive model—one defining how rises in cytoplasmic Ca2+ triggered the sequence of protein-protein interactions to effect membrane fusion—could not yet be formulated. On the other hand, electron microscopists and biochemists developed a satisfying model for retrieving vesicle membranes after exocytotic fusion, specifying the formation of clathrin-containing coated vesicles. As in previous chapters, prominent and convincing results emerged from examining favorable, experimentally accessible preparations. Here these included neuromuscular junctions, electric organs, and adrenal and mast cell vesicles. Analogously, certain experimental conditions were particularly rewarding, such as modified Ca2+ concentrations and rapid rates of stimulation. Generalizations in biology are always precarious, with prototypic representations inevitably failing somewhere. A critical issue, then, is the range of similarities. But establishing this scope is frequently frustrated when less favorable preparations must be studied. For example, did the exocytotic release that was demonstrable with high rates of stimulation also occur at physiological rates? Did full fusion of vesicles occur routinely, requiring then retrieval by clathrincoated vesicles, or were transient and reversible associations the rule normally? How did the quantal hypothesis accommodate nerve terminals having greater heterogeneity among their vesicles? Indeed, clear incompatibilities were documented in specific cases, as in certain instances of Ca2+-independent, nonvesicular release. Nevertheless, by 1990 models of vesicular/exocytotic release were generally acknowledged as the standard formulation.

Notes 1. Brown and Feldberg (1936b), p. 291. See also Feldberg and Guimarais (1936). 2. Dale (1938b), p. 420. 3. Brown and Macintosh (1939). Stimulating the presynaptic fibers would cause them to release more neurotransmitter. 4. Goodman and Gilman (1956), p. 399. 5. Fatt and Katz (1952a). 6. Fatt and Katz (1952c). This model followed an earlier proposal by Hodgkin and Huxley for the ion transfer during action potentials occurring by membrane carriers

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(although by this time Hodgkin and Huxley had withdrawn their proposal because of contrary evidence). 7. See Robinson (1997). 8. DeRobertis and Bennett (1954), p. 35. The observation and interpretation was repeated in DeRobertis (1958). 9. del Castillo and Katz (1955b), p. 410. 10. del Castillo and Katz (1957). The meeting at which they presented this paper was in 1955. 11. Katz (1962), p. 471. 12. de Duve (1963). 13. Hillarp et al. (1954), p. 162. 14. Carlsson and Hillarp (1956). 15. Ibid., p. 239. 16. von Euler (1966). 17. For example, Birks et al. (1960); Hubbard and Kwanbunbumpen (1968). 18. Active zones were depicted by Birks et al. (1960) and named by Couteaux and Pecot-Dechavassine (1970). 19. Jones and Kwanbunbumpen (1970). Parducz and Feher (1970), however, claimed that hemicholinium caused a breakdown of synaptic membranes due to the cell's consequent lack of choline for phospholipid biosynthesis. 20. For example, Birks (1971) stimulated at 20 Hz for 20 minutes without hemicholinium; Pysh and Wiley (1972) used interrupted trains of stimuli at 20 to 32 Hz for 150 to 190 minutes in the presence of hemicholinium. 21. Pysh and Wiley (1972), p. 192. See also Pysh and Wiley (1974). 22. Ceccarelli et al. (1972). See also Ceccarelli et al. (1973). 23. Holtzman et al. (1971) had earlier correlated peroxidase uptake with nerve activity. 24. Heuser and Reese (1973). 25. Heuser et al. (1974). See also Pfenninger et al. (1972); Peper et al. (1974). 26. For example, Birks et al. (I960); Couteaux and Pecot-Dechavassine (1970); Douglas et al. (1970). 27. Douglas et al. (1971) had already proposed a role for coated vesicles in neurosecretory cells of the pituitary. 28. Heuser et al. (1979), p. 275. They also stimulated transmitter release chemically to improve the yield of exocytotic processes. Previously, Birks et al. (1960) calculated that exocytotic processes should be too rare to record in micrographs consistently. See also Heuser and Reese (1981). 29. Torri-Tarelli et al. (1985). 30. Ibid., p. 1398. 31. Carlsson and Hillarp (1956). 32. Douglas et al. (1965). 33. Carlsson and Hillarp (1956). 34. Banks and Helle (1965). 35. For example, Blaschko et al. (1967); Kirshner et al. (1967); Schneider et al. (1967). 36. Viveros et al. (1968, 1969). 37. For example, Geffen et al. (1969); Weinshilboum et al. (1971). 38. von Wedel et al. (1981). 39. Capacitance changes linked to surface area changes had been studied with other cellular phenomena earlier. For example, Rothschild (1957) determined with this approach the cell surface changes that accompany fertilization.

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40. Gillespie (1979), p. 304. 41. Neher and Marty (1982). 42. Fernandez et al. (1984), p. 454. 43. Breckenridge and Aimers (1987). They used mast cells from a strain of mice having "giant" vesicles. See also Zimmerberg et al. (1987). 44. Chandler and Heuser (1980), pp. 666, 670. 45. Leszczyszyn et al. (1990), p. 14,736. 46. Mitchell and Silver (1963), p. 126. See also Fletcher and Forrester (1975); Vizi and Vyskocil (1979). 47. Katz and Miledi (1977), pp. 59-60. 48. Whittaker and Sheridan (1965); Wilson et al. (1973); Fletcher and Forrester (1975); Kuffler and Yoshikami (1975); Ohsawa et al. (1979). 49. del Castillo and Katz (1954a). 50. Wernig and Stirner (1977), p. 821. 51. Kriebel and Gross (1974); Kriebel at al. (1990). 52. Korn et al. (1981). 53. Birks and Macintosh (1961). 54. Kopin et al. (1968), p. 271. They compared "specific activities": radioactivity in the fraction/noradrenaline present in the fraction. See also Besson et al. (1969); Collier (1969). 55. For example, Dunant et al. (1972); Dunant and Israel (1979); Tauc (1982); Marchbanks (1978). 56. Zimmermann and Whittaker (1974). They also included electron microscopic evidence for exocytosis. 57. Zimmermann and Whittaker (1977). See also Suszkiw et al. (1978). 58. For example, Birks (1974); Marchbanks (1978). 59. Cohen and Van der Kloot (1983). 60. Van der Kloot (1978). 61. Israel et al. (1986). Birman et al. (1990) obtained the amino acid sequence of a 16 kDa subunit. This sequence was highly similar to that of the H+-transporting subunits of the vesicle ATPase involved in neurotransmitter storage. 62. Harvey and Macintosh (1940). See also Birks and Macintosh (1957). 63. Fatt and Katz (1952b), pp. 119, 120; (1952c). Only the latter cited Harvey and Macintosh. 64. del Castillo and Stark (1952), p. 515. 65. del Castillo and Katz (1954b). 66. Katz and Miledi (1967b). 67. Normally, Ca2+ enters passively, flowing down its electrochemical gradient into the cytoplasm. When the gradient is reversed, however, no net influx of Ca2+ can occur. 68. Fatt and Katz (1952c); del Castillo and Katz (1954a). 69. Hodgkin and Keynes (1957), p. 279. 70. Katz and Miledi (1967a), p. 543. See also Katz and Miledi (1967c). 71. Katz and Miledi (1969). Others subsequently studied Ca2+ currents extensively, for example, Llinas et al. (1976); Augustine et al. (1985). 72. Nowicky et al. (1985); Tanabe et al. (1987). The latter paper, however, described only one protein of the oligomeric channel complex. 73. Olivera et al. (1985). 74. Kerr and Yoshikami (1984). 75. Miledi (1973). An earlier attempt was unsuccessful (Miledi and Slater, 1966),

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reinforcing the possibility that Ca2+ acted on the surface of the membrane, not after crossing the membrane. 76. Llinas et al. (1972). They used aequorin, a protein that emits a flash of light when exposed to Ca2+. See also Llinas and Nicholson (1975). 77. For example, Fogelson and Zucker (1985); Simon and Llinas (1985). Moreover, the brief time interval between depolarization and release meant that the path of Ca2+ diffusion must be short (Llinas et al., 1981). 78. Heuser et al. (1974). 79. Pumplin et al. (1981). 80. Robitaille et al. (1990). 81. Eccles and Liley (1959), p. 103. 82. Banks (1966). See also Blioch et al. (1968). 83. Ueda et al. (1973). 84. Ueda and Greengard (1977). 85. Bloom et al. (1979). 86. DeCamilli et al. (1983a). Greengard subsequently distinguished a family of synapsins: see Siidhof et al. (1989). 87. Huttner et al. (1981); Kennedy and Greengard (1981). 88. Llinas et al. (1985). 89. DeCamilli et al. (1983b). 90. Bahler and Greengard (1987). See also Petrucci and Morrow (1987). 91. Hirokawa et al. (1989), p. 111. 92. Matthew et al. (1981). 93. Perin et al. (1991). 94. Perin et al. (1990). 95. Jahn et al. (1985); Wiedenmann and Franke (1985). 96. Siidhof et al. (1987); Leube et al. (1987). See also Buckley et al. (1987). 97. Thomas et al. (1988). 98. Trimble et al. (1988). 99. Baumert et al. (1989). 100. Scheer and Meldolesi (1985). 101. Ushkaryov et al. (1992). 102. Oyler et al. (1989). 103. See Wilson et al. (1991). 104. See Kelly (1988); Aimers (1990); Trimble et al. (1991). 105. Roth and Porter (1964). 106. Kanaseki and Kadota (1969). 107. Pearse (1975), p. 97. Later, she and others would identify a number of other functionally important proteins of the coated vesicles. 108. Crowther et al. (1976). 109. Ungewickell and Branton (1981). See also Kirchhausen and Harrison (1981). 110. Earlier, Pearse (1978) described 30-36 kDa proteins from the coat, but she did not calculate a 1:1 stoichiometry with the 180 kDa proteins. 111. Crowther and Pearse (1981). 112. Keen et al. (1979). See also Woodward and Roth (1978); Schook et al. (1979); Unanue et al. (1981). 113. For example, Pearse and Robinson (1984); Manfredi and Bazari (1987); Keen et al. (1987); Virshup and Bennett (1988). 114. Patzer et al. (1982).

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115. Schlossman et al. (1984). 116. Rothman and Schmid (1986). 117. Ellis (1987). 118. For example, Katz et al. (1969); Srinivasan et al. (1969). 119. See Cutler et al. (1971); Martin (1976). 120. The concentrations of solutes and electrical potentials required for reversal are readily calculable: see Attwell et al. (1993). 121. Haycock et al. (1978). 122. Sandoval (1980). She, however, argued for Ca2+-dependent release through Na+-induced liberation of mitochondrial Ca2+. 123. Lust and Robinson (1970). See also Carafoli et al. (1974). 124. Bernath and Zigmond (1988). 125. For example, Haycock et al. (1978); Raiteri et al. (1979); Fischer and Cho (1979). 126. Schwartz (1982). See also Yazulla and Kleinschmidt (1983). 127. Schwartz (1987). 128. For example, Ikeda et al. (1989). By this time, high concentrations of extracellular glutamate were known to cause neuronal damage. 129. Szatkowski et al. (1990). 130. Thoa et al. (1975). 131. Sulzer and Rayport (1990). For early and late reports of amphetamine affecting the catecholamine content of storage vesicles, see Schiimann and Philippu (1962) and Knepper et al. (1988). 132. Raiteri et al. (1979); Fischer and Cho (1979). 133. Edwards et al. (1985). 134. Vyskocil (1985).

11 FORMATION OF SPECIFIC SYNAPSES

Embryonic Development or Synaptic Connections

The Neuron Theory arose at the end of the nineteenth century as holistic conceptions of neural function were waning. Physiological and anatomical studies identified discrete pathways for voluntary and autonomic systems as well as for particular reflex circuits having sensory and motor limbs (chapters 1 and 2). Accordingly, formulations grounded in the Neuron Theory depicted neural pathways as cellular units linked through specific synapses. Such formulations, in turn, required accounts of how such linkages came to be. Embryology was then a flourishing science,1 led by a German school of descriptive embryologists that began in the early nineteenth century with Carl Ernst von Baer and embraced such pioneers as Rudolph von Koelliker and Wilhelm His. They were now joined by a new wave of German experimental embryologists, starting with Wilhelm Roux and Hans Driesch, who intervened in the developmental processes to demonstrate capabilities and alternative outcomes. They and their successors in the twentieth century, including Viktor Hamburger, Hans Spemann, and Paul Weiss, identified sequential transformations that were effected through cellular differentiations and migrations, with ancestral precursors giving rise to mature cells of distinct types and functions then assembled into specific tissues and organs. 273

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Explaining how such differentiations and migrations were directed and achieved became a compelling challenge through the twentieth century, although one incompletely satisfied. Nevertheless, experimental efforts incorporating anatomical, physiological, and biochemical approaches—and, notably, the techniques of molecular biology as these became available—provided by 1990 detailed accounts of complex causal chains linking genetic direction to cellular biochemistry, cellular biochemistry to intercellular associations, and intercellular associations to cellular biochemistry and genetic expression. Studies of neural development were an integral part of this effort and reflected the same conundra apparent in the formation of other organ systems: distinct cells types must assemble in proper order to construct a functional whole. This account, however, will consider a single topic: what guides a growing axon during development to a particular location and thereby establishes a specific synaptic connection.

Approaches ana Possible Mechanisms

His's studies on the outgrowth of nerve cell processes provided a central foundation for the Neuron Theory (chapter 1). These morphological studies also supported concepts of neural development through the extension of such processes to reach particular destinations. Santiago Ramon y Cajal pursued these issues with characteristic vigor and insight, in the process discovering a crucial participant, the growth cones at the termini of elongating axons (chapter 1). His's and Cajal's interpretations were based on microscopic examination of fixed, stained specimens taken from different neurons at different stages of development, requiring interpolations and extrapolations to reconstruct a continuous process from the collection of discontinuous images. Ross Harrison confirmed Cajal's inferences about amoeboid locomotion by watching axonal extensions from neurons grown in tissue culture (chapter 1). Harrison could thus follow the protoplasmic extension of growth cones, with processes extending to spin out an axon from the fixed cell body. Observations of live neurons in tissue culture would remain a valuable model for studying neural development through the twentieth century, despite the inherent artificiality of such isolated growth and development. Another opportunity for continuously monitoring axonal extensions was microscopic examination of neural development in (suitable) organisms. For example, in the 1930s Carl Speidel in Charlottesville took time-lapse photomicrographs of developing nerves in transparent tadpoles, recording the advances and retractions of motile growth cones on axonal sprouts.2 Although Speidel recorded responses of the outgrowing axons to some experimental

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manipulations, such as electrical shocks and assorted drugs, the consequences of more vigorous interventions could be examined better at other loci with fixed, stained specimens. Major manipulations, following the pattern of early successes in experimental embryology, included ablations and transplantations of neural precursors and/or their ultimate targets. These studies could then display not only histological evidence of the resultant connections, but also the functional consequences. (Neural regeneration after experimental interruptions was considered to be a reasonable approximation to the embryological processes responsible for initial innervations.) Another source of modifications was genetic mutation, either induced or occurring naturally; the consequent differences illustrated altered patterns of innervation and function and tied these differences to identifiable genetic controls. Different organisms provided certain experimental and interpretive advantages. Vertebrates, particularly mammals and especially humans, commanded the most attention but presented the greatest experimental difficulties. Avian embryos were simpler than mammalian and more accessible for study. Fish and amphibians, which regenerate limbs and connections from their central nervous systems, were favored organisms for more drastic manipulations. In all these animals, the most profitable systems for study turned out to be nervemuscle and eye-brain connections. With simple invertebrates anatomical complexities were vastly reduced, so that the life histories of identifiable neurons could be traced during development. In addition, a wealth of genetic information could be applied also to studies of neural development. And in the 1970s a roundworm, Caenorhabditis elegans, was chosen for intense genetic and developmental studies: it had the advantage of a rapid reproductive rate (allowing the selection of specific mutant strains) and notable structural simplicity (a total of 959 distinguishable cells in the adult, of which 302 are neurons). Through the twentieth century investigators used these approaches and organisms to examine several obvious possibilities for guiding axonal growth. His imagined a mechanical guidance, with the axon following the course of least resistance, turning, and even splitting, when faced with an obstacle. Cajal acknowledged a role for mechanical constraints but advocated strongly a specificity directed by chemotaxis (or chemotropism).3 The growth cone, Cajal argued, sensed chemicals secreted by the target, with this interaction favoring amoeboid growth up the concentration gradient to its source. (Chemotaxis had already been proposed and was by Cajal's time a prominent explanation for the migration of white blood cells to sites of inflammation.) At this time John Newport Langley independently suggested, from his studies on the organization of the autonomic nervous system (chapter 3), that chemical signals directed neuronal organization.4

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MECHANISMS OF SYNAPTIC TRANSMISSION Early Arguments Concerning Cnemotaxis (1890—1963)

In the decades after Cajal and Langley argued that chemical attractants guided axonal outgrowth, these proposals languished despite the eminence of their advocates. No evidence for attraction was forthcoming, and attempts to demonstrate it failed. For example, in 1934 Weiss, now in Chicago, reported that adding saline extracts of brain to discrete regions of the tissue culture medium neither attracted nor repelled axonal outgrowth.5 But even before this demonstration, Weiss had suggested an alternative mechanism. While still in Vienna he advanced in 1924 a Resonance Principle to account for the apparently specific connections between nerves and end organs, such as muscles and sense organs.6 When Weiss grafted an additional leg near a developing one in amphibia, this extra leg became innervated and then moved in concert with the natural one. Weiss believed that these "homologous responses" arose from homologous muscles responding specifically to (resonating with) particular frequencies of impulses carried by the nerves. Coded frequencies thus selected which muscle contracted: functional connections were effected not by activating particular pathways but by evoking particular patterns of impulses—in nerves broadly—to which only certain muscles would resonate/respond. When Edgar Adrian in Cambridge succeeded in recording from the individual fibers of nerves in 1928 (chapter 3), he found no such pattern of impulses keyed to particular muscles.7 Two years later C. A. G. Wiersma, while visiting Adrian in Cambridge, directly examined Weisss proposal. Wiersma found action potentials appearing not in all the fibers of a motor nerve, but only in those running to the particular muscle actually responding.8 So Weiss devised a new account, pushing back the site of frequency-dependent control to the central nervous system (although he retained the term Resonance Principle).9 Outgrowing axons, Weiss now argued, were initially unspecified functionally, but when axons reached a muscle, even if by random growth, they were modified by this contact: "converged] from indifferent into selective receivers specifically adapted" to activate this muscle.10 The motor nerve now resonated with the proper directives from the central nervous system; inside the central nervous system, Weiss believed, commands were encoded as particular patterns of frequencies.11 Weiss acknowledged that the modification or "modulation" of contacting axons may "plausibly be supposed to be a biochemical effect" of muscle on neuron, although he could not define those changes further.12 By 1934 Weiss favored a mechanical guidance of nerve directly to muscle. Twenty years earlier, Harrison had showed that axons in tissue culture required a solid support for growth and could follow surfaces within this support.13 Weiss, in the same paper where he discounted chemical guidance, described extensions of Harrison's studies. He aligned fibers within the matrix supporting neu-

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rons in tissue culture and found that outgrowing axons then followed this alignment.14 Earlier experiments had shown that growing tissues, such as transplanted legs in amphibia, attracted outgrowing axons.15 So in 1941 Weiss argued that growing tissues induced a tension in the intercellular matrix, thereby aligning molecular fibers of this matrix to form a pathway to the developing end organ.16 Weiss in 1941 also extended Harrisons notion of initial pathfinder neurons,1' adding a mechanism whereby the pathfinder neuron on reaching a target would be altered, as in his earlier Resonance Principle. Now, however, the alterations would include modifications of the pathfinder neuron's surface such that any neurons contacting it would grow alongside it. Consequently, neurons accumulating around the pathfinder would form a bundle ("fasciculus"). This "selective fasciculation" would thus assemble a group of axons running together to the same target. Contact with the target then assured that each incoming axon was modulated, or tuned, to receive the central commands activating this muscle specifically. Weiss, after receiving his doctoral degree in Vienna and working in Berlin, went to New Haven in 1929 to visit Harrison and then spent the years from 1933 to 1954 in Chicago. Among his graduate students there was Roger Sperry (Fig. 11-1A), who received his Ph.D. in 1941, the year Weiss's summation of pioneering neurons and selective fasciculation appeared. Sperry, however,

FIGURE 11-1. A, left, Roger W. Sperry (1913-1994). B, right, Rita Levi-Montalcini (1909-). (Courtesy of the National Library of Medicine.)

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began an independent course that edged him step by step away from Weiss s formulations. Sperry's dissertation described the consequences of surgically interchanging nerves to the extensors and flexors of rat leg muscles.18 Unlike Weiss s findings in amphibia (and some earlier reports on mammals), movements of the reinnervated rat leg were reversed, and this reversal was permanent: the aberrant responses could not be overcome by learning. Thus, the muscles, after being reinnervated by "incorrect" nerves, did not contract as they had before the interchange, a result contrary to Weiss s initial Resonance Principle that envisioned muscles responding to their characteristically coded impulses transmissible by any nerve. Sperry's results also did not support the revised Resonance Principle that envisioned muscles altering any arriving nerve so that it accepted and transmitted signals to the muscle from the appropriate centers in the central nervous system. Sperry suggested that, after their initial embryonic differentiation and synapse formation, mammalian motoneurons could no longer undergo muscle-specific modulation when regenerating. They were "no longer in a sufficiently labile state to be respecified by foreign muscles."19 By 1943 Sperry, now working in Cambridge, Mass., moved further from Weiss s formulations of functional rather than topographical connections.20 In amphibia the ganglion cells in the retina send axons through the optic nerve to a region at the top of the brain, called the tectum, that receives responses from the retina. Sperry now found that after cutting the optic nerve and rotating the eyeball 180°—so what was formerly up was now down—the amphibians visual world after the optic nerve regenerated was inverted also: the amphibian reacted to food placed above it as if the morsel were below. Sperry concluded that regeneration reestablished the original connections, which would now report the world upside down. He suggested that the guiding factors were chemicals, perhaps induced during embryonic differentiation, that directed regenerating fibers to their original targets in the tectum. By 1949 Sperry, back in Chicago, was advocating a "chemoaffiniry theory" to account for this specificity.21 Sperry's observations in the 1940s were functional, not histological, and he could not demonstrate that retinal neurons actually reconnected with their original targets. He continued to cite the coding aspects of the Resonance Principle also, acknowledging that such a mechanism could specify "the quality of the excitatory impulses which different kinds of fibers discharge," so that "functional organization [would] depend . . . not upon a specificity of fixed neuron connections, but upon the emission of qualitatively distinct excitatory agents [with] a selective receptivity to these discharges permitting functional precision."22 Consequently, there could be an "orderly recovery of precise functional relations despite random, indiscriminant" connections of retinal with tectal

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Not until the late 1950s did Sperry, now in Pasadena, undertake experiments showing in a convincing manner that regenerating retinal neurons did indeed reconnect to their original tectal loci.24 He destroyed the ganglion cells in one half of the retina of a fish and then cut the optic nerve; regeneration could only be from the half of the retina not destroyed. By destroying different halves (upper, lower, left, right) Sperry demonstrated that the residual neurons (from lower, upper, right, left halves) grew to distinguishably different regions of the tectum (Fig. 11-2). Moreover, ingrowing fibers would pass inappropriate tectal neurons to proceed, over their original courses, to the proper targets. How could these neurons find their way? Sperry, in an influential paper reviewing these studies in 1963, advocated a chemical specification.25 He imagined two or more chemical gradients that would determine by their relative strengths along opposing axes the latitude and longitude for a correspondingly receptive axon to follow. Support for this proposal would require identifications of these guiding chemicals and demonstrations of how outgrowing axons could respond to specific concentrations of chemical signals. Sperry himself did not pursue these demands (he soon became diverted by studies on split

FIGURE 11-2. Schematic representation of Sperry's experiment showing specific regrowth of retinal neurons. The figures on the left show that destroying the top half of the retina (lower drawing, blank segment) results in a regrowth from the bottom half of the retina to the upper half of the tectum (upper drawing, showing the distribution of ingrowing fibers). The figures on the right show the reverse case. A, P, D, V, M, and L refer to orientations: anterior, posterior, dorsal, ventral, medial, lateral. (From Attardi and Sperry [1963], Fig. 3, courtesy of Academic Press.)

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brain preparations, for which he was awarded a Nobel Prize). Those who did found the technical difficulties formidable, and progress on chemical guidance was delayed for some decades. In the meantime, other relevant concerns came into focus.

Cell Death and Neurotropnic Factors Cell Death in Morphogenesis

The death of certain cells, it was recognized early in the twentieth century, helps to shape organisms to their adult form. Significant losses of neural cells occur also. So selective innervation might reflect an early generalized innervation with a subsequent elimination of inappropriate connections. Indeed, evidence accumulating by mid-century pointed to such initial overproductions followed by selective exterminations. This appreciation emerged, however, from considering another issue: how did the numbers of neurons growing out from the spinal cord match the size of the end organ they innervated? The problem was illustrated clearly by Samuel Detweiler, a student of Harrison in New Haven. In 1920 Detweiler reported that grafting an extra limb bud in amphibia increased the size of the dorsal root ganglion innervating this region, whereas removing the limb bud decreased the size.26 In the early 1930s Victor Hamburger reexamined this phenomenon. Hamburger, who had been a student of Hans Spemann in Freiburg but was now visiting in Chicago, found that after destroying limb buds in avian embryos, the motoneuron region in the spinal cord decreased proportionally to the loss of muscle.27 He imagined that the initial axons contacting a muscle sent signals back to the spinal cord to increase or decrease the number of motoneurons being differentiated from their embryonic precursors. A different suggestion came from Rita Levi-Montalcini (Fig. 11-1B), working first in Turin as the war broke out and continuing alone after her flight to the countryside to escape German occupiers.28 Levi-Montalcini found, by counting cells at successive times, that the decrease after extirpating an end organ reflected a dramatic loss of differentiated neurons. After the war Hamburger invited her to St. Louis, where he was now working, and in 1949 they reported that while an end organ could increase or decrease the proliferation of neuronal precursors, it also "provides conditions for continued growth and maintenance of neurons" after the first outgrowth of axons.29 Nerve Growth Factor (NGF)

Identifying such conditions was a striking achievement in twentieth-century neuroscience, and although the active factors are of tangential significance here,

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the route to characterizing the first of these deserves mention. Elmer Bueker, who had studied with Hamburger but was now working in Washington, wanted to study axonal outgrowth in a rapidly growing but less complex tissue. For this purpose he tried transplanting several tumors into avian embryos and in the late 1940s found that a mouse sarcoma induced a pronounced enlargement of the spinal ganglia that sent sensory neurons to the transplant.30 He did not pursue these issues further, but Levi-Montalcini and Hamburger soon confirmed Bueker's observations. In 1951 they proposed that the sarcoma "producefd] specific growth promoting agents which stimulated the growth of sympathetic and of sensory . . . cells . . . but not of motor [cells]."31 Two years later they reported that sarcomas transplanted at a distance could still promote axonal outgrowths, so the factor(s) must be diffusible.32 The following year, 1954, they described a stimulated outgrowth from dorsal root and sympathetic ganglia cells in tissue culture, providing a means for quantitating the factor in vitro.33 Using this assay, Stanley Cohen, a biochemist who had joined them, could now show stimulation by cell-free homogenates of the sarcoma.34 While attempting to purify the factor, Cohen and Levi-Montalcini used snake venom preparations rich in phosphodiesterase activity to destroy nucleic acid polymers. To their astonishment, the snake venom alone stimulated axonal outgrowth spectacularly.35 Subsequently, they tried mouse salivary glands—homologs to snake poison glands—and these proved to be still richer sources. With this starting material Cohen purified a protein fraction in 1960 that they called nerve growth factor (NGF).36 Antibodies prepared against this material, which should block its actions, indeed destroyed sympathetic ganglia when injected into newborn mice. This was compelling evidence that NGF was necessary to maintain developing sympathetic postganglionic neurons. Using a new, highly sensitive assay, Hans Thoenen in Martinsreid was finally able to show in 1983 that target tissues actually contained NGF, present in amounts correlating with the density of their sympathetic innervation.37 The amino acid sequence, published in 1971, specified a 13 kDa peptide,38 with active NGF being a dimer of this peptide. (The three-dimensional structure followed twenty years later.39) For their seminal discoveries Levi-Montalcini and Cohen received the Nobel Prize in 1986. Demonstrating how NGF functioned was a more challenging quest. In the mid 1970s Leslie Iversen in Cambridge and Thoenen, then in Basel, showed that labeled NGF was taken up by sympathetic nerve endings and transported along the axons to their cell bodies, as might be expected for a growth factor secreted by end organs to regulate survival of innervating neurons.40 In 1979 Eric Shooter in Palo Alto described high- and low-affinity binding sites for NGF that might be receptors,41 but further identification proved difficult. (In 1991 groups in New York, Frederick, and Princeton showed that the high-affinity binding site corresponded to trk-A, a tyrosine kinase of previously unknown function.42 By this time several cellular growth factors, including

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insulin and epidermal growth factor, were known to bind to membrane receptors that then functioned as tyrosine kinases: phosphorylating proteins on the phenolic hydroxyl of tyrosine [rather than on the alcoholic hydroxyls of serine and threonine as did, for example, protein kinases A and C]. As in the responses of other tyrosine kinases, trk-A dimerized when it bound its activating ligand, NGF, and then catalyzed its own phosphorylation. Thus activated, trk-A next catalyzed the phosphorylation of certain other proteins, on their tyrosines, to initiate cascades of cellular second messenger systems.43 On the other hand, the identity of the low affinity binding sites remained uncertain, as did the functional consequences of NGF s transport to the cell body, which had been demonstrated earlier.) Levi-Montalcini and Hamburger showed that NGF affected sensory neurons from dorsal root ganglia and postganglionic sympathetic neurons. Thoenen found in 1979 that NGF also affected certain cholinergic neurons in the brain.44 But what modulated the growth and survival of other neurons? It seemed likely that many more growth factors operated; indeed, additional growth factors with distinctive targets but structures similar to NGF were identified during the 1980s (and more were characterized in the following decade).45

Eliminating Erroneous Connections by Neuronal Death Hamburger and Levi-Montalcini s demonstration of neuronal cell death during embryonic development renewed interest in this process and its consequences. One issue was competition among innervating axons for an end organ. In 1958 Hamburger interpreted this process as "a selective survival of those neurons which find an adequate peripheral milieu."46 A necessary trophic factor, if produced by the end organ in limited supply, could restrict the number of surviving neurons. Another issue, one addressed increasingly through the 1980s, was "programmed cell death," or "apoptosis"; reports that cell death in the roundworm C. elegans was controlled genetically furthered this interest.47 Here, however, the relevant issue is whether selective cell death was the means for assuring specificity, for various investigators had suggested over the decades that such a mechanism could prune away erroneous connections.48 Indeed, in some cases there was clearly a secondary elimination of synapses formed in embryos but not present in adults. For example, Maxwell Cowan in St. Louis showed in 1976 that axons growing out from paired brain nuclei to the retina innervated not only the eye on the opposite side from the innervating nucleus (as in adults) but also the eye on the same side.49 The neurons making the ipsilateral innervation then died, leaving the adult pattern of innervation. But, as is often the case in biological research, universal explanations were impossible. Even if cell death did revise synaptic connections at some sites, it was clear by the 1980s that axons at other sites grew to individual and appropriate destinations. Specific guidance systems must also function.

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Ckemical Guidance (1963-1990) Further Evidence for Specific Guidance

While Sperry was demonstrating regrowth to the tectum in the 1960s, additional examples of precise routings were also being cataloged.50 Further characterizations of the requisite mechanisms were not forthcoming at that time, however, but two later endeavors merit specific mention. Lynn Landmesser in New Haven found that motoneurons proceeding to limb muscles in avian embryos made few errors, with adjacent fibers growing to different and specific destinations. Landmesser concluded in 1981 that these "observations exclude models . . . in which there is a widespread testing of the environment with removal of projection errors by cell death."51 And Corey Goodman in Palo Alto was then mapping the outgrowth of specific neurons in insect embryos. Identifiable axons grew along discrete courses, making abrupt turns at characteristic loci to reach their particular end organs (Fig. 11-3). Goodman argued that they must follow labeled pathways.52

Diffusible Factors

Although Weiss s crude extract showed no neurotropic ability, approaches in the late 1970s used instead a specific substance that could be applied in concentrated form, NGF. (By this time NGF was a known trophic—nutritive— agent; these experiments tested it as a tropic—turning—agent.) Paul Letourneau in Palo Alto grew sensory neurons from embryonic dorsal root ganglia in tissue culture so that added NGF formed a concentration gradient within the supporting gel; axons, he reported in 1978, grew up the NGF gradient.53 The next year Ross Gundersen and John Barrett in Miami showed similar tropisms, with growth cones turning within minutes toward an NGF source.54 In 1983, however, Andrew Lumsden and Alun Davies in London argued that one or more other factors besides NGF attracted axonal outgrowth from sensory neurons.55 Their demonstration relied on younger sensory neurons, at a stage for mouse embryos before these neurons required NGF for survival (in contrast to Letourneau's and Gundersen and Barretts experiments). Axons still grew toward a target tissue present in the culture dish, and antibodies to NGF did not block this tropic effect. Five years later Lumsden, collaborating with Thomas Jessell and Marc Tessier-Lavigne in New York, reported clear tropic responses at another site.56 Cells from a region of the developing spinal cord called the floorplate, when grown in culture so substances diffusing from them would generate a gradient, attracted the outgrowing axons of cultured cells from a different region. Meanwhile, Edward Hedgecock in Nutley used genetic techniques to examine neuronal guidance in C. elegans. He selected mutant worms whose neurons failed

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FIGURE 11-3. Schematic map of axonal outgrowths from specific neurons of developing insects. The circles represent the cell bodies of neurons identified by the enclosed letters and numbers. The emerging lines show the pathways followed by their growth cones, turning at particular points to reach their distinctive targets, in some cases anteriorly, in others posteriorly, while other axons pass through these branch points without turning. In the figure anterior is up. (From Raper et al. [1983b], Fig. 11, ©1983 by the Society for Neuroscience.)

to grow along proper pathways and then identified the mutated genes responsible. Among those identified by 1990 was unc-6.57 (Continuations of these studies during the next five years—[1] by purifying active proteins secreted by floorplate cells and then determining their amino acid sequences by cDNA techniques and [2] by sequencing the unc-6 gene—

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identified a family of structurally related proteins named netrins.58 These netrins, when diffusing from a target cell, attracted the growth cones of certain outgrowing axons.)

Adhesion Molecules

Harrison, as noted above, showed that neurons in tissue culture required a supporting matrix for axonal outgrowth, and Weiss argued for mechanical guidance by such supports. On the other hand, the matrix might attract or repel by chemicals on its surface. Accordingly, Letourneau in the mid-1970s demonstrated hierarchies of adhesiveness to various surface materials guiding growth in vitro.5Q Others soon demonstrated gradients in the retina and tectum.60 But identifying the pertinent molecules was difficult: the scientific problem was obvious, but solving this problem—choosing a feasible system and devising successful methods—demanded insight and ingenuity. One attempt began in the early 1980s with investigations of proteins that form the extracellular matrix in the nervous system as well as in other body organs. For example, when one of these proteins, a laminin, was applied to the surface of tissue culture plates, it promoted axonal outgrowth over itself.61 Soon thereafter a family of cell membrane proteins, integrins, was recognized as receptors for laminins and other matrix proteins.62 Interactions between a particular integrin on a cell and an extracellular protein such as a laminin could thus favor growth selectively. Moreover, laminins had restricted localizations, and Letourneau, now in Minneapolis, identified in 1988 a laminin-coated pathway for axonal extension within the developing brain.63 On the other hand, this laminin pathway existed transiently, so other guides must operate as well.64 Additional candidates included members of two families of cell surface molecules identified during these decades. These membrane proteins interacted with like molecules (homophilic associations), in contrast to the heterophilic interactions between laminins and integrins. The first identified was a neural cell adhesion molecule (N-CAM). Gerald Edelman in New York, having deciphered essential properties of antibody molecules (Nobel Prize, 1972), described in 1977 structurally related molecules that promoted adhesion between neurons in avian embryos.65 These N-CAMs, like the antibody immunoglobulins, existed in a plethora of subtly different structures, and studies through the 1980s demonstrated specific interactions dependent on particular N-CAMs.66 The second family, N-cadherins, were identified in 1983 by Masatoshi Takeichi in Kyoto as Ca2+-dependent proteins that also produced intercellular adhesion.67 Further studies soon demonstrated that N-cadherins also represented a range of variants capable of selective associations.68 N-CAMS and N-cadherins could therefore promote association between neural processes. So it was clear by 1990 that multiple modes of interactions were involved in axonal guidance, including the steering of pioneer neurons along a novel course

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as well as the enlisting of follower neurons to form the nerve bundles. Weiss s notion of selective fasciculation was firmly supported. But in addition to attractions and adhesions, local interactions could also be negative. For example, Friedrich Bonhoeffer in Tubingen demonstrated in 1990 the collapse of growth cones induced by membranes from tectal cells.69 (In 1992 Goodman, now in Berkeley, identified, by using antibodies to screen for its effects, a cell surface protein that controlled axonal turning.70 The following year Jonathan Raper in Philadelphia identified a protein that induced growth cone collapse.71 Goodman then pointed out that these belonged to a structurally and functionally related family, named semaphorins, that included both membrane-bound and diffusible repellants.72)

Growtn Cone Motility

How do growth cones follow these cues? This question includes an inquiry about motility. Cajal likened advancing growth cones to amoebae extending pseudopodia. Such locomotion also appeared in many motile cells of higher organisms: cytoplasm apparently flowed into the advancing margins, often following fingerlike extensions. In axonal outgrowth, however, the cell body did not accompany the advancing growth cone but remained behind as the axon elongated. In addition, microscopy revealed tiny spikes ("filopodia") first extending from the leading edge of the growth cone (Fig. 11-4), with the web between adjacent filopodia then advancing, or the filopodia retracting and new ones extending elsewhere. Explanations for amoeboid movement reflected the mechanistic mode of the age. For example, in the 1930s, when colloidal processes seemed to underlie

FIGURE 11-4. Growth cone morphology. A. The growth cone at the axon terminus ends in numerous filopodia. B. The growth cone cytoplasm contains microtubules, mitochondria, and vesicles. Actin microfilaments are not depicted. (From Bray [1973], Figs. 1 and 2. Reprinted by permission of Nature, © 1973, Macmillan Magazines, Ltd.)

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a host of physiological functions, amoeboid movements were attributed to sol/gel transitions in the colloidal protoplasm.73 But with the recognition during the 1950s and 1960s of microscopic tubes and fibers in the cytoplasm, explanations centered on these components, construed as a cytoskeleton that controlled cell shape and motility. Investigations of axonal outgrowth then followed approaches successful with other motile cells and were grounded in conclusions drawn from their study. One relevant antecedent, it turned out, was the identification of actin microfilaments. By 1970 the protein actin had been characterized as a component both of muscle s contractile machinery (together with myosin) and of microfilaments present in many other cell types (identified microscopically by their binding to added myosin).74 Actin existed in two forms, monomeric G-actin (globular) and polymeric F-actin (filamentous), the latter constituting microfilaments. Cytochalasin, a toxin from fungi, inhibited cellular motility and disorganized microfilaments. In 1970 Norman Wessells in Palo Alto reported that adding cytochalasin to embryonic neurons caused their filopodia to retract and their growth cones to become spheroidal.75 He also described networks of microfilaments in growth cones, and cytochalasin disrupted these. The following year Dennis Bray in Cambridge identified actin from growing nerve cells as a prominently labeled band by SDS-PAGE.76 Bray thought that Wessels microfilaments were made of actin, and in 1972 Paul Burton in Lawrence demonstrated myosin binding to neuronal microfilaments.77 Robert Goldman in Cleveland then showed that this myosin binding extended to microfilaments within growth cones and filopodia.78 Visualizing microfilaments in growth cones and filopodia was difficult, but by the end of the decade Joel Rosenbaum in New Haven, using improved imaging techniques and new labeling reagents, found bundles of actin filaments running longitudinally within filopodia as well as a meshwork of microfilaments beneath the growth cone membrane.79 How could actin filaments cause locomotion? Bray in 1973 suggested interactions between strands of actin and myosin arranged longitudinally within filopodia.80 This scheme drew on the sliding filament model for muscle contraction elaborated in the 1950s: interdigitating filaments of actin and myosin slid past one another to effect shortening. Myosin was difficult to identify in growth cones, however, and even in the late 1980s, when its presence there seemed likely, myosin s role in growth cone movement remained unclear.81 Meanwhile, an alternative way for actin to induce movement was proposed. Studies of other motile cells suggested that a polymeric filament could advance by "treadmilling," adding subunits at its advancing end while removing subunits at the other.82 In 1988 Stephen Smith in New Haven demonstrated new actin subunits being added preferentially to distal microfilament ends adjacent to the membrane.83 Smith, following a new proposal by Bray, imagined a flow

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of actin from the leading edge of the growth cone back toward its center, where the filaments depolymerized; nevertheless, he still considered a role for myosin in propelling actin filaments.84 The actin microfilaments, moreover, seemed linked to the membrane through various actin-binding proteins; these in turn bound to integral membrane proteins that extended to the exterior.85 Consequently, adhesion of filopodia to the extracellular matrix could then be coupled to the cytoskeleton, allowing the tension development necessary for growth cone advancement.86 Such mechanisms had inherent problems,87 but notions of treadmilling and/or actin flow remained popular explanations as the decade closed. The second relevant antecedent was the identification in numerous cell types of microtubules, recognized as polymers of a protein subunit, tubulin, that formed structures several times the diameter of microfilaments.88 In neurons these microtubules ran longitudinally within axons. By 1970 microtubules were implicated in certain motile functions, including chromosome separation during cell division. Colchicine, a plant toxin, disrupted microtubules, providing a reagent for them akin to cytochalasin for microfilaments. In 1970 Wessells also reported that colchicine caused a retraction of elongated axons, although this response lagged behind the changes induced by cytochalasin; the next year Bray found that tubulin, like actin, was strongly labeled in growing nerve cells.89 Further studies through the 1980s showed that microtubules entered the growth cones but stopped short of the filopodia.90 In numerous cells, moreover, the microtubules appeared to advance by treadmilling, and in 1986 Bray, now in London, described the assembly of microtubules at the tips of growing axons, consistent with such a mechanism.91 Both actin and tubulin polymers thus seemed to participate in growth cone advancement. A further requirement for elongating axons was an addition of new material as the axon grew. This also seemed to occur distally. For example, Karl Pfenninger and Marie-France Pfenninger in New York argued in 1981 that during elongation new membrane appeared at the growth cone.92 For all these processes a critical issue was how guidance cues controlled outgrowth. Second messenger systems, triggered through receptors for diffusible agents or through cell adhesion molecules in the membrane, seemed likely participants.93 But by 1990 the identification of these systems was just beginning.

Synapse Formation

Not only must axons grow to their proper destinations, but they also must make functional synaptic contacts on arrival. Synapse development requires a transformation of growth cones into nerve terminals containing synthetic enzymes

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for appropriate neurotransmitters, synaptic vesicles with corresponding transporters, machinery for vesicular release, and reuptake transporters and/or neurotransmitter-degrading enzymes. Relevant receptors must appear postsynaptically (and often presynaptically as well). In addition, physical ties between pre- and postsynaptic elements must form, representing, at least in part, the action of selective cell adhesion molecules.94 This account, however, will merely cite studies on one prominent area of investigation, examinations of how arriving presynaptic terminals trigger an aggregation of cholinergic receptors at neuromuscular junctions. In the 1940s Fritz Buchtal, J. Lindhard, and Stephen Kuffler found that muscle cells responded to administered acetylcholine only in the highly restricted regions where the motor nerves terminated: evidently, acetylcholine receptors were concentrated at neuromuscular junctions (chapters 3 and 4). Then in 1959 S. Thesleff in Lund showed that after denervation, the area responsive to administered acetylcholine expanded to the whole surface of the muscle cell.95 But when the severed nerve grew back to reinnervate the muscle, sensitivity was again confined to the neuromuscular junctions, as Ricardo Miledi in London reported the next year.96 This denervated/reinnervated pattern also reflected the developmental course, as Miledi demonstrated in 1962.97 Fifteen years later Monroe Cohen in Montreal described the same sequence for embryonic muscle and nerve cells grown together in culture. Cohen, however, used fluorescently tagged a-bungarotoxin to monitor the receptor's location, since this toxin binds specifically to nicotinic cholinergic receptors (chapter 6). The labeled receptors, initially scattered over the muscle surface, aggregated at the sites where ingrowing neurons contacted muscle.98 But how did the approaching neurons initiate this redistribution of receptors in the muscle? Enveloping muscle cells—and intervening between nerve and muscle—is a porous sheath, the basal lamina, that is formed from collagen fibers of the extracellular matrix plus various other proteins, including cholinesterase and laminins. In 1979 U. J. McMahan in Palo Alto found that nerve-free patches of this basal lamina could promote receptor aggregation in denervated regenerating muscle cells at their original loci; five years later he described the ability of basal lamina preparations to promote cholinergic receptor aggregation in cultured muscle cells.99 A partially purified fraction of the basal lamina, McMahan reported in 1987, contained two peptides with molecular weights of 95 kDa and 150 kDa that could elicit receptor aggregation.100 He named these agrins. (The amino acid sequence, derived by cDNA techniques in 1991, specified a 200 kDa protein from which McMahan's smaller fragments had presumably been cleaved.101) McMahan also showed that motoneurons contained agrin (identified by antibodies to his purified peptides).102 Thus, motoneurons apparently synthesized

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agrins for release into the synaptic cleft, where these proteins became incorporated into the basal lamina. But how did extracellular agrins trigger aggregation of cholinergic receptors in muscle membranes? This question remained unanswered in 1990, but by then another likely participant had been identified. Acetylcholine receptors in muscle membranes that had been stripped of their basal lamina still did not diffuse within the membrane, suggesting that the receptors were linked to the muscle cytoskeleton. Jean-Pierre Changeux in Paris had identified a 43 kDa protein in cholinergic receptor preparations, and in 1982 he reported that a selective removal of this protein enabled the receptors to diffuse laterally in the membrane.103 (By this time S. J. Singer's fluid mosaic model for membranes104 was generally accepted: integral membrane proteins, like the acetylcholine receptor, would diffuse in the plane of the membrane unless specifically anchored, as by linkage to the cytoskeleton.) Jonathan Cohen in St. Louis derived the sequence of this 43 kDa protein, subsequently named rapsyn, by cDNA techniques in 1987.105 Identification of this DNA also allowed the corresponding mRNA and its encoded protein to be synthesized. In 1990 Stanley Froehner in Hanover and Jim Patrick in Houston described the expression in Xenopus oocytes of cholinergic receptors and rapsyn, singly and together.106 With receptors alone expressed, they distributed diffusely over the oocyte membrane. With receptors plus rapsyn expressed, however, the receptors formed clusters in the membrane. Evidently rapsyn promoted receptor aggregation. But there remained an obvious causal as well as spatial gap between agrins in the extracellular space and rapsyns associated with the cytoplasmic domains of the cholinergic receptor. As the decade closed the search for additional participants continued.

Conclusions

Forming functional connections between particular neurons is a critical requirement for nervous systems composed of discrete units. Such organization, as these decades of investigation revealed, employed multiple means for guidance. Outgrowing axons tended to follow paths of least resistance between obstacles. They also followed favorable surfaces, responding to attracting or repelling substances embedded therein. Outgrowing, axons also migrated toward attracting chemicals diffusing from targets and veered away from repelling chemicals. These various cues summed to steer developing neurons to predetermined connections. But in other cases axons grew to inappropriate destinations only to have the misconnections pruned away. In some instances this corrective cell death resulted from a failed competition with other neurons for a necessary growth factor. Outgrowth along specified pathways depended on the amoeboid advance of

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growth cones at the axon termini. This motility relied on actin and tubulin polymers of the cytoskeleton, although the precise mechanism was still uncertain in 1990. Also uncertain was the means by which extracellular cues modulated the advance to direct growth cones along designated courses. A final requirement was the establishment of synaptic function between apposed cells. Here, too, a number of necessary elements were known in 1990 while others remained unidentified. Despite such gaps, the explanatory accounts constructed by 1990 represent impressive achievements, sketching the nature of the processes and pointing toward likely areas for further exploration. The accounts emerged, as usual, by applying insights and techniques successful elsewhere, here notably from studies of other motile systems and of other developing cells. The accounts also resulted from new approaches and new techniques applied specifically to neuroembryology, permitting the examination of obvious questions. And the actual course to formulating these accounts included, as usual, proposals and generalizations that, after further scrutiny, failed. For example, an initial inability to demonstrate chemical guidance was construed as the absence of chemical guidance. Moreover, a bias toward unitary explanations discounted additional possibilities, blunting the quest for alternative mechanisms. Thus, Weiss had concluded it "could hardly be regarded as satisfactory to assume that in one instance mechanical [and] in another chemical. .. agents were the guiding principle."107 Also influencing experimental attention and explanatory formulations were general concepts of how the nervous system functioned: as a network of particular circuits or as a unitary system resonating to encoded frequencies. In all cases, as usual, every mechanistic proposal turned out, on closer examination, to be far more complex, involving additional entities interacting through additional processes.

Notes

1. See Gilbert (1994); Horder et al. (1986); Nyhart (1995). Purves and Lichtmanns text (1985) contains useful historical information. 2. Speidel (1933, 1941). These studies answered crtiticisms that Harrisons observations in vitro did not represent growth in vivo. 3. See Ramon y Cajal (1995), vol I, p. 532, and references cited. Forssman (1898) also advocated chemical guidance vigorously. 4. Langley (1895). 5. Weiss (1934). He also presented evidence against electrical guidance, which some then advocated. 6. Weiss (1924). 7. Adrian and Bronk (1928, 1929). 8. Wiersma (1931). 9. See Weiss (1936).

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10. Ibid., pp. 512-513. 11. Others then advocating such pattern-directed encoding included Karl Lashley (e.g., 1926). 12. Weiss (1936), p. 514. Weiss, however, suggested that selectivity might be due to different muscles responding to different neurotransmitters (chemical neurotransmission in 1936 being still an ill-defined process: see chapter 3). 13. Harrison (1914). 14. Weiss (1934). He stroked with a fine brush the forming clot of lymph that enveloped the neural tissue in culture, thereby aligning the fibers of the clot. 15. For example, Detweiller (1928). 16. Weiss (1941). 17. See Harrison (1935). 18. Sperry (1941). 19. Ibid., p. 16. 20. Sperry (1943). 21. Sperry and Miner (1949). 22. Sperry (1943), p. 46. 23. Ibid. 24. Attardi and Sperry (1963). They state the experiments began in 1958. 25. Sperry (1963). 26. Detweiler (1920). He found altered numbers of dorsal root ganglion cells, but he could not detect changes in numbers of motoneuron cells since they were not easily identifiable at that stage. 27. Hamburger (1934). 28. See her autobiography, Levi-Montalcini (1988). 29. Hamburger and Levi-Montalcini (1949), p. 498; italics in original. 30. Bueker (1948). 31. Levi-Montalcini and Hamburger (1951), p. 350. 32. Levi-Montalcini and Hamburger (1953). 33. Levi-Montalcini et al. (1954). 34. Cohen et al. (1954). 35. Cohen and Levi-Montalcini (1956). 36. Cohen (I960); Levi-Montalcini and Cohen (1960). 37. Korsching and Thoenen (1983). 38. Angeletti and Bradshaw (1971). 39. McDonald et al. (1991). 40. Hendry et al. (1974). 41. Sutter et al. (1979). 42. Kaplan et al. (1991); Klein et al. (1991). 43. See Reichardt and Farinas, in Cowan et al. (1997), pp. 220-263. 44. Schwab et al. (1979). See also Honegger and Lenoir (1982). 45. See Reichardt and Farinas, in Cowan et al. (1997), pp. 220-263. 46. Hamburger (1958), p. 399. 47. Ellis and Horvitz (1986). 48. For example, Cowan et al. (1984); Lamb (1977). 49. Clarke and Cowan (1976). 50. For example, Guth and Bernstein (1961); Hibbard (1965). 51. Lance-Jones and Landmesser (1981), p. 1. 52. Raper et al. (1983a, 1983b); Goodman et al. (1984).

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53. Letourneau (1978). See also Campenot (1977). 54. Gundersen and Barrett (1979). 55. Lumsden and Davies (1983). 56. Tessier-Lavigne et al. (1988). 57. Hedgecock et al. (1990). The abbreviation "unc" referred to uncoordinated behavior. 58. Ishii et al. (1992); Serafini et al. (1994). See also Goodman and Tessier-Lavigne, in Cowan et al. (1997), pp. 108-178. 59. Letourneau (1975). 60. For example, Bonhoeffer and Huf (1980); Gottlieb et al. (1976); Trisler et al. (1981, 1987). 61. For example, Baron van Evercooren et al. (1982); Manthorpe et al. (1983); Rogers et al. (1983). 62. See Hynes (1987). 63. Letourneau et al. (1988). 64. See Jessell (1988). 65. Thiery et al. (1977). 66. For example, Bixby et al. (1987); Doherty et al. (1989). 67. Hatta et al. (1985); Shirayoshi et al. (1983). 68. See Goodman and Tessier-Lavigne, in Cowan et al. (1997), pp. 108-178. 69. Cox et al. (1990). 70. Kolodkin et al. (1992). 71. Luo et al. (1993). 72. Kolodkin et al. (1993). 73. Mast and Prosser (1932). 74. For a history of early studies on muscle, see Needham (1971). See also Wessels et al. (1971). 75. Yamada et al. (1970). Experiments were on neurons grown in culture. 76. Fine and Bray (1971). They grew neurons in culture in the presence of a radioactive amino acid, labeling all proteins being synthesized. Actin was identified as a band of appropriate molecular weight. 77. Burton and Kirkland (1972). 78. Chang and Goldman (1973). 79. Kuczmarski and Rosenbaum (1979). 80. Bray (1973). 81. See Smith (1988); Bridgman and Dailey (1989). 82. See Wegner (1976); Hill and Kirschner (1982); Wang (1985). 83. Forscher and Smith (1988). 84. Bray and White (1988); Smith (1988). 85. Letourneau and Shattuck (1989); Sobue and Kanda (1989); Heidmann et al. (1990). 86. See Letourneau (1979). 87. For example, if the filament ended at the membrane to push it onward, how could a new subunit squeeze into this site? 88. See Olmsted and Borisy (1973). 89. Yamada et al. (1970); Fine and Bray (1971). 90. For example, Letourneau (1983); Forscher and Smith (1988). 91. Bamburg et al. (1986). See also Baas et al. (1987); Smith (1988). 92. Pfenninger and Maylie-Pfenninger (1981).

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93. For example, Bixby (1989); Schuch et al. (1989). 94. For example, Bixby et al. (1987). 95. Axelsson and Thesleff (1959). 96. Miledi (1960). 97. Diamond and Miledi (1962). 98. Anderson et al. (1977); Anderson and Cohen (1977). See also Frank and Fischbach (1979). 99. Burden et al. (1979); Godfrey et al. (1984). The first successful extract was from Torpedo electric organ, and the later successful purification was also from this rich source. However, McMahan showed that the active substance was in mammalian muscle, too. 100. Nitkin et al. (1987). 101. Magill-Solc and McMahan (1988). Agrins were also present in muscle, a localization that blurred the causal account at that time. See Reist et al. (1987); Fallen and Gelfman (1989). 102. Rupp et al. (1991). 103. Sobel et al. (1978); Rousselet et al. (1982). See also Barrantes et al. (1980). 104. Singer and Nicolson (1972). 105. Carr et al. (1987); Frail et al. (1987). 106. Froehner et al. (1990). 107. Weiss (1934), p. 394.

12 LEARNING

Background

Among the essential attributes of mind and brain is the ability to learn. It is also one of the most amazing. Experience leaves traces in our minds, so that we can both recapture events from the past and modify accordingly our thoughts and actions in the future. Yet the nature of these processes has long remained mysterious.1 Explanations in antiquity adopted various metaphors for learning and memory, such as impressing images on wax tablets and filing away notes among the pigeonholes of the mind's storehouse. Advancing beyond these speculative images to some underlying neural mechanism obviously required an appreciation of basic neural capabilities. Indeed, when the Neuron Theory directed attention to cellular relationships and properties, suggestions for neuronal mechanisms quickly followed. Experimental psychologists also began to define particular aspects of learning late in the nineteenth century, in the process developing useful methods for displaying and evaluating such behaviors. Early in the twentieth century Ivan Pavlov described conditioned responses, subsequently termed classical conditioning: pairing a conditioned stimulus (such as the ringing of a bell, which initially did not affect the behavior studied) with an unconditioned stimulus (the sight of food) capable of eliciting an unconditioned response (salivation), 295

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so that after a number of trials together the conditioned stimulus alone would elicit the response. At that time Edward Thorndike embarked on complementary studies using puzzle boxes, investigations developed prominently by B. F. Skinner as instrumental conditioning: animals in appropriate apparati associated chance behavior (such as stepping on a pedal) with a consequent reward (release of a food pellet), thereby learning how to gain the reward through specific actions. Such trial-and-error learning protocols included another favorite test system, the learning of successful routes through mazes by following environmental cues. In contrast to these associative modes of learning were two nonassociative processes: habituation, a decreasing response to a recurring stimulus, and sensitization, an increasing response. In addition, early experimenters distinguished between short-term memory, of limited duration (seconds to minutes), and long-term memory, which could persist for years in exquisite detail. Moreover, interventions that affected neural activity, such as anesthesia or electric shocks, could erase recent memories while leaving older memories largely unaffected. Formation of long-term memory apparently involved processes requiring a significant passage of time— minutes to hours—termed the consolidation period. Experimental psychologists explored a further characteristic of stored memories, their physical location within the brain. Contradicting earlier concepts of discrete localizations within the brain, such as "memory lobes," S.I. Franz and then notably Karl Lashley considered memories to be represented instead throughout the brain. In studies begun in the 1910s and continued through several decades, Lashley systematically examined the consequences of destroying particular regions of the brain. He found that even after a learned task was obliterated by sufficiently extensive lesions, that task could still be relearned. Moreover, the degree of loss of a learned task (measured, for example, as the number of trials required to relearn it) was proportional to the area destroyed. Such results, Lashley argued in 1926, "cannot be deduced from any explanatory system dependent upon the connections of particular neurons."2 He questioned formulations based on "low synaptic resistance between certain definite neurons arranged in more or less intricate fashion"; instead, he favored notions centered on "the ratio between [the] parts, and not the particular neurons excited."3 Later reinterpretations stressed merely a diffuse and redundant representation of learning within the brain.4 And -later criticisms questioned the extent of the lesions and hence Lashley s interpretation of generality.5 This chapter will ignore many crucial concerns in attempting to understand learning and memory, such as how memories are organized and generalized in storage and how specific memories can be retrieved on demand. Instead, it will focus on the cellular changes required to produce experience-dependent alterations in neural function. This account, as are those of previous chapters, is grounded on issues raised by Santiago Ramon y Cajal. His monumental trea-

Learning

29?

tise on neuroanatomy cataloged in 1909 a range of proposals for cellular mechanisms, including amoeboid movements of neuronal processes to facilitate conduction between particular neurons and his earlier conjecture (by then abandoned) for reversible interpositions of glial cell processes between neurons to regulate transmission. Cajal now envisaged a reinforcement of existing pathways through repetitive use as well as the creation of new pathways by a "continued branching and growth of dendritic and axonal arborizations."6 In 1917 C. U. Aliens Kappers in Amsterdam developed this notion of neuronal growth. Associative learning, he argued, resulted from the synchronous stimulation of neurons, with axons growing toward dendrites already active/ Learning required the same processes that he imagined to be involved in neural development. At midcentury the most precise and influential advocate of synaptic mechanisms was Donald Hebb in Montreal, although he had conducted his doctoral research at Harvard University under Lashley s direction. Hebb's 1949 book, The Organization of Behavior, proposed that if "an axon of cell A is near enough to excite cell B and repeatedly or persistently takes part in firing it, some growth process or metabolic change [will] take . . . place in one or both cells, such that As efficiency, as one of the cells firing B, [will be] increased."8 An essential component of Hebb's formulation was the consequent linkage of neurons into a distinct circuit that specifically underlay the learning: "repeated stimulation . . . will lead slowly to the formation of an 'assembly' o f . . . cells which can act briefly as a closed system after stimulation has ceased; this prolongs the time during which the structural changes of learning can occur and constitutes the simplest instance of a representative process (image or idea)."9 Rafael Lorente de No in New York had recently described loops of neurons in the brain and raised the possibility of self-reexciting, reverberatory neural circuits.10 Even though reverberatory circuits could not represent long-term memory, which persisted after interruption of electrical activity, they could serve, as Hebb suggested, to rehearse learning during the consolidation period. These synaptic models pointed to plausible mechanisms for the changes in neural activity required for learning. The actual demonstrations of such changes and the identifications of responsible entities, however, was delayed until the final decades of the century. They waited for the necessary conceptual and technical means to be developed.

Chemical Representations

Another way to preserve experience might be through encoding it in specific chemical representations, as Joseph Katz and Ward Halstead in Chicago suggested in 1950.n They imagined that stimulating a neuron could create a spe-

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cific nucleoprotein, which then acted as a template for constructing within this neuron a specific protein lattice. The lattice would govern transmission, so that organization of the first neuron then spread to adjacent neurons, each in turn forming the specific nucleoprotein and the consequent lattice (with the organizing chains halting wherever they encountered previously organized neurons). Thereafter, excitation would proceed only over pathways with identical nucleoprotein lattices. Learning would be represented by an assembly of neurons, but with the linkages established by molecules specific to a particular memory. At that time dozens of proteins had been distinguished, although the first report of a protein's amino acid content appeared only in 1945 and the first report of an amino acid sequence not until 1951.12 By 1960, however, Francis Crick had promulgated the "central dogma" of information flow from DNA to RNA to protein, and details of the underlying processes were accumulating rapidly. These new understandings then suggested new formulations for biochemical changes during learning. In 1960 Holger Hyden in Goteborg proposed that memory traces were formed through altering the nucleotide sequence of neural RNA.13 Wesley Dingman and Michael Sporn in Rochester independently argued that memory traces resulted from changing RNA structures and proceeded to examine one experimental consequence of such schemes.14 They found that administering to rats a nucleotide analog that is incorporated into RNA and that interferes with RNA's function, 8-azaguanine, retarded the rats' learning of a new maze without interfering with their performance in mazes previously mastered. And the next year, 1962, Hyden reported that teaching rats a balancing task altered, after four days' training, the composition of nucleotide bases in the RNA of Deiter's cells (which are involved in circuits controlling balance).15 The changes in RNA described in both studies could, by Cricks scheme, alter protein structure and function. So in 1963 Josefa and Louis Flexner in Philadelphia injected puromycin, a known inhibitor of protein synthesis, into mouse brains and found that such administration interfered with the animals' learning a new task.16 Subsequent studies by Samuel Barondes in New York showed that protein synthesis was required for preserving learned behavior for more than several hours and that this synthesis began within minutes of training.1' All these observations were interpretable as protein synthesis being necessary for forming long-term memories. But the synthesized proteins could be required either to facilitate synaptic transmission, as in Hebb's formulation, or to store specific memories in unique molecules.18 Moreover, actual identifications of the involved RNA or protein molecules were not forthcoming. At that time methods for separating and characterizing these macromolecules were not well developed. In any case, the pertinent changes would probably

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be swamped by irrelevant metabolic processes proceeding concomitantly throughout the brain. Nevertheless, an explicit claim for encoding memories chemically appeared at this time, a claim that enjoyed intense although short-lived publicity.19 James McConnell, as a graduate student in Austin, examined the then controversial question of whether invertebrates could learn. In 1955 he reported classical conditioning in planaria (flatworms): pairing a light flash (conditioned stimulus) with an electric shock (unconditioned stimulus) to produce a contraction of the worm or a turning of its body.20 The extent of learning, however, was modest, increasing from 2% to only 10% contracting and from 25% to 35% turning. Subsequently, a range of other learning tasks were used, such as choosing the designated branch of a T-maze, but always the improvements attributed to learning were small. Although others also reported learning by planaria,21 some failed and were severely critical.22 Such limitations in merely demonstrating the learning sharply undermined the value of McConnelPs approach when used as an assay in the more exotic experiments that followed. Planaria, when cut transversely or longitudinally, can regenerate whole worms from their fragments. McConnelFs next step, now in Ann Arbor, was to show in 1959 that learned contracting or turning could survive such ordeals (retention was measured as a decreased number of trials required to reach a criterion for learning).23 Indeed, McConnell found that learned behavior was retained even when worms regenerated from posterior fragments. One interpretation was that learning induced the formation of some molecule that was represented throughout the worm and that could replicate—and manifest— itself after any part of the worm regenerated.24 Another characteristic of planaria is cannibalism. So McConnell fed trained planaria to untrained ones, and the ingestors required fewer trials to learn what the ingestees had known than did planaria fed untrained ones.25 This research attracted considerable attention, enhanced by McConnelPs flair for publicity and consequent accounts in the popular press about potential information transfer through shots for learning history or pills for playing the piano.26 Meanwhile, Roy John in Rochester found in 1961 that including the enzyme that breaks down RNA, RNAse, in the water while bits of planaria were regenerating would obliterate the previously learned behavior in worms that arose from posterior fragments.27 Allan Jacobson, a former associate of McConnell now in Los Angeles, developed this notion, reporting in 1966 that RNA extracted from trained planaria would, when injected into untrained worms, reduce the number of trials the recipients needed to learn the task.28 Among the responses to such reports was a paper in 1966 signed by 23 scientists from seven institutions describing their inability to show that RNA extracts could transfer learning.29 Other claims for and against followed, but in diminishing numbers. This decline was rooted in imprecise experimental

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approaches30 as well as in the minimal, and therefore ambiguous, behavioral changes on which the conclusions had rested. Another reason was the intrinsic scientific implausibility of the claims. By this time RNAs role as the link between DNA and protein synthesis was becoming clear. Consequently, the proposed ability of planaria to use ingested or injected RNA to synthesize selectively new proteins for memory seemed wildly unrealistic, and McConnell did nothing to challenge or extend current concepts of RNA function in support of his formulation, let alone show how ingested/injected RNA affected cell physiology and biochemistry.31 The liklihood of a different protein for each memory also strained credeibility: human memory was then estimated32 to contain 1020 bits.31 Equally significant was the evidence then accumulating for quite different mechanisms, ones implicating synaptic changes rather than unique memory molecules.

Learning? in Aplysia

A more prudent strategy would be to select a simple organism in which detectable changes—physiological and biochemical—could be localized and one where learning could be demonstrated consistently.33 This wise course Eric Kandel (Fig. 12-1) followed profitably. Kandel received his medical degree in New York in 1956 and completed his psychiatric training in Boston in 1964, although this clinical course was interrupted by three years at NIH, where he worked on mammalian neurophysiology, and the year 1962-1963, spent with Ladislav Tauc in Paris, which launched his later investigations. Tauc was studying a mollusc, the sea slug Aplysia depilans, that has a simple nervous system with large neurons, some of which were already identifiable from animal to animal reliably. These experimental advantages allowed Kandel and Tauc to record the electrical responses of a particular neuron in an isolated abdominal ganglion after stimulating its excitatory inputs. Accordingly, they recorded from various neurons after exciting a nerve to the ganglion weakly with a "test stimulus," sufficient to elicit only a minimal e.p.s.p. This they paired 0.3 second later with strong excitation of a different nerve to the ganglion, to provide a "priming stimulus." After several minutes of paired excitations, the response to the test stimulus alone, when recorded from one of a small population of neurons within the abdominal ganglion, increased up to sevenfold. In 1965 they likened such changes to classical conditioning (with the priming stimulus as unconditioned stimulus and the test stimulus as conditioned stimulus).34 They also argued that this augmentation represented presynaptic facilitation: the priming stimulus potentiated neurotransmitter release from neurons conducting the test stimulus. These changes in synaptic properties—considered a manifestation of "synap-

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FIGURE 12-1. Eric Richard Kandel (1929-; courtesy of Eric Kandel.)

tic plasticity"—seemed a plausible analog of learning.35 But a more explicit representation would include altered functional responses elicited by stimulating sensory inputs, occurring through identified neural circuits, and with altered responses lasting longer than the half hour limit of these experiments. Beginning in the mid-1960s, Kandel, now in New York, pursued this quest. By 1970 Kandel described habituation of a behavioral reflex—gill retraction after tactile stimulation of the siphon (Fig. 12-2A)—and began characterizing the underlying neuronal changes.36 He recorded from motor neurons in the abdominal ganglia of Aplysia californica while stimulating with jets of water the skin on the siphon. Using isolated abdominal ganglia with their sensory nerves running to an attached patch of siphon skin, he could also record from the sensory neurons, whose receptors lay on the siphon skin and whose cell bodies were in the ganglia. Repeated stimulation did not affect activity in the sensory neurons but produced progressively smaller responses in the motor neurons: habituation (Fig. 12-2B). The diminished e.p.s.p.s, moreover, correlated with a decreased number of transmitter quanta released from the sensory nerves' presynaptic terminals.3' On the other hand, strong stimulation elsewhere on the mollusc could restore the reflex fully: dishabituation (Fig. 12-2B). By 1976 Kandel had identified in the ganglia 24 cell bodies of the sensory siphon neurons; these neurons excited by direct synaptic contact six motor neu-

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FIGURE 12-2. Habituation and dishabituation in Aplysia. A. Aplysia is viewed from above with the mantle shelf and parapodia retracted. The gill is contracted. The dotted line shows its extent when relaxed. B. The traces show gill contractions (upward deflections) decreasing with successive tactile stimuli (numbered) to the siphon. The interstimulus interval (ISI) was 1 min. At the arrow a strong tactile stimulus was applied to the neck; the original stimuli then elicited augmented responses. C. The proposed neural network for sensitization shows the cell body of a sensory neuron (SN) in the abdominal ganglion with its receptors on the siphon and its presynaptic terminals on the motor neuron to the gill (L7). Habituation results from decreased neurotransmitter release from SN onto L7. Another sensory neuron has its receptors on the head and its presynaptic terminals on the terminals of SN, such that it augments neurotransmitter release from SN onto L7. The neurons from the head may also activate L7 directly. The neural path from head to ganglion is uncertain, as indicated by the breaks in the line. (A and B from Pinsker et al. [1970], Figs. 1 and 2; C from Castellucci and Kandel [1976], Fig. 3. Reprinted by permission, ©1970, 1976, American Association for the Advancement of Science.)

rons to the gills whose cell bodies were also in the ganglia. Again using isolated abdominal ganglia with attached sensory nerves, he showed that stimulating sensory nerves from the head alone had no effect on the motor neurons. But stimulating nerves from the head augmented the motor neurons' response to sensory input from the siphon: sensitization.38 This augmentation represented an increased neurotransmitter release from presynaptic terminals of siphon sensory neurons. Accordingly, Kandel proposed a circuit whereby (1) neurons from the head nerve elicited presynaptic facilitation of (2) the siphon neurons' output onto (3) the motor neurons (Fig 12-2C). By surveying potential neuro-

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transmitters he found that added serotonin enhanced transmission between siphon neurons and motor neurons, suggesting that the sensitization produced by neurons from the head was mediated by their releasing serotonin onto the siphon neurons.39 How might released serotonin elicit presynaptic facilitation? Injecting cAMP into the siphon neurons also elicited this response. Moreover, adding serotonin or injecting cAMP increased Ca2+ influx into the siphon neurons.40 The elevated cytoplasmic Ca2+ in the terminals could thus trigger the vesicular release of neurotransmitter. In 1980 Kandel attributed the increased Ca2+ influx to a diminished K+ efflux through its voltage-gated channels (which would in turn increase Ca2+ influx through its voltage-gated channels by delaying repolarization of the neurons).41 That year Kandel, collaborating with Paul Greengard, showed that injecting protein kinase A into the siphon neurons also facilitated; later they found that inhibiting protein kinase A blocked presynaptic facilitation.42 Protein phosphorylation thus joined the causal chain. In the early 1970s Kandel demonstrated that both sensitization and habituation could persist for weeks, and a decade later two associates in New York, Craig Bailey and Mary Chen, described morphological changes accompanying such long-term learning.43 Active zones in the siphon neuron terminals contained fewer synaptic vesicles after long-term habituation and more after longterm sensitization. Five years later they also reported decreases and increases, respectively, in the number of synaptic contacts on the motor neurons.44 Dissociated sensory and motor neurons formed functional contacts in culture, and adding serotonin over a period of hours produced long-term sensitization in this simplified system, whereas a single application of serotonin produced short-term sensitization, paralleling responses in the intact ganglia. In 1986 Kandel reported that inhibitors of RNA and protein synthesis, when added with the serotonin, blocked the long-term but not the short-term sensitization.45 This was true for sensitization due to added cAMP as well.46 These repeated additions altered the labeling (i.e., synthesis) of a number of distinguishable proteins, although the functions of these proteins were not then identified.47 Apparently cAMP acted through CREB and CRE (chapter 7) to regulate protein expression: adding isolated, exogenous CRE, which would trap endogenous CREB and prevent its interaction with endogenous CRE, blocked long-term facilitation.48 At the end of the decade, Kandel summarized these interactions in a circuit that depicted sensitizing interneurons releasing serotonin onto the presynaptic terminals of siphon neurons (Fig. 12-3). The serotonin receptors then triggered a rise in cAMP within the siphon neurons that activated protein kinase A. Targets of subsequent phosphorylations included voltage-gated K+ channels and CREB (Fig. 12-4). Altered protein synthesis, mediated by CREB, could then stimulate the growth of synaptic contacts and affect protein kinase A activity.

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FIGURE 12-3. Expanded neural circuit for habituation and sensitization. This circuit adds facilitating interneurons between the sensitizing neurons (here from the tail rather than the head). The termini from these interneurons make synaptic contact with the presynaptic terminals of the sensory neurons from the siphon, and it is these interneuron termini that release serotonin to produce sensitization. (From Kandel et al. [1991], Fig. 65-3, reprinted by permission of the McGraw-Hill Companies.)

Earlier, James Schwartz, a major collaborator with Kandel, had identified an intermediate-term learning that did not require protein synthesis but instead reflected the loss of an inhibitory protein that regulates protein kinase A.49 Protein kinase A, in the absence of this inhibitory/regulatory protein, would then continue to act, even after cAMP levels fell. Such disinhibition of protein kinase A could maintain facilitation until protein synthesis replaced the lost inhibitor. One aspect of long-term facilitation might involve a continued disinhibition. Meanwhile, Kandel had described a pattern of stimulation that elicited classical conditioning of the gill reflex.50 This associative learning also involved presynaptic facilitation. Others—notably Daniel Alkon in Woods Hole— confirmed and elaborated on these studies of molluscan nervous systems,

FIGURE 12-4. Mechanisms for long- and short-term sensitization. The diagram of the sensory neuron from the siphon shows receptors for serotonin, released from the termini of interneurons (5-HT). These receptors are coupled to adenylate cyclase, and the consequent rise in cAMP levels activates protein kinase A. Targets for the protein kinase are (I) voltage-gated K+ channels (causing a delayed repolarization and thus a rise in Ca2+ influx through voltage-gated channels for Ca2+); (2) unspecified proteins involved in neurotransmitter release; and (3), in the nucleus, CREB, thereby facilitating CREB s binding to CRE. Possible consequences of the altered gene expression and protein synthesis are an increased neuronal growth and an increased protease activity that, by cleaving the endogenous inhibitor of protein kinase A, keeps its kinase activity elevated even after cAMP levels fall. (From Kandel et al. [1991], Fig. 65-5, reprinted by permission of the McGraw-Hill Companies.)

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adding evidence for the involvement of protein kinase C and CaM kinase as well as postsynaptic processes.51 More than one cellular mechanism could be involved in synaptic facilitation. But the relevance of changes in these simple nervous systems to learning in complex mammalian brains remained unestablished. Moreover, Kandel argued that his findings did not follow Hebb's formulation, which required that activation of cell A (here siphon neuron) be coupled with activation of cell B (motor neuron).52

Learning in Drosophila

Complementary evidence came from a different approach, studying genetic control of behavior and using a different invertebrate, the fruit fly Drosophila melanogaster, Beginning in the late 1960s, Seymour Benzer in Pasadena extended his acclaimed analyses to mechanisms of learning.53 He induced mutations chemically, bred colonies of flies expressing particular mutations selectively, and then tested the abilities of flies from these colonies to learn. Drosophila move toward light, and he trained them to avoid a passage to the light associated with one scent (coupled with an electric shock) but follow another passage associated with a different scent (no punishment). By the mid 1980s Benzer and his colleagues, who included Ronald Davis, Duncan Byers, and Yadin Dudai, plus William Quinn in Princeton, had identified two mutants with clear learning deficits and mapped the affected genes, dunce and rutabaga, by conventional genetic techniques.54 Flies bearing the mutant dunce had higher levels of cAMP and lower phosphodiesterase activity. In 1986 Davis, then in East Lansing, showed that dunce coded for phosphodiesterase.55 Flies bearing the mutant rutabaga had lower adenylate cyclase activity, associated with a decreased sensitivity to calmodulin.56 (In 1992 Davis, now in Houston, showed that rutabaga coded for a calmodulin-responsive adenylate cyclase.5') Both mutations altered cellular levels of cAMP, albeit in opposite directions. These ties between defective cAMP second messenger systems and impaired learning thus echoed Kandels findings with Aplysia. But the experiments did not demonstrate that synaptic facilitation relied in all cases on cAMP-sensitive processes, even with invertebrates.

Learning in Mammals: The Hippocampus and. Long-Term Potentiation (LTP)

Neither Benzers nor Kandels strategy seemed applicable to studying mammalian learning. The far longer generation times made the breeding of mutants protracted, and the far greater complexity of mammalian brains made the

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search for pertinent synapses and the definition of localized cellular changes seem unattainable. Hebb's formulation included synaptic changes that should be measurable, but even if such changes were found, how could they be linked causally and specifically to learning? Where among all the brains synapses should the changes be sought? If memory were broadly represented throughout the cortex—as Lashley s experiments seemed in the 1950s to imply—could a particular learning event be detected amongst the multitudes of other neural activities proceeding concomitantly? The path to resolving these uncertainties began with an enabling simplification: the recognition in the 1950s that, contrary to Lashley's views, some categories of learning required a distinct and restricted region of the brain. Examination of this region uncovered characteristic synaptic changes that could plausibly represent learning. And analyses of these changes then identified underlying cellular mechanisms. This progression, however, sprang fortuitously from some chance observations.

A Role tor the Hippocampus

Injuries to the brains temporal lobes (Fig. 12-5A) can result in seizures, and when such epilepsy is resistant to anticonvulsant medications, one therapeutic approach is removal of the damaged tissue. While developing this approach, Wilder Penfield, a celebrated neurosurgeon in Montreal, stimulated the brain in conscious patients to identify responsible areas and to avoid vital ones. He found, unexpectedly, that stimulating some parts of the temporal lobes could evoke vivid memories of isolated moments from the patients past, such as revisiting a certain scene or rehearing a particular song on a specific occasion.58

FIGURE 12-5. Temporal lobe and hippocampus. A. The lateral view of a human brain shows the left temporal lobe and, by the hatching, the location of the left hippocampus beneath the medial surface of the temporal lobe. B. The diagram of the hippocampus in cross section shows an excitatory input through the perforant pathway to granule cells. The granule cells then activate CAS neurons, whose axons send recurrent branches (Schaffer collaterals) to excite CA1 neurons. The arrows show the direction of impulse flow. Not shown are the large numbers of interneurons that modify activity.

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Penfield recruited a psychologist, Brenda Milner, to assess mental function before and after surgery, and in 1953 they reported that among a series of patients who had had one temporal lobe removed for epilepsy, two patients developed severe memory deficits postoperatively.59 They suggested that these two had a preexisting lesion in their opposite temporal lobe and that the memory loss was due to bilateral damage. At that time, before the introduction of effective drug therapies, one method for treating severe psychotic illness was surgical: removing the prefrontal cortex or cutting its connections to the rest of the brain. Because of the deleterious personality changes resulting from such operations and the limited improvements, William Scoville, a neurosurgeon in Hartford, tried destroying portions of the temporal lobes bilaterally, since these have connections to the prefrontal area. In 1953 he applied this procedure also to a 29-year-old man suffering from intractable epilepsy60 The operation successfully reduced the frequency of his seizures, but, as was apparent immediately, it also abolished his ability to form new memories. Although memories of events prior to the surgery remained, each moment thereafter was a fresh start. The patient could not recognize new faces, remember new conversations, or refind his way to new locations. When Scoville read Milner and Penfield s account, he invited Milner to Hartford to examine this patient, known as H.M., as well as the psychotic patients who had undergone similar operations. Scoville and Milner s paper in 1957 documented the memory changes and attributed them to bilateral destruction of the hippocampus (Fig. 12-5A), a structure in the medial portion of the temporal lobes well studied by anatomists (including Cajal) because of its prominent cells (Fig. 1-2) and striking cellular organization (Fig. 12-5B).61 Over the following decades Milner evaluated H.M., establishing the limits of his loss and its irreversibility. Memories formed prior to surgery were retained, so longterm storage was not within the hippocampus. Moreover, some types of learning could still occur, such as mastering new motor skills, even though H.M. could not remember the training sessions when he learned the skills.62 These observations seemed convincing, and they were widely noted. For example, they inspired early biochemical investigations of memory that focused on changes in the hippocampus, although those investigations did not then lead to further insights.63 But initial attempts to reproduce the memory deficits in animals failed. In 1977, however, David Gaffan in London questioned whether the learning assays then being used with experimental animals were appropriate.64 Indeed, Mortimer Mishkin in Bethesda reported the following year that destruction of the hippocampus bilaterally abolished a monkey s ability to recognize an object after a single exposure ("one-trial delayed matching").65 Subsequent studies through the 1980s confirmed this approach and localized the requisite region to the hippocampus and adjacent areas.66 The studies also confirmed the distinctions then being drawn between declarative memory, which

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depends on the hippocampus and records events and facts, and nondeclarative memory, which depends on other brain regions and participates in conditioned responses, attaining skills, and such.

Long-Term Potentiation (LTP)

The hippocampus6' was a favored structure among physiologists as well as anatomists, again because of its straightforward organization (Fig. 12-5B). A leading center for these studies was Oslo, where Per Andersen was characterizing hippocampal circuits. In the course of these investigations one of his students, Terje L0mo, identified in 1966 a notable feature, "frequency potentiation."68 Short trains of high-frequency stimuli to the perforant pathway augmented subsequent responses of the granule cells. Remarkably, this potentiation could last for hours after initial priming stimuli. L0mo interpreted this as a "plastic change . . . expressing itself as a long-lasting increase of synaptic efficiency." By then another form of synaptic facilitation, called "posttetanic potentiation," had attracted considerable interest. In 1941 T. P. Feng in Beijing described augmented muscle e.p.p.s after an initial strong ("tetanizing"69) stimulation.70 This augmentation was later observed at numerous synapses, including those in the central nervous system, and traced to an increased neurotransmitter release. The increased release was then attributed to the tetanizing stimuli producing elevated levels of cytoplasmic Ca2+ within the presynaptic terminal.'1 To those interested in mechanisms for learning, such potentiation was intriguing. But the potentiation was also disappointing, for it lasted only minutes. Still, posttetanic potentiation might prolong activity over reverberating circuits, then advocated as intermediates in memory formation. In any case, the far longer durations that L0mo described should have attracted great interest. Instead, L0mo's report was apparently overlooked.72 In 1968 Timothy Bliss came to Oslo to work with Andersen. Bliss was interested in learning, and Andersen suggested that he collaborate with L0mo in characterizing the hippocampal potentiation. Their study, published belatedly in 1973, described clearly increased responses of the granule cells (Fig. 12-6) that persisted for the duration of the experiment, up to 10 hours.'3 Subsequently Bliss, now back in Mill Hill, used electrodes implanted chronically in unanesthetized rabbits to show changes persisting for days.74 This phenomenon became known as long-term potentiation (LTP). Andersen reported, also in 1973, another site in the hippocampus where LTP occurred.75 Axons from CAS pyramidal neurons pass out of the hippocampus, but branches travel back (as "Schaffer collaterals") to innervate the CA1 pyramidal neurons as well (Fig. 12-5B). Trains of high-frequency stimuli to the Schaffer collaterals augmented responses of the CA1 neurons, just as stimulating the perforant pathway augmented responses of the granule cells.

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FIGURE 12-6. Long-term potentiation. Responses of the granule cells to test stimulation of the perforant pathway—measured by extracellular microelectrodes and expressed as e.p.s.p.s of the population (and normalized to initial values)—are plotted for experiments lasting six hours. High-frequency trains of stimuli to the perforant pathway were administered at the arrows. The open circles are control responses. (From Bliss and L0mo [1973], Fig. 4, courtesy of the Physiological Society.)

Unfortunately, LTP was not always easy to observe. A major advance, offering easier, more accessible, and more reproducible studies, was recording in vitro from isolated slices of the hippocampus, as described in 1975 by Philip Schwartzkroin and Knut Wester in Oslo.76 This approach was then widely adopted for examining and defining the process, although some significant concerns remained. Among these concerns was the locus of change. L0mo and Bliss noted that potentiation could result from alterations at pre- or postsynaptic sites, or at both. Evidence for all three possibilities was still being debated as the 1980s closed. Another significant concern was the dearth of evidence linking the observed synaptic changes to actual learning. Nevertheless, the enhanced synaptic efficacy made a causal connection seem highly plausible. A further capability enhanced this appeal. In 1979 William Levy in Charlottesville identified two pathways to the granule cells, the first of which produced LTP following highfrequency stimulation, whereas the second did not.77 But stimulating both pathways together produced LTP in the second pathway as well. Levy likened this phenomenon to associative learning at synapses, in accord with Hebb s formulation. In 1983 Thomas Brown in Duarte demonstrated the same association for CA1 neurons in hippocampal slices.78 During the 1980s evidence for LTP appeared at sites beyond the hippocampus, and LTP became recognized as a common physiological process

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throughout the nervous system.79 Moreover, in 1989 Patrick Stanton and Terrence Sejnowski in Baltimore described an associative long-term depression of synaptic activity, the converse of LTP.80 They adapted Levy's and Browns schemes, pairing stimuli over two pathways but with the stimuli out of phase; responses to test stimuli were then depressed for protracted periods.

Tke Role of NMDA Receptors

Deciphering one aspect of how LTP occurs began with a pharmacological study published in 1983.81 By that time three classes of glutamate receptors were distinguishable by their responses to various reagents and named for identifying agonists: kainate, quisqualate, and NMDA (chapter 6). Graham Collingridge, working with Hugh McLennan in Vancouver, extended this approach to hippocampal slices, chosen for the "wealth of information concerning [their] neurochemistry . . . anatomy . . . and electrophysiology [and their] amenability to investigations."82 He stimulated the Schaffer collaterals and monitored responses of CA1 neurons after administering various reagents to these cells microelectrophoretically. Specific antagonists for kainate and quisqualate receptors prevented e.p.s.p.s in CA1 neurons, but antagonists for NMDA receptors did not. Instead, NMDA antagonists blocked LTP. Collingridge concluded that different classes of glutamate receptors served distinct functions: kainate and quisqualate receptors mediated excitatory transmission between Schaffer collaterals and CA1 neurons, whereas NMDA receptors mediated LTP. That year Gary Lynch in Irvine argued that LTP required a rise in intracellular Ca2+: LTP no longer occurred after he injected into CA1 neurons a chelator of Ca2+.83 Also in 1983 Raymond Dingledine in Chapel Hill showed that administering NMDA to hippocampal neurons triggered a voltage dependent influx of Ca2+.84 Three years later groups in Bethesda and New Haven demonstrated that this influx occurred through NMDA receptors; meanwhile, groups in Paris, London, and Bethesda found that physiological concentrations of extracellular Mg2+ blocked ion fluxes through NMDA receptors but that depolarizing the cells relieved this block.85 Since the interiors of neurons at rest are electrically negative with respect to their extracellular environment, Mg2+ would be drawn inward through ion channels, where it could obstruct the channel's fluxes. Depolarizing the neuron would allow the Mg2+ to diffuse outward, thereby removing the obstruction. Holgen Wigstrom and Bengt Gustafsson in Goteborg synthesized these observations in 1985 into a model for the NMDA receptor and LTP that depended on "coincident pre- and postsynaptic activity." (Fig. 12-7 depicts an expanded model from 1991. )86 The initial activation (through kainate or quisqualate receptors conducting Na + and K + ) would depolarize the postsy-

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FIGURE 12-7. NMDA receptors and long-term potentiation. A. During normal synaptic transmission glutamate released from presynaptic terminals can bind to NMDA, quisqualate, and kainate receptors, but only the latter two classes can function as ligandgated ion channels since the NMDA receptor is blocked by Mg 2+ . B. When the postsynaptic dendritic spine is depolarized, however, Mg2+ can exit and relieve the blockade, allowing Ca2+ as well as Na + and K + to move through the NMDA ion channel. The elevated cytoplasmic Ca2+ can then activate various protein kinases and initiate short- and long-term consequences. These may include release of a retrograde messenger that affects the presynaptic neuron. (From Kandel et al. [1991], Fig. 65-11, reprinted by permission of the McGraw-Hill Companies.)

naptic cells, producing e.p.s.p.s. This depolarization could also relieve inhibition of the NMDA receptors by releasing Mg2+ temporarily from these receptors' channels. A second, closely associated activation would then allow Ca2+ influx through the unblocked NMDA receptors. Wigstrom and Gustafsson emphasized the parallel with Hebbs formulation, whereby "the synapse is strengthened only when the presynaptic volleys occur in conjunction with the firing of the postsynaptic cell."8'

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Just how the rise in intracellular Ca2+ could produce LTP was not specified, but by then Ca2+'s role as a second messenger was well established. Reports soon described increased protein kinase activity associated with LTP, although the proteins targeted for phosphorylation were not yet identified.88 Persistent LTP seemed likely to require protein synthesis, and as the decade ended searches were underway to demonstrate its causal dependence on gene transcription.89 Meanwhile, a convincing link between LTP and learning had appeared. In 1986 Lynch and Michel Baudry showed that NMD A antagonists given in vivo impaired the learning of a task that depended on the hippocampus.90 By contrast, learning a task that did not rely on the hippocampus (i.e., could still occur after the hippocampus was destroyed) was unimpaired. Nevertheless, NMDA receptors were present not only in the hippocampus, and, as noted above, LTP occurred in other regions of the brain as well. And although LTP occurred at a third synaptic junction in the hippocampus—between axons from the granule cells and CAS neurons—NMDA receptors were not involved here. Retrograde Messengers

Investigations of the NMDA receptors naturally focused on the postsynaptic changes associated with LTP, but other evidence implicated presynaptic changes as well. For example, Richard Tsien in Palo Alto described in 1990 an enhanced release of neurotransmitters that accompanied LTP.91 If these changes also depended on the activation of postsynaptic NMDA receptors, then some "retrograde messenger" must carry the signal from post- to presynaptic sites. Two candidates for such a messenger attracted interest. One was an arachidonic acid metabolite.92 The history of such compounds stretches back to Ulf von Euler's discovery of prostaglandins, their identification as metabolic products of the fatty acid arachidonic acid by Sune Bergstrom and Bengt Samuelsson, and John Vane's explanation of how aspirin achieves its therapeutic ends by inhibiting certain steps in arachidonic acid metabolism (Nobel Prizes, 1970 and 1982). Nevertheless, convincing evidence for the involvement of arachidonic acid metabolites in LTP was not available as the decade ended. The other candidate was nitric oxide.93 This compound also has a lengthy pharmacological history, being the active agent from drugs such as nitroglycerine and amyl nitrite that had long been used for treating angina pectoris. More recently Robert Furchgott found that blood vessel endothelia release a substance that can dilate blood vessels, and this substance was then identified as locally synthesized nitric oxide (Nobel Prize, 1998). But firm evidence for its involvement in LTP was not available either.

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Structural Changes

InAplysia, where learning could be induced at identifiable synapses, anatomists could search profitably for the structural changes that Cajal imagined. But in mammalian hippocampi the opportunities for observing such changes were vastly diminished. The continuous flow of new experiences presents the hippocampus with a stream of stimuli, so that a single learning event in vivo could not be recognized for individual study. Structural complexities also hampered efforts to identify potential sites that could be compared before and after learning in vitro, and dendritic dimensions were too small to monitor changes at any given site during learning. Nevertheless, studies in the 1970s revealed new junctions forming in the hippocampi of adult animals (although these followed experimental trauma), and other reports described changing synaptic contacts that accompanied LTP.94 After Crick, who turned to neuroscience during his golden years in La Jolla, suggested that learning might reflect movement ("twitching") of dendritic spines, contractile proteins were soon identified in these structures.95 Demonstrations that spinal motility participated in actual learning did not immediately follow, however. Some birds, on the other hand, undergo spectacular changes in brain structure that accompany their learning courtship songs. During the 1980s Fernando Nottebohm in New York described the generation of new neurons in regions that control singing and their linkage through new synapses.96 But such processes seemed irrelevant to mechanisms for mammalian learning. At that time the standard view proclaimed that no new neurons could arise in the brains of adult mammals.

Conorusions

Formulations of the Neuron Theory suggested that learning reflected altered interactions between the neurons governing the altered behavior. Demonstrating the functional changes responsible for this learning turned out to be a challenging task. And during a long stretch of the early twentieth century searches for such changes were discouraged by Lashley's influential interpretation of memory as a holistic process beyond cellular and molecular mechanisms. Nevertheless, Hebb revived neuronal models at midcentury, proposing that a simultaneous activation of presynaptic and postsynaptic cells could facilitate subsequent transmission between them. In the 1960s two experimental approaches then guided the analysis of relevant cellular and molecular manifestations. Kandel's successful strategy—rewarded by a Nobel Prize in 2000—began by choosing to study a simple invertebrate nervous system having neurons identifiable from animal to animal. Using Aptysia he could demonstrate physiolog-

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ical changes in a neural circuit that controlled a learned behavior, define the pharmacological characteristics of the synapses in the circuit, and specify the biochemical consequences of receptor activation. The studies implicated cAMP second messenger cascades that modified synaptic activation through protein phosphorylations, affecting ion channel conductivity and neurotransmitter release as well as the gene transcriptions required for long-term changes. Investigations using a different approach in another invertebrate—genetic studies of learning in Drosophila—confirmed a role for cAMP-mediated processes. The other course began with fortuitous observations of surgical patients that implicated a certain structure, the hippocampus, in some forms of learning. Independent explorations of hippocampal circuits revealed synaptic changes that suggested the learning mechanism Hebb had formulated. Long-term potentiation of transmission from presynaptic to postsynaptic neurons, occurring at distinguishable classes of synapses, followed two experimental protocols: trains of high frequency stimuli to presynaptic inputs or paired stimuli over two converging pathways. The latter seemed a credible representation of associative learning. Pharmacological investigations pointed to the participation of NMDA receptors for glutamate, the excitatory neurotransmitter at these synapses. NMDA receptors are ligand-gated ion channels that permit Ca2+ influx, but this conductivity requires not only glutamate but also the relief through a priming depolarization of the receptor's blockade, achieved by trains of high frequency stimuli or paired stimuli. The Ca2+ influx presumably triggered short- and long-term changes that mediated persisting potentiation, but many details remained unclear in 1990. Supporting the mechanistic plausibility of this scheme were studies showing that antagonists to NMDA receptors prevented learning dependent on the hippocampus. Both successful courses reflected a hierarchical sequence of investigations: characterization of synaptic changes in defined neural circuits followed by examinations of the molecular processes underlying these changes. One course rewarded a patient and thoughtful campaign incorporating ingenious approaches, and the other included astute recognitions of what independent observations implied and how they might be extended. By contrast, some other approaches in the 1960s skipped the identification of synaptic changes and attempted to correlate learning tasks with biochemical changes in the brain. These yielded no new mechanistic insights. Analytical techniques were imprecise and the multitudes of irrelevant activities obscured identifications. Common advice to neophyte scientists includes an endorsement of asking the right question. Here the global question was obvious: How does the brain change during learning? The questions that generated effective research strategies were more focused: Does learning modify activity over a circuit controlling the learned response? How is synaptic efficiency in such circuits altered? What regulates the variable efficiency? Progress with these questions also required an assessment of experimental practicality: What techniques are

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needed, and can these be adapted from available methods or be developed feasibly? Here a range of contemporary approaches—learning protocols, genetic analyses, microelectrode recordings, pharmacological interventions, biochemical explorations, electron microscopic examinations—were recruited to unravel causal strands. By 1990 the forms of several models had emerged, although the possibility remained that additional mechanisms for learning might also function.

Notes

1. See Finger (1994); Hunt (1993). 2. Lashley (1926), p. 45. 3. Ibid., pp. 42, 44. Lashley maintained these conclusions, asserting that his "results are incompatible with theories of learning by changes in synaptic structure" (1929, p. 176) and advocating "some sort of resonance among a very large number of neurons" (1950, p. 479). 4. See Hebb (1949); Young (1951). 5. See Kandel and Spencer (1968). In addition to doubts about the specificity of Lashley s lesioning, there was concern about the plurality of sensory systems involved in Lashleys learning tests. For example, if learning a maze involved smell, sight, and touch, then destroying a localized representation for one of these modalities could leave others, localized elsewhere, to direct the animals performance. The learned performance would thus appear to have a generalized representation, even though the learning of each modality was sharply localized. 6. Ramon y Cajal (1995), vol. II, p. 724. 7. Arie'ns Kappers (1917). 8. Hebb (1949), p. 62. 9. Ibid., p. 62. 10. Lorente de No (1939). 11. Katz and Halstead (1950). 12. For historical accounts, see Fruton (1999); Judson (1979); Morange (1998); Robinson (1997). 13. Hyden (1960). 14. Dingman and Sporn (1961). 15. Hyden and Egyhazi (1962). Deiter's cells are also quite large, which favored their selection for experiments studying single cells. 16. Flexner et al. (1963). See also Agranoff and Klinger (1964). 17. Barondes and Cohen (1968). 18. As Dingman and Sporn (1964) pointed out, Hyden's finding of altered nucleotide ratios could represent merely the transcription into RNA of different stretches of DNA, rather than the learning experience rearranging the nucleotide sequence in RNA, as Hyden originally proposed. 19. For an account of McConnell and his experiments, see Rilling (1996). 20. Thompson and McConnell (1955). 21. For example, Lee (1963). 22. For example, Bennett and Calvin (1964). 23. McConnell et al. (1959).

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24. A more modest interpretation was that some change, neural or nonneural, increased, at some point in the causal chain, responses to the conditioned stimulus. 25. McConnell (1962). 26. See Rilling (1996). 27. Corning and John (1961). 28. Jacobson et al. (1966). 29. Byrne et al. (1966). 30. Rilling (1996, p. 591) noted that "McConnell, an innovator, raced from one exciting phenomenon to the next without comprehensive experimental analysis or adequate controls." Bennett (1970, p. 150) complained that "in spite of ten years or more of research in this area . . . even Dr. Corning [who was active in this research] cannot point to a 100% procedural replication of any one training study." 31. Mechanisms necessary for McConnell's proposal contradicted emerging rules for protein expression. They also defied recognized difficulties that extracellular macromolecules, such as RNA, encountered in crossing the cell membrane (a barrier to both ingested and injected RNA). Moreover, if ingested RNA could direct protein synthesis in planaria, then feeding hamburger, with its complement of mRNA molecules, should force the worms to synthesize beef proteins: an unlikely but testable possibility. All these problems with McConnell's formulations were recognized at the time. 32. von Neuman (1958). 33. See Allport (1986) for an account that includes personalities and rivalries. 34. Kandel and Tauc (1965a, 1965b). The work was done in Arcachon, where Aplysia were available. The second paper concentrated on identifiable giant cells, although facilitation in these cells was not dependent on pairing the stimuli. 35. Kandel and Spencer (1968). 36. Pinsker et al. (1970); Kupfermann et al. (1970); Castellucci et al. (1970). 37. Castellucci and Kandel (1974). 38. Castellucci and Kandel (1976). 39. Brunelli et al. (1976). 40. Ibid. 41. Klein and Kandel (1980). 42. Castellucci et al. (1980, 1982). 43. Carew et al. (1972); Pinsker et al. (1973); Bailey and Chen (1983). 44. Bailey and Chen (1988a, 1988b). 45. Montarolo et al. (1986). 46. Schacher et al. (1988). 47. Barzilai et al. (1989). 48. Dash et al. (1990). 49. Greenberg et al. (1987). 50. Carew et al. (1981); Hawkins et al. (1983). 51. For example, Alkon (1984); Alkon and Nelson (1990); Crow and Alkon (1978); Lukowiak and Sahley (1981); Morielli et al. (1986); Walters and Byrne (1983). 52. Carew et al. (1984). But see Sahley (1985) and the letters following: Trends Neurosci. 9: 410^11 (1986). 53. Benzer (1967). Quinn et al. (1974) described the conditioning procedure. 54. Dudai et al. (1976); Duerr and Quinn (1982). Controls ruled out trivial alternatives, such as alterations in perception, sensitivity to shock, etc. 55. Byers et al. (1981); Chen et al. (1986). 56. Dudai et al. (1983); Livingstone et al. (1984). 57. Levin et al. (1992).

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58. See Penfield (1952). 59. Milner and Penfield (1955). See also Penfield and Milner (1958). 60. Haglund and Collett (1996a). 61. Scoville and Milner (1957). Lesions varied among the patients, including destruction of different amounts of the hippocampus and of surrounding regions. Evaluations focused on H.M.; testing psychotic patients was more problematic. 62. Milner (1962). 63. For example, Flexner et al. (1963). 64. Gaffan (1977). 65. Mishkin (1978). 66. Mishkin initially argued that the amygdala also was crucial. Later studies corrected that assignment, localizing critical regions to hippocampus, dentate gyrus, subicular complex, and entorhinal, perirhinal, and parahippocampal cortices. See Squire and Zola-Morgan (1991); Milner et al. (1998). 67. The name reflects a shape like a sea horse. Another name, Cornu Ammonis, is responsible for the "CA" designation of neurons and refers to a shape like that of the ram god's horn. 68. L0mo (1966). 69. A sustained, strong muscular contraction is known as "tetany." Stimuli capable of producing such contractions are "tetanizing" stimuli. 70. Feng (1941). 71. Katz and Miledi (1968). 72. Haglund and Brown (1995). 73. Bliss and L0mo (1973). 74. Bliss and Gardner-Medwin (1973). 75. Andersen et al. (1973). 76. Schwartzkroin and Wester (1975). 77. Levy and Steward (1979, 1983). The first pathway was from the ipsilateral side and the second from the contralateral. 78. Barrionuevo and Brown (1983). See also Kelso et al. (1986). 79. For example, Iriki et al. (1989); Artola et al. (1990). 80. Stanton and Sejnowski (1989). See also Artola et al. (1990). 81. Collingridge et al. (1983a, 1983b). See also Harris et al. (1984). 82. Collingridge et al. (1983a), p. 20. 83. Lynch et al. (1983). 84. Dingeldine (1983). 85. MacDermott et al. (1986); Nowack et al. (1984); Mayer et al. (1984). 86. Wigstrom and Gustafsson (1985), p. 519. 87. Ibid. 88. For example, Malinow et al. (1988); Malenka et al. (1989). 89. For example, Cole et al. (1989). 90. Morris et al. (1986). 91. Malinow and Tsien (1990). 92. Dumuis et al. (1988); Williams et al. (1989). 93. Garthwaite et al. (1988). See also O'Dell et al. (1991); Schuman and Madison (1991). 94. Matthews et al. (1976); Van Harreveld and Fifkova (1975); Lee et al. (1980). 95. Crick (1982); Matus et al. (1982); Caceres et al. (1983). 96. See Nottebohm (1986).

13 DISEASES AND THERAPIES

Deiiningf ana Developing

Scientists, while pursuing pure knowledge, are eager also for useful knowledge. In fact, much biomedical research is applied science, dedicated to defining diseases and developing therapies. In such endeavors the goals include recognizing distinct diseases, characterizing their pathophysiologies, and designing therapies to counteract these pathophysiologies.1 Medical practice has long included the gathering and sorting of symptoms into related groups that were then considered the manifestations of particular disorders. Often-associated symptoms,2 collected into syndromes, could designate diseases, and in many cases prominent alterations in form or function pointed to readily identifiable ills, such as rheumatism, convulsions, and dropsy. In other situations the dividing lines between apparent entities, and even the distinctions between normal and abnormal, were less clear. Moreover, subsequent investigations frequently subdivided what once had seemed a simple entity. The search for mechanisms underlying disease processes, which accompanied the development of scientific (and cellular) pathology during the nineteenth century, revealed different processes producing apparently similar symptoms. And while the search for pathophysiology aided in developing new therapies, different responses to a therapy could indicate different pathophysiologies and thus different diseases. 319

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Identifying diseases and discovering pathophysiologies has been particularly difficult for disorders of the nervous system, due in part to the complexity and inaccessibility of the brain and in part to a reliance often on patients' reports (of feelings, perceptions, emotions) rather than on manifestations directly observable by the physician. Furthermore, the split between neurological and psychiatric diseases seems to reflect a divide between disorders having lesions with detectable pathological changes, gross or microscopic, and disorders having no such manifestations, a divide echoing dualistic body-mind categorizations. Such divisions have also rationalized which mode of therapy (chemical or psychological) was deemed appropriate. In any event, the demarcations between psychiatric diseases—and even between psychiatric diseases and health—have been fiercely debated. One step toward distinguishing among possible psychiatric diagnoses was establishing pathognomonic characteristics, such as the American Psychiatric Association's Diagnostic and Statistical Manual of Mental Disorders (DSM). The third edition, published in 1980 (DSM-III), offered standardized criteria for defining disorders just as biological psychiatry began to flower. Although critics complained that diagnoses were then imposed by committee on political as well as scientific grounds,3 the latter decades of the twentieth century witnessed new demonstrations both of physical changes accompanying these clinical criteria and of altered physiological and biochemical function in many syndromes traditionally classified as psychiatric disorders. Extensive investigations in the latter half of the twentieth century have reshaped our understanding of both the pathology and therapy for a broad range of neural disorders. Here three disease complexes, one "neurologic" and two "psychiatric," may serve as examples of the growing clinical attention to synaptic transmission and its modifications.

Parkinson's Disease James Parkinson, a general practitioner on the outskirts of London, reported in 1817 the common features observed in six patients: "involuntary tremulous motion, with lessened muscular power," associated with a flexed posture and a gait that passed "from a walking to a running pace."4 He named the disorder shaking palsy (paralysis agitans) and described the patients' progressive deterioration, beginning in later life and advancing through the years, from initial mild tremor to ultimate immobility (akinesia). Although Parkinson recorded treatments with the standard remedies of the day—such as bleeding and blistering—he advocated caution "Until we are better informed respecting the nature of this disease."

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Jean-Martin Charcot, the most eminent Parisian neurologist of the nineteenth century, emphasized a characteristic muscular rigidity as well as the tremor and difficulty with initiating movements (hypokinesia), and in 1867 he attached Parkinson's name to the disorder.5 After trying available medicines Charcot recommended scopolamine, an atropine-like botanical product, for its ability to reduce the symptoms. Atropine-like anticholinergic agents (as they were later recognized) remained the standard therapy for a century. A significant step toward localizing the disorder and defining its pathophysiology was C. Tretiakoff's description—part of an extensive thesis on brain histopathology submitted in Paris in 1919—of lesions in the brains of nine patients with Parkinson's disease.6 Tretiakoff found a degeneration and loss of cells in the substantia nigra, a region in the midbrain named for its darkly pigmented neurons. Five years later S. A. Kinnier Wilson in London summarized clinical and experimental evidence for involvement of the caudate nucleus in Parkinson's disease, and in 1928 Armando Ferraro described how destroying the caudate led to degeneration of the substantia nigra, as would be expected if cell bodies in the substantia nigra sent axons to the caudate.7 Studies during the first half of the twentieth century characterized two interacting systems that govern motor control. One involves large pyramid-shaped neurons in a region of the cortex initiating voluntary movement; their axons pass down the spinal cord to activate ventral horn motoneurons. The other, named the extrapyramidal motor system, includes a loop passing through the basal ganglia. These ganglia are collections of cell bodies at the base of the cerebral hemispheres that receive impulses from the cortex and send impulses through intervening structures back to the cortex. The basal ganglia are separated spatially into caudate nucleus, putamen (together known as the striatum and containing morphologically similar cells), and globus pallidus. By the 1950s Parkinson s disease was classified as a disorder of the basal ganglia/extrapyramidal motor system, but precisely how malfunctioning over this circuit produced tremor, rigidity, and akinesia remained uncertain in 1990. The causes of Parkinson's disease also remained uncertain. Similar symptoms followed the encephalitis produced by certain viruses (von Economo's encephalitis lethargica) as well as the vascular changes in the brain that accompany aging. Most cases, however, were of unassignable origin: idiopathic Parkinson s disease. By midcentury doubts about these cases arising from a single cause grew, and some favored the general designation "parkinsonism," referring merely to the syndrome. After reserpine was introduced in the 1950s for the treatment of schizophrenia (see below), certain troubling side effects became apparent, including tremor, muscular rigidity, and akinesia. Fortunately, this drug-induced parkinsonism disappeared after the reserpine was discontinued. Moreover, examinations of reserpine's effects suggested a possible mechanism underlying these

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symptoms. Arvid Carlsson (Fig. 13-1A) had studied reserpine's ability to deplete serotonin from platelets while visiting Bernard Brodie in Bethesda. After Carlsson s return to Lund, he extended these investigations, demonstrating reserpine s ability to deplete catecholamines and serotonin in the brain (chapter 9). And in 1957 Carlsson reported that administering dopa, the metabolic precursor of dopamine and noradrenaline (Fig. 5-3), abolished the lethargic immobility that reserpine produced in mice.8 At that time dopamine was attracting attention as a likely neurotransmitter, and in 1958 Carlsson stressed parallels between reserpine-induced depletion of dopamine from the striatum and reserpine-induced parkinsonism.9 (For these and numerous other contributions, cited in this and in earlier chapters, Carlsson received the Nobel Prize in 2000.) Two years later Oleh Hornykiewicz in Vienna completed the argument. Hornykiewicz, while visiting with Hermann Blaschko in Oxford during the 1950s, had been introduced to dopamine as a likely neurotransmitter. Now he described a striking decrease in the dopamine content of the striatum from six deceased individuals who had suffered from parkinsonism; the noradrenaline content was unchanged.10 In 1963 Hornykiewicz reported a decreased dopamine content in the substantia nigra as well, and the next year Carlsson

FIGURE 13-1. A, left, Arvid Carlsson (1923-). B, right, Solomon H. Snyder (1938-; courtesy of Arvid Carlsson and Solomon Snyder).

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FIGURE 13-2. Connections of the basal ganglia. Hornykiewicz's diagram shows fibers from the substantia nigra (S.n.) innervating the putamen (Put.), caudate nucleus (N.C.), and perhaps the globus pallidus (Pall), which constitute the basal ganglia. In addition, fibers connect the cortex (areas 6ay and 4s), thalamus (Thai, with its nuclei L.po and V.o.a), and basal ganglia. (From Hornykiewicz [1966], Fig. 2, courtesy of the American Society for Pharmacology and Experimental Therapeutics.)

and associates used fluorescence microscopy to map a dopaminergic pathway from cell bodies in the substantia nigra to presynaptic terminals in the striaturn.11 Figure 13-2 shows Hornykiewcz's diagram from 1966 delineating these connections. Levoo.'op a

If Parkinson's disease reflected the loss of nigrostriatal neurons and their dopaminergic input to the striatum, then replacing the missing dopamine should provide symptomatic relief. But, as was recognized in the 1960s, amines like dopamine do not penetrate from blood to brain (at physiological pH they

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are largely ionized and thus cannot diffuse across the nonpolar blood-brain barrier). An obvious alternative was administering the immediate metabolic precursor, dopa, which does penetrate and does then increase brain dopamine levels (as Carlssons study indicated). So in 1961 Hornykiewicz injected intravenously into 20 patients with parkinsonism levodopa (L-dopa, the active L-stereoisomer).12 He reported a marked relief of symptoms: patients "who previously could not change from a prone position to ... sitting [were] able to run and jump." Independently, Andre Barbeau in Montreal effected similar improvements with oral administration of levodopa.13 Others, however, found only minimal improvements with low doses of levodopa, while high doses produced serious and limiting side effects, including nausea, vomiting, and elevated blood pressure and heart rate.14 But in 1967 George Cotzias in Upton described sustained improvement after administering severalfold higher doses, with side effects lessened by gradual increases in the dosage from initially low amounts.15 Further studies soon confirmed the striking improvement with high doses of levodopa, establishing Cotziass approach as a practical and effective therapy.16 But even with Cotziass regimen the side effects were troubling. A clever pharmacological ploy, however, minimized a significant class of toxicities, including the gastrointestinal and cardiovascular disturbances, toxicities arising from the conversion of levodopa to dopamine outside the brain ("in the periphery").17 During attempts to develop better antihypertensive drugs, the pharmaceutical industry synthesized various inhibitors of dopa decarboxylase (the enzyme responsible for converting dopa to dopamine; see chapter 9), and in 1966 Sidney Udenfriend in Bethesda noticed that one of these, later named carbidopa, unexpectedly increased dopamine levels in the brain.18 In 1969 Alfred Pletscher in Basel showed that carbidopa could not cross the blood-brain barrier and thus blocked the conversion of levodopa to dopamine in the periphery while allowing dopamine synthesis in the brain to continue.19 Clinical trials indeed demonstrated that administering carbidopa together with levodopa not only reduced the dose of levodopa required but also reduced the peripheral side effects.20 Merck then marketed this combination as Sinemet, which became a standard form of levodopa therapy. Other side effects, those attributed to altered brain function, were not diminished by carbidopa. Moreover, with prolonged administration of Sinemet as well as of levodopa, there could appear disabling involuntary movements plus abrupt transitions from mobility to immobility ("on-off effects"). And parkinsonian symptoms progressed even with continued dosages of these medicines, so the drugs became less effective as time passed. Although levodopa offered symptomatic relief to most patients, it did not halt the death of nigrostriatal neurons, which continued throughout the course of Parkinson s disease.

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Bromocriptine

A better approach might be to give dopaminergic agonists, since the nigrostriatal neurons that convert levodopa to dopamine are continually dying in Parkinson's disease, while the postsynaptic neurons with their dopamine receptors persist. Although unaware of its pharmacological mode of action, Robert Schwab in Boston tried apomorphine in 1951 and noted some transient relief of parkinsonian symptoms.21 Interest revived when in 1967 A. M. Ernst in Utrecht showed that apomorphine was a dopaminergic agonist.22 Apomorphine, however, had significant disadvantages clinically, including a propensity to cause vomiting and the inability to be administered orally. Then in 1974 Donald Calne in London found that another dopaminergic agonist, bromocriptine (Parlodel), which was being used with another dopamine-sensitive problem, could relieve parkinsonian symptoms.23 Further clinical trials confirmed the drug's utility.24 Unfortunately, bromocriptine was not the ultimate answer, either, for it seemed less potent than levodopa and had its own catalog of side effects. As the 1980s closed the search for better dopaminergic agonists continued. Amantadine

The preceding therapeutic approaches were developed through conscious efforts to correct an identified pathological defect. Meanwhile, another agent appeared fortuitously. Amantadine (Symmetrel) was introduced in the 1960s to ward off influenza, and in 1968 a patient with Parkinson s disease who was prescribed amantadine for this purpose noticed a remarkable decrease in her parkinsonian symptoms. The patient described this improvement to her neurologists in Boston, who included Schwab, and they quickly organized a sixmonth trial with 163 patients, two-thirds of whom enjoyed improvement.25 Further studies confirmed this efficacy and a generally low incidence of side effects. The therapeutic response was less than with levodopa, however, and sometimes disappeared after only a few months. How amantadine effected the clinical improvement remained uncertain in 1990. Early reports argued that amantadine increased dopamine release, but no increase in dopamine metabolites could be detected when amantadine was given to patients.26 Selegiline

In 1982 a neurologist practicing in San Jose, William Langston, discovered six young to middle aged adults who developed sudden and severe parkinsonism.27 All were intravenous drug abusers, and Langston traced the acute onset of their symptoms to injections of a synthetic opioid—a "designer drug" sold on

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the streets—contaminated with l-methyl-4-phenyl-l,2,5,6-tetrahydropyridine (MPTP).28 Three years earlier Irwin Kopin in Bethesda had described a single case of an intravenous drug abuser who also developed parkinsonism after injecting material contaminated with MPTP. This individual had died of a subsequent drug overdose and an autopsy demonstrated destruction of his substantia nigra specifically.29 Kopin's report attracted little attention, and he did not pursue the topic. But after Langston s discovery Kopin showed that MPTP could produce parkinsonism in primates, providing a useful animal model for studying this disorder.30 In 1984 groups in San Francisco, Bethesda, and Piscataway found that the actual toxin was not MPTP but a metabolite, l-methyl-4-phenylpyridinium (MPP+).31 Monoamine oxidase catalyzed the conversion of MPTP to MPP + , and the sensitivity of this oxidation to inhibitors of monoamine oxidase B indicated that this form of the enzyme was responsible. The next year Richard Heikkila in Piscataway showed that MPP+ inhibited mitochondrial processes essential for cellular survival.32 That year, 1985, Solomon Snyder in Baltimore reported that MPP+ was transported specifically into dopaminergic neurons by the dopamine re uptake system, accounting for its selective toxicity toward these neurons.33 Although the cause of idiopathic Parkinson s disease was unknown, a favorite candidate was some toxin, endogenous or exogenous, that could kill nigrostriatal neurons specifically. The findings with MPTP reinforced this notion and raised the possibility that the pathological agent might also require oxidative conversion by monoamine oxidase B. The best-studied specific inhibitor of this enzyme was selegiline (Eldepryl, originally named deprenyl), and before the identification of MPTP/MPP+ toxicity it had been used for some years in Europe to treat parkinsonism. The rationale was that monoamine oxidase B destroyed dopamine and hence preventing such destruction would augment neuronal dopamine stores.34 In the late 1980s several clinical trials were launched to determine whether selegiline could also slow or even stop the progression of cell death that occurs in Parkinson's disease. Selegiline did diminish the parkinsonian symptoms and did delay the need for levodopa treatment, and these studies confirmed selegiline as a useful therapeutic agent.35 Whether selegiline slowed the rate of nigrostriatal death was, however, argued.36

Anticnolinergic Agents

Clinical explorations in the nineteenth century identified as useful drugs certain botanical products that were later recognized as muscarinic antagonists. Synthetic anticholinergics, such as trihexyphenidyl (Artane) and benztropine (Cogentin), followed. None were as potent as levodopa, but they continued to

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be useful for treating patients before symptoms were severe and as adjuncts with other drugs. Experimental studies, moreover, provided a belated rationale. For example, injecting cholinergic agents into the basal ganglia of experimental animals produced parkinsonian symptoms that were relieved by administering levodopa.37 Such observations fostered the notion that parkinsonism reflected an imbalance between dopaminergic and cholinergic pathways.38

Other Therapeutic Approaches

As the 1980s ended two other therapeutic possibilities were being pursued. One involved transplanting dopaminergic cells into the brains of parkinsonian patients.39 The other explored the possible use of various neurotrophic factors to slow nigrostriatal death.40

Summary

Although the causes of neuronal death in Parkinson's disease remained unknown in 1990 and no proven means for halting the progressive deterioration was yet available, several palliative measures that enjoyed considerable clinical success had been developed, in many cases through rational searches. These therapies focused on suppressing cholinergic pathways and potentiating dopaminergic pathways of the extrapyramidal motor system. The latter approach included use of dopaminergic agonists, boosting dopamine synthesis by providing precursors to dopamine, and halting degradation with inhibitors of monoamine oxidase B.

Schizophrenia

Accounts of madness stretch back to ancient times, but only in recent centuries have descriptions focused on discriminating among its manifestations.41 In 1893 Emil Kraepelin in Heidelberg applied the term dementia praecox to a chronic and worsening disorder that began in late adolescence or early adulthood and was associated with a deterioration of rational thinking, often accompanied by hallucinations and bizarre behavior. This entity included groups earlier labeled hebephrenia, catatonia, and paranoia. Five years later Kraepelin distinguished between dementia praecox and manic-depressive illness, which involved exaggerated swings of mood, could begin at other ages, and did not necessarily have so dire an outcome. Although Kraepelin s accounts were largely descriptive, he endorsed the premise that altered behavior sprang from lesions in the brain.

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Eugen Bleuler in Zurich then refashioned Kraepelin's catalog, stressing thought disorders, inappropriate emotional responses, and social withdrawal, and he contemplated a family of diseases, whereas Kraepelin imagined a unitary illness. Influenced by psychoanalytical thought, Bleuler also presented psychological mechanisms. In 1911 he renamed the disorder schizophrenia, to signify a splitting among associations and between aspects of the personality. Through the twentieth century uncertainties about the diagnostic boundaries continued, with clear differences in diagnostic criteria appearing between national schools of psychiatry as well as among individual psychiatrists.42 Disagreements also persisted over the causal roles of psychological mechanisms—arising from individual, familial, or social dysfunction—and of neural mechanisms. During the first half of the twentieth century, psychiatry in English-speaking countries generally embraced psychoanalytical formulations of schizophrenia, culminating during the decade after World War II in explicit psychodynamic mechanisms.43 These schemes did not dwell on the likelihood of psychological trauma altering brain physiology, with altered physiology then manifesting as pathological behavior. And their proponents embraced psychological therapy, not dwelling on the likelihood of such therapy altering brain function. Inattention to neural causes and mechanisms was fostered not merely by commitments to the associated therapeutic mode. It also reflected failures to validate early accounts of pathological changes in the brains of schizophrenic patients.44 Further skepticism followed refutations through the 1960s of various claims for biochemical abnormalities.45 On the other hand, early X-ray studies supported a link between schizophrenia and enlargement of the brain's ventricles.46 And in the 1980s newer imaging techniques—computerized tomography, magnetic resonance imaging, and positron emission tomography— strengthened these conclusions and documented common deficits, including a decreased mass of the temporal lobes and a decreased metabolism in the frontal cortex.47 These changes might, of course, represent a secondary response to trauma, including psychological stresses. At midcentury hallucinogenic drugs attracted the attention of neuroscientists, at the same time furthering arguments for psychological symptoms arising from altered neural function. LSD produced striking visual hallucinations, and in 1953 John Gaddum in Edinburgh showed that LSD blocked responses to serotonin (albeit on smooth muscle).48 D. W. Wooley in New York then suggested that schizophrenic hallucinations might spring from deficient serotonergic activity in the brain.49 The psychotic behavior produced by LSD, however, did not resemble the symptoms of schizophrenia, in which auditory hallucinations predominate. More significant, the early antipsychotic drugs, reserpine and chlorpromazine, suppressed serotonergic systems, contrary to Wooleys proposal. By contrast, another hallucinogenic drug, amphetamine,

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induced a syndrome more closely resembling schizophrenia, including paranoia and auditory hallucinations.50 This capability supported a formulation that was highly influential through the remainder of the century. The most compelling evidence for a primary physical cause, however, came from genetic studies correlating the likelihood of schizophrenia among relatives with the closeness of their relationship.51 Comparisons of the incidence of schizophrenia between identical twins were particularly striking. Some criticized such conclusions by pointing out that individuals inherit environments as well as genes, but studies of adopted individuals then demonstrated that vulnerability followed the biological rather than the environmental ties.52 Nevertheless, the concordance for schizophrenia between identical twins was only about one half: other factors—perhaps infectious, metabolic, and/or social— must interact with an inherited vulnerability to produce illness. Even in cases with genetic predispositions, it seemed likely from the patterns of inheritance that multiple genes participated. This polygenic nature undoubtedly contributed to failures through the 1980s to demonstrate linkages between vulnerability and particular chromosomal loci.53 Furthermore, most schizophrenic patients had no family history of schizophrenia or similar psychiatric illnesses. This disparity reinforced the likelihood that the schizophrenic syndrome represented a collection of disorders differing in etiology and pathophysiology.

Reserpine

In 1931 two physicians in Calcutta, Gananath Sen and Kartick Bose, described the efficacy of snake root in treating "insanity" and high blood pressure.54 This root of an Indian bush (Rauwolfia serpentina) had been ingested since antiquity to treat a range of maladies, from snake bite to madness, and after Sen and Bose's report a flurry of publications, also in Indian journals, endorsed their findings. In 1949 a report in a British journal led to trials with hypertensive patients in the United States and to CIBA Pharmaceutical Products exploring the active ingredients in snake root.55 In 1952 they isolated reserpine and identified it as the component responsible for sedation; the following year Frederick Yonkman at CIBA coined the term "tranquilizer" to describe reserpine's soothing properties.56 An article in The New York Times reported in 1953 an award in India for the development of snake root to treat psychiatric illnesses. This account caught the eye of Nathan Kline, an energetic psychiatrist at Rockland State Hospital in Orangeburg. Kline, eager to identify effective remedies for psychiatric disorders, quickly organized trials on chronically ill inpatients of a snake root extract (Raudixin) provided by Squib and of purified reserpine (Serpasil) provided by CIBA. The following year, 1954, Kline described the preparations'

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abilities to sedate and alleviate anxiety, but he detected "no evidence that [they] in any way alter . . . the schizophrenic process itself."57 Kline noted, however, that the patients tested were severely deteriorated; later he was more enthusiastic, and subsequent investigations demonstrated the suppression of specific symptoms: diminished delusions and hallucinations plus normalization of the thought processes.58 Reserpine s efficacy, however, was less than that of other drugs then being introduced, and the latter became the predominant therapies for several decades. Reserpine also suffered from a tendency to induce psychological depression, an action that intrigued those pondering the mechanisms of mood disorders (see below). Reserpine was soon shown to cause depletion of catecholamine and serotonin stores in the body (chapter 9), and it became a widely used tool in pharmacological research. But because of its actions on this range of neurotransmitters, reserpine did not implicate any particular pathophysiology for schizophrenia. Cnlorpromazine ana Haloperiaol

Henri Laborit, a French naval surgeon stationed in Tunisia, was searching in the late 1940s for pharmacological means to blunt operative stresses.59 By 1951, when he was transferred to Paris, his regimen included an antihistaminic, promethazine (Phenergan), that induced a state of seeming indifference. Hoping for still more effective drugs, Laborit encouraged Rhone-Poulenc Laboratories to synthesize other antihistaminics with similar actions on the central nervous system. Simone Charpentier at Rhone-Poulenc showed that chlorpromazine (Thorazine, Fig. 13-3A), a compound newly synthesized in 1950 and having a phenothiazine ring like promethazine, produced a distinctive behavioral alteration: rats trained to climb a rope for food no longer did so after receiving chlorpromazine, not because they lost the ability but apparently because they lost interest. Laborit quickly incorporated chlorpromazine into his regimen and noted a similar blissful unconcern in his patients. Laborit encouraged psychiatrists to try this drug with agitated patients, too. In 1952 a prominent Parisian psychiatrist, Jean Delay, reported notable decreases in agitation, aggression, and hallucinations without a loss of mental alertness. (The standard treatment of agitated schizophrenic patients then included sedative drugs such as barbiturates, which produced lethargy and torpor.) By 1954 trials of chlorpromazine had spread across the globe, with the drug garnering broad acclaim. The success with chlorpromazine inspired the pharmaceutical industry to synthesize a host of variants based on the phenothiazine ring and its analogs. By 1964 there were 11 being prescribed, and that year a comprehensive test confirmed chlorpromazine s utility: more than 75% of acutely ill schizophrenic

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FIGURE 13-3. Structures of therapeutic drugs.

patients showed marked to moderate improvement, with relief of thought disorders, delusions, hallucinations, and agitation.60 A drug having similar effects but a different structure appeared soon after chlorpromazine. At the end of the second world war, Paul Janssen set out to build a research division for the pharmaceutical firm established in Turnhout by his father. Through screening a range of newly synthesized compounds for chlorpromazine-like behavioral responses, Janssen in 1958 selected haloperidol (Haldol, Fig. 13-3D). Clinical tests the following year were encouraging, and haloperidol became available for treatment in Europe in 1960 and in the United States in 1967. It, too, proved to be highly effective and has been widely used. Together these drugs have been called major tranquilizers,61 neuroleptics,62 and antipsychotics (I will use this last name). The phenothiazines and haloperidol seemed to produce similar therapeutic responses, although the incidence and severity of side effects differed among the individual drugs. Of these side

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effects, the most prominent were disorders of the extrapyramidal system, including a drug-induced, reversible parkinsonism. Examinations of the drugs' pharmacological actions also suggested how they relieved schizophrenic symptoms. These analyses, however, were complicated by the multitudes of the drugs' effects, particularly with the phenothiazines that variously oppose cholinergic, dopaminergic, noradrenergic, serotonergic, and histaminergic systems. Nevertheless, in 1963 Carlsson, now in Goteborg, proposed that antipsychotic drugs act by blocking catecholamine receptors.63 Mice given chlorpromazine or haloperidol excreted increased amounts of dopamine and noradrenaline metabolites, and Carlsson considered that these increases reflected an elevated release of the parent neurotransmitters to compensate for the (hypothesized) blockade of their receptors. Although Carlsson did not comment on whether the therapeutic response followed blockade of dopaminergic vs. noradrenergic receptors,64 J. M. van Rossum in Nijmegen soon did. He argued that "dopamine receptor blockade is an important factor" in the drugs' therapeutic activity since chlorpromazine and haloperidol produced extrapyramidal symptoms (he even suggested that "extrapyramidal side effects are a prerequisite" for antipsychotic activity).65 Van Rossum also cited the drugs' ability to suppress behavioral responses attributed to dopaminergic systems. A. Randrup in Roskilde had demonstrated earlier that chlorpromazine and haloperidol counteracted certain behaviors in rats induced by amphetamine, and Randrup now showed that these behaviors were attributable to dopaminergic systems.66 Nevertheless, other amphetamineinduced behaviors were attributable to noradrenergic systems. Antipsychotic drugs could also counteract amphetamine-induced psychoses in humans, so a significant question was whether this psychosis, said to resemble schizophrenia closely, reflected dopaminergic or noradrenergic actions of amphetamine. An ingenious approach to discriminating between these alternatives came from Snyder (Fig. 13-1B). Snyder had begun his stellar research career with Julius Axelrod in Bethesda, and after completing his training in psychiatry he returned to research, now in Baltimore. There he pursued a broad range of important problems with insight and vigor, and some of his numerous contributions are cited in previous chapters. Now Snyder approached the dopamine/noradrenaline quandary by noting that noradrenaline has two stereoisomers, only one of which is biologically active, whereas dopamine has no stereoisomers. Amphetamine also exists as two stereoisomers, and in 1969 Snyder showed that both stereoisomers of amphetamine were equally effective in eliciting dopaminergic responses in experimental animals, but only one stereoisomer could elicit noradrenergic responses.6' Samuel Gershon in New York then applied this discriminatory test to amphetamine-induced psychoses in volunteers. Both stereoisomers of amphetamine were equally effective, implying a dopaminergic action.68

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Thus "the dopamine theory of schizophrenia" emerged in the early 1970s, proposing that the disorder reflected excessive dopaminergic activity (as mimicked by amphetamine) and responded to dopaminergic receptor blockade (with chlorpromazine or haloperidol) or dopamine depletion (with reserpine). Accordingly, schizophrenic symptoms were worsened by administering amphetamine or dopa.69 Nevertheless, as Snyder warned in 1974, dopaminergic pathways might merely be modulating aberrant responses of another neurotransmitter system where the fundamental pathology lay.'° In 1971 Snyder showed that the three-dimensional structure of chlorpromazine resembled that of dopamine (Fig. 13-4), as expected if it blocked

FIGURE 13-4. Three-dimensional structures of chlorpromazine and dopamine. Drawings of the structures of chlorpromazine (A) and dopamine (B), determined by X-ray crystallography, are shown superimposed (C). (From Horn and Snyder [1971], Fig. 1, courtesy of Solomon H. Snyder.)

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dopamine receptors.71 The next year Paul Greengard in New Haven offered further support for dopamine theories, describing chlorpromazine's and haloperidol's inhibition of dopamine-activated adenylate cyclase activity in the basal ganglia.72 This observation suggested that drug-sensitive dopamine receptors were coupled to adenylate cyclase. Quantitative comparisons of receptor blockade, assayed as inhibition of dopamine-activated adenylate cyclase, correlated well with the therapeutic potencies of a series of phenothiazine antipsychotics. But, as Leslie Iversen in Cambridge pointed out in 1975, the correlation failed with haloperidol, a potent antipsychotic but a poor inhibitor of adenylate cyclase activation.73 Snyder, however, resolved this discrepancy the following year. He assayed receptor binding in vitro as the ability of various drugs to displace labeled haloperidol from a brain membrane fraction. Now there appeared an excellent correlation for haloperidol as well as the phenothiazines.74 Philip Seeman in Toronto independently obtained similar results, published as a convincing plot (Fig. 13-5).75

FIGURE 13—5. Correlation between clinical dose and affinity for dopamine receptors. The plot correlates clinical doses for treating schizophrenia with the drugs indicated (as milligrams of drug per day) on the x-axis, with affinity for the dopamine receptor (expressed as the ICso, the concentration of drug required for 50% inhibition of labeled haloperidol binding, measured as moles of competing drug per liter) on the y-axis. (From Seeman et al. [1976], Fig. 1. Reprinted by permission of Nature, © 1976, Macmillan Magazines, Ltd.)

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In 1979 John Kebabian and Calne, now in Bethesda, summarized studies on adenylate cyclase activation and receptor binding, from which they proposed two classes of dopamine receptors: DI, linked to adenylate cyclase activation (to which haloperidol binds weakly), and D2, not so linked (to which haloperidol binds strongly).76 The potency of antipsychotic drugs then correlated with binding to D£ receptors, and this property became a standard screening assay in searches for new antipsychotic medications. The amino acid sequence of D£ receptors, determined by cDNA methods, appeared in 1988.77 The hyperactivity postulated by dopamine theories of schizophrenia could result from increased dopamine release and/or increased sensitivity of dopaminergic receptors. Evidence for elevated dopamine turnover in the brains of schizophrenic patients was not clear-cut.78 On the other hand, dopamine receptor levels were demonstrably elevated, but this could merely represent compensatory changes after chronic blockade by antipsychotic drugs ("upregulation of receptors").79 In 1986 Snyder and associates, measuring ligand binding to D£ receptors in vivo with positron emission tomography, reported increased numbers of dopamine receptors in basal ganglia of schizophrenic patients who had not previously received any antipsychotic drugs.80 The next year, however, Lars Farde in Stockholm, using a different ligand for the technique, found no increase.81 As the 1980s ended this contradiction remained. Meanwhile, fluorescence microscopic techniques had delineated dopaminergic axons from the brain stem projecting not only to the basal ganglia but also to the frontal and temporal cortex.82 These were areas where newer imaging studies had by 1990 revealed structural and functional changes correlated with schizophrenia.83 Clozapine

Clozapine (Clozaril, Fig. 13-3C) was synthesized in 1958 by Wander AG during the pharmaceutical industry's rush to find new psychotherapeutic drugs.84 Although expected to have antidepressant properties, clozapine instead elicited chlorpromazine-like activities in animals. When clozapine was then tested in schizophrenic patients in the 1960s, it produced striking improvements even though it lacked one characteristic (if unwanted) attribute, the liability for causing extrapyramidal side effects.85 Indeed, responses seemed superior to those with earlier drugs. But in 1975 an infrequent but often lethal agranulocytosis was linked to clozaril use,86 and this toxicity led to clozariFs being withdrawn in many countries and sharply restricted in others. Due to impressions that clozapine was superior to the standard drugs, interest persisted. And in 1988 John Kane in Glen Oaks and Herbert Meltzer in Cleveland published a large-scale comparison of clozapine vs. chlorpromazine in patients unresponsive to haloperidol: 30% of these "drug-resistant" patients improved with clozapine vs. 4% with chlorpromazine.87 Subsequent studies

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confirmed clozapine's success in patients refractory to standard antipsychotic medications. Clozapine was then approved for use in the United States, but only with mandatory monitoring of the blood. (The agranulocytosis subsided if the drug was stopped immediately after changes became detectable.) By 1990 the pharmaceutical industry was avidly seeking new drugs with similar therapeutic capabilities but without the hematological toxicities. Why was clozapine superior to standard antipsychotic drugs? Surprisingly, it blocked D£ receptors only modestly.88 On the other hand, clozapine blocked serotonergic 5HT2 receptors, and enthusiasm for modulating this and other neurotransmitter systems revived.89 (In 1991 a fourth class of dopaminergic receptors, D4, was identified and sequenced. Clozapine, in contrast to standard antipsychotics, bound selectively to D4, suggesting that these receptors were the therapeutically significant entities; further research, however, soon undercut this proposal.90)

Summary

New remedies introduced in the 1950s proved dramatically superior to earlier medications for schizophrenia. These were derived from folk medicine and from synthesizing new substances based on a family of actions in animals, such as suppressing movement and interfering with conditioned responses. A common feature—among a vast range of pharmacological properties—was inhibition of dopaminergic systems: by depleting neuronal dopamine stores or blocking dopamine receptors, specifically D£ receptors. Conversely, agents promoting dopaminergic activity could elicit schizophrenic-like symptoms in volunteers and worsen the symptoms in schizophrenic patients. The dopamine hypothesis attributed schizophrenic symptomatology to such dopaminergic hyperactivity, while acknowledging that this hyperactivity might be relative to more fundamental defects in some as yet unidentified system(s). Correlations of drug efficacy with D£ receptor blockade pointed drug development to compounds having similar specificities, but a drug identified with superior clinical efficacy turned out to be relatively weak at D% receptors. Explanations for such "atypical antipsychotics" included specificity for different (more pertinent) dopaminergic receptors or blockade of other receptors (in addition or instead), such as serotonergic 5HT2 receptors. By 1990 interest was also turning to receptors for another neurotransmitter, although no practical drugs were yet available. Phencyclidine, an illicit hallucinogen notorious as "PCP" or "angel dust," could produce symptoms resembling schizophrenia, probably more closely than did amphetamine. The discovery that phencyclidine blocked NMDA receptors for glutamate then spurred investigations into glutamatergic systems' involvement in the pathophysiology of schizophrenia.91

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Depression ana Manic-Depressive Illness

Greek physicians characterized melancholia as an underactivity of mental functioning associated with debilitating grief and despair, contrasting it with the overactivity of raging frenzies.92 On the other hand, notions of a unitary soul discouraged subdivisions, stressing instead either sickness or health. Toward the end of the nineteenth century, however, Kraepelin distinguished between dementia praecox and manic-depressive illness. Elaborating on earlier formulations, Kraepelin described cycles of exaggerated mood in the latter disorder, with periodic swings from excitation and elation to hopelessness and despondency. Throughout the twentieth century the distinctions among disorders of mood shifted, to extend or restrict categories and to subdivide or enlarge them, with inclusions or exclusions based on the nature, severity, and duration of symptoms. In any event, classification had limited practical significance during the first half of this century, for prognoses were generally indistinguishable and medications generally ineffective. Nevertheless, Karl Leonhardt advocated a clinical distinction in the 1950s that later pharmacological and genetic studies confirmed:93 bipolar illness, with alternations between mania and depression, vs. unipolar illness, with recurrent bouts of depression. Thus, DSM-1II in 1980 included the categories "bipolar disorder" and "major depression." Still, arguments for heterogeneity within these categories persisted. The Greeks attributed melancholia to an excess of black bile (as the name denotes), and little further delineation of its etiology followed for two millennia. Absent firm evidence for the neural malfunctionings that nineteenth century psychiatrists had assumed, psychoanalytical formulations for depression flourished during the first half of the twentieth century, as with schizophrenia. As with schizophrenia, investigations after World War II of families, twins, and adopted children pointed toward genetic predispositions (and indicated distinctions between bipolar and unipolar disorders as well).94 And as with schizophrenia, newer imaging techniques disclosed characteristic structural abnormalities in the brain.95 The most prominent arguments for a neural disorder sprang, however, from discoveries of effective therapeutic agents that produced identifiable biochemical changes.96

Iproniazid

Researchers at Hoffman-La Roche identified in the early 1950s the potent antitubercular activity of isoniazid, which quickly became a major therapeutic agent against tuberculosis. Attempts to synthesize still better drugs soon produced iproniazid (Marsilid). Clinical use, however, revealed an unexpected ability to cheer these chronically ill patients. Although initial trials of iproniazid with hos-

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pitalized psychiatric patients were not promising,97 Kline in 1957 found that 70% of his depressed patients ultimately improved.98 Subsequent trials confirmed iproniazid's ability to elevate the mood of depressed patients.99 (Influenced by psychoanalytical theory, Kline imagined that depression reflected a loss of psychic energy to inappropriate realms of the mind. Effective therapy would release this energy to reenergize the psyche, although too much energy might result in schizophrenia. Accordingly, Kline termed iproniazid a "psychic energizer.") Earlier, Albert Zeller in Chicago had demonstrated that iproniazid inhibited monoamine oxidase in vivo.wo By the mid-1950s this enzyme was recognized as an important participant in metabolizing catecholamines and serotonin (chapter 9), so drugs that inhibited degradation should potentiate the effects of these biogenic amines. Studies with iproniazid, however, did not indicate which of these neurotransmitters must be affected to relieve depression. Unfortunately, iproniazid produced serious toxicities, and it was soon replaced by other drugs having the common ability to inhibit monoamine oxidase. This ability also provided the general name for such antidepressants: monoamine oxidase inhibitors. The newer drugs were not without side effects, either,101 and monoamine oxidase inhibitors were largely supplanted by another class of drugs that came into use at almost the same time.

Imipramine

Following the therapeutic (and commercial) success of chlorpromazine, Geigy, as did other pharmaceutical firms, sent its phenothiazine-like compounds to clinicians for testing. One of these, imipramine (Tofranil, Fig. 13-3B), differs chemically from chlorpromazine only slightly, but trials in the mid-1950s revealed negligible antipsychotic activity. In 1956, however, Roland Kuhn, a psychiatrist in Miinsterlingen, tested imipramine in depressed patients and found striking improvements.102 Confirmations of Kuhn s success followed,103 and imipramine was introduced in the late 1950s. Within a decade of Kuhn's initial report, five chemically similar compounds were in use. Because of their structures, these imipramine-like drugs became known as tricyclic antidepressants. The tricyclic antidepressants, too, had prominent side effects, including atropine-like symptoms and hypotensive responses, but these drugs were generally more acceptable to patients than the monoamine oxidase inhibitors. Clinical trials also demonstrated their therapeutic superiority,104 and the tricyclics became the standard medications for several decades. Initial studies demonstrated that imipramine did not inhibit monoamine oxidase appreciably. How then did it work? During his studies in the early 1960s on the fate of labeled noradrenaline, Axelrod showed that a significant fraction

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of administered noradrenaline was taken up by nerve endings and that imipramine blocked this uptake (chapter 9). These studies were on peripheral nerve and brain slices. To demonstrate this effect in the brain, Axelrod and a visitor from Paris, Jacques Glowinski, in 1964 injected labeled noradrenaline into the ventricles (bypassing the blood-brain barrier): tricyclic antidepressants blocked uptake into brain cells in vivo as well.105 Further studies confirmed the common ability of these drugs to block noradrenaline reuptake, the principal means for terminating the action of released noradrenaline (chapter 9). Accordingly, these "reuptake inhibitors" would potentiate the effects of noradrenaline released into the synaptic cleft. In 1965 Joseph Schildkraut, a young psychiatrist in Bethesda, summarized responses of patients, animals, and neurons to drugs producing or relieving depression. The resulting "catecholamine hypothesis" proposed that some if not all depressions are associated with an absolute or relative deficiency of catecholamines, particularly [noradrenaline]. . . . Elation conversely may be associated with an excess of such amines.106

Schildkraut noted that monoamine oxidase inhibitors and imipramine elevated the mood of depressed patients and could potentiate responses to noradrenaline. (Monoamine oxidase inhibitors could also block the metabolism of/potentiate responses to serotonin.) Conversely, reserpine could produce in some patients depression and in animals sedation, providing perhaps an animal model of depression. Reserpine depleted neuronal stores of catecholamines (and serotonin) and thus diminished adrenergic (and serotonergic) responses. Reserpine s effects—in patients and animals—could be diminished by treatment with monoamine oxidase inhibitors. And, as Carlsson had reported in 1957, metabolic precursors of catecholamines but not of serotonin reversed reserpineinduced sedation in animals. Two years later Alec Coppen in Epsom published a review that implicated decreased serotonergic as well as noradrenergic activity in depression, citing, for example, reports that feeding patients a precursor of serotonin potentiated antidepressant responses to monoamine oxidase inhibitors.107 That year evidence appeared for imipramine blocking the reuptake of serotonin as well as of noradrenaline (chapter 9). Accordingly, "biogenic amine hypotheses" emerged in the 1970s. These recognized that depression encompassed a "heterogeneous group of disorders," with noradrenergic activity diminished in some and serotonergic activity diminished in others.108 Various tricyclic antidepressants also differed in their relative potency toward noradrenaline vs. serotonin reuptake, and individual patients responded better to different drugs. In any case, interrelationships between noradrenergic and serotonergic systems were becoming apparent. For example, in 1980 George Aghajanian in New Haven showed that noradrenergic antagonists suppressed the activity of serotonergic

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neurons, consistent with noradrenergic neurons normally promoting the excitability of the serotonergic neurons that they contacted.109 These catecholamine and biogenic amine hypotheses generated broad interest. Not only did they specify pathophysiologies for depression, however incomplete, they also provided the first biological accounts of significant merit in the murky field of mental illnesses. The formulations thus pointed to the possibility of analogous accounts for the whole range of such disorders. And among the implications of Schildkraut's proposal were possible linkages between the levels of brain noradrenaline and its metabolites to the clinical state. Contradictory reports of such correlations followed, however, and by the 1980s confidence in a predictive/diagnostic role for such measurements had declined sharply.110 Attempts to correlate depression with diminished levels of serotonin or its metabolites also were contradictory.111 Failure to establish consistent, convincing ties to biogenic amine levels was joined with two other arguments to launch a third formulation of the pathophysiology of depression. As Fridolin Sulser in Nashville pointed out in 1978, antidepressant drugs affected neurotransmitter levels and responses within a day, but clinical improvement required several weeks.112 Sulser noted that manipulating neurotransmitter levels in the synaptic cleft caused changes in receptor-coupled responses and that these changes followed similarly protracted time courses. Further studies demonstrated that administering antidepressant drugs could indeed "downregulate" /3-adrenergic and serotonergic SHTg receptors (i.e., decrease their numbers).113 During the 1980s these "receptor theories of depression" were widely advocated.114 Such accounts stated that prolonged treatment with antidepressant drugs would alter responses to noradrenaline and/or serotonin compared to responses when the patient was first treated. But the arguments were unclear about what these altered responses were and how they would be therapeutic.115 Chronic administration of drugs could indeed change neuronal biochemistry, but just what the therapeutically important alterations were remained uncertain as the decade ended.

Fluoxetine

Several tricyclic antidepressants inhibited serotonin reuptake in vitro far more than noradrenaline reuptake, but in vivo these drugs were metabolized to compounds that blocked noradrenaline reuptake preferentially. Consequently, these drugs were not serotonin-specific in practice. In 1972, however, Carlsson and associates patented zimelidine, which was specific for serotonin reuptake in vivo; by 1980 clinical testing demonstrated its utility as an antidepressant, but toxicities led to its withdrawal the next year.116 Meanwhile, David Wong at Eli Lily reported in the mid-1970s that fluoxetine (Prozac, Fig. 13-3E), which did

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not resemble the tricyclic antidepressants structurally, was also highly specific in blocking the reuptake of serotonin both in vivo and in vitro, and he predicted that fluoxetine "may find clinical use . . . in mental depression."11' Fluoxetine indeed was an effective antidepressant,118 and when finally marketed in 1988 it quickly became a spectacularly popular drug, boosted by glowing accounts in the press as well as a best-selling book.119 Nevertheless, fluoxetine was no more effective against depression than the tricyclic antidepressants. Its advantage was fewer troubling side effects. (In addition, fluoxetine turned out to be effective with additional mental ills, including panic attacks and obsessive-compulsive disorder.) Other pharmaceutical companies soon marketed drugs with similar properties, establishing a class of "specific serotonin reuptake inhibitors" ("SSRIs").

Lithium

While searching in the 1940s for endogenous toxins that cause manic-depressive illness, John Cade, a psychiatrist in Bundoora, injected patients' urine into guinea pigs. He identified urea as one urinary constituent responsible for the resulting lethal convulsions, but urea levels were not elevated in patients' urine. Cade then added another urinary constituent, uric acid, choosing its most soluble salt, lithium urate. Instead of exacerbating the toxicity as he expected, lithium urate, when added to the patients' urine, suppressed the convulsions. Indeed, lithium salts alone produced marked lethargy in guinea pigs. Since Cade's focus was on mania, he tried the sedating lithium salts on 10 patients with manic-depressive illness. As he reported in 1949, lithium salts relieved manic symptoms astonishingly well.120 Cade's paper generated little enthusiasm, but five years later, in 1954, Mogens Schou in Risskov achieved similar success with controlled clinical trials.121 Moreover, Schou in 1967 described a prophylactic effect against both manic and depressive episodes of chronic treatment. Lithium extended the interval between episodes and blunted symptoms when they recurred.122 Others confirmed these successes.123 Although manic episodes might require added antipsychotic medications and depressive episodes added antidepressants, lithium for many patients was sufficient to prevent the devastating symptoms of this disorder. The U. S. Food and Drug Administration had banned lithium in 1949, the year of Cade's initial success. Lithium chloride had been used as a dietary salt substitute in hypertensive patients, but it was linked to lethal toxicities. Other lithium salts, such as carbonate and citrate, were less toxic, and in light of the accumulating evidence for their efficacy in bipolar disorder, these salts were approved in 1970. Still, the range of dangerous toxicities to lithium remained a serious concern, and therapeutic use required careful monitoring.

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The various amine hypotheses proposed that mania and depression were opposites, reflecting excesses and deficiencies. Accordingly, high doses of monoamine oxidase inhibitors could elicit manic behavior. The ability of lithium to forestall and blunt both manic and depressive symptoms was therefore unexpected. How could chemically simple lithium salts achieve such "normalizing" effects? Attempts to identify the pertinent cellular actions were frustrated by lithium's multitudes of effects, but during the 1980s a prominent proposal invoked lithiums ability to inhibit inositolphosphate metabolism,124 which would block this vital second messenger system (chapter 7). Critics of this proposal, however, argued that therapeutic levels of lithium did not alter brain levels of the second messengers significantly.125 This controversy was unresolved by 1990, and no convincing mechanism had then appeared to account for inositolphosphate controlling pathological mood swings.

Summary

Effective new medicines for depression and mania were identified through clinical observations of drugs expected to have other actions (iproniazid, imipramine) or serendipitously from explorations of hypothesized psychic toxins (lithium). The actions of these drugs soon inspired accounts of pathophysiology. Inhibition of monoamine oxidase or neurotransmitter reuptake, identified as consequences of antidepressant drug administration, would potentiate responses to endogenous noradrenaline and serotonin. (At this time anatomical studies—notably those exploiting fluorescence microscopy—were defining noradrenergic and serotonergic pathways that arise from relatively few neurons and then branch extensively to innervate the brain broadly. Such systems should modulate mental function broadly, as emotions seem to do.) The resulting catecholamine and biogenic amine hypotheses proposed that deficiencies of noradrenaline and/or serotonin were responsible for depression and excesses for mania. Newer drugs developed from these mechanistic principles, such as the specific serotonin reuptake inhibitors, yielded additional clinical successes. Although further studies failed to correlate amine levels with disease, the hypothesized deficiencies and excesses could be relative to more fundamental aberrations in other (unidentified) systems that influence or are influenced by the biogenic amines. On the other hand, the delayed onset of therapeutic benefits suggested that complex cellular changes participated, such as altered receptor numbers. How lithium's actions fit such schemes was unclear. In addition, evidence for other pathological aspects of mood disorders was also accumulating, implicating possible metabolic and endocrine malfunctionings.126 (Probably the most effective treatment was electroconvulsive therapy. Its administration altered neurotransmitter levels, receptor responses, and endo-

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crine function, but in 1990 the changes necessary for symptomatic remissions remained undefined.127)

Conclusions

Long-sought relief from the ravages of neurological and psychiatric illnesses arrived during the twentieth century in the form of new medicines. For parkinsonism, effective therapies were designed to counteract known deficits. For schizophrenia and depression, where the defects were not known, therapies came from a folk medicine (whose mechanism was unknown initially) and from noticing clinical responses to drugs administered for other purposes. The pharmacological properties of such empirically discovered drugs suggested ways these drug achieved their therapeutic ends, and these actions then suggested the malfunctionings that underlay the illnesses. Discoveries of effective drugs proceeded not just from crafting remedies to known defects and verifying observations from folk medicine or more recent clinical observations. Another important approach involved broad testing by the pharmaceutical industry. Their prolific chemists generated all possible variants of a successful drug in the hope of finding one still better. Consequently, the selection of useful screening assays was a vital requirement, although the screens were themselves often the fruits of serendipity. For psychiatric illnesses these screens ranged from behavioral assays (e.g., interference with conditioned avoidance for antipsychotics and with learned helplessness for antidepressants) to biochemical ones (e.g., blockade of D2 receptors for antipsychotics and of serotonin reuptake for antidepressants). Despite the pedigree of a compound and its success with particular screens, the clinical trial was crucial for determining a drug's utility against an actual illness. The methodology of clinical testing evolved, also, and its ideal form late in the twentieth century specified comparisons of responses to a new drug with those to an inactive placebo and/or a drug previously found effective. The drugs and placebos were assigned randomly to individuals from representative populations in numbers calculated for statistical merit, with knowledge of who received drug or placebo withheld from patient and physician until the study was completed.128 Although all testing methodologies have inherent shortcomings,129 the benefits of the new drugs described here were readily discernable, and the revolution in patient care that followed their introduction was apparent to almost all.130 Finally, two aspects of the drugs discussed in this chapter deserve emphasis. First, these drugs were palliative, not curative: they relieved symptoms, often for as long as the drugs were continued, but did not eradicate the under-

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lying disorder (which in all cases was unknown). Such is the case with much of medical practice, although curative drugs would obviously be better. And the search for better drugs would be facilitated by better knowledge of pathophysiologies. Second, all the drugs acted on synaptic transmission (as do the vast majority of drugs used to treat neurological and psychiatric illnesses), but in no case was there firm evidence for the illness being a primary disorder of synaptic transmission. The synapse, nonetheless, is an ideal target for palliative treatment since individual behaviors—whether mental or motor—represent activities over particular pathways, with the pathways formed from chains of neurons linked through synapses. The synapses are thus the sites controlling communication over the pathway. In addition, drugs that act on synaptic transmission may be relatively specific: critical synapses controlling a pathway may use only a few of the dozens of neurotransmitters employed elsewhere, and through only a few classes of receptors available to these defining neurotransmitters.

Notes

1. See Ayd and Blackwell (1970); Berrios and Porter (1995); Caldwell (1970); Shorter (1997). 2. Distinctions are often made between "symptoms" (that the patient feels and reports) and "signs" (that the physician observes). For convenience I will use "symptoms" to include both categories. 3. For example, Kirk and Kutchins (1992). 4. Abridged in Marks (1974), pp. 9-17. See also Langston and Palfremon (1995); Schiller (1967); Tyler (1991). 5. See Tyler (1991). 6. Translated in Marks (1974), pp. 21-28. 7. Wilson (1924); Ferraro (1928). 8. Carlsson et al. (1957). 9. Carlsson (1959). This was presented at a conference the previous year. 10. Translated in Marks (1974), pp. 47-56. 11. Hornykiewicz (1963). See also Anden et al. (1964); Poirier and Sourkes (1965). 12. Translated in Marks (1974), pp. 59-62. 13. Translated in Marks (1974), pp. 63-80. 14. For example, McGeer and Zeldowicz (1964); Fehling (1966). 15. Cotzias et al. (1967). 16. See Yahr et al. (1968). 17. Decarboxylation of dopa in the periphery floods the body with dopamine, which then acts on adrenergic receptors to produce cardiovascular responses and on the chemoreceptor trigger zone of the brain (which lies outside the blood-brain barrier) to produce vomiting. 18. Udenfriend et al. (1966). They believed, however, that the apparent rise in brain dopamine was an artifact due to the drug's conversion to a dopamine-like substance.

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19. Bartholini and Pletscher (1969). They had earlier studied a different peripheral inhibitor. 20. See Cotzias et al. (1969); Mars (1973). 21. Schwab et al. (1951). The motivation for clinical testing was apomorphine's ability to relieve rigidity in animals due to experimental lesions. 22. Ernst (1967). See also Anden et al. (1967); Cotzias et al. (1970). Apomorphine is formed from morphine, but its structure is drastically altered (apomorphine has no analgesic activity). 23. Calne et al. (1974). See also Corrodi et al. (1973). Bromocriptine was initially used to inhibit prolactin secretion. 24. See Lieberman et al. (1976). 25. Schwab et al. (1969). 26. For example, Grelak et al. (1970); Von Voigtlander and Moore (1971); Mawdsley et al. (1972). 27. Langston and Palfremon (1995). 28. Langston et al. (1983). 29. Davis et al. (1979). 30. Burns et al. (1983). 31. Chiba et al. (1984); Markey et al. (1984); Heikkila et al. (1984). 32. Nicklas et al. (1985). 33. Javitch et al. (1985). 34. Knoll (1978). He also cited other means by which selegiline could work. 35. Birkmayer et al. (1985); Elizan et al. (1989); Tetrud and Langston (1989); Parkinson Study Group (1989). 36. For example, see Letters to the Editors, Science 249: 303-304, 1990. 37. Connor et al. (1967). 38. See Hornykiewicz (1971). 39. Yurek and Sladek (1990). 40. Schults (1991). 41. See Berrios and Porter (1995); Howells (1991); Thompson (1987). 42. See Leff (1977). 43. For example, Fromm-Reichman (1948); Bateson et al. (1956). 44. See Dunlap (1924). 45. Seymour Kety in Bethesda carefully examined successive proposals for schizophrenias pathophysiology, only to find each seriously flawed (1959, 1967). 46. See Haug (1962). 47. See Bench et al. (1990); Lewis (1990); Suddath et al. (1990). 48. Gaddum (1953). 49. Wooley and Shaw (1954). 50. Connell (1958). 51. See Gottesman and Shields (1982); Kallman (1946). 52. Heston (1966); Kety et al. (1968). 53. See Karayiorgou and Gogos (1997). 54. Sen and Bose (1931). Astutely, they noted that reserpine relieved "maniacal" but not "morose" symptoms. For historical accounts, see Ayd and Blackwell (1970); Caldwell (1970); Healy (1996, 1997). 55. Vakil (1949); Wilkins et al. (1952). 56. Cited in Ayd and Blackwell (1970). 57. Kline (1954), p. 3.

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58. Barsa and Kline (1955); Lasky et al. (1962). 59. See Ayd and Blackwell (1970); Caldwell (1970); Swazey (1974). 60. Phenothiazine Treatment in Acute Schizophrenia (1964). 61. "Major" contrasts these drugs with pharmacologically distinct "minor tranquilizers" used to treat anxiety. 62. "Neuroleptic" has differing interpretations; the etymology refers to affecting the nervous system. 63. Carlsson and Lindqvist (1963). 64. They showed that haloperidol increased metabolites of dopamine but not noradrenaline, whereas chlorpromazine increased metabolites of both. They did not comment on this, however. 65. van Rossum (1966), p. 492. 66. Randrup et al. (1963); Scheel-Kriiger and Randrup (1967). 67. Coyle and Snyder (1969); Taylor and Snyder (1971). Snyder et al. (1974) cite some contradictory reports, however. 68. Angrist et al. (1971). 69. See Angrist et al. (1973). 70. Snyder et al. (1974). For other accounts, see Klawans et al. (1972); Matthysse (1973). 71. Horn and Snyder (1971). 72. Kebabian et al. (1972). 73. Iversen (1975). 74. Creese et al. (1976). 75. Seeman et al. (1976). 76. Kebabian and Calne (1979). 77. Bunzo et al. (1988). 78. For example, Bowers (1974); Sedvall and Wode-Helgodt (1980); Heritch (1990). 79. See Kornhuber et al. (1989). 80. Wong et al. (1986). 81. Farde et al. (1987). 82. Hokfelt et al. (1974); Swanson (1982). 83. See Bench et al. (1990); Lewis (1990); Suddath et al. (1990). 84. See Hippius (1989); McKenna and Bailey (1993). 85. Gross and Langer (1966). 86. Griffith and Saameli (1975). 87. Kane et al. (1988). 88. Farde et al. (1989). 89. Gelders et al. (1990). 90. Van Tol et al. (1991); Seeman et al. (1994). 91. See Javitt and Zukin (1991). 92. See Ayd and Blackwell (1970); Healy (1996, 1997); Johnson (1984); Pletscher (1991). 93. Leonhard (1957). 94. See McGuffin and Katz (1989). 95. See Jeste et al. (1988). 96. Amphetamine had been tried with depressed patients, but although it initially roused them, it then produced a rebound exacerbation of their depression. Opiates had proved more effective but carried severe liabilities with chronic use. 97. For example, Kamman et al. (1953). They examined behavioral changes in groups of psychiatric patients having unspecified diagnoses.

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98. Loomer et al. (1957); Kline (1958). Kline provided two rationales: iproniazid is a monoamine oxidase inhibitor (without specifying why this should be beneficial), and recent reports described excitation (in animals) after sequential treatment with iproniazid and reserpine. Kline initially planned to give iproniazid followed by reserpine (which he was then using with schizophrenics) but observed relief with iproniazid before he added reserpine. See also Crane (1957), who stressed "increased vitality" rather than antidepressant activity per se. 99. For example, Kiloh et al. (1960). 100. Zeller et al. (1952). 101. See Blackwell et al. (1967) for an explanation of why tranylcypromine (Parnate) produced dangerous hypertension in patients who ate cheese. 102. Kuhn (1957, 1958). He claimed he tested imipramine with depressed patients on behalf of "thoroughness" as well as a "conviction that it must be possible to find a drug effective in ... depression" (Ayd and Blackwell, 1970, p. 211). 103. For example, Lehmann et al. (1958). 104. Cole (1964). 105. Glowinski and Axelrod (1964). 106. Schildkraut (1965), p. 509. See also Bunney and Davis (1965). 107. Coppen (1967). He also reviewed evidence for changes in other factors. 108. For example, Maas (1975); Carver and Davis (1979). 109. Baraban and Aghajanian (1980). 110. For example, Agren (1982); Davis et al. (1988). 111. For example, Davis et al. (1988). There was, however, a correlation between low levels of a serotonin metabolite in the cerebrospinal fluid and suicide (Asberg, 1976), although low levels also occur in other psychiatric diseases. 112. Sulser et al. (1978). They also argued that new experimental drugs were effective without inhibiting monoamine oxidase or reuptake. See also Vetulani and Sulser (1975). 113. For example, Wolfe et al. (1978); Peroutka and Snyder (1980). 114. For example, Charney et al. (1981); Sugrue (1983); Stahl and Palazidou (1986). 115. Downregulation might overshoot the homeostatic set point, producing an absolute decrease in responses (although there was no precedent for this happening). Or autoregulatory receptors might be downregulated, producing a greater release of neurotransmitter. Or various classes of receptors might be downregulated to different degrees, with the altered balance between their responses producing the therapeutic change. Or. . . . 116. See Carlsson and Wong (1997). 117. Wong et al. (1974, p. 477, 1975). Fluoxetine was discovered by synthesizing analogs to an antihistaminic known to block neurotransmitter reuptake. It was one of 57 compounds then screened for specificity toward serotonin reuptake. 118. See Stark and Hardison (1985). 119. Prozac made the cover of Newsweek (26 March 1990) and starred in P. Kramers Listening to Prozac, New York: Viking Press (1993). 120. Cade (1949). 121. Schou et al. (1954). 122. Baastrup and Schou (1967). 123. For example, Coppen et al. (1971). 124. Berridge et al. (1982, 1989). 125. For example, Honchar et al. (1990). 126. See Honig and van Praag (1997), pp. 235-250.

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127. See Honig and van Praag (1997), pp. 397-412. 128. For the development of testing, see Healy (1997). 129. For example, in randomized double blind placebo trials, those receiving placebos may identify them through the absence of side effects that inevitably accompany an active drug. Moreover, double blind trials usually prevent adjusting dosages to individual needs. 130. There remain critics of the drugs, in principle and in practice. As with all revolutions, not all consequences were beneficial. Complaints of the overreliance on drugs have merit, as do complaints that patients were released from hospitals to the community without the community being prepared to care for them. (Coincident with the introduction of these drugs the population of mental hospitals dropped by four-fifths.)

14 EPILOGUE

Progress When formulated late in the nineteenth century, the Neuron Theory depicted discrete nerve cells interacting at their points of contact. Nerve impulses, then often identified with electrical signals traveling along neuronal processes, would pass electrically from neuron to neuron at these synaptic contacts. Over the next century, however, this view changed dramatically. Neurons could inhibit as well as excite other neurons; communication between cells was generally not electrical but achieved through the release of chemicals that then bound to specific receptors to elicit excitation or inhibition; there were dozens of distinct chemical neurotransmitters and multiple classes of receptors for each; receptors could initiate complex chains of metabolic alterations as well as elicit electrical responses; receptors were present on presynaptic terminals, also, modulating function at this site, too; neurotransmitters were not secreted after synthesis but stored in vesicles, from which they were released exocytotically as discrete quanta; transport back into presynaptic neurons terminated the actions of some neurotransmitters, whereas metabolic degradation terminated the actions of others. In addition, the formation of specific synapses during development and the alterations in synaptic transmission accompanying learning also relied on intricate chains of cellular modifications. 349

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Accumulating the detailed evidence for these entities and processes required approaches from anatomy, biochemistry, embryology, medicine, pharmacology, and physiology. Applying the techniques and concepts of these disciplines to the various issues then created a vast body of new knowledge now called neuroscience. But as accounts of neural structures and mechanisms accumulated, many of these capabilities were being identified in other cell types. For example, voltage-gated ion channels of nerve action potentials and ligand-gated ion channels of neurotransmitter receptors were initially described for neurons and their end organs, but subsequent investigations demonstrated that ion channels functioned in essentially all cell types, from bacteria to liver cells. Signaling within and between cells is an essential process that—across the biological realm—utilizes ion channels, chemical signals, receptors, and second messenger systems. Consequently, a complementary integration resulted, one that embraced neuroscience within general cell biology. By 1990 the formulations of cellular mechanisms were vastly richer in detail, with hosts of new entities linked through new processes. These understandings then directed and enabled new experimental manipulations for continuing explorations as well as for improving therapeutic interventions. But despite such spectacular progress, the overall picture was far from complete. Critical gaps remained and further investigations were continuing successfully when this history concludes.

Historical Accounts and Conclusions

Unlike many creative fields, science (according to its practitioners) progresses, with later formulations surpassing earlier ones. This account attempts to present activities and results in the context of their times. But it also places them on a path—twisting, tentative, fallible, but advancing overall—toward the present. "Whig histories" of social and cultural events are castigated for viewing the past as prelude to their authors' views of present virtue. Nevertheless, if scientific understanding is indeed proceeding toward better descriptions of the world, then progress toward this goal should be acknowledged.1 This account also illustrates an obvious characteristic of how science is done: pluralistically, in different ways for different reasons and in different places. Unitary explanations—algorithmic, economic, social—are inadequate. Like geographical explorations during the preceding centuries, individuals ventured into the unknown inspired by a range of human motivations: to satisfy curiosity, to seek fame and fortune, to honor God and country, to serve humanity. And like tales from geographical explorers, the scientists' reports also were colored by human aspirations as well as human frailties. But, just as the reality of the earths surface ultimately constrained the explorers' accounts, so the

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real world (assumed/inferred) ensures a coherence that overrides individual intentions and expectations. This reality, scientists argue, limits the alternatives and forces convergence. By contrast, historians who deny the role of a limiting, external world have been baffled by the consensus that scientific research builds.

Assumptions

Most scientists, having passed in adolescence through the requisite phases of solipsism and radical skepticism and recognizing the futility of maintaining such views in daily life, acknowledge the likelihood of a real external world of other individuals, other living creatures, and other material stuff—even if these might not satisfy personal desires.2 They also recognize the necessity for logic and the value of evidence in support of their scientific arguments, as in daily life. Indeed, scientific practice emerged (if somewhat unevenly) as a commonsense response to such assumptions and requirements: looking for reliable, reproducible evidence of regularities in nature, imagining entities and processes to account for these observations economically, checking the formulations and their consequences whenever feasible, and linking the accounts into a cohesive, causal explanation relating natural entities and natural processes. In due course new formulations were knitted into the fabric of established scientific knowledge, suggesting a unity in the universe and its workings. During the period covered here research was grounded on the general assumption that all biological processes are explicable in terms of chemical and physical laws. Vitalism was rejected.

Approaches

Scientific research is sometimes depicted as the testing of hypotheses. The necessity for scrutinizing the consequence of any formulation is clear, both to assure the validity of that hypothesis and to extend the scope of knowledge. Still, the utility of simple exploration and description should not be underestimated. The identification of new entities and processes is a vital component in the scientific enterprise, including such goals as recognizing new neurotransmitters or establishing the three-dimensional structures of receptors.3 In all cases a crucial factor in directing new investigations is the availability of requisite methods. General questions were frequently obvious (for example, how are neurotransmitters released?), but the routes to their solution were often tortuous and slow, awaiting the development of new methods for addressing each issue. On the other hand, significant and rapid advances followed the

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discovery of new instruments and techniques, such as electron microscopy, fluorescence microscopy, patch electrodes, and cDNA sequencing. But along with the new answers disclosed by these new methods appeared new questions, ones previously unanticipated. For example, what is the role of clathrin-coated vesicles for membrane retrieval? How are ion channel conductivities modified by second messenger systems? Are there common structural motifs appearing within the sequences of proteins that catalyze similar reactions? Often, however, the art of scientific practice includes the application of known techniques to accessible aspects of recognized questions. Research is directed pragmatically and opportunistically in light of what can be measured, isolated, or identified by methods currently available and concepts currently accepted. In this quest precedent suggests likely avenues for study, serving as a common, practical, albeit fallible guide. For example, if cAMP participates in hormonal regulation of glycogen breakdown in the liver, it might participate in noradrenalines actions on neurons. Furthermore, novel insights into hitherto unrecognized precedents could point research in directions previously unexpected; for example, appreciating parallels between noradrenaline uptake by neurons and Na+-dependent glucose transport in the intestine. Sometimes unanticipated results led serendipitously to new interpretations, new phenomena, and even new fields of study. For example, the unexpected discovery that iproniazid was both an effective antidepressant and an inhibitor of monoamine oxidase turned attention to the roles of catecholamines and serotonin in disorders of mood. Indeed, a host of significant entities and processes, ranging from m.e.p.p.s to calmodulin to LTP, were happened on during studies of other issues. Accompanying the various guides to further research were, of course, the full span of human motivations, including lures of fashion and urges of iconoclasm. But, as earlier explorers of the natural world learned, these expectations may not be fulfilled by the actual state of the world.

Goals

The histories of scientific investigations display numerous errors and misconceptions as well as inevitable oversimplifications. If aspects of a real world seem apparent, its details have been grasped with difficulty, often amid uncertainty and controversy, and with critical gaps still remaining. Arguments against direct, indubitable sensory access to reality range from the critiques of ancient philosophers to formulations of modern neurophysiology. The latter depict neuronal chains underlying perception that are not only susceptible to errors of processing but also subject to modulation from higher centers (and thus incipient bias).4 Furthermore, our senses receive direct information only through lim-

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ited channels sharply restricted in range. Instruments may confirm these observations and vastly extend them—as by electron microscopy—but at the price of theoretical justification and at the risk of artifact. These common recognitions endorse modest and circumspect goals for scientific research: the formulation of explanatory, causal models.5 These models, like the geographers' maps of the world,6 then attempt to display similarities to the real world in specified ways and at specified scales: models of behavior, of nervous systems, of neurons, of synaptic junctions, of receptors. The constraints on how such models can represent observation and experiment, moreover, reinforce the presumption that they reflect discoveries about a real world, even though they are human constructions. The models are, of course, subject to continuing amplification and correction in the light of further experiment and analysis. And they serve as guides for further exploration. Such causal models also embody an explanatory reductionism.7 For example, questions about how reserpine causes parkinsonism initiate a hierarchical chain: by depressing dopaminergic function in the basal ganglia; by depleting the stores of dopamine in nigrostriatal neurons; by binding to the dopamine transporter of the synaptic vesicles in these neurons and thereby inactivating it. The explanatory regress of biomedical research has a clear terminus, the laws of chemistry and physics (here, the theory of ligand binding to proteins). The regress also is a guide to chemical manipulation of responses distinguishable at higher levels, as in drug therapy of behavioral disorders. On the other hand, syntheses of complex wholes from models of their parts, while a proclaimed goal, is also a forbiddingly difficult one. Scientists often use terms far more casually than their critics, causing confusion over issues of "fact," "truth," "proof," and the like. These are indeed words that can be construed in various ways. But, assuming a real world, there should be facts about it, and descriptions that accurately describe these facts would be true. If such absolutes are unattainable, the scientific quest at least aims for closer and closer likenesses through its explanatory models, as demonstrated by experiment and formulated through interpretation. Accordingly, models of distinct neurons communicating through the release of chemical neurotransmitters seemed by 1990 far closer to the truth than the reticular models of Gerlach and of Golgi.

Generalities ana Exceptions

Scientists commonly strive for the simplest explanations until forced by experiment or interpretation into multiplying their entities and processes. The century of research depicted here displays a proliferation of detail that embellished initial representations. But this elaboration required the justification of each

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new entity and process. For example, arguments that inhibition could be presynaptic as well as postsynaptic were furthered by the discovery of anatomical and pharmacological correlates of the proposed physiological mechanism. The allegiance to parsimony also inspires the grouping together of individual entities and processes into common categories, such as neurons and receptors and protein phosphorylations. These generalizations, however, serve as prototypes having not only specified characteristics but also accepted ranges of deviation. For example, all receptors are not alike, and when examined more closely the designations are seen to embrace families of individuals distinguishable by the ligands they bind and respond to. Those binding and responding to noradrenaline are then further divided into functional (and later structural) classes such as a\, az, P\, and (3z, and these are again subdivided by degree of regulatory phosphorylation, and so on. Such designations and allocations have particular explanatory significance in specific contexts, including functional capabilities (for example, sensitivity to certain ligands, linkage to second messengers) and structural characteristics (for example, resemblances of their amino acid sequences to those of certain other receptors). In some instances, moreover, scientists are compelled to accept singular exceptions to otherwise consistent patterns. For example, postganglionic sympathetic fibers to the sweat glands release acetylcholine, as Dale acknowledged, in contrast to postganglionic sympathetic innervation of essentially all other end organs. Biological research is thus enriched (or plagued, depending on one's viewpoint) with identifiable prototypes that, on closer scrutiny, dissolve into populations of distinguishable individuals. Such diversity is, of course, understandable from the history of biological design. Mutations can cause a gene to duplicate as well as change, and after duplication each is then subject individually to further random changes, forming families of variants. Natural selection then chooses among these for distinct functional roles—as, for example, in accumulating families of receptors for different neurotransmitters. For the organism, this multiplicity of related structures offers adaptive advantages. For scientists, these relationships justify generalizations about classes of structures operating similarly, so that, for example, certain stretches of amino acids when appearing in distinct proteins can suggest a common function. Scientists' quest for simplicity also abetted tendencies to imagine all structures and functions in the form first established, for example, generalizing to all synapses the chemical transmission identified at certain sites, or generalizing all receptors as ligand-gated ion channels after the first classes of receptors were so identified. Although there has been a danger of seeing today only what one saw yesterday, further studies could still reveal exceptions, such as electrical transmission at certain loci and receptors instead coupled to second messenger systems.

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Conflict Resolution

The routes to resolving scientific conflicts are also various, including refutation of one hypothesis and confirmation of its rival. Here, too, the course is usually pragmatic and opportunistic, recognizing available capabilities. When experiments support and extend a given formulation, such confirmations of predicted results are usually considered persuasive arguments for the formulation s nearness to truth.8 Thus, Eccles accepted the principle of chemical transmission at neuromuscular junctions when confronted by extensive evidence favoring this notion, despite the absence of experiments explicitly refuting electrical transmission at this site. Katz bolstered his quantal hypothesis of neurotransmitter release from an additional physiological perspective by describing how Ca2+ affected m.e.p.p.s. Electron micrographs showing vesicles within presynaptic terminals then provided compelling arguments from an independent discipline. On the other hand, refuting a rival hypothesis is logically superior but often difficult technically. Claims that an entity does not exist or a process does not work may be contested by counterclaims that the attempted refutation was insufficiently sensitive or specific. For example, when Cajal argued that filaments did not cross from pre- to postsynaptic neurons, his critics complained that Cajal was merely unable to see what they saw. Cajal answered with new stains that revealed filaments but no continuity, satisfying many but not all. The development of electron microscopy, with its far higher resolution, confirmed Cajals refutation, albeit some decades later: fibrils were readily detectable within neurons, but none of these crossed the synaptic cleft that separated the neurons.9 Convincing refutations also occur in a more timely fashion, as in Eccles's demonstration that inhibition produced electrical changes that contradicted what he had predicted, and Wiersma's finding that impulses initiating contractions in a particular muscle passed only in nerve fibers to that muscle, contradicting Weiss's original Resonance Principle. (In addition, refuting alternative formulations is a daily part of scientific practice: experiments frequently include "controls" to eliminate conceivable alternatives.) Nevertheless, some critics claim that experimental evidence is inadequate for resolving scientific controversies, which instead are settled through social interests (political, economic, sexual, ethnic, etc.).10 Two philosophical issues bear on this question of experimental adequacy. (1) The notion that models/theories are underdetermined by evidence includes the recognition that an infinite number of hypotheses can account for any observation. This concern, however, is diminished by wielding Occam's razor and by acknowledging the web of scientific

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knowledge in which an issue is embedded. For example, a model relating administration of noradrenaline to cells and their subsequent production of cAMP depicts interacting entities—adrenergic receptors, G-proteins, and adenylate cyclase—for each of which there is independent evidence of its existence and function. An infinite number of alternative models having an infinite number of additional entities might be advocated, but even if such alternatives could not be explicitly disproved, the body of current biological knowledge renders such hypothetical alternatives unnecessary. (2) More serious is the notion of underdetermination included in the second issue. The Quine-Duhem thesis notes that hypotheses are not tested singly but in groups.11 For example, when Eccles impaled motoneurons and recorded hyperpolarizations that contradicted what his proposal specified, there were numerous ancillary hypotheses involved, including those concerning the identities of the impaled cell as a motoneuron and of the stimulated cell as a Renshaw cell, the electrodes recording the actual transmembrane potentials, the electronic circuits faithfully amplifying the electrode signals, and so on. Scientists are, of course, attuned to these concerns and aware of potential artifacts. Indeed, these are routinely addressed through independent calibrations, control experiments, the application of established principles (for example, the laws of electrochemistry), and the like.12 Consequently, Eccles was able to test with confidence the motoneurons potential changes and reject one hypothesis rationally. Similarly, his contemporary supporters and critics, who shared the same scientific knowledge of electrophysiological recordings and cellular identifications, could form rational, objective conclusions from the evidence presented. Unfortunately, critics who claim that such conclusions are impossible have not always understood the scientific issues and how scientists deal with them.13

Lessons

Broad generalizations about scientific practice are difficult. Some successful inquiries follow acknowledged avenues, while others progress through flouting accepted wisdom. Some begin through logical analysis, and others flourish through unanticipated results. Some attempt refutations and others confirmations. Indeed, a range of approaches and interests come together, and in so doing correct errors and reinforce accomplishments within the web of scientific knowledge. More is better. Effective scientific research requires technical and financial support. Correspondingly, the benefits of pluralism require open access to such support and

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to the forums for presenting and arguing the fruits of labor and inspiration. The hundred years covered here were blessed with growing opportunities and access, vital ingredients in attaining the achievements recorded here.

Notes

1. See Harrison (1987). For efforts to describe scientific practice scientifically, see Donovan et al. (1992). This book, however, selects cases as a reflection of its authors' interests rather than—as methodological dicta advise—through random (or at least representative) samplings of scientific practice. 2. For assorted arguments about scientific realism, see Leplin (1984). 3. Although most endeavors can be squeezed into the guise of testing some hypothesis, the most straightforward characterization of many explorations is simple empiricism. This is clearly the case, for example, in drug development by testing through various screening assays. 4. On the other hand, the principles of Darwinian evolution suggest that animals' nervous systems were selected to deal successfully with the gross aspects of a natural world: finding things to eat while avoiding being eaten. 5. See, for example, Cartwright (1983); Giere (1988, 1999). An interesting possibility is that more than one model could explain how the world works. But before taking this possibility seriously, many scientists would like to see such an independent alternative. 6. Geographical maps also are developed at different scales for different purposes and for emphasizing different aspects (e.g., physical, political, agricultural, climatic) while still representing a real world, even if not in all its particulars. 7. See, for example, Robinson (1986a, 1992). 8. Arguments about justifying hypotheses through corroborating evidence have a long history, which includes debates about the merits of induction. For many scientists corroborative evidence is considered to increase the likelihood that the tested hypothesis is (nearly) true, often on the basis of informal probabilistic assessments. 9. By the time evidence from electron microscopy was available, other arguments for discontinuity—including that from physiological and pharmacological approaches—had strongly confirmed Cajal's interpretation. 10. For example, "Despite the local and situated nature of scientific work, there appear to be some semblances of agreement, stabilizations, and continuities across situations and through time[, but while] scientific realists choose to interpret these as the outcomes of nature's guiding hand, most recent works in science studies take different views" (Clarke and Fujimura, 1992, p. 12); "scientists at the research front cannot settle disagreements through better experimentation, more knowledge, more advanced theories or clearer thinking" (Collins and Pinch, 1994, pp. 144, 145); and "Few controversies . . . centre on such epistemic factors as accuracy and predictive capacities[, instead the] overwhelmimg majority turn on matters of goal orientation, social interests, and stubbornly held metaphysical beliefs" (Shortland, 1988, p. 265). 11. For critical comments, see Laudan (1996). 12. See, for example, Franklin (1998). 13. For examples, see Robinson (1983, 1986b, 1992, 1997). Pertinent to the topics in this book is the account of learning in planaria, by Collins and Pinch (1994). For

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instance, in describing the chemical transfer of particular learned behaviors between planaria, they omitted discussions of the scientific improbabilities (e.g., that ingested RNA can redirect an animals protein synthesis), of the formulations of alternative models (e.g., Hebb's proposal), and of the successes in identifying memory encoded not in specific chemicals but in neural circuits (e.g., Kandels work on Aplysia). Consequently, they could then come to the astonishing conclusion in 1994 that "a determined upholder of the idea [of chemical transfer of learning] would find no published disproof [so that for] such a person it would not be unreasonable or unscientific to start experimenting [on chemical transfer] once more" (p. 25). Collins and Pinch's omissions, either deliberate or through inattention, should be disturbing to all interested in science and how it works.

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INDEX

Abel, J.J., 56 Acetylcholine as neurotransmitter, 59-68, 70-79, 95-98, 102, 106, 109-111, 119-123 metabolism, 229, 230, 238. See also Cholinesterase receptors (muscarinic and nicotinic), 153, 156-160, 177, 180, 186, 200-208, 211 storage, 225, 226 synthesis, 220, 221, 238 Actin, 88, 260, 287, 288, 291 Action potentials. See Impulse conduction Adenylate cyclase, 161, 174-177, 181-186, 209, 223, 306, 334, 335 Adrenaline (epinephrine) as neurotransmitter, 55-59, 69, 70, 76, 80, 119, 123, 124 storage, 226, 227 Adrian, E., 43, 75, 77, 79, 276 Affinity chromatography defined, 159 Aghajanian, G., 339 Ahlquist, R., 153, 154 Aldrich, T.B., 56 Alkon, D., 304 Alles, G., 229 Allosteric interactions, 88, 150-152, 179, 181

Aimers, W., 254 Amphetamine, 267, 328, 332, 333 Andersen, P., 309 Apathy, S., 21, 26, 109 Aprison, M., 133 Aliens, E.J., 147, 148, 150 Aliens Kappers, C.U., 297 Armstrong, M., 233 Arrhenius, S., 9 ATP (adenosine triphosphate) defined, 88, 89 Atropine, 60, 61, 63-65, 67, 71, 123, 211 Attwell, D., 266 Aurbach, G., 161 Avery, O., 88 Axelrod, J., 233-236, 238, 267, 332, 338, 339 Axonal conduction. See Impulse conduction Bacq, Z., 70, 113, 123 Bailey, C., 303 Bain, W.A., 65, 66 Banks, P., 253, 259 Barbeau, A., 324 Barker, L., 18 Barondes, S., 298 443

444

INDEX

Barrett, J., 283 Bartelmez, G., 50 Baudry, M., 313 Bayliss, W., 10, 44, 59 Beadle, G., 88 Beers, W., 156 Bell, C., 14, 34 Bennett, S., 107 Benzer, S., 306 Bergstrom, S., 313 Bernath, S., 266 Bernard, C., 34, 51, 72 Bernard, E., 206 Bernstein, J., 8-10, 49, 50, 89, 90 Berridge, M., 191, 192 Bertler, A., 126 Bethe, A., 10, 21, 23, 24, 26, 109 Betz, H., 206 Bichat, X., 51 Birks, R., 256 Birnbaumer, L., 183 Black, J., 153, 154 Blackburn, R.J., 237 Blaschko, H., 123, 125, 126, 221, 222, 226, 231, 232, 322 Blaustein, M., 188 Bleuler, E., 328 Bliss, T., 309, 310 Bloom, E, 126 Bodian, D., 50 Bonhoeffer, E, 286 Bonner, T., 211 Bose, K., 329 Boyd, I.A., 97 Bradford, H.F., 133 Branton, D., 263 Bray, D., 287, 288 Brock, L.G., 113 Brodie, B.B., 232, 233, 236, 237, 322 Brown, G.L., 74, 77, 155, 245, 246 Brown, T., 310, 311 Brown-Sequard, C.-E., 51 Buchtal, E, 74, 95, 289 Bueker, E., 281 Bulbring, E., 76 a-bungarotoxin, 159, 289 Bum, J.H., 76 Burton, P., 287 Butcher, R. 174 Byers, D., 306 Ca2+ as second messenger, 186—189, 191-193 Cade, J., 341

Cajal. See Ramon y Cajal Calmodulin, 188, 189 Calne, D., 325, 335 cAMP (cyclic AMP), 173-181, 183-186, 189, 193, 212, 214, 223, 224, 260, 303-306, 315 Cannon, W., 69, 70, 123, 153 Cantoni, G., 233 Capon, D., 211, 212 Carlsson, A., 126, 322, 324, 332, 339, 340 Caspars, H., 135 Cassel, D., 182 Catecholamine defined, 58 cDNA. See DNA, recombinant techniques Ceccarelli, B., 250, 252 cGMP (cyclic GMP), 177, 185 Chagas, C., 106, 158 Chang, H.C., 67, 71, 75 Changeux, J.-P, 151, 158, 159, 180, 205, 290 Channels, ion, 92, 112, 158, 159, 161, 163-166, 177, 180, 181, 186-188, 192, 193, 200, 204-208, 214, 258-261, 303-305, 311, 312, 315 Charcot, J.-M., 321 Charlton, M., 259 Charpentier, S., 330 Chemotaxis, 24, 275-280, 283-286 Chen, M., 303 Cheung, W.Y., 189 Chlorpromazine (Thorazine), 330-335, 338 Cholinesterase, 61, 67, 77-79, 229, 230 Clapham, D., 186 Clark, A.J., 144-148, 152, 154 Clathrin. See Coated vesicles Claude, A., 109 Clothia, C., 156 Coated vesicles, 248, 251, 262-265 Cocaine, 235, 237 Cohen, J., 290 Cohen, M., 289 Cohen, S., 281 Cole, K.S., 49, 90 Collingridge, G., 311 Collins, J., 189 Colquhoun, D., 163 Conformational changes. See Allosteric interactions Coombs, J., 113 Coppen, A., 339 Cori, C., 172, 173, 178, 194 Cori, G., 194 Cotman, C., 265 Co-transmitters, 228, 229 Cotzias, G., 324

We

Cowan, M., 282 Crane, R., 89, 227 CRE (cAMP response element), 214, 224, 303, 305 CREB (CRE binding protein), 214, 303, 305 Creed, R.S., 45 Crick, R, 88, 199, 298, 314 Curare, 72-75, 94-96, 106, 123 Curtis, D., 122, 125, 130, 132-134 Curtis, H.J., 90 Gushing, H., 45 Cytoskeleton, 260, 287, 288, 291 Dakin, H.D., 56 Dale, H.H., 57-59, 61, 63, 67-71, 74, 78, 79, 102, 113, 123, 124, 134, 146, 152, 153, 229, 245, 246, 354 Danielli, J., 89, 246 Davies, A., 283 Davis, R., 306 de Duve, C., 247 Deiters, O., 5, 6 Delay, J., 330 del Castillo, J., 97, 98, 122, 246, 247, 256, 258 Denny-Brown, D., 45 Depression and mania, 232, 233, 238, 330, 337-343 DeRobertis, E., 107, 109, 110, 128, 246 Descartes,R., 33 Detweiller, S., 280 Diacylglycerol, 189-193, 209, 223 Diamond, I., 180 Dikshit, B.B., 76 Dingledine, R., 311 Dingman, W., 298 Dixon, W., 59, 60, 63, 79 DNA, recombinant techniques, 199-203, 206-212 Dopamine as neurotransmitter, 58, 124-126, 177 receptors, 212, 332-336, 343 synthesis, 221-224, 238 Douglas, W., 187, 253, 258 Driesch, H., 273 du Bois-Reymond, E., 8, 9, 73 Dudai, Y., 306 Dunant, Y., 257 Durrell, J., 190 Duval, M., 22 Ebashi, S., 187-189 Eccles, J.C., 45, 77, 78, 93, 95-105, 108, 112-114, 123, 130, 134, 259, 355, 356

445

Eccles, R., 122 Edelman, G., 285 Edwards, C., 155, 267 Ehrlich, P., 11, 20 Einthoven, W., 41 Elliott, T., 56, 57, 59, 60, 63, 73, 79, 123, 124 Ellis, J., 265 Emmelin, N., 76 Enkephalins. See Opioids E.P.P.S (endplate potentials), 78, 93-98, 101, 171, 246, 254, 258 E.P.S.P.S (excitatory postsynaptic potentials), 101, 103-105, 171, 300, 301,311, 312 Erlanger, J., 76 Ernst, A.M., 325 Erspamer, V., 126, 127 Ewins, A., 61 Faber, D., 256 Falck, B., 125 Faraday, M., 9 Farde, L., 335 Fatt, P., 97, 101, 109, 114, 129, 246, 258 Feldberg, W, 68, 69, 71, 74-76, 79, 106, 113, 146, 220, 225, 226, 245, 246 Feng, T.P., 309 Ferraro, A., 321 Fessard, A., 106 Fischer, E., 178, 181, 193 Fleckenstein, A., 188 Flexner, J., 298 Flexner, L., 298 Florey, E., 129, 130, 132 Fluoxetine (Prozac), 238, 340, 341 Forbes, A., 44, 76 Forel, A., 3, 7, 10 Foster, M., 32, 33, 44, 51, 53, 79 Frank, K., 103 Franke, W., 261 Franz, S.I., 296 Froehner, S., 290 Fulton, J., 45 Furchgott, R., 150, 313 Furshpan, E., Ill, 112 GAB A (y-aminobutyric acid) as neurotransmitter, 105, 129-132 receptors, 206-208, 212 Gaddum, J., 67, 71, 75, 121, 123, 127, 134, 135, 146-148, 152, 328 Gaffan, D., 308 Galvani, L., 7, 8 Gap junctions, 112

446

INDEX

Gaskell. W., 31, 44, 51, 53, 56, 79 Gasser, H., 76 Gerard, R., 92 Gerlach, J. v., 3, 5, 7, 353 Gershon, S., 332 Gilbert, W., 201 Gillespie, J. I., 254 Gilman, A.G., 182, 183, 185, 193 Ginzel, K.H., 147 Glowinski, J., 339 Glutamate as neurotransmitter, 123, 131-133 receptors (kainate, metabotropic, NMDA, quisqualate), 206-208, 212, 311-315, 336 Glycine as neurotransmitter, 133, 134 receptors, 206-208 Goldman, R., 287 Goldstein, A., 136, 137, 162 Golgi, C., 3, 5-7, 12-18, 22, 353 Golgi stain, 1, 6, 11-21, 23 Goltz, R, 31, 34 Goodman, C., 283, 286 Gopfert, H., 93 G-proteins, 181-186, 192, 209-212, 215 Graham, J., 92 Granit, R., 45 Gray, E.G., 110 Gray, W., 259 Green, J.P., 120 Greengard, P., 177, 179, 180, 189, 194, 260, 261, 303, 334 Growth cone, 23, 274, 275, 286-288, 291 Grundfest, H., 74, 106 GTP (guanosine triphosphate) defined, 181 Gundersen, R., 283 Gustafsson, B., 311, 312 Hagiwara, S., 105 Hall, M., 34 Halliburton, W., 4 Haloperidol (Haldol), 330-336 Halstead, W., 297 Hamburger, V., 273, 280-282 Hare, M., 231 Harrison, R., 23, 274, 276, 277, 280-282, 285 Hasselbach, W., 187, 188 Hawes, R., 229 Heald, P.J., 179 Hebb, C., 109 Hebb, D.O., 297, 298, 306, 307, 310, 312, 314, 315

Hedgecock, E., 283 Heikkila, R., 326 Heilbrunn, V, 186 Held, H., 20 Helle, K., 253 Helmholtz, H. v., 8 Hemicholinium, 221, 249 Henle, E, 51 Hermann, L., 8-10 Heuser, J., 250-252, 254, 259 Hill, A., 19, 20, 22 Hill, A.V., 93, 144, 146-148, 152 Hillarp, N-A., 125, 226, 227, 247, 253 Hirokawa, N., 260 His, W., 2, 3, 7, 10, 23, 273, 274 Hodgkin, A., 50, 90-92, 112, 152, 188, 246, 258 Hofmann, E, 180 Hogeboom, G., 109 Hokin, L., 189, 190 Hokin, M., 189, 190 Hollmann, M., 207 Holmstedt, B., 120 Holtz, P., 124, 221 Hornykiewicz, O., 322-324 Howell, W.H., 9, 21 5-HT (5-hydroxytryptamine). See Serotonin Hubbard, J, 155 Hughes, J., 136, 224 Hunt, R., 60, 61 Huxley, A., 50, 88, 90-92, 112, 152, 188, 246, 258 Huxley, H., 88 Hyden, H., 298 Imipramine (Tofranil), 235, 237, 238, 338, 339, 342 Impulse conduction, 7-10, 49, 50, 89-93 Inositol phosphates, 189-193, 209, 342 Inositol phospholipids. See Phosphatidylinositols Iproniazid (Marsilid), 232, 337, 338 I.P.S.P.S (inhibitory postsynaptic potentials), 101, 171 Israel, M., 256, 257 Iversen, L. 130, 131, 237, 281, 334 Jacobsohn, D., 76 Jacobson, A., 299 Jahn, R., 261 Janssen, P., 331 Jasper, H., 133 Jenden, D., 120 Jessell, T., 283

Index

John, R., 299 Jolly, W.A., 41, 42

447

Kadota, K., 262, 263 Kalkkinen, N., 233 Kanaseki, T., 262, 263 Kandel, E., 300-306, 314 Kane, J., 335 Kanner, B., 237 Karlin, A., 151, 159 Karnovsky, M., 112 Katz, B., 78, 90, 93, 95, 97, 98, 101, 111-114, 122, 151, 152, 154, 163, 187, 238, 246, 247, 254-256, 258, 267, 268, 355 Katz, J., 297 Kebabian, J., 325 Keen, J., 263, 265 Kelly, R., 253 Kendrew, J., 88, 150 Kenny, J., 234 Kerr, L., 259 Kety, S., 234 Keynes, R., 92, 106, 258 Kinase. See Protein kinase Kinnier Wilson, S.A., 321 Kirshner, N., 226, 227, 253 Kline, N., 329, 338 Koch, R., 31 Kolliker, R. v., 2, 5, 10, 12, 51, 273 Kopin, L, 256, 326 Korn, H., 256 Koshland, D., 88 Kosterlitz, H., 136, 224 Kraepelin, E., 327, 328, 337 Kravitz, E., 130 Krebs, E., 177-179, 181, 193 Kriebel, M., 256 Krnjevic, K., 123, 131, 133, 135 Kuffler, S., 78, 93-95, 105, 112, 129, 289 Kuhn, R., 338 Kiihne, W., 73, 93 Kwanbunbumpen, S., 249

Larner, J., 179 Lashley, K., 296, 297, 307, 314 Learning chemical representations, 297-300 conditioning, 295, 296, 300, 304, 306 habituation, 296, 301-303 long-term, 296, 303 short-term, 296 synaptic changes, 22, 23, 296-316 Lee, C.-Y., 159 Leeman, S., 135 Lefkowitz, R.J., 161, 208-211, 213-215 Legallois, J., 33 Lehninger, A., 88 Lembeck, R, 135 Leonhardt, K., 337 Lester, H., 205 Letourneau, P., 283, 285 Levi-Montalcini, R., 280-282 Levitzki, A., 161 Levodopa, 323, 324, 327 Levy, W., 310, 311 Li, C.H., 225 Liddell, E.G.T., 45 Liley, A.W., 97 Lindhard, J., 74, 95, 289 Ling, G., 92 Lipkin, D., 174 Lipmann, E, 220 Liposome. See Membrane reconstitution Lithium, 341, 342 Llinas, R., 259, 260 Lloyd, D., 102 Loewi, O., 62-67, 69, 74, 75, 78, 124, 229, 237 L0mo, T., 309, 310 Lorente de No, R., 297 Lotze, R., 34 LTP (long-term potentiation), 306-315 Lucas, K., 10, 43, 44, 73, 74, 79 Lumsden, A., 283 Lust, D., 266 Lynch, G., 311, 313

Laborit, H., 330 Landmesser, L., 283 Lands, A.M., 154 Langer, S., 154, 155 Langley, J.N., 31, 44, 53-57, 59, 68, 70-73, 79, 93, 143, 144, 152, 275, 276 Langmuir, I., 146 Langston, W., 325, 326 Lapicque, L., 73, 74 Lapicque, M., 73, 74

MacDonald, J., 10 Macintosh, E.G., 75, 221, 257 Magendie, E, 14, 34 Mallet, J., 222 Mania. See Depression and mania Marchbanks, R., 257 Martin, A.R., 97 Matthes, K., 67 Matteucci, C., 8 Mauro, A., 250

448

INDEX

Maxam, A., 201 McConnell, J., 299, 300 McLennan, H., 122, 126, 129, 311 McMahan, U.J., 289 Meldolesi, J., 261 Meltzer, H., 335 Membrane, cell, 4, 20, 89, 108, 157, 158, 246, 290 Membrane reconstitution, 159, 160 Memory. See Learning Menten, M., 146 M.E.P.P.S (miniature endplate potentials), 97, 98, 106, 114, 246, 254, 256, 258 Michaelis, L., 146 Michell, R., 190 Microelectrode development, 90-93 Microtubules, 288 Miledi, R., 163, 187, 256, 258, 289 Milner, B., 308 Minz, B., 68, 71, 75, 76 Mishldn, M., 308 Mitchell, J., 121, 122, 131, 133, 255 Mitchell, P., 89, 227, 228, 237 Monoamine oxidase, 230-233, 238, 326, 338 inhibitors, 232, 233, 238, 326, 327, 338, 339, 342 Monod, J., 88, 151, 179 Montminy, M., 214 Morphine. See Opioids Muscarine and muscarinic effects, 60, 70, 71, 123, 153. See also Acetylcholine; Atropine Muschall, E., 155 Myosin, 88, 287 Nachmansohn, D., 106, 220, 221, 229, 230 Nagli, C., 4 Nastuk, W.L., 92 Neer, E., 186 Neher, E., 163, 164, 254 Nernst, W., 9, 10 Neuron Theory (Neuron Doctrine), 10, 21, 35, 55, 273, 274 Neurotransmitters. See Acetylcholine; Adrenaline; Dopamine; GABA; Glutamate; Glycine; Noradrenaline; Opioids; Serotonin; Substance P Release by exocytosis, 246-262, 267, 268 Transport. See Reuptake Neurotropic factors. See Chemotaxis NGF (nerve growth factor), 280-283 Nickerson, M., 149 Nicotine and nicotinic effects, 53, 71, 72, 75, 123, 153. See also Acetylcholine

Nishizuka, Y., 192 NMDA receptors. See Receptors, glutamate Nobili, L., 8 Noradrenaline (norepinephrine) metabolism, 230-234, 238 as neurotransmitter, 58, 70, 123-125 receptors (a and f3), 153, 160, 161, 175-177, 180, 181, 185, 208-214, 340 storage, 226, 227 synthesis, 221-224, 238 Noren, O., 234 Nottebohm, E, 314 Numa, S., 201-205, 211, 225 Obata, K., 131, 134 Oliver, G., 55, 56 Olivera, B., 259 Opioids metabolism, 234, 239 as neurotransmitters, 135-137 receptors, 161-163, 212 storage, 228, 239 synthesis, 224, 225, 239 Ostwald, W, 9 Otsuka, M., 135 Overton, E., 4, 9, 10 3, I., 127 Palade, G., 107, 109, 112 Palay, S., 107, 109, 246 Parkinson, J., 320, 321 Parkinsons disease and parkinsonism, 221, 233, 320-327, 332 Fasten, I., 263, 265 Patch electrode development, 163 Paton, W., 152, 155 Patrick, J., 203, 290 Pauling, L., 88 Pavlov, I., 295 Pearse, B., 263 Penfield, W., 45, 307, 308 Pernow, B., 135 Pert, C., 162 Perutz, M., 88, 150 Pfeffer, W, 4 Pfeiffer, C., 156 Pfenninger, K., 288 Pfenninger, M.-E, 288 Pfeuffer, T, 183 Pfliiger, E., 34 Phillips, D., 88 Phosphatidylinositols, 189-193, 209 Phosphodiesterase, 174, 175, 189, 306 Physostigmine, 67, 76-79, 96

Inde

Piccolino, M., 26 Picrotoxin, 105, 132, 134 Pletscher, A., 324 Popper, K., 113 Porter, K., 262 Potter, D., Ill, 130 Potter, J., 189 Potter, L., 159 Pourfour du Petit, R, 51 Protein kinases CaM-kinase (Ca2+/calmodulin dependent), 189, 193, 260, 306 phosphorylations identified, 174, 178-181 protein kinase A (cAMP dependent), 178, 180, 186, 193, 212-214, 223, 260, 303, 304 protein kinase C (diacylglycerol dependent), 192, 193, 213, 223, 261, 306 tyrosine kinases, 281, 282 Protein phosphatases, 179, 181 Protein phosphorylations. See Protein kinases Purkinje, J., 5, 6 Pysh, J.J., 250 Quantal responses and neurotransmitter release, 98, 101, 105, 106, 109, 155, 163, 246, 252, 255-258, 267, 268 Quastel, J., 220 Quinn, W., 306 Racker, E., 159, 160, 231 Radda, G., 227 Raftery, M., 159, 201, 202 Rail, T., 174 Ramon Cajal, P., 26 Ramon y Cajal, S., 1-3, 10-27, 31, 33, 35, 37, 40, 42, 50, 79, 274-276, 286, 296, 297, 308, 314, 355 Ramwell, P.W., 135 Randrup, A., 332 Raper, J., 286 Rapport, M., 127 Rasmussen, H., 186, 187 Raventos, J., 147 Receptors. See also Acetylcholine; Dopamine; GABA; Glutamate; Glycine; Noradrenaline; Opioid; Serotonin desensitzation, 151, 152 downregulation, 213, 214, 340 localization, 288-290 occupation theory, 143-152 proposed, 59, 143

449

purification, 157-165 rate theory, 152 unitary conductance, 163-165 upregulation, 214, 335 Reed, L., 179 Reese, T., 250-252, 259 Reich, E., 156 Reichard, L., 261 Remak, R., 51 Renshaw, B., 101, 102 Renshaw cells, 101, 102, 122, 123, 134, 137 Renyi, A.L., 237 Reserpine, 227-229, 235, 237, 321, 322, 329, 330, 333, 339 Resonance Principle, 276-278 Reticular theory, 3, 5-7, 12, 22 Retzius, G., 2, 10, 11, 23 Reuptake (neurotransmitter transport), 234-238, 339-343 inhibitors, 235-238, 339-343 Reuter, H., 188 Revel, J.-P, 112 Richter, D., 231, 232 Ringer, S., 186 Robbins, J., 132 Roberts, E., 129, 130 Roberts, P. J., 133 Robertson, J.D., 89, 107, 108 Robinson, J.D., 266 Robison, A., 174 Rodbell, M., 181-183, 193 Rodnight, R., 180 Roques, B., 234 Rosenbaum, J., 287 Rosenblueth, A., 70, 78, 153 Rosengren, E., 126 Ross, S.B., 237 Rothman, J., 265 Roux, W., 273 Rushton, W.A.H., 73, 74 Sakmann, B., 163, 164, 205 Salvaterra, P., 221 Samuelsson, B., 313 Sandow, A., 186 Samarro, L., 11 Sanger, E, 88, 201 Sano, I., 126 Scarpa, A., 228 Schaefer, H., 93 Schafer, E., 44, 56 Schatzmann, H.J., 188 Schayer, R., 232 Scheller, R., 261

450

INDEX

Schiff, M., 34 Schild, H., 154 Schildkraut, J., 339, 340 Schizophrenia, 177, 229, 327-336, 343 Schleiden, M., 4 Schmiedeberg, O., 60, 62, 78 Schou, M., 341 Schneider, W., 109 Schuldiner, S., 228 Schwab, R., 325 Schwann, T., 4, 23 Schwartz, E., 266 Schwartz, J., 304 Schwartz, J.-C., 234 Schwartzkroin, P., 310 Scoville, W, 308 SDS-PAGE defined, 158 Second messenger, defined, 175 Seeman, P., 334 Sejnowski, T., 311 Selegiline (Eldepryl), 233, 325, 326 Selinger, Z., 182 Sen, G., 329 Serotonin (5-hydroxytryptamine, 5-HT) metabolism, 230-233, 239 as neurotransmitter, 126-129 receptors, 206, 208, 212, 326, 340 synthesis, 224, 239 transport, 237, 238, 339-341 Setchenov, I., 34 Shaw, J., 135 Sherrington, C.S., 31-33, 35-45, 75, 77, 79, 93, 94, 99, 102, 112, 113, 130, 134, 179 Shooter, E., 281 Silman, I., 230 Simon, E., 162 Singer, S.J., 158, 290 Skinner, B.F., 296 Skou, J.C., 89 Smith, S., 287 Snyder, S., 133, 136, 162, 192, 326, 332-335 Sperry, R., 277-279, 283 Speidel, C., 274 Spemann, H., 273, 280 Sporn, M., 298 Stadtman, E., 223 Stanton, P., 311 Starke, K., 155 Starling, E., 44, 59, 63 Stedman, Edgar, 229 Stedman, Ellen, 229 Stephenson, R.P., 148-150, 154 Stern, P., 135

Stevens, C., 163 Straub, W., 147 Stroud, R., 160 strychnine, 8, 39, 102, 105, 132, 134 Stryer, L., 185 Substance P, as neurotransmitter, 134, 135 Siidhof, T., 261 Sulser, E, 340 Sulzer, D., 267 Sussman, J., 230 Sutherland, E., 172-179, 181, 183, 185, 193 sympathin, 69, 70, 123, 153 Synapse chemical transmission proposed, 55-76 cleft visualized, 106-108 delayed transmission, 40^12, 45, 77, 102 electrical transmission, 21-23, 76-78, 111, 112 named, 33 unidirectional transmission, 40, 45 vesicles, 109-111 Synaptosomes, 109-111 Takamine, J., 56 Takeichi, M., 285 Takeuchi, A., 97, 130, 132 Takeuchi, N., 97, 130, 132 Tasaki, I., 105 Tatum, E., 88 Tauc, L., 257, 300 Taylor, P., 230 Terenius, L., 136, 162 Tessier-Lavigne, M., 283 Thesleff, S., 289 Thoenen, H., 281 Thorndike, E., 296 Tilgmann, C., 233 Titus, E., 237 Transport membrane, 89, 92, 158, 187, 188, 190, 226-228, 234-238, 246, 247, 265, 267 neurotransmitter reuptake. See Reuptake vesicular. See Vesicular transport Trautwein, W., 180 Tretiakoff, C., 321 Tsien, R., 313 Tubulin, 260, 288 Udenfriend, S., 127, 222-225, 232, 324 Ungewickell, E., 263 Unwin, N., 203, 204 Valentin, G., 5 Van der Kloot, W., 257

Inie

Vane, J., 313 van Gehuchten, A., 10, 11 Van Harreveld, A., 132 van Rossum, J.M., 332 Verworn, M., 4 Vesicular transport, 226-229 Virchow, R., 5, 31 Vogt, M., 74, 125, 227 Volta, A., 7, 8 von Baer, K.E., 273 von Euler, U.S., 123-125, 134, 146, 238, 247, 313 von Gudden, B., 7 von Lenhossek, M., 10, 11, 23 Vyskocil, R, 267 Waldeyer, W. v., 2, 10, 18 Waller, A., 7, 45 Wang, J., 189 Watson, J., 88, 199 Waud, D.R., 152 Weber, Ernst, 34 Weber, Eduard, 34 Webster, R.A., 133 Weiner, N., 223 Weiss, P., 273, 276-278, 283, 285, 286, 291, 355

451

Wernig, A., 256 Wessels, N., 287, 288 Wester, K., 310 Whittaker, V., 109-111, 126, 128, 130, 257 Whytt, R., 33 Wiedenmann, B., 261 Wiersma, C.A.G., 276, 355 Wightman, M., 255 Wigstrom, H., 311, 312 Willis, T., 50, 51 Wilson, I.E., 230 Wilson, J.-B., 51 Wilson, M., 261 Wong, D., 340 Wooley, D.W., 328 Wosilait, W, 174 Wright, S., 76 Wylie, R.G., 250 Wyman, J., 151 Yonkman, R, 329 Yoshikami, D., 259 Young, J.Z., 90 Zeller, A., 231, 338 Zigmond, M.J., 266 Zimmermann, H., 257