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

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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


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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PREFACE

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

Prefa

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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.


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

Contents

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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



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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2


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-

Cajal and tke Neuron Tkeory (1889-1909)

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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


5

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

8


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

Cajal and the Neuron Theory (1889-1909)

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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).

Cajal and die Neuron Tkeory (1889-1909)

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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,

Skerrington and tlie Synapse (1890-1913)

33

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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39

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

42


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.)


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


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


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).


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


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


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


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


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

58


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


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.)

Chemical Transmission at Synapses (1895-1945)

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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-


Li

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

Chemical Transmission at Synapses (194

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