Joseph D. Robinson - Mechanisms of Synaptic Transmission - Bridging The Gaps (1890-1990) (2001)

MECHANISMS OF
SYNAPTIC TRANSMISSION
This page intentionally left blank
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 physi-
ologists, led by Santiago Ramon y Cajal, Wilhelm His, and Charles Sherring-
ton, 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 pre-
cise 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 inge-
nuity and interpretive insight, for initial efforts to answer them exposed a cas-
cade 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-
vii
viii 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, medi-
cine, pharmacology, and physiology. The composite field, neuroscience, that
emerged from these inquiries, however, was itself integrated within the main-
stream 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 prac-
tice 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 adjudi-
cation 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 his-
tory, either, although human interactions surely affected the course, and vari-
ous institutions facilitated or impeded research. Cities are listed, but chiefly to
illustrate the geographical extent of the investigators' activities. Whereas cer-
tain 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: scien-
tific advances are notably dependent on enabling techniques, and the devel-
opment of new methods permits the resolution of questions long asked but
unapproachable previously. This account, therefore, is intended as a framework
Prefa ix
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 com-
munication 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 Veen-
stra, 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 inter-
est, assistance, and an indulgent tolerance. Financial support from the Hen-
dricks 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
xi
xii 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 xiii
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
xiv 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
MECHANISMS OF
SYNAPTIC TRANSMISSION
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 Ger-
man 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 jour-
nal 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
1
2 MECHANISMS OF SYNAPTIC TRANSMISSION
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 publica-
tions, 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 identifica-
tion 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 accom-
plishments 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 repre-
sented to Cajal and his audience were single nerve cell bodies and their branch-
ing processes. What that interpretation centered on was a critical issue of struc-
Cajal and tke Neuron Tkeory (1889-1909) 3
FIGURE 1-2. Photomicrograph of a Golgi-stained pyramidal neuron of the hippocam-

pus. 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 cham-
pioned by His and by August Forel in Zurich. Theirs, however, was a minor-
ity 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.
Background: Cells, Nerve Cells, ana Nerve Impulses
Cells
Formulation of the cell theory is conventionally ascribed to Matthias Schlei-

den 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 authorita-
tively: "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 struc-
tures 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 pro-
toplasm abutted its nonliving environment. In fact, indications of a distin-
guishable 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 cell-
membrane is a general cell-constituent has completely disappeared."11 Corre-
spondingly, 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 indi-
cators 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, micro-
scopic examinations of the nervous system presented, instead, a confusing
complexity.
Transected nerves revealed, under microscopic examination, numerous cir-
cular profiles, interpretable as cross sections of tubes running longitudinally.
Those outlines were subsequently identified with "myelinated" fibers bearing
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 glob-
ular bodies containing nuclei. From ancient times nerves were thought to con-
tain hollow tubes through which animating spirits flowed, so the microscopic
fibers could be likened to such structures and functions. The nucleated glob-
ules 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 tear-
drops, some with branched tails (Fig. 1-3A; these subsequently were chris-
tened "Purkinje cells"). Elsewhere they identified globules with sharp outlines
containing granules and a nucleus, although Valentin concluded that these glob-
ules 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 dur-
ing life"; still, he considered that such cellular fibers were "an essential con-
stituent of the living nerves."14 In a posthumous publication of 1865 Otto Deit-
ers in Bonn took a major step further, distinguishing between branching
"protoplasmic processes" (later termed "dendrites"), which extended in vari-
able 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 develop-
ment 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, how-
ever, is on nerve cells themselves.)
In retrospect, Deiters's distinctions identify the tripartite nature of the pro-
totypic 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
FIGURE 1-3. Nerve cells and fibers. A. Purkinje's 1837 drawing of the cerebellar cor-
tex, 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 con-
nectivity of a reticulum. Golgi's stain, as noted above, showed distinct silhou-
ettes and allowed him to confirm Deiters s view of an axis cylinder distinguish-
able 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 there-
fore continuous—processes. What he actually saw, however, were merely
branchings into finer and finer processes: either with no termination visible in
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 appar-
ently free endings.
On the other hand, Golgi differed with Gerlach's view of protoplasmic pro-
cesses. Instead of the anastomoses that Gerlach specified, Golgi saw free end-
ings 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 pro-
toplasmic 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 con-
cluded that the nerve cell was a "genetic, nutritive, and functional entity" from
studying embryological development;21 moreover, he discovered that primor-
dial 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, empha-
sizing 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 "impon-
derable fluid" suggested its association with nerve impulses. At the end of that
century Luigi Galvani in Bologna furthered this notion by showing that elec-
trical 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 rail-
ing their muscles contracted. Galvani proposed a circuit for "animal electric-
ity" from muscle to iron railing to brass hook to spinal cord to nerve to mus-
cle; 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 stim-
ulating current. Volta confirmed his proposal of "metallic electricity"—arising
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 stim-
ulated by draping its nerve across a second muscle and its cut surface: the first
muscle then contracted. This "contraction without metals" Volta also chal-
lenged, 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, decap-
itated "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 mea-
sured a "resting current" in nerve as well as muscle; he also recorded the "neg-
ative variation"—the decrease in that resting current—during tetany. Du Bois-
Reymond 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 stimula-
tion 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 veloc-
ity, furthering its association with the nerve impulse. Moreover, Bernstein
reconstructed the timecourse of the negative variation by measuring its mag-
nitude at successive intervals: current fell and rose within milliseconds.
In the latter decades of the nineteenth century, Ludimar Hermann in Konigs-
berg, also a former student of du Bois-Reymond, vigorously attacked the notion
that resting currents were present normally. He found no such current flow-
ing 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) 9
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 envi-
ronment (the sea) by an insulating sheath. Hermann also rejected du Bois-
Reymonds 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 dif-
ferent 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 electrochem-
istry, in the process naming the positively charged ions ("cations") and nega-
tively 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 con-
centration 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, tran-
siently, a general increase in membrane permeability. This loss of selectivity
would decrease the transmembrane potential, transiently; the consequent cur-
rent 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 complemen-
tary 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 sug-
gested that excitation was associated with an exchange of intracellular K + for
extracellular Na + .
Subsequent developments had by mid-twentieth century validated Bern-
stein's thesis of selective permeabilities underlying the resting and action poten-
tials 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 uni-
formly 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
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, releas-
ing 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 bet-
ter 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 out-
lines—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 summa-
rizing synthesis by Wilhelm von Waldeyer, professor of anatomy in Berlin.
In a serialized review published in 1891, Waldeyer introduced the term neu-
ron to name as a discrete whole the cell body plus all its processes; the "Neu-
ron Theory" (or "Neuron Doctrine") explicitly enunciated this unity of parts
and separateness of cells.33 Although Cajal noted with some pique that Wal-
deyer "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 signifi-
cant. Not merely did he lend his personal prestige as an establishment author-
ity, Waldeyer assembled a careful compilation of the evidence leading to his
conclusion. (At that time His introduced dendrite to supplant protoplasmic pro-
cess, and Kolliker replaced axis cylinder with axon. These distinguishing new
names also helped to propel the Neuron Theory.)
Cajal's Contributions
Teennique
Cajal first viewed the Golgi stain in 1887, just two years before the Berlin meet-
ing. 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 "try-
ing 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 pro-
duced a dark red to black image of silver chromate.36 Nevertheless, stain-
ing was erratic, incomplete, and tedious. Cajal, who praised persistence
as "the virtue of the less brilliant,"37 set about perfecting the method, opti-
mizing concentrations, times, temperatures, and light exposure of the pho-
tosensitive silver salts. Through this careful and deliberate search Cajal
standardized a rapid staining method as well as developed a double-
impregnation 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 pro-
gressed 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 frac-
tion 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 selec-
tions 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 pro-
cesses 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 permit-
ted the use of such sections, which would have been opaque with older
techniques.)
3. Cajal used tissues from embryos and young animals. The neurons in these
animals are shorter (thereby increasing the likelihood that their termina-
tions would lie within the microscopic section), more separated from one
another (thereby minimizing the complexity of images), and not yet myeli-
nated (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 sim-
ilarities 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 prin-
ciple specified the direction in which nerve impulses move through neurons:
the dendrites and cell body conduct toward the axon, whereas the axon con-
ducts impulses away from the dendrites and cell body. This principle delin-
eated functional circuits that could be discovered by identifying points of con-
tact 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 conduct-
ing 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 hemi-
spheres displaying a structural pattern evident even by cursory histolog-
ical approaches. (Indeed, the strikingly regular architecture of the cere-
bellar 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 trans-
verse 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) 13
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 organi-
zation 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 Purk-
inje 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 longitudi-
nal section).41 In addition, his diagram includes axons of "granule cells"
rising toward the cerebellar surface, where they split: running longitudi-
nally as "parallel fibers" to contact the dendrites of Purkinje and basket
cells. The granule cells themselves receive axons called "mossy fibers"
(arising from cell bodies outside the cerebellum). Less diagrammatic

views were shown in CajaFs monumental atlas published in French trans-
lation 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 pro-
vide 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 den-
drites, 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 periph-
ery, 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 lat-
ter, 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 "effer-
ent 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
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 gan-
glion. 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 con-
tributing to the reticulum. The motor efferent fibers leaving through the
ventral roots Golgi identified as axis cylinders of large cells in the "ven-
tral horn" of the spinal cord gray matter ("ventral horn cells"). Since Golgi
believed that protoplasmic processes served only nutritive roles, connec-
tions 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 accommodat-
ing distinct functional responses, as well as exemplifying the uniform pat-
tern 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 mas-
sive 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 termina-
tions 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 dor-
sal 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 sen-
sory 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 motoneu-
ron; and (M) are nerve endings of the motoneuron axons on muscle. Arrows show the
direction that impulses travel. C. Corresponding diagram for indirect, diffuse, unilat-
eral 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 motoneu-
ron. (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.)
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 "interneu-
rons" then sent longer ascending and descending branches that termi-
nated on ventral horn motoneurons. Thus, by passing through interneu-
rons, the impulses from the periphery were spread to innervate additional
units. Moreover, the pathway could be modulated by other inputs affect-
ing 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 path-
way, 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 myeli-
nated axons through specific tracts in the white matter. Moreover, he pur-
sued 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 Purk-
inje 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. Persist-
ing 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 dur-
ing conversion of live cells into durable forms for sectioning, to staining arti-
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 bas-
kets 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 pro-
cessing. 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 natu-
ral 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 fur-
ther 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
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 cell-
system 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 dif-
ferent 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 dif-
ferent 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 sur-
face, 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 anas-
tomoses; 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
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 fila-
ments 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 del-
icate filaments."53 Cajal then developed a silver stain for the fibrils, and exam-
ining 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 res-
olution 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 prob-
lem by proposing that neurofibrils were the conducting elements of the ner-
vous system (comparisons were made to muscle fibers, which are the contrac-
tile elements of muscle). Thus, Howell's physiology textbook of 1905 likened
nerve conduction to transmission by a submarine cable. In each case a con-
ducting "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 empha-
sizing morphological discontinuity (Fig. 1-6B-D), although he did consider
that electrical conduction might cross a gap "by an induction effect, as in indue-
tion coils."58 Cajal also imagined a "granular cement, or special conducting sub-
stance" 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 connec-
tions 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 har-
mony 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 contrib-
utes," 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 under-
stand 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 neu-
rons serving as a "series of switches."68 Nevertheless, the neurofibrillary pro-
posal could include the notion that impulses passed along particular conduct-
ing strands within a vast reticulum and that the conductivities over specific fibrils
could be altered by prior experience. Indeed, Hill elaborated a mechanism by
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, particu-
larly 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 obser-
vations, 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 devel-
opmentally a single cellular unit—Cajal referred to as the "monogenist hypoth-
esis."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 dra-
matic 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 concav-
ity of a microscope slide and sealed the edges. With aseptic conditions the tis-
sue survived for a week or more, allowing him to observe continuously the
development of individual processes (unlike Cajal, who had to construct suc-
cessive 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 periph-
eral portion. The rival polygenist view, as exemplified notably by Bethe,73
argued that the regenerated axon arose instead from the chain of cells sur-
rounding its former course. (Schwann had described a series of bodies—
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 stud-
ies 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 mechani-
cal 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 funda-
mentally 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 degen-
erated 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 periph-
eral 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 dif-
fusely from the central portion of the nerve than the polygenist opponents had
imagined, and that these sprouts then coursed more widely in avoiding obsta-
cles. These conclusions sprang from the use of a new staining technique cou-
pled 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 uncon-
nected peripheral segment.)
An unanswered question pervading these studies of development and regen-
eration, however, was what guided the growing fibers to their proper destina-
tions. 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.
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, neu-
rons 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 strik-
ingly from prototypic neurons, such as Purkinje cells. Instead of a bipolar con-
figuration, 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 out-
ward from the cell body stalk, to be a dendrite even though it had "all the struc-
tural 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 develop-
ment, however, the peripheral segment assumes the appearance of an axon and
the cell body becomes separated by a stalk, achieving the unipolar configura-
tion. 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 accord-
ingly: dendrites conduct impulses toward the axon, and axons conduct impulses
away from the dendrites.78 (Cajal promulgated this formulation after physiol-
ogists had shown that both axons and dendrites could, when stimulated at a
point away from their termini, conduct impulses in both directions. Contra-
dictions 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 chap-
ters 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
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 Piccol-
ino 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 child-
hood 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 anatom-
ical study, although that schooling sparked continuing conflicts, as in his repu-
diating 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. Com-
ing thus from a country one contemporary dismissed as "remarkable for its bar-
renness in original research,"81 how did Cajal reach the pinnacle of scientific
greatness?
He toiled relentlessly. He developed techniques and used them wisely, choos-
ing specimens broadly and pursuing comparative, developmental, and func-
tional correlations. He enlisted his considerable artistic talents and promoted
his conclusions tirelessly. He persevered in his ambition.82 He grasped simi-
larities astutely,83 and he believed fervently in "the unity of biological laws,"84
which assured discoverable generalities. And he concluded that biology dis-
plays 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 con-
firmed 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-
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 attrib-
uted to insufficient resolution by light microscopy, inadequate fixing and stain-
ing, 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 mor-
phological and functional unit of the nervous system. The image of a proto-
typic neuron—with receptive dendrites and a transmitting axon—left unre-
solved, 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,
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 conse-
quent 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 anas-
tomoses" (quoted by Shepherd, 1991, pp. 36, 53, 54).
19. Golgi's initial paper of 1873 was largely overlooked; subsequent papers, begin-
ning 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 approach-
ing thunderstorm. (He had earlier shown that nearby electrical discharges could trig-
ger 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 homoge-
neous—with bimetallic contacts in the rod itself.
26. Electric fish had been known since antiquity, and both Galvani and Volta stud-
ied 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 per-
meable 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 exci-
tation on the frequency of stimulation by an alternating current. Lucas also cited a pro-
posal 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 cellu-
lar 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.
35. Ibid., pp. 308, 309. Cajal contrasts this with the tendency for investigators gen-
erally 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 descrip-
tions 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 rel-
egating 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; ital-
ics 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: "Elec-
tricity gives us ... innumerable examples of similar transmissions without direct conti-
nuity."
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.
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 enti-
ties (Ramon y Cajal, 1937, p. 334).
83. Sherrington reported that when Cajal included a specimen from birds to illus-
trate pyramidal tracts of the spinal cord, he protested that birds did not have pyrami-
dal 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 pro-
cesses "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. Prob-
ably 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 Sher-
rington (Fig. 2-1 ).1 Sherrington, then 37, was five years younger than Cajal
and quiet and reserved, whereas Cajal was voluble and dramatic. Both, how-
ever, 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 func-
tion. Initially, Sherrington's interests focused on cellular pathology and bacte-
riology. (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 sys-
tem 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
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 lec-
turer 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 physi-
ology, 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 junc-
tion between neurons, Cajal's point of contact without continuity. Earlier Sher-
rington 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 sug-
gested 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 theo-
retical interest.6 The issue here, however, is how their study influenced con-
templations 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 demon-
strations that reflections occurred in the spinal cord: he showed that certain
responses, such as movement of the leg after local irritation, persisted in decap-
itated 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 spe-
cific areas of sensation and movement were affected. Legallois concluded that
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 observa-
tions, establishing—and naming—the "reflex arc": an ingoing path to the cen-
tral 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 tran-
sected the spinal cord, finding that sensory inputs may evoke responses beyond
their segment. And he included responses of voluntary systems, such as skele-
tal musculature, as well as of involuntary and complex systems, such as sneez-
ing 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 pro-
tracted debates over a "spinal soul," an entity granting conscious sensation to
the spinal cord; principal protagonists were Eduard Pfliiger in Bonn (physiol-
ogist, 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 stim-
ulating 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 pro-
longed 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 inhi-
bition 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., dif-
ferences in stimulus intensity, duration, and mode) and inappropriate concep-
tual schemes (e.g., irrelevant criteria, such as classifying reflexes into superfi-
Skerrington and tke Synapse (1890-1913) 35
cial vs. deep). Moreover, vast areas of ignorance, anatomical as well as physio-
logical, prevented sharp distinctions between rival claims.
Snerringfton's Achievements
A half century after his death in 1952, Sherrington s influence continues to per-
vade 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 individ-
ual."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 com-
plexity 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 lec-
tures displayed this viewpoint through a wealth of experimental observations
unified by broad generalizations, summarizing a decade and a half of extraor-
dinary 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 speci-
fied neither the route nor the direction an impulse would travel through the
reticulum)—and technically by the diversity of stimuli employed and the vari-
ability 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 . . .
in the study of nervous reactions."10 And he bolstered that outlook with nec-
essary examinations of the sensory and motor connections, studies he consid-
ered "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 ven-
tral roots from the lumbosacral region of the spinal cord, stimulated electri-
cally 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 verte-
brae. 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 inspec-
tion alone.)
Correspondingly, in 1893 Sherrington published a 120-page paper describ-
ing 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 stimula-
tion 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 exam-
ined 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 Fur-
thermore, 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 [sen-
sory] 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
Sherrington and the Synapse (1890-1913) 37
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 pres-
ence 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 Sher-
rington'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 inner-
vation," 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 stimu-
lated, as that freed muscle shortened the antagonistic muscle still relaxed.22
Sherrington extended his studies to a variety of other reflexes, in the pro-
cess 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 hob-
bling 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 sec-
ond dorsal root ganglion cell a' carrying sensory information from the flexor F and pro-
ducing on the same side excitation of 8 to the flexor and inhibition of e to the exten-
sor. 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.)
38
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 demonstrat-
ing the functional processes present in reflex arcs. Indeed, some of these
differences remained unexplainable for decades, such as the greater sensitiv-
ity 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-
ference of potential with changes in surface-tension or in shape, or intervene

as a membrane between dilute solutions of electrolytes."26 Sherrington, how-
ever, 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 pro-
vided 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 hypo-
thetical "membrane" might be "more permeable in one direction than the
other."28 Again, direct evidence for a membrane having the requisite proper-
ties 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 flex-
ion 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 [evi-
dence] 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
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 flex-
ion 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 flex-
ion. 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 apply-
ing a stimulus—either striking the patellar tendon for the knee jerk or prick-
ing 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
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 (strik-
ing 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 record-
ing, 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 mus-
cle, 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 Sher-
rington listed involved disparities between stimuli and responses, such as dis-
TABLE 2-1. Response Times for Knee Jerk and Flexion Reflexes0
TIME (MILLISECONDS)
PROCESS 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
°The experiment is described in the text. (From Jolly [1911], p. 86. Used by permission of The
Physiological Society.)
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 "sublimi-
nal." 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 inter-
val, 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 sum-
mation 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 inter-
preted their results in terms of a brief period of "supernormal" excitability fol-
lowing 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 mag-
nified 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 integra-

tion is achieved: from hierarchical control manifested experimentally by spinal
shock and by decerebrate rigidity, to reciprocal innervation dependent on sen-
sory nerves from muscle, to the convergence of excitatory and inhibitory influ-
ences 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 exci-
tation, and was not due artifactually to experimental perturbations. (Thus, he
argued that spinal shock resulted from the loss of input above the point of sec-
tion rather than from the injury of sectioning, showing that a subsequent tran-
section 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
suggested that inhibition was "referable to a change in the conduction of the

synaptic membrane causing a block in conduction."41 Nevertheless, in an ear-
lier 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 immedi-
ately.42 He imagined that some process continued after the excitatory stimulus
ceased, a process that was susceptible to immediate inhibition. (If the contin-
uing 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, reflect-
ing qualitative differences between excitatory and inhibitory impulses,43 a pro-
cess 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 inti-
mate 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 perme-
ability 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 enter-
taining 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 estab-
lished 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 Denny-
Brown, 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 develop-
ments: clarifications, revisions, and extensions. Although aspects of Sherring-
ton's formulations were not without challenges, and although later decades saw
striking metamorphoses of such notions as synaptic transmission and the mech-
anisms 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 quan-
tifiable 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 apply-
ing newer techniques, he retained the virtues of clarity, simplicity, careful con-
trol 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 proper-
ties: acting as a one-way valve for impulse conduction between neurons, pro-
ducing 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 Sher-
rington'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.
3. Sherrington (1893b), p. 300.

4. The account is in Fulton (1938), where it is clear that Sherrington preferred "syn-
desm"; 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 Sci-
entific 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; how-
ever, 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 phys-
iological 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 photo-
graphic 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 stim-
ulus 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, asso-
ciated with the skeletal musculature, and peripheral inhibition, associated with the auto-
nomic 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 com-
pliquer 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, quan-
titating 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 perme-
ability, analogies with oxidation electrodes that changed potential with chang-
ing oxidation/reduction states, and even vaguer formulations implicating col-
loidal 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
membrane resistance during action potentials—consistent with Bernstein's the-

ory—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 pro-
posals 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 micro-
scope," 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 mor-
phological problem . . . will not be furthered . . . by the exclusive use of the
older . . . methods."7 Following Bartelmez, Bodian concentrated on better fix-
ation techniques and on correlating images from different staining procedures,
concluding in 1942 that at synapses "only one membrane can be resolved, pre-
sumably because of the intimacy of contact of the separate neuronal limiting
membranes"; the fused membrane separating two neurons he labeled a "synap-
tolemma."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 mid-
seventeenth 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 ("vis-
ceral") and voluntary ("somatic") actions. Willis also discovered that cutting cra-
nial 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 com-
municantes" 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 orig-
inated 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 Brown-
Sequard, a peripatetic investigator then in the United States, and Claude
Bernard in Paris demonstrated independently that electrical stimulation of sym-
pathetic 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 anatom-
ically as well as functionally in the 1880s.10 Gaskell had entered Trinity Col-
lege intending a career in clinical medicine, but while he was an undergradu-
ate 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 (slow-
ing) vs. stimulating fibers from the sympathetic trunk (accelerating), Gaskell
next turned to microscopic examinations of the spinal cord roots and rami com-
municantes, establishing five categories of motor fibers (Fig. 3-1). (1) Non-
myelinated 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 vol-
untary 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 sympa-
thetic 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, blad-
der, 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 under-
graduate 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 sym-
pathetic ganglia. After he painted a ganglion with nicotine solutions, a charac-
teristic response resulted that soon disappeared. Stimulating fibers leading from
the spinal cord to this ganglion then evoked no responses. By contrast, stimu-
lating fibers running/row this ganglion still produced the normal response. In
that manner Langley matched each root, ramus, and ganglion with its sympa-
thetic response.13 In the process he also provided new names, including "pre-
ganglionic 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 sym-
pathetic 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 PARASYMPATHETIC STIMULATION
(THROUGH (THROUGH
PROCESS THORACOLUMBAR FIBERS) 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
"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 gan-
glionic elements. Ganglia were collections of neuronal cell bodies, plus neu-
ronal 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 gen-
erally lay close to the spinal cord, so preganglionic fibers were short and post-
ganglionic fibers to innervated organs long. By contrast, ganglia of the parasym-
pathetic 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 pres-
ent in blood vessels and viscera; its contractions are not under voluntary con-
trol but are regulated—excited or inhibited—by the autonomic nervous sys-
tem. (Here, inhibition is "peripheral," through inhibitory nerves to the muscle.)
Striated muscle is present in somatic skeletal muscle subject to voluntary con-
trol. (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 neu-
rons; between autonomic motoneurons and the glands or smooth muscles they
innervate (commonly termed "neuroeffector junctions"); and between volun-
tary 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 sym-

pathetic 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,
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 vaso-
constriction occurred in isolated organs as well, they concluded that the re-
sponse was "due to the direct action of the active principle . . . upon the mus-
cular 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 pres-
sure; 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 auto-
nomic 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 for-
mula containing catechol, a secondary alcohol, and a methylated amine (Fig.
3-3A).24 That year H. D. Dakin in London synthesized this compound, show-
ing 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
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 degener-
ation 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: trans-
mission 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 sympa-
thetic . . . nerves."27 But Elliott did not repeat his earlier suggestion about sym-
pathetic 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 sym-
pathetic stimulation came from Henry Dale (Fig. 3-4), yet another who fol-
lowed 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 pharmaceu-
tical 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
FIGURE 3^t. Henry Hallett Dale (1875-1968; courtesy of the Wellcome Trust Med-
ical 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 [excita-
tory and inhibitory] are produced by different active principles."31
In 1910 Dale documented further divergences while comparing systemati-
cally 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 cat-
echol and amine constituents, they are known collectively as catecholamines.
Dale's critical observation was that the rank order of these three differed for
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 success-
ful 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 explo-
ration 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 recep-
tive substance."36 To account for inhibitory as well as excitatory responses, Lan-
gley 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 pro-
portion 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 demon-
strating 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,
began investigating the secretin system, generalizing that sequence to propos-

als 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 elec-
trically 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 mus-
carine 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 sub-
stance 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) 61
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, con-
stricting 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 sug-
gested that esterases in the body might split acetylcholine rapidly into acetate
and far weaker choline (Fig. 3-6A-B). Consequently, if acetylcholine were pres-
ent 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 per-
fect; 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 phar-
macology. His earlier studies in Marburg and Vienna ranged over carbohydrate
FIGURE 3-7. Otto Loewi (1873-1961).

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 devel-
oped 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 some-
thing 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 deter-
mine 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 with-
out. Both hearts were attached to ... canulas [sic] filled with a little Ringer solu-
tion.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 spe-
cific chemical substances which, in turn, cause the well-known modifications of
the function. . . . 55
The published reports, however, were less straightforward than Loewi's recol-
lections, and the consequent skepticism represented no unequivocal accept-
ance 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,
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 "Acceler-
ansstoff."
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 sym-
pathetic 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 exper-
iments in February and March, the latter experiments from April through
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 stim-
ulated (or, better yet, to stimulate electrically only parasympathetic or only sym-
pathetic 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 repro-
duced 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 diminu-
tion 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. Experi-
ments 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 disap-
peared. (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 appara-
tus 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 stimu-
lation ceased the donor heart resumed beating, as did the recipient heart. (From Bain
[1932], Figs. 1 and 2, courtesy of the Physiological Society.]
identified by acetylating the extract to form acetylcholine.66 This conversion

multiplied the extract's potency enormously, allowing him to establish its pres-
ence 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 physostig-
mine also augmented and prolonged the effects of both Vagusstoff and acetyl-
choline.68 And in 1930 both K. Matthes, who worked with Dale, and Loewi
found cholinesterase activity in blood that could be inhibited by physostig-
mine.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 acetyl-
choline was Dale's announcement the previous year that acetylcholine indeed
existed in animal tissues. In 1929 Dale, now at the National Institute for Med-
ical Research in Hampstead, reported the chemical identification of acetyl-
choline in bovine spleens.70 Measuring acetylcholine in other animal organs by
chemical means was, however, not possible for several decades. Instead, phar-
macologists relied on various bioassays, such as frog heart, rabbit blood pres-
sure, 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 multi-
tude 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 demon-
strating a fixed quantitative ratio of activities for an unknown substance to activ-
ities 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 requi-
site characteristics.72
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 jour-
neyed to Cambridge to work with Langley; after Langleys death he worked
with Dale in Hampstead before returning to Berlin in 1927. When he was dis-
missed 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 demon-
strated 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).
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 inac-
tivated Acceleransstoff, as it did adrenaline.76 A decade later he finally con-
vinced himself that Acceleransstoff was adrenaline: perfusates from stimulated
hearts, as well as extracts of these hearts themselves, showed the characteris-
tic 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 emo-
tional 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 adren-
als were removed, hearts denervated, and spinal cords transected.83 Again, the
only link between the site of stimulation and the affected organs was the blood-
stream: 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
as blocking of both by ergot extracts and increased sensitivity to both after den-
ervating tissues).
In 1933, however, Cannon and Arturo Rosenblueth argued that two varieties
of sympathin were formed: excitatory ("sympathin E") and inhibitory ("sym-
pathin 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 (sym-
pathin I); in this way an adrenaline-like substance became "differentiated for
positive and negative action."86 Now they added a further characteristic: neu-
rotransmitter 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 stim-
ulation having correspondingly diverse effects, but no one felt that two forms
of acetylcholine were required.87 Moreover, Zenon Bacq in Liege, who had col-
laborated 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 sub-
stances 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 nor-
adrenaline (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, con-
stricted the pupils, etc.—the well-known array that muscarine elicited and
atropine blocked. These Dale now classed as "muscarine actions" of acetyl-

choline (later called "muscarinic actions"). (2) But in the presence of atropine,
larger doses of acetylcholine produced the opposite effects: elevated blood pres-
sure, 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 differ-
ences. 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 pre-
ganglionic fibers and postganglionic neurons of both sympathetic and parasym-
pathetic divisions.94
The accumulating evidence for chemical transmission by postganglionic neu-
rons suggested that chemicals might mediate synaptic transmission by pregan-
glionic 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 acetyl-
choline, according to Dale, "have a long latency, rise slowly to a maximum with
repetitive stimulation of the nerve, and . . . outlast the period of such stimula-
tion"; by contrast, nicotinic responses to preganglionic neuronal activity "had
the appearance of a direct, unbroken, physical propagation [with a] transmis-
sion 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 pre-
ganglionic fibers to sympathetic ganglia released acetylcholine into the perfu-
sion 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 neu-
rons, and the nerves evoking adrenaline release from adrenal medullas corre-
spond to preganglionic fibers. Accordingly, Feldberg and Minz found that stim-
ulating these nerves released acetylcholine.100
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 mus-
carinic 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, sum-
marized in 1856,102 demonstrated that electrical stimulation of the nerves failed
to evoke muscle contractions in frogs injected with curare, although direct elec-
trical 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 axon-
endings but on the muscle itself . . . by combining with the receptive sub-
stance."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
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 vio-
lent excitation of the muscle takes place [after nerve stimulation], or the influ-
ence must be electric.108
Indeed, du Bois-Reymond then argued against electrical transmission on the-

oretical grounds.109
Despite du Bois-Reymond's stature as a founding father of physiology, sci-
entific 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 micro-
scopist, had observed what became known as the muscle "endplate," where
"[n]erves end blindly in the muscles," an apposition he thought favored elec-
trical transmission.110 And in the early decades of the twentieth century Louis
and Marcelle Lapicque in Paris developed an encompassing theory of electri-
cal transmission rooted in the relationship between the strength of stimulation
required to evoke responses and the duration of that stimulation.111 The min-
imal 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 dura-
tion 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 "hetero-
chronicity"—impeded transmission. They argued that curare blocked trans-
mission in just this fashion: by increasing the chronaxie of muscle.
Keith Lucas in Cambridge, however, obtained different results and drew dif-
ferent conclusions. Like the Lapicques, Lucas viewed neuromuscular transmis-
sion as an electrical process. But in 1907 he described triphasic plots of mini-
mal 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. Further-
more, he found no effect of curare on the strength-duration plots for muscle.
Lucas died during the war, and for decades thereafter the Lapicques force-
fully defended their observations of isochronic plots for nerve and muscle, the
effects of curare on these, and the consequent significance for junctional trans-
mission.113 But in 1930 W. A. H. Rushton in Cambridge reinvestigated Lucas's
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 dif-
ferent 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 Ger-
many, 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 convinc-
ingly, 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 poten-
tials.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 pro-
duced 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 con-
centration of cholinesterase" to achieve the transient response of a true mus-
cle "twitch."124 Brown supported this assertion with electrical recordings show-
ing 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
endplate region where motor nerve endings terminate.126 After a single admin-
istration 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 sug-

gested 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 transmis-
sion in the brain and spinal cord progressed slowly, due in large part to for-
midable difficulties. As Minz observed:
the extreme anatomical complexity . . . with its innumerable physiologically inter-

locking 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 acetyl-
choline 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 dif-
ferent portions of the brain and spinal cord.130 Such local variations accorded
with principles of neural localization attributing specific functions to particu-
lar areas of the brain: acetylcholine might mediate only certain functions in
certain locales. (What mediated other functions in other locales was a differ-
ent 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
given physostigmine intravenously, and these observations were subsequently

confirmed.131 Demonstrating a stimulated release was more difficult. Stimu-
lating sensory nerves generally produced no measurable increase in acetyl-
choline 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 adren-
aline 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 mim-
icking electrical stimulation of this region of the brain.135 Others cataloged var-
ious 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 trans-

mission 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 phe-
nomena. 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 poten-
tials in nerve trunks had complex wave forms, representing different fibers con-
ducting at different velocities; various agents suppressed specific responses,
allowing a classification of fibers by that sensitivity and also by conduction veloc-
Chemical Transmission at Synapses (1895—1945) 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 Sherring-
ton attributed to "an enduring central excitatory process," and he suggested
two possible mechanisms: that the "electrical processes of successive nerve-
impulses 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 reject-
ing Sherrington's second alternative was John Eccles, Sherrington's former stu-
dent and collaborator.145 Eccles was an ingenious and resourceful critic of
chemical transmission in ganglia, at neuromuscular junctions, and in the cen-
tral nervous system, although he accepted the principle of chemical transmis-
sion 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 inactiva-
tion 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 elec-
trical change in the postsynaptic cell that grew in a millisecond or so, account-
ing for the synaptic delay, and then disappeared within a few milliseconds, con-
sistent 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
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 pro-
longed responses to elecrical stimulation with higher concentrations of
physostigmine.150 On the other hand, Dale pointed out that high doses of acetyl-
choline could depress synaptic transmission, implying that too much physostig-
mine could interfere with cholinergic transmission as well as too little.151
Eccles in 1944, now returned to Australia, acknowledged that "the very pro-
longed 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 acetyl-
choline," 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 trans-
mission vigorously, drawing potential contours for model synapses that would
enable intercellular excitation, citing recent experiments demonstrating exci-
tation between experimentally apposed nerves, and accounting for unidirec-
tional electrical transmission at synapses by the local geometries.155
Concr
nclusions
Loewi and Dale shared the Nobel Prize in 1936 that rewarded their comple-
mentary achievements and careers. Loewi's scientific lineage stretched back to
Schmiedeberg, acclaimed as the "father of pharmacology," and to the nineteenth-
century titans of German physiology. Loewi progressed through German and
Austrian universities before a forced departure to England in 1938 and emi-
gration 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-
Chemical Transmission at Synapses (1895 — 1945) 79
macology comparable to Cajal's in neuroanatomy and Sherrington's in neuro-

physiology. Dale came from the Cambridge of Foster, Gaskell, Langley, Elliott,
Dixon, Lukas, and Adrian, but he abandoned academe for an industrial labo-
ratory 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 pub-
lished in 1921. Their general design, allegedly revealed in a dream, seem nowa-
days more trivially obvious than some subtle synthesis of protracted rumina-
tions, 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 ear-
lier, but his extraction procedure was too harsh for labile substances like acetyl-
choline; 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 hav-
ing low levels of cholinesterase activity and experimenting at colder tempera-
tures 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 trans-
mitting 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 alter-

natives, whereas Loewi met criticisms with reaffirmations, with a public demon-
stration in 1926 at the International Congress of Physiology in Stockholm, and
with improved experiments. Loewi also pursued the implications of his exper-
iments astutely. He showed that perfusates from stimulated hearts contained
a choline-like substance, that cholinesterase activity was present, that physostlg-
mine (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 transmis-
sion 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 gan-
glia and neuromuscular junctions. "From isolated facts of apparently moder-
ate significance there emerged general conclusions of high value."158 Acetyl-
choline was present, it was released by nerve stimulation, and its administration
mimicked the results of such nerve stimulation at synapses of postganglionic
parasympathetic neurons, of preganglionic autonomic neurons, and at neuro-

muscular junctions. Adrenaline (or some closely similar substance) was pres-
ent, was released, and mimicked stimulation of postganglionic sympathetic neu-
rons. 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: electri-
cal 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 pro-
tracted 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, depend-
ing 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 sup-
ply was offered to him (Fletcher, 1926).
13. Langley and Dickinson (1889); Langley (1893).
14. Langley (1893, 1898, 1905). Gaskell preferred "involuntary" to "autonomic."
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 dis-
appearance of adrenaline in the tissues. Here he refered to "the conjecture that [adren-
aline] 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 Lan-
gley (1901b), which compared the degree of responses to adrenaline vs. sympathetic
stimulation, and in Elliott (1905), which noted opposite effects, such as pupilary con-
striction 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 sub-
stances were neurofibrils.
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 blood-
borne 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 acetyl-
choline 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) pro-
posed 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 com-
pounds (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 extra-
cellular 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 irreg-
ularities as ... the frog's heart [preparation that Loewi used, with] grouped beats, spon-
taneous 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 sim-
ply 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
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 iden-
tified 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 search-
ing 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 dif-
ferent 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 concentra-
tions 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 contro-
versy concerning emotions and adrenal secretions (Barger, 1992).
83. Newton et al. (1931); Cannon and Bacq (1931).
84. Cannon and Rosenblueth (1933).
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 Feld-
berg 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 ingredi-
ent 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 elab-
orated 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).
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 neuromuscu-
lar 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). Cere-
brospinal 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 res-
piration.
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).
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 confi-
dence among the victors and to the euphoria of peace, in part to the imagina-
tive exploitation of technical capabilities developed for that conflict, and in part
to pent-up desires within the scientific community to resume interrupted inter-
ests.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 publi-
cized 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
87
for identifying metabolic interconversions. Analytical advances—notably

through chromatographic and electrophoretic separation methods and radioac-
tive tracer techniques—increased capabilities enormously and made measure-
ments 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 phos-
phorylation) and the trapping of liberated energy as an "energy-rich" com-
pound, 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 Bal-
timore showed that enzymes for the Krebs cycle and for oxidative phosphory-
lation 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 chemi-
cal embodiment of genetic information. Then in 1953 James Watson and Fran-
cis Crick in Cambridge proposed a double helical structure for DNA with far-
reaching 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 crys-
tallographic studies the three-dimensional structures of myoglobin, hemoglo-
bin, 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 cat-
alytic 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
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 phos-
phate, 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 asym-
metric 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 ear-
lier. First, impulses were conducted not in a decremental fashion but at a con-
stant, undiminished intensity in a self-propagating fashion. Second, impulses
were generated in an "all-or-nothing" manner: stimulus intensities below a crit-
ical 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 spec-
ifying 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:
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 cyto-
plasmic K + concentrations higher than those in the medium, the interior would
then be negative.) Since, according to Bernstein's formulation, the action poten-
tial reflected a transient loss of selective permeability, the membrane potential
should then fall toward 0 mV. The impulse then propagated by inducing cur-
rents, flowing in local circuits, that altered the permeability of the axon mem-
brane 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 approx-
imation involved measuring the "injury potential": one electrode was in the sur-
rounding 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 stimula-
tion 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 elec-
trode 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 convinc-
ingly.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 "Hodgkin-
Huxley 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 inter-
pretable 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
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 mem-
brane 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 trans-
membrane potential; impulse propagation reflected an action potential trig-
gering, through local circuits, the permeability change in adjacent regions of
the membrane.) The increased permeability to Na + then ceased and the per-
meability to K + increased, causing the downstroke of the action potential as
K + flowed down its electrochemical gradient out o/the cell. The action poten-
tial 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 cyto-
plasm 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 asym-
metric 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 con-
troversies 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 elec-
trodes, 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 Gra-
ham'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 mus-
cle 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<5—1965) 93
before relating their use in resolving the question of synaptic transmission in

the central nervous system, the earlier resolution of synaptic transmission at
neuromuscular junctions should be noted.
Transmission at Neuromuscular Junctions
In the nineteenth century Willy Kuhne described microscopically distinguish-

able regions on muscle surfaces where motor nerve fibers end in discrete, cir-
cumscribed areas. These regions became known as muscle "endplates," but
their detailed relationships could not be distinguished by light microscopy.21
In 1907 John Newport Langley localized the actions of curare and nicotine to
the region where nerve fibers terminated—and where the proposed reactive
substances would function.22 Then in the late 1930s Hans Schaefer and Her-
bert Gopfert in Bonn described characteristic electrical responses at the end-
plate region of frog muscles, evoked by stimulating the motor nerve: transient
depolarizations—decreases in the resting potential toward 0 mV.23 These they
measured in the presence of curare to reduce responses to nerve stimulation
(otherwise, induced muscle action potential would mask these depolarizations;
with higher concentrations of curare the depolarizations could be blocked
totally).
Through the 1940s and beyond John Eccles (Fig. 4-2), Bernard Katz (Fig.
4-3), and Stephen Kuffler developed such studies of "endplate potentials"
(e.p.p.s) to define functional properties and to reveal origins from neurotrans-
mitter actions, working first together in Sydney and then separately across the
globe. Eccles, after receiving his medical degree in Melbourne, went in 1925
as a Rhodes scholar to Oxford, where he was Sherrington's student and final
collaborator. After Sherrington's retirement, Eccles moved in 1937 to Sydney,
in 1944 to Otago, and in 1952 to Canberra. Katz received his medical degree
in 1934 in Leipzig, next studied with A. V. Hill in London, and then emigrated
to Sydney. Katz worked with Eccles from 1939 to 1942, when he joined the
Australian air force; Katz returned to London in 1946, where he remains. Kuf-
fler received his medical degree in 1937 in Vienna and left for Sydney in 1938,
moving to Chicago in 1945, Baltimore in 1947, and Boston in 1959.
Their joint paper of 1941 presented admirably the central properties of
e.p.p.s evoked by stimulating the motor nerves to frog and cat muscles: depo-
larizations were local and spread decrementally, their magnitude varied with-
out a threshold, and they could sum with another depolarization to form still
larger depolarizations.24 These characteristics thus differed sharply from those
of muscle action potentials, which propagated without loss of amplitude ("all-
or-nothing"). Eccles and associates also showed that when e.p.p.s exceeded a
critical threshold they elicited muscle action potentials: e.p.p.s served as elec-
trical stimuli to the adjacent muscle.
FIGURE 4-2. John C. Eccles (1903-1997; courtesy of the National Library of

Medicine).
The following year Kuffler described experiments with single frog muscle
fibers innervated by single nerve terminals. This preparation sharpened the
time resolution by reporting from a single endplate rather than a population
of them, and it also minimized distortions due to electrical shunting through
extraneous tissues.25 Kuffler added curare to the surrounding medium and
FIGURE 4-3. Bernard Katz (1911-; courtesy of Bernard

Katz).
could then record a progressive delay in the action potential's onset, eventu-
ally leaving only the e.p.p. when sufficient curare diffused into the region (Fig.
4—4A). Consequently, Kuffler could show individual endplate depolarizations
and the minimal amplitude sufficient to initiate action potentials in regions
adjacent to the endplate.
As noted in chapter 3, compelling evidence for the involvement of acetyl-
choline was accumulating at this time, including Fritz Buchtal and J. Lindhard's
demonstration in 1942 that endplate regions were 1000-fold more sensitive to
acetylcholine than the remaining muscle surface, observations confirmed and
extended by Kuffler in 1943.26 Nevertheless, Eccles resisted until 1948 the
notion that acetylcholine alone produced e.p.p.s. But that year he reported that
FIGURE 4-4. Muscle endplate potentials. A. Potentials at the endplate region of sin-
gle frog muscle fibers were measured with extracellular electrodes before adding curare
(bottom, showing the muscle action potential) and at successive times after adding curare
to the bathing medium, concluding with an endplate potential alone (top). Voltage is
displayed on the vertical axis (no calibration presented) and time on the horizontal axis.
B. Endplate potentials of frog muscles were measured with intracellular microelectrodes
in media containing low Na + concentrations (to prevent action potential formation) in
the absence (above) and the presence (below) of the cholinesterase inhibitor prostig-
mine. Voltage is displayed on the vertical axis and time on the horizontal. (A. from Kuf-
fler [1942], Fig. 5., courtesy of the American Physiological Society. B. from Fatt and
Katz [1951], Fig. 13, courtesy of the Physiological Society.)
both phases of the e.p.p.—the fast and slowly decaying components visible with
low levels of cholinesterase inhibitors such as physostigmine—"reacted simi-
larly to every test": adding curare or acetylcholine, changing the temperature,
and stimulating repetitively.27 Since he had previously acknowledged that
acetylcholine was responsible for the slow component, "this precise corre-
spondence indicates that the fast component is also due to acetylcholine."28

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

with intracellular microelectrodes, recorded (A) at the endplate region and (B) 2 mm
away (upper traces in each panel). The lower traces, at higher speed and lower ampli-
fication, show the response to nerve stimulation. Voltage is displayed on the vertical axis
and time on the horizontal. For the upper traces, the scale is 3.6 mV and 47 msec; for
the lower, 50 mV and 2 msec. (From Fatt and Katz [1952b], Fig. 1, courtesy of the
Physiological Society.)
domly in time.37 These m.e.p.p.s therefore seemed like spontaneous leaks of

neurotransmitter, but with a definite relationship to the evoked release of neu-
rotransmitter: Katz and del Castillo suggested that an e.p.p. was "built up sta-
tistically of small all-or-none units which are identical in size," so that m.e.p.p.s
"could be regarded as the least unit, or the 'quantum', of end-plate response."38
Nerve action potentials apparently increased by orders of magnitude the prob-
ability of such quanta being released.
Transmission in the Central Nervous System
Although Eccles conceded in the late 1940s that transmission at neuromuscu-

lar junctions was by chemical means, he remained convinced that transmission
in the central nervous system was electrical. One reason was the meager evi-
dence for acetylcholine functioning there: "it now seems probable that . . .
acetylcholine . . . plays no part whatsoever in central synaptic transmission."39
Another was his success in devising a scheme for inhibition at electrical
synapses, which had been an elusive goal.40 Eccles in 1947 depicted hypo-
thetical interneurons (which he termed "Golgi cells") shunting electric currents
into—and thereby depressing the excitability of—motoneurons (Fig. 4-6).
FIGURE 4-6. Model for inhibition at electrical synapses. A motoneuron (M) receives
synaptic contact from an excitatory neuron (E) and an inhibitory "Golgi" interneuron
(G), which is excited by another neuron (I). Excitation by I shunts electrical currents
through G to alter the excitability of M. (From Brooks and Eccles [1947], Fig. 1, ©
Macmillan Magazines Ltd., reprinted by permission.)
With the advent of intracellular microelectrodes, however, discriminating

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

+25 mV, like skeletal muscle and squid axons.
To examine synaptic potentials Eccles stimulated excitatory sensory nerves
(orthodromic stimulation) while he advanced the microelectrode. Just before
he impaled a motoneuron he could record, extracellularly, small potential
changes (Fig. 4-7A). But after impaling the motoneuron the potential changes
(1) were 20-fold larger, (2) had the opposite sign, and (3) had slower time
courses (Fig. 4-7B and C]). Moreover, presynaptic potentials recorded intra-
cellularly in the motoneuron (i.e., the initial deflections synchronous with the
stimulatory response) were no larger than presynaptic potentials recorded
extracellularly, so there was occurring "no special method of injecting current
across the post-synaptic membrane."43 Current flow from pre- to postsynaptic
FIGURE 4-7. Excitatory and inhibitory postsynaptic potentials. A. Potentials just out-
side the motoneuron were recorded extracellularly after a sensory nerve exciting the
motoneuron was stimulated. Voltage is displayed on the vertical axis and time on the
horizontal. (The breaks in the recording represent intervals of about 4 msec.) B. Poten-
tials in the motoneuron were recorded with an intracellular microelectrode after a sen-
sory nerve exciting the motoneuron was stimulated. In both B and C a downward deflec-
tion represents a depolarization. C. The trace in panel B is shown at lower amplification.
D. Potentials recorded intracellularly from a motoneuron are shown after stimulation
of a sensory nerve inhibiting the motoneuron (upper trace); the lower trace show poten-
tials recorded from the sensory nerve. Here and in E the more usual convention is fol-
lowed: a downward deflection represents a hyperpolarization. E. For comparison with
D, potentials are shown after stimulating a sensory nerve exciting the motoneuron (as
in panels B and C, but here with the conventional orientation). (From Brock et al.
[1952], Fig. 9A-C [A-C] and Fig. 12 C and D (D and E; courtesy of the Physiological
Society.)
cell was insufficient to produce the measured depolarizations in the postsy-

naptic cell. Eccles concluded: "The problem of [electrical] continuity versus
discontinuity at the synapse can now be regarded as decisively settled."44
When Eccles stimulated instead an inhibitory sensory input, the intracellu-
lar potential was hyperpolarized, that is, became more negative (Fig. 4—7D; for
comparison, the results of stimulating an excitatory sensory input were also
shown: Fig 4-7E). The observed hyperpolarization directly contradicted
Eccles's Golgi cell model for electrical inhibition (Fig. 4-5), which required
a depolarization of the motoneuron by currents shunted through the Golgi
cells. Eccles conceded that "experimental evidence has falsified the Golgi-cell
hypothesis . . . and left the chemical neurotransmitter hypothesis as the only
likely explanation."45
Eccles referred to the electrical changes as "post-synaptic potentials"
("p.s.p.s");46 these became subdivided into "excitatory postsynaptic potentials
("e.p.s.p.s") and "inhibitory postsynaptic potentials" ("i.p.s.p.s"). The graded
nature of e.p.s.p.s indicated how stimuli that were individually ineffective could
add to reach the threshold necessary for generating action potentials, and how
i.p.s.p.s. could sum algebraically with e.p.s.p.s to prevent action potentials. And
in 1963 Katz described miniature e.p.s.p.s from frog spinal motoneurons and
argued for the quantal release of neurotransmitters in the central nervous sys-
tem as well as at neuromuscular junctions.47
How chemical neurotransmitters could induce such depolarizations and
hyperpolarizations was less easy to study in the central nervous system than at
neuromuscular junctions, where the external environment can be manipulated
easily. Instead, Eccles altered the intracellular ion contents by injecting ions
through one barrel of a double-barreled micropipet (the other barrel he used
to measure electrical responses). Eccles concluded that e.p.s.p.s occurred like
e.p.p.s: through an increased permeability that short-circuited the resting
potential toward 0 mV.48 Defining ionic permeabilites to account for i.p.s.p.s
was less clear-cut, but Eccles argued for increased permeabilities to K + and/or
Cl~.49 In the meantime, evidence for inhibitory changes in ionic permeabili-
ties were reported at another site. Some invertebrate muscles are directly inner-
vated by both excitatory and inhibitory nerves (unlike mammalian skeletal mus-
cle, which is innervated solely by excitatory nerves), and in 1958 Fatt described
an increased permeability to Cl~ at crustacean neuromuscular junctions asso-
ciated with hyperpolarizing e.p.p.s.50
Soon after the identification of i.p.s.p.s, Eccles described interneurons in the
spinal cord whose neurotransmitter(s) hyperpolarized their target cells, pub-
lishing a model in 1954 (Fig. 4-8A) for the antidromic inhibition that Birdsey
Renshaw in New York had described in the 1940s.51 Stimulating ventral roots
can inhibit, for a brief interval, the firing of motoneurons whose axons run
through that root. Renshaw invoked two structures to account for this inhibi-
tion: branches of the stimulated axon that run back in the spinal cord ("recur-
rent collaterals") and interneurons stimulated by these collaterals. The causal
pathway thus lay from (1) antidromic stimulation of motoneuron axons in the
ventral root to (2) orthodromic spread along recurrent collaterals that (3) excite
interneurons that then (4) inhibit the motoneurons. The time course was appro-
priate for a two-neuron pathway, and Renshaw recorded discharges from in-
terneurons after antidromic stimulation of ventral roots, although he could not
connect interneuron firing to motoneuron inhibition. This Eccles did, using
intracellular microelectrodes to follow motoneuron potentials.52 He found that
stimulating motoneurons caused their own inhibition (hyperpolarization), and
he confirmed Renshaw's finding that action potentials in motoneuron axons
excite interneurons, presumably through recurrent collaterals.
Eccles named the interneurons "Renshaw cells" and showed, importantly, that
their activation was blocked by compounds such as dihydro-/3-erythroidine,
which block the effects of acetylcholine, whereas their activation was augmented
by cholinesterase inhibitors. By contrast, hyperpolarization of the motoneuron
was blocked by strychnine, a drug known to cause excessive motor discharges.
The major conclusions were (I) motoneurons release acetylcholine at neu-
romuscular junctions and, by their recurrent collaterals, on Renshaw cells, excit-
ing both; and (2) Renshaw cells inhibit motoneurons using an unknown inhib-
itory neurotransmitter whose actions are blocked by strychnine. Eccles stressed
that acetylcholine was released from all terminals of motoneurons, a manifes-
tation of what he termed "Dale's Principle": a neuron releases the same neu-
rotransmitter from all its terminals.53
A second invocation of interneurons concerned inhibition that was associ-
ated with the activation of sensors in muscle. For reciprocal innervation Sher-
rington had depicted sensory fibers from muscle exciting motoneurons to one
set of muscles while inhibiting motoneurons to the antagonistic set (Fig. 2-2).
In accord with this scheme, David Lloyd in New York argued for a "direct inhi-
bition," in which the sensory neuron acted without intervening interneurons
on the motoneuron.54 But in 1956 Eccles recalculated the latency between sen-
sory nerve activity and motoneuron inhibition and concluded that two synapses,
not one, intervened.55 Moreover, he found that stimulating sensory nerves acti-
vated certain interneurons (different from Renshaw cells). This scheme (Fig.
4-8B) differs from Sherrington's proposal not merely by including interneu-
rons but, more significantly, by restricting the influence of a neuron to either
excitation or inhibition at all its terminals.
But what is the excitatory neurotransmitter released by sensory neurons to
activate motoneurons and interneurons? Identifying that substance—as well as
the inhibitory substance released by the interneurons—remained a challenge
during the following decades (see chapter 5).
FIGURE 4-8. Proposed inhibitory circuits. A. Inhibition of a motoneuron (the large cell
at the bottom) occurs by means of a recurrent collateral, branching at a node from the
thick myelinated motoneuron axon, and exciting the small "Renshaw" interneuron,
which in turn inhibits the motoneuron. (Redrawn from Eccles et al. (1954), Fig. 18,
courtesy of the Physiological Society.) B. A sensory nerve from an extensor muscle sends
one branch to excite a motoneuron to that extensor muscle, while another branch excites
an inhibitory interneuron that in turn inhibits the motoneuron to the flexor muscle.
(From Eccles [1961], Fig. 1, courtesy of the Royal Society. This figure had been redrawn
Eccles et al. [1956], Fig. 13.)
Presynaptic Innimtion
Inhibition described in the preceding section was "postsynaptic": inhibitory

neurons hyperpolarized their target cells, hindering their activation by excita-
tory neurons. In 1957 Karl Frank in Bethesda described a different type of
inhibition, which he called "presynaptic."56 Frank found that stimulating cer-
tain inhibitory nerves alone did not inhibit a motoneuron but could reduce its
activation (i.e., reduce the magnitude of e.p.s.p.s in that motoneuron) evoked
through stimulating an excitatory nerve to that motoneuron. Thus, the inhibi-

tory nerve, when stimulated just before the excitatory nerve, diminished the
motoneurons response to that excitatory nerve.
Eccles confirmed these observations in 1961, describing decreases in evoked
e.p.s.p.s without changes in responsiveness of the motoneuron to electrical
FIGURE 4-9. Presynaptic inhibition in the spinal cord. A sensory nerve from an exten-
sor excites the motoneuron to that extensor (EM). Sensory nerves from a flexor and
from a flexor tendon excite interneurons (white) that excite a second-order interneu-
ron (black). This intemeuron makes synaptic contact with the sensory nerve terminal
from the extensor. The black neuron inhibits presynaptically by diminishing neuro-
transmitter release from the terminal it contacts. Pathways exciting and inhibiting the
motoneuron to the flexor (FM) are not shown. (From Eccles et al. [1962], Fig. 10, cour-
tesy of the Physiological Society.)
stimulation.5' These decreases in e.p.s.p.s he interpreted as due to inhibitory

neurons reducing neurotransmitter release from excitatory neurons. To explain
how one neuron could reduce neurotransmitter release by another, Eccles cor-
related the reductions with depolarizations of the presynaptic excitatory fibers,
arguing that depolarizing nerve terminals diminished their release of neuro-
transmitters.58 Although he could not demonstrate altered ionic permeabilities
to account for such presynaptic depolarization, he was able to show marked
differences between pre- and postsynaptic inhibition in the cat spinal cord:
strychnine blocked the latter, but picrotoxin blocked the former.59 Eccles also
incorporated presynaptic inhibition into a still more complex diagram of spinal
cord excitation and inhibition (Fig. 4-9).
At this time Kuffler demonstrated presynaptic inhibition at crayfish neuro-
muscular junctions.60 Stimulating inhibitory nerves not only reduced excitatory
e.p.p.s but also diminished the number of excitatory quanta: presynaptic inhi-
bition was associated with fewer quanta of excitatory neurotransmitter being
released.61 Moreover, administering y-aminobutyric acid (GABA), a known
inhibitory substance, reproduced these effects (see chapter 5).
Responses at Other Sites
Studies at three other sites were influential then and were developed subse-
quently.
1. Neurons of the stellate ganglion in squid form "giant synapses" with the
giant axons. These axo-axonal synapses are sufficiently large and accessi-
ble that microelectrodes can be advanced, guided visually (with a micro-
scope), through the giant axons and into the presynaptic terminals of the
stellate ganglion cells. In 1958 S. Hagiwara and Ichiji Tasaki in Woods
Hole described synaptic potentials in a postsynaptic giant axon when the
presynaptic stellate ganglion cells were stimulated.62 As they advanced
the microelectrode farther, the resting potential abruptly fell to zero and
synaptic potentials were replaced by far smaller deflections of the oppo-
site polarity. As they advanced it still farther, the resting potential reap-
peared; there they also recorded signals from stimulating the stellate gan-
glion cells. Hagiwara and Tasaki concluded that the electrode recorded
from three loci, and that "between the pre- and post-synaptic membrane
[there is] a small 'space' where the potential is close to that of the sur-
rounding sea water."63 Thus, they confirmed Eccles's 1952 interpretation
in a system where the entities could be seen with a microscope and where
potentials in each of the three compartments could be measured directly.
They also confirmed that current applied locally could not flow in signif-
icant amounts from one neuron to another.
2. Earlier, Alfred Fessard, working in Arachon, France on the electric ray

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

synapses with systems for liberating quanta of neurotransmitters from presy-
naptic elements. Anatomists had depicted terminals (or "boutons" or "endfeet")
but were unable, with light microscopes, to distinguish details. Electron micro-
scopes, with the potential for far greater resolution, were just beginning to be
marketed by Siemens in Germany and RCA in the United States when World
War II erupted; after the war some years were consumed in developing better
optics and better fixing, staining, embedding, and sectioning procedures. Then
several groups almost simultaneously published electron micrographs that cor-

related with physiological function, supplying entities for those processes.
In 1954 Sanford Palay (Fig. 4-10A), while visiting George Palade in New
York, described in an abstract the axo-dendritic synapses from rat cerebellum
and brainstem: between the plasma membranes surrounding presynaptic and
postsynaptic cells intervened a space approximately 200 A across.70 Not only
was there no continuity of cytoplasm between the cells, but a significant gap
separated them, a gap that came to be called the "synaptic cleft." A paper the
following year centered on the Nissl substance in neuronal cytoplasm, inter-
preted as endoplasmic reticulum with attached granules, a subject that Palade
was investigating vigorously in other cells.71 Not until 1956—after his return
to New Haven—did Palay describe fully the synaptic morphology, adding more
kinds of synapses at more locations in the central nervous system and includ-
ing a careful rationale for identifying synapses in the limited fields and thin
sections required by electron microscopy (Fig. 4-11 A).72
Meanwhile, Eduardo DeRobertis and Stanley Bennett in Seattle described
gaps of 100-150 A between presynaptic and postsynaptic cells of frog sympa-
thetic ganglia and earthworm nerve cord, again in an abstract in 1954; their
full paper appeared the following year.73 On the other hand, Robertson, then
in Cambridge, Mass., had noted in 1953 membranes "closely applied to one
FIGURE 4-10. A, left. Sanford L. Palay (1918-). B, right. Victor P. Whittaker (1919-).
(Courtesy of Sanford Palay and Victor Whittaker.)
FIGURE 4-11. Electron micrographs of synaptic junctions and of synaptosomes. A, on

the left. An axon terminal, containing mitochiondria "and numerous synaptic vesicles"
makes contact with a dendrite, with a synaptic cleft discernable between their cell mem-
branes. At x the "intrasynaptic space is enlarged . . . permitting . . . glial fibers to pass
between the terminal and the dendrite." B, on the right. An isolated synaptosome con-
tains a mitochondrion and numerous synaptic vesicles (sv). It is surrounded by a "thin
surface membrane" (tm). (A from Palay [1956], Fig. 2., reproduced by copyright per-
mission of the Rockefeller University Press. B from Gray and Whittaker [1962], Fig. 5,
courtesy of the Anatomical Society of Great Britain and Ireland.)
another" in squid synapses, but he felt that areas where the membranes seemed
to be separated were "probably ... a result of alterations associated with fixa-
tion and preparation."74 Robertson's further studies on membrane structure,
which culminated in his description of "unit membranes," clarified where the
edges of membranes lay among the stained images, and he then joined in advo-
cating synaptic clefts not only at neuronal synapses but also at neuromuscular
junctions.' 5
Eccles in 1957 remarked on the suitability of 200 A clefts: "There are two
conflicting requirements ... of the synaptic cleft: that it should be very narrow,
so that the . . . transmitter is applied . . . efficiently and . . . quickly ... to the
subsynaptic membrane; that it should be wide, so that the postsynaptic currents
flow as freely as possible."76 In 1964 he added that a fairly wide cleft would pro-
vide "a very effective shunt preventing electrical interaction between the presy-
naptic and postsynaptic components of a chemically transmitting synapse."7'
Palay identified neurofilaments in his micrographs, but while they were abun-
dant in dendrites and cell bodies and extended into axons, the neurofilaments
stopped short of the terminals.78 Neurofilaments seen by light microscopists
existed, but, contrary to arguments by Stefan Apathy, Albrecht Bethe, and their
successors (chapters 1 and 2), no continuity was found of neurofilaments across
synaptic clefts.
In 1954 Palay also reported a characteristic "agglomeration of mitochondria
and small vesicles (300-500 A)" in presynaptic terminals.79 Two years later he
described vesicles 200-650 A in diameter, some having a densely staining inte-
rior. He noted that these "vesicles may be considered as containing small units
of a chemical transmitter, like acetylcholine," and cited Fatt s recent review on
quantal release.80 In their 1954 abstract DeRobertis and Bennett also described
vesicles 200-500 A in diameter, naming them "synaptic vesicles" and suggest-
ing the following year that "[i]t is not unreasonable to speculate that active
compounds . . . might . . . be associated with . . . vesicles."81 DeRobertis, too,
promoted the notion that vesicles could represent quantal units of neuro-
transmitter, but his micrographs showed holes in the presynaptic membranes
and vesicles apparently in the cleft.82
Attempts to define mechanisms for the quantal release of neurotransmitter
from vesicles I will discuss in chapter 10, but here I will note initial experi-
ments showing that acetylcholine actually was present in synaptic vesicles.
These approaches continued earlier efforts at cell fractionation, begun suc-
cessfully by Albert Claude in New York and culminating in George Hogeboom
and Walter Schneiders four fractions obtained by centrifuging "homogenized"
tissues at successively greater speeds and longer times: (1) "nuclei and debris,"
(2) "mitochondria," (3) "microsomes," and (4) "supernatant."83 Indeed, a major
focus of Palade s research at this time was correlating biochemical and struc-
tural properties of these fractions with structures visible (by microscopy) in
cells and tissues.
Successful applications of such procedures to brain tissue came, however,
from England. In 1958 Victor Whittaker (Fig. 4-10B), who after biochemical
training in Oxford was now working with Catherine Hebb in Babraham, found
that most of the acetylcholine present in homogenates of rabbit and guinea pig
brains could be recovered, after such differential centrifugation, in the mito-
chondrial fraction.85 But when they separated that fraction further—by "dis-
continuous sucrose density gradient centrifugation," in which material is lay-
ered atop sucrose solutions of successively higher concentrations (thus of
increasing density from top to bottom) and then centrifuged—most of the
acetylcholine was in a fraction different from mitochondria.86
The following year Whittaker reported the presence of acetylcholine in a
similar subfraction, which he now identified as synaptic vesicles.8' Again, he
prepared a conventional mitochondrial fraction and then separated this by den-
sity gradient centrifugation, collecting material accumulating at the interface

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

Density Gradient Centrifugation0
RELATIVE SPECIFIC ACTIVITIES IN FRACTION
CHOLINE
ACETYL- CHOLINES- SUCCINIC
FRACTION ACETYLCHOLINE TRANSFERASE TERASE DEHYDROGENASE
O (no structures) 0.07 2.61 0.18 0.18

D (synaptic vesicles) 2.50 0.67 0.75 0.42
E (microsomes,
some synaptic
vesicles) 1.00 0.25 2.50 0.50
F (synaptosome
ghosts) 0.42 0.42 2.25 0.42
G (synaptosome
ghosts) 0.92 0.42 1.75 0.50
H (damaged
synaptosomes) 2.33 0.27 0.67 1.47
I (mitochondria,
shrunken
synaptosomes) 0.69 0.23 0.61 3.77
"The crude mitochondria! fraction prepared by differential centrifugation of brain homogenates

in 0.32 M sucrose was lysed osmotically by adding water. This material was then placed in cen-
trifuge tubes atop a series of five sucrose concentrations and centrifuged. Fraction O was mate-
rial remaining at the top; D, at the interface with 0.4 M sucrose; E, at the interface between 0.4
and 0.6 M; F, between 0.6 and 0.8 M; G, between 0.8 and 1.0 M; H, between 1.0 and 1.2 M; and
I, the pellet at the bottom of the centrifuge tube below 1.2 M sucrose. Fractions were character-
ized by electron microscopy and by measuring the relative specific activities of markers. Choline
acetyltransferase is the enzyme synthesizing acetylcholine, cholinesterase the enzyme hydrolyzing
it, and succinic dehydrogenase is found solely in mitochondria. Microsomes are small vesicles
formed during homogenization from the endoplasmic reticulum, an interlacing series of tubules
within cells. (From Whittaker et al. [1964], Tables 1 and 3. © the Biochemical Society; reproduced
by permission.)
Identifying Electrical Transmission
The preceding studies established chemical transmission convincingly at both

central and peripheral synapses. To explore further the relationships between
currents in presynaptic terminals and neurotransmitter release, Edwin Fursh-
pan and David Potter, visiting in Katz's laboratory, then examined synapses in
crayfish nerve cords. There "lateral giant fibers" (presynaptic) and "giant motor
fibers" (postsynaptic) are each large enough to impale with microelectrodes.
To Furshpan and Potters surprise, conduction at these synapses was, as they
reported in 1957, electrical yet unidirectional.98 Synaptic delays were quite
short, perhaps 0.1 millisecond. Current passed in a graded, decremental fash-

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

transmission energetically and then demonstrating chemical transmission deci-
sively. A skillful experimenter and imaginative interpreter—with significant
accomplishments beyond those cited here—Eccles was justly rewarded with a
Nobel Prize in 1963. (Others discussed in this chapter also so honored include
Hodgkin, Huxley, Katz, and Palade.) Nevertheless, Eccles was not chosen to
succeed Sherrington at Oxford. Instead, he returned to Australia in 1937 to
head a small research institute in the pathology department of a Sydney hos-
pital. Kuffler arrived as a pathologist, and Eccles diverted him to research. With
Katz they persevered admirably, although Eccles remembered them as "three

. . . huddled together in an alien world."103 The war and a change in hospital
administration made research impossible. With no academic positions available
in Australia, Eccles ventured alone to New Zealand, beginning at the Otago
medical school in 1944. Despite a consuming teaching load he attracted new
collaborators, notably L. G. Brock and Jack Coombs, and together they refuted
his model of electrical conduction. In 1951 Eccles returned to Australia again,
now to Canberra and the Australian National University, then being founded
as a graduate research institution. There, visited by dozens of scientists from
across the globe, he did what he considered his best work.
Eccles, like Sherrington, was a confirmed dualist and spent his final decades
pondering the mind-brain enigma.104 But the pervasive philosophical view
guiding his research was, by his account, Karl Popper's.105 Eccles learned of,
met, and was won over by Popper in 1944, when both were (temporarily) in
New Zealand. Eccles interpreted Popper's views as the methodological imper-
ative "to formulate clear hypotheses and then test them by rigorous experi-
ment."106 Accordingly, Eccles viewed his microelectrode study of postsynaptic
inhibition as "a clear test," since his electrical hypothesis predicted depolar-
ization and the chemical hypothesis hyperpolarization.107 That test, however,
refuted his hypothesis: "The result was repeatable, graded with stimulus
strength, and indubitable."108 (Perhaps Eccles considered Popper's views also
as consolation; Zenon Bacq quotes Eccles: "I can now rejoice in the falsifica-
tion [refutation] of a cherished theory, because even this is a scientific suc-
cess."109 Bacq also observed that "Eccles had changed mounts, the rider was
as tempestuous as ever, but the horse was much better."110 And in fairness
to Dale, Feldberg, et al., it is important to remember that there were good
reasons for believing synaptic transmission was chemical before Eccles's
experiments.)
Of course, admonitions about formulating hypotheses clearly and testing
them rigorously are routine scientific advice independent of Popper's writing,
even if this advice is not universally heeded. But Popper's interpretation of sci-
ence as conjecture and refutation accompanied his adamant rejection of induc-
tive inference. This distinctive aspect of Popper's teaching Eccles did not adopt
in practice. Eccles accepted chemical transmission at neuromuscular junctions
not after refuting electrical hypotheses but after assessing the positive evidence
in favor of chemical hypotheses. Instances of corroborative weight hailed
by the scientific community also included independent but mutual support
between physiological and anatomical studies: discovering synaptic clefts with
appropriate magnitudes and identifying synaptic vesicles suitable for storing
neurotransmitter quanta.
Scientific research is opportunistic not only in seizing available techniques
and ideas to make old problems soluble, but also in choosing to corroborate
one theory rather than refute another. When experiments to refute either
hypothesis for neuromuscular transmission were not apparent, Eccles instead
sought evidence favoring one. Definitive experiments can sometimes be
planned, as in Eccles's application of microelectrodes, even if the results are
unanticipated. Sometimes rewarding results appear by chance, as in Katz and
Fatt's discovery of m.e.p.p.s.
Moreover, initial generalizations derived from observations and corrobora-
tions are, at least in such biological research, inevitably modified. Intrinsic com-
plexities ensure that with closer scrutiny new entities and interactions will
appear. And biological diversity—reflecting random mutations and contingent
selections—makes exceptions likely. Generalizations are tied to specified
domains, as in recognizing that some neurons communicate electrically through
gap junctions while others communicate chemically across synaptic clefts. (Fur-
ther qualifications may then be required, specifying how particular instances
diverge from a prototypic manifestation.) But useful, instructive generalizations
can result nonetheless.
During these two decades new experimental techniques, notably those
exploiting intracellular microelectrodes and electron microscopes, did extend
the principle of chemical transmission from the autonomic nervous system to
neuromuscular junctions and the central nervous system. New general phe-
nomena were recognized, such as presynaptic inhibition, and new circuits for
reciprocal control were delineated. New details were added, such as the quan-
tal release of neurotransmitters packaged in synaptic vesicles and the identi-
ties of ionic currents responsible for membrane voltage changes. Still, many
questions remained unanswered, including the identities of the neurotrans-
mitters for sensory excitation and for central inhibition.
Notes
1. For general accounts, see Fruton (1999); Judson (1979); Morange (1998); Robin-
son (1997).
2. See Kornberg (1997).
3. For example, fluxes occurring during steady states—when there are no net
changes—cannot be measured by chemical analyses but can be followed as tracer fluxes
using radioactive isotopes added initially to one of the several compartments exchang-
ing materials.
4. For historical accounts, see Clarke and O'Malley (1968); Hodgkin (1992); Robin-
son (1997); Tasaki (1959).
5. Action potentials were identical for a given fiber and experimental conditions, such
as temperature, ionic milieu, etc. Stimuli below threshold values produced electrical
changes that were conducted decrementally.
6. The equilibrium distribution may be considered the result of K + , high in con-
centration within the cell, diffusing outward in the absence of accompanying negative
charges or of an exchange for positive charges diffusing inward. The resulting electri-
cal potential, representing the loss of positive charges within the cell, will oppose the
concentration gradient favoring a net outward diffusion. The Nernst equation states the
equilibrium potential and concentration ratio for such a system.
7. Young described the axons at a meeting attended by neurophysiologists (Young,
1936).
8. Cole and Curtis (1939); Cole and Hodgkin (1939). The experiments, performed
the previous summer, involved measuring transverse impedance to alternating current.
9. Hodgkin and Huxley (1939).
10. The calculation requires knowing the concentration of intracellular K + , which
was reported that year (Bear and Schmitt, 1939).
11. Hodgkin and Huxley (1945); their 1939 note did not attempt an explanation of
the overshoot.
12. Hodgkin and Huxley (1952) and preceding papers. These studies relied crucially
on an enabling new technique, the voltage clamp method (see Robinson, 1997).
13. The equilibrium potential across a membrane is that at which no net movement
of the ion occurs. Above this potential net movement of the ion occurs in one direc-
tion, below it in the opposite direction.
14. The term channel was introduced by Hodgkin and Keynes (1955); pore was a
common designation earlier.
15. Keynes and Lewis (1951) and preceding papers.
16. See Robinson (1997).
17. For historical accounts, see Frank (1986); Marshall (1987); Robinson (1997).
18. Graham and Gerard (1946). This represented the completion of work interrupted
by the war.
19. Ling and Gerard (1949).
20. Nastuk and Hodgkin (1950).
21. Even in the 1930s some argued about the continuity of such regions with mus-
cle cytoplasm: see Eccles and O'Connor (1939).
22. Langley (1907).
23. Gopfert and Schaefer (1938).
24. Eccles et al. (1941). These experiments, too, were made in the presence of curare.
Pertinent earlier work includes Gopfert and Schaefer (1938); Eccles and O'Connor
(1939); Feng (1940). Eccles (1977) stated that he was initially unaware of the work by
Gopfert and Schaefer and by Feng.
25. Kuffler (1942). With external electrodes at the endplates, e.p.p.s could be mea-
sured even without curare (Fig. 4-4E).
26. Buchtal and Lindhard (1942); Kuffler (1943).
27. Eccles (1948), p. 103.
28. Ibid., p. 104. At this time Kuffler announced that Eccles now believed that "the
electrical hypothesis cannot be reconciled with more recent experimental results" and
that his "evidence favors [acetylcholine] as the sole mechanism" (Kuffler, 1948, p. 445).
29. Eccles and MacFarlane (1949).
30. Katz (1942).
31. Fatt and Katz (1951).
32. Ibid., p. 362. Grundfest (1957b) emphasized the characteristic electrical inex-
citability.
33. del Castillo and Katz (1954b), p. 564.
34. Takeuchi and Takeuchi (1960).
35. Fatt and Katz (1952b).

36. Boyd and Martin (1956); Liley (1956).
37. Fatt and Katz (1952b). See also del Castillo and Katz (1954b).
39. Brooks and Eccles (1947), p. 760.
40. Ibid. Earlier attempts at explaining inhibition included schemes invoking the
axonal refractory period, a brief interval immediately following passage of an action
potential when that axon cannot again be excited (see Adrian, 1924).
41. Brock et al. (1952).
42. The cell body must be connected directly since, according to Cajal and Sher-
rington, antidromic impulses cannot cross synapses.
43. Brock etal. (1952), p. 455.
44. Ibid. p. 452.
45. Ibid. p. 455.
46. Eccles (1953), p. 130.
47. Katz and Miledi (1963). Earlier, Eccles had noticed random low-amplitude elec-
trical activity but attributed it to distant synaptic activity (Brock et al., 1952). After Fatt
and Katz's initial description, quantal release of neurotransmitters was then described
at several other sites, including autonomic ganglia (Nishi and Koketsu, 1960).
48. Coombs et al. (1955b).
49. Coombs et al. (1955a); Eccles et al. (1964).
50. Boistel and Fatt (1958).
51. Renshaw (1946) and preceding papers.
52. Eccles et al. (1954).
53. Ibid. See also Eccles (1976).
54. Laporte and Lloyd (1952). Eccles had considered such inhibition to be monosy-
naptic earlier (Bradley et al., 1953).
56. Frank and Fuortes (1957). The effect is still across a synapse, but the immedi-
ate target is the presynaptic terminal of the second neuron. Frank (1959) later termed
the phenomenon "remote inhibition," including an alternative mechanism.
58. Eccles inferred the depolarization from the greater excitability of these presy-
naptic fibers that he also observed. In addition, he cited Hagiwara and Tasakis demon-
stration (1958) that neurotransmitter release declined sharply as the presynaptic ter-
minal was depolarized.
59. Eccles (1964), Fig. 96.
60. Dudel and Kuffler (1961).
61. Similarly, Kuno (1964) described a decrease in the probability of neurotransmit-
ter release associated with presynaptic inhibition of spinal motoneurons.
62. Hagiwara and Tasaki (1958).
63. Ibid., p. 124.
64. Auger and Fessard (1938).
65. Feldberg and Fessard (1942). They also cited Elliott's attempt to extract the chem-
ical neurotransmitter at this site.
66. Altamirano et al. (1955a).
67. Keynes and Martins-Ferreira (1953).
68. Chagas et al. (1951); Altamirano et al. (1955b).
69. Nachmansohn (1961).
70. Palade and Palay (1954); Palay and Palade (1954). The second of these is chiefly
concerned with the Nissl substance.
71. Palay and Palade (1955). These granules were identified subsequently as ribo-
somes, the sites of protein synthesis.
72. Palay (1956). Palay argued from the prominence of mitochondria in presynaptic
terminals, visible by light microscopy, to the identification of nerve terminals by the
large numbers of mitochondria visible in electron micrographs. Recognizing synaptic
vesicles then added another identifying characteristic for presynaptic terminals.
Although electron microscopy showed small areas and great complexity (e.g., Fig.
4-10A), such comparisons established common identities.
73. DeRobertis and Bennett (1954, 1955).
74. Robertson (1963), pp. 221-222.
75. Robertson (1956, 1960). Birks et al. (1960) provided further detailed views of
neuromuscular junctions. Palade and Palay (1954) mentioned neuromuscular junctions,
but their account of an intervening space is vague.
76. Eccles (1957), p. 217.
77. Eccles (1964), p. 28.
78. Palay (1958).
79. Palade and Palay (1954), p. 355. Robertson (1956) saw circular profiles that he
interpreted as tubes cut in cross section; however, Palay (1956) argued that circular pro-
files were apparent after sectioning at assorted angles and thus the images represented
spheres not cylinders.
80. Palay (1956), p. 199.
81. DeRobertis and Bennett (1954); (1955), p. 55.
82. DeRobertis and Bennett (1955); DeRobertis (1956, 1958).
83. Hogeboom et al. (1948); Hogeboom (1955). The starting material was a
homogenate, prepared by grinding tissue with a cylindrical pestle turned inside a close-
fitting tube. This homogenate was then placed in plastic centrifuge tubes and spun at
successively higher speeds and longer times to obtain the characteristic four fractions.
(Homogenates originated as biochemists' preparations to provide a uniform stock from
which identical samples could be tested and analyzed.)
85. Hebb and Whittaker (1958). They modified Hogeboom and Schneider's scheme
somewhat, including the homogenizing of brain tissue in 0.32 M sucrose.
86. Mitochondria were recognized biochemically by the presence of an enzymatic
"marker," succinic dehydrogenase, an enzyme previously shown to be present solely in
mitochondria.
87. Whittaker (1959).
88. Whittaker measured the nitrogen content, which parallels the protein content
closely.
89. Whittaker (1959). p. 698.
90. Gray and Whittaker (1960).
91. Ibid., p. 35P.
92. Ibid. In contrast to earlier proposals that neurons were linked by conducting
fibers, this connection of pre- and postsynaptic elements is by extracellular materials
that do not participate in impulse conduction.
93. Gray and Whittaker (1962). In retrospect, it is obvious that small synaptic vesi-
cles should not have penetrated so far into the sucrose gradient as did the nerve ter-
minals.
94. Whittaker et al. (1964).
95. DeRobertis et al. (1961).

96. DeRobertis et al. (1963).
97. Whittaker et al. (1964) noted differences in distribution of markers, lower rela-
tive specific activities of acetylcholine, and claimed that DeRobertis s fixation technique
for electron microscopy caused "extensive disruption of the more organized structures
. . . leaving clumps of synaptic vesicles and membrane fragments" (p. 301).
98. Furshpan and Potter (1957, 1959). For a historical account, see Hagland and Col-
lett (1996b).
99. Furshpan (1964).
100. Robertson (1963).
101. Revel and Karnovsky (1967)
102. See Goodenough and Revel (1970); Makowski et al. (1977).
103. Eccles (1977), p. 5.
104. Eccles also expressed unorthodox views about "mind" influencing synaptic trans-
mission (see Robinson, 1987).
105. Eccles (1976). Popper's seminal book of 1934 was not translated into English
until much later (Popper, 1959).
106. Eccles (1976), p. 225.
107. Ibid.
108. Ibid.
109. Bacq (1975), p. 65. See also Eccles (1970), pp. 104-106.
110. Ibid.
5
IDENTIFYING
NEUROTRANSMITTERS
(1946-1976)
Scope and. Criteria
During the postwar decades the discovery of new neurotransmitters burgeoned

unexpectedly into an expanding enterprise. Earlier studies on the autonomic
nervous system had provided models for neural control through antagonistic
systems using two neurotransmitters, acetylcholine and adrenaline (chapter 3).
Each could be either excitatory or inhibitory, depending on the tissue, and two
neurotransmitters should be sufficient for such accelerator/brake control. But
despite this theoretical sufficiency, further experiments on the central nervous
system revealed that neither could fill prominent excitatory and inhibitory roles
(chapter 4).
If other neurotransmitters participated, what were they—and how could they
be identified? The criteria were obvious. The candidate neurotransmitter
should be present in the presynaptic terminal, be released when the presy-
naptic terminal was active, and, when applied experimentally, induce faithfully
the responses in the postsynaptic neuron.1 In practice, since central nervous
system neurons continuously integrate diverse excitations and inhibitions, the
last criterion was relaxed to demonstrating merely changes in such activity.
Not surprisingly, satisfying these criteria in the central nervous system was
frustrated by inherent complexities and impeded by imperfect techniques lack-
119
ing adequate precision and sensitivity. Nevertheless, efforts applied diligently

and imaginatively over the decades implicated substances in unanticipated
numbers: dozens upon dozens of likely neurotransmitters. Here I will illustrate
these quests by describing briefly some diverse courses by which nine putative
neurotransmitters gained credibility.
(Neurotransmitters are sometimes distinguished from "neuromodulators,"
the latter having a slower onset and longer duration of action and/or an indi-
rect effect through modifying responses to other agents.2 Here, however, neu-
rotransmitter is used inclusively for substances released by a neuron to affect
nearby cells acutely.)
Acetylcholine
By 1945 acetylcholine (Fig. 3-6A) was established as the excitatory neuro-

transmitter at neuromuscular junctions and autonomic ganglia and as the exci-
tatory or inhibitory neurotransmitter of postganglionic parasympathetic fibers.
Defining its role in the central nervous system was far less straightforward,
although satisfying the first criterion, demonstrating its presence, was soon
achieved. Not only was acetylcholine present in brain and spinal cord, it was
present in synaptic vesicles within nerve terminals (chapter 4). These analyses
were made by bioassays, however, and to skeptical chemists such identifica-
tions lacked—however close the correlations with authentic acetylcholine
across multiple systems—the assurance of molecular analyses. Measuring
acetylcholine chemically was a daunting challenge since it is intrinsically labile
(splitting to acetate and choline), exists among enzymes that catalyze this
hydrolysis, and.is present in minute quantities.3 Finally, in 1968 Bo Holmstedt
in Stockholm and Donald Jenden in Los Angeles collaborated to demonstrate
the molecule in mammalian brain, combining gas chromatographic methods
for separation and mass spectrometry for identification and quantitation.4
Alternative choline esters, if present, existed at far lower concentrations. The
following year Jack Green in New York reported the chemical identification of
acetylcholine released into the bath after he stimulated parasympathetic fibers
to isolated intestine.0
Still more difficult was correlating acetylcholine release with impulse trans-
mission across specific synapses in the brain. For autonomic ganglia and neu-
romuscular junctions, acetylcholine release into the surrounding bath or the
venous effluent could be matched to stimulation of the presynaptic fibers. For
the brain, however, release from one pathway might be commingled with, and
thus masked by, release from multitudes of other pathways if the total release
into bath or venous outflow were examined. More localized collections were
required to distinguish particular releases from the general background release.
Identifying Neurotransmitters (1946-1976) 121
This James Mitchell in Babraham achieved in the 1960s by catching released

acetylcholine in small cups on the cortical surface, formed by pressing hollow
plastic cylinders against the brain surface.6 Mitchell found that the spontaneous
release was "roughly proportional to the electrical activity of the brain"; more-
over, when he placed the cups over the sensory cortex, an increased release of
acetylcholine followed stimulation of peripheral sensory nerves or of sensory
pathways in the brain.7 By contrast, when he removed the cortex and placed
the cups on the underlying white matter, he could detect no acetylcholine
release.
In subsequent studies Mitchell found that acetylcholine release from the
visual cortex increased severalfold when the eyes were exposed to light or the
visual pathway was stimulated electrically.8 Severing the neural pathways elim-
inated light-evoked release. He also measured release into cups implanted in
conscious, unrestrained animals that correlated with their activity.9
Demonstrating release beneath the surface obviously required different
approaches. In 1961 John Gaddum (Fig. 5-1A), then also in Babraham,
described "push-pull" cannulas for perfusing deeper regions of the brain. Gad-
dum contributed broadly to neuropharmacology, just as he appears in numer-
ous chapters here. Now he inserted parallel hollow needles into brain tissue
FIGURE 5-1. A, left, John Henry Gaddum (1900-1965). B, right, Ulf S. von Euler
(1905-1983). (A courtesy of Wellcome Trust Medical Photography Library. B courtesy
of Leo von Euler.)
and then forced perfusion fluid through one, collecting it from the other.10 In
1964 Hugh McLennan in Vancouver applied this method to show acetylcholine
release within the caudate nucleus (a cellular region deep within the brain
known for its high acetylcholine content; see chapter 13); furthermore, acetyl-
choline release increased after he stimulated certain pathways to this nucleus.11
The previous year Mitchell had used push-pull cannulas to show that acetyl-
choline was released within the cortex but not from the white matter immedi-
ately beneath.12 But more discrete localizations were not possible by such tech-
niques.
The third criterion, mimicry of neurally evoked responses, was satisfied ear-
lier.13 In 1958 David Curtis and Rosamond Eccles in Canberra applied Bernard
Katz and Jose del Castillo's "microelectrophoretic" approach to spinal cord Ren-
shaw cells.14 Curtis and Eccles used five-barreled micropipets; the central pipet
was filled with concentrated NaCl to record electrical potentials, and the other
four contained acetylcholine and various analogs, acetylcholine antagonists, and
cholinesterase inhibitors (Fig. 5-2A). Each of these charged molecules could
be released electrophoretically by passing an electric current through its par-
ticular barrel.15 Since Renshaw cells are excited by recurrent collaterals of ven-
tral horn motoneurons (chapter 4), Curtis and Eccles identified these cells by
stimulating the ventral roots. Impulses passed antidromically back toward
motoneurons and then ortfoodromically along branching recurrent collaterals
to excite, across the synapse, a Renshaw cell. So they advanced the five-
barreled micropipet into the ventral horn until they recorded action potentials
correlating with this stimulation. At that point they could release, electro-
phoretically, each of the substances in the other four barrels near this Renshaw
FIGURE 5-2. Electrodes for electrical recording and for microelectrophoretic applica-
tion. A. Five barreled glass microelectrodes allowed one barrel to be used for measur-
ing electrical responses extracellularly and the surrounding barrels to release four test
substances extracellularly. Each barrel was about a micron in diameter. B. Concentric
glass microelectrodes contained an inner barrel for penetrating the cell, to permit trans-
membrane electrical measurements, and an outer barrel for releasing a test substance
extracellularly.
cell while continuing to monitor its activity. As John Eccles—with less precise
localization—concluded earlier, acetylcholine excited Renshaw cells. Curtis and
Eccles now showed that nicotine also excited, whereas the nicotinic antago-
nists curare and dihydro-/3-erythroidine blocked. Cholinesterase inhibitors
potentiated responses to acetylcholine.
With five-barreled micropipets both release of reagents and measurements
of potential were extracellular. To measure transmembrane potentials Curtis
used concentric pipets: the inner pipet penetrated to the cell interior, while
the outer released reagents extracellularly (Fig. 5-2B).
Shortly thereafter, Kresimir Krnjevic in Babraham applied these techniques
to neurons of the cerebral cortex.16 Acetylcholine excited only 15% of the thou-
sands of cortical neurons examined, although it excited 60% to 90% of the Betz
cells (large neurons of the motor cortex). By contrast, glutamate (see below)
excited all the cortical cells. And instead of the nicotinic responses of Renshaw
cells, the responses of cortical neurons to acetylcholine were muscarinic:
atropine blocked, but not curare or dihydro-/3-erythroidine. Moreover, responses
to acetylcholine were slow in onset and prolonged after administration ceased,
time courses reminiscent of muscarinic postganglionic parasympathetic
responses.
Others soon reported similar results at other sites in the brain, finding mus-
carinic responses predominantly.17 But these experiments reaffirmed the con-
clusion that acetylcholine played a far less pervasive role in central neuro-
transmission than it did in the periphery.
Noraarenaline
By 1945 adrenaline (Fig. 3-3A), too, seemed established as a neurotransmit-

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

acid, may be synthesized from another conventional amino acid, phenylalanine, A, by
hydroxylation of its benzene ring. Hydroxylation of tyrosine forms dopa, C, a third amino
acid but one not found in proteins (unlike phenylalanine and tyrosine). Decarboxyla-
tion of dopa forms dopamine, D, which, like the succeeding products, is a catecholamine.
(Catechol consists of a benzene ring with two adjacent hydroxyls.) Hydroxylation of
dopamine on its sidechain forms noradrenaline, E. Methylation of noradrenaline s amine
group forms adrenaline, F.
noradrenaline by comparing responses from a broad range of bioassays, which

he then supplemented with discriminating chemical analyses: the noradrena-
line content of sympathetic nerves was 50-fold that of adrenaline.20 Others soon
confirmed this reinterpretation.21 (In the meantime, P. Holtz in Rostock
detected noradrenaline in mammalian adrenals, although at far lower levels
than adrenaline.22) Dale, looking back in 1953 on his 1914 paper showing a
close parallel between sympathetic stimulation and added noradrenaline,
acknowledged: "I ought to have seen that noradrenaline might be the main
transmitter—that Elliott's theory might be right in principle and faulty only in
this detail."23
On the other hand, von Euler found adrenaline instead of noradrenaline in
frog hearts.24 Otto Loewi s identification in 1936 was correct for the species he
studied.
In 1946 von Euler also described noradrenaline s presence in brain extracts,
but he concluded that the low levels he found arose from postganglionic sym-
pathetic fibers accompanying blood vessels in the brain.25 Marthe Vogt, now
in Edinburgh, disagreed. In 1954 she reported that noradrenaline levels in
brain tissue were higher in some regions (notably the hypothalamus) than in
others, but there was no correlation between this uneven density and blood
vessel distribution.26 Moreover, after Vogt cut the sympathetic nerves to the
brain blood vessels and allowed the terminals to degenerate, she found no
change in the noradrenaline content of the hypothalamus.
Detailed localizations within the brain became possible with the develop-
ment of microscopic techniques for identifying catecholamine-containing neu-
rons. In the early 1960s Bengt Falck in Lund and Nils-Ake Hillarp in Gote-
borg found that treating fixed tissue sections with formaldehyde vapor produced
yellow-green fluorescent products from noradrenaline and dopamine.27 This
histochemical technique was then widely employed to define pathways using
noradrenaline, investigations of immense significance in understanding the
functional organization of the nervous system but lying beyond the bounds of
this history. It is, however, important to add that discriminating between nor-
adrenaline- and dopamine-containing neurons was initially based on responses
to pharmacological agents, but in 1968 Falck devised a microspectrofluoro-
metric method for distinguishing between these catecholamines that confirmed
earlier assignments.28
Unlike acetylcholine, noradrenaline is not rapidly destroyed in the brain, and
its metabolic products do not mix appreciably with general cellular metabo-
lites. Consequently, it is possible to add isotopically labeled noradrenaline and
follow it by means of this radioactivity. 3H-noradrenaline was used from the
early 1960s for such purposes, including both light and electron microscopic
investigations of cellular and subcellular distribution.29 Radioactive labeling also
facilitated demonstrations that noradrenaline, like acetylcholine, was present
in brain synaptosomes.30
Documenting noradrenaline release from sympathetic fibers followed soon
after von Euler's identification.31 Measuring noradrenaline release from the
central nervous system was technically more difficult, but by the mid-1960s
that, too, was achieved.32
Curtis s initial microelectrophoretic studies on the spinal cord failed to iden-
tify neurons responding to noradrenaline.33 Nevertheless, analogous studies by
others then cataloged and mapped neuronal responsiveness to noradrenaline
throughout the brain.34
Dopamine
Suggestions that dopamine (Fig. 3-3C) might serve as a neurotransmitter

emerged from the recognition that it was present in amounts greater than would
be expected for a mere metabolic precursor to noradrenaline. Blaschko showed
in 1956 that labeled dopa gave rise to labeled dopamine and noradrenaline, in
accord with the sequential conversion: dopa —» dopamine —» noradrenaline
(Fig. 5-3).35 But Blaschko, noting that some nerves contained as much
dopamine as noradrenaline, proposed the next year that "dopamine has some
regulatory functions of its own."36 In 1958 Arvid Carlsson in Lund found
dopamine levels in brain equivalent to noradrenaline levels; he, too, proposed
that such amounts "may indicate that the function of [dopamine] is not merely
that of a precursor."37
A. Bertler and E. Rosengren in Lund and I. Sano and associates in Osaka
then provided strong support for this proposal: they described in 1959 distri-
butions of dopamine in the brain that differed from those of noradrenaline.38
Combining chemical analyses with fluorescence microscopic techniques,
Swedish investigators then mapped dopamine-containing neurons in the brain
quite distinct from noradrenaline-containing ones.39 Concurrently, Victor Whit-
taker in Babraham identified dopamine in brain synaptosomal fractions.40
These mappings revealed prominent concentrations of dopamine in the cau-
date nucleus of the basal ganglia (see chapter 13). At the same time that McLen-
nan described the stimulated release of acetylcholine in the caudate nucleus
using push-pull cannulas, he also reported dopamine release that increased
when he stimulated a different pathway to the caudate.41 Floyd Bloom in Wash-
ington then showed that administering dopamine microelectrophoretically
inhibited certain caudate nucleus neurons.42
These experiments identified particular neuronal pathways containing
dopamine as their catecholamine, with the dopamine present in nerve endings
and released during stimulation. Administered dopamine affected the activity
of other neurons. But the enormous interest in dopaminergic systems that
developed subsequently represented further interests: changes in dopamine
levels accompanied certain diseases, and drugs effective in treating certain dis-
eases altered dopamine metabolism, transport, and receptors (chapter 13).
Serotonin
Scattered among the mucosal cells lining the gastrointestinal tract are "ente-
rochromaffin cells," named for their location plus their characteristic staining
by chromium salts. Vittorio Erspamer, first in Pavia and then Rome, began in
the 1930s efforts to identify the substances within these cells that were respon-
sible for the staining (as well as for the characteristic fluorescence and chem-
ical reactions).43 By 1940 he had prepared extracts that reproduced the fluo-
rescence and color reactions; in addition, these extracts caused various smooth
muscles to contract, notably the rat uterus and small intestine. From its
gastrointestinal origin and amine content, Erspamer named the substance
"enteramine," although he soon identified it in such disparate loci as mam-

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

tive serotonin allowed its subsequent localization by electron microscopy,54 as
with radioactive noradrenaline. Moreover, the fluorescence microscopic tech-
niques that formed yellow-green products with catecholamines formed yellow
products with serotonin.55 Mappings by this method demonstrated that
serotonin-containing cell bodies were localized in the midbrain, separate from
catecholamine-containing cell bodies. Again, these methods provided detailed
demonstrations of neuronal pathways and relationships essential to under-
standing brain function.
In the early 1960s Whittaker reported serotonin's presence in synaptosomal
fractions from the brain, like acetylcholine.56 Independently, Eduardo De-
Robertis also described serotonin-containing synaptosomes prepared by

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

sues, Eugene Roberts in St. Louis noticed a prominent spot on paper chro-
matograms of normal brain tissue. This he identified in 1950 as y-aminobutyric
acid (GABA; Fig. 5-4C).60 Roberts could detect GABA only in the central nerv-
ous system, where it was formed by enzymatic decarboxylation of glutamate
(Fig. 5-4D), one of the conventional a-amino acids that make up proteins;
GABA was then converted enzymatically to succinate, an intermediate in the
Krebs cycle for cellular metabolism. Since glutamate could be formed from
another intermediate in this cycle, Roberts interpreted the pathway forming
and degrading GABA as a metabolic "shunt," bypassing steps in the Krebs
cycle.61
Ernst Florey approached quite differently, from studying substances pres-
ent in the brain that elicited inhibitory responses.62 Favorite test systems for
inhibition were crustacean synapses, where inhibitory nerves, readily identifi-
able and accessible, participate.63 In 1956 Florey, then in Montreal, identified
GABA as the active substance in his "Factor I," an extract from beef brain that
inhibited crustacean synapses.64 Others soon joined in studying GABA as a pos-
sible inhibitory neurotransmitter. In 1958 Paul Fatt in London, while examin-
ing changeable ionic permeabilities at crustacean neuromuscular junctions
(chapter 4), showed that GABA affected ionic conductances in postsynaptic
neurons, as did stimulating inhibitory nerves.65 And Stephen Kuffler in Balti-
more found that the electrical responses of crustacean synapses were similar
when evoked by adding GABA or by stimulating inhibitory nerves.66
Nevertheless, Florey was having second thoughts about his identification. In
the mid-1950s Florey began collaborating with McLennan, then also in Mon-
treal; together they showed that Factor I inhibited various mammalian processes,
including synaptic transmission in autonomic ganglia.67 But after Florey identi-
fied Factor I as GABA, McLennan pointed to discrepancies—activities that Fac-
tor I possessed but GABA lacked—and reported that his preparation of Factor
I did not contain GABA.68 Florey agreed with the implicit assumption that Fac-
tor I should contain a single inhibitory substance displaying the entire range of
inhibitory responses, and in a review published in 1961 Florey declared that
"GABA is not the inhibitory neurotransmitter although it imitates the transmit-
ter action in many crustacean preparations."69
Florey also failed to find GABA in crustacean nerves, but Edward Kravitz
and David Potter did.70 The following year, 1963, they, together with Kuffler,
now also in Boston, reported that inhibitory but not excitatory nerves contained
GABA.71 They pointed out that Florey's argument against GABA being the
inhibitory substance could be countered by acknowledging that additional
inhibitory substances might be present in tissue extracts: the amount of GABA
present in extracts need not equal the total inhibitory capacity of the extract;
GABA might represent only part of the inhibitory capacity while being the
physiologically active substance. And in 1966 Kravitz found that stimulating
inhibitory nerves released GABA in amounts proportional to that stimulation;
by contrast, stimulating excitatory nerves released no GABA.72 The previous
year A. and N. Takeuchi in Tokyo described microelectrophoretic administra-
tion of GABA onto crustacean muscles: GABA evoked inhibitory electrical
responses when applied to neuromuscular junctions, but not elsewhere on mus-
cle surfaces or when injected into muscles.73 Thus, GABA was present specif-
ically in inhibitory nerves, it was released specifically from them, and its local-
ized application to neuromuscular junctions mimicked nerve stimulation.
Florey was won back to his original view.74
Building a comparable case for GABA in vertebrate nervous systems was
more protracted. GABA, as Roberts initially demonstrated, was present in the
central nervous system. Moreover, its uneven distribution there could reflect
diverse functions, as uneven distributions of established neurotransmitters were
so interpreted.75 But when Roberts examined the subcellular distribution in
1963, he found that most of the GABA was "free" in the supernatant fraction.
(The largest fraction of "bound" GABA, however, was associated with synap-
tosomes.76) At this time Roberts was still focusing on metabolic roles for GABA,
so this distribution was not crucial. Others interested in GABA as a neuro-
transmitter, including Whittaker, also found that most of the GABA was free,
but in 1971 Leslie Iversen in Cambridge argued persuasively that in vivo GABA
was largely confined to vesicles but was artifactually released during cell frac-
tionation.77
In the spinal cord, where Charles Sherrington and John Eccles had estab-
lished central inhibition decisively (chapters 2 and 4), GABA did not at first
satisfy expectations. In 1959 Curtis found that microelectrophoretically applied
GABA blocked excitation in the spinal cord but did not hyperpolarize the neu-
rons; moreover, strychnine, which both Sherrington and Eccles used to block
inhibition, did not alter GABAs action.78 Curtis concluded that GABA pro-
duced a nonspecific "depressant" effect, rather than the true inhibition ex-
pected of a neurotransmitter.
Studies on the brain were more encouraging. In 1963 Krnjevic reported that
applying GABA microelectrophoretically to the cerebral cortex inhibited both
spontaneous activity (Fig. 5-5A) and excitation induced by applied glutamate.79
Krnjevic subsequently found that applied GABA hyperpolarized cortical neu-
rons, producing i.p.s.p.s just as did stimulating inhibitory pathways. Moreover,
responses to applied GABA or to stimulation were similarly sensitive to injected
Cl~, indicating that similar changes in ionic permeability underlay each.80 Par-
ticularly persuasive were studies on the cerebellum, where earlier research
established Purkinje cells in the cerebellar cortex as prominent inhibitors of
neurons in subcortical nuclei and brainstem. In 1967 K. Obata in Tokyo dem-
onstrated that microelectrophoretically applied GABA hyperpolarized these
neurons.81 Obata also showed that Purkinje cells contained GABA and that
stimulating the cerebellum increased GABA release into the ventricle several-
fold.82 Mitchell and Iversen then collected GABA from cups placed on the
cerebral cortex: electrical stimulation of inhibitory pathways increased GABA
release here as well.83
FIGURE 5-5. Electrical responses to microelectrophoretic application of neurotrans-

mitters. A. Applying GABA extracellularly blocked spontaneous activity. Electrical activ-
ity of cerebellar neurons is represented by the vertical traces displayed against time
(marked in seconds below the trace), measured by one barrel of the electrode. When
a 60 nA current was passed through the barrel containing GABA (horizontal line beneath
the time trace), discharging GABA electrophoretically, the spontaneous activity ceased.
When the application of GABA stopped, the spontaneous activity resumed. B. Apply-
ing the L-stereoisomer of glutamate (L-Glut) by a 60 nA current through one barrel of
the electrode produced electrical activity in cortical neurons (vertical excursions,
recorded through a separate barrel of the micropipet) that began shortly after gluta-
mate was released and ceased abruptly when release was halted. Applying the D-stere-
isomer (D-Glut) produced no response. (A and B from Krnjevic and Phillis [1963a],
Figs. 17 and 4, respectively, courtesy of the Physiological Society.)
By 1968 Curtis realized that his multibarrel electrodes could damage neu-
rons, and he redesigned their configuration. He now found that applied GABA
indeed hyperpolarized spinal motoneurons.84 Strychnine, however, still did not
block the effects of GABA. On the other hand, earlier studies by Florey and
others demonstrated that picrotoxin, another well known convulsant derived
from plants, blocked inhibition at crustacean synapses as well as the effects of
Factor I and of GABA.85 Curtis surveyed an array of convulsants for a more
convenient reagent, and in 1970 he reported that bicuculline, another plant
product, blocked GABA's effects specifically.86 Bicuculline also blocked inhibi-
tion occurring at certain sites in the brain and spinal cord, but not at sites sen-
sitive to strychnine. There another inhibitory neurotransmitter must act.
Glutamate
With evidence accumulating that GABA served as an inhibitory neurotrans-

mitter in crustaceans, Jay Bobbins in New York screened dozens of other amino
acids for effects on crustacean synapses. In 1958 he reported that—alone among
these—glutamate (Fig. 5-4D) and aspartate excited crustacean muscles.87 Glu-
tamate was more potent, and Robbins suggested that it might be the neuro-
transmitter of excitatory nerves, opposing GABA released by inhibitory nerves.
Independently, Anthonie Van Harreveld in Pasadena identified glutamate as
the active substance in rabbit brain extracts that excited crustacean muscles.88
And in 1964 the Takeuchis used microelectrophoretic application to localize
glutamate s action to crustacean neuromuscular junctions.89 Glutamate excited
even after denervation, indicating direct effects on the endplate rather than
through the nerve. (Due to technical obstacles, glutamate release from excita-
tory nerves was not demonstrated until 1981.90)
Meanwhile, Curtis was curious about structural and metabolic relationships
between GABA and glutamate, and he found that microelectrophoretically
applied glutamate excited spinal cord neurons.91 But Curtis judged glutamate
not to be a neurotransmitter, in part because he considered antagonism between
applied glutamate and applied GABA as competition for a common receptor
(and he then considered GABA not to be a neurotransmitter) and in part
because he found the D- and L-stereoisomers of glutamate to be equally effec-
tive.92 Perhaps he also shared the contemporary skepticism about ordinary
amino acids serving as neurotransmitters, for in 1960 the likely neurotrans-
mitters (those considered above) were all specialized derivatives.
Nevertheless, even though glutamate as a conventional amino acid was a
component of all cells, its distribution in the nervous system was uneven, sug-
gesting a functional localization.93 Of particular note were glutamate concen-
trations in dorsal root ganglia and regions of the spinal cord where sensory dor-
sal root neurons terminate.94 This localization suggested a role in sensory limbs
of spinal cord reflex arcs, arcs where acetylcholine served in motor limbs.
Initial investigations of glutamate s subcellular distribution revealed—as with
GABA—that the greatest amount was free in the soluble fraction.95 In 1971,
however, Solomon Snyder in Baltimore found a portion of the neuronal gluta-
mate concentrated in synaptosomal fractions, and two years later H. F. Brad-
ford in London described a vesicular fraction containing glutamate.96
Demonstrating a stimulated release of glutamate was hindered by the high
background of "nonspecific" release, reflecting glutamate's multiple physiolog-
ical roles. In 1969, however, Herbert Jasper in Montreal described glutamate
release into fluid flowing over the brain surface that increased after electrical
stimulation.97 In 1972 P. J. Roberts and Mitchell, now in Bristol, reported a
stimulated release from the spinal cord and the following year a stimulated
release into cups on the visual cortex.98
Unlike Curtis, who found both stereoisomers of glutamate equally effective
on the spinal cord, Krnjevic showed in 1963 that the naturally occurring L-
stereoisomer was more effective on cortical neurons (Fig. 5-5B).99 Krnjevic
also found that glutamate excited spinal cord cells in regions where sensory
dorsal root neurons terminate.100 By 1972 Curtis agreed that glutamate was a
likely neurotransmitter in spinal cord and brain,101 exciting a far larger fraction
of neurons than any other putative neurotransmitter then known. But the delin-
eation of glutamatergic systems was hindered during this period by the lack of
specific blocking agents to assist in identifying such synapses.
Glycine
Glycine (Fig. 5-4B), too, is an ordinary a-amino acid present in all cells, mak-
ing its candidacy as a neurotransmitter "easy to deride."102 But in 1965 Mor-
ris Aprison in Indianapolis pointed out that glycine, measured during surveys
of amino acid content, was concentrated in the spinal cord gray matter, where
inhibitory interneurons and their terminals are localized.103 Two years later
Aprison furthered his proposal by describing sharp decreases in glycine con-
tent after destroying the spinal cord interneurons.104 Demonstrating glycine's
presence in synaptic vesicles, however, was not achieved during this period,
although Snyder provided circumstantial evidence for glycine's association with
synaptosomal fractions.105
Measuring a stimulated release was also challenging. In 1971 R. A. Webster
in London described an outflow of labeled glycine into fluid perfusing the cat
spinal cord, and this outflow increased when he stimulated peripheral nerves
electrically (he first loaded the spinal cord with labeled glycine by perfusing
with 14C-glycine.106) The following year Roberts and Mitchell, in experiments
paralleling their study on glutamate release, also reported an increased outflow

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

rotransmitters, including many previously identified as gastrointestinal hor-
mones (such as secretin). Here I will note the course to recognizing two novel
peptides.
Substance P
Working in Dale's institute in Hampstead, von Euler and Gaddum sought to

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

inite entity with actions distinguishable by certain bioassays. Again, uneven dis-
tributions—as reported by Gaddum, now in Edinburgh, Bengt Pernow in
Stockholm, and F. Lembeck in Graz—suggested specific functional roles in
brain and spinal cord.114 A notable concentration in dorsal roots and dorsal
horns of the spinal cord suggested that substance P participated in sensory
transmission. Moreover, in the early 1960s substance P was localized to synap-
tosomal fractions.115 Although Gaddum failed to find release from the spinal
cord using push-pull cannulas, in 1968 Jane Shaw and P. W. Ramwell in Shrews-
bury, Mass., reported that stimulatory drugs increased the release of substance
P into cortical cups.116 And although Krnjevic found only weak responses of
spinal cord neurons to microelectrophoretically applied substance P prepara-
tions, H. Caspars and P. Stern in Miinster described in 1961 an enhanced elec-
trical activity in the cortex after topical administration.117
Chemical identification, which permitted experiments using the pure sub-
stance and measurements of it, resolved and extended old issues. In the 1960s
Susan Leeman in Waltham began studying brain extracts that promoted sali-
vary secretion. This culminated in 1970 when she (1) isolated an active 11-
amino acid peptide of specified composition and (2) showed its pharmacolog-
ical identity to substance P.118 The next year Leeman and colleagues reported
the sequence of 11 amino acids and the chemical synthesis of this peptide.119
Antibodies to the pure peptide could now be used in highly sensitive analyti-
cal procedures to confirm the distribution of substance P in the brain and spinal
cord, again emphasizing a localization in dorsal root ganglion cells as well as
distinct regions of the brain.120 Masonori Otsuka in Tokyo then demonstrated
a stimulated release of substance P specifically121 And application of the pure
substance, by perfusion or microelectrophoresis, excited cortical neurons and
spinal cord ventral horn motoneurons.122 Whereas glutamate evoked rapid
responses of brief duration in the motoneurons, substance P caused more pro-
longed responses. Subsequent studies identified substance P as a major neu-
rotransmitter in sensory neurons mediating pain (so P stands not only for pow-
der and peptide, but also pain).
Enkepnalins
Opium, the dried exudate from seed capsules of the poppy, has been used to
relieve pain and induce euphoria for millennia. Early in the nineteenth cen-
tury morphine (Fig. 5-4F) and codeine were isolated from opium, and by mid-
twentieth century synthetic opioids123 were developed as well as specific antag-
onists. Precise structural requirements for morphine and its analogs to produce
analgesia and euphoria (and for antagonists to block the responses) suggested
that these compounds interacted with specific receptors in the body. In 1973
three groups identified such receptors in the brain (chapter 6). The existence
of specific receptors for exogenous opioids then suggested that endogenous
opioids might exist. So several investigators, including Lars Terenius in Upsala,
Avram Goldstein in Palo Alto, and Snyder, set about searching for substances
in brain extracts that bound to their receptor preparations and whose binding
could be displaced by known opioids and opioid antagonists.124
The initial success, however, was achieved through a traditional route. Hans
Kosterlitz had initially studied sugar metabolism and diabetes in his native Ger-
many and continued this research after fleeing to Aberdeen in 1934.125 After
the war he turned his attention to how the autonomic nervous system affects
digestion; this endeavor included examining morphine's inhibition of peristal-
sis,126 demonstrable on the isolated guinea pig ileum. On retiring in 1973
at age 70, Kosterlitz founded the Unit for Research on Addictive Drugs in
Aberdeen, where he and a young associate, John Hughes, exploited an
extremely sensitive and selective bioassay for opioids, the mouse vas deferens.
Electrical stimulation of sympathetic nerves to the vas caused it to contract;
morphine blocked this response, whereas opioid antagonists, such as naloxone,
prevented morphine's action.127 (Hughes and Kosterlitz also demonstrated that
morphine acted by preventing noradrenaline release from the excitatory nerve.)
Hughes and Kosterlitz then showed that pig brain extracts contained mate-
rial that blocked contractions of both the vas deferens and ileum; naloxone
antagonized the extracts' effects in both bioassays.128 The active substance,
named "enkephalin," had a molecular weight of roughly 1000, and its potency
was destroyed by enzymes cleaving peptide bonds. In 1975 they identified two
active peptides in the extract (and synthesized them): met-enkephalin (Fig.
5-4E) and leu-enkephalin; each contained five amino acids, differing in only
one of these.129 The following year Snyder reported that calf brain extracts also
contained met- and leu-enkephalin, whose purification he followed by assess-
ing binding to opioid receptor preparations.130 Both described uneven distri-
butions of enkephalins in the brain, extending beyond known pathways for pain
perception and implying that enkephalins played broader functional roles.131
In 1976 Snyder also reported that enkephalins were localized in synaptosomal
fractions.132
Demonstrating enkephlin release was technically difficult, in part due to free
enkephalins rapid destruction by endogenous peptidases. Within five years of
enkephalins discovery, however, descriptions of its release appeared.133
If exogenous opioids produce analgesia by acting on specific receptors in the
brain, then endogenous opioids should also be able to relieve pain. Indeed,
injecting enkephalins into brain ventricles produced analgesia.134 Reports also
published in 1976 described microelectrophoretic application of enkephalins
that altered the electrical activities of particular neurons in certain brain regions,
effects blocked by naloxone.135 (Although the structures of met-enkephalin
and morphine [Figs. 5-4E and F] seem at first glance dissimilar, molecular
models showed that strong resemblances in three-dimensional configurations
exist.136)
Meanwhile, Goldstein in 1975 found larger peptides in the pituitary that had
opioid activity both in bioassays and on receptor preparations.137 Within a
decade two further families of endogenous opioid peptides were characterized
in addition to enkephalins: endorphins and dynorphins, ranging from 16 to 31
amino acids.138
Concl
onciusions
After decades of concentration on acetylcholine and adrenaline/noradrenaline,

new studies shifted the focus. To the surprise of many, glutamate turned out
to be the major excitatory neurotransmitter in the brain as well as the sensory
neurotransmitter of dorsal root ganglion cells. And GAB A turned out to be
the major inhibitory neurotransmitter in the brain, with glycine a prominent
inhibitory neurotransmitter in the spinal cord (notably as the neurotransmitter
of Renshaw cells). Furthermore, considerations of pathologies and therapeu-
tics fostered an interest in many of the more newly established neurotrans-
mitters, notably dopamine, serotonin, GABA, and enkephalin.
In all cases, however, the pathways to validating these nine substances as
authentic neurotransmitters began from diverse personal interests that then
converged on commonly recognized steps: demonstrating presence, release,
and activity (mimicking physiological responses and pharmacological sensitivi-
ties to antagonists). Nevertheless, different candidate neurotransmitters posed
different conceptual and technical challenges, and viewing these quests as
stereotyped sequences of experiments would undervalue the skill and ingenu-
ity demanded. Instead, successes reflected directed efforts to develop new
strategies and modify old ones, although the cast here is so large that individ-
ual contributions blur together. Moreover, the successes represent communal
accomplishments. Some scientists concentrated on a few techniques applica-
ble to several neurotransmitters, while others examined only a few neuro-
transmitters but these extensively. Together, their findings corrected earlier
errors through adapting alternative approaches and extending efforts to further
issues and concerns.
These studies also illustrate the common perils of oversimplification and
overgeneralization. Thus, some investigators sought the inhibitory neurotrans-
mitter and so discarded evidence for an inhibitory neurotransmitter when the
candidate failed certain specifics. But an opposite extreme, assuming a new
entity for each phenomenon, would also lead to error. The effective course
required a reluctance to advocate new neurotransmitters and a tentative accept-
ance only as the weight of evidence accumulated. This evidence included not
only satisfying the criteria stated above but also displaying further attributes
recognized later. Important among these were identifications of receptors spe-
cific for the candidate neurotransmitter, receptors that, when occupied by these
substances or their close structural analogs, evoked demonstrable biochemical
and cellular responses (chapters 6 and 7).
During these decades evidence for and against other substances that might
serve as neurotransmitters also accumulated, but space does not allow discus-
sion of some later accepted, such as adenosine, and others that remained in
limbo, such as taurine. But it is important to recognize that the list of likely
neurotransmitters continued to grow throughout the period covered in this
book.
Notes
1. For a contemporary discussion, see Werman (1966). Werman also stated other cri-
teria that seemed obvious, too, such as the presence of enzymes for neurotransmitter
synthesis and degradation. The applicability of these to all neurotransmitters turned out,
nonetheless, to be limited (chapter 9).
2. See Cooper et al. (1996), pp. 4-5.
3. Using tracer techniques, such as radioactive labeling, is frustrated by the rapid
breakdown, with the products then being incorporated into multitudes of other cellu-
lar constituents.
4. Hammar et al. (1968).
5. Schmidt et al. (1969). They, too, used a gas chromatographic approach.
6. Mitchell (1963). Physostigmine was present in the Ringers solution in the cups;
acetylcholine was measured by bioassays. Acetylcholine concentrations in blood and
cerebrospinal fluid were lower, so the released acetylcholine could not simply have
leaked from these sources. This approach was first described in an abstract in 1953 by
Macintosh and Oborin in Montreal, but they did not pursue it.
7. Ibid., p. 114.
8. Collier and Mitchell (1966).
9. Collier and Mitchell (1967).
10. Gaddum (1961).
11. McLennan (1964).
12. Mitchell (1963).
13. Exact mimicry would be unlikely. For example, as McLennan (1970) pointed out,
much of the endogenous neurotransmitter is released onto the branched dendrites,
whereas acetylcholine was administered in such experiments onto the cell body, close
to the recording electrode.
14. del Castillo and Katz (1955a); Curtis and Eccles (1958a, b). These studies were
extended in Curtis et al. (1961). Rosamond Eccles is John Eccles's daughter.
15. The number of ions released is proportional to the amount of current passed
through the pipet. To prevent leakage from the pipets before release is desired, an
opposing "back current" is applied.
16. Krnjevic and Phillis (1961, 1962, 1963a, 1963b, 1963c).
17. For example, Spehmann and Kapp (1961); Spehmann (1963); Crawford and Cur-
tis (1966); McLennan and York (1966).
18. Blaschko (1942).
19. von Euler (1946). Von Euler also included chemical assays, but these could not
discriminate between noradrenaline and adrenaline when the quantities of each were
unknown (e.g., noradrenaline was stated to fluoresce more weakly than adrenaline).
20. von Euler (1948, 1949); von Euler and Hamberg (1949).
21. Bacq and Fischer (1947); Gaddum and Goodwin (1947); Holtz et al. (1947).
22. Holtz and Schumann (1948).
23. Dale (1965), p. 98 (a reprint of the 1953 edition). In 1914, however, noradrena-
line was not known to exist in mammalian tissues.
24. von Euler (1946).
25. Ibid. At that time sympathetic nerves were known to accompany blood vessels,
where they were importantly involved in maintaining blood pressure through activating
smooth muscles that controlled vessel diameter.
26. Vogt (1954).
27. Carlsson et al. (1962); Falck (1962); Falck et al. (1962). With adrenaline the flu-
orescence had a slower onset and was thus distinguishable.
28. For the pharmacological procedures, see Corrodi and Jonsson (1967). Bjorklund
et al. (1968) described treatment with hydrochloric acid that shifts the excitation spec-
tra between dopamine and noradrenaline, thereby making them distinguishable.
29. Samorajski and Marks (1962); Wolfe et al. (1962). In both these studies animals
were injected with 3H-noradrenaline and tissue sections then exposed to photographic
emulsions: the radioactive noradrenaline caused a deposition of silver grains in these emul-
sions that could be correlated with the cytological images from light or electron microscopy.
30. For example, Potter and Axelrod (1963); Glowinski et al. (1966).
31. For example, Peart (1949); West (1950).
32. For example, Anden et al. (1965); Baldessarini et al. (1967). Alternatively,
decreases in tissue content were reported (for example, Gunne and Reis, 1963).
33. Curtis et al. (1961).
34. For example, Bloom et al. (1963); Bradley and Wolstencroft (1962); Krnjevic and
Phillis (1963d).
35. Demis et al. (1956). They incubated adrenal medullary homogenates with 14C-
dopa and recovered 14C-dopamine and 14C-noradrenaline.
36. Blaschko (1957), p. 11.
37. Carlsson et al. (1958), p. 471.
38. Bertler and Rosengren (1959); Sano et al. (1959).
39. For example, Anden et al. (1964b, 1965); Hillarp et al. (1966).
40. Laverty et al. (1963). They found, however, considerable amounts of "free"
dopamine, that is, dopamine in the supernatant fraction.
41. McLennan (1964). He noted that dopamine "is not normally regarded as a likely
transmitter substance" (pp. 152-153).
42. Bloom et al. (1965). Dopamine excited a smaller number of caudate nucleus neu-
rons. See also McLennan and York (1967), who, citing recent reports, now considered
dopamine a likely neurotransmitter.
43. Vialli and Erspamer (1933); Erspamer (1940a, 1940b, 1940c).
44. Erspamer and Asero (1952), p. 801. Erspamer noted that far lower concentra-
tions of enteramine were required to affect the kidney than to raise blood pressure;
moreover, vessels leading to the glomeruli (the site of filtration where urine formation
begins) were most sensitive (Erspamer, 1953).
45. For autobiographical accounts, see Page (1976); Rapport (1987, 1997).
46. Blood consists of various blood cells plus liquid "plasma." After blood coagulates,
the clot contains trapped cells plus the clotting proteins. The remaining liquid is "serum,"
which is plasma minus the clotting proteins (it also contains substances liberated from
the cells during coagulation, such as serotonin).
47. Rapport (1948a, 1948b).
48. Rapport (1949).
49. Hamlin and Fischer (1951) at Abbott Laboratories; Speeter et al. (1951) at the
Upjohn Co.
50. Erspamer and Asero (1952).
51. Twarog and Page (1953).
52. Amin et al. (1954).
53. Bogdanski et al. (1957). The easy assay by fluorescence spectroscopy greatly facil-
itated further research.
54. Aghajanian and Bloom (1967).
55. Dahlstrom and Fuxe (1964); Anden et al. (1966). Procedures to distinguish sero-
tonin from catecholamines included pharmacological approaches and microspectroflu-
orescence methods (see Corrodi and Jonsson, 1967; Bjorklund et al., 1970).
56. Michaelson and Whittaker (1962, 1963).
57. DeRobertis et al. (1962); Zieher and DeRobertis (1963). DeRobertis's separation
of cholinergic from serotonergic synaptosomes was, however, controversial.
58. For example, Anden et al. (1964a); Beleslin and Myers (1970). Sometimes the
disappearance of serotonin and appearance of its metabolite was interpreted as release:
for example, Aghajanian et al. (1967).
59. For example, Bloom et al. (1963); Curtis et al. (1961); Krnjevic and Phillis (1963a).
60. Roberts and Frankel (1950). Awapara et al. (1950) independently identified GABA
in chromatograms of the brain.
61. Roberts (1956). The GABA shunt depicted a-ketoglutarate converted to gluta-
mate, which was then decarboxylated to GABA; GABA was next oxidized to succinate,
so the GABA shunt bypassed the steps in the Krebs cycle between a-ketoglutarate and
succinate.
62. Florey (1954).
63. Chief among these crustacean systems are neuromuscular junctions, where sep-
arate excitatory and inhibitory nerves innervate muscle, and stretch receptors (sense
organs in muscle), on which inhibitory nerves end.
64. Bazemore et al. (1956, 1957).
65. Boistel and Fatt (1958). Hagiwara et al. (1960) confirmed these observations.
66. Kuffler and Edwards (1958). Others studying these issues included Grundfest et
al. (1959) and Van der Kloot and Robbins (1959).
67. Florey and McLennan (1955).
68. McLennan (1957, 1958).
69. Florey (1961), p. 513.
70. Florey and Chapman (1961); Kravitz et al. (1962).
71. Kravitz et al. (1963). Their analyses were facilitated by using a highly sensitive
and specific enzymatic assay for GABA.
72. Otsuka et al. (1966).
74. Koidl and Florey (1975).
75. Baxter and Roberts (1959). See also Fahn and Cote (1968).
76. Weinstein et al. (1963).
77. Mangan and Whittaker (1966); Neal and Iversen (1969).

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

110. See Belcher et al. (1976).
111. Obata et al. (1970).
112. von Euler and Gaddum (1931).
113. Chang and Gaddum (1933); von Euler (1936).
114. Amin et al. (1954); Pernow (1953); Lembeck (1953).
115. Kataoka (1962); Cleugh et al. (1964).
116. Gaddum and Szerb (1961); Shaw and Ramwell (1968).
117. Galindo et al. (1967); Caspars and Stern (1961).
118. Chang and Leeman (1970).
119. Chang et al. (1971); Tregear et al. (1971).
120. Takahashi et al. (1974); Hokfelt et al. (1975).
121. Otsuka and Konishi (1976).
122. Konishi and Otsuka (1974); Phillis and Limacher (1974); Henry (1976).
123. Opioid refers to all substances with morphinelike properties, replacing terms
like opiate and narcotic.
124. Terenius and Wahlstrom (1975); Teschemacher et al. (1975); Pasternack et ai.
(1975).
125. For autobiographical reminiscences, see Kosterlitz (1979).
126. Opioids have been used to combat diarrhea also.
127. Henderson et al. (1972).
128. Hughes (1975); Hughes et al. (1975b).
129. Hughes et al. (1975a). One of the peptides contains leucine (leu), where the
other has methionine (met).
130. Simantov and Snyder (1976).
131. Hughes et al. (1975a); Simantov and Snyder (1976). With pure met-enkephalin
available, antibodies could then be prepared for use in immunohistological localization
(for example, Hokfelt et al., 1977).
132. Simantov et al. (1976).
133. For example, Henderson et al. (1978); Iversen et al. (1978); Glowinski (1981).
134. For example, Belluzzi et al. (1976); Biischer et al. (1976). Earlier, Liebeskind
found that electrical stimulation of certain brain regions produced analgesia, consistent
with a stimulated release of neurotransmitters in a pathway relieving pain (Mayer et al.,
1971).
135. For example, Frederickson and Norris (1976); Hill et al. (1976).
136. For example, Smith and Griffin (1978).
137. Teschemacher et al. (1975).
138. Frederickson (1984).
6
CHARACTERIZING
RECEPTORS
(1905-1983)
Essential Issues
John Newport Langley in 1905 introduced the notion that nerve activity, exci-
tatory drugs, and their antagonists all elicited cellular responses through "recep-
tive substances," hypothetical structures that soon became known by the shorter
title receptors (chapter 3).1 This and the following two chapters describe
attempts to answer essential questions raised by Langley s proposal: what, chem-
ically, are these receptors; how do neurotransmitters and drugs interact with
them; and how do such interactions trigger the cellular responses? These inves-
tigations also inspired various schemes for classifying receptor families, reveal-
ing generalities about evolutionary relationships and illustrating mechanistic
similarities—despite the multitudes of entities cataloged over the decades.
Moreover, demonstrations that certain neuronal constituents interacted with
specific receptors complemented the identifications of neurotransmitters that
were proceeding by other criteria (chapter 5).
Drugj—Receptor Interactions
One approach to discovering how receptors function lay in examining the

ways cells responded to various agents that (presumably) acted through these
receptors.
143
Interpreting Dose—Response Plots
Among such characteristics were the time courses and magnitudes of these
responses. In 1909 A. V. Hill, then in Cambridge, turned his formidable ana-
lytical powers to this issue, although only briefly.2 While examining Langley s
proposal quantitatively, Hill focused first on the time courses of nicotine-
induced muscle contraction and of curare-induced relaxation. Rather than the
time course representing merely the physical process of drug diffusion into or
out of the tissue, the response times, he concluded, reflected reversible inter-
actions, "a gradual combination of the drug with some constituent of the mus-
cle."3 As to the magnitude, Hill expressed the contraction height in terms of
such combinations between excitatory drug or "agonist," A, and receptor, R:
A + R <=> AR.
But his formulation included a threshold value, T, that AR must exceed before
contraction resulted; thus, the contraction magnitude was proportional to
(AR - T).
A. J. Clark, also educated in Cambridge, developed Hill's model of reversible
associations between drug and receptor. Clark's version, however, depicted the
response as proportional to the amount of this complex, without it having to
exceed some threshold level. In 1926 Clark, then in London, plotted the mag-
nitudes of frog heart and skeletal muscle contractions (response) against admin-
istered acetylcholine concentrations (dose), identifying the consequent "dose-
response plots" as rectangular hyperbolas.4 (With dose on the x-axis and response
on the i/-axis, the curves approached the horizontal asymptote of maximal
response at infinite dose.) For large concentration ranges it is more convenient
to plot the logarithm of the dose against the response, and rectangular hyper-
bolas are transformed in "log dose-response plots" into sigmoidal curves (Fig.
6-1 A). Clark noted that with both tissues the dose-response data "lie along a
curve which can be fitted closely by the equation [for a rectangular hyperbola
of] K.x = y/(lOO — y), where x = molecular concentration of the drug, y —
action produced as percentage of maximal action [i.e., 100]; K = constant" (the
line in Fig. 6-1A was drawn to this formula).5 He concluded that "the simplest
explanation [for this fit] is to suppose that a reversible monomolecular reaction
occurs between the drug and some receptor in the cells."6
An equivalent formula expresses the ratio of drug-receptor complex, AR, to
the maximal drug-receptor complex possible, ARmax, in terms of the binding
equilibrium A + R <^> AR:
where A is the concentration of free agonist, R the concentration of free recep-

tor, AR the concentration of agonist-receptor complex, and K^ the association
FIGURE 6-1. Acetylcholine concentration and muscle contraction. A. Acetylcholine, at
the concentrations indicated (logarithmically) on the x-axis, was added to baths con-
taining frog rectus abdominis muscles. The magnitude of contraction is plotted on the
y-axis, expressed as a percentage of the maximal contraction. The curve is that of a rec-
tangular hyperbola transformed in the semilogarithmic plot. B. In similar experiments
acetylcholine was added in the presence of no atropine (I) or 33 /iM (II), 100 ^iM (III)
or 1 mM (IV) atropine. Here, the logarithm of the normalized contraction is plotted on
the y-axis. (A and B from Clark [1926a], Fig. 6, and Clark [1926b], Fig. 4, respectively,
courtesy of the Physiological Society.)
145
constant for this binding. Response, according to Clark's principles, was then
linearly proportional to AR/ARmax.
Although Clark cited the similarities of his curves to those for oxygen's asso-
ciation with hemoglobin, he did not explicitly relate his equation to binding
formulas then known, such as those for Irwin Langmuir's adsorption curves or
for Leonor Michaelis and Maud Menten's substrate-velocity curves for enzyme
activity.7 Moreover, Clark did not explicitly identify his K as the association con-
stant, Ka, in the binding equilibrium.
Clark also described atropine's inhibition of these acetylcholine-induced con-
tractions: with successively higher concentrations of this antagonist, the log
dose-response curve was shifted further and further to the right (Fig. 6-1B).8
To describe this effect he inserted a factor in his equation, multiplying K by
the ratio of acetylcholine concentration to atropine concentration.9 This mod-
ification, however, had no theoretical justification, and he acknowledged that
"it only holds when atropine is present in a concentration sufficient to produce
a well-marked action," that is, at high atropine concentrations.10 Indeed, Clark
argued against atropine and acetylcholine competing for the same site on the
grounds that added acetylcholine did not accelerate atropine's dissociation from
the tissue.11
The next significant advance came from John Gaddum, who has appeared
in earlier chapters. He, too, was educated in Cambridge but then worked with
Henry Dale, Wilhelm Feldberg, and Ulf von Euler before beginning a series
of academic moves—from Cairo to London to Edinburgh (where he succeeded
Clark, who had moved there in 1927) and finally to Babraham. But it was while
in London in 1937 that Gaddum revised Clark's formulation for antagonism by
explicitly representing competition between agonist and antagonist for the same
receptor site:
where Kj and K% are the respective association constants for agonist GI and for
antagonist C% binding to the same receptor site.12 Again, the fractional response
was equated to the extent of agonist binding.
The Hill-Clark-Gaddum formulation stressed reversible binding to receptors
governed by distinct affinities for specific substances. Since this proposal may
seem trivially obvious to modern pharmacologists, it seems worth emphasizing
that alternative views were also expressed. For example, others, including Gad-
dum, reported sigmoidal log dose-response plots before Clark did, but they
had interpreted their plots differently: as agonists interacting with heteroge-
neous populations of units "whose susceptibility is distributed about a mean in
accordance with a probability curve [so that the dose-response] curve [is] the
integral of the normal distribution."13 This formulation was difficult to disprove
Characterizing Receptors (1905-1983) 147
before identical, pure receptors were available for study, but Clark in his influ-
ential review of 1937 disagreed on other grounds, judging the population argu-
ment "unfruitful, since, if the response to drugs is attributed to a peculiarity
of living tissue, there is no means of linking up such responses with the known
laws of physical theory."14
Even more dissimilar was Walther Straub's potential theory of 1907.15 He
considered that drugs exerted their effects not by combining with specific
receptors but through the concentration gradient of a drug from outside the
cell to inside. Straub derived his model from observing that added drugs could
produce large effects initially (when the gradient would be high), but that this
response would often decline slowly (presumably as the drug entered the cell
and the gradient dissipated). But, as Clark pointed out, many drugs do not show
decreased responses over time.16 Moreover, such time courses can be explained
by heterogeneous drug-receptor interactions, with receptors having different
rates of association and dissociation (falling back on the population argument).
Clark also attributed the decline to tolerance, a well-recognized phenomenon,
albeit one then without a clear mechanistic explanation.
Intrinsic Activity, Efficacy, Partial Agonists, and Spare Receptors
The Hill-Clark-Gaddum model specified that responses were linearly propor-

tional to the fractional occupancy of receptors by agonists, but this agreeably
simple relationship was not obeyed in many cases. In some instances the dis-
crepancies were attributable to extraneous complexities, such as metabolism of
the added drugs or their slow diffusion to the sites of action; actual concen-
trations at the receptors would then be unknown and equilibrium between drug
and receptor, on which the formulation relied, would not obtain. In other
instances paradoxical responses appeared, suggesting intrinsic disagreements.
For example, J. Raventos, working with Clark, reported in 1937 that a homol-
ogous series of compounds, varying in the length of the side chain bonded to
an amine group, excited when the side chains were short, but their maximal
responses declined with increasing chain length rather than remaining con-
stant.17 Moreover, at still longer chain lengths the compounds inhibited. Even
more puzzling were observations of K. H. Ginzel in Vienna. In 1951 he iden-
tified a member of such a homologous series that could act as either agonist
or antagonist.18
Such gradations of activity E. J. Ariens in Utrecht explained in terms of
"intrinsic activity." In 1954 Ariens proposed that response magnitudes did not
reflect the fractional occupancy of receptors alone, but the nature of the com-
plexes formed between agonist and receptor as well.19 Accordingly, substances
having high intrinsic activity would induce strong activation when occupying
their receptors and be strong agonists. Substances having lower intrinsic activ-
ity would produce weaker activation. These weak agonists could also act as
antagonists toward strong agonists by competing with them for receptor occu-
pancy: when weak agonists occupied the receptors instead of strong agonists,
they would evoke lesser responses, manifested as inhibition toward strong ago-
nists. Ariens formalized this proposal by including a factor to represent intrin-
sic activity, a, so responses would be proportional to a times the fractional
occupancy: a/(l + l/K^A).
Two years later R. P. Stephenson in Edinburgh called attention to further
discrepancies and added new hypotheses to account for drug—receptor inter-
actions.20 Stephenson pointed out that actual dose-response plots frequently
deviated from true hyperbolas. He also repeated Ariens's conclusions that a
homologous series of compounds could have different maximal responses (Fig.
6-2A) and that some compounds could have both agonist and antagonist prop-
erties. But Stephenson rejected the basic tenet of Hill, Clark, Gaddum, and
Ariens that responses must be linearly proportional to receptor occupancy.
Instead he introduced three hypotheses: (1) A maximum effect can be pro-
duced by an agonist when occupying only a small proportion of the receptors,
(2) The response is not linearly proportional to the number of receptors occu-
pied, and (3) Different drugs may have different capacities for initiating a
response.21 Stephenson named agonists that were unable to elicit maximal
FIGURE 6-2. Responses to acetylcholine homologs. A. Alkyl-trimethylammonium com-

pounds, containing ethyl, butyl, hexyl, heptyl, octyl, nonyl, or decyl groups as the alkyl
sidechains, were added—at the concentrations indicated (logarithmically) on the x-
axis—to baths containing guinea pig ileum segments. The magnitude of the contrac-
tions are plotted on the y-axis as a percentage of the maximal contraction. B. Responses
to the alkyl-trimethylammonium compounds calculated from the affinities and effica-
cies listed in Table 6—1, with response a hyperbolic function of the stimulus. (A and B
from Stephenson [1956], Figs. 1 and 10, respectively, courtesy Macmillan Magazines,
Ltd.)
responses, even at infinite dose, "partial agonists," and the excess of receptors,
unneeded when a full agonist acted, "spare receptors." The factor modifying
maximal response he named "efficacy," e, which he multiplied times fractional
occupancy (as had Ariens with intrinsic activity, a). Stephenson, however,
equated this product not to response, as had Ariens, but to "stimulus," S:
Whereas S directly represented the affinity of receptor for agonist and the effi-
cacy of agonist at receptor, response was now an unspecified function of S.
Stephenson illustrated these properties with a calculated family of curves for
a series of homologous compounds. These he derived from values for affinity,
which increased with side chain length, and for efficacy, which first rose with
side chain length and then fell (Table 6-1), and from a hyperbolic relationship
of response to S. These calculated curves mimicked experimental results: Fig-
ure 6-2B vs. 6-2A. Since responses were not necessarily linear functions of S
(or of fractional occupancy), Stephenson's formulation allowed dose-response
plots to deviate from true hyperbolic curves.
At this time Mark Nickerson in Winnipeg published results consistent with
Stephenson's formulation.22 Nickerson described how diphenhydramine irre-
versibly blocked responses to histamine over a slow time course (tens of min-
utes). During the early minutes diphenhydramine appeared to be a competi-
tive (reversible) antagonist: the maximal response to histamine was unchanged
but the apparent affinity decreased. Later in the time course, however, the
maximal response decreased. Nickerson interpreted these results in terms of
an excess of histamine receptors: initially enough (spare) receptors were avail-
able so that a maximal response could occur even though some were blocked
irreversibly, but the decreased number simulated a reduction in affinity. Later,
when the number of available receptors was decreased still further, too few
remained to generate a maximal response.
TABLE 6-1. Affinity and Efficacy of Chiolinergic Homologs*

ALKYL GROUP: ETHYL BUTYL HEXYL HEPTYL OCTYL NONYL DECYL
Affinity 0.63 3.8 19 41 63 110 190
Efficacy 31 200 21 2.2 1.4 1.0 0.6
"Affinities for the partial agonists (hexyl and longer derivatives) were estimated from the concen-
trations for half-maximal responses. Affinities for the shorter full agonists were estimated by extrap-
olating the plot for partial agonist affinity vs. chain length (Stephenson, 1956, Fig. 4). Values are
for the affinity constant X 10~3. Efficacies were then calculated from these affinity constants and
the magnitudes of the responses, using the concentrations for half-maximal responses. (From
Stephenson [1956], Table V. Used by permission of Nature Publishing Group.)
Stephenson considered the block by irreversible antagonists to be "sur-

mountable" as long as sufficient receptors remained to give maximal responses,
although with time, as more receptors were inactivated, the block would
become "insurmountable." This phenomenon of surmountable but irreversible
inhibition, Stephenson claimed, would be difficult to explain without the occur-
rence of spare receptors.
From independent investigations of irreversible antagonists, Robert Furch-
gott in New York also recognized that maximal responses might be obtainable
if agonists required only a tiny fraction of the total receptors present.23 Furch-
gott developed these considerations in parallel with Stephenson, and in 1966
he defined "intrinsic efficacy" as Stephenson's efficacy divided by the concen-
tration of receptors present, a term emphasizing the ability of an agonist to
produce a stimulus at a single receptor.24
These studies thus prompted appreciations of (1) receptor affinities for spe-
cific substances (agonists, antagonists, and partial agonists) that governed recep-
tor occupancy, with (2) the occupied receptor responding not merely by acti-
vation or inhibition but by degree of activation or inhibition (notions of intrinsic
activity and efficacy). Thus, receptor responses need not be linearly related to
receptor occupancy. (3) Tissue responses, such as smooth muscle contraction,
reflected the constraints of cellular capabilities as well as the sequence of steps
between receptor and response. Consequently, tissues might respond nonlin-
early to receptor occupancy and be unable to exceed certain maximal responses
even when further receptors were occupied by strong agonists (notions of stim-
ulus and of spare receptors). With weaker agonists (partial agonists), though,
tissues might never reach that maximum, despite the full complement of recep-
tors being occupied.
Two-State Models or Receptor Activation
Neither Ariens nor Stephenson explained how intrinsic activity or efficacy might
be manifested, and the link between hypothesized receptor occupancy and
observed tissue response remained mysterious as the 1950s ended. The chem-
ical nature of receptors was then also unknown, although many assumed that
receptors, like enzymes, were proteins. Such assumptions sanctioned further
speculations, founded on new insights into protein structures and their func-
tional changes.
By the early 1960s John Kendrew had determined with X-ray crystallogra-
phy the first three-dimensional structure of a globular protein, and Max Perutz
had reported the crystal structures of both oxygenated and deoxygenated hemo-
globin, revealing a distinct shift in protein conformation that accompanied oxy-
gen binding (chapter 4). Perutz's demonstration complemented recent studies
indicating that some enzyme activators and inhibitors bound to regulatory sites
distinct from catalytic sites ("allosteric" sites), thereby altering structure and
thus function. In 1965 Jacques Monod in Paris, together with Jeffries Wyman
and Jean-Pierre Changeux, presented a striking and influential model for
allosteric control of protein conformations.25 They proposed that proteins could
exist in two alternative, interconvertible forms, R and T, of which R was active
and T inactive (or significantly less active); moreover, if R and T had different
affinities for a ligand,26 L, its binding would shift the equilibrium between
R and T:
Consequently, if L bound more tightly to R than to T, L would shift the con-

formational equilibrium toward R and be an activator. If L bound more tightly
to T, then L would be an inhibitor.
Also incorporated in their model was the possibility for cooperative binding
among multiple sites: if ligand binding favored the conformation having a higher
affinity for this ligand, then binding of the first ligand would promote the bind-
ing of subsequent ones (as observed with oxygen binding to hemoglobin). Such
cooperativity would be represented by sigmoidal binding plots rather than
hyperbolic ones. (Log binding plots would also be sigmoidal, but steeper than
for noncooperative binding.)
In 1967 Arthur Karlin in New York adopted this model for drug interac-
tions with acetylcholine receptors, as did Changeux, visiting in New York, the
following year.27 Others soon followed.28 Not only did the Monod- Wyman-
Changeux model account for the apparent cooperativity of nonhyperbolic
dose-response curves, it also rationalized agonists, partial agonists, antago-
nists, and intrinsic activity/efficacy: full agonists bound preferentially to R
forms and shifted receptor molecules to these active conformations; antago-
nists bound preferentially to T forms and shifted receptors to inactive con-
formations; partial agonists bound to both, so significant amounts of both T
and R forms would be present and maximal activity unachieved even with all
binding sites occupied.
Desensitization
Pharmacologists in the 1930s noticed that when certain substance were added
to tissues at high concentrations, they initially excited but then prevented fur-
ther responses, so that subsequent additions were for some time thereafter
ineffective.29 Possible explanations included presumed depletions of cellular
energy stores during the initial excitation.30 But in 1957 Bernard Katz in Lon-
don accounted for this "desensitization" with a three-state model depicting

receptors: (1) in resting states capable of activation, (2) in active states, and
(3) in desensitized states incapable of activation.31 Katz considered various paths
among these states. Subsequent studies favored his cyclical scheme, which
rotated receptors through resting to active to desensitized and back to resting
states.32 Katz's model clearly harked back to Hodgkin and Huxley's 1953 model
for nerve impulse conduction (chapter 4), in which Na+-channels undergo a
voltage-dependent transition from closed to open states, followed by a sponta-
neous conversion to inactive (unexcitable) states before reverting to closed
(excitable) states. Moreover, Katz's three-state model for activation and desen-
sitization was compatible with proposals for conformational changes in recep-
tors, just as were two-state models then also being considered.
Rate Theory
William Paton, unlike the Langley, Hill, Clark, Gaddum succession, was edu-
cated in Oxford, although he worked briefly with Dale before returning to
Oxford. There in 1961 Paton published an ingenious alternative to these "occu-
pation theories," a "rate theory" proposing that "the stimulant effect produced
by a drug depends, not on the number of receptors occupied, but on their rate
of occupation."33 Maximal responses required maximal numbers of interactions,
so agonists should dissociate rapidly from receptors to permit further interac-
tions. Conversely, antagonists would block by dissociating only slowly, thereby
preventing agonists from interacting. Paton accumulated an impressive body
of corroborative evidence from studies on cholinergic stimulation of the guinea
pig ileum in the absence and presence of various inhibitors. But others were
less enthusiastic, and in 1968 D. R. Waud summarized criticisms.34 These
ranged from the lack of chemical precedent for such rate-dependent stimula-
tion to the lack of similar data at other loci where they were sought, including
neuromuscular junctions and adrenergic systems. Moreover, Waud provided
alternative explanations for Paton's observations, couched in terms of occu-
pancy theories and plausible assumptions that attributed Paton's time courses
not to rates of interaction with receptors but to rates of access to receptors,
with diffusion modified by tissue binding sites. In the face of these criticisms
and the continuing development of occupation theories, enthusiasm for Paton's
rate theory ebbed.
Receptor Classification
Another approach to understanding receptors was to sort them into meaning

ful categories, expressing similarities and exhibiting anomalies.
Classification by Agonist
One obvious scheme involved classifying neurotransmitter receptors by their

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

RANK-ORDER OF RESPONSES WITH
RESPONSE ADRENALINE NORADRENALINE ISOPROTERENOL
Vasoconstriction 1 2 3
Vasodilation 2 3 1
Intestinal contraction (inhibition) 1 2 3
Uterine contraction (inhibition) 2 3 1
"Rank-order of responses in rabbits to equimolar amounts of the three adrenergic agonists are tab-
ulated. Vasoconstriction was measured from blood pressure changes and vasodilation from coro-
nary artery flow. (From Ahlquist [1948], Table I. Used by permission of the American Physiolog-
ical Society.)
further subdivisions. In 1967 A. M. Lands in Rensselaer identified j3i and @2

receptors. Among other responses, the former was prominent in cardiac stim-
ulation, the latter in bronchodilation.37 The pharmaceutical industry—in which
both Black and Lands worked—then developed practical (3i and /3a agonists
and antagonists. For example, the fiz agonist terbutaline (Brethine) is useful in
treating asthma since it dilates bronchi without stimulating the heart; on the
other hand, the fii antagonist atenolol (Tenormin) is useful in treating hyper-
tension without affecting pulmonary airflow. Then in 1974 Salomon Langer in
Buenos Aires divided a-receptors into a\ and «£ after exploring different issues
(see below).
Many early classifications, such as Ahlquist's and Lands's, were based on ago-
nist responses. A better discriminator would be agonist affinity, avoiding pos-
sible variations in efficacy as well as the unknown links between responses and
stimuli (in Stephenson's sense). But direct measurements of agonist affinity
were not practical until labeled ligands of high specificity became generally
available in the 1970s. The third approach, advanced by Heinz Schild in Lon-
don, focused on affinities for competitive antagonists. Binding to receptors
could then be assessed without the confounding uncertainties of how responses
reflected receptor occupancy. Moreover, Schild in 1947 formulated a conven-
ient experimental protocol based on "dose ratios": determining the antagonist
concentration necessary to reduce (1) the response elicited by an x-fold dose
of some agonist to (2) the response of a single dose of this agonist in the absence
of antagonist.40 In 1959 he devised a simple plot of such data that revealed the
agonist association constant, a value that should be the same for all receptors
of a given class but would likely be different for receptors of other classes.41
These approaches both certified similarities between receptors in different
tissues and established distinctions between different receptor classes. Other
techniques supplemented these methods, including direct measurements of
agonist affinity, possible when suitably labeled ligands became available, and
examinations of receptor structures revealed by molecular biological techniques
(chapter 8). These approaches also culminated in the realization that multiple
receptors for each neurotransmitter were the rule; for example, 15 distinct sero-
tonin receptors were distinguished by 1994.
Classification by Cellular Localization
Since many neurotransmitters produced their effects quite rapidly, the respond-
ing receptors would seem to be on or near the cell surface, as Clark pointed
out in 1937.42 In 1955 Katz demonstrated just such a localization at neuro-
muscular junctions: acetylcholine released from micropipets near the muscle
cell surface excited responses, whereas acetylcholine released into the proto-
plasm did not.43 Later microelectrophoretic experiments confirmed this cell
Ckaracterizing Receptors (1905-1983) 155
membrane localization for many neurotransmitter receptors on many cell types

(chapter 5).
A different issue was where on the cell surface the receptors lay. Early mod-
els depicted the obvious localization at postsynaptic sides of synaptic clefts,
where receptors would receive neurotransmitters released from presynaptic
terminals, in accord with the principle of synaptic polarization. But further
studies uncovered further complexities. In 1963 Charles Edwards in Min-
neapolis described a decrease in the number of acetylcholine quanta released
at neuromuscular junctions when exogenous acetylcholine was added. He con-
sidered that this response was due to a "direct action [of acetylcholine] on the
presynaptic terminals."44 The following year John Hubbard in Canberra con-
firmed this phenomenon, which he interpreted as "a negative feed-back on fur-
ther acetylcholine release."45 Surprisingly, these observations were not pursued
at that time, and the notion of regulatory presynaptic receptors was developed
instead from studies on adrenergic synapses.
In 1957 G. L. Brown, then in London, reported that certain adrenergic antag-
onists, including phenoxybenzamine, increased noradrenaline outflow from
stimulated sympathetic nerve endings.46 Several explanations were advanced
in the following decade, including phenoxybenzamine s interference with mech-
anisms for noradrenaline disposal. As Langer pointed out in 1970, another pos-
sibility "cannot be excluded," that a-receptor antagonists such as "phenoxy-
benzamine may increase the release of [noradrenaline]."47 Klaus Starke in
Essen provided strong support for this alternative the following year, describ-
ing how a-receptor agonists reduced noradrenaline release—the converse of
antagonists enhancing release.48 In 1972 Starke reexamined responses to a-
adrenergic antagonists and concluded that "a-receptors are involved in the reg-
ulation of the release of noradrenaline from adrenergic nerve terminals."49
Langer that year reached a similar conclusion from similar studies, invoking a
"negative feedback mechanism regulating transmitter release"; in 1974 he
named the presynaptic receptors a2 and the postsynaptic receptors ai.50 Langer
and Starke also described differences in drug specificities between a\- and «£-
receptors.51 But these studies did not demonstrate directly that the regulatory
receptors were indeed presynaptic, although Langer soon provided indepen-
dent support, albeit also indirect: binding of a-adrenergic ligands decreased
after he destroyed sympathetic nerve endings in the heart.52 (But later studies
revealed a2~receptors postsynaptically as well as presynaptically.53)
On the other hand, Paton showed that added noradrenaline diminished
acetylcholine release from cholinergic nerve terminals, whereas E. Muschall in
Mainz found the converse: muscarinic agonists diminished noradrenaline
release from adrenergic nerve terminals.54 Such processes would augment
antagonisms between alternative systems, allowing one neurotransmitter (say,
acetylcholine) not only to direct the target organ oppositely from its rival neu-
retransmitter (say, noradrenaline) but also to diminish the release of its rival.
These presynaptic "heteroreceptors," which respond to neurotransmitters
released from different nerve terminals, thus contrast with presynaptic "auto-
receptors," which respond to the neurotransmitter released from their own
terminal.
Classification by Function
Langley imagined two classes of receptors, one eliciting excitatory and the other
inhibitory responses (chapter 3). Later studies, however, demonstrated that the
same receptor could mediate excitatory or inhibitory responses depending on
the cellular circumstances. A more rewarding classification considered classes
of receptor mechanisms, as further studies disclosed (chapters 7 and 8).
Structure—Activity Relationships
Receptor structures could not be determined directly with techniques then

available. Nevertheless, since neurotransmitters were thought to fit their recep-
tors like keys in locks, the receptors ligands should provide a complementary
image of its binding site. "Structure-activity" studies thus attempted to corre-
late ligand structures—accessible through contemporary chemical methods—
with receptor responses. A few steps in the analyses of cholinergic ligands will
exemplify such endeavors.
Acetylcholine is a flexible molecule, able to assume alternative conforma-
tions in solution. Some of its analogs, however, have more restricted motions,
so plausible common dimensions can be derived. This Carl Pfeiffer in Chicago
reported in a pioneering study published in 1948: he calculated common crit-
ical distances between a methyl group on the quaternary nitrogen and the
carbonyl and ester oxygens of various muscarinic agonists and antagonists (see
Fig. 3-6).55
Pfeiffer s distances came from molecular models, but in the following decades
X-ray diffraction and nuclear magnetic resonance techniques provided specific
dimensions. From these data Cyrus Clothia in London differentiated between
the muscarinic and nicotinic surfaces required for acetylcholine binding, which
he localized in 1970 to the methyl and carbonyl sides of the molecule (Fig.
6-3A).56 Selective muscarinic agonists had the methyl side accessible but the
carbonyl side blocked (Fig. 6-3B), whereas selective nicotinic agonists had the
methyl side blocked but the carbonyl side accessible (Fig. 6-3C). William Beers
and Edward Reich in New York inferred similar requirements from studying
molecular models: in addition to common interactions through the quaternary
nitrogen, muscarinic ligands interacted through the methyl group and ester
FIGURE 6.3. Muscarinic and nicotinic conformations. A. The acetylcholine structure

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

The receptors presumably had groups that fitted these surfaces and accom-
modated the ligands' polarities. Neither analysis, however, accounted convinc-
ingly for relative efficacies or revealed how ligand binding triggered receptor
responses.
Receptor Identification ana Purification
More direct and detailed information was needed, but to characterize recep-
tors chemically and mechanistically they must be isolated from contaminating
entities and confounding processes. Serious conceptual uncertainties and tech-
nical inabilities, however, impeded progress toward receptor purification. By
1960 neurotransmitter receptors were localized to cell membranes and thought
to be, at least in part, proteins.58 The structural organization of lipids and pro-
teins within membranes, however, was then unclear. The favored model de-
picted protein layers coating each surface of a lipid bilayer; not until late in the
1960s did S. J. Singer revive mosaic models, showing "intrinsic" membrane pro-
teins that extended across the bilayer (as would be appropriate for transporters,
channels, and receptors that must mediate between extracellular and intracel-
lular environments).59
Extracting such intrinsic proteins in their native state was also a daunting
challenge. During the 1960s methods appeared for fragmenting membranes
mechanically and for "solubilizing" their proteins with various detergents. The
detergent-solubilized proteins could then be purified by ultracentrifugation and
chromatography. As the 1970s began, a crucial new technique, electrophoresis
through polyflcrylamide gels of proteins solubilized in the detergent sodium
dodecylsulfate (SDS-PAGE), was being employed for separating membrane
proteins according to their molecular weights.
Membrane-bound enzymes could be followed through purification proce-
dures by assaying catalytic activity at successive steps. Following receptors was
less straightforward. The binding of specific ligands, such as receptor agonists
or antagonists, was obscured by "nonspecific" binding to other sites as well as
to irrelevant "specific" sites, including those on neurotransmitter transporters
and on synthetic and degradative enzymes. Moreover, purifying a ligand-
binding entity did not guarantee that this entity was the entire receptor.
Not surprisingly, some commentators were pessimistic about the prospects
for isolating intact receptors,60 and the piecemeal progress fostered skepticism.
Still, some successes accrued; three examples may illustrate the quests.
Nicotinic Cnolinergic Receptors
The first neurotransmitter receptor to be isolated was the nicotinic receptor,

although early attempts foundered for want of sufficiently discriminating
ligands. For example, Carlos Chagas in Rio de Janeiro tried to extract recep-
tors from eel electric organs—a wise choice since the organs are large and
richly innervated with cholinergic fibers—through their affinity for curare and
gallamine (a synthetic antagonist). By 1959 Chagas had separated a fraction
containing acidic mucopolysaccharides that bound radioactive derivatives of
these ligands tightly.61 But critics soon pointed out that the binding measured
in dilute solution in vitro was unlikely to occur in vivo, where higher salt con-
centrations would interrupt electrostatic binding between positively charged
ligands and negatively charged acidic mucopolysaccharides.62 Furthermore,
acidic mucopolysaccharides (and cholinergic ligand binding) were not confined
to innervated regions, as would be expected for neurotransmitter receptors. So
in 1962 Chagas modified his designation to "acceptor" sites.63
Changeux, then back in Paris, chose instead radioactive decamethonium, an
antagonist having a high affinity for nicotinic receptors. With this ligand,
Changeux in 1970 demonstrated binding to a detergent-solubilized membrane

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

("liposomes").72 By 1979 several investigators, including Racker, had reported
acetylcholine-stimulated Na + fluxes across liposome membranes that contained
purified receptors.73
Electron microscopic studies complemented these approaches. Early micro-
graphs showed rosettes on the surface of electric organs,74 and in 1982 Robert
Stroud in San Francisco published a model, based on examinations at higher
magnifications, that depicted a radial, pentameric array of subunits around a
central ion-conducting channel (Fig. 6-4).75 Finally, the availability of purified
receptor preparations permitted more direct studies on drug-receptor inter-
actions, demonstrating cooperative binding of acetylcholine to the two a-sub-
units and responses supporting multistate models for receptor activation and
desensitization.76
jS-Aarenergic Receptors
Success with other receptors was more elusive. Whereas fish electric organs pro-
vided large quantities and high densities of nicotinic receptors, no organs were
so richly endowed with receptors for any other neurotransmitter. This low den-
sity not only necessitated more extensive purification from extraneous materi-
als, it also required that labeling ligands have extremely high specific activities.77
Indeed, initial attempts with radioactive noradrenaline revealed only nonspe-
cific binding: antagonism by other ligands did not follow their pharmacological
ranking, and binding was neither saturable nor stereospecific.78
FIGURE 6.4. Model of the nicotinic receptor. The receptor is composed of (A) five
subunits traversing the lipid bilayer of the membrane and enclosing a (B) central ion-
conducting channel. (From Kistler et al. [1982], Fig. 6, courtesy of the Biophysical
Society.)
Ckaracterizing Receptors (1905-1983) 161
In 1974, however, experiments using radioactive /3-adrenergic antagonists

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

ther progress was relatively modest. In fact, purification was not achieved by
1983, when this account concludes. Interest instead reflected the importance
of morphinelike substances medically and socially, coupled to demonstrations
soon afterward that the body makes and uses morphinelike substances (chap-
ter 5). Investigations of opioid receptors also established general techniques
for labeling neurotransmitter receptors in the brain, launching a "grind and

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

that binding sites were (at least in part) protein.
These studies gained significance from the discovery of endogenous opioids
that soon followed (although, as noted in chapter 5, the initial isolation relied
on traditional bioassays). And although these studies did not culminate in recep-
tor isolation by 1983, they led to further revelations, including demonstrations
of how opioid receptors were distributed within the nervous system and allo-
cations of opioid receptors among several subclasses.93 Moreover, an intrigu-
ing difference in the interactions of receptors with agonists vs. antagonists also
supported proposals for alternative receptor conformations: Na + reduced the
binding of agonists but increased that of antagonists, implying that this cation
induced or selected protein conformations having different specificities for
these oppositely acting agents.94
Responses 01 Indiviaual Receptor Molecules
Models for neuromuscular transmission developed by Katz in the early 1950s

depicted populations of receptors that responded to discrete quanta of acetyl-
choline: to the spontaneous release of single quanta by miniature endplate
potentials and to the evoked release of multitudes of quanta by endplate poten-
tials (chapter 4). Twenty years later Katz and Ricardo Miledi advanced this
characterization a level further, identifying the responses of individual recep-
tors to administered acetylcholine.95 Since the "statistical variation of high-
frequency collisions between [acetylcholine] molecules and end-plate recep-
tors . . . might be discernable as an increase in membrane [electrical] noise,"
they recorded electrical fluctuations from endplates in the presence of con-
stant levels of acetylcholine.96 Katz and Miledi then analyzed these traces in
terms of discrete electrical pulses, representing abrupt transitions between non-
conducting and conducting receptor states. In 1972 they reported unitary con-
ductance changes on the order of 10 pS lasting roughly 1 ms.97 Others, notably
Charles Stevens in Seattle and David Colquhoun in Southampton, pursued the
"fluctuation analysis," calculating single-channel conductances of 25 pS and
mean open-channel lifetimes of 3 ms.98 But the high electrical background
from extraneous regions and processes required model-dependent statistical
analyses to uncover the individual events.
Through a spectacular technical triumph (celebrated with a Nobel Prize in
1991), Erwin Neher and Bert Sakmann (Fig. 6-5) in Gottingen solved the prob-
lems of reducing this background and of displaying the discrete events: they
devised a "patch electrode," a glass pipet with a polished tip 3-5 jjim in diam-
eter, that they pushed tightly against the carefully cleaned muscle surface. Since
FIGURE 6.5. A., left, Erwin Neher (1944-). B., right, Bert Sakmann (1942-). [A cour-
tesy of E. Neher, B courtesy of B. Sakmann.)
the patch was isolated by the tight seal from electrical events elsewhere, the
electrode responded prominently to ionic currents through the few channels
bounded by its orifice. So, with cholinergic agonists in the electrode bathing
the patch, Neher and Sakmann in 1976 recorded discrete electrical pulses (Fig.
6-6A), identifiable as multiples of a basic amplitude and interpretable as cur-
rents through channels that abruptly opened when agonist bound and later
abruptly closed." They calculated single-channel conductances of 22 pS and
mean open-channel lifetimes of 11 ms.100 Responses from a population of such
receptors would then sum to give the composite endplate currents previously
measured with intracellular microelectrodes. Neher and Sakmann improved
and extended their technique, and patch electrodes soon became widely used
to characterize channels throughout the biological realm.101
An alternative approach for recording responses from single receptor mole-
cules was through reconstituting purified receptors in lipid bilayers. In this case
the preferred system is a planar bilayer, formed across a tiny hole in a septum
that separates two solutions. With receptors incorporated in and penetrating
across this insulating bilayer, gross electrodes in the two solutions can then
monitor conductance through the receptor channels. By 1980 successful recon-
stitutions revealed the same discrete, abrupt steps in conductance, attribute-
FIGURE 6.6. Openings and closings of individual nicotinic receptor channels. A. Patch
electrodes containing the cholinergic agonist suberyldicholine were placed tightly
against the muscle fiber surface to measure transmembrane currents. Downward deflec-
tions record an increased current flow under the electrode and upward deflections a
decreased flow. These deflections were interpreted as agonist-induced openings of ion-
conducting channels with a unitary magnitude, followed by the return of the conduc-
tance to baseline values when the channel closed. In some cases the downward deflec-
tion is twice the unitary magnitude, interpreted as two channels under the electrode
being open at the same time. B. Gross electrodes were in two solutions separated by a
lipid bilayer containing purified nicotinic receptors. Currents measured between the
two electrodes then include ion flows through these receptors. In these tracings upward
deflections record an increased current. The occasional increases in conductance were
interpreted as spontaneous openings of receptor channels, producing currents of a uni-
tary magnitude. C. More frequent conductance increases occurred after the choliner-
gic agonist carbamylcholine was added to the solutions. These often appeared as mul-
tiples of the unitary conductance and were interpreted as multiple channels being open
at the same time. (A from Neher and Sakmann [1976], Fig. 3, reprinted by permission
of Nature, ©Macmillan Magazines, Ltd. B and C from Schindler and Quast [1980],
Figs. 4A and B, courtesy of H. G. Schindler.)
ble to the unitary conductance of single receptor molecules (Fig. 6-6B).102

Again, adding cholinergic agonists increased the probability of channel open-
ings (Fig. 6-6C). This method not only could check the functional capabilities
of purified receptors, it also allowed the easy pharmacological manipulation of
receptors through additions to the bathing solutions.
Conclusions
From initial notions of a neurotransmitter acting through a cellular receptor,

the studies cited here refined these concepts and extracted new generalities.
The studies also uncovered complexities and elaborations, including a growing
catalog of receptors. Indeed, one striking conclusion was that a single neuro-
transmitter could act through multiple classes of receptors, each dedicated to
controlling particular cellular processes. This realization had practical implica-
tions as well, for the multiplicity afforded opportunities for directed therapy:
through the design of new drugs aimed at distinct receptors modulating spe-
cific functions.
At this scale little can be said about individual scientists. A succession that
began once again in Cambridge at the turn of the century was soon joined by
other investigators across the globe. Effective communication and sharp com-
petition then merged individual styles into a cosmopolitan throng assimilating
new insights and extending new approaches. Indeed, their practice was marked
by the fruitful application of techniques devised for other inquiries—such as
detergent extraction of proteins, SDS-PAGE, affinity chromatography, and the
reconstitution of membrane proteins—as well as by the development of inno-
vative new techniques for these studies—such as patch electrodes (which were
then applied to studies of membrane channels at diverse loci).
Initial analyses of dose-response relationships revealed the fundamental
characteristic of receptor functioning: selective occupancy that initiated par-
ticular cellular changes. Further examinations of responses and broader explo-
rations with additional agonists and antagonists complicated the models but
merged the structures and functions into the mainstream of cellular biochem-
istry, with its protein conformational transitions and allosteric regulation.
The major achievement, however, was the isolation of functional receptors
and their recognition as integral membrane proteins. This feat rescued recep-
tors from the stigma of hypothetical entities, providing chemical identities and
structures that illuminated function. Purification of nicotinic receptors revealed
an oligomeric, funnel-shaped transmembrane unit surrounding a central chan-
nel for conducting ionic charges. By constrast, /3-adrenergic receptors had sim-
pler, quite different structures that—as the next chapter will relate—coupled
occupancy to the activation of enzymatic systems that then amplified responses
through the cell's interior.
Notes
1. Langley (1905, 1906). In fact, Langley enunciated the principle in 1878: "We may
. . . assume that there is some substance . . . in the nerve endings or gland cells with
which both atropin and pilocarpin are capable of forming compounds. On this assump-
tion then the atropin and pilocarpin compounds are formed according to some law of
which their relative mass and chemical affinity for the substance are factors" (p. 367).
From considering antiparasitic actions, Paul Ehrlich at the turn of the century also pos-
tulated that receptors mediated drug effects (see Bloch, 1994, chapter 5).
2. Hill (1909). For example, Hill described the onset of contraction as y = k(l —
e~ At ), where y is the magnitude of contraction, t the time since the beginning of the
contraction, and k and A constants.
3. Ibid., p. 372.
4. Clark (1926a).
5. Ibid., p. 535.
6. Ibid., p. 545.
7. Langmuir (1916); Michaelis and Menten (1913). The Michaelis-Menten analysis
proposed that enzyme velocity was proportional to the concentration of an enzyme-
substrate complex, ES, formed reversibly by the association of substrate, S, and enzyme,
E: E + S <=> ES. Their formula for velocity, v, in terms of these parameters, of maxi-
mal velocity, Vmax, and of the constant for ES dissociation, Km, is:
Clark's equation is formally equivalent, although it is expressed in terms of the associ-

ation constant, the reciprocal of the dissociation constant. In his 1937 review Clark made
this comparison.
8. Clark (1926b).
9. Clark justified his evaluation by citing a previous expression of this form for oxy-
gen vs. carbon monoxide binding to hemoglobin, as well as earlier pharmacological stud-
ies. But Clark presented no model of reversible antagonist binding to match his pro-
posal for reversible agonist interaction.
10. Clark (1926b), p. 555. When atropine concentrations are large compared to the
association constant for atropine, then Clark's formula approximates Gaddum's (1937,
cited below).
11. Clark (1926b). Clark's argument is mistaken. He came to his conclusion not from
analyzing competing equilibria but from reports that carbon monoxide accelerated oxy-
gen dissociation from hemoglobin. It was subsequently realized that hemoglobin has
multiple interacting sites, so that binding of carbon monoxide to one site can alter oxy-
gen affinity at another site; such interactions would not occur on receptors having inde-
pendent (noninteracting) sites. Clark repeated his argument in 1937, after Gaddum's
1937 formulation appeared.
12. Gaddum (1937). Gaddum's equation describes reversible, equilibrium binding to
receptors of antagonist, I, or agonist, A : I R < = > I + R + A<=> AR, where IR represents
the antagonist-receptor complex and the response is proportional to AR. This is for-
mally equivalent to earlier expressions of competitive inhibition of enzymes, expressed
in terms of dissociation constants, Km (for ES) and K, (for El):
When I is large relative to K,-, values of v/Vmax approximate those from Clark's for-
mulation for inhibition.
13. Gaddum (1926). See also Shackell et al. (1924).
14. Clark (1937), p. 205.
15. Straub (1907). Straub imagined that antagonists, such as atropine, inhibited by
preventing an agonist, such as acetylcholine, from entering the cell and thereby estab-
lishing a gradient.
16. Clark (1937).
17. Raventos (1937).
18. Ginzel et al. (1951).
19. Ariens (1954).
20. Stephenson (1956).
21. Ibid., p. 380.
22. Nickerson (1956).
23. Furchgott (1955).
24. Furchgott (1966).
25. Monod et al. (1965). Koshland et al. (1966) presented a somewhat different mul-
tistate model.
26. "Ligand" is a general term for a substance—atom, ion, or molecule—that binds.
27. Karlin (1967); Changeux and Podleski (1968). Changeux et al. (1967) earlier pro-
posed a related model in terms of cooperative effects transmitted through a membrane
lattice.
28. For example, Colquhoun (1973); Thron (1973); Pert and Snyder (1974).
29. For example, Barsoum and Gaddum (1935); Brown (1937).
30. For example, Cantoni and Eastman (1946).
31. Katz and Thesleff (1957).
32. For example, Rang and Ritter (1970).
33. Paton (1961), p. 23.
34. Waud (1968).
35. Ahlquist (1948).
36. Expressing dosages in molar terms allowed comparisons of the potency per mol-
ecule.
37. Ahlquist (1948), p. 595.
38. Black et al. (1965). At the time Ahlquist proposed his dichotomy, all known adren-
ergic antagonists affected a-receptors predominantly.
39. Lands et al. (1967a). A subsequent paper introduced the terms "/3-1" and "/3-2"
(Lands et al., 1967b).
40. Schild (1947). For example, the response to an agonist dose, A, will equal that
with twice the dose, 2A, in the presence of an antagonist, I, when I = 1/K, :
where Ka and K, are the association constants for agonist and antagonist binding.
41. Arunlakshana and Schild (1959).
42. Clark (1937).
43. del Castillo and Katz (1955a).
44. Ciani and Edwards (1963), p. 23.
45. Hubbard and Yokota (1964), p. 1073.
46. Brown and Gillespie (1957).
47. Langer (1970), p. 544.
48. Starke (1971).
49. Starke (1972), p. 19.
50. Enero et al. (1972), p. 672; Langer (1974). Delbarre and Schmitt (1973) sug-
gested splitting a-receptors into ai and 0.% categories but did not indicate what those
categories were.
51. Dubocovich and Langer (1974); Starke et al. (1975).

52. Story et al. (1979).
53. See Doxey et al. (1977).
54. Paton and Vizi (1969); Loffelholz and Muscholl (1969).
55. Pfeiffer (1948).
56. Chothia (1970). He was collaborating with Peter Pauling, the son of Linus
Pauling.
57. Beers and Reich (1970). Chothia and Pauling (1971) challenged a bond angle,
but Beers and Reich (1971) claimed that their model still fit when the angle was cor-
rected.
58. Nachmansohn (1953). Others proposed different compositions. For example,
Wooley (1958) argued for lipoidal receptors.
59. For the development of membrane models and experimental approaches, see
Robinson (1997).
60. For example, Waud (1968).
61. Chagas (1959).
62. Ehrenpreis and Kellock (1960).
63. Chagas (1962). He imagined that acceptor sites would, by binding acetylcholine,
increase the local concentration of acetylcholine near its receptor.
64. Changeux et al. (1970a, 1970b). He measured binding by equilibrium dialysis:
receptor fractions were confined inside dialysis sacks whose membrane pores prevented
proteins from escaping but allowed smaller ligands to enter from the bathing media.
Binding was calculated from the concentrations of ligands inside and outside the sacks
after equilibration. This approach gave precise values, but the prolonged exposures
allowed receptor desensitization. More rapid and convenient approaches used cen-
trifugation or filtration to separate, after an initial mixing, receptor-plus-ligand from
unbound ligand.
65. Chang and Lee (1963). a-bungarotoxin has a molecular weight of only 8 kDa, so
its mass contributed little to that of the receptor complex.
66. Miledi et al. (1971); Raftery et al. (1971). The electric organ of the ray, Torpedo
marmorata, has even higher densities of cholinergic innervation.
67. Changeux et al. (1971); Karlin and Cowburn (1973). Affinity-labeling uses a
reagent containing (1} a group that binds selectively but reversibly to the protein of
choice and (2) a highly reactive group that will bond irreversibly to proteins it contacts.
Thus, the affinity-group selects the protein for labeling with the reactive group.
68. These included Eldefrawi and Eldefrawi (1973); Karlin and Cowburn (1973);
Karlsson et al. (1972); Klett et al. (1973); Olsen et al. (1972); Schmidt and Raftery (1972).
69. Kalderon et al. (1970). Pedro Cuatrecasas, who developed this approach, also
applied it in 1972 to purifying insulin receptors.
70. Weill et al. (1974).
71. Reynolds and Karlin (1978); Raftery et al. (1971).
72. For a historical sketch and references, see Robinson (1997).
73. Changeux et al. (1979); Huganir et al. (1979); Wilson and Raftery (1979).
74. For example, Nickel and Potter (1973); Cartaud et al. (1978).
75. Kistleretal. (1982).
76. For example, Heidmann and Changeux (1980); Hess et al. (1983); Neubig et al.
(1982).
77. "Specific activity" refers to the radioactivity per molecule, expressed as curies/
mole; curies are measures of radioactive disintegrations per unit of time.
78. Cuatrecasas et al. (1974); Lefkowitz (1974); Maguire et al. (1974). Rank-order
for displacing radioactive noradrenaline did not follow rank-order for antagonizing nor-
adrenaline's effects at receptors; binding did not follow the hyperbolic "saturable"
response of dose-response plots but continued without apparent asymptote; and bind-
ing of the pharmacologically inactive stereoisomer was comparable to the active one.
79. Lefkowitx et al. (1974); Levitzki et al. (1974); Aurbach et al. (1974).
80. For example, Lefkowitz and Williams (1977); Bylund and Snyder (1976).
81. Caron and Lefkowitz (1976).
82. Shorr et al. (1981).
83. Shorr et al. (1982a).
84. Cerione et al. (1983).
85. DeLean et al. (1982).
86. Shorr et al. (1982b).
87. See Cozzens (1989); Goldberg (1988); Kanigel (1986).
88. Goldstein et al. (1971).
89. Goldstein et al. reported that stereospecific labeling increased at higher ligand
concentrations; as Pert and Snyder (1973a) noted, such a relationship is unexpected.
Moreover, Simon et al. (1973) stated that they could not replicate Goldstein's results.
Goldsteins subsequent attempts at isolation then resulted in a lipoprotein (Lowney et
al., 1974), inconsistent with later findings.
90. Terenius (1973a, 1973b).
91. Pert and Snyder (1973a, 1973b).
92. Simon et al. (1973). Simon had been pursuing opioid receptors since the mid
1960s (Van Praag and Simon, 1966), prior to efforts by Terenius and Snyder.
93. Kuhar et al. (1973); Martin et al. (1976); Lord et al. (1977).
94. Pert et al. (1973); Simon et al. (1973); Simon and Groth (1975).
95. Katz and Miledi (1970).
96. Ibid., pp. 963, 962.
98. Anderson and Stevens (1973); Colquhoun et al. (1975).
99. Neher and Sakmann (1976).
100. Since obtaining good electrode seals at neuromuscular junctions was difficult,
they used denervated muscle, which has "extrajunctional" receptors over its surface
where seals are easier. These extrajunctional receptors were previously known to have
responses about threefold longer.
101. Hamill et al. (1981). See also Robinson (1997).
102. Schindler and Quast (1980); Nelson et al. (1980).
7
SECOND MESSENGERS
(1951-1990)
Cyclic AMP
During the early 1950s Bernard Katz identified muscle e.p.p.s with increased
membrane permeabilities to Na + and K + induced by acetylcholine, while John
Eccles linked e.p.s.p.s and i.p.s.p.s of spinal cord motoneurons to increased
membrane permeabilities to these and other ions (chapter 4). The notion that
neurotransmitters excited or inhibited by altering specific ionic permeabilities,
which had been established at these sites, seemed applicable at all sites. But
the responses that Katz and Eccles studied had rapid onsets and brief dura-
tions, whereas responses at other sites were notably delayed and prolonged.
Indeed, such striking differences in time courses were one cornerstone for ear-
lier arguments against chemical transmission at neuromuscular junctions and
in the central nervous system (chapter 3). Then Eccles had contrasted rapid
responses at presumed electrical synapses with slow responses at acknowledged
chemical synapses (as with the vagal innervation of the heart). The latter, he
believed, reflected inevitable delays while released acetylcholine diffused to its
receptors and while cholinesterase inactivated it.
But if fast responses also relied on chemical mechanisms, indicating that
chemical transmission could be quite rapid, what produced the slow onset and
protracted time courses at other synapses? An unanticipated explanation for
171
such delays and prolongations (and much more) emerged from different inter-
ests. Earl Sutherland (Fig. 7-1) defined a new mode of cellular communica-
tion—and one that operates at many synapses—through an urge to understand
how hormones act.
Sutherland had received his medical training in St. Louis, and after military
service in World War II, he returned to work on the hormonal regulation of
glucose metabolism with the eminent biochemist Carl Cori. Cori was then
studying phosphorylase, a crucial en2yme in cellular metabolism that splits
glycogen, a polymerized storage form of glucose, into glucose-1-phosphate
subunits (Fig. 7-2A). Glucose-1-phosphate is next converted to glucose-6-
phosphate, which is either metabolized locally to supply cellular energy or
dephosphorylated to glucose, which is released into the bloodstream for metab-
olism elsewhere.1 Phosphorylase, however, exists in an active form, phospho-
rylase a, and an inactive form, phosphorylase b (Fig. 7-2B).
By 1951 two hormones, adrenaline and glucagon,2 were known to raise blood
glucose levels by promoting glycogen breakdown in the liver. That year Suther-
FlGURE 7-1. Earl Wilbur Sutherland, Jr. (1915-1974; courtesy of the National Library
of Medicine).
Second Messengers (1951-1990) 173
FIGURE 7-2. Control of glycogen breakdown to glucose-1-phosphate. A. Active phos-

phorylase a catalyzes the phosphorolysis of glycogen, forming glucose-1-phosphate with
inorganic phosphate (Pi). Glycogen synthesis is catalyzed by a distinct enzyme, glyco-
gen synthase. B. Phosphorylase kinase catalyzes the conversion of inactive phosphory-
lase b to active phosphorylase a through the transfer of a phosphate from ATP to a ser-
ine of phosphorylase. C. Protein kinase A catalyzes the conversion of inactive
phosphorylase kinase to active phosphorylase kinase through the transfer of a phosphate
from ATP to a serine of phosphorylase kinase. cAMP stimulates protein kinase A. D.
Adenylate cyclase catalyzes the conversion of ATP to cAMP and inorganic pyrophos-
phate (PPj); this enzyme is activated by, among other agents, glucagon and adrenaline.
cAMP is hydrolyzed to AMP by phosphodiesterase (i.e., one of the ribose-phosphate
bonds is hydrolyzed).
land and Cori identified phosphorylase as the rate-limiting step for glucose
release.3 They also demonstrated that adrenaline and glucagon increased glu-
cose release by stimulating the conversion of inactive phosphorylase b to active
phosphorylase a.
After Sutherland moved to Cleveland in 1953, he continued to study phos-

phorylase, aided over the years by a number of accomplished colleagues, includ-
ing Reginald Butcher, Theodore Rail, Alan Robison, and Walter Wosilait. There
he showed that activation involved phosphorylation of phosphorylase b, whereas
inactivation involved dephosphorylation of phosphorylase a. Moreover, adren-
aline and glucagon promoted the phosphorylation of phosphorylase b.4 (The
enzyme catalyzing the phosphorylation of phosphorylase Sutherland named
phosphorylase kinase. At that time kinase referred to an enzyme that activated;
it also carried the connotation of phosphorylation. The latter sense prevailed,
and enzymes later termed kinases cause inhibition as well as activation through
phosphorylations.)
For these experiments Sutherland used liver slices, very thin tissue sections
that allow oxygen to diffuse easily to the interior. Such slices were considered
"intact cell" preparations: although cells on the cut surfaces were damaged,
those in the interior remained undisturbed and functioning. At this time hor-
mone responses could be demonstrated with slices but not with "broken cell"
preparations, such as homogenates. Indeed, some claimed that intact cells—
or perhaps even groups of cells—were required for hormones to act.5 But the
contemporary biochemical approach to circumventing the impenetrable com-
plexities of whole cells was to study simplified systems, isolating and examin-
ing individual components.
Sutherland's first stellar achievement was to succeed with such simplifica-
tion, demonstrating in 1957 hormone responses in broken cell preparations:
added adrenaline or glucagon markedly increased phosphorylase activity in liver
homogenates.6 Moreover, he showed that this activation occurred in two steps
with two distinct cellular components. First, a membrane fraction, separated
from liver homogenates, formed a low molecular weight, heat-stable factor in
the presence of the hormones. Second, although the hormones had no effect
on supernatant fractions of the homogenate, adding this factor to the super-
natant fractions increased their phosphorylase activity.
Sutherland's ultimate triumph that year was identifying the activating factor
as cyclic adenosine monophosphate (cAMP; Fig. 7-3A), an unexpected sub-
stance with a structure not previously known in biological systems.7 Indepen-
dently, David Lipkin in St. Louis identified cAMP in barium hydroxide digests
of ATP and then synthesized cAMP chemically.8
Sutherland proceeded to characterize the enzyme catalyzing cAMP synthe-
sis from ATP, adenylate cyclase, and the enzyme destroying cAMP, phospho-
diesterase (Figs. 7-2D and 7-3A).9 Functional models thus depicted hormones
activating the membrane-bound adenylate cyclase to produce cAMP, with the
elevated concentrations of cytoplasmic cAMP promoting the conversion of
phosphorylase b to phosphorylase a. Cytoplasmic concentrations of cAMP then
returned to basal levels (and the hormone stimulation disappeared) as the phos-
FIGURE 7-3. ATP, GTP and some of their products. A. ATP can be hydrolyzed to ADP
and Pi by various ATPases. ATP can also be converted to cAMP and PPj by adenylate
cyclase (below). B. Analogously, GTP can be hydrolyzed by GTPases or converted to
cGMP by guanylate cyclase.
phodiesterase destroyed cAMP. In 1965 Sutherland designated cAMP a "sec-

ond messenger," since cAMP carried information from receptor to cell inte-
rior, while hormones, the "first messengers," brought information to the recep-
tor.10 The general process became known as "signal transduction."
Sutherland found adenylate cyclase activity in all organs; highest activities
were in the brain, where added adrenaline also stimulated cAMP formation.11
At the same time, adrenaline's ability to elevate cAMP levels was tied to cel-
lular responses beyond phosphorylase, for cAMP affected a wide array of cel-
lular properties sensitive to adrenaline, including other enzymes (e.g., inhibit-
ing glycogen synthase, the enzyme that opposes phosphorylases actions12) and
processes (e.g., water flow across the amphibian bladder wall13). The cataloging
of cAMP responses, however, was hindered by the inability of added cAMP to
cross cell membranes and reach its intracellular sites of action. Consequently,
the synthesis of a derivative that could gain entry more easily, dibutyryl-cAMP,
greatly facilitated these surveys.14 Subsequent studies then revealed cAMP's
participation in the whole gamut of cellular regulation, from cell membrane to
nucleus.
One prominent topic was adrenergic stimulation of the heart. In 1965 Suther-
land showed that adrenaline s excitatory effects were preceded by a sharp rise
in cAMP levels (Fig. 7-4A).15 Since /3-adrenergic agonists stimulated adenyl-
ate cyclase in the same rank order as they excited cardiac function, Sutherland
concluded that adenylate cyclase mediated these adrenergic responses. And
FIGURE 7-4. Effects of adrenaline on heart cAMP levels, contractile force, and phos-
phorylase activity. A. Adrenaline was added to perfused hearts, and after the times (in
seconds) indicated on the x-axis, the amount of cAMP (upper panel, expressed on the
y-axis as nmoles per gram of heart) was measured. Contractile force (middle panel,
expressed on the y-axis as percent of the unstimulated value) was also measured after
addition of adrenaline. Contractile force rose after cAMP rose. Phosphorylase activity
of the heart (bottom panel, expressed on the y-axis relative to the mean value before
adding adrenaline) also rose, but more slowly, peaking after the rise in contractile force.
These time courses suggested that the increased contractile force was not due solely to
increased phosphorylase activity. B. A model for the adrenergic receptor-adenylate
cyclase complex shows a catalytic subunit (C) regulated by inhibitory a-adrenergic
receptor subunits and by excitatory /3-adrenergic receptor subunits. (A from Robison
et al. [1965], Fig. 2, courtesy of the American Society for Pharmacology and Experi-
mental Therapeutics. B from Robison et al. [1967], Fig. 4, courtesy of the New York
Academy of Sciences.)
since a-adreneregic agonists decreased cAMP levels in some tissues and antag-
onized some /3-adrenergic effects, he suggested that a- and /3-receptors might
be linked to adenylate cyclase in an opposing fashion (Fig. 7-4B).16
By the early 1970s further investigations demonstrated that administering
dibutyryl-cAMP extracellularly or cAMP intracellularly (by microelectro-
phoresis) altered electrical responses that correlated with accelerated heart rate
and increased contractile force.17 These responses, which mimicked the addi-
tion of /3-adrenergic agonists, were then linked to alterations in particular K +
and Ca2+ currents. In later years the numbers of these currents (and of the
membrane channels conducting them) increased dramatically, and causal expla-
nations for adrenergic effects became correspondingly more complex. Never-
theless, a fundamental notion remained: adrenergic agonists affected cardiac
function through second messenger systems that altered specific channel
conductances.
Sutherland also noted that muscarinic cholinergic agonists decreased cardiac
cAMP levels, albeit modestly.18 Cholinergic antagonism to adrenergic stimula-
tion could then result from cholinergic agonists opposing the rise in cAMP due
to adrenergic agonists. But this explanation, too, was superseded by more com-
plex formulations (see below).
Meanwhile, it had become apparent that additional substances altered cel-
lular levels of cAMP. These included not only other hormones but also acknowl-
edged neurotransmitters, beginning with serotonin (initially demonstrated in
liver flukes, but in 1968 in mammalian brain).19 Then in 1971 Paul Greengard
in New Haven reported, first, that excitation of preganglionic fibers elevated
cAMP levels in sympathetic ganglia, and, second, that dopamine, released from
interneurons in the ganglia, was the stimulant to adenylate cyclase.20 The next
year Greengard demonstrated dopamine-stimulated adenylate cyclase activity
in the brain's basal ganglia, where dopamine plays a central role.21 Thus,
dopamine receptors were coupled to cAMP production in both ganglia and
brain, with cAMP apparently mediating the dopaminergic responses. And in
1974 he described the block of this dopaminergic stimulation of adenylate
cyclase by antipsychotic drugs, those used to treat schizophrenia but known
also to produce motor symptoms through actions on the basal ganglia (chap-
ter 13).22 Still missing from this account, however, are the mechanisms by which
cAMP induces its responses and the mechanisms by which receptors stimulate
or inhibit adenylate cyclase.
(A second cyclic nucleotide, cyclic guanosine monophosphate [cGMP, Fig.
7-3B], was discovered in 1967 during a survey of urinary constituents.23 Over
the next two decades the properties of cGMP were scrutinized, and although
significant physiological roles were discovered—notably in the chain of visual
responses in retinal cells—a definite participation in synaptic transmission was
not established.)
Protein Kinases and Pnospnatases
Glycogen breakdown to glucose is a primary step in liberating energy for mus-

cle contraction, also. While Sutherland was examining the hormonal activation
of phosphorylase b in liver, Edwin Krebs (Fig. 7-5A) was examining the acti-
FIGURE 7-5. A, left, Edwin G. Krebs (1918-). B, right, Edmond H. Fischer (1920-).
(A courtesy of E. G. Krebs. B courtesy of E. H. Fischer.)
vation of phosphorylase b in muscle. Krebs had initially studied medicine a

year behind Sutherland at Washington University, and after military service he,
too, worked with Cori in St. Louis. In 1948 Krebs moved to a faculty position
in Seattle, and there eight years later he described the conversion of inactive
phosphorylase b to active phosphorylase a, collaborating with Edmond Fischer
(Fig. 7-5B), who had emigrated from Geneva a few years earlier.24 This study
with cell-free muscle extracts was published the same year that Sutherland
reported the phosphorylation of liver phosphorylase b. Krebs and Fischer, how-
ever, proceeded to show in 1959 that the phosphorylating enzyme, phospho-
rylase kinase, also existed in two forms, activated and inactivated.25 Incubation
with ATP activated phosphorylase kinase, and adding cAMP augmented this
conversion. They offered the "speculative hypothesis" that "phosphorylase . . .
kinase itself exists in phosphorylated and dephosphorylated forms."26
Finding evidence for this suggestion consumed eight more years. During this
time Krebs showed that administering adrenaline (which stimulates muscle
phosphorylase just as it does liver phosphorylase) increased both phosphory-
lase activity and the level of cAMP in muscle.27 Three years later, in 1968,
Krebs reported that phosphorylation of phosphorylase kinase indeed accom-
panied its activation; moreover, cAMP promoted this phosphorylation.28 Later
that year he purified "phosphorylase kinase kinase" from muscle, the enzyme
catalyzing the phosphorylation of phosphorylase kinase.29 Subsequently this
enzyme became known as "cAMP-dependent protein kinase" or "protein kinase
A" (I will use the latter name).
The causal chain thus ran (Fig. 7-2): adrenaline => increased cAMP =>
active protein kinase A => active (phosphorylated) phosphorylase kinase =>
active (phosphorylated) phosphorylase a =$ glycogen breakdown. Two protein
kinases, protein kinase A and phosphorylase kinase, are involved, each trans-
ferring the terminal phosphate of ATP to a protein (onto the hydroxyl of the
amino acid serine, forming phosphoserine esters, as Krebs showed). Suther-
land described a phosphatase cleaving the phosphate from phosphorylase and
Krebs a phosphatase similarly dephosphorylating phosphorylase kinase.30 Acti-
vation could be turned off as well as on.
Independently, Joseph Larner in Cleveland was examining the opposing reac-
tion, the formation of glycogen from glucose catalyzed by glycogen synthase.
As noted above, adrenaline inhibited glycogen synthase while activating phos-
phorylase, a prime example of what came to be called reciprocal control (the
biochemical equivalent of Sherrington s reciprocal innervation). In 1963 Larner
showed that glycogen synthase also existed in two forms, with their intercon-
version reflecting phosphorylation/dephosphorylation of this enzyme, too.31
Moreover, cAMP activated a kinase that phosphorylated glycogen synthase to
produce the less active form.32 Evidently, cAMP bound to this glycogen syn-
thase kinase to activate its phosphorylating ability; Larner invoked Monod's
notion of allosteric control of protein function in describing the activation of
this kinase.33 Krebs then showed that the kinases were identical: phosphory-
lase kinase kinase and glycogen synthase kinase were the same enzyme (pro-
tein kinase A).34
In 1968 Krebs reported that protein kinase A phosphorylated several other
proteins in vitro.35 Whether it phosphorylated any of these in vivo was not
established, however, and all the enzymes then known to be regulated by cAMP
and protein kinases were involved with glycogen synthesis and breakdown. But
the next year Lester Reed in Austin showed that phosphorylation/dephospho-
rylation also regulated a key enzyme of intermediary metabolism, pyruvate
dehydrogenase.36 That year, 1969, Greengard proposed that "protein kinases
mediate all the diverse effects" of cAMP; indeed, he found protein kinase A
activity in every tissue he examined, concluding that cAMP through this kinase
"may play a role in the regulation of all animal tissues."37
In 1957 P. J. Heald in London had described an increased phosphorylation
of brain proteins following electrical stimulation in vitro; Heald related this
protein phosphorylation to transport mechanisms necessary to restore ionic bal-
ance after stimulation.38 Instead, Greengard in 1969 demonstrated receptor-
mediated increases not only in cAMP synthesis (see above) but also in protein
kinase activity; furthermore, he showed that protein kinases phosphorylated

particular, identifiable proteins in the brain.39 Independently, Richard Rod-
night in London reported in 1970 that cAMP stimulated the phosphorylation
of brain proteins and in 1973 that added noradrenaline as well as electrical
stimulation increased protein phosphorylations.40
Protein phosphorylation thus might play a major role in synaptic transmis-
sion, with likely targets including receptors and the ion channels facilitating
excitatory and inhibitory ionic fluxes. Accordingly, in 1977 Ivan Diamond in
San Francisco and Jean-Pierre Changeux in Paris reported that nicotinic acetyl-
choline receptors, present in postsynaptic membranes from fish electric organs,
could be phosphorylated in vitro by endogenous protein kinases.41 And in 1986
Greengard showed that phosphorylating the y- and 5-subunits of this receptor
altered its functional properties (increased its rate of desensitization).42 The
next year Greengard found that cAMP analogs promoted phosphorylation of
these receptors in muscle cells in vivo.43 Other studies demonstrated phos-
phorylation of ion channels and consequent changes in their properties. For
example, in 1980 Greengard described enhanced Ca2+ fluxes after he injected
protein kinase A into neurons.44 Similarly, W. Trautwein in Homberg and
F. Hofmann in Heidelberg found that injecting protein kinase A into heart cells
enhanced Ca2+ influx, as did adding noradrenaline.45 Noradrenaline's stimula-
FIGURE 7-6. The spreading recognition that multitudes of proteins are regulated
through phosphorylation/dephosphorylation. (Reprinted from Krebs [1994], ©1994,
with permission from Elsevier Science.)
tory effect on the heart was thus attributable to noradrenaline elevating pro-
tein kinase A activity through stimulation of cAMP formation; protein kinase
A would then phosphorylate Ca2+ channels to promote ion fluxes that stimu-
late cardiac activity.
Through the 1980s further evidence accumulated for protein phosphoryla-
tion throughout the organism, with such phosphorylations representing a ubiq-
uitous means for modifying protein function. Other protein kinases were soon
identified (see below), and the number of proteins known to be phosphory-
lated soared (Fig. 7-6).
Phosphorylations could participate in these regulatory processes only if the
phosphorylations were readily reversible. As cited above, Sutherland and Krebs
demonstrated the enzymatic dephosphorylation of phosphorylase and phos-
phorylase kinase. Protein phosphatase activity in the brain was identified soon
afterward.46 By 1983 four classes of protein phosphatases were established,4'
and further studies revealed that these phosphatases were themselves regu-
lated, in part by phosphorylation/dephosphorylation.
G-Proteins
Still missing from this account is the link between neurotransmitters binding
to receptors and the activation of adenylate cyclase to synthesize cAMP. By
1971 neither receptors nor enzyme had been isolated, but proposals centered
on complex systems with regulatory and catalytic subunits.48 Ligand-induced
conformational changes in receptor subunits could induce, through contiguity,
conformational changes to activate catalytic subunits, following the pattern of
allosteric enzymes then being described. But evidence for an intervening
entity—a mobile, amplifying entity—emerged from studies by Martin Rodbell
(Fig. 7-7A) in Bethesda.
Rodbell was born a decade after Sutherland, Krebs, and Fischer, with World
War II interrupting his education. He received his Ph.D. in biochemistry from
the University of Washington (where Krebs and Fischer were) in 1954, and in
1956 he began his career at the National Institutes of Health (NIH), studying
hormonal responses. In 1971 these investigations had evolved into examining
how glucagon stimulated adenylate cyclase. While measuring the binding of
labeled glucagon to liver cell membranes, Rodbell found that adding nucleo-
tides—most prominently guanosine triphosphate (GTP) and guanosine diphos-
phate (GDP) (Fig. 7-3B)—affected the process: as little as 50 nM GTP or GDP
decreased the affinity for glucagon.49 Since GDP as well as GTP was effective,
Rodbell considered that the nucleotides acted by binding to regulatory
(allosteric) sites rather than by phosphorylation.
In addition to these effects on binding, GTP also increased the rate of cAMP
FIGURE 7-7. A, left, Martin Rodbell (1925-1998). B, right, Alfred Goodman Oilman
(1941-). (A courtesy of the National Institutes of Health. B courtesy of A. G. Gilman.)
synthesis, and at very low substrate levels GTP was required for catalytic activ-
ity.50 Rodbell concluded that GTP "play a specific and obligatory role in the
activation . . . by glucagon."51 Moreover, Rodbell showed in 1974 that this acti-
vation by GTP occurred after glucagon bound to the receptor and that an ana-
log of GTP that was not hydrolyzed could induce a persisting activation.52 Such
analogs proved to be valuable both practically, because of this long-lasting acti-
vation, and conceptually, because binding rather than phosphorylation must
then be the essential process.
On the other hand, GDP did not activate (in contrast to its effects on glucagon
binding), and GTP's activation was transient, presumably because it was
hydrolyzed to inactive GDP. Others soon found that GTP and GTP analogs
activated adenylate cyclases from several sources following stimulation by sev-
eral ligands.53
Efforts then focused on identifying the component to which GTP bound and
on defining how the resulting complex activated adenylate cyclase. In 1976 Dan
Cassel and Zvi Selinger in Jerusalem described a distinct GTPase activity in
avian red blood cell membranes that was stimulated by adrenergic agonists;
they argued that GTP hydrolysis was required for returning the activated sys-
tem to its unstimulated state.54 Cassel and Selinger next showed that ligands
binding to their receptors promoted the release of GDP.55 (Rodbell initially

found that GDP decreased hormone binding to receptor; the necessary corre-
late is that hormone binding to receptor will decrease GDP binding to its sites.)
Meanwhile, Thomas Pfeuffer in Wiirzberg labeled a 42 kDa peptide from avian
red blood cell membranes using a reactive analog of GTP, and he also sepa-
rated this peptide by affinity chromatography using GTP complexed to a
matrix.56 Pfeuffer then demonstrated that when GTP bound to this peptide
(which he accordingly named "G-protein"), its association with—and thus acti-
vation of—adenylate cyclase was promoted.57
A model from 1981 (Fig. 7-8A) showed a complex of receptor, G-protein
(containing GDP), and adenylate cyclase. Ligands binding to the receptor facil-
itated the association between receptor and G-protein, with concomitant
release of GDP. The G-protein then bound GTP, permitting association with
and activation of adenylate cyclase. This activation ceased when the G-protein
hydrolyzed GTP to GDP, causing dissociation from the adenylate cyclase. The
model also indicated that the G-protein moved between receptor and adenyl-
ate cyclase.58 And implicit was the capacity for amplification: one receptor could
activate multiple G-proteins, and each G-protein could activate one adenylate
cyclase to form multiple cAMP molecules before the slow hydrolysis of GTP
terminated this activation.
Purification and further characterization of the G-proteins came prominently
from Alfred Goodman Gilman (Fig. 7-7B), destined by nature, nurture, and
name to be a pharmacologist: he was the son of one author of the standard
textbook of pharmacology and namesake of the other.59 Gilman was born a gen-
eration after Rodbell and received his M.D. and Ph.D. degrees in 1969 from
Case Western Reserve University, where Sutherland had identified cAMP. After
two years at NIH, he moved to Charlottesville and was soon resolving a num-
ber of significant issues. Pertinent here is his purification of the G-proteins.
Gilman approached this problem by exploiting various mutant tissue culture
cells that lacked different components of the receptor/G-protein/adenylate
cyclase system. Gilman could then characterize the components by reconsti-
tuting the functional system from constituents lacking certain capabilities.60
And he could use systems lacking certain capabilities (such as response to GTP)
to assay for components that restored function.
By 1980 Gilman isolated from mammalian liver three peptides that partici-
pated in the activation by GTP, having molecular weights of 52, 45, and
35 kDa.61 Avian red blood cells, however, contained only the 45 and 35 kDa
peptides; the 52 kDa peptide was subsequently recognized as a variant of the
45 kDa unit.62 And in 1984 Lutz Birnbaumer in Houston identified a 5 kDa
peptide (later corrected to 7 kDa).63 The native G-protein was thus a trimer
of a (45 kDa), ft (35 kDa), and y (7 kDa) subunits. Only a, however, bore the
GTP-binding site.
FIGURE 7-8. Models for G-protein actions. A. Receptor (R), G-protein (G), and adenyl-
ate cyclase (C) are in, or tethered to, the cell membrane. When an agonist binds to R,
G interacts with R and exchanges its bound GDP for GTP. With GTP bound, G inter-
acts with C, stimulating catalytic activity that continues until G hydrolyzes GTP to GDP.
This hydrolysis promotes dissociation of G from C, turning off the stimulation. B. The
exchange of GTP for GDP triggers a dissociation of a from /3y subunits of a het-
erotrimeric G-protein. Hydrolysis of GTP to GDP allows reassociation of the complex.
(A from Limbird [1981], Fig. 1, reproduced by permission of the author, ©The Bio-
chemical Society. B from Stryer [1988], Fig. 38-5, ©1988 by W. H. Freeman and Com-
pany, used with permission.)
184
Parallel studies on visual receptors in the retina revealed an analogous GTP-

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

of second messenger systems had to be revised during this decade, however.
Muscarinic receptors involved in the vagal slowing of the heart seemed to be
coupled to Gj-proteins,71 so when acetylcholine bound to the receptors G;a
would be liberated to inhibit adenylate cyclase. But in 1985 compelling evi-
dence appeared for G-proteins interacting directly with ion channels and
thereby modifying their conductances.72 Two years later Eva Neer and David
Clapham in Boston showed that liberated /3y subunits interacted directly with
a cardiac K + channel, opening it to cause a slowing of the heart.73
Previously, the /3y complexes had been thought to function solely through
their uniting with a subunits and—through formation of inactive a/3y
complexes—preventing a from binding to adenylate cyclase. Now distinctions
between various /3y subunits were stressed, as these subunits appeared to act
differentially on their own. Moreover, further studies in the succeeding years
provided additional evidence for both a and j3y subunits binding to and affect-
ing directly various cellular proteins beyond the second messenger systems.
Ca2+
By 1970 cyclic nucleotides were acknowledged as second messengers that car-

ried information from surface receptors throughout the cell interior. That year
Howard Rasmussen in Philadelphia proposed as a companion to these organic
molecules a quite different substance, inorganic calcium ions (Ca2+), provid-
ing cells with "two interrelated intracellular messengers" acting in concert.74
Rasmussen suggested that after cAMP triggered protein phosphorylations—as
shown by Krebs, Fischer, Greengard, and others—Ca2+ would then activate
the phosphorylated proteins. But he admitted that Ca2+ might also serve as a
signaling substance independent of cAMP.
Responses to Ca
Several roles for Ca2+ were then apparent. Longest established was the Ca2+
requirement for muscle contraction, noted in 1883 by Sydney Ringer in Lon-
don.75 In 1947 Victor Heilbrunn in Philadelphia showed that injecting Ca2+
into muscle provoked contraction, and in 1952 Alexander Sandow in New York
formulated a scheme for "excitation-contraction coupling" in which muscle
action potentials liberated Ca2+ intracellularly, with this Ca2+ then activating
the contractile proteins.76 During the 1950s and 1960s electron microscopists
identified both a tubular system within muscle cells and the association of this
"sarcoplasmic reticulum" with periodic imaginations of the muscle cell mem-
branes, "transverse tubules."77 Physiologists showed that these transverse
tubules conducted electrical signals to the cell interior,78 where Ca2+,

sequestered within the sarcoplasmic reticulum,79 would be released into the
cytoplasm. Meanwhile, Wilhelm Hasselbach in Heidelberg and Setsuro Ebashi
in Tokyo demonstrated in the early 1960s that muscle relaxation was effected
by removing Ca2+ from the cytoplasm: Ca2+ was reaccumulated within the sar-
coplasmic reticulum.80 The cycle of muscle contraction and relaxation thus fol-
lowed a cycle of Ca2+ release and sequestration.
A second role was in secretion. As an initial step toward defining how cholin-
ergic nerves to the adrenals evoke the secretion of adrenaline, William Dou-
glas in New York perfused the adrenals with acetylcholine in media containing
various ions. Acetylcholine, Douglas reported in 1961, caused adrenaline
release only when Ca2+ was also present in the perfusion media. Moreover, the
magnitude of the adrenaline release varied with the Ca2+ concentration.81 Since
Katz had shown that acetylcholine acts on the exterior of muscle cells to alter
permeability to Na + and K + , Douglas proposed that acetylcholine stimulated
secretion similarly: "by causing calcium ions to penetrate the adrenal... cells."82
Indeed, he found a marked increase in the uptake of labeled Ca2+ when he
added acetylcholine to the perfusion media.83 Douglas named the process
"stimulus-secretion coupling" to stress the parallel with muscle, and he sug-
gested that Ca2+ might participate analogously in noradrenaline secretion from
sympathetic nerves. He also demonstrated a Ca2+ requirement for secretion
by other glands.84
A third and related role was in neurotransmitter release, as delineated at
neuromuscular junctions and ganglionic synapses by Katz and Ricardo Miledi
in London. At these sites the absence of Ca2+ prevented synaptic transmission,
just as it prevented adrenaline secretion. In 1965 Katz and Miledi showed that
during perfusion with Ca2+-deficient media, the localized addition of Ca2+ at
motorneuron terminals, delivered by micropipets, promoted acetylcholine
release.85 This observation thus countered arguments that Ca2+ was required
merely for impulse propagation to the nerve terminals.
Rasmussen's 1970 proposal specified several additional roles for Ca2"1", includ-
ing several steps in the control of metabolism; others roles soon appeared.
But two salient issues loomed beyond the mere recognition that Ca2+ partici-
pated: how the local Ca2+ concentration was regulated—increased to initiate
responses and reduced to terminate them—and how Ca2+ actually caused these
responses.
Regulation or Cytoplasmic Ca Concentrations
Two sources for elevating cytoplasmic Ca2+ levels were illustrated in these stud-
ies, extracellular and intracellular. Katz and Miledi defined inward Ca2+ cur-
rents, attributable to Ca2+ flowing through transmembrane channels from
extracellular fluid to cytoplasm.86 Throughout the next decades a host of Ca2+

channels—present almost ubiquitously in cells across the biological universe—
were distinguished according to their electrical properties and sensitivities to
various inhibitors.87 These, like the Na+ and K + channels of the Hodgkin-Hux-
ley model for nerve action potentials (chapter 4), were voltage-gated channels:
their openings and closings depended on the transmembrane potential. Con-
sequently, the voltage-gated Ca2+ channels linked membrane potential changes
to the multitudes of cellular responses regulated by cytoplasmic Ca2+ concen-
trations. And in 1972 A. Fleckenstein in Freiburg demonstrated that certain
drugs known both to alter cardiac function and to be antagonized by higher
Ca2+ concentrations acted through a blockade of particular Ca2+ channels.88
These "calcium channel blockers"—including such drugs as nifedipine (Pro-
cardia) and diltiazem (Cardizem)—became widely used in treating angina, car-
diac arrhythmias, hypertension, and other disorders.
On the other hand, in muscle the sarcoplasmic reticulum was an intracellu-
lar reservoir of releasable Ca2+. The corresponding structure in nonmuscle
cells, the "endoplasmic reticulum," was later recognized as such a source in
these cells, too (see below).
For influxes of Ca2+ to alter the cytoplasmic concentration acutely, that con-
centration must normally be maintained at a lower level89 by Ca2+ pumps,
energy-consuming active transport systems capable of extruding Ca2+ against
its electrochemical gradient.90 In 1966 H. J. Schatzmann in Bern identified a
cell membrane transport system that used ATP as its energy source (it was a
Ca2+-ATPase).91 A few years later Harald Reuter in Bern and Mordecai
Blaustein in Cambridge described a parallel transport system that exchanged
extracellular Na+ for intracellular Ca2+, driven by the transmembrane elec-
trochemical gradient for Na + . 92
As noted above, Hasselbach and Ebashi identified a system that actively
transported Ca2+ from cytoplasm to sarcoplasmic reticulum. This was also a
Ca2+-ATPase, related to but distinct from the cell membrane Ca2+-ATPase.
The same Ca2+-ATPase was later recognized in the endoplasmic reticulum of
nonmuscle cells.
Mechanisms or Action
Accounts of how Ca2+ initiated its multitudes of responses were less tidy, evolv-
ing into a catalog of mechanisms. In some instances Ca2+ acted directly, as in
binding to, and thereby activating, the proteolytic enzyme calpain or various K +
and Ca2+ channels that control cellular function.93 In other instances Ca24" func-
tioned by binding to, and thereby activating, regulatory proteins, which in turn
affected the responses of further systems. During this time two important exam-
ples of such regulatory proteins were characterized, troponin and calmodulin.
Ebashi, while continuing his investigations on Ca2+ activation of muscle, sep-

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

further second messengers that were unrelated structurally to any previously
known. The paths to their recognition originated in Lowell Hokin and Mabel
Hokin's search in Montreal for an acetylcholine-stimulated labeling of nucleo-

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

sengers, diacyglycerol and inositol-frisphosphate. Phosphatidylinositol-Znsphosphate
contains a three-carbon, three-hydroxyl backbone, glycerol. The first two hydroxyls of
this alcohol are esterifed (acylated) with long chain fatty acids and the third with a phos-
phate group. This phosphate also forms an ester linkage with inositol, a six-carbon, six-
hydroxyl cyclic alcohol. Two other hydroxyls of inositol are esterifed with phosphate
groups. Phospholipase C cleaves between the third glycerol hydroxyl and the linking
phosphate, liberating glycerol with two fatty acids esterified (diacylglycerol) plus inosi-
tol with three phosphates esterified (inositol-frisphosphate).
ulated hydrolysis could "raise cell-surface permeabilities to Ca2+" and thereby

pass the signal on.114
Direct evidence came from an insect physiologist in Cambridge, Michael
Berridge. He had been studying the secretion of saliva by blowflies, which is
stimulated by serotonin, and he could measure secretion, Ca2+ influx, and phos-
phatidylinositol hydrolysis continuously.115 In accord with MichelPs hypothesis,
Berridge in 1979 found that when serotonin stimulated salivary secretion, it
increased both Ca24" entry into isolated salivary glands and phosphatidylinosi-
tol hydrolysis.116 On the other hand, removal of external Ca2+ prevented the
increased secretion without affecting phosphatidylinositol hydrolysis, as would
be expected if hydrolysis were a step between stimulation of receptor and Ca2+-

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

inositol-£mphosphate.128 Diacylglycerol thus served as a second messenger
between receptor occupancy and protein kinase C activation. Its actions were
then terminated by phosphorylation, which converted active diacylglycerol to
inactive phosphatidic acid.129
Receptor-stimulated hydrolysis of phosphatidylinositol-fozsphosphate thus
generates two second messengers. (1) Released diacylglycerol activates partic-
ular members of the protein kinase C family present locally, with consequent
phosphorylation of proteins that regulate processes from cell membrane to
nucleus. (2) Released inositol-£hsphosphate triggers Ca2+ efflux from stores in
the endoplasmic reticulum, elevating cytoplasmic Ca2+ levels. This Ca2+ then
acts directly or through binding to regulatory proteins such as calmodulin; its
targets include different protein kinases with different substrate specificities.
Concr
onclusions
The concept of a second messenger arose from studies on the hormonal con-
trol of glucose availability, but it matured into appreciations of general mech-
anisms for signal transmission within and among all cells. Receptors could trig-
ger an increase in the cytoplasmic concentrations of second messengers through
their formation (e.g., cAMP, inositol-fmphosphate, diacylglycerol) and through
their entry or release (e.g., Ca 2+ ). The second messengers could then alter cel-
lular function directly (e.g., Ca2+ affecting certain ion channels) or by activat-
ing certain enzymes (e.g., protein kinase A, CaM-kinase II, protein kinase C).
Protein kinases could phosphorylate particular proteins to regulate discretely,
if sometimes broadly, a range of cellular functions: cell division, growth, gene
expression, biochemical syntheses and degradations, motility, permeability,
excitation, and so on. In some cases G-proteins linked receptor occupancy to
second messenger formation (e.g., cAMP, inositol-fnsphosphate, diacylglyc-
erol). Moreover, G-proteins could also act directly on cellular components (e.g.,
ion channels).
These cascades of reactions amplified responses. They also directed them,
depending on the particular receptor occupied, the second messenger formed,
the effector activated, and the substrates available to that effector. The func-
tional consequences could be evanescent, reflecting the rapid removal of sec-
ond messenger and dephosphorylation of proteins, or prolonged as a result of
some long-lasting change initiated by the second messenger, such as the new
synthesis of some protein.
Defining the complexities of these systems consumed several decades and
demanded notable ingenuity and insight. Indeed, Nobel Prizes were awarded
to Sutherland (1971), Krebs (1992), Fischer (1992), Rodbell (1994), Gilman
(1994), and Greengard (2000) for their achievements—as well as to Carl and
Gerty Cori (1947), who laid the groundwork for the initial discoveries.
Appreciating the significance of these processes required time and flexibil-
ity, also. Through the 1950s neurotransmitter responses had been depicted as
depolarizations or hyperpolarizations elicited through the opening of channels
specific for certain ions. The subsequent investigations then added second mes-
senger systems, as primary effectors or as modulators of neurotransmitter
actions, to the earlier mechanisms for synaptic transmission. Moreover, this dis-
tinction between receptors opening channels and receptors acting through sec-
ond messengers complemented contemporary realizations that the structures
of neurotransmitter receptors also fall into two major classes (chapter 8).
Notes
1. At this time both the breakdown and the synthesis of glycogen were thought to be
catalyzed by the same enzyme, phosphorylase. (Leloir et al., 1959, demonstrated that a
different enzyme, glycogen synthase, was responsible for synthesis; this was a major con-
ceptual advance, establishing the pattern seen frequently thereafter of synthesis and degra-
dation following separate pathways, separately regulated.) Phosphorylase was so named
because the breakdown proceeds by phosphorolysis: breaking glucose-glucose bonds by
inserting a phosphate group (forming glucose-1-phosphate fragments). Only liver pos-
sesses glucose-6-phosphatase, so only liver can liberate free glucose from glycogen.
2. Hormones are regulatory substances released into the bloodstream. Adrenaline is
released by the adrenals, glucagon (then known as the "hyperglycemic-glycogenolytic
factor") by the pancreas.
3. Sutherland and Cori (1951).
4. Sutherland and Wosilait (1955); Rail et al. (1956).
5. See Sutherland (1972).
6. Rail et al. (1957).
7. Sutherland and Rail (1957).
8. Cook et al. (1957); Lipkin et al. (1959). Sutherland and Lipkin independently
approached Leon Heppel for a reagent, and he put them in touch with each other. Cook
et al.s original formula, containing two adenine nuclei, was corrected in their second
paper.
9. Sutherland et al. (1962); Butcher and Sutherland (1962). Alternative names for
adenylate cyclase are adenyl cyclase and adenylyl cyclase.
10. Sutherland et al. (1965).
11. Sutherland et al. (1962); Klainer et al. (1962).
12. Belocopitow (1961).
13. Handler et al. (1968).
14. Falbriard et al. (1967).
15. Robison et al. (1965).
16. Robison et al. (1967). See also Turtle and Kipnis (1967). By contrast, others at
this time were looking for adrenergic receptors elsewhere. For example, Honig and
Stam (1967) proposed that adrenaline acted directly on the contractile proteins of heart
muscle.
17. Yamasaki et al. (1974); Reuter (1974). See also Tsien (1974).
18. Murad et al. (1962).
19. Mansour et al. (1960); Kakiuchi and Rail (1968).
20. McAfee et al. (1971); Kebabian and Greengard (1971).
21. Kebabian et al. (1972).
22. Clement-Cormier et al. (1974).
23. Ashman et al. (1963).
24. Krebs and Fischer (1956).
25. Krebs et al. (1959).
26. Ibid., p. 2872.
27. Posner et al. (1965).
28. DeLange et al. (1968).
29. Walsh et al. (1968).
30. Sutherland and Wosilait (1955); Riley et al. (1968).
31. Friedman and Lamer (1963).
32. Rosell-Perez and Lamer (1964).
33. Huijing and Lamer (1966).
34. Solderling et al. (1970).
35. Walsh et al. (1968).
36. Linn et al. (1969).
37. Kuo and Greengard (1969), p. 1354.
38. Heald (1957, 1962).
39. Miyamoto et al. (1969); Ueda et al. (1973).
40. Weller and Rodnight (1970); Reddington et al. (1973).
41. Gordon et al. (1977); Teichberg et al. (1977).
42. Huganir et al. (1986).
43. Miles et al. (1987).
44. Kaczmarek et al. (1980); Castellucci et al. (1980).
45. Osterrieder et al. (1982).
46. For example, Weller and Rodnight (1971); Maeno and Greengard (1972).
47. Ingebritsen and Cohen (1983).
48. For example, Robison et al. (1967).
49. Rodbell et al. (1971b).
50. Rodbell et al. (1971a). In retrospect, it seems that RodbelFs ATP contained low lev-
els of contaminating GTP. Consequently, only with low concentrations of ATP (and thus
negligible adventitious GTP) did the requirement for added GTP become obligatory.
51. Ibid., p. 1877.
52. Rodbell et al. (1974); Londos et al. (1974).
53. For example, Bockaert et al. (1972); Wolff and Cook (1973).
54. Cassel and Selinger (1976).
55. Cassel and Selinger (1978).
56. Pfeuffer (1977).
57. Pfeuffer (1979).
58. By this time the reigning conception of membrane structure was the "fluid-
mosiac" model, which depicted proteins free to move laterally through the fluid lipid
bilayer (see Robinson, 1997). Consequently, G-proteins would be able to move in the
plane of the membrane from receptor to adenylate cyclase.
59. L. S. Goodman and A. Gilman's The Pharmacological Basis of Therapeutics, was
first published in 1941 (and continues to the present, although with different authors).
60. This reconstitution technique was previously applied to receptor and adenylate
cyclase by Orly and Schramm (1976).
61. Northup et al. (1980).
62. Hanski et al. (1981); Robishaw et al. (1986).
63. Hildebrandt et al. (1984).
64. Fungetal. (1981).
65. Manning and Gilman (1983).
66. Hamm (1998).
67. Hanski et al. (1981); Northup et al. (1982). See also Pfeuffer (1979).
68. Bokoch et al. (1983, 1984). Independently, Birnbaumer obtained similar results
(Hildebrandt et al. 1983). Arguments for the existence of inhibitory G-proteins existed
earlier (for example, Londos et al., 1981).
69. For example, Cassel and Pfeuffer (1978); Katada and Ui (1982).
70. Arguments for G; coupling to o^-adrenergic receptors included reconstitution
experiments by Cerione et al. (1986). See also Cotecchia et al. (1990).
71. Arguments for Gj coupling to muscarinic receptors included reconstitution exper-
iments by Florio and Sternweis (1985).
72. Pfaffinger et al. (1985); Breitwieser and Szabo (1985).
73. Logothetis et al. (1987). However, these experiments were disputed for some
time: see Birnbaumer et al. (1990); Schneider et al. (1997).
74. Rasmussen (1970), p. 409.
75. For historical accounts see Needham (1971); Robinson (1997).
76. Heilbrunn and Wiercinski (1947); Sandow (1952).
77. For example, Porter and Palade (1957); Franzini-Armstrong and Porter (1964);
Peachey (1965).
78. For example, Huxley and Taylor (1958); Freygang et al. (1964).
79. Costantin et al. (1965); Winegrad (1965).
80. Hasselbach and Makinose (1961); Ebashi and Lipmann (1962).
81. Douglas and Rubin (1961).
82. Ibid., p. 40.
83. Douglas and Poisner (1962).
84. For example, Douglas and Poisner (1963, 1964).
85. Katz and Miledi (1965). In these and other electrophysiological studies cited here,
the release of neurotransmitter was inferred from the presence of postsynaptic poten-
tials. Earlier proposals for Ca2+ involvement include Hodgkin and Keynes (1957) and
Birks and Macintosh (1957).
86. Katz and Miledi (1969). Hodgkin and Keynes (1957) identified a Ca2+ current
associated with the squid action potential, but this Ca2+ current was relatively tiny. Fatt
and Ginsborg (1958) showed that invertebrate muscle action potentials included a pre-
dominant Ca2+ current.
87. See Hagiwara and Byerly (1981); Bean (1989).
88. Kohlhardt et al. (1972); Spedding (1985).
89. See Hodgkin and Keynes (1957).
90. For a historical account, see Robinson (1997).
91. Schatzmann (1966).
92. Reuter asnd Seitz (1968); Blaustein and Hodgkin (1969).
93. See Melloni and Pontremoli (1989); Marty (1989).
94. Ebashi and Kodama (1965); Ebashi et al. (1967).
95. Greaser and Gergely (1971).
96. Collins et al. (1973).
Second Messengers (1951-1990) 19?
97. Cheung (1970). See also Kakiuchi and Yamazaki (1970).

98. Teo et al. (1973).
99. Dedman et al. (1978). Watterson et al. (1980) reported the complete sequence.
100. Cheung et al. (1978).
101. See Cheung (1981); Means et al. (1982).
102. Schulman and Greengard (1978); Kennedy and Greengard (1981). See also Gold-
enringet al. (1983).
103. See Colbran and Soderling (1990).
104. See Campbell (1983); Hille (1992).
105. For a historical account, see Robinson (1997).
106. M.R. Hokin and Hokin (1953, 1954).
107. L.E. Hokin and Hokin (1955).
108. L.E. Hokin and Hokin (1959).
109. M.R. Hokin and Hokin (1964a).
110. For example, Fisher and Mueller (1968).
111. M.R. Hokin and Hokin (1964b).
112. Durell et al. (1968, 1969).
113. Michell (1975).
114. Ibid., p. 137.
115. Berrdige showed that the rate-limiting step for Ca2+ appearance in the saliva
was Ca2+ influx into the gland cells; hence the rate of apppearance—which could be
monitored continuously—equaled the rate of influx. Berridge also equilibrated the gland
cells with radioactive inositol, which labeled the phosphatidylinositol; hence the subse-
quent release of radioactive inositol—which also could be monitored continuously—
reflected the hydrolysis of phosphatidylinositol.
116. Fain and Berridge (1979).
117. Berridge (1983).
118. Streb et al. (1983).
119. Berridge (1983); Storey et al. (1984).
120. Supattapone et al. (1988); Ross et al. (1989).
121. Ryu et al. (1987). See also Rhee et al. (1989).
122. For example, Cockcroft and Gomperts (1985); Smrcka et al. (1991); Taylor et
al. (1991).
123. Inoue et al. (1977).
124. Takai et al. (1979a).
125. Takai et al. (1979b).
126. See Kaczmarek (1987); Nishazuka (1988).
127. For example, Tanaka et al. (1984); DeRiemer et al. (1985).
128. Takai et al. (1979b).
129. Kaibuchi et al. (1983).
8
RECEPTOR
STRUCTURES AND
RECEPTOR FAMILIES
(1983-1990)
Molecular Biology ana Recomiinant DNA Techniques
In 1953 James Watson and Francis Crick proposed a double-helix model for
DNA, with helices linked by pairings between the complementary nucleotide
bases of each strand. The elaborations and extensions that followed in subse-
quent decades included demonstrations of how DNA specified protein struc-
ture.1 Successive triplets of the four nucleotide bases that constitute DNA (ade-
nine, guanine, thymine, and cytosine) designate successive amino acids of the
encoded protein. To direct protein synthesis, the sequence of nucleotide base
triplets is first "transcribed" into messenger RNA (mRNA) bearing the com-
plementary2 sequence of nucleotide bases. The mRNA then migrates from
nucleus to cytoplasm, where its complementary nucleotide triplets are "trans-
lated" into the sequence of amino acids.3 These triplets direct the stepwise link-
ing of amino acids into a peptide chain, with that assembly catalyzed by ribo-
somal enzymes. Information thus flows from DNA to mRNA to protein.
These understandings led also to the development of new techniques, includ-
ing powerful methods for examining protein structure and altering it in highly
specific ways. A range of restriction endonucleases were identified that cleave
DNA at distinct sites specified by characteristic sequences of nucleotide bases.
Various restriction endonucleases could thus generate specific fragments of
199
DNA. Also identified were DNA ligases that join together free ends of DNA
strands. Recombinant DNA could therefore be constructed by cutting native
DNA with restriction endonucleases and assembling fragments into new strands
with DNA ligases. Novel strands of DNA with desired sequences could be
formed from existing fragments or by incorporating short stretches of nucleo-
tides (oligonucleotides) synthesized chemically or enzymatically.
Important tools for generating multiple copies of a particular stretch of DNA
were bacterial plasmids, strands of DNA separate from the bacterial chromo-
some and serving as accessory chromosomes. These plasmids are replicated
during bacterial division like the single bacterial chromosome. Bacterial plas-
mids could be isolated, their DNA cut by restriction endonucleases, pieces of
new or altered DNA inserted with ligases, and the plasmid then replaced in
the bacteria ("transformation").
When a single bacterium containing a plasmid with a particular strand of
DNA undergoes successions of cell division, its progeny—a colony of cells
grown in vitro—will contain that DNA. These progeny are identical genetically
and thus are "clones"; in laboratory parlance the DNA is "cloned." This pro-
cedure provides a means for generating macroscopic quantities of a selected
or constructed DNA molecule.
Among the various "expression" procedures for synthesizing protein encoded
by DNA, the Xenopus oocyte system is particularly relevant here. First, the
DNA is transcribed into complementary mRNA. For this, tissue culture cells
may be "transfected" with plasmid cDNA, which then directs a massive tran-
scription. (In later experiments the mRNA was often synthesized by cell-free
systems.) Second, the resulting mRNA is extracted from the transfected cells
and injected into oocytes of the frog Xenopus laevis, which readily synthesize
protein from exogenous mRNA (translation or expression). The properties of
this newly synthesized protein, such as a neurotransmitter receptor that oocytes
ordinarily do not express—can then be studied in the injected cell.
Nicotinic Cholinergfic Receptors
Earlier investigators isolated and purified nicotinic cholinergic receptors from

fish electric organs, describing four protein subunits present as a pentameric
a$y8 complex that surrounded a central ion-conducting channel (chapter 6).
Specifying the chemical identity, however, requires knowledge of the sequence
in which amino acids appear in this protein and their spatial relationship (i.e.,
the three-dimensional structure of the protein). Mechanistic understandings
require, in addition, an explanation of how acetylcholine s binding to a sub-
units alters the overall structure and its conductivity: triggering the transition
from a closed, resting configuration to an open, active one.
Receptor Structures and Receptor Families (1983-1990) 201
Amino Acid Sequence
In 1980 Michael Raftery in Pasadena reported the amino acid sequences of

the first 50 or so amino acids from each of the isolated a, /3, y, and 8 subunits.4
These he determined by stepwise cleavages of the terminal amino acid from a
chain, identifying the liberated amino acid after each cycle. The identified
sequences, however, represented only a tenth of the total, and that procedure
could not progress significantly further. Although the cleavage efficiency at each
cycle is high, even tiny errors are multiplied progressively.
Frederick Sanger devised an approach for sequencing insulin in the early
1950s that used enzymatic and chemical means to cleave the peptide chain at
specific points, producing various overlapping fragments short enough to be
sequenced stepwise. Piecing together the individual sequences then revealed
the overall sequence. Sangers method, however, was laborious. It also was not
feasible with many intrinsic membrane proteins. Their transmembrane seg-
ments were highly hydrophobic (necessary for insertion within the surround-
ing lipid bilayer), and this hydrophobicity limited their compatibility with sol-
vents required for separatory and analytical procedures.
In 1977 Sanger in Cambridge, UK and Allan Maxam and Walter Gilbert in
Cambridge, Mass., independently developed rapid methods for determining
the sequence of nucleotide bases along a strand of DNA.5 The amino acid
sequence can then be read from the DNA sequence, following the rules for
which nucleotide triplet specifies which amino acid. These techniques greatly
facilitated protein sequencing, but they required an initial identification of the
particular DNA sequence encoding that protein.
Shosaku Numa (Fig. 8-1A) soon applied these methods to sequencing pep-
tide hormones, endorphins, and their precursors. Numa had received his M.D.
from Kyoto in 1953, and after three years of clinical training he was awarded
a Fulbright fellowship to work in biochemistry at Harvard University. After a
further three years examining enzymes of lipid biosynthesis in Munich, he
returned to Kyoto, where he continued with lipid biochemistry for a decade
and a half. But with the advent of molecular biological approaches, Numa
turned his efforts to these techniques, which he then adapted to studying nico-
tinic receptors. As the initial step in this venture, he reported in 1982 the amino
acid sequence for the a subunit.6
First, Numa isolated the mRNA from Torpedo electric organs, which spec-
ifies all the proteins being produced by the organ. From these mRNA mole-
cules he synthesized, enzymatically,' their complementary DNA (cDNA)
sequences. The resulting cDNA fragments thus encoded all the proteins being
produced in the organ. Numa next created a "cDNA library" by inserting all
these cDNA fragments randomly into plasmids, which he then incorporated
into bacteria so that an individual bacterium received randomly a particular
FIGURE 8-1. A, left, Shosaku Numa (1927-1992). B, right, Robert]. Lefkowitz (1943-).
(A courtesy of Osamu Hayashi. B courtesy of R. J. Leftowitz.)
fragment. The progeny of each transformed bacterium—its clones—would all

contain plasmids bearing a certain fragment of cDNA.
To find which bacterial clones contained the cDNA for the a subunit, Numa
then screened the cDNA library with probes based on Raftery's partial deter-
mination of the sequence. This screening involved searching, through
hybridization experiments,8 for complementary matches between the nucleo-
tide base sequences of clone and probe. One probe represented the pen-
tapeptide including amino acids 25-29 of the a subunit. However, more than
one nucleotide triplet may encode a given amino acid (the code is "degener-
ate"), so Numa was forced to use 32 different DNA oligonucleotides to repre-
sent all the ways that these five amino acids could be encoded. From 200,000
bacterial clones in the library, this probe selected 57. A second probe, repre-
senting amino acids 13-18 from Raftery's partial sequence and also containing
32 alternative representations, selected 20 clones from the first 57: these clones
thus contained DNA complementary to both probes.
Numa then sequenced the DNA from two of these clones.9 With this infor-
mation, plus Raftery's determination of the first 54 amino acids, Numa could
then deduce the sequence of all 437 amino acids in the a subunit.10 Numa
Receptor Structures ana Receptor Families (1983-1990) 203
identified four stretches of highly hydrophobic amino acids long enough to

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

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

exhaustive analyses, with images reconstructed statistically from the diffrac-
tion patterns of thousands of receptor molecules. The derived representation
provided convincing views of the receptor's contours, including cross-sections
depicting access routes to and from the transmembrane region (Fig. 8-2A).
Nevertheless, the course of the channel across the bilayer thickness could not
be defined at this resolution, nor could the individual amino acids of the pro-
tein be identified.
Meanwhile, Numa was providing complementary information about the
receptors functional structure using other new techniques of molecular biol-
FIGURE 8—2. Structures of ligand-gated ion channels. A. Section through the nicotinic
receptors of Torpedo electric organ, determined by electron microscopy/image recon-
struction. The figure depicts large lobes surrounding the extracellular access route to
the transmembrane channel (not resolved) as well as the smaller lobes surrounding the
intracellular egress route for Na + entering the cytoplasm. (K + moves along this route
in the opposite direction.) The elliptical image below the receptor was identified as an
extraneous protein (see Mitra et al., 1989). B. Diagram of the three rings of negative
charges flanking the transmembrane channel. A cation (circle enclosing +) is shown at
the extracellular mouth of the channel. This diagram is superimposed on Toyoshima
and Unwin's recontructed image. The chains of hexagons represent chains of sugars
covalently bonded to extracellular regions. C. Common folding pattern for subunits
of ligand-gated ion channels, showing the extracellular N-terminal and extracellular
C-terminal segments, four transmembrane a-helices that span the bilayer (M2 is
shaded), and the cystine loop in the N-terminal segment. (A from Toyoshima and Unwin
[1988], Fig. 4, reprinted by permission of Nature. © 1988, Macmillan Magazines, Ltd.
B from Hucho and Hilgenfeld [1989], Fig. 5C, courtesy of the Federation of European
Biochemical Societies. C from Ortells and Lunt [1995], Fig. 1, © 1995, by permission
of Elsevier Science.)
ogy: forming "chimeras," by joining strands of DNA from different sources to

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

positively charged cations (Fig. 8-2B). Models also included sites for acetyl-
choline on the N-terminal segment of a subunits.24 But the mechanism by
which acetylcholine s binding alters the structure—and in precisely what ways—
remained undetermined in 1990.
Ligfana-Gatea Ion Channels
Physiological and pharmacological studies had established a group of "ligand-

gated ion channels," so named because the binding of neurotransmitters
(ligands) opened gates to allow ion flow through receptor channels (chapter 6).
This group included receptors for GABA, glycine, and glutamate as well as
nicotinic receptors for acetylcholine; in 1988 one class of serotonin receptors,
SHTs, was shown to function as a ligand-gated ion channel also.25 But deter-
mining whether or not this common function reflected a common structure
required the newer approaches.
GABA ana Glycine Receptors
In 1987 Eric Bernard in Cambridge reported the sequences for two subunits
of the GABAA receptor and Heinrich Betz in Heidelberg that for one subunit
of the glycine receptor.26 They, too, determined these sequences by cDNA
techniques, using oligonucleotide probes based on partial amino acid analyses
of purified subunits. From their similar results they now stressed the chemi-
cal and structural similarities to nicotinic receptors and to each other: they
argued for the existence of a superfamily of related receptors. Indeed, there
was about 50% homology27 between GABAA and glycine receptors and about
25% homology with nicotinic receptors. All subunits were of similar size and
all had four hydrophobic stretches likely to be transmembrane segments (Fig.
8-2C). In fact, the homologies lay chiefly in the four putative transmembrane
segments, notably in M2. All had lengthy, extracellular N-terminal domains
where the binding sites for their neurotransmitters lay. These domains also
included two cysteines that were 15 amino acids apart, apparently forming a
loop through disulfide bonding between these cysteines.,
Differences among these receptors are to be expected, of course, for they
bind different ligands selectively. Moreover, GABA and glycine are inhibitory
neurotransmitters that promote Cl~ fluxes through their receptors, rather than
the Na + and K + fluxes of excitatory nicotinic receptors. Correspondingly, the
GABAA and glycine receptors had a cluster of positively charged amino acids
at each end of the presumed channel,28 where nicotinic channels had nega-
tively charged amino acids.
Based on the pattern of nicotinic receptors, the GABAA and glycine recep-
tors should consist of five subunits arranged around a central channel, although
the precise number of subunits for the GABAA receptor was debated for many
years. In any case, there appeared to be fewer kinds of subunits than for the
electric organ nicotinic receptor, although the GABAA receptor had multiple
varieties of each kind of its subunits.29
Glutamate Receptors
Certain glutamate receptors also function as ligand-gated ion channels, and these
had been subdivided pharmacologically according to their differential sensitiv-
ities to characteristic agonists, including kainate, quisqualate, and N-methyl-
D-aspartate (NMDA). It seemed likely, therefore, that these receptors would
be structurally similar to nicotinic, GABAA, and glycine receptors. These glu-
tamate receptors had not been purified, however, so there were no partial amino
acid sequences available and thus no guides for constructing oligonucleotide
probes.
Michael Hollmann in La Jolla instead sequenced the kainate receptor in 1989
by a different cDNA approach: "expression cloning."30 First he formed a cDNA
library from rat brain mRNA, representing all proteins synthesized in the brain
and consisting of 800,000 clones. He divided this library into 18 sublibraries of
44,000 clones each, and he then transcribed the cDNA of each sublibrary in
vitro, obtaining 18 pools of the corresponding mRNAs. He injected each of
these pools into a separate Xenopus oocyte, testing each oocyte for the kainate-
induced depolarizations that would signal the expression of functional recep-
tors. Hollmann next subdivided the positive cDNA sublibrary successively, test-
ing pooled mRNA from 4000 clones, then 400 clones, and finally 40 clones. At
this point he tested individually the mRNA from 12 clones of that final pool,
selecting clones having the longest stretches of cDNA. The mRNA from only
one of these clones elicited responses to kainate, and Hollmann sequenced the
corresponding cDNA, deducing an encoded protein with a molecular weight
of 100 kDa.
This protein was twice the size of the subunits from other known ligand-
gated ion channels. Moreover, there was no cysteine loop and "no significant
over-all homology" with the other receptors, although Hollmann did identify
four "candidates" for transmembrane segments.31 At best, it seemed that glu-
tamate receptors were "distant cousins" of the known ligand-gated ion chan-
nels.32 Sequences of other ligand-gated glutamate receptors, including NMDA
receptors,33 were determined soon afterward, and all these showed marked
similarities to this kainate receptor.
Structural ana Evolutionary Superramilies
Strong similarities in the sequences of nicotinic, GABAA, and glycine recep-

tors—and among the subunits of a given receptor—implied not only a close
structural and functional pattern (Table 8-1) but also a common ancestry: an
TABLE 8-1. Receptor Classes

LIGAND-GATED ION CHANNELS G-PROTEIN COUPLED RECEPTORS
nicotinic cholinergic muscarinic cholinergic

GABAA GABAB
glycine a- and /3-adrenergic
5HT3 5HTi and 5HT2
dopamine
glutamate metabotropic glutamate
kainate
quisqualate
NMDA
evolutionary descent from a primitive precusor.34 (The 5HTs receptor was

sequenced by expression cloning in 1991;35 its similarities placed it within this
superfamily. The 5HTs receptor conducts cations, and it is related more closely
to nicotinic receptors than to the anion-conducting GABAA and glycine recep-
tors. By contrast, further studies in the 1990s placed the glutamate receptors
in an unrelated superfamily. These studies also argued for only three trans-
membrane segments in glutamate receptor subunits.)
Aarenergfic Receptors
Robert Lefkowitz (Fig. 8-1B) had isolated /3-adrenergic receptors and recon-
stituted the purified proteins to form functional receptors—capable of stimu-
lating adenylate cyclase—as one step in his comprehensive study of adrener-
gic processes (chapter 6). Lefkowitz had received his M.D. from Columbia
University in 1966 at age 23, and after clinical training in internal medicine,
he spent two years in research at NIH before further training in cardiology at
Harvard University. In 1973 he was appointed associate professor of medicine
and assistant professor of biochemistry at Duke University, where he initiated
an active and productive research program.
Lefkowitz now obtained partial amino acid sequences from purified fiz-
adrenergic receptors and used these sequences to construct oligonucleotide
probes for screening a hamster library, identifying in 1986 the DNA that
encoded a protein containing 418 amino acids with a molecular weight of 46
kDa.36 The deduced sequence contained likely sites—"consensus sequences,"
since these appeared at demonstrated occurrences in other proteins—for phos-
phorylation by protein kinases and for glycosylation (covalent attachment of
sugars). Most notable, however, were seven stretches of hydrophobic amino

acids, each long enough to form a-helices that could span the membrane's lipid
bilayer. Lefkowitz pointed out that these putative transmembrane segments
were reminiscent of the seven putative transmembrane segments recently
deduced in the amino acid sequence of rhodopsin and the seven transmem-
brane a-helices visualized in bacteriorhodopsin.37 (Rhodopsin is the protein in
retinal rod membranes that bears the light-sensitive pigment retinal and that
couples with a G-protein to initiate its functional response to light. Bacteri-
orhodopsin is a retinal-containing, light-driven membrane pump that extrudes
H + from certain bacteria.) Lefkowitz suggested that (3 agonists might bind to
the receptor as retinal binds to rhodopsin, in both cases to activate a coupled
G-protein.
The next year, 1987, Lefkowitz reported the sequence of human /^-recep-
tors, presenting a model with seven transmembrane segments (Fig. 8-3).38 He
also reported the sequence for human jSi-receptors: this protein contained 477
amino acids (51 kDa) and had 54% homology with human /3£.39 The conserved
features between /3\, {$%, rhodopsin, and recently sequenced muscarinic recep-
tors (see below) included seven likely transmembrane segments containing
notable sequence homologies, plus consensus sites for phosphorylation on intra-
cellular domains and for glycosylation on extracellular domains.
Lefkowitz expressed fi\- and /^-receptors in Xenopus oocytes, obtaining
receptors specific for (3i and (32 agonists and antagonists.40 Next, he constructed
chimeras of /3i//32-receptors and expressed these also in oocytes. The fourth
transmembrane segment controlled specificity for (3\ vs. {$2 agonists, whereas
the sixth and seventh segments controlled specificity for (3i vs. /3£ antagonists.41
Both /3r and /^-receptors bind Gs, which stimulates adenylate cyclase,
whereas o^-receptors bind Gj, which inhibits it (chapter 7). So Lefkowitz
obtained sequences for o^-receptors by similar means and then constructed
a
2/Pz chimeras.42 The ability to couple with Gs lay in the third cytoplasmic loop
(between transmembrane segments 5 and 6). He refined this localization using
site-directed mutagenesis, implicating portions of the second intracellular loop
and of the intracellular tail as well as of the third intracellular loop.43
The ai-receptors activate, through a different G-protein, a phospholipase C
that splits phosphatidylinositol-tephosphate into two second messengers, inos-
itol-fnsphosphate and diacylglycerol (chapter 7). Lefkowitz determined the
sequence of this adrenergic receptor also, although he did not explore its prop-
erties at that time.44
These studies established a characteristic structure having seven transmem-
brane segments, with ligands binding in a pocket formed within the array of
transmembrane a-helices. Other approaches also contributed, including exam-
inations of ligand-binding sites using reactive analogs that would label adjacent
amino acids.45 Intracellular loops evidently should be involved with coupling
FIGURE 8-3. Amino acid sequence and folding pattern for the human /32-adrenergic
receptor. The amino acid sequence is depicted in the one-letter symbols for the amino
acids (A for alanine, R for arginine, N for asparagine, etc.). Black circles with white let-
ters indicate sites where the human and hamster receptors differ. The seven trans-
membrane a-helices, containing hydrophic amino acids, are shown, as well as two sites
for glycosylation on asparagines in the N-terminal extracellular segment. (From Kobilka
et al. [1987], Fig. 3, courtesy of Robert J. Lefkowitz.)
to G-proteins, but how ligand binding activated the coupled G-protein

remained undetermined.
G-Protein Coupled Receptors
In 1987 Lefkowitz proffered the "interesting speculation . . . that the seven

membrane-spanning . . . feature might be common to a l l . . . membrane recep-
tors . . . coupled to G proteins."46 The next few years justified his generaliza-
Receptor Structures and Receptor Families (1983—1990) 211
tion and affirmed the common name for this group (alternative names include
"heptahelical receptors" and "metabotropic receptors").
Muscarinic Cnolinergic, Serotonergic, ana Dopaminergic Receptors
The same year that Lefkowitz reported the first sequence for an adrenergic
receptor, 1986, Numa reported the first sequence for a muscarinic cholinergic
receptor.47 Muscarinic receptors had just been purified from pig brain, so Numa
obtained partial amino acid sequences by chemical means, produced oligonu-
cleotide probes based on these sequences, and then screened a pig brain cDNA
library with these probes. He identified the cDNA for a 51 kDa protein con-
taining 460 amino acids, with sequence homologies to rhodopsin and the /?2-
adrenergic receptor, including seven likely transmembrane segments. Expres-
sion in Xenopus oocytes produced characteristic electrical responses to
administered acetylcholine that were blocked by atropine.48
At that time pharmacological approaches distinguished two major classes of
muscarinic receptors, MI and MZ; the receptor classes also had different dis-
tributions in the brain. Numa identified his protein as MI based on the corre-
sponding localization of its mRNA in the brain as well as on the binding of a
diagnostic MI antagonist to oocytes expressing the cDNA.
The cDNA representing this receptor did not, however, encode the
sequences of certain peptide fragments present in proteolytic fragments of mus-
carinic receptors purified from pig brain. Numa concluded that these aberrant
sequences might belong instead to M£ receptors present along with MI in his
receptor preparation. Accordingly, he synthesized oligonucleotide probes cor-
responding to the aberrant sequences and screened cDNA libraries from brain
and from heart, where M2 receptors are prominent. The identified cDNA
encoded a protein of 466 amino acids (52 kDa) having sequence homology to
MI and containing seven likely transmembrane segments.49 Localization of
mRNA for this protein in the brain and heart indicated that it was M£. Daniel
Capon in San Francisco identified the cDNA encoding this receptor inde-
pendently, reporting the next year, 1987, that expression of this cDNA in
tissue culture cells led to the binding of a characteristic antagonist to M£
receptors.50
Also in 1987 Tom Bonner in Bethesda and Capon independently identified
two additional muscarinic receptors, MS and M^51 They used oligonucleotide
probes based on conserved sequences for screening libraries under conditions
of lowered stringency. The four encoded proteins that they detected—corre-
sponding to MI through M4—had similar sequences but distinctive cytoplas-
mic loops; when expressed individually, the proteins bound cholinergic ligands
with distinguishable affinities. (Their approach, searching for previously
unknown proteins through low-stringency screening, represented a powerful
exploratory mode then coming into prominence.)
In 1988 Capon showed that these receptors were coupled to particular sec-
ond messenger systems.52 He expressed each individually in tissue culture cells
that otherwise lacked muscarinic receptors: administering cholinergic agonists
then inhibited cAMP production in cells expressing M£ or M4 but increased
phosphatidylinositol-fozsphosphate hydrolysis in cells expressing MI and Ma.53
Bonner identified a fifth subtype in 1988; it also increased phosphatidylinosi-
tol-£>isphosphate hydrolysis.54
During these years similar approaches revealed sequences for serotonin
(SHTiA, 5HTic, and 5HT2) and dopamine (D£) receptors.55 These receptors,
too, had sequence homologies, seven likely transmembrane segments, and
responses mediated through G-protein coupled systems: SHTiA and Dg affect-
ing cAMP production and 5HTic and 5HT2 phosphatidylinositol-tephosphate
hydrolysis.
Structural ana Evolutionary Superramily
Common features—sequence homologies, seven transmembrane segments,

intracellular phosphorylation and extracellular glycosylation sites, and signalling
mediated through coupled G-proteins—indicated a structural and functional
superfamily (Table 8-1) as well as an evolutionary one. These genetic, struc-
tural, and functional analyses thus uncovered unifying generalizations despite
the plethora of neurotransmitters and their receptor subtypes. (Continued stud-
ies in the 1990s added further members to this superfamily, including GABAfi
and metabotropic glutamate receptors, plus receptors for enkephalins and other
peptides.)
Receptor Regulation
Earlier studies demonstrated that cells could adjust the responses of their
receptors, often in a compensatory, homeostatic manner. One well-recognized
process was desensitization, a diminishing response despite continued stimu-
lation. With some receptors this desensitization represented an obligatory con-
formational step between open and resting stages (chapter 6). Chronic admin-
istration of agonists could, however, alter responses in more complex fashions.
For example, adding /3-adrenergic agonists elevated cellular cAMP levels
acutely, but the cAMP levels then plateaued or even fell despite the continued
presence of agonist. Plausible explanations invoked feedback loops. In this case,
/3-adrenergic agonists would elevate cAMP levels and thereby activate protein
kinase A; protein kinase A would phosphorylate the receptor and thus dimin-
ish its response. When cAMP levels returned to basal levels, protein phos-
phatases would dephosphorylate the receptor, restoring its basal activity.
In agreement with this scheme, Lefkowitz correlated the desensitzation of

j8-adrenergic receptors with the receptors' phosphorylation by protein kinase
A, and the desensitization of ai-adrenergic receptors with their phosphoryla-
tion by protein kinase C.56 Phosphorylations by protein kinases A and C, how-
ever, affect not only the receptor being activated. Other cellular constituents
were phosphorylated, too, including other receptors (e.g., protein kinase A will
phosphorylate nicotinic cholinergic receptors, reducing their responses as
well).5' Thus chronic administration of a neurotransmitter could modify
responses not only at its own receptors but also at receptors for other neuro-
transmitters.
Contrasted with such "heterologous desensitization" was receptor-specific
"homologous desensitization."58 In 1986 Lefkowitz described a kinase that
phosphorylated—and inactivated—/3-adrenergic receptors preferentially.59
Moreover, this kinase phosphorylated only /3-adrenergic receptors that were
actually binding agonists. Evidently, the agonist induced the necessary confor-
mation in its receptor to make the receptor susceptible to this phosphoryla-
tion. The desensitization that followed this agonist-dependent phosphorylation,
however, required the presence of another protein, /3-arrestin, which Lefkowitz
identified in 1990.60 (This process paralleled one described earlier for
rhodopsin. Rhodopsin kinase phosphorylates light-activated rhodopsin and
thereby promotes its binding to arrestin; bound arrestin then blocks further
light-activated responses. Studies continuing in the 1990s grouped these kinases
into a family whose members phosphorylated various receptors specifically,
including receptors for acetylcholine, dopamine, serotonin, and enkephalin.61)
A second means for decreasing responses involved sequestering the recep-
tors within the cell.62 For example, administering adrenergic agonists reduced
the number of j8-receptors on the cell surface, as measured by the binding of
a labeled ligand that did not penetrate the cell membrane. The sequestered
receptors could be recovered in a membrane-bound fraction, separate from
the fraction containing surface receptors, after density-gradient centrifugation
of homogenized cells. But the total number of receptors, measured in broken
cells or by using ligands that penetrate membranes, did not change.
There appeared to be two distinct fates awaiting these sequestered recep-
tors. In some cases the sequestered receptors appeared to be "resensitized"
and returned to the surface.63 In other cases receptors disappeared, so that
new protein synthesis was required to replenish the cellular complement.64
"Downregulation" refers to the loss of receptors, as opposed to sequestra-
tion in which the total number is unchanged. Accounts of downregulatlon dur-
ing these years argued variously for receptor degradation and/or decreased syn-
thesis.65 Both processes were plausible, for receptors, like other cell proteins,
are continuously broken down and replaced. Different studies stressed one or
the other of these mechanisms, depending on the cell type, receptor, and ago-
nist studied. Although no general account was possible by 1990, interest was
growing in mechanisms for altering the cellular content of mRNAs encoding
these receptors. For example, direct measurements of the mRNA for /3-recep-
tors revealed agonist-induced decreases that paralleled downregulation.66 Mol-
ecules of mRNA are also continuously broken down and replaced, so decreased
mRNA levels could be due to decreased transcription or increased degrada-
tion. As the decade closed several studies favored agonist-mediated "destabi-
lizations" of mRNA—a shortening of their functional lifetimes—without a
change in transcription rates.67
"Upregulation," on the other hand, refers to a gain of receptors. Prolonged
administration of receptor antagonists could increase the number of recep-
tors,68 just as prolonged administration of agonists could decrease their num-
ber. In some instances the increase was confined to the particular receptor sub-
types to which that antagonist bound.69
But broader changes could also occur, as shown by responses to cAMP. Many
genes have regions in their DNA where particular proteins bind to enhance
the expression of that gene. In 1986 Marc Montminy in La Jolla identified such
a site for cAMP-stimulated protein synthesis. He named this region of the gene
the "cAMP response element" (CRE). 70 The next year he identified a CRE
binding protein (CREB), which was activated through phosphorylation by pro-
tein kinase A.71 The sequence thus ran: elevated cAMP => protein kinase A
=> phosphorylated CREB => activated CRE => mRNA transcription. In
1989 Lefkowitz showed that cAMP enhanced the transcription of mRNA for
^3-adrenergic receptors through such a mechanism.72 Consequently, it would
seem that any agonist elevating cAMP levels might increase the synthesis of
/3-receptors.
By 1990 it was clear that homeostatic processes could attenuate the ther-
apeutic responses to chronically administered agonist or antagonist drugs.
Furthermore, even when the agonist or antagonist was highly specific for a
given class of receptors, heterologous regulatory mechanisms could extend
the drugs' actions to affect other proteins, including receptors for other neuro-
transmitters.
Conclusions
New techniques of molecular biology rapidly disclosed the amino acid sequences
for an expanding roster of neurotransmitter receptors. These sequences iden-
tified the receptors chemically, even if they did not immediately reveal the pre-
cise mechanisms. Still, two major classes of receptor function as well as struc-
ture became apparent. (1) Oligomeric subunits surrounding an ion-conducting
pore formed a ligand-gated ion channel that opened when neurotransmitters
filled the binding sites. (2) Monomeric proteins with seven transmembrane seg-
Receptor Structures and Receptor Families (1983—1990) 215
ments formed receptors that activated a coupled G-protein. With the first class,
chemical signals (neurotransmitters) were converted to electrical signals. With
the second, chemical signals were converted to different chemical signals:
G-proteins that then interacted with various enzymes and channels to elicit
both chemical and electrical responses.
The reductionist program that delineated these structures and functions thus
disclosed unanticipated unities characterizing these classes, despite a prolifer-
ating catalog of distinguishable receptors.73 These unities, of course, reflected
evolutionary relationships. Indeed, such similarities are expected in evolution-
ary systems: random modifications in an ancestral protein enable new ligands
to bind (as in the divergence of serotonergic and nicotinic receptors) and thereby
activate a functional component, either similar (as in cation-conducting chan-
nels of serotonergic and nicotinic receptors) or modified (as in the appearance
of GABAergic and glycinergic anion-conducting channels).
This spate of discoveries and realizations was made possible by the devel-
opment of new methods, just as the great advances in understanding metabolic
syntheses and degradations in the 1940s and 1950s followed the introduction
of new separatory techniques and radioisotope labeling procedures. The new
methods were labor intensive, however, and one further consequence was the
creation of large research groups generating multiauthored papers (one cited
here had 16 authors).
These new methods also provided a new research strategy. As Lefkowitz
pointed out,' 4 the old paradigm directed a progression from physiological
phenomena to pharmacological characterization to biochemical specification
to molecular biological identification and manipulation. But a new paradigm
now emerged: probes corresponding to a given protein's amino acid sequence
could be used to screen DNA libraries at various degrees of stringency, uncov-
ering genes for related but previously unknown proteins having similar func-
tions as well as similar structures. Moreover, the growing compilations of pro-
tein sequences, structures, and functions also facilitated deductions of the
corresponding structures and functions when a new sequence was deter-
mined. The recognition of conserved motifs in amino acid sequences—again,
a characteristic to be expected with evolutionary changes—also aided the
assignment of functions to structures. But these new understandings of neuro-
transmitter receptors were advanced not only by the new techniques and prin-
ciples from molecular biology and biochemical genetics. Progress was assisted
also by concomitant advances in understanding hormonal and sensory recep-
tors and by the deciphering of further intracellular signalling and regulatory
processes.
Better understandings of receptor structure and function improved under-
standings of diseases as well (chapter 13), and certain pathologies could now
be attributed to precise aspects of receptor structure. For example, the mus-
cular weakness of myasthenia gravis is due to a failure in transmission at neu-
romuscular junctions: antibodies to muscle nicotinic receptors prevent acetyl-

choline from causing contractions. The sites to which the disabling antibodies
bind were now localized to specified regions on the extracellular N-terminal
segment of nicotinic receptor a subunits/ 5
Better understandings also promised better therapies by guiding the quest
for more precise interventions with fewer extraneous actions and side effects.
The recognition of multitudes of distinguishable receptor subtypes encouraged
this quest for specificity through the development of drugs acting on a single
subtype, leaving unaffected all other receptors. But dimming this prospect were
realizations of the cell's intrinsic homeostatic mechanisms. Although the initial
administration of a drug could evoke a particular response, the continued use
of that drug often resulted in altered responses. Studies during this decade
showed that after chronic administration the compensatory changes could
extend to other receptor species also.
Notes
1. For histories, see Fruton (1999); Judson (1979); Morange (1998).

2. "Complementary" refers to the structural fit that allows specific pairing between
particular bases on the two strands of DNA (in the double helix) and on a strand of
DNA and of RNA (in transcription): guanine with cytosine, thymine with adenine
(thymine is absent from RNA, where uracil takes it place, pairing with adenine).
3. Each nucleotide triplet has a specific meaning, although all triplets do not encode
an amino acid (e.g., some are "stop codons," signalling the end of a protein).
4. Raftery et al. (1980).
5. Both sequencing techniques rely on introducing base-specific breaks in the DNA.
Experimental conditions are chosen to produce only one or a few breaks per chain, but
with those breaks randomly distributed—in the population of chains—among the var-
ious occurrences of the particular base. With one end of the original chain labeled, the
breaks then produce a family of labeled fragments, each beginning at the labeled end
and extending to an occurrence of the particular base. These fragments are next sepa-
rated according to length (by gel electrophoresis, which resolves fragments differring
by as little as one nucleotide) and compared with the families of fragments for each of
the other three bases, forming a ladder of DNA fragments from which the base sequence
is read.
6. Noda et al. (1982b).
7. The enzyme "reverse transcriptase" transcribes mRNA to form cDNA. (The infor-
mation flow is opposite to that of the "Central Dogma" of molecular biology, from DNA
to mRNA.)
8. In hybridization experiments the two strands of the plasmid cDNA are separated
and fixed to a matrix. The labeled probe is added and allowed to bind to its comple-
mentary sequence. (If the probe is identical to the encoding sequence on one strand
of DNA, the probe will bind to the other, complementary DNA strand.) Labeled probe
that is not complementary, and therefore does not bind tightly, is washed away vigor-
ously ("high stringency" conditions). Labeled probe that survives such washing thus
identifies the plasmid cDNA bearing the complementary sequence.
9. Numa chose the two clones having the longest inserts and therefore the greatest
likelihood of containing the entire representation of the a subunit.
10. In addition, the cDNA encoded a preceding stretch of 24 amino acids that formed
a "signal sequence" of amino acids, which assists in inserting the peptide chain into the
membrane and is then removed.
11. Noda et al. (1983b, 1983c). Others at that time also sequenced particular sub-
units by similar methods: y by Claudio et al. (1983); a by Devillers-Thiery et al. (1983).
12. Mishina et al. (1984).
13. Noda et al. (1983a)
14. Boulter et al. (1986).
16. Brisson and Unwin (1984); Toyoshima and Unwin (1990); and intervening papers.
17. Imoto et al. (1986).
18. Ibid., p. 673.
19. Imoto et al. (1988).
20. Ibid., p. 648.
21. Ibid.
22. Leonard et al. (1988).
23. Giraudat et al. (1986); Revah et al. (1990). See also Hucho et al. (1986); Char-
net et al. (1990). Leonard et al. (1988) also used channel blocking agents to assess func-
tional changes produced by site-directed mutagenesis.
24. For example, Kao and Karlin (1986).
25. Yakel and Jackson (1988). See also Derkach et al. (1989).
26. Schofield et al. (1987); Grenningloh et al. (1987). GABAA receptors were distin-
guishable pharmacologically from GABAfi receptors, which are not ligand-gated ion
channels.
27. In contrast to an "identity" of amino acids between two sequences being com-
pared, a "homology" refers to the presence of the identical amino acid or a "conserva-
tive substitution": an amino acid of the same size and polarity.
28. Montal (1990); Stroud et al. (1990).
29. For example, Levitan et al. (1988). See also Montal (1990); Stroud et al. (1990).
30. Hollmann et al. (1989).
31. Ibid., pp. 646, 647.
32. Stroud et al. (1990), p. 11,017.
33. Moriyoshi et al. (1991).
34. See Ortells and Lunt (1995).
35. Maricq et al. (1991).
36. Dixon et al. (1986). Initially they screened a genomic library, which contained
the entire DNA content of the nucleus and thus encoded all the proteins the organism
was capable of synthesizing. By contrast, a cDNA library represents only the proteins
synthesized by the particular cell type at the particular time. Elliott Ross in Dallas
reported the sequence of a /3-receptor from turkey red blood cells also in 1986 (Yarden
et al., 1986).
37. Nathans and Hogness (1983); Henderson and Unwin (1975).
38. Kobilka et al. (1987).
39. Frielle et al. (1987). To locate a similar sequence they used the probe from /3g,
hybridizing with low stringency. This encoded a then unrecognized protein; however, a
probe based on the sequence of this unrecognized protein hybridized with the DNA
for the j3i-receptor. (The unrecognized protein turned out to be a serotonin receptor.
Fargin et al. [1988].)
40. Frielle et al. (1987).

41. Frielle et al. (1988).
42. Kobilka et al. (1987, 1988); Regan et al. (1988).
43. O'Dowd et al. (1988).
44. Cotecchia et al. (1988).
45. For example, Dohlman et al. (1988); Wong et al. (1988).
46. Kobilka et al. (1987), p. 49.
47. Kubo et al. (1986a).
48. The electrical responses were presumably manifested through altered channel
permeabilities mediated by G-protein-dependent processes. The electrical responses
were readily distinguishable from those of nicotinic receptors.
49. Kubo et al. (1986b).
50. Peralta et al. (1987b).
51. Bonner et al. (1987); Peralta et al. (1987a). See also Akiba et al. (1988). The num-
bering of muscarinic receptors 3 and 4 differ in these papers; that in Bonner et al. pre-
vailed.
52. Peralta et al. (1988). See also Fukuda et al. (1988).
53. Nevertheless, other studies suggested that a single receptor subtype might cou-
ple with different G-proteins (for example, Ashkenazi et al., 1987).
54. Bonner et al. (1988).
55. Fargin et al. (1988); Julius et al. (1988); Pritchett et al. (1988); Bunzow et al.
(1988); Neve et al. (1989).
56. Stadel et al. (1983); Leeb-Lundberg et al. (1987).
57. Huganir et al. (1986); Miles et al. (1987).
58. Sibley et al. (1987).
59. Benovic et al. (1986, 1987).
60. Lohse et al. (1990).
61. Pitcher et al. (1998).
62. For example, Chuang and Costa (1979); Maloteaux et al. (1983); Waldo et al.
(1983); DeBlasi et al. (1985).
63. For example, Feigenbaum and El-Fakahany (1985); Sibley et al. (1986).
64. For example, Mahan et al. (1985); Ray et al. (1989).
65. See Hausdorff et al. (1990); Hulme et al. (1990).
66. Hadcock et al. (1988).
67. For example, Collins et al. (1989); Hadcock et al. (1989).
68. See Creese and Sibley (1981).
69. For example, Hess et al. (1988). Nevertheless, more complex patterns of recep-
tor changes were also reported: see McGonigle et al. (1989).
70. Montminy et al. (1986). See also Comb et al. (1986); Short et al. (1986).
71. Montminy and Bilezikjian (1987).
72. Collins et al. (1989). This represents a positive feedback loop, but Collins et al.
attempted to reconcile the stimulated mRNA synthesis with an expected desensitiza-
tion of the receptor.
73. Nevertheless, the ability of reductionistic approaches to disclose unities has some-
times been challenged: see Robinson (1992).
74. Lefkowitz et al. (1989). See also Gilbert (1991).
75. Tzartos et al. (1988).
9
SYNTHESIS, STORAGE,
TRANSPORT, AND
METABOLIC
DEGRADATION OF
NEUROTRANSMITTERS
Steps in Chemical Neurotransmission
With the acceptance of chemical neurotransmission came the recognition that

synaptic transmission required a number of discrete processes: synthesis of the
neurotransmitter, its storage, release, and interactions with receptors (and the
consequences thereof), and the termination of its actions. All these steps were
obviously important functionally. Each of these steps, it also became apparent,
was a potential site of pathological malfunction. And each was a potential site
for therapeutic intervention.
Chapters 6 through 8 dealt with receptors and their associated signal trans-
duction systems. Chapter 10 will deal with neurotransmitter release into the
synaptic cleft. This chapter is concerned with synthesis, with metabolic degra-
dation, and with two transport steps, one for storing the neurotransmitter in
vesicles and one for terminating the responses to released neurotransmitter.
Because of the variety and complexity of these processes, I must restrict this
account to representative samplings.
Synthesis
If chemical neurotransmitters are present, then some means for their synthe-
sis must also be present, either locally or elsewhere in the body (or perhaps in
219
the body's foodstuffs). Delineations of these biosynthetic pathways sometimes

arose from, and usually became integrated with, general biochemical programs
addressing cellular metabolism.
Acetylcnoline
In the mid 1930s Wilhelm Feldberg in Hampstead amassed convincing evi-

dence for acetylcholine s participation in synaptic transmission in autonomic
ganglia (chapter 3). As part of these studies, Feldberg demonstrated that acetyl-
choline was being synthesized during periods of prolonged stimulation: the
amount of acetylcholine released during stimulation was five times the amount
initially present in the ganglia.1 That same year, 1936, Juda Quastel in Cardiff
described an increase in the acetylcholine content of brain slices that was
markedly stimulated by adding oxygen and glucose (or certain other metabolic
substrates).2
It seemed obvious that acetylcholine was formed by the condensation of
acetate with choline (Fig. 3-6), but such a reaction could not be defined using
whole cell preparations such as tissue slices. So in 1943 David Nachmansohn
in New York used brain homogenates to form acetylcholine in the presence of
the cholinesterase inhibitor physostigmine, choline, and, as a necessary energy
source, ATP.3 Adding acetate was not required—presumably there was enough
in the homogenate—nor was oxygen or any metabolizable substrate. Nach-
mansohn named the enzyme responsible for this synthesis choline acetylase,
although he did not then purify it further.
(Nachmansohn was studying acetylcholine not as a neurotransmitter—a role
he denied it—but as a component of the mechanism underlying the conduc-
tion of nerve impulses along axons. Despite convincing evidence in later
decades establishing both a different mechanism for axonal conduction and the
irrelevance of acetylcholine, Nachmansohn continued to proclaim his beliefs.4)
Acetylation reactions, then a topic of general biochemical interest, seemed
to require a modified form of acetate, termed—for lack of a more precise iden-
tification—"active acetate." Among those studying these acetylations was Fritz
Lipmann in Boston, and in 1945 he reported that acetylating homogenates con-
tained a necessary heat-stable factor.5 The following year Lipmann referred to
this factor as a coenzyme and demonstrated that it was also involved in acety-
lating choline.6 By 1949 Lipmann had characterized the factor chemically and
named it coenzyme A (CoA), for acetylation.7 The acetate-bound form, acetyl-
CoA, was soon recognized as an intermediate in numerous metabolic pathways,
including the synthesis of fatty acids and the formation of citrate in the Krebs
cycle (arising in this case from pyruvate, a product of glycolysis). A specific and
widely distributed enzyme synthesized acetylCoA from acetate, Co A, and ATP.
In 1951 Nachmansohn acknowledged that choline acetylase catalyzed only
Synthesis, Storage, Transport, ana Metabolic Degradation or Neurotransmitters 221
the ATP-independent condensation of acetylCoA with choline, leaving the ATP-

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

described in 1938 the decarboxylation of dopa (Fig. 9-1) by kidney extracts.15
Holz considered this reaction to be a step between the amino acid tyrosine and
its oxidized degradation products. The following year Hermann Blaschko (Fig.
9-1A) in Oxford proposed that this decarboxylation was instead a step in the
biosynthesis of physiologically active catecholamines (then recognized as adren-
aline and perhaps noradrenaline; see chapter 5). Blaschko, after leaving Ger-
many for Cambridge and then Oxford, enjoyed a long career examining the
syntheses and degradations of various catecholamines. He was also a convinc-
ing advocate of their functional roles. At this point he argued that decarboxy-
lation followed hydroxylation of tyrosine to dopa in this pathway (Fig. 9-2A)
since he found that decarboxylation of dopa was far faster than decarboxyla-
tion of tyrosine.16
FIGURE 9-1. A, left, Hermann Blaschko (1900-1993). B, right, Julius Axelrod (1912-).
(A courtesy of the department of pharmacology, University of Oxford. B courtesy of
J. Axelrod.)
In 1950 Blaschko reported that mammalian tissues did not decarboxylate the
dopa derivative having a hydroxylated side chain (dihydroxyphenylserine), so
decarboxylation must precede hydroxylation of the side chain.17 The reaction
sequence, he concluded, ran from tyrosine hydroxylation forming dopa, to dopa
decarboxylation forming dopamine, to dopamine hydroxylation forming nora-
drenaline (Fig. 9-2A). When radioactively labeled dopa became available,
Blaschko was able in 1955 to demonstrate this pathway: adrenal homogenates
rapidly converted labeled dopa into labeled dopamine, and after prolonged
incubation into labeled noradrenaline.18
Characterization of the responsible enzymes—tyrosine hydroxylase, dopa
decarboxylase, and dopamine 0-hydroxylase (Fig. 9-2A)—progressed during
the following decades.19 Here, however, I will include only some of the steps
taken in recognizing the properties of tyrosine hydroxylase.
In 1964 Sidney Udenfriend in Bethesda achieved a partial purification of
tyrosine hydroxylase from adrenals, but further purification was achieved only
slowly.20 Nevertheless, in 1985 Jacques Mallet in Gif-sur-Yvette determined the
amino acid sequence of a 65 kDa protein by cDNA techniques; the active
enzyme was a tetramer of these subunits.21
Synthesis, Storage, Transport, and Metabolic Degradation ot Neurotransmitters 223
FIGURE 9-2. Biosynthetic pathways. A. Synthesis of catecholamine can begin with

either phenylalanine or tyrosme, each a common amino acid. Hydroxylation of tyrosine
forms dopa, and decarboxylation of dopa forms dopamine. Noradrenergic neurons con-
tain an additional enzyme that hydroxylates dopamine to form noradrenaline. B. Syn-
thesis of serotonin (5-hydroxytryptamine) begins with hydroxylation of the common
amino acid tryptophan, followed by decarboxylation.
Udenfriend in 1964 also argued for tyrosine hydroxylase being the control-
ling enzyme of the biosynthetic pathway.22 It was the rate-limiting step in the
sequence (slowest in vivo), so changes in tyrosine hydroxylase activity—
increases or decreases—would change catecholamine production accordingly.
Earl Stadtman in Bethesda had recently stressed a principle of feedback con-
trol in biosynthetic pathways termed "end-product inhibition," whereby the
final biosynthetic product inhibits the first step in the pathway.23 Correspond-
ingly, Udenfriend showed that dopamine and noradrenaline inhibited tyrosine
hydroxylase.24 Moreover, Norman Weiner in Boston found that stimulating
sympathetic nerves increased tyrosine hydroxylase activity. He suggested that
the drop in noradrenaline levels, due to its release during stimulation, removed
noradrenaline s inhibition of the enzyme.25
Other regulatory modes soon became apparent. For example, cAMP in-
creased tyrosine hydroxylase activity acutely, and protein kinase A phosphory-
lated tyrosine hydroxylase.26 Protein kinase C also phosphorylated tyrosine
hydroxylase.27 But what signalled the rise in cAMP or diacylglycerol was still
being debated as the 1980s closed.28
Changes in total enzyme content could occur in addition to acute regulation
through feedback inhibition and enzyme phosphorylation. For example, deplet-
ing an animals stores of catecholamines with drugs or by stress (e.g., exposure
to cold) increased the amount of tyrosine hydroxylase present, with this change
following increases in mRNA for this enzyme.29 Added cAMP could also
increase the cellular content of tyrosine hydroxylase, and this change, too, was
associated with increased mRNA levels.30 Furthermore, the gene for tyrosine
hydroxylase, like the genes for certain adrenergic receptors (chapter 8), was
associated with a cAMP response element (CRE).31
The rate-limiting step in a biosynthetic pathway should be a prime target for
pharmacological control as well. But no practical activators of tyrosine hydrox-
ylase were discovered, and although an inhibitor, a-methyltyrosine, was stud-
ied, it found no significant role in therapy.
Serotonin
The biosynthetic pathway for serotonin resembles closely that for cate-
cholamines (Fig. 9-2B), and its elucidation sprang from parallel studies. In
1954 Udenfriend described the enzymatic decarboxylation of 5-hydroxytrypto-
phan, but he subsequently discovered, to his surprise, that this decarboxylat-
ing enzyme was identical to that for dopa.32 So in 1962 he suggested that a bet-
ter name for the common enzyme was aromatic amino acid decarboxylase.33
The hydroxylating enzyme, tryptophan hydroxylase, was identified in the
mid-1960s but not purified until the 1980s.34 Its amino acid sequence, obtained
by cDNA techniques in 1987, revealed a close structural and evolutionary rela-
tionship to tyrosine hydroxylase and phenylalanine hydroxylase.35 All three
hydroxylases commanded considerable attention because of an intriguing re-
action mechanism involving oxygen, iron, and a unique cofactor, tetrahydro-
biopterin.36
Tryptophan hydroxylase is the rate limiting step in the serotonin pathway, as
is tyrosine hydroxylase in the catecholamine pathway. It, too, is phosphorylated,37
but, unlike tyrosine hydroxylase, it is not subject to end-product inhibition.38
No therapeutically useful activators or inhibitors were developed by 1990,
but another route to manipulating serotonin synthesis was explored during
these decades. The cytoplasmic concentrations of tryptophan are insufficient
to saturate tryptophan hydroxylase, so increases or decreases in these levels
would increase or decrease 5-hydroxytryptophan formation (and serotonin also,
since 5-hydroxytryptophan is rapidly decarboxylated). Accordingly, feeding
large amounts of tryptophan could raise brain levels of serotonin.39 By 1990
clinical studies with tryptophan elicited some enthusiasm.40
Enkepnalins
When John Hughes and Hans Kosterlitz identified the first opioid peptides in
1975 (chapter 5), they noted that the amino acid sequence of met-enkephalin
Synthesis, Storage, Transport, ana Metabolic Degradation of Neurotransmitters 225
was present within a known pituitary hormone, /3-lipotropin.41 That presence

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

mented on a current controversy: whether nerve impulses triggered a new syn-
thesis of acetylcholine in the presynaptic neuron, which then interacted with
the postsynaptic cell, or instead triggered a release of previously synthesized

acetylcholine. In 1936 Feldberg acknowledged that "the theory of immediate
synthesis . . . affords an easier and more direct interpretation."52 But by 1945,
with acetylcholine measurements in tissues accumulating, Feldberg argued for
the storage of acetylcholine.53
Storage required that acetylcholine be protected from degradation by
cholinesterase. Feldberg rejected the possibility that acetylcholine was "present
in a diffusible form . . . but spatially separated from . . . cholinesterase."54 So he
concluded that acetylcholine was "bound in the tissue or cell granules to some
constituents" at synthesis, and in such linkage was "immune to cholinesterase."55
For over a decade the focus was on neurotransmitters stored in secretory gran-
ules through their binding to proteins or lipids or even nucleotides.56
A different model arose from examinations not of nerve or brain, but of
adrenals. This course began in 1953, when Blaschko and, independently, Nils-
Ake Hillarp in Stockholm separated catecholamine-containing "granules" from
adrenal homogenates.57 They used differential centrifugation, following pro-
cedures recently established for liver,58 and found 70%-90% of the tissue cat-
echolamines in what, with liver, would be the mitochondrial fraction. Blaschko
noted that this fraction contained mitochondrial enzymes but did not examine
its morphology. Hillarp failed to see "conventional mitochondria" but presented
electron micrographs showing large granules with diameters of 0.1-0.6 />tm; he
raised the possibility that these granules were "comparable to mitochondria."59
Subsequently both Blaschko and Hillarp used sucrose density-gradient cen-
trifugation to separate from mitochondria a pure fraction of catecholamine-
containing granules.60
By 1955 both Hillarp and Blaschko argued that the particles were bounded
by a semipermeable membrane (vesicles would then be a better name than
granules).61 But the prominent osmotic effect expected of catecholamines free
at high concentration within a semipermeable barrier was not apparent. On
the other hand, if the positively charged catecholamines were bound to con-
stituents of the granules, this osmotic potential would be diminished. Both
Hillarp and Blaschko then reported that high concentrations of negatively
charged ATP were present in the granules together with catecholamines.62 Such
binding might also account for the concentration differential between cate-
cholamines in granules and in cytoplasm.
In 1962 Norman Kirshner in Durham described an uptake of catecholamines
into isolated adrenal granules that was markedly stimulated by added ATP.63
Kirshner cited proposals that catecholamines and ATP were "associated in a
nondiffusable complex within the granules."64 He, however, argued for accu-
mulation through active transport rather than by binding: "catecholamines . . .
react with some component of the granule membrane [and] are transported
across the membrane and released into the [granule] interior."65 ATP was not
Synthesis, Storage, Transport, and Metabolic Degradation of Neurotransmitters 227
taken up with the catecholamines, and Kirshner, in accord with Hillarp s report
of ATPase activity in granules,66 proposed that ATP hydrolysis provided the
energy for active transport. Although binding within the granules could mini-
mize osmotic effects, active transport would account for the concentration
gradient.
Reserpine, a plant alkaloid recently established as an effective drug for treat-
ing schizophrenia (chapter 13), blocked ATP-dependent uptake. This was a sig-
nificant datum, since Marthe Vogt in Edinburgh had reported in 1956 that
reserpine caused a loss of catecholamines from brain and adrenals in vivo.67
Reserpine-inhibitable accumulation thus became the hallmark of catechol-
amine storage. (Adrenals could also accumulate serotonin in vitro, and reser-
pine blocked serotonin uptake in adrenals as well as serotonin uptake in the
brain.68)
A plausible mechanism for ATP-dependent transport would feature a cate-
cholamine-transporting ATPase, along the lines of the recently described
Na+/K+-ATPase that functions as the cell membrane Na'VK^-pump.69 The
Na+/K+-ATPase was the first transport ATPase to be characterized, and it estab-
lished the class of what became known as primary active transport systems:
these coupled metabolic energy (here, ATP) directly to transport (here, Na +
from and K + into the cell). But attempts to identify an analogous cate-
cholamine-transporting ATPase were unsuccessful.
The relevant precedent came instead from formulations of an alternative
mechanism, promulgated in the early 1960s by Robert Crane in St. Louis and
Peter Mitchell in Edinburgh.70 Their models of secondary active transport
depicted transmembrane gradients of one solute serving as energy sources for
transporting another solute. In these co-transport systems the energetically
downhill fluxes of one solute drove energetically uphill fluxes of the other, as
in the coupled influxes of Na+ and glucose that occur across the cell mem-
branes of the intestinal epithelia. Mitchell also incorporated this concept in his
chemiosmotic hypothesis for oxidative phosphorylation, whereby metabolic oxi-
dations established H+-gradients across mitochondrial membranes, and these
gradients then drove ATP synthesis. Mitchell proposed that a class of reagents
known as uncouplers of oxidative phosphorylation prevented ATP synthesis by
dissipating transmembrane H+-gradients.
More than a decade after Kirshner's description and Mitchell's proposal,
George Radda in Oxford reported that such uncouplers also blocked ATP-
driven catecholamine uptake by adrenal granules.71 Radda suggested that the
granule ATPase was a H+-transport pump that established H+-gradients across
granule membranes. Then catecholamine "transport could . . . occur by [a] co-
transport mechanism associated with the movement [of H+]."72 Two years later,
in 1977, Radda demonstrated an ATP-dependent acidification of the granule
interior, consistent with an ATPase transporting H + from cytoplasm to gran-
ule.73 Subsequent studies characterized this H+-transporting ATPase of the

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

quently, a neuron could release more then one neurotransmitter and thereby
evoke still more complex responses in the postsynaptic cells.
Altering synaptic transmission by affecting the storage of certain neuro-
transmitters was an obvious approach to therapy. Reserpine s ability to ame-
liorate the symptoms of schizophrenia centered attention on catecholamines
and serotonin as participants in the pathophysiology of this disease (chapter
13). Reserpine s ability to do so through blocking transport into synaptic vesi-
cles also suggested a particular mode of action for new drugs. Similar agents
did not prove clinically effective, however, and reserpine itself fell from favor,
being replaced by drugs that act instead on receptors.
Degradation
Cnolinesterase
The actions of a neurotransmitter could be terminated by destroying the neu-

rotransmitter, and just such a mechanism was established for acetylcholine in
its early years (chapter 3). Henry Dale in 1914 attributed the evanescence of
acetylcholine s actions to enzymatic destruction. Otto Loewi in 1926 showed
that frog hearts broke down Vagusstoff/acetylcholine but that physostlgmine
blocked this degradation. And they and their associates in 1930 found that blood
catalyzed a physostigmine-inhibitable hydrolysis of acetylcholine. In 1932
Edgar and Ellen Stedman in Edinburgh described acetylcholine hydrolysis by
blood serum, attributed to "choline-esterase."85 Further examinations revealed
different substrate preferences with cholinesterases from different sources. For
example, Gordon Alles and Roland Hawes in Los Angeles reported in 1940
that red blood cells hydrolyzed acetylcholine faster than other choline esters,
whereas serum hydrolyzed some other esters (notably butyrylcholine) faster
than acetylcholine.86 Nachmansohn in 1949 renamed the former activity—
present predominantly in neurons, muscle, and electric organs—acetylcholin-
esterase.87 (The activity in serum, glial cells, liver, and many other sites has
been known by several names, including butyrylcholinesterase. Later studies
revealed two distinct but related genes for these two catalytic specificities. The
differences are not essential here, and for continuity I shall use the general
term cholinesterase for acetylcholinesterase.)
Nachmansohn identified cholinesterase activity in eel electric organs in
193988 while in France, following medical studies in Berlin and before his fur-
ther emigration to the United States. In New York Nachmansohn proceeded
with purifications from this source, rich in cholinesterase as well as in choline
acetylase and nicotinic receptors. By 1947 he achieved a several hundredfold
purification, freeing the enzyme from competing activities.89 Nachmansohn s
associates continued this program through the 1960s, obtaining crystals of
apparently pure enzyme.90 In 1986 Palmer Taylor in La Jolla reported the amino
acid sequence for a 66 kDa protein from Torpedo electric organ, using cDNA
techniques.91 The enzyme had no transmembrane segments but was tethered
to the outside of the cell membrane through a covalently-attached lipid:
cholinesterase was thus sited effectively for cleaving released acetylcholine.
(In 1991 Joel Sussman and Israel Silman in Rehovot determined the three-
dimensional structure by X-ray crystallography.92)
The reaction mechanism attracted enzymologists' attention, not only because
it resembled the extensively studied mechanism of serine proteases93 but also
because it approached a "perfect enzyme" in catalytic efficiency: the reaction
rate was near the limit set by substrates diffusing to and by products diffusing
from the active site.
In the 1950s I. B. Wilson, initially collaborating with Nachmansohn, exam-
ined the enzyme s kinetic properties and proposed a model for its active site
bearing an "esteratic" or catalytic domain plus a negatively charged "anionic"
domain where the quaternary ammonium group of choline bound.94 Wilson
also formulated a two-step reaction sequence, with an initial acetylation of the
enzyme by acetylcholine, releasing choline, followed by hydrolysis of this
acetylenzyme, releasing acetate.95
Certain organophosphorus compounds inactivated cholinesterase by form-
ing nonhydrolyzable enzyme-phosphoryl adducts, corresponding to the acetyl-
enzyme intermediate.96 Radioactive inhibitors labeled a serine of the enzyme,
and Wilson in 1966 proposed that this serine was also acetylated by acetyl-
choline during hydrolysis.97
These organophosphorus compounds were first studied as insecticides in the
1930s, before their mode of action as cholinesterase inhibitors was recognized.
Then during World War II scientists in Germany, Britain, and the United States
developed organophosphorus compounds for chemical warfare.98 These also
became valuable reagents for studying the serine proteases as well as cholin-
esterase. And they continued to be important insecticides (e.g., parathion,
malathion) and potential war gases (e.g., tabun, sarin, soman). (Wilson in 1955
developed a reagent to reactivate cholinesterases blocked by these organophos-
phorus compounds, providing an antidote to such poisonings.99)
Reversible cholinesterase inhibitors—physostigmine and various relatives
both natural and synthetic—found continuing therapeutic uses, notably in treat-
ing glaucoma and myasthenia gravis. As the 1980s closed, new interest in such
drugs sprang from their potential benefits in treating Alzheimer's disease.
Monoamine Oxidases
The discovery of cholinesterase provided the precedent for neurotransmitter

actions being terminated by enzymatic destruction. Apparently in keeping with
Synthesis, Storage, Transport, ana Metabolic Degradation 01 Neurotransmitters 231
this mechanism, adrenaline, whose candidacy as a neurotransmitter was being

explored at the same time as acetylcholine's, was readily oxidized in vitro. Cor-
rect identification of the pertinent mode of oxidation is traceable to studies in
1928 by Mary Hare in Cambridge. She described, however, the oxidation by
liver extracts of a related compound, tyramine, an oxidation accompanied by
deamination.100
In 1937 Blaschko reported the oxidation of adrenaline, noradrenaline, and
dopamine by extracts of brain as well as of liver, kidney, and intestine.101 He
had just completed his Ph.D. studies at the University of Cambridge following
medical training in Germany, and now turned to catecholamines, collaborating
initially with Derek Richter, also in Cambridge. They noted the similarity to
Hare's observations and attributed their results to an enzyme, amine oxidase,
that catalyzed an oxidative deamination: forming an aldehyde and releasing
ammonia or an amine (Fig. 9-3). In 1949 Efraim Racker in New York described
an aldehyde dehydrogenase that oxidized a broad range of aldehydes to acids.102
For catecholamines the two enzymes acting sequentially would produce deam-
inated acid metabolites (Fig. 9-3). (In 1951 Albert Zeller in Chicago suggested
FIGURE 9-3. Metabolic degradation of noradrenaline. The pathway on the right shows
the more common sequence: methylation of one ring hydroxyl to form normetanephrine,
followed by oxidative deamination and further oxidation to the final methylated, deam-
inated acid. Either sequence can occur, however, and all products, except the reactive
aldehydes, can be identified in animals.
the name monoamine oxidase to distinguish this amine oxidase from enzymes
that oxidatively deaminated diamines, such as histamine.103)
Richter concluded in 1937 that the "most probable function of the amine
oxidase appears to be the destruction of toxic amines . . . but it may also have
a special role in ... the physiological inactivation of adrenaline."104 Both those
roles were subsequently verified, but here the focus is on the latter process.
Blaschko and Richter had identified the enzyme in the brain, and others sub-
sequently identified it in sympathetic nerves.105 Still, the relative slowness of
noradrenaline s and adrenaline's oxidations in vitro fostered considerable skep-
ticism about the physiological relevance of this degradation.106 But with the
advent of radioactive tracer techniques, Richard Schayer in Chicago was able
to show in the early 1950s that administered adrenaline was deaminated/
demethylated in vivo. He concluded that these results "indicate a major role
for amine oxidase in [adrenaline] metabolism in the intact animal."107 Bernard
Brodie in Bethesda then used a recently recognized inhibitor of monoamine
oxidase, iproniazid (Marsilid), to produce marked increases in noradrenaline
levels in the brain.108 This finding implied that monoamine oxidase degraded
noradrenaline in the absence of this inhibitor.
In 1955 Udenfriend discovered that monoamine oxidase acted on serotonin
also, catalyzing its oxidative deamination to an aldehyde.109 After the action of
aldehyde dehydrogenase, the ultimate product would then be 5-hydroxyin-
doleacetic acid. Brodie found that inhibiting monoamine oxidase in vivo
increased the brain levels of serotonin as well.110 Subsequent studies confirmed
the degradation of both catecholamines and serotonin by monoamine oxidase
in vivo, although they did not demonstrate that monoamine oxidase acted phys-
iologically to destroy neurotransmitters released at the synapse.111 In fact, the
enzyme was localized to mitochondria in 1952,112 indicating an mfracellular
site of degradation.
With the development of new monoamine oxidase inhibitors, different pat-
terns of susceptibility appeared, depending on the source of monoamine oxi-
dase and the particular substrate assayed. This diversity led to proposals in the
late 1960s that two (or perhaps more) forms of monoamine oxidase existed.113
Although purification of monoamine oxidases was difficult and fraught with
artifactual fragmentations, the amino acid sequences of two distinct monoamine
oxidases, A and B, were obtained by cDNA methods in 1988.114 Both were
oligomers with subunits of 60 and 59 kDa, respectively.
Even though other mechanisms for terminating the actions of catecholamines
and serotonin were recognized later (see below), interest in monoamine oxi-
dases continued because of the therapeutic potential of drugs that inhibited
these enzymes. In the mid-1950s the first effective drug for treating psycho-
logical depression, iproniazid, was discovered by chance; when its antidepres-
sant efficacy was attributed to inhibition of monoamine oxidase, a search for
further antidepressant inhibitors followed (chapter 13). These drugs were
Syntnesis, Storage, Transport, and. Metabolic Degradation or Neurotransmitters 233
prescribed extensively in the 1960s, but they fell from favor when newer
drugs, acting by different mechanisms and having fewer side effects, became
available.
The original monoamine oxidase inhibitors blocked both monoamine oxidase
A and B. In the 1970s, however, deprenyl (selegilene, Eldepryl) was shown to
inhibit monoamine oxidase B selectively. Rationales then appeared for using
this drug in the treatment of Parkinson's disease (chapter 13). The clinical suc-
cesses in the 1980s soon reinvigorated the search for clinically effective mono-
amine oxidase inhibitors.
Catecnol-O-Methyltransrerase
In 1957 Marvin Armstrong in Salt Lake City described a methylated compound

from human urine that he identified as a metabolite of noradrenaline.115 He
suggested that this substance, 3-methoxy-4-hydroxymandelic acid (Fig. 9-3),
could be formed through amine oxidation followed by O-methylation.
That finding turned Julius Axelrod's attention to a new route for cate-
cholamine metabolism. Axelrod (Fig. 9-1B) had just received his Ph.D. in 1955,
although he had worked for over a decade with Brodie—first in New York and
then in Bethesda—as an often-independent investigator. With his doctoral
degree, his own laboratory at NIH, and a background in drug metabolism, Axel-
rod now set about defining the pathways for catecholamine disposition. Giulio
Cantoni, then also in Bethesda, had found a few years earlier that S-adenosyl
methionine was the donor for some biochemical methylations.116 Accordingly,
Axelrod showed in 1957 that extracts of rat liver destroyed catecholamines when
incubated with S-adenosyl methionine. He then identified O-methylated
metabolites (metanephrine from adrenaline and normetanephrine from nora-
drenaline; Fig 9-3) in vitro and in vivo, and he partially purified the respon-
sible enzyme, catechol-O-methyltransferase.11' Subsequently, Carola Tilgmann
and Nisse Kalkkinen in Helsinki purified the enzyme fully and in 1990 pub-
lished the amino acid sequence determined by cDNA methods.118
Although Axelrod concluded in 1958 that O-methylation was the "principal
route" for metabolism—a larger fraction was methylated than deaminated—
uncertainty about the physiological function of this metabolism persisted.119
Clearly, catecholamines were usually methylated as well as deaminated before
they were excreted from the body, but whether catecholamines were methy-
lated to terminate their actions was less obvious. For example, inhibitors of cat-
echol-O-methyltransferase could prolong the responses to administered adren-
aline but not noradrenaline, and these inhibitors affected responses to nerve
stimulation only modestly.120 Moreover, the bulk of the enzyme was present in
the cytoplasm, as Axelrod showed initially, and this localization made a role in
terminating synaptic transmission unlikely.121 By 1990 no clinically useful
inhibitors of catechol-O-methyltransferase were available.122
Enkepnalin-Cleaving Proteases
As soon as enkephalins became available for study, it was apparent that their
actions in vivo were quite brief. Neural tissues rapidly destroyed activity in
vitro, apparently through enzymatic destruction, and in 1978 Bernard Roques
and Jean-Charles Schwartz in Paris identified a cleavage point between glycine
and phenylalanine (Fig. 5-4E).123 Roques and Schwartz named the responsi-
ble enzyme "enkephalinase," and they argued for a physiological role since
treating with morphine increased this enzyme s level, whereas inhibiting this
enzyme produced experimental analgesia.124 Four years earlier John Kenny in
Leeds had described a neutral endopeptidase125 in kidney, and in 1983 he
showed that enkephalinase was the same as this widely distributed enzyme.126
Moreover, this neutral endopeptidase, a membrane-bound enzyme belonging
to a large family of zinc-containing peptidases, had its active site oriented toward
the extracellular milieu, appropriate for a neurotransmitter-destroying enzyme.
In 1987 groups in Paris, Montreal, and San Francisco reported the amino acid
sequence, defining an 85 kDa protein with one transmembrane segment.127
Early studies also revealed the cleavage of tyrosine from the N-terminus of
enkephalin, reflecting the action of an aminopeptidase.128 In 1985 Kenny iden-
tified this aminopeptidase as membrane-bound aminopeptidase-N.129 This
enzyme, too, belonged to the family of zinc-containing peptidases and was
widely distributed throughout the body. In 1988 Ove Noren in Copenhagen
reported the amino acid sequence for a 110 kDa protein with a single trans-
membrane segment.130
These peptidases cleaved other peptides besides enkephalin, and other pep-
tidases could cleave enkephalin. Nevertheless, they were closely tied to
enkephalin destruction and considerable interest then focused on finding
inhibitors of the neutral endopeptidase and/or aminopeptidase-N. Such
inhibitors could produce analgesia,131 but by 1990 no clinically useful agent was
identified.
Transport ("Reuptake")
While extending his metabolic studies on catecholamines, Axelrod identified a

different mode of inactivation, one that for many neurotransmitters was more
significant physiologically. Seymour Kety, Axelrod's superior at NIH, had com-
missioned, for a study on schizophrenia, the custom synthesis of adrenaline
labeled to high specific activity, and Axelrod obtained some of the leftover com-
pound. He then described in 1958 the labeled metabolites in humans and the
following year in cats and mice.132 In all species O-methylated metabolites
appeared in the blood rapidly, followed by deaminated ones. But by using cats
and mice Axelrod was also able to examine tissue contents post mortem. Sev-
Synthesis, Storage, Transport, ana Metabolic Degradation 01 Neurotransmitters 235
eral organs, notably heart, spleen, and adrenal, took up significant amounts of
labeled adrenaline; furthermore, the loss of labeled adrenaline from these
organs was protracted, with an appreciable fraction retained for hours.133 Axel-
rod repeated this study with labeled noradrenaline, and an even larger fraction
was taken up and retained by tissues.134 This noradrenaline was in nerves, since
prior denervation markedly diminished uptake.135 Moreover, autoradiography
combined with electron microscopy—as well as cellular fractionation studies—
showed labeled noradrenaline in synaptic vesicles.136 And although intra-
venously administered noradrenaline did not appear in the brain (like many
polar substances, it is excluded by the "blood-brain barrier"), when labeled
noradrenaline was injected into the cerebral ventricles it entered brain cells,
too.137
In 1961 Axelrod showed that noradrenaline initially taken up by nerves was
released when he stimulated these nerves.138 His model (Fig. 9-4A) summa-
rized this observation, depicting both release from the nerve ending and uptake
into it.
Also noteworthy were the effects of drugs on noradrenaline uptake. Reser-
pine, cocaine, and the recently introduced antidepressant drug imipramine
(Tofranil) all decreased tissue levels of labeled noradrenaline but increased
blood levels. In 1960 Axelrod concluded that these drugs acted "presumably
by interfering with binding" and in 1961 "by altering the binding sites at the
nerve endings"; later that year he added that they could act by "preventing the
entry and/or the binding."139
To distinguish between drugs blocking entry from the circulation (uptake)
and drugs preventing storage in neurons (viewed as binding), Axelrod gave
drugs before or after injecting labeled noradrenaline into animals.140 Cocaine
and imipramine reduced tissue levels when given before but not after; Axel-
rod concluded that they "block the entry . . . into storage sites but do not cause
. . . release."141 Reserpine, on the other hand, acted when given after as well
as before.142 The experiment not only tied specific drugs to actions at particu-
lar sites, it also distinguished between uptake, soon assigned to transport at the
cell membrane (see below), and storage, soon attributed to transport within
vesicles (see above).143
These findings, confirmed and extended by others, established transport into
nerve endings as the major means for terminating the effects of released nora-
drenaline.144 Since the transport system retrieved noradrenaline for reuse,145
the term "reuptake," emphasizing this recycling process, appeared in the
1960s146 and became the prominent designation.
These findings also accounted for some previously unexplained phenomena.
For example, denervation was known to enhance the responses to certain neu-
rotransmitters, and such "denervation supersensitivity" was now attributable to
the loss of reuptake—and hence the loss of inactivation—that followed the
FIGURE 9-4. Fate of released noradrenaline. A. Noradrenaline released from the nerve
ending (the horizintal Y on the left) can react with the receptor or be O-methylated
(within cells) or be lost into the circulation. In addition, as Axelrod's experiments showed,
noradrenaline in the blood can be taken up by the nerve endings, which can also take
up released noradrenaline. B. Brodie's scheme shows, at the top, a ouabain-inhibitable
ATPase (the Na+/K+-ATPase) that pumps Na + out of and K + into the nerve ending,
thereby establishing an electrochemical gradient for Na + across the membrane. This
gradient then drives the influx of noradrenaline by the carrier (C, in the middle of the
figure), a cotransport system (symporter). The noradrenaline that is taken up into the
cytoplasm is next packaged in storage vesicles, safe from monoamine oxidase (MAO)
that destroys cytoplasmic noradrenaline. The bottom of the figure indicates a Ca2+-
activated release of noradrenaline into the synaptic cleft. (A from Herting and Axelrod
[1961], Fig. 2, reprinted by permission of Nature, © 1961, Macmillan Magazines, Ltd.
B from Bogdanski and Brodie [1969], Fig. 4, courtesy of the American Society for Phar-
macology and Experimental Therapeutics.)
236
destruction of nerve endings. And the potentiating effect of cocaine on admin-

istered adrenaline, which Loewi had shown in 1910, was now attributable to
cocaine preventing inactivation by blocking reuptake.147
In the 1960s similar studies demonstrated the reuptake into tissues and nerve
endings of serotonin and GAB A, then candidate neurotransmitters.148 Later
studies showed that glycine and glutamate also relied on reuptake to terminate
their synaptic actions.149 But attempts to demonstrate reuptake of acetylcholine
failed. This neurotransmitter, unlike the other amine and amino acid neuro-
transmitters, relies on metabolic degradation rather than transport. Choline,
however, is transported into the nerve terminal after it is cleaved from acetyl-
choline in the synaptic cleft (see above).
Meanwhile, experiments in vitro explored the mechanisms involved in reup-
take. In 1961 Elwood Titus in Bethesda described an uptake of labeled nora-
drenaline into brain slices that achieved higher concentrations in the tissue
than were in the medium.150 Cocaine, imipramine, and reserpine blocked
uptake, as did ouabain, an inhibitor of the Na+/K+-ATPase and thus of the
active transport of Na + . Five years later Leslie Iversen, then in Boston, reported
that noradrenaline uptake into isolated hearts required Na + in the medium;
independently Brodie reported a similar dependence for uptake into rat heart
slices.151 Both cited similarities to recent studies showing that sugar and amino
acid transport required extracellular Na+.152 Brodie s model in 1969 (Fig. 9-4B)
depicted a primary active transport system (the ouabain-sensitive Na + /K + -
ATPase) that established a Na+-gradient across the cell membrane, together
with a secondary active transport system, driven by this Na+-gradient, for
pumping noradrenaline across the membrane.153 The energetically downhill
flow of Na + into the cell drove an energetically uphill influx of noradrenaline
(in Mitchell's terms, noradrenaline was carried by a symporter). Noradrenaline
arriving in the cytoplasm could next be packaged into storage vesicles to pre-
vent destruction by monoamine oxidase. (Storage, as described above but shown
later, relied on another secondary active transport system, this one an antiporter
driven by the H+-gradient formed by a H+-ATPase.)
In 1967 R. J. Blackburn in Sandwich and S. B. Ross and A. L. Renyi in
Sodertalje found that ouabain, cocaine, and imipramine inhibited serotonin
uptake into brain slices.154 Brodie then showed that synaptosomes could accu-
mulate serotonin as well as noradrenaline by Na+-dependent processes.155
Subsequent studies characterized the affinities of distinguishable cell mem-
brane transporters for various neurotransmitters, their specific ionic require-
ments, and their inhibitors.156 Isolating these transporters proved difficult, but
in 1990 Baruch Kanner in Jerusalem and his associates reported the amino acid
sequence, obtained by cDNA methods, for the GABA transporter.15' (Sequences
for dopamine, noradrenaline, and serotonin transporters followed the next
year.158 These sequences identified a family of cell membrane transport proteins
238
having 12 transmembrane segments but distinct from the family of vesicle trans-
porters for these same neurotransmitters.159)
A major interest driving these investigations was the clinical efficacy of reup-
take inhibitors. Imipramine and its siblings—the tricyclic antidepressants
(named for their chemical structure)—supplanted monoamine oxidase
inhibitors as the first choice for treating depression by the mid-1960s because
of their greater efficacies and lesser toxicities (chapter 13). Then in the late
1980s fluoxetine (Prozac)—the first of the "specific serotonin reuptake
inhibitors" and which enjoyed still fewer side effects—supplanted in turn the
tricyclic antidepressants. The popularity of these drugs resulted not only from
their safety but also from their utility in treating a range of disorders: depres-
sion, obsessive-compulsive disorder, panic attacks, bulimia and eating disor-
ders, and more.
Conclusions
By 1990 various investigators across the globe had delineated specific steps in
neurotransmitter synthesis, storage, and disposition. In doing so they defined
particular processes and uncovered energetic and regulatory niceties, all in
accord with general mechanisms of cellular metabolism and transport. For his
contributions Axelrod shared the Nobel Prize in 1970 with Bernard Katz and
Ulf von Euler.
This chapter illustrates some diverse courses of discovery, citing studies on
acetylcholine, catecholamines, serotonin, and enkephalins. The resulting mod-
els depicted how:
(1) Acetylcholine is synthesized through the condensation—catalyzed by

choline acetylase—of acetylCoA, a common unit in metabolic pathways,
with choline. Newly synthesized acetylcholine is next transported into
storage vesicles by a H+-driven antiporter. Released acetylcholine is
hydrolyzed by cholinesterase, liberating acetate plus choline (which is
then transported into the cytoplasm of the presynaptic nerve ending by
a Na+-driven symporter).
(2) Dopamine is synthesized from the amino acid tyrosine by enzymatic
hydroxylation and decarboxylation. Dopamine is next transported into
storage vesicles by a H+-driven antiporter; dopamine is converted enzy-
matically to noradrenaline within adrenergic vesicles. The actions of
released dopamine and noradrenaline are terminated through transport
into the cytoplasm by a Na+-driven symporter (reuptake); there they
may be reincorporated into storage vesicles for reuse. Lesser amounts
of catecholamines are degraded—in neurons and glia as well as liver and
kidney—by catechol-O-methyltransferase and monoamine oxidase.
(3) Serotonin is synthesized from the amino acid tryptophan by enzymatic

hydroxylation and decarboxylation. Serotonin is next transported into
storage vesicles by a H+-driven antiporter. The actions of released sero-
tonin are terminated through transport into the cytoplasm by a Na + -
driven symporter, where it may be reincorporated into storage vesicles
for reuse. Lesser amounts are degraded by monoamine oxidase.
(4) Enkephalins, like other peptide neurotransmitters, are clipped enzy-
matically from precursor proteins. The precursor protein is translocated
within the cellular tubulo-vesicular system during its synthesis, and there
processing to the final peptide occurs. Enkephalins within storage vesi-
cles are then carried from the cell body to the nerve endings. The actions
of released enkephalins are terminated through destruction by pepti-
dases.
Practical interest in these processes sprang from their sensitivities to newly

discovered drugs effective against previously intractable disorders. Reserpine,
which depletes stores of noradrenaline and serotonin (and dopamine, too, as
shown later), relieves the symptoms of schizophrenia. However, it can also pro-
duce psychological depression. Monoamine oxidase inhibitors decrease the
metabolism of noradrenaline and serotonin (and dopamine, too, as shown later)
and relieve the symptoms of depression. Tricyclic antidepressants such as
imipramine block the reuptake of—and thus also potentiate the effects of—
noradrenaline and serotonin (to different degrees depending on the particular
drug) and relieve the symptoms of depression.
Clinical utility was often discovered before the biochemical mechanisms.
Recognition of these mechanisms then inspired new studies on schizophrenia
and depression based on catecholamine and/or serotonin malfunctionings
(chapter 13). Such recognition also prompted searches for better agents acting
in these manners (uncovering, for example, the specific serotonin reuptake
inhibitors such as fluoxetine [Prozac]).
Notes
1. Brown and Feldberg (1936a). They measured acetylcholine release into the veins
draining the ganglia, using a leech bioassay. Physostigmine was present to block acetyl-
choline destruction by cholinesterase.
2. Quastel et al. (1936). They, too, added physostigmine and measured acetylcholine
with a bioassay.
3. Nachmansohn and Machado (1943). They used homogenates initially but later
saline extracts of the homogenates; they also included fluoride to prevent depletion of
ATP by irrelevant ATPases. Lipmann (1941) had recently published his magisterial
review delineating the role of ATP and "energy-rich" phosphate bonds.
4. Nachmansohn (1953, 1961).
5. Lipmann (1945).
6. Lipmann and Kaplan (1946). Nachmansohn was also pursuing a cofactor (Nach-
mansohn and Berman, 1946), as was Feldberg (Feldberg and Mann, 1946).
7. Kaplan and Lipmann (1949). It was Feodor Lynen in Munich, however, who
described the chemical structure of acetylCoA (see Lynen, 1953).
8. Korey et al. (1951).
9. Korkes et al. (1952).
10. Itoh et al. (1986).
11. See Jope (1979).
12. Macintosh et al. (1956).
13. See Jope (1979).
14. See Johnston et al. (1992).
15. Holtz et al. (1938).
16. Blaschko (1939). See also Blaschko (1942); Holtz (1939).
17. Blaschko (1950).
18. Demis et al. (1955, 1956). Blaschko was then visiting with Arnold Welch in New
Haven, following Welch's visit with Blaschko in Oxford. See also Hagen (1956); Uden-
friend and Wyngaarden (1956); Kirshner (1957).
19. Dopaminergic neurons lack dopamine-/3-hydroxylase, which is present in adren-
ergic neurons. The adrenal contains in addition an N-methyl transferase that converts
noradrenaline to adrenaline.
20. Nagatsu et al. (1964); Shiman et al. (1971); Haavik et al. (1988).
21. Grima et al. (1985).
22. See also Levitt et al. (1965).
23. Stadtman (1963). See also Bonner (1961).
24. Nagatsu et al. (1964).
25. Alousi and Weiner (1966).
26. For example, Goldstein et al. (1973); Harris et al. (1974); Joh et al. (1978); Meli-
geni et al. (1982).
27. For example, Albert et al. (1984); McTigue et al. (1985).
28. See Zigmond et al. (1989).
29. For example, Mueller et al. (1969); Thoenen (1970); Black et al. (1985); Faucon
Biguet et al. (1986).
30. For example, Kumakura et al. (1979); Lewis et al. (1987).
31. Lewis et al. (1987). They also showed that glucocorticoids, which are released
with stress, interact with the gene for tyrosine hydroxylase through a response element
to which the steroid receptor binds.
32. Clark et al. (1954); Lovenberg et al. (1962). See also Sumi et al. (1990), who
showed that the expressed cDNA exhibited both catalytic activities.
33. Lovenberg et al. (1962).
34. For example, Grahame-Smith (1964); Lovenberg et al. (1967); Nakata and Fuji-
sawa (1982); Cash et al. (1985).
35. Grenett et al. (1987).
36. See Hufton et al. (1995).
37. For example, Hamon et al. (1978); Ehret et al. (1989).
38. For example, Jequier et al. (1969).
39. For example, Green et al. (1962); Wang et al. (1962).
40. For example, Boman (1988).
41. Hughes et al. (1975a).
42. Steiner (1977).

43. Li and Chung (1976).
44. Although Simon had suggested "endorphin" as a generic name for all endoge-
nous opioids, the name instead became attached to this family. A third family of endoge-
nous opioids, the dynorphins, arises from a third precursor.
45. Nakanishi et al. (1979). The pituitary secretes adrenocorticotropic hormone
(ACTH) to regulate adrenal release of cortical hormones and melanocyte-stimulating
hormone (MSH) to control pigmentation.
46. Rossier et al. (1977); Lewis et al. (1978).
47. Lewis et al. (1980). They cleaved the presumed precursors with proteases and
identified released enkephalins chromatographically and by their actions on opioid
receptors.
48. Rossier et al. (1980).
49. Noda et al. (1982a). See also Gubler et al. (1982); Comb et al. (1982).
50. For example, Fricker and Snyder (1982); Hook and Eiden (1984); Seidah et al.
(1990).
51. For example, Temple et al. (1990).
52. Brown and Feldberg (1936a), p. 282.
53. Feldberg (1945).
54. Ibid., p. 614. Feldberg argued that acetylcholine was still protected from
cholinesterase after homogenizing the tissue (he assumed homogenization would rup-
ture all membranes).
55. Ibid., pp. 614, 615.
56. For example, Green (I960); Green et al. (1961); Burack et al. (1961).
57. Blaschko and Welch (1953); Hillarp et al. (1953). Welch was visiting Blaschko in
Oxford; shortly thereafter Blascko visited Welch in New Haven, where they studied the
formation of labeled catecholamines from labeled dopa (ref. 18).
59. Hillarp et al. (1954), p. 166.
60. Blaschko et al. (1957); Hillarp (1958b).
61. Hillarp and Nilson (1954); Blaschko et al. (1955). A semipermeable membrane
would permit diffusion of water but not catecholamines, allowing the catecholamines
to exert osmotic effects.
62. Hillarp et al., (1955); Blaschko et al. (1956).
63. Kirshner (1962). See also Carlsson et al. (1963).
64. Ibid., p. 2311.
65. Ibid., p. 2316.
66. Hillarp (1958a).
67. Holzbauer and Vogt (1956). Earlier that year Brodie described a reserpine-
induced "release" of serotonin from its stores in brain (Pletscher et al., 1956).
68. Bertler et al. (1960). See also Paasonen and Vogt (1956); Bertler (1961); Bog-
danski et al. (1968); Phillips (1974).
69. For the historical background, see Robinson (1997).
70. Ibid.
71. Bashford et al. (1975).
72. Ibid., p. 155.
73. Casey et al. (1977). See also Flatmark and Ingebretsen (1977).
74. See Robinson (1997). The ATP synthase can run backwards, in this mode hydrolyz-
ing ATP to pump H + .
75. Schuldiner et al. (1978). See also Johnson et al. (1978); Phillips (1978).
76. Jonasson et al. (1964). See also Kanner et al. (1979).
77. Johnson et al. (1979). See also Knoth et al. (1980). The gradient is an electro-
chemical gradient, dependent on both the active transport pump and the passive per-
meabilities of the membrane.
78. Stern-Bach et al. (1990).
79. Liu et al. (1992); Erickson et al. (1992).
80. For example, Anderson et al. (1982); Naito and Ueda (1985); Kish et al. (1989).
For early studies on vesicles from peripheral nerve, see von Euler and Lishajko (1963).
81. See Mains et al. (1990).
82. See DeCamilli and Jahn (1990).
83. Hokfelt et al. (1980).
84. For reports of enkephalins localized with amine neurotransmitters, see Charnay
et al. (1982); Hunt and Lovick (1982); Altschuler et al. (1983).
85. Stedman et al. (1932).
86. Alles and Hawes (1940).
87. Augustinsson and Nachmansohn (1949).
88. Nachmansohn and Lederer (1939).
89. Rothenberg and Nachmansohn (1947).
90. Kremzner and Wilson (1963); Leuzinger and Baker (1967).
91. Schumacher et al. (1986). There are, however, alternative forms and sequences:
the enzyme exists as an oligomer of different multiples and the mRNA is processed
("alternative splicing") to produce different sequences from the same gene.
92. Sussman et al. (1991). Their structure featured an active site serine deep within
a catalytic "gorge" and adjacent to a histidine adjacent to a glutamate, forming a charge-
relay system analogous to that of the serine proteases.
94. Wilson and Bergmann (1950). The crystal structure, however, showed that the
"anionic site" was not composed of negatively charged amino acids but of aromatic amino
acids, whose 7r-electron clouds serve the same function.
95. Wilson et al. (1950); Wilson (1951).
96. See Burgen (1949); Aldridge (1950).
97. Wilson (1966).
98. For a historical account, see Holmstedt (1963).
99. Wilson and Ginsburg (1955). See also Childs et al. (1955).
100. Hare (1928). Tyramine is the decarboxylation product of tyrosine, analogous to
dopamine but having a single phenolic hydroxyl. Hare reported that her enzyme did
not affect adrenaline, apparently because enzmatic oxidation of adrenaline is far slower
than oxidation of tyramine, whereas the spontaneous oxidation of adrenaline is rapid.
101. Blaschko et al. (1937a, 1937b); Richter (1937). The key to their success was
blocking other routes of oxidation with cyanide. For an autobiographical reminiscence,
see Blaschko (1972).
102. Racker (1949).
103. Zeller (1951). This repeated a less accessible naming in German during the war
(Zeller et al., 1940).
104. Richter (1937), p. 2028.
105. For example, Holtz and Westermann (1956) found monoamine oxidase activity
in adrenergic nerves, while Burn and Robinson (1952) and Stromblad (1956) reported
the disappearance of such activity after nerve degeneration.
106. See Kopin (1972). Richter (1940) had also expressed doubts about the func-
Synthesis, Storage, Transport, and Metabolic Degradation or Neurotransmitters 243
tional role, and Blaschko (1952) wondered whether an active termination mechanism
was necessary in light of the prolonged effects of peripheral sympathetic stimulation.
107. Schayer (1951a, 1951b); Schayer and Smiley (1953). Schayer did not demon-
strate deamination directly but showed a cleavage between the methyl carbon of adren-
aline and the a carbon of the side chain.
108. Spector et al. (1958); see also Pletscher (1957). These experiments were with
rabbits and rats; with some other species monoamine oxidase inhibitors produced lesser
effects (Kopin, 1972).
109. Sjoerdsma et al. (1955). Blaschko (1952) had reported the oxidation of serotonin
by tissue extracts earlier.
110. Spector et al. (1958).
111. For example, Grout (1961) found that inhibiting monoamine oxidase in vivo did
not affect cardiovascular function. See also Kopin (1972).
112. Hawkins (1952).
113. For example, Maitre (1967); Johnston (1968); Squires (1968).
114. Bach et al. (1988); Hsu et al. (1988); Ito et al. (1988).
115. Armstrong et al. (1957).
116. Cantoni (1953).
117. Axelrod (1957); Axelrod et al. (1958); Axelrod and Tomchick (1958); LaBrosse
et al. (1958).
118. Tilgmann and Kalkkinen (1990); Salminen et al. (1990).
119. LaBrosse et al. (1958), p. 593. See also Kopin (1972); Guldberg and Marsden
(1975).
120. Wylie et al. (1960).
121. Depending on the organism, there could be a substantial fraction of membrane-
bound catechol-O-methyltransferase, although this enzyme, too, acted on cytoplasmic
catecholamines. See Broch and Fonnum (1972); Roth (1980, 1992).
122. In the late 1990s an inhibitor of catechol-O-methyl transferase, tolcapone (Tas-
mar), showed promise in the treatment of Parkinson s disease.
123. Malfroy et al. (1978). See also Sullivan et al. (1978); Gorenstein and Snyder
(1979).
124. Malfroy et al. (1978); Roques et al. (1980).
125. Peptidases that cleave the terminal amino acids are exopeptidases; aminopepti-
dases and carboxypeptidases cleave the N- and C-terminal amino acids, respectively.
Endopeptidases cleave peptide bonds father within.
126. Kerr and Kenny (1974); Matsas et al. (1983). The designation refers to the opti-
mal pH for catalysis and distinguished this endopeptidase from the well-known one that
cleaves optimally at acidic pHs.
127. Devault et al. (1987); Malfroy et al. (1987).
128. Hambrook et al. (1976).
129. Matsas et al. (1985).
130. Olsen et al. (1988).
131. For example, Roques et al. (1980); de la Baune et al. (1982); Waksman et al.
(1985).
132. LaBrosse et al. (1958); Axelrod et al. (1959). Axelrods methods for separating
adrenaline and its metabolites were crucially important for these studies.
133. Earlier Burn (1932) had inferred an uptake of adrenaline into tissues, and sub-
sequent investigators, measuring unlabeled adrenaline, also argued for an uptake (e.g.,
Raab and Humphries, 1947; Nickerson et al., 1950).
134. Whitby et al. (1961).
135. Hertting et al. (1961a). Fluorescence techniques also revealed the uptake of
unlabeled catecholamines into neurons (Hamberger et al., 1964).
136. Wolfe et al. (1962); Potter and Axelrod (1963).
137. Glowinski et al. (1961).
138. Hertting and Axelrod (1961). They gave labeled noradrenaline to animals, load-
ing nerve endings in the spleen. They then removed the spleen and perfused it. When
they stimulated the nerves to the spleen, labeled noradrenaline appeared in the per-
fusate.
139. Whitby et al. (1960), p. 605; Axelrod et al. (1961), p. 384; Hertting et al. (1961b),
p. 152.
140. Axelrod et al. (1962).
141. Ibid., p. 297.
142. Reserpine was then thought to cause depletion of stores, an effect later attrib-
utable to enzymatic destruction of catecholamines that had been prevented by reser-
pine from entering the storage vesicles.
143. Iversen (1965) identified a second uptake system, designated uptake£, with lower
affinity for catecholamines. This system was later localized to non-neuronal cells, where
it uses quite different transporters; these can nevertheless function importantly in neu-
rotransmitter disposal.
144. For example, Stromblad and Nickerson (1961); Iversen (1967). Moreover, Kopin
et al. (1962), using perfused rat hearts, found less than half as much labeled noradren-
aline metabolized as taken up into reserpine-sensitive stores. Correspondingly, Grout
(1961) demonstrated that even when administered together, the inhibitors of
monoamine oxidase and of catechol-O-methyltransferase altered adrenergic responses
minimally.
145. See Brown (1965).
146. For example, Folkow et al. (1967).
147. Frohlich and Loewi (1910). MacMillan (1959) proposed that cocaine blocked
uptake into neural stores, as did Muscholl (1960).
148. For example, Marchbanks (1966); Blackburn et al. (1967); Ross and Renyi
(1967); Iversen and Neal (1968); Kuriyama et al. (1969).
149. For example, Neal and Pickles (1969); Logan and Snyder (1971).
150. Dengler et al. (1961, 1962); Dengler and Titus (1961).
151. Iversen and Kravitz (1966); Bogdanski and Brodie (1966).
153. Bogdanski and Brodie (1969).
154. Blackburn et al. (1967); Ross and Renyi (1967).
155. Bogdanski et al. (1968).
156. See Kanner and Schuldiner (1987). Although the electrochemical Na+-gradient
was the crucial driving force for these transporters, many had various requirements for
other ions, too.
157. Guastella et al. (1990).
158. Pacholczyk et al. (1991); Usdin et al. (1991); Hoffman et al. (1991); Kilty et al.
(1991); Blakely et al. (1991); Shimada et al. (1991).
159. See Schloss et al. (1992). Glutamate transporters belong, however, to a differ-
ent family (see Worrall and Williams, 1994).
10
NEUROTRANSMITTER
RELEASE
Proposals
Schemes for chemical neurotransmission needed explanations also of how neu-

rotransmitters could pass from presynaptic to postsynaptic neuron. Pertinent
issues included identifications of the source from which the neurotransmitter
was released, descriptions of the molecular machinery responsible for release,
and characterization of the regulatory systems that controlled release. Defin-
ing the underlying mechanisms proved to be a particularly formidable task: the
machinery operated rapidly and intermittently, within only minute regions of
the neuron, yet depended on complex molecular arrays.
By the mid-1930s arguments for neurotransmitters being stored in presy-
naptic neurons focused attention on how release from such stores occurred and
what triggered it. G. L. Brown and Wilhelm Feldberg in Hampstead found
that adding K + to the fluid perfusing sympathetic ganglia augmented the
response of postganglionic fibers to stimulation of preganglionic fibers, and in
1936 they suggested that K + "might be the agent responsible for the discharge
of [acetylcholine]."1 Two years later Henry Dale endorsed this suggestion, not-
ing that the "mobilization" of K + accompanying an action potential could
"release . . . acetylcholine from its depot at the nerve ending."2 In 1939, how-
ever, Brown showed that adding K + stimulated nerve fiber activity, indicating
245
a different source for the augmented response,3 and by 1950 Alan Hodgkin
and Andrew Huxley had demonstrated that during action potentials K + flowed
from the cell (chapter 4). Nevertheless, Brown, Feldberg, and Dales sugges-
tion persisted for some years. For example, a prominent pharmacology text-
book stated in 1956 that K + "released during passage of the nerve impulse . . .
may liberate free acetylcholine from a precursor protein complex, perhaps by
cation exchange."4
On the other hand, Bernard Katz and Paul Fatt in London found that a
decrease in the external Na + concentration diminished e.p.p.s at neuromus-
cular junctions.5 So they suggested in 1952 that the inward flow of Na + dur-
ing action potentials stimulated acetylcholine release. Their model of Na+
exchanging for acetylcholine invoked a membrane carrier that could alternately
transport Na + inward and acetylcholine outward across cell membranes.6
(The notion of membrane carriers ferrying polar solutes across nonpolar
membrane barriers was then in vogue.7 James Danielli had developed a model
for cell membranes in the 1930s that featured layers of lipid molecules flanked
by surface proteins. To account for the ready diffusion into and from cells of
small ions, such as K + , or small polar molecules, such as water, Danielli later
invoked pores through his model membrane. In the 1940s and 1950s selective
"carriers" were also included: such lipoidal carriers would form nonpolar com-
plexes that could diffuse across the lipid barrier, transporting larger polar sub-
stances, such as sugars and amino acids. No carriers had been identified by
1952, but Katz and Fatt's suggestion met the spirit of these conjectures.)
Then in 1954 Katz and Jose del Castillo described a quantal model for neuro-
transmitter release (chapter 4). They identified the smallest e.p.p. evoked at neu-
romuscular junctions—achieved by lowering the external Ca2+ concentration—
with the smallest spontaneously occurring m.e.p.p.s, and they interpreted this
"unit potential" as the response to one quantum of released acetylcholine. Ordi-
nary e.p.p.s thus represented the summed responses to hundreds of such fun-
damental quanta.
That year Eduardo DeRobertis in Seattle and Sanford Palay in New York
independently described electron micrographs of nerve endings filled with
numerous small vesicles (chapter 4). DeRobertis, moreover, reported that
"some of these vesicles seem to perforate the presynaptic membrane, so that
portions of the vesicles seem to lie in the intermembranal space [i.e., the synap-
tic cleft] and come into direct contact with the post-synaptic membrane."8
The obvious identification of physiologists' quanta with microscopists' vesi-
cles (or "particles") led Katz and del Castillo in 1955 to "imagine a mechanism
by which each particle loses its charge of [acetylcholine] in an all-or-none man-
ner when it collides with, or penetrates, the membrane of the nerve terminal."9
(They also presented evidence against Katz's earlier model for Na+/acetylcholine
Neurotransmitter Release 247
exchange via a membrane carrier.) Later in 1955, however, Katz and del Castillo
specified a more restricted process that became the standard model for neu-
rotransmitter release through 1990 (and beyond): vesicles might bind to reac-
tive sites on the membrane and there fuse with the membrane, opening a path-
way to the exterior through which the vesicle contents could enter the synaptic
cleft (Fig. 10-1A).10 In his Croonian Lecture of 1961, Katz specified that
"We do not believe that . . . vesicles are discharged bodily from the nerve
terminal."11
Soon afterward, Christian de Duve named the process of vesicles fusing with
cell membranes "exocytosis"; the complementary process—pinching off invagi-
nations of the membrane to form vesicles—he named "endocytosis".12 Accord-
ingly, neurotransmitter release achieved through fusion of vesicle with cell
membrane became known as "exocytotic release."
But even if neurotransmitters were stored in synaptic vesicles, other mech-
anisms for release were conceivable. Indeed, some investigators claimed that
vesicles dispensed their neurotransmitter into the cytoplasm of the presynap-
tic neuron. Release into the synaptic cleft would then occur from the cytoplasm
through pores or carriers in the cell membrane. For example, Nils-Ake Hillarp
in Lund reported in 1954 that after catecholamines were released from the
adrenal, the storage vesicles (or "granules") remained in the cells "apparently
unchanged in number"; he concluded that "secretion is not accompanied by
the discharge of these granules from the cells, but rather [catecholamines are]
liberated and then secreted."13 Later, he found no appreciable loss of adrenal
granule proteins during catecholamine release, in accord with his contention
that the entire granule was not discharged.14 Since less of the nonmembrane
("soluble") protein of the granules was lost than would occur if the entire gran-
ule contents were discharged, he also concluded that catecholamines were
released first into the cytoplasm, from which they would "have little difficulty
in permeating the cellular membrane."15 By contrast, the soluble proteins would
be retained within the cell.
Arguments for neurotransmitter release from the cytoplasm were reinforced
in the 1960s by comparisons of the neurotransmitter content of isolated synap-
tic vesicles with the content of free, unbound neurotransmitter (chapter 4).
Appreciable amounts of neurotransmitter appeared to be free in the cytoplasm,
and although some of this was clearly due to losses from the vesicles that
occurred during their isolation, significant amounts of cytoplasmic neurotrans-
mitter still seemed to occur in the native cell. Moreover, studies on neuro-
transmitter synthesis, storage, and reuptake demonstrated that neurotransmit-
ters passed through the cytoplasm to gain entry to the vesicles (chapter 9).
Correspondingly, neurotransmitters might pass from vesicles to cytoplasm en
route to the synaptic cleft. Thus, in 1966 Ulf von Euler in Stockholm referred
248
to an extravesicular pool of noradrenaline that was refilled by reuptake as well

as from the vesicular stores, and he identified this pool as the source of released
neurotransmitter.16
Evidence for Exocytotic Release
Morphological Studies
If synaptic vesicles fuse with the cell membrane to release neurotransmitters,

then stimulating presynaptic neurons should deplete their complement of
vesicles—unless the process of replacement keeps pace with exocytosis. Stim-
ulating presynaptic neurons should also increase the surface area of these nerve
endings through the addition of vesicle membranes—unless the process of
retrieval keeps pace as well. On the other hand, attempts to outrace replace-
ment and retrieval by stimulating at high rates for long times might reveal
changes due to neural exhaustion rather than physiological release.
Through the 1960s studies on various systems under various conditions gen-
erated reports that vesicle numbers did or did not change with stimulation.17
In the 1970s, however, more convincing evidence for exocytosis appeared. Thus,
Suthiwan Kwanbunbumpen in Canberra described in 1970 changes in the num-
bers of synaptic vesicles at the "active zones" of neuromuscular junctions
(regions of the presynaptic ending filled with vesicles and lying opposite post-
synaptic folds of the muscle18). Kwanbunbumpen found reduced vesicle num-
bers if, before stimulating, he administered hemicholinium, which halts acetyl-
choline synthesis by blocking choline transport (chapter 9).19 Without
hemicholinium, however, stimulation increased vesicle numbers. He inter-
preted this contrary observation as a physiological recruitment of new vesicles
for the increased activity. Soon afterward several investigators found decreases
FIGURE 10-1. Proposals for exocytotic release. A. Katz and del Castillo's version, show-
ing as dots the molecules on each membrane that interact to assure fusion of vesicle
with presynaptic membrane, followed by exocytotic release. B. Heuser and Reese's cycle
at neuromuscular junctions, showing the presynaptic terminal lying beneath a (striped)
Schwann cell and above the (striped) muscle. On the left, synaptic vesicles move toward
the membrane and fuse for exocytotic release. On the right, vesicle membrane is
retrieved as coated pits and coated vesicles, enclosed within clathrin baskets and bound
for intraterminal cisternae from which new synaptic vesicles bud. C. Breckenridge and
Aimer's sequence of reversible and irreversible steps, showing the initial formation of
a fusion pore followed by full exocytosis. (A from Fig. 5 of the report of a meeting held
in 1955 [del Castillo and Katz, 1957]. B from Heuser and Reese [1973], Fig. 36, repro-
duced by permission of Rockefeller University Press. C from Breckenridge and Aimers
[1987], Fig. 6, courtesy of Wolfhard Aimers.)
in synaptic vesicle numbers in sympathetic ganglia following intense stimula-

tion.20 Furthermore, J. J. Pysh and R. G. Wylie in Chicago described an increase
in the nerve ending surface, too, consistent with vesicle membrane being added
during exocytosis: "initial calculations indicate that the estimated quantity
of vesicle membrane lost agrees with the . . . increase of membrane in axon
endings."21
Also in 1972 Bruno Ceccarelli, visiting Alexander Mauro in New York,
reported decreased stores of acetylcholine accompanying decreased numbers
of vesicles at neuromuscular junctions.22 These changes followed moderate
rates of stimulation for 6-9 hours in the absence of hemicholinium. After 4
hours of stimulation the number had not changed, but by this time the nerve
endings contained the enzyme peroxidase, which he had included in the bathing
medium to demonstrate endocytosis. (Peroxidase, like other proteins, cannot
diffuse across membranes but can enter cells by endocytosis: it can be taken
up within vesicles newly formed from the cell membrane. The presence of per-
oxidase is demonstrable in electron micrographs through its oxidation of added
substrates to form electron-opaque deposits.) Ceccarelli interpreted these
observations as exocytotic release in the first several hours being matched by
an endocyiotic retrieval of vesicle membranes that maintained vesicle num-
bers.23 Only with prolonged stimulation did retrieval and reformation of vesi-
cles lag behind release.
John Heuser and Thomas Reese in Bethesda confirmed and extended these
observations. In 1973 they described a depletion in synaptic vesicles at neuro-
muscular junctions after stimulating for 1 minute at high frequencies, and this
depletion was balanced by an increase in the cell membrane so the total mem-
brane content remained constant.24 After stimulating for 15 minutes, there was
an even larger depletion accompanied now by the appearance of numerous
membrane-bounded cisternae within the nerve ending. After allowing the nerve
to rest for 15 minutes, these cisternae disappeared and new vesicles appeared.
When they included peroxidase in the bathing media, this tracer first entered
the cisternae and later the synaptic vesicles. When they stimulated the nerve,
the tracer disappeared from the synaptic vesicles. Accordingly, Heuser and
Reese proposed a scheme with neurotransmitter release via exocytosis, followed
by vesicle retrieval through endocytosis and the subsequent transfer of mem-
brane through cisternae to new vesicles (Fig. 10-1B).
Heuser and Reese next applied a technique recently developed for studying
intramembrane components (such as intrinsic membrane proteins), freeze-
fracture electron microscopy. They split frozen tissues mechanically, and some
of the cleavages passed between the leaflets of the membrane lipid bilayer.
Electron microscopy could then reveal structures extending through the mem-
brane interior. They found "dimples" appearing in the presynaptic membranes
after nerve stimulation, which they interpreted as exocytotic channels occur-
ring at the site of vesicle fusion.25 By conventional thin-section electron

microscopy they also found profiles identifiable as vesicles fused with the mem-
brane (Fig. 10-2). (Others had already reported such profiles of fused vesicles.26)
An obvious problem was distinguishing between exocytotic and endocytotic
images. Heuser and Reese argued that exocytosis occurred at the active zones,
whereas endocytosis occurred beyond this region. Moreover, they identified
endocytosis with "coated pits" and "coated vesicles," so named because of their
fuzzy surfaces (Figs. 10-1B and 10-2).27
FIGURE 10-2. Electron micrographs of exocytosis and endocytosis at neuromuscular

junctions. Figures 23 through 25 show, at the arrows, profiles of exocytotic release. Fig-
ure 26 shows, at the arrow, a coated pit undergoing transformation into a coated vesi-
cle. (From Heuser et al. [1974], Figs. 23-26, courtesy of Kluwer Academic Publishers.)
A further problem lay in the poor time resolution of these approaches. So

Heuser and Reese developed a quick-freeze technique that fixed tissues within
a few milliseconds, sufficient to resolve a single nerve impulse. This approach
allowed them in 1979 to photograph nerve endings "caught in the act of exo-
cytosis" (Fig. 10-3); moreover, they correlated the number of exocytotic fig-
ures with the quanta of transmitter discharged, equating one vesicle with one
quantum.28 And in 1985 Ceccarelli, back in Milan, improved the time resolu-
tion to less than 1 millisecond: he could now show that vesicles fused with the
membrane at the same time that quanta were released.29
FIGURE 10-3. Freeze-fracture electron micrographs of quick-frozen neuromuscular

junctions, from Heuser et al. (1979). Figure 7 shows the array of intramembrane par-
ticles, later identified as Ca2+ channels, at the active zone of an unstimulated neuro-
muscular junction. Figure 8 shows an active zone after stimulation, with transmembrane
openings (one within a box) considered to be "exocytotic stomata." The arrows point to
regions interpreted as collapsed vesicles incorporated into the presynaptic membrane.
(Reproduced by permission of Rockefeller University Press.)
Ceccarelli, however, argued against a collapse of vesicle into the cell mem-
brane and for a "quick direct removal of the vesicle membrane without it flat-
tening into the [cell membrane]."30 Through the 1980s controversies persisted
about the fate of the vesicle membrane: whether release required coalescence
with the cell membrane or involved merely a transient opening between vesi-
cle interior and synaptic cleft, followed by withdrawal of the emptied vesicle.
Biochemical Studies
Adrenal vesicles contain ATP as well as catecholamines, and in 1956 Hillarp

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

(with antibodies) in the presynaptic membrane.38
Electrical Studies
The quantal hypothesis arose from studying electrical depolarizations in post-

synaptic cells (e.p.p.s and m.e.p.p.s). Two other modes of electrical recordings
also provided continuous records of rapidly changing events, bolstering the
static morphological and biochemical observations.
Exocytotic fusion would increase the surface of presynaptic neurons by adding
vesicle membrane to cell membrane. Among the consequences of this increased
area would be an increased electrical capacitance.39 Accordingly, J. I. Gillespie
in Plymouth reported in 1979 that stimulation increased the capacitance of
presynaptic terminals at squid giant synapses, changes he identified with "the
incorporation of synaptic vesicles into the presynaptic membranes."40 Although
Gillespie could not quantitate the changes, Erwin Neher in Gottingen applied
his patch electrodes (chapter 6) successfully to this task, describing in 1982
discrete steps of capacitance changes during stimulation of adrenal cells.41
These he attributed to successive exocytotic fusions of catecholamine-containing
vesicles.
To achieve still better resolution, Neher next studied exocytosis from mast
cells (cells present throughout the body that participate in inflammatory
responses and that contain large histamine-containing vesicles available for
release). With these cells he could detect an initial flickering of the capacitance
values that he interpreted as the reversible formation of an "aqueous pore
between the secretory vesicle and [cell] membrane, before formation of the
(irreversible) fused state."42 And in 1987 Wolfhard Aimers in Seattle combined
patch electrode recordings with fluorescence measurements of exocytotic
release from mast cells (Fig. 10-4).43 He described the flickering capacitance
changes as the reversible openings of "fusion pores" linking vesicle interior to
cell exterior prior to the irreversible fusion of membranes (Fig. 10-1C). Neher s
and Almers's studies thus supported Heusers characterization of "narrow-
necked pores" in electron micrographs of mast cells, a process that began "with
formation of a narrow orifice" before the ultimate fusion of vesicle with cell
membrane.44 These studies provided strong evidence for exocytotic release
from mast cells, but comparable studies on neurons were not technically fea-
sible by 1990.
A second realm of electrical studies used voltammetry to monitor neuro-
transmitter release. Katz's identification of quantal release—and subsequent
electrophysiological studies—equated presynaptic release with postsynaptic
depolarizations. These postsynaptic responses, however, need not represent
release precisely, for they depended on the intervening ability of postsynaptic
FIGURE 10-4. Capacitance and fluorescence studies of release from mast cell vesicles.
The upper tracing shows an initial flickering of the capacitance changes (in pF) after
stimulation of release. The lower trace, over the same time course, shows the fluores-
cence change (F), representing the release of a fluorescent compound from the vesi-
cles, beginning after the period of flickering. (From Breckenridge and Aimers [1987],
Fig. 3B, courtesy of Wolfhard Aimers.)
receptors to mirror every change in neurotransmitter level. Obviously, a bet-

ter approach would be to measure the released neurotransmitter directly. For
neurotransmitters that are readily oxidized, such as catecholamines and sero-
tonin, voltammetry provided such an alternative. (In this technique current is
passed through electrodes and the flow measured at various voltages. Oxida-
tions at the electrode tip alter this current flow at characteristic voltages.) This
technique had been used for over a decade to record neurotransmitter release
in vivo when in 1990 Mark Wightman in Chapel Hill placed microelectrodes
near adrenal cells and found current transitions that he interpreted as the
"direct chemical measurement of single exocytotic events."45
Complexities ana Criticisms
By 1990 a wealth of studies provided firm support for exocytosis. Still, evidence
for exocytosis was most compelling for secretory cells, such as adrenal and mast
cells; for neurons evidence was strongest at neuromuscular junctions. Gener-
alizations from these cases to all chemical synapses satisfied most investigators,
but sufficient complexities were apparent to provoke continuing criticisms from
an active few.
First, not all neurotransmitter release was quantal. In the absence of stim-
ulation, even acetylcholine release at neuromuscular junctions failed this cri-
terion: in 1963 John Mitchell in Babraham measured release by bioassay and
found that at rest only a small fraction was attributable to quantal release, with
"a large proportion . . . derived from some other source."46 Indeed, Katz and
Ricardo Miledi argued in 1977 that "a steady leakage . . . from the terminals
[being monitored could] exceed the . . . quantal discharge by at least an order
of magnitude."47 Leakage, however, did not increase during stimulation, so that
quantal release then became far larger.
Second, newer studies revealed less clear-cut characteristics of quanta!/
vesicular release than initial measurements at neuromuscular junctions had
implied. The number of acetylcholine molecules per vesicle, calculated from
brain and sympathetic ganglia preparations, was 1000-2000, whereas the num-
ber of molecules per m.e.p.p. (or quantum) was 6000-10,000; in the far larger
vesicles of electric organ preparations, the number was greater than 100,000.48
These imperfect correlations between vesicle and quantum accompanied
renewed uncertainty about statistical analyses of release. Katz and del Castillo
calculated in 1954 that the number of quanta available for discharge was roughly
200.49 This was far smaller than the number of vesicles present in the region
being studied, and in 1977 A. Wernig in Munich argued that "a quantum might
be due to the simultaneous discharge of several or all the vesicles in one 'active
zone.' "50 Wernig's interpretation not only provided a value comparable to Katz
and del Castillo's estimate, it also fitted descriptions by Mahlon Kriebel in Syra-
cuse of postsynaptic responses smaller than m.e.p.p.s (together with multiples
of these sub-m.e.p.p.s).51 KriebePs observations raised the possibility that
m.e.p.p.s—and conventional quanta—were composed of subunits: each m.e.p.p.
could require the release of more than one vesicle, and one vesicle (or less)
could elicit a sub-m.e.p.p. Moreover, Henri Korn in Paris and Donald Faber
in Buffalo analyzed release at brain synapses as independent all-or-none pro-
cesses at each active zone, which could represent the coordinate discharge from
an invariant number of vesicles.52
Third, neurotransmitters seemed to be present in at least two pools, but the
functions and subcellular locations of these pools were debated. On the one
hand, Richard Birks and F. C. Macintosh in Montreal described the stimulated
release of acetylcholine from sympathetic ganglia in 1961, interpreting their
data as the sequential outflow from two intraneuronal pools.53 They suggested
that the smaller, more readily releasable pool corresponded to vesicles close to
the presynaptic membrane. The larger pool might then serve as a reservoir for
replacing acetylcholine in emptied vesicles of the smaller pool. Indeed, stud-
ies in the late 1960s using radioactive tracers supported models depicting a
readily releasable pool that was preferentially replenished. For example, Irwin
Kopin in Bethesda administered a labeled precursor of noradrenaline and then
compared the radioactivity in the noradrenaline released by stimulation from
an adrenergic nerve to that remaining in the nerve: he concluded that "newly
synthesized [noradrenaline] is selectively released."54
On the other hand, these labeling experiments did not actually identify the
pool of readily releasable neurotransmitter as that in vesicles adjacent to the
presynaptic membrane. And in the 1970s some—notably Maurice Israel in
Paris, Y. Dunant in Geneva, Ladislav Tauc in Gif-sur-Yvette, and Roger March-

banks in London—concluded that the newly synthesized and readily releasable
pool was cytoplasmic, not vesicular.55 By this time the synthesis of many neu-
rotransmitters was known to occur in the cytoplasm, and cell fractionation stud-
ies revealed significant amounts of cytoplasmic neurotransmitter. These critics
noted that vesicle numbers did not always change in parallel with neurotrans-
mitter content and that after stimulation the cytoplasmic neurotransmitter
could change more—in absolute amount and in degree of labeling—than the
vesicular.
In 1974 Herbert Zimmermann, visiting Victor Whittaker in Cambridge,
found, contrary to the report of Dunant and Israel, that after stimulating nerves
to Torpedo electric organ, the acetylcholine content of the vesicles initially fell
in parallel with the decline in transmission; only after prolonged stimulation
were the vesicles depleted of acetylcholine.56 And in 1977 Zimmermann and
Whittaker, now in Gottingen, described the acetylcholine content of vesicles
that had undergone exocytosis/endocytosis. They stimulated electric organ tis-
sue in media containing sucrose as well as a labeled precursor of acetylcholine,
so that sucrose was taken up by the endocytosis that followed exocytosis. When
they next isolated the vesicles by density-gradient centrifugation, those vesicles
containing sucrose were denser and thus separable from vesicles that had not
undergone the exocytotic/endocytotic cycle, and the denser vesicles contained
manyfold higher amounts of radioactivity.57 Thus, the emptied vesicles were
preferentially filled with new acetylcholine, in accord with the exocytotic model.
Advocates of cytoplasmic release also had to struggle to imagine a mecha-
nism that could discharge neurotransmitters in quantal packets. One sugges-
tion was for release through voltage-gated channels that would open transiently
to allow a given number of neurotransmitter molecules to diffuse out.58 But
William Van der Kloot in Stony Brook showed that quantal size was not altered
by changes in the membrane potential of the nerve ending, as would be
expected if acetylcholine release were governed by such a channel.59 Alterna-
tively, channel openings for given periods of time might produce quantal
release. But Van der Kloot found that altering neurotransmitter concentrations
in the cytoplasm (by increasing or decreasing the water content of the cell, con-
centrating or diluting cytoplasmic solutes) also failed to change quantal size.60
Nevertheless, Israel continued through the 1980s to advocate release by chan-
nels, even isolating a protein that he believed was this channel.61
Triggering or Release
In 1940 Macintosh, then in Hamptead, reported that extracellular Ca2+ was

required for acetylcholine release from sympathetic ganglia.62 His observation
extended earlier studies stretching back to the nineteenth century that showed
extracellular Ca2+ was necessary for nerves to elicit muscle contractions. Oth-
ers, however, advocated roles for K + and Na + in promoting neurotransmitter
release, as noted above.
During investigations of such cation effects, Katz and Fatt included exami-
nations of how Ca2+ affected neuromuscular transmission, and in 1952 they
uncovered a "curious effect": lowering extracellular Ca2+ "reduces the e.p.p.
in definite 'quanta,'" that is, in a stepwise fashion.63 That year del Castillo
reported correlations between Ca2+ concentrations and e.p.p. amplitude, sug-
gesting that "the amount of acetylcholine released . . . is a function of the con-
centration of calcium ions."64 Two years later Katz and del Castillo recast such
interpretations as Ca2+ increasing the quantal content of e.p.p.s, and they
argued that Ca2+ acted by raising the probability of quantal release.65
Katz and Miledi then used ganglionic synapses of squid giant axons, where
neuronal elements are large enough for direct electrical manipulations, to dem-
onstrate that graded electrical depolarizations, induced presynaptically, could
elicit graded responses postsynaptically as long as extracellular Ca2+ was pres-
ent.66 But when they raised the transmembrane potential of the presynaptic
terminals so that Ca2+ influx could no longer occur,67 there was no sign of neu-
rotransmitter release.
Douglas's work on secretory cells in the early 1960s, from which he prom-
ulgated the concept of Ca2+-dependent "stimulus-secretion coupling" (chap-
ter 7), thus complemented the conclusions of Katz and his collaborators. Taken
together, these studies promoted the notion that exocytotic release required
Ca2+ and that a requirement for Ca2+ implied exocytotic release.
The mechanism by which Ca2+ acted was less clear, however. Katz and col-
laborators first proposed an acetylcholine carrier in the membrane that must
initially contain Ca2+.68 On the other hand, Hodgkin and Richard Keynes, cit-
ing the disruptive effects of Ca2+ on cytoplasmic structures, suggested in 1957
that intracellular Ca2+ might be involved "in breaking up the intracellular vesi-
cles near the membrane . . . and releasing acetylcholine from them."69 Although
it was soon clear that Ca2+ must enter the cytoplasm to act, what happened
next would remain uncertain during the next 30 years.
Defining the entry process progressed more rapidly. In 1967 Katz and Miledi
concluded that "depolarization opens a 'gate' to calcium," and since pulses of
extracellular Ca2+ were effective only at brief intervals during a stimulus, nerve
activity must open such gates transiently.70 They also measured currents, attrib-
utable to Ca2+ influxes, that were associated with neurotransmitter release.71
The Hodgkin-Huxley model for nerve action potentials relied on voltage-
gated channels for Na + and for K + that opened in response to membrane depo-
larizations. Voltage-gated Ca2+ channels were identified soon thereafter. In
1985 voltage-gated Ca2+ channels were allocated among several classes, based
on electrical properties and sensitivities to particular inhibitors, and the amino
acid sequence of one class of channels was defined in 1987.72 Meanwhile, Bal-
domero Olivera and William Gray identified a toxin from cone snails that caused
muscle paralysis, a peptide that they named w-conotoxin.73 The previous year,
1984, Lynne Kerr and Doju Yoshikami, also in Salt Lake City, had described
this toxin's ability to block neurotransmitter release by preventing Ca2+ entry.74
The w-conotoxin was subsequently confirmed as a specific blocker of the per-
tinent class of Ca2+ channels and became a valuable reagent for studying
the role of Ca2+ in neurotransmitter release as well as for identifying these
channels.
Miledi succeeded in 1973 in showing that Ca2+ injected into presynaptic ter-
minals could evoke neurotransmitter release, confirming a cytoplasmic role.75
A year earlier Rodolfo Llinas in Rochester, Minnesota demonstrated, using a
light-emitting Ca2+-indicator, that cytoplasmic Ca2+ levels within presynaptic
terminals rose following stimulation, in accord with electrical measurements of
Ca2+ currents through transmembrane channels.76 Further studies with a vari-
ety of indicators confirmed such rises, but the limited spatial and temporal res-
olution of these techniques were inadequate for precise characterization of the
cytoplasmic Ca2+ changes. Nevertheless, calculations showed that, due to local
binding, the regions of elevated Ca2+ would be tightly circumscribed after a
localized influx.77
This spatial restriction was pertinent to the placement of calcium channels
relative to neurotransmitter release sites. In 1974 Heuser and Reese suggested
that the rows of intramembrane particles visible in freeze-fracture micrographs
of synaptic active zones (Fig. 10-3) might be Ca24" channels.78 Llinas, now in
New York, supported this identification in 1981 by correlating the number of
such particles, the conductance of Ca2+ channels (previously determined), and
the measured current flow across presynaptic terminals.79 And in 1990 Milton
Charlton in Toronto used labeled w-conotoxin to demonstrate that these Ca2+
channels were present exclusively at the active zones.80 This localization would
therefore allow the efficient interaction of incoming Ca2+ with synaptic vesi-
cles arrayed at the active zones, poised—according to the exocytotic model—
to discharge their neurotransmitter into the synaptic cleft.
Mechanism of Release
How does this localized Ca2+ influx trigger exocytosis and how does the exo-
cytotic machinery work? Early explanations invoked simple solutions. John
Eccless suggestion in 1959 took the form of a question: "Is it possible that
vesicles are positively charged and hence are greatly accelerated . . . across a
depolarized membrane?"81 In 1966, however, Banks demonstrated that adre-
nal vesicles bore instead net negative charges, incompatible with such an elec-
trophoretic extrusion.82 Banks then proposed that divalent Ca2+ could link neg-
atively charged vesicle membranes to (presumably) negatively charged cell
membranes, thereby promoting fusion and exocytosis, although he did not spec-
ify how the latter events occurred. Defining the basic processes, it was clear,
would require a better understanding of the components. It was also clear in
1990 that the identification of these components was still incomplete.
Synapsin
The first participating protein to be identified was synapsin I, although it was

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

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

protein in black widow spider venom, a-latrotoxin, causes a massive exocytotic
release of neurotransmitter even in the absence of Ca2+, and in 1985 J. Mel-
dolesi in Milan identified a protein in synaptic membranes that bound to
a-latrotoxin.100 It was subsequently included in a family of integral membrane
proteins called neurexins.101 And Michael Wilson in La Jolla found a 25 kDa
protein by screening a cDNA library, which in 1989 he named SNAP-25 (for
synaptosomal associated protein, plus its molecular weight).102 SNAP-25 did

not contain a likely transmembrane segment, although it was tightly bound to
membranes. No more could be said about its physiological role by 1990.
Vesicular Transport in Non-Neural Systems
While these studies were in progress, considerable success rewarded exami-

nations of intracellular transport between membrane-bounded cisterns of the
cellular Golgi system as well as secretory systems of various nonneural cells
(notably yeast).103 These processes represented "membrane trafficking" through
the budding off of vesicles from one membrane and their fusion with another:
endocytosis and exocytosis. As the 1990s began interest was growing in the par-
allels between exocytotic fusions at diverse cellular sites.104
(During the 1990s further progress was achieved in identifying additional
components of the synaptic exocytotic process, in drawing parallels with enti-
ties and processes among disparate secretory systems, and in constructing mod-
els showing how interacting entities accomplished exocytotic release. Never-
theless, no definitive model was available as the century ended.)
Enaocytotic Retrieval or Vesicles
Given the exocytotic explanation of neurotransmitter release, there remained

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

pentagons and hexagons like the seams on a soccer ball (Fig. 10-5).106 They
proposed that transformations of hexagons into pentagons caused the curva-
tures required to form first pits and then vesicles from the relatively planar
presynaptic membrane.
Barbara Pearse in Cambridge, UK, then developed an effective procedure
for isolating coated vesicles from brain. The protein component of this puri-
fied preparation, analyzed by SDS-PAGE, consisted of "essentially just one pro-
tein species" having a molecular weight of 180 kDa; this she named clathrin.107
The following year, 1976, Pearse concluded from electron micrographs that
these coated vesicles were bounded by 12 pentagons plus a variable number
of hexagons, depending on the vesicle diameter.108 She suggested that three
clathrin molecules met at each vertex of the polygons.
In 1981 Ernst Ungewickell and Daniel Branton in Cambridge, Mass., dis-
sociated the baskets into three-legged structures with pronounced knees, which
they named triskelions (Fig. 10-6).109 Each triskelion contained three clathrin
molecules (180 kDa) plus three smaller proteins (33—36 kDa); these became
known as the clathrin heavy and light chains, respectively.110 That year Pearse
depicted rearrangements of triskelions to convert hexagons to pentagons (Fig.
10-7),m but the mechanism for such transformations was still unresolved when
the decade ended.
Meanwhile, James Keen and Ira Pasten in Bethesda showed that clathrin
baskets could be released from coated vesicles by solutions of high ionic
strength, fragmenting the baskets into filaments.112 Remarkably, this transfor-
mation was reversed when they reduced the ionic strength sharply, with the
filaments reassembling into empty baskets. This spontaneous reconstitution of
FIGURE 10-5. Baskets of coated vesicles, from Kanaseki and Kadota (1969). Figure 24
shows a soccer ball composed of hexagonal and pentagonal panels, while Figure 25
shows a model of a coated vesicle with similar hexagons and pentagons outlining its sur-
face. Figure 26 is an electron micrograph of a coated vesicle from the brain, also show-
ing outlines of hexagons and pentagons. (Reproduced by permission of Rockefeller Uni-
versity Press.)
FIGURE 10-6. Electron micrographs of clathrin triskelions from brain coated vesicles.
(From Ungewickell and Branton [1981], Fig. 3a, reprinted by permission of Nature,
©1981 Macmillan Magazines, Ltd.)
FIGURE 10-7. Proposal for the conversion of clathrin hexagons to pentagons through
"small distortions of the triskelion." (From Crowther and Pearse [1981], Fig. 4. Repro-
duced by permission of Rockefeller University Press.)
264
Neurotransmrtter Release 265
the baskets did not occur at cytoplasmic ionic strengths, but Keen and Fasten
identified a fraction from coated vesicles—containing a 110 kDa protein—that
mediated the reconstitution under physiological conditions. They named this
the basket assembly factor. Subsequent studies in the 1980s revealed additional
assembly proteins with molecular weights of roughly 50 and 20 kDa; together
with the 110 kDa protein, they formed complexes capable not only of con-
trolling the size of the coated vesicles but also of selecting which areas of the
membrane surface would be transformed into coated pits and vesicles.113 More-
over, the assembly protein complexes were themselves regulated by phospho-
rylation/dephosphorylation cycles.
Before endocytotic vesicles can fuse with membranes their coats must be
removed. But whereas coat assembly proceeded without an extrinsic energy
source, coat removal required added ATP. In 1982 James Rothman in Palo Alto
found that a cytoplasmic factor promoted dissociation of the baskets at physi-
ological ionic strengths if ATP were present.114 ATP was hydrolyzed during the
dissociation process, and Rothman then purified the "uncoating ATPase," iden-
tifying a 70 kDa cytoplasmic protein that stripped the coats away.115 In 1986
he grouped this enzyme with a family of 70 kDa proteins mediating cellular
responses to stress (called heat shock proteins because of the stress first stud-
ied).116 The following year John Ellis in Coventry included these proteins in
his category of molecular chaperones, proteins that assist other proteins in fold-
ing and unfolding.11' But what spurred the uncoating ATPase into action was
not then apparent.
Ca -Independent Non-Exocytotic Release
The critics of exocytotic release noted above were concerned with Ca2+-
dependent processes. Different issues arose with the recognition by 1970 that
release could also occur in the absence of extracellular Ca2+.118 If such Ca2+-
independent release implied nonexocytotic mechanisms, then an obvious can-
didate for carrying neurotransmitters from cytoplasm to synaptic cleft would
be the cell membrane reuptake transporter (chapter 9) running in reverse.119
These Na + cotransport systems are normally driven by Na4" flowing down its
electrochemical gradient from outside the cell to the inside, carrying the neu-
rotransmitter in the same direction. But if the gradients were reversed—by
electrical depolarization and/or lower ratios of extracellular to intracellular Na +
and/or lower ratios of extracellular to intracellular neurotransmitter—the Na +
cotransport system could carry cytoplasmic Na + plus neurotransmitter out of
the cell.120
Accordingly, in 1978 Carl Cotman in Irvine reported that depolarizing synap-
tosomal preparations with high concentrations of extracellular K + could trig-
ger the release of GABA even in the absence of extracellular Ca2+.121 More-
over, this Ca2+-independent release appeared to come from a different intra-
cellular pool of GABA (as reflected in the degrees of labeling) than did the
Ca2+-dependent release that could also be evoked. If they first incubated the
synaptosomes in Na+-free media with agents that facilitated Na+ fluxes, then
the subsequent Ca2+-independent release was diminished; this was explainable
as the manipulations causing a loss of intracellular Na + , which would magnify
the Na + gradient from out to in. Conversely, raising intracellular Na + by facil-
itating influx or by hindering efflux, which would minimize the gradient, pro-
moted Ca2+-independent GABA release.122
Nevertheless, release in the absence of extracellular Ca2+ might still be
occurring through a Ca2+-dependent exocytotic process, but one sensitive to
elevated levels of intracellular Na + : in 1970 David Lust and Joseph Robinson
in Syracuse proposed that higher concentrations of intracellular Na + could free
into the cytoplasm Ca2+ sequestered within mitochondria.123 Consequently,
orthodox exocytotic release that requires a rise in intracellular Ca2+ could still
be operating in the absence of extracellular Ca2+ under these circumstances.
But in 1988 S. Bernath and M. J. Zigmond in Pittsburgh directly implicated
the cotransport system, showing that an inhibitor of the Na + -GABA cotrans-
porter blocked neurotransmitter release in the absence of extracellular Ca2+.124
Similar studies with other neurotransmitters also implicated Ca2+-independent
release in particular instances.125 Three categories of such Ca2+-independent
release were established by 1990, as illustrated below.
Physiological Release
Horizontal cells in the retina release GABA even though they do not contain
synaptic vesicles, and in 1982 Eric Schwartz in Chicago described release from
these cells in the absence of extracellular Ca2+.126 Subsequently, Schwartz
showed that such release occurred even when the intracellular Ca2+ concen-
tration was kept low with buffers to counteract any rise in Ca2+ contributed by
intracellular reservoirs; on the other hand, GABA release was blocked by an
inhibitor of the Na + cotransporter.12'
Pathological Release
Various deleterious conditions, such as anoxia, damage the brain by triggering

the release of glutamate, but this release does not require extracellular Ca2+.128
Anoxia and similar perturbations also elevate cytoplasmic Na+ concentrations,
due in part to the loss of active transport systems that normally extrude this
cation. So in 1990 David Attwell in London proposed that this glutamate release
was due to a reversal of the Na + cotransporter: experimentally elevated con-
centrations of intracellular Na + and glutamate promoted release, whereas ele-

vated concentrations of extracellular Na + and glutamate inhibited release.129
Pharmacological Release
Amphetamine acts in large part by promoting the release of catecholamines

and serotonin, but in 1975 Julius Axelrod in Bethesda demonstrated that such
release was not exocytotic.130 Electrical stimulation of adrenergic nerves
released not only noradrenaline but also dopamine-/3-oxidase, an enzyme nor-
mally present within the synaptic vesicles, so that its appearance extracellularly
signified exocytosis. By contrast, administering amphetamine released nora-
drenaline without dopamine-j3-oxidase, and this release occurred in the absence
of extracellular Ca2+. In 1990 David Sulzer in New York argued that amphet-
amine promoted the loss of catecholamines from the synaptic vesicles, with the
consequent rise in cytoplasmic levels of catecholamines then driving the Na +
cotransporters in reverse to release catecholamines.131 Indeed, inhibitors of the
cotransport systems diminished amphetamines ability to elicit release.132
These explanations of C a2+-independent release seemed plausible for neu-
rotransmitters having Na + cotransport systems, such as catecholamines, sero-
tonin, GAB A, and glutamate. But they were obviously inapplicable to acetyl-
choline, which has no such system. For this case Charles Edwards, visiting
Frantisek Vyskocil in Prague, noted that after exocytotic release of acetyl-
choline, the vesicular H+/acetylcholine exchanger would be incorporated by
exocytotic fusion into the presynaptic membrane.133 They suggested that these
exchangers could then mediate Ca2+-independent acetylcholine release across
the cell membrane until they were retrieved by endocytosis (they would then
be replaced by new exchangers from vesicles that fused subsequently). Corre-
spondingly, Vyskocil reported in 1985 that an inhibitor of the H+/acetylcholine
exchanger blocked Ca2+-independent release of acetylcholine.134
Conclusions
The quantal hypothesis, advanced by Katz in the early 1950s from studies at
neuromuscular junctions, attributed e.p.p.s to summations of m.e.p.p.s, each
representing the release of a uniform packet of neurotransmitter. Soon after-
ward electron microscopists described vesicles in nerve terminals, and Katz
identified his quanta with the contents of these vesicles. This elaboration trans-
formed his quantal hypothesis to an exocytotic one: release was achieved
through the fusion of neurotransmitter-containing vesicles with presynaptic
membrane. Evidence supporting and extending these models accumulated in
the following decades, with measurements of neurotransmitters within vesicles,
identifications of vesicular contents released exocytotically, micrographic

demonstrations of vesicle fusion and retrieval, and calculations of membrane
surface changes attendant on these fusions.
Other studies, again initiated by Katz and associates, tied quantal/vesicular
release to an influx of Ca2+ through specific channels in presynaptic mem-
branes. In the 1980s biochemists began identifying proteins in vesicle and
presynaptic membranes that were plausible components of the exocytotic
machinery. Essential entities were still unidentified as the decade closed, how-
ever, and a comprehensive model—one defining how rises in cytoplasmic Ca2+
triggered the sequence of protein-protein interactions to effect membrane
fusion—could not yet be formulated. On the other hand, electron microscopists
and biochemists developed a satisfying model for retrieving vesicle membranes
after exocytotic fusion, specifying the formation of clathrin-containing coated
vesicles.
As in previous chapters, prominent and convincing results emerged from
examining favorable, experimentally accessible preparations. Here these
included neuromuscular junctions, electric organs, and adrenal and mast cell
vesicles. Analogously, certain experimental conditions were particularly reward-
ing, such as modified Ca2+ concentrations and rapid rates of stimulation. Gen-
eralizations in biology are always precarious, with prototypic representations
inevitably failing somewhere. A critical issue, then, is the range of similarities.
But establishing this scope is frequently frustrated when less favorable prepa-
rations must be studied. For example, did the exocytotic release that was
demonstrable with high rates of stimulation also occur at physiological rates?
Did full fusion of vesicles occur routinely, requiring then retrieval by clathrin-
coated vesicles, or were transient and reversible associations the rule normally?
How did the quantal hypothesis accommodate nerve terminals having greater
heterogeneity among their vesicles? Indeed, clear incompatibilities were doc-
umented in specific cases, as in certain instances of Ca2+-independent, non-
vesicular release. Nevertheless, by 1990 models of vesicular/exocytotic release
were generally acknowledged as the standard formulation.
Notes
1. Brown and Feldberg (1936b), p. 291. See also Feldberg and Guimarais (1936).
2. Dale (1938b), p. 420.
3. Brown and Macintosh (1939). Stimulating the presynaptic fibers would cause them
to release more neurotransmitter.
4. Goodman and Gilman (1956), p. 399.
5. Fatt and Katz (1952a).
6. Fatt and Katz (1952c). This model followed an earlier proposal by Hodgkin and
Huxley for the ion transfer during action potentials occurring by membrane carriers
(although by this time Hodgkin and Huxley had withdrawn their proposal because of
contrary evidence).
8. DeRobertis and Bennett (1954), p. 35. The observation and interpretation was
repeated in DeRobertis (1958).
10. del Castillo and Katz (1957). The meeting at which they presented this paper was
in 1955.
11. Katz (1962), p. 471.
12. de Duve (1963).
13. Hillarp et al. (1954), p. 162.
14. Carlsson and Hillarp (1956).
15. Ibid., p. 239.
16. von Euler (1966).
17. For example, Birks et al. (1960); Hubbard and Kwanbunbumpen (1968).
18. Active zones were depicted by Birks et al. (1960) and named by Couteaux and
Pecot-Dechavassine (1970).
19. Jones and Kwanbunbumpen (1970). Parducz and Feher (1970), however, claimed
that hemicholinium caused a breakdown of synaptic membranes due to the cell's con-
sequent lack of choline for phospholipid biosynthesis.
20. For example, Birks (1971) stimulated at 20 Hz for 20 minutes without hemi-
cholinium; Pysh and Wiley (1972) used interrupted trains of stimuli at 20 to 32 Hz for
150 to 190 minutes in the presence of hemicholinium.
21. Pysh and Wiley (1972), p. 192. See also Pysh and Wiley (1974).
22. Ceccarelli et al. (1972). See also Ceccarelli et al. (1973).
23. Holtzman et al. (1971) had earlier correlated peroxidase uptake with nerve
activity.
24. Heuser and Reese (1973).
25. Heuser et al. (1974). See also Pfenninger et al. (1972); Peper et al. (1974).
26. For example, Birks et al. (I960); Couteaux and Pecot-Dechavassine (1970); Dou-
glas et al. (1970).
27. Douglas et al. (1971) had already proposed a role for coated vesicles in neu-
rosecretory cells of the pituitary.
28. Heuser et al. (1979), p. 275. They also stimulated transmitter release chemically
to improve the yield of exocytotic processes. Previously, Birks et al. (1960) calculated
that exocytotic processes should be too rare to record in micrographs consistently. See
also Heuser and Reese (1981).
29. Torri-Tarelli et al. (1985).
30. Ibid., p. 1398.
32. Douglas et al. (1965).
34. Banks and Helle (1965).
35. For example, Blaschko et al. (1967); Kirshner et al. (1967); Schneider et al. (1967).
36. Viveros et al. (1968, 1969).
37. For example, Geffen et al. (1969); Weinshilboum et al. (1971).
38. von Wedel et al. (1981).
39. Capacitance changes linked to surface area changes had been studied with other
cellular phenomena earlier. For example, Rothschild (1957) determined with this
approach the cell surface changes that accompany fertilization.
40. Gillespie (1979), p. 304.

41. Neher and Marty (1982).
42. Fernandez et al. (1984), p. 454.
43. Breckenridge and Aimers (1987). They used mast cells from a strain of mice hav-
ing "giant" vesicles. See also Zimmerberg et al. (1987).
44. Chandler and Heuser (1980), pp. 666, 670.
45. Leszczyszyn et al. (1990), p. 14,736.
46. Mitchell and Silver (1963), p. 126. See also Fletcher and Forrester (1975); Vizi
and Vyskocil (1979).
47. Katz and Miledi (1977), pp. 59-60.
48. Whittaker and Sheridan (1965); Wilson et al. (1973); Fletcher and Forrester
(1975); Kuffler and Yoshikami (1975); Ohsawa et al. (1979).
49. del Castillo and Katz (1954a).
50. Wernig and Stirner (1977), p. 821.
51. Kriebel and Gross (1974); Kriebel at al. (1990).
52. Korn et al. (1981).
53. Birks and Macintosh (1961).
54. Kopin et al. (1968), p. 271. They compared "specific activities": radioactivity in
the fraction/noradrenaline present in the fraction. See also Besson et al. (1969); Collier
(1969).
55. For example, Dunant et al. (1972); Dunant and Israel (1979); Tauc (1982); March-
banks (1978).
56. Zimmermann and Whittaker (1974). They also included electron microscopic evi-
dence for exocytosis.
57. Zimmermann and Whittaker (1977). See also Suszkiw et al. (1978).
58. For example, Birks (1974); Marchbanks (1978).
59. Cohen and Van der Kloot (1983).
60. Van der Kloot (1978).
61. Israel et al. (1986). Birman et al. (1990) obtained the amino acid sequence of a
16 kDa subunit. This sequence was highly similar to that of the H+-transporting sub-
units of the vesicle ATPase involved in neurotransmitter storage.
62. Harvey and Macintosh (1940). See also Birks and Macintosh (1957).
63. Fatt and Katz (1952b), pp. 119, 120; (1952c). Only the latter cited Harvey and
Macintosh.
64. del Castillo and Stark (1952), p. 515.
65. del Castillo and Katz (1954b).
66. Katz and Miledi (1967b).
67. Normally, Ca2+ enters passively, flowing down its electrochemical gradient into
the cytoplasm. When the gradient is reversed, however, no net influx of Ca2+ can occur.
68. Fatt and Katz (1952c); del Castillo and Katz (1954a).
69. Hodgkin and Keynes (1957), p. 279.
70. Katz and Miledi (1967a), p. 543. See also Katz and Miledi (1967c).
71. Katz and Miledi (1969). Others subsequently studied Ca2+ currents extensively,
for example, Llinas et al. (1976); Augustine et al. (1985).
72. Nowicky et al. (1985); Tanabe et al. (1987). The latter paper, however, described
only one protein of the oligomeric channel complex.
73. Olivera et al. (1985).
74. Kerr and Yoshikami (1984).
75. Miledi (1973). An earlier attempt was unsuccessful (Miledi and Slater, 1966),
reinforcing the possibility that Ca2+ acted on the surface of the membrane, not after
crossing the membrane.
76. Llinas et al. (1972). They used aequorin, a protein that emits a flash of light when
exposed to Ca2+. See also Llinas and Nicholson (1975).
77. For example, Fogelson and Zucker (1985); Simon and Llinas (1985). Moreover,
the brief time interval between depolarization and release meant that the path of Ca2+
diffusion must be short (Llinas et al., 1981).
78. Heuser et al. (1974).
79. Pumplin et al. (1981).
80. Robitaille et al. (1990).
81. Eccles and Liley (1959), p. 103.
82. Banks (1966). See also Blioch et al. (1968).
83. Ueda et al. (1973).
84. Ueda and Greengard (1977).
85. Bloom et al. (1979).
86. DeCamilli et al. (1983a). Greengard subsequently distinguished a family of
synapsins: see Siidhof et al. (1989).
87. Huttner et al. (1981); Kennedy and Greengard (1981).
88. Llinas et al. (1985).
89. DeCamilli et al. (1983b).
90. Bahler and Greengard (1987). See also Petrucci and Morrow (1987).
91. Hirokawa et al. (1989), p. 111.
92. Matthew et al. (1981).
93. Perin et al. (1991).
94. Perin et al. (1990).
95. Jahn et al. (1985); Wiedenmann and Franke (1985).
96. Siidhof et al. (1987); Leube et al. (1987). See also Buckley et al. (1987).
97. Thomas et al. (1988).
98. Trimble et al. (1988).
99. Baumert et al. (1989).
100. Scheer and Meldolesi (1985).
101. Ushkaryov et al. (1992).
102. Oyler et al. (1989).
103. See Wilson et al. (1991).
104. See Kelly (1988); Aimers (1990); Trimble et al. (1991).
105. Roth and Porter (1964).
106. Kanaseki and Kadota (1969).
107. Pearse (1975), p. 97. Later, she and others would identify a number of other
functionally important proteins of the coated vesicles.
108. Crowther et al. (1976).
109. Ungewickell and Branton (1981). See also Kirchhausen and Harrison (1981).
110. Earlier, Pearse (1978) described 30-36 kDa proteins from the coat, but she did
not calculate a 1:1 stoichiometry with the 180 kDa proteins.
111. Crowther and Pearse (1981).
112. Keen et al. (1979). See also Woodward and Roth (1978); Schook et al. (1979);
Unanue et al. (1981).
113. For example, Pearse and Robinson (1984); Manfredi and Bazari (1987); Keen
et al. (1987); Virshup and Bennett (1988).
114. Patzer et al. (1982).
115. Schlossman et al. (1984).

116. Rothman and Schmid (1986).
117. Ellis (1987).
118. For example, Katz et al. (1969); Srinivasan et al. (1969).
119. See Cutler et al. (1971); Martin (1976).
120. The concentrations of solutes and electrical potentials required for reversal are
readily calculable: see Attwell et al. (1993).
121. Haycock et al. (1978).
122. Sandoval (1980). She, however, argued for Ca2+-dependent release through
Na+-induced liberation of mitochondrial Ca2+.
123. Lust and Robinson (1970). See also Carafoli et al. (1974).
124. Bernath and Zigmond (1988).
125. For example, Haycock et al. (1978); Raiteri et al. (1979); Fischer and Cho (1979).
126. Schwartz (1982). See also Yazulla and Kleinschmidt (1983).
127. Schwartz (1987).
128. For example, Ikeda et al. (1989). By this time, high concentrations of extracel-
lular glutamate were known to cause neuronal damage.
129. Szatkowski et al. (1990).
130. Thoa et al. (1975).
131. Sulzer and Rayport (1990). For early and late reports of amphetamine affect-
ing the catecholamine content of storage vesicles, see Schiimann and Philippu (1962)
and Knepper et al. (1988).
132. Raiteri et al. (1979); Fischer and Cho (1979).
133. Edwards et al. (1985).
134. Vyskocil (1985).
11
FORMATION OF
SPECIFIC SYNAPSES
Embryonic Development or Synaptic Connections
The Neuron Theory arose at the end of the nineteenth century as holistic con-
ceptions of neural function were waning. Physiological and anatomical studies
identified discrete pathways for voluntary and autonomic systems as well as for
particular reflex circuits having sensory and motor limbs (chapters 1 and 2).
Accordingly, formulations grounded in the Neuron Theory depicted neural
pathways as cellular units linked through specific synapses. Such formulations,
in turn, required accounts of how such linkages came to be.
Embryology was then a flourishing science,1 led by a German school of
descriptive embryologists that began in the early nineteenth century with Carl
Ernst von Baer and embraced such pioneers as Rudolph von Koelliker and
Wilhelm His. They were now joined by a new wave of German experimental
embryologists, starting with Wilhelm Roux and Hans Driesch, who intervened
in the developmental processes to demonstrate capabilities and alternative out-
comes. They and their successors in the twentieth century, including Viktor
Hamburger, Hans Spemann, and Paul Weiss, identified sequential transfor-
mations that were effected through cellular differentiations and migrations,
with ancestral precursors giving rise to mature cells of distinct types and func-
tions then assembled into specific tissues and organs.
273
Explaining how such differentiations and migrations were directed and

achieved became a compelling challenge through the twentieth century,
although one incompletely satisfied. Nevertheless, experimental efforts incor-
porating anatomical, physiological, and biochemical approaches—and, notably,
the techniques of molecular biology as these became available—provided by
1990 detailed accounts of complex causal chains linking genetic direction to
cellular biochemistry, cellular biochemistry to intercellular associations, and
intercellular associations to cellular biochemistry and genetic expression.
Studies of neural development were an integral part of this effort and
reflected the same conundra apparent in the formation of other organ systems:
distinct cells types must assemble in proper order to construct a functional
whole. This account, however, will consider a single topic: what guides a grow-
ing axon during development to a particular location and thereby establishes a
specific synaptic connection.
Approaches ana Possible Mechanisms
His's studies on the outgrowth of nerve cell processes provided a central foun-
dation for the Neuron Theory (chapter 1). These morphological studies also
supported concepts of neural development through the extension of such pro-
cesses to reach particular destinations. Santiago Ramon y Cajal pursued these
issues with characteristic vigor and insight, in the process discovering a crucial
participant, the growth cones at the termini of elongating axons (chapter 1).
His's and Cajal's interpretations were based on microscopic examination of
fixed, stained specimens taken from different neurons at different stages of
development, requiring interpolations and extrapolations to reconstruct a con-
tinuous process from the collection of discontinuous images. Ross Harrison
confirmed Cajal's inferences about amoeboid locomotion by watching axonal
extensions from neurons grown in tissue culture (chapter 1). Harrison could
thus follow the protoplasmic extension of growth cones, with processes extend-
ing to spin out an axon from the fixed cell body. Observations of live neurons
in tissue culture would remain a valuable model for studying neural develop-
ment through the twentieth century, despite the inherent artificiality of such
isolated growth and development.
Another opportunity for continuously monitoring axonal extensions was
microscopic examination of neural development in (suitable) organisms. For
example, in the 1930s Carl Speidel in Charlottesville took time-lapse pho-
tomicrographs of developing nerves in transparent tadpoles, recording the
advances and retractions of motile growth cones on axonal sprouts.2 Although
Speidel recorded responses of the outgrowing axons to some experimental
Formation or Speciiic Synapses 275
manipulations, such as electrical shocks and assorted drugs, the consequences

of more vigorous interventions could be examined better at other loci with
fixed, stained specimens.
Major manipulations, following the pattern of early successes in experimen-
tal embryology, included ablations and transplantations of neural precursors
and/or their ultimate targets. These studies could then display not only histo-
logical evidence of the resultant connections, but also the functional conse-
quences. (Neural regeneration after experimental interruptions was considered
to be a reasonable approximation to the embryological processes responsible
for initial innervations.) Another source of modifications was genetic mutation,
either induced or occurring naturally; the consequent differences illustrated
altered patterns of innervation and function and tied these differences to iden-
tifiable genetic controls.
Different organisms provided certain experimental and interpretive advan-
tages. Vertebrates, particularly mammals and especially humans, commanded
the most attention but presented the greatest experimental difficulties. Avian
embryos were simpler than mammalian and more accessible for study. Fish
and amphibians, which regenerate limbs and connections from their central
nervous systems, were favored organisms for more drastic manipulations. In all
these animals, the most profitable systems for study turned out to be nerve-
muscle and eye-brain connections.
With simple invertebrates anatomical complexities were vastly reduced, so
that the life histories of identifiable neurons could be traced during develop-
ment. In addition, a wealth of genetic information could be applied also to
studies of neural development. And in the 1970s a roundworm, Caenorhabdi-
tis elegans, was chosen for intense genetic and developmental studies: it had
the advantage of a rapid reproductive rate (allowing the selection of specific
mutant strains) and notable structural simplicity (a total of 959 distinguishable
cells in the adult, of which 302 are neurons).
Through the twentieth century investigators used these approaches and
organisms to examine several obvious possibilities for guiding axonal growth.
His imagined a mechanical guidance, with the axon following the course of
least resistance, turning, and even splitting, when faced with an obstacle. Cajal
acknowledged a role for mechanical constraints but advocated strongly a speci-
ficity directed by chemotaxis (or chemotropism).3 The growth cone, Cajal
argued, sensed chemicals secreted by the target, with this interaction favoring
amoeboid growth up the concentration gradient to its source. (Chemotaxis had
already been proposed and was by Cajal's time a prominent explanation for the
migration of white blood cells to sites of inflammation.) At this time John New-
port Langley independently suggested, from his studies on the organization of
the autonomic nervous system (chapter 3), that chemical signals directed neu-
ronal organization.4
Early Arguments Concerning Cnemotaxis (1890—1963)
In the decades after Cajal and Langley argued that chemical attractants guided
axonal outgrowth, these proposals languished despite the eminence of their
advocates. No evidence for attraction was forthcoming, and attempts to dem-
onstrate it failed. For example, in 1934 Weiss, now in Chicago, reported that
adding saline extracts of brain to discrete regions of the tissue culture medium
neither attracted nor repelled axonal outgrowth.5
But even before this demonstration, Weiss had suggested an alternative
mechanism. While still in Vienna he advanced in 1924 a Resonance Principle
to account for the apparently specific connections between nerves and end
organs, such as muscles and sense organs.6 When Weiss grafted an additional
leg near a developing one in amphibia, this extra leg became innervated and
then moved in concert with the natural one. Weiss believed that these "homol-
ogous responses" arose from homologous muscles responding specifically to
(resonating with) particular frequencies of impulses carried by the nerves.
Coded frequencies thus selected which muscle contracted: functional connec-
tions were effected not by activating particular pathways but by evoking par-
ticular patterns of impulses—in nerves broadly—to which only certain muscles
would resonate/respond.
When Edgar Adrian in Cambridge succeeded in recording from the indi-
vidual fibers of nerves in 1928 (chapter 3), he found no such pattern of impulses
keyed to particular muscles.7 Two years later C. A. G. Wiersma, while visiting
Adrian in Cambridge, directly examined Weisss proposal. Wiersma found
action potentials appearing not in all the fibers of a motor nerve, but only in
those running to the particular muscle actually responding.8 So Weiss devised
a new account, pushing back the site of frequency-dependent control to the
central nervous system (although he retained the term Resonance Principle).9
Outgrowing axons, Weiss now argued, were initially unspecified functionally,
but when axons reached a muscle, even if by random growth, they were mod-
ified by this contact: "converged] from indifferent into selective receivers
specifically adapted" to activate this muscle.10 The motor nerve now resonated
with the proper directives from the central nervous system; inside the central
nervous system, Weiss believed, commands were encoded as particular pat-
terns of frequencies.11 Weiss acknowledged that the modification or "modula-
tion" of contacting axons may "plausibly be supposed to be a biochemical effect"
of muscle on neuron, although he could not define those changes further.12
By 1934 Weiss favored a mechanical guidance of nerve directly to muscle.
Twenty years earlier, Harrison had showed that axons in tissue culture required
a solid support for growth and could follow surfaces within this support.13 Weiss,
in the same paper where he discounted chemical guidance, described exten-
sions of Harrison's studies. He aligned fibers within the matrix supporting neu-
Formation or Speciric Synapses 277
rons in tissue culture and found that outgrowing axons then followed this align-
ment.14 Earlier experiments had shown that growing tissues, such as trans-
planted legs in amphibia, attracted outgrowing axons.15 So in 1941 Weiss argued
that growing tissues induced a tension in the intercellular matrix, thereby align-
ing molecular fibers of this matrix to form a pathway to the developing end
organ.16
Weiss in 1941 also extended Harrisons notion of initial pathfinder neurons,1'
adding a mechanism whereby the pathfinder neuron on reaching a target would
be altered, as in his earlier Resonance Principle. Now, however, the alterations
would include modifications of the pathfinder neuron's surface such that any
neurons contacting it would grow alongside it. Consequently, neurons accu-
mulating around the pathfinder would form a bundle ("fasciculus"). This "selec-
tive fasciculation" would thus assemble a group of axons running together to
the same target. Contact with the target then assured that each incoming axon
was modulated, or tuned, to receive the central commands activating this mus-
cle specifically.
Weiss, after receiving his doctoral degree in Vienna and working in Berlin,
went to New Haven in 1929 to visit Harrison and then spent the years from
1933 to 1954 in Chicago. Among his graduate students there was Roger Sperry
(Fig. 11-1A), who received his Ph.D. in 1941, the year Weiss's summation of
pioneering neurons and selective fasciculation appeared. Sperry, however,
FIGURE 11-1. A, left, Roger W. Sperry (1913-1994). B, right, Rita Levi-Montalcini

(1909-). (Courtesy of the National Library of Medicine.)
began an independent course that edged him step by step away from Weiss s
formulations.
Sperry's dissertation described the consequences of surgically interchanging
nerves to the extensors and flexors of rat leg muscles.18 Unlike Weiss s findings
in amphibia (and some earlier reports on mammals), movements of the rein-
nervated rat leg were reversed, and this reversal was permanent: the aberrant
responses could not be overcome by learning. Thus, the muscles, after being
reinnervated by "incorrect" nerves, did not contract as they had before the
interchange, a result contrary to Weiss s initial Resonance Principle that envi-
sioned muscles responding to their characteristically coded impulses transmis-
sible by any nerve. Sperry's results also did not support the revised Resonance
Principle that envisioned muscles altering any arriving nerve so that it accepted
and transmitted signals to the muscle from the appropriate centers in the cen-
tral nervous system. Sperry suggested that, after their initial embryonic differ-
entiation and synapse formation, mammalian motoneurons could no longer
undergo muscle-specific modulation when regenerating. They were "no longer
in a sufficiently labile state to be respecified by foreign muscles."19
By 1943 Sperry, now working in Cambridge, Mass., moved further from
Weiss s formulations of functional rather than topographical connections.20 In
amphibia the ganglion cells in the retina send axons through the optic nerve
to a region at the top of the brain, called the tectum, that receives responses
from the retina. Sperry now found that after cutting the optic nerve and rotat-
ing the eyeball 180°—so what was formerly up was now down—the amphib-
ians visual world after the optic nerve regenerated was inverted also: the
amphibian reacted to food placed above it as if the morsel were below. Sperry
concluded that regeneration reestablished the original connections, which
would now report the world upside down. He suggested that the guiding fac-
tors were chemicals, perhaps induced during embryonic differentiation, that
directed regenerating fibers to their original targets in the tectum. By 1949
Sperry, back in Chicago, was advocating a "chemoaffiniry theory" to account
for this specificity.21
Sperry's observations in the 1940s were functional, not histological, and he
could not demonstrate that retinal neurons actually reconnected with their orig-
inal targets. He continued to cite the coding aspects of the Resonance Princi-
ple also, acknowledging that such a mechanism could specify "the quality of
the excitatory impulses which different kinds of fibers discharge," so that "func-
tional organization [would] depend . . . not upon a specificity of fixed neuron
connections, but upon the emission of qualitatively distinct excitatory agents
[with] a selective receptivity to these discharges permitting functional preci-
sion."22 Consequently, there could be an "orderly recovery of precise functional
relations despite random, indiscriminant" connections of retinal with tectal
Formation or Specinc Synapses 279
Not until the late 1950s did Sperry, now in Pasadena, undertake experiments
showing in a convincing manner that regenerating retinal neurons did indeed
reconnect to their original tectal loci.24 He destroyed the ganglion cells in one
half of the retina of a fish and then cut the optic nerve; regeneration could only
be from the half of the retina not destroyed. By destroying different halves
(upper, lower, left, right) Sperry demonstrated that the residual neurons (from
lower, upper, right, left halves) grew to distinguishably different regions of the
tectum (Fig. 11-2). Moreover, ingrowing fibers would pass inappropriate tec-
tal neurons to proceed, over their original courses, to the proper targets.
How could these neurons find their way? Sperry, in an influential paper
reviewing these studies in 1963, advocated a chemical specification.25 He imag-
ined two or more chemical gradients that would determine by their relative
strengths along opposing axes the latitude and longitude for a correspondingly
receptive axon to follow. Support for this proposal would require identifica-
tions of these guiding chemicals and demonstrations of how outgrowing axons
could respond to specific concentrations of chemical signals. Sperry himself
did not pursue these demands (he soon became diverted by studies on split
FIGURE 11-2. Schematic representation of Sperry's experiment showing specific

regrowth of retinal neurons. The figures on the left show that destroying the top half
of the retina (lower drawing, blank segment) results in a regrowth from the bottom half
of the retina to the upper half of the tectum (upper drawing, showing the distribution
of ingrowing fibers). The figures on the right show the reverse case. A, P, D, V, M, and
L refer to orientations: anterior, posterior, dorsal, ventral, medial, lateral. (From Attardi
and Sperry [1963], Fig. 3, courtesy of Academic Press.)
brain preparations, for which he was awarded a Nobel Prize). Those who did
found the technical difficulties formidable, and progress on chemical guidance
was delayed for some decades. In the meantime, other relevant concerns came
into focus.
Cell Death and Neurotropnic Factors
Cell Death in Morphogenesis
The death of certain cells, it was recognized early in the twentieth century,
helps to shape organisms to their adult form. Significant losses of neural cells
occur also. So selective innervation might reflect an early generalized innerva-
tion with a subsequent elimination of inappropriate connections. Indeed, evi-
dence accumulating by mid-century pointed to such initial overproductions fol-
lowed by selective exterminations.
This appreciation emerged, however, from considering another issue: how
did the numbers of neurons growing out from the spinal cord match the size
of the end organ they innervated? The problem was illustrated clearly by
Samuel Detweiler, a student of Harrison in New Haven. In 1920 Detweiler
reported that grafting an extra limb bud in amphibia increased the size of the
dorsal root ganglion innervating this region, whereas removing the limb bud
decreased the size.26 In the early 1930s Victor Hamburger reexamined this
phenomenon. Hamburger, who had been a student of Hans Spemann in
Freiburg but was now visiting in Chicago, found that after destroying limb buds
in avian embryos, the motoneuron region in the spinal cord decreased pro-
portionally to the loss of muscle.27 He imagined that the initial axons contact-
ing a muscle sent signals back to the spinal cord to increase or decrease the
number of motoneurons being differentiated from their embryonic precursors.
A different suggestion came from Rita Levi-Montalcini (Fig. 11-1B), work-
ing first in Turin as the war broke out and continuing alone after her flight to
the countryside to escape German occupiers.28 Levi-Montalcini found, by
counting cells at successive times, that the decrease after extirpating an end
organ reflected a dramatic loss of differentiated neurons. After the war Ham-
burger invited her to St. Louis, where he was now working, and in 1949 they
reported that while an end organ could increase or decrease the proliferation
of neuronal precursors, it also "provides conditions for continued growth and
maintenance of neurons" after the first outgrowth of axons.29
Nerve Growth Factor (NGF)
Identifying such conditions was a striking achievement in twentieth-century

neuroscience, and although the active factors are of tangential significance here,
Formation of Specific Synapses 281
the route to characterizing the first of these deserves mention. Elmer Bueker,
who had studied with Hamburger but was now working in Washington, wanted
to study axonal outgrowth in a rapidly growing but less complex tissue. For this
purpose he tried transplanting several tumors into avian embryos and in the
late 1940s found that a mouse sarcoma induced a pronounced enlargement of
the spinal ganglia that sent sensory neurons to the transplant.30 He did not pur-
sue these issues further, but Levi-Montalcini and Hamburger soon confirmed
Bueker's observations. In 1951 they proposed that the sarcoma "producefd]
specific growth promoting agents which stimulated the growth of sympathetic
and of sensory . . . cells . . . but not of motor [cells]."31 Two years later they
reported that sarcomas transplanted at a distance could still promote axonal
outgrowths, so the factor(s) must be diffusible.32 The following year, 1954, they
described a stimulated outgrowth from dorsal root and sympathetic ganglia cells
in tissue culture, providing a means for quantitating the factor in vitro.33 Using
this assay, Stanley Cohen, a biochemist who had joined them, could now show
stimulation by cell-free homogenates of the sarcoma.34 While attempting to
purify the factor, Cohen and Levi-Montalcini used snake venom preparations
rich in phosphodiesterase activity to destroy nucleic acid polymers. To their
astonishment, the snake venom alone stimulated axonal outgrowth spectacu-
larly.35 Subsequently, they tried mouse salivary glands—homologs to snake poi-
son glands—and these proved to be still richer sources. With this starting mate-
rial Cohen purified a protein fraction in 1960 that they called nerve growth
factor (NGF).36 Antibodies prepared against this material, which should block
its actions, indeed destroyed sympathetic ganglia when injected into newborn
mice. This was compelling evidence that NGF was necessary to maintain devel-
oping sympathetic postganglionic neurons. Using a new, highly sensitive assay,
Hans Thoenen in Martinsreid was finally able to show in 1983 that target tis-
sues actually contained NGF, present in amounts correlating with the density
of their sympathetic innervation.37 The amino acid sequence, published in 1971,
specified a 13 kDa peptide,38 with active NGF being a dimer of this peptide.
(The three-dimensional structure followed twenty years later.39) For their sem-
inal discoveries Levi-Montalcini and Cohen received the Nobel Prize in 1986.
Demonstrating how NGF functioned was a more challenging quest. In the
mid 1970s Leslie Iversen in Cambridge and Thoenen, then in Basel, showed
that labeled NGF was taken up by sympathetic nerve endings and transported
along the axons to their cell bodies, as might be expected for a growth factor
secreted by end organs to regulate survival of innervating neurons.40 In 1979
Eric Shooter in Palo Alto described high- and low-affinity binding sites for
NGF that might be receptors,41 but further identification proved difficult.
(In 1991 groups in New York, Frederick, and Princeton showed that the
high-affinity binding site corresponded to trk-A, a tyrosine kinase of previously
unknown function.42 By this time several cellular growth factors, including
insulin and epidermal growth factor, were known to bind to membrane recep-
tors that then functioned as tyrosine kinases: phosphorylating proteins on the
phenolic hydroxyl of tyrosine [rather than on the alcoholic hydroxyls of serine
and threonine as did, for example, protein kinases A and C]. As in the responses
of other tyrosine kinases, trk-A dimerized when it bound its activating ligand,
NGF, and then catalyzed its own phosphorylation. Thus activated, trk-A next
catalyzed the phosphorylation of certain other proteins, on their tyrosines, to
initiate cascades of cellular second messenger systems.43 On the other hand,
the identity of the low affinity binding sites remained uncertain, as did the
functional consequences of NGF s transport to the cell body, which had been
demonstrated earlier.)
Levi-Montalcini and Hamburger showed that NGF affected sensory neu-
rons from dorsal root ganglia and postganglionic sympathetic neurons. Thoe-
nen found in 1979 that NGF also affected certain cholinergic neurons in the
brain.44 But what modulated the growth and survival of other neurons? It
seemed likely that many more growth factors operated; indeed, additional
growth factors with distinctive targets but structures similar to NGF were iden-
tified during the 1980s (and more were characterized in the following decade).45
Eliminating Erroneous Connections by Neuronal Death
Hamburger and Levi-Montalcini s demonstration of neuronal cell death dur-

ing embryonic development renewed interest in this process and its conse-
quences. One issue was competition among innervating axons for an end organ.
In 1958 Hamburger interpreted this process as "a selective survival of those
neurons which find an adequate peripheral milieu."46 A necessary trophic fac-
tor, if produced by the end organ in limited supply, could restrict the number
of surviving neurons. Another issue, one addressed increasingly through the
1980s, was "programmed cell death," or "apoptosis"; reports that cell death in
the roundworm C. elegans was controlled genetically furthered this interest.47
Here, however, the relevant issue is whether selective cell death was the
means for assuring specificity, for various investigators had suggested over the
decades that such a mechanism could prune away erroneous connections.48
Indeed, in some cases there was clearly a secondary elimination of synapses
formed in embryos but not present in adults. For example, Maxwell Cowan in
St. Louis showed in 1976 that axons growing out from paired brain nuclei to
the retina innervated not only the eye on the opposite side from the innervat-
ing nucleus (as in adults) but also the eye on the same side.49 The neurons
making the ipsilateral innervation then died, leaving the adult pattern of inner-
vation. But, as is often the case in biological research, universal explanations
were impossible. Even if cell death did revise synaptic connections at some
sites, it was clear by the 1980s that axons at other sites grew to individual and
appropriate destinations. Specific guidance systems must also function.
Ckemical Guidance (1963-1990)
Further Evidence for Specific Guidance
While Sperry was demonstrating regrowth to the tectum in the 1960s, addi-
tional examples of precise routings were also being cataloged.50 Further char-
acterizations of the requisite mechanisms were not forthcoming at that time,
however, but two later endeavors merit specific mention. Lynn Landmesser in
New Haven found that motoneurons proceeding to limb muscles in avian
embryos made few errors, with adjacent fibers growing to different and spe-
cific destinations. Landmesser concluded in 1981 that these "observations
exclude models . . . in which there is a widespread testing of the environment
with removal of projection errors by cell death."51 And Corey Goodman in Palo
Alto was then mapping the outgrowth of specific neurons in insect embryos.
Identifiable axons grew along discrete courses, making abrupt turns at charac-
teristic loci to reach their particular end organs (Fig. 11-3). Goodman argued
that they must follow labeled pathways.52
Diffusible Factors
Although Weiss s crude extract showed no neurotropic ability, approaches in

the late 1970s used instead a specific substance that could be applied in con-
centrated form, NGF. (By this time NGF was a known trophic—nutritive—
agent; these experiments tested it as a tropic—turning—agent.) Paul Letour-
neau in Palo Alto grew sensory neurons from embryonic dorsal root ganglia in
tissue culture so that added NGF formed a concentration gradient within the
supporting gel; axons, he reported in 1978, grew up the NGF gradient.53 The
next year Ross Gundersen and John Barrett in Miami showed similar tropisms,
with growth cones turning within minutes toward an NGF source.54
In 1983, however, Andrew Lumsden and Alun Davies in London argued that
one or more other factors besides NGF attracted axonal outgrowth from sen-
sory neurons.55 Their demonstration relied on younger sensory neurons, at a
stage for mouse embryos before these neurons required NGF for survival (in
contrast to Letourneau's and Gundersen and Barretts experiments). Axons still
grew toward a target tissue present in the culture dish, and antibodies to NGF
did not block this tropic effect.
Five years later Lumsden, collaborating with Thomas Jessell and Marc
Tessier-Lavigne in New York, reported clear tropic responses at another site.56
Cells from a region of the developing spinal cord called the floorplate, when
grown in culture so substances diffusing from them would generate a gradient,
attracted the outgrowing axons of cultured cells from a different region. Mean-
while, Edward Hedgecock in Nutley used genetic techniques to examine neu-
ronal guidance in C. elegans. He selected mutant worms whose neurons failed
FIGURE 11-3. Schematic map of axonal outgrowths from specific neurons of develop-
ing insects. The circles represent the cell bodies of neurons identified by the enclosed
letters and numbers. The emerging lines show the pathways followed by their growth
cones, turning at particular points to reach their distinctive targets, in some cases ante-
riorly, in others posteriorly, while other axons pass through these branch points without
turning. In the figure anterior is up. (From Raper et al. [1983b], Fig. 11, ©1983 by the
Society for Neuroscience.)
to grow along proper pathways and then identified the mutated genes respon-
sible. Among those identified by 1990 was unc-6.57
(Continuations of these studies during the next five years—[1] by purifying
active proteins secreted by floorplate cells and then determining their amino
acid sequences by cDNA techniques and [2] by sequencing the unc-6 gene—
Formation or Speciiic Synapses 285
identified a family of structurally related proteins named netrins.58 These

netrins, when diffusing from a target cell, attracted the growth cones of cer-
tain outgrowing axons.)
Adhesion Molecules
Harrison, as noted above, showed that neurons in tissue culture required a sup-
porting matrix for axonal outgrowth, and Weiss argued for mechanical guid-
ance by such supports. On the other hand, the matrix might attract or repel by
chemicals on its surface. Accordingly, Letourneau in the mid-1970s demon-
strated hierarchies of adhesiveness to various surface materials guiding growth
in vitro.5Q Others soon demonstrated gradients in the retina and tectum.60 But
identifying the pertinent molecules was difficult: the scientific problem was
obvious, but solving this problem—choosing a feasible system and devising suc-
cessful methods—demanded insight and ingenuity.
One attempt began in the early 1980s with investigations of proteins that
form the extracellular matrix in the nervous system as well as in other body
organs. For example, when one of these proteins, a laminin, was applied to the
surface of tissue culture plates, it promoted axonal outgrowth over itself.61 Soon
thereafter a family of cell membrane proteins, integrins, was recognized as
receptors for laminins and other matrix proteins.62 Interactions between a par-
ticular integrin on a cell and an extracellular protein such as a laminin could
thus favor growth selectively. Moreover, laminins had restricted localizations,
and Letourneau, now in Minneapolis, identified in 1988 a laminin-coated path-
way for axonal extension within the developing brain.63 On the other hand, this
laminin pathway existed transiently, so other guides must operate as well.64
Additional candidates included members of two families of cell surface mol-
ecules identified during these decades. These membrane proteins interacted
with like molecules (homophilic associations), in contrast to the heterophilic
interactions between laminins and integrins. The first identified was a neural
cell adhesion molecule (N-CAM). Gerald Edelman in New York, having deci-
phered essential properties of antibody molecules (Nobel Prize, 1972), described
in 1977 structurally related molecules that promoted adhesion between neurons
in avian embryos.65 These N-CAMs, like the antibody immunoglobulins, existed
in a plethora of subtly different structures, and studies through the 1980s dem-
onstrated specific interactions dependent on particular N-CAMs.66 The second
family, N-cadherins, were identified in 1983 by Masatoshi Takeichi in Kyoto as
Ca2+-dependent proteins that also produced intercellular adhesion.67 Further
studies soon demonstrated that N-cadherins also represented a range of vari-
ants capable of selective associations.68 N-CAMS and N-cadherins could there-
fore promote association between neural processes.
So it was clear by 1990 that multiple modes of interactions were involved in
axonal guidance, including the steering of pioneer neurons along a novel course
as well as the enlisting of follower neurons to form the nerve bundles. Weiss s
notion of selective fasciculation was firmly supported.
But in addition to attractions and adhesions, local interactions could also be
negative. For example, Friedrich Bonhoeffer in Tubingen demonstrated in
1990 the collapse of growth cones induced by membranes from tectal cells.69
(In 1992 Goodman, now in Berkeley, identified, by using antibodies to screen
for its effects, a cell surface protein that controlled axonal turning.70 The fol-
lowing year Jonathan Raper in Philadelphia identified a protein that induced
growth cone collapse.71 Goodman then pointed out that these belonged to a
structurally and functionally related family, named semaphorins, that included
both membrane-bound and diffusible repellants.72)
Growtn Cone Motility
How do growth cones follow these cues? This question includes an inquiry
about motility. Cajal likened advancing growth cones to amoebae extending
pseudopodia. Such locomotion also appeared in many motile cells of higher
organisms: cytoplasm apparently flowed into the advancing margins, often fol-
lowing fingerlike extensions. In axonal outgrowth, however, the cell body did
not accompany the advancing growth cone but remained behind as the axon
elongated. In addition, microscopy revealed tiny spikes ("filopodia") first
extending from the leading edge of the growth cone (Fig. 11-4), with the web
between adjacent filopodia then advancing, or the filopodia retracting and new
ones extending elsewhere.
Explanations for amoeboid movement reflected the mechanistic mode of the
age. For example, in the 1930s, when colloidal processes seemed to underlie
FIGURE 11-4. Growth cone morphology. A. The growth cone at the axon terminus
ends in numerous filopodia. B. The growth cone cytoplasm contains microtubules, mito-
chondria, and vesicles. Actin microfilaments are not depicted. (From Bray [1973], Figs.
1 and 2. Reprinted by permission of Nature, © 1973, Macmillan Magazines, Ltd.)
a host of physiological functions, amoeboid movements were attributed to

sol/gel transitions in the colloidal protoplasm.73 But with the recognition dur-
ing the 1950s and 1960s of microscopic tubes and fibers in the cytoplasm, expla-
nations centered on these components, construed as a cytoskeleton that con-
trolled cell shape and motility. Investigations of axonal outgrowth then followed
approaches successful with other motile cells and were grounded in conclu-
sions drawn from their study.
One relevant antecedent, it turned out, was the identification of actin micro-
filaments. By 1970 the protein actin had been characterized as a component
both of muscle s contractile machinery (together with myosin) and of microfil-
aments present in many other cell types (identified microscopically by their
binding to added myosin).74 Actin existed in two forms, monomeric G-actin
(globular) and polymeric F-actin (filamentous), the latter constituting micro-
filaments. Cytochalasin, a toxin from fungi, inhibited cellular motility and dis-
organized microfilaments.
In 1970 Norman Wessells in Palo Alto reported that adding cytochalasin to
embryonic neurons caused their filopodia to retract and their growth cones to
become spheroidal.75 He also described networks of microfilaments in growth
cones, and cytochalasin disrupted these. The following year Dennis Bray in
Cambridge identified actin from growing nerve cells as a prominently labeled
band by SDS-PAGE.76 Bray thought that Wessels microfilaments were made
of actin, and in 1972 Paul Burton in Lawrence demonstrated myosin binding
to neuronal microfilaments.77 Robert Goldman in Cleveland then showed that
this myosin binding extended to microfilaments within growth cones and filopo-
dia.78 Visualizing microfilaments in growth cones and filopodia was difficult,
but by the end of the decade Joel Rosenbaum in New Haven, using improved
imaging techniques and new labeling reagents, found bundles of actin filaments
running longitudinally within filopodia as well as a meshwork of microfilaments
beneath the growth cone membrane.79
How could actin filaments cause locomotion? Bray in 1973 suggested inter-
actions between strands of actin and myosin arranged longitudinally within
filopodia.80 This scheme drew on the sliding filament model for muscle con-
traction elaborated in the 1950s: interdigitating filaments of actin and myosin
slid past one another to effect shortening. Myosin was difficult to identify in
growth cones, however, and even in the late 1980s, when its presence there
seemed likely, myosin s role in growth cone movement remained unclear.81
Meanwhile, an alternative way for actin to induce movement was proposed.
Studies of other motile cells suggested that a polymeric filament could advance
by "treadmilling," adding subunits at its advancing end while removing sub-
units at the other.82 In 1988 Stephen Smith in New Haven demonstrated new
actin subunits being added preferentially to distal microfilament ends adjacent
to the membrane.83 Smith, following a new proposal by Bray, imagined a flow
of actin from the leading edge of the growth cone back toward its center, where
the filaments depolymerized; nevertheless, he still considered a role for myosin
in propelling actin filaments.84
The actin microfilaments, moreover, seemed linked to the membrane
through various actin-binding proteins; these in turn bound to integral mem-
brane proteins that extended to the exterior.85 Consequently, adhesion of filopo-
dia to the extracellular matrix could then be coupled to the cytoskeleton, allow-
ing the tension development necessary for growth cone advancement.86 Such
mechanisms had inherent problems,87 but notions of treadmilling and/or actin
flow remained popular explanations as the decade closed.
The second relevant antecedent was the identification in numerous cell types
of microtubules, recognized as polymers of a protein subunit, tubulin, that
formed structures several times the diameter of microfilaments.88 In neurons
these microtubules ran longitudinally within axons. By 1970 microtubules were
implicated in certain motile functions, including chromosome separation dur-
ing cell division. Colchicine, a plant toxin, disrupted microtubules, providing a
reagent for them akin to cytochalasin for microfilaments.
In 1970 Wessells also reported that colchicine caused a retraction of elon-
gated axons, although this response lagged behind the changes induced by
cytochalasin; the next year Bray found that tubulin, like actin, was strongly
labeled in growing nerve cells.89 Further studies through the 1980s showed
that microtubules entered the growth cones but stopped short of the filopo-
dia.90 In numerous cells, moreover, the microtubules appeared to advance by
treadmilling, and in 1986 Bray, now in London, described the assembly of
microtubules at the tips of growing axons, consistent with such a mechanism.91
Both actin and tubulin polymers thus seemed to participate in growth cone
advancement.
A further requirement for elongating axons was an addition of new material
as the axon grew. This also seemed to occur distally. For example, Karl Pfen-
ninger and Marie-France Pfenninger in New York argued in 1981 that during
elongation new membrane appeared at the growth cone.92
For all these processes a critical issue was how guidance cues controlled out-
growth. Second messenger systems, triggered through receptors for diffusible
agents or through cell adhesion molecules in the membrane, seemed likely par-
ticipants.93 But by 1990 the identification of these systems was just beginning.
Synapse Formation
Not only must axons grow to their proper destinations, but they also must make
functional synaptic contacts on arrival. Synapse development requires a trans-
formation of growth cones into nerve terminals containing synthetic enzymes
Formation or Specific Synapses 289
for appropriate neurotransmitters, synaptic vesicles with corresponding trans-

porters, machinery for vesicular release, and reuptake transporters and/or neu-
rotransmitter-degrading enzymes. Relevant receptors must appear postsynap-
tically (and often presynaptically as well). In addition, physical ties between
pre- and postsynaptic elements must form, representing, at least in part, the
action of selective cell adhesion molecules.94 This account, however, will merely
cite studies on one prominent area of investigation, examinations of how arriv-
ing presynaptic terminals trigger an aggregation of cholinergic receptors at neu-
romuscular junctions.
In the 1940s Fritz Buchtal, J. Lindhard, and Stephen Kuffler found that mus-
cle cells responded to administered acetylcholine only in the highly restricted
regions where the motor nerves terminated: evidently, acetylcholine receptors
were concentrated at neuromuscular junctions (chapters 3 and 4). Then in 1959
S. Thesleff in Lund showed that after denervation, the area responsive to
administered acetylcholine expanded to the whole surface of the muscle cell.95
But when the severed nerve grew back to reinnervate the muscle, sensitivity
was again confined to the neuromuscular junctions, as Ricardo Miledi in Lon-
don reported the next year.96
This denervated/reinnervated pattern also reflected the developmental
course, as Miledi demonstrated in 1962.97 Fifteen years later Monroe Cohen
in Montreal described the same sequence for embryonic muscle and nerve
cells grown together in culture. Cohen, however, used fluorescently tagged
a-bungarotoxin to monitor the receptor's location, since this toxin binds specif-
ically to nicotinic cholinergic receptors (chapter 6). The labeled receptors, ini-
tially scattered over the muscle surface, aggregated at the sites where ingrow-
ing neurons contacted muscle.98 But how did the approaching neurons initiate
this redistribution of receptors in the muscle?
Enveloping muscle cells—and intervening between nerve and muscle—is a
porous sheath, the basal lamina, that is formed from collagen fibers of the extra-
cellular matrix plus various other proteins, including cholinesterase and
laminins. In 1979 U. J. McMahan in Palo Alto found that nerve-free patches
of this basal lamina could promote receptor aggregation in denervated regen-
erating muscle cells at their original loci; five years later he described the abil-
ity of basal lamina preparations to promote cholinergic receptor aggregation
in cultured muscle cells.99 A partially purified fraction of the basal lamina,
McMahan reported in 1987, contained two peptides with molecular weights of
95 kDa and 150 kDa that could elicit receptor aggregation.100 He named these
agrins. (The amino acid sequence, derived by cDNA techniques in 1991, spec-
ified a 200 kDa protein from which McMahan's smaller fragments had pre-
sumably been cleaved.101)
McMahan also showed that motoneurons contained agrin (identified by anti-
bodies to his purified peptides).102 Thus, motoneurons apparently synthesized
agrins for release into the synaptic cleft, where these proteins became incorpo-
rated into the basal lamina. But how did extracellular agrins trigger aggregation
of cholinergic receptors in muscle membranes? This question remained unan-
swered in 1990, but by then another likely participant had been identified.
Acetylcholine receptors in muscle membranes that had been stripped of their
basal lamina still did not diffuse within the membrane, suggesting that the
receptors were linked to the muscle cytoskeleton. Jean-Pierre Changeux in
Paris had identified a 43 kDa protein in cholinergic receptor preparations, and
in 1982 he reported that a selective removal of this protein enabled the recep-
tors to diffuse laterally in the membrane.103 (By this time S. J. Singer's fluid
mosaic model for membranes104 was generally accepted: integral membrane
proteins, like the acetylcholine receptor, would diffuse in the plane of the mem-
brane unless specifically anchored, as by linkage to the cytoskeleton.) Jonathan
Cohen in St. Louis derived the sequence of this 43 kDa protein, subsequently
named rapsyn, by cDNA techniques in 1987.105 Identification of this DNA also
allowed the corresponding mRNA and its encoded protein to be synthesized.
In 1990 Stanley Froehner in Hanover and Jim Patrick in Houston described
the expression in Xenopus oocytes of cholinergic receptors and rapsyn, singly
and together.106 With receptors alone expressed, they distributed diffusely over
the oocyte membrane. With receptors plus rapsyn expressed, however, the
receptors formed clusters in the membrane. Evidently rapsyn promoted recep-
tor aggregation. But there remained an obvious causal as well as spatial gap
between agrins in the extracellular space and rapsyns associated with the cyto-
plasmic domains of the cholinergic receptor. As the decade closed the search
for additional participants continued.
Conclusions
Forming functional connections between particular neurons is a critical

requirement for nervous systems composed of discrete units. Such organiza-
tion, as these decades of investigation revealed, employed multiple means for
guidance. Outgrowing axons tended to follow paths of least resistance between
obstacles. They also followed favorable surfaces, responding to attracting or
repelling substances embedded therein. Outgrowing, axons also migrated
toward attracting chemicals diffusing from targets and veered away from
repelling chemicals. These various cues summed to steer developing neurons
to predetermined connections. But in other cases axons grew to inappropriate
destinations only to have the misconnections pruned away. In some instances
this corrective cell death resulted from a failed competition with other neu-
rons for a necessary growth factor.
Outgrowth along specified pathways depended on the amoeboid advance of
growth cones at the axon termini. This motility relied on actin and tubulin poly-
mers of the cytoskeleton, although the precise mechanism was still uncertain
in 1990. Also uncertain was the means by which extracellular cues modulated
the advance to direct growth cones along designated courses.
A final requirement was the establishment of synaptic function between
apposed cells. Here, too, a number of necessary elements were known in 1990
while others remained unidentified.
Despite such gaps, the explanatory accounts constructed by 1990 represent
impressive achievements, sketching the nature of the processes and pointing
toward likely areas for further exploration. The accounts emerged, as usual, by
applying insights and techniques successful elsewhere, here notably from stud-
ies of other motile systems and of other developing cells. The accounts also
resulted from new approaches and new techniques applied specifically to neu-
roembryology, permitting the examination of obvious questions. And the actual
course to formulating these accounts included, as usual, proposals and gener-
alizations that, after further scrutiny, failed. For example, an initial inability to
demonstrate chemical guidance was construed as the absence of chemical guid-
ance. Moreover, a bias toward unitary explanations discounted additional pos-
sibilities, blunting the quest for alternative mechanisms. Thus, Weiss had con-
cluded it "could hardly be regarded as satisfactory to assume that in one instance
mechanical [and] in another chemical. .. agents were the guiding principle."107
Also influencing experimental attention and explanatory formulations were gen-
eral concepts of how the nervous system functioned: as a network of particu-
lar circuits or as a unitary system resonating to encoded frequencies. In all
cases, as usual, every mechanistic proposal turned out, on closer examination,
to be far more complex, involving additional entities interacting through addi-
tional processes.
Notes
1. See Gilbert (1994); Horder et al. (1986); Nyhart (1995). Purves and Lichtmanns
text (1985) contains useful historical information.
2. Speidel (1933, 1941). These studies answered crtiticisms that Harrisons observa-
tions in vitro did not represent growth in vivo.
3. See Ramon y Cajal (1995), vol I, p. 532, and references cited. Forssman (1898)
also advocated chemical guidance vigorously.
4. Langley (1895).
5. Weiss (1934). He also presented evidence against electrical guidance, which some
then advocated.
6. Weiss (1924).
7. Adrian and Bronk (1928, 1929).
8. Wiersma (1931).
9. See Weiss (1936).
10. Ibid., pp. 512-513.

11. Others then advocating such pattern-directed encoding included Karl Lashley
(e.g., 1926).
12. Weiss (1936), p. 514. Weiss, however, suggested that selectivity might be due to
different muscles responding to different neurotransmitters (chemical neurotransmis-
sion in 1936 being still an ill-defined process: see chapter 3).
13. Harrison (1914).
14. Weiss (1934). He stroked with a fine brush the forming clot of lymph that
enveloped the neural tissue in culture, thereby aligning the fibers of the clot.
15. For example, Detweiller (1928).
16. Weiss (1941).
17. See Harrison (1935).
18. Sperry (1941).
19. Ibid., p. 16.
20. Sperry (1943).
21. Sperry and Miner (1949).
22. Sperry (1943), p. 46.
23. Ibid.
24. Attardi and Sperry (1963). They state the experiments began in 1958.
25. Sperry (1963).
26. Detweiler (1920). He found altered numbers of dorsal root ganglion cells, but he
could not detect changes in numbers of motoneuron cells since they were not easily
identifiable at that stage.
27. Hamburger (1934).
28. See her autobiography, Levi-Montalcini (1988).
29. Hamburger and Levi-Montalcini (1949), p. 498; italics in original.
30. Bueker (1948).
31. Levi-Montalcini and Hamburger (1951), p. 350.
32. Levi-Montalcini and Hamburger (1953).
33. Levi-Montalcini et al. (1954).
34. Cohen et al. (1954).
35. Cohen and Levi-Montalcini (1956).
36. Cohen (I960); Levi-Montalcini and Cohen (1960).
37. Korsching and Thoenen (1983).
38. Angeletti and Bradshaw (1971).
39. McDonald et al. (1991).
40. Hendry et al. (1974).
41. Sutter et al. (1979).
42. Kaplan et al. (1991); Klein et al. (1991).
43. See Reichardt and Farinas, in Cowan et al. (1997), pp. 220-263.
44. Schwab et al. (1979). See also Honegger and Lenoir (1982).
45. See Reichardt and Farinas, in Cowan et al. (1997), pp. 220-263.
46. Hamburger (1958), p. 399.
47. Ellis and Horvitz (1986).
48. For example, Cowan et al. (1984); Lamb (1977).
49. Clarke and Cowan (1976).
50. For example, Guth and Bernstein (1961); Hibbard (1965).
51. Lance-Jones and Landmesser (1981), p. 1.
52. Raper et al. (1983a, 1983b); Goodman et al. (1984).
53. Letourneau (1978). See also Campenot (1977).

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

94. For example, Bixby et al. (1987).
95. Axelsson and Thesleff (1959).
96. Miledi (1960).
97. Diamond and Miledi (1962).
98. Anderson et al. (1977); Anderson and Cohen (1977). See also Frank and Fisch-
bach (1979).
99. Burden et al. (1979); Godfrey et al. (1984). The first successful extract was from
Torpedo electric organ, and the later successful purification was also from this rich
source. However, McMahan showed that the active substance was in mammalian mus-
cle, too.
100. Nitkin et al. (1987).
101. Magill-Solc and McMahan (1988). Agrins were also present in muscle, a local-
ization that blurred the causal account at that time. See Reist et al. (1987); Fallen and
Gelfman (1989).
102. Rupp et al. (1991).
103. Sobel et al. (1978); Rousselet et al. (1982). See also Barrantes et al. (1980).
104. Singer and Nicolson (1972).
105. Carr et al. (1987); Frail et al. (1987).
106. Froehner et al. (1990).
107. Weiss (1934), p. 394.
12
LEARNING
Background
Among the essential attributes of mind and brain is the ability to learn. It is
also one of the most amazing. Experience leaves traces in our minds, so that
we can both recapture events from the past and modify accordingly our
thoughts and actions in the future. Yet the nature of these processes has long
remained mysterious.1 Explanations in antiquity adopted various metaphors for
learning and memory, such as impressing images on wax tablets and filing away
notes among the pigeonholes of the mind's storehouse. Advancing beyond these
speculative images to some underlying neural mechanism obviously required
an appreciation of basic neural capabilities. Indeed, when the Neuron Theory
directed attention to cellular relationships and properties, suggestions for neu-
ronal mechanisms quickly followed.
Experimental psychologists also began to define particular aspects of learn-
ing late in the nineteenth century, in the process developing useful methods
for displaying and evaluating such behaviors. Early in the twentieth century
Ivan Pavlov described conditioned responses, subsequently termed classical
conditioning: pairing a conditioned stimulus (such as the ringing of a bell, which
initially did not affect the behavior studied) with an unconditioned stimulus
(the sight of food) capable of eliciting an unconditioned response (salivation),
295
so that after a number of trials together the conditioned stimulus alone would
elicit the response. At that time Edward Thorndike embarked on comple-
mentary studies using puzzle boxes, investigations developed prominently by
B. F. Skinner as instrumental conditioning: animals in appropriate apparati asso-
ciated chance behavior (such as stepping on a pedal) with a consequent reward
(release of a food pellet), thereby learning how to gain the reward through spe-
cific actions. Such trial-and-error learning protocols included another favorite
test system, the learning of successful routes through mazes by following envi-
ronmental cues. In contrast to these associative modes of learning were two
nonassociative processes: habituation, a decreasing response to a recurring stim-
ulus, and sensitization, an increasing response.
In addition, early experimenters distinguished between short-term memory,
of limited duration (seconds to minutes), and long-term memory, which could
persist for years in exquisite detail. Moreover, interventions that affected neu-
ral activity, such as anesthesia or electric shocks, could erase recent memories
while leaving older memories largely unaffected. Formation of long-term mem-
ory apparently involved processes requiring a significant passage of time—
minutes to hours—termed the consolidation period.
Experimental psychologists explored a further characteristic of stored mem-
ories, their physical location within the brain. Contradicting earlier concepts of
discrete localizations within the brain, such as "memory lobes," S.I. Franz and
then notably Karl Lashley considered memories to be represented instead
throughout the brain. In studies begun in the 1910s and continued through
several decades, Lashley systematically examined the consequences of destroy-
ing particular regions of the brain. He found that even after a learned task was
obliterated by sufficiently extensive lesions, that task could still be relearned.
Moreover, the degree of loss of a learned task (measured, for example, as the
number of trials required to relearn it) was proportional to the area destroyed.
Such results, Lashley argued in 1926, "cannot be deduced from any explana-
tory system dependent upon the connections of particular neurons."2 He ques-
tioned formulations based on "low synaptic resistance between certain definite
neurons arranged in more or less intricate fashion"; instead, he favored notions
centered on "the ratio between [the] parts, and not the particular neurons
excited."3 Later reinterpretations stressed merely a diffuse and redundant rep-
resentation of learning within the brain.4 And -later criticisms questioned the
extent of the lesions and hence Lashley s interpretation of generality.5
This chapter will ignore many crucial concerns in attempting to understand
learning and memory, such as how memories are organized and generalized in
storage and how specific memories can be retrieved on demand. Instead, it will
focus on the cellular changes required to produce experience-dependent alter-
ations in neural function. This account, as are those of previous chapters, is
grounded on issues raised by Santiago Ramon y Cajal. His monumental trea-
Learning 29?
tise on neuroanatomy cataloged in 1909 a range of proposals for cellular mech-

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

chemical representations, as Joseph Katz and Ward Halstead in Chicago sug-
gested in 1950.n They imagined that stimulating a neuron could create a spe-
cific nucleoprotein, which then acted as a template for constructing within this
neuron a specific protein lattice. The lattice would govern transmission, so that
organization of the first neuron then spread to adjacent neurons, each in turn
forming the specific nucleoprotein and the consequent lattice (with the organ-
izing chains halting wherever they encountered previously organized neurons).
Thereafter, excitation would proceed only over pathways with identical nucle-
oprotein lattices. Learning would be represented by an assembly of neurons,
but with the linkages established by molecules specific to a particular memory.
At that time dozens of proteins had been distinguished, although the first
report of a protein's amino acid content appeared only in 1945 and the first
report of an amino acid sequence not until 1951.12 By 1960, however, Francis
Crick had promulgated the "central dogma" of information flow from DNA to
RNA to protein, and details of the underlying processes were accumulating
rapidly.
These new understandings then suggested new formulations for biochemi-
cal changes during learning. In 1960 Holger Hyden in Goteborg proposed that
memory traces were formed through altering the nucleotide sequence of neu-
ral RNA.13 Wesley Dingman and Michael Sporn in Rochester independently
argued that memory traces resulted from changing RNA structures and pro-
ceeded to examine one experimental consequence of such schemes.14 They
found that administering to rats a nucleotide analog that is incorporated into
RNA and that interferes with RNA's function, 8-azaguanine, retarded the rats'
learning of a new maze without interfering with their performance in mazes
previously mastered. And the next year, 1962, Hyden reported that teaching
rats a balancing task altered, after four days' training, the composition of nucle-
otide bases in the RNA of Deiter's cells (which are involved in circuits con-
trolling balance).15
The changes in RNA described in both studies could, by Cricks scheme,
alter protein structure and function. So in 1963 Josefa and Louis Flexner in
Philadelphia injected puromycin, a known inhibitor of protein synthesis, into
mouse brains and found that such administration interfered with the animals'
learning a new task.16 Subsequent studies by Samuel Barondes in New York
showed that protein synthesis was required for preserving learned behavior
for more than several hours and that this synthesis began within minutes of
training.1'
All these observations were interpretable as protein synthesis being neces-
sary for forming long-term memories. But the synthesized proteins could be
required either to facilitate synaptic transmission, as in Hebb's formulation,
or to store specific memories in unique molecules.18 Moreover, actual identi-
fications of the involved RNA or protein molecules were not forthcoming. At
that time methods for separating and characterizing these macromolecules
were not well developed. In any case, the pertinent changes would probably
Learning 299
be swamped by irrelevant metabolic processes proceeding concomitantly

throughout the brain.
Nevertheless, an explicit claim for encoding memories chemically appeared
at this time, a claim that enjoyed intense although short-lived publicity.19 James
McConnell, as a graduate student in Austin, examined the then controversial
question of whether invertebrates could learn. In 1955 he reported classical
conditioning in planaria (flatworms): pairing a light flash (conditioned stimu-
lus) with an electric shock (unconditioned stimulus) to produce a contraction
of the worm or a turning of its body.20 The extent of learning, however, was
modest, increasing from 2% to only 10% contracting and from 25% to 35%
turning. Subsequently, a range of other learning tasks were used, such as choos-
ing the designated branch of a T-maze, but always the improvements attrib-
uted to learning were small. Although others also reported learning by pla-
naria,21 some failed and were severely critical.22 Such limitations in merely
demonstrating the learning sharply undermined the value of McConnelPs
approach when used as an assay in the more exotic experiments that followed.
Planaria, when cut transversely or longitudinally, can regenerate whole
worms from their fragments. McConnelFs next step, now in Ann Arbor, was to
show in 1959 that learned contracting or turning could survive such ordeals
(retention was measured as a decreased number of trials required to reach a
criterion for learning).23 Indeed, McConnell found that learned behavior was
retained even when worms regenerated from posterior fragments. One inter-
pretation was that learning induced the formation of some molecule that was
represented throughout the worm and that could replicate—and manifest—
itself after any part of the worm regenerated.24
Another characteristic of planaria is cannibalism. So McConnell fed trained
planaria to untrained ones, and the ingestors required fewer trials to learn what
the ingestees had known than did planaria fed untrained ones.25 This research
attracted considerable attention, enhanced by McConnelPs flair for publicity
and consequent accounts in the popular press about potential information trans-
fer through shots for learning history or pills for playing the piano.26
Meanwhile, Roy John in Rochester found in 1961 that including the enzyme
that breaks down RNA, RNAse, in the water while bits of planaria were regen-
erating would obliterate the previously learned behavior in worms that arose
from posterior fragments.27 Allan Jacobson, a former associate of McConnell
now in Los Angeles, developed this notion, reporting in 1966 that RNA
extracted from trained planaria would, when injected into untrained worms,
reduce the number of trials the recipients needed to learn the task.28
Among the responses to such reports was a paper in 1966 signed by 23 sci-
entists from seven institutions describing their inability to show that RNA
extracts could transfer learning.29 Other claims for and against followed, but in
diminishing numbers. This decline was rooted in imprecise experimental
approaches30 as well as in the minimal, and therefore ambiguous, behavioral

changes on which the conclusions had rested. Another reason was the intrin-
sic scientific implausibility of the claims. By this time RNAs role as the link
between DNA and protein synthesis was becoming clear. Consequently, the
proposed ability of planaria to use ingested or injected RNA to synthesize selec-
tively new proteins for memory seemed wildly unrealistic, and McConnell did
nothing to challenge or extend current concepts of RNA function in support
of his formulation, let alone show how ingested/injected RNA affected cell
physiology and biochemistry.31 The liklihood of a different protein for each
memory also strained credeibility: human memory was then estimated32 to con-
tain 1020 bits.31 Equally significant was the evidence then accumulating for
quite different mechanisms, ones implicating synaptic changes rather than
unique memory molecules.
Learning? in Aplysia
A more prudent strategy would be to select a simple organism in which

detectable changes—physiological and biochemical—could be localized and
one where learning could be demonstrated consistently.33 This wise course Eric
Kandel (Fig. 12-1) followed profitably. Kandel received his medical degree in
New York in 1956 and completed his psychiatric training in Boston in 1964,
although this clinical course was interrupted by three years at NIH, where he
worked on mammalian neurophysiology, and the year 1962-1963, spent with
Ladislav Tauc in Paris, which launched his later investigations.
Tauc was studying a mollusc, the sea slug Aplysia depilans, that has a sim-
ple nervous system with large neurons, some of which were already identifi-
able from animal to animal reliably. These experimental advantages allowed
Kandel and Tauc to record the electrical responses of a particular neuron in
an isolated abdominal ganglion after stimulating its excitatory inputs. Accord-
ingly, they recorded from various neurons after exciting a nerve to the ganglion
weakly with a "test stimulus," sufficient to elicit only a minimal e.p.s.p. This
they paired 0.3 second later with strong excitation of a different nerve to the
ganglion, to provide a "priming stimulus." After several minutes of paired exci-
tations, the response to the test stimulus alone, when recorded from one of a
small population of neurons within the abdominal ganglion, increased up to
sevenfold. In 1965 they likened such changes to classical conditioning (with the
priming stimulus as unconditioned stimulus and the test stimulus as condi-
tioned stimulus).34 They also argued that this augmentation represented presy-
naptic facilitation: the priming stimulus potentiated neurotransmitter release
from neurons conducting the test stimulus.
These changes in synaptic properties—considered a manifestation of "synap-
Learning 301
FIGURE 12-1. Eric Richard Kandel (1929-; courtesy of Eric Kandel.)
tic plasticity"—seemed a plausible analog of learning.35 But a more explicit rep-

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

above with the mantle shelf and parapodia retracted. The gill is contracted. The dot-
ted line shows its extent when relaxed. B. The traces show gill contractions (upward
deflections) decreasing with successive tactile stimuli (numbered) to the siphon. The
interstimulus interval (ISI) was 1 min. At the arrow a strong tactile stimulus was applied
to the neck; the original stimuli then elicited augmented responses. C. The proposed
neural network for sensitization shows the cell body of a sensory neuron (SN) in the
abdominal ganglion with its receptors on the siphon and its presynaptic terminals on
the motor neuron to the gill (L7). Habituation results from decreased neurotransmit-
ter release from SN onto L7. Another sensory neuron has its receptors on the head and
its presynaptic terminals on the terminals of SN, such that it augments neurotransmit-
ter release from SN onto L7. The neurons from the head may also activate L7 directly.
The neural path from head to ganglion is uncertain, as indicated by the breaks in the
line. (A and B from Pinsker et al. [1970], Figs. 1 and 2; C from Castellucci and Kan-
del [1976], Fig. 3. Reprinted by permission, ©1970, 1976, American Association for the
Advancement of Science.)
rons to the gills whose cell bodies were also in the ganglia. Again using isolated
abdominal ganglia with attached sensory nerves, he showed that stimulating
sensory nerves from the head alone had no effect on the motor neurons. But
stimulating nerves from the head augmented the motor neurons' response to
sensory input from the siphon: sensitization.38 This augmentation represented
an increased neurotransmitter release from presynaptic terminals of siphon sen-
sory neurons. Accordingly, Kandel proposed a circuit whereby (1) neurons from
the head nerve elicited presynaptic facilitation of (2) the siphon neurons' out-
put onto (3) the motor neurons (Fig 12-2C). By surveying potential neuro-
Learning 303
transmitters he found that added serotonin enhanced transmission between

siphon neurons and motor neurons, suggesting that the sensitization produced
by neurons from the head was mediated by their releasing serotonin onto the
siphon neurons.39
How might released serotonin elicit presynaptic facilitation? Injecting cAMP
into the siphon neurons also elicited this response. Moreover, adding serotonin
or injecting cAMP increased Ca2+ influx into the siphon neurons.40 The ele-
vated cytoplasmic Ca2+ in the terminals could thus trigger the vesicular release
of neurotransmitter. In 1980 Kandel attributed the increased Ca2+ influx to a
diminished K+ efflux through its voltage-gated channels (which would in turn
increase Ca2+ influx through its voltage-gated channels by delaying repolar-
ization of the neurons).41 That year Kandel, collaborating with Paul Greengard,
showed that injecting protein kinase A into the siphon neurons also facilitated;
later they found that inhibiting protein kinase A blocked presynaptic facilita-
tion.42 Protein phosphorylation thus joined the causal chain.
In the early 1970s Kandel demonstrated that both sensitization and habitu-
ation could persist for weeks, and a decade later two associates in New York,
Craig Bailey and Mary Chen, described morphological changes accompanying
such long-term learning.43 Active zones in the siphon neuron terminals con-
tained fewer synaptic vesicles after long-term habituation and more after long-
term sensitization. Five years later they also reported decreases and increases,
respectively, in the number of synaptic contacts on the motor neurons.44
Dissociated sensory and motor neurons formed functional contacts in cul-
ture, and adding serotonin over a period of hours produced long-term sensiti-
zation in this simplified system, whereas a single application of serotonin pro-
duced short-term sensitization, paralleling responses in the intact ganglia. In
1986 Kandel reported that inhibitors of RNA and protein synthesis, when added
with the serotonin, blocked the long-term but not the short-term sensitiza-
tion.45 This was true for sensitization due to added cAMP as well.46 These
repeated additions altered the labeling (i.e., synthesis) of a number of distin-
guishable proteins, although the functions of these proteins were not then iden-
tified.47 Apparently cAMP acted through CREB and CRE (chapter 7) to reg-
ulate protein expression: adding isolated, exogenous CRE, which would trap
endogenous CREB and prevent its interaction with endogenous CRE, blocked
long-term facilitation.48
At the end of the decade, Kandel summarized these interactions in a circuit
that depicted sensitizing interneurons releasing serotonin onto the presynaptic
terminals of siphon neurons (Fig. 12-3). The serotonin receptors then triggered
a rise in cAMP within the siphon neurons that activated protein kinase A. Tar-
gets of subsequent phosphorylations included voltage-gated K+ channels and
CREB (Fig. 12-4). Altered protein synthesis, mediated by CREB, could then
stimulate the growth of synaptic contacts and affect protein kinase A activity.
FIGURE 12-3. Expanded neural circuit for habituation and sensitization. This circuit
adds facilitating interneurons between the sensitizing neurons (here from the tail rather
than the head). The termini from these interneurons make synaptic contact with the
presynaptic terminals of the sensory neurons from the siphon, and it is these interneu-
ron termini that release serotonin to produce sensitization. (From Kandel et al. [1991],
Fig. 65-3, reprinted by permission of the McGraw-Hill Companies.)
Earlier, James Schwartz, a major collaborator with Kandel, had identified an

intermediate-term learning that did not require protein synthesis but instead
reflected the loss of an inhibitory protein that regulates protein kinase A.49 Pro-
tein kinase A, in the absence of this inhibitory/regulatory protein, would then
continue to act, even after cAMP levels fell. Such disinhibition of protein kinase
A could maintain facilitation until protein synthesis replaced the lost inhibitor.
One aspect of long-term facilitation might involve a continued disinhibition.
Meanwhile, Kandel had described a pattern of stimulation that elicited clas-
sical conditioning of the gill reflex.50 This associative learning also involved
presynaptic facilitation. Others—notably Daniel Alkon in Woods Hole—
confirmed and elaborated on these studies of molluscan nervous systems,
FIGURE 12-4. Mechanisms for long- and short-term sensitization. The diagram of the
sensory neuron from the siphon shows receptors for serotonin, released from the ter-
mini of interneurons (5-HT). These receptors are coupled to adenylate cyclase, and the
consequent rise in cAMP levels activates protein kinase A. Targets for the protein kinase
are (I) voltage-gated K+ channels (causing a delayed repolarization and thus a rise in
Ca2+ influx through voltage-gated channels for Ca2+); (2) unspecified proteins involved
in neurotransmitter release; and (3), in the nucleus, CREB, thereby facilitating CREB s
binding to CRE. Possible consequences of the altered gene expression and protein syn-
thesis are an increased neuronal growth and an increased protease activity that, by cleav-
ing the endogenous inhibitor of protein kinase A, keeps its kinase activity elevated even
after cAMP levels fall. (From Kandel et al. [1991], Fig. 65-5, reprinted by permission
of the McGraw-Hill Companies.)
305
adding evidence for the involvement of protein kinase C and CaM kinase as
well as postsynaptic processes.51 More than one cellular mechanism could be
involved in synaptic facilitation.
But the relevance of changes in these simple nervous systems to learning in
complex mammalian brains remained unestablished. Moreover, Kandel argued
that his findings did not follow Hebb's formulation, which required that acti-
vation of cell A (here siphon neuron) be coupled with activation of cell B (motor
neuron).52
Learning in Drosophila
Complementary evidence came from a different approach, studying genetic

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

Long-Term Potentiation (LTP)
Neither Benzers nor Kandels strategy seemed applicable to studying mam-

malian learning. The far longer generation times made the breeding of mutants
protracted, and the far greater complexity of mammalian brains made the
Learning 307
search for pertinent synapses and the definition of localized cellular changes
seem unattainable. Hebb's formulation included synaptic changes that should
be measurable, but even if such changes were found, how could they be linked
causally and specifically to learning? Where among all the brains synapses
should the changes be sought? If memory were broadly represented through-
out the cortex—as Lashley s experiments seemed in the 1950s to imply—could
a particular learning event be detected amongst the multitudes of other neu-
ral activities proceeding concomitantly?
The path to resolving these uncertainties began with an enabling simplifi-
cation: the recognition in the 1950s that, contrary to Lashley's views, some cat-
egories of learning required a distinct and restricted region of the brain. Exam-
ination of this region uncovered characteristic synaptic changes that could
plausibly represent learning. And analyses of these changes then identified
underlying cellular mechanisms. This progression, however, sprang fortuitously
from some chance observations.
A Role tor the Hippocampus
Injuries to the brains temporal lobes (Fig. 12-5A) can result in seizures, and
when such epilepsy is resistant to anticonvulsant medications, one therapeutic
approach is removal of the damaged tissue. While developing this approach,
Wilder Penfield, a celebrated neurosurgeon in Montreal, stimulated the brain
in conscious patients to identify responsible areas and to avoid vital ones. He
found, unexpectedly, that stimulating some parts of the temporal lobes could
evoke vivid memories of isolated moments from the patients past, such as revis-
iting a certain scene or rehearing a particular song on a specific occasion.58
FIGURE 12-5. Temporal lobe and hippocampus. A. The lateral view of a human brain
shows the left temporal lobe and, by the hatching, the location of the left hippocampus
beneath the medial surface of the temporal lobe. B. The diagram of the hippocampus
in cross section shows an excitatory input through the perforant pathway to granule
cells. The granule cells then activate CAS neurons, whose axons send recurrent branches
(Schaffer collaterals) to excite CA1 neurons. The arrows show the direction of impulse
flow. Not shown are the large numbers of interneurons that modify activity.
Penfield recruited a psychologist, Brenda Milner, to assess mental function

before and after surgery, and in 1953 they reported that among a series of
patients who had had one temporal lobe removed for epilepsy, two patients
developed severe memory deficits postoperatively.59 They suggested that these
two had a preexisting lesion in their opposite temporal lobe and that the mem-
ory loss was due to bilateral damage.
At that time, before the introduction of effective drug therapies, one method
for treating severe psychotic illness was surgical: removing the prefrontal cor-
tex or cutting its connections to the rest of the brain. Because of the deleteri-
ous personality changes resulting from such operations and the limited
improvements, William Scoville, a neurosurgeon in Hartford, tried destroying
portions of the temporal lobes bilaterally, since these have connections to the
prefrontal area. In 1953 he applied this procedure also to a 29-year-old man
suffering from intractable epilepsy60 The operation successfully reduced the
frequency of his seizures, but, as was apparent immediately, it also abolished
his ability to form new memories. Although memories of events prior to the
surgery remained, each moment thereafter was a fresh start. The patient could
not recognize new faces, remember new conversations, or refind his way to
new locations.
When Scoville read Milner and Penfield s account, he invited Milner to Hart-
ford to examine this patient, known as H.M., as well as the psychotic patients
who had undergone similar operations. Scoville and Milner s paper in 1957 doc-
umented the memory changes and attributed them to bilateral destruction of
the hippocampus (Fig. 12-5A), a structure in the medial portion of the tem-
poral lobes well studied by anatomists (including Cajal) because of its promi-
nent cells (Fig. 1-2) and striking cellular organization (Fig. 12-5B).61 Over the
following decades Milner evaluated H.M., establishing the limits of his loss and
its irreversibility. Memories formed prior to surgery were retained, so long-
term storage was not within the hippocampus. Moreover, some types of learn-
ing could still occur, such as mastering new motor skills, even though H.M.
could not remember the training sessions when he learned the skills.62
These observations seemed convincing, and they were widely noted. For
example, they inspired early biochemical investigations of memory that focused
on changes in the hippocampus, although those investigations did not then lead
to further insights.63 But initial attempts to reproduce the memory deficits in
animals failed. In 1977, however, David Gaffan in London questioned whether
the learning assays then being used with experimental animals were appropri-
ate.64 Indeed, Mortimer Mishkin in Bethesda reported the following year that
destruction of the hippocampus bilaterally abolished a monkey s ability to rec-
ognize an object after a single exposure ("one-trial delayed matching").65 Sub-
sequent studies through the 1980s confirmed this approach and localized the
requisite region to the hippocampus and adjacent areas.66 The studies also con-
firmed the distinctions then being drawn between declarative memory, which
Learning 309
depends on the hippocampus and records events and facts, and nondeclarative
memory, which depends on other brain regions and participates in conditioned
responses, attaining skills, and such.
Long-Term Potentiation (LTP)
The hippocampus6' was a favored structure among physiologists as well as

anatomists, again because of its straightforward organization (Fig. 12-5B). A
leading center for these studies was Oslo, where Per Andersen was character-
izing hippocampal circuits. In the course of these investigations one of his stu-
dents, Terje L0mo, identified in 1966 a notable feature, "frequency potentia-
tion."68 Short trains of high-frequency stimuli to the perforant pathway
augmented subsequent responses of the granule cells. Remarkably, this poten-
tiation could last for hours after initial priming stimuli. L0mo interpreted this
as a "plastic change . . . expressing itself as a long-lasting increase of synaptic
efficiency."
By then another form of synaptic facilitation, called "posttetanic potentia-
tion," had attracted considerable interest. In 1941 T. P. Feng in Beijing
described augmented muscle e.p.p.s after an initial strong ("tetanizing"69) stim-
ulation.70 This augmentation was later observed at numerous synapses, includ-
ing those in the central nervous system, and traced to an increased neuro-
transmitter release. The increased release was then attributed to the tetanizing
stimuli producing elevated levels of cytoplasmic Ca2+ within the presynaptic
terminal.'1 To those interested in mechanisms for learning, such potentiation
was intriguing. But the potentiation was also disappointing, for it lasted only
minutes. Still, posttetanic potentiation might prolong activity over reverberat-
ing circuits, then advocated as intermediates in memory formation. In any case,
the far longer durations that L0mo described should have attracted great inter-
est. Instead, L0mo's report was apparently overlooked.72
In 1968 Timothy Bliss came to Oslo to work with Andersen. Bliss was inter-
ested in learning, and Andersen suggested that he collaborate with L0mo in
characterizing the hippocampal potentiation. Their study, published belatedly
in 1973, described clearly increased responses of the granule cells (Fig. 12-6)
that persisted for the duration of the experiment, up to 10 hours.'3 Subse-
quently Bliss, now back in Mill Hill, used electrodes implanted chronically in
unanesthetized rabbits to show changes persisting for days.74 This phenome-
non became known as long-term potentiation (LTP).
Andersen reported, also in 1973, another site in the hippocampus where LTP
occurred.75 Axons from CAS pyramidal neurons pass out of the hippocampus,
but branches travel back (as "Schaffer collaterals") to innervate the CA1 pyram-
idal neurons as well (Fig. 12-5B). Trains of high-frequency stimuli to the Schaf-
fer collaterals augmented responses of the CA1 neurons, just as stimulating the
perforant pathway augmented responses of the granule cells.
FIGURE 12-6. Long-term potentiation. Responses of the granule cells to test stimula-
tion of the perforant pathway—measured by extracellular microelectrodes and expressed
as e.p.s.p.s of the population (and normalized to initial values)—are plotted for exper-
iments lasting six hours. High-frequency trains of stimuli to the perforant pathway were
administered at the arrows. The open circles are control responses. (From Bliss and
L0mo [1973], Fig. 4, courtesy of the Physiological Society.)
Unfortunately, LTP was not always easy to observe. A major advance, offer-
ing easier, more accessible, and more reproducible studies, was recording in
vitro from isolated slices of the hippocampus, as described in 1975 by Philip
Schwartzkroin and Knut Wester in Oslo.76 This approach was then widely
adopted for examining and defining the process, although some significant con-
cerns remained.
Among these concerns was the locus of change. L0mo and Bliss noted that
potentiation could result from alterations at pre- or postsynaptic sites, or at
both. Evidence for all three possibilities was still being debated as the 1980s
closed.
Another significant concern was the dearth of evidence linking the observed
synaptic changes to actual learning. Nevertheless, the enhanced synaptic effi-
cacy made a causal connection seem highly plausible. A further capability
enhanced this appeal. In 1979 William Levy in Charlottesville identified two
pathways to the granule cells, the first of which produced LTP following high-
frequency stimulation, whereas the second did not.77 But stimulating both
pathways together produced LTP in the second pathway as well. Levy likened
this phenomenon to associative learning at synapses, in accord with Hebb s for-
mulation. In 1983 Thomas Brown in Duarte demonstrated the same associa-
tion for CA1 neurons in hippocampal slices.78
During the 1980s evidence for LTP appeared at sites beyond the hip-
pocampus, and LTP became recognized as a common physiological process
Learning 311
throughout the nervous system.79 Moreover, in 1989 Patrick Stanton and Ter-
rence Sejnowski in Baltimore described an associative long-term depression of
synaptic activity, the converse of LTP.80 They adapted Levy's and Browns
schemes, pairing stimuli over two pathways but with the stimuli out of phase;
responses to test stimuli were then depressed for protracted periods.
Tke Role of NMDA Receptors
Deciphering one aspect of how LTP occurs began with a pharmacological study
published in 1983.81 By that time three classes of glutamate receptors were
distinguishable by their responses to various reagents and named for identify-
ing agonists: kainate, quisqualate, and NMDA (chapter 6). Graham Collin-
gridge, working with Hugh McLennan in Vancouver, extended this approach
to hippocampal slices, chosen for the "wealth of information concerning [their]
neurochemistry . . . anatomy . . . and electrophysiology [and their] amenabil-
ity to investigations."82 He stimulated the Schaffer collaterals and monitored
responses of CA1 neurons after administering various reagents to these cells
microelectrophoretically. Specific antagonists for kainate and quisqualate
receptors prevented e.p.s.p.s in CA1 neurons, but antagonists for NMDA
receptors did not. Instead, NMDA antagonists blocked LTP. Collingridge con-
cluded that different classes of glutamate receptors served distinct functions:
kainate and quisqualate receptors mediated excitatory transmission between
Schaffer collaterals and CA1 neurons, whereas NMDA receptors mediated
LTP.
That year Gary Lynch in Irvine argued that LTP required a rise in intracel-
lular Ca2+: LTP no longer occurred after he injected into CA1 neurons a chela-
tor of Ca2+.83 Also in 1983 Raymond Dingledine in Chapel Hill showed that
administering NMDA to hippocampal neurons triggered a voltage dependent
influx of Ca2+.84 Three years later groups in Bethesda and New Haven dem-
onstrated that this influx occurred through NMDA receptors; meanwhile,
groups in Paris, London, and Bethesda found that physiological concentrations
of extracellular Mg2+ blocked ion fluxes through NMDA receptors but that
depolarizing the cells relieved this block.85 Since the interiors of neurons at
rest are electrically negative with respect to their extracellular environment,
Mg2+ would be drawn inward through ion channels, where it could obstruct
the channel's fluxes. Depolarizing the neuron would allow the Mg2+ to diffuse
outward, thereby removing the obstruction.
Holgen Wigstrom and Bengt Gustafsson in Goteborg synthesized these
observations in 1985 into a model for the NMDA receptor and LTP that
depended on "coincident pre- and postsynaptic activity." (Fig. 12-7 depicts an
expanded model from 1991. )86 The initial activation (through kainate or
quisqualate receptors conducting Na + and K + ) would depolarize the postsy-
FIGURE 12-7. NMDA receptors and long-term potentiation. A. During normal synap-
tic transmission glutamate released from presynaptic terminals can bind to NMDA,
quisqualate, and kainate receptors, but only the latter two classes can function as ligand-
gated ion channels since the NMDA receptor is blocked by Mg 2+ . B. When the post-
synaptic dendritic spine is depolarized, however, Mg2+ can exit and relieve the block-
ade, allowing Ca2+ as well as Na + and K + to move through the NMDA ion channel.
The elevated cytoplasmic Ca2+ can then activate various protein kinases and initiate
short- and long-term consequences. These may include release of a retrograde mes-
senger that affects the presynaptic neuron. (From Kandel et al. [1991], Fig. 65-11,
reprinted by permission of the McGraw-Hill Companies.)
naptic cells, producing e.p.s.p.s. This depolarization could also relieve inhibi-
tion of the NMDA receptors by releasing Mg2+ temporarily from these recep-
tors' channels. A second, closely associated activation would then allow Ca2+
influx through the unblocked NMDA receptors. Wigstrom and Gustafsson
emphasized the parallel with Hebbs formulation, whereby "the synapse is
strengthened only when the presynaptic volleys occur in conjunction with the
firing of the postsynaptic cell."8'
Learning 313
Just how the rise in intracellular Ca2+ could produce LTP was not specified,
but by then Ca2+'s role as a second messenger was well established. Reports
soon described increased protein kinase activity associated with LTP, although
the proteins targeted for phosphorylation were not yet identified.88 Persistent
LTP seemed likely to require protein synthesis, and as the decade ended
searches were underway to demonstrate its causal dependence on gene tran-
scription.89
Meanwhile, a convincing link between LTP and learning had appeared. In
1986 Lynch and Michel Baudry showed that NMD A antagonists given in vivo
impaired the learning of a task that depended on the hippocampus.90 By con-
trast, learning a task that did not rely on the hippocampus (i.e., could still
occur after the hippocampus was destroyed) was unimpaired. Nevertheless,
NMDA receptors were present not only in the hippocampus, and, as noted
above, LTP occurred in other regions of the brain as well. And although LTP
occurred at a third synaptic junction in the hippocampus—between axons
from the granule cells and CAS neurons—NMDA receptors were not involved
here.
Retrograde Messengers
Investigations of the NMDA receptors naturally focused on the postsynaptic

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

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

interactions between the neurons governing the altered behavior. Demon-
strating the functional changes responsible for this learning turned out to be a
challenging task. And during a long stretch of the early twentieth century
searches for such changes were discouraged by Lashley's influential interpre-
tation of memory as a holistic process beyond cellular and molecular mecha-
nisms. Nevertheless, Hebb revived neuronal models at midcentury, proposing
that a simultaneous activation of presynaptic and postsynaptic cells could facil-
itate subsequent transmission between them. In the 1960s two experimental
approaches then guided the analysis of relevant cellular and molecular mani-
festations.
Kandel's successful strategy—rewarded by a Nobel Prize in 2000—began by
choosing to study a simple invertebrate nervous system having neurons iden-
tifiable from animal to animal. Using Aptysia he could demonstrate physiolog-
Learning 315
ical changes in a neural circuit that controlled a learned behavior, define the
pharmacological characteristics of the synapses in the circuit, and specify the
biochemical consequences of receptor activation. The studies implicated cAMP
second messenger cascades that modified synaptic activation through protein
phosphorylations, affecting ion channel conductivity and neurotransmitter
release as well as the gene transcriptions required for long-term changes. Inves-
tigations using a different approach in another invertebrate—genetic studies
of learning in Drosophila—confirmed a role for cAMP-mediated processes.
The other course began with fortuitous observations of surgical patients that
implicated a certain structure, the hippocampus, in some forms of learning.
Independent explorations of hippocampal circuits revealed synaptic changes
that suggested the learning mechanism Hebb had formulated. Long-term
potentiation of transmission from presynaptic to postsynaptic neurons, occur-
ring at distinguishable classes of synapses, followed two experimental proto-
cols: trains of high frequency stimuli to presynaptic inputs or paired stimuli
over two converging pathways. The latter seemed a credible representation of
associative learning. Pharmacological investigations pointed to the participation
of NMDA receptors for glutamate, the excitatory neurotransmitter at these
synapses. NMDA receptors are ligand-gated ion channels that permit Ca2+
influx, but this conductivity requires not only glutamate but also the relief
through a priming depolarization of the receptor's blockade, achieved by trains
of high frequency stimuli or paired stimuli. The Ca2+ influx presumably trig-
gered short- and long-term changes that mediated persisting potentiation, but
many details remained unclear in 1990. Supporting the mechanistic plausibil-
ity of this scheme were studies showing that antagonists to NMDA receptors
prevented learning dependent on the hippocampus.
Both successful courses reflected a hierarchical sequence of investigations:
characterization of synaptic changes in defined neural circuits followed by
examinations of the molecular processes underlying these changes. One course
rewarded a patient and thoughtful campaign incorporating ingenious
approaches, and the other included astute recognitions of what independent
observations implied and how they might be extended. By contrast, some other
approaches in the 1960s skipped the identification of synaptic changes and
attempted to correlate learning tasks with biochemical changes in the brain.
These yielded no new mechanistic insights. Analytical techniques were impre-
cise and the multitudes of irrelevant activities obscured identifications.
Common advice to neophyte scientists includes an endorsement of asking
the right question. Here the global question was obvious: How does the brain
change during learning? The questions that generated effective research strate-
gies were more focused: Does learning modify activity over a circuit control-
ling the learned response? How is synaptic efficiency in such circuits altered?
What regulates the variable efficiency? Progress with these questions also
required an assessment of experimental practicality: What techniques are
needed, and can these be adapted from available methods or be developed fea-
sibly? Here a range of contemporary approaches—learning protocols, genetic
analyses, microelectrode recordings, pharmacological interventions, biochem-
ical explorations, electron microscopic examinations—were recruited to unravel
causal strands. By 1990 the forms of several models had emerged, although
the possibility remained that additional mechanisms for learning might also
function.
Notes
1. See Finger (1994); Hunt (1993).

2. Lashley (1926), p. 45.
3. Ibid., pp. 42, 44. Lashley maintained these conclusions, asserting that his "results
are incompatible with theories of learning by changes in synaptic structure" (1929,
p. 176) and advocating "some sort of resonance among a very large number of neurons"
(1950, p. 479).
4. See Hebb (1949); Young (1951).
5. See Kandel and Spencer (1968). In addition to doubts about the specificity of
Lashley s lesioning, there was concern about the plurality of sensory systems involved
in Lashleys learning tests. For example, if learning a maze involved smell, sight, and
touch, then destroying a localized representation for one of these modalities could leave
others, localized elsewhere, to direct the animals performance. The learned perfor-
mance would thus appear to have a generalized representation, even though the learn-
ing of each modality was sharply localized.
6. Ramon y Cajal (1995), vol. II, p. 724.
7. Arie'ns Kappers (1917).
8. Hebb (1949), p. 62.
9. Ibid., p. 62.
10. Lorente de No (1939).
11. Katz and Halstead (1950).
12. For historical accounts, see Fruton (1999); Judson (1979); Morange (1998); Robin-
son (1997).
13. Hyden (1960).
14. Dingman and Sporn (1961).
15. Hyden and Egyhazi (1962). Deiter's cells are also quite large, which favored their
selection for experiments studying single cells.
16. Flexner et al. (1963). See also Agranoff and Klinger (1964).
17. Barondes and Cohen (1968).
18. As Dingman and Sporn (1964) pointed out, Hyden's finding of altered nucleo-
tide ratios could represent merely the transcription into RNA of different stretches of
DNA, rather than the learning experience rearranging the nucleotide sequence in RNA,
as Hyden originally proposed.
19. For an account of McConnell and his experiments, see Rilling (1996).
20. Thompson and McConnell (1955).
21. For example, Lee (1963).
22. For example, Bennett and Calvin (1964).
23. McConnell et al. (1959).
learning 317
24. A more modest interpretation was that some change, neural or nonneural,
increased, at some point in the causal chain, responses to the conditioned stimulus.
25. McConnell (1962).
26. See Rilling (1996).
27. Corning and John (1961).
28. Jacobson et al. (1966).
29. Byrne et al. (1966).
30. Rilling (1996, p. 591) noted that "McConnell, an innovator, raced from one excit-
ing phenomenon to the next without comprehensive experimental analysis or adequate
controls." Bennett (1970, p. 150) complained that "in spite of ten years or more of
research in this area . . . even Dr. Corning [who was active in this research] cannot point
to a 100% procedural replication of any one training study."
31. Mechanisms necessary for McConnell's proposal contradicted emerging rules for
protein expression. They also defied recognized difficulties that extracellular macro-
molecules, such as RNA, encountered in crossing the cell membrane (a barrier to both
ingested and injected RNA). Moreover, if ingested RNA could direct protein synthesis
in planaria, then feeding hamburger, with its complement of mRNA molecules, should
force the worms to synthesize beef proteins: an unlikely but testable possibility. All these
problems with McConnell's formulations were recognized at the time.
32. von Neuman (1958).
33. See Allport (1986) for an account that includes personalities and rivalries.
34. Kandel and Tauc (1965a, 1965b). The work was done in Arcachon, where Aplysia
were available. The second paper concentrated on identifiable giant cells, although facil-
itation in these cells was not dependent on pairing the stimuli.
35. Kandel and Spencer (1968).
36. Pinsker et al. (1970); Kupfermann et al. (1970); Castellucci et al. (1970).
37. Castellucci and Kandel (1974).
38. Castellucci and Kandel (1976).
39. Brunelli et al. (1976).
40. Ibid.
41. Klein and Kandel (1980).
42. Castellucci et al. (1980, 1982).
43. Carew et al. (1972); Pinsker et al. (1973); Bailey and Chen (1983).
44. Bailey and Chen (1988a, 1988b).
45. Montarolo et al. (1986).
46. Schacher et al. (1988).
47. Barzilai et al. (1989).
48. Dash et al. (1990).
49. Greenberg et al. (1987).
50. Carew et al. (1981); Hawkins et al. (1983).
51. For example, Alkon (1984); Alkon and Nelson (1990); Crow and Alkon (1978);
Lukowiak and Sahley (1981); Morielli et al. (1986); Walters and Byrne (1983).
52. Carew et al. (1984). But see Sahley (1985) and the letters following: Trends Neu-
rosci. 9: 410^11 (1986).
53. Benzer (1967). Quinn et al. (1974) described the conditioning procedure.
54. Dudai et al. (1976); Duerr and Quinn (1982). Controls ruled out trivial alterna-
tives, such as alterations in perception, sensitivity to shock, etc.
55. Byers et al. (1981); Chen et al. (1986).
56. Dudai et al. (1983); Livingstone et al. (1984).
57. Levin et al. (1992).
58. See Penfield (1952).

59. Milner and Penfield (1955). See also Penfield and Milner (1958).
60. Haglund and Collett (1996a).
61. Scoville and Milner (1957). Lesions varied among the patients, including destruc-
tion of different amounts of the hippocampus and of surrounding regions. Evaluations
focused on H.M.; testing psychotic patients was more problematic.
62. Milner (1962).
63. For example, Flexner et al. (1963).
64. Gaffan (1977).
65. Mishkin (1978).
66. Mishkin initially argued that the amygdala also was crucial. Later studies cor-
rected that assignment, localizing critical regions to hippocampus, dentate gyrus, subic-
ular complex, and entorhinal, perirhinal, and parahippocampal cortices. See Squire and
Zola-Morgan (1991); Milner et al. (1998).
67. The name reflects a shape like a sea horse. Another name, Cornu Ammonis, is
responsible for the "CA" designation of neurons and refers to a shape like that of the
ram god's horn.
68. L0mo (1966).
69. A sustained, strong muscular contraction is known as "tetany." Stimuli capable of
producing such contractions are "tetanizing" stimuli.
70. Feng (1941).
72. Haglund and Brown (1995).
73. Bliss and L0mo (1973).
74. Bliss and Gardner-Medwin (1973).
75. Andersen et al. (1973).
76. Schwartzkroin and Wester (1975).
77. Levy and Steward (1979, 1983). The first pathway was from the ipsilateral side
and the second from the contralateral.
78. Barrionuevo and Brown (1983). See also Kelso et al. (1986).
79. For example, Iriki et al. (1989); Artola et al. (1990).
80. Stanton and Sejnowski (1989). See also Artola et al. (1990).
81. Collingridge et al. (1983a, 1983b). See also Harris et al. (1984).
82. Collingridge et al. (1983a), p. 20.
83. Lynch et al. (1983).
84. Dingeldine (1983).
85. MacDermott et al. (1986); Nowack et al. (1984); Mayer et al. (1984).
86. Wigstrom and Gustafsson (1985), p. 519.
87. Ibid.
88. For example, Malinow et al. (1988); Malenka et al. (1989).
89. For example, Cole et al. (1989).
90. Morris et al. (1986).
91. Malinow and Tsien (1990).
92. Dumuis et al. (1988); Williams et al. (1989).
93. Garthwaite et al. (1988). See also O'Dell et al. (1991); Schuman and Madison
(1991).
94. Matthews et al. (1976); Van Harreveld and Fifkova (1975); Lee et al. (1980).
95. Crick (1982); Matus et al. (1982); Caceres et al. (1983).
96. See Nottebohm (1986).
13
DISEASES AND
THERAPIES
Deiiningf ana Developing
Scientists, while pursuing pure knowledge, are eager also for useful knowledge.
In fact, much biomedical research is applied science, dedicated to defining dis-
eases and developing therapies. In such endeavors the goals include recogniz-
ing distinct diseases, characterizing their pathophysiologies, and designing ther-
apies to counteract these pathophysiologies.1
Medical practice has long included the gathering and sorting of symptoms into
related groups that were then considered the manifestations of particular disor-
ders. Often-associated symptoms,2 collected into syndromes, could designate dis-
eases, and in many cases prominent alterations in form or function pointed to
readily identifiable ills, such as rheumatism, convulsions, and dropsy. In other
situations the dividing lines between apparent entities, and even the distinctions
between normal and abnormal, were less clear. Moreover, subsequent investiga-
tions frequently subdivided what once had seemed a simple entity. The search
for mechanisms underlying disease processes, which accompanied the develop-
ment of scientific (and cellular) pathology during the nineteenth century, revealed
different processes producing apparently similar symptoms. And while the search
for pathophysiology aided in developing new therapies, different responses to a
therapy could indicate different pathophysiologies and thus different diseases.
319
Identifying diseases and discovering pathophysiologies has been particularly

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

in 1817 the common features observed in six patients: "involuntary tremulous
motion, with lessened muscular power," associated with a flexed posture and a
gait that passed "from a walking to a running pace."4 He named the disorder
shaking palsy (paralysis agitans) and described the patients' progressive dete-
rioration, beginning in later life and advancing through the years, from initial
mild tremor to ultimate immobility (akinesia). Although Parkinson recorded
treatments with the standard remedies of the day—such as bleeding and
blistering—he advocated caution "Until we are better informed respecting
the nature of this disease."
Diseases and Therapies 321
Jean-Martin Charcot, the most eminent Parisian neurologist of the nine-

teenth century, emphasized a characteristic muscular rigidity as well as the
tremor and difficulty with initiating movements (hypokinesia), and in 1867 he
attached Parkinson's name to the disorder.5 After trying available medicines
Charcot recommended scopolamine, an atropine-like botanical product, for its
ability to reduce the symptoms. Atropine-like anticholinergic agents (as they
were later recognized) remained the standard therapy for a century.
A significant step toward localizing the disorder and defining its pathophys-
iology was C. Tretiakoff's description—part of an extensive thesis on brain
histopathology submitted in Paris in 1919—of lesions in the brains of nine
patients with Parkinson's disease.6 Tretiakoff found a degeneration and loss of
cells in the substantia nigra, a region in the midbrain named for its darkly pig-
mented neurons. Five years later S. A. Kinnier Wilson in London summarized
clinical and experimental evidence for involvement of the caudate nucleus in
Parkinson's disease, and in 1928 Armando Ferraro described how destroying
the caudate led to degeneration of the substantia nigra, as would be expected
if cell bodies in the substantia nigra sent axons to the caudate.7
Studies during the first half of the twentieth century characterized two inter-
acting systems that govern motor control. One involves large pyramid-shaped
neurons in a region of the cortex initiating voluntary movement; their axons
pass down the spinal cord to activate ventral horn motoneurons. The other,
named the extrapyramidal motor system, includes a loop passing through the
basal ganglia. These ganglia are collections of cell bodies at the base of the
cerebral hemispheres that receive impulses from the cortex and send impulses
through intervening structures back to the cortex. The basal ganglia are sepa-
rated spatially into caudate nucleus, putamen (together known as the striatum
and containing morphologically similar cells), and globus pallidus. By the 1950s
Parkinson s disease was classified as a disorder of the basal ganglia/extrapyra-
midal motor system, but precisely how malfunctioning over this circuit pro-
duced tremor, rigidity, and akinesia remained uncertain in 1990.
The causes of Parkinson's disease also remained uncertain. Similar symptoms
followed the encephalitis produced by certain viruses (von Economo's
encephalitis lethargica) as well as the vascular changes in the brain that accom-
pany aging. Most cases, however, were of unassignable origin: idiopathic Parkin-
son s disease. By midcentury doubts about these cases arising from a single
cause grew, and some favored the general designation "parkinsonism," refer-
ring merely to the syndrome.
After reserpine was introduced in the 1950s for the treatment of schizo-
phrenia (see below), certain troubling side effects became apparent, including
tremor, muscular rigidity, and akinesia. Fortunately, this drug-induced parkin-
sonism disappeared after the reserpine was discontinued. Moreover, examina-
tions of reserpine's effects suggested a possible mechanism underlying these
symptoms. Arvid Carlsson (Fig. 13-1A) had studied reserpine's ability to

deplete serotonin from platelets while visiting Bernard Brodie in Bethesda.
After Carlsson s return to Lund, he extended these investigations, demon-
strating reserpine s ability to deplete catecholamines and serotonin in the brain
(chapter 9). And in 1957 Carlsson reported that administering dopa, the meta-
bolic precursor of dopamine and noradrenaline (Fig. 5-3), abolished the lethar-
gic immobility that reserpine produced in mice.8 At that time dopamine was
attracting attention as a likely neurotransmitter, and in 1958 Carlsson stressed
parallels between reserpine-induced depletion of dopamine from the striatum
and reserpine-induced parkinsonism.9 (For these and numerous other contri-
butions, cited in this and in earlier chapters, Carlsson received the Nobel Prize
in 2000.)
Two years later Oleh Hornykiewicz in Vienna completed the argument.
Hornykiewicz, while visiting with Hermann Blaschko in Oxford during the
1950s, had been introduced to dopamine as a likely neurotransmitter. Now he
described a striking decrease in the dopamine content of the striatum from six
deceased individuals who had suffered from parkinsonism; the noradrenaline
content was unchanged.10 In 1963 Hornykiewicz reported a decreased
dopamine content in the substantia nigra as well, and the next year Carlsson
FIGURE 13-1. A, left, Arvid Carlsson (1923-). B, right, Solomon H. Snyder (1938-;
courtesy of Arvid Carlsson and Solomon Snyder).
Diseases ana Therapies 323
FIGURE 13-2. Connections of the basal ganglia. Hornykiewicz's diagram shows fibers
from the substantia nigra (S.n.) innervating the putamen (Put.), caudate nucleus (N.C.),
and perhaps the globus pallidus (Pall), which constitute the basal ganglia. In addition,
fibers connect the cortex (areas 6ay and 4s), thalamus (Thai, with its nuclei L.po and
V.o.a), and basal ganglia. (From Hornykiewicz [1966], Fig. 2, courtesy of the American
Society for Pharmacology and Experimental Therapeutics.)
and associates used fluorescence microscopy to map a dopaminergic pathway

from cell bodies in the substantia nigra to presynaptic terminals in the stria-
turn.11 Figure 13-2 shows Hornykiewcz's diagram from 1966 delineating these
connections.
Levoo.'op a
If Parkinson's disease reflected the loss of nigrostriatal neurons and their

dopaminergic input to the striatum, then replacing the missing dopamine
should provide symptomatic relief. But, as was recognized in the 1960s, amines
like dopamine do not penetrate from blood to brain (at physiological pH they
are largely ionized and thus cannot diffuse across the nonpolar blood-brain
barrier). An obvious alternative was administering the immediate metabolic
precursor, dopa, which does penetrate and does then increase brain dopamine
levels (as Carlssons study indicated). So in 1961 Hornykiewicz injected intra-
venously into 20 patients with parkinsonism levodopa (L-dopa, the active
L-stereoisomer).12 He reported a marked relief of symptoms: patients "who
previously could not change from a prone position to ... sitting [were] able to
run and jump." Independently, Andre Barbeau in Montreal effected similar
improvements with oral administration of levodopa.13
Others, however, found only minimal improvements with low doses of levo-
dopa, while high doses produced serious and limiting side effects, including
nausea, vomiting, and elevated blood pressure and heart rate.14 But in 1967
George Cotzias in Upton described sustained improvement after administer-
ing severalfold higher doses, with side effects lessened by gradual increases in
the dosage from initially low amounts.15 Further studies soon confirmed the
striking improvement with high doses of levodopa, establishing Cotziass
approach as a practical and effective therapy.16
But even with Cotziass regimen the side effects were troubling. A clever
pharmacological ploy, however, minimized a significant class of toxicities,
including the gastrointestinal and cardiovascular disturbances, toxicities arising
from the conversion of levodopa to dopamine outside the brain ("in the periph-
ery").17 During attempts to develop better antihypertensive drugs, the phar-
maceutical industry synthesized various inhibitors of dopa decarboxylase (the
enzyme responsible for converting dopa to dopamine; see chapter 9), and in
1966 Sidney Udenfriend in Bethesda noticed that one of these, later named
carbidopa, unexpectedly increased dopamine levels in the brain.18 In 1969
Alfred Pletscher in Basel showed that carbidopa could not cross the blood-brain
barrier and thus blocked the conversion of levodopa to dopamine in the periph-
ery while allowing dopamine synthesis in the brain to continue.19 Clinical tri-
als indeed demonstrated that administering carbidopa together with levodopa
not only reduced the dose of levodopa required but also reduced the periph-
eral side effects.20 Merck then marketed this combination as Sinemet, which
became a standard form of levodopa therapy.
Other side effects, those attributed to altered brain function, were not dimin-
ished by carbidopa. Moreover, with prolonged administration of Sinemet as
well as of levodopa, there could appear disabling involuntary movements plus
abrupt transitions from mobility to immobility ("on-off effects"). And parkin-
sonian symptoms progressed even with continued dosages of these medicines,
so the drugs became less effective as time passed. Although levodopa offered
symptomatic relief to most patients, it did not halt the death of nigrostriatal
neurons, which continued throughout the course of Parkinson s disease.
Bromocriptine
A better approach might be to give dopaminergic agonists, since the nigrostri-

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

efforts to correct an identified pathological defect. Meanwhile, another agent
appeared fortuitously. Amantadine (Symmetrel) was introduced in the 1960s
to ward off influenza, and in 1968 a patient with Parkinson s disease who was
prescribed amantadine for this purpose noticed a remarkable decrease in her
parkinsonian symptoms. The patient described this improvement to her neu-
rologists in Boston, who included Schwab, and they quickly organized a six-
month trial with 163 patients, two-thirds of whom enjoyed improvement.25 Fur-
ther studies confirmed this efficacy and a generally low incidence of side effects.
The therapeutic response was less than with levodopa, however, and sometimes
disappeared after only a few months.
How amantadine effected the clinical improvement remained uncertain in
1990. Early reports argued that amantadine increased dopamine release, but
no increase in dopamine metabolites could be detected when amantadine was
given to patients.26
Selegiline
In 1982 a neurologist practicing in San Jose, William Langston, discovered six

young to middle aged adults who developed sudden and severe parkinsonism.27
All were intravenous drug abusers, and Langston traced the acute onset of
their symptoms to injections of a synthetic opioid—a "designer drug" sold on
the streets—contaminated with l-methyl-4-phenyl-l,2,5,6-tetrahydropyridine

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

tain botanical products that were later recognized as muscarinic antagonists.
Synthetic anticholinergics, such as trihexyphenidyl (Artane) and benztropine
(Cogentin), followed. None were as potent as levodopa, but they continued to
Diseases and Tnerapies 327
be useful for treating patients before symptoms were severe and as adjuncts
with other drugs.
Experimental studies, moreover, provided a belated rationale. For example,
injecting cholinergic agents into the basal ganglia of experimental animals pro-
duced parkinsonian symptoms that were relieved by administering levodopa.37
Such observations fostered the notion that parkinsonism reflected an imbal-
ance between dopaminergic and cholinergic pathways.38
Other Therapeutic Approaches
As the 1980s ended two other therapeutic possibilities were being pursued.
One involved transplanting dopaminergic cells into the brains of parkinsonian
patients.39 The other explored the possible use of various neurotrophic factors
to slow nigrostriatal death.40
Summary
Although the causes of neuronal death in Parkinson's disease remained

unknown in 1990 and no proven means for halting the progressive deteriora-
tion was yet available, several palliative measures that enjoyed considerable
clinical success had been developed, in many cases through rational searches.
These therapies focused on suppressing cholinergic pathways and potentiating
dopaminergic pathways of the extrapyramidal motor system. The latter ap-
proach included use of dopaminergic agonists, boosting dopamine synthesis by
providing precursors to dopamine, and halting degradation with inhibitors of
monoamine oxidase B.
Schizophrenia
Accounts of madness stretch back to ancient times, but only in recent centuries
have descriptions focused on discriminating among its manifestations.41 In 1893
Emil Kraepelin in Heidelberg applied the term dementia praecox to a chronic
and worsening disorder that began in late adolescence or early adulthood and
was associated with a deterioration of rational thinking, often accompanied by
hallucinations and bizarre behavior. This entity included groups earlier labeled
hebephrenia, catatonia, and paranoia. Five years later Kraepelin distinguished
between dementia praecox and manic-depressive illness, which involved exag-
gerated swings of mood, could begin at other ages, and did not necessarily have
so dire an outcome. Although Kraepelin s accounts were largely descriptive, he
endorsed the premise that altered behavior sprang from lesions in the brain.
Eugen Bleuler in Zurich then refashioned Kraepelin's catalog, stressing thought

disorders, inappropriate emotional responses, and social withdrawal, and he
contemplated a family of diseases, whereas Kraepelin imagined a unitary ill-
ness. Influenced by psychoanalytical thought, Bleuler also presented psycho-
logical mechanisms. In 1911 he renamed the disorder schizophrenia, to signify
a splitting among associations and between aspects of the personality.
Through the twentieth century uncertainties about the diagnostic bound-
aries continued, with clear differences in diagnostic criteria appearing
between national schools of psychiatry as well as among individual psychia-
trists.42 Disagreements also persisted over the causal roles of psychological
mechanisms—arising from individual, familial, or social dysfunction—and of
neural mechanisms. During the first half of the twentieth century, psychiatry
in English-speaking countries generally embraced psychoanalytical formula-
tions of schizophrenia, culminating during the decade after World War II in
explicit psychodynamic mechanisms.43 These schemes did not dwell on the
likelihood of psychological trauma altering brain physiology, with altered phys-
iology then manifesting as pathological behavior. And their proponents
embraced psychological therapy, not dwelling on the likelihood of such ther-
apy altering brain function.
Inattention to neural causes and mechanisms was fostered not merely by
commitments to the associated therapeutic mode. It also reflected failures to
validate early accounts of pathological changes in the brains of schizophrenic
patients.44 Further skepticism followed refutations through the 1960s of vari-
ous claims for biochemical abnormalities.45 On the other hand, early X-ray stud-
ies supported a link between schizophrenia and enlargement of the brain's
ventricles.46 And in the 1980s newer imaging techniques—computerized
tomography, magnetic resonance imaging, and positron emission tomography—
strengthened these conclusions and documented common deficits, including a
decreased mass of the temporal lobes and a decreased metabolism in the frontal
cortex.47 These changes might, of course, represent a secondary response to
trauma, including psychological stresses.
At midcentury hallucinogenic drugs attracted the attention of neuroscien-
tists, at the same time furthering arguments for psychological symptoms aris-
ing from altered neural function. LSD produced striking visual hallucinations,
and in 1953 John Gaddum in Edinburgh showed that LSD blocked responses
to serotonin (albeit on smooth muscle).48 D. W. Wooley in New York then sug-
gested that schizophrenic hallucinations might spring from deficient seroton-
ergic activity in the brain.49 The psychotic behavior produced by LSD, how-
ever, did not resemble the symptoms of schizophrenia, in which auditory
hallucinations predominate. More significant, the early antipsychotic drugs,
reserpine and chlorpromazine, suppressed serotonergic systems, contrary to
Wooleys proposal. By contrast, another hallucinogenic drug, amphetamine,
Diseases ana Tnerapies 329
induced a syndrome more closely resembling schizophrenia, including para-

noia and auditory hallucinations.50 This capability supported a formulation that
was highly influential through the remainder of the century.
The most compelling evidence for a primary physical cause, however, came
from genetic studies correlating the likelihood of schizophrenia among rela-
tives with the closeness of their relationship.51 Comparisons of the incidence
of schizophrenia between identical twins were particularly striking. Some crit-
icized such conclusions by pointing out that individuals inherit environments
as well as genes, but studies of adopted individuals then demonstrated that vul-
nerability followed the biological rather than the environmental ties.52 Never-
theless, the concordance for schizophrenia between identical twins was only
about one half: other factors—perhaps infectious, metabolic, and/or social—
must interact with an inherited vulnerability to produce illness. Even in cases
with genetic predispositions, it seemed likely from the patterns of inheritance
that multiple genes participated. This polygenic nature undoubtedly contrib-
uted to failures through the 1980s to demonstrate linkages between vulnera-
bility and particular chromosomal loci.53
Furthermore, most schizophrenic patients had no family history of schizo-
phrenia or similar psychiatric illnesses. This disparity reinforced the likelihood
that the schizophrenic syndrome represented a collection of disorders differ-
ing in etiology and pathophysiology.
Reserpine
In 1931 two physicians in Calcutta, Gananath Sen and Kartick Bose, described
the efficacy of snake root in treating "insanity" and high blood pressure.54 This
root of an Indian bush (Rauwolfia serpentina) had been ingested since antiq-
uity to treat a range of maladies, from snake bite to madness, and after Sen
and Bose's report a flurry of publications, also in Indian journals, endorsed their
findings. In 1949 a report in a British journal led to trials with hypertensive
patients in the United States and to CIBA Pharmaceutical Products exploring
the active ingredients in snake root.55 In 1952 they isolated reserpine and iden-
tified it as the component responsible for sedation; the following year Freder-
ick Yonkman at CIBA coined the term "tranquilizer" to describe reserpine's
soothing properties.56
An article in The New York Times reported in 1953 an award in India for
the development of snake root to treat psychiatric illnesses. This account caught
the eye of Nathan Kline, an energetic psychiatrist at Rockland State Hospital
in Orangeburg. Kline, eager to identify effective remedies for psychiatric dis-
orders, quickly organized trials on chronically ill inpatients of a snake root
extract (Raudixin) provided by Squib and of purified reserpine (Serpasil) pro-
vided by CIBA. The following year, 1954, Kline described the preparations'
abilities to sedate and alleviate anxiety, but he detected "no evidence that [they]
in any way alter . . . the schizophrenic process itself."57 Kline noted, however,
that the patients tested were severely deteriorated; later he was more enthu-
siastic, and subsequent investigations demonstrated the suppression of specific
symptoms: diminished delusions and hallucinations plus normalization of the
thought processes.58 Reserpine s efficacy, however, was less than that of other
drugs then being introduced, and the latter became the predominant therapies
for several decades. Reserpine also suffered from a tendency to induce psy-
chological depression, an action that intrigued those pondering the mechanisms
of mood disorders (see below).
Reserpine was soon shown to cause depletion of catecholamine and sero-
tonin stores in the body (chapter 9), and it became a widely used tool in phar-
macological research. But because of its actions on this range of neurotrans-
mitters, reserpine did not implicate any particular pathophysiology for
schizophrenia.
Cnlorpromazine ana Haloperiaol
Henri Laborit, a French naval surgeon stationed in Tunisia, was searching in

the late 1940s for pharmacological means to blunt operative stresses.59 By 1951,
when he was transferred to Paris, his regimen included an antihistaminic,
promethazine (Phenergan), that induced a state of seeming indifference. Hop-
ing for still more effective drugs, Laborit encouraged Rhone-Poulenc Labora-
tories to synthesize other antihistaminics with similar actions on the central
nervous system. Simone Charpentier at Rhone-Poulenc showed that chlorpro-
mazine (Thorazine, Fig. 13-3A), a compound newly synthesized in 1950 and
having a phenothiazine ring like promethazine, produced a distinctive behav-
ioral alteration: rats trained to climb a rope for food no longer did so after
receiving chlorpromazine, not because they lost the ability but apparently
because they lost interest. Laborit quickly incorporated chlorpromazine into
his regimen and noted a similar blissful unconcern in his patients. Laborit
encouraged psychiatrists to try this drug with agitated patients, too. In 1952 a
prominent Parisian psychiatrist, Jean Delay, reported notable decreases in agi-
tation, aggression, and hallucinations without a loss of mental alertness. (The
standard treatment of agitated schizophrenic patients then included sedative
drugs such as barbiturates, which produced lethargy and torpor.) By 1954 tri-
als of chlorpromazine had spread across the globe, with the drug garnering
broad acclaim.
The success with chlorpromazine inspired the pharmaceutical industry to
synthesize a host of variants based on the phenothiazine ring and its analogs.
By 1964 there were 11 being prescribed, and that year a comprehensive test
confirmed chlorpromazine s utility: more than 75% of acutely ill schizophrenic
)iseases ana Therapies 331
FIGURE 13-3. Structures of therapeutic drugs.
patients showed marked to moderate improvement, with relief of thought dis-

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

including a drug-induced, reversible parkinsonism.
Examinations of the drugs' pharmacological actions also suggested how they
relieved schizophrenic symptoms. These analyses, however, were complicated
by the multitudes of the drugs' effects, particularly with the phenothiazines
that variously oppose cholinergic, dopaminergic, noradrenergic, serotonergic,
and histaminergic systems. Nevertheless, in 1963 Carlsson, now in Goteborg,
proposed that antipsychotic drugs act by blocking catecholamine receptors.63
Mice given chlorpromazine or haloperidol excreted increased amounts of
dopamine and noradrenaline metabolites, and Carlsson considered that these
increases reflected an elevated release of the parent neurotransmitters to com-
pensate for the (hypothesized) blockade of their receptors.
Although Carlsson did not comment on whether the therapeutic response
followed blockade of dopaminergic vs. noradrenergic receptors,64 J. M. van
Rossum in Nijmegen soon did. He argued that "dopamine receptor blockade
is an important factor" in the drugs' therapeutic activity since chlorpromazine
and haloperidol produced extrapyramidal symptoms (he even suggested that
"extrapyramidal side effects are a prerequisite" for antipsychotic activity).65 Van
Rossum also cited the drugs' ability to suppress behavioral responses attributed
to dopaminergic systems. A. Randrup in Roskilde had demonstrated earlier
that chlorpromazine and haloperidol counteracted certain behaviors in rats
induced by amphetamine, and Randrup now showed that these behaviors were
attributable to dopaminergic systems.66 Nevertheless, other amphetamine-
induced behaviors were attributable to noradrenergic systems.
Antipsychotic drugs could also counteract amphetamine-induced psychoses
in humans, so a significant question was whether this psychosis, said to resem-
ble schizophrenia closely, reflected dopaminergic or noradrenergic actions of
amphetamine. An ingenious approach to discriminating between these alter-
natives came from Snyder (Fig. 13-1B). Snyder had begun his stellar research
career with Julius Axelrod in Bethesda, and after completing his training in
psychiatry he returned to research, now in Baltimore. There he pursued a broad
range of important problems with insight and vigor, and some of his numer-
ous contributions are cited in previous chapters. Now Snyder approached the
dopamine/noradrenaline quandary by noting that noradrenaline has two
stereoisomers, only one of which is biologically active, whereas dopamine has
no stereoisomers. Amphetamine also exists as two stereoisomers, and in 1969
Snyder showed that both stereoisomers of amphetamine were equally effec-
tive in eliciting dopaminergic responses in experimental animals, but only one
stereoisomer could elicit noradrenergic responses.6' Samuel Gershon in New
York then applied this discriminatory test to amphetamine-induced psychoses
in volunteers. Both stereoisomers of amphetamine were equally effective,
implying a dopaminergic action.68
Thus "the dopamine theory of schizophrenia" emerged in the early 1970s,

proposing that the disorder reflected excessive dopaminergic activity (as mim-
icked by amphetamine) and responded to dopaminergic receptor blockade
(with chlorpromazine or haloperidol) or dopamine depletion (with reserpine).
Accordingly, schizophrenic symptoms were worsened by administering amphet-
amine or dopa.69 Nevertheless, as Snyder warned in 1974, dopaminergic path-
ways might merely be modulating aberrant responses of another neurotrans-
mitter system where the fundamental pathology lay.'°
In 1971 Snyder showed that the three-dimensional structure of chlorpro-
mazine resembled that of dopamine (Fig. 13-4), as expected if it blocked
FIGURE 13-4. Three-dimensional structures of chlorpromazine and dopamine. Draw-

ings of the structures of chlorpromazine (A) and dopamine (B), determined by X-ray
crystallography, are shown superimposed (C). (From Horn and Snyder [1971], Fig. 1,
courtesy of Solomon H. Snyder.)
dopamine receptors.71 The next year Paul Greengard in New Haven offered
further support for dopamine theories, describing chlorpromazine's and
haloperidol's inhibition of dopamine-activated adenylate cyclase activity in the
basal ganglia.72 This observation suggested that drug-sensitive dopamine recep-
tors were coupled to adenylate cyclase. Quantitative comparisons of receptor
blockade, assayed as inhibition of dopamine-activated adenylate cyclase, cor-
related well with the therapeutic potencies of a series of phenothiazine antipsy-
chotics. But, as Leslie Iversen in Cambridge pointed out in 1975, the correla-
tion failed with haloperidol, a potent antipsychotic but a poor inhibitor of
adenylate cyclase activation.73 Snyder, however, resolved this discrepancy the
following year. He assayed receptor binding in vitro as the ability of various
drugs to displace labeled haloperidol from a brain membrane fraction. Now
there appeared an excellent correlation for haloperidol as well as the pheno-
thiazines.74 Philip Seeman in Toronto independently obtained similar results,
published as a convincing plot (Fig. 13-5).75
FIGURE 13—5. Correlation between clinical dose and affinity for dopamine receptors.
The plot correlates clinical doses for treating schizophrenia with the drugs indicated (as
milligrams of drug per day) on the x-axis, with affinity for the dopamine receptor
(expressed as the ICso, the concentration of drug required for 50% inhibition of labeled
haloperidol binding, measured as moles of competing drug per liter) on the y-axis. (From
Seeman et al. [1976], Fig. 1. Reprinted by permission of Nature, © 1976, Macmillan
Magazines, Ltd.)
In 1979 John Kebabian and Calne, now in Bethesda, summarized studies on

adenylate cyclase activation and receptor binding, from which they proposed
two classes of dopamine receptors: DI, linked to adenylate cyclase activation
(to which haloperidol binds weakly), and D2, not so linked (to which haloperi-
dol binds strongly).76 The potency of antipsychotic drugs then correlated with
binding to D£ receptors, and this property became a standard screening assay
in searches for new antipsychotic medications. The amino acid sequence of D£
receptors, determined by cDNA methods, appeared in 1988.77
The hyperactivity postulated by dopamine theories of schizophrenia could
result from increased dopamine release and/or increased sensitivity of dopamin-
ergic receptors. Evidence for elevated dopamine turnover in the brains of schiz-
ophrenic patients was not clear-cut.78 On the other hand, dopamine receptor
levels were demonstrably elevated, but this could merely represent compen-
satory changes after chronic blockade by antipsychotic drugs ("upregulation of
receptors").79 In 1986 Snyder and associates, measuring ligand binding to D£
receptors in vivo with positron emission tomography, reported increased num-
bers of dopamine receptors in basal ganglia of schizophrenic patients who had
not previously received any antipsychotic drugs.80 The next year, however, Lars
Farde in Stockholm, using a different ligand for the technique, found no
increase.81 As the 1980s ended this contradiction remained.
Meanwhile, fluorescence microscopic techniques had delineated dopamin-
ergic axons from the brain stem projecting not only to the basal ganglia but
also to the frontal and temporal cortex.82 These were areas where newer imag-
ing studies had by 1990 revealed structural and functional changes correlated
with schizophrenia.83
Clozapine
Clozapine (Clozaril, Fig. 13-3C) was synthesized in 1958 by Wander AG dur-

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

medications. Clozapine was then approved for use in the United States, but
only with mandatory monitoring of the blood. (The agranulocytosis subsided if
the drug was stopped immediately after changes became detectable.) By 1990
the pharmaceutical industry was avidly seeking new drugs with similar thera-
peutic capabilities but without the hematological toxicities.
Why was clozapine superior to standard antipsychotic drugs? Surprisingly, it
blocked D£ receptors only modestly.88 On the other hand, clozapine blocked
serotonergic 5HT2 receptors, and enthusiasm for modulating this and other
neurotransmitter systems revived.89 (In 1991 a fourth class of dopaminergic
receptors, D4, was identified and sequenced. Clozapine, in contrast to standard
antipsychotics, bound selectively to D4, suggesting that these receptors were
the therapeutically significant entities; further research, however, soon under-
cut this proposal.90)
Summary
New remedies introduced in the 1950s proved dramatically superior to earlier

medications for schizophrenia. These were derived from folk medicine and
from synthesizing new substances based on a family of actions in animals, such
as suppressing movement and interfering with conditioned responses. A
common feature—among a vast range of pharmacological properties—was inhi-
bition of dopaminergic systems: by depleting neuronal dopamine stores or
blocking dopamine receptors, specifically D£ receptors. Conversely, agents pro-
moting dopaminergic activity could elicit schizophrenic-like symptoms in vol-
unteers and worsen the symptoms in schizophrenic patients. The dopamine
hypothesis attributed schizophrenic symptomatology to such dopaminergic
hyperactivity, while acknowledging that this hyperactivity might be relative to
more fundamental defects in some as yet unidentified system(s).
Correlations of drug efficacy with D£ receptor blockade pointed drug devel-
opment to compounds having similar specificities, but a drug identified with
superior clinical efficacy turned out to be relatively weak at D% receptors. Expla-
nations for such "atypical antipsychotics" included specificity for different
(more pertinent) dopaminergic receptors or blockade of other receptors (in
addition or instead), such as serotonergic 5HT2 receptors.
By 1990 interest was also turning to receptors for another neurotransmitter,
although no practical drugs were yet available. Phencyclidine, an illicit hallu-
cinogen notorious as "PCP" or "angel dust," could produce symptoms resem-
bling schizophrenia, probably more closely than did amphetamine. The dis-
covery that phencyclidine blocked NMDA receptors for glutamate then spurred
investigations into glutamatergic systems' involvement in the pathophysiology
of schizophrenia.91
Depression ana Manic-Depressive Illness
Greek physicians characterized melancholia as an underactivity of mental func-

tioning associated with debilitating grief and despair, contrasting it with the
overactivity of raging frenzies.92 On the other hand, notions of a unitary soul
discouraged subdivisions, stressing instead either sickness or health. Toward
the end of the nineteenth century, however, Kraepelin distinguished between
dementia praecox and manic-depressive illness. Elaborating on earlier formu-
lations, Kraepelin described cycles of exaggerated mood in the latter disorder,
with periodic swings from excitation and elation to hopelessness and despon-
dency.
Throughout the twentieth century the distinctions among disorders of mood
shifted, to extend or restrict categories and to subdivide or enlarge them, with
inclusions or exclusions based on the nature, severity, and duration of symp-
toms. In any event, classification had limited practical significance during the
first half of this century, for prognoses were generally indistinguishable and
medications generally ineffective. Nevertheless, Karl Leonhardt advocated a
clinical distinction in the 1950s that later pharmacological and genetic studies
confirmed:93 bipolar illness, with alternations between mania and depression,
vs. unipolar illness, with recurrent bouts of depression. Thus, DSM-1II in 1980
included the categories "bipolar disorder" and "major depression." Still, argu-
ments for heterogeneity within these categories persisted.
The Greeks attributed melancholia to an excess of black bile (as the name
denotes), and little further delineation of its etiology followed for two millen-
nia. Absent firm evidence for the neural malfunctionings that nineteenth cen-
tury psychiatrists had assumed, psychoanalytical formulations for depression
flourished during the first half of the twentieth century, as with schizophrenia.
As with schizophrenia, investigations after World War II of families, twins, and
adopted children pointed toward genetic predispositions (and indicated dis-
tinctions between bipolar and unipolar disorders as well).94 And as with schiz-
ophrenia, newer imaging techniques disclosed characteristic structural abnor-
malities in the brain.95 The most prominent arguments for a neural disorder
sprang, however, from discoveries of effective therapeutic agents that produced
identifiable biochemical changes.96
Iproniazid
Researchers at Hoffman-La Roche identified in the early 1950s the potent anti-
tubercular activity of isoniazid, which quickly became a major therapeutic agent
against tuberculosis. Attempts to synthesize still better drugs soon produced
iproniazid (Marsilid). Clinical use, however, revealed an unexpected ability to
cheer these chronically ill patients. Although initial trials of iproniazid with hos-
pitalized psychiatric patients were not promising,97 Kline in 1957 found that
70% of his depressed patients ultimately improved.98 Subsequent trials con-
firmed iproniazid's ability to elevate the mood of depressed patients.99 (Influ-
enced by psychoanalytical theory, Kline imagined that depression reflected a
loss of psychic energy to inappropriate realms of the mind. Effective therapy
would release this energy to reenergize the psyche, although too much energy
might result in schizophrenia. Accordingly, Kline termed iproniazid a "psychic
energizer.")
Earlier, Albert Zeller in Chicago had demonstrated that iproniazid inhibited
monoamine oxidase in vivo.wo By the mid-1950s this enzyme was recognized
as an important participant in metabolizing catecholamines and serotonin
(chapter 9), so drugs that inhibited degradation should potentiate the effects
of these biogenic amines. Studies with iproniazid, however, did not indicate
which of these neurotransmitters must be affected to relieve depression.
Unfortunately, iproniazid produced serious toxicities, and it was soon
replaced by other drugs having the common ability to inhibit monoamine oxi-
dase. This ability also provided the general name for such antidepressants:
monoamine oxidase inhibitors. The newer drugs were not without side effects,
either,101 and monoamine oxidase inhibitors were largely supplanted by another
class of drugs that came into use at almost the same time.
Imipramine
Following the therapeutic (and commercial) success of chlorpromazine, Geigy,

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

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

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

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

than noradrenaline reuptake, but in vivo these drugs were metabolized to com-
pounds that blocked noradrenaline reuptake preferentially. Consequently, these
drugs were not serotonin-specific in practice. In 1972, however, Carlsson and
associates patented zimelidine, which was specific for serotonin reuptake in
vivo; by 1980 clinical testing demonstrated its utility as an antidepressant, but
toxicities led to its withdrawal the next year.116 Meanwhile, David Wong at Eli
Lily reported in the mid-1970s that fluoxetine (Prozac, Fig. 13-3E), which did
Diseases and. Therapies 341
not resemble the tricyclic antidepressants structurally, was also highly specific
in blocking the reuptake of serotonin both in vivo and in vitro, and he pre-
dicted that fluoxetine "may find clinical use . . . in mental depression."11' Flu-
oxetine indeed was an effective antidepressant,118 and when finally marketed
in 1988 it quickly became a spectacularly popular drug, boosted by glowing
accounts in the press as well as a best-selling book.119 Nevertheless, fluoxetine
was no more effective against depression than the tricyclic antidepressants. Its
advantage was fewer troubling side effects. (In addition, fluoxetine turned out
to be effective with additional mental ills, including panic attacks and obses-
sive-compulsive disorder.) Other pharmaceutical companies soon marketed
drugs with similar properties, establishing a class of "specific serotonin reup-
take inhibitors" ("SSRIs").
Lithium
While searching in the 1940s for endogenous toxins that cause manic-depres-
sive illness, John Cade, a psychiatrist in Bundoora, injected patients' urine into
guinea pigs. He identified urea as one urinary constituent responsible for the
resulting lethal convulsions, but urea levels were not elevated in patients' urine.
Cade then added another urinary constituent, uric acid, choosing its most sol-
uble salt, lithium urate. Instead of exacerbating the toxicity as he expected,
lithium urate, when added to the patients' urine, suppressed the convulsions.
Indeed, lithium salts alone produced marked lethargy in guinea pigs. Since
Cade's focus was on mania, he tried the sedating lithium salts on 10 patients
with manic-depressive illness. As he reported in 1949, lithium salts relieved
manic symptoms astonishingly well.120
Cade's paper generated little enthusiasm, but five years later, in 1954,
Mogens Schou in Risskov achieved similar success with controlled clinical tri-
als.121 Moreover, Schou in 1967 described a prophylactic effect against both
manic and depressive episodes of chronic treatment. Lithium extended the
interval between episodes and blunted symptoms when they recurred.122 Oth-
ers confirmed these successes.123 Although manic episodes might require added
antipsychotic medications and depressive episodes added antidepressants,
lithium for many patients was sufficient to prevent the devastating symptoms
of this disorder.
The U. S. Food and Drug Administration had banned lithium in 1949, the
year of Cade's initial success. Lithium chloride had been used as a dietary salt
substitute in hypertensive patients, but it was linked to lethal toxicities. Other
lithium salts, such as carbonate and citrate, were less toxic, and in light of the
accumulating evidence for their efficacy in bipolar disorder, these salts were
approved in 1970. Still, the range of dangerous toxicities to lithium remained
a serious concern, and therapeutic use required careful monitoring.
The various amine hypotheses proposed that mania and depression were
opposites, reflecting excesses and deficiencies. Accordingly, high doses of
monoamine oxidase inhibitors could elicit manic behavior. The ability of lithium
to forestall and blunt both manic and depressive symptoms was therefore unex-
pected. How could chemically simple lithium salts achieve such "normalizing"
effects? Attempts to identify the pertinent cellular actions were frustrated by
lithium's multitudes of effects, but during the 1980s a prominent proposal
invoked lithiums ability to inhibit inositolphosphate metabolism,124 which
would block this vital second messenger system (chapter 7). Critics of this pro-
posal, however, argued that therapeutic levels of lithium did not alter brain lev-
els of the second messengers significantly.125 This controversy was unresolved
by 1990, and no convincing mechanism had then appeared to account for inos-
itolphosphate controlling pathological mood swings.
Summary
Effective new medicines for depression and mania were identified through clin-
ical observations of drugs expected to have other actions (iproniazid, imipra-
mine) or serendipitously from explorations of hypothesized psychic toxins
(lithium). The actions of these drugs soon inspired accounts of pathophysiol-
ogy. Inhibition of monoamine oxidase or neurotransmitter reuptake, identified
as consequences of antidepressant drug administration, would potentiate
responses to endogenous noradrenaline and serotonin. (At this time anatomi-
cal studies—notably those exploiting fluorescence microscopy—were defining
noradrenergic and serotonergic pathways that arise from relatively few neurons
and then branch extensively to innervate the brain broadly. Such systems should
modulate mental function broadly, as emotions seem to do.) The resulting cat-
echolamine and biogenic amine hypotheses proposed that deficiencies of nora-
drenaline and/or serotonin were responsible for depression and excesses for
mania. Newer drugs developed from these mechanistic principles, such as the
specific serotonin reuptake inhibitors, yielded additional clinical successes.
Although further studies failed to correlate amine levels with disease, the
hypothesized deficiencies and excesses could be relative to more fundamental
aberrations in other (unidentified) systems that influence or are influenced by
the biogenic amines. On the other hand, the delayed onset of therapeutic ben-
efits suggested that complex cellular changes participated, such as altered
receptor numbers. How lithium's actions fit such schemes was unclear. In addi-
tion, evidence for other pathological aspects of mood disorders was also accu-
mulating, implicating possible metabolic and endocrine malfunctionings.126
(Probably the most effective treatment was electroconvulsive therapy. Its
administration altered neurotransmitter levels, receptor responses, and endo-
crine function, but in 1990 the changes necessary for symptomatic remissions
remained undefined.127)
Conclusions
Long-sought relief from the ravages of neurological and psychiatric illnesses

arrived during the twentieth century in the form of new medicines. For parkin-
sonism, effective therapies were designed to counteract known deficits. For
schizophrenia and depression, where the defects were not known, therapies
came from a folk medicine (whose mechanism was unknown initially) and from
noticing clinical responses to drugs administered for other purposes. The phar-
macological properties of such empirically discovered drugs suggested ways
these drug achieved their therapeutic ends, and these actions then suggested
the malfunctionings that underlay the illnesses.
Discoveries of effective drugs proceeded not just from crafting remedies to
known defects and verifying observations from folk medicine or more recent
clinical observations. Another important approach involved broad testing by
the pharmaceutical industry. Their prolific chemists generated all possible vari-
ants of a successful drug in the hope of finding one still better. Consequently,
the selection of useful screening assays was a vital requirement, although the
screens were themselves often the fruits of serendipity. For psychiatric illnesses
these screens ranged from behavioral assays (e.g., interference with conditioned
avoidance for antipsychotics and with learned helplessness for antidepressants)
to biochemical ones (e.g., blockade of D2 receptors for antipsychotics and of
serotonin reuptake for antidepressants).
Despite the pedigree of a compound and its success with particular screens,
the clinical trial was crucial for determining a drug's utility against an actual ill-
ness. The methodology of clinical testing evolved, also, and its ideal form late
in the twentieth century specified comparisons of responses to a new drug with
those to an inactive placebo and/or a drug previously found effective. The drugs
and placebos were assigned randomly to individuals from representative pop-
ulations in numbers calculated for statistical merit, with knowledge of who
received drug or placebo withheld from patient and physician until the study
was completed.128 Although all testing methodologies have inherent short-
comings,129 the benefits of the new drugs described here were readily dis-
cernable, and the revolution in patient care that followed their introduction
was apparent to almost all.130
Finally, two aspects of the drugs discussed in this chapter deserve empha-
sis. First, these drugs were palliative, not curative: they relieved symptoms,
often for as long as the drugs were continued, but did not eradicate the under-
lying disorder (which in all cases was unknown). Such is the case with much
of medical practice, although curative drugs would obviously be better. And
the search for better drugs would be facilitated by better knowledge of patho-
physiologies. Second, all the drugs acted on synaptic transmission (as do the
vast majority of drugs used to treat neurological and psychiatric illnesses), but
in no case was there firm evidence for the illness being a primary disorder of
synaptic transmission. The synapse, nonetheless, is an ideal target for palliative
treatment since individual behaviors—whether mental or motor—represent
activities over particular pathways, with the pathways formed from chains of
neurons linked through synapses. The synapses are thus the sites controlling
communication over the pathway. In addition, drugs that act on synaptic trans-
mission may be relatively specific: critical synapses controlling a pathway may
use only a few of the dozens of neurotransmitters employed elsewhere, and
through only a few classes of receptors available to these defining neurotrans-
mitters.
Notes
1. See Ayd and Blackwell (1970); Berrios and Porter (1995); Caldwell (1970); Shorter
(1997).
2. Distinctions are often made between "symptoms" (that the patient feels and
reports) and "signs" (that the physician observes). For convenience I will use "symp-
toms" to include both categories.
3. For example, Kirk and Kutchins (1992).
4. Abridged in Marks (1974), pp. 9-17. See also Langston and Palfremon (1995);
Schiller (1967); Tyler (1991).
5. See Tyler (1991).
6. Translated in Marks (1974), pp. 21-28.
7. Wilson (1924); Ferraro (1928).
8. Carlsson et al. (1957).
9. Carlsson (1959). This was presented at a conference the previous year.
11. Hornykiewicz (1963). See also Anden et al. (1964); Poirier and Sourkes (1965).
14. For example, McGeer and Zeldowicz (1964); Fehling (1966).
15. Cotzias et al. (1967).
16. See Yahr et al. (1968).
17. Decarboxylation of dopa in the periphery floods the body with dopamine, which
then acts on adrenergic receptors to produce cardiovascular responses and on the
chemoreceptor trigger zone of the brain (which lies outside the blood-brain barrier) to
produce vomiting.
18. Udenfriend et al. (1966). They believed, however, that the apparent rise in brain
dopamine was an artifact due to the drug's conversion to a dopamine-like substance.
19. Bartholini and Pletscher (1969). They had earlier studied a different peripheral
inhibitor.
20. See Cotzias et al. (1969); Mars (1973).
21. Schwab et al. (1951). The motivation for clinical testing was apomorphine's abil-
ity to relieve rigidity in animals due to experimental lesions.
22. Ernst (1967). See also Anden et al. (1967); Cotzias et al. (1970). Apomorphine is
formed from morphine, but its structure is drastically altered (apomorphine has no anal-
gesic activity).
23. Calne et al. (1974). See also Corrodi et al. (1973). Bromocriptine was initially
used to inhibit prolactin secretion.
24. See Lieberman et al. (1976).
25. Schwab et al. (1969).
26. For example, Grelak et al. (1970); Von Voigtlander and Moore (1971); Mawds-
ley et al. (1972).
27. Langston and Palfremon (1995).
28. Langston et al. (1983).
29. Davis et al. (1979).
30. Burns et al. (1983).
31. Chiba et al. (1984); Markey et al. (1984); Heikkila et al. (1984).
32. Nicklas et al. (1985).
33. Javitch et al. (1985).
34. Knoll (1978). He also cited other means by which selegiline could work.
35. Birkmayer et al. (1985); Elizan et al. (1989); Tetrud and Langston (1989); Parkin-
son Study Group (1989).
36. For example, see Letters to the Editors, Science 249: 303-304, 1990.
37. Connor et al. (1967).
38. See Hornykiewicz (1971).
39. Yurek and Sladek (1990).
40. Schults (1991).
41. See Berrios and Porter (1995); Howells (1991); Thompson (1987).
42. See Leff (1977).
43. For example, Fromm-Reichman (1948); Bateson et al. (1956).
44. See Dunlap (1924).
45. Seymour Kety in Bethesda carefully examined successive proposals for schizo-
phrenias pathophysiology, only to find each seriously flawed (1959, 1967).
46. See Haug (1962).
47. See Bench et al. (1990); Lewis (1990); Suddath et al. (1990).
48. Gaddum (1953).
49. Wooley and Shaw (1954).
50. Connell (1958).
51. See Gottesman and Shields (1982); Kallman (1946).
52. Heston (1966); Kety et al. (1968).
53. See Karayiorgou and Gogos (1997).
54. Sen and Bose (1931). Astutely, they noted that reserpine relieved "maniacal" but
not "morose" symptoms. For historical accounts, see Ayd and Blackwell (1970); Cald-
well (1970); Healy (1996, 1997).
55. Vakil (1949); Wilkins et al. (1952).
56. Cited in Ayd and Blackwell (1970).
57. Kline (1954), p. 3.
58. Barsa and Kline (1955); Lasky et al. (1962).

59. See Ayd and Blackwell (1970); Caldwell (1970); Swazey (1974).
60. Phenothiazine Treatment in Acute Schizophrenia (1964).
61. "Major" contrasts these drugs with pharmacologically distinct "minor tranquiliz-
ers" used to treat anxiety.
62. "Neuroleptic" has differing interpretations; the etymology refers to affecting the
nervous system.
63. Carlsson and Lindqvist (1963).
64. They showed that haloperidol increased metabolites of dopamine but not nora-
drenaline, whereas chlorpromazine increased metabolites of both. They did not com-
ment on this, however.
65. van Rossum (1966), p. 492.
66. Randrup et al. (1963); Scheel-Kriiger and Randrup (1967).
67. Coyle and Snyder (1969); Taylor and Snyder (1971). Snyder et al. (1974) cite
some contradictory reports, however.
68. Angrist et al. (1971).
69. See Angrist et al. (1973).
70. Snyder et al. (1974). For other accounts, see Klawans et al. (1972); Matthysse
(1973).
71. Horn and Snyder (1971).
72. Kebabian et al. (1972).
73. Iversen (1975).
74. Creese et al. (1976).
75. Seeman et al. (1976).
76. Kebabian and Calne (1979).
77. Bunzo et al. (1988).
78. For example, Bowers (1974); Sedvall and Wode-Helgodt (1980); Heritch (1990).
79. See Kornhuber et al. (1989).
80. Wong et al. (1986).
81. Farde et al. (1987).
82. Hokfelt et al. (1974); Swanson (1982).
83. See Bench et al. (1990); Lewis (1990); Suddath et al. (1990).
84. See Hippius (1989); McKenna and Bailey (1993).
85. Gross and Langer (1966).
86. Griffith and Saameli (1975).
87. Kane et al. (1988).
88. Farde et al. (1989).
89. Gelders et al. (1990).
90. Van Tol et al. (1991); Seeman et al. (1994).
91. See Javitt and Zukin (1991).
92. See Ayd and Blackwell (1970); Healy (1996, 1997); Johnson (1984); Pletscher
(1991).
93. Leonhard (1957).
94. See McGuffin and Katz (1989).
95. See Jeste et al. (1988).
96. Amphetamine had been tried with depressed patients, but although it initially
roused them, it then produced a rebound exacerbation of their depression. Opiates had
proved more effective but carried severe liabilities with chronic use.
97. For example, Kamman et al. (1953). They examined behavioral changes in groups
of psychiatric patients having unspecified diagnoses.
98. Loomer et al. (1957); Kline (1958). Kline provided two rationales: iproniazid is a
monoamine oxidase inhibitor (without specifying why this should be beneficial), and
recent reports described excitation (in animals) after sequential treatment with iproni-
azid and reserpine. Kline initially planned to give iproniazid followed by reserpine (which
he was then using with schizophrenics) but observed relief with iproniazid before he
added reserpine. See also Crane (1957), who stressed "increased vitality" rather than
antidepressant activity per se.
99. For example, Kiloh et al. (1960).
100. Zeller et al. (1952).
101. See Blackwell et al. (1967) for an explanation of why tranylcypromine (Parnate)
produced dangerous hypertension in patients who ate cheese.
102. Kuhn (1957, 1958). He claimed he tested imipramine with depressed patients
on behalf of "thoroughness" as well as a "conviction that it must be possible to find a
drug effective in ... depression" (Ayd and Blackwell, 1970, p. 211).
103. For example, Lehmann et al. (1958).
104. Cole (1964).
105. Glowinski and Axelrod (1964).
106. Schildkraut (1965), p. 509. See also Bunney and Davis (1965).
107. Coppen (1967). He also reviewed evidence for changes in other factors.
108. For example, Maas (1975); Carver and Davis (1979).
109. Baraban and Aghajanian (1980).
110. For example, Agren (1982); Davis et al. (1988).
111. For example, Davis et al. (1988). There was, however, a correlation between low
levels of a serotonin metabolite in the cerebrospinal fluid and suicide (Asberg, 1976),
although low levels also occur in other psychiatric diseases.
112. Sulser et al. (1978). They also argued that new experimental drugs were effec-
tive without inhibiting monoamine oxidase or reuptake. See also Vetulani and Sulser
(1975).
113. For example, Wolfe et al. (1978); Peroutka and Snyder (1980).
114. For example, Charney et al. (1981); Sugrue (1983); Stahl and Palazidou (1986).
115. Downregulation might overshoot the homeostatic set point, producing an
absolute decrease in responses (although there was no precedent for this happening).
Or autoregulatory receptors might be downregulated, producing a greater release of
neurotransmitter. Or various classes of receptors might be downregulated to different
degrees, with the altered balance between their responses producing the therapeutic
change. Or. . . .
116. See Carlsson and Wong (1997).
117. Wong et al. (1974, p. 477, 1975). Fluoxetine was discovered by synthesizing
analogs to an antihistaminic known to block neurotransmitter reuptake. It was one of
57 compounds then screened for specificity toward serotonin reuptake.
118. See Stark and Hardison (1985).
119. Prozac made the cover of Newsweek (26 March 1990) and starred in P. Kramers
Listening to Prozac, New York: Viking Press (1993).
120. Cade (1949).
121. Schou et al. (1954).
122. Baastrup and Schou (1967).
123. For example, Coppen et al. (1971).
124. Berridge et al. (1982, 1989).
125. For example, Honchar et al. (1990).
126. See Honig and van Praag (1997), pp. 235-250.
127. See Honig and van Praag (1997), pp. 397-412.

128. For the development of testing, see Healy (1997).
129. For example, in randomized double blind placebo trials, those receiving place-
bos may identify them through the absence of side effects that inevitably accompany
an active drug. Moreover, double blind trials usually prevent adjusting dosages to indi-
vidual needs.
130. There remain critics of the drugs, in principle and in practice. As with all rev-
olutions, not all consequences were beneficial. Complaints of the overreliance on drugs
have merit, as do complaints that patients were released from hospitals to the commu-
nity without the community being prepared to care for them. (Coincident with the intro-
duction of these drugs the population of mental hospitals dropped by four-fifths.)
14
EPILOGUE
Progress
When formulated late in the nineteenth century, the Neuron Theory depicted
discrete nerve cells interacting at their points of contact. Nerve impulses, then
often identified with electrical signals traveling along neuronal processes, would
pass electrically from neuron to neuron at these synaptic contacts. Over the
next century, however, this view changed dramatically. Neurons could inhibit
as well as excite other neurons; communication between cells was generally not
electrical but achieved through the release of chemicals that then bound to
specific receptors to elicit excitation or inhibition; there were dozens of dis-
tinct chemical neurotransmitters and multiple classes of receptors for each;
receptors could initiate complex chains of metabolic alterations as well as elicit
electrical responses; receptors were present on presynaptic terminals, also,
modulating function at this site, too; neurotransmitters were not secreted after
synthesis but stored in vesicles, from which they were released exocytotically
as discrete quanta; transport back into presynaptic neurons terminated the
actions of some neurotransmitters, whereas metabolic degradation terminated
the actions of others. In addition, the formation of specific synapses during
development and the alterations in synaptic transmission accompanying learn-
ing also relied on intricate chains of cellular modifications.
349
Accumulating the detailed evidence for these entities and processes required
approaches from anatomy, biochemistry, embryology, medicine, pharmacology,
and physiology. Applying the techniques and concepts of these disciplines to
the various issues then created a vast body of new knowledge now called neu-
roscience. But as accounts of neural structures and mechanisms accumulated,
many of these capabilities were being identified in other cell types. For exam-
ple, voltage-gated ion channels of nerve action potentials and ligand-gated ion
channels of neurotransmitter receptors were initially described for neurons and
their end organs, but subsequent investigations demonstrated that ion chan-
nels functioned in essentially all cell types, from bacteria to liver cells. Signal-
ing within and between cells is an essential process that—across the biological
realm—utilizes ion channels, chemical signals, receptors, and second messen-
ger systems. Consequently, a complementary integration resulted, one that
embraced neuroscience within general cell biology.
By 1990 the formulations of cellular mechanisms were vastly richer in detail,
with hosts of new entities linked through new processes. These understand-
ings then directed and enabled new experimental manipulations for continu-
ing explorations as well as for improving therapeutic interventions. But despite
such spectacular progress, the overall picture was far from complete. Critical
gaps remained and further investigations were continuing successfully when
this history concludes.
Historical Accounts and Conclusions
Unlike many creative fields, science (according to its practitioners) progresses,

with later formulations surpassing earlier ones. This account attempts to pre-
sent activities and results in the context of their times. But it also places them
on a path—twisting, tentative, fallible, but advancing overall—toward the pres-
ent. "Whig histories" of social and cultural events are castigated for viewing the
past as prelude to their authors' views of present virtue. Nevertheless, if sci-
entific understanding is indeed proceeding toward better descriptions of the
world, then progress toward this goal should be acknowledged.1
This account also illustrates an obvious characteristic of how science is done:
pluralistically, in different ways for different reasons and in different places.
Unitary explanations—algorithmic, economic, social—are inadequate. Like geo-
graphical explorations during the preceding centuries, individuals ventured into
the unknown inspired by a range of human motivations: to satisfy curiosity, to
seek fame and fortune, to honor God and country, to serve humanity.
And like tales from geographical explorers, the scientists' reports also were
colored by human aspirations as well as human frailties. But, just as the real-
ity of the earths surface ultimately constrained the explorers' accounts, so the
Epilogue 351
real world (assumed/inferred) ensures a coherence that overrides individual

intentions and expectations. This reality, scientists argue, limits the alternatives
and forces convergence. By contrast, historians who deny the role of a limit-
ing, external world have been baffled by the consensus that scientific research
builds.
Assumptions
Most scientists, having passed in adolescence through the requisite phases of

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

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

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

ceptions as well as inevitable oversimplifications. If aspects of a real world seem
apparent, its details have been grasped with difficulty, often amid uncertainty
and controversy, and with critical gaps still remaining. Arguments against direct,
indubitable sensory access to reality range from the critiques of ancient philoso-
phers to formulations of modern neurophysiology. The latter depict neuronal
chains underlying perception that are not only susceptible to errors of pro-
cessing but also subject to modulation from higher centers (and thus incipient
bias).4 Furthermore, our senses receive direct information only through lim-
Epilogue 353
ited channels sharply restricted in range. Instruments may confirm these obser-
vations and vastly extend them—as by electron microscopy—but at the price
of theoretical justification and at the risk of artifact.
These common recognitions endorse modest and circumspect goals for sci-
entific research: the formulation of explanatory, causal models.5 These models,
like the geographers' maps of the world,6 then attempt to display similarities
to the real world in specified ways and at specified scales: models of behavior,
of nervous systems, of neurons, of synaptic junctions, of receptors. The con-
straints on how such models can represent observation and experiment, more-
over, reinforce the presumption that they reflect discoveries about a real world,
even though they are human constructions. The models are, of course, subject
to continuing amplification and correction in the light of further experiment
and analysis. And they serve as guides for further exploration.
Such causal models also embody an explanatory reductionism.7 For exam-
ple, questions about how reserpine causes parkinsonism initiate a hierarchical
chain: by depressing dopaminergic function in the basal ganglia; by depleting
the stores of dopamine in nigrostriatal neurons; by binding to the dopamine
transporter of the synaptic vesicles in these neurons and thereby inactivating
it. The explanatory regress of biomedical research has a clear terminus, the
laws of chemistry and physics (here, the theory of ligand binding to proteins).
The regress also is a guide to chemical manipulation of responses distinguish-
able at higher levels, as in drug therapy of behavioral disorders. On the other
hand, syntheses of complex wholes from models of their parts, while a pro-
claimed goal, is also a forbiddingly difficult one.
Scientists often use terms far more casually than their critics, causing con-
fusion over issues of "fact," "truth," "proof," and the like. These are indeed
words that can be construed in various ways. But, assuming a real world, there
should be facts about it, and descriptions that accurately describe these facts
would be true. If such absolutes are unattainable, the scientific quest at least
aims for closer and closer likenesses through its explanatory models, as dem-
onstrated by experiment and formulated through interpretation. Accordingly,
models of distinct neurons communicating through the release of chemical neu-
rotransmitters seemed by 1990 far closer to the truth than the reticular mod-
els of Gerlach and of Golgi.
Generalities ana Exceptions
Scientists commonly strive for the simplest explanations until forced by exper-
iment or interpretation into multiplying their entities and processes. The cen-
tury of research depicted here displays a proliferation of detail that embellished
initial representations. But this elaboration required the justification of each
new entity and process. For example, arguments that inhibition could be presy-
naptic as well as postsynaptic were furthered by the discovery of anatomical
and pharmacological correlates of the proposed physiological mechanism.
The allegiance to parsimony also inspires the grouping together of individ-
ual entities and processes into common categories, such as neurons and recep-
tors and protein phosphorylations. These generalizations, however, serve as pro-
totypes having not only specified characteristics but also accepted ranges of
deviation. For example, all receptors are not alike, and when examined more
closely the designations are seen to embrace families of individuals distin-
guishable by the ligands they bind and respond to. Those binding and respond-
ing to noradrenaline are then further divided into functional (and later struc-
tural) classes such as a\, az, P\, and (3z, and these are again subdivided by
degree of regulatory phosphorylation, and so on. Such designations and allo-
cations have particular explanatory significance in specific contexts, including
functional capabilities (for example, sensitivity to certain ligands, linkage to sec-
ond messengers) and structural characteristics (for example, resemblances of
their amino acid sequences to those of certain other receptors).
In some instances, moreover, scientists are compelled to accept singular
exceptions to otherwise consistent patterns. For example, postganglionic sym-
pathetic fibers to the sweat glands release acetylcholine, as Dale acknowledged,
in contrast to postganglionic sympathetic innervation of essentially all other end
organs.
Biological research is thus enriched (or plagued, depending on one's view-
point) with identifiable prototypes that, on closer scrutiny, dissolve into popu-
lations of distinguishable individuals. Such diversity is, of course, understand-
able from the history of biological design. Mutations can cause a gene to
duplicate as well as change, and after duplication each is then subject individ-
ually to further random changes, forming families of variants. Natural selec-
tion then chooses among these for distinct functional roles—as, for example,
in accumulating families of receptors for different neurotransmitters. For the
organism, this multiplicity of related structures offers adaptive advantages. For
scientists, these relationships justify generalizations about classes of structures
operating similarly, so that, for example, certain stretches of amino acids when
appearing in distinct proteins can suggest a common function.
Scientists' quest for simplicity also abetted tendencies to imagine all struc-
tures and functions in the form first established, for example, generalizing to
all synapses the chemical transmission identified at certain sites, or generaliz-
ing all receptors as ligand-gated ion channels after the first classes of receptors
were so identified. Although there has been a danger of seeing today only what
one saw yesterday, further studies could still reveal exceptions, such as elec-
trical transmission at certain loci and receptors instead coupled to second mes-
senger systems.
Epilogue 355
Conflict Resolution
The routes to resolving scientific conflicts are also various, including refutation
of one hypothesis and confirmation of its rival. Here, too, the course is usually
pragmatic and opportunistic, recognizing available capabilities.
When experiments support and extend a given formulation, such confirma-
tions of predicted results are usually considered persuasive arguments for the
formulation s nearness to truth.8 Thus, Eccles accepted the principle of chem-
ical transmission at neuromuscular junctions when confronted by extensive evi-
dence favoring this notion, despite the absence of experiments explicitly refut-
ing electrical transmission at this site. Katz bolstered his quantal hypothesis of
neurotransmitter release from an additional physiological perspective by
describing how Ca2+ affected m.e.p.p.s. Electron micrographs showing vesi-
cles within presynaptic terminals then provided compelling arguments from an
independent discipline.
On the other hand, refuting a rival hypothesis is logically superior but often
difficult technically. Claims that an entity does not exist or a process does not
work may be contested by counterclaims that the attempted refutation was
insufficiently sensitive or specific. For example, when Cajal argued that fila-
ments did not cross from pre- to postsynaptic neurons, his critics complained
that Cajal was merely unable to see what they saw. Cajal answered with new
stains that revealed filaments but no continuity, satisfying many but not all. The
development of electron microscopy, with its far higher resolution, confirmed
Cajals refutation, albeit some decades later: fibrils were readily detectable
within neurons, but none of these crossed the synaptic cleft that separated the
neurons.9
Convincing refutations also occur in a more timely fashion, as in Eccles's
demonstration that inhibition produced electrical changes that contradicted
what he had predicted, and Wiersma's finding that impulses initiating con-
tractions in a particular muscle passed only in nerve fibers to that muscle, con-
tradicting Weiss's original Resonance Principle. (In addition, refuting alterna-
tive formulations is a daily part of scientific practice: experiments frequently
include "controls" to eliminate conceivable alternatives.)
Nevertheless, some critics claim that experimental evidence is inadequate
for resolving scientific controversies, which instead are settled through social
interests (political, economic, sexual, ethnic, etc.).10 Two philosophical issues
bear on this question of experimental adequacy.
(1) The notion that models/theories are underdetermined by evidence

includes the recognition that an infinite number of hypotheses can
account for any observation. This concern, however, is diminished by
wielding Occam's razor and by acknowledging the web of scientific
knowledge in which an issue is embedded. For example, a model relat-

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

inquiries follow acknowledged avenues, while others progress through flouting
accepted wisdom. Some begin through logical analysis, and others flourish
through unanticipated results. Some attempt refutations and others confirma-
tions. Indeed, a range of approaches and interests come together, and in so
doing correct errors and reinforce accomplishments within the web of scien-
tific knowledge. More is better.
Effective scientific research requires technical and financial support. Cor-
respondingly, the benefits of pluralism require open access to such support and
Epilogue 357
to the forums for presenting and arguing the fruits of labor and inspiration.
The hundred years covered here were blessed with growing opportunities and
access, vital ingredients in attaining the achievements recorded here.
Notes
1. See Harrison (1987). For efforts to describe scientific practice scientifically, see
Donovan et al. (1992). This book, however, selects cases as a reflection of its authors'
interests rather than—as methodological dicta advise—through random (or at least rep-
resentative) samplings of scientific practice.
2. For assorted arguments about scientific realism, see Leplin (1984).
3. Although most endeavors can be squeezed into the guise of testing some hypoth-
esis, the most straightforward characterization of many explorations is simple empiri-
cism. This is clearly the case, for example, in drug development by testing through var-
ious screening assays.
4. On the other hand, the principles of Darwinian evolution suggest that animals'
nervous systems were selected to deal successfully with the gross aspects of a natural
world: finding things to eat while avoiding being eaten.
5. See, for example, Cartwright (1983); Giere (1988, 1999). An interesting possibil-
ity is that more than one model could explain how the world works. But before taking
this possibility seriously, many scientists would like to see such an independent alter-
native.
6. Geographical maps also are developed at different scales for different purposes
and for emphasizing different aspects (e.g., physical, political, agricultural, climatic)
while still representing a real world, even if not in all its particulars.
7. See, for example, Robinson (1986a, 1992).
8. Arguments about justifying hypotheses through corroborating evidence have a long
history, which includes debates about the merits of induction. For many scientists cor-
roborative evidence is considered to increase the likelihood that the tested hypothesis
is (nearly) true, often on the basis of informal probabilistic assessments.
9. By the time evidence from electron microscopy was available, other arguments for
discontinuity—including that from physiological and pharmacological approaches—had
strongly confirmed Cajal's interpretation.
10. For example, "Despite the local and situated nature of scientific work, there
appear to be some semblances of agreement, stabilizations, and continuities across sit-
uations and through time[, but while] scientific realists choose to interpret these as the
outcomes of nature's guiding hand, most recent works in science studies take different
views" (Clarke and Fujimura, 1992, p. 12); "scientists at the research front cannot set-
tle disagreements through better experimentation, more knowledge, more advanced
theories or clearer thinking" (Collins and Pinch, 1994, pp. 144, 145); and "Few con-
troversies . . . centre on such epistemic factors as accuracy and predictive capacities[,
instead the] overwhelmimg majority turn on matters of goal orientation, social inter-
ests, and stubbornly held metaphysical beliefs" (Shortland, 1988, p. 265).
11. For critical comments, see Laudan (1996).
12. See, for example, Franklin (1998).
13. For examples, see Robinson (1983, 1986b, 1992, 1997). Pertinent to the topics
in this book is the account of learning in planaria, by Collins and Pinch (1994). For
instance, in describing the chemical transfer of particular learned behaviors between

planaria, they omitted discussions of the scientific improbabilities (e.g., that ingested
RNA can redirect an animals protein synthesis), of the formulations of alternative mod-
els (e.g., Hebb's proposal), and of the successes in identifying memory encoded not in
specific chemicals but in neural circuits (e.g., Kandels work on Aplysia). Consequently,
they could then come to the astonishing conclusion in 1994 that "a determined upholder
of the idea [of chemical transfer of learning] would find no published disproof [so that
for] such a person it would not be unreasonable or unscientific to start experimenting
[on chemical transfer] once more" (p. 25). Collins and Pinch's omissions, either delib-
erate or through inattention, should be disturbing to all interested in science and how
it works.
REFERENCES
Adam, H.M., R.A. McKail, S. Obrador, and W.C. Wilson. Acetylcholine in cerebro-spinal
fluid. /. Physiol. 93: 45P^6P, 1938.
Adrian, E.D. Some recent work on inhibition. Brain 47: 399-416, 1924.
Adrian, E.D., and D.W. Bronk. The discharge of impulses in motor nerve fibres./. Phys-
iol. 66: 81-101, 1928.
Adrian, E.D., and D.W. Bronk. The discharge of impulses in motor nerve fibres./. Phys-
iol. 67: 119-151, 1929.
Adrian, E.D., and K. Lucas. On the summation of propagated disturbances in nerve
and muscle. /. Physiol. 44: 68-124, 1912.
Aghajanian, G.K., and F.E. Bloom. Localization of tritiated serotonin in rat brain by
electron-microscopic autoradiography. /. Pharmacol. Exp. Ther. 156: 23-30, 1967.
Aghajanian, G.K., J.A. Rosecrans, and M.H. Sheard. Serotonin: Release in the forebrain
by stimulation of midbrain raphe. Science 156: 402-403, 1967.
Agranoff, B.W., and P.D. Klinger. Puromycin effect on memory fixation in the goldfish.
Science 146: 952-953, 1964.
Agren, H. Depressive symptom patterns and urinary MHPG excretion. Psychiat. Res.
6: 185-196, 1982.
Ahlquist, R.P. A study of the adrenotropic receptors. Amer. ]. Physiol. 153: 586-600,
1948.
Akiba, I., T. Kubo, A. Maeda, H. Bujo, J. Nakai, M. Mishina, and S. Numa. Primary
structure of porcine muscarinic acetylcholine receptor III and antagonist binding
studies. FEES Lett. 235: 257-261, 1988.
Albert, K.A., E. Helmer-Matyjek, A.C. Nairns, T.H. Muller, J.W. Haycock, L.A. Greene,
M. Goldstein, and P. Greengard. Calcium/phospholipid-dependent protein kinase
359
(protein kinase C) phosphorylates and activates tyrosine hydroxylase. Proc. Natl.

Acad. Sci. USA 81: 7713-7717, 1984.
Aldrich, T.B. A preliminary report on the active principle of the suprarenal gland. Am.
J. Physiol. 5: 457-461, 1901.
Aldrich, T.B. Adrenalin, the active principle of the suprarenal gland./. Am. Chem. Soc.
27: 1074-1091, 1905.
Aldridge, W.N. Some properties of specific cholinesterase with particular reference to
the mechanism of inhibition by diethyl p-nitrophenyl thiophosphate (E605) and
analogs. Biochem. ]. 46: 451-460, 1950.
Alkon, D.L. Calcium-mediated reduction of ionic currents: A biophysical memory trace.
Science 226: 1037-1045, 1984.
Alkon, D.L., and T.J. Nelson. Specificity of molecular changes in neurons involved in
memory storage. FASEB J. 4: 1567-1576, 1990.
Alles, G.A., and R.C. Hawes. Cholinesterases in the blood of man./. Biol. Chem. 133:
375-390, 1940.
Allport, S. Explorers of the Black Box. New York: Norton, 1986.
Aimers. W. Exocytosis. Annu. Rev. Physiol. 52: 607-624, 1990.
Alousi, A., and N. Weiner. The regulation of norepinephrine synthesis in sympathetic
nerves: Effect of nerve stimulation, cocaine, and catecholamine-releasing agents.
Proc. Natl. Acad. Sci. USA 56: 1491-1496, 1966.
Altamirano, M., C.W. Coates, and H. Grundfest. Mechanisms of direct neural excitabil-
ity in electroplaques of electric eel. /. Gen. Physiol. 38: 319-360, 1955a.
Altamirano, M., C.W. Coates, H. Grundfest, and D. Nachmansohn. Electrical activity
in electric tissue. III. Biochim. Biophys. Acta 16: 449-463, 1955b.
Altschuler, R.A., M.H. Parakkal, and J. Fex. Localization of enkephalin-like immunore-
activity in acetylcholinesterase-positive cells in the guinea-pig lateral superior oli-
vary complex. Neurosci. 9: 621-630, 1983.
Amin, A.H., T.B.B. Crawford, and J.H. Gaddum. The distribution of substance P and
5-hydroxytryptamine in the central nervous system of the dog./. Physiol. 126: 596-
618, 1954.
Anden, N.E., A. Carlsson, A. Dahlstrom, K. Fuxe, N.-A. Hillarp, and K. Larsson.
Demonstration and mapping out of nigro-striatal dopamine neurons. Life Sci. 3:
523-530, 1964.
Anden, N.E., A. Carlsson, N.A. Hillarp, andT. Magnusson. 5-hydroxytryptamine release
by nerve stimulation of the spinal cord. Life Sci. 3: 473^178, 1964a.
Anden, N.E., A. Carlsson, A. Dahlstrom, K. Fuxe, N.A. Hillarp, and K. Larsson. Demon-
stration and mapping out of nigro-neostriatal dopamine neurons. Life Sci. 3: 523-
530, 1964b.
Anden, N.E., A. Carlsson, N.A. Hillarp, and T. Magnusson. Noradrenaline release by
nerve stimulation of the spinal cord. Life Sci. 4: 129-132, 1965.
Anden, N.E., A. Dahlstrom, K. Fuxe, and K. Larsson. Further evidence for the pres-
ence of nigro-neostriatal dopamine neurons in the rat. Amer. J. Anat. 116: 329-336,
1965.
Anden, N.E., A. Dahlstrom, K. Fuxe, K. Larsson, L. Olson, and U. Ungerstedt. Ascend-
ing monoamine neurons to the telencephalon and diencephalon. Acta Physiol.
Scand. 67: 313-326, 1966.
Anden, N.E., A. Rubenson, K. Fuxe, and T. Hokfelt. Evidence for dopamine receptor
stimulation by apomorphine. /. Pharm. Pharmacol. 19: 627-629, 1967.
Andersen P., T. Teyler, and K. Wester. Long-lasting change of synaptic transmission in
a specialized cortical pathway. Acta Physiol. Scand. Suppl. 396: 34, 1973.
Reierences 3ol
Anderson, C.R., and C.F. Stevens. Voltage clamp analysis of acetylcholine produced
end-plate current fluctuations at frog neuromuscular junction. J. Physiol. 235: 655-
691, 1973.
Anderson, D.C., S.C. King, and S.M. Parsons. Proton gradient linkage to active uptake
of [3H]acetylcholine by Torpedo electric organ synaptic vesicles. Biochemistry 21:
3037-3043, 1982.
Anderson, H.K. The action of eserine and atropine upon the denervated sphincter iridis.
J. Physiol. 31: xxii-xxiv, 1904.
Anderson, M.J., and M.W. Cohen. Nerve-induced and spontaneous redistribution of
acetylcholine receptors on cultured muscle cells. J. Physiol. 268: 757-773, 1977.
Anderson, M.J., M.W. Cohen, and E. Zorychta. Effect of innervation on the distribution
of acetylcholine receptors on cultured muscle cells. /. Physiol. 268: 731-756, 1977.
Angeletti, R.H., and R.A. Bradshaw. Nerve growth factor from mouse submaxillary
gland: Amino acid sequence. Proc. Natl. Acad. Sci. USA 68: 2417-2420, 1971.
Angrist, B., G. Sathananthan, and S. Gershon. Behavioral effects of L-dopa in schizo-
phrenic patients. Psychopharmacol 31: 1-12, 1973.
Angrist, B.M., B.Shopsin, and S. Gershon. Comparative psychotomimetic effects of
stereoisomers of amphetamine. Nature 234: 152-153, 1971.
Apathy, S. Das leitende Element des Nervensystems und seine topographischen
Beziehungen zu den Zellen. Mitheil. Zool Stat. Neapal. 12: 495-748, 1897.
Aprison, M.H., and R. Werman. The distribution of glycine in cat spinal cord and roots.
Life Sci. 4: 2075-2083, 1965.
Ariens, E.J. Affinity and intrinsic activity in the theory of competitive inhibition. Arch.
Int. Pharmacodyn. 99: 32-49, 1954.
Ariens Kappers, C.U. Further contributions on neurobiotaxis./. Comp. Neurol. 27: 261-
298, 1917.
Armstrong, M.D., A. McMillan, and K.N.F. Shaw. 3-methoxy-4-hydroxy-D-mandelic
acid, a urinary metabolite of norepinephrine. Biochim. Biophys. Acta 25: 422-423,
1957.
Artola, A., S. Brocher, and W. Singer. Different voltage-dependent thresholds for induc-
ing long-term depression and long-term potentiation in slices of rat visual cortex.
Nature 347: 69-72, 1990.
Arunlakshana, O., and H.O. Schild. Some quantitative uses of drug antagonists. Brit.
]. Pharmacol. 14: 48-58, 1959.
Arvanitaki, A. Effects evoked in an axon by the activity of a contiguous one. J. Neuro-
physiol 5: 89-108, 1942.
Asberg, M., L. Traskman, and P. Thoren. 5-HIAA in the cerebrospinal fluid: A suicide
predictor. Arch. Gen, Psychiat. 33: 1193-1197, 1976.
Asher, H., and N. Scheinfinkel. Studien iiber antagonistische Nerven. Pfliigers Arch.
217: 184-197, 1927.
Ashkenazi, A., J.W. Winslow, E.G. Peralta, G.L. Peterson, M.I. Schimerlik, D.J. Capon,
and J. Ramachandran. An M2 muscarinic receptor subtype coupled to both adenyl
cyclase and phosphoinositide turnover. Science 238: 672-675, 1987.
Ashman, D.F., R. Lipton, M.M. Melicow, and T.D. Price. Isolation of adenosine 3', 5'-
monophosphate and guanosine 3',5'-monophosphate from rat urine. Biochem. Bio-
phys. Res. Commun. 11: 330-334, 1963.
Attardi, D.G., and R.W. Sperry. Preferential selection of central pathways by regener-
ating optic fibers. Exp. Neurol. 7: 46-64, 1963.
Attwell, D., B. Barbour, and M. Szatkowski. Nonvesicular release of neurotransmitter.
Neuron 11: 401-407, 1993.
Auger, D., and A. Fessard. Interpretation de la latence observee dans la stimulation de

fragments d'organe electrique. C.R. Soc. Biol. 128: 1067-1070, 1938.
Augustine, G.J., M.P. Charlton, and S.J. Smith. Calcium entry and transmitter release
at voltage-clamped nerve terminals of squid. /. Physiol. 369: 163-181, 1985.
Augustinsson, K.B., and D. Nachmansohn. Distinction between acetylcholine-esterase
and other choline ester-splitting enzymes. Science 110: 98-99, 1949.
Aurbach, G.D., S.A. Fedak, C.J. Woodward, J.S. Palmer, D. Hauser, and F. Troxler.
Adrenergic-receptor: Stereospecific interaction of iodinated blocking agent with
high affinity site. Science 186: 1223-1224, 1974.
Awapara, J., A.J. Landua, R. Fuerst, and B. Scale. Free y-aminobutyric acid in brain.
/. Biol. Chem. 187: 35-39, 1950.
Axelrod, J. O-methylation of epinephrine and other catechols in vitro and in vivo. Sci-
ence 126: 400-401, 1957.
Axelrod, J., G. Hertting, and L. Potter. Effect of drugs on the uptake and release of 3H-
norepinephrine in the rat heart. Nature 194: 297, 1962.
Axelrod, J., S. Senoh, and B. Witkop. O-methylation of catechol amines in vivo. J. Biol.
Chem. 233: 689-701, 1958.
Axelrod, J., and R. Tomchick. Enzymatic O-methylation of epinephrine and other cat-
echols. /. Biol. Chem. 233: 702-705, 1958.
Axelrod, J., H. Weil-Malherbe, and R. Tomchick. The physiological disposition of H3-
epinephrine and its metabolite metanephrine. /. Pharmacol. Exp. Ther. 127: 251-
256, 1959.
Axelrod, J., L.G. Whitby, and G. Hertting. Effect of psychotropic drugs on the uptake
of H3-norepinephrine by tissues. Science 133: 383-384, 1961.
Axelsson, J., and S. Thesleff. A study of supersensitivity in denervated mammalian skele-
tal muscle. /. Physiol. 147: 178-193, 1959.
Ayd, F.J., and B. Blackwell (eds.). Discoveries in Biological Psychiatry. Phiadelphia: J.B.
Lippincott, 1970.
Baas, P.W., L.A. White, and S.R. Heideman. Microtubule polarity reversal accompanies
regrowth of amputated neurites. Proc. Natl. Acad. Sci. USA 84: 5272-5276, 1987.
Baastrup, P.C., and M. Schou. Lithium as a prophylactic agent. Arch. Gen. Psychiat.
16: 162-172, 1967.
Bach, A.W.J., N.C. Lan, D.L. Johnson, C.W. Abell, M.E. Bembenek, S.W. Kwan, PH.
Seeburg, and J.C. Shih. cDNA cloning of human liver monoamine oxidase A and
B: Molecular basis of differences in enzymatic properties. Proc. Natl. Acad. Sci.
USA 85: 4934^938, 1988.
Bacq, Z.M. Recherches sur la physiologic du systeme nerveux autonome. Arch. Int.
Physiol. 36: 167-246, 1933.
Bacq, Z.M. La pharmacologie du systeme nerveux autonome, et particulierement du
sympathetique, d'apres la theorie neurohumorale. Ann. Physiol. 10: 467-528, 1934.
Bacq, Z.M. Chemical Transmission of Nerve Impulses. Oxford: Pergamon Press, 1975.
Bacq, Z.M., and P. Fischer. Nature de la substance sympathomimetique extraite des
nerfs ou des tissues des mamiferes. Arch. Int. Physiol. 55: 73-91, 1947.
Bahler, M., and P. Greengard. Synapsin I bundles F-actin in a phosphorylation-depend-
ent manner. Nature 326: 704-707, 1987.
Bailey, C.H., and M. Chen. Morphological bases of long-term habituation and sensiti-
zation in Aplysia. Science 220: 91-93, 1983.
Bailey, C.H., and M. Chen. Long-term memory in Aplysia modulates the total number
of varicosities of single identified sensory neurons. Proc. Natl. Acad. Sci. USA 85:
2373-2377, 1988a.
Reierences 363
Bailey, C.H., and M. Chen. Long-term sensitization in Aplysia increases the number of
presynaptic contacts onto the identified gill motor neuron L7. Proc. Natl. Acad.
Set. USA 85: 9356-9359, 1988b.
Bain, W.A. A method of demonstrating humoral transmission of the effects of cardiac
vagus stimulation in the frog. Quart. J. Exp. Physiol. 22: 269-274, 1932.
Bain, W.A. The mode of action of vasodilator and vasoconstrictor nerves. Quart. J. Exp.
Physiol. 23: 381-389, 1933.
Baker, J.R. The cell-theory. /. Microsc. Sci. 93: 157-190, 1952.
Baldessarini, R.J., and I.J. Kopin. The effect of drugs on the release of norepinephrine-
H3 from central nervous system tissues by electrical stimulation in vitro. J. Phar-
macol. Exp. Ther. 156: 31-38, 1967.
Bamburg, J.R., D. Bray, and K. Chapman. Assembly of microtubules at the tip of grow-
ing axons. Nature 321: 788-790, 1986.
Banks, P. An interaction between chromaffin granules and calcium ions. Biochem. J.
101: 18c-20c, 1966.
Banks, P., and K. Helle. The release of proteins from stimulated adrenal medulla.
Biochem. J. 97: 40C-41C, 1965.
Baraban, J.M., and G.K. Aghajanian. Suppression of firing activity of 5-HT neurons of
the dorsal raphe by alpha-adrenoreceptor antagonists. Neuropharmacol. 19:
355-363, 1980.
Barcroft, H., and J.F. Talbot. Oliver and Schafer's discovery of the cardiovascular action
of suprarenal extract. Postgrad. Med. J. 44: 6-8, 1968.
Barger, A.C. Cannons emergency theory: The controversy with Stewart and Rogoff.
News Physiol. Sci. 7: 80-88, 1992.
Barger, G., and H.H. Dale. Chemical structure and sympathomimetic action of amines.
/. Physiol. 41: 19-59, 1910.
Barker, L.F. The Nervous System and its Constituent Neurones. New York: Appleton,
1899.
Baron van Evercooren, A., H.K. Kleinman, S. Ohno, P. Marangos, J.P. Schwartz, and
M.E. Dubois-Dalcq. Nerve growth factor, laminin and fibronectin promote nerve
growth in human fetal sensory ganglia cultures. /. Neurosci. Res. 8: 179-193, 1982.
Barondes, S.H., and H.D. Cohen. Memory impairment after subcutaneous injection of
acetoxycyclohexamide. Science 160: 556-557, 1968.
Barrantes, F.J., D.C. Neugebauer, and H.P. Zingsheim. Peptide extraction by alkaline
treatment is accompanied by rearrangement of the membrane-bound acetylcholine
receptor from Torpedo marmorata. FEBS Lett. 112: 73-78, 1980.
Barrionuevo, G., and T.H. Brown. Associative long-term potentiation in hippocampal
slices. Proc. Natl. Acad. Sci. USA 80: 7347-7351, 1983.
Barsa, J.A., and N.S. Kline. Treatment of 200 disturbed psychotics with reserpine.
/. Amer. Med. Assn. 158: 110-113, 1955.
Barsoum, G.S., and J.H. Gaddum. The pharmacological estimation of adenosine and
histamine in blood. /. Physiol. 85: 1-14, 1935.
Bartelmez, G.W., and N.L. Hoerr. The vestibular club endings in Ameiurus. J. Comp.
Neural 57: 401-428, 1933.
Bartholini, G., and A. Pletscher. Effect of various decarboxylase inhibitors on the cere-
bral metabolism of dihydroxyphenylalanine. J. Pharm. Pharmacol. 21: 323-324,
1969.
Barzilai, A., T.E. Kennedy, J.D. Sweatt, and E.R. Kandel. 5-HT modulates protein syn-
thesis and the expression of specific proteins during long-term facilitation in Aplysia
sensory neurons. Neuron 2: 1577-1586, 1989.
Bashford, C.L., R.P. Casey, G.K. Radda, and G.A. Ritchie. The effect of uncouplers on
catecholamine incorporation by vesicles of chromaffin granules. Biochem. J. 148:
153-155, 1975.
Bateson, G., D.D. Jackson, J. Haley, and J.H. Weakland. Towards a theory of schizo-
phrenia. Behav. Sci. 1: 251-264, 1956.
Baumert, M., P.R. Maycox, F. Navone, P. DeCamilli, and R. Jahn. Synaptobrevin: An
integral membrane protein of 18000 daltons present in small synaptic vesicles of
rat brain. EMBO ]. 8: 379-384, 1989.
Baxter, C.F., and E. Roberts. Elevation of y-aminobutyric acid in rat brain with hydrox-
ylamine. Proc. Soc. Exp. Biol. Med. 101: 811-815, 1959.
Bayliss, W.M. Principles of General Physiology. London: Longmans, Green, 1920.
Bayliss, W.M., and E.H. Starling. The mechanism of pancreatic stimulation./. Physiol.
28: 325-353, 1902.
Bazemore, A.W., K.A.C. Elliott, and E. Florey. Factor I and y-aminobutyric acid. Nature
178: 1052-1053, 1956.
Bazemore, A.W., K.A.C. Elliott, and E. Florey. Isolation of Factor I. J. Neurochem. 1:
334-339, 1957.
Bean, B.P Classes of calcium channels in vertebrate cells. Annu. Rev. Physiol. 51:
367-384, 1989.
Bear, R.S., and F.O. Schmitt. Electrolytes in the axoplasm of the giant nerve fibers of
the squid. /. Cell. Comp. Physiol. 14: 205-215, 1939.
Beers, W.H., and E. Reich. Structure and activity of acetylcholine. Nature 228: 917-922,
1970.
Beers, W.H., and E. Reich. Structure of acetylcholine. Nature 232: 422^23, 1971.
Belcher, G., J. Davies, and R.W. Ryall. Glycine-mediated inhibitory transmission of
group lA-excited inhibitory interneurones by Renshaw cells. J. Physiol. 256:
651-662, 1976.
Beleslin, D.B., and R.D. Myers. The release of acetylcholine and 5-hydroxytryptamine
from the mesencephalon of the unanesthitized rheusus monkey. Brain. Res. 23:
437-442, 1970.
Belluzzi, J.D., N. Grant, V. Garsky, D. Sarantakis, C.D. Wise, and L. Stein. Analgesia
induced in vivo by central administration of enkephalin in rat. Nature 260:
625-626, 1976.
Belocopitow, H. The action of epinephrine on glycogen synthetase. Arch. Biochem. Bio-
phys. 93: 457-458, 1961.
Bench, C.J., R.J. Dolan, K.J. Friston, and R.S.J. Frackowiak. Positron emission tomog-
raphy in the study of brain metabolism in psychiatric and neuropsychiatric disor-
ders. Brit. J. Psychiat. 157 (suppl. 9): 82-95, 1990.
Bennett, E.L. Commentary. In: Byrne, W.L. (ed.) Molecular Approaches to Learning
and Memory. New York: Academic Press, 1970. p. 150.
Bennett, E.L., and M. Calvin. Failure to train planarians reliably. Neurosci. Res. Prog.
2: 3-24, 1964.
Benovic, J.L., F. Mayor, C. Staniszewski, R.J. Lefkowitz, and M.G. Caron. Purification
and characterization of the jS-adrenergic receptor kinase. J. Biol. Chem. 262: 9026-
9032, 1987.
Benovic, J.L., R.H. Strasser, M.G. Caron, and R.J. Lefkowitz. /3-adrenergic receptor
kinase: Identification of a novel protein kinase that phosphorylates the agonist-
occupied form of the receptor. Proc. Natl. Acad. Sci. USA 83: 2797-2801, 1986.
Benzer, S. Behavioral mutants of Drosophila isolated by countercurrent distribution.
References 365
Berl, S., and H. Waelsch. Determination of glutamic acid, glutamine, glutathione and
y-aminobutyric acid and their distribution in brain tissue./. Neurochem. 3:161-169,
1958.
Bernard, C. Analyse physiologique des proprietes des systemes musculaire et nerveux
au moyen du curare. Compt. Rend. Acad. Sci. 43: 825-829, 1856.
Bernath, S., and M.J. Zigmond. Characterization of [3H]GABA release from striatal
slices: Evidence for a calcium-independent process via the GABA uptake system.
Neurosci. 27: 563-570, 1988.
Bernstein, J. Untersuchungen zur Thermodynamik der bioelektrischen Strome. Pflugers
Arch. 92: 521-562, 1902.
Bernstein, J. Zur elektrochemischen Grunlage der bioelektrischen Potentiale. Biochem.
Z. 50: 393-401, 1913.
Berridge, M.J. Rapid accumulation of inositol trisphosphate reveals that agonists hydrol-
yse polyphosphoinositides instead of phosphatidylinositol. Biochem. ]. 212: 849-
858, 1983.
Berridge, M.J., C.P. Downes, and M.R. Hanley. Lithium amplifies agonist-dependent
phosphatidylinositol responses in brain and salivary glands. Biochem. J. 206: 587-
595, 1982.
Berridge, M.J., C.P. Downes, and M.R. Hanley. Neural and developmental actions of
lithium: A unifying hypophesis. Cell 59: 411-419, 1989.
Berrios, G.E., and R. Porter. A History of Clinical Psychiatry. New York: New York
University Press, 1995.
Bertler, A. Effect of reserpine on the storage of catechol amines in brain and other tis-
sues. Acta Physiol. Scand. 51: 75-83, 1961.
Bertler, A., A.M. Rosengren, and E. Rosengren. In vivo uptake of dopamine and 5-
hydroxytryptamine by adrenal medullary granules. Experientia 16: 418-419, 1960.
Bertler, A., and E. Rosengren. Occurrence and distribution of catechol amines in brain.
Acta Physiol. Scand, 47: 350-361, 1959.
Besson, M.J., A. Cheramy, P. Feltz, and J. Glowinski. Release of newly synthesized
dopamine from dopamine-containing terminals in the striatum of the rat. Proc.
Natl Acad. Sci. USA 62: 741-748, 1969.
Bethe, A. Uber die Neurofibrillen und die Ganglienzellen von Wirbeltieren. Arch. Mikr.
Anat. 55: 513-558, 1900.
Birkmayer, W., J. Knoll, P. Riederer, M.B.H. Youdim, V. Hars, and J. Marton. Increased
life expectancy resulting from addition of L-deprenyl to Madopar® treatment in
Parkinson's disease: A longterm study. /. Neural Trans. 64: 113-127, 1985.
Birks, R.I. Effects of stimulation on synaptic vesicles in sympathetic ganglia as shown
by fixation in the presence of Mg 2+ . /. Physiol. 216: 26P-28P, 1971.
Birks, R.I. The relationship of transmitter release and storage to fine structure in a sym-
pathetic ganglion. /. Neurocytol. 3: 133-160, 1974.
Birks, R., H.E. Huxley, and B. Katz. The fine structure of the neuromuscular junction
of the frog. /. Physiol 150: 134-144, 1960.
Birks, R.I., and F.C. Macintosh. Acetylcholine metabolism at nerve endings. Brit. Med.
Bull. 13: 157-161, 1957.
Birks, R., and F.C. Macintosh. Acetycholine metabolism of a sympathetic ganglion. Can.
J. Biochem. Physiol 39: 787-827, 1961.
Birman, S., F.M. Meunier, B. Lesbats, J.P. LeCaer, J. Rossier, and M. Israel. A 15 kDa
proteolipid found in mediatophore preparations from Torpedo electric organ pre-
sents high sequence homology with the bovine chromaffin granule protonophore.
FEBS Lett. 261: 303-306, 1990.
Birnbaumer, L., J. Abramowitz, A. Yatani, K. Okabe, R. Mattera, R. Graf, J. Sanford,

J. Codina, and A.M. Brown. Roles of G proteins in coupling of receptors to ionic
channels and other effector systems. Crit. Rev. Biochem. Mol. Biol. 25: 225-244,
1990.
Bishop, G.H. The relation of bioelectric potentials to cell function. Annu. Rev. Physiol.
3: 1-20, 1941.
Bishop, G.H., P. Heinbecker, and J.L. O'Leary. The function of the non-myelinated
fibers of the dorsal roots. Amer. ]. Physiol 106: 647-669, 1933.
Bixby, J.L. Protein kinase C is involved in laminin stimulation of neurite outgrowth.
Neuron 3: 287-297, 1989.
Bixby, J.L., R.S. Pratt, J. Lilien, and L.F. Reichardt. Neurite outgrowth on muscle cell
surfaces involves extracellular matrix receptors as well as Ca2+-dependent and
-independent cell adhesion molecules. Proc. Natl. Acad. Sci. USA 84: 2555-2559,
1987.
Bjorklund, A., B. Ehinger, and B. Falck. A method for differentiating dopamine from
noradrenaline in tissue sections by microspectrofluorometry. /. Histochem.
Cytochem. 16: 263-270, 1968.
Bjorklund, A., B. Falck, and U. Stenevi. On the possible existence of a new intraneu-
ronal monoamine in the spinal cord of the rat./. Pharmacol. Exp. Ther. 175: 525-
532, 1970.
Black, I.E., D.M. Chikaraishi, and E.J. Lewis. Trans-synaptic increase in RNA coding
for tyrosine hydroxylase in a rat sympathetic ganglion. Brain Res. 339: 151-153,
1985.
Black, J.W., W.A.M. Duncan, and R.G. Shanks. Comparison of some properties of
pronethalol and propranolol. Brit. J. Pharmacol. 25: 577-591, 1965.
Blackburn, K.J., P.C. French, and R.J. Merrills. 5-Hydroxytryptamine uptake by rat brain
in vitro. Life Sci. 6: 1653-1663, 1967.
Blackstad, T.W. Mapping of experimental axon degeneration by electron microscopy of
Golgi preparations. Z. Zellforsch. 67: 819-834, 1965.
Blackwell, B., E. Marley, J. Price, and D. Taylor. Hypertensive interactions between
monoamine oxidase inhibitors and foodstuffs. Brit. J. Psychiat. 113: 349-365, 1967.
Blakeley, R.D., H.E. Berson, R.T. Fremeau, M.G. Caron, M.M. Peck, H.K. Prince, and
C.C. Bradley. Cloning and expression of a functional serotonin transporter from
rat brain. Nature 354: 66-70, 1991.
Blaschko, H. The specific action of l-dopa decarboxylase./. Physiol. 96: 50p-51p, 1939.
Blaschko, H. The activity of l(-)-DOPA decarboxylase./. Physiol. 101: 337-349, 1942.
Blaschko, H. Substrate specificity of amino-acid decarboxylases. Biochim. Biophys. Acta
4: 130-137, 1950.
Blaschko, H. Amine oxidase and amine metabolism. Pharmacol. Rev. 4: 415-458, 1952.
Blaschko, H. Metabolism and storage of biogenic amines. Experientia 13: 9-12, 1957.
Blaschko, H. Introduction and historical background. Adv. Biochem. Psychopharmacol.
5: 1-10, 1972.
Blaschko, H., G.V.R. Born, A. DTorio, and N.R. Eade. Observations on the distribu-
tion of catechol amines and adenosinetriphosphate in the bovine adrenal medulla.
/. Physiol. 133: 548-557, 1956.
Blaschko, H., R.S. Comline, F.H. Schneider, M. Silver, and A.D. Smith. Secretion of a
chromaffin granule protein, chromogranin, from the adrenal gland after splanch-
nic stimulation. Nature 215: 58-59, 1967.
Blaschko, H., P. Hagen, J.M. Hagen, and H.J. Schumann. Observations on the intra-
cellular distribution of pressor amines in the chromaffine cell. Arch. Int. Pharma-
codyn. 110: 128-130, 1957.
References 367
Blaschko, H., D. Richter, and H. Schlossmann. The inactivation of adrenaline./. Phys-

iol. 90: 1-17, 1937a.
Blaschko, H., D. Richter, and H. Schlossmann. The oxidation of adrenaline and other
amines. Biochem. J. 31: 2187-2196, 1937b.
Blaschko, H., and A.D. Welch. Localization of adrenaline in cytoplasmic particles of the
bovine adrenal medulla. Naunyn-Schmiedebergs Arch. 219: 17-22, 1953.
Blaschko, H., P. Hagen, and A.D. Welch. Observations on the intracellular granules of
the adrenal medulla. /. Physiol. 129: 27-49, 1955.
Blaustein, M.P., and A.L. Hodgkin. The effect of cyanide on the efflux of calcium from
squid axons. /. Physiol. 200: 497-527, 1969.
Blioch, Z.L., I.M. Glagoleva, E.A. Liberman, and V.A. Nenashev. A study of the mech-
anism of quantal release at a chemical synapse. J. Physiol. 199: 11-35, 1968.
Bliss, T.V.P., and A.R. Gardner-Medwin. Long-lasting potentiation of synaptic trans-
mission in the dentate area of the unanaesthetized rabbit following stimulation of
the perforant path. /. Physiol. 232: 357-374, 1973.
Bliss, T.V.P., and T. L0mo. Long-lasting potentiation of synaptic transmission in the den-
tate area of the anaesthetized rabbit following stimulation of the perforant path.
/. Physiol. 232: 331-356, 1973.
Bloch, K. Blondes in Venetian Paintings, the Nine-Banded Armadillo, and Other Essays
in Biochemistry. New Haven: Yale University Press, 1994.
Bloom, F.E., E. Costa, and G.C. Salmoiraghi. Anesthesia and responsiveness of indi-
vidual neurons of the caudate nucleus of the cat to acetylcholine, norepinephrine
and dopamine administered by microelectrophoresis./. Pharmacol. Exp. Ther. 150:
244-252, 1965.
Bloom, F.E., A.P. Oliver, and G.C. Salmoiraghi. The responsiveness of individual hypo-
thalamic neurons to microelectrophoretically administered endogenous amines.
Int. ]. Neuropharmacol. 2: 181-193, 1963.
Bloom, F.E., T. Ueda, E. Battenberg, and P. Greengard. Immunocytochemical local-
ization, in synapses, of protein I, an endogenous substrate for protein kinases in
mammalian brain. Proc. Natl. Acad. Sci. USA 76: 5982-5986, 1979.
Bockaert, J., C. Roy, and S. Jard. Oxytocin-sensitive adenylate cyclase in frog bladder
epithelial cells. /. Biol. Chem. 247: 7073-7081, 1972.
Bodian, D. The structure of the vertebrate synapse. J. Comp. Neurol. 68: 117-160,
1937.
Bodian, D. Cytological aspects of synaptic function. Physiol. Rev. 22: 146-169, 1942.
Boeke, J. Nerve endings, motor and sensory. In: Penfield, W. (ed.), Cytology & Cellu-
lar Pathology of the Nervous System, Vol. 1, New York: Hafner Publishing Co.,
1965. p. 243-315.
Bogdanski, D.F., and B.B. Brodie. Role of sodium and potassium ions in storage of nor-
epinephrine by sympathetic nerve endings. Life Sci. 5: 1563-1569, 1966.
Bogdanski, D.F., and B.B. Brodie. The effects of inorganic ions on the storage and
uptake of H3-norepinephrine by rat heart slices. J. Pharmacol. Exp. Ther. 165: 181-
189, 1969.
Bogdanski, D. F., A. Tissari, and B.B. Brodie. Role of sodium, potassium, ouabain and
reserpine in uptake, storage and metabolism of biogenic amines in synaptosomes.
Life Sci. 7: 419-428, 1968.
Bogdanski, D.F., H. Weissbach, and S. Udenfriend. The distribution of serotonin,
5-hydroxytryptophan decarboxylase, and monoamine oxidase in brain. /. Neuro-
chem. 1: 272-278, 1957.
Boistel, J., and P, Fatt. Membrane permeability change during inhibitory transmitter
action in crustacean muscle. /. Physiol. 144: 176-191, 1958.
Bokoch, G.M., T. Katada, J.K. Northup, E.L. Hewlett, and A.G. Oilman. Identification
of the predominant substrate for ADP-ribosylation by islet activating protein.
/. Biol. Chem. 258: 2072-2075, 1983.
Bokoch, G.M., T. Katada, J.K. Northup, M. Ui, and A.G. Gilman. Purification and prop-
erties of the inhibitory guanine nucleotide-binding regulatory component of adeny-
late cyclase. /. Biol. Chem. 259: 3560-3567, 1984.
Boman, B. L-Tryptophan: A rational anti-depressant and a natural hypnotic? Austral.
NZJ. Psychiat. 22: 83-97, 1988.
Bonhoeffer, F., and J. Huf. Recognition of cell types by axonal growth cones in vitro.
Nature 288: 162-164, 1980.
Bonner, D.M. Control Mechanisms. New York: Ronald Press, 1961.
Bonner, T.I., N.J. Buckley, A.C. Young, and M.R. Brann. Identification of a family of
muscarinic acetylcholine receptor genes. Science 237: 527-532, 1987.
Bonner, T.I., A.C. Young, M.R. Brann, and N.J. Buckley. Cloning and expression of the
human and rat m5 muscarinic acetylcholine receptor genes. Neuron 1: 403-410,
1988.
Boulter, J., K. Evans, D. Goldman, G. Martin, D. Treco, S. Heinemann, and J. Patrick.
Isolation of a cDNA clone coding for a possible neural nicotinic acetylcholine recep-
tor a-subunit. Nature 319: 368-374, 1986.
Bowers, M.B. Central dopamine turnover in schizophrenic syndromes. Arch. Gen. Psy-
chiat. 31: 50-54, 1974.
Boyd, I.A., and A.R. Martin. Spontaneous subthreshold activity at mammalian neuro-
muscular junctions. /. Physiol. 132: 61-73, 1956.
Bradley, K., D.M. Easton, and J.C. Eccles. An investigation of primary or direct inhi-
bition. /. Physiol. 122: 474-488, 1953.
Bradley, P.B., and J.H. Wolstencroft. Excitation and inhibition of brain-stem neurones
by noradrenaline and acetylcholine. Nature 196: 840, 873, 1962.
Bray, D. Model for membrane movements in the neural growth cone. Nature 244:
93-96, 1973.
Bray, D., and J.G. White. Cortical flow in animal cells. Science 239: 883-888, 1988.
Brazier, M.A.B. The historical development of neurophysiology. In: Field, J., H.W.
Magoun, and V.E. Hall (eds.) Handbook of Physiology, (Section 1), Neurophysiol-
ogy. Vol. 1. Washington, DC: American Physiological Society, 1959, pp. 1-58.
Breckenridge, L.J., and W. Aimers. Final steps in exocytosis observed in a cell with giant
secretory granules. Proc. Natl. Acad. Sci. USA 84: 1945-1949, 1987.
Breitwieser, G.E., and G. Szabo. Uncoupling of cardiac muscarinic and /3-adrenergic
receptors from ion channels by a guanine nucleotide analogue. Nature 317: 538-
540, 1985.
Bridgman, PC., and M.E. Dailey. The organization of myosin and actin in rapid frozen
growth cones. /. Cell Biol. 108: 95-109, 1989.
Brinkman, R., and M. Ruiter. Die humorale Ubertragung der neurogenen Skelett-
muskelerregung auf den Darrn. Pfliigers Arch. 204: 766-768, 1924.
Brinkman, R., and E. van Dam. Die chemische Ubertragbarkeit der Nervenreizwirkung.
Pflugers Arch. 196: 66-82, 1922.
Brisson, A., and P.N.T. Unwin. Tubular crystals of acetycholine receptor. /. Cell Biol.
99: 1202-1211, 1984.
Broch, O.J., and F. Fonnum. The regional and subcellular distribution of catechol-
O-methyltransferase in the rat brain. J. Neurochem. 19: 2049-2055, 1972.
Brock, L.G., J.S. Coombs, and J.C. Eccles. The recording of potentials from motoneu-
rones with an intracellular electrode. J. Physiol. 117: 431-460, 1952.
References 369
Brooks, C.M., and J.C. Eccles. An electrical hypothesis of central inhibition. Nature
159: 760-764, 1947.
Brown, G.L. Action potentials of normal mammalian muscle. Effects of acetylcholine
and eserine. J. Physiol. 89: 220-237, 1937.
Brown, G.L., H.H. Dale, and W. Feldberg. Reactions of the normal mammalian mus-
cle to acetylcholine and to eserine. J. Physiol. 87: 394-424, 1936.
Brown, G.L., and J.C. Eccles. The action of a single vagal volley on the rhythm of the
heart beat. J. Physiol. 82: 211-241, 1934a.
Brown, G.L., and J.C. Eccles. Further experiments on vagal inhibition of the heart beat.
/. Physiol. 82: 242-257, 1934b.
Brown, G.L., and W. Feldberg. The acetylcholine metabolism of a sympathetic gan-
glion. J. Physiol. 88: 265-283, 1936a.
Brown, G.L., and W. Feldberg. The action of potassium on the superior cervical gan-
glion of the cat. J. Physiol. 86: 290-305, 1936b.
Brown, G.L., and J.S. Gillespie. The output of sympathetic transmitter from the spleen
of the cat. /. Physiol. 138: 81-102, 1957.
Brown, G.L., and F.C. Macintosh. Discharges in nerve fibres produced by potassium
ions. /. Physiol. 96: 10P-11P, 1939.
Brown, L. The release and fate of the transmitter liberated by adrenergic nerves. Proc.
Roy. Soc. 162B: 1-19, 1965.
Brunelli, M., V. Castellucci, and E.R. Kandel. Synaptic facilitation and behavioral sen-
sitization in Aplysia: Possible role of serotonin and cyclic AMP. Science 194:
1178-1181, 1976.
Brunton, T.L. On the nature of inhibition, and the action of drugs upon it. Nature 27:
419-422, 1883.
Buchtal, E, and J. Lindhard. Transmission of impulses from nerve to muscle fibre. Acta
Physiol. Scand. 4: 136-148, 1942.
Buckley, K.M., E. Floor, and R.B. Kelly. Cloning and sequence analysis of cDNA encod-
ing p38, a major synaptic vesicle protein. /. Cell Biol 105: 2447-2456, 1987.
Bueker, E.D. Implantations of tumors in the hind limb field of the embryonic chick
and the developmental response of the lumbosacral nervous system. Anat. Rec.
102: 369-385, 1948.
Bulbring, E., and J.H. Burn. Observations bearing on synaptic transmission by acetyl-
choline in the spinal cord. /. Physiol. 100: 337-368, 1941.
Bunney, W.E., and J.M. Davis. Norepinephrine in depressive reactions. Arch. Gen. Psy-
chiat. 13: 483-494, 1965.
Bunzow, J.R., H.H.M. Van Tol, D.K. Grandy, P. Albert, J. Salon, M. Christie, C.A.
Machida, K.A. Neve, and O. Civelli. Cloning and expression of a rat D£ dopamine
receptor cDNA. Nature 336: 783-787, 1988.
Burack, W.R., P.R. Draskoczy, and N. Weiner. Adenine nucleotide, catecholamine and
protein contents of whole adrenal glands and heavy granules of reserpine-treated
fowl. J. Pharmacol. Exp. Ther. 133: 25-33, 1961.
Burden, S.J., P.B. Sargent, and U.J. McMahan. Acetylcholine receptors in regenerating
muscle accumulate at original synaptic site in the absence of the nerve. /. Cell Biol.
82: 412-425, 1979.
Burgen, A.S.V. The mechanism of action of anticholinesterase drugs. Brit. J. Pharma-
col. 4: 219-228, 1949.
Burn, J.H. The action of tyramine and ephedrine./. Pharmacol. Exp. Ther. 46: 75-95,1932.
Burn, J.H., and J. Robinson. Effect of denervation on amine oxidase in structures inner-
vated by the sympathetic nerves. Brit. J. Pharmacol. 7: 304—318, 1952.
370 MECHANISMS OP SYNAPTIC TRANSMISSION
Burns, R.S., C.C. Chiueh, S.P Markey, M.H. Ebert, D.M. Jacobowitz, and I.J. Kopin.
A primate model of parkmsonism: Selective destruction of the dopaminergic neu-
rons in the pars compacta of the substantia nigra by N-methyl-4-phenyl-1,2,3,6-
tetrahydropyridine. Proc. Natl. Acad. Sci. USA 80: 4546-4550, 1983.
Burton, P.R., and W.L. Kirkland. Actin detected in mouse neuroblastoma cells by bind-
ing of heavy meromyosin. Nature New Biol 239: 244—246, 1972.
Biischer, H.H., R.C. Hill, D. Romer, F. Cardinaux, A. Closse, D. Hauser, and J. Pless.
Evidence for analgesic activity of enkephalin in the mouse. Nature 261: 423-425,
1976.
Butcher, R.W., and E.W. Sutherland. Adenosine 3', 5'-phosphate in biological materi-
als. /. Biol. Chem. 237: 1244-1250, 1962.
Byers, D., R.L. Davis, and J.A. Kiger. Defect in cyclic AMP phosphodiesterase due to
the dunce mutation of learning in Drosophila melanogaster. Nature 289: 79-81,
1981.
Bylund, D.B., and S.H. Snyder. Beta adrenergic receptor binding in membrane prepa-
rations from mammalian brain. Mol. Pharmacol. 12: 568-580, 1976.
Byrne, W.L., D. Samuel, E.L. Bennett, M.R. Rosenzweig, E. Wasserman, A.R. Wag-
ner, F. Gardner, R. Galambos, B.D. Berger, D.L. Margules, R.L. Fenichel, L. Stein,
J.A. Corson, H. Enesco, S.L. Chorover, C.E. Holt, PH. Schiller, L. Chiappetta,
M.G. Jarvik, R.C. Leaf, J.D. Dutcher, Z.P. Horovitz, and PL. Carlson. Memory
transfer. Science 153: 658-659, 1966.
Caceres, A., M.R. Payne, L.I. Binder, and O. Steward. Immunocytochemical localiza-
tion of actin and microtubule-associated protein MAP2 in dendritic spines. Proc.
Natl. Acad. Sci. USA 80: 1738-1742, 1983.
Cade, J.F.J. Lithium salts in the treatment of psychotic excitement. Med. J. Aust. 36(11):
349-352, 1949.
Caldwell, A.E. Origins of Psychopharmacology. Springfield, 111: Charles C Thomas,
1970.
Calma, I., and S. Wright. Action of acetylcholine, atropine and eserine on the central
nervous system of the decerebrate cat. /. Physiol. 103: 93-102, 1944.
Came, D.B., P.F. Teychenno, L.E. Claveria, R. Eastman, J.K. Greenacre, and A. Petrie.
Bromocriptine in parkinsonism. Brit. Med. J. 4: 442-444, 1974.
Campbell, A.K. Intracellular Calcium. Chichester: John Wiley, 1983.
Campenot, R.B. Local control of neurite development by nerve growth factor. Proc.
Natl. Acad. Sci. USA 74: 4516-4519, 1977.
Cannon, D.F. Explorer of the Human Brain. New York: Henry Schuman, 1949.
Cannon, W.B. The story of the development of our ideas of chemical mediation of nerve
impulses. Amer. J. Med. Sci. 188: 145-159, 1934.
Cannon, W.B., and Z.M. Bacq. Studies on the conditions of activity in endocrine organs.
XXVI. Amer. J. Physiol 96: 392-412, 1931.
Cannon, W.B., and A. Rosenblueth. Studies on conditions of activity in endocrine organs.
XXIX. Amer. }. Physiol. 104: 557-574, 1933.
Cannon, W.B., and J.E. Uridil. Studies on the conditions of activity in endocrine glands.
Amer. J. Physiol. 58: 353-364, 1921.
Cantoni, G.L. S-adenosylmethionine; a new intermediate formed enzymatically from
L-methionine and adenosinetriphosphate. /. Biol. Chem. 204: 403-416, 1953.
Cantoni, G.L., and G. Eastman. On the response of the intestine to smooth muscle
stimulants. /. Pharmacol. Exp. Ther. 87: 392-399, 1946.
Carafoli, E., R. Tiozzo, G. Lugly, F. Crovetti, and C. Kratzing. The release of calcium
from heart mitochondria by sodium. J. Mol. Cell. Cardiol. 6: 361-371, 1974.
References 371
Carew, T.J., R.D. Hawkins, T.W. Abrams, and E.R. Kandel. A test of Hebb's postulate
at identified synapses which mediate classical conditioning in Aplysia. J. Neurosci.
4: 1217-1224, 1984.
Carew, T.J., H.M. Pinsker, and E.R. Kandel. Long-term habituation of a defensive with-
drawal reflex in Aplysia. Science 175: 451-454, 1972.
Carew, T.J., E.T. Walters, and E.R. Kandel. Classical conditioning in a simple with-
drawal reflex in Aplysia californica. J. Neurosci. 1: 1426-1437, 1981.
Carlsson, A. The occurrence, distribution and physiological role of catecholamines in
the nervous system. Pharmacol. Rev. 11: 490-493, 1959.
Carlsson, A., B. Falck, and N.A. Hillarp. Cellular localization of brain monoamines. Acta
Physiol. Scand. 56: supplement 196, 1962.
Carlsson, A., and N.-A. Hillarp. Release of adenosine triphosphate along with adrena-
line and noradrenaline following stimulation of the adrenal medulla. Acta Physiol.
Scand. 37: 235-239, 1956.
Carlsson, A., N-A. Hillarp, and B. Waldeck. Analysis of the Mg ++ -ATP dependent stor-
age mechanism in the amine granules of the adrenal medulla. Acta Physiol. Scand.
Suppl. 215: 1-38, 1963.
Carlsson, A., and M. Lindqvist. Effect of chlorpromazine or haloperidol on formation
of 3-methoxytyramine and normetanephrine in mouse brain. Acta Pharmacol. Tox-
icol 20: 140-144, 1963.
Carlsson, A., M. Lindqvist, and T. Magnusson. 3,4-Dihydroxyphenylalanine and 5-
hydroxytryptophan as reserpine antagonists. Nature 180: 1200, 1957.
Carlsson, A., M. Lindqvist, T. Magnusson, and B. Waldeck. On the presence of 3-hydrox-
ytyramine in brain. Science 127: 471, 1958.
Carlsson, A., and D.T. Wong. A note on the discovery of selective serotonin reuptake
inhibitors. Life Set. 61: 1203, 1997.
Caron, M.G., and R.J. Lefkowitz. Solubilization and characterization of the /3-adrener-
gic receptor binding sites of frog erythrocytes. /. Biol. Chem. 251:2374-2389,1976.
Carr, C., D. McCourt, and J.B. Cohen. The 43-kilodalton protein of Torpedo nicotinic
postsynaptic membranes: Purification and determination of primary structure. Bio-
chemistry 26: 7090-7102, 1987.
Cartaud, J., E.L. Benedetti, A. Sobel, and J.-P. Changeux. A morphological study of the
cholinergic receptor protein. /. Cell Sci. 29: 313-337, 1978.
Cartwright, N. How the Laws of Physics Lie. Oxford: Oxford University Press, 1983.
Caspars, H., and P. Stern. Die Wirkung von Substanz P auf das Dendritenpotential und
die Gleichspannungskomponente des Neocortex. Pfliigers Arch. 273: 94-110,1961.
Casey, R.P., D. Njus, G.K. Radda, and PA. Sehr. Active proton uptake by chromaffin
granules: Observation by amine distribution and Phosphorus-31 nuclear magnetic
resonance techniques. Biochemistry 16: 972-977, 1977.
Cash, C.D., P. Vayer, P. Mandel, and M. Maitre. Tryptophan 5-hydroxylase. Eur. J.
Biochem. 149: 239-245, 1985.
Cassel, D. and T. Pfeuffer. Mechanism of cholera toxin action: Covalent modification
of the guanyl nucleotide-binding protein of the adenylate cyclase system. Proc.
Natl. Acad. Sci. USA 75: 2669-2673, 1978.
Cassel, D., and Z. Selinger. Catecholamine-stimulated GTPase activity in turkey ery-
throcyte membranes. Biochim. Biophys. Acta 452: 538-551, 1976.
Cassel, D., and Z. Selinger. Mechanism of adenylate cyclase activation through the
/3-adrenergic receptor: Catecholamine-induced displacement of bound GDP by
GTP. Proc. Natl. Acad. Sci. USA 75: 4155-4159, 1978.
Castellucci, V.F., and E.R. Kandel. A quantal analysis of the synaptic depression under-
lying habituation of the gill-withdrawal reflex in Aplysia. Proc. Natl. Acad. Sci. USA
71: 5004-5008, 1974.
Castellucci, V, and E.R. Kandel. Presynaptic facilitation as a mechanism for behavioral
sensitization in Aplysia. Science 194: 1176-1178, 1976.
Castellucci, V.F., E.R. Kandel, J.H. Schwartz, F.D. Wilson, A.C. Nairn, and P. Green-
gard. Intracellular injection of the catalytic subunit of cyclic AMP-dependent pro-
tein kinase stimulates facilitation of transmitter release underlying behavioral sen-
sitization in Aplysia. Proc. Natl. Acad. Sci. USA 77: 7492-7496, 1980.
Castellucci, V.F., A. Nairn, P. Greengard, J.H. Schwartz, and E.R. Kandel. Inhibitor of
adenosine 3':5'-monophosphate-dependent protein kinase blocks presynaptic facil-
itation in Aplysia. J. Neurosci. 2: 1673-1681, 1982.
Castellucci, V.F., H. Pinsker, I. Kupfermann, and E.R. Kandel. Neuronal mechanisms
of habituation and dishabituation of the gill-withdrawal reflex in Aplysia. Science
167: 1745-1748, 1970.
Ceccarelli, B., W.P. Hurlbut, and A. Mauro. Depletion of vesicles from frog neuro-
muscular junctions by prolonged stimulation. J. Cell Biol. 54: 30-38, 1972.
Ceccarelli, B., W.P. Hurlbut, and A. Mauro. Turnover of transmitter and synaptic vesi-
cles at the frog neuromuscular junction. /. Cell Biol. 57: 499-524, 1973.
Cerione, R.A., J.W. Regan, H. Nakata, J. Codina, J.L. Benovic, P. Gierschik, R.L. Somers,
A.M. Spiegel, L. Birnbaumer, R.J. Lefkowitz, and M.G. Caron. Functional recon-
stitution of the o^-adrenergic receptor with guanine nucleotide regulatory proteins
in phospholipid vesicles./. Biol. Chem. 261: 3901-3909, 1986.
Cerione, R.A., B. Strulovici, J.L. Benovic, R.J. Lefkowitz, and M.G. Caron. Pure
/3-adrenergic receptor: The single polypeptide confers catecholamine responsive-
ness to adenylate cyclase. Nature 306: 562-566, 1983.
Chagas, C. Studies on the mechanism of curarization. Ann. N.Y. Acad. Sci. 81: 345-357,
1959.
Chagas, C. The fate of curare during curarization. Ciba Fdn. Study Grp. 12: 2-10, 1962.
Chagas, C., A. Couceiro, and H. Martins-Ferreira. Resultats de quelque experiences de
perfusion de 1'organe electrique d'Electrophorus electricus L. C.R. Soc. Biol. 145:
247-251, 1951.
Chan-Palay, V, and S.L. Palay. High voltage electron microscopy of rapid Golgi prepa-
rations. Z. Anat. Enttoickl.-Gesch. 137: 125-152, 1972.
Chandler, D.E., and J.E. Heuser. Arrest of membrane fusion events in mast cells by
quick-freezing. /. Cell Biol. 86: 666-674, 1980.
Chang, C.C., and C.Y. Lee. Isolation of neurotoxins from the venom of Bungarus mul-
ticinctus and their modes of neuromuscular blocking action. Arch. Int. Pharmaco-
dyn. 144: 241-257, 1963.
Chang, C.M., and R.D. Goldman. The localization of actin-like fibers in cultured neu-
roblastoma cells as revealed by heavy meromyosin binding./. Cell Biol. 57: 867-874,
1973.
Chang, H.C., and J.H. Gaddum. Choline esters in tissue extracts./. Physiol. 79: 255-285,
1933.
Chang, H.-C., W.-M. Hsieh, T.-H. Li, and R.K.S. Lim. Humoral transmission of nerve
impulses at central synapses. Chinese J. Physiol. 13: 153-166, 1938.
Chang, M.M., and S.E. Leeman. Isolation of a sialogogic peptide from bovine hypo-
thalamic tissue and its characterization as substance P. /. Biol. Chem. 245:
4784-^790, 1970.
Chang, M.M., S.E. Leeman, and H.D. Niall. Amino-acid sequence of substance P.
Nature Netv Biol. 232: 86-87, 1971.
References 373
Changeux, J.-R, T. Heidmann, J.L. Popot, and A. Sobel. Reconstitution of a functional

acetylcholine regulator under defined conditions. FEES Lett. 105: 181-187, 1979.
Changeux, J.-R, M. Kasai, M. Huchet, and J.C. Meunier. Extraction a partir du tissue
electrique de gymnote d'une proteine presantant plusieurs proprietes caracteris-
tiques du recepteur physiologique de 1'acetylcholine. C.R. Acad. Set. 270 D:
2864-2867, 1970a.
Changeux, J.-R, M. Kasai, and C.Y. Lee. Use of a snake venom toxin to characterize the
cholinergic receptor protein. Proc. Natl. Acad. Sci. USA 67: 1241-1247, 1970b.
Changeux, J.-R, J.C. Meunier, and M. Huchet. Studies on the cholinergic receptor pro-
tein of Electrophoms electricus. Mol. Pharmacol. 7: 538-553, 1971.
Changeux, J.-R, and T.R. Podleski. On the excitability and cooperativity of the electro-
plax membrane. Proc. Natl. Acad. Sci. USA 59: 944-950, 1968.
Changeux, J.-R, J. Thiery, Y. Tung, and C. Kittel. On the cooperativity of biological
membranes. Proc. Natl. Acad. Sci. USA 57: 335-341, 1967.
Charnay, Y., L. Leger, F. Dray, A. Berod, M. Jouvet, J.F. Pujol, and P.M. Dubois. Evi-
dence for the presence of enkephalin in catecholaminergic neurones of cat locus
coeruleus. Neurosci. Lett. 30: 147-151, 1982.
Charnet, P., C. Labarca, R.J. Leonard, N.J. Vogelaar, L. Czyzyk, A. Gouin, N. David-
son, and H.A. Lester. An open-channel blocker interacts with adjacent turns of
a-helices in the nicotinic acetylcholine receptor. Neuron 4: 87-95, 1990.
Charney, D.S., D.B. Menkes, and G.R. Heninger. Receptor sensitivity and the mecha-
nism of action on antidepressant treatment. Arch. Gen. Psychiat. 38: 1160-1180,
1981.
Chen, C.N., S. Denome, and R.L. Davis. Molecular analysis of cDNA clones and the
corresponding genomic coding sequences of the Drosophila dunce+ gene, the
structural gene for cAMP phosphodiesterase. Proc. Natl. Acad. Sci. USA 83: 9313-
9317, 1986.
Cheung, W.Y. Cyclic 3', 5'-nucleotide phosphodiesterase. Demonstration of an activa-
tor. Biochem. Biophys. Res. Commun. 38: 533-539, 1970.
Cheung, W. Y. Discovery and recognition of calmodulin: A personal account. /. Cyclic
Nuckotide Res. 7: 71-84, 1981.
Cheung, W.Y., T.J. Lynch, and R.W. Wallace. An endogenous Ca2+-dependent activa-
tor protein of brain adenylate cyclase and nucleotide phosphodiesterase. Adv.
Cyclic Nucleotide Res. 9: 233-251, 1978.
Chiba, K., A. Trevor, and N. Castagnoli. Metabolism of the neurotoxic tertiary amine,
MPTP, by brain monoamine oxidase. Biochem. Biophys. Res. Commun. 120: 574-
578, 1984.
Childs, A.F., D.R. Davies, A.L. Green, and J.P. Rutland. The reactivation by oximes and
hydroxamic acids of cholinesterase inhibited by organo-phosphorus compounds.
Brit. J. Pharmacol 10: 462-465, 1955.
Chothia, C. Interaction of acetylcholine with different cholinergic nerve receptors.
Nature 225: 36-38, 1970.
Chothia, C., and P. Pauling. Molecular models. Nature 229: 281, 1971.
Chuang, D.M., and E. Costa. Evidence for internalization of the recognition site of
j8-adrenergic receptors during receptor subsensitivity induced by ( — )-isoproterenol.
Chute, A.L., W. Feldberg, and D.H. Smyth. Liberation of acetylcholine from the per-
fused cat's brain. Quart. J. Exp. Physiol. 30: 65-72, 1940.
Ciani, S., and C. Edwards. The effect of acetylcholine on neuromuscular transmission
in the frog. /. Pharmacol. Exp. Ther. 142: 21-23, 1963.
Clark, A.J. The reaction between acetyl choline and muscle cells./. Physiol. 61: 530-546,
1926a.
Clark, A.J. The antagonism of acetyl choline by atropine./. Physiol. 61: 547-556, 1926b.
Clark, A.J. General pharmacology. In: Heubner, W., and J. Schiiller (eds.) Handbuch
der Experimentellen Pharmakologie. Viertel Band. Berlin: Verlag Springer, 1937.
Clark, C.T., H. Weissbach, and S. Udenfriend. 5-Hydroxytryptophan decarboxylase:
Preparation and properties. J. Biol. Chem. 210: 139-148, 1954.
Clarke, A.E., and J.H. Fujimura. What tools? Which job? Why right? In: Clarke, A.E.,
and J. H. Fujimura (eds.) The Right Tools for the Job. Princeton: Princeton Uni-
versity Press, 1992. pp. 3^4.
Clarke, E., and L.S. Jacyna. Nineteenth-Century Origins of Neuroscientific Concepts.
Berkeley: University of California Press, 1987.
Clarke, E., and C.D. O'Malley. The Human Brain and Spinal Cord. Berkeley: Univer-
sity of California Press, 1968.
Clarke, P.G.H., and W.M. Cowan. The development of the isthmo-optic tract in the
chick, with special reference to the occurrence and correction of developmental
errors in the location and connections of isthmo-optic neurons. /. Comp. Neural.
167: 143-164, 1976.
Claudio, T., M. Ballivet, J. Patrick, and S. Heinemann. Nucleotide and deduced amino
acid sequences of Torpedo californica acetylcholine receptor y subunit. Proc. Natl.
Acad. Sci. USA 80: 1111-1115, 1983.
Clement-Cormier, Y.C., J.W. Kebabian, G.L. Petzold, and P. Greengard. Dopamine-
sensitive adenylate cyclase in mammalian brain: A possible site of action of antipsy-
chotic drugs. Proc. Natl. Acad. Sci. USA 71: 1113-1117, 1974.
Cleugh, J., J.H. Gaddam, A.A. Mitchell, M.W. Smith, and V.P. Whittaker. Substance P
in brain extracts. /. Physiol. 170: 69-85, 1964.
Cockcroft, S., and B.D. Gomperts. Role of guanine nucleotide binding protein in the
activation of polyphosphoinositide phosphodiesterase. Nature 314: 534-536, 1985.
Cohen, I.S., and W. Van der Kloot. Effects of low temperature and terminal membrane
potential on quantal size at frog neuromuscular junction. J. Physiol. 336: 335-344,
1983.
Cohen, Lord, of Birkenhead. Sherrington: Physiologist, Philosopher, Poet. Liverpool:
Liverpool University Press, 1958.
Cohen, S. Purification of a nerve-growth promoting protein from the mouse salivary
gland and its neuro-cytotoxic antiserum. Proc. Natl. Acad. Sci. USA 46: 302-311,
1960.
Cohen, S., R. Levi-Montalcini, and V. Hamburger. A nerve growth-stimulating factor
isolated from sarcomas 37 and 180. Proc. Natl. Acad. Sci. USA 40: 1014-1018,
1954.
Cohen, S., and R. Levi-Montalcini. A nerve growth-stimulating factor isolated from
snake venom. Proc. Natl. Acad. Sci. USA 42: 571-574, 1956.
Colbran, R.J., and T.R. Soderling. Calcium/calmodulin-dependent protein kinase II.
Curr. Topics Cell. Reg. 31: 181-221, 1990.
Cole, A.J., D.W. Saffen, J.M. Buraban, and P.F. Worley. Rapid increase of an immedi-
ate early gene messenger RNA in hippocampal neurons by synaptic NMDA recep-
tor activation. Nature 340: 474-476, 1989.
Cole, J.O. Therapeutic efficacy of antidepressant drugs. J. Amer. Med. Assoc. 190:
448-455, 1964.
Cole, K.S., and H.J. Curtis. Electric impedence of the squid giant axon during activity.
/. Gen. Physiol. 22: 649-670, 1939.
References 375
Cole, K.S., and A.L. Hodgkin. Membrane and protoplasm resistance in the squid giant
axon. /. Gen. Physiol. 22: 671-687, 1939.
Collier, B. The preferential release of newly synthesized transmitter by a sympathetic
ganglion. /. Physiol. 205: 341-352, 1969.
Collier, B., and J.F. Mitchell. The central release of acetylcholine during stimulation of
the visual pathway. /. Physiol. 184: 239-254, 1966.
Collier, B., and J.F. Mitchell. The central release of acetylcholine during consciousness
and after brain lesions. /. Physiol. 188: 83-98, 1967.
Collingridge, G.L., S.J. Kehl, and H. McLennan. Excitatory amino acids in synaptic
transmission in the Schaffer collateral-commissural path of the rat hippocampus.
/. Physiol. 334: 33-46, 1983a.
Collingridge, G.L., S.J. Kehl, and H. McLennan. The antagonism of amino acid-induced
excitations of rat hippocampal CA1 neurons in vitro.]. Physiol. 334: 19-31, 1983b.
Collins, H., and T. Pinch. The Golem, second edition. Cambridge: Cambridge Univer-
sity Press, 1994.
Collins, J.H., J.D. Potter, M.J. Horn, G. Wilshire, and N. Jackman. The amino acid
sequence of a rabbit skeletal muscle troponin C. FEES Lett. 36: 268-272, 1973.
Collins, S., M. Bouvier, M.A. Bolanowski, M.G. Caron, and R.J. Lefkowitz. cAMP stim-
ulates transcription of the /32-adrenergic receptor gene in response to short-term
agonist exposure. Proc. Natl. Acad. Sci. USA 86: 4853^857, 1989.
Colquhoun, D. The relation between classical and cooperative models for drug action.
In: Rang, H.P. (ed.) Drug Receptors. Baltimore: University Park Press, 1973. pp.
149-182.
Colquhoun, D., V.E. Dionne, J.H. Steinbach, and C.F. Stevens. Conductance of chan-
nels opened by acetylcholine-like drugs in muscle end-plate. Nature 253: 204-206,
1975.
Comb, M., N.C. Birnberg, A. Seasholtz, E. Herbert, and H.M. Goodman. A cyclic AMP-
and phorbol ester-inducible DNA element. Nature 323: 353-356, 1986.
Comb, M., P.H. Seeburg, J. Adelman, L. Eiden, and E. Herbert. Primary structure of
the human Met- and Leu-enkephalin precursor and its mRNA. Nature 295:
663-666, 1982.
Connell, P.H. Amphetamine Psychosis (Maudsley Monographs No. 5). Oxford: Oxford
University Press, 1958.
Connor, J.D., G.V. Rossi, and W.W. Baker. Antagonism of intra-caudate carbachol tremor
by local injections of catecholamines./. Pharmacol. Exp. Then 155: 545-551, 1967.
Cook, W.H., D. Lipkin, and R. Markham. The formation of a cyclic dianhydrodiadenylic
acid (I) by the alkaline degradation of adenosine-5'-triphosphoric acid (II)./. Amer.
Chem. Soc. 79: 3607-3608, 1957.
Coombs, J.S., J.C. Eccles, and P. Fatt. The specific ionic conductances and ionic move-
ments across the motoneuronal membrane that produce the inhibitory post-synap-
tic potential. /. Physiol. 130: 326-373, 1955a.
Coombs, J.S., J.C. Eccles, and P. Fatt. Excitatory synaptic action in motoneurones.
/. Physiol. 130: 374-395, 1955b.
Cooper, J.R., F.E. Bloom, and R.H. Roth. The Biochemical Basis of Neuropharmacol-
ogy. New York: Oxford University Press, 1996.
Coppen, A. The biochemistry of affective disorders. Brit.]. Psychiat. 113:1237-1264,1967.
Coppen, A., R. Noguera, J. Bailey, B.H. Burns, M.S. Swani, E.H. Hare, R. Gardner,
and R. Maggs. Prophylactic lithium in affective disorders. Lancet 2: 275-279, 1971.
Corning, W.C., and E.R. John. Effect of ribonuclease on retention of conditioned
response in regenerated planarians. Science 134: 1363-1364, 1961.
Corrodi, H., K. Fuxe, T. Hokfelt, P. Lidbrink, and U. Ungerstedt. Effect of ergot drugs
on central catecholamine neurons: Evidence for a stimulation of central dopamine
neurons. /. Pharm. Pharmacol. 25: 409-412, 1973.
Corrodi, H., and G. Jonsson. The formaldehyde fluorescence method for the histo-
chemical demonstration of biogenic monoamines. J. Histochem. Cytochem. 15:
65-78, 1967.
Costantin, L.L., C. Franzini-Armstrong, and R.J. Podolsky. Localization of calcium-accu-
mulating structures in striated muscle fibers. Science 147: 158-160, 1965.
Cotecchia, S., B.K. Kobilka, K.W. Daniel, R.D. Nolan, E.Y. Lapetina, M.G. Caron, R.J.
Lefkowitz, and J.W. Regan. Multiple second messenger pathways of a-adrenergic
receptor subtypes expressed in eukaryotic cells. J. Biol. Chem. 265: 63-69, 1990.
Cotecchia, S., D.A. Schwinn, R.R. Randall, R.J. Lefkowitz, M.G. Caron, and B.K.
Kobilka. Molecular cloning and expression of the cDNA for the hamster ai-adren-
ergic receptor. Proc. Natl. Acad. Sci USA 85: 7159-7163, 1988.
Cotzias, G.C., P. Papavasiliou, C. Fehling, B. Kaufman, and I. Mena. Similarities
between neurologic effects of L-dopa and of apomorphine. New Eng. /. Med. 282:
31-33, 1970.
Cotzias, G.C., P.S. Papavasiliou, and R. Gellene. Modification of parkinsonism—chronic
treatment with L-dopa. New Eng. J. Med. 280: 337-345, 1969.
Cotzias, G.C., M.H. Van Woert, and L.M. Schiffer. Aromatic amino acids and modifi-
cation of parkinsonism. New Eng. J. Med. 276: 374-379, 1967.
Couteaux, R., and M. P^cot-Dechavassine. Vesicules synaptique et poches au niveau
des "zones actives" de la jonction neuromusculaire. C.R. Acad. Sci. Paris 271D:
2346-2349, 1970.
Cowan, W.M., J.W. Fawcett, D.D.M. O'Leary, and B.B. Stanfield. Regressive events in
neurogenesis. Science 225: 1258-1265, 1984.
Cowan, W.M., T.M. Jessel, and S.L. Zipursky (eds.) Molecular and Cellular Approaches
to Neural Development. New York: Oxford University Press, 1997.
Cox, E.G., B. Miiller, and F. Bonhoeffer. Axonal guidance in the chick visual system:
Posterior tectal membranes induce collapse of growth cones from the temporal
retina. Neuron 2: 31-37, 1990.
Coyle, J.T., and S.H. Snyder. Catecholamine uptake by synaptosomes in homogenates
of rat brain: Stereospecificity in different areas. J. Pharmacol. Exp. Ther. 170:
221-231, 1969.
Cozzens, S.E. Social Control and Multiple Discovery in Science: The Opiate Receptor
Case. Albany: State University of New York Press, 1989.
Crane, G.E. Iproniazid (Marsilid®) phosphate, a therapeutic agent for mental disorders
and debilitating diseases. Psychiat. Res. Rpts. 8: 142-154, 1957.
Crawford, J.M., and D.R. Curtis. Pharmacological studies on feline Betz cells./. Phys-
iol. 186: 121-138, 1966.
Creed, R.S., D. Denny-Brown, J.C. Eccles, E.G.T. Liddell, and C.S. Sherrington. Reflex
Activity of the Spinal Cord. London: Oxford University Press, 1932.
Creese, I., D.R. Burt, and S.H. Snyder. Dopamine receptor binding predicts clinical
and pharmacological potencies of antischizophrenic drugs. Science 192: 481-483,
1976.
Creese, I., and D.R. Sibley. Receptor adaptations to centrally acting drugs. Annu. Rev.
Pharmacol. Toxicol. 21: 357-391, 1981.
Crick, F. Do dendritic spines twitch? Trends Neurosci. 5: 44-A6, 1982.
Grout, J.R. Effect of inhibiting both catechol-O-methyl transferase and monoamine oxi-
dase on cardiovascular responses to norepinephrine. Proc. Soc. Exp. Biol. Med.
108: 482^84, 1961.
References 377
Crow, T.J., and D.L. Alkon. Retention of an associative behavioral change in Hermis-
senda. Science 201: 1239-1241, 1978.
Crowther, R.A., J.T. Finch, and B.M.F. Pearse. On the structure of coated vesicles.
/. Mol. Biol 103: 785-798, 1976.
Crowther, R.A., and B.M.F. Pearse. Assembly and packing of clathrin into coats./. Cell
Biol. 91: 790-797, 1981.
Cuatrecasas, P. Affinity chromatography and purification of the insulin receptor of liver
cell membranes. Proc. Natl. Acad. Sci. USA 69: 1277-1281, 1972.
Cuatrecasas, P., G.P.E. Tell, V. Sica, I. Parikh, and K.J. Chang. Noradrenaline binding
and the search for catecholamine receptors. Nature 247: 92-97, 1974.
Curtis, D.R., A.W. Duggan, D. Felix, and G.A.R. Johnston. GABA, bicuculline and cen-
tral inhibition. Nature 226: 1222-1224, 1970.
Curtis, D.R., A.W. Duggan, D. Felix, G.A.R. Johnston, and H. McLennan. Antagonism
between bicuculline and GABA in the cat brain. Brain Res. 33: 57-73, 1971.
Curtis, D.R., A.W. Duggan, D. Felix, G.A.R. Johnston, A.K. Tebecis, and J.C. Watkins.
Excitation of mammalian central neurones by acidic amino acids. Brain Res. 41:
283-301, 1972.
Curtis, D.R., and R.M. Eccles. The excitation of Renshaw cells by pharmacological
agents applied electrophoretically./. Physiol. 141: 435-445, 1958a.
Curtis, D.R., and R.M. Eccles. The effect of diffusional barriers upon the pharmacol-
ogy of cells within the central nervous system. /. Physiol. 141: 446-463, 1958b.
Curtis, D.R., L. Hosli, and G.A.R. Johnston. Inhibition of spinal neurones by glycine.
Nature 215: 1502-1503, 1967.
Curtis, D.R., L. Hosli, G.A.R. Johnston, and I.H. Johnston. The hyperpolarization of
spinal motoneurones by glycine and related amino acids. Exp. Brain Res. 5: 235-
258, 1968.
Curtis, D.R., J.W. Phillis, and J.C. Watkins. The depression of spinal neurones by
y-amino-n-butyric acid and /3-alanine. /. Physiol. 146: 185-203, 1959.
Curtis, D.R., J.W. Phillis, and J.C. Watkins. The chemical excitation of spinal neurones
by certain acidic amino acids. /. Physiol. 150: 656-682, 1960.
Curtis, D.R., J.W. Phillis, and J.C. Watkins. Cholinergic and non-cholinergic transmis-
sion in the mammalian spinal cord. /. Physiol 158: 296-323, 1961.
Curtis, D.R., and J.C. Watkins. The excitation and depression of spinal neurones by
structurally related amino acids./. Neurochem. 6: 117-141, 1960.
Cutler, R.W.P., J.P. Hammerstad, L.R. Cornick, and J.E. Murray. Efflux of amino acid
neurotransmitters from rat spinal cord slices. Brain Res. 35: 337-355, 1971.
Dahlstrom, A., and K. Fuxe. Evidence for the existence of monoamine-containing neu-
rons in the central nervous system. Acta Physiol. Scand. Suppl. 232: 5-55, 1964.
Dakin, H.D. The synthesis of a substance allied to adrenalin. Proc. R. Soc. 76B: 491-497,
1905.
Dale, H. Chemical transmission of the effects of nerve impulses. Brit. Med. ]. 1: 835-841,
1934.
Dale, H. Transmission of nervous effects by acetylcholine, Harvey Lect. 32: 229-245,
1937.
Dale, H. Acetylcholine as a chemical transmitter of the effects of nerve impulses. I.
/. Mt. Sinai Hosp. 4: 401-415, 1938a.
Dale, H. Acetycholine as a chemical transmitter of the effects of nerve impulses. II.
/. Mt. Sinai Hosp. 4: 416-429, 1938b.
Dale, H. Accident and opportunism in medical research. Brit. Med. J. 2: 451-455, 1948.
Dale, H. Autobiographical sketch. Persp. Biol. Med. 1: 125-137, 1958.
Dale, H.H. On some physiological actions of ergot./. Physiol. 34: 163-206, 1906.
Dale, H.H. The action of certain esters and ethers of choline and their relation to mus-
carine. J. Pharmacol. Exp. Ther. 6: 147-190, 1914.
Dale, H.H. Nomenclature of fibres in the autonomic system and their effects./. Phys-
iol. 80: 10P-11P, 1933.
Dale, H.H. Thomas Renton Elliott. Biogr. Mem. Fellows R. Soc. 7: 53-74, 1961.
Dale, H.H. Adventures in Pharmacology. London: The Wellcome Trust, 1965.
Dale, H.H., and H.W. Dudley. The presence of histamine and acetylcholine in the spleen
of the ox and the horse. /. Physiol. 68: 97-123, 1929.
Dale, H.H., and W. Feldberg. The chemical transmission of secretory impulses to the
sweat glands of the cat. /. Physiol. 82: 121-128, 1934.
Dale, H.H., W. Feldberg, and M. Vogt. Release of acetylcholine at voluntary motor
nerve endings, /. Physiol 86: 353-380, 1936.
Dale, H.H., and J.H. Gaddum. Reactions of denervated voluntary muscle, and their
bearing on the mode of action of parasympathetic and related nerves. J. Physiol.
70: 109-144, 1930.
Dash, P.K., B. Hochner, and E.R. Kandel. Injection of the cAMP-responsive element
into the nucleus of Aplysia sensory neurons blocks long-term facilitation. Nature
345: 718-721, 1990.
Davenport, H.W. Epinephrin(e). The Physiologist 25: 76-82, 1982.
Davenport, H.W. Early history of the concept of chemical transmission of the nerve
impulse. The Physiologist 34: 129, 178-190, 1991.
Davidoff, R.A., R.P. Shank, L.T. Graham, M.H. Aprison, and R. Werman. Is glycine a
neurotransmitter? Nature 214: 680-681, 1967.
Davis, J.M., S.H. Koslow, R.D. Gibbons, J.W. Maas, C.L. Bowden, R. Casper, I. Hanin,
J.I. Javaid, S.S. Chang, and P.E. Stokes. Cerebrospinal fluid and urinary biogenic
amines in depressed patients and healthy controls. Arch. Gen. Psychiat. 45: 705-
717, 1988.
Davis, G.C., A.C. Williams, S.P. Markey, M.H. Ebert, E.D. Caine, C.M. Reichert, and
I.J. Kopin. Chronic parkinsonism secondary to intravenous injection of meperidine
analogues. Psychiatry Res. 1: 249-254, 1979.
DeBelleroche, J.S., and H.F. Bradford. Amino acids in synaptic vesicles from mam-
malian cerebral cortex: A reappraisal. /. Neurochem. 21: 441-451, 1973.
DeBlasi, A., M. Lipartiti, H.J. Motulsky, P.A. Insel, and M. Fratelli. Agonist-induced
redistribution of /3-adrenergic receptors on intact human mononuclear leukocyte
redistributed receptors are nonfunctional. J. Clin. Endocrinol. Metab. 61: 1081-
1088, 1985.
DeCamilli, P., R. Cameron, and P. Greengard. Synapsin I (protein I), a nerve termi-
nal-specific phosphoprotein. I. /. Cell Biol. 96: 1337-1354, 1983a.
DeCamilli, P., S.M. Harris, W.B. Huttner, and P. Greengard. Synapsin I (protein I), a
nerve terminal-specific phosphoprotein. II. /. Cell Biol. 96: 1355-1373, 1983b.
DeCamilli, P., and R. Jahn. Pathways to regulated exocytosis in neurons. Annu. Rev.
Physiol. 52: 625-645, 1990.
Dedman, J.R., R.L. Jackson, WE. Schreiber, and A.R. Means. Sequence homology of
the Ca2+-dependent regulator of cyclic nucleotide phosphodiesterase from rat testis
with other Ca2+-binding proteins. /. Biol. Chern. 253: 343-346, 1978.
De Duve, C. Footnote. In: de Reuck, A.V.S., and M.P. Cameron (eds.) Lysosomes.
Boston: Little, Brown, 1963, p. 126.
De la Baune, S., C. Gros, C.C. Yi, P. Chaillet, H. Marcais-Collado, J. Costentin, and J.C.
Schwartz. Selective participation of both "enkephalinase" and aminopeptidase activ-
ities in the metabolism of endogenous enkephalins. Life Sci. 31: 1753-1756, 1982.
References 379
DeLange, R.J., R.G. Kemp, W.D. Riley, R.A. Cooper, and E.G. Krebs. Activation of
skeletal muscle phosphorylase kinase by adenosine triphosphate and adenosine
3',5'-monophosphate./. Bio/. Chem. 243: 2200-2208, 1968.
Delbarre, B., and H. Schmitt. A further attempt to characterize sedative receptors acti-
vated by clonidine in chickens and mice. Eur. J. Pharmacol. 22: 355-359, 1973.
del Castillo, J., and B. Katz. The effect of magnesium on the activity of motor nerve
endings. /. Physiol. 124: 553-559, 1954a.
del Castillo, J., and B. Katz. Quantal components of the end-plate potential. /. Physiol.
124: 560-573, 1954b.
del Castillo, J., and B. Katz. The membrane change produced by the neuromuscular
transmitter. J. Physiol. 125: 546-565, 1954c.
del Castillo, J., and B. Katz. On the localization of acetylcholine receptors. J. Physiol.
128: 157-181, 1955a.
del Castillo, J., and B. Katz. Local activity at a depolarized nerve-muscle junction.
/. Physiol. 128: 396-411, 1955b.
del Castillo, J., and B. Katz. La base "quantale" de la transmission neuro-musculaire.
In: Microphysiologie Comparee des Elements Excitables. Paris: Centre National de
la Recherche Scientifique, 1957, pp. 245-254.
del Castillo, J., and L. Stark. The effect of calcium ions on the motor end-plate poten-
tials./. Physiol. 116: 507-515, 1952.
DeLean, A., A.A. Hancock, and R.J. Lefkowitz. Validation and statistical analysis of
a computer modeling method for quantitative analysis of radioligand binding
data for mixtures of pharmacological receptor subtypes. Mol. Pharmacol. 21: 5-16,
1982.
Demis, D.J., H. Blaschko, and A.D. Welch. The conversion of dihydroxyphenylalanine-
2-C14 (dopa) to norepinephrine by bovine adrenal medullary homogenates./. Phar-
macol. Exp. Ther. 113: 14-15, 1955.
Demis, D.J., H. Blaschko, and A.D. Welch. The conversion of dihydroxyphenylalanine-
2-C14 (DOPA) to norepinephrine by bovine adrenal medullary homogenates.
/. Pharmacol. Exp. Ther. 117: 208-212, 1956.
Dengler, H.J., I.A. Michaelson, H.E. Spiegel, and E. Titus. The uptake of labeled nor-
epinephrine by isolated brain and other tissues of the cat. Int. J. Neuropharmacol.
1: 23-38, 1962.
Dengler, H.J., H.E. Spiegel, and E.O. Titus. Uptake of tritium-labeled norepinephrine
in brain and other tissues of cat in vitro. Science 133: 1072-1073, 1961.
Dengler, H.J., and E.O. Titus. The effect of drugs on the uptake of isotopic norepi-
nephrine in various tissues. Biochem. Pharmacol. 8: 64, 1961.
Denny-Brown, D. The Sherrington school of physiology. J. Neurophysiol. 20: 543-548,
1957.
DeRiemer, S.A., J.A. Strong, K.A. Albert, P. Greengard, and L.K. Kaczmarek. Enhance-
ment of calcium current in Aplysia neurones by phorbol ester and protein kinase
C. Nature 313: 313-316, 1985.
Derkach, V., A. Surprenant, and R.A. North. 5-HTa receptors are membrane ion chan-
nels. Nature 339: 706-709, 1989.
DeRobertis, E. Submicroscopic changes of the synapse after nerve section in the
acoustic ganglion of the guinea pig./. Biophys. Biochem. Cytol. 2: 503-512, 1956.
DeRobertis, E. Submicroscopic morphology and function of the synapse. Exp. Cell Res.
Suppl. 5: 347-369, 1958.
DeRobertis, E.D.P., and H.S. Bennett. Submicroscopic vesicular component in the
synapse. Fed. Proc. 13: 35, 1954.
DeRobertis, E.D.P., and H.S. Bennett. Some features of the submicroscopic morphol-
ogy of synapses in frog and earthworm. J. Biophys. Biochem. Cytol. 1: 47—57, 1955.
DeRobertis, E., A. Pellegrino de Iraldi, G. Rodriguez, and C.J. Gomez. On the isola-
tion of nerve endings and synaptic vesicles./. Biophys. Biochem. Cytol. 9: 229-235,
1961.
DeRobertis, E., A. Pellegrino de Iraldi, G. Rodriguez de Lores Arnaiz, and L. Salgan-
icoff. Cholinergic and non-cholinergic nerve endings in rat brain./. Neurochem. 9:
23-35, 1962.
DeRobertis, E., G. Rodriguez de Lores Arnaiz, L. Salganicoff, A. Pellegrino de Iraldi,
and L.M. Zieher. Isolation of synaptic vesicles and structural organization of the
acetylcholine system within brain nerve endings./. Neurochem. 10: 225-235, 1963.
Detweiler, S.R. On the hyperplasia of nerve centers resulting from excessive peripheral
loading. Proc. Natl. Acad. Sci. USA 6: 96-101, 1920.
Detweiler, S.R. Further experiments upon alteration of the direction of growth in
amphibian spinal nerves. /. Exp. Zoo/. 51: 1-35, 1928.
Devault, A., C. Lazure, C. Nault, H. LeMoual, N.G. Seidah, M. Chretien, P. Kahn,
J. Powell, J. Mallet, A. Beaumont, B.P. Roques, P. Crine, and G. Boileau. Amino
acid sequence of rabbit kidney neutral endopeptidase 24.11 (enkephalinase)
deduced from complementary DNA. EMBO J. 6: 1317-1322, 1987.
Devillers-Thiery, A., J. Giraudat, M. Bentaboulet, and J.-P. Changeux. Complete mRNA
coding sequence of the acetylcholine binding a-subunit of Torpedo marmorata
acetylcholine receptor. Proc. Natl. Acad. Sci. USA 80: 2067-2071, 1983.
Diagnostic and Statistical Manual of Mental Disorders. Washington, DC: American Psy-
chiatric Association, third edition, 1980.
Diamond, J., and R. Miledi. A study of foetal and new-born rat muscle fibres./. Phys-
iol. 162: 393-408, 1962.
Dikshit, B.B. Action of acetylcholine on the brain and its occurrence therein, /. Phys-
iol. 80: 409-421, 1934a.
Dikshit, B.B. The production of cardiac irregularities by excitation of the hypothalamic
centres./. Physiol. 81: 382-394, 1934b.
Dingledine, R. N-Methyl aspartate activates voltage-dependent calcium conductance in
rat hippocampal pyramidal cells. /. Physiol 343: 385-^05, 1983.
Dingman, W., and M.B. Sporn. The incorporation of 8-azaguanine into rat brain RNA
and its effect on maze-learning by the rat: An inquiry into the biochemical basis
of memory./. Psychiat. Res. 1: 1-11, 1961.
Dingman, W., and M.B. Sporn. Molecular theories of memory. Science 144: 26-29,1964.
Dixon, R.A.F., B.K. Kobilka, D.J. Strader, J.L. Benovic, H.G. Dohlman, T. Frielle, M.A.
Bolanowski, C.D. Bennett, E. Rands, R.E. Diehl, R.A. Mumford, E.E. Slater, I.S.
Sigal, M.G. Caron, R.J. Lefkowitz, and C.D. Strader. Cloning of the gene and
cDNA for mammalian /3-adrenergic receptor and homology with rhodopsin. Nature
321: 75-79, 1986.
Dixon, W.E. Vagus inhibition. Brit. Med. J. 2: 1807, 1906.
Dixon, W.E. On the mode of action of drugs. Medical Magazine 16: 454-457, 1907.
Dixon, WE., and T.G. Brodie. Contributions to the physiology of the lungs./. Physiol.
29: 97-173, 1903.
Dixon, WE., and P. Hamill. The mode of action of specific substances with special ref-
erence to secretin. /. Physiol. 38: 314-336, 1909.
Dodge, R. Theories of inhibition. Psychol Rev. 33: 106-122, 167-187, 1926.
Doherty, P., C.H. Barton, G. Dickson, P. Seaton, L.H. Rowett, S.E. Moore, H.J. Gower,
and F.S. Walsh. Neuronal process outgrowth of human sensory neurons on mono-
References 381
layers of cells transfected with cDNAs for five human N-CAM isoforms. J. Cell
Biol 109: 789-798, 1989.
Dohlman, H.G., M.G. Caron, C.D. Strader, N. Amlaiky, and R.J. Lefkowitz. Identifi-
cation and sequence of a binding site peptide of the /3£-adrenergic receptor. Bio-
chemistry 27: 1813-1817, 1988.
Donovan, A., L. Laudan, and R. Laudan (eds.) Scrutinizing Science. Baltimore: Johns
Hopkins University Press, 1992.
Douglas, W.W., J. Nagasawa, and R. Schulz. Electron microscopic studies on the mech-
anism of secretion of posterior pituitary hormones and significance of microvesi-
cles ('synaptic vesicles'). Mem. Soc. Endocrinol. 19: 353-377, 1970.
Douglas, W.W., J. Nagasawa, and R.A. Schulz. Coated microvesicles in neurosecretory
terminals of posterior pituitary glands shed their coats to become smooth "synap-
tic" vesicles. Nature 232: 340-341, 1971.
Douglas, W.W., and A.M. Poisner. On the mode of action of acetylcholine in evoking
adrenal medullary secretion: Increased uptake of calcium during the secretory
response. /. Physiol 162: 385-392, 1962.
Douglas, W.W., and A.M. Poisner. The influence of calcium on the secretory response
of the submaxillary gland to acetylcholine or to noradrenaline. /. Physiol. 165:
528-^541, 1963.
Douglas, W.W., and A.M. Poisner. Calcium movement in the neurohypophysis of the
rat and its relation to the release of vasopressin. J. Physiol. 172: 19-30, 1964.
Douglas, W.W., A.M. Poisner, and R.P. Rubin. Efflux of adenine nucleotides from per-
fused adrenal glands exposed to nicotine and other chromaffin cell stimulants.
/. Physiol 179: 130-137, 1965.
Douglas, W.W., and R.P. Rubin. The role of calcium in the secretory response of the
adrenal medulla to acetylcholine. J. Physiol. 159: 40-57, 1961.
Doxey, J.C., C.F.C. Smith, and J.M. Walker. Selectivity of blocking agents for pre- and
postsynaptic a-adrenoceptors. Brit. ]. Pharmacol. 60: 91-96, 1977.
Dubocovich, M.L., and S.Z. Langer. Negative feed-back regulation of noradrenaline
release by nerve stimulation in the perfused cat's spleen. J. Physiol. 237: 505-519,
1974.
du Bois-Reymond, E. Gesammelte Abhandlungen zur allgemeinen Muskel- und Ner-
venphysik. Vol.2. Leipzig: Veit, 1877.
Dudai, Y., Y.N. Jan, D. Byers, W.G. Quinn, and S. Benzer. dunce, a mutant of Drosophila
deficient in learning. Proc. Natl. Acad. Sci. USA 73: 1684-1688, 1976.
Dudai, Y., A. Uzzan, and S. Zvi. Abnormal activity of adenylate cyclase in the Drosophila
memory mutant rutabaga. Neurosci. Lett. 42: 207-212, 1983.
Dudel, J., and S.W. Kuffler. Presynaptic inhibition at the crayfish neuromuscular junc-
tion./. Physiol. 155: 543-562, 1961.
Duerr, J.S., and W.G. Quinn. Three Drosophila mutations that block associative learn-
ing also affect habituation and sensitization. Proc. Natl. Acad. Sci. USA 79: 3646-
3650, 1982.
Duggan, A.W., and G.A.R. Johnston. Glutamate and related amino acids in cat spinal
roots, dorsal root ganglia and peripheral nerves. /. Neurochem. 17: 1205-1208,
1970.
Dumuis, A., M. Sebben, L. Haynes, J.P. Pin, and J. Bockaert. NMDA receptors activate
the arachidonic acid cascade system in striatal neurons. Nature 336: 68-70, 1988.
Dunant, Y., J. Gautron, M. Israel, B. Lesbats, and R. Manaranche. Les compartiments
d'acetylcholine de 1'organe electrique de la torpille et leur modifications par la stim-
ulation. /. Neurochem. 19: 1987-2002, 1972.
Dunant, Y., and M. Israel. When the vesicular hypothesis is no longer the vesicular
hypothesis. Trends Neurosci. 2: 130-132, 1979.
Dunlap, C.B. Dementia praecox. Amer. J. Psychiat. 3: 403-421, 1924.
Durell, J., J.T. Garland, and R.O. Friedel. Acetylcholine action: Biochemical aspects.
Science 165: 862-866, 1969.
Durell, J., M.A. Sodd, and R.O. Friedel. Acetylcholine stimulation of phosphodiester-
atic cleavage of guinea pig brain phosphoinositides. Life Sci. 7: 363-368, 1968.
Ebashi, S., F. Ebashi, and A. Kodama. Troponin as the Ca++-receptive protein in the
contractile system. J. Biochem. 62: 137-138, 1967.
Ebashi, S., and A. Kodama. A new protein factor promoting aggregation of tropomyosin.
/. Biochem. 58: 107-108, 1965.
Ebashi, S., and F. Lipmann. Adenosine triphosphate-linked concentration of calcium
ions in a particulate fraction of rabbit muscle. J. Cell Biol 14: 389-400, 1962.
Eccles, J. The nature of central inhibition. Proc. R. Soc. 153B: 445-476, 1961.
Eccles, J. From electrical to chemical transmission in the central nervous system. Notes
Rec. R. Soc. 30: 219-230, 1976.
Eccles, J.C. The discharge of impulses from ganglion cells.J. Physiol. 91: 1-22, 1937a.
Eccles, J.C. Synaptic and neuro-muscular transmission. Physiol. Rev. 17: 538-555,
1937b.
Eccles, J.C. The nature of synaptic transmission in a sympathetic ganglion. J. Physiol.
103: 27-54, 1944.
Eccles, J.C. An electrical hypothesis of synaptic and neuro-muscular transmission. Ann.
N.Y Acad. Sci. 47: 429-455, 1946.
Eccles, J.C. Conduction and synaptic transmission in the nervous system. Annu. Rev.
Physiol 10: 93-116, 1948.
Eccles, J.C. The Neurophysiological Basis of Mind. Oxford: Oxford University Press,
1953.
Eccles, J.C. The Physiology of Nerve Cells. Baltimore: Johns Hopkins University Press,
1957.
Eccles, J.C. The development of ideas on the synapse. In: Brooks, C.M., and P.F. Crane-
field, The Historical Development of Physiological Thought. New York: Hafner Pub-
lishing Co., 1959, pp. 39-66.
Eccles, J.C. The Physiology of Synapses. New York: Academic Press, 1964.
Eccles, J.C. Facing Reality. New York: Springer-Verlag, 1970.
Eccles, J.C. My scientific odyssey. Annu. Rev. Physiol. 39: 1-18, 1977.
Eccles, J.C., R.M. Eccles, and M. Ito. Effects produced on inhibitory postsynaptic poten-
tials by the coupled injections of cations and anions into motoneurons. Proc. R.
Soc. 160B: 197-210, 1964.
Eccles, J.C., R.M. Eccles, and F. Magni. Central inhibitory action attributable to presy-
n^ptic depolarization produced by muscle afferent volleys./. Physiol. 159:147-166,
1961.
Eccles, J.C., P. Fatt, and K. Koketsu. Cholinergic and inhibitory synapses in a pathway
from motor-axon collaterals to motoneurones. J. Physiol. 126: 524-562, 1954.
Eccles, J.C., P. Fatt, and S. Landgren. Central pathway for direct inhibitory action of
impulses in largest afferent nerve fibres to muscle. J. Neurophysiol. 19: 75-98,
1956.
Eccles, J.C., and W.C. Gibson. Sherrington: His Life and Thought. Berlin: Springer
International, 1979.
Eccles, J.C., B. Katz, and S.W. Kuffler. Nature of the "endplate potential" in curarized
muscle. J. Neurophysiol. 4: 362-387, 1941.
References 383
Eccles, J.C., B. Katz, and S.W. Kuffler. Effect of eserine on neuromuscular transmis-
sion./. Neurophysiol. 5: 211-230, 1942.
Eccles, J.C., P.G. Kostyuk, and R.F. Schmidt. Central pathways responsible for depo-
larization of primary afferent fibres./. Physiol. 161: 237-257, 1962.
Eccles, J.C., and A.W. Liley. Factors controlling the liberation of acetycholine at the
neuromuscular junction. Amer. J. Phys. Med. 38: 96-103, 1959.
Eccles, J.C., and W.V. MacFarlane. Actions of anti-cholinesterases on endplate poten-
tial of frog muscle./. Neurophysiol. 12: 59-80, 1949.
Eccles, J.C., and W.J. O'Connor. Responses which nerve impulses evoke in mammalian
striated muscles. J.Physiol. 97: 44-102, 1939.
Eccles, J.C., and J.J. Pritchard. The action potential of motoneurones. /. Physiol. 89:
43P-45P, 1935.
Edwards, C., V. Dolezal, S. Tucek, H. Zemkova, and F. Vyskocil. Is an acetylcholine
transport system responsible for nonquantal release of acetylcholine at the rodent
myoneural junction? Proc. Natl. Acad. Set. USA 82: 3514-3518, 1985.
Ehrenpreis, S., and M.G. Kellock. The interaction of quaternary ammonium compounds
with hyaluronic acid. Biochim. Biophys. Acta 45: 525-528, 1960.
Ehret, M., C.D. Cash, M. Hamon, and M. Maitre. Formal demonstration of the phos-
phorylation of rat brain tryptophan hydroxylase by Ca2+/calmodulin-dependent
protein kinase. /. Neurochem. 52: 1886-1891, 1989.
Eldefrawi, M.E., and A.T. Eldefrawi. Purification and molecular properties of the acetyl-
choline receptor from Torpedo electroplax. Arch. Biochem. Biophys. 159: 362-373,
1973.
Elizan, T.S., M.D. Yahr, D.A. Moros, M.R. Mendoza, S. Pang, and C.A. Bodian. Selegi-
line use to prevent progression of Parkinsons disease. Arch. Neurol. 46:1275-1279,
1989.
Elliott, K.A.C., and E. Florey. Factor I—inhibitory factor from brain. /. Neurochem. 1:
181-191, 1956.
Elliott, T.R. On the action of adrenalin./. Physiol. 31: xx-xxi, 1904.
Elliott, T.R. The action of adrenalin. /. Physiol. 32: 401-467, 1905.
Elliott, T.R. The adrenal glands. Brit. Med. J. 1: 1393-1397, 1914.
Ellis, J. Proteins as molecular chaperones. Nature 328: 378-379, 1987.
Ellis, R.E., and H.R. Horvitz. Genetic control of programmed cell death in the nema-
tode C. Elegans. Cell 44: 817-829, 1986.
Emmelin, N., and D. Jacobsohn. Some effects of acetylcholine, eserine and prostigmine
when injected into the hypothalamus. Acta Physiol. Scand. 9: 97-111, 1945.
Enero, M.A., S.Z. Langer, R.P. Rothlin, and F.J.E. Stefano. Role of the a-adrenocep-
tor in regulating noradrenaline overflow by nerve stimulation. Brit. J. Pharmacol.
44: 672-688, 1972.
Engelhart, E. Der humorale Wirkungsmechanismus der Oculomotoriusreizung.
Pflugers Arch. 227: 220-234, 1931.
Engelhart, E., and O. Loewi. Fermentative Azetylcholinspaltung im Blut und ihre Hem-
mung durch Physostigmin. Naunyn-Schmiedebergs Arch. 150: 1-13, 1930.
Erickson, J., L. Eiden, and B. Hoffman. Expression cloning of a reserpine-sensitive
vesicular monoamine transporter. Proc. Natl. Acad. Sci. USA 89: 10,993-10,997,
1992.
Erlanger, J. The initiation of impulses in axons. /. Neurophysiol. 2: 370-379, 1939.
Erlanger, J., H.S. Gasser, and G.H. Bishop. The compound nature of the action cur-
rent of nerve as disclosed by the cathode ray oscillograph. Amer. J. Physiol. 70:
624-666, 1924.
Ernst, A.M. Mode of action of apomorphine and dexamphetamine on gnawing com-

pulsion in rats. Psychopharmacologia 10: 316-323, 1967.
Erspamer, V. Pharmakologische Studien iiber Enteramin. I. Naunyn-Schmiedebergs
Archiv 196: 343-365, 1940a.
Erspamer, V. Pharmakologische Studien iiber Erteramin. II. Naunyn-Schmiedebergs
Archiv 196: 366-390, 1940b.
Erspamer, V. Pharmakologische Studien iiber Enteramin, III. Naunyn-Schmiedebergs
Archiv 196: 391-407, 1940c.
Erspamer, V. Pharmacological studies on enteramine (5-hydroxytryptamine). Arch. Int.
Pharmacodyn. 93: 293-316, 1953.
Erspamer, V, and B. Asero. Identification of enteramine, the specific hormone of the
enterochromaffin cell system, as 5-hydroxytryptamine. Nature 169: 800-801, 1952.
Ewins, A.J. Acetylcholine, a new active principle of ergot. Biochem. J. 8: 44-49, 1914.
Fahn, S., and L.J. Cote. Regional distribution of y-aminobutyric acid (GABA) in brain
of rhesus monkey. /. Neurochem. 15: 209-213, 1968.
Fain, J.N., and M.J. Berridge. Relationship between hormonal activation of phos-
phatidylinositol hydrolysis, fluid secretion and calcium flux in blowfly salivary gland.
Biochem. J. 178: 45-58, 1979.
Falbriard, J.G., T. Posternak, and E.W. Sutherland. Preparation of derivatives ofadeno-
sine 3',5'-phosphate. Biochim. Biophys. Acta 148: 99-105, 1967.
Falck, B. Observations on the possibilities of the cellular localization of monoamines by
a fluorescence method. Acta Physiol. Scand. 56: supplement 197, 1962.
Falck, B., N.-A. Hillarp, G. Thieme, and A. Torp. Fluorescence of catechol amines and
related compounds condensed with formaldehyde. /. Histochem. Cytochem. 10:
348-354, 1962.
Fallon, J.R., and C.E. Gelfman. Agrin-related molecules are concentrated at acetyl-
choline receptor clusters in normal and aneural developing muscle. /. Cell Biol.
108: 1527-1535, 1989.
Farde, L., F.A. Wiesel, H. Hall, C. Halldin, S. Stone-Elander, and G. Sedvall. No D2
receptor increase in PET study of schizophrenia. Arch. Gen. Psychiat. 44: 671-672,
1987.
Farde, L., F.A. Wiesel, A.L. Nordstrom, and G. Sedvall. Dl- and D2-dopamine recep-
tor occupancy during treatment with conventional and atypical neuroleptics. Psy-
chopharmacol. 99: suppl. S28-S31, 1989.
Fargin, A., J.R. Raymond, M.J. Lohse, B.K. Kobilka, M.G. Caron, and R.J. Lefkowitz.
The genomic clone G-21 which resembles a /3-adrenergic receptor sequence
encodes the 5-HTiA receptor. Nature 335: 358-360, 1988.
Fatt, P., and B.L. Ginsborg. The ionic requirements for the production of action poten-
tials in crustacean muscle fibres./. Physiol. 142: 516-543, 1958.
Fatt, P., and B. Katz. An analysis of the end-plate potential recorded with an intra-cel-
lular electrode./. Physiol. 115: 320-370, 1951.
Fatt, P., and B. Katz. The effect of sodium ions on neuromuscular transmission./. Phys-
iol. 118: 73-87, 1952a.
Fatt, P., and B. Katz. Spontaneous subthreshold activity at motor nerve endings./. Phys-
iol. 117: 109-128, 1952b.
Fatt, P., and B. Katz. Some problems of neuro-muscular transmission. Cold Spr. Harb.
Sym. Quant. Biol. 17: 275-280, 1952c.
Faucon Biguet, N., M. Buda, A. Lamouroux, D. Samolyk, and J. Mallet. Time course
of TH mRNA in rat brain and adrenal medulla after a single injection of reserpine.
EMBO]. 5:287-291, 1986.
Rererences 385
Fearing, F. Reflex Action. New York: Hafner Publishing Co., 1964.

Fehling, C. Treatment of Parkinsons syndrome with L-dopa. A double blind study. Arch.
Neurol. Scand. 42: 367-372, 1966.
Feigenbaum, P., and E.E. El-Fakahany. Regulation of muscarinic cholinergic receptor
density in neuroblastoma cells by brief exposure to agonists. J. Pharmacol. Exp.
Ther. 233: 134-140, 1985.
Feldberg, W. Die Empfindlichkeit der Zungenmuskulatur und der Zungengefasse des
Hundes auf Lingualisreizung und auf Acetylcholin. Pfliigers Arch. 232: 75-87,1933a.
Feldberg, W. Der Nachweis eines acetylcholinahnlichen Stoffes im Zungenvenenblut
des Hundes bei Reizung des Nervus lingualis. Pfliigers Arch. 232: 88-104, 1933b.
Feldberg, W. Present views on the mode of action of acetylcholine in the central nerv-
ous system. Physiol. Rev. 25: 596-642, 1945.
Feldberg, W. The early history of synaptic and neuromuscular transmission by acetyl-
choline: Reminiscences of an eye witness. In: Hodgkin, A.L. (ed.) The Pursuit of
Nature. Cambridge: Cambridge University Press, 1977. pp. 65-83.
Feldberg, W., and A. Fessard. The cholinergic nature of the nerves to the electric organ
of the Torpedo (Torpedo marmorata). ]. Physiol. 101: 200-216, 1942.
Feldberg, W., and J.H. Gaddum. The chemical transmitter at synapses in a sympathetic
ganglion. /. Physiol 81: 305-319, 1934.
Feldberg, W., and J.A. Guimarais. The liberation of acetylcholine by potassium. /. Phys-
iol 86: 306-314, 1936.
Feldberg, W., and O. Krayer. Das Auftreten eines azetylcholinartigen Stoffes im Harzve-
nunblut von Warmbliitern bei Reizung der Nervi vagi. Naunyn-Schmiedebergs
Arch. 172: 170-193, 1933.
Feldberg, W., and T. Mann. Properties and distribution of the enzyme system which
synthesizes acetylcholine in nervous tissue. /. Physiol. 104: 411-425, 1946.
Feldberg, W., and B. Minz. Das Auftreten eines acetylcholinartigen Stoffes im Neben-
nierenvenenblut bei Reizung der Nervei splanchnici. Pfliigers Arch. 233: 657-682,
1933.
Feldberg, W., B. Minz, and H. Tsudzimura. The mechanism of the nervous discharge
of adrenaline. /. Physiol. 81: 286-304, 1934.
Feldberg, W., and H. Schriever. The acetylcholine content of the cerebro-spinal fluid
of dogs. /. Physiol. 86: 277-284, 1936.
Feldberg, W., and A. Vartiainen. Further observations on the physiology and pharma-
cology of a sympathetic ganglion. /. Physiol. 83: 103-128, 1934.
Feng, T.P. Studies on the neuromuscular junction. XVIII. Chinese J. Physiol. 15: 367-
404, 1940.
Feng, T.P. Studies on the neuromuscular junction. XXVI. Chinese J. Physiol. 16: 341-
372, 1941.
Fernandez, J.M., E. Neher, and B.D. Gomperts. Capacitance measurements reveal step-
wise fusion events in degranulating mast cells. Nature 312: 453-455, 1984.
Ferraro, A. The connections of the pars suboculomotoria of the substantia nigra. Arch.
Neurol. Psychiat. 19: 177-179, 1928.
Fessard, A. Reflexions dans le prolongement d'une discussion sur les mecanismes synap-
tique. Arch. Int. Physiol. 59: 605-618, 1951.
Fick, G.R. Henry Dale's involvement in the verification and acceptance of the theory
of neurochemical transmission: A lady in hiding./. Hist. Med. All Sci. 42: 467-485,
1987.
Fine, R.E., and D. Bray. Action in growing nerves. Nature New Biol 234: 115-118,
1971.
Finger, S. Origins of Neuroscience. New York: Oxford University Press, 1994.

Finger, S. Minds Behind the Brain. Oxford: Oxford University Press, 2000.
Finkleman, B. On the nature of inhibition in the intestine./. Physiol 70: 145-157, 1930.
Fischer, J.F., and A.K. Cho. Chemical release of dopamine from striatal homogenates:
Evidence for an exchange difussion model. J. Pharmacol. Exp. Ther. 208: 203-209,
1979.
Fisher, D.B., and G.C. Mueller. An early alteration in the phospholipid metabolism of
lymphocytes by phytohemagglutinin. Proc. Natl. Acad. Set. USA 60: 1396-1402,
1968.
Flatmark, T., and O.C. Ingebretsen. ATP-dependent proton translocation in resealed
chromaffin granule ghosts. FEBS Lett. 78: 53-56, 1977.
Fletcher, W.M. John Newport Langley. In memoriam./. Physiol. 61: 1-27, 1926.
Fletcher, P., and T. Forrester. The effect of curare on the release of acetylcholine from
mammalian motor nerve terminals and an estimate of quantum content. /. Phys-
iol. 251: 131-144, 1975.
Flexner, J.B., L.B. Flexner, and E. Stellar. Memory in mice as affected by intracerebral
puromycin. Science 141: 57-59, 1963.
Florey, E. An inhibitory and an excitatory factor of mammalian central nervous system,
and their action on a single sensory neuron. Arch. Int. Physiol. 62: 33-53, 1954.
Florey, E. Comparative physiology: Transmitter substances. Annu. Rev. Physiol. 23:
501-528, 1961.
Florey, E.. and D.D. Chapman. The non-identity of the transmitter substance of crus-
tacean inhibitory neurons and gamma-aminobutyric acid. Comp. Biochem. Phys-
iol. 3: 92-98, 1961.
Florey, E., and H. McLennan. The release of an inhibitory substance from mammalian
brain, and its effect on peripheral synaptic transmission. J. Physiol. 129: 384-392,
1955.
Florio, V.A., and P.C. Sternweis. Reconstitution of resolved muscarinic cholinergic
receptors with purified GTP-binding proteins./. Biol. Chem. 260: 3477-3483,1985.
Fogelson, A.L., and R.S. Zucker. Presynaptic calcium diffusion from various arrays of
single channels. Biophys. J. 48: 1003-1017, 1985.
Folkow, B., J. Haggendal, and B. Lisander. Extent of release and elimination of nora-
drenaline at peripheral adrenergic nerve terminals. Acta Physiol. Scand. Suppl.
307: 1-38, 1967.
Forbes, A. Reflex inhibition of skeletal muscle. Quart.]. Exp. Physiol. 5: 149-187,1912.
Forbes, A., and C. Thacher. Amplification of action currents with the electron tube in
recording with the string galvanometer. Amer. J. Physiol. 52: 409-471, 1920.
Forscher, P., and S.J. Smith. Actions of cytochalasins on the organization of actin fila-
ments and microtubules in a neuronal growth cone. /. Cell Biol. 107: 1505-1516,
1988.
Forssman, J. Uber die Ursachen welche die Wachstumsrichtung der peripheren Ner-
venfasern bei der Regeneration bestimmen. Beitr. Pathol. Anat. 24: 56-100, 1898.
Foster, M. A Textbook of Physiology. Seventh ed. vol. 3. London: Macmillan, 1897.
Frail, D.E., J. Mudd, V. Shah, C. Carr, J.B. Cohen, and J.P. Merlie. cDNAs for the post-
synaptic 43 kDa protein of Torpedo electric organ encode two proteins with dif-
ferent carboxyl termini. Proc. Natl. Acad. Sci. USA 84: 6302-6306, 1987.
Frank, E., and G.D. Fischbach. Early events in neuromuscular junction formation in
vitro. /. Cell Biol 83: 143-158, 1979.
Frank, E., M. Nothmann, and H. Hirsch-Kaufmann. Uber die "tonische" Kontraktion
References 387
des quergestreiften saugetiermuskels nach Ausschaltung des motorischen Nerven.

Pfliigers Arch. 197: 270-287, 1922.
Frank, K. Basic mechanisms of synaptic transmission in the central nervous system.
IRE. Trans. Med. Electron. ME-6: 85-88, 1959.
Frank, K., and M.G.F. Fuortes. Presynaptic and postsynaptic inhibition of monosynap-
tic reflexes. Fed. Proc. 16: 39-40, 1957.
Frank, R.G. The Columbian exchange: American physiologists and neuroscience tech-
niques. Fed. Proceed. 42: 2665-2672, 1986.
Franklin, A. Avoiding the experimenters' regress. In: Koertge, N. (ed.) A House Built
on Sand. New York: Oxford University Press, 1998, pp. 151-165.
Franzini-Armstrong, C., and K.R. Porter. Sarcolemmal invaginations constituting the T
system in fish muscle fibers. /. Cell Biol 22: 675-696, 1964.
Frederickson, R.C.A. Endogenous opioids and related derivatives. In: Kuhar, M., and
G. Pasternak (eds.) Analgesics: Neurochemical, Behavioral, and Clinical Perspec-
tives. New York: Raven Press, 1984, pp. 9-68.
Frederickson, R.C.A., and F.H. Norris. Enkephalin-induced depression of single neu-
rons in brain areas wirh opiate receptors—antagonism by naloxone. Science 194:
440-442, 1976.
French, R.D. Some concepts of nerve structure and function in Britain, 1875-1885.
Med. Hist. 14: 154-165, 1970.
Freygang, W.H., D.A. Goldstein, D.C. Hellam, and L.D. Peachey. The relation between
the late after-potential and the size of the transverse tubular system of frog mus-
cle. /. Gen. Physiol. 48: 235-263, 1964.
Fricker, L.D., and S.H. Snyder. Enkephalin convertase: Purification and characteriza-
tion of a specific enkephalin-synthesizing carboxypeptidase localized to adrenal
chromaffin granules. Proc. Natl. Acad. Sci. USA 79: 3886-3890, 1982.
Friedman, D.L., and J. Lamer. Studies on UDPG-ct-glucan transglucosylase. III. Bio-
chemistry 2: 669-675, 1963.
Frielle, T, S. Collins, K.W. Daniel, M.G. Caron, R.J. Lefkowitz, and B.K. Kobilka.
Cloning of the cDNA for the human /3i-adrenergic receptor. Proc. Natl. Acad. Sci.
USA 84: 7920-7924, 1987.
Frielle, T., K.W. Daniel, M.G. Caron, and R.J. Lefkowitz. Structural basis of /3-adren-
ergic receptor subtype specificity studied with chimeric j3i//32-adrenergic recep-
tors. Proc. Natl. Acad. Sci. USA 85: 9494-9498, 1988.
Froehner, S.C., C.W. Luetje, P.B. Scotland, and J. Patrick. The post synaptic 43K pro-
tein clusters muscle nicotinic acetylcholine receptor in Xenopus oocytes. Neuron
5: 403-410, 1990.
Frohlich, A., and O. Loewi. Uber eine Steigerung der Adrenalinempfindlichkeit durch
Cocain. Naunyn-Schmiedebergs Arch. 62: 159-169, 1910.
Frohlich, A., and C.S. Sherrington. Path of impulses for inhibition under decerebrate
rigidity. /. Physiol. 28: 14-19, 1902.
Fromm-Reichman, F. Notes on the development of treatment of schizophrenics by psy-
choanalytic psychotherapy. Psychiatry 11: 263-273, 1948.
Fruton, J.S. Proteins, Enzymes, Genes. New Haven: Yale University Press, 1999.
Fukuda, K., H. Higashida, T. Kubo, A. Maeda, I. Akiba, H. Bujo, M. Mishina, and
S. Numa. Selective coupling with K + currents of muscarinic acetycholine recep-
tor subtypes in NG108-15 cells. Nature 335: 355-358, 1988.
Fulton, J.F. The Physiology of the Nervous System. Oxford: Oxford University Press,
1938.
Fung, B.K.K., J.B. Hurley, and L. Stryer. Flow of information in the light-triggered
cyclic nucleotide cascade of vision. Proc. Natl. Acad. Sci. USA 78: 152-156, 1981.
Furchgott, R.F. The pharmacology of vascular smooth muscle. Pharmacol. Rev. 7: 183-
265, 1955.
Furchgott, R.F. The use of /3-haloalkylamines in the differentiation of receptors and in
the determination of dissociation constants of receptor-agonist complexes. Adv.
Drug Res. 3: 21-55, 1966.
Furshpan, E.J. "Electrical transmission" at an excitatory synapse in a vertebrate brain.
Science 144: 878-879, 1964.
Furshpan, E.J., and D.D. Potter. Mechanism of nerve-impulse transmission at a cray-
fish synapse. Nature 180: 342-343, 1957.
Furshpan, E.J., and D.D. Potter. Transmission at the giant motor synapses of the cray-
fish. /. Physiol. 145: 289-325, 1959.
Gaddum, J.H. The action of adrenaline and ergotamine on the uterus of the rabbit.
/. Physiol. 61: 141-150, 1926.
Gaddum, J.H. The quantitative effects of antagonistic drugs. ].Physiol. 89: 7P-8P, 1937.
Gaddum, J.H. Antagonism between lysergic acid diethylamide and 5-hydroxytryptamine.
/. Physiol. 121: 15P, 1953.
Gaddum, J.H. Push-pull cannulae. /. Physiol. 155: 1P-2P, 1961.
Gaddum, J.H., and L.G. Goodwin. Experiments on liver sympathin. /. Physiol. 105:
357-369, 1947.
Gaddum, J.H., and H. Schild. A sensitive physical test for adrenaline. /. Physiol. 80:
9P-10P, 1934.
Gaddum, J.H., and J.C. Szerb. Assay of substance P on goldfish intestine in a micro-
bath. Brit. J. Pharmacol. 17: 451-463, 1961.
Gaffan, D. Monkeys' recognition memory for complex pictures and the effect of fornix
transection. Q. J. Exp. Psychol 29: 505-514, 1977.
Galindo, A., K. Krnjevic, and S. Schwartz. Micro-iontophoretic studies on neurones in
the cuneate nucleus. /. Physiol. 192: 359-377, 1967.
Garten, S. Wird die Funktion des markhaltigen Nerven durch Curarin beeinflusst?
Naunyn-Schmiedebergs Arch. 68: 243-274, 1912.
Garthwaite, J., S.L. Charles, and R. Chess-Williams. Endothelium-derived relaxing fac-
tor release on activation of NMDA receptors suggests role as intercellular mes-
senger in the brain. Nature 336: 385-388, 1988.
Garver, D.L., and J.M. Davis. Biogenic amine hypotheses of affective disorders. Life
Sci. 24: 383-394, 1979.
Gaskell, W.H. On the structure, distribution and function of the nerves which inner-
vate visceral and vascular systems. J. Physiol. 7: 2-80, 1886.
Gaskell, W.H. On the relation between the structure, function, distribution and origin
of the cranial nerves; together with a theory of the origin of the nervous system of
vertebrata. /. Physiol. 10: 153-212, 1889.
Gaskell, W.H. The Involuntary Nervous System. London: Longmans, Green and Co.,
1916.
Gasser, H.S. Contractures of skeletal muscle. Physiol. Rev. 10: 35-109, 1930.
Gasser, H.S., and J. Erlanger. A study of the action currents of nerve with the cathode
ray oscillograph. Amer. J. Physiol. 62: 496-524, 1922.
Geffen, L.B., B.C. Livett, and R.A. Rush. Immunological localization of chromogranins
in sheep sympathetic neurones, and their release by nerve impulses. /. Physiol.
204: 58P-59P, 1969.
Gelders, Y., S. Heylen, G. Vanden Busche, A. Reyntjens, and P. Janssen. Serotonin 5HT2
Rererences 389
receptor antagonism in the treatment of schizophrenia. Clin. Neuropharmacol. 13:

182-183, 1990.
Gerst, J.W., and R.A. Brumback. Neuromuscular transmission: Early historical devel-
opment of the concept. In: Brumback, R.A., and J.W. Gerst, The Neuromuscular
Junction. Mt. Kisco, NY: Futura Publishing Co., 1984, pp. 1-23.
Giere, R.N. Explaining Science. Chicago: University of Chicago Press, 1988.
Giere, R.N. Science without Laws. Chicago: University of Chicago Press, 1999.
Gilbert, S.F. (Ed.) A Conceptual History of Modern Embryology. Baltimore: Johns Hop-
kins University Press, 1994.
Gilbert, W. Towards a paradigm shift in biology. Nature 349: 99, 1991.
Gillespie, J.I. The effect of repetitive stimulation on the passive electrical properties of
the presynaptic terminal of the squid giant synapse. Proc. Roy. Soc. 206B: 293-306,
1979.
Ginzel, K.H., H. Klupp, and G. Werner. Die Wirkung einiger aliphatischer a-n-
Bis-quarternarer Ammonium-Verbindungen auf die Skelettmuskulatur. Naunyn-
Schmiedebergs Arch. 213: 453-466, 1951.
Giraudat, J., M. Dennis, T. Heidmann, J.Y. Chang, and J.-P. Changeux. Structure of the
high-affinity binding site for noncompetitive blockers of the acetylcholine recep-
tor: Serine-262 of the 8 subunit is labeled by [3H]chlorpromazine. Proc. Natl. Acad.
Set. USA 83: 2719-2723, 1986.
Glowinski, J. In vivo release of transmitters in the cat basal ganglia. Fed. Proc. 40:
135-141, 1981.
Glowinski, J., and J. Axelrod. Inhibition of uptake of tritiated-noradrenaline in the intact
rat brain by imipramine and structurally related compounds. Nature 204:
1318-1319, 1964.
Glowinski, J., I.J. Kopin, and J. Axelrod. Metabolism of [3H]norepinephrine in the rat
brain. /. Neurochem. 12: 25-30, 1965.
Glowinski, J., S.H. Snyder, and J. Axelrod. Subcellular localization of 3H-norepineph-
rine in the rat brain and the effect of drugs./. Pharmacol. Exp. Ther. 152: 282-292,
1966.
Glynn, I. An Anatomy of Thought. London: Weidenfeld & Nicolson, 1999.
Godfrey, E.W., R.M. Nitkin, B.C. Wallace, L.L. Rubin, and U.J. McMahan. Compo-
nents of Torpedo electric organ and muscle that cause aggregation of acetylcholine
receptors on cultured muscle cells. /. Cell Biol. 99: 615-627, 1984.
Goldberg, J. Anatomy of a Scientific Discovery. Toronto: Bantam Books, 1988.
Goldenring, J.R., B. Gonzalez, J.S. McGuire, and R.J. DeLorenzo. Purification and char-
acterization of a calmodulin-dependent kinase from rat brain cytosol able to phos-
phorylate tubulin and microtubule-associated proteins./. Biol. Chem. 258: 12,632-
12,640, 1983.
Goldstein, A., L.I. Lowney, and B.K. Pal. Stereospecific and nonspecific interactions of
the morphine congener levorphanol in subcellular fractions of mouse brain. Proc.
Natl. Acad. Sci. USA 68: 1742-1747, 1971.
Goldstein, M., B. Anagnoste, and C. Shirron. The effect of trivastal, haloperidol and
dibutyryl cyclic AMP on [14C]dopamine synthesis in rat striatum. /. Pharm. Phar-
macol. 25: 348-351, 1973.
Golgi, C. The neuron doctrine—theory and facts. In: Nobel Lectures. Physiology or
Medicine. 1901-1921. Amsterdam: Elsevier Publishing Co., 1967.
Goodenough, D.A., and J.P. Revel. A fine structural analysis of intercellular junctions
in the mouse liver. /. Cell Biol. 45: 272-290, 1970.
Goodman, C.S., M.J. Bastiani, C.Q. Doe, S. du Lac, S.L. Helfand, J.Y. Kuwada, and
J.B. Thomas. Cell recognition during neuronal development. Science 225: 1271-
1279, 1984.
Goodman, L.S., and A. Gilman. Pharmacological Basis of Therapeutics. Second ed. New
York: Macmillan, 1956.
Gopfert, H., and H. Schaefer. Uber den direkt und indirekt erregten Aktionsstrom und
die Funktion der motorischen Endplatte. Pfliigers Arch. 239: 597-619, 1938.
Gordon, A.S., C.G. Davis, D. Milfay, and I. Diamond. Phosphorylation of acetylcholine
receptor by endogenous membrane protein kinase in receptor-enriched mem-
branes of Torpedo californica. Nature 267: 539-540, 1977.
Gorenstein, C., and S.H. Snyder. Two distinct enkephalinases: Solubilization, partial
purification and separation from angiotensin converting enzyme. Life Sci. 25:
2065-2070, 1979.
Gottesman, 1.1., and J. Shields. Schizophrenia: The Epigenetic Puzzle. Cambridge: Cam-
bridge University Press, 1982.
Gottlieb, D.I., K. Rock, and L. Glaser. A gradient of adhesive specificity in developing
avian retina. Proc. Natl. Acad. Sci. USA 73: 410-414, 1976.
Graham, J., and R.W. Gerard. Membrane potentials and excitation of impaled single
fibers./. Cell. Comp. Physiol. 28: 99-117, 1946.
Graham, L.T., R.P. Shank, R. Werman, and M.H. Aprison. Distribution of some synap-
tic transmitter suspects in cat spinal cord. /. Neurochem. 14: 465-472, 1967.
Graham, L.T., R. Werman, and M.H. Aprison. Microdetermination of glutamate in sin-
gle cat spinal roots. Life Sci. 4: 1085-1090, 1965.
Grahame-Smith, D.G. Tryptophan hydroxylation in brain. Biochem. Biophys. Res. Com-
mun. 16: 586-592, 1964.
Granit, R. Charles Scott Sherrington. Garden City, NY: Doubleday & Co., 1967.
Gray, E.G., and V.P. Whittaker. The isolation of synaptic vesicles from the central nerv-
ous system. /. Physiol. 153: 35P-37P, 1960.
Gray, E.G., and V.P. Whittaker. The isolation of nerve endings from brain: An electron-
microscopic study of cell fragments derived by homogenization and centrifugation.
/. Anat. 96: 79-88, 1962.
Greaser, M.L., and J. Gergely. Reconstitution of troponin activity from three protein
components./. Biol. Chem. 246: 4226-4233, 1971.
Green, H., S.M. Greenberg, R.W. Erickson, J.L. Sawyer, and T. Ellison. Effect of dietary
phenylalanine and tryptophan upon rat brain amine levels./. Pharmacol. Exp. Ther.
136: 174-178, 1962.
Green, J.P. Binding of some biogenic amines in tissues. Adv. Pharmacol. 1: 349-422,
1960.
Green, J.P, J.D. Robinson, and M. Day. Interaction between cerebroside sulfate and
amines. /. Pharmacol. Exp. Ther. 131: 12-17, 1961.
Greenberg, S.M., V.F. Castellucci, H. Bayley, and J.H. Schwartz. A molecular mecha-
nism for long-term sensitization in Aplysia. Nature 329: 62-65, 1987.
Greer, C.M., J.O. Pinkston, J.H. Baxter, and E.S. Brannon. Nor-epinephrine as a pos-
sible mediator in the sympathetic division of the autonomic nervous system./. Phar-
macol. Exp. Ther. 62: 189-227, 1938.
Grelak, R.P, R. Clark, J.M. Stump, and V.G. Vernier. Amantadine-dopamine interac-
tion: Possible mode of action in parkinsonism. Science 169: 203-204, 1970.
Grenett, H.E., F.D. Ledley, L.L. Reed, and S.L.C. Woo. Full-length cDNA for rabbit
tryptophan hydroxylase: Functional domains and evolution of aromatic amino acid
hydroxylases. Proc. Natl. Acad. Sci. USA 84: 5530-5534, 1987.
References 391
Grenningloh, G., A. Rienitz, B. Schmitt, C. Methfessel, M. Zensen, K. Beyreuther, E.D.

Gundelfinger, and H. Betz. The strychnine-binding subunit of the glycine recep-
tor shows homology with nicotinic acetylcholine receptors. Nature 328: 215-220,
1987.
Griffith, R.W., and K. Saameli. Clozapine and agranulocytosis. Lancet 2: 657, 1975.
Grima, B., A. Lamouroux, F. Blanot, N. Faucon Biguet, and J. Mallet. Complete cod-
ing sequence of rat tyrosine hydroxylase mRNA. Proc. Natl. Acad. Sci. USA 82:
617-621, 1985.
Gross, H., and E. Langer. Das Wirkungsprofil eines chemisch neuartigen Breitband-
Neuroleplikums der Dibenzodiazepingruppe. Wien. Medizin, Wochenschr. 116:
814-816, 1966.
Grundfest, H. Excitability of the single fibre nerve-muscle complex./. Physiol. 76: 95-
115, 1932.
Grundfest, H. Excitation at synapses. /. Neurophysiol. 20: 316-324, 1957a.
Grundfest, H. Electrical inexcitability of synapses and some consequences in the cen-
tral nervous system. Physiol. Rev. 37: 337-361, 1957b.
Grundfest, H., J.P. Reuben, and W.H. Rickles. The electrophysiology and pharmacol-
ogy of lobster neuromuscular synapses. /. Gen. Physiol. 42: 1301-1323, 1959.
Guastella, J., N. Nelson, L. Czyzyk, S. Keynan, M.C. Miedel, N. Davidson, H.A. Lester,
and B.I. Kanner. Cloning and expression of a rat brain GABA transporter. Science
249: 1303-1306, 1990.
Gubler, U., P. Seeburg, B.J. Hoffman, L.P. Gage, and S. Udenfriend. Molecular cloning
establishes proenkephalin as a precursor of enkephalin-contaming peptides. Nature
295: 206-208, 1982.
Guldberg, H.C., and C.A. Marsden. Catechol-O-methyltransferase: Pharmacological
aspects and physiological role. Pharmacol. Rev. 27: 135-206, 1975.
Gundersen, R.W., and J.N. Barrett. Neuronal chemotaxis: Chick dorsal-root axons turn
toward high concentrations of nerve growth factor. Science 206: 1079-1080, 1979.
Gunn, J.A. Walter Ernest Dixon. /. Pharmacol. Exp. Ther. 44: 3-21, 1932.
Gunne, L.M., and D.J. Reis. Changes in brain catecholamines associated with electri-
cal stimulation of amygdaloid nucleus. Life Sci. 2: 804-809, 1963.
Guth, L., and J.J. Bernstein. Selectivity in the re-establishmant of synapses in the supe-
rior cervical sympathetic ganglion of the cat. Exp. Neurol. 4: 59-69, 1961.
Haavik, J., K.K. Andersson, L. Petersson, and T. Flatmark. Soluble tyrosine hydroxy-
lase (tyrosine 3-monooxygenase) from bovine adrenal medulla. Biochim. Biophys.
Acta 953: 142-156, 1988.
Hadcock, J.R., and C.C. Malbon. Down-regulation of jS-adrenergic receptors: Agonist-
induced reduction in receptor mRNA levels. Proc. Natl. Acad. Sci. USA 85: 5021-
5025, 1988.
Hadcock, J.R., H.Y. Wang, and C.C. Malbon. Agonist-induced destabilization of
0-adrenergic receptor mRNA. /. Biol. Chem. 264: 19,928-19,933, 1989.
Hagen, P. Biosynthesis of norepinephrine from 3,4-dihydroxyphenylethylamine
(dopamine)./. Pharmacol. Exp. Ther. 116: 26, 1956.
Hagiwara, S., and L. Byerly. Calcium channel. Annu. Rev. Neurosci. 4: 69-125, 1981.
Hagiwara, S., K. Kusano, and S. Saito. Membrane changes in crayfish stretch receptor
neuron during synaptic inhibition and under action of gamma-aminobutyric acid.
/. Neurophysiol 23: 505-515, 1960.
Hagiwara, S., and I. Tasaki. A study of the mechanism of impulse transmission across
the giant synapse of the squid./. Physiol. 143: 114-137, 1958.
Haglund, K., and W. Brown. Landmarks. /. NIH Res. 7: 59-67, 1995.

Haglund, K., and C. Collett. Landmarks. /. NIH Res. 8: 42-51, 1996a.
Haglund, K., and C. Collett. Landmarks. /. NIH Res. 8: 51-57, 1996b.
Halliburton, W.D. Hand-book of Physiology. Philadelphia: Blakiston, 1904.
Hamberger, B., T. Malmfors, K.A. Norberg, and C. Sachs. Uptake and accumulation of
catecholamines in peripheral adrenergic neurons of reserpinized animals, studied
with a histochemical method. Biochem. Pharmacol. 13: 841-844, 1964.
Hambrook, J.M., B.A. Morgan, M.J. Ranee, and C.F.C.Smith. Mode of deactivation of
the enkephalins by rat and human plasma and rat brain homogenates. Nature 262:
782-783, 1976.
Hamburger, V. The effects of wing bud extirpation on the development of the central
nervous system in chick embryos. /. Exp. Zoo/. 68: 449-494, 1934.
Hamburger, V. Regression versus peripheral control of differentiation in motor hypopla-
sia. Amer. ]. Anat. 102: 365-409, 1958.
Hamburger, V., and R. Levi-Montalcini. Proliferation, differentiation and degeneration
in the spinal ganglia of the chick embryo under normal and experimental condi-
tions./. Exp. Zool. Ill: 457-500, 1949.
Hamill, O.P., A. Marty, E. Neher, B. Sakmann, and F.J. Sigworth. Improved patch-clamp
techniques for high-resolution current recording from cells and cell-free mem-
brane patches. Pflugers Arch. 391: 85-100, 1981.
Hamlin, K.E., and F.E. Fischer. The synthesis of 5-hydroxytryptamine./. Amer. Chem.
Soc. 73: 5007-5008, 1951.
Hamm, H.E. The many faces of G protein signaling./. Biol. Chem. 273: 669-672, 1998.
Hammar, C.G., I. Hanin, B. Holmstedt, R.J. Kitz, D.J. Jenden, and B. Karlen. Identi-
fication of acetylcholine in fresh rat brain by combined gas chromatography-mass
spectrometry. Nature 220: 915-917, 1968.
Hamon, M., S. Bourgoin, F. Hery, and G. Simonnet. Activation of tryptophan hydrox-
ylase by adenosine triphosphate, magnesium and calcium. Mol. Pharmacol. 14:
99-110, 1978.
Handler, J.S., R. Bensinger, and J. Orloff. Effect of adrenergic agents on toad bladder
response to ADH, 3',5'-AMP, and theophylline. Amer. J. Physiol. 215: 1024-1031,
1968.
Hanski, E., P.C. Sternweis, J.K. Northup, A.W. Dromerick, and A.G. Gilman. The reg-
ulatory component of adenylate cyclase./. Biol. Chem. 256: 12,911-12,919, 1981.
Hare, M.L.C. Tyramine oxidase. Biochem. J. 22: 968-979, 1928.
Harris, E.W., A.H. Ganong, and C.W. Cotman. Long-term potentiation in the hip-
pocampus involves activation of N-methyl-D-aspartate receptors. Brain Res. 323:
132-137, 1984.
Harris, H. The Birth of the Cell. New Haven: Yale University Press, 1999.
Harris, J.E., V.H. Morgenroth, R.H. Roth, and R.J. Baldessarini. Regulation of cate-
cholamine synthesis in the rat brain in vitro by cyclic AMP. Nature 252: 156-158,
1974.
Harrison, E. Whigs, prigs, and historians of science. Nature 329: 213-214, 1987.
Harrison, R.G. Observations on the living developing nerve fiber. Anat. Rec. 1: 116-118,
1907.
Harrison, R.G. Embryonic transplantation and development of the nervous system.
Anat. Rec. 2: 385-410, 1908.
Harrison, R.G. The reaction of embryonic cells to solid structures. /. Exp. Zool. 17:
521-544, 1914.
Reterences 393
Harrison, R.G. On the origin and development of the nervous system studied by the
methods of experimental embryology. Proc. Roy. Soc. 118B: 155-196, 1935.
Harvey, A.M., and F.C. Macintosh. Calcium and synaptic transmission in a sympathetic
ganglion./. Physiol. 97: 408^16, 1940.
Hasselbach, W., and M. Makinose. Die Calciumpumpe der "Erschlaffungsgrana" des
Muskels und ihre Abhangigkeit von der ATP-Spaltung. Biochem. Z. 333: 518-528,
1961.
Hatta, K., T.S. Okada, and M. Takeichi. A monoclonal antibody disrupting calcium-
dependent cell-cell adhesion of brain tissues: Possible role of its target antigen in
animal pattern formation. Proc. Natl. Acad. Set. USA 82: 2789-2793, 1985.
Haug, J.O. Pneumoencephalographic studies in mental disease. Acta Psychiat. Scand.
Suppl. 165: 1-104, 1962.
Hausdorff, W.P., M.G. Caron, and R.J. Lefkowitz. Turning off the signal: Desensitiza-
tion of /3-adrenergic receptor function. FASEB J. 4: 2881-2889, 1990.
Hawkins, J. The localization of amine oxidase in the liver cell. Biochem. J. 50: 577-581,
1952.
Hawkins, R.D., T.W. Abrams, T.J. Carew, and E.R. Kandel. A cellular mechanism of
classical conditioning in Aplysia: Activity-dependent amplification of presynaptic
facilitation. Science 219: 400^05, 1983.
Haycock, J.W., W.B. Levy, L.A. Denner, and C.W. Cotman. Effects of elevated [K + ] 0
on the release of neurotransmitters from cortical synaptosomes: Efflux or secre-
tion?/. Neuwchem. 30: 1113-1125, 1978.
Heald, P.J. The incorporation of phosphate into cerebral phosphoprotein promoted by
electrical impulses. Biochem. J. 66: 659-663, 1957.
Heald, P.J. Phosphoprotein metabolism and ion transport in nervous tissue: A suggested
connexion, Nature 193: 451-454, 1962.
Healy, D. The Psychopharmacologists. London: Altman, 1996.
Healy, D. The Antidepressant Era. Cambridge, Mass.: Harvard University Press, 1997.
Hebb, C.O., and V.P Whittaker. Intracellular distributions of acetylcholine and choline
acetylase. /. Physiol. 142: 187-196, 1958.
Hebb, D.O. The Organization of Behavior. New York: John Wiley, 1949.
Hedgcock, E.M., J.C. Culotti, and D.H. Hall. The unc-5, unc-6, and unc-40 genes guide
circumferential migrations of pioneer axons and mesodermal cells on the epider-
mis of C. Elegans. Neuron 2: 61-85, 1990.
Heidemann, S.R., P. Lamoureux, and R.E. Buxbaum. Growth cone behavior and pro-
duction of traction force./. Cell Biol. Ill: 1949-1957, 1990.
Heidmann, T, and J.-P. Changeux. Interaction of a fluorescent agonist with the mem-
brane-bound acetylcholine receptor from Torpedo rnarmorata. Biochem. Biophys.
Res. Commun, 97: 889-896, 1980.
Heikkila, R.E., L. Manzino, F.S. Cabbat, and R.C. Duvoisin. Protection against the
dopaminergic neurotoxicity of l-methyl-4-phenyl-l,2,5,6-tetrahydropyridine by
monoamine oxidase inhibitors. Nature 311: 467-469, 1984.
Heilbrunn, L.V., and F.J. Wiercinski. The action of various cations on muscle proto-
plasm. /. Cell. Comp. Physiol. 29: 15-32, 1947.
Henderson, G., J. Hughes, and H.W. Kosterlitz. A new example of a morphine-
sensitive neuro-effector junction: Adrenergic transmission in the mouse vas defer-
ens. Brit. J. Pharmacol. 46: 764-766, 1972.
Henderson, G., J. Hughes, and H.W. Kosterlitz. In vitro release of leu- and met-
enkephalin from the corpus striatum. Nature 271: 677-679, 1978.
Henderson, R., and P.N.T. Unwin. Three-dimensional model of purple membrane

obtained by electron microscopy. Nature 257: 28-32, 1975.
Hendry, LA., K. Stockel, H. Thoenen, and L.L. Iversen. The retrograde axonal trans-
port of nerve growth factor. Brain Res. 68: 103-121, 1974.
Henry, J.L. Effects of substance P on functionally identified units in cat spinal cord.
Brain Res. 114: 439-451, 1976.
Heritch, A.J. Evidence for reduced and dysregulated turnover of dopamine in schizo-
phrenia. Schizophrenia Bull. 16: 605-615, 1990.
Hertting, G., and J. Axelrod. Fate of tritiated noradrenaline at the sympathetic nerve-
endings. Nature 192: 172-173, 1961.
Hertting, G., J. Axelrod, I.J. Kopin, and L.G. Whitby. Lack of uptake of catecholamines
after chronic denervation of sympathetic nerves. Nature 189: 66, 1961a.
Hertting, G., J. Axelrod, and L.G. Whitby. Effect of drugs on the uptake and metabo-
lism of H3-norepinephrine. /. Pharmacol. Exp. Ther. 134: 146-153, 1961b.
Hertwig, O. The Cell New York: Macmillan, 1895.
Hess, G.P., D.J. Cash, and H. Aoshima. Acetylcholine receptor-controlled ion translo-
cation. Annu. Rev. Biophys. Bioeng. 12: 443-473, 1983.
Hess, E.J., A.B. Norman, and I. Creese. Chronic treatment with dopamine receptor
antagonists: Behavioral and pharmacological effects on DI and D% dopamine recep-
tors. /. Neurosci. 8: 2361-2370, 1988.
Heston, L.L. Psychiatric disorders in foster home reared children of schizophrenic moth-
ers. Brit.}. Psychiat. 112: 819-825, 1966.
Heuser, J.E., and T.S. Reese. Evidence for recycling of synaptic vesicle membrane during
transmitter release at frog neuromuscular junction. J. Cell Biol. 57: 315-344, 1973.
Heuser, J.E., and T.S. Reese. Structural changes after transmitter release at the frog
neuromuscular junction. J. Cell Biol. 88: 564-580, 1981.
Heuser, J.E., T.S. Reese, M.J. Dennis, Y. Jan, L. Jan, and L. Evans. Synaptic vesicle
exocytosis captured by quick freezing and correlated with quantal transmitter
release. /. Cell Biol. 81: 275-300, 1979.
Heuser, J.E., T.S. Reese, and D.M.D. Landis. Functional changes in frog neuromus-
cular junctions studied with freeze-fracture./. Neurocytol. 3: 109-131, 1974.
Hibbard, E. Orientation and directed growth of Mautimer's cell axons from duplicated
vestibular nerve roots. Exp. Neurol. 13: 289-301, 1965.
Hildebrandt, J.D., J. Codina, R. Risinger, and L. Birnbaumer. Identification of a y sub-
unit associated with the adenylyl cyclase regulatory proteins Ns and Nj. J. Biol.
Chem. 259: 2039-2042, 1984.
Hildebrandt, J.D., R.D. Sekura. J. Codina, R. lyengar, C.R. Manclark, and L. Birn-
baumer. Stimulation and inhibition of adenylyl cyclases mediated by distinct reg-
ulatory proteins. Nature 302: 706-709, 1983.
Hill, A. The chrome-silver method. Brain 19: 1-42, 1896.
Hill, A. Considerations opposed to the "neuron theory." Brain 23: 657-690, 1900.
Hill, A.V. The mode of action of nicotine and curari, determined by the form of the
contraction curve and the method of temperature coefficients. /. Physiol. 39:
361-373, 1909.
Hill, R.G., C.M. Pepper, and J.F. Mitchell. Depression of nociceptive and other neu-
rones in the brain by iontophoretically applied met-enkephalin. Nature 262:
604-606, 1976.
Hill, T.L., and M.W. Kirschner. Subunit treadmilling of microtubules or actin in the
presence of cellular barriers: Possible conversion of chemical free energy into
mechanical work. Proc. Natl. Acad. Sci. USA 79: 490-494, 1982.
References 395
Hillarp, N.-A. Enzyme systems involving adenosinephosphates in the adrenaline and

noradrenaline containing granules of the adrenal medulla. Acta Physiol Scand. 42:
144-165, 1958a.
Hillarp, N.-A. Isolation and some biochemical properties of the catechol amine gran-
ules in the cow adrenal medulla. Acta Physiol Scand. 43: 82-96, 1958b.
Hillarp, N.-A., K. Fuxe, and A. Dahlstrom. Demonstration and mapping of central neu-
rons containing dopamine, noradrenaline, and 5-hydroxytryptamine and their reac-
tions to psychopharmaca. Pharmacol. Rev. 18: 727-741, 1966.
Hillarp, N.-A., B. Hokfelt, and B. Nilson. The cytology of the adrenal medullary cells
with special reference to the storage and secretion of the sympathomimetic amines.
Acta Anat. 21: 155-167, 1954.
Hillarp, N.-A., S. Lagerstedt, and B. Nilson. The isolation of a granular fraction from
the suprarenal medulla, containing the sympathomimetric catechol amines. Acta
Physiol. Scand. 29: 251-263, 1953.
Hillarp, N.-A., and B. Nilson. The structure of the adrenaline and noradrenaline contain-
ing granules in the adrenal medullary cells with reference to the storage and release
of the sympathomimetic amines. Acta Physiol. Scand. Suppl. 113, 79-107, 1954.
Hillarp, N.-A., B. Nilson, and B. Hogberg. Adenosine triphosphate in the adrenal
medulla of the cow. Nature 176: 1032-1033, 1955.
Hille, B. Ionic Channels of Excitable Membranes. Sunderland: Sinauer Associates, 1992.
Hippius, H. The history of clozapine. Psychopharmacol. 99: S3-S5, 1989.
Hirokawa, N., K. Sobue, K. Kanda, A. Harada, and H. Yorifuji. The cytoskeletal archi-
tecture of the presynaptic terminal and molecular structure of synapsin 1. J. Cell
Biol. 108: 111-126, 1989.
Hodgkin, A. Chance and Design. Cambridge: Cambridge University Press, 1992.
Hodgkin, A.L., and A.F. Huxley. Action potentials recorded from inside a nerve fibre.
Nature 144: 706-707, 1939.
Hodgkin, A.L., and A.F. Huxley. Resting and action potentials in single nerve fibres.
/. Physiol. 104: 176-195, 1945.
Hodgkin, A.L., and A.F. Huxley. A quantitative description of membrane current and
its application to conduction and excitation in nerve./. Physiol. 117: 500-544,1952.
Hodgkin, A.L., and R.D. Keynes. The potassium permeability of a giant nerve fibre.
/. Physiol. 128: 61-88, 1955.
Hodgkin, A.L., and R.D. Keynes. Movements of labelled calcium in squid giant axons.
/. Physiol. 138: 253-281, 1957.
Hoff, H.E. The history of vagal inhibition. Bull. Hist. Med. 8: 461-496, 1940.
Hoff, H.E., and P. Kellaway. The early history of the reflex. /. Hist. Med. 7: 219-249,
1952.
Hoffman, B.J., E. Mezey, and M.J. Brownstein. Cloning of a serotonin transporter
affected by antidepressants. Science 254: 579-580, 1991.
Hogeboom, G.H. Fractionation of cell components of animal tissues. Meth. Enzymol.
1: 16-19, 1955.
Hogeboom, G.H., W.C. Schneider, and G.E. Palade. Cytochemical studies of mam-
malian tissues. /. Biol. Chem. 172: 619-635, 1948.
Hokfelt, T, J.O. Kellerth, G. Nilsson, and B. Pernow. Substance P: Localization in the
central nervous system and in some primary sensory neurons. Science 190: 889-890,
1975.
Hokfelt, T, A. Ljungdahl, K. Fuxe, and O. Johansson. Dopamine nerve terminals in the
rat limbic cortex: Aspects of the dopamine hypothesis of schizophrenia. Science
184: 177-179, 1974.
Hokfelt, T., A. Ljungdahl, L. Terenius, R. Elde, and G. Nilsson. Immunohistochemical

analysis of peptide pathways possibly related to pain and analgesia: Enkephalin and
substance P. Proc. Natl. Acad. Sci. USA 74: 3081-3085, 1977.
Hokfelt, T., J.M. Lundberg, M. Schultzberg, O. Johansson, A. Ljungdahl, and J. Rehfeld.
Coexistence of peptides and putative transmitters in neurons. Adv. Biochem. Psy-
chopharmacol 22: 1-23, 1980.
Hokin, L.E., and M.R. Hokin. Effects of acetylcholine on the turnover of phosphoryl
units in individual phospholipids of pancreas slices and brain cortex slices. Biochim.
Biophys. Acta 18: 102-110, 1955.
Hokin, L.E., and M.R. Hokin. Evidence for phosphatidic acid as the sodium carrier.
Nature 184: 1068-1069, 1959.
Hokin, M.R., and L.E. Hokin. Enzyme secretion and the incorporation of P32 into phos-
pholipides of pancreas slices./. Biol. Chem. 203: 967-977, 1953.
Hokin, M.R., and L.E. Hokin. Effects of acetylcholine on phospholipides in the pan-
creas. /. Biol. Chem. 209: 549-557, 1954.
Hokin, M.R., and L.E. Hokin. The synthesis of phosphatidic acid and protein-bound
phosphorylserine in salt gland homogenates./. Biol. Chem. 239: 2116-2122, 1964a.
Hokin, M.R., and L.E. Hokin. Interconversions of phosphatidylinositol and phospha-
tidic acid involved in the response to acetylcholine in the salt gland. In: Dawson,
R.M.C., and D.N. Rhodes (eds.) Metabolism and Physiological Significance of
Lipids. London: Wiley, 1964b, pp. 423-434.
Hollmann, M., A. O'Shea-Greenfield, S.W. Rogers, and S. Heinemann. Cloning by func-
tional expression of a member of the glutamate receptor family. Nature 342:
643-648, 1989.
Holmstedt, B. Structure-activity relationships of the organophosphorus anti-
cholinesterase agents. Handb. Exper. Pharmakol. 15: 428-485, 1963.
Holmsted, B. Pages from the history of research on cholinergic mechanisms. In: Waser,
P.G. (ed.) Cholinergic Mechanisms. New York: Raven Press, 1975, pp. 1—21.
Holmstedt, B., and G. Liljestrand. Readings in Pharmacology. New York: Macmillan,
1963.
Holtz, P. Dopadecarboxylase. Naturwissenschaften 27: 724-725, 1939.
Holtz, P., K. Credner, and G. Kroneberg. Uber das sympathicomimetische pressorische
Prinzip des Harns. Naunyn-Schmiedebergs Archiv 204: 228-243, 1947.
Holtz, P., R. Heise, and K. Liidtke. Fermentativer Abbau von 1-Dioxyphenylalanin
(Dopa) durch Niere. Naunyn-Schmiedebergs Arch. 191: 87-118, 1938.
Holtz, P., and H.J. Schumann. Arterenol—ein neues Hormon des Nebennierenmarks.
Naturwissenschaften 35: 159, 1948.
Holtz, P., and E. Westermann. Uber die Dopadecarboxylase und Histidindecarboxylase
des Nervengewebes. Naunyn-Schmiedebergs Arch. 227: 538—546, 1956.
Holtzman, E., A.R. Freeman, and L.A. Kashner. Stimulation-dependent alternatives in
peroxidase uptake at lobster neuromuscular junctions. Science 173: 733-736, 1971.
Holzbauer, M., and M. Vogt. Depression by reserpine of the noradrenaline concentra-
tion in the hypothalamus of the cat./. Neurochem. 1: 8-11, 1956.
Honchar, M.P., G.P. Vogler, B.C. Gish, and W.R. Sherman. Evidence that phospho-
inositide metabolism in rat cerebral cortex stimulated by pilocarpine, physostig-
mine, and pargyline in vivo is not changed by chronic lithium treatment. /. Neu-
rochem. 55: 1521-1525, 1990.
Honegger, P., and D. Lenoir. Nerve growth factor (NGF) stimulation of cholinergic
telencephalic neurons in aggregating cell cultures. Dev. Brain Res. 3: 229-238,
1982.
References 397
Honig, A., and H.M. Van Praag (eds.). Depression: Neurobiological, Psychopathologi-
cal and Therapeutic Advances. Chichester: John Wiley & Sons, 1997.
Honig, C.R., and A.C. Stam. The influence of adrenergic mediators and their structural
analogs on cardiac actomyosin systems. Ann, NY Acad, Sci. 139: 724—740, 1967.
Hook, V.Y.H., and L.E. Eiden. Two peptidases that convert 125I-Lys-Arg-(Met)enkephalin
and 125I-(Met)enkephalin-Arg6, respectively, to 125I-(Met)enkephalin in bovine adre-
nal medullary chromafin granules. FEES Lett. 172: 212-218, 1984.
Horder, T.J., J.A. Witkowski, and C.C. Wylie (eds.). A History of Embryology. New York:
Cambridge University Press, 1986.
Horn, A.S., and S.H. Snyder. Chlorpromazine and dopamine: Conformational similar-
ities that correlate with the antischizophrenic activity of phenothiazine drugs. Proc.
Natl. Acad. Sci. USA 68: 2325-2328, 1971.
Hornykiewicz, O. Die topische Lokalisation und das Verhalten von Noradrenalin und
Dopamin (3-Hydroxytyramin) in der Substantia nigra des normalen und Parkin-
sonkranken Menschen. Wien. Klin. Wschr. 75: 309-312, 1963.
Hornykiewicz, O. Dopamine (3-hydroxytyramine) and brain function. Phanmacol. Rev.
18: 925-964, 1966.
Hornykiewicz, O. Neurochemical pathology and pharmacology of brain dopamine and
acetylcholine. In: McDowell, F.H., and C.H. Markham (eds.) Recent Advances in
Parkinson's Disease. Philadelphia: F.A. Davis, 1971, pp. 33-65.
Howell, W.H. A Text-book of Physiology. Philadelphia: W.B. Saunders, 1905.
Howell, W.H., and W.W. Duke. The effect of vagus inhibition on the output of potas-
sium from the heart. Amer. ]. Physiol. 41: 51-63, 1910.
Howells, J.G. (ed.) The Concept of Schizophrenia: Historical Perspectives. Washington,
DC: American Psychiatric Press, 1991.
Hsu, Y.P., W. Weyler, S. Chen, K.B. Sims, W.B. Rinehart, M.C. Utterback, J.F. Pow-
ell, and X.O. Breakefield. Structural features of human monoamine oxidase A
elucidated from cDNA and peptide sequences. /. Neurochem. 51: 1321-1324,
Hubbard, J.I., and T. Yokota. Direct evidence for an action of acetylcholine on motor-
nerve terminals. Nature 203: 1072-1073, 1964.
Hucho, F., W. Oberthur, and F. Lottspeich. The ion channel of the nicotinic acetyl-
choline receptor is formed by the homologous helices Mil of the receptor sub-
units. FERS Lett. 205: 137-142, 1986.
Hucho, F, and R. Hilgenfeld. The selectivity filter of a ligand-gated ion channel. FEES
Lett. 257: 17-23, 1989.
Hufton, S.E., I.G. Jennings, and R.G.H. Cotton. Structure and function of the aromatic
amino acid hydroxylases. Biochern. J. 311: 353-366, 1995.
Huganir, R.L., A.H. Delcour, P. Greengard, and G.P. Hess. Phosphorylation of the nico-
tinic acetylcholine receptor regulates its rate of desensitization. Nature 321:
774-776, 1986.
Huganir, R.L., M.A. Schell, and E. Racker. Reconstitution of the purified acetylcholine
receptor from Torpedo californica. FERS Lett. 108: 155-160, 1979.
Hughes, A. A History of Cytology. London: Abelard-Schuman, 1957.
Hughes, J. Isolation of an endogenous compound from the brain with pharmacological
properties similar to morphine. Life Sci. 88: 295-308, 1975.
Hughes, J., T.W. Smith, H.W. Kosterlitz, L.A. Fothergill, B.A. Morgan, and H.R. Mor-
ris. Identification of two related pentapeptides from the brain with potent opiate
agonist activity. Nature 258: 577-579, 1975a.
Hughes, J., T. Smith, B. Morgan, and L. Fothergill. Purification and properties of
enkephalin—the possible endogenous ligand for the morphine receptor. Life Set.
16: 1753-1758, 1975b.
Huijing, F., and J. Larner. On the mechanism of action of adenosine 3', 5' cyclophos-
phate. Proc. Natl. Acad. Sci. USA 56: 647-653, 1966.
Hulme, E.G., N.J.M. Birdsall, and NJ. Buckley. Muscarinic receptor subtypes. Annu.
Rev. Pharmacol. Toxicol. 30: 633-673, 1990.
Hunt, M. The Story of Psychology. New York: Doubleday, 1993.
Hunt, R. Further observations on the blood-pressure-lowering bodies in extracts of the
suprarenal gland. Amer. J. Physiol. 5: vi-vii, 1901.
Hunt, R., and R. deM. Taveau. On the physiological action of certain cholin derivatives
and new methods for detecting cholin. Brit. Med. ]. 2: 1788-1791, 1906.
Hunt, R., and R. deM. Taveau. On the relationship between the toxicity and chemical
constitution of a number of derivatives of choline and analogous compounds.
/. Pharmacol. Exp. Ther. 1: 303^339, 1909.
Hunt, S.P., and T.A. Lovick. The distribution of serotonin, met-enkephalin and
/3-lipotropin-like immune reactivity in neuronal perikarya of the cat brainstem. Neu-
rosci. Lett. 30: 139-145, 1982.
Huttner, W.B., L.J. DeGennaro, and P. Greengard. Differential phosphorylation of mul-
tiple sites in purified protein I by cyclic AMP-dependent and calcium-dependent
protein kinases. /. Biol. Chem. 256: 1482-1488, 1981.
Huxley, A.F., and R.E. Taylor. Local activation of striated muscle fibres./. Physiol. 144:
426-441, 1958.
Hyden, H. The neuron. In: Brachet, J. and A.E. Mirsky (eds.) The Cell, vol. IV. New
York: Academic Press, 1960. pp. 215-237.
Hyden, H., and E. Egyhazi. Nuclear RNA changes of nerve cells during a learning
experiment in rats. Proc. Natl. Acad. Sci. USA 48: 1366-1373, 1962.
Hynes, R.O. Integrins: A family of cell surface receptors. Cell 48: 549-554, 1987.
Ikeda, M., T. Nakazawa, K. Abe, T. Kaneko, and K. Yamatsu. Extracellular accumula-
tion of glutamate in the hippocampus induced by ischemia is not calcium depend-
ent—-in vitro and in vivo evidence. Neurosci. Lett. 96: 202-206, 1989.
Imoto, K., C. Busch, B. Sakmann, M. Mishina, T. Konno, J. Nakai, H. Bujo, Y. Mori,
K. Fukuda, and S. Numa. Rings of negatively charged amino acids determine the
acetylcholine receptor channel conductance. Nature 335: 645-648, 1988.
Imoto, K., C. Methfessel, B. Sakmann, M. Mishina, Y. Mori, T. Konno, K. Fukuda,
M. Kurasaki, H. Bujo, Y. Fujita, and S. Numa. Location of a S-subunit region deter-
mining ion transport through the acetylcholine receptor channel. Nature 324:
670-674, 1986.
Ingebritsen, T.S., and P. Cohen. Protein phosphates: Properties and role in cellular reg-
ulation. Science 221: 331-338, 1983.
Inoue, M., A. Kishimoto, Y. Takai, and Y. Nishizuka. Studies on a cyclic nucleotide-inde-
pendent protein kinase and its proenzyme in mammalian tissues. /. Biol. Chem.
252: 7610-7616, 1977.
Iriki, A., C. Pavlides, A. Keller, and H. Asanuma. Long-term potentiation in the motor
cortex. Science 245: 1385-1387, 1989.
Ishii, N., W.G. Wadsworth, B.D. Stern, J.C. Culotti, and E.M. Hedgecock. UNC-6, a
laminin-related protein, guides cell and pioneer axon migrations in C. Elegans.
Neuron 9: 873-881, 1992.
Israel, M., N. Morel, B. Lesbats, S. Birman, and R. Manaranche. Purification of apresy-
naptic membrane protein that mediates a calcium-dependent translocation of
acetylcholine. Proc. Natl. Acad. Sci. USA 83: 9226-9230, 1986.
References 399
Ito, A., T. Kuwahara, S. Inadome, and Y. Sagara. Molecular cloning of a cDNA for rat
liver monoamine oxidase B. Biochem. Biophys. Res. Commun. 157: 970-976, 1988.
Itoh, N., J.R. Slemmon, D.H. Hawke, R. Williamson, E. Morita, K. Itakura, E. Roberts,
J.E. Shively, G.D. Crawford, and P.M. Salvaterra. Cloning of Drosophila choline
acetyltransferase cDNA. Proc. Natl. Acad. Sci. USA 83: 4081-4085, 1986.
Iversen, L.L. The uptake of catechol amines at high perfusion concentrations in the rat
isolated heart: A novel catechol amine uptake process. Brit. J. Pharmacol. 25:18-33,
1965.
Iversen, L.L. The Uptake and Storage of Noradrenaline in Sympathetic Nerves. Cam-
bridge: Cambridge University Press, 1967.
Iversen, L.L. Dopamine receptors in the brain. Science 188: 1084—1089, 1975.
Iversen, L.L., S.D. Iversen, F.E. Bloom, T. Vargo, and R. Guillemin. Release of
enkephalin from rat globus pallidus in vitro. Nature 271: 679-681, 1978.
Iversen, L.L., and E.A. Kravitz. Sodium dependence of transmitter uptake at adrener-
gic nerve terminals. Mol. Pharmacol. 2: 360-362, 1966.
Iversen, L.L., J.F. Mitchell, and V. Srinivasan. The release of y-aminobutyric acid dur-
ing inhibition in the cat visual cortex. J. Physiol. 212: 519-534, 1971.
Iversen, L.L., and M.J. Neal. The uptake of [3H]GABA by slices of rat cerebral cortex.
/. Neurochem. 15: 1141-1149, 1968.
Jacobs, M.H. Early osmotic history of the plasma membrane. Circulation 26:1013-1021,
1962.
Jacobson, A.L., C. Fried, and S.D. Horowitz. Planarians and memory. Nature 209:
599-600, 1966.
Jacobson, M. Foundations of Neuroscience. New York: Plenum Press, 1994.
Jahn, R., W. Schiebler, C. Ouimet, and P. Greengard. A 38,000-dalton membrane pro-
tein (p38) present in synaptic vesicles. Proc. Natl. Acad. Sci. USA 82: 4137-4141,
1985.
Jasper, H.H., and I. Koyama. Rate of release of amino acids from cerebral cortex in the
cat as affected by brainstem and thalamic stimulation. Canad. ]. Physiol. Pharma-
col. 47: 889-905, 1969.
Javitch, J.A., R.J. D'Amato, S.M. Strittmatter, and S.H. Snyder. Parkinsonism-inducing
neurotoxin, N-methyl-4-phenyl-l,2,3,6-tetrahydropyridine by dopamine neurons
explains selective toxicity. Proc. Natl Acad. Sci. USA 82: 2173-2177, 1985.
Javitt, D.C., and S.R. Zukin. Recent advances in the phencyclidine model of schizo-
phrenia. Amer. J. Psychiat. 148: 1301-1308, 1991.
Jeannerod, M. The Brain Machine. Cambridge, Mass.: Harvard University Press, 1985.
Jequier, E., D.S. Robinson, W. Lovenberg, and A. Sjoerdsma. Further studies on tryp-
tophan hydroxylase in rat brainstem and beef pineal. Biochem. Pharmacol. 18:
1071-1081, 1969.
Jessell, T.M. Adhesion molecules and the hierarchy of neural development. Neuron 1:
3-13, 1988.
Jeste, D.V., J.B. Lohr, and F.K. Goodwin. Neuroanatomical studies of major affective
disorders. Brit. J. Psychiat. 153: 444-459, 1988.
Joh, T.H., D.H. Park, and D.J. Reis. Direct phosphorylation of brain tyrosine hydroxy-
lase by cyclic AMP-dependent protein kinase. Proc. Natl. Acad. Sci. USA 75:
4744-4748, 1978.
Johnson, F.N. The History of Lithium Therapy. London: Macmillan, 1984.
Johnson, J.L., and M.H. Aprison. The distribution of glutamate and total free amino
acids in thirteen specific regions of the cat central nervous system. Brain Res. 26:
141-148, 1971.
Johnson, R.G., NJ. Carlson, and A. Scarpa. ApH and catecholamine distribution in iso-
lated chromaffin granules. /. Biol. Chem. 253: 1512-1521, 1978.
Johnson, R.G., D. Pfister, S.E. Carty, and A. Scarpa. Biological amine transport in chro-
maffin ghosts. /. Biol. Chem. 254: 10,963-10,972, 1979.
Johnston, J.P. Some observations upon a new inhibitor of monoamine oxidase in brain
tissue. Biochem. Pharmacol. 17: 1285-1297, 1968.
Johnston, M.V., R.L. MacDonald, and A.B. Young (eds.) Principles of Drug Therapy in
Neurology. Philadelphia: F.A. Davis, 1992.
Jolly, W.A. On the time relations of the knee-jerk and simple reflexes. Quart. ]. Exp.
Physiol 4: 67-87, 1911.
Jonasson, J., E. Rosengren, and B. Waldeck. Effects of pharmacologically active amines
on the uptake of arylalkylamines by adrenal medullary grannules. Acta Physiol.
Scand. 60: 136-140, 1964.
Jones, E.G. The neuron doctrine 1891. /. Hist. Neurosci. 3: 3-20, 1994.
Jones, S.F., and S. Kwanbunbumpen. The effects of nerve stimulation and hemicholin-
ium on synaptic vesicles at the mammalian neuromuscular junction. J. Physiol. 207:
31-50, 1970.
Jope, R.S. High affinity choline transport and acetylCoA production in brain and their
role in regulation of acetylcholine synthesis. Brain Res. Rev. 1: 313-344, 1979.
Jordan, C.C., and R.A. Webster. Release of acetylcholine and 14C-glycine from the cat
spinal cord in vivo. Brit. J. Pharmacol. 43: 441P, 1971.
Judson, H.F. The Eighth Day of Creation. New York: Simon and Schuster, 1979.
Julius, D., A.B. MacDermott, R. Axel, and T.M. Jessel. Molecular characterization of a
functional cDNA encoding the serotonin Ic receptor. Science 241: 558-564, 1988.
Kaczmarek, L.K. The role of protein kinase C in the regulation of ion channels and
neurotransmitter release. Trends Neurosci. 10: 30-34, 1987.
Kaczmarek, L.K., K.R. Jennings, F. Strumwasser, A.C. Nairn, U. Walter, F.D. Wilson,
and P. Greengard. Microinjection of catalytic subunit of cyclic AMP-dependent
protein kinase enhances calcium action potentials of bag cell neurons in cell cul-
ture. Proc. Natl. Acad. Sci. USA 77: 7487-7491, 1980.
Kahn, R.H. Uber humorale Ubertragbarkeit der Herznervenwirkung. Pfliigers Arch.
214: 481-498, 1926.
Kaibuchi, K., Y. Takai, M. Sawamura, M. Hoshijima, T. Fujikura, and Y. Nishizuka. Syn-
ergistic functions of protein phosphorylation and calcium mobilization in platelet
activation. /. Biol. Chem. 258: 6701-6704, 1983.
Kakiuchi, S., and T.W. Rail. The influence of chemical agents on the accumulation of
adenosine 3', 5'-phosphate in slices of rabbit cerebellum. Mol. Pharmacol. 4:
367-378, 1968.
Kakiuchi, S., and R. Yamazaki. Calcium dependent phosphodiesterase activity and its
activating factor (PAF) from brain. Biochem. Biophys. Res. Commun. 41: 1104-
1110, 1970.
Kalderon, N., I. Silman, S. Blumberg, and Y. Dudai. A method for the purification of
acetylcholinesterase by affinity chromatography. Biochim. Biophys. Acta 207: 560-
562, 1970.
Kallman, F.J. The genetic theory of schizophrenia: An analysis of 691 schizophrenic twin
index families. Amer. J. Psychiat. 103: 309-322, 1946.
Kamman, G.R., J.G. Freeman, and R.J. Lucero. The effect of 1-isonicotynyl 2-isopropyl
hydrazide (IIH) on the behavior of long-term mental patients./. Nerv. Ment. Dis.
118: 391-407, 1953.
Kanaseki, T., and K. Kadota. The "vesicle in a basket."/. Cell Biol. 42: 202-220, 1969.
References 401
Kandel, E.R., J.H. Schwartz, and T.M. Jessell. Principles of Neural Science. Third ed.
New York: Elsevier, 1991.
Kandel, E.R., and W.A. Spencer. Cellular neurophysiological approaches in the study
of learning. Physiol. Rev. 48: 65-134, 1968.
Kandel, E.R., L. Tauc. Heterosynaptic facilitation in neurones of the abdominal gan-
glion of Aplysia depilans. J. Physiol. 181: 1-27, 1965a.
Kandel, E.R., and L. Tauc. Mechanism of heterosynaptic facilitation in the giant cell of
the abdominal ganglion of Aplysia depilans. J. Physiol. 181: 28-47, 1965b.
Kane, J., G. Honigfeld, J. Singer, and H. Meltzer. Clozapine for the treatment-resistant
schizophrenic. Arch. Gen. Psychiat. 45: 789-796, 1988.
Kanigel, R. Apprentice to Genius. Baltimore: Johns Hopkins University Press, 1986.
Kanner, B.I., H. Fishkes, R. Maron, I. Sharon, and S. Schuldiner. Reserpine as a com-
petitive and reversible inhibitor of the catecholamine transporter of bovine chro-
maffin granules. FEES Lett. 100: 175-178, 1979.
Kanner, B.I., and S. Schuldiner. Mechanisn of transport and storage of neurotransmit-
ters. CRC Crit. Rev. Biochem. 22: 1-38, 1987.
Kao, P.N. and A. Karlin. Acetylcholine receptor binding site contains a disulfide crosslink
between adjacent half-cystinyl residues. /. Biol. Chem. 261: 8085-8088, 1986.
Kaplan, D.R., B.L. Hempstead, D. Martin-Zanca, M.V. Chao, and L.F. Parada. The trk
proto-oncogene product: A signal transducing receptor for nerve growth factor. Sci-
ence 252: 554-558, 1991.
Kaplan, N.R., and F. Lipmann. The assay and distribution of coenzyme A./. Biol. Chem.
147: 37-44, 1949.
Karayiorgou, M., and J.A. Gogos. A turning point in schizophrenia genetics. Neuron 19:
967-979, 1997.
Karlin, A. On the application of "a plausible model" of allosteric proteins to the recep-
tor for acetylcholine. /. Theoret. Biol. 16: 306-320, 1967.
Karlin, A., and D. Cowburn. The affinity-labeling of partially purified acetylcholine
receptor from electric tissue of Electrophorus. Proc. Natl. Acad. Sci. USA 12: 3636-
3640, 1973.
Karlsson, E., E. Heilbronn, and L. Widlund. Isolation of the nicotinic acetylcholine
receptor by biospecific chromatography on insolubilized Naja naja neurotoxin.
FEBS Lett. 28: 107-111, 1972.
Katada, T., and M. Ui. ADP ribosylation of the specific membrane protein of C6 cells
by islet-activating protein associated with modification of adenylate cyclase activ-
ity. /. Biol. Chem. 257: 7210-7216, 1982.
Kataoka, K. The subcellular distribution of substance P in the nervous system. Jap. J.
Physiol. 12: 81-96, 1962.
Katz, B. Impedance changes in frog's muscle associated with electrotonic and "end-
plate" potentials. /. Neurophysiol. 5: 169-184, 1942.
Katz, B. The transmission of impulses from nerve to muscle, and the subcellular unit
of synaptic action. Proc. Roy. Soc. 155B: 455-477, 1962.
Katz, B., and R. Miledi. A study of spontaneous miniature potentials in spinal motoneu-
rones. /. Physiol. 168: 389^22, 1963.
Katz, B., and R. Miledi. The effect of calcium on acetylcholine release from motor nerve
terminals. Proc. Roy. Soc. 161B: 496-503, 1965.
Katz, B., and R. Miledi. The timing of calcium action during neuromuscular transmis-
sion. /. Physiol. 189: 535-544, 1967a.
Katz, B., and R. Miledi. A study of synaptic transmission in the absence of nerve
impulses. /. Physiol. 192: 407-436, 1967b.
Katz, B., and R. Miledi. The release of acetylcholine from nerve endings by graded elec-
trical pulses. Proc. Roy. Soc. 167B: 23-38, 1967c.
Katz, B., and R. Miledi. The role of calcium in neuromuscular facilitation. J. Physiol.
195: 481-492, 1968.
Katz, B., and R. Miledi. Tetrodotoxin-resistant electric activity in presynaptic terminals.
/. Physiol. 203: 459-487, 1969.
Katz, B., and R. Miledi. Membrane noise produced by acetylcholine. Nature 226:
962-963, 1970.
Katz, B., and R. Miledi. The statistical nature of the acetylcholine potential and its
molecular components. J. Physiol. 224: 665-699, 1972.
Katz, B., and R. Miledi. Transmitter leakage from motor nerve endings. Proc. Roy. Soc.
196B: 59-72, 1977.
Katz, B., and O.H. Schmitt. Electric interaction between two adjacent nerve fibres.
/. Physiol. 97: 471-488, 1940.
Katz, B., and S. Thesleff. A study of the 'desensitization' produced by acetylcholine at
the motor end-plate. /. Physiol. 138: 63-80, 1957.
Katz, J.J., and W.C. Halstead. Protein organization and mental function. Comp. Psy-
chol. Monogr. 20: 1-38, 1950.
Katz, R.I., T.N. Chase, and I.J. Kopin. Effect of ions on stimulus-induced release of
amino acids from mammalian brain slices. J. Neurochem. 16: 961-967, 1969.
Kawagoe, R., K. Onodera, and A. Takeuchi. Release of glutamate from the crayfish neu-
romuscular junction./. Physiol. 312: 225-236, 1981.
Kebabian, J.W., and D.B. Calne. Multiple receptors for dopamine. Nature 277: 93-96,
1979.
Kebabian, J.W., and P. Greengard. Dopamine-sensitive adenyl cyclase: Possible role in
synaptic transmission. Science 174: 1346-1348, 1971.
Kebabian, J.W., G.L. Petzold, and P. Greengard. Dopamine-sensitive adenylate cyclase
in caudate nucleus of rat brain, and its similarity to the "dopamine receptor." Proc.
Natl. Acad. Sci. USA 69: 2145-2149, 1972.
Keen, J.H., M.H. Chestnut, and K.A. Beck. The clathrin coat assembly polypeptide
complex. /. Biol Chem. 262: 3864-3871, 1987.
Keen, J.H., M.C. Willingham, and I.H. Pastan. Clathrin-coated vesicles: Isolation, dis-
sociation and factor-dependent reassociation of clathrin baskets. Cell 16: 303-312,
1979.
Kelly, R.B. The cell biology of the nerve terminal. Neuron 1: 431-438, 1988.
Kelso, S.R., A.H. Ganong, and T.H. Brown. Hebbian synapses in hippocampus. Proc.
Natl. Acad. Sci. USA 83: 5326-5330, 1986.
Kennedy, M.B., and P. Greengard. Two calcium/calmodulin-dependent protein kinases,
which are highly concentrated in brain, phosphorylate protein I at distinct sites.
Kerr, L.M., and D. Yashikami. A venom peptide with novel presynaptic blocking action.
Nature 308: 282-284, 1984.
Kerr, M.A., and A.J. Kenny. The purification and specificity of a neutral endopeptidase
from rabbit kidney brush border. Biochem. J. 137: 477-488, 1974.
Kety, S.S. Biochemical theories of schizophrenia. Science 129: 1528-1532, 1590-1596,
1959.
Kety, S.S. Current biochemical approaches to schizophrenia. New Eng. J. Med. 276:
325-331, 1967.
Kety, S.S., D. Rosenthal, P.H. Wender, and F. Schulsinger. The types and prevalence
of mental illness in biological and adoptive families of adopted schizophrenics.
/. Psychiat. Res. 6: 345-362, 1968.
Reierences 403
Keynes, R.D., and P.R. Lewis. The sodium and potassium content of cephalopod nerve
fibres./. Physiol. 114: 151-182, 1951.
Keynes, R.D., and H. Martins-Ferreira. Membrane potentials in the electroplates of
the electric eel. /. Physiol. 119: 315-351, 1953.
Kibjakow, A.W. Uber humorale Ubertragung der Erregung von einem Neuron auf das
andere. Pflugers Arch. 232: 432-443, 1933.
Kiloh, L.G., J.P. Child, and G.A. Latner. A controlled trial of iproniazid in the treat-
ment of endogenous depression./. Ment. Sci. 106: 1139-1144, 1960.
Kilty, J.E., D. Lorang, and S.G. Amara. Cloning and expression of a cocaine-sensitive
rat dopamine transporter. Science 254: 578-579, 1991.
Kirchhausen, T., and S.C. Harrison. Protein organization in clathrin trimers. Cell 23:
755-761, 1981.
Kirk, S.A., and H. Kutchins. The Selling ofDSM. New York: Aldine de Gruyter, 1992.
Kirshner, N. Pathway of noradrenaline formation from dopa. /. Biol. Chem. 226: 821-
825, 1957.
Kirshner, N. Uptake of catecholamines by a particulate fraction of the adrenal medulla.
/. Biol. Chem. 237: 2311-2317, 1962.
Kirshner, N., H.J. Sage, and W.J. Smith. Mechanism of secretion from the adrenal
medulla. Mol. Pharmacol. 3: 254-265, 1967.
Kish, P.E., C. Fischer-Bovenkerk, and T. Ueda. Active transport of y-aminobutyric acid
and glycine into synaptic vesicles. Proc. Natl. Acad. Sci. USA 86: 3877-3881, 1989.
Kistler, J., R.M. Stroud, M.W. Klymkowsky, R.A. Lalancette, and R.M. Faircloth. Struc-
ture and function of an acetylcholine receptor. Biophys. J. 37: 371-383, 1982.
Klainer, L.M., Y.M. Chi, S.L. Freidberg, T.W. Rail, and E.W. Sutherland. Adenyl cyclase.
IV. /. Biol Chem. 237: 1239-1243, 1962.
Klawans, H.L., C. Goetz, and R. Westheimer. Pathophysiology of schizophrenia and the
striatum. Dis. Nerv. Syst. 33: 711-719, 1972.
Klein, M., and E.R. Kandel. Mechanism of calcium current modulation underlying
presynaptic facilitation and behavioral sensitization in Aplysia. Proc. Natl. Acad.
Sci. USA 77: 6912-6916, 1980.
Klein, R., S. Jing, V. Nanduri, E. O'Rourke, and M. Barbacid. The trk proto-oncogene
encodes a receptor for nerve growth factor. Cell 65: 189-197, 1991.
Kleinzeller, A. Exploring the cell membrane. Comp. Biochem. 39: 1-90, 1995.
Klett, R.P., B.W. Fulpius, D. Cooper, M. Smith, E. Reich, and L.D. Possani. The acetyl-
choline receptor. /. Biol. Chem. 248: 6841-6853, 1973.
Kline, N.S. Use of Rauwolfia serpentina benth. in neuropsychiatric conditions. Ann.
N.Y. Acad. Sci. 59: 107-132, 1954.
Kline, N.S. Clinical experience with iproniazid (Marsilid). /. Clin. Exp. Psychopath. 19
(2, suppl 1): 72-79, 1958.
Knepper, S.M., G.L. Grunewald, and C.O. Rutledge. Inhibition of norepinephrine trans-
port into synaptic vesicles by amphetamine analogs. /. Pharmacol. Exp. Ther. 247:
487-494, 1988.
Knoll, J. The possible mechanisms of action of ( — )deprenyl in Parkinsons disease.
/. Neural Trans. 43: 177-198, 1978.
Knoth, J., K. Handloser, and D. Njus. Electrogenic epinephrine transport in chromaf-
fm granule ghosts. Biochemistry 19: 2938-2942, 1980.
Kobilka, B.K., R.A.F. Dixon, T. Frielle, H.G. Dohlman, M.A. Bolanowski, I.S. Sigal,
T.L. Yang-Feng, U. Franke, M.G. Caron, and R.J. Lefkowitz. cDNA for the human
/32-adrenergic receptor: A protein with multiple membrane-spanning domains and
encoded by a gene whose chromosomal location is shared with that of the recep-
tor for platelet-derived growth factor. Proc. Natl. Acad. Sci. USA 84: 46-50, 1987.
Kobilka, B.K., T.S. Kobilka, K. Daniel, J.W. Regan, M.G. Caron, and R.J. Lefkowitz.
Chimeric a2-//32-adrenergic receptors: Delineation of domains involved in effector
coupling and ligand binding specificity. Science 240: 1310-1316, 1988.
Kobilka, B.K., H. Matsui, T.S. Kobilka, T.L. Yang-Feng, U. Francke, M.G. Caron,
R.J. Lefkowitz, and J.W. Regan. Cloning, sequencing, and expression of the gene
coding for the human platelet o^-adrenergic receptor. Science 238: 650-656,
1987.
Kohlhardt, M., B. Bauer, H. Krause, and A. Fleckenstein. Differentiation of the trans-
membrane Na and Ca channels in mammalian cardiac fibres by the use of specific
inhibitors. Pfliigers Arch. 335: 309-322, 1972.
Koidl, B., and E. Florey. Factor I and GABA: Resolution of a long-standing problem.
Comp. Biochem. Physiol 51C: 13-23, 1975.
Kolodkin, A.L., D.J. Matthes, and C.S. Goodman. The semaphorin genes encode a fam-
ily of transmembrane and secreted growth cone guidance molecules. Cell 75:
1389-1399, 1993.
Kolodkin, A.L., D.J. Matthes, T.P. O'Connor, N.H. Patel, A. Admon, D. Bentley, and
C.S. Goodman. Fasciclin IV: Sequence, expression, and function during growth
cone guidance in the grasshopper embryo. Neuron 9: 831-845, 1992.
Konishi, S., and M. Otsuka. Excitatory action of hypothalamic substance P on spinal
motoneurones of newborn rats. Nature 252: 734-735, 1974.
Kopin, I.J. Metabolic degradation of catecholamines. Handb. Exp. Pharmakol. 33:
270-282, 1972.
Kopin, I.J., G.R. Breese, K.R. Krauss, and V.K. Weise. Selective release of newly syn-
thesized norepinephrine from the cat spleen during sympathetic nerve stimulation.
Kopin, I.J., G. Hertting, and E.K. Gordon. Fate of norepinephrine-H3 in the isolated
perfused rat heart. /. Pharmacol. Exp. Ther. 138: 34-40, 1962.
Korey, S.R., B. deBraganza, and D. Nachmansohn. Choline acetylase. V. J. Biol. Chem.
189: 705-715, 1951.
Korkes, S., A. delCampillo, S.R. Korey, J.R. Stern, D. Nachmansohn, and S. Ochoa.
Coupling of acetyl donor systems with choline acetylase. /. Biol. Chem. 198: 215-
220, 1952.
Korn, H., A. Triller, A. Mallet, and D.S. Faber. Fluctuating responses at a central
synapse: n of binomial fit predicts number of stained presynaptic boutons. Science
213: 898-901, 1981.
Kornberg, A. The NIH did it! Science 278: 1863, 1997.
Kornhuber, J., P. Riederer, G.P. Reynolds, H. Beckmann, K. Jellinger, and E. Gabriel.
3
H-spiperone binding in post-mortem brains from schizophrenic patients: Rela-
tionship to neuroleptic drug treatment, abnormal movements, and positive symp-
toms. /. Neural Trans. 75: 1-10, 1989.
Korsching, S., and H. Thoenen. Nerve growth factor in sympathetic ganglia and corre-
sponding target organs of the rat: Correlation with density of sympathetic inner-
vation. Proc. Natl. Acad. Sci. USA 80: 3513-3516, 1983.
Koshland, D.E., G. Nemethy, and D. Filmer. Comparison of experimental binding data
and theoretical models in proteins containing subunits. Biochemistry 5: 365-385,
1966.
Kosterlitz, H.W. The best laid schemes o' mice an' men gang aft agley. Annu. Rev. Phar-
macol. 19: 1-12, 1979.
Kravitz, E.A., S.W. Kuffler, and D.D. Potter. Gamma-aminobutyric acid and other block-
ing compounds in Crustacea./. Neurophysiol. 26: 739-751, 1963.
References 405
Kravitz, E.A., D.D. Potter, and N.M. van Gelder. Gamma-aminobutyric acid and other
blocking substances extracted from crab muscle. Nature 194: 382-383, 1962.
Krebs, E.G. The growth of research on protein phosphorylation. Trends Biochern. Sci.
19: 439, 1994.
Krebs, E.G., and E.H. Fischer. The phosphorylase b to a converting enzyme of rabbit
skeletal muscle. Biochim. Biophys. Acta 20: 150-157, 1956.
Krebs, E.G., D.J. Graves, and E.H. Fischer. Factors affecting the activity of muscle
phosphorylase b kinase. /. Biol. Chem. 234: 2867-2873, 1959.
Kremzner, L.T., and I.E. Wilson. A chromatographic procedure for the purification of
acetylcholinesterase. /. Biol. Chem. 238: 1714-1717, 1963.
Kriebel, M.E., and C.E. Gross. Multimodal distribution of frog miniature endplate
potentials in adult, denervated, and tadpole leg muscle./. Gen. Physiol. 64: 85-103,
1974.
Kriebel, M.E., J. Vautrin, and J. Holsapple. Transmitter release: Prepackaging and ran-
dom mechanism or dynamic and deterministic process. Brain Res. Rev. 15: 167-
178, 1990.
Krnjevic, K., and J.W. Phillis. Sensitivity of cortical neurones to acetylcholine. Experi-
entia 17: 469, 1961.
Krnjevic, K., and J.W. Phillis. Excitation of Betz cells by acetylcholine. Experienta 18:
170-171, 1962.
Krnjevic, K., and J.W. Phillis. lontophoretic studies of neurones in the mammalian cere-
bral cortex. /. Physiol. 165: 274-304, 1963a.
Krnjevic, K., and J.W. Phillis. Acetylcholine-sensitive cells in the cerebral cortex./. Phys-
iol. 166: 296-327, 1963b.
Krnjevic, K., and J.W. Phillis. Pharmacological properties of acetylcholine-sensitive cells
in the cerebral cortex. /. Physiol. 166: 328-350, 1963c.
Krnjevic, K., and J.W. Phillis. Actions of certain amines on cerebral cortical neurones.
Brit. J. Pharmacol. 20: 471^90, 1963d.
Krnjevic, K., and S. Schwartz. The action of y-aminobutyric acid on cortical neurones.
Exp. Brain Res. 3: 320-326, 1967.
Kubo, T, K. Fukuda, A. Mikami, A. Maeda, H. Takahashi, M. Mishina, T. Haga, K.
Haga, A. Ichiyama, K. Kangawa, M. Kojima, H. Matsuo, T. Hirose, and S. Numa.
Cloning, sequencing and expression of complementary DNA encoding the mus-
carinic acetylcholine receptor. Nature 323: 411-416, 1986a.
Kubo, T, A. Maeda, K. Sugimoto, I. Akiba, A. Mikami, H. Takahashi, T. Haga, K, Haga,
A. Ichiyama, K. Kangawa, H. Matsuo, T. Hirose, and S. Numa. Primary structure
of porcine cardiac nuscarinic acetylcholine receptor deduced from the cDNA
sequence. FEBS Lett. 209: 367-372, 1986b.
Kuczmarski, E.R., and J.L. Rosenbaum. Studies on the organization and localization of
actin and myosin in neurons. /. Cell Biol. 80: 356-371, 1979.
Kuffler, S.W Electric potential changes at an isolated nerve-muscle junction. /. Neuro-
physiol. 5: 18-26, 1942.
Kuffler, S.W. Physiology of neuro-muscular junctions: Electrical aspects. Fed. Proc. 7:
437-446, 1948.
Kuffler, S.W, and C. Edwards. Mechanism of gamma aminobutyric acid (GABA) action
and its relation to synaptic inhibition./. Neurophysiol. 21: 588-610, 1958.
Kuffler, S.W, and D. Yoshikami. The number of transmitter molecules in a quantum.
/. Physiol. 251: 465-482, 1975.
Kuhar, M.J., C.B. Pert, and S.H. Snyder. Regional distribution of opiate receptor bind-
ing in monkey and human brain. Nature 245: 447-450, 1973.
Kuhn, R. liber die Behandlung depressiver Zustande mit einem Imidodibenzylderiva-

tiv (G22355). Schweiz. Med. Wchnschr. 87: 1135-1140, 1957.
Kuhn, R. The treatment of depressive states with G22355 (imipramine hydrochloride).
Amer. J. Psychiat. 115: 459-464, 1958.
Kiihne, W. On the origin and the causation of vital movement. Proc. R. Soc. 44: 427-448,
1888.
Kiilz, F. Zur Humoralphysiologie des Froschherzens. Naunyn-Schmiedebergs Arch. 134:
252-256, 1928.
Kumakura, K., A. Guidotti, and E. Costa. Primary cultures of chromaffin cells: Molec-
ular mechanisms for the induction of tyrosine hydroxylase mediated by 8-Br-cyclic
AMP. Mol. Pharmacol. 16: 865-876, 1979.
Kuno, M. Mechanism of facilitation and depression of the excitatory synaptic potential
in spinal motoneurones./. Physiol. 175: 100-112, 1964.
Kuno, M., and P. Rudomin. The release of acetylcholine from the spinal cord of the cat
by antidromic stimulation of motor nerves. /. Physiol. 187: 177-193, 1966.
Kuo, J.F., and P. Greengard. Cyclic nucleotide-dependent protein kinases, IV. Proc. Natl.
Acad. Set. USA 64: 1349-1355, 1969.
Kupfermann, I., V. Castellucci, H. Pinsker, and E. Kandel. Neuronal correlates of habit-
uation and dishabituation of the gill-withdrawal reflex in Aplysia. Science 167:
1743-1745, 1970.
Kuriyama, K., B. Haber, B. Sisken, and E. Roberts. The y-aminobutyric acid system in
rabbit cerebellum. Proc. Natl. Acad. Sci. USA 55: 846-852, 1966.
Kuriyama, K., H. Weinstein, and E. Roberts. Uptake of y-aminobutyric acid by mito-
chondrial and synaptosomal fractions from mouse brain. Brain Res. 16: 479—492,
1969.
LaBrosse, E., J. Axelrod, and S.S. Kety. O-methylation, the principal route of metabo-
lism of epinephrine in man. Science 128: 593-594, 1958.
Lamb, A.H. Neuronal death in the development of the somatotopic projections of the
ventral horn in Xenopus. Brain Res. 134: 145-150, 1977.
Lance-Jones, C., and L. Landmesser. Pathway selection by chick lumbosacral motoneu-
rons during normal development. Proc. Roy. Soc. 214B: 1-18, 1981.
Lands, A.M., A. Arnold, J.P. McAuliff, F.P. Luduena, and T.G. Brown. Differentiation
of receptor systems activated by sympathomimetic amines. Nature 214: 597-598,
1967a.
Lands, A.M., F.P. Luduena, and H.J. Buzzo. Differentiation of receptors responsive to
isoproterenol. Life Sci. 6: 2241-2249, 1967b.
Langdon-Brown, W. W.H. Gaskell and the Cambridge Medical School. Proc. R. Soc.
Med. 33: 1-12, 1939.
Langer, S.Z. The metabolism of [3H]noradrenaline released by electrical stimulation
from the isolated nictitating membrance of the cat and from the vas deferens of
the rat. /. Physiol. 208: 515-546, 1970.
Langer, S.Z. Presynaptic regulation of catecholamine release. Biochem. Pharmacol. 23:
1793-1800, 1974.
Langley, J.N. On the physiology of the salivary secretion. /. Physiol. 1: 339-369, 1878.
Langley, J.N. Preliminary account of the arrangement of the sympathetic nervous sys-
tem, based chiefly on observations upon pilo-motor nerves. Proc. R. Soc. 52:
547-555, 1893.
Langley, J.N. Note on regeneration of pre-ganglionic fibres of the sympathetic./. Phys-
iol. 18: 280-284, 1895.
References 407
Langley, J.N. On the union of cranial autonomic (visceral) fibres with the nerve cells of
the superior cervical ganglion. J. Physiol. 23: 240-270, 1898.
Langley, J.N. On the stimulation and paralysis of nerve-cells and of nerve-endings.
/. Physiol. 27: 224-236, 1901a.
Langley, J.N. Observations on the physiological action of extracts of the supra-renal bod-
ies. /. Physiol. 27: 237-256, 1901b.
Langley, J.N. On the reaction of cells and of nerve-endings to certain poisons, chiefly
as regards the reaction of striated muscle to nicotine and to curari. J. Physiol. 33:
374-413, 1905.
Langley, J.N. On nerve endings and on special excitable substances in cells. Proc. R.
Soc. 78B: 170-194, 1906.
Langley, J.N. On the contraction of muscle, chiefly in relation to the presence of "recep-
tive" substances. ].Physiol. 36: 347-384, 1907.
Langley, J.N., and W.L. Dickinson. On the local paralysis of peripheral ganglia, and on
the connexion of different classes of nerve fibres with them. Proc. R. Soc. 46:
423-431, 1889.
Langmuir, I. The constitution and fundamental properties of solids and liquids./. Amer.
Chem. Soc. 38: 2221-2295, 1916.
Langston, J.W., P. Ballard, J.W. Tetrud, and I. Irwin. Chronic parkinsonism in humans
due to a product of meperidine-analog synthesis. Science 219: 979-980, 1983.
Langston, J.W., and J. Palfreman. The Case of the Frozen Addicts. New York: Pantheon
Books, 1995.
Lapicque, L. Definition experimentale de 1'excitabilite. Compt. Rend. Soc. Biol. 67:
280-283, 1909.
Lapicque, L. L'Excitabilite en Fonction du Temps. Paris: Les Presses Universitaires de
France, 1926.
Lapicque, L. Has the muscular substance a longer chronaxie than the nervous sub-
stance?/. Physiol. 73: 189-214, 1931.
Lapicque, L. Neuro-muscular isochronism and chronological theory of curarization.
/. Physiol 81: 113-145, 1934.
Lapicque, M. et Mme. Sur le mecanisme de la curarisation. Compt. Rend. Soc. Biol.
65: 733-735, 1908.
Laporte, Y., and D.P.C. Lloyd. Nature and significance of the reflex connections estab-
lished by large afferent fibers of muscular origin. Am. J. Physiol. 169: 609-621,1952.
Lashley, K.S. Studies of cerebral function in learning./. Comp. Neurol. 41: 1-48, 1926.
Lashley, K.S. Brain Mechanisms and Intelligence. Chicago: University of Chicago Press,
1929.
Lashley. K.S. In search of the engram. Symp. Soc. Exp. Biol. 4: 454-482, 1950.
Lasky, J.J., C.J. Klett, E.M. Caffey, J.L. Bennett, M.P. Rosenblum, and L.E. Hollister.
Drug treatment of schizophrenic patients. Dis. Nero. Syst. 23: 698-706, 1962.
Laudan, L. Beyond Positivism and Relativism. Boulder, Colo.: Westview Press, 1996.
Laverty, R., LA. Michaelson, D.F. Sharman, and V.P. Whittaker. The subcellular local-
ization of dopamine and acetylcholine in the dog caudate nucleus. Brit. J. Phar-
macol 21: 482^90, 1963.
Lee, K.S., F. Schottler, M. Oliver, and G. Lynch. Brief bursts of high-frequency stimu-
lation produce two types of structural change in rat hippocampus. /. Neurophys-
iol. 44: 247-258, 1980.
Lee, R.M. Conditioning of a free operant response in planaria. Science 139: 1048-1049,
1963.
Leeb-Lundberg, L.M.F., S. Cotecchia, A. DeBlasi, M.G. Caron, and R.J. Lefkowitz.

Regulation of adrenergic receptor function by phosphorylation. Proc. Natl. Acad.
Sci. USA 262: 3098-3105, 1987.
Leff, J. International variations in the diagnosis of psychiatric illness. Brit. J. Psychiat.
131: 329-338, 1977.
Lefkowitz, R.J. Molecular pharmacology of beta-adrenergic receptors—A status report.
Biochem. Phartnacol. 23: 2069-2076, 1974.
Lefkowitz, R.J., B.K. Kobilka, and M.G. Caron. The new biology of drug receptors.
Biochem. Pharmacol. 38: 2941-2948, 1989.
Lefkowitz, R.J., C. Mukherjee, M. Coverstone, and M.G. Caron. Stereospecific [3H]( —)-
alprenolol binding sites, /3-adrenergic receptors and adenylate cyclase. Biochem.
Biophys. Res. Commun. 60: 703-709, 1974.
Lefkowitz, R.J., and L.T. Williams. Catecholamine binding to the /3-adrenergic recep-
tor. Proc. Natl. Acad. Sci. USA 74: 515-519, 1977.
Lehmann, H.E., C.H. Cahn, and R.L. deVerteuil. The treatment of depressive condi-
tions with imipramine (G 22355). Can. Psychiat. Assoc. J. 3: 155-164, 1958.
Leloir, L.F., J.M. Olavarria, S.H. Goldemberg, and H. Carminatti. Biosynthesis of glyco-
gen from undine diphosphate glucose. Arch. Biochem. Biophys. 81: 508-520,1959.
Lembeck, F. Zur Frage der zentralen Ubertragung afferenter Impulse. Naunyn-
Schmiedebergs Arch. 219: 197-213, 1953.
Leonard, R.J., C.G. Labarca, P. Charnet, N. Davidson, and H.A. Lester. Evidence that
the M2 membrane-spanning region lines the ion channel pore of the nicotinic
receptor. Science 242: 1578-1581, 1988.
Leonhard, K. Aufteilung der Endogenen Psychosen. Berlin: Akademie-Verlag, 1957.
Leplin, J. (ed.) Scientific Realism. Berkeley: University of California Press, 1984.
Leszczyszyn, D.J., J.A. Jankowski, O.H. Viveros. E.J. Diliberto, J.A. Near, and R.M.
Wightman. Nicotinic receptor-mediated catecholamine secretion from chromaffin
cells. /. Biol. Chem. 265: 14,736-14,737, 1990.
Letourneau, P.C. Possible roles for cell-to-substratum adhesion in neuronal morpho-
genesis. Dev. Biol. 44: 77-91, 1975.
Letourneau, P.C. Chemotactic response of nerve fiber elongation to nerve growth fac-
tor. Dev. Biol. 66: 183-196, 1978.
Letourneau, P.C. Cell-substratum adhesion of neurite growth cones, and its role in neu-
rite elongation. Exp. Cell Res. 124: 127-138, 1979.
Letourneau, P.C. Differences in the organization of actin in the growth cones compared
with the neurites of cultured neurons from chick embryos./. Cell Biol. 97: 963-973,
1983.
Letourneau, PC., A.M. Madsen, S.L. Palm, and L.T. Furcht. Immunoreactivity for
laminin in the developing ventral longitudinal pathway of the brain. Dev. Biol. 125:
135-144, 1988.
Letourneau, PC., and T.A. Shattuck. Distribution and possible interactions of actin-
associated proteins and cell adhesion molecules of nerve growth cones. Develop-
ment 105: 505-519, 1989.
Leube, R.E., P. Kaiser, A. Seiter, R. Zimbelmann, W.W. Franke, H. Rehm, P. Knaus, P.
Prior, H. Betz, H. Reinke, K. Beyreuther, and B. Wiedenmann. Synaptophysin:
Molecular organization and mRNA expression as determined from cloned cDNA.
EMBOJ. 6: 3261-3268, 1987.
Leuzinger, W., and A.L. Baker. Acetylcholinesterase, I. Large-scale purification, homo-
geneity, and amino acid analysis. Proc. Natl. Acad. Sci. USA 57: 446-451, 1967.
Levi-Montalcini, R. In Praise of Imperfection. New York: Basic Books, 1988.
References 409
Levi-Montalcini, R., and S. Cohen. Effects of the extract of the mouse submaxillary sali-
vary glands on the sympathetic system of mammals. Ann. N.Y. Acad. Sci. 85: 324-
341, 1960.
Levi-Montalcini, R., and V. Hamburger. Selective growth stimulating effects of mouse
sarcoma on the sensory and sympathetic nervous system of the chick embryo.
/. Exp. Zool. 116: 321-351, 1951.
Levi-Montalcini, R., and V. Hamburger. A diffusible agent of mouse sarcoma, produc-
ing hyperplasia of sympathetic ganglia and hyperneurotization of viscera in the
chick embryo. /. Exp, Zool. 123: 233-278, 1953.
Levi-Montalcini, R., H. Meyer, and V. Hamburger. In vitro experiments on the effects
of mouse sarcomas 180 and 37 on the spinal and sympathetic ganglia of the chick
embryo. Cancer Res. 14: 49-57, 1954.
Levin, L.R., P.L. Han, P.M. Hwang, P.G. Feinstein, R.L. Davis, and R.R. Reed. The
Drosophila learning and memory gene rutabaga encodes a Ca2+/calmodulin-
responsive adenyl cyclase. Cell 68: 479-489, 1992.
Levitan, E.S., P.R. Schofield, D.R. Hurt, L.M. Rhee, W. Wisden, M. Kohler, N. Fujita,
H.F. Rodriguez, A. Stephenson, M.G. Darlison, E.A. Bernard, and PH. Seeburg.
Structural and functional basis for GABAA receptor heterogeneity. Nature 335:
76-79, 1988.
Levitt, M., S. Spector, A. Sjoerdsma, and S. Udenfriend. Elucidation of the rate-limit-
ing step in norepiniphrine biosynthesis in the perfused guinea-pig heart. /. Phar-
macol. Exp. Ther. 148: 1-8, 1965.
Levitzki, A., D. Atlas, and M.L. Steer. The binding characteristics and number of
/3-adrenergic receptors on the turkey erythrocyte. Proc. Natl. Acad. Sci. USA 71:
2773-2776, 1974.
Levy, W.B., and O. Steward. Synapses as associative memory elements in the hip-
pocampal formation. Brain Res. 175: 233-245, 1979.
Levy, W.B., and O. Steward. Temporal contiguity requirements for long-term associa-
tive potentiation/depression in the hippocampus. Neuroscience 8: 791-797, 1983.
Lewis, E.J., C.A. Harrington, and O.M. Chikaraishi. Transcriptional regulation of the
tyrosine hydroxylase gene by glucocorticoid and cyclic AMP. Proc. Natl. Acad, Sci,
USA 84: 3550-3554, 1987.
Lewis, R.V., S. Stein, L.D. Gerber, M. Rubinstein, and S. Udenfriend. High molecular
weight opioid-containing proteins in striatum. Proc. Natl. Acad. Sci. USA 75: 4021-
4023, 1978.
Lewis, R.V., A.S. Stern, S. Kimura, J. Rossier, S. Stein, and S. Udenfriend. An about
50,000-Dalton protein in adrenal medulla: A common precursor of [Met]- and
[Leu]enkephalin. Science 208: 1459-1461, 1980.
Lewis, S. Computerized tomography in schizophrenia 15 years on. Brit. J. Psychiat. 157:
16-24, 1990.
Li, C.H., and D. Chung. Isolation and structure of an untriakontapeptide with opiate
activity from camel pituitary glands. Proc. Natl. Acad. Sci. USA 73: 1145-1148,
1976.
Liddell, E.G.T. The Discovery of Reflexes. Oxford: Oxford University Press, 1960.
Lieberman, A., M. Kupersmith, E. Estey, and M. Goldstein. Treatment of Parkinson's
disease with bromocriptine. New Eng. J. Med. 295: 1400-1404, 1976.
Liley, A.W. An investigation of spontaneous activity at the neuromuscular junction of
the rat. /. Physiol. 132: 650-666, 1956.
Limbird, L.E. Activation and attenuation of adenylate cyclase. Biochem. J. 195: 1-13,
1981.
Ling, G., and R.W. Gerard. The normal membrane potential of frog sartorius fibers.
/. Cell. Comp. Physiol 34: 383-396, 1949.
Linn, T.C., F.H. Pettit, F. Hucho, and L.J. Reed. a-Keto acid dehydrogenase complexes,
XI. Proc. Natl. Acad. Sci. USA 64: 227-234, 1969.
Lipkin, D., W.H. Cook, and R. Markham. Adenosine-3':5'-phosphoric acid: A proof of
structure. /. Amer. Chem. Soc. 81: 6198-6203, 1959.
Lipmann, F. Metabolic generation and utilization of phosphate bond energy. Adv. Efizy-
mol. 1: 99-162, 1941.
Lipmann, F. Acetylation of sulfanilamide by liver homogenates and extracts. /. Biol.
Chem. 160: 173-190, 1945.
Lipmann, F, and N.O. Kaplan. A common factor in the enzymatic acetylation of sul-
fanilamide and of choline. /. Biol. Chem. 162: 743-744, 1946.
Liu, Y., D. Peter, A. Roghani, S. Schuldiner, G.G. Prive, D. Eisenberg, N. Brecha, and
R.H. Edwards. A cDNA that suppresses MPP+ toxicity encodes a vesicular amine
transporter. Cell 70: 539-551, 1992.
Livingstone, M.S., P.P. Sziber, and W.G. Quinn. Loss of calcium/calmodulin respon-
siveness in adenylate cyclase of rutabaga, a Drosophila learning mutant. Cell 37:
205-215, 1984.
Llinas, R., J.R. Blinks, and C. Nicholson. Calcium transient in presynaptic terminal of
squid giant synapse: Detection with aequorin. Science 176: 1127-1129, 1972.
Llinas, R., T.L. McGuinness, C.S. Leonard, M. Sugimori, and P. Greengard. Intrater-
minal injection of synapsin I or calcium/calmodulin-dependent protein kinase II
alters neurotransmitter release at the squid giant synapse. Proc. Natl. Acad. Sci.
USA 82: 3035-3039, 1985.
Llinas, R., and C. Nicholson. Calcium role in depolarization-secretion coupling: An
aequorin study in squid giant synapse. Proc. Natl. Acad. Sci. USA 72: 187-190,
1975.
Llinas, R., I.Z. Steinberg, and K. Walton. Presynaptic calcium currents and their rela-
tion to synaptic transmission: Voltage clamp study in squid giant synapse and the-
oretical model for the calcium gate. Proc. Natl. Acad. Sci. USA 73: 2918-2922,
1976.
Llinas, R., I.Z. Steinberg, and K. Walton. Relationship between presynaptic calcium
current and postsynaptic potential in squid giant synapse. Biophys. J. 33: 323-352,
1981.
Loewi, O. Uber humorale Ubertragbarkeit der Herznervenwirkung. I. Pflugers Arch.
189: 239-242, 1921.
Loewi, O. tJber humorale Ubertragbarkeit der Herznervenwirkung. II. Pflugers Arch.
193: 201-213, 1922.
Loewi, O. The humoral transmission of nervous impulse. Harvey Lect. 27: 218-232,
1934.
Loewi, O. Quantitative und qualitative Untersuchungen iiber den Sympathicusstoff.
Pflugers Arch. 237: 504-514, 1936.
Loewi, O. Chemical transmission of nerve impulses. Amer. Sci. 33: 159-174, 1945a.
Loewi, O. Aspects of the transmission of the nervous impulse. /. Mt. Sinai Hosp. 12:
803-816, 1945b.
Loewi, O. Introduction. Pharmacol. Rev. 6: 3-6, 1954.
Loewi, O. An autobiographical sketch. Persp. Biol. Med. 4: 3-25, 1960.
Loewi, O., and E. Navratil. Uber humorale Ubertragbarkeit der Herznervenwirkung.
VI. Pflugers Arch. 206: 123-134, 1924.
References 411

X. Pflugers Arch. 214: 678-688, 1926a.
XI. Pflugers Arch. 214: 689-696, 1926b.
Loffelholz, K., and E. Muscholl. A muscarinic inhibition of the noradrenaline release
evoked by postganglionic sympathetic nerve stimulation. Naunyn-Schmiedebergs
Arch. 265: 1-15, 1969.
Logan, W.J., and S.H. Snyder. Unique high affinity uptake systems for glycine, glutamic
and aspartic acids in central nervous tissue of the rat. Nature 234: 297-298, 1971.
Logothetis, D.E., Y. Kurachi, J. Galper, E.J. Neer, and D.E. Clapham. The ]8y subunits
of GTP-binding proteins activate the muscarinic K + channel in heart. Nature 325:
321-326, 1987.
Lohse, M.J., J.L. Benovic, J. Codina, M.G. Caron, and R.J. Lefkowitz. j8-arrestin: A pro-
tein that regulates /3-adrenergic receptor function. Science 248: 1547-1550, 1990.
L0mo, T. Frequency potentiation of excitatory synaptic activity in the dentate area of
the hippocampal formation. Acta Physiol. Scand. Suppl. 277: 128, 1966.
Londos, C., D.M.F. Cooper, and M. Rodbell. Receptor-mediated stimulation and inhi-
bition of adenylate cyclases. Adv. Cyclic Nucleotide Res. 14: 163-171, 1981.
Londos, C., Y. Salomon, M.C. Lin, J.P. Harwood, M. Schramm, J. Wolff, and M. Rod-
bell. 5'-Guanylylimidodiphosphate, a potent activator of adenylate cyclase systems
in eukaryotic cells. Proc. Natl. Acad. Sci. USA 71: 3087-3090, 1974.
Loomer, H.P., J.C. Saunders, and N.S. Kline. A clinical and pharmacodynamic evalua-
tion of iproniazid as a psychic energizer. Psychiat. Res. Rpts. 8: 129-141, 1957.
Lord, J.A.H., A.A. Waterfield, J. Hughes, and H.W. Kosterlitz. Endogenous opioid pep-
tides: Multiple agonists and receptors. Nature 267: 495-499, 1977.
Lorente de No, R. The synaptic delay of the motoneurones. Amer. J. Physiol. Ill:
272-281, 1935.
Lorente de No, R. Transmission of impulses through cranial motor nuclei. /. Neuro-
physiol 2: 402-464, 1939.
Lovenberg, W., E. Jequier, and A. Sjoerdsma. Tryptophan hydroxylation: Measurement
in pineal gland, brainstem, and carcinoid tumor. Science 155: 217-219, 1967.
Lovenberg, W., H. Weissbach, and S. Udenfriend. Aromatic L-amino acid decarboxy-
lase. /. Biol. Chem. 237: 89-93, 1962.
Lowney, L.I., K. Schulz, P.J. Lowery, and A. Goldstein. Partial purification of an opiate
receptor from mouse brain. Science 183: 749-753, 1974.
Lucas, K. On the optimal electric stimuli of muscle and nerve./. Physiol. 35: 103-114,
1907a.
Lucas, K. The analysis of complex excitable tissues by their response to electric cur-
rents of short duration. /. Physiol 35: 310-331, 1907b.
Lucas, K. The excitable substances of amphibian muscle./. Physiol. 36:113-135,1907c.
Lucas, K. The process of excitation in nerve and muscle. Proc. R. Soc. 85B: 495-524,
1912.
Lukowiak, K., and C. Sahley. The in vitro classical conditioning of the gill withdrawal
reflex of Aplysia californica. Science 212: 1516-1518, 1981.
Lumsden, A.G.S., and A.M. Davies. Earliest sensory nerve fibres are guided to periph-
eral targets by attractants other than nerve growth factor. Nature 306: 786-788,
1983.
Luo, Y, D. Raible, and J.A. Raper. Collapsin: A protein in brain that induces the col-
lapse and paralysis of neuronal growth cones. Cell 75: 217-227, 1993.
Lust, W.D., and J.D. Robinson. Calcium accumulation by isolated nerve ending parti-
cles from brain. II. Factors influencing calcium movements. J. Neurobiol. 1: 317-
328, 1970.
Lynch, G., J. Larson, S. Kelso, G. Barronuevo, and F. Schottler. Intracellular injections
of EGTA block induction of hippocampal long-term potentiation. Nature 305:
719-721, 1983.
Lynen, F. Acetyl coenzyme A and the "fatty acid cycle." Harvey Lect. 48: 210-244, 1953.
Maas, J.W. Biogenic amines and depression. Arch. Gen. Psychiat. 32: 1357-1361, 1975.
MacDermott, A.B., M.L. Mayer, G.L. Westbrook, S.J. Smith, and J.L. Barker. NMDA-
receptor activation increases cytoplasmic calcium concentration in cultured spinal
cord neurones. Nature 321: 519-522, 1986.
Macdonald, J.S. The structure and function of nerve fibres. Proc. R. Soc. 76B: 322-350,
1905.
Macintosh, F.C. The distribution of acetylcholine in the peripheral and central nerv-
ous system./. Physiol. 99: 436-442, 1941.
Macintosh, F.C., R.I. Birks, and P.B. Sastry. Pharmacological inhibition of acetylcholine
synthesis. Nature 178: 1181, 1956.
Macintosh, F.C., and P.E. Oborin. Release of acetylcholine from intact cerebral cortex.
Abstracts, XIX International Physiological Congress, 580-581, 1953.
MacMillan, W.H. A hypothesis concerning the effects of cocaine on the action of sym-
pathomimetic amines. Brit. J. Pharmacol. 14: 385-391, 1959.
Maeno, H., and P. Greengard. Phosphoprotein phosphatases from rat cerebral cortex.
/. Biol. Chem. 247: 3269-3277, 1972.
Magill-Solc, C., and U.J. McMahan. Motor neurons contain agrin-like molecules./. Cell
Biol. 107: 1825-1833, 1988.
Maguire, M.E., P.H. Goldmann, and A.G. Gilman. The reaction of [3H]norepinephrine
with particulate fractions of cells responsive to catecholamines. Mol. Pharmacol.
10: 563-581, 1974.
Mahan, L.C., A.M. Koachman, and P.A. Insel. Genetic analysis of /3-adrenergic recep-
tor internalization and down-regulation. Proc. Natl. Acad. Sci. USA 82: 129-133,
1985.
Mains, R.E., I.M. Dickerson, V. May, D.A. Staffers, S.N. Perkins, L. Ouafik, E.J. Hus-
ten, and B.A. Eipper. Cellular and molecular aspects of peptide hormone biosyn-
thesis. Front. Neuroendc-crin. 11: 52-89, 1990.
Maitre, L. Monoamine oxidase inhibiting properties of SU-11,739 in the rat. /. Phar-
macol. Exp. Ther. 157: 81-88, 1967.
Makowski, L., D.L.D. Caspar, W.C. Phillips, and D.A. Goodenough. Gap junction struc-
tures. /. Cell Biol. 74: 629-645, 1977.
Malenka, R.C., J.A. Kauer, D.J. Perkel, M.D. Mauk, P.T. Kelly, R.A. Nicoll, and M.N.
Waxham. An essential role for postsynaptic calmodulin and protein kinase activity
in long-term potentiation. Nature 340: 554-557, 1989.
Malfroy, B., P.R. Schofield, W.J. Kuang, P.H. Seeburg, A.J. Mason, and W.J. Henzel.
Molecular cloning and amino acid sequence of rat enkephalinase. Biochem. Bio-
phys. Res. Commun. 144: 59-66, 1987.
Malfroy, B., J.P. Swerts, A. Guyon, B.P. Roques, and J.C. Schwartz. High-affinity
enkephalin-degrading peptidase in brain is increased after morphine. Nature 276:
523-526, 1978.
Malinow, R., D.V. Madison, and R.W. Tsien. Persistent protein kinase activity underly-
ing long-term potentiation. Nature 335: 820-824, 1988.
Rererences 413
Malinow, R., and R.W. Tsien. Presynaptic enhancement shown by whole-cell record-
ings of long-term potentiation in hippocampal slices. 'Nature 346: 177-180, 1990.
Maloteaux, J.M., A. Goussuin, P.J. Pauwels, and P.M. Laduron. Short-term disappear-
ance of muscarinic cell surface receptors in carbachol-induced desensitization.
FEES Lett. 156: 103-107, 1983.
Manfredi, J.J., and W.L. Bazari. Purification and characterization of two distinct com-
plexes of assembly polypeptides from calf brain coated vesicles that differ in their
polypeptide composition and kinase activities./. Biol. Chem. 262: 12,182-12,188,
1987.
Mangan, J.L., and V.P. Whittaker. The distribution of free amino acids in subcellular
fractions of guinea-pig brain. Biochem. J. 98: 128-137, 1966.
Manning, D.R., and A.G. Gilman. The regulatory components of adenylate cyclase and
transducin. /. Biol. Chem. 258: 7059-7063, 1983.
Mansour, T.E., E.W. Sutherland, T.W. Rail, and E. Bueding. The effect of serotonin (5-
hydroxytrypamine) on the formation of adenosine 3',5'-phosphate by tissue parti-
cles from the liver fluke, Fasciola hepatica. J. Biol. Chem. 235: 466-470, 1960.
Manthorpe, M., E. Engval, E. Ruoslahti, F.M. Longo, G.E. Davis, and S. Varon. Laminin
promotes neuritic regeneration from cultured peripheral and central neurons.
}. Cell Biol 97: 1882-1890, 1983.
Marchbanks, R.M. Serotonin binding to nerve ending particles and other preparations
from rat brain. /. Neurochem. 13: 1481-1493, 1966.
Marchbanks, R.M. The vesicular hypothesis questioned. Trends Neurosci. 1: 83-84,
1978.
Maricq, A.V., A.S. Peterson, A.J. Brake, R.M. Myers, and D. Julius. Primary structure
and functional expression of the SHTs receptor, a serotonin-gated ion channel. Sci-
ence 254: 432-437, 1991.
Markey, S.P., J.N. Johannessen, C.C. Chiueh, R.S. Burns, and M.A. Herkenham. Intra-
neuronal generation of a pyridinium metabolite may cause drug-induced parkin-
sonism. Nature 311: 464-467, 1984.
Marks, J. The Treatment of Parkinsonism with L-Dopa. New York: Elsevier Publishing
Co., 1974.
Mars, H. Modification of levodopa effect by systemic decarboxylase inhibition. Arch.
Neurol. 28: 91-95, 1973.
Marshall, L.H. Instruments, techniques, and social units in American neurophysiology.
In: Geison, G. (ed.) Physiology in the American Context, 1850-1940. Washington,
DC: American Physiological Society, 1987. p. 351-369.
Martin, D.L. Carrier-mediated transport and removal of GABA from synaptic regions.
In: Roberts, E., T.N. Chase, and D.B. Tower (eds.) GABA in Nervous System Func-
tion. New York: Raven Press, 1976, pp. 347-386.
Martin, W.R., C.G. Eades, J.A. Thompson, R.E. Huppler, and PE. Gilbert. The effects
of morphine- and nalorphine-like drugs in the nondependent and morphine-
dependent chronic spinal dog. /. Pharmacol. Exp. Ther. 197: 517-532, 1976.
Marty, A. The physiological role of calcium-dependent channels. Trends Neurosci. 12:
420-424, 1989.
Mast, S.O., and C.L. Prosser. Effect of temperature, salts, and hydrogen-ion concen-
tration on rupture of the plasmagel sheet, rate of locomotion, and gel/sol ratio in
Amoeba proteus./. Cell. Comp. Physiol 1: 333-354, 1932.
Matsas, R., I.S. Fulcher, A.J. Kenny, and A.J. Turner. Substance P and [Leu]enkephalin
are hydrolyzed by an enzyme in pig caudate synaptic membranes that is identical
with the endopeptidase of kidney microvilli. Proc. Natl. Acad. Sci. USA 80: 3111-
3115, 1983.
Matsas, R., S.L. Stephenson, J. Hryszko, A.J. Kenny, and A.J. Turner. The metabolism
of neuropeptides. Biochem. J. 231: 445-449, 1985.
Matthes, K. The action of blood on acetylcholine./. Physiol. 70: 338-348, 1930.
Matthew, W.D., L. Tsavaler, and L.F. Reichardt. Identification of a synaptic vesicle-
specific membrane protein with a wide distribution in neuronal and neurosecre-
tory tissue./. Cell Biol. 91: 257-269, 1981.
Matthews, D.A., C. Cotman, and G. Lynch. Electron microscopic study of lesion-
induced synaptogensis in the dentate gyrus of the adult rat. Brain Res. 115: 23-41,
1976.
Matthysse, S. Antipsychotic drug actions: A clue to the neuropathology of schizophre-
nia. Fed. Proc. 32: 200-205, 1973.
Matus, A., M. Ackermann, G. Pehling, H.R. Byers, and K. Fujiwara. High actin con-
centrations in brain dendritic spines and postsynaptic densities. Proc. Natl. Acad.
Sci. USA 79: 7590-7594, 1982.
Mauro, A. The role of the voltaic pile in the Galvani-Volta controversy concerning ani-
mal vs. metallic electricity. /. Hist. Med. 24: 140-150, 1969.
Mawdsley, C., I.R. Williams, LA. Pullar, D.L. Davidson, and N.E. Kinloch. Treatment
of parkinsonism by amantadine and levodopa. Clin. Pharmacol. Ther. 13: 575-583,
1972.
Mayer, M.L., G.L. Westbrook, and P.B. Guthrie. Voltage-dependent block by Mg2+ of
NMDA responses in spinal cord neurones. Nature 309: 261-263, 1984.
Mayer, D.J., T.L. Wolfe, H. Akil, B. Carder, and J.C. Liebeskind. Analgesia from elec-
trical stimulation in the brainstem of the rat. Science 174: 1351-1354, 1971.
McAfee, D.A., M. Schorderet, and P. Greengard. Adenosine 3', 5'-monophosphate in
nervous tissue: Increase associated with synaptic transmission. Science 171: 1156-
1158, 1971.
McConnell, J.V. Memory transfer through cannibalism in planarians. /. Neuropsychiat.
suppl., pp. 42-48, 1962.
McConnell, J.V., A.L. Jacobson, and D.P. Kimble. The effects of regeneration upon
retention of a conditioned response in the planarian./. Comp. Physiol. Psychol. 52:
1-5, 1959.
McDonald, N.Q., R. Lapatto, J. Murray-Rust, J. Gunning, A. Wlodawer, and T.L. Blun-
dell. New protein fold revealed by a 2.3-A resolution crystal structure of nerve
growth factor. Nature 354: 411-414, 1991.
McDougall, W. The nature of inhibitory processes within the nervous system. Brain 26:
153-191, 1903.
McGeer, P.L., and L.R. Zeldowicz. Administration of dihydroxyphenylalanine to parkin-
sonian patients. Canad. Med. Assoc. J. 90: 463-466, 1964.
McGonigle, P., S.J. Boyson, S. Reuter, and P.B. Molinoff. Effect of chronic treatment
with selective and nonselective antagonists on the subtypes of dopamine receptors.
Synapse 3: 74-82, 1989.
McGuffin, P., and R. Katz. The genetics of depression and manic-depressive disorder.
Brit. J. Psychiat. 155: 294-304, 1989.
Mclntyre, A.R. Curare. Chicago: University of Chicago Press, 1947.
McKail, R.A., S. Obrador, and W.C. Wilson. The action of acetylcholine, eserine and
other substances on some motor responses of the central nervous system. /. Phys-
iol. 99: 312-328, 1941.
References 415
McKenna, P.J., and P.E. Bailey. The strange story of clozapine. Brit. J. Psychiat. 162:
32-37, 1993.
McLennan, H. A comparison of some physiological properties of an inhibitory factor
from brain (Factor I) and of y-aminobutyric acid and related compounds. /. Phys-
iol. 139: 79-86, 1957.
McLennan, H. Absence of y-aminobutyric acid from brain extracts containing Factor
I. Nature 181: 1807, 1958.
McLennan, H. The release of acetylcholine and of 3-hydroxytyramine from the caudate
nucleus. /. Physiol. 174: 152-161, 1964.
McLennan, H. Synaptic Transmission. Philadelphia: W.B. Saunders, 1970.
McLennan, H., and D.H. York. Cholinergic mechanisms in the caudate nucleus./. Phys-
iol. 187: 163-175, 1966.
McLennan, H., and D.H. York. The action of dopamine on neurons of the caudate
nucleus. /. Physiol. 189: 393-402, 1967.
McTigue, M., J. Cremins, and S. Halegoua. Nerve growth factor and other agents medi-
ate phosphorylation and activation of tyrosine hydroxylase. /. Biol. Chem. 260:
9047-9056, 1985.
Means, A.R., J.S. Tash, and J.G. Chafouleas. Physiological implications of the presence,
distribution, and regulation of calmodulin in eukaryotic cells. Physiol. Rev. 62: 1-39,
1982.
Meligeni, J.A., J.W. Haycock, W.F. Bennett, and J.C. Waymire. Phosphorylation and
activation of tyrosine hydroxylase mediate the cAMP-induced increase in cate-
cholamine biosynthesis in adrenal chromaffin cells. /. Biol. Chem. 257: 12,632-
12,640, 1982.
Melloni, E., and S. Pontremoli. The calpains. Trends Neurosci. 12: 438-444, 1989.
Meyer, A. Historical Aspects of Cerebral Anatomy. New York: Oxford University Press,
1971.
Michaelis, L., and M.L. Menten. Die Kinetik der Invertinwirkung. Biochem. Z. 49:
333^369, 1913.
Michaelson, I.A., and V.P. Whittaker. The distribution of hydroxytryptamine in brain
fractions. Biochem. Pharmacol. 11: 505-506, 1962.
Michaelson, I.A., and V.P. Whittaker. The subcellular localization of 5-hydroxytrypta-
mine in guinea pig brain. Biochem. Pharmacol. 12: 203-211, 1963.
Michell, R.H. Inositol phospholipids and cell surface receptor function. Biochim. Bio-
phys. Acta 415: 81-147, 1975.
Miledi, R. Properties of regenerating neuromuscular synapses in the frog. /. Physiol.
154: 190-205, 1960.
Miledi, R. Transmitter release induced by injection of calcium ions into nerve termi-
nals. Proc. Roy. Soc. 183B: 421-425, 1973.
Miledi, R., P. Molinoff, and L.T. Potter. Isolation of the cholinergic receptor protein of
Torpedo electric tissue. Nature 229: 554-557, 1971.
Miledi, R., and C.R. Slater. The action of calcium on neuronal synapses in the squid.
/. Physiol. 184: 473-498, 1966.
Miles, K., D.T. Anthony, L.L. Rubin, P. Greengard, and R.L. Huganir. Regulation of
nicotinic acetylcholine receptor phosphorylation in rat myotubes by forskolin and
cAMP. Proc. Natl. Acad. Sci. USA 84: 6591-6595, 1987.
Milner, B. Les troubles de la memoire acompagnant les lesions hippocampiques
bilaterates. In Physiologic de L'Hippocampe. Paris: C.N.S.R., 1962, pp. 257-
272.
Milner, B., and W. Penfield. The effect of hippocampal lesions on recent memory. Trans.
Amer. Neurol. Assoc. 80: 42-48, 1955.
Milner, B., L.R. Squire, and E.R. Kandel. Cognitive neuroscience and the study of mem-
ory. Neuron 20: 445-468, 1998.
Minz, B. Pharmakologische Untersuchungen am Blutegelprapart, zugleich eine Meth-
ode zum biologischen Nachweis von Azelylcholin bei Anwesenheit anderer phar-
makologisch Wirksamer korpereigener Stoffe. Naunyn-Schmiedebergs Arch. 168:
292-304, 1932.
Minz, B. Sur la liberation, par la moelle epiniere, d'un corps du type de 1'acetylcholine.
Compt Rend. Soc. Biol. 122: 1214-1216, 1936.
Minz, B. Humoral Agents in Nervous Activity. Springfield, 111.: Charles C Thomas, 1955.
Mishina, M., T. Kurosaki, T.Tobimatsu, Y. Morimoto, M. Noda, T. Yamamoto, M. Terao,
J. Lindstrom, T.Takahashi, M. Kuno, and S. Numa. Expression of functional acetyl-
choline receptor from cloned cDNAs. Nature 307: 604-608, 1984.
Mishkin, M. Memory in monkeys severely impaired by combined but not separate
removal of amygdala and hippocampus. Nature 273: 297-298, 1978.
Mitchell, J.F. The spontaneous and evoked release of acetylcholine from the cerebral
cortex./. Physiol. 165: 98-116, 1963.
Mitchell, J.F., and A. Silver. The spontaneous release of acetylcholine from the dener-
vated hemidiaphragm of the rat./. Physiol. 165: 117-129, 1963.
Mitchell, J.F., and V. Srinivasan. Release of 3H-y-aminobutyric acid from the brain dur-
ing synaptic inhibition. Nature 224: 663-666, 1969.
Mitra, A.K., M.P. McCarthy, and R.M. Stroud. Three-dimensional structure of the nico-
tinic acetylcholine receptor and location of the major associated 43-kD cytoskele-
tal protein, determined at 22 A by low dose electron microscopy and X-ray dif-
fraction to 12.5 A. /. Cell Biol. 109: 755-774, 1989.
Miyamoto, E., J.F. Kuo, and P. Greengard. Adenosine 3',5'-monophosphate-dependent
protein kinase from brain. Science 165: 63-65, 1969.
Monnier, A.M., and Z.M. Bacq. Recherches sur la physiologie et la pharmacologie du
systeme nerveux autonome. Arch. Int. Physiol. 40: 485-510, 1935.
Monod, J., J. Wyman, and J.-P. Changeux. On the nature of allosteric transitions: A plau-
sible model./. Mol. Biol. 12: 88-118, 1965.
Montal, M. Molecular anatomy and molecular design of channel proteins. FASEB J. 4:
2623-2635, 1990.
Montarolo, P.G., P. Goelet, V.F. Castellucci, J. Morgan, E.R. Kandel, and S. Schacher.
A critical period for macromolecular synthesis in long-term heterosynaptic facili-
tation in Aplysia. Science 234: 1249-1254, 1986.
Montminy M.R., and L.M. Bilezikjian. Binding of a nuclear protein to the cyclic-AMP
response element of the somatostatin gene. Nature 328: 175-178, 1987.
Montminy, M.R., K.A.Sevarino, J.A. Wagner, G. Mendel, and R.H. Goodman. Identi-
fication of a cyclic-AMP-responsive element within the rat somatostatin gene. Proc.
Natl. Acad. Sci. USA 83: 6682-6686, 1986.
Morange, Michel. A History of Molecular Biology. Cambridge, Mass.: Harvard Uni-
versity Press, 1998.
Morielli, A.D., E.M. Matera, M.P. Kovac, R.G. Shrum, K.J. McCormack, and W.J. Davis.
Cholinergic suppression: A postsynaptic mechanism of long-term associative learn-
ing. Proc. Natl. Acad. Sci. USA 83: 4556-4560, 1986.
Moriyoshi, K., M. Masu, T. Ishii, R. Shigemoto, N. Mizuno, and S. Nakanishi. Molec-
ular cloning and characterization of the rat NMDA receptor. Nature 354: 31-37,
1991.
References 417
Morris, R.G.M., E. Anderson, G.S. Lynch, and M. Baudry. Selective impairment of

learning and blockade of long-term potentiation by an N-methyl-D-aspartate recep-
tor antagonist, AP5. Nature 319: 774-776, 1986.
Mueller, R.A., H. Thoenen, and J. Axelrod. Increase in tyrosine hydroxylase activity after
reserpine administration. /. Pharmacol. Exp. Ther. 169: 74-79, 1969.
Murad, F.,-Y.M. Chi, T.W. Rail, and E.W. Sutherland. Adenyl cyclase. V.J. Biol. Chem.
237: 1233-1238, 1962.
Muscholl, E. Die Hemmung der Noradrenalin-Aufnahme des Herzens durch Reser-
pin und die Wirkung von Tyramin. Naunyn-Schmiedebergs Arch. 240: 234-241,
1960.
Nachmansohn, D. Metabolism and function of the nerve cell. Harvey Lect. 49: 57-99,
1953.
Nachmansohn, D. Chemical factors controlling nerve activity. Science 134: 1962-1968,
1961.
Nachmansohn, D., and M. Berman. Studies on choline acetylase. III. /. Biol. Chem.
165: 551-563, 1946.
Nachmansohn, D., and E. Lederer. Sur la biochimie de la cholinesterase. Bull. Soc.
Chim. Biol. 21: 797-813, 1939.
Nachmansohn, D., and A.L. Machado. The formation of acetylcholine. A new enzyme:
"Choline acetylase." /. Neurophysiol. 6: 397-403, 1943.
Nagatsu, T, M. Levitt, and S. Udenfriend. Tyrosine hydroxylase. /. Biol. Chem. 239:
2910-2917, 1964.
Naito, S., and T. Ueda. Characterization of glutamate uptake into synaptic vesicles.
/. Neurochem. 44: 99-109, 1985.
Nakanishi, S., A. Inoue, T. Kita, M. Nakamura, A.C.Y. Chang, S.N. Cohen, and S. Numa.
Nucleotide sequence of a cloned cDNA for bovine corticotropin-/3-lipotropin pre-
cursor. Nature 278: 423-427, 1979.
Nakata, H., and H. Fujisawa. Purification and properties of tryptophan 5-monooxyge-
nase from rat brain stem. Eur. J. Biochem. 122: 41-47, 1982.
Nastuk, W.L., and A.L. Hodgkin. The electrical activity of single muscle fibers. /. Cell.
Comp. Physiol. 35: 39-73, 1950.
Nathans, J., and D.S. Hogness. Isolation, sequence analysis, and intron-exon arrange-
ment of the gene encoding bovine rhodopsin. Cell 34: 807-814, 1983.
Neal, M.J., and L.L. Iversen. Subcellular distribution of endogenous and [3H]y-
aminobutyric acid in rat cerebral cortex. J. Neurochem. 16: 1245-1252, 1969.
Neal, M.J., and H.G. Pickles. Uptake of 14C glycine by spinal cord. Nature 222: 679-680,
1969.
Needham, D.M. Machina Carnis. Cambridge: Cambridge University Press, 1971.
Neher, E., and A. Marty. Discrete changes of cell membrane capacitance observed under
conditions of enhanced secretion in bovine adrenal chromaffin cells. Proc. Natl.
Acad. Sci. USA 79: 6712-6716, 1982.
Neher, E., and B. Sakmann. Single-channel currents recorded from membrane of den-
ervated frog muscle fibres. Nature 260: 799-802, 1976.
Nelson, N., R. Anholt, J. Lindstrom, and M. Montal. Reconstitution of purified acetyl-
choline receptors with functional ion channels in planar lipid bilayers. Proc. Natl.
Acad. Sci. USA 77: 3057-3061, 1980.
Neubig, R.R., N.D. Boyd, and J.B. Cohen. Conformations of Torpedo acetylcholine
receptor associated with ion transport and desensitization. Biochemistry 21: 3460-
3467, 1982.
Neve, K.A., R.A. Henningsen, J.R. Bunzow, and O. Civelli. Functional characterization
of a rat dopamine D-2 receptor cDNA expressed in a mammalian cell line. Mol.
Pharmacol. 36: 446-451, 1989.
Newton, H.F., R.L. Zwemer, and W.B. Cannon. Studies on the conditions of activity in
endocrine organs. XXV. Amer. J. Physiol. 96: 377-391, 1931.
Nickel, E., and L.T. Potter. Ultrastructure of isolated membranes of Torpedo electric
tissue. Brain Res. 57: 508-517, 1973.
Nickerson, M. Receptor occupancy and tissue response. Nature 178: 697-698, 1956.
Nickerson, M., J. Berghout, and R.N. Hammerstrom. Mechanism of the acute lethal
effect of epinephrine in rats. Amer. J. Physiol. 160: 479-484, 1950.
Nicklas, W.J., I. Vyas, and R.E. Heikkila. Inhibition of NADH-linked oxidation in brain
mitochondria by l-methyl-4-phenyl-pyridine, a metabolite of the neurotoxin,
l-methyl-4-phenyl-l,2,5,6-tetrahydropyridine. Life Sci. 36: 2503-2508, 1985.
Nishi, S., and K. Koketsu. Electrical properties and activities of single sympathetic neu-
rons in frogs. /. Cell. Comp. Physiol. 55: 15-30, 1960.
Nishizuka, Y. The molecular heterogeneity of protein kinase C and its implications for
cellular regulation. Nature 334: 661-665, 1988.
Nitkin, R.M., M.A. Smith, C. Magill, J.R. Fallen, Y.M. Yao, B.C. Wallace, and U.J.
McMahan. Identification of agrin, a synaptic organizing protein from Torpedo elec-
tric organ. /. Cell Biol. 105: 2471-2478, 1987.
Noda, M., Y. Furutani, H. Takahashi, M. Toyosato, T. Hirose, S. Inayama, S. Nakanishi,
and S. Numa. Cloning and sequence analysis of cDNA for bovine adrenal pre-
proenkephalin. Nature 295: 202-206, 1982a.
Noda, M., Y. Furutani, H. Takahashi, M. Toyosato, T. Tanabe, S. Shimizu, S. Kikyotani,
T. Kayano, T. Hirose, S. Inayama, and S. Numa. Cloning and sequence analysis of
calf cDNA and human genomic DNA encoding a-subunit precursor of muscle
acetylcholine receptor. Nature 305: 818-823, 1983a.
Noda, M., H. Takahashi, T. Tanabe, M. Toyosato, Y. Furutani, T. Horose, M. Asai,
S. Inayama, T. Miyata, and S. Numa. Primary structure of the a-subunit precur-
sor of Torpedo californica acetylcholine receptor deduced from cDNA sequence.
Nature 299: 793-797, 1982b.
Noda, M., H. Takahashi, T. Tanabe, M. Toyosato, S. Kikyotani, Y. Furutani, T. Hirose,
H. Takashima, S. Inayama, T. Miyata, and S. Numa. Structural homology of Tor-
pedo californica acetylcholine receptor subunits. Nature 302: 528-532, 1983b.
Noda, M., H. Takahashi, T. Tanabe, M. Toyosato, S. Kikyotani, T. Hirose, M. Asai,
H. Takashima, S. Inayama, T. Miyata, and S. Numa. Primary structure of /3- and
5-subunit precursors of Torpedo californica acetylcholine receptor deduced from
cDNA sequences. Nature 301: 251-255, 1983c.
Nonidez, J.F. The present status of the neurone theory. Biol. Rev. 19: 30-39, 1944.
Northup, J.K., M.D. Smigel, and A.G. Gilman. The guanine nucleotide activating site of
the regulatory component of adenylate cyclase./. Biol. Chem. 257: 11,416-11,423,
1982.
Northup, J.K., PC. Sternweis, M.D. Smigel, L.S. Schleifer, E.M. Ross, and A.G. Gilman.
Purification of the regulatory component of adenylate cyclase Proc. Natl. Acad. Sci.
USA 77: 6516-6520, 1980.
Nottebohm, F. Neuronal replacement in adulthood. Ann. N.Y. Acad. Sci. 457: 143-161,
1986.
Nowak, L., P. Bregestovski, P. Ascher, A. Herbert, and A. Prochiantz. Magnesium gates
glutamate-activated channels in mouse central neurones. Nature 307: 462-465,
1984.
References 419
Nowycky, M.C., A.P. Fox, and R.W. Tsien. Three types of neuronal calcium channel
with different calcium agonist sensitivity. Nature 316: 440-443, 1985.
Nyhart, L.K. Biology Takes Form: Animal Morphology and the German Universities,
1800-1900. Chicago: University of Chicago Press, 1995.
Obata, K. Gamma-aminobutyric acid in Purkinje cells and motoneurones. Experientia
25: 1283, 1969.
Obata, K., M. Ito, R. Ochi, and N. Sato. Pharmacological properties of postsynaptic
inhibition by Purkinje cell axons and the action of y-aminobutyric acid on Deiters
neurones. Exp. Brain Res. 4: 43-57, 1967.
Obata, K., and K. Takeda. Release of y-aminobutyric acid into the fourth ventricle
induced by stimulation of the cat's cerebellum./. Neurochem. 16:1043-1047,1069.
Obata, K., K. Takeda, and H. Shinozaki. Further study on pharmacological properties
of the cerebellar-induced inhibition of Deiters neurones. Exp. Brain Res. 11: 327-
342, 1970.
O'Dell, T.J., R.D. Hawkins, E.R. Kandel, and O. Aranio. Tests of the role of two dif-
fusible substances in long-term potentiation: Evidence for nitric oxide as a possi-
ble early retrograde messenger. Proc. Natl. Acad. Sci. USA 88:11,285-11,289,1991.
O'Dowd, B.F., M. Hnatowich, J.W. Regan, W.M. Leader, M.G. Caron, and R.J.
Lefkowitz. Site-directed mutagenesis of the cytoplasmic domains of human /?£-
adrenergic receptor. /. Biol. Chem. 263: 15,985-15,992, 1988.
Ohsawa, K., G.H.C. Dowe, S.J. Morris, and V.P. Whittaker. The lipid and protein con-
tent of cholinergic synaptic vesicles from the electric organ of Torpedo marmorata.
Brain Res. 101: 447-457, 1979.
Oliver, G. On the therapeutic employment of the suprarenal glands. Brit. Med. J. 2:
653-655, 1895.
Oliver, G., and E.A. Schafer. The physiological effects of extracts of the suprarenal cap-
sules. /. Physiol. 18: 230-276, 1895.
Olivera, B.M., W.R. Gray, R. Zeikus, J.M. Mclntosh, J. Varga, J. Rivier, V. DeSantos,
and LJ. Cruz. Peptide neurotoxins from fish-eating snails. Science 230: 1338-1343,
1985.
Olmstead, J.B., and G.G. Borisy. Microtubles. Annu. Rev. Biochem. 42: 507-540, 1973.
Olsen, J., G.M. Cowell, E. K0nigsh0fer, E.M. Danielsen, J. M011er, L. Lausten, O.C.
Hansen, K.G. Welinder, J. Engberg, W. Hunziker, M. Spiess, H. Sjostron, and O.
Noren. Complete amino acid sequence of human intestinal aminopeptidase N as
deduced from cloned cDNA. FEES Lett. 238: 307-314, 1988.
Olsen, R.W., J.C. Meunier, and J.-P. Changeux. Progress in the purification of the cholin-
ergic receptor protein from Electrophorus electricus by affinity chromatography.
FEBS Lett. 28: 96-100, 1972.
Orly, J., and M. Schramm. Coupling of catecholamine receptor from one cell with adeny-
late cyclase from another cell by cell fusion. Proc. Natl. Acad. Sci. USA 73: 4410-
4414, 1976.
Ortells, M.O., and G.C. Lunt. Evolutionary history of the ligand-gated ion-channel
superfamily of receptors. Trends Neurosci. 18: 121-127, 1995.
Osterrieder, W, G. Brum, J. Heschler, W. Trautwein, V. Flockerzi, and F. Hofmann.
Injection of subunits of cyclic AMP-dependent protein kinase into cardiac myocytes
modulates Ca2+ current. Nature 298: 576-578, 1982.
Otsuka, M., L.L. Iversen, Z.W. Hall, and E.A. Kravitz. Release of gamma-aminobutyric
acid from inhibitory nerves of lobster. Proc. Natl. Acad. Sci. USA 56: 1110-1115,
1966.
Otsuka, M., and S. Konishi. Release of substance P-like immunoreactivity from isolated
spinal cord of newborn rat. Nature 264: 83-84, 1976.
Overtoil, E. Beitrage zur allgemeinin Muskel- und Nervenphysiologie. Pfliigers Arch.
92: 114-280, 346-386, 1902.
Oyler, G.A., G.A. Higgins, R.A. Hart, E. Battenberg, M. Billingsley, F.E. Bloom, and M.C.
Wilson. The identification of a novel synaptosomal-associated protein, SNAP-25, dif-
ferentially expressed by neuronal subpopulations. /. Cell Biol. 109: 3039-3052,
1989.
Paasonen, M.K., and M. Vogt. The effect of drugs on the amounts of substance P and
5-hydroxytryptamine in mammalian brain./. Physiol. 131: 617-626, 1956.
Pacholczyk, T., R.D. Blakely, and S.G. Amara. Expression cloning of a cocaine- and anti-
depressant-sensitive human noradrenaline transporter. Nature 350: 350-353, 1991.
Page, I. H. The discovery of serotonin. Persp. Biol. Med. 20: 1-8, 1976.
Palade, G.E., and S.L. Palay. Electron microscope observations of interneuronal and
neuromuscular synapses. Anat. Rec. 118: 335-336, 1954.
Palay, S.L. Synapses in the central nervous system./. Biophys. Biochem. Cytol. 2 suppl:
193-201, 1956.
Palay, S.L. The morphology of synapses in the central nervous system. Exp. Cell Res.
Suppl. 5: 275-293, 1958.
Palay, S.L., and V. Chan-Palay. Cell form as an expression of neuronal function. In: San-
tini, M. (ed.) Golgi Centennial Symposium. New York: Raven Press, 1975. pp.
51-59.
Palay, S.L., and G.E. Palade. Electron microscope study of the cytoplasm of neurons.
Anat. Rec. 118: 336, 1954.
Palay, S.L., and G.E. Palade. The fine structure of neurons./. Biophys. Biochem. Cytol.
1: 69-88, 1955.
Parducz, A., and O. Feher. Fine structural alterations of presynaptic endings in the
superior cervical ganglion of the cat after exhausting preganglionic stimulation.
Experientia 26: 629-630, 1970.
Parker, G.H. The neurofibril hypothesis. Quart. Rev. Biol. 4: 155-178, 1929.
Parkinson Study Group. Effect of deprenyl on the progression of disability in early
Parkinsons disease. New Eng. ]. Med. 321: 1364-1371, 1989.
Pasternack, G.W., R. Goodman, and S.H. Snyder. An endogenous morphine-like factor
in mammalian brain. Life Sci. 16: 1765-1769, 1975.
Paton, W.D.M. A theory of drug action based on the rate of drug-receptor combina-
tion. Proc. Roy. Soc. 154B: 21-69, 1961.
Paton, W.D.M., and E.S. Vizi. The inhibitory action of noradrenaline and adrenaline on
acetylcholine output by guinea-pig ileum longitudinal muscle strip. Brit. J. Phar-
macol. 35: 10-28, 1969.
Patzer, E.J., D.M. Schlossman, and J.E. Rothman. Release of clathrin from coated vesi-
cles dependent upon a nucleoside triphosphate and a cytosol fraction. /. Cell Biol.
93: 230-236, 1982.
Peachey, L.D. The sarcoplasmic reticulum and transverse tubules of the frog's sarto-
rius./. Cell Biol 25(2): 209-231, 1965.
Pearse, B.M.F. Coated vesicles from pig brain: Purification and biochemical character-
ization. /. Mol. Biol. 97: 93-98, 1975.
Pearse, B.M.F. On the structural and functional components of coated vesicles. /. Mol.
Biol. 126: 803-812, 1978.
Pearse, B.M.F., and M.S. Robinson. Purification and properties of 100-kd proteins from
coated vesicles and their reconstitution with clathrin. EMBOJ. 3: -1951-1957, 1984.
References 421
Peart, W.S. The nature of splenic sympathin. /. Physiol. 108: 491-501, 1949.
Penfield, W. Memory mechanisms. Arch. Neurol. Psychiat. 67: 178-198, 1952.
Penfield, W., and B. Milner. Memory deficit produced by bilateral lesions in the hip-
pocampal zone. Arch. Neurol. Psychiat. 79: 475-497, 1958.
Peper, K., F. Dreyer, C. Sandri, K. Akert, and H. Moor. Structure and ultrastructure of
the frog motor endplate. Cell Tiss. Res. 149: 437-455, 1974.
Peralta, E.G., A. Ashkenazi, J.W. Winslow, J. Ramachandran, and D.J. Capon. Differ-
ential regulation of PI hydrolysis and adenyl cyclase by muscarinic receptor sub-
types. Nature 334: 434-437, 1988.
Peralta, E.G., A. Ashkenazi, J.W. Winslow, D.H. Smith, J. Ramachandran, and D.J.
Capon. Distinct primary structures, ligand-binding properties and tissue-specific
expression of four human muscarinic acetylcholine receptors. EMBO J. 6: 3923-
3929, 1987a.
Peralta, E.G., J.W. Winslow, G.L. Peterson, D.H. Smith, A. Ashkenazi, J. Ramachan-
dran, M.I. Schimerlik, and D.J. Capon. Primary structure and biochemical prop-
erties of an M2 muscarinic receptor. Science 236: 600-605, 1987b.
Perin, M.S., V.A. Fried, G.A. Mignery, R. Jahn, and T.C. Sudhof. Phospholipid binding
by a synaptic vesicle protein homologous to the regulatory region of protein kinase
C. Nature 345: 260-263, 1990.
Perin, M.S., PA. Johnston, T. Ozcelik, R. Jahn, U. Franke, and T.C. Sudhof. Structural
and functional conservation of synaptotagmin (p65) in Drosophila and humans.
/. Biol. Chem. 266: 615-622, 1991.
Perl, E. The 1944 Nobel prize to Erlanger and Gasser. FASEB J. 8: 782-783, 1994.
Pernow, B. Studies on substance P. Acta Physiol Scand. 29 suppl: 105, 1953.
Peroutka, S.J., and S.H. Snyder. Long-term antidepressant treatment decreases
spiroperidol-labeled serotonin receptor binding. Science 210: 88-90, 1980.
Pert, C.B., G. Pasternak, and S.H. Snyder. Opiate agonists and antagonists discrimi-
nated by receptor binding in brain. Science 182: 1359-1361, 1973.
Pert, C.B., and S.H. Snyder. Opiate receptor: Demonstration in nervous tissue. Science
179: 1011-1014, 1973a.
Pert, C.B., and S.H. Snyder. Properties of opiate-receptor binding in rat brain. Proc.
Natl. Acad. Sci. USA 70: 2243-2247, 1973b.
Pert, C.B., and S.H. Snyder. Opiate receptor binding of agonists and antagonists affected
differentially by sodium. Mol. Pharmacol. 10: 868-879, 1974.
Petrucci, T.C., and J.S. Morrow. Synapsin I: An actin-bundling protein under phos-
phorylation control. /. Cell Biol 105: 1355-1363, 1987.
Pfaffinger, P.J., J.M. Martin, D.D. Hunter, N.M. Nathanson, and B. Hille. GTP-binding
proteins couple cardiac muscarinic receptors to a K channel. Nature 317: 536-538,
1985.
Pfeiffer, C.C. Nature and spatial relationship of the prosthetic chemical groups required
for maximal muscarinic action. Science 107: 94-96, 1948.
Pfenninger, K., K. Akert, H. Moor, and C. Sandri. The fine structure of freeze-
fractured presynaptic membranes. /. Neurocytol 1: 129-149, 1972.
Pfenninger, K.H., and M.F. Maylie-Pfenninger. Lectin labeling of sprouting neurons.
II. Relative movement and appearance of glycoconjugates during plasmalemmal
expansion. /. Cell Biol 89: 547-559, 1981.
Pfeuffer, T. GTP-binding proteins in membranes and the control of adenylate cyclase
activity. /. Biol. Chem. 252: 7224-7234, 1977.
Pfeuffer, T. Guanine nucleotide-controlled interactions between components of adeny-
late cyclase. FEBS Lett. 101: 85-89, 1979.
Phenothiazine treatment in acute schizophrenia. Arch. Gen. Psychiat. 10: 246-261,

1964.
Phillips, J.H. Steady-state kinetics of catecholamine transport by chromaffin-granule
'ghosts.' Biochem. ]. 144: 319-325, 1974.
Phillips, J.H. 5-Hydroxytryptamine transport by the bovine chromaffin-granule mem-
brane. Biochem. ]. 170: 673-679, 1978.
Phillis, J.W., and J.J. Limacher. Substance P excitation of cerebral cortical Betz cells.
Brain Res. 69: 158-163, 1974.
Piccolino, M. Cajal and the retina. Trends Neurosci. 11: 521-525, 1988.
Piccolino, M. Luigi Galvani and animal electricity. Trends Neurosci. 20: 443-448, 1997.
Pinsker, H.M., W.A. Hening, T.J. Carew, and E.R. Kandel. Long-term sensitization of
a defensive withdrawal reflex in Aplysia. Science 182: 1039-1042, 1973.
Pinsker, H., I. Kupfermann, V. Castellucci, and E. Kandel. Habituation and dishabitu-
ation of the gill-withdrawal reflex in Aplysia. Science 167: 1740-1742, 1970.
Piper, H. Uber die Leitungsgeschwindigkeit in den markhaltigen, menschlichen Ner-
ven. Pfliigers Arch. 124: 591-600, 1908.
Pi-Suner, A., and J. Pi-Suner. Ramon y Cajal and the physiology of the nervous system.
/. Nerv. Ment. Dis. 84: 521-537, 1936.
Pitcher, J.A., N.J. Freedman, and R.J. Lefkowitz. G protein-coupled receptor kinases.
Annu. Rev. Biochem. 67: 653-692, 1998.
Pletscher, A. Wirkung von Isopropyl-isonicotinsaurehydrazid auf den Stoffwechsel von
Catecholaminen und 5-Hydroxytryptamin im Gehirn. Schweiz. Med. Wschr. 78:
1532-1534, 1957.
Pletscher, A. The discovery of antidepressants: A winding path. Experientia 47: 4-8,
1991.
Pletscher, A., P.A. Shore, and B.B. Brodie. Serotonin as a mediator of reserpine action
in brain./. Pharmacol. Exp. Ther. 116: 84-89, 1956.
Poirier, L.J., and T.L. Sourkes. Influence of the substantia nigra on the catecholamine
content of the striatum. Brain 88: 181-191, 1965.
Popper, K.R. The Logic of Scientific Discovery. New York: Basic Books, 1959.
Porter, K.R., and G.E. Palade. Studies on the endoplasmic reticulum. /. Biophys.
Biochem. Cytol. 3: 269-300, 1957.
Posner, J.B., R. Stern, and E.G. Krebs. Effects of electrical stimulation and epineph-
rine on muscle phosphorylase, phosphorylase b kinase, and adenosine 3', 5'-phos-
phate. /. Biol Chem. 240: 982-985, 1965.
Potter, L.T., and J. Axelrod. Subcellular localization of catecholamines in tissue of the
rat. /. Pharmacol. Exp. Ther. 142: 291-305, 1963.
Pritchett, D.B., A.W.J. Bach, M. Wozny, O. Taleb, R. DalToso, J.C. Shih, and PH. See-
burg. Structure and functional expression of cloned rat serotonin 5HT-2 receptor.
EMBO J. 7: 4135^140, 1988.
Pumplin, D.W., T.S. Reese, and R. Llinas. Are the presynaptic membrane particles the
calcium channels? Proc. Natl. Acad. Sci. USA 78: 7210-7213, 1981.
Purves, D., and J.W. Lichtman. Principles of Neural Development. Sunderland: Sinauer,
1985.
Pysh, J.J., and R.G. Wiley. Morphologic alterations of synapses in electrically stimulated
superior cervical ganglia of the cat. Science 176: 191-193, 1972.
Pysh, J.J., and R.G. Wiley. Synaptic vesicle depletion and recovery in cat sympathetic
ganglia electrically stimulated in vivo. /. Cell Biol. 60: 365-374, 1974.
Quastel, J.H., M. Tennenbaum, A. Herbert, and M. Wheatley. Choline ester formation
in, and cholinesterase activities of, tissues in vitro. Biochem. J. 30:1668-1681,1936.
Rererences 423
Quinn, W.G., W.A. Harris, and S. Benzer. Conditioned behavior in Drosophila

melanogaster. Proc. Natl. Acad. Sci. USA 71: 708-712, 1974.
Raab, W. Adrenaline and related substances in blood and tissues. Biochem. ]. 37: 470-
473, 1943.
Raab, W, and R.J. Humphreys. Drug action upon myocardial epinephrine-sympathin
concentration and heart rate (nitroglycerine, papaverine, priscol, dibenamine
hydrochloride). /. Pharmacol. Exp. Ther. 89: 64-76, 1947.
Racker, E. Aldehyde dehydrogenase, a diphosphopyridine nucleotide-linked enzyme.
/. Biol Chem. 177: 883-892, 1949.
Raftery, M.A., M.W. Hunkapillar, C.D. Strader, and L.E. Hood. Acetylcholine recep-
tor: Complex of homologous subunits. Science 208: 1454-1457, 1980.
Raftery, M.A., J. Schmidt, D.G. Clark, and R.G. Wolcott. Demonstration of a specific
a-bungarotoxin binding component in Electrophorus electricus electroplax mem-
branes. Biochem. Biophys. Res. Commun. 45: 1622-1629, 1971.
Raiteri, M., F. Cerrito, A.M. Cervoni, and G. Levi. Dopamine can be released by two
mechanisms differentially affected by the dopamine transport inhibitor nomifen-
sine. /. Pharmacol. Exp. Ther. 208: 195-202, 1979.
Rail, T.W., E.W. Sutherland, and J. Berthet. The relationship of epinephrine and
glucagon to liver phosphorylase. IV. J. Biol Chem. 224: 463-475, 1957.
Rail, T.W., E.W. Sutherland, and W.D. Wosilait. The relationship of epinephrine and
glucagon to liver phosphorylase. III. /. Biol. Chem. 218: 483-495, 1956.
Ramon y Cajal, S. La fine structure des centres nerveux. Proc. R. Soc. 54: 444-468,
1894.
Ramon y Cajal, S. Les preuves objectives de 1'unite anatomique des cellules nerveuses.
Travaux Lab. Rech. Bio. Cajal 29: 1-137, 1934.
Ramon y Cajal, S. Recollections of My Life. Philadelphia: American Philosophical Soci-
ety, 1937.
Ramon y Cajal, S. The structure and connexions of neurons. In: Nobel Lectures. Phys-
iology or Medicine. 1901-1921. Amsterdam: Elsevier Publishing Co., 1967.
Ramon y Cajal, S. New Ideas on the Structure of the Nervous System in Man and Ver-
tebrates. Cambridge: MIT Press, 1990.
Ramon y Cajal, S. Degeneration and Regeneration of the Nervous System. New York:
Oxford University Press, 1991.
Ramon y Cajal, S. Histology of the Nervous System of Man and Vertebrates. New York:
Oxford University Press, 1995.
Randrup, A., I. Munkvad, and P. Udsen. Adrenergic mechanisms and amphetamine
induced abnormal behaviour. Acta Pharmacol. 20: 145-157, 1963.
Rang, H.P., and J.M. Ritter. On the mechanism of desensitization at cholinergic recep-
tors. Mol. Pharmacol. 6: 357-382, 1970.
Raper, J.A., M. Bastiani, and C.S. Goodman. Pathfinding by neuronal growth cones in
grasshopper embryos. I. Divergent choices made by growth cones of sibling neu-
rons. /. Neurosci. 3: 20-30, 1983a.
Raper, J.A., M. Bastiani, and C.S. Goodman. Pathfinding by neuronal growth cones in
grasshopper embryos. II. Selective fasciculation onto specific axonal pathways.
/. Neurosci. 3: 31-41, 1983b.
Rapport, M. Historical memoir: Detection of the indole nucleus. Einstein Quart. J. Biol.
Med. 5: 113-115, 1987.
Rapport, M.M. Serum vasoconstrictor (serotonin). V. /. Biol. Chem. 180: 961-969,
1949.
Rapport, M.M. The discovery of serotonin. Persp. Biol. Med. 40: 261-273, 1997.
Rapport, M.M., A.A. Green, and I.H. Page. Partial purification of the vasoconstrictor
in beef serum./. Biol. Chem. 174: 735-741, 1948a.
Rapport, M.M., A.A. Green, and I.H. Page. Crystalline serotonin. Science 108: 329-330,
1948b.
Rasmussen, H. Cell communication, calcium ion, and cyclic adenosine monophosphate.
Science 170: 404-412, 1970.
Raventos, J. Pharmacological actions of quaternary ammonium salts. Quart. J. Exp. Phys-
iol. 26: 361-374, 1937.
Ray, P., W. Middleton, and J.D. Berman. Mechanism of agonist-induced down-regula-
tion and subsequent recovery of muscarinic acetylcholine receptors in a clonal neu-
roblastoma x glioma hybrid cell line. /. Neurochem. 52: 402-409, 1989.
Reddington, M., R. Rodnight, and M. Williams. Turnover of protein-bound serine phos-
phate in respiring slices of guinea-pig cerebral cortex. Biochem. J. 132: 475-482,
1973.
Regan, J.W., T.S. Kobilka, T.L. Yang-Feng, M.G. Caron, R.J. Lefkowitz, and B.K.
Kobilka. Cloning and expression of a human kidney cDNA for an o^-adrenergic
receptor subtype. Proc. Natl. Acad. Sci. USA 85: 6301-6305, 1988.
Reist, N.E., C. Magill, and U.J. McMahan. Agrin-like molecules at synaptic sites in nor-
mal, denervated, and damaged skeletal muscles./. Cell Biol. 105: 2457-2469, 1987.
Renshaw, B. Central effects of centripetal impulses in axons of spinal ventral roots.
/. Neurophysiol. 9: 191-204, 1946.
Reuter, H. Localization of beta adrenergic receptors, and effects of noradrenaline and
cyclic nucleotides on action potentials, ionic currents and tension in mammalian
cardiac muscle. /. Physiol. 242: 429-451, 1974.
Reuter, H., and N. Seitz. The dependence of calcium efflux from cardiac muscle on
temperature and external ion composition. /. Physiol. 195: 451-470, 1968.
Revah, R, J.L. Galzi, J. Giraudat, P.Y. Haumont, F. Lederer, and J.-P. Changeux. The
noncompetitive blocker [3H]chlorpromazine labels three amino acids of the acetyl-
choline receptor y subunit. Proc. Natl Acad, Sci. USA 87: 4675-4679, 1990.
Revel, J.P., and M.J. Karnovsky. Hexagonal array of subunits in intercellular junctions
of the mouse heart and liver./. Cell Biol. 33: C7-C12, 1967.
Reynolds, J.A., and A. Karlin. Molecular weight in detergent solution of acetylcholine
receptors from Torpedo californica. Biochemistry 17: 2035-2038, 1978.
Rhee, S.G., P.G. Suh, S.H. Ryu, and S.Y. Lee. Studies of inositol phospholipid-specific
phospholipase C. Science 244: 546-550, 1989.
Richter, D. Adrenaline and amine oxidase. Biochem. J. 31: 2022-2028, 1937.
Richter, D. The inactivation of adrenaline in vivo in man./. Physiol. 98: 361-374, 1940.
Riesser, O., and S.M. Neuschloss. Physiologische und kolloidchemische Untersuchun-
gen iiber den Mechanismus der durch Gifte bewirkten Kontraktur quer gestreifter
Muskeln. Naunyn-Schmiedebergs Arch. 91: 342-365, 1921.
Riley, W.D., J. DeLange, G.E. Bratvold, and E.G. Krebs. Reversal of phosphorylase
kinase activation. /. Biol. Chem. 243: 2209-2215, 1968.
Rilling, M. The mystery of the vanished citations. Amer. Psychol. 51: 589-598, 1996.
Robbins, J. The effects of amino acids on the crustacean neuro-muscular system. Anat.
Rec. 132: 492-493, 1958.
Robbins, J. The excitation and inhibition of crustaceasn muscle by amino acids./. Phys-
iol. 148: 39-50, 1959.
Roberts, E. Formation and utilization of y-aminobutyric acid in brain. Prog. Neurobiol.
1: 11-25, 1956.
References 425
Roberts, E., and S. Frankel. y-Aminobutyric acid in brain: Its formation from glutamic
acid. /. Biol Chem. 187: 55-63, 1950.
Roberts, P.J. Glutamate, GABA, and the direct cortical response in the rat. Brain Res.
49: 451-455, 1973.
Roberts, P.J., and J.F. Mitchell. The release of amino acids from the hemisected spinal
cord during stimulation. /. Neurochem. 19: 2473-2481, 1972.
Robertson, J.D. Ultrastructure of two invertebrate synapses. Proc. Soc. Exp. Biol. Med.
82: 219-223, 1953.
Robertson, J.D. The ultrastructure of a reptilian myoneural junction. J. Biophys.
Biochem. Cytol 2: 381-393, 1956.
Robertson, J.D. Electron microscopy of the motor end-plate and the neuromuscular
spindle. Am J. Phys. Med. 39: 1-43, 1960.
Robertson, J.D. The occurence of a subunit pattern in the unit membranes of club end-
ings in Mauthner cell synapses in goldfish brains. /. Cell Biol. 19: 201-221, 1963.
Robinson, J.D. Identifying error types on behalf of better science. Phil. Sci. 50: 643-647,
1983.
Robinson, J.D. Reduction, explanation, and the quests of biological science. Phil. Sci.
53: 333-353, 1986a.
Robinson, J.D. Appreciating key experiments. Brit. J. Hist. Sci. 19: 51-56, 1986b.
Robinson, J.D. Physics, philosophy, and the nature of biological sciences. Persp. Biol.
Med. 30: 384-398, 1987.
Robinson, J.D. Aims and achievements of the reductionist approach in biochem-
istry/molecular biology/cell biology: A response to Kincaid. Phil. Sci. 59: 465-470,
1992.
Robinson, J.D. Moving Questions: A History of Membrane Transport and Bioenerget-
ics. New York: Oxford University Press, 1997.
Robishaw, J.D., D.W. Russell, B.A. Harris, M.D. Smigel, and A.G. Gilman. Deduced
primary structure of the a subunit of the GTP-binding stimulatory protein of adeny-
late cyclase. Proc. Natl. Acad. Sci. USA 83: 1251-1255, 1986.
Robison, G.A., R.W. Butcher, I. 0ye, H.E. Morgan, and E.W. Sutherland. The effect
of epinephrine on adenosine 3', 5'-phosphate levels in the isolated perfused rat
heart. Mol. Pharmacol. 1: 168-177, 1965.
Robison, G.A., R.W. Butcher, and E.W. Sutherland. Adenyl cyclase as an adrenergic
receptor. Ann. N.Y. Acad. Sci. 139: 703-723, 1967.
Robitaille, R., E.M. Adler, and M.P. Charlton. Strategic location of calcium channels at
transmitter release sites of frog neuromuscular synapses. Neuron 5: 773-779,1990.
Rodbell, M., L. Birnbaumer, S.L. Pohl, and H.M.J. Krans. The glucagon-sensitive adenyl
cyclase system in plasma membranes of rat liver. V. J. Biol. Chem. 246: 1877-1882,
1971a.
Rodbell, M., H.M.J. Krans, S.L. Pohl, and L. Birnbaumer. The glucagon-sensitive adenyl
cyclase system in plasma membranes of rat liver. IV./. Biol. Chem. 246: 1872-1876,
1971b.
Rodbell, M., M.C. Lin, and Y. Salomon. Evidence for interdependent action of glucagon
and nucleotides on the hepatic adenylate cyclase system./. Biol. Chem. 249: 59-65,
1974.
Rogers, S.L., PC. Letourneau, S.L. Palm, J. McCarthy, and L.T. Furcht. Neurite exten-
sion by peripheral and central nervous system neurons in response to substratum-
bound fibronectin and laminin. Dev. Biol. 98: 212-220, 1983.
Roques, B.P., M.C. Fournie-Zaluski, E. Soroca, J.M. Lecomte, B. Malfroy, C. Llorens,
and J.-C. Schwartz. The enkephalinase inhibitor thiorphan shows antinociceptive

activity in mice. Nature 288: 286-288, 1980.
Rosell-Perez, M., and J. Larner. Studies on UDPG-a-glucan transglucosylase. Bio-
chemistry 3: 81-88, 1964.
Rosenblueth, A., and F.A. Simeone. The action of eserine or prostigmin on the supe-
rior cervical ganglion. Amer. J. Physiol. 122: 708-721, 1938.
Ross, C.A., J. Meldolesi, T.A. Milner, T. Satoh, S. Supattapone, and S.H. Snyder. Inos-
itol 1,4,5-trisphosphate receptor localized to endoplasmic reticulum in cerebellar
Purkinje neurons. Nature 339: 468-470, 1989.
Ross, S.B., and A.L. Renyi. Accumulation of tritiated 5-hydroxytryptamine in brain slices.
Life Sci. 6: 1407-1415, 1967.
Rossier, J., J.M. Trifaro, R.V Lewis, R.W.H. Lee, A. Stern, S. Kimura, S. Stein, and S. Uden-
friend. Studies with pSjmethionine indicate that the 22,000-dalton [Metjenkephalin-
containing protein in chromaffin cells is a precursor of [Met]enkephalin. Proc. Natl.
Acad. Sci. USA 77: 6889-6891, 1980.
Rossier, J., T.M. Vargo, S. Minick, N. Ling, F.E. Bloom, and R. Guillemin. Regional dis-
sociation of /3-endorphin and enkephalin contents in rat brain and pituitary. Proc.
Natl. Acad. Sci. USA 74: 5162-5165, 1977.
Roth, J.A. Presence of membrane-bound catechol-O-methyltransferase in human brain.
Biochem. Pharmacol. 29: 3119-3122, 1980.
Roth, J.A. Membrane-bound catechol-O-methyltransferase: A reevaluation of its role in
the O-methylation of the catecholamine neurotransmitters. Rev. Physiol. Biochem.
Pharmacol. 120: 1-29, 1992.
Roth, T.F., and K.R. Porter. Yolk protein uptake in the oocyte of the mosquito Aedes
aegypti L. /. Cell Biol. 20: 313-332, 1964.
Rothenberg, M.A., and D. Nachmansohn. Studies on cholinesterase./. Biol. Chem. 168:
223-231, 1947.
Rothman, J.E., and S.L. Schmid. Enzymatic recycling of clathrin from coated vesicles.
Cell 46: 5-9, 1986.
Rothschild, L. The membrane capacitance of the sea urchin egg. /. Biophys. Biochem.
Cytol 3: 103-110, 1957.
Rousselet, A., J. Cartaud, P.F. Devaux, and J.P. Changeux. The rotational diffusion of
the acetylcholine receptor in Torpedo marmorata membrane fragments studied
with a spin-labelled a-toxin: Importance of the 43000 protein(s). EMBO J. 4:
439-445, 1982.
Rupp, F, D.G. Payan, C. Magill-Solc, D.M. Cowan, and R.H. Scheller. Structure and
expression of a rat agrin. Neuron 6: 811-823, 1991.
Rushton, W.A.H. Excitable substances in the nerve-muscle complex. /. Physiol. 70:
317-337, 1930.
Rushton, W.A.H. Lapicques theory of curarization./. Physiol. 77: 337-364, 1933.
Ryu, S. H., K.S. Cho, K.Y. Lee, P.G. Suh, and S. G. Rhee. Purification and characteri-
zation of two immunologically distinct phosphoinositide-specific phospholipase C
from bovine brain./. Biol. Chem. 262: 12,511-12,518, 1987.
Sahley, C.L. Co-activation, cell assemblies and learning. Trends Neurosci. 8: 423-424,
1985.
Salminen, M., K. Lunstrom, C. Tilgmann, R. Savolainen, N. Kalkkinen, and I. Ulma-
nen. Molecular cloning and characterization of rat liver catechol-O-methyltrans-
ferase. Gene 93: 241-247, 1990.
References 427
Samorajski, T., and B.H. Marks. Localization of tritiated norepinephrine in mouse brain.
/. Histochem. Cytochem. 10: 392-399, 1962.
Sandoval, M.E. Sodium-dependent efflux of [3H]GABA from synaptosomes probably
related to mitochondrial calcium mobilization. /. Neurochem. 35: 915-921, 1980.
Sandow, A. Excitation-contraction coupling in muscular responses. Yale J. Biol. Med.
25: 189-201, 1952.
Sano, I., T. Gamo, Y. Kakimoto, K. Taniguchi, M. Takesada, and K. Nishinuma. Distri-
bution of catechol compounds in human brain. Biochim. Biophys. Acta 32:586-587,
1959.
Schacher, S., V.F. Castellucci, and E.R. Kandel. cAMP evokes long-term facilitation in
Aplysia sensory neurons that requires new protein syntheses. Science 240: 1667-
1669, 1988.
Schatzmann, H.J. ATP-dependent Ca++ extrusion from human red cells. Experientia
22: 364-365, 1966.
Schayer, R.W. Studies on the metabolism of /3-C14-d/-adrenalin. /. Biol. Chem. 189:
301-306, 1951a.
Schayer, R.W. The metabolism of adrenalin containing isotopic carbon. J. Biol. Chem.
192: 875-881, 1951b.
Schayer, R.W., and R.L. Smiley. The metabolism of epinephrine containing isotopic car-
bon. III. /. Biol. Chem. 202: 425-430, 1953.
Scheel-Kriiger, J., and A. Randrup. Stereotype hyperactive behaviour produced by
dopamine in the absence of noradrenaline. Life Sci. 6: 1389-1398, 1967.
Scheer, H., and J. Meldolesi. Purification of the putative a-latrotoxin receptor from
bovine brain synaptosomal membranes in an active binding form. EMBO J. 4:
323-327, 1985.
Schild, H.O. The use of drug antagonists for the identification and classification of drugs.
Brit. J. Pharmacol. 2: 251-258, 1947.
Schildkraut, J.J. The catecholamine hypothesis of affective disorders: A review of sup-
porting evidence. Amer. J. Psychiat. 122: 509-522, 1965.
Schiller, F. The vicissitudes of the basal ganglia. Bull. Hist. Med. 41: 515-538, 1967.
Schindler, H., and U. Quast. Functional acetylcholine receptor from Torpedo marmorata
in planar membranes. Proc. Natl. Acad. Sci. USA 77: 3052-3056, 1980.
Schloss, P., W. Mayser, and H. Betz. Neurotransmitter transporters. A novel family of
integral plasma membrane proteins. FEBS Lett. 307: 76-80, 1992.
Schlossman, D.M., S.L. Schmid, W.A. Braell, and J.E. Rothman. An enzyme that
removes clathrin coats: Purification of an uncoating ATPase. /. Cell Biol. 99:
723-733, 1984.
Schmidt, J., and M.A. Raftery. Use of affinity chromatography for acetylcholine recep-
tor purification. Biochem. Biophys. Res. Commun. 49: 572-578, 1972.
Schmidt, D.E., P.I.A. Szilagyi, D.L. Alkon, and J.P. Green. Acetylcholine release from
neural tissue and identification by pyrolysis-gas chromatography. Science 165:
1370-1371, 1969.
Schneider, F.H., A.D. Smith, and H. Winkler. Secretion from the adrenal medulla: Bio-
chemical evidence for exocytosis. Brit. J. Pharmacol. 31: 94-104, 1967.
Schneider, T, P. Igelmund, and J. Hescheler. G protein interaction with K + and Ca2+
channels. Trends Pharmacol. Sci. 18: 8-11, 1997.
Schofield, PR., M.G. Darlison, N. Fujita, D.R. Burt, FA. Stephenson, H. Rodriguez,
L.M. Rhee, J. Ramachandran, V. Reale, T.A. Glencorse, PH. Seeburg, and E.A.
Bernard. Sequence and functional expression of the GABAA receptor shows a

ligand-gated receptor super-family. Nature 328: 221-227, 1987.
Schook, W., S. Puszkin, W. Bloom, C. Ores, and S. Kochwa. Mechanochemical prop-
erties of brain clathrin: Interactions with actin and a-actinin and polymerization
into basketlike structures or filaments. Proc. Natl. Acad. Sci. USA 76: 116-120,
1979.
Schou, M., N. Juel-Nielsen, E. Stromgren, and H. Voldby. The treatment of manic psy-
choses by the administration of lithium salts. /. Neurol. Neurosurg. Psychiat. 17:
250-260, 1954.
Schuch, U., M.J. Lohse, and M. Schachner. Neural cell adhesion molecules influence
second messenger systems. Neuron 3: 13-20, 1989.
Schuldiner, S., H. Fishkes, and B.L. Kanner. Role of a transmembrane pH gradient in
epinephrine transport by chromaffin granule membrane vesicles. Proc. Natl. Acad.
Sci. USA 75: 3713-3716, 1978.
Schulman, H., and P. Greengard. Ca2+-dependent protein phosphorylation system in
membranes from various tissues, and its activation by "calcium-dependent regula-
tor." Proc. Natl. Acad. Sci. USA 75: 5432-5436, 1978.
Schults, C.W. Current roles and future applications of trophic factors. In: Olanow, C.W.,
and A.N. Lieberman (eds.) The Scientific Basis for the Treatment of Parkinson's
Disease. Park Ridge, NJ: Parthenon Publishing Group, 1991, pp. 257-274.
Schumacher, M., S. Camp, Y. Maulet, M. Newton, K. MacPhee-Quigley, S.S. Taylor, T.
Friedmann, and P. Taylor. Primary structure of Torpedo californica acetyl-
cholinesterase deduced from its cDNA sequence. Nature 319: 407-409, 1986.
Schuman, E.M., and D.V. Madison. A requirement for the intercellular messenger nitric
oxide in long-term potentiation. Science 254: 1503-1506, 1991.
Schumann, H.J., and A. Philippu. Release of catechol amines from isolated medullary
granules by sympathomimetic amines. Nature 193: 890-891, 1962.
Schwab, M.E., U. Otten, Y. Agid, and H. Thoenen. Nerve growth factor (NGF) in the
rat CNS: Absence of specific retrograde axonal transport and tyrosine hydroxylase
induction in locus coeruleus and subststantia nigra. Brain Res. 168: 473-483, 1979.
Schwab, R.S., L.V. Amador, and J.Y. Lettvin. Apomorphine in Parkinson's disease. Trans.
Amer. Neurol. Assoc. 76: 251-253, 1951.
Schwab, R.S., A.C. England, D.C. Poskanzer, and R.R. Young. Amantadine in the treat-
ment of Parkinson's disease./. Amer. Med. Assoc. 208: 1168-1170, 1969.
Schwartz, E.A. Calcium-independent release of GAB A from isolated horizontal cells of
the toad retina./. Physiol. 323: 211-227, 1982.
Schwartz, E.A. Depolarization without calcium can release y-aminobutyric acid from a
retinal neuron. Science 238: 350-355, 1987.
Schwartzkroin, P.A., and K. Wester. Long-lasting facilitation of a synaptic potential fol-
lowing tetanization in the in vitro hippocampal slice. Brain Res. 89: 107-119, 1975.
Schweitzer, A., and S. Wright. The action of eserine and related compounds and of
acetylcholine on the central nervous system. /. Physiol. 89: 165—197, 1937.
Scoville, W.B., and B. Milner. Loss of recent memory after bilateral hippocampal lesions.
/. Neurol. Neurosurg. Psychiat. 20: 11-21, 1957.
Sedvall, G., and B. Wode-Helgodt. Aberrant monoamine metabolite levels in CSF and
family history of schizophrenia. Arch. Gen. Psychiat. 37: 1113-1116, 1980.
Seeman, P., T.Lee, M. Chau-Wong, and K. Wong. Antipsychotic drug doses and neu-
roleptic/dopamine receptors. Nature 261: 717-719, 1976.
Seeman, P., C. Ulpian, G. Chouinard, H.H.M. Van Tol, H. Dwosh, J.A. Lieberman,
References 429
K. Siminovitch, I.S.C. Liu, J. Waye, P. Voruganti, C. Hudson, G.R. Serjeant, A.S.

Masibay, and M.V. Seeman. Dopamine D4 receptor variant, D4glycinel94, in
Africans, but not in Caucasians: No association with schizophrenia. Amer. ]. Med.
Gen. 54: 384-390, 1994.
Seidah, N.G., L. Caspar, P. Mion, M. Marcinkiewicz, M. Mbikay, and M. Chretien.
cDNA sequence of two distinct pituitary proteins homologous to Kex2 and furin
gene products: Tissue-specific mRNAs encoding candidates for pro-hormone pro-
cessing proteinases. DNA Cell Biol. 9: 415-424, 1990.
Sen, G., and K.C. Bose. Rauwolfia serpentina, a new Indian drug for insanity and high
blood pressure. Indian Med. World 2: 194-201, 1931.
Serafini, T., T.E. Kennedy, M.J. Galko, C. Mirzayan, T.M. Jessell, and M. Tessier-
Lavigne. The netrins define a family of axon outgrowth-promoting proteins homol-
ogous to C. Elegans UNC-6. Cell 78: 409-424, 1994.
Shackell, L.F., W. Williamson, M.M. Deitchman, G.M. Katzman, and B.S. Kleinman.
The relation of dosage to effect. /. Pharmacol. Exp. Ther. 24: 53-65, 1924.
Shaw, F.H. The estimation of adrenaline. Biochem. J. 32: 19-25, 1938.
Shaw, J.E., and P.W. Ramwell. Release of a substance P polypeptide from the cerebral
cortex. Amer. J. Physiol. 215: 262-267, 1968.
Sheehan, D. Discovery of the autonomic nervous system. Arch. Neurol. Psychiat. 35:
1081-1115, 1936.
Sheehan, D. The autonomic nervous system prior to Gaskell. New Eng. J. Med. 224:
457-460, 1941.
Shepherd, G.M. Foundations of the Neuron Doctrine. New York: Oxford University
Press, 1991.
Shepherd, G.M., and S.D. Erulkar. Centenary of the synapse: From Sherrington to the
molecular biology of the synapse and beyond. Trends. Neurosci. 20: 385-392, 1997.
Sherrington, C. The Integrative Action of the Nervous System. Second ed. New Haven:
Yale University Press, 1947.
Sherrington, C.S. Notes on the arrangement of some motor fibres in the lumbo-sacral
plexuses. /. Physiol. 13: 621-772, 1892a.
Sherrington, C.S. Note toward the localisation of the knee-jerk. Brit. Med. J. 1: 545,
1892b.
Sherrington, C.S. Addendum to note on the knee-jerk. Brit. Med. J. 1: 654, 1892c.
Sherrington, C.S. Experiments in examination of the peripheral distribution of the fibres
of the posterior roots of some spinal nerves. Phil. Trans. R. Soc. 184: 641-763,
1893a.
Sherrington, C.S. Note on the spinal portion of some ascending degenerations. /. Phys-
iol. 14: 255-302, 1893b.
Sherrington, C.S. Note on the knee-jerk and the correlation of action of antagonistic
muscles. Proc. R. Soc. 52: 556-564, 1893c.
Sherrington, C.S. On the anatomical constitution of nerves of skeletal muscles; with
remarks on recurrent fibres in the ventral spinal nerve-root./. Physiol. 17: 211-258,
1894.
Sherrington, C.S. On reciprocal innervation of antagonistic muscles. Proc. R. Soc. 60:
414_417, 1897.
Sherrington, C.S. Experiments in examination of the peripheral distribution of the fibres
of the posterior roots of some spinal nerves. Phil. Trans. R. Soc. 190: 45-186,1898a.
Sherrington, C.S. Decerebrate rigidity, and reflex coordination of movements. J. Phys-
iol. 22: 319-332, 1898b.
Sherrington, C.S. On reciprocal innervation of antagonistic muscles. Proc. R. Soc. 76B:

269-297, 1905.
Sherrington, C.S. Reflex inhibition as a factor in the co-ordination of movements and
postures. Quart. J. Exp. Physiol. 6: 251-310, 1913.
Sherrington, C.S. Remarks on some aspects of reflex inhibition. Proc. R. Soc. 97B:
519-545, 1925.
Shimada, S., S. Kitayama, C.L. Lin, A. Patel, E. Nanthakumar, P. Gregor, M. Kuhar,
and G. Uhl. Cloning and expression of a cocaine-sensitive dopamine transporter
complementary DNA. Science 254: 576-578, 1991.
Shiman, R., M. Akino, and S. Kaufman. Solubilization and partial purification of tyro-
sine hydroxylase from bovine adrenal medulla./. Biol. Chem. 246:1330-1340,1971.
Shimidzu, K. Die Bildung von vegetativen Reizstoffen im tatigen Muskel. Pfliigers Arch.
211: 403-413, 1926.
Shirayoshi, Y., T.S. Okada, and M. Takeichi. The calcium-dependent cell-cell adhesion
system regulates inner cell mass formation and cell surface polarization in early
mouse development. Cell 35: 631-638, 1983.
Shore, P.A., J.A.R. Mead, R.G. Kuntzman, S. Spector, and B.B. Brodie. On the physi-
ologic significance of monoamine oxidase in brain. Science 126: 1063-1064, 1957.
Shorr, R.G.L., S.L. Heald, P.W. Jeffs, T.N. Lavin, M.W.Strohsacker, R.J. Lefkowitz, and
M.G. Caron. The /3-adrenergic receptor: Rapid purification and covalent labeling
by photoaffinity crosslinking. Proc. Natl. Acad. Aci. USA 79: 2778-2782, 1982a.
Shorr, R.G.L., R.J. Lefkowitz, and M.G. Caron. Purification of the /3-adrenergic recep-
tor. /. Biol Chem. 256: 5820-5826, 1981.
Shorr, R.G.L., M.W. Strohsacker, T.N. Lavin, R.J. Lefkowitz, and M.G. Caron. The /3r
adrenergic receptor of the turkey erythrocyte. /. Biol. Chem. 257: 12,341-12,350,
1982b.
Short, J.M., A. Wynshaw-Boris, H.P. Short, and R.W. Hanson. Characterization of the
phosphoenolpyruvate carboxykinase (GTP) promoter-regulatory region. J. Biol.
Chem. 261: 9721-9726, 1986.
Shorter, E. A History of Psychiatry. New York: John Wiley & Sons, 1997.
Shortland, M. Book review. Brit. J. Hist. Sci. 21: 264-267, 1988.
Shuster, L. Readings in Pharmacology. Boston: Little, Brown and Company, 1962.
Sibley, D.R., J.L. Benovic, M.G. Caron, and R.J. Lefkowitz. Regulation of transmem-
brane signaling by receptor phosphorylation. Cell 48: 913-922, 1987.
Sibley, D.R., R.H. Strasser, J.L. Benovic, K. Daniel, and R.J. Lefkowitz. Phosphoryla-
tion/dephosphorylation of the /3-adrenergic receptor regulates its functional cou-
pling to adenylate cyclase and subcellular distribution. Proc. Natl. Acad. Sci. USA
83: 9408-9412, 1986.
Simantov, R., A.M. Snowman, and S.H. Snyder. A morphine-like factor "enkephalin" in
rat brain: Subcellular localization. Brain Res. 107: 650-657, 1976.
Simantov, R., and S.H. Snyder. Morphine-like peptides in mammalian brain: Isolation,
structure elucidation, and interactions with the opiate receptor. Proc. Natl. Acad.
Sci. USA 73: 2515-2519, 1976.
Simon, E.J., and J. Groth. Kinetics of opiate receptor inactivation by sulfhydryl reagents:
Evidence for conformational change in presence of sodium ions. Proc. Natl. Acad.
Sci. USA 72: 2404-2407, 1975.
Simon, E.J., J.M. Hillier, and I. Edelman. Stereospecific binding of the potent narcotic
analgesic [3H]etorphine to rat-brain homogenate. Proc. Natl. Acad. Sci. USA 70:
1947-1949, 1973.
Simon, S.M., and R.R. Llinas. Compartmentalization of the submembrane calcium activ-
References 431
ity during calcium influx and its significance in transmitter release. Biophys. ]. 48:
485^98, 1985.
Simonart, A., and E.F. Simonart. L'acetylcholine et le muscle strie normal de mam-
mifere. Arch. Internat. Pharmaco. Ther. 49: 302-328, 1935.
Singer, S.J., and G.L. Nicolson. The fluid mosaic model of the structure of cell mem-
branes. Science 175: 720-731, 1972.
Sjoerdsma, A., T.E. Smith, T.D. Stevenson, and S. Udenfriend. Metabolism of 5-hydrox-
ytryptamine (serotonin) by monoamine oxidase. Proc. Soc. Exp. Biol. Med. 89: 36-
38, 1955.
Smith, G.D., and J.F. Griffin. Conformation of [Leu5]enkephalin from x-ray diffraction:
Features important for recognition at opiate receptor. Science 199:1214-1216,1978.
Smith, H.W. The plasma membrane, with notes on the history of botany. Circulation
26: 987-1012, 1962.
Smith, R. Inhibition. Berkeley: University of California Press, 1992.
Smith, S.J. Neuronal cytomechanics: The actin-based motility of growth cones. Science
242: 708-715, 1988.
Smrcka, A.V., J.R. Hepler, K.O. Brown, and P.C. Sternweis. Regulation of polyphos-
phoinositide-specific phospholipase C activity by purified Gq. Science 251: 804-807,
1991.
Snyder, S.H., S.P. Banerjee, H.I. Yamamura, and D. Greenberg. Drugs, neurotrans-
mitters, and schizophrenia. Science 184: 1243-1253, 1974.
Sobel, A., T Heidmann, J. Hofler, and J.P. Changeux. Distinct protein components from
Torpedo marmorata membranes carry the acetylcholine receptor site and the bind-
ing site for local anesthetics and histrionicotoxin. Proc. Natl. Acad. Sci. USA 75:
510-514, 1978.
Sobue, K., and K. Kanda. a-Actinins, calspectin (brain spectrin or fodrin), and actin
participate in adhesion and movement of growth cones. Neuron 3: 311-319, 1989.
Solderling, T.R., J.P. Hickenbottom, E.M. Reimann, F.L. Hunkeler, D.A. Walsh, and
E.G. Krebs. Inactivation of glycogen synthase and activation of phosphorylase
kinase by muscle adenosine 3',5'-monophosphate-dependent protein kinases.
/. Biol. Chem. 245: 6317-6328, 1970.
Spacek, J. Dynamics of the Golgi method. /. Neurocytol. 18: 27-38, 1989.
Spector, S., D. Prockop, P.A. Shore, and B.B. Brodie. Effect of iproniazid on brain lev-
els of norepinephrine and serotonin. Science 127: 704, 1958.
Spedding, M. Calcium antagonist subgroups. Trends Pharmacol. Sci. 6: 109-114, 1985.
Speeter, M.E., R.V. Heinzelmann, and D.I. Weisblat. The synthesis of the blood serum
vasoconstrictor principle serotonin creatinine sulfate./. Amer. Chem. Soc. 73: 5514-
5515, 1951.
Spehmann, R. Acetylcholine and prostigmine electrophoresis at visual cortex neurons.
/. Neurophysiol. 26: 127-139, 1963.
Spehmann, R., and H. Kapp. Die Wirkung lokaler Mikroelektrophorese von Acetyl-
cholin auf einzelne Neurone des visuellen Cortex. Pfliigers Arch. 274: 37-38, 1961.
Speidel, C.C. Studies of living nerves. Amer. J. Anat. 52: 1-75, 1933.
Speidel, C.C. Adjustments of nerve endings. Harvey Lect. 36: 126-158, 1941.
Sperry, R.W. The effect of crossing nerves to antagonistic muscles in the hind limb of
the rat./. Comp. Neurol. 75: 1-19, 1941.
Sperry, R.W. Visuomotor coordination in the newt (Triturus viridescens) after regener-
ation of the optic nerve. /. Comp. Neurol. 79: 33-35, 1943.
Sperry, R.W. Chemoaffinity in the orderly growth of nerve fiber patterns and connec-
tions. Proc. Natl. Acad. Sci. USA 50: 703-710, 1963.
Speriy, R.W., and N. Miner. Formation within sensory nucleus V of synaptic associa-
tions mediating cutaneous localization. /. Comp. Neurol. 90: 403-423, 1949.
Squire, L.R., and S. Zola-Morgan. The medial temporal lobe memory system. Science
253: 1380-1386, 1991.
Squires, R.F. Additional evidence for the existence of several forms of mitochondrial
monoamine oxidase in the mouse. Biochem. Pharmacol. 17: 1401-1409, 1968.
Srinivasan, V., M.J. Neal, and J.F. Mitchell. The effect of electrical stimulation and high
potassium concentrations on the efflux of [3H]y-aminobutyric acid from brain
slices. /. Neurochem. 16: 1235-1244, 1969.
Stadel, J.M., P. Nambi, R.G.L. Shorr, D.F. Sawyer, M.G. Caron, and R.J. Lefkowitz.
Catecholamine-induced desensitization of turkey erythrocyte adenylate cyclase is
associated with phosphorylation of the j3-adrenergic receptor. Proc. Natl. Acad. Set.
USA 80: 3173-3177, 1983.
Stadtman, E.R. Enzyme multiplicity and function in the regulation of divergent meta-
bolic pathways. Bacterial. Rev. 27: 170-181, 1963.
Stahl, S.M., and L. Palazidou. The pharmacology of depression. Trends Pharmacol. 7:
349-354, 1986.
Stanton, P.K., and TJ. Sejnowski. Associative long-term depression in the hippocampus
induced by hebbian covariance. Nature 339: 215-218, 1989.
Stark, P., and D. Hardison. A review of multicenter controlled studies of fluoxetine vs
imipramine and placebo in outpatients with major depressive disorder. J. Clin. Psy-
chiat. 46: (3, sect. 2): 53-58, 1985.
Starke, K. Influence of a-receptor stimulants on noradrenaline release. Naturwis-
senschaften 58: 420, 1971.
Starke, K. Alpha sympathomimetic inhibition of adrenergic and cholinergic transmis-
sion in the rabbit heart. Naunyn-Schmiedebergs Arch. 274: 18-45, 1972.
Starke, K., E. Borowski, and T. Endo. Preferential blockade of presynaptic a-adreno-
ceptors by yohimbine. Eur. J. Pharmacol. 34: 385-388, 1975.
Stedman, E., E. Stedman, and L.H. Easson. Choline-esterase. An enzyme present in
the blood-serum of the horse. Biochem. J. 26: 2056-2066, 1932.
Stehle, R.L., and H.C. Ellsworth. Dihydroxyphenyl ethanolamine (arterenol) as a pos-
sible sympathetic hormone. J. Pharmacol. Exp. Ther. 59: 114—121, 1937.
Steiner, D.F. Insulin today. Diabetes 26: 322-340, 1977.
Stell, W.K. Correlation of retinal cytoarchitecture and ultrastructure in Golgi prepara-
tions. Anat. Rec. 153: 389-398, 1965.
Stephenson, R.P. A modification of receptor theory. Brit. J. Pharmacol. 11: 379-393,1956.
Stern-Bach, Y., N. Greenberg-Ofrath, I. Flechner, and S. Schuldiner. Identification and
purification of a functional amine transporter from bovine chromaffin granules.
/. Biol. Chem. 265: 3961-3966, 1990.
Stolz, F. tiber Adrenalin und Alkylaminocetobrenzcatechin. Ber. Deutsch. Chem. Ges.
37: 4149-4154, 1904.
Storey, D.J., S.B. Shears, C.J. Kirk, and R.H. Michell. Stepwise enzymatic dephospho-
rylation of inositol 1,4,5-trisphosphate to inositol in liver. Nature 312: 374-376,
1984.
Story, D.D., M.S. Briley, and S.Z. Langer. The effects of chemical sympathectomy with
6-hydroxydopamine on a-adrenoceptor and muscarinic cholinoceptor binding in
rat heart ventricle. Eur. J. Pharmacol. 57: 423-426, 1979.
Straub, W. Zur chemischen Kinetik der Muskarinwirkung und des Antagonismus
Muskarin-Atropin. Pflugers Arch. 119: 127-151, 1907.
Streb, H., R.F. Irvine, M.J. Berridge, and I. Schulz. Release of Ca2+ from a nonmito-
References 433
chondrial intracellular store in pancreatic acinar cells by inositol-l,4,5-trisphos-

phate. Nature 306: 67-69, 1983.
Stromblad, B.L.R. Supersensitivity and amine oxidase activity in denervated salivary
glands. Acta Physiol Scand. 36: 137-153, 1956.
Stromblad, C.R., and M. Nickerson. Accumulation of epinephrine and norepinephrine
by some rat tissues. /. Phannacol. Exp. Ther. 134: 154-159, 1961.
Stroud, R.M., M.P. McCarthy, and M. Shuster. Nicotinic acetylcholine receptor super-
family of ligand-gated ion channels. Biochemistry 29: 11,009-11,023, 1990.
Stryer, L. Biochemistry. New York: W.H. Freeman. Third edition, 1988.
Suddath, R.L., G.W. Christison, and E.F. Torrey. Anatomical abnormalities in the brains
of monozygotic twins discordant for schizophrenia. New Eng. ]. Med. 322: 789-794,
1990.
Siidhof, T.C., A.J. Czernik, H.T. Kao, K. Takei, P.A. Johnston, A. Horiuchi, S.D. Kanzir,
M.A. Wagner, M.S. Perin, P. DeCamilli, and P. Greengard. Synapsins: Mosaics of
shared and individual domains in a family of synaptic vesicle phosphoproteins. Sci-
ence 245: 1474-1480, 1989.
Siidhof,Y., F. Lottspeich, P. Greengard, E. Mehl, and R. Jahn. A synaptic vesicle pro-
tein with a novel cytoplasmic domain and four transmembrane regions. Science
238: 1142-1144, 1987.
Sugrue, M.F. Do antidepressants possess a common mechanism of action? Biochem.
Pharmacol 32: 1811-1817, 1983.
Sullivan, S., H. Akil, and J.D. Barchas. In vitro degradation of enkephalin: Evidence
for cleavage at the gly-phe bond. Commun. Psychophartnacol. 2: 523-531, 1978.
Sulser, F, J. Vetulani, and PL. Mobley. Mode of action of antidepressant drugs. Biochem.
Pharmacol. 27: 257-261, 1978.
Sulzer, D., and S. Rayport. Amphetamine and other psychostimulants reduce pH gra-
dients in midbrain dopaminergic neurons and chromaffin granules: A mechanism
of action. Neuron 5: 797-808, 1990.
Sumi, C., H. Ichinose, and T. Nagatsu. Characterization of recombinant human aro-
matic L-amino acid decarboxylase expressed in COS cells. J. Neurochem. 55:
1075-1078, 1990.
Supattapone, S., P.F. Worley, J.M. Baraban, and S.H. Snyder. Solubilization, purifica-
tion, and characterization of an inositol trisphosphate receptor. J. Biol. Chem. 263:
1530-1534, 1988.
Sussman, J.L., M. Harel, F. Frolow, C. Oefner, A. Goldman, L. Toker, and I. Silman.
Atomic structure of acetylcholinesterase from Torpedo californica. Science 253:
872-879, 1991.
Suszkiw, J.B., H. Zimmermann, and V.P. Whittaker. Vesicular storage and release of
acetylcholine in Torpedo electroplaque synapses. /. Neurochem. 30: 1269-1280,
1978.
Sutherland, E.W. Studies on the mechanism of hormone action. Science 177: 401-408,
1972.
Sutherland, E.W., and C.F. Cori. Effect of hyperglycemic-glycogenolytic factor and epi-
nephrine on liver phosphorylase. J. Biol. Chem. 188: 531-543, 1951.
Sutherland, E.W., I. 0ye, and R.W. Butcher. The action of epinephrine and the role of
the adenyl cyclase system in hormone action. Rec. Prog. Horm. Res. 21: 623-646,
1965.
Sutherland, E.W., and T.W. Rail. The properties of an adenine-ribonucleotide produced
with cellular particles, ATP, Mg ++ , and epinephrine or glucagon. /. Amer. Chem.
Soc. 79: 3608, 1957.
Sutherland, E.W., T.R. Rail, and T. Menon. Adenyl cyclase. I./. Biol Chem. 237: 1220-
1227, 1962.
Sutherland, E.W., and W.D. Wosilait. Inactivation and activation of liver phosphorylase.
Nature 175: 169-170, 1955.
Sutter, A., R.J. Riopelle, R.M. Harris-Warrick, and E.M. Shooter. NGF receptors: Char-
acterization of two distinct classes of binding sites on chick embryo sensory gan-
glia cells. /. Biol. Chem. 254: 5972-5982, 1979.
Swanson, I.W. The projections of the ventral tegmental area and adjacent regions: A
combined fluorescent retrograde tracer and immunofluorescence study in the rat.
Brain Res. Bull. 9: 321-353, 1982.
Swazey, J.P. Reflexes and Motor Integration. Cambridge, Mass.: Harvard University
Press, 1969.
Swazey, J.P. Chlorpromazine: A Study in Therapeutic Innovation. Cambridge, Mass.:
MIT Press, 1974.
Szatkowski, M., B. Barbour, and D. Attwell. Non-vesicular release of glutamate from
glial cells by reversed electrogenic glutamate uptake. Nature 348: 443-446, 1990.
Takahashi, T., S. Konishi, D. Powell, S.E. Leeman, and M. Otsuka. Identification of the
motoneuron-depolarizing peptide in bovine dorsal root as hypothalamic substance
P. Brain Res. 73: 59-69, 1974.
Takai, Y., A. Kishimoto, Y. Iwasa, Y. Kawahara, T. Mori, and Y. Nishizuka. Calcium-
dependent activation of a multifunctional protein kinase by membrane phospho-
lipids. /. Biol. Chem. 254: 3692-3695, 1979a.
Takai, Y, A. Kishimoto, U. Kikkawa, T. Mori, and Y. Nishizuka. Unsaturated diacyl-
glycerol as a possible messenger for the activation of calcium-activated, phospho-
lipid-dependent protein kinase system. Biochem. Biophys. Res. Commun. 91:1218-
1224, 1979b.
Takamine, J. Adrenalin the active principle of the suprarenal glands and its mode of
preparation. Amer. J. Pharmacy 73: 523-531, 1901.
Takeuchi, A., and N. Takeuchi. On the permeability of end-plate membrane during the
action of transmitter. /. Physiol. 154: 52-67, 1960.
Takeuchi, A., and N. Takeuchi. The effect on crayfish muscle of iontophoretically applied
glutamate. /. Physiol 170: 296-317, 1964.
Takeuchi, A., and N. Takeuchi. Localized action of gamma-aminobutyric acid on cray-
fish muscle. /. Physiol 177: 225-238, 1965.
Tanabe, T., H. Takeshima, A. Mikami, V. Flockerzi, H. Takahashi, K. Kangawa,
M. Kojima, H. Matsuo, T. Hirose, and S. Numa. Primary structure of the recep-
tor for calcium channel blockers from skeletal muscle. Nature 328: 313-318, 1987.
Tanaka, C., K. Taniyama, and M. Kusunoki. A phorbol ester and A23187 act synergis-
tically to release acetylcholine from the guinea pig ileum. FEBS Lett. 175: 165-169,
1984.
Tansey, E.M. What's in a name? Henry Dale and adrenaline, 1906. Med. Hist. 39: 459-
476, 1995.
Tansey, E.M. Not committing barbarisms: Sherrington and the synapse, 1897. Brain
Res. Butt. 44:211-212, 1997.
Tasaki, I. Conduction of the nerve impulse. In: Field, J., H.W. Magoun, and V.E. Hall
(eds.) Handbook of Physiology, (Section 1), Neurophysiology. Vol. 1. Washington,
DC: American Physiological Society, 1959, pp. 75-121.
Tauc, L. Nonvesicular release of neurotransmitter. Physiol Rev. 62: 857-893, 1982.
References 435
Taylor, K.M., and S.H. Snyder. Differential effects of D- and L-amphetamine on behav-
ior and on catecholamine disposition in dopamine and norepinephrine containing
neurons of rat brain. Brain Res. 28: 295-309, 1971.
Taylor, S.J., H.Z. Chae, S.G. Rhee, and J.H. Exton. Activation of the f31 isozyme of phos-
pholipase C by a subunits of the Gq class of G proteins. Nature 350: 516-518,1991.
Teichberg, V.I., A. Sobel,and J.-P. Changeux. In vitro phosphorylation of the acetyl-
choline receptor. Nature 267: 540-542, 1977.
Temple, A., J.A. Kessler, and R.S. Zukin. Chronic naltrexone treatment increases expres-
sion of preproenkephalin and preprotachykinin mRNA in discrete brain regions.
/. Neurosci. 10: 741-747, 1990.
Teo, T.S., T.H. Wang, and J.H. Wang. Purification and properties of the protein activa-
tor of bovine heart cyclic adenosine 3',5'-monophosphate phosphodiesterase.
/. Biol Chem. 248: 588-595, 1973.
Terenius, L. Stereospecific interaction between narcotic analgesics and a synaptic plasma
membrane fraction of rat cerebral cortex. Acta Pharmacol. Toxicol. 32: 317-320,
1973a.
Terenius, L. Characteristics of the "receptor" for narcotic analgesics in synaptic plasma
membrance fraction from rat brain. Acta Pharmacol. Toxicol. 33: 377-384, 1973b.
Terenius, L., and A. Wahlstrom. Morphine-like ligand for opiate receptors in human
CSF. Life Sci. 16: 1759-1764, 1975.
Teschemacher, H., K.E. Opheim, B.M. Cox, and A. Goldstein. A peptide-like substance
from pituitary that acts like morphine. Life Sci. 16: 1771-1776, 1975.
Tessier-Lavigne, M., M. Placzek, A.G.S. Lumsden, J. Dodd, and T.M. Jessell.
Chemotropic guidance of developing axons in the mammalian central nervous sys-
tem. Nature 336: 775-778, 1988.
Tetrud, J.W., and J.W. Langston. The effect of deprenyl (selegiline) on the natural his-
tory of Parkinson s disease. Science 245: 519-522, 1989.
Thiery, J.P., R. Brackenbury, U. Rutishauser, and G.M. Edelman. Adhesion among neu-
ral cells of the chick embryo. /. Biol Chem. 252: 6841-6845, 1977.
Thoa, N.B., F. Wooten, J. Axelrod, and I.J. Kopin. On the mechanism of release of nor-
epinephrine from sympathetic nerves induced by depolarizing agents and sympa-
thomimetic drugs. J. Pharmacol. Exp. Ther. 11: 10-18, 1975.
Thoenen, H. Induction of tyrosine hydroxylase in peripheral and central adrenergic neu-
rones by cold-exposure of rats. Nature 228: 861-862, 1970.
Thomas, K.B. Curare. Philadelphia: J.B. Lippincott, 1963.
Thomas, L., K. Hartung, D. Langosch, H. Rehm, E. Bamberg, W.W Franke, and H.
Betz. Identification of synaptophysin as a hexameric channel protein of the synap-
tic vesicle membrane. Science 242: 1050-1053, 1988.
Thompson, C. (ed.) The Origins of Modern Psychiatry. New York: John Wiley, 1987.
Thompson, R., and J. McConnell. Classical conditioning in the planarian, Dugesia doro-
tocephala. J. Comp. Physiol. Psychol 48: 65-68, 1955.
Thron, C.D. On the analysis of pharmacological experiments in terms of an allosteric
receptor model. Mol. Pharmacol. 9: 1-9, 1973.
Tilgmann, C., and N. Kalkkinen. Purification and partial characterization of rat liver sol-
uble catechol-O-methyltransferase. FEBS Lett. 264: 95-99, 1990.
Torri-Tarelli, F., F. Grohovaz, R. Fesce, and B. Ceccarelli. Temporal coincidence
between synaptic vesicle fusion and quantal secretion of acetylcholine. /. Cell Biol.
101: 1386-1399, 1985.
Toyoshima, T., and N. Unwin. Ion channel of acetylcholine receptor reconstructed from
images of postsynaptic membranes. Nature 336: 247-250, 1988.
Toyoshima, T., and N. Unwin. Three-dimensional structure of the acetylcholine recep-
tor by cryoelectron microscopy and helical image reconstruction./. Cell Biol. Ill:
2623-2635, 1990.
Tregear, G.W., H.D. Niall, J.T. Potts, S.E. Leeman and M.M. Chang. Synthesis of sub-
stance P. Nature New Biol 232: 87-88, 1971.
Trimble, W.S., D.M. Cowan, and R.H. Scheller. VAMP-1: A synaptic vesicle-associated
integral membrane protein. Proc. Natl Acad. Sci. USA 85: 4538-4542, 1988.
Trimble, W.S., M. Linial, and R.H. Scheller. Cellular and molecular biology of the presy-
naptic nerve terminal. Annu. Rev. Neurosci. 14: 93-122, 1991.
Trisler, D., and F. Collins. Corresponding spatial gradients of TOP molecules in the
developing retina and optic tectum. Science 237: 1208-1209, 1987.
Trisler, G.D., M.D. Schneider, and M. Nirenberg. A topographic gradient of molecules
in retina can be used to identify neuron position. Proc. Natl. Acad. Sci. USA 78:
2145-2149, 1981.
Tsien, R.W. Mode of action of chronotropic agents in cardiac Purkinje fibers. J. Gen.
Physiol. 64: 320-342, 1974.
Turner, W.A. On recent applications of Golgi s method to the study of the nervous sys-
tem. Brain 16: 259-285, 1893.
Turtle, J.R., and D.M. Kipnis. An adrenergic receptor mechanism for the control of
cyclic 3' 5' adenosine monophosphate synthesis in tissues. Biochem. Biophys. Res.
Commun. 28: 797-802, 1967.
Twarog, B.M., and I.H. Page. Serotonin content of some mammalian tissues and urine
and a method for its determination. Amer. J. Physiol. 175: 157-161, 1953.
Tyler, K.L. A history of Parkinsons disease. In: Koller, W.C. (ed.) Handbook of Parkin-
son's Disease. New York: Marcel Dekker, Inc., 1991, pp. 1-33.
Tzartos, S.J., A. Kokla, S.L. Walgrave, and B.M. Conti-Tronconi. Localization of the
main immunogenic region of human muscle acetylcholine receptor to residues
67-76 of the a subunit. Proc. Natl. Acad. Sci. USA 85: 2899-2903, 1988.
Udenfriend, S., and J.B. Wyngaarden. Presursors of adrenal epinephrine and norepi-
nephrine in vivo. Biochim. Biophys. Acta 20: 48-52, 1956.
Udenfriend, S., P. Zaltzman-Nirenberg, R. Gordon, and S. Spector. Evaluation of the
biochemical effects produced in vivo by inhibitors of the three enzymes involved
in norepinephrine biosynthesis. Mol. Pharmacol. 2: 95-105, 1966.
Ueda, T., and P. Greengard. Adenosine 3':5'-monophosphate-regulated phosphopro-
tein system of neuronal membranes. J. Biol. Chem. 252: 5155-5163, 1977.
Ueda, T., H. Maeno, and P. Greengard. Regulation of endogenous phosphorylation of
specific proteins in synaptic membrane fractions from rat brain by adenosine 3':5'-
monophosphate. /. Biol. Chem. 248: 8295-8305, 1973.
Unanue, E.R., E. Ungewickell, and D. Branton. The binding of clathrin triskelions to
membranes from coated vesicles. Cell 26: 439-446, 1981.
Ungewickell, E., and D. Branton. Assembly units of clathrin coats. Nature 289: 420-422,
1981.
Usdin, T.B., E. Mezey, C. Chen, M.J. Brownstein, and B.J. Hoffman. Cloning of the
cocaine-sensitive bovine dopamine transporter. Proc. Natl. Acad. Sci. USA 88: 11,
168-11,171, 1991.
Ushkaryov, Y.A., A.G. Petrenko, M. Geppert, and T.C. Siidhof. Neurexins: Synaptic cell
surface proteins related to the a-latrotoxin receptor and laminin. Science 257:
50-56, 1992.
Rererences 437
Vakil, R.J. A clinical trial of Rauwolfia serpentina in essential hypertension. Brit. Heart
]. 2: 350-355, 1949.
Van der Kloot, W. Quantal size is not altered by abrupt changes in nerve terminal vol-
ume. Nature 271: 561-562, 1978.
Van der Kloot, W.G., and J. Bobbins. The effects of y-aminobutyric acid and picrotoxin
on the junctional potential and the contraction of crayfish muscle. Experientia 15:
35-36, 1959.
Van Harreveld, A. Compounds in brain extracts causing spreading depression of cere-
bral cortical activity and contraction of crustacean muscle. J. Neurochem. 3:
300-315, 1959.
Van Harreveld, A., and E. Fifkova. Swelling of dendritic spines in the fascia dentata
after stimulation of the perforant fibers as a mechanism of post-tetanic potentia-
tion. Exp. Neurol. 49: 736-749, 1975.
Van Praag, D., and E.J. Simon. Studies on the intracellular distribution and tissue bind-
ing of dihydromorphine-7,8-H3 in the rat. Proc. Soc. Exp. Biol Med. 122: 6-11,1966.
van Rossum, J.M. The significance of dopamine-receptor blockade for the mechanism
of action of neuroleptic drugs. Arch. Int. Pharmacodyn. 160: 492^494, 1966.
Van Tol, H.H.M., J.R. Bunzow, H.C. Guan, R.K. Sunahara, P. Seeman, H.B. Niznik,
and O. Civelli. Cloning of the gene for a human dopamine D4 receptor with high
affinity for the antipsychotic clozapine. Nature 350: 610-614, 1991.
Verworn, M. General Physiology. London: Macmillan, 1899.
Vetulani, J., and F. Sulser. Action of various antidepressant treatments reduces reactiv-
ity of noradrenergic cyclic AMP-generating system in limbic forebrain. Nature 257:
495-496, 1975.
Vialli, M., and V. Erspamer. Cellule enterocromaffini e cellule basigranulose acidofile
nei Vertebrati. Z. Zellforsch. Microsk. Anat. 19: 743-773, 1933.
Virshup, D.M., and V. Bennett. Clathrin-coated vesicle assembly polypeptides: Physi-
cal properties and reconstitution studies with brain membranes. /. Cell Biol. 106:
39-50, 1988.
Viveros, O.H., L. Arqueros, and N. Kirshner. Release of catecholamines and dopamine-
/3-oxidase from the adrenal medulla. Life Sci. 7: 609-618, 1968.
Viveros, O.H., L. Arqueros, and N. Kirshner. Quantal secretion from adrenal medulla:
All-or-none release of storage vesicle content. Science 165: 911-913, 1969.
Vizi, E.S., and F. Vyskocil. Changes in total and quantal release of acetylcholine in the
mouse diaphragm during activation and inhibition of membrane ATPase. /. Phys-
iol 286: 1-14, 1979.
Vogt, M. The concentration of sympathin in different parts of the central nervous sys-
tem under normal conditions and after the administration of drugs./. Physiol. 123:
451-481, 1954.
von Euler, U.S. Untersuchungen iiber Substanz P, die atropinfeste, darmerregende und
gefasserweiternde Substanz aus Darm und Him. Naunyn-Schmiedebergs Archiv.
181: 181-197, 1936.
von Euler, U.S. A specific sympathomimetic ergone in adrenergic nerve fibres (sympa-
thin) and its relations to adrenaline and nor-adrenaline. Acta Physiol. Scand. 12:
73-97, 1946.
von Euler, U.S. Identification of the sympathomimetic ergone in adrenergic nerves of
cattle (sympathin N) with laevo-noradrenaline. Acta Physiol Scand. 16: 63-74,
1948.
von Euler, U.S. The distribution of sympathin N and sympathin A in spleen and splenic
nerves of cattle. Acta Physiol. Scand. 19: 207-214, 1949.
von Euler, U.S. Release and uptake of noradrenaline in adrenergic nerve granules. Acta
Physiol. Scand. 67: 430^40, 1966.
von Euler, U.S., and J.H. Gaddum. An unidentified depressor substance in certain tis-
sue extracts./. Physiol. 72: 74-87, 1931.
von Euler, U.S. and U. Hamberg. Colorimetric determination of noradrenaline and
adrenaline. Acta Physiol Scand. 19: 74-84, 1949.
von Euler, U.S., and F. Lishajko. Effect of adenine nucleotides on catecholamine release
and uptake in isolated adrenergic nerve granules. Acta Physiol. Scand. 59: 454-461,
1963.
von Neumann, J. The Computer and the Brain. New Haven: Yale University Press, 1958.
Von Voigtlander, P.F., and K.E. Moore. Dopamine: Release from the brain in vivo by
amantadine. Science 174: 408-410, 1971.
von Wedel, R.J., S.S. Carlson, and R.B. Kelly. Transfer of synaptic vesicle antigens to
the presynaptic plasma membrane during exocytosis. Proc. Natl. Acad. Sci. USA
78: 1014-1018, 1981.
Vyskocil, F. Inhibition of non-quantal acetylcholine leakage by 2(4-phenylpiperidine)
cyclohexanol in the mouse diaphragm. Neurosci. Lett. 59: 277-288, 1985.
Waksman, G., R. Bouboutou, J. Devin, S. Bourgoin, F. Cesselin, M. Hamon, M.C.
Fournie-Zaluski, and B.P. Roques. In vitro and in vivo effects of kelatorphan on
enkephalin metabolism in rodent brain. Eur. J. Pharmacol. 117: 233-243, 1985.
Waldo, G.L., J.K. Northup, J.P. Perkins, and T.K. Harden. Characterization of an altered
membrane form of the /3-adrenergic receptor produced during agonist-induced
desensitization. /. Biol. Chem. 258: 13,900-13,908, 1983.
Waller, A.D. On the physiological mechanism of the phenomenon termed "tendon
reflex."/. Physiol. 11: 384-395, 1890.
Walsh, D.A., J.P.Perkins, and E.G. Krebs. An adenosine 3',5'-monophosphate-depend-
ent protein kinase from rabbit skeletal muscle. /. Biol. Chem. 243: 3763-3765,
1968.
Walters, E.T., and J.H. Byrne. Associative conditioning of single sensory neurons sug-
gests a cellular mechanism for learning. Science 219: 405-408, 1983.
Wang, H.L., V.H. Harwalkar, and H.A. Waisman. Effect of dietary phenylalanine and
tryptophan on brain serotonin. Arch. Biochem. Biophys. 97: 181-184, 1962.
Wang, L.Y. Exchange of actin subunits at the leading edge of living fibroblasts: Possi-
ble role of treadmilling./. Cell Biol. 101: 597-602, 1985.
Watterson, D.M., F. Sharief, and T.C. Vanaman. The complete amino acid sequence of
the Ca2+-dependent modulator protein (calmodulin) of bovine brain./. Biol. Chem.
255: 962-975, 1980.
Waud, D.R. Pharmacological receptors. Pharmacol. Rev. 20: 49-88, 1968.
Wegner, A. Head to tail polymerization of actin. /. Mol. Biol 108: 139-150, 1976.
Weill, C.L., M.G. McNamee, and A. Karlin. Affinity-labeling of purified acetylcholine
receptor from Torpedo californica. Biochem. Biophys. Res. Commun. 61:997-1003,
1974.
Weinshilboum, R.M., N.B. Thoa, D.G. Johnson, I.J. Kopin, and J. Axelrod. Proportional
release of norepinephrine and dopamine-/8-hydroxylase from sympathetic nerves.
Science 174: 1349-1351, 1971.
Weinstein, H., E. Roberts, and T. Kakefuda. Studies of sub-cellular distribution of
y-aminobutyric acid and glutamic decarboxylase in mouse brain. Biochem. Phar-
macol. 12: 503-509, 1963.
Weiss, P. Die Funktion transplantierter Amphibienextremitaten. Aufstellung eine Res-
References 439
onanztheorie der motorischen Nerventatigkeit auf Grund abgestimmter Endor-

gane. Arch. Mikrosk. Anat. Entwicklungsmech. 102: 635-672, 1924.
Weiss, P. In vitro experiments on the factors determining the course of the outgrowing
nerve fiber. /. Exp. Zool 68: 393-448, 1934.
Weiss, P. Selectivity controlling the central-peripheral relations in the nervous system.
Biol Rev. 11: 494-531, 1936.
Weiss, P. Nerve patterns: Mechanics of nerve growth. Growth 5(suppl.): 163-203,1941.
Weiss, U., and R.A. Brown. An overlooked parallel to Kekules dream./. Chem. Ed. 64:
770-771, 1987.
Weller, M., and R. Rodnight. Stimulation by cyclic AMP of intrinsic protein kinase activ-
ity in ox brain membrane fractions. Nature 225: 187-188, 1970.
Weller, M., and R. Rodnight. Turnover of protein-bound phosphorylserine in membrane
preparations from ox brain catalysed by intrinsic kinase and phosphatase activity.
Biochem. ]. 124: 393^06, 1971.
Werman, R. Criteria for identification of a central nervous system transmitter. Comp.
Biochem. Physiol. 18: 745-766, 1966.
Werman, R., R.A. Davidoff, and M.H. Aprison. Inhibition of motoneurones by ion-
tophoresis of glycine. Nature 214: 681-683, 1967.
Wernig, A., and H. Stirner. Quantum amplitude distributions point to functional unity
of the synaptic 'active zone'. Nature 269: 820-822, 1977.
Wessells, N.K., B.S. Spooner, J.F. Ash, M.O. Bradley, M.A. Luduena, E.L. Taylor, J.T.
Wrenn, and K.M. Yamada. Microfilaments in cellular and developmental processes.
Science 171: 135-143, 1971.
West, G.B. Further studies on sympathin. Brit. J. Pharmacol. 5: 165-172, 1950.
Whitby, L.G., J. Axelrod, and H. Weil-Malherbe. The fate of H3-norepinephrine in ani-
mals. /. Pharmacol. Exp. Ther. 132: 193-201, 1961.
Whitby, L.G., G. Hertting, and J. Axelrod. The effect of cocaine on the disposition of
noradrenaline labelled with tritium. Nature 187: 604-605, 1960.
Whittaker, V.P. The isolation and characterization of acetylcholine-containing particles
from the brain. Biochem. J. 72: 694-706, 1959.
Whittaker, V.P, LA. Michaelson, and R.J.A. Kirkland. The separation of synaptic vesi-
cles from nerve-ending particles ('synaptosomes'). Biochem. J. 90: 293-303, 1964.
Whittaker, V.P, and M.N. Sheridan. The morphology and acetylcholine content of iso-
lated cerebral cortical synaptic vesicles. /. Neurochem. 12: 363-372, 1965.
Whitteridge, D. The controversy over synaptic transmission. News Physiol. Sci. 8:
135-136, 1993.
Wiedenmann, B., and W.W. Franke. Identification and localization of synaptophysin, an
integral membrane glycoprotein of Mr 38,000, characteristic of presynaptic vesi-
cles. Cell 41: 1017-1028, 1985.
Wiersma, C.A.G. An experiment on the "resonance theory" of muscular activity. Arch.
Neerland. Physiol. 16: 337-345, 1931.
Wigstrom, H., and B. Gustafsson. On long-lasting potentiation in the hippocampus: A
proposed mechanism for its dependence on coincident pre- and postsynaptic activ-
ity. Acta Physiol. Scand. 123: 519-522, 1985.
Wilkins, R.W., WE. Judsons, and J.R. Stanton. Preliminary observations on Rauwolfia
serpentina in hypertensive patients. New Eng. Cardiovasc. Soc. Proc. 10: 34, 1952.
Williams, J.H., M.L. Errington, M.A. Lynch, and T.V.P. Bliss. Arachidonic acid induces
a long-term activity-dependent enhancement of synaptic transmission in the hip-
pocampus. Nature 341: 739-742, 1989.
Wilson, C.S., and M.A. Raftery. Carbamylcholine-induced rapid cation efflux from
reconstituted membrane vesicles containing purified acetylcholine receptor.
Biochem. Biophys. Res. Commun, 89: 26-35, 1979.
Wilson, D.W., S.W. Whiteheart, L. Orci, and J.E. Rothman. Intracellular membrane
fusion. Trends Biochem. Sci. 16: 334-337, 1991.
Wilson, I.E. Mechanism of hydrolysis. II. New evidence for an acylated enzyme as inter-
mediate. Biochim. Biophys. Acta 7: 520-525, 1951.
Wilson, I.E. The inhibition and reactivation of acetylcholinesterase. Ann. A/.Y. Acad. Sci.
135: 177-183, 1966.
Wilson, I.E., and F. Bergmann. Studies on cholinesterase. VIII. The active surface of
acetylcholine esterase derived from effects of pH on inhibitors. J. Biol. Chem. 185:
479-489, 1950.
Wilson, I.E., F. Bergmann, and D. Nachmansohn. Acetylcholinesterase. X. Mechanism
of the catalysis of acylation reactions. /. Biol Chem. 186: 781-790, 1950.
Wilson, I.E., and S. Ginsburg. A powerful reactivator of alkylphosphate-inhibited acetyl-
cholinesterase. Biochim. Biophys. Acta 18: 168-170, 1955.
Wilson, S.A.K. The old motor system and the new. Arch. Neurol. Psychiat. 11: 385-404,
1924.
Wilson, W.S., R.A. Schulz, and J.R. Cooper. The isolation of cholinergic synaptic vesi-
cles from bovine superior cervical ganglion and estimation of their acetylcholine
content. /. Neurochem. 20: 659-667, 1973.
Winegrad, S. Autoradiographic studies of intracellular calcium in frog skeletal muscle.
/. Gen. Physiol. 48: 455-479, 1965.
Wofsey, A.R., M.J. Kuhar, and S.H. Snyder. A unique synaptosomal fraction, which accu-
mulates glutamic and aspartic acids, in brain tissue. Proc. Natl. Acad. Sci. USA 68:
1102-1106, 1971.
Wolfe, B.B., T.K. Harden, J.R. Sporn, and P.B. Molinoff. Presynaptic modulation of beta
adrenergic receptors in rat cerebral cortex after treatment with antidepressants.
/. Phannacol. Exp. Ther. 207: 446-457, 1978.
Wolfe, D.E., L.T. Potter, K.C. Richardson, and J. Axelrod. Localizing tritiated norepi-
nephrine in sympathetic axons by electron microscopic autoradiography. Science
138: 440-442, 1962.
Wolff, J., and G.H. Cook. Activation of thyroid membrane adenylate cyclase by purine
nucleotides. /. Biol. Chem. 248: 350-355, 1973.
Wong, D.T., F.P. Bymaster, J.S. Horng, and B.B. Molloy. A new selective inhibitor for
uptake of serotonin into synaptosomes of rat brain: 3-(p-trifluoromethylphenoxy)-
N-methyl-3-phenylpropylamine. /. Phannacol. Exp. Ther. 193: 804-811, 1975.
Wong, D.T., J.S. Horng, F.P. Bymaster, K.L. Hauser, and B.B. Molloy. A selective
inhibitor of serotonin uptake: Lilly 110140, 3-(p-trifluoromethylphenoxy)-N-
methyl-3-phenylpropylamine. Life Sci. 15: 471-479, 1974.
Wong, D.F., H.N. Wagner, L.E. Tune, R.F. Dannals, G.D. Pearlson, J.M. Links, C.A.
Tamminga, E.P. Broussolle, H.T. Ravert, A.A. Wilson, J.K.T. Toung, J. Malat, J.A.
Williams, L.A. OTuama, S.H. Snyder, M.J. Kuhar, and A. Gjedde. Positron emis-
sion tomography reveals elevated D2 dopamine receptors in drug-naive schizo-
phrenics. Science 234: 1558-1563, 1986.
Wong, F.K.S., C. Slaughter, A.E. Ruoho, and E.M. Ross. The catecholamine binding
site of the /3-adrenergic receptor is formed by juxtaposed membrane-spanning
domains. /. Biol. Chem. 263: 7925-7928, 1988.
Wooley, D.W. Serotonin receptors. I. Extraction and assay of a substance which ren-
ders serotonin fat-soluble. Proc. Natl. Acad. Sci. USA 44: 1202-1210, 1958.
References 441
Wooley, D.W., and E. Shaw. A biochemical and pharmacological suggestion about cer-
tain mental disorders. Proc. Natl. Acad. Sci. USA 40: 228-231, 1954.
Woodward, M.P., and T.F. Roth. Coated vesicles: Characterization, selective dissocia-
tion, and reassembly. Proc. Natl. Acad. Sci. USA 75: 4394-4398, 1978.
Worrall, D.M., and D.C. Williams. Sodium ion-dependent transporters for neuro-
transmitters: A review of recent developments. Biochem. J. 297: 425^36, 1994.
Wylie, D.W., S. Archer, and A. Arnold. Augmentation of pharmacological properties of
catecholamines by O-methyl transferase inhibitors. /. Pharmacol. Exp. Ther. 130:
239-244, 1960.
Yahr, M.D., R.C. Duvoisin, M.H. Hoehn, M.J. Schear, and R.E. Barrett. L-Dopa
(L-3,4-dihydroxyphenylalanine)—its clinical effects in parkinsonism. Trans. Amer.
Neurol. Assoc. 93: 56-63, 1968.
Yakel, J.L., and M.B. Jackson. 5-HTs receptors mediate rapid responses in cultured hip-
pocampus and a clonal cell line. Neuron 1: 615-621, 1988.
Yamada, K.M., B.S. Spooner, and N.K. Wessells. Axon growth: Roles of microfilaments
and microtubules. Proc. Natl. Acad. Sci. USA 66: 1206-1212, 1970.
Yamasaki, Y., M. Fujiwara, and N. Toda. Effects of intracellularly applied cyclic 3',5'-
adenosine monophosphate and dibutyryl cyclic 3',5'-adenosine monophosphate on
the electrical activity of sinoatrial nodal cells of the rabbit. J. Pharmacol. Exp. Ther.
190: 15-20, 1974.
Yarden, Y., H. Rodriguez, S.K.F. Wong, D.R. Brandt, D.C. May, J. Burnier, R.N. Harkins,
E.Y. Chen, J. Ramachandran, A. Ullrich, and E.M. Ross. The avian /3-adrenergic
receptor: Primary structure and membrane topology. Proc. Natl. Acad. Sci. USA
83: 6795-6799, 1986.
Yazulla, S., and J. Kleinschmidt. Carrier-mediated release of GAB A from retinal hori-
zontal cells. Brain Res. 263: 63-75, 1983.
Young, A.B., and S.H. Snyder. Strychnine binding associated with glycine receptors of
the central nervous system. Proc. Natl. Acad. Sci. USA 70: 2832-2836, 1973.
Young, J.Z. Structure of nerve fibres and synapses in some invertebrates. Cold Spring
Harb. Symp. Quant. Biol. 4: 1-6, 1936.
Young, J.Z. Growth and plasticity in the nervous system. Proc. Roy. Soc. 139B: 18-37,
1951.
Yurek, D.M., and J.R. Sladek. Dopamine cell replacement: Parkinsons disease. Annu.
Rev. Neurosci. 13: 415^40, 1990.
Zeller, E.A. Oxidation of Amines. In: Sumner, J.B., and K. Myrback (eds.) The Enzymes.
New York: Academic Press, 1951. pp. 536-558.
Zeller, E.A., J. Barsky, E.R. Berman, and J.R. Fouts. Action of isonicotinic acid hydrazide
and related compounds on enzymes involved in the autonomic nervous system.
Zeller, E.A., R. Stern, and M. Wenk. Uber die Diamin-Diamin-oxydase-Reaktion. Helu.
Chim. Acta 23: 3-17, 1940.
Zieher, L.M., and E. DeRobertis. Subcellular localization of 5-hydroxytryptamine in rat
brain. Biochem. Pharmacol. 12: 596-598, 1963.
Zigmond, R.E., M.A. Schwarzschild, and A.R. Rittenhouse. Acute regulation of tyro-
sine hydroxylase by nerve activity and by neurotransmitters via phosphorylation.
Annu. Rev. Neurosci. 12: 415-461, 1989.
Zimmerberg, J., M. Curran, F.S. Cohen, and M. Brodwick. Simultaneous electrical and
optical measurements show that membrane fusion precedes secretory granule
swelling during exocytosis of beige mouse mast cells. Proc. Natl. Acad. Sci. USA
84: 1585-1589, 1987.
Zimmermann, H., and V.P. Whittaker. Effects of electrical stimulation on the yield and
composition of synaptic vesicles from the cholinergic synapses of the electric organ
of Torpedo. J. Neurochem. 22: 435-450, 1974.
Zimmermann, H., and V.P. Whittaker. Morphological and biochemical heterogeneity of
cholinergic synaptic vesicles. Nature 267: 633-635, 1977.
INDEX
Abel, J.J., 56 Aimers, W., 254

Acetylcholine Amphetamine, 267, 328, 332, 333
as neurotransmitter, 59-68, 70-79, 95-98, Andersen, P., 309
102, 106, 109-111, 119-123 Apathy, S., 21, 26, 109
metabolism, 229, 230, 238. See also Aprison, M., 133
Cholinesterase Aliens, E.J., 147, 148, 150
receptors (muscarinic and nicotinic), 153, Aliens Kappers, C.U., 297
156-160, 177, 180, 186, 200-208, 211 Armstrong, M., 233
storage, 225, 226 Arrhenius, S., 9
synthesis, 220, 221, 238 ATP (adenosine triphosphate) defined, 88,
Actin, 88, 260, 287, 288, 291 89
Action potentials. See Impulse conduction Atropine, 60, 61, 63-65, 67, 71, 123, 211
Adenylate cyclase, 161, 174-177, 181-186, Attwell, D., 266
209, 223, 306, 334, 335 Aurbach, G., 161
Adrenaline (epinephrine) Avery, O., 88
as neurotransmitter, 55-59, 69, 70, 76, 80, Axelrod, J., 233-236, 238, 267, 332, 338,
119, 123, 124 339
storage, 226, 227 Axonal conduction. See Impulse conduction
Adrian, E., 43, 75, 77, 79, 276
Affinity chromatography defined, 159 Bacq, Z., 70, 113, 123
Aghajanian, G., 339 Bailey, C., 303
Ahlquist, R., 153, 154 Bain, W.A., 65, 66
Aldrich, T.B., 56 Banks, P., 253, 259
Alkon, D., 304 Barbeau, A., 324
Alles, G., 229 Barker, L., 18
Allosteric interactions, 88, 150-152, 179, 181 Barondes, S., 298
443
444 INDEX
Barrett, J., 283 Cajal. See Ramon y Cajal

Bartelmez, G., 50 Calmodulin, 188, 189
Baudry, M., 313 Calne, D., 325, 335
Bayliss, W., 10, 44, 59 cAMP (cyclic AMP), 173-181, 183-186,
Beadle, G., 88 189, 193, 212, 214, 223, 224, 260,
Beers, W., 156 303-306, 315
Bell, C., 14, 34 Cannon, W., 69, 70, 123, 153
Bennett, S., 107 Cantoni, G., 233
Benzer, S., 306 Capon, D., 211, 212
Bergstrom, S., 313 Carlsson, A., 126, 322, 324, 332, 339, 340
Bernath, S., 266 Caspars, H., 135
Bernard, C., 34, 51, 72 Cassel, D., 182
Bernard, E., 206 Catecholamine defined, 58
Bernstein, J., 8-10, 49, 50, 89, 90 cDNA. See DNA, recombinant techniques
Berridge, M., 191, 192 Ceccarelli, B., 250, 252
Bertler, A., 126 cGMP (cyclic GMP), 177, 185
Bethe, A., 10, 21, 23, 24, 26, 109 Chagas, C., 106, 158
Betz, H., 206 Chang, H.C., 67, 71, 75
Bichat, X., 51 Changeux, J.-P, 151, 158, 159, 180, 205, 290
Birks, R., 256 Channels, ion, 92, 112, 158, 159, 161,
Birnbaumer, L., 183 163-166, 177, 180, 181, 186-188, 192,
Black, J., 153, 154 193, 200, 204-208, 214, 258-261,
Blackburn, R.J., 237 303-305, 311, 312, 315
Blaschko, H., 123, 125, 126, 221, 222, 226, Charcot, J.-M., 321
231, 232, 322 Charlton, M., 259
Blaustein, M., 188 Charpentier, S., 330
Bleuler, E., 328 Chemotaxis, 24, 275-280, 283-286
Bliss, T., 309, 310 Chen, M., 303
Bloom, E, 126 Cheung, W.Y., 189
Bodian, D., 50 Chlorpromazine (Thorazine), 330-335, 338
Bonhoeffer, E, 286 Cholinesterase, 61, 67, 77-79, 229, 230
Bonner, T., 211 Clapham, D., 186
Bose, K., 329 Clark, A.J., 144-148, 152, 154
Boyd, I.A., 97 Clathrin. See Coated vesicles
Bradford, H.F., 133 Claude, A., 109
Branton, D., 263 Clothia, C., 156
Bray, D., 287, 288 Coated vesicles, 248, 251, 262-265
Brock, L.G., 113 Cocaine, 235, 237
Brodie, B.B., 232, 233, 236, 237, 322 Cohen, J., 290
Brown, G.L., 74, 77, 155, 245, 246 Cohen, M., 289
Brown, T., 310, 311 Cohen, S., 281
Brown-Sequard, C.-E., 51 Cole, K.S., 49, 90
Buchtal, E, 74, 95, 289 Collingridge, G., 311
Bueker, E., 281 Collins, J., 189
Bulbring, E., 76 Colquhoun, D., 163
a-bungarotoxin, 159, 289 Conformational changes. See Allosteric
Bum, J.H., 76 interactions
Burton, P., 287 Coombs, J., 113
Butcher, R. 174 Coppen, A., 339
Byers, D., 306 Cori, C., 172, 173, 178, 194
Cori, G., 194
Ca2+ as second messenger, 186—189, Cotman, C., 265
191-193 Co-transmitters, 228, 229
Cade, J., 341 Cotzias, G., 324
We 445
Cowan, M., 282 Eccles, R., 122

Crane, R., 89, 227 Edelman, G., 285
CRE (cAMP response element), 214, 224, Edwards, C., 155, 267
303, 305 Ehrlich, P., 11, 20
CREB (CRE binding protein), 214, 303, 305 Einthoven, W., 41
Creed, R.S., 45 Elliott, T., 56, 57, 59, 60, 63, 73, 79, 123,
Crick, R, 88, 199, 298, 314 124
Curare, 72-75, 94-96, 106, 123 Ellis, J., 265
Curtis, D., 122, 125, 130, 132-134 Emmelin, N., 76
Curtis, H.J., 90 Enkephalins. See Opioids
Gushing, H., 45 E.P.P.S (endplate potentials), 78, 93-98,
Cytoskeleton, 260, 287, 288, 291 101, 171, 246, 254, 258
E.P.S.P.S (excitatory postsynaptic potentials),
Dakin, H.D., 56 101, 103-105, 171, 300, 301,311, 312
Dale, H.H., 57-59, 61, 63, 67-71, 74, 78, Erlanger, J., 76
79, 102, 113, 123, 124, 134, 146, 152, Ernst, A.M., 325
153, 229, 245, 246, 354 Erspamer, V., 126, 127
Danielli, J., 89, 246 Ewins, A., 61
Davies, A., 283
Davis, R., 306 Faber, D., 256
de Duve, C., 247 Falck, B., 125
Deiters, O., 5, 6 Faraday, M., 9
Delay, J., 330 Farde, L., 335
del Castillo, J., 97, 98, 122, 246, 247, 256, Fatt, P., 97, 101, 109, 114, 129, 246, 258
258 Feldberg, W, 68, 69, 71, 74-76, 79, 106,
Denny-Brown, D., 45 113, 146, 220, 225, 226, 245, 246
Depression and mania, 232, 233, 238, 330, Feng, T.P., 309
337-343 Ferraro, A., 321
DeRobertis, E., 107, 109, 110, 128, 246 Fessard, A., 106
Descartes,R., 33 Fischer, E., 178, 181, 193
Detweiller, S., 280 Fleckenstein, A., 188
Diacylglycerol, 189-193, 209, 223 Flexner, J., 298
Diamond, I., 180 Flexner, L., 298
Dikshit, B.B., 76 Florey, E., 129, 130, 132
Dingledine, R., 311 Fluoxetine (Prozac), 238, 340, 341
Dingman, W., 298 Forbes, A., 44, 76
Dixon, W., 59, 60, 63, 79 Forel, A., 3, 7, 10
DNA, recombinant techniques, 199-203, Foster, M., 32, 33, 44, 51, 53, 79
206-212 Frank, K., 103
Dopamine Franke, W., 261
as neurotransmitter, 58, 124-126, 177 Franz, S.I., 296
receptors, 212, 332-336, 343 Froehner, S., 290
synthesis, 221-224, 238 Fulton, J., 45
Douglas, W., 187, 253, 258 Furchgott, R., 150, 313
Driesch, H., 273 Furshpan, E., Ill, 112
du Bois-Reymond, E., 8, 9, 73
Dudai, Y., 306 GAB A (y-aminobutyric acid)
Dunant, Y., 257 as neurotransmitter, 105, 129-132
Durrell, J., 190 receptors, 206-208, 212
Duval, M., 22 Gaddum, J., 67, 71, 75, 121, 123, 127, 134,
135, 146-148, 152, 328
Ebashi, S., 187-189 Gaffan, D., 308
Eccles, J.C., 45, 77, 78, 93, 95-105, 108, Galvani, L., 7, 8
112-114, 123, 130, 134, 259, 355, 356 Gap junctions, 112
446 INDEX
Gaskell. W., 31, 44, 51, 53, 56, 79 Hedgecock, E., 283
Gasser, H., 76 Heikkila, R., 326
Gerard, R., 92 Heilbrunn, V, 186
Gerlach, J. v., 3, 5, 7, 353 Held, H., 20
Gershon, S., 332 Helle, K., 253
Gilbert, W., 201 Helmholtz, H. v., 8
Gillespie, J. I., 254 Hemicholinium, 221, 249
Gilman, A.G., 182, 183, 185, 193 Henle, E, 51
Ginzel, K.H., 147 Hermann, L., 8-10
Glowinski, J., 339 Heuser, J., 250-252, 254, 259
Glutamate Hill, A., 19, 20, 22
as neurotransmitter, 123, 131-133 Hill, A.V., 93, 144, 146-148, 152
receptors (kainate, metabotropic, NMDA, Hillarp, N-A., 125, 226, 227, 247, 253
quisqualate), 206-208, 212, 311-315, Hirokawa, N., 260
336 His, W., 2, 3, 7, 10, 23, 273, 274
Glycine Hodgkin, A., 50, 90-92, 112, 152, 188, 246,
as neurotransmitter, 133, 134 258
receptors, 206-208 Hofmann, E, 180
Goldman, R., 287 Hogeboom, G., 109
Goldstein, A., 136, 137, 162 Hokin, L., 189, 190
Golgi, C., 3, 5-7, 12-18, 22, 353 Hokin, M., 189, 190
Golgi stain, 1, 6, 11-21, 23 Hollmann, M., 207
Goltz, R, 31, 34 Holmstedt, B., 120
Goodman, C., 283, 286 Holtz, P., 124, 221
Gopfert, H., 93 Hornykiewicz, O., 322-324
G-proteins, 181-186, 192, 209-212, 215 Howell, W.H., 9, 21
Graham, J., 92 5-HT (5-hydroxytryptamine). See Serotonin
Granit, R., 45 Hubbard, J, 155
Gray, E.G., 110 Hughes, J., 136, 224
Gray, W., 259 Hunt, R., 60, 61
Green, J.P., 120 Huxley, A., 50, 88, 90-92, 112, 152, 188,
Greengard, P., 177, 179, 180, 189, 194, 260, 246, 258
261, 303, 334 Huxley, H., 88
Growth cone, 23, 274, 275, 286-288, 291 Hyden, H., 298
Grundfest, H., 74, 106
GTP (guanosine triphosphate) defined, 181 Imipramine (Tofranil), 235, 237, 238, 338,
Gundersen, R., 283 339, 342
Gustafsson, B., 311, 312 Impulse conduction, 7-10, 49, 50, 89-93
Inositol phosphates, 189-193, 209, 342
Hagiwara, S., 105 Inositol phospholipids. See Phosphatidyl-
Hall, M., 34 inositols
Halliburton, W., 4 Iproniazid (Marsilid), 232, 337, 338
Haloperidol (Haldol), 330-336 I.P.S.P.S (inhibitory postsynaptic potentials),
Halstead, W., 297 101, 171
Hamburger, V., 273, 280-282 Israel, M., 256, 257
Hare, M., 231 Iversen, L. 130, 131, 237, 281, 334
Harrison, R., 23, 274, 276, 277, 280-282,
285 Jacobsohn, D., 76
Hasselbach, W., 187, 188 Jacobson, A., 299
Hawes, R., 229 Jahn, R., 261
Heald, P.J., 179 Janssen, P., 331
Hebb, C., 109 Jasper, H., 133
Hebb, D.O., 297, 298, 306, 307, 310, 312, Jenden, D., 120
314, 315 Jessell, T., 283
Index 447
John, R., 299 Larner, J., 179

Jolly, W.A., 41, 42 Lashley, K., 296, 297, 307, 314
Learning
Kadota, K., 262, 263 chemical representations, 297-300
Kalkkinen, N., 233 conditioning, 295, 296, 300, 304, 306
Kanaseki, T., 262, 263 habituation, 296, 301-303
Kandel, E., 300-306, 314 long-term, 296, 303
Kane, J., 335 short-term, 296
Kanner, B., 237 synaptic changes, 22, 23, 296-316
Karlin, A., 151, 159 Lee, C.-Y., 159
Karnovsky, M., 112 Leeman, S., 135
Katz, B., 78, 90, 93, 95, 97, 98, 101, Lefkowitz, R.J., 161, 208-211, 213-215
111-114, 122, 151, 152, 154, 163, 187, Legallois, J., 33
238, 246, 247, 254-256, 258, 267, 268, Lehninger, A., 88
355 Lembeck, R, 135
Katz, J., 297 Leonhardt, K., 337
Kebabian, J., 325 Lester, H., 205
Keen, J., 263, 265 Letourneau, P., 283, 285
Kelly, R., 253 Levi-Montalcini, R., 280-282
Kendrew, J., 88, 150 Levitzki, A., 161
Kenny, J., 234 Levodopa, 323, 324, 327
Kerr, L., 259 Levy, W., 310, 311
Kety, S., 234 Li, C.H., 225
Keynes, R., 92, 106, 258 Liddell, E.G.T., 45
Kinase. See Protein kinase Liley, A.W., 97
Kinnier Wilson, S.A., 321 Lindhard, J., 74, 95, 289
Kirshner, N., 226, 227, 253 Ling, G., 92
Kline, N., 329, 338 Lipkin, D., 174
Koch, R., 31 Lipmann, E, 220
Kolliker, R. v., 2, 5, 10, 12, 51, 273 Liposome. See Membrane reconstitution
Kopin, L, 256, 326 Lithium, 341, 342
Korn, H., 256 Llinas, R., 259, 260
Koshland, D., 88 Lloyd, D., 102
Kosterlitz, H., 136, 224 Loewi, O., 62-67, 69, 74, 75, 78, 124, 229,
Kraepelin, E., 327, 328, 337 237
Kravitz, E., 130 L0mo, T., 309, 310
Krebs, E., 177-179, 181, 193 Lorente de No, R., 297
Kriebel, M., 256 Lotze, R., 34
Krnjevic, K., 123, 131, 133, 135 LTP (long-term potentiation), 306-315
Kuffler, S., 78, 93-95, 105, 112, 129, 289 Lucas, K., 10, 43, 44, 73, 74, 79
Kuhn, R., 338 Lumsden, A., 283
Kiihne, W., 73, 93 Lust, D., 266
Kwanbunbumpen, S., 249 Lynch, G., 311, 313
Laborit, H., 330 MacDonald, J., 10

Landmesser, L., 283 Macintosh, E.G., 75, 221, 257
Lands, A.M., 154 Magendie, E, 14, 34
Langer, S., 154, 155 Mallet, J., 222
Langley, J.N., 31, 44, 53-57, 59, 68, 70-73, Mania. See Depression and mania
79, 93, 143, 144, 152, 275, 276 Marchbanks, R., 257
Langmuir, I., 146 Martin, A.R., 97
Langston, W., 325, 326 Matthes, K., 67
Lapicque, L., 73, 74 Matteucci, C., 8
Lapicque, M., 73, 74 Mauro, A., 250
448 INDEX
Maxam, A., 201 Nishizuka, Y., 192

McConnell, J., 299, 300 NMDA receptors. See Receptors, glutamate
McLennan, H., 122, 126, 129, 311 Nobili, L., 8
McMahan, U.J., 289 Noradrenaline (norepinephrine)
Meldolesi, J., 261 metabolism, 230-234, 238
Meltzer, H., 335 as neurotransmitter, 58, 70, 123-125
Membrane, cell, 4, 20, 89, 108, 157, 158, receptors (a and f3), 153, 160, 161,
246, 290 175-177, 180, 181, 185, 208-214, 340
Membrane reconstitution, 159, 160 storage, 226, 227
Memory. See Learning synthesis, 221-224, 238
Menten, M., 146 Noren, O., 234
M.E.P.P.S (miniature endplate potentials), Nottebohm, E, 314
97, 98, 106, 114, 246, 254, 256, 258 Numa, S., 201-205, 211, 225
Michaelis, L., 146
Michell, R., 190 Obata, K., 131, 134
Microelectrode development, 90-93 Oliver, G., 55, 56
Microtubules, 288 Olivera, B., 259
Miledi, R., 163, 187, 256, 258, 289 Opioids
Milner, B., 308 metabolism, 234, 239
Minz, B., 68, 71, 75, 76 as neurotransmitters, 135-137
Mishldn, M., 308 receptors, 161-163, 212
Mitchell, J., 121, 122, 131, 133, 255 storage, 228, 239
Mitchell, P., 89, 227, 228, 237 synthesis, 224, 225, 239
Monoamine oxidase, 230-233, 238, 326, 338 Ostwald, W, 9
inhibitors, 232, 233, 238, 326, 327, 338, Otsuka, M., 135
339, 342 Overton, E., 4, 9, 10
Monod, J., 88, 151, 179
Montminy, M., 214 3, I., 127
Morphine. See Opioids Palade, G., 107, 109, 112
Muscarine and muscarinic effects, 60, 70, Palay, S., 107, 109, 246
71, 123, 153. See also Acetylcholine; Parkinson, J., 320, 321
Atropine Parkinsons disease and parkinsonism, 221,
Muschall, E., 155 233, 320-327, 332
Myosin, 88, 287 Fasten, I., 263, 265
Patch electrode development, 163
Nachmansohn, D., 106, 220, 221, 229, 230 Paton, W., 152, 155
Nagli, C., 4 Patrick, J., 203, 290
Nastuk, W.L., 92 Pauling, L., 88
Neer, E., 186 Pavlov, I., 295
Neher, E., 163, 164, 254 Pearse, B., 263
Nernst, W., 9, 10 Penfield, W., 45, 307, 308
Neuron Theory (Neuron Doctrine), 10, 21, Pernow, B., 135
35, 55, 273, 274 Pert, C., 162
Neurotransmitters. See Acetylcholine; Perutz, M., 88, 150
Adrenaline; Dopamine; GABA; Pfeffer, W, 4
Glutamate; Glycine; Noradrenaline; Pfeiffer, C., 156
Opioids; Serotonin; Substance P Pfenninger, K., 288
Release by exocytosis, 246-262, 267, 268 Pfenninger, M.-E, 288
Transport. See Reuptake Pfeuffer, T, 183
Neurotropic factors. See Chemotaxis Pfliiger, E., 34
NGF (nerve growth factor), 280-283 Phillips, D., 88
Nickerson, M., 149 Phosphatidylinositols, 189-193, 209
Nicotine and nicotinic effects, 53, 71, 72, Phosphodiesterase, 174, 175, 189, 306
75, 123, 153. See also Acetylcholine Physostigmine, 67, 76-79, 96
Inde 449
Piccolino, M., 26 purification, 157-165

Picrotoxin, 105, 132, 134 rate theory, 152
Pletscher, A., 324 unitary conductance, 163-165
Popper, K., 113 upregulation, 214, 335
Porter, K., 262 Reed, L., 179
Potter, D., Ill, 130 Reese, T., 250-252, 259
Potter, J., 189 Reich, E., 156
Potter, L., 159 Reichard, L., 261
Pourfour du Petit, R, 51 Remak, R., 51
Protein kinases Renshaw, B., 101, 102
CaM-kinase (Ca2+/calmodulin Renshaw cells, 101, 102, 122, 123, 134, 137
dependent), 189, 193, 260, 306 Renyi, A.L., 237
phosphorylations identified, 174, 178-181 Reserpine, 227-229, 235, 237, 321, 322,
protein kinase A (cAMP dependent), 178, 329, 330, 333, 339
180, 186, 193, 212-214, 223, 260, 303, Resonance Principle, 276-278
304 Reticular theory, 3, 5-7, 12, 22
protein kinase C (diacylglycerol Retzius, G., 2, 10, 11, 23
dependent), 192, 193, 213, 223, 261, Reuptake (neurotransmitter transport),
306 234-238, 339-343
tyrosine kinases, 281, 282 inhibitors, 235-238, 339-343
Protein phosphatases, 179, 181 Reuter, H., 188
Protein phosphorylations. See Protein Revel, J.-P, 112
kinases Richter, D., 231, 232
Purkinje, J., 5, 6 Ringer, S., 186
Pysh, J.J., 250 Robbins, J., 132
Roberts, E., 129, 130
Quantal responses and neurotransmitter Roberts, P. J., 133
release, 98, 101, 105, 106, 109, 155, Robertson, J.D., 89, 107, 108
163, 246, 252, 255-258, 267, 268 Robinson, J.D., 266
Quastel, J., 220 Robison, A., 174
Quinn, W., 306 Rodbell, M., 181-183, 193
Rodnight, R., 180
Racker, E., 159, 160, 231 Roques, B., 234
Radda, G., 227 Rosenbaum, J., 287
Raftery, M., 159, 201, 202 Rosenblueth, A., 70, 78, 153
Rail, T., 174 Rosengren, E., 126
Ramon Cajal, P., 26 Ross, S.B., 237
Ramon y Cajal, S., 1-3, 10-27, 31, 33, 35, Rothman, J., 265
37, 40, 42, 50, 79, 274-276, 286, 296, Roux, W., 273
297, 308, 314, 355 Rushton, W.A.H., 73, 74
Ramwell, P.W., 135
Randrup, A., 332 Sakmann, B., 163, 164, 205
Raper, J., 286 Salvaterra, P., 221
Rapport, M., 127 Samuelsson, B., 313
Rasmussen, H., 186, 187 Sandow, A., 186
Raventos, J., 147 Samarro, L., 11
Receptors. See also Acetylcholine; Sanger, E, 88, 201
Dopamine; GABA; Glutamate; Glycine; Sano, I., 126
Noradrenaline; Opioid; Serotonin Scarpa, A., 228
desensitzation, 151, 152 Schaefer, H., 93
downregulation, 213, 214, 340 Schafer, E., 44, 56
localization, 288-290 Schatzmann, H.J., 188
occupation theory, 143-152 Schayer, R., 232
proposed, 59, 143 Scheller, R., 261
450 INDEX
Schiff, M., 34 Stevens, C., 163

Schild, H., 154 Straub, W., 147
Schildkraut, J., 339, 340 Stroud, R., 160
Schizophrenia, 177, 229, 327-336, 343 strychnine, 8, 39, 102, 105, 132, 134
Schleiden, M., 4 Stryer, L., 185
Schmiedeberg, O., 60, 62, 78 Substance P, as neurotransmitter, 134, 135
Schou, M., 341 Siidhof, T., 261
Schneider, W., 109 Sulser, E, 340
Schuldiner, S., 228 Sulzer, D., 267
Schwab, R., 325 Sussman, J., 230
Schwann, T., 4, 23 Sutherland, E., 172-179, 181, 183, 185, 193
Schwartz, E., 266 sympathin, 69, 70, 123, 153
Schwartz, J., 304 Synapse
Schwartz, J.-C., 234 chemical transmission proposed, 55-76
Schwartzkroin, P., 310 cleft visualized, 106-108
Scoville, W, 308 delayed transmission, 40^12, 45, 77, 102
SDS-PAGE defined, 158 electrical transmission, 21-23, 76-78, 111,
Second messenger, defined, 175 112
Seeman, P., 334 named, 33
Sejnowski, T., 311 unidirectional transmission, 40, 45
Selegiline (Eldepryl), 233, 325, 326 vesicles, 109-111
Selinger, Z., 182 Synaptosomes, 109-111
Sen, G., 329
Serotonin (5-hydroxytryptamine, 5-HT) Takamine, J., 56
metabolism, 230-233, 239 Takeichi, M., 285
as neurotransmitter, 126-129 Takeuchi, A., 97, 130, 132
receptors, 206, 208, 212, 326, 340 Takeuchi, N., 97, 130, 132
synthesis, 224, 239 Tasaki, I., 105
transport, 237, 238, 339-341 Tatum, E., 88
Setchenov, I., 34 Tauc, L., 257, 300
Shaw, J., 135 Taylor, P., 230
Sherrington, C.S., 31-33, 35-45, 75, 77, Terenius, L., 136, 162
79, 93, 94, 99, 102, 112, 113, 130, 134, Tessier-Lavigne, M., 283
179 Thesleff, S., 289
Shooter, E., 281 Thoenen, H., 281
Silman, I., 230 Thorndike, E., 296
Simon, E., 162 Tilgmann, C., 233
Singer, S.J., 158, 290 Titus, E., 237
Skinner, B.F., 296 Transport
Skou, J.C., 89 membrane, 89, 92, 158, 187, 188, 190,
Smith, S., 287 226-228, 234-238, 246, 247, 265, 267
Snyder, S., 133, 136, 162, 192, 326, 332-335 neurotransmitter reuptake. See Reuptake
Sperry, R., 277-279, 283 vesicular. See Vesicular transport
Speidel, C., 274 Trautwein, W., 180
Spemann, H., 273, 280 Tretiakoff, C., 321
Sporn, M., 298 Tsien, R., 313
Stadtman, E., 223 Tubulin, 260, 288
Stanton, P., 311
Starke, K., 155 Udenfriend, S., 127, 222-225, 232, 324
Starling, E., 44, 59, 63 Ungewickell, E., 263
Stedman, Edgar, 229 Unwin, N., 203, 204
Stedman, Ellen, 229
Stephenson, R.P., 148-150, 154 Valentin, G., 5
Stern, P., 135 Van der Kloot, W., 257
Inie 451
Vane, J., 313 Wernig, A., 256

van Gehuchten, A., 10, 11 Wessels, N., 287, 288
Van Harreveld, A., 132 Wester, K., 310
van Rossum, J.M., 332 Whittaker, V., 109-111, 126, 128, 130, 257
Verworn, M., 4 Whytt, R., 33
Vesicular transport, 226-229 Wiedenmann, B., 261
Virchow, R., 5, 31 Wiersma, C.A.G., 276, 355
Vogt, M., 74, 125, 227 Wightman, M., 255
Volta, A., 7, 8 Wigstrom, H., 311, 312
von Baer, K.E., 273 Willis, T., 50, 51
von Euler, U.S., 123-125, 134, 146, 238, Wilson, I.E., 230
247, 313 Wilson, J.-B., 51
von Gudden, B., 7 Wilson, M., 261
von Lenhossek, M., 10, 11, 23 Wong, D., 340
Vyskocil, R, 267 Wooley, D.W., 328
Wosilait, W, 174
Waldeyer, W. v., 2, 10, 18 Wright, S., 76
Waller, A., 7, 45 Wylie, R.G., 250
Wang, J., 189 Wyman, J., 151
Watson, J., 88, 199
Waud, D.R., 152 Yonkman, R, 329
Weber, Ernst, 34 Yoshikami, D., 259
Weber, Eduard, 34 Young, J.Z., 90
Webster, R.A., 133
Weiner, N., 223 Zeller, A., 231, 338
Weiss, P., 273, 276-278, 283, 285, 286, 291, Zigmond, M.J., 266
355 Zimmermann, H., 257

Joseph D. Robinson - Mechanisms of Synaptic Transmission - Bridging The Gaps (1890-1990) (2001)

Uploaded by

Copyright:

Available Formats

You might also like

Joseph D. Robinson - Mechanisms of Synaptic Transmission - Bridging The Gaps (1890-1990) (2001)

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Joseph D. Robinson - Mechanisms of Synaptic Transmission - Bridging The Gaps (1890-1990) (2001)

Uploaded by

Copyright:

Available Formats

MECHANISMS OF

Oxford New York

Copyright © 2001 by Oxford University Press.

Library of Congress Cataloging-in-Publication Data

Our understanding of how the nervous system accomplishes its wondrous

Syracuse, N.Y. J.D.R.

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

2. Beginnings: Snerrington ana the Synapse (1890—1913), 31

3. Chemical Transmission at Synapses (1895—1945), 49

Chemical Transmission in the Autonomic Nervous System, 55

4. Chemical Transmission at Synapses (1945—1965), 87

5. Identifying Neurotransmitters (1946-1976), 119

6. Ckaracterizing Receptors (1905-1983), 143

7. Second Messengers (1951-1990), 171

8. Receptor Structures and Receptor Families (1983 — 1990), 199

9. Synthesis, Storage, Transport, ana Metabolic Degradation

10. Neurotransmitter Release, 245

11. Formation or Specific Synapses, 273

Chemical Guidance (1963-1990), 283

12. Learning, 295

13. Diseases ana Therapies, 319

14. Epilogue, 349

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

too distracted by anticipation to listen. But on the day for demonstrations he

FIGURE 1-2. Photomicrograph of a Golgi-stained pyramidal neuron of the hippocam-

Background: Cells, Nerve Cells, ana Nerve Impulses

Formulation of the cell theory is conventionally ascribed to Matthias Schlei-

Cell Theory ana the Nervous System

the mass of images, or with terminations attributed to the fiber diameters

Nerve Impulse Conduction

Another pertinent concern is the process of nerve conduction.23 The ancients

from the moist contact of dissimilar metals—by constructing a bimetallic "pile"

cited Albrecht Bethe's proposal for neurofilaments as core conductors.29 Keith

Proclamation or the Neuron Tneory

Cajal's Principle 01 Dynamic Polarization

Before considering his anatomical investigations further, a crucial functional

Relating Form ana Function

Two examples of Cajal's anatomical studies can exemplify his meticulous

(arising from cell bodies outside the cerebellum). Less diagrammatic

B. For indirect, diffuse, unilateral reflexes, Cajal showed the dorsal

Continuations, Criticisms, ana Responses

Within a few years of Cajal's visit to Berlin, a number of leading neuroanatomists

Fixing and Staining Artiiacts

which the first impulse conducted by an interneuronal fibril "creates an open

Development ana Regeneration

subsequently named "Schwann cells"—that formed a sheath about myelinated

1. For historical accounts—in many cases pertinent, as well, to other topics—see

69. Hill (1900), p. 685.

Snerrington, Reilexes, and trie Synapse

When Santiago Ramon y Cajal journeyed to London in 1894 to deliver his

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

sought out Sherrington to write the section on neurophysiology. That challenge

Shortened to synapse, that term was soon accepted widely.

Reflexes—involuntary, automatic, but characteristic responses to particular

each segment—associated with a pair of nerves emerging from the spinal

Synapses ana the Rerlex Arc

In his Silliman Lectures Sherrington listed 11 ways that the conduction of

ference of potential with changes in surface-tension or in shape, or intervene