International Review of Neurobiology Volume 13

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INTERNATIONAL REVIEW OF

Neurobiology VOLUME 13

Associate Editors

W. Ross ADEY

H. J. EYSENCK

D. BOVET

G. HARRIS

Josh DELGADO

C. HEBB

SIR JOHN ECCLE~

0. ZANGWIU

Consultant Editors

V. A m s m

K. KILLAM

MURRAYB. BORNSTEIN

C. KORNETSKY

F. TH. BRUCKE

A. LAJTHA

P. DELL

B. LEBEDEV

J. ELKES

SIR AUBREYLEWIS

W. GREYWALTER

VINCENZOLONGO

R. G. HEATH

D. M. MACKAY

B. HOLMSTEDT

STENMARTENS

P. A. J.

F. MORRELL

s. KETY

JANSSEN

H. OSMOND STEPHENSZARA

INTERNATIONAL REVIEW OF

Neurobioloav Edited by CARL C. PFEIFFER New Jersey Neuropsychiatric Institute Princeton, New Jersey

JOHN R. SMYTHIES Department of Psychiatry University of Edinburgh, Edinburgh, Scotland

VOLUME 13

1970

ACADEMIC PRESS

New York

San Francisco

A Subsidiary of Harcourt Brace Jovanovich, Publishers

London

COPY RIG^ @ 1970,

BY

ACADEMICPRESS,INC.

ALL RIGHTS RESERVED NO PART OF THIS BOOK MAY BE REPRODUCED IN ANY FORM, BY PHOTOSTAT, MICROFILM, RETRIEVAL SYSTEM, OR ANY OTHER MEANS, WITHOUT WRITTEN PERMISSION FROM THE PUBLISHERS.

ACADEMIC PRESS, INC. 111 Fifth Avenue, New York, New York 10003

United Kingdom Edition published b y ACADEMIC PRESS INC. (LONDON) LTD. 24/28 Oval Road, Londonf N W 1

LIBRARY OF CONGRESS CATALOG CARDNUMBER:59-13822

PRINTED IN THE UNITED STATES OF AMERICA

CONTENTS CONTHBUTORS .

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ix

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1 4 12 23 24

Of Pattern and Place in Dendrites MADGEE . SCHEIBELAND ARNOLDB. SCHEIEEL

I. I1. I11. IV .

Historical Introduction . . . . Specific Patterning in Dendritic Domains Specificity of Synaptic Position . . Conclusions . . . . . . References . . . . . .

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The Fine Structural Localization of Biogenic Monoamines in Nervous Tissue

FLOYD E . BLOOM I. Introduction . . . . . . . . . . . I1. Model Tissue Experiments and the “Ideal Localizing Paradigm” I11. The Binding of Biogenic Monoamines to Cellular Organelles . IV . Localization of Monoamines in Nervous Tissue . . . V . Conclusions . . . . . . . . . . . References . . . . . . . . . . .

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27 28 39 41 61 62

Brain Lesions and Amine Metabolism

ROBERTY . MOORE

I. Introduction . . . . . . . . I1. Subcortical Lesions and Brain Amine Levels . I11. Interpretation of Lesion Effects on Brain Amines IV . Conclusions . . . . . . . . References . . . . . . . .

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67 69 72 88

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Morphological and Functional Aspects of Central Monoamine Neurons

KJELL FUXE.TOMAS HOKFELT.AND URBANUNCERSTEDT I. Morphology of Central Monoamine Neurons I1. Function of Central Monoamine Neurons . I11. Some Conclusions . . . . . . References . . . . . . . V

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93 104 119 121

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CONTENTS

Uptake and Subcellular Localization of Neurotransmitters in the Brain

SOLOMONH . SNYDER.MICHAELJ . KUHAR. ALAN I . GREEN. JOSEPH T . COYLE. AND EDWARD G. SHASKAN

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I Introduction . . . . . . . . . . . I1. Catecholamine Uptake . . . . . . . . . I11. Serotonin Uptake . . . . . . . . . . IV . Amino Acid Uptake and Subcellular Localization . . . V . Separation of Catecholamine-Storing Synaptosomes in Different Brain Regions . . . . . . . . . . VI . Separation of Synaptosomes Storing Different Transmitters . VII Conclusions . . . . . . . . . . . References . . . . . . . . . . .

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127 128 139 143

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146 149 156 156

160 166 171 178

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Chemical Mechanisms of Transmitter-Receptor Interaction JOHN

T. GARLANDAND

JACK

DURELL

I. Studies to Elucidate the Chemical Nature of the Receptor I1. Biochemical Effects of the Transmitters . . . . I11. Theories of Transmitter-Receptor Interaction . . . References . . . . . . . . . .

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The Chemical Nature of the Receptor Site

A Study in the Stereochemistry of Synaptic Mechanisms J . R SMYTHIES

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I. I1. I11. IV

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VI . VII. VIII IX

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Introduction . . . . . . . . . . . Possible Role of RNA in Excitable Membrane . . . . Prostaglandin-Ribonucleoprotein Complex . . . . . Specification of the Cholinergic Receptor . . . . . Specification of the Catecholamine Receptors . . . . Specification of the Serotonin Receptors . . . . . Amino Acid Transmitters . . . . . . . . The Disulfide Bond . . . . . . . . . Miscellaneous Compounds . . . . . . . . Some Stereochemical Relations between Membrane-Active Drugs and Antibiotics . . . . . . . . . . RNA and the Sodium Pump Mechanism . . . . . References . . . . . . . . . . . Note added in proof . . . . . . . . .

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181 184 189 192 201 202 207 210 212 215 217 220 221

Molecular Mechanisms in Information Processing

GEORCESUNCAR

I. Introduction . . . . . I1. Neural Coding . . . . I11. Evidence for Molecular Mechanisms IV . Molecular Hypotheses . . .

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223 227 231 242

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CONTENTS

V . Concluding Remarks References

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248 250

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255 256 268 279 280 288

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289 290 296 313 316 319 321

325 327 333 335 338 339 340

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The Effect of Increased Functional Activity on the Protein Metabolism of the Nervous System

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B. JAKOUBEK AND B SEMIGINOVSK~

I. Introduction . . . . . . . . . . . I1. Studies Performed at the Tissue Level . . . . . I11. Protein and Nucleic Acid Metabolism in Neurons and Glial Cells IV . Conclusions . . . . . . . . . . . References . . . . . . . . . . . Note added in proof . . . . . . . . .

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Protein Transport in Neurons

RAYMOND J . LASEK I. I1. I11. IV . V. VI .

Introduction . . . . . . . . . . . . Site of Neuronal Protein Synthesis . . . . . . . Axonal Transport . . . . . . . . . . . Physiological and Pathological Changes which Effect Axonal Transport Possible Mechanisms Underlying Axonal Transport . . . . Conclusions . . . . . . . . . . . . References . . . . . . . . . . . . Neurochemical Correlates of Behavior

M . H . APRISONAND J . N . HINCTGEN I. Introduction . . . . . . . . . . I1. Behavioral Depression and Increases in Brain Serotonin . I11. Behavioral Depression and Decreases in Brain Serotonin . IV . Behavioral Excitation and Decreases in Brain Acetylcholine V. Methodological Problems . . . . . . . VI . summary . . . . . . . . . . References . . . . . . . . . .

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Some Guidelines from System Science for Studying Neural Information Processing

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DONALD0 WALTERAND MARTINF GARDINER I. Basic Concepts . . . . . . . . . . I1. New Insights Arising from the Information Systems Viewpoint 111 Summary and Prospect . . . . . . . . . . References . . . . . . . . . . .

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AUTHORINDEX.

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. SUBJECTINDEX. . CONTENTS OF PREVIOUS VOLUMES.

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343 356 369 372 375 392 399

This Page Intentionally Left Blank

CONTRlB UTORS Numbers in parentheses refer to the pages on which the authors’ contributions begin.

M. H. APRISON,Section of Neurobiology, The Institute of Psychiatric Research and Departments of Psychiatry and Biochemistry, Indiana University Medical Center, Indianapolis, Indiana ( 325) FLOYDE. BLOOM,Laboratory of Neuropharmacology, Division of Special Mental Health Research, National Institute ~f Mental Health, Saint Elizabeths Hospital, Waslzington, D. C . (27)

T. C O Y L E ,Departments ~ of Pharmacology and Experimental Therapeutics and Psychiatry and the Behavioral Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland ( 127)

JOSEPH

JACK

DURELL, Section on Membrane Chemistry, Laboratory of Cerebral Metabolism, National Institute of Mental Health, Bethesda, iMaqland, and Center for the Study of Behavioral Biology, Psychiatric Institute Foundation, Washington, D. C . (159)

K JELL FUXE,Department of Histology, Karolinska Institutet, Stockholm, Sweden (93)

T. GARLAND,+ Section on Membrane Chemistry, Laboratory of Cerebral Metabolism, National Institute of Mental Health, Bethesda, Maryland, and Center for the Study of Behavioral Biology, Psychiatric Institute Foundation, Washington, D. C. (159)

JOHN

MARTINF. GARDINER, Departments of Physiology and Anatomy, Brain Research Institute, University of California, Los Angeles, California ( 343 ) ALANI. GREEN,Departments of Pharmacology and Experimental Therapeutics and Psychiatry and the Behavioral Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland (127) J. N. HINGTGEN, Section of Neurobiology, The Institute of Psychiatric Research and Departments of Psychiatry and Biochemistry, Indiana University Medical Center, Indianapolis, Indiana ( 325)

TOMASHOKFELT,Department of Histology, Karolinska Institutet, Stockholm, Sweden (93)

* Present address: Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, Maryland f Present address: Washington University School of Medicine, Department of Internal Medicine, Metabolism Division, St. Louis, Missouri. ix

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CONTRIBUTORS

B. JAKOUBEK, Institute of Physiology, Czechoslovak Academy of Sciences, Prague, Czechoslovakia and Institute of Pathophysiology, Medical Faculty of Charles University, Pizen, Czechoslovakia ( 255) MICHAELJ. KUHAR, Departments of Pharmacology and Experimental Therapeutics and Psychiatry and the Behavioral Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland ( 127)

RAYMOND J. LASEK,*Department of Neurosciences, University of California San Diego, La Jolla, California (289) ROBERTY. MOORE,Department of Pediatrics and Medicine (Neurology) and the Joseph P . Kennedy, Jr. Mental Retardation Research Center, The University of Chicago, Chicago, Illinois (67) ARNOLDB. SCHEIBEL, Departments of Anatomy and Psychiatry and Brain Research Institute, U.C.L.A. Center for the "Health Sciences, Los Angeles, California (1) MADGEE. SCHEIBEL, Departments of Anatomy and Psychiatry and Brain Research Institute, U.C.L.A. Center for the Health Sciences, Los Angeles, California ( 1 ) B. SEMIGINOVSK+, Institute of Physiology, Czechoslovak Academy of Sciences, Prague, Czechoslovakia, and Institute of Pathophysiology, Medical Faculty of Charles University, Pizen, Czechoslovakia ( 255) EDWARD G. SHASKAN, Departments of Pharmacology and Experimental Therapeutics and Psychiatry and the Behavioral Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland ( 217)

J. R. SMYTHIES,Department of Psychiaty, University of Edinburgh, Edinburgh, Scotland ( 181) SOLOMON H. SNYDER,Departments of Pharmacology and Experimental Therapeutics and Psychiatry and the Behavioral Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland ( 127) GEORGES UNGAR,Baylor College of Medicine, Houston, Texas (223) URBAN UNGERSTEDT, Department of Histology, Karolinska Institutet, Stockholm, Sweden (93) DONALD 0. WALTER,Departments of Physiology and Anatomy, Brain Research Institute, University of California, Los Angeles, California (343) * Present address: Department of Anatomy, School of Medicine, Case Western Reserve University, Cleveland, Ohio.

OF PATTERN AND PLACE IN DENDRITES1f2 By Madge E. Scheibel and Arnold 8. Scheibel Departments of Anatomy and Psychiotry and Brain Research Institute, U.C.L.A. Center for the Health Sciences, Lor Angeles, California

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I. Historical Introduction 11. Specific Patterning in Dendritic Domains . . . . . . . A. Straight, Unbranched Pattern . . . . . . . . . B. Convoluted, Field-Specific Patterns . . . . . . . . C. Some Thoughts on Dendrite Patterning . . . . . . . 111. Specificity of Synaptic Position A. Dendrites as Monoreceptive Elements . . . . . . . B. Dendrites as Polyreceptive Elements . . . . . . . C. Some Though6 on the Significance of Synaptic Position in Complex Cortical Systems i . . . IV. Conclusions References

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I. Historical Introduction

The dendritic extensions of neurons have come to occupy a central role in all considerations of synaptic activity and integrative operations undertaken by nerve cells. Such consideration is not inappropriate in view of the extensive receptive surface provided to inccming volleys and the very large proportion of total neural tissue mass made up by these structures ( Aitken and Bridger, 1961). It is almost exactly a century since their existence was first reported by Gerlach in carmine stains (Gerlach, 1871), and present knowledge of the intrinsic structure and physiological properties peculiar to these elements may hardly seem commensurate to the period during which they have been hown. It is our purpose to review briefly the more significant contributions in this area and to present several bodies of data relevant to organization of the dendrite mass and to the arrangement of presynaptic ensembles along their surfaces. The epochal quality of Gerlach's discovery was mitigated by his assumption that the processes which he could see leaving the cell bodies 'Based in part on the twelfth George H. Bishop Lecture in Experimental Neurology delivered at Washington University School of Medicine, St. Louis, Missouri, April 14, 1967. 'Experimental studies of the authors were supported by the U. S. Public Health Service, grants NB-01063 and HD-00972.

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MADGE E. SCHEIBEL AND ARNOLD B. SCHEIBEX

reunited to form a net or reticulum marked by protoplasmic fusion. Camillo Golgi's use of chrome silver techniques established the free nature of dendritic terminals (Golgi, 1903) and set a cornerstone for the neuron theory eyen though he was later to err in maintaining reticularism as a general theory of neuronal organization, and nutrition as the specific role of the dendrites. Cajal's studies of the nervous system, using the impregnation methods of Golgi and ancillary techniques, capped the initial phases of dendrite studies with the discovery of spines, and the conclusion that, in the dynamically polarized neuron, dendrites received afferent volleys and conducted them toward soma and axon. A number of dendrite patterns were described by Cajal (1909, 1911) at various levels of the nervous system, but the first attempt to provide a reasonable set of physiological consequences for some of these pattern variations was that of Lorente de Nb (1939, 1947). In advancing the theses of open and closed fields, he related dendritic morphology to the probable distribution paths of current and suggested possible effects on adjacent neuronal ensembles. Elaboration and further applications of these ideas can be found in more recent studies by Mannen (1966), Leontovitch and Zhukova ( 1963), Ram6n-Molinar and Nauta ( 1966), and ourselves (Scheibel and Scheibel, 1958a). The majority of these workers relate pattern to the phylogenetic age of the neural subsystem, long radiating and unramified dendrite systems being equated with more archaic mechanisms. However, we have pointed to the relatively high degree of structural complexity in pre- and postsynaptic morphology present even in invertebrate forms of great antiquity (Scheibel and Scheibel, 1962). We prefer to consider the structural characteristics of dendrite systems rather as a function of the morphology of the presynaptic components, and as an expression of postsynaptic integrative mechanisms peculiar to those neural elements. We shall examine this problem in some detail below. The semiquantitative approach of Sholl (1956) to the problem of cortical dendritic organization and pre- and postsynaptic relations was predicated on the statistical irrelevance of individual connections. His studies set a trend in establishing possibilities for a mathematically more rigorous approach, At the same time, the possible importance of circuit patterns and place-specific effects was deemphasized. In light of more recent studies, this research orientation has not seemed entirely justified. Among the attempts to develop conceptual models of dendritic systems, the studies of Rall ( 1962, 1964, 1967) bear special mention. Expanding on the dendrite segment counting techniques first suggested by Bok (1959) and Sholl (1956), Rall developed a theoretical treatment, drawing on geometric properties and potential theory to enable certain predictions

OF PATTERN AND PLACE IN DENDRITES

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about mechanisms operating along dendrites to be made. His studies have led to the theoretical prediction of functional distinctions between somatic and dendritic synapses. The latter would be expected to dominate slow changes in the background level of excitation, while somatic synapses would be best situated for triggering of impulses. These predictions have proven reasonably congruent with data available from microphysiological studies by a number of workers. However, a number of features of the relatively passive, cable-like dendrites, which Rall invokes to facilitate model building, are clearly oversimplifications. More recent studies of membrane impedance changes that accompany development of excitatory and inhibitory postsynaptic potentials have provided documentation for a characteristically somatic position for inhibitory nerve endings--in spinal cord at least (Smith et al., 1967). Electron microscopy has added an important dimension of data to dendrite studies, with enhancement of resolution of visible detail, often by several orders of magnitude, though at the price of all sense of relative position and orientation. Aside from the dense covering of synaptic terminals and glial processes extending to the dendrite tips, a finding already indicated by Golgi impregnation studies, perhaps the greatest surprise has been the increasingly homogeneous, electron-transparent nature of the endoplasm in areas progressively further from the cell body. While granule-rich silhouettes of endoplasmic reticulum extend out limited distances from the soma-dendrite junction, these soon disappear, leaving only the smooth contours of occasional nongranular endoplasmic bodies and the ubiquitous longitudinally running dendritic tubules. Whether these tubelike profiles, 100A or less in diameter, actually serve a conduit function, or rather represent structural skeleton, or even fixation artifact, is still uncertain (Gray, 1964). The discovery of dendritic projections or spines (thorns, gemmules) by Cajal (1909) using rapid Golgi and methylene blue techniques has been supported by subsequent studies (Fox and Barnard, 1957; Scheibel and Scheibel, 1955; and others) and only recently confirmed by electron microscopic analysis (Gray, 1959). The function of these small ( 1 3 p ) substructures remains enigmatic, but it now seems likely that they serve purposes beyond that of simply increasing dendritic surface area (Colonnier, 1967). The discovery by Gray ( 1959) of intraspinous coiled canalicular structures would suggest functions of a more specific nature. More recently, Colonnier (1964) has demonstrated their dependence on the presynaptic influx by showing their degeneration following loss of the terminal boutons synapsing upon them. Furthermore, studies of Walberg (1963) suggest that the spines ingest such terminal bouton fragments, perhaps by a pinocytotic-like mechanism, prior to their own

4

MADGE E. SCHEIBEL AND ARNOLD B. SCHEIBEL

degeneration. We have used this close relationship between pre- and postsynaptic structures in exploring terminal presynaptic topography in a complex environment such as cerebral cortex, as will be described later. Although it does not seem appropriate at this time to review developments in dendritic physiology over the past thirty years, several contributions should be mentioned as pertinent to our own consideration. Bishop and Clare (1852, 1953) were among the first to suggest the additive, nonpropagating nature of dendritic potentials and their possible relation to the electroencephalograph on the one hand, and to modulation of the firing threshold of the soma1 trigger zone on the other. Such suggestions reflected empirical development of earlier notions of Gesell and his co-workers (1954) on the dynamic polarization of neurons and the effects of current flow between dendrite and soma. Experiments with neurons in tissue culture raised some questions as to whether decremental conduction actually characterizes the total repertoire of dendritic responses (Hild and Tasaki, 19.62). In any case, the ideas of Bishop and Clare have done much to enrich our concepts of the neuronal model and free it from the confines of all-or-nothing physiology. Dendritic excitation has been attributed exclusively to chemogenic processes by some workers ( Grundfest, 1957). Recent information has necessitated alterations in so monistic a view. Data from several sources stresses the uniqueness of the subsynaptic patch as the unit of functional activity along the dendritic membrane, determining the actual effect of the presynaptically released transmitter. The same transmitter substance may have different effects on different membrane patches (Tauc and Gerschenfeld, 1961; Kandel et al., 1967). Thus it remains possible to operate within the confines of Dale’s hypothesis (1935) (all terminals of all branches of a single neuronal axon release the same transmitter substance) and still obtain contrasting effects from different terminals of an axon synapsing upon different neuron systems. We have recently identified such a situation in cat spinal cord, where branches of a single primary afferent collateral supply both extensor and flexor motoneuron pools without intercalated internuncials ( Scheibel and Scheibel, 1969). II. Specific Patterning in Dendritic Domains

If it is assumed that structure reflects function, the opulence of dendritic patterns in neurons of the vertebrate nervous system bears witness to the range of operations subserved by dendritic membrane. While it seems safe to assume that reception and/or integration of presynaptic impulses (with the added possibility of spike generation in some dendrite systems under certain conditions) represent the principal dendritic role, specific problems of local topography and physiological dynamics vary widely.


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Figure 1 illustrates a few dendritic patterns found in the brainstem. The most significant variations exist in (1) the amount of branching of the individual dendrite shaft, ( 2 ) the course of the individual element, and ( 3 ) the size and shape of the total dendrite domain. Meaningful interpretation of these variables requires correlative information about the presynaptic input systems and, if possible, the nature of the information involved in the synaptic transaction. A. STRAIGHT,UNBRANCHED PATI-ERNS Those cells making up the greater part of the reticular core of the brainstem are characterized by limited numbers of long, relatively straight and poorly ramified dendrite shafts. The individual shaft may have as few as 2 or as many as 6 or 7 branch points aIong its course, and extend for as much as 500 to 7 5 0 p from the cell body of origin. The total diameter of the dendritic domain may accordingly exceed 1 mm, and we have calculated that between 4000 and 5000 similar nerve cell bodies may lie in such a tissue volume (Scheibel and Scheibel, 195813).

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B A / \

C

D

FIG.1. Dendrite patterns of a group of neurons in the brainstem. A: Neuron of the kitten axial core (reticular formation) showing straight, relatively unbranched dendrite shafts; B: neuron of the kitten ventrobasal nucleus of the thalamus showing multiple, richly branched dendrites producing a tufted appearance; C: neuron of the inferior olive in an infant with branched and convoluted dendrites forming a dense spherical domain; D: four neurons of the medial portion of the superior olive in a kitten showing paired dendrite domains of limited extent. Drawn from Golgi-stained preparations at varying magnifications.

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Several statements of fact can be made about the nature of this dendrite pattern, and several questions should be raised as to the nature of their functional role. Histological studies clearly show that the presynaptic influx to this type of dendrite system consists of axonal preterminal segments of varying lengths which run along the dendrites, often for appreciable distances, establishing repetitive terminal synaptic contacts along the way. This parallel apposition of pre- and postsynaptic components appears characteristic of the linear, poorly ramified dendrite shaft in most locations ( Scheibel and Scheibel, 19%). In most cases, these elements receive a number of synaptic inputs, not only from different presynaptic preterminal fibers, but also from different fiber systems, thereby resulting in appreciable convergence of heterogeneous afferent information along the soma-dendrite membrane of the individual cell (Scheibel et al., 195.5). The nature of the ensuing integrative process has been examined both theoretically and experimentally. For spinal motoneurons, at least (Smith et al., 1967; Burke, 1967; Rall, 1967; Rall et al., 1967), it may be sufficient to consider the dendrites as essentially passive conductors, The excitatory process which then develops in the region of the somal trigger zone is a moment-tomoment function of the size, geographical position, and sign of the multiple discreet local disturbance (postsynaptic potentials, PSPs ) along the entire domain membrane system. PSPs generated near the dendrite tips may be “invisible” to the intrasomal microelectrode but may still exert tonic shaping effects of some magnitude on somal output. Dendritic locations increasingly close to the soma presumably produce PSPs of progressively greater amplitude, decreased temporal distortion, and enhanced effectiveness in programming spike activities at the trigger zone. This quasi-linear model may represent an oversimplification of the behavior of reticular neurons where dendrites penetrate through adjacent nuclei and tracts of diverse function. Unpublished data (Scheibel et al., 1961) suggest that in this case, the locus of spike generation may wander along the soma-dendrite membrane, thereby raising the question of independent spike induction in dendrites. The penetration of long dendrite shafts through diverse nuclei and tracts brings up another variable of still largely unknown dimensions, i.e., the effect of variations in Iocal field potentials upon a passing dendrite element. It is known that potentials of the order of 1 millivolt can be measured in the immediate surround of an activated spinaI motoneuron (Nelson and Frank, 1964) and that closely adjacent motoneurons


7

can show consequent threshold variations of the order of 10 to 30%(Nelson, 1966). There are, as yet, no corresponding measurements for reticular neurons, but it seems reasonable to assume qualitatively similar effects in this locale. We can, therefore, conceive of the widely spreading, linear dendrite system of the reticular formation as a pattern suited to sampling information from heterogeneous and diversely situated afferent sources, directly through synaptic interaction, and more indirectly through impingement of local standing or evoked field potential phenomena imposed via nonsynaptic (ephaptic) means.

B. CONVOLUTED, FIELD-SPECIFIC PATTERNS In contrast with the unramified structure of axial reticular dendrite systems, a number of examples of highly convoluted, densely branched and/ or tufted, or otherwise uniquely configured dendrite domains can be cited. Some of the most striking of these can be found in sensory “relay” nuclei such as the ventrobasal complex of the thalamus and the superior olive of the upper medulla. Equally unique examples are seen in inferior olive and cerebellar cortex, related structures of more ambiguous functional significance. The ganglion cells of the retina differ from all of these in having a range of dendrite domain patterns, to each of which a putative function has recently been assigned. 1. Neurons of the Ventrobasal (Somatic Sensory) Thalamus Thalamic ventrobasal cells (Fig. 1B) are representative of neurons in sensory transmission systems, where a significant measure of mode and/or locus specificity is maintained ( Mountcastle, 1961). The dendrites are relatively thick and short, and quickly break up into dense masses of secondary branchlets forming tufts or brushes [the bzcschzellen or cellules en buisson of Cajal (1911)l. The rather small, densely configured domain resulting from this structural design overlaps and partially interpenetrates a group of immediately adjacent dendrite fields. Superimposed upon this matrix of interlocking postsynaptic elements is a group of presynaptic axonal systems, each forming a neuropil plexus of unique configuration. Of these, the medial lemniscus, carrying sensory information from the periphery, terminates in a set of conical arbors, also partly overlapped. In concert, the pre- and postsynaptic structures form a complex substrate well adapted to ensure maximal security and adequate fidelity of transmission (Purpura and Cohen, 1962). Unquestionably, the limited spread and elaborate branching of the dendrite systems of these cells ensures activation of the soma1 trigger zone

8


consequent to volleys arriving at a highly restricted portion of the ventrobasal field (Scheibel and Scheibel, 1966a).

2. Neurons of the Superior Olive Superior olive neurons bear dendrite patterns which are appreciably different from ventrobasal elements, but equally well adapted to their functional role. The cells are aligned, long axes parallel, around the periphery of the nuclear sac, each neuron being essentially bipolar, and carrying one main central and one peripheral-pointing dendritic shaft. These immediately generate a group of secondary branches, limited in number and extent, forming oval dendritic fields flanking each neuron soma ( Cajal, 1909). These dendritic domains are immersed in elongated, cone-shaped, terminal afferent fields, apparently tagged for several auditory variables including pitch. The highly restricted lateral dimensions of each dendritic field presumably restrict the frequency range monitored by each cell. A further, and even more dramatic specialization exists in the medial superior olive (Fig. 1D) in that the two major dendritic domains of each neuron sample inputs from opposite sides ( Goldberg and Brown, 1969). Several groups of physiological responses appear in response to high frequency binaural stimuli including excitatory-excitatory ( EE ), and excitatory-inhibitory ( EI ) effects. In addition, the same, or similar cells discharge in periodic or phase-linked manner to low frequency stimulation of either ear. In this case, discharge rate and degree of phase-locking are determined by the relative timing of the stimuli arriving from the two ears. Thus, the double dendritic domain of each superior olive neuron appears to serve as substrate to paired neural mechanisms participating in sound localization.

3. Neurons of the Inferior Olive Another type of highly developed dendritic domain is that of the inferior olive neuron (Fig. 1C). Here a number of main dendrites from each cell are multiply branched and convoluted around the soma, forming a dense, spherical field of impressive complexity. This very obvious attempt to limit the geographical range of the postsynaptic membrane is matched by presynaptic conical arbors of equal structural elegance. As in the case of the ventrobasal complex, the dendritic domains are partially overlapped and interpenetrating, thereby forming a dense postsynaptic matrix upon which presynaptic terminal neuropil of several patterns ( Scheibel and Scheibel, 1955) delimits secondary domains. Despite its obvious size and prominence as a brainstem landmark, the function of the inferior olive remains enigmatic, thereby seriously compromising any attempt to relate structure to function.


9

4. The Purkinje Neuron of the Cerebellum The Purkinje cell offers another example of a dendritic field of unique shape and dimension, configured to a specific relationship with the presynaptic influx. As Fig. 2 shows, the Purkinje dendrite domain develops from one side of the cell body, while the axon develops from the other (or from a primary dendrite branch). In the mammalian vertebrate, the dendrite system is made up of a series of primary, secondary, and tertiary branchlets compressed into a bidimensional domain placed perpendicular to one of the two major cerebellar inputs, the parallel fiber system. The surface area of tertiary branchlets together with the spines which they bear (as many as 120,000 per domain) far exceed that of primary and secondary systems together, and provide a veritable screen or filter system through which information in the parallel fiber ensemble must percolate (Cajal, 1911; Fox et aZ., 1967). The Purkinje dendrite domains of nonmammalian vertebrate brains show a basic pattern which is similar to that of mammals, but with certain obvious simplifications ( Fig. 2 ) . The distinction between primary, secondary, and tertiary components, while still recognizable in birds, can no longer be made out in fish where spines have become difficult

FIG.2. Varying dendrite patterns in cerebellar Purkinje cells of various vertebrate species, A: sturgeon; B: perch; C: turtle; D: pigeon; E: man. In the last, 1, 2, and 3 refer to primary, secondary, and tertiary dendrites respectively. Based on Golgi-stained sections drawn at varying magnifications.

10

MADGE E. SCHEIBEL AND ARNOLD B. SCHEJBEL

to identify, or have disappeared entirely. The number of branches, and consequently of branch points, has also noticeably diminished in these forms, presumably decreasing the range of possible dendritic operations. Nonetheless, even in cerebellar forms relatively as simple as that of the sturgeon, the restricted number of dendrite shafts characterizing these Purkinje cells form a domain whose orientation and silhouette do not differ greatly from that of man (Fig. 2A,E), In each case, the shaping factor would appear to be the presence of a system of parallel fibers crossing the sequence of Purkinje domains at right angles. Although a number of attempts have been made to interpret the meaning of this synaptic configuration ( Eccles et al., 1967), no definitive conclusions are yet possible save that it clearly represents an efficient mode of spreading information over very large numbers of similar cells in sequence. The success of this structural paradigm is attested by the similarity of the circuit throughout the vertebrate line despite interspecies variation in detail.

5. Ganglion Cells of the Retina Neurons of the ganglion cell layer of the retina deserve consideration at this point because of a group of physiological correlations which have tentatively been established with the varied dendritic domains which they show. The inclusion of these cells is appropriate since the retina, though thought of as part of the peripheral nervous system, is embryologically a derivative of the diencephalic vesicle (thalamus) from which it emigrated at an early stage in ontogenetic development. In the frog, five major dendritic patterns were described by Cajal (1894) and are illustrated in Fig. 3. These include ( a ) small cells with very constricted dendritic ramifications spread out in a single bushy slab at an inner level of the plexiform layer (Fig. 3C); ( b ) large cells with a single, very extensive planar domain spread along the outer margin of the plexiform layer (Fig. 3D); ( c ) large cells with multilevel “ H type

A

B

C

D

E

FIG.3. Different forms of retinal ganglion cells of the frog. A: An “ H cell with extensive multilevel dendrite domain; B: an “E” cell with multilevel and parcellated dendritic domain; C: small cells with constricted, one-level domains; D: large (parasol type) cell with extensive one-level dendrite domain; E: large cell with diffuse dendrite domain. Based on drawings of Cajal (1894) from Golgi-stained material.

OF PATI’ERN AND PLACE IN DENDRITES

11

distribution of dendritic masses (Fig. 3A); ( d ) cells with multilevel “E” type distribution of dendritic fields (Fig. 3B); and ( e ) cells with a diffuse, nonplanar dendritic domain (Fig. 3E). These dendrite systems receive information from the immediately overlying bipolar cells, The horizontal spread of these ganglionar dendrite systems and their position in the lamina largely determine the number and identity of bipolar cells with which they connect. Lettvin and co-workers (1961) have attempted to correlate these patterns with a group of physiological properties which they have isolated in optic nerve fibers of the frog visual system. The small, onelevel constricted elements are assigned a role in boundary detection where the receptive field extends over no more than 2 to 4 degrees. The large cells with extensive, single-layer dendritic domains are interpreted as dimming detectors (off fibers), Here, spatial resolution is poor, and relative rather than absolute values of illumination are apparently involved. The multilevel “ H type domain cell is conceived as primarily sensitive to “contrast, movement, and change” ( on-off detectors), functioning as a detector of events signalled by any combination of changes in distribution of light and boundaries over its surface. The multilevel “ E type domain neuron is associated with “movement-gated, dark convex boundary detection.” The multiple compartmentation of the horizontal fields is thought to provide a number of small receptive segments capable of determining the degree of dimming in any one such area combined with the amount of boundary in that area. Thus the “ E type is seen as combining a group of “H’ functions with a higher degree of spatial (boundary) resolution. Finally, the diffuse dendritic type is matched, by exclusion, with general light level measuring properties. The correlations suggested by Lettvin and his associates, though intuitive and conceptually satisfying, cannot yet be considered as proven. Nevertheless, they underline the possibility, also demonstrated in studies by Hubel and Wiesel (1959) on mammalian visual cortex, that various neural elements are preprogrammed for certain varieties of input which, in combination, may represent potentially meaningful environmental happenings. Their relevance to this review lies in the fact that the various dendrite patterns involved apparently determine those characteristics of visual input to which each cell will respond, and thereby represent a putative link between neural structure and epistemology.

C. SOMETHOUGHTS ON DENDRITE PATTERNING In examining a selected group of dendrite patterns, we obviously have omitted many others. Those selected illustrate a range of domain

12


patterns from the multipolar, long, straight, relatively unbranched systems characteristic of the brainstem reticular core, through the highly branched, convoluted and/ or tufted elements of the ventrobasal thalamus and inferior olive, to the consistently stylized, bidimensional fields of cerebellar Purkinje cells and the complex geometry of retinal ganglion cells. Some workers ( Ram6n-Molinar and Nauta, 1966) have stressed the existence of a pattern continuum linking the unramified “reticular” dendrite pattern of the “isodendritic core” of the brainstem through a heterogeneous group of domains of varying complexity ( allodendritic patterns) to the most highly specialized patterns such as those of the inferior olive and sensory thalamic fields (idiodendritic patterns). There are, unquestionably, appealing features to this mode of categorization. However, it would also appear to have certain limitations, such as the difficulty of including cortical cells-cerebral, cerebellar, or hippocampal-in the sequence. There is also the implication that reticular (isodendritic) cells represent the most primitive (i.e., least developed) dendrite pattern, a notion that does not seem justified when they are compared with phylogenetically more primitive neuron patterns in many nonvertebrate forms. In another attempt to bring some degree of functional relevance to dendrite patterning, Lorente de N6 (1939, 1947) introduced the concepts of open and closed nuclei, based on dendrite patterning and the consequent distribution of electrical sources and sinks. Although this classification may have some predictive value in the analysis of potential fields, it would appear to represent an oversimplification of the data. The limited roster of dendrites shown by any Golgi stain does not exist in vacuo, nor do they more than epitomize some of the patterns etched by the far greater numbers left unstained. Furthermore, the number and complexity of membrane profiles which pack the interdendritic space make any prediction as to field shape and current distribution, at best, an approximation. Ill. Specificity of Synaptic Position

The significance of synaptic position along the soma-dendrite membrane has become increasingly clear over the last few decades as the dynamics of membrane and field have been elucidated. WhiIe the properties of dendrites remain largely enigmatic, our models can no longer be fully satisfied by the assumption that they are passive, undifferentiated samplers of synaptic drive on the one hand, and extensions of the soma1 membrane for ion flux (source-sink) operations on the other. The hierarchical aspects of dendrite membrane organization and the contrasting roles of proximal and distal portions emphasize the impor-


13

tance of synaptic locus. Position becomes even more significant as the role of branch points along each dendrite shaft comes under increasing scrutiny. Are they points of potential block for membrane disturbances; or do they perhaps operate as valves, allowing transmission in one direction only? Alternatively; might they function as selective loci for spike initiation along the dendrites, or conceivably, even as integrators and/ or differentiators of the totality of local disturbances ( PSPs) generated distally? Clearly, if any of these putative roles could be proven, one more reason would be added to a rapidly growing roster for developing the fullest possible acquaintance with patterns of synaptic topography on various neurons.

A. DENDRITES AS MONORECEPTIVE ELEMENTS With the exception of a few nuclear pools, most cells located in the axial core of the central nervous system tend to show a comparatively simple pattern of synaptic localization along the soma-dendrite membrane. Most commonly, an entire dendrite or segment of dendritic domain tends to be dedicated to a certain type of input, suggesting (though not necessitating) a relatively uniform functional role for certain shafts, with the final integrative processes presumably occurring in the area of the soma1 trigger zone.

1. Neurons of the Brainstem Reticular Core Despite the complex organization of the brainstem reticular formation with its densely intermixed fields of cells, local fiber systems, and tracts, we are convinced that dendrite systems in large numbers of reticular cells are organized to receive certain inputs in relatively pure culture. As an example, we will cite reticular neurons located in Olszewski’s ventral subnucleus of the nucleus medullae oblongatae centralis ( Olszewski, 1954). As we have shown elsewhere, such cells may spread their dendrites over % to % of the cross-sectional area of the brainstem, as seen in transverse sections, while showing little development along the rostrocaudal axis, leading to a two-dimensional or chip-shaped domain module ( Scheibel and Scheibel, 1958b, 196813). Careful examination of shaft organization and impinging presynaptic afferent systems suggests definite concentrations of certain inputs on certain shafts (Fig. 4). Ventrally directed shafts appear preferentially loaded with terminals from pyramidal tract collaterals while ventrolateral projecting branches receive, in the main, collaterals streaming in from the spinothalamic bundles and their associated tract nuclei, the so-called lateral reticular nucleus. Dorsolateral ranging shafts appear to receive axonal termina-

14


FIG.4. Slightly schematized drawing showing the relation of specific presynaptic afferents to the monoreceptive dendrite system of a neuron of the brainstem reticular core. Left side: afferent terminals pictured here include (1) collaterals from vestibular nuclear complex, ( 2 ) collaterals and terminals of the descending root of the fifth nerve, (2’) axon collaterals from neuron of the nucleus of the fifth nerve, ( 3 ) collaterals from spinothalamic tract, ( 4 ) collaterals of pyramidal tract fibers, ( 5 ) collaterals and/or terminals from medial longitudinal fasciculus. Right side: large reticular cell in the ventral reticular complex showing various dendrite systems; ( a ) medial longitudinal fasciculus, ( b ) vestibular complex, ( c ) descending fifth nerve complex, ( d ) spinothalamic collaterals, ( e ) pyramidal collaterals.

tions from descending vestibular and/ or spinal tigeminal systems while dorsomedial branches, if any, may receive synaptic contributions from collaterals of fibers running in the medial longitudinal fasciculus and occasionally from contralateral systems. There is often some degree of admixture and dilution by boutons en passage from the rostrocaudal z o n a l elements which characterize all parts of the reticular formation. However, it is not possible to predict the location of such terminals; they do not appear to develop appreciable density at any point along the shafts. For the purposes of our working model, it seems quite appropriate to conceive of each major dendrite shaft as under presynaptic control of one predominant system. This picture may well be complicated by the as-yet-unexplored effects of local field phenomena present in the various nuclei and tracts which these typically long dendrites may penetrate in the tightly packed core. Such mechanisms, if significant, would probably develop their effects upon the dendrite shaft by extrasynaptic (ephaptic) means.


15

2. Spinal Neurons in the Dorsal Horn Another interesting example of segregation of presynaptic terminals upon individual dendrite shafts of single neurons is found in the large lamina IV neurons deep to the gelatinosal complex in the dorsal horn. The substantia gelatinosa is largely made up of the terminal elaborations of axons innervating the cutaneous surface of the body. In the usual transverse preparations stained by the chrome-silver methods ( Cajal, 1909), these terminal plexuses or lobules (Szentagothai, 1964) present a series of domains shaped like tall thin Italian cypresses. Sagittal preparations reveal that this appearance is owing primarily to viewing the plexuses “edge-on,” since they actually consist of long neuropil plates of minimal thickness extending hundreds or thousands of microns in the rostrocaudal plane. Similarly, the small gelatinosal cells which are immersed in this presynaptic matrix actually possess extensive dendritic domains, again developed almost entirely along the longitudinal axis (Scheibel and Scheibel, 1968a). Just ventral to this highly organized synaptic area is a layer of large neurons (lamina IV of Rexed, 1952) whose dendrites appear to show three principal orientations; lateral, dorsal, and medial (Fig. 5). The dorsal dendrites ascend into the cutaneous terminal plexuses just described, in diverging conical array and apparently receive virtually their entire presynaptic load from this source. These dendrites bear long convo-

FIG.5. Slightly schematized drawing showing the relations between the dendrites of a spinal cord lamina IV neuron and the three major sets of afferents which it receives. Coarse cutaneous afferent fibers (1) synapse on dorsal dendrites, d; collaterals and/or terminals of the lateral corticospinal fibers ( 2 ) synapse on lateral dendrites (1); collaterals and/or terminals of fibers from ventral part of midline white matter (dorsal columns) synapse on medial dendrites, m. Based on Golgistained sections of spinal cord.

16

MADGE E. SCHEIBEL AND ARNOLD B. SCHELBEL

luted spines similar to those found on gelatinosal cells, in marked contrast to the smoother, horizontally oriented shafts. In combination with similar types of data from other areas, it is difficult to avoid concluding from this observation that the nature of the presynaptic input helps determine the morphology (and perhaps even the presence or absence) of dendritic spines. The lateral dendrites are largely recipient to collaterals and terminals of the lateral corticospinal tract ( Scheibel and Scheibel, 1966b) descending from the sensorimotor strip and adjacent areas of frontal, parietal, and temporal neocortex. The medial are mainly receptive to terminal fibers from the medioventral portion of the dorsal columns, an area of intraspinally derived projection elements. These three dendrite systems clearly sample inputs of highly contrasting significance. The dorsal system presumably extracts information from a series of parallel neuropil plates acting as a first level of representation for parallel cutaneous fields upon the surface of the skin. The integrative operations of this system are presumably capable of modulation by cortical ( suprasegmental) and spinal (segmental) mechanisms via synaptic arrays on lateral and medial dendrites, respectively. It has, in fact, been demonstrated that spinal afferent conduction in the dorsal horn is susceptible to cortical control (Hagbarth and Kerr, 1954) and to manipulation of spinal input at remote levels (Wall, 1967).

B. DENDRITES AS POLYRECEPTIVE ELEMENTS 1. The Purkinje Cell One of the classic neurohistological examples of precise synaptic placement can be found in the Purkinje ( P ) cell of the cerebellum. Inasmuch as this is the principal efferent element of cerebellar cortex, it is not surprising that both of the major corticipetal afferent systems impinge upon this neuron. The climbing fiber, distributed essentially one per P cell, ascends along the primary and secondary dendrite branches only. The individual axon components divide and fuse a number of times in their course, thereby forming a kind of anastomotic vine partly enveloping the subjacent dendrite system. These climbing fiber elements “ride” the primary-secondary dendrite system virtually to the pial surface, thereby ensuring a continuous and intensive type of contact with the nonspine-bearing portions of the dendrite domain. Electrophysiological analysis reveals that the climbing fiber is, in consequence, a powerful and reliable activator of the individual P cell (Eccles et al., 1966a). The second major input to the Purkinje neuron is the parallel fiber system, arising from the subjacent granule cell complex. These elements,


17

running in enormous numbers parallel to the surface of cerebellar cortex, traverse the bidimensional P cell dendrite domains at right angles, effecting synaptic contact exclusively with the spines of the tertiary dendrite system. Fox and his co-workers (1967) have estimated that there may be as many as 120,000 spines on a single P cell and allowing, on the average, two presynaptic contacts per spine, it is obvious that the number of discreet impingements by parallel fibers on the tertiary dendrite system of a P cell is very great. This system has also been found to develop excitatory synaptic pressure on the P cell, although the response train is rapidly quenched by combined feed forward and feedback inhibitory effects also mobilized by parallel fiber activation of adjacent elements ( Eccles et al., 1966b). A third system contributing significantly to the presynaptic input is the axon system of cerebellar basket cells. These neuronal elements, which exist in moderate concentrations in the lower third of the molecular layer, extend their dendrites in tridimensional array into the parallel fiber stream, their main synaptic source of excitation. Their axons extend for many hundreds of microns at right angles to the course of parallel fibers, generating dense axonal terminal baskets about the axon hillockinitial segment areas of several rows of Purkinje cells. In effect, they usually envelop the P cell soma, the normal locus of the axon hillock. However, in the 5 to 10%of cases where the P cell axon issues from a primary dendrite, the basket cell terminal is generated here, instead. In either case, the electron microscope reveals the exclusive presence of symmetric synaptic profiles with flattened vesicles, a pattern now generally believed associated with inhibition ( Uchizono, 1965). Physiological data suggest that the basket cell input is an essential link in the inhibitory system of the cerebellar cortex (Eccles et al., 1967). Its position about the soma1 trigger zone is optimal for engendering a rapidly developed phasic control over neuronal spiking behavior (Eccles, 1964) and serves as an effective foil to the powerful synaptic driving effects mediated by the climbing fiber and parallel fiber systems farther out along the dendrites.

2. The Hippocampal Pyramid Another classic example of the degree of precision with which presynaptic terminals may be organized along neurons in a laminated environment is that of the hippocampal pyramid cell. Like the cerebellum, the hippocampus provides an example of a relatively simple 3-layered cortex where the source and termination of afferent elements are known and the major intracortical circuitry adequately delineated (Cajal, 1911; Lorente de N6, 1934). The pyramids are organized in a

18


single layer with their basal dendrites extending toward the fimbrial surface of the hippocampus, the apical shafts toward the deep boundary of separation from the dentate gyrus. Thus the dendritic system of each hippocampal pyramid traverses the entire thickness of cortex, offering maximal opportunity for sampling the synaptic milieu. As in the case of cerebellar Purkinje cells, and as we shall see below in the case of cerebral cortex pyramids, the hippocampal pyramidal soma is enshrouded by a rather dense axonal basket, generated by axons of adjacent local circuit or “basket cells,” terminating in symmetric synaptic profiles with flattened vesicles. Once again, this critical perisomatic position should offer maximal phasic control over the spike-initiating zone of the axon hillock-initial segment. It is physiologically appropriate, and consonant with current theoretically derived models, that this input proves to be inhibitory in nature (Andersen and Eccles, 1965), as the experimental data, indeed, demonstrate. Since the parent basket cells are driven, in part, by synaptic terminals generated by the outflow axons of many of these hippocampal pyramids, this seems to offer a unique example in mammalian neurohistology where both the synapses and pathway of a recurrent inhibitory system can be traced (Andersen, 1965). Along the comparatively short course of the basal dendrites of each hippocampal pyramid are a group of synaptic terminals apparently generated by axon collaterals of adjacent pyramids and of other nonbasket-producing local circuit cells. The precise role of these synaptic endings remain to be determined, although it seems not unlikely that those which are closely adjacent to the soma may be inhibitory, and those located farther toward the dendrite tips, excitatory. The apical dendrite system provides a rich example of synaptic termini, hierarchically ordered from soma to dendrite tip. Figure 6A identifies them from soma outward as basket cell endings, mossy fibers (originating in dentate pyramids), association paths (probably from septum and contralateral hippocampus ) , and Schaeffer collaterals (from hippocampal pyramids of the C A, region), all interspersed with terminals of short axoned (local circuit) cells. The latter apparently continue out onto the terminal twigs of the apical arches where endings from extrahippocampal afferents may also be found. The studies of Andersen (1965) and of Andersen and Eccles (1965) emphasize the preponderance of inhibitory effects exerted by most of these terminal systems with the apparent exception of (1) those along the apical twigs and ( 2 ) the endings of the Schaeffer collaterals. The latter terminate densely about the apical shaft, some 400 to SOOp from the cell body, in a virtually continuous cuff of asymmetrical synaptic profiles rich in


19

FIG. 6. Arrangement of presynaptic terminals on the polyreceptive dendrite systems of a hippocampal pyramid ( A ) and a large fifth layer pyramid of cerebral cortex ( B ) . A: Synaptic terminals include ( a ) collaterals of afferent fibers of presumably extrahippocampal source; ( b ) Schaeffer collaterals; ( c ) septa1 afferents; ( d ) mossy fibers; ( e ) basket cell terminals; ( f ) collaterals from other pyramidal cells. B: Synaptic terminals include ( a ) nonspecific thalamic and brainstem reticular core; ( b ) specific afferent radiation (i.e., geniculocalcarine radiation); ( c ) contralateral (callosal) afferents; ( d ) basket cell axons of adjacent stellate cells; ( e ) recurrent collaterals of other pyramidal cells. Based on Golgi-stained material.

spherical vesicles ( Hamlyn, 1963). Activation of this system apparently generates a considerable conductance change across the dendritic membrane, coeval with a large extracellular wave as index of depolarization of the apical shaft. All other terminal systems along the shaft, with even a fair degree of proximity to the soma, appear inhibitory in nature, their effectiveness decreasing roughly as a function of their distance from the trigger zone (Andersen, 1965). With the exception of the already rather remotely situated cuff of Schaeffer collaterals, all excitatory endings must therefore be considered ineffectively placed to exert any action save a rather low level tonic one upon the soma1 trigger zone.

3. Pyramids of the Cerebral Cortex We have saved until last a consideration of specificity of the placement of synapses along cortical pyramids, for several reasons. For one, this multilaminate structure is more complex, by several orders of magni-

20


tude, than any other system. For another, most of the relevant data have been developed in our own laboratory over the past few yearssome still unpublished-and are accordingly subject to modification with further work and to more experience with other species. Finally, despite the intriguing nature of the data, and the extensive literature on electrical studies of neocortex, functional correlations still must remain speculative in nature, attesting, once more, to the complexity of cortical organization. Our findings are based largely on the use of Golgi techniques following section of selected corticipetal afferent systems in newborn animals. Because of studies of Colonnier (1964) and Walberg (1963),it has been established that following loss of the relevant presynaptic afferent system, the spines along that section of dendrite receptive to the afferent system, degenerate. It follows that, upon a suitable interval (30 days), spine counts along dendrites of suspected terminal areas should show diminution relative to nondenervated control areas, a presumption borne out by experiment. Using this method, a rather specific arrangement of afferent terminals upon cortical pyramids has been demonstrated and we shall use, as a paradigm, the fifth layer pyramid which characteristically spans most of the thickness of cortex. 1. Specific afferent fiber systems as epitomized by the geniculocalcarine (visual) radiation terminate directly upon the central third of the apical shaft ( Globus and Scheibel, 1967a). It has been known since the time of Cajal (1909, 1911) that such fibers generate a dense plexus in cortical layer 4,the site of densest accumulation of stellate or granule (local circuit, Golgi type 11) cells. It was accordingly inferred that these local circuit elements were the primary receptors for specific corticipetal projections, and only secondarily transferred the excitation to cortical pyramids, the major outflow elements of cortex. The loss of spines along apical shafts of the pyramids strongly indicates the presence of direct projections without intercalated cortical elements, especially in the absence of any noticeable changes in the local circuit cells, which might suggest a transsynaptic degenerative process. This conclusion is supported by our correlative structure-function studies in cortices of newborn animals, where it is clear that, at the perinatal phase of cortical development in some species, primary afferent fibers and pyramids are present and at least rudimentarily active at a time when the stellate elements of layer 4 are not sufficiently developed to serve as relays (Scheibel and Scheibel, 1W).Our data do not rule out the possibility that these stellate cells may eventually receive synaptic termini from the specific sensory radiation. However, this synaptic arrangement can no longer be considered either primary or exclusive. 2. The projection of axial nonspecific systems from brainstem or thalamus upon cortex has received abundant physiological documenta-

OF PA"J3RN AND PLACE I N DENDRITES

21

tion (Moruzzi and Magoun, 1949; Magni and Willis, 1963; and others), but anatomical support has proven more elusive. The pluriareal afferent system described by Lorente de N6 (1943) has been suggested as part of the terminal cortical elaboration of this system and has received critical reappraisal by Nauta and Whitlock (1954). Recent studies by Skinner and Lindsley ( 1967) have identified, by electrophysiological means, the rostra1 path of the thalamic nonspecific system, via the inferior thalamic peduncle, onto the orbitofrontal cortex. At approximately the same time, we have had the opportunity to study orbitofrontal tissue following unilateral lesions to the inferior thalamic peduncle. Spine couhts suggest a low density synaptic innervation along the entire length of apical shafts of orbitofrontal cortex (Scheibel and Scheibel, unpublished). This agrees with direct Golgi observation of projection fibers from the anterior third of the thalamic nonspecific system (Scheibel and Scheibel, 1967). It is likely, though not yet proven, that the projections from the brainstem reticular core (posterior to thalamus) upon cerebral cortex, both direct and indirect, are similarly applied to the apical shafts of pyramids, 3. Axons from a specific locus on one cortical hemisphere project through the corpus callosum to a contralateral mirror image point. This commissural projection terminates upon the oblique branches of apical shafts and apparently upon no other site (Globus and Scheibel, 196%). 4. Recurrent collateral systems from the axons of pyramidal cells constitute a massive intracerebral system of local circuits. Terminal synaptic sites are found upon the basilar dendrite shafts and apical arches of surrounding pyramids up to 3 to 5 mm distant (Globus and Scheibel, unpublished). Since the function of this system appears to be largely inhibitory ( Kameda et al., 1969), it provides cortical elements with an inhibitory surround probably devoted, at least in part, to the enhancement of representational interfaces. 5. As in previously described cortical structures, a basket cell system whose terminals are applied closely about the pyramidal somata has also been described by a number of authors (Cajal, 1911; Colonnier, 1965; Marin-Padilla, 1969). In this case, the pericellular envelope of terminals appears built up of synaptic arrays from axons of a number of different layer 4 stellate cells. On the basis of considerations previously noted, the system may be considered inhibitory in function although this conclusion must still be considered tentative.

C. SOMETHOUGHTS ON THE SIGNIFICANCE OF SYNAPTIC POSITION IN COMPLEX CORTICAL SYSTEMS Several interesting conclusions can be drawn from this brief examination of synaptic loci along cortical pyramids. Afferents of extracortical

22

MADGE E. SCHEJBEL AND ARNOLD B. SCHEIBEL

origin (specific and nonspecific projections) are applied only to the vertical components (apical shafts) of the neurons, while afferents of intracortical origin terminate exclusively on horizontal components (basilar dendrites, oblique branches, and tips of apical arches ) . We have found that for a given cortical area, the maximal horizontal breadth of the dendrite domain (as expressed by the spread of the apical arches, basilar dendrites, and oblique branches) of any pyramid approximates a constant figure within 5 to 7% (Globus and Scheibel, 1967~).The length or height of the domain depends on the position of the pyramidal cell body which can vary from a location in the superficial part of lamina 2 to the depths of layer 6. Thus the height of the domain may vary from less than 200 p to more than 2000 p , If we assume a rough correspondence between the amount of dendritic surface and the number of synaptic terminals contained thereon, it becomes clear that for any cortical area, each pyramid bears approximately the same number of terminals of intracortical origin, while the number of extracortically derived terminals will vary as a function of the position (depth) of the soma. In this way, the relative precision with which synaptic place is maintained along cortical pyramids helps to maintain at a constant value the total influx of stored (intracerebral) information available to all cells of an area, Similarly, since the depth of each pyramid in cortex determines the length of the apical shaft and, pari passu, the total influx of extracortical information, we might speculate that each cell in a given cortical sector is thereby enabled to compute a set of correlation functions unique to itself, on the basis of its position. Another conclusion growing out of the consistent arrangement of synaptic terminals concerns the relative efficacy of the various afferent systems. Drawing largely on the mathematical predictions of Rall ( 1964), supported by physiological data where available, it would appear that in the cerebral cortex, as in other cortical structures we have examined, the most powerful type of phasic control is exerted directly on the somal trigger zone via basket endings by adjacent local circuit elements. It is probably not an exaggeration to suggest that the cortex thereby maintains absolute control over the output performance of its intrinsic elements. The most powerful synaptic driving influence that can be exerted upon cortical pyramids is undoubtedly that of the specific afferent fibers which densely cuff the central portion of the apical shafts of fifth layer pyramids in an almost continuous collar of asymmetric, round vesicle-containing synaptic profiles. Although this location is usually several hundred microns from the somal trigger zone, the powerful and well-localized zone of depolarizing postsynaptic potentials produced here is unquestionably strongly reflected at the spike trigger zone. In contrast, the low

OF PATTERN AND PLACE. IN DENDRITES

23

density, linear type of synaptic continuum, along most of the shaft and apical arches produced by nonspecific afferents, is more likely to exert a long-term, low-grade tonus upon the trigger zone. Such an input may function less as a drive mechanism than as a bias adjust, continuously readjusting the l e ~ ofl response sensitivity of cortical pyramids to the moment-to-moment fluctuations in the responsiveness or alertness of the organism. Modulation of still greater complexity may be conceived as resulting from the other systems already cited. IV. Conclusions

We have shown in several neuropil fields of increasing complexity that there is some degree of precision in the ordering of the synaptic scale of terminating afferents. In some cases, such as the large lamina IV cells of the spinal cord, specific dendrites appear allocated to specific inputs. In much more complex fields, such as those of neocortex, there appears to be a hierarchy of preferential sites, each afferent system represented in maximal density of terminals along one dendritic segment. Formerly we would have believed that, upon this hierarchy of place, there could be superimposed a hierarchy of values with a single slope, i.e., the closer to the soma, the more effective the postsynaptic potentials generated along that segment of dendrite in modulating the spikeinitiating mechanism of the soma1 trigger zone. Such ideas deserve reevaluation in the light of evidence that there may be more than one trigger zone on a neuron and that, in some cases at least, spikes may develop and travel along portions of dendrite toward soma and axon hillock (Green and Petsche, 1961; Spencer and Kandel, 1961; Purpura et aE., 1966; and others). Under these conditions, distance from the cell body may no longer be the sole criterion of importance. Instead, the position of the terminals in relation to dendritic trigger zones in various positions throughout the dendrite field become of increasing significance, and a new responsibility devolves upon the investigator to devise recording techniques of sufficient resolution to identify such subsidiary foci of spike-initiating activity. No short review is likely to do justice to the extensive literature on dendrites, their manifold patterns, the range of their physiological activity, and the increasingly venturesome models proposed in explanation of their roles in the processing of information. As in any rapidly progressing field, experimental data demand verification, and hypotheses must face the challenge of time. In limiting ourselves to an examination of the morphology of dendritic domains, and the arrangement of various presynaptic systems within them, we have sought to present data which can reasonably be expected to remain valid and of value to theories of

24

MADGE E. SCHEBEL AND ARNOLD B. SCHELBEL

dendrite function, whatever the ultimate models propose. Without making “form follows function” an article of blind faith, we are powerfully persuaded that this now-trivial phrase remains of far-from-trivial import. If we are to interpret successfully the functional displays culled from studies of dendrites in action, then field shape and orientation as well as synaptic distribution must provide the background for such assays. We have chosen examples where pattern and form are clearly etched. Other equally compelling examples might have been selected, or added to this roster. The interests and prejudices of the writers determine the specifics of selection and exclusion. However, we would like to believe that the lessons which they illustrate are sufficiently general and compelling to transcend the specific examples, whether we have chosen wisely or ill. REFERENCES Aitken, J. T., and Bridger, J. F. (1961). 3. Anat. 95, 38. Andersen, P. (1965). In “The Brain and Conscious Experience” (J. C. Eccles, ed.), p. 89. Springer Verlag, New York. Andersen, P., and Eccles, J. C. (1965). Symp. Biol. Hung. 5, 219. Bishop, G. H., and Clare, M. H. (1952). J. Neurophysiol. 15, 201. Bishop, G. H., and Clare, M. H. (1953). J. Neurophyviol. 16, 1. Bok, S. T. (1959). “Histonomy of the Cerebral Cortex.” Elsevier, Amsterdam. Burke, R. E. (1967). 1. Neurophysiol. 30, 1072. Cajal, S. R. (1894). “Die Retina der Wirbelthiere.” Bergmann, Wiesbaden. Cajal, S. R. (1909). “Histologie du Systhme Nerveux de 1’Homme et des Verthbrhs,” Vol. 1. Maloine, Paris. Cajal, S . R. (1911). “Histologie du Systhme Nerveux de l’Homme et des VerthbrBs,” Vol. 2. Maloine, Paris. Colonnier, M. (1964). J . Anat. 98, 47. Colonnier, M. (1965). In ‘The Brain and Conscious Experience” (J. C. Eccles, ed.), p. 1. Springer Verlag, New York. Colonnier, M. ( 1967). Personal communication. Dale, H. H. (1935). PTOC.Roy. Soc. Med. 28, 319. Eccles, J. C. (1964). “The Physiology of Synapses.” Springer, Berlin. Eccles, J. C., Llinb, R., andISasaki, K. ( 1966a). J. Physiol. (London) 182, 268. Eccles, J. C., Llinb, R., and Sasaki, K. (1966b). Exptl. Brain Res. 1, 82. Eccles, J. C., Ito, M., and Szentagothai, J. (1967). “The Cerebellum as a Neuronal Machine.” Springer Verlag, New York. Fox, C. A., and Barnard, J. W. (1957). 3. Anat. 91, 299. Fox, C. A., Hillman, D. E., Siegesmund, K. A., and Dutta, C. R. (1967). Progr. Brain Res. 25, 174. Gerlach, 0. (1871). Von dem Ruckenmark in Strickers Handbuch. Quoted in Cajal (1909), p. 54. Gesell, R., Brassfield, C. R., and Lillie, R. H. (1954). J. Comp. Neurol. 101, 331. Globus, A,, and Scheibel, A. B. (1967a). Erptl. Neurol. 18, 116. Globus, A.,and Scheibel, A. B. (1967b). Science 156, 1127. Globus, A., and Scheibel, A. B. ( 1 9 6 7 ~ )J.. Comp. Neurol. 131, 155.

OF PATlXRN AND PLACE IN DENDRITES

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Goldberg, J. M., and Brown, P. B. (1969). j . Neurophysiol. 32, 613. Golgi, C. ( 1903). “Opera Omnia,” Vols. 1 and 2. Ulrico Hoepli, Milan. Gray, E. G. (1959). J. Anat. 93, 420. Gray, E. G. ( 1964). In “Electron Microscopic Anatomy” ( S . M. Kurtz, ed.), p. 369. Academic Press, New York. Green, J. D., and Petsche, H. (1961). EZectroencephalog. Clin. Neurophysiol. 13, 868. Grundfest, H. (1957). Physiol. Reu. 37, 337. Hagbarth, K. E., and Kerr, D. I. B. (1954). 3. Neurophysiol. 17, 295. Hamlyn, L. H. (1963). I. Anat. 97, 189. Hild, W., and Tasaki, 1. (1962). J. Neurophysiol. 25, 277. Hubel, D. H., and Wiesel, T. N. ( 1959). 3. Physiol. (London) 148, 574. Kameda, K., Nagel, R., and Brooks, V. B. (1969). J. Neurophysiol. 32, 540. Kandel, E. R., Frazier, W. T., and Coggeshall, R. E. (1967). Science 155, 346. Leontovitch, T. A., and Zhukova, G. P. (1963). J. Comp. Neurol. 121,347. Lettvin, J. Y., Maturana, H. R., Pitts, W. H., and McCulloch, W. S. ( 1961). In “Sensory Communications” ( W. Rosenblith, ed.), p. 757. MIT Press and Wiley, New York. Lorente de N6, R. (1934). I. Pyschol. NIWO~. 46, 113. Lorente de N6, R. ( 1939). 1. Neurophysiol. 2, 402. Lorente de N6, R. (1943). Cited in Fulton, J. F. (1943). “Physiology of the Nervous System,” p. 274. Oxford Med. Publ., Oxford. Lorente de N6, R. (1947). 3. Celluhr Comp. Physiol. 29, 207. Magni, F., and Willis, W. D. ( 1963). Arch. Itul. B i d . 101, 667. Mannen, H. ( 1966). In “Correlative Neurosciences. Part A: Fundamental Mechanisms’’ (T. Tokizane and J. P. Schadb, eds.), p. 131. Elsevier, Amsterdam. Marin-Padilla, M. (1969). Bruin Res. 14, 633. Moruzzi, G., and Magoun, H. W. ( 1949). Electroencephulog. Clin. Neurophysiol. 1, 297. Mountcastle, V. B. ( 1961). In “Sensory Communication” (W. A. Rosenblith, ed.), p. 403. MIT Press and Wiley, New York. Nauta, W. J. H., and Whitlock, D. G. (1954). In “Brain Mechanisms and Consciousness” (J. F. Delafresnaye, ed.), p. 81. Thomas, Springfield, Illinois. Nelson, P. G. (1966). J. Neurophysiol. 29, 275. Nelson, P. G., and Frank, K. (1964). 3. Neurophysiol. 27, 913. Olszewski, J. (1954). In “Brain Mechanisms and Consciousness” (J. F. Delafresnaye, ed.), p. 54. Thomas, Springfield, Illinois. Purpura, D. P., and Cohen, B. (1962). J. Neurophysiol. 25, 621. Purpura, D. P., McMurtry, J. G., Leonard, C. F., and Malliani, A. (1966). I. Neurophysiol. 29, 954. Rall, W. (1962). Ann. N.Y. Acad. Sci. 96, 1071. Rall, W. (1964). In “Neural Theory and Modeling” (R. F. Reiss, ed.), p. 73. Stanford Univ. Press, Stanford, California. Rall, W. (1967). J. Neurophysiol. 30, 1138. Rall, W., Burke, R. E., Smith, G. T., Nelson, P. G., and Frank, K. (1967). J. Neurophysiol. 30, 1169. Ram6n-Molinar, E., and Nauta, W. J. H. (1966). J. Comp. Neurol. 126, 311. Rexed, B. (1952). 1. Comp. Neurol. 96, 415. Scheibel, M. E., and Scheibel, A. B. (1955). J. Comp. Neurol. 10%77.

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MADGE E. SCHEUBEL AND ARNOLD B. SCHEIBEL

Scheibel, M. E., and Scheibel, A. B. (1958a). Electroencephalog. Clin. Neurophysiol. Suppl. 10, 42. Scheibel, M. E., and Scheibel, A. B. (1958b). In “Reticular Formation of the Brain” (H. Jasper et al., eds.), p. 31. Little, Brown, Boston, Massachusetts. Scheibel, M. E., and Scheibel, A. B. (1962). In “Interhemispheric Relations and Cerebral Dominance” (V. B. Mountcastle, ed.), p. 26. Johns Hopkins Press, Baltimore, Maryland. Scheibel, M. E., and Scheibel, A. B. (1963). Ekctroencephalog. Clin. Neurophysiol. Suppl. 24, 235. Scheibel, M. E., and Scheibel, A. B. (1966a). In “The Thalamus” (D. P. Purpura and M. Yahr, eds.), p. 13. Columbia Univ. Press, New York. Scheibel, M. E., and Scheibel, A. B. (196613). Brain Res. 2, 333. Scheibel, M. E., and Scheibel, A. B. (1967). Brain Res. 6, 60. Scheibel, M. E., and Scheibel, A. B. (1968a). Brain Res. 9, 32. Scheibel, M. E., and Scheibel, A. B. (1968b). In “The Neurosciences” ( G . Quarton, T. Melnechuk, and F. Schmitt, eds.), p. 577. Rockefeller Univ. Press, New York. Scheibel, M. E., and Scheibel, A. B. (1969). Brain Res. 13, 417. Scheibel, M. E., Scheibel, A., Mollica, A., and Moruzzi, G. (1955). J. Neurophysiol. 18, 309. Scheibel, M. E., Uchiyama, K., Uchiyama, T., and Scheibel, A. B. (1961). Unpublished observations. Sholl, D. A. (1956). “The Organization of the Cerebral Cortex.” Methuen, London. Skinner, I. E., and Lindsley, D. B. (1967). Bruin Res. 6, 95. Smith, T. G., Wuerker, R. B., and Frank, K. (1967). 1. Neurophydol. 30, 1072. Spencer, W. A., and Kandel, E. R. ( 1961). J. Neurophysiol. 24, 272. Szentagothai, J. (1964). J. Comp. Neurol. 122, 219. Tauc, L., and Gerschenfeld, H. M. (1961). Nature 192, 366. Uchizono, K. ( 1965). Nature 207, 642. Walberg, F. ( 1963). Exptl. Neurol. 8, 112. Wall, P. D. (1967). J. Physiol. (London) 188, 403.

THE FINE STRUCTURAL LOCALIZATION OF BIOGENIC MONOAMINES IN NERVOUS TISSUE By Floyd E. Bloom laboratory of Neuropharmacology, Division of Special Mental Health Research, National Institute of Mental Health, Saint Elizobeths Hospital, Washingfon, D. C.

I. Introduction . . . . . . . . . . . . 11. Model Tissue Experiments and the “Ideal Localizing Paradigm” . A. Criteria for the Cytochemical Localization of Monoamines . . B. Reactivity of Biogenic Amines with Electron Microscope Fixatives . . . . . . . . . and Stains in Vitro . C. Model Experiments in Vivo . . . . . . . . D. Constraints on the Use of Autoradiography . . . . . 111. The Binding of Biogenic Monoamines to Cellular Organelles . . A. Background . . . . . . . . . . . . B. Monoamine Binding Reactions to Tissue Components . . . IV. Localization of Monoamines in Nervous Tissue . . . . . A. Peripheral Sympathetic Nerve Terminals . . . . . . B. Central Nerve Terminals . . . . . . . . . C. Monoamine Cell Bodies and Nonterminal Axons . . . . V. Conclusions . . . . . . . . . . . . References . . . . . . . . . . . .

27 28 28 29 35 38 39 39

40 41 41 53 59 61 62

I. Introduction

The recent history of biological electron microscopy (see Barrnett and Tice, 1963; Zobel and Beer, 1 9 6 ) indicates the increasing efforts which have been made to develop techniques imparting chemical as well as morphological information. Despite these efforts, electron cytochemical reactions are at best able to identify a select few nonenzymatic cellular components, most of which fall into the category of macromolecules such as DNA, glycogen, glycoproteins, lipids, and certain partially characterized charged proteins (Beer, 1965; Bloom and Barrnett, 1970). In neuropharmacological research, special attention has been devoted to the problem of high resolution cytochemical methods for the identification of synaptic transmitter substances in intact nervous tissue, as this essential criterion must be satisfied in order to identify a substrate as the likely transmitter for particular defined synaptic contacts (see McLennan, 1963; Paton, 1958; Bloom and Giarman, 1968a,b; Bloom, 1969). In t h i s review, the currently available electron cytochemical 27

28

FLOYD E. BLOOM

methods for the biogenic monoamines, norepinephrine ( NE ), dopamine (DA), and serotonin (5-HT) will be critically analyzed. The electron microscopic details of monoamine-storing nervous tissues has already been reviewed many times in the recent past (Grillo, 1966; Bloom and Aghajanian, 1968a; Bloom and Giarman, 1968a; Hokfelt, 1968a,b; Tranzer et al., 1969; Jaim-Etcheverry and Zieher, 1 9 6 9 ~ ) .Particular emphasis will now be placed on the state of knowledge of the cytochemical reactions involved in the localizations. Hopefully this will provide an interpretive base for the data at hand and stimulate further experimentation. II. Model Tissue Experiments and the "Ideal localizing Paradigm"

A. CRITERIAFOR THE CYTOCHEMICAL LOCALIZATION OF MONOAMINES In order to establish the identity and specificity of a cytochemical reaction designed to localize a substance as small as NE, DA, or 5-HT, the following criteria may be of value: 1. The precipitate or electron stain can be shown to selectively react with the desired substance in vitro. The relevant chemical reactions on this area are reviewed below (Section 11, B and C ) . 2. The distribution of the reactive tissue sites agrees with light microscopic histochemical and cellular biochemical analyses of the substance. Almost all quantitative ultrastructural evaluations of staining methods for monoamines are predicated upon the fluorescence histochemical data (see Van Orden et al., 1966; Bloom and Aghajanian, 1968a; Hokfelt, 1968a,b). The data on nerve ending particles, synaptic vesicle fraction analyses, and fluorescence histochemistry of monoamines are discussed separately (Section 111). 3. The amount of the reactive material should fluctuate in proportion to the amount of amine in the tissue when pharmacological depletion studies, loading studies, or catabolic inhibition cause fluctuations in the biochemically measurable levels. Such studies have formed the bulk of the data in which electron microscopic analyses of peripheral and central nervous tissues have been studied after pharmacological manipulations. This data will be discussed in detail (Section V ) . 4. The tissue elements reacting to the staining method should be confirmed by autoradiographic studies in which radioactive amine is physiologically introduced into the tissue pool of monoamine, and then serves as a separate marker for the sites binding the substance. This criterion has several subrequirements of its own, and is discussed separately ( Section 11, D ) . 5. Destruction of the nerve pathways which give rise to the mono-

LOCALIZATION O F MONOAMINES

29

amine-containing fibers should remove all the axons which gave a positive staining reaction. This criterion would appear to be relatively easily established for the peripheral nervous system, where the classical neuroanatomy is well established. However, in actual practice, it has been applied only irregularly there and quite rarely in the central nervous system (Section V ) . 6. The staining reaction should be confirmed by auxiliary cytochemical methods for the demonstration of synthetic or catabolic enzymes relevant to the monoamines. This last criterion is mere wishful thinking at the present time, since none of the synthetic enzymes, and only monoamine oxidase (Boadle and Bloom, 1969) of the catabolic enzymes, appear to be approachable from this standpoint, even by light microscopy. However, since enzymes are dynamic macromolecules, they may be localizable either by formation of an electron-opaque catabolite as is the standard approach for such enzymes as adenosine triphosphatases (see Barrnett and Tice, 1963) or cholinesterases (Koelle, 1969), or by electron microscope immunocytochemistry ( Sternberger, 1967).

B. REACTIVITY OF BIOGENIC AMINESWITH ELECTRON MICROSCOPE FIXATIVES AND STAINSin Vitro An extensive armamentarium of light microscopic reactions are available to identify the monoamines histochemically in adrenal medullary chromaffin cells, intestinal enterochromaffin cells, and mast cells. These reactions were developed empirically and have only become understood as the identification of the particular monoamines involved became clarified. However, inasmuch as the majority of these reactions depend upon the development of specific colored end-products, they did not appear directly applicable to the electron microscope. As applied to nervous tissues, prior to 1965, the only available cytochemical reactions on the nature of synaptic vesicle contents were based upon the peculiar variations in electron opacity of the vesicles after using OsO, as a combined fixative and electron stain (de Robertis and Pellegrino de Iraldi, 1961a,b; Grill0 and Palay, 1962; Richardson, 1958, 1962, 1963, 1964). The basis for accepting an osmiophilic subcellular precipitate as an indicator of monoamine content stemmed almost entirely from the untested assumption that the monoamines were “known to be potent reducing substances” (de Robertis and Pellegrino de Iraldi, 1961a,b). While this statement has indeed been borne out by subsequent independent experimentation, we shall see that interpretation of the reactions and their applicability to different types of amine-storing tissues is still quite an open field, eagerly awaiting new approaches. If tissue monoamine stores can be approximated by a solution of the

TABLE I REACTION OF MONOAMINES WITH STAININQ FIXATIVES ~~~

~~~~

~~~

~~

~

~~~~

~~

~~

~

~~

Test for reaction

Amine Norepinephrine Epinephrine Dopamine Serotonin 5-OH dopaminei 6-OH dopamine a-Methyl norepinephrine Metaraminol

0 ~ 0 4 "

+ + +

+ 0 0 0

Formaldehydeb

Glutaraldehydec

0 0 0 0

+ + +

+

KMn04d

++ ++ ++ ++ ++ ++ ++ ++

Glutal. K2Cr20p

Glutal. Agf

++++ ++

Van Orden et aZ. (1966). Hopsu and Makinnen (1966). Wood and Barrnett (1964), Conpland et al. (1964), Solcia et al. (1969). Hokfelt and Jonsson (1968). Wood and Barrnett (1964), Jaim-Etcheverry and Zieher (1968a, 1969~). f Tramezzani et al. (1964), Cannata et al. (1968). Solcia et al. (1969). * Coupland et al. (1964), Machado (1967), Tranzer and Thoenen (1967), Tranzer et al. (1969). a Tranzer and Thoenen (1967). a

++ + + +

+

+

Glutal. ninhydrino 0

0

+

Glutal. OsOah

+ + +

?

3

m

P0 0

z

LOCALIZATION OF MONOAMINES

31

amine, then test tube reactions may help us understand the reactivity of these substances with the commonly used fixatives (Table I ) , These tests indicate that a very large spectrum of naturally occurring and synthetic catecholamines and indolealkylamines will “reduce” Oso, and KMnO, to form grossly visible precipitates. However, from the reported data, it is uncertain whether such precipitates indicate insoluble polymers of the oxidized amines, or of the reduced metallic oxidants, or of some coordination complex between the two. Since it is the electron opacity of the precipitate which is most crucial, this information is obviously needed to interpret the qualitative chemical analyses.

1. Reactions of OsO, When Oso, is used as a fixative, it is ordinarily maintained in buffered solutions at pH 7-7.5, and as a 1-2%solution, for periods of 1 4 hr. Two questions essential to the problem at hand are retention of the monoamine in its tissue site during the fixation process with oso, and the subsequent oxidation-reduction reaction indicated by the test tube experiments. Because of its slow penetration, oso, is commonly used in immersion fixation at temperatures in the 3’5°C range, by which it is hoped to slow autolysis of cellular organelles. However, the cooler temperature is also likely to influence the reactivity of the 0 s and of the available reactive groups in the tissue, as well as the relative extraction of reactive tissue components. The reactions undergone by oso, were originally thought to be reduction of the 0 s to “osmium black,” a finely granular form of metallic osmium (Hanker et al., 1967). Despite the large number of investigations into the nature of the reaction of osmium in vitro and with tissue components (see Adams et al., 1967), it is still not certain which of the many reactions contribute to the electron opacity of the tissues and which to the reduction of osmium, The most recent data suggest that oso, is able to form several types of coordination compounds with reactive tissue components (Hanker et al., 1964,1966,1967; Seligman et al., 1966; Adams et al., 1967; Litman and Barmett, 1969), some of which may continue to form polymeric compounds leading to opaque precipitates within the tissue structure. In addition to double bonds, oso4 is reduced by mercaptyl and thiocarbamyl groups (Hanker et al., 1964, 1967), of which only the former occtws in animals. Proteins and polysaccharides are said to react poorly with Os, although proteins are broken up by it and “leeched” from the tissues during prolonged exposures ( Hake, 1965). This latter property is likely to be the cause of some of the high electron contrast of membranes of Os0,-fixed tissues (Pease, 1964).

32

FLOYD E. BLOOM

2. Other Oxidizing Substances

The only other metallo-oxidants of proven or suggested value as fine structural fixatives or electron strains are KMnO, (and other alkali metal salts of Mn0,- such as Na or Li) and K2Cr20,.Of the two, Mn0,has the somewhat greater redox potential (1.51, as compared to 1.33for Oso,). In both cases, the redox potential is increased by use of acidic unbuffered solutions (Cotton and Wilkinson, 1966), favoring the formation of the dioxide forms of each metal ( CrO, and MnOz). The tissue groups reacting with these substances are not well characterized aside from being “reducing substances” ( see Hake, 1965; Pearse, 1969) , although tissue proteins are thought to be particularly broken down by Mn0,- (Lenard and Singer, 1968). MnOz occurs in nature as a grayblack precipitate which would be expected to be somewhat more electron opaque than chromium oxide, although the latter has been found in tissue (Pearse, 1969) and filter residues (Arnold and Hager, 1968a,b), while the former has not. Since MnO, is readily attacked by reducing solutions, it should be possible to remove its contribution to electron opacity by this means and thus lead to an evaluation of the opacity of pigments it may have formed from the monoamines. As to the preservation of fine structure, Mn0,- salts in concentrations of 343% (Richardson, 1966; Hokfelt, 1967b,c, 1968a,b; Grillo, personal communication) can yield acceptable results on small blocks of tissue immersed for 2-4 hr at 0°C. The “fixation” with Crz072-is poor unless previously well fixed with aldehyde (Wood and Bannett, 1964; Tranzer and Snipes, 1968). 3. Metallic Oxidation after Primary Exposure to Aldehyde Fixatives a. Reactions with Formaldehyde in Solution. Although the standard choice for most optical microscopists, formalin was initially bypassed as a fixative for electron microscopy (see Pease, 1M4). Nevertheless, as demonstrated in freezeldry fluorescence histochemistry, the condensation of DA, NE (reactions 1 and 2), and 5-HT (reaction 3) with formaldehyde vapor rapidly produces an insoluble form of the amine (see Corrodi and Jonsson, 1968) which should be available for electron staining. On

33


the other hand, with respect to test tube precipitate formations, there is generally little to be seen between the NE, DAY5-HT, and formaldehyde in solution (Eranko, 1967; Hopsu and Makinnen, 1966; Laties et al., 1967). Provided a condensation of the Pictet-Spengler type did occur (see Corrodi and Jonsson, 1968), the resultant heterocyclic compounds ( quinolines and carbolines ) could effectively reduce metallic oxidants, although the exact nature and extent of the reaction remains to be studied. b. Reaction with Glutaraldehyde. The dialdehyde of glutaxic acid was first reported to be a superlative fixative for the fine structure by Sabatini et al. (1963). When solutions of this fixative are mixed with monoamines in the test tube, obvious yellowish precipitates can be seen to form with each of the 3 monoamines which are primary amines, but not with epinephrine, which is a secondary amine (Table I ) . The form of this reaction is believed to be (4)

A

B

1

As a dialdehyde, polymer formation by this reaction might also proceed (see Coupland and Hopwood, 1966) and would account for the majority of the yellow precipitate mentioned above. However, each NE molecule has only one reactive site, and each glutaraldehyde only two; therefore, if the ratio of 1:1 in the final precipitate is valid ( Coupland and Hopwood, lW),the exact structure of such a polymer is difficult to understand. The reaction of the monoamines with glutaraldehyde does not alter the reducing power of these substances (provided that excess aldehyde is removed) nor does it alter the reactivity of the pyrrolic indole of 5-HT ( Solcia et al., 1969), suggesting that the condensation must not close the terminal side chain into a ring form as occurs with formaldehyde, Hence, we would expect (no direct published data exist) that the monoamine-glutaraldehyde polymer should also be capable of reacting with the oxidants (reactions 4,B, C, D ) , although the relative amount of coordination compound formation which would occur must also be

34

FLOYD E. BLOOM

evaluated. Clearly the complex of aldehyde and monoamine can reduce alkaline silver salts to the metal (Tramezzani et al., 1964; Cannata et aZ., 1968; see Fig. 2).

4. Oxidation of the Monoamines to Insoluble Pigments A great body of rather precise organic chemical data and histochemical observations is available on the formation of pigments from catecholamines, because this is the main route for the formation of melanins (see Pearse, 1961) and aminochromes (see Heacock, 1965). In the case of the naturally occurring catecholamines, the reaction is believed to be ( 5 )

The aminochromes are of further interest in several regards. First, they represent the natural form of catechol-polymers such as melanin, which are known to be electron opaque in electron microscopic investigations of melanophores (Riley and Fortner, 1963). This indicates that equivalently electron-opaque products might be expected to occur from monoamines if the proper cytochemical production method were available. Second, the precise chemical changes during the oxidative cyclization reaction (see reactions 5, A-F) have recently been worked out (Harrison et al., 1968). These data indicate the reaction to be p H dependent, increasing in direct relation to H+ concentration, and that the hydrogens removed during the oxidation are most likely to have come from the two phenolic groups and the amino group. Furthermore, the reaction was noticeably catalyzed by Zn". Of pertinence to the interpretation of studies on the tissue retention of dl-73H-norepinephrine radioactivity with various fixatives (see below), no hydrogens were removed from the 7 ethylamine carbon during the oxidation. A naturally occurring plasma transhydrolase which would remove this side chain H+has been reported (reaction 5, G; Pichler et al., 1968). Similar information on the nature of the 5-HT oxidation would be very useful.


35

C. MODELEXPERIMENTS in Vivo 1. Catecholumine Test Tissues Regardless of the success of in vitro tests, only with application to tissues can the relative sensitivity of the method be evaluated. Thus, with the first attempts at specific localizations of catecholamines by electron microscopy, the tissue tested was the adrenal medullary cell, in which the high concentrations of catecholamines, the Iight microscopic success of the chromaffin reaction, and the rather extensive data on isolated chromaffin granules, made the chances for cytochemical localization of NE likely. Wood and Barrnett (1964) were, in fact, able to show that adrenal medulla fixed in glutaraldehyde and exposed to acidic solutions of K,Cr,O, resulted in selective opacification of the chromaffin granules of NE-containing cells, but not those of epinephrine cells. The latter remained unstained until the pH approached neutrality. Subsequently, Coupland et a2. (1964; Coupland and Hopwood, 1966) showed the same cytochemical separation between NE cells and epinephrine cells occurred if neutral Oso, (Fig. 1) were substituted for K,CrzO,. The latter reaction appears superficially to be less selective, since the Oso, also makes multiple organelles and membranes electron opaque, whereas the former method results in much greater opacification of chromaffin granules than other cytoplasmic structures. In both cases the reaction seems to be based upon the selective retention of the primary amine, NE, in the tissue by the glutaraldehyde, while epinephrine is lost either in the acidic rinses or during the exposure to osmium. In the reaction system proposed by Coupland, it should be noted that the “negative” reaction given by the epinephrine-containing granules is not completely negative, but that the granules exhibit a fibrillar, moderately electron-opaque, central matrix. The condensation of glutaraldehyde with NE in the granule also appears to account for the positive staining of the slightly opaque chromaffin granules exposed to ammoniacal silver after glutaraldehyde fixation (Tramezzani et al., 1964; Cannata et al., 1%8; Fig. 2). On the other hand, adrenal medullary tissue fixed in KMnO, shows no electron opacity within the chromaffin granules of either cell type (Duncan and Yates, 1967), although Hokfelt has obtained some precipitate in the NE cells of rat (personal communication).

2. lndolealkylamine Test Tissues While the adrenal medulla provides an effective test system for the localization of NE, there appeared to be no equivalent system for 5HT,

36

FLOYD E. BLOOM

FIG.1. Adrenal medulla of normal rat, after fixation with glutaraldehyde/OsOa.


37

FIG. 2. Norepinephrine-containing adrenal medullary cell from cat showing positive glutaraldehyde/silver reaction over chromaffin granules. All other cytoplasmic organelles, nucleus, and epinephrine-containing cells remain unreactive X 18,000. Contributed by Drs. S. R. Chiocchio and J. H. Tramezzani (see Tramezzani et al., 1964; Cannata et al., 1968).

until Tranzer et al. ( 1966) reported that rabbit platelets (which normally contain about 350 pg of 5-HT per 1O’O platelets) fixed in glutaraldehyde and exposed to OsO, exhibit unique electron-opaque organelles which can only be seen after this type of preparation. This organelle appears as a membrane-bounded vacuole in which the osmium exposure after glutaraldehyde produces an extremely electron-opaque deposit. The “osmiophilic” material in the vacuole is decreased if the platelets are exposed to reserpine to block 5-HT storage, and it can be restored if the platelets are incubated in rather high concentrations of 5-HT ( 1 mg/ml) . The same type of granule can be produced in guinea pig platelets incusequence (see Coupland et al., 1964). Very dense electron-opaque deposits are present in chromaffin granules of norepinephrine cell at top, while those of epinephrine-containing cell at bottom are much less electron opaque. The cells are separated by an intercellular space in which molls exhibiting large granular vesicles (arrows) may also be seen X 24,000. Insets show each type of chromaffin granule at higher power. Insets X 108,000 ( Bloom, unpublished).

38

FLOYD E. BLOOM

bated in 5-HT and can be seen in human platelets from patients having abnormally high platelet 5-HT levels (Boullin et al., 1969; Hattori, 1967). Platelets have also served as a model system for a triple fixation sequence method for the localization of 5-HT (Jaim-Etcheverry and Zieher, 1968a,c, 1969a). The reaction of Wood and Barmett (1964) for NE is blocked by initial fixation in liquid formaldehyde, after which glutaraldehyde exposure followed by K,Cr,O, results in selective opacification of the platelet granules (Tranzer et al., 1966), and of similarly appearing granular material in cells of the endocrine pancreas and thyroid (JaimEtcheverry and Zieher, 1968b,c). In each case, the exclusive independent criterion by which the reactive material is identified as 5-HT is based upon the in vitro reaction and upon the ability of reserpine to deplete the reactive material as it depletes the 5-HT. ON THE USEOF AUTORADIOGRAPHY D. CONSTRAINTS

Although electron microscopic autoradiography is often regarded as “high resolution,” with all commercially available emulsions of halide crystal sizes between 50 and 300 mp, the true resolution can be no greater than 80-150 mp, (Saltpeter et al., 1969). Therefore, autoradiography under the best of circumstances would only be able to distinguish certain types of axons and structures the size of chromaffin granules, while completely unable to distinguish among adjacent synaptic vesicles. Despite these disadvantages in resolution, autoradiography can still be a valuable adjunct to other cytochemical methods in localizing tissue chemical components, provided certain constraints are met. In the general circumstance in which the radioactive material is taken up by the cells and incorporated into a macromolecule, one needs only to ascertain that the amount of material injected as a tracer label did not alter the cellular activity of the cells, and that the radioactivity which remains in the tissue is not translocated from the sites labeled before fixation (see Stumpf and Roth, 1968). In the case of neurotransmitters, such as the monoamines in the case at hand, it must be ascertained that the amount of monoamine injected does not overload the relatively specific axonal processes for intra-axonal accumulation (see Iversen, 1967; Fuxe and Ungerstedt, l W ) , that radioactive metabolites of the injected substance are retained in the tissues to a substantially lower extent, and that the main, if not exclusive source of tissue radioactivity after fixation, is in fact due to the substance to be localized. Thus, Taxi and Droz (1969) have shown that only the catecholamine is retained by the peripheral sympathetic tissues after their preparative procedures. Similarly, Aghajanian and I showed that there was minimal retention of 3H-NE radioactivity after acute treatment of the central nervous system with


39

reserpine, suggesting that the unbound metabolites of the injected monoamine do not result in falsely positive autoradiographic localization ( 196713) I l l . The Binding of Biogenic Monoamines to Cellular Organelles

A. BACKGROUND By virtue of the tremendous amount of data collected by biochemical neuropharmacologists ( Glowinski et al., 1965, 1966; de Robertis, 1966; Whittaker, 1966; Pellegrino de Iraldi et al., 1968), cytochemists have a great deal of background with which to approach the problem of fine structural localization. The subcelhlar distribution of tissue norepinephrine was first studied on homogenates of the adrenal medulla (Blaschko and Welch, 1953) and splenic nerve (von Euler and Hillarp, 1956). In these tissues and in several sympathetic organs analyzed later (see Stjarne, 1966; Potter, 1967; Iversen, 1967), the monoamine content was richest in the microsomal fragments containing particulate structures on the order of 30-200 mp in size by electron microscopy. This binding of monoamines to particulate elements in honiogenates has thereafter been referred to trivially as the “storage granule” or granular amine fraction, in reference to the chromaffin granules. When subcellular fractionation studies were extended to the central nervous system (see de Robertis, 1966; Whittaker, 1966), the catecholamines could be found in those fractions of the homogenate which contained pinched-off nerve endings and in which synaptic vesicles could be seen. The greatest enhancement of specific concentration of acetylcholine (ACh) occurred when rather pure collections of the synaptic vesicles were obtained from ACh-rich nerve ending fractions. Highly purified vesicle fractions containing monoamines have not been prepared (see Maas and Colburn, 1965), due in part to lack of methods to retain the amines within the particles during the separation procedures ( Michaelson and Whittaker, 1963; Michaelson, 1967). Nevertheless, it is widely considered that the major portion of the particulate fraction monoamine is contained within synaptic vesicles, and that those portions of the axon in which vesicles are most heavily concentrated are also revealed by fluorescence histochemistry at the light microscopic level of resolution (see Dahlstrom and Fuxe, 1965; Fuxe, 1965a; Fuxe, 1965b). Recently, fluorescence histochemical data have been interpreted to indicate that this method may actually be most successful in demonstrating the monoamine which lies outside the presumptive storage vesicles but still within the axoplasm (Van Orden et al., 1969). While the above data and numerous indirect supportive observations relate monoamine storage to subcellular particles in either homogenates

40

FLOYD E. BLOOM

or tissue elements observed by light microscopy (see Iversen, 1967), electron microscopic resolution is required to relate the particulate storage fraction to specific nerve terminals and to specific organelles within the nerve terminals. REACTIONS TO TISSUE COMPONENTS B. MONOAMINEBINDING Before proceeding to the data contributing directly to this solution, let us examine the possible modes of binding of tissue monoamines to the subcellular organelles. Most of this type data have arisen from two sources: the study of adrenal chromaffin granules and of the similar but clearly not identical “granules” isolated from bovine splenic nerves. The chroma5n granules contain, in addition to the monoamines, large amounts of adenosine triphosphate ( ATP) as well as proteins, lipids, and possibly ribonucleic acids ( Schumann, 1966). During physiological release of the adrenal catecholamines, the ATP and at least two granule specific proteins are also released (Douglas, 1968; Kirschner, 1969). In both chromaffin granules and splenic nerve granules, the ratios of the monoamine to ATP are usually quite constant in a ratio of 1 ATP to 4 NE. This has lead to the speculation that the catecholamine may be stored in the granule by a form of coordination complex, in which an endogenous metal, such as Cu, may act to form a ternary complex (Maas and Colburn, 1965; Colburn and Maas, 1965). Such conclusions have been tested by simulation of the systems chemically in uitro and demonstrating monoamine binding. Present data are unclear regarding the amounts of protein that can be found in isolated brain synaptic vesicles thought to store norepinephrine (Colburn and Maas, 1965; Potter, 1967), and the contribution of protein to the formation or stabilization of the ternary complexes thus cannot yet be established. Furthermore, based on in vitro data, phospholipids such as phosphatidylinositol and phosphatidylserine can also be shown to exhibit binding affinity for catecholamines (Formby, 1968). Since these phospholipids also occur within the synaptic vesicles, they may provide some physiological binding as well. However, the main body of evidence on subcellular binding chemistry suggest that the catecholamine is held inside the vesicle by the formation of a complex between the metal (Ca++and/or Cu++) and the ring (phenolic) hydroxyls ( Maas and Colburn, 1965). This is of particular importance when we consider the variations between the effects of fixative on reactivity with monoamines in vitro and in oivo, and the differences between reactivity of different tissues to the same fixative. Clearly if it is the phenolic hydroxyls which must be oxidized to form the metallic or organometallic electron-opaque product, the availability


41

of these groups must be established by local tissue conditions and fixative-ligand interactions. IV. localization of Monoamines in Nervous Tissue

A. PERIPHERAL SYMPATHETIC NERVETERMINALS The possibility that sympathetic nerve terminals could be morphologically distinguished from other types of nerve terminals arose when multiple types of synaptic vesicles were observed in the nerves to the pineal (de Robertis and Pellegrino de Iraldi, 1961a,b) and to intestine (Grillo and Palay, 1962). The vesicles newly described were distinguishable from the routinely observed spherical electron-lucent synaptic vesicles on the basis of size and electron opacity of the contents after fixation with Os04. 1. Small Granular Vesicles (SGV)

These vesicles measure 40-60 mp and exhibit variable degrees of internal electron opacity, dependent upon the type of fixation and the tissue (Figs. 3-7). The SGV appear to be most easily demonstrated in the nerves of the pineal, and can be seen with approximately decreasing ease in this order: pineal (Milofsky, 1957; de Robertis and Pellegrino de Iraldi, 1961a,b; Wolfe et al., 1962; Fig. 7A; Pellegrino de Iraldi and de Robertis, 1963; Wolfe, 1965; Bondareff, 196-5, 1966; Bondareff and Gordon, 1966; Bloom and Giarman, 1967; Kelly, 1967; Machado, 1967; Machado et al., 1968; Wartenberg, 1968; Bloom and Giarman, 1970; Fig. 3A, B; Pellegrino de Iraldi and Gueudet, 1969; Fig. 5A, B) > vas deferens (Richardson, 1962; Clementi, 1965; Bloom and Barrnett, 1966; Hokfelt, 1966b; Van Orden et al., l966,1967a,b; Farrell, 1968; Tranzer and Thoenen, 1967a,b, 1968b; Tranzer et al., 1969; Fig. 4A, B) >iris ( Richardson, 1964, 1966; Duncan and Micheletti, 1966; Hokfelt, 1966a, 1968a,b; Fig. 6A, B, C ) > nictitating membrane (Van Orden et al., 1967c; Esterhuizen et al., 1968b) > intestine (Grillo and Palay, 1962; Gabella, 1967; Taxi, 1969) > blood vessels (Devine, 1967; Devine and Simpson, 1967, 1968; Ruskell, 1967; Graham et al., 1968; Fig. 7B; Lever et al., 1968; Siggins and Bloom, 1969; Bloom, unpublished observations ) > heart ( Wolfe and Potter, 1963; Thaemert, 1966; Potter, 1967) > endocrine pancreas (Legg, 1967; Esterhuizen et al., 1968a; Shorr and Bloom, 1970). These tissue differences can only be partially explained in terms of tissue content and relative extent of innervation, although these factors must obviously be of importance, However, the main known distinguishing feature of these tissues (aside from the relative turnover rates of norepinephrine and

FIG.3. Sympathetic axons after fixation with either glutaraldehyde/OsO, sequence (A, Bloom, unpublished) or KMnOa (B, Hokfelt, unpublished). Both methods show electron-opaque deposits in the LGV and SGV, but more granular material is seen with KMn04. A, X 108,000; B, X 115,000. 42

FIG.4. Rat vas deferens showing influence of fixation on the electron-opaque deposits of the SGV. A, fixation with triple fixation sequence (paraformaldehydeglutaraldehyde, KzCr20r,Os04) described by Tranzer and Snipes ( 1968). Empty vesicles at left are surface invaginations of smooth muscle fibers ( m ) ; B, fixation with OsO,; X 55,000. Contributed by Dr. J. P. Tranzer. 43

FIG.5. Rat pineal after treatment with p-Cl-phenylalanine; pineal divided and halves fixed either in glutaraldehyde/OsOd sequence ( B ) or with OsOI only ( A ) . Note the relative paucity of SGV in B (compare with Fig. 3 A ) with respect to A. A, X 60,000; B, X 45,000 (from Pellegrino de Iraldi and Gueudet, 1969; contributed by Dr. A. Pellegrino de Iraldi).

44

FIG. 6. A. Cat iris &ed with glutaraldehyde/Os04 sequence after four intraperitoneal injections of 5-hydroxydopamine (20 mg/kg) over 48 hr. Of the three nerve terminals in this field, the vesicles of two are completely filled with an electron-opaque material. The synaptic vesicles of the other axon (presumably 45

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FLOYD E. BLOOM

neural activity under the condition prior to and during fixation) appears to be the method for fixation used in their study. In the same tissues, the ease of demonstrating the granular deposits in the peripheral autonomic nerve vesicles appears to occur in the following decreasing order: 3% KMnO, (Richardson, 1966; Hokfelt, 1967a, 1968a,b; Van Orden et al., 1969; Bloom and Giarman, 1970; Figs. 3B and 6A) > glutaraldehyde/dichromate/OsO, (Tranzer et al., 1969; Fig. 4A, B) > glutaraldehyde/ Oso, (Bloom and Barrnett, 1966; Van Orden et al., 1966, 1967a,b,c; Machado, 1967; Bloom and Giarman, 1970; Pellegrino de Iraldi and Gueudet, 1969; Figs. 3A, 4A, and 5A) > formaldehyde Oso, (Bondareff, 1965) > OsO, (d e Robertis and Pellegrino de Iraldi, 1961a,b; Grillo and Palay, 1962; Grillo, 1966; Figs. 4B, 7A). These differences cannot be explained on the extent to which the fixative retains the tissue monoamine (Hokfelt and Jonsson, 1968; Bloom and Giarman, 1970; Taxi and Droz, 1969) or on the relative preservation of tissue fine structure (Devine and Laverty, 1968). Does the presence of SGV (Figs. 3,5,6,7,8A) serve to indicate monoamines? We can start by answering a more general question, but one which is equally helpful, by stating that peripheral nerves which exhibit the SGV are almost certainly and exclusively the norepinephrine-containing axons. This conclusion is established by the observations that with the proper fixation, such nerves can be seen in all sympathetically innervated tissues (Richardson, 1966; also see reviews by Bloom and Giarman, 1968a,b; Jaim-Etcheverry and Zieher, 1969c; Tranzer et al., 1969). Such nerves disappear when the sympathetic axons are surgically severed (Pellegrino de Iraldi et al., 1965; Van Orden et al., 1 9 6 7 ~). These axons are not seen after immunosympathectomy (Richardson et al., cited by Sjoqvist et al., 1965) or when degeneration is due to 6-hydroxydopamine (Devine and Laverty, 1968; Tranzer and Thoenen, 196%; Devine, 1969; Siggins and Bloom, 1969). Such nerves can be localized independently of fixation by electron microscopic autoradiography after labeling with 3H-NE (Wolfe et al., 1962; Fig. 7A; Devine and Simpson, 1967; Taxi and Droz, 1966b; Esterhuizen et al., 1968a,b; Graham et al., 1968; Lever et al., 1968; Fig. 7B; Budd and Saltpeter, 1969) or 3H-dihydroxyphenylalanine (Taxi and Droz, 1966a,b). Not all of these latter cholinergic) remain completely electron-lucent. X 50,000 (from J. P. Tranzer and Thoenen, 1967; contributed by Dr. J. P. Tranzer). B and C. Rat iris dilator muscle after pretreatment with a norepinephrine synthesis inhibitor. Tissues of irides B and C were handled identically except that nerves to iris B were stimulated electrically (20 stimuli/sec for 90 min) before fixation in KMn04. B, X 36,000; C , X 31,000 (modified from Hokfelt, 196813; contributed by Dr. T. Hokfelt).


47

FIG. 7. A. Electron microscopic autoradiograph localizing radioactive norepinephrine over nerve terminals of die rat pineal. Tissue fixed with intravascular

48

FLOYD E. BLOOM

experiments satisfy the dosage and retention criteria cited previously (see Section 11, D ) but all are mutually supportive. Finally, the SGV seen with KMnO, fixation can be depleted with nerve stimulation (Richardson, 1963; Hokfelt, 1968a,b; Fig. 6B, C ) , as well as drugs. The next question that arises is whether the deposit which makes these vesicles morphologically unique from other synaptic vesicles of the same size range (such as those seen in neuromuscular junction and other nonadrenergic peripheral junctions) is in itself indicative of the relative monoamine content. The observations which favor equating the granular deposit with catecholamine storage are derived from the observation that the relative numbers of granular vesicles per nerve terminal tend to decrease with amine depletion by reserpine (Pellegrino de Iraldi and de Robertis, 1961, 1963; Richardson, 1963; Clementi, 1965; Hokfelt, 196610; Van Orden et al., 1966), metaraminol (Bondareff and Gordon, 1966), or a-methyl-rn-tyrosine (Bloom and Barmett, 1966; Van Orden et al., 1966) and can be enhanced or restored by monoamine oxidase inhibitors (Pellegrho de Iraldi and de Robertis, 1963; Van Orden et aZ., 1967a) with or without dihydroxyphenylalanine ( Pellegrino de Iraldi and de Robertis, 1964; Clementi, 1965) or by loading with NE (Van Orden et al., 1966), a-methyl-NE (Bondareff, 1966; Hokfelt, 1967a,c, 1968a,b), or &OH DA (Tranzer and Thoenen, 1967b). The data which tend to refute equating the granular deposits with catecholamine content in the peripheral vesicles stem first from the observations that success or failure in the demonstration of the vesicle granularity is not proportional to the amount of radioactive catecholamine retained in the tissue (Devine and Simpson, 1968; Hokfelt and Jonsson, 1968; Taxi, 1968; Taxi and Droz, 1969; Bloom and Giarman, 1969) or in homogenate fractions (Michaelson and Whittaker, 1963; Michaelson et al., 1968; Whittaker, 1W; Michaelson, 1967; Potter, 1967). Second, by autoradiography, heavy deposits of silver grains can frequently be seen over axons in which the granular vesicles exhibit minimal granularities (Lever et al., 1968; Devine and Simpson, 1968; Esterhuizen et al., 1968a,b; Budd and Saltpeter, 1969; Taxi, 1969) indicating, at the least, that catecholamines can occur within the vicinity of the vesicles without the vesicles osmium tetroxide 30 min following the injection of 12.5 pg of radioactive norepinephrine. The silver grains are concentrated over the nerve terminals in which small granular vesicles can be clearly seen. X 37,380 (contributed by Dr.David E. Wolfe). B. Autoradiographic localization of n~repinephrine-~H over sympathetic nerves to cat pancreatic arteriole. Nerves labeled in vivo by intra-arteriolar perfusion of approximately 100 pg norepinephrine over 25 min and fixed in glutaraldehyde/Os04 45 min later. Note, despite large number of silver grains over nerve, only the LGV have any electron opacity, A few grains overlay the cytoplasm of the fibroblast ( f ) . X 50,000.Contributed by Dr. J. D. Lever (see Lever et al., 1968).

FIG. 8. A. Sympathetic ganglion of rat fixed in KMn04, showing electron-opaque deposits in both LGV and SGV in one of the two nerve processes X 100,000. Contributed by Dr. T. Hokfelt (see Hokfelt, 1967b, 1968a,b). B. Normal rat hypothalamus fixed by perfusion with glutaraldehyde and exposed to OsO4 in solution at

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FLOYD E. BLOOM

being granular. These arguments could be explained away most simply by proposing that the type of fixation was adequate to bind the catecholamine to the tissue, but that the correct conditions for producing the electron deposits with the metallic oxidant (see Section 11, B, 4) have not been found. However, after KMnO, fixation, the small granular vesicles seen exhibit fewer autoradiographic grains than do other portions from identical tissue prepared with glutaraldehyde/ OsO,. The latter exhibit multiple autoradiographic grains but fewer granular vesicles (Bloom and Giarman, 1970; Taxi and Droz, 1969); were it not for these observations, there would be no reason to doubt the conclusion that the presence of the intravesicular granularity is indicative of the catecholamine. However, these as yet unexplained observations indicate the need for caution before reaching such a conclusion, particularly when considered in the light of observations on NE in the central nervous system and in neuronal cell bodies (see Section IV, B and C). 2. Distinguishing N E and 5-HT in SGV An additional feature complicating the quantitative interpretation of the SGV precipitate arises from observations that certain “adrenergic” nerves may also store 5-HT (Bloom and Giarman, 1967, 1970; JaimEtcheverry and Zieher, 1968a,b,c; Pellegrino de Iraldi and Gueudet, 1969; Fig. 5A, B). On the basis of biochemical experiments, it can be estimated that more than half of the monoamine contained in the nerves to the rat pineal may be 5-HT rather than NE (Neff et al., 1969). This may be one explanation for the ease with which the SGV in the pineal nerves are demonstrated with various fixatives (see above, Section IV, A, 1). It is, therefore, of considerable interest that depletion of the 5-HT following synthesis inhibition with p-C1-phenylalanine ( Koe and Weissman, 1966; Lovenberg et al., 1968) produces loss of the SGV with glutaraldehyde/ OsO, fixation but not with primary fixation to either OsO, (Pellegrino de Iraldi and Gueudet, 1969; Fig. 5A, B ) or KMnO, (Bloom and Giarman, 1970). By the use of the triple staining sequence formaldehyde-glutara1dehyde-K2Cr,O7 ( Jaim-Etcheverry and Zieher, 1968a,b,c, 1969a,c), differential staining of 5-HT can also be achieved with only partial loss of sensitivity. With this technique, it is possible to demonstrate storage of 5-HT in the nerves to the rat vas deferens (Snipes et al., 1968) if there has first been depletion of the NE by 6OOC. In contrast to results with standard glutaraldehyde/OsO, sequence on brain, numerous electron-opaque deposits are seen in both the small and large synaptic vesicles in this terminal. X 50,000 (unpublished micrograph from Bloom and Aghajanian, 1 9 6 8 ~ ) .


51

synthesis inhibition ( Jaim-Etcheverry and Zieher, 1969a) . Such observations thus provide the possibility of untold complicating factors in the production of SGV precipitates in the brain (see below) where the knowledge of possible transmitters stored in vesicles is still primeval. 3. Large Granular Vesicles

In addition to the SGV (40-60 mp) which exhibit homogeneous opaque granular content after oso,, Grillo and Palay also described a larger vesicle, 80-120 mp in size, which exhibited a more fibrillar electron-opaque deposit, but of less opacity than that seen in the SGV. The LGV constitute about 1 5 % of the vesicles in various peripheral adrenergic nerve terminals (Bondareff, 1965; Van Orden et al., 1966, 1967a,b), but it is also seen in nerve terminals classically considered to be cholinergic, such as the preganglionic axons to a variety of sympathetic ganglia (Taxi, 1961;Grillo and Palay, 1962; Grillo, 1966; Clementi, 1965). The same type of large granular vesicle appears to be the main form of granular synaptic vesicle seen in the retina (Pellegrino de Iraldi and Jaim-Etcheverry, 1967b) and in the nervous systems of invertebrates (Best and Noel, 1969; Rude et al., 1969; Van Orden et al., 1969). The granular contents of the larger vesicle appear to be unrelated in relative electron opacity to the amount of monoamine in the tissue (Bondareff, 1965; Bloom and Barmett, 1!366), and statistical interpretation of autoradiographs of pineal nerves indicates no relatively increased number of grains over these vesicles with respect to the SGV (Budd and Saltpeter, 1969). Nevertheless, despite these negative correlations there is still some evidence, recently obtained, which suggests that the large granular vesicles may be participants in the monoamine storage process of peripheral nerves. These data come from the observations that loading tissue with 5-hydroxydopamine and examining it after glutaraldehydel OsO, fixation reveals increased electron opacity over the LGV in nerve terminals if they are terminal adrenergic axons, but not in preganglionic cholinergic axons ( Tranzer and Thoenen, 1968). Similar selective increases in the electron opacity of the LGV of adrenergic nerves have been seen after loading and KMnO, fixation (Hokfelt, 1968a,b) and after 5-HT loading using the triple sequence for 5-HT reactivity (JaimEtcheverry and Zieher, 1968a). These observations need not reflect differences among the LGV of chemically different nerve types, but may simply indicate the uptake specificities of the axonal membrane (Tranzer and Thoenen, 1967; Tranzer and Thoenen, 1968). In addition, when ligature constrictions of peripheral nerve trunks cause accumulation of biochemically and histochemically demonstrable norepinephrine (Dahlstrom, 1967), mainly LGV are found to be increased

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FLOYD E. BLOOM


53

in number by electron microscopic observation (Kapeller and Mayor, 1969a,b). An interesting clue to the possible function of LGV is that they are found in “cholinergic” rat neuromuscular junction during its morphogenesis, but are rare thereafter (Kelly and Zacks, 1969). The LGV are also the first vesicles seen in nerves to developing pineal (Machado et al., 1968).

B. CENTRAL NERVETERMINALS Knowing of the positive correlation between small granular vesicles and catecholamine in the peripheral nervous system, and the near negative correlation with the LGV, the noncombatant reader may find it difficult to understand the cause of the confusion over the localization of central monoamines, and which has only recently been partially clarified. Because small granular vesicles could not be demonstrated in the central nervous system, prior to 1967, all attention was devoted to the central LGV (Figs. 8B-10). These vesicles were presumed to be potential indicators of central monoamines, owing to the positive reaction of some LGV with the Wood-Barrnett procedure (Wood, 1966). They also appear in synaptosome fractions obtained from NE-rich areas of the hypothalamus (de Robertis et al., 1965) and according to some authors they alter their frequency after depletion of central stores (Pellegrino de Iraldi et al., 1963; Bak, 1965, 1967; Ishii et al., 1965; Matsuoka et al., 1965; Shimizu and Ishii, 1965; Halaris et al., 1967; Ishii, 1967) and increase in frequency after monoamine oxidase inhibitors and monoamine precursors (Pellegrino de Iraldi et al., 1963; Hashimoto et al., 1965; Ishii, 1967; Pellegrino de Iraldi and Jaim-Etcheverry, 1967a; Pfeiffer et at., 1968; Zambrano, 1968).Others disagree (Fuxe et al., 1965; Bloom and Aghajanian, 1968a). Further evidence supporting the relationship between the central LGV and monoamine storage was their presence in the majority of nerve terminals identified by autoradiography as containing NE-sH ( Aghajanian and Bloom, 1966, 1967a; Lenn, 1967; Descarries and Droz, 1968a,b; Fig. QA, B) or 3H-5-HT ( Aghajanian and Bloom, 1967b; Fig. lo). Furthermore, LGV are seen in the nerve terminals which degenerate after lesions of monoamine pathways (Aghajanian et aZ., 1969; Raisman, 1969). Finally, LGV are observed in FIG. 9. Autoradiographic localization of norepinephrine-’H in rat brainstem neuropil. Rat pretreated with a monoamine oxidase inhibitor and given 500 pC (specific activity 8 C/mM) intraventricularly 3 hr before fixation with glutaraldehyde/OsO, sequence. A and B are adjacent thin sections, in both of which the same axon terminal can be seen to be heavily labeled; LGV, but no SGV can be seen. X 10,OOO. Contributed by Drs. L. Descarries and B. Droz ( s e e Descarries and Droz, 1968a,b).

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55

the majority of brainstem and hypothalamic nerve terminals which degenerate after intracisternal injection of 6-hydroxydopamine ( Bloom et al., 1969). However, with the successful application of Mn0,- fixation to the central nervous system ( Hokfelt, 1967a,c, 1968a,b; Hokfelt and Jonsson, 1968), large granular vesicles could no longer be considered to be the only potential storage organelle for central monoamines. By using Mn0,on thin slices of tissue, Hokfelt was able to observe small granular vesicles in nerve terminals in hypothalamus, locus coeruleus, and median eminence of untreated rats. After incubation of the slices in NE, 5-HT, or a-methyl-NE (1-10 pg/ml), SGV could be seen frequently in terminals from caudate nucleus, locus coeruleus, and suprachiasmatic nucleus; their appearance could be blocked if the animals or the incubating media offered exposure to drugs capable of blocking the uptake or storage processes. Furthermore, nerves capable of binding a-methylNE disappear from the caudate after lesions of the substantia nigra ( Hokfelt and Ungerstedt, 1969). While these data on the effects of Mn0,- fixation in the central nervous system were being collected, Aghajanian and I reported statistical evaluations on the significance of the central large granular vesicles (Bloom and Aghajanian, 1968a). We were able to show that the distribution of central nerve terminals containing several large granular vesicles vaned throughout the rat central nervous system in relatively good correlation with the total reported values for regional content of NE and 5-HT, although not for DA. However, we were unable to observe any profound alteration in the electron opacity (with glutaraldehyde/ OsO,) of the central large granular vesicles after manipulating the monoamine levels. By using electron-opaque stains for proteins which did not depend on an oxidation-reduction reaction (Bloom and Aghajanian, 1966, 1968b), we concluded that while the LGV were likely to occur in nerve endings containing monoamines, most of their electron opacity after glutaraldehyde/ OsO, was due to a proteinaceous component in their internal matrix. This conclusion was also directly applicable to the peripheral LGV (also see Jaim-Etcheverry and Zieher, 1969b), although as we have already seen, such vesicles in the periphery do not appear to be exclusive possessions of adrenergic nerves. Furthermore, FIG. 10. Autoradiographic localization of 5-HT-’H in pontine raphe nucleus of normal rat, Tissue prepared with standard glutaraldehyde/Os04 sequence 2 hr after intracisternal injection of 50 pC (specific activity 5.5 C/mM) generally labeled 5-HT. Several grains are clustered over one terminal in which electron-lucent elliptical synaptic vesicles and 2 LGV are seen. X 108,000 (Unpublished micrograph from Bloom and Couch).

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FLOYD E. BLOOM


57

terminals with KMn04 SGV (Hokfelt, 1968a) can be seen in less than half the number of terminals showing LGV with glutaraldehyde/Os04 (Bloom and Aghajanian, 1 W a ) when the same brain regions are quantitated. We might conclude at this point that the localization problem has now been finally solved with respect to both central and peripheral granular vesicles. The frustrating truth is that we must still attend to some annoying complications. As in the peripheral nervous system, there was a disconcerting discrepancy in the brain between the retention of the amine and the appearance of the SGV (see above, Section V, A, 1). Here, there is the additional problem that two types of central small granular vesicles appear not to correlate with monoamine content: such vesicles can be seen in the rabbit brain, despite reserpine treatment (Tranzer et al., 1969) and can be produced in glutaraldehyde-fixed brain by exposure to Oso, at elevated temperatures (Bloom and Aghajanian, 1968c; Fig. 8B), although this procedure appears to “leech” out the radioactivity. Furthermore, the “hot osmium” SGV persist despite prolonged and repeated administrations of reserpine, and do not increase in frequency after inhibition of monoamine oxidase. The success of the “hot osmium” technique may depend upon the fact that osmium vapors are more effective sources of electron-opaque “osmium black” precipitates (Hanker et al., lW),but their persistence despite depletion of amines suggest they react with some vesicle component having reducing or coordinating potency, but which is distinct from the monoamine. Furthermore, since the latter substance resists reserpine, this alleged reducing component would not be likely to fluctuate in proportion to the monoamine, as do the MnO, deposits described by Hokfelt (1968a,b). Nevertheless, the appearance of SGV alone cannot be any more conclusive evidence for the occurrence of monoamines than can the mere observation of LGV. Both types of granular vesicles should be subjected to tests for the satisfaction of the criteria proposed above (Section 11, A) . Hopefully, these fixations can eventually be tested in turn against other cytochemical methods which will not depend solely upon the unknown

FIG.11. Effects of hation on the electron opacity of vesicles in rat sympathetic ganglioneurons. A, Axodendritic synapse in which LGV are seen in the nerve terminal and small vesicles with electron-opaque deposits are clustered in the cytoplasm of the dendrite (arrows). Fixation with OsO,; X 62,000, inset X 110,000. B, Portions of two adjacent ganglioneurons after fixation with NaMnOa. Numerous SGV are seen clustered throughout the cytoplasm of both cells (arrows), X 16,000, inset X 55,000. Unpublished micrographs contributed by Dr. M. Grillo (see Grillo, 1966)

.

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FIG.12. ( top ) Autoradiographic localization of n~repinephrine-~H over perikaryon


59

chemical reactance of undefined “polymers” of monoamines and metallic oxidants.

C. MONOAMINE CELLBODIESAND NONTERMINAL AXONS Since there appears to be a rather reasonable association between SGV and stores of monoamines in the nerve terminals, one might expect that the same type of vesicle would account for the amine present in the cell body and along the axons. From fluorescence histochemical observations (Dahlstrom and Fuxe, 1965), the cell bodies normally fluoresce after gaseous formaldehyde, but this fluorescence is greatly augmented by pretreating the animals with monoamine oxidase inhibitors. This experiment has been performed by two separate groups using electron microscopy to search for cellular organelles which increased following such treatment; in both cases no such organelle could be found (Lenn, 1965; Fuxe et al., 1966). When sympathetic ganglioneurons are subjected to subcellular fractionation, little or no particulate storage form of NE is found (Fischer and Snyder, 1965). Furthermore, no matter which of the fixative procedures are successful in revealing the SGV of the terminals, it is rarely found possible to see such vesicles within the cytoplasm of the cell body unless MnO, salts are used (cf. Grillo, 1966 and Fig. 11; Hokfelt, 1968a,b; Tranzer et al., 1969; Van Orden et al., 1969). On the other hand, the cell bodies of both central and peripheral NE neurons do exhibit autoradiographic labeling with 3H-NE ( Descarries and Droz, 1968a,b; Fig. 12; Taxi, 1969), particularly when monoamine oxidase has been inhibited. However, in the latter cases, the labeling appears to be somewhat random with respect to perikaryal organelles, although the activity within the cells is clearly greater than the surrounding tissues. Descarries has suggested (personal communication) that some type of macromolecular material may be present within such cells to bind the NE once it has entered the cell. Such a macromolecule could represent an “extra vesicular” type of binding, such as that seen in longer sympathetic axons. Similar suggestions were also put forward to account for in locus coeruleus of untreated rat, fixed 30 min after intraventricular injection (500 pC, 7.3 C/mM). Generalized labeling of all cytoplasmic and nuclear volume is observed with no specific association of label with any organelles. X 8,000. Unpublished micrograph contributed by Drs. L. Descarries and B. Droz (see Descarries and Droz, 1968a,b). FIG. 13. ( bottom) Autoradiographic localization of n~repinephrine-~H after in uivo labeling of bovine splenic nerve, routinely fixed with glutaraldehyde/Os04 sequence. Grains are seen over several axons, but less than half of these exhibit LGV in the same plane of the section. X 20,000 (modified from Stjarne et al., 1969).

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the autoradiographic localization of NE-3H over bovine splenic nerve axons in which no vesicles (neither LGV or SGV) could be seen (Stjarne et al., 1969; Fig. 13). No loading of cell body organelles with 5-hydroxydopamine has been seen ( Tranzer, personal communication) . Thus, for the present, we must conclude that the monoamine cell bodies are even more diEEicult sites in which to localize these substances. Despite both SGV and LGV occurring in and around the Golgi apparatus of the cell bodies, as well as in isolated areas of both the perikaryon and dendrites, in no case do they seem to be present in sufficient numbers to account for the content measurable by biochemical or fluorescence histochemical approaches. While special techniques are required to selectively identify 5-HTstoring organelles in some mammalian tissues ( see Jaim-Etcheverry and Zieher, 1969c), it is interesting that the spectrofluorometrically identified 5-HT in the colossal cell of the leech ganglion can be seen with routine

FIG.14. The electron-opaque deposits in the vesicles associated with the Golgi apparatus of the Collossal cell of the Leech ganglion. These deposits are correlated with microspectrofluorometric histochemically identified 5-HT. Fixation with glutaraldehyde/OsO, sequence. X 60,000 (modified from Rude et al., 1969; contributed by Dr. R. Coggeshall).


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methods of fixation (Rude et al., 1969; Fig. 14). Again, this indicates the wide tissue and species variability encountered in such localizations. Since the enzymes for the synthesis of the monoamines are carried to the terminals after synthesis by the rough endoplasmic reticulum (see Dahlstrom, 1967; Barondes, 1968), some of the perikaryonal monoamine content could be accounted for on the basis of the activity of these enzymes after they are synthesized and while enroute to the nerve terminals. Pulse labeling autoradiographic experiments during the recovery from synthesis inhibition would be most interesting in this regard, particularly with p-C1-phenylalanine inhibition of tryptophan hydroxylase (Koe and Weissman, 1966; Lovenberg et al., 1968), because new enzyme must be synthesized for recovery to occur after this treatment.

V.

Conclusions

On the basis of a large number of experimental approaches which combine various neurocytological and histochemical observations, the association of NE-storing nerve terminals in the peripheral sympathetic nervous system is well correlated with intra-axonal SGV. The LGV occurring in these nerves seem to play only a minor role in the storage; although they do participate, the LGV are also found in other types of nerve fibers and thus their basic cellular function remains unclear. The same conclusions relating SGV to central monoamine stores could well be valid. However, there seems to be as wide a variation between the requirements for demonstrating these vesicles in the brain as there is among various peripheral axon systems. These differences in staining do not seem to be solely explicable on the basis of promptness or adequacy of fixation and may reflect some underlying differences among the manner or lability of the binding within storage vesicles. A nonvesicular binding material may be ubiquitously present throughout the monoamine perikaryon and portions of the axons of these cells. Finally, the exact composition of the SGV granule and its qualitative and quantitative relation to the monoamine believed to be present in the vesicle remains to be established. ACKNOWLEDGMENTS While the lnal responsibility for the views and interpretations presented here are properly borne by the author, I was privileged to have a most cooperative correspondence with many of my colleagues. In addition to the very kind contributions of numerous unpublished electron micrographs from Drs. Chiocchio, Coggeshall, Descarries, Droz, Grillo, Hokfelt, Lever, Pellegrino de Iraldi, Trammezzani, and Tranzer, prepublications and personal communications were also received from Professors Taxi and Wood. MIS. E. Batenberg and Miss J. Kuprys were of immense assistance in the tissue

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BRAIN LESIONS AND AMlNE METABOLISM By Robert Y. Moore Department of Pediatrics and Medicine (Neurology) and fhe Joseph P. Kennedy, Jr. Mental Retardation Research Center, The University of Chicago, Chicago, Illinois

I. Introduction . . . . . . . . 11. Subcortical Lesions and Brain Amine Levels . A. The Medial Forebrain Bundle . . . . B. The Midbrain Tegmentum . . . . 111. Interpretation of Lesion Effects on Brain Amines A. The Peripheral Model B. The Central Nervous System . . . . IV. Conclusions . . . . . . . . References . . . . . . . .

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67 69 69 71 72 72 73 88 89

I. Introduction Finally I should like to speak a word of warning against the present tendency to ascribe very complex functions to the thalamus and brainstem. (Lashley, 1951, p. 36)

The studies to be described here have utilized the techniques of localized tissue destruction in the central nervous system to define the specific neuronal elements with which the biogenic monoamines, serotonin, norepinephrine, and dopamine, are associated in the brain. When the studies were begun, nearly ten years ago, few data were available on the biogenic amines in brain beyond designation of their gross tissue localization and some effects of pharmacological manipulation of amine levels. But over the intervening years a number of experimental programs using the lesion technique, electrical or chemical stimulation, pharmacological manipulation, and combinations of these have demonstrated a role for the biogenic amines in such activities as central autonomic control, neuroendocrine regulation, maintenance of sleep patterns, control of extrapyramidal motor function, elaboration of affective behavior, and learning. Most commonly these programs have analyzed the organization of the central nervous system in the context of the concept of localization of function which holds that specific functions are controlled by specific components of the nervous system. Our own studies were also conceived in this framework, as they arose from attempts to explain the behavioral effects of some subcortical lesions. Following the important theoretical papers of Herrick (1933) and 67

68

ROBERT Y. MOORE

Papez (1937) and the pioneering experimental work of Kluver and Bucy (1937), there has been a steady growth of interest in the subcortical telencephalic nuclei and related areas of the diencephalon and brainstem. Among the subsequent contributions were many that focused upon alterations of behavior resulting from stimulation or ablation of subcortical areas. One example of these was the “rage” syndrome observed by Brady and Nauta (1953) with ablation of the septal nuclei in the rat. Bilateral destruction of the nuclei causes a transient state of irritability in the animals characterized by an exaggerated startle response, ready assumption of a fighting posture upon stimulation, and excessive struggling, vocalization, and biting in response to handling. From the anatomical studies of Nauta (1956, 1958) it was evident that the septal nuclei comprised part of a system which included the hippocampus, the lateral hypothalamus, and a region of the dorsal and medial midbrain tegmentum designated by Nauta (1958) the “limbic midbrain.” In a series of experiments based upon these observations we found that lesions in the hippocampus and lateral hypothalamus also produced a transient irritability similar to but less marked than that observed in septal rats. Rats with lesions in the dorsomedial midbrain tegmentum, in contrast, were easily handled and exhibited none of the irritability of the septal animals but did show severe attack behavior (Heller et al., 1962; Harvey et al., 1964). This behavior occurred only when the animals were housed together. Animals with this lesion appeared normal when alone but attacked each other so persistently when together that both animals sustained numerous bites, and one often died unless they were separated (Heller et al., 19s2). From these observations it was evident that two lesions within the same system might have quite different behavioral sequelae and that these were not explicable in terms of the anatomical variables alone. At the same time that these experiments were being done, attempts to modify the behavioral effects of the lesions provided a new direction. Both septal animals and animals with lesions in the dorsomedial midbrain tegmentum showed a marked sensitivity to certain drugs, particularly barbiturates (Heller et al., 1%0; Harvey et al., 1964). Each of these lesions produced a fourfold increase in barbiturate sleeping time, whereas lateral hypothalamic, hippocampal, and other lesions had no significant effects upon the response to barbiturates (Harvey et al., 1964). This sensitivity to barbiturates could not be explained on the basis of lesion effects on brain or body weight, temperature regulation, drug metabolism, adrenal function, or brain levels of the drug (Heller et al., 1960; Harvey et al., 1964). The changes in behavior noted with the two effective lesions, septal and dorsomedial tegmental, combined with the

69

BRAIN LESIONS AND AMINE METABOLISM

alteration in sleeping time suggested another possibility, that the effects of the lesions might be related to a change in cerebral metabolism of the amine serotonin. The sedative effect of reserpine had been related to decreases in brain serotonin, and it was proposed that this amine represented a central neurotransmitter, possibly mediating parasympathetic activities such as sleep (Brodie et at., 1956; Brodie and Shore, 1957). In addition, there was considerable speculation regarding the role of derangements in serotonin metabolism in the pathogenesis of severe behavioral abnormalities such as the functional psychoses ( Woolley, 1962). Because of this we elected to investigate the effects of subcortical lesions on brain serotonin levels in an attempt to provide a correlation for the behavioral effects and the responses to drugs noted above. II. Subcortical lesions and Brain Amine levels

A.

THEMEDIALFOREBRAIN BUNDLE

As in the behavioral and pharmacological studies outlined above, a series of bilateral electrolytic lesions were placed in telencephalic, diencephalic, and midbrain areas of adult male rats. Whole brain serotonin levels were determined 35 days postoperatively. The results of our first study are presented in Table I. Four of the lesions used produced significant decreases in brain serotonin. Lesions in the septa1 area, in the ventral midbrain tegmentum (adjacent to the interpeduncular nucleus), TABLE I EFFECT OF BILATERAL LESIONSON WHOLEBRMNSEROTONIN LEVELSIN THE RAT"

Lesion group

Percent change in serotonin level from normal controls

Sham operated Caudate nucleus Hippocampus Septa1 area Medial hypothalamus Medial forebrain bundle (lateral hypothalamus) Habenula Ventromedial midbrain tegmentum Dorsomedial midbrain tegmentum Pontine tegmentum

+3 0 -5 -1 2 -7 -36b -7 - 15b - 14b f 7

From Heller et al. (1962). Differences from control statistically significant, p were not significant, p > .05. Q

< .01.

All other differences

70

ROBERT Y. MOORE

and in the dorsomedial midbrain tegmentum reduced brain serotonin levels by 12-151 in comparison to normal, unoperated controls. Lesions in the lateral hypothalamus had a more marked effect, reducing serotonin levels by 36%.The changes in brain serotonin content observed with sham operation or other electrolytic lesions were not significantly different from control values. The observed changes in amine levels did not correlate well with the behavioral effects of the lesions or with sensitivity to barbiturates, but the lesions which affected brain serotonin levels did share one feature. Consideration of the morphological relations of the areas which, when destroyed, led to decreases in brain serotonin levels prompted the conclusion that the integrity of one neural system was necessary for the maintenance of normal serotonin levels in the rat brain. The most marked lesion effect upon brain serotonin (-36%) was caused by lesions in the lateral hypothalamus. This area of the hypothalamus contains a few, scattered cells and a complex tract, the medial forebrain bundle, which interconnects the basal telencephalon, the hypothalamus, and the midbrain reticular formation ( Guillery, 1957; Nauta, 1958, 1963). The septal, ventral midbrain tegmentum and dorsomedial midbrain tegmentum lesions also reduced brain serotonin levels, and each of these lesions destroyed areas receiving fibers from and contributing significant numbers of fibers to the medial forebrain bundle in the lateral hypothalamus. In contrast, the five lesions which did not alter brain serotonin did not destroy numbers of medial forebrain bundle projections. The medial hypothalamic lesions were confined to the medial nuclear region and spared the medial forebrain bundle. The caudate nucleus and pontine tegmentum lesions each transected a number of long fiber systems and ablated a nuclear area but none related to the medial forebrain bundle. The habenular and hippocampal lesions were of particular interest because each of these nuclei is closely related to the medial forebrain bundle system although neither contributes fibers into it at the level of the lateral hypothalamus. Thus, each lesion which significantly reduced brain serotonin levels in contrast to those which did not, either transected the medial forebrain bundle or destroyed areas contributing fibers to that tract in the lateral hypothalamus. On this basis it was suggested that the loss of brain serotonin produced by the lesions was the result of degeneration of serotonin-producing fibers within the medial forebrain bundle ( Heller et al., 1962). Subsequent experiments provided confirmatory data. The medial forebrain bundle is predominantly an uncrossed pathway, originating and terminating on the same side of the brain ( Guillery, 1957). Unilateral destruction of the medial forebrain bundle produced a fall in brain serotonin half that observed with bilateral lesions. But, when the effect was studied in brain halves, it was found to occur only on the side containing the lesion and to an extent

71


equivalent to the bilateral lesion effect (Harvey et al., 1963). Further experiments provided similar information regarding the control of brain norepinephrine levels ( Heller and Harvey, 1963), indicating that the integrity of the medial forebrain bundle was essential to the maintenance of normal levels of both amines in brain. And, although as noted before, there was considerable information available on the regional distribution or serotonin and norepinephrine in brain (Bogdanski et al., 1957; Kuntzman et al., 196l), the experiments outlined here first demonstrated the association of these amines with a specific group of central neurons.

B. THE MIDBRAINTEGMENTUM In the previous experiments it was evident that the exact localization of the lesion was critical to the effect on amine levels. Lateral hypothalamic lesions which failed to transect the fibers of the medial forebrain bundle, for example, had much less effect upon brain amine levels than ones which did interrupt the tract (Heller and Moore, 1965). Similarly, anatomical analysis of the tegmentum lesions indicated that there were critical areas for production of neurochemical effects. The most effective lesions were pIaced at the level of the isthmus, and, at this level, lateral lesions appeared to have more effect upon norepinephrine whereas medial lesions affected both amines. To test this further another experiment was carried out in which a number of different bilateral lesions were made, and the brains assayed for content of both serotonin and norepinephrine (Heller and Moore, 1985). In this experiment the location of each lesion was verified anatomically. The results of the experiment are presented in Table 11. Medial forebrain bundle and dorso-

SELIKTIVE

TABLE 11 EFFECTS OF BIL~TERAL LESIOXS ON BRAIN SER OT ONIN A N D NOREPINSPHRINE I N THE RAT^ ~~~

~~

Percent difference from sham opersted group

Lesion group

Serotonin

Caudate nucleus Medial hypothalamus hledial forebrain bundle Dorsomedial midbrain tegrnentum Ventrolateral midbrain tegmentum Central gray

$2 -2 -33D - 28b -3

Eroni Heller and Moore (19G5). Differences statistically significant, p cant, p > 3 5 .

- 18b

Norepinephrine -9

-7 -26b -24b -32* -7

a

< .01. All other

differences were not signifi-

72

ROBERT Y . MOORE

medial tegmental lesions caused significant decreases in both serotonin and norepinephrine. The two other tegmental lesions, however, demonstrated selective effects upon brain amine content. Ablation of a part of the ventrolateral tegmentum [in the paralemniscal field of the tegmentum (Berman, 1968)] resulted in a decrease in norepinephrine without altering serotonin. Central gray lesions, on the other hand, affected only serotonin. As in previous studies, the other lesions did not significantly alter brain levels. From the morphological analysis of the lesions it was evident that the dorsomedial tegmental lesions, which produced falls in both amines, involved both tegmental areas. We concluded from this that the paralemniscal tegmental field and its associated fiber bundles was essential for the maintenance of brain norepinephrine and the central gray for brain serotonin. Since both of these areas project into the medial forebrain bundle in the lateral hypothalamus, this conclusion was in accord with the view that the medial forebrain contained monoaminecontaining fibers, perhaps originating in the midbrain. Ill. Interpretation of Lesion Effects on Brain Amines

A. THEPERIPHERALMODEL The basis for the interpretation of the central lesion effects described above was a series of studies on the peripheral nervous system in which denervation was used as a tool to demonstrate the neural origin of transmitter substances. For example, destruction of preganglionic sympathetic fibers causes a marked reduction in acetylcholine content of the superior cervical ganglion (Brown and Feldberg, 1936; MacIntosh, 1938), and tissue catecholamines show a similar decrease following postganglionic sympathetic denervation (Cannon and Lissak, 1989; von Euler and Purkhold, 1951; Sidman et al., 1962; Sedvall, 1963).The loss of the amine in these instances is restricted to the denemated tissue and takes place rapidly, within 1 to 4 days (Kirkepar et al., 1%2; Trendelenberg, 1963; Malmfors, 19%). The time course over which amine is lost is in accord with electron microscopic observations on the rate at which the postganglionic axon terminals degenerate following axon section ( Roth and Richardson, 1969). These data on peripheral lesions conclusively demonstrate that section and subsequent degeneration of a peripheral axon is accompanied by loss of the appropriate neurotransmitter from the denervated tissue. In view of the obvious similarity in experimental design and results, we concluded that this model probably represented the mechanism by which central lesions affected brain monoamine levels. It should be noted, however, that the peripheral effects of lesions are not always as simple as outlined above. Preganglionic sympathetic denerva-


73

tion, for example, does not alter norepinephrine levels in some tissues (Rehn, 1958; Fischer and Snyder, 1965); whereas in others, such as the pineal, amine levels are substantially decreased following such a lesion (Zieher and Pellegrino de Iraldi, 1966; Wurtman et al., 1967). The alteration of a tissue norepinephrine level by preganglionic denervation represents an effect of a lesion on amine metabolism mediated across a synapse in a cell which is presumably morphologically intact. The relevance and importance of this will be discussed in subsequent sections, B. THECENTRAL NERVOUSSYSTEM

1. Time Course of Amine Changes after Central Lesions From the many studies demonstrating denervation effects in the peripheral nervous system two major points were clear. First, section of a peripheral nerve causes a loss of neurotransmitter from innervated structures and, second, the time course of this loss correlates well with that for degeneration of the transected axons. To support the view that decreases in brain amine content produced by central lesions were owing to a direct denervation effect, we examined the time course over which the decreases in amine occurred and their distribution in the brain. A summary of one time course experiment is shown in Fig. 1. With unilateral medial forebrain bundle lesions there is a loss in brain serotonin and norepinephrine which first becomes evident on the second postoperative

POSTOPERATIVE DAY

FIG. 1. Time course of amine changes in the rat brains following unilateral medial forebrain bundle lesions. The serotonin and norepinephrine content of the brain half containing the lesion is expressed as a percent of the control side amine content, The original data from which the figure was prepared was presented by Moore and Heller (1967).

74

ROBERT Y. MOORE

day and the effect gradually increases until the twelfth day, when the amine levels stabilize at approximately 60%of control values. This time course differs from that following peripheral denervations in two ways: It is much more prolonged and there are no points of abrupt, large changes as are usually observed in the periphery. Attempts to correlate this time course with degeneration of the transected axons was only partially successful (Moore and Heller, 1967). In accord with the early part of the time course, degenerating axons were first noted following a medial forebrain bundle lesion on the second postoperative day. These increased in number until the fifth day but beyond that point no further changes were appreciated. Such changes might have occurred, however, and not have been demonstrated by the technique employed, the Nauta silver impregnation method for degenerating axons. The medial forebrain bundle contains many unmyelinated axons of which a substantial bundle proportion are less than 0.5 p in diameter (Moore, unpublished observations) and beyond the resolution of the light microscope. Unlike the situation in the periphery, there have not been as yet any studies reported on electron microscopy of the medial forebrain bundle following lesions transecting it. These would allow a direct comparison to be made between the time of onset of degenerative changes in the medial forebrain bundle and decreases in brain amine content. The data now available suggest that the time course for the amine changes differs from what one would expect with a direct denervation effect. 2. Regional Distribution Studies

The direct denervation interpretation also requires that the decreases in amine content occur from regions of the brain innervated by the transected axons. The connections of the medial forebrain bundle were well known (cf. Moore et al., 1965; Moore and Heller, 1967, for reviews), and experiments were designed to compare the areas of amine loss with the information on these connections. Initially, in order to carry out the regional distribution studies it was necessary to use an animal with a larger brain, the cat. Subsequently, refinements in analytical procedures permitted similar experiments on the rat. The data obtained from the two species are nearly identical (Moore and Heller, lW, 1967; Heller et al., 1964, 1966a,b; Moore et al., 1965, 19%) and, in themselves, indicate the generality of the findings. After transection of the medial forebrain bundle in the lateral hypothalamus there is a decrease in both serotonin and norepinephrine content throughout the telencephalon, whereas amine levels in the diencephalon and brainstem are unchanged. An example of the changes in regional brain serotonin content in the rat brain is shown in Table 111. In the telencephalic areas the fall in amine


75

TABLE I11 REGIONAL EFFECTSOF MEDIALFOREBRAIN BUNDLELESIONSO N SEROTONIN LEVELSI N RAT BRAIN^ Percent difference from control

Region assayed

-66'

Cortex Septum Striatum Amygdala Hippocampus Brainstem From Moore and Heller (1967). Differences statistically significant, p

-49b -51b -54b -6gb $4

< .002.

content is from 49%to 68%below control values. The cerebral cortex was assayed as a single sample in this study, but in other experiments on the cat (Moore et al., 1965; Heller and Moore, 1968) the effect was found to occur in all areas of the cortex. A summary 'of another representative experiment on the cat is shown in Table IV. Large decreases in norepinephrine were observed in all areas of the cerebral cortex as well as in all subcortical, telencephalic areas. These vaned from 51%in the parietal cortex to 71%in a sample made up of the amygdaloid complex and hippocampal formation together. In this series of regional studies it was found that the magnitude of the lesion effect on amine levels was largely dependent upon the size and location of the lesion. The larger TABLE I V REGION i L EFFECTS O F ,\IEDI.4L F O R E B R ~ BUNDLE IN LESIONSO N NOREPINEPHRINE LEVELSI N THE CAT BRAIN" Region assayed

Percent difference from control

Frontal cortex Parietal cortex Occipital cortex Septum and caudate Amygdala and hippocampus Diencephalon Nidbrain Pons and medulla

-63' -51b -67b -63b -71b - 10 -6 +3

~

From Heller et at. (1966a). Differences statisticoally significant, p cant, p > .05. a

< .01. All other differences were not signifi-

76

ROBERT Y. MOORE

TABLE V REGIONAL EFFECTSOF VENTROLATERAL TEGMENTAL LESIONSON SEROTONIN AND NOREPINEPHRINE I N THE R.ATBRAINn ~

~~

Percent difference from control Region assayed Neocartex Septum Striatum Amygdala Hippocampus Diencephalon a

Ir

From Heller and Moore (1968). Differences statistically significant, p

Serotonin -2 -5

+4 -3 -12 +5

Norepinephrine -43b -53h -31b - 44b - 43b -47b

< .05.

the proportion of lateral hypothalamus destroyed by the lesion the greater the fall in amine content, to approximately a 70%level. The widespread effects of the medial forebrain bundle lesions on telencephalic amine levels were not explicable on the basis of our knowledge of projections of the axons destroyed. Similar observations were also made with tegmental lesions (Table V ) , Following ventrolateral tegmental lesions there is a decrease in norepinephrine content both in the diencephalon and the telencephalon. Brainstem norepinephrine was unaffected by the lesions. This effect of tegmental lesions on norepinephrine, like that of medial forebrain bundle lesions on serotonin and norepinephrine, occurred in all areas of the brain rostral to the lesion and was nearly equal in each area. As with the medial forebrain bundle effects, this provided problems of interpretation which may be emphasized by a review of the relevant anatomy. 3. Anatomical Considerations Two possible interpretations were immediately apparent. First, the amine loss following lesions could be viewed as a result of neuronal degeneration and, second, it could be interpreted as a form of “trophic” effect occurring in morphologically intact neurons. A review of the connections of the areas whose destruction produced decreases in amines brought forth several problems for the first interpretation. These areas lay in a core of tissue extending from the septa1 area rostrally through the lateral preoptic and hypothalamic areas and into the basal and medial midbrain caudally. In our experience no lesion rostral to the septum or caudal to the isthmus has altered brain amine levels, And, as noted above (Section II,A), the pathway crucial to these effects is the medial fore-


77

brain bundle. This phylogenetically old tract provides the primary source of afferent and efferent connections of the hypothalamus (Bleier et al., 1966; Nauta, 1963) and contains large numbers of reciprocal interconnections between the basal telencephalon and a part of the basal and medial midbrain tegmentum ( Nauta, 1958, 1963; Guillery, 1957). Although considerable data were available on the connections of the medial forebrain bundle ( 6 .Nauta, 1958, 1963; Guillery, 1957; Wolf and Sutin, 1966), we elected to trace the exact distribution of the axons transected by our medial forebrain bundle lesions. Studies were carried out on both rats and cats in which lesions were made in the medial forebrain bundle or in the tegmentum in the same manner as in the amine experiments. Animals were killed at varying postoperative survival periods (rats, 2-12 days; cats, 5-14 days), and a series of sections from each brain was prepared by the Nauta-Gygax method (Nauta, 1957) for the selective impregnation of degenerating axons. Subsequently, a few brains have been studied using the Fink-Heimer technique (Fink and Heimer, 1967) which demonstrates degenerating axon terminals. The descending projections of the medial forebrain bundle are not immediately relevant to interpretation of the amine loss, and therefore will not be discussed. Ascending axons from a medial forebrain bundle lesion form a compact group in that tract. A few fibers enter the adjacent medial hypothalamus, but the majority are confined to the lateral hypothalamus where a number of terminal and preterminal axons are scattered among the cells of the lateral hypothalamic nucleus. At the level of the anterior hypothalamus some fibers leave the medial forebrain bundle to terminate in the anterior amygdaloid area. The main group of degenerating axons traverses the lateral preoptic area to enter the ascending arm of the diagonal band of Broca and, then, the septal nuclei. Numerous terminals are present along the course of the pathway in the lateral preoptic area, the nucleus of the diagonal band, and the medial and lateral septal nuclei. A few fibers run beyond the septal nuclei, over the rostrum of the corpus callosum, to enter the hippocampal rudiment. None of these could be traced for any distance caudally, and we did not observe projections from the medial forebrain bundle to either the neocortex or to telencephalic areas other than the septum, amygdaloid complex, and hippocampal rudiment. This holds true for both the Nauta and Fink-Heimer material, but final conclusions must be reserved because we have not as yet sampled a full series of postoperative survival times with the latter method. The hypothalamic and telencephalic projections observed with tegmental lesions were similar, particularly in that no fibers could be traced to neocortex. The discrepancy between the distribution of amine falls and the distribution of degenerating axons following the lesions raised an obvious diffi-

78

ROBERT Y. MOORE

culty in the interpretation of the results. Three alternatives were available. First, we could dismiss the neurochemical effects of the lesions on the grounds that they were the result of some nonspecific alteration of neuronal metabolism. This seemed unlikely because of the restricted distribution of the effects and their dependence upon exact localization of the lesions. Second, we could maintain the hypothesis that all of the effects were due to section and degeneration of amine-producing neurons. Third, we could propose an effect mediated, at least in some areas, across one or more intermediate neurons which may or may not be similarly affected. Such changes we have termed “transsynaptic” neurochemical effects of central lesions. Before discussion this further, it should be pointed out that the second alternative has been accepted by another group of workers on the basis of experiments using a recently developed histochemical method for the demonstration of monoamines in brain.

4. Fluorescent Histochemical Studies Coincident with our initial studies of lesion effects on brain serotonin and norepinephrine, Falck and Hillarp and their associates in Sweden developed a powerful histochemical method for the demonstration of monoamines in brain and other tissues (Falck et al., 1962; Carlsson et al., 1962; Falck, 1962). This method, now known as the Falck-Hillarp method, utilizes the condensation of monoamines with formaldehyde vapor in frozen-dried or dried tissue to form an intense fluorophor which can be visualized under appropriate conditions in the light microscope. Details of the development of the technique and its chemical basis have been reviewed recently by Corrodi and Jonsson (1967). In brain, serotonin, norepinephrine, and dopamine appear to be the principal monoamines demonstrated. On the basis of the early studies employing the method, the generalization was made that serotonin exhibited a yellow fluorescence and the catecholamines a greenish-yellow fluorescence (Falck et al., 1W2; Carlsson et al., 1962). Under certain conditions, however, the catecholamines may exhibit a yellow or, even, orangeyellow color and be indistinguishable from serotonin by visual inspection (Corrodi and Jonsson, 1967). The more recent development of microspectrophotofluorometry, which permits the determination of excitation and emission spectra from the histochemical preparations, makes possible an objective differentiation of the catecholamine and tryptamine fluorophors (Van Orden et aZ., 1965; Caspersson et al., 1966; Ritzen, 1966, 1967; Bjorklund et al., 1968). Combining fluorescence microscopy, microspectrophotofluorometry, the use of lesions, and pharmacological manipulation, then, allows the differential localization of the primary catecholamines and serotonin to specific neuronal elements in brain. With certain


79

reservations the Falck-Hillarp can be viewed as the most useful technique available for studying the tissue localization of monoamines. The reservations arise primarily over two problems, resolution and sensitivity. The technique utilizes light micoscopy so that the limits of resolution are set at about 0 . 5 p For higher resolution, other techniques such as electron microscopic autoradiography are required. The sensitivity of the Falck-Hillarp method is great, but the lower limits of its application in tissue have not been well established. For this reason negative resuIts are difficult to interpret conclusively. In this regard Fuxe (1965) has noted, “It must consequently be kept in mind that areas which . . . are said to contain no CA [catecholamine] or 5-HT [serotonin] terminals may contain such terminals not visible in the fluorescence microscope.” Obviously absence of fluorescence could indicate an amine concentration below the sensitivity of the method, improper application of the method, or some peculiarity of the tissue which prevented proper application of the method. With these reservations in mind, we can now review the information obtained from the use of the Falck-Hillarp method in studying the central nervous system. In material from the brain and spinal cord, cell bodies are identified as neuronal on the basis of their size and configuration. “Terminals” are identified on the basis of their similarity in appearance to known axon-terminals in the peripheral sympathetic system ( autonomic ground plexus ) and probably correspond to bouton terminaux or bouton en passage. In general, dendrites and preterminal axons are not demonstrated unless some specific experimental manipulation has been performed. The first application of the Falck-Hillarp method to the central nervous system was made by Carlsson et al. (1962) who observed catecholamine-containing terminals in some nuclei of the medial hypothalamus. Carlsson et al. (1964) and Dahlstrom and Fuxe (1965) found both serotonin- and norepinephrine-containing terminals in the spinal cord and demonstrated that these belong largely to a group of bulbospinal neurons with cell bodies in the medullary reticular formation and axons traversing the reticulospinal tracts to terminate in the spinal cord gray matter. Subsequent studies on the histochemical localization of monoamines in brain has been based on two important papers. In the first of these, Dahlstrom and Fuxe (1964) examined the distribution of monoamine-containing cell bodies. Catecholamine neurons were present primarily in the ventral and lateral reticular formation of the medulla, pons, and midbrain. For the most part these appeared to be norepinephrine containing, but dopamine-containing cells were identified in the substantia nigra and adjacent ventral tegmental area (groups A9 and A10 of Dahlstrom and Fuxe, 1964) and in the arcuate nucleus of the hypothala-

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ROBERT Y. MOORE

mus. Serotonin-containing cells were found almost exclusively in the medial reticular formation, particularly in the raphe nuclei, from medulla through the midbrain. With the exception of the arcuate nucleus, no monoamine cells were demonstrated in the diencephalon, telencephalon, or spinal cord. In contrast to this restricted distribution of monoaminecontaining perikarya, 1.arge numbers of terminals were found in nearly all areas of the central nervous system from the neocortex (Fuxe, 1965; Fuxe et al., 1968a) and subcortical telencephalic nuclei through the diencephalon, midbrain, pons, and medulla (Fuxe, 1965), and the spinal cord (Dahlstrom and Fuxe, 19%). The conclusions that could be drawn from this data were suggested by the work of Dahlstrom and Fuxe (1965) on the bulbospinal monoamine neurons. Transection of the spinal cord resulted in loss of histochemically demonstrable terminals distal to the lesion. Proximally, fluorescent material accumulated in the axon stumps, and this finally came to outline the entire neuron above the section. The application of this to the remaining cell bodies and terminals would then suggest that the terminals in the brainstem, diencephalon, and telencephalon are all derived from the cell bodies located in the brainstem. The first experimental test of this was provided by A n d h et al. (1964, 1965a, 1966) on a system of dopamine-containing neurons which appeared to be identical to the nigrostriatal pathway of the older neuroanatomical literature. Interest in this pathway had developed rapidly following the demonstration that dopamine was present in the neostriatum in high concentration (Bertler and Rosengren, 1959) and that the brains of individuals suffering from Parkinson’s disease had markedly reduced dopamine levels in the neostriatal nuclei and substantia nigra ( Ehringer and Hornykiewicz, 1960; Hornykiewicz, 1962). Since the primary pathology of Parkinson’s disease is present in the substantia nigra, these observations suggested that the pathophysiology of the disease involved disturbance of a dopaminergic nigrostriatal pathway ( Hornykiewicz, 1966). Experimental destruction of the substantia nigra results in a loss of dopamine from the neostriatum (Poirier and Sourkes, 1965; Poirier et al., 1967; Anden et al., lW),but attempts to demonstrate a nigrostriatal pathway by conventional anatomical methods have not been successful (Carpenter and McMasters, 1964; Cole et al., 1964). Using the Falck-Hillarp method, however, AndCn et al. (1964, 1965a, 1966) have provided substantial evidence for a nigrostriatal pathway in the rat brain. Lesions in the substantia nigra cause a substantial loss in catecholamine fluorescence from the caudate-putamen complex ( AndCn et al., 1964). Ablation of the neostriatum results in an accumulation of fluorescent material in axons of the internal capsule and cells of the pars compacta of the substantia nigra (AndBn et al., 1965a). The course of


81

the projection appears to be through the subthalamic region and the retrolenticular part of the internal capsule (AndCn et al., 1965a, 1966). Our own observations in the cat are in accord with these and provide conclusive anatomical and chemical evidence for a nigrostriatal, dopaminergic pathway. As an extension of our studies on the medial forebrain bundle, we performed one experiment in which caudate nucleus dopamine content was determined following lesions in the region of the lateral hypothalamus in the cat (Moore and Heller, 1969; Table VI). After destruction of the medial forebrain bundle and the adjacent medial internal capsule, there is nearly total depletion of dopamine in the ipsilateral caudate nucleus. Associated with this there is severe retrograde degeneration in the pars compacta of the substantia nigra and the adjacent ventral tegmental area, If the lesion is restricted to the medial internal capsule, however, the decrease in dopamine is less, and retrograde degeneration is observed only in the substantia nigra, pars compacts. Subsequent studies have demonstrated also that there is a loss of the enzymes tyrosine hydroxylase and dopa decarboxylase in the caudate nucleus which is directly proportional to the loss of dopamine (Moore et al., 1969). This situation, then, is nearly identical to that seen with peripheral denervations and provides presumptive evidence that there is a nigrostriatal pathway arising from the ventral tegmental area and the pars compacta of the substantia nigra and traversing the medial internal capsule and adjacent medial forebrain bundle on its path to the caudate nucleus. Anatomical evidence for this was obtained in another study using the Fink-Heimer method ( Moore, 1969). Following restricted lesions in the substantia nigra and ventral tegmentum, degenerating axons were traced through the ventral tegmentum into the ventral portion of the field H or Forel. At rostra1 tuberal levels of the hypothalamus, the fibers came to run in the medial internal capsule and adjacent medial TABLE VI CAUDATE NUCLEUS DOPAMINE CONTENT FOLLOWING UNILATERAL DESTROYING COMPONENTS O F THE NIGROGTRIATAL PATHWAY" Percent difference in dopamine content from control caudate nucleus

Area destroyed Medial hypothalamus Medial internal capsule Medial internal capsule and medial forebrain bundle a

From Moore and Heller (1967). significant, p

* Differences statistically

< .01.

-5.1 -62.4* -92.3'

82

ROBERT Y. MOORE

FIG. 2. Terminal degeneration in the cat caudate nucleus following unilateral destruction of the nigrostriatal pathway is shown in A. B is taken from the control caudate nucleus, The large black area is an impregnated blood vessel. Fink-EIeimer stain, X960.

BRAIN LXSIONS AND AMINE METABOLISM

83

forebrain bundle before distributing ventrolaterally through the entopeduncular nucleus and globus pallidus into the putamen or dorsomedially into the caudate nucleus. Using this impregnation method with a 5-day survival period, large numbers of degenerating terminals were demonstrated in the neostriatum (Fig. 2 ) . The combination of the anatomical findings (from both anterograde and retrograde degeneration) and the neurochemical data, thus, gives full confirmation for the observations using the histochemical method. The demonstration of the pathway by anterograde degeneration is also of importance because it illustrates that a monoaminergic pathway may be shown by conventional neuroanatomic techniques. Following their observations on the nigrostriatal pathway the Swedish workers undertook an extensive series of experiments examining the effects of lesions on brain monoamines ( A n d h et al., 196513, 1966, 1967; Fuxe et al., 1968b; Hillarp et al., 1966). In each case these were taken to demonstrate that the serotonin and norepinephrine present in the forebrain occurred in terminals projecting to these areas from cell bodies in the brainstem. The proposed pathway for these neurons has been schematized (Andkn et al., 1966; Fig. 10). From the lateral brainstem cell bodies norepinephrine-containingaxons ascend in the medial forebrain bundle to distribute to the hypothalamus, thalamus, striatum, limbic forebrain, and neocortex. An identical pathway is proposed for serotonin except that the cell bodies are placed more medially. The evidence for the pathways is the same. Following a lesion in the tegmenturn or in the medial forebrain bundle, there is a loss of terminals distal to the lesion (i.e., in the forebrain) and an appearance of accumulation of fluorescent material in proximal cell bodies. Because these events are similar to those occurring with lesions in the nigrostriatal tract and the reticulospinal system, it was inferred that the mechanism of amine loss following tegmental and medial forebrain bundle lesions also represented degeneration of amine-containing neurons.

5. Direct Denewation vs. Transsynaptic Efects From our studies, and those of the Swedish group, it was evident that restricted subcortical lesions could produce widespread losses of monoamines from the forebrain. Yet, despite the amount of study devoted to this problem, the mechanism by which the amine loss takes place is not entirely understood. In our earlier studies we proposed the view that this represented degeneration of amine-containing neurons, but a failure to find anatomical correlates for the distribution of the amine loss led us to suggest that the effects were, at least in part, transsynaptic. The basis for this was, of course, the negative evidence regarding direct neural pro-

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ROBERT Y. MOORE

jections from the areas destroyed by the lesion, particularly to the neocortex. But, as Bloom (1968) has noted, both groups have had to depend on negative evidence. The conclusions of the Swedish group have largely been based on their failure to demonstrate monoaminecontaining cell bodies above the brainstem, save for those innervating the median eminence. The absence of diencephalic and telencephalic monoamine cell bodies has been taken to indicate that none exists and, hence, that the terminals shown by the Falck-Hillarp method in the forebrain arise from brainstem neurons. This raises again the question of the sensitivity of the technique; that is, whether all monoamine-containing perikarya are demonstable in the brain even with pharmacological manipulation. There is no definitive answer to this question at present, but it is well to bear in mind Fuxe’s (1965) point that failure to demonstrate monoamine-containing neurons does not mean that they do not exist. Another question which can be raised concerns the exploitation of the technique. Several years ago catecholamine-containing cell bodies were observed in the olfactory bulb (Bertler et al., 1%6), and more recently Fuxe and Hokfelt (1969) have reported cell bodies in the subthalamic region. Those in the olfactory bulb are of particular interest in that they are the only ones thus far described in the telencephalon, and removal of the olfactory bulb produces changes in brain monoamines which have been interpreted to be transsynaptic (Pohorecky et al., 1969). It was evident from our initial experiments that the most compelling model for the central lesion effects has been the peripheral denervation studies. In the central nervous system the nigrostriatal pathway has conformed best to this model. The major points of similarity are as follows. Destruction of the pathway either at its origin in the substantia nigra or along its course causes a loss in neostriatal dopamine which is proportional to the number of nigrostriatal fibers transected. Complete transection of the pathway results in nearly complete loss of dopamine from the neostriatum (Moore and Heller, 1969). The loss of amine is accompanied by a loss of synthetic enzymes (tyrosine hydroxylase, dopa decarboxylase) which is also proportional to the extent of the lesion as well as to the amine loss (Moore et al., 1969). The time course of the amine loss in this system is not known, but it can be presumed to be in accord with that for degeneration of the fiber (about 5 days; Moore, 1!368). Finally, the origin, course, and distribution of the pathway can be demonstrated by conventional anatomical methods ( Moore, 1969). These observations can be compared with the situation which holds for the effects of medial forebrain bundle or tegmental lesions. After such lesions the loss of serotonin or norepinephrine never significantly exceeds 75%in any area of the forebrain regardless of the size or location of the

BRtlIN LESIONS AND AMINE METABOLISM

85

lesion. Of itself, this observation would imply that approximately 25%of the total monoamine pool is present in neurons with both cell bodies and terminals in the forebrain. Other evidence supporting this view has come from the studies of Parent et al. (1969) who observed only partial depletion of hypothalamic norepinephrine with extensive, bilateral lesions in the midbrain tegmentum. From their observations these authors concluded that the synthesis of hypothalamic norepinephrine takes place independently of central pathways to the hypothalamus. Further evidence that hypothalamic catecholamine- and tryptamine-containing terminals do not all arise from the midbrain tegmentum of the periventricular and arcuate nuclei has been presented by Bjorklund et al. (1970) in an elegant analysis of the monoaminergic innervation of the median eminence. In the analysis of the nigrostriatal pathway, the use of neostriatal lesions facilitated a description of the pathway by producing an accumulation of fluorescent material in the proximal axon (And& et aZ.,1965b, 1966). The application of this technique to the purported projections from medial forebrain bundle to the neocortex appears to present some problems. In our studies (Moore and Heller, 1967) as well as those of others (Wolf and Sutin, 1966), axons have been traced from the medial forebrain bundle through the septum into the hippocampal rudiment above the corpus callosum. Anden et al. (1967) have proposed that this pathway constitutes the route by which monoamine-containing neurons project terminals onto the neocortex. There is, as yet, no direct anatomical evidence for a pathway beyond the hippocampal rudiment, but the proposal is open to experimental test. To do this, we produced large, bilateral lesions in the septa1 area (cf. Harvey et al., 1964; Heller et al., 1962; for full descriptions) which completely transected all ascending fibers from the medial forebrain bundle into the septum. The effects of these lesions on telencephalic monoamines are shown in Table VII. The decreases in neocortical amines are quite modest and less than those observed with medial forebrain bundle lesions, and the only marked change was in hippocampal serotonin content, Two conclusions might be drawn from this data: first, that the pathway proposed by AndCn et a2. (1967) was incorrect or, second, that the decreases in amine content produced by these and other lesions are the result of mechanisms other than direct degeneration of amine-containing neurons. The time course of the amine falls produced by medial forebrain bundle lesions is also of interest in this context (cf. Section 111,B; Fig. 1).Unlike the time course for the loss of tissue catecholamines following peripheral denervation, central amines are lost slowly and, indeed, the process takes longer than would be expected for the axons destroyed to

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ROBERT Y. MOORE

TABLE VJI REGIONAL EFFECTSOF SEPTALAREA LESIONSON SEROTONIN AND NOREPINEPHRINE I N THE RAT BRAIN. ~

~-

Percent difference from control Region assayed

Serotonin

Norepinephrine

Neocortex Amygdala Hippocampns Diencephalon Brainstem

-15b $2 -40b

- 26b

- -

a

From Heller and Moore (1968). Differences statistically significant, p

+14 +9

-10 -9 +10 +2

< .05.

undergo terminal degeneration. In general, central terminal degeneration takes place rapidly (Heimer, 1967) and in the cerebral cortex, for example, Colonnier ( 1964) has observed complete degeneration and phagocytosis of terminals within 9 days after all afferents were transected by undercutting. This can be compared with the 12 days required for complete amine fall following medial forebrain bundle lesions. The medial forebrain bundle does contain many very small axons, but the evidence now available indicates that these would degenerate more rapidly than larger fibers (Bloom, 1968). A further difference between the nigrostriatal lesion effects and the tegmental or medial forebrain bundle effects is apparent in contrasting amine and enzyme data from the two types of lesion. With nigrostriatal lesions, the loss of amines and synthetic enzymes occurs equally and proportional to the amount of pathway destroyed. A similar analysis on the other lesions is shown in Table VIII. With the medial forebrain bundle lesions the loss of dopa decarboxylase is much less than that of either amine. After lateral tegmental lesions, dopa decarboxylase is unaffected. This could be explained if the enzyme was present primarily in serotonin-producing neurons, but the general picture in this study was one of a diversity of effects. It should be noted, however, that the telencephalon is a very heterogeneous tissue in comparison with the neostriatum and the diversity of effects may reflect this. All of the information just outlined has contributed to the problem of interpreting the effects of medial forebrain bundle and tegmental lesions on brain amines. Nevertheless, the major discrepancy has arisen from anatomical considerations, and these should be reviewed carefully. The direct denervation hypothesis requires that there be direct projections from the lateral hypothalamus or tegmentum to diffusely and homogene-

87


TABLE VIII DIFFERENTIAL EFFECTS OF M E ~ I AFOREBRAIN L BUNDLE AND VENTROLATERAL MIDBRAIN TEGMENTUM LESIONSO N TELENCEPHALIC MONOAMINES AND ENZYMES IN THE RAT^ Percent difference in telencephalic level from sham operated control

Lesion group Medial forebrain bundle Ventrolateral midbrain tegmen turn

Serotonin

Dopa Tyrosine Norepinephrine decarboxylase hydroxylase

-66b - 12

-51b -7Bb

-2Sb -5

-44b -30b ~

From Heller and Moore (1968). I , Differences statistically significant, p .05.

< .01. All other values not

significant, p

>

ously innervate the entire forebrain. Attempts to demonstrate these projections using conventional anatomical techniques have failed thus far (Moore and Heller, 1967; Heller and Moore, 1968). The use of a newer technique, the Fink-Heimer method (Fink and Heimer, 1967), has allowed demonstration of the nigrostriatal pathway and a wider distribution of medial forebrain bundle axons in the amygdala and septum than was previously known. We have not been able, however, to show axons to the neocortex, but this negative data must be taken skeptically until more extensive studies are carried out. It seems likely that some conventional anatomical technique would allow demonstration of at least the terminals of a medial forebrain bundle-neocortical pathway if such existed, as the terminals should be well within the resolving power of the light microscope (Guillery and Ralston, 1964; Alksne et al., 1966; Heimer, 1967; Heimer and Peters, 1968).In view of the recent demonstration of the nigrostriatal pathway, it cannot be stated that monoamine-producing neurons are refractory to conventional anatomical techniques. Electron microscopy is not a useful tool in surveying an area for axon terminals ( Alksne et aZ,, 1966), but it may be quite important if a localized area can be analyzed ( Aghajanian et al., 1969) and the electron microscopic criteria for axonal degeneration are carefully reviewed (Cohen and Pappas, 1969) , Although a reasonable doubt must be maintained, we have taken the bulk of available evidence to support the view that the loss of monoamines from the forebrain after medial forebrain bundle or tegmental lesions occur, in part, in neurons not directly damaged by the lesion. This, then, is an effect which is mediated across at least one synapse and

88

ROBERT Y. MOORE DIRECT DENERVATION EFFECT AREA OF AMINE LOSS LESION

TRANS-SYNAPTIC EFFECT 0

I -El

AREA OF AMINE LOSS

LESION

FIG.3. Diagrammatic representation of two possible mechanisms for amine loss following a brain lesion. In direct denervation the lesion results in degeneration of the destroyed elements. A transsynaptic mechanism proposes an alteration in amine metabolism in neurons whose innervation has been altered by a distant lesion.

which we have termed transsynaptic. The alternative views are represented diagrammatically in Fig. 3. It should be emphasized that this mechanism would account for only part of the amine loss; there is ample histochemical, chemical, and anatomical evidence to interpret the loss of amines from septum, for example, as a result of direct denervation. If the transsynaptic effect is accepted for part of the amine loss, two interesting questions arise. First, what is the state of the affected neurons? Transneuronal degeneration is not unknown in the central nervous system, but it occurs principally in sensory systems and there is no histological evidence to suggest it here. The most attractive hypothesis is that, as in the decentralized sympathetic neuron with a decreased norepinephrine content, the functional state of the central monoaminergic neuron is altered, but it is morphologically intact. The nature of the functional alteration would remain, then, open to investigation. The second question would relate to the possibility of returning the altered metabolic status of the affected neurons to normal. If this were possible, it would add another facet to the understanding and treatment of functional deficit resulting from central lesions. IV. Conclusions

Regardless of the interpretation of lesion effects on brain amine metabolism, the effects themselves are striking and should have marked functional consequences. The diffuse distribution of the effects of these subcortical lesions is of particular interest, for it returns us to Lashley’s


89

warning noted at the beginning of this review. Lashley was concerned that very complex functions were being ascribed to primitive subcortical structures, and he felt that such functions would be mediated through participation of both subcortical and cortical systems. The importance of the subcortical regions focused upon by this review is without question. They are critical, for example, to the maintenance of food and water intake (Teitlebaum and Epstein, 1962; Morgane, 1969), the mediation of reinforcement mechanisms (Olds, 1962), and the control of sleep patterns (Jouvet, 1969). In all likelihood amine metabolism plays only a part in these functions, but the studies on subcortical control of forebrain amine metabolism reviewed here provide a firm foundation for understanding the interdependent participation of cortical and subcortical structures in the mediation of complex functions, ACKNOWLEDGMENTS The work reported here was supported by research grants MH-04954, NS05002, and HD-04581 from the National Institutes of Health, USPHS, and was carried out in collaboration with Dr. Alfred Heller, Department of Pharmacology, The University of Chicago. The author has been supported by a Research Career Development Award (K3-NB-7, 389) from the National Institute of Neurological Diseases and Blindness and is a John and Mary R. Markle Scholar in Medical Science. REFERENCES Aghajanian, G. K., Bloom, F. E., and Sheard, M. D. (1969). Brain Res. 13, 266. Alksne, J. F., Blackstad, T. W., Walberg, F., and White, L. E., Jr. (1966). Ergeb. Anut. Entwicklungsgeschichte 39, 1. AndBn, N. E., Carlsson, A., DahIstrom, A., Fuxe, K., Hillarp, N. A., and Larsson, K. ( 1964). Life Sci. 3, 523. AndBn, N. E., Dahlstrom, A., Fuxe, K., and Larsson, K. (1965a). Am. J . Anat. 116, 329. AndBn, N. E., Dahlstrom, A., Fuxe, K., and Larsson, K. (1965b). Life Sci. 4, 1275. AndBn, N. E., Dahlstrom, A., Fuxe, K., Larsson, K., Olson, L., and Ungerstedt, U. (1966). Actu Physiol. Scund. 67, 313. And&, N. E., Fuxe, K., and Ungerstedt, U. (1967). Experientia 23, 838. Berman, A. L. (1968). “The Brain Stem of the Cat: A Cytoarchitectonic Atlas with Stereotaxic Coordinates.” Univ. of Wisconsin, Madison, Wisconsin. Bertler, A., and Rosengren, E. (1959). Erperientiu 15, 10. Bertler, A., Falck, B., Owman, C., and Rosengren, E. (1966). Phurmcol. Rev. 18, 369. Bjorklund, A,, Ehinger, B., and Falck, B. (1968). J. Hktochem. Cytochem. 16, 263. Bjorklund, A,, Falck, B., Hromek, F., Owman, C., and West, K. A. (1970). Bruin Res. 17, 1. Bleier, R., Bard, P., and Woods, J. W. (1966). J . Comp. Neurol. 128, 255. Bloom, F. E. (1968). Adoun. Pharmcol. 6A, 207. Bogdanski, D. F., Weissbach, H., and Udenfriend, S . (1957). J . Neurochem. 1, 272. Brady, J . V., and Nauta, W. J. H. (1953). J. Comp. Physiol. Psychol. 46, 339.

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MORPHOLOGICAL AND FUNCTIONAL ASPECTS OF CENTRAL MONOAMINE NEURONS By Kjell Fuxe, Tomas Hokfelt, and Urban Ungerstedt Department of Histology, Karolinska Inrtitutet, Stockholm, Sweden

I. Morphology of Central Monoamine Neurons . . . . A. Methods to Map Out Monoamine Pathways . . . B. DA Pathways . . . . . . . C. NA Pathways . . . . . . . D. 5-HT Pathways . . . . . . E. Electron Microscope Studies on Central Monoamine Neurons 11. Function of Central Monoamine Neurons . . . A. DA Pathways . . . . . . . . . B. NA Pathways . . . . . . C. 5-HT Pathways . . . . 111. Some Conclusions . . . . . . . . A. Nigro-Neostriatal DA Neurons . . . . B. The Tubero-Infundibular DA Neurons . . . . C. The Central NA Neurons . . . D. The Central 5-HT Neurons . . . . References . . . .

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In recent years several review articles have been published on the central monoamine neurons (see articles by Fuxe et aZ., 1968b, 1969a, 1970a, 1970c; A n d h et aZ., 1969a,b; Fuxe and Hokfelt, 1969). The present article will deal with work carried out in 1968 and 1969, especially in our laboratory, which has not been summarized in previous review articles. A major part of the work has been done in cooperation with the department of Pharmacology, Goteborg, Sweden (Head: Prof. A. Carlsson). I. Morphology of Central Monoamine Neurons

The detailed distribution of CA' and 5-HT nerve cell bodies and nerve terminals in the mammalian central nervous system has been given in previous articles (Dahlstrom and Fuxe, 1964b; Fuxe, 1965; Fuxe et aZ., 1968b, 1969a) using the histochemical fluorescence technique ( Falck et al., 1962; Falck, 1962; Hillarp et al., 1966; Corrodi and Jonsson, 1967). The cell bodies have a low amine concentration, whereas the terminals Abbreviations: CA = catecholamine( s ) , DA = dopamine, 5-HT = 5-hydroxytryptamine, &OH-DA = 6-hydroxy-dopamine, NA = noradrendine. 93

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have high concentrations of monoamines. The axons, on the other hand, have very low amine concentrations so that they are barely visible in the normal animal. The vast majority of the NA cell bodies are localized in the pons and the medulla oblongata mainly in the lateral part of the reticular formation, whereas the DA cell bodies are exclusively localized in the mesencephalon with the exception of some DA cell body groups in the hypothalamus (see Fuxe and Hokfelt, 1969). The 5-HT cell bodies are localized mainly in the raphe complex of the reticular formation. The localization of the monoamine cell bodies has been confirmed for the cat by Jouvet’s group (Pin et al., 1968). The NA and 5 H T nerve terminals have a widespread distribution in the brain and spinal cord. The NA nerve terminals are found in high densities in many areas of the hypothalamus, the preoptic area, the limbic system, and the lower brainstem (see Fuxe, 1965; Fuxe et al., 1969a). The 5-HT nerve terminals have a similar distribution but are more difficult to detect than the NA nerve terminals, especially in the di- and telencephalon mainly because of the low sensitivity of the histochemical fluorescence method for 5-HT compared to that for CA (see Jonsson, 1967; Fuxe and Jonsson, 1967). Thanks to the discovery that 6-hydroxytryptamine (Jonsson et al., 1969), which gives a stronger fluorescence reaction than 5-HT (Jonsson and Sandler, 1%9), has a high affinity for 5-HT nerve terminals and can be made to accumulate only in the 5-HT and not in the CA neurons with the help of pharmacological tools, it has now become possible to perform detailed mapping of the distribution of 5-HT nerve terminals also (see e.g., Jonsson et al., 1969; Hokfelt and Fuxe, 1969).

A. METHODSTO MAP OUTMONOAMINE PATHWAYS Before describing these pathways in some detail the principal methods used to map out monoamine neurons will be mentioned. 1. A common method is to damage the axons with the help of precise stereotaxic lesions and to study the subsequent anterograde and retrograde degeneration of the neurons (see A n d h et al., 1966a). The anterograde degeneration involves the destruction of the amine-storage mechanism in the granules after 2-4 days, somewhat preceded by the breakdown of the membrane pump mechanism in the nerve cell membrane (see And& et al., 1966a; Hokfelt and Ungerstedt, 1969; Fuxe et al., unpublished data). Similar results have been observed in studies on the anterograde degeneration of peripheral adrenergic neurons ( Malmfors and Sachs, 1965). 2. Of great help in tracing the pathways has also been the fact that

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monoamines accumulate in the proximal part of the axons (cell body side) after an electrothermic lesion, owing to the rapid transport of amine storage granules from the cell bodies to the terminals via the axons (Dahlstrom and Fuxe, 1964a,b; Dahlstrom, 1965; Kapeller and Mayor, 1967). An accumulation of aniines in the axons can also be obtained by injecting colchicine over the monoamine axons and cell bodies in the peripheral nervous system ( Dahlstrom, 1968). Thus, stereotaxic injections of colchicine into various pathways can also be used to map out various monoamine neuron systems ( Ungerstedt, unpublished data). 3. A new tool of importance for mapping out monoamine neurons is 6-OH-DA. This drug was discovered by Tranzer and Thoenen (1968) to cause a degeneration of peripheral adrenergic neurons. Later on, Ungerstedt (1968) and Uretsky and Iversen (1969) found that 6-OH-DA could cause degeneration of central NA neurons. Thus, Ungerstedt (1968) also found a complete degeneration of the nigro-neostriatal DA neurons after intracerebral injections of 6-OH-DA into the substantia nigra. The nigral DA cell bodies markedly accumulate 6-OH-DA by way of the membrane pump, after which they .rapidly degenerate, possibly owing to the fact that 6-OH-DA is a strong reducing agent. Similar injections close to NA pathways cause anterograde degenerations of the NA neurons also. Furthermore, at the site of injection a damming up of fluorescent material can be seen in the NA axons on the cell body side offering possibilities of tracing the pathways (see above). The 5-HT neurons do not seem to be seriously affected by 6-OH-DA. 4. During the early postnatal period (Maeda and Dresse, 1968; Olson and Fuxe, to be published) there is an increased fluorescence intensity in the CA axons, which makes it easier to trace them also in the untreated animal, i.e., without performing studies of the types described in the previous three points. 5. Of great help in mapping out the axonal pathways have also been in uitro studies on brain slices with monoamines in the incubation bath. Owing to the reserpine-resistant uptake-concentration mechanism at the nerve cell membrane (Hillarp and Malmfors, 1964; Fuxe and Hillarp, 1964; Carlsson and Waldeck, 1965; Hamberger and Masuoka, 1965), the axons will become strongly fluorescent and easy to trace (Hamberger, 1967; Hokfelt and Fuxe, 1969, and unpublished data) provided that the intraneuronal monoamine oxidase (MAO) has been inhibited, or a-methylated amines have been used. 6. A new approach which recently has been used in our laboratory is to stimulate electrically the various monoamine pathways and cell body groups on one side of the brain and to study the increased amine depletion obtained in the terminals belonging to the stimulated mono-

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amine neurons after inhibition of amine synthesis (Arbuthnott et al., unpublished data). 7. A pure pharmacological approach can also be helpful to evaluate the localization of the pathways. Thus, nialamide or other M A 0 inhibitors will cause a marked accumulation of amine in the central 5-HT neurons. Similarly, after nialamide-dopa treatment the CA nonterminal axons become clearly fluorescent owing to accumulation of CA formed from dopa (Dahlstrom and Fuxe, 196413, 1965; Fuxe, 1965).

B. DA PATHWAYS 1. Nigro-neostriatal DA neurons (And& et al., 1964a, 1965a, 1966a,b; Poirier and Sourkes, 1965; Ungerstedt, 1968; Hokfelt and Ungerstedt, 1969). 2. Mesolimbic DA neurons (And& et al., 1966a; Dresse, 1966). 3. Tubero-infundibuhr DA neurons (Fuxe, 1963, 1964; Fuxe and Hokfelt, 1966, 1967, 1969; Lichtensteiger and Langemann, 1966; Bjorklund, 1970). 1. The main nucleus of the nigro-neostriatal DA neurons is the zona compacta of the substantia nigra. Most of the cell bodies in this zone are DA nerve cell bodies (group A9; Dahlstrom and Fuxe, 196410). Some DA cell bodies are also found in the zona reticulata and the pars lateralis of the substantia nigra, which also belong to the nigroneostriatal DA neurons. Recently, studies after removal of n. caudatus putamen (NCP) also suggest that the CA cell bodies in the ventrolateral part of the midbrain tegmentum (group A8; Dahlstrom and Fuxe, 1964b) belong to this large uncrossed neurons system, inasmuch as they show marked reductions in fluorescence intensity and signs of atrophy after such operations ( Ungerstedt, unpublished data). These CA cell bodies may therefore also contain DA. In agreement with this view these CA cell bodies, together with most of the DA cell bodies of the substantia nigra, are the ones that show the most marked increases in fluorescence intensity following treatment with dopa (Butcher et al., 1970). Furthermore, after removal of the neostriatum there are no longer any marked increases in the fluorescence intensity of the DA cell bodies in groups A 9 and A 8 after dopa treatment but only in those surrounding the n. interpeduncularis (group A10) (Dahlstrom and Fuxe, 1964b) giving evidence for the view that both these CA cell body groups give rise to the nigro-neostriatal DA pathway. Evidence has been obtained by Poirier’s group ( Poirier, personal communication) that there may exist a medial and lateral nigro-neostriatal DA pathway innervating the cranial medial and caudal lateral part of the neostriatum respectively. In support of this view are the facts that the DA cell bodies in the


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medial part of the zona compacta and the DA terminals of the medial part of the neostriatum do not show as marked an increase in fluorescence intensity after dopa as do the DA-containing cell bodies and terminals in the lateral part of the zona compacta and the neostriatum respectively (Butcher et al., 1WO). A rough quantitative study of the nigro-neostriatal DA neurons (And& et al., 1966b) has revealed that the total number of DA cell bodies on one side was about 3500. The average amount of DA in each cell body was 2.5 pg with a concentration around 200 pglgm. Each of the DA varicosities of the terminals contained 2.5 x lo-' pg with a concentration of 8000 pg/gm wet weight. These figures are of the same order as those calculated for the peripheral adrenergic neurons (Dahlstrom, 1966). The calculations furthermore suggested that the total terminal system of each neuron was about 65 cm in length and contained on the average 500,000 varicosities, illustrating the high degree of divergence in the innervation pattern. This must be of high functional importance, since it allows nerve impulse activity in one DA neuron to influence a large number of nerve cells. The DA fibers to the neostriatum are caudally aggregated first medial to the crus cerebri. Then they diverge into the rostra1 crus cerebri and the retrolenticular part of the capsula interna and spread into the NCP by way of the fibrae capsulae internae. The DA nerve terminals are densely packed and very fine (Fuxe et al., 1964). Electron microscopically about 15%of all boutons in this nucleus have been found to belong to monoamine neurons (Hokfelt, 1968a). The varicosities are mainly 0.5-1 p in diameter . 2. The mesolimbic DA neurons have their cell bodies in the area surrounding the n. interpeduncularis (Anden et al., 1966a). After destruction of this CA cell body group (group A10; Dahlstrom and Fuxe, 1964b), the DA nerve terminals in the tuberculum olfactorium, n. accumbens, the dorsolateral part of the n. interstitialis striae terminalis and in the n. amygdaloideus centralis degenerate. Furthermore, several months after destruction of these parts of the limbic system, the DA cell bodies of group A10 have disappeared or appeared shrunk and atrophied with a very low fluorescence intensity. Caudally the DA fibers of this system run medially to the nigro-neostriatal DA fibers, whereas cranially they mainly lie ventral to the nigro-neostriatal DA fibers. In the tuberculum olfactorium the DA nerve terminals are found in somewhat higher density in the area surrounding the outer cell body layer. With the exception of the part of the n. accumbens close to the medial side of the anterior limb of the commissura anterior, the DA nerve terminals in the tuberculum olfactorium and n. accumbens have a somewhat lower

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intensity than those present in the neostriatum. Because the number of the DA cell bodies in group A10 are of about the same order as that found in group A9 and 80% of the brain DA is found in the neostriatum, the mesolimbic DA neurons probably contain less amounts of DA than the nigro-neostriatal DA neurons. This is probably due to the fact that the terminal network of each mesolimbic DA neuron is much smaller than that of the nigro-neostriatal DA neurons. 3. The tubero-infundibular DA neurons have their cell bodies mainly localized to the anterior part of the n. arcuatus. The axons run ventrally toward the lateral border of the median eminence. Some of them enter directly the external layer caudally. In the external layer the axons give rise to a densely packed plexus of DA nerve terminals, which probably exert an axo-axonic influence in this layer (Hokfelt, 196713, 1968b). The DA terminal plexus is present also in the external layer of the infundibular stem. Some may be found also in the posterior lobe and in the pars intermedia (Bjorklund, 1968; Fuxe and Hokfelt, 1969).

C. NA PATHWAYS 1. Ascending NA Neurons a. Dorsal NA pathway ( Fuxe, 1965; Ungerstedt, unpublished data). In the first publications it was discovered that there existed a distinct fascicle of bundles of NA axons with varicose-like enlargements in the dorsolateral tegmentum lateral and slightly ventral to the fasciculus longitudinalis medialis, which abruptly turned ventrally at the border between di- and telencephalon to pass laterally of the fasciculus retroflexus into the subthalamus (mainly zona incerta and Forel’s field H I ) and into the lateral hypothalamic area. By consecutive unilateral sections of the brain at various levels this pathway has now been mapped out in detail ( Ungerstedt, unpublished data), and evidence was obtained that this bundle mainly originated from the NA cell bodies of the locus coeruleus. Part of the bundle could be traced to the most cranial part of the medial forebrain bundle. Further evidence for this view has been obtained in studies on rats in the postnatal period (Olson and Fuxe, unpublished data) and on brain slices after incubation with CA (Hokfelt and Fuxe, unpublished data). Also in these experiments it was possible to trace some axons of the dorsal bundle into the most cranial part of the medial forebrain bundle. Furthermore, most of the axons of the dorsal bundle could be directly traced back to the NA cell bodies of the locus coeruleus. It should be pointed out that these NA cell bodies probably have some large axon branches which participate in the innervation of the lower brainstem. Recently, unilateral lesions of the

ASPECTS OF CENTRAL MONOAMINE NEURONS DORSAL PATHWAY

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FIG.1. Schematic drawing of the principal construction of the central NA pathways. The huge collateral system of these NA pathways is illustrated. The dorsal ascending NA pathway mainly innervates the cortex cerebri and the hippocampal formation, and the ventral NA pathway mainly innervates the hypothalamus and the ventral parts of the limbic system. dorsal bundle has been made in the tegmentum (Ungerstedt, unpublished data). The results indicate mainly degeneration of the NA nerve terminals in the cortex cerebri and the hippocampal formation. In agreement with this, it has been found that electrical stimulation of the dorsal bundle will cause preferential release of amine from the NA nerve terminals in the cortex cerebri and the hippocampal formation (Arbuthnott et al., unpublished data). b. Ventral N A pathway (Anddn et al., 1966a,b; Fuxe, 1965; Ungerstedt, unpublished data). This pathway probably arises mainly from the NA cell bodies in the n. reticularis lateralis ( A l ; Dahlstrom and Fuxe, 1964b), in tr. rubrospinalis at the level of the superior olive (A5; Dahlstrom and Fuxe, 1964b), and in the reticular formation ventral to the peduncularis cerebelleris superior at the level of the n. motorius dorsalis n. vagi (A7; Dahlstrom and Fuxe, 1964b). Recently, this pathway has been described in detail by Ungerstedt (unpublished data). By use of 6-OH-DA injections it was possible to trace the bundles of axons from their site of origin (groups Al, A5, and A7) all the way into the medial forebrain bundle. The fibers came together in the pons where they aggregate medial to the outgoing fibers of the facial nerve. On their ascent they pass medial to motor nucleus V lying in the lateral part of the reticular tegmentum and turn medially along the dorsal surface of

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the lemniscus medialis at the level of the caudal third of the substantia nigra. Before reaching the n. interpeduncularis they again turn rostrally and reach the medial forebrain bundle via the area ventralis tegmenti ( Ungerstedt, unpublished data). From lesion experiments ( Ungerstedt, unpublished data) there is strong support for the view that these axons mainly innervate the hypothalamus, the preoptic area, and many parts of the limbic system (n. interstitialis striae terminalis, ventral part; septa1 area). In agreement with this, it has been found that electrical stimulation of the ventral NA bundle at the level of n. interpeduncularis in the lateral part of the reticular formation causes mainly release of amines from NA nerve terminals in the hypothalamus, preoptic area, and the ventral parts of the limbic forebrain ( Arbuthnott et aZ., unpublished data ) .

2. Descending NA Neurons (Carlsson et al., 1964; Dahlstrom and Fuxe, 1965) The NA axons, which arise mainly from NA cell bodies in the n. reticularis lateralis ( A l ) , descend in the lateral and anterior funiculi. During their descent down the spinal cord they give off a large number of collaterals innervating the gray matter at various levels of the spinal cord. Inasmuch as many of the NA cell bodies also give rise to ascending axons (see above), it is probable that at least some of the NA cell bodies of group A1 have two main axons, one ascending and one descending axon.

3. NA Neurons Innervating the Lower Brainstem Since most of the NA cell bodies in the brain show retrograde cell body changes following lesions of the ascending NA pathways, it is likely that most of NA nerve terminals in the lower brainstem derive from collaterals of NA pathways ascending in the lower brainstem. It is, of course, possible that there exists a number of NA neurons which exclusively or mainly innervate the lower brainstem. The innervation of cortex cerebelli (Hokfelt and Fuxe, 1969) is, in many aspects, similar to that of cortex cerebri (Fuxe et d.,1968a) and hippocampal formation (Blackstad et aK, 1967). Thus, it is characterized by its diffuseness of innervation reaching almost all parts of the cortex cerebelli and lack of stratification in its innervation patterns. The morphological characteristics of the NA neurons are very similar to the reticular nerve cells described by Scheibel and Scheibel (1958) in Golgi preparations. These cells also have descending and/or ascending axons giving off a large number of collaterals to various nuclei of the lower brainstem. The functional significance of such nerve cells must be


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that these nerve cells can immediately influence neurotransmission in a large number of areas situated far apart at various levels of the brain and the spinal cord. In this way it would be possible to trigger off complicated behavioral or autonomic responses by increasing impulse traffic in the central NA neurons. Thus, the NA cell bodies of group A1 could immediately influence the transmission in the sympathetic lateral column, the dorsal motor nucleus V and the hypothalamus areas which all belong to the central autonomic system.

D. 5 H T PATHWAYS 1. Ascending 5-HT Neurons (Dahlstrom and Fuxe, 1964b; Fuxe, 1965; Heller and Moore, 1965; And& et al., 1966a) After treatment with nialamide, the 5-HT cell bodies in the raphe nuclei of the mesencephalon (n. raphe dorsalis, n. raphe rnedianus) and in the ventromedial reticular tegmentum can be seen to give rise to large numbers of ascending 5-HT axons, which run in the middle third of the tegmentum. First the axons run ventrally and then turn rostrally when approaching the n. interpeduncularis. The majority of the axons seem to lie close to the midline and become aggregated in a bundle lying medial to the fascicuIus retroflexus on the border between the mes- and diencephalon. These fibers enter the medial forebrain bundle by passing laterally close to the ventral outline of the fasciculus retroflexus, and most of them become aggregated close to the lateral surface of the fornix. In the lateral hypothalamic area there are also observed two other 5-HT bundles lying laterally which may originate from the lateral 5-HT bundles in the mesencephalon. One lies just beneath (ventral to) the most ventral part of the crus cerebri, the other one just dorsal to the lateral part of the optic tract. With the help of lesions of the lateral hypothalamic area and of the raphe nuclei, it has been possible to show that this pathway innervates large parts of the tel- and diencephalon, e.g., limbic forebrain structures and hypothalamus (And& et al., 196513, 1966a; Heller and Moore, 1965; Poirier et aZ., 1969). Also, after lesions of the raphe nuclei, electron microscopic signs of degeneration ( Aghajanian et aZ., 1969) have been observed in the suprachiasmatic nucleus, which is densely innervated by 5-HT nerve terminals (Fuxe, 1965). In agreement with this, it has been possible to show that electrical stimulation of the raphe nuclei results in increased formation of 5-hydroxyindoleacetic acid (5-HIAA) in the telencephalon ( Sheard and Aghajanian, 1968; Kostowski et al., 1969). Furthermore, indications of an increased release of 5-HT from the suprachiasmatic nerve terminals can be demonstrated histochemically after electrical stimulation provided that the amine synthesis is inhibited ( Arbuthnott et al., unpublished data).

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2. Descending Bulbospinal5-HT Neurons (Carlsson et al., 1964; Dahlstrom and Fuxe, 1965) This pathway ascends in the most superficial part of the lateral and anterior funiculus probably arising mainly from 5-HT cell bodies in the raphe nuclei of the medulla oblongata (n. raphe obscurus, B1 according to Dahlstrom and Fuxe, 1964b; n. raphe pallidus; B2) and the 5-HT cell bodies surrounding the pyramidal tract at the cranial level of the medulla oblongata. The 5-HT fibers, just like the NA fibers, give rise to large numbers of collaterals innervating the gray matter of the spinal cord. A particularly large projection of 5-HT axons exists to the lumbal and sacral part of the spinal cord. It should be mentioned that many of the 5-HT cell bodies of the medulla oblongata are innervated by NA nerve terminals as are also, but to a lesser extent, the NA cell bodies of group A l . These aminergic links should be remembered when discussing brain circuitary and interactions between NA and 5-HT neurons.

3. 5-HT Neurons Innervating the Lower Brainstem As discussed in the section on NA neurons, two possibilities exist. The 5-HT nerve terminals could derive from axons belonging to short 5-HT neurons or derive from collaterals of long 5-HT neurons. Probably both types of 5-HT neurons exist. The number of short 5-HT neurons compared to long 5-HT neurons is not known.

MICROSCOPE STUDIESON CENTRAL E. ELECTRON MONOAMINENEURONS Early electron microscopic studies on peripheral nervous tissues revealed the presence of so-called granular or dense core vesicles in probable noradrenergic neurons ( Lever and Esterhuizen, 1961; De Robertis and Pellegrino de Iraldi, 1961a,b; Taxi, 1961a,b; Richardson, 1962). At least two groups could be distinguished according to Grillo and Palay (1962)-small (diameter about 5008, type 2 and 3) and large (diameter about 1000 A, type 1) granular vesicles. Autoradiographic (Wolfe et al., 1962) and pharmacological (Pellegrino de Iraldi and DeRobertis, 1W; Richardson, 1963; Bloom and Barrnett, 1966; Bondareff and Gordon, 1966; Hokfelt, 1966) studies indicated that the small granular vesicles were the main intraneuronal storage sites for NA and also that they seemed to be specific for NA neurons. The role of the large granular vesicles remained obscure since they seemed to be present also in non-noradrenaline neurons (Coupland, 1965; Fuxe et aZ., 1965, 1966; Clementi et al., 1966; Grillo, 1966). In subsequent studies with routine fixation techniques ( glutaraldehyde and/or osmium tetroxide) on central nervous tissue only large


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granular vesicles could be demonstrated in addition to large numbers of the classic so-called synaptic vesicles, which have a clear interior (see e.g., Sjostrand, 1953; DeRobertis and Bennett, 1954; Palade and Palay, 1954). The large granular vesicles were then suggested to represent the central NA storage sites (Pellegrino de Iraldi et al., 1963). Although autoradiographic studies ( Aghajanian and Bloom, 1966a,b, 1967; Lenn, 1967; Descarries and Droz, 1968) disclosed that the large granular vesicles indeed often were present in boutons belonging to monoamine neurons, pharmacological and regional distribution studies have added new information on the significance of the large granular vesicles as revealed after glutaraldeli yde-osmium tetroxide fixation ( Fuxe et al., 1965, 1966; Bloom and Aghajanian, 1968) : 1. Large granular vesicles are not exclusively present in monoamine neurons and thus cannot be used as a criterion for identification of these neurons. 2. The dense core of the large granular vesicles does not mainly reflect the intraneuronal amine levels, and thus the effect of drugs cannot be properly investigated on the basis of the large granular vesicles. 3. Although the large granular vesicles in monoamine neurons probably contain amines, the main intraneuronal storage site for monoamines is made up of small vesicles which with glutaraldehyde and/or osmium tetroxide fixation appear as agranulas vesicles. The introduction of potassium permanganate (KMnO,) as a fixative for the demonstration of monoamine storage sites by Richardson (1966) opened up new possibilities for investigating monoamine neurons at the ultrastructural level. With this fixative, m a l l granular vesicles could be visualized both in the peripheral and central nervous system (Hokfelt, 1967a,b, 1968a,b, 1969, 1970; Hokfelt and Jonsson, 1968; Hokfelt and Ungerstedt, 1969). These studies have revealed that DA, NA, and 5 H T neurons contain both small and large granular vesicles, which seem to be present in all parts of the neuron. The highest numbers of vesicles are found in the boutons, and most of the vesicles are of the small type-in certain peripheral adrenergic neurons the large ones constitute only a few percent of all vesicles ( Hokfelt, 1969). The presence of small granular vesicles in central monoamine boutons has also been shown in glutaraldehyde-dichromate-osmium tetroxide tissue after intraventricular injections of 5-hydroxydopamine (Tranzer et al., 1969). This dopamine analog, which is taken up into catecholamine neurons and accumulated in the storage vesicles, seems to facilitate and enhance the demonstration of the dense core in the granular vesicles (Tranzer and Thoenen, 1967). The fact that monoamine boutons can be identified at the ultra-

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structural level has made it possible to estimate the monoaminergic input to various brain regions quantitatively (Hokfelt, 1968a). Thus, in the caudate nucleus about 15%,in the hypothalamic periventricular region about 4%, and in the hypothalamic suprachiasmatic nucleus about 5%of all boutons belong to monoamine neurons. It has also been possible to identify dopamine nerve endings in the median eminence (Hokfelt, 1967b). These nerve endings belong to the tubero-infundibular dopamine neurons and are probably involved in the regulation of gonadotropin secretion from the anterior pituitary (Fuxe and Hokfelt, 1969), probably via an axo-axonic influence in the median eminence ( Hokfelt, 1968b). The synaptic relations between monoamine boutons and other nervous structures are so far incompletely known (see Hokfelt, 1968a,b). In the hypothalamic areas probable NA and 5-HT boutons are associated, to a certain extent, with synapses of type 1 (Gray, 1961). Such synapses seem, however, to be comparatively rare in connection with probable DA boutons in the caudate nucleus. Test tube studies (Hokfelt and Jonsson, 1968) and pharmacological studies performed on brain slices in vitro (Hokfelt, 1968a,b) have given strong evidence for the view that the dense core in both the small and the large granular vesicles as revealed after KMnO, fixation is formed by a reaction between the amines and the fixative. Thus, the dense core directly reflects the presence of an amine at the moment of fixation. This opens up the possibility of studying the action of psychoactive drugs at the ultrastructural level.

II. Function of Central Monoamine Neurons

A. DA PATHWAYS 1. Nigro-Neostriatal D A Neurons

a. Motor Functions. This system is of great importance for normal movements and postures. Thus, the cause of the symptoms rigidity, hypokinesia, and tremor found in parkinsonism is probably due mainly to the degeneration of the nigro-neostriatal DA system (see Hornykiewicg 1966). In agreement with this it has been found possible to restore a relatively normal motor function in Parkinsonian patients by way of treatment with high doses of L-dopa (Cotzias et al., 1967, 1969; Duvoisin et al., 1969; Weiner et al., 1969; Anden et al., 1970). From studies on rats (Bartholini et al., 1967; Bartholini and Pletscher, 1968; Butcher and Engel, 1969; Butcher et al., 1970), we know that after treatment with dopa following peripheral dopa decarboxylase inhibition

ASPEmS OF CENTRAL MONOAMINE NEURONS

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only the intraneuronal DA levels are increased, whereas there is a slight decrease in the NA stores (Butcher and Engel, 1969; Butcher et al., 1970) and a marked decrease in the 5-HT stores (Bartholini et al., 1968; Butcher and Engel, 1969; Butcher et al., 1970), probably due to displacement by DA formed from dopa in the NA and 5-HT neurons. Since locomotion is markedly increased following dopa treatment (200 mglkg) in combination with a peripheraI dopa decarboxylase inhibitor, there is good evidence that the increased DA levels in the DA neurons result in increased DA neurotransmission (Bartholini et al., 1969; Butcher et al., 1970). The locomotion is mainly composed of stereotyped movements (Butcher et al., 1970). Studies on rats with the neostriatum removed on one side have also given evidence that neostriatal DA neurotransmission is increased following dopa treatment (Anden et al., 1966a; Ungerstedt, unpublished data). We know, for example, from previous studies using this model in rats and from intraneostriatal injections of apomorphine and DA (Ungerstedt et al., 1969) that the nigro-neostriatal DA neurons on one side aim to turn the animal toward the opposite side, but that this effect of the DA neurons is counteracted by other afferent pathways to the same neostriatum. However, after dopa administration in combination with the Peripheral dopa decarboxylase inhibitor the rats turn toward the operated side, probably because of the increased DA receptor activity induced by the increased amounts of DA formed in the terminals from dopa and which leak out onto the receptor sites. The increased turning can be quantitatively measured by counting the turns per minute (Ungerstedt and Arbuthnott, unpublished data) in a specially built rotometer. In this way a quantitative estimation of the efferent output from the NCP can be obtained. From the above it is obvious that dopa treatment should increase DA transmission in normal human beings. However, it still remains to be explained why dopa is so effective in treatment of parkinsonism, inasmuch as a considerable degree of degeneration of the nigro-neostriatal DA neurons exists with decreased dopa decarboxylation. The following factors could be of importance when trying to explain the beneficial effects of dopa. 1. The remaining DA neurons have enough dopa decarboxylase activity left to convert dopa into DA. Some formation of DA (10-20%) may also occur in the pericytes of the capillary walls. However, the functional significance of the DA formed extraneuronally is probably not large, since it seems as if the symptoms of patients with complete degeneration of the nigro-neostriatal DA neurons are not alleviated by the dopa treatment. Dopa itself is probably not active, since after central

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dopa decarboxylase inhibition it is no longer active, at least not in the rat. Furthermore, improvement after dopa is correlated with increased homovanillic acid ( HVA) levels in the CSF ( Weiner et al., 1969). 2. Because of a decreased DA neurotransmission in the neostriatum the receptor sites have developed a denervation supersensitivity, and therefore even minute amounts of DA formed can have functional significance. This view is strongly supported by studies in rats on nigroneostriatal DA neurons undergoing degeneration following SOH-DA injection into the substantia nigra (Ungerstedt, unpublished data) or following blockade of transmission by reserpine ( Fuxe and Ungerstedt, 1969; Ungerstedt, unpublished data). Thus, in these rats the effect of apomorphine, which stimulates DA receptors (Ernst, 1967; A n d h et al., 1967b), dopa, and amphetamine are markedly potentiated ( Ungerstedt, unpublished data; Fuxe and Ungerstedt, 1969). In many cases of parkinsonism, treatment with dopa has resulted in side effects such as nausea and vomiting. An interesting side effect has been the appearance of choreo-athetoid movements, which usually are parallelled by marked increases of HVA in the cerebrospinal fluid (Weiner et al., 1969). One explanation of these hyperkinetic side effects may therefore be that too much DA is being formed in the remaining DA nerve terminals, so that DA receptors in certain parts of the neostriatum are being overstimulated. This may be of particular significance in cases when mainly the medial or lateral nigroneostriatal DA pathway has degenerated. In these cases dopa will cause an overstimulation of the remaining pathway, which could lead to development of hyperkinesias. This possibility is now being tested with lesions in the substantia nigra and local injection of apomorphine into various parts of the NCP. Recently, certain antiparkinsonian drugs ( Fuxe et aZ., unpublished data) such as certain antihistamine agents ( diphenylpyraline and dexbrompheniramin) and adamantine have been found to potentiate the effects of dopa in the turning model (Ungerstedt and Arbuthnott, unpublished data, see above). Adamantine has been found not to cause a blockade of the membrane pump in the DA and NA neurons, but it must act via another mechanism to potentiate the dopa response ( Goldstein et al., unpublished data). In contrast, diphenylpyraline and dexbrompheniramin may cause a certain blockade of the membrane pump in the DA neurons (Fuxe and Hamberger, unpublished data). These drugs have previously been shown to be potent blockers of the membrane pump in the central NA and especially the 5-HT neurons ( Carlsson and Lindqvist, 1969). When dopa is given together with a peripheral dopa decarboxylase


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inhibitor there is a marked and rapid fall in the intraneuronal levels of 5-HT (Bartholini et al., 1 W ; Butcher et al., 1970)) and the 5-HT cell bodies become clearly green-fluorescent (Butcher et al., 1970). The main mechanism for this is probably rapid displacement of 5-HT by newly formed DA. The possible role of this released 5-HT is unknown, but it cannot be of critical importance for the therapeutic effects, since a second dose of dopa given when the effects of the first dose have subsided and the 5-HT levels still are low causes marked locomotion (Fuxe et al., unpublished data). In view of the results obtained in rats, it is probable that treatment with dopa in patients results in depletion of brain 5-HT, which also may be of importance for the elicitation of the hyperkinesias. There exists a dense cholinergic innervation of the NCP (Shute and Lewis, 19166), and anticholinergic agents have been used for a long time in the treatment of parkinsonism. The DA and the cholinergic afferents to the NCP are probably antagonistic in function (see review by Randrup and Munkvad, 1968). When the DA receptor activity is low in comparison with the cholinergic receptor activity, as in parkinsonism, the end results will be an increased a-motoneuron activity (Steg, 1966, 1969). In agreement with this hypothesis, it has been found that the increased locomotion found with dopa is potentiated with the help of anticholinergic drugs such as atropine and benztropine (Fuxe et al., unpublished data). The same holds true for this drug combination in treatment of parkinsonism. On theoretical grounds it would seem warranted to combine dopa treatment with at least two drugs, an anticholinergic agent and a DA membrane pump blocker. Benztropine seems to have both these properties (Coyle and Snyder, 1969; Farnebo et al., and Fuxe et al., unpublished). b. Mental Functions (the role of mesolimbic DA neurons will also be discussed), It is now generally accepted that stereotyped behavior induced by apomorphine or by amphetamine [amphetamine probably acts by releasing extragranular CA stores (Carlsson et al., 1966a)b;Fuxe and Ungerstedt, 1968, 1969)] is owing to increased DA receptor activity in the neostriatum (see Randrup and Munkvad, 1968; Fuxe and Ungerstedt, 1969). Such stereotyped behavior is often seen in patients with mental disorders such as schizophrenia, and it has therefore been assumed that an abnormally increased activity in the nigro-neostriatal DA neurons at least partly could be responsible for the psychotic state. This view has become strengthened by the fact that neuroleptic drugs of the phenothiazine type, such as perphenazine and of the butyrophenone type, such as spiroperidol, which mainly block DA receptor sites in the brain (Corrodi and Fuxe, 1969; Anddn et al., un-

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published data) are potent antipsychotic drugs, but at the same time they cause marked Parkinson-like side effects. Spiroperidol has been found to be the most potent blocker of neostriatal DA receptors so far. The rotation obtained by giving amphetamine to rats having a 6-OH-DA induced degeneration of the left nigro-neostriatal DA system is blocked by a dose as low as 0.005 mg/kg (Ungerstedt and Fuxe, unpublished data). Because the dopa-induced increase in the flexor reflex of spinalized rats, which is highly dependent on NA receptor activity ( Andkn et al., 1967a), is not clearly blocked with as high a dose as 10 mg/kg of spiroperidol, the NA receptor sites are probably not blocked with the doses used of this drug. There is little doubt that there exists a correlation with antipsychotic activity, and blockade of neostriatal DA receptors. However, it has not been sufficiently considered that these potent neuroleptic drugs also cause blockade of the DA receptors in the limbic forebrain, which are innervated by the mesolimbic DA neurons. Thus, these DA neurons also show an increase in turnover following treatment with clothiapin or spiroperidol (Corrodi and Fuxe, 1969, unpublished data; see below). It cannot in any way be excluded that part of or the main antipsychotic effect of neuroleptic drugs is mediated via blockade of transmission in the mesolimbic DA neurons. This can be the explanation for the recent findings with pimocide, a new neuroleptic drug (Janssen et al., 1968). This drug is said to give a good antipsychotic effect without causing marked parkinsonian-like side effects. However, it is possible that the lack of parkinsonian-like side effects with pimocide may be owing to the slow onset of action by pimocide compared to that seen with haloperidol or spiroperidol. We have found that pimocide in a dose as low as 0.1 mg/kg blocks DA receptors in the neostriatum and the limbic forebrain, whereas there is no blockade of NA receptors with as high a dose as 20 mg/kg (And& et al., unpublished data). We are now testing if doses from 0.01 to 0.1 mglkg cause a preferential effect of turnover in the mesolimbic DA neurons. Until inter aha these studies are completed, it is not possible to decide whether or not dysfunction in the activity of the mesolimbic DA neurons or the nigro-neostriatal DA neurons is the most important one in relation to mental disorders. It should be pointed out that also in the parts of the limbic system, rich in DA nerve terminals, there is a large input of presumably cholinergic nerve terminals (Shute and Lewis, 1966). Thus, in the limbic system there also may be a balance between dopaminergic and cholinergic activity. With the help of turnover studies it has been possible to obtain some support for this view. Thus, after treatment with oxotremorine, a cholinergic agent, there is an increase in the DA turnover in the two large ascending DA systems. This increase can be blocked with the


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help of atropine (Corrodi et at., 1967). When anticholinergic hallucinogenic drugs such as ditran and sernyl are given, there is instead a decrease in DA turnover in the two ascending DA systems (Andkn et al., unpublished data). These findings can be interpreted to mean that after oxotremorine there is a compensatory increased release of DA in the neostriatum and the limbic forebrain, possibly induced by nervous feedback onto the DA cell bodies causing increased nervous activity. After ditran and sernyl, on the other hand, there is a compensatory decreased release of DA, possibly induced by nervous feedback. It is thus possible that the hallucinogenic properties of drugs such as ditran are owing to an imbalance in activity between cholinergic and DA neurons innervating the limbic system and/ or the neostriatum. It is clear from the above that we know little about the function of the mesolimbic DA neurons. Studies on rats with bilateral electrocoagulations of neostriatum reveal after treatment with apomorphine in combination with cataprezan, a NA receptor stimulating agent ( Andkn et al., 1970b), a peculiar behavior, which consists of jerky, very rapid movements with periods of complete rest. Because this behavior could not be elicited without apomorphine, it may be dependent on the DA receptor activity in the limbic system (Fuxe and Ungerstedt, 1969). The ascending two systems of DA neurons may thus, play a role in motor functions and in mental functions. c. Function in Thermoregulation. It has recently been found in mice that apomorphine decreases body temperature probably via a central action (Fuxe and Sjoqvist, unpublished data). This effect can be blocked by haloperidol in a dose which mainly blocks central DA receptor sites. These findings are of great interest, since they can suggest a role of the DA neurons in thermoregulation. Hitherto, only NA and 5-HT neurons have been discussed in this respect. d. Autonomic Function. Another finding illustrating the multiple functions of the ascending DA neurons is the decrease in blood pressure (Bolme and Fuxe, unpublished data) observed with potent DA receptor blocking agents such as spiroperidol and pimocide, which do not block NA receptor sites ( Andkn et al., unpublished data; Ungerstedt and Fuxe, unpublished data). Thus, the DA neurons may play a role also in centrd vasomotor mechanisms. In our laboratory ( Ungerstedt, unpublished observations), some results have been obtained indicating that the nigro-neostriatal DA neurons are necessary for normal eating and drinking behavior. Thus, after bilateral 6-OH-DA injections into the substantia nigra causing complete degeneration of the nigro-neostriatal DA neurons the rats will become immobilized and develop a complete adipsia and aphagia and

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die after 5-7 days. The interpretation given is that the latter effects are secondary to the effects on motor function (akinesia, rigidity). The blockade of eating and drinking found by many authors by lesioning the lateral hypothalamic area may be due to the interruption of the nigro-neostriatal DA pathway.

2. Tubero-lnfundibulur D A Neurons The possible function of this system has been discussed extensively in previous review articles (Fuxe and Hokfelt, 1969, 1970a,b; Fuxe et al., 1970b). All the findings obtained in this laboratory so far support the view that the DA released in the median eminence acts locally on terminals storing luteinizing hormone releasing factor ( LHRF ) to inhibit the release of LHRF from the median eminence. This system also probably participates in mediating the negative feedback action of estrogen and testosterone on gonadotropin secretion, because estrogen and testosterone markedly increase the turnover of the tubero-infundibular DA neurons of castrated rats, resulting in increased release of DA in this area (Fuxe et al., 1967, 1969b). It has also been found recently that synthetic estrogens such as mestranol, ethinyl-estradiol which are used as antifertility steroids, markedly increase the turnover in this system. The blockade of ovulation found with these steroids may at least partly be mediated via activation of the tubero-infundibular DA neurons (Fuxe and Hokfelt, 1970a,b, and unpublished data). The system is also highly sensitive to prolactin, which also markedly increases the turnover in these neurons (Fuxe and Hokfelt, 1970a,b). This effect of prolactin probably explains the marked activation of the system seen in pregnancy, pseudopregnancy, and lactation (Fuxe et al., 1967, 1969c; Fuxe and Hokfelt, 1967). The prolactin secretion from the anterior pituitary is probably controlled indirectly by the central NA neurons, the activation of which increases secretion of prolactin inhibitory factor ( Fwe and Hokfelt, 1970a,b; Fuxe et al., 1970b).In this way, activity changes in NA neurons can, inter alia via prolactin, influence the activity in the tubero-infundibular DA neurons and thereby LH secretion. Of course, the possibility cannot be excluded that the central NA neurons may also directly influence LHRF secretion without involvement of changes in prolactin secretion and in changes of release of DA in the median eminence.

B. NAPATHWAYS 1. Ascending NA Neurons a. Fundion in Behavior and in Wakefulness. For many years antidepressive drugs of the imipramine type have been used in the treatment


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of depressive states. Some of these drugs such as desipramine and protriptyline have been found to be potent blockers of the membrane pump in the NA neurons (Carlsson et al., 1966b; see GIowinski and Baldessarini, 1966), to be weak blockers of the membrane pump in the 5-HT neurons, and to have no blocking effects at all on the membrane pump of the central DA neurons (Carlsson et al., 1966b, 1969a,b). Many psychiatrists believe that the main effect of these two drugs in patients is an increase in drive, i.e., the patients become activated and alert. It is, therefore, possible that this activating effect of desipramine and protriptyline is owing to the membrane pump blockade in the NA neurons, leading to increased amounts of NA reaching the receptor sites and to an increase in NA receptor activity. This view is supported by the fact that, for example, protriptyline in rats potentiates the effect of dopa combined with peripheral dopa decarboxylase inhibition on exploratory behavior. Increased rearing and frequent walking about the floor of the cage are observed. In a recent study (Fuxe and Ungerstedt, 1969), the evidence supporting the view that exploratory behavior is highly dependent on NA receptor activity has been summarized. A potent dopamine-/3-oxidase inhibitor (FLA 63; Corrodi et al., unpublished data) or a NA receptor blocking agent, phenoxybenzamine A n d h et al., 1967a), have both been found to block markedly the exploratory behavior seen after amphetamine, whereas the stereotyped behavior seen after amphetamine which is mainly dependent on DA receptor activity (see above) is not decreased. In agreement with this view, it has been found that a NA receptor stimulating agent, cataprezan (And6n et al., 1970b), if combined with apomorphine, a DA receptor stimulating agent, will induce marked behavioral hyperactivity (rapid running around and out of the cage) and with marked signs of aggressiveness (defense postures and biting). Furthermore, increased alertness (increased sensitivity to stimuli) seems to appear after cataprezan treatment. Rage behavior induced either by amygdaloid stimulation (Fuxe and Gunne, 1964; Gunne and Lewander, 1966) or by high decerebration (Reis and Fuxe, 1968, 1969) has also been found to be associated with an increased turnover of brain NA. All these findings suggest that these phylogenetically old NA systems may be of importance for the survival of the animal. Marked activation of the NA neurons may be required to meet situations involving defense and attack behavior. Of course, DA receptor activity has to be present, since otherwise no movements can be elicited. It may be (see above) that the limbic DA receptors also play a role in the performance of aggressive behavior. The involvement of neurons such as NA neurons with their enormous collateral networks to trigger off defense-attack reactions makes it possible immediately and concomitantly to influence

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various parts of the central nervous system. Thus, rapid general somatic and autonomic reactions can be immediately elicited if required (see above). It seems logical to assume that increased alertness is mainly due to activation of the dorsal ascending N A pathway, whereas the autonomic signs and the aggressiyeness may mainly be induced by activation of the ventral ascending N A pathuay innervating, for example, the hypothalamus and the limbic system. The descending NA pathway is probably also of importance for this type of behavior, inasmuch as they participate in regulating reflex activity and activity in preganglionic autonomic nerve fibers via an influence on the sympathetic lateral column. Drugs blocking central NA neurotransmission such as dopamine-Poxidase inhibitors and phenoxybenzamine causes sedation ( decreased alertness ) and decreased exploratory behavior. Furthermore, neuroleptic drugs such as chlorpromazine and thioridazine, which have potent blocking actions on NA as well as on DA receptors, also cause sedation (Fuxe et al., unpublished data). These results give further support for the view presented above, that the state of alertness depends on N A receptor actiuity. Carlsson and Lindqvist (1967) have recently found in mice normal exploratory and locomotor activity after selective protection of the DA stores against reserpine-induced amine depletion with the help of repeated doses of m-tyrosine and protriptyline, which block the membrane pump mechanism in the NA but not the DA neurons (Carlsson et al., 196613, 1969a). In rats, such protection experiments are, on the whole, less effective in counteracting reserpine-induced amine depletion and behavioral depression ( Butcher and Fuxe, unpublished data). However, in rats protection of both DA and NA stores leads to considerably less sedation and less decrease in locomotor and exploratory behavior than protection of DA stores alone. Therefore, it may be that in rats central NA neurons are of more importance for these behavioral activities than in mice. On the other hand, it should be pointed out that locomotion, mainly stereotyped behavior, can occur in rats in the absence of any known stores of NA. Thus, it has been found that apomorphine in reserpine-a-methyltyrosine pretreated rats can induce locomotion mainly in the form of stereotyped behavior (mainly sniffing). Exploratory activity, however, cannot be induced and is, thus, as pointed out previously dependent on the activity in NA neurons. For maintenance of conditioned avoidance behavior both central D A and N A neurons probably play an important role. Thus, it has been found that disruption of conditioned avoidance behavior in rats (conditioned lever pressing response on a discriminated avoidance schedule)


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by H44/68 is connected with signs of increases in both NA and DA turnover in most parts of the brain (Fuxe and Hansson, 1967). In agreement with this it has been found that disulfiram, a dopamine-P-oxidase inhibitor, blocked the dopa reversal of reserpine-induced conditioned avoidance behavior ( Seiden and Paterson, 1968). Furthermore, it has been shown (Butcher and Andh, 1969) that the DA receptor stimulating agent, apomorphine, is less effective than amphetamine, which releases “extragranular” stores of both DA and NA (Carlsson et al., 1 W b ; Fuxe and Ungerstedt, 1968) in restoring lever-pressing in rats on a variable interval schedule of food reinforcement after tetrabenazineinduced depression of that behavior. It is possible that the requirement for N A and not only D A receptor activity in exploratory behavior and conditioned behavioral processes may be the result of decreased alertness in response to the decreased N A receptor activity. A high degree of alertness may be of critical importance, not only in the conditioned behavior but also in exploratory behavior, because high demands are put on the responsiveness of the animal to stimuli. Such a condition does not exist in ordinary locomotion. Accordingly the “minor tranquilizers” such as chlordiazepoxide and diazepam have been found to cause a blockade of the stress-induced NA acceleration of turnover in all parts of the brain plus a reduction of NA turnover in the cortex cerebri and cerebelli compared to normal rats (Corrodi et al., unpublished data). Also in agreement with the above discussion, self-stimulation behavior has been found to be highly dependent on NA receptor activity (Stein and Wise, 1967; Wise and Stein, 1969). Thus, after dopamine-j3-oxidase inhibition the self-stimulation performance is suppressed, and intraventricular injections of 1-NA but not of d-NA, 5-HT or DA can restore self-stimulation. Furthermore, electrical stimulation of the medial forebrain bundle known to cause reward has been found to cause release of NA, as shown in local perfusing experiments on the amygdala and the hypothalamus. b. Autonomic Function. As mentioned above, results have recently been obtained that cataprezan stimulates NA receptor sites in the central nervous system ( AndCn et aE., 1970b). For several years it has been known that this drug is an antihypertensive agent ( Hoefke and Kobinger, 1966). It is, thus, possible that NA neurons participate in central vasomotor regulation. Evidence for this view (Bolme and Fuxe, unpublished data) has recently been obtained by showing that the decrease in blood pressure caused by cataprezan can be blocked by haloperidol in a dose sufficiently high to block NA receptors (1 mglkg) and by phenoxybenzamine. Furthermore, the decrease in blood pressure caused by cataprezan has been found to be potentiated by pretreatment with

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pimocide, a selective blocker of central DA receptors. Thus, probably both noradrenergic and dopaminergic mechanisms participate in central vasomotor mechanisms, and a combination of blockade of DA receptor activity and stimulation of NA receptor activity may result in a pronounced fall in blood pressure (Bolme and Fuxe, unpublished data). c. Neuroendocrine Function. As for the possible role of the central NA neurons in neuroendocrine regulation, this has been discussed in detail in previous reviews (Fuxe and Hokfelt, 1969, 1970a,b; Fuxe et al., 1970a). It is probable that mainly the ventral NA pathway is involved in the regulation of neuroendocrine responses, inasmuch as it primarily innervates the hypothalamus and the limbic system. The dorsal NA pathway, on the other hand, mainly innervates the cortex and the hippocampal formation is probably mainly involved in, for example, regulation of wakefulness (see above). Many findings suggest that central NA neurons may participate in the regulation of prolactin secretion (Coppola et al., 1965; Fuxe and Hokfelt, 1970a,b), probably by increasing secretion of prolactin inhibitory factor from the median eminence. In support of this view is the fact that many drugs blocking central CA neurotransmission cause lactation and pseudopregnancy. There are a number of findings showing that there is an increased NA turnover in connection with a stress-induced increase in adrenocorticotropic hormone ( A C T H ) secretion (see review by Fuxe et al., 1970a). Furthermore, adrenalectomized rats will also have an increased NA turnover compared to controls (Javoy et al., 1968; Fuxe et al., 1970a). This increase in NA turnover is blocked by cortisol (Fuxe et al., 1970a). Furthermore, many drugs interfering with central CA neurotransmission have been found to change ACTH secretion. Thus, it is probable that central NA neurons at least in some situations may play an important role in regulation of corticotropic releasing factor secretion from the hypothalamus. The work of Miiller et al. (1967) suggests that the central N A neurons also participate in regulating growth hormone secretion, because, for example, injections of small amounts of NA intraventricularly leads to increased release of growth hormone releasing factor. In support of the view is the fact that there is an increased turnover of NA after insulin-induced hypoglycemia in young rats ( Hokfelt et al., unpublished data).

2. Descending N A Neurons From the widespread distribution of NA nerve terminals in the posterior and anterior horn and in the intermediate part of the spinal


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cord, mainly the sympathetic lateral column, it is obvious that the descending NA pathways influence spinal reflexes and autonomic reflexes at various levels. Thus, both the afferent nerve cells in the posterior horn, the interneurons, and the motoneurons in the anterior horn are probably directly influenced. From experiments with nialamide-dopa on spinalized rats (see review by Anden et al., 1969a), it is known that activity in the central NA nerve terminals will increase the naturally evoked flexor reflex markedly. This increase is probably the result of the fact that repetitive activity in the flexor reflex afferents will cause a long-lasting, long-latency discharge in flexor motoneurons ( A n d h et al., 1966d). It has been postulated that the NA neurons produce these changes by inhibiting the short latency effects of activity in flexor reflex afferents (see also And& et al., 1966c), probably by way of an action on interneurons. It may be emphasized that at least some NA neurons innervating the spinal cord also give rise to large ascending axons and to collaterals to the lower brainstem, so that spinal reflex activity may be influenced at the same time as, for example, reflexes at the rhombencephalic level.

C. 5-HTPathways 1. Ascending 5-HT Neurons a. Mental Functions. It has been found that the most potent antidepressive drugs of the imipramine type, imipramine, amitriptyline, and particularly chlorimipramine are able markedly to block predominantly the uptake of 5-HT into the central 5-HT neurons (Carlsson et al., 1968; Fuxe and Ungerstedt, 1968; Carlsson et al., 196913). Inasmuch as these drugs seem to be able to cause an elevation of mood, it may be that the mood-elevating effects of these drugs are related to their ability to block the 5-HT membrane pump. This blockade probably results in an increased 5-HT neurotransmission, because the effects of tryptophan and 5-hydroxytryptophan ( 5-HTP ) on the spinal reflexes are potentiated (see below and Meek et al., 1970). Thus, a decrease in central 5-HT neurotransmission may be an important part in the depression syndrome, and the mood-elevating action of the antidepressant drugs may be caused by increasing 5-HT neurotransmission. In accordance with this view, 5-hydroxyindoleacetic acid ( 5-HIAA ) levels are decreased in the cerebrospinal fluid of depressed patients (Ashcroft and Sharman, 1960; Dencker et al., 1966; Ashcroft et al., 1966), and tryptophan administration also causes an alleviation of depression especially when combined with M A 0 inhibition (see Coppen, 1967; Persson and ROOS, 1967). Furthermore, recently it has been found that dexamethasone treatment

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enhances the effects of tricyclic antidepressants ( McClure and Cleghorn, 1968). This is in agreement with the view given above, because glycocorticoids have been found to increase tryptophan hydroxylase activity ( MacLean, personal communication). In this connection it should also be pointed out that a-ethyltryptamine (monase), which has antidepressive properties ( Robie, 19f3l), is capable of directly stimulating 5-HT receptors in low doses (down to 1 mg/kg) (Meek and Fuxe, unpublished data). Its receptor stimulating effect and not its MA0 inhibiting effect is probably the cause of its antidepressive properties. Thus, many findings support the so called “indolealkylamine theory” to explain the mechanism of depression (see Coppen, 1967).

The manic phase of manic-depressive illness should also be discussed. A potent antimanic agent and also a prophylactic agent against recurrent depression has been found to be lithium (Baastrup and Schou, 1967). Prolonged treatment with lithium has been found to decrease the 5-HT turnover in the central nervous system (Corrodi et al., 1969). This may be due to the fact that lithium ions decrease the release of 5-HT, since lithium chloride given in v i m or added in vitro has been found to diminish the release of tritiated 5-HT from brain slices evoked by mild electrical stimulation (Katz et al., 1968). Thus, it seems possible that lithium therapy may partly exert its beneficial effects by decreasing 5-HT neurotransmission, at least with regard to its antimanic effects. When discussing the possible mental functions of the 5-HT neurons the effects of psychotomimetic drugs on central 5-HT neuro-transmission should also be considered. Evidence has recently been found that lysergic acid diethylamide ( LSD ) directly stimulates 5-HT receptor sites in the brain (see Andkn et al., 1968). Thus, LSD increases markedly the extensor hind limb reflex of the spinal rats independent of presynaptic stores of 5-HT. This reflex is known to be highly dependent on 5-HT receptor activity, ar,d similar results are obtained when 5-HTP is given (see Andkn, 1968). Furthermore, LSD was observed to produce a marked decrease in 5-HT turnover in practically all parts of the brain and the spinal cord. The decreased turnover may have been caused by a nervous feedback onto the 5-HT cell bodies induced by the increased 5-HT receptor activity, resulting in a decreased nervous impulse flow in the 5-HT neurons. It is known that the amine turnover is highly dependent on the nervous impulse flow (see review by Anden et al., 1969b). From extracellular recordings in the raphe areas of the midbrain rich in 5-HT cell bodies, evidence has in fact been observed that LSD causes a decreased firing rate in these neurons (Aghajanian et al., 1968). It cannot as yet, however, be in any way excluded that changes in post-


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synaptic activity directly in the synapse can influence release from the presynaptic bouton. Thus, both a nemous and a local biochemical feedback may exist. Further studies on the effect of hallucinogenic drugs of the indolealkylamine type of central 5-HT neurotransmission have revealed that psilocybin, dimethyltryptamine (DMT), and to a certain extent tryptamine (but not bufotenine) stimulate 5-HT receptors directly and cause a decrease in 5-HT turnover, which probably is induced by the increased 5-HT receptor activity in a way similar to that described for LSD ( A n d h et al., 1970a). The decreased 5-HT turnover observed after these drugs cannot be explained as a result of MA0 inhibition, because a dose of, for example, pargylin causing the same reduction of 5-HT turnover as, for example, DMT inhibits intraneuronal MA0 in contrast to DMT. Hallucinogenic drugs such as DMT can probably also cause release of extragranular 5-HT, at least after artificial increase of this pool by MA0 inhibition (Fuxe et al., 1968b). However, this presynaptic effect cannot explain the present results, since such an action would rather result in an increased 5-HT turnover and not a decreased 5-HT turnover as was observed. Recently, evidence has also been obtained that hallucinogenic amphetamines ( Snyder et al., 1967, 1968), 4-methoxyamphetamine and especially 2,5-dimethoxy-4-methylamphetamine, can directly stimulate 5-HT receptor sites and cause a decrease in 5-HT turnover (Andkn et al., unpublished data). The increase in knee-jerk activity found in man after LSD, psilocybin, etc. (Isbell et aE., 1956; Isbell, 1959), is probably owing to an increased 5-RT receptor activity in view of the discussion given above. Inasmuch as the effects on the knee-jerk by these drugs are correlated with their hallucinogenic properties, it is possible that the central 5-HT receptor stimulation induced by these drugs is responsible also for the hallucinations caused by these drugs. In view of the discussion given above, overstimulation of central 5-HT receptor sites must be considered as one explanation for the cause of hallucinations found in psychotic states, Thus, not only overstimulation of DA receptors or a disruption of the balance between cholinergic and dopaminergic receptor activity in the neostriatum and/ or the limbic forebrain (see section on central DA neurons) should be considered. From the work of Jouvet’s group (see reviews by Jouvet, 1968, 1969), there is good evidence that the 5-HT neurons induce slow-wave sleep events. Thus, after extensive lesions of the raphe nuclei or after treatment with p-chlorphenylalanine, a blocker of tryptophan hydroxylase (Koe and Weissman, 1967) total sleep is almost completely suppressed.

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After p-chlorphenylalanine treatment there is a good correlation between decrease of 5-HT concentrations and decrease of slow-wave sleep. Furthermore, electrical stimulation of the raphe nuclei in the mesencephalon has produced behavioral signs of calmness and an EEG pattern similar to that found in sleep (Kostowski et al., 1989). Sheard and Aghajanian (1968) have also found a lack of habituation to external stimuli after such stimulation. Such electrical stimulations increase brain 5-HIAA levels and cause a decrease of 5-HT levels in the brain ( Aghajanian et al., 1967; Kostowski and Giacolone, 1969). Furthermore, lesions of this area in rats results in marked locomotion and disappearance of the slow-wave sleep pattern (Kostowski et al., 1968, 1969). From these results it is obvious that the sleep disturbances found in depressive states may be due to low 5-HT neurotransmission. Thus, in many respects the NA (see section on function of NA neurons) and 5-HT neurons seem to exert effects, for example, on behavior and wakefulness that are antagonistic to one another. This view is also supported by the fact that the behavioral effects of increased central NA neurotransmission obtained, for example, with help of cataprezan are potentiated by mean of tryptophan hydroxylase inhibition and inhibited by 5-HT receptor stimulation caused by LSD (Fuxe et al., unpublished data). b. Function in Sexual Behavior. From the work of Meyersson (1966) it seems that ascending 5-HT pathways can mediate inhibition of estrous behavior. Thus, the progesterone-induced estrus in estrogen-pretreated ovariectomized rats is markedly inhibited by increased monoamine levels, especially after seIective increases of 5-HT levels ( Meyersson, 1964). Furthermore, it has been found that the antidepressive drugs imipramine and amitriptyline inhibit estrous behavior in doses which do not prevent reserpine-induced sedation or induce exploratory behavior (Meyersson, 1966). These effects on estrous behavior are probably related to the blocking effects these drugs exert on the 5-HT membrane pump. The 5-HT nerve terminals mainly responsible for these effects are probably those lying in the retrochiasmatic area and the preoptic areas which are known to play an important role in the regulation of sexual behavior. c. Neuroendocrine Functions. The role of 5-HT neurons in neuroendocrine regulation has been summarized in previous reviews (Fuxe and Hokfelt, 1970b; Fuxe et at., 1970a,b). The work of Kordon et al. (1968; Kordon, 1969), clearly suggests that central 5-HT mechanisms in the hypothalamus act by inhibiting secretion of luteinizing hormone. Thus, not only sexual behavior but also ovulation is probably inhibited


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by increased activity in 5-HT neurons. 5-HT neurons may also participate in regulation of ACTH secretion ( Fuxe et al., 1970a).

2. Descending 5-HTNeurons The 5-HT nerve terminals in the spinal cord are found in the largest densities in the lumbal and sacral part of the spinal cord (Fuxe, 1965), where also the highest 5-HT concentrations in the central nervous system are found (And&, personal communication). The 5-HT nerve terminals are found in all parts of the gray matter, anterior horn, posterior horn, and intermediate zone, and they surround both small and large nerve cell bodies. In the acutely spinalized rat, it has been found that treatment with tryptophan or 5 H T P in combination with monoamine oxidase inhibition or with blockade of the 5-HT membrane pump results in a marked potentiation of the extensor hind limb reflex (AndCn, 1968; Meek et al., 1970). Furthermore, after treatment with 5-HTP there is an increased excitability of the a-motoneurons (Anden et al., 1964a), and many of the a-motoneurons are, in fact, surrounded by plexi of 5-HT nerve terminals. With regard to spinal autonomic function, the 5-HT nerve terminals in the sympathetic lateral column may inter alia act to inhibit activity in the sympathoadrenal neurons, since the insulin-induced adrenaline secretion is inhibited by 5-HTP (AndCn et al., 1964b). Ill. Some Conclusions

A. THENIGRO-NEOSTRLATAL DA NEURONS

1. Motor functions: Their degeneration will cause hypokinesia and rigidity. Overstimulation of neostriatal DA receptors causes stereotyped behavior. 2. Mental functions: Experiments especially with neuroIeptic drugs may indicate that normal activity in nigro-neostriatal DA neurons and/or mesolimbic DA neurons are required for normal thought processes. B. THETUBERO-INFUNDIBULAR DA NEURONS These neurons probably act by way of inhibiting the release of LHRF from the median eminence. NA NEURONS C. THECENTRAL

1. Function in wakefulness: Increased NA receptor activity probably results in alertness and increased drive. Possibly NA receptors in the

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cortex cerebri are mainly involved. The NA terminals belong mainly to the dorsal NA pathway. 2. Function in emotion: Increased NA receptor activity results in aggression. Possibly NA receptors in the limbic system and the hypothalamus are mainly involved. The NA nerve terminals belong mainly to the ventral NA pathway. 3. Function in behavior: Increased NA receptor activity results in marked locomotor hyperactivity with a strong component of exploratory activity provided DA receptor activity is also present, The NA neurons probably also are essential for conditioned processes as are the DA neurons. 4. Neuroendocrine function: The ventral NA pathway innervating mainly the hypothalamus and the limbic system may participate in the control of prolactin, ACTH, and GH secretion from the anterior pituitary and in the control of oxytocin and antidiuretic hormone secretion from the posterior pituitary. 5. Function in reflex activity: The descending bulbospinal NA neurons participate in the control of somatic and autonomic reflex activity. They decrease transmission in the short latency pathways from the flexor reflex afferents, probably by acting at an interneuronal level, hereby producing an increased flexor reflex.

D. THECENTRAL 5-HT NEURONS 1. Function in mood: There is good evidence that depressive states may partly be the .result of a decreased 5-HT neurotransmission. 2. Function in wakefulness: Destruction of the 5-HT neurons results in insomnia. 3, Function in thought processes: There are indications that high 5-HT receptor activity may cause hallucinations, inasmuch as the psychotomimetic drugs LSD, psilocybine, dimethyltryptamine, and pmethoxyamphetamine are potent stimulators of central 5-HT receptors. 4. Function in behavior: The 5-HT neurons probably inhibit sexual behavior and other types of hyperactive motor performance such as those seen after simultaneous stimulation of DA and NA receptor activity. 5. Neuroendocrine function: The 5-HT neurons may play an important role in inter alia regulation of LH secretion from the anterior pituitary. 6. Function in reflex activity: The descending bulbospinal 5-HT neurons participate in control of autonomic and somatic reflex activity especially in the lumbosacral part. In the spinalized rat the extensor hind limb reflex is markedly increased by an increase in 5-HT receptor activity.


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ACKNOWLEDGMENT This work has been supported by the Swedish Medical Research Council ( 14X-71504A,05B; 14X-1015-04,05; B70-14X-2887-01; B70-40X-2457-03), by a small Mental Health Grant (1R03 MH16825-01) from National Institute of Health and by grants from 0. och E. Ericssons Stiftelse, M. Bergwalls Stiftelse, Svenska Livforsakringsbolags namnd for medicinsk forskning, and from T. Svenssons Minne. Dr. Arbuthnott was supported by a Fellowship from the Wellcome trust.

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UPTAKE AND SUBCELLULAR LOCALIZATION OF NEUROTRANSMITTERS IN THE BRAIN' By Solomon H. Snyder, Michael J. Kuhar,' Alan 1. Green,s Joseph T. C ~ y l e , ~ and Edward G. Shaskan5 Departments of Pharmacology and Experimental Therapeutics and Psychiatry and the Behavioral Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland

I. Introduction . . . . . . . . . . . . 11. Catecholamine Uptake . . . . . . . . . . A. Uptake Kinetics . . . . . . . . . . . B. Stereospecificity . . . . . . . . . . . C. Antiparkinsonian Drug Action . . . . . . . . 111. Serotonin Uptake . . . . . . . . . . . IV. Amino Acid Uptake and Subcellular Localization . . . . V. Separation of Catecholamine-Storing Synaptosomes in Different Brain . . . . . . . . . . . . Regions . VI. Separation of Synaptosomes Storing Different Transmitters . . VII. Conclusions . . . . . . . . . . . . References . . . . . . . . . . . .

. . . . . . .

. .

. .

127 128 128 134 137 139 143 146 149 156 156

1. Introduction

Chemical mediation of synaptic transmission has been postulated for many years ( DuBois Reymond, 1877). One of the major problems in neurobiology has been to identify neurotransmitters and delineate their functions and interactions with pharmacological agents. In the peripheral nervous system this task is feasible, because physiological end points (muscular contraction, glandular secretion) exist so that effects of nerve stimulation can be compared with the application of the putative neurotransmitter. In the central nervous system neurophysiological attempts to mimic transmitter actions (Eccles, 1964) have met with only limited success. 'This work was supported by USPHS grants 1-R01-NB-07275 and 1-Pol-GM16492 and by U. S. Army Contract DADA 17-694-9144 and Justice Department Contract J-68-35. Solomon H. Snyder is a recipient of NIMH Research Career Development Award K3-MH-33128. Predoctoral fellow supported by NIH Training Grant GM-716. ' Year 5 Medical Student. * Present address: Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, Maryland. 'Postdoctoral fellow of NIH USPHS (GM-01183). 127

128

SOLOMON H. SNYDER ET AL.

Histochemical and neurochemical techniques have provided the principal criteria for assessing if a given chemical has a “synaptic” role, and the application of extremely rigorous criteria for neurotransmitter action has not been possible. Recent histofluorescent techniques have delineated specific neuronal systems for serotonin, norepinephrine, and dopamine in the brain (Hillarp et al., 1966). Another major advance has been the application of subcellular fractionation techniques culminating in the isolation of “synaptosomes,”nerve terminals pinched off from their axons during homogenization ( Whittaker, 1965; DeRobertis, 1967). Synaptosomal localization of a given chemical has come to be an important criterion for its candidacy as a putative neurotrysmitter. In theory, subcellular fractionation should be capabqe of resolving synaptosomes into populations specific for each neurotransmitter. Because the major anatomical features of nerve terminals are maintained when they are converted by homogenization into synaptosomes, it might become possible to identify the neurotransmitter used by a given nerve ending simply by its electron microscopic appearance. Although DeRobertis et al. ( 1962) separated synaptosomes into cholinergic and noncholinergic populations, and Michaelson and Whittaker ( 1963 ) observed a small separation of serotonergic and cholinergic endings, these and other attempts have not been very successful. The investigators measured endogenous contents of the putative neurotransmitters, usually worked with whole brain and with discontinuous sucrose gradients. Such procedures were fairly laborious and precluded the resolution of sucrose density gradients into more than 5 8 fractions. Clearly, sensitive, rapid procedures with high resolving powers would be useful in attempts to fractionate brain into transmitter-specific nerve terminals. Specific uptake mechanisms for several putative neurotransmitters in the brain have been described in recent years. By elucidating the kinetics of these uptake systems, we have been able to label synaptosomes storing specific neurotransmitters. Our studies of these uptake systems and their use in labeling and separating synaptosomes storing different putative neurotransmitters is the subject of this review. II. Catecholamine Uptake

A. UPTAKEKINETICS The concept of transmitter uptake by nerve terminals originated with norepinephrine, which occurs in specific neurons in the brain and is established as the neurotransmitter at postganglionic sympathetic nerve endings. There is considerable evidence that this neuronal reuptake of norepinephrine ( N E ) serves to terminate its synaptic actions ( Axelrod,

NEUROTRANSMITTERS IN THE BRAIN

129

1965; Iversen, 1967). This reuptake process appears to function similarly in the peripheral nervous system and in the brain (Glowinski and Baldessarini, 1966). In the peripheral sympathetic nervous system, NE uptake is almost totally abolished by sympathetic denervation, establishing that NE is taken up specifically into sympathetic nerve terminals. The brain, however, is a heterogeneous structure so that only considerable experimentation can establish the specificity of the labeling of endogenous catecholamines by the exogenous amine. The uptake of NE and dopamine (DA) in the brain has been studied in vivo by intraventricular injection of the amine ( Glowinski and Iversen, 1966), by incubation of brain dices with catecholamines (Dengler et al., 1962; Snyder et al., 1968a,b), and in isolated synaptosomes (Davis et al., 1967; Bogdanski et al., 1968). In experiments with isolated synaptosomes, extensive preparation is required before uptake studies can be initiated. Accordingly, we evaluated catecholamine uptake into synaptosomes in homogenates of brain tissue (Snyder and Coyle, 1969; Coyle and Snyder, 1969a). Instead of initially purifying synaptosomes, we utilized a nucleifree homogenate for uptake studies. After incubation in modified KrebsHenseleit (1932) solution at 37" under an atmosphere of 95%o,-% CO,, the homogenates were centrifuged at 48,000 x g for 30 minutes and the radioactivity in the precipitate determined. In experiments in which the resultant pellet was layered on a continuous sucrose density gradient and centrifuged to apparent density equilibrium, radioactive catecholamines were specifically associated with the synaptosomal fraction ( Fig. 1) . This indicates that catecholamine accumulated in these homogenates is taken up specifically into nerve terminals. The relative proportion of DA and NE terminals differs in various areas of the brain. The corpus striatum, comprising the caudate nucleus, putamen, and globus pallidus, contains large quantities of DA with very little NE (Hornykiewicz, 1966). Thus nerve terminals in this area are almost exclusively dopaminergic. In other areas of the brain, the predominant catecholamine is NE, although the density of NE terminals in various areas differs. When synaptosomal uptake of NE-3H and DA-3H was examined in different areas of rat brain, there were marked regional differences in accumulation. Catecholamine accumulation was greatest in the corpus striatum, which took up ten times more catecholamine than any other region (Coyle and Snyder, 1969a). Experiments with monkey brain slices (Gfeller et al., 1968; Snyder et al., 1969) (Table I ) showed that the high striatal catecholamine uptake was largely confined to the caudate nucleus and putamen. The corpus striatum possesses large numbers of the finest catecholamine nerve terminals in the brain; their considerable surface area could account for the high catecholamine

130

SOLOMON H. SNYDER ET At.

+ *

7

W

2 6 a I

0. W

z 5

n W

a

0

? 4 I

n

k t-

2

z W V

a t W n

0 0

10 20 FRACTION NUMBER

30

FIG.1. Sedimentation characteristics of n~repinephrine-~H,potassium, and monoamine oxidase in rat brain homogenates on continuous sucrose gradients, Homogenates prepared from rat hypothalamus were incubated with norepinephrine'H (0.2 b M ) . The pellets obtained from centrifugation at 48,000 X g were resuspended in 0.32M sucrose and were centrifuged on a linear sucrose gradient ( 1.46-0.32 M ) . Monoamine oxidase, potassium, and tritium were determined on the same gradient. Results are expressed in terms of the percentage of total monoamine oxidase activity, tritium, and potassium in each fraction. In three separate experiments the tube with peak activity for each component did not vary by more than one or two fractions. accumulation. Interestingly, in all brain regions, DA uptake was more than double that of NE. This raises the question of whether DA and NE enter the same nerve terminals. To study the specificity of catecholamine uptake, we measured uptake at varying concentrations, and, as uptake was saturable, we could

131


TABLE I TISSUE:MEDIUM RATIOSOF NOREPINEPHRINE-SH AND SEROTONIN 8H I N BRAINSLICESOF THE SPIDERMONKEY (Ateles geo$royi)= Tissue:medium ratio Brain region Cerebral cortical areas Motor cortex Insular cortex Basal ganglia Caudate nucleus Putamen Globus pallidus Amygdaloid nucleus Hypothalamus Midbrain areas Substantia nigra Periaqueductal gray Cerebellum Neocortex Vermis

Norepinephrine-SH

Serotonin-SH

2.25 f 0.12 3.17 _+ 0.21

3.00 f 0.36 3.76 5 0.33

9 . 4 0 f 0.66 9.14 f 0.52 3 . 0 0 0.11 4.13 f 0.52 5.05 k 0.61

5.37 5.61 3.84 5.18 5.77

2.96 f 0 . 3 2 4.05 0 . 3 4

*

4.71 f 0.18 6.38 f 0.65

2.47 f 0.23 2.45 zk 0.26

2.43 f 0.25 2.49 f 0 . 2 0

*

f 0.46 f 0.44 5 0.62 f 0.21 f 0.78

a Single blocks of tissue (2 X 3 X 3 mm) weighing 20-30 mg were incubated a t 37°C in 2 ml of modified Krebs-Henseleit (1932) medium under 95% 02-50/, GOz with ethylenediaminetetraaeetic acid (0.05 mg/ml), ascorbic acid (0.2 mg/ml), nialttmide (1.25 x 10-KM)and norepinephrine-SH (0.1 p M ) or serotonin-?H (0.1 p M ) for 20 minutes. Tissue:medium ratios were calculated as mpcuries of tritium accumulated in 20 minutes per gram tissue to mpcuries of tritium per millimeter of medium (Snyder el al., 1969). Data presented are the mean values f S.E.M. for groups of 5 monkeys.

estimate affinity constants for NE in a number of regions of the brain. In all areas except the striatum, double reciprocal plots converged on a single point, indicating the same K , (Fig. 2 ) about 4 x lo-' M. In the striatum, on the other hand, the K, for NE accumulation was 5 times higher, about 2 x M (Fig. 3). This was our h s t indication that there were fundamental differences in the behavior of the DA and the NE neurons. To look at this in more detail, we measured DA uptake at varying concentrations in the striatum (Fig. 3) as well as in other areas of the brain (Figs. 4 and 5 ) . In the striatum, DA had a lower K, than did NE, indicating that DA had a greater affinity for striatal neurons than did NE. Since DA is presumably the catecholamine transmitter in the striatum, this was not a surprising observation. In other areas of the brain, double reciprocal plots of DA uptake could not be described by a straight line. The curves that we observed, however, could be resolved into two straight line components which gave K, values of 0.8 x M

132


CEREBELLUM

3.0 0 MIDBRAIN 0 CEREBRAL CORTEX

0 HYPOTHALAMUS 2.5

2.0 I/U

I .5

I .o

0.5

-5

0

15

10

5

{NE]

X

20

M

FIG.2. Graphic analysis of the reciprocals of n~repinephrine-~H concentration ( [NE] ) and its accumulation into particulate fractions isolated from homogenates of different brain areas. Homogenates were incubated in triplicate for 5 minutes with n~repinephrine-~H concentrations ranging from 0.005 p M to 0.80 pW. Amine uptake ( v ) is expressed as mpmoles of 'HH/gm pellet per 5 minutes. and 1.4 x M , respectively. The two components of DA uptake were the same in hypothalamus, cerebral cortex, midbrain, medulla oblongatapons, and cerebellum. One of these DA uptakes (Uptake a; K,, = 0.8 x M ) had a fivefold greater affinity for DA than for NE in extrastriatal brain regions, while the other (Uptake b; K , = 1.4 x M ) had onefourth the affinity of the NE uptake system. Which of these DA uptake systems might represent DA entering NE sites? We tried to resolve this question by measuring the mutual competition of these two catecholamines for uptake into extrastriatal brain areas.

133


0.3 -

ST R IAT UM

NOREPINEPHRINE- 3H Km = 2.0 x loA6M

0.2

-

I/v

-5

0

5

10

15

20

I / S x 10-6M

FIG.3. Graphic analysis of the reciprocals of norepinephrine-'H and dopamine3H concentrations ( S ) and their uptake into particulate fractions isolated from homogenates of the striatum. Homogenates were incubated in triplicate for 5 minutes with concentrations of norepinephrine-*H or dopamine-'H ranging from

0.05 p M to 0.80 p M . Amine uptake (u) is expressed as mpmoles of 'H/gm pellet per

5 minutes.

In this type of experiment, the competitive inhibition by DA of NE-3H uptake in extrastriatal areas should be owing to DA entering NE uptake sites so that the inhibitory constant ( Ki ) ought to be the same as the affinity constant ( K , ) of DA for the NE transport system. In these experiments the Ki for inhibition by DA of NE-3H uptake in nonstriatal areas was 0.8 x M ,the same as the affinity constant for Uptake a of DA, showing that the high affinity Uptake a of DA takes place into NE neurons. These results indicate that DA has more affinity for the NE transport

134


I

CEREBRAL CORTEX

1.4

-

DOPAMINE-3H

Km, = 0.8 x 10-7M

I

0.8

t-

i

II S x

20

M

FIG. 4. Graphic analysis of the reciprocals of dopamine-’H concentrations ( S ) and its uptake into particulate fractions isolated from homogenates of the cerebral cortex. Homogenates were incubated in triplicate for 5 minutes with dopamine-’H (0.05 p M to 0.8 yM). Amine uptake ( 0 )is expressed as mymoles of ‘H/gm pellet per 5 minutes.

system than does NE itself. This is not altogether unexpected, since the work of Iversen (1967) suggests that DA also has greater affinity than NE for peripheral sympathetic nerve endings. The role of Uptake b of DA is puzzling. It could conceivably interfere with attempts to label NE neurons in nonstriatal areas with radioactive DA. Calculations from the Michaelis-Menten equation indicate that at low concentrations of DA-3H relatively little would enter Uptake b, but that at concentrations above M , more DA-”Hwould enter Uptake b than Uptake a.

B. STEREOSPECIFICITY Clearly, both dopaminergic and noradrenergic endings in the brain can take up either catecholamine. Af€inity constants for noradrenergic endings seem to be the same in a large number of brain regions and differ from the dopaminergic neurons of the striatum. Recently (Coyle and Snyder, 1969a), we observed striking differences in stereospecificity of the dopaminergic and noradrenergic endings. NE, because of its phydroxyl group, has two stereoisomers, and Z-NE is the naturally occurring isomer. Because the inhibitory constants for mutual inhibition of


5.0 4.0-

135

CEREBRAL CORTEX DOPAMINE-3H (low concentrations) Km =

0.8 x IO-'M

catecholamine uptake are the same as their affinity constants, we were able to determine the affinity of NE and DA uptake systems for d- and ZNE. In nonstriatal areas of the brain, in rat, guinea pig, and monkey (Table 11) the naturally occurring I-NE had 4 times more affinity than d-NE for the uptake process. In the striatum, on the other hand, where DA, a molecule with no stereoisomers, is the catecholamine transmitter, d- and I-NE had equal affinities. While the stereoisomers of NE differ at the ,&carbon, the two stereoisomers of amphetamine differ at the &-carbon.In a nonstriatal region of rat brain, d-amphetamine was 10 times as potent an inhibitor of NE uptake as Z-amphetamine (Table 111). It has been known for many years that d-amphetamine is a more potent central stimulant than Z-amphetamine. In recent dose-response studies on locomotor stimulation provoked in rats by d- and Z-amphetamine (Taylor and Snyder, 1970), we observed that d-amphetamine was exactly 10 times as potent as Lamphetarnine, a result which parallels the tenfold difFerence in ability to inhibit NE uptake. There have been many theories to explain the central stimulant action

136


TABLE I1 AFFINITYCONSTANTS FOR d- AND Z-NOREPINEPHRINE IN DIFFERENT BRAIN REGIONS"

KmdlJH-NE Region Rat cerebral rortex Rat hypothalamus Rat medulla oblongata-pons Rat cerebelliim Rat corpus striatrim Monkey cerebral cortex Guinea pig cerebral cortex

Ki d-NE

(x 1 0 - 7 ~ )

(x 1 0 - 7 ~ )

3 3 3 3 16 4 10

11 11 10 11 16 11 22

8-4 2 9 4 3 7-4 0 9 4 1 9-19 5 0-4 4 1-12 0

3-12 5-12 9-11 6-12 0-20 0-13 1-24

0 1 9 8 0 1 6

K , I-NE

(x 1 0 - 7 ~ ) 7-3 0 9-3 1 0-3 3 0-3 4 6-19 2 0-3 3 i 1-8 0

3 2 3 3 15 3

K , values were determined by graphic analysis using the method of Lineweaver and Burk (1934). K , values were determined both by the niethod of Lineweaver and Burk (1934) and by the method of I k o n (1953). Values preseated are the ranges obtained in four determinations. The tissue preparation employed was synaptosomes in brain homogenates incubated with catecholamines-3H and nonradioactive amines as described earlier (Snyder and Coyle, 1969; Coyle and Snyder, 1969a).

of the amphetamines, including synaptic release of NE, monoamine oxidase inhibition, and a direct receptor action. Our results suggest that inhibition of NE uptake may be the major mechanism of action. In contrast to the differential effects of the amphetamine isomers in extrastriatal areas, d- and t-amphetamines were equally potent inhibitors of catecholamine uptake in the striatum and were both three times more effective than was d-amphetamine in the nonstriatal areas. The possible clinical significance of this finding will be apparent after our discussion of antiparkinsonian drug effects. TABLE I11 CATECHOLAMINE UPTAKE I N RAT CEREBRAL CORTEX CORPUS STRIATUM BY d- A N D 1- AMPHETAMINE^.^

INHIBITION O F AND

Ki d-Amphetamine ( x 10-7 M )

K i l-Amphetamine

Region Cerebral rortex Corpus striatum

2.9-3.1 0.85-1.2

27-32 0.95-1.25

( x 10-7 M )

u Brain homogenates were incubated with n~repinephrine-~H in the presence of amphetamine as described elsewhere (Snyder and Coyle, 1969; Coyle and Snyder, 196%). Ki values were determined by graphic analysis with the method of Dixon (1953). Values presented are a range of four determinations.

137


C. ANTIPARKINSONIAN DRUG ACTION In our search for agents that could discriminate between DA and NE neurons, we found that a large number of antiparkinsonian drugs (Table IV) were potent inhibitors of striatal catecholamine uptake (Coyle and Snyder, 1969b).Because many antiparkinsonian drugs are anticholinergic agents, it had often been postulated that acetylcholine receptor blockade was their mechanism of action. However, some antiparkinsonian drugs such as the antihistamine diphenhydramine (Benadryl) are fairly weak anticholinergic agents. We observed that a variety of antiparkinsonian agents, be they anticholinergics, antihistamines, indole derivatives, or phenothiazines, were all active inhibitors of striatal catecholamine uptake. Some of these drugs, such as benztropine and trihexyphenidyl, were 10-20 times more potent inhibitors of catecholamine uptake in the striatum than in the hypothalamus. Their preferential action on dopaminergic neurons contrasts strikingly with the selective effect on NE neurons of some antidepressants. Desmethylimipramine, for instance, is up to loo0 times more active in inhibiting catecholamine uptake by noradrenergic than by dopaminergic neurons ( Hamberger, 1967; Coyle and Snyder, 1969b ) . TABLE IV CATECHOLAMINE UPTAKE I NTO STRIATAL B N D HYPOTHALAMIC SYNAPTOSOMES BY ANTIPARKINSONIAN DRUGSO

IN H IB I T IO N OF

ID-50 Corpus striatum d~pamine-~H ( x 10-7 M )

Drug -

~~

~

Hypothalamus NorepinephrineJH

(x 10-7~)

~~

Benztropine (Cogentin) Trihexyphenidyl ( Artane) Orphenadrine (Disipal) Diphenhydramine (Benadryl) Phenindamine (Thephorin) Diethasine (Diparcol)

2 7 6 4 4 8

0 0 0 6 8 0

4.0 4.7 4.3 4.2 4.5 7.8

Homogenates from the striatum and the hypothalamus were incubated with drugs ranging in concentrations from 5 x 111 t o 5 X 10-9 M and with 0.1 p M concentration of the respective catecholamine-sH. Values are presented as the molar concenaccumulation and trations of drugs that produced 507' inhibition of ~atecholamine-~H were determined on logarithmic probability paper. Data presented are the means of three independent determinations for which the f3.E.M.s were not greater than 10% of the ID 6 0 value. Brand names of drugs are in parentheses. (I

138


The brains of patients with Parkinson’s disease appear to be depleted of their DA content (Hornykiewicz, 1966). The therapeutic efficacy of L-dopa in this condition (Cotzias et al., 1969) may be due to a partial repletion of the DA deficiency. As already discussed, catecholamine uptake appears to terminate their synaptic actions, We proposed that antiparkinsonian drugs owe their therapeutic eficacy to inhibition of striatal DA uptake (Coyle and Snyder, 1969b). By inhibiting reuptake of DA, the antiparkinsonian drugs would potentiate the effects of DA released at striatal synapses. This hypothesis can resolve some apparent paradoxes in the drug treatment of Parkinson’s disease. (1) Patients with increasingly severe Parkinson’s disease become progressively refractory to drug therapy. In such patients presumably there is less DA available for potentiation by antiparkinsonian drugs. (2) Antiparkinsonian drugs are more effective in the treatment of phenothiazineinduced Parkinson’s disease than in idiopathic Parkinson’s disease. Patients with the drug-induced syndrome should have intact dopaminergic normal systems so that adequate amounts of DA are available for potentiation. Recent evidence suggests that phenothiazine drugs which induce parkinsonism block DA receptors resulting in enhanced DA synthesis and turnover, findings which explain their parkinsonian side effects (Roos, 1965; DaPrada and Pletscher, 1966; Nyback and Sedvall, 1968). In light of this hypothesis for the mechanism of action of antiparkinsonian drugs, the absence of stereospecificity of amphetamine in its action on DA neurons appears to have therapeutic implication. d-Amphetamine has been employed in the therapy of Parkinson’s disease, but its central stimulant effects have limited dosage. Our model suggests that the therapeutic actions of d-amphetamine in this disease are related to inhibition of catecholamine uptake in the striatum. Inasmuch as d- and Z-amphetamine are equally potent inhibitors of catecholamine uptake in the striatum, Lamphetarnine should have equal antiparkinsonian activity. However, it could be administered in higher doses with fewer central stimulant side effects than d-amphetamine, and accordingly, should be a more powerful therapeutic agent. In support of this prediction, we found d- and l-amphetamine to be equally active in preventing the tremor and rigidity produced in mice by oxotremorine, a compound whose effects in animals resemble Parkinson’s disease ( George et aE., 1962). More recently we contrasted two behavioral effects of d- and Zamphetamine in rats ( a ) locomotor stimulation, ( b ) a stereotyped, compulsive gnawing syndrome. There is strong evidence that amphetamineinduced locomotor stimulation involves NE neurons ( Stein, 1964; Weiss-

NEUROTRANSMITTEXS IN THE BRAIN

139

man et al., 1966; Coyle and Snyder, 1969a). The stereotyped compulsive, gnawing behavior has been attributed to stimulation of dopaminergic mechanisms in the striatum, because it is abolished by removal of the corpus striatum, and can be elicited by direct implantation of DA or dopa into the corpus striatum (Ernst, 1967,1969; Randrup and Munkvad, 1967). While d-amphetamine was 10 times as potent as l-amphetamine in provoking locomotor stimulation, it was only twice as active in initiating the stereotyped, compulsive gnawing. Moreover diphenpyraline, an antihistamine which is a potent inhibitor of striatal DA uptake (Table IV), is as active as amphetamine in inducing the compulsive gnawing syndrome (Taylor and Snyder, 1970). Ill. Serotonin Uptake

All the above evidence indicates considerable specificity of catecholamine uptake into dopaminergic and noradrenergic neurons both on kinetic evidence and on the basis of drug action. Moreover, it seems possible to relate effects of drugs on catecholamine uptake to their therapeutic actions as central stimulants, as antidepressants (Schildkraut and Kety, 1967; Snyder, 1967), and as antiparkinsonian agents. Much less is known about serotonin (5-hydroxytryptamine ) uptake. Several workers described serotonin accumulation in brain which did not appear to be specific and could simply represent ion exchange processes ( Schanberg, 1963; Robinson et al., 1965). More recently, investigators (Blackburn et al., 1967; Ross and Renyi, 1967; Chase et al., 1969) using low concentrations of serotonin found evidence for an energy-requiring specific uptake of serotonin by brain slices. In support of a serotonin-specific uptake, we found that the regional localization of serotonin uptake in monkey brain slices differs from that of NE (Gfeller et al., 1968; Snyder et al., 1969). Also, small doses of intraventricularly administered ~erotonin-~H appear to localize selectively in serotonergic neurons ( Aghajanian and Bloom, 1967). However, there has been evidence that serotonin is accumulated by catecholaminergic neurons in the vas deferens (Thoa et al., 1969), in the pineal gland ( Bertler et al., 1964; Neff et al., 1969), and in the brain (Lichtensteiger et al., 1967; Fuxe and Ungerstedt, 1968). To clarify the specificity of serotonin uptake in the brain, we compared the kinetics of serotonin and NE uptake in slices from different regions of rat brain (Shaskan and Snyder, 1970). Serotonin uptake was evaluated in finely chopped slices of rat brain by methods similar to that described for catecholamines by Hendley and Snyder (1968) under conditions in which uptake was linear with tissue weight and for the duration of the incubation period and in which a

140


- 10

0

10

20

'/@-HT] X

30

40

50

M

FIG. 6. Graphic analysis of the reciprocals of serotonin-'H concentration and its uptake into striatal slices. Slices were incubated in quadruplicate for 4 minutes with ~erotonin-~H concentrations varying from 0.02 p M to 4 p M . Amine uptake ( 0 )is expressed as mpmoles of 'H/4 minutes per gm. Top: Serotonin concentrations from 0.02 pM to 0.4 p M . Bottom: Serotonin concentrations from 0.4 p M to 4 pM.

monoamine oxidase inhibitor was included to prevent metabolic degradation. Under these conditions with a modified Krebs-Henseleit ( Krebs and Henseleit, 1932) medium incubated under an atmosphere of 95% 0,5%COa, no metabolism of serotonin took place. As had been found

141


for DA uptake in nonstriatal areas of the brain, the reciprocals of serotonin uptake could not be described by single straight line plots, but could be resolved into two straight line components (Fig. 6 ) . In the striatum, hypothalamus, cerebral cortex, medulla oblongata-pons and midbrain, the K , values for the high affinity system (Uptake 1) and low affinity system (Uptake 2) were 2 x lo-' M and 8 x 1CP M , respectively. Several lines of evidence suggested that the low affinity components (Uptake 2 ) , which predominated at high serotonin concentrations, represented accumulation of serotonin by catecholaminergic endings ( Table V ) . (1) The catecholamines were most efficient inhibitors of serotonin uptake when higher concentrations of serotonin were employed. ( 2 ) In the striatum d- and Z-NE were equally potent in inhibiting serotonin uptake, while in the hypothalamus, LNE was 2-4 times more potent than d-NE. ( 3 ) Both in the striatum and in the hypothalamus, DA was a more potent inhibitor of serotonin uptake than was NE. We calculated, from the Michaelis-Menten equation the extent to which serotonin should enter the specific serotonergic uptake system (Uptake 1 ) or the catecholaminergic system (Uptake 2) at various concentrations of serotonin (Fig. 7 ) . At concentrations above 1W M , serotonin should enter catecholaminergic endings to a greater extent that it would enter serotonin endings. The tendency of serotonin to enter catecholaminergic endings should be greater in the striatum than in the hypothalamus. Despite the tendency for high concentrations of serotonin to enter catecholamine endings, at low concentrations it appears to be accumuTABLE V INHIBITION BY CATECHOLAMINES OF SEROTONIN-%

ACCUMULATION INTO

STRIATUM AND HYPOTHALAMUS~ ID-50 ( M )

Region and ~erotonin-~H concentration ~~~

~

Striatum (5 x 10-8 M ) Striatum (I x 10-6il.I) Hypothalamus (1 1 0 - 7 ~ )

+

~

&Norepinephrine ~~~

GNorepinephrine

Dopamine

~

~

40-50 X lo-'

1-2

x

2540 X

10-6

40-50 X lo-'

4-6 X

1-2

x

10-6

0 3-0 5 X lo-'

8-10

x

10-5

2.54 0

x

10P

Slices were incubated for 5 minutes in the presence of the inhibitor before serofor both areas are indicated in tonin-3H was added. Concentrations of ~erotonin-~H parentheses. IDdO values (concentration of catecholamine to reduce ~erotonin-~H uptake 50%) were derived from log probit plots of percent inhibition a t 3 or 4 concentrations of inhibitor in quadruplicate. Data presented are the range of 2 4 independent determinations. Q

142


806040

-

STRIATUM

6.020

\ 0

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t

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I x 10-8

Y

I x lo-' IXlOd 5-HT CONCENTRATION ( M )

1x10-6

HYPOTHALAMUS

3-

2-

1x10-8

Ix lo-' 1x10-6 5-HT CONCENTRATION (MI

I x 10-5

FIG. 7. Velocity of serotonin uptake by Uptake 1 and Uptake 2 at varying serotonin concentrations. Kinetic constants for the two serotonin uptake systems were ( K,/S)]. K, and V,,, values substituted in the Michaelis equation, u = V,,,/[l were determined graphically. Serotonin concentrations ( S ) were selected from 0.01 p M to 10 p M . Top: Uptake of serotonin-'H into striatum. Bottom: Uptake of serotonin-'H into hypothalamus.

+

lated specifically by serotonergic endings, The uptake of low serotonin concentrations is resistant to inhibition by catecholamines and, in monkey brain slices, shows a different regional localization than NE (Table I ) (Snyder et al., 1969). These results emphasize the caution required when using exogenous

NEUROTRANSMITTERS I N THE BRAIN

143

serotonin to label serotonin nerve endings. Serotonin nerve terminals will be labeled selectively only if very low concentrations of radioactive serotonin are employed. IV. Amino Acid Uptake and Subcellular localization

Of a variety of amino acids that have been examined as possible neurotransmitters in the mammalian central nervous system, the most impressive electrophysiological evidence exists for y-aminobutyric acid (GABA) (Krnjevic and Schwartz, 1967). There have been numerous studies of GABA uptake by mammalian brain (Elliott and Van Gelder, 1958; Sisken and Roberts, 1964; Iversen and Neal, 1968). GABA uptake by brain slices is highly specific and is not inhibited by other amino acids or amines. It resembles catecholamine and serotonin uptake in being temperature sensitive, requiring sodium, and being a saturable process that obeys Michaelis-Menten kinetics (Iversen and Neal, 1968). Although some studies suggested that GABA was uniformly distributed within the cytoplasm of brain neurons (Mangan and Whittaker, 1966), we found exogenous GABA localized in synaptosomes (Iversen and Snyder, 1968). Recently, Neal and Iversen (1969) showed that endogenous and exogenous GABA had identical subcellular distributions. The failure of earlier experiments to reveal a synaptosomal localization for GABA was attributed to a rapid leakage of GABA from nerve terminals during the subcellular fractionation process. Thus, of the five best candidates for a neurotransmitter role in the brain (acetylcholine, NE, DA, serotonin, GABA) all but acetylcholine possess specific uptake processes such that the endogenous synaptosomal transmitter pool may be labeled by the exogenous compound. Several workers recently described acetylcholine uptake by brain slices (Polak and Meeuws, 1966; Schuberth and Sundwall, 1967; Liang and Quastel, 1969). There is ample evidence that reuptake of NE terminates its synaptic actions, at least in the peripheral nervous system (Iversen, 1967; Axelrod, 1965). One might argue by analogy that reuptake terminates synaptic actions of GABA and serotonin as well as of the catecholamines. For acetylcholine, however, much evidence accumulated over the years favors enzymatic hydrolysis by acetylcholinesterase as the primary mode for terminating synaptic activities of acetylcholine. Many electrophysiological studies indicate that other amino acids, especially glutamic acid and glycine, may be central nervous system transmitters (Curtis and Crawford, 1969). Inasmuch as synaptosomal localization constitutes one criterion for central neurotransmitters, Mangan and Whittaker (1966) evaluated the subcellular localization of a large number of amino acids. All were distributed like cytoplasmic

144


markers, suggesting that none had a specific synaptic role. These authors pointed out, however, that most amino acids have prominent functions in protein synthesis and intermediary metabolism besides any conceivable neurotransmitter role, and a small synaptosomally localized “transmitter pool” of any given amino acid might not be detected when measuring the subcellular localization of the total amino acid content. But how is one to label the “transmitter pool” of an amino acid? Perhaps it would be subject to specific synaptosomal uptake, as occurs with other putative neurotransmitters. To examine this possibility, we incubated rat brain slices with 18 different amino a ~ i d s - ~and H measured their subcellular localization (Table VI) (Kuhar and Snyder, 1970). Most of the amino a ~ i d s - ~ were H distributed primarily in the supernatant fluid. Glutamic a ~ i d - ~and H gly~ine-~H, the principal neurotransmitter candidates on electrophysiological grounds, were particulate in localization, similar to NE-3H, as were some small neutral amino acids and certain basic amino acids, especially arginine-3H and ly~ine-~H. To obtain a more precise idea of the subcellular localization of glutamic a ~ i d - ~ H a, crude mitochondria1 pellet obtained by differential centrifugation was layered on a continuous sucrose gradient and centrifuged to density equilibrium (Fig. 8 ) (Kuhar and Snyder, 1970). Glutamic a ~ i d - ~was H TABLE VI PARTICULATE:~UPERNATANT FLUID RATIOS OF AMINOACIDS-~H IN RAT CEREBRAL CORTICAL SLICES” ~

Compounds Putative neurotransmitters ~~Norepinephrine-~H oL-Glutamic acid-3H Gly~ine-~H L-Aspartic a ~ i d - ~ H Basic amino acids bArginine-3H ~-Lysine-~H ~~-Ornithine-~H ~Histidine-~H Small neutral amino acids ~-Alanine-SH L-Serine-3H ~-Threonine-aH

Compounds 0.99 0.84 0.62 0.18

t 0.04 k 0.017 f. 0.033 5 0.011

1.07 t 0.13 1.07 f 0.007 0.81 f. 0.058 0.65 f 0.052

PIS 0.39 0.42 0.41 0.32 0.23 0.20 0.19 0.19 0.08 0.077

f 0.028 f. 0.008 t 0.009 5 0.006 5 0.043 f 0.093 t 0.017 0.0001 5 0.009 5 0.0001

*

0.50 f 0.005 0.60 k 0.021 0.81 t 0.013

Cerebral cortical slices (4 slices, 20 mg each) were incubated with tritiated compounds (0.1 p M ) and homogenized in 0.32 M sucrose. Homogenates were centrifuged a t 100,000 X g for 1 hour to form a total particulate fraction (P) and a soluble supernatant fluid (S). P/S ratios were calculated as counts per minute in particulate to counts per minute in soluble supernatant fluid. Results are expressed as the mean of four experiments f S.E.M.

145


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TUBE NUMBER

FIG.8. Subcellular distribution of glutamic acid-'H, potassium, and monoamine oxidase in a continuous sucrose gradient, Tissue slices incubated with glutamic acid'H (0.1 p M ) were homogenized in 0.32 M sucrose and crude mitochondria1 pellets were obtained by differential centrifugation. After suspension in 0.32 M sucrose the pellets were centrifuged on 0.32 to 1.46 M continuous gradients, Monoamine oxidase, potassium, and tritium were determined on the same gradients, and the results shown represent a typical one of three gradients. Monoamine oxidase activity is expressed as mpmoles of indole acetic acid per 20 minutes.

localized in a fraction that showed the same distribution as endogenous potassium ( a marker for cytoplasm occluded within synaptosomes) and was separable from the peak of monoamine oxidase activity (an enzyme marker for free mitochondria). Electron micrographs confirmed that the glutamic a ~ i d - ~peak H was the area of the gradient most enriched in also was highly synaptosomes (Kuhar and Snyder, 1970). Gly~ine-~H localized to synaptosomes with a profile similar to that of glutamic acid3H. In experiments in which slices were labeled with glutamic a ~ i d - ~ H

146


TABLE VII SUBCELLULAR LOCALIZATION OF EXOGENOUS AND ENDOGENOUS GLUTAMIC ACID, AND NOREPINEPHRINE-aH, LACTICACID DEHYDROGENASE, POTASSIUM IN RAT CEREBRAL CORTEX‘

Endogenous glutamic acid Glutamic acid-8H Norepinephrine-aH Lactic acid dehydrogenase Potassium+

P1

P2

S

3.1 4.3 5.3 5.0 3.8

14.2 28.1 44.1 16.0 16.7

82.8 67.6 50.6 79.0 79.5

a Tissue slices were incubated with tritiated compounds (0.1 p M ) and homogenized in 0.32 1 11sucrose. The P1 pellet was obtained by centrifuging the homogenate at 1000 X g for 10 minutes and washed by resuspending in 0.32 M sucrose and recentrifuging. The resulting supernatant fluid was centrifuged a t 17,000 X g for 20 minutes to form a pellet (P2) and supernatant fluid (S). Results are the average of two experiments and are expressed as percent of total content of the homogenate.

and NE-l‘C, both compounds were localized to synaptosomes, but the NE pattern of distribution was narrower and confined to a denser region of the synaptosomal area. Endogenous and exogenous glutamic acid showed different subcellular localizations in differential centrifugal experiments ( Table VII ) Exogenous glutamic acid tended to be particulate while the endogenous glutamic acid was distributed like the cytoplasmic markers lactic acid dehydrogenase and potassium. To support our hypothesis that glutamic a ~ i d - ~labeled H a “transmitter pool” of the endogenous glutamic acid, it would be important that the small amount of endogenous glutamic acid that was particulate should have the same subcellular localization as the exogenous amino acid. Indeed, sucrose density gradient centrifugation revealed that the synaptosomal moieties of exogenous and endogenous glutamic acid showed identical subcellular localizations (Fig. 9). Because our centrifugation procedures can separate different populations of synaptosomes (Iversen and Snyder, 1968; Kuhar and Snyder, 1970), we concluded that the exact superimposition of the patterns of exogenous and endogenous glutamic acid in our gradients indicated that the exogenous amino acid had mixed with the endogenous stores.

.

V. Separation of Catecholamine-Storing Synaptosomes in Different Brain Regions

The studies described above showed that it was possible to label endogenous pools of neurotransmitters with the exogenous compounds.

147


0

5

10 15 20 TUBE NUMBER

25

30

FIG.9. Subcellular distribution of exogenous and endogenous glutamic acid. Tissue slices incubated with glutamic acid-*H (0.1 p M ) were homogenized in 0.32 it4 sucrose and crude mitochondria1 pellets were obtained by differential centrifugation. After suspension in 0.32 M sucrose, pellets were centrifuged on 0.32 ti3 1.46 M continuous sucrose gradients. Exogenous and endogenous glutamic acid were determined on the same gradients, and the results shown represent a typical one of three gradients.

Of all the putative neurotransmitters studied, the best evidence for valid labeling was obtained with the catecholamines. As mentioned in the Introduction, workers who had tried to resolve different populations of synaptosomes had measured the endogenous transmitter content using

148


fairly large samples with relatively few fractions. Such procedures had limitations of sensitivity, resolving power, and rapidity of execution. To overcome these difficulties our strategy was to incubate single brain slices with two different putative transmitters or brain regions, one labeled with 14C and the other with 3H. The high sensitivity of radioactive counting enabled us to monitor as many as 30-35 fractions from each density gradient, and the use of two isotopes in a single gradient helped to resolve subtle differences in sedimentation properties reproducibly. Since, under the conditions of incubation, we had found no metabolic transformation of radioactive serotonin, NE, DA, GABA, or glutamic acid, we needed only to estimate total radioactivity. In this way chemical manipulations could be accomplished rapidly and as many as twelve experiments (two runs of six gradients each) could be completely processed in a day. Our initial experiments examined the subcellular localization of catecholamines in different areas of rat brain. In the hypothalamus, by using a sensitive enzymatic-isotopic technique to measure endogenous NE, endogenous and exogenous hypothalamic NE could be measured in 18 fractions of a sucrose density gradient (Fig. 10) (Green et al., 1969). Endogenous and exogenous catecholamines had identical subcellular localizations. When hypothalamic slices were incubated with catecholamine labeled with one isotope and striatal slices with another, the catecholamine endings in these two areas could be separated when homogenates were centrifuged on continuous sucrose gradients to apparent density equilibrium (Fig. 11) (Iversen and Snyder, 1968). When striatal slices were labeled with DA-3Hand hypothalamic slices with NE-I'C, hypothalamic N E particles were distributed in a less dense layer above the striatal DA particles. When the order of labeling was reversed (NE-14C, striaturn; DA-3H, hypothalamus ), the hypothalamic and striatal peaks of labeled amines separated as before; in this case, however, hypothalamic particles labeled with DA were recovered above the striatal particles containing NE. In experiments in which slices of striatum were labeled with DA-3H and NE-14Cboth amines were recovered in identical regions of the gradients, confirming that the separations obtained with different areas of the brain were not simply isotopic artifacts. Similar experiments were performed with varying combinations of four different areas of the brain (Fig. 12). Our results indicated that the catecholamine synaptosomes could be ranked in order of increasing sedimentation density as follows: hypothalamus < medulla oblongata-pons < cerebral cortex < striatum. Do these differences in sedimentation properties possess morphological correlates? In our experiments the most dense terminals occurred in


5

10

149

15

FRACTION NUMBER

FIG. 10. Sedimentation characteristics of exogenous and endogenous norepinephrine in density gradients from the hypothalamus. Hypothalamic dices were incubated with norepinephrine-aH (0.2 p M ). These and other unlabeled hypothalamic slices were homogenized and centrifuged on identical linear sucrose gradients ( 50-11%), and the fractions were assayed for 'H- or for endogenous norepinephrine. This experiment was repeated three times.

the striatum and the least dense in the hypothalamus. Histochemical studies indicate that catecholamine endings in the striatum and cortex are extremely fine, whereas in the hypothalamus there are many thick terminah (Fuxe, 19%; Fuxe et al., 1968). Catecholamine terminals in the medulla oblongata-pons of the rat tend to be intermediate in thickness between those in the hypothalamus and those in the striatum and cerebral cortex. It thus appears that the finer catecholamine terminals (e.g., in the striatum) sediment in denser sucrose than the thicker terminals (e.g., in the hypothalamus). VI. Separation of Synaptosomes Storing Different Transmitters

To separate synaptosomes storing different neurotransmitters, we used single regions of the brain labeled with two putative transmitters. In our first experiments, whole homogenates of striatal slices of rat brain incubated with differently labeled GABA and NE were labeled on continuous

91 STRIATUM 3 DOPAMINE- H

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0

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FIG. 11. Synaptosomes storing radioactive catecholamines in the striatum and hypothalamus. ( a ) Striatum labeled with norepinephrine-"C and d~pamine-~H. ( b ) Striatum labeled with dopa~nine-~H and hypothalamus with norepinephrine-"C. ( c ) Striatum labeled with norepinephrine-"C and hypothalamus with dopamine-'H. Each experiment was repeated five times.

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FIG. 12. Separation of catecholamine-storing synaptosomes in different areas of rat brain on continuous sucrose density gradients. Brain slices were incubated with norepinephrine-'H (0.2 p M ) or norepinephrine-14C ( 1.0 p M ) , combined, homogenized, and layered on linear sucrose gradients (50-11%). After centrifugation for 90 minutes at 130,000 X g, fractions were collected and assayed for aH or I4C. Each experiment was repeated at least five times and was also performed with reversed labeling of the slices.

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153


sucrose density gradients and centrifuged to apparent density equilibrium. Reproducible separations of GABA-storing and catecholaminesstoring synaptosomes were obtained, the catecholamine particles being distributed in a denser portion of the gradient (Iversen and Snyder, 1968).To eIiminate celldar debris of whole homogenates, in subsequent experiments we initially isolated a crude mitochondria1pelIet and layered different amounts of it on sucrose density gradients which were again centrifuged to density equilibrium (Fig. 13) ( Kuhar et al., 1970). While the larger amounts of tissue showed the same separation as in experiments with whole homogenates, with small amounts of tissue, the catecholamine and GABA particles migrated to identical portions of the

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10 20 FRACTION NUMBER

30

FIG. 15. Determination of potassium, norepinephrine-*H, and GABA-“C in continuous sucrose gradients. Crude mitochondrial pellets were layered on 40-ml continuous 1.46-0.32 M sucrose. Tritium and “C were measured in an aliquot of each of 33 fractions collected after centrifuging for 20 minutes at 100,000 X g. Pairs of consecutive fractions were combined to give a total of 16 fractions and potassium content was measured in each. The experiment was performed three times.

gradient. In the gradients with smaller amounts of tissue, GABA particles migrated to a denser portion of the gradient than in gradients with higher tissue content so that their distribution pattern was superimposed on the catecholamine synaptosomes. This suggested that larger amounts of tissue had “retarded the movement of the GABA particles down the gradient. Thus in the experiments which separated GABA and catecholamines the GABA particles had only been “approaching” density equilibrium but had not attained it as had the catecholamine particles. These results suggested that incomplete density equilibrium sedimentation might be a productive approach. With gradient centrifugation at 100,OOO x g for only 15-25 minutes an improved separation of GABA and catecholamine synaptosomes was obtained ( Fig. 14). In some experiments these particles could be separated with no overlap. The enhanced resolution of the two synaptosomal populations occurred despite the use of a “small” mitochondrial pellet, which in 90-minute centrifugations (to apparent equilibrium density) would have failed to separate GABA and catecholamine particles. These findings raise a number of questions. (1) In what part of the sucrose gradient are the bulk of the striatal synaptosomes located? A biochemical approach to this question can be provided by measuring potassium, a marker for cytoplasm occluded within synaptosomes (Fig. 1 5 ) . The peak localization of synaptosomal potassium was between the

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FIG. 16. Electron micrographs of GABA and catecholamine synaptosome proEles in continuous sucrose density gradients. Gradients were prepared in the same manner as for Fig. 15. The contents of each fraction were fixed in buffered 1%KMnO, and embedded in Araldite. Magnification of all electron micrographs was 35,OOOX. Upper left: Synaptosomes from the catecholamine peak (fraction 9 of Fig. 15) showing a dense cytoplasm, mitochondrion and synaptic vesicles of 300-400 A diameter. Upper right: Synaptosomes from the catecholamine peak showing an intact synaptic cleft with adhering subsynaptic web. The postsynaptic element adhering to the synaptosome to the right may represent a dendritic spine. Upper right: Synaptosome from the GABA peak (fraction 16 of Fig. 15) showing vesicles of different size, some of which are synaptic vesicles. Next to the mitochondrion microtubular profiles can be identified. Lower right: Large synaptosomes from fractions of the GABA peak showing a large number of synaptic vesicles and two mitochondria1 profiles.

catecholamine and GABA peaks. Electron microscopy of different fractions from these gradients (Kuhar et al., 1970) confirmed that the largest number of synaptosomal profiles occurred in the area between the GABA and catecholamine peaks. ( 2 ) Most important, are there morphological differences among the

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various synaptosomal fractions? Electron microscopic studies, indeed, revealed unique synaptosomal morphology for different areas of the gradients ( Kuhar et al., 1970). Catecholamine particles predominated in the most dense portion of the gradients where synaptosome profiles were few and interspersed with large numbers of free mitochondria. The synaptosomes in this region had fairly uniform appearance (Fig. 16). Their diameter was smaller, and frequently their post-synaptic membrane was adherent. In the synaptosomes with intact junctional complexes the postsynaptic element stained densely with lead hydroxide. In the region of the GABA peak, synaptosomes had varying diameters with some very large synaptosomal profiles. In this region there were few free mitochondria and a high concentration of synaptosomes. However, synaptosomes with adherent postsynaptic membranes were infrequent. The morphological descriptions cited above certainly cannot be ascribed specifically to catecholamine or to GABA synaptosomes. It is quite possible that synaptosomes storing other known and unknown transmitters exist in all gradient fractions. However, in the most “catecholamine” area of the gradient, the uniform appearance of synaptosomes suggests that they constitute a single population and might be a fairly pure population of dopaminergic nerve terminals. Experiments are in progress to ascertain the localization of synaptosomes storing other transmitters, specifically serotonin, acetylcholine, and glutamic acid. VII. Conclusions

Nerve terminals in the brain possess specialized uptake mechanisms for a variety of putative neurotransmitters, such as DA, NE, serotonin, GABA, and possibly, for glutamic acid and glycine. Kinetic studies have elucidated optimal conditions for nerve endings in brain slices or in isolated, pinched-off form ( “synaptosomes”) to accumulate selectively their specific transmitter. We have used these uptake systems to label the endogenous transmitter pools with exogenous, radioactive neurotransmitters, labeling one class of synaptosomes with 3H-transmitter and another with I4C-transmitter. In this way, with density gradient centrifugal procedures, we have separated synaptosomal populations in the same brain region storing different transmitters or in different brain areas storing the same transmitter. Electron microscopic studies have revealed specific morphological features for the varying synaptosomal populations. REFERENCES Aghajanian, G., and Bloom, F. (1967). J. PhurmacoE. Exptl. Therap. 156, 23. Axelrod, J. (1965). Recent Progr. Hormone Res. 21, 597. Bertler, A., Falck, B., and Owman, C. (1964). Actu Physiol. Scand. SuppE. 239.


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Blackburn, K. J., French, P. C., and Merrills, R. J. (1967). Life Sci. 6, 1653. Bogdanski, D. F., Tissari, A., and Brodie, B. B. (1968). Life Sci. 7, 419. Chase, T. N., Katz, R. I., and Kopin, I. J. (1969). 1. Neurochem. 16, 607. Cotzias, G. C., Papavasilou, P. S., and Gellene, R. (19691). New Engl. J. Med. 280, 337. Coyle, J. T., and Snyder, S. H. (1969a). J. Pharmacol. Exptl. Therap. 170, 221. Coyle, J. T., and Snyder, S. H. (1969b). Science 166, 899. Curtis, D. R., and Crawford, J. M. (1969). Ann. Reu. Phurmacol. 9, 209. DaPrada, M., and Pletscher, A. (1966). Experientiu 22, 465. Davis, J. M., Goodwin, F. K., Bunney, W. E., Jr., Murphy, D. L., and Colburn, R. W. ( 1967). Pharmacologist 9, 184. Dengler, J. J., Michaelson, 1. A., Spiegel, H. E., and Titus, E. (1962). Intern. J. Neuropharmucol. 1, 23. DeRobertis, E. ( 19167). Science 156, 907. DeRobertis, E., Pellegrino de Iraldi, A., Rodriguez de Lores Arniaz, G., and Salganicoff, L. (1962). 1. Neurochem. 9, 23. Dixon, M. (1953). Biochem. J. 55, 170. DuBois Reymond, E. (1877). Ges. Abhandl. Allgem. Muskel. Neruenphysik. 2, 700. Eccles, J. C. (1964). “The Physiology of Synapses.” Springer, Berlin. Elliott, K. A. C., and Van Gelder, M. N. (1958). J. Neurochem. 3, 28. Emst, A. M. (1967). Psychophurmucologia 10, 316. Ernst, A. M. (1969). Acta Physiol. Pharmacol. Need 15, 141. Fuxe, K. (1965). Actu Physiol. Scand. Suppl. 241, 39. Fme, K., and Ungerstedt, U. (1968)).Histochemie 13, 16. Fuxe, K., Hamberger, B., and Hokfelt, T. (1968). Brain Res. 8, 125. George, R., Haslett, W. L., and Jenden, D. J. (1962). Life Sci. 8, 361. Gfeller, E., Green, A,, and Snyder, S. H. (1968). Brain Res. 11, 263. Glowinski, J., and Baldessarini, R. I. (1966). Phurmucol. Reu. 8, 1201. Glowinski, J., and Iversen, L. L. (1966). J. Neurochem. 13, 655. Green, A. I., Snyder, S. H., and Iversen, L. L. (1969). J. Phurmacol. Exptl. Therap. 168, 264. Hamberger, B. ( 1967). Acta Physiol. Scand. Suppl. 295, 7. Hendley, E. D., and Snyder, S. H. (1968). Nature 220, 1330. Hillarp, N. A., Fuxe, K., and Dahlstrom, A. (1966). Phurmucot. Rev. 18, 727. Hornykiewicz, 0. (1966). Phurmucol. Reu. 18, 925. Iversen, L. L. (1967). “The Uptake and Storage of Noradrenaline in Sympathetic Nerves.” Cambridge Univ. Press, London and New York. Iversen, L. L., and Neal, M. J. (1968). J . Neurochem. 15, 1141. Iversen, L. L., and Snyder, S. H. (1968). Nature 220, 796. Krebs, H. A., and Henseleit, K. (1932). 2. Physiol. Chem. Hoppe-Seykrs 210, 33. Kmjevic, K., and Schwartz, S. (1967). Exptl. Brain Res. 3, 320. Kuhar, M. J., and Snyder, S. H. (1970). I. Pharmucol. Exptl. Therap. 171, 141. Kuhar, M. J., Green, A. I., Snyder, S. H., and Gfeller, E. (1970). Brain Res. (in press). Liang, C. C., and Quastel, J. H. (1969). Biochem. Ph~rmacoZ.18, 1169. Lichtensteiger, W., Mutzner, U., and Langemann, H. (1967). J. Neurochem. 14, 489. Lineweaver, H., and Burk, D. (1934). J. Am. Chem. SOC. 56, 685. Mangan, J. L., and Whittaker, V. P. (1966). Biochem. J. 98, 128. Michaelson, 1. A,, and Whittaker, V. P. (1963). Biochem. Pharmacol. 12, 203. Neal, M. J., and Iversen, L. L. (1969). J. Neurochem. 16, 1245.

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Neff, N.'H., Barrett, R. E., and Costa, E. ( 1969). European J. Pharmucol. 5, 348. Nyback, H.,and Sedvall, G. (1968). 1. PharmacoZ. Exptl. Therap. 162, 294. Polak, R. L., and Meeuws, M. M. (1966). Biochem. Pharmacol. 15, 989. Randrup, A., and Munkvad, I. (1967). Psychopharmacologia 11, 300. Robinson, J. D., Anderson, J. H., and Green, J. P. (1965). J. Phamnacol. ExptZ. Therap. 147, 236. Roos, B. E. (1965). 1. Phamz. Pharmacol. 17, 820. Ross, S. B., and Renyi, A. L. (1967). Life Sci. 6, 1407. Schanberg, S. M. (1963). 1. Pharmacol. Exptl. Therap. 139, 191. Schildkraut, J. J., and Kety, S. S. (1967). Science 156, 21. Schuberth, J., and Sundwall, A. ( 1967). J. Neurochem. 14, 807. Shaskan, E. G., and Snyder, S. H. (1970). J. Phurmucol. Exptl. Therap. (in press). Sisken, B., and Roberts, E. (1964). Biochem. Pharmucol. 13, 95. Snyder, S. H. (1967). Am. J. Orthopsychiat. 37, 864. Snyder, S. H., and Coyle, J. T. (1969). J. Pharmucol. Exptl. Therap. 165, 78. Snyder, S. H., Green, A., Hendley, E. D., and Gfeller, E. (1968a). Nature 218, 174. Snyder, S. H., Green, A,, and Hendley, E. D. (196813). J. Pharmacol. Exptl. Therap. 164, 90. Snyder, S. H., Hendley, E. D., and Gfeller, E. (1969). Brain Res. 16, 469. Stein, L.( 1964 ) . Federation PTOC.23, 836. Taylor, K., and Snyder, S. H. (1970). Federation Proc. 29, 1280. Thoa, N. B., Eccleston, D., and Axelrod, J. (1969). J. Phamnacol. Exptl. Therap. 169, 68. Weissman, A., Koe, B. K., and Tenen, S. S. (1966). J. Phamacol. Exptl. Therap. 151, 339. Whittaker, V. P. (1965). Progr. Biophys. Mol. Biol. 15, 39.

CHEMICAL MECHANISMS OF TRANSMITTER-RECEPTOR INTERACTION By John T. Garland1 and Jack Durell Section on Membrane Chemistry, laboratory of Cerebral Metabolism, National Institute of Mental Health, Bethesda, Maryland, and Center for the Study of Behavioral Biology, Psychiatric Institute Foundation, Washington, D. C.

I. Studies to Elucidate the Chemical Nature of the Receptor A. Transmitter Structure-Activity Relationships (SAR) B. Receptor Inactivation-Reactivation . . . . C. Receptor Isolation . . . . . . . D. Summary . . . . . . . . . 11. Biochemical Effects of the Transmitters . . . . . . . . . A. Effects on Adenyl Cyclase . B. Changes in Lipids . . . . . . . C. Miscellaneous Studies . . . . . . 111. Theories of Transmitter-Receptor Interaction . . A. Models Requiring Only Conformational Changes . B. Models Requiring Chemical Changes . . . C. Interrelationships of Theories . . . . . References . . . . . . . . .

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Long ago man recognized that somehow it is man’s brain that sets him apart from other animals, but its remarkable complexity has made the detailed analysis of its mechanisms a slow process. Anatomical and physiological investigation revealed a vast array of intercommunicating cells. Early in this century more precise measurements led to the recognition of a delay in transmission that corresponded to the gaps between cells that had been seen by neuroanatomists. This led to the postulate that a chemical mediator carries the message across these gaps. Though the controversy continues, generally it is believed that at most synapses the electrical message is transmitted by a chemical intermediary. Little is known of the molecular mechanisms by which this occurs. Several substances have been proposed as transmitters, and there may well be several different systems; some, however, prefer to postulate a basic acetylcholine ( ACh) system with the other “transmitters” reIegated to the role of modulating the sensitivity of the postsynaptic cell to this main transmitter. We shall not attempt to critically review the identification Present address: Washington University School of Medicine, Department of Internal Medicine, Metabolism Division, 660 South Euclid, St. Louis, Missouri. 159

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of transmitters. The criteria for certain identification are quite rigorous ( McLennon, 1963), and in most cases there is continuing controversy due to the lack of adequate data. Rather, we shall address ourselves to the studies that contribute to the elucidation of the possible molecular mechanism of action of the neural transmitters. No attempt will be made to present an exhaustive listing of all related work; we shall, however, present pertinent examples and try to integrate the available information as much as possible. One of the drawbacks to progress in this area is that we lack unifying theories to test, and against which to measure the significance of experimental results. It is hoped that we may partially overcome this obstacle with this review. In the first section we shall consider the chemical nature of the receptors. Then we shall examine the biochemical effects of the transmitters in the postsynaptic cell. With this as background, we can then discuss the possible nature of the transniitter-receptor interaction. I. Studies to Elucidate the Chemical Nature of the Receptor

A. TRANSMIITER STRUCXURE-ACTIVITY RELATIONSHIPS ( SAR ) A frequently employed tool for the study of the chemical nature of receptors is the use of a series of closely related compounds-preferably differing by only small progressive changes in one parameter-to study the resulting changes in transmitter (or inhibitor) activity. If the relationship between these structural changes and the changes in activity is consistent, inferences may be drawn concerning the molecular structure of the complementary portions of the receptor. In these evaluations it is important to distinguish between the affinity (or binding tendency) and the intrinsic activity (or ability to produce the characteristic effects) of either an agonist (receptor activator) or an antagonist. For example, a critical change in structure might convert an agonist to an antagonist while producing only modest changes in binding.

1. Adrenergic The nature of the receptors for adrenergic drugs has been repeatedly studied by the SAR technique. Amid the reports of countless trials of multiple drugs on different tissues, Alquist (1948), on the basis of sequences of relative potency, postulated two types of receptors (alpha and beta). This allowed rational classification of compounds as agonists or antagonists for both alpha and beta receptors. Target tissues could also be classed as having predominantly alpha, beta, or a mixture of receptors. Often they are mixed in such a manner that alpha and beta

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stimulation provoke opposite physiological responses. Thus varying the proportions of epinephrine and norepinephrine allows for “finer tuning” of the blood pressure, owing to their differing effects on receptor activation. This concept, which brought order to a vast array of data, has been extremely useful. Increasing the size of the N-alkyl moiety of adrenergic drugs decreases alpha activity, whereas the addition of a large substituent (especially aralkyl groups) on the N gives an alpha blocker. Thus, the chemical properties of the amino group have special significance for activity with the alpha receptor ( Ariens, 1967). A small branched structure on the N is better for beta activity. The catechol nucleus-especially the phenolic hydroxyl groups-similarly have special significance for intrinsic activity in interaction with a beta receptor, and halogen substitution of the phenolic hydroxyl gives increased affinity for beta blockers. Further separation of the catechol nucleus from NH, gives a relatively more potent beta blocker. This suggests that the interaction of beta blocking compounds with accessory receptor areas contributes to this affinity (Ariens, 1967). Since some drugs (e.g., epinephrine) act as both alpha and beta stimulators we would e-pect considerable similarity between these receptors. Later (Section 111, C ) we will consider the implications of these facts for the actual structure of these receptors when we discuss Behau’s proposed mechanisms of action of adrenergic drugs.

2. Cholinergic It has been demonstrated that the postganglionic parasympathetic receptors in guinea pig ileum (an ACh receptor) require the 4-carbonyl group (presumably the onium group) for affinity and the 3 ether oxygen atom and the trimethylammonium for efficacy (Barlow et al., 1963). Bartles ( 1965), using intracellular recording from monocellular electroplax, confirms the importance of the carbonyl by demonstrating that cyclohexyl or phenyl substitution of the methyl on the carbonyl (which would alter the electron distribution) decreases the depolarizing action by a factor of $00. Replacement of the carbonyl carbon by sulfur to give methane sulfonyl choline (MSC) allows substitution for ACh in ganglionic (nicotinic) stimulation, but MSC has only a weak muscarinic actiondecreased 1000-fold ( Eckhardt and Schueler, 1963).This dissociation of these two major effects of ACh lends support to the possibility that these receptor sites are at least partially different chemically. The duality of ACh receptors (nicotinic arld muscarinic) is analogous

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to that of adrenergic receptors ( alpha and beta). The physiological advantage of this duality for the ACh receptor (AChR) is not clear. It is interesting to note that the receptors for serotonin (5-HT) have also been classified into two types: The M (nervous) type-as found in calf tracheal muscle-is antagonized by morphine or atropine and potentiated by acetylcholinesterase ( AChE) inhibitors, but LSD has no effect. The D (muscular) type-as found in the rat stomach-fundus strip-is antagonized by LSD, and AChE inhibitors have no effect (Offermeier and Ariens, 1966). The technique of studying transmitter SAR’s has provided a tool for reevaluating the role of acetylcholinesterase in ACh action. More than thirty years ago Roepke (1937) suggested that AChE was in fact the ACh receptor. Because of the high degree of complementarity for ACh, its localization at the postjunctional site, and its ability to react with many, if not all, of the compounds that influence cholinergic events, this seemed a reasonable hypothesis (Ehrenpreis, 1967), but there is increasing evidence to the contrary. With a series of esters of tropine and psi-tropine, Friess et al. (1964) related electronegativity to the toxic intravenous dose in mice and to the activity on rat phrenic nerve-diaphragm preparations, showing a steep linear relationship. Their studies with AChE showed exactly the reverse relation for inhibition of AChE, suggesting that AChE differs from the AChR. Kimura (1965) showed a difference in the order of potency of organophosphorylcholne derivatives reacting with the AChR in frog rectus abdominus compared to their inhibition of both AChE and ChE. Though he did find similar pK, values for the anionic sites, these data do support a difference between the esteratic site of AChE and the corresponding site of AChR. Nachmansohn (1964) notes that the dissociation constant for AChR in isolated electroplax is 2.4 x lo-? M and that at “this low concentration AChE is hardly affected even in solution.” Podleski ( 1967) , using intracellular recording on isoIated monocellular electroplax, showed that inhibition of the esteratic site of AChE by methane sulfonyl fluoride did not abolish depolarization by subsequent ACh, trimethylammonium, or 3-OH-phenyltrimethylammonium. Also, the inhibitory effects of l-methyl-7-hydroxylquinolinium were not affected. Similarly, preincubation of AChE with para-chloromercuribenzoate or dithiothreitol caused no significant effect upon the K , or ,TF of AChE under conditions which would give strong inhibition of the electrical response of intact electroplax to ACh (Karlin, 1967a). Thus it seems that the AChR is not AChE. This is not to say that AChE is unimportant, however, for it may be that its inactivation of ACh allows regeneration of the “resting” state of the receptor, permitting it to react again.

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B. RECEPTOR INACTIVATION-REACTNATION Another approach to the elucidation of the chemical composition of the receptor has been to determine what compounds can inactivate the receptor or restore its activity when it has been inactivated. From these data and a knowledge of basic chemical interactions, one may infer the corresponding chemical characteristics of the receptor.

1. c h o l i ~ r g i c Okada (1967) has shown a variety of effects of alcohols on the neuromuscular junction of the frog. Probably alcohols increase the permeability of the presynaptic membrane and decrease the effective resistance of the muscle membrane. Acetone will also cause some of these changes, but ether and chloroform will not. These findings suggest that the receptor structure includes lipid, but the results are not conclusive. Nastuk (1967) has shown competitive inhibition of the muscle postjunctional receptors by Ca++,Mg", or U02++,the latter at much lower concentrations, Since UO,++has a high affinity for phosphate, the anionic sites of the receptor may include phosphoryl ligands. Karlin and Winnik ( 1968) have utilized 4-( N-maleimido)phenyltrimethylammonium iodide (MPTA) for specific alkylation of the electroplax AChR (after its reduction by dithiothreitol). Specificity is indicated by the extremely rapid inactivation of the receptor by MPTA-more than 300-fold faster than N-ethyl maleimide ( NEM)-whereas MPTA's rate of reaction with cysteine is only slightly greater than that of NEM. Also, hexamethonium (HM) competes with MPTA but does not affect alkylation by NEM. Reaction with MPTA changes the AChR so that subsequent treatment with HM results in activation rather than the usual blockade ( suggesting some conformational change in the AChR itself). Using a molecular model of MPTA, the authors estimated that the disulfide group is about 10 A from the anionic subsite that binds N'. Together, these studies point to a complex receptor including a lipid, a phosphoryl group, and a disulfide bond near the anionic site. The disulfide probably is on a protein. 2. Serotonergic

For the serotonin (5-HT) receptor, Woolley and Gommi (1964) showed inactivation by a combination of neuraminidase plus EDTA ( ethylenediaminetetraacetate ) and reactivation by crude lipid extracts or purified gangliosides. Later studies ( Woolley and Gommi, 1965) have shown marked variation among different gangliosides, and the significance of this is not clear, though they suggest that different receptors

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may have differing lipid components. Offermeier and Ariens (1966) have criticized these results, for they have found that incubation with only a M N-acetylneuraminic sialic acid also restores 5-HT action (e.g., 3 x acid ( NANA ), N-glycolyl-NA, or N-carbobenzoxy-NA for 15-30 min) . This may not rule out a significant role for gangliosides, however, for one could postulate that these additions serve as a stimulus for endogenous ganglioside synthesis that then allows restoration of the 5-HT receptor. Similarly this possibility could allow the spontaneous restoration that both groups noted in 2-3 hours. Their other criticism-that EDTA alone will suffice to produce inactivation-is not so easily dismissed, and it suggests that the loss of Ca++is perhaps more important than the loss of sialoside. Further support for the sialoside role is provided by Wesemann and Zilliken (1968). They have found similar relationships between the 5 H T induced contraction and sialic acid metabolism in the liver Auke Fasciola hepatica and the rat stomach-fundus. They showed that exogenous sialic acids or gangliosides increase the height of contraction. An inhibitor of NANA-9-P synthetase reversibly decreased the 5-HT induced contraction of both liver fluke and rat stomach-fundus. These experiments suggest that the synthesis of gangliosides is necessary for 5-HT induced contraction. It is not clear, however, whether they are actually a part of the 5-HT receptor per se or whether they are only necessary for something else (e.g., structural integrity) required for the measurable end resultcontraction.

C . RECEPTORISOLATION Another method of studying the interaction of transmitter and receptor, free of the complexity inherent in studies involving the intact nervous system, is through attempts at isolation of the receptor. 1. Adrenergic Lewis and Miller ( 1966) used tritiated phenoxybenzamine (PhB3H), an irreversible inhibitor, to label the alpha receptors of the rat seminal vesicle, They compared the radioactivity in tissues treated with an agonist (to prevent PhB-3H binding on the alpha receptors) to that in unprotected controls. The PhB blockade was verified by cumulative dose-response curves, and the specificity was demonstrated by showing that prior exposure to phentolamine, a reversible inhibitor, prevented the irreversible blockade. After two extractions with 2 : 1 chloroform-methano1 ( C / M ) the counts due to receptor binding (i.e., those sites protected by the agonist) remained in the residue, This suggested to the authors that the receptor is nonlipid; it should be noted, however, that some


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phospholipids are not readily extractable in neutral C/M (e.g., phosphatidylinositol phosphate and phosphatidylinositol diphosphate ) , 2. Cholinergic Many efforts have been made to isolate the AChR, and these have been reviewed by Ehrenpreis (1967) and by Hasson-Voloch (1968). In spite of initial encouragement, in each of these studies, after all of the appropriate controls had been done, the binding was shown to lack sufficient specificity. For example, the d-tubocurarine ( dTC ) precipitable phospholipoprotein which Ehrenpreis and his group had isolated and had purified considerably is found in the membrane of both the innervated and noninnervated sides of electroplax. Also, the dTC binds to several different classes of partially purified cellular components ( Ehrenpreis, 1967). DeRobertis and his group ( DeRobertis, 1967; Fiszer and DeRobertis, 1967; DeRobertis et al., 1967a,b) have taken a different approach that seems quite promising. With differential and density gradient centrifugation, they isolate “synaptosomes.” By treating these with a nonionic detergent (Triton X-100) they were able to free these of much of the nonjunctional membrane as well as the contents of the synaptosomes. They demonstrated this by showing that the binding of cholinergic blockers ( dimethyl-dTC-14C and methyl-HM-l‘C ) is preserved even though there is considerable loss of AChE and NaK-ATPase. Takagi et al. (1965),using only differential centrifugation, were able to prepare AChR-rich fractions, as shown by the binding of 3H-dibenamine to receptors that had been blocked by atropine. When they applied this “receptor” to a Sephadex column it behaved like a high molecular weight compound. It was degraded by Pronase to lower molecular weight components, and this suggests a protein constituent. Thus we have evidence for a protein component of AChR and a method that carries subcellular fractionation one step past the synaptosome. It will be interesting to determine whether the effect of ACh on 32Pincorporation into phosphatidic acid (PA), which has been localized to the synaptosomes (Durell and Sodd, 19%), occurs in the “isolated junctional membranes.” 3. Serotonergic Marchbanks (19%) has shown 5-HT binding to nerve ending particles in rat brain. This high affinity binding is absent from liver and inhibited by M d-LSD. Because the binding factor can be extracted and reprecipitated from n-butanol while retaining its binding properties (including inhibition by d-LSD), it may be a lipid. It is destroyed by neuraminidase, which is further evidence that a ganglioside may be involved.

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D. SUMMARY With the above studies in mind, we can now draw a few conclusions about the chemical nature of the receptors. There are at least two types of adrenergic receptors which differ chemically, at least in part; different chemical characteristics of the catecholamines are critical in alpha as opposed to beta activity. There are some studies suggesting that lipids do not play a major role, but these are inconclusive as noted in Section I, c, 1. There are also two types of AChR. The cholinergic receptor is probably not AChE, though the anionic sites are similar. The presence of a disulfide and digestion by Pronase both suggest a protein component. A phosphoryl group is also likely, based on the tight binding of UO,". The evidence suggests that the serotonergic receptor is also comprised of two closely related but chemically different receptors. In these, the lipid component seems to be more firmly established. Gangliosides are especially important-at least at some step prior to the measurable end result, contraction. Considering the receptors for all of these compounds, one may infer that, in general, receptors are probably complex mixtures of lipid and protein. There are at least two types of receptor for each of these transmitters. I I . Biochemical Effects of the Transmitters

Studies of the biochemical effects of the neural transmitters have focused primarily on two areas. Most productive have been studies of the effects of catecholamines on the adenyl cyclase system. Other workers have studied the effects of cholinergic compounds or nerve stimulation upon phospholipids, proteins, or nucleic acids. Because of the paucity of data in other areas, we shall review primarily the adenyl cyclase and phospholipid studies.

A. EFFECTS ON ADENYLCYCLASE Murad et al. (1962) using preparations from liver and cardiac muscle found that catecholamines cause an increase in the concentration of cyclic 3',5'-adenosine monophosphate (CAMP),beginning at concentrations as low as 1W M. The order of decreasing potency was isopropylnorepinephrine, &epinephrine, norepinephrine-suggesting a beta adrenergic receptor, Turtle and Kipnis (1967) have confirmed an adrenergic receptor mechanism for CAMP, in three in vitro systems: isolated pancreatic islets, isolated fat cells, and toad bladders. They found that in the presence of theophylline (to block the phosphodiesterase that hydro-


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lyzes CAMP) beta stimulators increased and alpha stimulators decreased the levels of CAMP. These changes paralleled the usual hormonal effects seen with the drugs in these tissues. Acetylcholine or carbamylcholine, but not acetate or choline alone, decreases the formation of cyclic AMP in cardiac muscle and liver by 30% (Murad et al., 1962). Atropine will prevent this decrease, though it has no effect alone. Serotonin has been shown to activate phosphorylase and cause a rapid and specific increase in the formation of CAMP as catalyzed by a particulate fraction of the liver fluke (Mansour et al., 1960). Several other hormones have been shown to influence cAMP in their target organs (Sutherland et al., 1965). The first change associated with increased cAMP was increased activity of phosphorylase, and at first it was thought to be the mechanism for all CAMP action. Different pathways may be affected, however. Beuding et al. (1966) have shown that cAMP in intestinal smooth muscle may be increased without changes in hexose phosphates, i.e., with no activation of phosphorylase or phosphofructokinase. Also, it is possible to use a very small dose of epinephrine to increase the inotropic force of the heart without an increase in heart phosphorylase ( Robison et al., 1987). When assessing the significance of such changes in CAMP, one must keep in mind that the physiological parameters measured may often be several steps removed from the primary effect. Even if the changes are highly correlated, there need not necessarily be a direct causal relationship. An illustration of this divergence is found in the study of the cell membrane permeability in frog skin ( Cuthbert and Painter, 1968). These authors separately recorded skin resistance and the potential across the inner and outer facing membranes of both normal and current-clamped frog skin. Here antidiuretic hormone ( ADH) and theophylline reduce skin resistance, but cAMP increases it. ADH increases the potential across the outer membrane, theophylline reduces it, and cAMP gives a biphasic potential change. These differences are in striking contrast to the indistinguishable effects of these substances on sodium transport (Baba et al., 1967; Orloff and Handler, 1961, 1962,1964).If these studies are confirmed they will have great significance, for they show a divergence of the effects of a hormone and CAMP, where formerly the hormone was thought to act via the adenyl cyclase system. Also of note here is the divergence of the effects of theophylline and CAMP;if one uses the former to block the degradation of the latter, one must show that it has no independent effect on the tissue under study. Almost every tissue is capable of forming CAMP, and most show variation in the rate of synthesis with one or more hormones (Sutherland et al., 1965). The brain has an especially high concentration of cyclic

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3’,5’-nucleotide phosphodiesterase (Butcher and Sutherland, 1962) and adenyl cyclase (Sutherland et al., 1962). The latter is very sensitive to stimulation, for Klainer et al. (1962) have shown a half-maximal increase M Z-epinephrine. Studies of in cerebellar adenyl cyclase with 5 x the subcellular distribution of these enzymes following density gradient separation and osmotic shock show especially high concentrations in nerve ending membranes which include postsynaptic fragments ( Azcurra and DeRobertis, 1967). Thus brain has high concentrations of the necessary enzymes located appropriately for cAMP to be a mediator in the action of neural transmitters. Though catecholamines have been most extensively studied, both ACh and 5-HT can affect CAMP, as noted above. Nevertheless, there is no direct evidence that cAMP plays any role in neural transmission.

B. CHANCES IN LIPIDS Using pancreas slices, M. R. Hokin and L. E. Hokin (1953) first demonstrated that ACh stimulates the incorporation of radioactive inorganic orthophosphate ( 32Pi ) into the phospholipids. Studies on slices of a number of other ACh sensitive glands and on several preparations of neural tissue have shown similar effects (Hokin and Hokin, 1955, 1958a,b, 1960; Durell and Sodd, 1964). Phosphatidylinositol (PI) and phosphatidic acid (PA), a precursor in the biosynthetic pathway of PI (Paulus and Kennedy, 1960), are the phospholipids whose synthesis is most consistently stimulated. The physiological significance of these observations has been enhanced by the studies of Larrabee and Leicht (1965). They have studied autonomic nerves and ganglia using electrical stimulation and analyzing the tissue and/ or the media for chemical changes. Ganglia containing synapses are required to demonstrate the increased 32Pincorporation that accompanies electrical stimulation of nerves. After stimulation, the radioactivity of PI is increased 70-140% in the superior cervical ganglia of several species. This increased incorporation is inhibited by dTC, and the inhibition is proportional to the decrease in action potential. They suggest that PI may be involved in postsynaptic events preceding permeability alterations. They note that for PI involvement to occur during the later steps (propagation or the restoration of the resting potential) one should have observed increased incorporation during stimulation of the nerves without ganglia, since these later steps also occur normally in nonganglionic portions of the nerves. Later studies ( Larrabee, 1968) have shown similar results in naturally stimulated superior cervical ganglia. These lipid changes are not unique to nervous tissue, for similar

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changes are found during some hormone actions and other alterations in basic metabolic processes. For example, with phagocytosis both polymorphonuclear and mononuclear white blood cells show increased incorporation of 32Pinto the PI plus PS ( phosphatidylserine) fraction (Karnovsky et al., 1966). Thyroid slices show increased incorporation of 32Pinto phospholipids, which is most marked in PI, when stimulated with TSH or ACh ( Altman et al., 1966). In salt gland slices (L. E. Hokin and Hokin, 1963a; M. R. Hokin and Hokin, 1964) and brain homogenates (Redman and Hokin, 1964) it has been observed that low concentrations of ACh stimulate primarily the synthesis of PI-32P,while at higher concentrations of ACh the amount of 32Pjincorporated into PA can far exceed the amount incorporated into PI. Brain is the only tissue where the ACh effect has been demonstrated in subcellular fractions. Durell and Sodd (1966) have shown that the ACh-stimulated incorporation of 32Pi into PA in brain homogenates is observed maximally in the brain subcellular fraction containing a high concentration of “synaptosomes.” This corroborates a possible relation to synaptic transmission, as had been suggested by Larrabee. Further evidence for a postsynaptic site is provided by L. E. Hokin (1966) who found that ACh stimulated 32Pincorporation into PI is not affected by denervation. The increase in the incorporation of 32Piinto PA is reduced and that into PC is abolished by denervation-suggesting that these increments were presynaptic. (The change in PA is still significant, however.) To account for the observed kinetics of the Pi incorporation into PA and PI as well as the close link between synaptic transmission and the metabolism of PI, Durell (1967; Durell et at., 1969) has proposed that the primary biochemical event may be the hydrolysis of phosphoinositides and perhaps other phospholipids. This hypothetical reaction sequence is shown in Table I. It is assumed that the diglyceride moiety is stable and is bound to the membrane throughout the sequence. TABLE I HYPOTHETICAL REACTION SEQUENCE”

+

1. Diglyceride-P-X 2. Diglyceride AT32P 3. Digly~eride-~~PCTP 4. Digly~eride-~~P-P-C In

+

~

~

~

+

> >

+

~

> _

~

_

_

_

Diglyceride P-X Digly~eride-~~PADP Dig1y~eride~~P-P-C PP Digly~eride-~~P-In CMP

+

+ +

~

Diglyceride-P-X represents any phosphatide except phosphatidic acid; diglyce r i d e 3 T represents 32P-phosphatidic acid; digly~eride-~~P-P-Crepresents beta 32PCDP-diglyceride; I n represents L-myoinositol; and digly~eride-3~P-In represents 32Pphosphatidylinositol. ADP and ATP, adenosine di- and triphosphates; CMY, CDP, and CTP, cytidine mono-, di-, and triphosphates. a

170

JOHN T. GARLAND AND JACK D W L L

The primary assumption is that, in the intact excitable membrane, reaction ( 1 ) is rate limiting and is stimulated by ACh. It is further assumed that reaction ( 2 ) is rapid as compared to reactions (3) and (4). Under conditions of minimal stimulation of reaction ( 1 ) , it would remain the rate-limiting reaction, and newly synthesized PA-32P would not accumulate, but PA-32Pwould be converted to PI-32P (assuming also that there is no significant accumulation of the nucleotide intermediate). Low concentrations of ACh would therefore result primarily in increased incorporation of 32Pinto PI, with only small changes in PA-32P-perhaps because of the changes in the steady state level of a rapidly turning over fraction of PA. A t higher concentrations of ACh, reaction (1) may be accelerated sufficiently so that reactions ( 3 ) and ( 4 ) become rate limiting, and PA-3zP would accumulate. This sequence and these kinetic assumptions are consistent with the effects of ACh on PI and PA, noted above. Any phosphatide except PA could be the substrate for reaction ( l ) ,for if PA were the substrate then the ratio of PA-32Pto PI-32Pwould be highest at the lowest concentration of ACh. If PI were the substrate, then the reactions would form a cycle; if phosphatidylinositol phosphate (PIP) or phosphatidylinositol diphosphate (PIPP) were the substrate then the cycle would include one or two phosphorylation steps after reaction ( 4 ) . The observation by Hayashi et al. (1962) that electric convulsions increase the labeling of a lipid in rabbit brain (which is thought to be PI) while decreasing the labeling in another phospholipid resembling PIPP would support the longer cycle. ACh stimulation of the hydrolysis of inositol phosphatides has been demonstrated (Durell et al., 1W; Garland et al., 1970), by measuring the in Vitro liberation of 3H-inositol phosphates from endogenously M ) caused a stimulabeled inositides. ACh ( M ) plus eserine ( lation of 46%compared to the control. Choline could not substitute for ACh, and the effect was blocked by atropine. In summary the metabolism of some specific phospholipids-especially PI-is intimately related to the events that take place postsynaptically during normal synaptic transmission and to the stimulation of cells by various physiological stimuli, including the “neural transmitters.” In homogenates of cerebral tissue these changes have been localized primarily to the synaptosomes. C. MISCELLANEOUS STUDIES Grampp and Edstrom (1963) showed an increase in adenine:uracil and purine :pyrimidine ratios with nervous activity in lobster stretch receptor organ, but the significance of this is unknown. Heald (1957, 1962) proposed a primary change in phosphoproteins due to electrical


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stimulation of CNS slices. The significance of this work has been questioned by L. E. Hokin and Hokin (1963b) for the following reasons:

1. Phosphoserine accounts for only 10-15% of the 32Pin his acid hydrolysate. 2. If brain slices are fixed in an organic solvent prior to phosphoprotein isolation there is no evidence of increased labeling with electrical pulses. 3. Enzyme levels have not been shown to be adequate. 4. They (Hokin and Hokin) failed to find a change in the specific activity of phosphoprotein under conditions that increase the labeIing of PA and PI in salt gland.

L. E. Hokin et al. (1965) did show that a COOH group is involved in the first step of the ATP hydrolysis by Na+, K+-ATPase,but as they noted carbohydrate can also have an acyl phosphate. Also, this involvement with Na+, K+-ATPasesuggests a general membrane phenomenon rather than a specific synaptic mechanism, since Na+, K+-ATF’ase is distributed along nonsynaptic membrane too. 111. Theories of Transmitter-Receptor Interaction

There are two major approaches to explaining the mechanism of action of the neural transmitters. One is that the transmitters directly produce conformational changes in one or more membrane constituents, and that this conformational change per se causes the functional changes observed. The other is :hat a chemical change (i.e., the making or breaking of covalent bonds) is necessary for the functional change to occur. We shall consider each of these in turn. A priori one would expect that receptors are located on the outside of the postsynaptic membrane, so as to be easily accessible to the transmitter. This has not been conclusively demonstrated, however, and Paton (1967), while summarizing the evidence for and against an intracellular site of action for adrenergic drugs, notes that, “There are a few items [of evidence], although none are decisive.” The implications of an intracellular site of action include the introduction of considerable uncertainty into the interpretation of the significance of structure-activity studies. This imposition of a barrier through which a drug must pass before having a chance to interact with the receptor would be expected to influence considerably the chemical structure of a transmitter, and therefore deductions as to the chemical nature of an intracellular receptor would be more difficult. It is also possible that there are both types of action, for there may

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be one basic neural transmitter (e.g., ACh) that acts on a receptor on the outside of the postsynaptic membrane while the other putative transmitters act intracellularly as modulators of the sensitivity of the postsynaptic cell to the action of the main transmitter. In the following discussion, it will be assumed for simplicity that the transmitters act on the cell membrane, but some of the theories would not require this. A. MODELSREQUIRING ONLYCONFORMATIONAL CHANGES In considering the AChR mechanism, Nachmansohn (1955, 1964) proposed that ACh is bound to a macromolecule in the resting state; stimulation is thought to release the ester, which then reacts with a receptor, producing the conformational changes that cause increased membrane permeability. This concept has been extended by Karlin (1967b) and by Changeux and Podleski (1968). The latter authors have measured the response of the transmembrane potential of the isolated monocellular electroplax to a variety of ligands. They have confirmed earlier work by showing a sigmoid dose-responsive curve, which suggests cooperativity. Additional evidence for cooperativity is provided by their observation that when two activators ( carbamylcholine and decamethonium) are employed together, the presence of one alters the shape of the dose responbe curve of the other. To explain the cooperativity they have applied the model for allosteric transitions formulated for regulatory enzymes by Monod et al. (1965) to the interaction of ACh and its receptor. Using this allosteric model, they explain the cooperativity of ACh‘s effects by postulating two states for a hypothetical protom& ( P D ) where P represents the state of the protomer when the membrane is polarized and D represents the state of the protomer when the membrane is depolarized. The membrane potential is determined by the fraction of protomers which are in a given state, and depolarization reflects the transition of a sufficient number of protomers from the P state to the D state. Antagonism between activators and inhibitors is then described in terms of differential stabilization of either the D or the P conformation, respectively. The cooperative response seen with activators is due to the transition of any one protomer favoring identical transitions in neighboring protomers. The relatively low degree of cooperativity demonstrated for AChR (Hill coefficient 1.8) may be explained either by strong interactions restricted to small clusters of protomers or by weak interaction over a larger field of protomers.

’ “Protomer” is the term used by Monod et al. (1965) for the identical subunits associated within a protein.


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Belleau and Lavoie (1968) have used AChE as a model receptor to try to define the biophysical basis of ligand-induced activation of excitable membranes and associated enzymes. In spite of the obvious difficulties inherent in their choice of AChE, which, as noted in Section I, A, 2, is probably not the AChR, their approach is worth noting. For ligands of the trimethylammonium (TMA) series they found a constant A F of binding, but there were compensated variations in AH and AS. To explain these observations they postulate that “a ligand-induced modulation of the physical variable of ice melting from the binding cleft may control the position of the conformational equilibrium between the resting state P , and the energized state P, of the receptors” ( P D in Changeux and Podleski’s notation). Watkins (1965) has provided a more detailed model for a conformational mechanism, though he did not discuss its relation to cooperative interaction. He cites the similarities in structure and charge distribution of the polar head portions of several lipids and possible neurotransmitters, e.g., phosphatidylcholine-ACh, phosphatidylethanolamine-7-aminobutyric acid; phosphatidylserine-glutamine. He also reviews the evidence that biological membranes have breaks in the nonpolar lipid layer. To show how these breaks may control permeability, he hypothesizes that at restricted regions of greater vulnerability or “polar discontinuities,” these transmitters replace the polar head portion of the corresponding lipid by combining with a lipid-protein complex, causing a change in membrane permeability. When he applies this model to ACh action the proposed membrane structure involves: 1. Electrostatic binding of the quaternary ammonium group of phosphatidylcholine (PC ) to an anionic protein side chain. 2. A divalent metal ion bridge between the lipid phosphate group and a second anionic group of the protein. 3. Coordinate bonding between the double-bonded oxygen atom of the phosphate group and a peptide group of the protein chain. ACh competes with PC for two of these sites (1 and 3 ) and a conformational change takes place in the protein as it binds with the ACh. This weakens the binding to the membrane of the divalent cation which can then be displaced by a univalent cation, If this PC-protein complex controlled the ionic permeability of that region, the “conformational changes and liberation of anionic groups on both lipid and protein could easily open up cation-selective channels and increase the permeability of the region to sodium and potassium ions. . . .” Differing effects with analogs could depend upon whether they block the new channel themselves when

174


binding. Ehrenpreis (1967) proposes that the protein undergoing the conformational change in this model is in fact AChE, but as noted in Section I, A, 2, there is considerable doubt about this correspondence.

B. MODELSREQUIRINGCHEMICAL CHANGES 1. Beta Adrenergic Receptors The changes induced in the adenyl cyclase system discussed in Section 11, A can be considered as either occurring at a later step in a series of events triggered by the transmitter-receptor interaction or as the immediate result of interaction at the receptor itself. Robison et al. (1967) suggest that there may be two parts to the affected enzyme (e.g., adenyl cyclase), a variable regulatory portion and a consistent catalytic portion. [Gerhart and Schactman’s (1965) deft elucidation of this mechanism for aspartic transcarbamylase provides a good precedent for this.] Though such a regulatory enzyme could be free within the cell, or within some compartment of it, it might be a cell membrane constituent with the catalytic portion on the inside and the regulatory portion on the outside, where it is readily accessible to the transmitters. Unpublished work from DeRobertis’ group (cited by Azcurra and DeRobertis, 1967) shows localization of adenyl cyclase in isolated synaptic membranes. Furthermore, the catalytic portions of the various receptors could be in different compartments such that stimulation of specific receptors might give differing actions even though all resulted in the stimulation of adenyl cyclase. Alternatively, as Robison et al. point out, the alpha receptor type of regulatory subunit could inhibit adenyl cyclase while the beta receptor stimulates it. This latter is easier to reconcile with Turtle and Kipnis’ experiments (see Section 11, A ) , which showed a decrease in CAMP following alpha stimulation. Belleau (1967) has developed a model for beta adrenergic receptors, which provides for the direct catalysis by catecholamines of a phosphoryl group transfer. The p- and y-phosphates of ATP are coordinated with the side chain OH of the catecholamine and a water molecule (note that the D absolute configuration of the p-carbon as found in 1-epinephrine is required for an ideal fit). The catechol ring is accommodated on the lipoprotein surface. The environment of the a-phosphate (ribose and adenine) is lipophilic and would favor the binding of agonists that incorporate nonpolar substituents on the N of NE, as has been observed (Ariens et al., 1964). This binding of the nonpolar substituent on N would help to insure the close approach of the N+ of the catecholamine to the a-phosphate and the catalysis of the binding of the &-phosphateof ATP to the 3’ OH to give CAMP.


175

2. Alpha Adrenergic Receptors Belleau has also proposed a stereochemical model for the alpha adrenergic receptor which makes it an ATPase and part of a lipoprotein complex containing phosphatidylcholine. Though ATPases are enzymes that are localized primarily in membranes, they are not restricted to synaptic areas. One would be in a better position to defend this as the mechanism if ATPase action was restricted to the synaptic area, but this specificity could be provided for by differing activators or merely by the lack of accessibility of the general membrane ATPase to neural transmitters. The postulate of a lipoprotein complex is in accord with our earlier general conclusions about the chemical nature of the receptors (Section I, D). In the resting alpha receptor, Belleau postulates that the a- and pphosphates of ATP protrude but are coordinated with other unspecified regions of the surface through Mg"; this leaves the y-phosphate to undergo H-bonding to a histidine residue and to coordinate with the phosphate of phosphatidylcholine through Ca++.A carbonyl group also projects into the area from a protein area, as does a hydroxyl group. The Ca++is then coordinated with the N+ of histidine, the OH, the COO-, and water molecules. This neutralization of charge on the COO- and the simultaneous neutralization of that on the y-phosphate of A T P allows an easier nucleophilic attack on the y-phosphate. The ease of this phosphoryl transfer would be enhanced by a specific source of protons, which can be provided by catecholamines (see Fig. 1).This provides a mechanism for ti

FIG.1. Hypothetical model of norepinephrine cocatalysis of phosphoryl group transfer (Belleau).

176


cocatalysis by Ca++and the alpha agonists, as suggested by Edman and Schild ( 1%3), This model allows for selective interaction of the aromatic ring with imidazole and H-bonding of the oxygen to the para OH. The Ca++(as coordinated above) stereospecifically provides for bonding of the beta OH through the rapid exchange of a coordinated water for the OH. Then the residual anionic charge on the y-phosphate could interact electrostatically with the animonium group of the catecholamine from which a proton would be abstracted. As Belleau points out, this mechanism, which relies upon the activity of water in several instances, is supported by the observation that F- stimulates alpha adrenergic receptors, for F- exerts an overall structuring effect upon water. [Poton mobility is 60-100 times greater in ice than in liquid water (Kavanau, 1964), so an increase in the structuring would enhance mobility.] Proton mobility and accessibility in liquid water clusters is also altered by temperature changes and these are known to alter the reactivity of receptor components (Keatinge, 1964).

3. Cholinergic Receptors The hypothesis that a phospholipid hydrolysis is the initial biochemical step in the postsynaptic action of neural transmitters has been extended to suggest that this reaction might directly produce the increased permeability and depolarization ( Durell et al., 1969). The hypothesis is very simple and it requires only that the neurotransmitter activate an enzyme. As discussed in Section 11, B, any phospholipid except PA itself could be the substrate for the reaction (1) and still allow the observed labeling of PI and PA with ACh stimulation. If we assume the closed cycle including PIP and PIPP, then greater flexibility is obtained. PI, PIP, and PIPP may have differing susceptibility to hydrolysis; thus interconversions among them could alter the membrane’s response to ACh. These interconversions might be modulated by other putative transmitters. This inositide-diglyceride cycle could allow for the release of PIP and PIPP bound Ca++at the time of hydrolysis. If this free Ca++were required for later steps, then this model would provide for co-catalysis by Ca++and ACh (as Belleau’s does for the alpha adrenergic receptor). Concerning the actual mechanism for the increased permeability, it is not difficult to imagine that the release of inositol phosphates from inositides could leave a “pore” that would allow the passage of Na+ and K+. Perhaps in the resting state the Ca++helps to keep the adjacent fatty acids of neighboring phosphatides together, thus maintaining a relatively impermeable membrane (as suggested by Tobias and Nelson, 1959). The


177

release of inositol phosphates from PIP and PIPP would decrease the binding of Ca++and allow greater penetration of the membrane by Na+ and K’. Phosphorylation of the diglyceride (to form PA) might terminate the period of increased permeability, but complete restoration of the resting state and excitability (and susceptibility to phosphodiesterase ) would require the addition of the inositol and binding of the Ca++as well. OF THEORIES C. INTERRELATIONSHIPS

Having presented several theories of the mechanism of receptortransmitter interaction, it is worthwhile considering how they may be reconciled with one another. The general concept of cooperativity in the ACh-AChR interaction is compatible with each of the other theories. It may readily be applied to Watkins’ model of ACh-induced conformational change by suggesting that the individual, local changes are somehow relayed to a neighboring area (which could even include part of the same protein) where they favor corresponding conformational changes in another protomer. Since the theories requiring a chemical change probably involve the activation of an enzyme, one need only suggest that this activation involves cooperative conformational changes in the enzyme’s subunits. Conversely, Watkins’ theory makes unnecessary any chemical reaction, by providing directly for the permeability changes. Furthermore, since ACh merely competes reversibly with PC for the binding sites on the protein moiety of the receptor, the restoration of the resting state would not directly involve chemical reactions either, although degradation of ACh by AChE would favor termination of the effects. There are no glaring incompatibilities among the theories that require a chemical change, for they involve different transmitters and receptors. Considering the adrenergic receptors, Belleau’s models provide continuity with the ATPase. It is especially appropriate in that system since there are active compounds that physiologically have both alpha and beta activity. Durell’s hypothesis that the transmitter causes increased permeability by increasing the rate of hydrolysis of a phosphoinositide lends itself easily to generalization. There could be one phosphodiesterase that can be activated by several different transmitters or there could be several isozymes, each activated by a specific transmitter. There could also be differing substrate specificities for isozymes that respond to the different transmitters, One of the advantages inherent in the phosphodiesterase theory is that it provides a mechanism for synaptic action that is restricted to the synapse, for the inositide effect is not seen in nonsynaptic regions (see Section 11, B). The conformational changes proposed by

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Watkins on the other hand, might be expected to occur in the membrane of axons generally. The ATPase theory also lacks localizing specificity since Na+, K-ATPase is found in axonal membrane too, although there has been no suggestion that adrenergic compounds act in these membranes. It is obvious that the data are quite incomplete, and our knowledge of the mechanisms of neural transmitter-receptor interaction is fragmentary. It is hoped that this review will stimulate critical thought and creative experimentation that will lead to increased understanding of this important area. REFERENCES Alquist, R. P. (1948). Am. 3. Physiol. 153, 586. Altman, M.,Oka, H., and Field, J. B. (1966). Biochim. Biophys. Acta 116, 586. Ariens, E. J. ( 1967). Ann. N . Y. Acud. Sci. 139(3 ) , 606. Ariens, E. J., Simonis, A. M., and Van Rossum, J. M. (1964). Mol. Phurmacol. 1, 216. Azcurra, J. M., and DeRobertis, E. (1967). Intern. 1. Neuropharmucol. 6, 15. Baba, W. I., Smith, A. J., and Townshend, M. M. (1967). Quart. J. Exptl. Physiol. 52, 416. Barlow, R. B., Scott, K. A., and Stephenson, R. P. (1963). Brit. J. Pharmacol. 21, 509. Bartles, E. (1965). Biochim. Biophys. Acta 109, 194. Belleau, B. (1967). Ann. N . Y. Acad. Sci. 139(3),580. Belleau, B., and Lavoie, J. L. (1968). Can. J. Biochem. 46, 1397. Beuding, E., Butcher, R. W., Hawkins, J., Timms, A. R., and Sutherland, E. W., Jr. (1966). Biochim. Biophys. Acta 115, 173. Butcher, R. W., and Sutherland, E. W., Jr. (1962). J. Biol. Chem. 237, 1244. Changeux, J. P., and Podleski, T. R. (1968). Proc. Natl. Acad. Sci. U.S . 59, 944. Cuthbert, A. W., and Painter, E. (1968). J. Physiol. (London) 199, 593. DeRobertis, E. (1967). Science 156, 907. DeRobertis, E., Azcurra, J. M., and Fiszer, S. (1967a). Brain Res. 5, 45. DeRobertis, E., Rodriguez De Lores Arnaiz, G., Alberici, M., Butcher, R. W., and Sutherland, E. W., Jr. (1967b). J. Biol. Chem. 242, 3487. Durell, J. (1967). Neurosci. Res. Program Bull. 5, 41. Durell, J., and Sodd, M. A. (1964). J. Biol. Chem. 239, 747. Durell, J., and Sodd, M. A. (1966). J. Neurochem. 13,487. Durell, J., Sodd, M. A,, and Friedel, R. 0. (1968). Life Sci. 7, 363. Durell, J., Garland, J. T., and Friedel, R. 0. (1969). Science 165, 862. Eckhardt, E. T., and Schueler, F. W. (1963). j . Phurmacol. Exptl. Therap. 141, 343. Edman, K. A. P., and Schild, H. 0. (1963). J. Physiol. (London) 169, 404. Ehrenpreis, S. ( 1967). Ann. N . Y. A d . Sci. 144(2 ) , 720. Fiszer, S., and DeRobertis, E. (1967). Bruin Res. 5, 31. Friess, S. L., Baldridge, H. D., Jr., and Durant, R. C. (1964). Toxicol. Appl. Pharmacol. 6, 459. Garland, J. T., Brown, J. D., and Durell, J. (1970). In preparation. Gerhart, J. C., and Schactman, H. K. (1965). Biochemistry 4, 1054. Grampp, W.,and Edstrom, J. E. (1963). J. Neurochem. 10, 725. Hasson-Voloch, A, (1968). Nature 218, 330.


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Hayashi, K., Kanoh, T., Schimizu, S., Kai, M., and Yamazoe, S. J. (1962). J. Biochem. (Tokyo) 51, 72. Heald, P. J. ( 1957). Biochem. J. 66, 659. Heald, P. J. (1962). Nature 193, 451. Hokin, L. E. (1966). J. Neurochem. 13, 179. Hokin, L. E., and Hokin, M. R. (1955). Biochim. Biophys. Acta 18, 102. Hokin, L. E., and Hokin, M. R. ( 1958a). J . Biol. Chem. 223, 822. Hokin, L. E., and Hokin, M. R. (195813). J. Biol. Chem. 233, 818. Hokin, L. E., and Hokin, M. R. (1960). J. Gen. Physiol. 44, 61. Hokin, L. E., and Hokin, M. R. (1963a). Federation Proc. 22, 8. Hokin, L. E., and Hokin, M. R. (1963b). Ann. Reu. Biochem. 32,553. Hokin, L. E., Sastry, P. S., Galsworthy, P. R., and Yoda, A. (1965). Proc. Natl. Acad. Sci. U. S. 54, 177. Hokin, M. R., and Hokin, L. E. (1953). J. Biol. Chem. 203, 967. Hokin, M. R., and Hokin, L. E. (1964). In “Metabolism and Physiological Significance of Lipids” ( R . M. C. Dawson and D. N. Rhodes, ed~.),pp. 423-434. Wiley, New York. Karlin, A. (1967a). Biochim. Biophys. Acta 139, 358. Karlin, A. (196713). J. T h o r e t . Biol. 16, 306. Karlin, A., and Winnik, M. (1968). PTOC.Natl. Acad. Sci. U. S. 60, 668. Karnovsky, M. L., Shafer, A. W., Cagan, R. H., Graham, R. C., Karnovsky, M. J., Class, E. A., and Saito, K. (1966). Trans. N . Y. Acad. Sci. 28, 778. Kavanau, J. L. (1964). “Water and Water-Solute Interactions,” p. 50. Holden-Day, San Francisco, California. Keatinge, W. R. (1964). J. Physiol. (London) 174, 184. Kimura, M. (1965). Chem. Pharm. Bull. (Tokyo) 13, 1. Klainer, L. M., Chi, Y.-M., Freidberg, S. L., Rdl, T. W., and Sutherland, E. W., Jr. ( 1962). J. Biol. Chem. 237, 1239. Larrabee, M. G. (1968). J. Nezlrochem. 15, 803. Larrabee, M. G., and Leicht, W. S. (1965). J. Neurochem. 12, 1. Lewis, J. E., and Miller, J. W. (1966). J. Phurmcol. Exptl. Therap. 154, 46. McLennon, H. ( 1963). “Synaptic Transmission,” p. 23. Saunders, PhihdeIphia, Pennsylvania. Mansour, T. E., Sutherland, E. W., Jr., Rall, T. W., and Beuding, E. (1960). J. Biol. Chem. 235, 466. Marchbanks, R. M. (1966). J . Neuroche~?~. 13, 1481. Monod, J., Wyman, J., and Changeux, J. P. (1965). J. Mol. Biol. 12, 88. Murad, F., Chi, Y.-M., Rall, T. W., and Sutherland, E. W., Jr. (1962). J. Biol. Chem. 237, 1233. Nachmansohn, D. (1955). Harvey Lectures Ser. 49, 57. Nachmansohn, D. ( 1964). In “New Perspectives in Biology” ( M . Sela, ed.), p. 176. Elsevier, Amsterdam. Nastuk, W. L. (1967). Federation PTOC.26, 1639. Offermeier, J., and Ariens, E. J. (1966). Arch. Intern. Pharmacodyn. 164, 192. Okada, K. (1967). Japan. J. Physiol. 17, 245. Orloff, J., and Handler, J. S. (1961). Biochem. Biophys. Res. Commun. 5, 63. Orloff, J., and Handler, J. S. (1962). J. Clin. Invest. 41, 702. Orloff, J., and Handler, J. S. (1964). Cellular Functions Membrane Transport, Symp. SOC.Gen. Physiologists, Ann. Meeting, Woods Hole, Mass., 1963 pp. 251-268. Paton, W. D. M. (1967). Ann. N. Y. Acad. Sci. 139(3 ) , 632.

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Paulus, H., and Kennedy, E. P. (1960). J. B i d . Chem. 235, 1303. Podleski, T. R. (1967). Proc. Natl. Acad. Sci. U.S. 58( l ) ,268. Redman, C. M., and Hokin, L. E. (1964). J. Neurochem. 11, 155. Robison, G. A., Butcher, R. W., and Sutherland, E. W., Jr. (1967). Ann. N. Y. Acad. Sci. 139(3), 703. Roepke, M. H. (1937). J. Pharmacol. Exptl. Therap. 59, 264. Sutherland, E. W., Jr., Rall, T. W., and Menon, T. (1962). J. Bid. Chem. 237, 1220. Sutherland, E. W., Jr., @ye, I., and Butcher, R. W. (1965). Recent Progr. Hormone Res. 21, 623. Takagi, K., Akao, M., and Takahashi, A. (1965). Life ,Sci. 4, 2165. Tobias, J. M., and Nelson, P. G . (1959). Symp. Mol. B i d , Uniu. Chicago p. 248. Turtle, J. R., and Kipnis, D. M. (1967). Biochem. Biophys. Res. Commun. 2 8 ( 5 ) , 797. Watkins, J. C. (1965). J. Theoret. Bid. 9, 37. Wesemann, W., and Zilliken, F. (1968). Biochem. PhamacoE. 17, 471. Woolley, D. W., and Gommi, B. W. (1964). Nature 202, 1074. Woolley, D. W., and Gommi, B. W. (1965). Proc. Natl. Acad. Sci. U. S . 53, 959.

THE CHEMICAL NATURE OF THE RECEPTOR SITE A Study in the Stereochemistry of Synaptic Mechanisms By J. R. Srnythies Department of Psychiatry, University of Edinburgh, Edinburgh, Scotland

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I. Introduction 11. Possible Role of RNA in Excitable Membrane . . . . . . 111. Prostaglandin-RibonucleoproteinComplex IV. Specification of the Cholinergic Receptor . . . . . . . A. The Muscarinic Site . . . . . . . . . . . B. The Nicotinic Site . . . . . . . . . . . C. Cholinergic Antagonists V. Specification of the Catecholamine Receptors VI. Specification of the Serotonin Receptors VII. Amino Acid Transmitters . . . . . . . . . . VIII. The Disulfide Bond . . . . . . . . . . . IX. Miscellaneous Compounds . . . . . . . . . . A. Veratridine and Tetrodotoxin B. Reserpine X. Some Stereochemical Relations between Membrane-Active Drugs and Antibiotics XI. RNA and the Sodium Pump Mechanism References Note added in proof

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181 184 189 192 192 193 198 201 202 207 210 212 212 213

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1. Introduction

Certain portions of the neuronal membrane are specialized so that they can bind transmitters with the consequences that ionic channels are opened. If these channels conduct sodium the membrane becomes depolarized, and if they conduct chloride or potassium the membrane becomes hyperpolarized, resulting in excitation or inhibition of the neuron, respectively. A good deal is now known about the molecular specificity of transmitters, but almost nothing is known about the molecular specification of receptor sites. Because it has not yet proven technically possible to enucleate the receptor sites and subject them to chemical analysis, the approach to determining their nature has had to be indirect. Structure-activity relationship studies of drugs active at receptor sites can tell us something about the possible nature of the molecular configuration that could accept such molecules, but this approach has not yielded much in the way of concrete results. The difficulties are many. The activity or lack of activity of a molecule may depend on many 181

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factors besides its precise functional fit of the receptor, e.g., its effects on transmitter uptake, metabolism and release, its own uptake and metabolism, its capacity to cross membranes, and the presence of “silent” receptors. This makes deductions from the chemical relation between various agonists and antagonists to the possible nature of the receptor site tentative at best. Such arguments would be more cogent if anything were known, on independent grounds, of the chemical nature of the receptor site. Unfortunately very little is known. It is generally assumed that the receptor must be located in protein, or lipoprotein, or perhaps glycoprotein, and the binding of the transmitter is supposed to induce some confonnational change in the macromolecule that opens the ionic channel. This limits the elements that could be present in the site to those groups present in protein, lipid, or carbohydrate, but this is not much help owing to the wide range of such groups and the endless different conformations that they can adopt. Thus the solution of the problem would appear to be very difficult and would depend on developing the necessary heroic biochemical techniques, or on the use of new techniques such as those of immunochemistry. However, it now appears possible that the assumption that the receptor must be located in protein, lipid, or polysaccharide may be mistaken. Protein is certainly an important constituent of membrane, and there is evidence that agents known to react with protein can interfere with synaptic function. But protein may be an essential part of the receptor without actually forming the primary binding site for the transmitter. It is probably a mistake to think of the receptor as composed simply of the few groups to which the transmitter binds directly. Around these there may well be a most complex organization concerned with doubtless the most intricate mechanism for opening and closing the ionic channel. This may require the cooperative action of more than one type of macromolecule. Thus it becomes of interest that there have been reports in the recent literature that ribonucleic acid is present in membrane, and thus it is possible that the primary receptor site may include ribonucleic acid (RNA) in its makeup, There are certain logical similarities between a transmitter binding at a receptor and a substrate binding to an enzyme. This, however, does not prove that the receptor site for transmitters must be in protein. Likewise, the role of RNA in protein synthesis is impressive. But this in no way means that RNA may not have some other quite different roles in the cell for which its molecular structure and properties make it suitable. Proteins themselves are used for a large number of different purposes-compare, for instance, keratin with hemoglobin, or insulin with an immunoglobulin,

CHEMICAL NATURE OF THE RECEPTOR SITE

183

One theme to be developed in this essay is to explore the consequences of the basic speculative hypothesis that the primary receptor site for transmitters is membrane RNA as adumbrated by Smythies et al. (1970a)b). The possible mechanisms of action of RNA will be discussed first and then how this could be affected by transmitters. A great advantage is immediately gained because the molecular structure is known, and so it is much easier to explore possible interactions between transmitters and an RNA-based receptor because the variations permitted are strictly limited. The method to be followed is a stereochemical analysis using CPK ( Corey-Pauling-Koltun) models. On this basis it has been possible to construct a coherent and internally consistent theory of the nature of the receptor sites for most of the putative transmitters. This theory is consistent with the (large) proportion of the structure-activity relationship data that it has been possible to test. The strength of such a stereochemical analysis depends on the mutual support of a large number of independent formulations. Any one of these might be a mere coincidence, but a large number of such “coincidences”might suggest rather that they were separate manifestations of a common underlying structure. The hypothesis accounts for a large number of previously disparate facts and provides a unifying hypothesis for the molecular basis of transmitter action. As the molecular specifications are clear and unambiguous, this model hypothesis suggests a number of critical experiments in a number of disciplines. This paper then presents an outline of the basic hypotheses. A more detailed account is to be published later with particular attention to the stereochemistry of a wide range of membrane active drugs. The ideas expressed in this paper originated during a sabbatical term I spent as a Resident Scientist at the Neurosciences Research Program (NRP) of M.I.T. at the kind invitation of Professor Francis 0. Schmitt and in particular consequence of the NRP Work Session on the Mechanism of Action of Hallucinogenic Drugs, of which I was Chairman. The report of this Work Session has recently been published in the NRP Bulletin (Smythies, 1970b). The molecular models on which most of this work was based were kindly loaned by the Ealing Corporation, Cambridge, Mass., and the work was supported by a grant from the Mental Health Research Fund. I am grateful to the following for the benefit of many helpful discussions: Fuad Antun, R. B. Barlow, Fred Benington, George Boyd, Melvin Calvin, Ruth and George Clayton, Donald Eccleston, Watson Fuller, Paul Janssen, Seymour Kety, Michael Levitt, Ulrich Loening, Harden McConneIl, Peter Pauling, SoIornon Snyder, Michael Waring, and Lemone Yielding. The idea that a spirally wound piece of RNA could act as a spiral “chute” through the membrane was suggested

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by Theodore Melnechuk, who has been an unfailing source of information in the many branches of neurosciences on which this hypothesis impinges. 11. Possible Role of RNA in Excitable Membrane

The traditional picture of the membrane has depicted it as composed of a lipid-protein bilayer with a coating of glycoprotein on the outside. However, several workers (Kasper and Kashing, 1969; King and Fitschen, 1968; Tagaki and Ogata, 1968; Tulegenova et al., 1968) have reported the presence of RNA in membrane, and Morgan and Austin (1968) have suggested that RNA may be present in neuronal membrane. A review of earlier work on this subject has been published by Hendler

FIG.1. Helical RNA in the extended configuration required to bind prostaglandins. Note the tilted base pairs and the hydroxyl groups in the minor groove.


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(1968). There are indications that this RNA is different from ribosomal RNA, which would tend to occur as a contaminant of subcellular fractionations of membrane. It has been estimated that up to 58 of membrane may consist of RNA, and it becomes of interest to enquire what function it might have. RNA might be concerned with local protein synthesis or it might have some other function. It is possible that RNA may play some part in the ion-conducting mechanisms and in the receptor sites controlling them. Namba and Grob (1967) have reported that they have been able to extract a ribonucleoprotein from muscle that binds tubocurarine, and that this binding is inhibited by acetylcholine ( ACh) . Both the RNA and protein components are necessary for this binding. They suggest that the ribonucleoprotein may be concerned in the cholinergic receptor. Recently the EIM (excitability inducing material) fraction from bacteria (that renders an artificial lecithin membrane electrically excitable and capable of gating ions) has been reported to be a ribonucleoprotein (Kushnir, 1968). If the RNA and protein are separated, activity is lost; this is partly restored if the two components are recombined. Helical nucleic acids have several properties that could be relevant to this postulated function, They can exhibit extensive conformationaI changes and they have interesting electrical properties, The following mechanisms are suggested:

1. If a segment of double-stranded helical RNA (Fig. 1) runs across a membrane, it will form a potential channel across it (Fig. ,?a). When the hydrogen bonds joining the two strands are formed the channel would be closed, but it would be opened were these hydrogen bonds to be disrupted as indicated in Fig. 2b and c. Naturally the RNA could only form part of the side wall of such a channel, and the rest might be filled in by protein or stabilized by polyamine. Furthermore such a channel would not have to run completely across the membrane. Such an RNAbased channel could consist of a small portion of helical RNA acting as a shutter across the top of a channel through the proteolipid portion of the membrane. The function of the transmitter would be to bind to subsites on the receptor and disrupt the Watson-Crick hydrogen bonds. For example, the quaternary N+ of acetylcholine could disrupt a guaninecytosine hydrogen bond on the minor groove by attracting the cytosine oxygen group and repelling the guanine NH group. The disruption of this hydrogen bond in such manner might set up a cooperative disruption of the rest, in which case the RNA would act like a zip-fastener. However, it might appear that the RNA helix in its ordinary fully contracted ( A ) configuration would possess sufficient stabilization energy from the 7 cloud ( stacking) interaction to resist denaturation following the mere

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FIG.2. Models of RNA in membrane and their possible configurations: ( a ) a segment of helical RNA acting as a channel across an ion channel. a and b are alternative positions for the polyamine struts; ( b ) a top view of the RNA based shutter in the closed position; ( c ) the same in the “open” position following disruption of the hydrogen bonds; ( d ) the “spiral chute” model. This represents a cross section through the channels, the shaded portion being the RNA molecule in cross section with the two grooves A-A and B-B converted into channels by the laterally placed polyamine struts.

disruption of one Watson-Crick hydrogen bond. In this case, the RNA would have to be in an extended configuration in which the base pairs were no longer in T cloud contact. The RNA could only remain in this configuration if maintained in it by the protein part of the ribonucleoprotein bound presumably to the phosphate groups. Thus the ribonucleo-


187

protein would be in a metastable state vulnerable to denaturation in the manner suggested. 2. A second mechanism is suggested by the Hoffman-Ladik theory of carcinogenesis (Hoffman and Ladik, 1961). This is based on the fact that nucleic acids are semiconductors. Normally they are insulators, but if they are located in an electric field across their own length and a powerful electron donor (or acceptor) intercalates between the base pairs and donates (or removes) an electron to the T cloud of the adjacent base, this will set up a migration of T electrons along the nucleic acid molecule, provided the base pairs are in their stacked configuration. The molecule now becomes a conductor and the ends will become charged. In this case, the base pairs will take on the same charge (positive at one end and negative at the other end of the molecule-or that portion of the molecule within the electric field) and will repel each other, resulting in disruption of the hydrogen bonds joining them. H o h a n and Ladik used this property to base their theory of the mechanism of action of carcinogenic hydrocarbons. The theory can also be applied to explain how transmitters like serotonin, which are powerful electron donors, could act. In this case the electric field would be provided by the resting potential across the membrane. The flow of ions through the opened channel would depolarize the membrane and so the RNA molecule would again become an insulator and the charge on the bases would be dissipated. The base pairs would re-form their hydrogen bonds and the channel would close. 3. A third possible mechanism is suggested by Fig. 2d. This might be called the “spiral chute” model as opposed to the “internal shutter” model just described. If the two grooves in the side of the helical RNA molecule were roofed in or stabilized by a continuous array of polyamine molecules strung between the phosphate oxygens on adjacent ridges, this would convert the grooves into two spirally wound tubes running through the membrane. In the minor groove spermine is long enough to bridge between two adjacent ridges, making a channel some 8 8 in diameter. In the major grove spermine is not long enough to reach from one phosphate oxygen across to the phosphate oxygen on the other side of the groove, but it will reach the base oxygen (of guanine or uracil) in the middle and convert half the major groove into a channel with a diameter of some 6 A wide. This mode of attachment of the spermine molecules may be preferred owing to the extensive lipophilic interactions as well as N: HN interactions between the parallel spermine molecules it allows. 4. A fourth possible mechanism locates the actual channel not down the middle of the disrupted double helix, as in the case of the first two mechanisms suggested, but in protein at the side of the RNA. The protein

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layer would be threaded with RNA molecules, each with a segment exposed at the surface. The conformational change induced in the RNA would be transmitted to the protein, resulting in opening of the channel. 5. RNA has been shown to possess ferroelectric properties (Stanford and Lovey, 1968), and it could thus alter its configuration in response to changing electric fields. RNA will also alter its configuration in response to changes in its ionic environment. Clearly both these properties might be relevant to the postulated role of RNA in controlling ionic fluxes through membrane. The ingenious model maker could doubtless make up many such speculative channels based on the facts that RNA possesses semiconductor and ferroelectric properties, and a form that makes it a potential tube across the membrane in one of two ways. However, the fact that this model, or one like it, may represent the true state of affairs is suggested by the close and specific stereochemical relationship between many membrane active drugs and helical RNA to be detailed. For our present purposes it will be sufficient to consider the hypothesis that the receptor site is a segment of helical RNA, perhaps in association with some other molecule, exposed at the external surface of the membrane of excitable cells. The transmitter binds to this and initiates channel opening by initiating some conformational change in the RNA possibly by disrupting the hydrogen bonds or possibly by some other mechanism. There is no reason to believe that the ionic channels controlled by one transmitter are any different from those controlled by another transmitter. Thus the specification of the receptor site as to whether it is muscarinic, nicotinic, adrenergic, etc., should be a function of differences between the segments of RNA concerned. Inspection of the molecular model of helical RNA suggests two potential binding sites for transmitters capable of disrupting the WatsonCrick hydrogen bond: ( a ) the spare NH group and spare oxygen orbital on the hydrogen-bonded base pairs (Fig. 3 ) and ( b ) the lipophilic intercalation site. The negatively charged phosphate groups would ordinarily be expected to attract positively charged molecules such as ACh. However, it is difficult to see how this would bring about the desired configurational change in the helix. Moreover, it is possible that these phos-

FIG.3. The arrangement of the spare NH group and 0 orbital on the WatsonCrick hydrogen-bonded base pairs. Note the specific bond angles.


189

phate groups are masked by the binding of basic protein in the ribonucleoprotein. Furthermore, we have evidence ( Smythies and Antun, 1969) that the lipophilic transmitter serotonin does not bind to so hydrophilic a region. Factors that could digerentiate one segment of helical RNA from another, and thus specify the site, include the following: ( a ) the particular base pairs concerned, ( b ) the particular groove involved, ( c ) the degree of torsion of the helix, and ( d ) the degree of involvement of other molecules bound to the RNA. The most difficult of these variables to specify is the degree of torsion of the helix, As noted above, the preferred configuration of RNA itself is the A form with a tightly wound helix and base pair stacking. But it was noted above that this configuration was likely to be too stable for the purpose, and that any RNA in this context would be likely to be in an extended configuration maintained by attached protein. If we are concerned with trying to match putative transmitters with possible binding sites on the RNA, it would be necessary to know the degree of torsion of the helix, as the interatomic distances between these groups and the relative bond angles will be entirely dependent on the degree of torsion of the helix, Fortunately, the degree of torsion may be specified within reasonable limits if we make the further postulate that the receptor consists not of naked RNA or even ribonucleoprotein, but of a prostaglandin-ribonucleoprotein complex ( PG-RNA complex for short ) , I I I . Prostaglandin-Ribonucleoprotein Complex

The prostaglandins are thought to play a role in synaptic function without actually being transmitters themselves ( Horton, 1968). They are found widely distributed in brain, and, when applied to neurons by the iontophoretic method, they can modulate the firing of the neurons ( Bradley, 1968). Synaptic activity leads to PG release. Comparison of the molecular model of PGs with the RNA model showed that with a particular degree of torsion of the helix, the two molecules bear a specific stereochemical relationship to each other such that two PGs can bind to a four-base-pair segment of helical RNA by four hydrogen bonds as illustrated in Figs, 4 and 5. It will be observed that the two PGs form a lipophilic rhomboid around the bonding groups on the two middle base pairs exposed in the floor of the deep cup-like site. The purpose of such a n attachment would appear to be to provide specific points of attachment for transmitters by van der Waals’ and lipophilic interactions as will be detailed below. This aids the binding of the transmitters and makes this binding far more specific, as molecules that might bind to the RNA by itself can no longer do so when the PGs are in position. The lipoph3ic

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FIG. 4. A drawing of the prostaglandin-ribonucleoprotein complex. The protein moiety must be imagined as bound to the external phosphate groups. The 5-6 double bond in cis.

PGs might also have some action on the interaction of water with the primary binding site. It may be noted that the PGs, in order to bind to the RNA, take up an energetically favorable configuration with fully staggered side chains and excellent bond angles. The different PGs require different base pair sequences. If we number the base pairs from 1 to 4, the transmitter is postulated to bind to 2 and 3 and the PGs to 1 and 4. In the case of the majority of PGs (i.e., those with 20 carbon atoms), the carbonyl 0 (of -COOH) binds to the ribose OH of base pair 3; and the 9 OH (or 0 ) binds to the ribose hydroxyl of base pair 1.In PG E the 11 OH now binds to the guanine NH of base pair 1 and the 15 OH binds to the complementary cytosine 0. In PG F the base pair must be reversed and the 11 OH binds to cytosine 0 and the 15 OH to the guanine NH. PGs A and B, which lack an 11 OH group might bind to adenine-uracil (AU) base


191

FIG. 5. Molecular models illustrating the postulated PG-RNA complex in the GC :GC muscarinic configuration.

pairs. The interesting feature of the dinor prostaglandins will be discussed later. The stereochemistry of the PGs is taken from Ramwell et al. (1969). Any 19 OH group is correctly located to bind to the adjacent ribose hydroxyl on the other strand, and the cis double bond between C-5 and C-6 that characterizes the PG2s could serve to shorten the distance between the 9 OH and the carboxy 0 and provide a &binding site.

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1-

C

G

(Cl

FIG. 6. A diagrammatic representation of the arrangement of the bonding sites on the base pairs in the minor groove: ( a ) GC:GC (muscarinic); ( b ) CG:CG (nicotinic); ( c ) GC:CG (glutamate only). The wavy lines indicate steric hindrance by the PG. IV. Specification of the Cholinergic Receptor

Owing to the eccentric locus of the helical axis in RNA, the binding sites on the bases are arranged differently in the major and minor grooves. This is shown in Figs. 6 and 7 ; note the greater degree of staggering in the minor groove, The AU base pair is unlikely to act as a primary receptor in the minor groove, as it lacks a cationic site (adenine has no NH, group here) and there is no hydrogen bond at risk. SITE A. THE MUSCARINIC Muscarine is highly stereospecific, and the active form is shown in Fig. 8. Molecular models indicate that this can bind only to a GC:GC

G

A

F

l

C

I

l o ;

I

l

l

u

G

u

W

/

C

I

10:"

A

I

FIG.7. A diagrammatic representation of the arrangement of the binding atoms on the base pairs in the major groove.


193

FIG.8. The active stereoisomer of muscarine.

base pair combination in the minor groove as follows: two hydrogen bonds (ring 0 to guanine NH of base pair 2; OH to PG AC13-14) and one ion-dipole bond (N’ to cytosine 0 of base pair 3 disrupting this GC hydrogen bond) as well as extensive van der Waals’ interactions with the prostaglandin hydrocarbon chain. None of the other stereoisomers of muscarine fit, and the helix must be wound right handed. Note that the molecule is tilted in the site by the direction of the bonded orbital on the ring 0. This tilt locates the hydroxyl in the right location for binding as indicated (Fig. 9 ) . Acetylcholine binds in this site in a manner similar to muscarine. This gives extensive van der Waals’ contact between the methyl group and the “left” side of the ACh molecule and the upper PG hydrocarbon chain. The PG carboxy 0- at the end of this chain will form an ionic bond with the N+ of ACh (Fig. 10). The best fit is with a PG E. This fit means that the only ACh a or /3 H that could be substituted by a methyl group corresponds to the active /3 isomer. The lower (“right”) PG may be either E or F ( A = E throughout ).

B. THENICOTINICSITE Similar considerations indicate that the nicotinic site may be specified as a CG:GC combination in the minor groove. This locates the two cytosine 0 atoms correctly to form hydrogen bonds with optimum bond angles to the protonated pyrimidine ring N and the 6 2 carbon atom of the pyridine ring of nicotine. This allows extensive van der Waals’ contact between the hydrocarbon portion of the pyrimidine ring and the adjacent PG hydrocarbon side chain (Fig. 11).ACh binds in this site “upside down” by its carbonyl 0 to guanine NH (BP 2), its N+ to cytosine 0 (BP 3) (both 5 A apart) as well as the extensive lipophilic and van der Waals’ contacts as detailed for the muscarinic site-only in this case this PG must be F. The “ether” 0 is not used as may be seen in Fig, 12. This is because of the rhomboid shape of the site and the different bond angle on the guanine NH (BP 2 ) as between the “muscarinic” GC :GC and the “nicotinic” CG :GC sites. Muscarone (which has both muscarinic and nicotinic properties) can

+

194

J. R. SMYTHLES

FIG.9. Muscarine bound in its site (small molecule atoms are marked with a dot).

bind in the “muscarinic” site as follows: ring 0 to guanine NH (BP 2 ) ;

N+ to cytosine 0 of BP 3. Further evidence detailed elsewhere from the stereochemistry of nicotinic antagonists ( especially toxiferine, tubocurarine, and isoxazolidine ganglion blockers ) clearly suggests that the neuromuscular junction


195

FIG. 10. Acetylcholine bound in the GC:GC muscarinic site. The hydrogen that can be replaced by methyl is marked with two dots.

has two F prostaglandins (and hence a base pair specification GC:CG: GC :CG) whereas the ganglionic ACh receptor has an upper PG F and a lower PG E and a base pair specification GC:CG:GC:GC). Likewise there can be two types of muscarinic receptor-one with two PG Es (CG :GC: GC :GC ) and one with an upper PG E and a lower PG F

196

J. R. SMYTHIES

FIG.11. The binding of nicotine in the CG:CG nicotinic site.

(CG:GC:GC:CG). This specification allows us to account for much structure-activity relationship data for both agonists and antagonists. A selection will be given here but space does not permit a full treatment of the subject which will be presented elsewhere. 1. In compounds of the type R-C=C-CH,-N+-( CH,),, muscarinic activity is maximum when R = 2 and nicotinic when R = 3. This suggests that the former is associated with a PG E (and hence a smaller site) and the latter with a PG F (and hence a larger site). It would need a methyl group as R to attain van der Waals’ contact with the PG hydrocarbon chain in the former case and an ethyl group to do this in the latter case. R as an ethyl group in the muscarinic site leads to steric hindrance. 2. The model also explains why a meta substitution by hydroxyl in


197

FIG. 12. The binding of acetylcholine in the nicotinic site,

phenyltrimethylammonium (Fig. 13a) leads to a 100-fold increase in nicotinic activity whereas a para substitution leads to no increase (Cavallito and Gray, 1960). A meta substitution gives intermediate results. A methoxy group in the meta position leads only to a threefold increase in

198

J. R. SMYTHIES

activity. Molecular models show that a meta hydroxyl group hydrogen bonded to the cytosine 0 on base pair 2 locates the N+ much closer to the cytosine 0 on base pair 3 than does an ortho or para substitution. In addition, the guanine NH on base pair 2 can hydrogen bond to the benzene ring 7 cloud. If a methoxy group is substituted, this can bind only to the adjacent guanine NH which gives a poorer fit. 3. The model also explains why compounds of the form shown in Fig. 13b are inactive if R = H or C2H, and active when R = CH,. Only in the latter case does the terminal CH, group attain effective van der Waals’ contact with the PG hydrocarbon chain which makes up the containing wall of the site. If R = H the molecule does not reach, and if R = C,H, the end wall is overlapped leading to steric hindrance. 4. Similar considerations apply to the other end of the molecule since the site is symmetrical about the diagonal. In the compound shown in Fig. 13c (which is not very active by itself), activity is increased by breaking the ring at €3 > A > C. Again the break at B gives the optimum 2 carbon distance between the bonding group and the end wall of the site. The methyl and ethyl groups of pilocarpine could exert a similar function.

C. CHOLINERGIC ANTAGONISTS With atropine (Fig. 14a), the molecular models indicate an unambiguous mode of binding as follows (Fig. 15): “ether” 0 to guanine NH on base pair 2; N+ (protonated) to cytosine 0 of base pair 3; hydroxyl H to the PG carbonyl 0-. This locates the benzene ring of atropine in the deep lipophilic pocket under the cyclopentane ring at the acute comer of the site. This lipophilic pocket arises because of the mode of attachment of the PG molecules to the RNA. Due to the relationship between the

FIG.13. ( a ) 3-Hydroxytrimethylammonium. ( b )( c ) Some cholinergic compounds.


199

y 4 2

“JI (CH,), I

OH

TH2 +N CH3 I

( C2H5)2

(g)

HN , QCO.O.CH,.CH,.

N (c,H,)~

FIG.14. ( a ) Atropine; ( b ) benactyzine; ( c ) diphenhydramine; ( d ) perphenazine; ( e ) banthine; ( f ) chlorisondamine; ( g ) procaine.

hydrogen bonding sites on the two molecules, the PG overhangs the RNA to a considerable extent, especially in the region of the cyclopentane ring. In the pocket, a benzene ring can be accommodated either horizontally tucked under the apex of the PG, or vertically intercalated between the base pairs. In compounds like benactyzine (Fig. 14b) and diphenydramine (Fig. 14c), the two benzene rings form a T-shaped configuration that fits into the lipophilic pockets. In banthine (Fig, 14e) and chlorisondamine (Fig. 14f) the lipophilic ring system is too large to fit into the horizontal pocket, but its relation to the side chain is such that it can intercalate between base pairs in the manner of a phenothiazine (see below). The steric fit of the horizontally placed benzene ring is so tight that any substitution would tend to prevent binding, whereas there is more scope for a lipophilic substitution on a vertically placed ring in certain locations. However, any hydrophilic group in either location would tend to inhibit binding, as in the case of the NH, group of procaine (Fig, 14g) or the laterally placed hydrophilic group in cocaine. The G C : C G site. In this, both oxygen atoms are sterically hindered

200

J. R. SMYTHIES

FIG.15. The mode of binding of atropine in the muscarinic site.

by the overlying PG chain and in any case are very far apart (about 11A). The two NH groups are some 6 A apart and could bind glutamate (see below). The CG: CG site. Owing to the symmetry of the site this is equivalent to the muscarinic GC:GC site.


201

V. Specification of the Catecholamine Receptors

The intercalation site of helical RNA has a complex shape. When looked at from the side it is rhomboidal, whereas when looked at from on top it is tetrahedral, reflecting the eccentric mode of attachment of the bases to the ribose-phosphate backbone of RNA. This means that any phenothiazine (which are known to intercalate into nucleic acidsOhnishi and McConnell, 1965) with a bulky constituent on the 2 position (such as C1 or CF,), that gives the molecule a tetrahedral shape, can intercalate only from the side of the major groove. This allows the long side chain of a drug like perphenazine (Fig. 14d) to run down the floor of the major groove. This compound has three charged groups (N, N, 0) in the side chain. In order for any atom to hydrogen bond with an element of the bases, it must present a vertically oriented bond angle. Thus the piperazine ring of perphenazine must be in the boat configuration in order to give the two N atoms the right bond angles. In which case the three bonding groups of perphenazine will be 3 8 apart, which cor-

FIG.16. The mode of binding of NE in the major groove site.

202

J. R. SMYTHIES

responds to the distance between the bonding groups in the major groove if the helix is fully wound up with full base pair stacking. This indicates

that there can be no PG on the other side of the RNA since the minimum distance between these groups when the PG is in place is 4 A. Because the phenothiazines are potent antagonists of NE (norepinephrine) in the brain, this suggests that the binding site for NE may be in the major groove of a tightly wound helix. The molecular models indicate the following mode of binding for NE and dopamine. The two ring hydroxyls of a catecholamine can bind across one base pair as follows: 3 hydroxyl H to base pair 0; 4 hydroxyl 0 to base pair NH. The use of the 3 hydroxyl proton is indicated by the fact that 0-methylation in this position gives the inactive compound normetanephrine, during the normal course of NE metabolism. The amine molecule is now completely fixed relative to the RNA. Both base pairs possess a hydrogen bond in the major groove, and therefore, there is nothing to indicate whether the base pair is GC or UA. So we can only indicate the atom concerned, i.e., N (of adenine or cytosine) and 0 (of guanine or uracil). The active stereoisomer of norepinephrine requires the following base pair specification ON :ON: NO: NO which bind to the amino group ( protonated) ; phydroxyl: x cloud (to N H ) ; and the ring hydroxyls respectively as shown in Fig. 16. Apomorphine is a dopamine agonist (on certain receptors at any rate) whose rings contain the dopamine molecule in fixed form. This gives the dopamine receptor as ON :NO: NO. VI. Specification of the Serotonin Receptors

Experimental evidence for the suggestion made by Smythies et al. ( 1970a,b) that 5-HT intercalates into helical nucleic acid has been pro-

vided by Smythies and Antun ( 1969). Wagner (1969) and Yielding and Sterglanz (1968) have shown the same for LSD. Figure 17 illustrates the molecular model of 5-HT intercalated and bound in helical RNA (in the PG-RNA complex). The binding is by a x-7r interaction, two hydrogen

FIG.17. A model of the binding of 5-HT to the PG-RNA complex. This shows the side view of the site between the two pairs. The electrostatic bonds are depicted as barred rectangles.


203

bonds and two ion-dipole bonds as illustrated. This applies to the middle intercalation site in the PG-RNA complex. In the two end sites there is only one hydrogen bond and one ion-dipole bond possible, because ribose hydroxyls in these loci are engaged in binding the PGs. In this position, 5-HT might have two effects depending on the particular base pairs. We have seen previously that the RNA helix must be stabilized in this configuration by external protein, and there is evidence to be considered later (in the section on veratradine) that the RNA does not contract even when the channel is opened and the RNA is separated into its two strands. Thus, although the 5-HT would replace the missing stacking energy in the RNA helix if 3 molecules were to intercalate, this may not be very significant. However, 3 molecules of 5-HT also tie each strand of the RNA together by 4 hydrogen bonds and 4 ion-dipole bonds. This would clearly stabilize the double-stranded configuration and, if the site were cholinergic, 5-HT would act as an antagonist. However, 5-HT can also act as an excitatory transmitter. This suggests that it can also disrupt the helix. Because 5 H T is a powerful electron donor, as it donates the electron from the 2 position on the indole ring, and because cytosine is a good acceptor, serotonin could disrupt the PG-RNA complex by polarizing a base pair (or base pairs) by a charge transfer reaction, if its 2 carbon atom was touching a molecule of cytosine so that the transfer of the electron is consonant with the direction of the electric field-here provided by the resting potential of the membrane (Fig. 18). In a previous communication we suggested that the diethylamide side

t FIG.18. The postulated charge transfer process mediated by 5 HT.

204

J. R. SMYTHIES

chain of d-LSD made contact with the helical backbone of the nucleic acid (Smythies et al., 1970a). However, in the PG-RNA complex this is not possible, and the PG side chain gives a much better fit. Recently Chothia and Pauling ( 1969) have determined the preferred configuration

FIG.19. The postulated mode of binding of d-LSD in the intercalation site. Only one molecule of d-LSD is shown, but another could occupy the other “end” site.


205

of d-LSD which suggests a remarkable manner of binding to the PGRNA complex (Fig. 19). The skew in the D ring of LSD and the fixed C-N amide bond cause the two ethyl groups of d-LSD, in the preferred conformation, to form a small ellipsoid clump tilted at right-angles to the plane of the indole ring. This means that the D ring and diethylamide grouping together form a delta-shaped group (with the N-methyl group at the apex and the two ethyl groups forming the base of the triangle), which looks remarkabIy like the complex B and C ring system of Agtetrahydrocannabinol (THC) described below. If the indole ring is intercalated between the base pairs, this delta-shaped mass fits closely into the angle between the two limbs of the PGs with extensive van der Waals’ contact. The length of the D ring (N-methyl, C-8) and (2-32 lie along one side chain of the PG (C-13 to C-20) and the diethyl grouping lies along the other side chain with the C-32 methyl group at the apex of the acute angle between the two limbs of the PG. The protonated N+ of the D ring will form an ion-dipole bond with the guanine spare electron pair or cytosine 0 and the 6- charge associated with the D ring double bond will form an electrostatic bond with the guanine NH. If any of the other inactive isomers of LSD are tested in the site, the diethylamide group now causes complete steric hindrance. The FG must be F as the site provided by two PG Es is too small to admit the large superstructure of d-LSD. Ag-Tetrahydrocannubino2.In the case of THC examination of the molecular model (Fig. 20) shows that this has essentially the same shape as d-LSD different as the molecules may look in the more familiar line drawings. The B and C rings, because of the skew in the B ring and the exact location of the methyl groups, form a single smooth delta-shaped clump tilted at an angle of some 20’ to the plane of the benzene ring. If the benzene ring is intercalated between the base pairs and the hydroxyl hydrogen is hydrogen bonded to the ribose ring oxygen in the corner of the site, the delta-shaped mass now fits the angle of the PG exactly as the same shaped clump does in d-LSD except that the mass is somewhat smaller and the PGs must be E. It would appear that the ring 0 of THC is not used for binding. This fit of d-LSD and THC suggests that the 5-HT receptor is in a PG-RNA complex. This mechanism also explains why the 11,2’-dirnethylheptyl homolog of THC is some 70 times as active as THC (Adams et al., 1949). The two extra methyl groups on either side of the side chain maintain the %at configuration optimum for intercalation and materially increase the area of lipophilic contact, because the first two carbons are still sandwiched between the base pairs whereas the last three carbons are free: the 3l-methyl and 4’methyl analogs are no more active than THC. However, the extra termi-

206

J. R. SMYTHIES

FIG.20. The mode of binding of THC in the site (PG atoms are marked with a dot).

nal methyl group is also necessary as the 11,21-dimethylbutyl and propyl derivatives are only some 3 to 4 times as active as THC. Presumably the terminal methyl group in the heptyl compound achieves a lipophilic contact with the missing other wall of the site. Figure 21 illustrates the essential similarity in the mechanism of blocking of d-LSD and THC. But, since d-LSD is a more potent electron donor than 5-HT, the question arises as to why it should act as an antagonist and not an agonist. The answer may be that d-LSD can only intercalate into GC: GC :CG: CG site in a manner that its indolic 2 carbon atom is touching only guanines and so no charge transfer reaction can take place (Fig. 18). Now Boakes at al. (1969) have shown that, in the brain, d-LSD has no effect on iontophoretic sites where 5-HT has an inhibitory action, and acts only to inhibit sites where 5-HT has an excitatory action. The latter sites are fired also by glutamate but not by ACh. This suggests that this site will have a GC:GC:CG:CG base pairing. Although 5-HT and N methyl-5-HT both stimulate the 5-HT receptor in the autonomic ganglia, the hallucinogenic compound N,N-dimethyl-tryptamine stimulates the nicotinic receptors in the ganglia in preference to the serotonin receptors. The base pair sequence here will be GC:CG:GC:GC. When d-LSD


207

------mr U FIG.21. Diagram of the manner of binding of d-LSD and THC in the PG-RNA complex. PG, prostaglandin; A, indole ring of d-LSD or benzene ring of THC; B, ( B plus C ) ring system of THC or the D ring plus diethylamide side chain system of d-LSD; BP, base pair.

intercalates into this base pair sequence, not only does its 2-C touch cytosine on each side but its protonated N will be applied against the two GC hydrogen bonds of base pairs 2 and 3 and it should therefore act as an agonist. Possibly the detailed interaction of these compounds may also depend on subtle factors of 7 cloud interactions. Thus d-LSD would appear to be able to block the excitatory 5-HT receptor by its extremely detailed fit of the complex receptor site. The lack of ability of d-LSD to block the ACh (muscarinic) receptor would appear to depend on the fact that the latter has PG Es which preclude d-LSD entry. PG As are equivalent to PG Es in this context. VII. Amino Acid Transmitters

The present status of the amino acid transmitters (Fig. 22) is that glycine is thought to be the inhibitory transmitter in the cord and GABA has a similar function elsewhere. Glutamate and aspartate may have excitatory actions, Many other membrane active amino acids are known but their precise function in vivo is uncertain. Glutamate. The base pairing suggested for glutamate (Fig. 22a) is GC:CG but as not much is known about glutamate antagonists no confident specification as to PGs can be made. However, the molecule of picrotoxin, a possible GABA (Fig, 22c) antagonist, suggests that it acts at a site involving two PG Es. Glycine. This short compound (Fig. 22b) could only bind across one base pair (0-to NH and NH’ to 0),which it has the stereochemical

208

J. R. SMYTHIES

6t5A0+

&-

S-

FIG.22. Some amino acid transmitters; ( a ) glutamate; ( b ) glycine; ( c ) GABA.

capacity to do. With only one base pair GC would appear to be equivalent to CG as the site is symmetrical. As Fig. 4 clearly shows two ordinary PG molecules could not delineate a primary receptor site with less than two base pairs in it. There is no evidence to suggest that two molecules of glycine could fire an ordinary ACh receptor. However, if we construct the complex site using two molecules of a dinor prostaglandin, which will have only 5 carbon atoms in the “vertical” side chain, two molecules of these could bind as described previously for the other PGs but to a three base pair segment of helical RNA and not a four base pair segment. This would delineate a site containing only one base pair, which could correspond to only one transmitter, namely, glycine (and perhaps /3-alanine also). Strychnine (Fig. 23), which is known specifically to block glycine receptors, has a precise stereochemical relationship to this postulated glycine site which is illustrated in Figs. 24 and 25. The strychnine molecule is essentially a thick, flat, lumpy plate of rhomboidal form that fits

FIG.23. The absolute configuration of strychnine.


209

FIG.24. Strychnine bound in the glycine site (PG hydrogens are marked with a dot).

precisely into the cavity of the glycine site like a divot carved by an inexpert golfer in a thick clay turf. In addition to the wide area of lipophilic contact the N+ of strychnine could make an ion-dipole bond with the carboxy hydroxyl 0 on the PG on the other side.

FIG.25. A diagrammatic representation of strychnine bound in the glycine site; ( b ) top view.

( a ) side view;

210

J. R. SMYTHIES TABLE I OF THE POSTULATED RECEPTOR SITESFOR SPECIFICATION VARIOUS TRANSMITTERS Base pair

1

2 3 4 Groove PG

ACh (M)

ACh (N)

NE

DA

Glycine

CG GC GC

GC CG GC

ON ON NO NO Ma 0

-

GC GC CG Mi dinor

rl

*

Mi 20c

Mi 20c

ON

NO

NO Ma

0 ~

Base pair 1 2 3

4 Groove PG

Glutamate

* GC CG

*

Mi 20c

~

~-

Serotonin (Excitatory) GC GC CG CG Intercalation 20c

The base pair specification depends on the particular PG as described in the text.

Toxiferine can make a similar fit into the larger ACh site. It too is a flat rhomboid-shaped molecule, as might be expected, because it is made of two strychnine molecules sewn together as it were. It has the right size and shape to fit in the ACh receptor cavity like a larger divot. In addition, its two quaternary N+ atoms are adjacent to the PG carboxyl 0 atoms and could make two ionic bonds, and the two hydroxyl groups can insert themselves into two hydrophilic corners of the site and hydrogen bond to the ribose ring oxygen therein. Tubocurarine likewise closely resembles a plaster cast of the inside cavity of the PG-RNA complex. ydminobutyric acid. No specific receptors have yet been worked out for the other amino acids nor for histamine. Vlll. The Disulfide Bond

Karlin and Winnik (1968) have shown that a disuEi.de link is located 8-1OA from the primary anionic site in the cholinergic receptor in the electroplax organ of Electrophorus electricus. If this receptor is treated with dithiothreitol (DTT), a potent reducing agent, it will no longer react with ACh. Reaction can easily be restored by an oxidizing agent. Thus it is probable that this concerns the reduction of a disulfide bond.


211

The reduced disulfide group can be alkylated by 4-(N-ma1eido)phenyltrimethylammonium iodide ( MPTA ) leading to the irreversible blockade of ACh. If a molecular model of MPTA is placed in the PG-RNA “muscarinic” site, it will bind as follows: N’ to cytosine 0 on base pair 3 and one carboxyl 0 to guanine NH on base pair 2. The disulfide group can now be located by the MPTA double bond in the missing “roof” of the corner of the site located presumably in the supporting protein. This is directly over or even within the upper left hydrophilic pocket in the obtuse corner of the site. Presumably, when reduced, one sulfhydryl group could hydrogen bond to the adjacent PG carboxy 0and so prevent the binding of ACh, or else it could offer steric hindrance to the ACh molecule. In this same paper Karlin and Winnik report further that hexamethonium, which is normally a readily reversible competitive inhibitor of the ACh receptor in the electroplax (as well as blocking nicotinic sites), becomes an activator (depolarizer) of the receptor in the reduced state. The molecular model shows that hexamethonium could bind in the normal site diagonally across it with its two trimethylammonium groups attached by ion-dipole links to the cluster of electronegative atoms here located (S, ribose hydroxyl 0, cytosine 0). On reduction of the S-S bond, the hydrophilic pocket in the corner of the site (ribose and phosphate 0 atoms), which might normally be sterically blocked by the S-S grouping, might become vacated for occupation by one of the trimethylammonium groupings of hexamethonium. The model now shows that whichever hydrophilic is occupied by the trimethylammonium grouping in the nicotinic site, the trimethylammonium grouping on the other end of the molecule will be located directly over a cytosine 0 and the hexamethonium would now be an agonist. In the muscarinic site, this is true for one side but not the other, but the hexamethonium would still act as an agonist, although not so powerfully. Decamethonium could act as an agonist at the nicotinic site if it bent in the middle with one N+ over the CG bond and the other over the GC bond. This allows maximum lipophilic contact between the hydrocarbon chains of decamethonium and the PG F. It should prove possible to design an alkylating agent that would hydrogen bond to the sites available in the PG-RNA complex and then alkylate, not the reduced disulfide bond, but the adjacerat prostaglandin carboxyl. If both the alkylating agent and the PG were differentially radioactively tagged, the conjoint molecule could be dislodged from the RNA by urea and the co-valent linkage demonstrated.

212

J. R. SMYTHIES

IX. Miscellaneous Compounds

A. VERATRIDINE AND TETRODOTOXIN Veratridine is known to act by potentiating and prolonging Na+ fluxes through membrane. Its molecular model suggests very clearly how it might do so, as shown in Figs. 2629. Its array of oxygen atoms are located on a strip down the convex side of the molecule and are complementary to an eight base segment of polyguanine (some of which can be replaced by other bases). Thus veratridine could bind to singlestranded RNA. It will be recalled that the hypothesis suggests that the wall of the “open” ionic channel contains single-stranded RNA, and veratridine could thus prevent closure of the channel by blocking one strand of the RNA and so preventing the other complementary strand of RNA from re-forming the Watson-Crick hydrogen bonds. Levitt (personal communication) has pointed out that the RNA to which veratridine could bind is also in the extended configuration, meaning that, when the hydrogen bonds are disrupted by the transmitter and the helical RNA denatured, the RNA stays on the protein and is maintained in the extended configuration. This suggests that veratridine would not act on ionic channels controlled by the catecholamines, as in these the initial state of the RNA is in the fully contracted configuration. Tetrodotoxin and its simpler homolog amiloride both contain groups capable of forming Watson-Crick hydrogen bonds with nucleic acid bases, They act by blocking a sodium channel, and their molecular

FIG. 26. Line drawing of the veratridine molecule.


213

FIG. 27. Molecular model of veratridine.

form suggests they could do so by getting in between two complementary strands of RNA when just separated and preventing their further separation by hydrogen bonding on one side of the molecule to one strand and on the other side to the other strand, thus sticking the two strands together.

B. RESERPINE Schiimann (1Q66) has shown that adrenal chromaffin granules contain RNA and that the spontaneous release of ATP and catecholamines is accompanied by the release of an equivalent percentage of RNA, Mg++, and Ca++.Incubating the granules with increasing amounts of RNase produced a dose-dependent release of catecholamines, ATP, and RNA, each to the same degree; there was no release of protein. He therefore suggested that RNA may be involved in the storage complex for amines, together with Ca++,Mg', and ATP and that the spontaneous release of these might be owing to the action of intragranular RNase. He found that medullary granules obtained from discontinuous density gradients contained sufficient RNase activity for the purpose. Ca++released ATP and

214

J. R. SMYTHIES

Frc. 28. Diagram of the postulated mode of binding of veratridine in the open Na' channel. Seven or eight bases can bind.

NE but not RNA and therefore could not act by activating RNase. RNase, reserpine, and phenylamine each released catecholamines, ATP, Caw, and Mg++in the same percentage. Tyramine released catecholamines only. In this context the stereochemical relationship between reserpine and helical RNA (Smythies, 1970a) may be interpreted as follows: If the RNA in the synaptic vesicle is largely composed of poly U, it could act as the core to which molecules of ATP can hydrogen bond, If to each U there is paired one molecule of ATP by Watson-Crick hydrogen bonding, and if each ATP molecule hydrogen bonds to its neighboring ATP molecule (first phosphate to the 3 hydroxyl) a conformation much like RNA results; this conformation can take the left-handed helical conformation necessary to bind reserpine in the manner previously indicated ( Smythies, 1970a). This complex could be further stabilized by Ca++and Mg++.The amines could normally be released by the action potential


215

FIG.29. Molecular model of the binding of single-strand RNA and veratridine by no less than 11 hydrogen bonds.

causing a conformational change in the ferroelectric RNA. One molecule of reserpine binding to the complex could initiate a cooperative disruption of a large segment of the complex. A wider interaction between reserpine and nucleic acids is suggested by the report that reserpine inhibits protein synthesis ( Alivisatos et d.,1969). X. Some Stereochemical Relations between Membrane-Active Drugs and Antibiotics

The main class of chemicals known to act upon nucleic acids are the antibiotics and antitumor agents. If transmitters and their agonists and antagonists also act upon nucleic acids, we might expect to see some chemical relationships between these two classes of compound. The following compounds are therefore of interest in this regard. Mitomycin (Fig. 30a). This is a close relative of serotonin and interestingly enough it has the same pattern of substitution on its benzene ring as is found in the most potent of the amphetamine hallucinogensDOM (2,5-dimethoxy-4-methylamphetamine). Mitomycin is known to attack both DNA and RNA forming, it is thought, co-valent cross links between the 0 - 6 of the guanines on adjacent sites in opposing chains by

216

J. R. SMYTHIES

FIG.30. ( a ) Mitomycin; ( b ) violacein; ( c ) quinacrine.

its two very reactive groups that have been grafted onto the molecule (the cleavage points of these are indicated in the figure). Violacein (Fig. 30b). This is an even closer derivative of serotonin, with the side chain of the latter held by ring formation in the extended configuration required by binding in the intercalation site in nucleic acids. If the rest of the molecule takes up the configuration shown in the figure, the 0 can bond to an NH group on the base pair below. Quinacrine (Fig. 3Oc). This is remarkably similar to d-LSD, with respect to a possible location of its diethylamide group relative to the aromatic system. This compound frequently produces an acute psychotic reaction as a complication of malarial chemotherapy. Vinblastine (Fig. 31a). This most complex molecule is known to bind to nucleic acid and is used in the treatment of neoplasm. It is of particu-

FIG. 31. ( a ) Vinblastine; ( b ) ibogaine.


217

lar interest owing to the chemical relationship between its lipophilic core and the hallucinogenic drug ibogaine (Fig. 31b). If the ibogaine-like moiety is intercalated between base pairs, the rest of the molecule forms a T-shaped strut across the helix, Because of the peculiar twists in the ring system, the four oxygens marked with a star are located close together and can bind to elements in the base pairs below the site; the other four oxygens can make a similar connection to equivalent groups on the base pairs above the site. The antitumor effect of vinblastine is reported to be hindered by tryptophan. Other antibiotics with activity on transmitter function are streptomycin ( a curarizing agent thought to act by inhibiting ACh release), patulin (which inhibits the action on smooth muscle of ACh, serotonin, histamine, and PG) and citrinin (which has been reported to have nicotinic action). All these have clear-cut stereochemical relations to RNA, and the last to the nicotinic site as specified above. (See Eliasson, 1958 and Chu, 1946.) Xi. RNA a n d the Sodium Pump Mechanism

The stereochemical relationship among ouabain, erythrophlamine, and the spermine-RNA complex described below suggests that doublehelical RNA may also be concerned in the active Na pump mechanism linked to Na+,K+ dependent ATPase. If we take the “double spiral chute” model of the RNA-spermine complex outlined earlier, one channel could conduct Na’ out of the cell and the other could conduct K+ in. All theories of ionic transport through membranes have to make allowance for the fact that water in membrane may be highly ordered (ice-like) and so relatively impermeable to Na’. A channel made by running spermine molecules across the roof of the minor groove (between the nearest phosphate oxygens on each side to which are attached the terminal nitrogens of spermine) will have a diameter of approximately 8 A , ample to admit the hydrated Na’ ion (7.16 A maximum and 6.4 A minimum diameters). A possible manner of blockade of such a tube by ouabain and erythrophlamine is illustrated in Figs. 32 and 33. On the major groove side spermine could complete a similar but smaller channel if run from phosphate 0 to the base pair 0 on the small hump in the middle of the floor of the groove. Such a tube would have a diameter of some 6 A, large enough to admit the smaller hydrated K+ ion (6.62 A maximum and 4 A minimum diameter). This tube would be precisely blocked by tetraethylammonium, not only because this round, charged molecule physically blocks the channel but also by an electrostatic attraction between its positive charge and the negative sites in the walls of the

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FIG.32. Molecular model of ouabain blocking a spermine-RNA channel. The ouabain hydrogens are marked with a dot. The sugar component is not included. Two of the four hydrogen bonds that ouabain can make with RNA are visible and all three with spermine ( 3 dots).

tube (unshared electron pairs of guanine and the middle spermine N atoms). In the absence of any other factors both these tubes should be filled with water molecules hydrogen bonded to these charged sites on the walls. It is here that the different nature of the hydration shells of Na+ and K may be important. In Na+.3H,O, the water shell forms an A region, that is, the water molecules are tightly bound and the hydrated Na+ ion tends to increase the ordering of water surrounding the A region by further hydrogen bonding. In the case of Kt*2H,O, the water forms a B region, in which the water molecules are only lightly held. Ions with B shells can actually disrupt hydrogen bonds between polymers and water molecules that they come in contact with. Thus K - 2 H 2 0 would pass more easily through a tube in a macromolecule filled with water than could Na+.3H,O. The former would tend to dissolve the ice-like water filling the tube, whereas the latter would make it more ice-like. Thus the hydrated Na+ion would pass much more easily through a membrane if it could be dehydrated first and could be protected from rehy-


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FIG. 33. Postulated mode of binding of erythrophlamine.

dration during its passage through the membrane. The following mechanism is suggested as capable of effecting this function. The ATPase is located on the inner aspect of the membrane; if it were located over the inner aspect of the minor groove channel described, and if one of its enzymatic functions were to remove the water of hydration from Na+.3H,O, it could deliver naked Na+ ions into the start of the Na+ channel. The first Na+ ions would immediately be rehydrated at the expense of the water bound to the sides of the tube. Further Na+ ions would be bound to negatively charged sites in the wall. The rest would slip past these and eventually would escape at the outer orifice of the tube to be rehydrated by the outside water, The lipophilic tube would prevent the Na+ ions from being rehydrated by external water en route. The hydrated Na+ ion could not get through the K+ channel K+.2H20 would be unlikely to enter the Na+ channel against the stream of emerging Na'. Another factor can be invoked to help keep these channels relatively clear of ordered water. Since amine groups reduce the ordering of water in their vicinity, the particular base pairs in the RNA portion of the channel will be relevant, Since the GC base pair possesses an amine group in the minor groove and the AU pair does not, RNA with a high GC content should provide an optimum Na' channel. Likewise, the spermine molecule on the major groove should run from phosphate 0 to guanine 0 SO as to include the cytosine NH inside the tube.

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The sodium pump mechanism must be much more complex than this account of the possible mode of action. If the actual driving mechanism of the pump is ATPase, the spermine-RNA complex postulated here can be regarded as supplying merely the plumbing. It is, of course, quite possible that pores through a leaky protein sieve might be the same size and have an array of hydrogen bonding sites similar to those on the inside of a minor groove spermine-RNA channel, and thus would be blocked by ouabain. However, the erythrophlamine molecule is quite different from ouabain. Yet they are both complementary to different features of the spermine-RNA complex. Thus if any Na+ transport system is blocked by both drugs, it would be a remarkable coincidence if any protein channel happened by chance to have the additional specific bonding sites required to bind erythrophlamine as well. It would seem more reasonable to suppose that any channel blocked by both ouabain and erythrophlamine is based on an RNA-spermine or similar complex. REFERENCES Adam, R., Harfenst, and Loewe, S. (1949). I. Am. Chem. SOC. 71, 1624. Alivisatos, S. G. A., Ungar, F., and Seth, P. K. (1969). Federation Proc. 28, 577. Boakes, R. J., Bradley, P. B., Briggs, I., and Dray, A. (1969). Brain Res. 15, 529. Bradley, P. B. (1968). In “Recent Advances in Pharmacology” (J. M. Robson and R. S. Stacey, eds.), 4th Ed., p. 311. Churchill, London. Cavallito, C. J., and Gray, A. P. (1960). Progr. Drug Res. 2, 135. Chothia, C., and Pauling, P. (1969). Proc. Natl. Acnd. Sci. U . S. 63, 1063. Chu, W. (1946). I. Lab. Clin. Med. 31, 72. Eliasson, R. (19’58).Experentia 14, 460. Hendler, R. W. (1968). Protides BioZ. Fluids, Proc. Colbq. 15, 37. Hoffman, T. A., and Ladik, J. (1961). Cancer Res. 21, 474. Horton, E. W. (1968). In “Recent Advances in Pharmacology” (J. M. Robson and R. S. Stacey, eds.), 4th Ed., p. 185. Churchill, London. Karlin, A,, and Winnik, M. (1968). Proc. Natl. Acad. Sci. U. S. 60, 668. Kasper, C. B., and Kashing, D. M. ( 1969). Federation Proc. 28, 404. King, H. W. S., and Fitschen, W. (1968). Biochim. Biophys. Acta 155, 32. Kushnir, L. D. (1968). Biochim. Biophys. Acta 150, 285. Morgan, I. G., and Austin, L. (1968). J. Neurochem. 15,41. Namba, T., and Grob, D. (1967). Ann. N . Y. Acad. Sci. 144, 772. Ohnishi, S., and McConnell, H. (1965). 1. Am. Chem. SOC. 87, 2293. Pullman, A., and Pullman, B. (1968). Aduan. Quantum Chem. 4 , 267. Ramwell, P. W., Shaw, J. E., Corey, E. J., and Andersen, N. (1969). Nature 221, 1251. Schumann, H. J. (1966). Phurrnacol. Rev. 18, 432. Smythies, J. R. (1970a). Commun. Behauior Biol. 3, 263. Smythies, J. R. (197Ob). Neurosci. Res. Program Bull. 8, 123. Smythies, J. R., and Antun, F. (1969). Nature 223, 1061. Smythies, J. R., Benington, F., and Morin, R. D. (1970a). Intern. Reu. Neurobiol. 12, 207.


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Smythies, J. R., Benington, F., and Morin, R. D. (197Ob). Neurosci. Res. Program Bull. 8, 117. Stanford, A. L., and Lovey, R. A. (1968). Nature 219, 1250. Tagaki, M., and Ogato, K. (1968). Biochem. Biophys. Res. Commun. 33, 55. Tulegenova, L. S., Rodionova, N. P., and Shapov, V. S. (1968). Biochim. Biophys. Acta 166, 265. Wagner, T. E. (1969). Nature 222, 1170. Yielding, K. L., and Sterglanz, H. (1968). Proc. SOC.Exptl. Biol. Med. 128, 1096.

Note added in proof: The hypothesis, being highly specific, may readily be tested by experiment in a number of fields. For example, it can predict a certain pattern of PG release and the blockade of this release from active tissues. The release of only PC Es, blocked by atropine, indicates activation of a CC:GC:GC:GC type muscarinic receptor. Release should also be blocked by THC but not LSD or toxiferine. Release of only PG Fs could come from an ACh (neuromuscular junction type) receptor, blockable by toxiferine, or a 5-HT receptor blockable by LSD. Release should not be blocked by atropine or THC. A release of PG E and PG F in approximately equal proportions suggests activation of ( a ) a nicotinic ganglion-type ACh receptor blockable by isoxazolidine, or ( b ) a muscarinic receptor blockable by atropine, or ( c ) , of course, a mixture of pure E and pure F receptors (as above) or various mixtures of pure E, pure F,and mixed EF receptors, the proportions of which can be teased out by the various blockers. (PG A is equivalent to PG E in this analysis. ) The specification of the action of the antagonist is required to exclude a purely metabolic origin of the PGs, or possible other actions such as on storage sites or metabolic processes, or even from sites where PGs would have a primary actionfor example, a base pair combination-GC :AU :AU :CG would bind PG Fs but not ACh or glutamate and this binding might result in an effective conformational change in the ribonucleoprotein. Likewise, PG E and PG F should be differentially inactivated by 0-methylation. The account given above indicates that, for inactivation, the 15-OH of PG E (but not the 11-OH) would be methylated, whereas the reverse is the case for PG F. The complex forming the receptor could also be constructed of a double helical single stranded RNA (ribonucleoprotein )-nucleotide complex as described above ( p. 214). The nucleotide could itself form part of a phospholipid and the mechanism of opening and closing of the sodium channel, triggered by ACh, would be a function of the amount of the lipid hydrocarbon chain diving down into the lipid layer of the membrane bilayer. Thus the action of ACh in Durell’s theory would be to make the nucleotide-phospholipid (specified in stages 3 and 4 of his hypothesis: p. 169) available to the reaction (stage 4 ) from its “storage” in a metabolically inactive form as a part of the primary receptor site. Such a complex would require divalent ions for stabilization. Thus the receptor could well be a prostaglandin-ribonucleoproteinpucleotide-phospholipid complex. This complex exemplifies all the stereochemical relations described above as well as double stranded RNA itself does. A complex constructed entirely from nucleotides is unsatisfactory as it cannot explain the precise ordering of the bases required by the theory. However a very similar complex can be constructed by replacing the single strand of RNA in the RNA-nucleotide complex with a polypeptide, replacing guanine with arginine and cytosine with glutamate. Arginine can form two hydrogen (or ion-dipole) bonds

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with cytosine and glutamate likewise with guanine. Every alternate amino acid comes off the same side of the polypeptide chain and are the right distance apart to bind to the bases to give the required stereochemical relation. The PG on this side now binds as follows: COOH to carbonyl 0 (hydrogen bond from the unionized form) or COO- to peptide NH: 9 OH (likewise 9 0 ) to peptide NH: 11 OH to glutamate 0- and 15 OH to guanine NH plus quite extensive lipophilic interactions. The other PG binds to the RNA-like complex of nucleotides as before, The resulting structure is remarkably like the PG-RNA complex (on the “inside” of the site). The only observable differences are a slight widening of the (cytosine) 0 to the adjacent (ribose) OH distance when changing to the (glutamate) 0 to adjacent (polypeptide) 0 and the replacement of the 6in one comer of the intercalation site (ribose and phosphate 0 s ) by a carbonyl (polypeptide) 0 s - . The intercalation site is of course considerably changed, as a purine (or pyrimidine) B cloud has been lost to be replaced by CHz-CHzCH-NH.C( and CH-CHz*C< respectively. However, the arguments as to the possible effects of LSD and 5HT would still hold; the 5HT can still mediate a G + C electron transfer and d-LSD cannot, now in a C:C:G:G base sequence (on the right). The top B H T molecule can donate its electron to the adjacent C (and so disrupt the C-glutamate bond) whereas LSD cannot as before. In this case, only one of the connecting links will be broken. Otherwise the stereochemistry is the same, except that in GC:CG and CG:GC combinations one guanine spare electron pair is replaced by -CHabut this group does not feature to any extent in the interactions. All the stereochemical relations described for the PG-RNA complex hold for the PG-PNPL ( -protein-nucleophospholipid ) complex. The translation required to turn formulations made in terms of the PG-RNA complex is as follows: on one side of the complex vepluce G by arginine and C b y glutamate. Thus GC :GC becomes either arginine-cytosine :arginine-cytosine or guanine-glutamate :guanine-glutamate and so on. Likewise, the RNA in the postulated ionic channels associated with the Na+ pump could as well be an RNA-like complex of stacked nucleotides with an essentially similar stereochemistry to which the polyamines attach as described. The alkylating agent adumbrated on page 211 may have been developed by Edwards et al. (J. Memb. Biol. 2, 119, 1970). The disulfide bond (see p. 210) may now be explained if amino acid number 2 of the polypeptide is cystine. Thus the acid specification for the ACh neuromuscular junction ( Electroplax ) is arg:cyst:glu:?:arg:?:glu; the complementary bases being C:G:C:G and both PGs are F. Further investigations indicate that the PG-PNPL complex can explain the differential effects of hexamethonium and decamethonium on the receptors in the neuromuscular junction and ganglia, whereas the PG-RNA complex cannot. Furthermore, the latter can account simply and directly for the evidence linking a phospholipid, protein and calcium ions to the receptor, whereas these can only be accounted for indirectly in the case of the PG-RNA comp!ex. Prostaglandins themselves could act independently of ACh and other transmitters as follows: PGs may inhibit adenyl cyclase by stabilizing ATP molecules as a complex-hence, metabolically inactive. The amino acid complementary to ATP is glutamine. Hence, the required complex could consist of a glutamine-containing polypeptide strand with e.g. amino acids 1,3,5,7:glu :g1utamine:arg. This would bind two PGEs and two ATT molecules and would not be affected by any transmitter as adenine lacks an amino group of this side.

MOLECULAR MECHANISMS IN INFORMATION PROCESSING1 By Georges Ungar Baylor College of Medicine, Houston, Texas

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I. Introduction A. Neural Information Processing . . . . B. Learning and Memory . . . . . 11. Neural Coding . . . . . . . . A. Electrical Activity . . . . . . B. Structural Code . . . . . . . C. Chemical Codes . . . . . . . 111. Evidence for Molecular Mechanisms . . . A. Chemical Correlates . . . . . . B. Drug Actions . . . . . . . C. Bioassay Methods . . . . . . IV. Molecular Hypotheses A. Nonspecific Hypotheses . . . . . B. “Tape-Recorder’’ Molecules . . . . C. Hypotheses Based on Chemospecific Pathways V. Concluding Remarks . . . . . . References

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I. Introduction

It is now generally admitted that the essential function of the nervous system is the processing of information. As it often happens for established concepts, the widespread use of the term seems to have made its definition or even its explanation superfluous; it is treated as if it were self-evident and axiomatic. In a symposium devoted to this subject (Gerard and Duyff, 1962), there is no discussion on the meaning of information processing. Before entering into a detailed consideration of the possible molecular mechanisms in information processing, a brief consideration of the whole concept is in order. After all, the term “information” has gained acceptance in science only in the last twenty years when it “started to replace energy as the common experimental currency for biological systems” (Rosenblith, 1962). It is certainly worth examining how this shift came about and assessing its validity and usefulness. ‘The research work referred to in this review is supported by U.S.P.H.S. grant MH-13361. 223

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Ever since information, as an independent entity, emerged from the realm of communication engineering in the late forties (Shannon, 1948; Wiener, 1949), it has been steadily gaining ground in the biological sciences. Under its influence, the whole life process appeared as the creation of order from disorder. This paradox, contradicting the second law of thermodynamics, was explained by Schrodinger (1948) by the assumption that living matter “feeds on negative entropy.” Because negative entropy was identified with information, it became evident that the organization and operation of living systems is based on information. This idea contained the germ of the whole vast field of molecular biology, undoubtedly one of the turning points in our understanding of life. Living beings develop according to the genetic information stored in the parental germ cells just as an edifice arises from a heap of building materials obeying the architect’s blueprint.

A. NEURAL INFORMATION PROCESSING If the organization of living matter is based on genetic information, its relations with the environment are controlled by messages it receives from the outside world. This “experiential” information has increased in importance in the course of evolution and, in animals, has become the almost exclusive domain of the nervous system. The only notable exception is the immune mechanism, which has retained its autonomy. Thus, the nervous system came to be conceived as a machine which gathers information, “processes” it, and enables the organism to respond to the conditions of the environment by the appropriate adaptive means. In the absence of this machinery, survival is limited to those environmental conditions that could be foreseen by the inherited “knowledge.” The next step in evolution was the ability of the system to preserve information so as to change its behavior permanently, or at least lastingly. Retention of information increases survival since it enables the organism to learn from experience and to anticipate the proper adaptive response. At this stage, however, learning and memory are still limited to the individual, and every young organism has to learn anew all that its forebears have acquired through experience. Transmission of acquired information from parents to offspring exists to a rudimentary extent in some higher vertebrates, birds, and mammals, whose young require prolonged postnatal care, protection, and instruction. It is, however, man alone, the only “culture-making animal,” who, through the possession of symbolic communication, is capable of transmitting from generation to generation a whole store of knowledge, behavioral patterns, skills, customs, and beliefs. This latest step in the evolutionary ladder acquired an even greater significance with the invention of writing, which

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increased the efficiencyand accuracy of the cultural process by replacing oral tradition with history. The succession of these evolutionary steps corresponds to the gradual development of the central nervous system, culminating in the human brain. The most obvious change that has taken place is the increase of the number of units, the neurons, with the corresponding multiplication of the connections among them and the rising complexity of their organization. There can be no doubt of the close correlation between the complexity of behavior and that of the central nervous system. The former reflects an increasing refinement in information processing of which the latter is an essential condition. In its most elementary form, information processing is a simple transmission of the excited state from the sensory receptor to an appropriate effector, muscle, or gland. This elementary process persists in the simple reflexes of higher organisms. Sherrington (1906) considered the simple reflex as the unit reaction of nervous integration. His concept of integrative function of the nervous system was the first explicit statement of what we now call information processing. The term information, however, was never used by Sherrington, who believed that . . . our thinking is . . . an outcome of ‘energy.’” As we understand it today, it is information that is being integrated by the nervous system, and energy plays only a secondary role as a carrier. Information processing, therefore, may be considered as a new, more fashionable and perhaps more accurate name for integration. It serves as a general designation for a series of operations of widely varying complexity going from the simpIe reflex to the processes of creative thinking, Probably, the most intensively studied of these processes is perception, the mechanism by which stimulation of individual sensory receptors, say in the retina, is capable of forming in the brain a visual pattern, an image corresponding to an aspect of the outside world. Similar patterns are perceived after stimulation of receptors in all the sensory fields and the information supplied by all the relevant receptors is combined. Elements of information are thus being added together, integrated, organized, “processed” to produce a representation of the universe. At the other end of the system, a coordinated motor response is also the end product of the interplay of many components. Even the simplest muscular contraction requires the intervention of reciprocal inhibition to be effective. The extreme complexity of information processing that goes into such commonplace activities as walking, grasping an object-not to mention bicycle riding or violin playing-is well known. The real terra incognita is the vast area between the projection of “

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the universe into our brain and the output of the effector response. This is the area designated as higher nervous activity, mental function, mind, or soul, according to our philosophical beliefs. This activity apparently takes place through the same elementary units, only organized into more complex structures. It may, therefore, be profitable, until proof to the contrary, to assume that mental activity is the result of the same processing of information, involving higher and higher levels of integration. If percepts are the results of the convergence of many individual neurons, each carrying a parcel of information, these percepts, in turn, can become units for further processing. As integration proceeds, percepts become objects, objects become ideas (in the Platonic sense), then these are combined into concepts, which form systems of knowledge and belief, and so on. One important aspect of information processing in the nervous system is that new information is being constantly added to, combined and compared with already existing knowledge. This involves acquisition of new information (learning) and its integration into the store of innate and previously acquired information (memory). Since learning and memory are widely studied in connection with molecular mechanisms, I should like to discuss briefly their places in information processing.

B. LEARNING AND MEMORY There is no universally acceptable definition of learning. An operational definition that most experimental psychologists would agree with is that learning has taken place when behavior is modified as a result of experience. In the context of the present paper, learning could be defined as the acquisition and retention of information. This implies that all information input does not necessarily lead to learning; the piece of information gained has to combine with others, either innate or previously or simultaneously acquired. Elementary learning is based on innate stimulus-response relations and the newly acquired information, in turn, is used to learn a more advanced behavior, thus building up a whole hierarchy of patterns, more and more remote from the original instinctive behavior and improving the adaptation to environmental conditions. Hebb (1949) believed that “perception . . . is not immediately given but slowly acquired through learning.” Perception, that is, integration of information acquired either simultaneously or in a temporal sequence can, therefore, be considered learning. Although this extension of the meaning of learning has been criticized ( Grossman, 1970), it is compatible with observed facts and conceptually makes sense. Ever since the study of learning became a respectable pursuit for


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biological scientists, there has been a demand for a tightening of the concept. This is, no doubt, a legitimate reaction of psychologists to the broader use of the term by the intruders. However, the sharp distinctions proposed for what should and what should not be called learning are often arbitrary, as shown in the example given above. The tendency to deal with verbal abstractions rather than experimental facts indicates that the old “faculty psychology” is still with us. It is probable that, outside of the textbooks, there is no such thing as “pure” learning. On the one hand, learning is the result of the integration of the basic processes of neural activity and, on the other hand, it is part of “a more inclusive phenomenon, namely, cognition” (Von Foerster, 1970). It is not in the scientific tradition to delay action until the semantics are clarified. The scientific attitude could be characterized by paraphrasing Admiral Farragut: “Damn the definitions; full speed ahead.” This may be deplored on logical grounds but has been justified by experience. The problem of memory also conceals a trap. The term “engram,” coined by Semon (1904) to designate the hypothetical material trace that a given experience leaves in the brain, instead of as a metaphor, has been taken literally. It launched Lashley (1950) on his famous unsuccessful “search of the engram.” In spite of this failure, the search is still on, this time by biochemists chasing the molecular engram. There are reliable indications that there is no engram and that memory is represented in the brain by a set of functionally connected, but topographically widely dispersed, pathways along which traveled the nerve impulses fired by the original experience and which are recreated at each evocation. Memory is the projection of the universe into the brain. Each of us has, of course, a particular representation of our particular universe, depending on the genetic limitations of the species and the individual and on the extent of our experience. It must include also a self-image and its relations to the universe, dictating the behavior appropriate to the various situations to which the organism has learned to respond. In all these operations, the brain acts like a computer that would “simulate” the universe. II. Neural Coding

Information must be encoded in order to be processed. Information handling or communication systems, be they computers or brains, do not deal with real objects but with signals or symbols which substitute for them. Codes consist of a set of such signals and the rules for their use. In this sense, languages, alphabets, magnetic tapes, maps, and many

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other devices are codes. All systems that handle codes have an encoding and decoding device; a microphone changes vibrations of air into electric currents which can be reconverted into sound. Application of the coding concept to the processing of genetic information is now widely accepted. The DNA code, its transcription into RNA, and translation into a protein code are now taken for granted. Introduction of the same concept into neural function, however, has not been conspicuously successful and even its relevance has been questioned (Roberts, 1965; Moore et al., 1966). The main reason for this is the abundance of signals that present-day electronic methods can detect in the brain. In spite of intensive research in this area, we still do not know which aspects of neural activity carry information and which represent the background noise. Averaging techniques and other computation procedures may be able to help in detecting some regularities but the results are disappointing. Another cause of confusion is that an important coding principle, the information content inherent in the structure of the central nervous system, has been grossly neglected. A. ELECTRICAL ACTIVITY Two types of electrical activity have been recorded from the nervous system: the rapid firing of neuronal elements and the slower events detected in masses of neural tissue. Among the many parameters studied ( Perkel and Bullock, 1968), the only one that definitely functions as a code is the frequency which was shown by Adrian (1928) to transmit information on stimulus intensity. Ideally, one could assume that, as integration proceeds, impulse patterns join together and form new patterns of increasing complexity corresponding to perception and the other products of information processing. Unfortunately, however, nothing in our present store of knowledge suggests that this impulse code operates like radio waves, for example, which can be decoded and “played back” to give a reproduction of the information encoded at their source. As for the slow wave potentials, such as EEG, evoked potentials and steady potentials, which may represent information in “nonimpulse”form, their meaning is just as baffling as it was over a century ago when they were first observed by Caton. Harmon (quoted by Perkel and Bullock, 1968) considered the study of brain waves equivalent to examining a computer with a probe six feet in diameter and trying to guess its function from the oscilloscopic image obtained. This state of affairs seemed to justify the question asked in the discussion reported by Perkel and Bullock, “Are brain waves signals for the brain itself?” Most of the waves represent the noise of background activity and the only significant


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features of the record are caused by gross perturbation of the normal flow of impulses. Considerable efforts have been made, by means of the most sophisticated techniques, to detect changes in wave patterns that could be correlated with learning but no clear-cut results have been forthcoming. We can conclude with Uttal (1969) that only “stimulus codes,” generated at the receptor level, have been definitely established. “Systemic codes,” which may be introduced at higher levels and should represent integrated information, are at present purely hypothetical. Further examination of this problem would not be relevant to our subject matter, and the reader is referred to reviews by John (1967) and Adey ( 1%9) and to the excellent discussion already mentioned ( Perkel and Bullock, 1968).

B. STRUCTURAL CODE Information processing systems have codes built into their structure which represent their basic “program.” In the nervous system, this coding principle is based on the genetically determined organization of the centers and connecting pathways. It is implicit in the “law of specific energies” stated by Johannes Miiller in 1826. Here also, as in the conception of Sherrington quoted above, energy should be replaced by information. In present-day terms, one could formulate the law as follows: The type of information carried by a given neuron is independent of the nature of the stimulus and is determined by the central connections of the neuron. The specificity of the information is not limited to the sensory modality; it also has a “local sign.” Fibers of the optic nerve correspond to definite points on the retina, those of cutaneous nerves to a circumscribed skin area. On the other hand, in the olfactory, glossopharyngeal, and auditory nerves, individual fibers correspond to specific smells, tastes, and sound frequencies. When a given neuron fires, it carries information on the sensory modality of the stimulus, its quality and its localization. The only information that has to be added is the intensity of the stimulus. Even this can be carried by the structural code in a group of several fibers with different thresholds. There are at present two conflicting views on the relevance of structure for the processing of acquired information: a. The connectionist theory assumes that acquisition of new information can take place within the framework of preset channels through the creation of new connections between existing pathways. b. The field or mass theory denies the importance of specific neural connections and assumes that information is processed by diffuse electric fields across the bulk of nerve tissue.

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For a detailed discussion of the opposing views see reviews by Morrell ( 1961), John ( 1967), and Kandel and Spencer ( 1968). The controversy has an important bearing on the chemical coding problem since infonnation-carrying molecules must play an entirely different role whether they are assumed to code specific pathways or to “record experience” without reference to organized structure. A structural code is meaningless in the field theory, whereas its existence is essential to the connectionist approach.

C. CHEMICAL CODES Operation of chemical codes in neural information processing can be considered at three levels of increasing complexity. At the level of the neuron, the mechanism of firing involves ion movements closely linked with probable chemical alterations of some membrane constituents. The nature of these modifications, with possible changes in protein conformation, has been discussed elsewhere (Ungar and Irwin, 1968). The information content of these processes, particularly that of the ion movements, is limited to that of the single neuron. There have been, however, hypotheses emphasizing the role of ions in memory and these will be discussed below. The next level is that of the synaptic junction. The introduction of the principle of chemical mediation of synaptic transmission, the first intrusion of chemistry into the nervous system, which until then was believed to be a purely physical process, created a controversy which lasted almost twenty years. Today not only is it the established creed but it pervades our thinking to an extent that is probably disproportionate with its real importance. Synaptic function is more complex than is commonly believed and includes other processes than the mere transmission of impulses (for further discussion, see Ungar and Irwin, 19168). One thing the mediators can do is to provide chemical labels for certain pathways, particularly in the autonomic domain. A simple code based on chemical transmitters could also direct the traffic of impulses and, thereby, process information. A given neuron can accept signals only from those neurons that release a transmitter for which it possesses receptors. Transmitters can function like red and green traffic lights, and such a binary code could carry quite complicated routing schedules. It is doubtful, however, that such a simple system is adequate for the higher levels of integration, This consideration and the weight of experimental evidence which will be discussed below require the assumption of a third level of chemical coding. This code is often called macromolecular because the initial hypotheses postulated nucleic acids and proteins as the depositories of information, as in the genetic code.


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Research in this area has been influenced by the successful application of a molecular coding concept to genetic information. This influence explains some of the naive aspects of the initial studies bent on faithfully applying the principles of the genetic code to the machinery of the brain. We shall see, in fact, that comparatively small molecules, perhaps with a molecular weight of not more than 10410 to 5000, are sufficient to code all the information required. Except for a few speculations based on the field theory of brain activity, molecular codes have not been conceived to replace the two coding principles mentioned above but were meant to complement them. In his first hypothesis, Hydkn (1959) assumed that the impulse pattern was able to modify the base sequence of RNA in neurons. The new messenger RNA was then able to promote the synthesis of a protein which contained the information coded in the original impulses. This hypothesis was later amended in order not to violate the “central dogma” of molecular genetics, which does not admit RNA changes not transcribed from DNA. Hydkn (1969) still holds that the chemical code is a translation of the impulse code. In most molecular hypotheses, the coded material is believed to act at synaptic junctions to control the traffic of impulses. Information is processed according to the connectionist view; the chemical signal serves only to assure the permanence or, at least, the durability of the newly established connection (Katz and Halstead, 1950; Szilard, 1964; Ungar, 1968; Best, 1968). Molecular theories did not come about to liquidate the established concepts but to fill a gap in our understanding of learning and memory since the permanence of acquired information could not be explained on conventional anatomical and functional grounds. Ill. Evidence for Molecular Mechanisms

While there is widespread agreement on some sort of molecular mechanism playing a role in information processing, most of the evidence produced to support such a mechanism remains highly controversial. The main reason for this is the extreme difficulty inherent in detecting and characterizing the chemical correlates of learning and memory in such a complex system as the brain. It is probable that the processing of a particular set of information engages only an infinitesimal fraction of the neurons so that the chemical changes taking place in these neurons are practically impossible to demonstrate against the background of the ongoing chemical reactions in the mass of brain tissue. The situation is similar to the initial phases of the studies on the chemical nature of synaptic transmission. There were no chemical

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methods available for the direct demonstration of the release of transmitters at presynaptic endings even if their composition had been known, Indirect evidence could be supplied by the imaginative use of pharmacological tools: application of antagonists, of potentiating agents, and above all of bioassay methods. We are in a somewhat better position today because of the remarkable progress made in analytical biochemistry and development of micromethods of extraordinary sensitivity and accuracy. We also have at our disposal a greater array of drugs that affect behavior and we are aware of their biochemical effects. On the other hand, the complexity of the problem is many orders of magnitude greater than, for example, in Laewi’s experiments on the chemical transmission of the vagal stimulation of the heart. The evidence produced up to date can conveniently be divided as follows: ( a ) detection of chemical changes correlated with information processing; ( b ) drug actions; and ( c ) bioassay methods.

A. CHEMICAL CORRELATES These studies have the same basic design: submit animals to a training procedure and analyze the brain for possible chemical changes by comparing the results of the analysis with those obtained in control animals. The principle seems simple enough but its difficulties appear when a decision has to be made for the nature of the changes to look for, the probable location of the changes in the brain, and the appropriate controls to use for limiting the process to the acquisition of a given set of information.

1. Changes in RNA and Protein For reasons mentioned above, the first attempts at finding chemical correlates of learning were directed to possible quantitative and qualitative changes of brain RNA. Over the past decade, HydCn and his associates used extremely elegant microtechniques to determine the amount of nuclear RNA per cell and the proportions of the four bases, usually expressed in terms of base ratios. All the analyses were made in single or small groups of neurons or glial cells. From the changes in base ratios HydCn concluded that learning changed the composition of nuclear RNA, shifting it toward the “chromosomal” or “DNA-like”type. There was a general increase in the amount of nuclear RNA, but this was also observed in the controls, submitted to an equal amount of stimulation without learning. The results were interpreted first as indicating the synthesis of a new RNA species ( HydBn, 1959) and later as an increase


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in messenger-type RNA (Hyddn, 1967). This was observed at early stages of learning, while at a later stage there was an increase in ribosomal-type RNA. More recent experiments by Glassman’s group in North Carolina used a technique involving sampling of various regions of the brain and estimating incorporation of radioactive uridine with double labeling. After injection of uridine, the experimental animals were trained for 15 minutes in an avoidance task and killed. It was found that incorporation of uridine increased by about 50% in the trained animals as compared with the yoked controls which were submitted to the same stimuIi but were not supposed to learn. No base ratio changes were observed in these experiments ( Glassman, 1969). Bowman and Strobel ( 1969) made similar observations using 3H-labeled cytidine in animals trained in a Y-maze with positive reinforcement. Shashoua (1968) studied RNA changes in goldfish trained to acquire a new swimming skill. The fish were injected with labeled orotic acid, a precursor of uridine and cytidine, and the findings indicate a considerable increase of the uridinelcytidine ratio in the trained fish, as in Hydkn’s long-term experiments. Early attempts at correlating protein changes with learning were unsuccessful. Altman ( 1966), using autoradiography after injection of labeled amino acids, failed to find a reliable difference between incorporation in the brain of trained and control animals. More recent studies obtained positive results. Hydkn and Lange ( 1968), after administration of l e ~ c i n e - ~ H found , increased incorporation in cells supposed to be involved in their handedness training schedule. Brain proteins were fractionated by polyacrylamide gel microelectrophoresis and the increased incorporation was observed in two fractions containing proteins specific for the brain. Similar results were found with labeled lysine in chicks submitted to imprinting ( Bateson et al., 19169). With autoradiography, Beach et al. (1969) found greater uptake of l e ~ c i n e - ~in H the nuclei of neurons of rats trained for conditioned avoidance than in control animals. Bogoch (1968) made an extensive study of brain glycoproteins, “mucoids,” in pigeons. He was able to separate about fifty fractions by DEAE-cellulose chromatography. After training, characteristic changes were observed in a few of these fractions. The analysis was further refined by disc electrophoresis. Bogoch places particular emphasis on the carbohydrate moiety of these molecules and believes that nonprotein end groups are more likely to be involved in coding than the protein backbone.

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2. Location of the Chemical Changes The experiments done up to date vary from single cell studies to chemical analysis of the whole brain. The first experiments in Hyddn’s laboratory (Hyddn, 1959) were done in Deiters’ cells of rats trained to climb on a wire. The choice of these cells, which are equivalent to a spinal ganglion, was criticized since it is unlikely that they are involved in the learning process. In later experiments ( H y d h and Egyhazi, 1964), involving handedness training, neurons were collected for RNA analysis from a cortical location which is believed to be involved in the use of the forepaws. Booth (1970) criticizes the lack of physiological verification of the relevance of the cells analyzed. In the experiments done in Glassman’s laboratory, grossly dissected regions of the brain were analyzed separately. Significantly higher incorporation took place in the diencephalon and the limbic system in general. There was a comparative decrease in the cortex because the yoked controls showed an even higher rate of synthesis. Beach et al. (1969), who used avoidance training, like the authors just mentioned, found increased leucine incorporation in the limbic system, using autoradiographic techniques. Bowman and Strobe1 ( 1969) found significant changes in the hippocampus, which was also the site of increased amino acid incorporation in the handedness experiments of HydCn and Lange ( 1968). Whole brain analyses were done by Shashoua (1968) and Bogoch (1968). The magnitude of the changes was of the same order in these experiments as in those sampling limited areas of the brain. This is rather embarrassing if we do not keep in mind that much of the evidence points to a rather diffuse localization of acquired information, even if we believe that the hippocampus may have some special function in the recording of memory. The participation of the limbic system suggests the importance of emotional factors in avoidance learning. Subcellular localization of the RNA or protein fraction that exhibited the change correlated with learning has not produced any consistent result. Hydkn found significant changes only in nuclear RNA. Bogoch believes that learning-related increase takes place in membrane proteins but his evidence is sketchy. Delweg et al. (1968) found increase of the polysomes in the brains of rats trained to balance on a wire. Interesting observations were made by Hyden and Lange (1965) on the changes observed in glial cells simultaneously with the neuronal modifications. Interpretation of these results suggests an exchange of RNA and proteins between glia and neurons ( HydCn, 1969).


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3. Critical Factors The most vulnerable aspect of these experiments is the problem of their precise relationship with information processing. All the changes described, briefly summarized in the preceding pages, could have been caused by simple increase in neural activity without actual retention of information. The chemical correlates of neural activity were discussed by Ungar and Irwin (1968) and some aspects of the problem were more recently reviewed by Glassman (1969) and by Ungar (1970d). The reader is referred to these reviews for detailed information. Sensory or electrical stimulation was observed to increase RNA and protein synthesis in a number of structures. The same changes were observed after increased motor activity. In most cases, the initial increase was followed by reduced synthesis or increased destruction. These effects were best documented in the visual system where reduced light input caused decrease in RNA and protein content. Synthesis of macromolecules was augmented when normal light input was reestablished. Similar results were obtained after acoustical and vestibular stimulation. Changes in RNA base ratios were also reported after stimulation without learning in the usual sense. Grampp and Edstrom (1963) found that physiological stimulation of the stretch receptor of the crayfish altered the base ratio. The most puzzling results were obtained by Rappoport and Daginawala (1968), who applied a number of olfactory stimuli to the isolated head of the catfish. Some of these stimuli were novel since they were unlikely to exist in the normal environment of these animals. Among these, morpholine and amyl acetate produced definite changes in base ratios. Extracts of shrimp and redfish, certainly familiar to the experimental animals, caused similar alterations. Other novel stimuli, like camphor and menthol, failed to elicit any change. We have seen that in almost all the experiments dealing with learning-related chemical changes, controls were used which were supposed to receive the same amount of stimulation as the experimental animals but under conditions that did not induce learning. The question really is whether it is at all possible to make a sharp distinction between information input without learning and with learning. Theoretically, learning has taken place when the information transmitted by the stimulus is retained and can be retrieved. In practice, most psychologists do not think that learning can occur without motivation and the proper positive or negative reinforcement. It is, however, probable that the line cannot be drawn as distinctly between the two cases. Human experience shows the definite possibility of “spontaneous” learning,

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apparently devoid of motivation or reinforcement. There is no doubt that these factors do facilitate learning, but they are not indispensable for it. There are many gradations between the firmly anchored memory of a well-learned set of information and the almost instantaneous evanescence of an unreinforced and unmotivated stimulus input. With this in mind, we can perhaps interpret the results of the quest for chemical correlates of learning. They probably indicate a quantitative difference between information firmly retained and added to the existing record and the information accidentally acquired and only faintly and imperfectly processed. The changes may, in fact, be caused by increased neural activity which is more intense and widespread when the information is fully processed than in the case of stimuli leaving a vague and unstable memory. The question is now whether, in the present state of our knowledge, it is possible to demonstrate some chemical change that is specific for learning. Conceptually, such a change should be specific, not for the learning process in general, but for what was learned, that is, for the retention of some particular set of information. There are at present only two analytical methods that can promise this result. The recent attempts at DNA-RNA hybridization (Machlus and Gaito, 1969), if they are confirmed and can be successfully extended, could prove that information is recorded in RNA sequences. The second method is the production of antibodies to brain protein ( Mihailovii. and JankoviL, 1961; JankoviE et al., 1968; Rosenblatt, 1970). If an antibody can be produced that abolishes one particular piece of learned information, we can be reasonably certain that it was recorded in the structure of the molecule which served as antigen. There is, of course, a third method, based on the bioassay principle, which is capable of supplying the same evidence. It will be discussed in Section C.

B. DRUGACTIONS There is extensive literature dealing with the effect of drugs on learning and memory. There are drugs known to inhibit learning and others that seem to facilitate it ( McGaugh and Petrinovitch, 1965; Kelleher and Morse, 1968). I shall limit this discussion to those drug actions that are directly related to known chemical processes such as synaptic transmission and the synthesis of RNA and proteins.

1. Action on Synaptic Transmission Most of the work in this area is concerned with cholinergic mechanisms. Facilitation of memory consolidation by drugs acting on the


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adrenergic system, like amphetamine and probably magnesium pemoline, is usually attributed to their alerting action. Recent work (Essman, 1968) also implicates serotonin in information processing. In the cholinergic system, contradictory results have been published on the effect of cholinesterase inhibitors ( diisopropylfluorophosphate, eserine) and cholinergic blocking agents ( atropine, scopolamine). It has been known for several years that the latter category of drugs inhibited the learning process. It seemed logical to conclude that cholinergic transmission plays an important role in acquisition of information. It was, however, found that cholinesterase inhibitors, which theoretically should have enhanced learning by sustaining cholinergic transmission, had in fact variable effects, Deutsch (1966) proposed a hypothesis which attempts to explain these paradoxical results. He found that anticholinesterases enhance learning at the early and late stages, that is, when the memory trace is either imperfectly consolidated or is nearing extinction. The same drugs, however, have an inhibitory effect at the peak of learning. This suggested that learning requires an optimum concentration of acetylcholine at the synapses. When it is low, as in incipient fixation and during extinction, its increase by inhibition of cholinesterase facilitates the process. When information processing is at its maximum, further increases in acetylcholine may block the synapses and, thereby, inhibit learning. The hypothesis is in agreement with our knowledge of cholinergic mechanisms at neuromuscular junctions and, if it proves valid in the brain, it can explain some drug actions. Other agents involved in synaptic transmission have also been shown to affect learning. Strychnine, believed to be an inhibitor of the hypothetical inhibitory transmitter of the Renshaw cell, enhances memory consolidation ( McGaugh and Petrinovitch, 1965). Nicotine, a stimulant of ganglionic transmission, was found by Bovet et al. (1963) to have a facilitatory effect on learning. Earlier studies had shown that higher doses and more prolonged administration produce the opposite effect. On the whole, these drug studies confirm the common sense feeling that in an optimal state of stimulation of neural function and of synaptic transmission learning is enhanced. When stimulation is suboptimal or excessive, learning is slower and more erratic. These observations also suggest that synaptic transmitters may represent an elementary molecular coding system. It is known, for example, that application of cholinergic agents to certain areas of the brain may make rats behave as if they were thirsty while adrenergic substances applied to some of these sites elicit hunger-motivated behavior ( Miller, 1985). Although these observations cannot explain more compIex behavioral patterns, they may supply a simplified model for them.

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2. Action on R N A and Protein Synthesis This area has been studied in the last few years by several groups of investigators with the intent of finding a correlation between macromolecular synthesis and information processing. This research was facilitated by the discovery during this period of agents which inhibit transcription of DNA into RNA (8-azaguanine, actinomycin D ) and translation of RNA into protein, by blocking the growth of peptide chains (puromycin) or the supply of amino acids by tRNA (cycloheximide and acetoxycycloheximide) . Studies on the effects of all these substances have been reviewed by Glassman ( 1969) and Cohen ( 1970). There is general agreement on the fact that none of these drugs has an effect on short-term memory, that is, the acquisition and temporary retention of information. There is considerable disagreement on the duration of this temporary retention. According to the task to be learned, the animal species, the intensity of the reinforcing stimulus, the criteria of remembering, and the observers, the duration varies from a few seconds to 18 hours. It is possible that the very short times correspond to “reverberating circuits,” if these do indeed take place, or to conformational changes in existing macromolecules (Ungar, 1963), while longer durations of retention may involve chemical reactions not requiring increased synthesis of RNA or protein. There are contradictory observations on the effect of 8-azaguanine and actinomycin D on learning. Because of their high toxicity, these agents do not allow prolonged administration, so that retention for several days could not be explored. Agranoff et al. (1967) have, however, found that actinomycin D can inhibit avoidance learning in the goldfish, and Squire et al. (quoted by Cohen, 1970) made similar observations in rats Most of the work in this area was done with puromycin and with cycloheximide (or its acetoxy derivative). There is good agreement on the probability that both agents act by blocking the conversion of shortterm into long-term memory (Agranoff, 1965), suggesting, therefore, that this conversion depends on the synthesis of protein. The results obtained with puromycin in mice became difficult to interpret when it was found that the blocking effect of puromycin could be erased by cycloheximide (Flexner and Flexner, 1966; Barondes and Cohen, 1967) or by a simple injection of saline (Flexner and Flexner, 1968). These findings were interpreted by the assumption that puromycin acts by producing abnormal peptide fragments having an inhibitory effect. The effect of cycloheximide is probably more direct. It causes a prolonged memory deficit in mice if these are partially trained (three


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out of four correct responses in a Y maze), but it has no effect or causes only a transient deficit with more prolonged training (nine out of ten correct responses) (Barondes and Cohen, 1968; Flexner et d.,1966). In most cases, injection of these agents was done in localized areas of the brain. Flexner and Flexner (1969) believe that early suppression of memory can be produced by bitemporal injections affecting primarily the hippocampus. Later on, more extensive administration over larger cortical areas is required. T’his extension of the “memory trace” takes place even when the hippocampus is initially inhibited by puromycin. There have been a few attempts at enhancing learning by accelerating RNA synthesis. Tricyanoaminopropene, an impurity found in malononitrile preparations, was shown to produce increased RNA and protein synthesis (Egyhazi and HydCn, 1961). This drug was found to facilitate certain types of learning (Chamberlain et d.,1963; Schmidt and Davenport, 1967) but was without effect in others (McNutt, 1967; Otis and Pryor, 1W).Another drug, magnesium pemoIine, was promoted on the ground of its activating effect of RNA polymerase (Glasky and Simon, 1966). Neither this chemical action (Stein and Yellin, 1967) nor its behavioral effect ( Goldberg and Ciofalo, 1967) have been unequivocally confirmed. Whatever effect it may have on learning may be due to an amphetamine-like stimulation of activity (Beach and Kimble, 1967). On the whole, the approach to the chemical mechanisms of learning through inhibitors of RNA and protein synthesis has been useful. With the likelihood of better and more selective drugs becoming available in the future, it may provide clearer and less ambiguous results. Looking back on the development of our ideas on chemical transmitters, which I have chosen as a sort of a model in the quest for the molecular coding of acquired information, it is clear that it was considerably helped by the availability of pharmacological inhibitors and potentiators.

C. BIOASSAY METHODS The two approaches just summarized strongly suggest that information processing is accompanied by an increased synthesis of RNA and proteins, and that it can be impaired if these synthetic processes are inhibited. The results obtained up to now do not, however, prove that the material synthesized during learning represents a code for the information processed. They can be interpreted in the more conventional way as indicating a raised metabolic level known to occur in all tissues when their activity increases. With the exception of two approaches mentioned above (Section 111, B, 3 ) which are still at a preliminary stage, the only direct evidence for a molecular code of acquired information has been supplied by the bioassay method.

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The principle of this method is to detect the possible chemical changes that have taken place during learning, not by chemical analysis of the brain, but by administration of suitable brain preparations to naive animals whose behavior is then tested for similarities with the behavior of the trained donors. The basic design includes the following steps: (1) training of donor animals, ( 2 ) preparation of extracts from the donors, ( 3 ) administration of the extract to recipient animals, and (4)testing of the recipients and evaluation of the results. The first experiments of this type were done in planarian worms (McConnell, 1962). The donors, conditioned to respond to a light stimulus by a contraction which is originally elicited only by electric shock, were cut into pieces and fed to untrained recipients. These showed subsequently a higher ratio of responses to light than the controls which had ingested fragments of untrained worms. These experiments received wide publicity and stimulated a lively controversy which questioned more the interpretation of the results than their reality. The whole problem has been reviewed by Corning and Riccio (1970) and McConnell and Shelby (1970). Experiments in mammals based on the same principle were first published in 1965 almost simultaneously by four groups of workers ( ReiniS, 1965; Fjerdingstad et al., 1965; Ungar and Oceguera-Navarro, 1965; Babich et al., 1965). The work of the last-named group became widely publicized in the United States, and several attempts were made to replicate it. Most of these attempts resulted in failure and a brief summary of these negative experiments, signed by twenty-three workers from six laboratories (Byrne et al., 1966), was supposed to put an end to the issue. In one laboratory, however (Rosenblatt et al., 1966), positive results were obtained and the attack was broadened. Also, one of the signatories of the collective paper referred to above subsequently obtained positive results ( Byrne and Samuel, 1966; Byrne and Hughes, 1967). Three of the four original teams, unimpressed by the quality of the negative experiments and the arguments raised against the principle of the method, were joined by new groups of workers. At the time of this writing, there are sixty-six publications from twenty-two laboratories which have reported positive effects of brain extracts from trained donors on naive recipients. As for the opposition, since the onslaught of 1965-1966, seven papers appeared describing negative results. It would be impossible, in this review, to examine these experiments in detail; the reader is referred to the several summaries published or scheduled to appear early in 1970: a volume edited by Byrne (1970) and


2.41

reviews by Gurowitz ( 1969), Rosenblatt ( 1970), and Ungar ( 1970a,b,c). They include an analysis of the factors which determine the success or failure of the assay and a discussion of the significance that can be attached to the results obtained up to date. I should like to limit the discussion here to a few points relevant to molecular coding in information processing. Discussion of whether these experiments really represent transfer of memory, learning, or other less well-defined processes are probably sterile and irrelevant at this point. They undoubtedly do represent transfer of some sort of acquired information which must be encoded in terms of molecular structure because all other forms of information have been destroyed. There is still undoubtedly a problem of specificity that has not been entirely resolved. The recipients of brain from donors trained not to respond to a sound stimulus lose their responses to sound but retain them to an air puff and vice versa (Ungar, 1967). Similarly, there was no cross-transfer between two passive avoidance paradigms, avoidance of the dark, and avoidance of step-down from a platform (Ungar, 1970a). There is somewhat less evidence for specificity within the same sensory modality, although the recent experiments of Zippel and Domagk (1969) in goldfish suggest that color and taste discrimination can be transferred. There has been some uncertainty as to the chemical nature of the coding molecules active in the brain extracts. Out of the twenty-two groups of workers who obtained active extracts, nine assumed that the active substances were RNA, four thought they were proteins or peptides, and the others made no assumptions. In recent work in my laboratory (Ungar and Fjerdingstad, 1970; Ungar, 1970d), we found that the active material capable of inducing dark avoidance (Ungar et aZ., 1968) was present in both RNA and peptide preparations. Since it was inactivated by trypsin but not by ribonuclease, it seemed probable that the substance was a peptide attached to an RNA molecule. We were able to dissociate the RNA-peptide complex at low pH and found the whole activity in the diffusible, low-molecular material. This has been purified by gel filtration and thin-layer chromatography and is at present active at the dose of 0.1 pg per mouse. It has been identified as a pentadecapeptide with the following amino acid sequence: Ser-Asp-Asn-AsnGlu-Gln-Gly-Lys-Ser-Ala-Glu-Gln-Gly-Gly-TyrNH,. The structure is now being verified by synthesis. Three other active substances have been partially purified in my laboratory; they are all destroyed by trypsin or chymotrypsin or both; they are all dialyzable and on gel filtration appear to have a molecular weight between 1000 and 2000. Although application of the bioassay method is obviously at a very

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early stage, it suggests more than any other set of data produced so far that learned information does have chemical correlates in the brain and that these correlates represent in all probability a molecular code.

IV. Molecular

Hypotheses

There have been many speculative interpretations of the role of molecular mechanisms in neural information processing. It is probably appropriate to distinguish among them three main groups of hypotheses, each based on a different understanding of the operation of the nervous system and of the nature of information coding.

A. NONSPECIFIC HYPOTHESES The most common and conventional explanation of the chemical changes is that they simply reflect increased neural function. As in all tissues, increased function requires heightened metabolism and accelerated turnover of most cellular constituents. If protein is used up faster, RNA synthesis is stepped up by a negative feedback to increase the rate of protein synthesis. This hypothesis is compatible with all the data supplied by chemical analysis, even with the change in RNA base ratios, which means an increase in the production of mRNA and subsequent stimulation of protein synthesis. It could also explain the blocking effect of metabolic inhibitors on learning since, in the absence of an augmentation of RNA and protein synthesis, the increase in function could not be maintained. Increase in protein synthesis can be interpreted as an indication of the growth of new synapses which has been one of the favorite hypotheses since Tanzi (1893). Hebb (1949) and Eccles (1965), among others, have favored the idea that the new synaptic connections necessary for learning are etablished by local growth of the neurons. Another interpretation, which may be either an alternative or a complement to the growth hypothesis, is the production of synaptic facilitation by an increase in the availability of transmitters at the presynaptic ending or by a modification of receptor efficiency at the postsynaptic site. Increased RNA and protein synthesis would then mean a higher supply of the enzymes necessary for transmitter synthesis (Briggs and Kitto, 1962). Paradoxically, the only enzyme whose increased presence was observed under conditions of learning or, at least, increased neural activity is cholinesterase, which, of course, inactivates a transmitter (Bennett et al., 1964). It is possible that synthesis of cholinesterase is induced by the presence of its substrate, and Smith (1962) believes this to be the most important biochemical event in learning. Electrolyte shifts have been proposed as nonspecific correlates of


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learning. Overton (1959) developed a theory in which Ca++ions played a decisive role. He assumed that when acetylcholine is hydrolyzed, the acetate ion forms a soluble salt with Caw so that increased activity drains the cation away from the cells and this produces increased synaptic facilitation. Sachs ( 1961) observed that intraventricularly injected K+ facilitates learning, and Ca++inhibits it. More recently, he linked these data with the growth hypothesis by claiming that increased extracellular K‘ stimulates the formation of new synaptic junctions. The results obtained by the bioassay techniques are incompatible with a nonspecific interpretation of the chemical correlates. If a material extracted from the brain of trained donors can reproduce their behavior in the recipients, the information on which the behavior is based must be coded in molecular terms. The chemical correlates therefore represent either a direct storage of “experience” or some indirect code in which information is recorded.

B. “TAPE-RECORDER’’ MOLECULES Most of the hypotheses of this category are based on the field theory. They give the impression that the molecular memory trace floats in that ‘bowlful of porridge” which, according to Hebb (1949), represents all the structure that the field theory allows to the brain. The most extreme of the nonstructural molecular hypotheses is probably held by McConnell ( 1965), who expressed several times the opinion that “experience” is recorded in RNA as on a magnetic tape and is equally distributed, not only in all the neurons, but probably in all the cells of the body, like genetic information. There has been no suggestion as to the way in which acquired information reaches the cell nucleus (where RNA synthesis takes place) and how it is retrieved from there. Hypotheses formulated by Landauer (1964) and Robinson (1966) tend to replace “neural nets” with what Schmitt (1962) called “molecular nets.” Both assume that impulses are propagated in the brain by spreading through the bulk of the tissue instead of following regular neuronal pathways. Landauer thinks that a pattern of impulses may give rise to the synthesis in the glia of new molecular species of RNA which are later transferred to the neurons and sensitize them to future occurrences of the same impulse pattern. In Robinson’s hypothesis, information is processed by “purely non-neural means.” In the presence of a given pattern of firing, a number of widely distributed cells, called “pattern neurons” fire randomly. Each of these cells contains a set of identical “pattern molecules” and when these fire, they release some of these molecules into the extracellular environment. Here, they are picked up by “storage units” (probably glial cells) where they give rise to the

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synthesis of complementary molecules (“pattern co-molecules”) . When the same firing pattern occurs again, the pattern neurons, when they release their pattern molecules, will recognize the co-molecules and react with them. The main contention and also the greatest difficulty of these hypotheses is the transduction of electrical impulses into molecular structure. The explanations offered up to now include the assumption that RNA configuration can be changed by electric fields. This change is not completely reversible and exhibits hysteresis ( Katchalsky and Oplatka, 1966; Stanford and Lorey, 1968). This would not require changes in base sequence and could therefore take place independently of DNA but it is not known how much information the change could carry and for how long. Other hypotheses assume the possibility of methylation or demethylation of the bases in RNA (Hechter and Halkerston, 1964) or DNA (Griffith and Mahler, 1969). Such a modification could, of course, radically change the nature of the protein molecules synthesized and thereby store new information. The basic flaw of most of the hypotheses just mentioned is that they represent the molecular code as a transcription of the information recorded in impulse or wave patterns. As it appears from the discussion in Section 11, there is no evidence that these patterns have any meaning when they are taken out of the context of the pathway on which they travel. It is probable that the field theory, even supported by the modern lore of wave potentials, is not the proper framework for the study of information processing. f i e structural unit of information processing is the aggregate of neurons connected by synapses (the “cell assembly” of Hebb) not the bulk of brain tissue in which heterogeneous elements are crowded together without regard to functional interdependence.

C. HYPOTHESES BASEDON CHEMOSPECIFIC PATHWAYS The hypotheses most likely to make a correct guess at the molecular code are those that recognize the existence of specific pathways in the central nervous system and consider it as the basic coding principle. It is important, however, to modify the rigid “one-to-one” specificity and introduce a broader, more plastic concept with, perhaps, a probabilistic element, at least at the cortical level. Neural pathways going from sensory receptors to centers and from centers to effector organs are not linear structures eliciting always an expected response. This situation may exist in lower organisms with a predominantly instinctive behavior or in the spinal reflexes of vertebrates. At higher levels, however, the responses are less predictable because each of the neurons may be connected to thousands of other neurons so that


M A 5

the simplest stimulus, besides triggering a flow of impulses in the direct channel, gives rise to a wide peripheral area of excitation and inhibition. With the increase of acquired information, these peripheral areas gain more and more importance and increase the uncertainty of the response to a given stimulus. The widespread network of connections, characteristic of all highly developed nervous systems, does not, however, invalidate the principle of specificity. The common feature of all the hypotheses of this class is that they consider synaptic facilitation or inhibition as the focal point of information processing. In Katz and Halsteads speculations (1950), th’is was assumed to take place by changes of nucleoprotein molecules in the membranes. Since this hypothesis was formulated before the DNA-RNAprotein coding system was fully elucidated, some of it is obsolete now but the idea of alteration of membrane proteins induced by learning is still very much alive. With HydCn ( 1959), the emphasis shifted from the membrane to the nucleus where electrical impulse patterns were supposed to be transduced into RNA sequences. More recent views of H y d h (1967, 1969) recognize, however, that proteins are the “executive molecules” and play their role probably at synaptic junctions. In 1964, Szilard published an entirely speculative paper based an the assumption that “neurons which differ from each other in their response-specificity contain a different set of certain proteins in their cell membrane.” He then postulated that to each of these “specific membrane proteins” corresponds a complementary molecule which can combine with it in the manner of the antigen-antibody linkage. The efficacy of a synapse between two neurons A and B would depend on the number of AB “dimers” at the synaptic site. To explain learning, Szilard distinguished, in the nervous system, “congenitally determined neurons and “memory” neurons. The former were provided with their specific proteins during embryonic development. When they contact a memory neuron and both neurons fire simultaneously, the protein of the congenitally determined neuron penetrates into the memory neuron and induces in it “the complementary specific membrane protein just as an antigen induces its antibody.” By this process, called “transprinting,” new synaptic connections could be created. Synthesis of the complementary molecules is supposed to result from derepression of the appropriate RNA sequences. One of the theories of Griffith (1966) is based on a similar transprinting process, but the material present at the synapse is rather nonspecific and may be the same for all synapses. Furthermore, the passage of material that creates new synaptic bridges is assumed to take place

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from the soma or dendrite to the axonal ending, a highly improbable direction. Rosenblatt’s hypothesis ( 1967) follows very closely Szilards ideas. Synaptic junctions are established by “the production of an adhesive molecular complex specifically coded for both the presynaptic and postsynaptic membranes of the appropriate cell pairs, and made up of constituents released by the pre- and postsynaptic cells when they are jointly active.” These coded molecules are supposed to be located in “intrasynaptic filaments” and consist of complementary molecules. The hypothesis I formulated (Ungar, 1968) makes similar assumptions but emphasizes the continuity between the chemospecificity of pathways which controls the development and differentiation of the nervous system (Sperry, 1958; Jacobson, 1969) and the processing of acquired information as it takes place during adult life. I submitted that the labeling process which organizes synaptic connections in the nervous system is merely a refinement of the identification code by which cells of the same type recognize each other and form specific aggregates (Moscona and Moscona, 19433). By the operation of this process “the nervous system is chemically coded before any learning takes place . . . and serves to elicit the inborn responses.” It was postulated that “the genetically determined neural organization includes all that is required for learning: built-in pathways, inborn stimulus-response relations on which new patterns of behavior can be founded, and a fully developed molecular coding system which maintains the synaptic connections between the neurons of each functionally different pathway.” Learning was assumed to take place by the creation of new junctions between preexisting pathways. Establishment of these connections was explained by a mechanism similar to Szilard’s transprinting, that is, the passage of coded molecules at points of contact between simultaneously firing neurons. To make the hypothesis compatible with the results of the bioassay experiments, it was postulated that the coded molecules form complexes called “connectors.” These incorporate the substances coding the pathways that meet at a particular set of synapses. As a result of the intensive training necessary for the successful transfer of information, comparatively large amounts of these connectors are synthesized in the brain of the donors. When they are extracted and injected into the recipients, thanks to their high affinity for the specific sites at which they had been synthesized, they bind to homologous sites in the recipient brains and reconstitute the synaptic connections corresponding to the learned response. A substantially similar theory was formulated shortly after by Best ( 1968) based on a “neuron identification code” ( NIC) . According to


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the number of pathways involved in a learned experience, “polyNIC” molecules are formed which represent the record of the given experience. A problem common to all these hypotheses is the explanation of the mechanism by which new synaptic links can be formed in a tightly organized system. They use the concept of contiguity that has been much debated in psychology; it became with Hebb (1949) the central principle of the learning process. It means that, if anatomically contiguous but functionally unconnected cells fire simultaneously or almost simultaneously in an appropriate sequence, sooner or later they will become linked together by a functional synapse. Just how this spatial and temporal contiguity creates the synaptic bridge or “closure” is still a matter of speculation. Elul ( 1966) proposed a physical mechanism based on the suggestion that electric fields may cause a deformation of the cell surfaces and narrow the distance between two adjacent neurons. The possibility of chemical changes is suggested by a whole series of recent data. The existence of a proximodistal axoplasmic flow (Barondes and Samson, 1967) raised the question of the fate of the transported material at the axonal ending (Droz, 1969). Labeled material was seen to leave nerve endings and enter muscle cells (Korr et al., 1967). Intracellular communication of various types has been studied by Loewenstein (1866).It seems that when two cells “recognize” each other as belonging to the same type, the junction between them becomes permeable to comparatively large molecules (up to 10,000 molecular weight), There has been progress also in the study of the material occupying the intersynaptic space (Kennedy, 1967; Bloom and Aghajanian, 1968), with increasing probability of its playing some functional role. The whole problem has recently been discussed at a Neurosciences Research Program Work Session ( Schmitt and Samson, 1969). The mechanism of synaptogenesis has been studied by Glees (1966) and Aghajanian and Bloom (1967), suggesting the possibility of new synapses being formed in the adult brain. The hypothetical mechanism, based on the contiguity principle assumes that when two cells in close enough proximity fire simultaneously, the increased permeability associated with the active site facilitates the passage of material between them. The direction of axoplasmic flow suggests that the passage is from the presynaptic toward the postsynaptic site. This would also explain the necessity for the conditioned stimulus to precede the unconditioned stimulus by a critical interval. There is much that remains unknown on the extent to which the differentiation and coding of the brain pathways is completed under genetic control. The specificity of sensory and motor pathways is well established but there are elements of the cortex, such as the “neuropile”

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of Herrick ( 1956), which remain unorganized, uncommitted, and perhaps uncoded until they are utilized for the processing of acquired information. “The neuropile is considered by many as the prime locus for changes responsible for conditioning and learning” ( Livingston, 1966). A frequently voiced objection against the chemospecificity of pathways and the hypotheses derived from it is that the number of different molecular species they require is beyond the information content of the genome. The objection cannot at present be answered with any degree of certainty. Sehmitt and Samson (1969) invoked the possibility of information being carried in the tertiary and quaternary structures of proteins and their combinations with polysaccharides. Roberts and Flexner ( 1966) proposed an ingenious process involving “self-induction” and “sequential induction” capable of producing a large number of molecular markers using a limited number of genes. One can also suppose that many different peptides may be split off from a small number of protein precursors by differential enzyme actions. One can assume that the specific molecular labeling of neural pathways has evolved from a simple system perhaps analogous to the specification provided by the chemical transmitters. Recent developments (Aprison and Werman, 1968) which ascribe some role in synaptic function to individual amino acids may also give a clue to the principle of the coding system. The hypotheses summarized in this section, however naive they may appear as our knowledge advances, fulfil a useful role. m e y point to gaps in our knowledge, reveal inconsistencies in our thinking, and adjust it to new experimental facts. Their role in the scientific method was outlined by Claude Bernard (1865). “Les thkories sont comme des degrks successifs que monte la science en klargissant de plus en plus son horizon, parce gue les thkories reprksentent et comprennent nkcessairement d’autant plus de faits qu’elles sont plus avanckes. Le vrai progrbs est de changer de thkorie pour en prendre de nouvelles qui aillent plus loin que les premibres, jusqu’8 ce qu’on en trouve une qui soit assise sur un plus grande nombre de faits.” V. Concluding Remarks

The idea that living systems are chemical machines has not sufficiently permeated our understanding of the nervous system. The century-long tradition of studying neural processes by means of electrical stimulation and electronic recording devices created the more or less conscious feeling that the nervous system works on the same principles. This feeling underlies the thinking of many neurobiologists in spite of the knowledge


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that bioelectric phenomena are of an entirely different nature than their instrumental counterparts based on the flow of electrons in metallic conductors and controlled by electron tubes or transistors. A. V. Hill (1954) realized this and warned us that “ultimately the machinery itself is chemical in nature, the fuel it uses for its recovery processes is chemical, the ‘acid and the ‘plates’ of the ‘accumulators’ are chemical, the free energy of chemical change provides the mechanical work and various enzymes prescribe the course of the reactions.” Let us add that the switches, that is, the synaptic junctions, are also chemical, and the whole point of this review is to suggest that the machinery of information processing may be based on chemical principles. Introduction of the computer as a model for the brain would, superficially, seem to accentuate the physical nature of neural function further. But computers do not have to be electronic; they can work on mechanical, hydraulic, optical, and many other principles. In living systems, computation is done by chemical means, as exemplified by the genetic code. The replication of DNA, its transcription into RNA, translation into proteins, and the enzyme-substrate recognition are examples of molecular computation. Similar processes take place in the nervous system where the decision made by the neuron to fire or not to fire is the outcome of molecular computation. Behavioral response to a stimulus is the overall result of such computations by many thousands of these units “wired together in intricate circuits. The most distinctive feature of the system is its ability to reprogram itself by creating new connections. Weiss (1965) suggested that learning consists in the formation of new words with the letters of the innate alphabet or, perhaps more accurately, new sentences with the words of the innate vocabulary. If we accept that these new words are code names for pathways of functionally connected neurons, we can represent a behavioral pattern by a “sentence,” that is, a sequence of words naming the successive pathways on which impulses have to travel for the expression of the behavior. (Similarly, one could define a chemical by a code listing the successive steps of a synthetic process or a geographic location by naming the roads which lead to it.) If we go now one step further and assume that the words or names of pathways which make up the sentence are molecular markers we can conclude that the end-product of information processing-behavior, skill, or memory-can be adequately represented by a molecular complex encoding the sentence. The genetic code is a set of instructions for synthesizing chemicals. The code for acquired information could be seen as a set of itineraries for nerve impulses. There is, of course, a considerable gap between such a code and the highest expressions of creative thought. This gap is no

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greater, however, than the one between the syntheses specified in DNA and a newborn baby. Both will take a long time to be bridged, but the problem of neural information processing is both more complex and methodologically less advanced than the genetic code. A survey of the three main approaches to the chemical aspects of the problem showed that, up to now, only the results of the bioassay methods were sufficiently specific to suggest the existence of a molecular code. Two other promising techniques, using immunological identification of neural proteins and DNA-RNA hybridization are still in the preliminary stage. The bioassay method, however imperfect it may be, has provided us with means for guiding the isolation, purification, and identification of the substances which may represent the molecular code of acquired information. Accomplishing this for a small number of these substances, out of the many millions postulated by present hypotheses, would open the way for the decipherment of the code and lead to a better understanding of the way in which information is processed in the brain. REFERENCES Adey, W. R. (1969). Neurosci. Res. Program Bull. 7, 75. Adrian, E. D. (1928). “The Basis of Sensation. The Action of Sense Organs.” Christophers, London. Aghajanian, G. K., and Bloom, F. E. (1967). Brain Res. 6, 716. Agranoff, B. W. (1965). Perspectives Biol. Med. 9, 13. Agranoff, B. W., Davis, R. E., Casola, L., and Lim, R. (1967). Science 158, 1600. Altman, J. (1966). Protides Biol. Fluids, Proc. Colloq. 13, 127. Aprison, M .Ha, and Werman, R. (1968). In “Neurosciences Research” (S. Ehrenpreis and 0. C. Solnitzky, eds.), Vol. 1, p. 143. Academic Press, New York. Babich, F. R., Jacobson, A. L., Bubash, S., and Jacobson, A. (1965). Science 149, 656. Barondes, S . H., and Cohen, H. D. (1967). Commun. Behauwrd Biol. 1, 337. Barondes, S. H., and Cohen, H. D. (1968). Science 160, 556. Barondes, S. H., and Samson, F. E., Jr. (1967). Neurosci. Res. Program Bull. 5 , 307. Bateson, P. P. G., Horn, G., and Rose, S. P. R. (1969). Nature 223, 535. Beach, G,, and Kimble, D. P. (1967). Science 155, 698. Beach, G., Emmens, M., Kimble, D. P., and Lickey, M. (1969). Proc. Natl. A d . Sci. U. S. 62, 692. Bennett, E. L., Diamond, M. C., Krech, D., and Rosenzweig, M. R. (1964). Science 146, 810. Bernard, C. (1865). “Introduction Q l’6tude de la mkdecine exp4rimentale.” Baillibre et Fils, Paris. Best, R. M. (1968). Psychol. Rep. 22, 107. Bloom, F. E., and Aghajanian, G. K. (1968). J . Ultrastruct. Res. 22, 361. Bogoch, S. (1968). “The Biochemistry of Memory,” Oxford Univ. Press, London and New York. Booth, D. A. (1970). In “Molecular Mechanisms in Learning and Memory” (G. Ungar, ed.), p. 1. Plenum Press, New York.


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THE EFFECT OF INCREASED FUNCTIONAL ACTIVITY ON THE PROTEIN METABOLISM OF THE NERVOUS SYSTEM By B. Jakoubek and B. Semiginovsk9 Institute of Physiology, Czechoslovak Academy of Sciences, Prague, Czechoslovakia, and Institute of Pathophysiology, Medical Faculty of Charles University, Plzen, Czechoslovakia

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I. Introduction 11. Studies Performed at the Tissue Level A. Brain Energy Metabolism in Relation to Metabolism of Amino Acids during Excitation B. Breakdown and Synthesis of Nervous System Proteins . . . C. Changed Hormonal Balance in Alterations of Protein Metabolism 111. Protein and Nucleic Acid Metabolism in Neurons and Glial Cells . A. Methodological Approaches . . . . . . . . B. Quantitative Cytochemical Methods for Measuring RNA and Proteins . . . . . . . . . . . . C. Neuronal and Glial RNA D. Alteration of Neuronal and Glial Proteins and RNA during Stimulation E. Neuroglial Relationships during Increased Functional Activity . F. Axonal RNA and Proteins and Their Alteration during Stimulation IV. Conclusions References Note added in proof

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255 256 256 261 266 268 268 269 271 273 276 277 279 280 288

I. Introduction

The relationship beiween increased functional activity and the metabolism of protein and nucleic acids has been investigated for more than 60 years. At first cytochemical or biochemical methods were used, but rarely in combination. The experiments based on these methodological approaches had different aims and different limitations; this review will thus follow the two main lines of this area of research separately. The discrepancy of results, which is quite remarkable in the problem discussed here, is at least partially due to the different degrees of adequacy of the methods used. For this reason it was considered reasonable to stress the methodological approaches and to make clear the advantages and limitations particularly of those methods, which have been used in the analysis of protein and nucleic acid metabolism on a cellular level and which are not so commonly known. 255

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B. JAKOUBEK AND B. SEMIGINOVSKY

The term “increased functional activity” is so broad that some limitations are absolutely necessary. In this review the only results that are summarized are those in which the experimental conditions used were likely to increase the rate of firing of nerve cells or to enhance the propagation of action potentials along the nerve trunk. However, because these parameters have been directly measured only rarely, this criterion does not hold completely for most of the reviewed papers. The clearly pathological states (e.g., epileptic seizures) as well as the large body of evidence indicating the role of brain macromolecules in special functions of the brain (e.g., in memory mechanisms and sleep) have not been included. Even with these restrictions, it has been possible to include in this review only a proportion of the relevant literature. However, several excellent reviews (cited in the corresponding chapters of this review) are available, which deal mainly with the general aspects of protein and nucleic acid metabolism in the nervous tissue. II. Studies Performed at the Tissue Level

A. BRAINENERGY METABOLISM IN RELATIONTO METAB~LISM OF AMINOACIDSDURING EXCITATION The energy-producing reactions in the nerve cells depend on their level of functional activity, the intensity of which is determined by the AMP/ADP/ATP ratio (Shapot, 1957; Atkinson, 1966). On the other hand, the alteration of both energy metabolism and the metabolism of amino acids should be considered in the interpretation of experiments, as these demonstrate the altered kinetics of protein synthesis during the period of increased functional activity. The purpose of this chapter is thus to outline in a general way the main connections between the energy metabolism and the metabolism of amino acids. For exhaustive reviews of this topic the reader is referred to Coxon (1957), Geiger (1957), Strecker ( 1957), Tower ( 1959), Gaitonde et al., ( l W ) , and McIlwain (1966). 1. ATP, Glucose, and Amino Acid Metabolism in the Nervous System Increased functional activity is closely related to the energy metabolism in the nervous system. For example, seizures, induced either by various convulsant drugs or by electrical stimulation, are associated with increased neuronal activity and consequent increased energy expenditure ( McIlwain, 1966; King and Carl, 1969). The adenylate control hypothesis tries to provide an answer to the question of how the energy metabolism of the cell is regulated. A schematic illustration of the adenylate control

INCREASED FUNCTIONAL ACTIVITY AND PROTEIN METABOLISM

257

system, as applied to glycolysis and to the tricarboxylic acid cycle is depicted in Fig. 1. It is proposed that the balance of ATP/ADP/AMP concentration is a primary control factor. The diminished levels of phosphocreatine and ATP during increased functional activity are generally observed. However, negative results have also been reported ( FolbergrovL et al., 1969). Under similar conditions alterations of glucose and glycogen in the brain tissue are not unequivocal. An increase (Folbergrovi et al., 1969), a decrease (Sacktor et al., 1966; King et al., 1967), and no change

-

-- --_- - - - - - - - I A-

Glucose -1- P

Ii

\

I

Glycogen starch ’

-7Glucose-6-P)------”

-. II I I

I

FIG. 1. Schematic illustration of the adenylate control hypothesis as applied to glycolysis and the citric acid cycle (Atkinson, 1966) and the main relations between glucose and amino acids metabolism in the nervous system. Supply of electrons to the electron transport phosphorylation system is indicated by the symbol e-. Positive modifier action is indicated by an arrow, and negative modifier action by a crossmark ( X ) .

258


(Gershoff et al., 1949) of the concentrations of these substrates have been reported. Glucose metabolism is closely connected, via intermediary products of the tricarboxylic acid cycle ( a-ketoglutaric acid, oxalacetic acid, pyruvic acid, and succinic semialdehyde ) , with the synthesis of amino acids, namely of glutamic and aspartic acids, alanine, and 7-aminobutyric acid. As observed in isotopic experiments, the amount of glucose that is converted into amino acids is quite high. Bush (1W) demonstrated that 53% of the total 14C radioactivity, administered as 2 - ~ y r u v a t e - ~ ~ C remained in the glutamate fraction of the brain 3 minutes after the intravenous injection. A similar rapid labeling of amino acids in the brain taken from (U-"C)-glucose has been shown by Bush et al. ( 1960) and Vrba ( 1962). Beloff-Chain et al. (1955) found, after 1 hour incubation of brain slices with g l u c o ~ e - ~that ~ c , 56%of the total 14Cactivity was recovered in glutamate, 1% in GABA, 15%in aspartic acid, and 10%in alanine. Similar results were obtained by Tower (1960). Recently this problem has been attacked by Rose (1968a). The brain differs in this respect from other organs examined (Gaitonde et al., 1964). Hence, glutamate dehydrogenase ( EC 1.4.1.2), catalyzing the reaction L-glutamate

+ H20 + NAD+

or-ketoglutarate

+ NHI + NADH

(1)

occupies a key position. The presence of glutamate dehydrogenase in the brain was established 30 years ago (Dewan, 1938; von Euler et al., 1938).These investigators recognized the reversibility of the reactions catalyzed by this enzyme, but they emphasized its function in the synthesis of glutamic acid and in the metabolism of ammonia and amino acids group. This reaction sequence in uivo was more likely to be concerned with synthesis rather than with breakdown of the glutamic acid ( Weil-Malherbe, 1957), and it was concluded that glutamate dehydrogenase does not normally operate in uiuo in the direction of glutamic acid oxidation. Breakdown of glutamate as a potential substrate of energy metabolism is not realized in the pathway of direct oxidation. The activity of glutamate dehydrogenase was considerably increased at all stages of preconvulsive excitation and, to the same extent, during the convulsive phase of experimental cerebral seizures ( Wiechert and Gollnitz, 1969a). 2. Transamination The other potential source of glutamic acid is transamination reactions.

INCREASED FUNCTIONAL

ACTIVITY AND PROTEIN METABOLISM

259

Glutamic oxalacetic transaminase ( aspartate aminotransferase, EC 2.6.1.1.) which catalyzes the reaction sequence baspartate

+ a-ketoglutarate + tglutamate + oxalacetate

(2)

is active in the brain (Cohen and Hekhuis, 1941). Transamination between glutamic acid and pyruvic acid proceeds at only about onehundreth OE the rate found with oxalacetic acid. In the brain, reactions between aspartic and pyruvic acids are more active than between glutamic and pyruvic acids ( McIlwain, 1959). A less usual transamination occurs in cerebral tissue between aketoglutarate and y-aminobutyric acid with the formation of succinic semialdehyde (Bessman et al., 1953). This reaction sequence is catalyzed by 7-aminobutyric acid aminotransferase y-aminobut.yric acid

+ a-ketoglutarate

tglutamate

+ succinic semialdehyde

(3)

This transamination reaction is considered to be the major pathway of y-aminobutyric acid breakdown. The variations in the activity of transaminases, which insure the transfer of the amino group from glutamic acid to different a-keto acids during electrocorticographically controlled “spike” activity, were studied by Constantinescu and Steriade ( 1966). Focal strychnine spikes or eserine induced low voltage and fast rhythm are associated with diffuse decrease in the activity of aspartate and alanine aminotransferase ( alanine aminotransferase EC 2.6.1.2) in the neocortex. However, these findings were only partially supported by similar studies of Wiechert and Gollnitz (1969a). When conditions require that glutamate be oxidized it seems reasonable to suggest that a-ketoglutarate is formed from glutamate by the following series of reactions (Strecker, 1957).

+

+

>glutamate oxalacetate L-aspartate a-ketoglutarate a-ketoglutarate i 0 2 + oxalacetate COZ HzO

+

+

+

(4)

(5)

..........................................................................................................

I.-glutamate

+ 4 02 -+

taspartate

+ COZ+ HzO

(6)

Quastel and Wheatley (1932) and Krebs (1935a) have stated that L-glutamic acid is the only amino acid capable of maintaining the respiration of the brain and retina; in view of this fact and of the role of glutamic acid in transamination reactions, it may be concluded that amino acids may abstract potential energy from the tricarboxylic acid cycle and return it if necessary. This means that in normal living conditions a considerable amount of glucose is converted into amino acidsunits of structural metabolism; on the other hand, in the period of

260


increased energetic requirement, the amino acids may be broken down for the use of energy metabolism. Glutamic acid, which is situated at a metabolic crossroads between glucose and energy metabolism on one hand and the metabolism of nitrogen, amino acids, and proteins on the other, plays a unique role in this process.

3. Ammonia Formation during Zncreased Functional Activity Cerebral ammonia undergoes rapid changes with the changes of the cerebral activity. Tashiro ( 1922) first observed the increased production of ammonia following electrical stimulation of peripheral nerve in vitro. Later it was found that many stimuli (mechanical, photical, chemical, thermal) have the same effect (Winterstein and Hirschberg, 1925; Gerard and Meyerhoff, 1927; Pravdicz-Neminskij, 1932; Halter, 1933; Richter and Dawson, 1948). The “resting” value of ammonia is 0.28 mg/100 gm of brain tissue. After 1second of electrical stimulation, or 1second following the decapitation, the level was almost doubled (McIlwain, 1959; Folbergrod et al., 1969). Conditioned stimuli are also capable of increasing the ammonia level (Vladimirova, 1957). The main sources of ammonia are (1) glutamine and adenylic acid ( adenosine-5-phosphate) ( Muntz, 1953; Kleijn, 1957); ( 2 ) deamination and further degradation of proteins, deamination being accelerated both in the central (Vrba, 1955) and in the peripheral nervous systems (Jakoubek et al., 1963.a) by intensive motor activity; ( 3) the dehydrogenation of glutamic acid, which seems to be responsible for a minor fraction of total ammonia production. The main route of removal of ammonia is the formation of glutamic acid, with glutamate dehydrogenase catalyzing the binding of toxic ammonia (for the reaction sequence, see above). Another important enzyme for the removal of ammonia is glutamine synthetase [EC 6.3.1.2. L-glutamate: ammonia ligase (ADP)I, which catalyzes the reaction L-glutamate

+ NH, + ATP

$ L-glutamine

+ ADP +

Pi

(7)

The high concentration of brain glutamate and the fact that the adenosine polyphosphate system in the brain is almost entirely present as ATP (JSratzing and Narayaswami, 1953) strongly favors the forward reaction. Wiechert and Gollnitz (1969b) did not find changes in the activity of this enzyme during experimental brain seizures, but the activity of glutaminase ( EC 3.5.1.2. L-glutamine aminohydrolase), which mediated the reaction L-glutamine

+ HsO -+

L-glutamate

+ NHI

(8)


261

was substantially enhanced. Its properties were recently tested by WeilMalherbe ( 1969). It is generally accepted that glutamine level increases during the excitation process in the brain. Glutamine synthesis is an energy-requiring reaction ( Krebs, 1935b). Glutamine has been considered as a possible transport form of glutamic acid across the bloodbrain barrier (Meister, 1956), but the deamination of endogenous glutamine to glutamic acid does not appear to occur normally to any measurable extent in in vivo conditions. On the other hand, exogenously supplied glutamine appears to be readily metabolized (Tower, 1959). The studies of Vrba (1955) suggest that during cerebral activity proteinbound glutamine may be liberated and then resynthesized during recovery. The general question of whether the ammonia as a by-product only accompanies the excitation process in the nervous system, or whether it has a special biological role has not yet been answered.

B. BREAKDOWN AND SYNTHESIS OF NERVOUS SYSTEMPROTEINS 1. Central Nervous System The following reviews bear a close relation to the topic discussed in this chapter: Gerard (1932, 1955); McIlwain (1959); Waelsch (1957, 1962); Richter (1959, 1965); Schmidt (1956); Geiger (1958); Rossiter ( 1955); Waelsch and Lajtha (1960, 1961); Palladin (1962), Coxon ( 1963); L,ajtha ( 1964a,b); Davison (1967); Lodin and Rose (1968); Schmitt and Samson ( 1968); Droz ( 1969). The first experiments in this field were performed at the beginning of this century, when the relation between different functional states and the rate of proteolysis was investigated (Faure and Soula, 1913; Hirschberg and Winterstein, 1919). Gerard (1932) summarized the evidence suggesting that during nervous activity substrates other than carbohydrates are utiIized. Direct evidence for the cleavage of nitrogenous components from brain lipids, nucleic acids, and proteins due to the stimulation of sensorimotor cortex was obtained by Geiger ( 1958) and Geiger et al. (1956, 1960). Similar conclusions were drawn from the experiments of Vrba and Folbergrovd (1959), where the effect of intensive muscular activity on the protein and nucleic metabolism was investigated. Because the existence of a renewal of at least a great majority of proteins in the nervous system during the life span of an organism is generally accepted, many authors (see Table I ) have studied the effects of increased (or decreased) functional load on the protein turnover in

262


TABLE I EFFECT OF

STIMULI AND CHANGES O F INCORPORATION O F INTO PROTEINS OF

CENTR.412 NERVOUS

PRECURSORS

SYSTEM ~~

Stimnlus

Precursor

Motor activity

Methionine Methionine

Fatigue Light stimulation

Methionine Valine Lysine

Spreading depression

Leucine

Electrical stimulation

Methionine Proline Methionine

Eff ecta

+-

+

+.

Biphasic response 0

-

+ +

Methionine and glycine 32P

Pharmacologicnl Narcotics

Excitants

Hypnotics

~~

Reference Krawcxvnski (1961) Jakohek and Gutmann (1969 Shapot (1957) Talwar et al. (1966) Rose (196713. 1968b) Bennett and Edelman (1969) RnEIE8k (1961) Dingman et al. (1959) Gaitonde and Richter (1956) Vladimirov and Urinson (1957) Heald (1960)

Methionine

-

Glycine

-

Methionine Methionine Methionine Methionine

-

++

Gaitonde and Richter (1956) Vladimirov and Urinson (1957) Vladimirov (1958) Palladin et al. (1957) Shapot (1957) Zacharov and Orljanskaja

Methionine and glycine

-

Nechaeva et a2. (1957)

(1960)

+

a The symbols are : increased incorporation of precursor observed; - : decreased incorporation of precursor observed; 0: no change of incorporation observed.

the central nervous system, However, the interpretation of these results is complicated by several factors. The relationship between the rate of incorporation of amino acids and the rate of the synthesis of proteins can according to Schultze ( 1968), be expressed as s.a. Prot = a

II,"

s.a. AA

where s.a. Prot is the specific activity of proteins, s.a. AA is the specific activity of intracellular amino acids, tl is the time of the application of labeled amino acids, t2 is the time the animal was killed, a is the velocity


263

constant. The integral value of the specific activity of the labeled amino acid ( s ) has rarely been calculated because of methodological difficulties encountered particularly in experiments in duo. Instead, only one measurement of the specific activity of amino acid( s ) is usually performed (e.g., at t 2 ) .This involves a presumption that the s.a. AA, as measured (e.g., at t z ) is the representative value also for the time interval tl - tz. However, this assumption is not necessarily valid, because the changes of blood circulation in brain induced by stimulation (see e.g., Sokoloff, 1960; Meyer et al., 1966) are not of the same nature at the beginning and at the end of stimulation (Travis and Clark, 1965). It follows that even if, for example, the specific activities of both TCAsoluble and insoluble fractions, as determined at the end of the experiments, are the same in control and experimental animals, the rate of the protein synthesizing process may not be necessarily the same. The converse is also true. The difficulties arising from the different time course of specific activity of pool may be avoided if the descending part of the radioactivity-time curve is used, providing of course that no significant reutilization of the products takes place. A further limitation in the interpretation is the considerable heterogeneity of turnover rates of CNS proteins, as shown both by incorporation studies (Lajtha and Furst, 1957; Furst et al., 1958; Clouet and Richter, 1958/1959; Gaitonde, 1961) and experiments investigating the decay of radioactivity of labeled proteins (Piha et al., 1966a,b; Jakoubek et al., 1968a). The average half-life of 90%of cerebral proteins is 10-20 days (Lajtha, 1964a; Lajtha and Toth, 1966) as measured in proteins extracted from the whole brain. The half -life of proteins of nervous subcellular organelles is roughly in the same range (von Hungen et al., 1968).Proteolipids (Furst et al., 1958; Mokrash and Manner, 1963) and histones (Piha et d.,1966a) possess substantially longer half-lives. On the other end of the scale, there are some proteins with half-lives of seconds ( Clouet and Richter, 1958/1959). Besides the possibilities of different patterns of protein metabolism in neurons and glia (Koenig, 1958), this wide range of turnover rates is caused by several extrinsic factors (see Lajtha, 1964a). However the heterogeneity of turnover rates was observed also with spinal motor neurons of rats of the same age, using the method of direct counting of radioactivity from nerve cells isolated free hand (Jakoubek and Gutmann, 1968; Jakoubek et al., 1968a). Because of this large spectrum of turnover rates, the time interval tl-tz determines which part of the spectrum of turnover rates is measured. This seems to be important with regard to the possible relationship between the increased functional activity and the turnover of neuronal protein,

264


since the results of any experiment, in which a single time interval between injection and killing the animals was used, can be properly applied only to a certain fraction of the protein spectrum of the nervous tissue; the generalization of either positive or negative results may be hazardous. The possibility that other mechanisms (e.g., hormonal) also cooperate in the changed kinetics of protein synthesis during the increased functional activity is discussed in Section 11, C. The interference of these factors was ruled out in experiments in uitro, in which, after electrical stimulation of brain slices, both RNA biosynthesis (Orrego, 1967) and the synthesis of protein from g l u c o ~ e - ~(Orrego ~C and Lipmann, 1967) was found to be inhibited. However, even when the limitations mentioned above are taken into account, the experimental evidence (see Table I and Section 111, D ) indicates that in many cases there is a close relationship between the increased functional activity and the altered protein metabolism, even if the underlying mechanisms are not clear. One of the possible mechanisms can be the restricted supply of energy for protein and nucleic acid synthesis during and after the period of increased functional demands, since the reestablishment of ionic gradients is of primary importance ( McIlwain, 1959; Richter, 1959, 1965). The proportionality existing between the rate of RNA synthesis and ATP concentration in brain slices suggests that the cell content of available ATP may be actually the regulating factor (Orrego, 1967; Itoh and Quastel, 1969), especially since the continual formation of ATP for optimal amino acid incorporation by microsomal and ribosomal preparations was found to be more critical in cerebral cortex than in analogous liver systems (Zomzely et al., 1964). The unusual sensitivity of brain polysomes to variations in ion concentrations, especially in Na', K+, and Mg+, may be closely related to the passage of neuronal impulses, which are accompanied by ionic variations (Zomzely et al., 1968). The stimulation-induced changes of dynamic equilibrium between the polysome (as the most active protein synthesizing units) and ribosomes in the nervous system were described ( Vesco and Giuditta, 1968; Dellweg et al., 1963). Diminution of polysomes associated with environmental deprivation and their increase, associated with stimulation, indicated the participation of some control mechanisms at the translational level. The availability of mRNA was suggested to be the rate-limiting factor for protein synthesis in the brain both in vivo and in uitro experiments ( Appel et al., 1967). A close relation between the metabolism of phosphoproteins and stimulation was found (see Heald, 1960; Rodnight, 19f35). The specific activity of 32Pin phosphorylserine (the single phosphorylated amino acid


265

present in hydrolysate of brain phosphoproteins ) was observed to increase after stimulation (Heald, 1957, 1958). The increased turnover of 32Pis associated with changes and metabolism of potassium ions, phosphocreatine, ATP, and GTP (Heald and Stancer, 1962). The sensitive phosphoprotein is localized mainly in structures sedinienting with microsoma1 fractions (Trevor et al., 1965). The rapid turnover of 3zPin phosphoprotein seems to be connected with the maintenance and restoration of ionic gradients in stimulated tissue (Heald, 1961). 2. Peripheral Nervous System The participation of axoplasmic proteins in the generation and propagation of nerve impulses was suggested by Segal (1958); Nasonov (1959); and Ungar (1962). (For the role of axonal membrane proteins see Section 111, F.) Methodological approaches to these questions included both the investigation of protein metabolism in whole nerves (reviewed here) and studies of single axons (see Section 111, F). Changes in the ultraviolet absorption spectrum of proteins extracted from stimulated nerves (Ungar et al., 1957; Luxoro, 1959) and the changes of fluorescence of nerve fibers during stimulation (Ungar and Romano, 1962) were interpreted as stimulation-induced changes of protein configuration ( Ungar, 1962). This special kind of denaturation (reversible only at the beginning of the stimulation) might be connected with increased proteolytic activity of the peripheral nerve, observed after electrical stimulation (Ungar et al., 1957) or long-lasting muscular effort (Jakoubek et al., 1968b; Jakoubek and Gutmann, 1968). VodiEka (1960) described the decrease of total N and P in the sciatic nerve of rats after intensive muscular activity. Analysis of the changes of protein metabolism in sciatic nerves of rats under similar conditions showed a decrease of protein concentration, which can be partially explained by the increased deamination, by the increased activity of neutral proteinase and enzyme ( s ) splitting the leucyl-p-nitroanilide substrate, and by the inhibition of protein synthesis in sciatic nerves (Jakoubek et aZ., 1963a,b, 1968b). However, the investigation of the last mechanism is complicated by the concomitant changes of blood supply, which occur in the peripheral nerves during intensive muscular activity (Jakoubek et al., 1963a,b). Because xylocaine blockade of impulse propagation prevents the described decrease of protein concentration in the sciatic nerve (Jakoubek et al., 1%5),it is possible to suppose that the alteration of protein metabolism in peripheral nerve mentioned above has apparently some relation to impulse propagation along the nerve trunk.

266


The acceleration of amino acid utilization during excitation of frog nerve was observed by Mullins (1953). The increased turnover of proteins in the sciatic nerve of frog during stimulation in vitro was shown by Luxoro ( 1960). The reported experiments performed on desheathed axons do not allow us to localize the alteration of protein metabolism; rapid changes of the incorporation rate of labeled amino acids during stimulation are apparently too quick to be attributable to the autochthonous proteosynthetic machinery in axons (see Section 111, F). Similarly the enzyme( s ) splitting the leucyl-p-nitroanilide substrates are restricted mainly to the Schwann cells (Adams, 1968). The results of Causey and Stratmann ( 1956), dealing with the effect of stimulation on changes of nucleic acid metabolism in the peripheral nerve, can be explained only if the participation of nonaxonal constituents of the peripheral nerve is taken into account, For these reasons, even if the experimental evidence is very indirect, the possibility that increased propagation of nerve impulses modifies protein metabolism, especially in Schwann cells, cannot be neglected.

C. CHANGED HORMONAL BALANCE IN ALTERATIONS OF PROTEIN METABOLISM Review of experiments which investigated the effect of various stimuli applied in vivo to protein and nucleic acid metabolism (see Sections 11, B and 111, D ) indicates that the great majority of these stimuli can perform-besides their specific action-as stressors, which are known to increase protein catabolism. The possibility that the corticosteroids, which readily penetrate into the brain tissue (Peterson and Chaikoff, 1963), play a similar role in regulation of protein and nucleic acid metabolism in nervous tissue as in other organs (Hechter and Halkerston, 1965) cannot be excluded. Papers investigating the effects of hormones on protein or nucleic acid metabolism of the brain were reviewed by Lajtha (1964a,b), but only few studies dealt with the effect of corticosteroids (Piha et al., 196613) or catecholamines (Pevzner, 1965; Cambell et aE., 1966) on the metabolism of brain macromolecules. According to the author's opinion, the participation of corticosteroids in stimulation-induced changes of protein synthesis is highly probable. This assumption is based on experiments in which the synthesis of proteins in cortical brain slices, prepared from ACTH-treated animals, was investigated (see Table 11). The marked inhibition of incorporation of leucine-l'c into proteins was observed ( Jakoubek and Semiginovskk, 1970). Similar inhibition of in vitro utilization of labeled amino acids

CHANGES

IN

UPTAKE

TABLE I1 DL-LEUCINE-14c I N T O CORTICAL SLICES, P R E P A R E D FROM RATS SUBJECTED STRESS,ANTICIPATION STRESS,OR PREVIOUS TREATMENT WITH ACTHa OF

TO

RESTRAINT ~

~~

Uptake of leucine-14Cb Experimental group Restraint stress control stressed Anticipation stress control stressed ACTH control experimental

Plasma level of corticosteroids

1 5 . 3 zk 5 . 5 88.3 f 7.6 15.8 f 2 85.2 9

*

Significance

p p

<
100 mm/day 250-350 mm/day

400-500 mm/day

Author Droz and Leblond (1963) Lasek (1968b)

Bray and Austin (1968, 1969) ; Peterson et al. (1968) Ochs and Johnson (1969)

> 10 mm/day 1 . 3 mm/day

1 mm/day

500 mm/day

Lasek (1968a)

410 mm/day

Ochs et al. (1969)

Taylor and Weiss (1965)

Goldfish retinal ganglion cells

Protein

Rabbit retinal gangiion ceils

Protein

Monkey retinal ganglion cells Cat sympathetic ganglion cells Toad olfactory ganglion cells Cat ulnar nerve

Protein Protein Protein Protein

Hypoglossal motor Protein neurons Guinea pig sciatic Phosphoprotein nerve Rabbit vagus motor Phospholipids neurons Rabbit hypoglossal motor neurons

Phospholipids

Rabbit vagus and hypoglossal motor neurons Chicken anterior horn cells

RNAa

Orotic acid, RNA.

Lei~cine-~H injected into eye; radioautography and radioisotope counting Lei~cine-~H injected into eye; radioautography arid radioisotope counting Le~icine-~H injected into eye; radioautography L e u ~ i n e - ~injected H into coeliac ganglion; radioisotope counting L e ~ c i n e - ~administered H to olfactory epithelium Methionina 35Sand g l y ~ i n e - ~in H jected int,o the subarachnoid space; radioisotope counting Lysine3H administered to floor of fourth ventricle; radioautography Orthophosphate-32P injected systemically; radioisotope counting Orthophosphate32P administered t o floor of fourth ventricle; radioisotope counting 0rthophosphatge-32Padministered to floor of fourth ventricle; radioisotope counting Orthophosphate-J2P administered to floor of fourth ventricle Orotic a ~ i d - ~injected H into lumbar spinal cord

> 10 mm/day

Grafstein (1967); McEwen and Grafstein (1968) Karlsson and Sjosirand (1968); Sjostrand and Karlsson (1969) Hendrickson (1969)

120 mm/day

Livett et al. (1968)

0 . 4 mm/day

40 mm/day

1.5-2 mmjday

110-150 mmjday

1 mm/day 1 mm/day

Weiss and Holland

6 5 mm/day

(1967) Koenig (1958)

20 i4 9

E% 4

Korr et al. (1967)

3 mm/day

Samuels et al. (1951)

>72 mm/day

Miani (1963)

>40 mm/day

Miani (1963)

Miani et al. (1966)

Bray and Austin (1968); Peterson et al. (1968)

0 3

e

2

TABLE I1 (Continued) Velocity of transport ~~

Species and neuronal system

Axoplasmic component

Experimental evidence

~

Slow component

Fast component

Author

~~~~~

R a t retinal ganglion cells Cat sympathetic ganglion cells

5 hydroxytryptophan" Norepinephrine"

Rat sympathetic ganglion cells

Norepinephrine

Cat sympathetic ganglion cells

Nor-

Rat caudate nucleus and septum neurons Snail neurons

Dopaminee

Frog motor neurons

Glutamate

Dog sympathetic ganglion cells

Dopamine-phydroxylase

epinephrine

Glutamate

Hydroxytryptophan-3H injected into eye; radioautography Norepinephrine-3H injected into coeliac ganglion; radioisotope counting Norepinephrine accumulated in axons proximal to a constriction in the sciatic nerve Norepinephrine accumulated in axons proximal to a constriction in the sciatic nerve Dopamine injected into caudate nucleus and septum; traced movement by histochemical fluorescence Snail ganglion bathed in glutamate-'*C and ganglion stimulated electrically; glutamate 14Cmeasured in muscle perfusate Spinal cord bathed in glutamate-'*C and electrically stimulated; radioactivity measured in muscle perfusate Dopamine-p-hydroxylase activity increased in splenic nerve proximal t o a ligature

120 mm/day

O'Steen and Vaughn (1968) Livett et al. (1968)

120 mm/day

Dahlstrom (1965)

240 mm/day

Dahlstrom (1965)

?

3 'r'

Routtenberg et al. (1968)

720 mm/day

Kerkut et al. (1967)

120 mm/day

Kerkut et al. (1967)

72 mm/day

Laduron and Belpaire (1968)

r

5

R

Number of different Acetylcholinesvertebrates terase, choline acetylase oxidative enzymes Goldfish neurons of Neurosecretory preoptic nucleus granules Dog hypothalamic Vasopressin neurons which innervate the posterior pituitary Neurosecretory cells Neurosecretory in a number of granules different animals R a t sciatic nerve

Mitochondria

R a t sciatic nerve

Mitochondria

R a t neurons

Mitocondrial proteins and soluble proteins Chick embryo dorsal Axoplasmic root ganglion cells organelles

Increased enzyme act,ivit,yin axons proximal to constriction or cut in peripheral nerves

Depletion of secretion granules in less than minute after stimulating the neurons Methi0nine-3~Sinjected systemically; specific activity of vasopressin in hypothalamus and pituitary compared Histological demonstration that neurosecretory material accumulates in axons proximal to a cut in the axon and is dep!eted distally Mitochondria accumulated proximal to a constriction of the sciatic nerve Mitochondria accumulated in axons a t both ends of a n isolated segment and were reduced in the middle of the segment Leucine-14C injected into brain; labeling of synaptosomal mitochondria lagged behind total brain mitochondria Neurons stimulated with nerve growth factor in vitro were observed by cinemicrophotography

Reviewed by Lubihska (1964)

2000 mm/day

Jasinski et al. (1967)

much faster than 1 mm/ day

Sachs (1960)

Reviewed by Bargrnan (1966)

1 mm/day much faster than 1 mm/ day

Weiss and Pillai (1965) Zelenh et al. (1968)

Barondes (1966, 1968a)

8.4-12 p/sec (700-1000 mm/day)

Burdwood (1965)

TABLE TI (Continued) Velocity of transport Species and neuronal system

Axoplasmic component

Experimental evidence

Chick embryo spinal Mitochondria cord

Neurons were observed by microcinemaphotography

Chick embryo dorsal Pinocytotic root ganglion vesicles Goosefish h y p e Neurosecretory tha.lamus neurons granules

Pinocytotic vesicles were observed ascending axons in vitro Direct observation of excised goosefish pituitary stalk

The component which was transported in the axon was not identified.

Slow component

Fast component 0.1-2 p/sec (7-140 mm/ day) 0.02-0.2 p/sec 1.7-3.4 p/sec (144-288 mm/ day)

Author

?+4

Pornerat et aZ. (1967)

? Hughes (1953); Nakai (1956) Carlisle (1959)

+i

ti

R

305

PROTEIN TRANSPORT IN NEURONS 0

40

00

120

160 2 0 0 240

80

40

0

120

20 Days

10 Doys

.. P c

.

e

* 9

0

40

00

0

40

00

1

..

4

5 I

120 160 200 240 Distance from cell body Imm) 120

.: ...'* .

c

N=5

C

160 2 0 0 240

160 200 240

6

N=6 1007

I

I

1

.

, > , 120 160 2 0 0 240 Distance from cell body (mm) 1

,

v

I

1

I

0

40

80

0

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FIG.2. See legend of Fig. 1 for details. Figure taken from Lasek (1968a).

horn cells (Bray and Austin, 1969). Measurements of the rate at which the front moves along the axon have produced the most accurate estimates of the velocity of the fast component of axonal transport [i.e., 250350 mm/day in the chicken anterior horn cells (Bray and Austin, 1969) and 410 mm/day in cat dorsal root ganglion cells (Ochs et al., 1969)]. Other estimates based on the distribution of labeled proteins along the axon at intervals of 12 hoursrl day after injection are less accurate but consistent with those based on the rapidly moving front (Table 11). The data summarized in Table I1 indicate that proteins and other axonal components are transported in the axon at velocities of from 100 to 500 mm/day. The variation in the velocities of fast axonal transport which have been esti-

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mated is in part due to the different methods used to calculate the transport velocities. However, some variation in results may be a result of species differences and differences in the neurons analyzed.

2. Slow Component Another component of labeled proteins has been identified which traverses the axon somatofugally at a velocity of approximately 1 mm/ day. This slow component is evidenced in radioisotopic studies of protein transport as a peak of labeled proteins. Examples of this migrating peak are illustrated in Figs. 2 and 3. This peak moves along the axons at approximately 1 mm/day and has been demonstrated in a large number

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FIG.3. L e ~ c i n e - ~was H injected into the lumbar spinal cord of adult rats and the rats were sacrificed at 20, 30, and 40 days after injection. The 'H content of 5-mm segments of lumbar-4 and lumbar-5 ventral roots and the sciatic nerve was plotted against the distance of the segments from the spinal cord. The figure illustrates the proximo-distal shift of a peak of labeled proteins along the axons of the nerve at a velocity of 1 mm/day. This figure was compiled from the data of Lasek (1968b).

PROTEIN TRANSPORT IN NEURONS

307

of neurons (Table 11). Taylor and Weiss (1965) have suggested that the slow component is comparable to the slowly moving column of axoplasm which was found to move at 1 mm/day by Weiss and Hiscoe (1%8). It should be noted that in studies of the transport of RNA in the axon, no peak of labeled RNA has been found which corresponds to the slow component of axonal protein transport ( Bray and Austin, 1968). This may indicate that the slow component does not contain RNA. 3. lntermediate Components The presence of a fast and slow component of axonal protein transport is well documented. It has been suggested that intermediate velocities of axonal transport also occur in the axon (Lasek, 1968a,b). In the cat dorsal root ganglion cells, a large amount of the labeled proteins appear to move somatofugally within the axon at velocities well in excess of 1 mm/day but considerably less than several hundred mmlday. Between 1 and 10 days following the injection of Ie~cine-~H, the distributions of labeled proteins in the dorsal root ganglion cell axons changes substantially (Figs. 1 and 2 ) . As the injection-sacrificeinterval increased from 1 to 1.0 days the amount of radioactivity in the distal segments gradually increased suggesting that a proximo-distal shift of the labeled proteins occurred within the axons. The velocity or velocities of this proximo-distal shift cannot be well defined but range from about 5 mml day to 50 mm/day. This proximo-distal shift at intermediate velocities is best demonstrated in long axons such as those of the cat lumbar ganglion cells (Lasek, 1968a). The suggestion that certain components may be transported in the axon at a velocity which is intermediate between the fast and slow component does not necessarily imply that these proteins are continuously moving along the axon at one velocity. The intermediate component of transported axoplasmic constituents may represent net somatofugal movement at 5-50 mmlday; that is, the intermediate component is possibly an average velocity resulting both from the exchange of rapidly transported components along the axon with stationary components (Miani, 1963) and the retrograde transport of components which are rapidly transported somatofugally within the axon and subsequently back toward the cell body (Section 111, D ) . Other factors beside intermediate transport velocities could explain the proximo-distal shift of radioactivity found in the axon, and it will be necessary to characterize the proteins which are transported in the axon in order to determine whether the proteins of the intermediate component are different from the fast or slow component.

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The evidence indicates that two distinct velocities of axonal transport occur in the axon: ( a ) a fast component with a velocity ranging between 100 and 500 mm/day and ( b ) a slow component with a velocity of approximately 1 mmlday. Intermediate velocities may also be present in the axon. The intermediate component which is probably the resultant of several different factors influencing the net transport of the fast component has not been characterized.

C. THE COMPONENTS TRANSPORTED IN THE AXON When the slow-moving component of axonal transport is pulse labeled, it appears as a distinct peak of labeled proteins which slowly migrate along the axons. This peak does not appear to diminish or flatten out even after periods as long as 60-80 days (Lasek, l W a , b ) . Therefore, the proteins of the slow-moving component appear to have a very long half-life, at least in neurons with long axons. The fact that the labeled peak does not flatten out may indicate that the proteins of the slow component are bound to a relatively stationary part of the axoplasm, possibly the matrix of the axoplasm. It has been suggested that the neurofilaments and neurotubules are part of the slow component (Schmitt, 1968; McEwen and Grafstein, 1968). On the basis of the morphology of the axoplasm, the linearly oriented filaments and tubules are likely candidates as the fixed structures of the axoplasm. Electron microscope radioautographic studies indicate that the slowly migrating proteins may be associated with the neurotubules and neurofilaments ( Droz, 196513). Ochs et al. (1967) have found that the labeled proteins of the axons in the ventral root at 14 days after injecting I e ~ c i n e - ~include H soluble and particulate proteins in a ratio of about 3: 1. By selecting only the ventral roots, Ochs et al. (1967) reduced the relative amount of contamination from long-lived rapidly transported proteins. Much lower ratios of soluble to particulate proteins have been found when whole nerves were analyzed at intervals of 12-44 days after injection ( McEwen and Grafstein, 1968; Sjostrand and Karlsson, 1M9; Bray and Austin, 1969). The presence of a substantial amount of soluble proteins in the slow component is consistent with the possibility that the neurotubules and neurofilaments contribute to the slow component. McEwen and Grafstein (1968) have noted that under the conditions used to fractionate nerves the neurotubule subunit is presumably solubilized. While only proteins have definitely been identified in the slow component, a number of axonal constituents have been found to be rapidly transported in the axon (Table 11).The fast component of axonal transport is thought to move within the axoplasmic matrix (Miani, 1963; Taylor and Weiss, 1965), and it is tempting to suggest that the mito-


309

chondria and other membrane-bound organelles are included in the fast component. This possibility is supported by direct observations on living axons in oitm (Carlisle, 1959), which indicate that mitochondria and neurosecretory granules move at velocities of several microns per second. If these velocities are extrapolated from y/sec to mm/day the resulting velocities are on the order of several hundred mm/day, which is comparable to the fast component (Table 11). Studies on subcellular fractions of nerve indicate that the particulate constituents of the axoplasm are enriched with rapidly transported proteins ( McEwen and Grafstein, 1968; Sjostrand and Karlsson, 1969; Ochs et al., 1969). Weiss and Pillai ( 1965) have suggested that mitochondria are transported somatofugally at 1 mm/day. However, the studies of ZelenB et al. (1968; Zelenb, 1968) indicate that mitochondria are transported within the axon at velocities far in excess of 1 mm/day. Labeled mitochondria1 proteins appear to be transported from the soma via the axon into the axon terminals ( Barondes, 1966, 1968). Evidence has accumulated which indicates that catecholamine granules of sympathetic nerves are transported from the cell body toward the axon terminals at velocities of 250 mm/ day ( Dahlstrom and Haggendal, 1966). The fast component of axonal transport also consists in part of soluble proteins and possibly polypeptides ( Kidwai and Ochs, 1969). Evidence suggests that glutamate appears to be transported somatofugally at velocities up to 900 mmlday in snail neurons (Kerkut et aE., 1967). It is not clear whether the glutamate is transported as a free molecule or bound to a carrier protein. Studies on vasopressin indicate that this octapeptide is synthesized in the soma and transported somatofugally via the axons of the hypothalamo-hypophysial tract into the posterior pituitary at velocities far in excess of 1mm/day (Sachs, 1960). Vasopressin is apparently bound to a carrier protein in the axons and may be contained within the neurosecretory granules ( Sachs, 1967). The wide array of components which appear to be transported within the axon has complicated attempts to differentiate between axonal constituents which are transported in the slow component or in the fast component. However, the evidence supports the postulate that the slow component of transport represents the axoplasmic matrix and that the neurofilaments and neurotubules contribute substantially to this matrix. To date, only proteins have been identified in the slow component. For example, RNA does not appear to be included in the slow component. The fastest component of axoplasmic transport appears to contain a considerable amount of particulate material. This finding coupled with other evidence indicates that mitochondria and possibly membranebound vesicles are transported rapidly in the axon. Neurosecretory pro-

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teins, polypeptides, and small molecules such as amino acids may also be rapidly transported in the axon. It is not clear whether these lower molecular weight components are bound to carrier molecules during transit within the axon.

D. BIDIRECTIONAL AXONALTRANSPORT The possibility that certain axoplasmic components are transported somatofugally and also in the retrograde direction (from the axon terminal toward the neuron soma) could potentially be of great importance in understanding the mechanics of neuronal function. The evidence presented above conclusively demonstrates somatofugal axonal transport, but the study of retrograde axonal transport has, unfortunately, been the subject of much less investigation. If a nerve is severed at two places several centimeters apart, the following axoplasmic components accumulated in the axons at both ends of the isolated segment while they are concomitantly reduced in the middle of the segment: norepinephrine ( Dahlstrom, 1965, lam), mitochondria ( Blumcke et al., 1966; Kapeller and Mayor, 1967b), AChase (Lubihska, 1964; Kasa, 19%; SkangielKramska et al., 1969; Zeleni et al., 1968), labeled axonal proteins (Lasek, 1967), and labeled phospholipids ( Miani, 1M).One explanation for this redistribution of axoplasmic components in an isolated segment is that the components are transported in the axons from the middle of the segment toward the ends of the isolated segment. In light of the evidence supporting axonal transport, it seems reasonable to suggest that the piling up of axoplasmic components represents an interruption of axonal transport ( Lubihska, 1964). However, it has also been suggested that the redistribution of axonal components in the isolated segment is due entirely to traumatic changes in the axon (Schlote, 1966). Friede ( 1%4a,b) has presented evidence that the accumulation of organelles in severed axons results from microelectrophoresis within the axon. An example of an experiment in which the accumulation of labeled proteins was demonstrated proximal and distal to the point of section is illustrated in Fig. 4. L e ~ c i n e - ~ was H injected into the lumbar-7 dorsal root ganglion of adult cats and the sciatic nerve severed in two places at 6 days after the injection of isotope, Twenty-four hours after the nerve was severed, labeled proteins accumulated on both sides of the points of section. The labeled proteins were found to be localized within the axons by radioautography ( Lasek, 1967). Translocation of the labeled proteins must have occurred over a distance of many millimeters because the segments adjacent to points iGd in Fig. 4 did not show a decrease in radioactivity which could account for the accumulation in segments a-d. The accumulation of catecholamines in axons on both sides of a con-

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D i s t a n c e from C e l l Body (mrn) FIG.4. Leucine-’H was injected into the lumbar-7 dorsal root ganglia of adult cats. The sciatic nerve of adult cats was severed in two places approximately 60 to 110 mm from the lumbar-7 dorsal root ganglion at 6 days after the injection. The cats were sacrificed 1 day after severing the nerves and 5-mm segments were prepared as described in Fig. 1. Segments a, b, c, and d are directly adjacent to the points where the nerve was severed. The triangles represent control values of homologous segments from preparations in which the nerves were not severed, and the animals were sacrificed 6 days after injection (Fig. 1). This figure was taken from Lasek (1967).

striction in rat and cat sciatic nerves has been reported by Dahlstrom (1965, 1967). Dahlstrom has suggested that the accumulation of norepinephrine on the distal side of the constriction indicates retrograde transport of catecholamines. This interpretation has been questioned by Kappeler and Mayor (1967a), who found that very few axons distal to

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a constriction of guinea pig hypogastric nerve contained accumulated catecholamines. Furthermore, these axons appeared to emanate from cell bodies located in the nerve distal to the constriction. The retrograde transport of norepinephrine in sympathetic axons has also been questioned by Geffen et al. (1969), who did not find any increase of radioactivity in the splenic nerve distal to a cut after infusing the spleen with n~repinephrine-~H. Kerkut et al. (1967) have found that gl~tamine-*~C is transferred from frog gastrocnemeus muscle via the sciatic nerve to the spinal cord after incubating the muscle in glutamine-14Cfor 24 hours. The velocity of transfer was approximately 15 mm/ day. Because of the prolonged incubation for 24 hours, extra-axonal diffusion of the glutamine-l*C could not definitely be ruled out as a possible explanation for the movement of the label. Using radioautography, Watson ( 1968) has found that following the injection of l y ~ i n e - ~into H the geniohyoid muscle labeled proteins were present in the axons of the geniohyoid nerve but not in the hypogIossal nerve at 6 hours after injection. At 3 days after injection, certain axons of the hypoglossal nerve were labeled. If the nerve was constricted between 12 and 24 hours after injection, radioactivity was found in the axons distal to the constriction but not proximally. These experiments represent the most direct evidence for retrograde axonal transport, and experiments of this type should prove extremely useful in elucidating the characteristics of retrograde axonal transport. Direct observation of neurons in tissue culture have provided considerable information concerning the movement of organelles within the axon. The following observations have been made in axons of dorsal root ganglion cells cultured in vitro (Burdwood, 1965). Some particles moved through the portion of the axon which was observed at velocities up to 9 p/sec in either direction. The movement of many of the particles was erratic. These particles accelerated in one direction parallel to the axis of the axon, stopped, and often reversed their direction. The rapid bidirectional movements of organelles in the axoplasm are similar to the streaming movements observed in the axon-like processes of some unicellular organisms (Jahn and Rinaldi, 1959). Rehbun (1967) has suggested that the rapid movements of particles in the axon fall into the category of saltatory movements which have been observed in a number of cells. The method of microscopic observation of the axons of living neurons limits the investigator to very small portions of the axon; however, this method enables the resolution of movements within the axoplasm which would be averaged out in studies with radioisotopes. In fact, the averag-


313

ing out of transitory movements of axoplasmic components within the axon might explain the relatively uniform distribution of radioactively labeled proteins along the axon between 6 to 10 days after radioactive labeled precursors were injected (Fig. 1 ) . Bidirectional axoplasmic transport could represent a feedback loop which links the neuron soma with its long processes. While somatofugal transport represents the supply line on which essential commodities are carried into the axon, retrograde transport may close the loop and enable the soma to monitor the biochemical profile of its remote axonal processes. IV. Physiological and Pathological Changes Which Effect Axonal Transport

A. PHYSIOLOGICAL CHANGES The passage of electrical activity along a nerve has been associated with metabolic changes within the nerve (Gerard, 1932). If local metabolic changes occur along the axon in association with the conduction of action potentials, it might be expected that axonal transport would also be modified by the passage of action potentials, The studies of Kerkut et al. (1967) indicated that the transfer of glutamate-14Cfrom snail brain into the attached nerve trunk and subsequently into a perfusate of buccal muscle was effected by electrical stimulation. When the brain was stimulated, glutamate-’*C appeared in the muscle perfusate much earlier than in unsiimulated preparations. The appearance of glutamate-14Cin the muscle perfusate of the stimulated preparation was significantly delayed if the preparation was cooled to 0°C or if nembutal or xylocaine were applied to the nerve. These effects may have resulted from changes in the velocity of transport of glutamine from the brain to the muscle. However, glutamate may be a neurotransmitter in this system, and it is possible that alterations of the rate at which glutamate was released at the neuromuscular junction accounts for the effects of stimulation and anesthetics on the rate of appearance of glutamate in the muscle perfusate (Kerkut et al., 1967): The amount of 22Nawhich migrated from one end of an isolated nerve segment distally was increased by electrical stimulation ( Akcasu and Salafsky, 1967). However, it is not clear whether the 22Na was transported in the axon or in the perineural spaces. Stimulation of an isolated segment of sciatic nerve at 110 cycles/second (Jankowska et al., 1969) had no effect on the accumulation of AChase at either the proximal or distal stump of the nerve. The accumulation of norepinephrine proximal to a cut in the splenic nerve was not effected by decentralizing the postganglionic neurons (i.e., severing the splachnic nerves) (Geffen

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and Rush, 1968).The evidence which is available does not conclusively demonstrate whether or not the level of excitation along an axon modifies axonal transport. The velocity of the slow component of axonal transport is significantly faster in young animals than in adult animals. In the axons of adult rat peripheral nerves the velocity of labeled proteins transported in the slow component was 0.6-0.9mm/day, while the same nerves of young rats the velocity of the slow component was 2.0-2.5 mmlday (Droz, 1965a). In the peripheral axons of cat dorsal root ganglion cells, the velocity of the slow component in ad& was 1.3 mm/day and in young growing cats 2.9 mm/day (Lasek, 197%). The transport velocity of the fast component has not been compared in young and adult animals, Studying regeneration of the goldfish retinal ganglion cell axons, Grafstein and Murray (1969) have noted that after sectioning the optic tract the velocity of transport of the slow component was increased threefold from the normal velocity of 0.4 mm/day. About 2 weeks after the fibers had reinnervated the optic tectum, the rapidly transported proteins moved at twice the normal velocity of 40 mm/day. Ochs et al. (1960) were unable to demonstrate any effect of regeneration on the transport of orthopho~phate-~~P in cat anterior horn cells. The increased velocity of axonal transport during growth or regeneration is most probably correlated with the elongation of the axon, In the adult animal the axon is in a steady state. The soma supplies an amount of protein which is equivalent to that which is catabolized. During regeneration or growth the volume of the axon increases. The increase in the velocity of axonal transport provides a possible mechanism, whereby the soma supplies the axon which is present and in addition supplies extra material for the elongation of the axon. Other more subtle mechanisms may be available to the neuron for controlling the amount of axonal components which are transported within an axon. For example, in the axons of the dorsal root ganglion cells which bifurcate into a central process and a peripheral process, the net transport of axonal protein is greater in the peripheral process than in the central process (Lasek, 1968a). It has been suggested that the transport of less material into the central process of the neuron than into the peripheral process might be related to differences in the volumes of these processes (Lasek, 1968a). An interesting point is that the dorsal root ganglion cells do not undergo retrograde chromatolysis when the dorsal root is severed but that chromatolysis does occur if the peripheral processes are severed. This might be explained if the reaction of the cell body to axon section represents a compensatory response to the loss of a sizable portion of the neuronal cytoplasm (i.e., axoplasm). The loss


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of the smaller central process of the dorsal root ganglion cell might represent less of an insult to the metabolism of the neuron than loss of the larger cytoplasmic volume of the peripheral process.

B. PATHOLOCICAL CHANGES Several pathological conditions have been described in which axoplasmic organelles are found to accumulate at loci in axons or within the neuron soma (Herman et al., 1969).One of the possible explanations for this accumulation of axoplasmic organelles is that a defect in axonal transport results in the piling up of organelfes proximal to the site of the defect. Other possibilities are that the amumulation results from uncontrolled synthesis of the neuronal components which accumulate or from reduced catabolism of these components. 'In neurofibrillary degeneration induced by vincristine sulfate, vinblastine sulfate, colchicine, and other metaphase-blocking antimitotic drugs, the evidence suggests that interference with axonal transport may be responsible for the accumulation of neurofilaments in the neuron soma (Wisniewski et al., 1968; Shelanski and Wisniewski, 1969). If high doses of the drugs were administered to experimental animals the neuron soma of certain neurons become filled with filaments, and the axons of the peripheral nerves show signs of classic Wdlerian degeneration within 2 weeks of injecting the drugs. However, if lower doses of the drug are administered, complete morphological recovery of the axons occurred, and the accumulated neurofilaments were replaced by neurotubules. The following evidence suggests that colchicine interferes with axonal transport. AChase and NAD tetrazolium reductase ( a mitochondrial enzyme) accumulated in axons proximal to a site where the sciatic nerve was crushed. However, if colchicine was injected into the sciatic nerve at the same time as the nerve was crushed, the accumulation of AChase and NAD tetrazolium reductase proximal to the crush was substantially reduced ( Kreutzberg, 1969). The accumulation of these enzymes at the site proximal to a crush is considered evidence of somatofugal transport. Therefore, colchicine may have reduced the accumulation by interfering with axonal transport. This possibility is further supported by the demonstration that injection of low doses of colchicine into sciatic nerve resulted in the accumulation of AChase (Kreutzberg, 1!369) and norepinephrine (Dahlstrom, 1969) in axonal swellings proximal to the site of injection. Dahlstrom (1969) has also found that injection of colchicine into cat lumbar sympathetic ganglia resulted in a reduction of catecholamine fluorescence m the axons of the sciatic nerve and accumulation in the ganglion cell bodies. In the case of experimental neurofibrillary degeneration, evidence is

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accumulating which suggests that the mitotic spindle inhibitors such as colchicine exert their effect on the neuron by interfering with axonal transport, possibly by interacting with the neurotubule subunit. A colchicine-binding protein has been identified in pig brain which is thought to be a subunit of the neurotubules (Borisy and Taylor, 1967). This discovery may significantly assist us in understanding diseases such as Alzheimer’s disease for which neurofibrillary degeneration may be an experimental model. It seems likely that in the future pathological conditions will be characterized in which more subtle changes in axonal transport are involved, possibly even situations in which retrograde transport is disturbed. V. Possible Mechanisms Underlying Axonal Transport

Time-lapse motion pictures of myelinated axons in vitro indicate pulsatile activity and wavelike movements along the surface of the axon ( Weiss et al., 1962; Pomerat et aE., 1967). Weiss ( 1967a) has suggested that these pulsatile movements are generated at the surface of the axon and that these peristaltic movements may represent the driving force for axonal transport. However, it is equally probable that these wavelike movements at the surface of the axon reflect distortions of the axoplasmic mass produced by amoeboid forces within the axon. Amoeboid movement of the regenerating axon tip of growing axons in vitro (Pomerat et d., 1967) and in vivo (Speidel, 1933) is well known. The growing axon tip advances by continuous extensions and retractions of numerous fingerlike filopodia. The velocity of movement of the growing axon tip is on the same order of magnitude as the velocity of the slow component, approximately 1-3 mmiday (Lubihska, 1964). It seems reasonable to postulate that the mechanism responsible for the amoeboid movements at the growing axon tip is identical to that which underlies the transport of the slow component. Other mechanisms have been proposed to explain the movement of the axonal column at 1 mm/day. Young (1945) suggested that the cell body produces a pressure force which drives the axoplasm along the axon. The failings of this theory have been discussed in detail, and it is not generally accepted ( Weiss, 1967a). Lubihska (1964) has proposed that axonal transport at velocities of 1 mmlday might be explained by net movement which is the resultant of continuous bidirectional axoplasmic streaming. She suggests that axoplasmic components are continually conveyed from the soma to the axon terminals and cycled in another stream back to the soma. Growth of the axon at a few millimeters per day is accomplished by the convection of more material to the axon terminals than is carried back toward the soma, the resultant being


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somatofugal movement of the axon tip. This theory was proposed before the demonstration of the slow-moving peak of labeled proteins was identified, and, although the hypothesis of axoplasmic streaming can account for elongation of the axon, the presence of a slow-moving peak of labeled proteins is not consistent with this theory. The slow component of somatofugal axonal transport may represent the axoplasmic matrix, and the fast component of axonal transport may be transported within the slow-moving matrix (Weiss, 1967a; Miani, 1963). Weks (1967a) has proposed the existence of fluid channels in the axoplasrn between the neurofilaments and tubules where axoplasm components could be transported at high velocities. It has also been suggested that the tubules themselves represent channels for the movement of molecules within the axon. The neurotubules of the axoplasm are approximately 250A in diameter, and the space within the tubules is large enough to accomodate large proteins (Fawcett, 1968; Metuzals, 1966, 1967). The possibility that proteins and possibly other molecules are transported within the neurotubules is given impetus by the demonstration that proteins are apparently conveyed into the outer segment of the retinal rod through the connecting cilium (Young, 1968). A colchicine-binding protein which has been identified in pig brain and squid axoplasm is thought to be a subunit of the neurotubules and is similar to the protein subunits isolated from cilia and flagella (Borisy and Taylor, 1967; Weisenberg et al., 1968). This subunit shares many characteristics with actin from muscle (Weisenberg et al., 1968). It was suggested above that the movement of the slow component of axonal transport is identical to the amoeboid extension of the growing axon tip, Although the axon has a net movement on the order of 1 mml day, this movement is the net result of more rapid extensions and retractions of fingerlike processes from the axon tip. Neurofilaments and tubules are thought to be a constituent of the slow component (Section 111, C ) . The proteins of the slow component are apparently constrained in the axoplasmic matrix, as evidenced by the fact that the peak of labeled proteins which identifies the slow component does not become progressively diminished after periods as long as 60 to 80 days after pulse labeling the slow component ( Lasek, 1%8a,b). Cross bridges between the neurofilaments have been demonstrated in vertebrate and invertebrate axons ( Metuzal, 1966, 1967; Peters and Vaughn, 1967). These cross bridges may represent the constraining force which binds the matrix of the axoplasm together. The similarities between the cholchicine binding 6 S protein of brain cilia and flagella to actin has led to a model which incorporates the sliding filament concept of Hanson and Huxley (1955) to explain

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the high velocity movement of vesicles and other organelles in the axoplasm (Schmitt, 1968). Schmitt has formulated a model in which the actin-like molecules in the neurotubules interact with an ATPase-bearing myosin-like partner in the protein coat of vesicles. In this model, the

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FIG.5. An idealized segment of an axon is illustrated which includes neurofilaments ( Nf ) linked together by cross bridges (Cb) and profiles of mitochondria (Mit). To simplify the diagram, only four filaments (1-4) were included as a representation of the large number of filaments which are found in many axons. The parts A-D represent the same short segment of the axon at varying intervals of time. The diagram illustrates a possible mechanism for converting high velocity sliding movements of individual filaments relative to the mass of the axoplasm into slow unidirectional translocation of the entire bundle of filaments. For example, filament 1 shifts proximo-distally by sliding along the surrounding filaments in A and B. Subsequently, from B to C filament 3 shifts relative to the mass and in I: to D filament 4 is translocated proximo-distally. The net result of these individual sliding movements of filaments 1, 3, and 4 is movement of the bulk of filaments. The velocity of the net movement must be slower than that of the individual filaments and is dependent both upon the velocity and distance of the individual shifts and the interval between individual translocations. It is suggested that the filaments interact with one another through the cross bridges and that the cross bridges are broken and closed as the filament moves.


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vesicles move along the outside of the tubule much as the actin filaments are thought to slide along myosin in muscle. I would like to propose a plausible model which incorporates the sliding filament concept to also explain the movement of the slow component. This model provides for the conversion of the high velocity sliding movements of filaments into the slow transport of the axoplasmic matrix somatofugally at approximately 1mm/day. If, as depicted in Fig. 5, individual filaments shifted at high velocities relative to the mass of filaments, the net result of a large number of these individual jumps could be slow movement of the bundle of filaments. In order for this model to work, the short jumps of individual filaments would have to be preponderantly unidirectional, It is suggested that the cross bridges open and close as the filament moves along adjacent filaments. Because the mass of the combined filaments in a bundle is very great relative to any single filament, the sliding movement of one filament relative to the surrounding filaments would result in the directional translation of that filament and only slight recoil of the mass of filaments. The velocity of the net movement of the bundle of filaments must be considerably slower than the velocity of the individual shifts of the filaments. The resultant velocity is additive and is dependent upon the average velocity of the individual jumps, the average distance traversed by individual filaments, the interval between jumps, and the number of filaments in the bundle. Although this theory is unquestionably very speculative it is attractive in that it incorporates a mechanism which could conceivably underly both rapid axonal transport and slow axonal transport. VI. Conclusions

The synthesis of neuronal components in the soma and the subsequent transport of 'many of these components into the axon is well documented. The mechanism of axonal transport is selective and certain neuronal components such as ribosomes do not appear to be transported from the soma into the axon. Axonal proteins which are synthesized in the soma and transported somatofugally into the axon appear to consist of at least two primary components: ( a ) a fast component with a velocity of 100500 mm/day, and ( b ) a slow component with a velocity of 0.4-3.0 mml day. While the slow component is transported in only one direction in the axon from the soma toward the axon terminals, certain elements of the fast component may be transported bidirectionally in the axon (i.e., both somatofugally and in the retrograde direction from the terminals toward the soma). The fast component of axonal transport appears to be characterized by a substantial content of particulate material, and it has been suggested that mitochondria and neurosecretory granules are constituents of the

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fast component, Lower molecular weight substances such as glutamate and polypeptides also may be present in the rapidly moving sector of the axoplasm. However, it is not clear whether smaller molecules are free in the axoplasm or bound to larger carrier molecules. In contrast to the diffuse nature of the fast component, the slow component of axonal transport is constrained within the axoplasm. The slow component may be associated with the axoplasmic matrix and circumstantial evidence indicates that the neurofilaments and neurotubules are part of the slow component. It may be significant that cross bridges have been identified between neurofilaments in the axoplasm. Although the neuron soma is the primary source of axonal protein synthesis, evidence supports the synthesis of some axonal proteins at the level of the axon. It is not yet clear whether some axonal protein synthesis occurs in the Schwann cells followed by transfer of the proteins into the axon. It has been demonstrated that protein synthesis does occur in the axoplasm; however, it has not been ascertained whether axoplasmic protein synthesis is completely mitochondrial. A substantial amount of evidence indicates that the metabolism of the neuron soma and axoplasm are closely integrated. In order for the soma to be responsive to the requirements of the axon, information on the biochemical status of the axon must continually reach the soma. This information must be fed back to the soma rapidly, if the soma and the axon are closely coupled. Retrograde transport of axonal components represents a possible mechanism (albeit unproven) by which the soma may receive information from the axon, potentially enabling the soma to monitor the biochemical profile of its long cytoplasmic processes. Flexibility is an important component of any system in the living organism. The neuron appears to have certain options available for controlling the movement of components within the axon; for example: ( a ) alteration of the velocity of axonal transport, and ( b ) alteration in the capacity of the transport channel. Evidence is available which indicates that the velocity of somatofugal axonal transport in growing or regenerating neurons is faster than in normal mature neurons. An increase in somatofugal axonal transport most probably results in transport of an increased volume of axonal components within the axon. It seems possible that the size of the axon might also effect the volume of material which is conveyed. However, the evidence on this point is merely suggestive and hardly conclusive. The importance of axonal transport in the metabolism of the neuron is stressed by the indications that defects in axonal transport may underlie certain neuropathological conditions. The best studied example of such involvement is experimentally induced neurofibrillary degeneration. This


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pathological state is produced by metaphase-blocking agents such as colchicine, vinblastine, and vincristine and results in the accumulation of neurofilaments in the soma of certain neurons in affected animals. The evidence strongly supports the view that the filaments accumulate in the neuron soma where they are synthesized, due to the failure of their transport into the axon. Axonal transport has provided a plausible mechanism for integration of the metabolism of the neuron, a cell type which has the longest processes of any cell in the animal kingdom. For example, in whales the axon of many neurons reaches lengths of several meters. The concept of molecular transport within the axon has broadened our understanding of the role of the neuron. Not only are the processes of the neuron active electrical conductors, but the axon also represents a channel for molecular transport. This linear routing of neuronal components provides for both the maintenance of the axonal channel and possibly as a molecular information channel. This latter function of the axon is least understood but possibly one of the most important to the animal. It is probable that biochemical information is transferred from the controlling center of the neuron soma, via the axon, to the structures which the neuron innervates. Thus, the innervated structure potentially receives molecular communication from the neuronal soma, and in turn the axon may act as a channel for the transmission of molecular information from the innervated structure back to the soma. This postulate seems almost essential to any comprehensive theory on the function of the neuron and enables us to bring some order to much of the data available on the interaction of elements of the nervous system. REFERENCES Akcasu, A., and Salafsky, B. (1967). Exptl. Neurol. 18, 49. Austin, L., and Morgan, I. G. ( 1967). I. Neurochem. 14, 377. Autilio, L. A,, Appel, S. H., Pettis, P., and Gambetti, P. L. (1968). Biochemistry 7, 2615. Bargman, W. (1966). Intern. Reu. Cgtol. 19, 183. Barondes, S. H. (1966). J . Neurochem. 13,721. Barondes, S . H. (1967). Neurosci. Res. Program Bull. 5, 311. Barondes, S. H. (1968). J. Neurochem. 15, 343. Blumcke, S., Niedorf, A. R., and Rode, J. (1966). Acta Neuropathol. 7, 44. Borisy, G. G, and Taylor, E. W. (1967). 1. Cell Biol. 34, 525. Bray, J. J., and Austin, L. (1968). J. Neurochem. 15, 731. Bray, J. J., and Austin, L. (1969). Brain Res. 12, 230. Burdwood, \Y. 0. (1965). J. Cell Biol. 27, 115A. Carlisle, D. B. (1959). In “Comparative Endocrinology” (A. Gorbman, ed.), p. 146. Springer, Berlin. Clouet, D. €I., and Waelsch, H. (1961). J. Neurochm. 8, 201. Dahlstrijm A. ( 1965). J. Anat. 99, 677.

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Dahhtrtip, A. (1967). Acta Physiol. Scand. 69, 158. Dahlstllijm, A. (1969). European J. Pharmucol. 5, 111. Dahlstrom, A., and HaggendaI, J. (1966). Acta Physiol. Scand. 67, 278. Droz, B. (1965a). In “The Use of Radioautography in Investigating Protein Synthesis,” Symp. Intern. SOC.Cell Biol. (C. P. Leblond and K. B. Warren, eds.), Vol. 4, p. 159. Academic Press, New York. Droz, B . (1965b). 1. Microscopie 6, 201. Droz, B., and Barondes, S. H. (1969). Science 165, 1131. Droz, B., and Leblond, C. P. (1963). J. Comp. Neurol. 121, 325. Droz, B., Koenig, H. L., and Young, R. W. Electron Microscopy, Proc. 4th European Regional Conf., Rome, p. 523. Edstrom, A. (1964a). J . Neurochem. 11, 309. Edstrom, A. (196413). J. Neurochem. 11, 557. Edstrijm, A. (1966). I. Neurochem. 13, 315. Edstrom, A. (1967). J. Neurochm. 14,239. Edstrom, A., and Sjostrand, J. (1969). J. Neurochem. 16, 67. Edstriim, A., Edstriim, J. E., and Hokfelt, T. (1969). 1. Neurochem. 16, 53. Fawcett, D. W. (1968). Neurosci. Res. Program Bull. 6, 173. Fischer, S., and Litvak, S. (1967). I. Cell. Physiol. 70, 69. Fischer, S., Gariglio, P., and Tarifeno, E. (1969). J. Cell. Physiol. 74, 155. Friede, R. L. ( 1964a). Acta Neuroputhol. 3, 217. Friede, R. L. ( 1964b). Acta Neuropathol. 3, 229. Geffen, L. B., and Rush, R. A. (1968). J. Neurochem. 15, 925. Geffen, L. B., Hunter, C., and Rush, R. A. (1969). J. Neurochem. 16, 469. Gerard, R. W. (1932). Physiol. Reu. 12, 469. Giudetta, A., Dettbarn, W. D., and Brzin, M. ( 1968). Proc. Natl. Acad. Sci. U . S . 59, 1284. Globus, A., Lux, H. D., and Schubert, P. (1968). Brain Res. 11, 440. Goldberg, S., and Kotani, M. (1967). Anat. Record 158, 325. Gordon, M. W., and Deanin, G. G. (1968). J. Biol. Chem. 243, 4222. Grafstein, B. (1967). Science 157, 196. Grafstein, B. (1969). In “Advances in Biochemical Psychology” (E. Costa and P. Greengard, eds.), p. 11, Raven, New York. Grafstein, B., and Murray, M. (1969). ExptZ. Neurol. 25, 494. Gramp, W., and Edstrom, A. (1963). J. Neurochem. 10, 725. Hanson, J., and Huxley, H. E. (1955). PTOC.SOC. Exptl. Biol. Med. 91, 228. Hendrickson, A. ( 1969). Science 165, 194. Herman, M. H., Huttenlocher, P. R., and Bensch, K. G. (1969). Arch. Neurol. 20, 19. Hoy, R. R., Bittner, G. D., and Kennedy, D. (1967). Science 156, 251. Hughes, A. (1953). J. Amt. 87, 150. Jahn, T. L., and Rinaldi, R. A. (1959). Biol. Bull. 117, 100. Jankowska, E., Lubinska, L., and Niemerko, S. (1969). Comp. Biochem. Physiol. 28, 907. Jasinski, A., Gorbman, A., and Hara, T. J. (1967). Science 154, 776. Kapeller, K., and Mayor, D. ( 1967a). J. Physiol. (London) 191, 70. Kapeller, K., and Mayor, D. ( 1967b). Proc. Roy. SOC. (London) B167,282. Karlsson, J. O., and Sjostrand, J. (1968).Brain Res. 11, 431. Kasa, P. (1968). Nature 218, 1965. Kerkut, G. A., Shapiro, A., and Walker, R. J. (1967). Comp. Biochem. Physiol. 23, 729.


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NEURQCHEMICAL CORRELATES OF BEHAVIOR1 By M. H. Aprison and J.

N.Hingtgen’

Section of Neurnbiology, The Institute of Psychiatric Research and Departments of Psychiatry and Biochemistry, Indiana University Medical Center, Indianapolis, Indiana

I. Introduction . . . . . . . . . . 11. Behavioral Depression and Increases in Brain Serotonin 111. Behavioral Depression and Decreases in Brain Serotonin IV. Behavioral Excitation and Decreases in Brain Acetylcholine V. Methodological Problems . . . . . . . VI.Summary . . . . . . . . . . . References . . . . . . . . . .

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I. Introduction

Our motivation for the development of a research program to investigate relationships between central nervous system ( CNS ) transmitters and behavior stems from a desire to learn more about the neurobiological mechanisms which underlie abnormal behavior. If the brain is the source of the events which finally govern the behavior of an organism, then impaired behavior may be the result of biophysical, biochemical andl or anatomical lesions in that part of the nervous system. When “key” neurons are affected by the lesions, their most important function, the transfer of information to other cells, is often disrupted or impaired. Since it is generally accepted that mediation of synaptic excitation and inhibition (transfer of information) within the mammalian nervous system is mainly chemical, we choose to concentrate on the study of these neurochemical mechanisms and their effect on behavior. While we have restricted our immediate goals to looking for neurochemical correlates of behavior in lower animals, it is our hope that these data will some day be useful for understanding the specific biochemical mechanisms which may cause certain types of abnormal human behavior. Our studies were initiated in 1954 when Aprison, Nathan, and Himwich found asymmetric acetylcholinesterase ( AChE ) activities in the right and left cerebral cortices and caudate nuclei from rabbits exhibiting compulsive circling after a unilateral intracarotid injection of a potent anticholinesterase drug, diisopropylfluorophosphate ( Aprison et al., 1954). Subsequent studies measuring the transmitter levels instead Supported in part by grant MH-03225 from National Institute of Mental Health, U.S.P.H.S.

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M. H. APRISON AND J. N. HINGTGEN

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of the enzyme activities showed that the rate of turning correlated with the asymmetric accumulation of acetylcholine ( ACh) in the cerebral cortices ( Aprison, 1958, 1965). These early studies led to the formulation of a theoretical framework which suggested possible neurobiological mechanisms operating in the emission of atypical or abnormal behavior (Fig. 1). Whatever type of ‘lesion” occurs due to general causes such as diet, stress, and drugs, the effect is invariably on the ability of various nerve cells to function normally. Thus, two main defects are produced: transmitter system defects and nontransmitter system defects. We have been interested in the former, because much evidence has accumulated which suggests that a number of specific nitrogen-containing compounds suspected of acting as neurotransmitters or modulators ( Aprison, 1962; Aprison and Takahashi, 1965)2 have major effects on the behavior of *The authors in these references as well as the present authors prefer to distinguish between transmitters and modulators. A modulator as defined by us is a compound which once released from specific nerve endings may influence the effective transmitter concentration at certain synapses by competing with it for the receptor site. It may even influence the release or uptake of the true transmitter thus also affecting the latter’s influence at that receptor. This definition allows for the possibility that a modulator can also function as a transmitter at other synapses within the same organism or within other animals.

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living organisms. We think these effects are due to the individual or grouped action of these substances after their release from the presynaptic neurons. The action is at receptor sites or sensitive portions of the postsynaptic membrane located in the synapses of various neuronal pathways being activated or utilized. Deviation from stable emission of a learned response, which we define as abnormal behavior, may therefore be due to the change in the effective concentration of the free transmitter acting at the postsynaptic receptor sites of a group of important synapses involved in key neuronal pathways utilized for the “generation” or performance of that particular behavior. If we can affect one or more of the systems labeled 1 to 5 in the last line of Fig. 1, we should be able to simulate abnormal behavior. This can be done in a number of ways including the following: ( u ) injection of known transmitter precursors; ( b ) injection of drugs known to affect components of systems involved in 1 to 5 (Fig. 1); ( c ) diet; ( d ) direct production of tissue pathology. The quantitative measurement of both the specific neurochemical changes in discrete areas and the changes in behavior of the same animal during periods of simulated abnormal behavior present the possibility of making meaningful correlations during a critical time period. Such correlations, when they occur, may help in a formulation of some general behavioral-neurochemical relationships applicable not only to animals but also to humans. We would like to describe in some detail our research program which involves the measurement of changes in levels of transmitter or modulator suspects such as ACh, 5-hydroxytryptamine (serotonin or 5-HT) and norepinephrine ( N E ) as well as their associated enzyme systems in specific brain areas, and their temporal relationship to changes in the quantitatively measured behavior of these animals. Although we d o not suggest this program serves as a model, we do feel that the general approach would be of interest to other researchers in neurobiology. II. Behavioral Depression and Increases in Brain Serotonin

The first experiments began in 1959 with the 5-HT-monoamine oxidase (MAO) system because of the keen interest that had developed in this subject in the five preceding years. The early interest in 5-HT developed when it was shown in pharmacological studies on smooth muscle that d-lysergic acid diethylamide, a compound which provokes schizophreniolike states in man, is a 5 H T antagonist at certain doses (Gaddum, 1954), but acts synergistically at lower doses (Costa, 1956). Studies with antimetabolites of 5-HT also added impetus for work in this area (Woolley and Shaw, 1954a,b). All these studies suggested to many investigators that 5 H T may have a role in brain function. The metabolic

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steps involved in the formation and destruction of 5-HT indicate that the level of 5-HT can be elevated by ( a ) injecting its precursor 5-hydroxytryptophan (5-HTP); ( b ) injecting an MA0 inhibitor such as iproniazid; or ( c ) injecting 5-HTP after iproniazid pretreatment. Once these pharmacological manipulations were performed and the increase in 5-HT measured, it became necessary to obtain equally quantitative behavioral measures for the animals in which the biochemical correlate was sought. In studies involving the correlation of changes in brain chemistry with changes in behavior, the behavioral measurements should match the neurochemical measurements for objectivity, reliability, and sensitivity. One such behavioral methodology makes use of operant conditioning techniques (Skinner, 1938; Ferster and Skinner, 1957; Honig, 19%). The operant behavior measured in our studies was emitted under either an approach or avoidance schedule of reinforcement. The approach schedule used with pigeons, maintained at 80% of free-feeding weight, consisted of a multiple fixed-ratio 50, fixed-interval 10 (Mult FR 50 FI l o ) , in which key pecking was reinforced with grain. For rats, the approach schedule was a variable ratio 40 (VR 40), in which lever pressing was reinforced with chocolate milk. The avoidance schedule, used only with rats, consisted of a response-shock interval of 40 sec and a shock-shock interval of 20 sec (RS40 SS20). In this latter schedule a lever press postponed an electric shock (1.6 mA, 0.5 sec duration) for 40 sec, but, during periods of no responding, shocks were presented at 20 sec intervals. Our initial attempt to assess the behavioral changes resulting from increased levels of brain 5-HT consisted of establishing a dose-response relationship following injections of 5 - H P into pigeons working on a Mult FR 50 FI 10 schedule of reinforcement. Intramuscular (I.M.) inP the pigeons' jections of %, 50, and 75 mg/kg doses of D L - ~ H Tlowered rate of responding whereas doses below 25 mg/kg had little or no effect; since only the L isomer readily passes the blood-brain barrier, the effective dose is one half that shown (Aprison and Ferster, 1960, 1961c). The duration of the effect was usually over before the 6-hour experimental session ended. Since little 5-HT crosses the blood-brain barrier under normal physiological conditions compared to its precursor 5HTP (Costa and Aprison, 1958), a study was made to determine the behavioral effect of an I.M. injection of 5-HT on pigeons performing on a multiple FR 50 FI 10 schedule (Aprison and Ferster, 1960, 1961~). A comparable behavioral effect to 5-HTP was produced but with a much smaller dose of 5-HT; this effect was thought to be due principally to the peripheral actions of 5-HT. Since small doses of 5 H T had large behavioral effects, it might be


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supposed that the behavioral effects of 5-HTP were also due to 5-HT being formed peripherally. There is evidence that in spite of the fact that SHTP acting peripherally may affect the animal's behavior, its major influence is through the CNS. It was hypothesized that the behavioral effects were due mainly to a central mechanism involving free or physiologically effective 5-HT. Iproniazid, which is a much better central than peripheral MA0 inhibitor in vivu, enhanced the behavioral effects of injected 5-HTP. This greater behavioral disruption is best explained in terms of elevated brain 5-HT levels resulting from reduced brain M A 0 activity. When the same dose of 5-HTP was injected into iproniazid-pretreated pigeons at different intervals over a long period of time, both the behavior and brain MA0 activity returned to normal levels in about 35 days, whereas liver MA0 activity was back to normal in only 12 days; the return of the behavioral effect was readily correlated with the recovery of the brain M A 0 activity (Aprison and Ferster, 1961a,b). In studies where the dose of 5-HTP was varied and the dose of iproniazid held constant, it was found that at any level of brain MA0 activity (during the recovery period), the greatest behavioral effect was obtained at the highest 5-HTP dose injected. Even more important, these data show that at any given dose of 5-HTP, the greatest behavioral effect is obtained at the lowest brain M A 0 level during the recovery period ( Aprison and Ferster, 1961a,b), The free 5-HT in brain, which apparently is involved in the production of the behavioral effect (as a transmitter or modulator), probably comprises the 5-HT found at any instant in the synaptic cleft plus that of a labile storage pool within the presynaptic nerve endings. The latter pool can be thought of as a source of readily available 5-HT at a specific synapse and is probably in equilibrium with a second pool of 5-HT, the firmly bound storage pool. Serotonin re-entering the presynaptic nerve ending either enters the labile storage pool or is destroyed by MA0 in the mitochondria. Since an identical amount'of 5-HTP was injected at various times during the period that brain MA0 activity was returning to normal in the above mentioned experiments, the observed behavioral change should be due to action of available cerebral 5-HT formed from its precursor and released at the appropriate synapses. As the M A 0 activity increased, more 5-HT was destroyed and less 5-HT was available to produce physiological and behavioral effects. The changes seen in the birds' behavior appeared due to the action of increased free 5-HT in brain with the concentration controlled by the level of its catabolic enzyme located in the presynaptic neurons. It therefore became imperative to study the kinetic relationship between the 5-HTP induced behavioral change and brain 5-HT concentration in

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animals not given an M A 0 inhibitor. Serotonin was measured in four specific brain areas, as well as in liver, heart, lung, and blood in pigeons (Aprison et al., 1962) sacrificed at various time intervals during the period of measurable behavioral disruption, T ( Hingtgen and Aprison, 1965). Since the time course of the behavioral effect in any given animal is relatively invariant with a constant dose of 5-HTP while there is marked variation in the time course of response from animal to animal, we made use of a unique method of treating our data. The 5-HT data were plotted against the percent of the behavioral effect (in time) rather than length of time after 5-HTP administration. In this way, the variation in behavioral effect of the same dose of 5-HTP in each pigeon was weighted, thus markedly reducing the variability of the data. Only in the telencephalon and diencephalon (plus optic lobes) did the 5-HT concentrations return to normal when the behavior of the pigeons returned to normal. The time course of change in both parameters was therefore remarkably similar and confirms the hypotheses and our original explanation of the brain 5-HT-MAO-behavior correlation ( Aprison and Ferster, 196la,b; Aprison et al., 1962). It became evident that dopamine ( D A ) and NE levels in the four brain areas under investigation might also change after the injection of 5-HTP because the enzyme which synthesizes 5 H T , S-hydroxytryptophan decarboxylase, is thought to be the same as 3,4-dihydroxyphenylalanine decarboxylase, the enzyme which synthesizes DA, the precursor of NE. Consequently, another group of trained pigeons was given injections (I.M.) of 50 mg/kg 5-HTP. The average period of depressed behavior ( T ) following these injections was measured for each bird and at various percentages of T, the pigeons were sacrificed by decapitation, and the four brain parts were assayed for DA and NE (these data as well as 5 H T for three brain areas are shown in Fig. 2 ) . We found no significant changes in DA and NE concentrations in the telencephalon (Aprison and Hingtgen, 1964, 1965). Changes were found in the NE levels of the diencephalon (plus optic lobes) , pons-medulla oblongata and cerebellum, but these changes were not related to the observed behavioral changes. The NE levels of the pons-medulla oblongata in the experimental birds were depressed below the normal range both FIG.2. Serotonin, dopamine, and norepinephrine concentrations in telencephalon, diencephalon plus optic lobes, and pons plus medulla oblongata of pigeons during the complete period of atypical behavior ( T ) and after behavior had returned to normal. Each point represents a single determination from a pigeon working on a multiple FR 50 FI 10 schedule of reinforcement, injected with 50 mg/kg 5-HTP (I.M.) and killed at a percentage of its previously determined mean period of abnormal behavior. On the abscissa scale N.B. refers to normal behavior.

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during and after the period of behavioral disruption. Changes in DA occurred in diencephalon (plus optic lobes), pons-medulla oblongata, and cerebellum, but only in the first two brain parts is a temporal correlation with behavioral changes suggested. Whether this change in DA concentation contributes to the disruption of behavior, or reflects instead a fall in the NE precursor level, remains to be determined. In this regard, it should be noted that the DA concentration in the telencephalon, which contains the basal ganglia, is approximately 5 times higher than that in the midbrain and pons-medulla oblongata. The 5HT, DA, and NE concentrations in the telencephalon, diencephalon plus optic lobes, cerebellum, and pons plus medulla oblongata after 5-HTP administration suggest that the depressed approach behavior noted can best be explained by the action of free 5-HT on appropriate sites in the brain, but the role of DA in the midbrain and pons-medulla oblongata is still to be clarified. To verify these findings in another species, rats working on a VR 40 schedule of reinforcement were injected with 50 mg/kg D L - ~ H T P ( Aprison and Hingtgen, 1966a). Similar correlations over the period of disruption were found between the 5-HT changes in the telencephalon and changes in behavior. The elevated 5 H T level in the brain stem returned to normal before the behavior returned to normal. During the period of behavioral disruption, the NE concentration in the telencephalon did not vary from normal values. Further, after the behavior returned to normal, the NE concentration in the brainstem was slightly depressed. In general, these data on rats agree remarkably well with the data found in pigeons. Few studies have contained both behavioral and biochemical data on the same experimental animal, and these usually involve data from a single behavioral schedule. Experiments including measurements of two or more neurohumoral agents in specific brain areas from experimental animals working in two or more behavioral situations known to be differentially disrupted by either the injection of a transmitter precursor or drugs are even fewer in number. With such data, correlations between neurochemical and behavioral changes should occur just as in the single behavioral situation. Furthermore, these data could provide a basis for determining whether a specific biochemical system (i.e., serotonergic, noradrenergic, cholinergic) is involved in the specific production of the behavioral response (i.e., approach, avoidance). It was therefore of extreme interest to us to determine whether rats were equally affected by 5-HTP when working on either an approach or on an avoidance schedule of reinforcement. No behavioral effect followed an injection of 50 mg/kg DL-S-HTP


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into rats working on a RS40 SS20 schedule, although there was a comparable increase in brain 5-HT in these rats to that found in the approach rats ( Aprison and Hingtgen, 1966a). Furthermore, the avoidance rats still had elevated 5-HT levels beyond the time the levels in the approach rats returned to normal. These data suggest that avoidance behavior may not be as sensitive to brain 5-HT changes as is approach behavior, but that avoidance schedules may impose certain stresses on the rats to prolong the period of 5-HT elevation following 5-HTP administration. Ill. Behavioral Depression and Decreases in Brain Serotonin

Inasmuch as incremes in brain 5-HT are followed by disruptions in approach behavior, the question naturally arises: What behavioral changes would follow decreases in brain 5-HT? One method of reducing brain 5HT uses clrugs which deplete bound stores in nerve endings. Although many drugs, such as reserpine, tetrabenazine, a-methyl-m-tyrosine ( aMMT), cause a fall in more than one transmitter, a differential fall in any one compound over a reasonably long time period can be used to advantage by the investigator. For instance, after the injection of a-MMT in various species including the pigeon, brain 5-HT, NE, and DA levels are differentially depleted. Taking advantage of this differential depletion, we investigated the relationships between quantitatively measured behavioral changes after the injection of a-MMT (100 mglkg) into pigeons (Hingtgen and Aprison, 1963) and the brain levels of NE, DA, 5-HT, and a-mei-hyl-m-tyramineplus aramine, the decarboxylation products of a-MMT (Aprison and Hingtgen, 196613). The data indicate that both the change in 5-HT concentration and the change in mean behavioral response rates, including the return to normal levels of both parameters, followed the same time course (Fig. 3). This relationship was not true in the case of the other neurohumoral agents measured nor the decarboxylation products of a-MMT. In addition it was found that the a-methyl-rn-tyramines return to normal levels in pigeon brain at approximately 2-4 days after an injection of a-MMT. However, during the 8-hour period of decreased behavioral response rates following a-MMT, the almost stoichiometric displacement of DA and NE by their methyl analogs suggested that DA and NE changes are not directly responsible for the behavioral change since the a-methyl-rn-tyramines may function at the Dii and NE receptor sites (Carlsson, 1964). Although the suggestion had been made that an anorexic side effect was responsible for the behavioral results with pigeons (Carlton and Furgiuele, 1965), this explanation was shown to be inadequate since pigeons working on a multiple FR 50 FI 10 schedule of reinforcement as well as naive pigeons

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TIME A F T E R 1 0 0 m g / k g N - M M T (IM)

FIG.3. Temporal changes in behavioral response rates and brain monoamines in pigeons injected with 100 mg/kg cu-methyl-m-tyrosine (I.M. ). The behavioral data were obtained from pigeons working on a multiple FR 50 FI 10 schedule of reinforcement. [Reprinted with permission from Life Sciences 5, 1971 ( 1966).]

(both groups being maintained at 80%of free-feeding weight) consumed equivalent amounts of food in control sessions and sessions following the injection of 100 mg/kg a-MMT (Hingtgen and Aprison, 1966). At the present time we are not sure whether the behavioral disruption is due to SHT, dopamine, both of these compounds, or some unknown transmitter. However, the first lead that must be tested further is the one which indicates that a temporal relationship exists between the fall in both the total 5-HT levels and the behavioral responding. We have shown that increased total (free plus bound) brain 5-HT following an injection of 5-HTP produced a period of depressed responding in the pigeon working on a Mult FR 50 FI 10 schedule, whereas after a-MMT administration, decreased total brain 5-HT is also followed by a period of depressed responding on the same behavioral schedule. One explanation for the two opposite biochemical situations which result in virtually the same behavioral changes is found in the concept that a neurohumoral agent or neurochemical modulator must be free (to


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diffuse to the proper receptor site) rather than bound (in synaptic vesicles) in order to produce its physiological effect. Although the time course of the behavioral effects due to an injection of SHTP and a-MMT are distinctly different, one can offer the hypothesis that in both these cases more free 5-HT is released than in the normal state. Therefore, a physiological situation can exist in the brains of these pigeons where greater amounts of the free form are available at synapses to produce its effect. If one assumes that free and total transmitter levels follow the same time course, this relationship could be tentatively explained in terms of the corresponding changes in the free component during the behavioral disruption. The possibility must be supported by additional data. Another drug, tetrabenazine ( TBZ ) , has reserpine-like depressive effects on brain monoamines (Quinn et al., 1959) and on both approach and avoidance operant behavior ( Aprison and Hingtgen, 1966a) . The 5-HT and NE data from the TBZtreated ( 2 mg/kg S.C.) rats working on a VR 40 approach schedule indicate that this drug caused a depletion of both 5-HT and NE from the telencephalon and brain stem of all the rats at 30 and 80%T. By 100%T (return to normal behavior), the 5-HT concentration in these brain areas returned to normal levels as did the NE concentration in the brain stem. Although disruptions in approach behavior appear to be correlated with 5-HT changes (and possibly brain stern NE) following TBZ, the disruptions in avoidance behavior did not correlate with any of the transmitters measured ( Aprison and Hingtgen, 1966a). These data suggest that another transmitter system, possibly the cholinergic system, may be involved in disruptions of certain types of avoidance behavior. IV. Behavioral Excitation and Decreases in Brain Acetylcholine

By administering specific drugs alone or in combination to animals it is possible to induce different behavioral patterns of normal, depressed, or enhanced responding. Using a RS40 SS20 avoidance schedule, we injected 2 mg/kg TBZ in rats and found decreased response rates (depression). When rats were preinjected with 50 mg/kg iproniazid before the same dose of TBZ was given, the response rates increased for a finite length of time (excitation). Since numerous studies show that the ACh concentration varies inversely with the degree of functional activity of the brain, ACh is a logical compound to measure in the brain of animals exhibiting enhanced or depressed behavioral response rates. Therefore we repeated the psychopharmacological experiments and killed trained rats by freezing during each behavioral state as well as in several control conditions. ACh concentrations were measured and found

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to be lowered in the telencephalon, midbrain and ponsrmedulla oblongata of rats exhibiting increased avoidance response rates, whereas, in depressed rats, only the midbrain ACh concentration increased (Tom et al., 1966). In a later study we extended these measures to include the complete period of excitation and have measured the changes in ACh plus 5-HT and NE concentrations in all three brain areas as well as in changes in increased response rates in the same rats (Aprison et al., 1968). Acetylcholine concentration in all three brain areas decreased and returned to normal levels at different times (Fig. 4). The time course of increased response rates correlated best with the changing ACh levels in the telencephalon. Both the 5-HT and NE concentrations remained elevated similar to the iproniazid control values during the period of behavioral excitation. However, the NE concentration in the midbrain showed a continuous decreasing trend toward naive control levels, the latter being approximately 61%of the iproniazid controls. These data suggested that changes in a cholinergic system in the telencephalon and a noradrenergic system in the midbrain probably operate together in the maintenance of the behavioral excitation ( Aprison et al., 1%8), with the former appearing to be more important. Since the ACh concentration changes follow the behavioral excitation so closely during most of the time period, a possible reason is suggested for the lack of correlation between disruptions of avoidance behavior and the changes in 5-HT, NE, and DA reported in the earlier studies. These data open a new dimension to our understanding of excitation produced under the above-mentioned circumstances because most studies in the past referred only to NE as the transmitter involved in the behavior described above ( Aprison et al., 1%8). The duration of behavioral excitation following iproniazid plus TBZ administration varies from animal to animal. It was noted that a small number of rats in our experiments did not exhibit excitation following the usual sequence of administration of these drugs. When nonexcited rats were killed at times comparable to the times when excited rats were killed, the ACh levels in the three brain areas did not vary significantly from control values ( Aprison and Hingtgen, 1969). These data support the suggestion that excitatory behavior and lowered ACh levels in brain ( especially telencephalon) are related. The question naturally arises: Why were some of these animals not excited? One explanation comes from the assumption that the cholinergic, serotonergic, and noradrenergic systems are interrelated in some way. The fall in brain ACh is probably due to the increased release of ACh (free) and its subsequent destruction by AChE in the postsynaptic membrane. Possibly the release of ACh is due to the biochemical changes

337

NEUROCHEMICAL CORRELATES OF BEHAVIOR 50 200

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FIG.4. Temporal variations in acetylcholine, serotonin, and norepinephrine concentration in the telencephalon (T), midbrain ( M B ), and pons-medulla oblongata (P-M ) and avoidance response rates in rats preinjected with 50 mg/kg iproniazid (S.C.) 16 hours before being given 2 mg/kg tetrabenazine (S.C.). Each point represents the biochemical or behavioral measure obtained from the same group of rats killed a t a specific time after injection. The abscissa axis refers to time (in ininutes) after the tetrabenazine injection.

in one or both of the other systems. The NE and 5-HT levels in the brains of the nonexcited rats may not have been elevated sufficiently to release ACh following one injection of iproniazid and TBZ. If ACh was not released in increased quantities at cholinergic synapses, its destruction by AChE would remain normal, and the levels of this neurotransmitter in brain tissue would not decrease. Therefore, a second

338


injection of iproniazid was given to rats which did not show the typical elevated avoidance response rates ( excitation) following the usual iproniazid and TBZ injection sequence. The second iproniazid dose reversed the TBZ-induced depression of avoidance behavior and the animals response rates increased to above normal levels. In our laboratories, successive injections of 50 mg/kg iproniazid into rats at 12-hour intervals do not produce excitation until after the third injection. Therefore, a second dose of iproniazid could not account for the excitation now seen. However, in combination with the TBZ injection, the excitation of the previously nonexcited rats may be due to a change in the critical levels of 5-HT and NE in one or more brain areas which in turn cause a release of ACh, or ACh changes may produce a release of more NE. Our most recent studies indicate that the period of excitation can be prolonged or curtailed depending on the dose of atropine injected and the time sequence of the injections. Low doses (0.1 mg/kg) given 60 to 120 minutes before TBZ but after iproniazid caused an increase in the duration of excitation. Higher doses (0.8 mglkg) of atropine decreased the excitation period. These data with atropine support the involvement of the cholinergic system in behavioral excitation since low doses enhance the effect whereas high doses block it (Hingtgen and Aprison, 1970).

V.

Methodological Problems

Although the introduction of quantitative behavioral measures and the development of specific transmitter or modulator assays have facilitated the study of neurochemical-behavioral relationships, many methodological problems still remain. The ultimate experimental design for the determination of neurochemical correlates of behavior would involve the measurement of the neurochemical and behavioral parameters in the same animal at the same time. For example, in a study in which relationships between behavioral excitation and transmitter levels were being correlated, a minute by minute record of the excitatory behavior would be compared to minute by minute analyses of ACh, 5-HT, NE, etc., concentrations m discrete areas of the brain. Although our behavioral measures are kinetic, we are not yet able to assay CNS tissue in the live animal. The method of second choice, which we have described in various studies above, is to kill animals during various periods of behavioral responding, “freezing” the chemical changes at this point, and then determining levels following subsequent dissection and neurochemical assay. Another methodological shortcoming is that in our studies using concomitant measures of behavioral and neurochemical transmitter concentration changes, the best correlations should be obtained when the


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free or physiologically effective pool of the transmitter is measured. Unfortunately, neurochemists have accurately measured only totd neurohumoral concentrations in excised brain parts or metabolic turnover rates of a specific transmitter. Examination of such turnover rates of transmitter concentrations might provide additional insights into neurochemical-behavioral relationships. On the other hand, if turnover rate measurements reflmt the turnover of the total metabolic and functional pools rather than the turnover of only the free transmitter pool, these measures may not provide any better correlations with behavioral changes than were noted in our studies. It is assumed that an important factor in the stable emission of a specific learned response is the change in concentrations of the transmitters acting at the postsynaptic membrane of a group of important synapses involved in key neuronal pathways utilized for the performance of that particular behavior. If one is measuring the “correct” transmitter under conditions where specific drugs, metabolites, or enzyme inhibitors are injected, and the change in the effective concentration of its free pool follows the same time course as the change in the total transmitter level, then explanations as we have suggested become meaningful. Maximal efforts in our current work are being directed to overcoming the problem of measuring the change in the free transmitter pool in behavioral experiments where the response rates are continuously being measured. We hope that our studies reviewed here will stimulate many more collaborative efforts in this frontier area of neurochemicalbehavioral research.

VI.

Summary

A research strategy has been developed which continues to serve as a basis of designing new experiments in the field of neurochemical correlates of behavior. This strategy is based on the fact that evidence has accumulated to suggest that a number of nitrogen-containing compounds called neurotransmitters or modulators have major effects on the behavior of living organisms by their individual or grouped action on the central nervous system. It therefore becomes important to study how abnormal levels of such compounds have major effects on behavior and also to attempt to correlate temporal changes in their levels in different structures of the brain with concomitant changes in the behavior of the organism. This has been done by employing techniques which permit the investigator to measure quantitatively both the levels of the specific neurochemical agent in the brain and the behavior in the same experimental animal. Rats and pigeons were trained to emit stable response rates under various approach and avoidance schedules of reinforcement. Following the

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injection of a number of different biogenic amine precursors and drugs known to affect neurotransmitter or modulator levels, animals were killed at specific times, brains removed and dissected into specific areas and then assayed for one or more of the following compounds: acetylcholine, norepinephrine, serotonin, etc. Several correlations between biochemical (brain) and behavioral parameters have been found during the period of atypical or abnormal responding. Injections of the serotonin precursor, 5-hydroxytryptophan, produced a behavioral disruption in pigeons or rats working on approach schedules of reinforcement and caused an elevation of serotonin levels in the forebrain structures during the same time period that the behavior was depressed, with both parameters returning to normal IeveIs at the same time. However, the behavior of rats on an avoidance schedule was not changed after a 5-hydroxytryptophan injection even though the brain serotonin was elevated. The administration of a-methyl-m-tyrosine to pigeons on approach schedules resulted in a differential depression of brain serotonin, norepinephrine, and dopamine levels along with a reduction in response rates to about 20%of normal. The time course of the behavioral change appeared to follow the time course of the serotonin change more closely than that of the other amines measured. In additional studies on avoidance behavior, acetylcholine content of brain (parts) rapidly decreased during the excitation period following administration of tetrabenazine to iproniazid pretreated rats. f i e duration of increased response rates correlated best with the duration period of lowered acetylcholine levels in the telencephalon. In addition, serotonin and norepinephrine concentrations remained elevated except in the midbrain where norepinephrine showed a slow continuous decreasing trend toward naive control levels. These data suggest that changes in a telencephalic cholinergic system and a midbrain noradrenergic system probably operate in the maintenance of the behavioral excitation. Recent data with atropine support the involvement of the cholinergic system in behavioral excitation, since low doses enhance the effect whereas high doses block it. REFERENCES Aprison, M. H. (1958). J. Neurochem. 2, 197. Aprison, M. H. (1962). Recent Aduan. Biol. Psychiat. 4, 133. Aprison, M. H. (1965). Progr. Brain Res. 16, 48. Aprison, M. H., and Ferster, C. B. (1960). Expen'entia 16,159. Aprison, M. H., and Ferster, C . B. (1961a). J. Neurochem. 6, 350. Aprison, M. H., and Ferster, C. B. (1961b). Recent Advan. Biol. Psychiat. 3, 151. Aprison, M. H., and Ferster, C. B. (1961~).J. PhamcoZ. EzptZ. Therap. 131, 100. Aprison, M. H., and Hingtgen, J. N. (1964). Federation Proc. 23, 456.


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Aprison, M. €I., and Hingtgen, J. N. (1965). J. Neurochem. 12, 959. Aprison, M. H., and Hingtgen, J. N. (1966a). Recent Advan. Biol. Psychiut. 8, 87. Aprison, M. H., and Hingtgen, J. N. (1966b). Life Sci. 5, 1971. Aprison, M. H., and Hingtgen, J. N. (1969). Biol. Psychiat. 1, 87. Aprison, M. €I., and Takahashi, R. (1965). J. Neurochem. 12,221. Aprison, M. H., Nathan, P., and Himwich, H. E. (1954). Science 119, 158. Aprison, M. H., Wolf, M. A., Poulos, G. J., and Folkerth, T. L. (1962). J. Neurochem. 9, 575. Aprison, M. H., Kariya, T., Hingtgen, J. N., and Tom, M. (1968). J. Neurochem. 15, 1131. Carlsson, A. (1964). Progr. Brain Res. 8, 9. Carbon, P. I,., and Furgiuele, A. R. (1965). Life Sci. 4, 1099. Costa, E. (1956). Proc. SOC. Exptl. Biol. Med. 91, 39. Costa, E., and Aprison, M. H. (1958). Am. J . Physiol. 192, 95. Ferster, C. B., and Skinner, B. F. (1957). “Schedules of Reinforcement.” AppletonCenturyCrofts, New York. Gaddum, J. 13. (1954). Ciba Found Symp. Hypertension; Humoral Neurogenic Factors p. 75. Hingtgen, J. N., and Aprison, M. H. ( 1963). Science 141, 169. Hingtgen, J. N., and Aprison, M. H. (1965). Recent Aduan. Biol. Psychiat. 7, 163. Hingtgen, J. N., and Aprison, M. H. (1966). Life Sci. 5, 1249. Hingtgen, J. N., and Aprison, M. H. (1970). Neuropharmacol., in press. Honig, W. (1966). “Operant Behavior: Areas of Research and Application.” Appleton-Century-Crofts, New York. Quinn, G. P., Shore, P. A., and Brodie, B. B. (1959). J . Phumcol. Exptl. Therap. 127, 103. Skinner, B, €7. ( 1938 ). “Behavior of Organisms.” Appleton-Century-Crofts, New York. Tom, M., Hingtgen, J. N., and Aprison, M. H. (1966). Life Sci. 5, 181. Woolley, D. W., and Shaw, E. (1954a). Proc. Natl. Acad. Sci. U.S. 40, 228. Woolley, D. W., and Shaw, E. (1954b). Brit. Med. J . ii, 122.


SOME GUIDELINES FROM SYSTEM SCIENCE FOR STUDYI NG NEURAL INFORMATION PROCESSING By Donald 0. Walter and Martin F. Gardiner Departments of Physiology and Anatomy, Brain Research Institute, University of California, Lor Angeler, California

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I. Basic Concepts A. Systems Concepts B. Information: Its Definition and Properties C. Information Processing 11. New Insights Arising from the Information Systems Viewpoint A. Competing System Models for Representing Weak Evoked Responses B. Selected Experiments on Neural Information Processing 111. Summary and Prospect A. The Utility of Explicit System Models . . . . . B. The Utility of Information Measurements within System Models C. How Do Brains Process Information? References

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Intuitively, we know that nervous systems do process information; just what do we mean by saying that, and how do they go about it? What quantities of information does a brain process under different conditions? Which kinds of experiments contribute to answering these questions? These are the ideas which we will study in this review, first by suggesting definitions of some presently somewhat fuzzy concepts, then by reviewing some particular experiments in that light. I. Basic Concepts

We will define only simple cases of our concepts (such as the concept “System X is processing information”) ; not because more complicated cases are not important (or that more compIex systems are not processing information), but because we are not able to say much of value concerning them. Nevertheless, our simple cases include many of the systems which have been studied in biology from the informational viewpoint. A.

SYSTEMS CONCEPTS

1. System and System Model Our discussions will be couched in terms of a System, usually consisting of the set-up for an experiment, including the human or animal 343

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DONALD 0. WALTER AND MARTIN F. GARDINER

subject. The system is surrounded by an Enuironment, with which it can exchange matter and energy. The system contains Subsystems as parts, in accord with some System Model, which is a schema of some kind, written down or perhaps only potential in our minds, as to what are the important parts or partitions of the system, for the purpose of explaining or understanding the current experiment. Translation Rules between System and System Model need to be explicitly provided by the experimenter when he writes up the experiment, if they are not obvious to the reader. A system model often emphasizes only one aspect of system function to the exclusion of others, as when a computer is considered as merely a heat generator, for the purpose of air-conditioning design. 2. Variables, Inputs, and Outputs

A Variable, for us, is a list (or, if the variable is continuous-valued, a range ) of possibilities-its “values”-exactly one of which is actualized any time the variable is observed or measured. A Pure Input to a system is a variable which is controlled by the environment of the system; thus, a variable over which the system under study has no influence, or at most an influence which is ignored in the current system model. Commonly, an input has a mechanical advantage over the input part of the system; or else the input may be amplified as the first step of system processing. An example of a pure input having a mechanical advantage is air vibration affecting the ear; according to recent work, a pure input which is amplified is the light which falls on the rods or cones of the retina. Of course, if one is interested in pupillary action, light intensity modulation by the pupil would precede amplification by retinal absorberamplifiers. Consequently, in the system model for a study of pupillary action, retinal illumination would not be a pure input; on the other hand, if we were considering a system model directed toward rods or cones which were illuminated, rather than toward the general illumination of the retina as a whole, such light would be a pure input. Thus, as noted, different system models may totally ignore aspects essential to another model of the same system. The effects of a change in an input can be detected only within the system after at least some slight lag, a lag which may or may not be of importance to the system model. But, as we will illustrate later (Section 11, C), an input may have occurred long before its effect on output can be detected. Input variables are not restricted to physical dimensions alone, as that same example shows.

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A Pure Output is defined symmetrically as a variable whose value is controlled by the system, with no effect or a negligible one from the environment. Commonly, the output subsystem has a mechanical advantage over the environment. 3. An Illustrative Application from Neurophysiology: Cross-Spectra of EEGs Cross-spectral analysis of EEG channels is an area in which inappropriate, or at least unexamined, system modelling has led to questionable interpretations. Cross-spectra ( or, what is equivalent for present purposes, cross-correlation functions ) between any two time series having the same duration can be viewed (Bendat and Piersol, 1966; Walter, 1963; Walter and Adey, 1963) as a numerical transformation of the numbers representing the two series. Calculation of cross-spectra makes sense only if theory predicts a relationship between the time series, either between simultaneous pairs of values, or at an approximately known lag. (See Menger, 1955, p. 177 ff., for the importance of the idea of the pairing of observations.) Certainly there is reason to predict some relationships between EEG channels at lags up to perhaps a second, and such cross-spectra have been estimated by various workers (Krendel, 1959; Walter and Adey, 1963; Walter et al., 1966; Dumermuth and Fluehler, 1967; Brazier, 1967a). a. The Filter Model. Interpretation of the cross-spectra depends on the system model which the experimenter had in mind, but sometimes that model is not clearly formulated. A model used in engineering, at the time when cross-spectral calculations were being imported from that field, is that of a generalized filter. Originally, the word “filter” was applied only to electronic circuits designed for isolating a single frequency band out of a mixture, but a more recent concept, which we call a generalized filter, considers the frequency-emphasizing or -attenuating characteristics of any circuit, at all frequencies. If a generalized filter is linear, then the cross-spectrum between input and output equals the square of the frequency-response function of the filter (Bendat and Piersol, 1966, 98 ff.). But the input and output of such a generalized filter must be in fact a pure input and pure output, as defined above, although that point is not often emphasized in the engineering texts. Unfortunately, one can speak as if the brain were a generalized filter. If this were the case, the cross-spectrum between two EEG channels would have that simple relationship to the frequency-response function of that filter; but frequently the supposition that what is recorded is a pure input or pure output cannot be verified, and consequently a confused use of the idea of

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DONALD 0.WALTER AND MARTIN F. GARDINER

“filtering” has sometimes resulted. There are three major alternatives to this probably inappropriate model. b. Alternative Models. We may consider the filter model as adequate except that there are two filters, one having part of channel A’s activity as input, and part of channel B’s as output; the other filter is oppositely directed. In this case what we record in either channel is neither a pure input nor a pure output, and Granger and Hatanaka (1964) have shown that interpretation of the cross-spectrum in this feedback case can be quite different from the single-filter case. Another alternative model discards the concept of lumped operations, and assumes that the brain acts more like a drum-head than like a telephone conversation; that is, that the electrodes which record EEG are collecting activity from areas which are merely parts of a larger area, within which activity is g,enerated and spreads in a gradually transformed way. Yet a third plausible system model (Walter and Adey, 1970) supposes that there are lumped signal input sources as in the first models, but that signals from quite a few of them are recordable in both channels; that relative strengths of signal in the two channels differ for different sources, because of differing locations relative to recording electrodes; and that each source usually acts for a brief period, perhaps a second, at one time. Spectra and crossspectra are well known to be time averages, so this model would propose that they are “merely” averages of “actually pointillistic” brain activity. Each of these possible brain models has something to recommend it, and some counter-indications against it. To distinguish among them, one may consider using stepwise complex demodulation (Walter and Adey, 1970), or approximate biophysical modeling ( Rkmond, 1968; Joseph et al., 1969; Walter, 1970). The purpose of introducing so many alternative brain models here was to dispel the impression that “filtering” is the only clear brain model compatible with cross-spectral calculations.

4. Properties of System Variables a. Informative Dimensions. Not all describable aspects of an input or other system variable contribute to the system’s information processing. An important part of system modeling is specifying which aspects one hypothesizes to contribute. For example, when considering sensory inputs, it has sometimes been tacitly assumed that general physical description of the variable is sufficient. But which physical aspects are critical? Sutton’s discussion (1969) puts this question forcefully, in a psychological context. Strong warnings that physical dimensions may be insufficient have been issued by MacKay (1956 and personal communication) in his suggestion that what sense


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organs yield is “clues to coping,” presumably without regard for physical tidiness of describability. Somewhat similar viewpoints have been expressed by others (e.g., Schmitt, 1959; Bullock and Horridge, 1965; Lettvin et nl., 1959; Barlow, 1963). Similarily our system model can incorporate hypotheses concerning informative aspects of variables within the system. Thus, for example, the question “What is the informative aspect of a spike train?” has received much attention (MacKay and McCuIloch, 1952; Wall et al., 1956; Bullock, 1962; Moore et al., 19%; Segundo et nl., 1966). An important feature of the system model, then, is the specification of hypotheses concerning information-bearing aspects of system variables. b. Coding. Having chosen hypotheses as to information-bearing aspects of system variables, we still have to attempt to describe the structure of each variable, in relationship to the system model, and the hypothesized transformations or relationships between variables. Such structural description may be thought of as specifying “coding” of the variables within the system model. One aspect of coding, for example, is the specification of graduation of the scale of a variable. If a variable is discrete, such as the number of nerve spikes in response to one stimulus, then its coding may be simply that number; or our system model might lead us to lump together “less than 10 spikes” vs. “more than 10.” If the variable is inherently continuous, such as an EEG voltage, but we choose to treat it discretely (for example by digitizing it), then we should show that the coding used was fine enough not to lose phenomena essential to our system model “in between the meshes.” If a very small wave, relative to the whole EEG signal, is to be studied, adequate fineness of digitization may be questioned; however, if the studied waves are relatively independent of the larger irrelevant waves, it can be shown that the little ones will be “carried through the digital divisions in such a way as to make their recovery from the digitized voltages as easy as from the original continuous voltages. If both input and output of a system or subsystem are continuous, then coding amounts only to specifying a sensitivity, or gain, function for the subsystem. It is important to emphasize the need to distinguish conceptually between the coding hypothesized to be operating within an information processing system, and the coding which we, as observers, choose to use in measuring and describing that system (Cherry, 1956). Our external observers’ coding may be chosen purely as a computational device to allow for calculations using informational measures; as is explained below (Section I, C, 3, c ) , the quantity of information seen by us is more easily inferred to be active in the system, than is our particular choice of coding or of informative dimension.

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B. INFORMATION: ITSDEFINITION AND PROPERTIES 1. Two Major Technical Meanings We will make little use of the various informal meanings of the word information. The first of our technical definitions will mostly follow one of Shannon’s (1948; Shannon and Weaver, 19491, while our second, covered in a later section, will attempt to define it as used in the phrase “information processing.” Shannon’s concept can perhaps be qualified as “selective information,’’ and it was first defined in terms of a system consisting of an encoder, a one-way transmission line, a decoder, and a fixed alphabet or repertoire of symbols, together with a known set of probabilities of occurrence for each symbol. If we translate this into a one-way teletype communication system, a typical input to the “encoder” is a message consisting of a string of letters, which it translates into a sequence of current pulses, which are then sent over the transmission line, where they may be “corrupted by extraneous currents sometimes called “noise.” The noisy signal is received by the “decoder” or teletype receiver, which, if the received current pulses are close to one of the patterns it is designed to respond to, will print a letter (though, of course, not necessarily the letter transmitted); if they are not very close, it may not print any letter. Important features of Shannon’s theory, before reaching its explicit numerical calculations, are the now-familiar terms of his discussion of information transmission by such a system. The information content of a particular symbol is viewed solely in probability terms, as representing a selection of one from among the set of possible symbols that could have been sent. The information transmission of a system, as a whole, is expressed in bits per symbol: a logarithmic transform of probabilities, which can be interpreted, if desired, as the average number of yes-no questions required to distinguish between different symbols. In biological applications of the theory, one does not often have a clear analog for each element of Shannon’s system, particularly for the letters and messages, even if one tries to employ the artifices used by Shannon for converting continuous signals into “message units.” There is one biological application, however, in which detailed correspondences with elements of Shannon’s original system are apparently not a prerequisite to calculation (though they remain so, to interpretation), These are applications in which mutual information can be defined, and it is to these that we turn. 2. Mutual Information and Entropy The theory of selective information, as extended to analysis of multiple stochastic variables ( McGill, 1954; Garner and McGill, 1956; Ashby,


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1964), has proved a useful computational tool in psychology (Miller, 1956; Attneave, 1959; Gamer, 1962); the same theory offers promise as a tool for biological systems study. For our purposes, a stochastic variable (S.V.) is a variable whose probability distribution function we either know or wish to discuss (Cornlke and Walter, 1970); this implies no need for a stochastic variable to be unpredictable. For example, “110-Volt alternating current” has a voltage which obeys

V(2) = 21’2(110)sin[2~(60t)] whose probability distribution function is prob(Vo I V

5 V o4-AV)

= (AV)(2/~)/[2(110)~ -

V02]1’2 If two S.V.’s are paired (Menger, 1955), we can define their joint distribution function (Dixon and Massey, 1969, pp. 395-399), and from that, their mutual information (Garner, 1962, Ch. 2). Also, we can define a function called entropy, for each of the stochastic variables. If we have a stochastic variable named X , whose two values x1 and x2 occur with probabilities 0.5 and 0.5, and another S.V. named Y, whose values yl and yz occur with probabilities 0.75 and 0.25, respectively, we still have not enough information (in the informal sense) by which to know whether any relation exists between X and Y. If we are told in addition that whenever x1 is observed, y1 is also, and that x2 is observed with yl or yz equally often, we can construct the table of distribution functions shown: X

Y1 Yz

0.5 0

0.25 0.25

0.75 0.25

0.5

1.00

~

0.5

Mutual information between X and Y is given by

P b l , Wz>

P(XZ, WZ)

+ P b l , YZ) 1g P h )P(Y + p(xz, yz)lg p(xz)p(yz) 0.5 0.25 + 0.25 lg - Oa5 Ig (0.5 X 0.75) (0.5 X 0.75) 2)

+O k O

0.25

-k 0*25Ig (0.5 X 0.25) = 0.20 - 0.15 0 0.25 = 0.30 bits/observation where lg means logarithm to the base 2.

+ +

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The entropies of X and Y are - P ( 4 k P(XZ> lg 4 - 4 lg 3 = 0.5 0.5 = 1 bit/observation H ( Y ) = -0.75 Ig 0.75 - 0.25 lg 0.25 = 0.30 0.50 = 0.80 bits/observation

H ( X ) = - P ( 4 k P(Zd =

+

-4

+

The minimum value of mutual information is zero bits per observation, which can only occur if the S.V.’s are statistically independent. Nonzero values for mutual information indicate that the variables are not statistically independent; I ( x , y ) achieves its maximum value (equal to the lesser of the entropies of the two stochastic variables) when the behavior of the variable having lesser entropy is completely represented in the values of the other variable. Values of I ( x, y ) intermediate between those extremes reflect intermediate degrees of relationship between the variables. Thus I ( x , y ) may be considered as a measure of amount of relationship between two variables (Garner, 1962, Ch. 2 ) . The similarities and differences between this measure and another common measure of relationship, the correlation coefficient, will be considered in the next section. There are two important caveats that should be emphasized. The first is that the phrase “statistically independent” should not obscure the fact that two variables, which appear independent by calculations based on a particular system model, might be seen to be nonindependent, when analyzed in terms of a more adequate model. For example, suppose we treat Y and 2 as stochastic variables, with

+

+

Y = sin(2x 0.001) 2 = sin(3x O.OOl), (where the 0.001 is added merely for convenience in tabulating the distribution functions); and suppose that we categorize Y and 2 into positive and negative only; finally, suppose that x is sampled in steps of 12. Under these conditions, the distribution functions will be

T/

2

y

+

-

-1

-1

1 which is apparently a case of “statistical independence,” whereas a finer graduation of the scales for the two variables would show that they are distributed along a three-leaved rose, and far from “statistically independent.” This example, though artificially contrived, illustrates the 2

2


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difficulty of attempting to infer particular models from statistical measurements alone. The second caveat is that mutual information and entropy are both averages over a set of observations; attempts to attribute their values to individual observations must contend with this averaged character. Another awkward fact that must be taken into account, in going from average bitslobservation for a single isolated observation out of a series to the entropy for two such observations in a row, is that if pairs of observations are statistically independent, then the total entropy is twice that for one; but if successive observations are nonindependently distributed, the total entropy of the two of them is less than that. Thus, the information transmission on particular trials may not be independent of previous trials; it can be different for different stimulus levels among the levels used, and so forth. These problems can be tested by multivariate techniques including multivariate information calculations (Garner, 1953, 1962).

3. Mutual Information Compared to Correlation Another example will illustrate the contrast between mutual information and a different, widely used indicator of relationship, the coefficient of correlation. If the joint distribution function of two paired stochastic variables is a close approximation to one of the family of two-dimensional Gaussian shapes, then the mutual information and the (square of the) correlation are functions of each other, and either one can fully summarize the strength of relationship [the function relating them is given by Kullback, 1959, p. 8,as I = --4 lg( 1 - r 2 )1. If the stochastic variables are far from joint-Gaussian distributed, however, this relationship breaks down; a particularly clear example of this occurs for a variable which has an approximately sinusoidal form, which is paired with values of its derivative ( which will be approximately co-sinusoidal). The correlation between a sine and a cosine is essentially zero (as one can see analytically by averaging their product), while the mutual information is 1 bit. Zero correlation indicates no linear relationship, but is often interpreted loosely to indicate no relationship; one bit in this example is consistent with the fact that if one knows the value of the sinusoidal variable, one knows that its derivative has one of two values, and not any value in between. The reverse situation, in which mutual information suggests (by equaling 0 ) no relationship, while the correlation is not zero, is mathematically impossible, Further, the definition of mutual information includes, without any change, the relations between variables having only nominal scales, or combinations of numerical with nominal-in short, any pair which has a joint distribution function or table has a mutual informa-

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tion, while correlation is difficult or impossible to define in many such cases. Thus, mutual information is a more comprehensive parameter for summarizing relationships than is correlation. A consequence of the above statements is that reports of correlation between variables can be translated into a value of mutual information (or a minimum value of it, since correlation may have missed something); but the reverse is not always possible, as the sine and cosine show. Calculations with entropies and mutual information can be considered as similar to the analysis of variance, an analogy we have already mentioned and which has been clearly illustrated by Garner and McGill ( 1956) and in Garner ( 1962).

4. Information Transmission Now if the mutual information between two S.V.’s is nonzero (hence necessarily positive), we will say, with Garner and McGill and Ashby, that information is “transmitted” between the S.V.’s. This is merely a restatement of the observation that the behavior of the S.V.’s is not independent of one another. It does not, in itself, tell us anything about the direction or pathway of information transmission. Thus, one variable may be influencing the other, both may be influencing each other, or both could be reflecting the influence of a common, unmeasured causal agent outside the system. In the special case of a system where we have identified certain variables as inputs, and certain other variables as outputs, those identifications do specify the directions of information flow, and that the information flow passes through the system. Generalization of mutual information to sets of more than two S.V.’s (provided that compatible pairings are clear) are also considered by Garner (1962, Ch. 3); the parameter which naturally generalizes mutual information is called by Garner the total constraint of the set. If the total constraint of a set of S.V.’s is nonzero (which again implies positivity), we will say that information is being transmitted among the S.V.’s. If all the S.V.’s are either pure inputs or pure outputs, and if the total constraint of the full set is greater than the sum of the total constraints of the input subset and that of the ouput subset, we will say that information is being transmitted from inputs to outputs. In analogy with the bounds on mutual information, the total constraint cannot be greater than either the sum of the entropies of the input stochastic variables, or the sum of the output entropies, whichever is less. Now, a fundamental theorem about Shannon’s selective information is that its amount never increases through a processing or transmission step, and usually decreases. Kimura (1961) has shown how selective information can be locally increased by natural selection. But such


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processes enter only slightly, and at very restricted points in the system models we will be considering. Thus, nongrowth of information will be an important constraint for our studies here.

5. An Illustrative Neurophysiological Application of Information Calculations Suppose that we calculate that, between some sensory input and a particular behavioral output, information is transmitted at the rate of, say, 5 bits per stimulus, and we observe some internal variable (or, more strictly, an output which is controlled by one such variable) which we hypothesize on anatomical grounds to carry, or at least reflect, all of the observed transmission. But suppose that we calculate (on the basis of the coding hypothesis which is part of our system model) that the information transmitted from sensory input to the internal variable is only 3 bits per stimulus. In accordance with the nongrowth of selective information, then, information about the input, passing through this internal variable, to the behavioral output (this is equal to the “total constraint” of input, internal variable, and output, considered as stochastic variables) cannot be greater than 3 bits per stimulus; so either our coding hypothesis is insufficient, or else our hypothesis about this variable carrying or reflecting all that transmission is contradicted. If, by revising our coding hypothesis, we can now recognize that 5 bits per stimulus are being transmitted throughout our three variables, we can then conclude that the transmission hypothesis is not contradicted (though, of course, there may be parallel channels not covered in our anatomical model). But this by no means shows that the animal or human subject uses the same coding we have used; it only shows that 5 bits per stimulus can be reasonably hypothesized to be transmitted through the internal variable whose correlate we have observed. This distinction between a variable which is informative to us as observers, according to a coding scheme which we develop, and a variable which can be hypothesized to be informative to the animal, but perhaps with an unknown coding scheme, is a distinction to be borne in mind continuously.

6. Variability of l n f o m t i o n Measure Estimates Although information measures that we have discussed have the advantage of generality, it must also be borne in mind that they, like other measurements on biological function, are likely to contain some sampling variability. The theory of such variability has been worked out for some simple cases (Miller and Madow, 19%);and, in the case of underlying normal distribution, direct calculation of information mea-

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sures from correlation coefficients is possible. However, in more general situations, especially where correlation does not apply, sampling variability of information coefficients is unknown. If we would like to use or estimate information measures in an experiment where the known sampling theory does not apply very well, we must seek for “physiological significance” rather than statistical significance. Physiological significance is defined mainly by the ability to replicate the finding, by making related findings which tend, although to an incalculable degree, to support the first finding, and by the nonappearance of the finding in control experiments or control analyses-in short, by all classical scientific tests, without direct appeal to refined statistical theory of the distributions of the variables studied. Nonparametric statistical tests can often be applied to increase the power of such classical replication and control procedures; but to counsel, as we do, nondependence on refined distributional tests of statistical significance, is merely to accept the fact that experiment will almost always be somewhat beyond the range of perfectly applicable statistical theory. C. INFORMATION PROCESSING

1. Definition Now we are ready to define “System X processes information” in a special case: If we have (1) a system model of X which has just one input and one output, ( 2 ) a hypothesis as to what dimension of the input is informative, and as to its coding, and similarly for the output, and ( 3 ) we consider the input and output as stochastic variables (S.V.’s) and hypothesize a pairing or range of possible pairings between them, and finally (4) we calculate, or infer from some equivalent experimental evidence, that information is transmitted from the input S.V. to the output S.V., we will assert that system X processes information. The extension to the case of several inputs and/or outputs requires only the substitution of the appropriate plurals into the special definition, so we have defined our statement in general. Thus, for example, we can assert that the brain processes sensory information, because sensory inputs and motor outputs are known, codings for them have been proposed, the times over which pairings might be expected to be meaningful have been suggested, and relationships surely translatable into mutual information between some of these inputs and outputs have been demonstrated,

2. Apparent Multivariate Causality In biological systems, information flow may be more complex than in the special systems discussed above. In particular, one must take care


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to be explicit in formulating what system is being considered when discussing systems which seem to have nonpure inputs and outputs. If we are considering the general illumination of the retina as an input, in a system wherein the modulating action of the pupil is important, we can perhaps agree on a system model in which general illumination impinges on the cornea as a pure input, which is then acted upon by the pupil, and only then reaches the retina; hence we have defined a system in which the input is pure, and internal operations affect it. To take a slightly more difficult example, we may consider the inputs to the human temperaturecontrol system. If we formulate a system model in which heat flow from outside the body surface is an input, which is then transmitted to the tissues and the bloodstream, along with endogenous heat, which only then affects any hypothesized hypothalamic temperature receptor, then the heat flow from outside is a pure input, in this system model. Thus, by taking care to define the system’s boundaries, and to stick with those definitions, reasonable approximations to pure inputs and outputs can usually be defined. The situation is somewhat different inside a system, however, where feedback may be an essential feature. Granger and Hatanaka (1964) have formulated a careful discussion of concepts closely related to information flow, in feedback situations. Unfortunately it is too technical and complex to develop here, but we can make use of two concepts shown by Granger to be important in that discussion: the importance of time delays in systems and system models, and the concept of apparent multivariate causality. As we remarked in defining inputs, the effect of a change of input can be felt in an output only with some delay. Although this is perhaps a truism, Granger shows its unexpected power in organizing the system model, when, because of feedback, it might otherwise fall into confusion. Zadeh and Desoer (1963) have also emphasized the essentiality of this fact in system theory generally. Even feedback of a subsystem output as part of its own input must involve some delay of response, in order to avoid intractable instabilities. Granger exploits this and other concepts concerning greater partial predictability of some variables’ values by knowing the past values of other variables, to define his concept of apparent multivariate causality. Briefly, if our ability to predict the future values of variable A from A’s own present and past values are improved by knowing the past values of variable H, Granger argues for asserting apparent causality between them. Then, by ingenious exploitation of this concept, he reduces to their minimum complexity the paths of apparent causation among sets of many variables. In cases in which causality can be tested by other methods (such as by direct manipulation of a variable and observation of results), tests

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such as Granger discusses would be of only academic interest. On the other hand, one is often confronted in biological systems with situations where variables cannot be manipulated. Causality cannot be tested in such situations in direct ways; under these situations Granger’s tests become of practical interest. Testing actual systems for apparent multivariate causality is often impractical, at least according to Granger’s general method, because of its apparent requirement for large amounts of data representing a single state, though not necessarily in continuous segments. Thus, while Granger’s concept is a clear unified approach in principle, in practice the requisite calculations must be done in indirect ways, through such devices as more explicit or more particular hypotheses about the forms of relationship than would seem theoretically to be required. II. New Insights Arising from the Information Systems Viewpoint

Early evoked response experiments mapped areas of obvious responses to repeated, fairly strong stimuli and showed the apparent time course of the brain’s “handling” of those stimuli. Less noted at that time were parallel changes in background activity, which may just as easily represent any information processing by the brain concerning such stimuli, as do the foreground responses. Indeed, regional differentiation in background changes is at least as distinctive as foreground differences (Walter et al., 196713; Adey et al., 1967), and with the addition of automatic reliability screening (Walter et al., 1967a) perhaps more so.

A. COMPETING SYSTEMMODELSFOR REPRESENTING WEAKEVOKED RESPONSES More recently, of course, weaker and more differentiated stimuli have become feasible, because weaker responses can be detected and characterized; but this leads to new problems. 1. Interpretation of Weak Responses It is desirable to attenuate stimuli so far that single response waves are no longer easily detectable, in order to approximate more closely a physiological range of stimulus intensities. It is worth a moment’s contemplation from the evolutionary point of view that the stimuli ordinarily encountered in real life are received by the brain with small electrical responses: only exaggerated stimuli produce easily distinguishable disturbances. The difficulty which arises, when this more natural range of intensities is used in experiments, is that another class of system models becomes plausible, and must be considered along with the simpler


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models adequate to stronger waves. This raises the question of how to choose among possibilities. a. Fixecl-Respome-Plus-Noise Model. The usual models assume the existence of an entity called “the response,” observable as a wave in the EEG. To this fixed response wave are added irrelevant waves, that is, waves possibly representing some other brain function but not related to our stimuli. Such waves are often called “noise,” which conforms adequately to one usage of this word in engineering. If this model is assumed, then averaging the observed waves should produce an increasingly accurate picture of “the response.” This is the system model which lies behind the temptation to ignore background activity. It is well known that long-continued averaging often produces a somewhat changing picture. Although it is not always explicitly calculated how much of the change could be due to residual effects of noise, it is sometimes explicitly assumed that the response changes “slowly.” b. Variable-Response-PIus-NoiseModel. A different model, however, can apparently explain the data just as satisfactorily from the statistical viewpoint, while accounting for a wider class of brain activity. This model assumes that each response to a stimulus of weak intensity is one of a large collection of different possible responses. Precisely which one is produced in a particular case is due to what we will call random natural selection. One characteristic of this large collection is that its mean response function equals the single fixed response of the first model; but aside from that, its members may have almost any waveform within certain bounds of amplitude (so they will not be larger than background). Irrelevant activity is identical to the description in the former model. In response to stimuli of increasing intensity, the variability of the collection of responses decreases, so that just to the extent that its members become visible against the irrelevant activity, they become more similar to one another and to their average (Adey, 1965, 1967; Brazier, 1963). This model may seem at first to be merely gratuitous complication, and to be rejected by a cut of Occam’s rusty old razor. But if we consider the connection between grossly recorded evoked potentials and the cellular activity which may plausibly be assumed to underlie them, it is natural to think that, in response to a weak stimulus, there will be certain inherent variability in the degree to which different cells are activated. The effect of threshold for spike generation certainly provides a rationale for expecting some variability in participation of lower-level afferent pathway cells in at least some phases of producing an evoked response. If one further accepts the idea that responses in cells on the liminal fringe at all levels may be influenced by minor features of their preceding

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activity, one has a reasonable substrate for predicting that single evoked potentials to stimuli of ordinary intensity will be somewhat variable in shape, a variability which will decrease with increase of stimulus intensity. But if this second, superficially more complex, but more widely explanatory model is accepted for evaluation, a number of consequences follow, some of them perhaps unexpected. The first is that there is no longer any “real” response, which the average approximates. The average response is merely the average of a collection of possible responses. It still has some descriptive importance, but it is no longer necessarily a good representative of each single response. Any single response may crudely resemble the average, but it may have variations of its own, which may be as important for its further processing as are its resemblances to the average response. A second consequence of the new model is that the variance about the average response has to be attributed in part to activity of irrelevant structures, as with the previous model, but part of it (and an unknown proportion, at present) must be attributed to response variability. Thus, response variability becomes as interesting a neurophysiological parameter as is the average response. (This idea is not unique to this model; see, e.g., Brazier, 1964, 1967a.) We are not limiting our concept of response variability to those components attributable to uncontrolled or unmeasured confounding inputs (Donchin, 1966; Sutton, 1969) or which might signal change of state of information processing by the organism (Brazier, 1963, 1967b; Ritter and Vaughan, 1969; Gardiner, 1969). Both these sources of response variability can play important roles in influencing responses observed in a given evoked response experiment, and interpretation of variability, in terms of such factors, can form a significant part of a system model. We wish in addition to suggest the existence of sources of variability which may be inherently impossible to control. An improved general computation procedure for enhancing responses to weak stimuli, which could be applied to this question, is explained in Section 11, A, 2. Recalling that we spoke of random natural selection of participant cells as an aspect of the variable-response model, we can infer a final consequence of the model, based on two ideas argued elsewhere by Walter (1968). One, that because it will never be possible to measure enough of the parameters of the liminal neurons to predict accurately which form of response will occur, there is a fundamental indeterminism in the operation of the brain. If, as argued in that paper, the scale of this indeterminacy is great enough to invalidate predictions of future brain state frequently, then we should be building models based on the ideas


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of fundamental uncertainty which have been used by quantum physicists. If this viewpoint seems to the reader to be too extreme, because the uncertainties are “not that large,” and predictions based on averages are “good enough” for all needed purposes, we invite that reader to establish his viewpoint by an explicit “error theory” for brain models. The second idea argued by Walter in this connection is that whereas such indeterminacy would seem to leave us impaled on the “chaotic” horn of the chaos/ determinism dilemma, in fact there are many levels of selection, from natural selection of wavelets, or of participant cells, to gradually more nearly conscious selection of preferred thoughts. In the multiple levels of selection (described more fully in that article) resides the substrate for emergence of creative thought (analogously to the emergence of new species in evolution of animals by natural selection), and for holding the person who selects thoughts partially responsible for his action, as in fact we do now, in legal and social practice. 2. Autoregressive Enhancement of Weak Responses If we could subtract the irrelevant activity, evoked responses could be studied individually, allowing clear visualization of their form. A mathematical approximation to the irrelevant activity can be derived from Wiener’s theory of filtering predictors or, as is discussed below, from applying autoregressive prediction to the prestimulus activity. Depending on the closeness of approximation that is possible, the method may allow us to approach this ideal. a. System Model. Suppose we hypothesize that the “background activity” is unaffected by a stimulus, which merely adds its wave to the record. This is simulated by a system model in which the background activity is produced as pure output by a subsystem which has no input (as far as this experiment is concerned). The stimulus is modeled as a pure input: to a subsystem ordinarily inactive, but which in response to the stimulus emits a wave that is added to the output of the background subsystem. While the brain probably does not act precisely like this, it is an adequate approximation for purposes of modeling responses to small, repeated, uninteresting stimuli. To implement this system model numerically, we employ the method of autoregressive prediction. b. Improved Calculution Method. Let us visualize the data from a single EEG channel as a series of digitized values, sampled every 0.01 sec. We wish to assemble a battery of autoregressive predictors for all time shifts up to the duration of the response we are trying to enhance. The predictors will be applied to the data simultaneously, at the instant of stimulus, to predict the whole course of what the EEG “might have been” if no stimulus had occurred. Thus, each of these autoregressive

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predictors will have as its input the EEG values just preceding the stimulus, and each will be predicting a different time shift into the future. But before we can apply these predictors, they must be derived on the basis of the background activity between responses. Each predictor will be derived by a separate application of a multiple regression program; the best choice would be a stepwise multiple nonlinear regression program (such as BMD02R, see Dixon, 1968). This program also calculates a quality index for each regression, telling us how trustworthy each value will be on the average. The data supplied as input values for this fitting should be representative of the state of the EEG during stimulation (see Section 11, A), but data during response to stimuli should not be included; thus this method is only directly applicable to experiments where either the EEG state is not changed by stimulation, or the stimuli are infrequent enough for the regression fitting to be possible using data occurring between stimuli. First we derive a predictor for 0.01 sec time shift, This corresponds to a simple autoregressive study of the EEG ( Fenwick, 1969). When that function has been derived, a separate run of the regression program must be made for lag 0.02 sec; here the independent variables are values of voltage at least 0.02 sec prior to the dependent value. When this function is known, the process is repeated with a minimum lag of 0.03 sec, etc., up to the duration of the assumed response. This collection of autoregressive functions is then applied to predict the whole course of the EEG following each stimulus. If this set of predictions is made in the vicinity of each stimulus, and the predicted values are simply subtracted from the observed values, the result will be an improved version of the response evoked by each stimulus. Because of the quality index developed for each predictor, we will also know how much trust to put in the calculated response values, under our explicit hypothesis that stimuli do not affect background. Thus, if the prediction is very accurate throughout the duration of the response, we can treat the corrections as giving us an almost perfect picture of each response, and variability can be studied directly. If the predictors are moderately good over some of the duration of response (accounting, say, for half the observed variance), then we will have to treat matters a little more circumspectly, and draw our conclusions in terms of analysis of variance. If the predictors account for essentially none of the variance, this method will have failed in its goal of sensitization. But only trial can show which situation is the actual one in each real experiment. Of course, there is no reason in principle to limit the predictors in the above study to linear ones, or to the channel being predicted. In a nonlinear study a stepwise program must be used, since the number of


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potential predictors grows rapidly; of course, statistical problems concerned with optimality of coefficients also increase. c. Control Models. However, such technical details should not be allowed to interfere with reconsideration of the scientific problem, which is not limited to “Is the response well approximated by a fixed-response model, or must we assume large inter-response variability?” This was our question in Section 11, A, but the theory of brain activity is in too underdeveloped a state for us to rest content with a dichotomous interpretation of results of such a calculation; we must also compare with other plausible models, The most obvious modification of our proposed system model would be to allow the stimulus to have some effect on the “background” box; in other words, to make the stimulus an input to that box, .too. Another possibility, which in a way is more compatible with the conception that neurons may be diverted from their previous tasks into making responses, would be to make the output of the stimuIus box an input to the background box. These models could perhaps be distinguished by more refined statistical analyses of the predictions proposed for the previous study; but the feeling arises that perhaps this is the level of analysis at which “boxes” at this scale may become inappropriate, and a distributed model would be closer to physiological reality (or at least to obvious histological suggestions). Considerations such as the above, in which not control observations, but control models are considered, are essential to avoiding premature specificity (Hardin, 1957), given the present state of development of brain research

B. SELEC~ED EXPERIMENTS ON NEURAL INFORMATION PROCESSING 1. Stark‘s Experiment on Neural Coding in the Crayfish As one particular example of a valuable attempt to apply Shannon’s theories to information processing in neural systems, we consider Stark‘s treatment of a “Nerve Impulse Code” (summarized in Stark, 1968) for light intensity in the caudal ganglion of a crustacean. We have selected this work for critical treatment because it is important work by a mathematically sophisticated neurophysiologist. However, we feel that Stark‘s conclusioii that he has supported one hypothesis about the nerve impulse code, and contradicted another, is not adequately justified by his discussion because of his failure to provide adequate translation rules between system model and experimental variables. Nevertheless, this is otherwise a good example of applying information calculations to a neural system. a. System Model. Stark accepts the conclusion that light intensity is the informative dimension of input from the animal’s environment in this

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system, and we agree. He proposes (Stark, 1!368, p. 44) a system model which focuses on the functioning of the two similar photoreceptor cells in the ganglion. Their pure input is light intensity, which affects a single Coder (why he diagrams a single Coder is not clear, since the cells do not seem anatomically to be closely connected). The Coder has two similar pure outputs, which are strings of letters on two parallel channels (presumably, though we are not told so directly, representing the cells’ axons). The two channels reach a single Decoder, which has one pure output, walking movements. In the free crayfish, these walking movements would change the light intensity by taking the animal to a new location, but Stark has negated this feedback for easier study, by fixing the animal’s location, Returning to the Coder‘s two outputs, they are represented as producing five letters per minute each, with every fifth letter equal in the two outputs, while the other four are unequal. How we are to relate these letters to nerve impulse recordings is not clear, for Stark immediately proposes two general coding hypotheses, without suggesting translation rules which allow us to infer the identity of the ‘letters” which, as just described, are the output of the Coder in his systems model. It is Stark’s discussion of these two coding schemes which we feel does not support his conclusion about them, partly because of this lack of translation rules. b. Coding S.chemes. One scheme uses “detailed pulse patterns” as its informative dimension, and the other uses average frequency of pulses. Let us first follow his argument concerning the average-frequency hypothesis. Stark‘s only guidance toward translating the average rate into the letters of his system model is, “we must now decide what can be considered the average duration of a symbol. It seems reasonable to assume that the system can establish or measure the average firing frequency for a time approximately equal to its time constant.” This leads to a duration estimate of 1.2 sec. This estimate seems plausible as an order of magnitude, but would lead to an estimated 50 symbols per minute, rather than the 5 of his system model. He then investigates the relation between the two cells’ firings (which was represented in his system model by having one letter out of five equal) and reports a cross-coincidence function between the two pulse trains. This function is similar to a cross-correlation function (Moore et al., 1966); either function tests the correlation between spike trains, for approximately simultaneous observations, and at many specific time off sets. Stark says “ . . . it is clear . . . that the [cross-coincidence] function does not show any significant deviations from [its] mean. We feel that this is the crucial experimental result.” He apparently feels that this statement establishes the independence of the two trains. In broad terms


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we agree, but let us expand on the steps in this argument, in order to examine his conclusion more closely. We gave earlier (Section I, B, 3 ) and example of two variables which were uncorrelated but far from statistically independent. Could something similar apply to Stark‘s two pulse trains, so that, although uncorrelated according to his cross-coincidence function, they would actually be far from independent? Our example was only for simultaneously observed variables; the result is different if one examines, as the crosscoincidence function does, many time offsets in the pairing between variables. If the probability of firing in one cell were modulated by a sine wave, while the other was modulated by the corresponding cosine wave (this assumption is compatible with Stark‘s reported auto-coincidence functions for these trains ) , their cross-coincidencefunction would be at its mean value (meaning zero cross-covelation) for simultaneous observations. But the cross-coincidence would reach a high value for a time offset corresponding to a quarter cycle of the sine wave, a low value for three-quarters of a cycle, and so on, oscillating just like the sine wave. Stark‘s cross-coincidence function does not seem to oscillate so, and therefore his conclusion about independence seems reasonable, But how is this result to be related to his system model, in which two strings of letters have exactly one in five letters equal? If, as Stark (1968) suggests (p. 46), we are to assume that either nerve has a repertoire of approximately 20 letters (though this assumption is scarcely discussed), independent coding, such as we have just agreed upon, would lead to an average of one coincidence per 20 letters. Thus, the lack of translation rules between experimental variables and system model makes Stark‘s conclusions very difficult to follow. We also mentioned Stark‘s alternative hypothesis of coding by “detailed pulse patterns,” presumably analogous to Morse Code or the like, represented by something like long and short bursts of pulses. Stark‘s only guidance as to translation rules from pulse trains to letters, under this hypothesis, is the clause, “Taking the average symbol duration b to be approximately 50 msec. , . . ” Since he assumes the same repertoire of letters for this scheme as for the other, it is solely the ratio between the hypothesized symbol durations which makes this coding appear to transmit information 33 times faster than the other. In a final argument concerning this code, Stark states, “If the detailed pattern were the code through which the information is transmitted, a high correlation between the two pulse trains at the level of the detailed pattern should be observed.” But if one were to take seriously the hypothesis that the coding operations in a crayfish photoreceptor cell represented input having a high rate of information transmission, one

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would be forced at the minimum to include in one’s experiment and in one’s system model some input or inputs also displaying that rate. If one imagines a photoreceptor cell actually coding at such a rate, it does not seem particularly implausible that two similar cells could do coding at that rate, but using different codes, Of course, this would suggest that the decoding is well adjusted to each of these codes-unless one wishes to model the conception that the photoreceptor cells do much more elaborate coding than is ever used by the whole animal. All such complicated schemes seem relatively implausible on the general grounds that the crayfish‘s behavior does not seem to require such high information rates. But until such hypotheses are taken seriously enough to be explicitly tested by experiments with appropriate system models as well as control models, we will not know whether they might be correct.

2. Mountcastle and Werner’s Sensory Transmission Experiment A series of interesting studies from Mountcastle’s laboratory illustrates a variety of uses of information theory in a neurophysiological investigation. The basic aim of the series of studies (Mountcastle, 1967) is to test hypotheses regarding coding and information transmission in the human somatosensory system, by comparing information-handling performance in psychophysical test situations with the ability of the investigator to match that performance by proper observation of signals recorded from the nervous system of the unanesthetized cat or monkey. We will concentrate here on one of their earlier papers (Werner and Mountcastle, 1965),which presents some basic information calculations. The input to their system model is brief skin indentation over an Iggo capsule, and the output is the firing of the capsule’s nerve fiber; these clearly are pure input and output in our terms. They chose the stimulus pattern with unusual care, seldcting discrete levels for skin indentation. One advantage of a discrete scale is strictly computational: discrete information calculations are often within the range of hand calculation, whereas continuous ones often are not. It might be thought that a second advantage is that discrete scales avoid questions about the informative dimension-whether indentation, pressure, or some other. But if the variance in the relation between these dimensions is small in comparison to their ranges, the variables are interchangeable. The transformation between them cancels out of the calculation, yielding equal information values for either one. a. Coding. As their coding hypothesis, Werner and Mountcastle chose to count the number of spikes occurring between stimulus onset and a fixed time delay after onset, which was varied as one of the analysis parameters over the range 20 to 500 msec. They had shown earlier that


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the typical firing pattern for this nerve preparation was a short ontransient burst, followed by a relatively steady, lower-rate plateau. They state that the rate of firing during the on-transient does not appear to be sensitive to the degree of skin indentation, while the firing rate within the plateau region does depend on skin indentation. Nevertheless, they choose to measure “rate” as the number of spikes from stimulus onset, apparently accepting the approximation that the number of spikes in this initial transient will be a relatively constant error in each rate estimate, b. I n f o r n t h Transmission Calculationr. The basic calculation of this experiment was to measure average information transmission between controlled discrete indentations and nerve fiber output “rate.” Following the procedure used in parallel human psychophysical experiments (Garner, 1962, Ch. 2), the ability of the system to transmit information was measured first with a low degree of stimulus uncertainty (we would say low entropy; i.e., with only a small number of different values of input to be distinguished), then with increasingly higher degrees of stimulus uncertainty, until a point of apparent saturation in information transmission was reached. The authors report that 31 discrete levels of indentation were chosen for each experiment, and output “rate” was recoded into “bins” two spikes wide. At stimulus uncertainties less than 5 bits, it is not clear whether only a subset of these discrete levels was used, or if the data were obtained by pooling observations from adjacent values of indentation. If a subset of the authors’ original list of levels was used, it is also not made explicit whether the levels were distributed evenly or randomly over the whole range of indentation. Their finding is that the system transmits adequately at low stimulus uncertainties, and begins to saturate when stimulus uncertainty reaches 2.5 to 3 bits (i.e, 6 to 8 stimulus values). c. Disczcssim. This experiment demonstrates and quantifies the lack of precise correspondence between skin indentation and observed output firing in single trials. Interpretation and generalization from the author’s finding of saturation of information transmission would be facilitated by knowing their data acquisition and computation procedures in greater detail. For instance, we could then better interpret the contrast between the single-trial result just mentioned and the same author’s finding, reported earlier in the same paper of more exact functional relation, if several trials’ rates at equal indentations are averaged together. The authors call attention to an apparent parallel between their single-neuron findings and human visual and auditory psychophysical experiments (Garner, 1962; Miller, 1956). In those experiments individuals are asked to make absolute discriminations ( classifications) on stimuli which differ along a single (physical) stimulus dimension; infor-

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mation transmission between stimuli and classifications usually saturates at a level of 2 to 3 bits. In an important effort to bring together previously completely disparate fields of experimentation, they suggest that their single-neuron finding is compatible with the hypothesis that the source of this saturation is variability of output by the first-order neurons. This extrapolation must, however, be viewed with some caution, as it entails additional assumptions. For instance, it seems necessary to assume that human visual and auditory neurons have just the same information saturation behavior as monkey somatosensory neurons. The recording conditions in the Werner-Mountcastle experiment restrict neural activity elicited to single first-order neurons, or to small populations of first-order neurons at most. Thus Werner and Mountcastle in effect suggest that the information-transmitting capacity of neurons en masse is no better than that of individual elements. The authors themselves indicate that this may not necessarily be a valid assumption, in another section of the same paper: They suggest that the ability of human subjects to make fine relative discriminations between stimuli does depend on averaging across populations of neurons. It is not stated explicitly why absolute discrimination ability should depend on single-unit limitations, while relative discriminations should be able to make use of average activity within populations. Other studies from Mountcastle’s laboratory ( Mountcastle et al., 1962, 1963, 1969; Werner and Mountcastle, 1963; Talbot et al., 1968) have attempted to explore animal-human parallels in other ways. These studies appear in some cases to be impeded by the difficulty of studying directly the integration of the activity of naturally stimulated groups of neurons; nevertheless Werner and Mountcastle’s extrapolation to human psychophysics is in some cases quite challenging. In any case, their method of approach to information calculations about single-receptor information transmission are worthy of emulation.

3. Gardiner’s Differential Information Processing Experiment a. Description. A variety of recent studies have shown that evoked responses, recorded from human scalp during tasks which require a subject to make decisions based on some or all of the stimuli presented, can be influenced not only by the physical characteristics of particular stimuli, but also by their relationship to the decision task (Davis, 1964; Spong et al., 1965; Satterfield, 1965; Debecker and Desmedt, 1966; Cohen and Walter, 1966; Donchin and Cohen, 1967; Sutton et al., 1967; Mast and Watson, 1988; Ritter and Vaughan, 1969). The intent of such studies is to offer insight into central mechanisms of information processing in


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man. The tools of information processing analysis urged in this review will now be applied to such work performed by one of us (Gardiner, 1969; Gardiner and Walter, 1968, 1W9). Previous studies had shown differences between evoked potentials elicited by task-relevant stimuli when compared either to evoked potentials elicited by other, task-irrelevant stimuli presented during the same experiment, or to evoked potentials elicited by physically similar stimuli, in situations where the stimuli did not have special task relevance. Gardiner sought to investigate further the effects of decision tasks on evoked activity by comparing evoked potentials elicited by physically identical stimuli, in two task situations which both required the subjects to make use of information supplied by the same stimuli, but which differed in the physical characteristic of the stimulus which supplied relevant information in each task situation. The stimulus set consisted of four possible tones against a moderate noise background: high and loud, high and soft, low and loud, and low and soft, The pitch and intensity differences were very small and were adjusted so as to be difficult for each subject to detect, as well as being about equally difficult across subjects, according to the observed error rates. Runs of 48 tones were presented, containing in randomized order equal numbers of each of the four possibilities. In some runs the subject was instructed to write down pitch ( H or L ) of each tone, after its presentation, while in others he wrote down loudness ( L or S ) . (In some control experiments the letters were A, B, C, D.) Vertex-mastoid EEGs were recorded; in addition, a pulse signalling the moment of initiation of writing was recorded, which allowed compensation for any premotor potentials. The evoked potentials were evaluated by a stepwise discriminant analysis program ( BDM07M, see Dixon, 1968) which selected timessince-stimulus at which the voltages differed most reliably between groups of responses. It also calculated a weighting function for combining voltages at those times, whose value gives the program’s best guess as to which group a given single response should be assigned to. This program was applied to decide whether there were reliable correlates of a number of input dimensions; for instance, correlates of task without regard to stimulus pitch or stimulus loudness, or, as another example, stimulus pitch without regard to task or stimulus loudness, as well as correlates of more complex combinations of dimensions. The original publications detail the precautions, replications, and controls needed to ensure that the claimed effects are not likely to be due to sampling fluctuations. The results showed specific components in the evoked potential which appeared reliably different in the two task situations.

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Other evoked potential components were identified as correlates of loud or soft stimuli; components reflecting interaction between task and stimulus loudness were also found. Correlates of stimulus pitch were less reliable, even though pitch and loudness discriminations had been adjusted for approximately equal difficulty. Interpretation of neurophysiological or neuroanatomical detail must await further experimentation, but we feel that Gardiner has identified correlates of differential information processing in his subjects, relatively freed from effects of levels of attentiveness and of quantitative rates of information transmission. In order to justify this assertion, we give next a translation of his experimental and computational design into the terms of quantitative information theory. b. Identification and Measurement of Inputs and Outputs. Gardiner’s inputs are stimulus loudness (loud or soft), stimulus frequency (high or low), and task instructions [pitch classification ( P ) or loudness classification ( V ) 1. Each of these input variables has an entropy of 1 bit, since each has two values with equal probabilities (see the formulas in Section I, B, 2); they are also statistically independent in all combinations (that is, have zero mutual information), as part of the experimental design. One of the output variables is the letter written by the subject, whose values, in most runs, were limited to S, H, or L, so the joint distribution function of task with letter would be Letter Task

S

H

P V

O c

a o

L b d

1

2

z 1

b t d Gardiner’s other output variables, of course, were the voltage values as a function of time-since-stimulus, constituting the evoked responses. The discriminant analysis work quantifies the information transmission between various combinations of input variables on one hand, and particular re-codings of the voltage values on the other hand. The components of the evoked activity which were reliable correlates of input parameters (or of interactions between them) exhibit the information transmission from those inputs. The linear combinations of voltage variables computed by the discriminant analysis program constitute suggested recodings, each of which creates a new, continuous-valued output variable, dependent on the primary voltage variables. These new variables are also converted by the program to a discrete-valued variable, which is the machine’s c

a


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categorization of each response. Thus, in studying transmission from input variables to machine category, we can treat the linear combinations as intervening variables (Section I, B, 4). c. Discussion. The two tasks described above require close to the same quantity of attention by the subjects, to the same single modality of stimulation. The difficulty of the tasks was said by the subjects, and shown by the scores, to be both fairly high and close to equal. The stimuli were always from the same limited set, and the forms of motor response were very similar. Finally, the probabilities of each sensible letter response were always equal, so the entropy of the stimulus dimension to be discriminated was unvarying. By thus equalizing or removing a number of common confounding variables, and by using a sensitive detector of reliable differences between evoked response populations, Gardiner was able to study the information transmission from task to its correlates in the evoked response, a finding we believe to be new and promising for elucidation of cerebral information processing. Ill. Summary and Prospect

A. THEUTILITY OF EXPLICIT SYSTEMMODELS We hope to have illustrated that the formulation of an explicit system model, representing the ideas in the experimenter’s mind, often helps to clarify both what the experiments have proved, and what they may have left untouched. In one published experiment (Section 11, B, l ) ,a model of neural coding provided by the experimenter did not seem translatable into a description of his experimental system. In our last example (Section 11, B, 3), formulation of the system model helped to clarify the rather complex relations between shifting groupings of auditory stimuli, and an unfamiliar method of analyzing the response evoked. Another great value of this method of formulation is that it naturally suggests alternative models which might not otherwise occur to the experimenter. Such related ideas, when viewed as “control models,” are not easily disposed of by the sometimes helpful criterion of maximal simplicity. An example (Section 11, A, 1) was the common model of an unchanging, or slowly changing evoked response to a repeated stimulus, which seems most simple when only the average responses recorded by an external macroelectrode are considered. In contrast, the more complicated model of randomly varying responses unites the observations of macroelectrode average responses, and of somewhat variable cellular responses to the same stimuli recordable by microelectrode. Thus “simplicity’’ depends on the context accepted for explanation; and clear

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DONALD 0. WAL”J3R AND MARTIN F. GARDINER

differentiation between competing system models may require improved experimental or analytical methods (Section 11, A, 2). Finally explicit system models are good communicative and pedagogical devices, and allow us to concentrate on particular subsystems without losing sight of likely interactions from other subsystems.

B. THEUTILITY OF INFORMATION MEASUREMENTS WITHIN SYSTEM MODELS In spite of some measurement difficulties it seems that bits of information per observation or per stimulus have definite value in neurophysiology for two reasons: The nongrowth theorem puts helpful bounds on what can be transmitted between regions; and the amounts of selective information, in themselves, seem to catch some important features of what the nervous system is transacting between its regions. For example, by providing a quantitative summary of important aspects of unicellular responses to sensory inputs ( Section 11, B, 2), information calculations bring into sharper focus our ignorance of most interactions between neighboring cells subserving parallel functions. The concurrent difficulties of recording such interactions in mammalian preparations, and of the usual methods of analyzing them, have apparently discouraged work in this area. Since information calculations are very well adapted to handling this kind of data, it is to be hoped that experimenters may take up the technical challenges again. ( Multiple-unit recordings, in which individual cells are not separated, contribute very little to this question, because interaction between individual cells is usually completely obscured. ) By quantifying some aspects of a rather complex experimental design (Section 11, B, 3), information calculations have made clear another lack in present theory: a method of treating sensory qualities which would be as satisfactory as the information-theory treatment of quantitative sensory information flow.

C. How Do BRAINSPROCESS INFORMATION? We do not feel that we, or neurophysiologists generally, have arrived at clear conceptions of how brains do process their information. But we do feel that the time is ripe for models and experiments on the subject to be brought together. Several reviews have appeared in recent years on the subject of neural information processing (e.g., Adey, 1965, 1967; Fields and Abbott, 1963; Gerard and Duyff, 19612; MacKay, 1969; Moore et al., 19%) which are valuable in calling attention to the vast variety of observable phenomena that contribute to information processing, and of


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possible models to explain them. As in other areas of biological experimentation, much can be and has been done by nonnumerical discussion and exploitation of phenomena; but the questions unresolved at that level can profitably be treated numerically, if an appropriate mathematical model is available. We believe that the numerical study of information transmission and transformation, which we have illustrated in this review, is the appropriate model for many phenomena of neural information processing. It may be thought that the parts of the brain whose function we have been formulating are merely subservient to some higher center, perhaps inhabited by a “little green man,” who really runs that brain. Although we are not able as yet to propose explicit, yet testable models of higher function, we do claim that the kinds of models we have been proposing, if suitably adapted to incorporate the epistemological taboos of quantumlike uncertainty, are adequate to model all of brain function. To paraphrase Laplace, we have not found the concept of a higher and qualitatively different type of brain function to be a necessary hypothesis. Elaborate and ingenious proposals for information processing by brains have been put forward by students of artificial intelligence. Unfortunately, these suggestions seem to be hard to translate into biological experiments or, at least, into experiments which are accessible with present teclmiques. In some few cases this has led to deliberate seeking for new experimental techniques by the theorist; but in most cases, we feel that the models should have been developed with experimental testability more firmly held in the theorist’s mind. Is this nontestability merely due to ultrasophistication on the part of the artificial intelligencers, or to ultraprovincialism by the neurophysiologists? We think there is some justice in both accusations; but also, that the frustratingly slow progress of such collaboration is due to real, inherent difficulty of both fields. An important impediment which can, as we have proposed above, begin now to be removed, is the habit of using deterministic models of brain function. Fluctuating, stochastic, but information-based, models would be less of a caricature of the protean variety, yet teleonomic effectiveness of that highest output of the human brain, creative thought. ACKNOWLEDGMENTS Partially supported by USPHS Grants 3-R01-NB-02501 and 5T1 MHO 6415-13, and by AFOSR Contract AF 49( 838)1387. Bibliographical support by the UCLA Brain Information Service, a member of the NINDS Neurological Information Network, is also gratefully acknowledged.

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374


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AUTHOR INDEX Numbers in italics refer to the pages on which the complete references are listed. A. .

Abbott, W. C., 370, 372 Abdulla, Y. H., 31, 62 Abe, S . , 269, 286 Adams, C. W. M., 31, 62, 266, 280 Adams, R., 205, 220 Adey, W. R., 229, 250, 345, 346, 356, 357, 370, 372 Adler, E., 258, 287 Adrian, E. D., 228,250 Aghajanian, G. K., 27, 28, 50, 53, 55, 57, 62, 87, 89, 101, 103, 116, 118, 121, 122, 125, 139, 156, 247, 250 Agranoff, B. W., 238, 250 Aitken, J. T., 1, 24 Akao, M., 165,180 Akcasu, A., 313, 321 Alberici, M., 165, 178 Alexandrovskaya, M. M., 277, 280 Algeri, S., 55, 62 Alivisatos, S. G. A,, 215, 220 Alksne, J. F., 87, 89 Alquist, R. P., 160, 178 Altman, J., 233, 250, 274, 280 Alfman, K. I., 273, 282 Altman, M., 169, 178 And&, N.-E., 80, 81, 83, 85,89, 93, 94, 96, 97, 99, 101, 104, 105, 106, 108, 109, 111, 113, 115, 116, 117, 119, 121, 122, 125, 126 Andersen, N., 191, 220 Andersen, P., 18, 19, 24 Anderson, J. €I., 139, 158 Aniun, F., 189, 202,220 Appel, S. H., 264, 280, 295, 321 Aprison, M. €I., 248, 250, 325, 326, 328, 329, 331, 332, 333, 334, 335, 336, 338,340, 341 Ariens, E. J., 161, 162, 184, 174,178, 179 Arimura, A., 114, 125 Arnold, M., 32, 62 Aschheim, E., 265,287 Ashby, W. R., 349,372 Ashcroft, G. W., 115,121

Atkinson, D. E., 256, 257, 280 Attneave, F., 349, 372 Austin, L., 184, 220, 278, 285, 293, 294, 295, 297, 298, 300, 301, 302, 305, 307, 308, 321, 323 Autilio, L. A., 295, 321 Axelrod, J., 39, 41, 46, 63, 66, 73, 91, 102, 126, 129, 139, 143, 156, 158 Azcurra, J. M., 165, 168, 174, 178

B Baastrup, P. C., 116, 121 Baba, W. I., 167,178 Babich, F. R., 240, 250 Babickf, A., 263,283 Bachmann, L., 38, 65 Baggio, G., 274, 282 Bailey, H. Z., 273, 276, 284 Bak, I. J., 53, 62 Baker, P. F., 278, 280 Baldessarini, R. J., 111, 124, 129, 157 Baldridge, H. D., Jr., 162, 178 Bard, P., 77, 89 Bargman, W., 303, 321 Barlow, H. B., 347, 372 Barlow, R. B., 161, 178 Barnard, J. W., 3, 24 Barondes, S . H., 61, 62, 238, 239, 247, 250, 272, 273, 280, 290, 298, 303, 309,321,322 Barr, M. L., 276, 280 Barrett, R. E., 50, 65, 104, 123, 126, 139, 158 Barrnett, R. J., 27, 28, 29, 30, 31, 32, 33, 35, 38, 41, 46, 48, 51, 62, 64, 65, 66, 102,122 Bartholini, G., 104, 105, 107, 122 Bartles, E., 161, 178 Bass, A., 278, 284 Basurmanova, 0. K., 272, 276, 280 Bates, H. M., 104, 122 Bateson, P. P. G., 233, 250 Bayliss, 0. B., 31, 62 Beach, G., 233, 234, 239, 250

375

376

AUTHOR INDEX

Beck, C., 274,280 Bedard, P., 101, 125 Beer, M., 27, 62, 66 Belik, J. V., 262, 285 Belleau, B., 173, 174, 178 Belleville, R. E., 117,,124 Beloff-Chain, A., 258, 280 Belpaire, F., 302, 323 Bendat, J. S., 345, 372 Benington, F., 183, 202, 204, 220, 221 Bennett, E. L., 240, 242, 250, 251 Bennett, G. S., 262, 280 Bennett, H. S., 103, 123 Bensch, K. G., 33, 41, 46, 48, 51, 65, 66, 315, 322 Berger, B. D., 240, 251 Bergman, R. A., 31, 57, 63 Berman, A. L., 72,89 Bernard, C., 248, 250 Bertler, A., 80, 84, 89, 139, 156 Bertram, E. G., 276, 280 Bessman, S. P., 259, 280 Best, J. B., 51, 62 Best, R. M., 231, 246, 250 Beuding, E., 167, 178, 179 Bevitz, A., 279, 280 Bhatnagar, R. K., 74, 81, 84, 91 Bieth, R., 269, 282 Bignami, G., 237, 251 Bishop, G. H., 4, 24 Bittner, G. D., 296, 322 BjorMund, A., 78, 85, 89, 96, 98, 122 Blackburn, K. J., 139, 157 Blackstad, T., 100, 122 Blackstad, T . W., 87, 89 Blaschko, H., 39, 62 Bleier, R., 77, 89 Bloom, F. E., 27, 28, 29, 30, 38, 41, 46, 48, 50, 51, 53, 55, 57, 59, 60, 62, 66, 84, 86, 87, 89, 101, 102, 103, 121, 129, 139, 156, 247, 250 Bloom, M. G., 31,63 Blum, J., 105, 122 Blumcke, S., 310, 321 Boadle, M. C., 29, 62 Boakes, R. J,, 206, 220 Bocci, V., 269, 280 Bodian, D., 277, 280 Bogdanski, D. F., 71, 89, 91, 129,157

Bogoch, S., 233,234,250 Bok, S.' T., 2, 24 Bondareff, W., 41, 46, 48, 51, 62, 102, 122, 302, 323 Bondeson, C., 279,280 Bondy, S., 272, 273, 280 Booth, D. A., 234,250 Borisy, G. G., 316, 317, 321, 324 Boroviagin, V. L., 274, 281 Botturi, M., 102, 122 Boucher, R., 80, 91 Boucher, T., 101, 125 Boullin, D. J., 38, 62 Bouvier, G., 80, 91 Bovet, D., 237, 251 Bowman, R. E., 233, 234, 251 Boyarsky, L. L., 301, 324 Bradley, P. B., 189, 206, 220 Brady, J. V., 68, 89 Brassfield, C. R., 4, 24 Brattgard, S. O., 270, 272, 273, 274, 280, 281, 284 Bray, J. J., 278, 285, 293, 294, 297, 298, 300, 301, 3U5, 307, 308, 321, 323 Brazier, M. A. B., 345, 357, 358, 372 Bridger, J. F., 1, 24 Briggs, I., 206, 220 Briggs, M. H., 242, 251 Brodie, B. B., 69, 71, 90, 91, 129, 157, 335, 341 Brodskij, B. I., 270, 274, 276, 280, 281 Brodskij, V. J., 274, 287 Brooks, V. B., 21, 25 Brown, D., 345, 374 Brown, D. M., 264, 288 Brown, G . L., 72, 90 Brown, J. D., 170, 178 Brown, P. B., 8, 25 Brust, M., 301, 324 Brzin, M., 278, 282, 294, 295, 322 Bubash, S., 240, 250 Bucy, P. C., 68, 91 Budd, G. C., 46, 48, 51, 62 Bullock, T. H., 228, 229, 251, 347, 372 Bunney, W. E., Jr., 129, 157 Burdwood, W. O., 303, 312, 321 Burhard, W. P., 104, 122 Burk, D., 136, 157 Burke, J. P., 36, 46, 51, 59, 66

AUTHOR INDEX

Burke, R. E., 6, 24, 25 Bush, H., 258, 280 Butcher, L. L., 96, 97, 104, 105, 107, 113, 122, 126 Butcher, R. W., 165, 167, 168, 174, 178, 180 Butcher, S. G., 105, 126 Byrne, W. L., 240,251

C Cagan, R. H., 169, 179 Cajal, S. R., 2, 3, 7, 8, 9, 10, 15, 17, 20, 21, 24 Cammermeyer, J., 271, 281 Campbell, M. K., 266, 280 Cannata, M. A., 30, 34, 35, 37, 62 Cannon, W. B., 72, 90 Capella, C., 30, 33, 66 Carl, J., 256, 254 Carlisle, D. B., 304, 309, 321 Carlson, P. L., 240, 251 Carlsson, A., 78, 79, 80, 89, 90, 93, 95, 96, 97, 100, 102, 106, 107, 111, 112, 113, 115, 119, 121, 122, 333, 341 Carlton, P. L., 333, 341 Carnay, L., 279,287 Carpenter, M., SO, 90 Casola, L., 238, 250 Caspersson, T., 78, 90, 270, 281 Catarzano, R., 258, 280 Causey, G., 266, 278, 281 Cavallito, C. J., 197, 220 Cellino, M., 278, 281 Cervoni, P., 72, 91 Chaikoff, L. L., 266, 285 Chain, E. B., 258, 280 Chamberlain, T. J., 239, 251 Changeux, J. P., 172, 178, 179 Chapas, A. F., 276,281 Chase, T. N., 116, 124, 139, 157 Chaubal, K. A,, 270, 285 Chentsov, I. IJ. S., 274, 281 Cherry, C., 347, 372 Chi, Y.-M., 166, 167, 168, 179 Chiappetta, L., 240, 251 Chiocchio, S. R., 30, 34, 35, 37, 62, 66 Chopra, S. P., 262, 287 Chornock, F. W., 247, 252, 298, 301, 323 Chorover, S. L., 240, 251

377

Chothia, C., 204, 220 Chu, W., 217, 220 Ciofalo, V. B., 239, 251 Clare, M. H., 4,24 Clark, L. C., 263, 287 Clark, R. H., 241, 253 Cleghorn, R. A,, 116,125 Clementi, F., 41, 48, 51, 62, 102, 122 Clouet, D. H., 263, 281, 294, 321 Coggeshall, R. E., 4, 25, 51, 80, 61, 65 Cohen, B., 7, 25 Cohen, E. B., 87, 90 Cohen, H. D., 238, 239, 250, 251 Cohen, J., 366, 372 Cohen, L., 366, 372 Cohen, L. B., 279, 281 Cohen, P. P., 259,281 Colburn, R. W., 39, 40, 62, 64, 129, 157 Cole, M., 80, 90 Collins, G. H., 276, 284 Colonnier, M., 3, 20, 21, 24, 86, 90 Constantinescu, E., 259, 281 Copeland, J. S., 31, 63 Coppen, A., 115, 116, 128 Coppola, J., 114, 122 Corey, E. J., 191, 220 Cornke, J., 349, 372 Corning, W. C., 240, 251 Corrodi, H., 32, 33, 62, 78, 90, 93, 104, 107, 108, 109, 111, 112, 113, 114, 115, 116, 117, 118, 119, 121, 122, 123 Corson, J. A., 240, 251 Costa, E., 38, 50, 55, 62, 65, 139, 158, 327, 328, 341 Cotton, F. A., 32, 62 Cotzias, G. C., 104, 122, 138, 157 Coupland, R. E., 30,33, 35, 37, 62, 102, 122 Cowden, R. R., 276, 284 Coxon, R. W., 256, 261, 281 Coyle, J. T., 107, 122, 129, 134, 136, 137, 138, 139, 157, 158 Crawford, J . M., 143, 157 Crawford, T. B. B., 115, 121 Cremer, J. E., 269, 281 Cuknod, M., 263, 266, 285, 286 Cumings, J. N., 272, 285 Cummins, J., 268, 281

378 Curtis, D. R., 143, 157 Cuthbert, A. W., 167, 178

AUTHOR INDEX

D'Monti, B., 262, 287 Domagk, G. F., 241,253 Donchin, E., 358, 366, 372 Douglas, W. W., 40, 63 Dray, A., 206, 220 Dresse, A,, 95, 96, 108, 123, 124, 125 Droz, B., 38, 46, 48, 50, 53, 59, 63, 66, 103, 123, 247, 251, 261, 278, 281, 290, 291, 292, 298, 300, 308, 314, 322, 323, 324 DuBois Reymond, E., 127, 157 Duggan, F., 53, 65 Dumermuth, G., 345, 372 Duncan, D., 35, 41, 63 Durant, R. C., 162, 178 Durell, J., 165, 168, 169, 170, 176, 178 Dutcher, J. D., 210,251 Dutta, C. R., 9, 17, 24 Dutton, G. R., 272, 281 Duvoisin, R., 104, 123, 126 Duyff, J. W., 223, 251, 370, 373 Dyakonova, T. L., 272, 276, 280, 281

D Daginawala, H. F., 235, 252 Dahlstriim, A., 39, 51, 59, 61, 63, 79, 80, 81, 83, 85, 89, 90, 93, 94, 95, 96, 97, 99, 101, 102, 105, 119, 121, 122, 123, 124, 128, 157, 302, 309, 310, 311, 315, 321, 322 Dale, H. H., 4, 24 Daneholt, B., 272, 273, 281 Da Prada, M., 37, 38, 66, 105, 107, 122, 138, 157 Darian-Smith, I., 366, 374 Das, N. B., 258, 287 Davenport, J. W., 239, 253 Davies, R. K., 262, 281 Davis, H., 366, 372 Davis, J. M., 129,157 Davis, R. E., 238, 250 Davis, W., 264, 280 Davison, P. F., 261, 281 E Dawson, D. M., 272, 281 Eccles, J. C., 10, 16, 17, 18, 24, 127, 157, Dawson, R. M. C.,260,286 242, 251 Deanin, G. G., 295, 322 Eccleston, D., 115, 121, 139, 158 Deb, C., 31, 63 Eckhardt, E. T., 161, 178 Debecker, J., 366, 372 Edds, M. V., Jr., 297, 324 Dellweg, H., 264, 281 Edelman, G. M., 262, 280 Delwyg, H., 234,251 Edman, K. A. P., 176, 178 DeMyer, W. E., 314, 323 Edstrom, A., 270, 278, 279, 280, 281, Dencker, S. J., 115, 123 293, 294, 295, 322 Dengler, J. J., 129, 157 DeRobertis, E., 29, 39, 41, 46, 48, 53, Edstrom, J. E., 170, 178, 270, 271, 272, 275, 276, 278, 281, 282, 283, 286, 63, 65, 102, 103,123, 125, 128, 157, 293, 322 165, 168, 174,178 Egyhazi, E., 234, 239, 251, 252, 272, Descarries, L., 53, 59, 63, 103, 123 275, 283 Desmedt, J. E., 366, 372 Ehinger, B., 78, 89 Desoer, C. A., 355, 374 Dettbarn, W. D., 278, 282, 294, 295, 322 Ehrenpreis, S., 162, 165, 174, 178 Ehringer, H., 80, 90 Deutsch, J. A., 237,251 Eichner, D., 270, 271, 275, 278, 281 Devine, C. E., 41, 46, 48, 63 Einarson, L., 271, 276, 281 Dewan, J. G., 258, 281 Eliasson, R., 217, 220 Diamond, M. C., 242,250 Di Girolamo, A,, 278, 285, 294, 301, 323 Elliott, K. A. C., 143, 157 Di Girolamo, M., 278, 285, 294, 301, 323 EM, R., 247, 251 Emaizumi, R., 53, 64 Dingman, W., 262, 281 Emmens, M., 233, 234,250 Dixon, M., 136, 157 Enesco, H . E., 240, 251 Dixon, W. J., 349, 360, 367, 372

379

AUTHOR IiVDEX

Engel, J., 96, 97, 104, 105, 107, 122 Engstrom, A., 269, 278, 281, 285 Epstein, A., 89, 91 Eranko, O., 33, 63 Ernst, A. M., 106, 123, 139, 157 Essman, W., 237, 251 Esterhuizen, A. C., 41, 46, 48, 63, 102, 125 Estrom, J. E., 235, 251

F Facillace, L., 117, 125 Falck, B., 78, 79, 84, 85, 89, 90, 93, 100, 102, 122, 123, 139, 156 Faltin, J., 275, 277, 285 Farini Duggan, H., 103, 125 Farrell, K. E., 41, 63 Farrow, J. T., 240, 253 Faure, C., 261, 273, 281 Fawcett, D. W., 317,322 Feldberg, W., 72, 90 Fenichel, R. LA,,240, 251 Fenwick, P. B. C., 360,372 Ferster, C. B., 328, 329, 331, 340, 341 Field, J. B., 16.9, 178 Fields, W. S., 370, 372 Fink, R. P., 77, 87, 90 Fischer, J., 263, 283 Fischer, J. E., 59, 63, 73, 90 Fischer, S., 278, 281, 293, 294, 322 Fiszer, S., 165, 178 Fitschen, W., 184, 220 Fjerdingstad, E. J., 240, 241, 251, 253 Flexner, J. B., 238, 239, 251 Flexner, L. El., 238, 239, 248, 251, 252 Fluehler, H., 345, 372 FolbergrovL, J., 257, 260, 261, 281, 287 Folkerth, T. I,., 331, 341 Foote, W., 116, 121 Ford, D. H., 272, 281 Formby, F., 40, 63 Fortner, J. G., 34, 65 Fox, C. A., 3, 9, 17, 24 Frank, K., 3, 5 , 6, 25, 26 Frazer, H. F.. 117, 124 Frazier, W. T., 4, 25 French, P. C., 139, 157 Freshman, A., 271, 288 Freyzs, L., 269, 282

Friedberg, S. L., 168, 179 Friede, R. L., 310, 322 Friedel, R. O., 169, 170, 176, 178 Friess, S. L., 162, 178 Fujivara, C., 258, 280 Furchgott, F., 72, 91 Furgiuele, A. R., 333, 341 Furst, S., 263, 282, 284 Fuxe, K., 38, 39, 53,59, 63, 79, 80,81,83, 84, 85, 89, 90, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 121, 122, 123, 124, 125, 126, 128, 139, 149,157

G Gabella, G., 41, 63 Gaddum, J. H., 327,341 Gaito, J., 236, 252 Gaitonde, M. K., 256, 258, 262, 263, 282 Galambos, R., 240, 251 Galsworthy, P. R., 171, 179 Galvan, L., 241, 253 Gambetti, P. L., 295, 321 Garattini, S., 101, 118, 125 Gardiner, M. F., 358, 367, 372 Gardner, R., 240, 251 Gariglio, P., 278, 281, 293, 322 Garland, J. T., 169, 170, 176, 178 Garner, W. R., 348, 349, 350, 351, 352, 365, 373 Geffen, L. B., 301, 302, 312, 314, 322, 323 Geiger, A., 256, 261, 282 Gejnishman, Y. Y., 270, 277, 280, 282 Gellene, R., 104, 122, 138, 157 George, R., 138, 157 Gerard, R. W., 223, 239, 251, 260, 261, 282, 289, 301, 313, 322, 324, 370, 373 Gerber, G., 273, 282 Gerhart, J. C., 174, 178 Gerlach, O., 1, 24 Gerner, R., 234,251, 264, 281 Gerschenfeld, H. M., 4, 26 Gershoff, S. N., 258, 282 Gesell, R., 4, 24 Gfeller, E., 129, 139, 142, 157, 158

380

AUTHOR INDEX

Giacalone, E., 101, 118, 124, 125 Giacobini, E., 268, 271, 276, 282, 287 Giarman, N . J,, 28, 30, 41, 46, 48, 50, 51, 59, 60,62, 66, 78, 91 Giudetta, A., 294, 295, 322 Giuditta A., 264, 269, 273, 278, 282, 287 Glasky, A. J., 239, 251 Glass, E. A., 169, 179 Glassman, E., 233, 235, 238, 251 Glees, P., 247, 251 Globus, A,, 20, 21, 22, 24, 298, 322 Glowinski, J., 39, 63, 111, 114, 118, 124, 129, 157 Goel, B. K., 262,287 Gollnitz, G., 258, 259, 260, 288 Goldberg, J. M., 8, 25 Goldberg, M. E., 239,251 Goldberg, S., 298, 322 Golgi, C., 2, 25 Gomirato, G., 273, 274, 282 Gommi, B. W., 163,180 Goodwin, F. X., 129, 157 Gorbman, A,, 303, 322 Gordon, B., 41, 48, 62, 102, 122 Gordon, M. W.,295, 322 Gotoh, F., 263, 285 Grafstein, B., 290, 291, 297, 298, 301, 308, 309, 314, 322, 323 Graham, J. D. P., 41, 46, 48, 63, 64 Graham, R. C., 169, 179 Grampp, W., 170, 178, 235, 251, 270, 276, 281, 282, 293, 322 Grande, F., 278,282 Granger, C . W . J., 346, 355, 373 Gray, A. P., 197, 220 Gray, E. G., 3, 25, 104, 124 Green, A., 129, 139, 157, 158 Green, A. I., 148, 153, 155, 156, 157 Green, J. D., 23, 25 Green, J. P., 139, 158 Green, M. R., 278, 286, 293, 324 Griffith, J. S., 244, 245, 251 Grillo, M., 102, 124 Grillo, M. A., 28, 29, 41, 46, 51, 57, 59, 63 Grob, D., 185, 220 Gropetti, A., 55, 62 Grossman, S. P., 228, 251 Gruber, C. P., 264,288

Grundfest, H., 4, 25 Giinther, G., 258, 287 Gueudet, R., 41, 44, 46, 50, 65 Guillery, R. W., 70, 77, 87, 90 Gunne, L. M., 111, 123, 124 Guroff, G., 272, 282 Gurowitz, E. M., 241,251 Gutmann, E., 260, 262, 263, 265, 268, 274, 278, 282, 283, 303, 309, 310, 324

H Haggendal, J., 93, 115, 121, 309, 322 Hagbarth, K. E., 16,25 Hager, G., 32, 62 Haidarliu, S. H., 270, 276, 277, 282, 285 Haider, M., 366, 373 Hkjek, I., 2s0, 265, 271, 283, 284 Hake, T., 31, 32, 63 Halaris, A., 53, 63 Halick, P., 239, 251 Halkerston, I . D. K., 244, 251, 266, 282 Hall6n, O., 270, 275, 282 Halstead, W. C., 231, 245, 252 Halter, K., 260, 282 Hamberger, A., 268, 269, 275, 277, 282 Hamberger, B., 80, 90, 94, 95, 97, 99, 100, 107, 111, 112, 113, 121, 122, 123, 124, 137, 149, 157 Hamberger, C. A., 275, 282 Hamlyn, L. H., 19,25 Hammer, G., 275, 282 Hammer, W., 109, 122 Handler, J. S., 167, 179 Hanker, J. S., 31, 57, 63, 65 Hanson, J., 317,322 Hansson, L., 113, 123 Hara, T. J., 303, 322 Hardin, G., 361, 373 Harfenst, K., 205, 220 Harrison, W., 104, 106, 126 Harrison, W. H., 34, 63 Hartman, H. A., 278,286 Hartman, J,, 270, 275, 277, 284, 285 Harvey, J . A., 68, 69, 70, 71, 85, 90 Hashimoto, Y., 53, 64 Haslett, W. L., 138, 157 Hasson-Voloch, A., 165, 178 Hatanaka, M., 346, 355, 373

381

AUTHOR INDEX

Hattori, A., 38, 64 Hawkins, J., 167, 178 Hayashi, K., 170, 179 Heacock, R. A., 34, 64 Heald, P. J., 170, 179, 262, 264, 265, 282 Hebb, D. O., 226, 242, 243, 247, 251 Hechter, O., 244, 251, 266, 282 Heimer, L., 77, 84, 86, 87, 90, 91 Hekhuis, G. L., 259, 281 Heller, A,, 68, 69, 70, 71, 73, 74, 75, 76, 81, 84, 85, 86, 87, 90, 91, 101, 124 Hendelman, W. J., 304, 316, 323 Hendler, R. W., 185, 220 Hendley, E. D., 129, 139, 142, 157, 158 Hendrickson, A., 298, 301, 322 Henseleit, K., 129, 131, 140, 157 Herblin, W. F., 240, 253 Herman, M . H., 315,322 Herrick, C. J., 67, 90, 248, 251 Herriman, I. D., 273, 282 Hertting, G., 34, 65 Hild, W., 4, 25 Hill, A. V., 249, 252 Hill, B. J., 34, 63 Hillarp, N.-A., 39, 66, 78, 79, 80, 83, 89, 90, 93, 95, 96, 97, 100, 102, 119, 121, 122, 123, 124, 128, 157 Hille, B., 279, 281 Hillman, D. E., 9, 17, 24 Hillman, H., 268, 269, 282, 283 Himwich, H. E., 325, 341 Hingtgen, J. N., :331, 332, 333, 334, 335, 336 338, 340, 341 Hirschberg, E., 260, 261, 283, 288 Hiscoe, H. B., 289, 290, 307, 324 Hodgkin, A. L., 278, 280 Hoefke, W., 113, 124 Hoehn, M., 104,123,126 HGkfelt, B., 104, 109, 111, 113, 121 Htikfelt, T., 28, 30, 32, 41, 46, 48, 49, 51, 53, 55, 57, 59, 63, 64, 80, 83, 84, 90, 93, 94, 95, 96, 97, 98, 99, 100, 102, 103, 104, 106, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 121, 122, 123, 124, 149, 157, 278, 281, 293, 322 Hoffman, T. A., 187,220 Hogans, A. F., 272, 282

Hokin, L. E., 168, 169, 171, 179, 180 Hokin, M. R., 168, 169, 171, 179 Holland, Y., 301, 324 Hollister, L., 117, 125 Holt, C. E., 111, 240, 251 Honig, W., 328, 341 Hopsu, V. K., 30, 33, 64 Hopwood, D., 30, 33, 35, 37, 62 Hori6kovSr, M., 265, 274, 283 Horn, G., 233, 250 Hornykiewicz, O., 80, 90, 104, 124, 129, 138, 157 Horovitz, Z. P., 2.40, 251 Horridge, G. A., 347,372 Horton, E . W., 189, 220 Horvat, J., 236, 252 Horvath, N., 261, 282 Howell, R. R., 273, 285 Hoy, R. R., 296, 322 Hromek, F., 85, 89 Hubel, D. H., 11, 25 Hughes, A., 240, 251, 304, 322 Hunt, H. F., 68,85,90 Hunter, C., 312, 322 Hunter, G. D., 273, 282 Hurwitz, R. M., 297,323 Huttenlocher, P. R., 315, 322 Huxley, H. E., 317, 322 HydAn, H., 231, 232, 233, 234, 239, 245, 251, 252, 261, 268, 270, 271, 272, 273, 274, 275, 276, 277, 280, 281, 282, 283, 285 Hyvarinen, J., 366, 373

1 Ichii, S., 53, 65 Imaizumi, R., 53, 64, 65 Irwin, L. N., 230, 235, 253 Isbell, H., 117, 124 Ishii, S., 53, 64, 65 Ito, M., 10, 17, 24 Itoh, T., 264, 283 Iversen, L. L., 38, 39, 40, 64, 95, 126, 129, 134, 143, 146, 148, 153, 157 J Jacob, M., 269, 273, 282, 283 Jacobson, A,, 240,250 Jacobson, A. L., 240, 250

382

AUTHOR INDEX

Jacobson, M., 246, 252 Jacobwitz, D., 33, 64 Jahn, T. L., 312,322 Jahr, M., 104, 123 Jaim-Etcheverry, G., 28, 30, 38, 39, 46, 50, 51, 55, 60, 64, 65 Jakoubek, B., 260, 262, 263, 265, 266, 268, 269, 274, 278, 282, 283, 284, 286 Jankovii., B. D., 236,252 Jankowska, E., 313, 322 Janssen, P. A. J., 108, 124 Jarlsted, J., 269, 275, 284 Jarvik, M. E., 240, 251, 273, 280 Jasinski, A., 303, 322 Javoy, F., 114, 118,124 Jenden, D. J., 138, 157 Jequier, E., 50, 61, 64 John, E. R., 229, 230, 252, 366, 374 Johnson, J., 297, 298, 299, 300, 305, 308, 309, 323 Johnson, T. C., 272, 284 Johnston, P. V., 268, 269, 281, 284, 286 Jones, B., 94, 125 Jonsson, G., 30, 32, 33, 46, 48, 55, 62, 64, 78, 90, 93, 94, 103, 104, 110, 114, 116, 118, 119, 122, 123, 124 Joseph, B. S., 298, 323 Joseph, J. P., 346, 373 Jouvet, M., 89, 91, 94, 117, 124, 125 Judes, C., 269, 273, 282, 283 Jukes, M. G. E., 115,121 Jund, R., 273, 283

K Kachmann, R., 314, 323 Kado, R. T., 356,372, 374 Kai, M., 170, 179 Kameda, K., 21, 25 Kandel, E. R., 4, 23, 25, 26, 230, 252 Kanoh, T., 170,179 Kapeller, K., 53, 64, 95, 124, 310, 311, 322 Kariya, T., 336, 341 Karlin, A., 162, 163, 172, 179, 210, 220 Karlsson, J. O., 291, 297, 301, 308, 309, 322, 324 Karnovsky, M. J., 169, 179 Karnovsky, M. L., 169, 179

Kasa, P., 310, 322 Kashing, D. M., 184, 220 Kasler, F., 31, 63 Kasper, C. B., 184, 220 Katchalsky, A., 244, 252 Katz, I., 116,124 Katz, J. J., 231, 245, 252 Katz, R. I., 139, 157 Kavanau, J. L., 176, 179 Kawakita, Y., 261, 282 Kazachasvili, M., 270, 275, 277, 285 Keatinge, W. R., 176,179 Kelleher, R. T., 236, 252 Kelly, A. M., 53, 64 Kelly, D. E., 41, 64 Kennedy, D., 296, 322 Kennedy, E. P., 168,180, 247, 252 Kerkut, G. A., 278, 284, 302, 309, 312, 313, 322 Kerr, D. I. B., 16, 25 Kerr, L. M., 258, 280 Kety, S. S., 139, 158 Keynes, R. D., 279, 281 Khan, A. A., 272,284 Kidwai, A. M., 298, 309, 323 Kiefer, G., 271, 284 Kiefer, R., 271, 284 Kimble, D. P., 233, 234, 239, 250 Kimura, M., 162, 179, 352, 373 King, H. W. S., 184, 220 King, L. J., 256, 257, 284 Kipnis, D. M., 166, 180 Kirkepar, S. M., 72, 91 Kirschner, N., 40, 64 Kitto, G. B., 242, 251 Klainer, L. M., 168, 179 Klarvans, H., 104, 106, 126 Klatzo, I., 277, 284 Kleijn, E. E., 260, 284 Kliiver, H., 68, 91 Kobinger, W., 113,124 Koe, B. K., 50, 61, 64, 139, 158 Koe, K. B., 117, 124 Koelle, G. B., 29, 64, 294, 323 Koenig, E., 270, 271, 278, 284, 293, 294, 323 Koenig, H., 263, 273, 284, 301, 323 Koenig, H. L., 298, 322, 323 Kopin, I. J., 39, 63, 116, 124, 139, 157

AUTHOR INDEX

Kordon, C., 11.4, 118, 124 Kornhuber, H. H., 366, 374 Korr, I. M., 247, 252, 298, 301, 323 Kostowski, M., 101, 118, 124, 125 Kotani, M., 298, 322 Koval, V. A., 276,285 Krachko, J . L ,262,285 Kratzing, C. C., 260, 284 Krawczynski, J., 262, 284 Krebs, H. A,, 129, 131, 140, 157, 259, 261, 284 Krech, D., 242, 250 Krendel, E . S., 345, 373 Kreutzberg, G. W., 315, 323 Krnjevic, K., 143, 157 Krogh, E., 276, 281 KufHer, S. W., 276, 277, 284 Kuhar, M. J., 144, 145, 146, 153, 155, 156, 157 Kulenkampff, H., 277, 284 Kullback, S., 351, 373 Kuntzman, R., 71, 91 Kushnir, L. D., 185, 220 Kutschin, A. A., 276, 285

1 Ladik, J., 187, 220 Laduron, Y., 302, 323 Lajtha, A., 261, 263, 266, 282, 284, 287, 292, 323 Landauer, T. K., 243, 252 Lange, P., 233, 234, 252 Lange, P. W., 271, 275, 283 Langemann, H., 96, 125, 139, 157 Langer, S. Z., 41, 46, 51, 66 Larocelle, P., 80, 91 Larochelle, L., 101, 125 Larrabee, M. G., 168, 179 Larsson, K., 80, 81, 83, 85, 89, 94, 96, 97, 99, 101, 105, 119, 121 Lasek, R. J., 291, 292, 293, 296, 297, 298, 299, 300, 305, 306, 307, 308, 310, 311, 314, 317, 323 Lashley, K. S., 67, 91, 227, 252 Laties, A. M., 33, 64 Laverty, R., 46, 63 Lavoie, J. L., 173, 178 Layne, 11. C., 259, 280

383

Leaf, R. C., 240, 251 Leblond, C. P., 290, 292, 300, 322 Legg, P. G., 41, 64 Leicht, W. S., 168, 179 Lenaerts, F. M., 108, 124 Lenard, J., 32, 64 Lenn, N. J., 53, 59, 64, 103, 125 Leonard, C. F., 23, 25 Leonardi, R., 114, 122 Leontovitch, T. A., 2, 25 Ledvre, N., 346, 373 Lettvin, J. Y., 11, 25, 347, 373, 374 Lever, J. D., 41, 46, 48, 63, 64, 102, 125 Levi, J. U., 276, 284 Lewander, T., 111, 124 Lewis, J. E., 164,179 Lewis, P. R., 107, 108, 125 Liang, C. C., 143, 157 Libet, B., 301, 324 Lichtensteiger, W., 96, 125, 139, 157 Lickey, M., 233, 234, 250 Lillie, R. H., 4, 24 Lim, R., 238, 250 Lindqvist, M., 106, 107, 111, 112, 113, 122 Lindsay, R., 297, 323 Lindsley, D. B., 21, 26, 366, 373 Lindstrom, B., 269, 270, 278, 281, 284, 285 Lineweaver, H., 136,157 Lipmann, F., 264, 285 Lippmann, W., 114, 122 Lissak, K., 72, 90 Litman, R. B., 31, 64 Litvak, S., 294, 322 Liu, C. 273, 276,284 Livett, B. G., 301, 302, 323 Livingston, R. B., 248, 252 LlinLs, R., 16, 17, 24 Lodin, Z., 261, 270, 271, 275, 277, 284, 285 Loeb, J. M., 273, 285 Loewe, S., 205, 220 Loewenstein, W. R., 247, 252 Logan, C. R., 117, 124 Lorente de N6, R., 2, 12, 17, 25 Lorey, R. A., N4,253 Lovenberg, W., 50, 61, 64 Lovey, R. A., 188, 221

384

AUTHOR INDEX

Lowry, 0. H., 257, 260, 269, 281, 284, 285 Lubiriska, L., 278, 285, 290, 296, 303, 309, 310, 313, 316, 322, 323, 324 Lund, R., 33, 64 Lundberg, A., 115, 121 Lux, H. D., 298,322 Luenburg, J., 273, 285 Luxoro, M., 265, 266, 285 Lyons, R., 261, 282

M Maas, J. W., 39, 40, 62, 64 McClure, D. J., 116, 125 McConnell, H., 201, 220 McConnell, J. V., 240, 243, 252 McCulloch, W. S., 11, 25, 347, 373, 374 McEwen, B., 271, 283, 285, 297, 298, 301, 308,309, 323 McGaugh, J. L., 236, 237, 252 McGeer, E. G., 101, 125 McGeer, P. L., 101, 125 McGill, W. J., 348, 352, 373 Machado, A. B. M., 30, 41, 46, 53, 64 Machado, C. R. S., 41, 53, 64 Machlus, B., 236, 252 McIlwain, H., 256, 259, 260, 261, 264, 285 MacIntosh, F. C., 72, 91 MacKay, D. M., 346, 347, 370, 373 McLennan, H., 27, 65 McLennon, H., 160, 179 McMasters, R. E., 80, 90 McMurtry, J. G., 23, 25 McNutt, L., 239, 252 Madow, W. G., 353,373 Maeda, T., 95,125 Magni, F., 21, 25 Magoun, H. W., 21,25 Mahler, H. R., 244, 251, 263, 266, 272, 280, 281, 287 Makinnen, E. O., 30, 33, 64 Malliani, A., 23, 25 Malm, U., 115, 123 Malmfors, T., 72, 91, 94, 95, 124, 125 Mandel, P., 269, 273, 282, 283 Mangan, J. L., 143, 157 Mannen, H., 2,25 Manner, P., 263, 285

Mansour, T. E., 167, 179 Marchbanks, R. M., 165, 179 Marchi, S. A,, 256, 258, 282 Margules, D. L., 240, 251 Marin-Padilla, M., 21, 25 Marks, N., 292,323 Masi, I., 258, 280 Massey, F. J., 349, 372 Massey, J. F., 304, 316, 323 Mast, T. E., 366, 373 Masuoka, D., 95, 124 Mats, V. N., 277, 280,285 Matsuoka, M., 53, 64, 65 Maturana, H. R., 11, 25, 347, 373 Matussek, N., 53, 63 Mayor, D., 53, 64, 95, 124, 310, 311, 322 Meek, J., 115, 119, 125 Meeuws, M. M., 143,158 Mehler, W. R., SO, 90 Meister, A., 261, 285 Menger, K., 345, 349, 373 Menon, T., 168, 180 Merrills, R. J., 139, 157 Metuzals, J., 317, 323 Meyer, J. S., 263, 285 Meyerhoff, O., 260, 282 Meyersson, B., 118, 125 Miani, N., 278, 285, 294, 301, 307, 308, 310, 317, 323 Michaelson, I. A., 39, 46, 48, 65, 66, 128, 129, 157 Micheletti, G., 41, 63 Michler, H., 274, 280 Mihailovib, L., 236, 252 Miller, G. A., 349, 353, 365, 373 Miller, J. W., 164, 179 Miller, N. E., 237, 252 Milofsky, A., 41, 65 Miquel, J., 277, 284 Mokrash, L. C., 263, 285 Mollica, A., 6, 26 Monod, J., 172, 179 Montegazza, P., 102,122 Moor, W. J., 266, 280 Moore, G. P., 228, 252, 347, 362, 370, 373 Moore, R. Y., 68, 69, 70, 71, 73, 74, 75, 76, 81, 84, 85, 86, 87, 90, 91, 101, 124

385

AUTHOR INDEX

Moore, W. J., 263, 287 Morgan, I. G., 184, 220, 295, 321, 323 Morgane, P. J., 89, 91 Morin, R. D., 183, 202, 204, 220, 221 Morrell, F., 230, 252 Morse, W. H., 236, 252 Moruzzi, G., 6, 21, 25, 26 Moscona, A. A., 246, 252 Moscona, M. H., 246, 252 Mountcastle, V. B., 7, 25, 364, 366, 373, 374 Miiller, E., 114, 125 Miiller, J., 270, 275, 277, 285 Mullins, L. J., 266, 285 Munkvad, I., 107, 125, 139, 158 Muntz, J. A., 260,285 Murad, F., 166, 167,179 Murphy, D. L., 129, 157 Murray, M., 314, 322 Mutzner, U., 139,157

N Nachmansohn, D., 162, 172, 179 Nagel, R., 21, 25 Nakai, J., 304, 323 Nakamura, T., 273, 286 Namba, T., 185, 220 Narayaswami, A,, 260,284 Nasonov, D. M., 265, 285 Nastuk, W. L., 163, 179 Nathan, P., 325, 341 Nauta, W. J. H., 2, 12, 21, 25, 68, 70, 77, 80, 89, 90, 91 Neal, M. J., 143, 157 Neraeva, N. V., 274,287 Nechaeva, G . A,, 262, 285 Neff, N. H., 50, 65, 139, 158 Nelson, P. G., 6, 7, 25, 176, 180 Newell, G. W., 258, 282 Ng, M. H., 297, 298, 308, 323 Nicholls, J. G., 276, 277, 284, 288 Niedorf, A. R., 310, 321 Niemerko, S., 310, 313, 322, 324 Niemeyeers, C. J. E., 108, 124 Nievel, J. C., 272, 285 Nilsson, O., 53, 59, 63, 97, 102, 103, 110, 123 Nissen, T., 240, 251 Nissl, F., 271, 285

Noel, J., 51, 62 Nurnberger, J. A., 278, 285 Nyback, H., 138,158

0 O’Brien, R. A,, 38, 62 Oceguera-Navarro, C., 240, 253 Ochs, S., 278, 285, 290, 297, 298, 299, 300, 305, 308, 309, 314, 323 Bye, I., 167, 180 Offermeier, J., 162, 164, 179 Ogato, K., 184, 221 Ohi, Y., 53, 64 Ohnishi, S., 201, 220 Oka, H., 169, 178 Okada, K., 163,179 Okros, I., 53, 65 Olds, J., 89, 91 Olivier, A,, 80, 91 Olson, L., 80, 81, 83, 85, 89, 94, 96, 97, 99, 101, 105,121 Olszewski, J., 13, 25 Oplatka, A., 244, 252 Orljanskaja, R. L., 262, 288 Orloff, J., 167, 179 Orrego, F., 264, 285 O’Steen, W. K., 302,323 Otis, L. S., 238, 252 Overton, R. K., 243, 252 Owman, C., 84, 85,89, 139, 156

P Painter, E., 167, 178 Pakkenberg, H., 271, 285 Pakkenberg, M., 274, 275, 287 Palade, C. E., 103, 125, 272, 285, 291, 323 Palay, S. L., 29, 41, 46, 51, 63, 102, 103, 124, 125, 272, 285, 291, 323 Palladin, A. V., 261, 262, 285 Papavasiliou, P. S., 104, 122 Papavasilou, P. S., 138, 157 Papez, J. W., 68, 91 Pappas, G. D., 87,90 Parent, A., 85, 91 Passonneau, J. V., 257, 260, 281, 284 Paterson, D., 113, 125 Paton, W. D. M., 27, 65, 171, 179 Pauling, P., 204, 220

386

AUTHOR INDEX

Paulus, H., 168, 180 Pearse, A. G. E., 32, 34, 65 Pease, D. C., 31, 32, 65 Pellegrino de Iraldi, A., 29, 39, 41, 44, 46, 48, 50, 51, 53, 63, 65, 73, 91, 102, 103, 123, 125, 128, 157 Perkel, D. H., 228, 229, 252, 347, 362, 370, 373 Perkins, M., 72, 91 Perrine, J., 114, 122 Persson, T., 115, 125 Peters, A., 87, 90, 317, 323 Peterson, J. A., 278, 285, 294, 297, 300, 301, 323 Peterson, N. A., 266, 285 Peterson, R. P., 297, 323 Petrinovitch, L. F., 236, 237, 252 Petsche, H., 23, 25 Pettis, P., 295, 321 Pevzner, L. Z., 266, 270, 271, 276, 277, 285 Pfeiffer, A. K., 53, 65 Philipson, B., 270, 284 Pichler, H., 34, 65 Piersol, A. G., 345, 372 Pigon, A., 271, 272, 277, 281, 283 Piha, R. S., 263, 266, 285, 286 Pillai, A., 303, 309, 316,'324 Pilng, J., 270, 284 Pin, C., 94, 125 Pinchard, A,, 108, 124 Pitts, W. H., 11, 25, 347, 373, 374 Pletscher, A., 37, 38, 66, 69, 90, 104, 105, 107, 122, 138, 157 Pocchiari, F., 258, 280 Podleski, T. R., 162, 172, 178, 180 Poggio, G. F., 366, 373 Pohorecky, L. A,, 84, 91 Poirier, L. J., 80, 85, 91, 96, 101, 125 Polak, R. L., 143, 158 Polkouits, M., 53, 65 Pomerat, C. M., 304, 316, 323 Porcher, W., 74, 90 Potter, D. D., 277, 284 Potter, L. T., 39, 40, 41, 46, 48, 65, 66, 102, 126 Poulos, G. J., 331, 341 Pravdicz-Neminskij, V. V., 260, 286 Prescott, D., 268, 286

Pryor, G. T., 239, 252 Psychoyos, S., 265, 287 Pullman, A., 220 Pullman, B., 220 Purkhold, A., 72, 91 Purpura, D. P., 7, 23, 25 Pyper, A. S., 30, 35, 37, 62

Q Quastel, J. H., 143, 157, 259, 264, 283, 286 Quastler, H., 272, 288 Quinn, G. P., 335, 341

R Rahman, H., 278,286 Rahmann, H., 291,323 Raiborn, C. W., 304, 316, 323 Raisman, G., 53, 65 RakiQ L., 236, 252 Rall, T. W., 166, 167, 168, 179, 180 Rall, W., 2, 6, 22, 25 Ralston, H. J., 87, 90 Ram6n-Molinar, E., 2, 12, 25 Ram6n y Cajal, S., 289, 323 Ramwell, P. W., 191, 220 Randrup, A., 107, 125, 139, 158 Rapaport, D., 264, 288 Rappoport, D. A,, 235,252 Redman, C. M., 169,180 Rehbun, L. I., 312,323 Rehn, N. O., 73,91 ReiniS, S., 240, 252 Reinius, S., 59, 63, 102, 103, 123 Reis, D., 111,125 RGrnond, A., 346, 373 Renyi, A. L., 139, 158 Revuelta, A., 55, 62 Rexed, B., 15, 25 Rhines, R., 272, 281 Rhodes, J. M., 345, 356, 374 Riccio, D., 240, 251 Richards, J. G., 28, 30, 41, 46, 57, 59, 66, 103, 126 Richardson, K. C., 29, 32, 41, 46, 48, 65, 66, 72, 91, 102, 103, 125, 126 Richter, D., 256, 258, 260, 261, 262, 263, 264, 278, 281, 282, 286 Rieger, H., 346, 373

387

AUTHOR INDEX

Riesen, F., 274, 286 Rigler, R., 271, 286 Riley, V., 34, 65 Rinaldi, R. A., 312, 322 Ringborg, W.,272, 286 Ringler, I., 114, 322 Ritter, W., 358, 366, 373 Ritzen, M., 78, 9 4 91 Roberts, E., 143, 158, 228, 252 Roberts, R. B., 239, 248, 251, 252 Roberts, S., 264, 288 Robie, T. R., 116, 125 Robinson, C. E., 243, 252 Robinson, J. D., 139, 158 Robison, G. A., 167, 174, 180 Robustelli, F., 237, 251 Rode, J., 310, 322 Rodionova, N. P., 184, 221 Rodnight, R., 264, 265, 286, 287 Rodriguez de Lores Arnaiz, G., 53, 63, 128, 157, 165,178 Rgjigaard-Petersen, H. H., 240, 251 Roepke, M. H., 162, 180 Rogers, A. W., 268,286 Romano, D. V., 2,65,287 Roos, B. E., 138, 158 Roos, B-E., 115, 123, 125 Roots, B. I., 268, 269, 281, 284, 286 Rose, S. P. R., 233, 250, 258, 261, 262, 269, 284, 286 Rosenblatt, F., 236, 240, 241, 246, 252, 253 Rosenblith, W. A., 223, 253 Rosencrans, J., 118, 121 Rosengren, B., 270, 282 Rosengren, E., 80,84,89 Rosenzweig, M. R., 240, 242, 250, 251 Ross, S. B., 139, 158 Rossen, J., 259, 280 Rossiter, R. J., 261, 286 Roth, C. D., 72, 91 Roth, L. J., 38, (26, 68, 85, 90 Roth, R. H., 59, 60, 66 Routtenberg, A,, 302, 323 Rubensson, A., 106, 321 Rude, J., 51, 60,61, 65 RuBEQk, M., 262, 286 Rush, R. A., 312, 314,322 Ruskell, G. L., 41, 65

Ruther, E., 53, 63 Rydin, H., 104, 109, 111, 113, 121

S Sabatini, D. C., 33,65 Sabri, M. I., 297, 298, 299, 300. 305, 309, 323 Sachs, C., 94, 125 Sachs, E., 243, 253 Sachs, H., 303, 309, 323 Sacktor, B., 257, 286 Sadikova, V. N., 262,285 Saint-Jacques, C., 85, 91 Saito, K., 169, 179 Sakata, H., 366, 373 Salafsky, B., 313, 321 Salganicoff, L., 128, 157 Sdpeter, M., 294, 324 Saltpeter, E. E., 38, 65 Saltpeter, M. M., 38, 46, 48, 51, 62, 65 Samec, J., 273, 283 Sampietro, R., 30,33, 66 Samson, F. E., 261, 286 Samson, F. E., Jr., 247, 248, 250, 253 Samuel, D., 240, 251 Samuels, A. J., 30.1, 324 Sandlin, R., 279, 287 Sandritter, W., 271, 284 Sarvano, S., 114, 125 Sasaki, K., 16, 17, 24 Sastry, P. S., 171, 179 Satake, M., 269, 286 Satterfield, J. H., 366, 373 Schactman, H. K., 174, 178 Schaefer, J. M., 39, 46, 51, 59, 66 Schally, A., 114, 125 Schanberg, S. M., 139,158 Schaper, W. K. A., 108, 124 Schear, M., 104, 123, 126 Scheibel, A. B., 2, 3, 4, 5, 6, 8, 13, 15, 16, 20, 21, 22, 24, 25, 26, 100, 125 Scheibel, M. E., 2, 3, 4, 5, 6, 8, 13, 15, 16, 20, 21, 25, 26, 100, 125 Schellekrus, K. H. L., 108, 124 Schiffer, L. M., 104, 122 Schild, H. O., 176, 178 Schildkraut, J. J., 139, 158 Schiller, P. H., 240, 251 Schimizu, S., 170, 179

388

AUTHOR INDEX

Schlote, W., 310, 324 Schmidt, C.G., 261,286 Schmidt, M. J., 239, 253 Schmitt, F. O., 243,.247, 248, 253, 261, 286, 308, 318, 324 Schmitt, 0. H., 347, 373 Schou, M., 116,121,122 Schrodinger, E., 224, 253 Schubert, P., 298, 322 Schuberth, J., 143, 158 Schueler, F. W., 161, 178 Schiimann, H. J., 213,220 Schiirmann, F. W., 278,286 Schultz, D. W., 257, 260, 281 Schultze, B., 262, 268,286 Schumann, H. J., 40,65 Schwartz, A., 265, 287 Schwartz, S., 143,157 Scott, D., 278, 284 Scott, F. H., 289, 324 Scott, K. A., 161, 178 Scott, S., 264, 280 Seaman, H. R., 31, 57, 63 Sedvall, G., 72, 73, 91, 138, 158 Segal, J., 265,286 Segundo, J. P., 228, 252, 347, 362, 370, 373 Seiden, L., 113, 125 Seiden, L. S., 74, 75, 90 Seligman, A. M., 31, 57, 63, 65 Semiginovsv, B., 266, 268, 278, 283, 284, 286 Semon, R., 227, 253 Sensebrenner, M., 269, 282 Seth, P. K., 215, 220 Shafer, A. W., 169, 179 Shannon, C. E., 224, 253, 348, 373 Shapira, A., 278, 284 Shapiro, A., 302, 309, 312, 313, 322 Shapot, U. S., 256, 262, 286 Shapov, V. S., 184, 221 Sharman, D. F., 115,121 Shashoua, V. E., 233, 234, 253 Shaskan, E. G., 139,158 Shaw, E., 327,341 Shaw, J. E., 191,220 Shaw, T. I., 278, 280 Sheard, M. D., 87, 89

Sheard, M. H., 53, 62, 101, 116, 118, 121, 125 Shelanski, M. L., 315, 324 Shelby, J. M., 240, 252 Sherman, F. G., 272,288 Sherrington, C. S., 225, 253 Shimada, M., 273, 286 Shimizu, N., 53, 64, 65 Shimizu, U., 53, 65 Sholl, D. A,, 2, 26 Shore, P. A,, 69, 71, 90, 91, 335, 341 Shorr, S. S., 41, 66 Shute, C. C. D., 107, 108, 125 Sidman, R. L., 72,91 Siegesmund, K. A., 9, 17, 24 Siggins, G. R., 41, 46, 66 Simon, L. N., 239,251 Simonis, A. M., 174,178 Simpson, F. O., .41, 46, 48, 63 Singer, I., 279, 287 Singer, M., 278, 286, 293, 294, 295, 324 Singer, S. S., 32, 64 Singh, P., 80, 91 Sisken, B., 143, 158 Sjijqvist, F., 46, 66, 109, 122 Sjoerdsma, A., 50, 61, 64 Sjostrand, F. S., 103, 125 Sjostrand, J., 274, 278, 281, 286, 291, 294, 295, 297, 301, 308, 309, 322, 324 Skangiel-Kramska, J,, 310, 324 Skinner, B. F., 328, 341 Skinner, I. E., 21, 26 Skvortsevich, V. A,, 262, 285 Sladek, J., 302, 323 Slagel, D. H., 278, 286 Smith, A. J., 167, 178 Smith, C. E., 242, 253 Smith, G. T., 3, 5, 6, 25, 26 Smythies, J. R., 183, 189, 202, 204, 214, 220,221 Snipes, R. L., 28, 30, 32, 41, 43, 46, 50, 57, 59, 66, 103,126 Snyder, S. H., 39, 59, 63, 73, 90, 107, 117, 122, 125, 129, 134, 135, 136, 137, 138, 139, 142, 143, 144, 145, 146, 148, 153, 155, 156, 157, 158 Sodd, M. A., 165, 168, 169, 170, 178 Sokoloff, L., 263, 286

AUTHOR INDEX

Solcia, E., 30,33, 66 Soula, C., 261, 273, 281 Sourkes, T. L., 80, 91, 96, 125 Speidel, C. C., 316, 324 Spencer, W. A., 23, 26, 230, 252 Sperry, R. W., 246,253 Spiegel, H. E., 129, 157 Spong, P., 366, 373 Sporn, M. B., 262, 281 Spriggs, T. L. B., 41, 46, 48, 63, 64 Stancer, H. C., 265, 282 Stanford, A. L., 188,221 Stanford, A. L., Jr., 244, 253 Stark, L., 361, 332, 363, 374 Steg, G., 107, 125 Stein, H. H., 239, 253 Stein, L., 113, 125, 126, 138, 158, 240, 251 Stephenson, R. P., 161, 178 Sterglanz, H., 202, 221 Steriade, M., 259, 281 Sternberger, L. A., 29, 66 Stevenin, J., 273, 283 Stjarne, L., 39, 59, 60, 66 Stoller, W., 276, 286 Stone, W. E., 258,282 Stratmann, C. J., 266, 281 Strecker, H. J., 2.56, 259, 286 Strobel, D. A., 233, 234, 251 Stumpf, W. E., 38, 66 Suko, J., 34, 65 Sundwall, A., 143, 158 Sutherland, E. W., Jr., 165, 166, 167, 168, 174, 178, 179, 180 Sutin, J., 77, 85, 91 Suttan, S., 346, 358, 366, 374 Svensson, T., 104, 109, 111, 113, 121 Swift, H., 270, 287 Syrov9, I., 260, 265, 283 Szato, D., 53, 65 Szentagothai, J., 10, 15, 17, 24, 26 Szilard, L., 231, 245, 253

T Tagaki, M.. 184, 221 Takagi, K., 165, 180 Takahashi, A., 165, 180 Takahashi, R., 326, 341 Talbot, W. H., 366, 373, 374

389

Talwar, G. P., 262, 287 Tanzi, E., 242, 253 Tarifeno, E., 293, 322 Tasaki, I., 4, 25, 279, 287 Tashiro, S., 260,287 Tauc, L., 4, 26 Taxi, J., 38, 41, 46, 48, 50, 51, 59, 66, 102,125 Taylor, A. C., 291, 297, 298, 300, 307, 308, 316, 324 Taylor, E. W., 316, 317, 321, 324 Taylor, K., 135, 139, 158 Taylor, P. W., 46, 48, 65, 66 Teitlebaum, P., 89, 91 Tellez-Nagel, I., 278, 281 Tenen, S. S., 139, 158 Terry, R. D., 315, 324 Tewari, S., 266, 280 Thaemert, J. C., 41, 66 Thieme, G., 78, 90, 93, 123 Thoa, N. B., 139,158 Thoenen, H., 28, 30, 41, 45, 46, 48, 50, 51, 57, 59, 66, 95, 103, 125, 126 Thomsen, E., 274, 275, 287 Tice, L. W., 27, 29, 53 Tiekert, C. G., 257, 286 Timms, A. R., 167,178 Tissari, A,, 129, 157 Titus, E., 46, 48, 65, 66, 129, 157 Tobias, J. M., 176, 180 Tomita, M., 263, 285 Tomkins, G. M., 273, 285 Torp, A., 78,90, 93, 123 Toru, M., 336, 341 Toschi, G., 276, 287 Toth, J., 263, 284 Tower, D. B., 256, 258, 261, 287 Townshend, M. M., 167, 178 Tramezzani, J. H., 30, 34, 35, 37, 62, 66 Tranzer, J. P., 28, 30, 32, 37, 38, 41, 43, 45, 46, 48, 50, 51, 57, 59, 66, 95, 103,125,126 Travis, R., 263, 287 Trendelenburg, U., 41, 46, 51, 66, 72, 91 Trevor, A. J., 265, 269, 281, 287 Tueting, P., 366, 374 Tulegenova, L. S., 184, 221 Turtle, J. R., 166, 180

390

AUTHOR INDEX

U Uchiyama, K., 6, 26 Uchiyama, T., 6, 26 Uchizono, K., 17, 26 Udenfriend, S., 71, 89, 272, 282 Ungar, F., 215, 220 Ungar, G., 230, 231, 235, 238, 240, 241, 246,253,265,287 Ungerstedt, U., 38, 55, 63, 64, 80, 81, 83, 85, 89, 90, 93, 94, 95, 96, 97, 99, 101, 103, 104, 105, 106, 107, 109, 111, 113, 115, 117, 121, 122, 123, 124, 126, 139, 157 Uretsky, N. J., 95, 126 Urinson, A. P., 262, 287 Utina, J. A., 274, 287 Uttal, W. R., 229, 253

V Valzelli, L., 101, 118, 125 Van Gelder, M. N., 143, 157 Van Neuten, J. M., 108, 124 Van Orden, L. S., 28, 30, 39, 41, 46, 48, 51, 59, 60, 61, 65, 66, 78, 91 Van Rossum, J. M., 174, 178 Van Woerf, M. H., 104, 122 Vas, F., 273, 287 Vassent, G., 118, 124 Vaughan, H. G., Jr., 358, 366, 373 Vaughn, G. M., 302,323 Vaughn, J, E., 317,323 Veno, H., 31, 57, 63 Venson, V., 257,284 Veprintsev, B. N., 272, 276, 280, 281 Verbruggen, F. J., 108, 124 Vesco, C., 264, 273, 287 Veskov, R., 236, 252 Vladimirov, G. E., 262, 287 Vladimirova, E. A,, 260, 287 VodiEka, Z., 265, 287 Volpe, P., 269, 273, 287 von Euler, H., 258, 287 von Euler, U. S., 39, 66, 72, 91 Van-Foerster, H., 227, 253 von Hahn, H. P., 271,287 von Hungen, K., 263, 287 Vrba, R., 258, 260, 261, 287 Vugman, I., 41, 46, 66, 78, 91 Vyklicky, L., 115, 121

W Wacker, A., 234, 251, 264, 281 Waelsch, H., 261, 263, 268, 272, 280, 282, 285, 286, 287, 294, 321 Wagner, A. R., 240, 251 Wagner, R. P., 295, 324 Wagner, T. E., 202, 221 Walberg, F., 3, 20, 26, 87, 89 Waldeck, B., 95, 122 Walker, P., 270, 287 Walker, R. J., 278, 284, 302, 309, 312, 313, 322 Wall, P. D., 16, 26, 347, 374 Walter, D. O., 345, 346, 349, 356, 358, 367,372,374 Walter, W. G., 366, 372 Wang, H., 297,324 Wartenberg, H., 41, 66 Wasserkrug, H. L., 31, 63, 65 Wasserman, E., 240, 251 Wasserman, J. H., 30, 34, 35, 37, 66 Watanabe, A., 279, 287 Watkins, J. C., 173, 180 Watson, C. S., 366, 373 Watson, W., 273, 275, 287 Watson, W. E., 312, 324 Waxman, S. G., 277, 287 Wayner, M. J., 276, 286 Weaver, W., 348, 373 Weil-Malherbe, H., 258, 261, 287, 288 Weiner, N., 72, 91 Weiner, W., 104, 106,126 Weingartner, H., 117, 125 Weisenberg, R. G., 317, 324 Weiss, L. P., 31, 57, 63 Weiss, P., 249, 253, 289, 290, 291, 295, 297, 298, 300, 301, 303, 307, 308, 309, 316,317,324 Weissbach, H., 71, 89 Weissman, A,, 50, 61, 64, 117, 124, 139, 158 Welch, A. D., 39, 62 Werdinius, B., 115, 123 Werman, R., 248, 250 Werner, G., 278, 281, 364, 366, 373, 374 Wesemann, W., 164,180 West, K. A., 85, 89 Wheatley, A. H. M., 259, 286 Whisler, W. W., 34, 63

391

AUTHOR INDEX

White, L. E., Jr., 87, 89 Whitlock, D. G., 21, 25, 298, 323 Whittaker, V. P., 39, 48, 65, 66, 128, 143, 157,158 Wiechert, P., 258, 259, 260, 288 Wiener, N., 224,253 Wiesel, T. N., 11, 25 Wikler, A., 117, 124 Wilkinson, G., 32, 62 Wilkinson, P. N., 247, 252, 298, 301, 323 Willis, W. D., 21, 25 Wilson, J. E., 257, 272, 284, 286 Windle, W. F., 273, 276, 284 Winnik, M., 163, 179, 210, 220 Winterstein, H., 260, 261, 283, 288 Wise, C., 113, 125, 126 Wisniewski, H., 315, 324 Wolf, G., 77, 85, 91 Wolf, M. A., 331, 341 Wolfe, D. E., 41, 46, 66, 102, 126, 277, 288 Wong, S. L. R., 74, 75, 91 Wood, J. G., 30, 32, 35, 38, 53, 66 Woods, J. W., 77, 89 Woolley, D. W., 69, 91, 163, 180, 327, 341 Wragg, I,.E., 41, 53, 64 Wuerker, R. B., 3, 5, 26 Wiistenfeld, E., 277, 284 Wulff, V. J., 271, 272, 288

Wurtman, R. J,, 73, 84, 91 Wyman, J., 172, 179

Y Yahr, M., 126 Yamasaki, S., 261, 282 Yamazoe, S. J., 170, 179 Yates, R., 35, 63 Yellin, T. O., 239, 253 Yielding, K. L., 202, 221 Yoda, A., 171, 179 Young, J. Z., 316,324 Young, R. W., 291, 298, 317, 322, 324

Z Zacharov, N. V., 262,288 Zacks, S. I., 53, 64 Zadeh, L. A., 355, 374 Zambrano, D., 53, 66 Zelenh, J., 303, 309, 310, 324 Zhukova, G. P., 2, 25 Zieher, L. M., 28, 30, 38, 39, 40, 46, 50, 51, 53, 55, 60,63, 6S, 73, 91 Zigmond, M. J., 84, 91 Zilliken, F., 164, 180 Zippel, H. P., 241, 253 Zobel, C.R., 27, 66 Zomzely, C. E., 264,288 Zubin, J., 366, 374

SUBJECT INDEX Antibiotics, stereochemical relations to membrane-active drugs 215-217 Antiparkinsonian drug, effect on brain catecholamine uptake, 137-139 ATP, metabolism, in brain during excitation, 256-258 Atropine, binding in muscarinic site, 200 Axon( s ) diagram of, 318 nonterminal, monoamine cell bodies and, 59-61 proteins in, synthesis in soma, 295-296 Axon, protein synthesis in, 292-296 protein transport in, 2 9 6 3 1 3 RNA and proteins in, stimulation effects on, 277-279 Axoplasmic organelles, somatofugal transport of, 303

A

Acetycholine binding of in muscarine site, 195 in nicotinic site, 197 in brain, decreases related to behavioral excitation, 33-38 as neurotransmitter, 143 interaction with receptor, 172-174 Acetylcholinesterase, somatofugal transport of, 303 Acoustic stimulation, effect on brain RNA and proteins, 274275 Adenosine monophosphate, cyclic (CAMP), transmitter effects on, 166-168 Adenyl cyclase, transmitter effects on, 166-168 Adrenal medulla, as test tissues for localB ization of biogenic monoamines, 3538 Behavior Adrenergic receptors central serotonin neuron rule in, 120 chemical nature of, 180-101 neurochemical correlates of, 325-341 isolation of, 164-165 acetylcholine changes, 335-338 transmitter-receptor interaction in, methodological problems, 338-339 174-176 serotonin changes, 327-335 Aldehyde fixatives, for biogenic rnonoanoradrenaline neuron role in, 110-114, mines, 32-34 120 Amines, brain lesions and metabolism of, Brain 67-91 acetylcholine and serotonin levels reAmino acids lated to behavior, 325-341 metabolism, in brain during excitation, energy metabolism related to amino 256-258 acid metabolism, 256261 as transmitters, receptor sites for, 207information processing in, 37&371 210 molecular mechanism in, see Molecuptake and subcellular localization in ular mechanisms in information brain, 143-146 processing y-Aminobutyric acid, as neurotransmitter synaptosomes in, see Synaptosomes in brain, 143-156 Brain lesions and amine metabolism, 67synaptosomes storing, 149-156 91 Ammonia, increase in brain during excitaamine changes after central lesions, tion, 260-261 73-74

392

SUBJECr INDEX

anatomical considerations, 7 6 7 8 direct denervation vs. transsynaptic effects in, 8-8 fluorescent histochemical studies on, 78-83 interpretation of, 72-88 central nervous system, 73-88 peripheral model, 72-73 subcortical lesions, 69-72 in medial forebrain bundle, 69-71 in midbrain tegmentum, 71-72 Brainstem reticular core, neurons of, 1314

C Catecholamine( s ) ( CA ) specifications for receptors of, 201-202 storage synaptosomes for in different brain regions, 146149 test tissues for, 35 uptake in brain, antiparkinsonian drug action, 137138 kinetics of, 128-134 stereospecificity, 134-136 Central monoamine neurons electron microscopy of, 102-104 function of, 104-119 monoamine pathways, methods of mapping out, 94-96 morphological and functional aspects of, 93-126 Central nervous system, breakdown and synthesis of proteins in, 261-265 stimuli effects on, 262 Cerebellum, Purkinje neuron of 9-10 Cerebral cortex, pyramids of, 19-21 Chemical codes, for neural coding, 230231 Chemospecific pathways, in hypothesis of information processing, 244-248 Choline acetylase oxidative enzymes, somatofugal transport of, 303 Cholinergic antagonists, 198-200 Cholinergic receptors chemical nature of, 161-162 inactivation-reactivation of, 163 isolation of, 165 specification of, 192200

393

transmitter-receptor interaction in, 176-177 Citrinin, stereochemical relations to membrane-active drugs, 217 Codes, in neural coding, 227-231 Coding, in information processing, 347 Colchicine, effect on axonal transport, 315-316 D Dendrites as monoreceptic elements, 13-16 pattern and place in, 1-26 as polyreceptive elements, 16-21 specific patterning in, 4-12 convoluted field-specific types, 7-1 1 straight, unbranched types, 5-7 specificity of synaptic position of, 1223 synaptic position in complex cortical systems, 21-23 Depression (behavioral) central serotonin neuron role in, 120 decrease in brain serotonin and, 333335 increase in brain serotonin and, 327333 Disulfide bond, in receptor site, 210-211 Dopa decarboxylase, brain lesions and brain level of, 87 Dopamine ( DA ) in brain, lesions and, 81 fine structural localization in nervous tissue, 27-66 neurons autonomic functions of, 109-110 mental functions of, 107-109, 119 motor functions of, 104-107, 119 nigro-neostriatd type, 104-110, 119 in thermoregulation, 109 tubero-infundibular type, 110, 119 pathways of, 9 6 9 8 protein transport in, 302 uptake in brain, 128-139 Dopamine-P-hydroxylase, somatofugal transport of, 302 Dorsal horn, spinal neurons of, 15-16 Drugs, effect on learning and memory, 236-239

394

SUBJECT INDEX

E Electrical activity in neural coding, 228229 Electrical stimulation, effect on brain RNA and proteins, 275-276 Electroencephalograms, cross-spectral analysis of, 345-346 Electron microscopy of central monoamine neurons, 102104 of monoamines, 29-34 Emotion, noradrenaline neuron role in, 120 Engram, 227 Epinephrine, fine structural localization in nervous tissue, 27-66 Excitable membrane, RNA role in, 184189 Excitation ( behavioral), decreases in brain acetylcholine and, 335-338

F Formaldehyde, as stain for biogenic monoamines, 30, 32-33

G Gardiner’s differential processing experiment, 366-369 Glial cells, protein and nucleic acid metabolism in during increased activity, 261-266 Glucose, metabolism, in brain during excitation, 256-258 Glutamate receptor site for, 207 somatofugal transport of, 302 Glutaraldehyde, as stain for biogenic monoamines, 30, 33-34 Glycine receptor site for, 207-210 somatofugal transport of, 300 Granular vesicles (large) (LGV), localization of monoamines in, 51-53 Granular vesicles (small) ( SGV), localization of monoamines in, 41-51

H Hippocampal pyramid, organization of, 17-19

Hormones, effect on brain protein metabolism, 266-268 5-HT, see Serotonin Hydroxy dopamines fine structural localization in nervous tissue, 27-66 use to map out monoamine neurons, 95 5-Hydroxytryptamine ( 5-HT ) , see Serotonin 5-Hydroxytryptophan, somatofugal transport of, 302

1 Indolealkylamine, test tissues for, 35-38 Inferior olive, neurons of, 8 Information definition and properties of, 348-354 transmission of, 352-353 Information processing definition of, 354-356 explicit system models, 369-371 molecular mechanisms in, see Molecular mechanisms in information processing system models for representing weak evoked responses, 356-361 system science for study of, 342-374 basic concepts of, 343-356 systems concepts for, 343-347

1 Learning definition of, 226-227 drug effects on, 236-239 Lesions, in brain, see Brain lesions Light stimulation, effect on brain RNA and proteins, 274 Lipids, effects on transmitters, 168-170 LSD, binding to PG-RNA complex, 203205, 207

M Medial forebrain bundle, amine levels and lesions of, 69-71 Membrane-active drugs, stereochemical relations to antibiotics, 215-217 Memory definition of, 227 drug effects on, 238-239

395

SUBJECT INDEX

Mental functions of dopamine neurons, 107-109, 119 of serotonin neurons, 115-118 Metaraminol, fine structural localization in nervous tissue, 27-66 Methyl norepinephrine, fine structural localization in nervous tissue, 27-66 Midbrain tegmentum, amine metabolism and lesions of, 71-72 Mitochondria, somatofugal transport of, 303-304 Mitomycin, stereochemical relations to membrane-active drugs, 215-216 Molecular mechanisms in information processing, 223-253 evidence for, 231-242 bioassay methods, 239-242 chemical correlates, 232-236 critical factors, 235 location of chemical changes, 234 RNA and protein changes, 232-233 learning and memory, 226-227 molecular hypotheses of, 24S248 based on chemospecific pathways, 244-248 nonspecific, 242-243 “tape-recorder” molecular, 243-244 neural coding in, 227-231 chemical code, 23&231 electrical activity, 228-229 structural code, 229-230 neural information processing, 224-226 Monoamines. (See also Amines) autoradiography of, constraints in use, 38-39 binding to cellular organelles, 39-41 to tissue components, 40-41 central neurons for, see Central monoamine neurons cytochemical localization of, 28-29 electron microscopy of, reactivity with stains, 29-34 fine structural localization of in nervous tissue, 27-86 central nerve terminals, 53-59 nonterminal axons, 59-61 peripheral sympathetic nerve terminals, 41-53

model tissue experiments and “ideal localizing paradigm,” 28-39 oxidation to insoluble pigments, 34 pathways of, methods of mapping out, 94-96 Mood, central serotonin neuron role in, 120 Motor activity, effect on brain RNA and proteins, 273-274 Motor functions, of dopamine neurons, 119 Mountcastle and Werner’s sensory transmission experiment, 364366 Muscarine, receptor site for, 192-195

N Nerve terminals central type, see Central nerve terminals peripheral type, see Peripheral sympathetic nerve terminals Nervous system, protein metabolism in, see Protein metabolism of nervous system Nervous tissue, fine structural localization of biogenic amines in, 27-68 Neural coding of information, 227-231 Neural information processing, 224-226 Gardiner’s differential processing experiment, 366-369 Mountcastle and Werner’s sensory transmission experiment, 3fj4-366 Stark‘s experiment on, 361-364 Neuroendocrine function of noradrenaline neurons, 114, 120 of serotonin neurons, 118-119, 1u) Neurons central monoamine type, see Central monoamine neurons protein and nucleic acid metabolism in during increased activtiy, 268-279 protein synthesis in, 290-296 in soma, 290-292 protein transport in, 289-324 Neurosecretory granules, somatofugal transport of, 303-304 Neurotransmitters in brain amino acid uptake and, 143-146

396

SUBJECT INDEX

catecholamine-storing synaptosomes in different brain regions, 146-149 catecholamine uptake and, 128-139 serotonin uptake and, 139-143 uptake and subcellular localization of, 127-158 Nicotine, receptor site for, 193-198 Nigro-neostriatal dopamine neurons, function of, 104-110, 119 Noradrenaline neurons in behavior and wakefulness, 110114, 119-120 functions of, 110-115 neuroendocrine function of, 114, 120 pathways of, 98-101, 110-115 ascending NA neurons, 98-100, 110114 descending NA neurons, 100, 114115 innervating lower brainstem, ll& 101 Norepinephrine ( NE) binding in major groove site, 201 in brain, lesions effect on, 71, 75, 76, 86,87 fine structural localization in nervous tissue, 27-66 somatofugal transport of, 302 storage nerve terminals in intra-axonal SGV, 61 synaptosomes storing, 149 uptake in brain, 128-139 Nucleic acids measurement methods for, 269-273 metabolism of in neurons and glial cells during increased activity, 268-279

0 Orotic acid, somatofugal transport of, 301 Osmium tetroxide, as stain for biogenic monoamines, 30, 31-32 Osmotic stimulation, effect on brain RNA and proteins, 275

P Patulin, stereochemical relations to membrane-active drugs, 217

Peripheral nervous system, breakdown and synthesis of proteins in, 265-266 Peripheral sympathetic nerve terminals localization of monoamines in, 4 1 4 3 in large granular vesicles, 51-53 in small granular vesicles, 41-51 Phospholipids, somatofugal transport of, 301 Phosphoprotein, somatofugal transport of, 301 Pincytotic vesicles somatofugal transport of, 304 Potassium permanganate, as stain for biogenic monoamines, 30, 32 Prostaglandin-ribonucleoprotein complex, in receptor site, 189-191, 221-222 Protein( s ) in brain information processing, 232233 metabolism of in glial cells and neurons, 268-279 in nervous system during increased activity, 261-266 Protein metabolism of nervous system amino acid metabolism during, 256261 breakdown and synthesis of nervous system proteins, 261-266 hormonal effects, 266-268 increased functional activity effects on, 255-288 neuroglial relationships in, 276-277 stimulation effects on, 273-276 studies performed at tissue level, 256268 Protein transport in axons, 296-313 bidirectional transport, 310-313 components transported in, 308-310 mechanisms for, 316-319 physiological and pathological changes affecting, 313-316 somatofugal type, 29&298 velocity of, 298-308 in neurons, 284324 Purkinje cell, 16-17 of cerebellum, 9-10

SUSJECr INDEX

Q Quinacrine, stereochemical relations to membrane-active drugs, 216

397

somatofugal transport of, 301 synthesis of, drug effects on, 238-239 Rotation, effect on brain RNA and proteins, 275

R S Receptor site Serotonergic receptors for catecholamines, 201-202 chemical nature of, 181-222 inactivation-reactivation of, 163-164 disulfide bond in, 216211 isolation of, 1% specification of, 202-207 prostaglandin-ribonucleoprotein comSerotonin (5-HT) p k x and, 189-191 reserpine effects on, 213-215 in brain, lesions effect on, 69, 71, 75, 76, 86, 87 RNA and, 182-187 decrease of, in behavioral depression, in excitable membrane, 184-189 stereochemical relations between mem333335 fine structural localization in nervous brane-active drugs and antibiotics, 2J5-217 tissue, 27-66 increase of, in behavioral depression, tetrodotoxin effects on, 212-213 veratridine effects on, 212-213 327-333 Receptors neurons adrenergic type, 160-161 behavior and, 1%) chemical nature of, 160-166 mental functions of, 115-118, 120 cholinergic type, 161-162, 163 mood and, 120 inactivation-reactivation of, 163-164 neuroendocrine functions of, 118119,120 isolation of, 164-165 reaction with transmitters, see Transreflex activity and, 120 in sexual behavior, 118 mitter-receptor interaction thought processes and, 120 serotonergic type, 163-164 transmitter structure-activity relationpathways of, 101-102 ascending 5-HT neurons, 101, 115ships and, 160-162 Reflex activity 119 central serotonin neuron role in, 120 descending bulbospinal 5-HT neunoradrenaline neuron role in, 120 rons, 102, 119 Reserpine, effects on receptor sites, 212innervating lower brainstem, 102 uptake in brain, 139-143 213 Retina, ganglion cells of, 10-11 Sexual behavior, serotonin neuron role in, RNA 118 SGV (small granular vesicles), localizain axon, 292-294 tion of monoamines in, 41-51 in brain information processing, 232Sodium pump mechanism, RNA and, 233 217-220 in excitable membrane, 184-189 measurement, methods for, 269-271 Soma, protein synthesis in, 290-292 in neurons and glial cells Somatofugal axonal transport of proteins, concentration and distribution of, 296-313 Stains, for biogenic monoamines, 30-34 271-273 stimulation effects on, 273-276 Stark's experiment on neural coding in in receptor site, 182-184 crayfish, 361-364 sodium pump mechanism and, 217- Streptomycin, stereochemical relations to 220 membrane-active drugs, 217

398

SUBJECT INDEX

Strychnine, as glycine receptor block, 208-210 Structural code, for neural coding, 229230 Subcortex, amine metabolism and lesions in, 69-72 Superior olive, neurons of, 8 Synaptic transmission, drug effects on, 23&237 Synaptosomes catecholamine-storage type in different brain regions, 146-149 separation storing different transmitters, 149-156

T Tape-recorder molecule hypothesis, of information processing, 2.43-244 Thalamic ventrobasal cells, neurons of, 7-8 Tetrahydrocannabinol, binding to PGRNA complex, 206, 207 Tetrodotoxin, effects on receptor sites, 212-213 Thermoregulation, dopamine neuron role in, 109 Thought processes, central serotonin neuron role in, 120 Transamination, in brain during excitation, 258-260 Transmitters, ( See also Neurotransmitters ) biochemical effects of, 166-171

on adenyl cyclase, 166-168 on lipids, 168-170 Transmitter-receptor interaction, 159-180 biochemical effects of transmitters in, 166-171 chemical mechanisms of, 159-180 receptors in, see Receptors theories of, 171-178 interrelationships of, 177-178 models requiring chemical changes, 174-177 models requiring only conformational changes, 172-174 Tubero-infundibular DA neurons, functions of, 110, 119 Tyrosine hydroxylase, brain lesions and brain level of, 87

V Veratridine, effects on receptor sites, 212-213 Vinblastine, stereochemical relations to membrane-active drugs, 216-217 Violacein, stereochemical relations to membrane-active drugs, 216

W Wakefulness ascending noradrenaline neuron role in, 110-114 central noradrenaline function in, 119120 central serotonin neuron role in, 120

CONTENTS OF PREVIOUS VOLUMES Volume 1

Recent Studies of the Rhinencephalon in Relation to Temporal Lobe Epilepsy and Behavioral Disorders W. R. A&y Nature of Electrocortical Potentials and Synaptic Organizations in Cerebral and Cerebellar Cortex Dorninick P. Purpura Chemical Agents of the Nervous System Catherine 0. Hebb Parasympathetic Neurohumors; Possible Precursors and Effect on Behavior Carl C . Pfeifer Psychophysiology of Vision G . W . Granger Physiological and Biochemical Studies in Schizophrenia with Particular Emphasis on Mind-Brain Relationships Robert G . Heath Studies on the Role of Ceruloplasmin in Schizophrenia S . Mdrtens, S . Vallbo, and B. Melandm Investigations in Protein Metabolism in Nervous and Mental Diseases with Special Reference to the Metabolism of Amines F. Georgi, C . G . Honegger, D. Jordan, H . P . Rieder, and M . Rottenberg AUTHOR INDEX-SUB JECT INDEX

Volume 2

Regeneration of the Optic Nerve in Amphibia 8.M . Gaze Experimentally Induced Changes in the Free Selection of Ethanol Jorge Mardones The Mechanism of Action of the Hemicholiniums F . W. Schuebr 399

400

CONTENTS OF PREVIOUS VOLUMES

The Role of Phosphatidic Acid and Phosphoinositide in Transmembrane Transport Elicited by Acetylcholine and Other Humoral Agents Lowell E . Hokin and Mabel R. Hokin Brain Neurohormones and Cortical Epinephrine Pressor Responses as Affected by Schizophrenic Serum Edward 1. Walmxek The Role of Serotonin in Neurobiology Erminw Costa Drugs and the Conditioned Avoidance Response Albert Herx Metabolic and Neurophysiological Roles of y-Aminobutyric Acid Eugene Roberts and Eduardo Eidelberg Objective Psychological Tests and the Assessment of Drug Effects H . 1. Eysenck AUTHOR INDEX-SUB JECT INDEX

Volume 3

Submicroscopic Morphology and Function of Glial Cells Eduardo De Robertis and H . M . Gerschenfeld Microelectrode Studies of the Cerebral Cortex Vahe E . AmusAn Epilepsy Arthur A. Ward, IT. Functional Organization of Somatic Areas of the Cerebral Cortex Hiroshi Nakahuma Body Fluid Indoles in Mental Illness €3. Rodnight Some Aspects of Lipid Metabolism in Nervous Tissue G . R. Webster Convulsive Effect of Hydrazides : Relationship to Pyridoxine Harry L. Wiltiams and j a m s A. Bain The Physiology of the Insect Nervous System D. M . Vowles AUTHOR INDEX-SUBJECT

INDEX


401

Volume 4

The Nature of Spreading Depression in Neural Networks Sidney Ochs Organizational Aspects of Some Subcortical Motor Areas Werner P. Koelb Biochemical and Neurophysiological Development of the Brain in the Neonatal Period Williamina A. Himwich Substance P: A Polypeptide of Possible Physiological Significance, Especially within the Nervous System F. Lembeck and G. Zelter Anticholinergic Psychotomimetic Agents L. G . Abood and J. H . Biel Benzoquinolizine Derivatives: A New Class of Monamine Decreasing Drugs with Psychotropic Action A. Pbtscher, A. Brossi, and K . F . Gey The Effect of Adrenochrome and Adrenolutin on the Behavior of Animals and the Psychology of Man A. H o f e r AUTHOR INDEX-SUB JECT INDEX

Volume 5

The Behavior of Adult Mammalian Brain Cells in Culture Ruth S . Geiger The Electrical Activity of a Primary Sensory Cortex: Analysis of EEG Waves Walter J. Freeman Mechanisms for the Transfer of Information along the Visual Pathways Koiti Motokawa Ion Fluxes in the Central Nervous System F . J. Brinley, Jr. Interrelationships between the Endocrine System and Neuropsychiatry Richard P . M i c h l and J a m s L. Gibbons Neurological Factors in the Control of the Appetite Andre' Souluirac

402


Some Biosynthetic Activities of Central Nervous Tissue R. V. Coxon Biological Aspects of Electroconvulsive Therapy Gunnar Holmberg AUTHOR IN'DEX-SUB JECT INDEX

Volume 6

Protein Metabolism of the Nervous System Abel Lajthu Patterns of Muscular Innervation in the Lower Chordates Quentin Bone

The Neural Organization of the Visual Pathways in the Cat Thomas H . Meikb, IT.and James M . Sprague Properties of Afferent Synapses and Sensory Neurons in the Lateral Geniculate Nucleus P. C . Bishop Regeneration in the Vertebrate Central Nervous System Carmine D. Clemente Neurobiology of Phencyclidine ( Sernyl), a Drug with an Unusual Spectrum of Pharmacological Activity Edward F . Domino Free Behavior and Brain Stimulation JosB M . R. Delgado AUTHOR INDEX-SUB JECT INDEX

Volume 7

Alteration and Pathology of Cerebral Protein Metabolism Abel Laitha Micro-Iontophoretic Studies on Cortical Neurons K . KrnjeviL Responses from the Visual Cortex of Unanesthetized Monkeys John R. Hughes Recent Developments of the Blood-Brain Barrier Coiicept Ricardo Edstrom


403

Monoamine Oxidase Inhibitors Gordon R. Pscheidt The Phenothiazine Tranquilizers : Biochemical and Biophysical Actions Paul S. Guth and Morris A. Spirtes Comments on the Selection and Use of Symptom Rating Scales for Research in Pharmacotherapy J. B. Wittenborn Multiple Molecular Forms of Brain Hydrolases Joseph Bernrohn and Kevin D. Barron AUTHOR INDEX-SUB JECT INDEX

Volume 8

A Morphologic Concept of the Limbic Lobe Lowell E. White, IT. The Anatomophysiological Basis of Somatosensory Discrimination David Bowsher, with the collaboration of Denise Albe-Fessard Drug Action on the Electrical Activity of the Hippocampus Ch. Stumpf Effects of Drugs on Learning and Memory James L. McGaugh and Lewis F. PetrinovicJt Biogenic Amines in Mental Illness Giinter G. Bmne The Evolution of the Butyrophenones, Haloperidol and Trifluperidol, from Meperidine-Like 4-Phenylpiperidines Paul A. 1. Jamsen Amplitude Analysis of the Electroencephalogram (Review of the Information Obtained with the Integrative Method) Leonide Gold.stein and Raymond A. Beck AUTHOR INDEX-SUB JECT INDEX

Volume 9

Development of “Organotypic” Bioelectric Activities in Central Nervous Tissues during Maturation in Culture Stanby M . Crain The Unspecific Intralaminary Modulating System of the Thalamus P. Krupp and M . Monnier

404


The Pharmacology of Imipramine and Related Antidepressants Laszlo Gyermek Membrane Stabilization by Drugs : Tranquilizers, Steroids, and Anesthetics Philip M . Seeman Interrelationships between Phosphates and Calcium in Bioelectric Phenomena L. G . Abood The Periventricular Stratum of the Hypothalamus Jerome Sutin Neural Mechanisms of Facial Sensation I . Darian-Smith AUTHOR INDEX-SUB JECT INDEX

Volume 10

A Critique of Iontophoretic Studies of Central Nervous System Neurons G. C . Salmoiraghi and C . N . Stefanis Extra-Blood-Brain-Barrier Brain Structures Werner P. Koella and Jerome Sutin Cholinesterases of the Central Nervous System with Special Reference to the Cerebellum Ann Silver Nonprimary Sensory Projections on the Cat Neocortex P. Buser and K . E . Bignull Drugs and Retrograde Amnesia Albert Weissman Neurobiological Action of Some Pyrimidine Analogs Harold Koenig

A Comparative Histochemical Mapping of the Distribution of Acetylcholinesterase and Nicotinamide Adenine Dinucleotide-Diaphorase Activities in the Human Brain T . Ishii and R . L. Friede Behavioral Studies of Animal Vision and Drug Action Hugh Brown


405

The Biochemistry of Dyskinesias G. Curxon AUTHOR INDEX-SUB JECT INDEX

Volume 11

Synaptic Transmission in the Central Nervous System and Its Relevance for Drug Action Philip B. Bradley Exopeptidases of the Nervous System Neville Marks Biochemical Responses to Narcotic Drugs in the Nervous System and in Other Tissues Doris H . Clouet Periodic Psychoses in the Light of Biological Rhythm Research F. A. JennetEndocrine and Neurochemical Aspects of Pineal Function B81a Mess The Biochemical Investigations of Schizophrenia in the USSR D. V. Lowvsky Results and Trends of Conditioning Studies in Schizophrenia J. S a u m Carbohydrate Metabolism in Schizophrenia Per S , Lingjaerde The Study of Autoimmune Processes in a Psychiatric Clinic S . F . Semenov Physiological Foundations of Mental Activity N . P. Bechtereva and V. B . Gretchin AUTHOR INDEX-SUB JECT INDEX

Cumulative Topical Index for Volumes 1-10 Volume 12

Drugs and Body Temperature Peter L w x Pathobiology of Acute Triethyltin Intoxication R. Torack, 3. Gordon, and J . Prokop

406


Ascending Control of Thalamic and Cortical Responsiveness M. Steriade Theories of Biological Etiology of Affective Disorders John M . Davis Cerebral Protein Synthesis Inhibitors Block Long-Term Memory Samuel H. Barondes The Mechanism of Action of Hallucinogenic Drugs on a Possible Serotonin Receptor in the Brain 1. R. Smythies, F. Benington, and R. D. Morin Simple Peptides in Brain Zsamu Sam The Activating Effect of Histamine on the Central Nervous System M. Monnier, R. Sauer, and A. M . Hatt Mode of Action of Psychomotor Stimulant Drugs Jacques M. van Rossum AUTHOR INDEX-SUB JECT INDEX

International Review of Neurobiology Volume 13

International Review of Neurobiology, Volume 6

International Review of Neurobiology Volume 25

International Review of Neurobiology Volume 38

International Review of Neurobiology Volume 18

International Review of Neurobiology Volume 31

International Review of Neurobiology Volume 14

International Review of Neurobiology, Volume 23