International Review of Neurobiology Volume 11

INTERNATIONAL REVIEW OF Neurobiology VOLUME 1 1 Associate Editors W. Ross ADEY H. J. EYSENCK D. BOVET G. HARRIS ...

Author: Carl C Pfeiffer

14 downloads 963 Views 19MB Size Report

This content was uploaded by our users and we assume good faith they have the permission to share this book. If you own the copyright to this book and it is wrongfully on our website, we offer a simple DMCA procedure to remove your content from our site. Start by pressing the button below!
Report copyright / DMCA form

DOWNLOAD PDF

INTERNATIONAL REVIEW OF

Neurobiology VOLUME 1 1

Associate Editors

W. Ross ADEY

H. J. EYSENCK

D. BOVET

G. HARRIS

Tosf DELCADO

C. HEBB

SIR JOHN ECCLES

0. ZANCWILL

Consultant Editors

V. AMASSLAN

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 B. HOLMSTEDT

D. M. MACKAY

P. A. J.

F. MORRELL

JANSSEN

S. KETY

STEN M ~ R T E N S

H. OSMOND STEPHENSZARA

INTERNATIONAL REVIEW OF

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

J O H N R. SMYTHIES Deportment of Psychiatry University of Edinburgh, Edinburgh, Scotland

VOLUME 11

1968

ACADEMIC PRESS

New York and London

COPYRIGHT@ 1988,

BY

ACADEMICPRESS,INC.

ALL RIGHTS RESERVED. NO PART OF THIS BOOK MAY BE REPHODUCED IN ANY FORM,

BY PHOTOSTAT, MICROFILM, OR ANY OTHER MEANS, WITHOUT WRITTEN PERMISSION FROM THE PUBLISHERS.

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

United Kiiigdom Edition published by ACADEMIC PRESS, INC. (LONDON) LTD. Berkeley Square House, London W.1

LIBRARY OF CONGRESS CATALOG CARDNUMBER: 59-13822

PRINTED IN THE UNITED STATES OF AMERICA

CONTRIBUTORS Numbers in parentheses refer to the pages on which the authors' contributions begin.

N . P. BECHTEREVA, Institute of Experimental Medicine, Leningrad, USSR (329)

PHILIP B. BRADLEY,Department of Experimental Neuropharmacology, The Medical School, Birmingham, England (1) DORISH. CLOUET,New York State Research Znstitute for Neurochemistry and Drug Addiction, Ward's Island, and Columbia University College of Physicians and Surgeons, New York, New York (99)

V. B. GRETCHIN, Institute USSR (291)

of

Experimental Medicine, Leningrad,

F. A. JENNER, M.R.C. Unit for Metabolic Studies in Psychiatry, Middbwood Hospital, and University Department of Psychiatry, W h i t e b y Wood Clinic, Shefield, England (129)

PER S. LINGJAERDE, Department of Clinical Chemistry, Akershus Central Hospital, Nordb yhagen, Norway ( 259) D. V. LOZOVSKY, Institute o f Psychiatry of the USSR Academy of Medical Sciences, Moscoto, USSR ( 199)

NEVILLE MARKS,New Y m k State Research Institute for Neurochemistry and Drug Addiction, Ward's Island, New York, New York (57) B ~ L AMESS,Department of Anatomy, University Medical School, Pe'cs, Hungary (171)

J. SAARMA,Department of Psychiatry, Tartu State University, Tartu, USSR (227) S. F. SEMENOV,Moscow Research Institute of Psychiatry, Moscow, USSR (291)

V

This Page Intentionally Left Blank

PREFACE In this volume we are very pleased to welcome for the first time review articles by distinguished Soviet scientists on various aspects of work in the neurobiological sciences in the Soviet Union, with particular emphasis on psychiatric research. Professor Bechtereva gives an overall review of the current search for physiological correlates of mental processes. Two contributions are concerned with the considerable Soviet investment into biological aspects of schizophrenia-Professor Saarma deaIs with conditioning studies and Dr. Lozovsky with biochemical research. Finally, Professor Semenov discusses the autoimmune aspects of various neuropsychiatric disorders including, again, schizophrenia. These papers not only present succinct accounts of recent Soviet work but also provide access to a wide range of references. Our policy is still to cover neurobiology, to bring into focus new and interesting developments in the basic sciences as well as in psychiatric and neurological research. CARL C. PFEIFFER JOHN R. SMYTHIES October 1968

vii


CONTENTS CONTR~BUTORS .

.

.

.

.

.

.

.

.

.

.

.

PREFACE.

.

.

.

.

.

.

.

.

.

.

.

vii

VoLuhrEs

.

.

.

.

.

.

.

.

xiii

.

2 4 17 25 29 31 34 43 53

.

.

CONTENTSOF Pmvious

V

Synaptic Transmission in the Central Nervous System and Its Relevance for Drug Action

PHILIPB. BRADLEY I . Introduction . . . . . . . I1. Acetylcholine . . . . . . . 111. Monoamines . . . . . . . IV . Amino Acids . . . . . . . V . Other Potential Transmitters . . . . . . . V I . Multiple Effects on Neurons . VII . Effects of Centrally Acting Driigs . . . VIII . Conclusions and Summary . . . . References . . . . . . . .

.

.

.

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

.

. . . .

. . . .

. . . .

. . . . .

.

.

. . . .

.

Exopeptidases of the Nervous System

NEVILLEMARKS I . Scope of Review and Introduction

.

.

.

.

I1. a-Aminopeptide Amino Acid Hydrolases (E.C.3.4.1)

I11. Dipeptide Hydrolases (E.C.3.4.3) . . . . IV. Arylamide Amino Acid Hydrolases . . . . V . a-Carboxypeptide Amino Acid Hydrolases (E.C.3.4.2) VI . Exopeptidases in the Different Areas of the CNS . . . VII . Peripheral Nerve . . . . . . . . . VIII. Conclusions . . . . . . . . . . References . . . . . . . . . . .

. . . .

57 61 64 69 73 77 83 85 90

. .

.

. .

Biochemical Responses to Narcotic Drugs in the Nervous System and in Other Tissues

DORISH . CLOUET

I . Introduction . . . . . . I1. Metabolic Disposition of Narcotic Drugs ix

.

.

.

.

.

.

.

.

.

.

99 101

CONTENTS

X

. Effects on General Metabolic Systems . . . . . . . Effects on Specific Metabolic Reactions . . . . . . . Serum and Brain Factors . . . . . . . . .

I11 IV V VI .

Conclusions References .

. .

. .

. .

. .

. .

. .

. .

. .

. .

. .

. .

108 115 121 122 124

Periodic Psychoses in the Light of Biological Rhythm Research

F. A .

.

TENNER . . .

I Introduction . . . . . I1. Evidence for the Existence of Periodic Psychoses . I11. Nosology and Periodic Psychoses . . . . IV. Periodic Illnesses in General . . . . . V Richter’s Hypotheses . . . . . . . VI . Circadian Rhythms . . . . . . . VII Cellular Studies . . . . . . . . VIII . Mathematical Considerations . . . . . IX Survival Value . . . . . . . . X The Menstrual and Estral Clocks . . . . XI Estrogens, Androgens. and Animal Behavior . . XI1. Estrogens. Androgens. and Human Behavior . . XI11 Light and the Menstrual Cycle . . . . XIV Thyroid Activity and Periodic Psychoses . . XV. Vasopressin and Periodic Psychoses . . . XVI . Early Work on Periodic Psychoses . . . . XVII Gjessing’s Studies . . . . . . . XVIII The Adrenal Cortex and Periodic Psychoses . . XIX Catecholamines . . . . . . . . XX Autonomic Concomitants of Periodic Psychoses . XXI Electroencephalography . . . . . . XXII . Lithium and Periodic Psychoses . . . . References . . . . . . . . .

. . . . . . .

. . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . .

. . . .

. . . .

. . . . . .

. . . . . .

. . . . . .

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

129 130 132 133 137 138 139 139 140 141 142 144 148 149 151 152 152 154 156 157 157 158 160

Endocrine and Neurochemical Aspects of Pineal Function

B~LA MESS I . Structure and Metabolism of the Pineal Gland

.

.

.

.

V. Biorhythm of Melatonin and Serotonin Production . . . . . . VI . Concluding Remarks . References . . . . . . . . .

.

.

.

. .

. .

. .

I1. Effect of Pineal Function on the Endocrine System . . . 111. Biosynthesis and Metabolism of Melatonin and Serotonin . . IV Effect of Light and Sympathetic Innervation on Pineal Activity

.

171 174 183 187 191 194 194

xi

CONTENTS

The Biochemical Investigations of Schizophrenia in the USSR

D . V. LOZOVSKY

I . Current Trends . . . . . . I1. Pathogenesis . Major Syndromes . . I11. Pathogenesis . Some Other Concepts . IV. Classification . . . . . . V. Biochemical Investigations at the Institute VI . Summary . . . . . . . References . . . . . . .

.

.

.

.

.

199

. .

. .

. .

. .

. .

.

.

.

.

.

204 207 214 216 222 222

.

.

.

.

.

. .

.

.

.

.

.

of Psychiatry

Results and Trends of Conditioning Studies in Schizophrenia

J . SAARMA

I . Introduction . . . . . . . . . . . I1. Some Methodological Problems in HNA Studies . . . . I11. HNA Alterations in Schizophrenia . . . . . . . IV . Special Features of the HNA in Various Forms and Stages of Schizophrenia . . . . . . . . . . . V. Changes of the HNA in Schizophrenia under Treatment . . VI . Some Theoretical and Practical Conclusions . . . . . VII . Summary . . . . . . . . . . . . References . . . . . . . . . . . .

227 229 232

235 238 241 248 248

Carbohydrate Metabolism in Schizophrenia

PER S . LINCJAERDE I. Introduction . . . . . . . . . . I1. Glucose Tolerance Tests . . . . . . . . I11. Insulin Tolerance Tests . . . . . . . . IV. Lactate, Pyruvate. and Citric Acid Cycle Intermediates . V. Brain Metabolism . . . . . . . . . VI . Enzymes . . . . . . . . . . . VII . Red Cell Metabolism . . . . . . . . . VIII . Serum Factors and Carbohydrate Metabolism . . . IX . Concluding Remarks . . . . . . . . . References . . . . . . . . . . .

. . . . . . . . . .

259 261 262 264 265 266 267 279 284 286

The Study of Autoimmune Processes in a Psychiatric Clinic

S . F. SEMENOV

I . Introduction . . . I1. Schizophrenia . . . I11. Vascular Diseases of Brain IV . Neurosyphilis . . .

.

.

.

.

.

.

.

.

. . .

. . .

. . .

. . .

. . .

. . .

. . .

. . .

291 296 306 310

xii

CONTENTS

V. Residual Phenomena of Various Organic Affections of the Brain . . . . . . . . . and Psychic Trauma . References . . . . . . . . . . . .

310 325

Physiological Foundations of Mental Activity N . P . BECHTEREVA AND V. B . GRETCHIN

. . .

I Introduction . . . . . . . . . . . I1. EEG in Conditioning and Mental Tests . . . . . . I11 Some New Approaches to Physiological Investigation of Mental . . . . . . . . . . . . Activity IV Some Theoretical Considerations on the Structure-Functional . . . . . . . . Basis of Mental Activity . References . . . . . . . . . . . .

329 330 334

342 346

AUTHOR INDEX

.

.

.

.

.

.

.

.

.

.

.

.

SUBJECTINDEX

.

.

.

.

.

.

.

.

.

.

.

.

376

.

.

.

.

.

382

CUMULATIVETOPICALINDEX FOR VOLUMES1-10

353

CONTENTS OF PREVIOUS VOLUMES Volume 1

Recent Studies of the Rhinencephalon in Relation to Temporal Lobe Epilepsy and Behavioral Disorders W. R. Adey Nature of Electrocortical Potentials and Synaptic Organizations in Cerebral and Cerebellar Cortex Dominick P . Purpura Chemical Agents of the Nervous System Catherine 0. Hebb Parasympathetic Neurohumors; Possible Precursors and Effect on Behavior Carl C . Pfeiffer 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 RoIe of CerulopIasmin in Schizophrenia S . M&rtens, S . Vallbo, and B. Melander Investigations in Protein Metabolism in Nervous and Mental Diseases with Special Reference to the Metabolism of Amines F . Georgi, C . G . Honegger, D . .lordun, H . P. Rieder, and hl. Rottenberg AUTHOR INDEX-SUBJECT INDEX

Volume 2

Regeneration of the Optic Nerve in Amphibia R. M . Gaze Experimentally Induced Changes in the Free Selection of Ethanol Jorge Mardones xiii

xiv

CONTENTS OF PREVIOUS VOLUMES

The Mechanism of Action of the Hemicholiniums

F . W . Schueler 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 J . Walaszek The Role of Serotonin in Neurobiology

Erminio Costa Drugs and the Conditioned Avoidance Response Albert Hem Metabolic and Neurophysiological Roles of 7-Aminobutyric Acid

Eugene Roberts and Eduardo Eidelberg Objective Psychological Tests and the Assessment of Drug Effects H . J . Eysenck AUTHOR INDEX-SUB JECT INDEX

Volume 3

Submicroscopic Morphology and Function of Glial Cells Eduurdo De Robertb and H . M . Gerschenfeld Microelectrode Studies of the Cerebral Cortex Vahe E . A m & n Epilepsy

Arthur A. Ward, Jr. Functional Organization of Somatic Areas of the Cerebral Cortex

Hiroshi Nakahamu Body Fluid Indoles in Mental Illness R . Rodnight Some Aspects of Lipid Metabolism in Nervous Tissue G. R. Webster Convulsive Effect of Hydrazides : Relationship to Pyridoxine Harry L. Williams and James A. Bain


xv

The Physiology of the Insect Nervous System D . M . Vowles AUTHOR INDEX-SUB J E C r INDEX

Volume 4

The Nature of Spreading Depression in Neural Networks Sidney Och Organizational Aspects of Some Subcortical Motor Areas Werner P . Koella 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. Pletscher, A. Brossi, and K . F . Gey The Effect of Adienochrome and Adrenolutin on the Behavior of Animals and the Psychology of Man A. Hofer AUTHOR INDEX-SUB J E C r 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 Ko iti Motokawa

xvi


Ion Fluxes in the Central Nervous System F. J . Brinley, Jr. Interrelationships between the Endocrine System and Neuropsychiatry Richard P . Michael and James L. Gibbons Neurological Factors in the Control of the Appetite Andre' SoulaiTac Some Biosynthetic Activities of Central Nervous Tissue R. V. Coxon Biological Aspects of Electroconvulsive Therapy Gunnar Holmberg AUTHOR INDEX-SUB JECT INDEX

Volume 6

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

The Neural Organization of the Visual Pathways in the Cat Thomar H . Meikle, Jr., and James M . Sprague Properties of Merent 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--SUBJECT INDEX


Wii

Volume 7

Alteration and Pathology of Cerebral Protein Metabolism Abel Lajtha Micro-Iontophoretic Studies on Cortical Neurons K . Krnjeuik Responses from the Visual Cortex of Unanesthetized Monkeys John R. Huglws Recent Developments of the Blood-Brain Barrier Concept Ricardo Edstrom 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 Bernsohn and Kevin D . Barron AUTHOR INDEX-SUB JECT INDEX

Volume 8

A Morphologic Concept of the Limbic Lobe Lowell E. White, Jr. 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 . Petrinovich Biogenic Amines in Mental Illness Giinter G . Brune

xviii


The Evolution of the Butyrophenones, Haloperidol and Trifluperidol, from Meperidine-Like 4-Phenylpiperidines Puul A. J. Janssen Amplitude Analysis of the Electroencephalogram (Review of the Information Obtained with the Integrative Method) Leonide Goldstein and Raymond A. Beck AUTHOR INDEX-SUBJECT

INDEX

Volume 9

Development of “Organotypic” Bioelectric Activities in Central Nervous Tissues during Maturation in Culture Stanley M . Crain The Unspecific Intralaminary Modulating System of the Thalamus P . Krupp and M . Monnier 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 1. Darian-Smith AUTHOR INDEX-SUBJECT

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


XiX

Cholinesterases of the Central Nervous System with Special Reference to the Cerebellum Ann Silver Nonprimary Sensory Projections on the Cat Neocortex P. Buses and K . E . Bignall Drugs and Retrograde Amnesia Albert Weisman Neurobiological Action of Some Pyrimidine Analogs Harold Koenig

A Comparative Histochemical Mapping of the Distribution of Acetylcholinesterase and Nicotinamide Adenine DinucleotideDiaphorase Activities in the Human Brain T . lshii and R. L. Friede Behavioral Studies of Animal Vision and Drug Action Hugh Brown The Biochemistry of Dyskinesias G. Curzon AUTHOR INDEX-SUB JECT INDEX


SYNAPTIC TRANSMISSION IN THE CENTRAL NERVOUS SYSTEM AND ITS RELEVANCE FOR DRUG ACTION

.

By Philip 9 Bradley Department of Experimental Neuropharmacalogy. The Medical School. Birmingham. England

I. Introduction . . . . . . . . I1 Acetylcholine . . . . . . . A . Biochemical Evidence . . . . . B. Histochemical Evidence . . . . C. Actions on Neurons . . . . . D . Release . . . . . . . . I11. Monoamines . . . . . . . A . Biochemical Evidence . . . . . B. Histochemical Evidence . . . . C. Actions on Neurons . . . . . . D . Release . . . . . . . . IV . Amino Acids . . . . . . . A . Biochemical Evidence . . . . . B. Actions on Neurons . . . . . C. Release . . . . . . . . . . . . V. Other Potential Transmitters A . Histamine . . . . . . . B . Substance P . . . . . . . C. Ergothioneine . . . . . . D. Prostaglandins . . . . . . . . . . VI . Multiple Effects on Neurons A . Actions of Acetylcholine. Noradrenaline. and 5-Hydroxytryptamine . . . . . . . VII . Effects of Centrally Acting Drugs . A . Central Depressant and Sedative Drugs . . . . . B. Central Stimulant Drugs C. Tranquilizers . . . . . . . D. Psychotomimetic Drugs . . . . . . . . VIII . Conclusions and Summary References . . . . . . . .

.

1

.

.

.

.

. . .

. . .

. . .

. . .

. . . .

2 4 4

5

. .

. .

. .

. .

.

.

.

.

6 14 17 17 18 19

. .

. .

. .

. .

25 25 25

.

.

.

.

. . .

. . .

. . .

. . .

.

.

.

.

.

.

.

.

.

.

.

. .

. .

. . .

. . .

. .

. . .

.

.

.

.

.

.

. . . .

. .

.

. . . . . .

.

.

26 28 29 29 30 30 30 31 32 34 34 36 38 42 43 53

2

PHILIP B. BRADLEY

I. Introduction

It is now generally accepted that synaptic transmission in the vertebrate central nervous system is mediated chemically, i.e., that the passage of a nerve impulse along a presynaptic axon results in the liberation of a substance from the nerve terminals which diffuses to the postsynaptic receptor site, causing a change in membrane permeability and hence in the level of polarization. Many substances have been postulated as transmitters in mammalian brain but there is some divergence of opinion as to the criteria which need to be fulfilled in order that any particular substance may be classed as a synaptic transmitter. Some, for example acetylcholine and the catecholamines, are obvious choices because of their role as peripheral transmitters, since it seems likely that some of the mechanisms found in peripheral structures may also be present in the central nervous system. Others, 5hydroxytryptamine, amino acids, substance P, ergothioneine, prostaglandins, etc., have been suggested for other reasons, e.g., their presence in brain tissue, known pharmacological or biochemical actions, or involvement directly or indirectly with changes in mental function. While there has been a considerable amount of investigation, discussion, and speculation in relation to the role of these substances as synaptic transmitters, comparatively little attention has been paid to the possibility that their actions might be closely related to, and even possibly explain, the effects of many centrally acting drugs. The main purpose of this contribution is to discuss such a possibility. Since the vertebrate neuromuscular junction has been the subject of intensive study and is probably the best-documented example of chemical transmission at a synapse (for a full account, see Eccles, 1964a; Katz, 1966), there has been a tendency to use this structure as a model when considering transmission at other sites in the nervous system (Curtis, 1961). Although there are important differences in the organization of synapses in the central nervous system as compared with the neuromuscular junction, it may be useful to consider the criteria which are satisfied by acetylcholine as the transmitter at this site: 1. The transmitter substance must be present in s d c i e n t quantities in the presynaptic terminals (which must therefore contain a synthesizing enzyme system and storage facilities) and be released on nerve stimulation. 2. It must act on the postsynaptic membrane to cause depolar-

SYNAPTIC TRANSMISSION IN THE CNS

3

ization, and the action of the locally applied transmitter must be identical with synaptic action. 3. There must be an inactivating enzyme in the region of the synaptic cleft and specific antagonists to the transmitter substance must also block its synaptic action, i.e., the pharmacology of synaptic transmission and of the postsynaptic action of the locally applied transmitter substance must be similar. These criteria are useful when attempting to enumerate the requirements for chemical transmission in the central nervous system, but we must also consider what modifications may be necessary from our knowledge of the morphology and physiology of central synapses. The first important difference is that, whereas the neuromuscular junction operates as a simple relay by which an impulse is transmitted without fail and with the minimum delay from a single nerve cell to a large number of muscle fibers, neuronal synapses in the central nervous system transcribe impulses arriving at the presynaptic terminals, into graded subthreshold changes in potential of the postsynaptic membrane. Furthermore, whereas the nerve/muscle junction operates exclusively as an excitatory synapse, both excitation and inhibition can take place at synapses in the central nervous system. Thus, the membrane of a central neuron is the principal site at which integration of converging impulses, both excitatory and inhibitory, can take place. The transmission of information across synapses in the central nervous system will depend therefore upon the spatiotemporal distribution of excitatory and inhibitory postsynaptic potentials. If the intensity and area of the excitatory potentials is sufficient to overcome any inhibition present and exceed the postsynaptic threshold, an action potential will be propagated. We may therefore restate the criteria for chemical transmission in terms of central synapses: 1. The substance must be present at the presynaptic terminals and be released on nerve stimulation (it need not necessarily be synthesized at this site but could be transported there). 2. It must act on the postsynaptic membrane causing a local depolarization (excitatory) or hyperpolarization ( inhibitory) and the action of locally applied transmitter, e.g., by iontophoresis, must mimic the effect of nerve stimulation. 3. There must be a mechanism for its removal or inactivation, and specific antagonists must block its synaptic action. We can now proceed to consider how far these requirements are

4

PHILLP B. BRADLEY

fulfilled by various substances which have been proposed as synaptic transmitters in the central nervous system and how far the effects of certain centrally acting drugs may be related to the actions of these substances. I I . Acetylcholine

A. BIOCHEMICAL EVIDENCE The presence in the central nervous system of acetylcholine and of the two enzymes responsible respectively for its synthesis and degradation, together with evidence for their differential distribution in many structures, has been known and studied extensively for a long time (Feldberg and Vogt, 1948) and need not be reviewed here. In summary it can be stated that all three substances are found throughout the central nervous system and that the concentration varies in different structures. There is evidence that levels of acetylcholine change in different functional states (Richter and Crossland, 1949) and that certain aspects of behavior may be related to levels of cholinesterase activity in the cerebral cortex (Bennett et al., 1964). The most recent and important evidence from biochemical studies of the distribution of acetylcholine and its related enzymes in brain has come from subcellular fractionation studies, which enable preparations of isolated nerve endings to be obtained (De Robertis, 1964; Whittaker, 1964). In these studies, two main techniques are used: first, differential centrifugation of homogenates of brain tissue, and second, hypoosmotic shock. When brain homogenates are subjected to centrifugation at different speeds it has been found that the acetylcholine, which exists in brain mainly in a bound form (Feldberg, 1957), is present in the fraction containing the mitochondria. However, when this primary fraction is separated further by density gradient centrifugation, the bound acetylcholine is recovered from a fraction of intermediate density, the particles of which contain detached nerve endings. These nerve endings, which were identified morphologically, have been found to contain mostly synaptic vesicles together with some small mitochondria (Whittaker, 1964). Further centrifugation of the nerve-ending particles with hypotonic sucrose solution results in their disruption by hypoosmotic shock. Acetylcholine has been found to be present mainly in the fraction containing the isolated synaptic vesicles and in the

SYNAPTIC TRANSAIISSION IN THE CNS

5

fraction containing disrupted nerve endings. On the other hand, choline acetylase was located mainly in the soluble cytoplasm fraction of the nerve endings, and cholinesterase has a different distribution again, being recovered from the microsomal fraction. The evidence from these studies therefore supports a role for acetylcholine as a transmitter in the central nervous system since the bound acetylcholine seems to be associated with synaptic vesicles and the cholinesterase is localized postsynaptically.

B. HISTOCHEMICAL EVIDENCE One of the main advances in neurological techniques in recent years has been the application of fluorescence histochemistry for identifying and studying the morphological distribution of elements containing amines in the nervous system (Falck, 1952, 1964). There are as yet no such methods for the histochemical examination of acetylcholine itself, but the hydrolytic enzyme acetylcholinesterase can be demonstrated histologicall>i by the thiocholine method (G. B. Koelle and Friedenwald, 1949). Since it is generally thought that the presence of this enzyme may be an indicator for the presence of acetylcholine and therefore of cholinergic mechanisms, cholinesterase staining has been used to delineate histochemically some cholinergic pathways in the central nervous system. However, the method shows the enzyme to be present in many areas of the brain, not only in cell bodies but dong the length of the axons, including nerve terminals. It has been used by Shute and Lewis ( 1963, 1967), in combination with lesions which interrupt cholinesterase-containing axons to cause a buildup of the enzyme on the cell body side of the lesion. In this way it was possible to determine the distribution and polarity of cholinesterase-containing tracts and these workers have produced evidence for an ascending cholinergic pathway in the brain of the aIbino rat, arising in the reticular formation of the brain stem and projecting to most cortical and subcortical structures. They believe that this pathway coincides with the ascending reticular activating system, the existence of which has been demonstrated by mainly physiological evidence. Shute and Lewis have also shown that many afferents to the cerebellum are probably cholinergic and that lesions of the cerebellar peduncles produced a pileup of acetylcholinesterase on the side remote from the cerebellum. Cholinergic elements have also been described for the cerebellar cortex (Phillis, 196%; KBsa and Csillik, 1965). How-

6

PHILIP B. BRADLEY

ever, it is important to remember that many of these studies have been carried out in the rat and the findings may not necessarily be applicable to other species.

C. ACXIONSON NEURONS Although studies in which pharmacologically active substances are injected systemically are useful in indicating how they may modify function in the nervous system, particularly in terms of behavior, they throw little light on their possible role as synaptic transmitters. Thus, many of the effects observed may be due to indirect actions. Furthermore, it is known that some of the substances which may be transmitters in the nervous system do not readily penetrate the blood-brain barrier when injected into the bloodstream, e.g., noradrenaline and 5-hydroxytryptamine. The changes in neuronal activity observed in studies in which acetylcholine, adrenaline, or noradrenaline were injected intravenously or intra-arterially while recording with microelectrodes from single cells in the brain stem (Bradley and Mollica, 1958) may therefore have been due to indirect effects, rather than to direct actions on the nerve cells under study. Direct evidence for actions by substances suspected to be synaptic transmitters on neurons in the central nervous system has awaited the development of suitable techniques by which minute amounts could be applied in the vicinity of a single neuron. Such a technique is that of microiontophoresis or microelectrophoresis in which the ions of the active substance, dissolved in aqueous solution, are made to pass out of the tip of a fine glass micropipette by means of a suitably directed electric current. This technique was originally developed by Nastuk (1953) and was used by him and also by Del Castillo and Katz (1955) for the application of acetylcholine to the neuromuscular junction. In their experiments two separate pipettes were used, controlled by separate micromanipulators, one of which contained acetylcholine which was electrophoresed close to the end plate and the other pipette was used for intracellular recording of the end-plate potential. Since it is not normally possible to observe the neuron which is being recorded in the central nervous system, separate pipettes for recording neuronal activity and iontopboresis cannot be used, and therefore multibarrelled micropipettes have been developed for this purpose. The technique of microiontophoresis used by most workers is similar to

SYNAPTIC TRAKSMISSION I S THE CXS

7

that described by Curtis (1964) and has recently been the subject of critical review ( Salmoiraghi and Stefanis, 1967). Since the current used to eject the active ions from the micropipette can itself affect the activity of the neurons being recorded, it is advisable when using the technique of microiontophoresis for a control of current effects to be carried out. For this purpose one barrel of the micropipette is usually filled with an inactive substance, such as sodium chloride, so that the same current as that used to eject the active ions can be passed through this barrel to determine the effects, if any, of the current alone. The technique of microiontophoresis has now been successfully used for examining the effects of various substances, including acetylcholine, suspected as being synaptic transmitters, on neuronal activity in the spinal cord, cerebellum, cerebral cortex, thalamus, brain stem, and hippocampus. Neurons sensitive to iontophoretic application of acetylcholine have been found in all regions of the central nervous system where its actions have been tested so far. In the spinal cord, Renshaw cells are cholinoceptive and are excited by electrophoretically administered acetylcholine (Curtis and Eccles, 1958). In fact, the effects of acetylcholine so closely mimic those of synaptic excitation that it is generally accepted that the synapse between collaterals of motor axons and Renshaw cells of the anterior horn is cholinergic. A feature of the action of acetylcholine is that it is of rapid onset and ceases shortly after the end of application. Furthermore, this action as well as that of synaptic excitation is blocked by dihydroP-erythroidine (DH-P-E ) and potentiated by eserine. The actions of acetylcholine on Renshaw cells are mimicked by nicotine and, in its pharmacological properties, this cholinergic synapse appears to be nicotinic. Whereas in the spinal cord acetylcholine produces only excitation of neuronal activity, this is not true for other parts of the central nervous system; in the brain both excitatory and inhibitory effects have been observed. Furthermore, acetylcholine is not universally active. In the cerebral cortex, for example, less than 30%of the cells are cholinoceptive (Krnjevih and Phillis, 1963a,b); the majority of these are excited by acetylcholine, inhibitory effects being comparatively rare (Randih et d.,1964). However, apart from the fact that the majority of cortical neurons are unaffected by acetylcholine, there are other differences between the action of this substance on

8

PHILIP B. BRADLEY

Renshaw cells and its actions on cholinoceptive cortical neurons. For example, the rapid onset of action observed in the spinal cord is not seen, and excitation of cortical neurons by acetylcholine is relatively slow in onset and may also be considerably prolonged. Furthermore, while the acetylcholine receptors in the spinal cord appear to be strongly nicotinic, those in the cerebral cortex, for the excitatory response at least, appear to be muscarinic. Thus, excitatory actions of acetylcholine can be mimicked by muscarinic agents such as muscarine and acetyl-p-methylcholine, whereas nicotine has either a slight depressant action or produces effects completely unlike those of acetylcholine. The response to acetylcholine can be blocked by atropine and hyoscine but it is not greatly altered by antagonists of nicotine. These cholinoceptive neurons appear to be mainly confined to the third layer of the cerebral cortex, which includes Betz cells. Since there is histochemical evidence for the presence of cholinesterase-staining fibers linking the neocortex to subcortical projections from the striatum and septum, it has been suggested ( Krnjevi6, 1964) that the cortical cholinoceptive cells are probably innervated by cholinergic radiations from the brain stem reticular formation. In the thalamus, the regions that have been mainly studied with the iontophoretic technique are the lateral geniculate nucleus and the ventrobasal thalamus, consisting of the nuclei ventralis posterolateralis and ventralis posteromedianus. Acetylcholine and related cholinomimetic substances have been found to be weak excitants of lateral geniculate neurons (Curtis and Davis, 1963). On the other hand, neurons in thalamocortical relays were extremely sensitive to acetylcholine (Andersen and Curtis, 1964). The time course of this action was slow in onset and prolonged in duration, resembling that of acetylcholine at the cerebral cortex rather than on Renshaw cells. However, the effects were abolished by dihydro-p-erythroidine and also by atropine, and both nicotine and muscarine mimicked the effects of acetylcholine, although they were less potent. The excitation of thalamic neurons by stimulation of the medial lemniscus or optic tract was not blocked by these antagonists to acetylcholine. Thus, Curtis (1966) concludes that although acetylcholine could be a synaptic transmitter in the thalamus, there is no pathway which converges on the thalamus and which can be regarded as cholinergic. He suggests though, that since there is evidence that pathways derived from the reticular formation and

9

SYNAPTIC TRANSMISSION I N THE CNS

midline thalamic structures may be cholinergic, effects of acetylcholine on thalamic neurons may be related to reticular influences. This view is not held universally, however (see Davis, 1966; McCance et al., 1966). The pharmacology of neurons in the brain stem, particularly the t

60 -

50 -

FIG.1. Graphs of the frequency of discharge (f), plotted against time, for two different neurons in the mesencephalorl of a decerebrate cat, showing the relationship between the primary and secondary responses to intracarotid injections of acetylcholine ( ACh)., (Bradley and Mollica, 1958.) (-) unit; ( - - )

-

blood pressure.

pons and medulla, has been the subject of more extensive investigation than any other part of the brain and it is the findings from these studies which appear to be most relevant to mechanisms of action of drugs. Although effects on neuronal activity had been observed with intra-arterial injections of acetylcholine close to the head (see Fig. I ) , these could have been due to indirect actions

10

PHILIP B. BRADLEY

(Bradley and Mollica, 1958). In fact, the first studies in this region with microiontophoresis (Curtis and Koizumi, 1961) pointed to indirect actions. However, subsequent investigations using this method (Bradley and Wolstencroft, 1962, 1964, 1965; Bradley et al., 1966b) have shown that more than half the neurons in the medulla and pons of the unanesthetized decerebrate cat are cholinoceptive. Furthermore, while inhibition of neuronal activity appears to be rare in other regions of the brain where the effects of acetylcholine have been investigated, in the brain stem inhibitory effects are commonly observed and appear to have a different pharmacology. This suggests that the inhibitory action of acetylcholine in the brain stem is a specific action and not an indirect effect as has been suggested for inhibitory effects at the cerebral cortex (Krnjevib, 1964). In a sample of more than 600 neurons in the pons and medulla, tested with iontophoretic application of acetylcholine, and in which almost all were spontaneously active (Bradley et al., 1966b), 35.5% were excited by acetylcholine and 22%inhibited (Table I ) . In both TABLE I EFFECTS OF SUSPECTED TRANSMITTERS A N D DRUGS ON BRAINSTEM NEURONS ~~

Excitation

Inhibition

~~~

KOresponse ( %)

Suhst ance

(%)

(%I

Acetylcholine Noradrenaline 5-Hydroxytrypt.arnine Histamine

35.5 20

44.5

40 G

22 GO 49 58

Prostaglandin E, Prostaglandin E2 Prostaglandin Fzo

26 27.5 26

2.5 0 10

71.5 72.5 64

Pentobarbi tone Chlorpromazine Amphetamine LSD 25

2 11 0

0

20 11

36

100 69

0 29

50 30

70

39

cases, graded effects were observed when different current strengths were used to release different quantities of acetylcholine (Fig. 2 ) . The latency of these two actions of acetylcholine was between 2 and 10 seconds and the effects often persisted for 10-15 seconds after the current had been switched off. Cholinomimetic substances with


11

a nicotinic action, such as nicotine, l,l-dimethyl-4-phenylpiperazinium iodide (DMPP), and choline phenyl ether (TM1) were found to have excitatory actions on neurons which were also excited by acetylcholine but had no actions on neurons inhibited by acetylcholine (Fig. 3 ) . On the other hand, muscarinic agents such as muscarine and bethanechol mimicked both the excitatory and inhibitory actions of acetylcholine (Fig. 4), but muscarine was found to have a more prolonged and powerful inhibitory action than acetylcholine itself (Fig. 5 ) . None of these substances had any effect on neurons which did not respond to acetylcholine. The anticholinesterases, eserine and neostigmine, potentiated the actions of acetylcholine but, in addition, they had excitatory actions of their own which appeared to be unrelated to the action of acetylcholine (Fig. 6). Both excitatory and inhibitory actions of acetylcholine were antagonized by atropine (Fig. 7 ) , hyoscine, gallamine, and hexamethonium, whereas dihydro-/?-erythroidine, the nicotinicblocking agent, antagonized only excitatory responses ( Fig. 8). Apart from its antagonism to effects of acetylcholine, atropine was observed to have a nonspecific, depressant action on brain stem neurons, an effect which has also been observed with spinal cord neurons (Curtis and Phillis, 1960). From these findings it appeared that the excitatory and inhibitory actions of acetylcholine have different pharmacological properties and it has been suggested that the receptors for these effects may be different. Thus, the receptor for excitatory responses by neurons to acetylcholine appears to be mixed nicotinic and muscarinic but that for inhibitory responses is exclusively muscarinic. Nevertheless, other explanations for these effects must be considered (see p. 45). In studies of the responses of neurons in the hypothalamus, Bloom et al. (1963) found that 30%were cholinoceptive and that excitatory and inhibitory effects occurred in roughly equaI numbers. In the nuclei of the dorsal column (nucleus cuneatus and nucleus gracilis), acetylcholine was found to affect only a few neurons and the proportion of excitatory to inhibitory effects was about equal (Steiner and Meyer, 1966). Both the caudate nucleus and the hippocampus are unique in comparison with other regions of the brain in that most spontaneously active cells respond to application of acetylcholine, Thus, in the caudate nucleus more than 80%of the neurons tested in unanesthetized animals responded to acetylcholine (Bloom et al., 1965)

12

PHILIP B. BRADLEY

ACh

15

I

I

I

I

I

I

246810

1

,

I

20

I

40

30

50

Seconds

(A)

AC h

201

60

30

Seconds

(8)

FIG.2. Graded effects on the frequency of discharge ( f ) of two neurons in the brain stem of a decerebrate cat, with iontophoretic application of acetylcholine, using different current strengths. A: neuron excited by acetylcholine ( ACh); 3: neuron inhibited by acetylchohe (currents as in A). (Bradley et al., 1966b.)

and excitation was predominant. In the hippocampus the proportion of cholinoceptive neurons is about the same as in the brain stem, but only excitatory effects have been observed (Biscoe and Straughan, 1966). There is some evidence that cholinoceptive cells in the hippocampus may be muscarinic but this is not conclusive and

13


Inch

Nicotine

L

0

30

I

I

0

60

1

I

l

1

,

60 Seconds

30

(A)

k-, 0

30

Nicotine

I

I

,

60

0

,

I

I

30

,

,

60 Seconds

‘B)

FIG. 3. A comparison of the actions of acetylcholine and nicotine, in which the freqnency changes have been averaged for five Brain stem neurons. The substances were applied with currents of 100 nA. A: nemons excited by acetylcholine; B: neurons inhibited by acetylcholine. (Bradley et d.,1966b.)

not all the observations that have been made support this suggestion. In the cerebellum it has been found that some 75%of Purkinje cells are excited by iontophoretically applied acetylcholine (Crawford et al., 1966). However, whereas these authors believe that this action of acetylcholine is unlikely to be related to a cholinergic transmitter action, others (Phillis, 1965a) believe that transmission

14

PHILLF B. BRADLEY

- AC h 25

Methacholine

Muscarine 25

-

25

__

..

.A_v L

x

2

I

s I

d

2

Pdlnutes

FIG.4. The effects of niuscarine and acetyl-p-methylcholine ( methacholine ) on the impulse frequency of a neuron in the brain stem which was excited b y acetylcholine. Iontophoretic currents as shown (25 nA). (Bradley et d., 1966b.)

between some afferent mossy fibers to the cerebellum and granule cells may be mediated by acetylcholine.

D. RELEASE One of the requirements for a substance to be considered as a synaptic transmitter is that it be released from presynaptic nerve f 100-

4020

-

NoCl ,50

,

2

Me-line

,

5

Acetylcholine

9

, 5

,

, 10

Minutes

FIC.5. The effects of niusciirine and methacholine on R brain stem neuron which was inhibited by acetylcholine. A current control ( NaCI) is also shown. (Bradley et al., 19681~)

SYNAPTIC THANSMISSlON I N THE CNS

15

FIG 6. The excitatory effect of physostigmine (eserine) on a brain stem neuron which was inhibited by acetylcholine. The inhibition is potentiated by eserine. (Bradley and Wolstencroft, 1967.)

terminals by nerve stimulation. In the central nervous system it is, of course, virtually impossible to demonstrate, with the techniques available at present, the release of active substances at nerve terminals. Nevertheless, studies made on a grosser scale show release of acetylcholine, particularly from the cerebral cortex. Of particular interest in this connection is the observation by MitchelI (1963) that acetylcholine was released continuously from the cerebral cortex of sheep, cats, and rabbits to which an anticholinesterase f

10

L_1 -

L -

FIG. 7. Antagonism by atropine (applied for 1 minute with a current of 75 n A ) to the inhibitory action ( A ) of acetykholine on a neuron in the brain stem of the cat; B: application of acetylcholine 2 minutes after; and C: 28 minutes after cessation of the atropine application. (Bradley et al., 1966b.)

16

PHILIP B. BRADLEY

2(

I1

AC h 50

AC h I

I

2

Mi nu tes

FIG.8. Antagonism by dihydro-P-erythroidine (DH-P-E ) (applied for 1 minute with a current of 75 nA) to an excitatory action of acetylcholine on a brain stem neuron. The second application of acetylcholine is 30 seconds after, and the third 5 minutes after the DH-P-E application was terminated. (Bradley et al., 1966b.)

had been topically applied, and that the rate of release was roughly proportional to the electrical activity of the cortex. Furthermore, electrical stimulation of the cortex, or excitation produced by transcallosal or peripheral stimulation, increased release. An increased release of acetylcholine from the primary receiving areas of the cerebral cortex of the rabbit has been reported upon stimulation of sensory pathways (Collier and Mitchell, 1966) but, as the authors point out, this effect could be a consequence of indirect activation of ascending pathways from the reticular formation and electrical stimulation of this structure has been found to result in an increased acetylcholine output from all areas of the cerebral cortex (Kanai and Szerb, 1965). This idea is supported b y the findings of Celesia and Jasper (1966) who used unanesthetized animals and were able to correlate the release of acetylcholine from the cerebral cortex with the state of activation. It was found that the average release of acetylcholine in waking animals was about 3 ng/min/cm2, and during light natural sleep this fell to an average of 2 ng/min while


17

during barbiturate anesthesia there was a further decrease to 1 ng/min/cm2. Arousal from light sleep by application of brief natural stimuli and also by stimulation of the reticular formation was accompaiiied by an increasc in the release of acetylcholine. An increase also occurred during Metrazol-induced seizures and following administration of atropine. These authors conclude that cholinergic mechanisms play an important part in the desynchronized activation of the cortex characteristic of states of wakefulness or alertness, On the other hand, from experiments on anesthetized cats, which involved acutc undercutting of cerebral cortex and stimulation of various structures, Szerb ( 1967) concludes that, while the projections responsible for EEG activation and increased release of acetylcholine originate in the mesencephalic tegmentum, they follow divergent paths on their way to the cortex. AcetylchoIine release has also been shown from the cerebellar cortex, although the amount present is approximately one tenth that in the cerebral cortex (Phillis and Chong, 1965), and from the caudate nucleus with an increase on low frequency stimulation of the nucleus ventralis anterior of the thalamus ( McLennan, 1964). Ill. Monoamines

A. BIOCHEMICAL EVIDENCE Unlike acetylcholine, which has a wide distribution in the central nervous system, the catecholamines and 5-hydroxytryptamine appear to be mainly concentrated in the hypothalamus and brain stem (Vogt, 1954). There is considerably more noradrenaline in the brain than adrenaline and it has been suggested that the uneven distribution of noradrenaline in various brain areas supports the idea of a role for this catecholamine in central adrenergic transmission, other than as a transmitter at vasomotor sympathetic nerve endings on blood vessels in the brain. Another catecholamine. dopamine ( 3,4-hydroxyphenylethylamine), is also present in thc brain in roughly the same total amount as noradrenaline, but does not have the same distribution. Thus, dopamine may have an independent role apart from that of a precursor of noradrenaline, an idea which is supported by the results of fluorescence studies (see below ) . Studies on the suhcellnlar localization of monoamines in brain using centrifugation fractionation techniques have shown that

18

PHILIP B. BRADLEY

noradrenaline and dopamine are present mainly in the “mitochondrial” fraction, which contains intact nerve endings in addition to mitochondria and myelin (De Robertis, 1966). Some 402 of the noradrenaline is found in the supernatant, but it has been suggested ( D e Robertis, 1964) that the noradrenaline is localized in synaptic vesicles which are more sensitive to shock than those containing acetylcholine. Certainly, the disruption of the nerve endings by osmotic shock shows that the synaptic vesicles have the highest concentration of noradrenaline and dopamine. De Robertis ( 1967) believes that the synaptic vesicle is the main store for noradrenaline and dopamine, as well as for acetylcholine. Vesicles from the anterior hypothalamus contain 5 to 6 times more noradrenaline than those from the cerebral hemispheres. 5Hydroxytryptamine ( 5-HT ) is also found in the synaptic vesicle fraction (De Robertis, 1964). Furthermore, the enzymes dopa decarboxylase and 5-hydroxytryptophan decarboxylase, which synthesize dopamine and 5-HT, are contained within the nerve-ending fraction but dopamine-phydroxylase, which converts dopamine to noradrenaline, has not been localized to the synaptic complex ( D e Robertis, 1966). On the other hand, the two enzymes which inactivate catecholamines, monoamine oxidase and catechol-O-methyltransferase, are localized to mitochondria and nerve endings, respectively, and this is inconsistent with a possible postsynaptic action of these enzymes.

B. HISTOCHEMICAL EVIDENCE The use of the method of fluorescence histochemistry for the cellular localization of monoamines in the central nervous system has demonstrated that there are specific neuronal systems which form and store dopamine, noradrenaline, and 5-hydroxytryptamine, respectively. Furthermore, it has been postulated ( Dahlstrom and Fuxe, 1965) that these systems function by releasing the amines as neurotransmitters at their synaptic terminals. In addition it has been shown that there are close morphological, biochemical, and pharmacological similarities between central neurons containing monoamines and peripheral adrenergic neurons. A good deal of this work has been concentrated on the mammalian hypothalamus. Fibers containing noradrenaline fluoresce with an intense green to yellow-green color and are particularly prevalent in certain areas of the hypothalamus, although they also occur in other regions of the brain and spinal cord. Nerve terminals 1-2 p in diameter and containing noradrenaline appear to arise

SYNAPTIC TRAAShiISSlON I N THE CKS

19

from groups of neurons wit11 cell bodies in the brain stem. Nerve terminals containing dopamine are smaller, less than 1p in diameter, and more circumscribed in their distribution, being particularly abundant in the caudate nucleus and adjacent striatal areas. The striatal dopamine-containing nerve terminals appear to arise from cell bodies situated in the substantia nigra. There is a good deal of similarity between neurons containing catecholamines in the central nervous system and noradrenaline-containing neurons in the peripheral sympathetic nervous system. In the central nervous system, noradrenaline and dopamine are most highly concentrated in the nerve terminals and have much lower concentrations in other parts of the cell. Furthermore, their terminal regions are very much branched and have a varicose appearance similar to that of peripheral adrenergic nerves. There is also a close parallel in the metabolic pathways for synthesis and degradation, and in the enzymes. Neurons containing 5-hydroxytryptamine exhibit a yellow fluorescence, in contrast to catecholamine-containing neurons which are green fluorescent. Like monoamine-containing neurons, the cell bodies are mainly concentrated in the brain stem, but terminals containing 5-hydroxytryptamine have been found in the neocortex and hippocampus. The value of the fluorescence biochemical methods has been considerably enhanced by combination with other techniques such as the placing of lesions in specific pathways so that degeneration can be followed in terms of fluorescence studies and also by pretreatment of experimental animals with drugs: for example, pretreatment with reserpine, which depletes stores of catecholamines and 5-hydroxytryptamine; or with monoamine-oxidase inhibitors, which help to show up 5-hydroxytryptamine in cell bodies wherc the fluorescence intensity is normally very weak. The A uorescence histochemical studies leave little doubt that specific systems of catecholamine- and 5-HT-containing neurons exist in the central nervous system. The existence of separate neuronal systems containing noradrenaline and dopamine suggests an independent role for these two substances. C . ACTIONSON NEURONS

1. Catecholamines Both noradrenaline and dopamine are present in the spinal cord and effects have been obscrved with iontophoretic application of

20

PHILIP B. BRADLEY

these substances, particularly with interneurons. Noradrenaline has been found to have an inhibitory action on Renshaw cells in the spinal cord which are excited by acetylcholine (Biscoe and Curtis, 1966; Weight and Salmoiraghi, 1966a). However, these authors do not postulate an inhibitory transmitter action. At the cerebral cortex catecholamines such as dopamine, adrenaline, isoprenaline, and noradrenaline all cause depression of neuronal activity, although in many cases these effects have been observed against a background of excitation by glutamate in neurons which were not spontaneously active. Dopamine was found to be the most effective and noradrenaline least effective ( KrnjeviG and Phillis, 1 9 6 3 ~ ) . In the lateral geniculate nucleus of the thalamus, catecholamines have been found to cause depression of the spontaneous activity of the neurons as well as their responses to volleys in optic nerve fibers (Curtis and Davis, 1962). This action is shown by 5-HT (see below) which is also more potent than catecholamines ( dopamine, adrenaline, and noradrenaline) and other phenylethylamine derivatives. The action appeared to be present for all neurons tested. On the other hand, synaptic excitation of neurons in the ventrobasal thalamus was not blocked. Different effects from those observed in other parts of the brain have been found in the brain stem. Here, both excitation and inhibition of neuronal activity has been observed, whereas in all other regions so far studied depressant effects predominate. The results from early investigations (Bradley and Wolstencroft, 1962, 1965) showed that almost half the neurons in the medulla and pons responded to application of noradrenaline, 298 showing excitation and 19% inhibition. However, these figures have been modified from the results of more recent studies, in which it was found that 80%of brain stem neurons responded to noradrenaline, 20%being excited and 60%inhibited ( see Table I ) (Bradley and Wolstencroft, 1966). The two types of response to noradrenaline differed in their time courses. Excitation was almost invariably long-lasting and delayed in onset (Fig. 9A ) , while inhibition was comparatively rapid in onset and recovered soon after the applying current was switched off (Fig. 9B).Occasionally another form of inhibition was observed similar in time course to the excitatory response; this inhibitory response lasted much longer than the period of application and often reached its peak after the current had been switched off. The

21


excitatory response showed varying degrees of desensitization with repeated applications but this was not observed with inhibition. L-Noradrenaline was the most effective of the monoamines tested on brain stem neurons. Dopamine and adrenaline frequently had no effect on neurons which responded to noradrenaline, and where they had effects these were weaker. D-Noradrenaline inhibited neurons on which L-noradrenaline had an inhibitory action, but where the latter had an excitatory action, the effects of D-noradrenNA

f

m

35 -

-

30 -

NA

25 -

20 -

15-

10-

5-

I

0

L

30 (A)

60

0

X,

40

60 Seconds

(B)

FIG.9. The effects of L-noradrenaline,applied iontophoretically with a current of 50 nA, on two different neurons in the brain stem of a decerelmte cat. A: neuron excited by noradrenaljne; B: neuron inhibited. (Bradley and Wolstencroft, 1966.)

aline were either weak or absent. Thus, the excitatory effect appears to show stereospecificity while the inhibitory effect does not (Boakes et al., 1968). Attempts to block the actions of noradrenaline on brain stem neurons with various known antagonists of its peripheral actions, both of the 01- and /?-type, have so far proved unsuccessful but certain effects have been antagonized with chlorpromazine (Bradley et al., 1 9 6 6 ~ ) . In other regions of the brain, the actions of catecholamines on neuronal activity are mainly inhibitory with the exception of Deiters’

22

PHILIP B. BRADLEY

nucleus where noradrenaline has been found to have almost exclusively excitatory effects (Yamamoto, 1967). In the hypothalamus, for example, noradrenaline-sensitive cells have been found in all areas and the effects were predominantly depressant (Bloom et al., 1963). Dopamine has been found to inhibit most cells in the cuneate and gracilis nuclei of the dorsal column (Steiner and Meyer, 1966) and in the caudate nucleus 85%of neurons responded to noradrenaline, the effect being primarily one of depression, and 64% to dopamine, about a quarter of which showed excitation (Bloom et uZ., 1965). A somewhat surprising feature of the latter experiments was that administration of anesthetics did not seem to modify neuronal responses to catecholamines, although responses to acetylcholine were markedly altered. Inhibitory effects with noradrenaline have been observed in the hippocampus and caudate nucleus. In the olfactory bulb of the rabbit, noradrenaline has been found to depress the activity of mitral cells, and these responses, together with the inhibition produced by electrical stimulation of the lateral olfactory tract, were reduced following administration of a-antagonists, dibenamine and phentolamine ( Salmoiraghi et al., 1964). These authors suggest that a component of the inhibitory responses of mitral cells in the rabbit olfactory bulb is mediated by adrenergic synapses. 2. 5-Hydroxytryptarnine

Like the catecholamines, this substance is more highly concentrated in the hypothalamus and brain stem than in other regions, but it is also found in high concentrations in tissues outside the nervous system. Interest in the possibility that 5-hydroxytryptamine is important in the function of the central nervous system, possibly as a synaptic transmitter, was stimulated by the finding that the potent synthetic psychotomimetic drug D-lysergic acid diethylamide (LSD 25) antagonized the action of 5-HT on peripheraI tissues. Considerable discussion and speculation has ensued relating to the possibility that an interference with the actions of 5-HT in the central nervous system might be the mechanism by which LSD 25 produces its psychological effects. The evidence against this idea is related to the observation that a derivative of LSD, 2-bromolysergic acid diethylamide (BOL 148), which is without psychotomimetic actions, is an equally potent 5-HT antagonist. Nevertheless, it seems probable that 5-HT has an important role in the function

SYNAPTIC THAXSMISSION I N THE CNS

23

of the central nervous system and its actions when applied micro-

iontophoretically have been studied in various regions. No actions have been found on spinal interneurons, motoneurons, or Renshaw cells in the spinal cord. 5-Hydroxytryptamine was found to have a depressant action on most neurons in the cerebral cortex on which it was tested (Kmjevie and Phillis, 1963d). This effect was present both with neurons which were not spontaneously active, but excited by application of glutamate, and unit responses evoked by peripheral stimulation. In rare cases a paroxysmal excitation sometimes occurred when large currents were used and after a delay, and it has been suggested that this was a nonspecific effect. Recent investigations with 5-HT (Roberts and Straughan, 1966) on cortical neurons in the unanesthetized cat encbpphale isole' preparation have shown that iontophoretic application of this substance can produce excitation of certain cells. Out of the 80% of neurons which responded to application of 5-HT in their experiments, approximately 30% were excited and 50% inhibited. Excitation could be prevented temporarily by systemic injection of a barbiturate while the excitatory effects of 5-HT were antagonized by LSD 25, methysergide, and BOL 148. Antagonism of the inhibitory effects of 5-HT was rare. In the brain stem, the action of 5-HT appears to be more widespread than in any other region and for any other substance (with the exception of amino acids). In the medulla and pons 90% of neurons responded to application of this substance, 40% being excited and 49%inhibited (Table I ) ( Bradley and Wolstencroft, 1965). The time course for these responses was similar to those for noradrenaline (see Fig. 10). In the case of this substance, the effects obtained were in some ways dependent upon the salt used for iontophoretic application of 5-hydroxytryptamine, When used in its common form, 5-HT creatinine sulfate, it was found that creatinine itself had excitatory actions on neurons (Bradley and Wolstencroft, 1965). The use of this salt was therefore abandoned and the bimaleinate employed. Differences in the responses of cortical neurons to these two salts of 5-HT have also been observed (Roberts and Straughan, 1967). In the thalamus, 5-hydroxytryptamine is thought to be involved in transmission in the lateral geniculate nucleus. Thus, S-hydroxytryptamine and closely related tryptamine derivatives, together with lysergic acid and certain phenylethylamine derivatives, depress the

24

PHILIP B. BRADLEY

excitation of neurons in the IateraI geniculate nucleus by optic tract impulses but without affecting the excitability of these neurons tested antidromically or by iontophoretic application of excitant amino acids (Curtis and Davis, 1962, 1963). It has been suggested that these substances may block the access of an excitatory transmitter released from optic nerve terminals to subsynaptic receptors

- -

(A)

5-HT

NA

3010

20 40

--

0

20

40

0

?o 40 60 Seconds

FIG. 10. The efTects of acetylchoIine (ACh), noradrenaline ( N - - , an1 5-hydroxytryptamine ( 5-HT), applied iontophoretically to the same neuron. A: neuron excited by all three substances; B: neuron inhibited by all three (all applications were made with a current of 50 nA). (Bradley et al., 1966a.)

on geniculate neurons or prevent the release of this transmitter and, further, that the transmitter might be a compound structurally related to 5-hydroxytryptamine. So far, however, no transmitter has been identified although many indole and tryptamine derivatives have been tested. Neurons in the ventrobasal thalamus showed abolition of spontaneous discharges with application of 5-HT, 4-HT, and dopamine but synaptic excitation by impulses in cutaneous


25

fibers was only mildly depressed even when high electrophoretic currents were used (Curtis, 1966). It is suggested that these effects of 5-HT (and dopamine) arc due to a nonspecific depression of neuron excitability which is unrelated to synaptic mechanisms.

D. RELEASE Since neither the catecholamines nor 5-hydroxytryptamine or its derivatives are present in the cerebral cortex in significant amounts, their release from this structure would hardly be expected, and examination of superfusates from the unanesthetized cerebral cortex has confirmed that monoamines are not present in detectable quantities (Bradley and Samuels, 1967). Dopamine, together with acetylcholine, was found to be present in perfusates of caudate nucleus ( McLennan, 1964).The quantity of dopamine increased with electrical stimulation of the nucleus centromedianus ( C M ) but not with stimulation of ventralis anterior (VA), while the reverse was true for acetylcholine. From these findings, McLennan postulates a cholinergic final synapse in the VA-caudate pathway and a dopaminergic one in the CM-caudate pathway. IV. Amino Acids

Considerable attention has been directed towards a possible role for certain amino acids in the function of the central nervous system. This has stemmed partly from the fact that they have very potent actions as excitants and depressants of neuronal activity and that one, y-aminobutyric acid (GABA), has been found to be an inhibitory transmitter in certain invertebrates.

A. BIOCHEMICALEVIDENCE The free amino acid content of mammalian brain is nearly 8 times that of blood plasma. In subcellular fractions of brain homogenates the three amino acids found in highest concentrations are also those which possess the most potent actions on neuronal activity ( glutamic, aspartic, and y-aminobutyric acids ) ( Whittaker, 1964). However, the distribution in these fractions contrasts with that of acetylcholine and monoamines in that most of the amino acids (6276%of the total amount recovered) are found in the high-speed soluble cytoplasmic fraction and very little associated with particulate fractions following differential centrifugation (Ryall, 1964). Whittaker (1964) concludes that the free amino

26

PHILIP B. BRADLEY

acids do not appear to be specifically localized in nerve endings. On the other hand, two enzymes which are concerned with the synthesis and breakdown of y-aminobutyric acid in brain, glutamic acid decarboxylase and y-aminobutyric acid aminotransferase, have been found in submitochondrial fractions of brain homogenates and the two enzymes have different localizations in these fractions. Thus, glutamic acid decarboxylase, which catalyzes the formation of GABA from L-glutamic acid, was found in “nonaminergic” nerve endings and GABA aminotransferase, which catalyzes the transamination of GABA to succinic semialdehyde, was localized to neuronal mitochondria ( Salganicoff and De Robertis, 1965). From this evidence De Robertis (1967) suggests that GABA is the transmitter at inhibitory nonaminergic synapses at the cerebral cortex. There are no techniques available at present for histochemical localization of amino acids in nervous tissue.

B. ACTIONSON NEURONS Of the various amino acids whose actions have been investigated, the most important in the vertebrate central nervous system are L-glutamic acid and GABA. The actions of amino acids in the spinal cord have been extensively investigated by Curtis and his colleagues (see Curtis and Watkins, 1965, for review). A number of amino acids, e.g., glutamic, aspartic, and cysteic, have been found to excite interneurons, Renshaw cells, and motoneurons in the spinal cord. Apart from those amino acids which are endogenous in nervous tissue, a number of structurally related synthetic acids have been tested and found to be effective. In some cases these synthetic compounds are even more potent than the naturally occurring ones. Excitatory amino acids produce their effects by membrane depolarization. It is thought that amino acids are unlikely to be excitatory transmitters in the mammalian spinal cord because enzymatic removal does not appear to determine the duration of action and it would be expected that specific enzymes should occur near to excitatory synapses for destruction of synaptically released transmitters ( Curtis et al., 1960). Furthermore, the action is nonspecific with regard to the functional types of neurons tested. Somewhat conflicting results have been obtained in studies of the action of GABA on the spinal cord. In their early reports Curtis and his colleagues (1959) found that GABA and p-alanine de-


27

pressed the activity of spinal interneurons, motoneurons, and Renshaw cells. However, as they could find no evidence of a change in membrane potential, they concluded that GABA was not a transmitter at inhibitory synapses in the spinal cord, since to fill this role it would have to hyperpolarize the membrane. A further argument used was that strychnine, administered intravenously, did not prevent the action of GABA (or p-alanine). More recently Curtis et al., (1967) have found that GABA can cause hyperpolarization of motoneurons. On the other hand, recent evidence suggests that glyche may be an inhibitory transmitter in the spinal cord. Its distribution appears to be related to the presence of inhibitory interneurons, since experimentally induced reduction in the numbers of these cells was associated with a significant decrease in the level of glycine but not of GABA (Davidoff et al., 1!367); and glycine has been found to cause hyperpolarization of spinal motoneurons when applied iontophoretically ( Werman et QZ., 1967). Curtis et ul. ( 1967) found that while strychnine antagonized the inhibitory action of glycine it did not affect the action of GABA, and they suggest that glycine and GABA therefore interact with different postsynaptic receptors. However, this assumes that strychnine acts by occupying postsynaptic inhibitory receptor sites, for which the evidence is not conclusive. One problem to be solved is why enzyme inhibitors which affect the metabolism of glycine do not modify its action on interneurons. Another region in which the actions of amino acids have been studied in great detail is the cerebral cortex. Here the most potent of the naturally occurring amino acids is L-glutamic, which is also present in the brain (Berl and Waelsch, 1958).A detailed analysis of the actions of this substance has been made by Krnjevi6 and Phillis (1963d). One feature of its action is that every neuron to which it is applied shows a response. The effect usually has a short latency (less than 1 second) and an even more rapid termination, and the excitation is maintained during a prolonged release. The action is graded and the rate of firing can often be controlled by the current strength used to release the glutamate. Glutamate has been used by many workers to evoke activity from otherwise quiescent neurons and then to examine the effects of other pharmacologically active substances, including possible synaptic transmitters, against a background of this evoked activity. The interpretation of such findings is made more hazardous by the fact that we do not

28

PHILIP B. BRADLEY

know precisely how glutamate itself produces its effects. It appears that sensitivity to glutamate varies considerably and it is suggested (Krnjevi6, 1964) that this is related to the stability of resting potentials, Krnjevi6 regards glutamate as a possible cortical excitatory transmitter on the basis of the rapid onset of its action and restricted duration. It is suggested that it may be removed from its site of action by absorption into the cell rather than by enzymatic destruction. GABA, which produces inhibition of cortical neuronal activity, also has a rapid onset, high potency, and restricted duration of action. Its inhibitory action can block most types of activity in the cortex, including spontaneous activity, responses produced by peripheral nerve stimulation, and discharges initiated by local application of glutamate and acetylcholine. Crawford and Curtis (1964) argued from their experimental data against GABA being an inhibitory transmitter at the cerebral cortex, as they did for the spinal cord. However, Kmjevib and Schwartz (1967) recorded intracellular potentials from cortical neurons and found a drop in resting membrane resistance with application of GABA and a reduction or abolition of all inhibitory and excitatory postsynaptic potentials, as had been seen with spinal cord neurons. However, they found that GABA regularly had a hyperpolarizing action which occluded inhibitory postsynaptic potentials. These effects disappeared soon after the end of the release of GABA and were repeatable. It was noted that GABA always shifted the membrane potential in the same direction as the inhibitory postsynaptic potential, even when this was reversed by polarizing the neuron. They therefore consider that, since the action of GABA on cortical neurons is very similar to that of synaptic inhibition, it must be seriously considered as a possible inhibitory transmitter at the cortex. In other parts of the brain, e.g., thalamus, brain stem, and hippocampus, neurons appear to be excited by glutamate and inhibited by GABA, although the actions of these substances have not been analyzed in such great detail as they have for the spinal cord and cortex. C. RELEASE

There is little information in the literature about release of amino acids from the brain but a recent report (Jasper and Koyama, 1968) indicates that certain amino acids are released from the

29


cerebral cortex of unanesthetized animals and that the amount increases with arousal. V. Other Potential Transmitters

Among the various substances which, for various reasons, have been considered as possible candidates for synaptic transmission in the central nervous system are: A. HISTAMINE This substance has been proposed as a possible transmitter (Gaddum, 1963) as it is present in the same parts of the brain as f NA

20

Histomine

40

60

80

100

120

140

Seconds

FIG. 11. Inhibition of neuronal activity, produced by both noradrenaline and histamine, applied in succession to the same neuron with currents of 50 nA. ( Bradley et al., 1966a. )

the monoamines and in similar concentrations (Adam, 1961 ) . Furthermore, its amino acid precursor, histidine, is also present. Histamine has been found to be without effect when applied iontophoretically to neurons in the spinal cord (Curtis et al., 1961) and thalamus (Curtis and Davis, 1962). Some effects have been observed with brain stem neurons (Bradley et al., 1966a), though these were mainly depressant (Table I ) . Thus, more than half the neurons to which histamine was applied iontophoretically showed inhibition (Fig. l l ) , but excitation was rare. A weak depressant action on cortical neurons has been reported for histamine and histidine (Krnjcvii. and Phillis, 1 9 6 3 ~ ) .

30

PHlLIP B. BRADLEY

B. SUBSTANCEP This active polypeptide is found only in vertebrates and has a specific distribution in mammalian brain (Lembeck and Zetler, 1962). It has many of the properties which would be expected of a transmitter and has been found to have a similar subcellular distribution to that of acetylcholine (Ryall, 1964). However, there is no evidence of it having any action on nerve cells or of it being liberated by nerves. A possible carrier function for substance P in relation to acetylcholine in nerve endings has been considered but there is no evidence to support this idea. C. ERGOTHIONEINE This substance was found to be the active constituent of extracts of cerebellar tissue from several mammalian species ( Crossland et al., 1964). The extracts of so-called cerebellar factor were characterized by having excitatory effects on the cerebellar cortex (Crossland and Mitchell, 1956; Crossland, 1960). It was thought that this substance might be a noncholinergic transmitter at central excitatory synapses. When applied iontophoretically, ergothioneine was not found to have any significant effects on the excitability of cells of the cerebellar cortex and no effects on cells of the cerebral cortex (Krnjevit! et al., 1965). However, some actions on neurons in the brain stem have been reported (Avanzino et al., 1 W a ) . Further information about its actions is required before serious consideration can be given to the possibility of ergothioneine being a synaptic transmitter in the central nervous system.

D. PROSTAGLANDINS These substances, which are long-chain, unsaturated fatty acids, first identified in extracts of sheep prostate gland, would seem to be very unlikely candidates for consideration as possible synaptic transmitters. However, they have recently been found to be present in the mammalian central nervous system (Coceani and Wolfe, 1965; Horton and Main, 1966) and in superfusates of cerebral cortex (see below). Furthermore, certain prostaglandins (PG) have been found to have potent effects on neurons in the brain stem when they were applied iontophoretically ( Avanzino et al., 196613). Three prostaglandins, PGE1, PGE,, and PGF,, were tested and found to have actions on approximately 3045%of the neurons ex-


31

amined (Fig. 12, Table I ) . PGE, produced excitation in 26%and inhibition in 2.5%,while PGE, caused only excitation (27.5%).With PGF,, more inhibition was observed (10%) although the number of neurons excited was about the same (26%).Desensitization occurred with both the excitatory and inhibitory effect, but was specific for each compound. No relationship was found between actions of prostaglandins and those of acetylcholine or noradrenaline. Thus it seems that the prostaglandins are unlikely to be involved in cholinergic or adrenergic transmission in the nervous system; the precise elucidation of their role in neuronal mechanisms must await the results of further studies, particularly those concerned with their synthesis and degradation.

30 seconds

FIG. 12. Excitatory actions of prostaglandins El and Fz,, released with a current of 100 nA for 30 seconds, on the discharge rate of a neuron in the nucleus reticularis gigantocellularis in the cat. Current control ( NaCl) also 100 nA. (Avanzino et d.,1967.)

The prostaglandins are released from the cerebral cortex, both in anesthetized animals ( Ramwell and Shaw, 1966) when peripheral nerve stimulation caused an increase in the release, and in unanesthetized preparations (Samuels et nl., 1967) where the level was increased by stimulation of the reticular formation, leading to activation of the cerebral cortex. This increase was depressed by drugs such as pentobarbitone and chlorpromazine which aIso depressed the spontaneous release. VI. Multiple Effects on Neurons

The possibility that neurons in the central nervous system might be capable of responding to more than one potential transmitter has received relatively little attention. This is probably due to the

32

PHILIP B. BRADLEY

fact that the peripheral mechanisms on which models of receptors in the central nervous system have been based, are usually of one pharmacological type. However, although this is true for the neuromuscular junction, there is now evidence suggesting that both acetylcholine and noradrenaline may participate in transmission at sympathetic nerve endings (Burn and Rand, 1962). Another factor which has influenced our attitude towards the pharmacological classification of neurons in the central nervous system is that the information from fluorometric studies has pointed towards there being unitary types. Thus, according to the users of these histochemical techniques, neurons containing monoamines are either noradrenergic, dopaminergic, serotonergic, or adrenergic (Falck, 1964). While it may be true that receptors of one kind may predominate, the sensitivity of the fluorescence methods is not so high that it precludes the possibility of other, chemically different receptors being present on the same neuron. The more complex morphology and physiology of neurons in the central nervous system rather suggests that we should not expect them to fall into simple unitary classifications pharmacologically. A. ACTIONSOF ACETYLCHOLINE, NORADRENALINE, AND

5-HYDROXYTRYPTAMINE So far, the information on the actions of different substances, particularly these three, when applied to the same neuron is limited to certain regions of the central nervous system. Studies of the responses of Renshaw cells to noradrenaline (Weight and Salmoiraghi, 1966a) have been described (see p. 20). Responses of spinal interneurons to acetylcholine, noradrenaline, and 5-HT have also been studied (Weight and Salmoiraghi, 1966b) and some neurons TABLE I1 EFFECTSOF ACETYLCHOLINE (ACH), NORADRENALINE (NA) A N D 5-HYDROXYTRYPTAMINE (5-HT) APPLIED TO TLlE SAME NEURON^ ACh

(+I

ACh (-)

a11d 5-HT

(+I NA

+ 0

(1

(-1

ACh

and 5-HT (0)

(+I

(-1

(0)

and 5-HT (0)

(+I

(-1

(0) ~~

16 13 11

3 3 0

6 3 6

2 2 1

1 9

1

1 0 0

7 6 6

2 8 3

6 1

8

The figures represent the number of neurons showing each type of response:

+ = excitation, - = inhibition, o = no effect.

33


have been found which responded to all three substances with either facilitation or depression; some responded to one, others to two, but no correlation was found between the direction of these responses. Similar effects have been observed with the iontophoretic application of acetylcholine, noradrenaline, and 5-hydroxytryptamine to neurons in the brain stem (Bradley and Wolstencroft, 1965). Of a random sample of neurons in the medulla and pons, many were

10

A&

-

AC h I

I

5- HT I

I

I

I

I

NA I

,

L

A

I

,

I

,

FIG. 13. Responses of a neuron in the paramedian reticular nucleus of the decerebrate cat to iontophoretic application of acetylcholine ( ACh ), 5-hydroxytryptamine (5-HT) and noradrenaline ( N A ) with currents of 50 nA. (Avanzino et nl., 1966d.)

found which responded to all three substances and various combinations of excitation and inhibition occurred (Table 11). In some all these actions were excitatory (Fig. 1OA) and in others, all were inhibitory (Fig. 10B). A number were excited by one compound and inhibited by the other two and vice versa (Fig. 13). Others again were only affected by two of the substances with various combinations of excitation and inhibition and a third group only responded to application of one substance (see Table 11). However, when neurons in a particular nucleus, the paramedian reticular nucleus, were studied it was found that more consistent effects were observed (Avanzino et al., 1 9 6 6 ~ ) .These neurons responded to all three substances; acetylcholine consistently caused

34

PHILIP B. BRADLEY

excitation, noradrenaline inhibition, and 5-hydroxytryptamine excitation (Fig. 13). The neurons were identified anatomatically as projecting to the cerebellum and appear to have more uniform pharmacological properties than a random sample of brain stem neurons. Neurons which respond to application of acetylcholine, noradrenaline, and 5-HT have also been found in the hypothalamus (Bloonl et al., 1963). It is possible that mixed effects may yet be found in other regions of the brain. VII. Effects of Centrally Acting Drugs

The actions of drugs, known to modify the functions of the central nervous system, for example, by producing changes in consciousness, or effects manifested in behavioral or psychological changes, have been studied by a variety of methods in order to determine their sites of action. Thus, the combination of electrophysiological recording techniques with observations of behavior, together with other methods, such as placement of lesions to interrupt certain pathways and electrical stimulation of others, have all led to the formulation of hypotheses for sites of action of drugs in the brain. In some cases these hypotheses are well established; in others, they are more tenuous. However, a site of action is usually defined in terms of a particular structure which may be comprised of many thousand or even million neural elements. Thus we cannot decide, from the results of such studies, whether a drug, when administered systemically, produces its effects by a direct action on the neurons in these structures, or by some indirect action on more remote mechanisms which then influence neuronal activity in the region under study. It is therefore important to know whether centrally acting drugs modify the activity of neurons in the regions of the brain where they are believed to act, and if they do, whether these effects are related in any way to synaptic transmission at these sites. Thus, a drug might act by mimicking the actions of a transmitter or it might interfere with transmitter action. Some light is thrown on these problems by the results of recent investigations using the method of microiontophoresis.

A. CENTRAL DEPRESSANT AND SEDATIVE DRUGS The barbiturates, which produce sedation, loss of consciousness, and anesthesia, have a direct depressant action on arousal mecha-


35

nisms in the brain stem reticular formation (French et aZ., 1953; Bradley and Key, 19%). This action is thought to be the neurological basis of the anesthetic state. Since many investigations into the actions of iontophoretically applied substances on neuronal activity in various parts of the brain have been carried out against a background of barbiturate anesthesia, it is difficult to assess the precise effects which these compounds produce, or how far the effects observed may be modified by their presence. Reference has already been made to the studies of Bloom et al. (1965) on the responses of neurons in the caudate nucleus to acetylcholine, noradrenaline, and dopamine in relation to presence of anesthesia (see p. 22). They found that not only was spontaneous activity markedly reduced by light chloralose or barbiturate anesthesia but that facilitation by acetylcholine, which was common in the unanesthetized state, was either suppressed or changed to inhibition in the presence of anesthesia. Responses to noradrenaline and dopamine, which were mainly depressant, were relatively unchanged. Similarly, excitatory effects of 5-hydroxytryptamine on cortical neurons were lost when a barbiturate was administered ( Roberts and Straughan, 1967) and increasing the depth of anesthesia has been found to reduce excitatory responses of cortical neurons to acetylcholine ( Krnjevib and Phillis, 1963a). These findings may be of considerable importance in the interpretation of the results of iontophoretic application of suspected transmitters in anesthetized animals. In view of the depressant action of barbiturates on brain mechanisms responsible for arousal responses, which had been demonstrated from other studies, Bradley and Wolstencroft (1965) investigated the actions of one of these compounds, pentobarbitone, applied iontophoretically to neurons in the brain stem in unanesthetized decerebrate cats. Of the forty neurons tested, all showed inhibition of their spontaneous activity (Table I ) , although there appeared to be considerable variation in sensitivity. Thus, in some cases, the activity was completely suppressed within 5 seconds of the application, while in others there was a gradual reduction in activity to about 30%after a long delay, sometimes up to 60 seconds. In a few cases it was possible to test the response of the neurons to acetylcholine (Fig. 14) and 5-hydroxytryptamine before and after iontophoretic application of pentobarbitone, but these responses appeared to be unchanged (Bradley and Wolstencroft,

36

PHILIP B. BRADLEY

1966). Possible interaction between the effects of pentobarbitone and noradrenaline was not examined in these experiments. The fact that, in spite of the small number, all units tested were depressed by pentobarbitone suggests that this effect may be related to the anesthetic action of this drug. The variation in sensitivity is interesting, however, and needs further investigation, as do the interactions of this drug with suspected transmitters. f 90 -

80 70 60 50

-

40 30 -

2o

t

‘.I

-

0

I

I

- PB

ACh

2

3

ACh

- 4

5

6

Minutes

FIG.14. Effects of acetylcholine and pentobarbitone, applied with currents of 50 nA, on the activity of a neuron in the brain stem of a decerebrate cat. The excitatory response to acetylcholine (ACh) is still present after the pentobarbitone application (PB ) which causes depression of activity but with a slow onset. (Bradley and Wolstencroft, 1966.)

B. CENTRAL STIMULANTDRUGS The drug in this group which has been investigated in most detail is amphetamine. This drug produces its central excitant effects, resulting in increased alertness, by a direct facilitatory action on the brain stem reticular formation (Bradley and Elkes, 1953,’ 1957; Bradley and Key, 1958). Investigations with the iontophoretic application of amphetamine to neurons in the brain stem (Bradley and Wolstencroft, 1965) showed that the substance had predominantly inhibitory effects and acted on almost half (47%)

SYNAI'TIC 'I'RANSMISSION

37

IN THE CNS

of thc iicurons tested. However, in inore recent experiments, it has been found that amphetamine possesses both inhibitory and excitatory actions on brain stem neurons, and that these follow very closely the actions of noradrenaline. In a sample of 106 brain stem neurons to which D-amphetamine was applied iontophoreticalIy, 12 (11%)showed increased activity, 53 (50%) were inhibited, and 41 (3%) were unaffected (Table I). Thus, the proportion of neurons inhibited by amphetamine was approximately the same as in the earlier experiments. When the responses of these neurons to both amphetamine and noradrenaline were examined it was found that all those affected by iontophoretic application of D-amphetamine were sensitive also to iioradrenaline (Bradley et al., 1967a) TARTJC 111 EFFECTS OF NORADRENALINE A N D AMPHETAMINE APPLJED TO TIIE SAME NEURON" Noradrenaline

+

+

-

-

0

o

+-

~

~

0 0

4

+

0

-

-

Number of neurons responding 12 45 29 3

0

+ a

Aniphetaniine

-

+

0

1 1 0

+ = excitation, - = inhibition, o = no effect.

and in many cases, the direction of the two effects was the same (Fig. 15). Thus, in a sample of 95 neurons (Table 111), simiIar effects were produced by these two substances in 86 (90%).There were 7 neurons which were sensitive to noradrenaline but unaffected by amphetamine, only 1 where the reverse was true, and 1 on which the two substances had opposite effects. No such relationship has been found between the effects of amphetamine and histamine and it therefore appears that the central stimuIant action of amphetamine may be related to its sympathomimetic properties. Furthermore, the close parallel between the effects of amphetamine and those of noradrenaline lends support to the idea that the drug may act centrally by releasing noradrenaline from its storage sites.

38

PHILIP B. BRADLEY

IOL

--

0

30

60

30

0

60 Seconds

(A)

. ,1

, 0

30

60

NA 0

u

0

30

60 Seconds

[B) FIG. 15. Excitatory and inhibitory effects of D-amphetamine and L-noradrenaline on brain stem neurons. A: a neuron excited by both amphetamine and noradrenaline; B: a neuron inhibited by both substances. Note the similarity in the time courses of the effects in both cases. (Bradley et al., 1967b.)

C. TRANQUILIZERS Many compounds in this category have been investigated for their actions on the central nervous system, but the one which is not only the oldest and probably most widely used, but also has been investigated extensively by workers using electrophysiological, biochemical, and psychological techniques, is chlorpromazine. Its

39


-

CP7

I

I

NA

I

I

I

I

Minutes

FIG. 16. The effects of acetylcholine, noradrenaline, and chlorpromazine (CPZ), applied iontophoretically with currents of 50 nA on a neuron in the brain stem reticular formation. The inhibitory actions of acetylcholine and noradrenaline are not modified by the action of chlorpromazine, which also causes inhibition. (Bradley et al., 1966d.)

TABLE IV EFFECTS OF NORADRENALINE AND CHLORPROMMXNE APPLIED TO THE 8.4% NEURON" Xioradrenaline

Clrlnrpromazine

Number of neurons responding 0

47 10

4 9 0 0 6 0

+ = excitation, -

=

inhibition. o

= no

efi'ert.

action, in producing a state of indifference and unresponsiveness to the environment and to sensory stimulation, is believed to be due to a depressant action reIated to the collateral afferent input to the reticular formation of the brain stem (Bradley and Hance, 1957; Bradley and Key, 1958; Bradley, 1963). However, although the

40

PHILLP B. BRADLEY

effects of chlorpromazine have been studied on responses of single neurons in the reticular formation (Bradley, 1957), it was not possible to determine whether these effects were direct or indirect since the drug was injected systemically. The microiontophoretic technique has yielded some new information on the central effects of this substance (Bradley et al., 1 9 6 6 ~ )Chlorpromazine, . applied by iontophoresis, has been found to have a predominantly inhibitory

-

2

NA

10-

I

I

NA I

1

I

- Ach

I

NoCl

I

I

I

FIG. 17. The effects of noradrenaline and acetylcholine before and after iontophoretic application of chlorpromazine, on a neuron in the reticular formation of the cat. The excitatory response to noradrenaline is no longer present after chlorpromazine has been applied, but is replaced by a weak inhibitory effect. The excitatory response to acetylcholine is not significantly altered. All applications (including the current control ) were with currents of 50 nA; the chlorpromazine was applied for 1 minute. (Bradley et al., 1968c.)

action on neuronal activity in the brain stem reticular formation (Table I ) . Furthermore, when its effects were compared with those of possible transmitter substances (acetylcholine, noradrenaline, and 5-hydroxytryptamine ) it was found that chlorpromazine acted on neurons which were also affected by noradrenaline, and had no action on neurons unaffected by noradrenaline (Table IV) . Thus, inhibition by noradrenaline was almost invariably accompanied by inhibition by chlorpromazine and in many instances neurons excited by noradrenaline were inhibited by chlorpromazine. Since, in its peripheral actions, chlorpromazine is known to antagonize adrenaline, acetylcholine, 5-hydroxytryptamine, and histamine, its actions

41

SYNAPTIC 'I'RANSMISSION IN THE C N S

as a possible antagonist to these substances centrally were examined with iontophoretic application. No consistent antagonistic actions were found for the excitatory or inhibitory effects of acetylcholine, 5-hydroxytryptamine or histamine, nor was there any antagonism to the inhibitory actions of noradrenaline (Fig. 16), but the excitatory effects of noradrenaline were consistently abolished or reduced following chlorpromazine application (Fig. 17) and in some cases

'

CPZ m NA

%I

TH ; 5

NA

52T I

Minutes

FIG.18. The effects of noradrenaline and 5-hydroxytryptamine on the activity of a neuron in the reticular forniation of the cat, before and after iontophoretic application of chlorpromazine. The excitatory response to 5-HT is maintained while that to noradrenaline is lost and replaced by an inhibitory effect following chlorpromazine application (currents 50 nA; chlorpromazine 1960d.) applied for 1 minute). (Bradley et d.,

replaced by a weak inhibitory effect (Fig. 18). There was no action by chlorpromazine on excitatory effects of glutamate. Thus, it is suggested (Bradley et al., 1966c) that noradrenaline may be an inhibitory transmitter at some sites in the central nervous system and an excitatory transmitter at others and that chlorpromazine is an antagonist to the transmitter actions of noradrenaline at those synapses where it is excitatory. Furthermore, these effects can be related to neurons in the brain stem with rostrally projecting axons and which may therefore be concerned in the arousal mechanisms of the brain. This hypothesis, if confirmed, may help to explain many of the central actions of chlorpromazine, including its clinical effects.

42

PHILIP B. BRADLEY

D. PSYCHOTOMIMETIC DRUGS The two drugs in this category which have received most attention are the synthetic hallucinogen, D-lysergic acid diethylamide (LSD 25) and the naturally occurring one, mescaline. The possible relevance of the peripheral action of LSD 25 as an antagonist to 5-hydroxytryptamine has already been referred to (p. 22). LSD 25 has been found to have an action restricted to the brain stem reticular foimation (Bradley and Elkes, 1957), and, as in the case of chlorpromazine, this action is closely related to the influence of the afferent collateral input to the brain stem; in the case of LSD 25 it is facilitatory (Bradley and Key, 1958). Investigations into the behavioral and electrophysiological effects of this drug (Key and Bradley, 1960; Key, 1961, 1965) have shown that its action, at the brain stem level, is related in a highly specific manner to the neurophysiological mechanisms controlling the flow and integration of sensory information. As yet these mechanisms are undefined but they must involve a balance between facilitation and inhibition (Key, 1965), and a disturbance of this balance is probably responsible for disturbances in perception and for hallucinations. Investigations into the actions of LSD 25, when applied iontophoretically to single neurons have demonstrated only depression of activity (Table I ) . In anesthetized animals, LSD 25 and other derivatives of lysergic acid were found to cause prolonged depression of glutamate-evoked activity of cortical neurons (Krnjevi6 and Phillis, 1 9 6 3 ~ )Iontophoretic . application of LSD 25 to neurons in the brain stem of unanesthetized cats showed inhibition of spontaneous activity in 30%(Fig. 19) (Bradley and Wolstencroft, 1965). On the other hand, this substance has been found to antagonize excitatory actions of 5-hydroxytryptamine on cortical neurons (Roberts and Straughan, 1966) but this antagonism is not specific for LSD 25 since it is also shown by brom-LSD (BOL 148) which has no psychotomimetic properties. Depression of activity in cortical neurons by 5-HT was not blocked by LSD 25 (Legge et al., 1966). However, the existence of an antagonistic action at the cortex between LSD 25 and 5-HT is unlikely to help in explaining effects of LSD 25 in the brain stem. While it would be attractive to interpret the depressant action of LSD 25 on brain stem neurons as a possible depression of inhibitory mechanisms, much more investigation is needed before such a hypothesis can be made.

43

SYNAPTIC 1’RANSMlSSION IN THE CNS

Although mescaline is a much older hallucinogenic drug than LSD 25, it has received less attention from investigators. However, some experiments have been carried out in animals with this drug (Bradley and Elkes, 1957) and it has recently been subjected to extensive investigation of structure/ activity relationships ( Smythies and Sykes, 1967; Smythies et al., 1967). Roberts and Straughan (1967) have compared the effects of mescaline with those of noradrenaline and a nonhallucinogenic isomer of mescaline, all applied iontophoretically, on the activity of cortical neurons in unanesthetized cats. They found that both mescaline and its 2,3,Bisomer

-

LSC 25-

f

1

I

I

30

60

90

I20

150

I

180 Seconds

FIG. 19. The effects of iontophoretic application of D-lysergic acid diethylamide (LSD 25) on the response of a neuron in the brain stem of the cat to stimulation of the ipsilateral superficial radial nerve. (Bradley and Woktencroft, 19G4.)

had similar profiles of action, exciting about 30%and depressing 14% of the spontaneously active cells, Most cells tested with both mescaline and noradrenaline responded in the same direction, though mescaline had only half the potency of noradrenaline and a more prolonged effect. Some cells responded in opposite directions to these two compounds. Since the effects of mescaline and its nonhallucinogenic isomer were similar it is not possible to relate these findings to the hallucinogenic actions of mescaline. VIII. Conclusions a n d Summary

The best documented example of synaptic transmission mediated by acetylcholine in the central nervous system is that provided between collaterals of motor axons and Renshaw cells in the anterior

44

PHILIP B. BRADLEY

horn of the spinal cord. Here, liberation of acetylcholine from terminals of motor axon collaterals excites the Renshaw cell and causes recurrent inhibition of motor neurons. Local application of acetylcholine to the Renshaw cells causes a similar effect to synaptic excitation, and both types of excitation are blocked by acetylcholine antagonists. This cholinergic synapse is predominantly nicotinic but it can be argued that since the synapses on Renshaw cells are the peripheral endings of motor nerves, and since all synapses formed by one axon are mediated by the same chemical transmitter (Dale, 1935), it is inevitable that cholinergic endings should be found on Renshaw cells as the peripheral endings of motor axons are undoubtedly cholinergic. In the brain, the evidence for cholinergic transmission is largely circumstantial. The first criterion for chemical transmission is met, i.e., the presence of the suspected transmitter at presynaptic sites, together with the appropriate enzyme systems. Acetylcholine has a wide distribution in the brain, as have the enzymes for its synthesis and destruction, and fractionation studies have demonstrated that it is associated with nerve endings containing synaptic vesicles. Also, histochemical methods utilizing cholinesterase staining indicate that certain pathways have an affinity for acetylcholine. However, it has proved impossible to demonstrate in the brain that local application of acetylcholine can mimic synaptic excitation or that stimulation of presynaptic fibers causes liberation of acetylcholine. The complexity of the central nervous system mitigates against the likelihood of this being possible with the techniques available at present, although it is conceivable that future developments in neurohistochemistry might render it possible to demonstrate the release of suspected transmitters from nerve terminals. However, the fact that acetylcholine is released from the surface of the cerebral cortex, and that the amount released seems to be related to cortical function in terms of arousal, together with the demonstration of a cholinergic element in the ascending reticular activating system, although not providing information directly related to the role of acetylcholine in synaptic mechanisms, is certainly suggestive of such a role. The technique of iontophoresis, which provides the nearest approach that can be made at present to the local application of acetylcholine and other substances to synapses in the central nervous system, has demonstrated that cholinoceptive cells are present in


45

many parts of the brain. What is perhaps somewhat surprising is that the proportion of neurons affected by iontophoretic application of acetylcholine is relatively small (30%or less in the cortex and 57% in the brain stem, but 7580%in the caudate nucleus and hippocampus), In most regions, the action of acetylcholine is excitatory and, where the pharmacology is known, e.g., the cortex, the response appears to be muscarinic. In the brain stem, however, some neurons are excited and others are inhibited by acetylcholine; whereas the excitatory response appears to have mixed nicotinic and muscarinic properties, the inhibitory response is exclusively muscarinic. In interpreting the data derived from iontophoretic application of substances to single cells in the brain, it must be remembered that the effects observed could be due to: ( a ) an action on the postsynaptic membrane, mimicking, potentiating, or blocking the action of the transmitter; ( b ) an action on presynaptic terminals, causing release, or blocking release of the transmitter; ( c ) an action on nonsynaptic membranes, causing changes independent of synaptic processes; (cl) an action on a neighboring neuron, thus producing an indirect effect. We have at present no definite evidence to suggest which of the first three of these possibilities is likely to be true, but there is some data which makes it unlikely that ( d ) is important. This will be discussed below. One interesting feature of the actions of acetylcholine (and of some other substances when applied iontophoretically is that in the brain stem some neurons respond with excitation and others with inhibition. Obviously, one possibility which must be considered is that one action, e.g., the excitatory response, is synaptic and the other is due to some nonspecific or indirect effect. An indirect action might be due to the substance diffusing to another, smaller neuron, where it had an excitatory action, and this neuron had an inhibitory influence on the one being recorded, i.e., a Renshaw-like neuron. If this was the case then we should expect (1) a different time course for the excitatory and inhibitory effects, which is not consistently seen; ( 2 ) that moving the micropipette might alter the direction of the effects, and this has not been observed; ( 3 ) by recording with microelectrodes with smaller tips we should pick up the small, Renshaw-like cells more easily and this would aIter the proportion of cells excited to those inhibited. Experiments along these lines (Bradley et a/., 1967c) have shown that this is not the case.

46

PHILIP B. BRADLEY

A further possibility is that the excitatory actions of acetylcholine might be synaptic and the inhibitory action due to some nonspecific depressant effect, e.g., a local anesthetic action. If this were true we might expect (1) the inhibitory action would be universal, as it is with atropine, for example, but this is not the case (see Table I ) ; ( 2 ) the neurons would not respond to glutamate when inhibited, but in fact they still do. A third possibiIity is that the excitatory action might be postsynaptic and the inhibitory presynaptic oi- vice versa. This is related to the idea of a dual neurohumoral role for acetylcholine which has been proposed by G. B. Koelle (1962) and which will be discussed further. There remains the possibility that acetylcholine is an excitatory transmitter at some sites and an inhibitory transmitter at others, i.e., that there are two types of receptor for acetylcholine in the central nervous system, at least in the brain stem. This idea is supported by the fact that the excitatory and inhibitory responses have a different pharmacology. In fact, there is good evidence that in some invertebrates acetylcholine can act as an excitatory transmitter at some synapses and as an inhibitory transmitter at others (Tauc and Gershenfeld, 1961, 1962; Kerkut and Cottrell, 1963). Thus, because at those sites in the vertebrate nervous system where acetylcholine has been proved to be the transmitter, i.e., the neuromuscular junction and the Renshaw cell, its action is always excitatory, we rnay have been too easily led to believe that its role in the brain as a transmitter is likely to be exclusively excitatory. As a result of this the search has concentrated on different substances as the transmitters at excitatory and inhibitory synapses in brain. If both the actions of acetylcholine on brain stem neurons prove to be postsynaptic, and it should be possible to show this by intracellular recording, then a mechanism such as that proposed by Eccles (1964b) could explain the dual action. According to this hypothesis, excitatory or inhibitory effects would be the result of the transmitter opening pores of different sizes in the postsynaptic membrane. The opening of small pores would allow only potassium or chloride ions to pass through the membrane, resulting in inhibitory postsynaptic potentials, while the opening of larger pores could allow the free passage of sodium ions, thus producing excitatory postsynaptic potentials. The dual effects of acetylcholine could then be explained by ( a ) some neurons having a preponderance of all or


47

one type of receptor, or ( b ) that the two receptors have a different distribution on different parts of the cell, e.g., cell body, axon hillock, and dendrites, in which case the position of the micropipette relative to the neuron might determine the effect observed. HOWever, in the latter case moving the microelectrode would be expected to change the direction of the response in some cases and this has never been observed. Thus we are left with the possibility that the two receptors have a cliff erent distribution on cholinoceptive cells in the brain stem. Although subcellular fractionation studies provide the best evidence so far for the localization of acetylcholine to synaptic vesicles in the central nervous system, these studies have been carried out on homogenates of whole brain, whereas it is quite clear from other biochemical studies, as well as from cholinesterase staining and iontophoretic application, that cholinergic mechanisms are not evenly distributed throughout the brain. In addition, consideration must be given to the fact that homogenization and centrifugation are fairly violent processes to which to subject nervous tissue. With these reservations, it seems fairly certain that before very long evidence will be forthcoming to confirm the role of acetylcholine as a synaptic transmitter in certain pathways in the brain. In fact, it is difficult to conceive of a role for acetyIchoIine in neuronal mechanisms, other than as a transmitter. In all probability, the diffuse projections from the reticular activating system to the cerebral cortex will be shown to be cholinergic, or to contain a cholinergic link, and the caudate nucleus, hippocampus, cerebellum, and also certain nuclei of the thalamus will be found to be cholinersic, at least in part. The evidence for actions in the mammaIian brain by the monoamines ( catecholamines and 5-HT) as synaptic transmitters is rather less complete than for acetylcholine. Here, subcellular fractionation studies do not provide such conclusive evidence of localization, and noradrenaline, the most likely candidate among the catecholamines, is found partly in the supernatant, though this has been explained on the basis of the synaptic vesicles containing noradrenaline being more sensitive to ‘‘Shock.” However, the best evidence for the presence of amines conies from fluorescence histochemistry. The demonstration that adrenaline, noradrenaline, dopamine, and S-hydroxytryptamine are present in different neurons, mainly concentrated in the nerve terminals, and responding in a predictable manner to

48

PHILIP B. BRADLEY

lesions and drug treatments, has led to the postulate of noradrenergic, dopaminergic, and serotonergic neuron systems, on the basis of this evidence alone (Fig. 20). The present difficulty is to correlate these findings with those from other studies, e.g., from iontophoretic application of amines.

FIG. 20. Schematic diagram showing the proposed monoamine-containing neuron systems in the central nervous system, as determined by fluorescence histochemistry. (Fuxe and Anden, 1965.)

The actions of the monoamines on neurons are mainly inhibitory, except for 5-HT at the cortex and both 5-HT and noradrenaline in the brain stem, where both inhibitory and excitatory effects have been found. A characteristic feature of the response to iontophoretically applied amines is a slow onset and recovery, in contrast


49

to the response to acetylcholine (see Fig. 13). However, it is ~ O S sible that factors such as diffusion from the tip of the micropipette to the active site might account for these long latencies. Nevertheless, it is clear that, apart perhaps from the mitral cells of the olfactory bulb, where the evidence for noradrenaline being a transmitter is reasonably good, a considerable volume of further data is required to confirm and support the ideas originating from fluorescence studies. That the catecholamines and 5-hydroxytryptamine have important functions in the central nervous system cannot be disputed, even if it subsequently materializes that these substances are not directly concerned with synaptic transmission. Certainly, drugs which modify their mctabolism, and especially those which cause depletion of amines, e.g., reserpine, have profound effects on brain function. The universaI actions of amino acids, both excitatory and inhibitory, has led to considerable speculation as to their possible role as synaptic transmitters. One argument which has been used to favor L-glutamate as a possible excitatory transmitter at the cerebral cortex is the very short latency and rapid cessation of its action. However, nothing is known of the mechanisms for its inactivation. The main arguments against the amino acids as synaptic transmitters are their lack of specificity of action and the fact that they are cytoplasmic constituents of the neuron and are distributed evenly throughout the cell instead of being localized to nerve endings. On the other hand, some of the enzymes concerned in their synthesis appear to be localized to nei-ve endings. The recent Endings of a hyperpolarizing action by glycine in the spinal cord support the concept of a transmitter role for amino acids, although in the absence of enzyme systems for inactivation of glycine it has been necessary to postulate n removal from the extraneuronal environment by rapid intracellular transfer. No doubt the already considerable volume of literature on the central actions of amino acids will be extended by future investigations which may throw more light on their role in the central nervous system. How can we explain the fact that in some parts of the brain there are neurons which respond to more than one substance? For example, in the brain stem reticular formation where acetylcholine, noradrenaline, and 5-hydroxytryptamine produce mixed effects and various combinations of excitation and inhibition are seen (see Table 11). It would be tempting to intcrpret these findillgs as indi-

50

PHILIP B. BRADLEY

cating that receptors of different pharmacological types are present on the same neuron, perhaps with a differential distribution on different parts of the cell. Although the evidence from fluorescence histochemical studies of neurons containing monoamines points to there being three distinct types, containing dopamine, noradrenaline, or 5-hydroxytryptamineYit does not necessarily follow that the receptors on these neurons are exclusively of one type, but that one kind of receptor probably predominates. Thus, where acetylcholine, noradrenaline, and Shydroxytryptamine are all effective, though not necessarily producing similar effects, there may be three pharmacologically distinct receptors on the same neuron. However, there are other possible explanations which must be considered. First, it is possible that one substance is acting synaptically and the other two are producing effects indirectly, e.g., via neighboring neurons to which they diffuse, as has been considered for the dual action of acetylcholine. However, the same arguments against this possibility apply here. Thus, by moving the electrode we should expect to change the direction of the effects in some instances, and recording with electrodes with very fine tips ought to alter the relative proportions of the different types of responses, but in neither case has this been found to be true. However, the slower time course of action, which is observed on some occasions with noradrenaline and 5-hydroxytryptamine applications, does favor the possibility of indirect actions. A second possibility is that one substance, for example, acetylcholine, may be acting synaptically and that the other two (noradrenaline and 5-HT) have effects on cell excitability which are unrelated to synaptic transmission. If these latter two substances had a universal depressant action, then such a nonspecific effect on excitability, e.g., a local anesthetic action, would be a strong possibility. However, it is difficult to imagine that an action of this kind could produce excitatory responses in some cases and inhibitory responses in others. Nevertheless, it is conceivable that the excitatory actions of some, or all, of these substances might be related to synaptic transmission, while the inhibitory responses are due to indirect or nonspecific effects. Furthermore, where the actions of noradrenaline and 5-HT are similar, it is possible that they may be acting on the same receptors, but this can hardly be true where their effects are opposite or where only one is effective. A third possible explanation for multiple effects on neurons is related to the concept of primary and secondary transmitters. This


51

arose through the finding of considerable quantities of acetylcholinesterase in presynaptic terminals in sympathetic ganglia ( W. A. Koelle and G. B. Koelle, 1959). To explain this it was proposed (G. B. Koelle, 1962) that acetylcholine is liberated in very small quantities at the presynaptic terminal as a result of the arrival of a nerve action potential, and this in turn acts on a cholinergic receptor site on the presynaptic terminal, which then liberates a further quantity of acetylcholine which diffuses across the synaptic cleft to produce a postsynaptic potential. The function of the presynaptic cholinesterase is to terminate the action of the initially liberated acetylcholine and also to protect the presynaptic membrane against the effects of spontaneously liberated acetylcholine. The possibility that such a mechanism might explain excitatory and inhibitory actions of acetylcholine has already been mentioned. As an extension of this hypothesis, G. B. Koelle (1962) has proposed that the concentrations of acetylcholinesterase may be indicative of the relative importance of cholinergic transmission at different neural sites. Thus, it is suggested that in adrenergic transmission in sympathetic ganglia, acetylcholine is the primary transmitter which acts presynaptically to release noradrenaline as the secondary transmitter. This is consistent with the findings of Burn and Rand (1982), who showed that a cholinergic mechanism is involved in the release of noradrenaline by postganglionic sympathetic nerve fibers. If a similar mechanism is found to operate at certain synapses in the central nervous system, and there is as yet no evidence whatsoever for this, then we may have a relatively simple explanation for multiple actions by acetylcholine, noradrenaline, and 5-hydroxytryptamine in the brain stem, and possibly elsewhere. However, it might be necessary in this case to postulate not only the existence of primary and secondary transmitters, but tertiary ones as well. Although the neurons in the brain-stem reticular formation show many different types of response to suspected transmitters and the number of combinations of excitatory and inhibitory effects is somewhat bewildering (Table I I ) , there is some evidence of homogeneity of pharmacological properties in the neurons of one nucleus, the paramedian reticular nucleus. Here the majority of neurons show similar responses to iontophoretically applied acetylcholine, noradrenaline, and Shydroxytryptamine and it is possible that similar findings may be obtained for other anatomically and physiologically distinct nuclei in the brain stem. It will he interesting to

52

PHILIP B. BRADLEY

see whether multiple responses are found for neurolls in other regions of the brain or whether this is a property peculiar to reticular neurons, perhaps associated with the complexity of physiological functions subserved by this region. Some of the precautions which need to be observed when interpreting experimental results obtained with the technique of microiontophoresis have recently been reviewed ( Salmoiraghi and Stefanis, 1967). One thing which is certain is that today’s findings may not be true tomorrow, and differences in techniques, not only between different groups, but in different series of experiments by the same workers may influence the findings considerably (Bradley et al., 1!363). Furthermore, negative results must always be treated with great reserve as there are now many documented cases of substances which were first thought to be inactive and later found to have effects on neuronal activity (e.g., noradrenaline and its effects on brain stem neurons). There is evidence that anesthetics of various types can influence the responses of nerve cells to iontophoretically applied test substances, and this is especially true for acetylcholine, yet much of the data available about its actions has been obtained from anesthetized preparations where spontaneous activity is largely absent, necessitating the use of glutamate to evoke responses. Thus, the slow time course of the responses of cortical neurons to acetylcholine, which has been used as an argument against a transmitter action at this site, might be related to the presence of anesthesia in the experimental animals from which these responses were obtained. The use of the technique of microiontophoresis for studying actions on neurons of centrally acting drugs, particularly those used clinically for their effects on mental function, is still in its infancy. The results obtained so far must be regarded as tentative and preliminary, and must depend in many cases on further evidence of the nature of chemical transmission before more definite interpretations can be made. However, the approach is promising and may provide important information on the mechanisms of action of drugs. Nevertheless, whatever the results obtained by applying these substances directly to neuronal surfaces by iontophoresis, it must not be forgotten that they produce their characteristic effects on brain function when they are administered systemically. Thus, various factors, such as passage through the blood-brain barrier, possible chemical changes before reaching the brain, etc., have to be con-

SYNAPTIC TFL4NSMISSION IN THE CNS

53

sidered. In fact, it will be necessary in many cases to show that effects obtained with local iontophoretic injection can also be observed with systemic administration of the same substance. Only in this way can the data obtained from studies of neural mechanisms at the single neuron level be related to function of the central nervous system as a whole, as manifested in the behavior of the organism. ACKNOWLEDGMENT

I am grateful to my co-workers for allowing me to use much of our unpublished material in this review. REFERENCES Adam, H. M. (1961). In “Regional Neurocheniistry” ( S . S. Kety and J. Elkes, eds.), p. 293. Pergamon Press, Oxford. Anderson, P., and Curtis, I>. R. (1964). Acta Physiol. Scand. 61, 85. Avanzino, G. L., Bradley, P. B., Comis, S. D., and Wolstencroft, J. H. ( 1966a). Intern. J . Neuropharmacol. 5, 331. Avanzino, G. L., Bradley, P. B., and Wolstencroft, J. H. (1966b). Brit. J . Pharmacol. 27, 157. Avanzino, G. L., Bradley, Y. B., and Wolstencroft, J. H. ( 1 9 6 6 ~ )Experientia . 22, 410. Avanzino, G. L., Bradley, P. B., and Woktencroft, J. H. (1966d). Unpublished observations. Avanzino, G. L., Bradley, P. B., and Woktencroft, J. H. (1967). Progr. Biocheni. Pharmacol. 3, 136. Bennett, E. L., Diamond, M. C., Krech, D., and Rosenzweig, M. R. (1964). Science 146, 610. Berl, S., and Waelsch, H. (1958). J. Neurochem. 3, 161. Biscoe, T. J., and Curtis, D. R. (1966). Science 151, 1230. Biscoe, T. J., and Straughan, D. W. (1966). J. Physiol. (London) 183, 341. Bloom, F. E., Oliver, A. P., and Snlmoiraghi, G. C. (1963). Intern. J. Neuropharmacol. 2, 181. Bloom, F. E., Costa, E., and Salmoiraghi, G. C. (1965). J . Pharmacol. Exptl. Therap. 150, 244. Boakes, R., Bradley, P. B., Brookes, N., and Wolstencroft, J. H. (1968). Brit. 1. Pharmacol. 32, 417P. Bradley, P. B. (1957). In “Psychotropic Drugs” ( S . Garattini and V. Chetti, eds. ), p. 209. Elsevier, Amsterdam. Bradley, P. B. (1963). Physiol. Pharmcol. 1, 417. Bradley, P. B., and Elkes, J. (1953). J. Physiol. (London) 120, 13P. Bradley, P. B., and Elkes, J. ( 1957). Brain 80, 77. Bradley, P. B., and Hance, A. J. (1957). Electroencephalog. Clin. Neurophysiol. 9, 191. Bradley, P. B., and Key, B. J. (1958). Electroencephalog. Clin. Neurophysiol. 10, 97.

54

PHILIP B. BRADLEY

Bradley, P. B., and Mollica, A. (1958). Riu. Arch. ltal. B i d . 96, 168. Bradley, P. B., and Samuels, G. M. R. (1967). Unpublished data. Bradley, P. B., and Wolstencroft, J. H. (1962). Nature 196, 840. Bradley, P. B., and Wolstencroft, J. H. (1964). In “Neuro-psychopharmacology” (P. B. Bradley, F. Fliigel, and 1’. Hoch, eds.), Vol. 3, p. 237. Elsevier, Amsterdam. Bradley, P. B., and Wolstencroft, J. H. (1965). Brit. Med. Bull. 20, 15. Bradley, P. B., and Wolstencroft, J. H. (1967). Ann. N.Y. Acad. Sci. 142, 15. Bradley, P. B., Dhawan, B. N., and Wolstencroft, J. 11. (1963). J. Physiol. (London) 170,59P. Bradley, P. B., and Wolstencroft, J. H. ( 1966). Unpublished data. Bradley, P. B., Hosli, L., and Wolstencroft, J. H. (1966a). Unpublished observations. Bradley, P. B., Dhawan, B. N., and Woktencroft, J. H. (1966b). J . Physiol. (London) 183,658. Bradley, P. B., Wolstencroft, J. H., Hosli, L., and Avanzino, G. L. ( 1 9 6 6 ~ ) . Nature 21% 1425. Bradley, P. B., Wolstencroft, J. H., Hosli, L., and Avanzino, G. L. (1966d). Unpublished observations. Bradley, P. B., Hosli, L., and Wolstencroft, J. H. (1967a). Brit. J. Pharmucol. 29, 121. Bradley, P. B., Hosli, L., and Wolstencroft, J. H. (196%). Unpublished data. . Bradley, P. B., Brookes, N., and Wolstencroft, J. H. ( 1 9 6 7 ~ ) Unpublished data. Bum, J. H., and Rand, M. J. (1962). Aduan. Pharmacol. 1 , l . Celesia, G. G., and Jasper, H. H. (1966). Neurology 16, 1053. Coceani, F., and Wolfe, L. S. (1965). Can. J . Physiol. Pharmacol. 43, 445. Collier, B., and Mitchell, J. F. ( 1966). Nature 210, 424. Crawford, J. M., and Curtis, D. R. (1964). Brit. J. Pharmacol. 23, 313. Crawford, J. M., Curtis, D. R., Voorhoeve, P. E., and Wilson, V. J. ( 1966). J . Physiol. (London) 186, 139. Crossland, J. (1960). J . Pharm. Pharmacol. 12, 1. Crossland, J., and Mitchell, J. F. (1956). J. Physiol. (London) 132, 391. Crossland, J., Woodruff, G. N., and Mitchell, J. F. (1964). Nature 203, 1388. Curtis, D. R. ( 1961). In “Nervous Inhibition” (E. Florey, ed.), p. 342. Pergamon Press, Oxford. Curtis, D. R. (1964). Phys. Tech. B i d . Res. 5, 144. Curtis, D. R. (1966). In “The Thalamus” (D. P. Purpura and M. D. Yahr, eds.), p. 183. Columbia Univ. Press, New York. Curtis, D. R., and Davis, R. (1962).Brit. J. Pharmacol. 18, 217. Curtis, D. R., and Davis, R. (1963). J . Physiol. (London) 165, 62. Curtis, D. R., and Eccles, R. M. (1958). J . Physiol. (London) 141, 435. Curtis, D. R., and Koizumi, K. ( 1961). J. Neurophysiol. 24, 80. Curtis, D. R., and Phillis, J. W. (1960). J . Physiol. (London) 153, 17. Curtis, D. R., and Watkins, J. C. (1965). Pharmacol. Rev. 17, 397. Curtis, D. R., Phillis, J. W., and Watkins, J. C. (1959). J. PhysioE. (London) 146, 185. Curtis, D. R., Phillis, J. W., and Watkins, J. C. (1960). J . Physiol. (London) 150, 656.


55

Curtis, D. R., Phillis, J. W., and Watkins, J. C. (1961). J . Physiol. (London) 158, 296. Curtis, D. R., Hosli, L., and Johnston, G. A. R. (1967). Nature 215, 1502. Dahlstrom, A., and Fuxe, K. (1965). Acta Pliysiol. Scund. 64, 1. Dale, H. H. (1935). Proc. Roy. SOC. Med. 28, 319. Davidoff, R. A., Shank, R. P., Graham, L. T., Aprison, hl. H., and Werman. R. (1967). Nature 214, 680. Davis, R. (1966). In “The Thalamus” (D. P. Purpura and M. D. Yahr, eds.), p. 193. Columbia Univ. Press, New York. Del Castillo, J., and Katz, B. (1955). J . Physiol. (London) 128, 157. De Robertis, E. (1964). Progr. Bruin Res. 8, 118. De Robertis, E. (1966). Pharmacol. Reo. 18,413. De Robertis, E. (1967). Science 156, 907. Eccles, J. C. (1964a). “The Physiology of Synapses,” p. 316. Springer, Berlin. Eccles, J. C. (1964b). Science 145, 1140. Falck, B. (1962). Acta Physwl. Scand. 56, 1. Falck, B. ( 1964). Progr. Brain Res. 8, 28. Feldberg, W. (1957). In “Metabolism of the Nervous System” (D. Richter, ed.), p. 493. Pergamon Press, Oxford. Feldberg, W., and Vogt, M. ( 1948). J . Physiol. (London ) 107, 372. French, J. D., Verzeano, M., and Magoun, H. W. (1953). A.M.A. Arch. Neurol. Psychiut. 69, 519. F u e , K., and Anden, N-E. (1965). In “Biochemistry and Pharmacology of the Basal Ganglia” (E. Costa, L. J. Cote, and M. D. Yahr, eds.), p. 123. Raven Press, New York. Gaddum, J.H. (1963). Nature 197,741. Horton, E. W., and Main, I. H. M. (1966). J. PhysioZ. (London) 185, 36P. Jasper, H. H., and Koyama, I. ( 1968). Electroencephabg. Clin. Neurophysiol. 24, 292. Kanai, T., and Szerb, J. C. (1965). Nature 205, 80. Kba, P., and Csillik, B. (1965). Nature 208, 695. Katz, B. (1966). “Nerve, Muscle and Synapse,” p. 193. McGraw-Hill, New York. Kerkut, G. A., and Cottrell, G. A. (1963). Comp. Biochem. Physiol. 8, 53. Key, B. J. (1961). Psychopharmacologia 2, 352. Key, B. J. (1965). Brit. Med. Bull. 21, 30. Key, B. J., and Bradley, P. B. ( 1960). Psychopharmucologia 1,450. Koelle, G . B. (1962). J. Pharm. Pharmacol. 14, 65. Koelle, G. B., and Friedenwald, J. S. (1949). PTOC.SOC. Exptl. Biol. Med. 70, 617. Koelle, W. A., and Koelle, G. B. (1959). J . Pharmacol. Ex&. Therap. 126, 1. Kmjevi6, K. (1964). Intern. Rev. Neurobiol. 7, 41. Krnjevih, K., and Phillis, J. W. (1963a). J. Physiol. (London) 166, 296. Krnjevi6, K., and Phillis, J. W. (196313). J. Physiol. (London) 166, 328. . J. Pharmacol. 20, 471. Kmjevi6, K., and Phillis, J. W. ( 1 9 6 3 ~ )Brit. Kmjevib, K., and Phillis, J. W. (1963d). J . Physiol. (London) 165, 274. Kmjevi6, K., and Schwartz, S. (1967). Exptl. Bruin Res. 3, 320. Krnjevi6, K., Randi6, M., and Stranghan, D. W. (1965). Nature 205,603.

56

PHILIP B. BRADLEY

Legge, K. F., Randib, M., and Straughan, D. W. (1966). Brit. J. Phurmmol. 26. 87. Lembeck, F., and Zetler, G. (1962). Intern. Reu. Neurobiol. 4, 159. McCance, I., Phillis, J. W., and Westerman, R. A. (1966). Nature 209, 715. McLennan, H. (1964). 1. Physiol. (London) 174,152. Mitchell, J. F. (1963). J . Physiol. (London) 165, 98. Nastuk, W. L. (1953). Federation Proc. 12, 102. Phillis, J. W. (1965a). Brit. Med. Bull. 20,26. Phillis, f. W. ( 196513). Experientiu 21,266. Phillis, J. W., and Chong, G. C. ( 1965). Nature 207,1253. Ramwell, P. W., and Shaw, J. E. (1966). Am. J. Physiol. 211, 125. Randib, M.,Siminoff, R., and Straughan, D. W. (1964). Exptl. Neurol. 9,236. Richter, D., and Crossland, J. (1949). Am. J. Physiol. 159, 247. Roberts, M. H. T., and Straughan, D. W. (1966). J. Physiol. (London) 188, 27P. Roberts, M. H. T., and Straughan, D. W. (1967). J. Physiol. (London) 193, 269. Roberts, M. H. T., and Straughan, D. W. (1968b). Arch. Erptl. Pathol. Pharmkol. 259, 191. Ryall, R. W. ( 1964). J. Neurochem. 11,131. Sdganicoff, L., and De Robertis, E. (1965). J. Neurochem. 12,287. Salmoiraghi, G . C., and Stefanis, C. N. (1967). Intern. Rev. Neurobiol. 10, 1. Salmoiraghi, G. C., Bloom, F. E., and Costa, E. (1964). Am. J. Physiol. 207, 1417. Samuels, G. M. R., Shaw, J. E., and Bradley, P. B. (1967). Brit. J. Pharmucol. 30, 2. Shute, C. C. D., and Lewis, P. R. (1963). Nature 199, 1160. Shute, C. C. D., and Lewis, P. R. (1967). Brain 90,497. Smythies, J. R., and Sykes, E. A. (1967). In “Amines and Schizophrenia” (H. E. Himwich, S. S. Kety, and J. R. Smythies, eds.), p. 5. Pergamon Press, Oxford. Smythies, J. R., Johnston, V. S., Bradley, R. J., Benington, F., Morin, R. D., and Clark, L. C., Jr. (1967). Nature 216, 128. Steiner, F. A., and Meyer, M. (1966). Experientia 22, 58. Szerb, J. C . (1967). J. Physiol. (London) 192, 329. Tauc, L., and Gershenfeld, H. M. ( 1961). Nature 192,366. Tauc, L., and Gershenfeld, H. M. (1962). J. Neurophysiol. 25, 236. Vogt, M. (1954). J . Physiol. (London) 123,451. Weight, F. F., and Salmoiraghi, G. C. (1966a). J. Pharmacol. Exptl. Therap. 1% 391. Weight, F. F., and Salmoiraghi, G. C. (1966b). I. P h u m c o l . Exptl. Therap. 153, 420. Werman, R., Davidoff, R. A., and Aprison, M. H. (1967). Nature 214, 681. Whittaker, V . P. (1964). Progr. Brain Res. 8, 90. Yamamoto, C. (1967). J. Pharmacol. Exptl. Therap. 156, 39.

EXOPEPTIDASES OF THE NERVOUS SYSTEM By Neville Marks N e w York State Research Institute for Neurochemistry and Drug Addiction. Word's Islond. N e w York. N e w York

I. Scope of Review and Introduction . . . . . . Comment on the Classification of Exopeptidases . . . I1. a-Aminopeptide Amino Acid Hydrolases (E.C.3.4.1) . . A . Leucine Aminopeptidase ( LAP) (E.C.3.4.1.1) . . . B. Aminotripeptidase ( E.C.3.4.1.3 ) . . . . . . I11. Dipeptide Hydrolases (E.C.3.4.3) . . . . . . A. Glycyl-Glycine Dipeptidase (E.C.3.4.3.1) . . . . B. Carnosinase, Anserinase, and Cysteinyl-Glycine Dipeptidases . . . . . . . . . (E.C.3.4.3.3-5) C . Imido- and Iminodipeptidase ( E.C.3.4.3.&7) . . . D . e-Peptidases . . . . . . . . . . . . . E . Distribution in Brain Subcellular Fractions IV . Arylamide Amino Acid Hydrolases . . . . . . A . Arylamidase A . . . . . . . . . B . Arylamidase B . . . . . . . . . C . Arylamidase N . . . . . . . . . V . a-Carboxypeptide Amino Acid Hydrolases (E.C.3.4.2) . . A . Carboxypeptidase A (E.C.3.4.2.1) . . . . . B . Carboxypeptidase B (E.C.3.4.2.2) . . . . . VI . Exopeptidases in the Different Areas of the CNS . . . A . Pituitary . . . . . . . . . . . B. Hypothalamus . . . . . . . . . C. Pineal Gland . . . . . . . . . . D . Cerebral Spinal Fluid . . . . . . . . E . Spinal Cord . . . . . . . . . . . . . . . . . . . VII . Peripheral Nerve . VIII . Conclusions . . . . . . . . . . . A . Exopeptidases and Hormone Activity . . . . . B. Exopeptidases and Disease Processes . . . . . . . . . C . Exopeptidases and Transport Processes D. Exopeptidases and Protein Turnover . . . . . References . . . . . . . . . . .

.

. .

. .

. .

. .

.

. .

. . .

.

.

. . .

. .

. .

.

.

. . . .

.

57

58 61 61 63 64 65

66 67 68 68 69 70 71 72 73 74 76

77 77 79 81 81 82 83 85 85 85 86 88 90

.

I Scope of Review and Introduction

It is now generally accepted that the major portion of proteins of the nervous system is metabolically active . Protein turnover 57

58

NEVILLE MARKS

involves synthesis and breakdown, both proceeding concurrently. There have been many outstanding advances in our knowledge of the mechanism for protein synthesis, but only limited information is available on the mechanisms concerned with protein catabolism. Proteolytic enzymes are the chief agents responsible for the degradation of protein and these are widely distributed in all animal tissues. Based on the classic work of Bergmann (1942), proteolytic enzymes are divided into two classes: the endopeptidases or proteinases that hydrolyze peptide bonds regardless of their location within the protein or polypeptide molecule, and exopeptidases that act terminally on structures with free amino acid or carboxy end groups. Some recent reviews that discussed the occurrence and function of cerebral endopeptidases are those of Lajtha (1961, 1964a), Waelsch and Lajtha ( 1961), and Marks and Lajtha ( 1969). Consequently, the present account is limited to brain exopeptidases, a subject never previously reviewed. Indeed, it was commented on by some early investigators, E. Abderhalden and Ceaser ( 1940), and Hanson and Tendis ( 1954), that only scanty data existed in this area. Based on the literature survey for the present review, these observations are still valid. Presumably, progress in the brain parallels that in other tissues where considerable dif6culty has been experienced in the isolation and characterization of most exopeptidases (for the early literature, see reviews of E. L. Smith, 1955, 1960; Hanson, 1963, 1966). With the exception of the endocrine glands, closely linked with brain function, there are no large stores of free peptides other than glutathione in the brain. With the development of new techniques, the existence of other peptide compounds present in trace quantities have come to light, and scope for the existence of others undoubtedly exists. It is not clear whether the peptides reported arise simply as artifacts of extraction or represent intermediates in the degradation of protein. Some peptides such as glutathione may arise from synthetic mechanisms unrelated to those of protein synthesis. COMMENT ON THE CLASSIFICATION OF EXOPEPTIDASES For the purpose of this review, it is proposed to adhere to the conventional groupings now adopted by the recently instituted Enzyme Commission (see International Union of Biochemistry report in Dixon and Webb, 1964; Florkin and Stolz, 1964). Exopeptidases that have been studied in the brain are emphasized but

59

EXOPEPTIDASES OF THE NERVOUS SYSTEM

TABLE I CLASSIFICATION O F EXOPEPTIDASES (E.C.3.4) E.C. List,ing 3.4.1

3.4.1.1 3.4.1.2 3.4.1.3 3.4.2 3.4.2.1 3.4.2.2 3.4.3 3.4.3.1 3.4.3.2 3.4.3.3 3.4.3.4

3.4.3.5

3.4.3.6 3.4.3.7

Systematic name

Trivial name

a-Aminopeptide amino acid hydro 1ase Leucine-aminopeptidase iZminopeptidase Aminotripeptidase a-Carboxypeptide amino acid hydrolase Carboxypep tidase A Carboxypeptidase B Dipeptide hydrolaaes GIycyl-gly cine dipeptidase Glycyl-leucine dipeptidase AminoacylCarnosinase histidine hydrolase Aminoacyl-1Anserinase methyl histidine hydrolase Cysteinylglycine dipep t idase Iminodipeptidase Imitlodipeptidase (prolidase)

Typical substrate (metal requirement) -

Leu-NHz

(M++)

Leu-Gly-Gly

2-GI y-Phe

(Zn++)

Hippurylarginine

(Zn++)

Gly-Gly

Go++)

GIy-Leu

p - Ala-His

W++)

p-Ma-m ethy1 histidine

(Zn++)

CYS-GIY

(Mn++)

Pro-Gly

(M++)

Gly-Pro

(Mn++)

some description is accorded to enzymes present in other tissues that may be important to the function of the central nervous system (CNS). Exopeptidases are classified on the basis of those that require substrates with free amino groups ( aminopeptidases ) or

60

NEVILLE MARKS

carboxyl groups (carboxypeptidases) or those that are specific for dipeptide substrates with both terminal groups free ( dipeptidase hydrolases) (Table I). It must be emphasized that simple schemes of this nature can be misleading since many exopeptidases (especially aminopeptidases ) are unavailable in a satisfactory state of purity and frequently exhibit a broader range of specificity than implied by the classification. The use of more than one synthetic substrate and the consideration of other criteria are often essential for the differentiation of exopeptidases. Some confusion has existed in the past due to the practice of naming exopeptidases on the basis of unspecific polypeptide and protein substrates. To cite just a few examples still current in the literature: oxytocinase, protaminase (now named carboxypeptidase B), and glutathionase. These terms will be avoided in keeping with the recommendations of the Enzyme Commission. As noted by the commission, exopeptidases other than those listed undoubtedly exist but the allotment of system numbers must await further characterization of the enzymes involved. Since the completion of this report there seems to be good justification for the inclusion of enzymes that hydiolyze amino-acylated naphthylamines since these are distinct in their properties from the classic aminopeptidases. These are referred to by the trivial name of arylamidases and are described in the present review under the heading “arylamide amino acid hydrolases.” The introduction of synthetic substrates by Bergmann and associates (see review, 1942) facilitated the characterization of new exo- and endopeptidases. Nevertheless, many puzzling questions remain to be resolved concerning their role and the physiological substrates within the cell. The simple hydrolysis of naturally occurring proteins and polypeptides in oitro, especially by exopeptidases with broad specificity, may not correspond with the situation within the cell. Several factors should be considered as important to physiological activity; activation of possible zymogen precursor forms, multiple forms of the enzyme, accessibility of the substrate within the cell, the effect of cofactors on the mechanisms of hydrolysis. An attempt is made in the present review to assess previous and current work in relation to some of these unresolved problems. Special consideration is given to the different brain areas (pituitary, pineal gland, hypothalamus) that contain large amounts of physiologically active peptides. There is also some description accorded to the peptidase activities in the peripheral nerve and in the cerebrospinal fluid ( CSF) .

EXOPEPTIDASES OF TIIE NERVOUS SYSTEM

61

As seen in Table I, exopeptidases are classified by means of specific peptide substrates. In cases where the substrate is unequivocally known the chemical structure is quoted in the text. Due to the broad specificity of exopeptidases, the hydrolysis of specific substrates by crude extracts is only an indication of the probable presence of a specific exopeptidase group. To prevent unnecessary duplication all substrates quoted are of the L-configuration unless otherwise indicated. II. a-Arninopeptide Amino Acid Hydrolases (E.C.3.4.1 )

The peptidases comprising this group are summarized in Table I. These enzymes release N-terminal amino acids from suitable peptide substrates. A good deal of the earlier work with this group of enzymes was done with crude tissue extracts; this work is difficult to interpret and does not receive detailed description. A. LEUCINEAMINOPEPTIDASE(LAP) (E.C.3.4.1.1) The ability of this enzyme to hydrolyze a vast number of substrates including polypeptides and proteins has attracted interest as a potential tool for analysis of protein structures (Hill, 1965). This property, the hydrolysis of proteins, may be involved in brain protein turnover but studies with brain as a source of enzyme are practically nonexistent. Presumably, progress is related to the difficulties experienced in the purification, enzyme stability, and the apparent enzyme multiplicity shown by LAP from other sources (Patterson et al., 1963, 1965). In most respects the problems related to LAP are representative of the entire group of aminopeptidases; since information of the properties of aminopeptidases is not well represented in the literature, some attention is given to LAP below. Linderstrgm-Lang ( 1929) reported the presence in erepsin preparations of an enzyme that hydrolyzes the substrates m-LeuGly, and DL-Len-Gly-Gly. Much effort has been expended since that time on enzymes that hydrolyze leucine-containing peptides (see E. L. Smith and Hill, 1960; Hanson, 1966). The substrates most specific for LAP activity are the amino-acyl substituted amides, in particular Leu-, Norleu, Norval-NH, (E. L. Smith and Spackmann, 1955). Purified LAP preparations show a preference for substrates with a hydrophobic side chain; all amino acids in peptide linkage are susceptible to LAP hydrolysis, although with some the reaction rates are very slow, notably proline and cysteine. Thcre has been

62

NEVILLE MARKS

some doubt as to the purity of LAP preparations prepared in the laboratory or obtained from commercial sources. Frater et al. (1965) warned that LAP should be used with caution for the sequence determination of protein structures since most preparations contain prolidase and endopeptidase contaminants. It was reported by Spector and Mechanic (1963) in the case of purified bovine lens LAP that the cleavage of insulin A and B chains was not accompanied by any detectable endopeptidase activities. LAP, like several other exopeptidases, is a metal-dependent enzyme requiring Mn++or Mg++for its activation. There is insu5cient evidence at present to decide whether LAP is a metalloenzyme or if the metal is required as a cofactor for the formation of the enzyme-substrate complex. One property of particular interest is the esterolytic activity of LAP, equivalent on a molar basis to 10%of the peptide bond hydrolase activity (Fittkau et al., 1961; WoM and Resnick, 1963; Spector and Mechanic, 1963). A number of proteolytic enzymes exhibit both peptidase and esterase activities but the functional significance of this dual role is unknown. The esterolytic function exhibited in vitro may be limited within the cell by the factors that affect intracellular enzyme activities, as discussed in the introduction. Many early studies reported that brain extracts hydrolyzed leucine-containing peptides ( E. Abderhalden and Ceaser, 1940; Kies and Schwimmer, 1942; Hanson and Tendis, 1954; Uzman et al., 1961, 1962). Due to the overlap of specificities of aminopeptidases, these data are not conclusive for the presence of LAP in the brain. In an attempt to characterize this enzyme, Patterson et al. (1965) determined the ratio of activities for liver LAP with LeuGly, Leu-Gly-Gly, and Leu-NH, as substrates. The ratio for these activities evidently varies with LAP from different tissues: the ratio for liver was 0.85:1.2:1.0 compared with 2.5:2.0:1.0 for muscle LAP (Joseph and Sanders, 1966). Other differences between LAP from different sources have been amply documented (E. L. Smith, 1960; Bryce and Rabin, 1964a,b; Hanson, 1963, 1966). Comparable studies with purified brain enzymes that hydrolyze specific LAP substrates have not been undertaken. Brecher (1963) reported that hydrolysis of Leu-NH, in crude brain mitochondria1 and microsomal fractions which was activated by Mn++and to a With Tyr- or Phe-NH, as the sublesser extent by Mg++and CO++. strates, the activity was higher in the postmicrosomal supernatant


63

fractions with activation by Mn'+ and Cot+.I n our own studies we observed only low activities in the presence of Leu-NH, in brain extracts but very high activities with Leu-Gly, and Leu-Gly-Gly. The ratio of the three substrates in supernatant fractions in the same order as that considered for muscle and liver was 2.0:0.6:0.05 (Datta et al., 1968a). B. AMINOTFUPEPTIDASE (E.C.3.4.1.3)

Although this enzyme has not yet been obtained in purified form, much is known concerning its specificity (E. L. Smith, 1955). This enzyme is widely distributed in animal tissues. The tripeptidase is specific for tripeptides containing neutral amino acids, splitting off the N-terminal residue, It is readily distinguished from LAP and many other exopeptidases; there appears to be no metal-ion requirement, nor does it possess a thiol group essential for activity. The tripeptidase can hydrolyze a wide variety of tripeptides, but not typical dipeptides, tetrapeptides, or arylamide substrates. With tripeptides, the point of hydrolysis is the bond adjacent to the free amino group which must be a and of the L-configuration; the carboxy terminal residue can be /? or of the D-configuration. Hydrolysis does occur in some unusual dipeptides where the distance between the free NH, and the -COOH groups approximates the distance found in tripeptides, as is the case for glycyl-S-aminovalerate, and glycyl-p-aminobenzoate (Davis and Smith, 1955). It is reported that tripeptides are inhibited by Cd++ and by a large number of drugs including local anesthetics (D. Ellis and Fruton, 1951; Ziff and Smith, 1952). In recent work, we have shown the presence in the brain of tripeptidases with marked specificity for Leu-Gly-Gly and AlaGly-Gly, but no activity with triglycine ( Marks, 1967). Unlike tripeptidases described for other tissues, the brain enzyme also could hydrolyze tripeptides containing lysine as was the case with trilysine or to a lesser extent with Lys-Gly-Gly. The enzyme is readily separated from other brain exopeptidases by elution from DEAE-cellulose columns with a low concentration of NaCl (Fig. 1).Purified brain tripeptidase is not metal dependent and is not inhibited by puromycin (see arylamidases, Section IV) . Enzymes that hydrolyze tripeptides are localized chiefly in the soluble supernatant fractions (55%,Table 11);the activity associated with crude

64

NEVILLE MARKS

mitochondria1 fractions (10%)is localized in the synaptosome subfractions (‘Table 111) (Datta et al., 1968c; Marks et af., 1968a). I l l . Dipeptide Hydrolases (E.C.3.4.3)

The early surveys for dipeptidase activity relied exclusively on the measurement of activity in crude dispersions with a variety of

Fraction number

FIG.1. Distribution pattern of some exopeptidases from rat brain extracts after passage through DEAE-cellulose at pH 7.6. Tris-HC1 buffer, pH 7.6, containing 1 mM dithiothreitol was employed. Peaks were eluted with a linear gradient of NaCI: I1 represents brain aminotripeptidase; I11 represents arylamidase B with Arg-P-NA as the substrate; IV represents a mixed arylamidase activity with the different substituted arylamides indicated. (From Marks et aZ., 1968a,b. )

dipeptides; for the reasons already enumerated, these studies are only briefly reviewed. An additional difficulty in making comparisons is the lack of attention paid to the possible cofactor requirements, especially the effects of metal ions. Blum et d.(1936) were the first to observe “dipeptidase” activities in brain extracts; E. Abderhalden and Ceaser (1940) observed the hydrolysis of a series of dipeptides with the highest activity in the gray compared to the white matter. Kies and Schwimmer

65


TABLE I1 DISTRIBUTION OF AMINOPEPTIDASE, CARBOXYPEPTIDASE, AND ARYLAMIDASE I N BRAINSUBCELLULAR FRACTIONS~*~ Relative enzyme activities" Substrate Aminopeptidase Leu-Gly Cly-Gly Leu-Gly-Gly Leu-Leu-Leu Carboxypep tidase Z-Leu-Tyr Arylamidase a-Asp-p-NA y-Glu-&NA Lys-P-NA Arg-8-N A Leu-8-N A Ma-p-N A Met-@-NA Gly-p-NA Phe-8-NA Ser-Tyr-6-N A

€I

Mt

, I :

MG

Supt,.

100 18

7 11

61 1

100 30

10

55 25

5

5

2

2

0

1

3 3 83 70

0 0 5

0 1 10

1

3 3

11

50

77 63 16 13 15

5 4

46 15

0 0 6 4 7 12 6

1 1

5

2

3

1

1

1 1

6

1

37 35 26 21 20 16 3 5

From Marks et al. (196%) and Datta et al. (1968a). Values are relative to activity in the homogenate (H) with Leu-Gly as substrate. Assays were done a t pH 7.6 with 2 mM peptide or 0.5 mM arylamide and incubated 30 minutes at 37°C. = Key: H, homogenate; N, nriclcr; Mt mitochondria; Mc, microsomes; Supt ., superns t an t . a

~

(1942) reported higher dipeptidase activity in calf brain compared with muscle extracts. In a comparison of many body tissues, Price et al. (1947) also reported higher activities in the brain compared to muscle extracts but lower activity when compared to the spleen, kidney, liver, and lung with Gly-Ala as the substrate. A. GLYCYL-GLYCINE DIPEPTIDASE ( E.C.3.4.3.1)

Van den Noort and Uzman (1961) considered that a separate enzyme in the brain was responsible for the hydrolysis of Gly-Gly. This enzyme has not been fully characterized in the brain but can be distinguished from other dipeptidases by its sensitivity to activation by Co++. Specific Co++activated diglycinases are known to

66

NEVILLE MARKS

occur in the calf thymus (Fruton et al., 1948; D. Ellis and Fruton, 1951). In the cleavage of higher peptides containing diglycine, the release of some constituent amino acids can inhibit glycyl-glycine dipeptidase (Uzman et al., 1963). This may represent an interesting control mechanism for the release of glycine, an amino acid that recently was shown to be an “inhibitory” compound in neuronal function (Werman et al., 1967; Davidoff et al., 1967). In our own studies, the activity with Gly-Gly was 18%of that with Leu-Gly as the substrate. The highest activity was in the mitochondria1 preparations (11%) with lesser amounts in the nuclear, microsomal, and supernatant fractions (Table 11). In all fractions the activity was markedly increased by low concentrations of Co++.

B. CARNOSINASE, ANSERINASE, AND CYSTEINYL-GLYCINE DIPEPTIDASES ( E.C.3.4.3.3-5) The development of new techniques has led to the identification in the brain of a number of new dipeptides. In particular, the typical muscle components, carnosine ( p-alanyl-histidine) and anserine (P-alanyl-methyl histidine) have been reported as present in the TABLE I11 DISTFLIBUT~ON OF ENZYMXSIN MYELIN,NERVEENDING, AND MITOCHONDRIAL SUBFRACTIONS~*~ Percent recovery in the subfractions

Substrate Aminopeptidase Leu-Gly-Gly Leu-Leu-Leu Carboxypeptidase Z-Gly-Phe Z-Leu-T yr Arylamidase Leu-P-NA Ala-p-N A Arg-j3-N A

+

(Pz)

Myelin A

Synaptosomes BCD

Mt lysosomes E

100 75

21 9

42 24

10

11 6

4 2

7

100 50 98

6 6 7

42 22 57

Mt

3

6

1 1

3 3 3

a From Marks et al. (1968b) and Datta et al. (1968a). bCrude mitochondria (P2) and subfractions prepared by the methods of Marks and Lajtha (1963). Values used for comparison are in italics. See Table I1 for other details.

EXOPEI'TIDASES OF THE NERVOUS SYSTEM

67

brain (Hosein and Smart, 1960). There is also evidence for the trace quantities in the human brain of homocarnosine (y-aminobutryl histidine) (Pisano et al., 1961; Abraham et al., 1962). Specific dipeptidases that hydrolyze carnosine and anserine are found in the liver and the kidney (Meister, 1965a) but their presence in the brain has not been investigated. It is noteworthy, that the utilization of carnosine for growth in animals has been attributed to the prior enzymatic hydrolysis to form histidine (du Vigneaud et al., 1937). A dipeptidase specific for cysteinyl-glycine is present in rat and swine kidney ribosomes (Brinkley, 1961). Its presence in the brain has not been investigated; this enzyme may have importance because of the high concentration of glutathione; a peptide containing the cysteinyl-glycine residue. C. IMIDOAND IMINODIPEPTIDASE ( E.C.3.4.3.6-7) Despite the relatively high concentration of proline in the brain (0.3 compared to 0.73 pmoles/gm protein for glutamate) (Lajtha and Toth, 1968) the enzymes involved in proline metabolism have not been studied in any detail. Enzymes that are specific for proline containing dipeptides have been obtained in purified form from other tissues, from erythrocytes (E. S. Adams and Smith, 1952), and from the intestinal mucosa and the kidney (Davis and Smith, 1953; E. L. Smith, 1960). The enzyme specific for dipeptides with a free a-imino end group is termed iminodipeptidase (E.C.3.4.3.6); in dipeptides with the proline bound to the imino group the term imidodipeptidase or prolidase is employed (E.C. 3.4.3.7). The hydrolysis of Pro-Gly in the brain was observed by Hanson and Tendis ( 1954) and of Gly-Pro by Uzman et al. (1961) . Imino- and imidodipeptidases are Mn++-dependentand are specific only for proline and hydroxyproline in the form of a dipeptide linkage. They can be distinguished from other peptidases that are specific for N-terminal proline residues in proteins (Sarid et al., 1962). This enzyme has been termed iminopeptidase and is believed to be involved in collagen metabolism. There are only trace quantities of collagen in the brain and this is probably derived from non-neuronal elements (Lowery et al., 1941). Consequently, there are no detailed studies of collagen metabolism in the brain although proteins containing hydroxyproline are present in the plasma of man and animals (Kaplan et al., 1964). It has been sug-

68

NEVILLE MARKS

gested that the hydroxylation occurs subsequent to the incorporation of proline from polypeptide precursors (Hutton et al., 1967).

D. 6-PEPTIDASES The hydrolysis of dipeptides coupled to r-amino group of lysine has been reported in extracts of rat and hog kidney (Padayatty and von Kley, 1966). It has been proposed that tissues contain specific 6-peptidases; this enzyme remains tentative until confirmed by characterization studies on purified preparations. Peptide bonds with an r-amino group occur in bovine growth hormone (Li, 1957) and in collagen (Mechanic and Levy, 1959). Substrates for this category of enzyme could arise from the degradation of basic proteins; acid extracts of pig brain, bovine spinal cord, and bovine brain white matter contain relatively high concentrations of lysine (Nakoa et al., 1966; Tomasi and Kornguth, 1967). Some of the basic proteins and peptides are involved in the induction of experimental allergic encephalitis (Einstein et at., 1968). E. DISTRIBTJTIONIN BRAINSUBCELLULAR FRACTIONS The subcellular localization of many enzymes has supplied a guide to the possible functional roles within the cell. For example, it is well known that many degradative enzymes are present in lysosomal organelles; in particular the hydrolases, acid proteinase (cathepsin), and acid phosphatase (De Duve d al., 1962). The status of lysosome organelles in brain dispersions is not clear; they have been identified on a morphological and histochemical basis but have never been isolated by a satisfactory biochemical procedure (Beaufay et al., 1957; Pearse and Wachtler, 1968). In studies in our laboratory, fractions containing relatively high concentration of acidic proteinases were found to be associated with subfractions of crude brain mitochondria (Marks and Lajtha, 1963). Hanson and Tendis (1954) showed that the supernatant fractions contained the highest dipeptidase activity compared with the crude nuclear and mitochondria1 fractions. A similar distribution pattern was observed by Brecher (1963) for rat brain fractions with also some trace activities with dipeptide substrates containing D-amino acids. In OUT own studies, some 60% of all dipeptidase activity appeared in the postmicrosomal fraction, with 20%in the nuclear mitochondrial and microsomal fractions (Table 11). It is evident that this category of exopeptidases is not located exclusively in lysosomal

EXOPEI’TIDASES

OF *IHE NERVOUS SYSTEhl

69

particles but is present in varying amounts in all cellular fragments. There have been many studies on dipeptidase distribution in other tissues, in view of the difficulties in the interpretation of these results, it is not within the scope of this review to comment on these in any detail (Hanson and Blech, 1959; De Duve et d., 1962). Microchemical and Anatomical Studies There have been several investigations concerned with anatomical variation of dipeptidase in rat somatosensory cortex employing the quantitative microchemical procedures of Linderstr6mLang (1939) and Holler (1952). With DL-Ah-Gly as substrate the highest concentration was observed in the intralaminer layers 11, IV, Vb, and Vlb of rat and I, II-VI of man (Pope and Anfinsen, 1948; Pope, 1952, 1959). Activity in the human frontal isocortex was three times that in rat. Since these layers are relatively rich in nerve cell bodies, it was suggested that neuronal perikarya are the principal intracortical sites of dipeptidase activity. Based on the author’s data, the activity in white matter equals that of the cortex, if expressed in terms of dry weight, this would indicate that dipeptidase activity is present in cytoplasmic expansions of both neurons and glia cells ( Friede, 1966). A similar microchemical technique was employed by the late Dr. Uzman and his group in studies of the specific requirements of a large variety of glycyl substituted dipeptides ( Uzman et al., 1961, 1962). In glycerolphosphate extracts of histological slices, activity was favored with substrates having a lipophilic side chain and an aliphatic or aromatic C-terminal amino acid. Racemic peptides were equally active compared with those of L-configuration but studies on absolute stereospecificity requirements were incomplete. IV. Arylamide Amino Acid Hydrolases

Gomori ( 1954) first introduced chromogenic substrates for the histological detection of aminopeptidases. The hydrolysis by peptidases released P-naphthylamine (coupled to amino acids in the C-terminal position ) which formed azo dyes with diazonium compounds (see Burstone, 1962). At first, it was believed that these substrates were hydrolyzed by typical aminopeptidases such as LAP. It was shown by Patterson ct 01. (1963, 1965) that LAP differed in many of its most important properties from enzylnes hydrolyzing arylnmides. Arylamidcs differ from the normal amino-

70

NEVILLE MARKS

peptidase substrates in having a -CH2grouping between the peptide bond and the aromatic nucleus. No official E.C. listing is available for this particular group of hydrolases, but the trivial name “arylamidases” has been proposed (Patterson et al., 1963; E. E. Smith and Rutenburg, 1966). Substrates other than the substituted naphthylamides have been employed: nitroanilides ( Erlanger et al., 1961; Tuppy et al., 1962; S. Ellis, 1963), and aminonitriles (Szewczuk et al., 1965). Most tissues were found to contain relatively high concentrations of enzymes that hydrolyzed these substrates. Many of these enzymes were shown to possess different substrate specificities that were conferred by the substituted amino acid moiety. The classification based on the terminal amino acid must be regarded as tentative and refers only to the monosubstituted arylamide analogs. There is some evidence to suggest that enzymes hydrolyzing the dipeptidyl arylamide analogs belong to a different category of enzymes (S. Ellis and Perry, 1966; S. Ellis and Nuenke, 1967). A. ARYLAMIDASE A

There are a number of reports for the presence in tissues of enzymes specific for arylamidases containing acidic amino acids (Glenner and Folk, 1961; Glenner et al., 1962; Nagatsu et al., 1965; Nagatsu and Haru, 1967). Kidney microsomal extracts and sera contain such enzymes, activated by Ca++ ions. The most active substrates appear to be a-Glu- and a-Asp-P-NA. Arylamidases with specific acid functions are of special interest to brain in view of the high concentration of glutamate, aspartate, and acetylaspartate in the nervous system (Tallan et al., 1954, 1958). In rat brain, for example, the level of free glutamate, 12 pmoles/gm, exceeds the concentration of leucine by some 200-fold. The possibility exists that the high acidic amino pool in brain is primed by the degradation of brain proteins and polypeptides by specific enzymes. Recent studies from our laboratory have shown the presence of arylamidase A activity in brain homogenates and in some subcellular fractions. The activity observed with a-Gl~i,a,P-Asp-P-NA was less than 5%of that observed with neutral and basic analogs (Table 11). This activity was activated by Ca++,but unlike other arylamidases, this activity was not easily solubilized with hypotonic buffer or detergent treatments. The highest activity was associated with the mitochondrial fractions. Detailed study of this interesting class of arylamiclases must await the availahility of purified preparations.


71

Metabolism of y-Glutamyl Peptides Prior to the recent advancement in the knowledge of the mechanisms of protein synthesis, the view was held that proteolytic enzymes played an important role in peptide synthesis (Fruton et al., 1953; Wurtz et al., 1962). The pathways considered involved transpeptidation or transamidation such as the transfer of the y-glutamyl moiety to peptide or protein acceptors. Peptides containing y-glutamate occur in high concentration in the brain; glutathione, for example, is present at a level of 3-4 pmoles/gm in the rat, which represents one third of the total nonprotein extractable nitrogen (McIlwain and Trezize, 1957). More recently traces of y-Glu-Glu, 7-Glu-Gly, and ~-GIu-G~u-NH, were reported at a level of about 7 pg/gm (Kakimoto et aL, 1964; Kanazawa et al., 1965). In our own studies we observed some activity with y-Glu-D-NA which may represent transpeptidation rather than arylamidase A activity (Table 11). 7-Glutamyl transpeptidases occur in a number of tissues including that of the brain (Hanes ,et al., 1950; Fodor et al., 1953; Glenner and Folk, 1961; Albert et al., 1966). In the brain, the transpeptidase is some 100-fold lower than in the kidney (Orlowski and Meister, 1963, 1965). It is noteworthy that the histochemical location of y-glutamyl transpeptidase is distinct from that of arylamidase A (Glenner and Folk, 1961; Albert et al., 1966). There has been some confusion regarding the role of other enzymes involved in the metabolism of y-glutamyl peptides. One such enzyme, y-glutamyl transferase, appears to represent a reversal of the glutamine synthetic pathway with specific binding sites for glutamate, adenosine triphosphate ( ATP), NH, (Berl, 1966; Lajtha, 1966). This enzyme is quite distinct from transglutaminase that mediates the transfer of the protein or peptide bound 7-glutamyl moiety to a wide range of amine acceptors including ammonia, serotonin, histamine, cadaverine, and insulin ( Waelsch, 1962) .

B. ARYLAMIDASEB Good evidence for this group of enzymes was supplied by Hopsu and co-workers (1966a,b). They have isolated an enzyme from rat liver in a high state of purity that is specific only for basic substituted arylamides. Previously, Nachlas et af. ( 1962), in an evaluation of the arylamidases in different tissues, reported the hydrolysis of Arg-p-NA in brain homogenates. Recently, S. Ellis and his associates have been able partially to purify enzymes from the

72

NEVILLE MARKS

pituitary gland that were specific for basic substituted arylamides (see Section VI). In terms of the anatomical location of this enzyme, there have been very few studies; in the liver Mahadevan and Tappel (1967) showed that enzymes hydrolyzing Arg-P-NA were largely associated with the lysosomes. These authors suggested that the activity attributed to soluble fractions by other workers may have occurred by leakage from lysosomes. In our own work we have shown that the distribution in brain is Werent from that of the liver. The highest activity was associated with the sohMe supernatant fraction (37%),followed in descending order by crude mitochondria ( lo%),microsomes ( 6%), and nuclei (4%) (Table I1 ). Further fractionation of crude mitochondria revealed a high association of the arylamidase B activities in vesicle and synaptic structures (6%)rather than in the myelin (1%), or in the fractions which are considered to contain true mitochondria and lysosomes (Table 111) (Marks and Lajtha, 1963; Marks et al., 1967; Datta et al., 1967a,c). Unlike the purified liver enzyme, the brain arylamidase B activity was not halide dependent and was strongly activated by metal ions. Brain enzymes were stabilized by the sulfhydryl reagent, dithiothreitol, and activated by cysteine and P-mercaptoethanol. One of the most outstanding features of the brain enzyme was the strong inhibition by very low concentrations of puromycin ( K , , 2 x lo4). This inhibitory effect is of interest in terms of the possible behavioral effects known to occur on the direct injection of this antibiotic into the brain (Flexner et al., 1964). Arylamidases are the only enzymes known to be inhibited by puromycin (E. Ellis, 1963; Behal et al., 1966). This inhibitory property of puromycin can be used to differentiate arylamidases from other exopeptidases (Marks et a!., 1967; 1968a).

C. ARYLAMIDASE N Early studies with neutral substituted analogs were done in relation to possible LAP activity (see Section II,A), Since there is good evidence for separate enzymes hydrolyzing acidic and basic analogs it has been concluded that a separate category is responsible for the hydrolysis of the neutral substrates. C. W. M. Adams and Glenner (1962) reported the hydrolysis of Leu-P-NA in extracts of corpus callosum and frontal gray matter of adult and newborn rats. The hydrolysis of Leu-P-NA in other body tissues has been amply documented (Burstone, 1962; Nachlas et al., 1962;

EXOFEPTIDASES OF THE NERVOUS SYSTEM

73

E. E. Smith and Rutenburg, 1966; E. E. Smith et al., 1965; Behal et al., 1963, 1964, 1965; Wachsmuth et al., 1966a,b). In liver particulates, the distribution is similar to that reported for arylamidase B, with activity present in the lysosomal fractions (Mahadevan and Tappel, 1967). Arylamidase N activity was reported also in the microsomal and cytoplasmic fractions of the liver and kidney with some alteration in malignancy (Patterson et d.,1963; Pfeiderer et al., 1964; Sylven and Bois, 1964; Sylven and Lippi, 1965; Felgenhauer and Glenner, 1966). In the brain, the highest activity was associated with the supernatant fractions ( Leu-/?-NA, 43%), followed in descending order by mitochondria (22%),microsomes ( 13%) and nuclei (6%) (Table TI). In further subfractionation studies of mitochondria, the activity was shown in highest concentration in the synaptosome fractions (401%) rather than the other subfractions (Table 111). Attempts to purify arylamidase N activity from a variety of tissues have not met with the same degree of success as that for arylamidase B. In part, this difficulty may have arisen due to the presence in tissue of several distinct chromatographic and electrophoretic forms. These multiple forms have been observed in extracts of sera, spleen, pancreas, lymph node, small and large intestine (E. E. Smith and Rutenburg, 1966; Behal et al., 1965; Rybak et al., 1967). Arylamidase N activity has been partially purified from brain and was shown similar to that of other tissues (Marks et al., 1968a,b). Like the pituitary and liver enzymes, the brain arylamidase N activity required sulfhydryl compounds for maximum stability; the activity was inhihited by EDTA and reactivated by the addition of metal ions. The brain enzyme activity was the inhibition was strongly inhibited by puromycin ( K , , 1 x competitive and similar to that reported for the brain arylamidase B activity in the previous section (Table 111). V. a-Carboxypeptide Amino Acid Hydrolases ( E.C.3.4.2 )

The teim carboxypeptidase denotes an enzyme catalyzing the hydrolysis of -COOH terminnl end groups in proteins or peptides. Carboxypeptidases are of particular interest since the characterization of highly purified crystallinc forms has been possible. As such they have proved to be useful tools in the determination of protein structures. Carhoxypeptidases occur in high concentration in the pancreatic secretions which are the major source for the puri-

74

NEVILLE MARKS

fied enzymes. They have not been studied in any great detail in the nervous system. In the pancreas, most proteolytic enzymes occur in the inactive form in zymogen granules that give rise to a mixture of exo- and endopeptidases on activation. These granules appear to act as intracellular storage sites for newly synthesized enzyme (Keller and Cohen, 1961; Greene et al., 1963). The inactive forms of carboxypeptidases ( procarboxypeptidase A and B ) can be activated by endopeptidases. Activation of zymogens containing exopeptidases by endopeptidases may be an important mechanism in the regulation of peptide turnover within the cell. Precise information on the substrate specificity of carboxypeptidases has come from the study of highly purified crystallized forms ( Neurath, 1960). Two major groups of carboxypeptidases are recognized: carboxypeptidase A that hydrolyzes all peptides except those with a C-terminal proline and basic amino acids, and carboxypeptidase B that is specific for peptides with a C-terminal basic amino acid. Carboxypeptidases are metalloenzymes normally requiring Zn++ for activity. The synthetic substrates introduced by Bergmann (1942) serve as the chief means for the identification of carboxypeptidases. These peptides generally contain a carbobenzoxy ( abbr. Z ) -protected N-terminal residue. Other substrates for carboxypeptidase include N-halogen acylamino acids, and certain ester analogs of specific peptides ( Neurath, 1960). Enzymes hydrolyzing acylamino acids or peptides probably belong to the general class of carboxypeptidases although some workers consider them as a separate group of enzymes termed “acylases” (Hanson, 1966). Acylases may be involved in the metabolism of acylated amino acids such as N-acetylaspartate and its related compounds. Olson et al. (1967) recently reported traces of the dipeptide N-acetyl-aspartyl-glutamate in tissues that appeared to be a good substrate for kidney acylase enzymes liberating both glutamate and aspartate. Since there are no reported studies of these enzymes in the brain they are not considered further in this review.

A. CARBOXYPEPTIDASE A (E.C.3.4.2.1) This exopeptidase shows a preferential action on peptides which contain C-terminal aromatic acids, such as Phe, Tyr, or Try, or branched aliphatic amino acids, such as Leu or Ile. In all cases the C-terminal group must be unsubstituted. In addition to the lack of hydrolysis of Arg and Lys residues, carboxypeptidase A is inactive

EXOPEPHDASES 01.’ THE NERVOUS SYSTEM

75

toward proline and hydroxyproline as the terminal or penultirnate amino acid. Under physiological conditions the enzyme is likely to act on polypeptides and proteins; ribonuclease, a-lactalbumin, and proteins; ribonuclease, a-lactalbumin, and lysozyme serve as suitable substrates ( Fraenkel-Conrat et ul., 1955). Extracts of brain hydrolyze Z-Gly-Phe ( Brecher, 1963) but this activity is considcrably smaller than that of other exopeptidases (Datta et al., 1968a). The possibility that the low activity may represent the presence of stable but inactive zymogen forms has not been explored. In our own studies, the highest activity occurred with dipeptides similar to the sequences to be found in insulin B chain; Z-Phe-Phe, Z-ValPhe, Z-Gly-Phe and Z-Gly-Tyr (Marks and Lajtha, 1965; Datta et ul., 1968a). Brain carboxypeptidase A is largely tissue bound, with the highest concentration found in the nuclei and mitochondiia. In subfractions of mitochondria the highest activity occurred in the synnptosome fractions. Comment oft the hlechnni.mz of Action of Curboxypeptklase A

The availability of crystalline preparations in a high state of purity has led to some notable advances in the knowledge of exopeptidase structures and the mechanism of cataIysis (see Vallee, 1967; Neurath, 1960, 1967). Activation of zymogens has long constituted an effective approach to the elucidation of enzyme action, In the case of trypsinogen and chymotrypsinogen, activation occurs by proteolytic cleavage of a unique peptide bond in the amino terminal region of the zymogen. This primary chemical event is believed to result in conformational changes of the enzyme necessary for the catalytic mechanism. Similar investigations of bovine procarboxypeptidase A have been complicated by the existence of two or three tightly bound subunits; one subunit is the direct precursor of carboxypeptidase A, the second is the precursor of a chymotrypsinlike enzyme, the third remains to be identified (Brown et al., 1963). Succinylation results in the disaggregation of the subunits. The active sites appear to involve serine and histidine and are homologous with those of chymotrypsin. Interestingly, the endopeptidase subunit can combine with the active exopeptidase subunit to give a dimeric complex which displays carboxypeptidase activity (Brown et ul., 1963). The complex relationship between these enzymes is not fully understood. On the basis of N-terminal studies several different types of carboxypeptidnse are believed to exist: types A,,

76

NEVILLE MARKS

Ap, A, ( Sampath-Kumar et al., 1964). It appears that, contrary to previous expectations, carboxypeptidase A is not a uniquely defined protein but rather an assembly of allotropic variants. In other species, monomeric forms of the precursor zymogen have been reported, for example, in the Pacific spiny dogfish (Prahl and Neurath, 1966). The dogfish zymogen contains an inherent esterase activity that disappears during the activation process. Carboxypeptidases A and B of the bovine, porcine, dogfish pancreas are so similar in amino acid composition that the high degree of homology in structure suggests a common evolutionary origin. The full delineation of the complex activation process requires a knowledge of the amino acid sequence of the zymogen which is presently unavailable. Procarboxypeptidase A is a metallozymogen with Zn" as the metal bound to an -SH group and a second donor group that is unidentified. Replacement of Zn++in the active enzyme by C d + or Hg++leads to a loss of peptidase hydrolase activity with an increase in esterase activity (Vallee, 1964; Coleman et al., 1966). Similar functional changes occur on acylation with a series of mono- and dicarboxylic acid anhydride, tetranitromethane, photooxidation with methylene blue, hydrogen peroxide, UV-radiation. All these modifications indicate that tyrosyl residues are essential to activity; of the 19 tyrosines in carboxypeptidase A, 2 are essential for activity (Simpson et al., 1963; Simpson and Vallee, 1966). Peptide substrates can form stable apocarboxypeptidase complexes in absence of metals in distinct contrast to the binding of ester substrates (Coleman and Vallee, 1964). Many peptides act as competitive inhibitors and the competition of the inhibitor(s) for the metalbinding site on the enzyme may be significant to the control of enzyme activity within the cell.

R. CARBOXYPEPTIDASE B (E.C.3.4.2.2) A number of peptides with known biological functions form good substrates for carboxypeptidase B activity. Chief among these are the kinins, bradykinin, kalliden, angiotensin, etc. Kinins were first described by Rocha e Silva et al. (1949) and have important physiological properties; they alter cellular permeability and are involved in tissue inflammatory processes (Lewis, 1960; Rocha e Silva, 1963). Kinins are formed from plasma a-globulin; like the typical carboxypeptidase R substrate, hippuryl-L-argininc, they contain a C-terminal basic rcsidue (Habermann, 1963). Krivoy and

EXOWPTIDASES OF THE NERVOUS SYSTEM

77

Kroeger (1964) reported that the inactivation of bradykinin in brain extracts was by means of an enzyme similar in properties to pancreatic carboxypeptidase B. They observed this enzyme in extracts of rabbit, rat, porcine, and pigeon brain acetone powders. This enzyme, like the pancreatic carboxypeptidase B, was inhibited by phenothiazine compounds that also potentiate the activity of bradykinin in uioo. Some indication of the catalytic event was supplied by the formation of an inactive drug-enzyme complex in presence of k i t which did not interfere with the hydrolysis of bradykinin. Other peptidases are also believed to inactivate kinin compounds; these include imidopeptidase, endopeptidases, and carboxypeptidase-like enzymes different in properties from the two major A and B groups (Erdos and Yang, 1967; Erdos, 1966). Because of the unique biological function of kinins there have been a large number of studies in their formation and degradation (Boissanas et nl., 1960; Elliott et d., 1961; Erdos et of., 1964; Schrodcr and Hcmpel, 1964; Greenbaum cf LIZ., 1965). VI. Exopeptidases in the Different Areas of the CNS

The presence of peptides with physiological properties (particularly peptidyl hormones) in many specialized brain areas points to the possibility that exopeptidases may play a special role in thc regulation of many body activities. In most cases the full complement of peptides in the different anatomical locations is unknown. Also, there have been no extensive studies concerning the subcellular distribution, the pathways of biosynthesis, and degradation. A. PITUITARY

The pituitary consists of two regions containing different peptide components; the posterior lobe ( neurohypophysis ) and the anterior lobe (adenohypophysis). In mammals, the gland is recessed in a bony cavity and is not normally removed on excision of the brain. The peptide constituents vary in molecular size from those with 9 amino acids (vasopressin, oxytocin) to those with moi. wt. 2000 or higher in the anterior gland. Examples of the larger hormones are: the melanotropic agents ( ZOOO), ACTH (39 amino acids, mol. wt. 3500), thyroid-stimulating hormone ( lO,OOO), growth hormonr (45,000).Luteinizing and follicle-stimulating hormones are small glycoproteins. This scatter in s i x range indicates that degradation

78

NEVILLE MARKS

probably occurs by the combined effects of both endo- and exopeptidases. Newer methods of analysis have led to the identification of smaller peptides (Ramachandran and Winnich, 1957; Upton et al., 1966; Lande and Lerner, 1967). Since most of the new peptide components are not physiologically active, they may represent intermediates in the biosynthesis or degradation of known hormones. The peptide content of the posterior lobe represents 4% of the dry weight but many of these new peptide components are present in exceedingly small quantities; as an example, Lande et al. (1967) required as many as several million glands to prepare extracts for their identification. Except for the early observations of a Mn++activated dipeptidase in crude porcine extracts (E. S. Adams and Smith, 1951) there have been no extensive studies of amino- and carboxypeptidases in the pituitary (enzyme groups 3.4.13). Recent studies reported below have emphasized arylamide amino acid hydrolases.

Arylamiduses of the Pituitary S. Ellis (1963) has studied the spectrum of peptide hydrolases in the anterior pituitary. Extracts were shown to hydrolyze analogs of p-nitroanilide and of 0-NA in the following order of activity: Lys-, Arg-, Met-, Leu-, Phe-, Ah-. In a comparison between the anterior and posterior lobes, Jouan and Rocaboy (1966) reported higher activities in the posterior lobe homogenate with Ah-, Pheand Leu-, and equal activities in the two lobes with Thr-, Ser- and Val-p-NA. Vanha-Pertulla and Hopsu ( 1965a,b) separated five components from DEAE-cellulose columns that hydrolyze LeuP-NA; three components were inhibited by EDTA with different sensitivities on reactivation by Cot+, Mn++and Mg”; the other components were differentiated b y the pH optima and the effects of cysteine. Recent studies by S. Ellis and Perry (1966) show the presence of two thiol dependent exopeptidases in the nonparticulate fractions of the anterior pituitary; an arylainidase B specific for Lysand Arg-arylamides and an “aminopolypeptidase” that hydrolyzed a variety of arylamides but is preferentially active on the lysyl derivative. The aminopolypeptidase of the pituitary is similar in some properties to the brain arylamidases since it is strongly inhibited by puromycin. Pituitary arylamidase B hydrolyzed a variety of neutral and basic dipeptides whereas the aminopolypeptidase

EXOI’EPTIDASES

OF THE NERVOUS SYSTEhI

79

is principally active on oligopeptides such as A h r , Ala,, and Lyss. The complete hydrolysis of polypeptides was ascribed to the combined action of these enzymes to yield dipeptides that could be hydrolyzed subsequently by dipptidases to the constituent amino acids. Confirmation of this pathway must await the characterization of the enzyme aminopolypeptidase. Ellis and co-workers (S. Ellis and Perry, 1966; S. Ellis and Nuenke, 1967) have also described enzymes that are spccific for dipeptidyl arylamides. Three enzymes could be distinguished: (1) was -SH and C1- dependent and hydrolyzed Ser-Tyr at pH 4.0 but which also hydrolyzed His-Ser-, Ala-Ala-, Gly-Phe-, SerMet-p-NA; ( 2 ) an enzyme that hydrolyzed only Lys-Ala-p-NA at pH 5.5 which was -SH and halide dependent; ( 3 ) an enzyme specific for Arg-Arg-p-NA at pH 9.0 which also hydrolyzed oligopeptides of Ala or Lys containing four or more residues. The first enzyme resembled cathepsin C in its properties and cleaved the N-terminal dipeptide sequence of adrenocorticotropin ( McDonald et al., 1966). The existence of this family of arylamide aminoacid hydrolases with the capability to hydrolyze even- and odd-numbered oligopeptides may be highly significant to peptide turnover in the pituitary and in the brain.

B. HYPOTHALAMUS The hypothalmus does not contain any large pools of peptides (0.02% of fresh weight in the hog; Shome and Saffran, 1966). There is now good evidence that oxytocin and vasopressin are synthesized in the hypothalamic region and stored in the posterior pituitary; granules containing these peptides have been detected in the hypothalamic-hypophysial tract by histochemical methods and have been isolated by sucrose gradient techniques (Pardoe and Wetherall, 1955; Heller and Lederis, 1962; Sachs, 1960, 1963). The mechanism for the release of these peptides from the storage sites is not clear but could involve proteolytic enzymes; the situation may be analogous to the release of exopeptidnses from inactive pancrcatic zymogen precursors (see Section V ) . It has been established that under different physiological conditions, such as dehydration and lactation, there is an increased release of neurosecretory materia1 (see Ortmann, 1960) accompanied by increased levels of acid

80

NEVILLE MARKS

phosphatase ( Kawashima et al., 1964) and enzymes hydrolyzing Leu-P-NA ( Arvy, 1962; Arai and Kusama, 1965).

1. Inactivation of Oxytocin and Vasopressin Oxytocin and vasopressin are rapidly inactivated by a variety of body fluids and tissue homogenates (Heller, 1959). Tuppy (1959) considered that enzymes hydrolyzing Leu-, Ala-, Gly- and cystine-di-p-NA in sera were related to the exopeptidases that inactivated oxytocin and which increased several fold during pregnancy. Cystine-di-P-NA was considered to have some features that were akin to the structure of oxytocin since hydrolysis of the hormone by performic acid released cysteine by cleavage of the half-cystine residue adjoining tyrosine. The enzyme from retroplacentar serum hydrolyzing both the synthetic analog and oxytocin has been purified 4500-fold; the enzyme was inhibited by EDTA and most metal ions (Tuppy, 1959). An unusual feature of this enzyme was the increased level only in the sera of man and primates but not other species (Werle et al., 1950; Hooper, 1964). Thus, in other animals the hypothalamic exopeptidases may significantly affect the quantity of oxytocin released into the blood circulation. In a study of enzymes that inactivate oxytocin in pregnant and nonpregnant dogs, Hooper (1964) reported a changed distribution in subcellular fractions obtained from the hypothalamic region. The highest concentration of enzymes occurred in the mitochondrial and supernatant fractions with only low activities in the microsomes and nuclei. In pregnant dogs, the mitochondrial subfractions rich in myelin and synaptosomes contained a higher level of the inactivating enzyme compared to the subfractions obtained from the nonpregnant animals. Hypothalamic extracts are also capable of inactivating substance P, bradykinin, vasopressin ( Krivoy, 1957; Hooper, 1962). In a survey of six different brain areas, the enzymes inactivating vasopressin were the highest in the hypothalamus compared to the cerebellum, thalamus, cortex, caudate nucleus, and white matter. By way of contrast, enzymes inactivating bradykinin were eqiially distributed in all areas except in the white matter where it was in slightly lower concentration (Hooper, 1963). 2. €I-lypothnlamic-Releasinfi Factor It can be implied from the polypeptide nature of these factors that exopeptidases may be involved in their release and activation,


81

These factors arise from the hypothalamic region and are transported to the adenohypophysis by nieans of the circulation. Interruption of the blood supply has lecl to the identification of specific hormonal-releasing factors for a$-corticotropins, thyrotropic, follicle, luteinizing, and growth hormones (Schally et al., 1962, 1965; McCann and Ramirez, 1964). Extracts of the hypothalamus, especially the medial basal tuberal region (Watanabe and McCann, 1967), were active on the release of anterior pituitary hormones both in uitro and in viuo (Schally et al., 1967). As noted previously, the hypothalamus and sera contain exopeptidases that hydrolyze pituitary hormones and these may be involved in the levels of hormone-releasing factors. The adenohypophysis, unlike the posterior pituitary, is non-neuronal in origin and is linked to the hypothalamus only by means of the blood supply. C. PINEALGLAND Despite the known interrelationships between the pineal and pituitary glands (Reiss et al., 1963), studies concerning the pineal peptide composition and turnover are scanty. The enzymes studied in pineal gland tissue, especially the hydrolases and transferases, differ in properties from those of normal brain tissue (ThiCblot et nl., 1965). In a comparison of arylamidase N activities in the different brain areas, Jouan and Rocaboy (1966) reported that with Ala- and Phe-P-NA as the substrates, the activity in the pineal gland equaled that in the anterior and posterior pituitary but was less than that in whole brain tissue. With Leu-P-NA, the activity was the same in all four tissues; with Val-p-NA only trace activities were observed in the pineal compared with high activities in the pituitary gland extracts.

D. CEREBRAL SPINALFLUID The attempted correlations between disease states and the enzyme levels in the CSF are obscured by the large and unspecific increase in protein composition (Stern et al., 1950; Tourtellotte, 1967). Early studies showed that CSF could split polypeptides with increased polypeptidase activity in patients (Heyde, 1932). R. Abderhalden ( 1943) reported the hydrolysis of DL-Leu-Gly-Gly and in some cases DL-Leu-Gly in the CSF of a large group of patients. Later studies by Stern et al. (1950) showed that the activity with the tripeptides, Leu-Gly-Gly and triglycine, was activated by Cob+.

82

NEVILLE MARKS

In patients with brain tumors, Green and Perry (1955, 1963) reported increased levels of enzymes hydrolyzing Leu-P-NA, a substrate that would indicate the presence of LAP and arylamidase N activities. Chapman and Wolff (1959) reported in some disease states the appearance of vasodilator polypeptides in the CSF of man. The polypeptides had similar biological activities to bradykinin and its formation was attributed to the appearance of an unidentified proteolytic enzyme, Enzymes that inactivate the polypeptide were not explored but it would be of interest to determine if specific exopeptidases such as carboxypeptidase B are involved. In the most detailed study of exopeptidase activity in the CSF to date, Wiechert (1966) reported the rapid hydrolysis within 7 hours on intracisternal injection of the following peptides; Gly-Leu, Gly-D-Leu, Ala-Gly, Pro-Gly, Gly-Phe, triglycine, Leu-NH,, m-LeuLeu. The hydrolysis of the peptides in uitm was considerably slower with the best activity with the LAP substrate Leu-NH, and only trace activities with the iminodipeptidase substrate and with the glycine-glycine dipeptidase substrate Gly-Gly.

E. SPINALCORD The induction of acute experimental allergic encephalomyelitis (EAE ) by proteins and polypeptides of the spinal cord has been the subject of considerable interest (Nakao et al., 1966; Lumsden et al., 1966). Enzymatic degradation is considered to play an important role in the formation of the active immunological agents (Einstein et at., 1968). In a comparison of several exopeptidases in lobster spinal cord, we have shown hydrolysis of the following substrates; Leu-Gly-Gly ( aminotripeptidase ) , Arg-8-NA ( arylamidase B ), Leu-P-NA ( arylamidase N ), Z-Leu-Tyr ( carboxypeptidase B) (Table IV). The exopeptidase activities in the head and thorax regions of the spinal cord exceeded that in the abdomen and tail regions. This gradient of activity may be correlated with aspects associated with axonal flow. The relatively high concentration of neutral and acid proteinases present in the spinal cord (Datta et al., 1968b), together with the high exopeptidase activities, probably indicates a large protein breakdown and turnover in lobster spinal cord. In the rat, Beck and Smith (1967) reported an association of arylamidase N activity ( Ala-P-NA) in the myelin that decreased in rats with EAE but increased in all other suibcellular fractions except the nuclei.

TABLE 1v EXOFEPTIUASES O F I'ERII'IIERAL

NERVE AND SPINAL C O R D "

Subfitrate (mpmoleslmg protein/minute) I,c!~I-Gl.V-

Gly

Site Lobster Spinal cord Head hhdomeii Tail Lobstcr Peripheral iierve Proximal Distal Rat Proximal Distal

h (j

ti

1" II

Z-Gly-Tyr

2 I 0

"

I.eu+NA

Arg-:-p-?\'h

4

1 0 3 0 4

3 2

7

:

~

0

-

0 G

2 s

:I

4

1

2

10

4

5

lcrom Dalta et al. (196%).

Spilzal Routs and Gangliu

There have been a number of studies of the exopeptidases in the dorsal roots, ventral roots, dorsal-root ganglia. Extracts from these different areas hydrolyzed bradykinin and substance P but not oxytocin and vasopressin (Eber and Lembeck, 19.56; Krivoy, 19.57; Inouye et al., 1961; Hooper, 1963). The concentration of enzymes inactivating substance P was similar in dorsal and ventral roots but not in the case of bradykinin where the concentration was higher in the dorsal-root ganglia followed by the dorsal and ventral roots. Hooper (1963) attributed this enzyme gradient to aspects associated with axonal flow. In a recent study Droz (1967) observed the migration of labeled materials into rat spinal ganglia by radioautography. VII. Peripheral Nerve

There appears to be no detailed studies on the peptide hydrolases of peripheral nerve although considerable information is available concerning other hydrolases, especially the esterase group (E.C.3.1).Most of the data available concerns the complex changes accompanying Wallerian degeneration. With transection of the peripheral nerve there is considerable clestrnction of all cellrilar

84

NEVILLE MAHKS

elements accompanied by an increased level of aniino-nitrogen compounds, notably the amino acids. In the hen, some amino acids increased 15330% at 14 days (Porcellati and Thompson, 1957) while in the rabbit the increase was in the range 2240%( McCaman and Robins, 1959a). McCaman and Robins (1959b) showed in a study of 12 different enzymes that maximum degeneration occurred at 14 days, with a large increase in dipeptidase activity with LeuAla as the substrate, and also acid phosphatase, isocitric dehydrogenase, etc. Since the Wallerian degeneration is accompanied by the cellular influx of macrophages and by the proliferation of Swann cells, it cannot be assumed that the increased levels of enzymes originated from the peripheral nerve itself. Samorajski (1957) showed that the time sequences for the peripheral nerve degeneration are parallel to the infiltration by non-nervous tissue. The role of exopeptidases in Wallerian degeneration is not clear; presumably they contribute to the increased levels of amino acids and the cessation of their activities may be required for regeneration. In rat sciatic homogenates, C . W. M. Adams and Glenner (1962) reported high arylamidase N activity with Leu-P-NA as the substrate. This enzyme differed in properties from that reported as present in brain; it was inhibited by -SH and ascorbate, CN-, Mn" and diisopropyl fluorophosphate (DFP) but not by metalchelating agents. Activity was present in the different anatomical areas, in the myelin sheath, neurilemma, perineurium, endoneurium, and in the Schwann cells. We also observed arylamidase N activity in sciatic nerve extracts of several species-the lobster, the crab, and in the rat. In the unmyelinated invertebrate nerve, the activity with Arg-p-NA was =-fold higher than with Leu-p-NA as the substrate. As in the lobster ventral spinal cord there was a gradient of exopeptidases on comparison of the distal and proximal segments (Table IV). The presence of peptide hydrolases along the nerve trunk suggests that degradation of axoplasmic proteins and peptides is not confined to the nerve-ending region. In all species the highest activity occurred with Leu-Gly-Gly as the substrate but also with appreciable carboxypeptidase A activity was detected in the rat sciatic nerve. A number of studies have shown that proteolytic enzymes injected into giant squid axoplasm interferes with the action potential without interference with the resting potential (Rogas and Luxoro, 1963). Since squid axoplasm contains 70%of its protein in the form of neurofilaments with an extremely

EXOPEF'TIDASES OF THE NERVOUS SYSTEM

85

high percentage of acidic amino acid residues, exopeptidases, especially acidic arylamidases, may be intimately linked to some physiological events (see Schmitt and Davison, 1964). VIII. Conclusions

The very multiplicity of peptide hydrolases, their ubiquitous distribution in all brain areas and cellular fractions, the broad specificity patterns, all suggest many different roles of exopeptidases in the nervous system. For purposes of description, it is intended to treat these possible roles under two major subheadings: (1) roles related to physiological processes such as activation of hormones or transport processes, and ( 2 ) relationship to the metabolic turnover and elimination of protein breakdown products. A. EXOPEPTIDASES AND HORMONE ACXIVITY Modification of proteins or peptides may result in the formation of physiologically active compounds from inactive precursors or alternatively in the inactivation of active proteins. A recent example supporting this concept was the formation of bradykinin-like materials from the polypeptide kalliden-10 by arylamidase B ( Hopsu-Harvu et al., 1966). The precursor polypeptide itself is obtained from serum-a-globulin by trypsin digestion and the production of this hormone serves as an example of the sequential action of both endu- and exopeptidases. Instances of the inactivation of physiological peptides by enzymes present in the hypothalamus and pituitary, or the relationship to the hormonal-releasing factors have been mentioned in the previous sections.

B. EXOPEPTIDASES AND DISEASE PROCESSES A large number of inborn metabolic disorders are associated with generalized aminoacidurias. No specific exopeptidase enzyme defects as such have been detected but a number of clinical disorders are associated with increased turnover and excretion of amino acids and peptides. In cerebromacular disease ( Tay-Sachs ), for example, there is an increased excretion of carnosine and anserine in addition to histidine and methylhistidine (Bessman and Raldwin, 1962). Cystathioniniiria is associated with increased urinary excretion of cystathione; hepatolenticl~lar degeneration (Wilson's disease) and the Fonconi syndrome also result in aminoaciduria and increased peptide excretion (for other examples, see

86

NEVILLE MARKS

Lajtha, 1964b; Scriver, 1962; Meister, 1965b). Some of these defects have been ascribed to faulty reabsorption in the renal tubules; aspects associated with transport are briefly described below. C. EXOPEFTIDASES AND TRANSPORT PROCESSES Peptides enter tissue cells at slow rates compared with the transport of free amino acids; in most cases peptides are excluded from passage into tissues (Christensen and Rafn, 1952). In the intestinal mucosa, the role played by exopeptidases is of special interest since there is evidence that some dipeptides are not transported, as such, but are hydrolyzed first to amino acids in the barrier membrane. There are two lines of evidence to support this finding: ( 1 ) Washed isolated intestinal loops devoid of all enzyme secretions can still transport peptides placed in the lumen with the appearance of the constituent amino acids; ( 2 ) people with pancreatic insufficiency hydrolyze substantial quantities of dietary peptides (Newey and Smyth, 1959; Gitler, 1964). Some dipeptides placed in washed intestinal loops are hydrolyzed quite rapidly; 8 minutes was sufficient for Ala-Phe, Leu-Tyr, but 60 minutes was required for Gly-Leu and Gly-Tyr. In bacteria it has been possible to demonstrate separate transport systems for some di- and tripeptides that are distinct from those of amino acids (Leach and Snell, 1960; Kessel and Lubin, 1963; Brock and Wooley, 1964). Glycylglycine, for example, is transported unchanged in some Escherichia coli mutants that are devoid of glycyl-glycine dipeptidase. In Streptococcus foecalis and Lactobacillus casei, Gly-Gly is transported unchanged but is believed to be hydrolyzed within the cell; the hydrolysis of Gly-Gly appeared to stimulate the incorporation of glycine by exchange reactions with external glycine. A number of studies have indicated that the epithelial cell brush border is the probable site of peptide hydrolysis in the mucosal lining (Robinson, 1963; Eichholz and Crane, 1967). This area also serves as a digestive-absorptive surface for carbohydrates and contains a high concentration of enzymes hydrolyzing Leu-j3-NA ( arylamidase N activity), Leu-NH,, Leu-Gly, and Leu-Gly-Gly. The ratio of the latter three substrates was 0.4:1.0:0.5 and is distinct from that of liver, muscle and brain (Section I1,A) indicating the considerable heterogeneity of LAP-type enzymes in different tissues. If exopeptidases are involved in transport phenomena, then it is important to establish whether these enzymes are associated with

87

EXOPEPTIDASES OF T f I E KERVOUS SYSTEM

TABLE V DISTRIBUTION OF ENZYME ACTIVITIESIN MICROSOIL~L FRACTIONS~ Perceiit. of activity present in microsomes

Substrate

Membranous fraction (DOC) 11 68

Leu-Gly Leu-Gly-Gly Leu-Leu-Leu Ah-Gly-Gly Z-Leu-T yr Arg-p-N A A Ly~ys-8-N Met-p-NA a

Ribosomal fraction

76 61 13 97 80

36

From Datta et al. (1968a).

brain membranous fractions. In preliminary studies we have shown this to be the case with membranes obtained from rat brain microsomes and mitochondria by treatment with detergents or hypotonic buffer (Datta et al., 196713, 1968c; Marks et al., 1968a). Microsome membranes obtained by deoxycholate detergent ( DOC) treatment gave high aminotripeptidase activity with Leu-Gly-Gly, and arylamidase activity with Arg-, Lys-, Met-p-NA (Table V ) . These enzymes were purified and shown to be similar in properties to the purified tripeptidase and arylamidase of whole brain. The arylamidase activities were stabilized by dithiothreitol,

Property

Trilwptidnse Ala-Gly-Gly

p H optima

K,

Ki (puromycin) Cysteine (%) pCMB, 0.1 mM (%) Co++,0.1 mM (%) Zn++, 0.1 mM (%I Cd++, 0.1 mM (%) Cu++,0.1 mhf (%) a

From Datta et al. (1968a).

7.0

42

x

10-4

?To effect 20

+

-40

0 0 -40 -40

Arylamidase N Leu-8-NA

6.5 i .0X 8.0x 10-6 50 -70

+

+100 +90 - 100

- 100

S8

NEVILLE MARKS

activated by divalent metals, and inhibited by puromycin (Table VI ). Only trace exopeptidase activities were observed in the ribosomal preparations. In the mitochondria1 preparations, the highest exopeptidase activities were associated with the purified subfractions containing synaptosomes. Mitochondria1 membranes obtained by treatment of the crude material with Triton-X-100 were also characterized by exopeptidase activities, especially with Leu-GlyGly as the substrate. This enzyme on purification was similar to the tripeptidase of whole brain previously described. There have also been some studies on the uptake of dipeptides in rat brain slices. Abraham et al. (1964) reported the active transport of carnosine but not homocarnosine in rat brain slices. Both these dipeptides are constituents of brain tissue (Pisano et al., 1961; Abraham et al., 1962). The uptake of carnosine was qualitatively similar to histidine but with different time sequelae; maximum values for histidine uptake occurred at 30 minutes compared with 4 hours for the dipeptide. This study provided good evidence for the presence of the enzyme carnosinase, since 30%of the dipeptide was hydrolyzed as shown by the appearance of labeled palanine. Since other peptides decreased the uptake of carnosine to a greater extent than amino acids, the authors suggested a separate general mechanism for the transport of dipeptides in the brain. The significance of exopeptidases in the brain membrane fractions to the transport mechanism is not readily apparent. The increase in pool size as a result of degradation of peptides could serve to drive the transport mechanism by the increase in the rate of exchange reactions.

D. EXOPEPTDASES AND PROTEIN TURNOVER The mechanisms of intracellular regulation of protein and peptide breakdown are not well understood at present. Measurement of the incorporation of labeled amino acids into protein has shown that most cerebral proteins are unstable with rapid turnover rates (see Lajtha, 1964a; Lajtha and Toth, 1966). Since brain cells are seemingly impervious to proteins, the rate of protein synthesis must be balanced by degradation to maintain the dynamic state. Lajtha (1964a) proposed a tentative scheme for brain protein turnover with separate pathways for synthesis compared with degradation. Protein breakdown required, as the first step, the formation of polypeptide intermediates by the hydrolysis of cerebral proteins. Our

EXOPEPTIDASES OF 1 H E NERVOUS SYSTEM

89

earlier studies showed that brain extracts were comparatively rich in neutral and acid proteinases that could account for the initial degradation of proteins (Marks and Lajtha, 1963, 1965; Lajtha and Marks, 19s6). It is believed that acid proteinases, or cathepsins, hydrolyze peptide bonds by simple hydrolysis without any requirement for metabolically derived energy. There is some evidence that neutral proteinases ( p H optima 7.6) require an energy source for the hydrolysis of prelabeled protein in liver slices and liver and brain mitochondria (Penn, 1960; also see Lajtha, 1964a). These and other findings suggest that protein breakdown proceeds in several steps that are quite distinct from those required for protein synthesis. This does not exclude the possibility that some proteolytic enzymes and exopeptidases may be involved in the synthesis of some oligopeptides, principally by the mechanisms of transamidation (see Section IV,A,l), Also, a separate role, independent from that of degradation, has been mooted for hydrolytic enzymes in the release of polypeptide chains from the ribosome-messenger RNA complex. Cuzin et al. (1967) recently reported a hydrolytic enzyme in extracts of E. coli that catalyzed the hydrolysis of diphenylalanyltRNA and N-substituted oligopeptidyl-tRNA. The mechanism is not fnlly understood, but it is believed to involve enzymes capable of hydrolyzing the ester linkage which might include exopeptidases or acylases ( Fry and Lamborg, 1967). Hitherto, it was believed that puromycin interfered with protein synthesis by interruption of peptide chain elongation (Darken, 1964). The potent inhibition of arylamidases by puromycin is of particular interest in connection with the possible mechanisms involved in the interference with the fixation of memory in experimental animaIs (see Flexner et al., 1967).

Pathways of Peptide Breakdoton Recent work has supplied some evidence for the possible pathways by which peptide breakdown is mediated. The localization of exopeptidases together with endopeptidases at identical sites suggests that these enzymes operate in sequence for the final degradation of proteins to peptides or amino acids. It is known that many exopeptidases can completely hydrolyze small proteins and polypeptides directly by cleavage of the terminal residues; in particular, LAP will completely hydrolyze all the peptide bonds of insulin A chain (Frater et al., 1965). Alternatively, proteins can be degraded

90

NEVILLE MARKS

by proteinases to yield large polypeptides followed by aminopolypeptidase and arylamidases to yield even- or odd-numbered oligopeptides. In the hypothetical scheme illustrated in Fig. 2, some arylamidases can split oligopeptides to form tripeptides which can be hydrolyzed subsequently by aminotripeptidase to yield dipepBrain proteins htracellular proteinases (PH3.8 or 7.6)

1

Polypeptides Aminopolypeptidase

I

t

%

\

Aminotripeptidase

"t.

Arylamidase &?idme) (Dipeptidyl

LAP

or c arboxypeptidase A, B

Dipeptides +- - ---- dipeptidase (arylamidase) Amino acids

FIG.2. Scheme for the breakdown of proteins and polypeptides to amino acids.

tides. E. Ellis and Nuenke (1967) suggested that tripeptides can be split also by dipeptidyl arylamidase I11 present in pituitary glands to form the amino terminal dipeptide. Dipeptides are hydrolyzed finally to amino acids by the action of brain dipeptidases. The liberated amino acids, in addition to the reutilization for protein synthesis, or utilization for energy production or amino formation, can be transported from the free pool to other portions of the nervous system. REFERENCES

E., ancl Ceaser, G . (1940). Fevmentforschung 16, 255. Abderhalden, R. ( 1943). Fermentfovschung 17, 173. Abraham, D., Pisano, J. J., and Udenfriend, S. (1962). Arch. Biochem. Bio-

Abderhalden,

phys. 99, 210.

EXOPEPTIDASES OF THE NEAVOUS SYSTEM

91

Abraham, D., Pisano, J. J., and Udenfriend, S. (1964). Arch. Biochem. Biophys. 104, 160. Adams, C. W. M., and Glenner, C. G. (1962). 1. Neurochem. 9, 233. Adams, E. S., and Smith, E. L. (1951). J. Biol. Chem. 191, 651. Adams, E. S., and Smith, E. L. (1952). J. Biol. Chem. 198,671. Albert, Z., Orlowski, M., Rzucidlo, Z., and Orlowska, J. (1966). Acta Histochem. 25, 312. Arai, Y., and Kusama, T. (1965). Japan. Acad. Sci. 41, 734. Arvy, L. (1962). In “Neurosecretion” ( H . Heller and R. B. Clark, eds.), pp. 215-225. Academic Press, New York. Beaufay, H., Berleur, A., and Doyen, A. (1957). Biochem. J. 66, 32P. Beck, C. S., and Smith, M. E. (1967). Federation Proc. 26, 840. Behal, F. J., Hamilton, R. D., Kanavage, C. B., and Kelly, E. (1963). Arch. Biochem. Biophys. 100, 308. Behal, F. J., Hamilton, R. D., Kanavage, C. B., Dawson, F. B., and Terrell, L. C. [ 1964). Arch. Bioch~rn.Biophp. 108, 207. Behal, F. J., Asserson, B., Dawson, F., and Hardman, J. (1965). Arch. Biuchem. Biophys. 111, 335. Behal, F. J., Klein. R. A., and Dawson, F. B. (1966). Arch. Biochem. Biophys. 115, 545. Bergmann, M. (1942). Adoan. Etizyinol. 2, 19. Berl, S. (1966). Biochemistry 5, 91F. Bessman, S. P., and Balclwin, R. (1962). Science 135, 789. Blum, E., Jakovtschuk, A. I., ~ n dJarmoschkewitsch, A. I. (1936). Bull. Biol. Med. Erptl. USSR 1, 17. Boissanas, R. A., Guttman, S., and Jaguenod, P. A. (1960). Helo. Chim. Acta 43, 1349. Brecher, A. S. (1963). J. N ~ ~ W O C ~10, C T1. IL Brinkley, F. (1961). J. Biol. Chem. 236, 1075. Brock, T. D., and Wooley, S. 0. (1964). Arch. Biochem. Biophys. 105, 51. Brown, J. R., Greenshields, R. N., Yamasaki, M., and Neurath, H. (1963). Biochemistry 2, 867. Bryce, G. F., and Rabin, B. R. ( 1964a). Biochem. J. 90,509. Bryce, G. F., and Rabin, B. R. (196413). Biochem. J. 90,513. Burstone, M. S. (1962). “Enzyme Histochemistry and its Application in the Study of Neoplasms.” Academic Press, New York. Chapman, L. F., and Wolff, H. C . (1959). A.M.A. Arch. Intenla1 Med. 103, 86. Christensen, H. N., and Rafn, h l . L. (1952). Gamer Res. 12, 495. Coleman, J. E., and Vallee, B. L. (1964). Biochemistry 3, 1874. Coleman, J. E., Pulido, P., and Vallee, B. L. (1966). Biochcmistry 5 , 2019. Cuzin, N., Kretchmer, N., Grtvnberg, R . E., Hurwitz, R., and Chapeville, F. (1967). Proc. Natl. Acad. Sci. U.S. 58, 2079. Darken, M. A. (1964). Pharmacol. Rev. 16,223. Datta, R. K., Marks, N., and Lajtha, A. (1967a). 1st Intern. Congr. Neurochem. Strassbourg, 1967, p. 49. Datta, R. K., h4ark.5, N., and Lajtha, A. (19671~).Federation Proc. 26, 452. . J. Riochem. 4, 37. Datta, R. K., Marks, N., and Lajtha, A. ( 1 9 6 7 ~ )Indian

92

NEVILLE MARKS

Datta, R. K., Marks, N., and Lajtha, A. (1968a). Unpublished results. Datta, R. K., Marks, N., and Lajtha, A. (1968b). J. Comp. Biochem. Physiol. (submitted for publication). Davidoff, R. A., Shank, R. P., Graham, L. T., Aprison, M. I€., and Werman, R. (1967). Nature 214, 680. Davis, N. C., and Smith, E. L. (1955).J. Biol. Chem. 214,209. De Duve, C., Wattiaux, R., and Baudhuin, P. (1962). Aduan. Enzymol. 24, 291. Dixon, M., and Webb, E. C. (1964). “Enzymes.” Academic Press, New York. Droz, B. (1967). J. Microscopie 6, 201. du Vigneaud, V., Sifferd, R. H., and Irving, G. W. (1937). J. B i d . Chem. 117, 589. Eber, O., and Lembeck, F. (1956). Arch. Exptl. Pathol. Pharmukol. 229, 1391. Eichholz, A., and Crane, R. K. ( 1967). Biochim. Biophys. Acta 135, 959. Einstein, E. R., Csejtey, J., Davis, W. J., Lajtha, A., and Marks, N. (1968). Intern. Arch. Allergy Appl. Immtoiol. 34 (in press). Elliot, D. F., Horton, E. W., and Lewis, G. P. (1961). Biochem. J. 78, 60. Ellis, D., and Fruton, J. S. (1951). J. B i d . Chem. 191, 153. Ellis, S. (1963). Bioclaem Biophys. Res. Commun. 12, 452. Ellis, S., and Perry, M. ( 1966). J. Bid. Chem. 241, 3679. Ellis, S., and Nuenke, J. M. (1967). J. B i d . Clzem. 242, 4623. Erdos, E. G. (1966). Aduan. Pharmacol. 4, 1. Erdos, E. G., and Yang, H. Y. T. ( 1967). Life Sci. 6,569. Erdos, E. G., Sloane, E. M, and. Wohler, I. M. (1964). Biochem. Phurmacol. 13, 893. Erlanger, B. F., Kowkowsky, N., and Cohen, W. (1961). Arch. Biochem. Biophys. 95, 271. Felgenhauer, K., and Clenner, G. G. (1966). J. Histochem. Cytochem. 14, 401. Fittkau, S., Glasser, D., and Hanson, H. (1961). Z. P h ~ s i o lChem. . 322, 101. Flexner, L. B., Flexner, J. B., Roberts, R. B., and De La Ilaba, G. (1964). Proc. Natl. Acad. Sci. US.52, 1165. Flexner, L. B., Flexner, J. B., and Roberts, R. B. (1967). Science 155, 1377. Florkin, M., and Stolz, E. H. ( 1964). “Comprehensive Biochemistry.” Elsevier, A m terdam. Fodor, P. J., Miller, A., Neidle, A., and Waelsch, H. (1953). J. B i d . Chem. 203, 991. Fraenkel-Conrat, H., Harris, J. I., and Levy, A. L. (1955). Methods Riochcnz. Anal. 2, 359. Frater, R., Albert, L., and Smith, E. L. (1965). J. Bid. Chem. 240, 253. Friede, R. L. (1966). “Topographic Brain Chemistry.” Academic Press, New York. Fruton, J. S., Smith, V. A., and Driscoll, E. (1948). J. Biol. Chem. 173, 457. Fruton, J. S., Hearn, W. R., Ingram, V. M., Wiggans, D. S., and Winitz, M. (1953). J. Bid. Chem. 204, 511. Fry, K. T., and Lamhorg, M. R. (1967).J. Mol. B i d . 28,423.

EXOPEPTLDASES OF THE NEHVOUS SYSTEM

93

Gitler, C. ( 1964). 1n “hlammaliaii l’iotcin Metabolism” ( H . N. hlunro and J. B. Allison, eds.), Vol. 1, pp. 35-69. Academic Press, New York. Glenncr, G. G., and Folk, J. E. (1961). Nature 192,328. Glenner, G. G., McMillan, P. J., and Folk, J. E. (1962). Nature 194, 867. Gomori, G. (1954). Proc. SOC. Exptl. Biol. Med. 87, 559. Green, J. B., and Perry M. (1955). Arch. Biochem. Biophys. 57, 458. Green, J. B., and Perry, M. (1963). Neurology 13, 924. Greenbaum, L. M., Yamafuji, K., and Hosoda, T. (1965). Biochem. Pharmucol. 14, 411. Greene, L. J., IIirs, C. H. W., and Palade, G. E. (1963). J. Biol. Chem. 238, 2055. Habermann, E. (1963). Atin. N.Y. Acad. Sci. 104, 1301. Hanes, C. S., Hird, F. J. R., and Isherwood, F. A. (1950). Bzochem. J. 56,417. Hanson, H. (1963). Abhandl. Saechs. Akad. Wiss. Leipzig, Math.-Naturw. K l . 48, 1-15. Hanson, H. ( 1966). In “Hoppe-Seyler Thieifelder Handbuch der Physiologisch- und Pathologisch-Chemische Analyse,” Vol. 6C, p. 1. Springer, Berlin. Hanson, II., and Blech, W. (1959) 2. Physiol. Chem. 315, 191. Hanson, H., and Tendis, N. (1954). Z. Grs. Inn. Med. Ihre Grenzgebiete 5, 1. Heller, H. ( 1959). I n “Polypeptidrs Which Affect Smooth Muscles and Blood Vessels” ( M . Schachler, ed.), p. 59. Pergamon Press, Oxford. IIeller, H., and Lederis, K. (1962). In “Neurosecretion” (H. Heller and R. B. Clark, eds.), p. 35. Academic Press, New York. Heyde, W. (1932). 2.Neurol. 138, 536. Hill, R. L. (1965). Aduan. Protein Chem. 20, 37. Holter, H. (1952). Aduan. Enzymol. 13, 1. Hooper, K. C. (1962). Biochem. 3. 83, 511. Hooper, K. C. (1963). Biochem. 1. 88, 398. Hooper, K. C. (1964). Biochem. J. 90,584. Hopsu, V. K., Makinen, K. K., and Glenner, G. G. ( 1966a). Arch. Biochem. Biophys. 114, 557. Hopsu, V. K., Makinen, K. K., and Glenner, G . G. (1966b). Arch. Biochem. Biophys. 114, 567. Hopsu-Harvu, V. K., Makinen, K. K., and Clenner, G. G. (1966). Nature 212, 1271. Hosein, E. A., and Smart, M. (1960). Con. J. Biochem. Physiol. 38, 569. Hutton, J. J., Kaplan, A., ancl Udenfriend, S. (1967). Arch. Biochem. Biophys. 121, 384. Inouye, A., Kataoka, K., and Tsujika, T. (1961). Japan. J. Physiol. 11, 319. Joseph, R. L., and Sanders, W. J. (1966). Biochem. J . 100, 827. Jouan, P., and Rocaboy, J. C. (1966). Compt. Rend. SOC. Biol. 160, 859. Kakimoto, Y., Nakajima, T., Kanazawa, A., Takesada, M., and Sano, I. (1964). Biochim. Biophys. Acta 93, 333. Kanazawa, A., Kakimoto, Y.,Nakajima, T., Shimuzu, H., Takesada, M., and Sano, I. (1965). Biochim. Bioplays. Acta 97, 460. Kaplan, A., Witkop, B., and Udenfrirncl, S. (1964). J. Bid. Chem. 239, 2559.

94

NEVILLE MARKS

Kawashima, S., Farrer, D. S., Kobayashi, H., Oksche, A., and Lorenzen, L. (1964). Z. Zellforsch. Mikroskop. Anut. 62, 149. Keller, J. P., and Cohen, E. (1961). J. Biol. Chem. 236, 1407. Kessel, D., and Lubin, M. (1963). Biochim. Biophys. Actu 71, 656. Kies, M. W., and Schwimmer, S. (1942). J. Biol. Chem. 145, 685. Krivoy, W. A. (1957). Brit. J. Pharmcol. 12, 361. Krivoy, W. A., and Kroger, D. (1964). Brit. J. Pharmucol. 22,329. Lajtha, A. (1961). In “Regional Neurochemistry” ( S . S . Kety and J. Elkes, eds.), p. 25. Pergamon Press, Oxford. Lajtha, A. (1964a). Intern. Reu. Neurobiol. 6, 1-98. Lajtha, A. (1964b). Intern. Reu. Newmbiol. 7, 1-40. Lajtha, A. ( 1966). In “Hoppe-Seyler Thierfelder Handbuch der Physiologischund Pathologisch-Chemische Analyse,” Vol. 6C, p. 740. Springer, Berlin. Lajtha, A., and Marks, N. (1966). Protides Biol. Flnids, Proc. Colloq. 13, 103. Lajtha, A,, and Toth, J. (1966). Bwchem. Biophys. Res. Commun. 23, 294. Lajtha, A., and Toth, J. (1968). Personal communication. Lande, S., and Lerner, A. B. ( 1967). Pharmacol. Reu. 19, 1. Lande, S., Lerner, A. B., and Upton, V. G. (1965). J. Biol. Chem. 240, 4529. Leach, F. R., and Snell, E. F. (1960).J . Biol. Chem. 235, 3523. Lewis, G. P. (1960). Nature 188, 999. Li, C. H. (1957). Cancer 10, 698. Linderstdm-Lang, Z. (1929). Z. Physiol. Chcnz. 182, 151. Linderstr6m-Lang, Z. ( 1939). Hame!/ Lectures 34, 214. Lowery, 0. H., Cilligan, D. R., and Katersky, E. M. (1941). J. Biol. Chem. 139, 795. Lumsden, C. E., Robertson, D. M., and Blight, R. (1966). J. Neurochem. 13, 127. McCaman, R. E., and Robins, E. ( 1959a). J. Neurochem. 5, 18. McCaman, R. E., and Robins, E. (195913). J. Neurochem. 5,32. McCann, S . M., and Ramirez, D. V. (1964). Progr. Hormone Res. 20, 131. McDonald, J. K., Ellis, S., and Reilly, T. J. (1966). J. Biol. Chem. 241, 1494. McIlwain, H., and Trezize, M. A. (1957). Biochem. J. 65, 288. Mahadevan, S . , and Tappel, A. L. (1967). J. B i d . Chem. 242,2369. Marks, N. ( 1967). Biochem. J. 103,40P. Marks, N., and Lajtha, A. (1963).Biochem. J. 89,438. Marks, N., and Lajtha, A. (1965). Biochem. J. 97, 74. Marks, N., and Lajtha, A. ( 1969). In “Handbook of Neurochemistiy” (A. Lajtha, ed.), Vol. IV. Plenum Press, New York (in press). Marks, N., Datta, R. K., and Lajtha, A. (1967). Proc. 7th Intern. Congr. Biochem., Tokyo, 1967. Abstract J. 275. Sci. Council Japan, Tokyo. Marks, N., Datta, R. K., and Lajtha, A. (1968a). J. Biol. Chem. 243, 2882. Marks, N., Datta, R. K., and Lajtha, A. (19681)). In “Metabolism of Nucleic Acids and Proteins of the Neuron” (Z. Loch, ed.), Exerpta Med. Found., Amsterdam. Mechanic, G . L., and Levy, M. (1959). J. Am. Chem. SOC. 81, 1889. Meister, A. (1965a). “Biochemistry of the Amino Acids,” 2nd ed., Vol. 1. Academic Press, New York.


95

hleister, A. (196513). “Biochemistry of the Amino Acicls,” 2nd ed., Vol. 2. Academic Press, New York. Nachlas, M . M., Coldstein, T. P., and Selignmn, A. M. (1962). Arch. Biochem. Biophys. 97, 223 Nagatsu, I., and Haru, J. (1967). N o h e 213, 206. Nagatsu, I., Gillespie, L., George, J. Ad., Folk, J. E., and Glenner, G. G. (1965). Biochem. P h a m c o l . 14, 853. Nakao, A., Davis, W. J., and Einstein, E. R. (1966). Biochim. Biophgs. Actn 130, 163. Neurath, H. ( 1960). In “The Enzymes” (P. D. Boyer, H. Lardy, and K. Myrback, eds.), Vol. 4, Part A, pp. 11-36. Academic Press, New York. Neurath, H. (1967). Proc. 7th Intern. Congr. Biochem., Tokyo, 1967. Symp. 111, p. 151. Sci. Council Japan, Tokyo. Newey, H., and Smyth, D. H. (1959). 3. Physiol. (London) 145, 48. Olson, E. J., Littleton, H. W., and Auditore, J. V. (1967). Arch. Biochem. Biophys. 119, 22. Orlowski, A. Z , and Meister, A. ( 1963). Biochim. Biophys. Actn 73, 679. Orlowski, A. Z., and Meister, A. (1965). J. Bid. Chem. 240, 338. Ortmann, R. ( 1960). In “Handbook of Physiology” Am. Physiol. Soc. (J. Field, ed.), Sect. 1, Vol. 11, p. 1034. Williams I% Wilkins, Baltimore, Maryland. Padayatty, J. D., and von Kley, H. (1966). Biochemistry 5, 1394. Pardoe, A. U., and Weatherall, M. (1955). J. Physiol. (London) 127, 201. Patterson, E. K., Hsiao, S. H., and Keppel, A. (1963). 3. Biol. Chem. 238, 3611. Patterson, E. K., Hsiao, S. H., Keppcl, A., and Soiot, S. (1965). 3. Biol. Chem. 240, 710. Pearse, A. G. E., and Wachtler, K. (1968). In “Metabolism of Nucleic Acids and Proteins and the Function of the Neuron” (Z. Lodin, ed.). Excerpta Med. Found., Amsterdam. Penn, N. W. (1960). Biochim. Biophys. Acta 37, 55. Pfeiderei, C., Celliers, P. G., Stanulovic, M., Wachsmuth, E. D., Determann, H., a d Braunitzer, G. (1964). Biochem. 2. 340, 552. Pisano, J. J., Wilson, J. D., Cohne, L., Abraham, D., and Udenfriend, S. (1961). J. B i d . Chem. 236,499. Pope, A. (1952). J. Neurophysiol. 15, 115. Pope, A. ( 1959). J . Neurochem. 4, 31. Pope, A., and Anfinsen, C. B. (1948). J. Biol. Chem. 173,305. Porcellati, G., and Thompson, R. H. S. ( 1957). J. Neurochem. 1, 348. Prahl, J. W., and Nenrath, H. ( 1966). Biochemistry 5, 4137. Price, V. E., hleister, A,, Gilbert, J. B., and Greenstein, J. P. (1947). 3. B i d . Cbem. 181, 535. Ramachanclran, L. K., and Winnicli, T. (1957). Biochim. Biophys. Acta 23, 533. Reiss, M., Mauer, I., Sideman, hl. B., Davis, R. H., and Plichta, E. S. (1963). J. Neurochem. 10, 851. Robinson, G. B. (1963). Biochrm. J. 88, 162. Rocha e Silva, M. (1963). In “Comparative Endocrinology” ( U . S . von Euler ancl H. Heller, eds.), Vol. 2, p. 64. Acaclemic Press, New York.

96

NEVILLE MARKS

Rocha e Silva, M., Beraldo, W. T., and Rosenfeld, G. (1949). Am. J. Physiol. 156, 261. Rogas, E., and Luxoro, M. (1963). Nuture 199, 78. Rybak, M., Petakova, M., and Simonianova, F. (1967). Collection Czech. Chem. Commun. 32, 1051. Sachs, H. (1960). J. Neurochem. 5,297. Sachs, H. (1963). J. Neurochem. 10,289 and 299. Samorajski, T. (1957). J. Histochern. Cytochem. 5, 15. Sampath-Kumar, K. S. V., Clegg, J. B., and Walsh, K. A. (1964). Biochemistw 3,1728. Sarid, S., Berger, A., and Katchalski, E. (1962). J. Biol. Chem. 237, 2207. Schally, A. V., Lipscomb, H. S., and Guillemin, R. (1962). Endocrinology 71, 164. Schally, A. V., Carter, W. H., Hearn, I. C., and Bowers, C. Y. (1965). Am. J. Physiol. 209, 1169. Schally, A. V., Muller, E., Sawano, S., Saito, T., and Redding, T. W. (1967). Federation Proc. 26, 365. Schmitt, F. O., and Davison, P. F. (1964). Ber. Bunsengesell. Physik. Chem. 68, 887. Schroder, E., and Hempel, R. (1964). Experientia 20, 529. Scriver, C. R. (1962). Progr. Med. Genet. 2, 83. Shome, B., and Saffran, M. (1966). J. Neurochem. 13,433. Simpson, R. T., and Vallee, B. L. (1966). Biochemistry 5, 1760. Simpson, R. T., Riodan, J. F., and Vallee, B. L. (1963). Biochemistry 2, 616. Smith, E. E., and Rutenburg, A. M. (1966). Science 152, 1256. Smith, E. E., Kaufmann, J. T., and Rutenburg, A. M. (1965). J. Biol. Chem. 240, 1718. Smith, E. L. (1955). In “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds. ), Vol. 2, p, 83. Academic Press, New York. Smith, E. L. (1960). In “The Enzymes” (P. D. Boyer, H. Lardy, and K. Myrback, eds.), Vol. 4, Part A, pp. 1-10. Academic Press, New York. Smith, E. L., and Hill, R. L. ( 1960). In “The Enzymes” (P. D. Boyer, H. Lardy, and K. Myrback, eds.), Vol. 4, Part A, pp. 37-62. Academic Press, New York. Smith, E. L., and Spackman, P. H. (1955). J. Biol. Chem. 212,271. Spector, A., and Mechanic, G. (1963). J. Biol. Chem. 238, 2358. Stem, K., Cullen, A. M., Barber, V. T., and Richer, R. (1950). Can. Med. Assoc. J . 63, 473. Sylven, B., and Bois, I. (1964). Histochemistry 4, 135. Sylven, B., and Lippi, U. ( 1965). Exptl. Cell Res. 40, 145. Szewczuk, A., Kochman, M., and Baranowski, T. (1965). Actu Biochim. Polon. 12, 357. Tallan, H. H., Moore, S., and Stein, W. H. (1954). J. Biol. Chem. 211, 927. Tallan, H. H., Moore, S., and Stein, W. H. (1958). J. Biol. Chem. 230, 707. Thibblot, L., Bastide, P., Blake, J., Boyer, J., and Dastugue, G. (1965). Ann. Endocrinol. 26, 313. Tomasi, L. G., and Kornguth, S. E. (1967). J. Biol. Chem. 242, 4933.


97

Tourtellotte, W. W. ( 1967). I n “Neurological Diagnostic Techniques” (W. J. Fields, ed.), p. 1. Thomas, Springfield, Illinois. Tuppy, H. (1959). 111 “Polypeptides Which Affect Smooth Muscles and Blood Vessels” ( M . Schachter, ed.), pp. 49-58. Pergarnon Press, Oxford. Tuppy, H., Wiesbauer, U., and Wintersberger, E. (1962). Z . Physiol. Chem. 329, 278. Upton, G . V., Lerner, A. B., and Lande, S. (1966). J . BWZ. Chem. 241, 5585. Uzman, L. L., Rumley, M. K., and Van Den Noort, S. ( 1961). j . Neurochem. 6, 299. IJzman, L. L., Van Den Noort, S., and Rnmley, M. K. (1962). J. Ncurochem. 9, 241. Uzman, L. L., Rumley, M. K., and Van Dtm Noort, S. (196.3).J. Nezrrochem. 10, 795. Vallee, B. L. (1964). Federation Proc. 23, Part 1, 8. Vallee, B. L. (1967). Proc. 7th Intern. Congr. Biochem., Tokyo, 1967. Symp. 111, p. 149. Sci. Council, Tokyo, Japan. Van den Noort, S., and Uzman, L. L. (1961). Proc. SOC.Exptl. B i d . Med. 108, 32. Vanha-Pertulla, T. P. J., and Hopsu, V. K. (1965a). Histochemistry 4, 372. Vanha-Pertulla, T. P. J., and Hopsu, V. K. (1965b). Ann. Med. Erptl. Biol. Fenniae (Helsinki) 43, 32. Wachsmuth, E.D., Fritze, I., and Pfleiderer, G. ( 1966a). Biochemistry 5, 169. Wachsmuth, E. D., Fritze, I., and Pfleiderer, G. (1966b). Biochemistry 5, 175. Waelsch, H. (1962). In “Monamines et System Nerveux Central,” pp. 93-104. Georg, Geneva, Switzerland. Waelsch, H., and Lajtha, A, (1961). Physiol. Reu. 41, 709. Watanabe, S.,and McCann, S. M. (1967).Federation PTOC.26, 365. Werle, E., Semm, K., and Enzenbach, R. (1950). Arch. Gynaekol. 177,211. Werman, R., Davidoff, R. A., and Aprison, M. H. (1967). Nature 214, 681. Wiechert, P. (1966). Acta Bid. Med. Ger. 16, 11. WOE, J. B., and Resnick, R. A. (1963). Biochim. Biophys. Acta 73, 588. Wurtz, H., Tanaka, A., and Fruton, J. S. (1962). J. B i d . Chem. 1,119. Ziff, M., and Smith, A. A. (1952).Proc. SOC. Exptl. B i d . Med. 80, 761.


BIOCHEMICAL RESPONSES TO NARCOTIC DRUGS IN THE NERVOUS SYSTEM AND IN OTHER TISSUES By Doris H. Clouet N e w York State Research Institute for Neurachemistry and Drug Addiction, Ward's Islond, and Columbia University College of Physicians and Surgeons, New York, New York

.

.

.

.

.

.

B. Biotransformations . . . . Effects on General Metabolic Systems . A. Intermediary Metabolism . . B. Protein Metabolism . . . . C. Nucleic Acid Metaholisni . . . . . D. Lipid Metabolism . Effects on Specific Metabolic Reactions A. Amines . . . . . . B. Hormones . . . . . . C. Calcium . . . . . . Serum and Brain Factors . . . A. Serum Factors . . . . . B. Brain Factors . . . . . Conclusions . . . . . . . . . . . . References

.

.

.

.

.

.

. . .

. . .

. . .

. . .

. . .

. . .

I. Introduction

.

.

.

.

.

.

11. h4etabolic Disposition of Narcotic Drugs . . . . . A. Drug Distribution and Transport in the Nervous System .

111.

IV.

V. VI.

.

.

.

.

.

.

.

.

. . .

. . .

. . .

. . .

. . .

. . .

.

.

.

.

.

.

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

.

.

.

.

.

.

.

.

.

.

.

.

99 101 101 104 108 108 110 112 115 115 115 118 120 121 121 121 122 124

I. Introduction

The administration of a narcotic drug to an animal results in a number of well-known physiological and pharmacological responses such as sedation and changes in awareness of pain, blood pressure, respiratory rate, and body temperature. With a second dose (or multiple doses) tolerance develops as shown by a decreased response to the drug. On the biochemical level, however, the responses to narcotic drug administration are not so well known, either as singular events, or as related to the biochemical basis for the observed pharmacological effects. It is the purpose of this review to collect and classify relevant information on the biochemical events seen after the administration of narcotic drugs to animals, 99

100

DORIS H. CLOUET

including man, and on the interaction of drugs and tissue in uitro. Because the nature of the pharmacological effects produced by narcotic drugs indicates that the nervous system is directly involved in most of the parameters of response, the emphasis wil be on the nervous system with lesser attention given to other tissues involved. The term “narcotic drug” will be limited here to the opium alkaloids (such as morphine, heroin, codeine), derivatives ( levorphanol, dihydromorphine) , and synthetic compounds with comparable pharmacological activity (meperidine, methadone), Morphine has been used by many workers as the prototype of the narcotic alkaloids. The chemistry of morphine and other related drugs has been discussed adequately elsewhere; the quantitative analysis of the small amounts of drugs found in biological samples has been described in detail (Workshop on Detection of Narcotics, 1966) as have drug structure :pharmacological activity relationships (Beckett et al., 1956; Bentley et al., 1965; Carabateas and Harris, 1W).The pharmacology of narcotic drugs has also been reviewed many times (see Seevers and Deneau, 1963). However, since the pharmacological state of animals under study is often of importance when one is correlating pharmacological and biochemical parameters, a brief description of these effects will be given here. A single effective dose of a narcotic drug produces many measurable reactions, mainly depressant, over a period of time that is related to drug and dose. Immediately afterward, there are, in succession, a short period of hyposensitivity (acute partial tolerance), a period of hypersensitivity, and a longer period of hyposensitivity (longterm partial tolerance) to the second injection of the same dose of drug. Each succeeding dose produces less reaction until there is no discernible response to drug injection. This latter state is usualIy described as tolerance. A group of symptoms called collectively the withdrawal syndrome develops slowly upon drug discontinuance, and more quickly when a narcotic antagonist (nalorphine, levallorphan )is administered to a tolerant animal. Thus, the experimental subjects in the studies reported here may be described as drug-treated and either nontolerant, tolerant, withdrawn, or unexposed to narcotic drugs. The main divisions of this chapter are the metabolic disposition of narcotic drugs including tissue levels and distribution, the uptake and transport of the drugs, and their biotransformations through metabolism. In these aspects the excellent review of Way and Adler

B I o c m h i I c A L RESPONSES

*ro NARCOTIC

DRUGS

101

(1962) on the biological disposition of morpliine and related compounds has been heavily relied upon (Section 11). The effect of narcotic drugs on general metabolic systems such as protein, ribonucleic acids, lipid and intermediary metabolism in brain and other tissue (Section 111). The effects of narcotic drugs on specific SYStems involving hormones, amines, and inorganic ions (Section IV). Some aspects peripherally related to biochemical response such as serological studies ( Section V ) , I I . Metabolic Disposition of Narcotic Drugs

A. DRUGDISTRIBUTION AND

TRANSPORT IN THE

NERVOUS SYSTEM

1. Distribution and Levels

The first studies of tissuc distribution using several narcotic drugs and various routes of drug administration in animals established that the level in the nervous system at no time approached that found in organs such as liver and kidney, unless the drug was introduced directly into the brain, and that the pharmacological responses occurred when the level of drug in brain was very low (see Way and Adler, 1962). Although the highest level of systemically administered narcotic drug never was above a few pglgm brain, both morphine and its antagonist nalorphine have been shown to remain in brain longer than in other organs. A 4-hour interval after the subcutaneous injection of morphine in dogs was sufficient for the plasma drug levels to fall below brain levels (Woods, 1959), and the same interval was long enough for blood levels of dihydromorphine to fall below that in nerve (Kosterlitz et nl., 1964). Attempts to relate pharmacological activity to drug levels in brain have led to a generally agreed-upon conclusion: there was no necessary rclationship between response and drug level. Morphine levels in brain were not related to the analgesic effect (Miller and Elliott, 1955) or to the physical activity of the animal (Szerb and McCurdy, 1956). A definitive study of this question in which 14C-labeledmorphine was administered to dogs which were killed at intervals after drug injection has shown that the labeled drug was distributed throughout the hrain, that the level was higher in gray than white matter, higher in cerehral cortex than in cerebellum, that maximal levels were reached within 4 hours after the injection,

102

DORIS H. CLOUET

and that there was no difference between tolerant and nontolerant dogs (Mu16 and Woods, 1962). In a study of the disposition of tritiated dihydromorphine in guinea-pig brain by Mule and his co-workers (1967) in which the brain was dissected in its anatomical divisions before analysis, the same conclusions were reached: there was no change in drug distribution in the nervous system in tolerant or withdrawn animals from that in animals receiving the first injection of dihydromorphine. In tolerant animals, the administration of nalorphine before m~rphine-*~C had no effect on label in brain, but increased the amount of labeled morphine remaining in brain when nalorphine was given 35 minutes after morphine (Mulk et al., 1962). These latter data provided no evidence that there was a displacement of the agonist by the antagonist during antagonism. It is, of course, possible in all of these studies that the reaction at receptor sites was masked by the relatively large amount of drug not at receptor sites in brain. Morphine administered in vivo was found mainly in the soluble fraction after the centrifugation of brain homogenates in order to isolate particulate and soluble fractions and had the same distribution when morphine was added to the homogenate before centrifugation (Kaneto and Mellett, 1960). Dihydromorphine was also found in highest concentration in the supernatant fraction of brain either in free form or lightly bound after administration in vivo, and there was no change in distribution during tolerance or nalorphine-provoked withdrawal (Van Praag and Simon, 1966).

2. Uptake and Transport Morphine and other narcotic drugs haw been found in the nervous system after subcutaneous, oral, intravenous, and intracisternal routes of administration. That there is a blood-brain barrier to the transport of narcotic alkaloids from blood to brain was suggested by studies concerning the relative pharmacological effects of morphine in experimental animals of various ages. The LD,, for 16-day-old rats was found to be 60 mgfkg, and for 3Zday-old rats 220 mglkg, increasing in the same time sequence as the development of the blood-brain barrier for other substances (Kupferberg and Way, 1963). However, the blood-brain barrier to morphine seems to develop independently from other narcotic drugs since the LD,, vaIues for morphine and codeine increase with age at different rates (Braeunlich, 1966), and no change with age have

BIOCHEMICAL RESPONSES TO NARCOTIC DRUGS

103

been seen for heroin or meperidine (Way, 1967). The development of a blood-brain barrier, at least for morphine, was confirmed by finding less drug in the brains from older rats than from younger rats given the same dose of drug (Kupferberg and Way, 1963). The choroid plexus, long considered one site of the blood-brain barrier system (see Lajtha, 1962), has been shown to take up morphine against a gradient (Takemori and Stenwick, 1966). The passage of narcotic drugs into brain was at first believed to be by way of passive diffusion. However, the uptake of morphine by choroid plexus in vitro was inhibited by the usual metabolic inhibitors such as dinitrophenol and ouabain, and was accumulated against a gradient, indicating active transport of the drug. The uptake of morphine was also inhibited by codeine and levallorphan in the same system (Takemori and Stenwick, 1966). Pieces of choroid plexus also accumulated dihydromorphine, nalorphine, levorphanol, and dextrorphan ( Hug, 1967). With these latter two drugs, which are stereoisomers, there are some indications of stereospecificity since the lev0 form had a higher uptake than the less active dextro form. The uptake of these drugs by plexus was very similar to that found in renal slices (Hug, 1967). Cerebral cortex slices had slightly less uptake in the same experimental situation than choroid plexus. Dihydromorphine was accumulated by cortex slices against a concentration gradient in vitro, with the uptake inhibited by dinitrophenol, ouabain, nitrogen atmosphere, and nalorphine (Scrafani and Hug, 1967). The inhibition of uptake of one narcotic drug by another was competitive, suggesting a single transport system. In the nervous system, there was no competitive inhibition by hexamethonium and other quaternary drugs, suggesting a separate transport of tertiary amines (Takemori and Stenwick, 1966). This was in contrast to transport in the kidney in which the cationic drug cyamine inhibited the uptake of morphine from plasma to glomerular filtrate (May et al., 1967). There is evidence that narcotic drugs affect the transport of other compounds into and out of the brain, not by competitive inhibition but by interference in the transport mechanism. In in vivo systems, the passage out of the brain of the amino acid leucine introduced into the cerebrospinal fluid was slower in morphinized rats than in untreated control animals (Clouet and Ratner, 1968). In in vitro systems using cerebral cortex slices, the uptake of lysine and a-isobutyric acid into slices, and the exit of the same compounds

104

DORIS H. CLOUET

from preloaded slices were inhibited in the presence of M morphine (Cherayil et al., 1967; Lajtha and Toth, 1965). This inhibition (11 to 21%)was less than the inhibition produced in the same systems by non-narcotic drugs such as proveratine, chlorpromazine, or tetraethyltin. I n summary, it has been shown that narcotic drugs reach the nervous system by active transport, are distributed throughout the nervous system in a heterogeneous fashion, are retained in the nervous system after plasma drug levels have fallen, and are found mainly in the soluble fraction of the cell, When the pharmacological state of the experimental animal is altered by narcotic drug administration, no gross changes in the distribution or levels of drug in the nervous system are found. If biochemical responses to drug administration are found to vary with the previous drug history of the animal, gross tissue levels of drug in the nervous system are not a cause, and alternative suggestions must be considered such as a change of drug level at the active biochemical receptor site( s ) in brain, or a change in sensitivity of the secondary mechanisms governing the pharmacological effects.

B. BIOTRANSFORMATIONS 1. Liver Drug-Metabolizing Enzymes In studies of the patterns of urinary excretion after the consumption of narcotic drugs, it was found that the metabolic reactions of the drugs included N-dealkylations, O-dealkylations, hydrolysis, and conjugations and in other studies that the site of this “detoxification” was the liver (Way and Adler, 1962). The enzymatic activity which catalyzed N-demethylation of morphine and other narcotics was localized in the liver microsomal fraction (Axelrod, 1955; La Du et al., 1955). It was soon established that narcotic drugs and antagonists such as nalorphine competed as substrates for dealkylation by liver microsomal preparations in vitro and that O-demethylation of codeine and N-dealkylation of nalorphine were catalyzed by the same liver microsomal preparations (Axelrod and Cochin, 1957). The emergence of the drug-metabolizing systems of the liver microsomal fraction as the major site of drug metabolism was a breakthrough in biochemical pharmacology which has been reviewed in excellent detail (Conney, 1967). It must be sufficient to say here that the biotransformations of narcotic drugs are similar

BIOCHEMlCAL RESl'ONSES

'lo N ARCOIIC

DRUGS

105

to those of drugs having other pharmacological activities in that the system requires oxygen, the generation of reduced triphosphopyridine nucleotide, and the microsomal respiratory chain (Axelrod, 1956; Clouet, 1964,1965). The metabolic reactions found in uitro in the presence of liver microsomal preparations isolated from many species of animals have also been demonstrated in vivo. The N-demethylated derivative, normorphine, has been recovered from urine after the administration of labeled morphine in the rat (Misra et al., 1961) and from liver in the cat (Tampier and Penna-Herreros, 1966). Normorphine has also been recovered as the N-dealkylated product of nalorphine from liver (Tampier and Penna-Herreros, 1966) and from brain (Milthers, 1962). Since the demethylation is oxidative, the oxidized methyl group appears as formaldehyde and subsequently as carbon dioxide. When morphine with the N-methyl group labeled by 14C was administered to animals, labeled carbon dioxide was found in expired air (March and Elliott, 1954; Adler, 1967). There are other reactions of narcotic drugs which have not been established as oxidative reactions involving the microsomal cytochrome system. The products of the hydrolysis of meperidine and normeperidine, meperidinic and normeperidinic acids, have been recovered from urine in free and conjugated forms (Plotnikoff et al., 1956). In guinea-pig liver slices the recovery of 15%of added methadone as the quaternary salt suggested that a methyl group may be added to the tertiary nitrogen (Schaumann, 1960). A hydroxylation of morphine and nalophine on the phenol ring by microsomal preparation from rabbit liver has been reported by Daly et at. ( 19%). Also in liver, the enzymes catalyzing glucuronide formation from morphine have been examined in detail: uridine diphosphate ( UDP) -glucose pyrophosphorylase and UDPglucose dehydrogenase were found in the supernatant fraction, and UDP-transferase in the microsomal fraction (Takemori, 1960). The O-deacetylation of heroin, first to Smonoacetylmorphine and then to morphine, occurred when heroin was incubated with homogenates from liver and brain or with blood (Way et al., 1960; Way, 1967). In tissues other than livrr, there have been few reports of metabolism of narcotic drugs. The N-dealkylation of morphine and nalorphine in the brains of living rats was described by Milthers ( 1962). However, no oxidative demethylation or even the presence

106

DORIS H. CLOUET

of the microsomal cytochrome P450 could be found in brain (Clouet, 1965; Inouye and Shinagawa, 1965). On the other hand, the remethylation of normorphine to morphine has been found in brain both in vitro and in vivo (Clouet et al., 1963; Clouet, 1963) and in lung (Axelrod, 1962). It has been assumed that metabolic transformations, particularly glucuronide formation, also occurred in the kidney, and evidence of such metabolism has been reported (May et al., 1967). The metabolism of drugs by tissues is, of course, one response by tissues to the administration of drugs. However, the bioconversions are catabolic, leading to excretion of metabolites. The importance of such reactions to this discussion is twofold: (1) The effective exposure of drug to tissue is defined by drug catabolism if the metabolite is less active or inactive, and ( 2 ) the active form of the drug may be identified. There have been two hypotheses concerning the mode of action of morphine based on N-demethylation, one suggesting the demethylating enzyme in liver as a model for brain receptors (Axelrod, 1956), the other suggesting dealkylation as a prerequisite for pharmacological activity ( Beckett et al., 1956). The finding that normorphine itself was a potent narcotic agent, especially when introduced into the cerebrospinal fluid (Lockett and Davies, 1958), seemed to preclude N-demethylation as a requirement for activity. However, the possibility of remethylation of normorphine in the nervous system (Clouet, 1963) has reopened the question of the importance of methyl transfer to activity. 2. The Effect of Chronic Drug Administration on Liver Enzymes In 1956 Axelrod reported that the chronic administration of morphine to male rats resulted in a substantial decrease in the activity of the liver drug-metabolizing enzymes catalyzing the N-demethylation of morphine in vitro. Since a change in drug catabolic rate might be related to decreasing pharmacological response ( tolerance), this observation touched off an explosion of research in many laboratories. The development of tolerance to chronic drug administration was found to parallel the decrease in liver demethylase activity (Cochin and Axelrod, 1959). Many narcotic drugs in addition to morphine were found to have the ability to decrease the activity of enzymes metabolizing the injected drug as well as other narcotic drugs (Remmer and Alsleben, 1958; Mannering and

BIOCHEMICAL RESPONSES I‘O NARCOTIC DRUGS

107

Takemori, 1959), and also to decrease the metabolism of nonnarcotic drugs such as hexabarbital by liver microsomal enzymes ( Remmer, 1960; Clouet and Ratner, 1964). This decrease in activity of the drug-metabolizing enzymes by narcotic drug administration was the reverse of the effect of the chronic administration of many non-narcotic drugs. The administration of phenobarbital, phenylbutazone, and many other drugs produced an increase in drugmetabolizing enzyme activity, called an induction ( see Conney, 1967). The administration of non-narcotic “inducer” drugs such as phenobarbital, reserpine, and meprobamate also induced an increase in the activity of liver microsomes in the N-demethylation of morphine, meperidine, acetylmethadol, and dihydromorphinone (Clouet and Ratner, 1964; McMahon et al., 1965; Shuster and Hannan, 1965). In quantitative terms, no correlation was found between liver enzyme activity and pharmacological response ( Herken et al., 1959). When the drug was discontinued, there was no parallelism in the recovery in the two parameters with liver enzyme activity, measured in vitro, returning to control values at a time when the analgesic response was only 40% of the initial level (Cochin and Econonom, 1959). The relationship of response in the two parameters was explored in a study in which the decrease in liver enzyme activity produced by daily morphine administration was prevented by the simultaneous administration of inducer drugs such as phenobarbital or reserpine (Clouet and Ratner, 1964). When the rate of tolerance development in rats receiving morphine and phenobarbital (liver demethylase activity 118%of control level) was compared to that of morphine alone (36% of control level), there was a very slight increase in the rate of tolerance development. This would be expected if the inducer, phenobarbital, increased the metabolism of morphine each day, so that the effective drug level was maintained for a shorter time each day. However, this effect on tolerance was minimal and indicated that the phenomenon of tolerance to morphine was not dependent on the level of activity in the liver of the drug-metabolizing enzymes. An explanation of the effect of chronic morphine administration on liver enzymes is probably related to a peculiarity of the activity of these enzymes in the liver of the male rat. In rats, a sex difference was found in the activity in liver, with higher activity in preparations isolated from the male rats (Axelrod, 1956; Remmer and Alsleben, 1958). The administration of the opposite sex hormone reversed

108

DORIS 1-1. CLOUET

the activity of enzymes metabolizing narcotic drugs in the sexes (Axelrod, 1956). Neither the sex differences nor the response of liver enzymes to morphine administration have been found in mice and guinea pigs (Kato and Onodo, 1966; Castro and Gillette, 1967). In summary, narcotic drugs are metabolized oxidatively by drug-metabolizing enzymes localized in the liver microsomal fraction. Although the level of these enzymes in liver may be increased by the administration of inducer drugs in many species of animal and may be decreased in the male rat by chronic morphine administration, the effect of these alterations in enzyme activity on pharmacological responses is minimal. A few other, possibly nonoxidative, reactions of narcotic drugs are found in liver and in nervous tissue and blood. There is no definitive evidence on the identity of the active form of the drug, or on the importance of any metabolic reaction to drug action. 111. Effects on General Metabolic Systems

A. INTERMFDIARY METABOLISM Many of the early studies on the effect of narcotic drugs on intermediary metabolism in the nervous system utilized tissue slices. When brain cortex slices were incubated in the presence of M morphine, there was no effect on oxygen consumption by the slice. However, the increase in the rate of oxygen consumption found M upon electrostimulation was inhibited in the presence of morphine and completely abolished in M morphine (Bell, 1958). Similarly, the increase in oxygen consumption due to potassium stimulation was inhibited in the presence of lC3M morphine (Takemori, 1961). In this latter study, a difference was found between brain slices from untreated and morphine-treated rats. The inhibition of increased oxygen consumption upon potassium stimulation in the presence of morphine in vitro was not found when the tissue was isolated from morphine-treated rats. During chronic morphine treatment, the recovery in brain cortex slices of response to potassium stimuIation in the presence of morphine took place graduauy ( Takemori, 196%) . There was cross-cellular adaptation of brain slices removed from morphine-treated rats to methadone and meperidine in vitro (Takemori, 1962b). The need for a low calcium-containing medium for inhibition of potassium stimulation of oxygen uptake in brain slices by morphine was shown by Elliott


109

et uZ. (1963), who pointed out that the level of calcium in the medium used by Takemori was lower than that specsed by McIlwain ( 1959). The increase in oxygen consumption in the presence of potassium in brain slices from tolerant animals was accompanied by an increase in glucose uptake b y the slice (Takemori, 1964). The glucose was utilized more rapidly as shown by the more rapid conversion of glucose-’*Cto carbon dioxide-”C by slices in the presence of morphine ( Takemori, 1967) . After the acute administration of a single dose of morphine to rats which were killed 1 hour later, there was an increase in the levels in brain of glucose, glucose-Sphosphate, adenosine triphosphate ( ATP), and phosphocreatin, as well as an increased activity of glucose-6-phosphate dehydrogenase in the cerebral hemispheres (Takemori, 1965; Dodge and Takemori, 1967). Dodge and Takemori (1967) have concluded that the first injection of morphine produced an increase in glucose uptake and utilization in brain, to which increase a tolerance developed during chronic morphine treatment. The administration of morphine or levallorphan to mice resulted in an increase in brain ATP levels and a decrease in adenosine diphosphate (ADP) levels 1 hour later, with a decrease in brain glycogen and phosphocreatin ( Estler, 1961). When morphine was administered to rats, ATP levels in brain were higher 1 hour later, but there was no change from control levels in glycogen (Estler and Ammon, 1964). These authors have attributed their specirsspecific results to a factor which must be considered in all of the studies reported here: that narcotic drugs have both depressant and excitant effects which vary with the dose and species, so that the varying biochemical responses may be related to the relative excitant and depressant effects in the animal (or vice versa). In isolated muscle very similar effects of morphine on glucose uptake were found. When rat diaphragm was incubated in the presence of morphine, the uptake of glucose was higher than in the absence of morphine in vitro ( Walsh et nl., 1964). However, when the muscle was isolated from addicted rats, there was less rather than more glucose uptake in the presence of morphine (Lee Pang and Walsh, 1962). The pathway of excess glucose utilization in the brains of morphine-treated rats has not become clear. In vitro, more glucose-

110

DORIS H. CLOUET

14C was oxidized to carbon dioxide in the presence of morphine: glucose labeled in the 2-carbon directly, and glucose labeled in the l-carbon or 6-carbon after a lag period (Takemori, 1967). In D~UO, the conversion of glucose-l'C to aspartate and 7-aminobutyric acid was slower in the brains of morphine-treated rats than in those from untreated animals ( Bachelard and Lindsay, 1965). Also in U ~ U O , there was a less rapid utilization of leucine-I4C injected into the cerebrospinal fluid in brains of morphine-treated rats, although the relative conversion of the labeled leucine to glutamate, glutamine, and y-aminobutyric acid was similar in treated and untreated animals (Clouet et al., 1967). One may conclude that morphine, at least in in vivo experiments, produced an increase in glucose utilization in brain, probably not via the pentose pathway of glucose oxidation, with a concurrent increase in brain ATP levels, with little of the energy going into the synthesis of such extrapathway compounds as y-aminobutyric acid or glutamic acid. The questions of which effect was the initial one, or whether the effects were direct or neurohormone-mediated have not been answered as yet. Some recent work in Escherichia coli and HeLa cells may be relevant. In whole bacteria grown in 3 x 10 M levallorphan, an immediate effect of exposure to the drug was an increased dephosphorylation of ATP to ADP with a subsequent leakage of ADP from the cells (Greene and Magasanik, 1967). By the process of elimination of other mechanisms, an activation of adenosinetriphosphatase ( ATPase ) was suggested as the mode of action of the narcotic drug. Many synthetic reactions requiring ATP were subsequently inhibited in E. coli as well as in cultured HeLa cells grown in the presence of levallorphan or levorphanol. It is possible that in the mammalian nervous system, too, an initial drop in ATP levels brings about an increase in glucose utilization and ATP synthesis with little diversion of available energy into other synthetic processes.

B. PROTEINMETABOLISM When rats which have received a single injection of morphine were killed at intervals thereafter, the protein-synthesizing system of microsomes isolated from liver showed a biphasic response in activity (Ratner and Clouet, 1964). From I to 2 hours after an injection of 30 mglkg morphine there was a substantial decrease in the ability of isolated microsomes to promote the incorporation


111

of amino acid into protein, followed at the 5- to 6-hour interval by an increase to 150%of control activity. The liver supernatant fractions (containing the activating enzymes, soluble ribonucleic acid (sRNA ), and transferases necessary for protein synthesis) were examined separately after morphine treatment and found to increase in activity 16 hours after a single injection of morphine. This inhibition persisted when liver ribosomes were examined for capacity to promote protein synthesis in uitro (Clouet and Ratner, 1968). In earlier work, the level of morphine and meperidine N-demethylase and other drug-metabolizing enzymes in liver microsomes was found to be 20% of control values in the period between 1 and 5 hours after a single injection of morphine in male rats (Clouet, 1964). Since male rats were also used in the studies of protein synthesis, it is possible that the biphasic response in protein synthesis in liver is related to changes in activity of the drug-metabolizing enzymes. The effects of morphine administration on protein synthesis in brain have also been examined. Two hours after the injection of 60 mg/kg morphine in rats, brain ribosomes had a decreased ability to incorporate labeled amino acid into protein in oitro, significantly different from the control level ( p < 0.01). At lower doses of morphine, the trend was toward significant inhibition in the protein-synthesizing capacity of brain ribosomes ( Clouet and Ratner, 1968). When morphine was added to the incorporation assay system at levels as high as A4, there was no inhibition of amino acid incorporation of amino acid into protein. A similar inhibition of protein synthesis in rat brain was observed after the administration of morphine to rats, when protein synthesis was measured in U ~ U O(Clouet and Ratner, 1967). Labeled leucine was injected directly into the cerebrospinal fluid and its incorporation into brain protein was measured after a 30-minute exposure to the label. In morphine-treated rats, there was a significant inhibition of leucine incorporation into the protein of the microsomal and soluble fractions of rat brain with maximal inhibition at 2 to 3 hours after a single injection of drug. When the turnover of whole brain proteins from untreated rats was compared to that from rats injected with morphine 2 hours earlier, there was a significant ( p < 0.001) decrease in turnover. The doses of morphine used in these experiments produced a hypothermia. Since the inhibition of protein synthesis in brain produced by the administration of chlor-

112

DORIS €I. CLOUET

promazine has been shown to be related to the hypothelmic effect of that drug (Shuster and Hannan, 1964), some experilnents were performed in which hypothermia was abolished by raising the ambient temperature to 30". In normothermic rats, there was also inhibition of protein synthesis in brain after the administration of morphine, so that the effect cannot be ascribed to a decrease in body temperature alone ( Clouet and Ratner, 1967). The rate of protein synthesis in whole brain of the rat returned to normal levels, or above, 20 hours after the administration of a single dose of morphine. In mice which had received chronic treatment with morphine, the protein level in the microsomal-supernatant fraction of whole brain was found to be significantly increased when the animals were killed the day after the last injection (Spoerlein and Scrafani, 1967) . In bacterial systems and in cell cultures, protein synthesis was inhibited in the presence of narcotic drugs. Escherichia coli grown in the presence of levorphanol had less protein synthesis (Simon and Van Praag, 1964), and HeLa cells grown in the presence of levallorphan showed a decrease to 10%of control level 1 hour after the drug was added to the culture medium ( Noteboom and Mueller, 1966). When the concentration of drug was increased to 3 x M, the inhibition in protein synthesis was apparent 2 minutes after exposure of cells to the drug (Greene and Magasanik, 1967). Because concurrent changes in the metabolism of nucleic acids have been found in rat brain and in E . coli and HeLa cells, the discussion of possible modes of action of the drugs on protein synthesis will be deferred until the end of the next section.

C. NUCLEKACID METABOLISM 1. Synthesis of R N A and DNA An inhibition of synthesis of RNA in E . coli in the presence of levorphanol was first reported by Simon (1963). The incorporation of 32Pinto RNA was inhibited when the cells were grown in the presence of 1.35 X hl levorphanol with the inhibition limited to ribosomal RNA. In HeLa cells grown in the presence of hl levallorphan, the incorporation of guanine into RNA was depressed with inhibition of the synthesis of ribosomal RNA (rRNA) soluble RNA, and rapidly labeled RNA (generally considered messenger RNA) (Noteboom and Mueller, 1966). These latter workers found


113

that the polysomes became dissociated upon exposure to levallorphan for 1 hour, and have ascribed the effect of the narcotic drug on RNA synthesis to a primary inhibition of protein synthesis. Coinpletc cessation of all incorporation of uracil-’ ’C into RN.4 and of thymidine-llC into deoxyribonuclcic acid ( D N A ) was found M levallorphan when E . coli were grown in the presence of 3 x (Greene and Magasanik, 1967). These workers have stressed the importance of drug level in the culture medium, ascribing Simon’s findings that only rRNA synthesis was inhibited to insufficient concentration of drug, so that a “shift down” in the bacterial metabolism occurred. (However, there is no evidence of the inhibition of specific RNA synthesis during shift down.) They have suggested that all of the effects of levallorphan on protein and nucleic acid synthesis in E . coli are due to an increased destruction of ATP in the drug-treated cultures. When the bacterial cell nucleotides were prelabeled by exposing the cells to labeled adenine before adding levallorphan, there was a shift from ATP to adenosine monophosphate ( A M P ) within the cells and a leakage of labeled nucleotides from the cells in the presence of narcotic drug (Greene and Magasanik, 1!367). In rat brain, the inhibition of protein synthesis found after the administration of morphine was attributed to a lack of stability of brain polyribosomes (Clouet, 1967). The synthesis of RNA in brain, measured by the incorporation of orotic acid-*% injected into the cerebrospinal fluid into brain RNA, was inhibited transiently by morphine treatment of the animal. Of more importance, perhaps, was a shift of RNA labeling toward heavier RNA, >28 S, presumably an indication of the synthesis of new rRNA. The inhibitory responses to narcotic drugs are very similar in the three types of organisms studied. However, it is not possible with the present state of information to reconcile the two biochemical modes of drug action suggested by the work which has just been described: (1) There is an interference with protein and RNA syntheses due to the interaction of drug and nucleic acid, and ( 2 ) the initial decrease in ATP levels leads to the subsequent inhibition of all synthetic processes. That narcotic drugs do react with nucleic acids has been shown by changes in the temperature of thermal denaturation of the nucleic acids in the presence of morphine, and in the quantitative precipitation of sRNA by levallorphan in the test tube (Clouet, 1967). The instability of polysomal forms in both rat

114

DORIS H. CLOUET

brain and IIeLa cells seems to support interference with RNA as a mode of action. In this regard, other alkaloids have been shown to inhibit the incorporation of uridine into RNA in cell culture (Creasey and Markiw, 1964). The inhibition in protein synthesis in HeLa cells resulting from exposure to ipecac alkaloids has been attributed to interference at the aminoacyl sRNA transfer step (Grollman, 1966). In favor of the second hypothesis is that a decrease in cellular ATP would result in an inhibition of protein and RNA synthesis such as has been seen in rat brain and cell cultures. It might also correlate with the observed changes in energy production in whole brain and cortex slices, if one postulates an increased destruction of ATP as stimulating the generation of energy for the renewal of ATP levels. 2. Tolerance and Inhibition of Protein and R N A Synthesis The question of whether adaptation in the nervous system to the chronic administration of narcotic drugs is a form of memory was introduced in the work of Cohen and his collaborators (1965). In these experiments, mice and rats which had received actinomycin D (an inhibitor of RNA, and consequently protein, synthesis) developed tolerance to the chronic administration of morphine at a rate significantly ( p < 0.001) slower than that in rodents receiving morphine alone. The use of another inhibitor of RNA synthesis, 8-azaguanine, also resulted in less tolerance, as measured by analgesia, in mice when given in conjunction with morphine than in mice receiving morphine alone (Yamamoto et aE., 1967; Spoerlein and Scrafani, 1967). Another pharmacological parameter of tolerance, the development of tolerance to the narcotic drug-induced lens opacity in mice, was used in the studies of Smith et al. (1966, 1967). These workers studied the effect of actinomycin D and puromycin (an inhibitor of protein synthesis) on the development of tolerance to a single injection of levorphanol and found that short-term tolerance was unaffected 2 hours after levorphanol administration, but that tolerance no longer existed or was significantly altered 6 hours after the drug was given. Both puromycin and actinomycin D blocked long-term tolerance for up to 20 days after inhibitor administration, The conclusion that protein and RNA synthesis are required in the nervous system for the development of tolerance to narcotic drugs is reasonable but premature, since the ubiquitous nature of


115

the biochemical effects of puroinycin and actinomycin I)is well known .

D. LIPIDMETABOLISM The only aspect of lipid metabolism in the nervous system which has been examined for response to narcotic drugs is the synthesis of phospholipids. When rat brain cortex slices were incubated in the presence of 2 x 10.’ A4 morphine, there was a 40% increase in the incorporation of a2P into phosphoinositides and phosphatidic acids (Brossard and Quastel, 1963). A closer examination of phospholipid metabolism in guinea-pig cerebral cortex slices revealed that the changes in synthetic rate in the presence of morphine depended on drug concentration and phospholipid studied ( Mulk, 1966). At the highest drug concentrations, the largest increases in synthesis were seen in the incorporation of 32Pinto di- and triphosphoinositides, phosphoinositol, phosphatidylethanolamine, and phosphatidic acid. At the same morphine concentration there was less incorporation of 32Pinto phosphotidylcholine in the presence, than in the absence of morphine. Similar results were obtained when glycer01-~~C or choline-14C were used as phospholipid precursors. The administration of morphine to guinea-pigs also increased the incorporation of 32Pinto brain phospholipids in eiuo (Mulk et al., 1967). It is premature to attempt to fit these results into a biochemical pattern. In the work with cortex slices, Mu16 found no difference in the specific activity of ATP-3zP in the presence or absence of morphine suggesting that the level of ATP was not limiting. Mulk (1966) suggested that the 1,2-diglyceride became limiting due to a shift in relative rates of synthesis of various phospholipids in the presence of morphine. IV. Effects on Specific Metabolic Reactions

A. AMINES

1. Catecholamines a. Brain. The observation that the administration of morphine produces a decrease in the level of norepinephrine was made first by Quinn and Brodie (1961). Six hours after the administration of 2S mg/ks morphine to cats, therc IYRS a 67%c1ecrc.ase in norepi-

116

DORIS H. CLOUET

nephrine levels with a return to control levels in 18 hours. The same phenomenon was found in the brains of dogs, rats, and rabbits (Maynert and Klingman, 1962; Maynert, 1967). The initial injection of morphine produced this pronounced decrease in norepinephrine levels, but after chronic drug treatment there was either no change or an increase in amine level. In rats which were tolerant to a high dose of morphine, there was a significant increase in brain catecholamine levels upon the injection of morphine (Sloan et al., 1963).In withdrawal, the response was reversed to a rapid depletion of brain catecholamines (Gunne, 1961, 1963). The response to nalorphine-induced withdrawal was similar to that of drug discontinuance: a decrease in brain levels of norepinephrine (Maynert and Klingman, 1962). In the hypothalamus, the level of catecholamines was decreased 1 hour after the injection of morphine into the cerebrospinal fluid in the cat (Moore et al., 1965). This depletion of norepinephrine was ascribed to a change in turnover since the level of precursor, dopamine, remained constant ( Laverty and Sharman, 1965). However, in whole brain, a decrease in dopamine was seen to precede the decrease in brain norepinephrine (Tagaki and Nakama, 1966). The modification by other drugs of morphine-induced analgesia has been related to their effects on brain catecholamine levels. The pretreatment of animals with reserpine in order to release catecholamines in nervous tissue effectively abolished the anaIgesic response to morphine (Verri et al., 1967). The analgesic response was potentiated by the concurrent administration of monoamine oxidase inhibitors such as iproniazid, which blocked the further metabolism of the released arnine (Gupta and Kulkarni, 19%). An injection of iproniazid every second day to rats actually prevented the development of tolerance to the chronic administration of morphine ( Chodera, 1963). h. Adrenal Gland. The administration of a single injection of morphine also caused a decrease in epinephrine levels in the adrenal (Maynert and Klingman, 1962). The levels of norepinephrine were more variable, but usually decreased after a single dose of morphine (Maynert and Levi, 1964). After chronic drug treatment, there was no longer a release of adrenal catecholamines. The patterns of urinary excretion of catecholamines followed the patterns of adrenal release: an increased excretion after a single dose of narcotic drug with subsequent lack of response to morphine


117

administration (Gunne, 1961, 1963). In man, too, an increase in urinary excretion of catecholamines was seen after the first injection of morphine, followed by a decrease during chronic treatment and another increase upon drug withdrawal ( Weil-Malherbe et al., 1965). Thus, an important response to the administration of narcotic drug to animals is release and depletion of catecholamines in the hypothalamus, whole brain, and the adrenal with a subsequent increase in urinary excretion. A n increased synthesis in order to maintain catecholamine concentrations during chronic drug consumption may result in normal or increased amine levels. Of course, many of the secondary pharmacological responses of narcotic drugs are mediated by the action of catecholamines. However, the exact biochemical link between the initial site of drug action and catecholamine release is now a subject for speculation. 2. Serotonin and 7-Aminobutyric Acid Neither of these neurohormones responded to the single or chronic administration of morphine ( Maynert et nl., 1962). 3. Acetylcholine and Acetylcholiiiesteiase

The application of morphine to the isolated ileum of guinea pig inhibited the release of acetylcholine upon electrostimulation (Schaumann, 1957). Adaptation to the presence of morphine was suggested by the return toward the level of response found in the absence of drug upon repeated stimulation ( Paton, 1957; Cox and Weinstock, 1966). In nervous tissue, the response to morphine was more complex. The release of acetylcholine from the superior cervical ganglion was inhibited by morphine (Pelikan, 1960). Infusion of morphine intraventricularly in the cat led to a decrease in acetylcholine release into the cerebrospinal fluid and to a higher level of acetylcholine in brain tissue ( Beleslin and Polak, 1965). After a single large dose of morphine, the rise in brain acetylcholine in mice reached a maximum 30 minutes after the injection ( Hano et al., 1964). During the development of tolerance to morphine, the rise in brain acetylcholine became smaller after each injection, and returned to the rame as the initial response 15 days after the drug was withdram. In brain slices, the addition of potassium chloride resulted in a release of acetylcholine from the slice, which was inhibited in the

118

DORIS H. CLOUET

presence of M morphine (Hano et al., 1964). The entrance of acetylcholine into brain cortex slices was also inhibited by morphine in the medium (Schuberth and Sundwall, 1967). In this study, the inhibition between acetylcholine and morphine was comM. petitive with a KI of 3 x There is no doubt that morphine and other narcotic drugs inhibit acetylcholinesterase (Schaumann, 1959; Johannesson, 1962; Lane et al., 1966; Dewey and Harris, 1967). An evaluation of the kinetic constants of inhibition suggested that the competition was mixed; for morphine and nalorphine the major component of inhibition was not competitive with acetylcholine at low drug level, and for levallorphan the competitive component was large at low drug level (Hein and Powell, 1967). When morphine and acetylcholine concentrations were the same ( M ), the inhibition of acetylcholinesterase was competitive (Johannesson, 1962). However, no relation was found between the inhibition of acetylcholinesterase in vitro and the pharmacological activity of various narcotic and related drugs (Lane et al., 1966; Hein and Powell, 1967). The activity of acetylcholinesterase in whole brain homogenates was no different whether the brains were from tolerant or nontolerant animals (Johannesson, 1962). The activity of choline acetylase in brain was not inhibited during the infusion of morphine into the cerebrospinal fluid (Beleslin and Polak, 1965). The inhibition of release and uptake of acetylcholine in nervous tissue by narcotic drugs, and the inhibition of acetylcholine hydrolysis by acetylchoIinesterase by narcotic drugs suggest competition at receptor sites for both transport and hydrolysis of the neurohormone by morphine and other narcotic drugs. In this regard, a compound more nearly like acetylcholine structurally, the quaternary salt of morphine, N-methylmorphine, produced no analgesia or hypothermia when injected systemically, but was very active in both parameters when injected into the hypothalamus (Foster et al., 1967).

B. HORMONES 1. Corticosteroids As might be predicted from the involvement of the pituitaryadrenal system in secondary responses to the administration of narcotic drugs, the efflux of corticosteroids from the adrenal was in-


119

creased by a single injection of morphine (Slusher and Browning, 1961). Chronic treatment of rats with morphine had a biphasic effect; in the earliest period, both aldosterone and hydroxysteroid levels in plasma were considerably lower than in untreated animals; after 140 days of treatment, the plasma corticosteroid levels were above control level (Paroli and Melchiorri, 1961a). In chronically addicted man, the plasma corticosteroid levels remained low, not changing with drug administration ( Eisenman et al., 1961) . However, in tolerant animals the adrenal glands were able to respond to the administration of trophic hormone with a release of corticosteroids ( Paroli and Melchiorri, 1961b; Eisenman et al., 1961). 2. Pituitary Hormones The release of corticosteroids from the adrenal gland described in the preceding section is one measure of the release of adrenocorticotropic hormone ( ACTH ) from the pituitary. Another measure is the depletion of adrenal ascorbic acid. In morphine-treated animals, there was a pronounced depletion of adrenal ascorbic acid by morphine, again indicating the mediation of pituitary hormones in the response to morphine (Burdette et al., 1961). The stressinduced increase in plasma corticosterone in rats has been used as an index of the action of the corticotropin-releasing factor in the hypothalamus. The administration of morphine inhibited this response to stress (Schally et al., 1965). The involvement of the thyrotropic hormone (TSH) in the secondary responses to drug administration was indicated by finding enlarged thyroid glands and a decreased ability to concentrate 1311in chronically morphinized animals (Sung et al., 1953). The uptake of I3lI was inhibited by many narcotic drugs administered in uiuo, hut was not inhibited in hypophysectomized animals (Redding et al., 1966). The release of l3lI from the thyroid gland, also a measure of TSH, was inhibited by morphine administration of morphine to rats (Lomax and George, 1966). The antidiuretic hormone ( A D H ) also was released from the pituitary upon morphine treatment ( George and Way, 1959). The blood level of this posterior pituitary hormone increased after a single injection of levorphanol, with adaptation in the response in chronic treatment ( Newsome et al., 1963). The excretory patterns of sodium in chronically morphinized rats reflected these changes in blood ADH (Marchand and Fujimoto, 1966). A depletion of

120

DORIS H. CLOUET

both ADH and oxytocin was demonstrated in the posterior pituitary following morphine injection (Rodeck and Braukmann, 1966). That an increase in the release of ACTH and ADH was compatible with a decrease in the release of TSH with morphine administration was indicated in the experiments of Lomax and George ( 1966). When lesions were made in the posterior hypothalamus, there was no inhibition of TSH release by morphine. Since the hypothalamus is known to have both stimulatory and inhibitory control of pituitary hormone release, Lomax and George have demonstrated that the effect of morphine on TSH is a stimulation of inhibition of release. The localization of the control of various pharmacological responses to narcotic drugs in the hypothalamus has been explored by microablation and intracerebral microinjection techniques (Lotti et al., 1965; Lomax and George, 1966). Ablation of the medial mammillary nuclei of the caudal hypothaIamus prevented the effect of morphine on TSH release, and the injection of morphine in the area of the anterior hypothalamic nuclei produced a hypothermic response.

c. CALCIUM In the studies of the effect of morphine on oxygen uptake in cerebral cortex slices, the importance of calcium ions to the inhibition of potassium-stimulated increase in oxygen was indicated by Elliott et al. (1963). When the cortex slices were prepared from brains of tolerant animals, there was no longer any inhibition of potassium stimulation in the presence of morphine. An explanation of this difference between the brains of tolerant and nontolerant animals may be related to the concentration of calcium ions in the brains. After the first injection of morphine to mice, there was a significant ( P < 0.05) drop in the level of brain Cayminimal in 30 minutes, and returning to control within 24 hours (Shikimi et al., 1967a). In chronically morphinized animals the level of Ca in brain did not change (Shikimi et al., 196%). The injection of Ca into the cerebrospinal fluid decreased the analgesic effect of morphine or meperidine, while Ca complexers such as sodium citrate or the sodium salt of EDTA similarly introduced enhanced the drug-induced analgesia (Kakunaga et al., 1966). Ca ion has been shown to be involved in the effect of morphine on the reIease of neurohormones in experiments in which there was


121

no inhibition of acetylcholine release from brain slices in the presence of morphine when the medium was Ca-free (Hano et al., 1964). The need for Ca in the catecholamine release in the adrenal as well as for the regulation of nervous tissue and maintenance of membrane integrity has long been known (see Brink, 1954; Douglas, 1966; Abood, 1966). One would suspect that the effect of narcotic drugs on the level of Ca is approaching the initial site of the biochemical action of the drugs. V. Serum and Brain Factors

A. SERUMFACTORS In the recent literature, there have been several reports concerning the effect of the injection of serum from tolerant animals on the response to narcotic drugs. In rats, the injection of serum from tolerant dogs or monkeys potentiated the analgesic action of morphine (Kornetsky and Kiplinger, 1963). Serum prepared from tolerant dogs or man also increased the analgesic activity of morphine in mice (Kiplinger and Clift, 1964). However, there was less pharmacological response to morphine when serum from tolerant rabbits was given to mice (Cochin and Kornetsky, 1964). It is possible that the time between drug treatment and blood removal may be important in resolving these differences in the effect of serum from tolerant animals since both hypo- and hyperreactivity to a second injection of morphine are found at various times after the first injection.

B. BRAINF A ~ O R S When a homogenate prepared from the brain of a tolerant rat was injected intrapentoneally into naive mice, there was less analgesic response to the administration of morphine than in mice receiving morphine alone ( Ungar, 1965). In similar experiments the injection of whole brain homogenates from tolerant animals had no effect on morphine-induced analgesia (Tirri, 1967). The question of whether tolerance can be transferred by brain extracts may be resolved by experiments such as those described in Section III,C, in which the involvement of protein or ribonucleic acid synthesis in the development of tolerance to chronic drug treatment was explored.

122

DORIS H. CLOUET

VI. Conclusions

An attempt to integrate all of the known biochemical consequences of the administration of narcotic drugs to animals into a single scheme of response is premature, because there is no conclusive experimental evidence on the two most important pieces of information needed in order to discern a pattern of response: the biochemical and the anatomical sites of the initial drug/receptor interaction. In the design of many of the experiments described in this chapter, the intention was to approach the initial biochemical lesion by starting with known reactions and backtracking step by biochemical step. In this way, some partial metabolic sequences of drug action have been demonstrated. A consideration of the chemical relationship between narcotic drugs and acetylcholine and between narcotic drugs and steroid hormones suggests that there may be at least two types of drug/receptor action in two distinct metabolic pathways. The widespread responses to drug administration in the central nervous system and in the peripheral nervous system, as well as in non-nervous tissue, are suggestive of multiple anatomical sites of drug/receptor action. Multiple anatomical sites may require many chemically similar reactions, or many reactions of drug/tissue components which are not related to each other metabolically. One, thus, may anticipate finding several initial sites of biochemical action. Narcotic drugs behave in the animal body like any other large ionizable lipid-soluble compound: the drugs are taken up through membrane barriers, bound in tissue against a concentration gradient with no apparent site of accumulation, metabolized by the liver detoxifying reactions into hydrophilic metabolites, and excreted. There are no firm clues as to the identity of the active metabolite or to the necessity of drug catabolism for pharmacological effects. A number of metabolic consequences of narcotic drug administration may be eliminated from consideration as the site of the initial biochemical interaction. The release of catecholamines and steroid hormones from the adrenal gland and the release of trophic hormones from the pituitary gland are controlled by hypothalamic mechanisms, which are directly affected by the presence of narcotic drugs, so that these release phenomena may be classified as seeondary reactions. The effects of narcotic drugs on IeveIs of acetyIchoIine in brain

BIOCHEMICAL RESPONSES

'ro

NARCOTIC DRUGS

123

and on activity of cholinesterases seem to be straightforward competitive reactions with the drug (possible in quaternary form) competing with acetylcholine transport or hydrolysis. In this response, the unknown factor is the importance of the reaction to the pharmacological effects, including tolerance, of drug administration to animals. There are a number of reactions which must be accommodated in any general scheme of response. The decrease in calcium levels in brain in morphinized animals may be related to the changes in transport of ATP seen in tissue cultured in the presence of narcotic drugs, and to the changes in phospholipid synthesis found in the brains of morphinized animals, since membrane phospholipids have been implicated in the permeability of membranes to cations with concurrent ATP hydrolysis and resynthesis. The levels of ATP and calcium in brain slices are also related to the utilization of oxygen and glucose by the slice, since the ATP/ADP ratio is a major controlling factor for energy production (and for the activity of individual enzymes of the citric acid cycle) and low levels of calcium are known to increase the oxygen utilization by brain slices (see Abood, 1966, for discussion of the relation of ATP and calcium in nervous tissue ) . Which reaction initiates the changes in metabolism, and whether all of these reactions can be demonstrated in a single tissue remain to be determined. The interference of narcotic drugs in nucleic acid metabolism of microorganisms and of mammalian brain may be dependent on an initiaI drug nucleic acid chemical interaction, or may be related to energy ( ATP) levels or to a decrease in the rate of the synthesis of a particular protein. On the other hand, the inhibition of protein synthesis seen in animals and in HeLa cells treated with narcotic drugs may be consequent to the inhibition of ribonucleic acid synthesis. For ribonucleic acid synthesis ATP is required, but the levels of nucleotides, including ATP, in the free nucleotide pool are controlled by the rate of nuclric acid synthesis. Thus, in this group of interrelated effects, neither the biochemical site of initial drug action nor the importance of the various biochemical effects to pharmacological activity has been elucidated. The scheme of biochemical response to narcotic drugs is incomplete, but progress has been made in identifying many biochemical events which seem to be interrelated, and which may be part of the biochemical basis of narcotic drug action. The challenge for future

124

DOFUS H. CLOUET

research in this area is the need €or the identification of a unique sequence( s ) of events which occurs when narcotic drugs react with drug receptors. REFERENCES Abood, L. G. (1966). Intern. Reu. Neurobiol. 9,223. Adler, T. K. (1967). 1. Pharmucol. Exptl. Therap. 156,585. Axelrod, J. (1955). J . Pharmucol. Erptl. Therap. 114,430. Axelrod, J. (1956). J. Pharmcol. E r p f l . Therap. 117,322. Axelrod, J. (1962). J. Pharmacol. Ezptl. Therap. 13628. Axelrod, J., and Cochin, J. (1957). J. Pharmucol. Exptl. Therap. 121, 107. Bachelard, H. S., and Lindsay, J. R. (1966). Biochem. Pharrnucol. 15, 1053. Beckett, A. H., Casy, A. F., and Harper, N. J. (1956). 1. Pharm. Pharmacol. 8, 874. Beleslin, D., and Polak, L. (1965). j . Physiol. (London) 177, 411. Bell, J. L. (1958). J. Neurochem. 2, 265. Bentley, K. W., Boura, A. L. A., Fitzgerald, A. E., Hardy, D. G., McCoubrey, A., Aikman, M. L., and Lister, R. E. (1965). Nature 206, 102. Braeunlich, H. (1966). Acta Biol. Med. Ger. 16, 178. Brink, F. (1954). Pharmacol. Aeu. 6, 243. Brossard, M., and Quastel, J. H. (1963). Biochem. Pharmacol. 12, 766. Burdette, B. H., Leeman, S., and Munson, P. L. (1961). J. Pharmacol. Exptl. Therap. 132, 323. Carabateas, P. M., and Harris, L. S. ( 1966). J. Med. Chem. 9, 6. Castro, J. A., and Gillette, J. R. (1967). Biochem. Biophys. Res. Commun. 28, 426. Cherayil, A., Kandara, J., and Lajtha, A. (1967). J. Neurochem. 14, 105. Chodera, A. (1963). Arch. Intern. Pharmacodyn. 142, 362. Clouet, D. H. (1963). Biochem. Pharmacol. 12,467. Clouet, D. H. (1964). J. Phrmacol. Exptl. Therap. 144, 354. Clouet, D. H. (1965). Life Sci. 4, 365. Clouet, D. H. (1967). Psychopharmacol. Bull. 4, 34. Clouet, D. H., and Ratner, M. (1964). J. Pharmacol. ErptZ. Therap. 144, 362. Clouet, D. H., and Ratner, M. ( 1967). Brain Res. 4, 33. Clouet, D. H., and Ratner, M. (1968). J . Neurochem. 15, 17. Clouet, D. H., Ratner, M., and Kurzmann, M. (1963). Biochem. Pharmacol. 12,957. Clouet, D. H., Okimoto, K. O., and Ratner, M. (1967). Proc. 7th Intern. Congr. Biochem., Tokyo, 1967 p. 976. Sci. Council Japan, Tokyo. Cochin, J., and Axelrod, J. (1959). J . Pharmacol. Exptl. Therap. 125, 105. Cochin, J., and Economon, S. ( 1959). Federation Proc. 18, 377. Cochin, J., and Kornetsky, C. ( 1964). J. Pharmacol. Exptl. Therap. 145, 1. Cohen, M., Keats, A. S., Krivoy, W., and Ungar, G. (1965). Proc. SOC. Exptl. Biol. Med. 119, 381. Conney, A. H. (1967). Pharmacol. Reu. 19, 317. Cox, B. M., and Weinstock, M. ( 1966). Brit. J . Phrmacol. 27, 81.


125

Creasey, W. A,, and Markiw, M. E. (1964). Biochim. Biophys. Acta 87, 593. Daly, J., Inscoe, J. K., and Axelrod, J. (1965). J. Med. Chem. 8, 153. Dewey, W. L., and Harris, L. S. (1967). Pharmacologist 9, NO. 259,230. Dodge, P. W., and Takemori, A. E. ( 1967). Pharmacologist 9, No. 19,218. Douglas, W. W. (1966).P h a m c o l . Reu. 18, 471. Eisenman, A. J., Fraser, H. J., and Brooks, J. W. (1961). J. Pharmacol. Exptl. Therap. 132, 266. Elliott, H. W., Kokka, N., and Way, E. L. (1963). Proc. SOC. Exptl. Biol. Med. 113, 1049. Estler, C. J. (1961). Bioclzem. Pharmucol. 8, 68. Estler, C. J., and Ammon, H. P. T. (1964). J. Neurochem. 11, 511. Foster, R. S., Jerden, D. J., and Lomax, P. (1967). J. Pharmacol. Exptl. Therap. 157, 185. George, R., and Way, E. L. (1959). J. Pharrnacol. Erptl. Therap. 125, 111. Greene, R., and Magasanik, B. (1967). Mol. Pharmacol. 3, 453. Grollman, A. P. (1966). Proc. Natl. Acad. Sci. U.S. 56, 1867. Gunne, L.-M. ( 1961). Psychopharmucologia 2,214. Gunne, L.-M. (1963). Acta Physiol. Scand. 58,204. Gupta, S. K., and KuIkami, H. J. (1966). J . Indian Med. Assoc. 46, 197. Hano, K., Kaneto, H., Kakunaga, T., and Moribayashi, N. (1964). Biochem. Pharmucol. 13, 441. Hein, G. E., and Powell, K. (1967). Biocheni. Pharmucol. 16,567. Herken, H., Neubert, D., and Timmler, R. (1959). Arch. Exptl. Pathol. Pharmacol. 237, 319. Hug, C. C. (1967). Biochem. Phnrinacol. 16,345. Inouye, A., and Shinagawa, Y. ( 1965). J. Neurochem. 12,803. Johannesson, T. (1962). Acta Pharmacol. Toxicol. 19, 23. Kakunaga, T., Kaneto, H., and Hano, K. (1966). J. Pharmucol. Exptl. Therap. 153, 134. Kaneto, H., and Mellett, L. B. (1960). Pharmacologist 2,98. Kato, R., and Onodo, K. (1966). Japan J. Pharmacol. 16,217. Kiplinger, G. F., and Clift, J. W. (1964). J . Phannacol. Exptl. Therap. 146, 139. Kornetsky, C., and Kiplinger, G. F. (1963). Psychopharmacologia 4, 66. Kosterlitz, H. W., Thompson, J. W., and Wallis, D. 1. (1964). Nature 201, 719. Kupferberg, H. J., and Way, E. L. ( 1963). J. Pharmucol. Exptl. Therap. 141,

105. La Du, B. N., Gaudette, L., and Trousof, N . (1955). J. Biol. Chem. 214, 741. Lajtha, A. (1962). In “Neurochemistry” ( K . A. C. Elliott, I. H. Page, and J. H. Quastel, eds.), p. 399. Thomas, Springfield, Illinois. Lajtha, A., and Toth, J. (1965). Biochem. Pharmacol. 14, 729. Lane, A. C., Macfarlane, I. R., and McCoubrey, A. (1966). Biochern. Pharmawl.15, 122. Laverty, R., and Shaman, D. F. (1965). Brit. J. Pharmacol. 24, 759. Lee Pang, C. H., and Walsh, E. O’F. ( 1962). Nature 196, 171. Lockett, M. F., and Davis, M. M. (1958). J. Pharm. Pharmacol. 10, 8 . Lomax, P., and George, R. (1966). Brain Res. 2, 361.

126

DORIS H. CLOUET

Lotti, V. J., Lomax, P., and George, R. (1965). 3. Pharmucol. Exptl. Therap. 150, 135. McIlwain, H. (1959). In “Biochemistry and the Central Nervous System,” 2nd ed., p. 71. Little, Brown, Boston, Massachusetts. McMahon, R. E., Culp, H. W., and Marshall, F. J. (1965). J. Pharmacol. Exptl. Therap. 149, 436. Mannering, G. J., and Takemori, A. E. (1959). J. Pharmacol. Exptl. Therap. 127, 187. March, C. H., and Elliott, H. W. (1954). P70C. SOC. Exptl. Biol. Med. 86, 494. Marchand, C. R., and Fujimoto, J. M. (1966). Proc. SOC. Exptl. Biol. Med. 123, 600. May, D. G., Fujimoto, J. M., and Inturrisi, C. E. (1967). J . Pharmacol. Exptl. Therap. 157, 626. Maynert, E. W. (1967). Federation Proc. 26, 111. Maynert, E. W., and Klingman, C. I. (1962). J. Pharmacol. Exptl. Therap. 135, 285. Maynert, E. W., and Levi, R. (1964). J. Pharmacol. Exptl. Therap. 143, 90. Maynert, E. W., Klingman, G. I., and Kaji, H. K. (1962). J. Pharmacol. Ezptl. Therap. 135, 296. Miller, J. W., and Elliott, H. W. (1955). J. Pharmacol. Exptl. Therap. 113,283. Milthers, K. ( 1962). Nature 195, 607. Misra, A. L., Mul6, S. J., and Woods, L. A. (1961). 1. Pharmacol. Exptl. Therap. 132, 317. Moore, K. E., McCarthy, L. E., and Borison, H. L. (1965). J . Pharmacol. Exptl. Therap. 148, 169. Mul6, S. J. (1966). 3. Pharmacol. Exptl. Therap. 154, 370. MuE, S. J., and Woods, L. A. ( 1962). 3. Pharmacol. Exptl. Therap. 136, 232. Mu]$ S. J., Woods, L. A., and Mellett, L. B. (1962). J. Pharmacol. Exptl. Therap. 136, 242. Mul6, S. J., Redman, C. M., and Flesher, J. W. (1967). J. Pharmacol. Exptl. Therap. 156, 93. Newsome, H . H., Tobin, W., and Fujimoto, J. M. (1963). I. Pharmacol. Exptl. Therap. 139, 368. Noteboom, W . D., and Mueller, G. C. (1966). Mol. Pharmacol. 2, 534. Paroli, E., and Melchiorri, P. (1961a). Biochem. Pharmacol. 6, 1. Paroli, E., and Melchiorri, P. (1961b). Biochcm. Pharmucol. 6, 18. Paton, W. D. M. (1957). Brit. J . Pharmacol. 12, 119. Pelikan, E. W. (1960). Ann. N.Y. Acad. Sci. 90,52. Plotnikoff, N. P., Way, E. L., and Elliott, H. W. (1956). J . Phurmacol. Exptl. Therap. 117, 414. Quinn, G. P., and Brodie, B. B. ( 1961). Med. Exptl. 4, 349. Ratner, M., and Clouet, D. H. (1964). Biochem. Pharmacol. 13, 1655. Redding, T. W., Bowers, C. Y., and Schally, A. V. (1966). Acta Endocrinol. 51, 391. Remmer, H. ( 1960). Arch. Exptl. Pathol. Pharmakol. 338, 35. Remmer, H., and Alsleben, B. (1958). Klin. Wochschr. 36, 332. Rodeck, H., and Braiikmann, R. (1966). Z. Zellforsch. Mikroskop. Anat. 75, 517.


127

Schally, A. V., Carter, W. H., Hearn, I. C., and Bowers, C. Y. (1965). Am. J. Physiol. 209, 1169. Schaumann, W. (1957). Brit. J. Pharmacol. 12, 115. Schaumann, W. ( 1959). Arch. Exptl. Pathol. Pharmokol. 237, 229. Schaumann, W. ( 1960). Arch. Exptl. Pathol. Phannakol. 239,311. Schuberth, J., and Sundwall, A. ( 1967). J. Neurochem. 14,807. Scrafani, J . T., and Hug, C. C. (1967). Pharmacologist 9, No. 192, 218. Seevers, M. H., and Deneau, G. A. (1963). Physiol. Pharrnacol. 1, F , 1. Shikimi, T., Kaneto, H., and Hano, K. (1967a). Japan. J. Pharmacol. 17, 135. Shikimi, T., Kaneto, H., and Hano, K. (196713). Japan. J. Pharmacol. 17, 136. Shuster, L., and Hannan, R. V. (1964). J. Biol. Chem. 239,3401. Shuster, L., and Hannan, R. V. (1965). Can. J. Biochem. 43, 899. Simon, E. J. ( 1963). Nature 198, 794. Simon, E. J., and Van Praag, D. ( 1964). Proc. Nut. Acad. Sci. U.S. 51, 877. Sloan, J. W., Brooks, J. W., Eisenmann, A. J., and Martin, W. R. (1963). Psychopharmacologia 4, 261. Slusher, M. G., and Browning, B. ( 1961). Am. J. Physiol. 200, 1032. Smith, A. A., Karmin, M., and Gavitt, J. (1966). Iliochem. Pharnzacol. 15, 1877. Smith, A. A., Karmin, M., and Gavitt, J. (1967). J . Pharmacol. Exptl. Therap. 156, 85. Spoerlein, M. T., and Scrafani, J. (1967). Life Sci. 6, 1549. Sung, C. Y.,Way, E. L., and Scott, K. G. (1953). J. Pharmacol. Exptl. Therap. 107, 12. Szerb, J . C., and McCurdy, D. H. (1956). J. Pharmacol. Exptl. Therap. 118, 446. Tagaki, H., and Nakama, M. ( 1966). Japan. J. PharniacoL 16, 483. Takemori, A. E. ( 1960). J . Pharmacol. Expll. Therup. 130, 370. Takemori, A. E. (1961). Science 133, 1018. Takemori, A. E. (1962a). J. Pharmacol. Exptl. Therap. 135, 89. Takemori, A. E. ( 1962b). J . Pharmacol. Exptl. Therap. 135, 252. Takemori, A. E. (1964). J . Pha~nmcol.Exptl. Therap. 145, 20. Takemori, A. E. (1965). J . Neurocheni. 12, 407. Takemori, A. E. (1967). BiochPm. Pharmacol. 16, 87. Takemori, A. E., and Stenwick, M. W. (1968). J. Phurmacol. Ezptl. Therap. 154, 586. Tampier, L., and Penna-Herreros, A. ( 1966). Arch. B i d . Med. Ezptl. 3, 146. Tirri, R. (1967). Experimentiu 23, 278. Ungar, G. (1965). Federation Proc. 24, 548. Van Praag, D., and Simon, E. J. (1966). Proc. SOC. Exptl. B i d . Med. 122, 6 Verri, R. A., Groeff, F. G . , and Corrado, A . P. (1967). J. Pharm. Pharmacol. 19, 264. Walsh, E. O’F., Lee Pang, C. H., and Ng, M . (1964). Nature 204, 698. Way, E. L. (1967). Federation Proc. 26, 11 15. Way, E. L., and Adler, T. K. (19fi2). Ru11. World Health Organ. 25, 227; 26,51 and 261; 27,359. Way, E. L., Kemp, J. W., Young, 3, M., and Grassetti, D. R. (1960). J. Pharmacol. Exptl. Therap. 129, 144.

128

DORIS H. CLOUET

Weil-Maherbe, H., Smith, E. R. B., Eisenman, A. J., and Fraser, H. F. (1965). Biochem. Phormacol. 14, 1621. Woods, L. A. (1959). J . Pharmacol. Exptl. Therap. 120,58. Workshop on Detection of Narcotics. (1966). Psychopharmcol. Bull. 3, 21. Yamamoto, I., Inoki, R., Tamari, Y., and Iwatsubo, K. (1967). Jopon. J . P h a m c o l . 17, 140.

PERIODIC PSYCHOSES IN THE LIGHT OF BIOLOGICAL RHYTHM RESEARCH By F . A . Jenner M.R.C. Unit for Metabolic Studies in Psychiatry. Middlewood Hospital. and University Department of Psychiatry. Whiteley Wood Clinic. Sheffield. England

I . Introduction . . . . . . . . I1. Evidence for the Existence of Periodic Psychoses I11. Nosology and Periodic Psychoses . . . . . . . . IV. Periodic Illnesses in General . V . Richter’s Hypotheses . . . . . . VI . Circadian Rhythms . . . . . . . VII. Cellular Studies . . . . . . . VIII . Mathematical Considerations . . . . . IX . Survival Value . . . . . . . . . . . X. The Menstrual and Estral Clocks . XI . Estrogens, Androgens. and Animal Behavior . XI1 . Estrogens. Androgens. and Human Behavior . XI11. Light and the Menstrual Cycle . . . . XIV. Thyroid Activity and Periodic Psychoses . . XV . Vasopressin and Periodic Psychoses . . . XVI. Early Work on Periodic Psychoses . . . XVII . Gjessing’s Studies . . . . . . . XVIII . The Adrenal Cortex and Periodic Psychoses . . XIX . Catecholamines . . . . . . . . XX . Autonomic Concomitants of Periodic Psychoses . XXI . Electroencephalography . . . . . . . . . XXII . Lithium and Periodic Psychoses . References . . . . . . . .

.

. . .

. .

.

. . .

. .

. . .

. .

. . . .

. .

. .

. .

. .

. .

. . . .

. . . .

. . . .

. . . . . .

.

. .

.

. . . . . .

.

. .

. . . . . .

.

129 130 132 133 137 138 139 139 140 141 142 144 148 149 151 152 152 154 156 157 157 158 160

I . Introduction

In this review some of the unsolved problems associated with the periodic psychoses will be emphasized. In addition the possible relationship between these problems and other aspects of biological rhythms will be explored. No attempt has been made to present every aspect of the field. In particular. Russian and other Slavonic work has been almost neglected . A survey of the available literature shows this latter omission to be especially unfortunate in view of the number of relevant titles located (see Lupandin. 1965; Burmistrova. 129

130

F. A. JENNER

1964; Stankevich, 1964; Zaimov et al., 1965; and in Rumanian, Constantinescu and Cristodorescu, 1965). The subject has also attracted considerable attention in Japan where one suspects some of the best current studies are being performed (see Takahashi, 1965; Wada et al., 1964a,b); fortunately many Japanese studies are available to us in English articles. For the purposes of this review, a periodic psychotic can be described as a patient whose gross psychiatric disturbance recurs at regular intervals; the validity of this definition can be questioned by anybody who has taken the trouble to observe and record the recurrences. Precisely timed psychoses are extremely rare, but as Abe (1965) points out, the nearly regular syndromes in psychiatry form one end of the spectrum of remitting psychoses in which no clearly defined boundaries can be drawn. Abe (1965) also shows that in the longitudinal statistical analyses of the records of many less convincingly periodic psychotics a tendency to be regular can often be discerned. For this and other reasons one hopes that insight gained from studying exotic clinical material might be of more general relevance and perhaps explain important factors controlling normal human mentation, as well as defects leading to illness. II. Evidence for the Existence of Periodic Psychoses

It is almost tautological to state that there can hardly be an inviolable biological clock. Evidence for the existence of clocks cannot be refuted, however, simply because they can be influenced. Indeed a number of examples of environmental factors affecting otherwise precise rhythms of psychotic behavior are to be found in the literature. Jenner ( 1963), for example, described the disruption of a rhythm of psychosis by moving a patient to a strange ward. On four occasions this patient with a 6-7-day cycle of psychosis, first described by Crammer (1959a), responded in a quite predictable manner. On each occasion the reestablishment of the rhythm took 5 to 6 months, but with the exception of these periods and times of pharmacological interference the cycle has been present for 18 years and still persists. In another patient who for 13 years had a 48-hour cycle (Jenner et al., 1967) the rhythms could be changed to a 44hour cycle by living to an abnormal timetable of 11hours light and social activity and 11 hours darkness and sleep (Jenner et al., 1968). R. Gjessing (1968) has also presented very clear evidence of the

PERIODIC PSYCHOSES

131

effects of diet on psychotic cycles. Further, the release of cycles of psychosis can occasionally appear to be explained in terms of psychological conflicts (Janssen, 1963) and less commonly the process can appear to be terminated by the resolution of psychodynamic problems. Despite all the above considerations and the possibility that stressful conditions may be necessary to maintain such rhythms, adequate psychodynamic explanations for the observed periodicities as such do not exist, This is true, of course, with the exception of annual rhythms which may represent anniversaries, disturbances at weekends, or other calendar-determined occasions. Psychiatrists impressed by these observations can only begin to attempt to unravel their mysteries by recording the facts, including the concomitant physical changes. At the same time they must speculate, perhaps a little wildly, but consciously guided by the possible implications of other research on biological rhythms. Work in the field is inevitably complicated by ethical factors and the currently available therapeutic procedures which make the natural history of the conditions even more difficult to discern. The same facts have, however, been repeatedly recorded by the most reliable psychiatric observers ( see Kraepelin, 1913; E. Bleuler, 1911; Papadimitriou, 1955; Guilmot and Stein, 1961; S. M. WOE et al., 1964; Mall, 1960; Menninger-Lerchenthal, 1960; Crammer, 1959a,b; Richter, 1960; Mayer-Gross, 1961; W. Kretschmer, 1964; and many others, especially R. Gjessing, 1968). Writers agree, and our studies confirm the fact, that the 48-hour psychotic rhythm can be the most persistent and regular one observed. Alternate days of mania and depression can persist for decades of a patient’s life. Other cycles of 72, 96, and 120 hours have also been frequently reported. The longer the cycle, however, the less precise the timing. Despite the obscurity of the physical factors, and despite the clear demonstration of the importance of the environmental factors, the persistence for decades of severe periodic disturbances of behavior and mood is enough to suggest that some physical factors are operative. It might alternatively be suggested that this is a learned response, but even so, any reason for learning such a pattern of behavior is unknown. Furthermore, the syndrome has been reported following cerebral catastrophes ( Scheiber, 1901) .

132

F. A. JENNER

Janssen (1963) presents evidence for the opposite point of view and rightly emphasizes the need to consider the psychodynamics of the patient very closely. I l l . Nosology and Periodic Psychoses

Clinical psychiatry is bedeviled by nosological difficulties. It is not appropriate to discuss these at length here, except as a guide to the confusing terminology which can be used. Even the regularly recurring periodic psychoses cannot necessarily be assumed to be a homogeneous group of illnesses. They are considered together here because they all seem to suggest hypothalamic timing mechanisms which one would like to understand and which are discussed below. Further, they must be considered together because of our limited ability to subclassify these states meaningfully. Classifications of psychiatric diseases (see Stengel, 1959) often leave one wondering whether they are overgeneralizations of the bacteriological concepts of infectious diseases, guides to prognosis, or simply the inevitable linguistic necessity of describing complex phenomena with brief expressions for letters to general practitioners. The precise periodic psychotic clearly shows the course of illness typical of the manicdepressive psychosis, but the content of the illness can be schizophrenic as described by R. Gjessing (1968) (hence “periodic catatonia”), or recurrent schizophrenia (Rey, 1957), or affective (Bunney and Hartmann, 1965), or epileptic (Bercel, 1964). The combination of symptoms has also led to many being called schizoaffective psychoses. As is well known, however, Wernicke rejected Kraepelin’s (1899) attempt to divide the major functional psychoses into two large groups and he started a line of distinguished pupils who want to have a third group. Many periodic psychotics could well belong to this third group. Unfortunately Wernicke’s disciples have adopted new names and would include the periodic psychoses in the phasic psychoses (phasische Psychose of Neele, 1949, phasophrenic psychoses (Phasophrenie of Kleist, 1921, 1928), cycloid psychoses ( zykloide Psychose of Leonhard, 1959), or atypical endogenous psychoses (atypische endogene Psyclzose of Leonhard, 1961, Pauleikhoff, 1957, and Elsasser, 1950), degeneration psychosis ( Degenerutionpsychose of Bonhoffer, 1907, Schrijder, 1926, and Binswanger, 1928) , or endogenous pseudoschizophrenia ( endogene Pseudoschizophrenie of Riimke, 1958). In other language groups we find the schizophreniform psychoses (schizophreniforme Psychose of

PERlODlC PSYCHOSES

133

Langfeldt, 1956), the stignzute psychiquc &s d&ge'ne'rks of Magnan (1893), the sdiizonzanie of Claude (1926), and the marginal or atypical psychoses of Mitsuda ( 1953). Mitsuda ( 1962) discusses the problem at length and the conflicting views as to whether there is a mixed genetic basis of manic-depressive and schizophrenic origin (see R. Gjessing, 196S), or perhaps a distinct gene for the syndrome itself. Fish (1964) discussed this field in some detail and deals with the further subdivisions of the so-called cycloid psychoses by Leonhard (1961) and Sawa's suggestion (1963) that at least some are epileptoid psychoses. Vaillant (1964) in addition has presented an interesting historical review of the remitting schizophrenias. The above discussion is only presented to illustrate the clinical confusion but at the same time to emphasize that in these patients almost all of the signs and symptoms of the major psychoses, including epilepsy, can appear and then recede to a timetable. Whatever the psychodynamic meaning of the contents of the psychoses may be, it is clear that without any environmental change some endogenous process can produce the symptoms and then make them disappear completely. Presumably homeostatic euthymic mechanisms stabilizing human mood and performance exist; these, however, can be made to oscillate by insults, or else they can become synchronized or entrained to a cyclical process occurring in the hypothalamus. The fact that the cycles of euthymic mechanisms can be affected by environmental factors does not mean they do not exist. IV. Periodic Illnesses in General

We know almost nothing about the mysterious processes underlying abnormal periodicity in medicine, but it is a feature of some nonpsychiatric pathological conditions. Reimann ( 1964) includes peritonitis, fever, edema, purpura, myelodysplasia, arthrosis, sialadenosis, paralysis, pancreatism; Bercel ( 1964 ) deals with epilepsy; Roper-Hall and Jenner (1968) describe strabismus in childhood which often may occur every other day. Menninger-Lerchenthal (1960) has been an assiduous collector of case histories of conditions showing periodicities but his writing is often more anecdotal than convincing. Richter ( 1960) and Ask-Upmark (1963) have also reviewed the field. Both the latter authors propose in addition to

134

F. A. JENNER

hypothalamic clocks the existence of serosal clocks producing periodic hydrarthrosis, and bone marrow clocks causing the periodic hematological conditions. The former point is elegantly demonstrated by the patient with bilateral periodic hydrarthrosis of the knee joint, but with each joint having a different periodicity ( Richter, 1960). In an interesting study of neuropituitary-adrenal activity in Cushing’s syndrome, Stan (1967) alludes to the cycles of urinary 17-hydroxycorticosteroid excretion which follow a 6- to 10-day course, and which occur with the commoner loss of the circadian pattern in these patients. As far as is known, nobody has tried to correlate these changes with any alterations in the associated psychic syndrome. The observations of Stan (1967) and of the many authors he quotes on the cyclical factor, though not incompatible with the hypothesis that the abnormal clock arises from disturbed timing mechanisms, seem to suggest that the cycles are the oscillations to be expected in a feedback mechanism, which could oscillate when disturbed without any necessity for a postulated special biologically useful or significant timing device. Winkler and Herrmann (1967) draw attention to the possibly related and widely distributed (in man and animals) 10-day rebound phenomenon of urinary corticosteroids which follows one injection of adrenocorticotropic hormone. The steroids rise, and are suppressed for 10 days, and then show a secondary peak before returning to normal. Cyclic neutropenia (Owren, 1949; Hahneman and Alt, 1958; Alestig, 1961; Reimann, 1963; Videbaeck, 1962; Barkve, 1967) is a particularly intriguing condition of a periodically recurring fall in the white blood cell count which seems to be inherited. The condition is often associated with recurrent mental changes of moodiness, irritability, and tiredness. The symptoms sometimes seem to respond to testosterone in high doses (500 mg once a week intramuscularly) (Barkve, 1967). In other cases splenectomy and prednisone are effective ( Owren, 1949; Reimann, 1963; Videbaeck, 1962). Perhaps more interestingly Morley (1966) has shown a cycle of 14-23 days duration of neutrophiles in normal people and he suggests that the disease state is simply an exaggeration of this normal cycle. At the present time, it is diEicult to even guess at the underlying mechanisms or significance of these phenomena. The author of this review, however, has frequently been impressed by the presence of such

PEWODIC PSYCHOSES

135

changes in periodic psychoses, and R. Gjessing (1968) has puhlished longitudinal studies on his patients. It has been suggested in periodic fevers of various etiologies ( Hodgkin’s, sarcoidosis, familial Mediterranean fever, etc. ) that etiocholanolone is perhaps the pyrogenic agent ( see Huhnstock et al., 1966) with perhaps the other 5-fl-H steroids. The field has recently been very well summarized by Reimann (1966) and by S. M. Wolff et al. (1967). By intramuscular injection, etiocholanolone does produce fever in humans and is more effective in males. It seems it can release pyrogens from human leukocytes and possibly estrogens affect this. However, though Bondy et al. (1958) reported a rise in the unconjugated plasma levels with the febrile episodes, this has not been easy to confirm. On the other hand, significantly higher levels are persistently found by some other workers in both phases of the illness, while the occasional patient with a very high plasma level has been afebrile. The need for further work and the possible relationship of these findings to other conditions discussed in this review is clear. Reimann (1966) is particularly anxious to separate periodic fever from periodic peritonitis, but he emphasizes the fact that both come from a group of disorders which are difficult to classify. As he says, they begin at any time in life, are heritable, and occur in families stigmatized by neuroses, migraine, epilepsy, and psychoses. The episodes can occur with incredible regularity, or be completely unpredictable. The conditions can persist for life or cease gradually or abruptly. In some there is a temporary relief during pregnancy, or following stress or steroids, in others this does not occur. Abnormal electroencephalograms and autonomic reactions can occur. Without further knowledge classification is difficult, but the probability exists that many of these conditions are controlled by hypothalamic activity which is itself influenced by the mysterious biorhythms which are currently so inexplicable. Like migraine and psychotic reactions, severe stress can release these responses; they can also synchronize in some patients with menstruation, but the attacks can also be regular and not synchronized with the menses. Such results clearly give rise to the suggestion that the hypothalamus could perhaps be thought of as analogous to a gearbox. By some means or other, and under certain conditions, various oscillations can be synchronized.

136

F. A. JENNER

Many periodic diseases seem to be due to sudden bursts of hypothalamic activity which produce the symptomatology via the autonomic nervous system, as, for example, in angioneurotic edema, the accompanying meningismus, pleocytosis, ocular disturbances, pulmonary edema; shock paresthesia and psychic changes of periodic illnesses emphasize the brain’s special rBle (Reimann, 1963). Selbach and Selbach (1955) produced an influential and speculative, if somewhat difficult, paper on the nature of epileptic attacks. They argued that the seizure is a rebound phenomenon serving to bring the organism back to its state of equilibrium from an extreme trophotropic state (fainting serves the same function from the extreme ergotropic state). From a consideration of the autonomic changes in periodic catatonia they suggest that the catatonic state serves the same function, though the epileptic fit is a rapid readjustment and the catatonic stupor or excitement is slow. It is particularly since this paper that emphasis has been placed on three phasic aspects of periodic disease (see, however, Jaspers, 1946): there is a preparoxysmal labile phase with increasing cholinergic activity; when this becomes extreme and the various parasympathetic functions are acting together there is the decornpensation phase, which can be followed by a fit or a catatonic state; and the compensation phase returns the organism to an adrenergic state. It is difficult to know how seriously to take these almost semantic suggestions, but it is important to be aware of them, especially because of their influence, particularly on German clinical studies ( see Lauter, 1964). The documentation of these writers has added much support for the view of the importance of higher autonomic nervous centers in many periodic syndromes. Klages (1954) and Lauter (1964) draw attention to welldocumented lesions ( traumatic and encephalitic) leading to pathological periodicity of behavior, and Biissow ( 1949) deals specifically with midbrain tumors producing periodic depression. The latter immediately reminds one of Richter’s (1957) rat found by accident with a spontaneous 15- to 22-day rhythm of activity. At autopsy this rat was shown to have a large hypothalamic tumor. Beringer (1942) also reported a 4-week cycle of mood and behavior thought to be the sequelae of epidemic encephalitis. Pipkorn (1947) and de Jonge (1964) describe very similar 4-week cycles of less certain etiology.

PEHIODLC: I’SYCEIOSES

137

V. Richter’s Hypotheses

It is not surprising that “clock-like processes” occur centrally (i.e., in the central nervous system) and in the periphery. The concurrent occurrence of menstruation, the diurnal steroid rhythm, hydrarthrosis, cardiac action, respiration, and the EEG, etc., all with different, even if striking periodicities, show how mistaken it can be to overuse the word “clock” for every oscillatoiy process, and further how foolish it would be to feel confident that they all depend on the same or similar processes. This is a mistake, however, which in a less crude form is presumably made repeatedly in the rest of this review, looking as it must for apparently analogous phenomena. In addition to ( a ) peripheral clocks producing hydrarthrosis or the Pel-Ebstein syndrome in Hodgkin’s disease [a peculiarly precisely recurring pyrexia (Ebstein, 1887)],Richter (1960) postulates ( b )homeostatic clocks based on the feedback of endocrine glands to the pituitary and hypothalamus; in this group he includes the estral and menstrual clocks and that controlling the periodic catatonic schizophrenic illnesses, and ( c ) central clocks located in the central nervous system and regulating the circadian or nearly %hour clocks, and also indirectly the 48-hour rhythms of somatic and mental illnesses. He feels that precision is the hallmark of the central as distinct from the homeostatic clock. His distinction between central and homeostatic clocks is not easy to accept at face value, as will be discussed below. The menstrual and estral rhythms, for example, may perhaps depend on the presence of estrogens and nevertheless remain central clocks. Another of Richter’s hypotheses (1960), the “shock phase hypothesis” is equally difficult to evaluate. He suggested that each part of the organism, e.g., each cell, might have a similar intrinsic rhythm and that the normal smooth functioning of an organ or of the organism might depend on the normal random phase relationship. The synchronization of the rhythmic processes might follow an insult and hence the whole organ or organism will oscillate pathologically. He developed this view after considering the experiments of Kalmus ( 1935) and Pittendrigh and Bruce ( 1959). These authors demonstrated that the pupae of drosophila emerge randomly when kept in the dark but in a regime of 12 hours of light and 12 hours of darkness the colony is synchronized and

138

F. A. JENNER

pupae emerge at 24-hour intervals. When kept in constant darkness just one flash of light can synchronize the whole colony which again emerges at %-hour intervals. It is, of course, well known that parts of organs are functional while other parts are quiescent. Richter (1965) cites the renal glomeruli (Smith, 1956), the follicles of the thyroid (Williams, 1938), the cells of the gastric mucosa (Bradford and Davies, 1950), and the neurons of cerebral centers (Franck, 1956), each of which could perhaps become synchronized. Perhaps the most interesting of all the relevant observations quoted by Richter (1965) is the work of Stxumwasser (1963) in which isolated cells were active for 8 out of every 24 hours. Strumwasser et al. (1965) also found synchronized changes in the electroencephalogram of the amygdala. Richter quotes Strumwasser as giving positive evidence of a rhythm in the isolated cell, but of such evidence there is no shortage, as is further demonstrated below (see in particular von Mayersbach, 1967, for a review of this subject). Despite the difIiculties in demonstrating its definite relevance the “shock phase hypothesis” remains one of the few important suggestions attempting to explain the origin of abnormal rhythms. VI. Circadian Rhythms

The field of study of abnormal rhythms is one in which C. P. Richter is not only the doyen but also the most important worker. Other workers have concentrated so much more on the study of the normal and perhaps all-important circadian rhythms. The longer periodicities, however, may well be based on circadian rhythms. Richter (1960) himself feels that this is so for the interesting 48-hour rhythms. Aschoff (1967) has shown that a normal, approximately 50-hour rhythm of sleep and wakefulness is not uncommon in humans studied under free running conditions. The study of circadian rhythms in biology even more than some other subjects has shown something more akin to an explosion than even a geometrical progression. The field has been repeatedly and well reviewed by, for example, Aschoff (1965), Biinning (1!364), Harker (1964a), Sollberger ( 1965), and most relevantly to human physiology, by Mills (1966). As has already been stated the true relevance of this work of such otherwise disparate phenomena as periodic psychoses, or indeed to the seasonal migration of birds ( Cloudsley-Thompson, 1961; Wolfson, 1959), or the remarkable

PERIODIC PSYCHOSES

139

28-45-year developmental cycles of bamboo plants ( Seifriz, 1950; Wangermann, 1965) is still conjectural. The ubiquity of the circadian rhythm, however, may suggest that like the tricarboxylic acid cycle, which is present from yeast to man, a process of very similar fundamental biological significance could be involved. It may well he used to time other processes. Currently one must assume that this fundamental biological chronometer is learned by life in evolution from the earths rotation around the sun. As Kalmus (1966) says, most organisms must have experienced about lo7 dawns during their existence. VII. Cellular Studies

The period length of the circadian rhythm (of 24 hours) is, however, long compared to any of the explained oscillating processes in the body’s chemistry. Pittendrigh (1960) and Harker (1964b) among others have presented evidence that even in complex organisms the biological clock must work at the cellular level. Many cellular aspects of circadian rhythms are discussed in a symposium edited by von Mayershach ( 1967) including circadian changes in nucleic acids (Eling, 1967; Jerusalem, 1967), and liver glycogen (Leske, 1967). Pye and Chance (1966) and Hess et al. (1966) have been able to show that cell-free extracts of Saccharomyces carlsbergensis are able to maintain sustained oscillations of the order of 0.2 cps of the reduced pyridine nucleotide-in this case rather specikally using trehalose as the substrate supplying glucose to the glycolytic pathway it) a regulated manner due to the action of trehalase. Double periodicities and beat frequencies were also shown to occur, and upon such phenomena longer periodicities might be built. These fascinating suggestions are being activcly wplore d .

VI II .

M a t hematical Considerations

B. C. Goodwin (1967) expresses the view that during evolution cellular processes would have to choose between tolandic (or oscillatory) and nonoscillatory states according to their adaptive value. By a mathematical analysis of closed feedback repression loops, the simplest negative feedback state possible based on “messenger-directed protein synthesis and the mechanism of repression’’ (see Gorini and Gundersen, 1961), the likelihood of a tolandic state seems established. Such an oscillatory state offers advantages

140

F. A. JENNER

over nonoscillatory states in giving the cell a basis for a variety of time measurements. As mathematical analog analysis can only be a grossly oversimplified representation and as complexity of dynamic systems favors oscillatory processes such systems are even more “inevitable” (see Cesari, 1958; H. Rubin and Sitgreaves, 1954). Morowitz (1966) essentially develops the same argument from the point of view of statistical mechanics. Perhaps the above is more simply expressed by Waddington’s suggestion ( 1957) that all regulatory processes might be appropriately looked at to see if they are directed toward true homeostasis or homeorhesis (i.e., an oscillatory state). The latter would appear so much commoner than Claud Bernard would have expected. Almost all mathematical models or physical analogs of biological rhythms are based on assumptions of the relevance of nonlinear van der Pol-type of relaxation oscillators. Such systems, while selfsustaining and capable of entrainment, have a “preferred” amplitude and waveform which will reemerge when freed from external influences. In addition, however, it seems necessary to propose “some self-entraining communities” or “generalized relaxation oscillators” and Winfree (1967) in particular has anaIyzed the possible temporal organization of such communities. His analyses, which must be read in the original, might be as relevant sociologically as metabolically and at least might help in the attempts to develop a conceptual framework from which to review the probIems of this field. It is well beyond the reviewer’s competence to evaluate truly this impressive work. IX. Survival Value

The natural selection and survival value of the circadian rhythm based on an internal oscillator synchronized by external events is obvious. The metabolism of the plant can be ready for photosynthesis before the light comes and the animal can anticipate its active or inactive phase ( Biinning, 1967). Furthermore, because the endogenous oscillation can be measured against day length its comparison with the changes in real day length can identify seasons. However, evidence already suggests that some circannual endogenous rhythms not dependent on day length may also exist (see Lofts, 1964). The loss of selection value of a circannual rhythm in domesticated animals seems to have Ied to their comparative disappearance (Ortavant et al., 1964). Richter

PEHIODIC PSYCHOSES

141

(1965) also seems to believe that man has now so controlled his own environment as to have escaped the need for cycles of performance of either somatic or mental function: a sweeping statement in view of the evidence of endogenous rhythms in man (see Halberg et al., 1965). Though too general to be true, Richter’s view emphasizes the fact that the most striking periodicities in man’s behavior, other than the sleep wakefulness cycle, are the products of pathological processes (see Reimann, 1963; Richter, 1965; Menninger-Lerchenthal, 1960; Roberts, 1965). Bunning ( 1967) deals with the signscance of synchronization and beat effects between the tidal changes of period length 12.4 and 24.8 hours and the 24-hour rhythm-inducing beats at 15 and 29 days, the latter being a circalunar cycle apparent especially in the discharge of the gametes in marine life (see also Brown, 1965). Bunning sees this as possibly analogous to the mechanism producing the periodic pathological behavior of one to several weeks in man. X. The Menstrual and Estral Clocks

The similarity of period length between menstrual cycles and lunar or tidal cycles is too striking not to be noted, and Halberg et al. (1965) also report a $-week cycle of human male 17-ketosteroids ( “circatrigintan”). In addition their fascinating study adduces evidence for circaseptan, circavigintan, and circannual cycles. The menstrual clock, so important in medicine and psychiatry, is still surrounded by much mystery, though recent work has fairly clearly established that it is in the central nervous system (G. W. Harris, 1964). Its importance in affecting the mental state is difficult to doubt, though it is easy to underrate the obvious psychological significance of menstruation to a woman. Southam and Gonzaga ( 1965) conveniently summarize the literature on changes during the menstrual cycle and Glick (1966) very critically reviews the literature on the related subject of the psychological effects of oral contraceptives (see Daly et al., 1967 and Andrews et al., 1966). As long ago as 1936 Pfeiffer suggested that at birth male and female rats have an undifferentiated mechanism capable of establishing an estrous cycle. Castration of the male at birth preserves this ability as can be shown by implanting ovaries. Giving testosterone to the female at birth destroys this ability. Various combinations of transplanting testes, ovaries, and pituitaries (see G. W.

142

I?. A. JENNER

Harris, 1964) establish the fact that the system damaged or preserved is not in the endocrine glands. Inou6 (1961) showed, by transplanting ovaries into the spleen where their output would go straight to the liver and be destroyed, that cyclical excretion of gonadotropins did not occur unless other ovarian tissue was also present in the body, whereupon cycles occurred in the intrasplenic ovary too. The estral and presumably the menstrual clock then either requires estrogens to work or is perhaps itself a negative feedback system depending on the effects of estrogens on the hypothalamus, or perhaps some tonic stimulus on the hypothalamus allows the estrous cycle to occur. The menstrual clock is more variable than most women state and like the rhythm of the periodic psychoses it is subject to disturbances by all manner of physical and mental insults (see Chiazze et al., 1968). The hypothalamic clock controlling menses might be analogous to some of the presumed clocks of periodic psychoses. Furthermore, the effects of gonadal secretions on behavior in animals are striking, and even in man some evidence suggesting their relevance to behavior exists. The timing of psychotic episodes in relation to menses in particular is discussed below. XI. Estrogens, Androgens, and Animal Behavior

Swanson (1967) has shown in the hamster that male-type nonsexual behavior (low exploratory behavior in the open field and delayed emergence from a closed box) can be produced in the female given androgens at birth. She quotes some preliminary evidence that play patterns of female monkeys even before puberty can be made of the male type by the same treatment (see Young et al., 1964). She concludes that such factors could perhaps do something similar in humans, but this is a hypothesis which is certainly extremely difficult to test. Cagnoni et al. (1967), however, even go so far as to suggest that anorexia nervosa is the so-called “early androgen syndrome” in women. They support this hypothesis by emphasizing the differences between the signs and symptoms of anorexia nervosa and other forms of chronic weight loss. (These symptoms are the early weight loss, hypertrichosis, hyperactivity, cold tolerance, lack of atrophy of breasts, lack of starvation edema, polycystic ovaries, increased 17-ketosteroids, reduced 17-hydroxysteroids, and the loss of the typical circadian rhythm of plasma

PERIODIC PSYCHOSES

143

cortisol. ) This is, however, against the whole current psychological evidence for the explanation of thc origin of anorexia nervosa and even in conflict with the signs seen by other workers (see H. P. Wolff et al., 1968). It has been known for a long time that female copulatory behavior is rare in castrated male rats unless they are given large doses of estrogens (Ball, 1939; Beach, 1941). Again, however, the time of castration is important as absence of androgens in the first five days of life leads to female behavior. In the same way testosterone given in the first days of life abolishes female mating behavior in the intact female. In contrast, after oophorectomy estrogens and progesterone lead to receptivity (G. W. Harris and Levine, 1962; Barraclough and Gorski, 1962). Meyerson (1968) and in a series of earlier studies (Meyerson, 1964a,b,c) has obtained evidence to suggest that pathways depending on monoamines mediate the inhibition of female mating behavior in rats. In the ovariectomized but estrogen- and progesteroneactivated female, estrus is inhibited by increased levels of cerebral monoamines. If, however, reserpine or tetrabenazine is given to estrogen-treated rats estral behavior is induced. Without such drugs, progesterone must also be given. Meyerson (1968) was also able to show that the amine-depleting drugs induced the female lordosis response to the intact male in neonatally castrated males treated with estrogens. This was a more marked effect than was achieved with estrogens and progesterone in these rats. Similar results were produced in postnatally androgentreated but intact female rats, though in this case reserpine or tetrabenazine was no more effective than progesterone. Martini et al. (1967) have demonstrated that estrogens implanted in the median eminence of the immature female rat lead to precocious puberty and reduction of pituitary luteinizing hormone stores. Implantation in the epithalamic region ( habenular nucleus and pineal gland) retards puberty and causes augmentation of luteinizing hormone stores. Hence estrogen feedback mechanisms stimulating or inhibiting gonadotropins would seem to exist. Melatonin and pineal extracts implanted into the median eminence and midbrain reticular formation also lead to inhibition of synthesis and release of luteinizing hormone, Richter has produced pseudopregnancy cycles ( 12-14-dav cycles) of activity in female rats by section of the pituitary stalk

144

F. A. JENNER

or posterior lobectomy, by almost total thyroidectomy, or prolonged treatment with 13'1 or sulfamerazine, thiourea, thiouracil, prophylthiouracil, or a-naphthyl thiourea; removal of the superior colliculi, removal of the pretectal area, and severe stress (Richter et al., 1953, 1959; Richter, 1933a,b, 1957, 1959, 1965). In the male, removal of the superior colliculi and damage to the pituitary stalk or posterior lobe alone lead to similar cycles. Males will, however, exhibit 16-20-day cycles of intake of 2.5% calcium lactate solutions after parathyroidectomy (Richter, 1965). The possibly special role of the parathyroid in some cyclic conditions is further illustrated by activity cycles which can be abolished by treatment of parathyroid deficiency (Richter et al., 1940). Removal of the uterus and its appendages but preserving the ovaries, or giving estradiol, can release 19-21-day rhythms of activity in female rats which Richter (1965) considers as pregnancy cycles. Castration of the wild, but not laboratory, male rat leads to 35-40-day active cycles (Richter, 1965). The domesticated rat becomes almost totally inactive after castration. Some normal desert rats will show cycles of activity in the activity cage of 30-25 days, and normal pocket mice will also demonstrate fairly regular cycles of inactivity of 2448 days, the blinded, but otherwise normal, ground squirrel will show 9-13-day cycles (Richter, 1965). Richter (1965) lists and demonstrates the above and similar fascinating findings. He is, however, often criticized for lack of statistical evaluations of his findings, and for the semantic problems which can arise from describing the above as clocks. The presumption being that some important timing mechanism is revealed by this behavior. It is perhaps equally likely that the oscillations arise de mvo with the lesions and are interesting but of little fundamental significance. This could be true of all periodic diseases. It is perhaps not surprising that like so many observations in the field of abnormal rhythm study, most of the above remain as curiosities, studied by hardly anyone, and almost impossible to theorize about in any currently helpful manner, XII. Estrogens, Androgens, and H u m a n Behavior

Human sexual behavior is clearly influenced by social and psychological factors and many would deny the importance of the endocrine system. Schon and Sutherland (1960),however, took the

PERIODIC PSYCHOSES

145

opportunity of assessing the effects of hypophysectomy for carcinoma of the breast on female sexiial desire, activity, and gratification. First they showed that neither loss of a breast nor of the ovaries had a profound effect despite the clcar reaction and sensitivity of feelings to the mutilation. The fact that removal of the pituitary led to a profound decline they attribute to loss of the trophic hormone leading to the stimulation of adrenal androgens. They further demonstrated that neither cortisone nor thyroid replacement was beneficial in this respect. Waxenburg et al. (1959) also showed that adrenalectomy reduced female sexual behavior even after steroid replacement. Furthermore, the literature contains much evidence that androgen administration stimulates libido ( e.g., Salmon and Geist, 1956; Loeser, 1940; Greenblatt, 1943; Foss, 1951) . Hatotani et al. (1962) showed that periodically disturbed women do tend to become psychotic in the postovulatory and premenstrual phase of the menstrual cycle. Abortion, parturition, puerperium, lactation, and the menopause can appear to be precipitating factors, but seldom pregnancy. The graphs they publish, however, would not seem at first sight to support Cookson et aZ.’s views (1967). Wakoh (1959) and Wakoh et al. (1960) report, and this is confirmed by Hatotani et al. (1962), that there is a biphasic fluctuation in estrogen excretion in normal women. This is often lost in periodic psychotics in their studies. Very extensive work impressed them with the changing ratios of the fractions of the 17-ketosteroids in their patients. The gonadal fraction androsterone tends to decrease with a relative increase in etiocholanalone. They use the so-called androgen index (A1 ) of androsterone over etiocholanalone and show that it tends to be less than unity during psychotic phases but about 1.5, which is normal in the intervals. As they point out, this is typical of liver disease; after injection of 50 mg of testosterone the catatonic patient tended to convert less to 17-ketosteroids than did the normal patient. Similarly they showed that the periodic psychotic tends to excrete more estrone than estriol, which is equally consistent with hepatic dysfunction. Finally, they report lowered pregnanediol excretion and low conversion rates of progesterone to pregnanediol. However, routine liver function tests were essentially normal except for Quick‘s test (hippuric acid synthesis) and Lugol’s reaction. Administration of LSD 25 produced similar steroid changes in 10 out of 14 subjects; in the 4 with no such reaction no mental symptoms were produced. Since the androgen index is low

146

F. A. JENNER

in Simmond's disease, in dwarfism, infantilism, etc., it was felt that a disorder in the hepatodiencephalic homeostatic interrelationship might be indicated. Hatotani et al. (1962) showed that some patients not responding to thyroxine did very well when given estrogens and progesterones. Mall (1960) in a somewhat similar study concluded that in periodic psychotic women the %hour estrogen excretion should be examined; when this is low, treatment is more effective with estrogens and androgens than with thyroxine. Froshaug (1958), who worked with Gjessing, was unimpressed by the temporal relationship between menstruation and periodic catatonia, as it was shown that in many patients the two rhythms occurred apparently independently. Cookson et a2. ( 1967) applied harmonic analyses to data from a female patient showing a 36-day rhythm of psychosis and a 24-day menstrual cycle. During the period of amenorrhea produced by continuous norethynodrel and mestranol administration significant 12- and 36-day rhythms of weight, sodium balance, urine magnesium, and 17-ketosteroid excretion occurred. The 17-ketosteroid rhythms persisted during menstiuation. The peaks of the 12-day rhythms coincided with menses and the mid-menstrual phase (ovulation); the 36-day peaks marked the onset of stupor. They suggested that the 17-ketosteroid cycles might be of etiological significance and produced by surges in gonadotropic activity not blocked by norethynodrel. Very significantly they point to the fact that in their study the 17-ketosteroid rise and peak precede the increase in 17-hydroxycorticosteroids by three or four days. Rises in 17-hydroxysteroidsin periodic psychoses and particularly in the depressive stage of manic-depressive psychoses are well established. That these are probably secondary ( t o mental state) is generally accepted. The fact, however, that gonadotropins in women have been reported to follow a biphasic pattern with rises mid-menstrually and menstrually (Fukishima et al., 1964) fits well with the hypothesis of Cookson et aE. (1967). The latter authors in particular show how the data from studies by Gornall et al. (1953) can be shown to be consistent with their hypothesis and with a little more imagination the longitudinal studies of Rowntree and Kay (1952) and Rey et al. ( 1961) as well. In one of the patients (they studied the same women) the psychotic cycle equaled two menstrual cycles. To explain periodic psychoses in males the authors refer to

PERIODIC I’STCIIOSES

147

Exley and Corker’s description (1966) of the 10-day cycles of estrogen, thought to be of testicular origin and possibly secondary to surges of gonadotropins. The claim of Cookson (1966) of successful treatment of periodic patients with clomiphene citrate strengthens his argument, but this requires considerable verification. The clearly hormonal basis of sexual behavior in the subprimate mammal (see Bard, 1940) makes their study exciting even if it is clear that in man such mechanisms have become submerged, or perhaps completely lost, with the greater development of the cerebral cortex. Nevertheless, male and female roles can be significantly reversed by neonatal administration of sex hormones in the rat, and Young et al. (1964) report somewhat similar results in the female rhesus monkey, Perhaps the hormones act via the lateral hypothalamic regions which Avar and Monos (1967) are able to show are vital for the maternal behavior of nest building, young retrieval, etc., in the rat, or perhaps via the median cortical (cingulate and retrosplenial) areas in the rat. Stamm (1955) showed damage to these areas profoundly influences maternal behavior without disturbing lactation. Lisk ( 1%6) also demonstrated that estrogens as well as light regulate the neurosecretory material of the median eminence. More probably these and other cortical areas are all involved in behavior, in a complicated and integrated way, as part of the limbic system (see Smythies, 1966; Kluver and Bucy, 1937, 1938, 1939; McCleary and Moore, 1965). It is of considerable interest that normal women have a tendency to higher D time sleeping (period of rapid eye movements during sleep) toward the end of the menstrual cycle (E. Hartmann, 1966). It also seems this may be more striking in the premenstrual tension syndrome, but this requires much more investigation. Since primates, including women, and only excepting baboons (Kummer, 1957) and chimpanzees (Yerkes, 1943), do not have estrous cycles, many of the implications of the above speculation for human subjects are dependent on very doubtful assumptions. However, even the rhesus macaques shows a clear menstrual cycle of social and sexual behavior (see Rowell, 1963). Furthermore, Wagner ( 1943), E. Kretschmer ( 1949), and Meyer (1955) all adduce evidence for the importance of an intact midbrain for normal human sexual behavior; this is based on autopsy and other studies of cerelml lesions. Finally, Dalton (1964) in

148

I?. A. JENNER

particular proclaims, and common experience confirms, the high incidence of emotional and behavioral problems premenstrually (see also Sutherland and Stewart, 1965). However, the psychodynamic facts and the social effects of being a woman (or a man) must not be overlooked, and from personal observation and the experience of most mature clinicians it has to be stated that behavioral disorders related to menstruation and most other periodic human behavior in fact always present the clinician with a complicated human story which can make him humbly aware of his intellectual difficulties. The role of the scientist can only be to highlight interesting and important facets, not to attempt complete explanations. That an inherited vulnerability of the diencephalon, or diencephalopituitary-hepatic relationships is involved in periodic psychoses is perhaps a mere clichk, but perhaps the best formulation now possible. XIII. light and the Menstrual Cycle

The idea that “lunacy” has something to do with the moon dies hard. However, little evidence exists to support this view (see Tromp, 1963). The biological significance of moonlight has nevertheless become increasingly recognized, especially its entrainment of sexual cycles ( see Cloudsley-Thompson, 1961 and Hauenschild, 1955). The average human and subhuman primate menstrual and fertility cycles are 29 days (Menaker and Menaker, 1959; Dewan, 1967), very near the lunar cycle of 29.5 days. Guenons monkeys living near the equator have also been reported as having menstrual discharge a t new moon, which might imply that ovulation occurs at full moon (see Ellis, 1936; Allen et aL, 1939). Dewan (1967), having been encouraged by no less than Wiener and Stanley Cobb, and being himself interested in a perfect rhythm method of contraception, has presented evidence that a mere 100watt lamp at the foot of the bed can effect ovulation in women. He suggests that light acting via the pineal might somehow induce a sudden rise of the luteinizing hormone output. This would induce ovulation from a ripe follicle. Light it seems also affects human behavior and renal function. Aschoff and Wever (1962) have shown that intensity of light might control the period of the free-running rhythm of man in an isolated environment; there is little other evidence available from human studies to demonstrate this effect in man. Nevertheless, it is well

PERIODIC PSYCHOSES

149

established that rhythms in the renal excretion of water and electrolytes in man are influenced by light. Sharp (1960) blindfolded one group and showed that this caused differences in sodium and water excretion not seen in otherwise similarly treated controls. Lobban and Tredre (1964) have shown that the totally blind have a comparative absence of normal renal rhythms when compared with the partially or normally sighted. This is most strikingly true if the subject has been blind from birth. Further, many of the indigenous population in Alaska, where light and darkness follow almost 6-month cycles, have reduced renal excretory rhythms (see Lobban, 1960, 1965). Wallner et al. (1963) and Ishisu ( 1962) also show effects of light on human renal excretion of 17-ketosteroids. Studies of how light affects metabolism and behavior have also been stimulated by the finding that light falling on the retina changes the concentration of melatonin, a derivative of 5-hydroxytryptamine which occurs in the pineal. There is good evidence available that this is achieved via neural pathways from the retina to the superior cervical ganglia and from there to the pineal. Much needs to be done to decide on the significance of this finding for humans. There is, however, considerable evidence that in rodents this pathway and mechanism lead to changes in activity and estral cycles. No doubt work is continuing throughout the world on these fascinating areas of humoral control of behavior, One can assume that these pathways are at least affected by large numbers of tranquillizing drugs, and we must be humbled by all the theories which could perhaps be added to our present knowledge of how the dnigs we use work (see Cohen, 1964). XIV. Thyroid Activity and Periodic Psychoses

R. Gjessing’s (1968) work and therapeutic success with thyroxine obviously focused attention on the role of thyroid function in periodic catatonia. R. Gjessing ( 1938) showed the clear relationship between changes in mental state and basal metabolic rate. The latter is elevated in the catatonic phase of stupor or excitement. Though Gornall et al. (1953) did not find changes in plasma-bound iodine levels other workers have found changes, including Libow and Durell (1963), Durell et al. (1967), and Maeda et al. (1968). Tenner et al. (1967) failed to find such changes in a 48-hour manicdepressive psychotic though J. C. Goodwin et aE. (1968) did h d the changes in a 6-day periodic psychotic, and several other patients.

150

F. A. JENNER

Plasma-bound iodine (PBI ) tends to rise at or near the beginning of a catatonic phase (see also Hatotani et al., 1962) and to drop quickly thereafter. It is interesting that the PBI is only raised at the onset of a catatonic phase as it has been suggested that neural mechanisms can only produce a transient rise or fall in blood thyroxine (see Andersson et al., 1962; Brown-Grant, 1957). After an initial response the thyroid pituitary feedback system tends to have an overriding effect. Though Durell et al. (1967) are cautious about the interpretation, their results would seem to suggest that l"1 uptake shows a similar cycle, not found, however, by Pover and Crammer (1960). Jenner et al. (1967) failed to find changes in thyroid-stimulating hormone assays but the technical problems are formidable and work on this, plus changes in metabolism of radioactively labeled thyroid hormones, would seem of importance for further studies in this field, as would considerations of the binding proteins and of the long-acting thyroid-stimulating substance ( LATS ) . The complexities and difficulties in studying these problems are formidable and bedeviled by pitfalls of interpretation. For example, Van Middlesworth (1960) has shown that in the rat fecal excretion of thyroxine is related to fecal mass. Bowel action in periodic catatonia is severely disturbed and periodic, while the parameters of thyroid function show only small changes. This system is, however, probably less important in man than in the rat. Presumably the thyroid function changes are secondary to hypothalamic changes, and R. Gjessing's (1968) use of enormous doses of thyroxine to cure some patients was never used as an argument to suggest abnormal thyroid function. The interrelationship between brain and thyroid function, however, is complex and obscure (see Cameron and O'Connor, 1964). Gjessing never stated that thyroid disease was etiological in periodic catatonia, but naturally his work opened up this field for special inquiry. This has been carefully considered by a number of writers. The review by Gibson (1962), however, concluded that thyroid function studies in psychiatric patients did not add much to knowledge of the interaction between emotion and thyroid function. M. Bleuler (1954) also points out the poor correlation between endocrine disease and mental functioning; looked at either way round there is no one-to-one relationship between the intensity of any endocrine condition and psychiatric symptomatology (see Petersen, 1967, on hyperparathyroidism). Nevertheless, Bleuler

PERIODIC PSYCHOSES

151

reports a much higher incidence of psycliiatric problems in 1000 patients following thyroidectomy than in 1000 following appendectomy, the incidence of psychiatric sequelae to treatment with radioactive iodine was also similar to that following thyroidectomy. Hertz (1964) has summarized some of the relevant literature. Durell et al. (1967) also made an important study of the relationship between psychosis and thyroid function in four periodic patients showing clear correlations and a fifth patient who was made hypothyroid by a thyroidectomy and whose mental state varied apparentIy in relation to her thyroid levels. Howard and Ziegler ( 1942), Carpelan (1957), and Brockman and Whitman (1952) are also workers who have presented case histories apparently demonstrating changes in mental state associated with thyroidectomy. In some complicated way a feedback system between mental state and thyroid function does seem to exist, and it is especially well illustrated by the periodic psychoses. This can become synchronized with changes in other hypothalamically controlled functions but the problem of how to study the nature of the feedback system and the coupling involved still presents difficulties. The first presumption must be that periodic psychoses are more likely to be hypothalamic diseases than endocrine disorders, but the hormonal consequences of hypothalamic function are easier to study and we are still involved in piecing these together. XV. Vasopressin and Periodic Psychoses

R. Gjessing (1968), among other workers, was impressed by the large changes in urine volume which can occur in periodic psychoses. However, as in his patients this was often contrary to the changes in body weight, he felt that they were compensatory for large losses by extrarenal routes. Crammer ( 1959a,b), however, showed that this is by no means always so, and J. C. Goodwin and Jenner ( 1967), extending Crammer’s studies, have presented evidence that the marked antidiuresis can be due to antidiuretic substances found in the urine. It is an enigma that this antidiuretic siibstance(s) (J. C. Goodwin and Jenner, 1967), though like vasopressin, cannot be destroyed by sodium thioglycolate and this, for technical reasons and despite some years of work, we have not been able to resolve. Goodwin and Jenner (1967) also point out how difficult it is currently to explain the concurrent changes in sodium balance in terms of available endocrinological knowledge.

152

F. A. JENNER

XVI. Early Work on Periodic Psychoses

There are numerous early writings on the periodic psychoses, but the formulation of many are quite unacceptable or almost incomprehensible with our modern thought forms; perhaps the recent study of this strange group of syndromes really begins with Pilcz in 1901. Pilcz (1901) also includes an interesting review of earlier work including the difficult-to-obtain work of Kim ( 1878). In Pilcz’s monograph one reads of the early attempts to do metabolic balance studies, and the painful realization of how difficult it is in fact to collect all the excreta from disturbed patients and simultaneously control their diets. Petren (1908), Urstein (1913), and Barnes and Francis (1909), unperturbed by the difficulties, continued the struggles and include interesting early observations on periodic catatonia including the excretion of nitrogen, urea, creatinine, uric acid, phosphate, sulfate, chloride, etc. The famous biochemists Folin and Shaffei (1902) also reported on phosphate excretion in a patient with a 48-hour manic-depressive cycle. The rhythm they reported has not been confirmed but it is difEcult to see any fallacy in their methods or analyses. No lesser authors than Emil Kraepelin (1913) and Eugen Bleuler (1911) report on 48-hour cycles, and even include observations on water balance and salivation. XVII. Gjessing’s Studies

The most important studies ever made, however, are those of Rolv Gjessing (1932a,b, 1935, 1938, 1939, 1953a,b,c,d, 196Oa,b, 1968). He studied periodic catatonia in the Kraepelinian sense, though he was constantly aware of the resemblance of the syndromes to the manic-depressive psychoses. In essence he intensively studied 32 patients and showed that in 14 nitrogen balance and mental state had the same periodicity. The phase relationship of the nitrogen balance and mental state changes were different for different individuals but constant for any one patient. The mental state changes were abrupt and this group he called the synchronoussyntonic or ss type. Ten of his patients had gradual changes and less striking signs and symptoms; these he called asynchronousasyntonic or aa types. Those between these extremes, 8 of his patients, he called dyssynchronous-dyssyntonic or dd types.

PERIODIC PSYCHOSES

153

The ss types he further divided into A, B, and C groups depending on the phase relationship l~etwecnnitrogen balance and mental state changes. He showed that periodic catatonic stupor and periodic catatonic excitement could lie considered, at least biochemically, analogous conditions. He implied that the stupor might be due to a greater excess of a postulated noxia. In his studies, however, type Ass cases had stupor phases with a negative nitrogen halance during the stupor; types Bss and Css had periodic excitement. Type C showed positive nitrogen balance in the psychotic phase. Type B showed nitrogen retention in the second half of the psychotic phase and the first half of the interval. Under what must have been the most rigorously controlled and standardized conditions of life in the history of medicine the patients were studied for months on end. L. R. Gjessing (1968) quotes his father’s results as follows: The basal metabolic rate fluctuates phasically although mostly within normal limits. The body temperature rises in the reaction phase. The respiratory quotient falls below 0.71 in the interval. Circulation is adequate though most patients are, as it were, out of training, i.e., they meet the increased demand by a n acceleration of pulse rate. Blood volume fluctuates, being lower in the reaction phase with a rise in erythrocytes and blood pigments. Fasting blood sugar falls in the interval, sometimes to 80 mg. %. In the reaction phase it rises to about 120 mg. I%. Protein metabolism fluctuates in the cycles which are of the same duration as the cycles of energy metabolism, though the two do not coincide in time, Fluctuations in N-balance vary with the individual patient (within a range of 15-35 g. N ) and, so far as excretion in urine is concerned, are accounted for almost entirely by the fluctuations in urea. The non-urea N fraction in urine is hardly affected by the amount of N-intake. The total pigment excretion in urine is at times abnormally high, particularly so in the reaction phase. Residual N i n plasma fluctuates considerably, up to *20%or more. The thiocyanate excretion in the reaction phase is large, relative to the total nitrogen. After successful intervention with thyroxine and dry thyroid, thiocyanate excretion became noimal. Electrolytes in blood and urine also show phasic swings. Excretion of sodium chloride is always greater in the interval than in the reaction phase. The acid base equilibrium in the interval shows a compensated alkalosis, and i n the reaction phase a compensated acidosis. Retention of urine occurs in the early part of the reaction phase and is apparently not affected by the amount of N being excreted. There is no evidence of morphological renal damage. Endocrine activity: Phasic swings in thyroid activity have been confirmed, These swings are apparently not governed By the thyroid gland itself, and

154

F. A. JENNER

probably not primarily by the pituitary. The successful use of thyroid hormone shows that thyroid activity is part of the vicious circle in periodic catatonia. Autonomic activity in the interval is predominantly cholinergic and in the reaction phase adrenergic, as is clearly seen in myclriasis, salivation, pallor, retention of urine, etc. Sleep is disturbed in the reaction phase which is understandable in view of the general adrenergic pattern. The reverse is true of the interval. Further, the electrocardiogram in stupor shows an isoelectric TI. Muscular rigidity is increased in the reaction phase as in extrapyramidal disorders. The caloric nystagmus is inhibited in the reaction phase. The psychomotor excitement or stupor occurs phasically and spontaneously, independently of all external influences. There does not seem to be any connection between psychopathology and body build. Reaction time to visual and auclitory stimuli is much longer in the reaction phase, no doubt because of lower powers of concentration. Looked a t more broadly, catatonic periods show the following salient features: ( 1 ) Regular rhythms of two phases, the interval and the reaction phase. ( 2 ) A reversal, usually very sudden, in the pattern of autonomic activity with changes in all autonomic fields and still more in the cerebral and psychological field. ( 3 ) Fluctuations in N-balance with phases that have the same duration as the autonomic phases though there is a lag time. ( 4 ) There is an apparently specific effect of the thyroid hormone in controlling all functional disturbances, both somatic and psychological.

Possibly the implication of much of R. Gjessing’s (1968) work on the therapeutic value of thyroxine is related to its effects on some special detail of nitrogen metabolism. Hardwick and Stokes (1941) performed interesting studies in periodic catatonia on protein-rich diets (25 gm of nitrogen per day) and showed that the cycle of mental state could then occur without any phase of negative nitrogen balance. R. Gjessing (1968) also showed that on 2-4 gm of nitrogen per day cycles of stupor could continue to occur regularly; by implication the nitrogen balance changes previously reported are only obvious if a normal or inadequate protein diet is given. Despite the intervening years the significance of these observations remains obscure. XVIII. The Adrenal Cortex and Periodic Psychoses

The relationship of adrenal function to the nitrogen changes also still remains relatively unexplored. Rowntree and Kay (1952) studied two female patients with “periodic schizophrenia” in whom 17-ketosteroids in urine were high

PERIODIC PSYCHOSES

155

at the peak of an attack or a little delayed. They postulated from their results that just before and during the attacks electrolytecontrolling factors peaked while during the attack androgens were high, and during recovery the “sugar-active” corticosteroids predominated. The same patients and one other similar patient were studied 8 years later by Rey et a?. (1961). They confirmed the 17-ketosteroid findings but found high glucocorticosteroids during the attack which were low during remission. They confirmed the reduced sodium excretion occurring during the reaction phases and showed that sodium and potassium balance were following the same course. Gornall et al. (1953) reported three patients with periodic catatonia. In one of the three 17-ketosteroids were reduced during the psychotic phases though all the corticosteroids increased strikingly in the remissions. Two patients had defective responses to adrenocorticotropic hormone, but the odd patient had an excessive steroid response. Gunne and Gemzell ( 1956) studied 17-hydroxycorticoids in their periodic catatonic patient. These were elevated in the disturbed phase, especially in the early days of excitement. This patient also showed a poor response to adrenocorticotropic hormone. The literature on the relationship between mental state and adrenal cortical activity is well reviewed by R. T. Rubin and Mandell ( 1966) and by Fawcett and Bunney ( 1967). The reciprocal influences of one on the other are clear, though the adrenal response to emotion is currently more predictable than is the emotional response to steroids. There is an increasing consensus of opinion in favor of the formulation made by Bunney et al. (1965a,b) that the depressed patients who show great distress, anxiety, and agitation are the ones with particularly high 17-hydroxycorticosteroid excretion. Gibbons (1964) has shown that the urinary increase of 17-hydroxycorticosteroids is associated with an increased adrenal secretion rate. Kurland (1964), however, suggests that there is a decrease in adrenal activity during depression, and the possibility of a block in the metabolic pathway leading to cortisol is perhaps supported by the fmding of high levels of compound S in depressed patients ( see Jakobson et al., 1966). Certainly depression can occur without elevation of urinary steroid output and mania usually does do so. Longitudinal studies on periodic patients have, however, almost invariably produced results showing simultaneous changes in steroid excretion and mood.

156

F. A. JENNER

Controlled studies of steroid therapy have not produced reliable evidence of any psychopathological consequences ( see Rees, 1953; Lidz et aE., 1952). Nevertheless, clinical studies of Cushing’s syndrome (Spillane, 1951; Trethowan and Cobb, 1952; Glaser, 1953; Harxthal and OSullivan, 1959; von Furger, 1961; Clark et al., 1952; Michael and Gibbons, 1963) tend to show that many of the patients are severely depressed. Similarly clinical studies of Addison’s disease (Addison, 1868; F. A. Hartmann et al., 1933; Cleghorn, 1951; Engel and Margolin, 1941) equally suggest that a change in affect also occurs. The peculiar fact that euphoria or depression would seem to arise in both hyper- or hypoadrenal corticalism is not easy to explain. Some other evidence, however, suggests that mania is a severe form of depression (Coppen, 1965) and also that catatonic stupor and excitement are similar states (R. Gjessing, 1968). Coppen (1965) finds residual sodium to be increased in depression and even more significantly increased in mania. For the student of periodicity the problem presented is to explain how at least in some patients the two possible reactions to the same illness can alternate quite regularly. The study of the pituitary adrenal axis dearly presents many possibilities for oscillations in feedback circuits which may be relevant. There is now evidence that adrenal steroids enter and indeed are concentrated in the brain (Touchstone et al., 1966; Eik-Nes and Brizzee, 1965). Presumably they play some role in controlling electrolyte distribution across cell membranes. Woodbury’s classic review (1958) also emphasizes the evidence for an increase of cerebral intracellular sodium and excitability following the administration of cortisol. Currently knowledge of the effects of steroids on cerebral biogenic amines is limited, but as stated by Fawcett and Bunney ( 1967), evidence elsewhere in thc body plus the work on the significance of monoamines in psychiatry makes further work in this field seem very attractive. XIX. Catecholamines

L. R. Gjessing has supplemented his father’s earlier studies by his work on the catecholamines. He has shown (L. R. Gjessing, 19Ma,b, 1965, 1967a,b) that the excretion of 3-methoxy-4-hydroxymandelic acid ( VMA ) , 3-O-methylated adrenaline, and noradren-

PERIODIC PSYCHOSES

157

aline are elevated during recurrent stupor or excited phases. Histamine, 5-hydroxytryptamine, and tyramine are not changed. The metadrenaline levels interestingly rise at the beginning of the psychotic phase but the rise is only temporary, whereas the normetadrenaline excretion correlates more strikingly with the level of disturbance throughout the abnormal phase. Monoamine oxidase inhibitors increase the 3-O-methyl-catecholamine excretion without significant psychiatric effect but reserpine or haloperidol abolishes the psychotic phases. a-Methyldopa causes a quantitative replacement of normetadrenaline excretion with a-methylnometadrenaline and an amelioration of the psychotic phases. These findings would seem to imply that catecholamine secretion reflects more central and important changes, and do not themselves explain the psychiatric state. XX. Autonomic Concomitants of Periodic Psychoses

L. R. Gjessing summarizes much of the literature by suggesting that During the quiet cholinergic interval the PBI and BMR goes clown to minimal normal levels and cholesterol increases to high levels. The liver is gradually stuffed with fat [and peptides? or lipoproteins?] reaching, at the onset of the psychotic attack, such a degree that the liver is disturbed. In this quiet but extreme cholinergic condition a mechanism is activated which switches the vegetative nervous system suddenly into a predominating adrenergic phase with a strong hyperactivity of the hypothalamus resulting in stimulation of thyroid stimulating hormone, gonadotrophins, mineral-corticoids, and glucocorticoids, as well as of the adrenals and the entire sympathetic nervous system, and concomitantly throwing the patient into a psychotic state. After a certain time this hyperactivity subsides to a normal level at the beginning of the interval. Then it decreases to a subnormal level again preparing for the next psychotic phase. Intervention with thyroid hormone prevents the subnormal level and especially the accumulation of fat and protein [possibly in the liver?] and thereby the psychotic phases. And on thyroid medication and without psychotic phases the patient recovers. XXI. Electroencephalography

Electroencephalographic changes in periodic psychoses have been reviewed by L. R. Gjessing et al. (1967). Changes in a-rhythm correlate well with changes in mental state in most individuals studied; in some slow wave activity also comes and goes or varies with the mental state. Though for an individual the correlation is usually striking, the direction and type of change cannot be pre-

158

F. A. JENNER

dicted from individual to individual. One can summarize the literature by saying that there is a tendency for depression to be associated with a decreased a-frequency and increased a-amplitude, while in mania the reverse holds true. In patients described as schizophrenic there is a tendency for the reaction phase to have the EEG of the manic phase of the manic-depressive (see Bonkalo et al., 1955; Anderson et al., 1964; Gunne and Holmberg, 1957; Hes, 1960; Rowntree and Kay, 1952; Harding et al., 1966; Winnik and Assael, 1966). The electroencephalographic studies do not seem likely to lead to an understanding of etiology. It is, however, surprising how few longitudinal studies have been made and it would seem that further attempts to correlate chemistry and encephalography might be profitable. L. R. Gjessing et al. (1967), for example, present evidence that the electroencephalographic changes in periodic catatonia are more likely to be associated with noradrenaline secretion than adrenaline secretion as the time course of the changes are compatible with this hypothesis. As it is known that electroencephalographic changes occur with steroid administration in humans (see von Euler et nl., 1959; Glaser et al., 1955), and in Cushing’s syndrome (Hoefer and Glaser, 1950) and in Addison’s disease (Hoffman et al., 1942), it would be of interest to know more about the adrenal electroencephalographic correlation in periodic psychoses. Perhaps the a-rhythm changes represent changes in arousal, which as far as current evidence goes is higher in mania than depression. However, few patients with agitated depression have been serially studied. XXII. Lithium and Periodic Psychoses

The fascination of the timing of the precisely recurring manicdepressive or periodic psychoses may detract from the fact that these patients are really typical of affective psychotics in general. Certainly the effects of lithium ions on the syndromes might imply this. Boyce et aZ. (1968) have clearly demonstrated the marked therapeutic effect of lithium ions on a classic example of the 48hour periodic psychosis which could be stopped and started by altering the lithium to sodium ratio of the diet. A torrent of papers have agreed that lithium ions are helpful in mania, and in approximately 70% of patients with manic-depressive psychoses lithium is of prophylactic value (see Cade, 1949; Schou et a!., 1954, 1955;

PERIODIC PSYCHOSES

159

Maggs, 1963; Wharton and Fieve, 1966; etc.); for a more complete list see Schou (1968). L. R. Gjessing (1967a) was essentially unimpressed by the effects of lithium loading on a patient with periodic catatonia; however, this was also a patient who did not respond to thyroxine (L. R. Gjessing, 1967b). Furthermore, the dosage used was not as high as can sometimes be necessary in the reviewer’s limited experience. Anne11 studying adolescents with periodic catatonia is reported to have achieved more impressive results (see Schou, 1968). Sletten and Gershon (1966) have claimed that lithium ions are also prophylactic against premenstrual tension. The efficacy of lithium ions is not easily explained though it is not difficult to postulate hypotheses. In view of work, like that of Crammer (1959a,b), Coppen (1967), and Shaw (1966), in which changes of sodium balance (Crammer) and residual sodium (Coppen and Shaw) have been demonstrated to occur in relation to both mood and behavior changes of manic-depressive patients, it is natural to wonder if the effects of lithium are to be explained in terms of its competition with sodium ions. Boyce et al. (1968) demonstrated that changing the sodium content of the diet could influence the mental state during lithium therapy. The increased dietary sodium might increase lithium excretion and this would leave less lithium available to act. Talso and Clarke (1951) do not find a significant effect of lithium on renal sodium excretion, nor did Schou et al. (1967) despite enormous sodium loads in humans. Schou (1959) does, however, report a clear correlation between lithium excretion and sodium intake in dogs and rats. Coppen et at. (1965) have demonstrated that lithium reduces residual sodium. Because of the ionic hydration and polarity of the lithium ion it might be expected to act biologically more like the divalent ions calcium and magnesium and hence produce its interference with their metabolism. It is reported to cause a rise in serum magnesium (Nielsen, 1964). C. A. Harris and Jenner (1968) also report a diminution of the effect of vasopressin on the rat’s renal tubule after lithium administration. As J. C. Goodwin and Jenner (1967) have reported a cycle of excretion of antidiuretic substance in a periodic psychotic, the above finding is possibly relevant to the mode of action of lithium in psychoses. Boyce et al. (1968) suggest that lithium leads to a hyperadrenal cortical state

160

F. A. JENNER

with mild diabetes insipidus (see Schou, 1959; Trautner et al., 1955; Gershon, 1968) , and hypothyroidism ( see Schou, 1968) . In terms of current speculations about biochemistry and mood, however, some effect of lithium on cerebral monamine metabolism might be postulated. This could be primary or secondary to any of the above responses. Each in its own turn, however, is likely to be a secondary effect. Schanberg et al. (2367) and Schildkraut et al. (1966) showed that lithium produces a marked shift of noradrenaline metabolism from 0-methylation to intraneuronal deamination. Corrodi et al. (1967) showed that lithium alone does not affect brain noradrenaline content. If in the presence of lithium ions tyrosine hydroxylase is given the noradrenaline content of the neuron falls more quickly. This was particularly true of cranial as distinct from spinal neurons and was related to lithium concentrations in the brain. No similar effects were produced on cerebral Shydroxyptamine nor on dopamine. This finding might be considered specially relevant to hypotheses particularly implicating noradrenaline in abnormal mental states (see Schildkraut and Kety, 1967; L. R. Gjessing, 1967a,b). Baastrup and Schou (1967), however, suggest that lithium is different from other drugs in the fact that it is primarily a prophylaxis with an “antiphasic action.” If so, it would perhaps be a mistake to use arguments for the relevance of various metabolites arising from the effects of other drugs, but it might also mean that lithium is a specially valuable tool to study the postulated underlying “clock” and its mechanisms. REFERENCES Abe, K. (1965). Psychiat. Neurol. 150, 129. Addison, J. (1868). New Sydenhurn SOC. 36, 211. Alestig, K. (1961). Acta Med. S c a d 169, 253. Men, E., Danforth, C. H., and Doisy, E. A. (1939). “Sex and Internal Secretions.” Williams & Wilkins, Baltimore, Maryland. Anderson, W. Mc., Dawson, J., and Margerison, J. H. (1964). CZin. Sci. 26, 323. Anderson, B., Ekman, L., Gale, C. C., and Sundsten, J. W. (1962). Acta Physiol. Scand. 56, 94. Andrews, M. C., Andrews, W. C., and Lettew, W. L. (1966). Am. 1. Obstet. Gynecol. 96, 48. Aschoff, J. ( 1965). ‘,although considered in the light of more recent data (Bradley, 1965) this observation might call for somewhat different interpretation. Events displayed in the EEG were occurring-with reference to the universe of a cell-over vast expanses of the brain, while those going on within the living human brain reached the investi-

PHYSIOLOGICAL FOUNDATIONS OF MENTAL ACTIVITY

333

gator’s awareness after penetrating through his preconceptions, as well as through the subject’s cranium, aponeurosis, scalp, etc. Thus, in reality, direct observation of the brain was still far from being actually direct. Evidence, assumed to represent the state of the brain, proved to need the primary adjustment of standardized conditions, if it could be expected to provide results of a uniform type. The persistent urge to study physiological changes within the brain associated with mental events was responsible for two main trends assumed by research. There appeared to be an endless variety and growing complexity of tests on the one hand. This trend was represented in contributions by Gastaut and Bert ( 1954), Jung ( 1954), R. D. Walter and Yeager (1956), Lelord (1957), Devoto (1958), Kooi and Boswell (1960), Slatter (1960), Mulholland and Runnals (1962), Mirsky and Roswald (1963), Kugler (1963), Shagass and Canter ( 1966), and Webb (19ss). On the other hand, attempts were also made-and sometimes simultaneously-to extract maximal amounts of information from available data through the use of analyzers, electronic computers (Livanov et al., 1966; Chapman, 1966), or other mathematical devices for treating EEG’s elaborated for this purpose (Genkin, 1963, 1966). It should be noted that EEG studies of conditioning observations of this type have provided a wealth of interesting information, particularly on the role of different cortical regions in performing certain mental acts, or on principles of interaction between large brain regions in the performance of mental activity. However, factors limiting closer insight into the problem were not restricted to properties of the electrical activity penetrating the teguments to be recorded as the EEG, or to properties inherent in this activity in general. It should be noted here, that “failure,” or rather only partial success in application of the EEG to studies on the physiology of the human brain, depends on the fact that the EEG is not merely a reflection of what is going on in the brain. Economical Nature would not have created such a class of phenomena “for the benefit of investigators.” It becomes more and more apparent, in the light of current research, that activity recorded as the EEG possesses a certain function of control (Bechtereva, 1967). Rather than a mere manifestation of changes of the state of the brain, electrical activity evidently serves to maintain a certain optimal condition, some optimal tuning of the brain to its

334

N. P. BECHTEREVA AND V. B. GRETCHIN

changing environment. The endless argument over priority of hen or egg is likely to find an ambivalent solution. 111. Some New Approaches to Physiological Investigation of Mental Activity

A. CONTINGENT NEGATIVE VARIATION (CNV) AND MENTALSTATE Qualitative advances in research on the physiology of the brain as the basis of mentality have been made along two lines. The first direction is governed by the principle of maintaining observations over the intact human brain and dates from Walter’s discovery (W. G. Walter et al., 1964) of the new phenomenon of CNV. The second has arisen with the possibility of establishing really direct contact with the human brain. The CNV, or expectancy wave, was revealed under special conditions of observation when two stimuli had been arranged so that presentation of the first predetermined, or made probable, the appearance of the second (W. G. Walter, 1965, 1966; R. Cooper et d., 1965; Low et al., 1966). After presentation of the first stimulus a sustained potential change was found to appear in the frontal region, the latter becoming electronegative with respect to a reference electrode or to deeper structures (the “negative variation” phenomenon). On closer investigation the phenomenon was found to accompany a variety of human activities, such as decision making under laboratory conditions or by a free-ranging subject with the aid of radiotelemetry ( W. G. Walter et al., 1967). The changes have been found to depend to a considerable degree on the mental and emotional state of the subject, and the state of his training, reflecting what Grey Walter terms “subjective probability” of an event. The constant appearance of the CNV under certain conditions makes it a practically ideal phenomenon for studying the process of formation of readiness for action, of the activity of a subject in a situation, and of interaction between man and his environment. These aspects of the phenomenon determine the likelihood of its being used in conditions when maximal rapidity is essential in carrying out a decision previously formed by the brain. The CNV may be assumed to represent at least one of the components of anticipatory excitation ( Anokhin, 1958). With the discovery of this phenomenon, the possibility has been demonstrated of an action being dependent on the participation of the electrical

PHYSIOLOGICAL FOUNDATIOSS O F MENTAL ACTIVITY

335

activity of the brain, as part of the switching “on” or “off system. This discovery provides another proof of the investigator’s creative potentialities. A broad approach to the neurophysiological basis of mental activity, however, necessitates a variety of methods for investigating the events taking place in different parts of the brain.

BASED ON DIRECTCONTACT WITH B. EVIDENCE

THE

HUMANBRAIN

1. Mental Effects of Electrical Stimulation of the Cerebral Cortex The contribution of Penfield to knowledge of functions of the human brain cannot be overemphasized. His somatotopic maps of the cortex are well known, Stimulation of the cortex during operations for epilepsy has shed a new light on the part of this formation in mechanisms of memory (Penfield, 1958a,b, 1959a,b,c; Penfield and Milner, 1958). The brain seemed to keep securely the traces of past experience which can be revived by the touch of an electrode, as well as of the web of natural associations. It would be out of place in this review to raise once again the debatable problem as to whether these facts are more characteristic of epilepsy or of memory. The important thing is not how far these properties are inherent in the brain, but that they may be elicited at all. Facts to this effect have been obtained in other similar observations (Adams et al., 1962; Feindel, 1964; Brazier, 1966; Talairach and Bancaud, 1966). 2. Emotional and Mental ,EfFects of Electrical Stimulation of Deep Structures of the Brain The late 1940’s were marked by the elaboration of a human version of the stereotaxic technique (Spiegel et al., 1947; Spiegel and Wycis, 1952), thus opening the way to the selective therapeutic block of deep structures of the human brain. Soon, the first reports on the use of indwelling electrodes in human patients (Pool, 1948) proved the advantage of the new method over that of one-stage stereotaxic operations when the target of intervention was not clear preoperatively. Stimulation of deep brain structures was sometimes found to provoke striking emotional responses in the patient ( Smimov, 1963, 1965, 1966a,b; Smirnov and Grachev, 1963; Urmancheeva and Diakonova, 1965; Heath, 1954, 1963; Heath et al., 1954, 1955; Heath and Mickle, 1960; Monroe and Heath, 1954; Spiegel and Wycis,

336

N. P. BECHTEREVA AND V. B. GFtETCm

1961; Delgado et al., 1954; Delgado, 1959, 1964; Ursin, 1960; Umbach, 1964, 1966; Van Buren, 1963a,b; D’Andrea and Paolozzi, 1963; W. G. Walter and Crow, 1964; Crow, 1965; Fiendel, 1964; Sano, 1966). In some of these cases, additional electrical stimulation conforming to the program of treatment might result in marked changes in behavior. These observations demonstrated the necessity and efficiency of performing psychological tests during these electrical stimulations as part of the safety and control measures. Observation of emotional and mental experiences accompanying electrical stimulation are thus supplemented by data on changes in tested mental activities under the same conditions. Thus, tests of recent memory during electrical stimulation of deep brain structures have revealed depression of the aptitude to perform tasks, with increasing numbers of errors during stimulation of the caudate nucleus and anterior thalamus (Gorelik, 1967; Bechtereva et al., 1965). Special investigation of emotional responses occurring in the same situations has shown that of the total amount of deep brain structures subjected to stimulation (about 400 points in our observations) some 10%of stimulations was accompanied by emotional responses of a positive or negative tone (Bechtereva and Smirnov,

1967). Thus, electrical stimulation at the depth of the brain has revealed active participation of deep brain structures in complex mental acts. Records of electrical stimulation provide information on some features of the structure-functional basis of brain performance in mental responses. It is clear, however, that a large number of observations must become available before one may expect to extract the amount of information necessary for designing a convincing structure-functional model of performance of mental activity by the brain.

3. Direct Observation of Physiological Manifestations of the State of the Brain During Mental Activity Considerations of therapeutic efficiency, as well as the search for an approach to principles governing performance of mental acts by the brain, naturally led to the elaboration of other methods of investigation that could never be detrimental to the patient, but should rather provide results profitable for his treatment. Confoming to this condition, it seemed rational to make use of really direct

337


contact with the brain for observing parameters of its activity during performance of mental tasks. In addition to the two above approaches to investigation of performance of mental acts by the brain, a third avenue has been opened with the advent of novel methods of treatment, particularly with those using indwelling electrodes. Investigation may now be directed to the nature and site of changes occurring within the brain when the brain has been induced to perform a particular action. Both of the previously available approaches involve much cooperation from the patient “betraying” the secrets of his brain by reporting subjective sensations. The second approach discloses the effects of finer, modulating influences. In the third case, up-to-date techniques of physiological investigation are apt to extract from the brain evidence that had been absolutely inaccessible before direct and multipoint contact with the brain could be established. a. EEG of Deep Brain Structures during Mental Tasks.Although the experience gained with the use of the EEG in various experiments involving conditioning and mental tests had only been partially successful, these were adopted first, when direct access to the brain became available. It was certainly no error of scientific judgment, since the possibility of direct contact with the human brain could be expected to supplement available knowledge on the neurophysiological basis of mental activity through the EEG, and especially on the evidence of the electrosubcorticogram ( ESCoG) ; it was also apt to enlarge our understanding of the EEG( ESCoG). If any error was committed, it was to entertain too bright expectations. The search for reproducible correlates set particularly difficult problems with reference to the polymorphic EEG( ESCoG ) in brain pathology. The hope of the investigator to analyze a complicated phenomenon and to detect its import, inapparent to the eye, that is, to extract information concealed below the surface--in our case information related to mental activity-prompted us to apply mathematical methods to treatment of the ESCoG, in particular statistical analysis of the ratio between durations of ascending and descending wave phases (Genkin, 1963). Another method of computer treatment was based on the principle of “specialist machine,” where the specialist analyzed the EEG( ESCoG) visually, conforming to a program; then data from hundreds and thousands

+

338

N. P. BECHTEREVA AXD V. B. GRETCHIN

of strips were processed by the computer (Moiseeva and Orlov,

1965). Treatment employing the former method confirmed the significance of differences between background ESCoG recorded during electrical stimulation and mental tests. These differences, however, were purely statistical, No reproducible ESCoG patterns were to be revealed on repeated mental testing, even when there was direct contact with the brain. Treatment by the “specialist machine” method generally tended to support the same principle: changes in the ESCoG were statistically significant. Another interesting fact, revealed by this method, appeared disappointing at first sight. Changes accompanying mental tasks were found to occur in the cortex and in a variety of deep brain structures, including those whose bilateral destruction had been known, according to worldwide experience, to result in no mental defect. It appeared that the parameter in use reflected some general changes, or that it might be too sensitive, but not optimal for investigating the structure-functional basis of mentality. At the same time, it has to be admitted without going beyond established facts that significant changes occur in all, or almost all, parts of the brain during such mental acts as recent memory. May it not be surmised, that such an exceedingly fine parameter as the EEG (or ESCoG) should reflect changes inherent to action, as well as those accompanying readiness to act? Comparison of these facts with those from Pavlov’s laboratories on external inhibition and with the concept of our mathematician Kolmogorov (1963) of thought as a discrete process has led to the suggestion of a hypothesis on the inability of performing simultaneously more than one complex mental action (Bechtereva et nl., 1965; Bechtereva, 1966). The illusory impression of two or more processes being performed at the same time is due to the great speed with which some people can shift from one activity to another, to a great mobility of their nervous processes creating a firm subjective belief that a number of actions are being performed simultaneously. Before concluding this brief description, another fact should be mentioned. In studying the electrograms recorded under various conditions, as during electrical stimulation or performance of mental tasks in epileptic patients, some forms of electrical activity were

+

PHYSIOLOGICAL I’OUNDAl‘lONS OF hiENTAL AClIVITY

339

found to become reiterated, sometimes as relatively simple or highly complicated, spatial-temporal patterns (Bechtereva, 196%~; Bechtereva et al., 1965). When these reiterative patterns had been recognized with such impressive clarity in epileptics, they could also be detected in the ESCoG of other patients. Special investigations indicated that this aspect of electrical activity could be correlated to clinically apparent traits of these patients, particularly excessive “stickiness” of their mentality ( perseveration). It seems reasonable to inquire whether these electrographic features may be somehow related to mechanisms of memory. It may be remarked in connection with this phenomenon, enhanced in epilepsy, that the search for reiterative features related to memory is far from being new. M. A. B. Brazier, whose name is known for many of the most rational techniques applied to EEG aspects of brain research, has described reiterative EEG features in a brilliant and highly convincing form (Brazier, 1962). According to our views, the reiterative features in EEG (or ESCoG ) represent a component of greatest importance in mechanisms of memory. In all probability, they subserve the processtransmitting information from recent to long-term memory, reflecting some inborn (or intrinsic) properties of the brain, the framework into which processes of long-term memory are built in. b. Changes in Steady Potential, Neuron Population Activity, and Aoailable Oxygen in Deep Brain Strzlctures during Mental Tasks. Studies where brain research has been supported by investigations of single cell activity, of steady potential, of available oxygen, etc., have been reported in some remarkable contributions to experimental neurophysiology (Brazier, 1963; Jasper et al., 1962; Wurts, 1967). It was shown that the results of these investigations could only be evaluated as mutually supplementary evidence in every sense, including that implied in quantum mechanics. Data obtained with the aid of different methods were often difficult to correlate. It was therefore deemed expedient and necessary to investigate the structure-functional and neurophysiological foundations of the liriman mind by applying a multitrxde of methods displaying the state of the brain under test conditions, rather than by simply varying the mental tests. Moreover, it could be assumed in advance that investigations should not be conducted only under standard conditions of laboratory “rest,” but that they should deliberately

340

N. P. BECHTEREVA AND V. B. CRETCHIN

involve additional factors, tending to imitate the actual complexity involved in the solution of human problems under the changing conditions of everyday human life. Tests for recent memory (similar to Binet tests) were presented under usual conditions of laboratory rest or during nonrhythmical photostimulation (mostly as trigger stimulation, with varying relations of signal to wave phase), the latter assuming the role of “interference” or “noise.” Patterns of variation of steady potential II

I =

FIG.1. Superimposition of steady potential changes ( A ) , superimposition of oxygen availability alterations ( B ) , and alterations of summed-up impulse activity ( C ) at different stages (I, 11, 111) of recent memory tests in ventrolateral thalamic nucleus. I: during test presentation; 11: during retaining in the memory; 111: during reproduction of the test.

and oxygen availability at different stages of test performance were evaluated by superimposing tracings while alterations in total impulse activity were evaluated in terms of the amount of spiking. As revealed by these studies (Bechtereva and Smimov, 1967; Bechtereva, 1965a,b,c, 1966, 1967; Bechtereva et al., 1966; Bechtereva and Trochatchev, 1966; Gretchin, 1966a,b, 1967) in the records of steady potential, available oxygen, and neuron population activity, reproducible patterns become clearly apparent on repeated presentation of recent memory tests (Fig. 1).Differences

PHYSIOLOGICAL

FOUNDATIONS OF MENTAL ACTIVITY

341

between mental activity and background were apparent in these records by comparing great numbers of “background” strips with great numbers of records made during mental activity, so that their dissimilarity was more than statistically significant. Each record made during a memory test proved to show even visually apparent differences from its immediately preceding background. Moreover, patterns of “mental activity” bore some similarity to each other and could be superimposed. In some cases, and as a rule in occasional

FIG. 2. Superimposition of alterations of oxygen availability at different stages (I, 11, 111) of recent memory tests in the ventrolateral thalamic nucleus. A: wrong performance of the tests; B: correct performance of the tests; I: during test presentation; 11: during retaining in the memory; 111: during reproduction of the test.

patients, these records displayed different patterns in certain structures depending on whether the test was performed correctly, or with errors. Repeated investigations in different patients under conditions of rest, or against a noisy background, were summarized and plotted as a structure-functional design representing the state of the brain during performance of recent memory tests. What has emerged from comparing charts plotted from records

342


obtained at rest with those obtained against a background of noise? There were differences, but not in every component of the chart. In some features, reproducible patterns were found to be absent, whereas they reappeared clearly at other points. At the same time, there were also zones of persistent change, brain regions where reiterative patterns appeared at rest and persisted against a noisy background. Studies of steady potential during emotional tests ( Avramov and Smirnov, 1965) have disclosed that under these conditions the steady potential tends to show the greatest changes at brain sites where electrical stimulation had evoked emotional responses. Tests provoking emotions were also found to cause marked and varied changes in levels of available oxygen, which tended to be reiterated on repeated presentations of the tests, and demonstrated the rate of oxygen uptake by brain structures with emotional experience (Fig. 2 ) . The results of different tests-in particular, those concerning emotion, as compared to recent memory tests-are hardly commensurate. Nevertheless, in either case, certain brain regions appear to be of major importance, being related closely or rigidly to a particular kind of activity (Figs. 3 and 4). On the other hand, changes in the parameters of brain function did not appear to be so marked or so reproducible in other brain regions. Here, more strictly standardized tests for recent memory would probably reveal the structure-functional relation to be flexible, rather than rigid. W . Some Theoretical Considerations on the Structure-Functional Basis of Mental Activity

The wealth of factual data gathered from these studies permits us to regard the basis of mental activity as a cerebral system made up of links of various degrees of rigidity, or-schematically-of rigid and flexible links (Bechtereva, 1966, 1967). This property of the system ensures economical efficiency of the brain's work, on the one hand, and, on the other, its reliability in the face of changing environmental conditions. It should be emphasized that the notion of rigid links is not meant to imply that a reaction may be strictly dependent on a single neuron. This is not the way a reaction is patterned, even at lower levels (rigid inborn patterns), let alone mental responses. One of


343

the mechanisms responsible for reliability of all reactions of the central nervous system depends on neurons functioning as neuronal groups, each link of the system being provided for by such a neuronal group. Extensive neurosurgical experience, particularly in pediatrics, supports the assumption that various rigid as well as flexible links may be attributed to ontogenetic mechanisms of development, depending on a determinate anatomical and physiological foundation. Mental activity apparently depends on great redundancy, probably inherent even in the rigid links of the system. In all probability, some of these mechanisms can be replaced, particularly after unilateral destruction of pertinent brain areas. The time has not come yet for judging how stable these zones of rigid and flexible relations between function and structure may be in different persons. Individual variants of brain anatomy, excessive fractionation of brain function, and the fact that information on the brain comes from pathological sources-these are but some of the limitations that should curb the imagination of the investigator. If, however, the evidence obtained thus far will be supported, the hypothetical system may stand the test of facts and subsequently grow up to the status of theory; it would also justify the approach adopted in these studies. What really takes place within the brain? The answer must be restricted to concrete facts in terms of the parameters and brain sites considered. There is a change in difference of potential at two points, situated at 3 mm distance; its absolute level also changes; there is an abrupt shift, mainly downward, in available oxygen levels; single cell activity increases during retention of a memory task and declines with recollection of the task; it tends to rise again on repetition, being reduced with subsequent retention of the task by memory. An endless array of queries for neurophysiological speculation. While structure-functional puzzles appear to fit into a hypothetical design, neurophysiological data should rather be presented as bare facts; as yet different parameters and different aspects of the brain’s activity being too incongruous for correlation. A hypothesis may serve as a lighthouse, but it may also act as blinders on a racehorse. Unless it is sure to shine as the former, it should be withheld, rather than being awkwardly guided by the latter. Where, then, are we in our intricate quest for the neurophysiological and structure-functional foundations of human mentality?

Eyes closed

A

Eyes opened

l

6 7 8 9 10

2

3 N.centralis 4 thalami 5

,

Eyes closed

Eyes opened

2

7

N. ventralis 3 lateralis thalami

8 9

[Pars bordering capsulae)

--

6

5

Light (trigger stimulation) The structure

Light (trigger stimulation) The structure is involved in the activity under altered conditions

10

1,2 .... tests used for the comparison

-Similar -Different

Steady potential shifts

on operative memory tests ( a t rest)

pattern pattern

Steady potential shifts on operative memory tests (on trigger stimulation)

FIG.3. A. Charts of the reproducibility dynamics of the steady potential changes during recent memory tests under different conditions. Left, eyes closed; middle, eyes open; right, during trigger stimulation. A comparison of the steady potential pattern for every test with that of the following test is made. Black squares mark a coincidental pattern of steady potential; white squares, noncoincidental; numerals, the number of test presentations. A regularity of steady potential changes in the central nucleus area can b e seen independent of the investigation conditions. Steady potential changes in the most external parts of the ventrolateral nucleus become reproducible with the eyes open and in light, and even more distinctly so with trigger stimulation. B. Scheme of the frontal brain plane at the level of thalamus. Cd, caudate nucleus; VL, ventrolateral nucleus; Pal, globus pallidus medialis; Pal.]., globus pallidus lateralis; Ped, pedunculi; H, hippocampus; VP, nucleus ventralis posterior; A, amygdala. Black squares mark areas of reproducible steady potential changes during operative memory tests. The tests were presented against a background of a minimal noise. In the right lower part ( C ) the same marks as in B. The test was presented along with trigger stimulation.

A

Hippocampus

Eyes closed

Eyes opened

1 2 3

6

4

9

13 14

5

10

15

Eyes closed

11 12

7 8

Eyes opened

6

Amygdala

B - 1.2 .....tests

used for the comparison

Oxygen availability alterations on operative memory tests ( 0 7 rest)

The structure is rigidly involved

Light (trigger st Imu lot Ion )

11 12 13 14 15

7 8 9 10

H-

Light (trigger stimulation)

The structure I s involved in the activity under altered conditions only -Similar

pattern

0- D i f f e r e n t

pattern

;qg., Ped .

Oxygen availability alterations on operative memory tests (on t r i g g e r stimulation)

FIG. 4. A. Charts of the reproducibility dynamics of oxygen availability alterations during recent memory tests under difEerent conditions. Left, eyes closed; middle, eyes open; right, during trigger stimulation. A comparison of the oxygen availability pattern for every test with that of the following tests was made. Black squares mark coincidental pattern of the oxygen availability alterations; white squares, noncoincidental. Numerals, the number of test presentations. A regularity of oxygen availability alterations in the hippocampus can be seen independent as of the investigation conditions. Oxygen availability alterations in the amygdala become reproducible with the eyes open and in light, and even more distinctly so with trigger stimulation. B. The same scheme as in the Fig. 3. Cd, caudate nucleus; VL, ventrolateral nucleus; VP, nucleus ventralis posterior; VPM, nucleus ventralis posteromedialis; dm, nucleus dorsomedialis; C, nucleus centralis; Pal., globus pallidus medialis; P a l l , globus pallidus lateralis; Ped., pedunculi; H, hippocampus; A, amygdala. Black squares mark areas of reproducible oxygen availability alterations on recent memory tests. The tests were presented against a background of a minimal noise. In the right lower part ( C ) the same as in B. The tests were presented along with trigger stimulation.

346


At the outset of a path, or may this quest prove to lead nowhere, as many have? But data are being verified in many laboratories the world over, so that we may trust that this time our quest will lead to a breakthrough. It should be kept in mind, however, that although what we know of the brain is much more than what was known yesterday, it is still infinitely less than what remains to be known. REFERENCES Abashev-Konstantinovski,A. L. ( 1961 ). Vopr. Neirokhirurgii 5, 45. Abashev-Konstantinovski, A. L. (1964). Z. Neoropatol. i Psikhiatr. 64, No. 1,

43. Abramovich, G. B., and Zakharovn, V. V. (1961). Zh. Gos. nazich.-Issled. Inst. inz. V. M. Bechterewa 21, 125. Adams, R. D., Collins, G . H., and Victor, M. (1962). In “Physiologie de I’hippocanipe,” pp. 273-298. Pergamon Press, Oxford. Aleksandrovskaia, M. M., Nevzorova, T. A., and Shpir, E. R. (1947). Zh. Nezjropatol. i Psikhiatr. 16, No. 2, 30. Alpers, B. J. (1937). A.M.A. Arch. Neurol. Psychiat. 38, 291. Anokhin, P. K. ( 1958 1. “Vnutrennee toimozhenie kak problema fiziologii (Internal Inhibition as a Problem of Physiology) ,” Medicine, Moscow. Ataev, M. M. (1955). Zh. Vvsshei Nerzjnoi Deyatel‘nosti im. 1. P. Pavlooa 5, No. 1, 104. Avramov, S. R., and Smirnov, V. M. (1965). Zn “Rol’ glubokikh struktur golovnogo mozga cheloveka v mekhanizmakh patologicheskikh reaktsii (Role of Deep Structures of the Human Brain in Mechanisms of Pathologic Responses)” (N. P. Bechtereva, ed.), pp. 12-17. Leningrad. Barbizet, J. (1963a). J. Neurol., Neurosurg., Psychiat. [N.S.] 26, 127. Barbizet, J. ( 1963b). Semaine Hop. Paris 39, No. 20, 935. Bechtereva, N. P. (1965a). In “Sovremennye problemy fiziologii i patologii nervnoi sistemy (Current Problems of Physiology and Pathology of the Nervous System)” (V. V. Parin, ed.), pp. 274-291. Nauka, Moscow. Bechtereva, N. P. ( 196%). In “Problemy sovremennoi neirofiziologii ( Problems of Current Neurophysiology)” (V. N. Chernigovski, ed.), pp. 100133.Nauka, Moscow-Leningrad. Bechtereva, N. P. ( 1 9 6 5 ~ ) .In “ROY glubokikh struktur golovnogo mozga cheloveka v mekhanizmakh patologicheskikh reaktsii ( Role of Deep Structures of the Human Brain in Mechanisms of Pathologic Responses)” ( N. P. Bechtereva, ed. )., pp. 2-5-30. Nauka, Leningrad. Bechtereva, N. P. (1966). In “Glubokie struktury golovnogo mozga cheloveka v nonne i patologii (Deep Structures of the Human Brain-Normal and Pathologic),” Contributions to a Symposium (N. P. Bechtereva, ed.), pp. 18-20. Nauka, Leningrad. Bechtereva, N. P. (1967). Trans. Inst. Exptl. Med., Leningrad 9, No. 1, 7. Bechtereva, N. P., and Orlova, A. N. (1957). “Trydy mezhoblastnoi konfer-


347

entsii neirokhirurgov Zapadnoi Sibiii I Utala,” pp. 113-117. Izd. Meditzina, Novosibirsk. Bechtereva, N. P., and Smirnov, V. hl. (1967). Vcstn. Akad. Alecl. Nnztk SSSR 1, 3. Bechtereva, N. P., and Trochatchev, A. I. (1966). 18th Intern. Congr. PAYchol., MOSCOW,1966, Symposium 6 (W. C . Walter, ed.), pp. 16-23. Bechtereva, N. P., Genkin, A. A., Moiseeva, N. I., and Smirnov, V. hl. (1965). Abstr. 6th Intern. Congr. EEG Clin. Neuiophysiol., Vienna, 1965, p. 8. Bechtereva, N. P., Smimov, V. M., and Trohatchev, A. I. (1966). I n “Konferentsia posviashchennaia problemam pamiati ( Conference on Memory) ,” pp. 7-8. Pushchino. Bechterew, W. M. (1900). Neurol. Zentr. 19, 990. Beritov, I. S., and Vorobiev, A. (1943). Trudy Inst. Fiziol. im. IS. Beritushvili, Tbilisi 5, 369. Bradley, P. B. ( 1965). In “Second Advanced Course in Electroencephalography,” pp. 157-161. Salzburg. Brazier, M. A. B. ( 1962). In “Elektroentsefalograficheskoe issledovanie vysshei nervnoi deyatel’nosti (EEG Investigation of Higher Nervons Activity),” pp. 341-352. MOSCOW. Brazier, M. A. B. ( 1963). I n “Brain Function” ( M. A. B. Brazier, ed.), Vol. 1, pp. 1-13. Pergainon Press, Oxford. Brazier, M. A. B. ( 1966). Rev. Rocinmine Nerrrol., 3, 29. Busch, E. (1940). Acta Psychiut. Neurol. Scarid. 15, 257. Chapman, L. F. (1966). 18th luterii. Congr. Psychol., Moscow, 1966, Symposium 6 ( W . C. Walter, ed.), pp. 60-82. Moscow. Cooper, J. S. ( 1961). “Parkinsonism. Its Medical and Surgical Therapy.” Thomas, Springfield, Illinois. Cooper, R., Winter, A. L., Crow, H. J., and Rebert, 0. S. (1965). Electmencephalog. Clin.Neurophysiol. 21, 538. Crow, H. J. ( 1965). Electroencephalog. Clin. Neurophysiol. 19, 531. DAndrea, F., and Paolozzi, F. ( 1963). Acta Neurol. (Naples) 18, No. 2, 184. Delgado, J. M. R. ( 1959). Electroencephalog. Clin. Neurophysiol. 11, 591. Delgado, J. M. R. (1964). Intern. Rcb. Nettrobid. 6, 399. Delgado, J. M. R., Roberts, W. W., and Miller, N. E. (1954). Am. 1. Physiol. 179, 587. Devoto, A. (1958). Aim. Mecl. Nac;ale Trop. 63, Suppl., No. 4, 53. Drachman, D. A. (1964). Arch. Neurd. 10, 411. Drachman, D. A., and Arbit, J. (1966). Arch. Neurol. 15, 52. Durup, G., and Fessard, A. (1935). Anti. Physiol. 36, 1. Feindel, W. (1964). In “Brain Function” ( M . A. B. Brazier, ed.), Vol. 2, pp. 277-298. Pergamon Press, Oxford. Foerster, O., and Gagel, 0. (1933). Z. Ges. Neurol. Psychint. 149, 312. Gastaut, H. A., and Bert, J. ( 19S4). Elc,ctroe,icepli~log.Clin. Neirrophysiol. 6, 433. Gastaut, 1%.A., ]us, A,, Morrell, F., Storm van Leeuwen, W., Bekkering, D., Kamp, A., and Werre, J. (1957a). Zli. Vysshei Nerunoi Deyatel’nosti im. 1. P . Pavlova 7. 25.

348


Gastaut, H. A., Roger, A., Dongier, S., and Regis, H. (1957b). Zh. Vysshei Nerimoi Deyatel‘nosti im. 1. P. Pavlova 7, 185. Gastaut, H. A., Naquet, R., Roger, A., Dongier, S., Regis, H., Morrell, F., Jus, A., and Jus, S. ( 1 9 5 7 ~ )Zh. . Vysshei Nervnui Deydel’nosti im. I . P . Pavlova 7, 2Q3. Gastaut, H. A., Jus, C., Morrell, F., Storm van Leeuwen, W., Dongier, S., Naquet, R., Regis, H., Roger, A., Bekkerin, D., Kamp, A,, and Werre, J. ( 1957d ) . Electroencephalog. Clin. Neurophysiol. 9, 1. Genkin, A. A. ( 1463). Dokl. Akad. Nauk SSSR 149,1460. 1966, SymGenkin, A. A. (1906). 18th Intern. Congr. Psychol., MOSCOW, posium 6 ( W. G. Walter, ed. ), pp. 86-90. Gersuni, G. V. (1955). Zh. vysshiNervnoi Deyatel‘nosti im. I . P. Pavlova 5, 665. Gersuni, G. V., and Korotkin, I. I. (1947). Dokl. Akad. Nauk S S S R 57, 417. Gersuni, G. V., Kozhevnikov, V. A., Maruseva, A. M., and Chistovich, L. A. (1948).Bull. Eksperim. Biol. i Med. 26, 205. Giannitrapani, D. (1966).Perceptual Motor SkiEls 22, 399. Glees, P., and G&th, H. B. (1952). Mautsschr. Psychiat. Neurol. 123, 193. Gorelik, L. I. (1967). In “Materialy k nauchnoi konferentsii molodykh uchenykh IEM (Contributions to Conference of Young Investigators, Inst. Exptl. Med.),” pp. 15-17. Leningrad. Gretchin, V. B. (1966a). In “Pervaia nauchnaia konferentsia molodykh uchenykh Petrogradskogo raiona ( 1st Conference of Young Investigators, Petrograd District),” pp. 97-98. Leningrad. Gretchin, V. B. (1986b). In “Glubokie struktury golovnogo mozga cheloveka v mekhanismakh patologicheskikh reaktsii (Deep Structures of the Human Brain in Pathologic Responses)” ( N . P. Bechtereva, en.), pp. 50-52, Nauka, Leningrad. Gretchin, V. B. (1967). In “Materialy k nauchnoi konferentsii molodykh unchenykh IEM (Contributions to Conference of Young Investigators, Inst. Exptl. Med.),” pp. 17-20. Leningrad. Griinthal, E. (1939). Confiniu Neurol. 2, 64. Griinthal, E. (1947). Monatsschr. Psychiat. Neurol. 113, 1. Heath, R. G. (1954). I n “Studies in Schizophrenia,” pp. 197-200. Harvard Univ. Press, Cambridge, Massachusetts. Heath, R. G. (1983).Am. J. Psychiut. 120, 571. Heath, R. G., and Mickle, W. A. (1960). In ‘‘Electrical Studies on the Unanesthetized Brain” (E. R. Ramey and D. S. O’Doherty, eds.), pp. 214247. New York, Harper. Heath, R. G., Peacock, S. M., Monroe, R. R., and Miller, W., Jr. (1954). In “Studies in Schizophrenia,” pp. 573-608. Harvard Univ. Press, Cambridge, Massachusetts. Heath, R. G., Monroe, R. R., and Mickle, W. A. (1955). Am. J . Psychiat. 11, 862. Jasper, H., and Shagass, C. (1941). J. Exptl. Psychol. 28, 503. Jasper, H., Riggie, H., and Down, B. (1962). In “Elektroentsefalogficheskoe issledovanie vysshei nervnoi deyatel’nosti ( EEG Investigation of Higher Nervous Activity)” ( G . D. Smirnov, ed.), pp. 129-146. Moscow.

PHYSIOLOGICAL FOUNDATIONS OV MENTAL ACTIVITY

349

Jung, R. ( 1954). In “Brain Mechanisms and Consciousness” ( J . F. Delafresmaye, ed.1, PP. 310-339. Oxford Univ. Press, London and New York. Jus, A., and JUS, S. (1954). Zh. Neuropatol. i Psikhiat. 54, 715. Knott, I. R. ( 1939). J. Ezptl. Psychol. 24,384. Knott, I. R., and Henry, C . E. (1941). J. Ezptl. Psychol. 28, 134. Kohogorov, A. N. (1963). In “Vozmoshnoe i nevozmoshnoe v kibernetike (Possibility and Impossibility in Cybernetics),” pp. 10-29. U.S.S.R. Acad. Sci. Publ. House, Moscow. Kooi, K. A., and Boswell, R. (1980). in “Recent Advances in Biological Psychiatry,” pp. 172-182. New York. Korsakoff, S. S. (1890). “Boleznennye rasstroistva pamiati i ikh diagnostika (Pathologic Disorders of Memory and their Diagnosis ),” Moscow. Kozhevnikov, V. M., and Maruseva, A. M. (1949). Izo. Akad. Nauk S S S R , Ser. Biol. 5, 560. Kratin, Yu. G. (1955). Fiziol. Zh. SSSR 41, 676. Kugler, J. (1963). Z. Exptl. Angew. Psychol. 10, 197. Lansing, R. W. (1957). Electroencephulog. Clin. Neurophysiol. 9, 497. Lebedinski, A. V., and Bechtereva, N. P. (1960). Fiziol. Zh. S S S R 46, 509. Lelord, G. ( 1957). Ekctroencephalog. Clin. Neurophysiol. 9, 561. Livanov, M. N., Gavrilova, N. A., and Aslanov, A. S. (1966). 18th Intern. Congr. Psychol., MOSCOW, 1966, Symposium 6 (W. G. Walter, ed.), pp. 31-38. Loomis, A. L., Harvey, E. N., and Hobart, G. (1935a). Science 81, 587. Loomis, A. L., Harvey, E. N., and Hobart, G. (1935b). Science 82, 198. Loomis, A. L., Harvey, E. N., and Hobart, G. (1936a). Science 83, 239. Loomis, A. L., Harvey, E. N., and Hobart, G. (193613). J. ErptZ. PsycTwZ. 19, 249. Low, M. D., Borda, R. P., Frost, J. D., and Kellaway, P. (1966). Neurology 16, 771. Luria, A. R. (1962). “Vysshie korkovye funktsii cheloveka (Higher Cortical Functions in Man),” Moscow. Luria, A. R. ( 1963). “Mozg cheloveka i psikhicheskie protsessy (Human Brain and Mental Processes) .” MOSCOW. Lurie, R. N., and Rusinov, V. S. (1955). Probl. Fiziol. Optiki 11, 113. Maiorchik, V. E., and Spirin, B. G. (1951 ). Vopr. Neirokhirurgii 3, 3. Maiorchik, V. E., Rusinov, V. S., and Kuznetsov, G. D. (1954). In “K fiziologicheskomu obosnovaniu neirokhirurgicheskikh operatsii ( Physiologic Foundation of Neurosurgical Operations ) ,” p. 48. Izd. Meditzina, MOSCOW. Makarov, P. 0. (1956). in “Voprosy fiziologii nervnoi i myshechnoi sistemy (Problems of Physiology of Nervous and Muscle Systems),” pp. 365-371. Thilisi. Menozzi, C., and Biliach, E. (1963). Sistema Nervoso 15,319. Minky, A. F.,and Roswald, H. ( 1963). In “Psychophamacological Methods,” pp. 309-315. Pergamon Press, Oxford. Mituosi, A. (1954). Tohoku J . Exptl. Med. 60, 47. Moiseeva, N. I., and Orlov, V. A. (1965). In “Rol’ glubokikh struktur golovnogo mozga cheloveka v mekhani7makh patologicheskikh reaktsii (Role of

350

N. P. BECHTEREVA AND V. B. CRETCHIN

Deep Structures of the Human Brain in Pathologic Responses)” (N. P. Bechtereva, ed. ), pp. 98-99. Izd. Meditzina, Leningrad. hlonroe, R., and Heath, R. G. (1954). In “Studies on Schizophrenia,” pp. 345360. Harvard Univ. Press, Cambridge, Massachusetts. Mulholland, T., and Runnals, S. (1962). 1. Psychol. 52,317. Mushkina, N. A. (1956). Zh. Vysshei Nervnoi Deyatel‘nosti im. I . P . Pavlova 6, 157. Novikova, L. A., and Sokolov, E. N. (1957). 21%. oysshei Nervnoi Deyutel’nosti im. I . P. Pavlova 7, 363. Orthner, H. (1957). 1st Congr. Intern. Sci. Neurol., 1957. Sec. J. Comm., pp. 77-96. Peimer, I. A., and Fadeeva, A. A. (1956). Fiziol. Zh. SSSR 42, 319. Penfield, W. (1958a). Res. Publ., Assoc. Res. Neroocis Mental Disease 36, 210. Penfield, W. (1958b). Med. News ( N . Y . ) June, 8. Penfield, W. ( 1959a). In “Memory, Learning and Language” ( W . Feindel, ed.), pp. 55-60. Univ. of Toronto Press, Toronto. Penfield, W. (1959b). Vestn. Akad. Nauk SSSR 12, 22. Penfield, W., and Milner, B. (1958). A.M.A. Arch. Neurol. Psychiat. 79, 475. Pool, J. L. (1948). Cited after Bengochea et 01. (1954) (see additional references ). Sano, K. (1966). Progr. Brain Res. 21B, 350. Scoville, W. B. ( 1954). J . Neurosurg. 11, 64. Scoville, W. B., and Milner, B. (1957). J. Neurol., Neurosurg., Psychiat. 20, 11. Seregina, T. A. (1956). Bull. Eksperim. Biol. i Med. 2, 7. Shagass, C. ( 1942). Exptl. Psychol. 31, 367. Shagass, C., and Canter, A. (1966). 18th Intern. Congr. Psychol., Aloacow, 1966, Symposium 6 ( W. G. Walter, ed. ), pp. 47-52. Slatter, K. H. (1960). Electroencephulog. Clin. Neurophysiol. 12, 851. Smirnov, V. M. (1963). Abstr. Comm. 2nd Congr. SOC. Physiologists, Moscow, 1963, p. 4. Smirnov, V. M. (1965). In “Rol’ glubokikh struktur golovnogo mozga cheloveka v mekhahizmakh patologicheskikh reaktsii (Role of Deep Structures of the Human Brain in Mechanisms of Pathologic Responses)” (N. P. Bechtereva, ed. ), pp. 100-111. Nauka, Leningrad. Smirnov, V. M. (1966a). In “Glubokie struktury golovnogo mozga cheloveka v nornie i patologii (Deep Structures of the Human Brain-Normal and Pathologic)” ( N . P. Bechtereva, ed.), International Symposium, pp. 134140. Nauka, Leningrad. Smirnov, V. h1. (1966b). Vopr. Psikhol. 10, 385. Smirnov, V. M., and Grachev, K. V. (1963). I n “Elektrofiziologia nervnoi systemy ( Electrophysiology of the Nervous System),” Contributions to 4th All-Union Electrophysiological Conference, Rostov-on-Don, p. 355. Spiegel, E. A. (1966). J. Neurosurg. 24, 433. Spiegel, E. A., and Wycis, H. T. (1952). “Stereoenephalotomy, Thalamotomy and Related Procedures.” Part 1. Methods and Stereotaxic Atlas of the Human Brain. Crune and Stratton, New York.

PHYSIOLOGICAL FOUNUATIOXS OF MENTAL ACTIVITY

351

Spiegel, E. A., and Wycis, H. T. (1961). I n “Electrical Stimulation of the Brain” (P. E. Sheer, ed.), pp. 487491. Thomas, Springfield, Illinois. Spiegel, E. A., Marks, M., and Lee, A. J. (1947). Science 106, 349. Spiegel, E. A., Wycis, H. T., Orchick, C., and Freed, H. (1956). Am. J. Psychiat. 113, 95. Stepien, L., and Sierpinsky, S. (1960). J. Neurol., Ncurosurg., Psychiat. 23, 334. Talairach, J., and Bancaud, J. (1966). Intern. J. Neurol. 5, 330. Terzian, H., and Dalle Ore, G. (1955). Neurology 5, 373. Umbach, W. ( 1964). Intern. Kongr. cortiko-uiscerule Thcrapie, Berlin, 1964, p. 108. Umbach, W. ( 1966). “Electrophysiologische und vegetative Phanomene bei stereotaktischen Himoperationen.” Spcinger, Berlin. Urnmancheeva, T. G., and Diakonova, I. N. (1965). In “Rol’ glubokikh strnktur golovnogo mozga cheloveka v niekhanizmakh patologicheskikh reaktsii (Role of Deep Structures of the Human Brain in Mechanisms of Pathologic Responses)” ( N . P. Bechtereva, ed.), pp. 118-119. Nauka, Leningrad. Ursin, H. (1960). Acta Psychiat. Ncurol. Scancl. 35, 373. Van Buren, J. M. ( 1963a). Electrocncephdog. C h i . Neurophysiol. 15, 1. Van Buren, J. M. (1963b). J. Neurosurg. 20, 148. Victor, M., Angevine, J. B., Mangall, E. L., and Fisher, C. M. (1961). Arch. Neurol. 5, 244. Voronin, L. G., and Sokolov, E. N. (1962). In “Elektroentsefalograficheskoe issledovanie vysshei nervnoi deyatel’nosti ( EEG Investigation of Higher Nervous Activity)” ( G . D. Smirnov, ed.), pp. 310-321. Publ. Acad. Sci. USSR, Moscow. Walter, R. D., and Yeager, C. L. (1956). Electroeticrpho7og. Clin. Ncuroph-ysiol. 8, 193. Walter, W. G. (1965). Brit. J. Physiol. Optics 22, 1. Walter, W. G. (1966). Current Psychiat. Therapy 6, 18. Walter, W. G., and Crow, H. J. (1964). Electroctice~~halog. Clin. Neurophysiol. 16, 68. Walter, W. G., Aldridge, V. J., Cooper, R., McCallum, W. C., and Cohen, J. (1964). Reu. Neurol. 111, 257. Walter, W. G., Cooper, R., Crow, H. J., McCallum, W. C., Warren, W. J., Aldridge, V. J., Storm van Leeuwen, W., and Kamp, A. (1967). Electroencephalog. Clin. Neurophysiol. 23, 197. Webb, W. B. (1966). 18th Ziitcrn. Congr. Psychol., Moscow, 1966, Symposium 6 ( W. G. Walter, ed.), pp. 96-102. Williams, hl., and Pennypacker, S. ( 1954). J. Ncurol., Ncurosurg., Psychiat. 17, 115. Wurts, R. H. ( 19137). Electroenccphalog. Clin. Neurophysiol. 22, 43. Zangwill, 0. L. (1963). Adwun. Sci. 20, 335. ADDITIONALREFERENCES

Bechtereva, N. P., Grachev, K. V., Orlova, A. N., and Yatzuk, S. K. (1963). Zh. Neuropatol. i Psikhiatr. 63, 3.

352


Bechtereva, N. P., Bondarchuk, A. M., Smirnov, V. M., and Trochatchev, A. I. ( 1967). In “Physiologia i patophysiologia glubokip struktur mozgo chiloveko” (Physiology and Pathophysiology of the Deep Brain Structures of Man). Izd. Meditzina, Leningrad-Moscow. Bengochea, F. G., Sachs, E. I., and Llewellyn, R. C. (1954).In “Studies in Schizophrenia,” pp. 124-141. Harvard Univ. Press, Cambridge, Massachusetts. Beyn, E. S., Zhirmunskaya, E. A., and Volkov, V. N. ( 1967).Neurophysiologiu 5, 203. Brazier, M. A. B. (1964).In “Brain Function” ( M . A. B. Brazier, ed.), Vol. 2,pp. 299-310.Pergamon Press, Oxford. Gretchin, V. B. (1968).Fiziol. Zh. S S S R 54(3), 380. Gretchin, V. B. ( 1968).In “Physiologia i patologia limbiko-reticulamago kompleksa” ( Physiology and Pathology of the Limbico-Reticular Complex 1. (P. K. Anokin and A. U. Vein, eds.), pp. 1213. Nauka, Moscow. Gretchin, V. B., and Smimov, V. M. (1967).In ‘Tsikhologocheskie metody issledovania v klinike ( Psychological Methods in Clinical Investigation )” (V. N. Uyasishev et ul., eds.), pp. 232238.Leningrad. Trohatchev, A. I. ( 1966). In “Glubokie struktury golovnogo mozga cheloveka v mekhanianakh patologicheskikh reaktsii (Deep Structures of the Human Brain in Mechanisms of Pathologic Responses ) ,” International Symposium, pp. 157-160.Leningrad. Trohatchev, A. I. (1967).Trans. Inst. Erptl. Med., Leningrad 9,No. 1, 16.

AUTHOR INDEX Numbers in parentheses are reference numbers and indicate that an author’s work is referred to, although his name is not cited in the text. Numbers in italics show the page on which the complete reference is listed. Altman, K. I., 269, 276, 278, 279, A 289 Abashev-Konstantinovski, A. L., 329, Altschule, M. D., 175, 181, 182, 187, 346 196, 197 Abderhalden, E., 58, 62, 64, 90 Altshule, M. D., 261, 264, 265, 286, Abderhalden, R., 81, 90 288, 289 Abraham, D., 67, 86, 90, 91, 95 Alsleben, B., 106, 107, 126 Abe, K., 130, 160 Amdisen, A,, 158, 167 Abood, L. G., 121, 123, 124 Abramovitch, G. B., 320, 325, 329, Ammon, H. P. T., 109, 125 Anden, N. E., 48, 55 346 Andersen, P., 8, 53 Adams, C. W. M., 72, 84, 91 Anderson, B., 160 Adam, E. S., 78, 91 Anderson, E., 172, 194 Adam, H. M., 29, 53 Anderson, W., 158, 160 Adams, R. D., 329, 346 Andrews, M. C., 141, 160 Addison, J., 156, 160 Andrews, W. C., 141, 160 Adler, R. C., 131, 168 Adler, T. K., 100, 101, 104, 105, 124, Andreyeva, V. N., 229(8), 248 Andronnikov, N. J., 232(9), 248 127 Anfinsen, C. B., 09, 95 Ado, A. D., 309, 325 Angevine, J. B., 329, 351 Aganyants, E. K., 240(1), 248 Angyal, L., 238( lo), 248 Aikman, M. L., 100, 124 Anokhin, P. K., 334, 346 Akabane, Y., 267, 290 Anokhina, I. P., 230( 123), N O ( 123), Akkerman, V. J., 234(2), 248 252 Albers, W. R., 184, 194 Antebi, R. N., 267, 286 Albert, L., 62, 92 Appel, K. E., 266, 288 Albert, Z., 71, 91 Arbit, J., 329, 347 Aldridge, V. J., 334, 351 Arnold, 0. H., 273, 274, 278, 279, Aleksandrovskaia, M. M., 329, 346 287, 288 Aleksandrovsky, J. A., 240( 88), 251 Meksanyants, R. A., 234( 3), 236( 3), Aprison, M. H., 27, 55, 56, 66, 92, 97 248 Arai, Y., 80, 91 Alekseyenko, N. J.. 231(4), 248 Armand-Delille, 293 Alertsen, A., 280, 290 Arnold, O., 220(62), 225 Alertsen, A. R., 280, 285, 286, 290 Aron, E., 178, 194 Alestig, K., 134, 160 Alexander, L., 238(5, 6, 7), 244(7), ArstiIa, A. U., 172, 194 hutjunov, D. H., 206(23), 223 248 Arutjunov, E. S., 239( 11, 12), 248 Allen, E., 148, 160 Alpers, B. J., 346 Arvy, L., 80, 91 Alt, H. L., 134, 163 Asagoe, Y., 181, 194 353

354

AUTHOR INDEX

Asatiani, L. M., 233( 1 3 ) , 248 Ashoff, J., 138, 148, 160 Ask-Upmark, E., 133, 160 Aslanov, A. S., 236(14, 1 5 ) , 246 ( 15), 248, 333, 349 Asratyan, E. A., 229( 16, 17), 248, 249 Assael, M., 158, 168 Asserson, B., 73, 91 Astmp, C., 229( 18-26), 230( 18-26), 231(22), 232(22), 233(22, 23), 234( 18-22), 235( 22, 23), 236 (22, 23, 26), 240( 22-24), 241 ( 2 2 - a ) , 242( 22-24), 243( 2223), 246(26), 249 Ataev, M. M., 331, 346 Auditore, J. V., 74, 95 Avakyan, R. V., 231(27), 249 Avramov, S. R., 344, 346 Avanzino, G . L., 21, 30, 31, 33, 39, 40, 41, 53, 54 Avar, Z., 147, 161 Axelrod, J., 104, 105, 106, 107, 108, 124, 125, 174, 175, 176, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198 B Baastrup, P. C., 161 Bachelard, €1. S . , 110, 124 Balazs, R., 282, 289 Baldwin, R., 85, 91 Ball, J., 143, 161 Balonov, L. J., 229(233-235), 230 (233-235), 232( 233, 234), 240 (233-235), 241(234-235), 256 Bamdas, B. S., 209(28), 224 Ban, T. A., 230( 29), 237( 110), 243 (110), 249, 252 Bancaud, J., 335, 351 Banshcikov, V. M., 229( 193), 255 Baranowski, T., 70, 96 Barber, V. T., 81, 96 Barbizet, J., 329, 346 Bard, P., 147, 161 Barkve, H., 134, 161 Barnes, G., 152, 161

Barnes, F. W., 301, 328 Barraclough, C. A., 143, 161 Bartlett, G. B., 271, 278, 279, 287 Baskina, N. F., 235(30), 249 Bassin, F. V., 229( 193), 255 Bastide, P., 81, 96 Baudhuin, P., 68, 69, 92 Bayerovi, G., 171, 174, 195 Bayrakci, C., 266, 287 Baker, P. C . , 183, 184, 195 Barbour, B. H., 183, 194 Barchas, J. D., 174, 183, 197 Bartter, F. C . , 183, 194 BaschiAri, L., 178, 194 Bastide, P., 197 Bauer, W., 156, 161 Bayer, A., 171, 174, 195 Bazanova, A. N., 239(31), 244(31), 249 Beach, F. A., 143, 161 Beattie, C. W., 172, 195 Beaufay, H., 68, 91 Bechtereua, N. P., 332, 333, 336, 338, 339, 340, 344, 346, 347, 349, 351, 352 Bechterew, W. M., 329, 347 Beck, C. S . , 82, 91 Beck, G . M., 156, 164 Beckett, A. H., 100, 106, 124 Beckett, P., 217(56, 57), 225, 281, 287 Beckett, P. G. S., 272, 273, 278, 279, 281, 282, 287, 288, 289 Beckhtereva, N. P., 228(32), 249 Behal, F. J., 72, 73, 91 Behrens, H., 295, 328 Bekkering, D., 331, 332, 347, 348 Beleslin, D., 117, 118, 124 Belestreri, R., 181, 195 Bell, J. L., 108, 124 Bengochea, F. G., 3, 352 Benington, F., 43, 56 Benjaminsh, L. A., 234(33, 3 4 ) , 236 (33, 3 4 ) , 238(33, 34), 249 Bennett, E. L., 4, 53 Bentley, K. W., 100, 124 Beraldo, W. T., 76, 96 Bercel, N. A,, 132, 133, 161

AUTHOR INDEX

Berg, S., 27, 53 Bergen, J . R., 283, 287 Beigcr, A., 67, 96 Bergmann, M., 58, 74, 91 Bergsman, A., 263, 287 Beringer, K., 136, 161 Beritov, I. S., 331, 347 Berl, S., 71, 91 Berleur, A., 68, 91 Berman, E. R., 285, 287 Bernsohn, J., 273, 278, 279, 287 Bert, J., 333, 347 Berthelay, J., 176, 178, 197 Beyn, E. S., 352 Bessman, S. P., 85, 91 Biesold, D., 275, 278, 279, 287 Biliach, E., 331, 349 Binswanger, O., 132, 161 Binswanger, L., 144, 166 Bishop, C., 274, 278, 279, 287 Biscoe, T. J., 12, 20. 53 Blaise, J., 81, 96 Blaise, S., 175, 176, 178, 197 Blech, W., 69, 93 Bleuler, E., 131, 161 Bleuler, M., 150, 152, 161 Blight, R., 82, 94 Bloom, F. E., 11, 22, 34, 35, 53, 56 Blnm, E., 64,91 Boakes, R., 21, 53 Bois, I., 73, 96 Boissanas, R. A,, 77, 91 Bondarchuk, A. M., 340, 352 Bondy, P. K., 135, 161 Bonhoffer, K., 132, 161 Bonkalo, A., 158, 161 Borda, R. P., 334, 349 Borecka, D., 323, 325 Borison, H. L., 116, 126 Borissova, M. K . , 232( 35), 249 Borud, O., 149, 165 Bostoganashvili, N. I., 2lO( 37), 224 Boswell, R., 333, 349 Boszormenyi-Nagi, I., 266, 272, 287 Boura, A. L., 100, 124 Bowers, C. Y., 81, 96, 119, 126, 127 Bowman, J. E., 266, 287 Boyer, J., 81, 96

355

Boyce, K., 158, 161 Boyd, v., 292, 325 Boyer, J., 197 Bradley, P. B., 6 , 9, 10, 12, 13, 14, 15, 16, 20, 21, 23, 24, 25, 29, 30, 31, 33, 35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 52, 53, 54, 55, 56, 332, 347 Bradley, R. J., 43, 56 Brady, R., 300, 328 Brodford, N. M., 138, 161 Brand, K., 139, 164 Braeunlich, H., 102, 124 Braukmann, R., 120, 126 Brauninger, C., 266, 287 Braunitzer, G., 73, 95 Brayer, F. T . , 142, 161 Brazier, M. A. B., 329, 335, 347, 352 Brecher, A. S., 68, 75, 91 Brems, U., 233(36, 37), 234(36), 235( 36, 37), 238( 36, 37 ), 249 Brewer, G. J., 266, 287 Brink, 121, 124 Brinkley, F., 67, 91 Brizzee, K. R., 156, 162 Brock, T. D., 86, 91 Brockman, D. D., 151, 161 Brodie, B. R., 114, 126 Broman, L., 273, 287 Bronisch, F. W., 238(38), 249 Broolies, N., 21, 45, 53, 54 Brooks, J. W., 116, 119, 125, 127 Brossard, M., 115, 124 Brown, F. A., 141, 161 Brown, J. D., 283, 289 Brown, J. J., 143, 168 Brown, J. R., 75, 91 Brown-Grant, K., 150, 161 Browning, B., 119, 127 Bruce, U . G., 137, 166 Bruns, F. H., 268, 270, 289 Bryce, C. F . , 62, 91 Bucy, P. C., 147, 164 Biinning, E., 138, 140, 141, 161 Biissow, H., 136, 161 Bnhler, D. R., 282, 287 Bunney, W. E., 132, 155, 156, 161, 162

356

AUTHOR INDEX

Chong, G. C., 17, 56 Christensen, H. N., 86, 91 Chu, E. W., 175, 176, 190, 195, 197, 198 Chuprikov, A. P., 30@,302, 311, 322, 323, 325, 327 Citterio, C., 234(42), 240(42), 249 Clark, L. D., 156, 161 Clarke, R. W., 159, 167 Claude, H., 133, 161 Clegg, J. B., 76, 96 Clegg, M. T., 188, 195 Cleghorn, R. A., 156, 162 Clementi, F., 176, 179, 182, 195, 196 C Clift, J . W., 121, 125 Cloudsley-Thompson, J. L., 138, 148, Cade, J. F., 158, 161 162 Cagnoni, M., 142, 161 Clouet, D. H., 103, 106, 107, 110, Cameron, M. P., 150, 161 111, 112, 113,124,126 Canter, A., 333, 350 Cobb, S., 156, 161, 168 Canzler, E., 275, 278, 279, 287 Coceani, F., 30, 54 Carabateas, P. M., 100, 124 Cochin, J., 104, 106, 107, 121, 124 Carlsson, A., 192, 195 Cohen, E., 74, 94 Carpelan, H., 151, 161 Cohen, G., 266, 287 Carter, J. D., 156, 165 Cohen, J., 334, 351 Carter, J. L., 266, 287 Cohen, M., 114, 124 Carter, W. H., 81, 96, 119, 127 Cohen, R. A., 149, 162 Cartier, P., 270, 287 Cohen, W., 70, 92 Case, J. O., 175, 183, 185, 196 Cohn, G. L., 135, 161 Casper, A. G. T., 183, 194 Cohne, L., 67, 88, 95 Castro, J. A., 108, 124 Coleman, J. E., 76, 91 Casy, A. F., 100, 106, 124 Collier, B., 16, 54 Ceaser, G., 58, 62, 64, 90 Collins, G. H., 329, 346 Cecotto, C., 265, 266, 289 Combescot, C., 178, 194 Cesari, L., 140, 161 Comis, S. D., 30, 53 Chance, B., 139, 166 Conney, A. H., 104, 107, 124 Celesia, G. G., 54 Constantinescu, G. N., 130, 162 Celliers, P. G., 73, 95 Chalisov, M. A., 206(2%25), 207 Cookson, B. A,, 145, 146, 147, 161 Coppen, A., 156, 158, 162 223 Coppen, A. J., 282, 289 Chapeville, F., 89, 91 Cooper, J. S., 330, 347 Chapman, L. F., 82, 91, 333, 347 Cooper, R., 334, 347, 351 Chedru, P., 270, 287 Corker, C. S., 147, 162 Chen, C. A,, 305, 326 Corrado, A. P., 116, 127 Cherayil, A,, 104, 124 Corrodi, H., 162 Chiazze, L., 142, 161 Costa, E., 11, 22, 35, 53, 56 Childs, H. M., 264, 289 Cottrell, G . A., 46, 55 Chistovich, L. A,, 331, 348 Cox, B. M., 117, 124 Chodera, A., 116, 124

Burdette, B. H., 119, 124 Burlina, A., 267, 287 Burmistrova, T. D., 129, 161 Bum, J. H., 32, 51, 54 Burnet, 292, 302 Burstone, M. S., 69, 72, 91 Busch, E., 347 Buskirk, E. R., 131, 168 Butorin, V. I., 229(39), 243(39), 249 Byers, L. M., 303, 326 Bykov, K. M., 229(40), 249 Bykov, V. D., 249

(w,

AUTHOR INDEX

357

Davis, R., 8, 9, 20, 24, 29, 54, 55 Davis, R. H., 81, 95 Davis, W, J., 68, 82, 92, 95 Dawson, F., 73, 91 Dawson, F. B., 72, 73, 91 Dawson, J., 158 Day, M., 193, 195 De Duve, C., 68, 69, 92 de Jonge, J., 136, 162 DeLaHaba, G., 72, 92 Del Castillo, J., 6, 55 Delgado, J. M. R., 336, 347 DeLuca, F., 178, 195 Demaret, J., 178, 194 de Martino, C., 178, 195 Demidova, L. P., 249 Demin, A. A., 325 Dniitriyev, A. S., 231(47), 236(46), 239( 46, 47), 250 Dimitriyev, L. I., 231(48, 49), 234 (48), 236(48), 239(48), 244 (48), 250 Deneau, G. A., 100, 127 D Denton, D., 182, 195 Dabchev, P., 130, 169 Dem, R. J., 266, 287 Dahlstriim, A,, 18, 55 De Robertis, E., 4, 18, 26, 55, 56 Dale, H. H., 44, 55 De Robertis, M. D., 172, 195 Dalle Ore, G., 329, 351 De Sousa, J. F., 266, 287 Dalton, K. D., 147, 162 Determann, H., 73, 95 Daly, J., 105, 124 De Verdier, C. H., 270, 271, 287 Daly, R. J., 141, 162 De Vergiliis, C., 176, 195 D'Andrea, F., 336, 347 Devoto, A., 333, 347 Danforth, C. H., 148, 160 Dewan, E. M., 162 Danlenko, E. T., 239(43, 44), 244 Dewan, J. G., 146, 148, 149, 155, (43, 44), 249 163 Darken, M. A., 89, 91 Dewey, W. L., 118, 125 Dastugue, G., 81, 96, 197 Dhawan, B. N., 10, 12, 14, 16, 52, Datta, R. K., 63, 64, 65, 66, 72, 73, 54 75, 82, 87, 91, 92, 94 Diakonova, I. N., 335, 351 Davidoff, R. A., 27, 55, 56, 66, 91, Diamond, M. C., 4, 53 97 Dietze, F., 269, 289 Davidson, P. F., 85, 96 Dische, Z., 270, 287 Davies, B., 303, 328 Dixon, M., 58, 92 Davies-Jones, A., 130, 149, 150, 164 Dneprovskaya, S. V., 231( 144), 236 Davies, R. E., 138, 161 (1441, 238( 1441, 241( 144), Davis, J. O., 182, 195 253 Davis, M. M., 106, 125 Dobrzanskaya, A. K., 234(50, 51), Davis, N. C., 67, 91 237(50, 51), 239(50, 51), 250

Cox, J. R., 130, 149, 150, 164 Cramarossa, L., 178, 195 Crammer, J. L., 130, 131, 150, 151, 159, 162, 166 Crandall, R. G., 281, 289 Crane, R. K., 86, 92 Crawford, J. M., 13, 28, 54 Creasey, W. A,, 114, 125 Crispell, K. R., 135, 161 Cristodorescu, D., 130, 162 Crossland, J., 4, 30, 54, 56 Crow, H. J., 334, 336, 347, 351 Csejtey, J., 68, 82, 92 Csillik, B., 4, 55 Cullen, A. M., 81, 96 Culp, H. W., 107, 126 Curtis, D. R., 2, 7, 8, 10, 11, 13, 20, 24, 25, 26, 27, 28, 29, 53, 54 Custod, J. T., 273, 278, 279, 287 Cuzin, N., 89, 91 Czajkowski, N. P., 217( 58), 225( 58), 281, 288

358

AUTHOR INDEX

Dodge, P. W., 109, 125 Doisy, E. A., 148, 160 Dokucayeva, 0. N., 233(52, 53), 250 Dongier, S., 331, 332, 348 Donovan, B. T., 174, 195 Douglas, W. W., 121, 125 Down, B., 339, 348 Doyen, A., 68, 91 Drachman, D. A., 329, 347 Drellich, M. G., 145, 168 Driscoll, E., 66, 92 Droz, B., 83, 92 Drummond, P., 158, 163 Diisterdieck, G. O., 143, 168 Duffy, B. J., 142, 161 Dwell, J., 149, 150, 151, 162, 165, 282, 283, 287, 289 Durup, G., 331, 347 du Vigneaud, V., 67, 92 Dyer, C. G., 263, 287 Dyfverman, A,, 273, 287 Dzhamdzhieva, T. S., 130, 169 E

Easterday, 0. D., 264, 287 Ebels, I., 174, 176, 195 Eber, O., 83, 92 Ebstein, W., 137, 162 Eccles, J. C., 2, 7, 46, 55 Eccles, R. M., 7, 54 Economon, S., 107, 124 Efimovich, N. G., 273, 287 Efroimson, V. P., 213 (52, 53), 214 (52, 53), 225 Eglitis, B., 146, 149, 155, 163 Eichholz, A., 86, 92 Eik-Nes, K. B., 156, 162 Einstein, E. R., 68, 82, 92, 95 Eisenman, A. J., 116, 117, 119, 125, 127, 128 Eisenstein, R. B., 266, 287 Ekelova-Bagaley, E. M., 236( 54), 243(54), 250 Ekman, L., 150, 160 Eling, W., 139, 162 Ellis, H., 148, 162 Elkes, J., 36, 42, 43, 53

Elliot, D. F., 77, 92 Elliot, H. W., 101, 105, 108, 120, 125, 126 Elks, D., 63,66, 92 Ellis, E., 72, 90 Ellis, S., 70, 71, 78, 79, 92, 94 Elsasser, G., 132, 162 Engel, G. L., 156, 162 Engeli, M., 141, 163 Enzenbach, R., 80, 97 Eraos, E. G., 77, 92 Erlanger, B. F., 70, 92 Estler, C. J., 109, 125 Ewing, J. A., 141, 162 Exley, D., 147, 162 Ezhkova, Z., 299, 307, 328

F Fachet, J., 182, 197 Faddeyeva, V. K., 230(55), 2

International Review of Neurobiology Volume 11

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