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

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)

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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

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CONTENTS CONTR~BUTORS .

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PREFACE.

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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 . . . . . . . .

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Exopeptidases of the Nervous System

NEVILLEMARKS I . Scope of Review and Introduction

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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 . . . . . . . . . . .

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

DORISH . CLOUET

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

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99 101

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

I11 IV V VI .

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108 115 121 122 124

Periodic Psychoses in the Light of Biological Rhythm Research

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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 . . . . . . . . .

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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

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

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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

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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 . . . . . . .

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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 . . . . . . . . . . .

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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 . . .

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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

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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

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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

CONTENTS OF PREVIOUS VOLUMES

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

CONTENTS OF PREVIOUS VOLUMES

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

CONTENTS OF PREVIOUS VOLUMES

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

CONTENTS OF PREVIOUS VOLUMES

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

CONTENTS OF PREVIOUS VOLUMES

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

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

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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 . . . . . . . .

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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

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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

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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

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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-

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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

SYNAPTIC TRANSMISSION I N THE CNS

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

SYNAPTIC TRANSMISSION I N THE CNS

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

SYNAPTIC TRANSMISSION I N THE CNS

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

SYNAPTIC TRANSMISSION I N THE CNS

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

SYNAPTIC TRANSMISSION IN THE CNS

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-

SYNAPTIC TRANSMISSION I N THE CNS

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

SYNAPTIC TRANSMISSION IN THE CNS

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-

SYNAPTIC TRANSMISSION IN THE CNS

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

SYNAPTIC TRANSMISSION IN THE CNS

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-

SYNAPTIC TRANSMISSION IN THE CNS

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

SYNAPTIC TRANSMISSION IN THE CNS

-

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

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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

SYNAPTIC TRANSMISSION IN THE CNS

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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.

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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

SYNAPTIC TRANSMISSION IN THE CNS

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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

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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

SYNAPTIC TRANSMISSION IN THE CNS

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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-

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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

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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

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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-

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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

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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

EXOPEPTIDASES OF THE NERVOUS SYSTEM

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

EXOPEPTIDASES OF THE NERVOUS SYSTEM

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-

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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.

EXOPEPTIDASES OF THE NERVOUS SYSTEM

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

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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-

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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,,

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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

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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,

EXOPEPTIDASES OF THE NERVOUS SYSTEM

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+.

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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

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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

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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

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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

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I. Introduction

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

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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

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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

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(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,

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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

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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

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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

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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

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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

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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

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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

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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-

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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

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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-

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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

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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

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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

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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-

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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

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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

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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-

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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

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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

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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.

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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

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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

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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 . . . . . . . .

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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

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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

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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) .

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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

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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

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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

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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.

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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.

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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

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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

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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

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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

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(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.

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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

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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

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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

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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

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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

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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

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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

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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.

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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

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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.

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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.

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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

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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

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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.

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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-

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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-

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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;

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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

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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). ‘
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Avar, Z., and Monos, E. (1967). Acla Med. Acad. Sci. Hung. 22, 255. Baastrup, P. C., and Schou, M. (1967). Arch. Gen. Psychiat. 16, 162. Ball, J. (1939). J. Comp. Psychol. 28, 273. Bard, P. (1940). Rcs. Publ., Assoc. Aes. Neruous Mental Disease 19, 190. Barkve, H. (1967). Acta Med. Scand. 182, 503. Barnes, G., and Francis, M. (1909). Am. J. Iizsaitity 69, 559. Barraclough, C. A., and Gorski, R. A. (1962). J. Endocriuol. 25, 175. Beach, F. A. (1941). Ettdocriirotogy 29, 409. Bercel, N. A. (1964). Ann. N.Y. Acud. Sci. 117, 555. Beringer, K. (1942). Nerucnarzt 15, 225. Binswanger, 0. ( 1928). Arch. Psychiat. Nervenkraizkh. 83, 299. Bleuler, E. ( 1911). “Dementia praecox oder Gruppe der Schizophrenien.” F. Deuticke, Halle. Bleuler, M. ( 1954 ). “Endokrinologische Psychiatrie.” Thieme, Stuttgart. Bondy, P. K., Cohn, G. L., Hermann, W., and Crispell, K. R. (1958). Yale J . Biol. Med. 30, 495. Bonhoffer, K. (1907). “Klinische Beitrage zur Lehre von den Degenerationpsychosen.” F. Deuticke, Halle. Bonkdo, A., Lovett Doust, J. W., and Stokes, A. B. (1955). Am. J. Psychiut. 112, 114. Boyce, K., Hanna, S. M., and Jenner, F. A. (1968). The treatment of a fortyeight hour periodic psychosis with lithium. In preparation. Bradford, N. M., and Davies, R. E. (1950). Biochem. J. 46, 414. Brockman, D. D., and Whitman, R. M. (1952). 1. Nemous Mental Disease 116, 340. Brown, F. A. ( 1965). In “Circadian Clocks” (J. Aschoff, ed.), p. 231. NorthHolland Publ., Amsterdam. Brown-Grant, K. (1957). Ciba Found. Colloq. Endocrinol. 10, 97. Bunney, W. E., and Hartmann, E. L. (1965). Arch. Gen. Psychiat. 12, 611. Bunney, W. E., Mason, J. W., and Hamburg, D. A. (1965a). Psychosomat. Med. 27, 299. Bunney, W. E., Mason, J. W., Roatch, J. F., and Hamburg, D. A. (196Sb). Am. J. Psychiut. 182, 72. Biinning, E. ( 1964). “The Physiological Clock: Endogenous Diurnal Rhythms and Biological Chronometry.” Springer, Berlin. Biinning, E. (1967). Ann. N . Y. Acad. Sci. 138, 515. Burmistrova, T. D. (1964). Tr. Inst. Normalin. Patol. Fiziol. 7, 27. Biissow, H. (1949). 2. Ges. Neurol. Psychiat. 124, 161. Cade, J. F. J. (1949). Med. J. Australia 36, 349. Cagnoni, M., Toccafondi, R., and Tarquini, B. (1967). Rass. Neurol. Vegetatiua 21, 145. Cameron, M. P., and O’Connor, M. (1964). Ciba Found. Study Group 18, 1. Carpelan, H. (1957). Acta Psychiuf. Neurol. Scand. 32, Suppl. 116, 1. Cesari, L. (1959). Ergeb. Math., No. 16. Chiazze, L., Brayer, F. T., Mascisco, J. J., Parker, M. P., and Duffy, B. J. ( 1968). I. Am. Med. Assoc. 203, 377. Clark, L. D., Bauer, W., and Cobb, S . (1952). New Engl. J. Med. 264, 205. Claude, H. (1926). Ann. Med-Psychol. 134, 332.

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ENDOCRINE AND NEUROCHEMICAL ASPECTS OF PINEAL FUNCTION By BBla Mess Department of Anatomy, University Medical School, Pks, Hungary

I. Structure and Metabolism of the Pineal Gland . . . . 11. Effect of Pineal Function on the Endocrine System . . . A. Influence of the Pineal Gland on the Genital Apparatus . B. Influence of the Pineal Gland on the Thyroid-Stimulating . . . . . . . . . . Hormone . C. Influence of the Pineal Gland on Adrenocortical Function . 111. Biosynthesis and Metabolism of Melatonin and Serotonin . . IV. Effect of Light and Sympathetic Innervation on Pineal Activity V. Biorhythm of Melatonin and Serotonin Production . . . VI. Concluding Remarks . . . . . . . . . References . . . . . . . . . . .

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Gland Research on the structure and function of the pineal gland has received a revived interest within the last decade. Previous investigations had considered the pineal to be a residual organ, a vestige of the third eye, constructed of glial elements and epitheloid cells. With advancing age, pigment depletion and calcification (so-called “acervuli cerebri”) were described as signs of age-related regression of the gland. This was all that was generally known about the histology of this small, insignificant organ located in the epithalamic region of the brain. Modern histochemical, electron microscopic, and biochemical studies have now provided new data about the structure and metabolism of the pineal gland in different species. The classification of pineal cell types has yet to be unified from the different histochemical and electron microscopic studies. On the basis of histochemical (Bayerovh and Bayer, 1960) and of fluorescence and polarizing microscopic investigations ( Bayerovh et al., 1962), two cell types were identified in the pineal gland: one containing finer granules of mucopolysaccharides and muco- and glycoproteins; I. Structure and Metabolism of the Pineal

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B

~ MESS A

the other, rough granulations, containing lipids. Gusek and Santoro (1961) described a single ultrastructural type of pinealocyte and some glial elements in the pineal gland of the rat, while Arstila and Hopsu (1964) discriminated three cell types within the same species. Orofino (1964) and Anderson ( 1965) found two different cell types in other mammalian species. In spite of these seemingly contradictory findings, most of the authors agree that the pineal gland has an active secretory function. According to Sano and Mashimo (1966) the Golgi vesicles are responsible for the production of the secretory granules, which are passed into the capillary lumina. A highly developed endoplasmic reticulum was also described in the pinealocytes (Gusek and Santaro, 1961; Anderson, 1965; Orofino, 1964), which has been considered to be histological evidence of secretory activity. De Robertis and Pellegrino de Iraldi (1961) described plurivesicular processes of the pineal cells containing two types of granules with different electron densities. These processes might be the site of active secretion in the pineal cell. The increase in the number and size of the osmophilic plasma granulations is the most important sign of the secretory activity of the pineal cell, according to the investigations of Milcou and Petrea (1964). The osmophilic granulation is produced by the endoplasmic reticulum; the depletion of these granules is enhanced by castration and inhibited by testosterone treatment. Arstila ( 1967) has published a comprehensive monograph on the ultrastructure and histochemistry of the pineal gland. According to this author, the pineal gland contains chief (parenchymal) cells, interstitial cells, and connective tissue elements. The so-called light and dark chief cells are due only to different functional activities of the same (chief) cell type. Enzymologically the chief cells show acid phosphatase, aryl sulfatase and thioacetic acid esterase activity, while no acetylcholinesterase and alkaline phosphatase activity was found in these cells. The vascular supply of the pineal gland is also characteristic for an actively secreting glandular tissue (Beattie and Glenny, 1966). The pineal blood flow per gram of pineal tissue of rats, determined by radioisotopic methods, exceeds that of most endocrine organs, and equals that of the neurohypophysis (Goldman and WuGnan, 1964). There is littIe doubt, on the basis of the foregoing data, that the pineal gland has an active glandular structure. There are, however,

STRUCTURE AND FUNCTION OF THE PINEAL GLAND

173

as mentioned above, some contradictory data. Quay ( 1965a), summarizing the present knowledge about the structure and function of the pineal gland, closes his excellent review with the following sentence: " . . . the diversity and complexity of pineal structure and composition, and the recent advances in knowledge reaffirm the wisdom of avoiding inflexible and narow concepts of pineal structure and function." The innervation of the pineal gland is also a particular one. In the lower vertebrates ( e.g., frog) sensory nerve-like cells, similar to the retinal receptors, have been observed (Oksche and Vaupelvon Harnack, 1965), which have been so modified in birds that they can no longer be considered as true sensory cells (Oksche and Vaupel-von Harnack, 1966). The axons of these sensory cells form bundles and make connection with the habenular and the caudal commissure. Practically nothing is known about the exact terminations of these afferent fibers (Kappers, 1965). From a functional point of view, a dense network of autonomic, particularly sympathetic, postganglionic nerve fibers plays the most important role in the pineal body. In primates, however, a few preganglionic fibers and intramural ganglion cells also have been described (Kappers, 1965). We shall return to the significance of this sympathetic innervation of the pineal body in Section IV of this chapter. The very active metabolism and synthetic processes of the pineal body also provide convincing evidence for the physiological role of this small, hidden organ. The pineal gland has a rapid 3zPuptake, and incorporates phosphorus isotope into lipids and nucleic acids (Player, 1958). Both lipid and amino acid metabolism are considerably active in the pineal body. Hellman and Larsson (1961) found a high degree of amino acid formation from I4C-labeled glucose in young goats. This would show, according to these investigators, that the pineal gland secretes a hormone of a protein nature. The same conclusion was drawn by Niemi and Ikonen (1960) on the basis of their findings that the pineal gland of rats has a high aminopeptidase activity. The lipid content of the pineal cells decreased following treatments (hypophysectomy, adrenalectomy combined with castration, treatment with angiotensin I1) , which influences the sodium balance of the body ( Hungerford and Panagiotis, 1962). The very potent uptake and degradation of '"'I-labeled triiodothyronine demonstrates the characteristics of an active glandular tissue, such as has been seen for the aclenohypophysis (Ford, 1965).

174

BkLA MESS

Pineal tissue is extremely rich in a variety of enzymes. Thikblot and his co-workers (1966a) stated that this gland contains a series of enzymes of the Krebs cycle, others taking part in protein and lipid metabolism, in addition to the so-called specific enzymes, which are related to the monoamine-synthesizing and metabolizing activity of the gland, The specific enzyme system of the pineal gland will be discussed more fully in Section 111 of this review. The significance of an age-related regression in pineal structure and function is not equivocally accepted. BayerovL and Bayer (1960, 1962) found an increase in cellular lipid with advanced age, while the oxygen consumption and protein-synthesizing activity was decreased (Hellman and Larsson, 1961). Contrary to these findings, Horhyi (1956, 1966) noticed no direct relation between age and histological character of the pineal gland. No age-related difference was found either in the aminopeptidase activity (Niemi and Ikonen, 1960) or in the serotonin concentration (Giarman et al., 1960) of this structure. The so-called specific enzyme system (see Section 111) of the pineal gland also shows no sign of an agerelated decrease ( Wurtman et d.,1964a). On the basis of our present knowledge, the calcification and changes in lipid or pigmentcontaining pinealocytes must be considered as changes not essentially involved in the functional activity of the pineal gland. II. Effect of Pineal Function on the Endocrine System

The view that the pineal gland influences other endocrine organs is at present unequivocally accepted. Stalsberg (1965) found no changes in weight and histological character of the pituitary gland, gonads, and thyroids in young chickens epiphysectomized in early embryonic life. He concluded that all the effects following epiphysectomy, as reported by different investigators, might be due to unspecific consequences of the epiphysectomy. Melatonin, the hormone of the pineal gland, failed to induce any effect on the rat gonad in the experiments of Ebels and Prop (1965). The monograph by Donovan and van der Werff ten Bosch (1965) on the physiology of puberty questions the physiological role of the pineal gland in the regulation of the sexual sphere. On the other hand, a bulk of experimental evidence has been published in the last 10 years, which indicates that the pineal gland plays a significant role in the regulation of different endocrine functions.

STRUCTURE AND FUKCTION O F THE PINEAL GLAND

A. INFLUENCE OF THE PINEAL GLAND ON

THE

175

GENITALAPPARATUS

Most authors agree that the pineal gland has an inhibitory influence on sexual function. Pinealectomy causes an ovarian hypertrophy (Kitay, 1954; Kitay and Altschule, 1954a; Wurtman et al., 1959), which can be inhibited by the injection of crude (Kitay and Altschule, 1954b), or protein-free pineal extract ( Wurtman et d., 1959). The incidence of the estrous phase was also significantly increased in pinealectoinized young rats. This effect could be reversed by multiple pineal transplants ( Gittes and Chu, 1965). Similar hypertrophy of the genital organs (testicles, seminal vesicles) was also found in male rats (Thikblot and Blaise, 1965) following epiphysectoiny. That pinealectomy influences adenohypophysial tissue, and that the peripheral effects of pinealectomy are, at least partly, due to probable pituitary changes, was shown by Thikblot and Blaise (1965), who observed a significant increase in the percent occurrence of acidophilic cells of the adenohypophysis in pinealectomized male or female rats. Basophils were only slightly increased. These changes in pituitary histology can be reversed by injecting pineal extract. Fraschini et al. (1967) found an increase in pituitary luteinizing hormone ( L H ) content of pinealectomized male rats, and the weights of the peripheral genital organs were also increased, indicating that the pineal body inhibits both LH synthesis and release. In prepubertal animals, premature opening of the vagina was also observed following pinealectomy (Kitay and Altschule, 1954a). On the other hand, protein-free pineal extracts arrest prolonged vaginal estrus, usually seen in rats of postreproductive age, but are not able to alter the estrous cycle in animals of reproductive age ( Meyer et al., 1961). Since Lerner and his co-workers detected the new indole derivative, melatonin (Lerner et al., 1959, 1960), which is present in the pineal gland of all mammalian species, research on pineal function took a new course. The effect of melatonin on reproductive activity, as well as on other endocrine functions, was thus investigated by different research groups. Daily microgram injections of melatonin have been shown to produce a delay in spontaneous vaginal opening, a significant decrease in ovarian weight, and a decrease in the incidence of vaginal estrus (Wurtman et nl., 1963a). The precursors of melatonin, such as serotonin, and N-acetylserotonin, or its metabolite,

176

B ~ L AMESS

6-hydroxymelatonin, were ineffective in altering vaginal estrus (Chu et ul., 1964). The direct relationship between melatonin and the vaginal cycle is clearly demonstrated by the results of Wurtman et ul. (1965),who found a twofold increase in melatonin synthetic activity of the pineal gland during the diestrous phase, falling to half of the diestric value during estrus and proestrus. The mechanism of the influence of melatonin on reproductive function is not clear, Thikblot et al. (1966b) described a stimulating effect of melatonin on ovarian luteinization. They concluded that melatonin enhances pituitary LH secretion, which leads to an anestrous state and inhibition of ovulation. However, Fraschini et al. (1967) assume that melatonin inhibits pituitary LH synthesis and release. This LH-inhibiting effect of melatonin is mediated through the central nervous system. Implantation of a homologous pineal gland, or of purified melatonin into the median eminence (ME) of the hypothalamus, or into the reticular substance ( R S ) of the mesencephalon, significantly depresses the castration-induced rise in pituitary LH-content (Table I ) . No change, however, was found following implantation of either pineal tissue or of melatonin into other brain areas, e.g., into the cerebral cortex, or directly into the anterior pituitary (Mess et al., 1966; Martini et ul., 1966), Furthermore pineal grafts, or melatonin implants in the ME or RS significantly inhibited the formation of castration cells in castrated male rats (Clementi et aZ., 1966a,b). These data indicate that the active component of the pineal body, melatonin, depresses pituitary LH-synthesis and release through circumscribed areas of the diencephalon and mesencephalon, and not via a direct pinealpituitary “negative feedback” effect. The fractionation of the pineal extract also provides new data about the gonadotropin-inhibiting effect of the pineal gland. Ebels et ul. (1965) found two biologically active fractions in pineal extract, using Sephadex G25 columns for fractionation. The so-called F, fraction inhibits in vitro the release of follicle-stimulating hormone (FSH) from the pituitary, while F, is capable of enhancing this release. Of the two factors, F, is the more potent, which might explain the inhibiting effect of total pineal extracts on sexual function. The pineal gland inhibits the pituitary synthesis and release of gonadotropins, according to Moszkowska (1965, 1966a,b), in three different ways. The F, fraction inhibits FSH release by a direct action on the pituitary; serotonin acts through the inhibition

EFFECTO F THE

TABLE 1 TISSUE AND O F MELATONIN INTO THE MEDIAN EMINENCE (ME), LATERaL RETICUI~AR SUBSTAXCE OF THE MIDBRAIN(RS), THE PITUlTARY (PIT) A N D THE CEREBRAL CORTEX(CC) OF MALE RATS"

IMPL.4NTS OF P I N E A L

Groups

No. of rats

Castrated controls ME-sham ME-pineal ME-melatonin RS-sham RS-pineal RSmelatonin Pit-melatonin CC-melatonin

24 28 32 14 14 14 21 10 10

'I

b

Final body weight (gm) 245.6 247.5 235.5 248.1 239.9 251.5 240.3 247.2 243.4

5 6.2 f 5.2 f 4.9 f 5.8 f 4.7 5.3 i: 6 . 0 4.1 k 5.3

*

Anterior pituit,ary weight (mg) 11.2 i 0.60 11.1 & 0.51 10.4 1 0 . 5 8 10.1 i 0.49 10.6 jI 0.53 10.8 k 0.46 10.9 1 0 . 5 1 10.8 f 0.47 10.7 i 0.50

Adrenal weight (mg)

Pituitary LH rglpita

KO.wf

44.3 f 2.7 44.5 k 2.5 2.9 41.2 36.3 f 2 . 1 ~ 41.4 rt 2 . 4 43.3 f 2 . 9 37.7 f 2 . 6 42.2 f 2 . 0 43.9 k 2.1

25.63 k 3.10 25.97 2.80 15.70 f 1.70 13.93 f 1.20 24.84 f 2.50 16.20 1.59 15.58 f 1.40 31.96 f 3.10 28.32 rt 2.70

+

G 4

*

3 3 3" 5

+

Values are means 5 SE. NIH-LH (S 11 ovine) per pituitary. (NIH = National Institute of Health.) p < 0.01 vs. ME-Sham. (Sham = Implantation of muscle t.issue.) p < 0.001 vs. RS-Sham.

fig equivalents of

assays

@

2

2

178

&LA

MESS

of the hypothalamic-releasing factors; and melatonin inhibits LH release “in a still unclarified way.” It seems to be evident on the basis of the experiments of Fraschini et al. (1967) that this unclarified way might be the inhibitory effect of melatonin on a specific receptor system within the diencephalon and mesencephalon. On the other hand. Thikblot and Blaise (1966) isolated seven fractions of peptides and amino acids from pineal extract by paper chromatography. Which of these fractions are responsible for the antigonadotropic activity of the pineal extract is not verified as yet. In contrast to the opinion of most of the authors, melatonin cannot be considered, according to the view of Thidblot, as one of the antigonadotropic components of the pineal gland. Summarizing the effect of the pineal gland, or of the supposed “pineal hormones” on gonadotropin secretion, it can be concluded that the pineal gland very probably inhibits both FSH and LH secretion, although some less pronounced enhancing effects of some of the fractions of the pineal extract were also reported. The mechanism of this gonadotropin-inhibiting effect presently occupies a focal point of pineal research.

B. INFLUENCE OF THE PINEAL GLAND ON THE THYROID-STIMULATING HORMONE The relationship between the pineal gland and thyroid function is much less clear than in the case of reproductive function. There are, however, a few data indicating some relationship between the two endocrine organs. Pinealectomy caused an activation in the histological character of the thyroid gland in male turtles (Aron et al., 1960); even more pronounced hypertrophy of the thyroid cells, namely, the formation of a real adenoma, was reported by Schpovic (1963) in pinealectomized rats. The l3II uptake of the thyroid gland was found to be increased in the same animals. Very high doses of bovine pineal extract decreased the weight and 1S1I accumulation of the thyroid gland, and partly inhibited the development of thiouracil-induced goiter in rats (De Luca et al., 1961). Baschidri et al. (1963) found the same effects following melatonin treatment; Thieblot et al. (1966c), however, reported just the opposite effect with melatonin. Miline (1963) proposed, on the basis of his previous experiments (Miline and Sc6povic, 1959; Miline and Nesic, 1959) a triple feedback system for the regulation of the thyroid activity. These

STRUCTURE AND FUNCTION 01*' 'I'I1E PINEAL GLAND

179

are pituitary-thyroid gland, pituitary-thyroid-hypothalamic paraventricular nucleus, and pituitary-thyroid-habenulopineal system. No change was found in the thyroid function of epiphysectoinized rats utilizing different parameters of thyroid activity (thyroid weight, histological character, T/S ratio,' 13'I-release, the biological half-life of I3'I) under different experimental conditions, such as CP tJ 300.

250.

200.

450.

400 .

50

. 1

I

2

3

4

5

6

I

0

Days

FIG.1. Thyroidal "'I-release curves of intact and pinealectomized rats kept a t 28" and 14°C environmental temperature. -, Intact rats at 28°C; ---, pinealectomized rats a t 28°C; *, intact rats at 14°C; - -, pinealectomized rats at 14°C.

different environmental temperatures, continuous light, or darkness (Mess, 1967; Mess and Clementi, 1966). The 1311-releasecurve is exactly the same in the pinealectomized animals as in the controls. The slight increase in the slope of '?'I-release was also similar in the control and pinealectomized groups following exposure to a mild decrease of environmental temperature ( Fig. 1 ) . No change could be observed in the T/S ratio of the epiphysectomized animals, and the reactivity of the thyroid gland to continuous light or darkness was also unaltered (Table 11). 'The ratio between the '"I content of 100 mg thyroid tissue and that of 0.1 i d blood serum.

TABLE I1

INFLUENCE OF CONSTANT ~ OF

G H T AND

DARKNESS ON THE THYROID WEIGHTAND T/S RATIO

NORMAL AND PINEALECTOMIZED RATS ~

First experiment

Experimental group Controls in normal day-night changes Pinealectomized animals in normal day-night changes Controls in constant light, Pinealectomized animals in constant light Cont,rolsin constant darkness Pinealectomized animals i n constant darkness

Second experiment

Thyroid weight (mg)

T/S ratio

Thyroid weight (mg)

7.7 5.6

16.4 f 0.85 19.7 f 1.27

5.8 5.9

30.7 rt2.38 29.7 f 0.86

6.4 6.4 7.1 6 6

14.9 f 1.28 16.9 rt 1.23 8 . 3 k 0.74 12.8 f 0.80

6.6 7.6 6.3 6.4

17.7 1.64 23.7 f 3.73 12.5 f 1.70 13.8 k 1.Fjl

T/S ratio

*

Mean Thyroid weight (mg)

T/S ratio

6.7 5.7

23.6 f 1.37 24.7 f 1.36

6.5 7.0 6.7 6.5

16.3 1.09 20.3 f 1.65 10.4 0.85 13.3 rt 0.68

STRUCTURE AND FUNCTION OF THE PINEAL GLAND

181

The data of the latter experiments do not exclude entirely the possibility of any relationship between the thyroid gland and pineal body. Using finer, and perhaps more sensitive parameters of the thyroid function, some minor changes might possibly be detectable. A good example for this possibility is presented by Ishibashi et al. (1966). They found an insignificant increase (5%)in food consumption and an 11.8%increase in the thyroid secretion rate (TSR) following pinealectomy, and similarly low decreases in both parameters after melatonin treatment. These data indicate that the pineal gland might have some, probably indirect, influence on thyroid activity, but this influence does not seem to play an essential role in the regulation of the thyroid gland. C. INFLUENCE OF THE PINEAL GLAND ON ADRENOCORTICAL FUNCTION Interest was focused on pineal-adrenocortical relations when Farrell ( 1959a,b) described a factor, termed glomerulotropin (later adrenoglomerulotropin), which specifically enhanced aldosterone production in the adrenal cortex. This factor was supposed to be produced by the pineal gland. Farrell (1960) also found a second factor in the pineal extract, which has an antagonizing effect on adrenoglomerulotropin and on adrenocorticotropic hormone ( ACTH). The quantitative relationship between the three factors might determine, according to Farrell's working hypothesis ( 1960), the intensity of adrenocortical hormone production. There is evidence showing that pinealectomy might have some influence on the regulation of adrenocortical functions. Asagoe and Hamamoto (1959) reported a decrease in adrenocortical, as well as in ovarian ascorbic acid, content following pinealectomy. Similar results were obtained in the same year by Wurtman et al. (1959): who found an adrenal hypertrophy in pinealectomized animals, while pineal extract reversed this change. ACTH secretion was also enhanced following ablation of the pineal gland (Juan, 1963).This effect, however, might be due to a diminution of corticoidogenesis. The pineal-adrenoglomerulotropin concept of Farrell ( 1959b), was fortified by the in vitro findings of Juan (1963) and Giordano and Belestreri (1963) showing that pineal extracts enhance the secretion of aldosterone. Recently, Lommer (1966) described a special adrenal-inhibiting effect of the pineal extract, which might be in agreement with Farrell's adrenal-inhibiting pineal factor ( 1960). Pineal extract inhibits the P-hydroxylation in the adrenal

182

B ~ L AMESS

cortex; therefore, the production of corticosterone, aldosterone, cortisol, and cortisone is inhibited, but not that of deoxycorticosterone. This effect is very similar to the effect of metyrapone (Metopirone). Some indirect evidence, demonstrating the role of the pineal gland in the maintenance of electrolyte balance (Clementi et al., 1962, 1965; Quay, 1965a,b), would also indicate the possible role of a pineal-adrenocortical functional relationship. Palkovits et al. (1964) and Palkovits ( 1965) demonstrated a significant role of the subcommissural organ on adrenocortical secretion and electrolyte balance. It was further suggested that the habenulopineal complex might also be involved. A considerable amount of data has been collected by different authors arguing against the existence of a pineal-adrenocortical interrelationship. Wurtman et al. (1960) could not affirm the aldosterone secretion-enhancing effect of the pineal gland, since neither pinealectomy nor pineal extract had any effect on the histology of the zona glomerulosa, on the urinary potassium excretion, or on the quantity of saline intake. Pinealectomy was ineffective in abolishing the secondary hyperaldosteronism induced in dogs by thoracic compression, while nephrectomy was able to suppress the increased release of aldosterone following the same stressful stimulus (Davis, 1961). A new hypothesis, that an aldosterone-stimulating hormone (ASH) is produced by the kidney, was proposed on the basis of these experiments. Denton (1961) also negates the role of the pineal gland in the maintenance of aldosterone secretion. He observed the restoration of salivary Na+/K+ balance in pinealectomized sheep with the same rapidity, when Na’ was resupplemented following a Na+-free diet. In an investigation by Quay (1965a) on the role of the pineal body in the homeostasis of brain composition, and in which increased adrenal weights and enlargement of the adrenocortical zones were observed in some instances in pinealectomized rats, the conclusion is made: “Although the adrenal cortex cannot be excluded as a possible cause or intermediary in the cerebral effect of pinealectomy, it is suggested that the demonstrated effect on cerebral composition may possibly be direct from the pineal.” Both aqueous and hexane extracts of bovine pineal body failed to increase the aldosterone, or corticosterone concentration of the adrenal venous blood. Even l-methyl-6-methoxy1,2,3,4-tetrahydroxy-2-carboline, isolated from the pineal body, which enhanced aldosterone secretion in the experilnents of

STRUCTURE AND FUNCTION OF THE PINEAL GLAND

183

Farrell and McIsaac (Mil),was unable to provoke increased aldosterone, or corticosterone release from the adrenal gland ( Barbour et al., 1965). On the basis of a series of well-founded experimental evidence from the literature, contradicting the existence of a direct pinealadrenocortical relationship, the concept of Farrell ( 1959a,b, 1960) has not been generally accepted. There is, of course, no equivocal opinion developed as yet in respect to the role of the pineal body in the regulation of the adrenal cortex, either of the aldosteroneproducing zona glomerulosa, or of the glucocorticoid-producing fascicular zone. Ill. Biosynthesis and Metabolism of Melatonin and Serotonin

When Lerner et al. (1959) isolated melatonin from the pineal gland, and its chemical structure was established as N-acetyl-5methoxytryptamine (Lerner et al., 1960), extended chemical and biochemical studies were already in progress to uncover the pathway for melatonin biosynthesis. The pineal gland has a considerable content of serotonin (Giarman and Day, 1959), which is the basic compound of melatonin biosynthesis. McIsaac and Page (1959) showed that the pineal gland is able to convert serotonin into N-acetylserotonin. In previous investigations, Axelrod ( 1957) stated that S-adenosylmethionine is necessary as a methyl donor for the enzymatic O-methylation of catecholamines, e.g., of acetylserotonin. One year later the enzyme, hydroxyindole-O-methyl transferase ( HIOMT ) , which is responsible for the O-methylation of acetylserotonin, was detected by Axelrod and Weissbach ( 1960). When N-acetylserotonin and S-adenosylmethionine were incubated with bovine pineal extract, a substance, identified as melatonin, was formed. This enzyme was later purified from bovine pineal gland (Axelrod and Weissbach, 1961). HIOMT is present in the pineal gland of all mammalian species (cat, cow, monkey: Axelrod and Weissbach, 1961; rat: Wurtman et al., 1963; man: Wurtman et al., 1964a). The pineal gland has a high melatonin-synthesizing capacity; 200 pg melatonin per gm pineal tissue per hour is the production rate of the human pineal gland ( Wurtman et al., 1964a). Later HIOMT activity and melatonin production were also found in birds (Axelrod et al., 1964), and in amphibians (Axelrod et al., 1965a). In the hen’s pineal body the melatonin synthetic rate

184

B ~ L AMESS

was found to be twice as high as in the monkey; the monkey has the highest degree of melatonin synthetic activity among the mammals, and 200 times higher than that of the rat (Axelrod et uE., 1964). HIOMT is present exclusively in the pineals of mammals (Axelrod and Weissbach, 1961; Axelrod et al., 1961) and in buds ( Axelrod et al., 1964), while in the amphibians, besides the pineal body, cerebral tissue also contains this enzyme (Axelrod et al., 1965a) . The fate of melatonin has been investigated by two different approaches. One is the distribution of melatonin in the different organs and the disappearance rate of exogenous melatonin. The other mode of approach was the biochemical investigation of the metabolism and degradation of the hormone. Melatonin, labeled in different positions and with different isotopes ( I4C, 3 H ) , shows equally a multiphasic, but generally quick disappearance curve following intravenous injections. In the first 10 minutes the disappearance of melatonin is very rapid, with a half-life of about 2 minutes. After 40 minutes the disappearance rate became slower, with a half-life of about 35 minutes. After 1% hours of intravenous injection of the labeled melatonin, less than 2%of the injected amount of radioactivity was present in the whole mouse (Kopin et al., 1961). The distribution of labeled melatonin within this relatively short time shows a characteristic anatomical localization. One hour following the intravenous injection of melat~nin-~H, the pineal body contains about 20 pc/lOO gm tissue, while the iris-choriod plexus concentrated only approximately 5 pc/lOO gm,and the ovary about 4.5 pc/lOO gm of labeled melatonin. The other endocrine organs had an uptake of labeled melatonin between 1.2 and 2.3 pc/lOO gm tissue. Most of the nonendocrine organs and tissues (liver, spleen, heart, brain, muscle, and adipose tissue, etc.) showed a radioactivity below 1 pc/lOO gm tissue ( Wurtman et al., 1964d). The metabolism of melatonin in the body begins practically immediately. Within 1 minute of an intravenous injection of labeled melatonin, only 70230%of the total radioactivity, as measured in the different organs, is caused by unchanged melatonin. After 30 minutes, melatonin accounted for only 3550% of the total radioactivity in the tissues. The depletion of melatonin is relatively slow in the adrenal gland and in the brain, where about 60% of the radioactivity was present as melatonin even after 30 minutes. On

STRUCTURE AND FUNCTION O F THE PINEAL GLAND

185

the other hand, the degradation of melatonin in the liver and spleen is extremely rapid. In these organs, unchanged melatonin accounted for only 10-20% of the total radioactivity (Kopin et al., 1961). The biochemistry of the metabolism of melatonin was studied both by in v i m and in vitro techniques. About 60-704& of the metabolites of melatonin are excreted in 48 hours by the urine and 15%by the feces. No unchanged melatonin is found in these excreted products. The main melatonin metabolites, separated from the urine, are the 6-hydroxymelatonin compounds, conjugated in major part (70-808) to sulfates, and in minor part (about 5%)to glucuronic acid (Kopin et al., 1961). The hydroxylation of melatonin into 6-hydroxymelatonin takes place in the liver. Szara and Axelrod ( 1959), as well as Jepson et al. (1959) have demonstrated that the microsomes of the liver contain an enzyme, which hydroxylates the indole derivatives in position 6. This enzyme is also responsible for the first step of the degradation of melatonin, i.e., for the hydroxylation of melatonin in position 6, as has been reported by Kopin et al. ( 1961). The course of synthesis and metabolism of melatonin, as well as the enzymes catalyzing these chemical procedures, are clearly visualized by the scheme presented in Wurtman and Axelrods publication ( 1965) (Fig. 2 ) . The methoxy derivative of melatonin, ( 5-methoxyindoleacetic acid; right part of the scheme), which is also present in the pineal body (Lerner et al., 1960), is formed by O-methylation of 5-hydroxyindoleacetic acid or by deacetylation and deamination of melatonin (Wurtman and Axelrod, 1965). Serotonin, as mentioned above, is the major substrate for melatonin biosynthesis. Since serotonin is by itself a considerably complicated indole derivative, the biosynthesis of this basic compound was also investigated. Snyder and Axelrod (1964) stated that the pineal gland has the highest concentration of 5-hydroxytryptophan decarboxylase (5-HTPD), the enzyme that plays an important role in the formation of serotonin by mammalian tissue. The other enzyme that takes part in the biosynthesis of serotonin is tryptophan hydroxylase. From this essential aminoacid, tryptophan, the hydroxylase catalyzes the formation of 5-hydroxytryptophan, which is decarboxylated by 5-HTPD to serotonin. Schein et 02. (1967) developing an in uitro method (pineal tissue culture), investigated the role of these two enzymes in the regulation of the dynamics of serotonin synthesis. When different amounts of tryptophan were

186

B ~ L AMESS

introduced into this in vitro system the serotonin production remained unchanged. In contrast, when the concentration of hydroxytryptophan was increased, the total amount of serotonin produced

-

H O ~ C H z C W H

HowcHzcHzm*

1

I H

I H

5-Hydroxyindoleacetic acid

Serotonin

H

H

N-Acetylserotonin

5-Methoxyindoleacetic acid 4

C % O Q . @ % ~ H , V H C O4 CH3

?

l3

1

C H 3 0 ~ C H z C & N H C O C H s

I

I H

H

Melatonin

[Sulfate] [Glucosiduronic acid]

,-

-

CH

3

0

m

l5

CHzCHzNHCOCH3

HO

I

H 6- Hydroxymelatonin

FIG.2. Pathways for the formation and metabolism of melatonin. 1, Monoamine oxidase; 2, N-acetylating enzyme; 3, hydroxyindole-0-methyl transferase; 4, deacetylating and deaminating enzyme; 5, microsomal hydroxylase. (From Wurtman and Axelrod, 1965.)

by the pineal tissue culture was significantly increased. These data indicate, on the one hand, that the activity of the decarboxylase enzyme is considerably greater than that of the hydroxylase, and

STRUCTURE AND FUNCTION OF THE PINEAL GLAND

187

on the other that the hydroxylation step determines the rate of serotonin production. The degradation of serotonin is catalyzed by an enzyme, monoamine oxidase (MAO), which can be inhibited by P-phenyl isopropylhydrazine. Treatment with this MAO-inhibitor agent abolishes the circadian rhythm of serotonin synthesis (Snyder and Axelrod, 1965). IV. Effect of Light a n d Sympathetic Innervation on Pineal Activity

The pineal gland, as mentioned in Section I of this review, is a phylogenetic derivative of the so-called third, or pineal, eye. Within the vertebrates, and especially with the higher vertebrates, this organ has been transformed into an endocrine gland. The function of the pineal body, however, has remained closely related to the reception of environmental light, transmitting the effect of this external stimulus to the endocrine system, primarily to gonad-target organ axis. Continuous light provokes a change in the histological picture of the pineal tissue characterized by a decrease in cell nuclear size and resultant cytoplasmic basophilia. Continuous darkness causes more or less the opposite effect (Roth et al., 1962). The weight of the pineal gland also decreases following exposure to continuous light ( Fiske et al., 1962). Besides these morphological changes, considerable functional modifications also occur in the pineal body, a consequence of the changes in environmental lighting. Exposure to constant illumination provoked an increase in ovarian and adrenal weights similar to that caused by pinealectomy ( Wurtman et al., 1961). The synthetic activity of the pineal gland hormone is under the control of light. Continuous darkness for 6 days produced a fivefold increase in the melatonin-synthesizing enzyme, HIOMT, compared with animals kept in continuous light. Pineal glands of rats maintained in constant light showed approximately a 50% decrease of HIOMT activity compared to animals kept in normal diurnal lighting. No change, however, was registered in the activity of another oxidative enzyme, monoamine oxidase (MAO) of the pineal gland, indicating that only the specific melatonin-forming enzyme system is affected by periods of continuous darkness or light ( Wurtman et al., 1963b; Axelrod et al., 1965b). In addition to melatonin biosynthesis being modified by the changes in environmental illumination, the production of serotonin,

188

&LA

MESS

the precursor of melatonin, is affected. The most important serotonin-forming enzyme, Ei-HTPD, increased twofold in the pineal gland in rats kept in constant light, compared to littermate controls in continuous darkness (Snyder et al., 1964, 1965a). A discussion of the possible causes of these inverse changes in melatonin and serotonin synthetic activity will be presented in Section V. Light impulses might act on the pineal body by several possible ways: ( a ) Light could penetrate the skull, as was demonstrated by the use of intracerebral photoelectrodes by Ganong et d. (1963), and in this way could act directly on the pineal body. ( b ) The action of light through the hypothalamohypophyseal system (Lisk and Kannwisher, 1964), modifying the levels of different circulating hormones, might influence the melatonin synthesis of the pineal body. ( c ) Light impulses might reach the pineal body by the retinal receptors and central nervous pathways. Considering this last possibility as most probable, the question arose as to whether or not the light-induced changes in the pineal activity also could be provoked in the blinded animal. The data of Wurtman et al. (1964b) did not support this supposition. They found a complete loss in the capacity of the pineal gland to respond to changes in environmental lighting, either in terms of pineal weight or HIOMT activity, following bilateral enucleation. Blinded animals also showed a moderate increase in pineal HIOMT activity, but the diurnal changes did not occur (Axelrod et al., 196513). The same was also the case in the 5-HTPD activity. Enucleated animals have the same 5-HTPD activity in the pineal gland under constant light as do controls under subjected to continuous darkness (Snyder et al., 1964). The afferent pathways, through which light impulses are transmitted from the retinal receptors to the sympathetic nervous system, giving rise to the secretory innervation of the pineal body, has led to some interesting neuroanatomical questions. Axelrod et al. ( 1966) reported that bilateral lesions in the lateral hypothalamus, which thus destroy the median forebrain bundle (MFB), have similar effects on pineal weight and HIOMT activity, as does enucleation, i.e., lateral hypothalamic lesions abolish light-induced changes in both parameters. A very elegant neuroanatomical study was performed recently by Moore et al. (1967) in order to locate the chain of neurons from the retinal receptors to the sympathetic cervical ganglion. Figure 3 presents a scheme for the different sites of

189

STRUCTURE AND FUNCTION O F THE PINEAL GLAND

transection of the optic pathways, which led to the following results: Unilateral blinding combined with ipsilateral transection of the MFB (Fig. 3, group 2 ) completely abolished the reactivity of the pineal HIOMT activity to changes in environmental illumination, similar to those induced by bilateral ehucleation (Fig. 3,

A

B

B

A

A

B

B

A

-&X Inferior accessory optic tract (MFB) tronsection

Orbital enucleatian

1)

4)

\

A

B

B

Optic tract tronsection

A

A

B

B

A

Transection of inferior occessocy optic tract and optic tracts

FIG.3. Scheme of the different sites of interruption of the optic pathways for the investigation of the afferent pathways through which light impiilses exert their influence of pineal activity, A, primary optic tract; B, inferior accessory optic tract; C, retinohypothalamic fibers, (From Moore at d.,1967.)

group 1).An intact pineal reactivity was observed after unilateral enucleation and bilateral transection of the primary optic tract running to the lateral geniculate body (Fig. 3, group 3 ) , in spite of the fact that animals of this group behaved as if completely blind. The combination of the second and third types of operative

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incidences (Fig. 3, group 4 ) also produced a complete extinction of the pineal HIOMT reaction to light, as was observed in group 2. Taking into consideration the fact that the inferior accessory optic tracts undergo a complete decussation, the interpretation of these findings is clear. These four different experimental groups equivocally demonstrate that in all cases, when the pathways of the MFB are disconnected on both sides, independent of the primary optic tract, light reactions of the pineal gland disappear. The afferent pathways, which mediate light impulses to the pineal gland, can be constructed as follows: retinal receptors, optic nerve and chiasma, contralateral inferior accessory optic tract, which runs inside the MFB to the rostra1 midbrain tegmentum. The pathway from this point to the lateral column of the spinal medulla, where the cervical sympathetic preganglionic nerves feed into the cervical superior ganglion, is as yet unknown ( Wurtman, 1966). The supposed retinohypothalamic fibers ( c of Fig. 3 ) have no importance in the mediation of light-induced changes in pineal secretory activity. Zweig et al. (1966) reported an effect of prolonged light periods on the diurnal rhythm of pineal serotonin content in very young (6-12 days) rats. This effect was observed even after enucleation or in animals prior to the opening of the eyes, or with artificial closure of the eyelids. This failed to occur in older (27 days) blinded animals. They suggested that in the perinatal period, before visual function can be demonstrated, a photoreceptive function of the pineal body still might persist. If an extraretinal photoreception in very young mammals has any role in the control of pineal function, it is restricted to such a very short period of life that this might not be of any major importance in the regulation of the light-induced changes in the function of the pineal-gonadreproductive target organ axis. The efferent pathways, mediating secretory impulses to the pineal body, are the sympathetic postganglionic fibers, originating from the superior cervical ganglia. Bilateral extirpation of this sympathetic ganglion eliminates the light-induced enhancement of vaginal estrus, as well as the hypertrophy of the ovaries and the uterus. In addition, ganglionectomy inhibits the reactive weight loss of the pineal body and the decrease of HIOMT activity following exposure to light (Wurtman et d.,1964b,c). The darkinduced diurnal increase of HIOMT-activity at midnight is also blocked by cervical ganglionectomy (Axelrod et al., 1965b). The

STRUCTURE AND FUNCTION OF THE PINEAL GLAND

191

synthesis of serotonin, the precursor of melatonin, is also under the control of the sympathetic nervous system. S-HTPD activity of the pineal gland did not increase under constant light following cervical ganglionectomy, or treatment with the sympatholytic drug, Brethylium (Snyder et al., 1964). The concentration of this serotonin-forming enzyme of the pineal gland is almost doubled in ganglionectomized rats under diurnal lighting conditions ( Snyder et d.,1965a). Summaries of these studies have been made by Axelrod and Wurtman (1966a) and by Wurtman and Axelrod ( 1966a) . V. Biorhythm of Melatonin a n d Serotonin Production

As was just shown in Section IV, the intensity of the synthesis of both melatonin and its precursor, serotonin, is regulated by the environmental lighting. The proposal logically arose as to whether the biorhythms of melatonin and serotonin synthesis are also regulated by environmental lighting conditions, or if this biorhythm is an endogenous rhythm not affected by environmental factors. The study of Axelrod and Wurtman (1966b) is pertinent, since they demonstrated that the pineal gland has the characteristics of a “biological clock.” MeIatonin production has a daiIy rhythm. The lowest HIOMT activity in the pineal gland has been observed at 6 P.M., i.e., at the end of the daylight period. This enzyme activity increases about twofoId at midnight. This 24-hour rhythm of HIOMT is abolished in blinded animals kept under a normal day-night light schedule. Moreover, prolongation of the light period until midnight inhibits the increase of melatonin synthesis characteristic for this period of the day ( Axelrod et nl., 1965b) . These results clearly show that the rhythm of HIOMT activity, i.e., of melatonin synthesis, is exogenous. It is completely under the control of environmental lighting conditions. Quite another biorhythm was found in the case of serotonin production. The serotonin synthetic rate ( 5 HTPD-activity) is the highest at midday and the lowest at midnight in mammals (Quay, 1963), in the pigeon (Quay, 1966a) and in the monkey (Quay, 1966b), which is exactly the reverse of that observed with the HIOMT activity. Not only are the time schedules of the intensity of melatonin and serotonin synthesis reversed, but the type of bio-

192

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rhythm of the two monoamines is also different. The rhythm of serotonin is endogenous, and persists even following complete blinding of the animals and in rats kept in continuous darkness (Snyder et al., 1965b). The biorhythm of serotonin production can be considered to be a true circadian rhythm. The exogenous, light-induced biorhythm of melatonin, and the true circadian (endogenous) rhythm of serotonin production, are both controlled by the sympathetic nervous system. Bilateral extirpation of the superior cervical sympathetic ganglion prevents the rhythmic changes of either the melatonin (Axelrod et aE., 1965b) or the serotonin (Fiske, 1964; Snyder et al., 196510) synthetic rate. TABLE I11 TWENTY-FOUR-HOUR BIOCHEMICAL RHYTHMS I N THE RAT P I N E A L GLAND" Serotonin

Normal Continuous light Continuous dark Blinding Ganglionectomy Decentralization MFBLc Reserpine a

HIOMTb

Day

Night

Day

High High High High Medium Medium Medium Medium

Low High Low Low Medium Medium Medium Medium

LOW

Night

Noradrenaline Day

Night

High Low High Low Low Low Low High High High High High High High High Medium Medium Low Low Not examined Not examined Medium Medium Not examined Not examined Not examined

From Axelrod and Wurtnian (1966b). HIOMT: hydroxyindole-0-methyltrsnsferase (melatonin-forming enzyme). MFBL: bilateral medium forebrain bundle lesion.

The pathways controlling the biorhythm of both monoamines run from the medial forebrain bundle (MFB) to the superior cervical sympathetic ganglion and then to the pineal body. The role of the MFB could be postulated from the following studies: Carlsson et aE. ( 1962) described serotonin- and noradrenaline-containing tracts in the central nervous system. Using stereotaxic methods Heller and Moore (1965), on the other hand, reported a reduced serotonin and noradrenaline content of the brain, following lesions in the MFB. Axelrod and Wurtman (1966b) summarize the present knowledge about the central-nervous control of the biorhythm of melatonin and serotonin production with the following words:

STRUCTURE AND FUNCIION OF THE PINEAL GLAND

193

“Thus it appears the exogenous HIOMT rhythm and the endogenous serotonin clock use the same biogenic amine-containing tracts in the central and peripheral nervous system.” Table I11 lists surveys of the biorhythm of serotonin, melatonin, and noradrenaline synthesis under different experimental conditions. It appears curious that the time schedule of the biorhythm of melatonin, and that of its precursor, serotonin, show inverse changes. This strange fact might have several possible explanations: ( 1 ) More serotonin is synthetized during the day, than at night. ( 2 ) The destruction of serotonin is more rapid at night. ( 3 ) More serotonin is transformed into melatonin during the night. (4)The rate of release of serotonin is quicker at night than during the day. The experiments of Quay (1963) suggested that serotonin might be released from a bound form during the night period, becoming accessible for destruction by MAO. This would favor the fourth possibility. Snyder and Axelrod ( 1965) reported that treatment with P-phenyl isopropylhydrazine, a MAO-inhibitor, prevented the nocturnal fall of the pineal serotonin content, but did not affect serotonin concentration during the day. This clearly showed that M A 0 is able to oxidase serotonin only in an unbound form that is rhythmically present at night. Noradrenaline is also present in a considerable amount in the pineal gland (Giarman and Day, 1959; Pellegrino de Iraldi and Zieher, 1966), and it has a biorhythm similar to that of melatonin synthesis ( Wurtman and Axelrod, 1966b). This biorhythm is also similar to that of HIOMT activity in that they are both exogenous rhythms. Any further discussion on the relationship between pineal noradrenaline content and its sympathetic innervation is beyond the scope of this paper. Summarizing the data presented in this section we see that the pineal gland functions as a biological clock. It consists of two different types of biorhythms. Melatonin and noradrenaline have an exogenous rhythm completely regulated by environmental lighting. The peak of melatonin and noradrenaline production is during the night. Serotonin, on the other hand, has a true circadian, i.e., an endogenous biorhythm, not affected appreciably by environmental lighting conditions. In contrast to the biorhythm of melatonin and noradrenaline, the maximum serotonin synthesis occurs during the daylight period.

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VI. Concluding Remarks

The epithelial structure of the pineal body has a glandular character. Many ultrastructural elements of the pineal cells show active secretory processes; the rich enzyme systems of these cells, as well as the intensity of blood flow and the type of vascularization of this organ provide convincing evidence for the endocrine nature of the pineal gland. A series of active hormone-like principles, isolated from pineal extracts ( i.e., melatonin, serotonin) fulfill one of the most important criteria of an endocrine organ, its hormone-secreting ability. On the basis of all of these facts, there remains little doubt that the pineal gland belongs to the endocrine system. A functional interplay between the pineal gland and practically all the endocrine organs has been demonstrated. The influence of pineal hormone-like substances on the reproductive system, elicited by environmental lighting conditions, seems to be the most important of the endocrine effects exerted by the pineal gland. REFERENCES Anderson, E. (1965). J. Ultrastruct. Res. Suppl. 8, 1. Aron, E., Combescot, C., and Demaret, J. (1960). Compt. Rend. Soc. B i d . 154, 1856. Arstila, A. U. (1W7).Neuroendocrinobgy 2. Suppl. 1. Arstila, A. U., and Hopsu, V. K. (1964). Ann. Acad. Sci. Feniiicae 113, 1. Asagoe, Y.,and Hamamoto, A. (1959). Yonago Acta Med. 3, 192. Axelrod, J. (1957). Science 126,400. Axelrod, J., and Weissbach, H. ( 1960). Science 131, 1312. Axelrod, J., and Weissbach, H. (1961). J. B i d . Chem. 236, 211. Axelrod, J., and Wurtman, R. J. ( 1966a). Res. Publ., ASSOC.Res. Nervous Mental Disease 43, 200. Axelrod, J., and Wurtman, R. J. (196%). Probl. Actueh Endocrhol. Nutr. 10, 201. Axelrod, J., McLean, P. D., Albers, W. R., and Weissbach, H. (1961). In “Regional Neurochemishy” ( S . S. Kety and J. Elkes, eds.), Vol. 2, pp. 307411. Pergamon Press, Oxford. Axelrod, J., Wurtman, R. J., and Winget, C. M. (1964). Nutare 201, 1134. Axelrod, J., Quay, W. B., and Baker, P. C. (1965a). Nature 208, 386. Axelrod, J,, Wurtman, R. J., and Snyder, S. H. (196513). 1. B i d . Chem. 240, 949. Axelrod, J., Snyder, S. H., Heller, A., and Moore, R. Y. (1966). Science 154, 898. Barbour, B. H., Slater, J. D. H., Casper, A. G. T., and Bartter, F. C. (1965). Life Sci. 4, 1161.

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THE BIOCHEMICAL INVESTIGATIONS OF SCHIZOPHRENIA IN THE USSR By D. V. Lozovsky Institute of Psychiatry of the USSR Academy of Medical Sciences, Moscow, USSR

I. Current Trends . . . . . . . . . 11. Pathogenesis. Major Syndromes . . . . . . A. Hypoenergetic Syndrome . . . . . B. Autointoxication Syndrome . . . . . . 111. Pathogenesis. Some Other Concepts . . . . . . . . . . . . . . IV. Classification . V. Biochemical Investigations at the Institute of Psychiatry . VI. Summary . . . . . . . . . . . . . . . . . . . . References

. . .

. . . . . .

.

199 204 . 2 0 4 . 205 . 207 . 214 . 216 . 222 . 222

.

I. Current Trends

There is a long history behind the biochemical investigations of schizophrenia in Soviet psychiatry. The first work in this field was carried out (1, 2 ) at the turn of our century. But in this article we do not intend to give a historical and chronological review of this work. We think it more expedient to try and outline the general features of the present status of development of the biochemical investigations of schizophrenia in our country, which can be called the “Soviet psychiatric stage” from the point of view of scientific concepts and organizational measures. This period has been noted for its rapid development of public health services and medical and biological science; at the same time a large number of scientific groups have been organized in a comparatively short period of time in the form of numerous psychiatric chairs at higher educational establishments and at specialized mental scientific research institutes, which continue to study the nature of schizophrenia, including its biology and in particular its biochemical aspects. From the ideological and scientific point of view this stage is characterized by a tendency in the studies on the nature of 199

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schizophrenia to go beyond the framework of empiric investigations, and to concentrate on certain theoretical conceptions, which to some degree, although they vary greatly, have a number of essential general features in common. In formulating these general features first of all it is necessary to state the generally accepted initial principle that the fundamental basis of the schizophrenic process is in disturbances of a biological nature and that schizophrenia is not only a disease of the brain but of the whole organism. It may also be assumed that a more or less full understanding of the nature and essence of schizophrenia cannot be obtained only with the aid of biological methods; it requires a comprehensive study including pathopsychological, clinical, and sociological investigations. Moreover, such an approach is deemed necessary within the biology of schizophrenia itself, because we presume that the pathological changes in the course of this disease do not occur only at one level (morphological, biochemical, physiological, etc. ) , but at many conjugated and interdependent levels of biological organization, each of which is necessarily included in the formation of the disease. Proceeding from this idea the overwhelming majority of Soviet investigators, working in the field of the biochemistry of schizophrenia and constructing their concepts about the pathogenesis of this disease, consider the biochemical disorders they find or suspect as being present to be only one link in a complicated pathogenetic chain. The next conception of Soviet investigators along these lines is that the biochemical changes during schizophrenia are manifested in the clinical picture because of the disorders in the mechanisms of the higher nervous activity, in the understanding of which the physiological ideas of I. P. Pavlov play an important role. Another important featuie of the biological and biochemical investigations of schizophrenia in Soviet psychiatry is the special attention paid to the clinical aspects of the investigations. Naturally this does not mean that in the Soviet Union there is complete unanimity in the clinical appraisal of schizophrenia. Although the majority of the Soviet investigators proceed from the premise of the nosological independence and unity of this disease, there are, nevertheless, essential differences in the conceptions of the limits of schizophrenia, its diagnostic criteria, classification, etc. But within the framework of their own clinical conceptions almost

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all the investigators try to select clinically similar groups of patients and to obtain their precise and detailed clinical psychopathological characteristics, as well as to compare the laboratory data with the clinical parameters, which they accept as most essential or as reflecting the corresponding biological changes. The study of the biochemistry of schizophrenia continues to develop in close connection with the general progress of ideas, tendencies, and methods of modern clinical and theoretical medicine and natural sciences in general, which have a steady influence on the theoretical understanding, direction, scientific level, methodological adequacy, and scientific-technological basis for the corresponding investigations. We shall attempt to present a brief outline of the trends and work being conducted by some of the leading scientific groups in our country in the field of the biochemical investigations of schizophrenia. First and foremost we must mention the work being carried out by V. P. Protopopov et a?. (at the Kharkov Psychoneurological Institute, and later in the department of psychiatry of the Kiev Institute of Clinical Physiology and the chair of psychiatry of the Kiev Institute for Advanced Medical Training). Protopopov is undoubtedly the founder of the first Soviet school of psychiatry which systematically and steadily continues to study the biological and the physiological and biochemical aspects of psychoses, mainly schizophrenia and manic-depressive psychosis. Protopopov ( 3 ) formulated the essence of his conception about the pathogenesis of schizophrenia, which he started to study back in the 1930s, in the following way. According to many investigators (De Krinis, Pfeiffer, Buscaino, Reiter, Gjessing, Yan, Mayer, and others), schizophrenia is based on an autointoxication, which in turn is caused by metabolic disorders, mainly of protein metabolism. Toxic products accumulate in the organism of the patient in a form which is as yet undefined, the derivatives of aromatic compounds taking an essential place among them. The toxic products, not adequately detained, affect the central nervous system, in particular, suppressing the oxidative enzymes, which Ieads to brain hypoxia, increased tissue breakdown, and other disturbances of metabolic processes of the brain. As a result the reactivity of the nervous cells, which are constitutionally weak to start with, is disrupted and they begin to function so as to produce the so-called hypnoid syndrome

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(described by Protopopov, on the basis of I. P. Pavlov’s understanding and his own investigations, as a special complex of disorders of conditioned reflexes ) . Protopopov regards the disorders of the higher nervous activity in the form of the hypnoid syndrome ( 3 ) as a special syndrome of cerebral pathology, which is characteristic for schizophrenia and is the most probable pathophysiological basis of its psychopathological symptoms. According to Protopopov, the cause for the toxicosis of schizophrenia lies in the constitutional peculiarities of the patients, who are characterized by a number of congenital defects. Among them an insufficiency of the endocrine system, in particular hypofunction of the thyroid gland, hypophysis, and genital glands, which leads to a reduction of the basic metabolism and, consequently, of the energy of oxidation processes. Another inborn defect is the malfunctioning of the nervous centers that regulate the metabolic processes. These deficiences culminate in a disturbance of protein metabolism and the accumulation of nitrogen-containing waste products. At the same time, Protopopov regards the detoxication protective systems of the liver to be constitutionally weak in schizophrenics. The glycogen content in the liver of the patients is reduced, and its enzymic functions are weaker. Schizophrenics have a reduced urea content and a higher ammonia level in the urine. A significant increase in the concentration of ammonia in the blood is also often observed ( 4 ) . Loading experiments show a decline in the glycogen- and urea-forming functions of the liver (5, 6 ) . In addition, the capacity of the liver to detoxicate toxic substances by forming conjugate ether-sulfuric and ether-glucuronic acids and rhodanate compounds is also impaired (7-10); as a result a high level of derivatives of the aromatic phenols can be demonstrated in the blood of schizophrenics during the active stages of the disease. The antitoxic function of the reticuloendothelial system is also reduced. Thus, according to Protopopov, schizophrenics have a number of peculiarities that create conditions for the development of autointoxication as a consequence of the pathology of intermediary metabolism. Protopopov further considers that the toxic products affect the brain by inhibiting the oxidative enzymes, thus reducing the oxygen consumption of the brain and leading to a condition bordering on

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anoxia. He considers that the data provided by Quastel and Wheatley (1Oa) support his own opinion because they show that the proteinogenic amines and their derivatives can suppress the consumption of oxygen by the brain to a considerable degree. In Protopopov’s hypothesis, the reduction of oxidative processes leads to various biochemical disorders, and, in particular, can serve as the most probable reason for increased tissue breakdown among the schizophrenics and the disturbances of the nitrogen, lipoid, and mineral metabolism of the brain. In subsequent years Protopopov’s hypothesis, which he expounded in 1946 ( 3 ) ,was supported by additional data. Gorodkova obtained data about the toxic properties of the cerebrospinal fluid of schizophrenics and the presence in it of phenol and cresol-like compounds (11). Zelinsky (12, 13) demonstrated that the sera of schizophrenics in the active period inhibit in vitro the oxygen consumption of the brain tissues of man and mouse using such substrates as glucose, lactic, and pyruvic acid. This same investigator discovered that there are disorders in the metabolism of certain vitamins (cofactors of a number of enzymes taking part in the processes of biological oxidation): a drop in the thiamine level and especially its phosphorylated fraction-cocarboxylase in the blood (but not in the urine) when enough of it is consumed with food, a reduction of the riboflavin content in the blood and urine, a low level of nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate (NAD and NADP) when the nicotinic acid content is normal ( 14). According to Protopopov, all these vitamin metabolic disorders, can promote the development of cerebral hypoxia. Finally, the presence of the latter in schizophrenia was, to a great degree, confirmed by data provided by Rasin and Kolchinskaya (S), on the reduction of the arteriovenous 0, difference in schizophrenics. Protopopov’s work had a great influence on the study of the biology of schizophrenia in our country. After his death in 1957 his work in the field of biochemical investigations of schizophrenia was continued by his group under the guidance of Polishchuk (department of psychiatry of the Kiev Institute for Advanced Medical Training). At the present stage of investigations, this group of scientists is adhering to the following concepts ( 15-18). Schizophrenia, the limitation of which is close to Kraepelin’s conception of dementia

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praecox, occurs on the basis of an hereditary-constitutional or acquired constitutional predisposition, and apparently under the influence of various unfavorabIe environmental factors. The biochemical investigations are not aimed at creating a “biochemical conception of schizophrenia,” but reflect the tendency to understand the complicated pathophysiological structure of psychosis. Moreover, the biochemical work is not planned to detect some specific substance that is responsible for the schizophrenic process, but is rather aimed at the study of the changes in the normal course of metabolism. According to Polishchuk, the actual biochemical disorders that have so far been discovered in psychiatry are a number of interconnected complex disorders and can thus be broken down into several biochemical syndromes (15), the leading ones being the syndrome of the disorder of the energetic metabolism, the autointoxication syndrome, the biochemical syndromes of disorders of the endocrinal vegetative regulation, and the biochemical manifestations of immunobiological disorders. For schizophrenia the characteristic phenomenon is a combination of all these syndromes. it. Pathogenesis. Maior Syndromes

It is beyond the scope of this article to detail the work that served as the foundation for the above-named conceptions, but we shall discuss several main points. A. HYPOENERGETIC SYNDROME According to Polishchuk, one of the major manifestations of the constitutional predisposition to schizophrenia is the syndrome of hypoenergism, which is basic to such clinical pathophysiological symptoms as hypotonia of consciousness ( Bertze), a reduction of the energetic potential (Conrad), cortical weakness (Pavlov), vital asthenia (Polishchuk) . The biochemical manifestations of hypoenergism are the disorders of the energetic metabolism in both the glycolytic and the aerobic phases with a diminution of the phosphorylation processes, a reduction of the basic metabolism, of the level of sugar in the blood and its arteriovenous difference, the low level of acid-soluble phosphorus in the blood, of hexosophosphate phosphorus, diphosphoglyceric acid, adenosine-5’-triphosphate ( ATP), and a sluggish course of tricarboxylic acid metabohm ( a low or a very high content of citric, ketoglutaric, and succinic acids) (15-17, 19,20).

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These manifestations of hypoenergism are most characteristic for patients with a sluggish process: during acute exacerbation of the disease with an abundance of active symptoms they may not be observed, apparently being masked by the adaptive reactions. The constitutional and typological nature of these changes is indicated by the fact that there are analogous or similar disorders during remissions, in the schizophrenic defect state, and in certain cases in the relatives of schizophrenics, and in people having an asthenic constitution in other diseases.

B. AUTOINTOXICATION SYNDROME The autointoxication syndrome is, according to Polishchuk, an important characteristic of the active stage of schizophrenia (15-18, 21). According to his data one manifestation of autointoxication is an abnormal amino acid metabolism. Of the greatest interest are the results concerning the transformation of phenylalanine and tryptophan after loading with these amino acids. The administration of phenylalanine leads to abnormal elevation of phenylalanine in the blood and of phenylpyruvic and homogentisinic acids in the urine. The administration of tryptophan to patients is followed by a higher than normal increase in the content of tryptophan and its metabolites in the urinc-antliranilic acid, kynurenin, 5-hydroxyindoleacetic acid, indican, as well as the products of phenylalanine metabolism, phenylpyruvic and homogentisinic acids and free phenols. Similar changes during the administration of amino acids can also be observed among the parents of schizophrenics, but they are less evident and are compensated for more rapidly. Consequently it is supposed that schizophrenic autointoxication results from defects or inadequacy of the enzyme systems, apparently of a genetic nature, which decompensate under the influence of unfavorable environmental conditions. In addition, hypoenergism can possibly cause an abnormal amino acid metabolism leading to a deficiency in the processes of biological synthesis and breakdown. But disorders in amino acid metabolism, according to Polishchuk, do not exhaust the manifestations of autointoxication in schizophrenics. Another factor is an impairment of the detoxication processes (7-10, 15, l 6 ) , which manifests itself in the active stages of the disease by the reduced formation of rhodanate compounds. Although the synthesis of conjugated ether-sulfuric and ether-

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glucuronic acids is increased, compared to the normal, it is still insufficient to ensure the fixation of toxic substances; the content of such substances as free phenols and cresols in the blood and urine is increased. Polishchuk considers the immunological changes during schizophrenia ( allergization, possible manifestations of autoaggression) to be the result of disorders of protein metabolism, and hormonal changes to be the sequel of disorders of the regulatory processes. In assessing the nature of the biochemical disorders among schizophrenics, Polishchuk stresses that they are widely diverse and diffuse indicating that this disease is based not on some isolated defect, “an enzyme block,” as is the case for a number of hereditary enzymopathies (inborn errors of metabolism), but on the decompensation of a genetically based general weakness, leading to the derangement of a variety of enzyme systems that control metabolism. Other scientific groups are also working in a field closely associated with that of Protopopov, Polishchuk, and their coworkers. Among them we should mention the work of Chalisov (department of psychiatry of the Minsk Medical Institute) and his group. His hypothesis of the pathogenesis of schizophrenia can be summarized as follows (22). Toxicosis is the main factor in the pathogenesis of schizophrenia. It appears as a result of the influence of a number of products of protein decomposition, such as the indole compounds, the derivatives of adrenaline or other substances whose chemical characteristics are as yet unknown. However, the mere appearance of toxic substances is not enough to cause toxicosis, and at least two other conditions are required: ( a ) an insufficiency in the systems of destruction or binding of toxic substances, and ( b ) a biological deficiency of the brain entailed by hereditary factors or by unfavorable influence of the environment, manifested by the retention in the brain of a number of substances, including toxic ones, which are excreted by the normal brain. This hypothesis, which is Chalisov’s special conception, was formulated by him on the basis of his investigations (23, 24) of the venous blood from the brain. In particular it was demonstrated that during the acute period of the disease there occurs a retention of nitrogen in the brain of the schizophrenics, in the form of urea, uric acid, and creatine-creatinine, and of aromatic compounds which are detected with the aid of the xanthoprotein reaction.

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Later (22, 25) Chalisov and his associates studied some aspects of metabolism in patients with various classic forms of schizophrenia (catatonic, hallucinatory-paranoid, and simple ) . A study of phosphorylated products in the blood and the level of oxidation processes shows that the results in principle coincide with the data of Polishchuk and that there is a disturbance of energetic metabolism, especially among patients with the catatonic form. In studying the free amino acids in the blood sera the most marked changes occur in catatonic and hallucinatory-paranoid forms during the acute stages of the disease; these changes consist in a drop of the content of cystine, cyteine, and glutamic acid, as well as glycine, leucine and isoleucine; those suffering from the hebephrenic and simple forms have a more pronounced drop in the level of free lysine. Chalisov supposes that the noted reduction of sulfurcontaining amino acids and glutamic acid in the blood is caused by their mobilization for the processes of detoxication, the level of which is higher in patients with the catatonic form of schizophrenia as compared to other forms. Sorokina ( 25a) investigated glutamic-alanine and glutamicaspartic transaminases in the sera of the schizophrenic patients and found that the activity was higher when the course was favorable; this finding is evaluated as a good prognostic sign. Since interest has been manifested lately in ceruloplasmin in schizophrenia and in the role of copper in vital processes, Tushkevich (25b) studied the copper level in the serum of over 200 schizophrenics. She found that the copper content is higher in catatonic and hallucinatory-paranoid forms, especially during the acute stage of the process. When the case takes a sluggish course, the copper level does not differ from the normal, and during the simple form it is often reduced. Chalisov considers the increase of the copper content in the serum of the patients as a favorable sign, reflecting the protective and adaptational reaction of the organism to the toxicosis. 111. Pathogenesis. Some Other Concepts

In close conceptual relation with the work of Protopopov’s and Chalisov’s schools are the investigations conducted by Sereysky et al. (department of psychiatry of the Moscow Institute for Advanced Medical Studies, subsequently, the Moscow Institute of Psychiatry of the Ministry of Health of the Russian Federation).

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Sereysky (26) proceeded from the toxohypoxic conception of the pathogenesis of schizophrenia. This concept is first of all related to the catatonic form of schizophrenia. In his opinion schizophrenia is a miscellaneous group of illnesses; the catatonic form is to a certain extent opposed to the paranoid form. Sereysky based his conception of the reduction of the oxidation processes in the brain on the existing literature and on his own investigations of the arteriovenous difference in sugar, oxygen, and carbon dioxide of the blood in the brachial artery and the jugular vein. According to these investigations ( 2 6 ) , the consumption of oxygen and sugar by the brain of the schizophrenics is reduced. At the same time the tissue hypoxia during schizophrenia is not limited to the brain. Data concerning the reduction of the redox potential of the blood, during the acute phase of schizophrenia, (mostly in the catatonic form) supports these findings ( 2 7 ) . Sereysky sees the cause for this hypoxia in the toxic influence of the products of a disturbed protein metabolism, the derivatives of the aromatic and heterocyclic amino acids, on the corresponding enzyme systems. He considers that insufficient detoxication of these products plays a definite role in the schizophrenic process. Sereysky personally conducted extensive investigations on protein metabolism during schizophrenia. He discovered that during the acute stages, especially during psychomotor excitation, schizophrenics often have an increased level of proteins in the blood, the most marked changes being in the ratio of protein fractions. For short periods of time, the patients may show significant fluctuations in the albumin-globulin ratio in the plasma with a predominance of the less stable globulins and fibrinogen. During the catatonic form of schizophrenia, the albumin-globulin ratio sometimes drops to 0.35. These deviations are not observed during the chronic stages of the disease ( 2 6 ) . It should be noted that Sereysky started a whole series of investigations dealing with changes in the biochemical indices of schizophrenics, which appear under the influence of various methods of active therapy ( insulin, electroconvulsive therapy, etc. ) . After Sereysky died, the biochemical investigations have continued at the Moscow Institute of Psychiatry of the Ministry of Health of the Russian Federation, and are headed by his co-worker Lando, whose efforts have concentrated mainly on the study of the extensive range of indices of protein metabolism in schizophrenics, and also how they change under treatment.

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Lando does much work in the clinical field, and her approach to this aspect of the problem differs from Sereysky’s. In accordance with the ideas formulated in Sovirt psychiatry by a number of leading Soviet clinicians ( Snezhnevsky, Sukharcva, Melekhov, and others), Lando bases the clinical differentiation of schizophrenia ( the classification of material and clinical and biochemical comparisons) on the dynamic criteria of the course of the disease. More will be said about this principle of classification later. The data obtained by Lando indicates that the changes in the indices of the protein metabolism in schizophrenics are closely connected with the type of course the disease takes, the number of attacks, and the mental state of the patients during the period of observation (28-33). Lando noticed the greatest changes in patients with the periodic course of disease, especially during catatonic excitation in the first acute attack of oneiroid catatonia. These changes include the reduced content of albumin in the serum and the albumin-globulin ratio, an increase of the a - l - and a-%globulin fractions and ammonia of the blood. According to these indices the periodic schizophrenic patients differed not only from the normals, but also from patients of two other types of schizophrenia. An increase of the general amino-nitrogen content in the blood was noted during periodic schizophrenia, as compared to the progressive course. A reduction in the glutamic acid and alanine content in the blood and an increase in the arginine and leucine content are observed during the acute catatonic state. Schizophrenics showed increased activity of the glutamic-aspartic and glutamic-alanine transaminases, which was also markedly expressed during the periodic type of disease. All groups of patients showed an increased glutamine content of the blood. The content in the blood of serotonin and the excretion of 5-hydroxyindoleacetic acid did not, on the average, differ from the normals regardless of the course of disease. A high serotonin content occurred relatively more often in patients with psychomotor excitation, regardless of its nature. The spontaneous excretion of tryptophan, and the products of the kynurenine pathway of its metabolism, was not changed in cases of periodic schizophrenics, while in the nuclear group, the excretion of tryptophan and the indole compounds was increased, and the excretion of kynurenine was reduced. When tryptophan was administered to patients with periodic

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schizophrenia there was a sharp increase in the excretion of kynurenine and xanturenic acid as compared to the normals, which was interpreted as indicating an increase in the intensity of breakdown of tryptophan, especially during the first stages of the kynurenine pathway.’ At the same time the nuclear schizophrenics showed a drop in the excretion of kynurenine and methylnicotinamide and an increase in the excretion of tryptophan, which was interpreted as an indication of the reduction in the use of the kynurenine route. Lando considers that a comparative analysis of these peculiarities shows that the changes in the protein metabolism in periodic patients are of a functional nature, while during nuclear schizophrenia they are more profound and “organic.” Lando does not consider that these changes in the patients are the causative factors of schizophrenia. However, if they appear as secondary phenomena (as, for instance, the accumulation of ammonia, and the reduction of the glutamic acid in the blood), they may be the cause of a number of changes in the metabolic processes and may thus become factors influencing the course of the disease. Another scientific group basing its investigations on the endotoxic conception of schizophrenia is working at the Tbilisi Institute of Psychiatry of the Georgian Ministry of Public Health, and is headed by Zurabashvili. Zurabashvili proceeds from the concept that the pathology of schizophrenia is based on a chronic course of toxicosis, occurring as a sequel of the formation of organic toxic substances, which causes an encephalopathy, affecting axodendritic synapses. This results in the disintegration of mental activity, which serves as the basis of the clinical-psychopathological manifestations of schizophrenia ( 35). A number of other investigators at this institute are studying the peculiarities of schizophrenic toxicosis. Sikharulidze ( 36) and Bostoganashvili ( 3 7 ) demonstrated that the intramuscular administration to dogs of schizophrenic plasma (especially from catatonic patients) induces a reduction of oxygen consumption by the brain tissue of the animals (on the basis of a study of the oxygen content Similar data about the disorder of the kynurenine pathway in the metabolism of tryptophan in schizophrenics, especially of the periodic type, when loading with this amino acid, were obtained earlier by Lozovsky in the Institute of Psychiatry of the USSR Academy of Medical Sciences (34). He concluded that there is an abnormal kynureninase reaction as a result of an endogenous deficiency of pyridoxal phosphate.

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in common carotid and internal jugular), as well as a number of neurodynamic disorders. Moreover, the development of hypoxia in the brain precedes the latter and can, consequently, be taken as their causative agent. Studying the toxic properties of blood plasma of schizophrenics, Zurabashvili (38, 40) found that the administration to dogs of plasma from catatonic patients results in disorders of nucleic acid and polysaccharide metabolism in the white blood cells, and to notable morphological changes (by means of histochemical, histological and electron microscopic observations ) . Besides studying the toxic effects of the blood plasma of schizophrenics Zurabashvili (39) discovered a number of changes in its biophysical properties: a reduction in the transfer of energy along the peptide links of plasma proteins, a decrease of its redox potential, an increase of electroconductivity of plasma in high frequencies, a decrease in surface buffer capacity, and a reduction of the speed of plasma stricture, while the surface tension remains unchanged. This, according to the author, points to the tendency of the protein micellae and colloid particles of the plasma to increased destruction and a drop in the adsorptive force of adhesion. Much work in the biochemical investigation of schizophrenia is done at the Kharkov Psychoneurological Institute. Khaimovich has investigated certain aspects of carbohydrate-energetic metabolism in schizophrenics ( 4 1 4 4 ) . She proceeded from the premise, advanced by Popov and developed by Tatarenko, that schizophrenia is based on changes in the functions of the central nervous system brought about by disorders of processes of stimulation and inhibition, with the latter predominating; the changes in the metabolic processes in schizophrenia are deemed to be dependent on the central nervous system. The main results obtained by Khaimovich can be summarized as follows. Carbohydrate metabolism in schizophrenics proceeds at a low level, namely, there is a reduction of the average sugar content in the blood, triosophosphoric and lactic acids, and bisulfite-binding compounds. At the same time the indices of carbohydrate metabolism are very unstable, depending on the clinical condition of the patient and are reversible. The greatest deflections are observed in the catatonic form of schizophrenia, especially during the peak of the disease. With the onset of remission the changes in the metabolism even out.

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According to Khaimovich, a change of certain blood enzymes occurs in schizophrenics: there are significant fluctuations and a drop of the average values of glycolytic activity while the adenosinetriphosphatase ( ATPase) activity of the erythrocytes is higher and there is an increase in the velocity constant of the catalase reaction; in remission these changes also even out. The investigations conducted by Khaimovich of the reactivity of certain biochemical indices showed that the administration of a significant quantity of saccharose (150 g ) entails, in the majority of patients, diabetic glycemic curves with a high hyperglycemic coefficient; at the same time some of the patients manifest hypoglycemic curves. Occasionally there was an equated or inverted reaction of the blood sugar in response to the administration of adrenaline and insulin: when saccharose was administered after pretreatment with adrenaline the elevation of blood sugar was equal or even less, as compared to the level when only saccharose was used; after pretreatment with insulin it was increased. In remission all these distortions of the glycemic reaction disappeared. The biochemical investigations of schizophrenia carried out at the Daghestan Medical Institute in the city of Makhachkala by Glazov have their own interesting features (45-49). Glazov belongs to that group of scientists who consider that schizophrenia (understood as the so-called nuclear group with the aspect of Krepelin’s dementia praecox), is a result of a congenital insufficiency of the nervous system in the form of primary disorders in the higher nervous functions (45). As a result the syndrome of disintegration of cerebral processes appears. Whereas all the other changes in the patient’s organism, including the biochemical ones, are of a secondary nature and are either explained by disorders of central regulation, or are held to be protective-adaptive reactions. However, the secondary biochemical disorders lead to the appearance of products of a disturbed metabolism, whose long-term effects on the nervous system can cause irreversible organic disorders of the brain and other organs. Therefore at a certain stage, the metabolic disorders may become the predominant factors in the development of the disease. The biochemical investigations carried out by Glazov et al. are mainly concerned with the study of certain biochemical indices in the leukocytes of schizophrenics, which are thought to be very sensitive indications of changes occurring in the internal mediiim of

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the organism. This group of scientists discovered that there is an increased content of tyrosine and phenolic inclusions in the segmented nuclear leukocytes of schizophrenics (47) and that the content of phenolic inclusions is especially elevated after the administration of 1 gm sodium salicylate. Moreover, there is an increased activity in the leukocytes of peroxidase and phenolase (49), and an increase of the content of sulfhydryl groups (48). Glazov et uZ. consider these changes as indicating the limitations of the protective-adaptive possibilities of the organism of schizophrenics. An important scientific group studying the biological aspects of schizophrenia is located at the Institute of Psychiatry of the USSR Academy of Medical Sciences, headed by Snezhnevsky. This institute has a long history, but we shall dwell only on the present stage of its activity, which began in 1962, when the institute became a scientific establishment dealing with only one problem-the comprehensive study of schizophrenia. The basic scientific direction and the organizational structure of the institute follow the principle of the broadest possible multidisciplinary investigations of this disease at various levels: sociological, clinical-psychopathological, psychological, neurophysiological and general pathological ( genetic, biochemical, endocrinological, biophysical, immunological, and morphological). Modern basic scientific achievements and corresponding methods are extensively used. Organizationally the latter are ensured by physicians and certain theoretical specialists who are invited to work at the institute as well as by fully equipped modern laboratories. At present this institute does not endorse any final general concept of the nature of schizophrenia, because it is considered that there are still not enough reliable and reproducible facts for this to be possible. But there are a number of initial positions that form the basis of the direction of the investigations in the field of the biology of schizophrenia. Some of these positions have already been presented in articles contributed by Vartanian ( 50-52). It is accepted that the etiology of schizophrenia is genetically conditioned, but is not fully linked with the genetic factor; rather it is a disease in which the hereditary factor is merely the pathological background for the appearance of the disease proper. This is based on the study of monozygotic twins having similar genotypes, whose pathological concordance was not 100% but 68-70%; the

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separation of such twins leads to a significant increase in discordance. Moreover certain data point to the fact that schizophrenia is a genetically heterogeneous disease with a multiple etiology, and the corresponding calculations show that there must be a great number of loci, whose mutational changes could entail a predisposition to schizophrenia (52, 53). From this it follows that the initial links of the pathological chain of schizophrenia, which are directly connected with the hereditary factor, could differ in various patients. This makes it necessary to stress the study of the families of the probands in investigating the biology of schizophrenia. It can further be supposed that different pathogenic mechanisms may play the decisive role in one and the same patient at different stages in the development of schizophrenia. Moreover in the course of the disease, these pathogenic mechanisms can lose their ties with the factor that caused them and develop independently (spontaneous self-development of the disease). Since such a possibility cannot be excluded for schizophrenia, it follows that the various data obtained by scientists in studying the biology of this disease can be compared only if they concern a definite stage of the disease; the only way of getting a more complete understanding of the nature of the pathogenic mechanisms is to pursue investigations throughout the whole course of the development of schizophrenia. It is now generally recognized that the clinical aspect is very important in studying the biology of schizophrenia. However, various scientists give different appraisals to the significance of one or another clinical category for biological investigations, as well as to their concepts about what clinical parameters are the most adequate for reflecting the main pathogenic biological mechanisms. Many investigators base their classification of schizophrenia, and consequently their selection of patients for clinical and biological comparisons, on the so-called classic forms ( catatonic, hebephrenic, paranoid, and simple). IV. Classification

The Institute of Psychiatry of the USSR Academy of Medical Sciences bases its biological investigations of schizophrenia on the clinical concept of the limits, criteria, and classifications that have been developed in the past few years by Snezhnevsky (54, 55). Accordingly, a diagnosis of schizophrenia is made if certain

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changes of personality, such as autism, changes in attitude to former life interests, a general change in emotionality, and a reduction in energetic potential, occur in the patient. Besides these negative symptoms, which may vary greatly in degree, the patients also manifest a number of positive disorders. These include neurotic disorders ( obsessions, depersonalization), depressive or manic conditions (which usually occur in conjunction with delusions), as well as hypochondriac, cenesthopathic,‘ oneiroid, paranoial, pseudohallucinatory, paraphrenic, and catatonic disorders. A diagnosis of schizophrenia is not made if the patient has a loss of memory or other manifestations of an organic syndrome, convulsive states, or syndromes of clouded consciousness (delirium, twilight states of consciousness ). As far as the classification of schizophrenia is concerned, the Institute of Psychiatry considers that the decisive criteria are not the static symptoms of “cross-section,’’ as is the case when the division is made into classic forms, but rather we stress the criteria of the type of development of the disease. From this point of view there are three main forms of schizophrenia: the progressive-translational course (progressive progredient schizophrenia ) , intermittent-translational course (in the form of “shifts” or shift-like schizophrenia ) and the recurrent course ( periodic or recurrent schizophrenia). Progressive progredient schizophrenia can again be subdivided, in accordance with the course, into several subgroups of which the most important would be sluggish, paranoid, and malignant ( juvenile or nuclear) schizophrenia. Periodic schizophrenia can take the course of oneiroid-catatonic, depressive-paranoid, and circular forms. The classification of schizophrenia according to the forms of its development reflects more completely the constant, more profound, and essential features and differences of the clinical prognosis and, correspondingly, the pathogenesis of the main variations of the schizophrenic process than the classic forms provide, because the latter give only a temporary, transitory, and incomplete characterization of the process at certain stages of its development. From this it follows that the classification according to forins of the development and a comparison of these forms should be adopted as Unusual, pathological, painful, bodily sensations of a vague and indefinite

kind.

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the primary basis for the selection of groups of patients and for clinical-biological correlations in the biological investigations of schizophrenia. We have adopted this principle from the start of our investigation and it has proved most fruitful in the multidisciplinary investigations of schizophrenia conducted by the Institute of Psychiatry. V. Biochemical Investigations at the Institute of Psychiafry

Since there is only a limited possibility of studying the processes taking place in the brain of schizophrenics, we must study the various properties of the biological fluids of the patients: first and foremost, of the blood, as well as of the urine and cerebrospinal fluid. Moreover, it is assumed that the disorders taking place in the brain are to some extent, directly or indir'ectly reflected in the whole organism, particularly in the humoraI media. A study of the properties of biological fluids of schizophrenics has been carried out for many years, and has revealed many facts, showing that there are certain anomalies, qualitative or quantitative, in the metabolites having biological activity. The nature and significance of these substances in the pathogenesis of the disease is still unknown, while the results obtained in this field by the various investigators do not easily lend themselves to comparison, since they were obtained on different test objects with the aid of various methods, using material obtained from different patients. Therefore for the last few years our institute has been studying the properties of humoral media of schizophrenics with the aid of a number of tests or bioindicators, which differ in the level of biological organization (cytological, biophysical, biochemical, general pathological, and iieurophysiological) , but which are conducted by one group of scientists on the same group of patients. There are three laboratories in the institute carrying out these investigations: pathomorphological ( headed by Romasenko) , neurophysiological (headed by Monakhov) and general pathophysiological (headed by Vartanian). The latter laboratory is the largest both with regard to the number of people working there and amount of equipment. It consists of four groups: biochemical (where the author of this article is working), biophysical, immunological, and neuroendocrinological. The biochemical investigations in this multidisciplinary complex have two main purposes: to establish the influence of the serum

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of patients on certain biochemical processes and to study the nature of this biological activity. As far as the first aim is concerned, ure have concentrated mainly on the effects of the sera of schizophrenics on the processes of carbohydrate-energetic metabolism. We are conducting these particular investigations because we share the view of a number of investigators that in the pathogenesis of schizophrenia, a great role is played by disorders of the intermediary carbohydrate-energetic metabolism, of which one manifestation could be an insufficiency of the system that ensures the mobilization of energy in conditions of functional stress (56, 57). For a direct biochemical bioindication of the effects of the blood serum of schizophrenics we have used the test developed by Frohman et al. (58); namely, the influence of the serum on the content and the ratio of lactic and pyruvic acid in the model system of chicken erythrocytes incubated in a glucose-containing medium. In full accord with Frohman’s data we have found (59, 60) that the serum of 242 patients causes a significant increase ( p < 0.001) of the lactate/pyruvate ratio ( l / p ) as compared to the serum of 69 normals and 82 patients with other mental disorders. The corresponding ratio values for these three groups were 7.3 ?Z 0.3, 4.9 t 0.2, and 5.3 t 0.4, respectively. The effect obtained is due both to some increase in the content of lactic acid, and, mainly, to the reduction of pyruvic acid. In comparing the data with the clinical features of the patients under investigation it was found that among the periodic-type patients the highest ratio was observed in oneiroid catatonia (8.9 -F0.6) and the lowest in the circular patients (6.3 f 0.7). In paranoid schizophrenia the highest ratio was noted at the paranoia1 stage of the disease (8.6 f 0.7), the lowest in the terminal stage (6.3 k 0.4). Among the patients with the progressive progredient form the highest ratio (7.8 -t 0.3) was noted during the malignant course of the disease (hebephrenic and early paranoid forms ). In cases of sluggish schizophrenia the ratio (5.4 -t 0.4) does not differ from normal values. In comparing the ratio values with the length of the disease it was seen that the patients with the periodic type had an l / p ratio which was highest during the first year of the disease, then there was a reduction, which came down to normal values in 5-7 years ( v = 0.34 when p < 0.001).

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In malignant progressive progredient schizophrenia, in the first years of the disease the ratio was in the range of normal values, and it reached the highest values in the 4-7 year-period of the disease, and then dropped, approaching normal values again in the 10-14year period ( q = 0.66 when p < 0.001). In sluggish cases there was no significant correlation between the ratio value and the length of the process. All this can be regarded as an indication of the possible ties between the l / p ratio value and such clinical parameters as the gravity of the process and the expression of active psychotic symptoms. At the same time it was shown that the state of stress of the patients under investigation, caused by a physical load or by the administration of ACTH, is also accompanied by an increase of the lactate/pyruvate ratio in experiments with the corresponding sera. When comparing the effect under study of the sera of schizophrenic patients with certain other manifestations of its biological activity it was discovered that there is a linear correlation between the effects of the schizophrenic serum on the lactate/pyruvate ratio and its ability to induce hemolysis of chicken erythrocytes under the same conditions ( r = 0.74 when p < 0.001). In periodic schizophrenic patients there was also a direct link between the activity of the serum on the lactate/pyruvate ratio in vitro and its capacity to raise the sugar level in the blood when administered to rabbits in uiuo. Finally there was linear correlation ( T = 0.36 when p < 0.01) between the lactate/pyruvate ratio and the presence in the corresponding sera of antibodies against antigens of water-saline extracts of the brain; this was discovered with the aid of the complement fixation test. The study of some properties of the biologically active factor of the serum that influences the transformation of glucose by chicken erythrocytes showed the following: The activity of the serum is thermolabile and unstable: Its effects disappeared when the serum was heated at $56" for 30 minutes, and at room temperature in 24 hours. At a temperature of +4", the serum preserved its activity for 10-12 days. The serum could be prevented from inactivation during its storage by the addition of certain reducing substances (ascorbic acid, cysteine). When frozen for 15 minutes at a temperature of -30" with consequent thawing, the serum was also inactivated. But the serum kept

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its activity when it was lyophilized. A serum inactivated by heating could be reactivated if we added fresh serum from a normal or a complement preparation (lyophilized serum of guinea pigs) ; this can serve as an indication of the two-component nature of the active principle. Experiments were conducted with ultrafiltration of the sera through semipermeable membrane, with the aim of finding out the nature of the active factor of the serum that affects the transformation of glucose. The activity of the serum was associated with its high molecular fraction. The nonprotein low molecular fraction turned out to be inactive. To obtain further information as to the nature of the active principle, the proteins of the serum were fractionated in a preparative scale. With the aid of an instrument for continuous electrophoresis in a supporting medium, the proteins of the serum were divided into five fractions: prealbumin, albumin, a-, P-, and y-globulins. The P-globulin serum protein fraction from schizophrenics was the most active as compared to similar fractions from normals. With the aid of absorption chromatography on columns with hydroxylapatite ( after preliminary albumin precipitation) the serum globulins were divided into four fractions that differed in absorption capacity. The 11 and IV globulin fractions of serum from schizophrenics were the most active. An analysis with the aid of paper electrophoresis and immunoelectrophoresis showed that ,8globulin is almost always discovered in the active fractions, as well as ,&lipoprotein, when stained for lipoprotein. Comparing our data on the properties and nature of the active factor of the serum with the data of Frohman, Bergen, and Pennell, it can be said that there is agreement of results in a number of essential indices. All this allows us to suppose that in our work and in the work of several groups of American investigators, despite the different tests and methods used, we are dealing with one and the same factor. As for the possible mechanism of the biochemical effect under study, it is known that anaerobic glycolysis with the accumulation of lactic acid occurs in the chicken erythrocytes when there is a lack of oxygen in the medium, or if it is wrongly utilized. In our case the incubation of the erythrocytes takes place under aerobic conditions. This means that under the influence of the active factor of the patient’s serum, there occurs n disturbance of the consumption of

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oxygen by the erythrocytes, possibly as a result of the effect of the serum factor on the transport of hydrogen in the respiratory chain. In this connection it could be expected that the serum of schizophrenic patients would inhibit tissue respiration. Corresponding experiments conducted by us using the manometric method on tissue slices of rat brain showed that such inhibition does in fact take place (61). Moreover, the electron microscopic investigations of the slices showed changes in the structure of the mitochondria (swelling, fragmentation, changes in the structure of the crysts ) . At present similar investigations using the polarographic method are being conducted on the mitochondria of the brain of cats. Naturally the question arises as to the effect of the factor under investigation on the organism of the patients and of the possible role of this phenomenon in the pathogenesis of the disease. To clarify this question we studied the effects of the schizophrenic serum not only in vitro but also in vivo (on rats, using intravenous administration) on the lactate/pyruvate ratio in brain tissue; we ascertained that schizophrenic serum causes significant increase of the ratio from 6.4 (in the case of serum from normals) to 8.3. Thus, these experiments show the possibility that the serum factor may have an effect on the transformation of glucose in the brain in the intact organism. When comparing the above-mentioned facts with the numerous data available in the literature pointing to the presence of hypoxia in the brain of schizophrenic patients, as well as the data of Arnold and Hoffman (62) concerning the connection of disturbances of the carbohydrate-energetic metabolism in schizophrenic patients with the genotype and the clinical peculiarities of the process, it can be supposed that the effect of the serum factor under study on the carbohydrate-energetic metabolism can play an essential role in the pathogenesis of schizophrenia. This supposition is to some extent supported by the work carried out in our laboratory in our investigations of the effect of the serum of relatives of schizophrenic patients (63) on the lactatel pyruvate ratio in in vitro experiments. This investigation showed that the occurrence of pathologically high values of the lactate/ pyruvate ratio in clinically healthy relatives of schizophrenic patients is higher than in the whole group of normals. The average values of the ratio for these two groups were respectively 6.2 0.6 and 4.9 f 0.3 ( p < 0.05).

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We also investigated the effect of the sera from seven pairs of dizygotic and nine pairs of monozygotic twins. In eight pairs of monozygotic twins, despite the similarity of the disease, it was noted that the partners had both high and low ratio values. In one monozygotic pair, who were discordant for schizophrenia, the ratio values of the twins was also different. Among the dizygotic pairs, the twins of only one pair were both ill; here the ratio was elevated in both cases. I n the other pairs there was no agreement in the ratio values. The results, which point to the increased activity of the serum of clinically healthy relatives of schizophrenics, support the supposition that this activity may reflect certain genetically determined features of their metabolism. However, the data from the study of the monozygotic twins force us to postulate that the presence or absence in the serum of this activity is conditioned not only by the genotype, but also by some other factors. In our laboratory we also undertook the study of the nature of another manifestation of the biological activity of the serum of schizophrenic patients that is detected by another test; namely, the cytopathological effect of the serum on the tissue culture of fibroblasts of chicken embryos ( 64 ) . With the aid of preparative electrophoresis, it was shown that the main activity of the serum, according to this index, is also contained in the P-globulin fraction (65). Other characteristic manifestations of the biological activity of schizophrenic sera (51) were also discovered by the biophysical, neuroendocrinological, and immunological groups of the general pathophysiological laboratory of the institute. They include the hemolytic activity of the serum (66) as an index of its effects on the cellular membranes, which may lie at the basis of many of the manifestations of the activity of the serum; the peculiar stressinducing properties of the serum, which differ essentially among patients with various types of schizophrenia (67-69); and the action of the serum on the mytotic activity of the tissue culture cells and the development of experimental animals ( 7 0 ). The methods so far used by us in the isolation of the serum proteins proved to be unsuitable for the solution of one of the central problem of this investigation, namely, the question of the unity or diversity of the material substrate, responsible for these various effects of the serum. This was because the activity of the serum, according to certain bioindicators, was too labile. Hence an

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attempt was made to study this question by the investigation of the changes in the various manifestations of the biological activity of the serum under certain physical and chemical influences (the effect of various storage periods under various temperatures, the influence of lyophilization, freezing, extraction by organic solvents, and the effect of adding a reducing substance, ascorbic acid). The results obtained (51, 66, 71) showed that the same physical and chemical influences affect the activity of the serum of schizophrenics differently, depending on the bioindicator used. These data support the hypothesis that there are a number of different biologically active factors in the serum. However, at present we cannot exclude another explanation, which states that the biologically active factor is single, but because of the difference in the threshold of sensitivity of the bioindicators used, the degree of its inactivation, caused by one or another influence, has different effects on the various manifestations of its activity. To clarify this problem, it is necessary to establish the threshold concentrations of the serum, which can still cause an effect on each test object, and to conduct a comparitive investigation on the influence of the physical and chemical effects on the various manifestations of activity at these threshold concentrations. VI. Summary

This account of the biochemical investigations of schizophrenia carried out in the Soviet Union is not of course exhaustive. An attempt has been made, however, to include the initial concepts of leading Soviet scientific groups about the nature of schizophrenia and the place of biochemical investigations in its multidisciplinary study, to characterize in general outlines the wide range in which these investigations are being approached in the Soviet Union, their modem theoretical and methodological levels, and the main results obtained so far. If this review will enable psychiatrists in other countries who are not well acquainted with our scientific publications to enrich their knowledge of this question and to get the necessary reference material for more detailed information on this subject, we will consider that this review will have served its purpose. REFERENCES 1. Omorokov, L. I. (1909). Obozr. Psiklaiat. 9, 518. 2. Yushchenko, A. I. (1903). Obozr. Psikhiat. 3.

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3. Protopopov, V. P. ( 1946). Iti “Patofiziologicheskie osnovy ratsionalnoi terapii shizofrenii.” Gosmedizdat, Kiev. 4. Leshchinsky, A. L. ( 1938). I n “Probleniy patofiziologii i terapi shizofrenii,” Part 10, p. 173. Gosmedizdat, Kharkov. 5. Rasin, C. D. ( 1948 ). “Funktsionalnoe sostojanie pecheni pri shizofrenii.” Candidate of Medicine’s Thesis, Kiev. 6. Rasin, C. D., and Kolchinskaya, A. Z. (1952). In “Kislorodnaja terapija i kislorodnaja nedostatochnost.” Gosmedizdat, Kiev. 7. Polishchuk, I. A. ( 1935). Sovet. Neoropatol., Psikhiatriya, Psikhogigieria 8, 95. 8. Polishchuk, I. A. ( 1937). 111 “Biokhiniichny doslidzhenija v psihiatrii.” Kharkov. 9. Polishchuk, I. A. ( 1938 ) , Sovet. Neoropatol., Psikhiatriya, Psikhogigiena 6, 87. 10. Polishchuk, 1. A. ( 1939 ) . In “Trudy tsentralnogo psikhonevrologicheskogo instituta,” Part 11. Gosmedizdat, Kharkov. 10a. Quastel, L., and Wheatley, A. (1933). Biochem. J . 27, 5. 11. Gorodkova, T. M. (1953). Vopr. Fiziol. 4, 211. 12. Zelinsky, S . P. (1953). Vopr. Fiziol. Ibid., 191. 13. Zelinsky, S. P. (1951). “The Influence of Schizophrenic Serum on the Brain Tissue.” Candidate of Medicine’s Thesis. Kiev. 14. Zelinsky, S . P. (1955). Fiziol. Zhur. Akad. Nauk. URSR 5, Part 1, 37. 15. Polishchuk, I. A. ( 1 x 7 ) . In “Biologicheskie issledovanija shizofrenii” (D. Lozovsky, ed.), p. 191. Pravda, Moskva. 16. Polishchuk, I. A. ( 1967). In “Biokhimicheskie sindromy v psikhiatrii.” Zdorovje, Kiev. 17. Polishchuk, I. A. (1965). Ti-. 4th Vses. Sjezda Neoropat. Psikhiat., Moskva, 1965 Vol. 111, Part I, p. 464. 18. Polishchuk, I. A. (1965). In “Problemy patologii vysshey nervnoy dejatelnosti, somaticheskikh narusheniy, kliniki i terapii psikhozov” ( A. Makartchenko, ed.), p. 244. Naukova dumka, Kiev. 19. Polishchuk, I. A. (1958). Zh. Nevropatol. i Psikhiatr. 58, 55. 20. Polishchuk, I. A. (1958). Zh. Nevropatol. i Psikhiatr. 58, 1163. 21. Polishchuk, I. A. (1962). Zh. Nevropatol. i Psikhiatr. 62, 1192. 22. Chalisov, M. A. ( 1967). In “Biologicheskie issledovanija shizofrenii” (D. Lozovsky, ed. ), p. 203. Pravcla, Moskva. 23. Chalisov, M. A., Volfson, N. M., and Arutjunov, D. H. (1932). “Biokhimichni doslidzhenija v psihiatrii,” p. 9. Kharkov. 24. Chalisov, M. A., and Volfson, N. M. (1940). Tr. Stalinsk. M e d . Inst. 4, 161. 25. Chalisov, M. A. (1965). Tr. 4th Vses. Sjezda Nevropatol, Psikhiat., Moskva, 1965 Vol. 111, Part I, p. 449. 25a. Sorokina, T. T. (1965). Tr. 4th Vses. Sjezda Nevropatol. Psikhiat., Moskva, 1965 Vol. 111, Part I, p. 477. 2%. Tushkevich, Z. R. (1965). Tr. 4th Vses. Sjezda Neuropatol. Psikhiat., Moskva, 1965 Vol. 111, Part I, p. 471. 26. Sereysky, M. Ya. (1958). In “Voprosy kliniki, patogeneza, lechenija shizofrenii” (V. Banshchikov, ed.), p. 107. Moskva.

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27. Sereysky, M. Ya., and Rotshteyn, G. A. (1939). Nevropatol. Psikhiatriya 8, 31. 28. Lando, L. I. (1967). “The Study of the Protein-nitric Metabolism in Schizophrenics and its Dynamics in the Treatment with Aminazin.” Doctor of Medicine’s Thesis, Moscow. 29. Lando, L. I. (1965). In “Problemy patologii vysshey nervnoy dejatelnosti, somaticheskikh narusheniy, kliniki i terapii psikhozov” (A. Makartchenko, ed.), p. 204. Naukova dumka, Kiev. 30. Lando, L. I,, Krupenina, L. B., and Zeleva, M. C. (1965). In “Problemy patalogii vysshey nervnoy dejatelnosti, somaticheskikh narusheniy, kliniki i terapii psikhozov” ( A . Makartchenko, ed.), p. 208. Naukova dumka, Kiev. 31. Lando, L. I. (1959). Zh. Nevropatol. i Psikhiatr. 59, 135. 32. Lando, L. I. (1962). Zh. Nmropatol. i Psikhiatr. 62, 1855. 33. Lando, L. I., Zakharjev, Ju. L., and Krupenina, L. B. (1962). Zh. Nevropatol. i Psikhiatr. 62, 99. 34. Lozovsky, D. V. (1962). Vopr. Med. Khim. 8, 616. 35. Zurabashvili, A. D. (1965). Tr. 4th Vses. Sjezda Nevropatol. Psikhiat. Moskva, I965 Vol. 111, Part I, p. 5. 36. Sikharulidze, A. I. (1965). Tr. 4th Vses. Siezda Nevropatol. Psikhiatr., Moskva, 1965 Vol. 111, Part I, p. 435. 37. Bostoganashvili, N. I. ( 1967). In “Problemy Shizofrenii” ( B . Naneyshvili, ed.), p. 154. Tbilisi. 38. Zurabashvili, Zig. A. (1967). In “Problemy Shizofrenii” (B. Naneyshvili, ed.), p. 196. Tbilisi. 39. Zurabashvili, Zur. A. ( 1967). In “ProbIemy Shizofrenii” ( B . Naneyshvili, ed.), p. 196. Tbilisi. 40. Naneyshvili, B., Sikharulidze, A,, and Zurabashvili, Zig. (1967). In “Biologicheskie issledovanija shizofrenii” ( D. Lozovsky, ed. ), p. 182. Pravda, Moskva. 41. Khaimovich, L. A. (1964). “Certain Features of Carbohydrates Metabolism in Schizophrenic Patients.” Doctor of Medicine’s Thesis, Kharkov. 42. Khaimovich, L. A. (1958). In “Voprosy lechenija shizofrenii v patofiziologicheskom osveshchenii” (P. Kovalenko, ed.), p. 127. Kharkov. 43. Khaimovich, L. A., and Podkamenny, B. H. ( 1 9 6 0 ) .In “Obmen veshchestv pri psikhicheskikh zabolevanijakh” (A. Sigrist, ed.), p. 47. Medgiz, Moskva. 44. Khaimovich, L. A. (1962). Zh. Neuropatol. i Psikhiatr. 62, 1905. 45. Glazov, V. A. (1965). TT. 4th Vses. Siezda Nevropatol. Psikhiut. Moskva, 1965 Vol. 111, Part I, p. 310. 46. Glazov, V. A. ( 1967). In “Biologicheskie issledovanija shizofrenii” (D. Lozovsky, ed.), p. 210. Pravda, Moskva. 47. Pipkin, 1. M. (1965). In “Problemy patologii vysshey nervnoy dejatelnosti, somaticheskikh narusheniy, kliniki i terapii psikhozov” ( A. Makartchenko, ed.), p. 248. Naukova dumka, Kiev. 48. Idrisov, I. M. (1965). In “Problemy patologii vysshey nervnoy dejatelnosti, somaticheskikh narusheniy, kliniki i terapii psikhozov” ( A. Makartchenko, ed.), p. 151. Naukova dumka, Kiev. 49. Ibragimova, A. A. (1965). TT. 4th Vses. Siezda Nevropatol. Psikhiat. Moskva, 1965 Vol. 111, Part I, p. 482.

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50. Vartanian, M. E. (1963). Zh. Neuropatol. i Psikhiatr. 63, 805. 51. Vartanian, M. E. (1966). Vestn. Akad. Med. Nauk SSSR 3, 11. 52. Efroimson, V. P., and Vartanian, M. E. (1964). Zh. Vses. Khim. Obshchestva im. D. I . Mendelesva 9, 462. 53. Efroimson, V. P. (1967). I n “Biologicheskie issledovanja shizofrenii” (D. Lozovsky, ed. ), p. 256. Pravda, Moskva. 54. Snezhnevsky, A. (1966). Vestn. Akad. Mcd. Nauk SSSR 3, 3. 55. Nadzharov, R. A. ( 1967). In “Biologicheskie issledovanja shizofrenii” (D. Lozovsky, ed.), p. 146. Pravd‘i, Moskva. 56. Gottlieb, J. S., Frohman, C. E., Beckett, P., Tourney, G., and Senf, R. (1959). Arch. Gen. Psychiat. 1, 243. 57. Frohman, C. E., Letham, L., Beckett, P., and Cottlieb, J. S. (1960). Arch. Gen. Psychiat. 2, 255. 58. Frohman, C. E., Czajkowski, N. P., Liiby, E., Cottlieb, N. D., and Senf, R. (1960). Arch. Gen. Psychiat. 2, 263. 59. Lozovsky, D. V. et al. ( 1967). In “Biologicheskie issledovanija shizofrenii” (D. Lozovsky, ed.), p. 151. Pravda, Moskva. 60. Krasnova, A. I. et al. (1966). Zh. Nevropatol. i Psikhintr. 66, 1538. 61. Tikhonov, V. H., Lozovsky, D. V., and Clezer, I. I. (1967). In “Biologicheskie issledovanija shizofrenii,” p. 156. Pravda, Moskva. 62. Arnold, O., and Hoffman, G . (1962). Wien. Z . Nervenheilk. Grenzg. 19, 1. 63. Krasnova, A. I. ( 1967). In “Biologicheskie issledovanija shizofrenii” (D. Lozovsky, ed.), p. 262. Pravda, Moskva. 64. Rapoport, R. N., and Vartanian, M. E. (1965). Tr. 4th Vses. Siezdu Neuropatol. Psikhiat., Moskva, 1965 Vol. 111, Part I, p. 379. 65. Lozovsky, D. V. (1965). Tr. 4th Vses. Sjezda Neuropatol. i Psikhiat., Moskva, 1965 Vol. 111, Part I, p. 412. 66. Lideman, R . R. (1966). Zh. Neuropatol. i Psikhiatr. 66, 891. 67. Orlovskaja, D. D. et a2. ( 1964). Zh. Neuropatol. i Psikhiatr. 64, 1396. 68. Orlovskaja, D. D. et al. (1966). Vopr. Med. Khim. 2, 150. 69. Orlovskaja, D. D. et al. (1967). In “Biologicheskie issledovanija shizofrenii” (D. Lozovsky, ed.), p. 142. Pravda, Moskva. 70. Kobrinsky, G. D. et al. (1967). In “Biologicheskie issledovanija shizofrenii” (D. Lozovsky, ed.), p. 233. Pravda, Moskva. 71. Vjugova, L. A. ( 1967). In “Problemy shizofrenii” (B. Naneyshvili, ed.), p. 164. Tbilisi.

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RESULTS AND TRENDS OF CONDITIONING STUDIES I N SCHIZOPHRENIA By J. Saarma D e p a r t m e n t of Psychiatry, Tartu S t a t e University, Tartu, USSR

I. Introduction . . . . . . . . . . . . 11. Some Methodological Problems in HNA Studies . . . . 111. 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

I . Introduction

In the living organism there are various levels of organization at different levels of complexity. Behavior as an integral whole is the highest and most complicated level; it is divisible into a great number of more elementary verbal, motor, vegetative, and endocrine components. The states of activity of different nervous structures can be regarded as the next stage; and the physicochemical processes in the nervous tissue form the basic level of the activities of an organism. For adequate investigation of these various levels corresponding methods have to be applied. Only on the basis of the total information gained by means of a great number of different methods is it possible to get an entire picture of the laws governing the activities of an organism. It would be a mistake to hope to obtain exhaustive information about the whole by means of only one or two methods that characterize only one form or level out of many complex activities. So one must keep in mind that basically different methods of investigation, as a rule, do not compete between themselves but complement one another, each contributing its corresponding quota to the elucidation of the basic laws governing the organism. The behavior of a human being can be regarded as a complex 227

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of reflex acts, as was pointed out by SeEenov in 1863. In more detail the general reflex character of the behavior of man, as well as the particular forms of the individual reflexes underlying it, have been investigated by Pavlov and his co-workers and followers. Following a long-term systematic study of the physiological laws governing the activity of the higher levels of the nervous system in animals, Pavlov from 1918 onward established a close cooperation with psychiatrists. On the basis of his first “Excursions of a Physiologist into Psychiatry” (131) Pavlov pointed out that the most general features of the higher nervous activity ( HNA), in health as well as in disease, are the same both in animals and man. Therefore the principles and methods for investigation of HNA are also appliable in man. HOWever, as Pavlov showed in his later works (131), some new cortical mechanisms and interactions are encountered in man, underlying verbal activities and social behavior. These mechanisms are directly involved in mental processes and so play a significant role in establishing the structure of pathological deviations and the ways in which they are brought about. The basic concepts proposed by Pavlov in regard to the pathophysiological background of various psychopathological phenomena served as a basis for further study of HNA in mental disorders. In the course of almost fifty years a great number of investigations have been carried out in this field. In addition to the Soviet investigators a steadily growing number of psychiatrists in other countries are applying concepts and methods of conditioning in their studies on HNA in psychoses and neuroses. The main principles in HNA investigations, as used by Soviet investigators are pronounced objectivity of the data collected; complexity of methods applied; many-sidedness of reflex acts investigated simultaneously; taking into account the leading role of the verbaI (second signaling) system in the integrated activities of the human being; and interpretation of HNA data supported by and carried out on the basis of clinical data ( 71-79,146,182,219-223,229232). Recently the fruitfulness of cooperation of conditioning and electrophysiological methods in the study of HNA in mental disorders has been repeatedly emphasized ( 32,84,99-101,236,251,252). One of the most exciting problems in psychiatry is schizophrenia, the etiology, pathogenesis, diagnostics, treatment, prognosis, and prevention of which are still awaiting an adequate solution, Quite understandably, schizophrenia has been one of the main objects for

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HNA studies. HNA investigations have already delivered, and we may expect them to continue to deliver, data and results contributing to the explanation of all the problems in schizophrenia mentioned above, with the exception only of etiology. I f . Some Methodological Problems in HNA Studies

The investigator studying the activities of the central nervous system of the human being must keep in mind that the psychic processes are determined both cerebrally and socially ( 193). The task of the HNA is to guarantee an adequate adaptation of the individual in his biological and increasingly sophisticated social surroundings. Conditioning, i.e., elaboration of temporary nervous connections, is the most flexible tool of the organism wherewith to manage this task (16,17,40,131).There are a great number of types of temporary connections, all based on unconditioned reflexes, which together constitute the HNA (40,95,131,238). Recent studies have shown that sometimes temporary connections that have been just elaborated do not find their expression immediately in behavior ( 95,199). Understandably, all this brings about many complications in studying HNA in mental disorders, especially in schizophrenia. There are two ways to apply conditioning principles in mental diseases. One of them leads to physiological interpretation of clinical phenomena-symptoms, syndromes, and diseases-on the basis of general laws gained from animal experiments. This is the way started by Pavlov himself ( 131 ), and it provides investigators with basic hypotheses, stimulating further concrete studies. The other way is the experimental investigation of various levels and mechanisms of HNA in patients. This provides investigators with objective data, allowing them to analyze elementary forms of HNA in mental disorders (8,18-26,71-79,146,147,162-166,226,229235). Protopopov (146,147) and Rushkevic (162-166) have demonstrated the possibility of studying even the most complicated processes of thinking by means of conditioning methods. Butorin (39) and others have shown that these methods are very suitable for the investigation of emotional reactions in man. Wortis (258) has pointed out the fruitfulness of conditioning methods in the investigation of intellectual abilities of children. Consequently, the experimental investigation is regarded as the basic way to study the HNA in man, both in health and mental disorders (71-79,146,226). A great number of methods have been applied in studies of

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HNA in man, There are some exhaustive surveys of the methods suitable for HNA examination in mental disorders [the latest: (in Russian) Faddeyeva (55); (in English) Astrup (22,23) and Ban ( 2 9 ) l . It must be emphasized that, in connection with the leading role of the second signaling system in the HNA of man, an investigation of HNA in mental disorders cannot be regarded as satisfactory without dealing with the interaction of signaling systems and of the second signaling system with their connections to and from vegetative regulatory mechanisms. As far as each single method corresponds mainly to one nervous mechanism, the complexity of the investigation can be guaranteed only by simultaneously applying a number of different methods (55,109,221,222,230). Another important point is that suitable methods must be able to provide the investigator with data characterizing the basic nervous processes (excitatory and inhibitory, the latter in its various forms) and their interaction in particular nervous mechanisms ( 16-26,55,98-101, 103, 219-224,229-235). The elementary connecting activity of the second signaling system can be examined by means of the word learning test, the data from which are interpreted from the standpoint of conditioning theory ( 184-191). The association test is suitable for the examination of basic nervous processes in the established verbal connections, i.e., in the connections of the second signaling system, elaborated during the previous social experience, as it was demonstrated by IvanovSmolensky (71-73), A great number of supplements and additions to this method have been introduced (80,96,103-105,261). Zurabashvily (261) has elaborated a graduated system of signal words. Kreindler (103) has shown the significance of the basic structure into the framework of which the signal word belongs. Besides the mean reaction time and the quality of the answers the importance of the mean deviation (184-188) and the amplitude of the deviations of the reaction time (103) have been pointed out. In recent years electroencephalogram ( EEG ) recording has been carried out in the course of an association test ( 100,101,123). The widely applied motor reflex test mainly characterizes the activities of the first signaling system. Motor reactions, elaborated either on the ground of the avoidance reflex (146) or by means of verbal reinforcement (78), provide us with data on both excitatory and inhibitory processes in the first signaling system. Many investi-

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gators have demonstrated the imp0 rtant role of the second signaling system in the motor reflex test wit11 verbal reinforcement. In many cases the inhibition from the second signaling system brings about the suppression of a newly elaborated motor reflex, although the connection is there, as demonstrated in EEG and in verbal reports ( 66,143,169,230,256). Consequently, it has been recommended that one should give a previous verbal instruction to the person before starting the elaboration of a motor reflex by means of verbal reinforcement ( 182). The so-called “phenomena of elective irradiation,” proposed by Ivanov-Smolensky (71-76) and Vinogradov and Reisser (246) to be a sign of interaction of both signaling systems, has not proved to be adequate (22,151,152,169). It has been demonstrated, by means of motor reflex tests, that it is not possible to study correctly the so-called law of power, proposing a high correlation between the force of a signal and the reaction time and intensity of the response ( 153,166). A great number of amendments and variations to the method of motor reflex test have been proposed. A detailed graphical recording of motor reactions (63), counting the average deviation of reaction times ( 176,182), simultaneous recording of electromyogram (EMG ) ( 192) and EEG ( 100,101,149), in combination with the eyelid reflex method (154), elaboration of motor reflexes on verbal stimuli of different complexity ( 147,16%166,248), as well as variations in the conditions of elaborating of new motor reflexes ( 4,27,47-49,132,144, 150-1S2,213,248)-alI these measures contribute greatly to a more effective application of the motor reflex test to study the HNA in man, especially in mental disorders. The motor reflex method is an adequate tool for examining the most complicated forms of motor behavior of man as well as for detwting deviations in it in even Flight mental disturbances. Recent studies have been able to demonstrate the promising results as to the characteristics of the HNA in man as gained by means of investigation of the single sensory apparatus (93,197). The orienting reflex, especially its vegetative components, has been an object of many-sided studi’es for a long time, the results of which have been interpreted on tlie basis of conditioning theory. Although there are some recent papers expressing doubt about the conditioning character of some vegetative components ( 116,121, 145), the galvanic skin reflex (GSR), in particular, still engages

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investigators dealing with HNA studies in man. By means of combinations of GSR recording with other parameters, e.g., motor reactions, vascular, or EEG patterns, many interesting data have been gained ( 100,101,118,20~,24~,~3,252), characterizing some special features of the HNA especially in mental disorders. Some new details have been explained as to vascular reflexes (22,160), the conditioning of which can be elaborated on the ground of both temperature and pain stimuli, but with different characteristics ( $1,233,234,247). The role of verbal stimuli in vascular reflexes has been pointed out (116). Conditioning of cardiac (9,233,234) as well as breathing activities (65) has been demonstrated, but only applied to a small extent in HNA studies in mental disorders. Recent studies have dealt with the influence of some supplementary factors, such as intensity of light (214), noise (35), etc., upon various reactions in animals and man. Results of these investigations contribute to avoiding these disturbances in HNA experimental studies. The same can be said about the correct statistical processing of experimental data ( 114,115,182,184,187,192). A placebo control is necessary in HNA dynamic studies of the action of various therapeutic methods ( 138,182,18~-191,196). Although a great number of research projects have been carried out which contribute to methodology, many serious problems still await solution. The standards of most parameters, registered by means of methods discussed above, have not yet been worked out. Even the physiological essence of these parameters is interpreted by various investigators in different ways. It would be advisable to set up a standard battery of methods suitable for investigating the HNA in all its complexity. These tasks can be solved only on the ground of a wide-scale research program at an international level. Ill. H N A Alterations in Schizophrenia

On the basis of his vast experience in normal and abnormal HNA in animals, Pavlov ( 131) gave a general neurophysiological interpretation to various schizophrenic symptoms. He proposed that the basis of stupor, negativism, delusions, and some hebephrenic features in the behavior of schizophrenic patients is an external, transmarginal inhibition,' taking place in different cortical mechanisms. The transmarginal inhibition is the protective inhibition resulting from overstrain of nervous processes.

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Sometimes the inhibition tends to irradiation, in other cases it brings about an induced excitation in zonnected mechanisms. Pavlov pointed out that the external inhibition appears mostly in the form of hypnotic phases, e.g., in some negativistic features as a paradoxical phase. Later it was possible to demonstrate that some negativistic symptoms in verbal communication of schizophrenic patients can be regarded as an appearance of the ultraparadoxical phase ( 167). Co-workers of Pavlov have contributed to the pathophysiological interpretation of schizophreraic symptoms, especially of delusions, in which a combination of inert excitation and transmarginal inhibition plays a major role (238). By means of the experimental methods discussed above, a great number of investigations have been carried out, contributing to the explanation of more concrete deviations and disturbances of the HNA in schizophrenia. The experimental investigation of thinking processes has been carried out mainly by Protopopov (147), Rushkevic ( ISZlSS), and their co-workers. Making use of a special variation of the motor reflex test as well as of artificially created concepts (words), Rushkevic pointed out the weakening of analytic and synthetic processes in schizophrenia. Both excitatory and internal inhibitory processes are weakened, and at the same time an inertia of nervous processes2 is to be noted. Further investigatioiis have confirmed these findings (94,105,224). Some studies have shown that the ability of schizophrenics to operate with essential stimuli either in comparison or in classification processes is significantIy disturbed and the accidental stimuIi dominate (96,119,12(1,130,156). A number of characteristic deviations have been found in the activities of the established connections of the second signaling system, investigated by means of different variations of the association test. Reaction times are lengthened, while a considerable deviation of them takes place (22,~,52,~,71-73,76,161,172,177,182,191, 261-263). At the same time a higf percentage of answers of low quality is obtained (52,53,71-73,76,133,148,172,177,182,191,~0).In some cases the quality of answers depends on the complexity of the signal words ( 13,BJOq ; mostly the disturbances appear unexpectedly (22,36,37,130). The influence of a dominant i.e., pathoInertia of nervoiis processes indicates the impaired ability to reverse positive or negative reflexes or to change patterns of conditioned response.

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dynamic structure (76,78), as a rule, does not appear clearly (1822,86,111). The activities of the first signal system in schizophrenic patients have been widely investigated by means of different experimental methods. Alterations have been found even in the elementary activities of the sensory apparatus, mainly in the form of decreased analytic and adaptation ability (68,93,197). In elementary motor activities, examined mainly by means of motor reflex tests, a great number of deviations and disturbances have been found. In many schizophrenics even what should be an established motor reaction, i.e., a movement upon corresponding command, can be absent ( 59,98,99,165,166,169,182,183).Elaboration of a new conditioned motor reflex is difficult and in some patients even impossible, and if elaborated, the reflexes are highly unstable (3,22,23,33,34,48,59,7~78,82,8~87,98,99,124,155-157,169, 182,183,191,195). The intensity and dynamics, but especially the reaction time of motor reflexes are markedly variable, the average reaction time being significantIy longer than in normals (22,23,42, 59,76,78,98,148,lsS,182,189,190,191,194,195,~8,2~). The correlation between the strength of the signal and the power of the motor response, i.e., the “law of power,” is disturbed (59,76,78,97,98),as well as the dependance of the reaction time on the timing of the stimulus with respect to the cu-rhythm ( 122,149). Schizophrenics manifest great difficulty in the elaboration of differentiation, conditioned, and other forms of internal inhibition in the course of the motor reflex test (33,34,36,50,51,67,82,92,118, 155-157,172,182,18~,191,208 ). The avoidance motor reflex appears irregularly in many schizophrenics; in some patients it is absent. To elaborate a conditioned motor reflex on this basis is quite difficult and, if elaborated, it is highly unstable (22,23,108,146). In the course of the motor reflex test, marked disturbances in the interaction of the first and second signaling systems have been found in schizophrenic patients. Many authors have pointed out the instability or even absence of the so-called elective irradiation of a new temporary connection in schizophrenics ( 2,33,34,98,246). It must be noted that this sign cannot be regarded as a characteristic one, since it appears even in a great number of normal persons (22, 23,169). Nevertheless, many other signs testify to serious deviations in the interaction of signaling systems in schizophrenia, e.g. absence

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of motor response on verbal command, deficient and inadequate verbal reports about the motor reflex test, deviations in the test response as a result of complex elective irradiation3 ( 22,23,90,91,169, 182,191,230,232). Furthermore, the insufficient generalization of the experience in the first signaling system, which becomes evident as an inertness of deviation and by disturbances during a number of investigations, can be regarded as a characteristic sign of the impaired interaction of two signaling systems (36,37,208). An examination of the orienting reflex shows that similar disturbances are present in the interaction of signaling systems and vegetative functions. As a rule, the vegetative component of the orienting reflex in schizophrenics is markedly weakened as compared with normals. This finding is evident in experiments with either nonverbal or verbal stimuli ( 22,23,64,99,107,159,171,180,191, 209,218). Both GSR and pupillary components are weak in most patients; in some patients an inertia of vegetative reactions can be noted (30,171,180,191,215,216,222,223,2~5). If a number of vegetative functions are recorded simultaneously, a marked dissociation in their reactions is found ( 100,101,181,18.2,191). Special investigations dealing with vegetative functions by means of simultaneous recording of a number of parameters (heart rate, pneumogram, electrical resistance of the skin, plethysmography, etc.) have been able to demonstrate a marked dissociation even in interaction of various vegetative activities. The correlation between the breathing and electrical resistance of the skin is disturbed (99-101), as well as correlation between breathing and heart rate ( i.e., respiratory arrhythmia) ( 174,181,182,189-191 ) and hetween cardiac and vascular activities ( 200). IV. Special Features of the HNA in Various Forms and Stages of Schizophrenia

Besides general deviations in the HNA of schizophrenic patients a great number of experimental investigations have been devoted to the examination of special disturbances in various forms and stages of schizophrenia. In paranoid and paranoid-hallucinatory forms of schizophrenia a Complex elective irradiation indicates the following: On both nonverbal and corresponding verbal signals a positive motor reflex is elaborated; then on rwersal of either the nonverbal or verbal signal to the negative one by means of verbal command, the other signal will be reversed without any command.

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transmarginal inhibition, as a rule, is of less intensity as compared with other forms. It appears episodically, in connection with the negative induction from an inert focus of excitation (14,15,59,7076,98,155-157,172,182,191,207). Internal inhibition is impaired, but not very severely ( 98,143,144,172,182,183,189,207).Only slight disturbances are found in the interaction of signaling systems (85,86). The most severe transmarginal inhibition of the HNA is met with in the catatonic form of schizophrenia. Fairly often even unconditioned reflexes as well as elementary responses on verbal commands are either absent or very causal and weak. It is difficult or even impossible to elaborate experimental conditioned reflexes; if present, they are extremely unstable ( 22,23,59,71-76,146,148,162166,172,182,183). Internal inhibition, especialIy differentia1 inhibition, is very underdeveloped and in addition a marked inertia of nervous processes is noted (71-76,98,202,259). In the interaction of two signaling systems severe disturbances have been pointed out (46,48,98). The intensity of the transmarginal inhibition is quite uniform during a long period of time, embracing mainly the cortical and subcortical motor structures, whereas the vegetative functions in some patients are less inhibited ( 173,176,182,218). In hebephrenic and simple schizophrenia some special features only have been mentioned on the ground of experimental investigations. In hebephrenic patients it is mainly the higher functions of the second signaling system that underlie the transmarginal inhibition, whereas more primitive functions often are disinhibited ( 128). In some hebephrenics vegetative orienting responses, especially the GSR, are even more weakened than in catatonic patients ( 218). In simple schizophrenia a stable, inert, transmarginal inhibition of the HNA is noted (3,237,263). The stimulating effect of verbal signals on the cortical activities is quite small (99). Many investigators have dealt with the application of experimental HNA data in the differential diagnosis of various forms of schizophrenia. Astrup (22,26) has pointed out that in catatonic and hebephrenic schizophrenia a fluctuating external inhibition, and in paranoid schizophrenia the inhibition of unconditioned reflexes, are the most characteristic disturbances. In patients with prevailing affective symptoms deviations in the interaction of cortical and vegetative mechanisms are reported ( 22,26,54,205,220-222). Tatarenko (220-222) has found the pupillary component of the orienting reffex to be less impaired in catatonics and hebephrenics than in other

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forms. Lehmann et al. (110), by means of a special study, were able to note a difference between various forms of schizophrenia only in the elaboration and appearance of the conditioned reflexes: in paranoid patients the disturbances were significantly less than in other forms. All other experimental parameters did not demonstrate any significant differences. Rushkevic (166) as well came to the conclusion that there is no marked correlation between the disturbances of thinking processes and clinical forms of schizophrenia. Gindis (63) carried out a special investigation by means of motor reflex tests to examine the differences between various forms of schizophrenia. He could point out many special features in almost all parameters, such as dynamics of motor responses, reaction time, stability of reflexes, interaction of signaling systems, and interaction of cortical and vegetative functions. He found the most severe external inhibition of cortical functions in catatonics, whereas in hebephrenics and simple schizophrenics there were only some characteristic deviations in vegetative functions and their reactivity. In summary it must be emphasized that only one or two special investigations have been carried out with the aim of examining the possibilities for differential diagnosis of special features in the HNA of different patients. Consequently, the correlations between the deviations in the HNA and clinical forms still await further investigations. Of course, lack of unified clinical criteria as to diagnostics of schizophrenia and its forms brings about serious difficulties in this work. On the other hand, there is hope that the experimental data on HNA could contribute to unifying the diagnostic criteria to a marked degree. Some pronounced differences in the disturbances of the HNA have been described in different stages of schizophrenia. The comparison has been mainly between acute and chronic schizophrenia. In the acute type, marked weakening of the excitatory and internal inhibitory processes, as well as diminishing of the mobility of nervA ous processes have been reported (33,34,50,Sl,SZ,l05,239). marked fluctuation and changeability of the intensity of external inhibition of the HNA and of the deviations of the internal inhibition are also typical. At the same time vegetative functions as well as subcortical motor mechanisms are not inhibited; in most cases they are even disinhibitcd, whereas the main disturbances appear in cortical activities (200). In chronic schizophrenics the intensity of external inhibition

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is lesser, but it shows a pronounced inertia. There is also a marked disturbance of the internal inhibition (33,34,36,37,90,91,106,130,239, 254,255). Deviations in the activities of the first signaling system in chronic stages are less than in acute schizophrenia (197). Inert external inhibition of moderate intensity covers both cortical and subcortical mechanisms (200). In some investigations the dynamics of the disturbances of the HNA in the course of the schizophrenic process have been examined. It has been reported that the intensity of the external inhibition diminishes, and becomes more uniform and inert ( 182,189, 190,221,222). The mobility of basic nervous processes in the course of the disease steadily decreases, and both excitatory and inhibitory processes become more and more inert (92,143,144,166,182,190). Although up to now the experimental data on HNA have not been applied in evaluating the actual stage of the schizophrenic process, there are some promising results in this direction. The task of further investigations is to devise special experimental studies to find out objective criteria for characterizing the different stages and the intensity of the disease process. These results would contribute to the more effective treatment of schizophrenic patients. V. Changes of the HNA in Schizophrenia under Treatment

From the very beginning HNA studies in psychoses, especially in schizophrenia, were associated with the examination of the effect of various therapeutic measures. These investigations have contributed to a better understanding of the mode of action of diEferent methods of treatment as well as of the ways in which the improvement is gained. One of the first and major objects for those studies has been insulin treatment, which at that time had not lost the place in the therapy of schizophrenia to the degree it has now (38,56,168,175, 178,182,200). Characteristic shifts in the HNA of schizophrenics under insulin hypoglycemia have been examined in detail (5-7,10, 112). A gradual deepening, as well as irradiation, of the transmarginal inhibition, first in the HNA and then also in the vegetative functions, has been described during deepening hypoglycemia, as well as the decrease and disappearance of the inhibition following the return of the blood sugar levels to normal. No certain conclusions as to the future effect of an insulin course could be drawn

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from the individual deviations in the shifts of HNA under insulin hypoglycemia. During a course of insulin treatment in most schizophrenic patients the disturbances of the HNA diminish as a step function. Both the intensity and extensity of the transmarginal inhibition decline, excitatory process become more and more adequate and stable, and elaboration of conditioned reflexes is less and less difficult ( 43,44,50,51,98,113,168,176,182,195,229,230).Various forms of internal inhibition improve, although it takes place later than other shifts in the HNA (43,44,168,182,210,245). In the verbal activities the quality of the responses improves first; subsequently the latency periods shorten ( 158,182,229,230). The vegetative component of orienting and avoidance responses becomes more and more intense and adequate, and in other patients its inertia decreases (11,12,107, 113,134,171,180,182,220). Some investigators have stated there is an obvious correspondence between clinical improvement and shifts in the HNA under insulin treatment (43,44,82,113,127,24A5). At the same time it has been pointed out that in many patients there is no parallel between the clinical and experimental changes (31,50,51,158), In those patients who are clinically improved but who still show marked disturbances in experimental performances, a relapse usually takes place quite soon after cessation of treatment ( 31,136,137,158,179, 182,185,186). On the other hand, in patients with improved HNA clinical recovery, as a rule, is stable and sometimes even continues after the end of insulin course ( 136,137,179,182,185,186). In most schizophrenic patients with a good and stabIe clinical recovery, some deviations in the HNA experimental data can still be found, mostly in the sophisticated activities of the second signaling system as well as in the most difficult forms of internal inhibition ( 11,12,46-48,98,182,186,230). Although those slight deviations do not become manifest in the everyday behavior of patients, it is useful to keep them in mind when administering the work and life regimen in order to avoid difficulties in adaptation. In the last decade a great number of investigations have been carried out on the influence of various psychotropic drugs in schizophrenia, including those using conditioning methods. The major object of research has been chlorpromazine (in the Soviet literature the name “aminazine” is used). It has been pointed out that under chlorpromazine a marked

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external inhibition of unconditioned reflexes takes place, whereas conditioned reflexes and cortical activities in general do not suffer from inhibition, with the exception of the motor reflexes (2S24, 233-235). The excitatory process tends to irradiation to a smaller extent than before treatment with chlorpromazine, and internal inhibition improves (22-24,189-191,235). Ivanov-Smolensky ( 78 ) has reported an inhibition and diminishing of the cortical activities under chlorpromazine, thus contributing to defensive tendencies in the brain cortex, Astrup (22-24), on the other hand, regards inhibition of subcortical structures as the most important feature in the therapeutic action of chlorpromazine in schizophrenia. The main point of action of chlorpromazine as to the HNA is still under discussion. Some authors suppose that the first and foremost action of chlorpromazine is the inhibition of cortical activities (1,39, 47). Other investigators postulate that the inhibition of subcortical and vegetative functions is the main effect ( ~2-~4,1OO,lOl,B4,235, 253). Recent experimental data in animals have demonstrated the dependence of the particular action on the dose of chlorpromazine: even the smallest dose brings about an inhibition in the cortical activities (1).Some EEG studies in schizophrenics also testify to the presence of cortical inhibition in the action of chlorpromazine (123). Comparative studies have shown marked differences in the action of chlorpromazine, if administered in a single dose or repeatedly. Under a single dose both excitatory and internal inhibitory processes in the HNA of schizophrenics weaken, whereas following a course of drug therapy the opposite takes place (189-191). In particular the reaction times become markedly shorter and more standard (42,189-191), which is quite different from the action of a single dose. As to the vegetative functions, the action of chlorpromazine can be interpreted as a shift toward vagotonia (212), although it produces a rise in heart rate. Under reserpine a marked external inhibition in both cortical and subcortical structures has been reported (2224,117). Inhibition of unconditioned reflexes clearly predominates ( 22-24) and this inhibition can be irradiated even to the vegetative conditioning mechanisms ( 227). HaloperidoI also causes a marked inhibition in the HNA in schizophrenics, especially in motor activities (88). It must be

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pointed out that this inhibitory effect is seen only following a single dose of haloperidol, whereas during ;i treatment course just the opposite change is found ( ls%191). Improvement of the internal inhibition appears under both single dose and chronic administration (189-191). So, as in the experiments with chlorpromazine, it is not possible to predict the action of a course of halperidol on the basis of changes produced in the HNA by a single dose. In contrast, trifluoperazine improves the cortical excitatory process in both a single dose and following a course (189-191). Following a course of trifluoperazine internal inhibition also becomes more adequate. The main emphasis in the general action of neuroleptic drugs upon the HNA of schizophrenic patients may be put on the inhibitory effect in avoidance reactions and on the emotionalvegetative components of responses (22-24). But one has to consider that the basic condition of the HNA plays an important role in establishing the effect of a drug (143,1441, as well as the specific features in the action of different neuroleptics (189-191). Besides neuroleptics some other drugs have been studied for their action on the HNA in schizophrenia. For instance, it has been shown that imipramine administered in schizophrenics with depressive features occasionally brings about a stimulating effect on unconditioned reflexes and secondarily also on cortical activities (234, 235). Adrenocorticotropic hormone ( ACTH ) in animals elicits an improvement of the cortical excitatory process, but in schizophrenics it brings about an intensification of transmarginal inhibition, especially in the second signaling system ancl also impairment of the internal inhibition ( 157). It must be pointed out that the results of investigations dealing with the action of various psychotropic drugs on the HNA in schizophrenia are quite difficult to interpret because only a few studies have been carried out using proper placebo control and with contemporary data processing (22-24,18%191,257). Furthermore, research in this field has to be methodologically scrupulous, otherwise it cannot contribute effectively to the solution of problems in schizophrenia.

VI.

Some Theoretical a n d Practical Conclusions

As a result of persevering and systematic research work by numerous investigators, much factual material has been collected

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on the characteristic deviations and disturbances of the HNA in schizophrenia. As a result, it is possible to arrive at some theoretical and practical conclusions. First of all some general considerations about the pathogenetic structure of schizophrenia from the point of view of the HNA concept. Pavlov (131) has emphasized the leading role of external, transmarginal inhibition of the HNA in schizophrenia. I n connection with the partly superficial and irregularly spread-out inhibition, he has made use of the term “chronic hypnoid state,” thus characterizing the main essence of the disturbances of the HNA in schizophrenia. Protopopov ( 146) has introduced the term ‘Xypnoid syndrome” in the same sense, All experimental investigations on the HNA in schizophrenia have demonstrated that transmarginal inhibition in the form of various hypnotic phases is the most general and characteristic feature in the HNA of these patients. Differences in the intensity and extensity of the external inhibition can be regarded as a neurophysiological background of various syndromes, clinical varieties, and stages of schizophrenic process ( 2224,104, 119,127,139-142,166,182,191,207222,263) , Pavlov and his co-workers have emphasized that the external inhibition of the HNA has an obvious defense role, while avoiding serious disturbances in nervous cells under extreme conditions. Many investigators have shown that in a number of schizophrenics the defensive, protective character of external inhibition is insufficiently elaborated. Therefore it does not protect the nervous system from further damage and schizophrenic deterioration occurs ( 141, 142,146,221,222). Besides external inhibition an inert excitation in certain cortical structures, as well as an insufficiency of internal inhibition, has been regarded as the background mechanisms for some schizophrenic symptoms, e.g., for delusional ideas, stereotypes, etc. ( 76,78,98,155157,204,238). The correlation between the disturbances of the HNA and deviations in vegetative functions have been under discussion for a long time. Some investigators regard the vegetative dysfunctions as a primary disturbance in schizophrenia ( .22,23,61,62,69,1S). Disturbances in the cortical activities would then be of a secondary character, developing on the basis of vegetative deviations. Other authors are of the opinion that cortical disturbances have a primary character and that the external inhibition in the higher nervous mechanisms secondarily brings about various deviations in the

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vegetative functions ( 76,78,197,198,200,221,222 ) , A special correlation analysis by means of a computer, processing a great number of parameters characterizing the activities of two signaling systems, their interactions, vegetative functions, and vegetative reactivity has shown that a central position among all disturbances is OCCUpied by those in the second signaling system (176,182). Some recent data on the electrical activity of the brain of schizophrenics also seem to support the concept of a leading role for cortical disturbances in the pathogenesis of schizophrenia ( 122). The problem of what is the basis of the weakness of cortical mechanisms in schizophrenic patients and which are the factors causing the profound disturbances in their HNA cannot be answered on the basis of data collected by HNA studies. Probably neurochemistry will provide the solution to these problems ( 129,241) . In all experimental investigations on the HNA in schizophrenia a great variety of disturbances have been reported and on this basis many attempts have been made to work out some objective criteria for purposes of differential diagnosis among the different clinical forms and stages of schizophrenia (2%24,S4,176,182,191,222,223). Until now it has been possible to demonstrate those differences only on a statistical level, but not to make use of them in individual cases. Systematic research in this direction will probably provide us with more useful results. The situation is almost the same in the case of the differential diagnosis of schizophrenia as a nosological entity, A number of investigators have elucidated characteristic disturbances in the HNA in many other mental disorders: in involutional and other depressions (39,188), in manic-depressive psychosis ( 166), in organic psychoses ( 166), and in psychopaths (201). Although these studies, too, demonstrate the major role of hypnotic phases and of the dissociation between various mechanisms of the HNA in schizophrenia (Z~,110,182,200,204,2~1), it has only a general value at statistical level. Until now no diagnostic model of HNA parameters has been elaborated for schizophrenia, nor for other mental diseases. Results gained are already quite promising and must stimulate further research in this direction. Without doubt, the parameters of the HNA must be closely combined with clinical, electrophysiological and biochemical studies, for only then can we hope to achieve real diagnostic models for practical purposes. The importance of the latter is evident. One field in which the experimental data on HNA may con-

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tribute to practical clinical psychiatry is in evaluating the actual level of improvement reached by means of therapy. Many investigators have pointed out that the HNA parameters tested in experiments are more sensitive indications of therapeutic improvement than is ordinary psychopathological examination ( 7,57,58,89,168,170, 249). Most papers report a clear correlation between clinical recovery and changes in the HNA (31,43,44,82,113,127,158,245). Other authors emphasize the possibility that a difference between these two signs can take place (48,100,101,179,191,23~).In these cases the condition of the HNA, as obtained from experimental data, is usually better able to characterize the real quality of the improvement. These findings make it desirable to investigate more fully the possibilities of applying HNA data in the clinical evaluation of the effect of treatment of schizophrenic patients. Such a preliminary investigation has been carried out recently in Tartu in connection with insulin, chlorpromazine, haloperidol, and trifluoperazine therapy ( 179,182,186,189-191). By means of a test battery 32 parameters characterizing various levels of the HNA and 11 parameters of vegetative functions were obtained in a sample of 224 schizophrenics with various forms and in different stages of the illness. Correlations between clinical recovery and changes in the HNA experimental data after a treatment course were calculated. The general results are given in Table I. It is interesting to note that the degree of restoration to normal of both excitatory and the internal inhibitory processes in various mechanisms of the HNA do not have an equal value for evaluating the actual quality of recovery under different methods of treatment. These differences would appear to be due to differences in the mechanisms of action of particular types of therapy. Although these results are so far only tentative, they seem to contribute to a certain extent to a better evaluation of the real level, as well as the further stability, of remission obtained. This will in its turn exercise a considerable influence on the subsequent fate of the patients, since it will be possible to work out a working and living regimen and to prescribe supportive therapy on a more rational basis, thus ensuring the prevention of relapses. Determination of the prognosis in schizophrenia before starting treatment and in relation to certain methods of therapy is an even more important aspect of the practical utilization of the data from

TABLE I CRITERIAFOR EVALUATING DEGREE OF RECOVERY IN SCH~ZOPHRENIA ON THE BASISOF HNA DATA Degree of recovery following treatment with Condition of the mechanisms of the HNA Connecting activity of second signaling system Excitation restored Internal inhibition restored Established connect,ions of second signaling system Excitation rest,ored Internal inhibhion restored Interaction (Jf signaling systems Excit.ation restored Internal inhibition restored Mechanisms of first signaling system Excit,ation restored Internal inhibition restored Vegetative functions Reduction in sympathetic tone Reduc.t,ioriin sympathetic react,ivitp

Insulin

Good

Chlorpromazine

Trifluoperazine

Haloperidol

Good Good

Good Good

Good

Good

Good Good

Good

Good

Good -

~

Good

-

-

Good Good

Good Good Urlfavorable

-

Good -

Unfavorable Good

Unfavorable -

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investigation of the HNA. HNA experimental data characterizing special pathogenetic and pathophysiological features in individual patients can also be useful in considering prognosis. There have been some attempts to make use of HNA experimental data in prognostic prediction as to therapy. IvanovSmolensky, on the basis of the condition of the HNA, has suggested new indications for sleep therapy in schizophrenics. Some general considerations as to prognostic value of the HNA data are pointed out in other papers (15,211). Astrup has carried out a special analysis (26) of prognostic prediction in schizophrenia on the basis of experimental HNA data and has reported promising results in this field. In view of the large and constantly increasing number of therapeutic agents in psychiatry, the development of objective differentiated prognostic criteria is taking on an ever greater importance for clinical practice. In the psychiatric clinic at Tartu the prognostic value of experimental data on the HNA obtained before starting treatment was calculated in the sample quoted above, with regard to insulin, chlorpromazine, trifluoperazine, and haloperidol therapy. Not only the immediate stage of recovery but also the stability of the remission over some years have been taken into account in estimating the prognostic significance of different variables of HNA (177,178, 182,187,189-191). The most general results are given in Table 11. As will be seen from the findings quoted, the functional condition of the basic nervous processes in different cortical mechanisms as well as of vegetative functions, observed before the beginning of treatment, can yield not only general prognostic information, but its significance differs depending on the method of therapy. These differences are due to different pathogenetic mechanisms of the disease in individual cases as well as to different mechanisms of action of various methods of treatment. These data not only make it possible to forecast more accurately the effect of treatment in general but in particular the effect of a particular method, Though the results quoted above are provisional, there can be no doubt that further systematic research in this field will open up wide prospects for the application of the therapeutic agents available in contemporary psychiatry on the basis of the pathogenesis and thus they can be used more effectively. In order to determine whether a certain method of treatment is effective for an individual patient, the development of accurate, differentiated, prognostic

TABLE I1 PROGNOSIS IN SCHIZOPHRENIA, BASEDON EXPERIMENTAL HNA DATA, WITH VARIOUS METHODSOF TREATMENTS Prognosis after treatment with Condition of the mechanisms of the HNA (before treatment) Connecting activity of second signaling system Intensive transmarginal inhibition Internal inhibition severely disturbed Established connections of second signaling system Intensive transmarginal inhibition Internal inhibition severely disturbed Interaction of signaling systems Intensive transmarginal inhibition Internal inhibition severely disturbed Mechanisms of first signaling system Intensive transmarginal inhibition Internal inhibition severely disturbed Vegetative functions Prevailing sympathetic tone Intensive sympathetic reactivity

Insulin

Chlorpromazine

Trifluoperazine

Haloperidol

-

Unfavorable

Unfavorable

-

Unfavorable Favorable

-

-

Unfavorable

-

Favorable Favorable

Unfavorable

-

Favorable Favorable Unfavorable

-

-

Unfavorable

-

-

-

-

Unfavorable -

-

Unfavorable

Favorable Favorable

-

-

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criteria is of highest importance. The task of further investigation is to work out such criteria on the ground of all the parameters available. As may be concluded from the above discussion the experimental data on the HNA may contribute to this aim. VII. Summary

Investigations on the HNA in schizophrenia have contributed to a better understanding of the pathogenetic and pathophysiological mechanisms involved in this severe mental disease and in its various forms and stages. Besides its theoretical interest, certain practical results of value have been demonstrated as to prognostic prediction and to evaluation of recovery. Further lines of research on the HNA in schizophrenia are suggested.

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CARBOHYDRATE METABOLISM IN SCHIZ 0PHRENIA By Per S. Lingjaerde Deparlment of Clinicol Chemistry, Akershus Central Hospital, Nordbyhagen, Norway

Introduction . . . . . . . . . . Glucose Tolerance Tests . . . . . . . Insulin Tolerance Tests . . . . . . . Lactate, Pyruvate, and Citric Acid Cycle Intermediates . Brain Metabolism . . . . . . . . Enzymes . . . . . . . . . . Red Cell Metabolism . . . . . . . . A. Normal Human Red Cell Metabolism . . . . B. Red Cell Metabolismin Schizophrenia . . . . VIII. Serum Factors and Carbohydrate Metabolism . . . IX. Concluding Remarks . . . . . . . . References . . . . . . . . . . I. 11. 111. IV. V. VI. VII.

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I. Introduction

Is schizophrenia a metabolic disorder? Is it a disorder of carbohydrate metabolism, amino acid metabolism, catecholamine metabolism, serotonin metabolism, or is it an autoimmune disease? These and similar questions have been raised over and over again, and numerous people have tried to give the answer; some even claimed they knew it. Exciting reports have appeared at intervals, but attempts to confirm the findings have usually failed. Lack of solid and properly controlled experimental work and sound biochemical judgment are not too infrequently observed in investigations of mental diseases. In many ways this type of research has been characterized by too much speculation and too little experimental verification. This review is restricted to carbohydrate metabolism, including the effects of the schizophrenic serum factor. Studies on carbohydrate metabolism in schizophrenia started many years ago, and has evolved from simple measurements of fasting blood sugar, through glucose and insulin tolerance tests, determinations of various carbohydrate intermediates in blood, radioisotope studies of energy metabolism and phosphate compounds in red cells, into the present259

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day studies of the effects of the plasma factor on metabolic parameters. The results presented so far are inconclusive and partly contradictory, and the key to the etiology of schizophrenia still seems well hidden. If disturbances in carbohydrate metabolism are involved in the etiology of schizophrenia, they are probably of a small order of magnitude or localized to peripheral metabolic pathways. One can hardly visualize a complete block in any of the major metabolic pathways that would only affect some mental functions. Diabetes, galactosemia, and the glycogen storage diseases, which are examples of major disturbances in carbohydrate metabolism, have much more serious physical consequences than are observed in schizophrenia. From a purely medical point of view the schizophrenic patient is not a sick person, and whether treated or untreated his condition is no direct threat to his life. The cause of schizophrenia may possibly be a disturbance of some finer regulating mechanisms of the organism? for instance? the delicately tuned interplay of glycolysis and gluconeogenesis, of insulin and glucocorticoids, etc. If disturbances of this kind are the key to the problem, one should look for a certain combination of small biochemical abnormalities, which per se may not be characteristic of schizophrenia-not even of mental diseases. The genetic studies of Karlsson (1966) led to the following conclusion: Two independent gene controlled personality characteristics are envisioned, one associated with a thought deviation, which can be demonstrated by certain psychologic tests, the other possibly associated with a state of nervous tension. The thought deviation is detemiined by the “dominant” gene and occurs in one person out of fifteen. The other condition results from the recessive gene which is homozygous in one person out of six. When both exist in the same individual, as would occur in 1 per cent of all persons, an incompatible situation arises, usually leading to schizophrenia.

One more factor must be considered: the compensating mechanisms of the organism. Thus, after completing his experimental work the researcher is left with the difficult task of deciphering what is related to the postulated genetic factors, which are the compensating mechanisms and what are of no importance for the condition under investigation. Several minor abnormalities of carbohydrate metabolism have

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been reported in schizophrenic patients. Statistical comparison of patient and normal control groups has invariably been necessary in order to prove the difference. Marked overlap of values is a frequent finding, and the etiological relationship of these abnormalities to schizophrenia is obscure. However, they should not be discarded for that reason-unless a questionable or unreliable technique has been used-but be kept in mind as possible bits of the great puzzle. II. Glucose Tolerance Tests

Glucose tolerance tests have been performed in schizophrenic patients by many people; the work up to the last war w7as reviewed by Holmgren and Wohlfahrt ( 1944). With regard to schizophrenia they concluded that most authors have reported a pathological glucose tolerance curve in the acute state of the disease, most pronounced in catatonic unrest. Shattock (1950) found a low curve in most schizophrenic patients, but a few showed a persistent tendency to sustained hyperglycernia. She further demonstrated that it was possible to modify the shape of the curve by varying the speed of sugar ingestion in direct duodenal feeding. R4eduna (1950) claimed that a diabetic type of reaction to the Exton-Rose two-dose glucose tolerance test was characteristic of a syndrome he has named “oneirophrenia,” but Mayer-Gross ( 1952) was unahle to confirm this. A modified Exton-Rose test was also performed by Simon and Garvey (1951), and about two thirds of the schizophrenics and most of the senile psychotics had an abnormal diabetic-like response. Henneman et al. (1954a) concluded their studies on a mixed population of schizophrenic, manic-depressive, and patients with involutional psychosis as follows: Changes observed in the patients after the ingestion of 100 gni of glucose overlap those found in the normal subjects, but there are significant differences in the changes in all blood constituent7 measrired: in the psychotic patients there is a lag in the return to the fasting level of the trrie blood glucose, an excessive elevation of lactic and pyruvic acids, a rise rather than a fall in citric and a-ketoglutaric acids, and an increased and prolonged fall in inorganic phosphate. Clinical status can be correlated with biochemical abnormality: patients with psychosis of recent onset manifest at least three and frequently all of the abnormalities, whereas those with chronic psychosis fail to exhibit the abnormal changes in blood lactic acid and inorganic phosphate but usually show the citric and a-ketoglutaric acid changes to an exaggerated degree. Remission following treatment is correlated with Iiinclremical changes toward the normal values.

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The meaning of the abnormal curves in mental patients is di5cult to evaluate. It may indicate a disturbance in the intestinal absorption of glucose. Shattock‘s observation with direct duodenal feeding points in this direction, The question would probably have been answered if the conventional oral glucose tolerance test had been compared with the intravenous test. On the other hand, a disturbance in the liver function, or more specifically the normal glycogenesis, could just as we11 explain the findings. A third possibility is the presence of an anti-insulin factor in the blood. Ill. Insulin Tolerance Tests

Meduna et al. (1942) and Meduna and McCulloch (1945) claimed that the group of schizophrenics designated by them as “oneirophrenia” was characterized not only by a decreased glucose tolerance, but also by insulin resistance and the presence in the blood of an anti-insulin factor. However, Goldner and Ricketts (1942) could not confirm the finding of an anti-insulin factor. Insulin resistance is nevertheless a frequent finding in schizophrenic patients, but the claim that this is characteristic of oneirophrenia has not been confirmed (Harris, 1942; Freeman et al. 1943; Freeman, 1946; Langfeldt, 1953; 0. Lingjaerde, 1953). 0. Lingjaerde (1953) has pointed out that the most pronounced finding with insulin tolerance tests in schizophrenic patients is a prolonged hypoglycemia (“hypoglycemia unresponsiveness”), and this was observed as frequently in nonschizophrenic as in schizophrenic patients. If the patients were supplied with an additional amount of carbohydrate, a normal insulin tolerance curve was always obtained, but the time required for normalization varied greatly. In some patients the extra supply of carbohydrates had to be continued for several weeks ( 0. Lingjaerde, 1964). The finding that hypoglycemia unresponsiveness is normaIized if patients are supplied with additional carbohydrate does not necessarily mean that undernourishment is the single and simple explanation of this phenomenon in mental patients. The findings of 0. Walaas and 0. Lingjaerde (to be discussed later) that plasma from some psychotic patients markedly inhibits glucose uptake in isolated rat diaphragm, has been restricted to those patients who responded to an insulin tolerance test with prolonged hypoglycemia. This observation points to a more fundamental disturbance than mere undernourishment.

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A delayed maximum fall in blood sugar after subcutaneous injection of insulin in schizophrenic patients was observed by Nadeau and Rouleau ( 1953). This abnormality disappeared, however, when a modified test-intravenous injection of glucose followed by intravenous insulin 30 minutes later-was performed in the same patients. P. S. Lingjaerde and Skaug (1959) carried out insulin tolerance tests in chronic schizophrenic patients, and determined the blood sugar values both with the Hagedorn-Jensen and the anthrone methods. The maximum blood sugar fall after insulin injection was significantly less in the patients than in normal controls with both methods. After 120 minutes the mean blood sugar value was lower in the patients than in the controls, although this difference was significant only with the Hagedorn-Jensen method. Bergsman (1959) stated that no definite differences were noted between the various psychotic groups or between these and healthy subjects. His tables, however, show that the 30-minute fall was much less in acute schizophrenics and patients with endogenous depression than in chronic schizophrenics and normal controls. Plasma hydrocortisone was also included in the study of P. S. Lingjaerde and Skaug. In normal fasting subjects the hydrocortisone response to a standard insulin tolerance test was as follows: a slight decrease (insignificant) 30 minutes after insulin injection, a significant increase after 60 minutes, and a progressive and significant fall in the course of the next 120 minutes (Skaug and Lingjaerde, 1960). This response of hydrocortisone to insulin was less in chronic schizophrenic patients. The hydrocortisone curve followed the same pattern as observed in normal controls, but the fluctuations were statistically insignificant (P. S. Lingjaerde and Skaug, 1959). Farstad and Skaug ( 1966) observed that prolonged insulin-induced hypoglycemia coincided with increased fasting blood sugar and free fatty acids, and with an insignificant increase in plasma hydrocortisone. Patients with increased fasting hydrocortisone values ( above 25 pg/lOO ml) had increased fasting blood sugar and free fatty acid values, a lack of adrenal response to insulin, and prolonged hypoglycemia after insulin injection. These authors further reported that a sign8cant correlation was found between fasting blood sugar levels and plasma hydrocortisone values, but in my own study of plasma hydrocortisone in a larger patient material there was no significant correlation between these two parameters (P. S. Lingjaerde, 1964).

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IV. lactate, Pyruvate, and Citric Acid Cycle Intermediates

As early as 1934 Looney and Childs demonstrated a significantly higher blood lactate level in schizophrenic than in normal men. Lactate and pyruvate are known to accumulate in the blood following bodily exercise. Easterday et al. (1952) found that this increase was significantly higher in schizophrenics than in normals. Altschule and his group (Henneman et al., 1959a) reported that fasting whole blood lactate was higher in patients than in normals, whereas the mean values for pyruvate were identical in the two groups. Following a glucose tolerance test, an excessive and prolonged rise in whole blood lactate was observed in the patients. This was observed almost exclusively in patients with psychosis of recent onset, whereas only 1 out of 7 patients with chronic psychosis had an elevated bIood lactate level after glucose administration. A favorable response to treatment was associated with the exhibition of a normal change in blood lactate after ingestion of glucose. Similar changes were observed in blood pyruvate concentrations, although there was a considerable overlap with the normal values. Oral and intravenous administration of fructose produced the same rise in blood lactate and pyruvate in normal and psychotic individuals (Henneman et al., 1954b). If hydrocortisone or ACTH was administered to chronic patients 3 days prior to a glucose tolerance test, the changes in lactate and pyruvate were similar to those commonly found in patients with psychoses of recent onset (Henneman et al., 1955). A statistically slower disappearance of intravenously injected sodium-d-lactate was observed in patients with schizophrenia or manic-depressive psychosis as compared to the normal controls. This disturbance was qualitatively similar to one found in many somatic diseases (Altschule rt al., 1956). If large amounts of glutathione were administered intravenously before the glucose tolerance test, the curves €or lactate and pyruvate became almost normal (Altschule et al., 1957). Administration of large doses of a-lipoic acid caused impairment of the lactate and pyruvate utilization in 5 of 6 chronic schizophrenics. This impairment accompanied clinical worsening (Altschule ct a!., 1959). When adrenaline was administered together with the glucose tolerance test, the blood lactate and pyruvate levels rose significantly in both normals and psychotics. The amount of change in the lactate level was not significantly different in the two groups, while the shapes of the pyruvate

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cllrves were different. In the controls pyruvate values continued to rise until the second hour, both with and without adrenaline. In the patient group maximum values occurred within the first hour in both tests (Perrin et d.,1959). The findings of Altschule et al. are not restricted to any particular group of mental patients, and there is considerable overlap between groups and normals. An interpretation of their data is difficult, but the findings with intravenous administration of Na-dlactate indicates that primarily the utilization of lactate and pyruvate is impaired. The normalizing effect of glutathione may indicate that some sulfhyclryl group-blocking substance is present in an abnormal amount in the blood of these patients. Glutathione is known to help maintain certain sulfhydryl-containing enzymes in the reduced state, wbich is essential for their activity. Glutathione has also been recognized as a prosthetic group of the glycolytic enzyme glyceraldehyde phosphate dehydrogenase. It seems a little puzzling that the adininistration of a-lipoic acid causes an impairment of the lactate and pyruvate metabolism in chronic schizophrenics. This substance is known to be an essential cofactor in the conversion of pyruvate to acetyl-CoA, requiring Mg++and thiamine pyrophosphate as cofactors. Altschule is of the opinion that the large doses used may have produced a relative deficiency of thiamine. The work of Altschule and his group also include citric and a-ketoglutaric acids, which did not differ in the fasting state in patients and controls. During a glucose tolerance test there was a steady fall in the concentration of these substances in the control subjects, whereas in the psychotics the values started to rise 1 hour after glucose ingestion. This rise was reversed to norma1 when chronic patients were treated with hydrocortisone or ACTH for 3 days prior to the glucose tolerance test. A similar normalizing effect was seen if the patients were treated with glutathione. V. Brain Metabolism

During the short era of prcfrontal lobotomy researchers had the possibility of studying h i n tissue from mental patients, but lack of normal controIs hampers the interpretation of the clata obtained. Hayashi (1959) reported that the glucose uptake of brain tissue removed from schizophrenics was 40%lower than in other patients, whereas the 0, uptake was similar in the two groups. Rabassini and

266

PER S. LINGJAERDE

Cecotto ( 1960) compared the concentrations of adenosine triphosphate ( ATP), adenosine diphosphate ( ADP), and adenosine monophosphate (AMP) in brain tissue from schizophrenics and patients with extensive lesions of the central nervous system. Despite the absence of normal controls, it was held that these substances did not differ from the normal. Studies on intact brain has revealed no differences in overall cerebral blood flow and oxygen consumption between schizophrenic patients and normal subjects (Kety et al., 1948), whereas Sacks (1959, 1961), utilizing “C-labeled glucose and lactate, found that in normal controls 56% of the CO, produced derived from glucose, whereas in schizophrenic patients this figure was only 36%.There was a difference in the utilization of lactate dependent upon which isomer he used: with D-lactate he found a greater than normal brain metabolism, but with the L-isomer he observed a decreased catabolism. VI. Enzymes

Surprisingly few enzymological studies have been performed on schizophrenic patients. It would appear logical to follow up the findings on red cell metabolism in schizophrenic patients with studies of enzyme activities, but this has not been done. There are a few studies of cholinesterase activity, but very few of carbohydrate metabolism enzymes. Boszormenyi-Nagy and Gerty ( 195513) reported that they had measured the activities of hexokinase, aldolase, triose phosphate dehydrogenase, enolase, adenosinetriphosphatase, and hexosediphosphatase in hemolysates from schizophrenic patients. No significant differences were observed in comparison with normal controls. However, these authors do not report the enzymological methods used or the number of patients studied, and no figures are presented. Until such information is given this work must be regarded as unreliable. Red cells from American Negro subjects show a high incidence of glucose-&phosphate dehydrogenase deficiency, and Dern et al. (1963) reported a significantly lower incidence of this enzyme defect in paranoid than in catatonic schizophrenics. However, the data were reevaluated in 1965 by Bowman et al., and no significant differences were found. This enzyme has also been studied by Fieve et uE. (1965) and De Sousa and Manso (1963), but no differences were found between schizophrenic patients and normal controls or within the schizophrenic subgroups.

CARBOHYDRATE METABOLISM IN SCHIZOPHRENIA

267

Takahashi and Akabaiie (1960) stated that brain hexokinase activity was lower in schizophrenic patients than in normal controls. It was reported by Seeman and OBrien (1963) that red cell membrane Na+-K+-activatedATPase was much higher in schizoph&nic patients than in normal controls. This finding was not confirmed by Parker and Hoffman (1964), who found no significant difference in total or strophantidine-sensitive ATPase activity between schizophrenics and normal controls. Seeman ( 1964) then reported that the increased enzyme activity might have been “induced by previous chlorpromazine treatment. My own work on red cell aldolase activity in mental diseases led to no significant findings in schizophrenic patients as compared to normal controls or other patient groups. There was, however, a significantly higher aldolase activity in inactive ( chronic) schizophrenic patients than in active (acute) schizophrenics and normal controls. But, this difference permits no definite conclusions as far as overall glycolytic rate is concerned. Whether the increase in aldolase activity is related to the primary biochemical mechanisms responsible for the development of schizophrenia, or only reflects the compensating mechanisms of the organism, is still an open question. Further, a high mean aldolase activity was not restricted to schizophrenic patients, but were observed in several patient groups, including patients with thyrotoxicosis without psychotic manifestations (P. S. Lingjaerde, 1966). Serum enzymes have been studied by several investigators, but increased activity of these enzymes are more related to cell damage than to changes in metabolic pathways. Juveth (1957) found an elevated aldolase activity in serum from schizophrenics, highest in patients under chlorpromazine treatment. This last observation is not surprising when we consider the well-known effect of chlorpromazine on liver cells. Several other glycolytic enzymes have been studied, but no significant differences were observed between schizophrenic patients and normal controls ( Antebi and King, 1962; Stanley and Melki, 1964; Burlina and Visentin, 1965). VII. Red Cell Metabolism

A. NORMAL HUMANRED CELLMETABOLISM The red cell has been extensively used in biochemical investigations of mental diseases. The reason for using these cells in studies of intracellular metabolism is mainly because they are

268

PER S. LINCJAERDE

easily obtained and separated from other cells, and disruption of the cell membrane is no problem. However, one must be aware of the limitations of red cell metabolism. Their main, and probably only, reason for existing is to exchange gases between the lungs and the peripheral tissues. Hence, their metabolic equipment has been restricted to that necessary for this task only. With maturation of the red cell, nucleus, mitochondria, and microsomes are lost, together with the ability to reproduce, synthesize proteins, and so on. This must be borne in mind when investigations of red cell metabolism in schizophrenia are discussed. Interesting results have been reported, but an interpretation of the data so far presented is difficult, and the reader should be warned against the fallacy of regarding findings in red cells as valid for other body cells. For that reason, and before discussing the published studies of red cell metabolism in schizophrenia, Figure 1 presents a brief sketch of the normal human red cell glycolysis and phosphate metabolism. Red cell glycolysis is restricted to the Embden-Meyerhof and pentose shunt pathways, whereas the enzymes necessary for the operation of the citric acid cycle are lacking. The synthesis of ATP in red cells is thus restricted to the phosphoglycerate kinase and pyruvate kinase steps. Under normal conditions 8590% of the metabolized glucose is recovered as lactate (Reinauer and Bruns, 1964). The total net amount of energy produced as ATP is 2 moles per mole of glucose, in contrast to cells with a complete citric acid cycle, which may have a net production of 38 moles of ATP per mole of glucose, It is thus obvious that the red cell has a very low energy demand. The energy is needed for ( a ) the phosphorylation of glucose and glucose-6-P, ( b ) the transport of Na+ into the cell and K+ out of the cell, ( c ) the preservation of cell integrity, and ( d ) the reduction of methemoglobin. Compared with other cells the red cell is unique in its very high content of 2,3-diphosphoglycerate, which makes up half the total acid-soluble phosphate of the cell. In other body cells this ester is found in trace amounts only, as a cofactor for the enzyme phosphoglycerate mutase. In contrast to 1,3-diphosphoglycerate, which is a high-energy phosphate compound, 2,3-diphosphoglycerate is a lowenergy compound. In the phosphoglycerate cycle, which appears to be unique for the red cell, 1,3-diphosphogIycerate is conveded to 2,3-diphosphoglycerate, which means a loss of energy, If the breakdown of glucose is shunted over the 2,3-diphosphoglycerate path-

CARBOHYDRATE METABOLISM IN SCHIZOPHRENIA

Outside

Membrane

GLUCOSE -

269

Inside

'

TGL" /ATP

I

1

n NADP NADPH,

ADP

p,

~

? CX-P

NADH,

3-PGA

I + 3 Naf-

FIG. 1. Diagram of glycolysis, phosphate metabolism, and cation pump in human red blood cells. Abbreviations: ADP, adenosine diphosphate; ATP, adenosine triphosphate; ATPase, adenosinetriphosphatase; DHAP, dihydroxyacetone phosphate; F-6-P, fructose-6-phosphate; F-1,6-P, fructose-1,6-diphosphate; CAP, glyceraldehyde-3-phosphate;C-6-P, glucose-6-phosphate; GLU, glucose; LAC, lactate; NAD, nicotinamide adenine dinucleotide; NADP, nicotinamide adenine dinucleotide phosphate; PI, inorganic phosphate; PEP, phosphoenol pyruvate; 2-PGA, 2-phosphoglycerate; 3-PGA, 3-phosphoglycerate; 1,3-PCA, 1,3-diphosphoglycerate; 2,3-PGA, 2,3-diphosphoglycerate; PYR, pyruvate; X 2 P, hypothetical primary phosphate acceptor.

way there will be no net gain of energy as ATP. It thus appears that the phosphoglycerate cycle is a key regulator step in the production of ATP, according to the needs of the cell. Rapoport et aE. (1964) have stated that under physiological conditions most of the glucose metabolized in red cells is shunted over 2,3-diphosphoglycerate. Prankerd and Altman (1954) have suggested that 2 , s

270

PER S . LINGJAERDE

diphosphoglycerate acts as a readily available phosphate store. It is apparent from Fig. 1that it is possible to maintain a certain production of ATP even if there is a block in the glycolysis above the Z,%diphosphoglycerate step, provided there is a sufficient amount of this ester, If red cells are incubated in their own plasma with no other substrates added the concentration of 2,3-diphosphoglycerate decreases progressively, but ATP is kept at a constant level as long as there is 2,3-diphosphoglycerate available ( Mills and Summers, 1959; Reinauer and Bruns, 1964). Dische ( 1964) assumes that 2,3-diphosphoglycerate is present in the cell in a free and a bound form, but little is known of the regulating mechanisms behind this. Presumably only the free form takes an active part in glycolysis. Dische and Igals (1963) reported that 2,3-diphosphoglycerate strongly inhibits both transaldolase and transketolase, two key enzymes in the pentose shunt pathway. They suggest that this ester may act as a regulator for glycolysis over the shunt or the Embden-Meyerhof pathway. Dische (1964) also reported that 2,3-diphosphoglycerate inhibits hexokinase, but this observation could not be confirmed by De Verdier and Garby ( 1965). In order to understand the mechanisms behind studies of phosphate metabolism in red cells one must know the pathways of phosphate from the plasma to the incorporation into various phosphate compounds. Inorganic phosphate most probably enters the cell by a passive diffusion (Passow, 1964; Cartier and Chedru, 1966), but there are indications that it is bound to an unknown compound in or at the inner surface of the celI membrane (Tachibana, 1958; Schauer and Hillmann, 1961). This unknown compound is marked xlP in Fig. 1, and it is assumed that it transfers the phosphate group to glyceraldehyde phosphate with resulting synthesis of 1,3-diphosphoglycerate, but the mechanism of such a transfer is not known. In the next step the high-energy phosphate group of 1,3-diphosphogIycerate is transferred to ADP and ATP is formed. The generation of ATP is thus dependent aIso on the availability of ADP. The glycolytic regulatory mechanisms in human red cells are not fully elucidated, but glucose concentration appears to play a negligible role in this picture (Reinauer and Bruns, 1964; Tsuboi, 1965). Tsuboi and Fukunaga (1965) observed that glycolysis increased with increasing inorganic phosphate concentration in the incubation

CARBOHYDRATE METABOLISM IN SCI-IIZOPHRENIA

271

medium, and suggest that inorganic phosphate stimulates phosphofructokinase. A key regulatory role is played by glucose-6-phosphate, which inhibits hexokinase ( Rose and OConnell, 19M). De Verdier and Garby (1965) have further found that glucose-6-phosphate is a competitive inhibitor of ATP toward hexokinase, and this inhibition is markedly diminished by phosphate ions.

B. RED CELLMETABOLISM IN SCHIZOPHRENIA Skaug and Hellem (1939, 1941) observed that patients who were under insulin coma treatment did not regain consciousness after oral ingestion of glucose unless inorganic phosphate was administered simultaneously. A still better effect was obtained if the patients were given phosphate at the beginning of the treatment, when still able to drink. This observation led them to study different phosphate fractions in the blood of these patients, and the main finding was a significantly lower hexose phosphate concentration in the patients than in normal controls. This work was followed some years later by other Scandinavian researchers, who introduced labeled phosphate (,*P) in the study of intermediary metabolism in red cells from mental patients (Goldkuhl and Orstrom, 1948; Orstrijm and Skaug, 1950; Kvamme, 1951a,b). These workers found a significantly lower specific activity of the ATP fraction in schizophrenic patients than in normal controls. The difference was most marked the first hour after the injection of 32Pinto the bloodstream, but in the course of 2 hours the difference between patients and controls disappeared. This indicated that phosphate turnover in red blood cells was slower in schizophrenics than in normal subjects, although a marked overlap was observed. Orstrom and Skaug (1950) reported that in patients with a very low specific activity in the ATP fraction 15 minutes after injection of 32P the highest activity appeared in an unknown fraction, later claimed by Orstrom (1951) to be phosphoglycolic acid. Skaug and Jellum (1959) have developed a specific method for the determination of phosphoglycolic acid, hut have been un;ihle to confirm the findings of Orstrom. Nor could Bartlett (1959) detect the presence of glycolic acid in human red cells. If this so-called “schizophrenic ester” of Orstrom and Skaug was not phosphoglycolic acid, one may speculate which other possibilities should be considered. me unknown primary phosphate acceptor suggested in Fig. 1 is of course

272

PER S. LINGJAEFDE

a strong candidate, but 2,3-diphosphoglycerate should also be considered, as this compound is very difficult to separate from phosphoglycolic acid (Skaug and Jellum, 1959). If the unknown membrane compound is involved, the findings in schizophrenics could mean that the transfer of phosphate from the unknown compound to glyceraldehyde phosphate and subsequently to ADP is impaired. This transfer is probably localized to the cell membrane, which makes it susceptible to external influence, for instance, the schizophrenic plasma factor. If the “schizophrenic ester” is identical with 2,3-diphosphoglycerate the findings of Orstrom and Skaug may possibly indicate that more l,%diphosphoglycerate is converted to 2,3-diphosphoglycerate in chronic schizophrenic patients than in normal subjects, and consequently the turnover of ATP may be slower than normal. We may then assume that the concentration of active (unbound) 2,3-diphosphoglycerate is higher in schizophrenic than normal subjects, and that an abnormal inhibition of shunt enzymes could occur. This possibility would fit in with the report of Frohman et al. ( 1960a) that schizophrenic patients appear to metabolize less glucose via the pentose shunt than normal subjects. A much cited paper, which claimed that the effect of insulin on red cell metabolism in schizophrenic patients was opposite to that found in normal controls, was published by Boszormenyi-Nagy and Gerty (1955a). These authors erroneously assumed that the citric acid cycle is operative in red cells, and used an incubation mixture consisting of fructose-l,6-phosphate as substrate, glutathione, NAD, AMP, orthophosphate, KCI, KF, and cytochrome c, and studied the effects of addition of pyruvate to this incubation mixture. When even the effect of insulin is added, an interpretation of the data reported becomes rather difficult. In 1957 Frohman et al. (1959) and Gottlieb et al. (1959a) stated that insulin produced a marked increase in the ATP turnover rate in normal red cells after 1 hour of incubation, whereas no increase or a marked decrease was observed in schizophrenic patients. To some extent insulin also affected the amount of ATP. Even ADP and AMP behaved differently in patients and normal controls, and differences between acute and chronic schizophrenics were observed with all the three adenine nucleotides. The only compound studied which clearly differentiated both acute and chronic schizophrenic patients from normal controls was fr~ctose-l,B-phosphate

CARBOHYDRATE METABOLISM I N SCHIZOPHRENIA

273

(FDP). The specific activity of this fraction was signscantly higher in both patient groups than in normal controls under basal conditions. After insulin there was a nearly fourfold increase in the specific activity for the control group, and a significant decrease in both the acute and the chroiiic patient groups (Gottlieb et uZ., 1959b). Dyfverman and Broman (1957) compared the paper chromatograms of phosphoric acid esters from red cells of schizophrenic patients under insulin coma treatment, untreated schizophrenics, and normal controls. Insulin treatment caused one or more unknown substances to appear, but no differences were found between the chromatographic patterns of normals and untreated schizophrenics. Efimovich (1961) reported that in schizophrenic patients under insulin hypoglycemia treatment who responded to treatment, the rise in blood ATP concentration began considerably later than in patients with infectious psychoses, and reached its peak only at the height of the hypoglycemia. In schizophrenics who did not respond to treatment, ATP concentration fell shortly after insulin injection, then rose, but did not exceed the initial level at the peak of the shock. Bemsohn et al. (1962) claimed that after 2 hours of incubation red cells from schizophrenic patients had a significantly higher ATP content than red cells from normal and psychiatric control subjects. It may be of importance that these patients were under chlorpromazine treatment. Honda (1963) incubated red cells with 32Pfor 60 minutes, and reported that both the concentration and specific activity of 2,3diphosphoglycerate were significantly higher in schizophrenic patients than in normal controls. Specific activity of A T P was higher in chronic schizophrenic than in acute schizophrenic patients and control subjects. There were no significant differences in specific activities of ADP and FDP between patients and controls. Arnold and Hofmann (1963) and Hofmann and Arnold (1967) have mainly concentrated on the effects of succinate on intermediary phosphate metabolism in red cells. There is no obvious rationale behind the use of succinate in such studies, as this compound has no known effects in human red cells, and it is quite unknown how succinate could influence red cell metabolism. The authors reporled that succinate treatment induced an increased concentration of ADP in red cells from scliimphrenic patients, as compared with

274

PER S. LINGJAERDE

control subjects, and a decreased ATPIADP ratio. They have further divided their patients into what they call “metabolic regulation types.” For that purpose they constructed a distribution diagram of mean changes in the specific activities of ATP, ADP, hexose diphosphate, and total trioses after succinate treatment (expressed as percent of initial values), which they claim falls into three peaks. Patients with a decrease in the mean specific activity of the substances mentioned are called “regulation type I” or “asthenic type,” and patients with an increase in these values are labeled “regulation types I1 and 111” or “sthenic type.” From a biochemical point of view it is difficult to see the meaning of this division into “metabolic regulation types,” and a more thorough biochemical interpretation of these data is not presented. As stated, the specific activity of ADP is included in the metabolic factors which make up the “regulation types.” However, the authors state that changes in ADP concentration have no connection with the regulation types, and make the following statement ( Hofmann and Arnold, 1967) : “In our opinion a change in ADP concentration (or a change in ATP/ ADP ratio) on the one hand and a change in overall metabolic factors have very little in common.” Overall metabolic factors obviously refer to the speci6c activities of ATP, ADP, FDP, and trioses. It is hard to see how ADP could change independently of these other factors. There is a close relationship between glycolysis and ATP levels in the mature red cell, and the relationship between ATP and ADP need no further comments. An increase in ADP levels could come about either by dephosphorylation of ATP, for instance, by the activity of membrane ATPase, or through the action of adenylate kinase which catalyzes the reaction AMP A T P e 2 A D P . In both instances a change in ADP would be reflected by a change in ATP, in the last instance also in AMP. A third possibility is a de n . 0 ~ 0synthesis of ADP. The mature red cell appears unable to synthesize purines and pyrimidines from small precursor molecules, but free adenine is readily incorporated into adenine nucleotides (Bishop, 1964). Tsuboi ( 1965) has shown that incorporation of label does not occur in adenine nucleotides from sources other than adenine. He also found that labeled adenine was equalIy distributed among AMP, ADP, and ATP, in support of their existence in a single metabolic pool. The possibility of a df?nova synthesis of ADP thus exists, but according to Tsuboi it is not evident that adenine is normally available to the cells in duo.

+

CARBOHYDRATE METABOLISM I N SCHIZOPIIRENIA

275

In any case it is hard to visualize a de nouo synthesis of ADP that is not reflected in ATP. In the paper of Hofmann and Arnold one further misses information about AMP. Hofmann and Arnold have also introduced the term “amentialike factor” ( Q-DPGS ) which is related to 2,3-diphosphoglycerate and expressed as the ratio of the specific activity of 2,3-diphosphoglycerate after and before succinate treatment divided by the ratio of the specific activity of ATP after and before succinate treatment. It is hard to see how a difference in this ratio of four variables could be interpreted. In biochemicaI work one should be very careful in introducing ratios instead of evaluating the single components as such. An attempt to verify the findings of Hofmann and Arnold have been performed by Biesold et al. (1965), who determined the blood levels of AMP, ADP, and ATP 5, 10, 15, and 20 minutes after succinate injection. There was no difference between their chronic schizophrenic patients and controls before succinate injection, whereas a more or less marked decrease occurred in all substances studied after snccinate both in patients and controls. The many reports referred to above do not give a clear picture of which-if any-abnormalities are characteristic of red cell metabolism in schizophrenia. The techniques used differ in many respects : Some patients were under treatment with psychopharmaceuticals; some workers have included nonschizophrenic patients in their control groups; and the conception of schizophrenia differs from country to country. I do not intend to discuss the term schizophrenia or the effects of psychopharmaceuticals on intermediary metabolism, but rather concentrate on the methodological aspects of the studies referred to. After all, this is the basis of any interpretation of biochemical data. A11 the Scandinavian workers reported that the specific activity of ATP was considerably lower in chronic schizophrenic patients than in normal controls, whereas Gottlieb et al. and Honda found a higher specific activity of ATP in chronic schizophrenics than in controls, and the study of Hofmann and Arnold revealed no significant differences. These discrepancies are not easily explained, but some important factors should be considered. First and foremost the experiments of the Scandinavian workers were performed in vivo, i.e., 32P was injected into the bloodstream, and bIood was withdrawn at different intervals and immediately sllbjected to

276

PER S. LINCJAERDE

phosphate ester analysis. The experiments of the other workers mentioned were all performed as in vitro incubations of red cells or whole blood with radiophosphate. Gottlieb et al. incubated for 60 minutes, Hofmann and Arnold for 15 minutes plus a preincubation of 10 minutes, and Honda for 60 minutes. All these incubations were performed at 37°C. In their paper Goldkuhl and Orstrom state that in a preliminary study they had measured the specific activity of ATP at more frequent intervals than reported in the paper, and this study showed that the maximum specific activity of ATP occurred 15 minutes after the injection of 32P.Kvamme recorded time-activity curves in individual patients, and demonstrated that whereas there was a steady fall in the specific activity from 30 minutes after the injection in normals (no values were taken earlier), there was a maximum value after 60 minutes, followed by a decline in chronic schizophrenics. Gottlieb et al. did not record a time-activity curve, which makes it difficult to tell if their finding of a significantly higher specific activity of ATP in schizophrenic patients reflects a slower or faster than normal turnover of ATP. If maximum specific activity of ATP had occurred in the normals earlier than after 60 minutes of incubation, and then started to decline, whereas 60 minutes was the time of maximum activity in the chronic schizophrenics, the conclusion would be a slower than normal turnover in the patients, in agreement with the findings of the Scandinavian groups. However, Prankerd and Altman (1954) claimed that the relative specific activity of ATP in normal human red cells incubated in vitro did not reach a maximum value until after 6 hours. Similarly, Gerlach (195s) showed that the specific activity of both the a-,p-, and y-P of All' increased steadily during a 5-60-minute incubation period. With these studies in mind, one would be apt to conclude that the values reported by Gottlieb et al. do indicate a faster than normal turnover of ATP in chronic schizophrenic patients, which is in striking contrast to the conclusions drawn by the Scandinavian groups. Metabolic changes occur rapidly in red cells after withdrawal from the blood vessels. It is particularly important to exclude any hydrolysis of ATP during the isolation procedures. Even if blood from normal controls and patients are treated in exactly the same manner, one cannot exclude a difference in the possible hydrolysis of labile compounds. The necessity for taking such a possibility into account is illustrated by the report of Seeman and O'Brien

CAFWOIIYDRATE METABOLISM I N SCHIZOPHRENIA

277

(1963) of an abnormally high Na+-K+-activatedATPase activity in red cells from schizophrenic patients. The Scandinavian workers performed their experiments at a time when subtle methods for separation of different phosphate fractions were not fully developed. Consequently, they did not isolate ATP, but analyzed the 10-minute hydrolyzable organic phosphate fraction. ATP is the major component of this fraction, but other acid-labile fractions are also included. The separation of inorganic phosphate and 10-minute hydrolyzable phosphate left a rest fraction, which mainly consisted of 2,3diphosphoglycerate. Gottlieb et at. isolated the different organic phosphate fractions by chromatography after precipitation of a trichloroacetic acid extract of the blood with barium. Hofmann and Arnold performed twodimensional paper chromatography on a perchloric acid extract of the red cells. The concentration of phosphate compounds may be expressed as mg/ 100 ml, mg P per unit volume (or weight), pmoles per unit volume, pmoles P per unit volume, etc. Concentrations expressed as mg/100 ml in one paper and pmoles/liter in another, are not readily comparable without recalculation of the data. In Table I are compiled some data on various phosphate compounds in human red cells, recalculated as pmoles/liter packed red cells. In some instances the original paper has expressed the concentration of phosphate compounds per gm or 100 gm red cell mass. In this instance the recalculations have been done assuming a specific weight of packed red cells of 1100. Values expressed per unit volume of whole blood have been recalculated as pmoles/liter packed red cells assuming a hematocrit of 47%. Table I shows that great discrepancies exist between different reports on red cell phosphate intermediates. One explanation is that some workers have incubated the red cells before phosphate analysis is performed, which alters the steady-state condition of red cells in vivo. In order to get a reliable picture of the in vivo conditions it is very important to chill the blood immediately upon withdrawal from the blood vessels, and carry out all extraction procedures in the cold. Still the method of phosphate analysis may cause some splitting of labile phosphate esters, and accordingly too low values for the labile compounds and too high levels for inorganic phosphate. Skaug and Natvig (1957) compiled data from the literature on inorganic phosphate in human red cells, and reported a

TABLE I Hux.4~ RED CELLPHOSPHATE COMPOUNDS'

Authors Skaug and Natvig (1957) Bartlett (1959) Gerlach and Lubben (1959) Prankerd and Altman (1954) Shafer (1965) Bishop et al. (1959) Goldkuhl and Orstrom (1948) Goldkuhl and Orstrom (1948) Hofmann and Arnold 1967) Hofmann and Arnold 1967) Bernsohn et al. (1962) Bernsohn el al. (1962) Gottlieb st al. (1959b) Gottlieb et al. (1959b) Got,tlieb et al.

(1959b)

Biesold et al. (1965) Biesold et u2. (1965) a

Diagnosis Normal Normal Normal Normal Normal Normal Normal Chronic schizophrenia Controls Schizophrenia

Normal Schizophrenia Normal Chronic schizophrenia Acute schizophrenia Normal Schizophrenia

Incubation time (minutes) 0 0 45 ? 5 0 0 0

Pi

AMP

ADP

ATP

FDP

87 28M80 10-20 120 310 1100-2100 700 30 13

190-245 900-1230 3600-5100 60-120 780 3700 460 330 190400 250-870 1900-2900 200 765 3500 45 100 960 850 4800 800 4500

145 2.5

240 256 100 105

1050 1000 1180 1230 190 195

60

42

82

190

0 0

20

135 135

1220 119.5

25 25 20 20 60 60

2,3-PGA

600 560 665 595

300 220

25

Data compiled from the literahre and recalculat,ed as pmoles per liter packed red cells.

3175 3370

170 215 55 100 88 68

70

CARBOHYDRATE METABOLISM IN SCHIZOPHRENIA

279

range of reported values from 1 to 21 mg/100 ml of cells. In their own work they used a method that was especially suited to avoid any splitting of organic phosphate compounds during the analytical procedure, and found an average value of 0.27 mg/100 ml ( =87 p,moles/liter). As seen from the table this is markedly lower than any values reported by other workers. Which, then, are the correct values for organic phosphate compounds in human red cells? Those reported by Bartlett (1959) and Bishop et al. (1959) are probably the most reliable. These figures show that the concentration of ADP is 10-20% that of ATP, and AMP is 1-28. Gerlach and Lubben (1959) and Shafer (1965) have both incubated the cells for 45 and 5 minutes, respectively, and, as expected, they have found lower ATP values and higher values for ADP and AMP. A surprisingly high value for inorganic phosphate was reported by Prankerd and Altman (1954). This is probably due to the fact that these workers performed their analyses on cells which had been frozen and thawed. The values of Goldkuhl and Orstrom (1948), who analyzed the blood immediately upon withdrawal from the vein, are in fairly good agrement with those considered most reliable. However, their technique was relatively crude, and other substances than ATP and 2,3-diphosphoglycerate are included in the respective fractions. Considering the incubation times used, the figures reported by Hofmann and Arnold (1967) and Bernsohn et al. ( 1WZ) are in the ranges expected, although the ATP values appear somewhat high for incubated cells. Biesold et al. (1965) chilled the blood immediately upon withdrawal and carried out the analyses without previous incubation. Their ADP values are surprisingly low. The determinations were carried out enzymatically. In sharp contrast stand the figures reported by Gottlieb et al. (1959b). First and foremost the ATP levels are only a fraction of what should normally be found, and second the AMP, ADP, and ATP concentrations are completely out of normal proportion to each other. The deviations can not be explained by the incubation time used (60 minutes), but more probably reflects the use of an unreliable technique. The reported concentrations of FDP show such great variation that one can hardly tell which value is the correct one. VIII. Serum Factors and Carbohydrate Metabolism

In order to test if the observed hypoglycemia unresponsiveness in schizophrenic patients could be due to the presence of some

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serum factor, 0. Walaas et al. (1954) studied the effects of sera on the glucose uptake by isolated rat diaphragm. Sera from normal controls significantly stimulated the glucose uptake, whereas the effects of sera from the patients were significantly less pronounced. Further experiments by this group (Haavaldsen et al., 1958) showed that the active factor in sera from schizophrenic patients was localized to the a,-globulin fraction. This fraction from patient sera inhibited glucose uptake by isolated rat diaphragm. In one patient the al-globulin fraction initially completely blocked the glucose uptake, but in three subsequent iiwestigations no significant effects were obtained. Simultaneously ketonuria, which was initially present, disappeared. In some few experiments the effects of plasma dialyzates were studied. Dialyzates from normal plasma stimulated the glucose uptake by diaphragm, and heat treatment of the dialyzate (lOO°C for 5 minutes) further increased the uptake. With serum dialyzates from some schizophrenic patients there was no stimulation, and after heat treatment there was either no effect or an inhibition of the glucose uptake was observed. The authors concluded that the decreased stimulation of glucose uptake in rat diaphragm by sera from schizophrenic patients was due to an abnormality in the protein fraction as well as in the dialyzable fraction (lack of stimulating factor). Subsequent experiments by E. Walaas et al. (1964) and 0. Walaas et al. (1965) revealed that the serum prealbumin fraction from schizophrenic patients had a pronounced multimetabolic effect on isolated rat diaphragm. This protein fraction reduced net glycogen synthesis, incorporation of glucose-*4C into glycogen and incorporation of phenylalanine-14Cinto proteins to half the control values. This finding was not restricted to schizophrenia, but even observed in a patient wih constitutional psychosis. However, serum prealbumin from a number of normal controls had no inhibitory effects on rat diaphragm metabolism. If the patients were treated with chlorpromazine, the inhibitory effects of the serum prealbumin fraction gradually disappeared. The authors assume that the effects observed are due to an unknown compound attached to the prealbumin fraction. In an attempt to identify this compound it was observed that the fluorescence intensity of the patient prealbumin at 340 nm was 10 times lower than for prealbumin from normal subjects. The studies of the Lafayette Clinic group logically led to the conclusion that a ‘toxic” factor in the sera of schizophrenic

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patients was responsible for the observed disturbances in carbohydrate metabolism. Frohman ct al. ( 1960a ) reported that insulin caused a signific;int drop in thr rate of incorporation of the l-carbon of glucose into CO, by blood from control subjects, but such a drop did not occur with blood from schizophrenics. The insulin effect disappeared when blood cells from control subjects were incubated with plasma from schizophrenic patients, so that these cells now metabolized in the same manner as cells from schizophrenic patients in their own plasma. Plasma from schizophrenics produced a lower pyruvate production and a higher lactate/pyruvate ratio in chicken red cells than did normal plasma (Frohman et al., 196Ob). Similar results were obtained if sera were incubated with isolated rat diaphragms. In a later paper Frohman et al. (1962) stated that only plasma from schizophrciiic patients had this effect on chicken erythrocytes, whereas plasma from patients with manic-depressive psychosis, alcoholism, or diabetes did not. The effect was not found with plasma from patients with “childhood schizophrenia” and who later, as grown-ups, revealed a classic schizophrenic picture. Attempts to isolate and identify the active factor gave the following results (Frohman et al., 196Oc, 1962): The factor is a protein or protein-bound molecule which electrophoretically migrates with the a,-globulin fraction. It is unstable above pH 9 or below pH 6, and is destroyed rapidly at room temperature. Loss of activity could be retarded by the addition of ascorbic acid or by placing the material under hydrogen. Freezing destroyed the activity completely. By immunoelectrophoresis and by inducing antibodies the factor was found to consist of six distinct fractions. About 2% times as much sialic acid was found in the active fraction from patients than from iiormal controls. In their first studies the Lafayette Clinic group used insulin as a stressor. In later investigations ( Frohman et aZ., 1961) they induced stress by a painful stimulus, and then incubated the plasma from the test person with chicken red cells for 1 hour. The painful stimulus caused a decrease in the specific activity of ATP in the blood of the controls but no change in the patient samples. The glucose-l14C/glucose-6-“C ratio found in the labeled CO, increased with pain in the controls, but decreased in the patients. The lactate/ pyruvate ratio was not affected b y any of the experimental parameters. In 1962 Latham et al. observed that the factor was only found after a certain amount of bodily exercise. Latham et al. (1963) have

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further shown that the differences in lactate/pyruvate ratio are not due to cell membrane permeability changes. With labeled sodium acetate Frohman et al. (1967) have studied the effects of schizophrenic plasma on chicken red cell tricarboxylic acid cycle intermediates. The specific activities of both a-ketoglutaric acid and lactic acid were significantly lower in red cells incubated with plasma from schizophrenic patients than with plasma from normal controls. It appeared that this finding was due to isotope dilution rather than a block in the tricarboxylic acid cycle. It turned out that red cells incubated with schizophrenic plasma contained twice as much lactate than cells incubated with normal plasma, and the same phenomenon was observed with both a-ketoglutarate and malate. The authors find it unlikely that this extra lactate comes from glucose, and suggest that it is being formed from amino acids. It is also reported that cells from chickens injected with adrenaline or noradrenaline gave much lower lactatelpyruvate ratio than cells from untreated chickens, and these cells failed to differentiate between schizophrenic and normal plasma. Finally, they report that plasma from schizophrenic patients even has an effect on the rate of esterification of inorganic phosphate in chicken red cells. Plasma from schizophrenic patients caused the inorganic phosphate to disappear much more rapidly than plasma from the control subjects. If mitochondria were substituted for intact cells the difference between control and schizophrenic plasma disappeared. Other researchers have tried to confirm the investigations of the Lafayette Clinic group. Mangoni et al. (1963) found no difference in lactate/pyruvate ratio between chicken red cells incubated in plasma from schizophrenic patients and normal controls. The reliability of this work,-however, was challenged by Durell and Ryan (1967). Buhler and Ihler (1963) studied the effects of plasma on the oxidation of g l u c o ~ e - l - ~and ~ C g l u ~ o s e - 6 - ~in ~ Cchicken red cells. Again, contrary to the Lafayette Clinic group, no significant differences were observed between schizophrenic and normal subjects, either with or without administration of insulin to the test subjects. However, plasma from schizophrenic patients consistently reduced the rate of oxidation of ''C-labeled glucose by chicken red cells below that observed for the same cells in the presence of buffer

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alone, whereas normal plasma induced a variable stimulation or inhibition. Krasnova (1965) found a high lactate/pyruvate ratio in chicken red cells incubated with serum from patients with different forms of epilepsy, but no difference in this ratio with serum from normal controls and psychiatric patients, and no differences among the various types of schizophrenia. The activity of serum was considerably diminished by heat treatment at 56°C for 30 minutes (which is used to inactivate complement ) . Preparative electrophoresis gave the highest activity in the /%globulin region. The biochemical mechanisms behind the changes in lactate/ pyruvate ratio with plasma from schizophrenic patients is unknown, but an intact cell membrane appears to be necessary in order to produce the effect. Ryan et al. (1966) and Durell and Ryan (1967) have shown that a close relationship exists between lactic acid production and hemolysis when chicken red cells are incubated with human plasma. There is no evidence of an active glycolysis in chicken red cells incubated in a plasma-free medium, but the addition of a number of factors may stimulate glucose metabolism. These factors include inhibitors of oxidative metabolism, saponin, mechanical disrupture, sensitization with rabbit anti-Forssman antibody, and all human plasmas tested. They further demonstrated a good correlation between the degree to which any human plasma stimulated the accumulation of lactic acid and the degree to which it produced lysis of the chicken cells. When human serum was preheated in order to inactivate complement the effect on hemoglobin liberation and lactate accumulation was prevented. Durrell and Ryan fully confirm the findings of the Lafayette Clinic group that human plasma affects the lactate/pyruvate ratio in chicken red cells, but in contrast to Frohman et al. they conclude that there is no etiological relationship between the heterogenic antibody-which they claim causes the lactate accumulation-and the diagnosis of schizophrenia. Bergen ( 1967) reported that dimethoxyphenethylamine elevated the lactate/pyruvate ratio of chicken red cells to twice the values without the drug. An attempt to study the effects of sera from schizophrenic patients on brain carbohydrate metabolism has been published by Seregina ( 1966). Rats were treated for 2 weeks with sera from acute schizophrenics or normal controls, and a third group of rats were

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given insulin coma therapy after 2 weeks of schizophrenic serum injections. Glycogen, sugar, and lactate were determined in the brains after decapitation and immediate fixation in liquid oxygen. Rats treated with serum from schizophrenic patients had considerably lower brain values for all the three parameters than brains of rats treated with normal serum. Insulin coma treatment tended to normalize the carbohydrate content in the brains of rats treated with serum from schizophrenics. IX. Concluding Remarks

If this review has left the reader a little confused, it may perhaps console him to know that the reviewer shares his feelings. The picture revealed is very complicated, and at the present time no competent worker in this field can give a plain “Yes” or “NO” answer to the simple question: “Is schizophrenia caused by a disturbance in carbohydrate metabolism?” Many reports, which have been published over the years, are no doubt unreliable, and should be forgotten. However, there are several interesting observations that appear founded on solid experimental work. Admittedly, many of the published differences between mental patients and normal controls are small, but they deserve careful consideration and ought to be studied by more delicate biochemical techniques. No doubt many schizophrenic patients do respond abnormally to glucose and insulin tolerance tests, although the deviations are negligible compared with that observed in a disease like diabetes. It is also fairly well established that phosphate metabolism in red blood cells from schizophrenic patients deviates from the normal. These abnormalities may very well be explained by the influence of an extracellular factor, even if this factor does not penetrate the cell membrane. We know little about the mechanism of a possible external influence upon red cell intracellular metabolism. Two glycolytic shunt mechanisms are operative in the red cell: The pentose and the diphosphoglycerate shunts, but how glycolysis is directed over one or the other of alternative pathways is unknown. If these regulating mechanisms are disturbed-for instance, by the unknown serum factor-there is every reason to expect a disturbance in phosphate metabolism. Based on many reports by independent research groups one is apt to accept the existence of an unknown plasma or serum factor in schizophrenia, but we still do not know if we are dealing with one

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or more substances. This review is restricted to studies of the effects of the factor on carbohydrate metabolism, and readers who are interested in a more thorough discussion of the whole serum factor problem should consult “Molecular Basis of Some Aspects of Mental Activity” ( 0. Walaas, 1967). What is the nature of this factor? Is it a protein or a small molecule attached to a protein? Is it a normal substance that occurs in abnormal amounts in the blood of schizophrenic patients, or is it a substance specific for schizophrenia (or mental diseases)? It may have relevance to this problem that Berman and Wertheimer (1960) found an unknown factor in the serum of fasting rats which inhibited glucose uptake and glycogen synthesis in the isolated rat diaphragm. 0. Walaas et al. (1965) have pointed out that the multimetabolic effects observed with the serum prealbumin fraction from schizophrenic patients are characteristic of the action of hormones or hormone-like substances, and they discuss the possibility that the primary action of the unknown factor is at the level of messenger RNA and enzyme synthesis. In my own work (P. S. Lingjaerde, 1966) I have discussed the increased plasma hydrocortisone levels observed in many mental patients in relation to the effects of this hormone on gluconeogenesis. Weber et al. (1965) assume that there is a continuous, slow discharge rate of glucocorticoid molecules from the adrenal cortex increasing the synthesis and a similar slow, continuous discharge of insulin from the pancreas, suppressing the synthesis of gluconeogenetic enzymes. In this fashion a dynamic oscillatory balance of inducer and suppressor molecules results in the homeostatic equilibrium in the biosynthesis of the pace-maker enzymes. A criticaI alteration in the equilibrium of inducer and suppressor results in a homeostatic imbalance affecting the biosynthesis of key enzymes which underlies the metabolic disorder at the molecular level,

Many of the findings in schizophrenic patients could be explained by a disturbance of gluconeogenetic regulation. Either there is an increased need of gluconeogenesis, and accordingly of increased adrenocortical activity, or an unknown suppressor molecule could act at the level of glucocorticoid action, which presumably would induce an increased glucocorticoid activity. Many discrepancies in biochemical findings in schizophrenia are probably due to differences in patient materials studied. It appears particularly important that some workers do not differentiate between acute and chronic schizophrenia. 0. Lingjaerde (1964) has

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for many years stressed the importance of separating “active” and “inactive” phases of schizophrenia. The “active” phase includes acute schizophrenia and acute exacerbations in chronic schizophrenia, and the “inactive” phase includes stationary defect schizophrenia and convalescent states. In the active phase the disease process is apparently in progress, whereas in the inactive phase it seems to have leveled off. From a biochemical and physiological point of view the two phases are quite different. The active phase reveals a picture of disturbed homeostasis, whereas the inactive phase is characterized by apparent stability. Significant differences in biochemical parameters have been observed between active and inactive phase or between acute and chronic schizophrenia (for further details, see P. S. Lingjaerde, 1966). It is perhaps not too unrealistic to regard the active phase as an uncompensated condition and the inactive phase as the compensated state. Some workers in the psychiatric field have performed biochemical investigations on patients under treatment. One should be strongly warned against such a course of action as is clearly illustrated by the finding of 0. Walaas et al. (1965) that the multimetabolic inhibitory effects of the prealbumin fraction from schizophrenic patients disappeared under chlorpromazine treatment. Many readers may find this review too technical. However, one should not refer to reported results without some considerations of the methods and techniques used. Some reports, which have been cited over and over again in psychiatric literature, do not stand the test of a careful methodological evaluation, and some workers in the field need to be reminded that a statistical signgcant difference cannot without further ado overrule a questionable biochemical finding. Biochemical investigations in mental patients should be as carefully evaluated as any other biochemical work. It should be warned against what Riebeling once called “klebrige Festhalten an einem positiven Resultatchen.” REFERENCES Altschule, M. D., Henneman, D. H., Holliday, P., and Goncz, R. M. (1956). A.M.A. Arch. Internal Med. 98, 35-38. Altschule, M. D., Henneman, D. H., Holliday, P., and Goncz, R. M. (1957). A.M.A. Arch. Internal Med. 99, 22-27. Altschule, M. D., Goncz, R. M., and Holliday, P. (1959). A.M.A. Arch. Znternal Med. 103, 72&729. Antehi, R. N., and King, J. (1962). J. Mental Sci. 108, 75-79.

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Arnold, 0. H., and Hofmann, G. (1963). Wien. Klin. Wochschr. 75, 593-602. Bartlett, G. B. (1959). J. Biol. Chem. 234, 449458. Bergen, J. R. (1967). In “Molecular Basis of Some Aspects of Mental Activity” (0. Walaas, ed.), Vol. 2, pp. 257-267. Academic Press, New York. Bergsman, A. (1959). Acta Psychiat. Neurol. Scand. Suppl. 133, 1-107. Berman, E. R., and Wertheimer, E. (1960). Am. J. Physiol. 198, 1075-1078. Bernsohn, J., Custod, J. T., Remenchik, A. P., and Talso, P. J. (1962). J. Neuropsychiat. 4, 22-27. Biesold, D., Weise, K., and Canzler, E. ( 1965). Psychiat. Neurol. Med. Psychol. 17,232-239. Bishop, C. ( 1964). In “The Red Blood Cell” (C. Bishop and D. M. Surgenor, eds. ), pp. 147-188. Academic Press, New York. Bishop, C., Rankine, D. M., and Talbott, J. H. (1959). J. Biol. Chem. 234, 1233-1237. Boszormenyi-Nagy, I., and Gerty, F. J. (1955a). Am. 1. Psychiat. 112, 11-17. Boszormenyi-Nagy, I., and Gerty, F. J. (1955b). J. Nervous Mental Dzkeuse 121, 53-59. Bowman, J. E., Brewer, G. J., Frischer, H., Carter, J. L., Eisenstein, R. B., and Bayrakci, C. (1965). J. Lab. Clin. Med. 65, 222-227. Buhler, D. R., and Ihler, G. S. (1963). 1. Lab. Clin. Med. 62, 306-318. Burlina, A., and Visentin, B. (1965). Riu. Anat. Patol. Oncol. 80, 380-386. Cartier, P., and Chedru, J. (1966). Bull. SOC. Chim. Biol. 48, 1421-1437. Dern, R. J., Glynn, M. F., and Brewer, G. J. (1963). J. Lab. Clin. Med. 62, 319-329. De Sousa, J. F., and Manso, C. (1963). Arquiu. Patol. (Lisbon) 35, 83-85. De Verdier, C.-H., and Garby, L. (1965). Scand. J. Haemutol. 2, 305417. Dische, Z. ( 1964). In “The Red Blood Cell” (C. Bishop and D. M. Surgenor, eds.), pp. 189-209. Academic Press, New York. Dische, Z., and Igals, D. ( 1963). Quoted by Dische ( 1964). Dwell, J., and Ryan, J. W. (1967). In “Molecular Basis of Some Aspects of Mental Activity” (0. Walaas, ed.), Vol. 2, pp. 283-302. Academic Press, New York. Dyfverman, A., and Broman, L. (1957). Scand. J . Clin. 6 Lab. Invest. 9, 356364. Easterday, 0. D., Featherstone, R. M., Gottlieb, J. S., Nusser, M. L., and Hogg, R. V. (1952). A.M.A. Arch. Neurol. Psychiat. 68,48-57. Efimovich, N. G. (1961). Probl. Psychiat. Neuropathol. ( U S S R ) (English Transl. ) 2, 160-178. Farstad, M., and Skaug, 0. E. (1966). Scand. J. Clin. 6. Lab. Invest. 18, 657665. Fieve, R., Brauninger, G., Fleiss, J., and Cohen, C. (1965). J. Psychiat. Res. 3, 255-262. Freeman, H. (1946). A.M.A. Arch. Neurol. Psychiat. 56, 74-78. Freeman, H., Looney, J. M., Hoskins, R. G., and Dyer, C. G. (1943). A.M.A. Arch. Neurol. Psychiat. 49, 195-203. Frohmann, C. E., Beckett, P. G. S., Tourney, G., and Gottlieb, J. (1959). 2nd Intern. Congr. Psychiat., Zurich, 1957. Congress Report, Vol. 11, pp. 289291.

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Frohman, C. E., Latham, K., Beckett, P. G. S., and Gottlieb, J. S. (1960a). A.M.A. Arch. Gen. Psychiat. 2, 255-262. Frohman, C. E., Czajkowski, N. P., Luby, E. D., Gottlieb, J. S., and Senf, R. (1960b). A.M.A. Arch. Gen. Psychiut. 2,263-267. Frohman, C. E., Luby, E. D., Tourney, G., Beckett, P. G. S., and Gottlieb, J. S. ( 1 9 6 0 ~ )Am. . J . Psychiat. 117,401-408. Frohman, C . E., Ward, V., Latham, K., Beckett, P., and Gottlieb, J. (1961). Federation Proc. 20, 79. Frohman, C. E., Goodman, M., Beckett, P. G. S., Latham, L. K., Senf, R., and Gottlieb, J. ( 1962). Ann. N.Y. Acad. Sci. 96, 438447. Frohman, C. E., Latham, L. K., Beckett, P. G. S., and GottIieb, J. (1967). In “Molecular Basis of Some Aspects of Mental Activity” (0. Walaas, ed.), Vol. 2, pp. 241-255. Academic Press, New York. Gerlach, E. (1958). &dioisotopes Sci. Res., Proc. Intern. Conf., Paris, 1957. Vol. 4, pp. 74-89. Pergamon Press, Oxford. Gerlach, E., and Liibben, K. (1959). Arch. Ges. Physiol. 269, 520-554. Goldkuhl, E., and Orstrom, A. (1948). Nord. Psykiat. Medlemblad. pp. 30-38. Goldner, M. G., and Ricketts, H. T. (1942). A.M.A. Arch. Neurol. Psychiat. 4 4 552-560. Gottlieb, J. S., Frohman, C. E., Tourney, G., and Beckett, P. G. S. (1959a). A.M.A. Arch. Neurol. Psychiat. 81, 506508. Gottlieb, J. S., Frohman, C. E., Beckett, P. G. S., Tourney, G., and Senf, R. (1959b). A.M.A. Arch. Gen. Psychiat. 1,243-249. Haavaldsen, R., Lingjaerde, O., and Walaas, 0. (1958). Confnia Neurol. 18, 270-279. Harris, M. M. (1942). A.M.A. Arch. Neurol. Psychiat. 48, 761-773. Hayashi, S. (1959). 2nd Intern. Congr. Psychiatry., Ziirich, 1957. Congress Report, Vol. 11, pp. 281-288. Henneman, D. H., Altschule, M. D., and Goncz, R. M. (1954a). A.M.A. Arch. Internal Med. 94,402416. Henneman, D. H., AItschuIe, M. D., and Goncz, R. M. (1954b). A.M.A. Arch. Neurol. Psychiat. 72, 696-704, Henneman, D. H., Altschule, M. D., and Goncz, R. M. (1955). A.M.A. Arch. Internal Med. 95, 241-246. Hofmann, G., and Arnold, 0. H. (1967). In “Molecular Basis of Some Aspects of Mental Activity” (0. Walaas, ed.), Vol. 2, pp. 381396. Academic Press, New York. Holmgren. H., and Wohlfahrt, S. (1944). Actu Psychiat. Neurol. Scand. Suppl. 31, 1 3 1 9 . Honda, 0. ( 1963). Supporo Igaku Zasshi 23,345457. Juveth, L. (1957). 2. Klin. Med. 154,569-579. Karlsson, J. L. (1966). “The Biologic Basis of Schizophrenia.” Thomas, Springfield, Illinois. Kety, S. S., Woodford, R. B., Harmel, M. H., Freyhan, F. A., Appel, K. E., and Schmidt, C. F. (1948). Am. J. Psychiat. 104, 765-770. Krasnova, A. I. (1965). Zh. Nevroputol. i Psikhiatr. 65, 12061211. Kvamme, E. (1951a). J. Oslo City Hosp. 1, 106-115. Kvamme, E. ( 1951b). J. Oslo City Hosp. 1, 152-160.

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THE STUDY OF AUTOIMMUNE PROCESSES IN A PSYCHIATRIC CLINIC By S.

F. Semenov

Moscow Research Institute of Psychiatry, Moscow, USSR

I. Introduction . . . . . . . . . . . . 11. Schizophrenia . . . . . . . . . . . A. Study of the Antibrain Antibodies . . . . . . . B. Possibility of Direct Influence of Antibodies on the Brain . . 111. Vascular Diseases of Brain . . . . . . . . . IV. Neurosyphilis . . . . . . . . . . . . V. Residual Phenomena of Various Organic Affections of the Brain and . . . . . . . . . . . Psychic Trauma A. Epilepsy . . . . . . . . . . . . B. Dynamics . . . . . . . . . . . . References . . . . . . . . . . . .

291 296 297 303 306 310 310 319 321 325

I. Introduction

Recently much attention has been paid by psychiatrists to the study of autoimmune processes. On the one hand, each new aspect of the study of mental diseases gives rise to the hope that it will throw some light on the problems of etiology and pathogenesis of the main mental diseases. On the other hand, there is the danger that this new direction of study will prove to be only a blind alley, as has often happened in our science. The same situation is involved in the study of autoimmune processes in psychiatry. Not long after the formulation of the theory of allergy, this concept was used to explain the pathogenesis and etiology of mental diseases. However, these explanations are now considered erroneous and these ideas have been abandoned as having no scientsc value. This occurred mainly because the comparative-descriptive method was chiefly used in psychiatry. This could establish only an analogy between some allergic states and mental diseases: direct proof of the allergic nature of mental disorders needed the combination of methods of clinical obseFvation with experimental research. The development of experimental medicine and the use of experiment in the study of psychiatric 291

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diseases have made it possible to launch new fruitful investigations in the study of this problem. The problem of autoimmune diseases is of great interest to psychiatry. The general feature of these diseases is the appearance of antibodies not to exogenous proteins and other allergens, but to the tissues and cells of the organism itself. Examples of autoimmune diseases are the collagen disorders, disseminated lupus, some diseases of thyroid gland, e.g., Hashimoto’s disease (struma lymphamatosa ), and experimental demyelinizing encephalomyelitis ( Boyd, 1949, 1960, 1963; Waksman, 1962; Burnet,, 1962). The main etiological factors in these diseases are supposed to be specific antibodies which interact with the tissue antigens and cause dysfunction of the organ (e.g., thyroid gland, in the case of Hashimoto’s disease, or collagen tissue, in the case of collagen disorders). One should bear in mind that there is a wide range of other diseases of different etiologies in which antibodies to the tissues and organs of the same organism are created. But, in the most of these cases, autoimmune processes only accompany the action of other etiological factors. Apparently in many cases the autoimmune reactions characterize destructive processes in the affected organ. The result of these destructive processes is that the tissues become alien to the organism and cause the appearance of antibodies. Thus antibodies to heart muscle are observed in the blood of the patients in cases of myocardial infarction, and antibodies to the tissues of liver are observed in cases of some liver diseases. It is natural to postulate that some mental diseases might be connected to some extent with autoimmune processes; this idea was expressed in many forms at the end of the nineteenth and the beginning of the twentieth centuries. A note of caution: in this relatively new sphere of knowledge there is still much inconsistent data, conflicting results, and disputes over theoretical interpretations. Consequently this article should be regarded not as exposition of the finished study of some definitive sphere of knowledge, but as a search for new outlooks in the study of biological factors in psychoses on the basis of the data which were found in adjacent branches of science, such as immunology, pathophysiology, and biochemistry. We have to take into consideration the fact that some scientists are rather skeptical toward immunological trends in mental disease research, but opposing this

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attitude are rnany facts that support thc. hypothesis of the immunopathogcnesis of the brain. The main fundamentals of the modern theory of autoimmune processes derive from Mechnikov's study of cytotoxins begun in 1899. In 1901 Mechnikov for the first time obtained neurotoxic serum during immunization of rats and guinea pigs with emulsion of brain and spinal cord of doves. In the same year in Mechnikov's laboratory Delezenne obtained immune rabbit serum toward brain tissue of guinea pig, and Centani prepared neurotoxic serum by immunization of sheep with emulsion of rabbit brain. The introduction of the serum into the brain of a rabbit caused convulsions and paralysis. Some researchers found that the introduction of neurotoxic serum into the brain of animals produced unmistakable histopathological changes ( Pirone, 1903; Armand-Delille, 1906; Khoroshko, 1910, 1912; Semenov et al., 1961a). This trend of research has been developed in the study of the modern problem of experiineiital demyelinizing encephalomyelitis. This disease of the central ncrvous system is artificially produced by repeated introduction of brain matter into the organism; with some limitations it may be used as a model of multiple sclerosis. It was demonstrated that along with the harmful cytotoxic and allergic effects, in certain doses neurotoxic serum containing antibrain antibodies has a defensive adaptive influence. Mechnikov and his pupils suggested (and later proved) the possibility of directing a therapeutic influence on definite organs by means of organospecific cytotoxic serum. We can assume that the mechanism of this therapeutic influence lies in the fact that the serum neutralizes neuroantigens. Possibly small doses of antibrain antibodies have a stimulating effect on the functioning of the central nervous system. The therapeutic use of neurotoxic serum was applied to the treatment of mental diseases, especially to schizophrenia using organospecific serum containing antibrain antibodies. Usually the scriim that was used for these therapeutic purposes was produced by immunization of a larger animal (often horse) with repeated introduction of brain antigens from the brain of a healthy person killed in some accident. The serum of the animal which contained antibodies to the brain of man was repeatedly injected into the patients. The authors who have used this method report positive therapeutic effects, which were greater the shorter the period of

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time elapsed after the onset of the disease (Livshitc, 1929; Shutova and Staritsin, 1934; Morozov et al., 1959; and others). There are experimental investigations which demonstrate that the introduction of antibrain serum stops or slows up the development of experimental encephalomyelitis. Finally a very important development in the study of autoimmune processes concerned the immunology of infectious disease. The etiology and pathogenesis of many infectious diseases are known to be connected with immunological and allergic processes which are to some extent caused by etiological infectious factors, and to some extent are secondary autoimmune processes developing as the result of damage by the infectious agent of specific tissues and systems of the organism. Immunological changes caused by infection have much in common with endogenous, noninfectious, autoimmune processes. This is true with such fundamental questions as the role of lymphoid tissues and y-globulin. So the modern hypothesis about the possible role of autoimmune processes in mental diseases is based on two main sources-Mechnikov’s theory of encephalomyelitis and infectious diseases and the modern theory of collagen disorders and other autoimmune diseases. All these sources demonstrate from different points of view that the brain is not merely a passive agent but that it may in certain cases become an active component in immune processes and function as an antigen of complex structure. Before giving the clinicoimmunological data, which is the main purpose of this article, it is necessary to dwell shortly on the antigenic structure of brain. The study of the antigenic nature of brain is of great interest in connection with the immunological origin of brain tissue, The relative heterogeneity of brain for the organism is caused by the early alienation of the brain from the immune systems of the organism in the process of embryogenesis (which is comparable to the same peculiarity of other tissues-crystalline lens, skin, thyroid gland, testis); this determines the fact that the brain has high organospecific immune qualities. Thus, the brain antigens are not tolerated in the case of contact with lymphoid tissue: the latter reacts to the antigens as to the action of aIien tissue generating organospecific antibrain antibodies. The causes of activation of the antigenic properties of brain may be ( a ) increased permeability of the blood-brain barrier; ( b ) a constitutional or acquired increased activity of the immune com-

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petent system; or ( c ) the entry of brain antigens into blood because of damage to brain tissue. The brain contains heterogeneous group and specific antigens and, in addition, it has organospecific antigenic properties which differentiate it from all the other tissues. In the brain of a man there are found antigens which are the same as the antigens of animal or bird brain and also antigens corresponding to human blood. The study of the antigenic properties of brain after processing of brain tissue with different chemical factors or with heat-processing has shown that the organo-specific antigen is heterogeneous and contains at least two factors: lipoid and lipoproteid (Witebsky and Steinfeld, 1928; Kuznetsova, 1961; Popova, 1962; and others). But the antigenic structure of the brain is not limited to the class of organospecific antigens similar or different in the organisms of different animals and men. Brain tissue has a very complex system of morphological, biochemical, and functional properties, which results in the differentiation of the antigenic properties of different brain subsystems. There have been a number of reports of immune differences between the gray and white matter of the brain (Korenevskaya, 1958, 1963; Sokolov, 1960; Semenov et al., 1961a,b). In the allergic skin reaction and in the complement fixation test the white substance as a rule is more active. The majority of reports on demyelinating encephalomyelitis concur that it is more easily produced by the introduction of a suspension of white matter. There are also papers reporting antigenic differences of the gray substance of the cortex, cerebellum, and thalamus (Semenov et al., 1961a,b). All attempts to demonstrate immune differences between the gray substance of the two hemispheres of the cortex have failed. There are reports indicating serological differences between the anterior and posterior hypophysis. The latter is similar in its properties to brain ( Witebsky and Behrens, 1931). Some authors report different antigenic properties of cortex and epiphysis ( Witebsky and Reichner, 1933), cortex and tuber cinereum (Filipov, 1957), cortex and the third ventricle (Zabrodin, 1964a,b). It is necessary to point out that some authors have failed to demonstrate any immune differentiation of different sections of the brain (Mikhailov, 1909; and others). The most general outline of the working hypothesis of autoimmune diseases suggested by many authors could be formulated as follows: Autoantibodies appearing in the blood or spinal fluid,

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and coupling with the brain antibodies play a defensive adaptive role and thus neutralizing any possible toxic influences of antigens on the brain and organism as a whole. Normal and pathologically changed tissues have similar antigenic properties. Consequently the antibody action is directed not only against antigens which caused their appearance but also against normal cells. Antibodies damaging the normal cells introduce into the pathological process new tissue antigens which in their turn generate antibodies. In this way, the specific character of autoimmune diseases is explained. Autoimmune diseases, once started, have a tendency to a relapsing and lingering course because they are kept up by the vicious circle of the pathoimmune process. The case becomes more complicated because the reaction of coupling antibodies with antigens produces free histamine, serotonin, adrenaline, acetylcholine, and other biologically active substances. Psychiatric research must take into consideration the complexity of immunobiological changes in the organism and should try to evaluate the role of the immunoallergic processes in their contradictory roles of adaptive defensive and damaging actions. This work has only just commenced in psychiatry. I I . Schizophrenia

The central problem of schizophrenia is whether the etiology is psychogenic or biological. According to some views schizophrenia is a result of psychic trauma, or emotional shocks during personality development because of adverse social and, more importantly, disordered family dynamics. This theory has many followers and many papers, especially in American psychiatry, are directed to the description of the role of the psychogenic factors in the development and therapy of schizophrenia. The adherents of this trend include under the term ‘‘schizophrenia” schizophrenic reaction types. This is not accepted by many psychiatrists including the Soviet and Scandinavian workers. In these cases of short-lived reactions rather than processes, we prefer to speak of “schizophrenoid reactive” conditions or schizophrenic ‘episodes’ provoked by the emotional stress. In the works which are based on the psychogenic theory, data are often presented claiming more frequent family disorders in childhood and other psychic traumas among the schizophrenics. There is no doubt that psychotherapy and work therapy, as well as social adaptation in general, produce positive effects on the

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patient and on the quality and duration of remissions. Unquestionably, psychogenic factors have an important though not decisive influence on the development of schizophrenia. However, biological factors are also important, e.g., the genetic data; the profound changes of personality with the phenomena of specific defects; the existence of malignant types of development, which sometimes become lethal (the “lethal catatonia” of old authors); the ineffectiveness of psychotherapy without special methods of active therapy; the patterns of development ( Sneznevsky) ; the toxicity of urine, blood, spinal fluid; morphological changes in the brain, etc. According to many authors, autointoxication and particularly autoimmune processes may play a leading role in the etiology and pathogenesis of schizophrenia ( Snesarev, 1934; Krasnushkin, 1920, 1963; Kerbikov, 1962; Sivadon, 1964; Semenov, 1964; Heath, 1967; and others). Autoimmune reactions may be disclosed with the help of different methods of investigation. A. STUDYOF

THE

ANTIBRAINANTIBODIES

Using this method we have found in many cases of schizophrenia the fundamental characteristic of autoimmune diseases-antibodies to the affected organ. The complement fixation test detects antibrain antibodies in the blood and spinal fluid of these patients. Although not many papers devoted to this question have appeared compared to the purely clinical literature, nevertheless this fact may be considered as firmly established. We can interpret this to indicate that the autoimmune processes characterize not the functional change, but the processes of tissue damage; in this sense schizophrenia approaches the organic diseases of the brain. Lehman-Facius was the first to work seriously on this problem; for this purpose he employed the method of floculation of brain lipoid in the presence of serum containing antibrain antibodies. He had previously used a similar method for the differential diagnosis of malignant tumors. In 1937 he published the results of the analysis of the sera of 238 men who had schizophrenia; 225 (95%) gave a positive result in the reaction with the spinal lipoid, whereas only 4% of his patients with organic damage and 0.3%of 35 patients with “other psychoses” and psychopathy were positive. Lehman-Facius ( 1938) could distinguish three groups of diseases on the basis of the floculation. In the first group (Huntington’s chorea, schizophrenia, multiple sclerosis, brain tumors, postencepha-

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litic brain damage) positive results appeared frequently. In the second group ( epilepsy, involutional psychosis ) only occasional positive results were found. In the third group (amyotrophic lateral sclerosis, poliomyelitis, concussion of the brain, hydrocephalus, arteriosclerosis, senile dementia, symptomatic psychoses, manic-depressive psychosis, and others) there were no positive results. A total of 1019 patients were investigated. Lehman-Facius compared the activity of the lipoid fraction from the brain tissue of a schizophrenic (catatonic form) with the activity of this fraction from the brain of a normal control. The serum from 22 schizophrenics reacted with the antigen from the catatonic brain, whereas this reaction with the normal brain occurred in only 7 of these cases. The results of absorption of the serum by both antigens in turn showed that only the absorption by the brain tissue of the catatonic led to the abolition of the ability of the serum to react with brain antigens. In addition, with the help of the serum of other patients known to have antibodies to the lipoids of the brain, the author found brain antigen in some of the schizophrenic cases. Nagel (1939) could not, however, confirm this report. He investigated 100 schizophrenics, and found a large number of negative and positive reactions. This led him to doubt the methodological validity of the floculation reaction. Read et al. (1939) used the complement fixation reaction for the investigation of blood of schizophrenics. Unfortunately, they did not give the details in their article. But they often observed positive reactions with brain lipoid. On the basis of experimental observations on lesions in the brain of rabbits receiving large doses of insulin, they came to the conclusion that the antibrain antibodies appear during schimphrenia as a reaction to the introduction of the products of brain decay resulting from insulin therapy. In 1941 Lehman-Facius published some further observations. He adduced the results of the investigations of other authors, some confirming, some refuting his data. Then, in order to prove the specificity of his determination of the serum antibrain antibodies, Lehman-Facius made the following experiment. The serum with antibodies were absorbed with the lipoid fraction of human liver, rabbit carcinoma, and human brain. The activity of the serum remained during absorption by the two first antigens but disappeared completely after absorption by brain. Furthermore, the author reported the results of blind experiments with the serum and

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cerebrospinal fluid of different neuropsychic patients. According to his observations the antibrain antibodies are found most often in schizophrenics, and more often in the serum than in the cerebrospinal fluid (88 and 68%,respectively). These antibodies could also be found, but rarely, in manic-depressive psychosis (4% in the serum, 0%in the cerebrospinal fluid) and even in psychopathy (138 in the serum, 4%in the cerebrospinal fluid). A total of 423 patients were investigated. The results of these experiments confirms the immunological activity of schizophrenic serum in the presence of the lipoid brain antigen. However, there is as yet no information as to whether the floculation reaction is specific for schizophrenia. Kuznetsova and Semenov ( 1961) used the complement fixation test (with cooled serum) to investigate the serum of 127 patients suffering from different neurological or psychiatric diseases and found positive reactions with an aqueous salt extract from the human brain in 33 cases. In the group of schizophrenics the number of positive reactors was especially numerous (22 out of 84). The authors concluded that the results they found were due to the existence of specific antibodies to the brain because serum prepared from human heart or liver had no such positive reaction. This conclusion was supported by the results of experiments using selective absorption of the sera by antigens from brain and liver. Clinical analysis of the data made by Semenov et al. (1961b) led the authors to the conclusion that the antibodies found in the blood of subjects suffering from schizophrenia was the result of the schizophrenic process itself and not any previous diseases and other additional noxious factors. Normal controls showed a much lower incidence of antibodies to the hrain ( 3 4 % ) . Czechoslovakian researchers ( Skalichkova et al., 1962) used the method of absorption of serum complement and obtained similar results in a study of 106 patients suffering from schizophrenia. According to their observations a reaction between the brain aqueous-salt extract and spinal fluid or serum of the patients was more frequent in cases with evident psychotic symptoms, Antibodies in the cases of chronic, deteriorated schizophrenics were absent in most of the cases. Bulgarian workers have also obtained evidence of possibility of the presence of antibrain antibodies in some cases of schizophrenia and other psychiatric diseases (Vylchanov and Popivanov, 1964). They used the complement fixation test

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and found a positive reaction in 9.1%cases among 186 schizophrenics examined (Vylchanov and Khadzhieva, 1964). Negative results were, however, obtained by Rubin (1965), who failed to End antibrain antibodies in the serum of psychotics, using Ouchterloni’s method of microdiffusion. In various papers Semenov ( 1961a,b, 1%2, 1964a,b,c, 1965, 1967), Semenov and Fedotov (1967), Semenov and Glebov ( 19655))Semenov and Mogilina (1966), Semenov et al. ( 1961a,b, 1962,1965), and his collaborators ( Nazarov, 1961a,b, 1962; Glebov, 1962, 1964a,b, 1965; Zabrodin, 1964a,b; Chuprikov, 1966, 1967; Mogilina, 1964, 1967; Savina, 1967) obtained positive results in their own immunological investigations, establishing a definite correlation between clinical and immunoallergic manifestations of different diseases, including schizophrenia. These investigations lead us to the conclusion that there are features of neuroallergy and neuroimmunity during the development of a number of neuropsychiatric diseases. Yokoyama and his collaborators ( 1962) investigated the antibodies to brain lipoids and obtained positive results in 3 out of 13 schizophrenics, and 8 out of 16 patients with virus encephalitis and in other diseases as well. Among schizophrenics examined in our clinic there were 141 (40.6%)positive cases with antibrain antibodies in the serum, and 206 (59.4%)negative cases. These last patients had no antibrain antibodies in blood serum. One can postulate that the number of positive cases may be even greater because there were many examples when antibrain antibodies were found only at the second or third, and sometimes the fourth or fifth investigations during periods of time from one to several months. Nevertheless there were patients who continued to show negative reactions for many months. The form and peculiarities of the development of schizophrenia are evidently reflected in the immunological data. According to the experimental data in more than half the cases antibrain antibodies are found in the paranoid or catatonic forms of schizophrenia. In the cases of secondary catatonia, simple schizophrenia, and hebephrenia, antibrain antibodies are found less often. The frequency of appearance of antibodies is apparently dependent on the duration of the illness. They appear in the serum later than brain antigens (Glebov), increase with the clinical development of the schizophrenic process and the appearance of psychopathological defect symptoms, and decrease again in the last stages of the process (defect state). The frequency of detection and the reac-

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tivity of antibodies to the homological antigens of human brain is not as great as the frequency and reactivity of complement reactions with the heterological brain of rat. From this fact we may conclude that antibodies to the specific human antigens develop later in the pathological process and that the first impact of the toxic factors is dealt with by the less specific interspecies brain antiboches. The solution of this problem depends on the further immrtnological study of the serum reaction to the presence of qualitatively diverse brain antigens and antibrain antibodies. Brain antigens in cases of schizophrenia have not yet been thoroughly studied. Williams et al. (1959) used the microprecipitation ring test with antibrain immune serum, found 29 samples from schizophrenic cases in which cerebral antigens reached a high titer ( 1 :320 and more). Antigens in blood serum have been systematically studied by Glebov (1965). He used the complement fixation test and, as the antibrain serum, rabbit serum obtained after immunization with 20%suspensions of human, rat, and dog brain, and in addition the serum of the patients with a high titer (1:320) of antibodies to the brain. It was found that the blood of patients suffering from schizophrenia, as well as organic affections of the brain, contains substances whose immunological characteristics make it possible to identify them with the brain antigens. In schizophrenia 58%of cases gave a positive reaction; in a control group of blood donors of the Institute of Blood Transfusion the frequency was 4.4%.This difference was statistically significant and shows that there exists some definite connection between antigenic phenomena and the schizophrenic process. At the same time it was found, after examination of 300 patients, that the most intensive reaction of complement fixation was observed in the serum of schizophrenics under investigation using anticerebral serum, whereas the reaction with immune serum against heterogeneous types was considerably weaker. These facts are important for the understanding of the organic basis of schizophrenia, i.e., the antigens of the brain are essentially the products of brain disintegration. All this is deduced on the basis of theoretical considerations-and this is in full accord with the concept that antigen production is a feature of a group of organic forms of psychotic diseases, schizophrenia being one of them. On the other hand, in reactive psychosis, neurosis, psychopathy, and other functional disorders of nervous activity, cerebral antigens are not typical biological features of the disease. The frequency of antigen formation

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in different forms of schizophrenia is approximately the same. This

circumstance emphasizes the above-mentioned fact that antibody production in different forms of schizophrenia is a factor which is dependent on the course and clinical manifestations of schizophrenia. Thus, for example, the simple form of schizophrenia is characterized by a relatively high intensification of antigen production and very feeble antibody production. There are many reasons to suppose that this correlation of antigens and antibodies determines to a considerable extent the insiduous course of the simple type of schizophrenia. The active interaction of the antigen-antibody complex during the more intense antibody production, together with the development of autoallergic reactions, is likely to result in a more florid symptomatology which sometimes may approach an exogenous psychosis in type. According to Semenov’s data, and providing evidence in favor of the genetic basis of autoimmunity (Burnet, 1962) anticerebral antibodies are most frequently found in schizophrenics whose nearest relatives suffered from psychosis (29 out of 141 patients, i.e., 20.6%),whereas the reaction was positive in fewer patients (26 out of 206, or 12.6%) where there was no family history (in close relatives). Neuroallergic processes in schizophrenia are characterized by skin-allergic reactions with cerebral antigens and the neutrophile reaction. The skin-allergy reaction with brain antigens of schizophrenics were used for the first time by Nazarov in our clinic. Using as antigens aqueous-salt extractions of brain tissues of a man who was killed during an accident, Nazarov investigated 352 schizophrenics and found a positive allergic skin reaction in 217 patients (i.e., 61%), whereas the reaction was positive only in 6 (7%)of normal controls. Here the most distinctive feature was the influence of the duration of schizophrenic process on the frequency of the reactions. Positive reactions were observed relatively infrequently during the terminal stages of schizophrenic defect states. The greatest frequency of the reactions was noticed for the most serious conditions (72%from 209 patients under study). In cases of the development of a defect state, an increased sensitivity to brain antigens was found only in 37%of 64 patients under investigation. These data were confirmed by Zabrodin ( 1964a,b), Mogilina ( 1964, 1967), and Chuprikov (1966, 1967). The authors note a considera-

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ble increase of the frequency and intensification of allergic skin reactions during periods of aggravation of the schizophrenic process. Allergic skin reactions occur in the form of reddening and edema of the delayed “tuberculin” type. This reaction lasts for 24-28 hours. The delayed reactions, according to modern ideas, are a result of antigen-antibody interaction, fixed in tissues, or to be more exact, with “immunologically competent” lymphocytes and monocytes, which accumulate in the region of antigen injection. Thus, for schizophrenics a delayed allergic type of reaction is typical. This is observed also in multiple sclerosis and experimentally induced autoimmune diseases, e.g., demyelinating encephalitis.

POSSIBIL~Y OF DIRECT INFLUENCE OF ANTIBODIES ON THE BRAIN Recently Heath and his collaborators (1967a,b; Heath and Krupp, 1967) conducted a series of investigations indirectly suggesting that the schizophrenic’s blood contains antibrain antibodies which are homogeneous to brain tissues and which can be observed by the ( Koon’s ) fluorescent-antibody method. The authors reported that brain tissues of psychotic patients suffering from schizophrenia contain globulin, which may contain antibodies fixed by brain antigens and which can be observed by the method of fluorescent microscopy. Antigenous areas in the case of schizophrenia were mainly localized in the septa1 region and the caudate nucleus. Introduction of globulin fractions of blood serum of acute and some chronic schizophrenic patients intravenously, or into the brain ventricles, caused definite changes in the behavior of animals and changes in the EEG. The authors interpret their data as supporting the autoimmune theory of schizophrenia. These reports could not, however, be confirmed by Whittingham ,et al. (1968). Connections between the ( neurophysiological) interference with neurodynamics and neuroallergic processes are suggested by parallel investigations of the immunoallergic reactions and physiological functions of different structures of brain. These indicated a correlation between clinical features of schizophrenic remissions, complicated by alcoholic intoxication with some pathophysiological and immunological indicators ( Zabrodin, 1964a,b) . He investigated the correlations between clinical features of stuporous syndromes during schizophrenia with the electromyographic and skin allergy reactions ( Mogilina, 1964, 1967). The results of these investigations indicate B.

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some functional interference with those anatomicphysiological systems of the brain which in their immunological respect, judging by the skin sample, are in a state of allergic reaction. For instance, patients with obvious vegetative-vestibular excitability (which can be established with the help of special methods of investigation) show a correlation with a high intensity of autoallergic processes (as compared with the reactions on the other antigens of the same patient) using the antigens from the nuclei in the complex vestibular nuclei on the floor of the fourth ventricle (Zabrodin, 1964a,b). Patients with catatonic supor have an increased frequency and intensity of allergic skin reactions if the antigens from the subcortical region are used [in combination with the obvious displays of subcortical insufficiency on the electromyographic curves (“packetlike” rhythm ) ]. The neuroallergic process does not proceed in isolation, but is accompanied by a number of changes in the organism, partly caused by interference with nervous function and partly brought about by the influence of autosensitization by brain antigens. In this respect a new method of investigation of neuroallergy, which was used by Chuprikov in a mental clinic, is of great interest. This method uses the reaction between neutrophiles and antigen (according to Fradkin) the gist of which is that neutrophiles, sensitized beforehand (by incubation with the corresponding antigen ) respond by irritative reactions, which are easily detected by microscopy if the preparation is stained for glycogen. It is known that neutrophiles, as compared with other blood cells, react much more easily to antigens, and take part in the immunological reactions of the phagocytosis type ( Mechnikov) among others. Neutrophiles are supposed to be carriers of antigens and specific materials to the lymphocytes, macrophages, and plasma cells. Nevertheless neutrophiles themselves are inactive from the point of view of antibody production. The allergic reactions of neutrophiles should reflect the current degree of sensitization of the organism. Chuprikov showed that alteration produced in the neutrophile was a function of the degree of acuteness of development and the type of the schizophrenic process. The cell is more often affected in the case of the remittent type than the continuous type of schizophrenia. It was shown at the same time that species-specificallergic and immune reactions to the brain change in opposite directions; allergic reactions in sensitized

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neutrophiles are more often obscrved in response to human brain antigens, whereas antibodies arc more often obscrved to heterogeneous antigens from the brain of a rat. It is established that antibodies and the neutrophiles do not coincide. This fact makes it possible to consider antibodies as defensive factors in establishing the constancy of homeostasis. The study of antibodies, as well as allergic skin reactions, showed that the neutrophiles reacted in a similar manner toward antigens from different regions of the brain. One may conclude that schizophrenia in its different clinical manifestations and types of development is characterized as a biological process, in which both harmful and adaptive defensive mechanisms of neuroimmune allergic reactions play a significant role. It is especially interesting to note that the treatment of patients with neuroleptics influences their autoimmune processes. The general result of these studies shows that the phenothiazines decrease the indications of neuroallergic reactiveness (as measured by the allergic skin and neutrophile tests), but they do not influence the dynamics of antibrain antibodies. Therapeutic failures of phenothiazines in schizophrenia may result from an insufficient influence on autoallergic processes. There is no doubt that autoimmune processes develop in close connection with general dysfunctions of metabolic processes. Thus in the case of schizophrenia, Protopopov and Lando (in Lando, 1960) have reported dysfunctions of protein metabolism, especially an increase of globulins. Fessel (1961, 1962) found that in 33%of patients suffering from schizophrenia the serum contained an increased amount of macroglobulins of the S,, class. Korenevskaya with the help of immunoelectrophoretic analysis of the serum reported an increasing content of y-globulins, p,-macroglobulins and p,,-globulins in cases of schizophrenia. The increase occurs in the group of protein fractions, which are classified as immunoglobulins, in which different antibodies are localized. Finally it is necessary to take into consideration the possibility of including in the autoimmune processes all the hormones, especially neurohormones, for which autoimmune and autoallergic reactions are possible. Patients suffering from schizophrenia showed an increased frequency of antibodies and hormones of the thyroid gland (Goodman et al., 1963) and positive allergic skin reaction

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with some hormones of suprarenals and gonads (Ismailov, 1964). Thus the study of the pathogenesis of schizophrenia is closely connected with the study of autoimmune processes, particularly with the problem of immunopathology of brain in connection with neuiopsychiatric and metabolic processes. It is important to note that the study of the histopathology of brain in cases of so-called “hypertoxic” schizophrenia made by Romasenko ( 1967) suggests a definite allergic process, although this was somewhat obscured by the decreased reactivity of the reticuloendothelial system of these patients. Ill. Vascular Diseases of Brain

The pathology of the vascular system plays an important role in neuropsychiatry and is responsible for a number of specific syndromes; for example, arteriosclerosis, hypertensive cerebral disease, and endarteritis in its different variants. The psychiatric symptoms include epileptic seizures and alterations in consciousness, temporary dysfunctions of speech and sight and other brain functions. In the case of a progressive development, especially with repeated strokes, dementia may ensue. The vascular system is the first physiological system that is involved in the pathological process in allergy and it becomes the main sphere in which the allergic reactions develop during the progress of allergic disease. Vascular dysfunctions also play a role in cases of rheumatism, schizophrenia, epilepsy, the psychosis of advanced age, and some other neuropsychic diseases. In the literature, in addition to the neurogenic and renal theories of the etiology of hypertension, ideas of its possible allergic genesis have been put forward. It has been noted that hypertensive disease develops after chronic or acute infections which are combined with definite allergic reactions. Arnold considers hypertensive disease the result of allergic reactions involving the vascular system. Veil discovered morphological changes of hyperengic (hypersensitive) character in cases of hypertensive disease. He put forward the hypothesis that constant hypertension as well as marked changes of arterial pressure causes destruction of the walls of small arteries with the development of products of protein catabolism which sensitize the organism. These protein breakdown products derived from the walls of arteries cause changes in the sensitized arteries of a hypoengic character. As the process continues and the

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cardiovascular system of the organism becomes more and more sensitized, an interaction develops between the neural regulation and blood pressure. In this way the neurogenic phase of hypertensive disease overlaps with the vascular allergic phase. Experiments carried out by Soviet authors and investigations at the Institute of the Rumanian People’s Republic have shown the possibility of developing constant hypertensive disease in previously sensitized experimental animals. Antigens of different organs and systems including antigens of the vascular system are specific and the organism reacts to their appearance by producing specific antibodies. Complex antigens are supposed to be especially active; experimental investigations by Levkova (1958) showed that an antigen from the vascular system in combination with a streptococcal organism has an enhanced harmful effect on the vascular system and its components. With collaborators he produced an experimental arterial hypertension (without affecting the kidneys) by sensitizing rabbits with a complex antigen containing a suspension of artery together with a culture of streptococci. It is possible that a complex infectiousallergic factor may play a role in the development of hypertension (or possibly a form of it) making it necessary to use antibacterial and desensitization therapy ( Lerner et al., 1963). The Czechoslovakian authors Skalichkova et d. (1962) studied the content of antibodies to the arteries and parenchyma of the brain in the serum of patients of advanced age. They established that antibodies to gray and white substance of the brain are more frequent than antibodies to vascular tissue. The authors suggested that this indicated that these patients had atrophic senile changes dominating the arteriosclerotic process. Evidence suggesting that the parenchyma of the brain was involved in the autoimmune process in the case of diseases of the vascular system of the brain was obtained by the study of the allergic skin reaction to the injection of brain antigens and the complement fixation test (using brain antigens and antibrain antibodies). All fonns of vascular affections of brain exhibited neuroiinmunoallergic reactions although the extent and frequency varied. Investigations conducted partially under the guidance of Semenova, by Lichodey, and partially by Semenova in collaboration with Semenov’s clinic, showed the following results. Of 43 patients, 12 in the age range of 40-60, suffering from cerebral form of hypertension, arteriosclerosis of the

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brain vessels, cerebral hemorrhage, and thrombosis, had antibrain antibodies in the blood ( 9 patients) or brain antigens. It was noted that in these cases the complement fixation test using heterogeneous antigens (from rat brain) was more frequently positive and more intensive. Antibodies are not very characteristic in the early stages of acute dysfunctions of the brain circulation. At this period it is more usual to find brain antigens in the blood. This suggests some suppression of immunological reactiveness during the earliest stages of cerebral vascular disorder. A single serological investigation showed autoimmune reactions with brain tissues in more than a third of cases, so it is noteworthy that with repeated investigations the number of positively reacting sera increased sharply. This fact was supported by longitudinal studies of patients suffering from other categories of disease. All these data point to the hypothesis that vascular diseases of brain should be considered the result of a disorder of neurovascular systems in the brain structures involving to a great extent the phenomenon of neuroallergy. In the case of failure of blood supply to the brain involving damage to the brain substance, the products of neuronal degeneration appear in the blood. These are detected by the complement fixation test. These antigens sensitize the organism and produce specific antibrain antibodies; these bind the antigens, neutralizing their toxic effect, but in their turn they may possibly intensify the pathological process affecting other brain structures, thus decreasing the defensive properties of neural tissues against harmful antigens and causing dysfunction of the neural regulation of the blood circulation. During the chronic phase of vascular disease after hemorrhage the possibility of autosensitization by brain antigens is still preserved. The condition of neuroallergy, or a propensity to it, thus becomes a part of the pathogenesis of chronic neuropathological disorders. Acute vascular disorders are very often connected with the appearance of brain antigens (Glebov), and later antibrain antibodies to the brain tissues of man and rat appear. After the appearance of antibodies the brain antigens as a rule disappear. Another dynamic law of antibrain antibodies may be formulated in the rule that antibodies to the heterologous brain (e.g., rat) appear only during late stages of the disease; when they appear antibodies to homologous brain (of man) are simultaneously detected. During periods of remission the antibodies to homologous brain disappear first; heterologous antibodies are more stable. It is evident that in some cases of chronic vascular diseases after

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hemorrhage autosensitization by brain antigens is preserved. This was further shown by Lichodey under the guidance of Semyonova. This author investigated 50 patients with hemiparesis after hemorrhage, using the skin test for detecting allergy to brain antigens. Of these 39 patients showed positive reaction with brain antigens. Thus a close correlation was demonstrated between the clinical picture of the later stages of vascular disease and allergic reactions of the skin showing sensitization of the organism to brain antigens. The control skin tests with liver or heart tissues produced negative results. It is interesting that localization of the affection also influenced the quality of allergic reactions. As a rule the allergic reaction on the paralyzed side was of the slow tuberculin type and lasted for 24-48 hours. On the unaffected side the reaction lasted only 6-18 hours and was less evident. It is possible that trophic changes, which were more evident on the paralyzed side, played a role in this asymmetry of the reactions. It is possible, however, that the allergic reaction was more active on the side on which the motor functions and sensation were more affected, because functional denervation increases allergic sensitivity ( Ado, 1929, 1959). The asymmetry of the neuroallergic responses between the affected and nonaffected hemispheres may possibly be due to the different developrncnt of allergic process in them. Evidently, according to the localization of the affected area, the organism may become differentially sensitized to the antigens of the affected area. This may be shown by using antigens from different areas of the brain. The most evident positive reaction with antigens from the thalamus was observed in 9 patients with expressive emotional dysfunctions in the form of abrupt change of mood, fatigue, excitement, memory dysfunctions, and immature behavior disorders. In 14 patients with subcortical hyperkinesis allergic skin reactions to the tissues of the caudate nucleus were the most marked. All these data may be considered as evidence for the hypothesis that hemorrhage with consequent hypoxia and other biochemical dysfunctions causes damage of the brain substance. The results of this damage are brain dysfunction, especially of motor function and sensation, and second, activation of antigenous properties of brain together with autosensitization of the organism by the products of neural tissue destruction and the development of neuroallergy. Possibly brain vessels of the patients are also sensitized. The increased vegetative and vascular reactiveness of the patients with vascular diseases, the tendency to relapse which is characteristic for

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these diseases, may, in the light of data given above, be considered as the results not only of the neurological changes but also of the autoallergic changes with the phenomena of neuroallergy. The postulated autoimmune mechanism of vascular pathogenesis might be considered as general for diseases involving destructive phenomena. Nevertheless this mechanism is activated differently by different etiological factors. This may be illustrated on the data obtained in the immunological investigation of neurosyphilis. IV. Neurosyphilis

Clinical observations and histopathological investigations suggest that the clinical picture of neurosyphilis is determined not only by the infection of brain by Treponema pallidurn but also by the allergic reaction which accompanies infection. A series of immunological investigations reporting autoimmune reactions in the case of different forms of neurosyphilis are based on the detection of antibrain antibodies in the spinal fluid and serum (Marchionini, 1931; Hildebrandt, 1934; Jezkova and Shcalickova, 1961). Many authors note that the presence of antibrain antibodies to the lipoid fractions of the brain are characteristic for the vascular forms of neurosyphilis. Antibodies to the parenchyma of brain are observed less frequently. Antibodies to brain tissue are more often observed in the case of general paralysis. It is possible that this immunological difference is partly responsible for the main psychiatric differences between patients suffering from general paralysis and vascular forms of neurosyphilis. In the first case we may observe dementia which corresponds to the more evident destructive changes in the neural elements of brain. Another characteristic of immune reactions in neurosyphilis according to our data is the frequent combination of antibrain antibodies with antibodies to the liver. This suggests that neurosyphilis is a hepatocerebral immunoallergic process, The association of autoimmune reactions with the on-going syphilitic process is shown by the fact that autoimmune reactions decrease or even disappear after successful therapy of syphilitic affection of brain (Hildebrandt, 1934). V. Residual Phenomena of Various Organic Affections of the Brain a n d Psychic Trauma

This problem requires some words of introduction. It was stated that autoimmune reactions accompany ongoing organic processes

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of disease; therefore we must explain the meaning of immunological investigations of the residual phenomena of brain injuries which no longer have the specific features of the processes that took place in the past. We must also explain how we can link together conditions with such m e r e n t etiological factors-organic brain disorder and psychological stress-as if they had something in common. An attempt will be made to show that immunological methods of investigations supplementing clinical observations, allow us to observe an element of regularity in some processes of brain disorder which arise in response to stress. In our clinic we tackled this problem with the aid of different immunological and allergic methods of investigation and obtained some interesting and sometimes unexpected results. Using immunological methods, we investigated 107 patients with residual phenomena of organic brain disorder; specific antibrain antibodies were found in 40 patients 37.3%).In the great majority of these cases antibodies to the heterogeneous brain (rat) were found; whereas antibodies to homologous brain of the man were found simultaneously in only 19 of them. This disproportion in the frequency of different antibodies probably confirms the suggestion already made, linking antibrain antibodies with severe organic processes as well as the connection between less serious processes with autoimmune reactions involving heterogeneous brain antigens less specific for man. The etiology of the residual phenomena of brain organic disorder was varied: traumatic encephalopathy ( 35 patients), postmeningitic and postencephalitic syndromes, and alcoholic encephalopathy. Psychoses were met with more frequently in the seropositive group than in the seronegative one. Thus it is evident that the clinical diagnosis of residual phenomena in these cases does not correspond to the immunological picture which gives evidence of an ongoing current process. Chuprikov (1967) confirmed the presence of an antibrain autoimmune reaction in these patients using the neutrophile test. In a number of cases the clinical picture testified to the intensification of the psychopathological process. Since all the patients were characterized by psychological stress, the problem of the possible psychogenic and functional nature of the disease arises. The purely functional nature of the disease is, however, excluded by the fact that all the patients were also characterized by phenomena of autoimmune reactions, i.e., features of destructive processes. It should be emphasized that in milder reactive functional states auto-

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immune processes do not as a rule appear. In these cases autoimmune reactions are observed with the same frequency as for the group of control healthy people, i.e., approximately in 4% of all cases. More severe and progressive disorders resulting after psychic trauma are, however, qualitatively different from short-lived psychogenic reactions. In our laboratory we have found evidence that prolonged reactive psychoses developing after psychic stress but which have lost the immediate psychological connection with the trauma are accompanied by neuroallergy; we can suppose that the further development of the process in these cases is essentially caused not only by the action of the psychic stress but by consequent biological disorders of the central nervous system: for example, the processes of the autosensitization of the organism by the antigens of the brain. In these cases the complete resolution of the traumatic situation does not lead to a recovery of normal psychic activity, and the development of the psychosis proceeds. In these cases immunological methods of investigation reveal the existence of an autoimmune process. We must assume that poignant disappointments experienced by the subject, the realization of his social inferiority, the fear of retribution, and dissatisfaction with his social activities and position may create definite prerequisites for the overstrain of biological mechanisms in the brain. In this connection the mobilization of autoimmune reactions, which under usual conditions are suppressed by the activities of suprarenal cortex and pituitary hormones ( ACTH), takes place. Attempts have also been made to correlate the action of the psychogenic factors with autoimmune processes on the basis of Freud's school of thought, but these attempts do not go further than superficial analogies and it is hardly possible that they are of any scientific value. Under certain definite conditions, for example, in the presence of residual brain disorder, psychogenic factors may cause serious disorders of the bioIogical processes of the organism, resulting in an intensification of the destructive changes. This situation may promote the development, under such conditions, of autoimmune reactions. This mechanism is also of great significance in the case of epilepsy and schizophrenia. The following observations ( Semenov and Mogilina, 1966) can be used as an illustration of the intensification of the development of symptoms in organic brain disorder after a grave psychotic trauma.

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The patient N.M., 38 years of age, was hospitalized on 16/11/60 in Professor Serbsky‘s Institute of Forensic Psychiatry for examination in connection with his behavior after being arrested 011 charges of hooliganism. In 1942 he suffered a head injury; in 1947 a perforated ulcer was operated upon. After the operation some symptoms of “traumatic psychoneurosis with hysterical disorders” were observed. In 1957 he again had a serious head injury complicated by a fracture of the right temporal bone and meningitic symptoms. Shortly thereafter a short-lived psychotic state developed which was diagnosed as a “traumatic psychosis.” In 1960, after excessive use of alcohol, he spent three weeks in the Kazstenko psychiatric hospital and complained of hearing voices. In the Serbsky Institute he again complained of voices, and a stuporous condition with some elements of Parkinson’s disease developed. The following were noted: greasiness of face, mutism, oral automatism, the grasping reflex, a mild paresis of the left arm with some suppression of movements in association with simultaneous movement of the right arm, and hyperkinesis in the form of counting movements of the first and second fingers. In the process of pharmacological disinhibition the hyperkinesis almost completely disappeared. The patient talked quite willingly and said that he visualized himself in the working environment of his shop. His abnormal finger movements were the (imagined) working operations which he performed when he made bags. He also said that somebody wanted to kill him and that he was surrounded by “spies.” When the drug wore off the patient’s state again became stuporous. Approximately 9 months later the patient became normal. Symptomatic treatment gave only insignificant improvement. From July to October the patient received 400-600 mg of aminazine (chlorpromazine) daily. During the year repeated investigation of the complement fixation reaction ( CFR ) with brain antigens showed an intense reaction. I t was also shown quite clearly that the patient’s serum reacted selectively with the antigens from different morphological formations of the brain (see Table I ) . The intensity of the CFR with brain antigens and the differential reaction of antibodies with antigens prepared from different parts of the brain is of interest. This fact is likely to account for the irregular course of neuroallergic processes which may be intensified in one structure and weakened in others. Definite correlations can be established between clinical and immunological indexes of the disease.

REACTIONOF SERUMOF PATIENT N. M. Date of blood sampling 8/11/60

13/11/60

ZO/l2/60

TO

TABLE 1 ANTIGENS FROM DIFFERENTMORPHOLOGICAL FORMATIONS OF THE BRAIN Serum solution

Antigens

1:40

1:80

1:160

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

Frontal lobe Thalamus Caudate nucleus Cerebellum White matter of the hemispheres Brain of rat +++(+) ++(-) Liver of man Liver of rat Frontal lobe Occipital lobe Corpus callosum +-I-++ 1:20 1.10

+ +++ +(+I ++ + ++++ ++++ ++++ -

++(+)

-

1:320

++ -

+ -

Psychotic state Stupor, mutism, auditory, and dreamlike hallucinations, sharp inhibition of spontaneous movements, suppression of sensory and motor functions of left arm

+(+I -

++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ 1:40 1:80

Frontal lobe Occipital lobe Thalamus Caudate nucleus

of stupor state; ++ ++(+I ++++ +++ + Intensification symptoms of hyperkinesis ++++ ++++ ++++ +++++ and other symptoms of + +(+) + Parkinsonism + + +(+)

Cerebellum Liver of man

15/ 2 /6 1

++ 1:40

+++ ++++ ++++ 1:80

1:160

1:320

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

17/3/61

& Repeated invest,igation, 3/6/61

20/7/61 and 9/1/62

End of January, 1962

By end of February, beginning Frontal lobe of March, 1962, sharp Thalamus ++(+I intensification of counting Caudate nucleus movements, deepening of Cerebellum White matter of the stupor, appearance of hemispheres grasping reflex with greater manifestation to the right Brain of rat Liver of man Liver of rat All reactions of fixation with antigens from different sections of central nervous system are negative Acute intensification of inhibiNo antibodies to the brain tion, deepening of the grasping reflexes arid hyperkinesis Antibodies to the cerebellum only From July, aminazine therapy with gradual convalescence after the period of dizziness and cerebellar neurological symptoms Cerebellar symptoms comNo brain antibodies pletely disappeared, no psychosis, some symptoms of asthenia

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Some correlations are given in Table I. The first investigation (8/11/60) gave a very intense specific immune reaction associated with the change of the antigenic properties of brain tissues. The concentration of antibodies in the blood to different parts of the brain, as measured by the complement fixation reaction, gave similar results. The complement fixation reactions to frontal lobe and cerebellar antigens were active. The complement fixation reaction with antigens from the optic nerve was apparently weaker and apparently still weaker was the reaction with antigens from the striatal-pallidal system. However in the latter two cases this result was probably due to a qualitative distortion (in low dilution, the absence of a reaction; in higher dilution its presence) of the reaction 0s a result of the intense antigen production in these regions. In these conditions, depending on the degree of dilution, some proportion of the antibodies in the serum are not fixed by the brain antigens pouring into the blood. When the dilution is not so great, the fixation of antibodies by their own antigens takes place (Kuznetsova). The finding of local antibodies points to the involvement of definite regions of the brain. These symptoms of disorder are evident in the clinical picture as well. The localization of the centers of disorder as determined by the clinical-physiological methods and immunological analysis coincides to a considerable degree. In the second investigation, which took place 5 days later (13/11/60), the antibodies were exposed to antigens from the corpus callosum and occipital lobe (all four solutions from 1:40 to 1:320-4 plus). These data may be compared with the results of clinical observation. The phenomenon mentioned above of the suppression observed with some patients was described with right-sided injuries of the occipital-parietal lobes in combination with disorder of the thalamocortical connections. The antibodies found to the thalamus and occipital lobes correspond to these data. In the past the patient had suffered trauma of the right hemisphere after which he had had a loss of orientation in space for a short time. The damage of the optic analyzers was clinically demonstrated by the hallucinatory reproduction of his working environment. We can describe these hallucinations as functional “oneiros” on the analogy with functional frenzy and delirium. The dreamlike states are more characteristic of injuries of the temporal region, which in the case of the patient under question is illustrated by his amnesia. The damage to the

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optical analyzer as well as to the cerebellum, which was indicated by the immunological reactions, is a function of his excessive use of spirits. In this connection the patient had undergone predelirious states in the past. As far as the antigenic properties of the corpus callosum are concerned, these are expressed clinically in the case of our patient by the symptoms of the suppressioll of movements and perceptivity of the left part of the body. This is in accordance with our understanding of the role of corpus callosum in the coordination of function in both hemispheres of brain. The complement fixation reaction with thalamic antigens became weaker, but its distorted character remained (i.e., in low dilution, the absence of the reaction; in higher, a positive CFR). During the fourth investigation ( 18/1/61 ) , almost a month later, the complement fixation reaction with thalamic tissues, the caudate nucleus, and white matter of the brain became negative. The reaction to the antigens of the corpus callosum remained highly positive in unusually high dilution (1:640). This time the reaction of serum with other sections of the brain was not investigated. Approximately one month later on February 15, 1961, during the fifth test, it was found that the complement fixation reaction remained highly positive to the frontal lobe and cerebellum antigens in all solutions, from 1:40 to 1:320. We observed the appearance of a very weak reaction with the thalamic antigens. At the same time the reaction with the striopallidary system tissues, which were at first the most active, appeared to be negative. The transition from the distorted immunological reaction to the striopallidary system antigens into the negative one, the disorder of which was shown clinically by the hyperkinesis, was the result of the intense antigen production in this region and fixation of antibodies, which could therefore not be found in the blood. On March 17, 1961, all complement fixation reaction to cerebral antigens appeared to be negative, including antigens of midbrain. The reaction to these at first had been very active. Thus during the period of 4 months and 9 clays the immuno1ogical reactions underwent definite dynamic changes and became negative. The weakening of the immunologicaI reactions took place against the background of the general deterioration of the patient’s state. On March 7 the treatment began and some improvement took place. Afteward the immunological reactions became negative

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(with the exception of the weakly expressed CFR with cerebellar antigens ) . During this period the cerebellum symptoms were not tested. Against the background of the increasing clinical improvement the highly positive complement fixation reaction with the majority of antibodies occurred. However, during this period there was a break in the treatment and patient’s state gradually but not noticeably deteriorated. During the examination on July 3, 1961, it was found that the phenomena of psychomotor inhibition had increased, the rare answers of the patient were angry and irritable, and grasping and “trunk” reflexes appeared. The hyperkinesis in the form of counting movements of the fingers increased to such a degree that the patient lost his ability to sign his name, which he had always been able to do (during the previous period he could even write to dictation). Perspiration of the palms increased and he lost weight. During that period repeated investigations revealed no antibodies to the cerebral tissues. Then the patient was treated with large doses of chlorpromazine and his state improved. By October, 1961, he had almost recovered. At this time some weakly positive immunological reactions to all regions of the brain appeared in his blood but sometime later these reactions disappeared. Gradually the patient responded appropriately to painful experiences, hypomimia decreased, and the patient met his doctor with a smile. Only some degree of psychomotor inhibition remained. Against this background the optical-vestibular symptoms were revealed; they probably had been of significance in the mechanism of development of his former psychotic phenomena. The patient noticed that during an abrupt turn of his head to the right he got dizzy, and all objects seemed to move from left to right. (The hallucinatory images had appeared in his right field of vision.) The cerebellar symptoms were likely to have occurred earlier during stupor, but they were not revealed because of the immobility of the patient. This conclusion is suggested by the fact that the immunological investigations of CFR with cerebellar antigens at this time (cf. tests 1,2,4,5on July 20, 1961, and on January 9, 1962) disclosed a positive reaction of complement fixation to cerebellar tissues; the reaction with other regions of the brain was negative. On January 13, 1962, careful neurological study of the patient disclosed distinct cerebellar symptoms-intention tremor and past pointing more marked to the left (Babinsky’s cerebellar sign). On January 22, 1962, the cerebellar

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and optical-vestibular symptoms almost completely disappeared. During that period the antibodies to cerebellum tissues could not be demonstrated. Thus the dynamics of cerebral processes is probably accompanied by the change of the antigenic properties of the brain and immunological disorders. In the case under question the patient, with his brain damage, complicated by the excessive use of alcoholic spirits, and his psychological stresses, developed a reactive psychotic state. However, it would be wrong to infer that this psychogenic reaction is a purely functional disease as is usually done. The psychic trauma not only intensified the organic brain inferiority, as some authors have described, but also caused neuroallergic process in the vulnerable and, probably, partly in more secure, anatomo-physiological structures. The process of allergization of traumatic encephalopathy under the influence of psychic trauma on the one hand determines the “organic” character of the clinical picture, and on the other hand the prolonged character of its course. Confirmation of the role of autoimmune processes of some cases of the residual syndromes following organic brain disorders is to be found in Chuprikov’s investigations. He found in some patients under study (their diagnosis being “residual syndromes following traumatic disorder of the brain”) the injury reaction of neutrophils which indicates a current process of autosensitization of the organism by cerebral antigens. A. EPILEPSY It is known that some forms of epilepsy are caused by allergy: for example, the epilepsy caused by certain foods to which the patient develops an idiosyncrasy, Such reactions are usually caused by milk and milk products, beans, strawberries, eggs, and sometimes meat products. In these cases the positive allergic skin reactions to the corresponding food allergens often allow us to find the patliogenic foods. Their elimination from the food diet stops the episodes. The specific character of allergic skin reactions to food is not, however, absolute and a positive allergic skin reaction can be found simultaneously to different foods. This complicates the search for the epileptogenic allergen to a considerable degree. Innoculations against rabies and other infectious diseases are also known to be the cause of seizures (Ravkin, 1963) of an allergic

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character. The repeated transplantation of nervous tissues into the organism of an animal, according to some authors’ observations, very often results in spasmodic seizures in connection with neurogenic sensitization of the organism (Khoroshko, 1910, 1912; Semenov et al., 1961). The relation of epileptic seizures to endogenous autoimmune processes was revealed in the appearance of epileptiform episodes in diseases which are based on the processes of autoallergy and autosensitivity. Epileptiform seizures occur in lupus, a typical autoimmune disease which is caused by immunologically active “prohibited” clones, according to Bumet, that are directed against the most essential part of the cell-the DNA. The nuclei of the cells of all systems and organs are thus damaged. Though many disorders may be observed during the later stages of the disease, epilepsy as well as other forms of nervous and psychotic disorders may precede the development of the clinical picture of systematic lupus. In cases of antibody production to elements of connective tissue, epileptic episodes may occur so often that some authors distinguish a condition of “rheumatic epilepsy.” There exist some experimental data indicating the role of allergical and particularly autoallergical processes in the genesis of epilepsy. Ckhoroshko suggested as long ago as 1912 that the products of nervous tissue disintegration may act as toxins, resulting in central nervous system poisoning and disturbing its functions. One clinical manifestation of this process is epilepsy, Speransky ( 1955) considered the disintegration products of brain substance (autoneurotoxins) to be of great significance in the genesis of epilepsy. In a number of his papers Shapiro (1961) discusses epilepsy in connection with immunobiological disorders. Shapiro et al. (1962) came to the conclusion that in children with immunological indices close to normal, the epileptic process tends to a favorable course and the most stable remissions. The course of the disease is more complicated, the remissions less stable, and the disease no longer responds to therapy when the children show altered immunobiological indices, with a considerable decrease of the normal antibodies in their blood. The gravest course is observed in cases with a complete absence of antibodies. The authors conclude that the higher the titer of the normal antibodies of the children as well as adults having epilepsy, the more favorable is the course of the disease. Abramovitch and Shapiro

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(196l), on the basis of their analysis of isoiinmunization in the pathogenesis of children’s nervous and psychotic diseases, suggested an immunopathological etiology of epilepsy. The study of epilepsy in our clinic with the aid of different methods has established the presence of autoimmune indexes in a number of cases of epilepsy. The following may be found in the cases of epilepsy: ( a ) a positive allergic skin reaction with brain antigens; ( b ) anticerebral antibodies in blood and cerebrospinal fluid; ( c ) brain antigens in serum (not found in cerebrospinal fluid); ( d ) the positive neutrophile injury reaction in the presence of brain antigen; ( e ) the phenomenon of leukocytes sticking together, particularly in the presence of brain antigen. All five methods give positive results in patients with frequent seizures, dysphoria, and conditions of epileptic psychosis, i.e., such forms of epilepsy which are characterized by a serious course of the disease. Up to now it has been difficult to determine the frequency of autoimmune reactions according to the results of the investigations by different methods, but according to our preliminary data they are found in less than one third of the cases. The other peculiarity is that genuine epilepsy is more often accompanied by autoimmune processes than is symptomatic epilepsy. Thus we can suggest that autoimmune processes play a considerable role in the pathogenesis of endogenous epilepsy rather than in epilepsy caused by exogenous agents.

R. DYNAMICS The antibrain antibodies of epileptic patients are characterized by their lability. During different stages of investigation antibodies frequently appear or disappear. The inconstant detection of such antibodies may also be observed in other nervous and psychotic diseases. In the case of epilepsy it attracts particular attention as a phenomenon, reproducing on the immunobiological level that instability of nervous and psychotic activity which is so characteristic of epileptic patients. The clinical course of these patients is notable for its episodic nature. Epilepsy is characterized by major and minor seizures, and their psychotic equivalents, a state of dysphoria, and other profound paroxysmal disturbances of nervous and psychic activity. But even when there are no seizures the apparently compensated condition of the patient is also characterized by fluctuations of cerebral functions. Thus we can demonstrate periodic

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fluctuations of efficiency in the process of labor therapy, in the solution of experimental and psychological problems, the daily oscillations of body temperature, of levels of biochemical indices of serum, etc. On the basis of all these data it is possible to suggest that the rhythm of alteration of excitation and inhibition, or speaking on a broader scale, the rhythm of many biological processes is disturbed in the course of epilepsy. This weakness in homeostasis is likely to be one of the factors of the inherent or hereditary predisposition to epilepsy ( Semenov and Fedotov, 1967). The fluctuations of antibody content seems to be one of the phenomena of instability of homeostasis which acts uniformly with the lability of the cerebral functions. The significance of the functional state of the central nervous system in antibody production is borne out by the possibilities of both neurogenic stimulation and immunogenetic depression. The possible connection of these fluctuations with neurogenic disorders is shown by the positive relationship of maximum levels of antibody production in epilepsy in periods of frequent seizures ( Semenov, 1964c, 1967; Torba, 1967), which characterize the most profound disturbances of nervous activity. However, one must recognize a second mechanism in the periodic increase of the level of antibodies, namely, the introduction of products of brain tissue destruction (antigens) into the organism. These antigens are also found in the blood of some epileptics, Thus in the fluctuation of the level of immune processes the central nervous system seems to play a dual role-producing both neurogenic (neurohumoral) and antigenic influence on immunogenesis. The investigation of the skin reaction with brain antigens also shows the predominance of positive tests before seizures and fewer allergic skin reactions immediately after-possibly a result of desensitization by the introduction of new antigen. The bursts of paroxysmal epileptic activity seem to arise from activation of the epileptogenic focus, which is caused not only by the disorders of the equilibrium and distribution in digerent cerebral structures of physiological processes of excitation and inhibition but also by activation of brain antigenic properties and the development of autoimmune reactions. The consequences of these processes in epilepsy can be traced by the aid of other immunological methods: the neutrophile reaction and possibly after the phenomenon of leukergy (the stickiness of leukocytes) (Chuprikov, 1967; Glazov, 1967).

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The neutrophile disorder reaction, according to Chuprikov, demonstrates the autosensitization of the organism and in particular the leukocytes by the cerebral antigens. In this connection leukocytes are disordered in vitro in the presence of brain tissues. However, it is more difficult to demonstrate this in the case of epilepsy as compared with schizophrenia. The reasons for this difference remain obscure. The leukergy reaction, the allergic nature of which is still questionable, is more typical for epileptic patients than for schizophrenics. It is of special interest, as the problem of the specific character of autoimmune reactions in different nosological disorders presents a complicated and still unsolved problem. Though from a theoretical point of view the general role of autoimmune processes in different diseases seems to be indisputable, we cannot yet single out any sample for revealing autoimmune processes which would be unconditionally specific for one nosological form. The phenomenon of leukergy (the capacity of leukocytes to stick together ) according to some authorities, is linked with allergic reactions (Fleck and Borecka, 1949; Chuprikov, 1967; Glazov and Chuprikov, 1966). The study of this phenomenon shows that its presence characterizes disorders of homeostasis and that it is likely to be connected with autoallergic and allergic processes typical for these patients. Longitudinal investigations of epileptic patients revealed that the periodic fluctuations of the different states of leukergy bear a definite relation to the number of seizures. Thus in the period between seizures, according to Glazov’s data ( 1967) the quantitative evaluation of leukergy was approximately 3%; in the period when the fits were more frequent or soon after a convulsive paroxysm, the quantity of agglomerated cells increased up to 20-25%. It is possible that this phenomenon is associated with current immunoallergic reactions, which are also changeable and are also directly related to seizure frequency. We should also point out that antibodies are rarely seen in the later stages of the epileptic process, probably in connection with the general decrease of reactivity. In the later stages of the development of epileptic process the peculiar immunological reactivity decreases, together with a general degredation of brain activity. A variety of immunological reactions in respect to different antigenic structures of the brain is now known to exist (Korenevskaya, 1958, 1963; Kuznetsova, 1961; Kuznetzova and Popova, 1962; Soko-

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lov, 1960; Semenov et al., 1961a) and this gives rise to the possibility of the existence of a cybernetic type of connection between the body fluids, with their qualitatively different antibodies, and brain tissues with their heterogeneous antigenic qualities as determined by the extreme variety of combinations of properties of their protein, lipoid, proteid, and neuroendocrinological constituents. If we regard antibodies as a biological reaction of defense against antigen from different sections of the brain, one may suggest that despite the general tendency of the brain in epilepsy to act as a uniform antigen against which antibodies are produced, there are still some qualitatively different anticerebral antibodies found in the blood. This may be a manifestation of a relative selectivity of antibody production, determined by the different antigenic structures of various regions of the brain. Therefore we may suppose that during epilepsy a diffused autoneuroallergic process takes place which foIlows a complex course in the central nervous system and is partially differentiated in relation to different anatomicophysiological systems. This reaction is relatively specific and only sometimes involves other internal organs (liver, heart, etc.). Thus epilepsy can be connected with two types of allergic processes. The first type is connected with the action of exogenous allergens such as food allergens (pollen), drugs, etc.; sometimes they are etiological factors. The demonstration of these factors with the aid of allergic skin reactions and the subsequent elimination of these factors often results in clinical improvement. These types are related to symptomatic epilepsy. The other type of association of epilepsy with allergic processes is the development of endogenous autoimmune reactions caused by the sensitization of the organism by brain antigens. These autoimmune processes can take place during genuine epilepsy. In the latter they may be of great importance in the development and course of the disease. However, the cause of the origin of the autoimmune processes is still unknown. Finally, a third source of development of autoimmune processes during epilepsy is the immunological conflict between the mother’s organism and the embryo during the different stages of its development. Here to a certain extent are included the cases of epilepsy resulting from an incompatibility of blood groups of mother and child. During nervous and psychotic diseases in the mother, antibrain antibodies may be found in her blood as well as in the fetus, It is

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probable that these antibodies penetrate into the fetus through the placenta from the mother’s blood, which as a rule in such cases contains the analogous antibodies to cerebral tissue. This immunological reaction in the fetus is likely to be the source of many disorders of nervous and psychotic activities during the postnatal period, including mental retardation in various forms, or epilepsy. This branch of early brain pathology is still only in the process of development but it is promising. The problem of the relationship of the psychopathies with autoimmune processes has not yet been studied adequately. However, the preliminary data at our disposal allow us to hope that the study of this aspect of psychiatry will be promising for the solution of many theoretical and practical problems. The further improvement of the methods of laboratory investigation and the intensified study of the correlations between laboratory and clinical observations are important conditions for the successful solution of the problem of the role of autoimmune processes in the treatment of psychiatric diseases. REFERENCES Abraiiiovitch, G. B., and Shapiro, A. N. (1961). Trans. Bekhtereu Inst. 24, 79. Ado, A. D. (1929). “Antigens as Extraordinary Irritants of the Nervous System.” Moscow. Ado, A. D. (1959). Klinich. Med. 12, 3. Boyd, V. ( 1949). “Principles of Imniunology.” Boyd, V. (1960). Zh. Patofiz. Eksper. Terap. 2, 3. Boyd, V. ( 1963). “Introduction to Immunochemical Specific.” Chnprikov, A. P. (1966). “A Comparative Study of Clinical and Neuro-allergic Symptoms in Schizophrenia.” Dissertatsiya kandidatskaya. Chuprikov, A. P. (1967). Zh. Neoropatol. i Psikhiatr. 6, 918. Demin, A. A., and Kolayev, V. A. (1959). In “Rheumatism and Parasitic Diseases.” Novosibirsk. Fessel, W. (1961). J. Neruoirs M u i t d Diseuse 132, 89. Fessel, W. ( 1962). Arch. G a t . Ps!/chiat. 6 , 132. Filipov, V. V. ( 1957 ). “Antituberal Neirrotoxic Serum.” LJl;~n-Ude. Fleck, L., and Borecka, D. ( 1949 ). Mcd. Doswiadczalna Mikrobiol. 1, 427. Glazov, A. V., and Chuprikov, A. P. (1966). Proc. 6th All-Union Sci. Conf. Lab. Doctors, Moscow, pp. 160-161. Glebov, V. S. (1962). In “Voprosy kliniki, patofiziologiy i iinmunologiy shizofreniy,” Vol. 2, p. 58. Moscow. Glel)ov, V. S. ( 1964 ). In “Problemy kliniki suclelmo-psikhiatricheskoy ekspertizy patofiziologiy i iinmunologiy shizofreniy,” Vol. 11, p. 58. Moscow. Glebov, V. S. (1905). Zh. Neoropntol. i Psikhiatr. 10, 1517.

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Goodman, M., Rosenblatt, M., Gottlieb, J. S., Muller, J.. and Chen, C. A. (1963). Heath, R. G., and Krupp, J. M. (1967). Arch. Gen. Psychiat. 16, 1. Heath, R. G., Krupp, J. M., Byers, L. M., and Liljekvist, J. G. (1967a). Arch. Gen. Psychiut. IS, 10. Heath, R. G., Krupp, J. M., Byers, L. M., and Lilekvist, J. G . (1967b). Arch. Gen. Psychiat. IS, 24. Hildebrandt, A. (1934). Dermatol. Wochschr. 98, 25. Ismailov, T. I. (1964). Med. Zh. Uzbekistana 9, 60. Ivanova, A. V., and Polyakova, A. G. (1961). Terapeut. Arkh. 4, 65. Jezkova, Z., and Shcalickova, 0. ( 1961). Cesk. Psychiat. 107, 85. Kerbikov, C. V. (1962). In “Shizofreniya,” p. 5. Moscow. Khoroshko, V. K. (1910). Zh. Neuropatol. i Psikhiatr. 5-6. Khoroshko, V. K . (1912). “The Reaction of the Organism to the Introduction of Nerve Tissue.” Moscow. Korenevskaya, V. A. (1958). Byull. Eksper i B i d . Med. 45, 93. Korenvskaya, V. A. (1963). Zh. Neuropatol. i Psikhiatr. 6 , 910. Korenevskaya, V. A. (1964). Zh. Neuropatol. i Psikhiat. 9, 1355. Krasnushkin, E. K. (1920). Klinich. Med. 1, 34. Krasnushkin, E. K. (1936). In “Voprosy sotcialnoy i klinicheskoy psikhonevrologiy,” Vol. 3, p. 5. Moscow. Kuznetsova, N. I. ( 1961 ). In “Voprosy kliniki, patofiziologiy i inimuniologiy shizofreniy,” Vol. 2, p. 142. Moscow. Kuznetsova, N. I., and Semenov, S. F. (1961). Zh. Neuropatol. i Psikhiatr. 6, 869. Kuznetsova, N. I., and Popova, N. N. (1962). In “Voprosy kliniki, patofiziologiy i immuniologiy shizofreniy,” Vol. 2, p. 19. Moscow. Lehman-Facius, H. (1937). Klin. Wochschr. 16, 1646. Lehman-Facius, H. (1938). 2. Ges. Neurol. Psychiat. 161, 515. Lehman-Facius, H. (1941). Allgem. 2. Psychiat., 2. Psych. H y g . 117, 222. Lerner, N. P., Pertselay, E. M., Plotnikova, E. P., and Yudovich, E. P. (1963). In “Questions Concerning Allergy in the Clinic.” Gosmedizat, U.S.S.R., Kiev. Levkova, N. A. (1958). Arkh. Patol. 5, 59. Livshitc, Y. G. (1929). Rum. Klin. 2, 57. Marchionini. ( 1931). Deut. Z. NeruenheiZk. 122, 55. Mechnikov, I. (1901). Russ. Arkh. Patol. 8, 101. Mikhailov, S. (1909). Med. Zh. Kharkou 8, 306. Mogilina, N. P. ( 1964). In “Problemy kliniki sudebno-psikhiatricheskoy ekspertizy patofiziologiy i immunologiy shizofreniy,” Vol. 3, p. 151. Moscow. Mogilina, N. P. ( 1967). “Clinico-physiological and Immuno-allergical Peculiarities of the Stuperous Syndrome in Different Types of Schizophrenia.” Dissertatsia kandidatskaya, Moscow. Morozov, G . B., Ursova, L. G., Yumasheva, Y. S., and Kuznetsova, N. I. (1959). In “Problemy sudebnoy psikhiatriy,” p. 458. Nagel, W. (1939). Monatsschr. Psychiat. Neurol. 101, 269. Nazarov, K. N. (1961a). In “Voprosy kliniki, patofiziologiy i immunologiy shizofreniy,” Trans. SerbktJ Insf., p. 210. Moscow.

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Nazarov, K. N. (1961b). “Specific Antigens of the Cerebrum and Skin Allergy Tests Using Them.” Dissertatsia kandidatskaya, MOSCOW. Nazarov, K. N. (1962). Zh. Ncuropatol. i Psikhiatr. 8, 211. Nevzorova, T. A. (1958). “Psychopathology in the Clinic for lntexnal Diseases and Neurological Aid.” Moscow. Pirone, P. G. (1903). Arkh. Biol. Nauk 10, 77. Popova, N. N. ( 1964). In “Problemy kliniki sudebno-psiakhiatricheskoy ekspertizy patofiziologiy, i immunologiy shizofreniy,” Vol. 3, p. 38. MOSCOW. Ravkin, I. G. (1963). Zh. Nevropatol. i Psikhiatr. 5, 722. Read, C., Heilbrunn, R., and Liehert, R. (1939). J . Neruoics Menfal Disease 90, 747. Romasenko, V. A. ( 1967). “Hypertoxic Schizophrenia.” Moscow. Rubin, R. T. (1965). Brit. 1. Psychiat. 3, 479 and 999. Savina, N. S. (1967). In “Voprosy kliniki patogeneza i sudebno-psikhiatricheskoy otsenki psikhicheskikh zabolevaniy,” p. 82. Vol. 7. Moscow. Semenov, S. F. (1961a). In “Voprosy kliniki, patofiziologiy i immunologiy shizofreniy,” Vol. 1, p. 156. Moscow. Semenov, S. F. (1961b). “Schizophrenia.” Kiev. Semenov, S. F. (1962). In “Shizofreniya,” p. 215. Moscow. Semenov, S. F. ( 1964a). In “Problemy kliniki sudebno-psikhiatricheskoy ekspertizy patofiziologiy, i immunologiy shizofreniy,” Vol. 3, p. 5. Moscow. Semenov, S. F. (1964b). Zh. Nevropatol. i Psikhiatr. 64, 398. . “Epilepsiya,” Vol. 1, p. 308. Moscow. Semenov, S. F. ( 1 9 6 4 ~ ) In Semenov, S. F. (1965). Trans. 4th All-Union Congr. Neuropathologists Psychiatrists, Moscow, p. 13. Semenov, S. F. (1967). In “Aktualny problemy epilepsiy,” p. 20. Moscow. Semenov, S. F., and Fedotov, D. D. ( 1967). In “Aktualny problemy epilepsiy,” p. 5. Moscow. Semenov, S. F., and Glebov, V. S. (1965). In “Voprosy sotsialnoy i klinicheskoy psikhonevrologiy,” p. 306. Moscow. Semenov, S. F., and Mogilina, N. P. (1966). In “Voprosy sovremennoy psikhonevrologiy,” p. 477. Bekhterev Inst. Leningrad. Semenov, S. F., Georgievsky, S. N., Nazarov, K. N., and Usik, V. D. (1961a). In “Voprosy kliniki, patofiziologiy i immuniologiy shizofreniy,” Vol. I, p. 156. Moscow. Semenov, S. F., Morozov, G. B., and Kuznetsova, N. I. (196lb). Z h . Nevropatol. Psikhiat. 8, 1211. Semenov, S. F., Morozov, G. B., Senienova, K. A., Kuznetsova, N. I., Popova, N. N., and Glebov, V. S. (1962). In “Voprosy kliniki, patofiziologiy i immunologiy shizofreniy,” Vol. 2, p. 5. Moscow. Semenov, S. F., Glebov, V. S., and Chuprikov, A. P. (1965). I n “Voprosy sotsialnoy i klinichevskoy psikhonevrologiy,” p. 298. Moscow. Shapiro, A. (1961). Trans. Bekhterev Inst. 24, 24. Shapiro, A., Yakoleva, M. K., and Shnirnian, N. V. (1962). Vopr. Psikhiat. i Neuropatol. 8, 121. Shutova, 0. N., and Staritsin, S. E. (1934). Z. Klin. Med. 11-12, 17G6. Sivadon, P. ( 1964). Ann. Med. Psychol. 122, 369.

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PHYSIOLOGICAL FOUNDATIONS OF MENTAL ACTIVITY By N. P. Bechtereva and V. B. Gretchin Institute of Experimental Medicine, Leningrad, USSR

I. Introduction

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111. Some New Approaches to Physiological Investigation of Mental Activity . . . . . . . . . . A. Contingent Negative Variation (CNV) and Mental State . B. Evidence Based on Direct Contact with the Human Brain . IV. Some Theoretical Considerations on the Structure-Functional . . . . . . . . Basis of Mental Activity References . . . . . . . . . . .

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I . Introduction

Many decades of efforts in brain research have enabled psychologists to draw a marvelous filigree design of features that are peculiar to the human mind. From cIinical and anatomical experience assimilated by clinical psychology many of the details of this design have been attributed to particular structures of the brain ( Luria, 1962, 1963; Zangwill, 1963). The everyday practice of neurology, psychiatry, and especially neurosurgery has brought additional confirmation of the classical claim on relationship between brain and mental activity (Korsakoff, 1890; Bechterew, 1900; Foerster and Gagel, 1933; Alpers, 1937; Grunthal, 1939, 1947; Busch, 1940; Aleksandrovskaia et al., 1947; Glees and Griffith, 1952; Williams and Pennybacker, 1954; Orthner, 1957; Abramovich and Zakharova, 1961; Abashev-Konstantinovski, 1961, 1964; Victor et al., 1961; Adams et al., 1962; Barbizet, 1963a,b; and many others). Many of these clinicoanatomical findings have been confirmed during operations for cpilepsy or hyperkinetic disorders (Terzian and Dalle Ore, 1955; Scoville, 1954; Scoville and Milner, 1957; Penfield and Milner, 1958; Stepien and Sierpinsky, 1960; Drachman, 1964; Drachman and Arbit, 1966). After destruction of certain brain structures (those of the mediobasal temporal 329

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lobe regions in particular) some essential part of the mechanism appeared to be missing, while gates admitting the traffic of information to memory stores were deadlocked. The world with its incessant demands on the human mind was still living its statistically invariably changing life; after such a bilateral operation, this no longer concerned the brain. Recovery of its aptitude to transfer information from recent to long-term memory failed to occur. It became evident from reviews of extensive experience of operations for hyperkinetic disorders that more or less irretrievable psychiatric sequelae tended to occur in a relatively high percentage of cases, particularly after bilateral destruction of subcortical structures (amounting to some 8%)(J. S. Cooper, 1961; Spiegel et al., 1956; Spiegel, 1966). However, neither the worldwide experience of psychologists nor the clinicoanatomical basis of clinical psychology has ever attempted to disclose within the brain the fine structure-functional pattern responsible for maintaining adequate communication between man and his human environment under natural conditions of daily life. What actually takes place within the brain to enable this process to be maintained has remained even more elusive. Naturally, the major burden of research into various physiological problems has been borne by experimental investigators. It was for them to pioneer a path, where the limitation to experimentation would depend only on the talent of the experimentor. However, whereas experimentation proves fully equal to the requirements of research into such biological factors as those controlling gastrointestinal activity, its results can hardly be extrapolated so easily when considering the intricate workings of the human brain with its qualitatively new aptitudes, inherent to the contemporary stage of its development. In the 1930’s the then young method of electroencephalography (EEG) ventured to associate the experimental approach created by Pavlov’s genius with almost direct observation of the human brain. It. EEG in Conditioning and Mental Tests

Investigation of changes in electrical activity with conditioned responses in humans began as early as the 1930’s in studies by

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Durup and Fessard (1935) and by Loomis et al. (1935a,b, 1936a,b). It was shown that the alpha rhythm could be blocked by a stimulus (as sound), indifferent for the response under consideration, after it had been associated several times with illumination. The effect was extinguished after two or three presentations of sound unreinforced by light. As confirmed later, depression of the alpha rhythm in the occipitoparietotemporal brain regions may be evoked by signals conditioned to photic “reinforcement” ( Knott, 1939; Knott and Henry, 1941; Mushkina, 1956; Jasper and Shagass, 1941; Shagass, 1942; Jus and JUS,1954; Gastaut et al., 1957a,b,c). Relative invariability in the effect of illumination prompted investigators to consider and to use light as a counterpart of an unconditioned stimulus. Conditioned changes in the alpha rhythm accompanying association of different stimuli with changes in illumination were studied by Beritov and Vorobiev (1943), Gersuni and his associates (Gersuni and Korotkin, 1947; Gersuni et al., 1948; Gersuni, 1955; Kozhevnikov and Maruseva, 1949), Ataev (1955), Lurie and Rusinov ( 1955), Makarov ( 1956), and Lansing ( 1957). Maiorchik and Spirin ( 1951) investigated conditioned EEG changes accompanying reinforcement with intermittent rather than constant illumination. Studies on changes in EEG rhythms accompanying conditioned signals were made with acoustic reinforcement by Mituosi (1954) and in greater detail with proprioceptive reinforcement ( Maiorchik et at., 1954; Peimer and Fadeeva, 1956; Seregina, 1956; Gastaut et al., 1957d; Novikova and Sokolov, 1957; Menozzi and Biliach, 1963; and a number of others). The use of proprioceptive reinforcement also provided the opportunity to reveal a more or less generalized depression of the alpha rhythm, or more local and sometimes unilateral depression of rolandic rhythm (Gastaut et al., 1957d). Dynamic patterns of EEG changes with conditioned inhibition have been investigated by Maiorchik and associates (1954), Kratin (1955), Seregina (1956), Peimer and Fadeeva (1956), Novikova and Sokolov (1957), Gastaut et al. ( 1957a-d), Voronin and Sokolov (1962), Jus and Jus (1954), and others. At first, an inhibitory stimulus was found to evoke in the EEG an initial response of similar sign to that caused by a positive stimulus. A steadily inhibi-

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tory stimulus may fail to result in the desynchronization of the EEG, rather tending to augment synchronization. With presentation of inhibitory signals slow oscillations have been recorded only in occasional cases. The EEG pattern accompanying conditioned inhibition is generally attributed by investigators chiefly to the incidental concentration of the process of inhibition. Studies of this type in human subjects, as well as numerous experiments in animals, based on the same principle, have confirmed, extended, and improved many features of the theory of conditioning. They have also provided ample data on changes occurring under these conditions against the fairly monotonous electrical background pattern of the human brain in health and becoming more intricate in disease. However, we can ask what really has been contributed by these investigations to the store of human knowledge of the brain? Years spent by one of the present authors in research into these problems may to some extent justify the harshness of this statement. The electrical activity of the brain, as revealed by the electroencephalogram, has been shown to change on performance of conditioned responses. Changes may differ with performance of positive in contrast to inhibitory responses. The actual type of change depends for the most part on the initial background activity. Against an initially moderately synchronized background in a normal human subject, positive conditioned responses bring about changes of the desynchronization type, established conditioned inhibitory responses being associated with enhanced synchronization (Gastaut et at., 1957a-d). Against a different initial background other types of change, up to their reversal, may occur, even in normal persons. Under these conditions polymorphous activity of a diseased brain reveals changes that may serve for topical diagnosis, or for assessing reactivity of the brain (Bechtereva and Orlova, 1957). Focal variations of slow activity in response to conditioned signals had appeared to represent evidence for summation of inhibition (Lebedinski and Bechtereva, 1960>,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-

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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

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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

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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,

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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

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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

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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

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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

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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

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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

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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

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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?

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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.

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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.

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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-

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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.

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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<50 Fadeeva, A. A., 331, 350 Falck, B., 4, 55, 192, 195 Fa&, J. L., 182, 197 Farrell, G., 181, 183, 195 Farrer, D. S., 80, 94 Farstad, M., 263, 287 Fawcett, J. A., 155, 156, 162 Featherstone, R. M., 264, 287 Fedotov, D. D., 322, 327 Feigenberg, I. M., 230(109), 252 Feindel, W., 335, 336, 347 Feldberg, W., 4, 55 Felgenhauer, K., 73, 92 Fessard, A., 331, 347 Fessel, W., 305, 325 Fieve, R. R., 159, 168, 266, 287 Filipov, V. V., 295, 325 Fischer, J. E., 188, 190, 191, 192, 197, 198 Fisher, C. M., 329, 351 Fish, F., 133, 162 Fiske, V. hl., 187, 192, 195 Fittkau, S., 62, 92 Fitzgerald, A. E., 100, 124 Fleck, L., 323, 325 Fleiss, J., 266, 287 Flesher, J. W., 115, 126

AUTHOR INDEX

Flexner, J. B., 72, 89, 92 Flexner, L. B., 72, 89, 92 Florkin, M., 58, 92 Fodor, P. J., 71, 92 Foerster, O., 329, 347 Folin, O., 152, 162 Folk, J. E., 70, 71, 9.3, 95 Ford, D. F., 173, 195 Foss, G. L., 145, 162 Fossum, A., 229( 25), 230( 25), 249 Foster, R. S., 118, 125 Fraenkel-Conrat, H., 75, 92 Fraser, H. J., 117, 119, 125 Francis, M., 152, 161 Franck, U. F., 138, 162 Fraschini, F., 143, 165, 175, 176, 178, 182, 195, 196 Frater, R., 62, 89, 92 Freed, H., 330, 351 Freedman, D. X., 174, 195 Freeman, H., 262, 287 Freierov, 0. E., 230( log), 252 French, J. D., 35, 55 Freund, J. D., 238(56), 250 Freyhan, F. A., 266, 288 Friede, R. L., 69, 92 Friedenwald, J. S., 4, 55 Frischer, H., 266, 287 Fritze, I., 73, 97 Froshaug, H., 148, 162 F,rohman, C., 281, 289 Frohman, C. E., 217(56-58), 22,5, 272, 273, 278, 279, 281, 282, 287, 288 Frost, J. D., 334, 349 Fruton, J. S., 83, 68, 71, 92, 97 Fry, K . T., 89, 92 Fujimoto, J. M., 103, 106, 119, 126 Fukishima, M., 146, 162 Fukunaga, K., 270, 290 Furukohri, T., 130, 168 Fuxe, K., 18, 48, 55, 162

G Gaddum, J. H., 29, 55 Gagel, O., 329, 347 Gale, C. C., 150, 160 Ganong, W. F., 188, 195

359

Gantt, C. L., 146, 162 Gantt, W. A. H., 244(57, 5 8 ) , 250 Garby, L., 270, 271, 287 Garestein, N. G., 234(59), 236(59), 2.50 Gamey, J. T., 261, 289 Gavrilova, N. J., 250 Gastaut, H . A., 331, 332, 333, 347, 348 Gaudette, L., 104, 125 Gavitt, J., 114, 127 Gavrilova, N. A., 333, 349 Geist, S. H., 145, 167 Gellhorn, E., 242(61, 62), 250 Gemzell, C. A., 155, 158, 163, 168 Genkin, A. A., 333, 336, 337, 338, 339, 348 George, J. M., 70, 95 George, R., 119, 120, 125, 126 Georgievsky, S. N., 293, 327 Gerlach, E., 276, 278, 279, 288 Gershenfeld, H. M., 46, 56 Gershon, S., 159, 162, 167, 168 Gershuni, G. V., 231( 27), 249 Gersuni, G. V., 331, 348 Gerty, F. J., 262, 266, 272, 287, 289 Ciannitrapani, D., 348 Giarman, N . J., 174, 193, 195 Gibbons, J. L., 146, 154, 156, 162, 165, 166 Gibson, J. G., 150, 156, 162 Gilbert, J. B., 65, 95 Gillespie, L., 70, 95 Gillette, J. R., 108, 124 Gilliam, J. J., 138, 167 Gilligan, D. R., 67, 94 Gindis, I. Z., 231( 631, 237( 63), 250 Giordano, G., 181, 195 Cider, C., 86, 93 Gittes, R. F., 175, 195 Cjessing, L. R., 130, 132, 149, 150, 153, 156, 157, 158, 159, 162, 163, 164, 165 Gjessing, R., 131, 133, 135, 149, 150, 151, 152, 154, 156, 163 Glasser, D., 62, 92 Glaser, G. H., 156, 158, 163, 164 Glazov, A. V., 322, 323, 325

360

AUTHOR INDEX

Glazov, V. A., 212(45-46), 224, 235 (a), 250 Glebov, V. S., 300, 301, 325, 327 Glees, P., 329, 348 Glenner, G. G., 70, 71, 72, 73, 84, 85, 91, 92, 93, 95 Clenny, F. H., 172, 195 Glezer, B. B., 220(61), 225 Glod, G. D., 209(28), 224 Glynn, M. F., 266, 287 Goldkuhl, E., 271, 278, 279, 288 Goldman, H., 172, 195 Goldstein, T. P., 71, 95 Golodov, I. I., 232(65), 250 Goniori, G., 69, 93 Gonzaga, F. P., 141, 167 Goodman, M., 281, 288, 305, 326 Goodwin, B. C., 139, 163 Goodwin, J. C.,130, 149, 151, 159, 163, 164 Goldner, M. G., 262, 288 Goncz, R. M., 261, 264, 265, 286, 288, 289 Gorelik, L. I., 336, 348 Gorini, L., 139, 163 Gornall, A. G., 146, 149, 155, 163 Gorodkova, T. M., 203(11), 223 Gorski, R. A,, 143, 161 Gottlieb, N. D., 217(58), 225 Gottlieb, J., 264, 272, 281, 282, 287, 288, 289 Gottlieb, J. S., 217(56, 57), 225, 272, 273, 278, 279, 281, 287, 288, 305, 326 Coy, R. W., 142, 147, 169 Grachev, K. V., 335, 350, 351 Graham, L. T., 27, 55, 66, 92 Grassetti, D. R., 105, 127 Grave, G., 182, 197 Green, J. B., 82, 93 Green, L. J., 74, 93 Greenbaiim, L. M., 77, 93 Greenberg, R. E., 89, 91 Greenblatt, R. B., 145, 163 Creene, R., 110, 112, 113, 125 Greenshields, R. N., 75, 91 Greenstein, J. P., 65, 95 Creep, R. O., 182, 197

Gretchin, V. B., 340, 348, 352 Griffith, H. B., 329, 348 Giindel, 0. M., 231(66), 250 Groeff, F. G., 116, 127 Grollman, A. P., 114, 125 Griinthal, E., 329, 348 Guillemin, R., 81, 96 Guilmot, P., 131, 163 Guk, E. D., 234(67), 250 Gundersen, W., 139, 163 Gunne, L. M., 116, 117, 125, 155, 158, 163 Gupta, S. K., 116, 125 Gusek, W., 172, 195 Gushansky, E. L., 234(68), 250 Guttman, S., 77, 91

H Habermann, E., 76, 93 Haavaldsen, R., 280, 288 Hahn, D. W., 181, 196 Hahneman, B. M., 134, 163 Haimovich, L. A., 211(4144), 224 Halberg, F., 141, 163 Hamamoto, A., 181, 194 Hamburg, D. A., 155, 161 Hamburger, C., 141, 163 Hamilton, R. D., 73, 91 Hance, A. J., 39, 53 Hanes, C. S., 71, 93 Hanna, S . M., 130, 150, 158, 159, 161, 163, 164 Hannan, R. V., 107, 112, 127 Hano, K., 117, 118, 120, 121, 125, 127 Hanson, H., 58, 61, 62, 67, 68, 74, 92, 93 Harding, G. F . A., 157, 158, 163 Hardman, J., 73, 91 Hardwick, S. W., 154, 163 Hardy, D. C . , 100, 124 Harker, J. E., 138, 139, 163 Harmel. hl. H., 266, 288 Halper, N . J., 100, 106, 124 Harris, A., 159, 163 Harris, G. W., 141, 142, 143, 163 Harris. 1. I.. 75. 92 Harris; L. S:, 160, 118, 124, 125

AUTHOR INDEX

Harris, M. M., 262, 288 Hartinann, E., 164 IIartmann, E. L., 147, 161 IIartmann, F. A., 156, 164 Haru, I., 70, 95 Harvey, E. N., 331, 349 Harxthal, L. M., 156, 164 Hatotani, N., 146, 150, 164 Hauenschild, C., 148, 164 Hayashi, S., 265, 288 Hazen, I. M., 209(28), 224 Hearn, 1. C., 71, 96, 119, 127 Hearn, W. R., 71, 81, 92 Heath, R. G., 303, 326, 335, 348, 350 Heilbrunn, R., 298, 327 Hein, G. E., 118, 125 Heinzelman, R. V., 175, 183, 196 Hellem, A. J., 271, 290 Heller, A., 188, 189, 192, 194, 196 Heller, H., 79, 80,93 Hellman, B., 173, 174, 196 Hempel, R., 77, 96 Henneman, D. H., 261, 264, 286, 288 Henry, C. E., 331, 349 Herken, H., 107, 125 Herman, W., 134, 135, 161 Hermann, M., 134, 168 Heitz, M., 151, 164 Hes, J. P. H., 158, 164 Hess, B., 139, 164 Heyde, W., 81, 93 Hildebrandt, A,, 310, 326 Hill, R. L., 58, 61, 93, 96 Hillarp, N. A., 192, 195 Hillman, D., 141, 163 Hillman, G., 270, 289 Hiramoto, K., 145, 146, 150, 164, 168 Hird, F. J. R., 71, 93 Hirs, C. H. W., 74, 93 Hobart, G., 331, 349 Hoefer, P. F. A., 158, 164 Hoffman, G., 220(62), 225(62) 273, 274, 278, 279, 287, 288 Hoffman, J. F., 267, 273, 289 Hoffman, W. C., 158, I64 Hogg, R. V., 264, 287 Holboe, R., 229(25), 230(25), 249

361

Holliday, P., 264, 265, 286, 289 IInlinberg, G., 158, 163 IIolmgren, H., 261, 288 Holingren, U., 175, 181, 197 kI(iIter, II., 69, 93 IIonda, O., 273, 288 Honeyman, W. M., 144, 166 Hooper, K. C., 80, 83, 93 Hopsu-Harvu, V. K., 71, 85, 93 Hopsu, V. K., 71, 78, 93, 97, 172, 194 Horhyi, B., 174, 196 Horton, E. W., 30, 55, 77, 92 Hokfeldt, T., 162 Hosli L., 13, 21, 24, 29, 38, 39, 40, 41, 54, 55 Holwitz, h.I. R., 156, 167 Hosein, E. A., 67, 93 Hoskins, R. G., 242(69), 250(69), 262, 287 Hosein, E. A., 67, 93 Howard, Q. M., 150, 164 Howells, G. W., 158, 163 Hsiao, S. H., 61, 62, 69, 70, 73, 95 Hug, C. C., 103, 125, 127 Hughes, P. A., 156, 167 Huhnstock, K., 135, 164 Hullin, R. P., 130, 149, 150, 164 Hnndevadt, E., 280, 290 Hungerford, G. F., 173, 196 Hunter, H., 144, 166 Hurwitz, R., 89, 91 Huszka, G., 145, 146, 162 Hutton, J. J., 68, 93 I Ihragimova, A. A., 212(49), 213(49), 224 Idrisov, I. M., 213( 48), 213( 48), 223 Igals, D., 270, 287 Ihler, G. S., 282, 287 Ikonen, M., 173, 174, 796 Ingram, V. M., 71, 92 Inoki, R., 114, 128 Inou6, S., 142, 164 Inouye, A, 83, 93, 106, 125 Inscoe, J. K., 105, 125 Intiirrisi, C. E., 103, 106, 126

362

AUTHOR INDEX

Irving, G. W., 67, 92 Isherwood, F. A., 71, 93 Ishibashi, T., 181, 196 Ishida, C., 146, 164 Ishisu, T., 149, 164 Ismailov, 306, 326 Issakova, T. P., 236( 70), 250 Ivanov-Smolensky, A. G., 228( 71-79), 229( 71-79), 230( 71-73, 78), 231(71-76), 233(71-73, 76), 234( 76-78), 236( 71-76), 240 (78), 2A2(76, 78), 243(76, 78), 250, 251 Ivanova, A. V., 326 Ivascenko, F. I., 230(80), 251 Iwatsubo, K., 114, 128

J Jaguenod, P. A., 77, 91 Jakobson, T., 155, 164 Jakoleva, M. I., 232(81), 251 Jakovtschuk, A. I., 64, 91 Janovic, F. P., 234(82), 237(82), 239( 82), 244( 82), 251 Janssen, D., 131, 132, 164 Jashvily, V. E., 251 Jasper, H., 331, 339, 348 Jasper, H. H., 28, 54, 55 Jarnioschkewitsch, A. I., 64, 91 Jaspers, K., 136, 164 Jeavons, P. M., 158, 163 Jefremov, D. I., 235(64), 250 Jellum, E., 272, 290 Jenner, F. A., 130, 133, 149, 150, 151, 157, 158, 159, 161, 163, 164, 166 Jepson, J. B., 185, 196 Jerden, D. J., 118, 125 Jerohhina, V. N., 231( 144), 236 (144), 238( 144), %1( 144), 253 Jeroshkin, I. G., 228(84), 251 Jerusalem, C., 139, 164 Jezkova, Z., 310, 326 Johannessen, N. B., 157, 158, 163 Johannesson, T., 118, 125 Johnston, G. A. R., 7, 55 Johnston, V. S., 43, 56 Jones, G. S., 144, 166

Jones, I. H., 303, 328 Joseph, R. L., 62, 93 Jouan, P., 78, 81, 93 Juan, P., 181, 196 Juel-Nielsen, M., 158, 167 Jung, R., 333, 349 ]us, A., 234(85), 236(86), 251, 331, 332, 347, 348, 349 Jus, C., 331, 332, 348 Jus, S., 331, 332, 348, 349 Juveth, L., 267, 288

K Kaji, H. K., 117, 126 Kakimoto, Y., 93 Kakunaga, T., 117, 118, 120, 121, 125 Kalashnikova, Z. S., 231(132), 253 Kalmus, H., 137, 139, 164 Kameneva, E. N., 234(86, 87), 236 (86), 251 Kamenskaya, V. M., 240(88), 251 Kamp, A., 331, 332, 334, 347, 348, 351

Kanai, T., 16, 55 Kanavage, C. B., 73, 91 Kanazawa, A., 93 Kandara, J., 104, 124 Kane, F. J., 141, 162 Kaneto, H., 102, 117, 118, 120, 121, 125, 127 Kangnr, V. A., 230(188), 243(188), 255 Kannwischen, L. R., 188, 196 Kaplan, A., 67, 68, 93 Kappas, A., 135, 168 Kappers, A. J., 173, 196 Karlsson, J. L., 260, 288 Karniin, M., 114, 127 Krisa, I?., 4, 55 Kasparow, M., 156, 167 Kataoka, K., 83, 93 Katchalski, E., 67, 96 Katersky, E. M., 67, 94 Katinas, V. J., 232(81, 89), 251 Kato, R., 108, 125 Kato, Y., 146, 150, 164 Katz, B., 2, 55

AUTHOR INDEX

363

Kaufnian, D. A., 229(234, 235), 230 Kolesnicenko, N. S., 233(94), 251 ( 234, 235), 232 ( 234 ) , 238 ( 90, Koltsova, M. M., 229(95), 251 91), 240(234, 235), 241(234, Koizumi, K., 10, 29, 54 235), 251, 256 Kolmogorov, A. N., 338, 348 Kaufmann, J. T., 73, 96 Konovalov, V. F., 232(243), 257 Kawashima, S., 80, 94 Kooi, K. A., 333, 349 Kay, W. W., 146, 154, 158, 166 Kooul, M. L., 230( 96), 233( 96), 251 Keats, A. S., 114, 124 Kopin, I. J., 167, 184, 185, 196 Kellam, S. G . , 149, 150, 151, 162 Korenevskaya, V. A,, 295, 323, 326 Kellaway, P., 334, 349 Korj, N. N., 230(96), 233(96), 234 Keller, J. P., 74, 94 (97), 251 Kelly, E., 73, 91 Kornetsky, C., 121, 124, 125 Kemp, J. W., 105, 127 Kornfeld, D. S., 158, 163 Keppel, A., 61, 62, 69, 70, 73, 95 Kornguth, S. E., 68,96 Kerbikov, C. V., 297, 326 Korotkin, I. I., 331, 348 Kerepesi, M., 149, 168 Korsakoff, S. S., 329, 349 Kerkut, G. A., 46, 55 Kostandov, E. A., 228(99-101), 230 Kessel, D., 86, 94 (98-101), 231( 100, 101), 232 Kety, S. S., 167, 266, 288 (100, 101), 234(98, 99), 235 Key, B. J., 35, 36, 39, 42, 53, 55 (99-101), 236( 15, 98, 99), 239 Khadzhieva, 300, 528 (98), 240( 100, 101), 242(98), Khoroshko, V. K., 293, 320, 326 244( 100, 101), 246( 15), 248, Kies, M. W., 62, 94 251, 252 Kimball, H. R., 135, 168 Kosterlitz, H. W., 101, 125 King, J., 267, 286 Koyama, I., 28, 55 Kiplinger, G. F., 121, 125 Kowkowsky, N., 70, 92 Kirpicenko, A. A., 234( 92), 238( 92), Kozhevnikov, V. M., 331, 348, 349 251 Kraepelin, E., 131, 132, 152, 164 Kirn, L., 152, 164 Krasnova, A. I., 217(60, 63), 220(60, Kisseljov, N. I., 231(93), 234(93), 63), 225, 283, 288 251 Krasnushkin, E. K., 297, 326 Kitay, J. I., 175, 196 Kratin, Yu. G., 331, 349 Klages, W., 136, 164 Krech, D., 4, 53 Klein, R. A., 72, 91 Kreindler, A., 230( 103), 252 Kleist, K., 132, 164 Kretchmer, N., 89, 91 Klingman, G. I., 116, 117, 126 Kretschnier, E., 147, 164 Kliiver, H., 147, 164 Kretschnier, W., 131, 165 Knight, R. P., 158, 163 Kritskaya, V. P., 130( 104), 232( 104), Knott, I. R., 331, 349 252 Kobayashi, H., 80, 94 Krivoy, W., 114, 124 Kobrinsky, G . D., 221(70), 225 Krivoy, W. A., 76, 77, 80, 83, 94 Kochman, M., 70, 96 Krnjevi6, K., 7, 8, 10, 20, 23, 27, 28, Koelle, G. B., 4, 46, 51, 55 29, 30, 35, 42, 55 Koelle, W. A., 51, 55 Kroger, D., 76, 77, 94 Kokka, N., 108, 120, 125 Kriick, F., 143, 168 Kolayev, V. A,, 325 Kolchinskaya, A. Z., 202( G ) , 203(6), Krupenina, L. B., 209( 30, 33), 244 Kr~ipp,J. M., 303, 326 223

364

AUTHOR INDEX

Kugler, J., 333, 349 Kuhn, D., 135, 164 Kulkarni, H. J., 116, 125 Kuniaresan, P., 181, 196 Kupferberg, H. J., 102, 103, 125 Kurzmann, M., 106, 124 Kusama, T., 80, 91 Kutalia, N. A., 130(105), 233( 105), 237(105), 252 Kutin, V. P., 238( 106), 252 Kuznetsov, G. D., 331, 349 Kuznetsova, N. I., 295, 299, 323, 326, 327 Kuznetsove, N. I., 294, 326 Kvamme, E., 271, 288 Kummer, H., 147, 165 Kurland, H. D., 155, 165 Kurosawa, R., 145, 168

1 La Du, B. N., 104, 125 Lajtha, A., 103, 104, 124, 125 Lajtha, A., 58, 63, 64,66, 67, 68,71, 72, 73, 75, 82, 86, 87, 88, 89, 91, 92, 94, 97 Lakosina, N . D., 235(107), 239 (107), 252 Lamborg, M. R., 89, 92 Lande, S., 78, 94, 97 Landkof, B. L., 234(108), 252 Lando, L. I., 209(28-33), 224 Lando, L. M., 209(28), 224 Lane, A. C., 118, 125 Langfeldt, G., 262, 289 Lansing, R. W., 331, 349 Larsson, S., 173, 174, 196 Latham, L. K., 281, 282, 288, 289 Latham, K., 272, 281, 287, 288 Lauter, H., 136, 165 Laverty, R., 116, 125 Lazo-Wasem, E. A., 175, 196 Leach, F. R., 86, 94 Lebedinskaya, E. I., 230(109), 252 Lebedinski, A. V., 332, 349 Lederis, K., 79, 93 Lee, A. J., 335, 351 Lee, H., 237(110), 243(110), 252 Leenian, S., 119, 124

Lee Pang, C. H., 109, 125, 127 Legge, K. F., 42, 56 Lehman-Facius, H., 297, 326 Lehmann, H. E., 237(110), 243 (110), 252 Lellep, K., 234( l l l ) , 252 Lelord, G., 333, 349 Lembeck, F., 30, 56, 83, 92 Leonhard, K., 132, 133, 165 Lerner, A. B., 78, 94, 97, 175, 183, 185, 196 Lerner, N. P., 307, 326 Leshehinsky, A. L., 202(4), 223 Leske, R., 139, 165 Letham, L., 217(57), 225 Lettew, W. L., 141, 160 Lever, A. F., 143, 168 Levi, L., 158, 168 Levi, R., 116, 126 Levine, S., 143, 163 Levkova, N. A., 307, 326 Levkovic, A. P., 209(28), 224 Levy, A. L., 75, 92 Levy, M., 68, 94 Lewis, B. I., 156, 165 Lewis, D. J., 155, 166 Lewis, G. P., 76, 77, 92, 94 Lewis, P. R., 4, 56 Lewis, R. A., 158, 164 Li, C. H., 68, 94 Liberman, A. E., 230(224), 233 (224), 256 Libow, L. S., 149, 150, 151, 162, 165 Licko, A. E., 238(112), 252 Lideman, R. R., 221(66), 222(66), 225 Lidz, T., 156, 165 Liebert, B., 298, 327 Liljekvist, J. G., 300, 326 LinderstrGm-Lang, Z., 61, 69, 94 Lindsay, J. R., 110, 124 Lingjaerde, O., 262, 280, 285, 286, 289, 290 Lingjaerde, P., 263, 267, 285, 286, 289, 290 Linsky, V. P., 234(113), 239(113), 244( 113), 252 Lippi, U., 73, 96

AUTHOR IKDEX

Lipscomb, H. S., 81, 96 Lisk, R. D., 147, 165, 188, 196 Lister, R. E., 100, 124 Littleton, H. W., 74, 95 Livanov, M. N., 333, 349 Livshic, M. P., 232(114), 252 Livshic, N. N., 232( 115), 252 Livshitc, Y. G., 294, 326 Llewellyn, R. C., 352 Lobban, M. C., 130, 149, 164, 165 Lockett, M. F., 106, 125 Lockhart, R. A., 231( 116), 232(116), 252 Loken, F., 280, 290 Loser, A. A,, 145, 165 Lofts, B., 140, 165 Lomax, P., 118, 119, 120, 125, 126 Lommer, D., 181, 196 Loncharich, K., 281, 289 Loomis, A. L., 331, 349 Looney, J. M.,262, 264, 287, 289 Lorenzen, L., 80, 94 Lotti, V. J , 120, 126 Lovett Doust, J. W., 158, 161 Low, M. D., 334, 349 Lowery, 0. H., 67, 94 Lozovsky, D. V., 210(34), 217(50), 220(61), 221(65), 224, 225 Lubin, M., 86, 94 Luby, E., 217(58), 225 Luby, E. D., 281, 288 Liibben, K., 278, 279, 288 Lumsden, C. E., 82, 94 Lupandin, V. M., 129, 165 Luria, A. R., 329, 349 Lurie, R. N., 331, 349 Luxoro, M., 84, 96 M McCallum, W. C., 334, 351 McCaman, R. E., 84, 94 McCance, I., 9, 17, 56 McCann, S. M., 81, 94, 97 McCarthy, L. E., 116, 226 McCleary, R. A., 147, 165 McCoubrey, A,, 118, 124, 125 McCulloch, W. S., 262, 289 McCurdy, D. H., 101, 127

365

McDawson, J., 158, 160 McDonald, J. K., 79, 94 Macfarlane, 1. R., 118, 125 McIlwain, H., 71, 94, 109, 126 McIsaac, W. M., 183, 195, 196 Mackay, 1. R., 303, 328 McLean, P. D., 184, 194 AlcLennan, H., 25, 56 McMahon, R. E., 107, 126 McMillan, P. J., 70, 93 Maeda, M., 146, 149, 150, 164, 165 Magasanik, B., 110, 112, 113, 125 Maggs, R., 159, 165 Magnan, V., 133, 165 Magoun, H. W., 35, 55 Mahadevan, S., 72, 73, 94 Main, I. H. M., 30, 55 Maiorchik, V. E., 331, 349 Makarov, P. O., 331, 349 Makinen, K. K., 85, 93 Maletsky, I., 252 Malkhov, B. B., 231( 249), 244( 249), 257 Mall, G., 131, 146, 165 Malleson, A., 159, 162 Mandell, A. J., 155, 167 Mangall, E. L., 329, 351 Mangoni, A., 282, 289 Mannering, G. J., 106, 107, 126 Manso, C., 266, 287 Marayev, V. A., 232( 118), 252 March, C. H., 105, 126 Marchand, C. R., 119, 126 Marchionini, 310, 326 Margerison, J. H., 158, 160 Margolin, S. G., 156, 162 Markiw, M. E., 114, 125 Marks, M., 335, 351 Marks, N., 58, 63, 64, 65, 66, 68, 72, 73, 75, 82, 87, 89, 91, 92, 94 Marshall, F. J., 107, 126 Martin, W. R., 116, 127 Martini, L., 143, 165, 175, 178, 178, 195, 196 Maruseva, A. M., 331, 348, 349 Mascisco, J. J., 142, 161 Mashimo, T., 172, 197 Mason, J. W., 155, 161

366

AUTHOR INDEX

Mauer, I., 81, 95 Mauleon, P., 140, 166 May, D. G., 103, 106, 126 Mayer-Gross, W., 131, 165, 261, 289 Maynert, E. W., 116, 117, 126 Mayer, S. K., 301, 328 Mechanic, G., 62, 96 Mechanic, G. L., 68, 94 Mechnikov, I., 326 Medaglini, E., 234( 42), 240( 42), 249 Meduna, L. J., 261, 262, 289 Meister, A., 65, 67, 71, 86, 94, 95 Melchiorri, P., 119, 126 Meleshko, T. K., 233( 119, 120), 242 (119), 252( 119, 120) Me&, S., 267, 290 Mellett, L. B., 102, 125, 126 Menaker, A., 148, 165 Menaker, W., 148, 165 Menninger-Lerchenthal, E., 131, 133, 141, 165 Menozzi, C., 331, 349 Merlin, V. S., 131(121), 252 Mess, B., 143, 165 Mess, B., 175, 176, 178, 179, 195, 196 Meyaura, V., 299, 307, 328 Meyer, C. J., 175, 198 Meyer, J. E., 147, 165 Meyer, M., 11, 22, 56 Meyerson, B. J., 143, 165 Michael, R. P., 156, 165 Mickle, W. A., 335, 348 Mikhailov, S., 295, 326 Milcou, S. M., 172, 196 Miline, R., 178, 196 Miller, A., 71, 92, 146, 149, 155, 163 Miller, N. E., 336, 347 Miller, J. W., 101, 126, 335, 348 Mills, G. C., 270, 289 Mills, J. N., 138, 165 Milner, B., 329, 334, 350 Milthers, K., 105, 126 Mirskaya, M. M., 231(144), 236 (144), 238(144), 241( 144). 253 Mirsky, A. F., 333, 349

Misra, A. L., 105, 126 Mitchell, J. F., 15, 16, 30, 54, 56 Mitsuda, H., 133, 165 Mituosi, A., 331, 349 Mogilina, N. P., 300, 302, 303, 312, 326, 327 Moiseeva, N. I., 336, 338, 339, 349 Mollica, A., 6, 9, 10, 54 Monakhov, K. K., 234(122), 243 ( 122), 252 Monos, E., 147, 161, 182, 197 Monroe, R., 335, 350 Monroe, R. R., 335, 348 Moore, K . E., 116, 126 Moore, R. Y., 147, 165, 188, 189, 192, 194, 196 Moore, S., 70, 96 Moravtseva, S., 299, 307, 328 Moribayashi, N., 117, 118, 121, 125 Morin, R. D., 43, 56 Morley, A. A., 134, 165 Morowitz, H. J., 166 Morowv, G. B., 294, 326, 327 Morozov, G. V., 230( 123), 240 (123), 252 Morozova, T. N., 234(124), 252 Morrell, F., 331, 332, 347, 348 Morris, R., 168 Moskvocenko, K. P., 246(211), 256 Moszkowska, A., 176, 195, 196 Motta, M., 143, 165, 176, 196 Movcan, N. P., 252 Mueller, G. C., 112, 126 Mu U,S . J., 102, 105, 115, 126 Mulholland, T., 333, 350 Muller, E., 81, 96, 182, 19.5 Muller, J., 305, 326 Munson, P. L., 119, 124 Muramoto, Y., 130, 168 Murovic, B. V., 126, 252 Mushkinn, N. A,, 331, 350

N Nachlas, M. M., 71, 72, 95 Nadeau, G., 263, 289 Nadzharov, R., 214( 55), 225 Nagatsu, I., 70, 95 Nagel, W., 298, 326

AUTHOR INDEX

Nakajima, T., 93 Nakama, M., 116, 127 Nakao, A,, 68, 82, 95 Naneyslivili, B., 211( 40), 224 Naquet, R., 331, 332, 348 Nastuk, W. L., 6, 56 Natvig, R. A., 277, 278, 290 Nazarov, K. N., 293, 300, 326, 327 Nebylicyn, V. D., 229(226), 256 Neele, E., 166 Negri, M., 178, 195 Neidle, A., 71, 92 Nesic, L., 178, 196 Neubert, D., 107, 125 Neurath, H., 74, 75, 76, 91, 95 Nevzorova, T. A., 327, 329, 346 Newey, H., 86, 95 Newsome, H. H., 119, 126 Ng, M., 109, 127 Nielsen, J., 159, 166 Niemi, M., 173, 174, 196 Nikitina, G. M., 249 Noack, C. H., 168 Nomura, J., 146, 150, 164 Noreik, K., 229(26), 230(26), 236 (26), 246(26), 249 Noteboom, W. D., 112, 126 Novikova, L. A,, 331, 350 Novikova, L. V., 239( 127), 242 (127), 244(127), 252 Nuenke, J. M., 70, 79, 90, 92 Nuller, J. B., 236( 128), 253 Nusser, M. L., 264, 287

0 O’Brien, E., 267, 276, 289 Obrucnik, M., 171, 195 O’Connell, E. L., 271, 289 O’Connor, M., 150, 161 Orstrom, A., 271, 278, 279, 288, 289 Oertel, G. W., 135, 164 Oikawa, K., 130, 168 Okimoto, K. O., 110, 124 Oksche, A., 80, 94, 173, 196 Oliver, A. P., 11, 22, 34, 53 Oliverio, A,, 178, 195 Olson, E. J., 74, 95 Omorokov, L. I., 199(1), 222

367

Onodo, K., 108, 125 Orchick, C., 330, 351 O~lov,V. A., 338, 349 Orlova, A. N., 332, 346, 351 Orlovskaja, D. D., 221(67-69), 225 Orlowska, J., 71, 91 Orlowski, A. Z., 71, 95 Orlowski, M., 71, 91 Ornesi, A., 182, 195 Orofino, G., 172, 197 Ortavant, R., 140, 166 Orthner, H., 329, 350 Ortmann, R., 79, 95 Osmond, H., 243(129), 253 O’Sullivan, J. B., 156, 164 Owren, P. A., 134, 166 P Padayatty, J. D., 68, 95 Page, I. H., 183, 196 Pais, V., 233(130), 238(130), 253 Palade, G. E., 74, 93 Palkovits, M., 182, 197 Panagiotis, N. M., 173, 196 Paolozzi, F., 336, 347 Papadimitriou, C . , 131, 166 Paramonova, N. P., 255 Pardoe, A. U., 79, 95 Pare, C. M. B., 184, 185, 196 Parker, J. C., 267, 289 Parker, M. P., 142, 161 Paroli, E., 119, 126 Passow, H., 270, 289 Paton, W. D. M., 117, 126 Patterson, E. K., 61, 62, 69, 70, 73, 95 Pauleikhoff, B., 132, 166 Pavlov, I. P., 228(131), 229(131), 232( 131), 242( 131), 253 Peacock, S. M., 335, 348 Pearse, A. G. E., 68, 95 Peimer, I. A., 331, 350 Pelikan, E. W., 117, 126 Pellegrino de Iraldi, A., 172, 193, 195, 197 Penfield, W., 329, 334, 350 Penn, N. W., 89, 95 Penna-Herreros, A., 105, 127

368

AUTHOR INDEX

Pennypacker, S., 329, 351 Perrin, G. M., 265, 289 Perry, M., 70, 78, 79, 82, 92, 93 Perry, S., 135, 168 Person, R. S., 231( 132), 253 Pertselay, E. M., 307, 326 Peruzy, A. D., 178, 195 Pervomaisky, B. J., 233(133), 253 Peskova, M. V., 239(134), 253 Petakova, M., 73, 96 Petrea, I., 172, 196 Petersen, P., 150, 166 Petren, A., 152, 166 Pfeiderer, G., 73, 95, 97 PfeifTer, C. A., 166 Pfister, H. O., 243(135), 253 Phillips, L. L., 187, 197 P h i b , J. W., 4, 7, 8, 11, 13, 17, 20, 23, 26, 27, 29, 35, 42, 54, 55, 56 Phoenix, C. H., 142, 147, 169 Picard-Ami, L., 174, 195 Pipkin, I. M., 212(47), 213(47), 224 Pilcz, A,, 152, 166 Pirone, P. G., 293, 327 Pipkorn, U., 136, 166 Pisano, J. J., 67, 88, 90, 91, 95 Pittendrigh, G. S., 137, 138, 166 Piva, F., 176, 196 Player, P., 173, 197 Plichta, E. S., 81, 95 Ploticer, A. I., 239( 138, 137), 253 Plotnikoff, N. P., 105, 126 Plotnikova, E. P., 307, 326 Podkamenny, B. H., 211(43), 224 Pogge, R. C., 232(138), 253 Polishchuk, I. A., 292(7-lo), 203 (15-18), 204( 15-20), 205( 1518, 21), 205(7-10, 15, 16), 223 Polak, L., 117, 118, 124 Polyakova, A. G., 326 Pool, J. L., 335, 350 Pope, A., 69, 95 Popivanov, R., 299, 328 Popov, E. A., 242(139-1411, 243 (142), 253 Popova, N. N., 295, 323, 326, 327 Porcellati, G., 84, 95

Potter, L. T., 184, 198 Pound, J., 187, 195 Pover, W. F. R., 150, 166 Povorinsky, J. A., 231(143, 144), 236( 143, 144), 238( 143, 144), Ul(143, 144), 253 Powell, K., 118, 125 Prahl, J. W., 76, 95 Prankerd, T. A. J., 269, 276, 278, 279, 289 Prescott, J. W., 231(145), 253 Price, V. E., 65, 95 Prop, N., 174, 176, 195 Protopopov, V. P., 201(3), 202(3), 203( 3), 223( 3 ) , 228( 146), 229 (147, 148), 230( 146), 231( 1479, 233( 1477, 234( 146), 236( 148), 242( 146), 243, 253 Prusenko, A. P., 233( 148), 234( 148), 236( 148), 253 Pulido, P., 76, 91 Pwanova, T. S., 130, 169 Puskina, V. G., 231(149), 234(149), 253 Putnam, J., 187, 195 Pye, K., 139, 164, 166

Q Quarrington, B., 145, 146, 162 Quastel, J. H., 115, 124 Quastel, L., 203( lOa), 223 Quay, W. B., 173, 182, 183, 184, 191, 193, 194, 197 Quinn, G. P., 115, 126

R Rabassini, A., 265, 266, 289 Rabin, B. R., 82, 91 Rabinovic, R. L., 231( 150), 253 Radno't, M., 149, 168 Rafn, M. L., 86, 91 Ramachandran, L. K., 78, 95 Ramirez, D. V., 81, 94 Ramwell, P. W., 31, 56 Rankine, D. M., 278, 279, 287 Rand, M. J., 22, 51, 54 Randi6, M., 7, 30, 42, 55, 56

369

AUTHOR INDEX

Rapoport, R. N., 221(64), 225 Rapoport, S., 269, 289 Rasin, C. D., 202( 5, 6), 203( 6 ) , 223 Ratner, K. S., 231( 151-154), 253 (151), 254 Ratner, M., 103, 107, 110, 111, 112, 124, 126 Ravkin, I. G., 319, 327 Rayeva, S. N., 233(156), 234(155157), 236( 155-157), 241( 157), 242( 155-157), 254 Read, C., 298, 327 Rebert, 0. S., 334, 347 Redding, T. W., 81, 96, 119, 126 Redman, C. M., 115, 126 Rees, L., 156, 166 Regis, H., 331, 332, 348 Reichner, H., 295, 328 Reilly, T. J., 79, 84 Reimann, H. A,, 133, 134, 135, 136, 141, 166 Reinauer, H., 268, 270, 289 Reiss, M., 81, 95 Reisser, L. A., 231( 246), 234( 246), 239( 158), 244( 158), 254( 158), 257 Remenchik, A. P., 273, 278, 279, 287 Remmer, H., 106, 107, 126 Resnick, R. A., 62, 97 Rey, J. H., 132, 146, 155, 166 Richter, C. P., 131, 133, 134, 136, 137, 138, 140, 141, 144, 166 Richter, D., 4, 56 Richter, R., 81, 96 Ricketts, H. T., 262, 288 Riggie, H., 339, 348 Rimon, R., 155, 164 Riodan, J. F., 76, 96 Rjazansky, B. V., 235(159), 254 Roath, J. F., 155, 161 Roberts, M. H. T., 23, 35, 42, 56 Roberts, R. B., 72, 89, 92 Roberts, S. K., 141, 166 Roberts, W. W., 336, 347 Robertson, D. M., 82, 94 Robertson, J. I. S., 143, I68 Robins, E., 84, 94 Robinson, G. B., 86, 95

Rocaboy, J. C., 78, 81, 93 Rocha e Silva, M., 76, 95, 96 Rodeck, H., 120, 126 Rogas, E., 84, 96 Roger, A., 331, 332, 348 Rogov, A. A., 232( 160), 254 Rohde, M., 233( 161), 254 Romasenko, V. A,, 306, 327 Root, R., 135, 168 Roper-Hall, hl. J., 133, 166 Roscher, S., 143, 168 Rose, I. A,, 271, 289 Rosenblatt, M., 305, 326 Rosenfeld, G., 76, 96 Rosenzweig, M. R., 4, 53 Roswald, H., 333, 349 Roth, W. D., 187, 197 Rotshteyn, G. A., 208(27), 224 Rouleau, Y., 263, 289 Rowell, T. E., 147, 166 Rowntree, D. W., 154, 158, I66 Rubin, H., 140, 167 Rubin, R. T., 155, 167, 300, 327 Riimke, H. C., 132, 167 Rumley, M. K., 66, 67, 69, 97 Runnals, S., 333, 350 Rushkevic, E. A,, 229( 147, 162-166), 231( 147, 162-166), 233( 147, 162-166), 234( 165, 1%), 236 (162-166), 237( 166), 238( 166), 242( 166), 243( l66), 253( 147), 254 Rusinov, V. S., 331, 349 Rutenburg, A. M., 70, 73, 96 Ryall, R. W., 25, 30, 56 Ryan, J. W., 282, 283, 287, 289 Rybak, M., 73, 96 Rzucidlo, Z., 71, 91

S Saarma, J. M., 228( 182), 230( 184190), 231(169, 176, 182), 232 (182, 184, 187, 189, 190), 233 (167, 172, 177, 182), 234( 169, 172, 182, 183, 189, 190), 235 (169, 171, 174, 180-182, 189, 190), 236(173, 176, 182, 183, 189), 238(168, 175, 178, 182,

370

AUTHOR INDEX

189, 190), 239(168, 171, 172, 176, 179, 180, 182, 185, 186), 240( 189, 190), 241( 189, 190), 242( 182), 243( 176, 182, 188), 244(168, 170, 179, 182, 186, 189, NO), 246(177, 178, 187, 189, 190), 254, 255 Saarma, M. M., 230(189-191), 231 (189), 232( 18%191), 233( 191), 234( 189-191), 235( 189-191), 236( 189, 191), 238( 189, 190), 240(189, 190, 191), 241(189191), 242(191), 243(191), 244 ( 189-191), 246( 189-191), 255 Sachs, E. I., 352 Sachs, H., 79, 96 Sacks, W., 266, 289 SafFran, M., 79, 96 Saito, T., 81, 96 Sakurada, S., 130, 168 Salganicoff, L., 26, 56 Salmoiraghi, G. C.,7, 11, 20, 22, 32, 34, 35, 52, 53, 56 Salmon, V. J., 145, 167 Samorajski, T., 84, 96 Sampath-Kuniar, K. S. V., 76, 96 Samsonova, V. G., 231(192), 232 (192), 255 Samuels, G. M. R., 25, 31, 54, 56 Sanders, W. J,, 62, 93 Sano, I., 93 Sano, K., 336, 350 Sano, Y., 172, 197 Santoro, A., 172, 195 Sarid, S., 67, 96 Sarkissov, S. A., 229(193), 255 Sasaki, J., 130, 168 Sauer, G., 269, 289 Savaneli, N. A., 234(194), 255 Savina, N. S., 300, 327 Sawa, M., 133, 167 Sawano, S., 81, 96 Scemama, A., 176, 195 S&povic, M., 178, 196, 197 Schally, A. V., 81, 96, 119, 126, 127 Schanberg, S. M., 167 Schauer, R., 271, 289 Schaumann, W., 105, 117, 118, 127

Scheiber, S. H., 131, 167 Schein, H. M., 185, 197 Schildkraut, J. J.. 167 Schlechte, F. R., 138, 167 Schmidt, C. F., 266, 288 Schmitt, F. O., 85, 96 Schou, M., 144, 167 Schou, M., 158, 159, 161, 162, 167 Schroder, E., 77, 96 Schroder, P., 132, 167 Schuberth, J., 118, 127 Schwartz, S., 28, 55 Schwimmer, S., 62, 65, 94 Scott, K. G., 119, 127 Scoville, W. B., 329, 350 Scrafani, J., 103, 112, 114, 127 Scriver, C. R., 86, 96 Seeman, P. M., 267, 276, 289 Seevers, M. H., 100, 127 Segal, J. E., 234( 195), 239( 195), 255 Seifriz, W., 139, 167 Selbach, C., 136, 167 Selbach, H., 136, 167 Seligman, A. M., 71, 95 Semenova, K. A., 294, 327 Semenov, S. F., 293, 295, 297, 299, 300, 312, 320, 322, 326, 327 Semm, K., 80, 97 Senf, R., 217(56), 225, 273, 278, 279, 281, 288 Sens, R., 217(58), 225 Seregina, L. M., 283, 289 Seregina, T. A., 331, 350 Sereysky, M. J., 208(26, 27), 223, 224 Shader, R. I., 149, 150, 151, 162 Shafer, A. W., 278, 279, 289 Shaffer, P, A., 152, 162 Shagass, C., 333, 348, 350 Shank, R. P., 27, 55, 66, 92 Shapherd, M. D., 188, 195 Sharman, D. F., 116, 125 Shapiro, A. K., 233(196), 255 Shapiro, A. N., 320, 325, 327 Sharp, G. W. G., 149, 167 Shattock, F. M., 261, 289 Shaw, D. M., 159, 162, 167

AUTHOR INDEX

Shaw, J. E., 31, 56 Shcalickova, O., 310, 326 Shcerbina, E. A., 231(197), 234 (197), 238( 197), 243( 197, 198), 255 Sheridan, M., 130, 158, 163, 164 Shibuki, K., 130, 168 Shicko, A. K., 229( 199), 255 Shikimi, T., 120, 127 Shimuzu, H., 93 Shinagawa, Y., 106, 125 Shnirman, N. V., 321, 327 Shome, B., 79, 96 Shostakovic, V. V., 235(200), 237 (200), 238(200), 242(200), 243 (200), 255 Shpacerman, M. D., 243(201), 255 Shpir, E. R., 329, 346 Shuster, L., 107, 112, 127 Shute, C. C. D., 4, 56 Shutova, 0. N., 294, 327 Sideman, M. B., 81, 95 Sierpinsky, S., 329, 351 Sifferd, R. H., 67, 92 Siharulidze, A. I., 210( 36), 211(40), 224 Siminoff, R., 7, 56 Simon, E. J., 102, 112, 127 Simon, W., 261, 289 Simonianova, F., 73, 96 Simpson, R. T., 76, 96 Singhal, R. L., 285, 290 Sinkevic, Z. L., 236( 202), 255 Sitgreaves, R., 140, 167 Sivadon, P., 297, 327 Sizan, E. P., 232(203), 255 Skalichkova, O., 299, 307, 328 Skaug, 0. E., 263, 271, 272, 277, 278, 287, 289, 290 Slater, J. D. H., 183, 194 Slatter, K. H., 333, 350 Slavikova, V., 299, 307, 328 Sletten, I. W., 159, 167 Sloan, J. W., 116, 127 Sloane, E. M., 77, 92 Slucevsky, I. F., 242( 204), 243( 204), 255 Slusher, M. G., 119, 127

371

Smart, M., 67, 93 Smetannikov, P. G., 236(205), 255 Smirnov, V. M., 335, 336, 338, 339, 340, 344, 346, 347, 350, 352 Smith, A. A., 63, 97, 114, 127 Smith, E. E., 70, 73, 96 Smith, E. L., 58, 61, 62, 63, 67, 78, 91, 92, 96 Smith, E. R. B., 117, 128 Smith, H. W., 138, 167 Smith, J. L., 138, 167 Smith, M. E., 82, 91 Smith, V. A., 66, 92 Smyth, D. H., 86, 95 Smythies, J. R., 43, 56, 147, 167 Snell, E. F., 86, 94 Snesarev, P. E., 297, 328 Snezhnevsky, A., 214( 54), 225 Snyder, S. H., 176, 185, 187, 188, 190, 191, 192, 193, 194, 197, 198 Sovik, O., 280, 285, 286, 290 Sokolov, A. V., 295, 323, 324, 328 Sokolov, E. N., 228(252), 232(252), 255(206), 257, 331, 350, 351 Sollberger, A., 138, 167 Sologub, J. L., 236( 207), 255 Solyom, L., 234( 208), 235(208), 255 Sorokina, T. T., 207( 25a), 223 Sorot, S., 61, 62, 69, 95 Southam, A. L., 167 Spackman, P. H., 61, 96 Spasskaya, N. D., 235(209), 256 Spector, A., 62, 96 Speransky, A. L., 320, 328 Spiegel, E. A., 330, 335, 350, 351 Spillane, J. D., 156, 167 Spirin, B. G., 231(66), 250, 331, 349 Spoerlein, M. T., 112, 114, 127 Srivastava, L.,181, 196 Srivastava, S. K., 285, 290 Stalsberg, H., 174, 197 Stamm, J. S., 147, 167 Stan, M., 134, 167 Stanishevskaya, N. N., 239( 210), 256 Stankevich, L. A., 130, 167 Stanley, D. G., 267, 290

372

AUTHOR INDEX

Stanulovic, M., 73, 95 Staritsin, S. E., 294, 327 Staritsyn, A. S., 246(211), 256 Stefanis, C. N., 7, 52, 56 Stein, F., 131, 163 Stein, W. H., 70, 96 Steinberg, S. M., 240(212), 256 Steiner, F. A., 11, 22, 56 Steinfeld, G., 295, 328 Steingart, K. M., 231(213), 256 Stenback, A., 155, 164 Stengel, E., 132, 167 Stenwick, M. W., 103, 127 Stepanova, T. S., 232(214), 256 Stepien, L., 329, 351 Stem, K., 81, 96 Stevens, V. C., 146, 162 Stewart, I., 148, 167 Stokes, A. B., 146, 149, 154, 155, 158, 161, 163 Stolz, E. H., 58, 92 Storm van Leeuwen, W., 331, 332, 334, 347, 348, 351 Strandstrom, L., 155, 164 Straughan, D. W., 7, 12, 23, 30, 35, 42, 43, 53, 55, 56 Strelcova, N . I., 235(215, 216), 256 Strom, G., 158, 168 Stromgren, E., 158, 167 Strumwasser, F., 138, 167 Sukharebsky, L. M., 240(217), 256 Summers, L. B., 270, 289 Sundsten, J. W., 150, 160 Sundwall, A., 118, 127 Sung, C. Y., 119, 127 Snrratt, C., 156, 165 Sutherland, A. M., 144, 145, 167, 168 Sutherland, H., 148, 167 Swanson, H. H., 142, 167 Sykes, E. A., 43, 56 Sylven, B., 73, 96 Szara, S., 197 Szerb, J. C., 16, 17, 55, 56, 101, 127 Szewczuk, A., 70, 96

T Tachibana, M., 270, 290 Tagaki, H., 116,127

Tait, H., 146, 154, 166 Takahashi, I., 175, 183, 185, 196 Takekoshi, A., 145, 146, 150, 164, 168 Takahashi, Y., 130, 167, 267, 290 Takemori, A. E., 103, 105, 106, 107, 108, 109, 110, 125, 126, 127 Takesada, M., 93 Talairach, J., 335, 351 Talbott, J. H., 278, 279, 287 Tallan, H. H., 70, 96 Talso, P. J., 159, 167, 273, 278, 279, 287 Tarnari, Y., 114, 128 Tampier, L., 105, 127 Tanaka, A., 71, 97 Tappel, A. L., 72, 73, 94 Tarquini, B., 142, 161 Tatarenko, N. P., 228(219, 223), 230 (219-222), 230( 224), 233( 224), 235( 222, 223), 236 ( 218, 220222), 238( 221-222), 239(220), 242( 221, 222), 243( 221-223), 256 Tauben, 1. J., 130, 164 Tauc, L., 46, 56 Tcerny, M., 235(225), 256 Tendis, N., 58, 62, 67, 68, 93 Teplov, B. M., 229(226), 256 Terrell, L. C., 73, 91 Terzian, H., 329, 351 Thibault, C., 140, 166 Thii.blot, L., 81, 96, 174, 175, 178, 178, 197 Thompson, J. W., 101, 125 Thompson, R. H. S., 84, 95, 131, 168 Thorn, G. W., 156, 158, 164 Tikhonov, V. H., 220(Sl), 225 Timniler, R., 107, 125 Tirkeltaub, J. A., 240(227), 256 Tirri, R., 121, 127 Tizard, J., 234(228), 256 Tobin, W., 119, 126 Toccafondi, R., 142, 161 Tomasi, L. G., 88, 96 Torba, V. A., 322, 328 Toth, J., 104, 125 Touney, G., 217(56), 225

373

AUIlIOB 1hUUE.X

Tourney, G., 272, 273, 278, 279, 281, 287, 288 Traugott, N. N., 228(229-232), 229 ( 227-235 ) , 230 ( 229-235 ) , 232 (233-234), 234(59), 235(230, 232), 236(59), 239(229-230), 240(233-235), 241( 234235), 244( 232), 250, 256 Trohatchev, A. I., 340, 347, 352 Trousof, N., 104, 125 Toth, J., 67, 88, 94 Touchstone, J. C., 156, 167 Tonrtellotte, W. W., 81, 97 T,rams, E., 300, 328 Trap-Jensen, J., 158, 167 Trautner, E. M., 168 Tredre, B., 149, 165 Trethowan, W. H., 156, 168 Trezize, M. A,, 71, 94 Trofimov, L. G., 228( 236), 257 Tromp, S. W., 148, 168 Tsiciashvily, S. I., 236(237), 257 Tsistovic, A. S., 229( 238), 233(238), 242(238), 257 Tsuboi, K. K., 270, 274, 290 Tsucinaryova, N. I., 237(239), 238 (239), 257 Tsujika, T., 83, 93 TUPPY,H., 70, 80, 97 Turner, C. W., 181, 196 Tushkevich, Z. R.,207(256), 223

U Udalcova, M. S., 233(240), 257 Udenfriend, S., 67, 68, 88, 90, 91, 93, 95, 185, 196 Umbacli, W., 336, 351 Ungar, G . , 121, 124, 127 Upton, G . V., 78, 94, 97 Unnancheeva, T. G., 335, 351 Urse, V. G., 262, 2S9 Ursin, H., 336, 351 Ursova, L. G., 294, 326 Urstein, M., 152, 168 Usik, U. D., 293, 327 Uzman, L. L., 65, 66, 67, 69, 97

V Vaillant, G. E., 133, 168 Vallee, B. L., 75, 76, 91, 96, 97 Van Buren, J . M., 336, 351 Van Den Noort, S., 65,66, 67, 69, 97 van der Werff ten Bosch, J. J., 174, 195 Van Middlesworth, L., 150, 168 Van Praag, D., 102, 112, 127 Vanha-Pertulla, T. P . J., 78, 97 Vardapetyan, G. A., 231(27), 249 Varga, E., 234(208), 235(208), 255 Vartanian, M. E., 213( 50, 51, 64), 221(50, 51, 64), 222(51), 225 Vartanyan, M. E., 243(241), 257 Vasilyeva, V. M., 232(%2, %3), 257 Vaupel-von Harnack, M., 173, 196 Vecsei, P., 143, 168 Venables, P. H., 234(228), 256 Verri, R. A., 116, 127 Vertogradova, 0. P., 257 Verzeano, M., 35, 55 Victor, M., 329, 346, 351 Videbaeck, Aa., 134, 168 Vinogradov, N. V., 231(246), 239 (245), 234( 246), 244( 2 4 5 ) , 257 Vinogradova, 0. S., 232(247), 257 Visentin, B., 267, 287 Vjugova, L. A., 222(71), 225 Vlasova, M. M., 231(248), 257 Volper, M. I., 231(249), 244(249), 257 Vogt, M., 4, 17, 55, 56 Voldby, H., 158, 167 Volfson, N. M., 206(23, 2 4 ) , 223 Volkov, V. N., 352 von Brunt, E. E., 188, 195 volt Euler, U. S., 158, 168 von Finger, R., 156, 168 von Kley, H., 68, 95 von Mayersbach, H., 138, 139, 168 Voorhoeve, P. E., 13, 54 Vorgs, N., 146, 162 Vorobiev, A., 331, 347 Voroncov, D. S., 257

374

AUTHOR INDEX

Werle, E., 80, 97 Werman, R., 27, 55, 56, 66, 92, 97 Werre, J., 331, 332, 347, 348 Wertheimer, E., 285, 287 Westerman, R. A., 8, 56 Wharton, R. N., 159, 168 Wheatley, A., 203(10a), 223 W Whitman, R. M., 150, 151, 161 Wachsmutli, E. D., 73, 95, 97 Whittaker, V. P., 4, 25, 56 Wada, T., 130, 168 Whittingham, S., 303, 328 Wiechert, P., 82, 97 Waddington, C. H., 140, 168 Wachtler, K., 68, 95 Wiesbauer, U., 70, 97 Waelsch, H., 27, 53, 58, 71, 92, 97 Wiggans, D. S., 71, 92 Wagner, W., 147, 168 Wikler, A., 241(257), 257 Wakao, T., 146, 150, 164 Williams, M., 329, 351 Wakoh, T., 145, 168 Willocox, D. R. C., 146, 154, 166 Waksman, B. H., 292, 328 Williams, C. H., 301, 328 Williams, R. G., 138, 168 Walaas, E., 280, 285, 286, 290 Walaas, O., 280, 285, 286, 290 Wilson, 3. D., 67, 86, 95 Wilson, V. J., 13, 54 Wall, J. R., 188, 195 Wallis, D. I., 101, 125 Winfree, A. T., 140, 168 Wallner, E., 149, 168 Winkler, G., 134, 168 Walsh, E. 0. F., 109, 125, 127 Winnich, T., 78, 95 Winnik, H. Z., 158, 168 Walsh, K. A., 76, 96 Winitz, M., 71, 92 Walter, R. D., 333, 351 Walter, W. C., 333, 334, 336, 351 Witebsky, E., 295, 328 Winget, C. M., 183, 183, 194 Wangermann, E., 139, 168 Winter, A. L., 334, 347 Ward, V., 281, 288 Wintersberger, E., 70, 97 Warner, K. A., 281, 289 Warren, W. J., 334, 351 Witkop, B., 67, 93 Wohler, I. M., 77, 92 Watanabe, S., 81, 97 Wohlfahrt, S., 261, 288 Watkins, J. C., 26, 54 Wolfe, L. S., 30, 54 Wattiaux, R., 68, 69, 92 Wolff, H. G., 82, 91 Waxenburg, S. E., 145, 168 Way, E. L., 100, 101, 102, 103, 104, Wolff, J. B., 62, 97 105, 108, 119, 120, 125, 126, Wolff, H. P., 143, 168 Wolff, S. M., 131, 135, 168 121 Wolfson, A., 138, 168 Weatherall, M., 79, 95 Wolstencroft, J. H., 10, 12, 13, 14, Webb, E. C., 58, 92 15, 16, 20, 21, 23, 24, 29, 30, 31, Webb, W. B., 333, 351 33, 35, 36, 37, 38, 39, 40, 41, Weber, G., 285, 290 42, 43, 45, 52, 53, 54 Wever, R., 148, 160 Woodbury, D. M., 168 Weight, F. F., 20, 32, 56 Weil-Malherbe, H., 117, 128 Woodford, R. B., 266, 288 Woodruff, G. N., 30,54 Weinstock, M., 117, 124 Woods, J. W., 144, 166 Weise, K., 275, 278, 279, 287 Weissbach, H., 183, 184, 185, 194, Woods, L. A., 101, 102, 105, 126, 128 196 Voronin, L. G., 228(251, 252), 232 (252), 257, 331, 351 Vorono, M. S., 238(254, %5), 257 Vosnesensky, B. B., 240( 253), 257 Vyhodov, G. F., 231(256), 257 Vylchanov, V. K., 299, 300, 328

375

AUTHOR INDEX

Wooley, S. O., 86, 91 Wortis, J., 229(258), 257 Wurtman, J., 191, 193, 194, 197 Wurtman, J . J., 187, 197 Wurtman, R. J., 172, 174, 175, 176, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 194, 195, 196, 197, 198 Wwts, R. H., 339, 351 Wurtz, H., 71, 97 Wycis, H. T., 330, 335, 350, 351 Y Yakoleva, M. K., 320, 327 Yamafuji, K., 77, 93 Yamamoto, C., 22, 56 Yamamoto, I., 114, 128 Yamasaki, M., 75, 91 Yang, H. Y. T., 77, 92 Yatzuk, S. K., 332, 351 Yeager, C. L., 333, 351 Yerkes, R. M., 147, 169 Yokoyama, M., 300, 328 Yoshimoto, K., 145, 146, 150, 164, 168 Yoshimoto, S., 145, 146, 150, 164, 168 Young, J. M., 105, 127 Young, W. C., 142, 147, 169 Yudovich, E. P., 307, 326 Yumasheva, Y. S . , 294, 326

Yura, R., 146, 150, 164 Yushchenko, A. J., 199(2), 222

Z Zabrodin, G. D., 295, 300, 302, 303, 304, 328 Zaharjev, Ju. L., 209(33), 224 Zaimov, K., 130, 169 Zakharova, N. N., 236(259), 257 Zakharova, V, Y., 329, 346 Zaltzmann, P., 185, 196 Zaugwill, 0. L., 329, 351 Zanoboni, A., 182, 195 Zarikov, N. M., 234(260), 257 Zeleva, M. C., 209(30), 224 Zelinsky, S. P., u)3( 12, 13, 14), 223 Zetler, G., 30, 56 Zhirmunskaya, E. A., 352 Ziegler, L. H., 151, 164 Zieher, L. M., 133, 197 Ziff, M., 63, 97 Zomukova, L. J., 231( 144), 236 (144), 238( 144),241( 144), 253 Zubarev, J. G., 231( 144), 236( 144), 238( 144), 241( 144), 253 Zurabashvili, A. D., 210( 35), 211 ( 3 8 4 0 ) , 224 Znrabashvily, A. D., 230( 26l), 233 ( 261-263), 236 ( 263), 242( 263 ) , 258 Zweig, M., 190, 192, 197, 198

SUBJECT INDEX A Acetylcholine, drug effects on, 117118 as synaptic transmitter, 4-17, 4351 actions on neurons, ~ 1 4 32-34 , biochemical evidence, 4-5 histochemical evidence, 5-6 release of, 15-17 Acetylcholinesterase, drug effects on, 117-118 Adrenal cortex, periodic psychoses and, 154-156 Adrenal gland, catecholamines in, drug effects on, 118-117 Adrenaline, presence and action in CNS, 17-25 Adrenocortical function, pineal &land effects on, 181-183 Adrenoglomerulotropin, 181 Allergy, epilepsy and, 319-321 Amines, drug effects On, 115-118 Amino acids, as synaptic transmitters, 2S29 actions on neurons, 26-28 biochemical evidence, 25-26 release of, 28-29 y-Aminobutyric acid, &g effects O n , 117 Aminopeptidase( s ) , 59-60 in brain, 65 histological detection, 69 in nervous system, 66 fi-hinopeptide amino acid hydrolases, classification of, 59 of nervous system, 61-64 aminotripeptidase, 63-84 leucine aminopeptidase, 61-63 Aminotripeptidase, classification of, 59 in nervous system, 83-64 Amphetamine, effects on CNS, 36-38 Androgens, animal behavior and, 142144

human behavior and, 144-148

Anserinase, in brain, 66-67 Antibrain antibodies, in epilepsy, 319-331 dynamics Of, 321325 Arylamidase A, in brain, 70-71 Arylamidase B, in brain, 71-72 Arylamidase N, in brain, 72-73 Arylamidases, in brain, 65 in nervous system, 66 in Pituibry, 78-79 Arylamide amino acid hydrolases, 60 in system, 64-73 in pituitary, 78-79 Autoimmune Processes, in neurosyphilis, 310 in schizophrenia, 297-306 studies of, 291328 Autointoxication syndrome, in schizophrenia, 205-207

B Barbiturates, effects on CNS, 34-35 Brain, antibrain antibodies in schizophrenia, 297-306 effects on brain, 303-306 catecholamines in, drug effects on, 115-116 deep structures of, emotional and mental effects of, 33-36 exopeptidase distribution in, 64, 65 mental activity studies on, 335-344 metabolism of, in schizophrenia, 265-266 organic affections, psychic trauma, and autoimmunity, 310-325 subcellular fractions, enzymes in, 68-69 vascular diseases of, autoimmune processes in, 306-310

C Calcium, drug effects on, 120-121 Carbohydrate, metabolism, in schizophrenia, 259-290

376

377

SUBJECT INDEX

Carboxypeptidase( s), 59-60 in brain, 65 in nervous system, 66 Carboxypeptidase A, classification of, 59 in CNS, 74-76 mechanism of action, 75-76 Carboxypeptidase B, classification of, 59 in CNS, 76 a-Carboxypeptide amino acid hydrolases, classification of, 59 in nervous system, 73-77 carboxypeptidase A, 74-76 carboxypeptidase R, 76-87 Carnosinase, in brain, 66-67 classification of, 59 Catecholamines, action on neurons, 19-22 drug effects on, 115-117 in periodic psychoses, 158-157 presence and action in CNS, 17-35 Central depressant drugs, mode of action, 34-36 Central nervous system, synaptic transmission in, and drug action, 1-56 acetylcholine, 4-17 amino acids, 25-29 monoamines, 17-25 Central stimulant drugs, effects 011 CNS, 36-38 Cerebral cortex, electrical stimulation, mental effects of, 335 Cerebral spinal fluid, exopeptidases in, 81-82 Chlorpromazine, effects on CNS, 3841 in schizophrenia therapy, effects on HNA, 239-240, 244-245, 247 Circadian rhythms, 138-139 c e h l a r aspects of, 139 mathematical considerations, 13% 140 survival value, 140-141 Contingent negative variation (CNV), mental state and, 334-335 Corticosteroids, drug effects on, 118119

Cysteinyl-glycine dipeptidases, in brain, 86-67 drolases, 73-77 in peripheral nerve, 83-85 in pineal gland, 81 in pituitary, 77-79 protein turnover and, 88-90 in spinal cord, 8 2 8 3 transport processes and, 86-88

D Djpeptide hydrolases, classification of, 59 in nervous system, 64-69 anserinase, 68-67 carnosinase, 68-67 cysteinyl-glycine dipeptidases, 66-67 glycyl-glycine dipeptidase, 65-66 imido- and iminodipeptidase, 67-

88 6-peptidases, 68 DNA synthesis, drug effects on, 112114 Dopamine, action on neurons, 19-22 presence and action in CNS, 17-25 Drugs, affecting CNS, mode of action, 31-43 narcotic type, see Narcotic drugs

E Electroencephalography ( EEG ), in conditioning and mental tests, 330-334 of deep brain structures during mental tasks, 337-339 Endocrine system, pineal gland effects on, 174-183 Endopeptidases, 58 Enzymes, drug-metabolizing, in liver, 104-106 role in schizophrenia, 268-267 Epilepsy, autoimmune processes in, 319-325 Ergothioneine, as possible synaptic transmitter, 30 Estral clock, 141-142 Estrogens, animal behavior and, 142, 144

378

SUBJECT INDEX

human behavior and, 144-148 Exopeptidases, in cerebral spinal fluid, 81-82 classification of, 58-61 hormone activity and, 85 in hypothalamus, 79-81 inborn metabolic errors and, 85-86 of nervous system, 57-97 a-aminopeptide amino acid hydrolases, 6%73 dipeptide hydrolases, 64-69 a-carboxypeptide amino acid hy-

Genital apparatus, pineal gland effects on, 175-178 Gjessing’s studies on periodio psychoses, 152-154 Glomerulotropin, see Andrenoglomerulotropin Glucose tolerance tests, in schizophrenia, 261-262 y-Glutamyl peptides, metabolism of, 71 Glycyl-glycine dipeptidase, classification of, 59 in nervous system, 65-66

H Haloperidol, in schizophrenia therapy, effects on HNA, 240-241, 244245, 247 Higher nervous activity ( HNA), studies, alterations in schizophrenia, 232235, 241-248 special features of, 235-238 under treatment, 238-241, 244 use to assess improvement, 244246 methodological problems in, 229232 Histamine, as possible synaptic transmitter, 29 Homocarnosine, in brain, 67 Hormones, activity of, exopeptidases and, 85 drug effects on, 118-119

3,4-Hydroxyphenyletliylamine, see

Dopamine 5-Iiydroxytryptamine, action on neurons, 22-25, 32-34 presence and action in CNS, 17-25 5-Hydroxytryptophan decarboxylase, in pineal gland, 185 Hypoid syndrome, in schizophrenia, 201-202, 242 Hypoenergetic syndrome, in schizophrenia, 204-.205 Hypothalamic-releasing factor, exopeptidases and, 80-81 Hypothalamus, exopeptidases of, 7% 81 I

Imidodipeptidase, in brain, 67-68 classification of, 59 Iminodipeptidase, in brain, 67-68 classification of, 59 Inborn metabolic disorders, exopeptidases and, 85-86 Institute of Psychiatry (USSR), schizophrenia studies at, 216-222 Insulin, in schizophrenia therapy, effects of HNA, 238-239, 244245,247 tolerance tests, in schizophrenia, 262-283 Intermediary metabolism, drug effects on, 108-110

K Kinins, 76-77

1 Lactate/pyruvate ratio, in schizophrenia, 217-221, 284-285, 281283 Leucine aminopeptidase (LAP), classification of, 59 in nervous system, 61-63 properties of, 62 Leukergy, in allergic reactions, 322, 323

379

SUBJECT INDEX

Light, effects on pineal activity, 187191 menstrual cycle and, 148-149 Lipid metabolism, drug effects on, 115 Lithium, in periodic psychoses, 158160 Liver, drug-metabolizing enzymes of, 104-106 LSD-25, effects on CNS, 4 2 4 3

h\ Melatonin, biosynthesis and metabbolism of, 183-187 biorhythm of, 191-193 pathways of, 186 effects on reproductive function, 175-176 Memory, brain studies and, 340-342 charts of reproductibility dynamics in studies on, 340442 EEG studies and, 339 Menstrual clock, 141-142 Menstrual cycle, light and, 148-149 Mental activity, brain studies on, 335344 contingent negative variation and, 334435 physiological foundations of, 329352 steady potential, neuron population activity and oxygen in brain in, mental tasks, 339-342 structural-functional basis of, 344-

346 Mescaline, effects on CNS, 42-43 Metabolism, drug effects on, 108-115 Microsomal fractions, enzymes in, 87 Mitochondria, enzymes in, 66 Monoamines, as synaptic transmitters, 17-25 actions on neurons, 19-25 biochemical evidence, 17-18 histochemical evidence, 18-19 release of, 25 Morphine, distribution and levels in nervous system, 101-102 uptake and transport in nervous system, 10S104 Myelin, enzymes in, 66

N P-Naphthylamine, in detection of aminopeptidases , 69 Narcotic drugs, biochemical responses to, 99-128 biotransformations of, 104-108 by liver drug-metabolizing enzymes, 104-106 effects of chronic drug administration on, 1Of3-108 distribution and transport in nervous system, 101-104 distribution and levels, 101-102 uptake and transport, 102-104 effects on, amines, 115-118 calcium, 120-121 general metabolic systems, 108115 hormones, 118-120 intermediary metabolisn~, 108-

110 lipid metabolism, 115 nucleic acid metabolism, 112-115 protein metabolism, 110-112 metabolic disposition of, 101-108 serum and brain factors, 121 Nerve endings, enzymes in, 66 Nervous system, exopeptidases of, 5997 narcotic drug effects on, 9S128 Neurons, effects on synaptic transmitters on, 31-34 Neurosyphilis, autoimmune processes in, 310 Noradrenaline, action on neurons, 1922, 32-34 presence and action in CNS, 17-25 Nucleic acid, metabolism, drug effects on, 112-115

0 Oxytocin, enzymatic inactivation of, 80

P Pentobarbitone, effects on CNS, 35-38 e-Peptidases, in nervous system, 88

380

SUBJECT I N D F X

Peptide breakdown, pathways of, 8990 Periodic illnesses, 133-136 Periodic psychoses, adrenal cortex and, 154-156 autonomic concomitants of, 157 biological rhythm and, 129-169 catecholamines in, 156-157 circadian rhythms and, 138-139 early work on, 152 electroencephalography in, 157 evidence for existence of, 130-132 Gjessing’s studies on, 152-154 lithium and, 158-160 menstrual and estral clock in, 142143 nosology and, 132-133 Richter’s hypotheses and, 137-138 thyroid activity and, 149-151 vasopressin and, 151 Peripheral nerve, exopeptidases of, 83-85 Phosphates, in schizophrenic and normal red blood cells, 278 Pineal gland, biorhythms in, 191-193 effects on, adrenocortical function, 181-183 endocrine system, 17418.3 genital apparatus, 175-178 thyroid-stimulating hormone, 178-181 exopeptidases in, 81 function of, endocrine and neurochemical aspects of, 171-198 light effects on, 187-191 melatonin and serotonin metabolism in, 183-187 metabolism of, 171-174 structure of, 171-174 sympathetic innervation effects on, 187-191 Pituitary, exopeptidases in, 77-79 arylamidases, 78-79 Pituitary hormones, drug effects on, 119-120 Prolidase, see Imidodipeptidase Prostaglanains, as possible synaptic transmitters, 3031

Protein, metabolism, drug effects on, 110-112 turnover, exopeptidases and, 88-90 Psychoses, periodic, see Periodic psychoses Psychotomimetic drugs, effects on CNS, 4 2 4 3

R Red cells, glycolysis and phosphate metabolism in, 269 metabolism, normal, 267-271 phosphates, 278 in schizophrenia, 271-279 Reserpine, in schizophrenia therapy, effects on HNA, 240 Rhythms, circadian, see Circadian rhrthms Richter’s hypotheses, 137-138 RNA, synthesis, drug effects on, 112115 Russia, see USSR

S Schizophrenia, 1-06 antibrain antibodies in, 297-303 autointoxication syndrome in, 205207 biochemical studies on, at Institute of Psychiatry, 216-222 in USSR, 199-225 brain metabolism in, 265-266 carbohydrate metabolism in, 259290 citric acid cycle intermediates in, 264-265 classification of, 214-216 conditioning studies in, 227-258 enzymes in, 266-267 glucose tolerance tests on, 261-262 hypnoid syndrome in, 201-202, 242 HNA studies on, 22&229, 241-248 in therapy, 238-241 of various stages, 235-238 hypoenergetic syndrome in, 204-205 insulin tolerance tests in, 262283 lactate/pyruvate ratio in, 217-221, 264-265, 281-283

381

SUBJECT INDEX

neuroallergic reactions in, 302-303 pathogenesis of, 204-214 prognosis, HNA data in, 245-247 recovery evaluation, using HNA data, 244-246 red cell metabolism in, 271-279 “schizophrenic ester” in, 271-272 serum factors in, 279-285 “toxic” factor in, 280-281 toxicosis of, 202 Sedative drugs, effects on CNS, 34-

36 Serotonin, biosynthesis and nietabo h i n of, 183-187 biorhythm of, 191-193 drug effects on, 117 Spinal cord, exopeptidases of, 82-83 Spinal roots and ganglia, exopeptidases of, 83 Substance P, as possible synaptic transmitter, 30 Synaptic transmission in CNS, 1-56

T Thyroid activity, periodic psychoses and, 149-151 Thyroid-stimulating hormone pineal gland effects on, 178-181 Tranquilizers, effects on, CNS, 3 8 4 1 Transport processes, exopeptidases and, 86-88 Trifluoperazine, in schizophrenia therapy, effects on HNA, 241, 244-245, 247

U USSR, biochemical studies on schizophrenia in, 199-225 V Vascular diseases of brain, autoimmune processes in, 306-310 Vasopressin, inactivation of, 80 in periodic psychoses, 151-152

Cumulative Topical Index, Volumes 1-1 A Acetylcholinesterase, histochemical mapping, in human brain, 231 Adrenochrome, effect on animals and man, 4, 307 Adrenolutin, effect on allimals and man, 4, 307 Afferent synapses, in lateral geniculate nucleus, 6, 191 Amines, metabolism of, in mental diseases, 1, 343 y-aminobutyric acid, metabolic and neurophysiological roles, 2, 279 Amnesia, retrograde, and drugs, 10, 167 Amphibia optic nerve, regeneration, 2,

1 Anesthetics, membrane stabilization by, 9, 145 Animal vision, and drug action, 10, 277 Anticholinergic psychotomimetic agents, 4, 217 Antidepressants, pharmacology of, 9,

95

Appetite control, neurological factors in, 5, 303

B Benzoqniriolizine derivatives, monamine decreasing drugs, 4, 275 Bioelectric activities, central nervous tissues, in culture, 9, 1 Bioelectric phenomena, phosphates and calcium in, 9, 223 Biogenic amines, in mental illness, 8,

197

0

neurophysiological, in neonatal period, 4, 117 Brain hydrolases, molecular forms, 7, 297 Brain stimulation, and free behavior, 6, 349 But~ro~henones,evolution froin meperdine-like 4-phenylpiperidines, 8, 221

C Central nervous system, cholinesterases of, 10, 57 ion fluxes in, 5, 183 neurons, iontophoretic studies, 10, 1 Central nervous tissue( s ), bioelectric activities during maturation, 9, 1 biosynthetic activities, 5, 347 Cerebral, and cerebellar cortex, 1, 47 C e d d cortex, microelectrode studies of, 3, 67 somatic areas of, 3, 187 Cerebral protein metabolism, pathology

of, 7, 1

Ceruloplasmin, role in schizophrenia, 1, 333 ChemicaI agents, of nervous system, 1, 165 Cholinesterases, of central nervous system, reference to cerebellnm, 10, 57 Conditioned avoidance response, and drugs, 2, 229 Cortical epinephrine Pressor responses, affected by schizophrenic serum, 2, 137 Cortical neurons, microiontophoretic studies, 7, 41

Biosynthetic activities of central nervous tissue, 5, 347 D Blood-brain barrier concept, 7, 153 Brain cells, adult manimalian, in Drugs, culture, 5, 1 and conditioned avoidance response, Brain development, biochemical and 2, 229 382

383

CUMULATIVE TOPICAL IN'DEX

effect on learning and memory, 8, 139 retrograde amnesia, 10, 167 Dyskinesias, biochemistry of, 10, 323

Ion fluxes, in central nervous system, 5, 183 Iontophoretic studies, of central nervous system neurons, 10, 1

E

L

EEG waves, analysis of, 5, 53 Electroconvulsive therapy, 5, 389 Electrocortical potentials, nature of, 1, 47 Electroencephalogram, amplitude analysis of, 8, 265 Endocrine system, and neuropsychiatry, 5, 243 Epilepsy, 3, 137 temporal lobe, 1, 1 Ethanol, induced changes in free selection, 2, 41 Extra-blood-brain-barrier brain structures, 10, 31

Lateral geniculate nucleus, afferent synapses and sensory neurons in, 6, 191 Limbic lobe, morphologic concept, 8, 1 Lipid metabolism, in nervous tissue, 3, 293 Lower chordates, muscular innervation, 6, 99

F Facial sensation, neural mechanisms of, 9, 301 Free behavior, and brain stimulation, 6, 349

G Glial cell function, and morphology, 3, 1

H Haloperidol, see Butyrophenones Hemicholiniums, mechanism of action, 2, 77 Hippocampus, electrical activity drug action on, 8, 77 Hydrazicles, convulsive effect of, 3, 319 Hypothalamus, periventricular stratum of, 9, 263

I Imipramine, and related anti-depressants, 9, 95 Indoles, body fluid, in mental illness, 3, 251 Insect nervous system, physiology of, 3, 349

M Membrane stabilization, by drugs, 9, 145 Memory, effects of drugs on, 8, 139 Mental illness, biogenic amines in, 8, 197 Mind-brain relationships, in schizophrenia, 1, 299 Monoamine oxidase inhibitors, 7, 191 Muscular innervation patterns, in lower chordates, 6, 99

N Neocortex, cat nonprimary sensory projections, 10, 111 Neonatal period, development of brain, 4, 117 Nervous system, chemical agents of, 1, 165 protein metabolism of, 6, 1 Nervous tissue, lipid metabolism in, 3, 293 Neural networks, spreading depression in, 4, 1 Neurobiology, role of serotonin in, 2, 175 Neurological control of appetite, 5, 303 Neuropsychiatiy, and endocrine system, 5, 243 Nicotinamade adenine dinucleotidediaphorase, mapping of, in human brain, 10, 231

384

CUMULATIVE TOPICAL INDEX

Nonprimary sensory projections, on cat neocortex, 10, 111

0 Optic nerve regeneration, in amphibia, 2, 1

P Parasympathetic Neurohnmors, and behavior, 1, 195 Periventricular stratum, of hypothalamus, 9, 263 Phencyclidine, drug, pharmacological activity of, 6, 303 Phenothiazine tranquilizers, biochemical and biophysical actions, 7,

231 Phosphates and calcium, in bioelectric phenomena, 9, 223 Polypeptide, see Substance P Protein metabolism, in mental diseases, 1, 343 of nervous system, 6, Psychological tests, and assessment of drug effects, 2, 333 Psychophysiology of vision, 1,245 Pyridoxine, relationship to hydrazides, 3, 319 Pyrimidine analogs, neurobiological action of, 10, 199

R Rhinencephalon, relation to temporal lobe epilepsy, 1, 1

S Schizophrenia, ceruloplasmin in, 1, 333 physiological and biochemical studies in, 1, 299 Schizophrenic serum, cortical epinephrine pressor responses affected by, 2, 137

Sensory cortex, electrical activity of, 5, 53 Sensory neurons, in lateral geniculate nucleus, 6, 191 Sernyl, see Phencyclidine Serotonin, in neurobiology, 2, 175 Somatosensory discrimination, anatomophysiological basis, 8, 37 Somatosensory discrimination, anatomophysiological basis, 8, 37 Spreading depression in neural networks, 4, 1 Steroids, membrane stabilization by,

9, 145 Subcortical motor areas, organizational aspects, 4, 71 Substance P, polypeptide, significance within nervous system, 4, 159 Symptom rating scales, use in pharmacotherapy research, 7, 279

T Thalamus, unspecific intralaminary modulating system of, 9, 45 Tranquilizers, membrane stabilization by, 9, 145 Transmembrane transport, phosphatidic acid and phosphoinositide in, 2, 99 Triffnperidol, see Butyrophenones

V Vertebrate central nervous system, regeneration, 6, 257 Vision, psychophysiology of, 1, 245 Visual cortex, responses, of unanesthetized monkeys, 7, 99 Visual pathways, neural, in cat, 6, 149 transfer of information, 5, 121