International Review of Neurobiology Volume 17

INTERNATIONAL REVIEW OF

Neurobiology VOLUME 17

Associate Editors W. R. ADEY

H. J. EYSENCK

D. BOVET

C. HEBB

Josh DELGADO

S. KETY

SIR JOHN ECCLES

A. LAJTHA

0. ZANCWILL

Consultant Editors M. BORNSTEIN

C. KORNETSKY

R. J. BRADLEY

B. A. LEBEDEV

F. BRUCKE

SIR AUBREYLEWIS

J. ELKES

V. LONGO

R. HEATH

D . M. MACKAY

B. HOLMSTEDT

S. MKRTENS

P. JANSSEN

F. MORRELL

K. KILLAM

H. OSMOND S. SZARA

INTERNATIONAL REVIEW OF

Neurobiology Edited by CARL C. PFEIFFER Brain Bio Center 1225 State Road Princeton, New Jersey

JOHN R. SMYTHIES Department ot Psychiatry and the Neurosciences Program University of Alabama Medical Center Birmingham, Alabama

VOLUME 17

1975

ACADEMIC PRESS

New York

San Francisco

London

A Subsidiary of Harcourt Brace Jovanovich, Publishers

COPYRIGHT 8 1975, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

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LIBRARY OF CONGRESS CATALOG CARDNUMBER:59-1 3822 ISBN 0-12-366817-4 PRINTED IN THE UNITED STATES OF AMERICA

CONTENTS

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CONTRIBUTORS

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Epilepsy and y-Aminobutyric Acid-Mediated Inhibition B. S . MELDRUM

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37 39 43 50 56

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I . Introduction I1. y-Aminobutyric Acid (GABA) and Inhibitory Transmission in the Central Nervous System . . I11. GABA Synthesis IV. GABA Receptor Blockade V . GABAUptake VI . GABA Metabolism VII GABA Analogs VIII . Anticonvulsant Drugs and the Functional Role of GABA . . References

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Peptides and Behavior GEOROES UNGAR

I . Introduction . . . I1. Peptides and Innate Behavior

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. . . . . . . I11. The Role of Peptides in Learned Behavior . . . . . . . IV . Is There a Peptide Code in the Nervous System? . . . . . . References . . . . . . . . . . . . . . Biochemical Transfer of Acquired information S . R . MITCHELL, J . M . BEATON,A N D R . J . BRADLEY I . Introduction . . . . . . . . . . . I1. Approaches to the Study of the Biochemistry of Learning . .

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Aminotransferase Activity in Brain

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M BENUCK A N D A LAJTHA

I . Introduction . . . . . . . . . . I1 . Chemical and Physical Properties of the Aminotransferases .

111. Regulationof Aminotransferase Activity . . . IV. Developmental Changes in Aminotransferase Activity V . Conclusions References . . . . . . . . .

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85 87 109 117 121 122

The Molecular Structure of Acetylcholine and Adrenergic Receptors: An All-Protein Model

J . R . SMYTHIES I . Introduction . . . . . . . . . . . . . 132 I I . Possible Molecular Complexes Involved in Receptors with a Particular Consideration of the Acetylcholine Receptors V

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137

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CONTENTS

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I11 Adrenergic References

Receptors . . . . . . . . . . . . . . . . . . . . . . . . .

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189 191 193 199 205

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174 186

Structural Integration of Neuroprotease Activity

ELENAGABRIELESCU

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I Introduction I1. History and Present Data I11. Neuroprotease System: Molecular Analysis IV . Structural Integration in the Subcellular Compartments V . Binding of Proteolytic Enzymes within the Structure . . . . . VI . The Functional Significance of Enzyme Integration in Subcellular Compartments VII . Integration in the Tissue Cellular Compartments: The Histochemistry of Neuroproteases VIII . The Implications of Neuroproteases in the Physiology and Pathology of the Nervous System References . . . . . . . . . . . . . .

217 230 232

On Axoplasmic Flow LILIANALUBIASKA Introduction . . . . . . . . . . . Aspects of Axoplasmic Flow as Revealed by Various Methods . Influence of Various Factors on Axoplasmic Flow . . . General Description of Axoplasmic Flow . . . . References . . . . . . . . . . .

I. I1 I11 IV

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241 242 273 278 287

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297 298 302 310 31 1 314

Schizophrenia: Perchance a Dream?

J . CHRISTIANGILLINA N D RICHARD J . WYATT I. Introduction

I1. I11 IV. V VI VII

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Major Concepts Previous Sleep Studies in Schizophrenics General Methods Study No 1: Longitudinal Sleep Study in Acute Schizophrenia Study No 2 : Experimental REM Deprivation in Psychiatric Patients Study No 3: REM Sleep of Depressed Patients after Withdrawal of Phenelzine VIII Study No 4 : Longitudinal Sleep Study of a Manic-Depressive Patient I X Discussion X . Summary References

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SUBJECTINDEX CONTENTS OF

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329 332 333 338 338 343 349

CONTRIBUTORS Numbers in parentheses indicate the pages on which the authors’ contributions begin.

J. M. BEATON,Neurosciences Program and Department of Psychiatry, University of Alabama in Birmingham, Birmingham, Alabama (61) M. BENUCK,New York State Research Institute for Neurochemistry and Drug Addiction, Ward’s Island, New York, New York (85) R. J. BRADLEY, Neurosciences Program and Department of Psychiatry, University of Alabama in Birmingham, Birmingham, Alabama (61) ELENAGABRIELESCU, rrDr. V . Babe$” Institute Genetics, Bucharest, Roumania ( 189)

of

Pathology and Medical

J. CHRISTIAN GILLIN,Saint Elizabeths Hospital, Washington, D.C. (297) A. LAJTHA,New York State Research Institute for Neurochemistry and Drug Addiction, Ward’s Island, New York, N e w York ( 8 5 )

LILIANALUBII~SKA, Department of Neurophysiology, Nencki Institute of Experimental Biology, Warsaw, Poland (241) B. S. MELDRUM, Department England ( 1 )

of

Neurology, Institute

of

Psychiatry, London,

S. R. MITCHELL,Neurosciences Program and Department of Psychiatry, University o/ Alabama in Birmingham, Birmingham, Alabama (61)

J . R. SMYTHIES, Department

of Psychiatry and the Neurosciences Program, University of Alabama Medical Center, Birmingham, Alabama (131 )

GEORGES UNGAR, Baylor College of Medicine, Houston, Texas ( 3 7 ) RICHARD J. WYATT, Saint Elizabeths Hospital, Washington, D.C. (297)

Vii

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EPILEPSY AND 7-AMINOBUTY RIC ACID-MEDIATED INHIBITION By B. S. Meldrum

Department of Neurology, Institute of Psychiatry, London, England

1. Introduction . 11. y-Aminobutyric Acid (GABA) and Inhibitory Transmission in the Central Nervous System A. Postsynaptic Inhibition B. Presynaptic Inhibition C. Functional Systems 111. GABA Synthesis . A. The GABA Shunt and Glutamic Acid Decarboxylase B. Inhibitors of Glutamic Acid Decarboxylase C. Pyridoxine Deficiency and Dependency . D. Other Factors Influencing GABA Synthesis E. Features of Seizures Associated with Glutamic A d d Decarboxylase Inhibition IV. GABA Receptor Blockade V. GABAUptake VI. GABA Metabolism Inhibitors of GABA Transaminase . VII. GABA Analogs VIII. Anticonvulsant Drugs and the Functional Role of GABA . References Note Added in Proof

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1. Introduction

Although y-aminobutyric acid (GABA) was identified in brain in 1950 (Awapara et al., 1950; Roberts and Frankel, 1950; Udenfriend, 1950), convincing evidence that it acts as an inhibitory transmitter in the central nervous system has become available only in the.last 7 years (see Section 11). However, a role for GABA in epileptic phenomena had been suggested before its physiological significance was established. Hayashi showed that direct application of GABA to the canine motor cortex could arrest a local epileptic discharge (Hayashi and Nagai, 1956; Hayashi, 1959). At about the same time it was shown that certain convulsant hydrazides inhibited glutamic acid decarboxylase, the enzyme synthesizing GABA in the brain (Killani and Bain, 1957). The observation that some seizures in infants were the result 1

2

B. S. MELDRUM

of a dietary deficiency of vitamin B, (Coursin, 1954; Hunt et al., 1954) was subsequently related to the requirement of pyridoxal phosphate as coenzyme for glutamic acid decarboxylase (Holtz and Palm, 1964) . Early studies of seizures after hydrazides or pyridoxal phosphate antagonists emphasized the ease of induction of reflex or sensory epilepsy (Reilly et al., 1953; Pfeiffer et al., 1956; Balzer et al., 1960), and it is now possible to offer a physiological explanation for this (see Section VIII) . Other convulsant drugs (picrotoxin and bicuculline) have been shown to block the inhibitory action of GABA at postsynaptic membrane sites, and also to diminish presynaptic inhibition on afferent pathways (see Section IV) . The functional significance of the uptake of GABA into nerve terminals or glial cells and of its subsequent metabolism is not well understood, but some drugs which block the further metabolism of GABA have an antiepileptic action (see Sections V and VI) GABA does not penetrate the blood-brain barrier, but various structural analogs do enter the brain and have effects on epileptic phenomena (see Section V I I ) . Early claims for a relationship between cerebral GABA metabolism and epilepsy led to much confusion and controversy. Now that more definitive biochemical and physiological data are available, it is possible to give a coherent account of the subject.

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II. y-Aminobutyric Acid (GABA) and Inhibitory Transmission in the Central Nervous System

The inhibitory action of short-chain w-amino acids (such as p-alanine, GABA, and taurine) on central neurons was clearly established by the microiontophoretic studies of Curtis and Watkins (1960, 1965). Additional data that have led to the widespread acceptance of the hypothesis that GABA is the inhibitory transmitter at numerous central sites include (1) the demonstration of high GABA content and glutamic acid decarboxylase (GAD) activity in synaptosome preparations (Weinstein et al., 1963; Kuriyama et al., 1968; Perez de la Mora et al., 1973) ; (2) evidence for the selective local release of GABA when known inhibitory systems are activated in the cerebral cortex or cerebellum (Obata and Takeda, 1969; Jasper et al., 1965; Mitchell and Srinivasan, 1969; Iversen et al., 1971); (3) evidence from intracellular recordings that the changes in membrane potential and in specific ionic conductances produced by physiological inhibitory inputs are similar to those produced by the iontophoretic application of GABA (Krnjevic' and Schwartz, 1967) ; (4) evidence that picrotoxin and bicuculline can block both natural inhibition and inhibition produced by iontophoretic application of GABA (but not that produced by glycine or catecholamines)

EPILEPSY AND 7’-AMINOBUTYRIC ACID-MEDIATED INHIBITION

3

(Galindo, 1969; Curtis et al., 1970a, 1971a,b; Curtis and Felix, 1971; MCLennan, 1971; Kelly and Renaud, 1973; Hill and Simmonds, 1973) ; ( 5 ) evidence for a high-affinity uptake system selective for GABA (Elliott and Van Gelder, 1958; Roberts and Kuriyama, 1968; Beart and Johnston, 1973; Snodgrass and Iversen, 1973).

INHIBITION A. POSTSYNAPTIC The principal sites where GABA produces hyperpolarization of the neuronal membrane, which resembles the physiologically induced inhibitory postsynaptic potential (IPSP) in its dependence on an enhanced chloride conductance, and where naturally induced inhibition and the action of applied GABA are blocked by picrotoxin or bicuculline, are the neocortex, the cerebellum, the hippocampus, and the thalamus. In the cerebellum, basket-cell endings on Purkinje cell bodies, and the terminations of Purkinje cell axons on nerve cell bodies in deep cerebellar nuclei or in the lateral vestibular nucleus, are GABA mediated (Obata et al., 1967; Obata and Highstein, 1970; Curtis and Felix, 1971). In the cerebral cortex several functional systems apparently employ GABA, but their anatomical specification is not entirely clear. These systems include the intrinsic inhibitory system activated by direct cortical stimulation (KrnjeviE et al., 1966; Krnjevif. and Schwartz, 1967; Curtis and Felix, 1971) . In the hippocampus, GABA mediates the local inhibition of basket cells on pyramidal neurons (Curtis el al., 1970c; Storm-Mathisen and Fonnum, 1971) . Collateral inhibition via interneurons in the thalamus also is blocked by bicuculline (Duggan and MCLennan, 1971). Some postsynaptic inhibition in the cuneate nucleus is GABA-mediated (Galindo, 1969; Kelly and Renaud, 1973).

B. PRESYNAPTIC INHIBITION A volley in primary afferent fibers entering spinal dorsal roots or cranial nerve nuclei is followed by prolonged depolarization of certain afferent terminals, associated with a reduction in afferent transmission. Both the inhibition (as tested by monosynaptic reflexes) and the depolarization are diminished or blocked by picrotoxin (Eccles et al., 1963; Banna and Jabbur, 1969; Levy and Anderson, 1972, 1973) and by bicuculline (Levy et al., 1971; Barker and Nicoll, 1972; Benoist et al., 1972). As picrotoxin and bicuculline are known to block postsynaptic inhibition, due to GABA, by receptor site competition, it appears that GABA may also be responsible for “presynaptic inhibition.” Supporting evidence for this includes the demonstration that

4

B. S. MELDRUM

inhibition of GABA synthesis by semicarbazide administration reduces dorsal root potentials and presynaptic inhibition in the cat (Bell and Anderson, 1972). There is some evidence that GABA produces the same ionic conductance changes in afferent terminals as does the natural transmitter (Barker and Nicoll, 1972), but definitive intracellular studies have not yet been performed. C. FUNCTIONAL SYSTEMS Whole-animal pharmacology studies relating to GABA-mediated transmission will make sense only if GABA is employed in systems whose function can be defined at the level of the whole nervous system, not merely at the cellular level. Fortunately, this appears to be the case in at,least two respects. Small interneurons (Golgi type I1 neurons or basket cells) in the cerebellar cortex, neocortex, hippocampus, and thalamus appear to be a major category of neurons releasing GABA. In each case they act on the cell bodies of neurons with long axons. Broadly their function appears to be, in somatotopic terms, to narrow or sharpen the area of activity and, in temporal terms, to shorten or terminate a period of activity. Thus, if epilepsy is defined as an excessive synchronicity of discharge in a neuronal mass, these GABAreleasing interneurons would appear to provide a major defense against the buildup or spread of epileptic activity. Presynaptic inhibition on afferent pathways probably achieves a similar spatial and temporal sharpening of activity, but also provides the basis for some features of reciprocal motor inhibition. Difficulties arise where GABA is employed more than once in a given functional system. The cerebellum is one system concerned with posture and movement, but it contains several anatomically distinct GABA-releasing synaptic systems (basket cells on Purkinje cells, Purkinje cells on neurons in deep cerebellar nuclei, and probably stellate cells in the molecular layer and Golgi cells in the granular layer). In such circumstances, the physiological effects of drugs modifying GABA-mediated transmission will not necessarily be consistent with their known pharmacological actions at the cellular level. Whole-animal studies of posture and movement are further complicated by the participation of the basal ganglia, among which the substantia nigra and globus pallidus have exceptionally high GABA content and GAD activity (Lowe et al., 1958; Albers and Brady, 1959; Fahn and C M , 1968). I t should also be borne in mind that the different GABA systems will not necessarily be pharmacologically similar. GABA receptors in the thalamus, the deep cerebellar nuclei and the spinal cord may differ as markedly as cholinergic receptors at the skeletal neuromuscular junction, cardiac muscle, and the Renshaw cell.

EPILEPSY AND Y-AMINOBUTYRIC ACID-MEDIATED INHIBITION

5

111. GABA Synthesis

A. THEGABA SHUNTAND GLUTAMIC ACID DECARBOXYLASE Neither GABA nor its precursor glutamic acid enter the brain from the blood in significant quantities under normal circumstances. The carbon chains of both derive from glucose via glycolysis, and the entry of pyruvate into the Krebs cycle (Vrba, 1962; Gaitonde et al., 1965). Transamination of a-ketoglutarate (amino group donors include aspartate and GABA itself) gives glutamate, further metabolism of which, via the GABA shunt, allows 'the carbon chain to return to the Krebs cycle as succinate (see Fig. 1). This shunt proceeds only in the direction a-ketoglutarate to succinate and yields three high-energy phosphate bond equivalents. Not all cerebral glutamate is metabolized via this pathway; some apparently in a separate metabolic compartment (Patel and Balhs, 1970; Watkins, 1972) forms glutamine. Most of the GABA is metabolized to succinate although many alternative pathways are available (see Baxter, 1970). For the whole brain, the flux through the GABA shunt appears to be about 10% of the flux through the Krebs cycle (Balhs et al., 1973), but within nerve endings the proportional flux is probably very much higher. I n anoxic conditions the synthesis of GABA proceeds, but its further metabolism is blocked because the dehydrogenation of succinic semialdehyde requires oxidized NAD. Not only is there an increase in cerebral GABA concentration during anoxia (Elliott and Van Gelder, 1960; Wood, 1967; Wood et al., 1968) and a marked postmortem irlcrease in cerebral GABA content (Love11 et al., 1963; Tews et al., 1963; Patel et al., 1970; Minard and Mushahwar, 1966), but hyperbaric oxygenation leads to a reduction in cerebral GABA content (Wood et al., 1966, 1969) (see Section 111, D ) . Glutamic acid decarboxylase (GAD) is a soluble or cytoplasmic enzyme found predominantly in cerebral gray matter. Studies of its regional distribution in man (Miiller and Langemann, 1962) show that its concentration is very high in the extrapyramidal motor system (globus pallidus, substantia nigra, and dentate nucleus), in the precentral and postcentral neocortex, and in the head of the caudate nucleus and is moderately high in the putamen, frontal neocortex, and cerebellar cortex. I n the monkey (Lowe et al., 1958; Albers and Brady, 1959) the distribution is similar, but the superior colliculus has very high GAD activity. I n subcellar fractions of brain homogenates, GAD is concentrated in the synaptosomal fraction, from which it can be released by hypoosmotic shock (Weinstein et al., 1963; Salganicoff and De Robertis, 1965; Fonnum, 1968; Kuriyama et al., 1968; Perez de la Mora et al., 1973).

6

B. a-Ketoglutarate

S. MELDRUM

HOOC-Ch-

CH,-CO-COOS

~-Aspartate:2-oxoglutarate aminotransferaee + PyP Glutamic acid

HOOC-CH,--

CHz-- CH(NH,)-COOH

L-Clutamic 1-carboxy-lyaee + PyP (Clutamic acid decarboxylase)

y-Aminobutyric acid

HOOC --CH,--

CH,---CH,--NH,

4-Aminobutyrate :2-oxoglutarate aminotransferase t PyP (CABA transaminase)

Succinic semialdehyde

HOOC -CH,-

CH,-CHO

Succinate semialdehyde :NAD+ oxido reductase (Succinate semialdehyde dehydrogenase)

Succinate

HOOC- C&-

C q - COOH

Fxo. 1. Enzymes of the y-aminobutyric acid shunt pathway. +PyP, pyridoxal phosphate-dependent enzyme. Parentheses under systematic names of enzymes enclose the common name used in this chapter.

M (Roberts The pH optimum is pH 6.4 or 7.2-7.5. The K, is 3-8 X and Simonsen, 1963; Susz et al., 1966). The purified enzyme has a molecular weight of 90,000 daltons, possibly composed of 15,000 dalton subunits (Matsuda et al., 1973). Preparations of GAD derived from invertebrate sources show end-product inhibition (Molinoff and Kravitz, 1968), but GAD from mammalian brain is inhibited only by very high concentrations of GABA (Susz et al., 1966). GAD is competitively inhibited by chloride ions at a concentration intermediate between that of extracellular fluid and cytoplasm (i.e., 50 mM), and this may provide a physiological control mechanism (Susz et al., 1966). GABA enhances membrane chloride conductance : a massive synaptic release of GABA would tend to reduce the rate of GABA synthesis (provided that the ionic action of GABA also affects the presynaptic membrane). GAD has an absolute requirement for the coenzyme pyridoxal phosphate, to which it is relatively loosely bound (see Sections 111, B and C ) . I t has been suggested that there are two forms of the enzyme (Haber et al., 1970) : GAD I (predominantly neuronal or synaptosomal, inhibited

EPILEPSY AND 7-AMINOBUTYRIC ACID-MEDIATED INHIBITION

7

by anions and by aminooxyacetic acid) and GAD I1 predominantly glial, activated by anions and aminooxyacetic acid)

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B. INHIBITORS OF GLUTAMIC ACIDDECARBOXYLASE Many compounds known to impair GAD activity in vivo do so by interfering with the synthesis or coenzymic function of pyridoxal phosphate (PyP) (Holtz and Palm, 1964; Baxter, 1970). PyP is synthesized from ATP and pyridoxal (the aldehyde form of vitamin Be) by pyridoxal phosphokinase (EC 2.7.1.35), an enzyme present in large excess in the brain. 1. Hydraides

The first group of GAD inhibitors to be identified (Killam and Bain, 1957), and the most intensively studied subsequently, comprised the hydrazides (Holtz and Palm, 1964; Medina et al., 1962; Medina 1963; Uchida and OBrien, 1964; Tapia et al., 1966; Wood and Abrahams, 1971; Wood and Peesker, 1972a,b; Perez de la Mora et al., 1973). Correlation of changes in brain GABA content or rate of GABA synthesis with the production of seizures by hydrazides has proved to be exceptionally difficult, probably because ( a ) different hydrazides inhibit GAD by different mechanisms, (b) they have variable additional biochemical actions (including inhibition of GABA transaminase), and (c) a variety of biochemical artifacts. Studies based on cerebral GABA content have led to much confusion because of elevation in GABA levels occurring during anoxia (in seizures or preterminally) or associated with drug-induced GABA transaminase inhibition, or occurring at postmortem. A critical reduction in GABA concentration in presynaptic terminals may be concealed in wholebrain content studies by a rise in GABA concentration in glial cells. There is no satisfactory method for determining GAD inhibition in vivo. The usual procedure is to administer an effective drug dose in vivo, then decapitate the animal in order to estimate GAD activity on a cerebral homogenate. Most assay methods involve the addition of extra substrate. This is satisfactory for irreversible enzyme inhibitors gaining unrestricted access to the brain, but results will be misleading with, for example, a competitive inhibitor of GAD, or a substance producing a relative deficiency of PyP in a limited part of the brain or particular subcellular compartment. The majority of the hydrazine derivatives listed in Table I form hydrazones with pyridoxal or with pyridoxal phosphate, according to the general reaction R1CH2CH0 NH2NHRZ= R1CH2CHN:NHRZ H 2 0 . This reaction absorbs a proportion of the available coenzyme. I t has been claimed that some of the PyP hydrazones formed can replace PyP as coenzyme for GAD (Tapia et al., 1967; Gonnard and Fenard, 1962), but data about

+

+

8

B. 9. MELDRUM

coenzymic mechanisms suggest that this is unlikely (Holtz and Palm, 1964; Snell, 1972). The most important consequences of this reaction, however, result from the powerful inhibition of pyridoxal kinase that the pyridoxal hydrazones produce (McCormick and Snell, 1959, 1961; Holtz and Palm, 1964). Tapia et al. (1969) and Perez de la Mora et al. (1973) have demonstrated in vivo inhibition of pyridoxal kinase in mice receiving various pyri. doxal phosphate hydrazones. After a convulsant dose of pyridoxal phosphatey-glutamylhydrazone they observed a reduction in the concentration of PyP in the synaptoplasm that correlated with the reduction in synaptosomal GAD activity. Doubly substituted hydrazines, such as Nfl-dimethylhydrazine, do not form hydrazones with pyridoxal and, in general, are not convulsants. However, NJ’-dimethylhydrazine inhibits GAD (Medina, 1963). This inhibition of GAD must depend on a mechanism other than the hydrazone formation which is common to the monosubstituted hydrazines. This is supported by the lack of correlation between convulsions and cerebral pyridoxal phosphate levels after administration of the first 4 hydrazines in Table I (Uchida and O’Brien, 1964). The values for GAD inhibition given in Table I will be too low if they depend on a selective focal reduction in PyP content that is lost on homogenization of the brain. They will also be too low if there is an element of competitive inhibition (that will be corrected by addition of extra substrate to the assay system, and by dilution of the hydrazide). In the table, doses of hydrazides associated with more than 40% inhibition of GAD activity are always associated with seizures. A lower inhibition (3540%) may be associated with convulsions if there is not concomitant (GABA transaminase inhibition. The high CABA transaminase inhibition may explain the CNS depression often seen after hydrazine. Some drugs, such as thiosemicarbazide, thiocarbohydrazide, and methyl dithiocarbazinate, at convulsant doses, apparently produce only about 20% inhibition of GAD activity. Possibly the mechanisms leading to GAD inhibition are such that the assay system underestimates the inhibition, or there is a convulsive mechanism additional to GAD inhibition operating with these sulfur-containing hydrazines. Compared with isoniazid and other hydrazines in mice, chicks, and baboons, thiosemicarbazide shows an atypical dose-response curve for seizure induction (Jenney and Pfeiffer, 1958; Wood and Abrahams, 1971; Meldrum et ul., 1970).

2. Other Carbonyl Trapping Agents Apart from the hydrazine derivatives, other carbonyl trapping agents have been shown to inhibit GAD activity in vitro, presumably by combining with the aldehyde group of pyridoxal phosphate (Roberts et al., 1964).

TABLE I HYDRAZINE DERIVATIVES~

Structure Hydrazine HZN:NH2 Monomethylhydrazine CHsNH:NH* Unsymmetrical dimethylhydrazine (CH&NNHz N,N’-Dimethylhydrazine C H Y H :NHCHr Semicarbazide NH,CH~NH:NHZ Thiosemicarbazide NH2CS.NH:NHz Methyl dithiocarbazinate CH6S:CNH:NHz Thiocarbohydrazide HJV:NHCSNH:NHt L-Glutamic acid 7-hydrazide HOOC4HzCHzCH (NH:NHz)COOH Pyridoxal phosphate; glutamyl hydrazone Hydrazinopropionic acid HOOCCHtCHJVH:NH2 Isonicotinic acid hydrazide (NCsH4)CONH:NH, Phenethylhydrazine CsHsCH2CHJVH:NHz

Dose (fimol-/kg) 1,600 800 1,600

7,m

GAD GABA-T inhibition inhibition (%) (%) 86.5 57 70

93 85 20

39

76

2 , m 2,510 82 130

42 20 37 20

11 3 10 0

225 11,700

24 82

49

5

Species

References

Rats Rats Rats Rats Chicks Chicks Rats Mice

Medina (1963) Medina (1963) Medina (1963) Medina (1963) Wood and Abraham (1971) Wood and Abraham (1971) Collins, 1973 B. S. Mddrum and R. W. Horton (unpublished) Wood and Abraham (1971) Tapia cf al. (1966, 1969)

940

25

205 164

42

28 0

20

80

Chicks Mice Mice Mice Mice

4,360

35

7

chicks

Wood and Abraham (1971)

275

6

42

Rats

Popov and Matthies (1969)

Tapia cf al. (1967, 1969) Van Gelder (1968)

Doses (and inhibitions) not associated with convulsions are italicized. The ‘‘wnVUlSant dose” is not always the minimal convulsant dose, but one that was experimentally convenient. GAD = glutamic acid decarboxylase: GABA-T = y-aminobutyric acid transaminase. W

10

B. 5 . MELDRUM

Cycloserine, hydroxylamine, and numerous o-substituted hydroxylamine derivatives are effective in concentrations of to M. Aminooxyacetic and aminooxypropionic acid are the most potent compounds, producing 95 and 97% inhibition at M (Dengler, 1962; Roberts et al., 1964). However, in vivo these compounds are much less effective GAD inhibitors than the hydrazine derivatives, and commonly rather selectively inhibit GABA transaminase. I n subconvulsant doses, they can be shown to possess anticonvulsant properties (see Section V I ) . Among other compounds capable of combining with the carbonyl group of pyridoxal phosphate is penicillamine (Jaffe, 1972). At 0.3 mg/kg this produces running fits and convulsions in mice, and inhibition of cerebral GAD activity is demonstrable (Matsuda and Makino, 1961; Roberts and Simonsen, 1963) . Many amino acids and amines react with the carbonyl group of pyridoxal phosphate to produce Schiff bases. This may be the explanation for the observation that in rats cerebral GAD activity is reduced by 24-56% 24 hours after the administration of L-3,4-dihydroxyphenylalanine ( L-DOPA) 1252500 mg/kg (Kurtz and Kanfer, 1971). 3. Pyridoxine Analogs (“Antimetabolites”) Structural analogs of vitamin B,, such as toxopyrimidine, 4-deoxypyridoxine, or 3-methoxypyridoxineJ have long been known to produce convulsions in rats and mice (Holtz and Palm, 1964). There are two main mechanisms of action. The analog either inhibits phosphokinase, blocking the synthesis of pyridoxal phosphate, or it is phosphorylated by phosphokinase and then competes with pyridoxal phosphate for binding sites on the apoezyme. The second mechanism predominates in the case of toxopyrimidine; 4deoxypyridoxine acts in both ways. Inhibition of GAD can be demonstrated in cerebral homogenates after the systemic administration of a convulsant dose of 4-deoxypyridoxine, and this inhibition is reversed by the addition of pyridoxal phosphate (Horton and Meldrum, 1973). Likewise, the seizures can usually be prevented by the simultaneous systemic administration of pyridoxine. As described above (Section 111, B, l ) , hydrazones formed by the combination of hydrazines and pyridoxal (or oximes formed from hydroxylamines and pyridoxal) are powerful inhibitors of phosphokinase. 4. Competitive and “Noncompetitive” Inhibitors of G A D

Competitive inhibition of cerebral glutamic acid decarboxylase by substrate analogs has been little investigated. Lamar (1970) and Horton and Meldrum (1973) have provided kinetic data showing that the convulsants

EPILEPSY AND ‘Y-AMINOBUTYRIC ACID-MEDIATED INHIBITION

11

3-mercaptopropionic acid and 4-mercaptobutyric acid are competitive inhibitors of cerebral GAD. The latent period to the onset of seizures is much shorter after 3-mercaptopropionic acid than after pyridoxine antagonists. Inhibition of cerebral GAD activity is demonstrable 2 minutes after its intraperitoneal injection in mice (Horton and Meldrum, 1973). Hydrazinopropionic acid may also be a competitive inhibitor of GAD activity, but it has a much more powerful effect on cerebral GABA transaminase (Van Gelder, 1968,1969). Allylglycine (2-amino-4-pentenoic acid) given systemically to rats, mice, or baboons produces a pattern of seizures similar to that seen after pyridoxine antagonists (McFarland and Wainer, 1965; Horton and Meldrum, 1973). In vitro it is only a weak inhibitor of cerebral GAD activity. Cerebral homogenates from mice given allylglycine 30-90 minutes earlier show up to 40% inhibition of GAD. Neither the in uitro nor the “in uiuo” inhibition is relieved by the addition of pyridoxal phosphate, The mechanism of GAD inhibition is not understood, but it may be indirect or dependent on a metabolite of allylglycine. Allylglycine also inhibits the synaptosomal incorporation of leucine into protein (Alberici de Canal and De Lores Arnaiz, 1972) and the uptake of leucine and proline into brain slices (Balcar and Johnston, 1974). Direct inhibitory effects of allylglycine on neuronal firing have also been demonstrated (Roper, 1970; Curtis et al., 1970b). Phenylalanine metabolites accumulate in the brain and elsewhere in phenylketonuria or after the administration of excess dietary phenylalanine. High concentrations of some of the metabolites lead to inhibition of GAD and other cerebral decarboxylases. Phenylacetic acid apparently acts as B competitive inhibitor of GAD (Hanson, 1958; Tashian, 1961; Schlesinger and Uphouse, 1972). A N D DEPENDENCY C. PYRIDOXINE DEFICIENCY

Convulsions occur in infants as a result of dietary deficiency of vitamin Be, when, for example, excess heat sterilization destroys the natural vitamin content of milk. Such seizures can be prevented or arrested by the injection of pyridoxine (Coursin, 1954, 1964). A relative dietary deficiency of vitamin B, in rats or mice makes them more susceptible to the convulsive action of pyridoxine antagonists (Schlesinger and Uphouse, 1972). As detailed in Section 111, E, strains of mice susceptible to audiogenic epilepsy are more vulnerable to dietary pyridoxine deficiency than are other mice. Pyridoxine dependency is a rare familial disorder in which normal vitamin Be intake and normal or high blood levels of Be vitamers are inadequate to ensure normal cerebral function (Hunt et al., 1954; Coursin, 1964). Affected children develop seizures in the neonatal period and subsequently

12

B. S. MELDRUM

show mental retardation, but the administration of high daily doses of pyridoxine prevents both the seizures and the mental deficit. D. OTHERFACTORS INFLUENCING GABA SYNTHESIS As mentioned in Section 111, A above, high concentrations of GABA or of C1- inhibit GAD activity. Such factors and pH changes could play a role in physiological mechansims late in seizure activity. As the pH optimum of GAD is around 6.5 and that of GABA transaminase around 8.2, increases in Pco, will increase cerebral GABA content. Another physiological variable influencing GAD activity is the partial pressure of oxygen. The increase in cerebral GABA content during hypoxia, which results from the impariment of the further metabolism of GABA, has been described above (Section 111, A ) . That hyperbaric oxygenation produces seizures in animals and man has been known for nearly 100 years (Wood, 1972). Activity of several cerebral enzyme systems is impaired, probably because of oxidation of SH groups. Antioxidants prevent or diminish the occurrence of seizures. Using different species (rats, mice, guinea pigs, chicks) and different partial pressures of oxygen, it is possible to demonstrate a very close correlation between the rate of decrease of cerebral GABA content and the susceptibility to seizures (Wood et al., 1966, 1969; Wood, 1972 ) . Hyperbaric oxygenation also produces reversible audiogenic seizure susceptibility in genetically nonsensitive rats (Wada et al., 1970). Enzyme induction by high substrate concentration has been demonstrated in the mouse (Kraus, 1968). Peak activity of cerebral GAD activity was seen 2-4 hours after the intraperitoneal injection of L-glutamate ( 1 gm/kg) Drug withdrawal convulsions may also be related to changes in GABA metabolism or GABA receptor properties. Animals and man addicted to alcohol, barbiturates, or opiates in the immediate withdrawal period often show spontaneous convulsions, and when these are not shown, seizures can often be induced by photic or auditory stimulation (Wulff, 1959; Crossland and Leonard, 1963). In barbiturate abstinence seizures in rats, Essig (1968) has shown a protective effect of aminooxyacetic acid. That chronic ethanol administration in rats produces a marked increase in cerebral GABA content and in GABA transaminase activity was shown by Sutton and Simmonds (1973), but these authors did not describe changes during withdrawal. I n rats habituated to barbitone, cerebral GABA content is unchanged 48 hours after drug withdrawal (Crossland and Turnbull, 1972). Various endocrine disorders are associated with seizures. Estrogens have been shown to inhibit GAD activity (Baxter, 1970) ; they can exacerbate epilepsy when administered systemically (Logothetis et al., 1959) and have an epileptogenic action when applied topically to the cortex of cats, rabbits,

.

EPILEPSY AND Y-AMINOBUTYRIC ACID-MEDIATED INHIBITION

13

and monkeys (Marcus et al., 1966; Hardy, 1970). However, there is no evidence correlating GAD inhibition with estrogen-induced seizure activity. Two disorders of movement in man associated with pathological changes in the basal ganglia, Parkinsonism and Huntington’s chorea, show a reduction in GAD activity in the affected brain areas (Bernheimer and Hornykiewicz, 1962; McGeer et al., 1971, 1973; Lloyd and Hornykiewicz, 1973; Bird et al., 1973). In Parkinsonism this reduction in GAD activity appears to be secondary to the abnormality in amine metabolism because prolonged L-DOPA therapy raises GAD activity to normal (Lloyd and Hornykiewicz, 1973). In Huntington’s chorea, it may be secondary to the local loss of neurons.

E. FEATURES OF SEIZURES ASSOCIATED WITH GLUTAMIC ACID DECARBOXYLASE INHIBITION 1. Reflex or Sensory Epilepsy Facilitation of the sensory induction of seizures after the administration of pyridoxine antagonists was observed in the 1950’s (see Introduction). If a natural syndrome of sensory or reflex epilepsy is present in man or animals, subconvulsive doses of pyridoxine antagonists will enhance the natural syndrome without modifying the nature of the seizures. Thus, in rodents with audiogenic epilepsy, low doses of isoniazid or thiosemicarbazide enhance the severity of sound-induced seizures (Lehmann, 1964). I n baboons with photosensitive epilepsy (Papio papio from the Casamance region of Senegal: Naquet and Meldrum, 1972), low doses of isoniazid or 4-deoxypyridoxine facilitate the appearance of generalized myoclonic responses to intermittent photic stimulation and increase the probability that they will become self-sustaining and lead to a brief tonic-clonic seizure (Meldrum et d., 1970; Meldrum and Horton, 1971; Meldrum and Balzamo, 1972). Although subconvulsant doses of pyridoxine antagonists do not change the clinical features of individual reflex seizures (i.e., audiogenic seizures in mice continue to show the usual evolution through a wild running phase, a clonic flexor seizure, a tonic extensor phase, and then motor and respiratory depression, and photically induced seizures in baboons progress from facial myoclonus with frontorolandic spikes and waves to generalized rhythmic myoclonus, a brief tonic phase, slow rhythmic myoclonus and postictal depression), they do shorten the normal refractory period that follows sensory induction of one seizure and prevents the induction of a second seizure (Meldrum et al., 1970). “Dilute” strains of mice (such as DBA/2J) showing a high susceptibility to audiogenic seizures are more vulnerable to a dietary deficiency of pyri-

14

B. S. MELDRUM

doxine than are “nondilute” strains with a low seizure susceptibility. Such deficiency impairs their growth and enhances the audiogenic seizures (Lyon et al., 1958; Schlesinger and Uphouse, 1972). Administration of supplemental pyridoxine to genetically susceptible mice reduces the severity of audiogenic seizures, Pyridoxine administration does not diminish photically induced myoclonic responses in Papio papio (Meldrum et al., 1970). I t is not only pyridoxine antagonists that enhance reflex epilepsy. Subconvulsant doses of allylglycine and 3-mercaptopropionic acid show the same effect (Horton and Meldrum, 1973). The GAD inhibitors that are also powerful GABA transaminase inhibitors (such as aminooxyacetic acid, hydroxylamine, and cycloserine) , tend, however, to diminish epileptic responses to sensory stimulation (Lehmann, 1964; Meldrum et al., 1970). I n humans or animals not normally showing reflex epilepsy, such syndromes are readily induced by subconvulsant doses of GAD inhibitors. They commonly resemble the naturally occurring syndromes but are not always identical. Thus, in rhesus monkeys, photically induced seizures occur after 4-deoxypyridoxine (Meldrum and Horton, 1971). In nonsensitive rats, in cats, and in monkeys “audiogenic” seizures can be induced after thiosemicarbazide (Wada and Asakura, 1969). In man, photically induced seizures are seen after isoniazid (25-35 mg/kg i.v.), or semicarbazide (25 mg/kg i.v.), thiosemicarbazide, thiocarbohydrazide, or 4-deoxypyridoxine (Reilly et al., 1953; Pfeiffer et al., 1956).

2. Seizures Following Systemic Administration Characteristically, after the systemic administration of a nonlethal convulsant dose of a pyridoxine antagonist or allylglycine, there is a latent period of 0.5-4 hours, then the abrupt onset of a brief generalized seizure lasting 1-3 minutes, then recovery to apparent normality of behavior and the EEG, then, after an interval of 5-90 minutes, recurrence of the brief seizure with a repetition of recovery and/or recurrence, until eventual complete recovery. Higher doses of the GAD inhibitors produce closer recurrence of the seizures with slower recovery so that the next seizure may start while the EEG shows diffuse delta activity. Close recurrence of seizures may merge into status epilepticus. In baboons and rhesus monkeys, these brief recurrent seizures start with horizontal nystagmus, lateral deviation of the eyes and turning of the head and neck to one side, with, on the EEG, rhythmic spikes occurring posteriorly in the opposite hemisphere (occipital and posterior parietal cortex), During a sequence of seizures, the side of seizure origin may alternate. Unilateral posterior cerebral onset of seizure discharges is seen consistently after pyridoxine antagonists and allylglycine, but not after the competitive GAD inhibitor, 3-mercaptopropionic acid, which produces diffuse spike discharges

EPILEPSY AND 7-AMINOBUTYRIC ACID-MEDIATED INHIBITION

15

and then the generalized onset of tonic-clonic seizures (Horton and Meldrum, 1973). 3. Seizures Following Focal Cerebral Administration Because GAD inhibitors are effective convulsants when given systemically their local cerebral application has not been greatly studied. However, application of semicarbazide (1.66% solution) to the neocortex in rhesus monkeys (Lowell et al., 1952; Pfeiffer et al., 1956) produces focal spiking after about 20 minutes and spreads in the next 25-40 minutes to give local, and finally generalized, seizure activity. Local injections also produce epileptic foci. I n cats thiosemicarbazide (100-600 pg) injected into one hippocampus produces focal discharges after 1-5 hours; changes in local excitability occur with a shorter latency (Baker and Kratky, 1967).

4. Regional Changes in G A D Activity The kind of seizures after drugs inhibiting GAD and their pattern of origin might depend not only on the relative importance of GABA-mediated inhibition in different systems, but also on relatively greater effects of the drug in particular regions. I n the rhesus monkey the occipital cortex has a higher GAD activity and a higher GABA content than the frontal or motor cortex (Albers and Brady, 1959; Fahn and Cat&,1968). The effects of drugs are not well documented. I n rabbits methoxypyridoxine produces preictally a rapid decrease in GABA concentration in the caudate, putamen, and superior colliculus, but little or no change in the sensorimotor cortex (Hassler et al., 1972). Enzyme inhibitors may be selectively accumulated in specific brain regions or by particular cell types. Electron microscopic autoradiography indicates that in the hippocampus thiosemicarbazide is selectively accumulated in glial cells (Knyihlr et al., 1971).

IV. GABA Receptor Blockade

1. Bicuculline Iontophoretic studies, as mentioned in Section I1 above, have shown that the convulsant alkaloid bicuculline can block the postsynaptic inhibitory action of GABA at numerous sites in the nervous system. Some authors have also described an enhancement or acceleration of the action of GABA (Godfraind et al., 1970; Hill et al., 1973b). Bicuculline (0.1-1.0 mM in vitro) does not modify cerebral GAD activity and has only a slight inhibitory action

16

B. S. MELDRUM

on GABA-transaminase (Svenneby and Roberts, 1973; Straughan et al., 1971 ; Beart and Johnston, 1972). Its antagonistic effects, when iontophoretically applied, apparently arise from direct competition with GABA for specific inhibitory receptor sites (Curtis et d., 1971a,b; Straughan et al., 1971; Hill and Simmonds, 1973; Hill et al., 1973b). I t does not block the inhibitory effects of glycine in the cat or rat spinal cord or of norepinephrine in the cat cerebellum (Curtis et al., 1971a,b). In the cat neocortex bicuculline blocks the inhibitory actions of GABA and “GABA-like” compounds (p-hydroxy-GABA, imidazoleacetic acid) , but not those of glycine. This selectivity for GABA-like inhibitoiy amino acids is less marked in the rat neocortex (Biscoe et al., 1972). As mentioned in Section 11, B above, presynaptic inhibition is also blocked by bicuculline when this is given systemically or when it is applied locally to the spinal cord or cuneate nucleus (Davidson and Southwick, 1971; Barker and Nicoll, 1972; Levy and Anderson, 1972, 1973; Davidoff, 1972b; Benoist et al., 1972; De Groat et al., 1972). Bicuculline methochloride ( “N-methylbicuculline” ) is more water soluble than bicuculline and is more potent as a blocker of GABA’s inhibitory effects when administered iontophoretically (Johnston et al., 1972). I t is less active as a systemic convulsant, probably because it crosses the blood-brain barrier less readily. I t has recently been shown that bicuculline and bicuculline methochloride are competitive inhibitors of mouse-brain acetylcholinesterase (Svenneby and Roberts, 1973). The relationship of this to the convulsant action of bicuculline is doubtful. Irreversible inhibitors of acetylcholinesterase are powerful convulsants, but bicuculline can block the inhibitory action of GABA without enhancing the effects of acetylcholine. Seizures induced by the intravenous administration of bicuculline occur after a very short latency (4-12 seconds) (Meldrum and Horton, 1971). Seizures are generalized from the onset; a brief tonic phase is followed by a clonic phase that may be extremely prolonged (up to 5 hours in unanesthetized, and up to 7 hours in paralyzed, animals (Meldrum and Horton, 1973; Meldrum et al., 1973). The minimal convulsant dose (0.2-0.4 mg/kg) is similar for rats, cats, monkeys, and baboons (Curtis et al., 1970a; Meldrum and Horton, 1971; Johnston and Davies, 1974). Rats 1-10 days old show epileptic responses to lower doses than do adult rats (Johnston and Davies, 1974). Local intracerebral injections of bicuculline can produce sustained focal discharges (J. Stevens, personal communication) . Intraarterial injection of bicuculline in the middle cerebral artery territory (L. Symon and B. S. Meldrum, unpublished) indicates that the neocortex is much less sensitive to bicuculline than the lower centers initiating the generalized seizures after

EPILEPSY AND Y-AMINOBUTYRIC ACID-MEDIATED INHIBITION

17

intravenous administration. Enhancement of photic epilepsy after subconvulsant doses of bicuculline has not been demonstrated (Meldrum and Horton, 1971). The short latency of seizure onset makes it difficult to correlate synaptic effects of the drug with the occurrence of seizures. When a cat is given a just-convulsive dose of bicuculline, the observed reduction in the effects of iontophoretically applied GABA may be due to a local pharmacological effect of bicuculline on cortical neurons, but it could also result from changes in the synaptic input to the neuron, secondary to an action of bicuculline in the brain stem or elsewhere.

2. Picrotoxin This mixture of picrotoxinin and picrotin produces convulsions when administered intravenously in doses of 1-5 mg/kg after a latent period much greater than that for bicuculline, but less than that of pyridoxine antagonists. Subconvulsant doses produce a fairly prolonged period of enhanced sensitivity to the induction of reflex epilepsy [l-3 hours for photosensitive epilepsy in baboons (Meldrum and Balzamo, 1972)l. Local application of picrotoxin to the cat cortex produces a focal discharge lasting several hours (Banerjee et al., 1970). The ability of picrotoxin to reduce presynaptic inhibition and dorsal root potentials has been recognized since 1963 (see Section 11, B) More recently it has been shown to block the postsynaptic inhibitory action of iontophoretically applied GABA at various sites in the rat, rabbit, and cat central nervous system (It0 et al., 1968; Obata and Highstein, 1970; Galindo, 1969; Bruggencate and Engberg, 1971; Engberg and Thaller, 1970; Hill et al., 1973a; Biscoe et al., 1972). Comparing the potency of compounds applied by iontophoresis is perhaps a debatable exercise, but the quantitative method devised by Hill and Simmonds (1973) indicates that picrotoxin and bicuculline are of similar potency against GABA (Hill et al., 1973b).

.

3. Penicillin Benzyl penicillin is a powerful local convulsant agent and “penicillin foci” are a standard tool for the research worker in epilepsy (Ward, 1969; Prince, 1972). Recently Davidoff ( 1972a) showed that penicillin antagonized the GABA-induced depolarization of dorsal root terminals in the frog spinal cord. When applied iontophoretically benzyl penicillin reversibly blocks the inhibitory action of GABA on cortical and spinal neurons (Curtis et al., 1972 ; Hill et al., 1973b) but is less potent than bicuculline. 4. Tubocurarine

Locally applied tubocurarine has a powerful convulsant action on the cerebral cortex or on the hippocampus (Bhargava and Meldrum, 1969;

18

B. S. MELDRUM

Banerjee et al., 1970). Iontophoretically applied tubocurarine is a potent GABA antagonist (Hill et al., 1973b), but the relationship of this effect to its very sustained local convulsant effects has not been established.

5 . Convulsive Activity and GABA Receptor Blockade The demonstration that a compound administered iontophoretically can block GABA-mediated inhibition does not prove that this is the cause of its systemic or local convulsant action. The case is strengthened if the pharmacological effect can be demonstrated with subconvulsant doses of the drug, or before the onset of focal or generalized seizures. Once seizure activity starts, the abnormal synaptic input will severely alter the responsiveness of the neuron. The evidence is best for picrotoxin; given intravenously, this produces changes in the effectiveness of GABA or of natural inhibitory systems in the absence of seizure activity (It0 et al., 1968), and will reduce primary afferent depolarization (De Groat et al., 1972). For bicuculline the evidence is less clear. A subconvulsive dose modifies the receptor field properties of occipital neurons ; those of complex and hypercomplex neurons change in a way that suggests that intracortical inhibitory processes are impaired (Pettigrew and Daniels, 1973). However, a close correlation between changes in the responsiveness of single cortical neurons and seizure onset after intravenous bicuculline is not to be expected, because this is probably not the major site of action of the drug. Close arterial injection of bicuculline suggests that the cortex is much less sensitive to the convulsive action of bicuculline than are some lower centers. Slow intravenous infusions of bicuculline or picrotoxin in cats have shown that both drugs reduce presynaptic inhibition in the cuneate nucleus before the onset of seizures (Hill et al., 1973b).

V. GABA Uptake The uptake of GABA by brain slices or particulate fractions was described by Elliott and Van Gelder (1958). Subsequently, the kinetic properties, specificity, and pharmacology of the uptake system have been very thoroughly explored (Gottesfeld and Elliott, 1971 ; Iversen, 1971; Iversen and Johnston, 1971; Iversen and Neal, 1968; Roberts and Kuriyama, 1968; Sano and Roberts, 1963; Kuriyama et al., 1969; Snodgrass and Iversen, 1973; Beart and Johnston, 1973; Johnston and Davies, 1974). Low-affinity cerebral uptake systems exist for many amino acids (Blasberg, 1968), but GABA and certain other putative neurotransmitters, such as glutamic and aspartic acids (in brain slices) and glycine (in spinal cord slices) show specific high-affinity uptake systems (Logan and Snyder, 1971; Bennett et al.,

EPILEPSY A N D Y-AMINOBUTYRIC ACID-MEDIATED INHIBITION

19

1973). The GABA-uptake system may be of functional importance in epileptic phenomena because it is probable that uptake into neurons and glia is the main method of terminating the postsynaptic inhibitory action of GABA after it has been released into the synaptic cleft. Autoradiographic studies have indicated that labeled GABA can be taken up into neurons, nerve terminals or glial cells (Bloom and Iversen, 1971; Neal and Iversen, 1972; Hokfelt and Ljungdahl, 1970, 1972). In brain slices, uptake into nerve terminals has been predominantly reported, 3 0 4 0 % of terminals taking up GABA actively (Iversen, 1971). However, slice studies may not give a true picture of the in v i m activity of glial cells. In cerebral and cerebellar cortex, uptake is predominantly into stellate, basket, and Golgi cells, i.e., inhibitory interneurons (Hokfelt and Ljungdahl, 1972; Ljungdahl et al., 1973; Schon and Iversen, 1972). Uptake of GABA by brain slices, homogenates, or synaptosomal fractions is Na' dependent (Sano and Roberts, 1963; Kuriyama et al., 1969; Bennett et al., 1973; De Feudis, 1973). Both energy-dependent and energy-independent processes are involved. Ouabain and protoveratrine inhibit the Na+dependent uptake (Gottesfeld and Elliott, 1971 ; Kuriyama et al., 1969). The pH optimum is 7.3-7.5, and the K,, is about 10 pA4 (Bond, 1973; Balcar and Johnston, 1973). The uptake system is specific for GABA and glycine; taurine, L-aspartate, and L-glutamate do not inhibit it (Balcar and Johnston, 1973). Various substituted GABA derivatives act as competitive inhibitors. 2-Hydroxy-GABA is active at a concentration of 1 p M ; 2-chloro-, 2- and 4-methyl-GABA are 50-100 times less active. Homohypotaurine, L-2,4-diaminobutyric acid and P-guanidinopropionic acid are also competitive inhibitors of uptake (Beart and Johnston, 1973; Harris et al., 1973). The enzyme inhibitor p-chloromercuriphenyl sulfonate is a potent inhibitor of GABA uptake at 0.1 mM (Harris et al., 1973; Iversen and Johnston, 1971). Various carbonyl agents (aminooxyacetic acid, D-cycloserine, p-hydrazinopropionic acid) if preincubated with rat brain slices in concentrations much higher than those required to inhibit GABA-transaminase block the uptake of GABA. 4-Deoxypyridoxine given in vivo or in vitro will also block GABA uptake (Snodgrass and Iversen, 1973). Many centrally acting drugs at concentrations of 0.1-1 .O mM, block GABA uptake in brain slices or homogenates. These include phenothiazines (chlorpromazine, prochlorperazine, fluphenazine), tricyclic antidepressants (desimipramine, amitriptyline) , butyrophenones (haloperidol), diazepam, and apomorphine (Harris et al., 1973). Diphenylhydantoin and anticonvulsant barbiturates do not modify GABA uptake, nor do convulsant agents such as bicuculline and leptazol, except for a weak inhibition produced by picrotoxin (Gottesfeld and Elliott, 1971; Harris et al., 1973). None of the

20

B. S. MELDRUM

centrally acting drugs specifically blocks GABA uptake; in fact, many inhibit the uptake of norepinephrine or 5-hydroxytryptamine at much lower concentrations ( Iversen, 1971) . Thus, GABA uptake is believed to be the major mechanism for inactivation of GABA at synapses, and it is readily modified pharmacologically in vitro. I n the anesthetized cat, administration of p-chloromercuriphenyl sulfonate prolongs the inhibitory action of iontophoretically applied GABA (Curtis et al., 1970b). However, so far there is no clear epileptic or antiepileptic drug effect that can be confidently related to induced changes in GABA uptake. VI. GABA Metabolism

The further metabolism of GABA is primarily via succinic semialdehyde and succinate (see Fig. 1) . GABA transaminase (Baminobutyrate:2-oxoglutarate aminotransferase, EC 2.6.1.19) requires pyridoxal phosphate as coenzyme and converts a-ketoglutarate GABA to succinic semialdehyde glutamate. Succinic semialdehyde dehydrogenase (SSAD, succinate semialdehyde :NAD ( P ) oxidoreductase, EC 1.2.1.16) converts succinic semialdehyde, oxidized nicotinamide-adenine dinucleotide water to succinic acid, reH'. The succinate is then further metabolized in the tricarduced NAD boxylic acid cycle. GABA-T and SSAD are both mitochondrial enzymes and are closely coupled (Salganicoff and De Robertis, 1965). SSAD has a very low K,, and succinic semialdehyde is present in the brain only in very low activity. Both enzymes have pH optima in the alkaline range (GABA transaminase, 8.0-8.6;.SSAD, 8.6-9.4) (Waksman and Roberts, 1965; Baxter, 1970). Isoenzymes of GABA transaminase can be separated and may differ in enzymic properties according to their cellular origins. As GABA transaminase is a mitochondrial enzyme, it is improbable that it plays a direct role in the inactivation of GABA after its release into the synaptic cleft. A powerful inhibitor of GABA transaminase (hydrazinopropionic acid, see below) when administered iontophoretically did not enhance the inhibitory action of GABA on single neuron firing (Curtis et al., 1970b). However, drugs inhibiting GABA transaminase or factors restricting the availability of NAD+, and thus inhibiting SSAD, could lead to an accumulation of GABA in presynaptic endings, which might be of functional significance.

+

+

+

+

+

INHIBITORS OF GABA TRANSAMINASE Various compounds known to inhibit cerebral GABA transaminase activity both in uitro and in vivo are listed in Table 11. Some of these have

TABLE I1 INHIBITORS OF GABA-TRANSAMINASE'

Inhibitor

Enzymic mechanism

Ki (M)

Dose inhibiting in vivo (mg/kg)

Hydroxylamine

PyP oxime (compet. GABA) Aminooxyacetic acid PyP oxime (compet. GABA)

25

1.4 x 10-3

200

m

Species

Seizures

Mice

Audiogenic, ECS, PTZ

Mice, baboons

ECS, drugs, photosensitive

Mice, Audiogenic, baboons, photosensitive, man petit ma1 -

Di-n-propylacetate

Compet. GABA

Hydrazinopropionic acid Ethanolamine O-sulfate

Compet. GABA Irreversible (active site)

4.4 X lo-'

(Intracisternal) 1

Mice

(Intracerebral) ECS

Cycloserine

Compet. GABA noncompet. Compet. GABA

2.3 x 10-4

4500

Mice

Audiogenic

5-Ethyl-5-phenyl-2pyrrolidone, yethyl-y-phenylGABA 0

+

2.35

x

10-7

10

m 2 r

Antiepileptic effect References Baxter and Roberts (1961); Lehmann (1964); Kohli and Kishor (1965) Wallach (1961); Da Vanzo

(1961);Kuriyama (1966);Meldrum ct 01. (1970) Simler et al. (1973);Patry and Naquet (1971);VBlzke and Doose (1973) Van Gelder (1968,1969) et al. ct al.

Fowler and John (1972); Fowler (1973);Baxter ct al. (1973) Dann and Carter (1964 Lehmann (1964) Carvajal ct al. (1964); Perez de la Mora and Tapia (1973)

'd

v1

.e

s-

2% 2

0

m C

2

5P E

z 5

E

2 0 z

ECS, electroconvulsive shock; PTZ, pentylenetetrazole; TSC, thiosemicarbazide; PyP, pyridoxal phosphate; compet. GABA, com-

petitor of y-aminobutyric acid.

r a c

22

B. S. MELDRUM

already been discussed as GAD inhibitors under the heading “Other Carbony1 Trapping Agents.” In vivo hydroxylamine, aminooxyacetic acid, and cycloserine depress GABA transaminase selectively in comparison to GAD. One possible explanation is that the pyridoxal phosphate derivative becomes tightly bound to the apoenzyme. However, inhibition by hydroxylamine may also be competitive with the substrate (Baxter and Roberts, 1961). Several GABA transaminase inhibitors show a structural similarity to GABA. Thus, some substances listed in Table I1 are also included in Fig. 2 as GABA analogs. Apparent GABA analogs which are competitive GABA transamiiiase inhibitors in vitro have little effect on cerebral GABA transaminase activity when given intraperitoneally (Baxter and Roberts, 1961; Carvajal et al., 1964) . Thus, 5-ethyl-5-phenyl-2-pyrrolidone. which like other lactams

2-Pyrrolldona

2-Pyrrolidone acetpmlde

1-Hydmxy-Samlnopyrrole

8-Hydroxybutyrtc acld

NH,CH,CH,CH,CH,COOH 6-Amlnovalertc acld

NH,C(NH)NHCH&H,CCOH I I1 HC,\C,CH

~-Cuantdlnoproplonlc aeld

H 1-Phenyl- S-methyl2-pyrmltdone

Nti,CH,,CCCOOH

5-Ethyl-5-phenyl2-pyrmlldone

4-Amlnotetrottc acid

h N *CH, * ?HI.COOH HCPCY!H I I1 HCa ,CH

H cONH, I

I&C-

I

c

O“40

NH,.O. CH, . COOH

H,c-CH, I

I

Amtmxyacettc acld

HaC.oXao

H

c1

NH,NHCH,CH,COOH

8-P-Chlomphenyl-y-

Cyclosertne

mminobutyrtc actd

Butymlactone

Hydrazlnoproptonlc acld

NH,CH,CH.CH(NH,)COOH -0

h-CH

r;’‘0 >-C+ Mumclmol

Mi:

Hoh2-CH II II N\ONc\FH-NH,

CCOH b t e n l c acld

Fro. 2. 7-Arninobutyric acid analogs.

L-2,4-MamlMbutyrtC acld

(CH,CH,CH,),CHCOOH

M-n-propylacetate

EPILEPSY AND Y-AMINOBUTYRIC ACID-MEDIATED INHIBITION

23

is converted by hydrolysis in uivo to a GABA derivative (y-ethyl-y-phenyl-yaminobutyric acid) has little apparent effect on in uiuo GABA transaminase activity (when given at 160 mg/kg to mice), although it and the GABA derivative are powerful competitive inhibitors of GABA transaminase in uitro (Carvajal et al., 1964). However, as with all “in uiuo” studies of competitive inhibitors where the assay is made on a diluted homogenate of brain, the inhibition present in vivo may have been concealed by the addition of substrate. Hydrazinopropionic acid is a GABA analog which is a very powerful competitive inhibitor of GABA transaminase. I t also inhibits tyrosine aminotransferase so that cerebral levels of tyrosine are markedly elevated (Van Gelder, 1969). As with aminooxyacetic acid, low doses ( 10-30 mg/kg) produce sedation, and higher doses aqe followed by seizures. Di-n-propyl acetate is a much weaker competitive inhibitor of GABA transaminase (Simler et al., 1973). Ethanolamine O-sulfate when incubated with partially purified cerebral GABA transaminase irreversibly inactivates it; apparently competing with GABA for the active site (Fowler and John, 1972). Ethanolamine O-sulfate does not cross the blood-brain barrier, but if it is given intracerebroventricularly to mice or intracisternally to rats, in vivo inhibition of GABA transaminase can be demonstrated (Fowler, 1973; Baxter et al., 1973). Table I indicates that hydrazine and many of its derivatives are powerful GABA transaminase inhibitors; most are also powerful GAD inhibitors and convulsant agents. However, a number that are also monoamine oxidase inhibitors, such as phenylethylhydrazine or phenylpropylhydrazine powerfully inhibit GABA transaminase in uivo and raise brain GABA content (Popov and Matthies, 1969) . Chronic administration of phenylethylhydrazine or of isonicotinic acid hydrazide to rats produces a sustained elevation of brain GABA content (Perry and Hansen, 1973). Intracerebroventricular injection of ethanolamine O-sulfate in mice gives partial protection against maximal electroconvulsive shock (Baxter et al., 1973). Parenteral administration of any of the other inhibitors of GABA transaminase listed in Table I1 (except hydrazinopropionic acid) protects against experimental epilepsy (either electroconvulsive shock, convulsant drugs such as pentylenetetrazole or thiosemicarbazide, audiogenic seizures in rodents or photosensitive epilepsy in baboons) . Among these drugs only di-n-propylacetate has been tried extensively in man. It is reported to be most effective in petit ma1 and in centrencephalic grand ma1 (Carraz et al., 1964; Volzke and Doose, 1973). Although it is not proved that antiepileptic activity possessed by compounds inhibiting cerebral GABA transaminase activity is due to this action alone, an increase in the amount of GABA available for release from syn-

24

B. S. MELDRUM

aptic endings is a possible explanation. Studies with ethanolamine O-sulfate indicate that 50% inhibition of GABA transaminase activity was required to elevate brain GABA content (Fowler, 1973). Problems with assay conditions prevent accurate assessment of percentage inhibition of GABA transaminase in uivo after other inhibitors. Some of these compounds may, in addition, act as “GABA analogs” at receptor sites, but as the following section shows, there is not yet adequate evidence to correlate such activity with an ticonvulsan t effects. VII. GABA Analogs

Compounds which are structural analogs of GABA may compete with GABA ( a ) at receptor sites on the neuronal surface, where they either mimic the action of GABA, or block it, or do both; (b) at carrier sites for uptake into neurons or glia; and (c) at the active site of the enzyme GABA transaminase. Theoretically any one compound could possess all these properties (and indeed other properties related to the excitatory amino acids or to cellular metabolism) However, this section is concerned with substances that have been proposed as physiological GABA analogs, i.e., “GABAmimetic” compounds. GABA itself when given systemically enters the brain little, if at all (Van Gelder and Elliott, 1958), and some of its antiepileptic effects, when it is given in high doses, probably result from dehydration of the brain (De Feudis and Elliott, 1967; De Feudis, 1971). There are two circumstances where the blood-brain barrier may be ineffective. In young chicks it is poorly developed, and moderately high doses of GABA ( 100-350 mg/kg) produce sedation and suppress pentylenetetrazole convulsions (Kobrin and Seifter, 1966). In monkeys with focal epileptogenic lesions produced by alumina cream, GABA ( 1 gm/kg intraperitoneally or 500 mg/kg intravenously) had some protective action against convulsions due to 4-methoxypyridoxine or pentylenetetrazole (Kopeloff and Chusid, 1965) . As GABA failed to protect monkeys without focal lesions, penetration into the site of the lesions may be the explanation for its antiepileptic effect. When given intracerebrally GABA has antiepileptic properties in many test systems, e.g., audiogenic seizures in mice (Schlesinger et al., 1969), pentylenetetrazole seizures (Schlesinger et al., 1969), and focal and general cortical discharges (Hayashi and Nagai, 1956; Hayashi, 1959). Thus, any GABAmimetic analog that crossed the blood-brain barrier might be a clinically useful antiepileptic agent. 2-Pyrrolidone is a lactam which, being relatively nonpolar, enters the brain more readily than GABA. Hawkins and Saret (1957) reported that it was a more effective anticonvulsant than GABA when given orally to mice treated with pentylenetetrazole or p-methyl p-ethylglutarimide (Megimide) .

.

EPILEPSY AND Y-AMINOBUTYRIC ACID-MEDIATED INHIBITION

25

The anticonvulsant ED,, was 150 or 200 mg/kg compared with 200 to 400 mg/kg of GABA. Subsequently much effort was devoted to the synthesis and testing of derivatives of 2-pyrrolidone. The structural formulas of three of these are shown in Fig. 2. The acetamide (Piracetam) protects rats against audiogenic seizures (at 30-300 mg/kg) and limits the spread of discharges from strychnine or penicillin cortical foci in rabbits (Moyersoons et al., 1969). The l-hydroxy-3-amino derivative of 2-pyrrolidone (HA 966) is a central depressant shown to possess anticonvulsant properties in several test systems (Bonta et al., 1969, 1971). Thus, 30-50 mg/kg given intraperitoneally in mice raises the threshold for strychnine or metrazole convulsions. This compound may undergo hydrolysis or metabolic conversion in the liver before exercising its central actions (Bonta et al., 1971) . However, microelectrophoretic studies show that l-hydroxy-3-amino-2-pyrrolidoneitself depresses the firing rate of cortical neurons with about half the potency of GABA (Davies and Watkins, 1972, 1973). Davies and Watkins have proposed that this action is at least in part due to blockage of excitatory amino acid receptor sites; i.e., it competes with glutamate or aspartate at postsynaptic sites. The 5-ethyl-5-phenyl derivative of 2-pyrrolidone has already been discussed as an inhibitor of GABA transaminase (Section V I ) . I t has undoubted anticonvulsant properties, but there is not yet any iontophoretic evidence to show that it, or its hydrolysis product, y-ethyl-7-phenyl-GABA, are true GABA-mimetic compounds. Russian authors (Motovilov and Kozhevnikov, 1968) have demonstrated anticonvulsant properties in some pyrazolidone derivatives (e.g., 1-phenyl-3methyl-5-pyrazolidone and 5-phenyl-3-pyrazolidone,25 mg/kg in rabbits given pentylenetetrazole) . Cycloserine also has structural similarities to 2-pyrrolidone and to GABA, and it has definite anticonvulsant properties, but here also there is no evidence for GABA-mimetic properties other than inhibition of GABA transaminase and blockade of GABA uptake at high doses. Butyrolactone is a central depressant which is hydrolyzed in vivo to form y-hydroxybutyric acid, whose properties are discussed below. Two centrally active compounds found in fungi of the genus Amanita, muscimol and ibotenic acid, have rather specific actions when applied iontophoretically to central neurons in the cat (Johnston et al., 1968a; Curtis et al., 1970a). Ibotenic acid is a more powerful excitant of spinal interneurons than is glutamic acid. Muscimol, the decarboxylated analog of ibotenic acid, depresses the firing of spinal or cortical neurons, having about half the potency of GABA. This inhibitory action is blocked by bicuculline, but not by strychnine. Thus, muscimol appears to be a true “GABA-mimetic.” Its anticonvulsant properties have not been extensively studied, partly because of its hallucinogenic properties (Theobald et al., 1968).

26

B. S. MELDRUM

There have been numerous compounds synthesized, in which GABA, or a related similarly active compound, is attached to a phenyl ring or other lipophilic structure to facilitate passage through the “blood-brain barrier.’’ Initial Russian studies (Khaunina, 1964, 1968) showed that central activity was greatest when the phenyl ring was attached to the p carbon of GABA rather than the CY or y carbons. p-Phenyl-7-aminobutyric acid potentiates narcosis and reduces motor activity and arecoline-induced tremor. Strychnine and pentylenetetrazole were not antagonized, but thiosemicarbazide was. Further potentiation can be obtained by halogen substitution, and Keberle and Faigle (1972) found that, among a series of such compounds, the most centrally active was p- (p-chlorophenyl) -7-aminobutyric acid (Lioresal, baclofen) . This compound is clinically effective against various forms of spasticity (Birkmayer et al., 1967; Knutsson et al., 1973; McLellan, 1973; Burke et al., 1971). Electrophoretic studies with p-phenyl-GABA derivatives (Davies and Watkins, 1974) show that such compounds depress the firing of central neurons but apparently do not act specifically on postsynaptic GABA receptors, as the inhibition is not blocked by bicuculline. In mice, baclofen antagonizes pentylenetetrazole or thiosemicarbazide convulsions, but not strychnine or electroconvulsive shock ( Bein, 1972). In baboons with photosensitive epilepsy, baclofen provoked the appearance of continuous spikes and waves on the EEG (Meldrum, 1974). In patients with epilepsy, baclofen administered for spastic conditions may provoke EEG deterioration or even the occurrence of grand ma1 seizures (Pinto et al., 1972; Bein, 1972). Thus, pphenyl-GABA derivatives may act on presynaptic inhibition to reduce spasticity, but an experimental test failed to demonstrate enhanced presynaptic inhibition (Ashby and White, 1973). The tendency of baclofen to exacerbate spontaneous epileptic phenomena may be completely independent of any actions at GABA receptor sites. Various straight-chain analogs of GABA are also shown in Fig. 2. Of these, 4-aminotetrolic acid inhibits spinal neurons with 0.5-0.2 times the potency of GABA, and this inhibition is reversibly blocked by bicuculline (Beart et al., 1971). Central actions of 4-aminotetrolic acid have not been described. Other w-amino acids, such as 8-aminovaleric acid and p-guanidinopropionic acid, are GABA-mimetic in several physiological systems (Curtis and Watkins, 1965), but have not been shown to be anticonvulsants when given systemically. y-Amino-P-hydroxybutyric acid was found to be more effective than GABA at blocking cortical seizure discharges in dogs, when given either into the carotid artery or into the cerebral ventricles (Hayashi, 1959). However, when applied iontophoretically to cortical neurons it is less potent than GABA (Crawford and Curtis, 1964; KrnjeviE. and Phillis, 1963).

EPILEPSY A N D 7-AMINOBUTYRIC ACID-MEDIATED INHIBITION

27

Butyrolactone and its hydrolysis product, 4-hydroxybutyric acid, are sedative and anesthetic agents in many species (Jouany et al., 1960; Jenney et al., 1962; Laborit, 1964). Both substances have been claimed to be normally present in brain (Bessman and Fishbein, 1963; Roth, 1970). However, Crawford and Curtis ( 1964) found that iontophoretically 4-hydroxybutyric acid was not a GABA-like inhibitor. Strangely, as with baclofen, 4-hydroxybutyrate (100-600 mg/kg in cats) produces sustained cortical spike and wave discharges (Winters and Spooner, 1965a,b). Three straight-chain “GABA-analogs” that inhibit GABA transaminase are illustrated in Fig. 2 (hydrazinopropionic acid, aminooxyacetic acid, and di-n-propylacetate) . Electrophoretically hydrazinopropionic acid is a weak GABA-like inhibitor of spinal neurons (Curtis et al., 1970b). ~-2,4-Diaminobutyricacid is a neurotoxic convulsant (O’Neal et al., 1968), that raises brain GABA content (Vivanco et al., 1966) and is a competitive inhibitor of GABA uptake (Beart and Johnston, 1973; Harris et ul., 1973). Thus, to date, there is no compound that has been clearly established as both a physiological GABA analog and as a generally effective anticonvulsant agent. There are numerous approaches that have not been fully investigated, and now that the structural specification for compounds reacting with the GABA receptor is a t least partially established (Kier and Truitt, 1970; Beart et al., 1971), further progress is to be expected.

VIII. Anticonvulsant Drugs and the Functional Role of GABA

The drugs that are most widely used clinically as anticonvulsants can be classified into six groups. These are hydantoins (diphenylhydantoin ) barbiturates (phenobarbitone, primidone), oxazolidinediones (trimethadione), succinimides (ethosuximide, phensuximide) , sulfonamides (acetazolamide) and benzodiazepines (diazepam, nitrazepam). For none of these categories is it possible to give a definitive explanation in molecular and cellular terms of the antiepileptic effect. It is clear that all six groups act differently. One group (the sulfonamides) has a distinctive biochemical action (inhibition of carbonic anhydrase activity). Although the anticonvulsant effect of sulfonamides may well be secondary to inhibition of carbonic anhydrase in glia and neurons, the physiological events underlying the antiepileptic effect have not yet been specified. It is possible that a change in intracellular pH modifies GABA metabolism. There is evidence that monoamines play an intermediate role in the anticonvulsant action (Gray and Rauh, 1971). The hydantoins have effects on various membrane phenomena, in particular the transport of sodium. Administration of diphenylhydantoin to rats

28

B. S. MELDRUM

and mice has been shown to increase cerebral GABA content and decrease glutamic acid content (Vernadakis and Woodbury, 1960; Woodbury, 1969; Saad et al., 1972). The barbiturates and the benzodiazepines both enhance presynaptic inhibition in the spinal cord (Schmidt, 1971). Eccles et al. (1963) showed that dorsal root potentials in the cat are markedly prolonged by pentobarbitone, and corresponding changes in presynaptic inhibition are demonstrable. Trimethadione and general anesthetics, including chloroform and ether, also prolong the time course of presynaptic inhibition (Miyahara et al., 1966). Diazepam markedly augments and prolongs dorsal root potentials and presynaptic inhibition (Schmidt et al., 1967; Schmidt, 1971). The site or sites at which barbiturates and diazepam act to augment presynaptic inhibition is not known. I t is a reasonable speculation that this mechanism contributes to the antiepileptic action of barbiturates and benzodiazepines. Both drugs are especially effective against reflex or sensory epilepsy (Lehmann, 1964; Stark et al., 1970; Meldrum et al., 1974). Barbiturates and benzodiazepines are also the most generally effective agents for arresting status epilepticus. I t may be that presynaptic inhibition plays a role in the mechanism by which prolonged epileptic activity becomes self-sustaining. An enhanced cerebral GABA content has been described in mice 2 hours after the intraperitoneal injection of 25-50 mg of phenobarbital per kilogram (Saad et al., 1972). A great many other neurophysiological effects have been described following the administration of barbiturates, benzodiazepines, and other anticonvulsants, but they are difficult to relate to either biochemical events at membranes and synapses or antiepileptic action, and will not be discussed here. In any neuronal aggregate when epileptic activity is displayed, the basic phenomenon is an excessively synchronous discharge of neurons. Although cerebral gray matter displays immense variety in its pattern of cellular organization, a universal feature is the use of inhibitory interneurons, activated by direct or recurrent collateral pathways, to prevent the synchronous discharge of large numbers of neurons. With the exception of the spinal cord and lower brain stem, where glycine is the transmitter producing postsynaptic inhibition, GABA is the principal inhibitory substance identified in the central nervous system. T o increase the effectiveness of the postsynaptic inhibition that normally prevents excessive synchronicity of firing, the ideal method would be to increase the amount of GABA released by activity in the inhibitory interneurons, or to slow its removal from the synaptic cleft. The use of a GABA-mimetic is theoretically not ideal because it will not act in the appropriate place at the right time, but will tend to depress all

EPILEPSY AND Y-AMINOBUTYRIC ACID-MEDIATED INHIBITION

29

activity. I n practice, although the perfect GABA-mimetic compound has not been found, nonspecific depression seems to be the main effect. Indeed, synchronous depression of responsiveness may enhance the appearance of rhythmic epileptiform discharges (as observed after butyrolactone or lioresal) . Drugs blocking GABA uptake do not appear to possess particular antiepileptic properties. Perhaps the right compound has not yet been found or tested. Possibly the reuptake of GABA into nerve terminals contributes to the maintenance of inhibitory function. Thus the best way to increase postsynaptic inhibition appears to be enhancement of the amount of GABA released from synaptic terminals. This may be the mode of anticonvulsant action of drugs inhibiting GABA-transaminase activity. Further study of this class of compounds may, in the future, provide the improved drug therapy required by so many of today’s epileptic patients. REFERENCES Alberici de Canal, M., and De Lores Arnaiz, G. R. (1972). Biochem. Pharmacol. 21, 133-136. Albers, R. W., and Brady, R. 0. (1959). J . Biol. Chem. 234, 926-928. Ashby, P., and White, D. G. (1973). J . Neurol. Sci. 20, 329-338. Awapara, J., Landua, A. J., Fuerst, R., and Seale, B. (1950). J . Biol. Chem. 187, 35-39. Baker, W. W., and Kratky, M. (1967). Arch. Int. Pharmacodyn. Ther. 170, 81-92. Balks, R., Machiyama, Y., and Patel, A. J. (1973). In “Compartmentation and the Metabolism of Gamma-aminobutyrate” (R. Balhs and J. Cremer, eds.), pp. 57-70. Macmillan, New York. Balcar, V. J., and Johnston, G. A. R. (1973). J . Neurochem. 20, 529-539. Balcar, V. J., and Johnston, G. A. R. (1974). Biochem. Pharmacol. 23, 821-827. Balzer, H., Holtz, P., and Palm, D. (1960). Naunyn-Schmiedebergs Arch. E x p . Pathol. Pharmakol. 239, 520-552. Banerjee, U., Feldberg, W., and Georgiev, V. P. (1970). Brit. J . Pharmacol. 40, 6-22. Banna, N. R., and Jabbur, J. J. (1969). Int. J . Neuropharmacol. 8, 299-307. Barker, J. L., and Nicoll, R. A. (1972). Science 176, 1043-1045. Baxter, C. F. (1970). I n “Handbook of Neurochemistry” (A. Lajtha, ed.), Vol. 3, pp. 289-353. Plenum, New York. Baxter, C. F., and Roberts, E. (1961). J . Biol. Chem. 236, 3287-3294. Baxter, M. G., Fowler, L. J., Miller, A. A., and Walker, J. M. G. (1973). Brit. J . Pharmacol. 47, 681P. Beart, P. M., and Johnston, G. A. R. (1972). Brain Res. 38, 226-227. Beart, P. M., and Johnston, G. A. R. (1973). J . Neurochem. 20, 319-324. Beart, P. M., Curtis, D. R., and Johnston, G. A. R. (1971). Nature (London) 234, 80-81. Bein, H. J. (1972). I n “Spasticity-a Topical Survey” (W. Birkmayer, ed.), pp. 76-89. Huber, Bern. Bell, J. A,, and Anderson, E. G. (1972). Brain Res. 43, 161-169.

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Note added in proof. Since this article was completed (November 1973) three reviews that, although not specifically concerned with epilepsy, discuss the role of GABA in the brain have appeared-Krnjevit, K. (1974). Physiol. Rev. 54, 418-540; Curtis, D. R., and Johnston, G. A. R. (1974). Ergeb. Physiol. Biol. C h e m . Exp. Pharmakol. 69, 97-188; and Roberts, E.’ (1974) Biochem. Pharmacol. 23, 2637-2649. I n addition, a definitive account of inhibitors of L-glutamate decarboxylase has been published-Wu, J.-Y. and Roberts, E. (1974). J . Neurochem. 2 3 , 759-767.

PEPTIDES AND BEHAVIOR' By Gcorgcr Ungar

Baylor College of Medicine, Houston, Texas

I. Introduction

.

11. Peptides and Innate Behavior

A. B. C. D.

.

Glutathione and the Feeding Behavior of Hydra Invertebrate Neuroseuetory Peptides Peptides in Eating and Drinking Behavior Territoriality and Aggressivity E. Peptides and Sleep F. BirdMigration III. The Role of Peptides in Learned Behavior A. Peptide Hormones and Their Derivatives B. Peptides in Learning and Memory IV. Is There a Peptide Code in the Nervous System? A. InnateMechanisms B. Formation and Labeling of Metaarcuits C. The Role of Peptides in Neural Coding References

.

.

.

.

.

. .

.

.

. .

.

. .

.

37 39 39

. 4 0

. . . .

41 41 * 42 43 43 43 . 4 4 50 52 53 55 56

. . . . . .

1. Introduction

The biological revolution of the mid-twentieth century started from the idea that living organisms require for their development and survival not only energy, but also information. I n the last twenty-five years we have learned a great deal about genetic information, but we are just beginning to be interested in the elaborate biological information processing systems which integrate the life of the many cells of each individual into a harmoniously functioning whole and assure the best adaptation of the organism to the changing conditions of the environment. Some of the agents of biological coordination, the hormones, have been known for a long time. Most of these belong to two chemically distinct groups: ( a ) steroids produced by the sex glands and the adrenal cortex and ( b ) peptides or amino acid derivatives, secreted by nerve cells, the ' T h e research work done in the author's laboratory referred to in this review is supported by a grant from the U.S. National Institute for Education.

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pituitary and the glands of the digestive tract. Most of the latter substances can directly or indirectly be traced back to the neurosecretory process. All information-carrying molecules can be characterized by two parameters: the message they convey and the “address” to which they have to deliver it. From what we know of cells at present, the messages reaching them can elicit a limited repertoire of responses. The apparent complexity of cellular responses to hormones and other messengers depends, therefore, not so much on the variety of messages they carry as on the specificity of their destinations. This specificity varies considerably from the sharply defined selectivity of the releasing and tropic hormones to the wide range of cells acted upon by, for example, insulin, growth hormone, or thyroxine. Specificity of the address is less critical for short-distance messengers, such as the neurotransmitters. Among the many millions of cells that could respond to them, they reach only those that are across the synaptic gaps into which they are released. This may explain the comparatively small number of transmitter substances, as opposed to the number of peptide hormones. I t should be emphasized here that all of the types of substances known to carry information (steroids, prostaglandins) or potentially capable of doing it (polysaccharides, complex lipids), peptides have by far the highest information content. Transplantation data suggest that there is a sufficient number of possible peptides to mark all the individuals of all the species of higher vertebrates that have ever existed on this globe. It is easy to understand, therefore, that peptides could carry specific messages from cell to cell or, more precisely, messages between specific cells, with the highest possible accuracy of address, even if each single cell were marked with a distinctive receptor. Such luxury of specificity is probably not necessary. Even in the nervous system, which has acquired the highest degree of differentiation of all the tissues, specificity is probably limited to cell groups that form a functionally differentiated circuit. This point will be discussed below in some detail. These considerations are of importance for the role of peptides in behavior. Behavior as “the sum total of the action of the effectors” (Young, 1938) implies integration or coordination, that is, some sort of communication between several neurons and neuronal circuits. The purpose of this review is to examine the role of peptides in the integration of innate and learned behavior and to discuss the mechanisms of this integration. The term peptide is used in this review in its widest meaning. Since there is no uniformly accepted boundary bstween peptides and proteins or between oligo- and polypeptides, the term peptide will be applied to any chemical entity that contains at least one peptide bond. Although I shall deal mostly with sequences of less than one hundred amino acids, in some

PEPTIDES AND BEHAVIOR

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cases larger molecules will be considered, since the active sites that determine their specificity may be comparatively short sequences.

II. Peptider and Innate Behavior

Peptide hormones control many innate behavioral patterns : sexual behavior is dependent on gonadotropin secretion; eating can be induced by insulin ; drinking can be inhibited by vasopressin; parturition can be triggered by oxytocin; etc. I shall, however, not discuss these well known behavioral effects and mention, instead, some examples that are less known but may supply clues to some basic mechanisms. A. GLUTATHIONE AND

THE

FEEDINGBEHAVIOR OF Hydra

Probably the simplest example of behavioral modification induced by a peptide is a phenomenon discovered by Loomis (1955) and studied by Lenhoff (1968) and Rushforth (1965). They showed that the feeding behavior of several species of Hydra and other coelenterates is controlled by the presence of glutathione in their environment. Hydra littoralis, on which most of the experiments were done, is a fixed animal whose tentacles capture any small prey that happens to come near. The contact elicits the response of nematocysts lining the tentacle which puncture and poison the animals. As soon as the body fluids of the wounded prey escape, the tentacles bend toward the mouth, the mouth opens and the prey is swallowed. Whereas the stimulus for the response of the nematocysts is probably mechanical, the tentacle bending and mouth opening are elicited specifically by the presence of glutathione in the fluid escaping from the wound. The specificity of reduced glutathione is well established; none of the three amino acids that make up the tripeptide are active alone; on the contrary, glutamic acid, glutamine, and cysteinylglycine act as inhibitors. The only derivatives capable of eliciting the feeding response are ophthalmic acid (y-glutamyl-cramino-n-butyrylglycine), norophthalmic acid ( 7-glutamylalanylglycine) , and S-methylglutathione. These well documented observations are of considerable interest for several reasons. The primitive nature of the organisms may afford an insight into the very foundations of hormonal mechanisms, even if the action of glutathione does not correspond to the definition of hormonal actions in higher animals. The study of the phenomenon may also afford some insight into the agonist-receptor relationship. Lenhoff (1969) has proposed a hypothesis in which glutathione would act as a “modifier” (in the sense of the model of Koshland et al., 1966) or as an “allosteric activator” (in

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the interpretation of Monod et al., 1963) of the receptor molecule. Another important consideration is that the role of glutathione is played in other coelenterate species by single amino acids, such as proline, valine, or glutamine. The possibility that peptides take over the functions of amino acids as the complexity of the organisms increases and their information input becomes diversified will be discussed later. There is no evidence at present, one way or the other, for the intervention of neural elements in the feeding behavior of coelenterates, because of their diffuse, noncentralized nervous system. The role of amino acids and peptides as the adequate stimuli for feeding behavior have, however, been demonstrated in higher organisms (annelids, arthropods) in which the nervous system has a definite control over behavior (Lindstedt, 1971). NEUROSECRETORY PEPTIDES B. INVERTEBRATE There is a great deal of literature on neurosecretion in invertebrates (see Barrington, 1964; Fingerman, 1970), but I shall mention only two examples of behavior-controlling peptides secreted by nerve cells. The first is the crustacean color-change hormone, identified by Fernlund I t has and Josefsson ( 1972) as pGlu-Leu-Asn-Phe-Ser-Pro-Gly-TrpNH2. been known since the early studies of Parker (1948) that this important behavioral component of some crustaceans, cephalopods, fishes, and amphibians is under neurohumoral control. According to the color of the background and the illumination, these animals can change their color so as to blend in with their environment. I n shrimps, the peptide, secreted by the nerve endings in the sinus gland located in the eyestalks, represents the efferent channel of a reflex whose afferent pathway is represented by neurons going from the eye to the gland. In vertebrates, the chromatophores are under more direct neural control and the color of the skin can be influenced by the known neurotransmitters. This, however, does not exclude the possible role of peptides, and vasopressin, oxytocin, and melanocyte-stimulating hormone (MSH) are known to have an effect on skin color in amphibians and fishes. The exact relationship between these effects and direct nervous control is not known; its elucidation could be of considerable importance for understanding the role of peptides in neural function. The second example is a “polypeptide” secreted by the “bag cells” of the parietovisceral ganglion of Aplysia (Toevs and Brackenbury, 1969). It can be released by electrical stimulation of the ganglion or by K+-induced depolarization. Injection of the isolated substance into a recipient animal induces egg laying (Kupfermann, 1967). Its molecular weight is estimated to be about 6000.

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Similar neurosecretory phenomena have been seen in ganglia of invertebrates from various groups (Scharrer and Weitzman, 1970). These observations, however, were based on histochemical criteria only, and no attempts have been made at isolating and characterizing the peptides. C. PEPTIDES I N EATING AND DRINKING BEHAVIOR Neural control of feeding behavior has been widely studied in the last decade. The role of neurotransmitters has received particular attention : norepinephrine in eating and acetylcholine in drinking (Grossman, 1960), but the ubiquitousness of the transmitters precludes them from being the specific agents in these behaviors. The transmitter effect varies according to species and more particularly according to the site of injection. Using the same method of intracranial injections, Epstein et a/. (1970) and Fitzsimons (1971) showed that angiotensin injected into the medial preoptic area produced drinking in nonthirsty rats. The amount necessary is just a few picomoles of the substance. It is assumed that hypovolemia induces secretion of renin in the kidney and increases production of angiotensin which would act on the thirst centers and activate drinking behavior. It is important to note that the peptide, unlike the amines, is active also by systemic administration at high doses. This fact underlines the fundamental difference in the specificity of the two types of active substances. Information supplied by the method of intracerebral injections has been supplemented by experiments measuring the release of specific substances in the brain of hungry or thirsty animals. I t has been shown (Yaksh and Myers, 1972) that the hypothalamus of the hungry rhesus monkey releases a substance that induces eating in a satiated animal. Conversely the brain of the satiated monkey releases a substance that inhibits feeding behavior. The chemical nature of the active material is unknown. The authors seem inclined to believe that the stimulating factor is a catecholamine, but some of the experimental data do not agree with this assumption. In any case, the inhibitory substance does not fit with the description of any of the neurotransmitter substances known.

D. TERRITORIALITY A N D AGGRESSIVITY Analysis of the territorial behavior of the Mongolian gerbil by Thiessen (1973) points to the possibility of the role of specific peptides or proteins. It has been established that territorial marking in this species is done principally by the secretion of a scent gland whose functioning is controlled by testosterone or progesterone. Thiessen and his colleagues have found that the effect on territorial marking of hormones implanted in the preoptic area

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of the hypothalamus can be inhibited by actinomycin D and puromycin, injected together with the hormone. This suggests that the hormone induces the synthesis of some protein necessary for the marking behavior. Yahr and Sanders (quoted by Thiessen) found a qualitative difference between proteins of the preoptic area in hormone-stimulated and control animals. There is good evidence that territoriality, as measured by making frequency, is related to social dominance and aggressivity. It is, therefore, possible that certain portions of the brain produce substances that enter into the mechanism of aggressive behavior. There is a whole area of study here that has hardly been touched yet in spite of the availability of an appropriate methodology. Active substances could be isolated by using test systems, such as territorial marking, measure of aggressivity, or other behavioral characteristics.

E. PEPTIDESAND SLEEP A humoral mechanism of sleep was the first proposed by Legendre and PiCron in 1910 and further supported by the experiments of Schnedorf and Ivy (1939). More recently, the study of this problem has been resumed in the laboratories of Monnier in Basle and Pappenheimer at Harvard University. Using electroencephalographic criteria of sleep in their assay systems, both authors have shown that intraventricular infusion of cerebrospinal fluid from sleep-deprived goats (Pappenheimer et al., 1967) or from rabbits put to sleep by electrical stimulation of the thalamic sleep center (Monnier and Hoesli, 1965) induced sleep in recipient animals. Both laboratories found that the active substance had a low molecular weight (around 500-700 daltons) and was inactivated by heating to looo, extreme variations of pH, and repeated freezing and thawing. Further attempts at characterization indicated that it was a peptide probably containing the following amino acids: Ala, Asp, Glu, Gly, Leu, Ser and Thr, and perhaps T r p (Schoenenberger et al., 1972). The material is highly active: it induces sleep at the dose of about 6 ng/kg when infused by intraventricular route. The experimental data suggest that the peptide may be released by the thalamic sleep center or from the reticular formation (Drucker-Colin et al., 1970), which in turn are sensitive to stimuli reaching them either from the periphery or some other parts of the central nervous system. The relation of this peptide to other chemical factors known to play a role in the mechanism of the sleep cycle (norepinephrine, serotonin) is at present completely unknown.

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F. BIRDMIGRATION The annual migrations of many species of birds are controlled by a complex mechanism based on the gradual shortening of daily illumination. This stimulates food intake necessary to accumulate the energy reserves required for the long flight. This stimulation by the information on daily illumination can be effectively replaced by injections of prolactin under well defined conditions (Meier and Dusseau, 1968; Farner et al., 1967). This effect of prolactin is potentiated by administration of corticosteroids (Meier and Martin, 1971). I n Farner’s interpretation, the secretion of prolactin is part of the timemeasuring device by which birds gain information on the gradual shortening of the days. The peptide would be the trigger both for the premigratory increase in feeding and for the initiation of the migration itself. In any case, this is a clear example of a peptide hormone capable of inducing a specific behavior.

111. The Role of Peptider in learned Behavior

The few examples just discussed of peptides playing a role in innate behavior represent only an extension of the well known hormonal function of these substances. There is nothing surprising in the possibility that hormonal peptides can control such elementary behavioral patterns as feeding, sleep, reproduction, adaptation to environment, territorial defense, all intimately linked to survival. Consideration of the possible role of peptides in the acquisition and preservation of new patterns of behavior, however, implies that they may have something to do with the mechanism of learning and memory. Since there is an apparent prejudice against using the traditional methods of endocrinology for the study of higher nervous activity, I shall endeavor to show that there is no fundamental difference between innate and learned neural mechanisms and, thereby, to justify the use of the same approaches to both. A. PEPTIDEHORMONES AND THEIR DERIVATIVES I t was first shown by Applezweig and Baudry in 1955 that hypophysectomized rats had difficulty in learning a conditioned avoidance task. Murphy and Miller (1955) found that this leaning deficiency could be corrected by administration of ACTH. This action was independent of the adrenocortical stimulating effect of the peptide since it was observed in adrenalectomized animals. ACTH had an effect on learning also in intact, non-

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hypophysectomized animals, in which it delayed significantly the extinction of learned behavior, suggesting a firmer consolidation of memory. These observations were developed by De Wied and his co-workers in several directions (De Wied, 1973). First, they found that the whole molecule of ACTH was not required for the behavioral effect: sequences 1-10 and 4-10 were equally effective, but sequences 11-24 and 25-39 were inactive. They found, furthermore, that a-MSH and P-MSH exerted similar effects. They explored the structural requirements for the behavioral action and found that substitution of D-Phe in position 7 of ACTH reversed the effect and produced a facilitation of extinction while similar substitution of D-amino acids in other positions was not critical. De Wied also found that posterior lobectomy, while not preventing the acquisition of avoidance responses, impaired consolidation and caused rapid extinction of the behavior. This deficiency could be corrected by Pitressin and later with synthetic vasopressin. The effect of this peptide, also observed in intact rats, was significantly more prolonged than that of ACTH and its derivatives. Since the behavioral effect of these peptides seemed independent of their hormonal properties, De Wied attempted to find substances that would have no hormone action, but would exhibit the behavioral effect alone. From anterior pituitary extracts, he isolated a peptide, desglycinamide lysine vasopressin, that produced a better and longer-lasting suppression of extinction than any of the hormones previously tested. Since these peptides have been tested primarily in avoidance situations, it has been proposed that they act by increasing fear, thereby maintaining and prolonging the effect of negative reinforcements. Weiss et al. (1969) proposed a hypothesis based on the antagonistic effect of corticosteroids, which decrease fear, and of ACTH, which increases it. The former would accelerate extinction of behavioral responses based on fear while the latter would delay it. The idea is far from being proved since ACTH can also inhibit extinction of appetitive responses (Leonard, 1969; Gray, 1971; Guth et al., 1971). I t seems probable that the hypothalamic and pituitary peptides have some nonspecific role in the motivational arousal which is an important factor in learning (Stratton and Kastin, 1973).

B. PEPTIDESI N LEARNING AND MEMORY This problem has been reviewed repeatedly (Ungar, 1970a,b, 1972, 1973b), and I shall give here only a brief sketch of its development and a discussion of recent data. Although there have been allusions to chemical mechanisms in the storage of acquired information in the 19th century literature, the problem could

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not be adequately formulated before sufficient knowledge was accumulated on the chemistry of the nervous system and before the concept of information processing by molecular mechanisms gained acceptance in biology. The first full-fledged hypothesis, proposed by Katz and Halstead ( 1950), was inspired by the early stages of molecular biology and assumed that information was stored in nucleoprotein molecules. The hypothesis had a profound influence on those who after an interval of over 10 years resumed the problem and initiated the first experimental approaches to it. It was assumed that, if there was a chemical process involved in learning and memory, it had to be “macromolecular” (Schmitt, 1962; Gaito, 1972; Ansell and Bradley, 1973). Three main experimental strategies have been used to attack the problem: (1) search for chemical correlates of memory; ( 2 ) effect of metabolic inhibitors on memory, and ( 3 ) biological assay methods. All three methods started with the assumption that RNA molecules played the primary role in the chemical mechanism of learning and memory (HydCn and Egyhazi, 1962; Dingman and Sporn, 1961; McConnell, 1962). In later years, however, the emphasis shifted to proteins ; evidence was produced for increased turnover of brain proteins in learning animals (reviews by Booth, 1970; HydCn, 1973) and memory impairment by inhibitors of protein synthesis (review by Cohen, 1970; Squire and Barondes, 1972). Among the particular types of proteins involved, emphasis was laid on glycoproteins by Bogoch (1968) and on the brain-specific S-100 proteins by HydCn (1973). In 1963, I discussed the reasons why comparatively simple peptides could be adequate for information processing and storage in the nervous system (Ungar, 1963). Subsequently, using the bioassay approach, my laboratory demonstrated the formation of peptides in the brain of rats submitted to a series of morphine injections making them tolerant to the drug (Ungar and Cohen, 1966) and of animals habituated to a sound stimulus (Ungar and Oceguera-Navarro, 1965). Identification of the active substances as peptides was based on two criteria: dialyzability and inactivation by proteolytic enzymes. Subsequently, other laboratories working on the molecular problem of memory reached similar conclusions (Rosenblatt et al., 1966; Chapouthier and Ungerer, 1969; Giurgea et al., 1971; Zippel and Domagk, 1969). However, a doubt remained in the mind of those workers who were able to demonstrate the presence of active material in RNA extracts of brain (Fjerdingstad et al., 1965; Adim and Faiszt, 1967; McConnell et al., 1968; among others). It is noteworthy that almost all the negative results were also obtained with RNA preparations (review by Ungar, 1971; Ungar and Chapouthier, 1971) . The apparent discrepancy was explained when RNA extracts of brain from rats trained for dark avoidance were dialyzed and the active material was found in the dialyzable fraction, while the RNAcontaining nondialyzable fraction was inactive. The dialyzable material was

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identified as a peptide loosely bound to RNA from which it could be separated at low pH (Ungar and Fjerdingstad, 1971). Some of the peptides were purified, and their structure was determined. 1. Scotophobin Between 1968 and 1970, we accumulated material for the purification and structural identification of the peptide that had been detected by bioassay in the brain of dark avoidance-trained rats (Ungar et al., 1968). Out of about 5 kg of rat brain, we isolated 300 pg of a pure peptide containing the following amino acids: Ala, Asp,, Glu,, Glys, Lys, Serz, Tyr. End-group analysis of the whole peptide and its tryptic fragments gave the partial sequence of Ser (Asp, Gln, Gly) Lys-Ser (Ala, Gln, Gly) Tyr-NH,. On the basis of these data and mass spectrometric analysis, a tentative sequence was proposed in which positions 3, 5, and l l remained uncertain. By synthesizing a number of variants and testing their behavioral activity, the following most probable sequence was arrived at : Ser-Asp-Asn-Asn-GlnGln-Gly-Lys-Ser-Ala-Gln-Gln-Gly-Gly-TyrNH2 (Ungar et al., 1972a). This pentadecapeptide was given the name scotophobin. In spite of criticisms (Stewart, 1972; Goldstein, 1973), the dark avoidance-inducing effect of synthetic scotophobin was confirmed in several laboratories (Guttman et al., 1972; Bryant et al., 1972; Malin and Guttman, 1972; Thines et al., 1973). It seemed that once the structure of scotophobin was elucidated it would be possible to develop a chemical method for its detection and quantitative determination. By means of a microdansylation procedure (Neuhoff et al., 1969), the thin-layer chromatographic characteristics of the peptide were established and allowed us to make quantitative determinations of scotophobin in brain (Ungar, 1973a,b). It has never been detected in untrained rat brain, but during dark avoidance training it appears and increases gradually until the sixth day (to a maximum of about 200 ng per gram of brain). Beyond this point it decreases gradually, and at 15 days it is no longer detectable. The same method allowed us to do preliminary experiments on the regional distribution of scotophobin in the brain. About two-thirds of the peptide is present in the cortex, one-fourth in the brain stem and cerebellum, and the remaining small amount is disseminated in subcortical areas. The fate of exogenous scotophobin injected intraperitoneally into mice was investigated by the same method. I t was found in the brain 15 minutes after administration and reached its peak in about 4 hours, after which it decreased gradually and could not be detected at 48 hours. The maximum found in the brain did not exceed 1% of the injected material. All these experiments are still preliminary and will be repeated with a more sensitive method, using 'H-labeled dansyl reagent.

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It is obvious that there are still some important questions to be answered regarding the specificity of the behavioral effect of scotophobin and the uniqueness of its structure. When scotophobin was synthesized by the solidphase method (Parr and Holzer, 1971), the final product had to be purified. The fraction selected by bioassay had the same dark-avoidance inducing potency and the same chromatographic characteristics as the natural material. However, when we tested a sample of scotophobin made in Dr. Weinstein’s laboratory by the classical method, we found that its activity was significantly lower than that of the substance purified from the solid-phase product. The possibility that natural scotophobin possesses some unusual linkages (involving perhaps the /3-hydroxyl of aspartic acid and the r-amine of lysine) is now being investigated. De Wied (1973) tested a number of synthetic scotophobin analogs and found that, like the ACTH fragments and vasopressin and its derivatives, they delayed the extinction of learned avoidance behavior.

2. Ameletin As mentioned above, in 1965 we published experiments suggesting the formation of a peptide in the brain of rats habituated to a sound stimulus (Ungar and Oceguera-Navarro, 1965). During a period of two and a half years, with the help of S. R. Burzynski and T . Innerarity, I have been attempting the isolation and identification of this substance. We started out with the hope that, with the experience acquired in the isolation of scotophobin, the task would be comparatively easy. In fact, the isolation proved more difficult and different schemes of extraction and purification had to be devised. Although we used a higher amount of starting material, the final yield of pure substance was considerably smaller, so that no quantitative amino acid analysis could be done. By microdansylation we detected the presence of six amino acids: Ala, Glu, Gly, Lys, Ser, Tyr. End group analysis was difficult because, as we learned later, the N-terminal was pyroglutamic acid, which is not detectable by the usual reagents unless it is converted to glutamic acid by alkali treatment. After estimating the size of the molecule by gel filtration and splitting the peptide by chymotrypsin, the following partial structure was proposed : Pglu ( Ala, Gly ) Tyr-Ser (Ala, Gly ) Lys (Ungar and Burzynski, 1973). Subsequently, by means of the enzyme dipeptidyl aminopeptidase (Callahan et al., 1972), the following dipeptides were identified : Glu-Ala; Gly-Tyr; Ser-Lys, suggesting the following tentative sequence : Pglu-Ala-Gly-Tpr-Ser-Lys. This hexapeptide was synthesized by Dr. B. Weinstein (University of Washington, Seattle) and Dr. H. Lackner (University of Giittingen) . The thin-layer chromatographic properties of the synthetic peptides are in general agreement with those of the natural material. Preliminary assays of the

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synthetic material indicate that it does reduce the startle responses of mice to the sound stimulus used in the habituation of the donor animals but the effect, although statistically significant, is less marked. Further work, including synthesis of an N-acetyllysine derivative and that of a cyclic form of the peptide, is being carried out. The natural peptide received the name ameletin (from the Greek ameletos, indifferent). 3. Other Peptides under Study Three other substances, whose behavioral effect was demonstrated by bioassay, are at present being isolated from goldfish brain. Two of them have been extracted from the brain of goldfish trained by color discrimination (Zippel and Domagk, 1969). One group of fish is trained to avoid the blue compartment of a tank by being submitted to electric shocks if they do not respond to the color cue within 20 seconds. Another group of fish is trained in the opposite direction by being shocked in the green compartment. Brain material has been accumulated from these donors for almost two years, and purification has followed the pattern established for scotophobin. The two peptides are somewhat smaller than scotophobin, around 12 amino acid residues. The blue-avoidance inducing peptide is inactivated by trypsin, and the green-avoidance inducing one by chymotrypsin (Ungar et al., 1972b). A third peptide is extracted from the brain of goldfish trained to adapt their swimming behavior to a float attached to them (Shashoua, 1968). Extract from trained fish shortens significantly the time required for adaptation (Heltzel et al., 1972). The active substance is a peptide somewhat larger than those mentioned previously (20-25 residues). It is inactivated by trypsin, and its activity is decreased by chymotrypsin. All three of these substances isolated from goldfish brain have been obtained in an almost pure state, and it is hoped that their structure will soon be elucidated. A number of other peptides have been demonstrated by bioassay to be formed in the brain of animals trained for various tasks: morphine tolerance (Ungar and Cohen, 1966; Ungar and Galvan, 1969), stepdown avoidance (Ungar, 1971), and maze learning (Radcliffe and Shelton, 1973). No attempts have yet been made at their isolation and chemical identification.

4. Emergence of a Methodology The suitability of the behavioral bioassay method for the study of chemical processes in learning and memory and the validity of its results have been the object of controversy for almost ten years. Bioassays are hardly in need of an apology; they laid the foundations of many of the most important concepts in biology; antibodies, hormones, vitamins, and neurotrans-

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mitters. In spite of their disadvantages, by their sensitivity and specificity they are uniquely suitable for the detection of small amounts of biologically active but chemically unknown substances in complex mixtures. The controversy over the application of bioassays to the chemical problem of learned behavior stems largely from the reluctance to accept the idea that learning can be dependent on information-specific chemical factors (Barondes, 1972). This contrasts with the ready acceptance of the hormonal control of innate behavioral patterns. The literature of the behavioral bioassay has been reviewed repeatedly (Rosenblatt, 1970; Dyal, 1971; Ungar, 1971; Ungar and Chapouthier, 1971 ; see also volumes edited by Ad6m, 1971 ; Fjerdingstad, 1971 ; Zippel, 1973), and the controversial points have recently been critically examined (Ungar, 1973a). I shall only mention here that successful behavioral bioassays have now been published from at least 42 laboratories, not counting the many unpublished reports that came to my attention. In any case, the bioassay is only the first step in the overall strategy that has emerged from the experience of the last decade. This strategy includes the following stages: I

a. Elaboration of an Assay System. It includes the definition, by trial and error, of the optimum conditions for the training of the donors and the testing of the recipients. At this point, a first approximation can be made to the chemical nature of the active substances, which up to the present have invariably proved to be peptides. b. Isolation and Purification. Donors are trained and their brains are collected in numbers estimated adequate for obtaining the amount of material necessary for structural identification. It is probable that these numbers will be steadily decreasing with the progress of methods of separation and advances of analytical peptide chemistry. Each purification step includes the identification of the active fraction by bioassay. c. Structural Identification. This includes amino acid composition, end group analysis, and sequence determination of the whole peptide and its tryptic or chymotryptic fragments. I mentioned above the particular usefulness of dipeptidyl aminopeptidase. Most of the well established methods work well only with millimolar amounts of peptides, but new methods are available for the nanogram and even the picogram levels. I mention among these the microdansylation procedures with or without isotope labeling and mass spectrometry. I n most cases, synthesis is a necessary part of this phase of the research, to confirm the structure and, especially, to make sufficiently large amounts of material available for further research.

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d . Development of Chemical Methods of Determination. Once the structure of the active substance has been elucidated, it is usually possible to devise chemical methods for its detection and quantitative determination. This eventually allows bypassing the bioassay, which seems to be most vulnerable to criticism. The fact that the presence of scotophobin can now be detected by the dansyl method invalidates most of the criticisms of the work leading to its characterization. I n some cases, radioimmunoassays may be necessary instead of chemical techniques. If histochemical methods can be devised, these could give significant information for the next stage of the research. e. Search for the Significance of the Substance and Its Place in an Overall Scheme, As indicated above, this phase has just been started for scotophobin and involves the kinetics of its formation and distribution. Future research, using the isotope-labeled peptide, will further specify the localization of the peptide in the brain, anatomically and perhaps at the subcellular level. Another line of research concerns the chemical specificity of the peptide; the effect of its analogs and derivatives, and the determination of the active site of the molecule. A third question is the behavioral specificity: Does scotophobin, for example, cause only dark avoidance or may it elicit other avoidance behaviors? Can its formation be induced by other types of trainings besides dark avoidance? Responses to all these and other questions will contribute to the solution of the final problem, the overall significance and function of these substances. This solution will come only when a number of peptides isolated from the brain have been chemically identified. The scheme just summarized is not different, in any significant respect, from what has been accomplished for the peptide hormones. There is no particular reason why it should be different if one accepts the idea that learning and memory can be studied by essentially the same methodology as innate behavior and physiological functions. IV. Is There a Peptide Code in the Nervous System?

Before discussing this point, I shall examine briefly the presence of peptides in the brain, the evidence for their being released, and their possible role in neural function. Besides the hypothalamic peptides, present in minute amounts but detectable only by their biological activity, a large variety of brain peptides have been identified chemically (Sano, 1970). These are mostly small peptides: N-acetylaspartylglutamic acid, glutamyl di- and tripeptides (Reichelt, 1970), lysyl peptides (Gatfield and Taller, 1971 ), and tryptophanyl peptides (Edvinsson et al., 1973). There are no estimates of the total peptide content

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of brain, except for the hog hypothalamus which may contain several grams per kilogram (Shome and Saffran, 1966). Figures of this type, however, are difficult to evaluate and compare with other tissues because of the loose definition of peptides in relation to proteins. Brain peptides are known to be released from neurons as neurosecretory products. Neurosecretory cells have been divided into type A, secreting peptides, and type B, producing biogenic amines (Knowles, 1967). The distinction between the two types, based on the electron microscopic appearance and dimensions of the neurosecretory granules has been admitted to be arbitrary, and in recent years the so-called “unitary concept” of neurosecretion has prevailed (De Robertis, 1964). This concept has been formulated by Zetler (1970) as follows: “All neurons, in addition to generating and spreading electrical phenomena, have secretory functions by which active substances are synthesized and released.’’ There is now evidence that the same cell can release both peptides and amines, thus abolishing the distinction between “peptidergic” and “aminergic” neurons (Bargmann at al., 1967; Owman et al., 1973). Release of neurosecretory substances from nerve endings has been demonstrated in vitro (Musick and Hubbard, 1972 ; Edwardson et al., 1972). Release of peptides by neurons being demonstrated, it remains to be seen what the peptide does once it is released. The well known cases are the hormones released into the general circulation (posterior pituitary hormones) or the hypothalamopituitary portal system (releasing hormones) . It should be noted that evidence is accumulating to suggest that all the peptide-secreting cells derive ultimately from the neural crest by migration into the skin and the digestive tract and its glands (Weichert, 1970; Le Douarin and Teillet, 1973). Much less is known about peptides released at actual synaptic or “synaptoid” junctions. Some peptides, such as substance P, have been mentioned as possible neurotransmitters (Zetler, 1970), but the evidence is at best fragmentary. If neurons can release transmitter amines and peptides at the same time, one has to assume that the latter must have some function other than synaptic transmission. “Whether or not polypeptides act on neurons as classical synaptic transmitters, they clearly can exert powerful effects on neuronal activity” (Bloom et al., 1972). This type of effect has been designated by the general term of “modulation.” A modulator has been defined by Florey (1967) as “any compound of cellular and non-synaptic origin that affects the excitability of nerve cells and represents the normal link in the regulatory mechanisms that govern the performance of the nervous system.” Several terms of this definition require explanation and correction : “cellular” origin may be interpreted as excluding modulation by hormones released from distant cells, and the exclusion of substances of synaptic origin would reject

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the possibility of modulators being released from presynaptic endings together with transmitters. Modulators may act on the presynaptic neuron by influencing the amount of available transmitters, the amount of transmitters released and the rate and time course of release. They may also act on the postsynaptic neuron by controlling the amount of available receptors and perhaps their affinity for the transmitters. We should probably include in modulation the slower effects that may control the type of protein being synthesized by the cell (Bloom et al., 1970). Any of these effects would clearly control neuronal connectivity by deciding which synaptic inputs are accepted by the neuron and which are ignored. A. INNATE MECHANISMS During development of the embryo, the organism acquires the neural and humoral control mechanisms necessary for survival. Regulation of visceral functions, reflex responses and, in some species, elaborate instinctive behavioral patterns elicited by certain stimuli depend on prewired neural circuits. These are obviously organized according to a genetic blueprint but the mechanism of the innate organization of the nervous system is imperfectly known. Over the past twenty years the concept of “chemospecificity of pathways” has emerged to explain the complex mechanism by which the neural pathways develop (Sperry, 1958; Jacobson, 1969; Gaze, 1970). It has been supported by experimental data suggesting that neurons destined to be part of the same innate pathway find each other and make synaptic connections by means of a molecular recognition system. The nature of the labels carried by the neurons has never been investigated, but circumstantial evidence suggests that they are proteins. Only proteins have the potential information content to encode the number of specific pathways formed in the mammalian brain (estimated to be of the order of lo7 in the human brain; Ungar, 1968). I t is probable that the neuronal recognition system is but a special case of the general mechanism of histogenesis by which cells of the same type aggregate to form tissues and organs. Because of the extreme complexity of the nervous system, the labeling process reaches in it a much higher degree of differentiation than in other tissues. It seems fairly well established that homotypic cell aggregation is controlled by specific glycoproteins (Moscona and Moscona, 1963). Recent observations of Garber and Moscona (1972) suggest that regional differentiation of the brain is controlled by similar substances. I t may be significant that aggregation of embryonic brain cells is inhibited by all general anesthetic drugs (Ungar and Keats, 1973).

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These recognition molecules do not fit the conventional definition of peptides, but their specificity depends in all probability on a limited peptide sequence and perhaps to some extent on carbohydrate prosthetic groups. Each innate pathway is probably marked by a peptide sequence which may be regarded as its code designation. These sequences need not be very long. There is a sufficient number of sequences in dipeptides to hexapeptides (6.7 X 10’) to label all the innate pathways in the human brain. We cannot be certain that this labeling system persists beyond embryonic life and does not disappear once the pathways are organized. It has been suggested, however, that “identity reactions could strengthen frequently used synapses if inducers were transmitted across synapses at simultaneous firings” (Roberts and Flexner, 1966). Similar opinions were expressed by Sperry ( 1963) and Jacobson ( 1969). B. FORMATION AND LABELING OF METACIRCUITS I t is widely assumed that learning, the acquisition of new behavior, involves the formation of specific neural circuits named cell assemblies by Hebb (1949) and metacircuits by Barbizet ( 1968). I t is probable that these metacircuits are formed initially by means of new connections between the innate pathways (which can be called protocircuits), and at higher levels by further connections between existing metacircuits. There are, therefore, nth order. metacircuits of first, second, third, If the labeling system of the protocircuits, mentioned in the preceding section, serves for the marking of the metacircuits, one can assume the existence of a whole hierarchy of peptide chains from the comparatively simple sequences that label the protocircuits to the increasingly complex molecules that code for the metacircuits of higher and higher order. The former are undoubtedly inscribed in the genome, since the structure of the brain is genetically determined, but the latter may be formed by a nongenetic process to deal with acquired information. RNA-directed peptide synthesis is now universally accepted, but, before the era of molecular biology, several other mechanisms were studied. They had been all but forgotten until recently, when the problem was revived by Lipmann (1971) and by Meister (1973). Lipmann described the biosynthesis of the bacterial peptides gramicidin or tyrocidine by complex enzyme systems. The amino acid is activated by ATP to form an aminoacyl adenylate-enzyme complex and is linked by the transpeptidation of a thioesterified carboxyl to the amino group of the next aminoacyl thioester. Meister showed that enzymic synthesis of peptides can take place also in eukaryotic organisms. The prototype of this process is the synthesis of glutathione, but more complex peptides could be formed by the same transpeptidation

- - -

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mechanism. T h e brain is rich in enzymes involved in this synthesis (Meister, 1973), and preliminary experiments in my laboratory suggest that the activity of one of them increases during behavioral training (Ungar, 1973b). The coding process could include the following steps (some of them illustrated in Fig. l ) : 1. Activation of the pathways involved in the behavioral training causes increased synthesis of their genetic labels. 2. Simultaneous firing of two or more protocircuits induces transsynaptic transfer of their labels ( U and C). This step, called “transprinting” in the hypothesis of Szilard ( 1964), may include the participation of interneuronal feedback systems. 3. At the same time, the peptide-synthesizing enzymes undergo activation in some of the cells involved and combine the genetic labels to form a new molecular species ( U C ). 4. The newly formed peptide is incorporated into the activated synaptic membranes and consolidates the new connection. This same process taking place a t a number of synapses creates the metacircuit corresponding to the new behavior. Some of the steps enumerated are hypothetical, but none are incompatible with the experimental data. Step 1 is supported by the observations of

ZI-.

~7 “F

FIG.1. Example of the possible formation of labeled metacircuits. An unconditioned stimulus ( U ), traveling through innately labeled pathways, elicits a preprogrammed response ( R ) . Presentation of a conditioned stimulus ( C ) activates a circuit that is not innately connected with circuit U , but if both C and U are applied in a definite temporal sequence some of the neurons involved in their respective circuits are simultaneously activated, together with a set of interneurons (which also play the role of a feedback system). This simultaneous or almost simultaneous firing, by activating the synaptic membranes and increasing their permeability, could allow penetration of the labels C and U into the interneurons. If these contain the enzyme systems necessary to combine the two peptide fragments C and U , a new molecular species CU could be synthesized and become the label or marker for the newly formed synaptic connection. Since the same process is repeated a t many other junctions, CU becomes the “codeword” for the whole metacircuit through which the newly acquired conditioned response is elicited.

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increase in RNA and protein synthesis in the brain of learning animals (Booth, 1970; HydCn, 1973) and impairment of learning by inhibitors of these syntheses (Squire and Barondes, 1972). Step 2 is supported by steadily accumulating data on the possibility of transsynaptic transport (Korr et al., 1967; Globus et al., 1968; Alvarez, 1970; Grafstein, 1971). Evidence for nongenetic peptide synthesis in step 3 was mentioned above. Analysis of our data on the life cycle of scotophobin in the brain of dark avoidance-trained animals (Ungar, 1973a,b) indicates that an excess of the peptide is synthesized during the first days of training, but subsequently it disappears from the brain in spite of the persistence of the learned behavior. It is possible that retention of information depends on a small amount of the coding material bound to some structure, presumably to synaptic membranes.

C . THEROLEOF PEPTIDESIN NEURALCODING Information processing is probably a great deal more complicated in the nervous system than in the genome. There are at least two types of neural coding systems :

1 . Coding Schemes Stimuli reaching sensory receptors are transduced into bioelectric wave patterns analogous to the “coding schemes” into which data have to be translated so that the computer can handle them. A similar transduction takes place also at each neuron to convert the chemical stimulus exerted by the synaptic transmitter into the appropriate axonal output. Although this coding scheme has a basic principle of frequency modulation, there may be as many varieties of it as there are neural pathways. It is generally agreed that the neural coding scheme cannot be a long-term information store.

2. Command Codes and Program Codes They represent the repertoire of elementary operations that the brain is capable of, the order in which the operations take place, and hold the key to the manner in which these operations can be changed and their repertoire enlarged. The brain is born with a genetically determined program that allows it to respond to stimuli in a stereotyped manner. Learning consists in changing this program, i.e., reprogramming the brain. If the innate program is chemical, as suggested by data mentioned in the preceding section, it is probable that the reprogramming also takes place by a chemical process. Nobody denies that the nervous system possesses “labeled lines” (Perkel and Bullock, 1968) ; the opinions differ only over the identity of the labeling substances. Labeling by means of the transmitter

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released at the axonal ending into cholinergic, adrenergic, dopaminergic, serotonergic, etc. neurons is implicit in the neurohumoral doctrine which also postulates a complementary labeling by means of receptors present at the postsynaptic surfaces of cholinoceptive, adrenoceptive, etc., neurons. This is genuine coding since only a neuron possessing cholinergic receptors can be excited by a cholinergic axon. We do not know at present whether this type of program code can be modified by learning, but, on the face of circumstantial evidence, this seems unlikely. One would be tempted to assume that the transmitter-linked program code controls pathways that remain immutable and are not involved in learning. Many of the generally recognized neurotransmitters are amines derived from amino acids by decarboxylation with or without additional chemical modification. I n recent years, evidence has been presented suggesting that unchanged amino acids can function as transmitters, and the role of glycine and glutamic acid has been demonstrated (Curtis and Crawford, 1969). It is not clear whether amino acid transmitters operate on their own or in association with amines. One can speculate that they represent the foundations of another chemical coding system starting with amino acids and, as the number of labeled lines increases, forming amino acid chains of increasing complexity. It has been suggested that “the response to single amino acids might only be a reflection of the endogenous affinity toward a larger molecule” (Bloom et al., 1972). A peptide code based on the twenty-letter amino acid alphabet has unlimited information content, sufficient to program the protocircuits and to operate their reprogramming into the metacircuits necessary to preserve all the information acquired in a lifetime. I t is hoped that the question mark in the title of this section is sufficient to warn the reader of the speculative nature of many of its propositions. A simpler and more elegant solution of the problem may be hidden to us by our present ignorance of many relevant facts. The fact that the molecular hypothesis is at present controversial should not discourage its further study. Controversy is a normal part of the dialectical process by which science advances, and there has hardly been any new idea that did not have to contend with it. A good example, close to our topic, is the neurohumoral doctrine, for which it took over a quarter of a century to be fully recognized as one of the basic principles governing the neurosciences. Controversy may discourage the faint-hearted, but it is a challenge to those who are determined to find out the truth. REFERENCES

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BIOCHEMICAL TRANSFER OF ACQUIRED INFORMATION By

S. R. Mitchell, J. M. Beaton, a n d R. J. Bradley

Neurorciences Program and Department of Psychiatry, University of Alabama in Birmingham, Birrninghorn, Alabama

I. Introduction

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II. Approaches to the Study of the Biochemistry of Learning A. B. C. D.

RNA and Rotein Synthesis Inhibitors Correlational Studies. Transfer Studies Recent Synaptic Models References

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61 62 62 63 63

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1. Introduction

No one knows what changes, or even what types of changes, take place in the brain when an organism acquires new information; nor is it known how this information, once stored, is retrieved and acted upon. What is known is that there exist lawful relationships between an organism’s past history and its subsequent behavior. What are known are some empirical rules of thumb for manipulating, for modifying behavior, and the overwhelming preponderance of work on learning thus far has addressed itself to the elucidation of these lawful relationships; to behavior, its description, quantification (where possible), and elaboration of the contingencies that affect it. Relatively little work has been done on the underlying physiological substrate of learning, on just what goes on inside an organism’s skull during learning. Early work on the problem centered on attempts to localize and characterize the elusive engram, the physical trace or representation of a stored event, the rationale for which went something like this: Given that all remembered events are physically stored in discrete locations in the brain, it should be possible, by removing very small amounts of brain tissue from a trained animal, to eventually determine just where specific behaviors are stored. That is, a dichotomous, an all-or-none sort of effect was expectedeither the learned behavior should be completely eliminated or it should remain unimpaired. Unfortunately for the engram searchers, the results were 61

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J . M. BEATON, A N D R. J . BRADLEY

not so clear cut. Rather it appeared that it was not so much the location of the ablated tissue that was crucial, but the amount. An additional complication was that damage to sensory or motor areas could not be ruled out. Thus, results from this early approach were at best equivocal, prompting Lashley, an advocate of this approach, to summarize his thirty-year effort by saying he sometimes thought that learning was just not possible (Lashley, 1950). Following this early failure, interest in and work on a physiological mechanism to mediate learning and memory began to lag, in part because the very delicate tools available today were nonexistent then and, perhaps even more important, because of the ascendancy of the Hullian framework (which aspired ultimately to reduce all mammalian behaviors to empirically derived mathematical expressions) over virtually every other approach. But, as inadequacies in Hullian formulations became apparent, and when it was seen that such an ambitious endeavor was premature and even presumptuous, and when the mechanism for the storage and transmisssion of genetic information was elaborated, speculation began to proliferate again, eventually providing the impetus for the three current approaches to the problem. II. Approaches to the Study of the Biochemistry of learning

A. RNA

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PROTEINSYNTHESIS INHIBITORS

The first general class of studies comprises those which use inhibitors of RNA or protein synthesis in an attempt to determine how information is stored and retrieved, by inactivating the posited relevant mechanisms. Typically an animal is trained on a task and the injection of the inhibitor is timed so that its effects are maximal at some specified time after learning. The animal is later tested for retention deficits. However, interpretation of results are complicated by a number of observations: first, the reduction in cerebral protein synthesis necessary to produce an effect on memory is massive (at least 90%: Barondes, 1970), and there are some indications that the observed deficit may be due to the aversiveness of the treatment in and of itself (Booth and Pilcher, 1973). Second, there is the inability of advocates of this approach to differentiate effects on specific learning mechanisms from the generalized debilitating effects of this procedure, which would wreak havoc with normal enzymic functioning. Cohen and Barondes (1967) observed that puromycin effects on memory were correlated with a marked impairment of cerebral electrical activity, and found that its amnesic effect could be countered by administration of an anticonvulsant, suggesting that some other action of puromycin may be responsible for the amnesic effect. Indirect support for such a notion comes from the finding that one anti-

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biotic, acetoxycycloheximide, evidently negates the effect of another, puromycin (Flexner and Flexner, 1966). Other complications include the observation that intracerebral injections of isotonic saline (Flexner and Flexner, 1967), as well as other chloriaes, an ultrafiltrate of blood serum, and distilled water (Flexner and Flexner, 1969), will suppress the amnesic effect of prior injections of puromycin. Flexner’s explanation in terms of the “flushing out” of toxic truncated peptides, produced by puromycin, is speculative at best. Finally, convincing explanations concerning the failure of puromycin to affect a positively reinforced response (Potts and Bitterman, 1967) as well as the environmental triggering effect (Davis and Agranoff, 1966) in which the lability of the consolidation mechanism is extended by leaving the subject in the training situation, have yet to appear. B. CORRELATIONAL STUDIES The second approach attempts to find biochemical correlates of behavioral change. However, again a number of problems emerge. Gaito (1971) pointed out that there are at least three possible relationships between RNA or protein changes and behavior. First, the effect may be primary; that is, RNA or protein changes may be a necessary and sufficient condition for concomitant changes in behavior. Second, the observed RNA or protein changes may be secondary, may be a consequence of, rather than a causal antecedent for, learning. Finally, behavior and RNA or protein changes may both be manifestations of an underlying CNS event. Additionally very few studies have been able to differentiate learning-specific changes from those resulting from differential motor activity or sensory stimulation. However, there are a number of promising new techniques, such as the DNA-RNA hybridization procedures which have presented evidence for the notion that unique species of RNA are produced during learning, as well as the reversal of handedness studies (HydCn, 1973b), which avoid criticisms of HydCn’s earlier work (Grossman, 1967) regarding the appropriateness of the selection of cells to be studied, as well as the adequacy of some control groups.

C. TRANSFER STUDIES 1. Early Work

The last and most recent phase of the experimental attack on the problem of how experiential information is stored and retrieved are the transfer studies. That learned behavior might be transferred from one organism to another, and that this transfer might be mediated by some biochemical sub-

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stance in the donor brain that was somehow modified by learning, was first suggested by McConnell ( 1962). McConnell fed classically conditioned flatworms to naive recipients and measured the number of responses to the conditioned stimulus made by the cannibals. Surprisingly, the cannibals made more responses than a control group fed untrained animals. I n the midst of the heated controversy that resulted over the question of whether planaria could learn, much less whether such learning could be transferred, work was begun to demonstrate transfer in vertebrates. The first, that of Fjerdingstad et al. (1965) , used a light-dark discrimination in a maze for water reinforcement with a 90% correct criterion. A cold phenol extraction procedure with an intracisternal injection was used and significant results were obtained. Reinis (1965) trained rats to push open a door for food in response to a tone or light discriminative stimulus. A description of his extraction procedure is not available, but injection of the extract was intraperitoneal, and the recipients learned the task significantly faster than a control group. These two studies, while demonstrating a transfer effect, were published in relatively obscure journals and failed to arouse the clamor that immediately accompanied the third paper. Babich et al. (1965) magazine-trained donor rats, then, using a phenol extraction procedure, injected recipients intraperitoneally. Their criterion, the appropriateness of which has been challenged, was whether or not the recipients would place their noses inside a 63 cmz area within 5 seconds after a magazine click. Testing was conducted double-blind, and significant results were again obtained. All three of these studies assumed that the transfer was mediated by RNA. The author of the last paper, Ungar (1965), on the basis of previous work with the transfer of morphine tolerance, maintained that a small polypeptide was responsible. This argument was based on the effects of proteolytic enzymes and RNase on the transfer factor. Ungar found that incubation with RNase had no effect, whereas a protease, chymotrypsin, eliminated activity. There are a number of reasons why the first three groups assumed that RNA was the transfer factor. RNA was the macromolecule central to many of the then prevalent theoretical models concerning a mechanism for the storage and retrieval of experiential information, although almost every other type of biochemical compound had been suggested, with varying force and precision. This notion originated in earlier work ,by molecular biologists on the role of nucleic acids in the transmission and regulation of genetic information. After the roles of polynucleotides had been elaborated it was a short leap to the notion that nucleic acids could somehow store experiential, as well as genetic, information. Characteristics of DNA seemed to preclude it (although DNA involvement survived in derepressor type hypotheses),

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so RNA was chosen as the next most likely mediator. A number of more or less plausible mechanisms were devised, most of them dependent on the fact that RNA is intimately involved in the transcription process for the synthesis of specific proteins. Some mechanism (for example, permanent alteration in particular RNA molecules or a process analogous to enzyme induction) was assumed selectively to enhance the formation of a protein which modifies the membrane to facilitate firing, although how this facilitation occurred and how this was related to learning was left unspecified. Thus the theories were, in large part, fragmentary models for components of the learning process, or rather how it was,envisioned to work. But psychology has often borrowed concepts and models from the physical sciences; field theory from classical mechanics ; homeostasis from Cannon, a physiologist ; and an information storage system from molecular biology seemed attractive. The early positive results in memory transfer were followed by a flurry of “failure to replicate” articles, many of which were attempting to repeat the results of Babich et al., since these were the best known at the time. Unfortunately, Babich et al. found results 6 hours after injection, and most attempts at replication assumed this to be a sufficient interval. It now appears that this value is at the lower limit, optimal results usually being found 24-48 hours after injection. Probably the most important of these failures was reported in the Byrne et al. (1966) article. This was a joint statement by twenty-three authors from seven laboratories, all of whom failed to find any transfer effect even though a number of training and extraction procedures were used. One of the most widely quoted of the failures to replicate studies is that by Luttges et al. (1966), largely because it describes a fairly extensive, carefully designed series of studies, all of which failed to support any kind of memory transfer phenomenon. Seven different sets of training and extraction procedures were used, each varying some apparently relevant variable. However, the most telling blow to the RNA-mediator hypothesis was the demonstration by Luttges et al. (1966) that RNA does not cross the blood-brain barrier intact and, further, that it is quickly excreted. The technique used was to inject radiolabeled RNA intravenously, wait a period of time, remove the brain, and measure the amount of radioactivity present. Almost none was found. Similarly, Enesco ( 1966), Eist and Seal (1965), Sved ( 1965), and Reinis ( 1971) all failed to find radiolabeled RNA in brain after systemic injection.

2. Factors Influencing Transfer After this period when virtually no positive results were reported, evidence began to accumulate in favor of an effect, often from those who had previously failed, notably Byrne (Byrne and Samuel, 1966; Byrne and

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Hughes, 1967), who later organized a symposium on the subject (Byrne, 1970). Characteristics common to successful attempts began to emerge, and most failures are explicable in terms of the lack of one or more of the requisite factors, The first and most obvious factor is simply familiarity with the extraction procedures, some of which are fairly complicated ; seemingly insignificant variations at any of several crucial points might destroy the effect. Dyal and Golub (1968) note that puzzling discrepancies in results from their laboratory were attributable to the fact that students who had gone through the procedure before obtained positive results, whereas those who were merely handed written procedures found nothing. Often initial or exploratory work fails to show an effect, although later, with increasing familiarity, if the experimenter is persistent enough, positive results are often attainable. I t must be noted that failures to show an effect are typically “one-shot” attempts rather than a series utilizing a range of dosages and response measures (Ungar, 1971a ) . There are also a number of incompletely understood relationships between recipient behavior, and the kind and amount of donor training, as well as dosage level. First, it seems that the nature of the task to be transferred may be significant. Rosenblatt et al. (1966b) maintained that tasks with definite cue stimuli and those that require less effort, especially choice or approach measures, are the most readily transferable. I t may well be that an inappropriate choice of task was responsible for a number of early failures. For example Weiss (1970) has criticized Luttges et al. (1966) for a choice of tasks in which any transfer effect may have been masked by fixated responses characteristically emitted by highly motivated animals. Indirect support for this supposition comes from observations by Laird et al. (1972), who found that extracts from the brain of stressed donors could either impair or facilitate acquisition of an avoidance task, depending on the level of training of recipients. I t also appears that interposition of intervals of no donor training may influence recipient behavior. Golub et al. ( 1970), while attempting to replicate earlier work using a series of acquisition, extinction, and reacquisition trials, found that their extract induced an impairment in acquisition. Finding the only apparent difference between the earlier and later studies to be a delay of several days after the series of extinction trials, they then deliberately introduced similar delays after extinction in half of a subsequent group and after acquisition in the other half. They found impairment in the first, relative to the latter, group. However, Dyal (1971) failed to replicate this effect. With respect to donor training, Ungar et al. (1968) have demonstrated a direct relationship between amount of training and potency of extract. Generally, optimal results are obtained when donors have just reached

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asymptotic performance levels (Rosenblatt et al., 1966b) with a lessened effect at either incomplete or overtraining. However, the likelihood that the relationship may be rather more complicated is indicated by the occasional appearance, in both invertebrate and vertebrate preparations, of inversion effects in which recipients of trained brain extract behave in a manner completely opposite to that of the donors, typically at very high or very low levels of training. A plausible explanation for the appearance of inversion phenomena from overtrained donors was offered by Rucker (1971), who cited observations by Mackintosh (1969) to the effect that overtraining on a reasonably difficult discrimination for relatively large rewards facilitates acquisition of reversed discrimination. An alternative explanation, offered by Rosenblatt (1970), suggests that one of the factors that is transferred is habituation to the correct response. This results in a reversal of induced preference because the incorrect response is a novel one, especially if, as is common, the recipient is not reinforced. Similar effects can be elicited by manipulating dosage levels, and again optimal results are found in the middle range (one to four brain equivalents) giving way to null or inverted effects at very high and very low dosages. Of all the variables that affect recipient behavior, dosage may well be the most critical. Ungar (1970a) notes that of all the studies that failed to find an effect, only 17% used more than one brain equivalent. Extensive and exhaustive dose-response curves have not been carried out although Rosenblatt and Miller (1966b) have offered a tentative curve over the lower portion of the dosage range, part of which has been replicated by Herblin (1970), but attempts to repeat this curve have yielded inconsistent results (Rosenblatt, 1970). Ungar has occasionally published curves with the effect plotted against the log of dosage; however, the variation in both behavioral and biochemical techniques prevents any but the most general statements to be made. Two related variables, degree of refinement and route of injection of the extract, have some effect. The early work, while largely maintaining that RNA was the transfer factor used at best crude RNA preparations, and as the biochemical sophistication of this group increased so did the purity of their preparations. Unfortunately, as the purity went up, paradoxically, to them at least, the potency went down. Most negative results used “RNA extracts” (Chapoutier, 1972) whereas those ignorant of prevailing dogma typically used crude brain homogenates, and more frequently found results. A number of routes of injection have been used; intracranial, intravenous, intraventricular and intraperitoneal, with the last being by far the most common. The only consistent differences between them seem to be that the intraperitoneal route has a much slower onset and requires higher dosages.

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Another very common characteristic. of studies which fail to find transfer is that they tend to test recipients once or twice soon after injection (Byrne et al., 1966; Gross and Carey, 1965; Gordon et al., 1968; Luttges et al., 1966) with only one dosage level. Typically those reporting positive results test the animals at widely spaced intervals, starting a few hours after injection, and continuing for several days.

3. Types of Behaviors Transferred and Characteristics Various learning tasks have been shown to be transferable: instrumental conditioning, spatial and brightness discrimination, conditioned avoidance, simple maze learning, color and taste discrimination, Umweg tasks. Oddly enough it appears that extinction in recipients can be facilitated by injections of extract from extinguished donors (Braud, 1970), suggesting that extinction may not be simply the gradual weakening of a learned response or the gradual dissipation of the storage macromolecule, but rather that an independent antagonistic response has been acquired. The demonstration by Braud and Braud ( 1972) that transpositional responding, in which subjects apparently respond to relationships between, rather than physical characteristics of, stimuli in discrimination tasks, can be transferred is the most complex response tested to date. I t is very difficult to imagine how recipient behavior of this type can be ascribed to general activation effects or to changes in overall learning ability since the recipients went unreinforced in the initial phase. A secondary post hoc observation was made concerning just this last question, that of the role of reinforcement. To counter contentions that what might be transferred was “merely” some substance that facilitated learning in general, many workers very early adopted the use of nonreinforced trials for recipient testing. When this was done, it was found that the effect of trained brain extract on recipient behavior was very different from that of original training on the task. First, the effects of the extract seemed very transient, dissipating in a matter of days; second, it was found (Zippel et al., 1971) that transferred behaviors were less resistant to extinction than normally acquired behavior, and finally the standard deviations of test scores for recipients of trained extract are generally much larger than for control groups which acquired the task by conventional means. Braud and Braud (1972) found that exposing recipients to reinforced trials after the effect had apparently dissipated boosted their scores greatly, suggesting that the apparent transient nature of transferred behavior might be due in part to motivational factors rather than to lability of the transfer factor. Unfortunately, the animals were followed only until 10 days after injection, and it is not known just how long the effects of the transfer factor can persist under optimal conditions. As to the finding that transferred behaviors are less resistant to extinction, beyond the initial statement, this has yet to be investigated.

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Concerning the variability in recipient scores, this is due in part to the fact that the transfer seems to “take” on some, but not on other, subjects. This may be due to the influence of some component of the extraction procedure, e.g., phenol. Second, injection of crude brain homogenate has been shown to induce a variety of debilitating effects, including transient decrease in food and water consumption and activity (Gurowitz, 1968) as well as a mild pyrogenic effect (Rosenblatt and Miller, 1966a). Effects on activity after saline injection have also been demonstrated (Beaton and Gilbert, 1968). These nonspecific effects may well explain some of the early failures, especially those with short injection-testing intervals and may account for a number of marginally significant results. Finally, Reinis and Mobbs (1970) maintain that food deprived recipients respond better to trained extract, suggesting that different rates of uptake may play a part, especially when the intraperitoneal route is used. Further support for variable uptake or utilization comes from pilot work in our laboratory where, using a darkavoidance task, it was found that an effect was apparent anywhere from 6 to 48 hours after injection. The few attempts which have been made to transfer classically conditioned behavior have failed. Leaf et al. (1966) associated a tone conditioned stimulus (CS) to an electric shock unconditioned stimulus in donors. Recipients were then tested with the tone for conditioned suppression of drinking. No significant effect was noted. Ward (1970), using a tone CS to facial electric shock in donors, found that recipients responded to the extract with a reduction in the rate of spontaneous eyeblinks, which suggests that a specific adaptive response rather than a stimulus-response contingency was being transferred. However, it has been shown that extract from classically conditioned donors can influence unreinforced preference as well as reinforced avoidance behavior in recipients (Braud and Laird, 1973 ; Laird and Braud, 1973). Similarly, attempts to transfer innate behaviors have typically not succeeded. Lagerspetz et al. (1968) attempted to modify activity level in mice by injecting them with extracts from donors which exhibited high or low spontaneous activity. Approximately 30% of rats spontaneously kill mice. Reinis and Mobbs (1970) failed to modify this muricidal behavior by injecting “killer” rats with extract from nonkillers or by injecting nonkillers with killer extract. Dyal and Golub (1970) identified two inbred mouse strains, DBA and C-57 which exhibited differential innate alcohol preference. Extract from drinkers apparently did not increase intake in nondrinkers, and nondrinker extract did not diminish consumption in drinkers. Zippel and Langscheid (1973) have apparently been able to induce imprinting to a dummy model mother in postcritical period fish by means of brain extracts from imprinted recipients. The authors contend that their

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success was due to the fact that they attempted to restore normal behavior rather than counter an innate tendency as previous workers had done. However, there is some question as to exactly what has been transferred. Behavior of recipients appeared to be more dependent on their age than on donor “training.” A number of response modifications not normally considered to be learning have also been shown to be transferable. Ungar’s early work demonstrated that morphine tolerance (Ungar, 1965) as well as habituation to sound and air puff could be transferred (Ungar and Ocequera-Navarro, 1965). Daliers and Giurgea (1971) have shown that the time course of fixation of postural asymmetry can be affected by brain extracts. Finally, Fjerdingstad ( 1973) has demonstrated a transfer of acoustic priming for audiogenic seizure in a normally resistant strain of mice. Less quantifiable, but perhaps no less significant, are the idiosyncratic behaviors observed in subjects receiving trained extract. In our laboratory we have noticed that animals injected with extract from donors shocked in a specific location in an apparatus approach that location with what could be described, somewhat anthropomorphically, as dread ; they oscillate at the entrance, make abortive entrances and often cower in a corner as far as possible away from the point a t which their unwilling donor received such harsh treatment. McConnell and Malin (1973) have recently begun attempts to quantify such behaviors, finding significant increases in scotophobin-treated animals relative to controls. I n another laboratory ( Weiss, 1971) plans are underway to study such behaviors using videotape and a one-to-one as opposed to pooled transfer. In the end it may be the induction of behaviors such as these that are the most convincing. 4. RNA-Protein Controversy

A variety of extraction procedures have been used. Early studies often used a crude brain homogenate with no attempt to refine any particular type of biomolecule. Later a number of techniques were devised, the most popular being a phenol extraction procedure, that of Laskov et al. (1959), which consists basically of first obtaining a cold phenol extract of brain tissue, which is then centrifuged. The aqueous phase is retained, after which the “RNA” is precipitated with MgCl, and ethanol. After evaporation of the solvent, the residue is taken up in saline for injection. Several variations on this basic technique yield similar results. Generally the precipitate is discarded and is largely ignored, in part because the “RNA” is found in the aqueous phase. But Rosenblatt and Miller (1966a) found that activity remained in the precipitate and further that prolonged exposure to solvents enhanced the effect of the solute and weakened the effect of the precipitate which suggests that the transfer factor is a membrane-bound substance. In-

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vestigations into the activity of different subcellular fractions lent further support to this notion when it was found that the mitochondria1 subfraction that also contained membrane fragments was the most potent. That the transfer factor is probably a membrane-bound substance bears upon several theoretical models (Ungar, 1968; Rosenblatt, 1967 ; Best, 1968; Szilard, 1964) as well as the RNA-protein controversy, if indeed a question that has been so widely ignored can be called controversial. The evidence for both RNA and protein may be briefly summarized. The evidence for a RNA-mediator hypothesis is indirect and hardly compelling, consisting primarily of the fact that brain extracts from donor animals trained on a specific task, which facilitate the acquisition of that task in recipient animals, contain RNA. The problem is that the extracts also contain other macromolecules ( polysaccharides, proteins, glycoproteins, polypeptides), which are referred to in the literature as “contamination.” No one has yet demonstrated a transfer effect using a solution which demonstrably contains only RNA. Rosenblatt et al. (1966a) duplicated the Babich procedure and found appreciable quantities of protein. Ungar ( 1970b) duplicated the Danish group’s procedure and found as much as 50% protein. Evidence that can be marshaled against the RNA-mediator hypothesis is much more impressive. First, a transfer effect is obtained even after an intraperitoneal injection, and the likelihood of intact RNA surviving this type of administration is remote. Second, activity has been shown to persist after RNase incubation (Ungar and Ocequera-Navarro, 1965). Third, Rosenblatt et al. (1966b) separated the extract by molecular weight and found greatest activity in the 1000-5000 molecular weight range, which is many times smaller than any known RNA molecule, but compatible with the notion that a small polypeptide mediates transfer. Fourth, Ungar (1971a) has found that activity is destroyed by proteases which should have no effect on RNA. Fifth, the active factor is soluble in phenol and insoluble in acetone, a finding that also is compatible with the polypeptide notion. Finally, the first and only time McConnell et al. (1970) subjected his extract to biochemical analysis, in a two-and-one-half-year study, he found no intact RNA in the extract, yet made no attempt to account for this supposed anomaly. I n contrast, the evidence for polypeptide mediation is rather impressive. Unlike RNA, peptides are soluble in phenol and insoluble in acetone. They do fall in the 1000-5000 molecular weight range, they are destroyed by proteases, and contrary to early results they are found in “RNA” extracts. Early workers tended to use a biuret test for protein which is negative for dosages under 1 mg. Rosenblatt et al. (1966a) have shown, using a Folin-Ciocalteau test, that protein is present in extracts prepared according to the descriptions given by those who failed to find proteinaceous materials and further that

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the amount of protein found was more than sufficient to account for the transfer phenomenon. These early reports of failure to find protein in extracts undoubtedly explain part of the subsequent lack of interest in the question of whether protein or RNA is the essential “carrier” of experiential information. Virtually all the early work on the problem was undertaken by two investigators, Ungar and Rosenblatt, both of whom concluded that the widespread acceptance of the RNA-mediator notion was unfounded. Up until 1971 about every third paper concerning memory transfer explicitly referred to RNA in the title, and the others either assumed it in the body of the paper or made no assumption whatever, especially those using crude brain extracts. Yet the notion persists, despite apparently convincing evidence against it.

5 . Scotophobin and Other Specific Behavior-Inducing Peptides Ungar (1971b) has suggested that part of this confusion may result from the fact that the active peptide is often bound together with RNA in a peptide-RNA complex, which was demonstrated by separating the active peptide from inactive RNA by dialysis at low pH. However, the crucial experiment, that of the elaboration of the structure and synthesis of a specific behavior-inducing peptide has now been done. Ungar et al. (1972) have reported the amino acid sequence and synthesis of scotophobin, a peptide which induces dark avoidance. And, as has often happened, the announcement touched off vigorous and vocal criticism. Stewart (1972), asked by Nature to referee the paper, sat on it for over a year and finally offered a lengthy critique of the work that had gone into the elaboration and synthesis of scotophobin, complete with a random sequence of amino acids, termed pseudoscotophobin, which was said to fit the spectroscopic data better than the sequence offered by Ungar. Stewart attacked virtually every aspect of the attempt, except for the relatively “trivial” consideration that synthetic scotophobin induces a reduction in the amount of time spent in the dark by rats (Guttman, 1973), mice (Malin and Guttman, 1972; Malin and Radcliffe, 1973), goldfish (Bryant et al., 1972; Guttman et al., 1972, 1973), and even roaches (Guttman and Hoffman, 1973). O n the behavioral front, Goldstein ( 1973) has attacked Ungar’s training procedures, attempting ‘to account for the observed effects in terms of stress, ignoring, or not aware of, earlier data from yoked controls. However, some aspects of Ungar’s dark-avoidance training are puzzling. The procedure used is very much like a one-trial learning paradigm, yet Ungar administers five trials a day for 5 days and typically after the first trial the animal must be forced into the dark box to be shocked, which sounds more like a classical conditioning than a passive avoidance paradigm. It is not at all clear what

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the animal is learning unless it is to associate the naturally preferred darkness with shock. It is also not clear just why twenty-five trials are necessary when the animals learn on the first trial. According to Ungar the additional trials are necessary for the transfer factor to build up to adequate levels, but this is extremely speculative. An obvious sort of compromise design would be to administer the one trial and then wait for the accumulation, if any, without any intervening training, but this has yet to be done. Gradually, as more scotophobin became available, some of its characteristics began to emerge. Guttman and Gronke (1971) noted a variable onset, duration, and magnitude of the effect with both the natural preparation and the synthetic polypeptide. In an attempt to locate the site of action of scotophobin Guttman et al. (1972) first injected radiolabeled ( Y )scotophobin into goldfish and subsequently differentiated responders, those whose time in the dark had been reduced to less than 100 seconds of a 180-second trial from preresponders whose time was over lo0 seconds. A ratio of specific activity of responders to preresponders was determined, and it was found that the greatest differential localization took place in the vagal lobes with smaller ratios in IIMSE, pituitary, and spinal cord. Comparing specific activities of trichloroacetic acid-soluble and insoluble fractions, Guttman suggested that scotophobin may exert its effects by complexing with neural membrane, a view which is certainly compatible with Ungar’s model ( 1968). One anomaly was a preferential association for kidney which could not be explained in terms of its vascularity since liver was not similarly affected. McConnell and Malin (1973) investigating the specificity of the effect of scotophobin, concluded that the polypeptide exerts its effects not by producing a generalized increase in recipient emotionality, but rather that the response is specific to the experimental situation used in the original training. Similarly Guttman et al. (1973) and Malin and Radcliffe (1973) both present evidence that scotophobin does not exert its effects through a generalized change in activity level. Further the latter authors have shown, using a visually cued escape task, that scotophobin does not as some critics have suggested act by modifying visual thresholds. Ungar ( 1973) tested scotophobin-treated animals for an effect on startle response, step-down passive avoidance, as well as maze learning. No effect was found. Similarly circadian rhythms, GABA activity, and smooth muscle preparations were unaffected. However, that nonspecific effects may be found with scotophobin is indicated by Thinnes et al. ( 1973), who showed that scotophobin-treated goldfish evidently exhibit greater light tolerance, and Bryant et al. (1972) demonstrated that scotophobin facilitates acquisition of a dark-avoidance task and impairs acquisition of a light-avoidance task. A possible explanation for this comes from the fact that rat scotophobin seems to be different from goldfish scotophobin.

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Ungar (1973) has described the development of a microanalytical technique for the determination of the concentration of scotophobin in brain. Use of this method revealed that the concentration rises to a peak at 6 days and gradually falls off thereafter, a time course which roughly corresponds to earlier work using a bioassay (Ungar et al., 1968). This test was also used to determine localization of scotophobin in various brain regions. It was found that scotophobin is primarily located (72%) in the cortex, with lesser amounts in brain stem, spinal cord (26%), and subcortical areas ( 2 % ) , results that agree with earlier observations (Rosenblatt et al., 1966b) that cortical extracts are more potent than subcortical. When the sequence of scotophobin was still somewhat uncertain various polypeptides which eventually turned out to be analogs were synthesized and tested. As it happened these analogs also exert an effect on behavior. de Wied (1973) obtained what he termed deacetylscotophobin (with Glu substituted for Gln in positions 5 and l l ) , a n 8-15 fragment of deacetyl scotophobin, and an 8-15 fragment of scotophobin. All three peptides were tested for effects on extinction of a pole-jumping avoidance latency, in a passive avoidance, light-dark preference and an open field test. All three peptides apparently increased resistance to extinction of the pole-jumping avoidance for 5-7 days. Effects on the passive avoidance were seen only if subjects were preshocked, and even then the effect was marginal. However, it must be noted that no data were offered, only results. de Wied maintains that the effect of scotophobin analogs are similar but of greater duration than ACTH analogs. Both analogs are effective in passive avoidance situations if, and only if, the subjects receive shock prior to injection. de Wied suggested that some variation in recipient behavior may be due to this effect, a question that may bear upon the use of nonreinforced recipient testing. However, de Wied admitted that natural or synthetic scotophobin might not have the same mode of action as its analogs. Guttman et al. ( 1973) obtained four compounds: 5,l l-deamidoscotophobin (with Glu substituted for Gln in positions 5 and 11, the same compound as that de Wied used), N-acetyldeamidoscotophobin, an 8-15 fragment of deamidoscotophobin and degraded scotophobin. These compounds were tested for their effects on dark avoidance. Only “low-grade, variable, and transient” activity was found. The sequence of a second polypeptide, termed ameletin, which appears to induce habituation to an abrupt intense auditory stimulus has now been determined ( Glu-Glu-Gly-Tyr-Ser-Lys) (G. Ungar, personal communication), and work is currently underway in Ungar’s laboratory to isolate and sequence the polypeptides that mediate color-discrimination in goldfish (Galvan and Ungar, 1973) and learned motor adaption (Heltzel and Ungar, 1973).

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Scotophobin and ameletin share only one dipeptide sequence (Gly-Tyr), but it if happens that common sequences are found in specific behaviorinducing peptides this will support Ungar’s model, in which the common sequence would presumably function to facilitate firing over a previously nonfunctioning synapse, and in which the unique portion of the polypeptide would function as a specific code or label for the particular pathways involved.

6. T h e Question

of

Specificity

After the reality of the transfer phenomenon had been demonstrated and the molecular nature of the transfer factor had been elucidated, at least one serious interpretive problem remained, that of just what gets transferred: Was it specific information relevant to the contingencies of the task, as advocates claimed? Was it just a general activation phenomenon induced by a stress-related hormone? Was it a nonspecific facilitation of any type of learning? O r could the effects be due merely to nutritional factors as the early memory facilitation by ingestion of yeast RNA seemed to be? Some of these questions could be countered immediately. Testing nonreinforced recipients answered the generalized facilitation of learning notion, while explanations in terms of changes in activity due to transfer of stressrelated substances were met with designs which either were activity free, such as discrimination tasks or used yoked control groups. That it was not a nutritional effect could be shown by setting up control groups which received identically prepared extracts from untrained animals. However, all this work was defensive, attempting to refute alternative explanations offered by critics. There has been considerable discussion of the importance of a clearcut demonstration of specificity, as well as the most expeditious and logically tight manner in which to go about it (Booth, 1967; Dyal, 1971), but remarkably little experimental work. This paucity is especially surprising in view of the fact that the most common technique, cross-transfer, is conceptually very simple. Essentially cross-transfer consists of first demonstrating that two tasks, A and B, can be transferred, then brain extracts of animals trained on task A are injected into a group which is then tested on task B and trained B extract is injected into a group run on A. Typically what is sought is evidence that injection of material from the brain of animals trained on an irrelevant task has no effect on acquisition of the other. Unfortunately such designs provide little new information. I t is already known that soniething is transferred. At best they provide suggestive, but not conclusive, evidence for the notion of specificity. They do not exclude interpretation in terms of, for example, a predisposition to attend, or respond, to one set of stimuli rather than another.

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What few studies there have been attempting to show that the transfer of specific information is responsible for the modified recipient behavior, have either used response modifications not normally considered learning, or have not been replicated. Additionally, none of the designs used thus far have permitted unambiguous support of the specificity notion. Jacobson et al. (1965) trained two donor p u p s to approach a food cup to either a click or light cue. Recipients, injected with either clicktrained or light-trained extract, were then exposed to a random presentation of twenty-five click and twenty-five light stimuli. It was found that, as expected, the group that received light-trained extract responded significantly more often to the light and recipients of click-trained extract responded significantly more to the click. However, Halas et al. (1966) in a partial replication in which only the click was used, failed to find similar effects, and de Balban Verster and Tapp (1967) found that both groups responded more to the click. Ungar and Ocequera-Navarro (1965) using habituation to sound and air puff, showed that although injection of sound-habituated extract facilitated habituation to sound, and air puff-habituated extract facilitated habituation to air puff, neither extract induced habituation to the other. Similarly Ungar ( 1971a) failed to demonstrate cross-transfer between step-down and dark-avoidance tasks, as well as between two complex mazes (Ungar, 1973). In an attempt to provide a test for response specificity, Dyal and Golub (1970) reinforced one group of donors for a bar-press response and another for magazine entry. Recipients received both a nonreinforced preference test and reinforced acquisition sessions, half of each recipient group being trained on either the same task as its donor or the alternate task. Results were not supportive of the specificity notion. Somewhat similar designs were used by Gurowitz (1968), who failed to find any evidence to support transfer or its specificity, and by Allen et al. (1966), who found that handled controls performed better than experimentals and that the split-transfer group did better than the straight-transfer group. Essman and Lehrer (1967) also used straight- and split-transfer groups, finding that recipients of left-trained extract who were trained to go left performed better than those trained to go right. However, no data are offered, only significance levels, and it is not clear from their abstract just how performance was evaluated ; e.g., there is no description of the criterion. Cartwright (1970) offered a novel approach to the problem, varying the proportion of left- and right-trained extract injected into recipients, then testing for differential performance. However, it is not clear whether or not recipients were reinforced. Unfortunately, the results obtained were equivocal, only one of five predictions of recipient behavior being confirmed, per-

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haps in part because recipient testing consisted of a single test period, 4 hours after injection. A somewhat similar study was reported by McConnell (1967) in which donor planarians were trained on a discrimination task in a T-maze. One recipient group was fed donors which had been trained to one cue, a second group was fed donors trained to another cue, and each member of the third group was fed a donor from both training conditions. A final control group received untrained donors. It was found that the first group learned faster than the other three, but also that the second learned faster than the last two. Finally, the third group learned slower than the control group. The author concluded that both specific and nonspecific effects had evidently been transferred. Additionally, indirect support for the notion of specificity comes from two other sources, enzymic digestion and immunology. The enzymic digestion work was primarily carried out by Ungar (1971a), who found that the transfer factors for different tasks were not all subject to attack by the same enzymes. The immunological studies bear upon both the question of specificity and the mechanism of transfer itself. Most of the tentative explanations of transfer assert that the transfer factor exerts its effects by selectively acting on homologous areas in the recipient. The immunological studies provide a plausible mechanism for the requisite recognition system implicitly assumed. Mihailovic and Jankovic ( 1961) began this work, showing that antibodies could be produced which selectively attack one brain region, the caudate nucleus. Later the same group (Jankovic et al., 1968) showed that brain-specific antibodies had behavioral effects. Similarly Dergachev (1972) has shown that antibodies specific to one task may be produced, and HydCn (1973a) has described an antibody specific for the S-100 protein, which also has behavioral effects. Finally Rosenblatt ( 1970) has described a technique, similar to that employed by Dergachev, by which antibodies of animals trained on a discrimination task were produced. It was found that these antibodies both interfered with and facilitated performance of recipients in the same task, leading Rosenblatt to speculate that transfer effects may be due to an antibody-induction process. Although it is appealing to invoke the commonalities of selective recognition in the immune system and the CNS in proffering new concepts or explanations of brain function, this is as yet quite speculative. The most significant applications of immunologic technology in neurobiology to date have been in the development of animal models of CNS disease by the autoinduction of antibodies to CNS proteins. Injection of basic brain protein from myelin in complete Freunds’ adjuvant causes progressive CNS demyelination, paralysis, and eventual death in a variety of species and is thought to mimic multiple sclerosis. Highly purified ACh receptor from the elec-

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troplax of Electrophorus electricus in Freund's complete adjuvant injected into rabbits results in neuromuscular blockade and suggests an autoimmune explanation for myasthenia gravis (Patrick and Lindstrom, 1973). It might well be worthwhile to extend previous work on the relationship between CNS-specific antibodies and learning. For example, if it is possible to induce an immunological response to a specific behavior-related polypeptide, such as scotophobin or ameletin, then there may exist concomitant behavioral changes. Immunology offers a powerful set of tools that will facilitate the indirect identification and comparison of CNS proteins without fully understanding their role. D. RECENTSYNAPTIC MODELS It seems that information may be retained synaptically until other macromolecular events take place. Regardless of the nature of any memory macromolecule a permanent alteration of synaptic function would surely be the most parsimonious explanation, and the synapse the most readily retrievable locus of learning. The state of the art allows us to speak substantively of the transmitter release mechanism and receptor function and the molecular nature of both is yielding to biochemical investigation on many fronts. Recently Kosower (1972) has offered a biophysical theory of how synaptic use may result in an increased synthesis of synaptopores with subsequent potential for increased transmitter release. It is suggested that this change is mediated by the alteration of dithiolate structures to disulfides during depolarization by the influx of calcium at the presynaptic membrane. The formation of a calcium dithiolate at the synaptopore causes an opening of the membrane due to the decreased sulfur-sulfur (S-S) distance. This dithiolate is now oxidized to an even shorter disulfide by glutathione disulfide and may then be returned to its original dithiolate form by reaction with glutathione, thus closing the pore again. These events could be responsible for short-term memory and increased incorporation of synaptomeric protein producing more synaptopores could underlie long-term storage. Kosower and Werman ( 1971) have suggested that the calcium-independent production of miniature end-plate potentials could be explained by the oxidation of the dithiol to disulfide by glutathione disulfide. Dodge and Rahamimoff (1967) have reported that the thiol-oxidizing agent diamide causes a marked increase in miniature end-plate potentials implying an acceleration of ACh release due to S-S bond formation. Kowsower proposed that postsynaptic mechanisms might also be involved in increasing junctional efficiency. He speculated that the neurotransmitter might have the added property of inducing aggregation of the receptor, thus increasing the probability that a given number of neurotransmitter molecules might have the desired effect

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at the postsynaptic membrane. The theory implied that increased efficiency would be caused by the permanent location of receptor aggregates in the membrane as compared to individual receptors which would be more mobile given the fluid-mosaic model of Singer and Nicolson (1972). This theory of aggregate formation could account for the spread of the ACh receptors away from the end-plate region after denervation and relocation after renervation as described by Miledi and Potter ( 1971). Meanwhile, in this laboratory attempts have been underway to study the biophysical properties of individual CNS receptors. This research has recently led to the extraction and purification of a nicotinic acetylcholine receptor (AChR) from mouse brain. The AChR was incorporated in artificial lipid bilayers and found to be channel forming (Goodall et al., 1974a). When acetylcholine couples with the receptor there is a change in conformation which allows ions to flow through a channel in the protein. These events may be measured by monitoring conductance across an artificial membrane to which the AChR has been added. The conductance of an individual mouse brain receptor was found to be 0.37 x 10-lo mho for sodium ions. A similar AChR was extracted from hog brain and found to have the same basic conductance (Romine et al., 1974) , However, these receptors were found to have more remarkable properties in that they very often turn on and off in aggregates rather than as individuals. This functional synchrony of aggregated receptors was promoted by carbachol and blocked by curare and atropine (Goodall et al., 1974b). It is interesting that these aggregates mostly occurred as multiples of four times the basic event, suggesting that the receptor is a tetramer. The data imply that aggregation of receptors in the plasma membrane has important functional significance, and the following theory was formulated by Goodall, Romine, and Bradley to explain their data (Goodall et al., 1974b).

1. ACh induces the receptor protomer to polymerize into an oligomer. 2. The oligomer may have intracooperative interactions such that, as one protomer turns on or off, it induces its neighbors to behave similarly. 3. Receptors may activate more efficiently or for longer when aggregated than when single. The whole may be greater than the sum of its parts. The individual receptor may make no substantive contribution to ion passage through the membrane on its own and most of the work may be done by receptor aggregates. 4. Fewer ACh molecules may be required to activate an oligomer than would be required to activate all its protomers individually. In fact the binding sites themselves may be different in location as well as in number or in efficacy. 5. If in fact ACh does induce the aggregation of AChR in the brain,

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and if these aggregates do subsequently operate more efficiently, as is evidenced by their simultaneous switching in the bilayer, then these facts would offer an explanation for memory at the postsynaptic membrane. If ACh were to increase the degree of aggregation then more receptors might be activated by the available ACh molecules, thus increasing the probability, intensity, or lifetime of the event. 6. The effective lifetime of the oligomer state might explain the many phenomena of learning and memory.

A plausible theory for the incorporation of new receptor units into the postsynaptic membrane has been proposed by Huttunen ( 1973). The neurotransmitter in question which may be in the process of forming a new connection is said to penetrate the postsynaptic membrane and couple with a newly synthesized receptor still bound to the polysome. This receptor is thus protected from enzymic breakdown by its neurotransmitter while migrating to the postsynaptic membrane, thus forming a new connection. These are the types of theory that can be subjected to experimental test in the near future, but which may be only necessary and not sufficient to explain the basic underlying mechanisms of learning. That the peptide code offers unlimited informational content does not, however, automatically qualify it for a synthetic role in memory storage. The problem of unraveling CNS function remains, and even though the locus and mode of action for behavior-specific peptides is presently unknown, they may ultimately provide the means for the explication of information storage and retrieval. REFERENCES Allen, A. R.,Grissom, R. J., and Wilson, C. L. (1966). Psychon. Sci. 15, 257. Babich, F. R., Jacobson, A. L., Bubash, S., and Jacobson, A. (1965). Science 149, 656. Barondes, S. H. (1970). In “Protein Metabolism of the Nervous System” (A. Lajtha, ed.), pp. 545-554. Plenum, New York. Beaton, J. M., and Gilbert, R. M. (1968). Nature (London) 218, 391. Best, R. M. (1968). Psychol. Rep. 22, 107. Booth, D. A. (1967). Psychol. Bull. 68, 149. Booth, D. A., and Pilcher, C. W. T. (1973). In “Macromolecules and Behavior” (C. B. Ansell and P. B. Bradley, eds.), pp. 105-112. Univ. Park Press, Baltimore, Maryland. Braud, L. W., and Braud, W. G. (1972). Science 176,942. Braud, W. G. (1970). Science 168, 1234. Braud, W. G., and Laird, P. V. (1973). SOC.Ncurosci., 3rd Annu. Meet. p. 131. Bryant, R. C., Santos, N. N., and Byrne, W. L. (1972). Science 177,635. Byrne, W. L., ed. (1970). “Molecular Approaches to Learning and Memory.” Academic Press, New York. Byrne, W. L., and Hughes, A. (1967). Fed. Proc., Fed. Amer. SOC. E x p . Biol. 26, 676.

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Byrne, W. L., and Samuel, D. (1966). Science 154, 418. Bryne, W. L., Samuel, D., Bennett, E. L., Rosenzweig, M. R., Wasserman, E., Wagner, A. R., Gradner, F., Galambos, R., Berger, B. D., Marqules, D. L., Fenichel, R. L., Stein, L., Corson, J. A., Enesco, H. E., Chovover, S. L., Holt, C. E., Schiller, P. H., Chiappetta, L., Jarvik, M. E., Leaf, R. C., Dutcher, J. D., Horovitz, Z. P., and Carlson, P. L. (1966). Science 153, 658. Cartwright, G. M. (1970). J . B i d . Psychol. 12, 53. Chapoutier, G. (1972). I n “The Physiological Basis of Memory” (J. A. Deutsch, ed.), pp. 1-25. Academic Press, New York. Cohen, H. D., and Barondes, S. H. (1967). Science 157, 333. Daliers, J., and Giurgea, C. (1971). I n “Biology of Memory” (G. Adam, ed.), pp. 191-198. Plenum, New York. Davis, R. E., and Agranoff, B. W. (1966). Proc. Nat. Acad. Sci. US.55, 555. de Balban Venter, F., and Tapp, J. T. (1967). Psychol. Rep. 21, 9. Dergachev, V. V. (1972). Znt. I. Psychobiol. 2, 321. de Wied, D. (1973). I n “Memory and the Transfer of Information” (H. Zippel, ed.), pp. 373-390. Plenum, New York. Dodge, F. A., and Rahamimoff, R. (1967). J . Physiol. (London) 193, 419. Dyal, J. (1971). In “Chemical Transfer of Learned Information” (E. J. Fjerdingstad, ed.), p. 219-258. Amer. Elsevier, New York. Dyal, J. A., and Golub, A. M. (1968). Psychon. Sci. 11, 13. Dyal, J. A., and Golub, A. M. (1970). In “Molecular Approaches to Learning and Memory” (W. Byrne, ed.), pp. 275-284. Academic Press. New York. Eist, H., and Seal, U. S. (1965). Amer. J . Psychiat. 122, 584. Enesco, H. E. ( 1966). Ex#. Cell Res. 42, 640. Essman, W. B., and Lehrer, G. M. (1967). Fed. Proc., Fed. Amer. SOC. Ex@. Biol. 26, 263. Fjerdingstad, E. J. (1973). In “Memory and the Transfer of Information” (H. Zippel, ed.), pp. 429-450. Plenum, New York. Fjerdingstad, E. J., Nissen, T., and Roigaard-Peterson, H. H. (1965). Scand. 1. Psychol. 6, 1. Flexner, J. B. and Flexner, L. B. (1967). Proc. Nat. Acad. Sci. US.57, 1651. Flexner, J. B., and Flexner, L. B. (1969). Yale J . B i d . Med. 42, 235. Flexner, L. B., and Flexner, J. B. (1966). Proc. Nat. Acad. Sci. U.S. 55, 369. Gaito, J. ( 1971 ) . “DNA Complex and Adaptive Behavior.” Prentice-Hall, Eaglewood Cliffs, New Jersey. Galvan, L., and Ungar, G. (1973). Proc. Soc Neurosci. 3, 130. Goldstein, A. (1973). Nature (London) 242, 60. Golub, A. M., Masian, F. R., Villan, T., and McConnell, J. V. (1970). Science 168, 392. Goodall, M. C., Bradley, R. J., Saccomani, G., and Romine, W.0. (1974a). Nature (London) 250, 68. Goodall, M. C., Romine, W. O., and Bradley, R. J. (1974). Znt. J. Quantum Chem. (in press). Gordon, M. W., Deanin, G. G., Leonhardt, H. L., and Gwynn, R. H. (1968). Amer. J. Psychiat. 11, 1. Gross, C. G., and Carey, F. M. (1965). Science 150, 1749. Grossman, S. P. (1967). “A Textbook of Physiological Psychology.” Wiley, New York. Gurowitz, E. M. (1968). Psychol. Rep. 23, 899.

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Guttman, H. N. ( 1973). Psychopharmacol. Bull. 9, 1. Guttman, H. N., and Gronke, L. (1971). Psychon. Sci. 24, 107. Guttman, H. N., and Hoffman, M. (1973). SOC.Neurosci., 3rd Annu. Meet. p. 193. Guttman, H. N., Matwyshyn, G., and Warriner, G. H. (1972). Nature ( L o n d o n ) N e w Biol. 235, 26. Guttman, H. N., Matwyshyn, G., and Weiler, M. (1973). I n “Memory and the Transfer of Information” (H. Zippel, ed.), pp. 391-418. Plenum, New York. Halas, E. S., Bradfield, K., Sandlie, M. E., Theye, F., and Beardsley, J. (1966). Physiol. B Behav. 1, 281. Heltzel, J. A., and Ungar, G. (1973). SOC.Neurosci., 3rd Annu. Meet. p. 131. Herblin, W. F. (1970). I n “Molecular Approaches to Learning and Memory” (W. Byrne, ed.), p. 243. Academic Press, New York. Huttunen, M. 0. (1973). Perspect. Biol. M e d . 17, 103. HydCn, H. (1973a). I n “Memory and the Transfer of Information” (H. Zippel, ed.), pp. 51 1-520. Plenum, New York. HydCn, H. (1973b). I n “Macromolecules and Behavior” (G. B. Ansell and P. B. Bradley, eds.), pp. 3-26. Univ. Park Press, Baltimore, Maryland. Jacobson, A. L., Babich, F. R., Bubash, S., and Jacobson, A. (1965). Science 158, 636.

Jankovic, B. D., Rakic, L., Veskov, R., and Horvat, R. (1968). Nature ( L o n d o n ) 218, 270. Kosower, E. M. (1972). Proc. Nat. Acad. Sci. U.S.69, 3292. Kosower, E. M., and Werman, R. (1971). Nature ( L o n d o n ) , New Biol. 233, 121. Lagerspetz, K. M. J., Ratis, P., Tirri, R., and Lagerspetz, K. Y. H. (1968). Scand. j . Psychol. 9, 225. Laird, P. V., and Braud, W. G. (1973). SOC.Neurosci., 3rd Annu. M e e t . p. 132. Laird, P. V., Braud, W. G., and Hoffman, R. B. (1972). j . Biol. Psychol. 14, 8. Lashley, K. S . (1950). Symp. SOC.Exp. Biol. 4, 454. Laskov, R., Margoliash, E., Littauer, V. Z., and Eisenberg, H. (1959). Biochim. Biophys. Acta 33, 247. Leaf, R. C., Dutcher, J. D., Horovitz, Z. P.,and Carlton, P. L. (1966). Supplementary material to Byrne et al. (1966). Luttges, M., Johnson, T., Buck, C., and McGaugh, V . (1966). Science 151, 834.

McConnell, J. V. (1962). j . Neuropsychiat. 3, 42. McConnell, J. V. (1967). I n “Chemistry of Learning” (W. C. Corning and S. C. Ratner, eds.), pp. 310-328. Plenum, New York. McConnell, J. V., and Malin, D. H. (1973). I n “Memory and the Transfer of Information” (H. Zippel, ed.), pp. 343-362. Plenum, New York. MoConnell, J. V., Shigehisa, T., and Salive, H. (1970). In “Molecular Approaches to Learning and Memory” (W. Bryne, ed.), pp. 245-274. Academic Press, New York. Mackintosh, N. J. (1969). 1. C o m p . Physiol. Psychol. 67, 1. Malin, D. H., and Guttman, H. N. (1972). Science 178, 1219. Malin, D. H., and Radcliffe, G. J. (1973). SOC.Neurosci., 3rd Annu. M e e t . p. 129. Mihailovic, L., and Jankovic, B. D. (1961). Nature ( L o n d o n ) 192, 665. Miledi, R.,and Potter, L. T. (1971). Nature ( L o n d o n ) 233, 599. Patrick, J., and Lindstrom, J. (1973). Science 180, 871. Potts, A., and Bitterman, M. E. (1967). Science 158, 1594. Reinis, S. (1965). Activ. Ncrv. Super. 7, 167.

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Reinis, S . (1971). I n “Chemical Transfer of Learned Information” (E. J. Fjerdingstad, ed.), pp. 109-142. Amer. Elsevier, New York. Reinis, S., and Mobbs, D. R. (1970). In “Molecular Approaches to Learning and Memory” (W. Bryne, ed.), pp. 189-194. Academic Press, New York. Romine, W. O., Goodall, M. C., and Bradley, R. J. (1974). Biochem. Biophys. Acta (in press). Rosenblattt, F. (1967). In “Computer and Information Sciences-11’’ (J. T. Tou, ed.), p. 33. Academic Press, New York. Rosenblatt, F. (1970). I n “Molecular Mechanisms in Memory and Learning” (G. Ungar, ed.), pp. 103-147. Plenum, New York. Rosenblatt, F., and Miller, R. G. (1966a). Proc. Nut. Acad. Sci. US.56, 1423. Rosenblatt, F., and Miller, R. G. (1966b). Proc. Nut. Acad. Sci. US.56, 1683. Rosenblatt, F., Farrow, J. T., and Rhine, S . (1966a). Proc. Nut. Acad. Sci. U.S. 55, 548. Rosenblatt, F., Farrow, J. T., and Rhine, S . (1966b). Proc. Nut. Acad. Sci. US. 55, 787. Rucker, W. B. (1971). I n “Chemical Transfer of Learned Information” (E. J. Fjerdingstad, ed.), pp. 97-108. Amer. Elsevier, New York. Singer, S. J., and Nicolson, G. L. (1972). Science 175, 720. Stewart, W. W. (1972). Nature ( L o n d o n ) 238, 202. Sved, S . (1965). Can. I . Biochem. 43, 949. Szilard, L. (1964). Proc. Nut. Acad. Sci. U.S.51, 1092. Thinnes, G., Domagk, G. F., and Schonne, E. (1973). I n “Memory and the Transfer of Information” (H. Zippel, ed.), pp. 363-372. Plenum, New York. Ungar, G. ( 1965). Fed. Proc., Fed. Amer. SOC.Exp. Biol. 25, 207. Ungar, G. (1968). Perspect. B i d . M e d . 11, 217. Ungar, G. (1970a). I n “Handbook of Neurochernistry” (A. Lajtha, ed.), p. 241. Plenum, New York. Ungar, G. (1970b). Int. Rev. Neurobiol. 13, 223. Ungar, G. (1971a). I n “Chemical Transfer of Learned Information” (E. J. Fjerdingstad, ed.), pp. 31-50. Amer. Elsevier, New York. Ungar, G. (1971b). I n “Methods in Pharmacology” (A. Schwartz, ed.), pp. 479-513. Appleton, New York. Ungar, G. (1973). I n “Memory and the Transfer of Information” (H. Zippel, ed.), pp. 317-342. Plenum, New York. Ungar, G., and Ocequera-Navarro, C. (1965). Nature ( L o n d o n ) 207, 301. Ungar, G., Galvin, L., and Clark, R. H. (1968). Nature ( L o n d o n ) 217, 1259. Ungar, G., Desiderio, D. M., and Parr, W. (1972). Nature ( L o n d o n ) 238, 198. Ward, N. (1970). Unpublished observations (quoted in Rosenblatt, 1970). Weiss, K. P. ( 1970). I n “Molecular Approaches to Learning and Memory” (W. Byrne, ed.), pp. 325-334. Academic Press, New York. Weiss, K. P. ( 1971) . I n “Chemical Transfer of Learned Information” (E. J. Fjerdingstad, ed.), pp. 85-96. Amer. Elsevier, New York. Zippel, H. P., and Langscheid, C. (1973). I n “Memory and the Transfer of Information” (H. Zippel, eds. ), pp. 45 1-470. Plenum, New York. Zippel, H. P., Bieck, B., and Rueckerk, K. (1971). I. Biol. Psychol. 13, 9.

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AMINOTRANSFERASE ACTIVITY IN BRAIN By M. Benuck and A. Lajtha

New York State Research Inrtilute for Neurochemirtry ond Drug Addiction, Ward'r Irlond, New York, New York

I. Introduction

.

II. Chemical and Physical Properties of the Aminotransferases A. B. C. D. E.

.

.

Aspartatem-KetoglutarateAminotransferase (EC 2.6.1 .I) Alanine:cr-Ketoglutarate Aminotransferase (EC 2.6.1.2) YAminobutyric Acid:a-Ketoglutarate Aminotransferase (EC 2.6.1.1 9) Branched-Chaii Amino Aad:a-Ketoglutarate Aminotransferase (EC 2.6.1.42) Transamination of Clutamine and Asparagine F. The Aromatic Aminotransferases G. 0rnithine:a-KetoglutarateAminotransferase (EC 2.6.1.1 3) H. G1ycine:a-Ketoglutarate Aminotransferase (EC 2.6.1.4) I. Phosphoserine:a-Ketoglutarate Aminotransferase (EC 2.6.1.52) J. Other Aminotransferase Activity in Neural Tissue. 111. Regulation of Aminotransferase Activity A. Glucose Metabolism and Aminotransferases 8. Conditions Resulting in Altered Aminotransferase Levels in Tissues C. Stability of Cerebral Aminotransferase D. Compartmentation in Nervous Tissue and Aminotransferase Activity E. Possible Role of Aminotransferase in Neurotransmitter Metabolism IV. Developmental Changes in Aminotransferase Activity. V. Conclusions. References

.

.

.

.

.

.

.

.

.

. .

. . .

.

85 87 87 94 96 99 101 103 107 108 108 109 109 109 110 113 114 115 117 121 122

1. Introduction

Transamination is defined as the transfer of an amino group from one molecule to another without the intermediate formation of ammonia. The enzymes that catalyze such a reaction are called transaminases or aminotransferases. Enzymic transfer of amino groups was initially described by Braunstein and Kritsmann (1937), who observed the transfer of amino groups from a number of amino acids to a-ketoglutarate and oxaloacetate in preparations from pigeon heart muscle. Early work suggested that most transamination reactions were catalyzed by two enzymes : aspartate :a-ketoglutarate aminotransferase (EC 2.6.1.1 ) and alanine: a-ketoglutarate aminotransferase (EC 2.6.1.2), the major amino acid substrates being asparate, glutamate, and alanine (Cohen, 1949; Braunstein, 1947). However, a number 85

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M. BENUCK A N D A. LAJTHA

of investigators soon found that the transamination reaction is one involving many amino acids and a number of enzymes (Awapara and Seale, 1952; Cammarata and Cohen, 1950; Meister and Tice, 1950; Feldman and Gunsalus, 1950). A wide variety of aminotransferases was reported in bacteria, plants, and animal tissue. Cohen and Hekhuis (1941) and Awapara and Seale (1952) reported the presence of these enzymes in brain tissue. A number of reviews have appeared covering the early work in this area, including those of Bonavita ( 1961) , Meister ( 1962), Guirard and Snell (1964), Sallach and Fahien ( 1969), and the comprehensive work of Meister (1965). The aminotransferases occupy a significant place in amino acid metabolism. They are the major degradative mechanism for the catabolism of many amino acids. Conversely, synthesis of nonessential amino acids occurs via the transamination of glutamic acid. These enzymes also provide a mechanism for the conversion of alanine, glutamate, and aspartate to pyruvate, a-ketoglutarate, and oxaloacetate, joining amino acid metabolism with carbohydrate metabolism. The aminotransferases are also crucial enzymes in other metabolic processes, such as urea synthesis in the liver, ammonia metabolism, or the degradation of neurotransmitter agents, such as y-aminobutyric acid (GABA) . A number of the aminotransferases have been extensively studied, and their properties have been well characterized. Most of these studies used tissues where the activity of these enzymes is high, such as liver or muscle. Until recently, little attention has focused on the aminotransferase activity in neural tissue. However, aminotransferase activity in brain is quite strong (Cohen and Hekhuis, 1941; Awapara and Seale, 1952; Alben et al., 1962; Benuck et al., 1971) . The enzyme aspartate: a-ketoglutarate aminotransferase (EC 2.6.1.1) is one of the most active enzymes in the brain, comparable to the more active enzymes of the glycolytic or respiratory cycles (BalAzs and Haslam, 1965; Balks, 1970). Other aminotransferases, such as alanine: a-ketoglutarate aminotransferase (EC 2.6.1.2), although of much lower activity, are as active as other crucial enzymes in neural metabolism, such as acetylcholinesterase or monoamine oxidase. Early studies of aminotransferase activity in brain concentrated on the transfer of amino groups from various amino acids to a-ketoglutarate. Such studies found that twelve to eighteen amino acids are transaminated by neural tissue from either vertebrates or invertebrates (Cohen and Hekhuis, 1941 ; Awapara and Seale, 1952; Yamada, 1959; Imai, 1959; Albers et al., 1962). These studies showed that the highest activity was obtained when either glutamate or aspartate was incubated with a-ketoglutarate or oxaloacetate. Significant activity was also obtained when neural tissue was incubated with neutral aliphatic or aromatic amino acids, whereas little or no activity was

AMINOTRANSFERASE ACTIVITY I N BRAIN

87

found when basic amino acids were incubated with btain preparations (Albers et al., 1962; Benuck et al., 1971). Recently a number of aminotransferases have been purified from brain. Among these are GABA :a-ketoglutarate aminotransferase (EC 2.6.1.19) (Schousboe et al., 1973) ; aspartate: a-ketoglutarate aminotransferase (Magee and Phillips, 197 1) ; the branchedchain amino acid aminotransferase (EC 2.6.1.42) (Aki et al., 1969) ; phenylalanine :a-ketoglutarate aminotransferase (George and Gabay, 1968) ; tyrosine:a-ketoglutarate aminotransferase (EC 2.6.1.5) (Aunis et al. 1971) ; and phosphoserine :a-ketoglutarate aminotransferase (EC 2.6.1.52) (Hirsch and Greenberg, 1967). I n this review we shall concentrate on the properties of the brain aminotransferases, comparing them with those of other tissues. The references cited are not meant to be comprehensive, but serve as examples of work done in specific areas. The authors apologize for any papers that were inadvertently omitted from this article.

II. Chemical and Physical Properties of the Aminotranrferarer

A. ASPARTATE :a-KETOGLUTARATE AMMINOTRANSFERASE (EC 2.6.1.1 ) This enzyme catalyzes the interconversion of aspartate and a-ketoglutarate with glutamate and oxaloacetate. I t is quantitatively the most important aminotransferase occurring in the cell and is closely associated with glutamate metabolism. Both liver and heart muscle are rich sources of this enzyme, with highest activity in heart muscle (Zimmerman et al., 1968; H e n feld and Greengard, 1971). The activity of the enzyme from brain is about 6040% that of the liver enzyme (Balhs and Haslam, 1965; Herzfeld and Greengard, 1971) (Table I ) . 1. Physical Properties

The enzyme was purified from pig heart muscle by Jenkins et al. (1959). I t is pyridoxal phosphate dependent, as is the case with aminotransferases generally, with 2 moles of coenzyme tightly bound to 1 mole of enzyme. The equilibrium constant has been reported as 6.1 or 7.1 in the direction of aspartate and a-ketoglutarate (Velick and Vavra, 1962a; Henson and Cleland, 1964). Early studies reported molecular weights for the enzyme of about 110,000 (Jenkins et al., 1959). More recent work indicates a somewhat lower molecular weight of about 80,000-90,000 (Banks et al., 1968; Magee and Phillips, 1971; Doonan et al., 1972). The enzyme exhibits highest activity between pH 8 and pH 9 (Jenkins et al., 1959). Structural studies indicate that the enzyme is composed of two polypep-

TABLE I A C T I V ~OF ~ YSEVERALAMXNOTRANBFERASES IN VARIOUSTISSUE HOMOCENATES OF

THE

Activity (junoles/min/gm tissue)

Enzyme Aspartate aminotransferase Soluble Particulate Alanine aminotransferase Branched-chain aminotransferase Valine Leucine Isoleucine Aromatic aminotransferase Phenylalanine Tryptophan Tyrosine Ornithine aminotransferase

Heart

191 103 2.2

Liver

47 85 36.4

Kidney

46 60 0.6

0.41 1.4 1.0

0.008 0.014 0.220

1.4 1.6 1.8

0.98 0.13 0.25

0.90 0.12 1.2

1.07 0.27 0.09

0.22

3.1

5.9male 2.9 female

Skeletal muscle

59 30 4.2

0.08 0.23 0.19

Brain

32 43 1.2

Reference

Herzfeld and Grcengard (1971) Hopper and Segal (1964); Benuck ct 41. (1971)

1.2 1.1 1.3

Ichihara and Koyama (1966); Benuck et 01. (1971)

-

0.25 0.07 0.15

-

0.22

Fuller ct al. (1972) Wada el nl. (1972) Wurtman and Larin (1968); Benuck ct 41. (1971) Herzfeld and Knox (1968)

a Activity for different enzymes measured using separate assay procedures. Most assays were performed a t 37OC, that for aspartate aminotransferase performed a t 25°C. Data of Benuck et 41. (1971) are for activity in brain homogenate for alanine aminotransferase, the branchcdchain aminotransferase, and tyrosine aminotransferase.

TABLE I1 AMINOACID COUWSITXON OF SELECTEDAUINOTRANSFERASESO Aspartate aminotransferase

Aspartate Threonine Serine Glutamate Proline Glycine Alanine 34-Cysteine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Tryptophan

Brain

Heart

Cytoplasmic Mitochondrial

Cytoplasmic Mitochondrial

77 36 41 66 43 52

55 6 47 12 26 63 20 43 36 13 38 17

65 34 45 74 33 66 62 12 52 18 35 59 22 31 50 17 35 11

74 44 46 71 38 47 53 9 50 8 30 64 20 36 35 14 41 18

64 26 35 69 30 58 59 8 45 19 45 48 19 32 50 19 34 16

Alanine aminotransferase (liver)

Ornithine aminotransferase (liver)

Tyrosine aminotransferaseb (liver)

73 31 48 166 73 84 104 34 88 31 41 109 32 46 22 10 56 4

25 15 13 78 17 24 22 4 19 10 16 27 11 9 17 7 13 4

95 34 68 115 6 62 70 28 60 25 55 94 27 33 49 18 46 9

a Amino acid content for brain aspartate aminotransferase is from Magee and Phillips (1971), given as residues per 80,000 molecular weight. For heart cytoplasmic aspartate aminotransferase, data are from Banks ct ul. (1968), given as residues per 78,600 molecular weight. For mitochondrial aspartate aminotransferase, data are from Martinez-Carrion and Tiemeier (1 967): residues are recalculated for 80,000 molecular weight. Data for alanine aminotransferase (residues per 11 4,000 molecular weight) ornithine aminotransferase (residues per 33,000 molecular weight) and tyrosine aminotransferase (residues per 115,000 molecular weight) are from Matsuzawa and Segal (1968), Paraino ct 41. (1969), and Valeriote ct 01. (1969),respectively. * The amino acid composition of tyrosine aminotransferase in rat brain has recently been described by P. Mandel and D. Aunis (1974) in “Aromatic Amino Acids in the Brain” (G .E. W. Wolstenholme and D. W. Fitzsimons, eds.), Ciba Foundation Symposium 22 (new series) pp. 67-83. Elsevier, Amsterdam.

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M. BENUCK AND

A. LAJTHA

tide chains, with two N-terminal alanine and two C-terminal leucine residues per mole of enzyme (Marino et al., 1966). The two polypeptide chains have identical primary structures and are held together through noncovalent linkages. The complete amino acid structure of the supernatant enzyme from pig heart has been recently reported (Doonan et al., 1972; Vinogradova et al., 1973). The two polypeptide chains each consist of about 400 amino acid residues. The combined total molecular weight of the residues is about 46,000, and that for the whole enzyme about 92,000; The amino acid composition of the enzyme is shown in Table 11. 2. Substrate Specificity The major amino acid substrates for this enzyme are glutamate and aspartate (Table 111). However, low activity has been obtained with other amino acids, such as alanine, methionine, and phenylalanine (Shrawder and Martinez-Carrion, 1972 ; Jenkins, 1961 ; Novogrodsky and Meister, 1964). Various substrate analogs, such as maleate, glutarate, p-hydroxyaspartic acid, and p-fluorooxaloacetate are competitive inhibitors of the enzyme (Jenkins et al., 1959; Kun et al., 1963) . Product inhibition has been demonstrated with all four substrates of the enzyme (Henson and Cleland, 1964). Substrate inhibition has been demonstrated with a-ketoglutarate, and, in rat brain, with oxaloacetate (Magee and Phillips, 1971) . Glyceraldehyde 3phosphate is a noncompetitive inhibitor of the enzyme in both liver and brain (Kopelovich et al., 1970; Magee and Phillips, 1971). Early studies of the pig heart enzyme, including a kinetic analysis of the mechanism of transamination, have been reviewed by Velick and Vavra (1962b). 3. Isozymes Aspartate aminotransferase was separated into two subforms by Fleisher et al. (1960). They obtained two protein fractions from this enzyme after subjecting pig heart preparations to an electric field at neutral pH values. The cationic form was subsequently associated with mitochondria, while the anionic form was localized in the soluble supernatant (Borst and Peeters, 1961). Since the mitochondria1 isozyme is more labile to heat treatment than is the supernatant isozyme (Nisselbaum and Bodansky, 1966), early studies of the purified enzyme most probably dealt with the supernatant form, since heating was included in the purification (Jenkins et al., 1959). The presence of these isozymes has been demonstrated in a variety of tissues from a number of species (Boyd, 1961 ; Bodansky et al., 1965; Henson and Cleland, 1964; Martinez-Carrion et al., 1967 ; Shrawder and MartinezCarrion, 1973; Magee and Phillips, 1971; Herzfeld and Greengard, 1971) . They have been purified and characterized, and can be differentiated in terms of structure, amino acid composition, and substrate affinity (Wada

TABLE I11 RELATIVESPECIFICACTIVITYOF SELECTED AMINOTRANSFERASES FOR THEIR AMINO ACID SURSTRATES~ ~

~~

Amino acid Enzyme Aspartate aminotransfer= Mitochondria1 Soluble Alanine glvoxylatc Gluraminc Phcnylalaninc Tyroaine Tyrosine Branched chain Soluble Mitochondria Soluble Dopa aminotransferasc Aminotrmsfvasc (11) Aminotranafcrasc (111)

Time

Glu Asp Tyr Trp Phe Ala His Leu Val Ile Am Gln Ser Arg Dopa Mcth Cys

Pig Heart

Shrawder and Martinez-Carrion (1972) 100 72

H u m a n liver Rat liver Hog brain Rat liver Rat brain Hog heart

Hog brain Rat brain

References

64 100

11 2

12 6

29 2 100 8

49

100 100

52 2

100 4 40

2100

77 7

13 2

20 46 16

35

56 71 89 8 100 33 31 100

84 2

90 50 81

100 100 100

100 7 11

2

Thompaon and Richardson (1967) Cooper and Meiner (1972) George and Gabay (1968) Jacoby and La D u (1964) Aunia rt al. (1971) Taylor and Jenkins (1966) Aki t I al. (1967) Aki rf al. (1969) Aki rf al. (1968) Fonnum and L a m (1965) Tangen I f al. (1965) Tangen rt 01. (1965)

8 Relative activity of various amino acids as rubstratea for aminotraosferasu purified from various tizaua. Activity is generally measured in direction of conversion of .,-kctoglutaratc to glutamate. Amino acid with highat activity for each enzyme is given value of 100. Where no value is given. activity was either low or not mcnrured. (Activity for glutamate is given only for aspartate aminotransfcrase.) Total activity for aspartate a m i n o d e r a s e is about 100-fold greater than most of the o r h v ensyma lined. Dopa aminot r d e r a x is similar to aminotranderase I. AminotrpluferaseI1 is aimilar to tyrosine aminotranafmsc of rat brain. Aminotranaferase111ia similar to tryptophan aminowansferase.

92

M. BENUCK A N D A. LAJTHA

and Morino, 1964; Martinez-Carrion et al., 1967; Magee and Phillips, 1971). The amino acid composition of the two isozymes is shown in Table I1 and their substrate affinity is shown in Tables I11 and IV. The supernatant isozyme has a much greater affinity for the keto acid substrate, whereas the mitochondrial isozyme has a greater affinity for the amino acid substrate. Inhibition by carboxylic acids is not similar, with 4-carbon compounds inhibiting the mitochondrial isozyme, whereas 4- or 5-carbon compounds inhibit the supernatant form (Michuda and Martinez-Carrion, 1970). The purified isozymes are also influenced by the presence of phosphate or sulfate ions in the assay medium. When assayed a t pH 7.4 in TrisSHC1 buffer, addition of phosphate or sulfate ions inhibits the cationic isozyme and enhances the activity of the anionic isozyme (Nisselbaum, 1968). Each isozyme has been separated into several subforms, which are chemically and immunologically similar. The subforms present in minor quantity may be breakdown products of the major subform (Martinez-Carrion et al., 1967; Magee and Phillips, 1971 ; Krista and Fonda, 1973). 4. Brain Aspartate Aminotransferase Early purification of the enzyme from human brain indicated that its properties were similar to that of the pig heart enzyme (Bonavita, 1959). The major differences noted by Bonavita (1959) were that the brain enzyme, in contrast to the enzyme from heart muscle, had a p H optimum of 7.0, and appeared to be less tightly bound to its cofactor, pyridoxal phosphate. Early studies also indicated that the brain enzyme consisted of two subforms. Sellinger and Rucker ( 1963), among others, found that homogenization of brain tissue in dilute phosphate buffer or in detergent would significantly increase the activity of asparate aminotransferase, reflecting the solubilization of the mitochondrial isozyme. Magee and Phillips ( 197 1) have recently purified both the mitochondrial and supernatant isozymes of aspartate aminotransferase from rat brain. Both forms have been obtained in a crystalline state, with molecular weights of about 80,000. As with the isozymes from other tissues, the brain isozymes are differentiated by their electrophoretic behavior at neutral pH. Their amino acid composition also differs, in correlation with their electrophoretic behavior (Table 11).Their substrate affinities are different: the cytoplasmic brain isozyme, for example, has a 10-fold lower K , value for asparate than does the soluble form (Table IV) . The cytoplasmic isozyme has also been purified almost 400-fold from beef brain (Krista and Fonda, 1973). Its properties are similar to those of the beef heart isozyme.

5. Subcellular Distribution The subcellular distribution of the two enzymes differs from tissue to tissue (Table I ) . In brain, the major isozyme appears to be that associated

TABLE IV COMPARISON OF MICHAELIS CONSTANTS OF -IRED AMINOTRANSFERASE FOR THEIR SUBSTRATES FROM BRAINvs LIVEROR HEARTOF MAMMALIAN Tm~e K,(mM) ~

~~

Branched-chain aminotransferase

Aspartate aminotransferase

Substrate

Rat braina

Rat h e r b

Mitochondrial Soluble

Mitochondrial Soluble

Tyrosine aminotransferase Rat brain.

Rat liverd

Hog brain, solubled

Hog heart

Leucine specific, Mitorat Soluble, chondrialu Iiverh

+

5 z

s

! cn 4

Dopa Leu Val Ile a-Ketoglutarate Oxaloacetate

1.3 0.10

0.15 0.11

1 .o -

Magee and Phillips (1971). bBoyd (1961). c Aunis ct al. (1971). d Hayashi et al. (1967)and Jacoby and La Du (1964). Aki et al. (1969). 1 Taylor and Jenkins (1966). 0 Aki ct al. (1967). Aki ct al. (1968).

-

0.2 -

0.04 -

2.6 0.7 -

0.56 1.4 0.67 0.57 -

3.8 11 3.8 0.63 -

0.4 1.3 0.4 2.2 -

25 0.065 -

(0

w

94

M. BENUCK AND A. L AJ TH A

with the particulate matter (Herzfeld and Greengard, 1971; Benuck et al., 1972). Other tissues, such as heart and muscle, contain a larger amount of the soluble isozyme, while liver and kidney contain more of the particulate form. The particulate isozyme in brain appears to be tightly bound to the mitochondria1 membrane (Salganicoff and De Robertis, 1965; van Kempen et al., 1965).

B. ALANINE :WKETOOLUTARATE AMINOTRANSFERASE (EC 2.6.1.2) This enzyme catalyzes the interconversion of alanine and a-ketoglutarate with glutamate and pyruvate. The highest concentration of this enzyme is in the liver (Zimmerman et al., 1968; Hopper and Segal, 1964) ; activity in brain is about 10% of the liver enzyme (Balbs and Haslam, 1965) (Table I ) . 1. Physical Properties Green et al. (1945) first purified this enzyme, separating it from aspartate aminotransferase activity of pig heart muscle. Subsequent purifications of the soluble enzyme have since been described by a number of investigators, including Segal et al. (1962) and Gatehouse et nl. (1967), who purified the enzyme from rat liver, and Saier and Jenkins (1967), who purified the enzyme from pig heart. The enzyme has been purified more than 2000-fold (Bulos and Handler, 1965) ; it has been crystallized and its amino acid composition determined (Table 11) (Matsuzawa and Segal, 1968). Its molecular weight is estimated at about 90,000-1 15,000 with approximately 1 mole of pyridoxal phosphate per mole of enzyme (Saier and Jenkins, 1967). The equilibrium constant of the rat liver enzyme is 1.6 (Segal et al., 1962) and of the beef heart enzyme 2.2 (Bulos and Handler, 1965) in the direction of alanine and a-ketoglutarate formation. The enzyme is highly specific for its substrates. a-Aminobutyrate is active as a substrate for the beef heart enzyme; its activity, however, is only 2% of that of alanine. The enzyme possesses a broad pH activity curve, with optimal activity between pH 7.7 and p H 8.4 (Segal et al., 1962). A number of substrate analogs, including a-aminobutyrate and a-aminoadipate, inhibit the enzyme. Various dicarboxylic acids, such as maleate and acrylate, are also inhibitory. Product inhibition by alanine and glutamate has also been observed. The enzyme, as well as other aminotransferases, is sensitive to compounds which react with sulfhydryl groups. Inhibition by p-mercuribenzoate can be reversed by addition of glutathione and pyridoxal phosphate. Other compounds, such as cycloserine, aminooxyacetic acid, and isonicotinic acid hydrazide, inhibit this enzyme and other aminotransferases (Segal et al., 1962 ; Hopper and Segal, 1964). Purification procedures for the pig heart and rat liver enzyme, and

AMINOTRANSFERASE ACTIVITY IN BRAIN

95

their properties, have recently been summarized by Segal and Matsuzawa (1970) and Jenkins and Saier (1970). 2. Isozymes In common with aspartate aminotransferase, this enzyme is present in the form of two isozymes, one associated with the mitochondrial compartment of the cell, and one associated with the soluble cytoplasm (Hopper and Segal, 1964; Swick et al., 1965). The soluble liver isozyme has been separated into subspecies (Gatehouse et al., 1967). The mitochondrial enzyme of liver is very labile and has been difficult to purify; its properties have been studied using alanine or stabilizing agents, such as glycerol. The affinity of the isozymes for the enzyme substrates differ; the mitochondrial isozyme possesses a smaller K,,, value for alanine than does the soluble isozyme.

3. Brain Alanine Aminotransferase Little work appears to have been done specifically on the purification and study of the chemical properties of the brain enzyme. Studies using crude homogenates of brain tissue indicate that the brain enzyme possesses a similar substrate specificity to that of the liver enzyme; its pH activity curve also resembles that of the liver enzyme (Benuck et al., 1971). The enzyme from brain is also present in the form of two isozymes; one is associated with the mitochondria and the other with the soluble supernatant (Salganicoff and De Robertis, 1965; Benuck et al., 1972). The major activity is associated with the soluble fraction, a distribution similar to that of the liver enzyme, where only 10-35% of the total activity is associated with the mitochondrial fraction (Hopper and Segal, 1964; Takeda et al., 1964).

4. Distribution of Aspartate and Alanine Aminotransferase in Neural Tissue There have been a number of studies dealing with the distribution of these two aminotransferases in neural tissue. May et al. (1959) measured the activity of aspartate aminotransferase in six areas of the brain. They found the highest activity in the cerebellum and thalamus, somewhat less activity in cerebral cortex and white matter, and lowest activity in the hypothalamus and medulla. The total activity in all areas was high, that of the hypothalamus and medulla being only about 15% lower than that of the cerebellum. A recent study by Johnson (1972a) measured the activity of aspartate aminotransferase in 20 areas of the nervous system. He found the activity of aspartate aminotransferase to be highest in central gray areas, lower in the medulla, and lowest in the dorsal and ventral roots of the spinal cord. Values in the central gray areas were about 2-fold higher than values

96

M. BENUCK AND A. LAJTHA

in white tissues, such as corpus callosum or the cerebral peduncle. Activity levels in the spinal cord were about one-fifth those in brain. Levels in the gray tissues of the brain did not vary greatly. The study of Johnson found aspartate aminotransferase activity to be higher in neurons than in glia. Rose (1968) measured the activity of this enzyme in neuronal and glial tissue, and also found that the neurons contained about twice as much activity as did the glial cells. The subcellular distribution of this enzyme also differed, as indicated by the enzyme distribution obtained in the presence and in the absence of the detergent Triton X-100. I n the absence of detergent, activity was similar in both types of tissue. However, with detergent, where activity in the particulate material was solubilized, higher levels were found in neuronal tissue. Johnson (1972a) found that the activity of alanine aminotransferase was the same in all the central gray areas, and only slightly lower in white tissue. In white tissue there was a small increase in activity passing from peripheral white to central white matter. The activity difference between the central gray areas and the tissues with lowest activity (dorsal and ventral root) was about 20%. Benuck et al. (1972) found a much higher activity in the cortical gray matter than either the midbrain or corpus callosum. Johnson (1972a) attempted to correlate enzyme activity with amino acid levels, in view of the possible role of aminotransferases in the regulation of glutamate or GABA transmitter pools in brain (Johnson, 1972b). However, the activities of these enzymes did not parallel the differences found in the levels of glutamic acid. Graham and Aprison (1969) also found that the levels of aspartate aminotransferase in the dorsal and ventral roots and in the gray and white tissue of the spinal cord did not correlate with the differences found in levels of free glutamic acid. Other studies of the distribution of this enzyme include that of Papadimitriou and Van Duijn (1970), who found enzymic activity associated with the surface of the neuronal perikarya, dendrites, periphery of myelin sheaths, and in the synaptic area and mitochondria. The distribution of this enzyme in the hippocampal region of the guinea pig has also been reported (Borre and Geneser-Jensen, 1972). However, in both these studies little correlation of activity to structure was made.

C. y-AMMINORUTYRIC ACID:~-KETOCLUTARATE AMINOTRANSFERASE (EC 2.6.1.19) Substantial evidence has accumulated concerning the metabolic and inhibitory role of GABA in neutral tissue (Roberts and Kuriyama, 1968; Krnjevic' and Schwartz, 1967; Baxter, 1970). Its formation from glutamate and its subsequent metabolism have been extensively studied. The enzymes

AMINOTRANSFERASE ACTIVITY IN BRAIN

97

involved, glutamic acid decarboxylase (EC 4.1.1.15) and GABA aminotransferase (EC 2.6.1.19), have been highly purified and well characterized (Schousboe et al., 1973; Wu et al., 1973). The aminotransferase and the decarboxylase form part of the “GABA shunt” in brain, whose turnover rate is estimated to be about 10% of that of the citric acid cycle (Balks et al., 1970). The “GABA shunt’’ represents an alternate pathway for the portion of the tricarboxylic acid cycle between a-ketoglutarate and succinate. 1. Physical and Chemical Properties The presence of this enzyme in brain and liver preparations was initially described by Bessman et al. (1953) and Roberts and Bregoff (1953). The enzyme reaction is reversible in vitro. However, succinic semialdehyde is rapidly oxidized to succinate, so that the reaction proceeds rapidly in the direction of GABA catabolism. Properties of GABA aminotransferase from brain were initially described by Baxter and Roberts (1958). The enzyme has been purified from the brain of the rat and the pig 150-400-fold over the original homogenate (Waksman and Roberts, 1965; Sytinsky and Vasilijev, 1970; Eremin et al., 1972). Recently, it has been purified over 1200fold from the brain of the mouse (Schousboe et al., 1973). Its molecular weight is about 109,000. In common with other aminotransferases, pyridoxal phosphate is required for a cofactor. Its pH optimum is between pH 8.0 and p H 8.1. The Michaelis constants for the enzyme from mouse brain were estimated to be 1.1 m M for GABA and 0.25 mM for a-ketoglutarate. Previous reports had given larger values for both substrates, ranging from 3-12 mM for GABA and 2-5 mM for a-ketoglutarate.

2. Substrate Specificity Of over 20 amino acids tested using purified enzyme from mouse brain, no a-amino acid gave activity. Only p-alanine was transaminated to the same extent as GABA. P-Aminoisobutyric acid and a-aminovaleric acid were about 50% as effective as GABA. A number of other GABA analogs are transaminated by brain tissue, but not necessarily by GABA aminotransferase (Beart and Johnston, 1973). 3. GABA-T Inhibitors Hydroxylamine, cycloserine, and aminooxyacetic acid are inhibitors of GABA aminotransferase as well as other aminotransferases (Wallach, 1961; Hopper and Segal, 1964; Dann and Carter, 1964). Among other inhibitors are substrate analogs which inhibit the enzyme in vitro, but not in vivo (Baxter and Roberts, 1961 ; Carvajal et al., 1964) ; compounds with a hydrazine structure, including isonicotinic acid hydrazide, y-glutamylhydrazine, and

98

M. BENUCK AND A. LAJTHA

phenylpropylhydrazine (Medina, 1963; Massieu et al., 1964; Wood and Peesker, 1972; Popov and Matthies, 1969;Perry and Hansen, 1973).

4. Isozymes Schousboe et al. (1973) found their purified preparations to be homogeneous as indicated by a single band on polyacrylamide gel electrophoresis. Previous workers, however, have found that the enzyme can be separated electrophoretically into at least four isozymic forms. These isozymes were found to have different pH optima, as well as a different subcellular distribution (Waksman and Bloch, 1968). Although GABA aminotransferase is a particulate enzyme, activity of the order of 10-20% of the total was reported in the soluble supernatant. The cationic isozymes were present in higher concentration in the mitochondrial compartment, while the anionic forms were present in higher concentration in the soluble compartment.

5 . Regional and Subcellular Localization in Brain GABA aminotransferase is localized in the mitochondria1 compartment of the cell (Salganicoff and De Robertis, 1963; van Kempen et al., 1965; Waksman and Rendon, 1968).The mitochondria of the postsynaptic region ( neuronal, dendritic, glial, and endothelial mitochondria) contain about 80% of the total activity, whereas the mitochondria of the presynaptic region contain about 20% of the total activity. Large regional variations in GABA-T activity have been found in brain, with activity concentrated in areas of gray matter. Activity in white matter is very low (Salvador and Albers, 1959). In the gray matter of monkey brain, GABA-T activity levels vary over a %-fold range (Salvador and Albers, 1959; Fahn and C M , 1968). In mouse brain, GABA-T activity is highest in the midbrain colliculi, and decreases in activity in the following order : medulla, cerebellum, cortex, spinal cord, pons, diencephalon, and olfactory lobe (Waksman et al., 1968). In rabbit cerebellum, activity is highest in structures related to inhibitory function (Kuriyama et al., 1966). I n the lobster, GABA-T activity is associated with inhibitory axons rather than excitatory axons (Hall et al., 1970).Other studies, such as those of Pitts et al. (1965)and Sheridan et al. (1967)indicate similar distribution of activity in cortical tissue of rat, mouse, guinea pig, and rabbit, and in human brain. Peripheral nerve contains little or no activity (Salvador and Albers, 1959;Benuck et al., 1972).Spinal cord contains low activity, concentrated in areas of gray matter (Salvador and Albers, 1959; Graham and Aprison, 1969). The activity of GABA aminotransferase is apparently not limited by the concentration of GABA, since GABA levels in various brain areas are higher than the relatively low K , value for GABA reported by Schousboe et al.

AMINOTRANSFERASE ACTIVITY I N BRAIN

99

( 1973). However, the concentration of a-ketoglutarate is somewhat lower than its K , value, and Schousboe et al. (1973) suggest that this keto acid may play a role in regulation of GABA levels in brain, especially in light of the finding that this keto acid is a strong inhibitor of glutamate decarboxylase (Wu, 1972). The regional variation in GABA levels does not coincide with the activity of the aminotransferase. Rather, GABA distribution parallels the activity of its synthesizing enzyme, glutamate decarboxylase (Sisken et al., 1961; Fahn and C8t6, 1968). The metabolic turnover of GABA is also related to the activity of glutamate decarboxylase rather than the aminotransferase (Collins, 1972). The subcellular localization of GABA differs from that of GABA aminotransferase; GABA is present throughout the neuron (Mangan and Whittaker, 1966) whereas the aminotransferase is localized in the mitochondria of the postsynaptic region (Salganicoff and De Robertis, 1963; Waksman and Rendon, 1968). As noted by Salganicoff and De Robertis ( 1963), such localization is suited for the degradation of GABA after crossing the synaptic cleft.

D. BRANCHED-CHAIN AMINOACID:a-KETOGLUTARATE AMINOTRANSFERASE (EC 2.6.1.42) The branched-chain aminotransferase catalyzes the transfer of amino groups from leucine, valine, and isoleucine to a-ketoglutarate and to the corresponding a-keto acids of the branched-chain amino acids. This enzyme exhibits its highest activity in kidney tissue, followed by skeletal and cardiac muscle tissue. Low activity is present in liver (Ichihara and Koyama, 1966; Mimura et al., 1968) (Table I ) . Activity in brain appears to be of the same order as that of alanine aminotransferase and GABA aminotransferase (Benuck et al., 1971). 1. Physical and Chemical Properties The soluble enzyme has been extensively purified from pig heart muscle (Taylor and Jenkins, 1966; Ichihara and Koyama, 1966). Its molecular weight is estimated at about 75,000, with 1 mole of pyridoxal phosphate per mole of enzyme protein. It has a sharp pH activity curve with optimum activity at p H 8.3. The enzyme is also found in the mitochondria1 fraction of the cell from pig heart muscle (Aki et al., 1967). Its physical properties are similar to those of the soluble enzyme. 2. Substrate Specificity and Inhibitors The major substrates for the soluble enzyme are the branched-chain amino acids (and glutamic acid) (Tables I11 and I V ) . Related compounds,

100

M. BENUCK AND A. LAJTHA

such as alloisoleucine and norvaline, are also transaminated by the enzyme. a-Ketoglutarate and the corresponding a-keto acids of leucine, valine, and isoleucine are the major keto acid substrates. Oxaloacetate and pyruvate may also serve as substrates. The enzyme will also react with other amino acids, including a-aminobutyrate, norleucine, methylcysteine, a-aminoadipate, a-aminopimelate, allylglycine, and methionine. The K , for leucine and a-ketoglutarate is about 5 mM. Like other aminotransferases, the enzyme is sensitive to sulfhydryl agents and inhibited by carboxylic acid substrate analogs and a variety of carbonyl agents. The mitochondrial enzyme has somewhat different substrate specificity. Norvaline and norleucine are less effective substrates for the mitochondrial isozyme. a-Ketoglutarate is the preferred keto acid acceptor for this isozyme ; however, neither oxaloacetate nor pyruvate are active as substrates. The K,,, values for the major substrates also differ; values of 1.3 mM, 0.4 mM, 0.4 mM, and 2.2 m M have been obtained for valine, leucine, isoleucine, and a-ketoglutarate, respectively. A summary of the physical and chemical properties of these enzymes and methods for their purification are given by Jenkins and Taylor ( 1970) and Aki and Ichihara ( 1970a,b).

3. Leucine Aminotransferase ( E C 2.6.1.6) Rat liver contains the branched-chain aminotransferase as well as a leucine aminotransferase located in the mitochondrial fraction of the cell (Aki and Ichihara, 1 9 7 0 ~ ;Aki et al., 1968). This enzyme is specific for leucine, has been found only in rat liver, and has a K , value for leucine of 25 mM. 4. Brain Enzyme

Aki et al. (1969) have recently purified the branched-chain aminotransferase from hog brain supernatant. Its properties generally are similar to those of the pig heart soluble enzyme. The molecular weight is about 39,000, one half that of the heart enzyme. The pH activity curve is broad, with optimum activity at pH 8.0. Its activity is limited to that of the branchedchain amino acids, glutamate, and the respective keto acids. It also reacts to a smaller extent with norleucine and norvaline. a-Ketoglutarate is the major keto acid acceptor; activity with oxaloacetate is less than 20% of the activity with a-ketoglutarate. The K, values are 0.56 mM, 1.4 mM, 0.67 mM, and 0.57 m M for leucine, valine, isoleucine, and a-ketoglutarate, respectively.

5. Regional and Subcellular Localization The enzyme in rat brain is associated primarily with the supernatant fraction of the cell; one-third of the total activity is associated with the

AMINOTRANSFERASE ACTIVITY I N BRAIN

101

mitochondria (Benuck et al., 1972). It is found in highest concentration in the midbrain area, with lower activity in cortical gray matter and little activity in the corpus callosum (Benuck et al., 1972).

6 . Isozymes The enzyme found in hog heart supernatant ( I ) and that found in brain supernatant (111) can be differentiated in terms of their elution pattern upon chromatography on DEAE-cellulose. The heart soluble enzyme is eluted with 0.02 M phosphate buffer, while the brain soluble enzyme is eluted with 0.2 M buffer. The brain supernatant contains only form (111) while the brain particulate fraction contains both ( I ) and (111). Hog heart supernatant contains only form ( I ) while both kidney and liver supernatant contain both ( I ) and (111), the latter present in greater amount. Tissues from rat, on the other hand, contain only form ( I ) , except for rat liver, which also contains the leucine specific enzyme ( 1 1 ) , and rat brain, which contains (111) and a trace of ( I ) .

E. TRANSAMINATION OF GLUTAMINE AND ASPARAGINE Separate aminotransferases catalyze the reversible transfer of the a-amino group of glutamine or asparagine to an a-keto acid, forming the corresponding L-amino acid and a-ketoglutaramate or a-ketosuccinamate (Meister and Tice, 1950; Meister et al., 1952; Meister and Fraser, 1954). Since the reaction is combined with deamination in vivo, it is essentially irreversible-with a-ketoglutaramic acid converted to a-ketoglutarate and a-ketosuccinamic acid converted to oxaloacetate. Both enzymes have wide substrate specificity. They are active with a large number of a-keto acids, as well as several amino acids. For example, glyoxylic acid, a-ketoglutarate, or pyruvate may serve as keto acid substrates, and leucine, methionine, histidine, and phenylalanine may serve as amino acid substrate. For summaries of earlier work done, the reader is referred to Meister (1962, 1965) or Cooper and Meister (1972). 1. Glutamine Aminotransferase (EC 2.6.1.15)

Recently, Cooper and Meister ( 1972) have purified glutamine aminotransferase from rat liver 900-fold over the original homogenate. The enzyme was free of w-amidase, aspartate aminotransferase, and alanine aminotransferase activities, contaminations which complicated earlier studies of the enzyme (e.g., Braunstein and T'ing Sen, 1960). The enzyme has a molecular weight of about 111,000 and consists of two subunits of molecular weight of about 54,000. Associated with the enzyme are 2 moles of pyridoxal phosphate tightly bound to 1 mole of enzyme.

102

M. BENUCK A N D A. LAJTHA

As previous studies indicated, the enzyme has a wide substrate specificity. Of the amino acids tested, glutamine is the most active substrate. However, substantial activity was observed with analogs of glutamine, methionine, and several amino acids related to methionine. The most active a-keto acid substrates are a-ketoglutaramate, a-keto-y-methiolbutyrate, P-mercaptopyruvate, glyoxylate, pyruvate, and p-hydroxypyruvate, Cooper and Meister ( 1973) have recently isolated glutamine aminotransferase from the kidney of the rat. The kidney enzyme has a similar molecular weight as the liver enzyme, consisting of two subunits with tightly bound pyridoxal phosphate. However, it differs from the liver enzyme in some of its physical properties, as well as its substrate specificity. T h e kidney enzyme has a narrow substrate specificity when compared to the liver enzyme; of the amino acids tested, only phenylalanine, tyrosine, and methionine could replace glutamine. The specificity toward a-keto acid substrates is broad, although markedly different from that of the liver enzyme. The best a-keto acid substrates are phenylpyruvate and a-keto-y-methiol butyrate. Glyoxylate is a poor substrate. When phenylpyruvate is used as keto acid substrate, highest activity of the enzyme is found in kidney (Kupchick and Knox, 1970). However, if glyoxylate is used as keto acid substrate, activity of the kidney enzyme is only about 10% of the liver enzyme (Cooper and Meister, 1973). 2. Brain Enzyme Little work has been done with the brain glutamine or asparagine aminotransferase. Glutamine aminotransferase activity has been reported in brain. DAbramo and Tomasos (1959) and Guha et al. (1958) found the activity to be associated with the mitochondrial fraction. The liver enzyme, on the other hand, is associated with the soluble fraction. Brain enzymic activity appears to be low when compared to that of the liver (Cooper and Meister, 1973) or the kidney (Kupchick and Knox, 1970). Benuck et al. (197 1, 1972) found that brain preparations would catalyze aminotransfer reactions from both asparagine and glutamine. Aminotransfer from glutamine was associated with mitochondrial preparations, as previously reported. However, aminotransfer from asparagine was associated with both the mitochondria and the soluble supernatant. Glutamine aminotransferase activity was most clearly demonstrated with glyoxylic acid as the keto acid substrate, similar to the liver enzyme. However, transamination of asparagine proceeded best with a-ketoglutarate or oxaloacetate as the a-keto acid substrate. Glyoxylic acid was not a substrate for asparagine aminotransferase (EC 2.6.1.14) (Benuck et al., 1971). The products produced by glutamine aminotransferase, notably serine, alanine, or glycine, inhibit rat liver glutamine synthetase (Tate and Meister, 1971). This enzyme may participate in a regulatory mechanism, as sug-

AMINOTRANSPERASE ACTIVITY I N BRAIN

103

gested by Cooper and Meister (1972), in which the products of transamination inhibit the synthesis of glutamine. Further, the broad keto acid substrate specificity may provide a mechanism for amination of a-keto acids, conserving their carbon chains and providing amino acids for protein synthesis (Cooper and Meister, 1972). F. THEAROMATICAMINOTRANSFERASES 1. Purification

Early studies indicated that the aromatic amino acids are transaminated

by more than one enzyme (Lin, et al., 1958; Lin and Knox, 1958). Tyrosine :a-ketoglutarate aminotransferase (EC 2.6.1.5) from the liver of dog and rat was the first of these enzymes to be purified (Canellakis and Cohen, 1956; Kenney, 1959). An enzyme from rat liver catalyzing the transamination of tryptophan and 5-hydroxytryptophan was partially punfied (Sandler et al., 1960; Spencer and Zancheck, 1960). Other aromatic aminotransferases include phenylalanine :pyruvate and phenylalanine :aketoglutarate aminotransferase (Fuller et al., 1972; Scandurra et al., 1967; George and Gabay, 1968) ; kynurenine aminotransferase (EC 2.6.1.7) (Ueno et al., 1963; Minatogawa et al., 1973) ; tryptophan aminotransferase (Wada et al., 1972) ; and histidine: pyruvate aminotransferase (Spolter and Baldridge, 1964). Of the various aromatic aminotransferases enumerated, the most intensively investigated has been tyrosine aminotransferase.

2. Tyrosine:a-Ketoglutarate Aminotransferase (EC 2.6.1 5 ) Activity of this enzyme occurs primarily in the liver, with lower levels in other tissues (Table I ) . Tyrosine aminotransferase activity in brain is about 2-5% of liver (Wurtman and Larin, 1968; Miller and Litwack, 1969; Zigmond and Wilson, 1973). The enzyme from liver has been purified by a number of authors (Jacoby and La Du, 1964; Hayashi et al., 1967; Granner et al., 1968; Valeriote et al., 1969). Valeriote et al. (1969) have achieved an enrichment of enzyme protein of 2500-fold over the original liver homogenate. The molecular weight is estimated to be about 115,000, with 4 moles of pyridoxal phosphate bound to 1 mole of enzyme protein. Its amino acid analysis is given in Table 11. Optimal activity is near pH 7.5. For a summary of methods for its purification and its properties, the reader is referred to Granner and Tomkins ( 1970). 3. Substrate Specificity Early studies indicated that this enzyme had a narrow substrate specificity, limited to tyrosine and a-ketoglutarate (Kenney, 1959). However, Ja-

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M. BENUCK A N D A. LAJTHA

coby and La D u (1964) found broader specificity for the enzyme with respect to the amino acid substrate. Besides tyrosine and monoiodotyrosine, dopa, phenylalanine, and tryptophan are transaminated by the enzyme, activity with the latter two amino acids being about 8.4% and 1.8% of that of tyrosine, respectively. Apparent Michaelis constants are 1.5 mM, 3.0 mM, and 8.0 m M for tyrosine, tryptophan, and phenylalanine, respectively. Hayashi et al. (1967) give K, values for tyrosine and a-ketoglutarate of 1.4 mM and 0.7 mM, respectively (Tables I11 and IV) Among the inhibitors of the enzyme are analogs of tyrosine, phenylalanine, and tryptophan. A variety of catechol compounds, including norepinephrine, 2,3-dihydroxyphenylamine, dopa, and dopamine, are effective competitive inhibitors of this enzyme in vitro (Jacoby and La Du, 1964; Black and Axelrod, 1969).

.

4. Subcellular Localization Tyrosine aminotransferase, as well as many of the other aromatic aminotransferases of the liver enumerated above, is localized primarily in the cytoplasmic portion of the cell. However, substantial activity has been found in the mitochondria-as much as one-third of the total activity (Rowsell et al., 1963; Fellman et al., 1969; Miller and Litwack, 1971). The mitochondrial enzyme has a broader substrate specificity than the soluble form with respect to both amino acid and keto acid substrates (Miller and Litwack, 1971; Tong et al., 1973). The liver enzyme was purified over 900-fold over the original homogenate by Miller and Litwack ( 1971), who suggested that the mitochondrial isozyme of tyrosine aminotransferase is not a separate entity, but that its activity is a result of the activity of the mitochondrial isozymes of aspartate and alanine aminotransferase. Evidence is based on substrate specificity; aspartate is its best substrate and its activity toward tyrosine is blocked by aspartate. Further, this enzyme can be precipitated by antibodies specific for aspartate aminotransferase. Of the other aromatic aminotransferases, kynurenine aminotransferase has been purified from liver, kidney, small intestine, and brain of the rat (Ueno et al., 1963; Minatogawa et al., 1973; Noguchi et al., 1973). Tryptophan aminotransferase has been purified from rat liver (Wada et al., 1972). Enzymic activity is associated with a specific tryptophan aminotransferase which is induced after the administration of tryptophan or cortisol. A similar enzyme is also present in heart and kidney. The presence of other aromatic aminotransferases in mammalian tissue has beeh inferred from induction studies similar to the early work of Lin and Knox (1958) or the recent study of Wada et al. (1972). For example, Fuller et al. (1972) have suggested that phenylalanine :pyruvate aminotransferase is a distinct enzyme, and is not attributable to the activity of other aminotransferases (e.g., Civen et al., 1967). Their suggestion is based on the different induction patterns

AMINOTIUNSFEIUSE ACTIVITY IN BRAIN

105

of the enzyme after glucagon injection or food deprivation. Induction patterns after injection with glucagon also suggest that phenylalanine :a-ketoglutarate aminotransferase and tyrosine aminotransferase of liver are two separate enzymes.

5 . Brain Aromatic Aminotransferases One of the first reports of transamination of aromatic amino acids in brain was that of Canellakis and Cohen (1956), who found that tyrosine could be transaminated by crude tissue extracts of dog brain. Haavaldsen (1962) found that extracts of rat brain could transaminate phenylalanine, tyrosine, tryptophan, dopa, and 5-hydroxytryptophan. Partial fractionation by acetone precipitation and heat treatment indicated the presence of three different aminotransferases that react with the aromatic amino acids in brain (Fonnum et al., 1964; Tangen et al., 1965). The three aminotransferases were purified 25-100-fold, and have greatest activity between pH 8.1 and pH 8.2. Aminotransferase I has a high affinity for dopa, and utilizes a-ketoglutarate as the keto acid substrate. Aminotransferase I1 has affinity for both phenylalanine and tyrosine, and utilizes a-ketoglutarate as the preferred keto acid substrate. Aminotransferase I11 has high affinity for tryptophan and 5-hydroxytryptophan, and utilizes oxaloacetate as the preferred keto acid substrate (Table 111). Fonnum and Larsen (1965) purified dopa aminotransferase (EC 2.6.1.49) from guinea pig brain 38-fold over the crude homogenate. The enzyme is specific for dopa as the amino acid substrate-with little or no activity toward tryosine and phenylalanine. The apparent Michaelis constant for dopa and a-ketoglutarate is 41 mM and 0.48 mM, respectively. This contrasts with the liver aminotransferase where tyrosine aminotransferase has a high affinity for dopa (Jacoby and La Du, 1964). George and Gabay ( 1968) purified phenylalanine :a-ketoglutarate aminotransferase from pig brain approximately 900-fold over the crude homogenate. The enzyme has a broad substrate specificity, utilizing tyrosine, dopa, tryptophan, 5-hydroxytryptophan, and histidine as well as phenylalanine as substrates. Activity with the former substrates in relation to phenylalanine is 4976, 46%, 5l%, 62%, and 35%, respectively. Tyramine, dopamine, norepinephrine, and epinephrine have no effect on the transamination of tyrosine by this enzyme, even when tested at a final concentration of 1.25 mM. The apparent Michaelis constant for phenylalanine and a-ketoglutarate is 50 m M and 0.7 mM, respectively. That for tyrosine, dopa, and tryptophan is 3.8 mM, 6 mM, and 15 m M , respectively. High concentrations of a-ketoglutarate are inhibitory. George and Gabay suggested that a single enzyme catalyzes the transamination of tyrosine and phenylalanine, similar to aminotransferase 11. However, the two activities were partially

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M. BENUCK AND A. LAJTHA

separated by heat treatment. Upon heating a partially purified preparation for 10 minutes at 6OoC, 80% of the tyrosine activity remained, while only 60% of the phenylalanine activity was present. Recently, tyrosine aminotransferase from rat brain has been purified over 2500-fold from that of the original homogenate (Aunis et al., 1971; Mandel and Aunis, 1972). The molecular weight is about 100,000, and the enzyme can be subdivided into four units of molecular weight of 25,000 each. The brain enzyme, in contrast to the liver enzyme, contains two pyridoxal phosphate units tightly bound to 1 molecule of enzyme protein. I t also differs in amino acid content, containing substantially more proline than the liver enzyme, fewer of the acidic and more of the basic amino acids (Mandel, 1972). Tyrosine and monoiodotyrosine are the most active substances. The apparent Michaelis constants for tyrosine, monoiodotryosine, phenylalanine, and a-ketoglutarate are 20 mM, 11 mM, 8 mM, and 0.04 mM,respectively (Table IV) Inhibitors of the enzyme include products of tyrosine metabolism, such as homogentisate, fumarate, and maleate. The brain enzyme is inhibited by norepinephrine only at high concentrations (0.25 m M ) . Aminotransferase I11 may be two separate enzymes. Evidence has been obtained by Gabay and Huang (1969) that the administration of P,P’iminodipropionitrile, a psychotropic agent, led to a decrease in the specific activity of 5-hydroxytryptophan aminotransferase without affecting the transamination of tryptophan, tyrosine, or phenylalanine. Semba and Civen (1970) found that the enzyme which acts upon 5-hydroxytryptophan is highly concentrated in the synaptic vesicles, in contrast to the other aromatic aminotransferases of brain. Thus, tryptophan and 5-hydroxytryptophan in brain may be acted upon by two separate aminotransferases.

.

6. Subcellular Localization The brain aromatic aminotransferases differ from the liver enzymes in their subcellular localization. Brain tyrosine aminotransferase, as well as the other brain aromatic aminotransferases, are associated with the mitochondrial fraction (Benuck et al., 1972; Miller and Litwack, 1969; Mandel and Aunis, 1972; Semba and Civen, 1970; Mark et al., 1970). Liver tyrosine aminotransferase, in contrast, is present mainly in the soluble fraction of the cell. With respect to the mitochondrial location of these enzymes, Shrawder and Martinez-Carrion ( 1972) have suggested that brain phenylalanine aminotransferase is the same as the mitochondrial isozyme of aspartate aminotransferase. Evidence that the phenylalanine aminotransferase isolated by George and Gabay (1968) and the mitochondrial isozyme of aspartate aminotransferase are the same is based in part on the similar affinities of the two enzymes for the aromatic amino acids. Second, antibodies

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107

against the heart isozyme of aspartate aminotransferase inhibit the brain enzyme with similar decrease in both aspartate and phenylalanine aminotransferase activity. The brain enzyme also has the same mobility as the heart mitochondrial isozyme on starch gel electrophoresis. This suggestion is similar to that of Miller and Litwack (1971) with respect to liver mitochondria1 tyrosine aminotransferases. 7. Regional Distribution in Brain The distribution of the aromatic aminotransferases appears to be relatively uniform in most areas of the brain. Oja (1968) found their activity to be higher by about 20-300/0 in the rat cerebellum than in other brain areas (cortex, brain stem, interior cerebrum) . Transamination of histidine, although very low in all areas studied, was highest in the cerebral cortex. Miller and Litwack (1969), Mark et al. (1970), and Vogel et al. (1973) have found a relatively uniform distribution of tyrosine aminotransferase activity in most areas of the brain. Webb and Gibb (1970) in contrast found a substantially higher activity of tyrosine aminotransferase in the pineal gland, followed by the caudate nucleus and the superior colliculus in the sheep brain.

G. ORNITHINE :WKETOGLUTARATE AMINOTRANSFERASE (EC 2.6.1.13 ) This enzyme catalyzes the conversion of ornithine and a-ketoglutarate to glutamic semialdehyde and glutamate. The reaction is almost irreversible, with an equilibrium constant of 73, due to the spontaneous cyclization of glutamic semialdehyde (Strecker, 1965). Highest activity is found in liver and kidney (Peraino and Pitot, 1963; Herzfeld and Knox, 1968). The enzyme has been highly purified from both of these tissues: 1000- to 10,000fold over the original homogenate. The molecular weight of the liver enzyme is estimated at 130,000 to 180,000 with 2 moles of pyridoxal phosphate per mole of enzyme (Peraino et al., 1969; Matsuzawa et al., 1968). That from pig kidney has a molecular weight of 248,000 with 4 moles of pyridoxal phosphate per mole of enzyme (Jenkins and Tsai, 1970; Sanada et al., 1970). The enzyme is active between pH 6 and pH 10, with optimal activity at p H 8.15 (Peraino, 1972). Despite the discrepancies in physical properties reported for the enzyme from kidney and liver, Sanada et al. (1970) found the chemical properties of the enzyme from kidney, liver, and small intestine to be similar, and they present evidence for a similar protein in all three tissues. Its amino acid composition is given in Table 11. The enzyme is specific for ornithine, with a-ketoglutarate as the best keto acid acceptor. Other keto acids, such as glyoxylate or pyruvate, serve as substrates for this enzyme (Strecker, 1965; Sanada et al., 1970). The

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M. BENUCK AND A. L AJ TH A

apparent Michaelis constant for ornithine is 2.8 mM; and for a-ketoglutarate, 0.28 mM. Among inhibitors of the enzyme are structurally related compounds such as canavanine and 8-aminovalerate. Cadaverine and putrescine were also tested as inhibitors of the compound, and at concentrations of 25 mM cause a 14% inhibition of the rat liver enzyme. All substrates of the reaction are inhibitory at certain concentrations (Strecker, 1965). Little work appears to have been done with the brain aminotransferase. Its presence in brain has been reported (Peraino and Pitot, 1963; Herzfeld and Knox, 1968), with activity in brain about 10% of that of liver. The enzyme in brain, as in liver and other tissues, is primarily mitochondrial. I t also appears to be similar to that of the liver in substrate specificity, for transamination from ornithine may occur using either glyoxylate, a-ketoglutarate, or pyruvate as keto acid acceptors (Benuck et al., 1971).

H. GLYCINE :(u-KETOGLUTARATE AMINOTRANSPERASE (EC 2.6.1.4) A number of aminotransferases, including asparagine, glutamine, and ornithine aminotransferase, are capable of converting glyoxylic acid to glycine. Nakada ( 1964) partially purified a specific glutamic-glycine aminotransferase from rat liver. Only liver, kidney, and spleen of the rat contained this enzyme in appreciable amounts. It appears to be specific for glutamate and glycine, and is irreversible, proceeding in the direction of glycine formation. An enzyme which transfers amino groups from alanine to glyoxylic acid has also been described in human liver (Thompson and Richardson, 1967; Richardson and Thompson, 1970). I n rat brain and the spinal cord of the cat, glutamate, alanine, and glutamine have been found to be active substrates for the enzymic production of glycine (Johnston and Valeria-Vitali, 1969; Johnston et al., 1970). Benuck et al. (1971) found that brain homogenates would catalyze the transfer of amino groups from ornithine, glutamic acid, glutamine, tyrosine, and alanine to glyoxylic acid. The reaction in brain, as in liver, is irreversible, proceeding in the direction of glycine formation.

I. PHOSPHOSERINE :~-KETOOLUTARATE AMINOTRANSFERASE (EC 2.6.1.52) Phosphoserine :a-ketoglutarate aminotransferase converts glutamate and 3-phosphohydroxpyruvate to serine-3-0-phosphate and a-ketoglutarate. The enzyme is present in tissues which possess the phosphorylated pathway of serine biosynthesis, including the liver, kidney, and brain of many vertebrate species (Walsh and Sallach, 1966). It is a major enzyme in the pathway for serine formation in the brain (Bridgers, 1965) and was purified from sheep brain 500-fold by Hirsch and Greenberg (1967). The molecular weight is estimated at about 96,000. The enzyme has a relatively sharp pH

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optimum, with maximal activity at p H 8.15. Glutamate is the major amino acid substrate; activity with alanine is only about 10% of the activity with glutamate.

TISSUE J: OTHERAMINOTRANSFERASE ACTIVITY I N NEURAL

To the authors’ knowledge, little is known concerning transamination of other amino acids, or amines, by neural tissue. Transfers from methionine have been reported (Albers et al., 1962; Benuck et al., 1971), which may be associated with the activity of glutamine aminotransferase (Cooper and .Meister, 1972). Other aminotransferases have been described in mammalian tissue, such as transamination of lysine in liver; histidine :pyruvate aminotransferase, and alanine :glyoxylate aminotransferase. However, possibly because of their low activity, if any, there is no evidence at present for the presence of these aminotransferases in neural tissue. Meister (1965) lists close to one hundred amino acids, amines, or their derivatives that undergo transamination. Among these are the transamination of cadaverine and putrescine by plants and bacteria. Although the latter substances are found in brain, no evidence of brain aminotransferase activity for these substances in the literature has been found by the authors. Cadaverine does inhibit ornithine aminotransferase of liver (Strecker, 1965), and similarly may inhibit brain ornithine aminotransferase. Recently, the aminotransferase which catalyzes the transamination of L-triiodothyronine has been purified from rabbit liver (Soffer et al., 1973). While kidney and liver had the highest total activity, some activity (about 1% that of the liver enzyme) was found in the brain soluble supernatant.

111. Regulation of Aminotransferase Activity

A. GLUCOSEMETABOLISM A N D AMINOTRANSFERASES I n brain, glucose metabolism is closely connected with the metabolism of amino acids, notably glutamate. Upon injection of labeled glucose into rats, a high rate of incorporation of the labeled carbon from glucose into brain amino acids and protein was observed. This phenomenon is fairly specific for brain since the labeling of amino acids such as glutamate is much lower in other tissues such as liver. Incorporation of labeled carbon from glucose and factors influencing their incorporation have been studied in detail (Vrba et al., 1962; Gaitonde et al., 1965; Vrba and Winter, 1972; Balbs and Haslam, 1965). Vrba et al. (1962) have proposed that the utilization of glucose by the brain proceeds largely through the synthesis and oxidation

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of amino acids such as glutamate, and that the glucose carbon is also used for the synthesis of proteins and lipids in the brain. However, as Balhs and Haslam (1965) suggest, the high labeling of glutamate and aspartate from glucose does not necessarily mean that these amino acids are on the main pathway of glucose oxidation in brain. A number of factors are involved which affect amino acid levels and metabolism in brain. The labeling of glutamate can be accounted for by the high rate of exchange transamination of glucose and a-ketoglutarate in brain (Albers and Jakoby, 1960). The rate of exchange transamination is of the same order in brain as in other tissues; however, other factors can account for the rapid labeling of amino acids in brain. Among these are the low glucose content in brain, its high content of glutamic acid, and the rate of glucose conversion to acetyl-CoA, which is more rapid in brain than in other tissues (Haslam and Krebs, 1963) . This indicates that even though the isotopic equilibrium between glucose and glutamate is more rapid in brain than in most other tissues, owing to rapid turnover and specific pool sizes, there is no justification to assume that the major pathways of glucose oxidation are different in brain as compared to other tissues.

RESULTING IN ALTEREDAMINOTRANSFERASE LEVELSI N B. CONDITIONS TISSUES The liver of the mammal is a major site of amino acid metabolism, as well as a major site for gluconeogenesis. Under conditions which lead to a high rate of amino acid degradation, many of the enzymes that catabolize amino acids increase in activity. The soluble isozymes of aspartate and alanine aminotransferase increase in activity after glucocorticoid injection, after the animal is fed a high protein diet, or if the animal is starved. The increase in activity is associated with enhanced gluconeogenesis, increased protein catabolism, and an increase in the amino acid pool of the liver (Rosen et al., 1959; Waldorf and Harper, 1963 ; Freedland et al., 1968). For a review, the reader is referred to Knox and Greengard (1965) and Sheid and Roth ( 1965). Two major functions have been assigned to the enzyme aspartate:aketoglutarate aminotransferase. The mitochondria1 isozyme is the key enzyme in the oxidation of glutamate (Balhs and Haslam, 1965). The mitochondrial and soluble isozymes together with malate dehydrogenase function as a shuttle system for the oxidation by the mitochondria of extramitochondrial NADH (Borst, 1963; Shrago and Lardy, 1966). This, for example, may provide reducing equivalents for extramitochondrial processes, such as gluconeogenesis in the liver. Other aminotransferases which are altered under various physiological conditions include the leucine-specific aminotransferase of rat liver, ornithine

111

AMINOTRANSPERASE ACTIVITY IN BRAIN

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aminotransferase, and the aromatic aminotransferases (Table V) The leucine aminotransferase enzyme increases in activity after a high protein diet and after the injection of corticosteroids (Mimura et al., 1968; Ichihara and Koyama, 1966; Shirai, 1970). In contrast, the branched-chain aminotransferase, found predominantly in muscle tissue, is not altered after a high protein diet. Ornithine aminotransferase of rat liver is induced upon force feeding a casein hydrolyzate, or mixture of amino acids including arginine and lysine, to protein-depleted rats (Peraino et al., 1965). Administration of cortisone, if accompanied by a high protein diet, does not induce the enzyme. I n fact cortisone administration inhibits dietary stimulation of ornithine aminotransferase. The activity of this enzyme in liver and kidney is affected in different ways by the addition or omission of various amino acids to the diet. For example, the liver enzyme is decreased by addition of arginine to the diet, and increased in arginine deficiency. The kidney enzyme is increased by the addition of glutamate to the diet. Volpe et al. (1969) suggest that this enzyme functions in the liver toward the synthesis TABLE V ON INFLUENCES ILLUSTRATIVE LIST OF HORMONAL AND DIETARY AHINOTRANSPERASE ACTIVITYIN THE LIVEROF THE ADULTRAT Enzyme Aspartate aminotransferase Alanine aminotransferase Leucine aminotransferase Ornithine aminotransferase

Phenylalanine: pyruvate aminotransferase Tryptophan aminotransferase Tyrosine aminotransferase

Stimulus High protein diet Hydrocortisone High protein diet Cortisol Cortisol High protein diet High protein diet Glucagon Dietary amino acids Glucagon

Inhibitor

Referencea

Estradiol

1,2

Growth hormone

3.4 5,6

Glucose Cortisone

Cortisol Growth hormone Hydrocortisone Giucagon Insulin Dietary amino acids High protein diet

7-9 10 11 12-17

Key to references; (1) Rosen ct al. (1959); (2) Herzfeld and Greengard (1971); (3) Hopper and Segal (1964); (4)Swickcfal. (1965);(5)Ichiharacf al. (1967);(6)Shirai (1970); (7) Peraino ef al. (1965);(8)Peraino (1968);(9)Peraino and Pitot (1964); (10) Fuller c# al. (1972); (11) Wada et al. '(1972); (12) Lin and Knox (1958); (13) Holten and Kenney (1967);(14) Hager and Kenney (1968);(15) Kenney (196713);(16)Kenney (1970); (17) Matsutaka cf al. (1971).

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of arginine, and functions in the kidney toward the synthesis of glutamate. One common response of the liver and kidney enzyme is a significant increase in activity in response to dietary methionine. A number of the aromatic aminotransferases increase in activity after administration of corticosteroids or other hormones. Lin and Knox (1958) first observed the response of these enzymes to corticosteroids. Tryptophan aminotransferase is induced by its substrate and by cortisone injection (Wada et al., 1972) and phenyla1anine:pyruvate aminotransferase is induced by glucagon (Fuller et al., 1972). One aromatic aminotransferase whose regulation has been extensively studied is the tyrosine aminotransferase of rat liver. The enzyme is easily induced and has a rapid turnover, with a half-life under 2 hours (Kenney, 1967a). Many workers have used this enzyme as a tool in the study of the regulation of protein synthesis and degradation. These studies are outside the scope of this article, and for a review the reader is referred to Kenney (1970). Lin and Knox (1958) were the first to report induction of this enzyme after glucocorticoid injection. The liver enzyme is induced by glucocorticoids, glucagon, and insulin (Greengard and Baker, 1966; Holten and Kenney, 1967). It is suppressed by growth hormone (Kenney, 1967b). Its substrate tyrosine, as well as the catecholamines, also induces liver tyrosine aminotransferase (Bartholini et al., 1970; Fuller and Snoddy, 1968; Black and Axelrod, 1968, 1969). Liver tyrosine aminotransferase shows a 4-fold change in activity over a 24-hour period. The diurnal variation in activity has been related to a number of factors, including fluctuations in corticosteroid levels (Govier et al., 1969), food intake of the animal (Fuller and Snoddy, 1968; Wurtman, 1970), and levels of norepinephrine or acetylcholine (Black and Axelrod, 1968; Black and Reis, 1971). Fuller and Snoddy (1968) were able to change the daily rhythm of tyrosine aminotransferase by changing the time of day when food is made available to the rat. Wurtman et al. (1968) suggested that it is the cyclic delivery to the liver of dietary amino acids which is a major factor in the generation of the diurnal cycle. Black and Axelrod (1969) have proposed that norepinephrine, by competing with the apoenzyme of tyrosine aminotransferase for its cofactor, plays a role in generation of the diurnal rhythm. Black and Reis (1971) further suggested that the daily variation in activity is regulated by cholinergic mechanisms as well as variations in levels of norepinephrine. Hardeland (1973) has suggested that the diurnal variation in tyrosine aminotransferase activity is due to a number of factors, each of which is effective at different points of the daily cycle. During the greater portion of the light period induction of the enzyme by corticosteroids or dietary

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amino acids (tryptophan) is suppressed, possibly caused by the inhibitory action of the sympathetic nervous system. At the end of the light period, when norepinephrine is secreted at a lower rate, corticosteroids and tryptophan become highly effective in inducing the enzyme. OF CEREBRAL AMMINOTRANSFERASE C. STABILITY

Whereas the aminotransferases of liver are affected by hormonal changes, dietary protein intake, and amino acid levels, aminotransferase activity in adult brain appears to be rather stable. Benuck and Lajtha (1974) fed to mice diets enriched with either methionine, valine, or lysine. While such diets would increase the activity of ornithine aminotransferase, alanine or aspartate aminotransferase of liver, no effect was found on the activity of these aminotransferases, nor of the branched-chain aminotransferases in brain. Wada et al. (1972) and Fuller et al. (1972) both observed induction by glucagon or by the substrate tryptophan on phenylalanine :pyruvate aminotransferase and tryptophan aminotransferase, respectively, in liver. However, no comparable effect was found in brain. Rosen et al. (1958) found that while alanine aminotransferase in liver is increased 2-%fold after cortisone treatment, there is less than a 20% increase in brain alanine aminotransferase activity. Likewise, Jacobsohn et al. (1965) found that after hypophysectomy marked changes in alanine and aspartate aminotransferase activity occur in tissues besides brain. Brain tyrosine aminotransferase does not undergo diurnal variation, nor is it induced by glucocorticoids or injection of catecholamine depleting agents (Fuller, 1970; Zigmond and Wilson, 1973). Kaplan and Pitot (1970) indicated that the affinity of amino acids for a number of the enzymes involved in their degradation is relatively low. The aminotransferases and other enzymes involved in the degradative process have K , values generally in the range of 1-10 mM. They suggested that in general amino acids tend to be conserved for protein synthesis rather than degraded, except when the amount of amino acid becomes excessive and potentially toxic to the organism. For example, Wurtman and Fernstrom (1972) suggested that the transamination pathway for tyrosine serves as a shunt for removal of excess and potentially toxic levels of the amino acid during periods of high food intake. The brain free amino acid pool remains fairly constant under normal physiological conditions. Thus, there would not be the same stimuli in brain to induce enzymic activity as in the liver. This plus the low affinity for a number of aminotransferases may account for the stability of enzymes in the adult brain. However, other factors are likely to be involved, for even when the amino acid level is altered upon feeding diets enriched in methi-

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onine, lysine, or valine, no change in cerebral aminotransferase activity of the adult brain was found. Brain aminotransferases d o not appear to be influenced by the same factors as the liver aminotransferases. Changes in activity have been noted after long periods on a protein-deficient diet. Rajalakshmi and co-workers (1965, 1969) placed adult rats on a protein-deficient diet for 4-6 months. At the end of the experimental period the activities of aspartic and alanine aminotransferase were significantly lower than in the control animals. Similar changes were noted with other enzymes as well. The levels of glutamate, aspartate, and alanine were altered as well, indicating a possible correlation with aminotransferase activity and their amino acid substrates in brain.

D. COMPARTMENTATION I N NERVOUS TISSUE AND A MINOTRAN sPERASE ACTIVITY Although no direct correlation between amino acid levels and aminotransferase activity has been found in brain, compartmentation of the enzymes may make them rate limiting at specific sites. Graham and Aprison (1969) suggest that enzyme activities may be related to amino acid levels in various metabolic or functional compartments, such as an accumulation of glutamate and aspartate in certain areas of the spinal cord for synaptic transmission. As Salganicoff and De Robertis (1963, 1965) and Sellinger and Rucker ( 1963) have suggested, compartmentation of aminotransferases may affect the function Qf the enzymes in many different ways: (a) the availability of the enzymes, and thereby their function, is restricted; (b) restriction of the enzymes to particular compartments also may determine their function; (c) compartmentation of the enzymes and their substrates may result in separate metabolic functions for the different compartments. For a review on metabolic compartmentation, the reader is referred to the symposium edited by Balhs and Cremer (1973). To summarize briefly a few points, there are assumed to be two tricarboxylic acid cycles, associated with two different types of mitochondria. Associated with the two different mitochondria are two glutamate pools. One pool is for synthetic purposes, the other for energetic purposes. The two cycles are connected by GABA and glutamine. GABA aminotransferase and glutamate dehydrogenase are associated with one type of mitochondria, and GABA and ammonia are precursors of the “small” glutamate pool. GABA aminotransferase is associated with the synthetic cycle. Aspartate aminotransferase, on the other hand, is associated with the “energy” cycle. Johnson (1972b) suggests that the tricarboxylic acid cycle activity is partitioned relative to the metabolic and transmitter roles of glutamate. The two cycles have also been related to the function of GABA as a

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neurotransmitter (BalAzs and Cremer, 1973) . While GABA aminotransferase is associated with the synthetic pool, glutamate decarboxylase is associated with the “energy” pool. The site of GABA formation is different from the site of degradation (Balbs et al., 1970), which is in agreement with the association of the two enzymes with two different tricarboxylic acid cycles. Two different tricarboxylic acid cycles may thus be related to the role of GABA as a neurotransmitter, in that the amino acid is formed on the presynaptic site of the synapse and metabolized on the postsynaptic site (van den Berg, 1973).

E. POSSIBLE ROLEOF AMMINOTRANSFERASE IN NEUROTRANSMITTER METABOLISM Aspartate aminotransferase in brain functions at a branch point between the citric acid cycle and the “GABA shunt.” The latter pathway, found only in nervous tissue, catalyzes the formation of GABA from glutamate. Since it has been suggested that glutamate may act as an excitatory neurotransmitter agent while GABA acts as an inhibitory agent (KrnjeviL, 1970), this enzyme, as well as those of the “GABA shunt,” may be of importance in regulating the levels of these amino acids. However, as noted previously, no correlation has been found between levels of glutamate and aspartate and aminotransferase activity in brain (Johnson, 1972b). Nor are GABA levels correlated with aminotransferase activity. Indeed, it is the decarboxylase activity which parallels GABA levels in brain (Fahn and C8t6, 1968; Sisken et al., 1961). GABA aminotransferase also is most likely saturated by the amino acid in brain, because of its low K , value (Schousboe et al., 1973). Numerous studies and hypotheses have been made concerning the place of GABA, GAD, and GABA-T in the etiology of seizures (Lovell, 1971). Some theories implicate changes in GAD activity (Tapia and Pasantes, 1972) or a combination of GAD inhibition and increased GABA levels (Wood and Peesker, 1972). Correlations have been found between the rate of hydrazide-induced seizures in chicks, cerebral GABA content, and inhibition of GAD activity (Tapia and Awapara, 1967; Wood and Abrahams, 1971) . However, agents which specifically inhibit GABA-T activity, such as the in v i m injection of glutamic acid y-hydrazide do not affect seizure activity. This may again be related to distribution of GABA, which parallels that of its synthesizing enzyme GAD rather than GABA-T. Gibb and Webb (1969) have suggested the possibility in brain of a regulatory mechanism whereby low levels of catecholamines would repress aminotransferase activity, thereby making available more tyrosine for hydroxylation to replenish catecholamine levels. Their suggestion is based, in part,

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on the finding that reserpine injection would lead to a decrease in brain tyrosine aminotransferase activity. However, a recent study by Zigmond and Wilson (1973) failed to confirm their findings. They found that the enzyme is not altered by a drug-induced increase or decrease in brain norepinephrine content, nor is it induced by in uitro addition of norepinephrine. 6-Hydroxydopamine, reserpine, and a-methyltyrosine, deplete the brain of norepinephrine. In liver, a-methyltyrosine injection led to a 32% rise in tyrosine aminotransferase activity. No alteration in activity was observed in brain. Phenyprazine, an agent that increases brain norepinephrine (by inhibition of monoamine oxidase), led to a 33% decrease in liver enzyme activity. Again, brain enzyme activity was not altered. In vitro addition of M norepinephrine to the brain enzyme at concentrations from to had no effect on the brain enzyme. Only the high concentration of 250 p M led to a slight inhibition of the brain enzyme. Aunis et al. (1971) also found that purified brain tyrosine aminotransferase is competitively inhibited by norepinephrine only at very high concentrations (1.75 m M ) . In contrast, the liver enzyme is inhibited by norepinephrine both in uivo and in uitro. In uitro inhibition occurs over a concentration range of to lo-' M. Other related compounds which inhibit liver tyrosine aminotransferase include dopamine, dopa, and 2,3-dihydroxyphenylethylamine (Black and Axelrod, 1969). The aromatic keto acids are at least as effective as a-ketoglutarate in accepting amino groups from aromatic amino acids, when tested at low concentrations (Lees and Weiner, 1973; Benuck et al., 1971). In diseases such as phenylketonuria, the aromatic keto acids may increase in brain to levels that would compete with a-ketoglutarate. Lees and Weiner (1973) suggested that, in such cases, tryptophan aminotransferase could compete with tryptophan hydroxylase for tryptophan, and this could result in a decrease in serotonin. Millard and GPl ( 1971) have suggested that aminotransferase activity may be of importance in 5-hydroxyindole metabolism, providing another regulatory site for the synthesis of serotonin, as well as other 5-hydroxyindoles. They, and Fonnum et al. (1964), have found also that serotonin inhibits the transamination of 5-hydroxytryptophan.

Vitamin Be Deficiency and Aminotransferase Activity I n a number of tissues there is generally a linear relationship between tissue levels of vitamin B, and aminotransferase activity. For references to previous studies, the reader is referred to the articles of Brin and Thiele (1967) and Shiflett and Haskell (1969). The aminotransferases of adult brain are less affected by such diets than are the enzymes of other tissues. For example, Brin and Thiele (1967) found that varying the dietary intake

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of vitamin B, in rats did not affect aspartate aminotransferase activity of brain, and affected only slighly the activity of alanine aminotransferase. Activity in other tissues, such as liver, muscle, heart, and kidney, was significantly different from control animals. Shiflett and Haskell (1969) fed a vitamin Be-deficient diet to chickens, and found a significant decrease in the activity of the branched-chain aminotransferase in liver and heart after 4 days on the diet. In brain, lower levels were found only after 9 days on the diet. Bayoumi et ul. (1972) noted only a moderate change in GABA aminotransferase in adult rats on a vitamin B,-deficient diet, and glutamic decarboxylase activity was significantly decreased. In the immature animal, activity of both GABA aminotransferase and glutamic decarboxylase was significantly decreased (Bayoumi and Smith, 1972).

IV. Developmental Changes in Aminotransferase Activity

Most studies of enzyme development have concentrated on changes in enzymic activity in the liver. The pattern of development in this tissue has been divided into three stages: the late fetal, neonatal, and late suckling stages (Greengard, 1971). During fetal life, hormonal factors are the primary stimuli for enzyme growth. After birth, nutritional factors gain in importance. The supply of glucose is no longer constant for the organism, and enzymes associated with gluconeogenesis, including the aminotransferases, increase in the liver. After weaning, the diet given to the animal differs from the mother’s milk, in that the diet is high in carbohydrate and low in fat and protein. Again, the aminotransferases are among the enzymes which develop at this stage. Among the aminotransferases which increase in the liver during the late fetal stage are histidine :pyruvate aminotransferase and phenylalanine :pyruvate aminotransferase (Makoff and Baldridge, 1964; Auerbach and Waisman, 1959) . Aspartate aminotransferase, tyrosine aminotransferase, and alanine :glyoxylate aminotransferase increase during the neonatal stage (Yeung and Oliver, 1967; Greengard and Dewey, 1967; Sereni et ul., 1959; Rowsell et al., 1969). Ornithine aminotransferase and alanine aminotransferase increase during the late suckling stage (Raiha and Kekomaki, 1968; Henfeld and Knox, 1968; Yeung and Oliver, 1967). The developmental pattern of aminotransferase activity varies from tissue to tissue; since the enzymes of each tissue are responsive to different environmental stimuli. For example, Henfeld and Greengard ( 197 1) have measured the activity of aspartate aminotransferase during gestation and postnatal development in the liver, kidney, heart, and mammary gland of the rat. In the liver, both isozymes of aspartate aminotransferase begin to increase in activity shortly before birth; soon after birth there is a short

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period where values are higher than the adult-the values subseqilently decline to reach adult values at about the twelfth day after birth. The isozymes of the heart and kidney, on the other hand, are low during fetal life, and gradually increase to adult levels during the first 3-4 postnatal weeks. In the mammary gland of the mother, levels of both isozymes increase between day 21 of pregnancy and the second day after birth-the period coinciding with rapid growth of the gland. The soluble isozyme continues to increase during lactation, and decreases with involution. The liver enzyme can be stimulated by the administration of thyroxine 3 days before birth or by hydrocortisone 2 days before birth. Herzfeld and Greengard (1971) suggested that thyroxine is the essential stimulus for the formation of aspartate aminotransferase and may be necessary for the rapid response of the enzyme to hydrocortisone. Another example of such a response to hormonal stimuli is that of tyrosine aminotransferase, which is stimulated in fetal liver by glucagon, and becomes responsive to hydrocortisone only after birth (Greengard, 1969). Estradiol inhibits the development of liver aspartate aminotransferase and, in the adult, also inhibits the induction of the soluble enzyme by hydrocortisone. Similar inhibition by estradiol has been noted with ornithine aminotransferase of liver (Herzfeld and Greengard, 1969). The levels of aspartate aminotransferase in kidney, brain, or heart show no response to injections of hydrocortisone, thyroxine, or estradiol, in contrast to the liver enzyme. Injection of glucagon into fetal rats 2 days before birth stimulates mitochondrial alanine :glyoxylate aminotransferase and the soluble isozyme of alanine :a-ketoglutarate aminotransferase. I n contrast, the cytosol alanine :glyoxylate enzyme and the mitochondria1 alanine :a-ketoglutarate enzyme do not respond to glucagon injection, but are stimulated by corticosteroids. While glucagon and cortisol stimulate different isozymes, thyroxine induces all the above isozymes (Snell and Walker, 1972). The leucine-specific aminotransferase, which appears in the liver during the neonatal period, and then gradually increases in activity, is also elevated under various gluconeogenic conditions, and the stimulus for its induction is related in part to glucocorticoid secretion. The branched-chain aminotransferase, on the other hand, is not responsive to hormonal stimuli and is present at levels higher than that of the adult during the neonatal period, gradually decreasing during growth (Ichihara and Takahashi, 1968). Glucocorticoid treatment stimulates the development of ornithine aminotransferase of rat liver (Raiha and Kekomaki, 1968). The hormone, however, has no effect on the kidney enzyme. O n the other hand, estradiol stimulates kidney enzyme development, and inhibits growth of the liver enzyme (Greengard, 197 1 ; Herzfeld and Greengard, 1969) . Generally, while glucagon, thyroxine, and the glucocorticoids influence the development of the

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liver enzyme, they apparently do not affect kidney enzyme development (Greengard, 1971). The development of ornithine aminotransferase in kidney is associated with a sex difference. The level is higher in the female than in the male; the difference in activity is eliminated after ovariectomy. Estradiol, when given to intact female rats, induces a further increase in activity, while testosterone has no effect on the enzyme (Herzfeld and Knox, 1968). A specific time period for enzyme induction during development has been noted for a number of aminotransferases. For example, glucagon stimulates tyrosine aminotransferase of liver if given at birth, but not if given 3 days before birth. This has been correlated with an increase in liver cyclic AMP through which induction of glucagon may be mediated (Greengard, 1969, 1971). A similar pattern of induction has been noted for glucocorticoid stimulation of liver tyrosine aminotransferase (Holt and Oliver, 1968; Litwack and Nemeth, 1965) as well as the stimulation of cytoplasmic alanine aminotransferase of liver by cortisol and glucagon (Snell and Walker, 1972). The enzyme may be repressed during gestation by various factors, including the repressive action of hypophysial growth hormone (Kenney, 1967b). A number of enzymes thus appear to be influenced by hormonal factors during fetal life. After birth, the changing patterns of nutrition introduce additional influences and further development is related to both hormonal and nutritional factors. For recent articles on enzymic development, the reader is referred to Greengard (1971) and Snell and Walker (1972).

Aminotransferase Development in Brain Studies of enzyme development in brain have centered around the critical period of morphological and functional development of the nervous system. I n the rat and mouse this occurs at 10-20 days after birth. During this period there is a striking increase in many of biochemical and functional components of neural tissue, including changes in aminotransferase activity. A number of workers have reported an increase in the activity of alanine and aspartate aminotransferase in brain. Amore and Bonavita (1965) found the specific activity of these two enzymes to increase rapidly to adult levels between day 17 and day 20 after birth in rat brain. The increase was associated with a decrease in the pyridoxal form of the enzyme compared to the pyridoxamine form, and was attributed to the shift in rat brain during development from glycolysis to respiration. Waksman and Rendon ( 1968) found that the soluble isozyme of aspartate aminotransferase increases 5-fold in activity starting at about 8 days after birth. Pasquini et al. (1967) and Garcia Argiz et al. (1967) found an 8-fold increase in activity of the mitochondrial isozyme of aspartate aminotransferase in rat brain, with the higher rate of increase occurring between day 15 and day 20 after birth.

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GABA aminotransferase activity also increases sharply during this period. Bloch-Tardy et al. (1971 ) and Sims et al. (1968) found an increase of 10to 15-fold in the specific activity of the enzyme. Van den Berg et al. (1965) and van Kempen et al. (1965) measured the activity of this enzyme as well as glutamic decarboxylase in the developing rat brain and found a sharp increase in activity for both enzymes beginning with day 5 after birth. Other workers have also noted similar developmental patterns for GABA aminotransferase and glutamate decarboxylase (Kuriyama et al., 1968). The development of these enzymes was correlated with the development of recognizable synaptic structures in the chick embryo cerebellum. Sisken et al. (1961) also found that maturation of the GABA system in the optic lobe of the chick paralleled the increase in size of the optic tectum as well as the differentiation of the major tectal neuronal elements. I n contrast to studies with aspartate, alanine, or GABA aminotransferase, Oja (1968) found that activity of the aromatic aminotransferases increased in a continuous and relatively slow fashion, with no rapid enzyme induction found at any stage of development. An approximately 5-fold increase in specific activity was noted. Benuck et al. (1971) measured the activity of a number of aminotransferases in the brain of the newborn and adult rat. The highest increase in activity was observed with alanine aminotransferase. Some enzymes such as the branched-chain aminotransferase showed little increase in activity from birth to adulthood. Transamination of methionine was lower in the adult when compared to the newborn animal. A recent study by Macaione et al. (1973) has related the development of aspartate, alanine, and tyrosine aminotransferases to the functional and morphological differentiation of the retina. All three enzymes increased with age, although differences in the pattern of development was noted. Increase in aspartate aminotransferase was related to the maturation of the photoreceptors of the retina. Development of alanine aminotransferase was related to a shift in retinal metabolism from glycolysis to respiration. This is similar to the result of G6mez and Ramirez de Guglielmone (1967), who found a high conversion of glucose to alanine in the immature rat cortex when compared to adult tissue. Development of both tyrosine aminotransferase and alanine aminotransferase differed from that of aspartate aminotransferase, in that activity of the former two enzymes decreased 15 days after birth. Macaione et al. (1973) suggested that changes in tyrosine aminotransferase may affect the synthesis of dopamine, the inhibitory neurotransmitter of these neurons. Little is known concerning regulatory factors associated with enzymic development in brain. The importance of the thyroid hormone in neural development is well established, and the effects of this hormone on aminotransferase activity have been investigated (Pasquini et al., 1967; Garcia Argiz et al., 1967). Neonatal thyroidectomy produces a significant decrease

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in the mitochondria1 isozyme of aspartate aminotransferase in rat brain. The decrease is first apparent on day 20 after birth (where the greatest increase in activity in the normal animal is noted) and is sustained throughout the period studied. No effect is observed on the supernatant isozyme. Thyroidectomy also suppresses GABA-T activity, the change from normal again coinciding with the period where the normal enzyme begins to sharply increase in activity. If thyroidectomized animals were given triiodothyronine or growth hormone starting on day 10 after birth, the alterations produced by neonatal thyroidectomy were reversed. However, if treatment was delayed until day 15 after birth, there was no restoration to normal activity (Krawiec et al., 1969). These alterations in activity are not necessarily specific to the aminotransferases, but reflect the general effect of thyroidectomy on protein synthesis in the immature brain. Thyroidectomy does not affect all developmental patterns of enzymic activity. For example, the supernatant isozyme of aspartate aminotransferase is not affected by neonatal thyroidectomy. Nor is the activity of alanine aminotransferase affected (Balkzs et d., 1969). Other regulatory factors involved may be amino acid levels in newborn and developing brain. For example, Wang and Lin (1967) found that injection of glutamic acid in chick embryos would alter the development of alanine aminotransferase activity during a specific time in embryonic development. The rat fetus has high levels of certain amino acids, such as alanine, which decrease after birth of the animal (Lajtha and Toth, 1973). These altered levels may affect the development of enzymes involved in amino acid metabolism. A second study which suggests that amino acid levels in immature brain may affect the development of aminotransferase activity is that of Arthur et al. (1973). They found that injections of monosodium glutamate or aspartate into mice had no affect on aminotransferase activity in brain or liver of weanling mice. However, when injected into newborn animals, both aspartate and alanine aminotransferase of brain and liver were significantly increased. They suggested that newborn mice, in contrast to weanling mice, do not have the ability to metabolize large amounts of aspartate or glutamate, and thus there is an induction of these aminotransferases in brain and in liver in response to the increased amino acid. In a similar study, Prosky and O’Dell (1972) fed a 10% monosodium glutamate diet to rats for four generations. Analysis of the brains of fourth generation neonatal rats showed that the glutamate-enriched diet had no effect on the activities of either aspartate or alanine aminotransferase. V. Conclusions Total aminotransferase activity in brain is high, and is comparable to that of other tissues. Most of the amino acids in brain are transaminated by these enzymes, with the exception of the basic amino acids. Aspartate

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aminotransferase is one of the most active enzymes in brain, and its properties are similar in brain and other tissues. Alanine aminotransferase of brain and of other tissues also possesses similar properties. Other enzymes differ, notably the aromatic aminotransferases. In the liver, these enzymes are located primarily in the supernatant fraction of the cell, while in the brain they are associated with the particulate fraction. The enzymes also differ in substrate specificity and other physical properties. The brain aminotransferases are less responsive to various physiological stimuli than are the enzymes of other tissues. While enzymes of the liver may be induced by hormones or dietary factors, brain enzyme activity remains relatively constant. The difference in response may reflect, in part, the stability of the amino acid pool in the brain. Further, aminotransferases are not usually saturated by the amino acid substrate at physiological concentration, so that the amount of enzyme protein is not the limiting factor in aminotransferase activity under most conditions. Aminotransferases may play a role in regulation of amino acid metabolism and amino acid concentration of brain, as well as in neurotransmitter synthesis. The stimuli which affect aminotransferase activity in other tissues during development and adulthood are both nutritional and hormonal and are related to tissue function. Little is known of the control of brain aminotransferase activity, or of the stimuli which lead to enzymic differentiation in brain. The elucidation of the control mechanisms for brain aminotransferases may emerge through further study of the stimuli which induce or inhibit their growth during maturation, a period where aminotransferase activity in brain appears more susceptible to external influence. REFERENCES Aki, K., and Ichihara, A. (197Oa). In “Methods in Enzymology” (H.Tabor and C. W. Tabor, eds.), Vol. 17A, pp. 807-81 1. Academic Press, New York. Aki, K., and Ichihara, A. (1970b). In “Methods in Enzymology” (H.Tabor and C. W. Tabor, eds.), Vol. 17A, pp. 811-814. Academic Press, New York. . “Methods in Enzymology” (H.Tabor and Aki, K., and Ichihara, A. ( 1 9 7 0 ~ ) In . C. W. Tabor, eds.), Vol. 17A, pp. 814-817. Academic Press, New York. Aki, K., Ogawa, K., Shirai, A,, and Ichihara, A. (1967). J. Biochcrn. ( T o k y o ) 62, 610. Aki, K., Ogawa, K., and Ichihara, A. (1968). Biochim. Biophyr. Acta 159, 276. Aki, K.,Yokojima, A., and Ichihara, A. (1969). J. Biochcrn. ( T o k y o ) 65, 539. Albers, R. W.,and Jakoby, W. B. (1960). In “Inhibition in the Central Nervous System and Gamma-Aminobutyric Acid” (E. Roberts, ed.) , pp. 468-470. Pergamon, Oxford. Albers, R. W.,Koval, G. J., and Jakoby, W. B. (1962). Exp. Ncurol. 6,85. Amore, G., and Bonavita, V. (1965). Life Sci. 4, 2417. Arthur, R. D., Komer, E. G., and Bloomfield, R. A. (1973). Proc. Soc. Exp. Biol. M c d . 144, 34. Auerbach, V. H.,and Waisman, H. A. (1959). J. Biol. Chcm. 234, 304.

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THE MOLECULAR STRUCTURE OF ACETYLCHOLINE AND ADRENERGIC RECEPTORS: A N ALL-PROTEIN MODEL By J. R. Smythies

Department of Psychiatry and the Neurosciences Program, University of Alabama Medical Center, Birmingham, Alabama

“All advances of scientific understanding, a t every level, begin with a speculative adventure, a n imaginative preconception of what might be true-a preconception which always, and necessarily, goes a little way (sometimes a long way) beyond anything which we have logical or factual authority to believe in. It is the invention of a possible world, or of a tiny fraction of that world. T h e conjecture is then exposed to criticism to find out whether or not that imagined world is anything like the real one. Scientific reasoning is therefore a t all levels an interaction between two episodes of thought-a dialogue between two voices, the one imaginative and the other critical; a dialogue, if you like, between the possible and the actual, between what might be true and what is in fact the case.” Sir Peter Medawar 1972

I. Introduction

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A. GeneralAspects B. A General Specification of Receptors C. The Acetylcholine Receptor in Neuromuscular Junction. D. The Acetylcholine Ganglionic Receptor E. The Acetylcholine Muscarinic Receptor F. Structure-Activity Relationship Data G . Nicotinic Agonists H. Neuromuscular Blocking Agents , . I. The Disulfide Bond J. The Ganglionic Receptor, Ganglionic Agonists, and Blockers K. The Acetylcholine Muscarinic Receptor L. MuscarinicAgonists M. Muscarinic Antagonists N. Do Agonists and Antagonists Bind t o the Same Receptor?

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137 137 141 143 146 146 147 149 151 156 157 162 162 166 172

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111. Adrenergic Receptors A. The Adrenergic @Receptor B. 0-Agonists C Otherp-Agonists . D. p-Blockers . E. Thecr-Receptor F. cr-Agonists . G . a-Blockers H. The Relationship between p-Adrenergic and Muscarinic Receptors References , Note Added in Proof .

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I. Introduction

This review will present an exploration of the molecular structure of receptors for transmitters, in particular acetylcholine and catecholamines. Reviews of the molecular structure of receptors for amino acid transmitters and of the sodium channel have appeared elsewhere (Smythies, 1974; Smythies et al., 1974a). The method that I have followed has been to try to deduce the nature of the molecular construction of receptors from a consideration of the molecular structure of the various compounds that act: either as agonists or antagonists, upon receptors. This general approach has been widely used in the past. Transmitters and other membrane active compounds have been likened to a series of keys that fit and open a series of locks-the receptors. I t is generally agreed that the molecular properties of a compound that determine which key it will open, or block, and how well, are its precise three-dimensional shape, its quantum chemical properties and the manner in which it orients its polar and hydrophobic groups in three-dimensional space. These groups can interact by ionic and hydrogen Bonding, dispersion forces (van der Waals and London forces), and liydrophobic binding with complementary groups in the receptor. Previous attempts to deduce the possible nature of the lock from a study of the nature of the keys that will fit it have suffered from two main disadvantages. First, the authors depended on their concepts of the nature of the molecules they were studying on their ordinary two-dimensional chemical formula rather than on space-filling three-dimensional models. Or if they did use models, they tended to be chemists and were thus familiar with Dreiding or Kendrew models, which are excellent for certain purposes but of less use when considering drug-receptor interactions. Many “maps” of possible receptor structures have been published which depict a flat receptor “surface” with loci marked on it indicating the postulated location of charged and hydrophobic groups (Fig. 1 ) . T h e attempt to depict in two dimensions a three-dimensional reality has proved to be a serious barrier to the construction of hypotheses, or working models, of the molecular structure of receptors. I have therefore studied three-dimensional molecular

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FIG. 1. Two-dimensional “map” of a “receptor.”

models of drugs, and of different protein structures, using Corey-PaulingKaltun (CPK) space-filling molecular models, which are the most accurate and convenient for this purpose. From a study of the location in space of the polar and hydrophobic groups on these models, together with a determination of the possible range of bond angles for hydrogen bond formation, it is possible to make hypotheses as to the possible location of the complementary polar and hydrophobic groups in the receptor. This process could not proceed very far, however, without developing procedures to reduce the very wide degrees of freedom generated. First, it is helpful to study large and complex and fixed, rather than small and simple and mobile, molecules. In some cases, such as strychnine, the molecule has no movable parts. In others, such as toxiferine, it has only minor movable parts. Others again, such as d-tubocurarine and platyphylline, have a number of possible conformations. Yet others, such as decamethonium, might appear to be very mobile in solution. However, even in the case of the latter types, recent quantum chemical calculations and X-ray crystallography have revealed certain preferred conformations which the molecule will take up in specified environments. These conformations are not foolproof, as some molecules may take up one conformation in crystals, another in water, and yet a third in a nonpolar environment, such as the lipid layer inside a membrane or some nonpolar environment in protein. However, it seems reasonable, when trying to deduce the nature of the receptor from a consideration of the nature of drugs, that one should start with the preferred conformation ( s ) revealed by these methods. Second, it is necessary to make some hypothesis about the molecular species that constitute the receptor--otherwise the degrees of freedom remain insufferably large; for example, one can look at protein, or phospholipid, or nucleotides, or prostaglandins, or various combinations of these, to see whether there is any coherent way in which they can provide the complementary groups in the receptor structure deduced from a study of the drugs. This complementary array of atoms can be generated only by a limited number of specifiable ways within any one species, but by a much larger number of ways if more than one species is involved. For example, a hydrophobic

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area can be provided by protein in the form of lysine > terminal NH,+ group of protein > histidine (Lewin, 1969). This is because of the cooperative resonance of the arginine-glutamate double ionic bond that results from the association of a positively charged resonating group (guanidinium) and a negatively charged resonating group (carboxylate) which increases the stability of the ionic bond. ( 2 ) Only the arginine-glutamate bond gives the correctly located bonding atoms with the right bond angles to bind the transmitters (the acetyl group of ACh or equivalent), as we will see.

3. Ion-Dipole Bonds Ionic groups can form ion-dipole bonds with any group capable of forming a hydrogen bond. These are intermediate in strength between ionic and hydrogen bonds. Thus a simple working hypothesis is that the basis of the receptor is provided by two parallel polypeptide chains in the p pleated-sheet conformation tied together by arginine-glutamate ionic bonds which in a p pleated sheet will be some 7 A apart. Barlow (1964) has suggested that the ACh receptor contains a grid of such charged points. This could be provided by a series of such arginine-glutamate bonds. Figure 2 shows a diagram of heptapeptide chains linked by four arginine-glutamate double ionic bonds arranged so that the successive charged groups form a grid or ladder some 7 A apart. The odd-numbered amino acids are either arginine or glutamate and the even-numbered ones are not specified-these come off the other side of the two chains. The ionic bond neutralizes the formal charges on the

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binding groups, but polar atoms remain-the spare proton (N H group) of arginine and the spare electron pair of the glutamate O-exposed a t the LLsurface”and capable of acting as hydrogen-bonding agents. However, this simple scheme provides no lipophilic area suitably placed to bind the lipophilic part of ACh and other transmitters and their antagonists. Moreover, the grid generated has no clear-cut three-dimensional structure related to molecules with an obvious three-dimensional structure, such as p-erythrodine, picrotoxinin, dibenzpetimide, ergotamine, etc. I n a previous publication (Smythies, 1971a) it was suggested that the required extra portion might be provided by prostaglandin (PG) as the PG molecule is complementary to an Arg-Glu grid of this type. That is to say, the polar groups on the PG molecule are so located and with bond angles such that each can bind to a complementary group in the grid with the correct alignment of hydrogen bonds. Moreover, PGE is complementary to a Glu-Arg rung in the grid and PGF is complementary to the reversed rung Arg-Glu (Fig. 14 in Smythies, 1971a). However, since that article was written I have determined how an equivalent structure can be constructed using protein only. The PG-protein complex was quite successful in “explaining” a quantity of structure-activity relationship data, but in certain key instances it has been possible to compare the PG-protein and the new all-protein models to the advantage of the latter in each case, as we will see. Experiments with a variety of different types of protein structure yielded the structures described in detail below. I will describe first the molecular specification of the ACh receptor and then indicate how various other receptors differ from it. For the general hypothesis suggests that a number of receptors for transmitters are variations on a common theme; that is, they share a common general molecular structure but differ in the arrangement of one or more details within it. This technique results in the specification of a particular protein structure that is capable of explaining the phenomena. That does not constitute proof that this is actually the protein structure concerned. However, the real receptor can only either differ in detail (e.g., some minor differences in conformation, or some conservative substitution in an amino acid) or differ in toto-that is some quite different protein structure is really involved-for example, an a-helix, or some other type of helix, or an irregular structure with several segments of protein chain running in different directions. I will return to this point later but will just say now that the variations on the simple B structure described provide good working models for a variety of receptors with great economy of detail and a certain elegance of form, whereas no such scheme can be based on any of the other known protein conformations. The model described here is certainly the simplest capable of accounting for these facts.

MOLECULAR STRUCTURE OF RECEPTORS

141

B. A GENERAL SPECIFICATION OF RECEPTORS I t will be convenient at this point to name some of the constituents of the model receptor. The basis is two primary chains in the p conformation linked by their opposing amino acids (Glu or Arg with possible conservative substitution) to give the Glu-Arg grid described. The amino acids coming off the outer sides of the two chains will not be specified at present. In this model three types of variability are immediately available : ( 1) the number of rungs in the grid (in all the model receptors I shall describe, this number is three or four) ; (2) the particular Arg-Glu sequence involved; and (3) the direction in which the protein chains are running. With four rungs, there are ten possible different Arg-Glu combinations (Fig. 3) for each of two possible arrangements of the protein chains (parallel and antiparallel) (Fig. 4), giving a possible total of twenty. Figure 5 shows a Corey-Pauling-Kaltun (CPK) model of one such grid. To the two primary chains are now added two secondary chains to form in part two pleated sheets. Part of the secondary chain forms this p pleated sheet with the primary chain, and part crosses one of the two outer rungs of the grid. There are three main types of secondary chain, each with possi-

(a)

(b)

(d)

(C)

(el

HBBBB

(1)

(9)

(h)

(i)

(jl

FIG. 3. Ten possible arrangements of a 4-runged Arg-Glu grid. Some of these form the basis of the following receptors: (acetycholine) muscarinic ( b ) ; ACh, neuromuscular junction ( i ) ; ACh ganglionic ( c ) ;7-aminobutyric acid ( d ) ; glutamate (j).

(0)

(b)

FIG. 4. Two different ways of running the protein chains: ( a ) parallel; ( b ) antiparallel.

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J . R. SMYTHIES

FIO. 5. A Corey-Pauling-Kaltun model of an Arg-Glu grid. Note the spare proton and the unused spare electron pair in the middle of each rung.

ble minor variations, which I have named after the prostaglandin with which they are homomorphic. F Sequence:' -x~-Gly~-x~ly~-Pro-x~-(Ile)-x~-(Asp)-x~(Val)

(His)

The segment xl-Pro runs along the top of an Arg-Glu rung in the primary structure with two hydrogen bonds (Glyl carbonyl 0 from Arg NH and Gly, peptide NH to Glu 0:) and extensive lipophilic (hydrophobic) and multiple weak interactions. The segment Pr-x5 forms an antiparallel p pleated sheet with one primary chain with six hydrogen bonds. The /3 pleated sheet has a rigidly determined structure, and the location of the amino acids coming off it is rigidly fixed (parallel to each other). This reduces the degrees of freedom in the site in a helpful manner. I n some cases position 9 has Asp and in some His, and position 7 may have Ile or Val. E sequence: -x~-(Asp)-x4-Val-Pro--Gly~-x~-Gly~(His)

The E sequence differs from the F sequence in two main respects: it is antiparallel to F and one amino acid (x3) has been deleted. It requires 'Pro here guarantees that the protein chain will make the right-angled bend required, but chains do not have to have Pro for this-in some cases Gly or some other substitution might be found.

MOLECULAR STRUCTURE OF RECEPTORS

143

a Glu-Arg rung to bind (in place of Arg-Glu) at position 1. The two hydrogen bonds are formed this time from x2 N H to Glu 0: and to Pro carbonyl 0 from Arg NH. If the amino acids on the grid are simply read from left to right note that an F sequence requires Arg-Glu at rung 1 but Glu-Arg at rung 4 (where it is binding upside down) to construct a receptor cup. Likewise an E sequence requires Glu-Arg at rung 1 and Arg-Glu at rung 4. DN sequence: Gly-x-Gly-Pro-x-(Asp)-x(His)

The DN sequence is associated only with the glycine receptor (Smythies, 1974). Thus the complete description of any receptor in this hypothesis requires the specification of the following: (1) the Arg-Glu sequence in the primary structure. This in turn determines which secondary chains can bind and the sense of the chains; and ( 2 ) the amino acid sequence in the secondary chain:i.e., whether Val or Ile, or His or Asp, at the loci given above. In some receptors Lys might substitute for Arg, or phosphoserine for Glu (these are stereochemically very similar) in the primary chains. C. THEACETYLCHOLINE RECEPTOR IN

THE

NEUROMUSCULAR JUNCTION

The primary structure required is shown in Fig. 3 ( i ) . The sequence (right-hand chain) is -Glu-x-Arg-x-Glu-x-Arg-, which can be shortened to Glu :Arg :Glu :Arg. This binds two F chains, and the direction of the primary chains is shown in Fig. 4b. An optimum “fit” is obtained with Ile and Asp in each F chain as shown in Fig. 6 and in diagrammatic form in Fig. 7. Note that a large rectangular cup is generated with walls -N-] with lined by lipophilic groups, peptide pseudo ?r clouds, [-C (a) one prominent negatively charged group on each side wall. The floor is composed of two Arg-Glu links each with one spare proton ( N H ) and one spare electron pair (0:) with specific bond angles flanked by hydrocarbon chains [-(CH,)3-for Arg and -(CH,)2for Glu]. There are also three intercalation sites in the floor (between rungs 1 and 2; 2 and 3, and 3 and 4) of which the walls are composed of the extensive pseudo T clouds of the resonating Arg-Glu bonds (the T electrons of which are smeared over the entire system to give a T cloud almost the size of the 7 cloud of a benzene ring) in the center, and on the periphery the hydrocarbon chains of the amino acids. The only variations possible with any given amino acid bond of Ile complement are provided by: (1) rotation at the C,Cp and Asp so that these can take up subtly different positions although one will normally be preferred and ( 2 ) rotation at the C,Cp bond of the Arg-Glu rungs 2 and 3. This allows the floor to be raised or lowered by

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J . R. SMYTHIES

Fro. 6. A Corey-Pauling-Kaltun model of acetylcholine receptor (neuromuscular junction). The amino acids in the secondary chain are numbered. 1, xl; 2, Gly; 3, x,; 4, Gly; 5, Pro; 7, Ile; 8, Cys; 9, Asp; A, Arg; G, Glu.

Fro. 7. A diagram of the acetylcholine receptor in the neuromuscular junction. Note the large rectangular cavity.

some 1.5 A. I n the lowered position, Arg presents two NH groups in the floor; in the raised position, only one. With an E chain only the lowered position is sterically possible. In the ACh (nicotinic) receptor in autonomic ganglia, the only difference is that the lower F chain is replaced by an E chain, and so the Glu-Arg

MOLECULAR STRUCTURE OF RECEPTORS

145

rung at 2 is reversed to Arg-Glu. Two E chains generate a much smaller irregularly shaped cavity, and one F and one E chain generate a trapezoidshaped cavity (see further below) . If we now consider the “fit” of acetylcholine in this model receptor and its possible function, the following is suggested. The relevant conformation is the synclinal, antiplanar conformation of Baker et al. (1971) proposed on the basis of X-ray data. The C,-Cp ( t z ) bond has a rotation of some -130O; and the CB-0 bond ( t 3 ) ,of some - 1 6 0 O . The molecule now binds as follows (Fig. 8) : (hydrogen bond) carbonyl 0 from Arg NH of rung 2. The bond angle aligns the ACh molecule in van der Waals contact with Ile with lipophilic binding between them. The onium head fits into the three-dimensional anionic pocket between Asp and Glu with an electrostatic link between the positive charge (which is smeared over the surface of the onium head) and the negative charge on Asp and the S- charge on Glu. This would tend to disrupt Arg-Glu link No. 3 by attracting Glu and repelling Arg. This might in turn tend to destabilize this particular protein conformation and set up some other one. Thus in Rang and Ritter’s terminology A

+R+

[A:R]+ A

+ RI

where A is the agonist and R and R, the two different conformations of

FIG.8. Postulated manner of binding of acethylcholine in its receptor in neuromuscular junction (see text).

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J . R. SMYTHIES

the receptor protein. The details of these changes together with a consideration of the metaphilic effect and desensitization will be dealt with elsewhere. When R + R,, the structure binding ACh is disrupted and the ACh molecule is released, taken up by acetylcholinesterase, and hydrolyzed. Then R -+ R, by the primary chains coming together again. When an Arg-Glu rung is reversed to Glu-Arg the location of the central NH group is not appreciably altered, but the bond angle is tilted through some 90°. The molecular models indicate that to bind ACh, if rung 2 is Glu-Arg, then the adjacent secondary chain must be F and ACh must bind by its carbonyl 0. This derives from the fact that the carbonyl 0 juts out farther from the flank of the ACh molecule than does the flush ester 0. If rung 2 is Arg-Glu, the adjacent secondary chain must be E and ACh must bind by its ester 0, as in the muscarinic receptor (see further below). I n some previous models of ACh receptors the distance between the two polar groups in the receptor binding ACh 0: and ACh N+ was set at the 0-N distance in ACh (center to center, around 3.6 A). However, as the charge on the onium head is distributed over its surface, a more relevant measurement might be 0 to the edge of the N methyl group.

D. THEACETYLCOLINE GANGLIONIC RECEPTOR The essential difference between this and the receptor in the neuromuscular junction is that rung 4 is Arg-Glu instead of Glu-Arg. This determines that one protein chain is F and the other now E. The receptor cup has a rhomboid shape instead of rectangular. The lower (Val) lipophilic area is some 2 A closer to the anionic site than in neuromuscular junction.

E. THEACETYLCHOLINE MUSCARINIC RECEPTOR This is considerably different from neuromuscular junction. Both seconary chains are now E, which gives a small narrow receptor cup (Fig. 9 ) . In addition rung 2 is reversed to Arg-Glu, and Asp on the lower left secondary chain is replaced by His. The constriction on the space available inside the receptor cup imparted by the E chain, as compared with the F chain, imposes a restriction on the rotation of Ile such that the ethyl group cannot point away from Asp. If the methylene chain is aligned alongside the peptide backbone (which seems likely as this provides the most hydrophobic environment) the space between Ile and Asp is now too small to accept even a part of the onium head of ACh. When the onium head, with ACh in the Chotia-Pauling conformation, binds in the anionic pocket, one methyl group must enter this pocket (Fig. 30). This is only possible for the E chain if Ile is replaced by Val. An even larger gap is left if Ile is replaced by Thr

MOLECULAR STRUCTURE OF RECEPTORS

147

Fro. 9. Model of the acetylcholine muscarinic receptor. Note the much smaller irregular cavity.

or Ala. However, experiments with a number of muscarinic agonists showed that replacement by Val produced a satisfactory model. Of course muscarinic receptors might differ in this regard in different areas. Leu, Met, and Phe can all be excluded. In the lower left chain, on the other hand, Ile is acceptable and even indicated by some data detailed below. In the muscarinic receptor ACh binds by its ester 0 and in a conformation which approximates to the mirror image of the nicotinic conformation. I will deal in turn with each of these receptors but will first illustrate how this specification allows us to explain the comparative structure-activity relationship data for ACh in the two types of receptor (nicotinic and muscarinic).

F. STRUCTURE-ACTIVITY RELATIONSHIP DATA 1. The acetyl methyl group of ACh can be replaced by ethyl or propyl groups in nicotinic receptors, but not muscarinic. A comparison of Fig. 6 and 9 shows the extra space available in the nicotinic receptor cup to accommodate the larger groups. The length of the hydrocarbon chain allowable is determined by the location of the upper arm of the secondary chain, that

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J . R. SMYTHIES

constitutes the upper wall of the receptor cup. In the muscarinic receptor, in contrast, the methyl group attains an extensive lipophilic contact with the end wall of the receptor cup as well as Val adjacent. This supports the postulate made by Chothia (1970) that this methyl group is important for muscarinic, but not nicotinic receptors.

+

2. I n the series R-N(CH,),

muscarinic activity is maximum when If the onium head is bound as for ACh, these chain lengths are what are required to run along Ile (or Val) and make an effective contact with the end wall of the site in the smaller muscarinic and the larger nicotinic sites, respectively.

R

= C,Hll; and nicotinic activity, when R = C6HIs and C,H,,.

+

3. In the series R-CO-CH,-CH,-N(CHs) any increase of R from CH,- in the muscarinic site greatly reduce (C,H,-) or abolishes activity (C,H,-) whereas maximum activity is attained in nicotinic receptors by the latter substitutions.

+

4. In the case of R-C=C-CH2-N(CHS),, muscarinic activity is maximum when R is ethyl and nicotinic when R is propyl.

+

5 . In compounds of the type R-O-CH,-CH,N(CHs), potent chowhere X is some linergic agonists are obtained when R = X-CH2-CHzunsaturated ring system such as imidazole, pyridine, or indole. This allows the ring to intercalate between the Arg-Glu rungs and possibly to achieve a charge transfer reaction with the adjacent Arg-Glu pseudo-* cloud. 6. The pattern of allowable substitution of the a and /3 methylenes of ACh is different in nicotinic and muscarinic receptors. Figure 9 shows the only methylene hydrogen ( p + ) that can be substituted by methyl. The p-hydrogen is apposed to Val on the one wall, and both the a hydrogens are apposed to Ile on the other wall. Note that Val here would not block the a-methyl groups which suggests that, as the a-substituted compounds are inactive, the amino acid here is Ile, not Val. 7. In either receptor, the replacement of one of the onium head methyl groups by ethyl reduces activity ( % ) ; and by two groups, practically abolishes it (1/300). This may be partly due to the weakening of the effective negative charge on the surface and partly to a strengthening of the lipophilicity of the head, which would tend to form a bond between the Asp and Ile methylenes on either side of the receptor cup. 8. Thiol esters equivalent to ACh (in which the ester 0 is replaced by S) lose muscarinic, but retain nicotinic, activity. The S atom carries a smaller charge than 0 and hence acts as a poorer hydrogen bond acceptor. The hypothesis suggests that the ACh molecule binds by its ester 0 in the muscarinic, but not in the nicotinic, receptor.

MOLECULAR STRUCTURE OF RECEPTORS

149

G. NICOTINIC ACONISTS 1. Nicotine

This compound (Fig. 10a) first stimulates and then blocks nicotine receptors, and both stereoisomers are active (but S > R) . If the basic N of the pyrrolidine ring binds to the anionic site (Glu 0 of rung 3), the pyridine ring nitrogen, which carries a 8- charge, makes an electrostatic bond with the N H of Arg of rung 2, the pyrrolidine ring 5 methylene group forms a lipophilic bond with the adjacent methylenes of Asp (above) and Glu (below), and the N-methyl group tucks into the lipophilic gap between Ile and Asp methylenes. The C H groups 4 and 5 of the pyridine ring make lipophilic contacts with the adjacent Ile. Both nicotinic receptors can bind two molecules of nicotine per receptor in this manner (head to tail) but the muscarinic receptor cannot. Possibly two molecules are required, which may explain why nicotine is inactive at the latter. The R isomer does not fit the model receptor as well as the S isomer does. The related compounds 8-pyridylethyltrimethyl ammoium and leptodactyline (Fig. 10b,c) bind in a comparable manner.

2. Decamethonium This compound first acts as an agonist of ACh, causing a contraction of voluntary muscle and then blockade. Comparison of the molecule of decamethonium and the receptor cup indicates that, as it is longer than the longest internal diameter of the cup, it must bend in the middle in order to fit. Each onium head now fits in one anionic cavity (Asp; Glu) forming two ionic bonds and there is also considerable lipophilic binding to Ile and both Glys. Decamethonium is actually not the most active bisonium agonist compound, which is C,, bis-TMA; the latter also gives the optimum fit of the receptor cup. The blocking action of decamethonium is described

OH (C)

FIG. 10. Formulas of some nicotinic agonists: ( a ) nicotine, ( b ) p-pyridylethyltrimethyl ammonium, ( c ) leptodactyline.

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J. R. SMYTHIES

further below. It entails binding in its fully extended conformation to R,-that is, to a primary chain with the sequence -Glu-x-Arg-xGlu-. In such a chain, the two Glus are 14 A apart-the interonium distance of decamethonium-which thus binds by two ionic bonds plus a lipophilic bond between its middle methylenes and the hydrocarbon chain of Arg underneath. Thus the hypothesis suggests that derivatives of decamethonium that cannot bend at right angles in the middle will have only antagonist activity (examples are shown in Fig. lla) whereas those that can will have agonist activity as well (Fig. 1lb ) and those that are fixed in this bent position will have only agonist activity. Gallamine ethiodide (Fig. 1l c ) can be regarded as a combination of these two forms of decamethonium. Two interesting compounds to test would be obtained by replacing the C,-C, methylenes in decamethonium by a C I C double bond. In this case, the trans isomer should have pure blocking activity and the cis isomer pure agonist activity. Compounds like polydecamethonium ( n = 37) are pure blocking agents. They are much too long to coil into one receptor cup but could fit Barlow’s anionic grid and thus should have only blocking action-which they do. Likewise decamethonium derivatives in which bulky hydrophobic groups are substituted for onium methyls will not fit into the receptor cup (see below) and thus will bind to R, rather than R. Rang and Ritter (1970, 1971) have named those antagonists that bind preferentially to R, (and whose activity is thus potentiated by pretreating the tissue with the agonist) “metaphilic” antagonists as opposed to “conventional” antagonists that bind preferentially to R. Prominent among them are compounds of the type just described. An interesting example of the difference between “bent” and “straight” derivatives of decamethonium is provided by the compounds studied by Barcompound tels et al. (1971) (Fig. 12), in which the trans (-N=N-) ( a ) has the bent shape and the cis compound is straight (b) , The former is a potent agonist (in electroplax) whereas the latter is inactive in this

Fro. 1 1 (a) Antagonist compounds; (b) agonist/antagonist compounds; ( c ) gallamine ethiodide.

MOLECULAR STRUCTURE OF RECEPTORS

151

(b)

FIG.12. Bartel’s compounds: ( a ) a potent acethylcholine agonist; ( b ) inactive. regard. The conformation of these compounds is fixed in the planar position owing to ?M interaction between the double N bond and the benzene rings. H. NEUROMUSCULAR BLOCKING AGENTS There are two main types of neuromuscular blocking drugs: conventional, which bid to R ; and metaphilic, which bind to R, (Rang and Ritter, 1971). A conventional antagonist must bind to the receptor in such a way as to prevent the binding of the transmitter; and also in some cases it may stabilize the R conformation of the protein. A metaphilic antagonist binds to R, and prevents the reaction R, + R. An examination of various types of neuromuscular blocker shows that they are all complementary, in different ways, to the model of the receptor proposed. 1. Curare Alkaloids

There are two main types of curare alkaloids-those related to tubocurarine (tube curare) and those related to toxiferine (calabash curare). The formula of toxifenne I is shown in Fig. 13. I t is a perfectly symmetrical molecule with a flat rectangular shape with, on each longer side, a benzene ring and a quaternary nitrogen atom separated by the -CH= CH,CH.OH group. I t forms a closely fitting lid on top of the receptor (Figs. 14 and 15) with lipophilic binding, on each side, between the benzene

+

ring and Ile; an ionic bond between N and Asp; and the -CH=CHCH,OH group fits neatly into the gap between Ile and Asp to hydrogen bond by its terminal O H group to the carbonyl 0 of x p . The contribution of these two hydrogen bonds may be estimated from the relative potency of C-alkaloid H (which lacks one) and C-alkaloid K which lacks both. These are approximately 0.5 and 0.25 as active as toxiferine I, respectively. There is also contact with the floor of the receptor. Toxiferine I1 (Fig. 16a)

152

J . R. SMYTHIES

Me

FIG. 13. Toxiferine I.

FIG. 14. Corey-Pauling-Kaltun model of toxiferine I blocking acetylcholine (neuromuscularjunction) receptor: note exact “lid” fit. has a subtly different shape and does not form so good a lid. Two feebly active compounds are caracurine I1 and C-alkaloid D (Fig. 16b,c) both of which have an entirely different folded-over shape with an interonium distance of only about 8 A instead of the required 12 A ( N to N ) . The conformation of d-tubocurarine itself in crystal form has been determined by Sobell et al. ( 1972). It has a more rectangular and more irregular shape than toxiferine I and fits deeper into the receptor cup than does toxiferine. Each N group (one quaternary, one protonated) forms an ionic link

MOLECULAR STRUCTURE OF RECEPTORS

153

OH

It1

FIQ. 15. Diagram of toxiferine I blocking ACh (neuromuscular junction) receptor. FIG. 16. ( a ) Toxiferine I1 (rest of molecule as for toxiferine I ) . ( b ) Caracurine I1 (rest of molecules as for toxiferine I ) . ( c ) C-alkaloid D (rest of molecule as for toxiferine I ) . to Asp, the methoxylated benzene rings fits into the lipophilic corners of the receptor, and the two linking benzene rings bind between the two Iles. There is also contact with the floor of the receptor.

2. Decamethonium Decamethonium exerts its blocking action by binding to R, as described above. The 14 A distance between its onium heads matches the 14 A distance between the two Glu’s in 41u-x-Arg-x-Gluwhich may be involved in R,. It is therefore of interest that the maximum blocking action is found in the C,, compound as compared with the C,, compound for agonist action. A variety of other bisonium blocking compounds have been described, some of which would appear to be curarelike blockers (binding preferentially to R ) and some metaphilic blockers (binding preferentially to R,) . Replacement of one onium methyl group by benzyl in decamethonium (at each end) leads to loss of agonist and maintenance of antagonist activity. Some other interesting blocking agents are detailed by Martin-Smith (1971) (Fig. 17). I n the case of 9-(p-methoxyphenyl) fluorene-2,7-bistrimethylammonium (Fig. 17a), the molecule takes an unusual diagonal line directly across the receptor cup, each onium head making an ionic bond with each Asp. The fixed methoxyl phenyl group now intercalates between rungs 2 and 3. In addition, one horizontally oriented benzene ring binds hydrophobically to the adjacent Ile and the other receives a hydrogen bond from the underlying Arg NH to its 7 cloud. Its action therefore should be conventional, not metaphilic. In the case of diplacine (Fig. 17b), the two complex ring systems at each end allow both hydroxyls on each ring to hydrogen bond to one carbonyl 0 (of x5) in the peptide chain of R, with

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J . R. SMYTHIES

OH.CH2 H o

~

~

$ OH

2

aD . C l $ . C l $ ~ ~ z ~ o H

Ibl

F I ~ .17. Some neuromuscular blockers: ( a ) 9-(p-methoxyphenyl) fluorene-2,7bistrimethylammonium; ( b ) diplacine. accessory lipophilic bonds to Glu methylenes as well. Thus diplacine should be a metaphilic antagonist, especially as the mid-chain benzene ring prevents the bent conformation required to bind to R. The various blocking agents based on tying two onium heads onto a steroid ring developed by MartinSmith and his colleagues (Martin-Smith, 1972) should all be strong metaphilic and weak conventional antagonists. Trisonium compounds, like gallamine ethiodide (Fig. 1lc) , which have decamethonium-like properties, can be regarded as a combination of both the bent and the straight forms of decamethonium in one molecule. Rang and Ritter classify gallamine ethiodide as a conventional antagonist because its effects are not promoted by previous exposure of the receptor to an antagonist. However, if gallamine ethiodide and decamethonium are their own agonists, this procedure would not detect the fact that they bind preferentially to R, as antagonists. The Russian workers Khromov-Borisov and Michelson ( 1966) reported the potent neuromuscular blocking action of compounds of the form

+

..

+

(Me) 3N-CH, CH, * 0 .CO NH (CH,) NH * CO.O.OH, CH,-N (Me)3, where blockade is a maximum if n = 6 or 7. These compounds bear a clearcut relation to R, as shown in Fig. 18, and they should thus be metaphilic antagonists.

3. 8-Erythroidine This is an unusual neuromucular blocker as it is a tertiary rather than a quaternary base. The dihydro derivative (Fig. 19) is more potent. Each receptor cup, with the floor in the lowered position, can accommodate two molecules of the drug which binds as follows (Fig. 20) : the carbonyl 0 and adjacent double bond (S-) bind to the two unused protons of Arg,

MOLECULAR STRUCTURE OF RECEPTORS

155

N

Fro. 18. Suggested mode of blockade of R, by compounds investigated by Khromov-Borisov and Michelson (1966) : +, hydrogen bond; @ ionic bond. Fro. 19. Dihydro-p-erythroidine, line diagram. Fro. 20. Diagram showing how two molecules of dihydro-p-erythroidine can block the acetylcholine (neuromuscular junction) receptor.

the basic protonated N+ to Asp-, and CH groups on the three nonlactam rings bind lipophilically to Ile and the two Glys. The molecule has a rectangular corner, which slots into the right-angled corner provided by Ile and the two Glys. The methoxy group intercalates between rungs 1 and 2 of the grid. The receptor cup can accommodate two molecules of the drug, which can bind lipophilically to each other.

4. Tropine Derivatives The compound shown in Fig. 21a can bind to R in the manner of curare. The compound shown in Fig. 21b cannot bind in this fashion, for to do this at least a C, bridge is required. Neither can it bind in the antagonist manner of decamethonium, for this requires at least a C , interonium chain. The only manner the model suggests for how this compound could bind is illusMe

RO

Me

OR

OR

Fro. 21. (a) Tropine neuromuscular blocker that can bind to R in the manner of curare. ( b ) Tropine neuromuscular blocker that may bind as shown in Fig. 22. Fro. 22. Binding of compound shown in Fig. 21b to neuromuscular junction.

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J.

R. SMYTHIES

trated in Fig. 22. In this the two sides of R, have a minimal or zero separation, and the blocker zigzags across two rungs binding to Glu’s by N+, and by tropine ring C H s to Glu methylenes and by the CO and OH groups to peptide NH and C O groups in the protein chain.

5. Laudexium Laudexium (Fig. 23) binds somewhat differently from tubocurarine, as the isoquinoline ring binds upside down (relative to tubocurarine) and the dimethoxybenzene rings intercalate between the rungs 1 and 2 and 3 and 4 of the grid. 6. Cobra Neurotoxin Neurotoxins from the venom of cobras and related snakes are potent and irreversible blockers of the ACh receptor in the neuromuscular junction. Their action is dealt with elsewhere (Smythies et al., 1974b). I. THEDISULFIDE BOND Karlin and his co-workers (Karlin and Winnik, 1968) have identified a disulfide bond some 10 A from the anionic site in the ACh receptor in electroplax (a modified neuromuscular junction). The receptor is first reduced by dithiothreitol (specific for disulfide links) and then alkylated by quaternary ammonium derivatives of N-ethylmaleimide. If the onium head of this compound binds to the anionic site, this locates the group attacking the SH group some 10 A away. Examination of my model of the receptor suggests that this disulfide bond is located at x, in the F sequence (between Ile and Asp). Each x4 is 10 A from one of the two anionic sites in the recep-

OCH3

Fro. 23. Laudexium.

MOLECULAR STRUCTURE OF RECEPTORS

157

tor, Alternatively the disulfide bond could be located in the primary chain at the x in the middle between Arg and Glu. The disulfide bond may be made from either of these two loci to another Cys in an adjacent chain, or possibly the primary and secondary chains are so linked to each other. The location of this disulfide bond outside the receptor cup explains why its reduction by dithiothreitol is not blocked by curare (Rang and Ritter, 1971) , which sits entirely inside the cup. The receptor in which the disulfide bond is reduced becomes much less sensitive to carbachol, whereas hexamethonium, which is normally a weak conventional antagonist (Rang and Ritter, 1971) develops agonist properties. I t is simplest to suppose that the reduction of the disulfide bond causes a reversible conformational change in the protein. The receptor can be restored to normal by oxidizing agents such as 5,5’-dithiobis ( 2-nitrobenzoic acid).

J. THEGANGLIONIC RECEPTOR, GANGLIONIC AGONISTS,A N D BLOCKERS The specification for this differs from that for the ACh receptor in neuromuscular junction in that one F sequence is replaced by an E sequence. This is associated with the switch of one end rung of the grid from Arg-Glu to Glu-Arg (Fig. 3 ) . Thus the primary sequence is Glu :Arg :Glu :Glu : and both secondary sequences have Asp (rather than His). This locates one Ile about 2 A closer to the adjacent anionic site. Acetylcholine binds in the same way as in neuromuscular junction, but the site will accommodate only one molecule of ACh with the onium head in the E corner. However, the pattern of stimulation and particularly blockade by other drugs is altered. 1. Agonists. Simple Onium Salts

Tetraethylammonium (TMA) (Fig. 24a) is much more active at ganglia than at the neuromuscular junction. The latter Ile is too far away from the anionic site for TMA to be able to bind to it by a lipophilic bond when “in place” on the anionic site. Whereas if the adjacent secondary chain is E, this is now possible. The other agonists mainly follow the pattern for the neuromuscular junction as agonists bind mainly to the F secondary chain, which is the same in both, rather than to the E chain. 2. Antagonists a. The blocking action of d-tubocurarine at the ganglionic receptor is about one-tenth that at the neuromuscular junction, but it is nevertheless quite high. However, the blocking action of toxiferine I, which attains a more perfect fit at the neuromuscular junction is only I/soth in the ganglion, which it fits much less well in the model.

158

J . R. SMYTHIES ft

Yo I Me-I-Mo I@ Me

I El-n-Et

lo

fl

la1

Ibl

(eI

Fro. 24. (a) Tetramethylammonium. ( b ) Tetraethylammonium. ( c ) Pempidine. ( d ) Mecamylamine. ( e ) fi-Trimethylammonium benzene diazonium.

b. Tetraethylammonium ( T E A ) , unlike TMA, is purely a blocking agent (Fig. 24b). Presumably this is due to two factors. The more diffuse negative charge on its surface would have a lesser tendency to disrupt the Arg-Glu link and the greater lipophilic binding to both sides of the receptor cup (Ile and Asp and Glu methylenes) would promote the stabilization of R by linking the two sides together by a lipophilic bridge. Blocking activity is increased if the area of lipophilic contact is increased as in diethyldiisopropyl ammonium and related compounds, and in compounds like pempidine and mecamylamine (Fig. 24c,d).

3. Bisonium Compounds The classical blocking agents at ganglia are hexamethonium and pentamethonium, which do not depolarize the receptor and thus act more like curare than decamethonium at the neuromuscular junction. A comparison of hexamethonium with my models of the neuromuscular junction and ganglionic receptors suggests why this should be so. In either receptor the mole-

Fro. 25. Previous model ( a ) and present model (b) of hexamethonium CS bistetramethylammonium binding to neuromuscular junction. ( c ) Cs bistetramethylammonium binding to acetylcholine ganglionic receptor.

MOLECULAR STRUCTURE OF RECEPTORS

159

cule of hexamethonium could cross the receptor cup diagonally binding to each Asp, but only in the latter can it attain a lipophilic contact with an adjacent Ile as it does so (Fig. 25). Barlow and Franks (1971) concluded on the basis of binding experiments that hexamethonium binds mainly by hydrophobic (lipophilic) bonding. When bound in this way the onium heads of hexamethonium do not contact the Arg-Glu bonds at risk. However, hexamethonium has some blocking action in neuromuscular junction (Rang and Ritter, 1971) and to some extent prevents curare binding (Ferry and Marshall, 1971). Previously, I suggested that it might bind in neuromuscular junction as shown in Fig. 25a, which still allows one molecule of ACh to bind alongside, but this requires one onium head to enter a lipophilic pocket, which is unlikely. A more plausible mode of binding is that shown in Fig. 25b. It is of particular interest that, whereas decamethonium is inactive at ganglia as a blocking agent, bis-TEA compounds with a chain length of between 15 and 17 are active at both neuromuscular junction and ganglia. The comparison of the primary chains in each case suggests why. Figure 26 shows the primary chains in ( a ) ganglion and ( b ) neuromuscular junction. The inter-Glu distance in ( a ) is 28 A whereas in ( b ) it is 14 A. The overall length of C,, bis-TEA is 28 A. An inspection of Fig. 26 explains why C, bis-TMA can bind in both loci, but in order to explain why C, bis-TMA does not bind to the ganglionic R, one must suppose that for some reason it is denied access to the other half of the grid, where there are two possible binding sites for it. A variety of partially rigid bridge systems can replace the hexamethylene chain of hexamethonium with retained activity. It would be of interest to test a rigid bridge, such as biphenyl. Activity may be increased by providing lipophilic groups that can intercalate between the rungs of the grid (e.g., chlorisondamine: Fig. 27a). The two onium heads connect the Glu 0 s (approximately 8 A apart) rather than the Asp 0 ' s (approximately 12 A apart), but the massive tetrachlorobenzene ring system intercalates between rungs 1 and 2 of effectively links the protein strands together.

FIG. 26. ( a ) Basis of R1 generated in ganglionic receptor (plus CISbistetramethylammonium). (b) Basis of R1 generated in neuromuscular junction (plus Cla bistetramethylammonium ) .

160

J . R. SMYTHIES

(b)

(0)

FIG. 27. ( a ) Chlorisondamine. (b) Ganglion-blocking isoxazolidine.

4. Zsoxarolidines The formula of a ganglion-blocking isoxazolidine is shown in Fig. 27b, and its suggested mode of blockade of the ganglionic receptor is as follows. The molecule fits into the E corner of the receptor cup with extensive lipophilic bonds and three polar bonds [N to Glu 0 and Asp 0 (of F chain) ; ring 0 from Arg NH of rung 3; O H to Glu 0 of rung 21.

5 . Choline Ethers Gyermek (1967) reported the remarkable effects of a series of branchedchain choline ethers (Table I ) . Compounds of the type R-0-CHz-CH,-N(Me), could bind to either Arg NH (Fig. 28a). If R = CH,, it is more likely to bind to the E corner (Fig. 28 lower) for the reasons detailed in Section 11, F ; and if R > CH,, then it is more likely to bind in the F corner (Fig. 28a, upper). T h e trapezoid shape of the receptor cup means that, if the molecule binds in the “lower” manner (Fig. 28a), then a butyl substitution on the p carbon will allow the terminal methyl group of the butyl group to reach across the receptor and contact the opposite wall slotting into the lipophilic gap between Ile and Asp (Fig. 28b). Whereas, if the molecule binds in the “upper” manner, the butyl group is not long enough and a heptyl group is now needed (Fig. 28c). This suggests that the activity pattern if R, = C,H,, would be with R = CH,+; R = CzH5++; R = C,H,++. TABLE I ACTIVITY OF SOME BRANCHED-CHAIN CHOLINE ETHERS’ R-O-CHRi-CHpN+(CHs)s

RI R CH3 CiHa C4Ho CHsCO

H

+

CH3 CaHa C3H7

-

+ ++ + + -

f

C4He

-

++ -

-

-

-

-

Predicted CSHII

f

++ ++ ++

0 Adapted from Gyermek (1967). “Drugs affecting the Peripheral Nervous System” (A. Burger, ed.), Vol. I, by courtesy of Marcel Dekker, Inc.

MOLECULAR STRUCTURE OF RECEPTORS

161

FIG. 28. ( a ) Two ways in which choline methyl ether could bind to the ganglionic receptor. ( b ) Diagram of how the butyl group contacts the opposite wall of the receptor lip if the compound binds as shown in 28a (lower). ( c ) If it binds as shown in 28a (upper), the butyl group now does not make contact.

6 . Zsoquinolines The compound shown in Fig. 29a is a potent ganglion blocker (10 X TEA). It is chemically very similar to the potent P-adrenergic agonist metaquinol, which suggests that there may be structural features in common between adrenergic and cholinergic receptors (see Section 111, H) . The catecholamine structure is suited to bind across an Arg-Glu bond. One hydroxyl binds by its proton to 0: of Glu, and the other accepts a hydrogen bond from the NH of Arg. This binding would be aided by the fact that the two hydroxyls normally orient facing in opposite directions owing to their dipole nature. The suggested mode of binding to the ganglionic receptor is that the two hydroxyls bind to the Arg-Glu rung 2 as described, the CH,-Ph group intercalates between rungs 2 and 3, the N+ binds to Asp (-) and Glu (S-), and there are additional lipophilic bonds to Ile as shown in the figure that are not attained in the neuromuscular junction owing to the different location of Ile in the two receptors.

7. Trimetaphan Trimetaphan (Fig. 29b) is rather an unusual but very potent (100 x TEA) ganglionic blocker. I t binds by a hydrogen bond from Arg NH of rung 3 to the carbonyl 0; the two benzene rings intercalate one on each side, and the lipophilic ring system wraps around Ile with an anionic bond from S+ to Asp. The extensive lipophilic binding explains the high potency of the compound.

"qy-h HO

6

(a1

I + ~ t ~ ~ - y / ~ ~ p -

"-FH

cYH2 (b)

FIG. 29. ( a ) Ganglion-blocking isoquinoline. (b) Trimetaphan.

162

J . R. SMYTHIES

K. THEACETYLCHOLINE MUSCARINIC RECEPTOR The required pattern of the primary chains is Arg: G1u:Glu:Glu. This determines that the secondary chains will both be E (Fig. 9 ) . The sequence in the two E chains is different. The upper right one has Val and Asp, and the lower left one Ile and His, on the inside of the lateral wall (Fig. 9 ) . This results in a much smaller receptor cup than in the case of the two nicotinic receptors I have described. The Glu 0 of rung 2 and both Arg NHs of rung 3 are occluded by the overlying hydrophobic amino acid (Val/Ile). Thus only one proton ( N H of Arg in rung 2 ) and one spare electron pair (0:of Glu in rung 3) are exposed in the floor of the site.

L. MUSCARINIC ACONISTS The “fit” of acetylcholine itself is illustrated in Fig. 30. The conformation required ( t 2 = +130°; t S = +150°) lies within the range designated by Baker et al. (1971) ( t . = +73O to +137O; t s = 180 35”).* It binds as follows: ( 1 ) ester 0 to the NH group (Arg) of rung 2 ; (2) its acetyl methyl

*

FIG. 30. Acetylcholine bound in its muscarinic receptor. ‘The angle t l is that between C, and the C8 atoms; C8 atom and the ester 0.

ta

is the angle between the

MOLECULAR STRUCTURE OF RECEPTORS

163

group to the end wall of the receptor cup; (3) the p(-) ( C ) H to the adjacent lipophilic amino acid (Val) ; (4) by both a (C)H’s to the lipophilic amino acid (Ile) on the other side of the receptor cup; and (6) the onium head tucks into the anionic pocket between Asp and Glu with one methyl group in the lipophilic gap between Val and Asp methylene. The lipophilic amino acid on this side cannot be Ile for, as we saw earlier, this leaves no gap (with an E chain) between it and Asp. T o bind with Ile present in this locus the angle tl has to be about +160°, well outside the Pauling range (see Baker et al., 1971). This applies also to the old prostaglandin-protein model, as the hydrocarbon chain of a PG does not have the required gap in this location. The gap is larger if the lipophilic amino acid is Thr or Ala. However, in all the stereochemical investigations that I have carried out, Val allows a reasonable fit. But different receptors in different loci might differ in just such a conservative substitution. This close fit entails that the acetyl methyl group cannot be increased to ethyl and that neither the p ( - ) nor either (Y hydrogen can be replaced by methyl [whereas the p(+) H can], otherwise steric hindrance results. A p( -) methyl group contacts the p ( C )H of the adjacent lipophilic amino acid (which can thus for this purpose be Ala) whereas methyl groups on the a C atom clash with the 6 C atom of the lipophilic amino acid on the other wall of the receptor cup-which must therefore be Ile, since both the p ( - ) and both a methyl derivatives of acetylcholine have lost their muscarinic potency to varying degrees. T h e /3 (+) compound, mecholyl, is as active as ACh. The fact that the S (IME compound is 8 times less active than the R isomer (Brimblecombe et al., 1970) indicates that the angle t 2 must be around +130°, as if it were around +70° [with greater entry of the onium head into the Val-Asp (or Ala-Asp) gap], the R isomer would be more occluded than the S. The +130° t z angle is compatible with Val adjacent, but a +70° angle leads to some steric crowding between the onium head and the a methyl group of Val, which replacement of Val by Ala or Thr would relieve. Thus the comparison of the R and S isomers of methyl ACh in muscarinic receptors in different tissues could be used as a molecular probe to test whether this lipophilic amino acid was Val or Ala (Thr) . (I-

1. Muscarine and Muscarone Only one of the four possible stereoisomers of muscarine is active (Fig. 31a) whereas both stereoisomers of muscarone are active (Fig. 31b). The stereoisomer of muscarone corresponding to the active isomer of muscarine is, however, only about one-third as active as its enantiomer. The active isomer of muscarine in the Pauling conformation (envelope with C, forming

164

J. R. SMYTHIES

FIQ.31. (a) Muscarine. (b) Muscarone. ( c ) Agonist dioxolane. ( d ) Oxotremorine.

the downward facing flap) has a square end, the corners of which are formed by the hydroxyl and the methyl groups. If the ring 0 binds to the NH of Arg of rung 2 by the spare electron pair cis to the hydroxyl group, this square end fits snugly into the right-angle between Val and the Gly-x-Gly upper wall of the receptor cup. The O H group forms a hydrogen bond with the A cloud of the peptide bond between x2 and Gly2, and the methyl group forms a lipophilic bond to x2 and the His methylene group. The C,aH and the C,H bind lipophilically to Val, the CaaH to Ile, and the onium head fits into the anionic pocket as for ACh. The 3 position is unhindered, and it is known that substitution here by phenyl is compatible with activity. The C,PH and the C,BH are also unhindered. The methyl group cannot be moved to the a locus as steric hindrance results, and moving the hydroxyl to the p locus results in the loss of the prescise fit and one hydrogen bond. The molecule could not bind by the other orbital in the ring 0 (to Arg N H ) , as this would lead to the occlusion of the 3 positions and disallow any phenyl substitution, In the case of muscarone, the molecule may bind by this other orbital on the ring 0. This allows the His moiety in the receptor to form a hydrogen bond with the carbonyl 0. The different ring structure (half-chair) allows the methyl group to be set at either locus on the 2 carbon. In the case of the more active R isomer, the methyl group attains a good lipophilic contact with Val, whereas in the case of the less active S isomer, the lipophilic bond to the end wall of the site is less extensive.

2. Dioxolane Only one of the four possible stereoisomers of the dioxolane series (Fig. 31C) is active. The molecule binds by the orbital on the ring 0 trans to the methyl group (as for muscarine). The methyl group attains a lipophilic contact with the end wall of the receptor cup. The C4crH, CsaH, and C2aH achieve a good lipophilic contact with Val and both C,Hs with Ile on the other side. The onium head fits into the anionic pocket.

MOLECULAR STRUCTURE OF RECEPTORS

165

3. Oxotremorine Earlier it was stated that the ACh muscarinic receptor cup, in this model, has an irregular cavity. The high activity of ,oxotremorine (Fig. 31d) may be related to the fact that it is, as it were, like a plaster cast of the inside of this cavity (Fig. 32). Each ring system fills one recess at each end with lipophilic contacts to Gly, Ile (Val), and Asp (His) methylenes (Fig. 33).

+

The basic N (protonated to NH) attacks Arg-Glu bond No. 3 binding to Glu and repelling Arg. The carbonyl 0 in the other ring accepts a hydrogen bond from His (hence a second Asp here will not d o ) , and the ring amide r cloud accepts a second hydrogen bond from the Arg NH of rung 2 underneath. The acetylenic bridge lies snugly in the “waist” of the cavity betwem Ile and Val.

Fro. 32. Diagram of close fit of oxotremorine into cavity of muscarinic receptor.

FIG.33. Model of oxotremorine in muscarinic receptor.

166

J. R. SMYTHIES

Bebbington and Brimblecombe ( 1965) have presented structure-activity relationship data on oxotremorine. The replacement of the pyrollidine ring by N(CH3), reduces activity lox and by N(C,H,), 1OOX. I n the former case lipophilic interaction with the receptor is reduced [N ( CH3), is too small], and in the second case there is steric hindrance [N(C2H6), is too large]. This suggests that N(CH,) (C,H,) might be more active. If the acetylenic bond is replaced by ethylenic, or -CH,-CH,-, or a benzene ring activity is lost, these molecules have quite different shapes and no longer fit the receptor. In the other (proline) ring, the ring can be broken to give C H 3 * C 0 * N ( C H 3 ) - with only a slight decline in activity. This is equivalent to the N(CHs) (C,H5) compound adumbrated above for the other end of the molecule. A bulkier group such as (CH3),N*CO*N(CH8)- leads to a 20X reduction of activity. If the proline ring of oxotremorine is replaced by a succinimide or maleimide ring a series of blocking agents is produced (Karlen et al., 1970). Blocking activity is particularly enhanced if the -CH,link between the ring system and the acetylenic group is replaced by - C H * C H 3 - or -C* (CH,) ,-. These methyl substitutes ensure that the carbonyl 0 can no longer get in the correct location to receive a hydrogen bond from His. The only way for the compound to bind to the receptor is if this ring system intercalates between rungs 2 and 3 of the Arg-Glu grid and bind by AT interaction. These rings generate quite an extensive A cloud. As- we shall see such w-r interactions are often associated with blocking activity, particularly if no charge transfer reactions are involved.

M. MUSCARINIC ANTAGONISTS The classical muscarinic antagonist is atropine, which is the racemic form of hyoscyamine, of which the S form is much more active than the R (Fig. 34a). This fits closely into the model receptor as follows (Fig. 35) : ( 1) ester 0: (right : ) hydrogen bond from Arg N H of rung 2; ( 2 ) hydroxyl hydrogen bond to His ( 2 N : ) ; (3) -CH,-(OH) lipophilic bond to - lipophilic bond to Val; (5) NH electrostatic bond x,CH; (4) (+)-CH to Asp 0: or r u n r 3 ; (6) ring methylenes-lipophilic bonds to Ile and Val on each side; ( t ) benzene ring intercalates between rungs 1 and 2; (8)the carbonyl 0 is not used directly.

*

This formation explains the importance of the stereochemistry at 4-Cwhich is due to the requirement that the benzene ring, OH and C H groups all match the position of the intercalation site, the His N : and Val in the receptor complex, respectively. The N can be quaternized, and if the extra group is -CH2-+-+ (as in gastropin) the biphenyl group can intercalate which could not). A normal between rungs 3 and 4 (as opposed to

-+-+,

MOLECULAR STRUCTURE OF RECEPTORS

167

(C)

FIO. 34. ( a ) Atropine (S-hyoscyamine). ( b ) General form of antimuscarinic glycolic acid esters. ( c ) Formula of a very potent antimuscarinic dioxolane.

N-butyl substitution (Buscopan) allows a good lipophilic contact with the end wall of the receptor. In the other stereoisomer of atropine in which the substitution at the 3 carbon atom is reversed to produce pseudotropine, the fit is similar except that the axial N-methyl group goes “down” instead of the ethelene bridge. These compounds are active.

Fro. 35. Fit of atropine into muscarinic receptor.

168

J. R. SMYTHIES

1. Glycolic Acid Esters Abood and Biel ( 1962) have reviewed the structure-activity relationship data of a number of anticholinergic glycolates, some of which have potent CNS activity producing a delirium. The general form of these compounds is given in Figure 34b. R, must be hydroxyl; H, C1, or Me here lead to inactive or very weak compounds. However, the O H group may be transferred to the 1 position on the cyclohexyl ring at R,. But there cannot be hydroxyls at both loci. R, is preferably phenyl, although propyl is compatible with anticholinergic but not psychotomimetic activity. R, must be phenyl, thienyl, cycloalkyl, or l-OH-cycloalkyl. The most potent is cyclopentyl. These compounds bind in a manner equivalent to atropine; i.e., R, intercalates, R, hydrogen bonds to His, and R, fits into the lipophilic pocket between Val, Gly, and x4CH (and their associated pseudo T clouds), If R, is H, a hydroxyl at the 1 position on the cyclohexyl ring can bind to His N : instead. But a hydroxyl at both loci will form an internal hydrogen bond that will interfere with the bond to His. When R, = R, = phenyl (+), Abood and Biel state that substitutions on completely destroyed the antithe phenyl ring, such as C1, Me, MeO, or cholinergic property of the compound. Examination of the model shows that any ortho substitution leads to internal steric hindrance so that the molecule cannot take up the proper conformation for binding. Second, the benzene ring that intercalates does so at an angle rather than vertically and so could not tolerate any m or p substitution either. However, the other benzene ring that lies on top of the secondary chain is unhindered in these loci, and thus m or p substitutions in one ring should be tolerated. Abood and Biel do not comment on such compounds. The essential (Ph) (cycloalkyl) ( C H ) group (R configuration) is found in other potent antimuscarinic agents, such as artane, a series of dioxolanes (Figs. 34c, 36), etc.

+,

Fro. 36. Model of very potent dioxolane blocker (hydrogen bonds in sifu).

MOLECULAR STRUCTURE OF RECEPTORS

169

2 . Dibenzpetimide This potent compound (Fig. 37) was developed by Janssen’s group (Spek et al., 1971). The S isomer is very potent and the R is quite inactive. This forms an extremely close fit of the model receptor as follows (Fig. 38). The basic NH binds to the anionic site (Glu 0 : or rung 3 ) , and both benzene rings intercalate, one between rungs 2 and 3 and the other between rungs 3 and 4 of the grid. One carbonyl 0 binds to the Arg N H of rung 3 and the other to His NH. There are also extensive lipophilic contacts between ring methylenes and Val and Ile. In the R isomer, one carbonyl 0 would approach Asp but not His, which is inappropriate.

3. Platyphylline The strength of an hypothesis of this type depends in part on its ability to explain a wide range of otherwise unconnected data. The compounds

51 FIG. 37. Dibenzpetimide.

FIG.38. Dibenzpetimide bound in muscarinic receptor.

170

J. R. SMYTHIES

of the type we have been discussing have for the most part borne some kind of relationship to each other. The hypothesis gains strength if it can also account for the action of active drugs of a totally different chemical structure on the same receptor. These new compounds may bind to groups in the receptor complex not utilized by the first class at all. Pomeroy and Raper (1971) have filled this gap for the muscarinic receptor by finding

FIG.39. Platyphylline (a) and helurine ( b )

Fro. 40. Platyphylline, Corey-Pauling-Kaltun model : hydrogen bonds in

situ.

MOLECULAR STRUCTURE OF RECEPTORS

171

that platyphylline and helurine (Figs. 39 and 40),both pyrrolizidine alkaloids, are good muscarinic antagonists. The entire molecular structure, of which the conformation has been deduced on the basis of X-ray and other data (Pomeroy and Raper, (1971) is important, as minor modifications lead to a loss of activity. For example, seneconine in which the 1,2C-C is replaced by -C=C--, is inactive. Platyphylline has a very close stereochemical relationship to my model receptor as illustrated in Fig. 41. This shows that, whereas most antagonists (e.g., atropine) lie with their long axis along the long axis of the receptor cup, in the case of platyphylline the long axis is at right angles to the receptor cup. The pyrrolizidine ring forms, as it were, a foot which binds to the anionic site and the surrounding lipophilic area. The rest of the molecule juts out of the receptor like a leg with the extensive bonds shown in Fig. 41. This includes one to a group not utilized by any other compound so far examined, the peptide NH of Glyl. Pomeroy and Raper (1971) suggested that platyphylline may be homomorphic with atropine with the following isoteric groups: basic N and basic N; ring 0 attached to C, of the pyrrolizidine ring and atropine ester 0; carbonyl 0 attached adjacent to C2 of the pyrrolizidine ring and atropine carbonyl 0; the adjacent hydroxyl and atropine hydroxyl ; and the lipophilic -C(CH3)-CH*CH3*CH2*C=CH(CH3)system and the atropine benzene ring. My model suggests that the first of these is correct, but the rest are not. Platyphylline covers a large part of the receptor not covered by atropine and does not cover the same part to which the ester 0 and benzene ring of atropine bind. The third group of platyphylline listed above binds to the same group as atropine OH, but the dipole (His) is reversed. If the C2-C3 is double, the superstructure now is located in a different locus, and the resulting compound (seneconine) is no longer active. The lipophilic superstructure of platyphylline may contact some groups in the vicinity of the receptor outside the receptor cup.

Fro. 41. Mode of binding of platyphylline to muscarinic receptor. Molecular model building will elucidate the number code.

172

J . R. SMYTHIES

4. Tricyclic Compounds Tricyclic compounds, such as imipramine (Fig. 42a), and certain phenothiazines, e.g., transergan (Fig, 42b), have anticholinergic properties. These cause annoying side effects in the treatment of depression with such agents. If the side-chain NH group of imipramine binds to Asp, the lipophilic chain runs up between Ile and Val, binding mainly to the former. One N-methyl group also contacts Ile. One phenyl ring intercalates between rungs 2 and 3, and the other makes a lipophilic contact with Val and part of the adjacent secondary chain. His forms an electrostatic link with the ( & ) N linking the two rings.

5 . Dibenamine My specification of the adrenergic receptors is similar to that of the muscarinic receptor (see Section 111). It is, therefore, of interest that dibenamine blocks muscarinic receptors as well as its well known effect in blocking a-adrenergic receptors. In the former case it probably acts by alkylating carboxyl or His (Beddoe et al., 1971). My model suggests that this group is likely to be Asp. The N H group binds to the anionic site (Glu 0:) and the two benzene rings intercalate one on each side. The alkalating group is now in contact with Asp. In the a-adrenergic receptor, dibenamine alkylates Arg rather than His.

N. Do AGONISTS A N D ANTAGONISTS BINDTO

THE

SAMERECEPTOR?

Brimblecombe et al. (1970) have pointed out certain differences in the structure-activity relationship requirements of cholinergic and anticholinergic drugs at muscarinic receptors and have argued that this indicates that the receptors for these two types of compound are at least in part different. In compounds of the type X-CO

+

*

CHR, * CHR, * N (Me) where X may

CO

N 1 CH3l2

I 0 I

y21, n(Etl, (a)

(b)

FIG. 42. ( a ) Imipramine; (b) transergan.

MOLECULAR STRUCTURE OF RECEPTORS

173

be CH, (agonist) or C.+.C,H,,-OH (antagonist) the following structure-activity relationship data have been found: (1) Replacement of the N methyl groups in the agonist by any other alkyl group leads to a great reduction in activity; whereas in the antagonist a wide range of such replacements is possible. ( 2 ) a-Methyl substitution in muscarhic agonists reduces activity (but the R compound is still 8 times as potent as the S) whereas in the antagonists such substitutions increase activity but stereospecificity is lost. (3) p-Methyl substitution reduces agonist activity greatly if R, but not if S. In antagonists activity is reduced but R=S. (4) If the ester 0 is replaced by S, agonist activity is greatly reduced, but not antagonist activity. ( 5 ) I n the agonist dioxolane, stereoisomerism at C4 is important, and in the antagonist dioxolanes it is the C, stereoisomerism that counts. In my model these facts may be explained as follows. The essential function of the agonist is to disrupt R to produce R,. I t does this by disrupting Arg-Glu link No. 3 by locating a sufficiently powerful electrostatic charge close enough to it-in this case a quaternary ammonium group. The agonist requires enough accessory binding groups to make it specific for this receptor and to enable it to locate the onium head correctly. The essential function of the antagonist is to bind as tightly as possible to the receptor complex without disrupting the Arg-Glu link. This involves as close a steric fit as possible to allow multiple weak interactions to summate, and as many hydrophobic and polar bonds as possible in addition. If the antagonist has a trimethylammonium head, then it must either locate this not touching the Arg-Glu bond at risk and/or provide other groups that will tie the two P pleated sheets that form the two halves of the receptor together. For this, hydrophobic binding and multiple weak interactions are especially effective. As noted above, replacing N-methyl groups by N-ethyl or larger groups encourages lipophilic binding and reduces the effective charge on the surface of the onium head-both of which will tend to promote antagonist action. However, the onium head must not get too large to fit inside the receptor cup. If one end of the antagonist molecule is firmly bound in the receptor by the C*+.C,H,, O H system, a- or P-substituted compounds can bind even if the ester 0-Arg NH hydrogen bond cannot be made. This is so because the ionic link between the onium head and Asp is nondirectional, like all ionic bonds, and so the onium head has a wide range of possible positions in the receptor cup; and because the energy lost by the loss of this hydrogen bond can be more than made up for the opportunity for a stronger hydrophobic bond offered by the extra methyl group (i.e., in the case of a substitution but not P ) . To contact both Asp and Glu, however, which is necessary for agonist action, there is only one possible location for the onium head, and this position is incompatible with an a- or p ( - ) -

174

J. R. SMYTHIES

methyl substitution. In antagonists the stereoisomerism at the -C * +*C,H,, OH system is all-important as the cyclohexane group is not suited for intercalation and the hydroxyl must be in the right location to bind to His. Thus the antagonist, in this formulation, binds essentially to the same receptor as the agonist but utilizes in part different portions of it and in a functionally different manner.

111. Adrenergic Receptors

Receptors responsive to epinephrine are found in a wide variety of tissues, often in association with the enzyme adenyl cyclase. Receptors responsive to norepinephrine are found, particularly in association with sympathetic nerve endings and in the brain, where norepinephrine is concerned with several important hypothalamic and limbic mechanisms. Dopamine receptors are prominent in the extrapyramidal system. Adrenergic receptors have been divided into @-receptors,which respond best to compounds like isoprenaline, and a-receptors, which respond best to norepinephrine and epinephrine. Different organs may have complex patterns of a- and p-receptors. A. THEADRENERGIC @-RECEPTOR An examination of the molecular structure of a range of @-agonistsand antagonists has led to the specification of the following molecular structure. The primary structure consists of only three rungs of the grid for which the sequence is 41u-x-Glu-x-Glu(Fig. 43). There is only one secondary chain ( F ) in the upper right position. The @ receptor has Ile and Asp on the inner aspect of the lateral wall and the a-receptor differs mainly in that Asp is replaced by a nonpolar amino acid, such as Ala or Gly. An alternative form uses Gln in place of Glu. The suggested binding of isoprenaline is shown in Fig. 43. The two phenolic hydroxyls bind across the Arg-Glu link No. 2 as described in Section 11, I, 6. The p hydroxyl proton binds to Glu 0: of pair three; the a-methylene group makes a close hydrophobic bond to the methylene of this Glu; the NH group binds ionically to Asp COO- and the isopropyl group slots into the hydrophobic gap mentioned previously between Ile and Asp methylene. The advantage of the isopropyl group on N is that it can easily insert one of the two terminal methyl groups into this lipophilic slot as can a terminal terbutyl group (also active). That is to say, whatever the rotational angle of the isopropyl group on the N may be as the molecule approaches

MOLECULAR STRUCTURE OF RECEPTORS

175

the receptor, only a small rotation a t most is needed to align the methyl group properly. Whereas, if the N substitution is ethyl, the terminal methyl component of this can slot in, but the required rotation may be much larger (see further on this below). This applies also to the previous PG-protein model, which does not have any comparable slot in its side wall as the PG hydrocarbon chain simply continues to the COO- group. In that model one would predict the n-propyl would be a more potent N substituent than isopropyl. As the opposite is the case, this evidence favors the present pure protein model rather than the PG-protein model. The function of epinephrine in such a receptor would be to maintain or promote the closed conformation of the receptor protein: R, A + [RIA] + [RA]. I t would do this by promoting the Arg-Glu link No. 2. The 3-OH group aligns Glu correctly to bind Arg and then the 4-OH group binds Arg. There may be an associated change in the local water structure; e.g., the benzene ring may disrupt the water shells around ionic Arg and Glu that tend to hinder the formation of ionic bonds in a water environment. Thus epinephrine in the p receptor would appear to be acting in a manner directly opposite to that of ACh in its receptors. Presumably the altered protein conformation consequent on epinephrine binding is transmitted through the protein molecule to an adjacent ionophore, or to the active site on adenyl cyclase, if the particular 8-receptor is an allosteric site on that enzyme.

+

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J. R. SMYTHIES

B. ~-AGONISTS Since the postulated function of epinephrine in the p-receptor is to promote the reaction R, + R, p-agonist activity will be increased by promoting the binding of the epinephrine-like molecule to the receptor-for example, by a suitable adjustment of the N-alkyl group. This fits in with the conclusion reached by George et al. ( 1971) that the function of the N-alkyl group in a p-agonist is to act directly on the receptor rather than by influencing the side-chain conformation relative to the catecholamine ring, or affecting the charge distribution around the N. Table I1 gives the relative potency of a number of N-alkylated catecholamines of the general form where R = Me is epinephrine. The hydrophobic contact between R and the Ile-Asp methyl system in the receptor for these different alkyl substituents is shown in Fig. 44. The detailed mode of interaction explains the observed potency of the compounds. Compound number (Table 11) : (1) (Epinephrine) The CH, group contacts Ile CH, (Fig. 44a) ; ( 2 ) The terminal CH, group can now slot into the Ile-Asp gap and form a tight bond by doing so (Fig. 44b). (3) The terminal methyl group cannot now slot into the gap which is too shallow to accept C,H,--. The propyl group can therefore only run along the Ile chain with a less extensive hydrophobic bond (Fig. 44c). (4) Th'is compound (isoprenaline) has already been discussed. ( 5 ) Compound 5 extends TABLE I1 OF DIFFERENT N-SUBSTITUTED RELATIVES OF POTENCY EPINEPHRINE Compound

R

1 2 3 4 5 6

-CHI -CiHs 11CsH7 -iCaH7 -nC4H7 11CsHii -cyclopentyl -(CHi)r-Ph -C.(CHJ)r--CHi-Ph -(CH2)a-ph -CH(CHs)-(CH2)-Ph -C (CH,) $-(CHI) S-Ph -CH(CH~)-CHI-P~OH -CH (CHa)-CHa-Ph-3,4-methylenedioxy

7 8 9 10 11 12 13

14

Activity 15 60 11 100

a 25 200 11 250 100 500 100 1000 800

MOLECULAR STRUCTURE OF RECEPTORS

177

FIG.44. The fit of various N-alkyl substituents on P-agonists to the AspIle-Gly system in the wall of the receptor cup (see text). the process started in 3, and only in compound 6 does activity increase for a similar reason that determines the “rule of five” in the case of the cholinergic muscarinic receptor-the terminal methyl group now connects the end wall of the receptor cup (Fig. 44d). (6-14) Compound 8 is equivalent to to Ile). 5, and compound 9 to 4 (with an additional bond from -CH,-Ph Compound 10 is equivalent to 6, with a more extensive contact from the terminal Ph to the end wall of the site. In compounds 9 and 11-14 the a-methyl group can slot into the crevice. In compound 14 (protokylol), the methylenedioxy group has the same function as this group in bicuculline and the dimethoxy group in curare, which is to convert the round contour of the benzene ring into a square corner to mortise into the square outline of the receptor cup inside the Ile-Gly corner. In compound 13, the most potent of them all, the para-hydroxyl group attains a good hydrogen bond with the carbonyl 0 of x4. The only obscure result is with compound 7, as the cyclohexyl ring is much too large to slot into the hydrophobic crevice: possibly its more extensive area of contact with Ile terminal Me is responsible for its high potency. The above data indicate that a methyl group fitting into a hydrophobic pocket that closely invests it gives much more potent binding than two hydrocarbon chains running parallel to each other. The conformation of epinephrine (or isoprenaline) required for this fit is shown in conventional form in Fig. 45a, where B = 300° (-60°), corresponding to the second preferred minimum energy conformation of George e t a l . (1971). The close contact between the a-methylene group of epinephrine and the /I-methylene group of Glu in the receptor explains why a-alkyl substitution of agonists usually leads to inactive products. However, there is one fascinating exception to this rule (Table 111, from AriEns, 1967). If R is

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J . R. SMYTHIES

(a 1

(b)

Fro. 45. (a) Required conformation of epinephrine to fit P-receptor. ( b ) Required conformation of norepinephrine to fit a-receptor. methyl or propyl ( i / n ) activity is very greatly reduced. Whereas if R is ethyl, activity is not reduced at all, but only if R, is isopropyl or cyclohexyl. It seems very difficult to think of a circumstance that would allow an ethyl, but neither the smaller methyl nor the large propyl, group. However, examination of the model suggests one way in which this remarkable pattern of activity may be explained. If the R group has the S stereochemical configuration and if the three hydroxyl groups bind as for epinephrine, the a-methyl group forces an anticlockwise rotation of t 2 ( C A B bond) such that the N+H and the isopropyl groups are hopelessly out of position. But if the R group has the R stereochemical configuration, the forced rotation is now clockwise. This means that the NH+ group can still contact Asp COO- and the isopropyl Me group now contacts the Asp methylene on the other side of the Asp moiety (as compared with the location of this group in isoprenaline). In this event if, and only if, R is ethyl can the terminal methyl group (of R) slot into the Ile-Asp crevice (Fig. 46). R cannot be methyl TABLE 111

EQUIPOTENT MOLARRATIOSOF ~~-ALKYL-SUBSTITUTED DERIVATIVES OF ISOPRENALINE ( = l)a (0H)r.Ph.CH (OH) C H R.NHR

Ri R

H CHa

CzHs nCsHi iCaH7 a

H

iCaHr

150 100 100 1000

1 1000 2.5 >loo0 1000

From Ariens (1967).

Cyclopentyl Cyclohexyl 0.6

> 1000 1 1000

10 100 7 > 1000

MOLECULAR STRUCTURE OF RECEPTORS

179

FIG. 46. R-a-ethyl isoprenaline bound in preceptor.

(too short), propyl (too long) or isopropyl. I n the latter case, as the two of the active ethyl compound are occluded by the H’s on the --CH,-part adjacent benzene ring, they could not tolerate methyl substitution to give isopropyl. Thus the model predicts that ( R )-a-ethylisoprenaline will be active and its (S) isomer inactive.

C. OTHERp-AGONISTS(Fig. 47) The 4-hydroxyl group seems essential for /3 activity (Brittain et al., 1970), but the 3-hydroxyl does not, as the pure p-agonist nylhydrin (Fig. 47a) lacks it. With soterenol (Fig. 47b) the meta NH group binds as does the meta O H group of epinephrine and the methyl group binds to Ile adjacent. The SO, group sticks upward into water. Salbutanol (Fig. 47c) group in place of the meta hydroxyl. This achieves has a -CH,OH a better bond angle to Glu 0: than O H does, but the methylene group is rotated away from the adjacent Ile and does not contact it. I n quintereno1 (Fig. 47d) the bonding is (hydrogen bond) OH to Glu 0: or rung 2 and N : from NH of rung 2 (electrostatic). There is also a confact with Arg of rung 3 (NH to w cloud; lipophilic between ben-

180

J . R. SMYTHIES

111

FIG. 47. Some p-agonists: (a) nylhydrin, ( b ) soterenol, ( c ) salbutanol, ( d ) quinterenol, ( e ) orcipenaline, ( f ) trimetoquinol. zene ring CH's and Arg methylenes). In orcipenaline (Fig. 47e) there are two meta OH groups. This compound could bind by one OH to Arg NH of rung 2 (the essential group to contact), but the second OH group could make only a very poor H bond to Glu 0:. Alternatively, one O H ,could bind to Glu 0: of rung 2 (well) and HN of Arg of rung 3, but the latter bond angle is again poor. Trimetoquinol (Fig. 47f) has been mentioned above in connection with the ganglionic ACh receptor, as it resembles closely one of the ganglionic blockers (see Fig. 27b). If the OH, OH, and NH groups bind as do the three OH groups of epinephrine, the large trimethoxybenzene ring system intercalates between rungs 2 and 3 of the grid. The NH+ group can also contact Asp if the latter rotates "down" at the C,Cb bond. The benzene ring is not essential and can be replaced by a fully saturated ring. In my model it functions purely as a carrier of functional groups (plus possibly some quantum chemical contribution to their charge) . No attempt has been made to determine what might be the structural basis of PI and p2 receptors as their real status seems uncertain (Buchner and Patil, 1971).

MOLECULAR STRUCTURE OF RECEPTORS

181

D. p-BLOCKERS (Fig. 48) If the function of an agonist in a p-receptor is to promote the reaction R, + R (that is to promote the closed conformation of the receptor), then clearly an antagonist will have to be able to bind to R, and prevent the change in the conformation of the receptor protein that leads to R, in this case closure of the Arg-Glu bonds to form the cross-linked p structure and formation of the hydrogen bonds from the secondary chain to Arg of rung 1. A prominent series of /3 blockers (Fig. 48) replace the ring hydroxyls of a compound like isoprenaline with large lipophilic groups, such as C1 or alkyl. The normal function of the 3-OH group in an agonist may be to orient the Glu carboxyl group correctly to bind Arg, and the function of the 4-OH group is to bind this Arg itself. Therefore reC I O C H I OH) CH, WHXH I CH,Iz CI lot 0.CHz.CHIOHt C H ~ . N H . C H I C H ~ t z

Ibt

H

FIG. 48. Some p-blockers (see

text).

placements of these polar groups by C1 or similar lipophilic groups will prevent closure of the Arg-Glu bonds. The 3 C1 group will bind to the hydrocarbon chain of Glu instead of the carboxyl group and align this wrongly, and the second C1 group will repel Arg. The lipophilic group does not have to be located on top of this Arg-Glu link that is to be inhibited. In compounds like propanolol and LB 46 (Fig. 48), the lipophilic ring system intercalates between rungs 2 and 3 and prevents access of Arg.

E. THEa-RECEPTOR The hypothesis suggests that this differs from the /%receptor mainly in that Asp in the secondary chain is replaced by a nonpolar amino acid, such as Ala or Gly, or perhaps by a basic one, such as His. It may also have two secondary chains (see below). The most potent a agonists are epinephrine and norepinephrine (which is weak at p-receptors) . The suggested mode of binding and mechanism of action of norepinephrine at the a-recep-

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J . R. SMYTHIES

FIG. 49. Norepinephrine bound in the a-receptor.

tor is as follows (Fig. 49). The phenolic hydroxyls bind as before across the Arg-Glu link of rung No. 2. In the absence of Asp alongside in the secondary chain (plus a possible active repulsion by His) the N'H, group of norepinephrine binds to the next closest &group which is the 0: of Glu on rung 3. I n this case the P-OH receives a hydrogen bond from Arg of rung 3. This requires slightly different bond angles in the hydrogen bonds of rung 2 so as to allow the molecule to swivel round through the 10' needed. The conformation of norepinephrine required for binding in this manner is shown in Fig. 45b, which is the preferred conformation as determined by Pedersen et al. ( 1971) , The function of norepinephrine here would be to disrupt Arg-Glu rung No. 3 by contact with the N+H, group. This is as it were an opposite effect to that exerted at the P-receptor, as R, is produced rather than R as a consequence of transmitter action.

Brittain et al. (1970) concluded from a study of the structure-activity relationship data that the important group in an a-agonist is the N'H,

MOLECULAR STRUCTURE OF RECEPTORS

183

FIG. 50. Some a-agonists: ( a ) phenylephrine, ( b ) naphthazoline, ( c ) zylometazoline, ( d ) tuaminoheptane.

group-as indicated above. If the para-OH of epinephrine is removed, a pure a-agonist (phenylephrine, Fig. 50a) results. Both hydroxyls may be removed and the catecholamine ring replaced by a group suited for intercalation, as in the case of naphthazoline or zylometazoline (Fig. 50b,c). In these the 8-ring N receives a hydrogen bond from the Arg N H underneath, and the positively charged ring NH disrupts the Arg-Glu link. Tuaminoheptane (Fig. 50d) is an a-agonist quite unrelated in structure to epinephrine but closely related to various cholinergic compounds, such as pentyl TMA. It is clearly adapted to lie along the top of the secondary chain in the /I-receptor binding to Asp ionically; to Ile and Pro lipophilically and inserting its a methyl group into the strategic Ile-Asp gap from above. Thus it would appear to convert the p-receptor into a passable imitation of the a-receptor so that NA binds to it in the manner promoting R + R, rather than R, + R. Thus, its a-agonist activity may be due not to any action on a-receptors but to the fact that it alters the ratio of alp-receptors in the tissues by converting the latter into surrogates of the former. G. a-BLOCKERS 1. Phenoxybenzamine

The relationship of dibenamine and phenoxybenzamine (Fig. 8a) to an Arg-Glu grid has already been noted. The N H binds to Glu 0: of rung 2; both benzene rings intercalate, one between rungs 1 and 2 and the other between rungs 2 and 3. The methyl group binds to Ile and Glu methylenes (but only if R : the R isomer is 14.5 times as potent as the S ) . The group alkylated will be Arg NH.

2. Ergotamine (Fig. 5 Z b ) Ergotamine is a large, complex, and mainly fixed molecule that is likely therefore to subject the model of the receptor to a stringent test. If both the lysergic acid and the benzene rings intercalate (between rungs 1 and

184

J. R. SMYTHIES

la1

Fro. 5 1. Some a-blockers: ( a ) phenoxybenzamine, (b) ergotamine, ( c ) emetine.

2 and 2 and 3, respectively), both carbonyl 0 s reach down from the cyclic polypeptide chain to receive hydrogen bonds from the Arg N H s of rungs 2 and 3. The proline methylenes now make an extensive lipophilic bond with Ile. Previously, I had supposed that the a-receptor might differ from the p-receptor in lacking any secondary chain. But in that case this Pro has no counterpart in the receptor. Besides, it is necessary to eliminate only Asp in the secondary chain, not the whole secondary chain itself, to effect what is required. The hydroxyl group of ergotamine achieves functional importance if the Asp of the secondary chain is replaced by His rather than Ala or Gly. This His can form a hydrogen bond with the hydroxyl group of ergotami ne . If the methyl group on ergotamine is replaced by ethyl to give ergokrystine, this leads to a considerable increase of activity. This suggests that the receptor has a lipophilic group in a complementary location to bind this. This in turn suggests that the a-receptor may have to secondary chains. If SO, the second must also be F to locate Ile (or Val) in the correct complementary relationship. Thus, one can very tmtatively suggest that the a recepwith Ile and His and the other with (Ile or tor has two F chains-ne Val) and Asp. This would not therefore bind curare, as the His in one corner would clash with the N+ of curare. There is no evidence from the structure of any agent acting on the p-receptor that this has two secondary chains. But again there is no direct evidence that it does not. It may be that new compounds may be discovered with actions on the p receptor that may suggest by their structure that a second secondary chain exists, But there is no such evidence at the moment.

MOLECULAR STRUCTURE OF RECEPTORS

185

3. Ernetine This is another hefty molecule of relatively fixed form (Fig. 51c) that has mainly a blocking action, but also affects p-receptors to some extent. Its structure also suggests that the a-receptor has two secondary chains. The stereochemistry of the benzoquinolizidine ring is given by Battersby and Garratt (1959), but they do not specify the arrangement at the C-1 position. If, however, the H here is a, the methoxy isoquinoline portion can bind as does the same group in d-tubocurarine, that is in the angle of the F chain with the N H group binding to Asp in the other seconary chain. The benzoquinolizidine portion can now intercalate between rungs 1 and 2, which it, plus its attached ethyl group, fills up very effectively. Its NH binds to the 6- C of the guanidinium group of the Arg of rung 1 (the formal charge on the guanidium group being neutralized by the ionic bond to Glu). BETWEEN P-ADRENERGIC AND H. THERELATIONSHIP MUSCARINIC RECEPTORS

I t has already been noted that some compounds act at both these receptors, and that there are close structural relations between other compounds active at these sites. Deshpande and Jadhar (1970) find that p-agonists (such as isoprenaline and nilhydrin) block ACh and histamine-stimulated activity in some tissues. It might be asked, if muscarinic and p-receptors are so similar, how specificity for the agonists is maintained. The relevant part of the p-receptor is postulated to consist of two rungs -Arg-Glu; Arg-Gluwith an adjacent F secondary chain. The muscarinic receptor has the same primary system, but the secondary chain adjacent is E. Specificity is maintained as follows. The muscarinic receptor cannot bind a catecholamine as the Val of an E chain occludes the Glu 0: adjacent so that the 3-hydroxyl cannot bind. The p-receptor cannot bind ACh for the reason already detailed when considering the relation of ACh to nicotinic and muscarinic receptors. The structure of the ACh molecule and the geometry of the secondary and primary protein systems entail that, to bind ACh with an upper F chain, the rung adjacent must be Glu-Arg (nicotinic) and not Arg-Glu. If the rung is Arg-Glu, then to bind ACh the adjacent secondary chain must be E (muscarinic) . However, some blocking agents have molecular structures such that they can block either receptor. ACKNOWLELKIMENTS This work owes much to the collaboration of Fuad Antun, Frederick Benington, Ronald J. Bradley, William F. Bridgers, Richard Morin, and William Romine; to helpful comments and advice from Roger Brimblecombe, Patrick Carnegie, Samuel T. Christian, George and Ruth Clayton, David Curtis, Kenneth Eakins, Martin Evans,

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Kjell Fuxe, Paul Janssen, Derek Leaver, William 0. McClure, Theodore Melnechuck, Eric Nelson, Peter Pauling, Alan Robison, A. David Smith, and Lemone Yielding; and in particular I am grateful to Sir John Eccles, Seymour Kety, and Francis 0. Schmitt for their unfailing help and support. I am also grateful to the Ealing Corporation for the generous loan of CPK molecular models. The research described in this review was supported in part by N.I.M.H. Grant No. MH21437-01.

ACKNOWLEDGMENTS I am grateful to Sir Peter Medawar and Methuen and Company, Ltd. for permission to reproduce material from “The Hope of Progress”; to Professor E. J. Ariens and the New York Academy of Sciences (for Table 4, Ann. N . Y . Acad. Sci. 139, 606) and to Dr. Lazlo Gyermek and Marcel Dekker Publications, Inc. (Table 47 from Chapter 4 of “Drugs Affecting the Peripheral Nervous System”).

REFERENCES Abood, L. G., and Biel, J. H. (1962). Znt. Rev. Neurobiol. 4, 217. Ariens, E. J. (1967). Ann. N . Y . Acad. Sci. 139, 606. Baker, R. W., Chothia, C. H., Pauling, P., and Petcher, T. J. (1971). Nature (London) 230, 439. Barlow, R. B. ( 1964). “Introduction to Chemical Pharmacology.” Methuen, London. Barlow, R. B., and Franks, F. (1971). Brit. J . Pharmacol. 42, 137. Bartels, E., Wassermann, N. H., and Erlanger, B. F. (1971). Proc. Nat. Acad. Sci. U.S.68, 1820. Battersby, A. R.,and Garratt, S. (1959). J , Chem. SOC.,London pp. 2704 and 3512. Bebbington, A., and Brirnblecombe, R . W. (1965). Advan. Drug. Res. 2, 143. Beddoe, F., Nicholls, P. J., and Smith, H. J. (1971). Biochem. Pharmacol. 20, 3367. Brimblecornbe, R. W., Green, D., and Inch, T. D. (1970). J . Pharm. Pharmacol. 22,951. Brittain, R. T., Jack, D., and Ritchie, A. C . (1970). Advan. Drug Res. 5, 197. Buchner, C. K., and Patil, P. N. (1971). J . Pharmacol. Exp. Ther. 176, 634. Chothia, C. (1970). Nature (London) 225, 36. De Robertis, E., Fiszer, S., and Soto, E. F. (1967). Science 158, 928. Deshpande, V. R., and Jadhar, J. H. (1970). J. Pharm. Pharmacol. 22, 101. Edwards, E., Bunch, W., Marfey, P., Marois, R., and Van Meter, D. (1970). J . Membrane Biol. 2, 119. Ferry, C. B., and Marshall, A. R. (1971 ). Brit. J. Pharmacol. 41, 380p. George, J. M., Kier, L. B., and Hoyland, J. R. (1971). Mol. Pharmacol. 7 , 328. Gill, E. W. (1965). Prog. Med. Chem. 4, 39. Goodall, M. C., Bradley, R. J., Sacomani, G., and Romine, W. O., Jr. (1974). Nature (London) 250, 68. Gyermek, L. (1967). Zn “Drugs Affecting the Peripheral Nervous System” (A. Burger, ed.), Vol. I. Dekker, New York. Karlen, B., Lindeke, B., Lindgren, S., Svensson, K-G., Dahlhom, R., Jenden, D. J., and Giering, J. E. (1970). J . Med. Chem. 23, 651. Karlin, A,, and Winnik, M. (1968). Proc. Nat. Acad. Sci. U.S.60, 668. Khromov-Borisov, N. V., and Michelson, M, J. (1966). Pharmacol. Rev. 18, 1051. Klett, R. P., Fulpius, B. W., Cooper, D., Smith, M., Reich, E., and Possani, L. D. (1973). J . Biol. Chem. 248, 6841. Kuznetsov, S. G., and Ghokov, S. N. ( 1962). “Synthetic Atropine-like Substances.” State Publicity House of Medical Literature, Leningrad (English translation, U.S.Dept. Comm. J. P. R. S.)

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Lewin, S . (1969). Biochem. /. 114, 83p. Liu, J. H., and Nastuk, W. L. (1966). Fed. Proc., Fed. Amer. [email protected]. 25, 570. Martin-Smith, M. (1972). In “Drug Design” (E. J. AriEns, ed.), Vol. 2, p. 453. Academic Press, New York. Miledi, R., Molinoff, P., and Potter, L. T. (1971). Nature (London) 229, 554. Moran, J. F., and Triggle, D. J. ( 1969). In “Fundamental Concepts in Drug-Receptor Interaction” (J. F. Danielli, J. F. Moran, and D. J. Triggle, eds.), p. 133. Academic Press, New York. Nastuk, W. L. (1967). Fed. Proc., Fed. Amer. SOC.E x p . Biol. 26, 1639. O’Brien, R. D., Bilmour, L. P., and Eldefrawi, M. E. (1969). Proc. Nut. Acad. Sci. US.65, 438. Pedersen, L., Hoskins, R. E., and Cable, H. (1971). /. Pharm. Pharmacol. 23, 216. Podleski, T., Meunier, J-C., and Changeux, J. P. (1969). Proc. Nut. Acad. Sci. US.63, 1239. Pomeroy, A. R., and Raper, C. (1971). Brit. 1.Pharmacol. 41, 683. Rang, H. P., and Ritter, J. M. (1970). Mol. Pharmacol. 6, 357 and 383. Rang, H. P., and Ritter, J. M. (1971). Mol. Pharmacol. 7 , 620. Smythies, J. R. (1970a). Neurosci. Res. Program, Bull. 8, No. 1. Smythies, J. R. (1970b). Int . Rev. Neurobiol. 13, 81. Smythies, J. R. (1971a). Znt. Reu. Neurobiol. 14, 233. Smythies, J. R. (1971b). Eur. /. Pharmacol. 14, 268. Smythies, J. R. (1974). Annu. Reu. Pharmacol. 14, 9. Smythies, J. R., Benington, F., Bradley, R. J., Bridgers, W. F., and Morin, R. D. (1974a). /. Theor. Biol. 43, 29. Smythies, J. R., Benington, F., Bradley, R. J., Bridgers, W. F., Morin, R. J., and Romine, W. O., Jr. (1974b). 1.Theor. Biol. (in press). Sobell, H. M., Sakore, T. D., Tavale, S. S., Canepa, F. G., and Pauling, P. (1972). Proc. Nut. Acad. Sci. US..69, 2212. Sokoll, M. D., and Thesleff, S. (1968). Eur. 1.Pharmacol .4, 71. Spek, A. L., Peedeman, A. F., Van Wijngaarden, I., and Soudijn, W. (1971). Nature (London) 232, 575.

Note added in proof: A similar analysis applied to possible prostaglandin (PG) receptors has yielded a series of model receptors for the different PGs. This in turn has led to the hypothesis, based on the degree of complementarity between the model receptors and the compounds concerned, that there exists a series of naturally occuring PG agonists (probably for the E series) ; these include chrysarobin, phorbol myristate acetate, colocynthin, ricinoleic acid, emodin, chrysophanic acid, rhein, gambogic acid, and rotterlin. These variously have PG-like action on the eye (inflamation), kidney, skin, gut (ion transport rather than smooth muscle) and the immune response (Werner Braun, personal communication). It also appears possible that there exists a similar “receptor” (differing in one amino acid-possibly for PG.A) which has an effect on cell division. Compounds acting here may be the cocarcinogens phorbol myristate acetate and anthralin (desmethyl chrysarobin) (as agonists) and a series of naturally occuring antitumor agents (as antagonists) which includes acronycine, maytansine, datiscoside, mycophenolic acid, hellebrigenin 3-acetate and hymenoflorin. Although these are chemically widely different, CPK models indicate that they are all complementary to the same pentapeptide segment of protein chain.

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STRUCTURAL INTEGRATION OF NEUROPROTEASE ACTIVITY By Elena Gabrielescu "Dr. V. Babel" Institute of Pathology and Medical Genetics, Bucharest, Roumania

I. Introduction

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II. History and Resent Data

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111. Neuroprotease System: Molecular Analysis

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A. Endopeptidases B. Exopeptidases IV. Structural Integration in the Subcellular Compartments A. Mitochondria1 Fraction B. MicrosomalFraction. C. PlasmaMembranes D. Nuclear Fraction V. Binding of Proteolytic Enzymes within the Structure VI. The Functional Significance of Enzyme Integration in Subcellular Compartments A. The Micromedium B. Enzyme-Substrate Relationship C. Enzyme-Inhibitor Relationships VII. Integration in the Tissue Cellular Compartments: The Histochemistry of Neuroproteases A. Methodological Problems B. The Topchemistry of Neuroproteases C. Investigations under Functional Stimulation and Stress. VIII. The Implications of Neuroproteases in the Physiology and Pathology of the Nervous System References.

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1. Introduction

The biological significance of the intracellular protease system has remained unknown for a long time, overshadowed by the deeply rooted concept concerning the role of these enzymes in the processes of cellular death. Known as far back as 1904 from the works of Hedin, and identified by Willstatter and Bamann (1929) under the name of cathepsins, intracellular proteases, owing to their action within the cell, could not be efficiently approached by the biochemical methodology used for enzymes of the extracellular space. 189

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The advance of cellular biology, the idea of metabolic and morphological compartmentation of enzymic activity, as well as the modern biochemical and histochemical methodology using specific synthetic substrates and determinations in cell fractions and subfractions, have opened new prospects in the analysis of the intracellular proteolytic system. Interest in these enzymes again arose after the development of investigations on protein metabolism in the nervous system. The rapid renewal of proteins, with stimulation of their synthesis and catabolism demonstrated during excitation by Geiger et al. (1956), Waelsch (1957), and Ungar et al. (1957), as well as the data concerning the intervention of proteolytic enzymes in regulation of the neurohormonal function of the hypothalamus (Hopper, 1962, 1964, 1966; Rinne, 1967; Tuppy, 1968) drew attention to the role of neuroproteases in the physiology of the nervous system. The pathological implications of these enzymes, are linked especially to demyelination (MacCaman and Robins, 1959; Porcellati and Curti, 1960; Adams, 1962, 1968; Benetato, et al. 1964c, 1965; Gabrielescu, 1968, 1969a,b,c). Their activity in the course of functional overloading raised the question of the role of neuroproteases in the pathogeny of stress (Gabrielescu et al., 1966a,b; Gabrielescu and Bordeianu, 1967b, 1968, 1971 ; Gabrielescu 1970a,b). The results obtained during the last ten years, based especially upon biochemical analysis (Lajtha, 1964; Marks and Lajtha, 1965, 1971; Marks, 1968; Datta et al., 1967, 1969; D’Monte et al., 1970a,b) show the widespread activity of proteases in the nervous tissue, the plurality of proteolytic enzymes being represented by both intracellular endopeptidases and exopeptidases, and their ubiquity in the cellular and subcellular structures. The lack of homogeneity of the nervous system and cellular and subcellular compartmentation determine the lack of homogeneity in the distribution of neuroproteases, reflected in their activity. In contrast to extracellular proteases whose activity is manifested in the extracellular compartment, the entire functional cycle of intracellular proteases takes place within the cell in structurally differentiated compartments, ensuring not only synthesis and storage but also their catalytic activity. This structural integration causes spacing of the biological cycle of these enzymes, suggesting a high degree of organization that explains the sequence of the reactions within the multienzymic systems, the regulation and modulation of their activity with respect to the metabolism and functions of the cell. The analysis of neuroproteases, viewed from the angle of the concept of structural integration, finds support in the theory of metabolic compartmentation of Waelsch (1959, 1961), who considered structural integration as a necessary postulate for the correlation between function and metabolism. Waelsch, assuming the tridimensional character of metabolism in the ner-

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vous tissue, tried to establish a correspondence between “metabolic pools and morphologic compartments.” These metabolic pools appear at all levels of organization, corresponding to the morphological units at cellular and subcellular level, specialized in the synthesis, storage, elimination, and degradation of chemical compounds (Waelsch, 1961; Tower et al., 1961). Thus, structural integration of the activity of neuroproteases, which may open up new vistas in our understanding of the biological role of this system, represent the subject of the present work.

II. History and Present Data

The presence of proteolytic processes in the nervous system were reported first by Soula (1912), then by Faure and Soula (1913), in connection with the excitation of the nerve centers stimulated electrically or by motor exhaustion. Under these conditions, increased aminogenesis was the first finding regarding functional proteolysis in the nervous tissue. Subsequently, increased proteolysis in the central nervous system was noted following photic (Gorodinskaia, 1926) or acoustic stimulation (Hydtn, 1955). I n general, hyperexcitation brings about a decrement of the proteins in the nervous system (Palladin, 1953; Geiger et al., 1956; Ungar et al., 1957). I n his theory on excitation, Ungar (1963) attributed a special role, not only to the transconformation of cerebral proteins, but also to proteolysis, which affects both the central and the peripheral nervous tissue. The enzymic mechanism of proteolysis in the nervous system remained obscure for a long time, the progress made in the identification of neuroproteases and their functional significance progressing very slowly. Analyzing the “role of enzymes in the phenomena of neuronal life,” Marinescu (1930) was the first to suggest the intracellular presence of certain enzymes “similar to those of the digestive tract” with proteolytic function in intraneuronal digestion and with a role in the pathogeny of certain demyelinating and degenerative diseases of the nervous system, such as Wallerian degeneration and amyotrophic lateral sclerosis. Biochemical identification of the proteolytic enzymes in the nervous system began in 1927 with the work of Takasaka, followed by Krebs (1931) and Edelbacher et af. (1934). The presence of peptidases was signaled by Blum et af. (1936) and by Abderhalden and Caesar (1940). In 1942, Kies and Schwimmer tried to draw up a biochemical characterization of the cerebral proteinases. Abderhalden and Elsaesser ( 1943) described the activity of halogenate peptidases (acylases) in the brain, in connection with development, and Zeller in 1945 tried to analyze cerebral peptidases from the philo- and ontogenetic viewpoints. According to Krimsky and Racker (1949)

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the rat brain contains an acid protease able to alter carbohydrate metabolism by the dissociation of phosphoglyceraldehyde dehydrogenase. Schurr et al. (1950) confirmed the presence of proteolytic enzymes in the nervous system. The work of Ansell and Richter (1954a,b) imposed the classification of neuroproteinases into acid proteases with a pH optimum of 3.8 and neutral proteases with a pH optimum of 7.4, marking an important moment in the analysis of enzymic specificity. I n the same year, Hanson and Tendis (1954a,b) studied the location of di- and tripeptidases in cerebral tissue fractions, finding a maximum proteolytic activity of these exopeptidases in the mitochondria1 fraction. By these researches, which offered the possibility of correlating enzymatic activities and their localization in the morphological structures, modern enzymology endeavored to determine proteases microchemically, at different levels of morphological integration : regional, cellular, subcellular. In general, the distribution of proteolytic activity in the white and gray matter depends upon the type of proteases. Thus, acid proteases are predominantly situated in the gray matter and neutral proteases bound to myelin in a significant proportion are to be found in the white matter (Ansell and Richter, 1954b; Guroff, 1964; Waelsch and Lajtha, 1961; Lajtha, 1961, 1964; Brecher, 1963; Friede, 1966). The exopeptidases, i.e., aminopeptidases and dipeptidases, appear to be equally distributed in the gray and white matter (Blum et al., 1936; Pope, 1959). According to Abderhalden and Caesar (1940), they are more frequent in the white than in the gray matter, as also confirmed by Adams and Glenner (1962). In the myelinated nerves, MacCaman and Robins ( 1959), Porcellatti and Curti (1960), and Porcellati et al. (1961) described the presence of a neutral protease at pH 7.4, similar to that observed in brain by Ansell and Richter and by Guroff, an enzyme incriminated in the secondary demyelination of Wallerian degeneration and in the demyelination produced by organophosphoric compounds. The works of Adams (1968), Adams and Tuqan (1961a), and Hallpike and Adams (1969) supplied bio- and histochemical evidence of the activity of acid protease (cathepsin), neutral protease, and aminopeptidases, bound to the lipoprotein components of the myelin sheath, and of their early intervention in demyelination. The presence of proteases and their high content in the white matter of the central nervous system (Adams, 1965), spinal cord (Porcellati et al., 1961; Serra et al., 1971), and myelinated nerves (Adams and Bayliss, 1961) have drawn attention to the myelin-bound proteolytic system. Adams et al. (1963, 1968), demonstrated the large amount of proteolytic enzymes in this lipoprotein plurimembranous structure, in contrast to the minor content of other enzymatic systems. For the central nervous system, the hypothalamus represents a center

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of regional integration of high proteolytic potential in connection with the regulation of the activity of hypothalamo-hypophysial polypeptide hormones. The capacity of brain homogenate to inactivate these hormones was demonstrated by Heller and Urban ( 1935) and Itoh and Kikuchi ( 1959). According to Hooper (1962, 1964, 1966), maximum inactivation appears to be localized in the hypothalamus owing to a thermolabile, nondialyzable aminopeptidase enzyme, found in the mitochondrial fraction, which shows variations in the course of pregnancy and lactation. Purified and analyzed by Tuppy and Wintersberger ( 1960) and Tuppy ( 1968), this enzyme is known under the name of oxytocinase, being implicated in the inactivation of oxytocin and vasopressin. Farner et al. (1961), who described the increase of catheptic activity in the median eminence induced by photic stimulation of the gonadotropic function in birds, assert that the hypothalamus also contains endopeptidases. Advance in our knowledge of the subcellular sites of neuropeptidases ran parallel to the development of the ultracentrifugation techniques and fractionation in a density gradient. In 1957, Beaufay et al. identified cathepsin in the sediment fractions of brain homogenates, showing its latency linked to the structure. Palladin (1961 ) , Palladin e’t al. ( 1963), Polyakova and Lishko (1962), Lishko (1963, 1965), and Tsariuk (1964) found a specific maximum proteinase activity in the mitochondrial fraction of the brain. Waelsch and Lajtha (1961) isolated a light mitochondrial fraction in which they identified acid protease, pH 3.5, neutral protease, pH 7.4, and peptidases. Isolation of lysosomes from the mitochondrial fraction led to the location of cathepsins B, C, and D (De Duve et al., 1955). In their investigations carried out between 1963 and 1971 (Lajtha, 1964; Marks and Lajtha, 1965, 1970, 1971; Marks et al., 1968, 1970; D’Monte et al., 1970a,b), concluded on the complex integration of proteolytic enzymes in the subcellular structures of the nervous system, with an ubiquitous and differentiated character. 111. Neuroprotease System: Molecular Analysis

The molecular analysis of neuroproteases led to the identification of “families” of proteolytic enzymes represented by endo- and exopeptidases. with pecularities linked to their tissue origin. A. ENDOPEPTIDASES 1. Acid Proteinases

Acid proteinases of the nervous tissue identified in 1942 by Kies and Schwimmer on the basis of their property to hydrolyze hemoglobin and

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casein at an acid pH appear to correspond to cathepsin D, an intracellular protease discovered in 1960 in the spleen by Press et al. and in the liver by Lapresle and Webb (1962) equivalent to the acid cathepsin studied by Anson (1939) in the liver and by Dannenberg and Smith (1955) in the lung.

a. Cathepsin D (EC 3.4.23.5). Cathepsin D is defined by its capacity to hydrolyze denatured protein substrates at an acid pH (3-5.6) and its complete incapacity to split synthetic substrates ; it was analyzed in brain by Marks and Lajtha (1965, 1971), who found many isoenzymic forms. Cathepsin D does not hydrolyze di- and tripeptides, the synthetic substrates specific for cathepsins A, B, or C, nor monoacyl- and dipeptidylaryl amides. I t is a thermostable enzyme, with a pH optimum of 3.2. It is not affected by metallic ions or by sulfhydryl agents or trypsin inhibitors; this underlines the fact that it does not belong to the group of SH-dependent, serine or metal-dependent proteases. In the nervous tissue, cathepsin D is a lysosomal enzyme bound to the structure and distributed according to Palladin (1961) in a proportion of 82% in the crude mitochondria1 fraction of the brain, 10% in the microsomal fraction, and 3% in the nuclei. The regional distribution of this enzyme differs from one species to another; in sheep its maximum activity was recorded in the cortex and cerebellum, then in the hypothalamus, spinal cord, white matter (Lajtha, 1961) ; in monkeys the most intense activity appeared in the lenticular nucleus, then in the cerebellum, brain, midbrain, pons, thalamus, white matter, spinal cord. The determinations of Brecher in a human material (1963) showed the following order: brain-white matter, brain-gray matter, cerebellum, midbrain, hypothalamus, pons, thalamus, spinal cord, corpus callosum.

b. Cathepsin A . The chemical determinations using synthetic substrates revealed the presence in the nervous system of proteases other than cathepsin D. According to Brecher et al. (1966) the brain also contains a pepsinlike cathepsin A, but this was not confirmed by other workers. Matsunga et al. (1969a,b) drew attention to a cathepsin involved in the metabolism of angiotensin at pH 5, which exhibits a certain similarity with cathepsin A but differs from it by its sensitivity to DFP. Marks and Lajtha (1965) reported on the presence of cathepsins B and C, identified in very small amounts, representing 1/50 of cathepsin D in the brain and 5% of the amounts in the spleen and corresponding to a proteolytic activity of 0.1-0.3 per milligram of protein per hour. The detection of such a low intracerebral activity might be accounted for by the great lability of these enzymes, especially of cathepsins B, and by the interference of tissue

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inhibitors released in the course of the determinations (Marks and Lajtha, 1971). c. Trypsinlike Cathepsins. These differ from trypsin by their acid pM (5.6), by their belonging to the group of SH-dependent hydrolases, and by their lack of sensitivity to trypsin inhibitors (DFP) . They are represented in the brain by two enzymes: cathepsin B, active upon the substrate benzoylarginine P-naphthylamide) (BANA) , which is poorly represented ; and cathepsin B', differing from cathepsin B by its specificity for the substrate benzoylarginine anilide (BAA), by its specificity with regard to the peptide bonds of the B chain of insulin, by its molecular weight and sensitivity to inhibitors, such as heavy metals, PMSF and TPCK, and to activators, such as EDTA and mercaptoethanol. d. Cathepsin C (EC 3.4.14.1) Cathepsin C is an SH- and C1- dependent enzyme ; it is characterized by its poor endopeptidase activity, compensated however, by hydrolysis of the dipeptidyl amides at p H 5-6, so that it bears the name dipeptidyl aminopeptidase I ; by its transpeptidation and polymerization capacity at a neutral pH, conferring upon it the name dipeptidyl transferase ; and by its esterification function, which suggested to some authors its equivalence to indoxylacetate inhibitor-resistant esterase. Holt (1963), Pepler and Pearse (1957), and Fishman and Hayashi (1962) found evidence of cathepsin C activity in the brain. Cathepsin C in brain, not yet purified, differs from that of other tissues by its preferential affinity for certain substrates, such as Ser-Tyr-P-naphthylamide, and by a reduced range of substrates. The crude brain preparations are able to hydrolyze the classical substrates of cathepsin C described by Fruton (1960) ; Gly-Phe-NH, at pH 5.6, Pro-Tyr-NH, at pH 7.6, as well as a number of dipeptidyl arylamides at neutral pH. By the removal of the N-terminal dipeptide sequences, this enzyme is involved in the inactivation of certain biologically active substances, such as somatotropin ( S T H ) , glucagon, the B chain of insulin, ACTH (8-corticotropin) , and angiotensin 11.

2. Neutral Proteinases Neutral proteinases, hydrolases active at a physiological or slightly alkaline pH, described for the first time by Ansell and Richter (1954a) in aqueous brain extracts, then studied by Lajtha (1961), Marks and Lajtha (1965), Belik et al. (1968), Guroff (1964), Penn (1961), Riekkinen and Clausen ( 1969), are characterized by extreme lability that renders their chemical analysis very difficult. The enzyme isolated from the brain and partly purified by Marks and Lajtha (1965) is able to degrade hemoglobin, casein, albumin, and a-globulin, at pH 7-7.8. It acts upon oxidated ribo-

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nuclease and the oxidated B chain of insulin, breaking down the tyrosylleucyl bonds, the same as chymotrypsin, from which it differs, however, in that it does not split the tyrosyl-threonyl or phenyl-alanyl bonds and by the absence of the inhibitory effect of DFP. I t is likely that a correlation exists between the proteolytic activity of neutral neuroproteases and the esterolytic activity of crude brain extracts on synthetic benzoylarginine methyl ester (BAME) and tosylarginine methyl ester (TAME) substrates, specific for trypsin (Oja and Oja, 1970). The plurality of the isoenzymic forms of neurtral proteases was revealed by Marks and Lajtha (1965) and Riekkinnen and Rinne (1968). Evidence of one of these neutral proteases in the central and peripheral nervous system was supplied by Gabrielescu (1970a) by means of the histochemical technique of Glenner and Cohen used for revealing the activity of mast cell tryptase. This is a trypsinlike tryptase with a specific action on the benzoylarginine P-naphthylamide substrate at neutral PH* The neutral protease isolated from the brain by Marks and Lajtha (1965) and by Guroff (1964) is activated by Ca2+and inhibited by Cuz+,O-iodosobenzoate, and PCMB. Activation with cysteine, mercaptoethanol and reduced glutathione demonstrates the presence of a free SH- group in the active center of the enzyme. The activity of neutral proteases in the brain is 0,-dependent, diminishing up to disappearance under anaerobic conditions. Especially hydrolysis of serum albumin is energy-dependent, being stimulated by the presence of the cofactors ATP and CoA (Penn, 1960; Simpson, 1953; Marks and Lajtha, 1971). According to Belik et al. (1968) 50% of neutral protease activity in the total brain homogenate is contained in the crude mitochondrial-synaptosoma1 fraction, being linked especially to the nerve endings and myelin. The nuclear fraction contains 24% and the microsomal supernatant 14%. In contrast to cathepsin, whose activity is 10-fold that of the protein turnover in the brain, neutral protease activity corresponds to the protein turnover, splitting 0.2% of protein per hour. Another characteristic of neutral proteases in brain is their linear activity which is n& random, being controlled by a microsomal inhibitor that appears to correspond to RNAs (Schlessinger and Ben-Hamida, 1966). In connection with the biological role of this group of enzymes, there are several postulates regarding their participation in physiological neuroprotein turnover, in synaptic function, in the release from ribosomes of the newly formed proteins and splitting of histones in nucleoprotein complexes (Marks and Lajtha, 1971). In the sphere of pathology, the implication of neutral protease in demyelinating processes has been reported ( Porcellati and Curti, 1960; Riekkinnen and Rinne, 1968).

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B. EXOPEPTIDASES Exopeptidases, richly represented in the nervous tissue, display numerous particularities as compared to those in other tissues (Marks, 1968). 1. T h e a-Aminopeptide-Amino Acid hydrolases or Aminopeptidases This group (EC 3.4.11) is represented in the nervous system by: a. Leucine Aminopeptidase ( E S 3.4.11.1). The activity of this enzyme was made evident by Abderhalden and Caesar ( 1940), Kies and Schwimmer ( 1942), and Uzman et al. ( 1961) by the capacity of the brain extracts to hydrolyze leucine-containing peptides (Leu-NH,) . According to Brecher (1963), this enzyme is located in the mitochondrial and microsomal fraction, being activated especially by Mn2+and, to a lesser extent, by Mgzt and Co'+, in contrast to another aminopeptidase present in the postmicrosomal supernatant, with an action on Tyr-NH, and Phe-NH, substrates and which is activated by Mgzt and Co2+.The preferential substrates of aminopeptidase in the brain appear to be, according to Marks (1968), Leu-Gly and LeuGly-Gly, its activity upon the specific substrate of the homologous enzyme in the other tissues, Leu-NH,, being very slight.

b. Aminotripeptidase (EC 3.4.1I . 4 ) . This enzyme is specific for tripeptides containing neutral amino acids and is inhibited by Cd2+and anesthetics. In the brain, the enzyme has a marked specificity for Leu-Gly-Gly and AlaGly-Gly, but does not hydrolyze Gly-Gly-Gly (Marks, 1968) . In contrast to tripeptidases in other tissues, that in the brain also acts upon the lysinecontaining tripeptides (trilysine and Lys-Gly-Gly) and is not inhibited either by metals or by puromycin. Most of the activity of the enzyme appears in the supernatant (55%), then in the mitochondrial fraction (lo%), being localized in ,the synaptosome subfraction (Datta et al., 1967, 1969).

2. Dipeptide Hydrolases The dipeptide hydrolases (EC 3.4.13) or dipeptidases were found in the brain as far back as 1936 by Blum, then by Abderhalden and Caesar (1940), Kies and Schwimmer (1942), and Price et al. (1947). In this group the following enzymes were identified : a. Glycylglycine Dipeptidase (EC 3.4.13.1). This has a specific action on the Gly-Gly substrate, activated by Cozt, and is important because of the release of glycine, an amino acid with an inhibitory role in the neuronal

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function. A similar enzyme, alanylglycine dipeptidase, was analyzed in the layers of the somatosensory cortex by Pope ( 1959).

b. Imidodipeptidase ( Prolidase) ( E C 3.4.13.9). This enzyme is specific for dipeptides containing proline bound to the imido group and was identified in brain by Hanson and Tendis (1954b) and Uzman et al. (1962). The subcellular location of dipeptidases appears to be especially in the supernatant (Brecher, 1963). Marks (1968) stated that 60% of their activity is to be found in the postmicrosomal fraction and the remaining activity in the nuclear, mitochondrial, and microsomal fractions. 3. Carboxypeptidases

Carboxypeptidases (carboxypeptide-amino acid hydrolases) (EC, 3.4.12) were detected in brain after hydrolysis by crude brain extracts of the peptide sequences: Z-Phe-Phe, Z-Val-Phe in the B chain of insulin. The following were identified : carboxypeptidase A (acylases) and carboxypeptidase B. Acylases, enzymes that hydrolyze peptides substituted in the N-terminal position by acyl, N-halogen acyl, or ester groups are of importance in the hydrolysis of acetyl aspartate and other acyl peptides, richly represented in the brain. Carboxypeptidase B, specific of peptides with C-terminal basic amino acids, is involved in the hydrolysis of kinins. Krivoy and Kroeger (1964) mentioned the presence in brain of an enzyme able to inactivate bradykinin, identical to carboxypeptidase B. 4. Arylamide-Amino Acid Hydrolases or Arylamidases

This is a group of exopeptidases not yet registered in the international classification. Differentiated by Patterson et al. (1965) and Smith and Rutenburg ( 1963) from the group of aminopeptidases, arylamidases have as substrate arylamides characterized by the presence of the -CH,- group between the peptide bond and the coupled aromatic nucleus (amino acids coupled with p-naphthylamine in the C-terminal position) . These enzymes have a rich activity in the brain, being represented by arylamidases A, B, and N (Marks, 1968; Marks and Lajtha, 1971; Adams and Glenner, 1962).

a. Arylamidase A ( A c i d ) . This enzyme is specific for the substrate containing acid amino acids of the a-glutamyl and a-aspartyl p-naphthyl amide type. I t is of particular interest in view of the rich brain content of peptides containing glutamate, aspartate, and acetyl asparatate from which the acid arylamidases release glutamic and aspartic acid, specific to the metabolism and functions of the nervous tissue. Marks (1968) identified this enzyme in the brain homogenate and brain subcellular fractions ; its maximum activity stimulated by Caz+is located in the mitochondrial fraction.

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b. Arylamidase B (Basic). This is specific for the hydrolysis of arylamides containing basic amino acids of the arginine, lysine type. It was found in brain homogenates which can hydrolyze arginine-P-naphthylamide (Nachlas et al., 1962). Activated by metallic ions, by cysteine, and by p-mercaptoethanol and intensely inhibited by puromycin, arylamidase B is found in a proportion of 60% in the microsomes, 10% in the mitochondria, 4% in the nuclei, 6% in the vesicles and synaptic structures, and 1% in myelin. It is characterized by a high solubility appearing in the supernatant in a proportion of 37% (Marks, 1968). Worthy of note is the role of this enzyme in the formation of bradykinin owing to its action on peptides (kallidin 10) obtained from the globin previously hydrolyzed by trypsin (Hopsu-Havu et al., 1966). c . Arylamidase N (Neutral). The enzyme acts upon substrates of the leucine-/3-naphthylamide type; it was found by Adams and Glenner (1962) in the frontal gray matter and corpus callosum of the rat brain, and by Arvy (1962) in the hypothalamic nuclei. After fractionation of the brain homogenate, 43% of the activity of the enzyme appeared in the supernatant, 22% in the mitochondria, 13% in the microsomes, and 6% in the nuclei. In the mitochondria1 fraction, 40% of arylamidase N activity belongs to the synaptosomes. Of particular importance for the neurosecretory function, neutral arylamidases in the hypothalamus interfere in the release and inactivation of the polypeptidic hormones. Thus, in states of dehydration and in lactation, an increase in the activity of these enzymes is noted in connection with the release of hormones from the neurosecretory granules (Arvy, 1962; Arai and Kusama, 1865). Exopeptidases of the Leu-Ala, Gly-, or cystine-di-pnaphthylamide type, localized in the hypothalamus inactivate oxytocin and vasopressin, being identical t o oxytocinase (Tuppy, 1968; Hooper, 1962, 1964, 1966). With a maximum location in the hypothalamus, the enzymes inactivating the polypeptide hormones are to be found in the cerebellum, thalamus, cortex, caudate nucleus, and white matter, where they also meet with the exopeptidases inactivating bradykinin and the P substance (Akira and Kiyoshi, 1962). The hydrolysis of bradykinin reaches a peak in the spinal ganglia and in the dorsal and ventral roots (Inouye et al., 1961).

IV. Structural Integration in the Subcellular Compartments

The biological activity of proteases is functionally integrated in morphological structures. This integration affects the synthesis and storage of enzymes for all types of proteases. With extracellular proteases, the morpho-

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logical structures are involved in the process of secretion and release into the extracellular compartment. Cathepsins display their entire functional cycle intracellularly, including latency, activation, inhibition, catalytic activity and its regulation, which depend upon the specific cellular and subcellular structures. Spacing of the biological cycle of proteases includes various levels of integration into the structures: at a supramolecular level, the enzymic molecule is complexed with the “structure unit” molecules; at a subcellular level, their specific localization is in the organelles; at the cellular level, quantitative and qualitative differences develop between the cytological enzymic types, which at the tissue and organ level are integrated within the particularities of functional specialization.

FRACTION A. MITOCHONDFUAL Analysis of the location and site of maximum activity of intracellular proteases revealed their nonhomogeneous distribution in the structures and the plurality of the structures in which they are integrated. I n the phase in which ultracentrifugation of the tissue homogenates allowed for enzymic analysis at the level of the subcellular fractions, an intense cathepsin activity was found linked to the complex mitochondrial fraction. Thus Finkenstaedt (1957), in the four fractions he obtained from the rat liver homogenate (nuclei, mitochondria, microsomes, supernatant) identified cathepsin B (pH 5.2) and cathepsin C activity in the mitochondrial fraction. I n the brain, Palladin (1961) likewise found maximum protease activity in the mitochondrial fraction. The mitochondrial fraction appears to be, according to Hanson and Tendis (1954a), also the site of di- and tripeptidases. The possibility of separating certain subcellular fractions into subfractions by ultracentrifugation in a sucrose density gradient has supplied further details concerning the subcellular location of proteases, together with the discovery of new cellular organelles. 1. Lysosomes Individualization of the lysosomes as structures of the vacuolar system of intracellular digestion has permitted the identification of cathepsins from among the lysosomal hydrolases. These cathepsins, described for the first time in the lysosomal fraction of the liver by De Duve and Beaufay (1959), were subsequently considered to be lysosomal hydrolases, assumed to be involved in intracellular, phagolysosomal, and autophagic digestion, in almost all the tissues. After identification of the activity of cathepsins A, B, and D by De Duve et al. (1962) and Beaufay et al. (1964) in hepatic lysosomes, Holt (1963) and Shibko and Tappel (1964) demonstrated lysosomal inte-

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gration of cathepsin C or indoxylacetate esterase, resistant at E 600. In the nervous tissue, liver, and spleen, cathepsin B and D activity is connected with the neurolysosomes (Marks and Lajtha, 1965). Koenig et al. (1964) observed the distribution of cathepsins in the subfractions rich in lysosomes isolated from the mitochondrial fraction of brain homogenate. I n the nervous system, neutral proteinase is partly present in the lysosoma1 fraction, but the greatest proportion is to be found in the other mitochondrial subfractions : the nerve endings and myelin. According to the quantitative table included in the work of Marks et al. (1968), the distribution in the brain is expressed by the following ratio : nerve endings 47.5 :lysosomes 15:myelin 9.5. From these data it results that all the intracellular endopeptidases are totally or partly represented in the lysosomal fraction. Exopeptidases, likewise distributed in several cellular fractions, have their representatives in the lysosomes. Arylaminopeptidase (leucine-p-naphthylamidase) is a lysosomal enzyme as demonstrated in the liver, kidney, intestine, and connective cells. The hepatic lysosomes contain arylamidase B in its totality (Mahadevan and Tappel, 1967) and most of the arylamidase N (Marks, 1968). According to MacCabe and Chayen ( 1965), arylarnidase A is likewise a lysosomal enzyme. On the other hand, in the nervous system arylamidases are very poorly represented in the lysosomes, being chiefly found in the nerve endings and then in myelin.

2. Nerve Endings, Synaptosomes, Myelin By ultracentrifugation of the mitochondria and separation in a density gradient, mitochondrial subfractions other than lysosomes were isolated ; these are of particular importance, especially for the nervous tissue, which exhibits an intense proteolytic activity. These are the three mitochondrial subfractions obtained by Marks and Lajtha (1965) and by Lajtha (1964) in a 0.32-1.2 M sucrose gradient: Al,, containing myelin and glial cell fragments; BP2, containing nerve endings and synaptic vesicles (synaptosomes) ; and CP,, corresponding to the dense lyosomal particles. Among the endopeptidases, acid proteinase, which is richly represented in the neurolysosomes, is reduced in the other two subfractions, the nerve endings containing less than half of the lysosomal activity and the myelin subfraction only a fifth part. I n contrast, neutral proteinase is predominantly contained in the subfraction of the nerve endings. The determinations of Lajtha (1964) showed that neutral proteinase activity is 150 in the nerve endings and synaptosomes, 165 in myelin, and 21 in lysosomes. According to Marks et al. (1968) the highest activity appears in the subfraction of the nerve endings, then in the lysosomes and myelin. The nerve endings-synaptosome subfractions-also contains a high proportion of exopeptidases of the tripeptidase type, leucine P-naphthylamidase, and other arylamidases, accounting for the intense pro-

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teolytic potential of these formations (Gordon et al., 1968). Oxytocinase, a hypothalamic aminopeptidase inactivating oxytocin and vasopressin, has been reported in the nerve endings and axonal, synaptosomal, and myelin fractions, in which it increases during pregnancy (Hooper, 1964). The synaptosomes likewise appear to be the site of carboxypeptidase A, rarely reported in the tissues (Marks and Lajtha, 1971) . Myelin, isolated biochemically both from the peripheral and from the central nervous tissue (Adams et al., 1965, 1968) is concentrated in one of the lighter subfractions of the mitochondrial ultracentrifugate and exhibits a marked proteolytic activity in spite of its relative enzymic inactivity (Adams et al., 1963). The myelin proteolytic enzymes are represented by neutral protease, leucine aminopeptidase, tripeptidase, arylamidase (Adams and Bayliss, 1968; Marks and Lajtha, 1970). Although present, acid protease has a more reduced activity than in the lysosomes. c. Mitochondria. Debate persists concerning the relationship between the mitochondrion considered as a morphological entity and proteolytic enzymes. The mitochondria organelles linked especially to energy metabolism, are notwithstanding involved in protein metabolism. Alberti and Bartley ( 1969) and Baird (1964) reported on the release of amino acids following the activation of proteolysis caused by the incubation of mitochondria in vitro. Penn (1961) supported the existence of a mitochondrial protease with an optimal neutral pH, activated by CoA and ATP, with a hydrolytic action upon serum albumins. After careful washing of the mitochondria in order to remove a casual contamination, Maggio (1957), Roodyn et al. (1961; and Roodyn 1967) confirmed the presence of a mitochondrial proteolytic activity, with particular reference to neutral protease. Similarly, worthy of note is the association of exopeptidases with the mitochondrial membrane fractions (Leu-Gly-Gly-ase) described by Datta et al. ( 1967, 1969).

B. MICROSOMAL FRACTION Another important compartment in which intracellular protease activity takes place is the ergastoplasm, including the ribosomes and plasma endoreticulum membranes; after ultracentrifugation of the homogenates, these formations sediment in the microsomal fraction. The microsomes are the site of cellular integration, especially of exopeptidases and to a lesser extent of endopeptidases. According to Datta et al. (1967) the microsomes within the nervous tissue do not contain acid or neutral proteinase activity. Lajtha (1964) and Palladin (1961) , however, consider that the microsomal fraction contains an appreciable endoproteinase activity. Thus, in the rat brain, the acid protease activity of this fraction is two-thirds and the neutral activity is one-third of that in the mitochondrial fraction. Although in small amounts,

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ribosomal neutral proteinase has a role in the release of newly formed proteins (Marks and Lajtha, 1965). The activity of exopeptidases is more broadly represented in the microsomes. In the microsomal fraction of the nervous system, Marks (1968) mentioned the presence of leucine aminopeptidase having the same activators and the same substrate as that in the mitochondria] fraction (lysosomal) . Hypothalamic oxytocinase is poorly represented in the microsomes. By isolation of the microsomal subfractions from the brain, Datta et al. (1969) found that the ribosomes contained only traces of aminopeptidase and arylamidase. The microsomal membranes, belonging to the plasma endoreticulum, have an intense aminopeptidase activity at p H 7 on substrates such as L-Leu-Gly-amine, L-Leu-Gly-Gly-amine, and L-Ala-Gly-Gly-amine, and they have an arylamidase activity at pH 6.5 on Arg-Lys-Meth- and Leu-P-naphthylamide substrates. The activity of dipeptidases, represented especially by glycylglycine dipeptidase, is likewise integrated substantially in the microsomal fraction, although owing to its high solubility, it appears in larger amounts in the supernatant. Thus, the microsomes, especially the endoreticulum membranes, appear as the site of morphological integration of a protease multienzymic system involved in the catabolism, synthesis, turnover, and transport of proteins. Conclusive data exist concerning the participation of this enzymic system in protein synthesis. These data refer for the moment to Escherichia coli ribosomes and polysomes whose proteolytic activity was demonstrated by Chaloupka (1961 ), Bolton and McCarthy (1959), and Matheson (1963). The proteolytic enzymes of these structures release polypeptide chains from the messenger-RNA complex (Elson, 1967). Cuzin et al. (1967) described in E. coli a protease that catalyzes the hydrolysis of diphenylalanyl-tRNA and N-substituted oligopeptidyl-tRNA. The arylamidases are assumed to participate in protein synthesis by favoring lengthening of the peptide chain. This process involved in memory is inhibited by puromycin, which inhibits arylamidases (Flexner et al., 1967). I n cell cultures tested in the phase of transition from exponential to stationary growth, Elson (1967) observed a decrease in proteolytic activity in the free ribosomes and an increase in the membranous subfractions of the microsomes (endoreticular membranes) in connection with the transfer of synthesized proteins: In the ribosomes of the nervous system, neutral protease appears to play a similar role, releasing the newly formed proteins (Lajtha, 1964; DMonte et al., 1970b). The role of proteases in the informational mechanism of protein synthesis was also made evident by Mano (1966) in the Hemicentrotus egg. After fertilization, the presence of a trypsinlike neutral protease was found in the iniormosomes. The neutral protease appears to release mtRNA from the informosomes, triggering its transfer to the ribosomes in order to form polyribosomes.

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C. PLASMAMEMBRANES The cell plasma membranes as the site of proteolytic enzymes have focused the attention of research workers since Robinson (1963) discovered the site of peptide hydrolysis in the brush border of the intestinal cells. The intestinal cell membrane is the site of intense peptide hydrolysis, representing the final stage of intestinal digestion (Newey and Smith, 1960; Ugolev, 1965). This “contact digestion” is performed by aminopeptidases and arylamidases bound to the surface of the cell membrane (Eicholz and Crane, 1967; Holt and Miller, 1961). In 1966, Coleman and Finean mentioned the presence of the specific activity of leucine 8-naphthylamidase in the liver, renal, and intestinal cell membranes. Emmelot et al. (1968) supplied evidence of the activity of L-leucine /3-naphthylamidase (arylamidase N ) in the plasma membrane of the liver cells, isolated in subcellular fractions. Leucine naphthylamidase is localized in repeating functional and structural supramolecular units that cover the surface of the plasma membranes, interfering locally in the specific selective permeability of these membranes. Considering myelin as a plurimembrane system, we should like to recall the work of Adams and Glenner, who in 1962 mentioned the presence of leucine aminopeptidase, leucine naphthylamidase, and neutral protease in this structure.

D. NUCLEAR FRACTION The nuclear fraction, which is the least known from the point of view of enzymic activity, should not be omitted from among the compartments of protease activity although the data concerning this problem are very limited. Lending support to this assumption are the studies of Lajtha (1964) , Marks (1968), and Marks and Lajtha (1971) on the nervous system attesting to the activity of acid proteinases in the brain nuclear fraction, second in intensity only to the mitochondria1 and microsomal fractions, and persisting even after separation of the nuclei from the residue. Neutral proteinase is not only present in the nuclear fraction but also shows maximum activity, maintaining itself integrally even after separation of the residue. According to Marks and Lajtha (1965) nuclear neutral proteinase plays a part in the release of basic proteins (histones) from the nucleoprotein complexes. Tappel et al. (1963) assumed that cytoplasmic proteases penetrate the nuclei through the nuclear pores, participating under certain conditions in the hydrolysis of histones. Integration of the proteolytic enzymes in the nuclear compartment, still questioned in view of a possible contamination, opens the prospect of researches on the role of these enzymes in the physiology of the cell nucleus.

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V. Binding of Proteolytic Enzymes within the Structure

The intracellular separation of proteases into compartments consequent to their integration in the subcellular structures should be considered as a dynamic phenomenon, adapted to the biological cycle of enzymes and closely related to the metabolic and functional activities of the cell. In the case of proteases, similar to that of other hydrolases, the connection with the structure impresses upon the enzymes a state of temporary latency, controlling at the same time the degree of activation and the site of action. The relationship between the bound and the free enzyme, between the latent and the active enzyme is the result of the lability of these bonds under the influence of the micromedium factors. Hence, the importance of analyzing this kind of labile physicochemical bonds between the enzymic molecules and the morphological structures. Three hypotheses may be taken into consideration regarding the integration of proteases into lysosomes, which are valid in general for all lysosomal hydrolases. 1. De Duve Hypothesis. De Duve and his school (1959, 1963), who consider the lysosomes as bags containing latent hydrolase enzymes, hold that the enzymic molecules are released within the organelle, the activity of the enzymes being controlled by the lysosomal membrane. The physical model outlined by De Duve and Beaufay ( 1959) and Beaufay and De Duve ( 1959) implies the existence within the lysosome of a hydrated matrix containing two distinct spaces: an osmotic space accessible only to water with the behavior of an osmometer; and sucrose space accessible to water and to solutions with a low molecular weight of the sucrose type, but impermeable to macromolecular substances. Hydrolases appear in the osmotic space of lysosomes. The single lysosomal membrane, of a lipoprotein nature, maintains the integrity of these spaces by its selective permeability, governing the relationship between the enzymes and their substrates. The studies carried out on the lysosomal fraction isolated from the tissue homogenate showed that altered permeability of the lysosomal membrane or its rupture, caused by physical and chemical agents in uitro or by injury in uiuo, bring about the release of hydrolases from the organelles with their passage into the supernatant. This release, in the opinion De Duve, is similar and is not differentiated for the various lysosomal hydrolases. Subsequent experimental data have shown, however, that the behavior of the various types of lysosomal hydrolases differs, the rate of release varying in terms of the activation stimulus and time of manifestation. This individual behavior in respect to factors that alter lysosomal permeability appears to reflect different kinds of bonds within the lysosomes. Thus arose the idea

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of a labile physicochemical binding either to the lysosomal membrane or to the matrix. 2. Desai Hypothesis. The hypothesis of Desai et al. (1964), concerning the mode of integration of hydrolases in the lysosomal structure postulates an ionic bond between the enzymic molecules and lysosomal membranes. After repeated freezing and thawing of the lysosomes, Tappel et al. ( 1963) were able to separate by ultracentrifugation the membranous subfraction from the lysosomal supernatant. They found that 27% of the ribonuclease and 45% of the acid phosphatase remains associated with the lysosomal membrane ; 457% of this enzyme bound to the membrane may be eluted with sucrose, buffer. or buffer sucrose, demonstrating that the lysosomal enzymes are bound to the membrane by electrostatic bonds. Chemical analysis of the lysosomal membranes showed the presence of 60-70% of the total lysosomal proteins, including the enzymic protein. Among the lysosomal proteases, cathepsin C has been found to be bound to the external surface of the lysosomal membrane (Shibko and Tappel, 1964). According to Tappel et al. ( 1963), the lysosomal membrane is not a mere semipermeable barrier, access to the enzyme substrate being due to translocation through the lysosomal membrane that depends upon the electric charge on the surface of the latter and electrovalent bonds with the polarized sites. The electric potential of the lysosoma1 membrane makes the transfer of molecules or ions possible, in the form of complexes translocated in the direction of the electrochemical gradient, which also explains the passage of large molecules. According to Lucy ( 1969), the stability of lysosomal membranes depends on the bimolecular bindings of the proteins and lipids. The micellar state of these structures induces membrane permeability. 3. Koenig Hypothesis. The theory of Koenig (1962, 1963, 1965, 1966, 1969) postulates supramolecular integration of hydrolases in the lysosomal matrix by electrovalent bonds. To De Duve’s concept of the lysosomal “osmotic bag,” Koenig opposes the concept to “matrix binding.” Micro- and histochemical analysis of the lysosomal matrix has demonstrated its glycolipoprotein nature. The presence of gangliosides and glycolipids rich in polyunsaturated fatty acids, in a complex with proteins, confers upon this matrix polyanionic properties that favor its interaction with cationic groups by electrostatic forces. Lysosomal hydrolases are assumed to be bound in a latent state to the anionic sites of the matrix by electrovalent bonds. The presence of N-acetylneuraminic acid in the matrix and the solubilization action of hydrolases exercised by neuraminidase on the lysosomal fraction of brain, liver, and kidneys demonstrates, in the opinion of Koenig et al. (1964), the participation of a ganglioside component in the ionic bonds of the enzymes. A similar release of the lysosomal enzymes is manifested by phospholipase C (Clostridium welchii) , which splits off the phosphatide phospho-

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esters from the proteolipid matrix, indicating as anionic site of the bond the phosphate phosphatide groups, belonging to the phosphoryl fraction of the phospholipids. The lysosomal matrix, owing to the polyanionic reaction conferred by gangliosides and phosphilipids, can fix in uivo and in uitro the basic cationic dyes. Binding of these substances by electrostatic forces, as well as of other cationic molecules, such as phenothiazine, chlorpromazine, protamine, polylysine (basic proteins), favors the release of lysosomal enzymes by a competitive action upon the matrix anion sites. This demonstrates integration of the lysosomal hydrolases at the anion sites of the glycoprotein matrix to which they are loosely bound, in a latent state, by electrostatic forces. These results were obtained for acid phosphatase and p-glucuronidase. Extrapolation to proteases still remains to be verified. Lending support to this theory was the fine biochemical analysis of renal lysosomes carried out by Goldstone et al. (1968, 1970), which offered valuable details on the chemical nature of the matrix anionic site. This is the C fraction separated by chromatography on DEAE-cellulose from the lysosoma1 proteins. In contrast to fractions A and B which contain enzymes, fraction C, which exhibits no enzymic activity, contains apart from phosphatides two anionic lipoproteins isolated by electrophoresis in polyacrylamide gel. These lipoproteins, which represent 30% of the soluble protein in the lysosomal matrix, are important components of the matrix, to which lysosomal hydrolases are bound electrostatically. Fraction A contains arylsulfatase, and fraction B contains additional acid phosphatase, p-glucuronidase, and cathepsins. Each fraction can be dissociated into cationic proteins by electrophoresis in polyacrylamide gel. Fraction B contains in addition a protein-associated nucleotide pyridine, responsible for the autofluorescence of the lysosomes. Therefore “cathepsin” is a cationic protein belonging to fraction B of the soluble lysosomal protein, probably bound, as are the other enzymes, to the anionic lipoproteins of fraction C. According to Dingle and Barrett (1967) and Barrett and Dingle (1967) the matrix component of the renal lysosomes is of lipid nature. Of medium molecular weight, it is intensely acid because of the presence of carboxylic or phosphate groups, rich in free or loosely combined aldehyde groups, and cannot be extracted from the lysosomes by freeze-thawing or ultrasonication ; it is not attacked by deoxyribonuclease, ribonuclease, hyaluronidase, neuraminidase, or pepsin. I t can be extracted with 0.170 Triton X-100; detergents and slightly concentrated saline solutions prevent the reaction with cationic substances. According to Dingle and Barrett, this lipophilic polyanion is the binding site of cations, and hence of hydrolases. I t is likely that all these theories are valid, since, as demonstrated by the lysosomal physiological and pathological data, both the integrity of the lysosomal membrane with maintenance of the osmotic and electrochemical

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functions and the polyanionic properties of the lysosomal matrix have a special role in the latency of lysosomal hydrolases, in the bound enzyme :free enzyme ratio, in enzyme-substrate accessibility, in the sequence of enzyme activation and release, aspects increasingly implicated in the metabolism, physiology, and pathology of the cell. Thus, analysis of the biological activity of acid proteases should be correlated with the state of integrity of the lysosomes and with the labilizing and stabilizing factors of the structure, which influence the activity and separation into compartments of the enzymes at the cellular level. This question is, however, but little known, the activation and release of cathepsins not always developing parallel to that of the other lysosomal hydrolases and being characterized by a differentiated behavior in terms of specific injuries to, and nature of, the tissues. There are certain conditions in which lysosomal cathepsins are released rapidly and in large amounts-for instance, under the influence of endotoxin (Janoff et at., 1962) ; in ischemia, which produces hypoxia and acidosis (De Duve et al., 1955) ; under conditions of high pressure hyperoxygenation (Hall and Sanders, 1966) ; in traumatic shock (Gahan, 1967), excess vitamin A (Dingle and Lucy, 1963), and vitamin E deficiency (Tappel et al., 1963). A particularly intense effect is that of the cytopathic viruses (Flangan, 1966; Allison, 1968). I n vitro, a low p H and a temperature of 37OC, as well as alteration of the lysosomal membrane by auto-oxidation of the lipids (Tappel et al., 1963), are micromedium conditions favoring the release of cathepsins. O n the other hand, freezing and thawing, Triton X-100, mechanical homogenization (Dannenberg and Bennett, 1964), and lecithinase (Dingle, 1961) exercise only a slight effect upon the release of these enzymes. Intoxication with CCl, releases hepatic lyosomal cathepsins later than acid phosphatase (Ugazio et al., 1964; Dianzani, 1963) just as UV light induces a greater release of acid phosphatase and P-glucuronidase than of cathepsins (Janoff et al., 1962). Larger doses of Triton WR 1339 are necessary for the release of cathepsin from the liver than for the release of acid phosphatase. This points to the necessity of a more detailed analysis of the relationship between the nature of lysosomal lability and cathepsiii activity, and the influence of various lyosomal labilizers and stabilizers on the release of cathepsins. It is likewise important to dissociate the direct effect upon the enzymic molecule, of these factors, as inhibitors and activators, from the indirect effect mediated by alteration of the lysosomal structure. Nothing is known as yet of the mode of binding and integration of proteases in the other cellular structures. Emmelot et d. (1968) described an aminopeptidase in the membrane which appears to be integrated at a supramolecular level in globular protein granules, irregularly distributed on the membrane surface, from which it can be released by papain but not by tryp-

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sin. Integration of the proteolytic enzymes in the myelin membranous system is far from clear; the enzymes appear to participate as soluble protein components, integrated by electrovalent bonds into the lipoprotein structure, with regard to which cholesterol is assumed to play the role of stabilizer (Adams and Bayliss, 1968). Without special reference to proteases, Wolman’s theory of effect of phase transitions in the membrane on bound enzymes (Wolman, 1968, 1969) opens interesting prospects on the active mode of intramembranous integration of enzymes. According to this theory, the membranes may be regarded as structures formed of biomolecular lipoprotein layers having a hydrophilic and a hydrophobic surface. The membrane enzymes may have an active site bound either to the hydrophilic surface in an oil-in-water system ( O / W ) or to the hydrophobic surface in a water-in-oil system ( W / O ) . These phase systems are the consequence of the interaction between the lipoprotein membranes, having an anionic character and various cations. They form O / W systems with monovalent cations and W /O systems with bivalent cations. The ionic alterations may result in phase transitions, influencing the activity of bound enzymes. Thus Caz+ (C1,Ca) inhibits the enzymes situated in the hydrophobic layer of the membrane in a O/W system, transforming the latter into a W / O system. This is the case for certain membrane proteases, for example, leucine aminopeptidase, cathepsin C , and other hydrolases, such as phosphomonesterase and adenosine triphosphatase. These enzymes are affected by harmful agents that act in the aqueous phase, being protected by factors able to produce a phase transition. Thus integration into the membrane structure appears not only as a simple spatial orientation, but also as the polarization of a specific phase system, a colloidal organization within which enzymic activity may be regulated by interaction with the phase exchange ions. VI. The Functional Significance of Enzyme Integration in Subcellular Compartments

Starting from the stepwise regulation of enzymic activity by kinetic change of the molecular stereostructure of the bound enzyme, whose active centers are changeably masked, and up to regulation of enzyme-substrate, enzyme-inhibitor, enzyme-activator accessibility, the entire activity of the proteolytic enzymes appears to be subordinated to intracellular spatial distribution and hence integrated into cellular cybernetics. A.

THE

MICROMEDIUM

A corollary of this integration is the specificity of the micromedium offered by the morphological compartments, drawing attention to the impos-

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sibility of superposing the intracellular activity of the enzyme upon its activity in the soluble humoral medium, or especially in the conventional medium of the enzymologic tests. The problem of the micromedium depending upon integration in the cellular structure includes (a) the relationship with the intracellular pH, and (b) the relationship with the energy potential and cofactors. 1. Relationship with the Zntracellular PH

Many works refer to the optimal acid pH of cathepsins, considered by some authors to be incompatible with physiological cellular activity. In a period in which the existence of neutral and alkaline proteases was not known, and in which testing on conventional media in vitro was correlated with maximum cathepsin activity at an acid pH (3.8-4.5), it was considered that ,these enzymes play a role only in postmortem autolysis when the tissue pH is acidified, denying any physiological role. This concept still persists with those who confound the optimal pH of the enzyme in vitro on conventional substrates with the optimal pH within the cell on natural intracellular substrates. The intracellular pH is not identical to that of the humors; within the cell, pH variations exist in terms of the electrochemical potential of the various subcellular structures. Aronson and Davidson (1967) and Mego (1971) demonstrated that the intracellular pH is more acid than in the blood, ranging between p H 6.4 and 7.2, with shifts down to p H 4.5. In the lysosomes, the pH is 5-6.9 owing to the acid glycolipid and lipoprotein components of these organelles (Barrett and Dingle 1967; Goldstone et al. 1970). The hyaloplasm’ has a pH of 7.0 on the border surfaces; on the membranes the protons are more highly concentrated, favoring intracellular spaces with a more acid pH (Bohley, 1968). In general, the subcellular particles isolated present a lower pH than the “physiological” pH (Marks, 1968) ; especially the secondary lysosomes ( phagolysosomes) have an acid pH (Allison, 1968). The optimal pH of the lysosomal enzymes differs, moreover, in terms of the ionic concentration, being neutral at a low ionic concentration and acid at a high ionic concentration. The membranes, as structures involved in the variations of the intracellular ionic concentrations, become active factors in the variations of the optimal intracellular pH of these proteolytic hydrolases which, as already mentioned, exhibit wide pH spectra. On the other hand, these pH variations within the structure may play the role of a modulator of enzymic activity, both as regards its action specificity and that of the substrate. It is known that an alkaline pH favors the shift of cathepsin activity toward transamidation and synthesis, whereas an acid pH favors proteolytic activity. It is likewise known that selection of the substrates attacked depends for the same cathepsin upon pH conditions. Moreover, cathepsin D which has

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a broad pH spectrum (2.5-6) hydrolyses bovine hemoglobin at pH 4.2 in vitro. I n the phagolysosomal structure, in which the pH is higher, catheptic digestion of hemoglobin takes place more slowly (Straw, 1964a,b). I n general, the rate of degradation of the endocyte proteins in the phagolysosomes is slow, facilitating partial proteolysis to biologically active fragments. The highly acid optimal pH found for cathepsins in vitro in soluble biochemical systems may also be accounted for in that this pH, apart from its action upon the enzymic molecule, favors the activity of the enzyme by secondary indirect effects: altering the lysosomal membranes, which allows for the release of enzymes; denaturation of protein substrate, favoring proteolysis; and inactivation of inhibitors.

2 . Energy Potential and Cofactors Structural integration also influences the energy conditions and contact with the cofactors necessitated by activity. Although enzymic proteolysis is considered in general as an economic reaction from the viewpoint of energy, in the absence of O2 and cofactors, analysis of protease activity at the cellular level revealed energy requirements both for the activity of a certain group of proteases and for the integration of enzymic activity in the subcellular compartments. According to Simpson ( 1953), the catabolism of endogenous proteins on liver slices at neutral pH depends upon the oxidative processes. Proteolytic activity at pH 7 is inhibited by anaerobiosis and dinitrophenol. Penn (1961) described in the mitochondria1 fraction of the liver and brain a group of neutral proteases, active at physiological pH, whose proteolytic activity on serum albumins depends upon the release of energy and is stimulated by ATP and CoA, needed for the synthesis of a cofactor. In the nervous system, Ansell and Richter (1954b) described a neutral protease, rapidly inhibited after death of the experimental animal owing to the absense of 0,. Guroff (1964) and Marks and Lajtha (1965) analyzed the tissue localization and activity of this enzyme, integrated in the maximum proportion in myelin and membranes and in lower amounts in the neurolysosomes, and they demonstrated its inactivation in anaerobiosis and stimulation in the presence of cofactors. In contrast to the neutral protease, acid cathepsin is potentiated by anaerobiosis, its activity being independent of the sources of energy. But for both categories of proteases, intracellular integration demands an indirect energy supply mediated by the morphological structures, whose formation and kinetics are energy dependent. Thus, the dependence upon energy is another consequence of the structural integration of proteases, which once more draws attention to the difference between the micromedium conditions necessary for proteolytic activity in the normal or dam-

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aged live cell, within the integrated cellular systems, and those required in the biochemical soluble systems.

B. ENZYME-SUBSTRATE RELATIONSHIP The enzyme-substrate relationship may be regarded from two points of view: (1) the activity of proteases on natural intracellular substrates, in the course of metabolism and cellular functions, as compared with the action of these enzymes on the conventional or synthetic substrates on which they were tested; (2) enzyme-substrate accessibility mediated by the cellular structures.

1. Intracellular Substrates The first aspect has been but little explored but is notwithstanding decisive for establishing the functional role of proteases in cellular metabolism (Gordon, 1973). The problem is founded upon analogous biochemical models using conventional substrates of protein nature, or synthetic substrates. The natural conventional, native or denatured substrates are hemoglobin, edestin, casein or serum albumin, macroproteins seldom present in the intracellular medium. The synthetic substrates are polypeptide esters, or the chromogenic complexes of polypeptides and of the amino acids introduced by Bergmann (1942) for analysis of the specificity of extracellular proteases and which represent analogous, but not identical, substrates with the intracellular ones. How do intracellular proteases act upon the intracellular proteins? There are few studies that have tried to answer this question, which is still further complicated by the heterogeneous character of the substrate proteins. Functionally, it appears important in the first stage to analyze two large categories of intracellular proteins : cellular homo- and heteroproteins. The cellular homoproteins are represented by structural proteins and functional proteins elaborated and stored in the cell (in general endogenous self-proteins) . The heteroproteins may be of exo- or endogeneous nature, being in general nonself-proteins, foreign or denatured. The action of proteases may take place within certain heterophagic or autophagic processes, I n superior organisms, physiologic intracellular proteolysis is very important. By this mechanism the amino acids necessary for resynthesis are released daily in amounts 3-fold those provided by food (San Pietro and Rittenberg, 1953; Waterlow, 1964).This process is reflected in vivo by the half-life of proteins. In cell cultures the renewal of proteins is 1% per hour irrespective of species (Eagle, 1961), as well as in the rat liver homogenate at p H 7 (Bohley, 1968).I n the isolated organelles (subcellular fractions), protein catabolism is more intense than in vivo, attaining a peak in the

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mitochondria and lysosomes (Bohley, 1968), in proportion to the protease content. The hyaloplasm presents a more reduced autoproteolysis owing to the very low content in proteases and the presence of proteolytic inhibitors. However, hyaloplasmic proteins may be catabolized in vitro by the organelles with a high proteolytic capacity (Bohley, 1968). Thus, the capacity of lysosomes to catabolize their own proteins at pH 7 (Alberti and Bartley, 1969; De Duve, 1959; De Duve and Wattiaux, 1966; Baird, 1964) as well as mitochondria1 and microsomal proteins, was demonstrated in vitro. I n vivo this process is linked to protein digestion in the autophagic vacuoles and hence to sublethal cytolysis (De Duve, 1963; Straus, 1967b). The phagolysosomal digestion of heteroproteins in uivo was demonstrated by Straus (1964a,b), Mego and McQueen (1965), De Duve et al. (1955, 1962), and Gordon and Jaques ( 1966). Extracellular proteins labeled with 1311 and [1311] As-arsonazoalbumin, pinocytosed in the liver cells, are degraded in the phagolysosomes, by lysosomal cathepsins (Mego and McQueen, 1965). Thus, the serum proteins (Gordon and Jaques, 1966; Schultze and Heremans, 1966), the immunoglobulins (Ghetie and Motaa, 1971), or the proteins of the interstitial connective tissue (Wynn, 1967; Woessner, 1967; Parakkal, 1969) may form substrates of intralysosomal proteolytic digestion in vivo. Another physiological substrate of intraphagolysosomal proteolysis is the precursor of hormonal proteins. Wollman et al. (1964) described the enzymic breakdown of thyroxine from the thyroglobulin macromolecule, a process that takes place in the phagolysosomes of the thyroid epithelia consequent to pinocytosis of the precursors in the follicular colloid (Belanger and Drovin, 1966). The protein hormones form a current substrate of the intracellular proteases, interfering both in their activation, within the macromolecular complexes (hypothalamoneurohypophyseal hormones), and in their degradation (oxytocin and vasopressin are inactivated by hypothalamic oxytocinase) (Tuppy, 1968; Hooper, 1966). Liver lysosomal cathepsins degrade glucagon, and the anteriorhypophysial ones split the corticotropic hormone molecule (Tuppy, 1968). According to several authors, intracellular proteolysis, as well as digestive extracellular proteolysis, appears to manifest a preference for denatured substrates (Freeman et al., 1958; Okunuki, 1961; Coffey and De Duve, 1968). This explains why the antigens are more rapidly destroyed after immunization (Sorkin and Boyden, 1959). I n v i m denaturation may be a prerequisite for proteolysis (Sriram and Marver, 1957; Hill, 1965) but is not always necessary (Beeken, 1965; Gordon, 1973). Within the structures, the degradation of the natural protein substrates sometimes takes on special characters, different from those manifested in the course of proteolysis in vitro in soluble systems. These characters are

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reflected in ( 1 ) the possible slow rate of proteolysis in the phagolysosomal structures (Straus, 1964a,b) ; ( 2 ) the limited and partial character of intracellular proteolysis which accounts for the appearance of biologically active polypeptide fragments, such as the polypeptide hormones, the activated enzymes of the precursors, the basic immunogen polypeptides, etc.; (3) a more restricted substrate specificity of the intracellular peptides than that of the respective extracellular enzymes. The incapacity of cathepsins to hydrolyze short peptide chains has also been reported. Some cathepsins lose their endopeptidase action, acting intracellularly as exopeptidases. Thus, cathepsin C acts as a dipeptidylpeptidase or transferase (Planta and Gruber, 1961) ; cathepsin A acts as a carboxypeptidase A (Jodice, 1967).

2. Intracellular Proteolysis The implications of structural compartmentation in the enzyme-substrate accessibility is one of the key problems of intracellular protein metabolism. The molecular contact between the enzyme and the substrate can take place only in two circumstances: either by penetration of the substrate in the structural compartment in which the enzymes are localized or by release of the enzymes from the compartment to which they are bound. The latter phenomenon was demonstrated for lysosomal enzymes by De Duve (1959, 1963, 1964) and his school in subcellular fractions in uitro when, in order to determine the activity of lysosome-bound hydrolases, the necessity appeared to release enzymes into the medium by lysosomal labilizing factors. The labilizing of lysosomes, which moreover favors the liberation of enzymes in the supernatant, was also noted as a consequence of the in viuo action of certain pathological agents. The free enzyme:bound enzyme ratio is considered to express lysosomal lability, an index of permeabilization of the lysosoma1 membrane for macromolecules. Under normal conditions in viuo, lysosomes are permeable only to small molecular substances and ions. Without assuming the identity of lysosome behavior in uitro with that in vivo the investigations on subcellular fractions have supplied valuable information concerning the relative permeability of lysosomes, their physicochemical instability, their lability under certain biological conditions. The degree of integrity or alteration of the integrative structures determines a change in the compartmentation of hydrolases, favoring the access of enzymes to the structural cellular substrates, whose degradation constitutes the cytochemical background of autolytic lesions, just as the unusual release of some lysosomal hydrolases into the humoral compartment signals cellular necrosis. Under conditions of diffusion of lysosomal hydrolases into the cytoplasm, the degradation of some subcellular organelles has been demonstrated, such as the mitochondria, endoreticulum, etc., with alteration of their functional potential, initiating the formation of autophagic vacuoles

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or cytolysomes (Slater, 1968; Gordon, 1973). But the usual functional mode of enzyme-substrate access takes place as a function of intracellular digestion, within the intracellular vacuolar system of the phagolysosomes (De Duve, 1969; Straus, 1964a,b, 1967a,b). Within this system the exogenous substrates are encompassed within the cell by endocytosis and transported within the cytoplasm by vacuoles designated by Straus with the generic name of phagosomes. As these phagosomes encompass substrates of exogenous origin, they bear the name of heterophagosomes, differing from the autophagosomes or autophagic vacuoles that contain a substrate of cellular endogenous origin, segregated within the cytoplasm. Both the hetero- and the autophagosomes fuse with the primary lysosomes, containing the hydrolase enzymes, and form the phagolysosomes (hetero- or auto-), according to Straus (1964a,b), or the secondary lysosomes, according to De Duve ( 1963). Within these formations the access of activated lysosomal enzymes and of the exogenous and endogenous substrates occur, as well as the consecutive, specific hydrolysis with a compartmental character. The undigested substrate residues (either because they are resistant to lysosomal hydrolases or because of a deficiency in the enzymic activity of the lysosomes) are stored in the lysosomes as myelin figures, lipofuscin pigment, etc., leading to the formation of telolysosomes or postlysosomes (De Duve and Wattiaux ( 1966). Concerning the access of cathepsin to the substrate within the lysosomes and phagolysosomes, indirect indications exist furnished by degradation of proteins labeled with isotopes such as [1311]albuminand [74As]arsono-azoalbumin, used in the experiments of Mego and McQueen (1965), attributed to a lysosomal cathepsin; or by dissociation of thyroglobulin into thyroxine due to the thyroid lysosomal proteases described by Wollman et al. (1964). The intracellular lytic cycle of hemoglobin and ferritin was followed up by Miller and Palade (1964) in the electron microscope in renal cells, confirming the phagolysosomal pathway of protein digestion. The encounter of lysosomal proteases with their substrates occurs along the pathways of heterophagic phagolysosomal digestion affecting the extracellular proteins that have penetrated within the cell by pinophagocytosis. The encounter of lysosomal cathepsins with the cell proteins proper appears to take place within the autophagolysosomal compartment, following upon segregation of certain areas in the altered cytoplasm containing mitochondria, ergastoplasm fragments, hyaloplasm. Digestion of the proteins in the autophage vacuoles (cytolysomes) may have a physiological significance under conditions of rapid protein renewal or differentiation, such as occurs in regeneration or metamorphosis, or more often, a pathological signifiance in autolytic lesions in which cathepsins play a primordial role. I n order to cover all the cell compartments in which the protease-sub-

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strate access occurs, mention must also be made of the proteolytic system afferent to the membranes, about which, however, very little is known. Starting from the findings of Ugolev (1965) and Newey and Smith (1960) concerning contact digestion that takes place at the level of the cell membranes of the intestinal epithelia, it may be assumed that, owing to the activity of the exopeptidases contained in the membranes, a similar mechanism exists for all cell membranes in general and for the intracellular membrane systems in particular. I n this way, proteases, bound to the cell membranes oriented toward the hydrophilic phase, may participate in the screening of cellular information and its eventual storage as memory (Flexner et al., 1967). C. ENZY M E-I N H IBITOR RELATION s HIPS Apart from regulation of the relationship with the specific substrates, the integration of cathepsins into structural compartments also intervenes in the enzyme-inhibitor relation of such importance for the control of intracellular proteolytic activity. In contrast to the extracellular endopeptidase systems, in most of which the specific inhibitor is bound to the enzyme molecule in the inactive proenzyme complex, intracellular endopeptidases are separated from the inhibitors by intracellular structures. Except for the natural inhibitors of extracellular proteases secreted by special glandular organs, such as the pancreas, salivary glands, seminal vesicles, or lymph nodes, spleen, and liver, and, except for the serum and urinary protease inhibitors, there also exist specific cathepsin inhibitors, localized and active within the cell. Finkenstaedt (1957) found in the rat liver homogenate supernatant an inhibitor of cathepsin B. Tokuda et al. (1960) observed during an antigen-antibody reaction in cell cultures, the late release from the cells of protease inhibitor, able to inhibit cathepsins and papain, but not trypsin. This was confirmed by Lebez (1961), who noted in the kinetics of proteolysis produced by liver homogenate, the onset of an inhibtory phase due to the release of an inhibitor, that forms together with cathepsin a dissociable soluble complex. Blackwood and Mandl (1962, 1964) found in the tissue homogenates of liver, kidney, spleen, and tumors a large amount of protease inhibitors, which inhibited both catheptic hydrolysis at an acid pH and hydrolysis of the trypsin or chymotrypsin type. According to some authors, the intracellular compartment of integration of these inhibitors is assumed to be the endoplasmatic reticulum. Penn (1960) observed the inhibitory action of a microsomal fraction, obtained from brain and liver homogenate, on neutral proteinase activated by ATP and CoA, localized in the mitochondrial fraction. The microsomal localization of the inhibitor was confirmed by Lajtha (1961) in nerve tissue homogenates, the inhibition being exercised upon the proteases of the mitochondrial fraction (Brecher and Quinn, 1967; D’Monte et al., 1970b). The role of inhibitor is played

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by aminoacyl-sRNA. In uiuo, cathepsins and their intracellular inhibitors, isolated in different structural compartments can come in contact only after alteration of the separating membranes, when the lytic action of the released enzyme is limited by interaction with the inhibitor. Under the pathological conditions of a third-degree cellular permeability, the cathepsins released into the humoral compartment come in contact with another category of specific protease inhibitors, the serum inhibitors. Among these serum protease inhibitors, specific cathepsin inhibitors have been identified apart from those affecting the extracellular proteolytic systems. Thus, Martin (1961 ) described in sheep serum an a-globulin inhibitor, specific for proteinase A. Evidence was found of serum protease inhibitors in the skin by Beloff (1946) and Wells and Babcock (1953). Ottoson and Sylven (1960) showed that blood plasma contains a powerful inhibitor of cathepsin identified by Snellman and Sylvdn (1967) with Cohn’s IV-b fraction, containing haptoglobins. This haptoglobin inhibitor specific for lysosomal cathepsin B and inefficient on cathepsin D or trypsin, increases parallel to the release of cathepsin B from the tissues (Laurel1 and Gronvall, 1962), preventing, within certain limits, the pathological effects produced by the inadequate presence of cathepsin in the Kumoral Compartment.

VII. Integration in the Tissue Cellular Compartments: the Histochemistry of Neuroproteases

The various aspects of cellular integration of proteases discussed in Section V I probably reflect only part of the implications of enzymic spacing in the heterogeneity of the morphological structures but are sufficient, we believe, to suggest the way in which biological levels of organization, superior to the molecular one, may influence the biological function of these enzymes, engaging them in the metabolism and specific functions of the tissues and organs. This explains the wealth of cellular proteases and their rapid activation in cells specialized for the degradation of proteins (e.g., macrophages, Kupffer cells) or in formations with a rapid protein turnover (liver, spleen, kidneys, nervous system). Their localization in some endocrine glands or in certain areas of the nervous system is correlated with their regulating function in the biology of protein hormones. Therefore the topographical criterion, the criterion of the nonhomogeneity of the tissues and organs, should be taken into account both in the methodology and the interpretation of the biology of these enzymes. From here stems the critical attitude of De Duve (1967), who, in a review of the contribution of enzymologic methods using homogenates and subcellular fractions, emphasizes the limitations of methods that do not realize the postulate of the integrity and homogeneity of the structures, producing alterations and contamination, not taking into

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account the natural micromedium of the enzyme and cooperation of the polyenzymic chains in the complexity of the structures. Hence “the necessity of integrating the analytical results a t the various hierarchical levels of organization.”

A. METHODOLOGICAL PROBLEMS Biochemical and histochemical methodologies differ in that the biochemical methodology deals with soluble systems, and the histochemical one with systems complexly integrated into morphological structural compartments. With most histoenzymologic methods the soluble substrate penetrates into the morphological structure in proportion to the permeability of the membranes, the enzyme-substrate encounter taking place within the subcellular structure, at the site of integration of the enzyme. Hence, histochemical methodology deals with the bound form of the enzyme, which can be activated within the respective subcellular structure. Changes in the permeability of the membranes that govern the enzymesubstrate access occur in states of functional stimulation as well as in pathological states and, for histochemical methodology, represent the cause of variations in the intensity of the reaction. Another methodological aspect of the integration of enzymic molecules into the structures is that of the relationships with the natural inhibitors of enzymes, localized in different structural compartments from those of the enzymes. This phenomenon in the course of histochemical reactions, with penetration of the substrate into the compartment of the enzyme, prevents the intervention of inhibition. With the biochemical methods, the encounter of the enzyme with the inhibitors in the supernatant may lower enzymic activity values. This phenomenon may also occur in histoenzymologic reactions on a film substrate in which the enzyme, released from the structures to act upon the underlying substrates, may be interfered with by the liberated inhibitors. The structural separation of the enzymes into compartments, representing a spatial organization of the enzymatic molecules that affects the steric conformation of the enzyme and sequence of its activity within an enzymic or multienzymic system, ensures to the histoenzymologic reactions a kinetics close to that of the biological conditions, permitting analysis of the reaction in its structural determinism.

B. THETOPOCHEMISTRY OF NEUROPROTEASES Histoenzymologic investigations permitting integrated structural research at the tissue and cellular level are still very few in the domain of neuropro-

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teases. I n contrast to other enzymic systems in the nervous system for which cytochemical maps exist with regional topographic indications and reference to the morphofunctional differentiation, the intracellular protease system has not been subjected to a systematic cyto- and myeloarchitectonic analysis. The investigations on aminopeptidases (Arvy, 1962) and cathepsin C (an inhibitor-resistant esterase) (Pepler and Pearse, 1957; Pearse, 1958) point to the significant representation of these enzymes in the hypothalamic nuclei and intensification of their activity following neurohormonal stimulation in states of dehydration and lactation. The myelin fibrillary structures, although rich in aminopeptidase activity as results from the biochemical determinations (Adams and Glenner, 1962; Adams et al., 1963), give an insignificant histochemical reaction, sometimes below the limit of optical microscopic visualization. This might be accounted for by the toxic action of diazo dyes used in the histochemical reaction to which neuroaminopeptidases are far more sensitive than those of other tissues (Wolfgram, 1961 ; Adams, 1968). In general, the histochemical reaction of this enzyme appears to be predominant in the glial elements, and more intensely stainable than in the neurons and nerve fibers (Onicescu and Cuida, 1970). Histochemical exploration of endopeptidases in the nervous system began in 1961 with the works of Adams and co-workers who, using a technique developed by them with argentic gelatin film (Adams and Tuqan, 1961b), reported on protease activity at pH 5.4 and 7.6 in the myelin nerves (Adams and Bayliss, 1961) and in the central nervous system, with predominant localization in the white matter (Adams and Tuqan, 1961a). Poorly represented in the normal nervous tissue, this reaction becomes more accentuated in Wallerian degeneration and at the periphery of the plaques in multiple sclerosis in connection with demyelination (Adams and Tuqan, 1961a; Adams et al., 1965; Adams 1968; Hallpike and Adams, 1969). Between 1964 and 1972, using at first the original method of Adams and Tuqan, then a technique of our own with a nuclear emulsion of argentic gelatin which allowed for a better resolution at cellular level, we studied the histochemistry of neuroproteases and of cathepsin D in brain and sciatic nerves in connection with functional stimulation and stress (Gabrielescu, 1966, 1970a,c; Gabrielescu and Bordeianu, 1967b, 1968, 1971; Gabrielescu et al., 1966a,b, 1972; Benetato and Gabrielescu, 1967a,b, 1968), as well as in the immunopathologic lesions of experimental demyelinating allergic encephalitis (EAE) (Benetato and Gabrielescu, 1964a,b; Benetato et af., 1965; Gabrielescu, 1966, 1968, 1969a,b,c ; Gabrielescu and Bordeianu, 1967a). I n our material, in the control sciatic nerves, the neuroprotease reaction (Adams-Tuqan method) appears at the limit of visualization in a small

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number of fibers, along which areas of fine granular digestion can be perceived. Using the same method, the control brain sections remain dark except for small areas of reactions in the subcortal white matter, striatum, and optical tract. I'n the cortex the cathepsin D reaction is manifested by a fine, discrete granular reaction in the neurons and in the segment of origin of some of the fibers (Fig. 1) , The presence of a remarkable activity of tryptase, a trypsinlike neutral endopeptidase, was reported for the first time in the nervous tissue by Gabrielescu (1970a, 1971), Gabrielescu and Bordeianu (1971) , using the histochemical method of Glenner and Cohen (1960) and as substrate N-benzoylarginine 8-naphthylamide, a t pH 7.2. Among the histochemical reactions for neuroproteases, the tryptase reaction is the most intense in the normal nervous tissue, integrating itself topochemically especially in the fibrillar structures of the white and gray matter (Fig. 2A, B, C ) . In the perikaryons, the activity of this enzyme is revealed by a fine, granular pale reaction, in contrast to the marked intensity of the reaction in the neuropyle (Fig. 2A,B) . I n general, however, in spite of its relative wealth in proteolytic enzymes, attested to by the biochemical determinations, the normal nervous system exhibits a very low neuroprotease histochemical reactivity, explicable both by the limits of the methodology and especially by the structure latency of these enzymes and their partly inductible character with regard to metabolic requirements. This explains why in states of functional or pathological stimulation, when the activation of latent enzymes is triggered, the histochemical reactions of neuroproteases become evident, and sometimes even strikingly intense, owing to permeabilization of the structures and to the mechanism of enzymic induction.

FIG. 1. Neural activity of cathepsin D (Gabrielescu method) in cerebral cortex of a control.

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UNDER FUNCTIONAL STIMULATION AND STRESS C. INVESTIGATIONS

1. Histochemical Alterations in Protease Activity in the Peripheral Nerves and Brain under the Influence of Electrophysiological Stimulation Studying the significance of protease activity in the course of excitation, we analyzed kinetically the histochemical reaction of these enzymes in the peripheral nerve, cortex, and hypothalamus under the influence of electric stimulation in uiuo, within functional limits, controlled by recording the action potential (Gabrielescu et al., 1966a,b; Benetato and Gabrielescu 1967a,b). The results of these investigations furnished evidence of the activation of acid and neutral proteases in nerve tissue (Adams-Tuquan method). The enzymic reaction appeared to be correlated with the functional state of the structures and proportional to the intensity, frequency, and duration of the stimuli, with a reversible character in the phase of recovery. More intense (20 V ) stimulations and of longer duration (20 minutes) have drawn attention to the importance of protease activation in states of functional overstrain of the nervous system. In these conditions, a positive protease reaction in the cerebrospinal fluid attests to the extracellular release of neural enzymes.

2 . T h e Histochemistry

of Neuroproteases under Conditions of Physical, Environmental, and Psychical Stress

The experimental model of electric stimulation has the great advantage of direct recording of the functional state of the nervous tissue, but also has the disadvantage of unphysiological conditions. Therefore, in subsequent investigation we resorted to various experimental models of stress in order to obtain overstrain of the nervous system due to the effort of adaptation of the entire organism to the unusual activity and environmental conditions (Gabrielescu, 1970a,b,c). The following experimental models were used : ( 1 ) stress caused by swimming up to the point of fatigue or exhaustion (Gabrielescu and Bordeianu, 1967b, 1968) ; ( 2 ) stress caused by adaptation to the environment under high temperature conditions (1 hour at 37-50°C) (Benetato and Gabrielescu, 1968) ; ( 3 ) gravitational overstrain caused by acceleration (6-10 g) (Gabrielescu, 1970a,c) ; (4) emotional stress produced in rats by the presence of a cat (Gabrielescu, 1970a; Gabrielescu and Bordeianu, 1971 ) . Histochemical analysis of neuroprotease activity under these varied stressing conditions revealed similar, common alterations irrespective of the nature of the stimulus. On control brain sections, the Adams-Tuqan method supplies evidence of only a very reduced proteolytic reaction, localized especially in the subcortical and periventricular white matter, in the striatum, fornix,

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FIG. 2. For legend see opposite page.

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FIG. 2. Histochemical reaction of tryptase (Glenner and Cohen method). ( A ) In a normal cerebral cortex. ( B ) In Amrnon’s horn. (C) In subcortical white matter.

and optic chiasma. These reactions are produced by the activity of proteases which, under incubation conditions, are liberated from the morphological structures of the section, acting upon the underlying film of argentic gelatin. Consequently, it may be asserted that the activity of the labile, releasable form of the enzymes, active extratissually, is reduced in the controls under the technical conditions used. I n stress, the reaction of proteases is intensified, enlarging the areas of proteolysis that permit the plotting of a map of the reactive nervous structures, affecting both the white and the gray matter. The intensity of the reaction appears to be proportional to the intensity of the stress. Medium stressing conditions ( I ) such as swimming during 1 hour for guinea pigs, hyperthermia of 37-40°C, emotional stress for 15 minutes, produce a moderate activation of the protease reaction. Intense stress (11) , such as swimming for 2-4 hours, 43O hyperthermia, 6 g acceleration for 1 hour, emotional stress for more than 30 minutes, produce an intense protease reaction. Very intense stress (111) such as swimming up to exhaustion, hyperthermia of 45-50°C, acceleration of 10 g for 1 hour, is characterized by a lower proteolytic activity than that induced by intense stress, developing up to negative results in the case of longitudinal acceleration of 10 g per hour, suggesting the

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FIG. 3. For legend see opposite page.

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FIG. 3. Increased reaction of cathepsin D during intense stress. ( A ) In neurons of cerebral cortex. ( B ) In neurons of Ammon’s horn. ( C ) In bundles of subcortical white matter.

liberation of enzymes in uivo and their passage into the humoral compartment. As the histochemical method of Adams-Tuqan supplies unselective evidence of a mixture of endopeptidases activating the gelatin substrate, differentiated only by their pH optimum (acid and neutral), we attempted to identify histochemically certain types of proteases on the basis of their substrate specificity and of their specific activators and inhibitors. Cathepsin D was identified histochemically in the central nervous system by means of a technique developed by us (Gabrielescu, 1970a) with a nuclear emulsion of argentic gelatin that offers resolution at cellular level. Incubation at a low pH of 3 . 4 4 . 5 and high temperature of 45-50°C differentiates cathepsin D from the other cathepsins which are thermolabile and from the proteinases with a neutral pH optimum. By this method, the reaction for cathepsin D is also slightly positive in the control neurons and the origin segment of the neuronal processes (Fig. 1 ) . In stress the hydrolysis areas are outlined in the perikaryons and fibrillar bundles, appearing, however, more intense intracellularly and in the vascular walls (Fig. 3 A-C) In contrast to the techniques on a film substrate (acid and neutral pro-

.

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teases and cathepsin D ) that assume the liberation of the enzyme from the structures, the techniques for tryptase and arylamylases, using a soluble substrate in the medium, presuppose penetration of the specific reactants into the morphological structures in which the enzyme is integrated, revealing the bound form of the protease activated within the lysosomes or the membranes. Histochemical analysis of specific proteases under stress conditions demonstrates, in the course of medium stress, intensification of the granular reaction in the perikaryons and neuropyle, both for trypsinlike tryptase (Glenner and Cohen technique, 1960) and for the aminopeptidases (Adams and Glenner technique, 1962). Worthy of note is the sharp reaction of proteases in the hypothalamic nuclei, even in the incipient phase of stress (emotional stress), characterized by activation of these enzymes within the lysosomes and membranes. With intensification of the stress, the images show an increase in the granular reaction, clustering of the final product of the reaction, accentuating the contour of the neuronal processes and fibrils (Fig. 4A,B). The reaction is generalized so that it includes not only the hypothalamus, but also the cortex, Ammon’s horn, and the thalamus. The protease reaction in the vascular walls concomitantly becomes more intense. I n intense stress (11) the heterogeneity of the degree of granular loading of the neurons may be noted within the same topographical formation, with a tendency to diffusion and bleaching of the reaction in some cells. I n very intense stress (111) the entire reaction becomes pale, suggesting a decrease in the activity of the enzymes probably due to enzymic discharge in vivo.

3. Action

of

Certain Mediators of Stress (Biologically Active Substances)

I n order to study the integration of the neuroprotease reaction in the general mechanisms of neurohumoral regulation, we followed up in a series of experiments the influence on the in vitro and in vivo activity of these enzymes of certain chemical mediators, polypeptide hormones, and biologically active substances, known for their participation in stress reactions. Of the substances administered in vivo and in vitro, norepinephrine and oxytocin appear to have no appraisable reaction, vasopressin and bradykinin exercise a moderate influence, whereas epinephrine and histamine in very small doses produce a violent activation of brain neuroproteases, appraised by the film-substrate method, suggesting the possibility of a correlation between the chemical mediators and intracellular proteolytic enzymes in stress reactions. 4. Correlations between the Intensity of the Histoenzymic Reactions and

Labiliration of the Structures a. I n Vitro Labilizations. With a view to furthering our study on the physicochemical mechanisms that interfere in the integration of hydrolase

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FIG.4. Tryptase reaction during intense stress. ( A ) Increased activity in cerebral cortex, involving perikaryons and neuropile. ( B ) Increased activity in perikaryons and processes of Ammon’s horn neurons.

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enzymes in morphological structures and in the change of their compartmentalization in the course of stress, a series of experiments was carried out. In these, brain sections were treated in uitro with substances that might change the permeability of the membranes or of lysosomal and myelin structures to which the enzymes are bound, or which might split the physicochemical bonds by which the enzyme is integrated into the macromolecular structure of the membrane or of the matrix or these organelles. After treatment of the sections with distilled water or fat solvents, the protease reaction on the gelatin film increases owing to release of the enzyme. I n the methods using a soluble substrate in the incubation medium (tryptase, cathepsin C, aminopeptidase, acid phosphatase) preincubation of the sections in vitro with substances that render the morphological membrane structures more permeable, such as acetone and normal saline, favor, in terms of the duration of their action, various degrees of activation of the enzymic reaction, from a more accentuated granular reaction at the level of the structures up to diffusion of the reaction in the tissue, or even extracellularly in the incubation medium. These results demonstrate the existence of varied linkages between the protease molecules and the morphological structures. 6. Lysosomal Lability Test. In acute experiments, the activation of hydrolases by lysosomal labilizations appears to represent the most rapid response with consequent histochemical alteration of the enzymic reactions. Using acid phosphatase as a histochemical “marker” of the presence of lysosomes and Bitensky’s lysosomal lability test ( 1963a; Bitensky and Gahan, 1962) as an index of the functional state of the lysosomes, we followed up the correlation between the histochemistry of proteases and lysosomal permeability in stress reactions. Bitensky’s test demonstrates the correlation between the intensity of the histochemical reaction for acid phosphatase and lysosomal permeability in the unfixed tissues, with maintained biologic reactivity. Acid phosphatase, as well as the other acid hydrolases contained in the lysosomes in a latent state, becomes active in terms of labilization of the lysosomal structure. Firstdegree permeabilization of the lysosomal membrane permits enzymesubstrate contact by intralysosomal penetration of the substrate (sodium /3-glycerophosphate with the technique used), whereas second-degree permeabilization corresponds in general to alteration of the membrane permitting extralysosomal liberation of the enzyme and a diffuse reaction. Follow-up of the degree of histochemical activity of the enzyme on serial sections-from the minimal incubation time necessary for the reaction to appear, to the optimal time necessary for reaching maximum intensity, and up to the moment of diffusion-permits kinetic testing of lysosomal reactiv-

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ity, the permeability of the membrane being greater the shorter is the minimal and the diffusion time. Under stress conditions, the functional histochemistry of acid phosphatase demonstrates changes common to most neurons, irrespective of the experimental model used. The positive reaction appears in the form of endproduct granules, randomly distributed throughout the perikaryon of the nerve cells. I n the controls, a positive reaction becomes evident after incubation for 30 minutes to 1 hour. Fifteen minutes’ incubation is not enough to reveal a granular reaction. Under the influence of stress, the reaction becomes positive after a minimal incubation of 15 minutes, demonstrating the increased permeability of the lysosomal membranes, that permits rapid penetration of the substrate within the lysosomes where it reacts with the enzymes. This phenomenon has been observed after prolonged physical effort up to exhaustion, under the influence of transverse, and especially of longitudinal, acceleration, as well as under vasopressin treatment. Another difference with regard to the controls is the increased number of the end-product granules, intensifying the intraneuronal reaction, as well as the increased proportion of strongly reactive neurons. Quantitative alteration of the reaction is accompanied by an increase in the size and staining intensity of the granules by coalescence of the structures in which they appear. This phenomenon develops with the intensity of the stress, being extremely evident after 3-4 hours of swimming, in hyperthermic stress at 43OC, and after 1 hour’s transverse acceleration at 6 g. The tendency to conglomeration appears especially toward the cone of origin of the axon and in general toward the origin segments of the neuronal processes, particularly manifest in rats subjected to a hyperthermic environment. In many instances, granular conglomeration at the origin of the processes is accompanied by a granular reaction along the processes, sometimes in segments distal to their origin, as may be noted in the cortex and hypothalamic neurons as well as in isolated cells of the lateral hypothalamus. I n many instances, the reaction of the neuronal processes coexists with a rich granular loading of the perikaryon, but under certain conditions, as in stress of longer duration or greater intensity, the perikaryon reaction is weak and runs parallel to a clustering of the granules in the emergence cone and their presence along the fibers, suggesting a true migration, an intraneuronal circulation of the enzyme carrier particles. Another noteworthy aspect in the functional histochemistry of acid phosphatases is the phenomenon of diffusion of the reaction that appears in the neurons of the animals subjected to greater stress. This diffusion, which may be accounted for by the release of the enzyme from the lysosomes into the neuroplasm, is brought about by alteration of the permeability of the lysosoma1 membranes, permitting exteriorization of the enzymic macromolecules

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in the course of a longer incubation. The enzymic origin of this diffuse reaction is proved by its inhibition by 0.01 M NaF, an acid phosphatase inhibitor. In another category of experiments in which the animals are exposed to unusual stress, a clinical state close to shock is produced and intraneuronal acid phosphatase activity falls sharply, becoming almost negative or paucigranular. This is the case in accelerations of 1 hour at 10 g ( = LDso), or hyperthermic stress at over 45O. I n longitudinal acceleration of 1 hour at 10 g, the lowest reaction was recorded after 1 hour’s incubation, reduced to a few granules in very rare neurons. Lysosomal permeabilization with normal saline in uitro (Gabrielescu, 1970a), which in the case of the intralysosomal presence of acid phosphatase produces an intensification of the reaction in the tissue, remains negative after longitudinal acceleration of 10 g for 1 hour, lending support to the hypothesis of the extraneuronal liberation of the enzyme in vivo.

VIII. The Implications of Neuroproteares in the Physiology and Pathology of the Nervous System

O n reviewing the main data concerning the proteolytic potential of the nervous system, the biological role of neuroproteases begins to be outlined increasingly clearly. One may thus perceive several sections of nervous physiology in which the implications of intracellular protease activity are evident and certain pathogenic mechanisms in which the role of these enzymes cannot be questioned. Represented by most of the known enzymic types, belonging to the intracellular endo- and exopeptidases, neuroproteases display their activity in supramolecular, subcellular, cellular, and regional morphologic-metabolic compartments within the framework of which they are integrated diff erentially in the metabolism and functions of the nervous system. Structural compartmentation and spacing appear to be essential for the reactivity of this enzymic system and for establishing the borderlines between their physiological and pathological implications. From the metabolic viewpoint, total proteolysis realized by the sequential action of intracellular proteases, normally taking place in the phagolysosomal compartment, represents the fundamental mechanism by means of which the intracellular catabolism of proteins takes place. In the balance of protein synthesis, stimulated by a feedback mechanism and by the increase in the free amino acid pool it furnishes, enzymic catabolism stimulates the turnover of neuroproteins and their rapid rate of renewal. In superior organisms and especially in the tissues with a high functional specialization, intracellular proteolysis represents one of the mechanisms of regulation of the enzymic

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systems by means of which the resynthesis of proteins from the released polypeptides and amino acids is possible, without any change in the cell mass (Bohley, 1968) Correlation of the processes of protein synthesis and catabolism are also reflected in the implications that proteases of the endoreticular compartment may have in polyribosomal assembly and in the transport of newly formed polypeptides, in the initiation of transcription by DNA derepression, or in lengthening of the polypeptide chains linked to memory. By partial proteolysis, the neuroproteases participate in the release and inactivation of some polypeptide hormones, interfering in the neurosecretory function and the regulation of neurohormonal mechanisms (Tuppy, 1968; Burgus and Guillemin, 1970). In the regulation of the permeability of the blood-brain barrier these enzymes may interfere by both activation and inactivation of certain vasoactive polypeptides (kinins, angiotensin, P substance) (Prado, 1968). The functional state of excitation of the nervous system is accompanied by the activation of neuroproteases (Ungar, 1963; Gabrielescu et al., 1966a,b; Jakoubek and Gutmann, 1968). The way in which this enzymic system participates in the genesis and transmission of the nervous inflow is still obscure. Prospects of a closer understanding may be foreseen following the research work of Bass and MacIlroy (1968) concerning the role of a membrane tryptase on the function of the ionic pump, by reversible proteolysis of a “gate protein,” and the suggestion of other authors regarding the axonal flow mechanism and function of the synaptosomes (Marks et al., 1970; Orrego, 1969; Gordon et al., 1968). Overloading of the nervous system produces stress conditions that favor increased neuronal protease activity correlated with an increase in lysosomal permeability and that of the membrane structures, into which they are integrated (Gabrielescu, 1970a,b ; Gabrielescu and Bordeianu, 1968, 1971) . Proportional to the intensity and duration of stress, the activation of enzymes presupposes various degrees of labilization with regard to the structure. Stimulation within functional limits favors the release of the enzyme by labilization of the electrovalent bonds with anionic sites of integration at supramolecular level, but with maintenance of subcellular compartmentation. In intense stress the altered membrane permeability permits intracytoplasmic diffusion of the enzymes with stimulation of the autophage process. Alteration of the subcellular compartments favors sublethal cytolysis, as an early stage of the lesion in the pathology of stress, bringing about, in case of repeated and prolonged stress, the phenomenon of wear and tear characterized by the accumulation of residual bodies. I n very intense stress, cellular compartmentation is compromised, alteration of the membranes permitting the release of neuroproteases in the extra-

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cellular space, with permeabilization of the blood-brain barrier and sometimes with the production of shock phenomena. I n the experimental pathology of the nervous system, encephalopathy due to vitamin E deficiency (Tappel et al., 1963) and that induced by hyperbaric hyperoxygenation (Hall and Sanders, 1966) constitute conditions in which there is a marked increase in catheptic activity consequent to alteration of the intracellular membranes by lipid peroxidation. I n the pathogeny of demyelination neuroproteases interfere at an early moment in the labilization and disintegration of myelin by the digestion of basic proteins, following of alteration of the morphological compartments (Adams, 1968; Gabrielescu, 1969a,b,c). The release of neuroproteases from the lysosomes of axons or glial cells or even from the lamellar structures of myelin, favors autolytic processes, unmasking antigenic polypeptides which under certain conditions may sensitize the lymphoid system triggering autoimmune reactions. Illustrated in the case of EAE, this mechanism may find favorable conditions in human pathology, in the course of multiple sclerosis, and especially in postviral demyelinating encephalomyelitis, where the intense activation of proteases induced by cytopathic viruses (Allison, 1968) may be encountered together with intraneural infiltration of mononuclear cells (see Benetato et al., 1960, 1961a,b, 1963a,b, 1966a,b). The as yet insufficient investigations give to the interpretations a hypothetical character; but even if at this stage it is not yet possible to define the moment and mode of action of neuroproteases, the existing research work allows us to foretell the place this enzymic system will have in the biology of the nervous system. Integrated complexly in the metabolism and functions of the nervous tissue, the reaction of neuroproteases represents a fundamental response to many types of injuries and stress, triggering chains of biological reactions at the uncertain, mobile borderline between functional and pathological. Differentiated structural integration, with metabolic and morphological compartmentation of these enzymes, is one of the conditions of compliance with this frontier line, ensuring the enzyme-substrate relation and equilibrium between synthesis, activation, and inhibition. REFERENCES Abderhalden, E., and Caesar, G. (1940). Fcrmcntforschung 16, 225. Abderhalden, R., and Elsaesser, K. H. (1943). Fcrmentforschung. 17, 178. Adams, C. W. M. (1962). In “Neurochemistry” (K. A. Elliott, I. H. Page, and J. H. Quastel, eds.), pp. 85-1 12. Thomas, Springfield, Illinois. Adams, C. W. M., ed. (1965). “Neurohistochemistryy.” Elsevier, Amsterdam. Adams, C. W. M. ( 1968). In “Macromolecules and the Function of the Neurone” (2.Lodin and S. P. R. Rose, eds.), pp. 111-120. Excerpta Med. Found., Amsterdam. Adams, C. W. M., and Bayliss, 0. B. (1961). I. Hisfochcm. Cyfochcm. 9,473.

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Ugolev, A. M. (1965). Physiol. Rev. 45, 555. Ungar, G. ( 1963). “Excitation.” Thomas, Springfield, Illinois. Ungar, G., Ascheim, E., Psychoios, S., and Romano, D. V. (1957). J . Gen. Physiol. 40, 635. Uzman, L. L., Rumley, M. K., and Van den Noort, S. (1961). J . Neurochem. 6, 299.

Uzman, L. L., Van den Noort, S., and Rumley, M. K. (1962). J . Neurochem., 9, 241. Waelsch, H. (1957). In “Metabolism of the Nervous System” (D. Richter, ed.), p. 431. Pergamon, Oxford. Waelsch, H. (1959). Proc. Int. Congr. Biochem., 4th, 1958, pp. 36-45. Waelsch, H. (1961). I n “Regional Neurochemistry” (S. S. Kety and J. Elkes, eds.), pp. 57-64. Pergamon, Oxford. Waelsch, H.,and Lajtha, A. (1961). Physiol. Rev. 41, 709. Waterlow, J. C. (1964). Tech. Rep. Ser., I n t . A t . Energy Ag. 45, 235. Wells, G. C., and Babcock, C. (1953). J . Invest. Dermatol. 21, 459. Willstatter, R., and Bamann, E. (1929). Hoppe-Seyler’s Z. Physiol. Chem. 180, 127.

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ON AXOPLASMIC FLOW By Liliana Lubihska

Department of Neurophyriology. Nencki Institute of Experimental Biology, Warsaw, Poland

I. Introduction

.

11. Aspects of Axoplasmic Flow as Revealed by Various Methods A. Miuoscopy of Living Neurons

B. Tracers in Intact Nerves

.

. .

C. Interruption of Axonal Pathways D. Peripheral and Central Effects of Nerve Section 111. Influence of Various Factors on Axopksmic Flow A. Separation from Cell Bodies B. Metabolic Factors C. Temperature D. Growth and Regeneration E. Nerve Activity IV. General Description of Axopksmic Flow A. Fast Flow B. Slow Flow References

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. .

. .

241 242 242 245 253 261 273 273 273 2 74 276 277 278 280 285 287

1. Introduction

The existence of “axoplasmic flow,” that is of the longitudinal translocation of materials in axons, is now generally recognized. The descriptions of this process and the proposed interpretations are, however, very discordant. Thus, for workers dealing with transparent neurons in tissue culture or in developing animals, axoplasmic flow is bidirectional and just. They see much movements directly under the microscope or on cinemicrophotographic films. O n the other hand, physiologists work mostly with nerves of mature animals. Such nerves are usually opaque, and axoplasmic flow in them eludes direct observations. Indirect methods have to be used to detect the flow in this material. The existence of movements of neuronal components in axons in situ was first inferred from the effects of nerve section and subsequent regeneration. Since the peripheral effects of nerve section are much more conspicuous than the central effects, attention was focused almost exclusively on the transport of substances from the cell body to the nerve end24 1

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LILIANA L U B I ~ S K A

ings. The timing of experiments was based on the anticipated velocity of translocation, which was believed to be approximately equal to that of elongation of regenerating fibers. Therefore, for a long time workers using opaque nerves considered axoplasmic flow to be unidirectional (somatofugal only) and slow. Although recently a fast somatofugal flow of some axonal constituents (by one or two orders of magnitude faster than the rate of nerve regeneration) has gained general recognition, the existence of flow in the somatopetal direction is still often disregarded or even denied by experimenters using adult animals.' Thus, the general impression created by the published data is that the pattern of axoplasmic flow is different in uitro and in vivo. It does not seem likely, however, that an essential intracellular process would in fact be fundamentally different under the two conditions. O n closer analysis the discrepancies seem to be caused largely by extraneous factors. The anatomical and physiological features of various experimental materials require selection of appropiate methods of analysis. These methods have various limitations and bring to light only certain facets of axoplasmic flow, influencing correspondingly the interpretation of results. Individual interests of observers, the known or presumed biological significance of migrating components, and current ideas about the working of nerve cells are other sources of discrepancies. The aim of the present essay is to reexamine the divergent descriptions of axoplasmic flow and the ways by which they were arrived at. The paper is composed essentially of two parts. I n the first, the advantages and shortcomings of various methods of detection and measurements of flow will be discussed in relation to results obtained by these methods. I n the second, an attempt will be made to give a provisional general description, quantitative where possible, of axoplasmic flow as it seems to emerge at present from the body of critically examined evidence. The paper is not intended to be a complete review of the published data. The number of papers dealing with axoplasmic flow of various components is constantly increasing. Only papers pertinent to the problem under discussion will be analyzed; for the rest the reader will be referred to the existing reviews. II. Aspects of Axoplamic Flow as Revealed by Various Methods

A. MICROSCOPY OF LIVING NEURONS The most extensive studies of living axons in uivo were made by Speidel (1933, 1935, 1936, 1942, 1964) on cutaneous nerves in transparent fins of Several papers describing somatopetal axoplasmic flow were published after this paper had been sent to the editor.

ON AXOPLASMIC F L O W

243

tadpoles. He observed individual axons for many successive days and described their growth, branchings, relation to Schwann cells at various stages of myelination, and intra-axonal movements of granules. Recently, Williams and Hall (1970, 1971) were able to observe for prolonged periods myelinated fibers in undisturbed mature mammalian nerve trunks with preserved circulation. Using oblique incident illumination and focusing progressively through the epineurial and perineurial sheaths, they examined the shape of internodes and of paranodal bulbs and the behavior of Schmidt-Lantermann incisures in normal fibers and after nerve crush. The details of axoplasmic content of fibers observed under these conditions are not resolved, and this very ingenious technique has not been used so far to study axoplasmic flow. .The bulk of microscopic documentation concerning axoplasmic movements was obtained on neurons in tissue culture (for reviews, see Willmer, 1960; Murray, 1965; Pomerat et al., 1967; Lumsden, 1968). O n time-lapse cinematographic films, particles are seen to pass from the cell body into the axon and from the axon into the cell body (Nakai, 1956, 1964; Pomerat et al., 1957; Pomerat, 1958; Shahar and Saar, 1970). I n the axons their itineraries are usually linear. Movements in both directions may be seen in a single fiber. Sometimes the particles switch tracks or reverse their course. The bidirectional migration of particles and the movements of pseudopods at the growth cones continue for some time in axons cut from their cell bodies (Levi and Meyer, 1936). The pinocytotic droplets taken up at the nerve endings move in the ascending direction and accumulate at the proximal end of the transected fiber (Hughes, 1953; Godina, 1963). In some cases the speed of individual particles was measured. Thus, ranges of 31-155 mm/day were observed by Pomerat et al. (1967). Pinocytotic droplets moved at velocities of 18-48 mm/day (Hughes, 1953). I n culture of dissociated neurons, the migration of particles is slower (Nakai, 1956). Lasek (1970a) quotes speeds of up to 1000 mm/day found by Burdwood (1965) in axons grown in the presence of nerve growth factor. Short-term observations of movements of axoplasmic granules were made also on surviving neurons in excised pieces of transparent tissues (Fedorov, 1935; Adamstone and Taylor, 1953; Carlisle, 1959; McMahan and Kuffler, 1971; Kirkpatrick et al., 1972; R. S. Smith, 1971; and others). The general pattern of movements in excised fibers of adult animals resembles that seen in tissue culture. The range of velocities is also similar. Thus, Carlisle (1959) describes velocities of 144-288 mm/day in the axons of the pituitary stalk of goosefish; Kirkpatrick et al. (1972), 110 mm/day in the sciatic nerve of chicken; and R. S. Smith (1971), 86 mm/day in the sciatic nerve of frog. The optical resolution afforded by microscopy of the living cell is not high, and the nature of migrating particles cannot be identified except for mitochondria and larger pinocytotic droplets. Since the speed of intra-axonal

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movements depends on the kind of neurons and conditions of explantation and is probably different for various organelles, it seems useful to confront the direct measurements of translocation of particles with more selective biochemical or autoradiographic methods applied to the same material. Such a combined study was made by Kirkpatrick et al. (1972). When studied at high temporal resolution, the progress of individual particles is not uniform (Rebhun, 1964). The particles move over a certain distance then stop, oscillate, and move again. Berlinrood et al. (1972) studied these movements in culture of embryonic fibers of several species of Amphibia. The particles moved in the same fibers in both directions at a variety of speeds. Discontinuity of advance is also suggested by inspection of autoradiographs of fibers labeled in culture (Utakoji and Hsu, 1965). The distribution of silver grains over the axons is uneven; short gaps, which do not show any clear periodicity, appear between heavily labeled portions of axoplasm. Probably the uneven distribution of organelles in adjacent regions of axoplasm seen in electron micrographs (Fillenz, 1970) also reflects the microirregularities of their movements. The average velocities of movements of particles. measured over longer time intervals and greater lengths of axons are, however, fairly constant and fall within the range of velocities found in axons in vivo by biochemical or autoradiographic methods. In tissue culture axoplasmic flow and elongation of growing or regenerating fibers may be studied in the same nerve cells. The relationship between the two processes is not a simple one. The growth of axons may be inhibited selectively by certain drugs, which do not affect axoplasmic flow. The velocity of particle movements is 10-50 times faster than that of the elongation of fibers (Hughes, 1953; Nakai, 1956; Utakoji and HSU,1965). A similar difference between the velocity of migration of some axonal components and the rate of regeneration was found in mammalian nerves in vivo (Lubihka, 1964). These results show that elongation of axons cannot be regarded as a simple outpouring of migrating axonal materials. According to Yamada et al. (1970, 1971) the elongation of growing fibers is brought about by at least two essential processes: axoplasmic flow and incessant activity of growth cones. Thus, both cytochalasin B, arresting the movements of growth cones and colchicine or Colcemid, interfering with axoplasmic flow, inhibit the elongation of axons (Daniels, 1968, 1972; Seeds et al., 1970; Yamada et al., 1971). According to Crooks and McClure (1972), cytochalasin B does depress the fast axoplasmic flow. Cyclic adenosine 3p-5p-monophosphate,on the contrary, enhances axonal elongation and reverses the effects of Colcemid. Roisen et al. (1972a,b) attribute this reversal to the stimulating effect of cyclic AMP on the assembly of microtubules from the preexisting subunit pool. The amount of neurotubule protein is not increased (Hier et al., 1972).

ON AXOPLASMIC F L O W

245

The mechanism by which the nerve growth factor (NGF) (Levi-Montalcini and Angeletti, 1968) produces the spectacular acceleration of outgrowth of axons has not yet been elucidated. According to Hier et al. (1972) NGF stimulates de novo synthesis of neurotubule subunit protein, and this effect precedes axon extension. Yamada and Wessells (1971), however, think that protein synthesis is not affected in the critical early phases of axonal outgrowth. They found a similar number of microtubules in axons in NGFtreated cultures and in controls and attribute the accelerated growth to the stimulating influence of NGF on the activity of growth cones. Yamada et al. (1971) proposed the following tentative description of elongation of nerve fibers. The perikaryal materials necessary for formation of additional portions of the axon are conveyed by axoplasmic flow and accumulate at the growth cone. It is there that the new surface membrane is assembled. The ultrastructure of growth cone and the possible role of its various components in the assembly of membrane as well as of its degradation are discussed by Yamada et al. (1971) and Bunge (1973). With the advance of the growth cone, the stretch of axon left behind does not move; if the fiber bifurcated at the growth cone, the point of branching remains at approximately constant distance from the cell body in spite of the further advance of the fiber tip. The new fiber surface does not show any distal motion (Bray, 1970, 1973). Many perikaryal materials may, however, travel to the tips of regenerating axons as preformed organelles. This appears to be the case, for example, for large dense-cored vesicles and clusters of small vesicles in sympathetic neurons. They accumulate gradually in front of the site of nerve transection and subsequently appear in regenerating axonal sprouts at a time when the number of these organelles in the cell bodies decreases significantly (Matthews and Raisman, 1972; Matthews, 1972).

B. TRACERS I N INTACT NERVES 1. Radioactive Tracers The methods that have provided most of the quantitative data on the. migration of axonal components in intact neurons in vivo involve the use of radioactive tracers. These are introduced systemically or into neuronal pools and, recently, also intracellularly into individual cell bodies (Lasher et al., 1970; Schubert et al., 1971). In the perikarya the tracers are incorporated into macromolecular components, and these freshly synthesized molcules are discharged into the axons, where their migration may be followed by various techniques. It should be stressed that such experiments will show only the somatofugal migration. Since the incorporation of radioactive pre-

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cursors is taking place in the perikarya, the components migrating in the somatopetal direction will not initially carry the label and their movement will not be detected. The study of labeled migrating materials is most often confined to macromolecules, mainly proteins, or to smaller molecules attached to macromolecular carriers. I n some cases the fate of small molecules or unincorporated precursors has also been studied (Akcasu and Salafsky, 1967; Kidwai and Ochs, 1969; James and Austin, 1970a; Di Giamberardino, 1971; Forman, 1971; Wolburg, 1972; Fischer and Schmatolla, 1972; and others). The newly synthesized proteins stay in the cell bodies for a variable time (Droz, 1965, 1967a). Some remain there for many days, others are rapidly discharged into the axons. Droz calls them “sedentary” and “migratory,” respectively. I n the axon the label appears first in the proximal part, then spreads somatofugally and finally reaches the nerve endings. The progress of label along axons may be followed by quantitative autoradiography or by radiometric counting techniques applied either to the whole nerve or to its subcellular fractions. In some experiments migration of individual labeled proteins (Miani et al., 1972; and others) has been investigated. Procedures have been worked out to eliminate from analysis the unincorporated precursors and labeling of nonaxonal cellular elements. The label migrates only in uninterrupted stretches of axons. When the nerve is crushed before injection, the labeled material does not spread beyond the site of injury but accumulates in front of it (Miani, 1964; Lasek, 1968). If the crushed nerve is allowed to regenerate, the label moves down as far as the tips of regenerating axons (Miani, 1964). If however, the nerve is crushed several days after injection of precursors, when the label has already spread throughout the length of axons, a progressive increase in radioactivity appears both on the proximal and on the distal side of the crushed region. The increase in radioactivity on the proximal side of the lesion is generally regarded as a result of “damming” of materials migrating in the proximodistal direction (Weiss and Hiscoe, 1948). The analogous increase on the distal side of the lesion may be similarly interpreted as damming of materials migrating in the distoproximal direction. This time, however, we are dealing not with materials freshly discharged from the perikarya, but with those which had already reached the distal parts of the axon before the crush was made and which are migrating in the reverse direction. These experiments on crushed nerves will be discussed in detail in the next chapter. The early measurements of the somatofugal migration of labeled proteins and phospholipids gave velocities of one to several millimeters per day (Samuels et al., 1951; Ochs and Burger, 1958; Weiss, 1961; Miani, 1962; Droz and Leblond, 1962; Ochs et al., 1962; Taylor and Weiss, 1965; and others).

ON AXOPLASMIC FLOW

247

Later work brought to light the existence of a much faster migration of the order of 100 mm/day (Miani, 1964; Lasek, 1966, 1968; Karlsson and Sjostrand, 1968; Livett et al., 1968; McEwen and Grafstein, 1968; Ochs and Ranish, 1969; Ochs et al., 1969; Chou, 1970; Sjostrand, 1970; CuCnod and Schonbach, 1971; Elam and Agranoff, 1971; and others). Intermediate velocities have also been described (for references, see Lasek, 1970a; Bradley et al., 1971). I t should be noted, however, that the measurements of flow were usually made on nerves containing a heterogeneous population of fibers of various diameters and functions. The calculated values thus represent average velocities in the nerve. If, as seems probable, the characteristics of axoplasmic flow are different in various types of nerve fibers (cf. Miani et af., 1972; Lubiriska and Waryszewska, 1972) rather large differences between nerves, even in the same animal, are to be expected.

2. Nonradioactive Tracers I n a few cases when the radioactive tracers were injected near the nerve endings (Watson, 1966) or in the muscle compartment of a spinal cordnerve-muscle preparation in vitro (Kerkut et al., 1967), the ascending migration of label, presumably not incorporated into macromolecules, was observed. However, the main body of evidence concerning the existence of somatopetal axoplasmic migration in intact neurons in uivo was obtained with nonradioactive macromolecular tracers. Several neurotropic viruses and toxins may spread to the spinal cord by neural routes when injected into the limb (Goodpasture, 1925; Meyer and Ransom, 1903; Bromeis, 1938; Howe and Bodian, 1942; Kryzhanovsky et al., 1961; Wildy, 1967; and others). When the humoral spread of these agents is prevented, their migration in nerves may be analyzed. Thus, Kryzhanovsky et al. (1961, 1971) studied the neural ascent of tetanus toxin in various mammals after neutralization of the circulating toxin by antitoxin (antitoxin does not enter the nerve). The tetanus toxin injected into the leg muscles of rats appears first in the distal parts of the sciatic nerve and successively invades the more proximal parts and ventral roots. The spread in the roots is also in the distoproximal direction. The cord is reached in 7-8 hours after injection, and it is only then that the electrophysiological changes appear in the injected muscle. The velocity of migration calculated from these experiments is about 200 mm/day. It is interesting to note that in discussions of whether the tetanus toxin migrates in the axons or in the perineurial spaces, the fast rate of translocation was considered to be an argument against the axonal route ( Payling Wright, 1955). At present the consensus is in favor of axonal spread (for references, see Wildy, 1967). Neumtropic viruses ascend the axons at velocities of a similar order of magnitude (Table I ) .

TABLE I ASCKNDING h O N A L M I G u - n o N

Reference GO0dpaStW-C (1925) Kristensson (1970), Krktensson ct al. (1971a)

Tracer

Herpa simplex

Animal Rabbit

Virus

Herpes simplex

Young mice

Virus

Bromeis (1938)

Tetanus toxin

Kryzhanovsky c t d . (1961)

Tetanus toxin

-

Rat

OF

NONRADIOACTIVE TRACERS Time of appearance incd bodies

r

I

E: > z

Velocity of migration (mm/24Hr)

Remarks

-

-

-

Spinal cord

Day 2

-

Leg muscles

Spinal cord

-

About 70

Gastrocnemius muscle

Sciatic, ventral roots, spinal cord

7-8 Hr

About 200

Ligation or keezing of the sciatic blocks the ascending migration Freezing of the sciatic prevented the a p pearance of symptoms Humoral spread blocked by antitoxin

Site of injection Cornea, mas-

seter muscle Intradermally into sole of foot

Site of detection

Pons, medulla

r C

z2rA

F

Bodian and Howe (1941), Howe and Bodian (1942) Nathanson and Bodian (1961) Kristensson (1970), Kristensson and Olsson (1971a,b) Kristensson ct of. (1971a,b,c)

LaVail and LaVail (1972)

Poliomyelitis virus

Monkey

Poliomyelitis virus, neurotropic strain Evans Blue albumin, horseradish peroxidase

Monkey

Evans Blue albumin, horseradish peroxidase

Rat, rabbit

Horseradish peroxidase

Chick 3-34 days old

Suckling mice

Cut end of the nerve dipped in the virus suspension Intramuscular

Gastrocnemius muscle

Spinal cord

58

Spinal cord

Spread blocked

Spinal cord, motoneurons

by freezing of the nerve In intramuscular axons reaction products seen after 30-90 minutes

Tongue muscles

Optic tectum

Hypoglossal neurons

Retinal ganglion cell bodies

10 Hr

5 Hr

120

72

Blocked by crushing of hypoglossalnerve and by arrest of circulation -

0 2:

&

s*

!? ij kl

250

LILIANA LUBII~SKA

I n the analysis of axoplasmic flow, the neurotropic viruses and toxins may be regarded as tracers revealing the existence of an ascending axonal migration. I t might be argued that such migration could have been provoked by these pathogenic agents. Recently, however, the somatopetal migration of nontoxic tracer proteins (Evans Blue albumin and horseradish peroxidase) has also been demonstrated (Kristensson, 1970; Kristensson and Olsson, 1971a,b; Sjostrand et al., 1971; Kristensson et al., 1971a,b). When the horseradish peroxidase was injected into the gastrocnemius muscle of the suckling mouse the reaction products were found within 30-90 minutes in membrane-bound organelles in the intramuscular axons. After 16-24 hours they were seen in the motoneurons clustered near the nuclei. Control experiments have shown that there was no vascular spread of the tracer. Evans Blue albumin injected into tongue muscles of adult rabbit could be detected in the hypoglossal neurons ab6ut 10 hours later. The velocity of its ascending migration was 120 mm/day. Crushing of the hypoglossal nerve or the arrest of circulation prevented the access of the tracer to cell bodies. Ascending axonal migration has also been observed in the central nervous system. LaVail and LaVail (1972) described the cellulipetal movement of horseradish peroxidase from nerve terminals to the cell bodies in two groups of neurons in the optic system of young chick. The enzyme injected into the optic tectum was transported to the retinal ganglion cell bodies at a rate of 72 mmjday. The authors observe that if injected into the eye, horseradish peroxidase did not move cellulifugally in the axons of retinal ganglion cells. The results described in this chapter show that in intact axons in vivo, as in tissue culture, the translocation of substances occurs both in the cellulifugal and in the cellulipetal direction.

3. Reliability of Estimates of Velocities of Axoplasmic Flow in Intact Nerves Velocity of translocation is calculated from the amount of label found along the nerve at varying distances and times after injection of the tracer and is usually referred to milligrams of protein of the nerve. Often the time of appearance of label at the nerve endings and the total length of axons are used for these calculations. Several factors that affect the reliability of estimates of velocity based on such measurements have to be considered. They concern both the label itself and the amount of other components of the nerve.

a. Loss of Label. The underlying assumptions are that the material under examination is not metabolized in transit along the axon nor lost through the axolemma or at the nerve endings. These assumptions may not always be correct.

ON AXOPLASMIC FLOW

25 1

b . Overlap of Fast and Slow Label. The fact that migration of labeled axoplasmic components occurs with at least two different velocities creates some technical difficulties in the determination of characteristics of the slow flow. Whereas it is relatively easy to calculate the velocity of the fast somatofugal migration by measuring the time taken by the radioactive label to reach either a certain point along the nerve or the nerve endings at a known distance from cell bodies, the situation for the analysis of the slow flow is more complicated. Much longer time intervals between the injection of the tracer and removal of the nerve are required for detection of the slow flow. At such periods the whole length of the nerve is already labeled by the rapidly migrating components. The velocity of the slow movement has been calculated in various ways: from the progressively varying slopes of activity, from the changing ratio of activity in the distal and proximal part of nerve, from displacements of the additional peak of radioactivity slowly moving along the labeled nerve, or from the course of accumulation of the label in the nerve endings. Estimates of velocity have varied according to the points on the curve of radioactivity which have been chosen for calculation. These difficulties have been discussed by Grafstein (1967) and Ochs (1970). c . Overlap of the Descending and Ascending Label. Another complication is introduced by the existence of the somatopetal flow. Here again the estimates of velocities of rapidly moving labeled materials will not be affected because the axons are examined soon after the injection of tracers when materials migrating in the distoproximal direction are not yet labeled. These usually become labeled in less than 24 hours (Miani, 1964; Lasek, 1968), and from this time onward the axons will carry both the fast moving label migrating in the ascending (and probably also in the descending) direction and the slowly migrating descending label. I t is difficult with the usual type of experiments to determine what proportion of the label in a piece of nerve under analysis had been migrating in one particular direction. What is calculated as velocity of the slow flow is, in most cases, the resultant of the bidirectional migration.

d . Varying Length of Stay of Label in the Perikarya. The time course of incorporation of label is different for various components, and the labeled macromolecules remain in the perikarya for varying lengths of time (Droz, 1967a). In most of the published papers the overall time between the injection of tracers and their appearance at a given point in the axonal path is used to calculate the velocity of migration. With this procedure no distinction is made between materials which are discharged late from the perikarya, but travel rapidly, and those which are discharged early, but migrate slowly. Whereas it is possible to confine observations to the behavior

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LILIANA L U B I ~ S K A

of the early discharge, by cutting the nerve near its origin after letting the first wave of label enter the axons (Ochs and Ranish, 1969; Heslop, 1971),

the fate of material staying longer in the cell bodies is more difficult to follow. I n some cases a study of the time course of incorporation and perikaryal residence of a specific precursor permitted reevaluation of the velocities calculated with omission of the time taken by perikaryal events, Thus, early work suggested that axonal migration of RNA in the optic tract was slow, whereas that of ribosomal proteins was fast (Bray and Austin, 1968; Casola et al., 1969; Bondy, 1971a,b). This conclusion was based on the finding that labeled RNA appeared in the optic nerve endings much later than labeled proteins. It was puzzling because RNA and ribosomal proteins are constituents of the same particle. In a subsequent work, however, Bondy (1972) has shown that the cytoplasmic RNA in retinal cells is labeled much more slowly than the cytoplasmic protein, and this time lag accounts for the delayed arrival of labeled RNA to the optic lobe. Bondy concluded that the slow velocity of RNA calculated from earlier results should be corrected and that both RNA and ribosomal proteins move in the axons by fast flow.

e. Axonal Synthesis and Exogenous Proteins. Axonal synthesis of small amounts of proteins was observed in the giant fibers of squid (Giuditta et al., 1968), in fibers of lower vertebrates and, occasionally, in mammals (for reviews, see Edstrom, 1970; Jakoubek and Semiginovskf, 1970; Koenig, 1972) , During embryonic development, the ribosomes are scattered throughout the length of axonal processes of motor and sensory neuroblasts (Lyser, 1964, 1968; Tennyson, 1965, 1979), but mature axons seemed to be devoid of ribosomes except in the initial segment (Palay et al., 1968; Peters et al., 1968). The site of the extramitochondrial axonal synthesis of proteins was therefore uncertain. Recently, however, ZelenA (1970, 1972), has found ribosomes in the sensory axons of rats beyond the initial segment. They were seen in the myelinated part of axons in their intraganglionic course and, in smaller numbers, in the proximal part of spinal nerves. Distal stretches were not examined, so it is not yet known over what distance from the perikarya the ribosomes do occur in the axons. In addition to proteins produced in the neuron, the axon may contain some admixture of exogenous proteins. Tracer proteins injected into the tissue may be taken up by various parts of the neuron. Pinocytotic vesicles containing tracer proteins were observed in the nerve terminals at central and neuromuscular synapses as well as in the perikarya and axons (Rosenbluth and Wissig, 1964; Brightman, 1965, 1968; Nickel et al., 1967; Waxman, 1968; KAsa, 1968a; Holtzman and Peterson, 1969; Zacks and Saito,

ON AXOPLASMIC FLOW

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1969). These data suggest that macromolecular materials may normally enter the axons from the intercellular spaces or from neighboring cells. The contribution of locally synthesized proteins to the overall balance of axonal proteins is generally considered to be small (Jakoubek and Semiginovskf, 1970). The quantitative importance of exogenous proteins in the axons has not been assessed so far.

C. INTERRUPTION OF AXONAL PATHWAYS Experimental interference with axoplasmic flow by interruption of the continuity of axons permits to analyze certain of its characteristics which are not easily detected in intact neurons. The following approaches have proved to be useful in this respect. a. The study of the accumulation of various materials near the cut ends in freshly interrupted nerves at a time when it may be justifiably assumed that the axoplasmic flow continues relatively unchanged in the preserved parts of axons beyond the disorganized zone. b. The time course of development of changes a t synapses and in the innervated cells after denervation and, to a much lesser extent so far, that of perikaryal changes in axotomized neurons. c. The progress of Wallerian degeneration in the peripheral stump of a transected nerve.

1. Early Morphological Changes near the Lesion Transection, crushing or ligation of nerves disturbs the structure of nerve fibers on each side of the lesion over some 100-500 pm, according to fiber diameter (Lubiliska, 1952a). The longitudinal arrangement of neurofilaments and microtubules is lost, the tip of the fiber rounds up and often becomes completely ensheathed by myelin (Lubihska, 1964; ZelenA et al., 1968). Very soon various organelles begin to pile up progressively at the disorganized zone and distend the terminal part of axons. The swelling is considerable in sympathetic fibers. Thick myelinated fibers are only slightly enlarged, The accumulating material tend to exhibit a certain degree of spatial organization, various kinds of organelles being arrayed predominantly in a particular way in various fibers (Schlote, 1964, 1966; Blumcke et al., 1966; Kapeller and Mayor, 1966, 1967, 1969; Mayor and Kapeller, 1967; ZelenA et al., 1968; Zelenh, 1969; Schlaepfer, 1971; Morris et al., 1972; Rodriguez-Echandia and Schoebitz, 1972 ; Matthews, 1973; and others) . The spatial organization may be detected also by histochemical methods. Thus, for example, NAD-diaphorase activity increases in the immediate

254

LILIANA

LUBIRSKA

proximity of the lesion whereas acetylcholinesterase (AChE) is scattered at some distance from the fiber tip (KBsa, 1968b; Martinez and Friede, 1970). Still farther away, the normal appearance of the axon is resumed. A detailed description of the course of accumulation and subsequent transformation of organelles collecting at the proximal end of transected sympathetic fibers and of concomitant changes in axotomized perikarya was given by Matthews ( 1973). I n the early stages of accumulation, the ultrastructural organization of the region resembles that of terminals of regenerating nerve sprouts (cf. Bliimcke and Niedorf, 1965; Lentz, 1967).

2 . Course of Accumulation of Axonal Materials at the Ends of Interrupted Fibers There are reasons to assume that most of the materials accumulating near the ends of interrupted fibers is brought there by axoplasmic flow. The increase in the amount of some components may be due also to a local, injury-induced synthesis of activation. An experimental procedure to test whether the accumulating substances have been imported or produced locally was described by Lubinska et al. (1964) and Skangiel-Kramska et al. (1969). For imported materials the course of accumulation of various axonal components near the ends of interrupted fibers may be used for inferences concerning the characteristics of their translocation along the axons. This method presents certain advantages, as well as weaknesses, as compared with that using radioactive tracers in intact nerves. Whereas radioactive precursors most often label a whole class of macromolecules (for example, proteins), individual compounds may be more easily sorted out at will from the accumulating materials by specific biochemical methods. Thus, migration of a selected kind of molecule may be compared with that of other molecular species under various physiological circumstances. Moreover, in contrast to natural nerve endings, unsuitable for a direct analysis of axoplasmic flow because of their complicated shape, functional specialization and intimate relations with postsynaptic cells, the artificial ends created by transection, crushing, or ligation of the nerve are made on a functionally uniform cyclindrical axon, easily accessible to analysis on both sides of the interruption. It is possible under these conditions to examine the course of accumulation of components migrating in the axons in either direction. The main limitation in the use of transected nerves for a quantitative study of axoplasmic flow is the relatively short time available for experiments. The peripheral stump may be studied only as long as the transport is not disrupted by the onset of Wallerian degeneration. This time varies in different types of fibers. In the central stump, migration continues indefinitely, but the reaction of cell bodies to axotomy alters profoundly their

ON AXOPLASMIC FLOW

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metabolism and may modify both the nature and the time of discharge of perikaryal products into the axons. The quantitative determination of characteristics of migration in the central stump has to be confined therefore to the period during which the materials reaching the ends of the nerve are those which had already been present in the axons at the moment of nerve section. In long axons the accumulation progresses linearly with time during the first day or longer (Dahlstrom and Haggendal, 1966; Lubiiiska and Niemierko, 1971; Banks and Helle, 1971). I t is perhaps safe not to exceed this period of linear growth. It permits one to analyze the fast migration only, the slow migration requiring much longer times to be detected. Even for fast migration, however, some caution is necessary. In this kind of investigation the underlying assumption is that the accumulated organelles are trapped at the disorganized zone and do not move back along the axon. This assumption is probably approximately correct for the early stages of accumulation. There are indications, however, that some of the accumulated components may leave the terminal pile and ascend the axon. Thus, the increase in AChE activity at the ends of interrupted axons spreads with time beyond the zone of close packing to which it seems to be confined in the initial hours after nerve section (Lubidska, 1964). Still more suggestive of return flow is the observation by Matthews (1973), who found, from about 2-7 days after nerve section, scattered dense bodies containing altered axonal organelles in nondistended proximal portions of axons of otherwise normal appearance. The presence of altered organelles in these dense bodies suggests that they had resided previously in the terminal zone, where digestion of accumulating organelles was initiated. Other limitations concern the nature of components which may be usefully studied by this method. As with the method of radioactive tracers, the investigated components should have a relatively long life-span as compared with the duration of the experiments and should not be synthesized in the axon or escape from it through the axolemma or at the site of injury. Moreover, when biochemical methods are used on nerve homogenates, it is important to know the cellular location of the substance under study. It should preferably be confined to axons. Most compounds are also present in the Schwann cells, and in such cases additional data are necessary for a correct interpretation of the increase observed at the ends of interrupted nerves (for discussion, see Skangiel-Kramska et al., 1969; Niemierko, 1972). The course of accumulation of axonal materials at the ends of interrupted fibers has been investigated by a variety of methods, some of them quantitative. The results obtained fall into two main categories. In one, the accumulation has been observed only on the proximal side of the interruption, in the second the investigated materials were seen to accumulate at the ends of both central and peripheral stumps. Whereas the accumulation

256

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on the proximal side of the lesion is generally regarded as a result of a damming of the proximodistal flow, there is a widespread tendency to interpret differently the accumulation on the distal side. The increase on the distal side is attributed by various authors to neuronal perikarya scattered along the nerve, to a direct local effect of the lesion, to leakage of substances from the proximal accumulation, or even to induction of enzymes across the lesion by substances released from the proximal stump (McLean and Burnstock, 1972). Although it is not clear why such factors should produce an increase exclusively on the distal side of the lesion, this attitude led to a certain neglect of the study of the peripheral stump. The majority of published data is therefore based on analyses of events occurring proximal to the site of nerve division and deals solely with somatofugal flow. Nevertheless, in many cases accumulation of axonal materials on both sides of the lesion was described. Besides the electron microscopic observations quoted earlier, biochemical histochemical and radiometric methods revealed a progressive accumulation on both sides of the lesion of labeled phospholipids (Miani, 1964), labeled proteins (Lasek, 1967, 1968; Watson, 1968b), oxidative enzymes (Kreutzberg, 1963; Roessmann and Friede, 1965; Banks et al., 1969), acid phosphatase (Holtzman and Novikoff, 1965), acetylcholinesterase (Zelen6 and Lubiriska, 1962; Lubifiska et al., 1963a, 1964; Waldron, 1969; Bray et af., 1971; for other references, see Lubiriska, 1964). Neurosecretory materials were found to accumulate on both sides of the lesion in some cases (Hild, 1951; Christ, 1962; Iturriza and Restelli, 1967; Dellman and Owsley, 1968; Dellmann and Rodriquez, 1970), and on the proximal side only in others (Hild and Zetler, 1953; Alvarez-Buylla et al., 1970). Similar discrepancies were observed with components of catecholamine storage granules. The prevalent opinion at present (Geffen and Livett, 1971; Dahlstrom, 1971b) is that they accumulate only on the proximal side. and it is inferred that they move in the axons only in the somatofugal direction. However, in many of the published data there are clear indications that noradrenaline accumulates on the distal side of the lesion too (Dahlstrom, 1965; Dahlstrom and Haggendal, 1966; Banks et al., 1969; Olson, 1969; Osborne and Cottrell, 1970). This increase in much weaker than that on the proximal side and disappears earlier. The conflicting results concerning accumulation of constituents of catecholamine storage and neurosecretory granules on the distal side of the lesion are probably due largely to the experimental situation, in particular to the time interval between transection and examination of the nerve. This possibility is shown in experiments of Dellmann and Rodriguez ( 1970). In the transected hypothalomohypophysial tract of the frog the accumulation of neurohormones is parallel on both sides of the lesion during the first day. Thereafter the level of hormones decreases considerably in the whole peripheral stump. The authors attribute

ON AXOPLASMIC FLOW

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this fall to Wallerian degeneration. According to whether the nerve is removed before the fall of hormone content in the peripheral stump or later, the accumulation on the distal side of the transection will be present or absent. Even if the level of the investigated component does not fall in the peripheral stump during incipient Wallerian degeneration, the axoplasmic flow is disrupted in disconnected axons at a period when it continues in the central stump. If the nerve is removed rather late after transection, the process of accumulation would have been going on for a much longer time in the central than in the peripheral stump, resulting in a larger increase on the proximal side. The observed difference may also reflect an actual disparity of the flux in each direction. Unless the time course of accumulation of a substance near the cut end of the nerve is examined systematically at close time intervals in the early period after nerve section, no safe inferences may be drawn about the character of its axonal migration. No such study seems to have been made so far on components of catecholamine storage granules. The question of whether they move in the axons in both directions or only somatofugally awaits, therefore, further experiments. Various components of single types of organelles such as noradrenaline P-dopamine hydroxylase, and chromogranine A of catecholamine storage granules (Livett et a!., 1968), or neurohormones and hypophysins of neurosecretory granules (Pickering et al., 1971; Norstrom, 1972) accumulate, as was to be expected, in a parallel way. Several apparent exceptions to this rule were clarified when improved techniques or better experimental design permitted reassessment of earlier results (Bondy, 1972 ; Jarrot and Geffen, 1972; Coyle and Wooten, 1972). Various types of organelles seem to migrate at distinct velocities (Banks et al., 1969; Laduron and Belpaire, 1968; Laduron, 1970; Jonason, 1970). Laduron thinks that a kind of subcellular fractionation of axonal materials might be achieved by differential arrival of various components to the end of interrupted fibers. This idea sounds overoptimistic at present. However, when the characteristic speed of an organelle is known, it may help sometimes to decide whether a component of doubtful subcellular localization is likely or not to be a constituent of this organelle. This point has been analyzed for tyrosine hydroxylase by Coyle and Wooten ( 1972).

3. Isolated Nerve Segments The size of the terminal pool of a substance brought to the blind end of a transected nerve fiber or to the natural nerve endings of an intact one by axoplasmic flow is determined by the amount of migrating substance and velocity of its translocation along axons. If the total contents of a substance is moving in a single direction, the size of the pool may be used directly to calculate the velocity of its flow. If, however, the flow is bidirec-

258

LILIANA L U B I I ~ S K A

tional and/or the substance is attached to two subcellular structures, one migratory and the other stationary, the estimates of velocity will be too low, because the observed increase will be calculated in relation to the total amount of the investigated axonal component instead of its mobile fraction migrating toward the analyzed end. For a correct description of axoplasmic flow it is necessary to determine what fraction of the total content is mobile and how much of it moves in either direction. This problem may be solved in some cases by the study of an- isolated nerve segment separated by two transections both from cell bodies and from nerve endings. Such a segment forms a relatively closed system in the sense that no substances carried by axoplasmic flow are arriving or leaving it during the experiment. I n the surviving segment the axoplasmic flow continues for a time, and the migrating materials are arrested at its ends. The general pattern of accumulation obtained in such experiments is similar for many axonal components. It is illustrated in Fig. 1 showing the distribution of labeled phospholipids (Miani, 1964), AChE (Lubihska and Niemierko, 1971), and cytochrome oxidase (Banks et al., 1969). The initial content of the investigated substance may be determined on small pieces of the nerve removed at the time of transection and measuring the length of the segment. If the total amount of the substance in the segment remains unchanged during the experiments, as is the case with AChE, for example (Lubihska et al., 1964), the increase observed at the ends is entirely accounted for by the longitudinal translocation, and it may be assumed that no synthesis or activation of the investigated component had taken place. To determine whether the total amount of AChE in the nerve is mobile and may accumulate at the cut ends, an attempt was made to deplete the segment by repeatedly removing, at 4-hour intervals, its terminal portions with the accumulated materials (Niemierko and Lubifiska, 1967). After several such removals, the number depending on the length of the segment, there was no further increase of AChE at the ends, suggesting that the mobile store was exhausted. At that time a considerable part of the enzyme remained in the segment, indicating the existence of a stationary (or slowly moving) fraction. The time during which the accumulation proceeds and the amount of AChE collected at the ends are proportional to the length of the isolated segment. Analysis of the time course of accumulation of AChE at the ends of isolated segments of varying lengths and of the central and peripheral stumps of the nerve permitted determination of both the velocity of translocation and the amount of rapidly moving enzyme (LubiAska and Niemierko, 1971). In the peroneal nerve of the dog, the velocity of somatofugal migration is 260 mm/day and that of somatopetal migration 130 mm/day. Only 15% of the AChE content of the nerve is migrating at these rates, about 10%

259

O N AXOPLASMIC FLOW

7

700.

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2

0

$ 600-

22hr

0

u

g

500.

E

400-

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300-

E

I-

y

200.

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100. 0

-C(mm)

300

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250

-

48 hr

-

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L

5 3 1-I

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P(mm)

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FIG. 1. Accumulation of axonal materials at the ends of central and peripheral stumps and of the isolated nerve segment. I. Acetylcholinesterase (AChE) activity in the peroneal nerve of dog, (as percent of control), 22 hours after transection, (Lubiriska and Niemierko, 1971). 11. Labeled phospholipids in the vagus nerve of rabbit (as percent of control), 24 hours after crushing, 7 days after labeling (recalculated from Miani, 1964). 111. Cytochrome oxidase activity (as micromoles of cytochrome c oxidized per minute) in the hypogastric nerve of cat, 48 hours after constriction (Banks et al., 1969). C, Central stump; P, peripheral stump, IS., isolated nerve segment. Abscissas: distances from points of interruption in millimeters, except in 111, where 0.8 mm are marked. Arrows show the sites of interruption.

260

LILIANA L U B I ~ ~ S K A

in the descending and 5% in the ascending direction. The rest is either stationary or moves slowly. A similar study was made by Partlow et al. (1972) on segments of frog sciatic nerve in uitro. About a quarter of the total AChE contents was mobile, half of it moving in each direction. At 22OC the velocities were 99 mm/day and 19 mm/day in the proximodistal and distoproximal directions, respectively. AChE in axons has at least two subcellular locations: at the axolemma and at tubules of the smooth endoplasmic reticulum (Brzin et al., 1966; Kokko et al., 1969; and others). Although no quantitative evaluations have been made, inspection of electron micrographs of normal fibers suggests that the axolemmal location is much richer. K h a (1968a) studied AChE in the region of accumulation of organelles 24 and 48 hours after ligation of the nerve. At the axolemma AChE activity seemed unchanged, but it had increased strongly in the axoplasm. One could speculate therefore that the stationary (or slowly moving) fraction of AChE is axolemmal and that the rapidly migrating one is connected with organelles in the axoplasm. Mitochondria in axons in tissue culture seem to move more slowly than smaller organelles (Pomerat et al., 1967). Their speed, however, still falls within the range of “fast” flow. This is also suggested by studies of translocation of mitochondrial enzymes in constricted nerves (Banks et al., 1969). Analysis of translocation of mitochondria is more complicated than that of AChE, which is confined to axons. Mitochondria are located both in axons and in the Schwann cells. Schwann cell cytoplasm overlying the infoldings of myelin in the paranodal regions is densely packed with mitochondria (Williams and Landon, 1963). In the axoplasm they are loosely scattered throughout the internodal length, 3 to 4 to a cross section of myelinated, and 0.5 to 0.9 of unmyelinated, nerve fibers in the sciatic nerve of the rat (ZelenP, 1968). In isolated nerve segments, axonal mitochondria pile up at both ends, and a concomitant progressive fall of their number in the middle part of the segment is observed (Zeleni, 1968). When mitochondrial movements are estimated from shifts of activity of mitochondrial enzymes along the nerve, it is particularly important to know what fraction of the activity corresponds to axonal mitochondria and may move throughout the length of the axon. Partlow et al. (1972) found in the isolated segments of frog sciatic nerve that only about 10% of the mitochondrial enzymes (hexokinase and glutamic dehydrogenase) are actually migrating, at a rate of 20-31 mm/day in the peripheral and of 11-20 mm/day in the central direction. Jeffrey et al. (1972) failed to detect fast flow of mitochondria in using 68Fe as a marker for mitochondrial cytochromes. However, their experiments show that incorporation of label into mitochondria is a very slow process and, at times chosen to detect the ac-

ON AXOPLASMIC FLOW

261

cumulation of radioactivity in front of the constriction of the nerve, axonal mitochondria had not yet been labeled. As to the soluble enzymes, the pattern of their distribution in isolated nerve segments is different from that of particulates and gives no indication that the enzyme had moved longitudinally along axons. Phosphoglucoisomerase activity increases initially at the ends of the segment, but the increase is slight, does not progress with time, and there is no corresponding depletion in the middle of the segment. This suggests that the observed increase is a local, injury-induced phenomenon (Skangiel-Kramska et al., 1969). The distribution of 6-phosphogluconic dehydrogenase and choline acetylase do not change in the isolated segment of a frog sciatic nerve for 96 hours (Partlow et al., 1972). In constricted nerves in vivo, in animals kept for 12 days, the authors detected an increase in choline acetylase activity on the proximal side and a fall on the distal side of the constriction. In assuming that all of the choline acetylase of the nerve moved in the proximodistal direction, a velocity of 0.34 mm/day was calculated. In view of the considerable decrease of activity observed after 12 days in fibers separated from the cell bodies, this calculation appears uncertain. Other studies concerning the axonal translocation of choline acetylase in various nerves are quoted by Saunders et al. (1973). The absence of progressive accumulation of soluble enzymes at the ends of isolated nerve segments seems to indicate either that these enzymes do not move by fast flow, or that they move but are not arrested at the blind ends as are particle-bound enzymes, so that their movements are not revealed by the study of isolated segments which proved to be adequate for components trapped at the ends of fibers.

D. PERIPHERAL AND CENTRAL EFFECTSOF NERVESECTION An intact nerve supply is nesessary to maintain the normal structure and function of the innervated cells. This dependence is presumably mediated by L‘trophic”substances produced in neuronal cell bodies and conveyed down the axons to the nerve endings (Scott, 1906; Gerard, 1932). For a long time it has been suspected that the trophic substances may pass directly from nerve terminals to the innervated cells. What appears to be the first direct proof of the possibility of such transcellular transfer was given by Korr et al. (1967) for passage of labeled proteins from the hypoglossal nerve to tongue muscles. Recently several other cases of transsynaptic transport in mature animals have been noted (Globus et al., 1972; Alvarez, 1970; Alvarez and Piischel, 1972; Grafstein, 1972; Miani, 1971). It is not certain, however, whether the transferred materials actually exert a trophic function. A different mechanism for the transcellular effects of denervation was re-

262

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cently contemplated by Ghetti and Wisniewski (1972; Cook et al., 1972), who found that degenerating optic terminals are engulfed within glial processes together with synaptic thickening and fragments of postsynaptic dendrites. The authors suggest that the resulting microtraumata of the postsynaptic cell may initiate its degeneration. The multiple effects of denervation on the structure and function of innervated cells have been observed in the central nervous system and in various peripheral structures. Of these the striated muscles have been analyzed most extensively (for reviews, see Gutmann, 1962; Guth, 1968; Close, 1972). Interruption of nerve supply alters the metabolism of muscle cells, their contractile characteristics, and properties of the muscle membrane. These changes may be reversed by reinnervation (Guth, 1969). Experiments with cross-innervation of muscles have shown that the pattern of innervation and the character of neuromuscular transmission depend on the nature of the innervating axon. Both the biochemical equipment and the characteristics of muscular contraction are modulated by reinnervation of the muscle by an alien nerve (Buller, 1970; Jiimanovd et al., 1971). The trophic influence of nerves on muscles may be exerted even without establishment of functional connections. Thus, reinnervation of a skeletal muscle by an adrenergic nerve prevents the appearance of fibrillations although neither contraction nor muscle action potentials appear on stimulation of this nerve (Mendez et al., 1970). Even implantation of a fast nerve into an innervated slow muscle, that is under conditions when practically no new end plates are formed, alters the properties of the muscle (Gutmann and Hanzlikovi, 1967; Fex, 1969; Fex and J i h a n o v i , 1969; Fex and Sonesson, 1970), presumably by some substances leaking out of the implanted nerve. I n vitro the degeneration of denervated motor end plates is greatly delayed when the muscle is cultured in the presence of a sensory ganglion which does not innervate directly the muscle fibers. Although motor nerve endings degenerate under these conditions, the junctional folds on the muscle surface and cholinesterase activity persist much longer than in cultures made without nerve explants (Lentz, 1972b). Extracts of nerve homogenates added to the medium have a similar delaying effect on cholinesterase activity of muscle in vitro (Lentz, 1971). A different type of influence of innervation on muscle cells has been described in the embryo (Wintrebert, 1920; Harris and Whiting, 1954) and in tissue culture (Crain, 1966) where the spontaneous myogenic muscle contractions are stopped on arrival of nerve fibers. I t is not certain whether these various effects are due to a single or to different trophic substances (for discussion, see Gutmann, 1968, 1970 ; Guth, 1968). Their chemical nature is at present unknown. The axonal migration of trophic substances may, nevertheless, be detected and measured by analy-

ON AXOPLASMIC FLOW

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sis of the time of appearance of disturbances after nerve section made at various distances from the innervated cells. 1. Eflerent Synapses and Innervated Cells The earliest effects of nerve section appear at synapses where functional failure always coincides with the degeneration of nerve endings and precedes, the changes that will appear later in axons in the nerve trunks. (Ranvier, 1878; Titeca, 1935; CoppCe and Bacq, 1938; Gutmann et al., 1955; Davidovich and Luco, 1956; Vera and Luco, 1958; Vaccarezza et al., 1970; Miledi and Slater, 1970; and others). I t has often been observed that degeneration of nerve terminals appears first in anatomical structures close to the level of section of axons and later invades progressively more distant regions (Torrey, 1934b; Birks et al., 1960; SzentAgothai, 1968) . This relationship suggests that longer peripheral stumps of axons contain a larger store of trophic substances, ensuring for a longer time the maintenance of the normal state of nerve endings. Experiments in which the relationship between the time of appearance of a particular synaptic or postsynaptic disturbance and the length of the peripheral stump of nerve fibers was analyzed permitted calculation of the velocity of axonal migration of the relevant trophic factors. The main results are summarized in Table 11. The velocities vary between 20 and 320 mm/day for various disturbances. The duration of survival after nerve section varies considerably with the type of synapse and the animal species (for references, see Table V in Lubidska, 1961). Even in homologous nerves of various mammals, unexpectedly large differences are sometimes observed. Thus, the time course of physiological disturbances following enucleation of the eye is different in cat and monkey. I n the cat synaptic transmission in the midbrain disappears in 2-3 days and nerve conduction in optic axons fails after 5 days. The corresponding figures in the monkey are 14 and 20 days (Fentress and Doty, 1971; Vaccarezza et al., 1970). The origin of this difference is unknown. One could speculate that the optic axons of monkeys contain a larger store of tropic substances or need less. 2. Wallerian Degeneration in Nerve Trunks There is a century-long controversy as to whether Wallerian degeneration of fibers in the nerve trunk spreads progressively toward the periphery or involves simultaneously the whole length of the peripheral stump. The consensus at present favors the latter opinion (cf. Ram6n y Cajal, 1928; Gutmann and HolubG, 1950; Sunderland, 1968) in spite of many scattered observations pointing to a proximodistal spread. They are listed in Table

INFLUENCE OF

THE

LENGTHOF

THE

TABLE I1 THE DEVELOPMENT OF SYNAPTIC AND POSISYNAPTIC DISTURBANCES"

PERIPHERAL STUMPON ~~

Rdaence Eyzaguirre ct al. (1 952)

Gutmann et al. (1955)

Vial (1955)

Animal Cat

Rabbit

Guinea Pig

Birh et al. (1960)

Frog

Slater (1966), Miledi and Slater (1970)

Rat

Nerve and innervated structure Nerve to tenuissimus muscle

Sciatic nerve to leg muscles

Experimental procedure Transection

High and low transection

Nerve to tibial anterior muscle Sciatic

High and low

Phrenic nerve diaphragm

Transection of the nerve at various distances from end plates

transection

-

~ _ _ _ _ _ _ ~

Disturbance Synaptic disturbances

Failure of transmission, disintegration of nerve endings Degeneration of end plates

Timeof appearance and progress ofchanges

Progress from loci close to the site of nerve section After 24 hr more intense with short stump

After 30 hr more intense with short stump Failure of neuro- Appears earlier muscular in the thigh than in the transmission lower part of the leg Failure of trans- Appears earlier mission, disapin end plates pearance of closer to the MEPP's, and site of transecdegeneration of tion. Onset at

ru Q,

-P

_______~~

Velocity of migration of the presumed trophic factor (mm/24hr)

48

-

Remarks End plates are distributed longitudinally inthismuscle Axonal excitability unchanged at that time

-

320

-

-c r

>

3 6' E

2-

F

Emmelin ef al.

Rat

(1966)

nerve endings in 50% of end plates Fall in ChAc activity in the diaphragm

Phrenic nerve diaphragm

Long and short

Nerve to EDL and tibial anterior muscles Optic nerve to lateral geniculate body

Cut at levels dif- Decrease in ChAc fering by activity in the about 35 mm musclcs to 50% Cut at levels dif- Dark degenerafering by about tion; neuro28 nun fibrillar hypertrophy at nerve endings; failure of transmission Cut close to the Disturbance of ganglion on transmission one side and far on the other Degeneration of nerve endings in the gray matter Transcction Fibrillation and hypersensitivity to ACh

stump

8 hr, no trans-

mission after 20 hr

Fall is much stronger with short stump at 1, 2, and 3 days

-

10 Hr

113

ChAc in the diaphragm is concentrated mainly in intramuscular nerve branches (Hebb r t al., 1964)

TuEek (1968)

Vaccarnza et al.

Rat

Cat

(1970)

Davidovich and Luco (1956)

SzentAgothai

Cat

-

Preganglionic nerve to superior cervical ganglion Dorsal roots

(1968)

Luco and Eyzaguirre (1955)

Cat

Nerve to tenuissimua muscle

0 1

Appear earlier with short stumps

About 20

-

48

End plates are distributed longitudinally inthismuscle

Appears earlier with short stump Appears earlier at levels close to the transected roots Progress from the loci close to the site of nerve section

(Continued)

G;

TABLE I1 ( C a t i w d )

AniReference

mal

Nerve and innervated structure

Experimental prOCedUrC

Gutmann (1962)

Rat

Sciatic nerve to leg muscles

High and low transection

Gutmann (1969)

Rat

Sciatic nerve to leg muscles

High and low transection

HAj& ct al. (1964)

Rat

Sciatic nerve to soleus and EDL muscles

High and low transection

Emmelii and Malm (1965)

Cat

Hypoglossal Long and short nerve to tongue stump muscles

Harris and Thddf (1972)

Rat

Sciatic nerve to EDL muscle

Hiiggendal and Dahlstr6m

Rat

Sciatic nerve to gastrocnemius muscle

(1971)

Disturbance Metabolic overshoot reactions in muscles Changes in glycogen synthesis in muscles Increase in proteolytic activity in muscles Hypersensitivity of muscles to ACh

Cut at levels dif- Appearance of fering by about TIX-resistant 30 mm action potenti& in muscle Cut at levels dif- Disappearance of fering by noradrenaline 30 nun in muscle

Time of appearance and progress of changes

Velocity of migration of the presumed trophic factor (mm/24 hr)

Appear earlier with short stump Appear earlier with low transsection Appears first in muscles with short stumps

-

At 4 days markedly higher with short stumps 36 Hr

-

Disappears earlier with short stump

30

-

120

240

Remarks

Emmelin (1968)

Cat

Torrey (1931)

Catfish

Parker (1932)

Catfish

Auriculotemporal Cut at levels difnerve to p a r e fering by tid gland 12-20 mm Nerve to taste Transection buds Nerve to lateral Transection line

Goldfish Nerve to sensory epithelium in the vestibular system Nerve to EDL Albuquerque ct al. Rat muscle (1971) Alvara and Piischd (1972)

Geffen and Hughes (1972)

Injection of labeled leucine or lysine Transection

Chicken Nerve to expansor Cut at levels difsecundariorum fering by muscle 50 mm

Degeneration secretion of saliva Degeneration of taste buds Degeneration of neuroepithdium Appearance of label in the sensory epithelium Decrease of muscle resting potential

27 Hr

125

Progressive along the barbel Appears earlier nearer the site of nerve section 6-12 Hr

10-20

15 0 2:

2 Hr; depends

on the length of intramuscular stump Sensitivity to ACh 24 Hr on extrajunctional membrane Transverse resis3 Days tance of muscle membrane Loss of transmis- 48-72 Hr sion and onset of degeneration contraction

-

Authors suggest that more than one trophic substance is responsible for thesechanges

24-48

a MEPP, miniature end-plate potential; ChAc, choline acetylase; ACh, acetylcholine; EDL, extensor digitorum longus; TTX, tetrodotoxin.

5

%

r

$ g

zL4

N

m ca

TABLE 111

PROXIMODJSTAL SPREAD OF AXONALCHANGES IN

THE

PERIPHERAL STUMPS OF DIVIDED NERVES ~

Reference

Animal

Parker and Paine (1931)

Catfish

Parker (1933)

Frog

Nerve

Experimental procedure

Nerve to lateral line

Transection

Sciatic

Transection

Titeca (1935)

Frog

Sciatic

Transection

Joseph and Whitlock (1972)

Toad

Sensory fibers in dorsal roots and CNS

Transection of 9th spinal dorsal root

Bubis and Wolman (1965)

Rabbit

Sciatic

Transection

Axonal change

~

~~

Time of appearance nearthe site of nerve lesion

11 Days Histological changes in nerve fibers 16 Days at Disappearance of 18°C impulses

Fatiguability of impulses; delayed recovery; increased staining with methylene blue Nauta-positive degeneration

2 Days

Increased activity of leucine aminopeptidase

4 Hr

Velocity of spread (mm/day) 20-30 at

Remarks

-

18°C Spread throughout the nerve length after 20 days 20 at 20°C Failure of nerve impulses after 22 days, simultaneous in the whole peripheral stump 2-4 at 20°C Calculated without subtraction of the latent period About 48 Spreads over 16 mm after 8 hours

E t: >

F C

E, v)

r

Causey and (Palmer 1953)

Rabbit

Phrenic

Transection or crushing

Remaction of myelin from the nodes of Ranvier

Lubihka and Waryszewska (1972)

Rat

Phrenic (left)

Transection

Breakdown into ovoids of fibers of various diameters small medium large largest Decrease of action potentials in A fibers

Rcsenblueth Cat and D d Pozo, 1943 Salafsky and Rabbit Jasinski, 1967

Peroneal

Peroneal

Transections at hip, middle of the thigh, and knee Transections made Disappearance of at distances difaction potentials at a fixed test fering by 40 mm point of the nerve

48 ~r

20 Hr 22 Hr 25 Hr 33 Hr 2 Days

PrOximodiStal spread and progressive increase of the myelinfree gap at the node

250 150

0 2:

100

About 40 Spreads periph-

h 8r

>

-

Eij, 53

Disappear after 54hrwithlow andafter72hr with high mansection

*J

270

LILUNA L U B I ~ S K A

111. (The numerous experiments suggesting a simultaneous degeneration in the whole peripheral stump are not shown in this table.) O n the hypothesis that the integrity of nerve fibers is maintained by substances produced in the perikarya and migrating down the axons, Wallerian degeneration in a severed nerve would presumably set in when the concentration or amount of these substances at a certain site would fall below a critical level. After interruption of the perikaryal supply this critical depletion would begin near the site of transection and spread toward the periphery. With fast axoplasmic flow of the trophic substances the change would sweep the nerve of a small animal in a few hours, and its advance would be easily missed with the usual timing of degeneration experiments. Wallerian degeneration of axons (Torrey, 1934a; Armstrong, 1950; Gamble et al., 1957) as well as the failure of neuromuscular transmission (Lubifiska, 1952b; Birks et al., 1960; Hoy et al., 1967) are greatly delayed at low temperatures. Probably because of the slower course of degeneration, its proximodistal spread was first measured in cold-blooded animals. Thus, in the transected sciatic nerve of a frog kept at 18OC (Parker, 1933) the fibers looked normal for 16 days. Then the changes appeared in the proximal part of the stump and spread throughout its length during the following 4 days. A similar course of events, except for the time scale, has been found recently (Lubifiska and Waryszewska, 1972) in the transected phrenic nerve of the rat. After a latent period of about 20 hours the fibers begin to degenerate near the site of nerve section, and the disturbance advances progressively in the distal direction (Fig. 2 ) . Both the latent period of degeneration and the rate of its advance depend on fiber diameter. The velocity of the proximodistal spread of degeneration varies from 270 mm/day in the thinnest myelinated fibers to about 40 mm/day in the thickest (9-1 1 pm) fibers of the phrenic nerve, suggesting that perikaryal materials responsible for the maintenance of integrity of nerve fibers travel in the axons at these velocities. The trophic substances ensuring the integrity of nerve endings and synaptic transmission in the phrenic-diaphragm preparation of the rat migrate at a velocity of the same order of magnitude (Slater, 1966; Miledi and Slater, 1970) although the latent period of synaptic changes is much shorter (Fig. 3 ) . It is not impossible, therefore, that the integrity of axons and that of nerve endings is maintained by the same substance, but the critical level of depletion is different at each site. Not only the nerve endings but also the preterminal branches of axons (Weddell and Glees, 1942) degenerate earlier than the unbranched fibers in the nerve trunks. As even in nerve trunks the thin fibers are the first to degenerate, a tentative conclusion may be drawn that the thinness of the axon makes for its fragility after nerve division. A thin fiber has a greater surface to volume ratio contributing to

271

ON AXOPLASMIC FLOW

22 hr

In a W

m 0

i

i

a W

W

w

% 1 6

16

16

36

46

6

16

26

36

46

6

16

26

36

46

6

16

26

36

DISTANCE FROM THE SITE OF NERVE SECTION Imml

Fro. 2. Longitudinal spread of Wallerian degeneration in the phrenic nerve of rat, 22, 26, 30 and 38 hours after nerve section. Percentage of degenerating fibers of Small; 0-0, medium; 0-0, large; 0-0, various diameters. A-A, largest (L. Lubidska and J. Waryszewska, unpublished).

TIME AFTER NERVE SECTION (HOURS)

Fro. 3. Time of appearance of end-plate failure and Wallerian degeneration of fibers in the trunk of the phrenic nerve cut in the neck. m-m, End plates at which miniature end-plate potentials had disappeared (redrawn from Miledi and Slater, 1970). A-A, -0, 0-0, Degenerated thin, medium, and thick fibers, 5 mm below the site of transection (L. Lubifiska and J. Waryszewska, unpublished).

46

272

LILIANA L U B I ~ S K A

the physical instability of the axoplasmic cylinder (cf. D’Arcy-Thompson, 1942; Kuhn, 1953). On the other hand, the Schwann cell cytoplasm is believed to play an important role in the process of degeneration (Ram6n y Cajal, 1928), and it is more abundant in thin fibers. Electron microscopic examination of the early stages of Wallerian degeneration led Singer and Steinberg (1972) to the conclusion that the adaxonal layer of the Schwann cytoplasm, activated by nerve section, releases cytolytic enzymes that erode the axolemma and destroy in patches the denuded axon. Some axons have a capacity to survive axotomy for very long periods (Van Crevel, 1958). In the crayfish the motor fibers in transected claw nerves do not degenerate for over 3 months when kept at about 16OC (Hoy et al., 1967; Nordlander and Singer, 1972). I t is interesting to note that under these conditions about a week after nerve section the regenerating nerve sprouts begin to grow from the central stump and ultimately establish functional connections with muscles in spite of the presence of the nondegenerated original peripheral stump (Nordlander and Singer, 1972).

3. Central Effects

of

Nerve Section

Nerve section gives rise also to a wide range of changes in the affected perikarya (for references, see Gutmann, 1968, 1970; Cragg, 1970; Lieberman, 1971 ; Matthews and Raisman, 1972). Transneuronal effects are also observed. Synaptic boutons withdraw from the perikaryal surface (Blinzinger and Kreutzberg, 1968; Hamberger et al., 1970). Adhesion between axotomized neurons and glia is loosened (Watson, 1966; Kirkpatrick, 1968). Sometimes glial processes extend along portions of the neuronal plasma membrane and may invaginate into the neuronal cytoplasm (Torvik and Skjorten, 1971 ; Price, 1972). Proliferation of glia (Pannese, 1963, 1964; Humbertson et al., 1969; Price, 1972) and an increased density of capillaries is seen in the neighborhood of the affected neuronal pool. There are indications that these phenomena might be due to, or triggered by, the interruption of supply of some substances normally carried from the periphery by the ascending axoplasmic flow or by ascent of a “wound” substance. I t has been observed by Marinesco (1896), Van Gehuchten ( 1903), Bodian (1947), and others that the intensity of perikaryal reaction and the time of its appearance depend on the distance between the site of nerve section and the cell bodies. This observation has often been confirmed by later workers, but very few measurements have been made. Watson (1968a) studied the time of appearance of nucleolar changes in the hypoglossal neurons of rats after transection of the nerve made at various distances from the perikarya. His results suggest that the factor preventing, or causing, the appearance of nucleolar changes ascends the axons at a velocity of 4-5 mm/day (calculations made by Cragg, 1970). In another series of experiments, Wat-

ON AXOPLASMIC F L O W

273

son (1968b) found in hypoglossal axons a velocity of 10-30 mm/day for ascending migration of label after injection of [SH]lysineinto the geniohyoid muscle. Contrary to the well established influence of the length of the peripheral stump on the time of survival of efferent synapses and the onset of changes in the denervated cells, quantitative information about the influence of the length of the central stump on various perikaryal changes is extremely scarce. The hypothesis that central effects of axotomy are due to interruption of supply of axonal materials from the periphery or to arrival of a “wound substance” carried by somatopetal axoplasmic flow cannot be considered as substantiated so far. Other hypotheses about the mechanism of axon reaction have been discussed by Cragg ( 1970).

111. Influence of Various Factors on Axoplasmic Flow

A. SEPARATION FROM CELLBODIES

Both in tissue culture (Section 11, A ) and in vivo (Lubidska et al., 1963a,b, 1964), axoplasmic flow continues for some time in axons severed from their cell bodies. Its stops rapidly in the cadaver and on asphyxiation (Ochs et al., 1969) but is maintained in nerves kept in oxygenated Ringer solution (Jankowska et al., 1969; Mayor and Banks, 1970; Edstrom and Mattsson, 1971; and others). The velocity of fast somatofugal flow seems unaffected by the interruption of the nerve. In using radioactive tracers in cats, Ochs and Ranish (1969) crushed the axons near their origin after allowing time for the rapidly migrating peak of radioactivity to move some distance down the axons. The further downward progress of the peak was the same as that seen in the contralateral intact nerve. Similar results were obtained by Heslop and Howes (1972) on a molluscan nerve. These observations, in addition to their theoretical interest, suggest that the characteristics of axonal migration determined on interrupted nerves are probably valid also for intact neurons.

B. METABOLIC FACTORS As suggested by its continuation after separation from the perikarya, fast axoplasmic flow is not directly dependent on protein synthesis. Inhibitors of protein synthesis do not interfere with axoplasmic flow (Peterson et al., 1967; McEwen and Grafstein, 1968; Barondes, 1969; and others). A similar independence of intracellular transport from protein synthesis was observed

2 74

LILIANA L U B I ~ S K A

by Jamieson and Palade (1968) on pancreatic cells. On the other hand, colchicine, which blocks axoplasmic flow, ,does not affect protein synthesis (Daniels, 1968; Karlsson and Sjostrand, 1968; James et al., 1970; Chang, 1972). The influence of various disturbances of metabolism on axoplasmic flow has been studied recently by Ochs and his co-workers (Ochs, 1971a,b 1972; Ochs and Hollingsworth, 1971; Ochs and Smith, 1971a; Sabri and Ochs, 1972) Interference with oxidative metabolism disrupts the fast axoplasmic transport in cats. The metabolic mechanism acts at every point along the axon. When a short stretch of nerve is asphyxiated, migration is arrested in the affected zone and materials accumulate in front of it. Migration may be resumed if oxygen is readmitted within about 1 hour. Molluscan nerves are much less sensitive to anoxia. The rate of fast axoplasmic flow in the connective of Anodonta does not change in animals kept in an atmosphere of nitrogen before and during the experiment (Heslop and Howes, 1972). The fast flow is inhibited by heavy water (Anderson et al., 1972). I t seems to be unaffected by some anesthetics, phenobarbital, pentobarbital, and halothane (Norstrom and Sjostrand, 1971a; Kennedy et al., 1972; Partlow et al., 1972) and by diisopropyl fluorophosphate poisoning (James and Austin, 1970b).

.

C. TEMPERATURE The survey of published data shows consistently lower velocities of axoplasmic flow in cold-blooded than in warm-blooded animals (cf. tables in Lasek, 1970a). Results of a few systematic studies 011 the influence of temperature on fast axoplasmic flow are shown in Table IV. Grafstein et al. (1972) tried also to determine the temperature coefficient of the slow flow. This has proved to be very difficult because arrival of the slow wave to the optic tectum was undetectable. The authors infer nevertheless from their experiments that the slow flow is relatively insensitive to temperature, the temperature coefficient being at most 1.4. The temperature dependence of fast flow described by Heslop and Hbwes (1972) in the connective of Anodonta presents a curious course (Fig. 4). The velocity of flow remains unchanged (about 40 mm/day) between 4O and 15OC. Between 15O and 28OC it increases with a Qlo of about 2.0. It is interesting to note that the rate of elongation of regenerating sciatic fibers in frogs and toads show a similar curve of temperature dependence (Lubihska and Olekiewicz, 1950). The published data concerning velocity of axoplasmic flow in other animals are insufficient to establish whether a rather wide range of temperatures within which the flow remains unchanged is a general phenomenon.

TABLE IV INFLUENCEOF TEMPERATURE ON THE FUTE

OF

FASTAXOPLASMIC FLOW

~~

Rcfcrcnn

ochs and Smith

Animal

Cat

(1971b) Koike ef al. (1972)

Hulop and How- (1972)

Grdstein cf al. (1972) Elam and Agranofl (1971)

Aplyrio

Axonal route

Recursor

Sciatic nerve in m'fro

['H]Leucine

Pleuroabdominal COMCCtive

['HICholine chloride

Andonfa Ccrcbrovisccral connective

(WlValinc

Site of injection

L7 dorsal root ganglion

Site of detection

Moving component

Temperature range ("C)

Along the nerve

Labeled protein

8, 18, 28, 38

Intraneuronal Along the giant cell R, nerve in the abdominal ganglion Into the cereAlong the nerve bra1 ganglion

Goldfish Optic axons

['H]Lcucinc

Intraocular

Goldfish Optic axons

[IHlProlinc

Intraocular

Optic iectum Optic tectum

Labeled acetylcholine

18-26 28-38 5, 15. and 22

QIO

Velocity (-/day)

Remarks

2.3 2.0 1.6

17 at l5OC

c 4-40 4-1 5 15-28

1 .o 2.0

Labeled protein

9 and 20.5

2.6

Labeled protein

10, 16, 23

Labeled protein

40

60 at 20.5OC. 20 at 9OC 2 . 5 70-100 at 23%; about 30 at 16%

From 30% on, disturbed flow probably due toheatdamage

$ r $, g

L4

r The finat level of labeling of the tectum is higher at 23O than a t 16% At 10DCthe plateau waa not reached in the investigated time interval of 22 hr

276

LILIANA

5

LUBIASKA

15

10

20

25

TEMPERATURE (“C)

FIO. 4. Influence of temperature on velocities of axoplasmic flow and elongation of regenerating nerve fibers. ( I ) Axoplasmic flow of labeled proteins in the connective of Anodonta (Heslop and Howes, 1972). (11) Elongation of regenerating fibers in the sciatic nerves of frog (0-0) and toad (0-0) (Lubidska and Olekiewicz, 1950).

D. GROWTHAND REGENERATION Axoplasmic flow during development was investigated in the optic system of the rabbit by Hendrickson and Cowan (1971). In an extremely careful study, where many relevant factors were examined, the authors found an increase in “fast” transport from 120 to 200 mm/day between the end of the first and fourth week, and a slight further increase thereafter. The length of the optic nerve doubled during this time. Hendrickson and Cowan attributed the observed acceleration of the fast flow to the functional maturation of the visual system, the establishment or maturation of synapses requiring a faster and more abundant delivery of materials involved in synaptic transmission. Velocity of the slow flow fell to about half during this period. On the other hand, in the maxillary, hypoglossal, and sciatic nerves of 3-week-old rats (Droz, 1965) and in the axons of dorsal root ganglia of kittens 2-4 months old (Lasek, 1970b), the slow flow was found to be 2-3 times faster in growing than in mature animals. It is not possible to tell whether the discrepancy is due to different stages of development examined by these authors, to species and type of fibers differences, or to a

O N AXOPLASMIC FLOW

277

different mode of calculation of velocity of the slow flow (cf. Section 11,

B, 3 ) . The published data about axoplasmic flow in regenerating nerve fibers are also discrepant. They were obtained on different materials at various periods of regeneration, so that it is difficult to give a general description. Ochs et al. (1960) found in regenerating ventral roots of cats a decreased velocity of flow between 3 and 15 days after nerve section. At later periods the velocity was mostly unchanged. Carlsson et al. (1971 ) found no changes in the slow migration and an increase in the fast migration at late stages of regeneration. Grafstein and Murray (1969) described an increased velocity of axoplasmic flow in the regenerating optic system of the goldfish. The “slow” flow increased three times, and the “fast” flow twice, as compared with normal fibers. Kreutzberg and Schubert (1971) did not find any change in velocity of “fast” flow in the regenerating hypoglossal nerve of guinea pig, although the labeling of the regenerating nerve was much heavier. Kristensson and Sjostrand (1972) described, at late stages of regeneration, an increased accumulation in the neuronal perikarya of tracer proteins injected near the nerve endings and conveyed by axonal route to the cell bodies. These data suggest that the velocity of fast axonal migration in regenerating fibers remains unchanged but that both the cellulifugal and cellulipetal flux of axonal materials is more intense than in normal nerve fibers (see also Section 11, A ) . Other interpretations of heavier labeling of axons and cell bodies are, however, possible.

E. NERVEACTIVITY Rather unexpectedly, the attempts to discover the influence of nerve activity on axoplasmic flow did not provide clear-cut results. This contrasts sharply with the close links between excitation and cytoplasmic movements observed in plant cells, where the movements are arrested by all kinds of mechanical or electrical stimulation (for reviews, see Kamiya, 1959; MacRobbie, 1971). The elongated cells of Cham and Nitella are particularly convenient for this type of study. Their action potentials last several seconds and the velocity of cytoplasmic streaming is 50-70 pm/second. Cytoplasmic streaming stops abruptly during the ascending phase of the action potential and is resumed progressively after its passage (Auger, 1931 ; Franck, and Auger, 1932 ; Vorobyev and Vorobyeva, 1963). I n axons, where the duration of action potentials is much shorter and axoplasmic flow slower, such temporal resolution would be very difficult to attain, but the cumulative effect of a great number of impulses on the accumulation of axonal materials at the ends of interrupted nerves has been studied. There was no arrest or slowing down of migration of AChE (Jankowska et al., 1969) nor of

278

LILIANA LUBII~SKA

noradrenaline (Dahlstrom, 197la) . Rather, a slight, statistically nonsignificant increase in velocity was observed for both components. No influence of nerve stimulation on the velocity of migration of radioactive tracers was found by Ramos (1955) . Antidromic stimulation of nerves produced an increased labeling of axons, but the velocity of migration of label remained unchanged (Lux et al., 1970). Similar results were obtained by Norstrom and Sjostrand (1971b) in the neurohypophysial tract after functional loading. Karlsson and Sjostrand kept animals before and after birth in a dark room and found the amount and velocity of migrating proteins the same as in controls kept under ordinary conditions. Geffen and Rush (1968), on the contrary, observed an increased velocity of translocation of noradrenaline in the splenic nerve after preganglionic denervation, that is, after suppression of impinging impulses. Here again the difference was not statistically significant. Akcasu and Salafsky ( 1967) describe a longitudinal translocation of **Na in stimulated nerves but are not certain whether the transport is axonal or interstitial. Kerkut et al. (1967) found that stimulation increased the amount of glutamate in the muscle compartment of their central nervous system-nerve-muscle preparation of. the snail, but it is possible that this was due to the release of glutamate from the nerve endings, not to the acceleration of its axonal migration.

IV. General Description of Axoplasmic Flow

As an object of study of intracellular movements, the nerve cell presents certain unique features. Few other cells are so extensive. In cells of usual dimensions the main sites of synthesis and of further biochemical elaboration of components are in close proximity to one another and to the cell periphery, but in the neurons they are segregated. The cell body is separated from, the nerve endings by a uniform and often very long process, the axon. Since the axon is largely devoid of synthetic activities, the traffic of selected materials, relatively uncomplicated by the intervening metabolic events, may be followed in the neuron over long distances. O n the other hand, the very length of the axonal process, traversing many anatomical regions, makes it difficult to study the movement of substances in the entire neuron. Special methods had been devised to study various aspects of intracellular movements in separate parts of the nerve cell. The methods employed range from a detailed biochemical analysis of the synaptosomal fraction of the whole brain removed at varying times after introduction of tracers (Barondes, 1969) to quantitative autoradiography of a selected presynaptic axon, its nerve ending, and the postsynaptic cell) (Koenig and Droz, 1971a). The results of the latter work are illustrated

O N AXOPLASMIC FLOW

279

in Fig. 5. They show, with a remarkable spatial and temporal resolution, the course of migration of label but give little indication of the biochemical nature of migrating components.* The biochemical composition may be studied extensively in the synaptosomal fraction of the brain, but the heterogeneity of neurons from which the synaptosomes are obtained and the very unequal length of their axons blur the characteristics of axonal migration of the components under investigation. A dynamic picture of neuronal cytophysiology has been built up progressively from a mosaic of results obtained by different experimental approaches. Often the interest has centered on metabolic problems or on the fate of various components, and observations on their axonal migration have

Fro. 5. Radioactivity (in silver grains/100 pm') in preganglionic axons, caliciform nerve endings and postsynaptic perikarya in the ciliary ganglion of chicks, 1, 3, 6, and 18 hours and 2 and 6 days after injection of ['H]leucine into the third ventricle. From Koenig and Droz (1971a).

' An important series of papers concerning the axonal migration, subcellular location, and fate of transported proteins and glycoproteins in the chicken ciliary ganglion appeared after this paper was prepared (Droz et al., 1973; Bennett et al., 1973; Di Giamberardino et al., 1973; Koenig et al., 1973).

280

LILIANA

LUBISJSKA

been only incidental. The interrelated problems of synthesis and turnover of neuronal constituents and their transfer to various sites of the nerve cell have been discussed in recent reviews (Droz, 1969; Droz and Koenig, 1971; Grafstein, 1969; Jakoubek and Semignovskf, 1970; A. D. Smith, 1971a,b; Axelrod, 1971; Geffen and Livett, 1971; Dahlstrijm, 1971b). Axoplasmic flow was detected in all types of axons of vertebrates and invertebrates in which it has been looked for. The velocities of flow seem to vary with the nature of moving components, type of nerve fibers and the animal species. As was discussed in foregoing chapters, however, the reliability of estimates of velocities is very unequal and many corrections of earlier results are constantly being published. I n particular, in many of the published data no clear-cut distinction is made between the velocity of translocation and the amount of material transported in unit time (cf. Section 111).Yet, probably these two magnitudes are not physiologically equivalent. I t is tempting to anticipate that the velocity of translocation is closely linked with the mechanism of transport and would be little affected by a wide range of conditions compatible with preservation of this mechanism. The amount of migrating components on the other hand, particularly of those involved in rapid functional changes, would be much more variable. It would presumably change with current functional load and previous activity and would be modified by all circumstances in which the equilibrium between the rate of production and that of utilization of the investigated component is disturbed. The available experimental evidence is not complete enough to test the correctness of these anticipations. The observed velocities of axoplasmic flow may be grouped into two main categories, of the order of 100 mm/day and of 1 mm/day. The huge difference between fast and slow flow suggests that the underlying mechanisms (and routes) are fundamentally different and so probably are the physiological and metabolic roles of materials traveling at these two rates. The two types of flow will be therefore considered separately.

Many constituents of small organelles, enzymes, neurotransmitters, and neurosecretory substances move in various nerve of warm-blooded animals at velocities ranging from about 40 to over 400 mm/day. Similar velocities have been found for components, such as “trophic substances” which are as yet undefined biochemically, but whose physiological role is known, and for the axonal migration of neurotropic viruses and toxins and other exogenous substances which are taken up by nerve endings and ascend the axons (Tables 1-111).

O N AXOPLASMIC FLOW

281

The fast transport is bidirectional. In living nerve fibers bidirectional movements of particles have been observed in the same axon. I n quantitative experiments on whole nerves the cellulipetal migration has been studied by methods used to analyze the cellulifugal migration : translocation of various tracers in intact neurons and accumulation of endogenous or tracer materials at the site of interruption of nerves. There are indications that even components destined for eventual export from the neuron such as neurotransmitters and neurosecretory materials may for some time (or in some proportion) circulate in the neuron before being released. 1. Change of Direction of Flow Whereas the bidirectional migration in the cylindrical part of the neuron seems clearly established, and in many cases the characteristics of such migration in each direction have been determined, it is not at present known how the change of direction occurs nor where exactly it happens. Probably the mechanism by which the flow proceeds does not operate at the very end of the nerve fiber. The differences in shape and ultrastructure between the cylindrical axon and the nerve endings suggest that different forces may be responsible for the movement of materials at these two locations. I n particular, the nerve endings, and also, in adrenergic nerves, the varicosities of terminal axons (Fillenz, 1971) , usually lack the longitudinal arrangement of neurofilaments and microtubules believed to play an important role in the mechanism of fast axoplasmic migration. It is possible that a portion of the moving material may reverse its direction of flow at the preterminal branching of axons. Such reversal was observed directly by Mahlberg (1964) at the branching of transvacuolar protoplasmic strands in cultured Euphorbia cells. The points of bifurcation of axons are known to exhibit special electrophysiological properties, such as block of impulses at side branches or transformation of a continuous series of impulses into an intermittent one (for references, see Waxman, 1972). But there is a complete lack of information about the distribution of migrating materials and possible alterations of characteristics of flow at these points. The local movements of organelles in the nerve endings of resting and stimulated cells have been studied extensively. The analysis of migration of synaptic vesicles toward the surface membrane and of their ultimate fate has contributed greatly to our insight into the mechanism of synaptic transmission (Katz, 1971 ; Kuno 1971) . Depolarization of nerve terminals also causes local movements of mitochondria and other organelles, changes the width of the synaptic gap and increases the area of apposition to the postsynaptic membrane (Landau and Kwanbunbumpen, 1969; Jones and

282

LILIANA L U B I ~ S K A

Kwanbunbumpen, 1970; Pysh and Wiley, 1972; Korneliussen, 1972; Akert, 1973). It is not known whether these movements have any influence on the course of migration in the axons. D. S. Smith (1971) thinks that synaptic events may modulate the transport of materials in distant regions of the neuron, including the perikarya. So far such modulation has not been detected experimentally (cf. Section 111), but the present methods of measurement are probably not sensitive enough for detection of small and transient changes of axoplasmic flow.

2. Biological Significance of Bidirectional Flow The amount of work devoted to the somatofugal axoplasmic migration reflects the importance of many migrating components in the control of the innervated cells. Somatopetal migration usually receives only marginal attention. Yet the ascending flow is probably one of the main mechanisms whereby the periphery influences the state of the innervating cell and sometimes that of neurons situated higher up the neuronal chain. In the central nervous system, destruction of certain regions may cause a retrograde degeneration of presynaptic neurons. This phenomenon could be due to the suppression of trophic influence of the destroyed neurons, but it is difficult to control other possible causes, such as interference with the normal traffic of impulses or disturbance of reflex relations. A retrograde trophic influence of the postsynaptic on the presynaptic neuron has been demonstrated directly in the superior cervical ganglion of rat and mouse, a less complex system and more amenable to experimental analysis (Black et al., 1972; Thoenen et al., 1972). In neonatal animals the adrenergic neurons in the ganglion were selectively destroyed by administration of 6-hydroxydopamine or antiserum to nerve growth factor (NGF). This procedure inhibited the development of choline acetylase activity in the preganglionic cholinergic fibers but had no effect on cholinergic fibers which do not make synaptic contact with adrenergic neurons. The arrest was long lasting. Choline acetylase activity was depressed for at least 2 months when the antiserum to NGF was administered a few days after birth. At the time of injection the organs innervated by the superior cervical ganglion had not yet received a functional nerve supply. Hence, it is unlikely that reflex effects would be responsible for the arrest of enzymic maturation of the preganglionic neuron, These results suggest that some materials coming from the adrenergic neurons in the superior cervical ganglion are normally taken up by the presynaptic endings and carried somatopetally to the cholinergic neuron, modulating its biochemical equipment. The physiological importance of somatopetal flow is particularly apparent in early development, when the trophic influence of peripheral structures on the innervating neurons is prominent. The periphery controls both pro-

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liferation and the continued growth and maintenance of neurons. I n many classes of animals extirpation of limb buds or amputation of limbs causes cell death in the corresponding nerve centers and sensory ganglia even when the operation is made before the establishment of functional innervation. Implantation of supernumerary limb buds, on the contrary, leads to an increase in number and volume of cells in the neuronal pools (for reviews, see Detwiler, 1933; Piatt, 1948; Hamburger and Levi-Montalcini, 1949, 1950). There are strong indications that these phenomena are determined by some substances stemming from the periphery and normally transported to the neuronal perikarya by an ascending axonal route (Prestige, 1967a,b, 1970).

3. Mechanism

of Fast Flow

NO consistent theory of fast axoplasmic flow is available at present. AS with intracellular movements in general, the proposed interpretations are tentative and fragmentary. It is generally felt that longitudinally oriented structures are operant in the propulsory mechanism of fast axoplasmic flow. Schmitt (1968) put forward the hypothesis that translocation of vesicles occurs by mechanochemical interaction between microtubules and membranes of vesicles. Two types of observations lend support to Schmitt’s hypothesis : the ultrastructural organization of axoplasm and the arrest of axoplasmic flow produced by agents disrupting the microtubules. Small organelles often occur in the axoplasm in association with microtubules (Smith et al., 1970; Banks et al., 1971b) and structural cross-bridges between microtubules and synaptic vesicles (D. S. Smith, 1971; D. S. Smith et al., 1970) and mitochondria (Hirano and Zimmerman, 1971; Raine et al., 1971) have been observed. It is not certain as yet whether these contacts are functional. The number of microtubules is very unequal in various fibers. Their spatial arrangement and relation to neurofilaments is different in the initial segment and farther down the axon (Kohno, 1964; Palay et al., 1968; Wuerker and Kirkpatrick, 1972). No information is available as to whether the intensity or other characteristics of flow are correlated with the number or distribution of microtubules in the axoplasm. Experiments with radioactive tracers show that rapidly migrating components are not randomly distributed in the axoplasm. Lentz (1972a), in regenerating nerves of newt, found a concentration of label in the annular region of the axon cross section, which was rich in microtubules and other organelles. The center of the fiber, where neurofilaments are concentrated, was poorly labeled. A similar distribution of label near the axolemma was observed by Hendrickson and Cowan (1971) in the optic fibers of rabbit. In the axons of leech a different topography of label was observed by Droz

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(1969). The central portion of the axon, rich in neurofilaments, was preferentially labeled. The arrest of both cellulifugal (McEwen and Grafstein, 1968; Dahlstrom, 1968; Haggendal and Dahlstrom, 1970; Kreutzberg, 1969; Schlaepfer, 1971; Banks et al., 1971a; Mayor et al., 1972; and others) and cellulipetal (Kristensson and Sjostrand, 1972) axoplasmic flow by local application of colchicine is generally attributed to disassembly of microtubules (Borisy and Taylor, 1967) caused by this drug. Recently, however, several uncertainties have arisen concerning this interpretation. O n the one hand, colchicine was found to bind not only to tubulin, but also to certain membrane proteins (Dahl et al., 1970; Stadler and Franke, 1972) ; on the other hand, several cases have been reported in which fast axoplasmic flow was arrested by doses of colchicine that apparently did not affect the microtubules (Karlsson et al., 1971; Fernandez et al., 1970, 1971; Norstrijm et al., 1971; Flament-Durand and Dustin, 1972). Chang (1972) found, on the contrary, that doses of colchicine which reduce considerably the number of microtubules in axons of cultured chick sensory ganglia only slightly diminish the rate of movements of particles. The influence of concentration of colchicine on the number of axonal microtubules and velocity of migration of noradrenaline was studied systematically by Banks and Mayor (1972) in hypogastric nerves of cats. A remarkably parallel course of changes of both was observed, suggesting strongly that microtubules are actually operant in the transport of norepinephrine. The authors have also found that some colchicine-binding activity is associated with the particulate fraction containing dense-cored and agranular vesicles and envisage the possibility that some microtubular protein may. be bound to noradrenaline storage granules. The discrepant results concerning the relationship between microtubules and axoplasmic flow need further elucidation (cf. Samson, 1971). It is possible that some organelles, such as catecholamine storage granules, move by interaction with microtubules whereas others are transported by a different mechanism. The experimental procedure may also play an important role. Microtubules are extremely labile structures and they rapidly disperse and reassemble again in vivo under various conditions. Thus, RodriguezEchandia et al. (1970) found that microtubules disappear from the sciatic fibers of the frog when the animal is kept at 2OC and reassemble in 20 minutes after return to room temperature. A similar effect of temperature on reassembly of microtubules in brain homogenates was observed by Weisenberg (1972). Therefore, in spite of many uncertainties, the microtubules remain a strong candidate for an important role in the transport of organelles. Although real proof is lacking, the ultrastructural appearance of axons,

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the almost linear tracks of axonal granules seen in living axons, and the arrest of fast flow in places where the longitudinal course of mirotubules and neurofilaments is disrupted by experimental intervention or by a pathological process (Suzuki and Terry, 1967; Terry and Wisniewski, 1970; and others), create an overwhelming impression that the longitudinal fibrous structures are operant in fast axoplasmic flow. The theories designed to explain fast flow (Ochs, 197 1b; Hejnowicz, 1970 ; Samson, 1971 ) are generally based on this assumption. B. SLOW FLOW The slow flow, a t a velocity of about 1 mm/day, has been observed to proceed in the proximodistal direction only. Quantitative data were obtained mainly by use of radioactive precursors, labeling rather wide classes of substances. Thus, a large fraction of “soIuble proteins” (including microtubule protein) was found to move slowly (Droz, 1967a; Ochs et al., 1967; McEwen and Grafstein, 1968; Bray and Austin, 1968; Kidwai and Ochs, 1969; Grafstein et al., 1970; Sjostrand, 1970; Barondes, 1971; and others). Migration of individual components of known physiological significance has been studied less often. The slow flow of soluble proteins raises the question of whether the solvent, axoplasmic water, also moves at a slow rate. There is an annoying lack of experimental data on this point. Water is by far the most abundant of axoplasmic components. In the giant fibers of the squid the water content is about 80% (Bear and Schmitt, 1939; Villegas and Villegas, 1960). In denuded axoplasmic cylinders of rat nerve fibers water occupies about 90% of the axonal volume (L. Lubihska and B. Jusihska, unpublished). In the axoplasmic matrix the proportion of water is probably higher still. In spite of its abundance much less is known about the intraaxonal movement of water than about that of some components, such as AChE, occupying only about one millionth of the axoplasmic volume. Axonal water may be structured (cf. Hovey et al., 1972). It presumably exchanges, to an unknown extent, with the extraaellular water, is involved in metabolic reactions, and may be incorporated into rapidly migrating organelles. The intensity and the time course of these processes are unknown. The study of the longitudinal translocation of water in the axons thus presents tremendous experimental difficulties. The clarification of this problem has probably to await techniques for the examination of nondehydrated tissues in the electron microscope. Then the autoradiography of axons after intraperikaryal injection of tracer amounts of tritiated water could presumably provide information about the fate and dynamics of axonal water, essential for the understanding of the mechanism of the slow flow.

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Weiss (1970) regards the slow flow as a unidirectional translocation of the “solid axonal matrix and presumably also part of the associated water moving as a single cohesive, viscous, somewhat plastic body within the Schwann (or glia) cell.” He proposed that the movement of the column is brought about by a peristaltic wave traveling on the surface of the axon. Weiss pointed out, however, that actual proof for this mechanism of propulsion is still lacking. The anatomy of the neuron makes it unlikely that axoplasm moves as a coherent body. This would require a constant volume of axoplasm per unit length at every point of the nerve fiber. In fact both narrowings and enlargements occur in various parts of the axonal path. In myelinated fibers the diameter of the axon is reduced at each node of Ranvier and enlarged at the paranodes. Longer stretches of narrowed axon are seen in the initial segment, between the axon hillock and the first myelinated internode and in the region of the pial ring in fibers leaving the spinal cord (Ranvier, 1882; Tarlov, 1937) . Other difficulties raised by the hypothesis of unidirectional flow of the whole axoplasmic mass were discussed by Lubifiska (1964). Perhaps some of the hypotheses proposed to interpret longitudinal translocation of solutes in plants (see MacRobbie, 1971) could be also applied to analyses of axoplasmic flow. Besides the intrinsic difficulties of experiments and interpretation, the slow flow presents some biological problems as to what type of molecules are likely to migrate slowly. T h e question is particularly pertinent to long axons. The average half-life of neuronal proteins varies from 10 to 20 days. Some proteins have a much shorter half-life (see Jakoubek and Semiginovskf, 1970). Exceptionally long persistence of protein labeling, up to 80 days, was observed by Lasek (1970a). If a protein molecule synthesized in the perikarya is to reach the terminal portions of the axon, the velocity of transport should not fall below a critical value determined by the length of the axonal path. A slow migration of short-lived components would be indicated by their presence in the proximal and their absence from the distal portions of the axon. Possibly the proximodistal gradients of some axoplasmic constituents are determined by the relation between the life-span of the molecule and the duration of the axonal transit (Lubiliska, 1971; Orrego, 1971) . The slowly moving substances stable enough to survive the time taken by the journey from the perikaryon to the end of the axon cannot be involved in any moderately rapid nervous function. In a motoneuron of a medium-sized animal, like the dog, such a journey would take over a year. This is a very long time in the life of an organism, and a variety of metabolic, hormonal and seasonal changes are bound to occur during this period.

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If there are substances migrating in long axons at velocities of the order 1 mm/day, their biochemical nature and functional role, as well the characteristics of their migration under various experimental conditions would merit a thorough study by finer and more diversified methods than those used so far. I n the meantime it is disturbing for a neurobiologist to think that the far end of a neuron would learn what is happening in its cell body only after a year’s delay. The significance of such a message for the nervous activity of the animal or for the life of the nerve cell itself seems puzzling. ACKNOWLEDGMENTS

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SCHIZOPHRENIA: PERCHANCE A DREAM? By J. Christian Gillin and Richard J. Wyatt The National Institute of Mental Health, Betherda, Maryland, and Saint Elizobethr Hospital, Worhington, D.C.

I. Introduction 11. Major Concepts

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A. Three States of Consciousness * B. Physiological and Biochemical Considerations C. Dreaming and REM Sleep D. The Phenomenology of Hallucinations and Dreams E. The REM Rebound * F. Individual Differences in REM Compensation * III. Previous Sleep Studies in Schizophrenics A. Cross-Sectional Studies * B. Longitudinal Studies. * C. Experimental REM Deprivation in Schizophrenia * D. Reservations * E. Purpose of the Present Study . IV. General Methods. * V. Study No. 1: Longitudinal Sleep Study in Acute Schizophrenia * A. Clinical Description of the Patients B. The Results of the Sleep Studies C. Comment VI. Study No. 2: Experimental REM Deprivation in Psychiatric Patients A. Methods B. Results C. Comment VII. Study No. 3: REM Sleep of Depressed Patients after Withdrawal of Phenelzine. A. Methods B. Results C. Comment VIII. Study No. 4: Longitudinal Sleep Study of a Manic-Depressive Patient . A. Clinical Description of the Patient. B. Results C. Comment IX. Discussion. X Summary. References .

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The purpose of this paper is to present the results of a clinical study which suggest schizophrenia is related to an abnormality in rapid eye move297

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ment (REM) sleep, the stage of sleep most closely associated with dreaming. The hallucinations of the psychotic have long been compared with the hallucinations experienced by everyone at night in dreams. Bleuler, for example, who based his observations of schizophrenia on years of living with patients, in 1908 compared the schizophrenic process and dreaming as follows: “In dreams, a similar dissociation of thinking occurs ; symbolism, condensations, predominance of emotions which often remain hidden, hallucinations-all these can be found in both states and in the same way, and in spite of the difference in genesis and in spite of the minor differences, it may well be possible to show the secondary symptomatology of schizophrenia as wholly identical with that of dreams” (Bleuler, 1950). Jung (1944) has said in an oft-quoted statement, “Let the dreamer walk about and act as one awakened, and we have the clinical picture of dementia praecox.” With the discovery that dreaming occurs during REM sleep, new hope arose that it would be possible to understand hallucinations as waking dreams. The pioneering efforts of Dement, Snyder, Vogel, Feinberg, Hawkins, and Mendels are relevant to this hypothesis. As we shall see, early results failed to establish a relationship between psychosis and REM sleep, but the studies did suggest that more subtle differences in REM sleep might exist between schizophrenics and other psychiatric patients or normals. This paper reexamines one of the issues raised by these studies: Do schizophrenic patients have a normal “rebound” of REM sleep following deprivation of REM sleep?

II. Maior Concepts

A. THREE STATESOF CONSCIOUSNESS After the discovery of Aserinsky and Kleitman in 1953 that there are two types of sleep, it was proposed that mammals alternate between three separate states of consciousness: REM sleep, non-REM (NREM) sleep, and wakefulness (Snyder, 1963; Jouvet, 1967). REM sleep is characterized by the presence of bursts of rapid eye movements, a low-voltage, mixed-frequency electroencephalogram (EEG) , atonia of the antigravity muscles, variability of autonomic functions, penile erections, and dreaming. NREM sleep is characterized by slower frequency, higher amplitude, synchronized EEG patterns, preservation of muscle tone, ocular quiescence, release of growth hormone in its early phases, and the relative absence of dreaming. Sleep normally begins with a period of NREM sleep lasting some 70-100 minutes. Following a relatively brief REM sleep period, NREM sleep reappears, and REM and NREM sleep alternate for the remainder of the night

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with a cycle length of about 90 minutes. Each REM period tends to be longer than the one that preceded it, and there are four or five REM periods per night. About 80-100 minutes are normally spent in REM sleep each night, accounting for 2 6 2 5 % of total sleep time. NREM sleep is divided into four separate stages on the basis of the EEG: stage 1, a brief, transitional stage characterized by a low-voltage, mixed-frequency EEG and muscle tone; stage 11, with its characteristic sleep EEG spindles and K complexes; and stages I11 and IV, characterized, respectively, by a moderate (20-50%) and by a large (greater than 50%) proportion of time spent in delta waves. Taken together, stages I11 and IV are referred to as delta sleep and occupy 1 6 2 0 % of the night’s sleep. B. PHYSIOLOGICAL AND BIOCHEMICAL CONSIDEIUTIONS

The physiological phenomena of REM sleep are divided into tonic and phasic events (Moruzzi, 1963). The tonic events, which persist throughout the REM period, include the characteristic low-voltage, mixed-frequency EEG and the loss of muscle tone. The phasic events, which occur periodically during REM and at times during NREM sleep, include the rapid eye movements themselves, changes in heart rate, respiration, and blood pressure, and, in cats, monophasic, sharp waves in the pons, lateral geniculate body, and occipital cortex (PGO spikes). There is some evidence in man that the phasic events of REM sleep are particularly associated with dreaming itself (Dement and Wolpert, 1958; Hobson et al., 1965; Molinari and Foulkes, 1969). Considerable work has been done on the biochemical aspects of sleep and has been reviewed recently (Jouvet, 1967; Wyatt, 1972). Of particular interest to the present discussion, the tonic events and the phasic events can be dissociated from each other in cats by administration of reserpine and p-chlorophenylalanine (PCPA). Both of these drugs deplete the brain of serotonin; the former by interfering with storage, the latter by inhibition of tryptophan hydroxylase, the rate-limiting enzymic step in the synthesis of serotonin. After treatment with these agents, phasic events, such as PGO spikes, occur in NREM sleep and wakefulness (Jouvet, 1967; Dement et af., 1969a,b).

C. DREAMING AND REM SLEEP Much of the research relating REM sleep to psychosis has been based on the assumption that dreaming occurs during REM sleep. In fifteen studies reviewed by Snyder and Scott (1972) the incidence of “dream reports” obtained on awakenings from REM ranged from 60 to 80% with a median

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of 74%. The exclusive relationship between REM sleep and dreaming was clouded, however, by the finding of some investigators that “dream reports” are frequently elicited upon awakenings from NREM sleep. I n particular, mental activity at sleep onset is often said to be indiscriminate from mental activity reported following REM awakenings (Foulkes et al., 1966; Foulkes and Vogel, 1965; Vogel et al., 1972). The significance of these NREM “dream reports” is unresolved since many problems of definition and methodology remain (Snyder and Scott, 1972), but in general, mental activity during REM sleep appears to be perceptual while that during NREM sleep tends to be more conceptual. If dreams do occur during NREM sleep, it may be in relation to phasic events. PGO spikes in cats are known to occur ocasionally during NREM sleep. A recently discovered phasic event in man, middle ear muscle activity, occurs predominantly in REM, but also during NREM, sleep (Pessah and Roffwarg, 1972). It is not known what cognitive events they are related to.

D. THE PHENOMENOLOGY OF HALLUCINATIONS AND DREAMS Although it is commonly believed that schizophrenic hallucinations are “auditory” and dreams are “visual,” the actual phenomenology of the two experiences appears to be remarkably similar, given the limited accessibility that any investigator has to these private events. A comparison of hallucinations and dreams can be made from the studies of Goodwin et al. ( 1971), who interviewed 116 hallucinatory patients, including 32 chronic and 13 acute schizophrenics, and of Snyder and Karacan (Snyder, 1970), who examined 635 REM reports from 250 subject nights, collected mostly froni young, normal adults. While Goodwin et al. found that 94% of the chronic schizophrenics and 69% of the acute schizophrenics reported hearing voices, Snyder found that speech was mentioned between about 52 and 100% of the time following REM wakenings, depending upon the length of the dream report and the vigor with which the subjct was questioned. Snyder accepted the subject’s report as a dream only if it contained evidence of organized perceptual imagery, usually visual. Since he did not report how often the subjects’ reports failed to meet this criterion, we cannot judge the frequency with which “dreams” might occur without visual imagery. Goodwin et al. reported that 72% of the chronic schizophrenics and 46% of the acute schizophrenics had visual hallucinations. The visual hallucinations reported by Goodwin et al. and the visual dreams reported by Snyder both appear to be very realistic, with content most frequently of normalappearing, normal-sized people and in natural color. Hallucinations and dreams differ, however, in other respects. The hallucinating patients reported tactile, olfactory, and gustatory experiences

SCHIZOPHRENIA

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much more frequently than the REM dreamers. In addition, it is important that schizophrenic hallucinations are not unique ; they are similar in many respects to those reported by patients with affective disorder, alcoholism, organic brain syndrome, and hysteria. I t would be surprising if dreams and hallucinations were identical. Hallucinations probably result from the simultaneous integration of perceptions from the external world and of internally generated stimuli, while dreams evolve with minimal simultaneous contribution from the external world. Nevertheless, the apparent similarity between dreams and hallucinations suggests that the physiological mechanisms producing the two processes could be related. E. THEREM REBOUND I n 1960, Dement discovered that most normal subjects attempt to “make up” for the loss of REM sleep. During and after deprivation of REM sleep by arousal at the onset of each REM period, subjects enter REM sleep sooner and more frequently; on the first night of undisturbed sleep following REM deprivation, subjects have greater than normal amounts of REM sleep; and on following nights, the amount of REM sleep declines each night to baseline levels. This increase in REM sleep above baseline levels during the postdeprivation period is commonly called the REM rebound or REM compensation. T h e finding of a REM rebound following REM deprivation has been amply confirmed in man and animals (Kales et al., 1965; Dement et al., 1966; Dement and Fisher, 1963; Agnew et al., 1967; Kiyono et al., 1965; Morden et al., 1967; Siege1 and Gordon, 1965). REM compensation follows not only specific deprivation of REM sleep, but nonspecific deprivation of REM sleep during partial (Sampson, 1965; Levitt, 1967) or total sleep deprivation (Berger and Oswald, 1962; Williams et al., 1964), though in the latter, the REM rebound may be delayed initially while delta sleep is markedly increased. In addition, after discontinuance of the administration of most drugs that suppress REM sleep, sleep usually is characterized by a large REM rebound (Rechtschaffen and Maron, 1964; Le Gassicke et al., 1965; Wyatt et al., 1971a,b). Early studies suggested that REM deprivation might be deleterious (Dement, 1960; Dement and Fisher, 1963), but better controlled studies did not confirm these observations (Kales et al., 1965; Dement, 1964) although subtle changes have been detected (Clemes and Dement, 1967; Greenberg et al., 1970; Cartwright and Ratzel, 1972). Furthermore, using the “flower pot” technique to REM-deprive cats for as long as 70 days, Dement failed to find adverse effects on behavior or performance (Dement

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et al., 1967). I n human studies, chronic administration of monoamine oxidase inhibitors completely suppresses REM sleep in depressed (Wyatt et al., 1969b, 1971b) and narcoleptic patients (Wyatt et al., 1971a) without evidence of adverse psychological effects, even though in some cases REM sleep has been completely suppressed for as long as a year. These results in animals and man suggest that REM suppression is not harmful per se and support the conclusion reached by Vogel (1968) that REM suppression does not cause psychosis.

F. INDIVIDUAL DIFFERENCES IN REM COMPENSATION Considerable individual differences in REM compensation exist (Dement, 1960; Kales et al., 1965; Sampson, 1965; Rechtschaffen and Maron, 1964; Cartwright et al., 1967). In Dement’s original study, for example, one of the subjects failed to have a REM rebound, even though he was the subject who had the greatest increase in the number of entries into REM sleep during the REM deprivation period. Early studies suggest that individual differences in REM compensation following short-term REM deprivation in normal subjects may be related to psychological factors. For example, field independent, “good dreamers” tend to be good “REM compensators” (Cartwright et al., 1967; Cartwright and Ratzel, 1972). These studies are in an early stage. The number of subjects studied so far is small and a test-retest study has not been done yet to determine whether magnitude of REM compensation is an enduring characteristic of the individual.

111. Previous Sleep Studies in Schizophrenics

A. CROSS-SECTIONAL STUDIES Contrary to early hopes and expectations after the discovery of REM sleep, initial studies failed to establish an abnormality of sleep in schizophrenia. No manifestation of REM sleep was found in waking schizophrenic patients (Rechtschaffen et al., 1964b), and no marked deviation in REM percent during sleep was found between chronic schizophrenic patients and normals (Dement, 1955; Feinberg et al., 1964; 1965; Hartmann et al., 1966; Caldwell and Domino, 1967; Caldwell, 1969; Jus et al., 1968; Vincent et al., 1968) or between hallucinating and nonhallucinating schizophrenics (Koresko et al., 1963). A number of investigators have reported reduced amounts of delta sleep in schizophrenics (Feinberg et al., 1969b; Caldwell and Domino, 1967; Caldwell, 1969; Lairy et al., 1965; Stern et al., 1969;

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Itil et al., 1970; Kupfer et al., 1970; Kunugi, 1970), but the meaning of this finding is unclear since reduced delta sleep has also been reported in mental retardates (Feinberg et al., 1969a), in patients with chronic brain syndrome (Feinberg, 1967), in the normal elderly (Feinberg et al., 1967), in students under stress (Lester et al., 1967), and in depressed patients (Diaz-Guerrero et al., 1946; Gresham et al., 1965; Castellotti and Pittaluga, 1966; Mendels and Hawkins, 1967a,b; Hawkins et al., 1967; Muratorio et al., 1968; Snyder, 1968, 1971; Lowy et al., 1971). Two studies of chronic schizophrenics did, however, find contrasting amounts of REM sleep. Gulevich et al. (1967) reported an increased amount of REM sleep in a group of 13 chronic, unmedicated schizophrenics in remission. O n the other hand, Azumi (1966) found less REM sleep in a group of 35 chronic schizophrenics than in 33 normals. This discrepancy between the two reports seems, however, to be compatible with the finding of Feinberg et al. (1964) that “short term” (less than one year) schizophrenics have significantly lower REM than “long term” (greater than one year) schizophrenics. Azumi’s patients appear to be more acute and more disturbed than the patients of Gulevich et al. (1967). Insomnia and loss of REM sleep have been reported during acute phases or at times of psychic turmoil. While Fisher and Dement (1963) reported a REM% of 50 in a patient at the beginning of an acute paranoid psychosis, Dement later reexamined the sleep records and concluded that the patient had very little REM sleep at that time (Dement, 1966). Nevertheless, the presence of low amounts of REM in disturbed patients was subsequently confirmed by Lairy et al. (1965) in 10 patients at the onset of an acute psychosis or delirium, by Vincent et al. ( 1968) in four disturbed patients, by Rogina et al. (1968) in three schizophrenics during an acute period, and by Kupfer et al. (1970) in six acute schizophrenic patients. Not all so-called acute schizophrenics, however, have low amounts of REM [the less disturbed patients of Vincent et al. (1968), the previously untreated patients of Jus et al. (1968), and 7 of the 8 acute patients of Stern et al. (1969)l. I t is not clear at this time whether these discrepant results reflect differences in symptomatology, severity or length of illness, past use of drugs, or other factors.

B. LONGITUDINAL STUDIES While the cross-sectional studies suggest, but do not prove, that REM sleep is reduced in schizophrenics at times of psychic turmoil, the few longitudinal studies to date suggest that schizophrenic patients who do suffer marked loss of REM sleep during an acute psychosis do not show REM compensation when they improve clinically, and total sleep time returns to normal. Kupfer et al. (1970) studied six acute schizophrenic subjects with

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nightly sleep recordings during ten psychotic episodes. Three of the patients were not placed on any medication during the study. A nursing research staff assessed the behavior and symptomatology of each patient each day, and each psychotic episode was divided into a waxing and waning phase. During the waxing period, the patients suffered a marked insomnia and disproportionate loss of REM sleep. As the psychotic behavior waned, both REM and NREM sleep gradually returned to normal values, although the REM sleep improved less rapidly than NREM sleep. In the postpsychotic and remission periods, the schizophrenics had relatively similar amounts as normal controls. Since the patients did suffer a marked loss of REM sleep during the waxing period, the absence of a REM rebound during the waning, postpsychotic, and remission periods was surprising, particularly since the few psychotically depressed patients studied with similar REM deficits seem to evidence large amounts of REM sleep as they improve clinically. Synder (1972) reported longitudinal data on two psychotically depressed patients who experienced profound insomnia and loss of REM sleep when most severely depressed. While improving clinically, they both had marked elevations of REM sleep, averaging between 125 and 150 minutes of REM per night for periods as long as 10 days at a time. I n contrast to the schizophrenic patients, these depressed patients showed REM patterns that increased more rapidly than the NREM patterns as they recovered. Similar REM rebounds during clinical improvement of depression have been reported by others (Hawkins et al., 1967; Mendels and Hawkins, 1971). The patients in the latter studies, however, unlike the two patients reported by Snyder, received electroshock therapy or antidepressants. No other nightly longitudinal sleep studies of schizophrenics during a psychotic episode have been reported, although several studies of sleep at intervals during the course of a psychosis have been reported. These studies also suggest that REM sleep is reduced during the early, acute phases of psychosis but that it is not elevated above normal values during recovery and remission (Lairy et al., 1965; and one patient studied longitudinally by Stern et al., 1969).

C. EXPERIMENTAL REM DEPRIVATION IN SCHIZOPHRENIA While this curious deficiency of REM compensation may be an important finding, several studies of experimental REM deprivation in schizophrenic patients have yielded conflicting results (Table I ) . Two studies report that schizophrenics have normal REM rebounds following REM deprivation. Vogel and Traub (1968) REM-deprived 5 chronic schizophrenics for 7 nights by awakenings and by administration of nighttime phenobarbital and amphetamines, drugs which partially suppress REM sleep. Four of the

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patients were on phenothiazines. All patients had an increase in REM above baseline levels during the recovery period. De Barros-Ferreira et al. (1973) REM-deprived eleven chronic schizophrenics for 3 nights and reported that all had a “REM rebound.” Eight of the patients were on phenothiazines. Two other studies, however, reported abnormal response to REM deprivation in schizophrenic subjects. Azumi et al. (1967) deprived three chronic schizophrenics of REM sleep for 5 nights. Only one of the patients had a “rebound” in REM sleep, and it was more marked than that of normal controls. Zarcone, Dement, and their collaborators (Zarcone et al., 1968, 1969) selectively REM-deprived actively ill schizophrenics, remitted schizophrenics, and nonschizophrenics for 2 nights, although in one actively ill patient REM deprivation was carried on for 8 nights with the aid of nighttime amphetamines. Most of their subjects received constant amounts of medications as clinically prescribed. During the postdeprivation period, the actively ill schizophrenics did not have a REM rebound, whereas the remitted schizophrenics had an exaggerated rebound. The nonschizophrenics were in between.

D. RESERVATIONS The results of these four studies of experimental REM deprivation in schizophrenia are inconclusive for several reasons. First, the clinical status of the patients a t the time of the study was not specified in three of the reports (Vogel and Traub, 1968; de Barros-Ferreira et al., 1973; Azumi et al., 1967). [In the Vogel and Traub study, however, unpublished psychological tests indicate that the patients were actively ill, although the degree to which they were was not stated ( G . W. Vogel, personal communication 1972).] As Zarcone et al. (1968) indicated, clinical status may be an important factor in response to REM deprivation. Second, the effects of psychoactive medication and amphetamines on REM compensation are not fully understood. I n cats, phenothiazines enhance the magnitude of REM compensation (Cohen et al., 1968). Small doses of chlorpromazine, however, do not affect the REM rebound in normal volunteers (Naiman et al., 1972). In humans, cessation of amphetamine administration results in a large REM rebound (Rechtschaffen and Maron, 1964). Third, in the de Barros-Ferreira et al. (1973) study the sleep records were not scored or analyzed according to standard criteria. Fourth, in the Vogel and Traub (1968) and de BarrosFerreira et al. (1973) studies reporting normal REM compensation, total sleep time increased from baseline to recovery periods. Verdone (1968) has shown in normal subjects that the proportion of total sleep spent in REM rises as total sleep time increases. Therefore, the REM rebound reported by Vogel and Traub may merely reflect an increase in total sleep following

TABLE I EXPERIMENTAL DEPRIVATION OF Rrsm EYE MOVEMENT @EM) SLEEP IN SCHIZOPHRENIC PATIENTS: PREVIOUS STUDIES~

First-recovery night

Study Azumi cf al. (1967)

Subject

Duration of Drug REM treatment deprivation

REM (minutes)

Ev1

Full recovery period

REM

Totalsleep

(%)

(minutes)

REM (minutes)

w

:

Changes in sleep from baseline to recovery period

REM

Totalsleep

(%)

(minutes)

8 =" 2

3 Ch. Sch. Sex: M Age: 34-56

No

3 Normals8 Sex: NS Age: 20-26

No

5 Ch. Sch.

Yes,4

Sex: 4F, 1M Age: 31-37

No, 1

5 Nights

5 Nights

NS

NS

+4.3% (1.5-8.3%)

NS

+5.7% (l.8-10.6%)

NS

NS

NS

+4.2%b (1.9-7.4%)

NS

+6.3%* (67%)

NS

> z

0

En

P r

Vogel and Traub (1968)

Zarcone ct al. (1968)

3 Ch. Sch.f (actively ill) Sex: M Age: 3638 6 Ch. Sch. (remitted)

Yes

7 Nights

2 Nights

+

80 (19-35)

+

32 (2-65)

+12.4% (1 .>22.1%)

+83 (-49-201)

NS

NS

+41" +6.3% (2W58) (-0.1-10.9%)

+3" (-9-16)

+0.1% (-1.7-5.3%)

+55

3> 4

- 12 (-64-27)

Sex: M Age: 21-50 4 In Pt. Controls Sex: M Age: 24-44 de Barros11 Ch. Sch. Ferreira Sex: F et 01. (1973) Age: 25-63

5 Normals Sex: NS Age: 21-25

+

Yes

2 Nights

82 (51-1 27)

NS

NS

+39" (26-50)

+6.9% (5-10.2%)

+21 (-8-58)

Yes

2 Nights

+34 (9-69)

NS

NS

+1lC (-3-20)

+1.5% (0.4-2.1%)

4-13 (-29-31)

No, 3

3Nights

NS

About +7%# About +60

NS

About +5%d About + l o

No

3 Nights

NS

About8%

NS

About 59Ld About +15

Yes,8

About+40

~~

Values for change in sleep parameters were determined by subtracting baseline from data on first recovery night or full recovery period. Abbreviations: Ch. Sch. = chronic schizophrenic, M = Male, F = female, NS = not stated, In Pt. = psychiatric inpatient. Parenthesis = range. * Means for H nights. Means for 5 nights. Means for 3 nights. A fourth normal subject was studied for 2 nights of REM and NREM awakening. Nine subjects have been studied; data for 3 have been reported. In addition, data have not been published for one subject REMdeprived for 8 nights. v Barros-Ferreira data estimated from figures.

W

0

U

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REM deprivation. As stated in their article, Vogel and Traub based their calculations on the assumption that total REM minutes vary directly with total sleep time. Fifth, none of the published studies report an adequate statistical comparison between the schizophrenics and a control group. Vogel and Traub (1968) did not study control subjects (although they did compare their results with published reports). Azumi et a1. (1967) did not study enough patients or controls to make meaningful comparisons. Zarcone et al. (1968-1969), have not published statistical comparisons between the actively ill schizophrenics, remitted schizophrenics, and controls. Their published data reveal considerable variability in amount of REM compensation within the actively ill schizophrenic and control groups. Moreover, in the three studies employing control subjects, nonspecific factors such as age, sex, medication, anxiety, and psychiatric hospitalization were not controlled. Finally, some apparently normal subjects fail to show REM compensation following REM deprivation, as was discussed earlier. The evidence that schizophrenics fail to have a REM rebound following sleep or REM deprivation has been criticized. Vogel (1972), in particular, has stated that this proposition rests on three poorly supported propositions. First, since the proportion of REM increases as the night progresses (Verdone, 1968), Vogel suggests that schizophrenics in the waxing phase of a psychosis actually have as much REM sleep as would be expected given that total sleep time is curtailed. Examination of the data of Kupfer et al. (1970), however, suggests otherwise. Their patients had a mean actual sleep period of 235 minutes and a mean REM time of 21 minutes during the waxing phase. According to Verdone, normal subjects with a total sleep time of 240 minutes should have had 32 minutes of REM sleep (Table 11, Verdone, 1968), or an increase of about 50% over that of schizophrenic patients. Vogel also states that Feinberg et al. (1964) failed to find a significant difference in REM time between short-term schizophrenics and nonschizophrenic controls. Examination of the data, however, reveals that average REM time was 100 minutes for the nonschizophrenic controls and 73 minutes for the short-term schizophrenics (which is significant, p < 0.05, Mann-Whitney U test, 2-tailed test, according to the calculations based on the data in Fig. 3, Feinberg et al., 1964). As we indicated earlier, however, not all studies of the sleep of acute schizophrenics agree that REM is curtailed. The important issues, however, are not whether the proportion of total sleep spent in REM is reduced, but whether the actual number of minutes spent in REM is reduced, and, if so, whether there is a REM rebound following curtailment of REM sleep. Second, Vogel rightly questioned whether there is sufficient evidence from studies of the sleep of depressed patients to conclude that they fail to have a REM rebound during clinical recovery. He also asserted that other studies indicate that REM time

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decreases as depression lifts (Hartmann et al., 1966; Bunney et al., 1972). While we agree that no firm conclusions can be reached until more longitudinal sleep studies of depressed patients have been published, the data that do exist suggest that depressed patients have a REM rebound as they recover. Snyder (1969, 1972) has published data on two psychotic patients, not one as stated by Vogel (1972). In both there was a REM rebound. In the study by Hartmann, cited by Vogel, the patients were studied at infrequent intervals, so that no conclusions regarding dynamic changes in sleep over time can be made. In the study of Bunney et al., no data was presented concerning sleep as depression lifts. Third, Vogel states that the Zarcone et al. (1968) and Azumi et al. (1967) data actually refute the theory that schizophrenics differ from normals in REM rebound because he found no statistically significant differences between the schizophrenics and controls. This observation only indicates that these two investigators have failed to find a significant difference, not that their data refutes the theory. As we mentioned earlier, the number of patients reported in the two studies is so small that meaningful statistical comparisons are impossible. Feinberg (1969) has also criticized the studies by Azumi et al. and Zarcone et al. because the patients and controls were not well matched for baseline sleep characteristics, age, and anxiety : measurements of eye movement activity (phasic events) were not included; and drugs or prior somatic treatment might have influenced the response to REM deprivation. To turn to the longitudinal study of sleep in acute schizophrenia by Kupfer et al. (1970), conclusions regarding the REM compensation hypothesis are difficult to make since no detailed data were presented from the 2-week period between the end of the waning phase of the acute psychosis and the beginning of the postpsychotic phase. E. PURPOSEOF

THE

PRESENT STUDY

The purposes of this study are to reexamine whether schizophrenic patients fail to have a REM rebound, to control for some of the ambiguities of previous studies, and to point to new directions in the understanding of REM compensatory phenomena in general. There are four parts to the study. In the first part, in order to obtain more information regarding REM compensation during naturalistic observation, longitudinal sleep data are presented for 2 schizophrenics undergoing acute psychotic episodes. I n the second part, the results of a study are presented during which 8 actively ill schizophrenics and 8 nonpsychotic psychiatric patients were partially REM deprived by the awakening technique for 2 nights. In the third part, in order to provide more data regarding REM compensation in nonschizophrenic patients, changes in sleep are examined in 7 depressed patients fol-

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lowing withdrawal of a monoamine oxidase inhibitor, a drug that completely suppresses REM sleep. And, finally, to provide further control data for the longitudinal sleep studies during acute schizophrenic psychosis, the results are presented of a longitudinal sleep study during manic psychosis. IV. General Methods

Twenty-six psychiatric patients were studied on two psychiatric research wards on the Bethesda and St. Elizabeths Hospital campuses of the National Institute of Mental Health. The patients were referred by local private psychiatrists, mental health clinics, and psychiatric hospitals. Patients were admitted because they required psychiatric hospitalization for reasons of severe depression, inability to care for themselves, and disruptive, bizarre, or unpredictable behavior. Patients were excluded from admission if they had significant physical illnesses, epilepsy, organic brain syndromes, alcoholism, addictions, or narcolepsy. After a suitable verbal explanation of the purpose, nature, and dangers of the various studies, informed consent was obtained from each patient. The right to withdraw at any time was respected. Since sleep studies may be stressful even for normal individuals, a careful attempt was made to explain the nature of sleep studies before admission and to create an atmosphere on the ward in which sleep studies were an accepted part of the routine. All patients on the wards participated in longitudinal sleep studies throughout long portions of their hospitalization. The wards were designed with cables connecting a plug at the bedside of each patient to a central monitoring station so that minimum inconvenience was caused. During sleep recordings, electrodes were attached for recording of the electroencephalogram (EEG), electrooculogram, and electromyogram. Napping during the day was prohibited and was prevented whenever possible by other patients and by the nursing staff. The sleep records were scored in blind, random order according to standard criteria (Rechtschaffen and Kales, 1968). The number of minutes spent in total sleep (TS), REM sleep, stages I11 and IV, and NREM sleep were calculated. In addition, the following sleep parameters were derived: REM latency (RL, the minutes of sleep from sleep onset to the beginning of the first REM period) ;REM % = (REM/TS) X 100; REM density ( R D ) = average amount of eye movement activity during REM sleep, scored on a scale of 0-8 per minute of REM sleep. Each patient received a constant number of identical capsules so that medications could be substituted for placebo without knowledge of the patient or nurses (when part of the experimental protocol), Behavioral assessments were made daily by the nursing staff using a rating scale that has previously been shown to have high reliability (Wyatt and Kupfer, 1968)

.

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31 1

V. Study No. 1: Longitudinal Sleep Study in Acute Schizophrenia I n order to expand the data already published by Kupfer et a / . ( 1970), nightly sleep recordings were obtained from two acutely psychotic schizophrenic patients throughout most of their hospitalization. A. CLINICAL DESCRIPTION OF THE PATIENTS

Patient L, a 19-year-old male with a diagnosis of acute schizophrenic reaction, undifferentiated type, was admitted 6 weeks after he suddenly felt he had received a message from God that he had a special mission to accomplish. Prior to admission, he received chlorpromazine with limited clinical effect. He was not medicated while hospitalized. During early hospitalization, he had loosening of associations, delusions that he was Christ, and considerable anger toward his parents. Although he remained depressed, on three occasions his psychosis appeared to be remitting slowly only to reappear in heightened degree following difficult visits at home. During these periods of psychosis, he was agitated, confused, incoherent, delusional, angry, and, at times, hallucinating. He eventually recovered and was discharged to a job. Patient R, a 29-year-old male with the diagnosis of schizophrenic reaction, acute, schizoaffective type, was admitted 3 days after he suddenly became fearful, overly talkative, unpredictable, and delusional. About 10 days after admission, he became grossly psychotic, with tangential, rambling patterns of speech, word salad, ideas of reference, hyperactivity, preoccupation with sexual themes, and delusions that the F.B.I. was about to shoot him. After treatment with chlorpromazine, which was started with low nocturnal doses, he improved slowly, eventually making a good recovery. B. THERESULTSOF

THE

SLEEPSTUDIES

Sleep records were made during three psychotic episodes in these two patients, as shown in Figs. 1 and 2. During the psychotic phases, each patient showed insomnia, varying in degree with the severity of the illness. Patient R, for example, averaged only 104 minutes of total sleep per night during a 16-day period starting at the onset of his psychotic episode; during that time, average REM sleep was only 17 minutes per night. Even after he began to improve clinically, the tenuous nature of his sleep was evidenced by occasional nights of total insomnia. I n contrast, patient L, who was less severely disturbed, suffered lesser degrees of insomnia and loss of REM sleep.

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J . CHRISTIAN GILLIN AND RICHARD J . WYATT

Fro. 1. Patient R: rapid eye movement (REM) and non-REM sleep time (min. utea) and psychosis rating during hospitalization for acute schizophrenia.

As the patients improved clinically, both showed increases in total sleep, REM, and NREM. Neither patient, however, showed sustained elevations of REM sleep as he improved, although each had nights on which REM was elevated. Patient R, in particular, appeared to have more elevations of REM above 120 minutes per night and to improve more rapidly after chlorpromazine 400 mg was discontinued at 1O:OO PM and was given daily at 8:00 AM. Nevertheless, during the time that chlorpromazine was administered at 8:OO AM., mean REM was 96 minutes per night, well within normal limits. Patient L, as he was recovering from the second psychotic episode, had three nights during which REM varied from 122 to 138 minutes per night, but on no other occasion did he show sustained elevations of REM sleep.

C. COMMENT These results illustrate some of the difficulties in interpretation of longitudinal sleep studies. Since the REM rebound is defined as an increase of REM above the individual’s normal levels, it is necessary to know the normal level of REM in order to determine whether an individual has had a REM rebound or not. Determination of this normal level is difficult if not impossible, however, during a longitudinal sleep study. Sleep records

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313

Fro. 2. Patient L : rapid eye movement (REM) and non-REM sleep time (minutes) and psychosis rating during hospitalization. The psychosis rating scale was the same for acute schizophrenia employed in the other cases reported here, but the values were multiplied by one hundred to eliminate fractions.

obtained during nonpsychotic or remitted periods provide inadequate information about the patients’ natural level of REM sleep. Although the psychosis is remitted, the patient may continue to be depressed, anxious, or otherwise disturbed. Furthermore, adequate control data with which to compare these results does not currently exist. Few longitudinal studies of sleep in depressed patients have been published and no sleep studies have been done on normal individuals who have attempted to simulate the insomnia of an acute psychotic episode or of depression. With these limitations in mind, it does not appear that either patient had a marked or sustained elevation of REM following the insomnia of the acute psychotic episode. The sleep records from these two patients ap-

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pear to be similar to those reported by Kupfer et al. (1970) suggesting no immediate REM rebound during recovery from an acute psychotic episode. In contrast, results from longitudinal sleep studies suggest that depressed patients, even psychotically depressed patients, exhibit prolonged elevation of REM sleep as they recover (Snyder, 1968, 1969, 1972; Mendels and Hawkins, 1971). I t is possible, however, that REM compensation may occur at a later time in recovered schizophrenic patients, as suggested by the data of Gulevich et al. (1967).

VI. Study No. 2: Experimental REM Deprivation in Psychiatric Patients

Experimental REM deprivation is one approach to overcoming the limitations of the longitudinal method. In this study, we investigated the effects of 2 nights of experimental REM deprivation in 8 actively ill schizophrenics and 8 nonpsychotic psychiatric control patients. With this technique, sleep during the postdeprivation, recovery period was compared with sleep during the predeprivation, baseline period. Thus, it was possible to determine whether REM sleep increased above baseline levels during the recovery period following REM deprivation. Furthermore, in order to clarify some of the ambiguities in previous studies of experimental REM deprivation in schizophrenic patients, we attempted to study patients who were relatively acute and unmedicated and to include subjectively distressed patients of other diagnostic categories in our control group. A. METHODS The 16 psychiatric inpatients, admitted for treatment to two psychiatric research wards, were chosen for study on the basis of diagnosis, clinical status at the time of the study, and willingness to participate. The patients were clinically stable and cooperative at the time of the study. Diagnosis was agreed upon by 3 ward psychiatrists. Eight patients were assigned to the group of actively ill schizophrenics and 8 patients to the group of nonpsychotic patients. The determinations were always made prior to knowing the results of the REM deprivation. This assignment was based on a review of the patient’s course in the hospital, clinical observation by treating psychiatrists, nurses, and social workers, and, in most cases, an intensive mental status examination a day or two before the first night of REM deprivation. Clinical data on each patient are summarized in Table 11. The actively ill schizophrenic patients evidenced loosening of associations, inappropriate affect, bizarre behavior, delusions and hallucinations. The control patients showed no evidence of psychosis at the time of the study.

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TABLE I1 CLINICAL DATAFOR PATIENTS WHO WEREEXPERIMENTALLY REM-DEPRIVED" Patient

No. Age Sex

Diagnosis

Length of illness

Clinical condition a t time of study

Actively ill schizophrenics F

Chronic schizophrenia, undifferentiated

4 Months

24

F

Chronic paranoid schizophrenia

4 Years

40

F

Chronic schizophrenia, undifferentiated

1.5 Years

33

F

15

F

Acute schizophrenic reaction Schizophrenic reaction, undifferentiated

4 Months

20

M

Chronic schizophrenia, catatonic type

3 Years

23

M

Chronic schizophrenia, undifferentiated

2.5 Years

19

M

Chronic schizophrenia, undifferentiated

3 Years

23

1

2

3

4 5

6

7 8

1 Year

Marked thought disorder, auditory hallucinations, ideas of reference, delusions, anxiety, preoccupation. Autistic fantasy world. Auditory hallucinations unfavorable to herself, belief that thoughts were controlled or read by others. Aloof, hypochondriacal. Unpredictable outbursts of anger. Autistic fantasy world. Belief that thoughts were controlled by others. Tangential thinking, flattened affect. Delusional, thought disorder, flattened affect. Moderate depression. Flagrant thought disorder, autistic fantasy world. Auditory hallucinations, somatic delusions. Concrete thinking. Flattened affect with inappropriate grimacing and autistic laughter. Auditory hallucinations. Echolalia, occasional physical immobility. Intrusive thought disorder. Unrealistic fantasies. Inappropriate behavior. Anxiety, irritability. Marked thought disorder, inappropriate affect. Bizarre behavior and grimacing.

Nonpsychotic (control) patients

9 26

F

27

M

23

F

10

11

Postpartum psychosis, recovered (2 weeks)

6 Weeks

Transient paranoid reaction, recovered (6 days) Borderline personality, depressed

3 Days

1 Year

Moderately depressed. Concerned about marriage and future. Occasionally belligerent. No thought disorder. Anxious and concerned about what had happened to him. No thought disorder. Moderately depressed. Very anxious, fearful, withdrawn. Difficulty concentrating. Serious suicide attempt 2.5 months later. (Continued)

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TABLE I1 (Continued) Patient No. Age Sex 12

Diagnosis

Length of illness

F

Hysterical personality, depressed

1

21

F

Manic-depressive, depressed

1 Year

22

F

34

M

24

M

Acute schizophrenic reaction, recovered (1 month) Chronic schizophrenic, undifferentiated, in remission Chronic paranoid schizophrenia, in remission

2 Months

28

13

14

15

16

(I

14 Years

3 Years

Clinical condition at time of study Depressed, dependent, presenting self as needy. Frequent anxiety attacks with dyspnea, crying. Retarded depression. No evidence of thought disorder. Five months later, sudden switch into mania. Mildly depressed. No evidence of thought disorder. Chlorpromazine, 100 mg t.i.d. Moderately depressed. No evidence of thought disorder. Awaiting discharge. Anxiety. No evidence of delusions or thought disorder. Awaiting discharge.

From Gillin ct 01. (1974).

There were five females and three males in each group of patients. The average age was 25 for the actively ill schizophrenic group and 26 for the control group. Two of the actively ill schizophrenic patients (patients 7 and 8) and 5 of the control patients (patients 9, 11, 13, 15, and 16) had had a previous psychiatric hospitalization. Except for control patient 14, none of the patients received psychotropic medication or drugs to induce sleep for at least 3 weeks prior to the beginning of the study. Patient 14 received chlorpromazine 300 mg/day before and during the study. Following a t least two adaptation nights, each patient was then studied for 9-11 consecutive nights: Baseline (4nights apiece for 14 of the subjects, 2 nights apiece for control subject 16 and for experimental subject 7 ) , 2 nights of partial REM sleep deprivation (D1and D2)and 5 nights of recovery sleep (R,-5). Subjects were encouraged, but not forced, to retire and arise at specific times. REM deprivation was accomplished by awakening the subject as soon as REM sleep was identified by polygraphic recording, usually with the first eye movement. Subjects were kept awake about 4 minutes before being allowed to return to sleep. For each patient, the mean baseline value for each parameter of sleep was calculated: total sleep, REM, REM o/o, REM latency, early REM, REM density, and stages I11 and IV. Early REM is the number of minutes of REM sleep during the first two REM periods, a figure chosen because each patient had at least two REM periods on each night of the study.

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Change (A) from baseline means were derived for each night of REM deprivation and recovery sleep by subtracting mean baseline sleep data for each parameter of sleep from the corresponding values on D1, D2, R1, R2, R3, R4, and R5. The baseline sleep data and the change from baseline sleep data for each group of patients were analyzed by student t tests and by multivariate analysis of variance (Finn, 1970). The recovery night data was also subject to analyses of covariance and to a principal components analysis. For each patient, data from each of the 7 sleep parameters from each of the 5 recovery nights were entered into the multivariate analysis of variance in the following order: A REM, A REM %, A total sleep, A REM latency, A early REM, A REM density, and A stages I11 and IV.

B. RESULTS 1. Baseline Period

During the baseline period, the actively ill schizophrenics and the control subjects had similar sleep patterns (Table 111).When the seven sleep variables (REM, REM %, total sleep, REM latency, early REM, REM density, and stages I11 and IV sleep) were tested, no statistically significant difference was found between the two patient groups. The only comparison which approached significance was early REM, the amount of REM in the first

TABLE 111

BASELINESLEEPDATA:ACTIVELYILL SCHIZOPHRENIC PATIENTS AND CONTROL PATIENTS' Subjects Actively ill schizophrenics Controls

REM

REM (%)

TS

RL

93 f 25 99 f 10

25 f 7.8 25.5 f 2.7

378 f 37 390 f 45

72 f 19 78 f 36

Subjects

REM D

ER

111 + I V

NREM

Actively ill schizophrenics Controls

1.8 f 0.9 1.9 f 0.8

42 f 14 54 f 12

37 f 33 32 f 27

284 f 49 291 f 41

Data are in minutes per night, except for REM % and REM D (the REM density). Values are expressed as mean f SD. From Gillin ef 01. (1974).

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two REM periods of the night ( p < 0.09,F = 3.367, df = 1, 14).To test the possibility that a multivariate pattern of sleep differences might be found between the two groups, a one-way multivariate analysis of variance using the seven variables mentioned above was done; a significant group difference was not found (p < 0.8,F = 0.531,df = 7, 8).

2. R E M Deprivation Period During the 2 nights of REM deprivation, the actively ill schizophrenics and control patients were deprived of similar amounts of REM sleep. The actively ill schizophrenic patients averaged 26.2 minutes REM per night and the control subjects averaged 26.5 minutes REM per night on each of the two nights of the REM deprivation. Actively ill patients lost an average of 71.8% of baseline REM on each deprivation night and the control patients lost an average of 72.8%. This is approximately the same level of REM deprivation achieved by other investigators (Vogel and Traub, 1968; de Barros-Ferreira et al., 1973; Azumi et al., 1967; Zarcone et al., 1968). Figure 3 illustrates that on the two REM deprivation nights the two groups were quite similar with respect to changes relative to baseline in REM time, REM %, total sleep, REM latency, REM density, and stages I11 and IV. In order to achieve these levels of REM deprivation, the control group had to be awakened an average of 17.5 times per night whereas the actively ill group had to be awakened only 14.0 times per night ( p 0.05,one-tailed t test). The two groups were awakened with similar frequency on the first deprivation night (Fig. 4 ) . On the second REM deprivation night, however, the control subjects entered REM significantly more frequently than the actively ill schizophrenics (19.8compared with 13.6 awakenings, p < 0.01). The control patients averaged 4.5 more awakenings on the second night than on the first, whereas the actively ill schizophrenic patients actually required slightly fewer awakenings on the second night than on the first,