International Review of Neurobiology Volume 15

INTERNATIONAL REVIEW OF Neurobiology VOLUME 15 Associate Editors W. Ross ADEY SIR JOHNECCLES D. BOVET H. J. EYSE...

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INTERNATIONAL REVIEW OF

Neurobiology VOLUME 15

Associate Editors

W. Ross ADEY

SIR JOHNECCLES

D. BOVET

H. J. EYSENCK

WILLIAMF. BRIDGERS

C. HEBB

Josh DELGADO

0. ZANCWILL

Consultant Editors V. AMASSIAN

K. KILLAM

MURRAYB. BORNSTEIN

C. KORNETSKY

F. TH. BRUCKE

A. LAJTHA

P. DELL

B. LEBEDEV

J. ELKES

SIR AUBREYLEWIS

W. GREYWALTER

VINCENZOLONCO

R. G. HEATH

D. M. MACKAY

B. HOLMSTEDT

STEN

P. A. J.

F. MORRELL

JANSSEN

S. KETY

MKRTENS

H. OSMOND STEPHENSZARA

INTERNATIONAL REVIEW OF

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

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

VOLUME 15

1972

ACADEMIC PRESS

New York and London

COPYRIGHT 0 1972, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. N O 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 F R OM THE PUBLISHER.

ACADEMIC PRESS, INC.

111 Fifth Avenue, New York, New York 10003

United Kingdom Edition published by ACADEMIC PRESS, INC. (LONDON) LTD. 24/28 Oval Road, London NWl

LIBRARY OF

CONGRESS C A T h L O o CARD

NUMBER: 59 - 13822

PRINTED IN THE UNITED STATES OF AMERICA

CONTENTS CONTRIBUTORS

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Projection of Forelimb Group I Muscle Afferents to the Cat Cerebral Cortex

INGMAR ROS~N I. Introduction . . . . . . . . . . . I1. Course and Relay Nuclei of the Pathway . . . . . I11. Cortical Neurons . . . . . . . . . . IV . Cortical Projection Areas . . . . . . . . V . Identification of Receptors . . . . . . . . VI . Synaptic Properties . . . . . . . . . VII . Spatial Patterns of Group I Convergence at Different Levels of . . . . . . . . . . . Pathway . VIJI . Convergence of Excitation from Other Types of Afferents . . IX. Afferent Inhibition . . . . . . . . . X . Cortical Control of Transmission . . . . . . . XI . Comparative Aspects . . . . . . . . . XI1 . Functional Considerations . . . . . . . . References . . . . . . . . . . .

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10 12 14 15 18 20 21

Physiological Pathways through the Vestibular Nuclei

VICTORJ . WILSON

I . Introduction . . . . . . . . . . . I1. Labyrinthine Input to the Vestibular Nuclei . . . . 111. The Vestibular Nuclei and the Cerebellum . . . . IV . Projections of the Vestibular Nuclei to the Spinal Cord . . V. Projections of the Vestibular Nuclei to Extraocular Motoneurons VI . Concluding Remarks . . . . . . . . . References . . . . . . . . . . . Note Added in Proof . . . . . . . . .

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Tetrodotoxin. Saxitoxin. a n d Related Substances: Their Applications in Neurobiology

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MARTIN H EVANS I . Introduction . . . . . . . . I1 Sources of the Toxins . . . . . . 111. Chemical and Physical Properties of the Toxins . IV . Assay Methods . . . . . . . V. Actions in Vitro . . . . . . . VI Actions in Vivo . . . . . . . VII . Summary and Concluding Remarks . . . References . . . . . . . . Note Added in Proof . . . . . .

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83 84 89 94 108 141 152 155

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vi

CONTENTS

The Inhibitory Action of y-Aminobutyric Acid.

A Probable Synaptic Transmitter KUNIHIKOOBATA

I . Introduction . . . . . . . . I1. Inhibitory Synaptic Transmission . . . . I11. Methods of Drug Administration . . . . IV. Electrical Changes in Neuronal Membranes . V . Antagonism b y Convulsants . . . . . VI . Chemistry of Synaptic Inhibition . . . . VII . Conclusion . . . . . . . . References . . . . . . . .

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167 168 171 173 178 181 183 184

Some Aspects of Protein Metabolism of the Neuron

MEI SATAKE I . Introduction . . . . . . . . . . . I1. Protein Metabolism in the Nerve Cell Perikaryon . . . I11. Protein Metabolism in the Axon . . . . . . . IV . Protein Metabolism in Nerve Endings . . . . . . V Protein Breakdown in the Neuron . . . . . . VI . Neuro-Neuronal and Neuro-Nonneuronal Transfer of Proteins VII . Proteins Specific to Neurons and Their Metabolism . . . VIII Neuronal Protein Metabolism Related to Functional Activity . IX . Conclusions . . . . . . . . . . . References . . . . . . . . . . .

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189 190 200 202 202 203 203 205 207 208

Chemistry and Biology of Two Proteins. 5-100 and

14.3.2. Specific to the Nervous System BLAKEW. MOORE I . Introduction . . . . . . . . . . 11. Preparation of Nervous System Proteins . . . . I11. Immunological Assays for Brain Proteins . . . . IV . Species Distribution of S-100 and 14-3-2 . . . . V . Distribution and Localization within the Nervous System . VI . Developmental Studies . . . . . . . . VII . Turnover Studies of $100 . . . . . . . VIII . Chemistry of S-100 . . . . . . . . IX . Function of S-100 . . . . . . . . . References . . . . . . . . . .

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I . Introduction . . . . . . . . . . . . I1. Scope of This Review . . . . . . . . . . I11. Unitary Sources of the EEG . . . . . . . . . IV . Site of Production of Wave Activity in Cortical Nerve Cells . . V . Biophysical Factors in Summation of Unitary Neuronal Waves .

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215 217 217 219 219 221 221 222 223 224

The Genesis of the EEG

RAFAELELUL

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228 228 230 24 1 245

vii

CONTENTS

. . . VI . Theoretical Analysis of System of Unitary Generators . VII . Experimental Analysis of Population Behavior of Cortical Wave Generators . . . . . . . . . . . . . VIII . Subcortical Control of the EEG and Possible Functional Implications . IX . Conclusion and Consequences for Evaluation of Gross EEG Activity . References . . . . . . . . . . . . .

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255 264 267 270

Mathematical Identification of Brain States Applied to Classification of Drugs

E. R.

JOHN.

P . WALKEH. D . CAWOOD. M . RUSH.AND J . GEHRMANN

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AUTHOR INDEX.

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SUBJECTINDEX.

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366

CONTENTS OF PREVIOUS VOLUMES.

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36Y

I . Introduction I1. Methods . I11. Results . IV . Discussion References

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273 276 284 339 346 349

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

D. CAWOOD, Department of Psychiatry, New York Medical College, New York, New York (273) RAFAEL ELUL,Department of Anatomy and Brain Research Institute, School of Medicine, University of Californiu at Los Angeles, Los Angeles, California (227) MARTIN H. EVANS,Agricultural Research Council, Institute of Animal Physiology, Babraham, Cambridge, England ( 8 3 )

J . GEHRMANN, Department of Psychiatry, New York Medical College, New York, New York (273) E. R. JOHN, Department of Psychiatry, New York Medical College, New York, New York (273) BLAKEW . MOORE,Department of Psychiatry, Washington University School of Medicine, St. Louis, Missouri (215 ) KUNIHIKOOBATA,Department of Pharmacology, Faculty of Medicine, Tokyo Medical and Dental University, Tokyo, Japan (167) INGMARROSBN, The Institute of Physiology, University of Lund, Lund, Sweden ( 1 )

M. RUSH, Department of Psychiatry, New York Medical College, New York, New York (273)

MEI SATAKE,Department of Neurochemistry, Brain Research Institute, Niigata University, Niigata, Japan ( 189) P. WALKER, Department of Psychiatry, New York Medical College, New York, New York (273) VICTOR J . WILSON, The Rockefeller University, New York, Netc York (27)

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PROJECTION OF FORELIMB GROUP I MUSCLE AFFERENTS TO THE CAT CEREBRAL CORTEX By lngmar R o s h The Institute of Physiology, University of Lund, Lund, Sweden

I. 11. 111. IV. V. VI. VII. VIII. IX. X. XI.

XII.

Introduction . . . . . . . . . . Course and Relay Nuclei of the Pathway . . . . Cortical Neurons . . . . . . . . . Cortical Projection Areas . . . . . . . Identification of Receptors . . . . . . . Synaptic Properties . . . . . . . . Spatial Patterns of Group I Convergence at Different Levels of the Pathway . . . . . . . . Convergence of Excitation from Other Types of Afferents Afferent Inhibition . . . . . . . . . Cortical Control of Transmission . . . . . . Comparative Aspects . . . . . . . . A. Hindlimb Pathway in Cat . . . . . . B. Group I Projection in Monkey . . . . . Functional Considerations . . . . . . . References . . . . . . . . . .

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I. Introduction

Information from muscle stretch receptors would seem important for integration of movements in the sensorimotor cortex. It is now well known that information from primary muscle spindle afferents and tendon organ afferents reaches the cerebellar cortex via various paths (Lundberg, 1964; Oscarsson, 1965; J. K. S. Jansen and Wallpre, 1969). On the other hand, earlier studies suggested that such information is not available to the sensorimotor cortex (Mountcastle et al., 1952; McIntyre, 1953, 1962a; Kruger, 1956). However, a brief report by Amassian and Berlin (1958) indicated that group I afferents originating from slowly adapting stretch receptors in the forelimb do project to the sensorimotor cortex. This observation was confirmed by Oscarsson and RosCn (1963) who, in addition, demonstrated that the forelimb group I path is activated by primary muscle spindle afferents and projects through the dorsal funiculus-medial leniniscus system to a small area located rostra1 to the postcruciate dimple in the posterior sigmoid gyrus. Since then a 1

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number of investigations have given a detailed description of the anatomical and functional organization of the synaptic relays in the cuneate nucleus and the thalamus as well as in the cortical projection area. Later investigations demonstrated that the same path projects to additional cortical areas, and a cortical projection of group I hindlimb afferents has also been discovered. Furthermore, observations on the monkey show that information from group I afferents also reaches the cerebral cortex. The organization of these pathways will be discussed in less detail on p. 19. II. Course and Relay Nuclei of the Pathway

A schematic diagram of the forelimb group I afferent pathway is shown in Fig. 1A. Although the group I path and the cutaneous path in the dorsal funiculus-medial lemniscus system follow the same general course to the sensorimotor cortex a separation between them is maintained at all levels including the cortical projection areas (Fig. 1B) (Oscarsson and Roshn, 1963, 1966; Mallart, 1964, 1968; Anderson et al., 1966; Landgren et al., 1967a; Roshn, 1969a,b). Three separate groups of neurons, monosynaptically activated by the ascending group I afferents, were identified by systematic recording from the dorsal column nuclei and the rostral cervical cord. The secondorder neurons of the group I pathway to the cerebral cortex ( 2 in Fig. 1A) were identified as cuneothalamic relay cells by antidromic stimulation of the medial lemniscus. They were localized in the deep part of the main cuneate nucleus (MCN of Fig. 1A) ventral to the nuclear region which is occupied by relay cells activated by cutaneous afferents (Roshn, 1969a,b). No group I-evoked responses were found in the most rostral part of the nucleus. Within the group I relay nucleus a slight tendency was found for afferents from proximal muscles to evoke maximal potentials ventral to the focus for distal muscle afferents but there was considerable overlap. These observations add to the picture of the dorsal column nuclei as highly differentiated in terms of modality segregation both in the dorsoventra1 and rostrocaudal directions (cf. Gordon and Jukes, 1964). In the ventral part of the cuneate nucleus the group I cuneothalamic cells Iie intermingled with cells which are excited from the cerebral cortex and which presumably do not send their axons through the medial lemniscus (Andersen et al., 1964c; RosBn, 1969b). The ventral part of the main cuneate nucleus differs anatomically from the more dorsal parts. It contains smaller and more multiform cells and receives a heavier termination of corticofugal fibers (Ram6n y Cajal, 1952; Walberg, 1957; Kuypers and Tuerk, 1964). Monosynaptic group I-evoked activity was found in the external

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GROUP I PATHWAY TO CEREBRAL CORTEX

\

POSTCRUCIATE

\ A

G&)q ! 1ECN

q

P-

B

IAL

FIG. 1A: Diagram of the forelimb group I afferent pathway to the cerebral cortex. The primary afferents ( 1) ascend in the dorsal funiculus to relays in the base of the dorsal horn of the rostra1 cervical spinal cord, the external cuneate nucleus ( E C N ) , which projects to the cerebellar cortex, and the ventral part of the main cuneate nucleus ( MCN ) which projects through second-order neurons ( 2 ) to the nucleus ventralis posterolateralis (VPL) of the thalamus (THAL). The thirdorder neurons ( 3 ) of the pathway project to the cortex. A hypothetical diagram of the neuronal interconnections in the region of the postcruciate dimple of the cerebral cortex is also shown (Oscarsson et ol., 1966; Grampp and Oscarsson, 1968) with fourth- (4a,b) and fifth-order ( 5 ) neurons of the pathway indicated. Inhibitory neuron black. PT indicates a pyramidal tract cell. B: Short latency cortical projection areas related to cutaneous (hatched) and group I muscle (stippled) afferents in the forelimb (Oscarsson and RosBn, 1966; Landgren et al., 1967a; Silfvenius, 1968). The ansate, coronal, cruciate, and suprasylvian sulci, and the postcruciate dimple are indicated. The thinner lines indicate the cytoarchitectonic fields, delimited by Hassler and Muhs-Clement ( 1964 ).

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ROSBN

cuneate nucleus (ECN of Fig. 1 A ) which projects to the cerebellar cortex through the cuneocerebellar tract ( J. Jansen and Brodal, 1958; Grant, 1962), the forelimb equivalent of the dorsal spinocerebellar tract (Holmqvist et al., 1963). The external cuneate nucleus was shown to be somatotopically organized with distal forelimb afferents projecting to medial parts and proximal afferents to more lateral parts of the nucleus (RosBn, 1969a; Cooke et al., 1971a). No activity was evoked in this nucleus by cutaneous afferent volleys, implying a separate relay nucleus for the component of the cuneocerebellar tract which is activated by cutaneous and high-threshold muscle afferents ( cf. Holniqvist et al., 1963). These cells have recently been localized in an adjacent part of the main cuneate nucleus (Cooke et al., 1971a). A third group of cells activated from group I forelimb afferents was encountered in the base of the dorsal horn of the rostral cervical cord (Fig. 1 A ) . These neurons were activated almost exclusively by afferents from distal muscles and were further characterized by monosynaptic excitation both from cutaneous and group I afferents (RosBn, 1969b). Although the axonal termination of these neurons is unknown, their localization to the medial part of Rexed's lamina VI is of interest since at the level of the first cervical segment the cuneate nucleus appears as a small cell aggregate in intimate contact with this zone (Rexed, 1954). Accordingly there appears to be a continuous column of cells activated from group I afferents throughout the caudal medulla and the rostral cervical cord. In the thalamus a narrow group I-activated zone was localized in the rostral two-thirds of nucleus ventralis posterolateralis ( VPL, Fig. 1A) dorsomedial to the area activated by cutaneous afferents (Mallart, 1964, 1968; Andersson et al., 1966).In this zone the third order, thalamocortical neurons of the group I afferent pathway ( 3 of Fig. 1A) were identified by antidromic excitation from the cerebral cortex ( Andersson et al., 1966; RosBn, 1 9 6 9 ~ ) . Ill. Cortical Neurons

The organization of cortical neurons influenced by volleys in group I afferents was studied with intracellular and extracellular recording technique. The investigations published so far have been concerned with neurons in the group I projection area in the posterior sigmoid gyrus rostral to the postcruciate dimple (Fig. 1B) (Oscarsson et al., 1966; Swett and Bourassa, 1967a; Grampp and Oscarsson, 1968). From the surface of the projection area, a group I-evoked thalainocortica1 volley could usually be recorded as a remarkably synchronous spikelike

GROUP I PATHWAY TO CEREBRAL CORTEX

5

potential which preceded the large cortical potential (Oscarsson and Rosen, 1963). The latencies of the responses evoked in cortical neurons could be measured relative to this volley, permitting identification of fourth- and fifth-order neurons of the pathway. The interconnections of cortical neurons have been analyzed by stimulation of cortical afferent and efferent fibers in the white matter below the cortex of the projection area (Grampp and Oscarsson, 1968). Group I-influenced cells were usually distributed 500-1500 ,U below the cortical surface with maxiniuni at about 1000 p (Oscarsson et al., 1966). This is consistent with the termination of specific thalamic afferents in cortical layer IV and in the deep part of layer I11 (Lorente de N6, 1949; Nauta, 1954). The vast majority of the cells which were activated by group I afferent volleys had a latency indicating a monosynaptic linkage with the thalainocortical fibers ( Oscarsson et al., 1966). It can be concluded that these cells belong to the fourth-order neurons in the group I projection system (4a,b of Fig. 1 A ) . In a few of the cells the e.p.s.p.’s were followed by i.p.s.p.’s after latencies which indicated a disynaptic linkage with the thalamocortical fibers. A second class of group I-influenced neurons received only inhibition which appeared after a latency, indicating a disynaptic linkage with the thalamic fibers. These cells constitute fifth-order neurons of the pathway (5 or Fig. 1 A ) . These observations indicate that at least some of the fourth-order neurons in the cortex are inhibitory (413 of Fig. 1A) (Oscarsson et al., 1966; Grampp and Oscarsson, 1968) . The group I-activated cortical neurons were not antidromically activated on stimulation of the contralateral pyramidal tract in the upper cervical region ( Oscarsson et al., 1966). However, group I-activated cells aiitidromically invaded from the pyramidal tract were reported by Swett and Bourassa (1967a) although some uncertainty is introduced by the relatively high stimulus strength used for identification of group I-activated cells (1.7 times the nerve threshold for the deep radial nerve) (cf. Roskn, 1969b). Local stimulation below the cortical area produced antidromic activation of only 5 out of 51 intracellularly recorded group I-activated cells (Grampp and Oscarsson, 1968) (4a of Fig. 1 A ) . In some of these neurons, antidromic invasion might have been blocked because of depolarization after impalement, but their results suggest that many of the neurons terminate intracortically. None of the exclusively inhibited neurons in this investigation was antidromically invaded, suggesting that they terminate intracortically. The finding that the majority of the group I-influenced cells terminate intracortically raises the question of how the group I-information leaves the projection area. It was suggested (Grampp and Oscarsson, 1968) that this occurs through

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INGMAR ROSEN

a modulating influence upon the activity of other types of neurons in the same cortical area. A third group of neurons (encountered in the group I projection area) was activated by volleys in cutaneous afferents and high-threshold muscle afferents (flexor reflex afferents, FRA) but was not influenced by group I afferent volleys (Oscarsson et al., 1966; Grampp and Oscarsson, 1968). These cells occurred at greater depths than the group Iactivated neurons. They could all be antidromically activated from the underlying white matter (Grampp and Oscarsson, 1968) and at least some of these cells send their axons through the corticospinal tract (Oscarsson et al., 1966; PT of Fig. 1A). It was suggested that the group I-influenced cells exert a modulating influence upon the cortical reflex system made up by the FRA-activated cells and their afferents. The group I-inhibited neurons described above might be the link between the two systems. They were not spontaneously active under barbiturate anaesthesia and could not be expected to produce any postsynaptic effects (Grampp and Oscarsson, 1968). A reinvestigation of the descending and intracortical connections of the cortical group I-influenced neurons on a preparation having a high level of spontaneous activity would be of interest. IV. Cortical Projection Areas

Forelimb group I afferent vollcys evoked large surface positive potentials in the sensorimotor cortex with a focus just rostral to the postcruciate dimple (Oscarsson and RosCn, 1963, 1966, Fig. 1 B ). Small but distinct potentials could also be recorded in a tongue of cortex extending rostrolaterally toward the lateral end of the cruciate sulcus. The surface potentials evoked from nerves of muscles acting at the wrist and digital joints occurred slightly lateral to those evoked by nerves innervating muscles at the elbow and shoulder joints, although the overlapping area was large (Oscarsson and RosBn, 1963; Oscarsson, 1966; Oscarsson et al., 1966). The pre-dimple group I projection area was distinctly separated from the two primary projection areas for cutaneous afferents which were situated rostrally and caudally to the group I area (Oscarsson and Roskn, 1966, Fig. 1 B). The cortical cytoarchitectonic fields delimited by Hassler and MuhsClement (1964) are shown in Fig. 1B in relation to the various projection areas. The caudal cutaneous projection in the first somatosensory area corresponds to the classical sensory cortex (fields 1, 2, and 3b), whereas the rostral cutaneous area lies mainly in the motor cortex (field 4). The pre-dimple group I projection area corresponds to field 3a and a caudal part of field 4 with the focus in field 3a. Field 3a is a

GROUP I PATHWAY TO CEREBRAL CORTEX

7

transitional zone between sensory and motor cortexes; it consists of granular .cortex but also contains some giant pyramidal cells ( Hassler and Muhs-Clement, 1964). From Nauta-Gygax studies after small cortical lesions, Jones and Powell have suggested that field 3a has intracortical commissural and thalamic connections so similar to those of the architectonic subdivisions of the first somatosensory area proper that it might constitute part of this area. Some uncertainty is, however, introduced by the fact that the lesions were not small enough to be confined to single architectonic fields (Jones and Powell, 1968a,b,c), Similar detailed studies with minimal lesions based on electrophysiological identification of projection areas would be of great interest. Within the pericruciate cortex two additional forelimb group I projections have been reported briefly ( Silfvenius, 1968). One of these lay in the rostra1 bank of the ansate sulcus, i.e., in the sensory cortex (field 2, see Fig. 1B) and the other, possibly indirect, projection lay in the forelimb motor cortex (field 4 ) . The latter projection is presumably the same as was earlier described as a rostrolateral extension from the pre-dimple area into the motor cortex (Oscarsson and Roskn, 1963, 1966). Field 3a has somatotopically organized connections with the motor cortex (field 4) (Jones and Powell, 1968a). This was recently supported by cortical microstimulation technique ( Thompson et al., 1970). The type of information forwarded by the connecting neurons remains unknown but the observed group I-evoked surface potentials in the forelinib motor cortex ( Oscarsson and Rositn, 1966; Silfvenius, 1968) might reflect cortically relayed group I-evoked activity in this area. With natural stimulation, neurons in the motor cortex have been shown to respond to joint movements but the receptors responsible were not identified (Welt et al., 1967; Asanuma et al., 1968, Sakata and Miyamoto, 1968). These effects might be evoked in part by group I muscle afferents. Recent experiments in baboon suggest, however, that group I-evoked activity is not directly transmitted to the motor cortex via a relay in area 3a (Phillips et al., 1971). Although group I-evoked potentials could not be recorded from the outer cortical surface in the region of the second somatosensory area, it was possible to invade group I-activated thalamic neurons antidromically by stimulation of the cerebral cortex in the region of the anterior suprasylvian sulcus as well as the postcruciate diniple (Anderson et al., 1966; Roskn, 1 9 6 9 ~ ) .Most of the neurons projecting to the suprasylvian sulcus could also be invaded from the diinple region, which indicates axonal branching. The group I projection in the second somatosensory area was localized in the lower bank of the suprasylvian sulcus (Fig. 1 R ) where it lies surrounded by cortical areas activated

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INGMAR ROSEN

by cutaneous, vestibular, and cochlear afferents in a pattern of overlapping but not identical fields (Landgren et al., 1967a,b). The existence in the sensorimotor cortex of multiple small cortical areas which receive a primaiy projection from the same type of receptor has been demonstrated not only for the group I afferents but also for joint and Pacinian receptors ( Korner and Landgren, 1969; Silfvenius, 1970). Presumably the thalamocortical axons branch to project to these various spots. A similar axonal branching has been shown among afferents to the cerebellar cortex (Oscarsson and Uddenberg, 1964; Armstrong et al., 1969). The branching axons provide the same kind of information to several cortical loci which presumably represent different cortical control and motor mechanisms. V. Identification of Receptors

The observation ( Oscarsson and RosBn, 1963) that the lowest threshold forelimb muscle afferents effectively contribute to the evoked cortical response suggests that primary muscle spindle afferents are responsible (Bradley and Eccles, 1953; Eccles et al., 1957; Laporte and Bessou, 1957; Coppin et nl., 1969). However, the separation of the group I afferent volley into a Ia component, originating from primary endings in muscle spindles, and a Ib component, originating from Golgi tendon organs, which was described for hindliinb muscle nerves, cannot be attained for distal forelimb nerves. Furthermore, the degree of admixture in the forelimb group I component of afferents from other receptors was unknown. Natural stimulation of receptors was necessary to investigate these problems in detail. The contribution of primary muscle spindle afferents to thc cerebral projection pathway was demonstrated by the following experiments (Oscarsson and Roskn, 1963). (1) A short pull to a forelimb muscle tendon, which presumably activated all group I spindle afferents but no tendon organ and very few group I1 spindle afferents, evoked a cortical potential after a short latency. ( 2 ) By using the fact that a continuous barrage of impulses in the projection path depresses the cortical potential evoked by nerve stimulation, it was shown that the receptors responsible had low threshold and slow adaptation to stretch with a marked dynamic response and that they were activated by succinyl choline. These expcrinients indicate that the pathway is activated by primary spindle afferents but they do not exclude the possibility that tendon organ afferents also project to the cerebral cortex. By recording from primary afferent fibers in the deep radial nerve which was left in continuity with four muscles, primary spindle afferents and tendon organ afferents were identified with natural stim-

GROUP I PATHWAY TO CEREBRAL CORTEX

9

ulation (stretch and contraction), succinyl choline injection, and sinusoidal longitudinal vibration ( cf. Matthews, 1964; Brown et al., 1967). Both types of afferents were shown to ascend through the dorsal funiculus to the rostra1 part of the cervical spinal cord. Thirty-four identified group I cuneothalamic cells (second-order neurons of the pathway) all responded to natural stimulation as if they had been activated exclusively from spindle affercnts (Roskn and Sjiilund, 1969, 1972a). The absence of group I relay cells that forwarded information mainly from tendon organs suggests that the forelimb group I path to the cerebral cortex is a pure spindle afferent pathway, The tendon organ afferents shown to ascend in the dorsal funiculus activate cells in the external cuneate nucleus and “non-relay cells” in the main cuneate nucleus. VI. Synaptic Properties

Like other projection pathways to the somatic cortical areas (Amassian, 1951; Mark and Steiner, 19S8; McIntyre, 1962b, Oscarsson and RosBn, 1966; cf. Hensel and Boman, 1960; Bourassa and Swett, 1967; McIntyre et al., 1967) the amount of spatial summation needed for evoking a discharge in the group I pathway is very small. This was demonstrated by the steep initial rise of the input-output curves constructed for the evoked mass discharge recorded from the medial lemniscus, the thalamocortical fibers, and the cerebral cortex ( Oscarsson and Roskn, 1963). Action potentials from cuneothalamic and thalaniocortical neurons and synaptic potentials from the cortical neurons could be recorded with stimulation of the nerve at, or just above, threshold indicating that activation of only a few primary affereiits is required for impulse transmission in the pathway (Oscarssoii et al., 1966; Roskn, 1969b,c). Large unitary e.p.s.p’s were observed both among the secondorder cuneate cells and the fourth-order cortical cells of the group I afferent pathway, and at both these levels the compound e.p.s.p.’s evoked by maximal afferent volleys were often formed by a relatively small number of unitary e.p.s.p.’s (Oscarsson et al., 1966; Grampp and Oscarsson, 1968; I. Roskn and B. Sjiilund, 1970, unpublished data). Unitary e.p.s.p.’s have not been studied in the group I-activated thalaniic cells but in other ventrobasal thalamic neurons large unitary potentials havc been observed (Anderson et a?., 1964e, 1966; Maekawa and Purpura, 1967). The group I-activated cuneothalamic cells often followed high frequencies of nerve stimulation for long periods. Almost half of the group I-evoked responses tested followed stimulus frequencies above 200 Hz ( R o s h , 196913). The responses of the cuiieatc neurons to muscle stretch of moderate amplitudes varied within the same frequency range as those of the primary afferent fibers ( R o s h and Sjdund, 1972a). Like

10

INGMAR R O S ~ N

the dorsal spinocerebellar neurons, the group I cuneothalamic relay cells seem well adapted to transfer signals over a wide frequency range (cf. Jansen et al., 1967b; Jansen and Wallge, 1969). Recurrent inhibition does not appear to play a significant role in the transmission through the dorsal column nuclei (Gordon and Paine, 1960; 1. RosCn, 1969, unpublished data). When tested by repetitive stimulation of nerves, the ability to transmit impulses at high frequencies is much less for the group I thalaniocortical and cortical neurons than for the cuneothalamic neurons ( Oscarsson and RosBn, 1963). This is presumably explained by recurrent inhibition in the thalamus evoked by the synchronous group I volleys (cf. Andersen et al., 1964e). The ability of high-frequency impulse transmission in these neurons during natural stimulation has not yet been studied. Many cuneothalamic relay cells responded to a group I afferent volley with high-frequency discharge consisting of two or three action potentials, whereas the thalainocortical neurons of the pathway responded with only one or sometimes two action potentials, and occasionally with a burst of a larger number of impulses ( RosCn, 1969b,c). The latencies of the responses in the thalamic cells often showed stepwise fluctuations. The distribution of latencies was bimodal, with one peak corresponding to a monosynaptic connection with the lemniscal fibers and the other peak about one millisecond later. The latency fluctuations and the longer latencies of the thalamic neuronal discharges are presumably explained by a summation of e.p.s.p.’s evoked by the consecutive volleys of impulses in the presynaptic cuneothalamic fibers. If this factor has any significance under natural conditions, high-frequency components of the muscle spindle responses would be transmitted to the cortex with a specially high efficacy. On natural stimulation many group I-activated cortical cells responded with only a transient discharge to muscle stretch of low amplitude whereas larger loads on the muscle tendon produced also maintained impulse activity throughout the period of stimulation ( RosBii and Asanuma, 1971, 1972b). VII. Spatial Patterns of Group I Convergence at Different Levels of the Pathway

The convergence of group I-evoked excitation of single neurons at the cuneate, thalamic, and cortical levels of the pathway was investigated with electrical stimulation of various forelimb nerves ( Oscarsson et al., 1966; RosBn, 1969b,c). The results are discussed in detail in a previous paper ( RosQn, 1 9 6 9 ~ )The . convergence of group I excitation from receptors in synergistic muscles was studied by stimulating three separate branches of the deep radial nerve innervating dorsiflexors

GROUP I PATHWAY TO CEREBRAL CORTEX

11

at the wrist and digits, and two branches of the triceps nerve innervating the long and lateral heads of the triceps muscle. Convergence of group I excitation from less-related muscle groups was studied by stimulation of three pair of nerves, each pair innervating extensors and flexors at the wrist (as well as digits), elbow, and shoulder. The results were obtained mainly by extracellular recording and reflect the pattern of impulse transmission evoked by synchronous group I afferent volleys. In order to study the detailed pattern of convergence onto the neurons at the different levels of the pathway the pattern of intracellularly recorded synaptic potentials should be compared with the pattern of impulse activity in the presynaptic neuronal elements. This was possible for cortical group I-activated cells and to a certain extent for the cuneate neurons but not for the thalamic neurons. However, the small need for spatial summation in the pathway and the fact that most of the observations were made on spontaneously active cells during many sweeps suggest that the functionally iniportant connections were revealed by extracellular recording. Two-thirds of the group I cuneothalamic relay cells tested were activated exclusively from one of the three deep radial nerve branches and only one out of 14 cells were activated from both the triceps branches (RosBn, 1969b). The same degree of spatial specificity was obtained by natural stimulation of individual muscles ( RosCn and Sjolund, 1969, 1972b). It is obvious that the second-order neurons of the group I afferent pathway to the cerebral cortex permit a high degree of spatial discrimination comparable to that of the neurons of the dorsal spinocerebellar tract ( Holmqvist et al., 1956; Lundberg and Winsbury, 1960; Eccles et al., 1961; J. K. S. Jansen and Wallge, 1969), and its forelimb equivalent, the cuneocerebellar tract ( Holmqvist et al., 1963; RosBn and Sjolund, 1969, 1972b; Cooke et al., 1971b). Some of the cuneate cells showed co-excitation froin two or three muscle groups in various combinations. These neurons might signal complex information concerning stages of movements or limb position. However, it is quite possible that this convergence represents, at least, in part, aberrations in the synaptic connections, since one of the contributing nerves often had a more effective synaptic linkage than the others (RosCn, 196913). This type of convergence does not occur in the group I-activated component of the cuneocerebellar path (Cooke et al., 1971b; R o s h and SjGlund, 1972b). Among the thalamocortical neurons of the pathway a significantly higher degree of convergence from synergistic muscles was observed than in the cuneate nucleus (RosCn, 1 9 6 9 ~ ) Two-thirds . of the cells tested were activated from more than one of the deep radial nerve

12

INGMAR R O S ~ N

branches and 10 out of 13 cells were activated from both the triceps branches. The convergence described in the previous section from a few unrelated muscle groups to some of the cuneate cells was mirrored by the convergence with an equal proportion of thalamic cells. Among the group I-activated cortical cells a dramatic change of pattern was found: few cortical cells were activated from one nerve only and more than half of the cells were activated from afferents in three or more muscle groups, in some cases from all the six groups studied. No significant change of convergence from afferents in the synergistic muscles appeared from a comparison with the thalamic level of the pathway. The results indicate that information from group I afferents is integrated in two main steps. The first step is the convergence from synergistic muscles occurring at the thalamic level. The second step is the wide convergence at the cortical level, which implies integration of information from afferents from various muscle groups of less-related function and also from antagonistic muscle groups. The results obtained from cortical cells do not, however, imply an absence of spatially specific patterns of activity at this level of the pathway. Some of the neurons were activated mainly from one or a few muscle groups. Under natural conditions the cortical cells with wide receptive fields might also show some spatial specificity as indicated by the different amplitudes of the synaptic potentials evoked from the various nerves. The somatotopic organization, which was found by surface recording, indicates that the relative amplitudes of the synaptic potentials evoked from different nerves vary systematically according to the relative positions of the neurons in the projection area. Natural stimulation of cortical group I-activated cells in awake cats recently revealed a larger restriction of the afferent input than was suggested by the results of electrical nerve stimulation (Roskn and Asanuma, 1971, 1972b). It is possible that in the unanaesthetized animal with intact connections from the muscle spindles, weak effects are depressed by surround inhibition at the thalamic or cortical level. The complex patterns of convergence obtained among the fourth-order cortical cells might represent a relatively final stage of the integration of the incoming signals, the information at this stage being used for modulation of the cortical efferent outflow as has been suggested (see p. 21). The convergence in the fourth-order neurons was mirrored by the convergence in the inhibited fifth-order neurons of the pathway (Oscarsson et al., 1966). VIII. Convergence of Excitation from Other Types of Afferents

Convergence of excitation from group I1 muscle afferents was observed in one-third of the group I cuneothalamic relay cells, In most

GROUP I PATHWAY TO CEREBRAL CORTEX

13

of these cases the convergeiice was seen as action potentials following those evoked by group I afferents in the same nerve ( RosBn, 1969113). The same type of convergeiice was also encountered among neurons of the dorsal spinocerebellar tract (Laporte et aZ., 1956; Lundberg and Oscarsson, 1956; Eccles et al., 1961) and the cuneocerebellar tract (Cooke et al., 1971b; Rosen and Sjolund, 1972b). Among the DSCT neurons the excitation from group I1 affereiits occurred preferentially in cells activated by Ia afferents (Luadberg and Oscarsson, 1956; Eccles et al., 1961). The combined effects upon single cells by groups I and I1 afferents in the same muscle nerve presumably represent a highly specific convergence from the two types of spindle afferents. It is remarkable that the group I1 muscle afferents, which should mediate information about muscle length and static y bias, do not seem to have any separate channel for transmission of information to the cerebral cortex. Although both the dorsal spinocerebellar tract and the cuneocerebellar tract have been suggested to contain neurons specifically activated by secondary muscle spindle afferents (Jansen and Rudjord, 1965; RosBn and Sjolund, 1972a), in most ascending pathways studied group I1 muscle afferents converge either to the group I-activated cells or to cells activated by the other components of the flexor reflex afferents (group 111 muscle, cutaneous, and high-threshold joint afferents) (cf. Eccles and Lundberg, 1959; Lundberg, 1964; Oscarsson, 1967; Roskn, 1969b). In the thalamus and the cerebral cortex group I1 afferent convergence to group I-activated cells was observed less often than in the cuneate . the slightly nucleus ( Oscarsson et al., 1966; RosBn, 1 9 6 9 ~ )Presumably delayed excitation from group I1 affereiits is blocked by recurrent inhibition produced in the thalamus by the synchronous group I-evoked activity (cf. Andersen et al., 1964e). Under natural conditions the group I1 effects might be effectively transmitted to the cortex via the group I afferent pathway. Twenty-four percent of the cuneothalamic cells and 3% of the thalamocortical cells of the group I afferent pathway were excited by stimulation of a cutaneous nerve (the superficial radial) (RosBn, 1969b,c; cf. Andersson et al., 1966). The excitation from the cutaneous nerve was usually weak, with action potentials evoked occasionally at varying and often long latencies. The excitation usually appeared only when the stimulus strength was above 1.25 times the nerve threshold, implying either a need for summation or that the lowest threshold cutaneous afferents did not contribute to the excitation. Part of these effects might be explained by a dorsal root or dorsal funiculus reflex (Aiidersen et al., 1964b; Roskn, 1969b). It was suggested that the excitation evoked in the thalamocortical cells by the cutaneous nerve volley might be explained by the excitation produced at the cuneate level of the pathway ( RosBn, 1 9 6 9 ~ ) .

14

INGMAR R O S ~ N

The superficial radial nerve evoked e.p.s.p.’s in the vast majority of the group I-activated cortical cells (Oscarsson et id.,1966; cf. also Swett and Bourassa, 1967a). The thresholds for these effects were often close to the nerve threshold. The synaptic potentials occurred consistently and had latencies suggesting that at least one more neuron was included in the pathway than in the group I afferent path to the same cortical cells. These observations cannot be explained by the excitation evoked by cutaneous afferents at the cuneate or thalamic levels of the pathway but indicate an excitation from a separate group of thalamocortical or corticocortical neurons activated by cutaneous afferent volleys ( RosCn, 1 9 6 9 ~ ) With . natural stimulation a few group I-activated cortical cells could be shown to be excited also by hair receptors in the paw region ( Rosdn and Asanuma, 1972b). Low threshold joint afferents and afferents from Pacinian corpuscles in the forelimb interosseus nerve have been shown to project to cortical areas partly overlapping the group I projection fields (Korner and Landgren, 1969; Silfvenius, 1970). Presumably the joint afferents and possibly the Pacinian afferents are important in motor integration. It is not known, at present, if these various types of information converge to the same cortical neurons in the primary projection areas. No convergence from the elbow joint nerve seems to occur at the thalamic level of the group I afferent pathway ( Andersson et al., 1966). IX. Afferent Inhibition

Group I cuneothalamic neurons which were activated by stretch of a given muscle could usually not be inhibited by stretching synergistic or antagonistic muscles (Roskn and Sjdund, 1969, 1972b). In contrast, with natural stimulation inhibitory effects were often found in the group I relays of the cuneocerebellar tract (Roskn and Sjolund, 1969, 1972b; cf. also Cooke et al., 1971b) and the dorsal spinocerebellar tract (Holmqvist et al., 1956; Laporte and Lundberg, 1956; Jansen et al., 1967a). Postsynaptic inhibitory potentials were only rarely observed in the group I main cuneate cells whereas large i.p.s.p.’s were often recorded in the cutaneous relay cells encountered in the same experiments [I. R o s h , 1969b, 1969, unpublished data; cf. also Andersen et al., 1964dI. Presynaptic depolarization of the group I afferent terminals in the main cuneate nucleus was observed after stimulation of group I muscle afferents, high-threshold muscle afferents, and cutaneous afferents and had no discernable topographic pattern ( Roskn, 1969b). Of these groups of afferents the cutaneous fibers were by far the most effective. The same pattern of inhibition was encountered by recording the mass activity in the medial lemniscus and single-cell discharges in the nucleus

GROUP I PATHWAY TO CEREBRAL CORTEX

15

after conditioning afferent volleys. This suggests that the inhibition observed at the cuneate level of the group I afferent pathway is mainly of presynaptic type. The organization of the presynaptic inhibition in the cuneate group I relay is different from that found for various groups of afferents at the segmental level of the cord where a much higher degree of modality specificity was observed (Eccles et al., 1962, 1963a,c; Janig et al., 1968). It is, however, reminiscent of the presynaptic inhibition in the dorsal spinocerebellar tract (Eccles et at., 1963b; Jankowska et al., 1965) where the group I afferent terminals were depolarized by flexor reflex afferents and Ib afferents. The flexor reflex afferents seemed, however, to be more effective in the cuneate group I relay than in the dorsal spinocerebellar tract. The observations discussed above suggest that a reciprocal inhibition corresponding to that described for motoneurones ( Lloyd, 1946a,b; Eccles and Lundberg, 1958) does not occur in the cuneate relay. The only inhibitory effects observed in the group I activated thalamic cells were those apparently due to recurrent inhibition (Roskn, 1 9 6 9 ~ )This . inhibition was presumably responsible for the depression of the cortical evoked potentials which lasted for about 100 msec following stimulation of group I afferents (Fig. 4A in Oscarsson and RosCn, 1963). It is presumably relatively unspecific. In the cortical group I-influenced neurons (fourth- and fifth-order neurons of the pathway) the patterns of synaptic potentials give no indication of a specific reciprocal organization (Oscarsson et al., 1966). X. Cortical Control of Transmission

The cortical influence on cells in the cuneate nucleus has been studied in several investigations which were concerned with neurons activated by cutaneous afferents (Jabbur and Towe, 1961; Towe and Jabbur, 1961; Andersen et al., 1964a-d; Levitt et al., 1964). The cortical effects on the transmission in the group I afferent pathway was studied (RosCn, 1969b,c) because this pathway forwards information which presumably plays no role in conscious perception (see below) and might be differently controlled from higher centers. Furthermore, the restricted cortical projection fields would make it possible to correlate these fields with the cortical areas responsible for corticofugal effects on the pathway. About half of the group I cuneothalamic cells were inhibited by cortical stimulation. The inhibition was further investigated by recording the group I-evoked mass discharge in the medial lemniscus after conditioning stimulation of the cortex at various spots (Roskn, 1969b). The focal area for the inhibition was found around the lateral end of the cruciate sulcus ( Fig. 2B, vertical hatching), corresponding

16

INGMAR R O S ~ N

u EXCITATION OF GROUP

NON-RELAY CELLS

I

EXCITATION INHIBITION

AND

111

OF

GROUP I RELAY CELLS

(-

: , . :...... ...:........ ............. II ........ ....... ...... ...... .....

EXCITATION OF CUTANEOUS CELLS

INHIBITION OF CUTANEOUS CELLS

FIG. 2. Areas in pericruciate cortex influencing cells in the cuneate nucleus. A: Distribution area of “best points” for cortical excitation of group I “non-relay cells” (dashed line). Most of the “best points” lay at the postcruciate dimple and at the lateral end of the cruciate sulciis (stippled area). Data from thirty-three neurons. Cortical stimulation by needle electrodes 1 111111 below cortical surface at fixed positions ( after R o s h , 1969b). B: Approximate areas for excitation ( horizontal hatching, 3 experiments) and inhibition (vertical hatching,5 experiments) of the group I-evoked mass discharge in the medial lemniscus. Bipolar cortical surface stimulation (after RosCn, 196911). C and D : Cortical areas for excitation ( C ) and inhibition ( D ) of cuneate cells activated by cutaneous afferents ( after Fig. 5A, B Levitt et al., 1964). Total effective cortical regions (dashed line), and the focal areas (stippled) are indicated. Bipolar surface stimulation. The maps are composite representations of mapping studies in several cats.

to the forelimb motor cortex (Livingston and Phillips, 1957; Hassler and Muhs-Clement, 1964; Asanuma and Sakata, 1967). Approximately the same area was described as the focus for the corticofugal inhibition of cells in the cuneate nucleus that were activated by cutaneous afferents (Levitt et al., 1964; shown in Fig. 2 0 ) . The separation of the inhibitory focus from the primary group I projection area (cf. Fig. 1B) demonstrates that the corticofugal inhibition of group I cuneothalamic cells does not represent a direct negative feedback mechanism from the projection area (cf. Darian-Smith and Yokota, 1966a,b). Under

GROUP I PATHWAY TO CEREBRAL CORTEX

17

physiological conditions corticofugal inhibition of the transmission through the cuneate nucleus has been shown to occur during bursts of rapid eye movements (REM) in desyiichroilized sleep (Carli et al., 1967a,b). A good correlation was found between the cortically evoked inhibition of the group I cuneate transmission, and the depolarization of the group I terminals in the main cuneate nucleus studied with Wall’s technique (cf. Wall, 1958). Thic does not exclude the possibility of additional postsynaptic inhibition which has been demonstrated in cuneate neurons activated by cutaneous afferents ( Andersen et al., 1964d) and in dorsal spinocerebellar tract neurons ( Hongo and Okada, 1967). A few (7.5%)of the group I cuneothalamic neurons could be excited from the cerebral cortex. In three experiments conditioning by cortical stimulation produced an early increase of the group I-evoked lemniscal test discharge followed by an inhibition. The cortical focus for the excitation lay caudal and lateral to the focus for the inhibition and close to the coronal sulcus (Fig. 2R horizontal hatching). The facilitatory area corresponds approximately to the rostrolateral part of area 3a (cf. Fig. 1 B ) . In this area, and buried in the upper bank of the coronal sulcus, neurons have been identified which project directly to the cuneate nucleus (Gordon and Miller, 1969). These observations indicate that although under barbiturate anaesthesia the cortical influence on the group I cuneothalamic cells is mainly inhibitory there is also a comv poiient of facilitat ion. In accordance with previous observatioiis on cuneate cells activated by cutaneous afferents ( Andersen et al., 1964c) cortically evoked excitation was produced in the majority (75%)of the group I cells which were not antidromically invaded from the medial lemniscus ( “non-relay cells”). The cortical “best points” for this excitation were rather widely spread over the forelimb sensorimotor cortex but the most effective points lay at the lateral end of the cruciate sulcus and at the postcruciate dimple (Fig. 2A). The total excitatory region corresponds to that delimited by Levitt et al. (1964, Fig. 2C) for excitation of cuneate cells activated by cutaneous afferents. Relay cells were not discriminated from “non-relay cells” in the latter investigation. The cortically excited group I “non-relay cells” differed from the cuneothalamic cells in receiving a strong excitation from group I1 muscle afferents in various nerves and from cutaneous afferents ( RosCn, 196913). The convergence of excitation to these cells from the cerebral cortex, group I afferents, and the flexor reflex afferents would be in accord with their possible role as interneurons in inhibitory pathways to the relay cells (cf. Andersen et al., 1964c,d). The possibility that at

18

INGMAR R O S ~ N

least some of the group I “non-relay cells” project to structures outside the ventrobasal thalamic complex is not excluded. Cortical stimulation produced an inhibition of group I thalamocortical cells which was presumably due to recurrent inhibition in the thalamus (Andersen et al., 1964e; RosCn, 1 9 6 9 ~ )In . addition an excitation of the thalamic relay cells was shown to occur at moderately high stimulus frequencies. Corticothalamic excitation has previously been described in a number in investigations (for references, see RosCn, 1 9 6 9 ~ and ) ~ has been shown to be most effectively produced by the cortical projection area of the thalamic cells under observation (Shimazu et d.,1965; Andersen et al., 1967). This observation can be correlated with the Nauta study of Jones and Powell (1968b) which showed that the corticothalamic fibers from the somatosensory cortical area were distributed according to the thalamic representation of the body surface. The thalamic group I relay zone (cf. Andersson et al., 1966) received excitation from a sharply confined cortical area identical with the group I projection area rostral to the postcruciate dimple ( RosCn, 1 9 6 9 ~ ) . The topographic specificity of this excitation might suggest a positive feedback mechanism. However, the interaction under natural conditions between this mechanism and the thalamic recurrent inhibition is completely unknown, as is the link between the cortical afferents and the corticothalamic neurons. XI. Comparative Aspects

A. HINDLIMB PATHWAY IN CAT Hindlimb group I afferents were recentIy shown to project to the cat cerebral cortex in a projection pattern in several respects similar to that of forelimb afferents (Landgren and Silfvenius, 1969a). The cortical potentials evoked in the cerebral cortex were much smaller than those produced by forelimb group I volleys of similar size. One of the projection fields lies rostromedial to the postcruciate dimple and overlaps the corresponding forelimb projection area. A second projection field lies on the medial side of the hemisphere and extends from area 3a into the sensory cortex (area 1 of Hassler and Muhs-Clement). Group I hindlimb responses were also reported in the region of the anterior suprasylvian sulcus ( Landgren et al., 1967a). The hindlimb pathway has one synaptic relay more than the forelimb pathway; the spinal path travels with the dorsal spinocerebellar tract or utilizes brainstem collaterals of this tract. The brainstem relay was localized in the nucleus z of Brodal and Pompeiano (1957; Landgren and Silfvenius, 1969b, 1971), rostral to the gracile nucleus. After lesions in nucleus z, degenera-

GROUP I PATHWAY TO CEREBRAL CORTEX

19

tion was found dorsolaterally in the rostra1 end of the lateral subdivision of nucleus ventralis posterolateralis (VPL) and in nucleus ventralis lateralis (Boivie et al., 1970). One further difference in organization might be indicated by the observation that both Ia and Ib components of the group I volley in hindlimb nerves contributed to the evoked cortical potentials (Landgren and Silfvenius, 1969a). However, natural stimulation will presumably be necessary to demonstrate a cerebral projection of hindlimb tendon organ afferents since it has been shown that stimuli to the semitendinosus nerve which excite group I fibers in the higher threshold range recruit to the volley not only fibers from tendon organs but also many from muscle spindles (Coppin et al., 1969; cf. also Laporte and Bessou, 1957; Sumner, 1961) .

B. GROUPI PROJECTION IN MONKEY In the macaque monkey evoked potentials were recorded both in the motor and sensory cortexes after group I afferent stimulation (AlbeFessard et al., 1965). The group I-evoked potentials were described as localized, although the projection fields were not indicated. Relatively large and repetitive group I afferent volleys were required to evoke clear-cut cortical potentials which might indicate that considerable summation is needed for impulse transmission in the afferent pathway. When the stimulus strength was increased to include group I1 muscle afferents, much larger and more-widespread cortical potentials were evoked. The projection of muscle spindle afferents (primary or secondary) was further demonstrated by using selective fusimotor stimulation ( AlbeFessard et al., 1966). Natural stimulation showed that cells in the motor cortex were driven by joint movements and the receptors were often found to be intramuscular ( Albe-Fessard and Liebeskind, 1966). However, effects from primary and secondary spindle afferents and from tendon organ afferents were not discriminated in these experiments. Complex patterns of excitation and inhibition, often of a reciprocal type, were found. The transitional zone between the sensory and motor cortexes, area 3a, which is the main projection area for group I afferents in the cat, is located in the depth of the central sulcus of the monkey (Roberts and Akert, 1963; Powell and Mountcastle, 1959). In recent investigations this area was shown to be the primary projection zone for group I afferents also in the monkey [Phillips et al., 1971 (baboon); RosQn and Asanuma, 1972b (Cebus)]. In none of these investigations could short-latency group I-evoked responses be recorded from area 4, i.e., the motor cortex. The study of Phillips and co-workers demonstrated that the group I

20

INGMAR ROSEN

projection from the baboon's forearm muscles to the cerebral cortex has many features in common with those earlier described in the cat. The spinal pathway ascends in the dorsal column. The transmission across the first-second- and second-third-order synapses were secure. Extracellular recording from group I-activated cells in area 3a showed less convergence from afferents in the deep radial and ulnar nerves than was the case in the similar experiments on cat (Oscarsson et al., 1966). None of the cells in area 3a could be antidromically activated from the lateral corticospinal tract. It would therefore seem that neither in the monkey nor in cat do the pyramidal tract cells receive a significant early activation from group I afferents (cf., however, Swett and Bourassa, 1967a). Microstimulation within the cortical projection area for group 1 afferents never produced any motor effects in the awake monkey ( Asanuma and RosCn, 1972; Ros6n and Asanuina, 197"). XII. Functional Considerations

What is the functional significance of a cortical projection of group I muscle spindle afferents? The information forwarded to the central nervous system by these afferents is complex: the impulse pattern is a function of muscle length, change of muscle length, and activity in dynamic and static y motoneurons (cf. Matthews, 1964). The activity of the y motoneurons is set by segmental reflexes and by various supraspinal control mechanisms ( cf. Grillner, 1969). Except for some recent reports, there is little evidence in the literature indicating that information from the group I afferent pathway contributes to specific or nonspecific sensory mechanisms. Stimulation of group I forelimb or hindlimb afferents in waking or sleeping cats did not cause any change of behavior or electroencephalogram (Giaquinto et al., 1963). Group I afferent volleys could not be used for triggering of instrumental conditioned reflexes ( Swett and Bourassa, 196713). In man, stretching of muscle tendons at tensions which apparently activated primary as well as secondary muscle spindle afferents failed to evoke sensations of change in muscle tension or joint position (Gelfan and Carter, 1967). In human experiments blockage of impulses from joint and skin receptors without affecting the transmission from muscle receptors was reported to eliminate kinesthesia and position sense ( Provins, 1958; Merton, 1964). Recent results with similar techniques indicated, however, that muscle afferents can contribute to kinesthesia if joint movement is comparably rapid ( 5-lO0/second) and particularly if the subject is allowed to tense the muscles influenced by the movement (Goodwin et al., 1 9 7 2 4 . It is therefore still conceivable that information from muscle spindle afferents adds to the information given by receptors around the joint. Paillard and Brouchon (1968) reported that the accuracy of limb local-

GROUP I PATIIWAY

‘ro CEHEBRAL CORTEX

21

ization after active movements was significantly higher than after passive movements. The increased accuracy might be due to information from muscle spindle afferents, activated via the y route concomitantly with the muscle contraction, the information from the joint and skin afferents being simiIar for active and passive movements, Furthermore, it was recently found that muscle vibration may induce an illusion of movement just as if the vibration-induced increase in muscle afferent firing had been perceived (Goodwin et al., 1972b). It was suggested (Oscarsson and RosCn, 1963, 1966) that the group I afferent projection to the cerebra1 cortex represents a feedback system used for adjusting the motor output from the cortex. This hypothesis receives some support from Evarts’ observations that, in the monkey, the impulse activity in many preccntral pyramidal tract cells during motor performance is strongly related to variation of force rather than displacement (Evarts, 1968, 1969). These results seem to imply a need for a feedback path to the cortex, providing information about the load encountered during the movements. Such information might be provided by primary and secondary muscle spindle aff erents and possibly tendon organ afferents. Various types of movements have been shown to be produced by coordinatcd activation of LY and y motoneurons (“-7 linkage of Granit, 1955, 1968; cf. von Euler, 1966; Severin et al., 1967; Vallbo, 1970, 1971). This mechanism prevents unloading of muscle spindles during extrafusal muscle contraction and would guarantee a segmental servoaction via the y loop, provided that the relative amounts of activity in LY and y inotoneurons are adjusted for the shortening “expected” ( Matthews, 1964). Discrepancies between the “intended” and actual movements should be signaled by the muscle spindle afferents and reduced by the “follow-up-length servo” at the segmental level ( Merton, 1953). These “error signals” i n the muscle spindle afferents are also rapidly transmitted to the cerebral cortex and might induce a ‘resetting” of the cortical motor activity to a level appropriate to overcome the load encountered. ACKNOWLEDGMENTS The article is a revised edition of a doctoral thesis presented at the University of Lund, Sweden, in 1970. Thanks are due to Dr. Olov Oscarsson, in whose laboratory the work was performed, for advice and criticism. The work was made possible by continuous support to the author from the Medical Faculty, University of Lund, and by grants to Dr. Oscarsson from the Swedish Medical Research Council.

REFERENCES Albe-Fessard, D., and Liebeskind, J. ( 1966). E x p . Brain Res. 1, 127. Albe-Fessard, D., Liebeskind, J., and Lamarre, Y. (1965). C . R. Acad. Sci. 261, 3891. Albe-Fessard, D., Lamarre, Y.,and Pimpaneau, A. (1966). J. Physiol. (Paris) 58, 443.

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Amassian, V. E. (1951). 1. Neurophysiol. 14, 445. Amassian, V. E., and Berlin, L. (1958). I. Physiol. (London) 143, 61P. Andersen, P., Eccles, J. C., Schmidt, R. F., and Yokota, T. (1964a). 1. Neurophysiol. 27, 78. Andersen, P., Eccles, J. C., Schmidt, R. F., and Yokota, T. (1964b). J. Neurophysiol. 27, 92. Andersen, P., Eccles, J. C., Schmidt, R. F., and Yokota, T. ( 1 9 6 4 ~ )J.. Neurophysiol. 27, 1080. Andersen, P., Eccles, J. C., Oshima, T., and Schmidt, R. F. (1964d). J. Neurophysiol. 27, 1096. Andersen, P., Eccles, J. C., and Sears, T. A. (1964e). J. Physiol. (London) 174, 370. Andersen, P., Anderson, S. A., and Landgren, S. (1966). Acta Physiol. Scand. 68, 72. Andersen, P., Junge, K., and Sveen, 0. (1967). Nature (London) 214, 1011. Anderson, S. A., Landgren, S., and Wolsk, D. (1966). J. Physiol. (London) 183, 576. Armstrong, D. M., Harvey, R. J., and Schild, R. F. (1969). J. Physiol. (London) 202, 106P. Asanuma, H., and Roskn, I. (1972). E r p . Brain Res. 14, 243. Asanuma, H., and Sakata, H. (1967). J. Neurophysiol. 30, 35. Asanuma, H., Stoney, S. D., and Abzug, C. (1968). J. Neurophysiol. 31, 670. Boivie, J., Grant, G., and Silfvenius, H. (1970). A d a Physiol. Scand. 80, 11A. Bourassa, C. M., and Swett, J. E. (1967). J. Neurophysiol. 30, 515. Bradley, K., and Eccles, J. C. ( 1953). J. Physiol. (London) 122, 462. Brodal, A,, and Pompeiano, 0. (1957). J. Anat. 91, 438. Brown, M. C., Engberg, I., and Matthews, P. B. C. (1967). J. Physiol. (London) 192, 773. Carli, G., Diete-Spiff, K., and Pompeiano, 0. (1967a). Arch. Ztal. Biol. 105, 52. Carli, G., Diete-Spiff, K., and Pompeiano, 0. (1967b). Arch. ltal. Bid. 105, 83. Cooke, J. D., Larson, B., Oscarsson, O., and Sjolund, B. (1971a). E r p . Brain Res. 13, 339. Cooke, J. D., Larson, B., Oscarsson, O., and Sjolund, B. (1971b). E r p . Brain Res. 13, 359. Coppin, C. M. L., Jack, J. J. B., and McIntyre, A. K. (1969). J. Physiol. (London) 203, 45P. Darian-Smith, I., and Yokota, T. (1966a). J. Neurophysiol. 29, 170. Darian-Smith, I., and Yokota, T. (19661~).1. Neurophysiol. 29, 185. Eccles, R. M., and Lundberg, A. (1958). J. Physiol. (London) 144, 271. Eccles, R. M., and Lundberg, A. (1959). Arch. Ital. Biol. 97, 199. Eccles, J. C., Eccles, R. M., and Lundberg, A. (1957). J. Physiol. (London) 136, 527. Eccles, J. C., Oscarsson, O., and Willis, W. D. (1961). J. Physiol. (London) 158, 517. Eccles, J. C., Magni, F., and Willis, W. D. (1962). I. Physiol. (London) 160, 62. Eccles, J. C., Schmidt, R. F., and WiIlis, W. D. (1963a). J. Neurophysiol. 26, 1. Eccles, J. C., Schmidt, R. F., and Willis, W. D. (19631,).I. Neurophysiol. 26, 635. . Neurophysiol. 26, 646. Eccles, J. C., Schmidt, R. F., and Willis, W. D. ( 1 9 6 3 ~ )I. Evarts, E. V. (1968). 1. Neurophysiol. 31, 14. Evarts, E. V. (1969). 1. Neurophysiol. 32, 375. Gelfan, S., and Carter, S. (1967). E r p . Neurol. 18, 469. Giaquinto, S., Pompeiano, O., and Swett, J. E. (1963). Arch. Ztal. Biol. 101, 133. Coodwin, G. M., McCloskey, D. I., and Matthews, P. B. C. (1972a). Brain Res. 37, 326.

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Goodwin, G. M., McCloskey, D. I., and Matthews, P. B. C. (1972b). Science 175, 1382. Gordon, G., and Jukes, M. G. M. (1964). J. Physiol. ( L o n d o n ) 173, 263. Gordon, G., and Miller, R. (1969). Quart. J. Erp. Physiol. Cog. Med. Sci. 54, 85. Gordon, G., and Paine, C. H. (1960). 1. Physiol. ( L o n d o n ) 153, 331. Grampp, W., and Oscarsson, 0. ( 1968). Struct. Funct. Inhibitory Neuronal Mech., Proc. Int. Meet. Neurobiol. 4th, 1966, p. 351. Granit, R. (1955). “Receptors and Sensory Perception.” Yale Univ. Press, New Haven, Connecticut. Granit, R. (1968). Proc. Roy. SOC. Med. 61, 69. Grant, G. (1962). Exp. Neurol. 5, 179. Grillner, S. (1969). Acta Physiol. Scand., Suppl. 327. Hassler, R., and Muhs-Clement, K. ( 1964). J. Himforsch. 6, 377. Hensel, H., and Bonian, K. K. A. (1960). J. Neiirophysiol. 23, 564. Holniqvist, B., Lundberg, A., and Oscarsson, 0. (1956). Acta Physiol. Scand. 38, 76. Holmqvist, B., Oscarsson, O., and RosCn, I. (1963). Acta Physiol. Scand. 58, 216. Hongo, T., and Okada, Y. (1967). E x p . Brain Res. 3, 163. Jabbur, S. J., and Towe, A. L. ( 1961). J. Neurophysiol. 24, 499. Janig, W., Schmidt, R. F., and Zinimerman, M. (1968). Erp. Brain Res. 6, 116. Jankowska, E., Jukes, M. G. M., and Lund, S. ( 1965). J. Physiol. ( L o n d o n ) 178, 17P. Jansen, J., and Brodal, A. (1958). In “Handbuch der mikroskopischen Anatomie des Menschen” ( W . von Mollendorff and W. Bergmann, eds.), Vol. 4, Part 8, pp. 1-323. Springer-Verlag, Berlin and New York. Jansen, J. K. S., and Rudjord, T. (1965). Science 149, 1109. Jansen, J. K. S., and Wallpre, L. (1969). I n “The Cerebellum in Health and Disease” ( W. S. Fields and W. D. Willis, eds.), p. 143. Warren H. Green, lnc., St. Louis, Missouri. Jansen, J. K. S., Nicolaysen, K., and Wallgje, L. ( 1967a). Acta Physiol. Scand. 70, 362. Jansen, J. K. S., Poppele, R. E., and Terzuolo, C. A. (1967b). Brain Res. 6, 382. Jones, E. G., and Powell, T. P. S. (1968a). Brain Res. 9, 71. Jones, E. G., and Powell, T. P. S. (1968b). Brain Res. 10, 369. Jones, E. G., and Powell, T. P. S. ( 1 9 6 8 ~ ) J. . Anat. 103, 433. Korner, L., and Landgren, S. (1969). Acta Physiol. Scand. 76, 5A. Kruger, L. (1956). Arner. J. Physiol. 186, 475. Kuypers, H. G. J. M., and Tuerk, J. d. ( 1964). J. Anat. 98, 143. Landgren, S., and Silfvenius, H. ( 1969a). J. Physiol. (London ) 200, 353. Landgren, S., and Silfvenius, H. (196913). Acta Physiol. Scand. 77, Srippl. 330, p. 190. Landgren, S., and Silfvenius, H. (1971). J. Physiol. ( L o n d o n ) 218, 551. Landgren, S., Silfvenius, H., and Wolsk, D. (1967a). J. Physiol. (London) 191, 543. Landgren, S., Silfvenius, H., and Wolsk, D. (1967b). J. Physiol. ( L o n d o n ) 191, 561. Laporte, Y., and Bessou, P. (1957). I. Physiol. ( P a r i s ) 49, 1025. Laporte, Y., and Lundberg, A. (1956). Acta Physiol. Scand. 36, 204. Laporte, Y., Lundberg, A., and Oscarsson, 0. (1956). Acta Physiol. Scand. 36, 188. Levitt, M., Cameras, M., Liu, C. N., and Chambers, W. W. (1964). Arch. Ital. Biol. 102, 197. Livingston, A., and Phillips, C. G. (1957). Quart. J. Exp. Physiol. 42, 190. Lloyd, D. P. C. (1946a). J. NecirophysioE. 9, 421. Lloyd, D. P. C. (194613). J. Neurophysiol. 9, 439.

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Lorente de Nb, R. ( 1949). 1s “Physiology of the Nervous System” (J. F. Fulton, ed.), 3rd ed., pp. 288-315. Oxford Univ. Press, London and New York. Lundberg, A. (1964). In “Physiology of Spinal Neurons” ( J . C. Eccles and J. SchadB, eds. ), p. 135. Elsevier, Amsterdam. Lundberg, A,, and Oscarsson, 0. (1956). Acta Physiol. Scand. 38, 53. Lundberg, A,, and Winsbury, G. (1960). Acta Physiol. Scand. 49, 165. McIntyre, A. K. (1953). Proc. Uniu. Otago Med. Sch. 31, 5. McIntyre, A. K. ( 1962a). In “Symposium on Muscle Receptors” ( 1). Barker, ed. ), pp. 19-30. Hong Kong Univ. Press, Hong Kong. McIntyre, A. K. (1962b). J. Physiol. (London) 163, 46. McIntyre, A. K., Holman, M. E., and Veale, J. L. (1967). E x p . Brain Rex 4, 243. Maekawa, K., and Purpura, D. P. ( 1967). 1. Nezrrophysiol. 30, 360. Mallart, M. A. (1964). C. R. Acad. Sci. 259, 1215. Mallart, M. A. (1968). 1. Physiol. (London) 194, 337. Mark, R. F., and Steiner, J. (1958). J. Physiol. (London) 142, 544. Matthews, P. B. C. (1964). Physiol. Rev. 44, 219. Merton, P. A. (1953). Spinal Cord, Ciba Fozrnd. Symp., 1952 p. 247. Merton, P. A. (1964). S y m p . SOC. E r p . B i d . 18, 387. Mountcastle, V. B., Covian, M. R., and Harrison, C. R. (1952). Res. Pirhl., Ass. Res. N e w . Ment. Dis. 30, 339. Nauta, W. J. H. (1954). Attat. Rec. 118, Z33. Oscarsson, 0. (1965). Physiol. Rev. 45, 495. Oscarsson, 0. (1966). Muscular Afferents Mot. Contr. Proc. Nobel Symp., l s t , 1965 p. 307. Oscarsson, 0. ( 1967). In “Neurophysiological Basis of Normal and Abnormal Motor Activities” ( M . D. Yahr and D. P. Purpara, eds.), p. 93. Raven, New York. Oscarsson, O., and RosBn, I. ( 1963). 1. Physiol. (London) 169, 924. Oscarsson, O., and RosBn, I. (1966). 1. Physiol. (London) 182, 164. Oscarsson, O., and Uddenberg, N. (1964). Acta Physiol. Scand. 62, 125. Oscarsson, O., RosBn, I., and Sulg, I. (1966). 1. Physiol. (London) 183, 189. Paillard, J., and Brouchon, M. ( 1968). In “The Neuropsychology of Spatially Oriented Behavior” ( S . J. Freedman, ed.), p. 37. Dorsey, Homewood, Illinois. Phillips, C. G., Powell, T. P. S., and Wiesendanger, M. (1971). 1. Physiol. (London) 217, 419. Powell, T. P. S., and Mountcastle, V. B. (1959). Johns Hopkins Hosp. Bull. 105, 108. Provins, K. A. ( 1958). I . Physiol. (London) 143, 55. Ram& y Cajal, S. (1952). “Histologie du systkme nerveux de I’homme et des vertc5brb.” Talleres Grhficos Montana, Madrid. Rexed, B. (1954). 1. Cornp. Neurol. 100, 297. Roberts, T. S., and Akert, K. (1963). Schweiz. Arch. Neurol., Neurochir. Psychiat. 92, 1. RosBn, I. (1969a). Brain Res. 16, 55. RosBn, I. (1969b). I. Physiol. ( L o n d o n ) 205, 209. RosBn, I. ( 1 9 6 9 ~ )1. . Physiol. ( L o n d o n ) 205, 237. RosBn, I., and Asanuma, H. (1971). Fed. Proc., Fed. Amer. SOC. E r p . Biol. 30, 664. RosBn, I., and Asanuma, H. (1972a). E x p . Brain Res. 14, 257. RosBn, I., and Asanuma, H. (197213). E x p . Brain Res. In press.

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Rosbn, I., and Sjolund, B. (1969). Acta Pltysiol. Scund. 77, Suppl. 330, p. 189. Roskn, I., and Sjolund, B. (1972a). Erp. Brain Res. In press. Roskn, I., and Sjolund, B. (19721)). E x p . Brain Res. In press. Sakata, H., and Miyamoto, J. (1968). Jup. J. Physiol. 18, 489. Severin, F. V., Orlovskii, G. N., and Shik, M. L. (1967). Biophysics ( U S S R ) 12, 575; Biofizika 12, 502 (1967). Shimazu, H., Yanagisawa, N., and Garoutte, B. (1965). Jail. J. PhysioZ. 15, 101. Silfvenius, H. (1968). Actu Physiol. S c a d . 74, 25A. Silfvenius, H. (1970). Acta Physio?. Scan&. 80, 196. Simmer, A. J. ( 1961). Proc. Uniu. Otugo Med. Sclz. 39, :3. Swett, J. E., and Bourassa, C. M. (1967a). J. Physiol. ( L o n d o n ) 189, 101. Swett, J. E., and Bonrassa, C. M. (1967b). J. Neurophysiol. 30, 530. Thompson, W. D., Stoney, S. D., and Asanuma, H. (1970). Bruin Res. 22, 15. Towe, A. L., and Jabbur, S . J. (1961). J. Netmphysiol. 24, 488. Vallbo, A. B. (1970). Acta Physiol. Scand. 78, 315. Vallbo, A. B. (1971). J. Physiol. (Londun) 218, 405. von Euler, C. (1966). Muscular A f e r e n f s Mot. Contr. Proc. Nobel Symp., I s t , 1965 p. 197. Walberg, F. ( 1957). Brain 80, 273. Wall, P. D. ( 1958). J. Physiol. ( London) 142, 1. Welt, C., Aschoff, J. C., Kameda, K., and Brooks, V. B. (1967). In “Neurophysiological Basis of Normal and Abnormal Motor Activities” ( M . D. Yahr and D. P. Purpura, eds.), p. 255. Raven, New York.

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PHYSIOLOGICAL PATHWAYS THROUGH THE VESTIBULAR NUCLEI By Victor J. Wilson' The Rockefeller University, New York, New York

I. Introduction . . . . . . . . . . . . 11. Labyrinthine Input to the Vestibular Nuclei . . . . . . A. Anatomical Studies of the Termination of Vestibular Nerve Fibers . . . . . . . within the Vestibular Nuclei . B. Electrophysiological Studies of the Labyrinthine Influence . . . . . . on Cells in the Vestibular Nuclei . 111. The Vestibular Nuclei and the Cerebellum . . . . . . A. Vestibulocerebellar Pathways . . . . . . . . B. Cerebellovestibular Pathways . . . . . . . . IV. Projections of the Vestibular Nuclei to the Spinal Cord . . . A. The Lateral Vestibulospinal Tract . . . . . . . B. The Medial Vestibulospinal Tract . . . . . . . V. Projections of the Vestibular Nuclei to Extraocular Motoneurons . A. Anatomical Background . . . . . . . . . B. Physiological Investigations . . . . . . . . VI. Concluding Remarks . . . . . . . . . . . References . . . . . . . . . . . . . Note Added in Proof . . . . . . . . . . .

. .

2

27 8

.

28

. .

30 40 40 41 42 42 58 66 66 68 76 77 81

.

. .

.

. . . . . . .

I. Introduction

The brainstem cell groups that constitute the vestibular nuclei are important in the control of posture and eye movement. Their influence is exerted in part by means of direct axonal projections to the spinal cord, extraocular neurons, contralateral vestibular nuclei, and the cerebellum. In addition, there is a close relationship between vestibular nuclei and the reticular formation, the Iatter acting to distribute nuclear output to various regions of the central nervous system. A major input to the vestibular nuclei comes from labyrinthine receptors, and an important role of some second-order vestibular neurons is to act as a relay between the labyrinth on the one hand and the spinal cord or extraocular neurons on the other. Stimulated by the comprehensive anatomical investigations of the

' Work in the author's laboratory supported by grants NS 02619 and NS 05463 from the National Institutes of Health, US. Public Health Service. 27

28

VICTOR J. WILSON

Oslo group of Brodal and his colleagues, there have been, in recent years, detailed electrophysiological studies of neurons in the vestibular nuclei. This review will examine some of this work, particularly that dealing with those pathways through the vestibular nuclei of the cat that originate in the labyrinth. First, the distribution and properties of the vestibular input to the nuclei will be described, then the projections of the nuclei. Some outputs of the nuclei, for example, the efferent processes to vestibular receptors (see Gacek, 1968) and those directed toward higher regions of the central nervous system (for example, see Fredrickson et al., 1966a; Landgren et al., 1967) will not be considered. Emphasis will be on the direct projections of vestibular neurons to the spinal cord and extraocular neurons, and on the inputs that govern the activity of projecting cells, Most of the material covered in this review will be physiological, but some of the necessary anatomical background will be discussed in each section. Anatomical references will not be complete, but rather will consist of recent or particularly pertinent papers only. I I . labyrinthine Input to the Vestibular Nuclei

The labyrinth is divided into parts sensitive to different stimuli. The utriculus is stimulated by linear acceleration (including gravity), The semicircular canals are stimulated by angular acceleration; each canal is particularly sensitive to acceleration in its own plane. The structure of the receptor cells, and their responses to natural stimuli have been described in numerous reviews (cf. Roberts, 1967; Wersall et al., 1967; Lindeman, 1969; Engstrom, 1970) that should be consulted for further references. Afferent impulses originating in labyrinthine cells are transmitted along the fibers of the vestibular nerve that, in the brainstem, terminate almost exclusively in the vestibular nuclei. The organization and subdivision of these nuclei has been studied in great detail by Brodal and Pompeiano (1957; see also Brodal et d . , 1962) whose analysis has recently been supplemented by the Golgi studies of Hauglie-Hanssen (1968). The boundaries of the nuclei as given by Brodal and Pompeiano will be used in this review, which will be concerned only with the four main cell groups: the superior, lateral ( Deiters’) , medial, and descending nuclei.

A. ANATOMICAL STUDIESOF THE TERMINATION OF VESTIBULARNERVE FIBERS WITHIN THE VESTIBULAR NUCLEI By destroying the labyrinth, allowing 5- to 19-day survival times, and impregnating degenerating fibers and terminals with the Glees and

VESTIBULAR PATHWAYS

29

Nauta-Gygax methods, Walberg et al. (1958) studied the distribution of vestibular nerve terminals in the vestibular nuclei of the cat. Some regions of the vestibular nuclei were free of degenerating terminals, which were, however, observed in the following areas: the central region of the superior nucleus; the ventral part of the lateral nucleus, very little degeneration seen in the dorsal part; the lateral part of the medial, degeneration decreasing caudally; the whole length of the descending nucleus, but little in its ventrolateral part. No primary fibers crossed the midline (the labyrinth on one side does influence the contralateral nuclei by means of indirect connections, which will be discussed below ). AS pointed out by the authors, even those areas of the nuclei not supplied by primary vestibular fibers may be influenced by labyrinthine activity, by means of pathways involving interneurons: anatomical methods show only direct connections. In addition, cells located in areas free of vestibular terminals may have dendrites extending into an area where primary fibers do terminate, receiving an input from the labyrinth in this manner, Golgi studies, however, have shown that “the vast majority of the dendrites are found within the confines of the subdivision where the soma is localized ( Hauglie-Hanssen, 1968, p. 76). In any case, the areas free of vestibular terminations may not be as extensive as first thought. For example, electron microscopic observations, some on animals with survival periods as short as two days, have revealed synaptic terminals on giant Deiters’ cells and in the peripheral part of the superior nucleus, both previously believed to lack vestibular input (Mugnaini et al., 1967; Gacek, 1969). Walberg et al. (1958) did not attempt to discriminate between afferents arising in different parts of the labyrinth. Such a study was first performed by Lorente de N6 (1933a), who made the important observation that in the brainstem the semicircular canals and the utricuIar and saccular maculae have a central representation that is partly different, partly common. These findings have been confirmed and extended by Stein and Carpenter (1967) in the monkey, and Gacek (1969) in the cat. Both investigations depended on localized damage to the vestibular ganglia, degeneration then being traced peripherally and centrally. The results of the two groups agree in many respects. The superior nucleus receives inputs only from the semicircular canals. Within this nucleus there is some difference and some overlap between termination of fibers from the posterior canal on the one hand and from the anterior and horizontal canals on the other (Gacek, 1969). The medial and descending nuclei also receive inputs from all three semicircular canaIs in their rostra1 regions, and perhaps caudally ( Gacek, 1969) ; within these two nuclei there are regions where the projections from different canals

30

VICTOR J. WILSON

overlap, others where they are relatively separate. The medium- to largesized rostroventral neurons of Deiters’ nucleus receive fibers from the utricular macula; the most medial region of the nucleus receives some fibers from all three canals (Gacek, 1969). Utricular afferents also terminate in the rostra1 regions of the medial and descending nuclei: endings in the descending nuclei are stressed by Stein and Carpenter, who consider this region the most important central representation of the utricular macula; Gacek considers the medial nucleus to be the main destination of utricular fibers. Anatomical findings thus demonstrate that no major subdivision of the vestibular nuclei is supplied by only one part of the labyrinth. Some separation exists between the termination of afferents from different parts of the receptor, and even where there is overlap there is often a quantitative difference between the terminations of the various nerves. Nevertheless, even on the basis of direct connections only, ample opportunity seems to exist for interaction between inputs arising in the utricular macula and the cristae of the three canals.

B. ELECTROPHYSIOLOGICAL STUDIESOF ON

CELLSIN

THE

THE

LABYRINTHINE INFLUENCE

VESTIBULAR NUCLEI

1. Experiments with Electrical Stimulution Stimulation of the vestibular nerve with single shocks excites some cells in the vestibular nuclei monosynaptically, others polysynaptically; some cells are inhibited. An additional group of cells is not affected by vestibular nerve stimulation. Among them may be cells with a vestibular input subthreshold for firing, missed in studies with extracellular recording. But there do seem to be cells devoid of significant vestibular input, located particularly in regions of the nuclei lacking an input from the vestibular nerve. a. Ipsilateral Actions

Precht and Shimazu (1965) made the first detailed analysis of singleunit activity and field potentials produced in the vestibular nuclei by stimulation of the vestibular nerve. The fields (Fig. 1) consist of a positive or positive-negative P wave, indicating the arrival of afferent impulses, followed by a negative N 1 potential consisting of monosynaptically-evoked activity. In some areas there is a later N2 potential, due to polysynaptic activity. The latencies of these components vary somewhat with recording location; that of the N1 potential usually ranges from O.gl.2 msec. The threshold of the N 1 potential is similar to the threshold of the largest vestibular nerve fibers. Because this potential

31

VESTIBULAR PATHWAYS a

b

-

C

I

FIG. 1. Field potentials and single-unit firing evoked in Deiters' nucleus by stimulation of the ipsilateral labyrinth. Each frame consists of several superimposed sweeps. a: l/sec. A cell is fired monosynaptically by labyrinthine stimulation and responds to every shock; there is a large P wave right after the stimulus artifact. b: 5/sec. Cell misses occasionally, revealing underlying N1 potential. c: 2O/sec. Cell no longer responds, but a sizable N1 remains. Time mark, insec; voltage calibration, 500 pV. Negative deflection upward (Wilson et ol., 1967a).

is easier to monitor than the P wave, the strength of vestibular nerve stimulation has usually been expressed as a multiple of N1 threshold ( XNlT). i. Monosynaptic responses of vestibular neurons. Stimulation of the vestibular nerve with single shocks results in short latency synaptic potentials and firing of vestibular neurons. Synaptic potentials with latencies of approximately 0.6-1.0 msec, and extracellular spikes superimposed on the N1 potential with latencies approximately 0.8-1.5 msec (Fig. 1), are monosynaptic. These monosynaptic effects have been studied in all four vestibular nuclei. In some of the investigations an important objective was a study of the properties of the synapses between vestibular afferents and second-order neurons; in others a correlation between responses to electrical and to natural stimulation; in still others a systematic mapping of the location of monosynaptically driven cells, and determination of the relation between vestibular input to the cell and destination of its axon. The distribution of monosynaptic responses wiIl be considered first. Location and prevalence of monosynaptically driven cells. The distribution of monosynaptic activity within the vestibular nuclei displays a pattern that is closely related to the pattern of vestibular nerve terminals observed by anatomists. Although techniques for electrical stimulation of the different ampullae (Cohen and Suzuki, 1963) and of the utricle or its nerve (Suzuki et al., 1969; Fluur and Mellstrom, 1971) have been described, they have so far not been used to map the location of

32

VICTOR J. WILSON

neurons responding to electrical stimulation of different parts of the labyrinth or to study convergence from these different parts onto individual neurons. Only the location of cells responding to electrical stimulation of the labyrinth or vestibular nerve as a whole has been studied.

DEITERS’ NUCLEUS. Wilson et al. (1967a), Peterson (1969, 1970), and R. M. Wylie and L. P. Felpel ( 1969, unpublished data), used extracellular recording to study monosynaptic responses of Deiters’ cells. The location of the cells was determined by referring to dye marks deposited at the bottom of electrode tracks. In their combined results, 97/249 (39%) of Deiters’ cells were driven monosynaptically by stimulation of the labyrinth. As extracellular recording was used, cells with a subthreshold vestibular input were classified as not driven by vestibular stimulation. Because the labyrinthine input was powerful and often caused firing of otherwise silent cells, it is unlikely that this limitation resulted in serious underestimation of the number of cells with monosynaptic input. In confirmation of this view, Ito et al. (1969a), who sampled cells for the presence of monosynaptic e.p.s.p.’s and did measure effects subthreshold for firing, found e.p.s.p.’s in 44/151 (29%) of Deiter’s neurons. Within Deiters’ nucleus, the monosynaptic vestibular input is found mainly ventrally rather than dorsally, on cells within a wide range of sizes, as shown both with extracellular recording (Table I and Fig. 2b, p. 45) and with intracellular sampling of synaptic potentials (It0 et al., 1969a). This result is in excellent agreement with anatomical findings (Walberg et al., 1958; Mugnaini et al., 1967). The functional consequences of this distribution will be discussed later, in connection with the lateral vestibulospinal tract. MEDIALNUCLEUS. Cells in this nucleus fired monosynaptically by vestibular nerve stimulation have been seen by Shimazu and Precht (1965), Wilson et al. (1968b), Markham (1968), and Peterson (1970), while TABLE I 1)ISTRIRUTION

O F CELLS D R I V E N B Y L . 4 B Y R I N T H I N E S T I M U L A T I O N WITHIN

DEITERS’NUCLEUS. Monosynaptic firing

Dorsal Deiters’ Ventral Deiters’ All cells

Polysynaptic firing

Ratio

Percentage

Ratio

Percentage

14/177 83/132 97/249

12 63 39

14/177 18/132 32/249

12 14 13

* Data derived from the results of Wilson d al. (1967a), Peterson (1970), and Wylie and Felpel (1969).

33

VESTIBULAR PATHWAYS

TABLE I1 DISTRIBUTION OF CELLSDRIVENBY LABYRINTHINE STIMULATION WITHIN THE MEDIALNUCLEUS~ Monosynaptic firing Number of cells Rostra1 third Middle third Caudal third All cells

64 143 46 253

Polysynaptic firing

Number

Percentage

Number

Percentage

31 51 4

48 36 9

55 41 54

86

34

35 59 25 119

47

a Data from the results of Wilson et al. (1968b). Cells that fired twice (33), monoand polysynaptically, are included in the totals of both columns.

monosynaptic e.p.s.p.’s have been recorded by Kawai et al. (1969). Studied extracellularly, 86/253 (34%)of medial nucleus neurons were fired monosynaptically (Wilson et al., 1968b). They were almost completely absent from the caudal third of the nucleus, and were most concentrated in its rostral half (Table 11). Once again these results are in good agreement with anatomical findings (Walberg et al., 1958; Stein and Carpenter, 1967). DESCENDING NUCLEUS. This nucleus has not been studied as exhaustively as the two previously mentioned, but the presence of monosynaptic input to some of its cells has been noted (Wilson et al., 1967b; Kawai et aZ., 1969; Peterson, 1969, 1970). Peterson (1970) observed that 26/49 (53%) of the cells were driven monosynaptically, and that these cells were located primarily in the more rostral region of the nucleus.

SUPERIORNUCLEUS. This is the least studied of all the vestibular nuclei. Cells excited monosynaptically were seen by Shimazu and Precht ( 1965), Markham (1968), Kawai et al. (1969), and Peterson (1969, 1970). Properties of the relay between vestibular afferents and neurons in the nucbi. In the cat synapses between vestibular afferents and second-order neurons are all excitatory, and all seem to be the chemical type (Eccles, 1964). The possibility exists that in other animals there may be electrical as well as chemical synapses between vestibular afferents and secondorder neurons. Some evidence for the presence of an electrical synapse in the pigeon has been presented recently (Wilson and Wylie, 1970). The monosynaptic labyrinthine input to neurons in the vestibular nuclei is a strong one that can dominate the behavior of the cells. In Deiters’ nucleus the mean firing threshold of extracellularly recorded spikes is 1.7 2 0.8 (mean k SD) x N1T and any cells fired at all can fire with a probability of 100%if the stimulus is strong enough. The

34

VICTOR J. WILSON

monosynaptic e.p.s.p. recorded in Deiters’ neurons is apparently due to convergence of a large number of afferent fibers (lower limit estimated to be 25 by Ito et al., 1969a). Cells may fire at stimulus strengths very near the afferent and N1 threshold, indicating either that only part of this converging input is required to reach threshold, or that many low-threshold fibers can be recruited even with very weak shocks. The same situation prevails, even to a greater extent, in the other vestibular nuclei. Unitary e.p.s.p’s in cells in the medial, superior, and descending nuclei tend to be larger than in Deiters’ neurons (Kawai et al., 1969), and firing threshold is reached with very weak stimuli (Precht and Shimazu, 1965; Wilson et al., 1968b). Most likely an important reason for the difference in unitary e.p.s.p. amplitude is that the cells in the other nuclei tend to be considerably smaller than Deiters’ neurons and therefore have a higher input impedance: the same synaptic input would produce a larger synaptic potential. Such a correlation between size of unit e.p.s.p., input impedance, and cell size has been reported in spinal motoneurons (cf. Kuno and Miyahara, 1969). ii. Polysynaptic responses of Vestibular neurons: Excitation. Singleshock electrical stimulation of the labyrinth or vestibular nerve evokes, in many neurons in all four nuclei, e.p.s.p.’s with a latency longer than 1 msec (Ito et al., 1969a; Kawai et al., 1969). These potentials lag behind monosynaptic e.p.s.p.’s by one synaptic delay, and are therefore disynaptic. In Deiters’ nucleus disynaptic e.p.s.p.’s differ from monosynaptic ones by having a longer time to peak and falling phase, and smaller amplitude. Their threshold, however, is similar to that of monosynaptic responses, indicating that they are not produced by activation of a population of afferent fibers with higher threshold and slower conduction velocity (Ito et al., 1969a). Later synaptic potentials, some apparently due to repetitive firing of the vestibular nerve, have also been observed (Ito et al., 1969a). Polysynaptic activation has also been studied extracellularly. Precht and Shimazu (1965) observed that some neurons in the superior and rostra1 medial nuclei fired later than monosynaptically driven ones, although a t similar threshold. With strong shocks many cells fired both mono- and polysynaptically. Medial nucleus neurons were also studied by Wilson et (11. (1968b) : 22% of neurons responded monosynaptically, 33%polysynaptically, and 13%mono- and polysynaptically (Table 11). Whereas the firing probability of all cells with monosynaptic input reached 100%with sufficient stimuli, that of many neurons with polysynaptic input did not. Firing probability of polysynaptic cells rose much more gradually with increases in stimulus strength than firing probability

VESTIBULAR PATHWAYS

35

of monosynaptic cells, suggesting a need for more spatial summation in the former. Both in medial nucleus and in Deiters’ cells (Wilson et al., 1967a) the average threshold of polysynaptically driven cells was 1.6 times the threshold of monosynaptically driven cells, another indication that more summation is required to fire the polysynaptic cells, a finding in agreement with the observation of Ito et al. (1969a) that polysynaptic e.p.s.p.’s are smaller than monosynaptic ones. The higher threshold of polysynaptically fired neurons, also observed by Cook et al. (1969a) for vestibular neurons projecting rostrally in the MLF, contrasts somewhat with Precht and Shimazu’s observations. Anesthesia does not seem to be responsible for this difference: although the animals of Wilson et al. (1967a, 1968b) were anesthetized with chloralose, those of Cook et al. (1969a), like those of Precht and Shimazu (1965), were decerebrate. The difference may be due to sampling, or to a different relationship between nerve and stimulating electrode. Polysynaptic activation of vestibular neurons is usually less powerful than monosynaptic activation, but it is more widely distributed within the vestibular nuclei. A significant number of polysynaptically fired neurons have been observed in all the vestibular nuclei (Peterson, 1969, 1970). In addition, a polysynaptic input has been observed in both dorsal Deiters’ and in the caudal medial nucleus (Tables I and 11), regions lacking significant vestibular terminals and monosynaptic input (Ito et al., 1969a; Wilson et al., 1968b). Inhibition. Stimulation of the vestibular nerve inhibits some neurons in the ipsilateral nuclei, apparently by activation of inhibitory interneurons located within the nuclei (Shimazu and Precht, 1966). All i.p.s.p.’s and inhibition of cell firing that have been observed in response to stimulation of the ipsilateral vestibular nerve are at least disynaptic (Ito et uZ., 1969a; Xawai et al., 1969; Wilson et al., 1968b). b. Contralateral Actions Stimulation of the vestibular nerve produces localized field potentials in the ventral part of the contralateral vestibular nuclei. These fields are evoked by stimuli too weak to cause significant activity in the reticular formation and are abolished by shallow midline cuts beneath the fourth ventricle ( Shimazu and Precht, 1966). Similar stimuli produce excitation and inhibition of single units in the contralateral nuclei. Shimazu and Precht ( 1966) studied commissural inhibition in cells affected by horizontal angular acceleration. As will be discussed below, two groups of cells affected in opposite fashion by horizontal acceleration

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VICTOR J. WILSON

are type I and type I1 cells, the former excited and the latter inhibited by activation of ipsilateral horizontal canal receptors. It is the type I units that are inhibited by natural or electrical activation of fibers from the contralateral horizontal canal. Shimazu and Precht suggested that the inhibition is executed by axons of vestibular nucleus neurons crossing the midline and terminating on cells of the vestibular nuclei on the other side. Commissural fibers have recently been observed and described in detail by Ladpli and Brodal ( 1968). Originally three synapses were postulated for commissural inhibitory pathways: between vestibular afferents and second-order neurons in the ipsilateral nuclei; between the commissural axons of these cells and inhibitory neuron- in the nuclei on the other side; and between these inhibitory cells and the target, type I neurons (Shimazu and Precht, 1966). The role of inhibitory neurons was filled by type I1 cells, activated by contralateral vestibular nerve stimulation early enough to account for inhibition of type I cells. Subsequent intracellular investigation has confirmed the existence of such a pathway (Kasahara et al., 1968). In addition, however, some commissural fibers themselves are inhibitory, and there is monosynaptic inhibition between the nuclei of the two sides (Wilson et al., 1968b; Kasahara, et al., 1968; Mano et al., 1968). Therefore there are inhibitory neurons in the vestibular nuclei with axons terminating beyond the confines of the nucleus of origin; some of these neurons appear to be in the medial nucleus (Mano et al., 1968).

Summary. Neurons in many regions of the nuclei, especiaIly those areas receiving primary vestibular fibers, are strongly activated monosynaptically by stimulation of the ipsilateral vestibular nerve. Some of these cells, and others, are activated polysynaptically. This input is not as strong as, but is more widespread than, the monosynaptic one, being found in regions devoid of vestibular nerve terminations as well as in regions where terminals are present. There are also cells not excited by stimulation of the ipsilateral vestibular nerve. Some neurons are inhibited by stimulation of the ipsilateral vestibular nerve, and the inhibitory pathway is always a t least disynaptic. Vestibular neurons are also influenced by pathways originating in the contralateral labyrinth. The first relay in these pathways is in the contralateral nuclei. Neurons in these nuclei give rise to commissural fibers that cross the midline. In some cases the commissural fibers are inhibitory; in others they are excitatory and activate inhibitory neurons. The ipsilateral and commissural connections provide the pathways linking the labyrinth with second- and higher-order neurons. Activation of these pathways by natural stimulation is considered next.

VESTIBULAR PATHWAYS

37

2. Experiments with Natural Stimulution

Adrian ( 1943) first observed responses of mammalian (cat) vestibular neurons to position change and to angular acceleration. Gernandt ( 1949) examined responses to horizontal acceleration in somewhat more detail. The comprehensive investigations of Duensing and Schaeffer ( 1958, 1959) on the rabbit demonstrated the variety of responses that could be observed in vestibular neurons and provided a classification of these responses that has been used by subsequent experimenters. Duensing and Schaeffer studied the effects of horizontal angular acceleration and of tilt. Considering acceleration toward the recording side (ampulla trailing, ampulopetal flow of endolymph) as ipsilateral, and acceleration away from the recording side as contralateral, the following response types were observed ( designates excitation, - desigcontra -; 11, ipsi - contra +; 111, ipsi + nates inhibition): I, ipsi contra f ; IV, ipsi - contra - (Duensing and Schaeffer, 1958).Type I units were the most common and there was a considerable number of type I1 units, while the other types were rare. Different response types were found distributed throughout the vestibular nuclei, but not in a uniform manner. A similar classification of responses to lateral tilt was also described ( Duensing and Schaeffer, 1959). Considering ipsilateral tilt around the naso-occipital axis to indicate that the recording side is tilted down, the classification of responses was as follows: a, ipsi + contra -; j3, ipsi - contra 7, ipsi + contra +; 8, ipsi - contra -. The a units were the most frequently encountered, and once again the different types of units were found throughout the vestibular nuclei. An interesting observation was that the same cells could be affected by both horizontal acceleration and lateral tilt. Because the responses of cells to electrical stimuli were not studied, the pathways responsible for this convergence remained unknown. More recently Duensing ( 1968a,b) has also studied and classified responses to vertical acceleration, due to activation of the anterior and posterior canals. Convergence from static receptors on neurons activated by vertical acceleration was demonstrated. Responses of vestibular neurons to natural stimulation, both acceleration and tilting, have now been explored in detail. The responses have been related to the nature of the pathway linking the labyrinth to neurons in the vestibular nuclei, and to the location of the cells within the nuclei.

+

+

+;

a. Responses t o Acceleration

Responses to horizontal acceleration have been described by Shirnazu and Precht (1965, 1966; Precht and Shimazu, 1965). They recorded

38

VICTOR J. WILSON

mainly in an area including the superior nucleus and the rostral part of the medial, some cells also being found in the ventral part of the lateral nucleus and the rostral part of the descending. Their search was not comprehensive and does not rule out the possibility of responses to horizontal acceleration in other regions of the nuclei, but the finding of many such responses in the superior and rostral medial nuclei is in excellent agreement with the observations of Stein and Carpenter (1967) and Gacek (1969). Of the cells studied by Shimazu and Precht (1965) 67%were type I neurons and 29% type I1 neurons. Type I cells were divided into two categories. A large fraction showed resting activity, low threshold to acceleration, and only moderate further increases in activity with higher rates of acceleration: these were tonic neurons. A smaller group showed little activity at rest, high threshold, and marked further activation a t higher rates of acceleration: these were kinetic neurons. The differences in behavior were related to the type of afferent input received by the cells: while separation was not absolute, most kinetic cells were activated monosynaptically by stimulation of the vestibular nerve, most tonic cells polysynapticaIly (Precht and Shimazu, 1965). It would be of interest to see whether tonic cells are present throughout the medial nucleus while kinetic ones are restricted to its rostral half, the distribution observed for polysynaptically and monosynaptically driven cells (Wilson et al., 1968b). The differences between the responses of tonic and kinetic neurons may, however, be due to factors other than differences in the vestibular afferent input during acceleration. Jones and Milsum (1970) observed that all type I and type I1 neurons respond homogeneously to sinusoidal horizontal rotation in the range of 0.251.7 Hz. Some of the differences in the responses to natural stimulation, as well as the higher threshold and lack of spontaneous activity of kinetic cells, may be due to the lower level of excitability of the latter. This leaves the question of the factors responsible for the higher excitability level of cells with polysynaptic input from the labyrinth. Type I1 cells, inhibited by ipsilateral acceleration, are still found when the ipsilateral labyrinth is destroyed: their input may come from the contralateral labyrinth (Shimazu and Precht, 1966). They may be activated by electrical stimulation of the contralateral vestibular nerve with a Iatency as brief as 3.2 msec, and many are located in the ventral part of the medial nucleus, a region supplied by commissural fibers (Shimazu and Precht, 1966; Ladpli and Brodal, 1968). As already described, some type 11 neurons appear to be inhibitory cells acting on type I cells. Comniissural inhibition reveals another difference between tonic and kinetic nciirons. The former are inhibited trisynaptically

VESTIBULAR P A T W A Y S

39

via type I1 neurons, the latter disynaptically by inhibitory comniissural fibers ( Shimazu, 1967; Kasahara et ul., 1968). It will be appreciated that ipsilateral horizontal acceleration increases the firing of type I neurons in two ways. increase in the activity of the ipsilateral receptors, causing monosynaptic or polysynaptic excitation of type I neurons; and decrease of activity of the contralateral receptors, leading to removal of contralateral inhibition, that is, to disinhibition. Very similar findings have been made with respect to the anterior canal, except that there are fewer units affected by the anterior than by the horizontal canal, by factor of 1:2 (Markham, 1968). Type I anterior canal neurons (excited by acceleration with the ampulla leading) were located in the superior nucleus and in the rostral part of the medial, often very near-and found in the same electrode track as-units affected by horizontal acceleration. This is consistent with the finding that the terminations of afferents from these two canals overlap within the vestibular nuclei (Stein and Carpenter, 1967; Gacek, 1969).

b. Responses to Position Change Responses of vestibular neurons to tilt, and the relation between these responses and the location of the neurons and the input they receive from the vestibular nerve, have been studied by Peterson (1969, 1970) in decerebellate cats. Cells affccted by lateral and by forward-backward tilting were found in all four vestibular nuclei. They were most likely to be found in ventral Deiters’ nucleus and in the rostral part of the descending nucleus, however, and it is in these regions that the responses to tilt were largest. Furthermore, the biggest responses were found in cells located in areas supplied by primary vestibular afferents, specifically in cells with monosynaptic input from the labyrinth. Peterson’s ( 1969, 1970) results indicate that utricular fibers terminate particularly in Deiters’ iiucleus and in the descending nucleus. There is therefore agreement with the anatomical findings of Stein and Carpenter (1967) rather than with those of Gacek ( 1969), who stressed the medial nucleus as an important destination of utricular afferents. Deiters’ cells strongly affected by tilting were found almost exclusively in the ventral half of the nucleus and showed either cr or ,8 responses to tilt (see also Fujita et al., 1968). Unlike the crista of the semicircular canals, where all the receptors ale oriented in a similar manner, the utricular inacula is so arranged that tilt to one side will produce cy responses of some hair cells and p responses of others (Flock, 1964). It is presumed that the response of monosynaptically driven Deiters’ neurons to tilt reflects the behavior of afferents from the utricle (Peterson, 1970). Deiters’ cells with polysynaptic input from the labyrinth are much

40

VICTOR J. WILSON

more weakly affected by tilt than monosynaptic cells. They are found in dorsal as well as ventral Deiters’ and show other responses in addition to the a and p type. Presumably such behavior is due to convergence of input from a and p monosynaptic cells as well as from more complex excitatory and inhibitory pathways, including those from the contralateral labyrinth. Summary. Responses to angular acceleration and to position change have been observed in various regions of the vestibular nuclei. In general these responses have been found in areas that could be predicted from anatomical work: units iduenced by horizontal and vertical acceleration especially in the superior nucleus and in the rostra1 part of the medial; units influenced by tilt especially in the ventral part of Deiters’ nucleus and in the rostral part of the descending. The properties of the response are related to the nature of the pathway linking the cells to the labyrinth. The difference between mono- and polysynaptically driven neurons with respect to their responses to naturaI stimulation is more marked for responses to position change than for responses to angular acceleration. Ill. The Vestibular Nuclei and the Cerebellum

The cerebellum exerts a most important inff uence on the activity of the vestibular nuclei. This is mediated in part by a two-way flow of impulses between labyrinth, vestibular nuclei, and cerebellum, carried by primary and secondary vestibulocerebellar fibers and by a powerful cerebellovestibular projection. Detailed consideration of cerebellar connections, structure, and function is beyond the scope of this review; the purpose of this brief section is to outline the anatomy, and excitatory or inhibitory nature, of the main links between vestibular centers and cerebellum. Some further details of the organization of these connections, as well as some aspects of their functional significance, will be discussed in the sections dealing with vestibular projections to the spinal cord and to extraocular motoneurons. A. VESTIBULOCEREBELLAR PATHWAYS

Vestibulocerebellar fibers are divided into two catagories: primary, originating in the labyrinth, and secondary, originating in the vestibular nuclei. The termination of primary vestibular afferents in the cerebellum is an indication of the close relation between the latter and vestibular centers, as such terminations are not present to any extent in any other structures outside the vestibular nuclei. The distribution of primary fibers in the cerebellum has most recently been traced, with

VESTIBULAR PATHWAYS

41

silver impregnation, by Brodal and HZivik (1964). That paper, as well as the monograph of Brodal et al. (1962) should be consulted for references to earlier work. The fibers end mainly as ipsilateral mossy fibers in the flocculus, paraflocculus, nodulus, and uvula, thus defining these regions as the vestibulocerebellum; there are also some terminations in the dentate nucleus. Secondary vestibulocerebellar fibers arise from the medial and descending nuclei (mainly from the latter); they terminate in the fastigial nucleus, and as mossy fibers (Precht and Llinis, 1969a) in parts of the vestibulocerebellum ( Brodal et aZ., 1962). Electrophysiological studies, however, indicate that the region of the cerebellum affected by labyrinthine activation is wider than might be expected from these anatomical findings. Electrical stimulation of the vestibular nerve produces ipsilateral and contralateral activation from the 3rd to the 7th cerebellar folia, and electrical activity in these regions is modified by both tilt and horizontal acceleration ( Ajala and Poppele, 1967). Whereas electrical stimulation of the vestibular nerve with single shocks excites Purkinje ceIls only via the mossy fiber pathway (Precht and Llinb, 19694, caloric and galvanic stimulation activate both mossy and climbing fiber pathways, the latter probably by a complex route (Ferin et al., 1971). There has been some single-unit work on the effects of natural vestibular stimulation in the frog, where primary vestibulocerebellar fibers terminate as both mossy and climbing fibers (Precht and Llinh, 1969a). Purkinje cells that behave like type I, 11, 111, and IV vestibular neurons to horizontal acceleration have been seen in various regions of the frog cerebellum (Precht and Llinis, 1969b). Further work along these lines in the cat is needed. B. CEREBELLOVESTIBULAR PATHWAYS As emphasized by Brodal (1964), these pathways are far more abundant than vestibulocerebellar ones. In addition, they arise from regions other than the vestibulocerebellum, anatomically the principal target of vestibulocerebellar axons.

1. Projections of the Cerebellar Cortex One region of the cortex projecting to the vestibular nuclei is the vestibulocerebellum (cf. Angaut and Brodal, 1967). Of the four nuclei, Deiters’ is poorly supplied whereas the others receive dense terminations. There is a somatotopic organization in the projection that will be considered to some extent in subsequent sections. Fibers to the vestibular nuclei also arise from the vermis of the anterior and posterior lobes, especially from the former. The main target of these fibers is (dorsal) Deiters’ nucleus, with significant terminations also seen in the

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VICTOR J. WILSON

descending nucleus (Brodal et al., 1962). Cells in these nuclei are therefore under the control of an area of the cerebellum whose main input is not vestibular, but rather somatic (Brodal, 1967). All the cortical projections exert a direct inhibitory influence on neurons in the vestibular nuclei. Wherever their action has been tested, Purkinje cells have been shown to inhibit monosynaptically the cells with which they synapse: they are inhibitoiy neurons (Ito and Yoshida, 1966; Ito et al., 1966, 1968, 1970a; Kidokoro, 1968). In the frog, Purkinje cells have also been shown to innervate vestibular hair cells and to inhibit them ( L l i n h and Precht, 1969). There is no evidence that Purkinje cells contribute to efferent fibers supplying vestibular receptors in the cat. 2. Projections of the Fastigial Nucleus This nucleus projects, in complex somatotopic fashion, to the ipsilateral and contralateral vestibular nuclei (Brodal et al., 1962). This is an excitatory projection (Ito et al., 1970d) by means of which a variety of inputs, including that from the cerebellar cortex, can regulate the activity of vestibular neurons. IV. Projections of the Vestibular Nuclei to the Spinal Cord

Stimulation of the labyrinth or vestibular nuclei evokes a variety of effects in the body musculature (cf. Brodal et al., 1962), some of which are produced by two separate projections linking the vestibular nuclei to the spinal cord. One is the vestibulospinal tract, originating in Deiters’ nucleus, sometimes called the lateral vestibulospinal tract ( Nyberg-Hansen, 1966, 1970). The other arises in the medial and descending nuclei and runs caudally in the medial longitudinal fasciculus (ML F ) . This group of fibers has been termed the medial vestibulospinal tract ( Nyberg-Hansen, 1966, 1970). These two descending systems will be considered in sequence with particular emphasis on their origin, their functional organization, and the synaptology of their connections with motoneurons. VESTBULOSPINAL TRACT A. THE LATERAL

1. Origin a d Functional Organization of the Tract The classical vestibulospinal tract ( VST ) originates entirely in Deiters’ nucleus, is strictly homolateral, and extends caudally as far as the lumbosacral cord (Brodal et al., 1962). On the basis of the location of cells showing retrograde changes following sections performed at different levels of the spinal cord, Pompeiano and Brodal (1957) concluded that Deiters’ nucleus was subdivided somatotopically. Although

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43

overlap existed, cells sending axons to the lunibosacral segments were in the dorsocaudal part of the nucleus, made up chiefly of giant neurons (“hindlimb area”); cells with axoils to the thoracic cord in the ventral region of the caudal third; and cells with axons to the cervical cord in the rostra1 third and the ventralmost part of the middle third (“forelimb area”). Considering the distribution of afferents within thc iiucleus such a somatotopic arrangement has predictable consequences: there should be a high probability of direct contact between labyrinthine afferents and ventral Deiters’ neurons projecting mainly to the ccivical thoracic cord; and between Purkinje cell axoiis and dorsocaudal Deiters’ neurons projecting mainly to the lumbosacral cord. The presence of a somatotopic division of Deiters’ nucleus is also indicated by electrophysiological investigations. It proved convenient to subdivide VST neurons into two groups: those projecting to the lumbosacral cord ( L cells) and those projecting to the cervicothoracic cord ( C cells). L cells have usually been activated by stimulation of the spinal cord at L1, or a t L3-L4; C cells have been identified by response to a stimulus at C3-C4, and lack of response to the lumbar stimulus. This subdivision has the drawback that cells supplying the cervical enlargement and thoracic cord are usually lumped together. A number of cells in Deiters’ nucleus do not respond antidromically to stimulation of the spinal cord (Wilson et ul., 1967a; Peterson, 1969, 1970). This does not necessarily indicate that there are cells not projecting to the cord: first because the thresholds of some axons at the levels stiniulated may not be reached, and second because stimulation at C3 or below will not affect the axons of cells ending above this point. Since the upper cervical segments control the neck musculature, which is closely tied to the labryriiith (see below), it must be assumed that axons or axon collaterals of a reasonable number of VST cells end in this region. Lack of information about these cells in all experiments that have been performed is regrettable. Antidromic activation of L cells produces negative field potentials in Deiters’ nucleus that are greatest in the dorsal half of the nucleus, whereas activation of C cells produces field potentials that are greatest in the ventral half (Fig. 4 in Ito et al., 1964a). Extracellular studies show that the dorsocaudal region of the nucleus consists inaiiily of L cells (Wilson et d.,19674. Overall, if the dorsal and ventral halves of the nucleus are considered without regard for rostrocaudal differences, the summed data of Wilson et ul. (1967a) and Peterson (1969) show the following distribution of cells: dorsal half 75L, 31C; ventral half 52L, 64C. Therefore L cells outnuinber C cells by a margin greater than 2: 1 dorsally, whereas C cells somewhat outnumber L cells ventrally.

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VICTOR J. WILSON

The soniatotopic arrangement is therefore rather blurred, although it becomes a little clearer when the dorsal and ventral halves are subdivided further (Fig. 2a). There is a variety of inputs to Deiters’ neurons, including those froin “higher centers” and the reticular formation ( Brodal et al., 1962). Relatively little is known about inputs other than those from the labyrinth, cerebellum, and periphery, whose effects can now be related to the two populations of VST cells.

a. Inputs from the Labyrinth The vestibular nerve makes monosynaptic contact with many VST neurons, as first shown by Ito and his colleagues (1964a; 1969a). Because monosynaptically activated neurons are found mainly ventrally in Deiters’ nucleus we might expect the monosynaptic input to be more prevalent among C than among L cells. This point has been investigated by Wilson et al. (1967a) who found that 51%of C cells, and only 22% of L cells, were fired at monosynaptic latency by stiniulation of the labyrinth. This difference was due entirely to the location of many L cells in the dorsal half of the nucleus, since C and L cells present in the ventral half were equally likely to receive a monosynaptic input. Polysynaptic labyrinthine activation is found in both dorsal and ventral Deiters’ nucleus ( 12%and 14%of neurons, respectively, Table I ) , and is seen in both L and C neurons (Wilson et al., 1967a). Not only is there a quantitative difference in the labyrinthine input to different types of VST neurons, but the activity of VST neurons in general is not homogeneously dependent on the labyrinth. Whereas destruction of Deiters’ nucleus strongly affects decerebrate rigidity as a whole (Brodal et al., 1962), destruction of the labyrinths in decerebratedecerebellate cats abolishes only opisthotonus, not limb rigidity ( Batini et al., 1957). It may be inferred that the Deitersian limb projection receives numerous inputs that maintain an adequate level of excitability in the absence of the labyrinth, whereas the neck projection is strongly labyrinth-dependent. How are the patterns of connections between labyrinth and VST neurons reflected in the response of these neurons to natural stimulation? There has been little work on the spinal projection of neurons influenced by horizontal rotation, except for the demonstration that some type I1 and type 111 cells (but not type I cells) can be fired antidromically by VST stimulation, but none by stimulation at the lumbar level: they are apparently C cells (Precht et al., 1967b). Peterson (1970) showed that C and L cells, as well as cells not responding to antidroniic stimulation, were affected by tilt. There was

45

VESTIBULAH PATHWAYS

a

D i s t r i D u t l o n of S p i n a l Projecilon Number of cells a t different depths

L

C

cells cells

-

A

9

2

14

9

5

10

I

6

Venlrol

Distribution of Labyrinthine Input

b

M e a n responses a 1 different d e p t h s

cI

. ".:// . . . Ventral

P r o j e c t ion

A

L cells C cells N cells

Mean response

+2/+2 +4/-3 +5/- I

-

+ 6 / -I

-

+a/-6

Labyrinthine inpul

Mean response

o POLY cells

+ 9/-7 t41-I

x N O N cells

t

MONO c e l l s

1/+1

FIG. 2. Relationship between anatomical organization of Deiters' nucleus and responses of Deiters' neurons to tilting. a : Distribution of 29 L cells and 27 C cells superimposed on a schematic lateral projection of Deiters' nucleus, Numbers of each type of cell in four dorsal-ventral segments of the nucleus are given at the right. b: Distribution of 74 cells classified as responding to labyrinthine stimulation monosynaptically ( MONO ), polysynaptically ( POLY ), or not at all ( N O N ). Alongside the section the mean responses to tilting of cells in four dorsal-ventral segments of the nucleus are expressed as fractions with mean response (change of firing rate in spikes per second) to 20' ipsilateral tilting over mean response to 20' contralateral tilting. c: Syinbols used in plotting locations of various types of cells are shown together with mean responses of those cell types to lateral tilting, expressed as in part b (Peterson, 1970).

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VICTOR J. WILSON

considerable variability in the responses of individual VST cells, but a pattern emerged when populations were considered (Fig, 2). The mean response of C cells had an a pattern (ipsi +, contra - ) : this resembled the pattern exhibited by cells located in ventral Deiters’ nucleus, driven monosynaptically by stimulation of the labyrinth. In contrast, the mean response of L cells was smaller and had a y pattern (ipsi +, contra + ): this is the average response pattein of cells in dorsal Deiters’ nucleus, fired by single shocks to the labyrinth either polysynaptically or not at all. Therefore the somatotopic organization of Deiters’ nucleus, combined with the differential distribution of vestibular afferents within the nucleus, leads to processing of tilt-evoked labyrinthine activity with the following results: activity with an pattern is relayed to segments controlling forelimb and back muscles, weaker activity with a y pattern to segments controlling hindlimb muscles. Because VST cells innervating the neck segments are probably located rostroventrally in Deiters’ nucleus, it is likely that strong activity with an pattern is relayed by this group of neurons. (Y

(Y

b. Inputs from the Cerebellum Deiters’ nucleus receives an inhibitory input from the cerebellar cortex and an excitatory one from the fastigial nucleus. Ipsilateral fastigial axons distribute over all of Deiters’ nucleus, but especially in its dorsal half; contralateral axons terminate in the ventral half of the nucleus (Brodal et al., 1962). In agreement with this arrangement fastigial stimulation activates ventral and dorsal cells in Deiters’ (Ito et al., 1970d). Therefore inputs activating the ipsi and contralateral fastigial nuclei should be able to modulate the activity of VST cells in general, presumably in a manner reflecting the somatotopic arrangement of the fastigiovestibular projection. There has been more investigation of the influence of the cerebellar cortex on Deiters’ cells. The most important part of this projection is that from the vermis of the anterior lobe, known to supply mainly the dorsal region of the nucleus (Brodal et al., 1962; Walberg and Mugnaini, 1969). An additional, smaller, projection is that from the vestibulocerebellum: a few fibers from the nodulus and uvula reach the dorsocaudal part of Deiters’, a somewhat greater number of floccular fibers reach the rostroventral part ( Angaut and Brodal, 1967). Ito and his colleagues (19sS) have studied the effect of anterior vermal stimulation on VST cells, but did not distinguish between C and L neurons. The results illustrated in their Fig. 5 (p. 318) show that most inhibited cells are located dorsally to the mean depth of all cells studied, almost all noninhibited cells ventrally. There are, however, a reasonable number of

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inhibited cells in the ventral half of their sampling area, and some are found almost as ventrally as the deeper noninhibited neurons. Inhibition resulting from activity of Purkinje cells in the anterior lobe vermis therefore should affect a large fraction of L cells, a smaller fraction of C cells. Wylie and Felpel (1971), studying VST neurons whose projection also was not identified more specifically, deduced from the effect of cerebellectomy on the distribution of spontaneous activity within Deiters’ nucleus and from the inhibitory effects of peripheral nerve stimulation (relayed uia the cerebellum) that most dorsal cells were under an inhibitory influence from the cerebellum, while many ventral cells were not. But some cells even in the most ventral part were inhibited, so the subdivision of the nucleus was not sharp. Their picture of the distribution of inhibition is therefore very similar to that obtained by Ito et aE. (1968) from stimulating the anterior lobe. It is reasonable to draw the inference that to a varying degree cerebellar inputs to Deiters’ nucleus, both excitatory and inhibitory, modulate the activity of vestibulospinal projections to all levels of the spinal cord even though there is no direct evidence from recent singleunit work. When Pollock and Davis (1927) described the behavior of animals decerebrated by the anemic method, they pointed out that opisthotonus of a degree never observed in ordinary decerebrates was present in decerebro-decerebellate animals. The optisthotonus, which was due to tonic vestibular reflexes as it disappeared on destruction of the labyrinths, was undoubtedly due to pathways relaying in Deiters’ nucleus (see p. 56). Pollock and Davis concluded that “the cerebellum as a whole inhibits in a general way tonic labyrinthine reflexes,” and their results suggest that even Deiters’ neurons innervating the neck segments are subject to this inhibition.

c. Somatosensory Inputs The activity of Deiters’ neurons can be modified by inputs from the periphery reaching the nuclei by means of pathways ascending the spinal cord. One route by which somatosensory inputs may reach VST cells consists of the spinovestibular fibers described by Pompeiano and Brodal (1957). These fibers arise mainly from the caudal region of the spinal cord, are few in number and terminate, among other places, in the dorsocaudal regions of the lateral vestibular nucleus ipsilateral to their spinal course. The localized origin and termination of spinovestibular fibers led to the suggestion that peripheral inputs should be somatotopically organized, selectively influencing the “hindlimb” region of Deiters’ nucleus (Pompeiano and Brodal, 1957). Early physiological investiga-

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tions, however, showed that individual neurons could be affected by converging inputs from wide areas of the body and it was believed that this broad influence was due to passage of impulses through the cerebellum (Brodal et al., 1962). This hypothesis was tested in decerebellate animals by Wilson et al. (1966, 1967a). In decerebellate cats the effect of stimulation of peripheral nerves on Deiters’ neurons was usually facilitatory, had a latency of 10-20 msec, and lasted 100-200 msec. The facilitation was not powerful enough to fire previously silent neurons, but among spontaneous ones 44/50 C cells and 76/82 L cells were facilitated. Furthermore, facilitation of any one cell could be produced by stimulation of hindlimb and forelimb nerves, ipsilateral and contralateral. Obviously there is a lack of somatotopic organization in this ascending system that is not due to cerebellar pathways. Presence of the cerebellum modifies the effect produced by peripheral stimulation. One important change is the presence of inhibition (Allen et al., 1971; ten Bruggencate et al., 1971; Wylie and Felpel, 1971). Whereas in the decerebellate animal facilitation is observed usually, and inhibition rarely, in intact animals the usual pattern of activity consists of facilitation followed by inhibition, sometimes followed by another period of enhanced activity. Apparently the facilitation is largely produced by brainstem pathways ( with collaterals of cerebellopetal pathways an important starting point: see Wilson et al., 1966; Ito et al., 1969b), and the inhibition is due to activation of Purkinje cells by the limb nerve projection to the cerebellar cortex. The final stage of enhanced excitability appears to be due to inhibition of Purkinje cells and disinhibition of Deiters’ neurons. In addition, presence of the cerebellum may introduce a somatotopic pattern into the peripheral influence on VST cells, judging by the distribution of inhibition (Allen et d., 1971; ten Bruggencate et al., 1971). The nature of the receptors whose activation would produce facilitation and inhibition of Deiters’ cells under normal conditions is of interest. Surprisingly, no effects are produced by stimulation of group I fibers either in decerebellate or in intact preparations (Giaquinto et al., 1963; Wilson et al., 1966, 1967a; Wylie and Felpel, 1971). Therefore the activity of Deiters’ cells, which have a strong influence on extensor motoneurons, is not affected by impulses from tendon organs or primary spindle receptors in the muscles that these motoneurons innervate. Stimulation of group I1 fibers in forelimb muscle nerves, and group I11 fibers in both fore- and hindlimb neives does affect Deiters’ neurons, although stimulation of mixed and cutaneous nerves is much more effective. Much of the peripheral influence of Deiters’ neurons may be due to joint receptors. Cells in Deiters’ nucleus were among those studied by Fredrickson et al. (1966b) and Fredrickson and Schwarz (1970). The

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activity of most of these cells was influenced by joint movement, whereas stimulation of the skin, or muscle pressure, was ineffective. Effects were obtained from movement of proximal limb joints, but the most prominent excitability changes were observed in response to manipulation of the neck and vertebral column, regions that are a particular target of VST axons (see p. 5 2 ) . Some more direct actions on Deiters’ neurons that may be relayed by spinovestibular fibers have also been seen; in a few cells stimulation of the ventral and lateral funiculi of the cervical spinal cord evokes shortlatency e.p.s.p.’s (Ito et al., 1964b, 1969b). But extracellular studies (Wilson et al., 1966, 1967a) have revealed little evidence of shortlatency excitability changes, which therefore cannot be considered to play an important role in the regulation of activity of VST cells.

Summary. The vestibulospinal tract projects to all levels of the spinal cord where, as will be described in the next section, it exerts facilitatory effects on extensor motoneurons. The somatotopic organization of Deiters’ nucleus, coupled with the pattern of termination of different afferents, leads in effect to a subdivision of the tract into different components. One way of subdividing is into the group of fibers terminating at neck, forelimb, and back levels ( C ) and the group terminating at the hindlimb level ( L ) . The former originates mainly in the ventral part of the nucleus, the latter from the ventral and dorsal parts but with a prominent dorsocaudal component. The labyrinthine input, a considerable part of which comes from the utricle, is relayed to the spinal cord by both C and L components. The cervicothoracic projection is more properly considered a labyrinthine relay, however: the utricular input to it is stronger (more cells involved; individual cells more powerfully affected), more direct, and reflects the pattern of activity of utricular afferents. The cerebellum exerts an important influence on the VST. Excitatory influence, reaching Deiters’ nucleus via the fastigial nucleus, is widespread. The inhibitory Purkinje cell input is found largely dorsally in Deiters’ nucleus. Most likely the L component of the VST is particularly influenced by this inhibitory control, and to some extent it may therefore be considered part of the cerebellar output system (see Chapter 14 in Eccles et ab., 1967). Inhibition originating in the cerebellar cortex, however, reaches more ventral regions of the nucleus also, and there is evidence that it exerts an influence even on the most rostrally projecting neurons of the C component. Any subdivision of the VST into components dominated by the labyrinth and cerebellar cortex respectively must be made with caution. The somatotopic organization of the nucleus is blurred, the separation

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of different afferents not rigorous, and in a number of instances direct inputs from labyrinth and cerebellar cortex have been found in the same ceIls (Eccles et al., 1967, p. 293; Walberg and Mugnaini, 1969; Wylie and Felpel, 1971). The VST as a whole is influenced by a broad, nonspecific, somatic input. Joint receptors may be an important source of this input, and there is a possibility of feedback loops between Deiters’ and the periphery.

1. InfEzlence of the Lateral Vestibulospinal Tract on Spin01 Neurons a. Vestibulospinal Fibers and Their Spinal Connections Stimulation of Deiters’ nucleus facilitates extensor muscles in the ipsilateral limbs (Brodal et al., 1962). We now consider the mechanism of this facilitation. Deiters’ cells of all sizes send their axons into the vestibulospinal tract (Pompeiano and Brodal, 1957), which contains small, medium-sized, and large fibers, the majority being rather thick ( Nyberg-Hansen and Mascitti, 1964). Electrophysiological measurements of conduction velocity confirm that the range of fiber size is wide: two different samples of L cells had velocities between 20 and 140 meters/sec, with the bulk of the values between 50 and 120 meters/sec (Ito et al., 1964a; Wilson et al., 1967a). Vestibulospinal fibers extend as far as the sacral segments of the spinal cord, terminating rather medially in the ventral horn (Schimert, 1938; Nyberg-Hansen and Mascitti, 1964; Petras, 1967). There is no anatomical evidence that the fibers synapse with spinal mononeurons ( at least with motoneuron somata), except perhaps in the thoracic cord ( Nyberg-Hansen and Mascitti, 1964; Nyberg-Hansen, 1969). Connections between VST fibers and motoneurons have been studied by anatomists at all levels of the cord except those controlling neck muscles. In view of physiological results that will now be described, this omission should be rectified. The effects of Deiters’ nucleus stimulation have been studied by means of intracellular recording from spinal motoneurons. Initial experiments, conducted on hindlimb motoneurons, showed that monosynaptic e.p.s.p.’s were produced in some by Deiters’ nucleus stimulation (Lund and Pompeiano, 1965,1968; Shapovalov, 1966). Subsequent investigations have been performed on axial and limb motoneurons. Axial motoneurons will be considered first. i. Neck and back motoneurons. Because of its connections Deiters’

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nucleus is well suited to be a link in the pathway mediating the important effects that the labyrinth exerts on the neck musculature (Magnus, 1926; Batini et al., 1957). Accordingly, Wilson and Yoshida (1969a) studied the effect of Deiters’ nucleus stimulation on a population of alpha motoneurons located in the C2 and C 3 segments and innervating dorsal muscles of the neck that act to extend the head. Stimulation of a region such as the brain stem, containing various ascending and descending fiber tracts traversing closely packed cell groups, is a hazardous procedure. To control the source of stimulation Wilson and Yoshida (1969a) used an array of steel electrodes placed, with the aid of antidromic field potentials, so that one or two were in Deiters’ nucleus, some were in other regions of the vestibular nuclei and in the MLF and nearby medial reticular formation. The nature and threshold of the synaptic actions produced by stimulation at these various locations could then be compared and those originating in Deiters’ nucleus identified. Stimulation of Deiters’ nucleus with single shocks produced in 54/92 motoneurons simple-shaped e.p.s.p.’s (Fig. 3) with an intrasegmental latency of 0 . 4 4 9 msec: the e.p.s.p.’s were monosynaptic (poly-

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FIG. 3. The e.p.s.p.’s evoked in neck extensor motoneurons by stimulation of Deiters’ nucleus, MLF, and labyrinth. a: The e.p.s.p.’s produced in a C3 splenius motoneuron by stimulation of Deiters’ nucleus (1) and MLF ( 2 ) with a pulse of 1 O V (0.33 mA). Response in ( 3 ) evoked by stimulation of ipsilateral labyrinth with a stimulus 0.46 V, 3.1 X NIT. h: Responses evoked in another C3 neck extensor from Deiters’ nucleus (1) and MLF (Z),with an 8.3 V (0.28 mA) stimulus. e.p.s.p. in ( 3 ) produced by stimulation of ipsilateral labyrinth with 0.9 V stimulus 1.5 X N1T. All records consist of several superimposed sweeps, and lower records of each pair show field potentials recorded juxtacellularly. Positive deflection upward (Wilson and Yoshida, 1969a).

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synaptic e.p.s.p.’s were also observed but were not studied in detail). Monosynaptic e.p.s.p.’s were recorded, in an even greater number of motoneurons, on stimulation of the MLF and nearby reticular formation (Fig. 3 ) . The vestibulospinal and reticular inputs were separate and not due either to stimulus spread from one region to the other or to stimulation of branches of the same fibers: summation took place when the two areas were stimulated simultaneously. Although the monosynaptic e.p.s.p.’s were small ( mean size of vestibulospinal e.p.s.p.’s 0.8 mV) it was concluded, because of the number of motoneurons in which they were seen, that Deiters’ nucleus and the MLF constitute an important source of excitation to neck extensor motoneurons. Neck flexor motoneurons have not been studied so far. Back extensor motoneurons also may be expected to receive a direct input from VST fibers: they are axial motoneurons, like neck motoneurons; VST fibers terminate in their vicinity, medially in the ventral horn ( Schimert, 1938; Nyberg-Hansen and Mascitti, 1964). Recent experiments (Wilson et al., 1970) have shown that stimulation of Deiters’ nucleus does produce monosynaptic e.p.s.p.’s in some of these cells, particularly in longissimus dorsi motoneurons in T1-T10. ii. Limb motoneurons. Whereas Lund and Pompeiano ( 1965, 1968) originally believed that VST fibers made monosynaptic contact with hindlimb extensor alpha motoneurons in general, a modified pattern of connections has emerged from more recent work. Wilson and Yoshida (1969a) studied limb motoneurons with the technique used in their neck experiments. Grillner et al. (1970) stimulated with a single fine metal electrode with which they tracked systematically in the brainstem, relating electrode location to antidromically evoked fields in Deiters’ nucleus and to threshold of descending volleys and field potentials in the ventral horn. In addition, the cord was transected in the thoracic region except for the ipsilateral ventral quadrant, to reduce possible contamination of the results by impulses in other descending tracts. Where the results of the two groups overlap they are essentially in agreement. Monosynaptic e.p.s.p.’s, of the same order of magnitude as, and similar in appearance to, those observed in neck motoneurons and illustrated in Fig. 3 are often found in the ankle and knee extensors gastrocnemius and quadriceps, rarely in other extensors, never in flexors. In many flexor cells, and in some extensors, there is monosynaptic excitation by fibers in the MLF. In contrast to neck extensor cells, however, hindlimb extensors do not receive a monosynaptic input from both Deiters’ nucleus and MLF: the two inputs are distributed reciprocally ( Grillner et al., 1968; Wilson and Yoshida, 1969a). Wilson and Yoshida (1969a) also studied the VST input to fore-

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limb motoneurons, including some of the elbow extensors, elbow flexors, and muscles of the wrist and digits. Surprisingly, no monosynaptic connections between Deiters’ nucleus and these motoneurons were observed, although as in the hindlimb there were monosynaptic connections between fibers in the MLF and many extensor and flexor motoneurons. Perhaps more extensive sampling in the motor nuclei already studied and in others might reveal monosynaptic contact between the VST and forelimb motoneurons. For the moment it must be concluded that such contact is not a prominent feature of the relation between Deiters’ cells and these motoneurons. Polysynaptic e.p.s.p.’s and i.p.s.p.’s are frequently seen in limb motoneurons when Deiters’ nucleus is stimulated with short trains of high-frequency stimuli, and sometimes even in responses to single shocks. Polysynaptic e.p.s.p.’s, growing with the frequency and number of shocks in stimulus trains, are seen in most extensor motoneurons and some flexors; i.p.s.p.’s are seen in many flexors, some extensors (Wilson and Yoshida, 196%; Grillner et al., 1970). iii. Gamma (fusimotor) neurons. Impulses in VST fibers do not influence only motoneurons. Carli et al. (1967) accelerated discharge in primary and secondary spindle aff erents by repetitive stimulation of Deiters’ nucleus, indicating a VST input to y motoneurons. Sometimes the input may be monosynaptic: Grillner et al. (1969) observed monosynaptic activation of y motoneurons as a result of brainstem stimulation. Electrodes either in Deiters’ nucleus or in the MLF were effective, and there is a suggestion that, as with motoneurons, reciprocal effects are evoked from the two regions. Deitersian (and MLF) monosynaptic activation of y motoneurons may be found in the same motor nuclei in which monosynaptic activation of a motoneurons takes place, and such a-y linkage provides another means by which the VST can regulate motor activity. (Y

Comment. The VST regulates motor activity by monosynaptic and polysynaptic connections with a and y motoneurons, and by modulating segmental reflexes by means of terminations on interneurons in their pathways (cf. Grillner et al., 1970). As a first approximation it may be stated that VST fibers, themselves excitatory, provide for excitation of many a extensor motoneurons and inhibition of flexors. In neck extensor cells monosynaptic excitation is common and this direct route should make possible accurate control of the neck musculature. In the limbs the direct route is either lacking (forelimb) or restricted to some extensor nuclei (hindlimb), part of a specialized input that should be studied further. An important excitatory route to limb extensors in gen-

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eral, probably usually responsible for much of the extensor activation seen when Deiters’ nucleus is stimulated, is the polysynaptic one, that produces reasonably large depolarizations in motoneurons even in animals under pentobarbital anesthesia ( Wilson and Yoshida, 196%). The polysynaptic pathway, under appropriate stimulating conditions ( or with sufficiently rapid firing of Deiters’ cells), is more powerful than the monosynaptic, but it does not permit as fine control of motoneurons, and the presence of segmenta1 interneurons exposes it to regulation by peripheral and descending activity. Obviously numerous factors must be considered when attempting to understand the significance of the monosynaptic pathway to some limb motoneurons, and the relative importance of mono- and polysynaptic pathways (cf. Grillner et al., 1970). Comparative studies of animals with different postures and means of locomotion may clarify these matters. It should be remembered that the VST effects on motoneurons are not activated only by labyrinthine impulses. Rather they are also under the control of impulses of cerebellar, somatic, and other origin, as described in the preceding section on the origin and functional organization of the tract.

b. Role of the Lateral Vestibulospinal Tract in Spinal Actions Produced by Electrical and Natural Stimulation of the Labyrinth Activation of the labyrinth results in pronounced effects on the body musculature due to labyrinthine reflexes (cf. Brodal et al., 1962; Roberts, 1967), but all of these effects are not necessarily due to the VST. Alternate pathways must be considered.

The descending nucleus. This nucleus receives a strong vestibular input and projects to the spinal cord. The projection has been detected electrophysiologically (Wilson et al., 196710; Kawai et al., 1969) but not anatomicaIly ( Nyberg-Hansen, 1964), and is apparently not extensive. What influence the descending nucleus exerts on motoneurons is not known. The role of the liucleus as a spinal relay niay be assumed to be minor on quantitative grounds, but remains a question mark. The medial nucbus. The role of this nucleus will be considered in detail in the section on the medial vestibulospinal tract. Evidence will be presented that the known action of this nucleus on motoneurons is inhibitory. Accordingly it may play no excitatory role in the production of labyrinth-evoked activity. The reticular formation. There are extensive connections between the vestibuIar nucIci and reticuIar formation (cf. Lorente de N6, 193313;

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Ladpli and Brodal, 1968) that will be considered again in the discussion of pathways between the labyrinth and extraocular nuclei. In turn, the pontine and medullary reticular formation have a major spinal projection (Brodal, 1957). Some reticular neurons can be influenced by natural stimulation of vestibular receptors ( Duensing and Schaeffer, 1960). On the basis of electrical stimulation it has been suggested ( Gernandt et al., 1957; Gernandt and Gilman, 1960) that reticulospinal fibers are an important relay for labyrinthine impulses, being responsible for activity recorded in lumbar ventral roots with a latency as short as 5 msec (but see Brodal et al., 1962; Cook, et al., 1969a). The interpretation of Gernandt and his colleagues has recently been tested directly by extracellular recording from cells in the medial reticular formation, some identified as reticulospinal cells ( Peterson and Felpel, 1971). Approximately 30%of the cells studied in N. pontis caudalis and N. gigantocellularis could be driven by stimulation of the ipsilateral labyrinth at latencies ranging from 1.8 to 40 msec. No cells were fired monosynaptically, and relatively few as early as 3 msec. Considering that the earliest latency of reticulospinal e.p.s.p.’s evoked in hindlimb motoneurons by brainstem stimulation is 3 msec (Wilson and Yoshida, 1969a), it is unlikely that activity appearing in ventral roots as early as 5 msec is due to a pathway such as labyrinth-vestibular nucleireticulospinal neuron. Nevertheless, the data of Peterson and Felpel indicate that a considerable number of neurons in the medial reticular formation of the lower brainstein, including reticulospinal neurons, are activated by electrical stimulation of the labyrinth. The reticular formation, therefore, remains a probable spinal relay for activity originating in the labyrinth. It should be possible to predict the result of activity in VST fibers. The most direct and strongest vestibular afferent connections are with the region of Deiters’ nucleus projecting to the cervical and thoracic cord. Furthermore there are more direct connections between VST fibers and axial, rather than limb, motoneurons. Effects on the latter will be carried out more by polysynaptic than by monosynaptic pathways, and by modulation of activity of reflex pathways. Conditions are therefore most favorable for strong, clear-cut labyrinthine actions on axial motoneurons, especially those of the neck, and least favorable for actions on hindlimb motoneurons. Overall the signs of activity should consist of excitation of ipsilateral extensor motoneurons and inhibition of flexors. i. Electrical stimulation. There are several reports of movement, muscular contraction, or potentials in peripheral nerves elicited by stimulation of the labyrinth or eighth nerve, but evidence pointing to

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the pathways involved is usually lacking. The most elegant work is that of Szentigothai and Gomory ( Szenthgothai, 1952) who stimulated discrete regions of the labyrinth (mechanically, not electrically) and produced reflexes in neck muscles that varied with the stimulus site. More generalized responses, usually consisting of ipsilateral extension and contralateral flexion, were observed in forearm muscles, but only when all canals were stimulated together. The authors stressed the likelihood that the pathways linking the labyrinth to limb motoneurons were more complex than the pathways to axial motoneurons. The extension of the ipsilateral forearm, and the differences in the responses of neck and forearm muscles, are consistent with the pattern of termination of the VST, which must be an important link in the pathways evoking the responses. The VST undoubtedly is also partially responsible for the ventral root discharges (cervical and lumbar) seen in response to stimulation of the eighth nerve (Gernandt et al., see, for example, Gernandt and Gilman, 1959; Gernandt et al., 1959) or one of its branches (Yamauchi and Kato, 1969), and for the movements that J. Suzuki and Cohen (1964) evoked by canal stimulation. There is direct evidence for the involvement of the VST in contraction of hindlimb extensors (gastrocnemius) produced by stimulation of the 8th nerve ( Diete-Spiff et al., 1967). Similar contraction can be produced by stimulation through an electrode located in Deiters’ nucleus; when a lesion is made with this electrode, the response to 8th nerve stimulation is abolished (Barnes and Pompeiano, 1970). Whatever the pathway, the above experiments demonstrate that connections exist not only between labyrinth and neck motoneurons but between labyrinth and limb motoneurons that are powerful enough to activate the latter in cats that are decerebrate or under chloralose anesthesia. Only the pathways to neck and back motoneurons function well under pentobarbital anesthesia. Wilson and Yoshida ( 1969a,b,c) stimulated the labyrinth with single shocks and with short trains of stimuli and made intracellular recordings from different types of niotoneurons. Disynaptic e.p.s.p’s and i.p.s.p.’s were often observed in ipsilateral and contralateral neck motoneurons in response to stimulation of one labyrinth. The e.p.s.p resembled those produced by Deiters’ stimulation and were probably due, at least in part, to the VST (Fig. 3 ) . Disynaptic potentials have also been observed in some back motoneurons (Wilson et al., 1970) but so far have been absent from limb motoneurons. This is further evidence that the pathway to limbs is complex and labile compared with the pathways to the axial musculature. ii. Natural stimulation. Tilting provides reasonably selective activation of the VST, because strong effects of tilt are seen mainly in Deiters’ nucleus and the descending nucleus (Peterson, 1970). As already in-

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dicated the effects of the latter on niotoneurons are unknown but probably scanty. Direct effects on spinal motor centers, therefore, must be exerted mainIy via the VST and perhaps through the reticular formation, since the activity of neurons in the latter can be modified by tilt (Duensing and Schaeffer, 1960). Peterson’s results ( 1970) showed that the mean response of Deiters’ cells projecting to the cervicothoracic cord consisted of facilitation by ipsilateral, inhibition by contralateral tilt (Fig. 212). The mean response of cells projecting to the lumbosacral cord was weaker, and consisted of facilitation by both ipsi- and contralateral tilt. A similar pattern has been seen in the musculature and in motoneurons. When studying the effect of tilt on the musculature it is necessary to eliminate neck reflexes that arise when the head is rotated in relation to the body, either by tilting the whole animal or by denervating the C1-C4 segments. When this is not done the results are due to combined neck and labyrinthine reflexes ( Ajala and Poppele, 1967; Poppele, 1967) and are difficult to analyze further. Effects of tilt on forelimb muscles were studied by Nagaki (1967) and Roberts (1968) in carefully controlled experiments. To obtain measurable labyrinthine reflexes Nagaki found it necessary to transect the spinal cord at T12 while Roberts did not, but otherwise the results agree: tension in forelimb extensors increases with ipsilateral tilt, decreases with contralateral tilt, In addition, tension is increased when the head is tilted nose down (Roberts, 1968). As indicated by Roberts (1967, 1968) such responses would counteract the tilt and restore the normal attitude, and the results are in conflict with Magnus (1926) claims that all limbs react in the same direction to a change in head position. A change in position also affects hind limb motoneurons. Roberts (1970) showed that the activity of hindlimb extensor muscles increases with nose up or ipsilateral tilt and decreases with nose down or contralateral tilt. Effects on hindlimb muscles were studied in more detail by Erhardt and Wagner (1970). The head was rotated about the longitudinal axis before and after denervation of the neck. When tonic labyrinthine reflexes alone were acting many extensor motoneurons were facilitated by ipsilateral tilt, while contralateral tilt produced weaker facilitation, or inhibition. The opposite was seen with flexors although the effects were more variable. The lack of clear reciprocity between the effects of ipsilateral and contralateral tilt is in agreement with Peterson’s observations ( 1970). When neck reflexes were functioning there were phasic effects on motoneurons during rotation, few tonic effects (cf. Poppele, 1967). Apparently the phasic action is clue to

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neck reflexes, while tonic neck and labyrinthine reflexes usually cancel each other (cf. Roberts, 1970). The excitability changes observed in limb muscles or motoneurons during tilt are consistent with the effects of tilt on Deiters’ neurons and with the known connections between the VST and motoneurons. They are not as consistent with the known connections between reticulospinal cells and motoneurons, since stimulation of the medial reticular formation produces excitation of flexors as well as extensors. It may be concluded that the effects of tilt are produced mainly by activation of the VST. This section has dealt mainly with an analysis of the results of tilting the animal, because this form of natural stimulation has been studied most carefully. Erhardt and Wagner (1970) also showed that horizontal acceleration modifies the activity of hindlimb motoneurons. The pathway for this is not certain. Summary. The synapses that lateral vestibulospinal fibers make with spinal motoneurons and interneurons are so distributed that activity of the VST generally leads to activation of extensor and inhibition of flexor motoneurons. Monosynaptic linkage predominates in the control of neck motoneurons whereas polysynaptic linkage seems at least equally important in the control of limb motoneurons. The VST influence on spinal motor centers can be activated by a variety of inputs including those from the cerebellum. A particularly important input is that from the labyrinth. Electrical and mechanical stimulation of the labyrinth evoke contraction of the body musculature, the most discrete and diverse responses being elicited in neck muscles. On the basis of direct and indirect evidence, mainly the latter, the VST can be assumed to play an important role in the production of these effects. Reasonably selective activation of the VST by natural stimulation (tilt) evokes a pattern of excitation and inhibition in limb muscles that fits with predictions made on the basis of the known utricular input to Deiters’ neurons and connections of VST fibers with motoneurons.

B. THEMEDIALVESTIBULOSPINAL TRACT 1. Origin, Course, and Termination of Fibers The name medial vestibulospinal tract (MVST) has been applied by Nyberg-Hansen (1966) to the group of fibers that originates in the vestibular nuclei, turns medially, and enters the MLF to descend to the spinal cord. One assumption underlying this terminology is that the

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tract originates entirely in the medial vestibular nucleus. This, however, is not the case. Anatomical evidence suggests that the medial nucleus does make an important, even the principal, contribution to the tract ( Nyberg-Hansen, 1964; McMasters et al., 1966; Petras, 1967). Participation of axons of medial nucleus neurons in the MVST has been confirmed electrophysiologicaIly. Antidromic activation of MSVT cells (previously called DMLF cells) reveals that they make up about 17% of all medial nucleus cells studied; that they are found mainly in the rostral two-thirds of the nucleus; and that the spinal-projecting fraction of the cell population increases steadily as more rostral regions are sampled (Wilson et al., 1968a). A contribution by the descending nucleus to the MVST is not ruled out on anatomical grounds (Brodal et al., 1962), although there is no recent anatomical evidence in support of this notion. Electrophysiological data, however, show that the cell bodies of some MVST neurons lie in the descending nucleus (Wilson et al., 1967b; Kawai et al., 1969). The tract is therefore heterogeneous in origin and may be heterogeneous in function. Despite this the term medial vestibulospinal tract will, for convenience, be used in this review. Lesions in the medial vestibular nucleus produce degeneration in the more dorsal regions of the ventral funiculus along the anterior median fissure ( Nyberg-Hansen, 1964). Degeneration is bilateral, but contralateral fibers are less numerous than ipsilateral ones. Overall there are considerably fewer MVST than lateral vestibulospinal axons. There is some uncertainty about the caudal extent of the MVST. Although electrophysiological findings suggest that it may reach lumbar segments (Precht et al., 1967b), anatomical evidence restricts the tract to the rostral half of the cord. Degeneration has been observed as far caudal as midthoracic segments, but it is particularly abundant above the cervical enlargement ( Nyberg-Hansen, 1964; McMasters et al., 1966; Petras, 1967). Some of the spinal-projecting fibers are branches of dichotomizing axons coursing both rostrally and caudally (Brodal et al., 1962; Wilson et al., 1968a). In the spinal cord, descending fibers terminate on large and small cells in the ventral horn, but not on motoneuron cell bodies ( Nyberg-Hansen, 1964). As with the VST, physiological results suggest the need for close examination of MVST termination in the upper cervical segments. 2. Inputs to Neurons of Om'gin Convergence of various inputs has been studied, especially for medial nucleus neurons, including MVST cells and neurons without long axons. We will consider mainly MVST neurons.

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a. Inputs from the Labyrinth

Antidromically identified MVST iieurons in the medial nucleus have been studied by Wilson et al. (196813). They are located in the region of the nucleus receiving primary vestibular afferents, and 80%can be fired monosynaptically by stimulation of the ipsilateral vestibular nerve. This very high percentage of driven cells exceeds that found among neurons projecting rostrally in the MLF (37%)or not projecting in either direction ( 25%), and suggests preferential terminations of vestibular afferents on one of several intermingled cell types. Conversely, polysynaptic firing, which is also seen in all types of medial nucleus neurons, is least frequent in MVST cells. The effect of stimulation of the contralateral labyrinth on MVST neurons has also been tested: all observed effects were inhibitory ( Wilson et al., 196% ) . MVST neurons were identified antidroniically in only one investigation utilizing natural stimulation. Few horizontal type I or type 11, and many type I11 neurons (excited by ipsilateral and contralateral acceleration) project to the spinal cord (Precht et al., 1967b). Acceleration in the plane of the vertical canals and tilt affect medial nucleus neurons (Markham, 1968; Peterson, 1970), but it is not known whether these are MVST cells. A high percentage (64%, n = 11) of descending nucleus MVST neurons are fired monosynaptically by labyrinthine stimulation. The effective form(s ) of natural stimulation for these neurons are not known, but tilt is a powerful stimulus for neurons in the descending nucleus (Peterson, 1970) and is most likely to be an important input to MVST cells in that nucleus.

b. Inputs from the Cerebellum The vermis of the anterior lobe does not project to the medial nucleus (Brodal et al., 1962), but both the vernial part (nodulus) and the hemisphere ( flocculus ) of the vestibulocerebellum do ( Angaut and Brodal, 1967). The descending nucleus is supplied by fibers from the vermis of the anterior lobe, as well as from the flocculus, nodulus, and uvula (Brodal et al., 1962; Angaut and Brodal, 1967). It is of particular interest that the region of the medial nucleus where fibers from the flocculus terminate is the region where MVST cells are found ( Angaut and Brodal, 1967). We do not know, however, whether the cerebellar fibers synapse with projecting neurons. Fibers from the fastigial nucleus tcrrninate in the ipsilateral and contralateral medial and descending nuclei. Stirnulatioil of the fastigia1 nucleus produces e.p.s.p.’s in descending neurons, but so far in none

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identified as MVST cells (Ito et al., 1970a). In the medial nucleus some type I neurons projecting to the cord, i.e., MVST cells, are excited from the fastigial nuclei on the two sides (Shimazu, 1967, p. 172). Type I1 neurons that are excited from the contralateral labyrinth are also excited from the contraIateraI fastigial nucleus, with many type I neurons inhibited by both stimuli ( Shiniazu, 1967). In summary, inhibitory and excitatory fibers originating in the cerebellar cortex and nuclei respectively terminate in the regions giving rise to the MVST and there is some cvidence that they participate in the regulation of its activity either directly or through interneurons. Compared with our knowledge of thc cerebellar regulation of the lateral vestibulospinal tract, however, alinost nothing is known about cerebellar influence on the MVST. This deserves further study. c. Somatic Inputs

Information about somatic inputs to the medial and desceiiding nuclei is scanty. A few spinuvestibular fibers end in the medial and descending nuclei (Brodal et al., 1962; Brodal and Angaut, 1967), and from the observation of Loreute de N6 (1924) it appears that collaterals of fibers to the cerebellum also terminate i n thc area. In their experiments 011 the medial nucleus, Wilson et al. (1968b) looked for synaptic activation of medial nucleus iieurons by stimulation with an electrode inserted near the midline a t about C3, and sometimes by stimulation of muscle nerves. A number of cells were excited transsynaptically with latencies ranging from 2 to 5 up to 15 nisec or more by cord stimulation, from 12 to 30 nisec by limb nerve stimulation. Only 1/36 cells projected into the MVST, however, and most were apparently interneurons. Peripheral influence on MVST cells is exerted indirectly. Fredrickson et al. (1966b) ~ n dFredrickson and Schwarz (1970) studied the effects of natural stimulation of vestibular neurons. Most of their cells were in the medial and descending nuclei, and they were particularly affected by movement of joints of vertebral column and neck, and of proximal linib joints.

d . Inputs from More Rostra1 Regions of the Central Nervous System In addition to the sources of cxcitation and inhibition already described, medial nucleus nc~ironsare subject to iiifluence by fibers dcscendiiig in the MLF, many of which originate in the interstitial nucleus of Cajal (Pompeiano and Walberg, 1957; Markham et al., 1966; Markham, 1968). Type I1 and type III cells arc excited by this input, type I cells are inhibited (presumably via type 11 cells). Some type 111 cells project to the spinal cord, but it is not known whether they are affected by

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stimulation of the interstitial nucleus. Wilson et al. (1968b) found 40 medial nucleus cells activated by stimulation of the MLF rostral to the medial nucleus, and only one of them was a MVST cell. Inputs from rostral brain regions therefore end on interneurons rather than on MVST cells, although they probably nevertheless participate in regulation of the activity of the latter. Summary. More is known about inputs to MVST cells in the medial nucleus than to those in the descending. For all of these cells, however, the input from the labyrinth is a very important one, found in a high proportion of the cells and apparently able to dominate their behavior. The tract therefore acts primarily to relay the labyrinthine inflow to the spinal cord, particularly to its most rostral levels. Fibers originating in the cerebellum, interstitial nucleus of Cajal, and periphery regulate the activity of MVST cells. At least in the case of the two latter inputs this is accomplished primarily by termination on interneurons, that in turn influence the activity of projecting cells.

3. Spinal Action of the MVST Until recently there had been no indication of the role of the MVST in motor control. Gernandt (1968) showed that section of the MLF did not affect the motor outflow produced at forelimb or hindlimb levels by vestibular stimulation, indicating that the MVST had no role in producing this outflow. With only the MLF intact in an otherwise transected brainstem, vestibular stimulation did evoke potentials in the radial nerve, with a latency of 7 msec or so. The MLF, however, contains reticulospinal fibers in addition to fibers of vestibuIar origins, and these may be activated polysynaptically by stimulation of the vestibular nerve (Peterson and Felpel, 1971). The function of the MVST therefore remained obscure. The MVST is distributed mainly to the most rostral cervical segments, i.e., those controlling the neck musculature. Wilson and Yoshida ( 196913) have studied the effect of brain stem stimulation on motoneurons in these segments, and their results indicate that the MVST is predominantly an inhibitory pathway. Procedures were similar to those used in studies of the lateral vestibulospinal tract (Wilson and Yoshida, 1969a). Arrays consisting of up to thirteen stimulating electrodes were used, in order to improve the localization of brainstem zones from which synaptic potentiaIs were produced in motoneurons. When stimulating electrodes were in the medial nucleus or MLF, short-latency i.p.s.p.’s were often evoked in neck extensor motoneurons (Fig. 4 ) . The latency of i.p.s.p.’s overlapped

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0

rnsec

lrl -

al

1.6

18 2 0 2 2

24

2.6

0

6 & n

Latency of disynoptic IPSPs evoked from labyrinth, msec

0.4

0.6 0.8

1.0

1.2

1.4

Latency difference between

I P S P s evoked from lobyrinth ond mediol n u c l e u s , msec

FIG. 4. The i.p.s.p.’s evoked in neck extensor motoneurons by stimulation of medial vestibular nucleus and labyrinth. a: Typical i.p.s.p evoked in motoneuron by stimulation of medial nucleus ( 1 ) and labyrinth ( 2 ) . Stimulus to medial nucleus was 6 V , threshold 2.4V (0.14 mA). Labyrinthine stimulus was 1.1V, threshold was 1.4 X NIT. b: Distribution of latencies of those labyrinthine i.p.s.p.’s considered disynaptic. Results from C2 and C3 are pooled in the histogram. c: Latency differences between i.p.s.p.’s evoked in the same cell by stimulation of medial nucleus and labyrinth (Wilson and Yoshida, 1969b).

with that of monosynaptic e.p.s.p.’s produced by Deiters’ nucleus or MLF stimulation (Wilson and Yoshida, 19f39a), and the mean interval between the earliest-arriving descending volleys and i.p.s.p. onset was 0.7 msec: the i.p.s.p.’s were monosynaptic. This finding provided the first demonstration of the existence of inhibitory fibers descending from supraspinal regions to the spinal cord. It had previously been thought that supraspinal inhibitory control was effected by descending excitatory fibers synapsing with segmental inhibitory interneurons. Effective inhibitory zones were delimited in the horizontal plane by the use of multielectrode arrays, and in the vertical plane by movement of the arrays. With moderate shock strength, monosynaptic i.p.s.p.’s were produced only by electrodes in the medial nucleus and in, or near, the MLF. This is consistent with the view that there are inhibitory neurons in the medial nucleus, since MVST axons originating in the latter de-

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scend to the cord via the MLF. All inhibitory fibers in the MLF, however, are not necessarily of vestibular origin, and the presence of inhibitory axons of reticular (It0 et al., 1970c) or other neurons is not ruled out. Vestibular origin of some of the inhibitory axons is confirmed by the results of labyrinthine stimulation: disynaptic i.p.s.p.’s are often seen in neck motoneurons (Fig. 4 ) , and, as stated previously, in the brainstem apparently only vestibular neurons are excited monosynaptically by vestibular afferents. Disynaptic inhibition of neck motoneurons by labyrinthine stimulation is blocked by strychnine, unaffected by bicuculline (Felpel, 1972). This is consistent with the view that glycine may be the inhibitory transmitter at this synapse. The inhibitory action of the MVST may also be exerted at more caudal spinal levels. Wilson et al. (1970) have evoked i.p.s.p.’s in axial back motoneurons by brainstem stimulation. Linear regression analysis shows that some of the i.p.s.p.’s are monosynaptic, being produced by fibers of medium conduction velocity ( 2 7 0 m/sec). Disynaptic i.p.s.p.’s can be produced be stimulation of the labyrinth, particularly the contralateral labyrinth, indicating a relay in the vestibular nuclei. Direct evidence of the origin of inhibition has not yet been obtained but a reasonable hypothesis would place the cells in the contralateral medial nucleus. The presence of some inhibitory fibers reaching the thoracic cord suggests that others (not necessarily of vestibular origin) may reach the lumbar cord (cf. Baldissera and Weight, 1969). The i.p.s.p.’s produced in hindlimb rnotoneurons by single shocks to the brainstem have previously been classified as disynaptic on the basis of long “segmental delay” (cf. Gernandt, 1968; Grillner et al., 1968; Lund and Pompeiano, 1968). It may be worthwhile to reexamine this classification. Comment. All the evidence about the spinal connections and actions of MVST fibers applies only to those originating from cells in the medial vestibular nucleus. Apparently inhibitory MVST fibers do not come from the descending nucleus (Wilson and Yoshida, 1969b), whose functions require detailed investigation. The probable function of the medial nucleus is to act together with Deiters’ nucleus to provide excitatory and inhibitory innervation of neck muscles by pathways originating in labyrinthine receptors, Deiters’ serving as an excitatory and the medial as an inhibitory relay. The two nuclei undoubtedly are an important part of the pathways involved in the execution of the variety of reflex actions produced in neck muscles by stimulation of different parts of the labyrinth (SzentLgothai, 1952). Exactly how the medial nucleus is involved in various postural re-

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flexes of the neck is not yet clear. Consider responses to position change. Utricular activation of medial nucleus neurons with unidentified axonal projection exists, but from available evidence it seems on the average less powerful than that of ventral Deiters’ neurons (Peterson, 1970) and may or may not be monosynaptic. Therefore, in response to position change, inhibition of neck motoneurons by medial nucleus neurons could be brought about by polysynaptic, or even monosynaptic, activation of the latter. On the other hand, the discharge of neck motoneurons during tonic position reflexes may be governed largely by facilitation and disfacilitation through the Deiters’-neck niotoneuron pathway, if the pathway is both active enough and potent enough. Medial nucleus inhibition may be reserved for faster reactions that follow canal activation. This problem can only be resolved by identification of those parts of the labyrinth responsible for the disynaptic effects observed in different groups of neck motoneurons. The interesting question still remains: are all cells in the medial nucleus inhibitory? Probably not, as the cell population of the nucleus is not homogeneous (Brodal et al., 1962; Hauglie-Hansen 1968) and weak shocks to the medial nucleus can produce monosynaptic e.p.s.p.’s in contralateral trochlear motoneurons (R. Baker et aE., 1971; Precht and Baker, 1972). We cannot even be certain that all spinal-projecting medial nucleus neurons, including dichotomizing cells with an ascending branch, are inhibitory, but this may be the simplest initial assumption. The medial and descending nuclei have been implicated in a wide variety of actions associated with REM sleep (cf. Pompeiano, 1967). In particular two spinal phenomena associated with REM are atonia of neck muscles (Jouvet, 1962) and presynaptic inhibition of spinal afferents (Pompeiano, 1967). The former may be readily explained by bursts of activity in MVST cells. Production of presynaptic inhibition in lumbar pathways is due to pathways that seem to include the medial nucleus, but involve relays in the reticular formation rather than the MVST (Cook et al., 1969b). Summary. The medial vestibulospinal tract originates in the medial and descending vestibular nuclei. The former makes by far the more important contribution to the tract, and it is this component of the MVST that has been studied. MVST fibers descend as far as the thoracic cord, but are distributed most markedly to the rostra1 cervical segments. Many MVST neurons receive a monosynaptic input from the labyrinth that is capable of dominating the activity of these cells, although impulses from other sources contribute to the regulation of this activity. The MVST therefore is suited to provide labyrinthine regulation of seg-

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mental activity of rostra1 spinal segments. Electrophysiological evidence indicates that this regulation is inhibitory, and is carried out by inhibitory MVST fibers that terminate directly on spinal axial motoneurons, those innervating the neck, and, perhaps, those innervating the back musculature. It is suggested that together with the excitatory lateral vestibulospinal tract, the MVST provides the pathways permitting accurate control of the neck musculature by labyrinthine receptors. V. Projections of the Vestibular Nuclei to Extraocular Motoneurons

The labyrinth exerts a powerful reflex influence on the extraocular muscles. Labyrinthine eye reflexes generally act to preserve the original field of vision: for example, the eyes deviate upward when the head is tilted down, or deviate to the right when the head is accelerated to the left. The reflexes can be divided into two categories, those producing compensatory movements and those producing compensatory positions (cf. Lorente de N6, 1932). Both categories are produced by neural pathways originating in the labyrinth and relaying first in the vestibular nuclei. As emphasized by Lorente de N6 (1933b) and discussed below, there are two types of pathways between vestibular nuclei and extraocular neurons. First are direct pathways ascending in the MLF, some now known to be monosynaptic. Others are more complex, involving relays in the reticular formation. Unlike vestibular pathways to the spinal cord, the projections to extraocular neurons are not conveniently divided into tracts originating in discrete nuclei. Because of this somewhat diffuse organization, this section will be presented differently from the preceding one. After an introductory discussion of anatomy, the labyrinthine actions on eye muscles will be described. Next we will consider the pathways that underlie the reflexes, first in general, then with a detailed analysis at the single-unit level of the pathway that is best understood. A. ANATOMICALBACKGROUND Brodal and Pompeiano (1958), using retrograde cell changes as a criterion, showed that all four vestibular nuclei project rostrally in the MLF. They concluded that each of the four nuclei supplies the oculomotor, trochlear, and abducens nuclei, and that the vestibulo-extraocular projection therefore, is rather diffuse. This matter was recently reinvestigated by Tarlov (1970), who made discrete lesions in different regions of the cat vestibular nuclei and studied preterminaI degeneration of second-order vestibular axons by means of the Nauta method. Contrary to the earlier suggestions that all four vestibular nuclei participate in the extraocular projection ( Brodal and Pompeiano, 1958; McMasters

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et al., 1966), Tarlov demonstrated that this projection originates only in the superior nucleus and in the rostral part of the medial. The two nuclei have widely overlapping as well as apparently separate projection fields, and there are strong indications of topical representation between different regions of the vestibular and extraocular nuclei (cf. Szentigothai, 1964). Tarlov ( 1970) confirmed the earlier findings that fibers from the lateral and descending nuclei also ascend in the MLF. He believes that these nuclei, however, project only to the interstitial nucleus of Calal and the nucleus of Darkschewitsch, cell groups that also receive fibers from the medial and superior nuclei. The interstitial nucleus of Cajal has recently been shown to influence the activity of vestibular neurons (Markham et al., 1966; Markham, 1968). As described by Tarlov (1970) vestibular axons to the extraocular nuclei arise from regions of the vestibular nuclei receiving a heavy input from the three semicircular canals, but not from those regions-Deiters’ nucleus and the rostral part of the descending-receiving the main input from gravity receptors. If this is so, an important consequence is that the utricle has less-direct connections with extraocular motoneurom than the cristae. Of course anatomical evidence only suggests the presence or absence of relatively direct pathways between some parts of the labyrinth and the extraocular nuclei, but does not prove that any rostrally projecting vestibular neurons actually receive a direct labyrinthine input. Monosynaptic connections between fibers innervating the cristae and vestibular neurons synapsing with extraocular motoneurons have now been demonstrated electrophysiologically ( see Section V, B ). Much less is known about vestibulo-extraocular pathways that involve the reticular formation. The importance of these pathways was demonstrated not only physiologically but also anatomically by Lorente de N6 ( 193313). For example, a typical abducens motoneuron (mouse) receives many more synaptic contacts froni reticular neurons on its dendrites outside the confines of the nucleus, than from secondary vestibular fibers within the confines of the nucleus (Lorente de N6, 1933b, p. 10). The exact origin of the reticular fibers, and the manner in which their activity is iiifluenced by the labyrinth, arc not clear. Since there is no anatomical or physiological evidence to suggest that primary vestibular fibers make significant synaptic coniicctions with reticular neurons ( Walberg et al., 1958, Hauglic-Hanssen, 1968; Peterson and Felpel, 1971) vestibular inputs to these cells must be relayed largely through second-order vestibular neurons. Collaterals to the reticular formation from axons of various types of vestibular neurons have been observed (cf. Lorente de N6, 1933b; Hauglie-Hanssen, 1968). Recent investigations by Ladpli and Brodnl (1968) show that such connections

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are extensive and that thc superior, lateral, and descending nuclei, and perhaps the medial, project to areas of the reticular formation known to have cells with ascending axons. More specific anatomical data on vestibulo-reticular-extraocular pathways are not available.

B. PHYSIOLOGICAL INVESTIGATIONS 1 . Labyrinthine Action on Extraoculur Muscles

To a certain extent compensatory positions and movements, produced by excitation and inhibition of extraocular muscles as a result of natural activation of labyrinthinc receptors, can be reproduced by other types of stimulation. Natural, caloric, mechanical, and electric stiinuli have been used to study the effects produced in extraocular muscles by activation of the labyrinth (cf. Fluur, 1959; Szenthgothai, 1952). Eye movement, electrical activity of extraocular muscles or nerves, and activity of single ~ieuronsof the extraocular nuclei have been used as test systems. Natural and electrical stimulation of the labyrinth, combined with single-unit recording from vcstibular and extraocular neurons, have been most useful in analyzing the pathways involved in the execution of labyrinthine reflexes. Studies of direct pathways have progressed considerably further than studies of pathways with reticular relays. Activation of individual semicircular canal nerves produces fairly stereotyped effects in extraocular muscles. These effects have been obtained in experiments involving mechanical activation of the receptors and isotonic recording from eye muscles in cats and dogs (Szentlgothai, 1950, 1952) and electrical stimulation of canal nerves together with EMG recording in cats (Fluur, 1959). Most recently a technique permitting individual stiniulation of different ampullary nerves in acute or chronic animals has been described by Cohen and Suzuki (1963) and this technique has been used in cats and monkeys in conjunction with various recording techniques (Cohen et al., 1964; Suzuki and Cohen, 1964). Results of all investigators agrec on the essential point that stimulation of an ampullary nerve always produced contraction of one niuscle in each eye, usually with inhibition of the antagonist (Table 111). Depending on the preparation and on the condition of the animal, stimulation of a canal also produces changes in the activity of other eye muscles. Those muscles listed in Table 111, showing the strongest, most repeatable, shortest-latency response to stimulation have been designated “prime movers” ( Coheii et al., 1964). In unanesthetized animals stimulation of one anipullary nerve activates two muscles in each eye besides the prime mover and any evoked eye movement is produced by a pattern of activity involviilg all eye muscles (Cohen et al., 1964; see also Lorente de N6, 1932). In alert animals the movements produced

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Muscles roiitracting

Horizontal

Ipsilateral medial rectus Coiitralateral lateral rect,us Ipsilateral superior oblique Contralateral inferior reotiis Ipsilateral superior rect.irs Contralateral inferior oblique

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69

by canal stimulation with short pulse trains are those compensatory movements that would be required to oppose rotation in the plane of that canal (Suzuki et al., 1964). This may be followed by a rapid return movement resembling the fast phase of nystagmus (Cohen et al., 1967). With longer and stronger stimulation, nystagmus results. Because of greater technical difficulties, there have been fewer experiments involving selective stimulation of the utricular macula or its nerve. Szentigothai ( 1952, 1964) succeeded in stimulating parts of the utricular macula meclianically and electrically in dogs, and observed reasonably reciprocal behavior in the vertical rccti and in the oblique muscles. Usually the muscles did not react exactly as expected from known tonic reflexes, probably, as suggested by Szentigothai, because only part of the macula was being stimulated. Coordinated eye niovements have recently been evoked in cats by electrical stimulation of different areas of the utricular and saccular maculae (Suzuki et al., 1969; Fluur and Mellstrom, 1971). There is a pronounced diff'erence between the inuscular responses evoked by stimulation (mechanical, electrical ) of cristae and maculae: the response to macular stimulation develops much more slowly (Szentigothai, 1964). It was suggested by SzentAgothai that whereas disynaptic reflex pathways connected ampullar receptors with extraocular motoneurons, the pathways connecting the rnacula with such neurons were more complex, presumably because monosynaptic connections between aff erents from gravity receptors and vestibular neurons projecting to the extracular nuclei were Iacking ( Szentdgothai, 1964 ) . Tarlov's observations ( 1970), discussed above, support this view.

2. Pathways of Vestibulo-Extraocular Reflexes Excitation of prime movers by afferent impulses from receptors in the cristae apparently takes place via pathways ascending in the MLF, and does not require more complex reticular pathways. The responses of these muscles to stimulation of individual canals disappeared if the

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VICTOR J. WILSON

MLF was cut, and remained if the whole brain stem other than the MLF was transected ( SzentQgothai, 1950). It was assumed that the response was brought about by a three-neuron arc consisting of first- and second-order vestibular neurons and extraocular motoneurons. Many second-order vestibular neurons with ascending axons were known to be located in regions of the vestibular nuclei receiving a primary vestibular input; it was also established that there is monosynaptic contact between fibers in the M L F (presumably originating in the vestibular nuclei) and some extraocular neurons: Lorente de N6 (1935) used this pathway for the first accurate measurements of central synaptic delay. Some details of the pathway have emerged from recent electrophysiological investigations, and these will be described below. There will be little discussion here of the pathways involved in the production of nystagmus, partly because information regarding these pathways is scant. The simplest reflex arcs with their pathways in the MLF are not sufficient for the production of nystagmus, which also requires a reticular substrate (Lorente de N6, 1933b). A particular region of the reticular formation has been related to nystagmus, namely, the pontine reticular formation between coordinates A0 and A2, between the midline and lateral 2-2.5. Rythmic potential changes in this area precede eye movement ( Cohen and Feldman, 1968), while lesions placed there cause loss of the quick phase of nystagmus (Cohen et al., 1968). It cannot be assumed that this is the only region of the reticular formation important in rythmic vestibuloocular reflexes, but this area certainly deserves further, more-detailed, investigation.

3. Single-Unit Analysis of Simple Vestibular-Extraocular Reflex Arcs This work has proceeded by means of intra- and extracellular recording from cells in the vestibular and extraocular nuclei. Vestibular units have been identified by their location, response to natural stimulation, and projection. Unfortunately all criteria have usually not been employed in the same experiments. In this section some experiments emphasizing the study of cells identified by location and projection will be described first. This will be followed by a detailed examination of the horizontal canal-abducens motoneuron reflex arc, studied in experiments that emphasized natural stimulation. Some pathways controlling this reflex arc will be considered briefly, as well as some other simple connections between labyrinths and extraocular motoneurons. a. Organization of the Medial Vestibular Nucleus

The medial nucleus is important in control of extraocular muscles, and its organization has recently been studied in some detail by means

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of extracellular recording (Wilson et al., 1968a,b ) . Cells were identified by their antidromic responses to stimulation of the MLF 1.5-2 nim rostral to Deiters’ nucleus, and their location was subsequently determined by reference to dye marks made in most electrode tracts. Of more than 350 cells, 41%were fired antidromically by MLF stimulation; the axons of many of these neurons can be presumed to terminate in the extraocular motor nuclei, No attempt was made to identify cells projecting to the different nuclei, nor has this been done in any other investigation. In agreement with some previous and subsequent aiiatomical evidence (Tarlov, 1970) rostrally projecting cells were mainly located in the rostral 70%of the medial nucleus, that is, in the region receiving a primary vestibular input (Table 11). A substantial fraction of the units with axons in the ascending h4LF could be fired at short latency by electrical stimulation of the labyrinth, 37% monosynaptically and 51% polysynaptically. This result provided the first definite physiological evidence of monosynaptic vestibular activation of rostrally projecting neurons, in other words of the first limb of the three-neuron arc postulated previously (see also Cook et at., 1969a), The regions of the labyrinth providing input to these cells were not determined, but almost certainly among the cells there were horizontal type I and type 111 units (Shimazu and Precht, 1965) as well as type I units of the anterior canal (Markham, 1968). Units of these two groups, also found in the superior nucleus, are present near each othcr in the medial: Markham (1968) noted some of each of the same electrode tracks. More than likely the sample used by Wilson and his colleagues (1968a,b) also included units receiving inputs from utricular receptors (Peterson, 1970) and from the posterior canal. Units with rostrally coursing axons were only infrequently excited by inputs other than that from the ipsilateral labyrinth (Wilson et al., 196810). For example, 40 medial nucleus cells were fired synaptically by MLF stimulation, but only one of these projected into the MLF. Similarly, only 4/34 units activated synaptically from the spinal cord, and 2/ 13 fired by stimulation of the contralateral labyrinth, projected into the MLF. Convergence of inputs was found predominantly on cells that could not be fired antidromically either from the MLF or from the spinal cord. This group of cells must include excitatory and inhibitory interneurons performing an integrating function and, as with MVST neurons, regulating the activity of projecting cells. The medial nucleus, then, contains cells driven a t short latency from the ipsilateral labyrinth-from at least two different regions of the receptor and perhaps from four-whose projection into the MLF suggests them as the first central link of some vestibulc-ocular reflex arcs.

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I?. The Horizontal Canal-Abclucens Reflex Arcs Aside from the uncertainty concerning the exact location of the vestibular neurons involved, part of one reflex pathway to extraocular neurons has now been worked out in detail. This is the network producing deviation of the eyes in response to activation of the horizontal canal. Ipsilateral rotation produces contralateral deviation of the eyes by means of contraction of the ipsilateral internal rectus and contralateral external rectus, and relaxation of their antagonists. It is the pathway between the horizontal canal and lateral rectus motoneurons in the abducens nucleus that has received particular attention. Ipsilateral horizontal acceleration must produce excitation of contralateral and inhibition of ipsilateral abducens motoneurons, with reverse effects during contralateral acceleration. Not surprisingly this is what has been found experimentally. The discussion of experimental results can be followed by referring to Fig. 5. Many abducens motoneurons fire tonically, as rapidly as 80/sec, and the rate of discharge of ipsilateral motoneurons increases during contralateral acceleration (Schaeffer, 1965; Precht et al., 1969). This increase is interrupted by a sharp decrease during the rapid phase of nystagmus, a phenomenon usually absent in decerebrate cats ( Schaeffer, 1965; Precht et al., 1969). Excitation of the motoneurons (type IIA cells) can also be achieved by stimulation of the vestibular nerve contralateral to them with shocks as weak as 1.3 x N1T (Precht et al., 1969). Such Midline Controloteral structures

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I

' L

FIG.5. Schematic representation of the siiaplest pathways producing excitation and inhibition of abducens motoneurons. Only connections to the abducens nucleus on the right side (ipsilateral nucleus) are illustrated. Shown are excitatory pathway activated by contraIateraI acceleration and inhibitory pathway activated by ipsilateral acceleration. Abbreviations: A, abducens nucleus; L, labryrinth; V, vestibular nerve; I excitatory (0) and inhibitory ( 0 ) type I neurons of the horizontal canal; II,, abducens motoneuron. The area enclosed in dashed lines corresponds approximately to the rostra1 medial nucIeus.

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stimulation evokes within the ipsilateral abducens nucleus units activity at 0.8-1.1 msec; these units are apparently commissural axons of contralateral type I neurons, fired monosynaptically from the contralateral vestibular nerve (Richter and Precht, 1968). Intracellular recording from ipsilateral abducens motoneurons reveals e.p.s.p.’s at 1.22.0 msec (Baker et al., 1969). Similar e.p.s.p.’s can be evoked by stimulation in the area of the contralateral rostral medial nucleus with latencies of 0.55-1.4 msec. Because the latency difference between e.p.s.p.’s evoked from the contralateral vestibular nerve and nuclei is usually 0.6-1.2 msec, the pathway between the vestibular nerve on one side and abducens motoneurons on the other is at its briefest disynaptic. According to Baker et al. (1969) the relay is apparently in the rostral medial nucleus or ventral lateral nucleus. One danger when the medial nucleus is stimulated is that the stimulus will activate commissural fibers originating in the descending nucleus and crossing through the medial nucleus. Because the effective region of the medial nucleus is its rostral part this is unlikely to have happened in the experiments of Baker et al. (1969): the commissural fibers tend to be more caudally located ( Ladpli and Brodal, 1968). Rostra1 medial stimulation could involve parts of the superior nucleus, however. In any case, some excitatory cells of the three-neuron arc originating in the horizontal canal on one side and terminating in the abduceiis nucleus on the other are located in or near the rostral medial nucleus. Anatomical evidence is of little help in fixing the location of these neurons more precisely, as there is little information on this pathway from Tarlov’s experiments ( 1970). These show a weak contralateral link between the superior and abducens nuclei. As pointed out by that author, his lesions in the medial and superior nuclei were incomplete; perhaps an important source of fibers to the contralateral nbducens nucleus was missed. During ipsilateral acceleration ipsilateral abducens motoneurons are inhibited (Precht et al., 1967a). Inhibition of unit activity can also be produced by low-intensity (shocks less than 2 X NLT) stimulation of the ipsilateral vestibular nerve; the inhibition is brought about by pathways restricted to the ipsilateral brainstem (Richter and Precht, 1968). Extracellular action potentials, presumed to be spikes of axons of ipsilateral type I vestibular neurons, can be recorded near the motoneurons 0.8-1.1 msec after the stimulus. With inhibition seen as early as 1.3-1.4 msec, this suggests the presence of a disynaptic inhibitory pathway, a suggestion confirmed by intracellular recording. Stimulation of the vestibular nerve produces i.p.s.p.’s in ipsilateral abducens motoneurons at 1.353.2 msec; stimulation of the vestibular nuclei produces i.p.s.p.’s at 0.65-2.5 msec; therefore the nuclei exert monosynaptic inhibitory

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action on abducens motoneurons of the same side (Baker et al., 1969), When localized stimulation of the vestibular nuclei is performed, the rostral region of the medial nucleus is shown to be the effective area (Baker et al., 1969). Although the conclusion must again be qualified by the dangers involved in electrical stimulation of such a complex area, it appears that a restricted region that includes the rostral part of the medial nucleus is an inhibitory relay area for connections to abducens motoneurons. Anatomical evidence indicates a strong ipsilateral connection between the medial vestibular and abducens nuclei ( Tarlov, 1970). Inhibition and excitation of abducens motoneurons as a result of horizontal acceleration can therefore be executed by simple reciprocal three-neuron arcs, together with longer-latency pathways of greater complexity. Rotation to one side activates the excitatory and inhibitory pathways originating in one labyrinth, and at the same time reduces the activity of antagonistic pathways originating in the receptors of the labyrinth on the other side. This reduction aids in the execution of the appropriate movement.

c. Control of the Horizontal Reflex Arc The three-neuron arcs just described are under the control of structures other than the labyrinth. As already described, inputs from rostral areas of the nervous system and from the spinal cord excite interneurons, including the type I1 cells of Shimazu and Precht (1965). These neurons, that are assumed to inhibit type I cells (Shimazu and Precht, 1966), are activated at short latency from the contralateral labyrinth (Sh‘imazu and Precht, 1966); from the interstitial nucleus of Cajal, a center that seems to be of particular importance in the suppression of the pathway for horizontal eye movements (Markham et al., 1966); from the contralateral fastigial nucleus (Shimazu, 1967). These are only some of the inputs available for control of the vestibulo-ocular pathway responsible for horizontal eye movement. Whereas this pathway is not affected by stimulation of the cerebellar vermis ( Shimazu, 1967), it undoubtedly can be inhibited from the vestibulocerebellum, that projects to the medial and superior nuclei (Angaut and Brodal, 1967).

d. Some Other Simple Vestibular-Extraocular Pathways Connections similar to those of the horizontal reflex arcs must link the other cristae to extraocular motor nuclei. Some observations on these pathways exist. IntracelIular recording from cat oculomotor neurons reveals short-latency e.p.s.p.’s and i.p.s.p.’s on stimulation of the vestibuIar nerve ( Sasaki, 1963). Similar potentials have recently been observed in

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rabbit oculomotor neurons: e.p.s.p.’s at 1.3-2.4 msec with a mean of 1.7; i.p.s.p.’s a t 1.3-2.4 msec with a mean of 1.9 (Ito et al., 1970b). These are disynaptic latencies. The synaptic potentials can also be produced by stimulation of the rostra1 part of the ipsilateral vestibular complex, with monosynaptic latency ( Ito et al., 1970b). Vestibular inhibition of oculomotor neurons is resistant to strychnine, but may be blocked by picrotoxin ( I t o et al., 1970b), suggesting GABA as the inhibitory transmitter. The potentials in oculomotor neurons are depressed at short latency by stimulation of the flocculus. The depression seems to be due to inhibition of relay cells in the vestibular nuclei, as i.p.s.p.’s are recorded in these neurons on stimulation of the flocculus, with a latency short enough so that the pathway may be monosynaptic (It0 et al., 19704. Recently disynaptic i.p.s.p.’s and e.p.s.p.’s have been evoked in trochlear motoneurons by stimulation of the ipsi- and contralateral vestibular nerve, respectively (R. Baker et d.,1971; Precht and Baker, 1972). Systematic localization of the excitatory and inhibitory areas was not attempted, but there were low-threshold points for inhibition in the ipsilateral superior, and for excitation in the contralateral medial nucleus. As we have seen, different lines of evidence suggest that the pathways linking the maculae to cxtraocular neurons may be more complex than three-neuron arcs, although the possibility of some pathways of this length is not excluded. Further investigation of these connections is particularly desirable. Whereas three-neuron chains are the simplest present in the vestibuloocular arc of mammals, even simpler situations exist in lower vertebrates. Stimulation of the eighth nerve in puffer fish evokes spikes in ipsilateral oculomotor neurons. The spikes arise in the niotoneuron dendrites and, because of their short minimal latency (1.0-1.5 msec), may be evoked monosynaptically, without a relay in the vestibular nuclei ( Kriebel et d.,1969). Cerebellar control of oculomotor neurons also is more direct in teleost than in mammal. Stimulation of the cerebellum produces i.p.s.p.’s in oculomotor neurons of the Japanese snake fish, and calculations reveal that the inhibitory pathway is monosynaptic ( Kidokoro, 1968). Further comparative studies in lower vertebrates, where the labyrinth plays a greater role in oculomotor regulation than in higher animals, may reveal other direct and powerful pathways.

e. Comment While the basic organization of the simplest mammalian vestibuloocular reflex arc is now clear, the unraveling of the detailed connections between different labyrinthine receptors and extraocular motoneurous

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remains to be done. The problem is of interest mainly from the point of view of the microorganization of the vestibular nuclei. Thus we can ask whether particular regions of the nuclei are connected with motoneurons innervating specific muscles, and whether there are rostrally projecting inhibitory and excitatory neurons in both the superior and medial nuclei. There may still be a possibility that all inhibitory cells are concentrated in one region, for example the medial nucleus. The cells in this nucleus appear to be involved in inhibition of spinal motoneurons, contralateral vestibular neurons, and ipsilateral abducens motoneurons, but the recent work of Precht and Baker (1972) on trochlear motoneurons suggests that inhibitory neurons are not restricted to the medial nucleus. This point should be tested more closely. Finally, one would like to know whether inhibitory fibers to neck motoneurons and some extraocular motoneurons arise from the same population of medial nucleus cells, and whether some are branches of dichotomizing axons. The pharmacological results of Felpel (1972) and Ito et al. (1970b) suggest that two different inhibitory transmitters are liberated by axons arising in the vestibular nuclei and point to at least two groups of inhibitory cells. It is not known whether all vestibulo-extraocular inhibitory pathways are blocked by the same pharmacological agents, and this certainly should be investigated. While attempting to answer such questions we must be careful not to think of the reflex arcs between given receptors and muscles as working in isolation, as the fuiictioning of the horizontal arc has just been described. Obviously few if any head movements take place only in the plane of one canal, and in normal function many reflex pathways, activated simultaneously, will influence each other. Nor can we separate reflexes originating in the cristae from those originating in the maculae: stimulation of a canal may evoke different results depending on the position of the head (cf. Lorente de Nb, 1932; Duensing and Haufe, 1968). The presence of such interactions is not surprising, since it is known that different parts of the labyrinth converge on cells in the vestibular nuclei, even though the nature of the converging pathways is not known in detail.

VI.

Concluding Remarks

Throughout this review some points of special interest have been discussed and areas in need of further study have been pointed out. A few comments may still be made. Considerable progress has been made in unraveling the functional organization of the vestibular nuclei and of their projections by means of closely correlated anatomical and physiological investigations. Both approaches have provided evidence for the subdivision of the nuclei. It

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is in this area that challenging questions remain, concerned, for example, with the way in which vestibular neurons projecting to different extraocular motoneuron nuclei are grouped, and with the organization of inhibitory projections to extraocular and neck motoneurons. Experiments with the type of microstimulation used in the motor cortex by Asanuma and his colleagues ( Asanuma and Sakata, 1967; Stoney et al., 1968) may be of value here. Evidence has been brought forward for the localized termination of various inputs to the vestibular nuclei, and this has been discussed in terms of the location of vestibular neurons projecting to different regions of the central nervous system. We must be careful not to overemphasize this “somatotopic” organization: anatomical work shows only direct projections, and much electrophysiological work emphasizes monosynaptic pathways at the expense of less-direct ones. The possibility for interaction between separate regions of the nuclei, and for the overwhelming of apparent boundaries by complex inputs, is great. The pathways discussed in this review are only a skeleton, that provides no more than a pattern for vestibular activity during normal function. The cerebellum must be a most important regulator of the activity of vestibular neurons. We have some knowledge, certainly incomplete, of the pathways linking cerebellum and vestibular nuclei and of the actions that these pathways are capable of executing. We remain ignorant of the way these pathways function to modify the vestibular activity that occurs in response to natural stimulation or movement. This should be a fruitful field for research. Finally, much of the work done so far has dealt with the static properties of vestibular pathways. There is need now for more analysis of their dynamic properties, a direction in which a beginning has already been made. It is here that the cerebellar influence way be most important. ACKNOWLEDGMENTS

I am grateful to my colleagues Drs. B. W. Peterson and L. P. Felpel for reading and commenting on various drafts of this review, and to Miss Mary Acquaviva for preparing the manuscript. REFERENCES Adrian, E. D. (1943). J. Physiol. (London) 101, 389. Ajala, G. F., and Poppele, R. E. (1967). I n “Neurophysiological Basis of Normal and Abnormal Motor Activities” ( M . D. Yahr and D. P. Purpura, eds.), p. 141. Raven, New York. Allen, G. I., Sabah, N. H., and Toyama, K. ( 1971). Brain Res. 25, 645. Angaut, P., and Brodal, A. (1967). Arch. ltal. Biol. 105, 441. Asanuma, H., and Sakata, H. (1967). J. Neurophysiol. 30, 35.

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TETRODOTOXIN. SAXITOXIN. AND RELATED SUBSTANCES: TH El R APPLICATIONS IN NEUROBIOLOGY By Martin

H . Evans

Agricultural Research Council. Institute of Animal Physiology

.

Babraham. Cambridge. England

I . Iiitroduction . . . . . . . . . I1. Sources of the Toxins . . . . . . . A Tetrodotoxin ( T T X ) . . . . . . . B . Saxitoxin (STX) and Related Coinpounds . . 111. Chemical and Physical Properties of the Toxins . . A . Tetrodotoxin . . . . . . . . B Saxitoxin . . . . . . . . . IV Assay Methods . . . . . . . . . A. Bioassay of Tetrodotoxin . . . . . . B. Bioassay and Chemical Assay of Saxitoxin . . C . Differentiation between Tetrodotoxin and Saxitoxin V. Actions in Vitro . . . . . . . . . A . Effects on Nerve . . . . . . . B . Effects on Muscle . . . . . . . C . Effects on Neuroniuscular Junctions and Synapses D. Effects on Receptor Organs . . . . . E . Effects on Other Tissues . . . . . . VI . Actions in Vivo . . . . . . . . A . Effects on Nearomuscrilar Systems . . . . B . Effects on the Cardiovascular System . . . . . C Effects on the Central Nervous System . . . . . VII . Summary and Concluding Remarks References . . . . . . . . . Note Added in Proof . . . . . . .

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. . . .

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. . . .

. . . .

. . . .

. .

. .

. .

. .

.

.

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83 84 84 86 89 89 92 94 95 101 106 108 108 122 133 137 139 141 141 144 147 152 155 166

I . Introduction

During the course of about ten years. tetrodotoxin has risen from relative obscurity to a position of importance . Once regarded as an exotic toxin. it is now commonly used in neurobiological research as a tool with a specific pharmacological action which is helpful during investigations into fundamental n e ~ r ~ n i ~ s c u Iphysiology ar . However. tetrodotoxin is not unique in its action; saxitoxin appears to possess very similar pharmacological propertks. and a number of other substances 83

s4

MARTIN H. EVANS

may be related chemically and pharmacologically. Saxitoxin and related compounds have not been so widely used as tetrodotoxin, because they are not yet available from a commercial source, whereas tetrodotoxin has been marketed for several years.' Saxitoxin will be given some prominence in this review, partly because it possesses certain advantages compared with tetrodotoxin, which could result in it becoming widely used, and partly because I have devoted much of my own research effort to an analysis of its biological effects. The usefulness of tetrodotoxin depends upon its ability to suppress the action potentials in skeletal muscles and in vertebrate (and some invertebrate) nerves. It acts on these tissues in a highly specific manner, preventing the voltage-dependent, regenerative, increase of permeability to sodium ions. The appropriate sections of this article will discuss the evidence for this, together with evidence that it is without effect on potassium conductance and without effect on the mechanisms of chemical transmission at synapses and the neuromuscular junction. The evidence that saxitoxin has very similar actions is more scanty, but nevertheless adequate. In general, the actions of tetrodotoxin and saxitoxin resemble each other so closely that they will not be treated separately in this article except for the sections that deal with their origins and physicocheniical properties. The similarity of their actions makes it difficult to distinguish between them in bioassays, so another section has been set aside for a discussion of the ways in which they can be differentiated in those cases where a worker is faced with the identification of an unknown agent. II. Sources of the Toxins

A. TETRODOTOXIN ( TTX) Certain species within two unrelated groups of animals, puffer fish, belonging to the order Tetraodontidae, and newts, belonging to the family Salamandridae, have been found to contain tetrodotoxin [abbreviated to TTX by Moore (1965) at the suggestion of K. S. Cole; this abbreviation is now widely used]. These animals seem to have evolved the ability to produce TTX through metabolic pathways and to build up considerable concentrations of it in their tissues. It has been suggested that the presence of this toxin may have a definite survival value for these species of fish (Steinbeck and Ricketts, 1941) and newts (Mosher et al., 1964). Nothing is known about the metabolic pathways through which TTX 'Originally by the Sankyo Co. Ltd., 7-12 Ginza 2-chome, Chuo-ku, Tokyo 104, Japan. It is now also available from several distributors.

TETRODOTOXIN AND SAXITOXIN I N NEUROBIOLOGY

55

is synthesized. It has been suggested that it might be acquired from a food source (Halstead, 1967), but there is little evidence to support this (Y. Hashimoto and Kamiya, 1970). Biosynthesis of TTX niay be linked with hormonally controlled breeding cycles, because in the puffer fish there appears to be considerable seasonal variation in toxicity. The TTX content of the tissues, especially the ovary, rises before spawning in May and June (Tani, 1945; Ogura, 1958; both cited by Ogura, 1971). Newt eggs also contain considerable quantities of TTX (Twitty and Johnson, 1934; Mosher et al., 1964). Not all species of puffer fish contain TTX. Among the Tetraodontidae, the most toxic species are in the genus Sphaeroides. In Japan, where the flesh of the pdffer fish (fugu) is regarded as an epicurean delicacy, the very toxic nature of Sphaeroides porphyreus ( m a fugu) and Sphaeroides rubripes (tora fugu) is well recognized. Arothron and Tetraodon species may also be poisonous. Detailed lists of toxic species are given by Kao (1966) and also in Halsteads beautifully illustrated monograph (1967); they both discuss tetraodontid poisoning from the public health viewpoint, with quotations from classical accounts of accidental poisoning. Kao also tabulates data (recalculated from Tani), showing the TTX content of individual organs from 14 species of puffer fish. The liver or ovaries of four Sphaeroides species contain up to 1 mg of TTX per gm of fresh tissue. (One mg is approximately 1 human lethal dose, taken by mouth.) Crystalline TTX was first isolated from S. rubripes by Yokoo (1950, 1952) who named it spheroidine. It is now available commercially in ampules containing 1mg of TTX (as the free base) with 5 mg citrate buffer to stabilize the pH at 4.8-4.9 when made up into solution. I t is important that users of the commercial product should be aware of the citrate content. Several workers have reported TTX effects, obtainable only at extremely high concentrations, that niay have been due to the citrate rather than the toxin. Kao (1966), Dettbarn (1971), and Gage (1971) emphasize caution in this respect. The toxicity of the Californian newt Taricha torosa has been known since Twitty and his colleagues performed organ transplantation experiments between embryos of T. tarosa and the salamander Ambystoma punctatum (Twitty and Elliot, 1934; Twitty and Johnson, 1934). Fuhrman and Mosher and their colIeagues succeeded in isolating the toxin in crystalline form, and they named it tarichatoxin (M. S. Brown and Mosher, 1963). Wakeley et al. (1966) found tarichatoxin in fairly large amounts in the skin, ovaries, and ova of T . torosa and in the blood of the male but not the female of the species. Twitty and Johnson (1934), on the other hand, found the blood of the female to be more toxic. Wakeley et al. (1966) found T . torosa females to have a mean content

86

MARTIN H. EVANS

of 47 pg of tarichatoxin per gm body weight (range 2 1 1 0 pg/gm); the males were less toxic, with a mean of 18 pg/gm. The related species Taricha rivularis and Tarichu granulosa contained comparable amounts, but other species of newts, salamanders, frogs, and toads contained little or none. Tarichatoxin was soon found to have pharmacological properties that were indistinguishable from those of TTX (Kao and Fuhrman, 1963), and it was not long before tarichatoxin was shown to be identical with TTX (Buchwald et al., 1964). It is now usual to refer to the newt toxin as tetrodotoxin. Halstead (1967) gives details of the extraction and purification techniques employed by a number of workers to obtain TTX from puffer fish. In addition to puffer fish and newts, TTX has been reported to occur in the goby Gobius criniger (Y.Hashimoto and Noguchi, 1971). TTX does not appear to have been synthesized yet, though progress is being made in this direction (Kishi et al., 1970a,b, 1971, personal communication). A number of derivatives and related chemicals have been prepared and their biological actions investigated ( Narahashi et al., 1967a; Deguchi, 1967; Moore and Narahashi, 1967; Ranney et al., 1968; Ranney and Fuhrman, 1968; Spiegelstein and Kao, 1970).

B. SAXITOXIN (STX) AND RELATED COMPOUNDS Saxitoxin appears to be identical with the active principle in moreor-less purified toxic extracts which have been known as paralytic shellfish poison, clam poison, or mussel poison, depending upon the source of the extracts. It has occasionally been called plankton poison and mytilotoxin ( b y Barstad, 1968). Brieger ( 1888, 1889) also prepared a substance which he called mytilotoxin, but it was probably not STX (Schantz, 1960, 1971b). The name saxitoxin was given by Schuett and Rapoport ( 1962) to the purified paralytic shellfish poison extracted from the Alaskan butterclam, SaxicZomus giganteus. Pure paralytic shellfish poison has also been obtained from the Californian sea-mussel Mytilus californianus and from the plankton dinoflagellate Gonyaulux catenella. The name saxitoxin is now applied to the poison from any of these three sources, because Schantz and his associates have evidence that their physical and chemical characteristics are identical (summarized by Schantz et al., 1966). Their biological effects are also indistinguishable (Evans, 19714. Saxitoxin is now often abbreviated to STX (Narahashi and Haas, 1967). Sommer et al. (1937) showed that there was a strong correlation between the occasional appearances of toxicity in the Californian mussels and the presence of large numbers of G. catenella in the plankton. They suggested that the mussels acquired their poison only when feeding

TETRODOTOXIN AND SAXITOXIN I N NEUROBIOLOGY

87

on a “bloom” of G. catenella. Further work substantiated this view (e.g., Riegel et aI., 1949) and the fascinating story of the food chain origill of “mussel poison” has been repeated inany times. Detailed accounts can be found in reviews by Kao (1966), Schantz (1971a), and Prakash et al. (1971), as well as in the classical paper by Somnier and Meyer (1937). In view of this evidence, together with the apparent chemical, physical, and pharmacological identity of the poison from M . californianus and G. catenella ( mentioned above and discussed in Section 111, B ) there can be little doubt that the Californian mussels acquire STX by feeding on “blooms” of G. catenella. The source of the STX in the clam S. giganteus is more problemat~cd. The clam STX is, as mentioned above, identical with the poison of the dinoflagellate G. catenella. However, several attempts to show the presence of this dinoflagellate in the seawater near areas where the clams are toxic have met with little success (Schantz and Magnusson, 1964). Clams show variations in toxicity according to their location and the degree of toxicity varies from time to time. Sommer and Meyer (1937) showed that in niussels half the STX disappear5 in about 10 days. In cIams, however, the half-life appears to be much longer. Schantz and Magiiusson transferred toxic clams to an apparently nontoxic environment and found that the STX took more than one year to disappear. Chambers and Magnusson (cited by Schantz and Magnusson, 1964) noted spontaneous loss of 5 6 8 0 % toxicity from clam syphons in 3-4 months. Hence clams would be able to accumulate STX slowly over long periods of time, even though there were few G. catenella in their food, but Schantz and Magnusson thought it doubtful whether there were enough G. catenella present in Alaskan waters to account foi the STX content of the clams even when allowance was made €or this cumulative effect. Kao (1966) lists 14 species of bivalve shellfish that have been reported to contain STX, as well as the sand crab Emerita analoga. It seems probable that all these acquire STX through a food chain from plankton organisms such as G. cateneTEn. More recently, Hashinioto and his colleagues have found three species of pacific island crab to be highly touic: Zosimus aeneus and Platypodia granulova ( Hashimoto et al., 1967) and Atergatis floridus (Inoue et al., 1968). The toxin is present mainly in the exoskeleton and, at least in the case of Z. aeneus, appears to be identical with STX (Konosu et al., 1968; Noguchi et al., 1969). I t has been suggested that the5e crabs might accumulate STX when feeding on coral reef algae (Hashinioto and Kamiya, 1970). The freshwater blue-green algae Aphanixomenon f7os-aqttae (or A. holsaticum) sometimes produce a substance that is very similar to, and

88

MARTIN H. EVANS

may be identical with STX. It can also produce other toxic substances similar to but not identical with STX (Sawyer et al., 1968; Jackim and Gentile, 1968; Gentile and Maloney, 1969). The dinoflagellate Gonyaulax tamarensis appears capable of producing two toxic substances, one of which closely resembles STX, while the other has similar though not identical properties ( Schantz, 1960; Prakash, 1967; Evans, 1970a). Poison from G . tamarensis has recently been purified and found to have a chemical structure different from STX ( E . J. Schantz, 1971, personal communication). Substances that are similar to, but not identical with STX, have also been reported by Casselinan et al. (1960) and Bannard and Casselman (1961a, 1962). Some of these may be degradation products of STX. Most attempts to prepare derivatives of STX result in considerable loss of toxicity (Schantz, 1960), but Bannard and Casselman (1961b) obtained a toxic substance by treating STX with a mixture of anhydrous nitiic and sulphuric acids, that probably produced an oxidative degradation product of STX. Sommer and Meyer (1937) mentioned other toxins from mussels, but these are clearly different from STX. Although many species of freshwater and marine algae manufacture toxic substances, some of which have effects on nervous tissue, these substances are not identical with STX (see Schantz, 1970). Not all species of Gonyaulax produce STX-like substances; Gonyaulax polyedra has been used as a major dietary constituent for rats with no evidence of toxic effects (Patton et al., 1967), although this species has occasionally been reported to produce toxin (see Loeblich, 1966; Schantz, 19714. Even the regularly toxic species vary in their ability to produce poison (Prakash, 1967). Nothing is known about the biochemical pathways that lead to the formation of STX by G. catenella, which can be grown in artificial seawater to produce STX under Iaboratory conditions (references and some details in Schantz, 1971a). An examination of the empirical formula (see Section 111, B) reveals the large proportion of nitrogen. About 33% of the molecular weight is due to the N content. This high N content makes it tempting to speculate that STX is a product, perhaps the end product, of a pathway for the excretion or sequestration of nitrogen. It might be linked with the metabolism of guanine, present in large amounts in some Gonyaulax, and which appears to be associated with luminescence (see Loeblich, 1966). While TTX offers protection against predators, to benefit those species of fish and newts that possess it, STX seems to be of no survival value to the dinoflagellates, because the bivalve shellfish which feed on the dinoflagellates are not themselves affected by STX (see Section V, B, 4 ) . It is probably not feasible to label either STX or TTX with radio-

TETRODOTOXIN AND SAXITOXIN I N NEUROBIOLOCY

89

active isotopes by the Wilzbach tcchniquc, because of thc severe destruction of toxin which is liable to occur during the rcaction, and in any case repurification of thc toxin would be necessary to remove the radioactive breakdown products formed. It should, however, bc possible to culture G. catenella in an artificial seawater in which an isotope is dissolved. The high N content of STX suggests the possibility that if G. catenella were grown in artificial seawater containing "N, this isotope would become concentrated and incorporated into the STX molecule. The labeled STX could be extracted from the culture and purified, using standard techniques such as those described by Schantz et nl. (19S7). Unfortunately I3N is not radioactive, so thc tracer would have to be followed by mass spectrometer techniques. I l l . Chemical and Physical Properties of the Toxins

Oidy those properties likely to be of practical importance to the experimental biologist are discusscd. For further details of the chemistiy, including the establishnient of the structural formulas, the reader should refer to reviews by Schantz (1960, 1971a,b), Bannard (1962), Ikuma (1964), Mosher et al. (1964), Scheuer (1964), Goto et nl. (1965), and Wong et al. (1971). Shorter papers, by Schantz et al. (1961, 1966), Brown and Mosher (1963), Buchwald et al. (1964), Tsuda et al. ( 1964a,b), and Furusaki et al. (1970) also give useful inform a t'1017.

A. TETRODOTOXIN The active principle in extracts of puffer fish was mined tetrodotoxin by Tahara (1911) but it WRS not obtained in reasonably pure form until it was crystallized by Yokoo (1950, 1952), who named it spheroidiiie. This name is not now used. Pure TTX is a white crystalline solid, darkening above 220°C without melting. The free base is not soluble in lipids, organic solvents, or in water, although if refluxed in water for several hours it will go into solution, through conversion to tctrodonic acid, a nontoxic derivative (Tsuda et al., 196413). TTX dissolves readily in wcak acids. It is a monoacidic base and the pK,, has bceii given as 8.5 (Woodward, 1964), 8.84 (Tsuda et al., 1964b), and 8.76 (Goto et al., 1965). In 50%ethanol the pK, is given as 9.54 (Tsuda et nl., 196413) and as 9.40 (Goto et al., 1965).In acid solutions TTX is reported partly to epinierize to anhydroepitetrodotoxin, ( pK. = 7.95) forming an equilibrium mixture in a ratio of about 4: 1 (Goto et al., 1965). Solutions of 7TX, in weak acids or buffered to pH 4 5 , arc reasonably stable if kept refrigerated. They can be relied on for at least a month

90

MARTIN H. EVANS

and can often be kept for a year or more at about 0°C with negligible loss of toxicity. However, toxicity is lost in alkaline solutions, and TTX is also less stable in strong acids than at pH 4-5, differing in this respect from STX. It is rapidly destroyed by boiling at pH 2 or under. There have been reports that different batches of commercially prepared TTX vary considerably in effectiveness on some biological responses ( Benolken and Russell, 1967; Hille, 1968; Solomon, 1969). The specific optical rotation [a12 is quoted to be -8.64" (Sankyo Co. data); -5.0 2 1.0" or -7.8 k 1.0" (Buchwald et ul., 1964). There is no characteristic ultraviolet ( UV) absorption spectrum, but the infrared ( I R ) spectrum shows several strong absorption bands. The IR spectra of TTX and tarichatoxin are identical, as shown in Fig. 1 (Buchwald et al., 1964; see also Section 11, A). The empirical formula of TTX is C,, H,iN,30, (molecular weight = 319.3). The structure was established independently by four groups of workers: T. Goto and his colleagues at Nagoya University, K. Tsuda of the University of Tokyo working with colleagues from Sankyo Co. Ltd., F. A. Fuhrnian, H. S. Mosher and their colleagues at Stanford University, and R. B. Woodward and his group at Harvard University. These four groups reported their results at the third IUPAC Symposium on the Chemistry of Nature Products (13th April 1964, Kyoto, Japan) and there was agreement that the structure of TTX is best represented as shown in Fig. 2a. Furusaki et aZ. (1970) have confirmed this molecular structure by means of three-dimensional X-ray analysis. Figure 2a shows the zwitterion form of the TTX molecule, with a positive charge on the guanidinium group and a negative charge at the hemilactal oxygen. TTX can also exist in two tautomeric cationic forms in equilibrium with each other, shown in Fig. 2b and 2c. It has been 3

4

5

6

7

10

0

15

12

TETRODOTOXIN

I

I

,

)

3400 3000

9

1700 1600

I

1400

I

1200

cm-' I

1000

I

000

600

FIG. 1. Infrared absorption spectra of taricliatoxin and tetrodotoxin. From BuchwaId et at. (1964). Reproduced from Science 143, 4 7 4 4 7 5 (1964) by permission of the holders of the copyright (1964), The American Association for the Advancement of Science.

TETRODOTOXIN AND SAXITOXIN I N NEUROBIOLOCY

(a)

91

0-

on

0

I

-CH20H

H

(b)

on (C)

FIG. 2. Structure of tetrodotoxin, showing the zwitterion, a, which is in equilibrium with two cationic forins, b and c.

pointed out that significant amounts of all three forms will be present at physiological pH values because the pK, of TTX is about 8.8. The activity of TTX in vitro is dependent on the p H and so it seems that one or both cationic forms are biologically more potent than the zwitterion. This has important implications when the mode of action of TTX is discussed in Section V, A, 1 (Camougis et al., 1967; Narahashi et al., 1969b). The entire structure of the TTX molecule appears to be critical for its action. A number of derivatives have been prepared and their toxicities measured on mice (Tsuda et al., 1964b; Deguchi, 1967; Ogura and Mori, 1968), on conduction of impulses in frog sciatic nerve (Deguchi, 1967), crayfish nerves (Ogura and Mori, 1968), and in the giant axons of lobster and squid (Narahashi et al., 1967a). None of the derivatives were as potent as TTX, and only deoxytetrodotoxin showed any substantial activity. However, Moorc and Narahashi (1967) found that the sample of deoxytetrodotoxin that they had used was contaminated with more than enough TTX to account for the potency of the sample. The other derivatives had less than 1%of the potency of TTX and again it is possible that at least some of this activity was due to contamination with small amounts of TTX. The derivatives produced effects that were, with a fcw cxceptions, qualitatively indistinguishable from those of TTX (Deguchi, 1967). In an attempt to elucidate the active structure, a number of synthetic guanidine esters have been

92

MARTIN H. EVANS

tested, but none of them approach the potency of TTX and they do not appear to have actions that closely resemble that of TTX (Fuhrman et al., 1968; Raiiney and Fuhrman, 1968; Ranney et al., 1968; Spiegelstein and Kao, 1970, 1971). Promising attempts are being made to synthesize TTX ( Kishi et al., 1970a,b, 1971, personal communication). At the time of writing, Kishi and his collaborators have reached the stage where 9-deoxytetrodotoxin has been synthesized and full synthesis of TTX may soon be achieved ( Kishi, 1971) .

B. SAXITOXIN Saxitoxin is iiornially purified in the form of the dihydrochloride salt, a white hygroscopic powder. It is difficult to crystallizc, usually being obtained as a glassy solid (Casselniaii et al., 1960; Schantz, 1960). I t is very soluble in water, somewhat soluble in ethanol, methanol, and glacial acetic acid, and insoluble in lipid solvents; quantitative solubilities have not been published. Two base functions have been reported, one at pK;, 8.1-8.3 and the other at about 11.5 (Schantz, 1960; Lhantz et al., 1961). The specific toxicity has been given as 5500 +- 500 mouse units ( M U ) per nig when in aqueous solution aiid tested by the standardized technique described in Section IV, B. The specific optical rotation [a]:: is + 130 +. 5" (Schantz et al., 1957). Pure STX does not fluoresce, nor does it show any distinct absorption of visible or ultraviolet above 220 nm. In alkaline solutions, however, nontoxic oxidation products are formed, which do absorb UV in the regions of 235 aiid 333 nm. The infrared absorption spectrum shows strong absorption bands at approximately 3.3, 5.8-6.3, 7.6, and 8.6-9.5 p m , as illustrated in Fig. 3. This curve shows soiiie similarity with the IR absorption spectrum of TTX, illustrated in Fig. 1, although the fine spectral detail present in Fig. 1 is absent in Fig. 3. It is recommended that stock solutions of STX should be in 0.1 N HCI, and these appear to retain their potency almost indefinitely if refrigerated, though some workers also add ethanol 1 5 2 0 %by volume to prevent the growth of microorganisms (Schantz et al., 1958). STX is SO stable in acid solution that it loses only 25-30% of its toxicity after 24 hours in 3 N HCI at 100°C. It differs markedly from TTX in this respect. At pH 5 it is less stable and in alkaline conditions it is degraded quite rapidly unless the temperature is kept close to 0°C (Somnier et al., 1948). The formula of saxitoxiii dihydrochloride has usually been given as CloH,iN,0,.2HC1 (molecular weight = 372, Schantz, 1960). However, Kao (1966) suggested that STX may be C,oH,,N,O,. The structure has been subjected to detailed investigation by Rapoport and his co-workers,

TETRODOTOXIN A N D SAXI'l'OXlN I N NEUROBIOLOGY

93

MICRONS

FIG.3. Infrared spectra of saxitoxin, piirified from Goiiyattlux cateiiellu ( solid h e ) and Mytiltrs calijorniantts (broken line), The spectrum of saxitoxin from Saxidonazts giganteus is identical with the latter. Froni Schantz et al. ( 1966). Reproduced froin Biochemistry 5, 1191-1195 ( l9G6). With perinission of the holders of the copyright ( 19GG), The American Chemical Society.

and a tentative structure was assigned to it by Rapoport et ul. (1964). The most recent studies indicate that the structure of the STX molecule is that shown in Fig. 4 ( H . Rapoport, 1971, personal communication); the chemical formula of this structure is C,,H1,N,O:,.2HC1(Wong et ul., 1971). The molecule can exist in two tautomeric forms of slightly differing toxicities, and these are normally in equilibrium with each other, the most toxic form predominating (Mold et ul., 1957; Schantz, 1960). The tautomeric forms were separated by means of countercurrent distribution techniques in a system composed of n-butanol, 95%ethanol, 0.1 M aqueous potassium bicarbonate and ,,-ethyl caproic acid. The aqueous phase was buffered at pH 8 and loss of toxicity by degradation in alkali was minimized by keeping the temperature below 10°C. Countercurrent techniques can be used in thc final stage of purdication of STX, though the paper chromatography and papcr electrophoresis techniques developed by Bannard and his colleagues are probably ii~orcsuitable for routine use. In t-butanol, acetic acid, and water ( 2 : l : l ) STX has an RE.

vt H+

CI- H,N+

T:. *)NH;

CI-

H

FIG. 4. Structure of the saxitoxin niolecule, shown as the dihydrochloride. The two guanidinium groups each carry a positive charge. This structural diagram was kindly provided by R:ipoport (1971 ); it has since been published by Wong et a!. (1971).

94

MARTIN H. EVANS

of 0.32 on paper (component 3 of Casselinan et al., 1960) or 0.39-0.46 on thin-layer silica gel (Jackim and Gentile, 1968), but cannot be separated from traces of a fluorescent impurity. Better purification is obtained by electrophoresis in 1N foiinic acid, when the STX migrates toward the cathode and can be made visible by spraying the paper with Weber’s nitroprusside reagent ( Bannard and Casselman, 1962). STX will also form colored complexes with aromatic nitro compounds, and Jaffe’s (1886) picric acid test for creatinine has been modified for the chemical assay of STX (McFarren et al., 1958, 1959; see Section IV, B, 2 ) . Before these final stages of purification can be attempted, it is necessary to extract STX from the source material and partially purify it to a specific toxicity of about 3000 MU/nig. Riegel, Schantz, Sommer, and their co-workers developed the techniques for extraction and partial purification from about 1933 onwards. A satisfactory method was published by Schantz et al. (1957). They extracted STX with 15%ethanol acidified to p H 2-3. The crude extract was concentrated by vacuum evaporation, the p H brought to 5.5, and the concentrate adsorbed onto a column of the sodium form of the carboxylic ion-exchange resin AmberIite IRC-50. An acetic acid-acetate buffer, p H 4.0, was used to wash unwanted material off the column and partially purified STX was then eluted with 0.5 M acetic acid. Further purification was accomplished by elution through columns of the hydrogen form of a similar resin (Amberlite XE-64) which yielded a material with a specific toxicity of 25003000 MU/mg. Chromatography on alumina in ethanol was used in the final stage of purification. This resulted in an increase of specific toxicity to 5000 t 500 MU/mg and a specific optical rotation of 130 2 5”, but the percentage recovery from the alumina was lower than that from the preceding ion-exchange stages. The technique described above has been used successfully to extract and partially purify a saxitoxin-like substance from the exoskeleton of the crab Zosimus aeneus (Konosu et al., 1968). However, it has not been successful in the case of “paralytic shellfish poison” extracted from scallops or mussels that have been feeding on Gonyaular tamarensis (Schantz, 1960; Evans, 1970a). The major poison from G. tamarensis is not retained by the carboxylic acid resin, which may indicate that it does not have pK, values similar to those of STX, although the biological actions of the two toxins are very similar (see Sections V and VI). IV. Assay Methods

In many practical situatioils it is essential to be able to estimate amount or concentration of TTX, STX, etc. Methods of estimation are of great importance to public health departments concerned with moni-

TETRODOTOXIN A N D SAXITOXIN Ih. NEUROBIOLOGY

95

toring seafoods for these toxins. Experimental work may require assay methods when following the toxin during chromatographic or other stages of purification, or even to estimate the amount of toxin taken up by tissues exposed to it. Methods of bioassay have been developed by many workers and some of their techniques are now widely accepted as standards. Physicochemical methods have, in general, been less satisfactory, but they are mentioned later in this section. Because of the similarity in the mode of action of STX and TTX they are indistinguishable in many tests. For this reason some space is devoted at the end of this section to a description of methods whereby STX and TTX can be distinguished.

A. BIOASSAY OF TETRODOTOXIN The commonest of methods that have been used for the estimation of TTX depend on the determination of the mouse LDso or LD,,,. A d2tailed account of an apparently satisfactory technique has been given by McFarren and Bartsch (1960) with further details by McFarren (1971). The method is based on the earlier techniques used by Sommer and Meyer (1937) to assay STX in mussels, etc. McFarren and Bartsch (1960) were concerned to find a method that would allow public health workeis to estimate the amount of TTX in food materials. They intended the results to be expressed as mouse units ( M U ) of TTX per 100 gm food, whcre the MU is defined as the LD,n for a 20-gm mouse dosed intraperitoneally (i.p.) under standard conditions. They found that the LD,, was 0.23 p g (11.5 pglkg), but other workers have found other values. Although Kao and Fuhrman (1963) found negligible variation in LDSn when testing tarichatoxin (TTX) on four strains of mice, it is known that with STX the LD,, figures can vary considerably with the strain of mice used ( McFarren, 1959). Therefore i t is obviously preferabIe to express TTX content directly in micrograms per 100 gni food. This can be done if every worker first determines the LD,n for his own strain of mice, using the pure TTX that is now available. Examples of LD,, values from various sources are given in Table I. It can be seen that the LD.,,,, expressed as microgranis per kilogram (pglkg), varies from one species to another, and also with the route of administration. Most assays arc based on i.p. administration to mice. McFarren and Bartsch ( 1960) carried out their assays on extracts (or solutions of TTX) in dilute acid. Minced tissue was extracted by boiling for 5 inin in an equal volume of 0.15N HCI, but this probably destroyed some of the toxin in their samples because TTX is unstable under these conditions ( Hashimoto and Migata, 1951; Waterfield and

96

MARTIN H. EVANS

TABLE I LETHALDOSEOF TBTRODOTOXIN, IN pg/kg BODY WEIGHT,FOR VARIOUS SUBJECTS: DOSES EXPRESSED AS LDso UNLIGSS OTHERWISE STATED ~

~~

~

~

~

~~

Route of administration Subject

l.v.

i.p.

2041 lob

11.5 8 6.11 12

8. 22a 11

9.0 13 8-10

11

8-10

11

Rat 11.Of

10

12

Rabbit 2

< 10 5-10 2a

< 10

Oral

References

Tsuda and Kawamura (1953) Murtha et al. (1958) 14 Ogura (1958), cited by Cheymol et al. (1961) McFarren and Bartsch (1960) Cheymol et al. (1961) Fleisher et al. (1961) Sakai et al. (1961) 16 332 50OC*' Fuhrrnan et al. (1963) Kao and Fuhiman (1963) 7.1'~~ Mosher et al. (1964) Tsuda et al. (196413) Cheymol et a/. (1965) Ishihara, recalculated by 8= Kao (1966) Deguchi (1967) 435 Evans (1970a), recalculated 180 Ogura (1963), cited by Ogura (1971) Sankyo Co. data sheet 13-15 180 14 Ogura (1958), cited by Cheymol et al. (1961) Fleisher et al. (1961) 2.70 Ishihara, recalculated by Kao (1966) Sankyo Co. data sheet; 14 147 Ogura (1971) Ishihara, recalculated by 8a Kao (1966) Sankyo Co. data sheet; 10 200 Ogura (1971) >200 Murtha et a/. (1958) Borison et al. (1963) Ogura (1963), cited by Ogura (1971) Ishihara, recalculated by 1Oa Kao (1966) 15" Ogura (1963), cited by Ogura (1971) 9-10a

Mouse

Cat

S.C.

TETRODOTOXIN AND SAXITOXIN I N NEUROBIOLOGY

97

TABLE I (Continued) Route of administration i.v.

Subject Chicken (chick) Chicken

i.p.

S.C.

6,'

5"

Rana pipiens Newt (Taricha torosa) Carp

References Cheymol et al. (1961)

3-4e

Frog

Goldfish (Carassius auratus) Killifish (Fundulus heteroclitus ) Man

Oral

500e

12"-14 >9O,OOW

Ishihara, recalculated by Kao (1966) Ishihara, recalculated by Kao (1966) Wakeley ct a/. (1966) Kao and Fuhrman (1967) Kao and Fuhrman (1967) Ishihara, recalculated by Kao (1966) Kao and Fuhrman (1967)

3u

<10"

R'IcLaughlni and Down (1969)

15",".0

30a e

Recalculated from Halstead (1967)

a Minimum lethal dose. * LD,, or LDlw. As tarichatoxin. d LD,,, see text. Approximate or estimated. TTX hydrochloride. 0 Injscted into the gas bladder.

Evans, 1972). The extracts were then adjusted to between p H 2 and p H 4 and centrifuged before being injected i.p. (in a volume of 1 ml) into mice weighing 20 gm. To determine the LDfiO,a series of dilutions was prepared, each one differing by a constant ratio (1.2 or 1.5) and 1 ml injected into each mouse. For each dilution tested 10 mice were used, and the results analyzed using Weil's (1952) tables. McFarren and Bartsch (1960) found that the mean death-time of mice dying from a dose of 1 x LD,, was approximately 30 inin, although other workers have found some variations from this figure when using other strains of mice. If groups of mice are given larger doses of TTX their mean death-times are shorter, and within a range of 5-30 min there appears to be a linear relation between the logarithm of the dose and the reciprocal of the mean death-time. This can be utilized to assay unknown amounts of T T X without having to determine the LD,, every time. McFarren and Bartsch (1960) published Table 11, giving the relationship between death-time in minutes and MU ( i.e., LDSomulti-

98

MARTIN H. EVANS

TABLE I1 RELATIONSHIP BETWEEN Dosl.; AND DEATH TIMEOF MICE INJPCTED INTR.4PERITONEALLY WITH TETHODOTOXIN" _____

~~

Death timeb

Mouse units

Death timeb

Mouse unit.s

Death timeb

Mouse units

Death timeb

Mouse units

2.00 2.30 3.00 3.30 4.00 4.30 5.00 5.15 5.30 5.45 6.00 6.15 6.30 6.45 7.00 7.15 7.30 7.45 8.00 8.15

12.00 8.00 6.00 5.00 4.00 3.50 3.10 2.95 2.80 2.70 2.60 2.50 2.40 2.30 2.20 2.15 2.10 2.05 2.00 1.95

8.30 8.45 9.00 9.15 9.30 9.45 10.00 10.15 10.30 10.45 11.00 11.15 11.30 11.45 12.00 12.15 12.30 13.00 13.30 14.00 14.30

1.90 1.85

15.00 15.30 16.00 16.30 17.00 17.30 18.00 18.30 19.00 19.30 20.00 20.30 21.00 21.30 22.00 22.30 23.00 23.30 24.00 24.30 25.00

1.30 1.29 1.28 1.27 1.26 1.25 1.24 1.23 1.22 1.21 1.20 1.19 1.18 1.17 1.16 1.15 1.14 1.13 1.12 1.11 1.10

25.30 26.00 26.30 27.00 27.30 28.00 28.30 29.00 29.30 30.00 31.00 32.00 33.00 34.00 35.00 36.00 37.00 38.00 39.00 40.00

1.09 1.08 1.07 1.06 1.05 1.04 1.03 1.02 1.01 1.00 0.98 0.96 0.94 0.92 0.90 0.88 0.86 0.84 0.82 0.80

1.80

1.76 1.72 1.68 1.64 1.61 1.58 1.55 1.52 1.50 1.48 1.46 1.44 1.42 1.40 1.37 1.34 1.32 1.31

11 From McFarreii and Bartsch (1960), reprinted from J. Ass. Ofic.Agr. Chem. 43, 548. By permission of the copyright holders, the Association of Official Analytical Chemists . * Given in minutes and seconds.

ples) from 2 min (12 MU) to 40 min (0.80 MU). The regression line of reciprocal minutes (min-') against log,, MU, calculated from their figures between 5 and 30 min, has an intercept at 1 MU = 0.0289 (34.6 min-') and a slope of 0.3317 niin-l per log MU, and is shown graphically in Fig. 5. With different strains of mice the intercept and slope may vary, and should be determined with his own strain of mice and with pure TTX by any worker intending to use this method of assay. If it is necessary to use mice that do not weigh 20 ? 1 gm, then the weight correction factors shown in Table I11 should be used. The MU's indicated by the assay should be multiplied by the correction factor corresponding to the weight of mice used, to obtain the result in standard MU's. The correction factor is not linear beyond 23 gin and heavier mice should not be used for accurate work. McFarren and Bartsch (1960) recommended that assays should be done with dilutions that give mean death-

TETRODOTOXIN AND SAXITOXIN I N NEUROBIOLOGY

99

TABLE 111 WEIGHTCORHWTION FACTORS~ Wt of Mouse mice (gm) units 10.0 10.5 11.0 11.5 1-2.00 12.5 13.0 13.5

0.45 0.48 0.51 0.54 0.57 0.60 0.63 0.66

W t of Mouse mice ( g m ) units 14.0 14.5 15.0 15.5 16.0 16.5 17.0 17.5 18.0

0.69 0.72 0.75 0.775 0.80 0.8% 0.85 0.875 0.90

Wt, of Morise mice (gm) units 18.5 19.0 19.5 20.0 20.5 21.0 21.5 22.0 22.5

0 !Y25 0.0.5 0.97 1.00 1.02 1.04 1.06 1.08 1.10

Wt of Mouse mice (gm) units 23.0 24.0 25.0 26.0 27.0 28.0 29.0 30.0

1.12 1.15 1.18 1 20 1.21 1.22 1.23 1.24

0 From McFarren and Bartsch (1960), reprinted from J . Ass. O j i c . Agr. Chem. 43, 548. By permission of the copyright, holders, the Aasoeiation of Official Analytical Chemists.

time in the range 10-15 min, or 20-25 min when using crude extracts that contain interfering salt, etc. A practical assay may be rendered inaccurate by the presence of interfering substances in the extracts. Salt can reduce the sensitivity of the assay; even 0.1%NaCl will reduce the TTX assay to 65%of the true value. Other substances present in crude extracts of fish muscle can also affect the assay, and if the injected material is less acid than pH 3 the assay results will be too low ( McFarren and Bartsch, 1960). Similar methods of assay have been used by other workers, e.g., Ogura ( 1958) and Tani ( 1945, cited by Kao, 1966, and by Ogura, 1971) . The latter worker defined his mouse unit as the amount of toxin per gram of mouse needed to kill a 15-20-gm mouse. Hence, the lethal dose for a 20-gm mouse would be 20 Taiii MU, so his MU represents approximately one-twentieth of the quantity of TTX defined as one MU by McFarren and his co-workers. Fuhrman and Mosher and their colleagues at Stanford University developed a similar assay for use during the estract:on of tarichatoxin (TTX) from newts, but their MU was defined as the amount of toxin that would kill a 20-gm mouse in 10 niin when injected subcutaneously (Horsburgh et al., 1940), or i.p. in a volume of 0.2 ml (Kao and Fuhrman, 1963). Horsburgh et al. (1940) gave figures for the minimum lethal dose that would be about 0.57 Stanford MU. Mosher et al. (1964) gave the specific activity of crystalline tarichatoxin as 7000 MU/mg, i.e., one of their mouse units is equivalent to 143 ng of toxin. AS their MU was defined as producing death in 10 min, it must be

100

hfARTIN H. EVANS m in 2 r 0.5r

0.4

-

4 0.2 6 0 -

10

-

0.1

15 -

30 01

I

I

1

I

I

1.o

0 5

0 I

L

1

2

3 M.U.

4

z

1

5

6

I

‘

<

,

7 0 9 1 0

FIG. 5. Graph of the relation between dose of poison and median death time, plotted from data in Tables I1 and IV. Regression lines are fitted to the linear portions of the curves. Ordinate: median death times, as a linear reciprocal scale. Abscissa: dose in multiples of 1 LDs0 on a log scale.

greater than 1 LDSo,which produces death in 30 min according to McFarren and Bartsch (1960) and McFarren (1967). McFarren (1971) estimated the Stanford MU as being approximately 1 LD,,, dose, and Kao (1966) estimated that one Stanford MU was roughly equal to 2.7 LD,, units, giving the extraordinarily low figure of 53 ng (2.65 pg/kg) as the LD,, of the crystalline tarichatoxin of Mosher et al. (1964). This figure seems seriously at variance with the figure of 10 pg/kg quoted by Kao and Fuhrman (1963) for crystalline tarichatoxin, and with similar figures obtained by other workers who used TTX, with which tarichatoxin is identical (see Table I ) . Murtha et al. (1958) gave the LD,, of TTX hydrochloride as 20 pg/kg, but this must have been more than 1 LD,, because their mice died in 3 .4 3 .7 niin. All this emphasizes the desirability of quoting toxicities in nanograms or micrograms rather than in some arbitrary “‘mouse unit,” and also tends to substantiate the views of some workers who believe that LD,, values have little real meaning (Russell, 1966).

TETRODOTOXIN Ah11 SAXI’IOXIN I N KEUROBIOLOGY

101

One of the disadvantages of the “inouse unit” typc of assay lies in the quantity of toxin needccl. It is true that as little as 200 lig of T T X can be detected by i.p. injc~tioninto a small niouse, but a minimum of 5 pg is needed for a reliablc LD,,, assay using four groups of six mice. Even when assayiiig on the close/ death-time graph of McFarren and Bartsch (1960), about 2 pg would bc needed to give a group of six mice a meail death-time in the rc~coininended range of 10-15 min. A different type of assay, developed by Ogura (1963, cited and discussed by Kao, 1966) allows much snialler quantities of TTX to be assayed. This in vitro method measures thc contractions of sniooth muscle in rat stomach, elicited by stimulation of the vagus iiervc at 5-minute intcrvals. TTX blocks conduction in the nerve fibers without affecting smooth inuscle contractility (see Sections V, A, 1 and V, B, 3 ) and inhibition of the vagally elicited conti-actions is propoitional to thc log of the TTX concentratioii between 1 and 20 ng/ml. Tissue extracts cannot be added to the organ bath without first removing some interfering substances. An extraction and partial prificatioi~of the TTX is therefote necessary (Ogura, 1958). Reduction of the amplitude of the compound action potential was used by Moore et ul. (1967a) and by Keynes et al. (1971) to bioassay very low c~ncentrationsof T T X (see Section V, A, 1). Unfortunately, no chemical methods have yet been developed which could be used to assay small quantities of TTX. Thc Molisch reaction is reported to give a strong positive rcwlt (Tsnda and Kawamura, 1953), but of course this reaction is b y no imeans specific.

B. BIOASSAY AND CHEMICAL ASSAYOF SAXITOXIN 1. Bioassay The first reliable bioassays for “paralytic shellfish poison” were developed by Dr. Hermann Sornmer and his associates at the Hooper Foundation of the University of California. Prinzmetal et ul. ( 1932) defined the average lethal dose as that which killed a 20-gm mouse in 10-20 min with characteristic symptoms. This has become known a s a “mouse unit” ( M U ) . Later workers (e.g., Schantz et al., 1958) narrowed the definition of the MU to the quantity that would kill a 20-gin mouse in 15 rnin, when given i.p. in a volumc of 1 1111 at pH 2-4. One MU, by this definition, need not be identical with 1 LD,,, yet in practice it has given satisfactory results. Sonimer and Meyer (1937) notcd that rnicc died more rapidly when given more than 1 MU, and they published a curve showing the re1at‘1011 between dose and iiiean or median death-time ( MDT). They also showed

102

MARTIN H. EVANS

that a correction was needed if the mice did not weigh 20 gm. Schantz et al. (1958) developed a reliable assay technique based on the dose/ MDT relation. They plotted a graph of the reciprocal of the MDT against log dose, and this is shown in Fig. 5. They noted that the graph was linear between 4 and 8 minutes MDT, the curve following the law: log,, dose = (145/t) - 0.2 where t = MDT in sec, between 240-480 sec. For best accuracy Schantz et al. (1958) recommended that the assay should be carried out using dilutions that gave a MDT between 5 and 7 min after i.p. injection in 1 ml. The injected solutions should be between pH 2-4 and free from salt and alcohol, which prolong survival times. Tables have been published giving the doses in M U (0.87SIOO) that result in MDT between 1 and TABLE I V RELATIONSHIP B E T W E E N DOSEA N D DEATH TIMEOF MICE INJECTED INTR.APERITONEALLY

Deat8h Mouse t,imeb units

l:oo 10 15 20 2.5 30 35 40 45 50 55 2:OO

100

66.2 38.3 26.4 20.7 16.5 13.9 11.9 10.4 9.33 8.42 7.67

05

7.04

10 15 20 25 30 35 40 45 50 55

6.52 6.06 5.66 5.32 5.00 4.73 4.48 4.26 4.06 3.88

WITH P.4RALYTIC SHELLFISH POISONa

Death time6

Mouse units

Death timeb

Mouse units

3:OO 05 10 15 20 25 30 35 40 45 50 55 4:00 05 10 15 20 25 30 35

3.70 3.57 3.43 3.31 3.19 3.08 2.98 2.88 2.79 2.71 2.63 2.56 2.50 2.44 2.38 2.32 2.26 2.21 2.16 2.12 2.08 2.04 2.00

4:55 5:OO 05 10 15 20 30 40 45 50 6:OO 15 30 45 7:OO 15 30 45 8:OO 15 30 45

1.96 1.92 1.89 1.86 1.83 1.80 1.74 1.69 1.67 1.64 1.60 1.54 1.48 1.43 1.39 1.35 1.31 1.28 1.25 1.22 1.20 1.18 1.16

40

45 50

0:oo

Death timeb 9:30

1o:oo 30 11:OO 30

12:OO 13 14 15 16 17 18 19 20 21 22 23 24 25 30 40 60

Mouse units

1.13 1.11 1.09 1.075 1.06 1.05 1.03 1.015 1.000 0.99 0.98 0.972 0.965 0.96 0.954 0.948 0.942 0.937 0.934 0.917 0.898 0.875

For 20-gm mice. From hlcFanen (1971),reprinted from Food Technol. 25,234.With permission of the holders of the copyright (1971)the Iustitute of Food Technologists. Given in minutes and seconds.

TETRODOTOXIN AND SAXITOXIN I N IiEUROBIOLOGY

103

60 min (McFarren, 1959, 1971). Table IV shows these values, and Table V shows the correction factors for mouse weight from the same publications. These tables are used in the same way that Tables I1 and 111 are used for the estimation of TTX. The points plotted in Fig. 5 are from part of Table IV, with the regression line calculated for the points between 4 and 8 minutes. If extrapolated this regression line intercepts the 1 MU dose at 0.0839 (11.92 min-’) and has a slope of 0.4115 min-l per log,, MU. Assays conforming to the rccommended technique and using this table of McFarren’s (1959) are sometimes known as AOAC (Association of Official Agricultural Chemists ) assays. Wiberg and Stephenson ( 1960, 1961) have investigated many of the factors that affect accuracy and have shown the influence of pH, NaCI, and other contaminating salts, the strain, and even the sex of mice, on the LDjo. Mice that survive these assays ought not to be reused, because traces of toxin linger in the body for several hours, producing a cumulative effect if a second dose is given within 12-24 hours. Later a degree of tolerance appears to develop ( McFarren et al., 1960). Recent publications assume that 1 MU, as defined above, is equal to 1 LD,,. This seems to be substantially correct in many cases (Schantz et a[., 1958). McFarren (1967) explicitly states that after 1 LD,, of “paralytic shellfish poison” (STX) those mice that die do so with a mean death-time of 15 min, or 30 niin in the case of TTX. It has already been pointed out that other workers have found values that differ slightly from 30 min for the intercept of the TTX dose/death-time regression line. Konosu et al. (1968) showed a regression line for STX that intercepted 1 MU near 12.5 niin and Evans (1970a) found the STX regression line to intercept 1 LD,, at 7.26 min. It is not clear to what

Wt of Mouse mice (gm) units

10 10.5 11

11.5 12 12.5 13

0.50 0.53 0.56 0.59 0.62 0.65 0.675

Wt. of mice (gm)

Mouse rinits

13.5 14 14.5 15 15.5 16 16.5

0.70 0.73 0.76 0.785 0.81 0 . 84 0.86

W t of Moiise mice (p) units 17 17.5 18 13.5 19 19.5 20

0 . S8 0.905 0.93 0.95 0.97 0.!)85

1 no0

W t of Mouse mice (gm) iinits 20.5 21 21.5 22 2’2.5 23

1.015 1.03 1.04 1.05 1.06 1 07

104

MARTIN H. EVANS

TABLE VI LI:THALn0SI.E OF s &XITOXIN, p g / k g BODYWEIGHT, FOR V.4RIOUS SUnJECTS: AS LDN UNLESS OTHERWISE STATED THEDOSESARE EXPRESSED Route of administration Subject

i.v.

i.p.

S.C.

Mouse

Oral

References

4003

Recalculated from Woodward (1955, cited by McFarren et al., 1960)

8.33-10"

3.4

263

6.5C, 8.010.0, 13, Od

(1960)

Cheymol et al. (1968) Recalculated from Noguchi et al. (1969) Evans (1970a) 2006 Recalculated from Woodward (1955, cited by McFarren et al., 1960) 309-588 Davis et al. (1956), cited by McFarren et al. (1960) 721-531 Watts et al. (1965) Cheymol and Thach Toan

7.1-9.0 10.fP 10.3

Rat

5.5'-10.5

> 12, < 15-18

(1969)

Rabbit,

Chicken Cat

2003

2-79 3-4d 3-4 3-4 2803

2-79

Frog Rana pipiens Rana

Recalculated from Schantz et al. (1957) Recalculated from McFarren (1959) Wiberg and Stephenson

Recalculated from Woodward (1955, cited by McFarren et al., 1960) Evans (1965) hlurtha (1960) Cheymol et al. (1968) Cheymol et al. (1968) Recalculated from Woodward (1955, cited by McFarren et al., 1960) Evans (1965) Kao and Fuhrman (1967)

75c

> 100

Evans (1964)

tem.poraria

Newt (Taricha

torosa)

>6000, <9oooc

Kao and Fuhrman (1967)

TETRODOTOXIN AND S A X I l O X I N IN “EUROBIOLOGY

105

TABLE VI (Continued) ~~

Subject

i.v.

Killifish (Fundulus heteroclitus )

Route of administration ~

i.p.

H.C.

Oral

Jackim and Gentile (196s) hlclaughlin and Down (1969)

2gh :3()h*c..

6 ,g h , c -

Man

References

McFiirren et a/. (1960)

15. 7b*c 5.7b.c

7*eC

Kao and Nishigama (1965b) Schnntz (1971b)

aAssayed as “mouse units.” See text. bApproximate or estimated on basis of c Minimum lethal dose. L1Ig9 or Ll>,Oo. e Toxin from Zosimus. Respiratory paralysis in anesthetized animals. h Toxin from J Newborn animals. Aphunimmenon. z Injected into the gas bladder. 1 MU = 0.2 rg.

extent these differences depend on the strain of mice, or modifications in technique, used by these workers. It does, however, serve to emphasize the necessity for any worker to construct his own dose/death-time regression line before using this technique for assay work. Different laboratories have found that, in their hands, 1 MU represented amounts of pure STX that varied from 162 k 9 ng to 254 -+ 18 ng (McFarren, 1959). Values obtained by various workers for the LD,, are shown in Table VI. Jackim and Gentile (1968) observed a darkening of pigmentation of killifish (Fundulus lieteroclitus) that had received a lethal dose of Aphanizomenon toxin. McLaughlin and Down ( 1969) reported that STX and TTX caused sectoral darkening when injected into the gas bladder of the killifish. They reported a sensitivity of about 0.1 pg STX in 0.1 ml, with a fish weighing not more than 6 gm. This melanophore response was suggested as an assay method with a somewhat better sensitivity than the AOAC mouse bioassay. Serological assay techniques have been developed by Johnson and Mulberry (1966). STX is not itself antigenic, but by haptenic conjugation with a protein, STX antigens can be induced in rabbits (Johnson et d.,1964). Johnson and Mulberry (1966) used these antigens to detect small amounts of STX in shellfish extracts, and reported sensitivity similar to or better than thc AOAC niousc bioassay technique. These serological techniques are likely to be more specific than mouse bioassay.

106

MARTIN H. EVANS

2. Chemical Assay Because of the disadvantages inherent in biological assays, a chemical assay has been developed, based upon a modification of the Jaffe (1886) color reaction with picric acid (McFairen et al., 1958, 1959; McFarren, 1960; McFarren and Campbell, 1961). The color is developed by warming the STX solution with picric acid and sodium hydroxide at 38°C for 20 min, and estimating the optical density with a spectrophotometer. The Jaffe reaction is very nonspecific and satisfactory assays can be achieved only by meticulous attentioil to detail. Other substances present in shellfish extracts also give color reactions that would interfere with the assay, and must be removed or compensated for. It is therefore necessary to purify crude STX extracts partially, using an ion-exchange column, and then to compensate for color-reaction products that form with interfering substances ( possibly histamine and glucosamine ) still present in the column eluate. This compensation can be done in one of two ways. The original (1958) recommendation was to heat a second sample to 99.5-100°C for 15 min, which increased the intensity of color due to interfering substances and decreased that due to STX, when measured at 510 nm. The modified technique (1959) suggested removal of unreacted picrate with 25% pyridine in ethyl acetate, followed by measurement at two wavelengths, 375 nm and 490 nm. The reaction products of interfering substances absorb more strongly at 375 nm than the STX color products do, whereas the reverse is true at 490 nm. STX is deteiinined by fitting absorbance measurements to graphs or equations published by McFarren et al. (1958, 1959). Although this chemical assay has some advantages over the AOAC biological assay, especially with regard to accuracy when dealing with extracts from weakly toxic clams, it has in practice proved difficult to apply. Schantz (1971a) considers the chemical assay to be of value in work with purified STX. C. DIFFERENTIATION BETWEEN TETRODOTOXIN AND SAXITOXIN

The biological actions of TTX and STX resembIe each other so closely that they are not readily distinguishable by their effects. For this reason they are discussed together in the remainder of this review, with occasional differences noted where necessary. However, because it may sometimes be essential to identify an unknown toxin, the following points of difference may be emphasized. It has been pointed out above that when LD,, determinations are made, those mice that die from 1 LD,, of TTX do so with mean deathtimes of about 30 min (McFarren and Bartsch, 1960). In the case of

TETRODOTOXIN AND SAXITOXIN I N NEUROBIOLOGY

107

STX the mean death-time is between 10-20 min (Sommer and Meyer, 1937). Furthermore, when mean death-times are measured at higher doses, there is a significant daerence between the slopes of the min per log,, LD,, regression lines, as shown in Fig. 5. McFarren (1967) suggested that TTX and STX could be distinguished by these differences, and his differential mouse assay has been used by Konosu et al. (1968) and by Evans ( 1970a) when investigating unknown \poisons. A very reliable method of distinguishing the toxins depends on the observation that species of puffer fish and newt containing TTX possess a natural resistance to administered TTX, but succumb to STX. Kao and Fuhrman (1967) studied this phenomenon and showed that isolated nerves from Sphaeroides muculatus and Taricha torosa continued to conduct normally in the presence of TTX: 0.5 pM (160 pg/liter) and 94 pM ( 30 mg/liter ) respectively. Higher concentrations of TTX caused some conduction block. Concentrations of STX began to block S. mculatus nerves at 30-80 nM (ca. 1 1 3 0 pg/liter) and T . torosa nerves were usually affected at 3 p M (1 mg/liter). This was confirmed by Evans (1970a) during an investigation of an unknown toxin from Mytilus edulis, which was distinguished from TTX by means of this test. The only disadvantages of this test are that rather high concentrations of toxin are needed to produce an effect, and living S. maculatus or T. torosa may not be readily available. It is unfortunate for our purposes that the common European newts contain negligible quantities of ?TX (Wakeley et al., 1966) and their nerves do not show much difference in the sensitivity to TTX and STX (C. J. Waterfield, 1970, personal communication). The different slopes and intercepts shown in Fig. 5 probably reflect different rates of absorption and/or elimination following i.p. administration. Similar differences in ability to diffuse through permeability barriers in the region of the neuromuscular junction probably account for the qualitative differences seen when the end-plate potential ( e.p.p. ) is recorded in the presence of T T X or STX. The e.p.p. disappears abruptly after a few minutes exposure to T T X (ca. 10 pglliter) due to a conduction block in the motor nerve axon (Furukawa et al., 1959). I n the presence of STX the e.p.p. falls gradually, due to an action on the presynaptic nerve terminals (Nishiyama and Kao, 1964). This difference was studied by Evans (196913) who suggested that it might serve to differentiate between the toxins. The tests can be done at very much lower concentrations than are necessary when using T . torosa nerves, but are not 100% reliable. Cheymol (1966) mentioned that STX resists boiling better than TTX does, and a comparison of their properties reported by other workers (see Section 111) also suggests a considerable difference between

108

MARTIN H. EVANS

their stabilities in acid solution. Waterfield and Evans (1972) compared their stabilities at 100°C in solutions with pH values between 0.64 and 5.0. They found that TTX solutions always lost toxicity more rapidly than STX solutions did, and at pH 2 and below the difference was so great that the toxins could be distinguished easily. They suggest that the method could be used successfully at about p H 1, and no morc toxin would be needed than about 3 pg-su5cient to assay on a few mice the toxicity remaining after 10-20 niin at 100°C. If fairly pure solutions of the toxins are available, they can be distinguished by chemical methods such as the Jaffe (1886) test. In this test, or modifications of it, alkaline picrates give colored complexes with STX but not with TTX (McFarren et al., 1958, 1959; McFarren and Bartsch, 1960). The test is not specific, as many organic compounds forni colored products (see Section IV, B, 2 ) . Other differences between the effects of these toxins are discussed by Kao (1967) and Cheymol and Bourillet (1972). V. Actions in Vitro

A. EFFECTSON NERVE 1. Actions of Tetrodotorin and Saxitorin on Nerve Membrane The effects of TTX and STX on the electrically excitable cell membranes of neurons and skeletal muscle fibers are without doubt the most important of their actions. The effect of TTX in blocking conduction of impulses along motor axons or sensory processes has been known since the end of the nineteenth century (Takahashi and Inoko, 1890; Ishihara, 1918; for further references, see Kao, 1966, and Halstead, 1967). Turner and Fuhrman (1947) demonstrated that TTX from newts suppressed the compound action potential in isolated frog sciatic nerve. Kellaway ( 1935) investigated the effects of STX on sensory and motor nerves. The axonal conduction block caused by thes- toxins can explain at least part of their paralyzing effect, but the mechanism of action has been made much clearer since 1960. Dettbarn et al. (1960) showed that pure toxins reversibly blocked conduction at the nodes of Ranvier of single frog axons in concentrations of 10-lo g/ml (about 5 x 10-lnM ) and suppressed responses in the electroplaques of Electrophorus at g/ ml without depolarization of the intracellular potential. Narahashi et aE. (1960), working with frog muscIe fibers, showed that TTX raised the threshold a t g/ml and abolished action potentials at lo-' g/nd, without changing the resting potential or membrane resistance. They

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suggested that TTX was inactivating the inward niovement of Na+ which is necessary for the generation of action potentials in frog skeletal muscle fibers. Voltage-clamp experiments with large-diameter invertebrate axom have supported the hypothesis of Narahashi et aE. (1960), showing it to apply to nerve also. In voltage clamping, a current is passed through the axolemnia, between an extracellular and an intracellular electrode. A separate pair of electrodes dctect the ntembrane potential, and rcgulate the current by means of a feedback circuit so as to keep the inembrane potential clamped at sonie chosen value. In such an experiment, if the clamping voltage is suddenly switched from somewhere near the normal resting potential (about - 70 mV) to a less-negative intracellular potential, this results in current being passed through the membrane by the feedback circuit in order to bring the membrane potential to its new value. Thcre is an initial rapid inward current flow. Alterations in the composition of the bathing fluid show that this early inward current is normally carried by sodium ions in nerves like squid giant axons, and if the axon was not voltage clamped this inward Na' current would generate the rising phase of an action potential. A few niilliseconds later this inward current is suppressed as the axon's sodium conductance ( g N U )returns to its resting value, and is succeeded in the voltage clamped axon by a delayed outward current carried by K+ as potassium conductance ( g K ) rises slowly (see Hille, 1970). A modification of the voltage-clamping technique has allowed small myelinated axons to be studied (Hille, 1966, 1968; Koppenhofer and Vogel, 1969). The curves in the upper part of Fig. 6 show a set of current records in a voltage-clamped frog nerve inimcdintely after a depolarizing pulse had been applied across the membrane, previously held at -75 mV. Moderate depolarizing pulses cvoke a large, transient inward flow of Na+ current, shown as downgoing peaks within the first millisecond. This Na' current is then inactivated and a n outward K current slowly appears, reaching a steady value 10-15 millisec after the start of the depolarization. Larger depolarizations show mainly the outward K+ current, as these depolarizations bring the membrane potential to a positive value close to or even above the equilibrium potential for Na', thereby preventing the Na+ ions from flowing inward down an electroconcentration gradient. Very large positive clamping pulses even cause a transient outward Na+ current. In the lower part of Fig. 6 the effect of TTX is shown: no movement of Na+ follows any depolarizing pulse, but the Iate outward K+ current is unaffected. Families of voltage clamp curves such as these are often plotted as a graph of the current-voltage

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-0

3 x 10- M Tetrodotoxin

FIG. 6. Voltage-clamp currents, measured in one node of Ranvier of a frog axon, plotted against time. The curves were plotted by a computer programmed to subtract leakage currents from the raw data, and show families of current curves for values of clamping potential ranging from -67.5 to +67.5 in steps of 7.5 mV. The upper family of curves shows the initial inward current carried by Na+, followed by the slow outward current carried by K’, when the node was in Ringer’s solution. The initial current reverses direction, becoming an outward current, when the voltage clamping is more positive than the electrochemical equilibrium potential for Na+, about + 5 0 mV in this case. The lower family of curves shows abolition of all initial Na’ current, both inward and outwaid, hy TTX, which does not affect the delayed K’ current. From Hille ( 1966). Reproduced from Nature (London) 210, 1220-1222 ( 1966). With permission of the copyright holders.

relationships (Fig. 7 ) . In these graphs it is usual to show separately the peak of the transient sodium current and the delayed outward potassium current after this has reached a “steady state” several niilliseconds after the start of the depolarizing clamp. ?TX has been found to suppress the early inward Na’ current, without affecting the delayed outward K+ current, in squid giant axons (Moore, 1965; Nakaniura et al., 1965a; Cuervo and Adelman, 1970), in lobster giant axons (Narahashi et al., 1964; Takata et al., 1966), in crayfish axons (Ozeki et al., 1966), insect giant axons (Pichon, 1969), and in the giant axon of the marine worm, Myxicola infundibulum (Binstock and Goldman, 1969). Under certain abnormal conditions, internally perfused giant axons develop abrupt prolonged depolarizations, which are suppressed by TTX (Tasaki et al., 1968). Similar phenomena have been reported for crayfish muscle and ecl electroplaque (see Section V, B ).

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Only part of the spike-generating current is carried by Na+ in Aplysia giant neurons, and only this part is suppressed by TTX (Geduldig and Junge, 1968; Geduldig and Gruener, 1970). The action of TTX is at least partially reversible by washing the nerve, but most workers have found recovery to be incomplete, especially after using high concentrations of the poison. Comparable studies of STX started a little later and have not been so detailed, but they have shown that STX behaves in essentially the same way. Evans (1964) showed that STX blocked the conduction of impulses in desheathed frog nerve in concentrations greater than 5 X g/ml and also prevented the generation of action potentials in muscle without depolarization. An STX-like poison from Aphanixomenon flosaquae renders nerve and muscle inexcitable without depolarizing the resting potential (Sawyer et al., 1968). Two STX-like poisons, extracted from mussels that had been feeding on Gonyaulax tamarensis, have similar effects (Evans, 1970a). Kao and Nishiyama ( 1965b) showed, in a detailed study, that STX g/ml inhibited action potentials in frog axons without depolarizing the membrane, and suggested that STX blocked inward sodium current, without affecting the permeability of the membrane to potassium or chloride. This was confirmed in voltage-clamp experiments by Hille (1966, 1968). STX also acts like TTX on invertebrate giant axons (Narahashi et al., 1967b; Narahashi and Haas, 1968). Figure 7 shows relations obtained by voltage clamping a lobster giant axon, and plotting the peak inward current (I,, circles) and steady state delayed outward current (I.?, triangles). The graph shows that the transient inward sodium current, which is maximal between - 40 and -50 mV, is abolished by STX 3 x lo-: M . The steady state outward currents are unaffected. It has been repeatedly stated i n the published literature that TTX and STX have no effect on “leakage” currents, carried by various ions through the membrane of nerve or muscle fibers. This conclusion has been reached from voltage clamp eupcrinients. However, there is now some evidence that a part of the leakage current, due to passive exchange of sodium across the membrane, may be reduced by the toxins. Clapham and Evans (1964) reported n slight reduction in passive sodium exchange when frog muscles were poisoned with STX. Narahashi (1964) noted a 2-3 mV hyperpolarization, possibly resulting from a reduction in inward leakage of Na+, when lobster giant axons were treated with TTX. Freeman ( 1969) measured hyperpolarizations averaging 4.9 mV in squid axons treated with TTX 3 x lo-: M . A similar hyperpolarization occurred when the axon was in sodiuni-free medium, and then TTX did not produce any further hyperpolarization. Raker et al. (1969)

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-8

FIG. 7 . Graph of currents recorded from a voltage-clamped lobster giant axon. The points are plotted from curves similar to those shown in Fig. 6 and indicate the peak of the transient Na' current (circles, marked I,,) and the steady outward K' current (triangles, L5). The dotted curves through the filled symbols show that STX eliminates the Na' current without significantly affecting the delayed K current. Ordinate: membrane current density. Abscissa: clamped value of membrane potential. From Narahashi et ol. ( 1967b). Reproduced from Science 157, 1441-1442 ( 1967). By permission of the holders of the copyright ( 1967), The American Association for the Advancement of Science.

found that the influx of sodiuni into resting squid axoiis was reduced glnil. Narahashi et a2. from 27.6 to 11.2 pmole/cm'sec by TTX (1971) have made use of this ability of TTX to reduce resting sodium leakage currents in a study of the effects of batrachotoxin (from frogs belonging to the genus Phyllobates) on nerve membrane. It now seems clear that TTX and STX do inhibit part of the leakage currents, but the TTX-sensitive fraction of the sodium leakage current is often too small a proportion of the total leakage for any significant effect to be noted on overall membrane potential or conductance.

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When low concentrations of TTX have reduced, but not abolished, the inward Na+ current, the remaining Na' current has normal kinetics of onset and inactivation (Takata et al., 1966; Moore et al., 1967b). Several workers have analyzed the actions of these toxins in terms of the equations in Hodgkin and Huxley's ( 1952) theory (Takata et al., 1966; Hille, 1966, 1968, 1970; Cuervo and Adelman, 1970). They have all concluded that the only significant action is on maximum sodium conductance (&). Cuervo and Adelnian (1970) noted a slight shift of peak sodium current along the voltage axis of the current-voltage curves, which is also commented upon by Narahashi et al. ( 1969b), Conti ( 1970) found the resonant frequency of squid axon membrane unchanged by TTX, or by changes in Na+ or K+ concentrations. Khodorov et al. (1970) studied theoretical effects of TTX in reducing the gNaterm in a mathematical model of axonal conduction. Some substances, such as aconitine, veratridine, and batrachotoxin depolarize neurons. They appear to act by greatly increasing the resting permeability of the membrane to Na', possibly by opening up the channels through which Na+ enters at the start of the action potential. TTX can prevent these substances from depolarizing the neurons, and can even restore the normal membrane potential after these substances have depolarized it ( Wellhoner, 1970; Narahashi et al., 1970; Albuquerque et al., 1971). The insecticides DDT and allethrin and some other poisons such as scorpion and Cond!llactis venoms greatly prolong action POtentials. The use of TTX and STX has helped to analyze the actions of these agents, which appear to act by delaying the inactivation of gs, (Hille, 1968; Narahashi and Haas, 1967, 1968; Narahashi, 1969; Narahashi et al., 1969a). Some electrogenic systems, discussed in later sections, depend on Ca2+rather than Na+ for the generation of action potentials. These systems are generally resistant to the action of TTX and STX. However, even in systems in which the action potential is sodium dependent, a raised extracellular concentration of Ca'+ can, to a slight extent, offset the action of liminal concentrations of TTX (see Takata et al., 1966; Albuquerque et al., 1969). Different tissues vary in their sensitivity to the toxins. The nodes of Ranvier of isolated frog axons are very susceptible; Dettbarn et al. (1960) reported a failure of electrical activity within 30 see of exposure to lo-'' glml (about 3 x M ) of either toxin, although they later stated that STX M blocked the node in 60 sec (Dettbarn et al., 1965). Furukawa et ul. (1959) stated the liminal concentration of TTX to be 5-10 X lo-!' g/ml and Kao and Fuhrman (1963) gave thc figure of g/ml for TTX from newts. Rather greater concentrations are needed

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to block conductioll of compound action potentials in desheathed nerves or spinal cord roots, and some invertebrate giant axons require TTX Concentrations between 1 and 5 x lo-‘ g/ml before most of the early inward Na+ current is blocked (Nakamura et al., 1965a; Narahashi et al., 1966). Ogura and Mori (1968) stated that TTX 3 X lo-’” M (about g/mI ) blocked crayfish iioiiniyeIinated axons, a value that is very different from the figure of lo-” g/ml given by Ozeki et al. (1966) for the motor axons of the crayfish. Toad nerves are said to be relatively resistant to TTX (Furukawa et al., 1959; Carter, 1969). The nerves of puffer fish and Taricha newts are extremely resistant to TTX, but less so to STX (see Section IV, C). The toxins act on the outer surface of the axolemma. Some early experiments suggested that intracellular injection of TTX into giant axons reduced the action potential, but more recent work has established that giant axons can be internally perfused with TTX concentrations up to M (3.2 mglliter) without effect on either the resting or the action potentials, threshold or rate of rise of these action potentials (Moore, 1965; Narahashi et al., 1966). Koppenhofer and Vogel (1969) also found TTX ineffective when diffused into the axoplasin of single myelinated Xenopus fibers. TTX is insoluble in lipids and appears unable to pass through the axolemnia. The axolemnia must therefore be asymmetrical, with TTX-susceptible sites on the outer surface but not on the intracellular surface. These TTX-susceptible sites may be thought of as being the outer “gates” to a channel or pathway whose sodium conductance rises early in the generation of an action potential (Narahashi and Moore, 1968a). Little is known of the molecular interactions which must take place when TTX or STX bind to these receptors at the “sodium channels.” Kao and Nishiyama (1965b) pointed out that both molecules appear to have guanidinium groups as part of their structure. Guanidine itself can substitute for Na+ as a charge carrier, to generate action potentials, and they suggested that positively charged guaiiidinium groups in the toxin molecules enter the “sodium channel,” the rest of the toxiii molecule blocking the channel because of its size. Cheymol and Bourillet (1972) offer the delightful simile of the toxin blocking the channels in the way that a champagne cork seals the neck of the bottle. The rest of the toxin molecule must come into a stereochemical relationship with parts of the membrane around the “sodium channel,” because the toxins are effective at very low concentrations, i.e., there is a powerful tendency to bind to these receptor sites. Hille (1968) found the equilibrium dissociation constant for STX, at which it reduced gNR to 5058, to be 1.2 X lO-’M for frog nodes; he suggested a value of about 3 x M for

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TTX (Hille, 1966). Cuervo and Adelman ( 1970) found 3.31 X 10F’hl for TTX on squid axon; they also calculated the rate constants for the reaction between TTX and the binding sites, but their figures ( k , = 0.202 x los M - l ; k, = 0.116 inin-’) may be distorted by the presence of diffusion barriers, because Hille ( 1963) reported complete abolition of sodium current in frog node of Ranvier by TTX and STX within 5 sec. Colquhoun and Ritchie ( 1972) calculated the equilibrium dissociation constant K for the binding of TTX to mammalian nonmyelinated newe fibers. They obtained values in the range 3-5 X lo-“ hl at 20°C, using an indirect approach based on the reduction in amplitude of the compound action potential in the rabbit vagus. At 12.8OC, K = 1.84 t 0.31 X M , at 31”C, K = 8.47 2 2.96 X hl. Wagner et al. (1972) found K = 3.5 x 1O-!’M for TTX binding to single nodes of Ranvier isolated from Rana esculenta. They were also able to calculate the rate constants and found k, = 4 x loFhf-lsec-l and k, = 1.5 x 10’ s e c t at 20-22OC. Data obtained at other temperatures suggest that their rate constants were not limited by diffusion barriers. The structure of the TTX molecule is usually shown in the zwitterion configuration (Fig. 2 a). On thc acid side of the pKt, (about 8.8, see Section 111, A) TTX is cationic, there being two tautonieric forms in equilibrium with each other (Fig. 2b and 2c). STX has two pK,, values, at about 8.2 and 11.5 (Section 111, B ) so at a physiological pH it too will be in cationic form, It has been shown that both TTX and STX are more potent at about pH 6-7 than at p H 8.6-10.1. Therefore these toxins bind best to the receptor sites when they are in their cationic form. The negative charges carried b y other parts of the molecule, when on the alkaline side of pK,, presumably iiiterfere with the sterie attractions to the receptor site near the ‘‘sodium channels” (Camougis et al., 1967; Hille, 1968; Ogura and Mori, 1968; Narahashi et al., 1969b). Hille (1966) postulated a negative charge in the membrane near the “sodium channel,” that would tend to repel an interacting molecule carrying a similar charge. Newt TTX was less effective in producing corneal anesthesia a t pH 5 than at 7.6 (Kao and Fuhrnxm, 1963) but this probably implies that the zwitterion form diffuses through tissues better than the cation forms (see Hille, 1966; Ogura and Mori, 1968; Ogura, 1971). The stereochemical relationship between the toxin molecules and the cell membrane, which allows binding at nanomolar concentrations, is evidently critical. Derivatives of ?TX are virtually inactive. Early reports suggested that a few derivatives, especially deoxytetrodotoxin, had appreciable toxicity and ability to reduce &,. However, later work showed that there was enough T T X contaminating the samples of these derivatives to account for all the activity found (see Narahashi et al.,

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1967a; Moore and Narahashi, 1967). A synthetic guanidinium ester, S-benzoyloxy-2-iminohexahydropyrimidine, is reported to have effects on action-potential generation, but it is much less potent and its actions differ from TTX and STX in some important respects (Spiegelstein and Kao, 1971). Hydrogenation of STX, which changes the empirical formula from C,,H,,N,O, to C,,H,,N,O, (b ut see the revised formula shown in Fig. 4, on p. 93), results in complete loss of toxicity (Schantz, 1960). Bannard and Casselnian (1961b) reported that treatment of STX with an anhydrous mixture of nitric and sulfuric acids resulted in a crystalline Weber-positive substance with a specific toxicity of 3060 MU/nig, which they thought was an oxidative degradation fragment. Unfortunately nothing further has been published about this interesting substance. As most of the derivatives of TTX and STX are not active it is evident that virtually the entire structure of their inolecules is of consequence for the specific binding. Camougis et al. (1967) have proposed various models for the molecular interaction between TTX and the receptor site, including hydrogen binding with an electronegative oxygen of a peptide chain or a phosphate group. Evans ( 1 9 6 9 ~ )pointed out that there are 7 hydrogen atoms adjacent to the guanidinium group of TTX, in an approximately planar triangle, and these might form hydrogen bonds with atoms in the membrane. Ogura and Mori (1968) proposed a model in which van der Waals forces, hydrogen bonding, and electrostatic forces all play a part (see also Ogura, 1971). Cuervo and Adelman (1970), having calculated the energies of the binding reaction between TTX and squid axon membrane, concluded that in addition to the guanidinium grouping at least one other binding site holds the toxin to the membrane. This other binding site has more energy than could be accounted for by a single hydrogen bond, but several hydrogen bonds could account for the binding energy. Smythies et al. ( 1971) and Smythies (1972) have studied the stereochemistry of TTX using molecular models, and concluded that a molecule of TTX could bind with two glutaniine moieties and one arginine moiety of protein and two phosphorylated cytidine nucleosides. They suggest that such amino acid-base pairs form a “gate” on the “sodium channel” and that when the “gate” is open a 7 T X molecule can enter and, by binding with the complementary structures, two bases on one side, and three amino acids on the other, block the “gate.” Thus Na+ ions are denied access to the “channel” underneath. This model would account for the loss of toxicity resulting from even minor structural changes in the TTX molecule, for all polar atoms on TTX bar one are involved, and for the stoichiometric ratio of one TTX molecule per channel, dis-

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cussed below. The model also accoiints for the actions of batrachotoxin and veratridine. Smythies and his co-workers have recently developed an alternative hypothesis of the nature of the TTX “receptor” ( J . R. Smythies, 1972, persona1 communication ) . A protein segment with the amino acid sequence -Asp-Pro-Asii,-Pro-Glii-Pro-Asn,-Pro-Asn~-Pro-As~i.,forms a cyclical structure (completed by a hydrogen bond between the aniide CO and NH groups of the terminal Asp and Asn,). Asp and Asnl are bound to each other by one hydrogen bond and a fixctl water niolecule. This forms a rectangular-shaped electronegative pore lined by an oxygen ring measuring 3 X 5.5 A (Hille, 1971). A m , , Asn,, and A m , form a triangular multiple dipole located prccisely under the three water molecules in thc hydration shell of N a + . ( 3 H , O ) .Their function could be to ion to rcmove three (or two) of these and allow the Na+ (Na+.lH,O) pass through the pore. This structurc is stereochemically complementary to the TTX molecule as follows. The TTX molecule lies on top of the pore into which its guanidinium group is inserted forming a double resonating ionic bond with Asp and an ion dipole bond to Asn.;. Gln is bound by two hydrogen bonds from the hydroxyl and methy1t:nc hydroxyl groups on C6. The other three hydroxyls are now located precisely in the right location and with the correct bond angles to bind to Asnl, Amz, and Am4. This strncture is also complementary to STX and to the barbiturates. McIlwain et al. (1969) noted similarities of structure in TTX, phosphatidylserine, and glutamic acid. The latter substaiiee is known to excite some central nervous systcm ( CNS ) neurons, and they suggested that TTX might bind with a phosphatidylserine group in a glutamate receptor. However, this would imply that TTX blocked chemical transmission in the CNS, whereas chemical transmission in peripheral junctions is known to be uiiaffected by TTX, which blocks only elcctrically excitable membranes (see Section V, C ) , An interaction with membrane cholesterol has also been suggested (see Section 17, A, 2 ) . It seems probable that only oiie molecule of toxin is needed to block one “sodium channel.” Hille (1968) plotted ,ijN,of a frog node as a function of S T S concentration, and found that the points matched the curve of a thermodynamic equation which assumed that one receptor complexes with one molecule of STX. Guttnian and Barnhill (1968) obtained a similar curve by plotting excitability of squid axon against ‘ITX concentration, although they required rather high concentrations (1.2-2.3 X lo-’ g/ml) to double thc rheobasc. Cuervo and Adelnian (1970) and Colquhouii and Ritchie (1972) also concluded that one molecule of TTX interacted with one receptor site.

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It also seems probable that the toxins do not bind indiscriminately with the nerve membrane, but do so only at the sites of the “sodium channels.’’ This conclusion is based on experiments in which it has been shown that very few toxin molecules become bound to the membrane at a concentration sufficient to suppress most of the &,. Moore et al. (1967a) soaked lobster nerve trunks in a small volume of TTX 3 x leiM until conduction failed, and then bioassayed the TTX remaining in solution. They estimated that 159 mg of nerve, with a total memcm?, bound 2.5 x lo-’? moles of TTX. This brane area of 1.11x gives a figure of 13 molecules TTX per pm’ axolemma-a value that is too low for it to be probable that TTX binds other than at the “sodium channels.” Keynes et al. (1971) used a variation of this approach, in which the bioassay of TTX or STX remaining in solution was done by comparing the size of the compound action potential, after exposure to a small volume of toxin, with action potentials from the same nerve in known concentrations of toxin. They also found that only a few molecules of toxin needed to bind to 1 pni? of membrane in order to reduce the ENa.to the level at which conduction of action potentials failed. From their data they calculated that lobster nerves have 36 binding sites/ pm2, crab nerves 49 sites/pm’, and rabbit vagus 75 siteslpn?. The figures obtained by Moore et al. (1967a) and Keynes et al. (1971) are of the same order as the estimates of the density of “sodium channels” in axolemma obtained by other methods (Chandler and Meves, 1965; Hille, 1970). Hafemann et al. (1969) estimated 120 sites/pmZ of cat CNS neuron, but several approximations had to be made during their calculations. McIlwain et al. (1969) arrived at a figure of 100 molecules TTX/pm2 in isolated slices of guinea pig cortex. Keynes et al. (1971) estimated, from their data, that the TTX-binding “sodium channels” in rabbit vagus must have a mean spacing of approximately 0.1 pm. Hille (1970), calculating from estimates of the conductance of “sodium channels,” arrives at figures of 0.6, 0.3, and 0.06 pm for squid, lobster, and frog nerves, respectively. This wide spacing between the “sodium channels” expIains why STX, with its two guanidinium centers, is not twice as potent as TTX. The two guanidinium centers in the STX molecule are separated by about 5 A , and obviously could not simultaneously occupy two sites at least 600 A apart. The term “sodium channel” has been used freely in the preceding paragraphs to describe the pathway or carrier through which Na’ enters the axon at the start of the action potential. The concept of “sodium channels,” “potassium channels,” and “leakage channels” has been widely used since Hodgkin and Huxley analyzed the action potential in the early 1950s ( summarized by Hodgkin, 1958, 1964). However, the early

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channel is not restricted to Na+; several other ions, including Li+ and some quaternary ammonium ions, can carry charges through this early channel in the absence of extracellular Na’. Moore et al. (1967b) found that TTX suppressed the inward transient lithium current, when Li+ was substituted for Na+ in the artificial seawater bathing their squid giant axons. Hille (1968) and Evans ( 19694, working with frog and rat nerves, respectively, found that action currents or potentials generated by Li+ were abolished by TTX and STX. Moore et al. (1967b) reported that under some circumstances one could obtain an early transient outward current carried primarily by Na’, and TTX suppressed this as effectively as it suppressed the noinial inward sodium current. There was no effect on the slow delayed current, whether it was inward or outward and irrespective of the ion which carried it. They therefore suggested that TTX acts by blocking an early transient-conductance path (along which Na+ normally enters at the start of the action potential) without affecting the late steady-conductance path ( along which K+ normally leaves the axon at the end of the action potential). Binstock and Goldman (1969) reported that TTX suppressed outward early transient current evoked in MyxicoZa axons by +160 mV voltageclamping pulses, as well as suppressing the normal early inward current evoked by smaller pulses. Tasaki and his colleague5 (Tasaki and Singer, 1966; Tasaki et al., 1966; A. Watanabe et al., 1967a) reported that under certain circumstances Ba2+, Sr2+, Ca2+, K , NH,’, hydroxylarnine, guanidinium, and hydraziniuin ions can substitute for Na’ to generate action potentials in squid giant axons, and that TTX abolishes these action potcntials as well as the normal Na+-generated action potential. They therefore argue that there is no such thing as a “sodium channel,” and suggest that the surface of the axoleminx functions a5 a cation exchanger with no distinct sodium and potassium channels. The argument in favor of separate sodium and potassium channels is now based on the often repeated observations, mentioned earlier i n this section, that T T X and STX can suppress the inward (noi-mally Na”) current early in the voltage-clamped pulse, without any effect on the delayed K+ or leakage currents (Nakamura et al., 196%). Rojas and Atwater ( 1967) added further support to the two-channel hypothesis by finding that under certain voltage-clamp conditions an outward current, carried partly by K , is detectable early in the clamped pulse and this early outward K+ current is also suppressed by TTX. Narahashi et al. (196713) found that STX blocked both inward and outward early transient currents in voltage clamped lobster giant axons. Rojas and Atwater ( 1967) used the expresion “fast-conductance channel” instead

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of “sodium channel,” to avoid the semantic differences that Watanabe et al. (1967a,b) object to. The points in favor of the two-channel hypothesis were summarized by Narahashi and Moore ( 1968b). Hille (1970) also discusses these semantic differences, but comes out in favor of “sodium channel” to describe the carrier or pathway activated early in the action potential or voltage clamp pulse. Whether or not Watanabe et al. (1967a,b) are correct in stating that the “channels” through which Na+ and K pass through the membrane are spatially identical remains to be settled. This may again prove to be a semantic rather than a real difference, because the early fast “sodium channel” is clearly distinguishable from the late “potassium channel” both in its fundamental characteristics as well as in its susceptibility to TTX, STX, and other agents. The late “potassium channel” can be blocked by tetraethylammonium, which does not interact with TTX and does not affect the early “sodium channel” (Hille, 1967, 1970). D D T affects the turning-off processes differently for sodium than for potassium cuirent (Narahashi and Haas, 1968). The proposal by Watanabe et al. that sodium and potassium channels are spatially identical would be untenable if it could be shown that the numbers of “potassium channels” per unit area of membrane was very different from the numbers of “sodium channels” calculated by Moore et al. (1967a), Hille (1970), and Keynes et al. (1971). This has not yet been done accurately, but Hille (1970) suggested that in squid axon there are more than 50 times as many “potassium channels” as “sodium channels.” Dettbarn et al. (1965) noted that STX M caused a transient increase in the amplitude of the action potential at a node of Ranvier, before blocking conduction in the frog nerve fiber. This correlates with Hille’s (1968) observation that ZPia may be 1&15%higher on recovery from exposures to low concentrations of STX. This effect does not seem to have been looked for in the case of TTX, although Keynes et aE. (1971) stated that “the impression was gained that sometimes at the lowest concentrations of TTX the size of the action potential (in crab nerve) was even slightly greater than the control value.” Other workers, using voltage-clamped nerves, may have missed seeing slight enhancement of gNn,though using only high concentrations of TTX. Wassermann and Holland (1969) noted that low concentrations of T T X often caused a transient increase in the contractile force of isolated heart muscle, and they suggested that this might have been caused by the TTX displacing calcium from binding sites. Cheyinol et al. (1961) saw similar effects on skeletal muscle in uivo. Kao (1966) thought that STX washed out more easily than TTX from some tissues, though Dettbarn et al. (1965) found STX rather irreversible in its actions on eel electroplaques. Narahashi et al. (1967b)

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compared the time constants for recovery of lobster nerves exposed to TTX or STX, both at lo-‘ M. They found that the average time constant for recovery from STX was about half of that for TTX. Cuervo and Adelman (1970) felt that a degree of irreversability, observed by most workers, was intrinsic in the TTX-membrane interaction kinetics. Hille (1968), working on the node of Ranvier of single frog axons, stated: “The effects of STX can always be reversed with 30 sec of washing, whereas frequently the effects of TTX are only partly reversed with several minutes of washing.” He recommended STX in preference to TTX because of the rapid reversibility. Takata et al. (1966) stated that giant axons recovered from newt TTX more rapidly and completely in the presence of high calcium (25 mM) in the media. 2. Actions on Other Nervous Elements

Although all the effects of TTX and STX on nervous tissue seem to be due to their actions on the electrically excitable surface membrane, several studies have been made of the actions of the toxins on cell fragments and enzyme systems. The work of Villegas and Camejo (1968), Camejo and Villegas (1969), and Villegas et al. ( 1970) has shown that M interacts with cholesterol monolayers so as to expand TTX 5 X the monolayer. The dissociation constant was found to be 2.6 X lo-’ M , which is much higher than the values obtained on axons, discussed in the previous section. Although there is some suggestive evidence that a TTX-cholesterol complex might occur at the sodium channels, it must be remembered that cholesterol is an abundant constituent of nerve membranes but that there are very few TTX-binding sites per pm2 of axolemma (see p. 118, and Adrian, 1!369). Itokawa and Cooper (1969) found that substances which affect Na+ movement across nerve membrane (acetylcholine, KCI, ouabain, ethylenediaminetetraacetate) result in labeled thiamine being released from perfused neural tissue or membrane fragments in homogenate fractions. TTX was specially potent, releasing thiamine when applied at 3 x lo-* M . They suggested that inward movement of Na’ through nerve membrane is an active process, involving thiamine pyrophosphate or thiamine triphosphate, which is blocked when TTX competitivcly displaces the thiamine at dephosphorylation sites. Hopkins and Herbst (1968) reported that STX inhibited ATPase in brain microsomes, but that TTX was without effect. This may be unrelated to the neurotoxicity of these substances, because the ATPase from rat liver mitochondria was slightly stimulated by these toxins. Lardy (cited by Schantz, 1971a) reported no inhibition of ionic movement across mitochondria1 membrane by STX, but Marumo et al. (1968)

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did report slight effect on mitochondria1 respiration and electron transM ) concentrations of TTX, port with exceptionally high (1.4 x loA4 when significant amounts of citrate would be present from the commercial buffer which is used to stabilize TTX. There have been several studies of the effects of TTX on tissue respiration at the cellular level, but these have generally given negative results (Fuhrman and Field, 1941; van Wagtendonk et al., 1942; Schantz, 1960; Fuhrman et al., 1963; Kao and Fuhrman, 1963; Nakazawa and Ogura, 1961, cited by Kao, 1966; see also Ogura, 1971). McIlwain (1967) reported that TTX prevented stimulation from increasing respiration and glycolysis in brain slices, but this can probably be explained by the abolition of evoked action potentials and does not necessarily imply a direct effect on intracellular oxidative metabolism. When the brain slices were depolarized by high extracellular K the increased respiration and glycolysis was unaffected by TTX. Similar comments apply to the work of Chan and Quastel (1967, 1970) and Joanny and Corriol (1969), who studied oxidative metabolism in stimulated brain slices, and that of Bull and Trevor (1970), who studied the effects of TTX and STX on Ca2+and K fluxes in rat brain slices. Fleisher et al. (1961) and Cheymol et aZ. (1962a,b) reported that acetylcholine release following tetanic stimulation was diminished after exposure of the rat phrenic-diaphragm preparation to TTX. Again, the results can be explained by the action of TTX in preventing nerve impulses from reaching the motor terminals, and it is not necessary to postulate any specific toxin action on transmitter synthesis or release. There have been several investigations of cholinacetylase ( choline acetyltransferase, EC 2.3.1.6), specific and nonspecific cholinesterase ( EC 3.1.1.7 and EC 3.1.1.8) in the presence of TTX or STX. Almost all workers report that these enzymes are unaffected by the toxins (Bolton et al., 1959; Dettbarn et al., 1960, 1965; Schantz, 1960; Fleisher et al., 1961; Cheymol and Bourillet, 1961; Kao and Fuhrman, 1963; Barstad, 1968; Teravainen, 1970). Young ( 1970) however, claims to have demonstrated = competitive inhibition of cholinesterase by TTX, with apparent K,,, 2.6 X M and Ki = 7.8 X 10-I' M. The reviews by Kao (1966) and by Cheymol and Bourillet (1966) tend to discount any probability of the toxins affecting the enzyme systems of neurons or other cells.

B. EFFECTSON MUSCLE 1. Actions of the Toxins on Skeletal (Striated) Muscle The direct paralyzing action of TTX on muscular contraction was noted by Yano (1937, cited by Kao, 1966, and by Halstead, 1967) and

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confirmed by Matsumura and Yamamoto (1954), who recorded the disappearance of the muscle action potential after application of the toxin. These findings have since been confirmed on frog and on mammalian muscles by innumerable other workers. Kellaway (1935) found that STX had a direct action on frog gastrocnemius muscle, although he erroneously believed that the main action resembled that of curare on the neuromuscular junction. Fingernail et al. (1953) also described the paralysis of frog muscle by STX, noting that end-plate potentials (e.p.p.) could sometimes be recorded from muscles paralyzed to direct and indirect stimulation. Cheymol et al. (1968) confirmed that STX caused paralysis of mammalian muscle to direct as well as to indirect stimulation. The biophysical analysis of the actions of these toxins on muscle can be said to date from 1960, when Narahashi et al. (1960) published their findings (already referred to in Section V, A. 1).They found that TTX ( g/ml) increased threshold, and higher concentrations paralyzed frog muscle with abolition of the action potential, without effect on resting membrane potential or resistance (due mainly to K and C1conductances). Delayed rectification, due to an increase in g K after stimulation, became evident after exposure to TTX, so they concluded that the toxin was inactivating the sodium-carrying mechanism only. The toxin differs from local anesthetics, like procaine, which reduce both and gK.Nakajima et al. (1962) obtained similar results, showing also that the potassium activation potential was unaffected by TTX (2 X g/ml). Kao and Nishiyama (1965a,b) found that STX had similar actions on frog muscle fibers. They suggested that the guanidinium groups in TTX and STX entered the sodium channels, which became closed by the remainder of the molecule. Hagiwara and Takahashi (1967), working on fish, found that TTX ( 5 x 1W) abolished the action potential without affecting resting potential in muscle fibers from Periophthalmodon, Hemiramphus, Parexocoetus and stingrays. However, action potentials in Tetradon ( probably Arothron immuculatus) muscle fibers were unaffected by concentrations of TTX 10,000 times greater. The action potentials in all these fish, including Tetradon, were Na+ dependent. The resistance of Tetradon muscle to TTX, which occurs naturally in fish of this genus, correlates with other observations on the resistance of these fish to 'lTX (see Section IV, C ) , but this specific resistance remains unexplained. The electric organs of gymnotid eels such as Ebctrophorms electricus are analogous to skeletal muscle fibers and they also generate action potentials. These are sodium dependent, though the electroplaque is unusual in developing a marked potassium inactivation when depolarized. When applied to the innervated surface of the electroplaque, both TTX

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aiid STX (10-‘-10 ” g/nil) abolish the electrogenesis of action potentials, eliminating early inward Na+ current without affecting permeability to K (Dettbarn et al., 1960, 1965; Nakaniura et al., 1964, 196513). However, ’ITX has no effect on the sustained depolarization of the innervated surface that can be evoked in calcium-free solutions ( Bartels, 1971) . Experiments in which skeletal muscle fibers have been controlled by voltage clamping have given results essentially similar to those on voltage-clamped axons. The early transient inward current ( “sodium CUTrent”) was abolished by TTX, while the delayed outward (“potassium”) current was unaffected (Kao and Stanfield, 1968; Rougier et al., 1968a; Heistracher and Hunt, 1969; Adrian et al., 1970a,b). It is of interest to note that although the toxins paralyze skeletal muscle by abolishing niuscle action potentials, they do not appear to interfere with the fundamental contractile mechanisms. Thus, focal depolarization of a paralyzed fiber evokes a localized, nonpropagating, contraction (Kao and Nishiyania, 196513, Kao and Stanfield, 1968; Heistracher and Hunt, 1969; Costantin, 1970; see also Ogura, 1971). If a muscle is denervated it becomes partially depolarized and then regenerative action potentials usually cannot be elicited. At the same time, acetylcholine-sensitive receptors appear to spread out from the original end-plate zone, and application of acetylcholine will elicit a contraction. If the denervated muscle is artificially hyperpolarized, regenerating spikes can be elicited by electrical stimulation. These spikes are Na+ dependent, and are propagated along the fiber. It is interesting to find that they are only partially affectcd by TTX ( M ) ( Redfern et al., 1970; Redfern and Thesleff, 1971), and the contractions induced in denervated rat diaphragm by acetylcholine are likewise unaffected by TTX or STX (lo-‘ g/ml) (ca. 3 X lo-’ M ; Freeman and Turner, 1969). The action potentials are also resistant to STX (Harris, 1971). These denervation changes arc delayed by actinomycin D (Grampp et al., 1971). The development of TTX-resistant spikes follows the course of spreading acetylcholine sensitivity, but Thesleff ( 1971) does not believe that these spikes are generated by the cholinergic receptor sites, although the acetylcholine receptors are known to be resistant to the toxins ( Section V, C ) . Thesleff has found that cholinergic blocking agents such as gallamine and curare do not affect the TTX-resistant action potentials, and receptors for chemical transmission are generally regarded as being electrically inexcitable ( Grundfest, 1957). Electrically generated, propagated, TTX-resistant spikes in vertebrate skeletal muscle are very unusual, and one wishes for an explanation of this phenomenon. Thesleff ( 1971, personal communication) believes that after denervation some action-potential-generating sites are altered in such a way that the TTX molecule no longer has access to them. It may be that the TTX-

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resistant nerves and muscles of puffer fish and certain newts function with sodium channels that are always inaccessible to the toxin which is present in their tissues. Batrachotoxin depolarizes inusclc nienibraiie and leads to contracture. This action is antagonized by TTX (Albuquerque and Warnick, 1970; see also Albuquerque et al., 1971). Crotaniine, from rattlesnake venom, causes a contracture of rat muscle which is inhibited by TTX (Cheymol et al., 1971), but the depolnrizing action of cobra venom is not prevented by TTX (J. Earl and B. Excell, 1971, personal communication). Caffeine induces action potentials and contracture of crayfish muscle, g/ml) has no effect on these (Chiarandini et a/., 1970) but TTX ( nor on the caffeine-induced contracture in frog sartorius muscle (J. M. O'Donnell, 1970, personal communication ) . This lack of effect correlates with current views which place the site of action of caffeine functionally close to the contractile processcs, at the sarcoplasmic reticulum or transverse tubular system (T-system), where caffeine appears to release Ca'+ (Chiarandini et al., 1970; Huddart and Oates, 1970; Luttgau, 1970). The normal muscle action potential appears to release Ca2+,and so initiate a twitch, either by electrotonic spread into the T-system or by regener at'ive conduction within the T-system (Adrian et al., 1969). There is some evidence that TTX suppresses regenerative Na+ conduction in the T-system ( Costantin, 1970). Substances siniilar to STX have been isolatcd from mussels M!/tilus edulis and from the blue-grrcn alga Aphnnizomenon flos-aquae. These STX-like toxins paralyze frog muscle with abolition of the action potential but without effect on the resting potential (Sawyer et d., 1968; Evans, 1970a). 2. Actions of the Toxins on Cardiac Muscle Although in viva the toxins procluce hypotension, the heart is not paralyzed, and in vitro experiments have indicated that cardiac muscle continues to beat spontaneously in the presence of moderate concentrations of TTX and STX (Murtha et al., 1958; Murtha, 1960; Li, 1963; Feinstein and Paimre, 1968; Pappano and Renibish, 1971). Low concentrations of TTX transiently increase the force of contraction in isolated rabbit atria (Wassermann and Holland, 1969), an effect analogous to that sometimes seen in skeletal muscle (see Section VI, A, 1) and on the nerve action potential (see Section V, A, 1, p. 120). High concentrations of the toxins weaken cardiac contractions although the pacemaker is relatively resistant ( Murtha, 1960; Kuriyania et al., 1966; Sano et aZ., 1966; Cheng et al., 1970). The amplitude of action potentials in a spontaneously beating strip of frog atrium could be

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reduced or abolished by TTX ( 1 0 - b 5X lo-' g/ml), without affecting pacemaker activity (Aceves and Erlij, 1967; Brown and Noble, 1969). Analysis of the effect of TTX on the intracellularly recorded cardiac muscle action potential has shown that both rate of rise and conduction velocity are reduced by TTX concentrations ranging from 3 x to M, depending upon the source of the cardiac muscle, without significant effect on the resting potential. Although the rate of rise was reduced, TTX usually had little or no effect on the overshoot nor on the plateau phase of the cardiac action potential, except at very high concentrations. Atrial muscle appears to be more resistant than ventricular, and mammalian hearts are usually affected by lower doses than are necessary for frog heart (Carmeliet and Vereecke, 1969; Dudel et al., 1967a,b; Kuriyama et al., 1966; Scholz, 1969; Hagiwara and Nakajima, 1965, 1966; Yanaga and Holland, 1970). Under certain circumstances, in calcium-free media, heart muscle becomes depolarized, and TTX will then induce a slight repolarization, suggesting that part of the steady depolarizing current passes through TTX-sensitive channels (Gamier et al., 1969). Dudel et al. (1967a), however, reported rather anomalous effects of high concentrations of TTX on sheep Purkinje fibers, including slight depolarization. Neurally mediated effects, such as vagal inhibition, are of course eliminated by low concentrations of TTX (Tomlinson and James, 1968; Hashimoto and Chiba, 1969; Endoh and Hashimoto, 1970; Pappano and Rembish, 1971). Reuter (1967) was among the first to apply voltage-clamp techniques to cardiac muscle. He noted that a slow inward current in the Purkinje fibers of mammalian hearts appears to be carried by Ca2+ions and was g/ml). Rougier et al. (196813) used a sucrose not affected by TTX ( gap technique to voltage clamp cardiac muscle. Working with trabecular muscles from frog atria, they noted that an initial fast inward current appeared to be carried by Na', and this current was abolished by TTX (1-5 X g/mI), which had no effect on delayed currents. Rougier et al. (1969) and Garnier et al. (1969) found further evidence for a slow inward current having a higher activation threshold than the spikelike initial current. This slow current, which presumably maintained the plateau phase of the cardiac action potential, was unaffected by TTX (5 X g/ml). In Ca2+-freesolutions it appeared to be Na' current, while in Na+-free solutions it seemed to be carried by Ca2+ions. It was inactivated with a time constant 10 to 20 times greater than that of the rapid initial current. This slow channel was thought to be the sole factor responsible for action potentials in the sinoatrial node, which would explain the relative resistance of this part of the heart to TTX.

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Manganese suppressed the current, an observation that is in accord with Hagiwara and Nakajima’s (1966) work on frog ventricle and on the Ca’+-dependent action potentials in barnacle muscle ( see Section V, B, 4 ) . The TTX-insensitive slow inward current has been analyzed by H. F. Brown and Noble (1969). They generally confirmed the findings of Rougier et al. (1968b, 1969), noting that this slow current was sometimes sufficient to support regenerative depolarization. However, thresholds were much higher when the fast initial current had been abolished by TTX (5 X lo-? g/ml). Beeler and Reuter (1970a,b) made a similar study of the slow inward current in ventricular muscle from dog hearts, g/ml, concluding that Ca2+carin ‘ITX concentrations up to 1.5 X ried the main charge inward during the plateau of the cardiac action potential. These experiments support hypotheses put forward by several workers (e.g., Niedergerke and Orkand, 1966; Reuter, 1967, 1968), in which it is suggested that the rapid depolarization during the rising phase of the cardiac action potential is due to inward current carried by Na+,whereas the plateau phase is maintained, at least in part, by Ca2+ions. There have been suggestions that the Na+ and Ca2+ions might interact, but it is clear that they do not do so in the case of these two separate phases of the cardiac action potential. TTX blocks the “channels” through which the early fast current enters, without affecting the “slow channels” through which either Na+ or Ca?+ions can pass. These “slow channels” are, however, blocked by manganese (Rougier et al., 1969). This is another illustration of the error of thinking that ‘ITX and STX are specific inhibitors of Na+ currents. The toxins block “channels” which may normally carry Na+ current, and which remain blocked even when other ions are offered that can normally substitute for Na+. Equally, Na+ current will not be abolished, if this current is carried through “channels” unaffected by the toxins. There have been very few investigations of the action of STX on cardiac muscle. Murtha (1960) reported that a perfused rabbit heart stopped beating spontaneously when STX was added to the perfusion fluid at rates of about 3 4 pglmin. Cat papillary muscle ceased to contract spontaneously in the presence of STX (6.7 X g/ml). Contractions elicited by electrical stimulation were weakened by concentrations and abolished by about lo-? g/ml. I n uiuo, STX greater than 1.3 X produces hypotension, although it is not as potent as TTX in this respect (Kao and Nishiyama, 1965b). Most workers agree that this hypotension is a peripheral effect and that the heart is not significantly affected by doses less than 7 pglkg (see Kao, 1966). Sapeika (1953), studying a crude mussel poison from Donax serra,

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reported that it produced cardiac arrest in isolated frog heart and severely depressed mammalian cardiac activity. However, the heart continued to beat in frogs that had received a paralytic dose. A substance similar to STX, isolated from Aphanizomenon flos-aquae by Sawyer et al. (1968), abolished spontaneous contractions in frog heart. Ventricular contractions failed before auricular, suggesting a block of A-V conduction. Heart muscle flutters or fibrillates after treatment with aconitine, and this rapid activity is sodium-dependent. In mammalian heart muscle fibrillation is reduced or abolished by TTX, 1-5 X g/ml (Kuriyama et al., 1966; Huang, 1970). Several groups of workers have recently used TTX to assist in the analysis of the actions of other substances on the heart (Wassermann and Holland, 1969; Hashimoto and Chiba, 1969; Bernstein, 1969; Pappano and Rembish, 1971; Lagier et al., 1971). 3. Actions of the Toxins on Smooth (Involuntary) Muscle

Kao (1966), reviewing the results of many workers, concluded that “The actions of tetrodoxin on these autonomic effector cells are limited to actions on neurally elicited responses, and do not extend to responses of the effector cells to direct stimulation by pharmacological agents.” “The various mammalian smooth muscles are quite resistant to tetrodotoxin.” This general view is still acceptable (Ogura, 1971), in spite of occasional observations that suggest some direct effects. Gershon ( 1966, 1967) made a detailed study of the responses of various rodent smooth muscles to drugs in the presence of TTX, and conchded that the toxin abolished all neurally mediated responses in doses of to 5 X lo-; g/ml. These concentrations were without effect on the responses arising from a direct effect of the drugs on the muscles. Kuriyama et al. (1966, 196%) came to similar conclusions. Avian smooth muscle was also unaffected by TTX, which abolished neurally mediated inhibitory junction potentials (Bennett, 1969). Bell (1968) showed that in a variety of smooth muscle preparations TTX prevented the action of nicotine without depressing the effect of tyramine, suggesting that the former acted via a neural mechanism while the latter released catecholamines by direct action on sympathetic nerve terminals. The responses of guinea pig ileum to angiotensin, and of colon to vasopressin, were depressed but not abolished by ‘ITX (Blair-West et al., 1967; Botting and Turmer, 1969). The smooth muscle of guinea pig ureter or bladder was unaffected by TTX (Washizu, 1966; Kuriyama et al., 1967a; Creed, 1971). Kuriyama et al. (1970) found that TTX (lo-’ to g/ml) reduced the frequency or abolished spontaneous spikes in guinea pig stomach muscle by inhibiting slow wave generation, but action potentials evoked by electrical stimulation were unchanged by TTX. Botting and Turmer

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(1969) and Hobbiger et al. (1969) noticed that l T X sometimes increased the tone of guinea pig intestinal smooth muscle preparations. The shape of the action potential in guinea pig taenia coli was unaffected g/ml), but sometimes an increase in rate of spontaneous by TTX ( discharge was seen by Kuriyaina et al. (1966). This action potential was apparently calcium dependent, but in Ca”-free solutions a sodiumdependent spike could be elicited, especially if a small amount of magnesium was present, and it was also TTX-resistant (Bulbring and Tomita, 1970a,b). Abe (1971) found intracellular action potentials in uterine muscle unchanged after 90 min in T T X (lo-’ g/ml). Intraocular administration of TTX causes mydriasis, probably by paralyzing the parasympathetic nerves, because the smooth muscle of the iris still responds to ACh (Fleisher et al., 1961). Many workers now use TTX routinely to allow smooth muscle physiology and pharmacology to be investigated in the absence of neural activity (e.g., Bulbring and Tomita, 1967; Biilbring and Gershon, 1967; Drakontides and Gershon, 1968; Schnieden and Weston, 1969; Kottegoda, 1969; Furness, 1970, 1971; Ambache et al., 1970; Schnieden and Small, 1971; Haffner, 1971; Paton et al., 1971). Abolition of nervous activity after addition of TTX ( 3 X g/ml), would account for a reduction in acetylcholine released from guinea pig ileum which was stimulated electrically, but it would not necessarily explain the significant fall in resting acetylcholine output g/ml). Beani reported by Ogura et al. (1966) after adding TTX ( et al. (1969) and Paton et ul. (1971) also found a fall in acetylcholine release from guinea pig intestine after adding TTX (lo-’ g/ml), which they interpreted as being due to an abolition of spontaneous neural activity. However, this effect was not seen in similar experiments by Schnieden and Weston ( 1969). There have been very few studies of STX effects on smooth muscle. Kellaway (1935) noted the hypotension produced in uiuo, but concluded that the vasodilatation was due to a central effect of the toxin, a conclusion contested by Kao (Kao and Nishiyama, 196513; Kao et al., 1967). Sapeika ( 1953), studying a saxitoxin-like poison from Donax serra mussels, concluded that it had no effect on intestinal or uterine muscle, and Prinzmetal et al. (1932) came to the same conclusion with a crude preparation of STX. There do not seem to have been any detailed reports of in vitro studies of smooth muscle in the presence of STX, but it seems likely that such studies would give results very similar to those obtained with TTX. Nagasawa et al. (1970, 1971) suggest that small doses of STX have a direct vasodilator action similar to that claimed for TTX. The behavior of vascular smooth muscle in the presence of these

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toxins is still somewhat controversial. Kao originally believed that any effects on vascular peripheral resistance in vivo could be accounted for by a block of axonal conduction in the autonomic nervous system (Kao and Fuhrnian, 1963; Kao and Nishiyama, 1965b; Kao, 1966; Kao et al., 1967). Most other workers continue to hold this view (K. K. Cheng and Li, 1966; Feinstein and Paimre, 1968; Hughes and Vane, 1970). Kao and his colleagues now, however, believe that TTX and STX also have a direct relaxant action on vascular smooth muscle (Lipsius et al., 1967, 1968; Nagasawa et al., 1970, 1971; Kao et al., 1971). Their argument in support of this view is based upon in vivo observations that very small doses of these toxins (‘ITX: 0.5-0.8 pg/kg) caused vasodilatation, and they do not believe that these doses caused any blockade in the autonomic nerves. Their circumstantial evidence that the nerves were unaffected needs to be supported by direct evidence showing that catecholamine release following sympathetic stimulation is not reduced by these doses. This would be technically difficult and has not yet been done. It must be borne in mind that several other workers have applied TTX to vascular muscle preparations in vitro and found no effect on spontaneous activity or on the shape of the action potentials even at very high concentrations (Su and Bevan, 1967; Keatinge, 1968; Holman et al., 1968; Golenhofen and Loh, 1970; Ito and Kuriyama, 1971). Catecholamines, histamine, and acetylcholine, which act directly on vascular muscle, and tyramine, which appears to act indirectly through the release of noradrenaline from nerve terminals, continued to evoke their usual responses in the presence of high concentrations of TTX (Bell, 1968; Feinstein and Paimre, 1968; Holman et al., 1968; Hughes and Vane, 1970; Kao and Fuhrman, 1963; Su and Bevan 1967; see Section VI, B for further references to experiments in uivo). Irrespective of any possible relaxant action that TTX might have on vascular smooth muscle, it is clear that smooth muscle in general remains excitable and can generate action potentials in the presence of massive concentrations of the toxin. This resistance of smooth muscle to doses of TTX that would paralyze skeletal muscle, or even cardiac muscle, is not yet fully understood. The muscles of some invertebrates are dependent upon Caz+ions for the generation of action potentials (see next Subsection). Hagiwara and Nakajima (1966) found that the Ca2+-spikeof barnacle muscle was unaffected by TTX, and suggested that TTX was a specific inhibitor of Na’ spikes. Other work, discussed in Section V, A, 1, has made it clear that these toxins should be regarded as inhibitors of membrane “channels” rather than inhibitors of specific ions. There is a growing acceptance of the hypothesis that the action potentials of intestinal and some other smooth muscles are generated, at least in part,

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by Ca’+ (Bulbring and Tomita, 1970a), and that this explains the insensitivity of these muscles to TTX. Keatinge (1968), on the other hand, showed that in the absence of Ca2+the action potential in arterial muscle was generated by Na+ ions, and this sodium spike was unaffected by ITX ( g/ ml). Bulbring and Toniita ( 1970b) have obtained similar results on intestinal smooth muscle. Perhaps one should simply regard the “channels” in smooth muscle membrane as being different from those in skeletal muscle, insofar as the smooth muscle “channels” lack binding sites for TTX and usually appear to be better adapted for carrying Ca2+than Na+. Rougier et al. (1969) implied this when they commented: “Such a system is probably responsible for the calcium action potentials (suppressed by Mn) dcscribed on the guinea pig taenia coli by Bulbring and Tomita (1968) and also for the sodium (but TTXinsensitive) action potentials observed by Keatinge ( 1968) on arterial smooth muscle.” 4. Actions of the Toxins on Some Itivertebrute Rduscles Many, but not all, invertebrate muscles are resistant to the toxins. The giant muscle fibers of the barnacle Balanus nubilus develop, under certain conditions, action potentials in which Ca” ions carry inward current (Hagiwara and Naka, 1964; Parnas and Abbott, 1964). These calcium-dependent spikes were unaffected by TTX ( 4 X g/ml), though they were suppressed by manganese (Hagiwara and Nakajima, 1966). Other niolluscs also appear to be resistant to the toxins. Feinstein and Paimre (1967) report snail muscle to be resistant. Filter-feeding bivalves, including several species of mussels, clams, and scallops, acquire very large amounts of STX when Gonyaulax dinoflagellates are present in the phytoplankton (see Section 11, B ) . These bivalves do not seem to be affected in any way by the large amounts of STX they can accumulate (Somnier and Meyer, 1937). I n Na+-free solutions Mytihs muscles generate calcium-dependent spikes which are not affected by g/ml) (Twarog and Hidaka, 1971). Calcium-dependent TTX ( spikes could account for the fact that mussels and other bivalve shellfish are not affected by the large amounts of STX that they sometimes accumulate. However, Twarog et al. (1972) do not believe that this can explain fully the high immunity of some bivalves to TTX and STX. Action potentials induced in the muscles of various species of crayg /ml). fish, by treatment with procaine, were unaffected by TTX ( Other electrical responses in crayfish muscle, including excitatory postsynaptic potentials ( e.p.s.p.’s) induced by microionophoretic application of glutamate at the excitatory neuromuscular junction, and spontaneous miniature p.s.p.’s, were also unaffected by high concentrations of TTX

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or STX (Ozeki and Grundfest, 1965; Ozeki et al., 1966). In calciumfree solutions, however, crayfish muscle becomes selectively permeable to sodium, developing large transient depolarizations or oscillations that are Na+ dependent and TTX sensitive (Reuben et al., 1967). Sawyer et al. (1968), studying a STX-like poison from Aphanizomenon flos-aquae, reported suppression of action potentials in crayfish muscle, which appears to be at variance with the findings of Grundfest and his co-workers. The nervous system of the crayfish is known to be sensitive to these toxins. Axonal conduction was blocked by l T X ( g/ml) or by STX (lo-* g/ ml ) [Ozeki et al., 1966; but note that Ogura and Mori (196S), gave the extraordinarily low figure of 3 X lo-’ p M : about 10-l3 g/ml] and spike electrogenesis was inhibited in the stretch receptor neurons by TTX, though the generator potential was not abolished (Loewenstein et al., 1963, Obara and Grundfest, 1968; Welhoner, 1970). The same is true of lobster nerves and stretch receptors (Takata et al., 1966; Albuquerque and Grampp, M a ) , but there is little published information about lobster muscle, in which the normally graded responses can be converted to all-or-none action potentials by treatment with procaine. It has been reported that, as in crayfish muscle, miniature p.s.p.’s persisted in the presence of STX and TTX (Reuben and Grundfest, 1960; Ozeki et al., 1966). The earthworm, Lumbricus terrestis, appears to be like the crayfish in having a nervous system that is sensitive to TTX, but resistant musculature (Feinstein and Paimre, 1967), and some insect muscles are also reported to be resistant (Nakamura et al., 1965a) though their nerves are not ( Pichon, 1969) . The susceptibility of Na+-spikes to low concentrations of toxins, contrasted with the immunity of Ca2+-spikes,led some workers to regard X and STX as specific inhibitors of Na+-dependent electrical activity. Comments in several preceding sections have made it clear that the toxins are not ion specific, but should be regarded as blocking agents for specific “channels” or “carriers” in the membrane. The “channels” that are easily blocked are those that normally pass Na+-current; other channels, which may be better adapted to passing Ca2+-current or K+current, appear to lack binding sites for these toxins. An apparent anomaly is raised by the work of Geduldig and Junge (1968) who found that Aplysia neurons generated TTX-resistant, calcium-dependent, action potentials in sodium-free media, but in calcium-free media the action potentials were sodium dependent and were suppressed by TTX (330 x M ) . However, other evidence suggests that two types of channel exist in Aplysia neuron membrane; a TTX-sensitive sodium channel and a resistant calcium channel. These channels are largely independent, and are therefore likely to be topographically as well as

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operationally distinct ( Geduldig and Gruener, 1970). There could be a similar explanation for the findings reported by Reuben et al. (1967) previously mentioned.

C . EFFECTS ON NEUROMUSCULAR JUNCTIONS

AND

SYNAPSES

Many early workers erroneously thought that the paralytic effects of TTX and STX were due to a curare-like action of these toxins. These errors arose largely because these workers were unable, for technical reasons, to observe the failure of conduction in motor nerve terminals, or else they neglected to test the direct excitability of muscle fibers after the toxins had caused paralysis to indirect stimulation. Kao (1966) and Halstead (1965, 1967) give detailed accounts of the early work in their reviews. Matsumura and Yamamoto (1954) concluded that 7TX had a direct paralyzing action on nerve and muscle, leaving the end-plate potential (e.p.p.) unaffected. Furukawa et al. ( 1959) used intracellular microelectrodes to study the action of TTX on the toad sartorius neuromuscular junction (n.m.j.). They found that the n.m.j. was unaffected by doses of TTX that abolished the muscle action potentials. In curarized preparations, TTX to g /ml) sometimes produced a slight diminution in e.p.p. amplitude before causing a sudden disappearance of the e.p.p., suggesting that the motor axon blocked after a latent period. Sometimes the e.p.p. decreased in steps, suggesting successive blockade at different branches of a multiply-innervated n.m.j. They noted little or no effect of TTX on the depolarization of the end-plate zone in a frog muscle, tested by adding acetylcholine ( ACh) to the bath fluid. This work suggested that TTX has little or no effect on the sensitivity of the motor end-plate to ACh, and that the paralytic effects of TTX are due entirely to its action on nerve and muscle (described in preceding sections). Their conclusions have been fully substantiated by later workers. Using classical pharmacological techniques, Cheymol et al. ( 1961) concluded that TTX had no curare-like action, but produced its paralyzing effects by direct action on nerve and muscle. It paralyzes nerve and muscle by inhibiting the entry of Na+ (Section V, A, 1 and B, l ) , and the e.p.p. and miniature e.p.p. (m.e.p.p.) are at least partly sodium dependent, yet it has been found that m.e.p.p.’s continued with normal amplitudes in the presence of high concentrations of TTX (Elmqvist and Feldman, 1965; Katz and Miledi, 1967a), that ACh applied microionophoretically continued to produce typical depolarizing potentials at the end-plate (Nishiyama, 1967; Feltz and Mallart, 1970; Evans, 1971b) and that e.p.p.’s could still be recorded in the presence of high concentration of TTX when ACh was released from motor terminals by electro-

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tonic depolarization down the motor axon (Katz and Miledi, 1965, 1966, 1967a, 1968a). Landau (1969) reported a 30% fall in m.e.p.p. frequency when rat diaphragms were exposed to TTX ( g/ml), but this was an effect on the motor terminals and not on postsynaptic receptor sites. Katz and Miledi (1967a) found no change in m.e.p.p. frequency when this concentration of TTX was applied to frog sartorius preparations. Electrotonic depolarization has been used to produce posttetanic potentiation in the presence of TTX ( Weinreich, 1970). Naturally, ACh release on stimulation of the motor nerve is reduced by TTX (Fleisher et al., 1961; Cheymol et al., 1962a,b) because the toxin prevents nerve impulses from reaching the terminals. Katz and Miledi ( 196Sb) succeeded in blocking individtral motor nerve terminals by microionophoretic application of TTX, and found that electrotonic spread of an action potential into rz blocked terminal did not release ACh in the frog, though electrotonic conduction of a large depolarizing pulse into motor terminals was effective in their experiments described above. Electrotonic spread of an action potential appears to be usual in some invertebrate and electroplaque junctions (see Ozeki et al., 1966). There have been several studies of ACh synthesis and hydrolysis in the presence of TTX. These have generally given negative results, and have been mentioned in Section V, A, 2. Kellaway (1935) and Fingerman et al. (1953) thought that STX had some curare-like actions, among its other effects. DAguanno ( 1959) and Bolton et al. (1959) felt that there was no curare-like action. Evans (1964, 1965), Kao and Nishiyama (1965b), and Cheymol et al. (1968) also disputed the curariform hypothesis, though the latter group did have evidence of an effect on the motor cnd-plate in uivo, in which the contraction of cat tibialis muscle evoked by intra-arterial ACh was suppressed by STX. Recent work on e.p.p.'s and ionophoretic application of ACh to the end-plate of frog muscle, in the presence of STX ( 3 x lo-" M ) has shown that in vifro STX has no significant effect on the postsynaptic structures responsible for the e.p.p., although m.e.p.p. frequency is raised by high concentrations of STX (Nishiyania, 1967, 1968; Evans, 1971b). Krauss et al. (1970) showed that ACh continued to evoke a release of norndrenaline from perfused cat spleen after TTX blocked the release to nerve stimulation and the antidromic volleys induced in the splenic nerve by ACh. It would appear therefore, that neither TTX nor STX have any effects in vitro on the cholinergic receptors of the motor end-plate, nor on the sodium conductance of the receptor cliaiinels at this site. However, qualitative differences in the behavior of the e.p.p. in presence of low concentrations of the toxins havc been reported. TTX, as described

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above, causes an abrupt disappearance of the e.p.p. (Furukawa et al., 1959) whereas STX brings about a graded fall of e.p.p. amplitude (Nishiyama and Kao, 1964; Kao and Nishiyama, 1965b). This difference between the effects of the toxins has been confirmed, and it is clear that TTX more readily causes axonal block in the preterminal part of the motor nerves, probably at a node of Ranvier, whereas STX tends to produce a progressive reduction in the extent to which the impulse invades the motor terminals, although it too will block the myelinated preterminal axons after longer periods of- exposure ( Evans, 1969b, 1971a,b). If TTX is applied focally to a nerve terminal by micro-ionophoresis, a graded fall in the e.p.p. is then observed (see Fig. 5 in Katz and Miledi, 1968b) as the toxin progressively reduces the amount of ACh released by the nerve impulses. A toxin, isolated from mussels that had fed on Gonyaulax tamarensis, resembled STX in some respects but acted more like TTX on the e.p.p., causing it to dimppear abruptIy after a latent period ( Evans, 1970a ) . In the isolated neurohypophysis of the rat, TTX blocked nervous transmission but did not interfere with the spontaneous resting release of hormones, nor with the increased release evoked by K’. These neurosecretory terminals can be regarded as being analogous to the neuroinuscular terminals of motor axons (Dreifuss et al., 1971). Iwasaki and Satow ( 1970, 1971) have studied the neurosecretory “X-organ” of the crayfish and here found that TTX abolished Na+-dependent action potentials, revealing A Ca*+-dependentelectrical response that may be linked with hormone release. A synthetic guanidine ester, 5-benzoyloxy-2-iminohexahydropyrirnidine HCI (HM-197) has some resemblances to TTX, but differs from the puffer fish poison in having definite blocking actions on the postsynaptic receptors of the n.ni.j. ( R a n n e y et al., 1968, Spiegelstein and Kao, 1970, 1971). Matsumura and Yamamoto (1954) found evidence that TTX blocked ganglion cells in the toad but spared the preganglionic fibers. The preganglionic fibers of the toad have been reported to be relatively resistant to ‘ITX ( Carter, 1969). On the other hand, Ozawa and Sugawara ( 1967) found that TTX affected responses to preganglionic stimulation more than those evoked by stimulation of postganglionic fibers, in agreement with the results of Kao and Fuhrman (1963). Ozawa and Sugawara also reported ganglion blocking actions of TTX in guinea pig vas deferens and in rabbit intestine, but it is difficult to exclude axonal blocking effects from their results. Koketsu and Nishi (1969) reported that lumbar sympathetic ganglion cells of the bullfrog R a m catesbeiana were rendered inexcitable when TTX ( 5 x l@:g/ml) was

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added to the Ringer’s solution. However, these cells can generate action potentials in sodium-free isotonic CaCI,, and in this medium the spikes were unaffected by TTX (5 X g/ml). Ogura (1971), reviewing the literature, thought that a ganglion blocking action might contribute to the well-known hypotension that TTX causes, but Kao and Fuhrman (1963) stated that cholinergic receptors in autonomic ganglion were not affected by TTX from newts. In a detailed study of synaptic transmission in parasympathetic ganglion cells, Dennis et al. (1971) recorded miniature e.p.s.p.’s from postganglionic neurons in frog heart. TTX (lo-:) blocked nerve conduction but did not affect either amplitude or frequency of the miniature e.p.s.p.’s. The toxins therefore do not appear to be ganglion blockers, in the sense that hexamethonium is, although their actions on axonal conduction would of course prevent transmission through ganglia. Conduction block within the ganglion probably explains earlier reports of a ganglion blocking action of TTX. A few studies have been made on invertebrate synapses and n.m.j.’s. Neither TTX nor STX affected excitatory or inhibitory responses in crayfish muscle fibers, to ionophoretically applied glutamate or y-aminobutyric acid (Ozeki et al., 1966). In crayfish and in lobster muscle fibers m.e.p.p.’s could still be recorded in the presence of TTX (5 x 1W g/ml) (Reuben and Grundfest, 1960; Ozeki et al., 1966). In the squid these toxins blocked the generation of action potentials in nerves (see Section V, A, 1) but TTX did not prevent depolarization of a giant presynaptic fiber, by current passed into the fiber through a microelectrode, from giving rise to postsynaptic potentials (p.s.p.’s) in the stellate ganglion (Bloedel et al., 1966, 1967; Katz and Miledi, 196%; Kusano et al., 1967). Miniature p.s.p.’s could also be recorded in this preparation in the presence of TTX (lo-‘ g/ml or more) (Miledi, 1966, 1969). Evidently, as in the vertebrate n.m.j., TTX does not interfere either with the release of transmitter from presynaptic terminals, except insofar as it prevents nerve impulses from being conducted to the terminals, nor does it block the action of the transmitter on the postsynaptic receptors, when it is applied in concentrations just sufficient to prevent action potentials from being generated. However, Kusano et al. (1967) reported a depression of p s . ~ . by ’ ~ high concentrations of TTX. The relative immunity of synaptic transmission to ?TX has allowed the latter to be used in experiments on the relation between presynaptic depolarization and transmitter release. It appears that the log of transmitter release is approximately proportional to the depolarization of the nerve terminal (see Gage, 1967). Katz and Miledi (1969, 1970) have evidence that in the presynaptic terminals of the squid stellate

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ganglion there is a calcium-dependent regenerative local response, which may be associated with the transniitter-releasing mechanism. This was unmasked by treating the terminals with tetraethyl ammonium ions and TTX. The electroplaque organ of Electrophorus eels is developmentally analogous to striated muscle and the junction is cholinergic. As in other vertebrate n.m.j.’s, the toxins do not prevent acetylcholine, or its analog carbamylcholine, from evoking a depolarizing response ( Dettbarn et at., 1965). When it was discovered that TTX abolished the action potential in the axon without interfering with cholinergic transmission at the neuromuscular junction, Katz and Miledi (1966) felt that this weakened the argument for a cholinergic mechanism for axonal conduction. I t is true that the old suggestions of special permeability barriers have become almost untenable, but the current views of those who favor the hypothesis of cholinergic axonal conduction are so far removed from the views of electrophysiologists that there is little common ground to be discovered (Nachmansohn, 1970, 1971, and 1971, personal communication). It is only reasonable to point out that abolition of axonal action potentials by toxins which do not affect cholinergic mechanisms does not, of itself, exclude the possibility that cholinergic mechanisms may play some role in axonal conduction at some point different from the site of action of these toxins. Nachmansohn’s hypothesis will therefore continue to stimulate neurobiologists to perform further experiments designed to test its validity.

D. EFFECTS ON RECEPTOR ORGANS Several workers have found that spike electrogenesis in afferent nerves can be blocked with TTX, leaving a nonpropagating generator or receptor potential recordable at the nerve terminals. For instance, Loewenstein et al. (1963), Ozeki and Sato (1965), and Nishi and Sato ( 1966) have recorded receptor potentials from decapsulated Pacinian corpuscles in which the propagated action potential was eliminated by TTX; Albuquerque et al. (1968, 1969) have recorded receptor potentials from frog muscle spindles under similar circumstances; Loewenstein et al. (1963), Albuquerque and Grampp (196S), Obara and Grundfest (1968), and Wellhoner (1970) have abolished spikes with TTX when recording generator potentials from crayfish or lobster stretch-receptor neurons; Millecchia et al. (1966) and Benolken and Russell (1967) used TTX when studying the sensory responses in Limulus eye; Katsuki et al. (1966) and Konishi and Kelsey (1968) found that local applica-

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tion of TTX to the inner ear of guinea pigs abolished nerve impulses but had little effect on the cochlear microphonic receptor potential. A few comments are necessary, however, on some of the results obtained. Loewenstein et al. (1963) had evidence that the generator potentials in crayfish stretch receptors and cat Paciiiian corpuscles were to 5 X lo-’ g/ml). Ozeki and Sat0 (1965) found unaffected by TTX ( little or no effect at a concentration of 1 5 x lo-; g/niI, which abolished the propagated spike, but found the receptor potential depressed by prolonged exposure to higher concentrations. In a more detailed examination of the behavior of the receptor potential, Nishi and Sat0 (1966) found that TTX ( 5 X 10.: g /m l) reduced the potential slowly to 40-70% of its control value. After this reduction had been effected, in 30-60 minutes, increasing the concentration to 1-2 X g/ml had little further effect. This discrepancy between the two groups of workers was noted by Kao (1966) who commented: “if 300 nM tetrodotoxin can affect a substantial part of the generator potential, it is strange that 30 p M should have no effect on it.” In seeking an explanation for the discrepancy, one can probably discount significant deterioration in the corpuscles studied by Nishi and Sato (1966), as their potentials often returned to control values when the TTX was washed out. Loewenstein (1971) has suggested that the responses studied by Nishi and Sat0 contained an abortive impulse coniponent. I t is possible that their generator potentials were boosted by a TTX-sensitive regenerative effect similar to the local subthreshold responses seen in axons stimulated with weak currents, but then one would expect the T T X to eliminate this as quickly as the action potential. It is more likely that permeability barriers hinder the diffusion of TTX to the receptor binding sites, so that Loewenstein et al. (1963) did not observe the decrease during the time course of their experiments. It is well known that the connective tissue sheaths of peripheral nerves offer an almost impenetrable barrier to these toxins and even the terminals seem to have some barriers, such as the sheath of Henle at the terminals of motor axons. Arden and Ernst ( 1969) described a receptor potential generated by cones in pigeon eyes. This response was sodium dependent, but was unaffected by TTX or STX. Byzov et al. (1970) reported that TTX suppressed spikes in the optic nerve of frog without affecting slow potentials of retinal origin. Retinula ceIls in honeybee eyes can generate slow-wave receptor potentials and regenerative spike potentials. The latter could be evoked by electrical stimulation, appeared to be sodium g/ml), which had no dependent, and were abolished by TTX ( significant effect on the slow-wave response evoked by light (Baumann, 1968). In the lateral eye of Limulus, TTX suppressed a transient photo-

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response without affecting the steady-state component ( Benolken and Russell, 1967). I n the ventral eye, however, the toxin was without effect on any of the photoresponses although these were sodium dependent (Millecchia et al., 1966; Millecchia and Mauro, 1969). The lateral line organs of teleost fish respond to Na’, K’, and some other monovalent cations with an increased frequency of discharge. This response was abolished by addition of T T X ( 10-7 g /m l) to the test solution. Addition of Ca’+ 46 mM also abolished the response (Katsuki et al., 1971). It is not clear whether the TTX was acting on the sensory receptors or on the afferent nerve fibers. There are no reports that the toxins decrease the generator potentials in frog muscle spindle or crustacean stretch-receptor neurons. However, in the latter, the resting membrane resistance was reported to fall within 1-2 min of exposure of TTX (Lowenstein et al., 1963; Albuquerque and Grampp, 1968), the resting potential hyperpolarized slightly, and the poststimulus hyperpolarization was reduced. Evidently TTX has some effects on the resting properties of the crustacean neuron, and an increased permeability to K’ and C1-, coupled with a decreased resting Na’ permeability would account for the observed effects (Albuquerque and Grampp, 1968). However, for the generator potentials to remain constant in the face of these changes in resting permeability, the generator current would have to be doubled in the presence of TTX. This would seem to conffict with all other observations on the blocking action of these toxins on Na’ channels; all the generator potentials examined in these studies are at least partly Na+ dependent, and lithium can substitute for sodium to a large extent (Diamond et a!., 1958; Higman and Bartels, 1962; Edwards et al., 1963; Ottoson, 1964; Obara and Grundfest, 1968). Calcium ions may make a small contribution (Albuquerque et al., 1969). These observations, and the discrepancies between different reports, indicate that it must not be assumed that the toxins are without effect on sensory receptors. Ottoson and Shepherd ( 1971) have made the interesting suggestion that the insensitivity of most sodium dependent receptor potentials to TTX may be due to an intracellular location of the sodium “gates.” More detailed work is needed in this field, to give a better understanding of the properties of these sensory receptors. E. EFFECTSON OTHERTISSUES

Kao and Fuhrman ( 1963) concluded that TTX (from newts) had no effect on the potential or short-circuit current of the frog skin preparation. Solomon (1969) found that one sample of TTX reduced both potential and short-circuit current, especially when applied to the mucosal side, but that two other samples of TTX were without effect.

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H e also obtained similar results on reabsorption of saline by rat kidney tubules, the first sample inhibiting reabsorption but the other two being without effect. He suggested that an active impurity was present in his first sample. Pullman et al. (1968) infused TTX into dog renal artery, and they obtained evidence that the toxin increased excretion of Na+, K+, Mg”, Ca2+,and C1-. They suggested that TTX might block tubular reabsorption of Na+, but could not exclude the possibility that the toxin was causing changes in the renal blood flow, through its known vasodilator effects, during their in vivo experiments. Ogura (1971) mentions an antidiuretic effect, but this could have been secondary to reduced glomerular filtration resulting from typical TTX-induced hypotension. Matsumura et al. (1968) noted the vasodilator action of TTX on vesical blood vessels, with no apparent effect on the bladder musculature. Marumo et al. (1968) claimed that TTX inhibited active sodium transport in the toad bladder preparation. They found some reduction in short-circuit current when the toxin was applied to the serosal side, but M . They usually used the only at concentrations greater than massive concentration of 3.6 x lo-‘ M , and if their toxin was the usual Sankyo product this would have introduced significant (3 mM) concentrations of citrate, apart from the possibility that an active contaminant was present, as suggested by Solomon (1969). Kao and Fuhrman (1963) tested their crystalline TTX (from newts) on toad bladder at concentrations up to approximately 3.8 x lo-’ M without seeing changes in potential or short-circuit current. Marumo et al. (1968) reported slight reduction in oxidative metabolism of rat liver mitochondria and toad bladder membrane, without effect on ATPase, again using massive concentrations of TTX that may have introduced significant amounts of citrate buffer or some other contaminant into their systems. Kao and Fuhrman (1963) failed to note any effect of comparable concentrations of newt TTX on oxidative metabolism of bullfrog nerves, confirming the findings of van Wagtendonk et al. (1942) who also failed to find significant effects on respiration of kidney and liver slices. E. J. Schantz and H. A. Neufeld found no effect of STX on oxygen consumption of mouse diaphragm muscle or on the ATP-induced contraction of isolated muscle fibers; J. O’Neill found no effect on the phosphorylation of ADP to ATP (unpublished work, cited by Schantz, 1960). Other experiments on oxidative metabolism and enzyme activity have been mentioned in Section V, A, 2. Cheymol et al. (1965) tested the effects of TTX on HeLa tumor cells growing in tissue culture. They found some effects on mitosis, with inhibition in metaphase, but only at very high concentrations of the g/ml). toxin ( 5 X

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Erythrocyte membranes are unaffected by TTX (see Ogura, 1971, and several “personal communications” in Moore and Narahashi, 1967) . V1. Actions in Vivo

A. EFFECTS ON NEUROMUSCULAR SYSTEMS 1. Locomotor Muscles and Peripheral Nervous System The paralytic effects of TTX a d STX (once known as paralytic shellfish poison) are the most outstanding actions of these substances, and these effects have been recognized from the start. However, there have been conflicting opinions regarding the site of action of the toxins. The early work is exhaustively reviewed by Cheymol and Bourillet (1966), Halstead (1965, 1967), and Kao ( 1966) and need not be repeated here, except to state that early theories in which the toxins were thought to paralyze by actions on the central nervous system (CNS) or by a curariform action on the neuromuscular junction have been abandoned by most workers. It is true that Cheymol et al. (1968) had evidence of a curare-like action of STX on the tibialis muscle of the cat, but such an action was not seen in the similar experiments by Kao and Nishiyama (1965b) and most workers have found that in vitro the motor end-plate is unaffected by the toxins (see Section V, C). The paralyzing effects of TTX and STX can be adequately accounted for in terms of their known actions on peripheral nerve and skeletal muscle (see Section V, A and B ) . Horsburgh et al. (1940) recognized that both nerve and muscle were affected. The comparative sensitivities of nerves and muscles to the paralyzing effects of the toxins have not been studied in detail. In general, i.v. administration of T T X or STX to rabbits blocks motor nerve conduction at about 5-20 pglkg, with loss of the indirectly stimulated muscle twitch. In rabbits the nerve block usually precedes loss of muscle excitability, tested by direct stimulation. The muscle itself becomes paralyzed more slowly, sometimes 20-30 min elapsing between i.v. injection and maximum paralytic effect, and usually a higher dose is needed. In cats and rats there is little difference between the sensitivity of nerve and muscle, though pale muscles (tibialis) appear to be slightly more resistant than red muscles (soleus). When the poisons are administered intra-arterially they are, of course, effective at lower doses : 0.5-4 pg/ kg. Subparalyzing doses sometimes cause a transient slight increase in maximal twitch amplitude; no explanation of this effect has been offered, but in vitro experiments on nerve, mentioned in Sec-

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tion V, A, 1, have sometimes noted an analogous effect on gNa (Murtha et al., 1958; Cheymol et al., 1961, 1968; Evans, 1965; Cheymol, 1966; Cheymol and Thach Toan, 1969). An ascending paralysis is said to follow TTX (5-6 pg/kg i.v.), with forelimb muscles proving more resistant than hindlimb muscles and the diaphragm intermediate in sensitivity ( Murtha et al., 1958). Cheng et al. (1968) found that TTX produced respiratory paralysis shortly before it paralyzed the forelimb biceps muscle or its nerve supply. Kao and Nishiyama (196%) found that in cat the respiratory muscles were more resistant to STX than the hindlimb tibialis anterior. Their claim that STX (0.75 pg/kg i.v.) caused some weakening of tibialis twitches should be accepted with caution, since these small doses appear to have been given after recovery from a larger dose. Due to the good safety factor present in the spike-generating mechanism of nerve and muscle there is a definite threshold dose with these toxins and apparently complete recovery will be seen when a significant, but subliminal, concentration of toxin remains in the tissues. Under these circumstances a small, normally ineffective, dose will bring the total concentration above threshold and a paralytic effect will ensue (Cheymol et al., 1961). Some workers have found that STX paralyzes respiratory muscles to indirect stimulation before the indirect responses of hindlimb muscles are lost (Evans, 1965; Cheymol et al., 1968; Cheymol and Thach Toan, 1969). When the toxins are given slowly i.v., nerve conduction fails in afferent fibers at the dorsal root ganglion before efferent fibers are blocked. This is probably due to a lower safety factor at the T junction where the pseudo-unipolar ganglion cell is joiiicd to the main fiber (Evans, 19674. There is no difference between the sensitivities of sensory and motor axons along the rest of their course between CNS and the periphery. The toxins are ineffective when applied topically to peripheral nerves, unless these are first desheathed. Topical application of very low concentrations of TTX and STX to the intradural portion of spinal roots will lead t o a conduction block, because these roots lack the thick coniiectivc tissue sheath of a peripheral nerve ( Evans, 1968a). Toxins administered i.v. gain access to the peripheral nerve fibers through the blood vessels that penetrate the multiple layers of perineural epithelial cells, which constitute the main permeability barrier in the connective tissue sheath (Krnjevii., 1954; G. H. Bourne, 1969, personal communication ) , 2. Respiratory System

Respiratory paralysis is undoubtedly the cause of death in poisoning by TTX or STX. In the normal course of events (i.e., in cases of

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ingestion of toxic food by humans or following i.v. administration to experimental animals ) the rcspiratory paralysis can be accounted for by the peripheral cffects of the toxins. Contrary to the opii~ionsof most early workers, actions on the brainstcm respiratory centers do not appear to contribute significantly to the lethal effects of T T X and STX. Kellaway ( 1935) , working on partially purified mussel poison, was one of the first to record phrenic nerve impulses in an animal whose rcspiratory movements were failing after administration of the poison. He showed that STX, given i.v. to a rabbit, led to a failure of ventilation without loss of nervc inipulscs desccnding the phrenic nerve from the CNS. The respiratory center, in fact, responded to the asphyxia by discharging massive bursts of impulses. With this evidence of a peripheral site of action it is hard to understand why Kellaway believed that STX depressed respiration centrally. Kao and Fuhrnian ( 1963) concluded that the systemic cffects of newt TTX could be accounted for by its peripheral actions. Kellaway’s results were confirmed by Evans ( 1965), using pure STX and better recording techniques. Again there was no evidence of central depression, the respiratory center continuing to respond to the increasing asphyxia for 1.5 niin after the last weak respiratory movement. Direct stimulation of respiratory muscles sometimes evoked a weak contraction, when no natural respiratory movements could b e seen. Evidently the toxin was affecting respiratory niuscles directly, but there was also wine effect in blocking conduction in the phrenic nerve below the cervical Icvel, where the activity from the respiratory center was being recorded. Recent work has virtually excluded curare-like actions (Section V, C ) so the probability remains that the toxin was blocking nerve conduction near the motor terminals of phrenic and intercostal nerves, as well as paralyzing the muscles by a direct action. It is known to affect motor terminals in the frog sartorius nerve-niuscle preparation ( Kao and Nishiyania, 1965b; Evans, 1969b). Similar experiments by Sakai et al. (1961), Wang et al. ( 1968), and Cheng et aE. (1968) showed that T T X also exerted its effects on the peripheral respiratory system, vigorous bursts of action potentials from the respiratory center being recorded in the phrenic nerve after the diaphragm was paralyzed and veiitilation had failed. Evans ( 1970a), studying two partially purified toxins derived from Gonyaulax tamarensis, concluded that they both paralyzed respiration by a peripheral action without any significant central depression. Deguchi ( 1967) reported respiratoiy depression produced by a number of TTX-derivatives, but it is difficult to exclude the possibility that these effects were due to contaniination of the derivatives by $mall amounts of l T X (see p. 91). Kao et al. (1967) and Wang et al. (1968) used a different technique to demonstrate that TTX and STX havc no significant central effects.

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In crossed circulation experiments they perfused the head only of one dog from the systemic circulation of a second donor animal. When the toxins were given i.v. into the donor’s circulation its blood pressure and respiration were severely depressed. Toxin carried with the donor’s blood through the head of the recipient did not depress the recipient’s blood pressure or respiration. On the contrary, the recipient’s respiration increased significantly, probably as a result of the donor’s blood becoming hypercapnic due to reduced ventilation in the donor animal. It is surprising that toxins having such potent actions on neurons should show no evidence of central effects when given intravenously. This is probably a reflection of their failure to cross the blood-brain barrier. There is some evidence that TTX given intra-arterially into a carotid ai-tery so as to achieve higher concentrations in the cranial circuIation than in the systemic circulation, might have a central effect on respiration (Murtha et aE., 1958). It is certainly true that very small quantities of TTX, in the range 0.2-0.5 pg, produce respiratoi-y arrest in cats when given direct into the cerebral ventricles (Borison et al., 1963) or in somewhat greater amounts into the cisterna (Li, 1963). When given intraventricularly TTX (Borison et al., 1963) and STX (M. H. Evans, 1967, unpublished data) often caused an apneustic arrest of respiration in sustained inspiration. Borison et al. (1963) suggested that this was due to the toxin reducing central sensitivity to CO, and disrupting supraniedullary modulation of the medullary centers.

B. EFFECTSON

THE

CARDIOVASCULAR SYSTEM

Almost all workers who have studied the effects of TTX and STX in viuo have noted the severe hypotension resulting from intravascular administration of either toxin. Kao states that TTX is more potent than STX in this respect: “with low, paralytic doses of the shellfish toxins, there is no significant hypotension; with tetrodotoxin hypotension always accompanies muscular paralysis, whatever the dose. Clinically hypotension is uncoinmon in paralytic shellfish poisoning but is an early sign portending grave prognosis in puffer fish poisoning” ( Kao and Nishiyama, 1965b). Murtha ( ISSO), however, thought STX to be more cardiotoxic than TTX. Early workers generally believed that the cardiovascular effects of these toxins resulted from central actions, in which cardiovascular centers in the brainstem were depressed (for references, see Halstead, 1965, 1967; Kao, 1966). Their evidence was, however, indirect and of doubtful validity. Most authorities now consider TTX or STX actions on cardiovascular centers in the CNS to be of negligible importance or even nonexistent (Feinstein and Paimre, 1968; Kao et a]., 1967).

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If central actions arc unimportant, then the hypotension caused by these toxins can only be due either to a reduction in cardiac output or to a decrease in vascular resistance through some peripheral effect. This peripheral effect might be a direct relaxant one on arteriolar smooth muscle, a ganglion blocking action, or a removal of sympathetic vasomotor tone through the known ability of these toxins to block axonal conduction, the block occurring in pre- or postganglionic sympathetic neurons. It is conceivable that more than one of these possible sites of action might be important. The severe hypotension resulting from intravenous administration of these toxins in doses of 1 to 5 pg/kg cannot be accounted for entirely by a reduction in cardiac output (Li, 1963). Slowing of the heart, probably due to suppression of sympathetic activity, is a common finding with either TTX or STX, and cardiac output is reduced by doses in the range 1-5 pg/kg (Cheng and Li, 1966; Feinstein and Paimre, 1968; Tomlinson and James, 1968; Hashinioto and Chiba, 1969) but the reduction is insufficient to account for the precipitous fall in arterial blood pressure, which may fall from 120-160 nim Hg to 40 mm Hg or less. Only in an animal whose CNS has been destroyed by pithing, to remove all vasomotor tone, can the fall in cardiac output account for the hypotension. In these animals large closes of toxin cause sinus bradycardia and heait block or fibrillation (Cheng et al., 1970). The toxins have little direct effects on heart muscle in vitro except at higher concentrations (Section V, B, 2 ) . As the reduction in cardiac output is insufficient to account for the severity of the hypotension produced by m a l l doses of the toxins, it is evident that these doses cause significant vasodilatation (Kao and Fuhrnian, 1963). Unfortunately, there is not yet general agreement about the site of action rcsponsiblc for this vasodilatation, and Murtha ( 1960) thought that STX did not cause much vasodilatation. Ganglion blocking effects can probably be discounted (Section V, C ) . Several Japanese workers had earlier suggested that the hypotension was due, at least in part, to conduction block in Sympathetic fibers (references in Murtha et al., 1958). Horsburgh et al. (1940) and Kao and Fuhiman (1963), working on newt TTX were o f the opinion that the hypotension was due to the axonal blocking action of the toxin. The latter workers concluded that preganglionic cholinergic axons werc more affected than postganglionic adrenergic ncrvcs, since the nictitating inenibrane sometiines responded to ganglionic stimulation when preganglionic stiinuli were ineffective. This effect was also seen by Ozawa and Sugawara ( 1967) following intra-arterial administration of TTX. Cheng et al. (1970) reported no direct dilator action on vascular

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muscle in perfused isolated rat hindlimb. Many workers have shown that even in the presence of high concentrations of TTX, one still obtains vasoconstriction in response to adrenaline, noradrenaline, and tyramine (Horsburgh et al., 1940; Kao and Fuhrman, 1963; Cheng and Li, 1966; Ozawa and Sugawara, 1967), and vasodilatation or diphasic responses to adrenaline, acetylcholine, histamine, nicotine, etc. ( Kao and Fuhrman, 1963; Cheng and Li, 1966; Koizumi et al., 1967; Feinstein and Paimre, 1968), and that after vascular a-receptors are blocked TTX no longer produces further vasodilatation ( Feinstein and Paimre, 1968). Therefore most workers have come to believe that TTX produces hypotension by blocking the conduction of vasomotor impulses in sympathetic axons, and not by a direct paralyzing action on vascular smooth muscle (see Section V, B, 3 for additional references to in uitm studies). Kao and his co-workers now believe, however, that the toxins have a direct action on arteriolar smooth muscle in addition to their effects on nerve conduction. Their evidence for this is based on experiments in which peripheral vascular resistance was measured in perfused hindlimb muscles. In dogs, small doses of ?TX caused a fall in perfusion pressure, indicating vasodilatation. They presented evidence that these small doses (0.8 pg/kg) did not block nerve conduction, but some caution is needed in accepting this evidence because there is only a small margin between these doses and those that do reduce sympathetic tone (1pg/kg) (Lipsius et al., 1967, 1968). Kellaway ( 1935) recorded vasodilatation due to STX. Nagasawa et al. (1970, 1971) reported that STX has dilator actions on arteriolar muscle similar to those claimed for TTX, but that reflex release of catecholamines can rapidly reverse the effects of small doses of STX. Feinstein and Paimre (1968) noted a vasodilatation caused by these small doses of TTX in perfused cat hindlimb, but disagree with Kao and his co-workers over the interpretation. Because TTX caused 110 additional vasodilatation after blockade of adrenergic endings with guanethidine or 2( Z,6-dimethylphenoxy)propyltrimethylammonium bromide, or blockade of a-receptors with phenoxybenzamine, Feinstein and Paimre (1968) concluded that the toxin had no direct action on vascular muscle, and that its effects on peripheral resistance were due solely to loss of sympathetic vasoconstrictor impulses. They found that 0.5 pg/kg was sufficient to reduce vasoconstrictor responses to nerve stimulation. These experiments were performed on cats, while Kao's group had been working on dogs. Species differences are not, however, the explanation of the difference of opinion between the two groups of workers, for Kao has repeated his experiments using cats and obtained

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essentially the same results as those which his group previously reported when using dogs as subjects (Kao et al., 1971). It is not easy to reconcire Kao’s claim that the toxins have direct vasodilator actions with the evidence presented above, and the in oitro results mentioned in Section V, B, 3, in which other workers have failed to find direct effects even at very high concentrations. Lipsius et al. (1968) suggest that the in vitro findings of other workers, using aortic strips, might not apply to an in vivo preparation, in which arteriolar muscle plays the major role and whose properties might differ from aortic muscle in vitro. They also claim that the vasodilator effects can be seen reliably only in their perfused gracilis muscle, and that when a hindlimb is perfused entire, as in Feinstein and Paimre’s experiments, the specific effects are submerged in other effects on mixed vascular beds (Kao et al., 1971). As mentioned in Section V, B, 3, the crucial experiment would seem to be a demonstration that stimulus-evoked catecholamine output from adrenergic terminals is not reduced by small doses of toxin that cause vasodilatation-described in the Note Added in Proof. (see p. 166 ) . Other workers have examined changes in particular regions of the cardiovascular system. OBrien ( 1969) reported significant increases in coronary sinus blood flow and cardiac output, following TTX ( 2 pg/kg i.v.). The latter effect is probably due to the fall in total peripheral resistance and total pulmonary resistance. Mean arterial blood pressure was not changed significantly at this dose but fell 41% at 4 pg/kg i.v. Matsumura et al. (1968) noted that TTX caused vasodilatation in vesical blood vessels without any effect on the smooth muscle of the urinary bladder. Pullman et al. ( 1968), in discussing the results of experiments in which TTX increased urinary excretion of several cations, noted that alterations in intrarenal blood flow might have taken place. Deguchi ( 1967) observed hypotension in response to i.v. administration of a number of TTX derivatives. However, this may have been due to traces of TTX contaminating the derivatives (see p. 91). Hypotensive effects were also seen by Evans (1 9 7 0 4 , working with two partially purified STX-like poisons extracted from shellfish which had been feeding on Gonyaulax tamarensis. C. EFFECTSON

THE

CENTRAL NERVOUS SYSTEM

Of all the physiological systems 011 which the effects of these toxins have been examined, none has given rise to more error and confusion than the CNS. Reference to detailed reviews, such as that by Kao (1966) and the monograph by Halstead (1965, 1967), reveals that the majority of early workers attributed almost all the effects of these toxins to actions on the CNS. However, even these workers recognized that the toxins

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also had peripheral actions, which they L I S L I ~termed ~ “curare-like” (Takahashi and Inoko, 1890; Kellaway, 1935). Now the current of opinion has reversed itself; in the two preceding sections I have mentioned that most authorities now discount central actions on the respiratory and cardiovascular “centers” of the brain. Nevertheless, there is a body of recent literature, concerned with the effects of these toxins on the CNS, which must be discussed. The literature specifically concerned with central effects on respiration and the cardiovascular system has already been dealt with in Sections VI, A, 2 and B, and although a few papers claiming to show central effects on these systems have not been mentioned, they are referred to in recent reviews by Kao (1966) and Ogura (1971). These brainstem centers will, therefore, not be specifically considered in this section. The chief difficulty in evaluating the results of experiments with TTX and STX on the CNS lies in the ability of these poisons to interfere with impulse traffic into and out of the CNS at very low doses. Even 1 pg/kg i.v. will produce significant failure of conduction in some peripheral nerves of experimental animals (Kao et al., 1967) and it is probable that a small percentage of peripheral nerve fibers will cease to conduct at even lower doses. A subtle aspect of this general difficulty is the fact that an afferent input to the CNS may fail in the region of the dorsal root ganglion with doses of toxins that do not seriously affect conduction along peripheral sensory and motor fibers (Evans, 1967a; see Section VI, A, 1). The existence of a permeability barrier, loosely referred to as the “blood-brain barrier,” appears to limit severely the passage of TTX and STX into the CNS after intravascular or i.p. administration. There have been several reports of changes in CNS activity following such adniinistration, but in most of these reports one cannot exclude the possibility that the effects described resulted mainly from the peripheral actions of these toxins on axonal conduction, or are secondary to arterial hypotension or respiratory depression. Failure to control these possible actions makes it hard to evaluate some reports of cortical depression (Frank and Pinsky, 1966) or depression of spinal reflexes (Murtha, 1960; Kuriaki et al., 1956) following i.v. administration of the toxins. Some workers have noted that hypotension could have caused at least some of the CNS effects (Frank and Pinsky, 1966; Murtha et al., 1958). Rudomin and Murioz-Martinez (1969) reported that TTX, given intra-arterially in doses up to 115 pg/kg, failed to abolish a dorsal root potential conducted electrotonically from a stimulation site in the dorsal horn of cat spinal cord. All reflex activity in the spinal roots was, of course, blocked at much lower doses through the usual peripheral action

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of the TTX, though antidromic action potentials in the dorsal root (central to the dorsal root ganglion ) were relatively resistant. They discussed various possible explanations for this TTX-resistant dorsal root potential, but do not seem to have considered the possibility that a permeability barrier might have prevented the toxin from penetrating the tissues of the spinal cord adequately. Blankenship ( 1968) studied the properties and activity of spinal motoneurons in the cat. When TTX ( 10-4-10-6 g/ml) was applied topically to the exposed cord, conduction failed in the spinal roots within 1-2.5 min and reflex excitation of the motoneurons failed simultaneously. A few minutes later the motoneurons failed to respond to stimulation of intraspinal pathways, and 10-30 niin after application of the poison the motoneuron threshold rose and it then became inexcitable, even by direct intracellular depolarization. The passive properties of the motoneuron membrane were unchanged, but “synaptic noise” or miniature synaptic potentials disappeared, as spontaneous activity in incoming neurons was abolished ( see also Blankenship and Kuno, 1968). Intravenous administration of the poison, on the other hand, was relatively ineffective. Massive doses (20-80 pg/kg) had to be given, and motoneurons were not noticeably affected until 5-10 niin after the roots and intraspinal pathways had become blocked. Crawford and Shibata ( 1968) also failed to obtain effects on the CNS visual pathway of rats when massive doses of TTX were administered i.p., but saw changes in the electroencephalogram ( EEG ) and abolition of photic-evoked cortical potential when TTX (28 pg) was given into the cerebral ventricles. Rech et al. (1964) obtained slight changes in the EEG of cats with intraventricular administration of TTX (0.1-0.2 pg), which also led to marked paralytic effects. No EEG changes were produced by i.v. administration of 2.5-5.0 fig/ kg. Koizumi et aE. (1967) made a careful study of the effects of TTX on spinal reflexes in anesthetized cats. In their experiments the afferent stimuli were given to peripheral nerves and the volley reaching the spinal cord was monitored centra1 to the dorsal root ganglion. They found that TTX (1-2 pg/kg i.v.) depressed mono- and polysynaptic reflexes recorded either from peripheral nerves or from central ends of divided ventral roots. Cortical evoked potentials were also depressed. The afferent volley reaching the cord did not seem significantly depressed by these doses, although conduction in the splanchnic nerve appeared to be blocked and there was a significant hypotension. They argued that the reflex depression was not due to the hypotension, nor to the respiratory depression resulting from the higher doses, However, it seems surprising that Blankenship ( 1968) and Crawford and Shibata (1968)

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failed to obtain CNS effects except at niuch greater i.v. doses. Evans (1968b, 1969d) also found little evidence for CNS effects, except occasionally after prolonged exposure to very high i.v. doses. In Evans' experiments artifacts due to hypotension and respiratory depression caused by the toxins were avoided by using artificially ventilated acute spinal cats. Slow i.v. administration of TTX or STX led to a loss of spinal reflexes, but the afferent volley was also reduced and an examination of the input-output characteristics of these reflexes showed that the loss of reflex activity was almost always correlated exactIy with the reduction in afferent input. Only in a few cases, when the responses were recovering at least one hour after giving a large dose of STX, was there some evidence of reflex depression due to intraspinal action of the toxin. The long intraspinal pathways were unaffected by the toxins until the dose rose to about 15 pg/kg or more. Arterial bloodpressure, already low in the acute spinal cat, was not affected by these doses of STX. However, it is possible that penetration of the toxin into the CNS was impeded by the low bIood pressure. It is difficult to reconcile these conflicting publications. All that can safely be said at present is that central depressant actions of these toxins appear to be unimportant compared to their peripheral effects, unless the toxins are administered direct into the CNS. When they are applied topically, or injected into the cerebral ventricles, then they undoubtedly produce dramatic effects at low doses. The work of Blankenship (1968) and of Crawford and Shibata (1968) has already been mentioned in this connection, as has the work of Borisoii et al. (1963) and Li (1963) on the respiratory center (Section VI, A, 2 ) . Koizumi et al. (1967) noted reduction of ventral root discharge or cortical evoked potentials by topical application of TTX g /m l). Frank and Pinsky (1966) depressed the responses to electrical stiniulation in an isolated slab of cortex by topical application of TTX ( 5 X lo-$ g/ml). Ochs and Ng (1966) and Ochs and Clark ( 1968) also employed topical application of TTX to the suprasylvian gyrus of cat cerebral cortex during an analysis of different components of the electrical responses to focal stimulation. All these workers have found 7TX to be an effective blocking agent, rendering CNS neurons inexcitable, when applied topically in adequate concentrations. Other workers who have used topical application of this poison to suppress neuronal activity in the CNS include Tarby et al. (1968), who obtained a reduction in EEG activity without change of cerebral impedance; Hafemann and Costin (1968) and Hafemann et al. ( 1969), who administered 0.001-0.250 pg by microinjection into the lateral geniculate body and hippocampus during a study of visual responses; Dudar and Szerb (1969), who found that it reduced ACh out-

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put from undercut slabs of cerebral cortex, and Richards and ter Keurs (1971) who used TTX to differentiate a mass excitatory postsynaptic potential from a compound action potential in the lateral olfactory tract. The paper by Hafeniann et al. (1969) includes a mathematical analysis of the diffusion of TTX in brain tissue, in which the diffusion coefficient was estimated to be 4 x cm2sec-’. After making a number of assumptions they calculated the density of TTX binding sites to be 120/pin? of lateral geniculate body neuron. A reduction in spontaneous neuronal activity and paralysis of evoked activity by topically applied TI’X and STX probably explains the effects seen by Rech et al. (1964), McIlwain (19671, Chan and Quastel (1967), McIlwain et u2. (1969), Bull and Trevor (1970), and Okamoto and Quastel ( 1970). Hubbard et al. ( 1967) used intracellular micropipette electrodes to record low-amplitude “noise” potentials in niotoneurons within the spinal cord of kittens. They found that topical application of TTX ( 3 X glml) to the segment of the cord from which the recording was being obtained led to a diminution of the “noise.” They concluded that in the absence of TTX their records showed “synaptic noise,” composed of random excitatory and inhibitory postsynaptic potentials arising from impulses impinging on the motoneuron from other neurons which were firing spontaneously, and that T T X was able to diffuse into the cord to abolish this spontaneous impulse activity in the other neurons. A few very small potentials persisted, at a low frequency, even after the TTX had acted. These may have been analogous to the m.e.p.p.’s seen in skeletal muscle, arising from the random release of single packets (“quanta”) of transmitter from presyiiaptic terminals. Blankenship (1968) and Blankenship and Kuno (1968) saw a similar reduction in “synaptic noise” in cat motonrurons, brought about by topical application of TTX ( 10-4-10-Gg/nil). In the isolated spinal cord of the frog, however, the “synaptic noise” was hardly affected, long after topical g /ml) had abolished imapplication of ?TX ( 2 x lo-: to 7 x pulse conduction in the lateral funiculus (Colomo and Erulkar, 1968). In this preparation it seems likely that most of the “synaptic noise” recorded from motoneurons is due to spontaneous random release of transmitter from presynaptic terminals (Katz and Miledi, 1963). S. Watanabe and Yuasa ( 1970) have recorded postsynaptic potentials in neurons within the cerebral cortex. Topical application of TTX ( 4 x lo-’ g l m l ) abolished most of this neuronal activity, with the rather surprising observation that it depolarized neurons in the visual cortex. They claimed that TTX caused a decrease in excitatory postsynaptic potentials without affecting the inhibitory postsynaptic potentials, but their published recordings are not clear in this respect.

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Vomiting is a frequent occurrence in TTX poisoning (see Kao, 1966; Ogura, 1971) but not in paralytic shellfish poisoning. The emetic action of TTX appears to be one of the few genuinely central effects, because in dogs it follows i.v. administration of 0.2-0.4 pg/kg within 2 5 min. Subcutaneous administration of 1 pg/ kg also regularly causes vomiting in conscious dogs, although this emetic response has a latency of 12-20 min, due to slower absorption of the toxin from the S.C.site. Emetic responses are abolished by ablation of the chemoreceptor trigger zone in the medulla (Hayama and Ogura, 1963) and are usually abolished by anesthetization of experimental animals (Murtha et al., 1958, but see Lipsius et al., 1968). Monkeys also vomit (Horsburgh et al., 1940). Cats are a little less sensitive to the emetic effects of intravenously administered TTX, but in other respects, Borison et al. (1963) obtained results similar to those of Hayama and Ogura (1963). Rather surprisingly however, vomiting was infrequent when the toxin was administered directly into the cerebral ventricles or cisterna. Hypothermia follows administration of TTX to cats. Doses of 5 pglkg i.v. cause body temperature to fall 1-2"C, as well as causing behavioral disturbances. A similar fall in temperature follows an intraventricular dose of 0.05 pg, and 0.2 pg by this route causes the temperature to fall more than 5"C, with concomitant nystagmus, loss of righting reflex, and loss of pupillary reflexes. After 1-2 hours the body temperature begins to recover, but shivering is not seen (Borison et al., 1963). VII. Summary and Concluding Remarks

It has been the intention in this review to demonstrate how TTX has become an established agent for neurobiological research, following the detailed analysis cariied out by many workers of its actions on electrically excitable membranes. In most respects, there have been fewer comparable studies of STX, but those that have been done suggest that STX could almost invariably be used for the same purposes for which TTX is now employed, and that STX has a few properties that give it significant advantages over ?TX for certain types of experiment. Unfortunately, there is some doubt a t present whether or not STX will contiiiue to be available. If the supplies of STX can be maintained, it will be assurcd of an important future in neurobiology. Most of the experimental analyses of the actions of these toxins in blocking the early transient channels in nerve and muscle cell membrane have been carried out using invertebrate giant fibers. These tissues, such as squid and lobster axons, offer obvious advantages to an experimenter wishing to use voltage clamp techniques or to inject material

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intracellularly. However, this cmphasis on invcrtcbratc preparations has meant that relatively little biophysical analysis has been carried out on mammalian tissues. Such information that is available suggests that the conclusions derived from squid giant axons, etc., is applicable to vertebrate nerve fibers, but more direct confirmation ought to be obtained. Techniques are now available that allow single vertebrate myelinated nerve fibers to be voltage clamped at a node of Ranvicr. These techniques have already been used to study the actions of the toxins on frog sciatic nerve fibers. Single mammalian myclinated axons can be dissected relatively easily from the roots of the spinal cord, where there is little connective tissue, and voltage-clamp experiments ought to be carried out on these axons to obtain confirmation of the action of TTX and STX on mammalian nerve fibers. The extraordinary resistancc of the nerves of Tetraodon puffer fish and Turicha newts to TTX remains a fascinating mystery. Little work has yet been done to try and explain this resistance. Two possible explanations come to mind. Either the gates to the sodium channels in the axolemma lack binding sites for TTX, or else there are special permeability barriers around these nerves. The resistance of the newt and puffer fish nerves could perhaps be clue to special permeability barriers, lacking in other vertebrate nerves. STX might be able to penetrate these barriers to some extent; it does appear to differ from TTX in its ability to diffuse through mesothelium and layers of connective tissue (see, for instance, the differences between the dose-survival times after i.p. administration of the toxins, and differences between their effects on motor nerve terminals at the neuromuscular junction), On the other hand, the resistance might be due to the T T X failing to bind at the sodium channels, if the molecular configuration of the membrane at the entrance to the channels were different in these nerves. For example, the gate to these channels might resemble the gate to those channels which normally pass calcium current in tissues such as barnacle muscle, which is also unaffected by TTX. The action potentids in Tetraodon and Taricha nerves are known to be sodium-dependent. Examples are known of sodium-dependent action potentials in other tissues ( e.g., cardiac and some smooth muscle), which are resistant to TTX. The sodium-dependent action potentials in cardiac and smooth muscle also appear to be resistant to STX, although there is very little information on this yet. The Tetruodon and Taricha nerves, however, are less resistant to STX than they are to TTX, although they will resist concentrations of STX considerably higher than are needed to block other vertebrate nerves. Perhaps the gates to the sodium channels in these puffer fish and newt nerves have a molecular configuration somewhat intermediate between the sodium

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channel gates of other vertebrate nerves and the calcium gates of resistant tissues. The difference between the sodium gates of most vertebrate nerves on the one hand and newt and puffer fish nerves on the other, might be sufficient to prevent TTX from binding to the resistant channels, yet insufficient to prevent STX from binding at relatively high concentrations. If TTX could be demonstrated within the nervous tissues of these puffer fish and newts it would suggest that these tissues are inherently different in their sodium channels, rather than relying on special permeability barriers to exclude the TTX that is present in such high concentration in other parts of the animal. It is probable that this would, in fact, be found to be the case, because Tetraodon muscle is known to contain TTX, the muscle action potentials are sodium-dependent, and are resistant to the toxin. Further work is also needed to resolve the current controversies about possible effects on the CNS and on vascular smooth muscle. The points for and against the opposing hypotheses in these two fields have been mentioned in the appropriate sections of this review, but for full details readers will have to refer to the publications cited. It is to be hoped that some readers will feel encouraged to try and resolve these controversies. In the opinion of the present author, T T X and STX cross the blood-brain barrier with such difficulty that central actions are unimportant, compared with the peripheral effects, if the toxins are administered into the circulation either directly or indirectly. Only when injected direct into the CNS or the cerebrospinal fluid are they able to produce unequivocal central effects. At the time of the outbreak of paralytic shellfish poisoning in Britain in 1968 some clinicians felt that their cases showed signs of central effects, not previously reported in cases of STX poisoning. In this 1968 outbreak most of the poison was one different from STX, being derived from a bloom of Gonyauhx tamarensis. It is conceivable that this new poison might cross the blood-brain barrier more easily than STX does, but much caution is needed before coming to a firm conclusion, because the history of research on TTX and STX is littered with examples of effects once ascribed to central actions, which are now known to be due to the peripheral actions of these toxins. It is to be hoped that this review will have given readers an overall introduction to those areas of neurobiology in which tetrodotoxin and saxitoxin have been used to further our understanding. The list of references cited here is by no means exhaustive. No attempt has been made to discuss the clinical aspects of paralytic shellfish poisoning or puffer fish poisoning. Others, especially Halstead ( 1965, 1967) and Kao ( 1966), have included a discussion of the clinical aspects in their writings. These and other reviewers have covered the early work in considerable

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detail, so the emphasis in the present review has been on work published during recent years. The toxins, especially TTX, are being inentioned so often in current publications that it has been impracticable to review the field exhaustively. I offer my apologies to those authors whose contributions to the field could not be discuss6d because of lack of space. Several reviews have been published recently in which one or both toxins have been discussed. The ones by Halstead and Kao, mentioned above, and one in French by Cheymol and Bourillet (1966), are excellent general reviews that cite many references. Shorter general reviews have been published by Mosher et al. (1964), Janiszewski and Skubalanka (1967, in Polish), Cheng and Li (1969), Evans (1969c), Fuhrman et al. (1969), Hashimoto and Kamiya (1970, in Japanese), Scheuer ( 1970), and Cheymol and Bourillet ( 1972). Semipopularized articles include those by Fuhrmaii (1967), Cheymol ( 1970, in French), and Evans (1970b). A book on neuropoisons edited by Simpson (1971) includes useful chapters on TTX and STX by Ogura, Schantz, Dettbarn, and Gage, Some reviews are more specialized in that the emphasis is upon particular research problems. Woodward (1964) and Goto et al. (1965) deal with the chemical structure of TTX. Moore and Narahashi (1967) and Hille (1970) deal with the role of TTX and STX in terms of membrane biophysics. Russell (1967, 1969) and Lane (1968) give considerable zoological information, while the reviews of Schantz ( 1967, 1970, 1971a) pay particular attention to the biocheniistry of STX and its origin in plankton dinoflagellates. Public health problems posed by TTX and STX and related poisons are dealt with in reviews by McFarren et al. ( 1960), Clark ( 1968), McFarren ( 1971) , and Prakash et al. ( 1971) . ACKNOWLEDGMENTS

I would like to express my gratitude to all those who sent me reprints, photocopies and prepnblication copies of their papers, to those who gave additional information in letters, much of which is cited as “personal communications,” and to those who gave peiinission for their puldished material to he used in this review. I am particularly indebted to my assistant Miss Jeanie Tnlloch for all her careful work in collecting and indexing material and typing a large pait of the manuscript. REFERENCES

Abe, Y. ( 1971). J. Physiol. (London) 214, 173. Aceves, J., and Erlij, D. (1967). Nature (London) 215, 1178. Adrian, R. H. ( 1969). I t i “The Molecular Basis of Membrane Function” ( D . C. Tosteson, ed. ), p. 249. Prentice-Hall, Englewood Cliffs, New Jersey. Adrian, R. H., Costantin, L. L., and Peachey, L. D. (1969). J. PhysioZ. (London) 204. 231.

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Note added in proof: Recent experiments have added fresh evidence to support Kao's hypothesis that TTX can cause vasodilatation by a direct relaxant action on arteriolar smooth muscle. These experiments have shown that in a perfnsed cat spleen the amount of catecholamine released on stimulation of the splenic nerve is not reduced by the addition of TTX 2 ng/ml to the perfrision saline, a concentration equivalent to an i.v. dose that causes systemic hypotension and vasodilatation in the gracilis muscle. See Kao and McCullough (1972), J. PhurmucoZ. Erp. Ther. (in press).

THE INHIBITORY ACTION OF 7-AMINOBUTYRIC ACID, A PROBABLE SYNAPTIC TRANSMITTER By Kunihiko Obata Department of Pharmacology, Faculty of Medicine, Tokyo Medical and Dental University, Tokyo, Japan

Introduction . . . . . . . Inhibitory Synaptic Transmission . . . Methods of Drug Administration . . . Electrical Changes in Neuronal Membranes Antagonism by Convulsants . . . . A. Strychnine . . . . . . . B. Picrotoxin . . . . . . . C. Bicuculline . . . . . . . VI. Chemistry of Synaptic Inhibition . . . VII. Conclusion . . . . . . . . . . . . . . References

I. 11. 111. IV. V.

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167 168 171 173 178 178 179 180 181 183 184

I. Introduction

Since the discovery in 1950 that 7-aminobutyric acid (GABA) is present in a high amount in the mammalian central nervous system (CNS) (Awapara et al., 1950; Roberts and Frankel, 1950; Udenfriend, 1950), the significance of GABA in nervous function has been investigated extensively (cf. Elliott and Jasper, 1959; Roberts, 1960; Curtis and Watkins, 1965; Kravitz, 1967). It has become clear that GABA, on the one hand, occupies a unique position in the metabolic pathways of the brain and, on the other, powerfully affects the electrical activities of nerve cells. As its known effects on neuronal activities are all inhibitory, the hypothesis has been put forward that GABA functions physiologically as a transmitter substance released from presynaptic nerve fibers at inhibitory synapses. Initially identity in the action of GABA and the neural transmitter could not be shown in the spinal cord in the pioneer work of Curtis et al. (1959), although GABA was established as an inhibitory transmitter in the Crustacea (Kravitz et al., 1963; Takeuchi and Takeuchi, 1965; Otsuka et al., 1966). It is only recently that experiments have been extended to a variety of neurons, and the above hypothesis is now considered plausible in many cases. Intra167

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cellular studies, for example, on Deiters’ neurons (Obata et al., 1967, 1970b; ten Bruggencate and Engberg, 1971) and cortical neurons (Knijevib and Schwartz, 1967; Dreifuss et al., 1969) have shown close similarity in the electrical changes induced by the synaptically released transmitter and the exogenously administered GABA. Both synaptic inhibition and GABA action on many neurons are blocked by picrotoxin and bicuculline but not by strychnine (see Table I, p. 170, for references). Specific distribution of GABA and its synthetic enzyme in inhibitory pathways and the release of GABA into perfusates during synaptic inhibition have been demonstrated, indicating involvement of GABA in the inhibition. It has been suggested that the principal inhibitory transmitter in the spinal cord is glycine rather than GABA (Wemian et al., 1968; Curtis et al., 1968a,b). In this essay the inhibitory actions of GABA and related amino acids on single cells of the mammalian CNS will be discussed in relation to their possible role as synaptic transmitters. I f . Inhibitory Synaptic Transmission

Although several extensive reviews have been published on synaptic transmission (e.g., Eccles, 1964; McLennan, 1970), it may be useful in the present context to review the characteristics of inhibitory transmitter action before the actions of GABA and the transmitters are compared closely. In postsynaptic inhibition, a change in membrane potential, usually a hyperpolarization (inhibitory postsynaptic potential, i.p.s.p. ) and an increase in the membrane conductance are the most obvious changes induced by a transmitter in the postsynaptic cell. These changes tend to counteract the depolarization arising ( 1 ) by excitatory synaptic action ( 2 ) due to antidroniic conduction of the action potential or ( 3 ) by artificial application of current; they, therefore, inhibit generation of the action potential at the initiaI segment of a neuron. The size and polarity of the i.p.s.p. are sensitive to changes in the resting potential, and the i.p.s.p. changes from a negative-going to a positivegoing potential change when the membrane is hyperpolarized beyond a certain value. This indicates that the transmitter works on the membrane by increasing the permeability to some species of ions; as a result, the membrane potential shifts toward a certain level (null potential for i.p.s.p., E , I ) . I, ) which is determined by the equilibrium potentials and membrane permeabilities of the relevant ions. Chloride is one of the ions whose permeability commonly increases during inhibition. Electrophoretic injection of Cl- into a neuron from an intracellular electrode can in such cases move E,,,, in the depolarizing direction, even to a

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level more positive than the resting potential. This reversal of the i.p.s.p. can be induced by injection of not only C1- but also other anions to which the activated postsynaptic membrane is permeable. Shemngton (1906) suggested blockade of inhibition as the mechanism of action of certain convulsant drugs, and strychnine, picrotoxin, and bicuculline have since proven very useful for the study of inhibitory transmission. As seen in Table I, many of the inhibitory synapses of the mammalian CNS can be divided on this basis into two categoiies: one is blocked by picrotoxin and bicuculline but not by strychnine; the other shows the converse effects. In addition to the inhibition of spinal motoneurons in Table I, centrifugal inhibition of the inner ear by the olivo-cochlear bundle (Desmedt and Monaco, 1960) and inhibitions of hypoglossa1 motoneurons ( Morinioto et al., 1968) and trigeminal niotoneurons (Kidokoro et al., 1968) are strychnine-sensitive. The spinal motoneurons have, in addition, stiychnine-resistant inhibition which is induced by stimulation of bulbar reticular formation (Llinls, 1964) and by stretching muscles ( Kellei-th and Szumski, 1966). Cerebellar Purkinje cells inhibit not only Deiters’ neurons but also some of the other vestibular neurons as well as those of intracerebellar nuclei. Each of these cases appears to have the same pharmacological properties. For example, picrotoxin blocks the inhibition 011 the superior vestibular nucleus (It0 et al., 1970b); reverberatory activity appears between the pontine and the intracerebellar nuclei after injection of picrotoxin (Tsukahara et al., 1971); and bicuculline depresses disfacilitatory effects of the cerebellum on spinal reflexes (Huffman, 1971)-these effects may be due to blockade of Purkinje cell inhibition on intracerebelh and Deiters’ neurons. Intravenous injection of subconvulsive or just-convulsive doses of strychnine and bicuculline blocks inhibitory transmission but usually a much higher amount of picrotoxin is necessary for the development of effects on inhibition. In spite of their relatively selective action on inhibitory synapses, these substances, in a higher dose, can alter membrane excitability and even affect autonomic functions such as blood pressure. Therefore, interpretation of the effects of intravenously applied drugs must be made cautiously. In several neurons the blockade of inhibition by these convulsant drugs has been demonstrated with focal electrophoretic application. Detailed intracellular studies have been made of the effects of strychnine on the electrical properties of spinal motoneurons (Curtis, 1962; Larson, 1969). The amount of strychnine just adequate to depress inhibition does not affect the resting potential, the threshold for spike generation, or the excitatory postsynaptic potential (e.p.s.p.). Tetanus toxin resembles strychnine in depressing the strychnine-sensitive inhibition (Brooks et aE., 1957) without effect on the

INHIBITORY SYNlPSES

WITH

TABLE I GABA OR GLYCINE AS

PROBABLE

Blocked by Postsynaptic neurons Spinal motoneurons Cuneate neurons Deiters' neurons Purkinje cells Granule cells of the cerebellum Oculomotor neurons Auditory neurons of the inferior colliculus Relay cells of the venhobasal thalamus Mital cells of the olfactory bulb Hippocampal pyramidal cells Cortical cells of the cerebrum

Presynaptic inhibitory neurons or pathways

, interneurons . cells Renshaw Via median nerve, pyramidal tract, etc. Purkinje cells Basket cells Golgi cells From superior vestibular nucleus Curtailing of spike discharge during sound stimulation Recurrent inhibition

Strychnine

Picrotoxin

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TRaNSMITTER

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+c

GABAu GABAz GABAaa

Bradley et al. (1953). b Eccles et al. (1954). c Curtis et al. (1970a). Curtis et al. (1968a). a Kelly and Renard (1971). f Galindo et al. (1967). Obata et al. (1967). Obata et al. (1970b). ' Curtis el al. (1970b). i ten Bruggencate and Engberg (1971). k Andersen et al. (1963). I Bisti et al. (1971). m Kawamura and Provini (1970). Magni et al. (1970). Ito et al. (1970a). p Obata and Highstein (1970). * Watanabe and Simada (1971). T. Watanabe (1971, personal communication). Duggan and McLennan (1971). Green et al. (1962). u Nicoll(l970). Felix and McLennan (1971). =' McLennan (1971). Curtis etal. (1970~).Y Crawford el al. (1966). Krnjevid etal. (1966). aa Dreifuss etal. (1969). Q

Q

O

7

INHIBITION BY GABA

171

strychnine-resistant inhibition (Krnjevik et al., 1966; M. Ito et al., cited in Obata et al., 1967). However, it does not block the action of externally applied glycine, a putative transmitter in the spinal cord (Davidoff et al., 1967; Curtis et al., 1968a,b) and is presumed to act on the presynaptic terminals by preventing transmitter release (Curtis and de Groat, 1968). Presynaptic inhibition refers to the decrease in release of excitatory transmitter from presynaptic terminals when they are affected by another transmitter, Although the mechanism has not been fully worked out, depolarization and excitability increase of the presynaptic fibers ( as well as the dorsal root potential and dorsal root reflex) can be detected concurrently with depression of the e.p.s.p. Picrotoxin antagonizes presynaptic inhibition (Eccles et al., 1963; Shende and King, 1967; Baniia and Jabbur, 1969) and recently bicuculline was shown to have the same effect (Curtis and Felix, 1971) . Electrically operated inhibitory synapses ( Furukawa and Furshpan, 1963) have not been demonstrated in the mammalian CNS. I l l . Methods of Drug Administration

When drug action on single neurons of the CNS is studied, several problems must be taken into consideration. Synapses are distributed widely on the membrane of the soma and dendrites. Systemic administration, therefore, has the advantage over microapplication of applying a substance more uniformly to the whole neuron and its synapses. For example, strychnine-blockade of the inhibition of spinal neurons ( Curtis et al., 1968a) and picrotoxin-blockade of the Purkinje cell inhibition of Deiters’ neurons (Obata et al., 1970b) are obtained more easily by intravenous injection than by microinjection. For systemic administration, a substance must be able to permeate the blood-brain barrier. The barrier is known to be impermeable to GABA (van Gelder and Elliott, 1958; Kuriyama and Sze, 1970). It is usually difficult to determine whether the changes induced by systemically administered substances are direct effects on the neuron under study or are secondary to changes in activities of other synaptically connected neurons. Microelectrophoretic administration of drugs to central neurons was introduced by Curtis and his collaborators (Curtis and Eccles, 1958; Curtis et al., 1959) and has the advantage of permitting quantitative and repeated administration of several kinds of substaiices to a small area while avoiding difficulties associated with the blood-brain barrier. A glass capillary with tip diameter a few micrometers in size is filled with a drug solution and current passage through it ejects the drug from the capillary into the medium. Ions are transported iontophoretically and nonionic substances are carried by electroosmotic flow of the

172

KUNIHIKO OBATA

solution. The amount of the released substance in each experiment can be estimated from the amount of charge flow if the ratio of the substance released to charge passed is known in advance. This “apparent transport number” for GABA was measured with pipettes which were filled with radioactive GABA and soaked in saline; it was 0.22 (moles/Faraday) for GABA cation in GABA-CI solution (1M , pH 2.5), 30.0 for electroosmotic release of GABA zwitterion from 1M solution with pH 7, and 0.04 for GABA anion in GABA-Na solution ( p H 11.5-12) (Obata et al., 1970a). However, when a pipette is inserted into tissue, the tip is sometimes clogged and its electrical resistance changes. This may modify significantly the transport situation, especially if the clogging material is electrically charged. In fact, transport of norepinephrine into the brain tissue is much lower than into saline (Hoffer et al., 1971a). Therefore, it may be appropriate simply to express the dose in terms of total current, without attempting a correction for transport number. After a substance is released from a pipette, it changes its ionic state according to the pH of the tissue; for example, GABA, released as a cation, becomes a zwitterion within the limit of the buffering capacity of the tissue. A broad intercellular space for rapid diffusion will be produced by the rather coarse tip of a pipette and the dihsion should be rapid even through intercellular spaces 200 A in width (Kuffler and Nicholls, 1966). Therefore, after the cessation of current, the substance will disappear rapidly from the neighborhood, if any special binding mechanism does not exist in the tissue. It is possible on excised tissue or tissue-cultured cells to administer the substance to only a small part of a single cell’s membrane by fine adjustment of the pipette under visual control (del Castillo and Katz, 1955; Takeuchi and Takeuchi, 1964, 1965; Harris et al., 1971) but this advantage cannot be utilized in the CNS. Usually the recording electrode is fixed immovably to the drug pipette. For extracellular recording, the orifice of all barrels are at the same plane ( a multibarrel pipette consisting of a recording barrel and 1-6 drug barrels) and for intracellular recording and concurrent extracellular drug application, the recording electrode extends beyond the drug barrels by 10-60 pm ( a coaxial or parallel pipette). About ten times more current is usually necessary to get comparable effects using coaxial or parallel electrodes than with multibarrel electrodes, since the drug barrels of the former cannot come so close to the cell. In electrophoretic application, an increasingly larger area above the minimum receptive area will be affected as the drug concentration is increased. Therefore, it must be remembered that changing doses means not only changing the concentration at each receptor but also the number of receptors involved. The meaning of the d o s e

INHIBITION BY GABA

173

response curve is further complicated when the drug action is gauged as a change in spike discharge rather than more directly as a change in membrane conductance (Curtis et al., 1971 for detailed discussion). Studies of antagonism between two drugs are complicated in electrophoretic-application experiments, because the areas affected by two drugs may not be of the same extent. For example, blockade by picrotoxin of GABA action on Deiters’ neurons is overcome by a large amount of GABA but this does not necessarily demonstrate competitive interaction of these substances (Obata et al., 1970b). The effect of a substance administered electrophoretically is usually considered to result from a direct action on the cell whose activity is recorded. But sometimes the actions on other cells can be detected. Glutamic acid (von Baumgarten et at., 1963) and DL-homocysteic acid (Felix and McLennan, 1971; McLennan, 1971), strong excitants on other neurons, depress the mitral cells of the olfactory bulb. This is a manifestation of excitation of the inhibitory granule cells which are present abundantly around the mitral cells and inhibit them. In the cerebellar cortex, electrophoretically released substances can diffuse to and affect cells 200300 pm distant from the drug pipette and with response-latencies of 15-50 seconds (Crawford et al., 1966). IV. Electrical Changes in Neuronal Membranes

It is generally agreed that aspartic acid, glutamic acid, homocysteic acid, and related substances excite mammalian central neurons. Conversely, glycine, p-alanine, GABA, and closely related substances are inhibitory (Curtis and Watkins, 1960, 1965). In almost all of the experiments the substances are administered electrophoretically onto the single neuron from micropipettes filled with concentrated solutions (ca. 1M). With extracellular recording, the spike discharge of a single cell can be recorded stably for a long time, whether the discharge is spontaneous or generated by one of the above excitatory substances; the inhibitory substances effectively depress spike generation ( Fig. la,b) . Among the inhibitory amino acids, GABA and glycine deserve primary attention because of their high potency and presence in large quantities throughout the CNS. The potencies of these substances can be compared in terms of the dectrophoretic currents required for obtaining comparable effects and, as shown in Table I, the relative potency of GABA to glycine is different among neuron types. However, the potency of a substance cannot be taken, by itself, as indication that it is a transmitter. With intracellular recording, the membrane is usually observed to be hyperpolarized (by up to 10 mV) during administration of the

174

e

KUNIHIKO OBATA

’’b

-1-

+wh

t

urnsec

u

2 rnV

1: 5 rnsec

u rnsec

LLLLLLL

msec

FIG. 1. Inhibitory action of GABA on Deiters' neurons of the cat. a and b, Unit spike discharges recorded extracellularly with one barrel ( 5M NaCl) of a multibarrel pipette. DL-Homocysteic acid ( DLH ) was administered with a pulse of 150 nA for 90 msec from the second barrel (0.2 M DLH-Na, pH 7.5); a, without, and b, during, application of GABA. GABA was ejected by a steady current of 12 nA from the third barrel ( 1M GABA, pH 7 ) (Obata et al., 1970b). c, Membrane potential recorded intracellularly with an inner barrel ( 2 M citrate-K) of a coaxial electrode. GABA was administered extracellularly with cationic current of 500 nA from an outer barrel ( 1M GABA-Cl, pH 2.5). This electrode had an artifact of 3 mV in the depolarizing direction during the GABA current, as it was also recorded extracellularly in d, and the dotted line in c indicates the net membrane potential after correcting the artifact. e and f, h t i d r o m i c action potential of the same cell as in c produced by stimulation of the spinal cord. Stimulus intensity was adjusted to the threshold and in about half of superimposed traces there was a failure of spike generation at the spinal cord. e, Before, and f, during, GABA application (500 nA). Left, the horizontal bars show the control level of spike height. An increase of 2 mV is seen in f. The oblique arrows point to the onset of after-depolarizations. In f, spike conduction to the cell soma was sometimes blocked. Right, recorded with higher amplification and slower sweep velocity to show the after-hyperpolarizations of action potential ( at the upward arrows). g and h, Intracellularly recorded e.p.s.p.'s induced by stimulation of the cerebellum and obtained with a coaxial electrode; g, Before, and h during, administration of GABA with a current of 500 nA from the outer barrel ( 1 M GABA-CI, pH 2.5). In g, action potentials were sometimes generated at the peak of the e.p.s.p. and i.p.s.p. followed the e.p.s.p. i-k, Cerebellar-induced i.p.s.p.'s recorded with a coaxial electrode; i, before, and j, during, GABA application (current, 500 nA; 1 M GABA-Cl, p H 2.5). k, Recorded just external to the cell membrane in the absence of GABA. Arrow indicates the field potential of the presynaptic spike (Obata et al., 1967).

HIB BIT ION BY GABA

175

inhibitory substances ( Fig. l c ) . Electrical conductance, measured by passing current through the neuronal membrane, is found to increase markedly during electrophoretic drug application; the maximum conductance increase by inhibitory amino acids is more than threefold for spinal motoneurons (Werman et al., 1968), up to sixfold for Deiters’ neurons (Obata et al., 1970b) and two- to sixfold for cortical neurons (Dreifuss et al., 1969). The hyperpolarization by amino acids is sensitive to the background level of membrane potential and changes over to a depolarization when the background level is experimentally made sufficiently negative. This “change-over” or “reversal” level can be compared with that for the neurally evoked i.p.s.p. Current-voltage relationships during current passage through the membrane are shown for a Deiters’ neuron in Fig. 2. Less-steep slopes during the i.p.s.p. ( 0 ) and GABA administration ( A) as compared to the resting membrane (0) indicate an increase in conductance. The membrane potential at which the current-voltage line measured during the i.p.s.p. or GABAaction intersects with the resting value indicates the respective reversal potential. It can be seen that reversal potential for GABA is at the same level as that for the i.p.s.p. Sometimes there is a discrepancy not exceeding a few millivolts but this is not considered significant, since the transmitter probably affects a greater area of neuronal membrane than does the administered substance (Curtis et ,d., 1968a). Similar effects were obtained in comparisons among glycine, p-alanine, GABA, and 8-aminovaleric acid (Curtis et al., 196th; ten Bruggencate and Engberg, 1971). Although Kelly and Krnjevib ( 1969) indicated statistically significant differences between GABA and glycine reversal potentials in the cortical neurons, exact comparison is problematical as the effect of glycine on these neurons is difficult to detect. Intracellular injection of various ions has effects on the GABA- or glycine-induced potential very similar to those on the i.p.s.p.; injection of C1- and several other anions reverses the amino acid-induced hyperpolarization into a depolarization (Werman et al., 1968; Curtis et al., 1968a; ten Bruggencate and Engberg, 1971). The time cowse of recovery from the effects of ion injection are surprisingly similar for the i.p.s.p. and amino acid-induced potential in spinal motonemons (Curtis et al., 1968a). In summary, changes in electrical properties induced by GABA and related substances are similar to those during the transmitter action in every respect thus far investigated. Concomitant with the hyperpolarization and conductance increase caused by inhibitory drugs, action potentials and synaptic potentials are depressed. The e.p.s.p. (Fig. lg,h) is depressed as a result of the conductance increase although hyperpolarization will have a slight facilitatory effect on the size of the e.p.s.p. If GABA and glycine act

176

KUNIHIKO OBATA

d

---

f

- LOI

- 20 I

I

+20

0 I

I

I

I

nA I

- 2.3

-16.7

L

+5.0

20 mV+ - 1

-

-

b

-2.5

-18.5

-

50 msec

4

- 110

FIG.2. Current-voltage relationships of a Deiters' neuron. A parallel pipette was used consisting of a double-barrelled intracellular electrode ( 2 M citrate-K, one for recording membrane potential and the other for passing current through the cell membrane) and an extracellular pipette filled with 1M GABA (pH 7 ) . a-c, Specimen records for D. During the period shown by the uppermost bar, polarizing currents were passed. Their intensity was indicated on each record in nA, with plus sign ( + ) representing outward currents through the membrane and minus ( - ) inward. The cerebellum was stimulated at the arrow to induce an i.p.s.p. duting each sweep in a and b; b, during application of CABA ejected by a current of 270 nA. c, Recorded extracellularly after the experiment, shows the artifacts due to interbarrel coupling; these were corrected for in plotting points in d. d, Abscissa, hleasured just before cerebellar cnrrents. Ordinate, membrane potential level. 0, stiinulation in a; 0 , a t the peak of the i.p.s.p. in a; A, as for 0 hut during GABA adininistration in I, (Obata ct al., 19701~).

on presynaptic terminals diminishing the release of the excitatory transmitter, as in the presynaptic inhibition (see later), this might contribute to depression of the e.p.s.p. Both hyperpolarization and conductance increase contribute to a depression of the i.p.s.p. (Fig. li,j,k). Furthermore if these amino acids react with the same receptors that the inhibitory transmitter reacts with, competitive interaction will also help to decrease the i.p.s.p. Usually generation of action potentials is suppressed but, when the dose of GABA is not sufficient to block, the size of the spike increases with concomitant increase in the after-depolarization and decrease in the after-hyperpolarization (arrows, Fig. le,f ) ; these are changes expected during membrane hyperpolarization.

INHIBITION BY GABA

177

During prolonged administration (30 sec. to several minutes), the effect of GABA and related amino acids sometimes decreases gradually. This does not necessarily indicate desensitization of the receptors as seen for the acetylcholine receptor at the neuromuscular junction (Katz and Thesleff, 1957). Prolonged increase in ionic conductance by the amino acids results in changes in intracellular ionic composition and accordingly decreases the difference between the resting potential and the “equilibrium” potential for the amino acid action (Curtis et d., 1968a). Interaction between GABA and glycine has not been investigated. GABA does not have any effect on nerve fibers of the ventral and dorsal roots even at the very high concentrations of 10-100 mg/ml (Curtis et al., 1959). However it has a marked effect on the afferent nerve terminals in the spinal cord and cuneate nucleus where presynaptic inhibition is known to be well-developed (Eccles et al., 1963; Curtis and Ryall, 1966; Galindo, 1969). In both regions GABA, applied either topically to the external surface of the cord or medulla, or electrophoretically near the axons, facilitates the dorsal root reflex just as seen in presynaptic inhibition. However the excitability of the terminals of the afferent fibers seems to decrease during electrophoretic application of GABA in contrast to the situation during presynaptic inhibition (Curtis and Ryall, 1966). Experimental difficulties prevent comparison of GABA and transmitter actions on the presynaptic terminals in mammals, while in the Crustacea it has been demonstrated that GABA decreases the transmitter release from the excitatory synapses just as in presynaptic inhibition (Takeuchi and Takeuchi, 1966). A depolariziiig action of GABA has been suggested in the sympathetic ganglion cells ( d e Groat, 1970b), although it has not been \howl1 whether the action is on the presynaptic nerve teiminals. It is very interesting to know whether sensitivity to the amino acid5 is restricted only to subsynaptic Inembrane or other specialized area, or exists generally on the neuronal membrane of the soma, dendrite, and axon terminal. Conversely it would be very useful to know whether sensitivity to a transmitter substance necessarily implies the presence of synapses of the corresponding type. In noimally innervated cells, known or putative transmitters appear to work only on the synaptic area: acetylcholine on skeletal muscles (del Castillo and Katz, 1955) and on parasympathetic ganglion cells ( Harris et al., 1971), glutamic acid (Takeuchi and Takeuchi, 1964) and GABA (Takeuchi and Takeuchi, 1965) on crustacean muscles. The molluscan neurons are exceptional in that the cell body lacks synaptic contact but is still sensitive to the putative transmitter, acetylcholine (Tauc and Gerschenfeld, 1962). The whole of the surface membrane is sensitive to acetylcholine in denervated muscles

178

K U N W K O OBATA

(Axelsson and Thesleff, 1959), in denervated neurons (Kuffler et aZ., 1971) and in cell bodies of tissue-cultured neuroblastoma which is presumed to have originated from sympathetic neurons (Harris and Dennis, 1970). V. Antagonism by Convulsants

In pathways so far investigated in the mammalian CNS, inhibitory transmission is blocked selectively either by strychnine on the one hand, or by picrotoxin and bicuculline on the other. These substances would, therefore, be expected to interfere with the actions of putative transmitters. Present evidence indicates that the action of GABA is blocked by picrotoxin and bicuculline but not by strychnine, while the converse holds for glycine. A. STRYCHNINE The depression of spinal motoneurons by GABA is not affected by strychnine (Curtis et al., 1959), and this has been taken to indicate that GABA is not a transmitter of the strychnine-sensitive I, and Renshaw inhibitions. Complementing these observations are recent investigations which have demonstrated that strychnine blocks the actions of glycine but not of GABA on several types of neurons: spinal motoneurons (Curtis et al., 1968a), spinal interneurons (Curtis et al., 1968b), cortical neurons (Curtis et al., 1968b), cuneate neurons (Galindo, 1969), Deiters’ neurons (ten Bruggencate and Engberg, 1969a, 1971; Obata et al., 1970b), oculomotor neurons ( Obata and Highstein, 1970, Fig. 3 ) , reticular neurons ( Hiisli and TebEcis, 1970), parasympathetic preganglionic neurons (de Groat, 1970a), mitral cells of the olfactory bulb (Nicoll, 1970; Felix and McLennan, 1971) and relay cells of the ventrobasal thalamus ( Duggan and McLennan, 1971) . In most of these experiments, spike potentials of single cells were recorded extracellularly with one barrel of a multibarrel pipette while drugs were administered electrophoretically from other barrels; depression of the spike frequency by GABA and glycine and its modification by strychnine were studied. It was shown on spinal interneurons by Curtis et al. (196813) that a number of convulsants with strychnine-like structure, although less potent, also block the action of glycine but not of GABA. Antagonism by strychnine of the actions of other depressant amino acids was also studied; p-amino acids such as p-alanine and taurine are strychnine-sensitive and 7- and higher @-aminoacids are strychnine-resistant (Curtis et al., 196813). In sufficiently high concentrations, strychnine is effective in reducing the inhibition of spinal neurons by GABA; however, at these levels the actions of norepineph-

179

INHIBITION BY GABA Picrotoxin D

-

6 0 - GABA Glycine

0-

I

I

...-.

4‘40

0

4’50

Strychnine I

1

-

0

1-

FIG. 3. Effects of picrotoxin and strychnine on the inhibition induced by GABA and glycine in a rabbit oculomotor neuron. The barrels of a six-barrel pipette were filled, respectively, with 5 M NaCl, 1 M glutamate-Na, 1M GABA (pH 7 ) , 1 M glycine ( p H 7 ) , 4.5 mM picrotoxin in 165 mM NaCl and 50 mM strychnine nitrate. Abscissa, time after beginning of the experiment. Ordinate, frequency of spontaneous spike discharge recorded extracellularly with the NaCl barrel. Each substance was administered a t the indicated time. Currents used to eject substances: GABA, 23 nA; glycine, 38 nA; picrotoxin, 25 nA, and strychnine, 10 nA. Glutamic acid was not used in this experiment. Note that a sufficient amount of picrotoxin for complete block of the GABA action could not be ejected from this pipette (Obata and Highstein, 1970).

rine and dopamine are also interfered with. This may result from modification of membrane permeability by strychnine (see above; Curtis et al., 1971).

B. PICROTOXIN As is well-known, picrotoxin blocks the action of GABA in Crustacea (Florey, 1957; Grundfest et al., 1959) but comparable activity of this drug in the mammalian nervous system was not initially observed (Kmjevi6 et al., 1966; Curtis et al., 1968b). More recently, however, picrotoxin has been demonstrated to block the action of GABA but not that of glycine on cuneate neurons (Galindo, 1969), oculomotor neu-

180

K U N W K O OBATA

rons (Obata and Highstein, 1970, Fig. 3 ) , Deiters’ neurons (ten Bruggencate and Engberg, 1969b, 1971; Obata et al., 1970b), spinal interneurons (Engberg and Thaller, 1970), mitral cells of the olfactory bulb ( Nicoll, 1970), and neurosecretory cells of the hypothalamus ( Nicoll, 1971). Picrotoxin also depresses the depolarization by GABA of the superior cervical ganglion ( de Groat, 1970b). The effects of picrotoxin seem much more difficult to demonstrate than those of strychnine. The p H of the solution decreases gradually and at both acidic and alkaline p H s the potency of picrotoxin deteriorates rapidly (Davidoff and Aprison, 1969). Only some of picrotoxin pipettes tested were found to be effective (Engberg and Thaller, 1970; Obata et al., 1970b). The mechanism of action of picrotoxin in the crustacean muscles has been studied by Takeuchi and Takeuchi (1969). GABA and picrotoxin were superfused over the whole surface of muscle fibers and changes in membrane conductance were measured. The analyses of the doseresponse curves showed that picrotoxin blocks GABA in a noncompetitive manner. The action of GABA on presynaptic terminals of the cuneate nucleus was antagonized by picrotoxin in some experiments ( Galindo, 1969).

C. BICUCULLINE Very recently, a number of investigations have been made of the interference by bicuculline with the action of the inhibitory amino acids. In general it has been found that the alkaloid blocks GABA action without any detectable effect on glycine action in the following cases : spinal neurons ( interneurons and Renshaw cells), cortical cells, Purkinje cells (Curtis et al., 1970a), Deiters’ neurons (Curtis et al., 1970b), hippocampal pyramidal cells (Curtis et al., 1970c), ventrobasal thalamic cells (Duggan and McLennan, 1971), mitral cells of the olfactory bulb ( Felix and McLennan, 1971; McLennan, 1971), cuneate neurons ( Kelly and Renard, 1971), neurosecretory cells of the hypothalamus ( Nicoll, 1971), and medullary reticular neurons (TebEcis et al., 1971). Kmjevik (1971) has observed bicuculline blockade of the cction of GABA only in some of cortical cells he tested. P-Alanine is “glycine-like” in its sensitivity to strychnine blockade, but “GABA-like” is being blocked by bicuculline (Curtis et al., 1970a,b; Duggan and McLennan, 1971; Felix and McLennan, 1971) . The differential antagonism by the convulsant drugs demonstrates clear differences between the actions of GABA and glycine but does not necessarily indicate the presence of two types of receptors. It is still possible that these amino acids act on the same membrane sites but with a minute difference in their modes of action, distinguishable by

INHIBITION BY GABA

181

the convulsants. The direct interaction of GABA and glycine has not yet been studied. VI. Chemistry of Synaptic Inhibition

GABA and glycine have been shown to mimic inhibitory transmitters in their actions and, at the same time, other criteria have been investigated

for their identification as transmitters-their selective presence in inhibitory synapses or neurons, and their release during synaptic transmission. In this connection, the concentrations of GABA and its synthetic enzyme, glutamic decarboxylase (GAD), are about a hundred times higher in the inhibitory axons and cell bodies of the Crustacea than in the excitatory ones (Kravitz et al., 1963; Otsuka et al., 1967; Hall et al., 1970) and GABA is released from the crustacean neuromuscular synapses during stimulation of the inhibitory axon ( Otsuka et al., 1966). Preliminary assays of GABA in the Purkinje cell layer of the cerebellum (Kuriyama et al., 1966) and isolated Purkinje cells (Obata, 1969) indicate that the substance is present in relatively high concentration in the Purkinje cells and/or the axon terminals ending on them. The Purkinje cell is known to inhibit cells on which it ends and has upon its soma inhibitory terminals from the basket cells. Recent degeneration experiments indicate that Purkinje cell axons may contain very high amounts of GABA (Otsuka et al., 1971). Purkinje cells send their axons to form inhibitory synapses in the intracerebellar nuclei and the dorsal part of Deiters’ nucleus but not in the ventral part (Walberg and Jansen, 1961; Ito et al., 1968). GABA concentrations of isolated Deiters’ neurons, on which there still remain presynaptic nerve endings as well as glial cells, are much higher in cells from the dorsal part (6.3 m M ) than in those from the ventral part (2.7 mM, Fig. 4). After destroying the cerebellum and waiting long enough to allow degeneration of Purkinje axon termiids, GABA in the dorsal cells is markedly reduced while remaining the same in the ventral cells. A comparable situation has been shown for the GABA synthetic enzyme, GAD, in Deiters’ nucleus (Fonnum et al., 1970). GAD activity in the dorsal part of Deiters’ nucleus is 2.5 times higher than in the ventral part, and decreases after degeneration of Purkinje cell axons to the same level as in the ventral. Intracerebellar nuclei cells, accompanied by axon terminals of Purkinje cells, and Purkinje cells with axonal arborization of inhibitory basket cells are high in GABA (6.0 mM for the foimer and 6.6 mM for the latter) (Otsuka et al., 1971). These values can be compared with low concentration of GABA in spinal motoneurons (0.9 mM) and spinal ganglion cells (0.2 mM) (Otsuka et al., 1971). GAD activities are high

182

KUNJHIKO OBATA

Ventral Deiters’ cells

Dorsal Deiters’ cells a 6-

(2.7f0.2mM)

(6.3f0.8rnM

Nonoperated cats

4-

4

2-

-

0

33

6 6 9

91

12

0-

0

3

6

9

1

2

IOd

8-

Operated cats

-D

(2.7f04mM)

6-

0 Q r

O

4-

L

D 0)

5 z 20-

0

3

6

9

1

GABA concentrotion (mM)

2

3

6

9

12

GABA concentrotion ( mM)

FIG. 4. CABA concentration in isolated Deiters’ neurons from normal and operated cats. In the latter (two cats), the cerebellar vermis was totally removed and the animals were kept alive for 9 and 40 days respectively. Neurons were isolated with a fine-tipped glass needle from faintly stained tissue. Cell volume was estimated from photomicrographs of each cell taken before the assay. GABA content was enzymatically assayed on single cells (maximum sensitivity, 2 x lo-’’ moles of CABA). Concentrations were separately calculated for each cell. Numbers in brackets represent the mean 2 S.E.M. (Otsuka et al., 1971).

in the Purkinje cell layer (Kuriyama et al., 1966), in the interpositus nucleus; one of the intracerebellar nuclei (Fonnum et al., 1970), and in the pyramidal cell layer, rich in inhibitory granule cells, of the hippocampus ( Storm-Mathisen and Fonnum, 1969). Demonstration that a substance is released from active synapses is required for rigorous identification of the substance as a transmitter. However, the difficulty in selective stimulation of a single synaptic pathway in the CNS makes the interpretation of release experiments ambiguous. The fourth ventricle of the cat was perfused while electrically stimulating the cerebellar cortex; GABA in the perfusate was assayed enzymatically (Obata and Takeda, 1969). GABA release during the

WHIBITION BY GABA

183

cerebellar stimulation was three times that of the resting value. As the intracerebellar and Deiters' nuclei are located near the wall of the fourth ventricle, the possibility was considered that the GABA release was associated with Purkinje cell inhibition in these nuclei. Direct perfusion of these nuclei was performed with push-pull cannulae (K. Obata and H. Shinozaki, 1970, unpublished results). In about half of the experiments, the GABA collected in the perfusates increased during cerebellar stimulation to about four times the control value. However, field potential;, recorded during the perfusion, indicated rather rapid deterioration of the perfused tissue and the correlation between the electrical activity of Purkinje cells and the amount of GABA release could not be demonstrated. GABA release from the cerebral cortex has been reported by Mitchell and Siinivasan ( 1969) and Iversen et al. (1971). The posterior lateral gyrus was superfused and the nearby cortex and lateral geniculate body were stimulated to activate the cortical neurons (including the inhibitory synapses). Release of both endogenous and "-labeled GABA increased significantly, up to 8.58 times, during the stimulation. An uptake mechanism for GABA is very active in the brain (Iversen and Neal, 1968). This was also shown by electron microscopic autoradiography in which GABA-3H was taken up fairly selectively by 27% of the total nerve terminals (Bloom and Iversen, 1971). It is intriguing possibility that these terminals are GABA-releasers. Recently Miyata and Otsuka (1972) diced the cat spinal cord into 200-pm cubes and measured their GABA content. The concentration of GABA was 2-2.6 mM in the dorsal horn and 0.6-1.3 mM in the ventral horn. Local lesions of blood circulation resulted in reduction of both the dorsal root potential and GABA in the dorsal horn where interneurons for presynaptic inhibition seem to be situated (Wall, 1964). On the other hand, Davidoff et al. (1967) reported that the GABA content of the dorsal horn does not change in spite of marked decrease in glycine after the aortic occlusion. Albers (1960) had reported earlier that, in monkey CNS, one of the highest recorded activities for GAD was in the dorsal horn: it was about four times that of the ventral horn. These observations may be related to the suggestion that GABA is also a transmitter mediating presynaptic inhibition ( Eccles et al., 1963). VII. Conclusion

Electrophoretically administered GABA and related amino acids can reproduce the effect of inhibitory transmitter( s ) on mammalian central neurons in hyperpolarizing the membrane and increasing membrane conductance. The inhibitory actions of GABA are blocked by picrotoxin and bicuculline and not antagonized by strychnine. In many

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types of neurons, mainly located at the supraspinal level, inhibitory synaptic transmission is blocked by picrotoxin and bicuculline but not by strychnine and, therefore, GABA is a leading candidate for a transmitter at these synapses. The inhibitory action of GABA is more potent on most of these neurons than that of glycine, a putative transmitter on spinal motoneurons. Several other lines of experimental evidence, including studies of presence, release, and uptake of substances, are also consistent with the transmitter role of GABA. In addition, the possibility must be mentioned that other substances are working as transmitters at some inhibitory synapses of the mammalian CNS. Neurons of the lateral hypothalamus of the rat are inhibited by electrophoretic application of acetylcholine. These neurons receive inhibitory input from the amygdala, and both the synaptic inhibition and the depression by acetylcholine are potentiated by eserine and blocked by atropine (Oomura et al., 1970). In the rat cerebellum, norepinephrine-containing fibers make synapses on Purkinje cells (Bloom et al., 1971), and electrophoretic administration of norepinephrine inhibits the activity of Purkinje cells (Hoffer et al., 1971b). ACKNOWLEDGMENT

I wish to thank Dr. E. J. Furshpan for reading the manuscript. This essay was prepared during my stay at Department of Neurobiology, Harvard Medical School, Boston, Massachusetts. REFERENCES

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SOME ASPECTS OF PROTEIN METABOLISM OF THE NEURON By Mei Satake Department of Neurochemistry, Brain Research Institute, Niigafa University, Niigota, Japan

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A. Methodological Approach . . . . . . . B. Fundamental Mechanisms of the Protein Metabolism in the Neuron . . . . . . . . . . C. Protein Metabolism in Neuronal Perikaryon as a Whole Protein Metabolism in the Axon . . . . . . . Protein Metabolism in Nerve Endings . . . . . . Protein Breakdown in the Neuron . . . . . . Neuro-Neuronal and Neuro-Nonneuronal Transfer of Proteins Proteins Specific to Neurons and Their Metabolism . . . Neuronal Protein Metabolism Related to Functional Activity . Conclusions . . . . . . . . . . . References . . . . . . . . . . .

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I. Introduction

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11. Protein Metabolisni in the Nerve Cell Perikaryon

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I. Introduction

Protein metabolism is fundamental to the life processes of the cell. Recently molecular mechanisins of catalytic action of enzyme proteins and of receptor function of membrane proteins have been elucidated. In addition to this fundamental role, proteins and their metabolism are supposed to play some or definite roles in the very complicated functions of the brain in the nervous system. It has been suggested that even “memory,” one of the most mysterious functions of the brain, is concerned with brain proteins and their metabolism. This proposal was based on the many works reported by HydCn and his co-workers, who employed microdissection and microanalytical methods, as well as on the recent papers on the effects of inhibitors of protein biosynthesis on the memory. Details of the inetabolisin especially of brain specific proteins in the neuron in both qualitative and quantitative senses are essential in understanding the complicated functions of the brain mentioned above. Elucidation of functions of the glia may be also essential in conjunction with those of the neuron. Recent advances in such methods 189

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as microdissection methods followed by microanalysis, autoradiography, and bulk preparation methods of brain cells or cell bodies have made it possible for neurobiologists to gain knowledge about protein synthetic activities and their functional correlation to a certain extent, though there have been limitations to interpretations of experimental results obtained by these methods. In this review, a biochemical character of protein synthetic machinery in the whole neuron, neuronal perikaryon, axon, and synapse will be discussed with emphasis on the metabolism of the neuron specific proteins. Comprehensive reviews by Lajtha (1964) on the protein metabolism in the nervous system and by Jakoubek and Semiginovsk9 (1970) on the correlation between functional activity and protein metabolism in the nervous system are available. From the anatomical point of view, a neuron could be divided into three distinct parts: perikaryon with dendrite, axon, and presynaptic ending. This artificial separation of the neuron is convenient for a description of the protein metabolism of the neuron, but the phenomenon of axoplasmic transport should be kept in mind for real understanding of the events. It. Protein Metabolism in the Nerve Cell Perikaryon

A. METHODOLOGICAL APPROACH The first difficulty which will be encountered in the study of the chemical or biochemical character of the neuronal perikaryon is that the volume occupied by the neuronal perikarya or by the neuronal perikarya plus the dendrites amounts to only about 5%and 30%, respectively, of that of the whole cerebral cortex (Shariff, 1953) and it is therefore very hard or almost impossible to know what happens in the nerve cell perikaryon from information on the whole brain. For the study of protein metabolism in the nerve cell perikaryon three kinds of methods are at present used: (1) autoradiography, ( 2 ) microdisscction of nerve cell bodies followed by microanalysis, and ( 3 ) bulk preparation of nerve cell bodies followed by analysis with the standard method. Interferencemicroscopical analysis is rarely used.

1. Autoradiography The autoradiographic method has been quite often used for the study of the protein metabolism in the central nervous system in the normal or pathological condition. Methionine-35SS, leucine-", or lysine-3H were used as the protein precursors (Droz and Waschawsky, 1963; Altman, 1963; Droz and Leblond, 1963). This method has the following

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advantages: ( 1 ) The site in the neuron of the active amino acid incorporation into proteins can be revealed. ( 2 ) Transportation or transfer of newly formed protein can be followed by time-sequential analysis. (3) The mean half-life of protein or protein group can be roughly estimated by counting the grain number. On the other hand, the disadvantages are: (1) The grade of density or the number of grains does not necessarily imply the degree of protein synthetic activity, but an integration of the degree of the protein synthetic activity, size of amino acid pool with respect to the precursor amino acid, and degree of the accessibility of the amino acid to the site of protein synthesis. ( 2 ) Owing to the semi-quantitative nature and the impossibility of getting the specific activity of protein (mole amino acid incorporated/gr protein/ time), comparison between specimens is difficult.

2. Microdissection and Microanalysis Samples (neuronal perikaryon and glial clump or neuropil) can be dissected freehand from fresh (Giacobini, 1956; Hydkn, 1959) or frozen dried (Lowry, 1953) materials. A micromanipulator is also available for Carnoy-fixed material (Edstrom, 1953). For analysis of protein metabolism of the neuronal perikaryon, protein content has been analyzed by X-ray microanalysis (Hydkn and Larsson, 1956). It can also be measured by interference microscopy (Lodin et al., 1967) or photomicrography at 230 mp of the protein solubilized by concentrated phosphoric acid at elevated temperature (Koenig, 1968). A minute amount of the cellular protein, lo-’” gr of each, is separated by microscale acrylamide gel electrophoresis ( McEwen and Hydkn, 1966a,b; Hydkn and McEwen, 1966; Hydkn and Lange, 1968). The radioactivity of the minute amount of the protein was measured by counting trapped 3HH,0after combustion (McEwen and Hydkn, 1966a,b) or 3H2 gas evolved by combustion followed by reduction ( Koenig, 1967) with low-background counter. The method of “microdissection and microanalysis” has given many experimental results and suggestions or hypotheses, for example, the proposed existence in the nervous system of a “neuron-glia unit” for higher nervous activities (Hydkn, 1967). The advantage of this method is that the data obtained are quantitative and the information is obtained from the cell body of individuals or from a group of cells with the same functions, so that it seems easy to correlate the biochemical information with the peculiar function of the neuron or of the “neuron-glia unit,” if any. The disadvantages are that (1) it needs special techniques, instruments, and moreover, a high degree of skill and patience and ( 2 ) not the whole neuron but only the

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neuronal perikaxyon with dendrites is isolated by the microdissection technique. A resting membrane potential of about 40 mV was recorded at room temperature ( Hillman and Hydkn, 1965) and the electron microscopically trilamellar image of plasma membrane was shown provided that the glutaraldehyde-osmic acid-fixed specimen was dehydrated in Durcupan instead of alcohol ( Bondareff and HydCn, 1969) . Scanning electron microscopic pictures of the surface of the isolated neuronal bodies revealed the existence of numerous spherical particles on it. This suggests that these particles are nerve terminals attached to it (Hamberger et al., 1970b). But the preservation of the neuronal plasma membranes of the dissected neuron seems to be still under debate (Johnston and Roots, 1965). 3. Bulk Isolation and Semimicro or Macroanalysis

Recently several methods were devised to isolate a large number of nerve cell perikarya with or without concomitant separation of the glial cells (Rose, 1965; Satake and Abe, 1966; Bocci, 1966; Flangas and Bowman, 1968; Satake et al., 1968; Blomstrand and Hamberger, 1969; Norton and Poduslo, 1970; Sellinger et al., 1971; Giorgi, 1971a). These methods are classified into two groups according to the procedures for disintegrating the brain tissue to make suspensions. Only mechanical disruption through bolting or metal mesh was used in one group (Rose, 1965; Satake and Abe, 1966; Bocci, 1966; Flangas and Bowman, 1968; Satake et al., 1968; Blomstrand and Hamberger, 1969; Sellinger et al., 1971) and enzymatic treatment (trypsin) in the other group, followed by mechanical disintegration as in the former group (Norton and Poduslo, 1970; Giorgi, 1971a). Various media were used for suspending and separating the cell body by centrifugation or sieving. Those were sucrose, Ficoll or sucrose plus Ficoll at various concentrations with or without Ca2+or EDTA. For the study of the protein metabolism in the neuron or neuronal body, ( 1 ) labeled precursors, mainly amino acids, were administered to animals before separation of the neuronal perikaryon from the brain (Satake and Abe, 1966; Blomstrand and Hamberger, 1969; Giorgi, 1971b; Sellinger et al., 1971), ( 2 ) neuronal perikaryon was separated from brain slices incubated with the precursors ( Blomstrand and Hamberger, 1970), and ( 3) isolated neuronal perikaryon was incubated with the precursor (Rose, 1968; Hamberger et al., 1971; Giorgi, 1971a,b; Kohl and Sellinger, 1971). Labeled neuronal perikaryon was purified with respect to its protein, and radioactivity and protein content were measured by the routine methods. In a word, the advantage of these methods is that common biochemical

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or analytical methods are applicable because of the fairly large amount of materials obtained. The disadvantage common to all methods is that as in the case of the “microdissection and microanalysis,” not the whole neuron but only the neuronal perikaryon and dendrites, in which most distal parts are usually lost, can be obtained by the present group of methods. Among the methods, there is wide diversity in the purity of the neuronal perikaryon fractions. Pure neuronal and neuron-enriched terms are used with less objectivity, which arouses rather critical discussion (Cremer et uZ., 1968; Rose, 1968; Johnston and Roots, 1970). As in the case of “microdissection and microanalysis,” there seems at present no definite method available to maintain cellular integrity, especially the preservation of plasma membrane during the preparation. A critical assessment of the prevailing methods is available (Johnston and Roots, 1970). The degree of integrity of the plasma membrane might affect the in vitro amino acid transport into neuronal perikaryon, thus the degree of amino acid incorporation into the protein of neuronal perikaryon. Preservation of active amino acid transport into the neuronal perikaryon has been suggested (Rose, 1968). As present, nerve cell perikaryon can be separated from the whole cerebral cortex, but in order to correlate the biochemical findings with the function of the nervous system, nerve ceIl perikaryon of special regions should be separated in good yield in the future. 4. Histochemical Analysis ( Interference Microscope )

This is a method for estimating protein content of a thin slice of fixed cell. Dry mass concentration after extraction of RNA and lipid is measured by interference microscopy, and multiplied by the cell volume (Pevzner, 1965). The advantage of this method is that it makes it possible to deal with the individual neuronal perikaryon in situ. Disadvantages are (1) large error inherent to the cytospectrophotometry, including errors in the thickness of the slice, ( 2 ) error due to cell volume estimation, and ( 3 ) as in other methods described above, not the whole neuron but only a part of the neuron, namely the neuronal perikaryon plus the proximal parts of the dendrites, can be managed.

B. FUNDAMENTAL MECHANISMS OF METABOLISM IN THE NEURON

THE

PROTEIN

Little is known of the precise mechanism of protein breakdown in the cell in connection with protein turnover in the ceII. On the nervous tissue, which is not an exception in this respect, a few reports are avail-

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able (Marks and Lajtha, 1963; Marks, 1970; Palladin and Belik, 1970). No report is at present available on the neuron except one showing the presence of proteolytic activity in the nerve endings (which should be derived from the neuron terminals) (Marks and Lajtha, 1963; Palladin and Belik, 1970). For this reason, this section will emphasize the protein synthesis rather than the protein breakdown, even though the latter seems important for the neuron and its activity.

1. Ribosomal System or Polysonial Sgstem Ribonucleic acid concentration is high in the nerve cell perikaryon ( Hydkn, 1960; Satake and Abe, 1966; Satake et al., 1968). The presence of ribosomal 5 S, 18 S, and 2 8 s RNA, and 4 s RNA species in the isolated nerve cell perikaryon was shown by gel electrophoresis (Jarlstedt and Hamberger, 1971; H. Okazaki and M. Satake, unpublished data). A large accumulation of ribonucleoprotein particles was shown to be present by electron microscopy in the nerve cell perikaryon. Ribosomal particles were also present in the proximal dendrite but absent in the axon distal to the initial segment (Palay and Palade, 1955; Peters et al., 1970). By using freehand dissection the presence of polysomes containing 4 to 5 ribosomes up to 15 ribosomes (some had a hundred ribosomes) and of messenger RNA strands was shown in neuronal perikaryon isolated from Deiters’ nucleus ( Ekholni and Hydkn, 1965). Ultracentrifugal analysis revealed that the population of free ribosomes exceeds the membranebound ribosomes by about five to six times in the cerebral cortex (Mertis et al., 1969; Andrews and Tata, 1971). Electron microscopy, showed that the number of free ribosomes in the nerve cell perikaryoii exceeded that of the membrane-bound ribosomes ( Peters et al., 1970). Thus, the presence of polysomes and transfer RNA in the nerve cell perikaiyon suggests that a ribosomal or polysomal protein synthetic system is operating in the neuron. However, there has been no report which characterizes the system in the neuron but several reports on the system in the whole brain or the cerebral coi-tex are available (Murthy, 1966; Takahashi et al., 1966; Campagnoni and Mahler, 1967; Zomzely et al., 1968; Roberts et al., 1970; Andrews and Tata, 1971). In the cerebral cortex, the volume occupied by the nerve cell perikarya is almost equal to that occupied by glia cells (Shariff, 1953), but electron microscopically, the ribosomal population is higher in the nerve cell perikaryon than in the glia cells. Thus the characteristics of the polysonial protein synthetic system of the neuronal perikaryon may be reflected in those of the whole cerebral cortex. However, the following considerations should be taken into account: (1) Variation in the cell population of the brain by age may reflect on the nature of the brain

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polysonies ( 2 ) as will be mentioned later, proteins specific to either neuron or glia seem to be synthesized in each species of the cell, and it may be possible that there will be qualitatively different polysonial protein biosynthetic systems in the neuron and the glia. The basic requirements for optimal protein synthesis in the brain polysomal system seem to be similar to those reported for other tissues and ribosomal system of the brain. Cell sap, ATP, an ATP-generating system, and GTP were required. Unusual sensitivity to variations in bn concentrations (Na', K+, Mg" and/or Ca"+) of both the protein synthetic activity of the brain polysomal system ( Campagnoni and Mahler, 1967) and the stability of detergent-treated brain polysomal preparation was reported (Zomzcly et al., 1968). If these characters of the polysonie and polysomal system in vitro were relevant in uiuo, protein synthesis in the neuron niight bc under the iiiflucnce of concentrations of the above ions. Thus a suggestion can be made that excitation or inhibition of a neuron results in a variation of protein metabolism through changes of concentration of ion in that neuron. As described above two kinds of polysomes, free and membranebound, are observed in the neuron as well as in all other kinds of cells. Much experimental evidence ( Siekevitz and Palade, 1960; Redman, 1968; Takagi and Ogata, 1968; Ganoza and Williams, 1969) supports the idea that all secretoiy proteins are synthesizcd exclusively on inembrane-bound ribosomes or polysomes. On the contrary, the nonsecretory protein ferritin was shown to be synthesized on free polysomes (Hicks et al., 1969; Redman, 1969). However, these facts do not necessarily imply that all proteins synthesized on membrane-bound polysornes are exclusively those for secretion, and nascent serine dehydratase, an intracellular hepatic enzyme, has been shown to be localized exclusively on rough endoplasniic reticulum (Pitot et al., 1969). A difference in vectors of discharge of nascent proteins synthesized on the membrane-bound polysomes between secretory and nonsecretory cclls has been suggested by an in vitro experiment, wherc most of the nascent proteins synthesized on membrane-bound polysomes of the brain and muscle were released into solution by treatment with puromycin, whereas those of the liver seemed to be released into lumen of the membranous vesicle (Andrews and Tata, 1971). In uitro, more incorporation of labeled amino acid was observed in mernbrane-bound than free polysomes of the brain (as in other tissues) (Andrews and Tata, 1971). As was described above, the proportion of membrane-bound polysomes was reported to be 14 to 23%in the brain, as compared to 69 to 80% in the liver (Mertis et al., 1969; Murthy, 1970; Andrews and Tata, 1971) . Variation in this proportion during the

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development of the brain was reported (Murthy, 1966; Roberts et al., 1970); but data cannot be applied to the neuron since the neuron/glia ratio also changes during development ( Brizzee et at., 1964). Accordingly the contribution of the neuronal polysomal system in the age-dependent rapid decline in the protein synthetic activity of the whole brain is obscure at present. Much work has been donc to characterize mRNA in the brain by employing sucrose-gradient ultracentrifugation and gel electrophoresis (Barondes and Jarvik, 1964; Vesco and Giuditta, 1965; Egyhazi and HydBn, 1966; Jacob et al., 1966, 1967; Kimberlin, 1967; Yamagami and Rappoport, 1967; Takahashi et at., 1969). The S values reported for the mRNA range widely; some are higher than 2 7 s and others between 4 s and 18 S. However, two DNA-like RNA's of which the S values were 8 S and 16 S were recently isolated by EDTA treatment of polysomes of the adult rat brain in a yield of 2%.These 8 S and 16 S RNAs were found to have template activities and calculated to be templates for proteins having molecular weights of 16,000 and 65,000, respectively ( Zonizely et al., 1970). Unknown neuronal or brain proteins are shown to correspond to these proteins. 2. Nuclei A report on protein synthesis in nuclei of the brain (Lajtha, 1961) was followed by several reports on protein synthesis in the isolated nuclei from the whole brain or from neuron and glia (Mase, 1966; Ghosh, 1967; Inamura et al., 1968; Burdman and Journey, 1969; Lgvtrup-Rein, 1970; Dravid and Burdman, 1971). The general characteristics of the system for protein synthesis in the cerebral nuclei seem to be similar to those reported on calf thymus nuclei (Alfrey et al., 1957) and rat liver nuclei (Logan et al., 1959; Rendi, 1960; Rees and Rowland, 1961). That is, the incorporation of amino acid into nuclear protein is dependent on the energy generated in the nuclei themselves and independent on the added ATP, ATP-generating system, and glucose. Puromycin is inhibitory to the incorporation. But contradictory results on the effects of Na', KA,and RNase were reported. Stimulation by optimal concentrations of Na' was reported to be high (Mase, 1966; Lplvtrup-Rein, 1970) and low (Inamura et al., 1968; Burdman and Journey, 1969). Potassium was strongly inhibitory ( Lgvtrup-Rein, 1970) or slightly stimulative (Inamura et al., 1968; Burdman and Journey, 1969). RNase inhibits incorporation markedly ( Mase, 1966), only slightly (Inamura et al., 1968), or not at all (Burdman and Journey, 1969). Contradictory findings on separated nuclei ( neuronal, astroglial, and oligodendroglial plus rnicroglial ) were obtained. Astroglial nuclei showed

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the highest amino acid-incorporating activity per protein, and neuronal nuclei and then oligodendro plus microglial nuclei followed ( LgvtmpRein, 1970), whereas the reverse order was reported by Burdman and Journey ( 1969). Differences in the ages of the animals and in the procedures or solutions used for preparation of nuclei might give rise to the contradictory results mentioned above namely, young rats were used by Mase (1966) whereas adult rats were used by other authors; isotonic siicrose was used by Burdmaii and Journey ( 1969), Triton by Mase (1966), and hypertonic sucrose solution by Inamura et al. (1968) and Lgvtrup-Rein (1970). The physiological significance of the protein-synthetic activity in the brain nuclei or in the neuronal nuclei is still obscure, although histones, supposed to be regulators in the transcription of DNA, are thought to be synthesized in the brain nuclei as well as in the calf thymus nuclei (Reid and Cole, 1964). Alternatively, synthesis in the cytoplasm of nuclear proteins, histones (Robbins and Borun, 1967), and proteins of the large pre-ribosomal particles ( Tsurugi et al., 1972) has been suggested.

3. Mitochondria The capacity of neuronal mitochondria to incorporate amino acids into their proteins in vitro was shown by using the particles isolated from the neuron-enriched fraction of the rabbit brain (Hambcrger et al., 1970a). The activity was higher than that of the glia-enriched fraction of the animal. Incorporation was stimulated by an ATP-generating system and addition of ammonium sulfate. The precise characters of this protein-synthesizing system in the neuron has not yet been reported, but has been described only for the whole brain (Bachelard, 1966; Campbell et al., 1966; Gordon and Deanin, 1968; D’Monte et al., 1970; Burdett and Work, 1971). Brain mitochondria as well as those isolated from other tissue contain DNA and RNA. The content, especially of DNA, differed in two reports (Austin and Morgan, 1967: RNA, 12 pg/mg protein, and DNA, 6.3 pg/mg protein; Balaz and Cocks, 1967: RNA 10 g / m g protein, and DNA 0.8 pg/mg protein). Moreover, as to the other tissue, the brain mitochondrial-protein-synthesizingsystem in vitro is inhibited by chloramphenicol and puromycin, but insensitive or almost insensitive to cycloheximide, RNase, and actinomycin D. No effects were reported from pH 5 enzymes and ATP. Some species of the brain mitochondria1 proteins seem to be synthesized by their own protein-synthesizing system, although some of them (e.g., cytochrome C ) have been shown to be transported from the polysomal system where it is made ( Gonzalez-Gadavid and Campbell,

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1967; Kadenbach, 1970; Davidian and Penniall, 1971). Active amino acid incorporation into mitochondrial proteolipid protein was observed using the brain mitochondrial system in vitro (Klee and Sokoloff, 1965; Mokrash, 1966). The half-life of rat brain mitochondrial proteins was reported to be 18 days for the soluble proteins, 31 days for insoluble proteins, and 26 days for total proteins. These values are relatively longer than those of the respective proteins of the kidney and the liver ( Beattie et al., 1967). But because of the various loci of brain mitochondria-neuronal perikaryon, axon, nerve endings and glia cells, as well as the heterogeneous preparation used in this experiment-it will be necessary to fractionate brain mitochondria before obtaining reliable half-life values.

C. PROTEIN METABOLISM IN NEURONAL PERIKARYON AS A WHOLE I have described the activity and character of each protein-synthetic system of the brain supposed to be operating also in the neuron. Studies on protein metabolism in the neuronal perikaryon as a whole, irrespective of each protein-synthetic system in it, have been carried out autoradiographically in situ ( Altman, 1963; Doroz and Leblond, 1963; Ford et al., 1965) or by microdissection and microanalysis procedures (BrattgArd et al., 1958; HydCn, 1970) mainly for elucidating the mechanism of brain function. In addition to these studies, bulk preparation-standard analysis procedures offer additional evidence. At present several reports are available (Satake and Abe, 1966; Rose, 1968; Blomstrand and Hamberger, 1969, 1970; Johnson and Sellinger, 1970, 1971; Hamberger et al., 1971; Giorgi, 1971a,b; Tiplady and Rose, 1971). The methods employed in these reports are different not only in their procedures for isolating nerve cell perikaryon but also in the manner of labeling the protein with the precursors. Neuronal perikarya were labeled in vivo (Satake and Abe, 1966; Blomstrand and Hamberger, 1969; Johnson and Sellinger, 1970, 1971; Giorgi, 1971b) and in vitro in slices (Blomstrand and Hamberger, 1970; Hamberger et al., 1971), or after isoIation (Rose, 1968; Hamberger et al., 1971; Giorgi, 1971a,b; Tiplady and Rose, 1971). The results revealed the high activity of protein synthesis in nerve cell perikaryon. However, there has been a discordance between these results with respect to the activity of neuronal perikaryon relative to that of non-neuronal perikaryon or glial cells. In one group of experiments, neuronal perikaryon showed higher specific activity of protein than non-neuronal elements or glial cells, irrespective of the labeling conditions (Satake and Abe, 1966; Rose, 1968; Blomstrand and Hamberger, 1969, 1970; Hamberger et al., 1971; Tiplady

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and Rose, 1971). The opposite result was obtained in another group of experiments (Johnson and Sellinger, 1970, 1971; Giorgi, 1971a,b), except in the case using young rat brain (Johnson and Sellinger, 1971). There are also differences between the two groups in the yields and specific activities of subcellular fractions of neuronal perikaryon which was labeled in vivo (Hamberger et al., 1971; Johnson and Sellinger, 1971). These contradictory results may reflect the present status of the method and indicate the necessity of a very careful consideration in the interpretation of the results obtained. Differences in homogeneity and preservation of the neuronal perikaryon in the experiments might be largely responsible for these confusing results. Moreover, even in one experiment, degrees of homogeneity, preservation and recovery of different cell type, neuronal and glial, seem very different (Rose, 1965; Cremer et al., 1968; Blomstrand and Hamberger, 19f39; Rose and Sinha, 1969; Johnson and Sellinger, 1971). When the differences in protein-synthetic activity of isolated nuclei in vitro of the different types of glia (Burdman and Journey, 1969; Lgvtrup-Rein, 1970) as described in Section 11, B, 2, are taken into consideration, there remains a probability that the activity is different in each species of glia. If this is true, the mean activity of glial fraction may be inffuenced by the recovery ratio of these glial species. The distribution of most of the silver grains was restricted largely to neuronal perikaryon, including the base of large dendrites, and the reaction was weak over the neuropil in the brain and the spinal cord at a relatively early time (30 minutes to 1 day) after the administration of labeled amino acids to the animal ( Altman, 1963; Droz and Leblond, 1963). This autoradiographic evidence supports the experimental results, which showed higher labeling in uivo of proteins of neuronal perikaryon than of the neuropil or glia cells. High labeling of protein of the cell with the precursor in viuo does not necessarily mean high activity in protein synthesis in the cell, but rather the product of accessibility of precursor amino acid to the cell, and pool size of the corresponding amino acid in the cell, in addition to the protein-synthetic activity itself. Moreover, axoplasmic transport must be taken into consideration. By the method in which neuronal perikaryon is isolated from the slice which has been incubated with labeled amino acid, it was shown that the amino acid incorporation into neuronal perikaryal protein is dependent on temperature and oxygen tension. It was stimulated by high concentration of Na+, inhibited by puromycin, inhibited more strongly by cycloheximide and dinitrophenol, but was insensitive to chloramphenicol ( Blomstrand and Hamberger, 1970). The uptake of labeled amino acid into the cerebra1 protein was agedependent in uiuo. Decrease in the turnover rate with development was

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also shown (Lajtha et al., 1957; Lajtha, 1959). Neuronal perikaryon, labeled in uiuo and isolated in bulk (Johnson and Sellinger, 1971) or by microdissection (Jakoubek et al., 1968), showed the same change of rate with development. However, similar turnover rates of proteins were observed between neuronal perikarya of adolescent and old rats by microdissection and microanalysis and autoradiographically (Jakoubek et al., 1968). When the descending part of the radioactivity-time curve of the proteins of neuronal perikaryon is used for calculating the turnover rate, consideration should be given to a large factor, the axoplasmic flow which may transport newly labeled protein out of the soma. The speed of axoplasmic flow of the slow component was shown to decrease with age in experiments with the peripheral nerve (Droz, 1965; Lasek, 1970b), but no report was made on the fast component. In taking account of the difference of the speed of axoplasmic flow at different ages, true turnover rates of the proteins of younger neurons (Jakoubek et al., 1968) may be calculated to be longer than is the case for older neurons. More experiments, especially by using younger animals than those in the above experiment, seem to be necessary before reaching a conclusion. I l l . Protein Metabolism in the Axon

Methodological difficulties encountered in the study of axonal protein metabolism in the brain are comparable to the difficulties encountered in the study of neuronal perikaryon. Most of the information has been obtained from work on peripheral nerves and giant axons. The lack of an evident proximo-distal gradient in the rate of restoration of acetylcholinesterase activity after irreversible inhibition of the enzyme with organophosphorus compounds supports the concept of axonal protein synthesis (Clouet and Waelsch, 1961; Koenig and Koelle, 1961) . Active amino acid incorporation into the axonal proteins in vitro was revealed after incubation of cranial nerve ( Koenig, 1967), Mauthner nerve fiber (Edstriim, 1966; Edstrom and Sjostrand, 1969), and squid giant axon (Fischer and Litvak, 1967; Giuditta et al., 1968) with labeled amino acids. In these experiments axonal segments were handdissected free from the surrounding tissues, including myelin, under the stereomicroscope. For quantitative estimation of the incorporation, the radioautographic-densitometric method ( Edstrom, 1966) or ultraviolet microscopic determination of minute amounts of protein was used (Koenig, 1967). Labeled amino acids were microinjected into the axon (Fischer and Litvak, 1967). Equivocal results were obtained for the effect of chloramphenicol and actinomycin D. There was very remarkable

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inhibition of the labeling in the squid axon (Fischer and Litvak, 1967; Giuditta et al., 1968) and no inhibition in the rabbit cranial nerve axon (Koenig, 1967) and Mauthner nerve fiber of goldfish (Edstrom and Sjostrand, 1969). In experiments with squid giant axon, axoplasm was labeled less than axonal sheath; in one of those experiments most of the labeled proteins of the axoplasm and about half of those of axonal sheath were recovered in a soluble fraction after high-speed centrifugation (Giuditta et al., 1968) and in the other experiment most of the labeled proteins were recovered in a fraction sedimented at low centrifugal force (Fischer and Litvak, 1967). These discrepancies in the experimental results may be explained partly by differences in degree of contamination of axon with non-axonal elements and by differences in the experimental procedures. It is difficult to reach an ultimate conclusion, but it seem$ probable that a certain amount of protein may be synthesized locally apart from the neuronal perikaryon. However, to what extent the protein-synthetic system of axonal mitochondria or that of periaxona1 tissue contributes to this local synthesis is unknown. The probability of the latter phenomenon of the transfer of macromolecules from periaxonal tissue to axon will be discussed later. No ribosomes have been shown electron microscopically in the axon (Peters et al., 1970) beyond the initial segment as described above. However, a certain amount of RNA was found to exist by microchemical, autoradiographic, or standard chemical methods in axons, Mauthner axon (Edstrom et al., 1962; Edstrom, 1964), giant axon of squid ( Lasek, 1970a), stretch receptor axon of crayfish ( Grampp and Edstrom, 1963), cat cranial nerve axon (Koenig, 1965), and rabbit cervical vagus and hypoglossal nerve (Miani et al., 1966), but the concentrations were very low, as compared with those of perikarya. It is suggested that the concentration of RNA is too low to show any ribosomes by electron microscopy. RNA species of ribosomal type, 28 S and 18 S RNA's, were detected in sheath fraction of the giant axon of squids (Fischer et al., 1969) and the whole nerve fibers of rabbit vagus and hypoglossal nerves (Miani et al., 1966). But only a low-molecular RNA of about 4 S was found isotopically in isolated axon of Mauthner (Edstrom et at., 1969). Axoplasmic transport of RNA from perikaryon was suggested by autoradiography (Peterson et al., 1968) but denied by Lasek (1970b). The participation of membrane RNA ( Shapot and Davidova, 1970), the very existence of which in the nervous system or axonal membrane has not yet been definitively established by experiments, in local synthesis remains to be clarified by further work.

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IV. Protein Metabolism in Nerve Endings

Several reports have shown the protein-synthesizing ability of isolated synaptic bouton or synaptic fraction in oitro (Bachelard, 1966; Autilio et al., 1968; Gordon and Deanin, 1968; Morgan and Austin, 1968, 1969; Austin et al., 1970). However, the synaptosomal fraction used in the experiments was not always homogeneous. Recent work in which the synaptosomal fraction labeled in vitro was analyzed autoradiographically revealed that only half of the resolvable grains originated from synaptosomes (Cotman and Taylor, 1971). In another study most of the labels arose from nonsynaptic structures, especially from those containing ribosomes, though a small but significant number of grains originated from synaptosomes ( Gainbetti et al., 1970), For this reason characteristics of the synaptosomal protein-synthesizing system, which has been reported to be similar to the ribosomal protein-synthesizing system except for the difference in the requirement for energy supply (Autilio et al., 1968; Gordon and Deanin, 1968; Morgan and Austin, 1968, 1969), should be reappraised in the light of possible contamination with the ribosomal protein-synthesizing system of perikaryon. The most interesting features of the synaptosomal protein-synthesizing system, which suggests the possibility of Na+, K+-linked ATPase involvement in the active transport of labeled amino acid, is that optimal concentration of Na+ and K+, similar to those for the maximal activity of Na+, K+-ATPase, were required for maximal activity, and that ouabain was inhibitory to this system (Autilio et al., 1968; Austin et al., 1970). Although, most of the synaptic proteins, including mitochondria, may be transported by axoplasmic flow (Droz and Leblond, 1963; Barondes, 1966, 1968; Droz and Koenig, 1970; Jakoubek et al., 1970; Weiss and Mayr, 1971), it is probable that a small amount of certain species of synaptic proteins of functional importance are synthesized locally by the system. The difference in the half-life of the soluble proteins from the whole brain and the synaptosomal fraction (Rodriguez et al., 1971) supports the hypothesis. V. Protein Breakdown in the Neuron

Metabolism of neuronal protein should be expressed as an integration of the dynamic state of protein synthesis and breakdown. No report on the protein breakdown in the neuron is at present available, except for reports of the presence in synaptosomal fraction of neutral proteinase (Marks and Lajtha, 1963; Palladin and Belik, 1970) and acid proteinase (Marks and Lajtha, 1963). Characterization of protein breakdown, especially of proteinases and

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peptidases has been carried out on whole brain (Uzman et al., 1961; Marks and Lajtha, 1963, 1965, 1970; Marks, 1967; Marks et al., 1968). The precise mechanism of cerebral protein breakdown remains to be elucidated, though a scheme has been proposed (Marks and Lajtha, 1970). Vl. Neuro-Neuronal

and Neuro-Nonneuronal

Transfer of Proteins

Uptake of extraneous macromolecules by cells has been shown using spermatozoa, cultured cells, or ascites cells. The macromolecules were DNA (Herriot, 1961; Kay, 1961; Beiidich et al., 1965; Meizel and Kay, 1966; Brackett et al., 1971), RNA (Kolodny, 1971; Schell, 1971), or chromosomes (Kato et al., 1971). Glia-to-neuron transfer of RNA in the mammalian brain and its physiological importance in brain function have been suggested by analyzing the quantity and base ratio of RNA of neuron and glia before and after nervous activity ( H y d h , 1967). Protein transfer related to function was also suggested by Pevzner (1965). By using exogenous proteins, peroxidase, albumin labeled with *Tor Evans blue, uptake into the axon of these proteins has been observed with giant axon (Giuditta et ul., 1971), mammalian peripheral nerve, and mammalian cranial nerve ( Kristensson, 1970; Olsson et al., 1971) by measuring the radioactivity or by microscopic observations. In experiments with mammalian nerves the macromolecules were administered around the nerve or into the target muscles, and retrograde transport, ‘won-to-perikaryon, was revealed after the transfer into the axon. Neuro-neuronal transfer of S-100 protein niediated by the satellite cells was shown by autoradiography in maminalian cranial nerves (Miani, 1971). With the giant axon the uptake of labeled albumin was strongly stimulated by applying electrical impulses (Giuditta et al., 1971). TO estimate the contribution of the transfer of proteins or proteinsynthesizing machinery to the protein metabolism of the neuron, much more data seem necessary. VII. Proteins Specific to Neurons and Their Metabolism

Since there is no conclusive evidence that any neuronal proteins are exclusively supplied by glia or through the blood, it may be reasonable to suppose that the neuron synthesizes all the proteins necessary to maintain its cellular integrity and its special functions. The following cerebral proteins have been shown to be specific to nervous tissue: S-100 protein (Moore, 1965; Moore and McGregor, 1965); 14-3-2 protein ( Moore and Perez, 1968); 10B protein (Bogoch, 1968); other proteins ( MacPherson and Liakopolou, 1966; Kosinski and Grabar,

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1967; Warecka and Baner, 1967; Bennett and Edelnian, 1968); and the encephalitogenic basic protein ( Kies and Alvord, 1959). The 14-3-2 proteins so called because of its position on DEAE cellulose and Sephadex during its purification, has a molecular weight of about 40,000 and has been shown to be primarily neuronal, although it is absent in the nervous system of fishes and reptiles (Perez et al., 1970; Moore and Cicero, 1971; Moore et al., 1971). On the other hand, experimental evidence suggests that S-100 protein is mainly of glial origin ( Hydkn and McEwen, 1966; Benda et al., 1969; Moore and Cicero, 1971). It is also found in neuronal nuclei (Hydkn and McEwen, 1966) and neuronal cytoplasm (Hydkn and Lange, 1970a) by immunofluorescence and immunodiffusion methods. Disparate values have been obtained by two groups concerning the half-life of S-100 protein-16 days ( Cicero and Moore, 1970) and 4.5-7 hours (McEwen and Hydkn, 1966a). In experiments carried out by the latter authors, S-100 protein increased in the pyramidal cell bodies of rat hippocampus after learning. The intraventricular injection of antiserum against $100 protein during the course of training prevented the rat from learning further (Hydkn and Lange, 1970a,b). The content of 14-3-2 and S-100 proteins in optic tectum increased by the same increment during chick developnient (Moore et al., 1971). Microtubule proteins are neither specific to the brain nor to the neuron, but rather are universal although a difference was noted in the composition of the proteins isolated from different tissues (Olmsted et al., 1971) , However, neurotubules synonymous with microtubules are very abundant in neuron and higher colchicine-binding capacity was found in the squid axoplasm and the brain than in other tissues (Borisy and Taylor, 1967a). The concentration of microtubule protein was 1 to 2% of the total protein (or 5-10% of the soluble protein) in the brain ( Borisy and Taylor, 1967a,b; Weisenberg et al., 1968) and 10 to 12%of the soluble protein in cultured neuroblastoma ( Olmsted et al., 1970). Microtubule protein has a molecular weight of 110,000 and consists of two heteropolymers with molecular weights of 56,000 and 53,000 (Bryan and Wilson, 1971; Feit et al., 1971; Olmsted et al., 1971). The dimer has a capacity to bind to colchicine and guanosine triphosphate ( Weisenberg et al., 1968). After subcellular fractionation of the brain, half of the colchicine bound to protein was recovered in the cell sap fraction and a small amount of it in the synaptosomal fraction (Lagnado et al., 1971), though no microtubules were found by electron microscopy in synaptic endings (Peters et d., 1970) and it is not likely that microtubule proteins are intrinsic to nerve terminal. It has been suggested that microtubules in the axon

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may serve as intra-axonal channels for proxinio-distal transport of macromolecules on the basis of electron microscopy (Schmitt, 1968) and experiments where colchicine blocked a release of neurotransmitters at synapses (Spoor and F e r g ~ o i i 1965). , They may also act in the proximodistal transport of amine granules ( Dahlstrbn, 1971) or acetylcholine esterase (Kreutzberg, 1969). There are only a few reports concerning the metabolism of the microtubule proteins of brain (Dutton and Barondes, 1969; Feit and Barondes, 1970). Some 3040% of solublc proteins in mouse brain (labeled with hot leucine for one day) bound colchicine in thc young animal, and 15-20% in the adult. The half-life calculated was 4 days. Of the niRNA's isolated from cerebral polyribosonies of rats, one with a sedimentation coefficient of 1 6 s was calculated to code for protein with molecular weight of about 65,000 (Zomzely et al., 1970). It is very interesting that the molecular weights of the monomers of microtubule protein are 56,000 and 53,000, and are close to those presented to a hypothetical protcin which is supposed to be translated 011 the mRNA mentioned above. Many attempts have recently been made to isolated receptor protein for the chemical transmitters acetylcholine and serotonin. The electric organ of electric eels and the synaptic menibranc fraction of the cerebral cortex have been used for the sources of acetylcholine receptor protein and labeled cholinergic ligaiids as markers during the preparations (Changeux et al., 1969; De Robertis et al., 1971; Eldefrawi et al., 1971; Miledi et al., 1971). The receptor protein seems not to be fully purified as yet, but it could be separable from acetylcholinesterase (Miledi et al., 1971). Its minimal molecular weight was said to be 80,000 (Miledi et al., 1971) or 40,000 ( De Robertis et al., 1971). The latter authors claimed that the receptor proteins for acetylcholine and serotonin (Fiszer and De Robertis, 1969) are of proteolipid species. No report on the metabolism of such receptor proteins for chemical transmitters has been published. The probable participation of special membrane protein or proteins in the impulse conduction has been indicated (Tasaki et al., 1968, 1971) by using a photometer connected with a computer in the giant axon labeled with fluorescent dye. VIII.

Neuronal Protein Metabolism Related to Functional Activity

This article is described as a supplement to the extensive review appearing in this serial publication (Jakoubek and Semiginovsk9, 1970). In the search for the effects of stimulation on the protein metabolism in neuron, the autoradiography, microdissection and microanalysis, bulk preparation-standard analysis, and histochemical methods described in Section 11, A, have been used. However, as mentioned above, no

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methods can clarify all events which might occur in a neuron. The radioautographical method, which is semiquantitative, can deal only with neuronal perikaryon and not with axon or synapse. Moreover, it is conceivable that mechanisms other than those concerned with protein metabolism may be influenced by the functional activity of the neuron as in the case of the axonal transport of glutamate by the snail brain, which was accelerated by applying electrical stimuli (Kerkut et al., 1967). This transport of glutamate was significantly delayed when the preparation was cooled to 0°C or when Nembutal or Xylocaine was applied to the nerve. By using sciatic nerve, the transport of the fast component was shown to depend on the energy supply (Ochs, 1971). Further movement of the fast component labeled in vivo was blocked by exposure of the sciatic nerve to nitrogen, NaCN, or dinitrophenol in vitro. Moreover, the functional activity very well affect amino acid transport into the neuron. Employment of brain slices as a model showed a very complicated dependence of uptake of various amino acids on the ATP level of the tissue ( Banay-Schwartz and Lajtha, 1971). In the review of Jakoubek and Semiginovskf (1970) experimental results were presented on the alteration of neuronal protein metabolism during various kinds of stimulation, such as motor activity, light stimulation, acoustic stimulation, rotation, osmotic stimulation, and electrical stimulation, but not during learning or memory. The authors supported the postulated relationship between a high rate of synthesis of macromolecules (including protein) and excitation, based on the large body of experimental evidence mentioned in their review. They considered the relationship to be indirect, mainly because no evidence of any direct or quantitative correlation was available. Furthermore, negative results had been obtained in some of the experiments. Several mechanisms which might operate as intermediates between functional activity and alteration of protein metabolism were suggested, e.g., changes in the intracellular pool of free amino acid caused by changes of the blood flow; permeability changes of cell membranes induced by stimulation; changes in intracellular kinetics of macromolecular synthesis, which might be caused by the limited availability of energy sources; extraneuronal factors of hormonal character induced by excitation. However, recent experimental results rather agree with the presence of a direct correlation between the stimulation of neuronal activity and macromolecular synthesis in the neuron and nervous system, provided the stimulus is applied transsynaptically. Transsynaptic stimulation of ganglion cell of Mollusca produced an increase in the amount of labeled RNA, whereas direct stimulation had no effect (Kernel1 and Peterson, 1970; Price et al., 1970) as shown in the crustacean stretch-

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receptor cell, where the base ratios and labeling of neuronal RNA did not change after direct stimulation of the cell (Grampp and Edstrijm, 1963; Edstjrom and Grampp, 1965). Dopamine P-hydroxylase activity in sympathetic ganglion increased by administratioii of reserpine, and this induction was abolished by surgical decentralization, by ganglion blocking agents, or by cycloheximide. The biochemical characteristics of the enzyme activity induced suggested de w v o synthesis of the enzyme by the transsynaptic stimulus (Molinoff et al., 1971). In this connection it is of interest to note that in superior cervical sympathetic ganglia the content of Y,S-adenosine monophosphate increased severalfold following stimulation of the preganglionic nerve fibers but not by postganglionic stimulation (McAfee et al., 1971). In the liver, T,S-adenosine monophosphate induced the synthesis of various enzymes (Wicks et al., 1969), probably acting at the step of release of nascent protein from polysomes (Chuah and Oliver, 1971). Adenyl cylase and cyclic phosphodiesterase (responsible for the formation and degradation of 3,sadenosine monophosphate) were shown to be present in high levels in the brain ( Butcher and Sutherland, 1962; Sutherland et al., 1962), especially in nerve endings and synaptic membranes ( D e Robertis et al., 1967; Gaballah and Popoff, 1971). The postulated physiological role of cyclic 3',5'-adenosine monophosphate in the synaptic area seems not to be necessarily limited to presynaptic events, namely transmitter release (Breckenridge et al., 1967), but may also participate in postsynaptic events. Reports on the 3,s-adenosine monophosphate-dependent protein kinase of the brain are available (Miyamoto et al., 1969; Goodman et al., 1970). Microtubule protein was shown to be a substrate for this enzyme (Goodman et al., 1970). Studies on the modification of neuronal protein may be expected to elucidate neuronal function in relation to the macromolecules of the neuron. In summary, it is at present very probable that neuronal activity produces an effect on neuronal protein metabolism directly through changes in the ionic environment of the neuron, which influences polysoma1 stability and synthetic activity, as well as by affecting the concentration of Y,S-adenosine monophosphate, and indirectly through many factors suggested by the former reviewers ( Jakoubek and Semiginovskf, 1970). IX. Conclusions

Many unequivocal data are now available on the protein metabolism and its relationship to function of whole brain, even though many details, especially of catabolism, remain to be clarified. On the other hand, gather-

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CHEMISTRY AND BIOLOGY OF T W O PROTEINS, S-1 00 AND 14-3-2, SPECIFIC T O THE NERVOUS SYSTEM By Blake W. Moore Deportment of Psychiatry, Washington University School of Medicine, St. Louis, Missouri

I. 11. 111. IV. V. VI. VII. VIII. IX.

Introduction . . . . . . . . . . Preparation of Nervous System Proteins . . . . Immunological Assays for Brain Proteins . . . . Species Distribution of S-100 and 14-3-2 . . . . Distribution and Localization within the Nervous System Developmental Studies . . . . . . . . Turnover Studies of S-100 . . . . . . . Chemistry of S-100 . . . . . . . . Function of S-100 . . . . . . . . . References . . . . . . . . . .

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I. Introduction

The nervous system is characterized by many unique forms and functions, for example, its two major cell types, neurons and glia; the characteristic structures of neurons, axons, and dendrites, and their synaptic complexes with specialized membranes and vesicles; the ordering of the nervous system with its specific connections; the propagation of action potentials; the transmission of information from cell to cell at synapses, with all the characteristic functions of such transfer such as metabolism, release, and reception of transmitters; and learning and memory. Since proteins have long been known to participate in biological functions which require specificity of direction, such as enzyme-catalyzed reaction sequences, immunological reactions, hormone effects, membrane functions, and genetic repressor function, it is reasonable to assume that specific proteins direct the specific functions of the nervous system, and are responsible for specific structures. In other words differentiation of the nervous system in terms of form and function should be expressed, at least in part, by the appearance in the nervous system of specific proteins. One way of looking for such proteins is to try to find the specific 215

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proteins connected with certain specific forms and functions. This method has been successful in studying specific proteins of myelin (basic protein, proteolipid), the proteins associated with microtubules, and, so far less successfully, proteins connected with synaptic transmission, and learning and memory. Another method would be to screen proteins of the nervous system to try to find those proteins which are unique to the nervous system, that is, absent from other organs, and which are present in all nervous systems; in other words, to find nervous-systein-specific, species-nonspecific proteins. Such proteins have, of course, long been known in other tissues, such as the hemoglobin in red cells. We now present a general description of the method which has been used in our laboratory in the search for brain-specific, species-nonspecific proteins. 1. “Protein maps” (Moore and McGregor, 1965) are prepared by fractionating the proteins of aqueous extracts of brain and other organs, such as liver. The proteins are first separated into about 15 fractions by ion-exchange chromatography (on DEAE-cellulose ) and then each fraction is further separated by acrylamide gel electrophoresis. The result is a “map” showing 100-200 separate protein bands. When brain “protein maps” are compared to those from liver or other organs many protein bands seem to be specific to brain, especially a number of relatively acidic, small-molecular-weight proteins. It is obvious that such a procedure is neither a highly specific n u a greatly sensitive test for uniqueness of a protein to the nervous system, since (1) different proteins could overlap on the patterns; ( 2 ) the sensitivity of detection in organs other than brain is not great, since it depends on visualization of stained proteins on the gels. 2. Such “presumptively” nervous-system-specific proteins are purified from brain by a sequence of steps; ( a ) ammonium sulfate fractionation, ( b ) ion-exchange chromatography, ( c ) gel permeation chromatography, ( d ) preparative gel electrophoresis, to give a protein which is pure by the standard criteria, ultracentrifugation, gel electrophoresis at several p H values, and immunological tests. 3. Antiserum to the pure protein is prepared by immunizing rabbits and the antiserum is tested for monospecificity by agar diffusion against the pure protein and against brain extract. 4. The antiserum is then used in an immunological assay for the protein, complement fixation. Immunoassays, especially complement fixation, are known to be highly specific and sensitive assays for antigens, being able to detect, in some cases, single amino acid substitutions, and to measure the antigens down to the picogram range. With such an assay

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it is possible to determine rigorously with high specificity and sensitivity whether the protein can be detected in nonnervous tissues, and at what level it is present in brain and peripheral nerve. It can also be determined whether there are cross-reactions with brain extracts of different species. The immunoassay can also be used to measure the protein in areas of brain, down to a single ccll level, during development, degeneration, and under different functional conditions in order to localize and determine its function. II. Preparation of Nervous System Proteins

All of the proteins to be describcd arc relatively acidic, small-niolecular-weight proteins; that is, they migrate rapidly at neutral or alkaline pH on gel electrophoresis and are tightly bound to anionic ion exchangers. Six such proteins have been found and prepared in pure fomi from beef brain by the “protein map” and purification procedures described above. Good antisera have been made to two of these, called S-100 and 14-3-2, and, as shown below, these two have been shown to be nervous-systemspecific and species-nonspecific. The two proteins, S-100 and 14-3-2, are prepared (Moore, 1965) by a combination of salt precipitation and column chromatography. Brain is homogenized in four parts of 5 m M tris phosphate, pH 7.2, and after the extract is centrifuged, the clear supernatant fluid is fractionated as shown in Fig. 1 to give pure S-100 and 14-3-2. These proteins are pure by acrylamide gel electrophoresis at several pH values and by ultracentrifugation. Ill. Immunological Assays for Brain Proteins

Levine and Moore (1965) first made antiserum to beef S-100 by complexing the antigen with methylnted bovine serum albumin before immunization of rabbits. The antisera to both S-100 and 14-3-2 show single precipitin bands by double agar diffusion ( Ochterloney method) against either pure antigen or brain extract. There is no cross-reaction between S-100 and 14-3-2. A complement fixation assay, adapted from the method of Wasserman and Levine, is used to measure S-100 and 14-3-2 (Moore et al., 1968). The assay has been scaled down to the picogram level (Moore and Perez, 1966), a sensitivity sufficient for measurements in single cells. Both S-100 and 14-3-2 were found, using the C’-fixation assay, to be specific to the nervous system, that is, brain contained at least 10,000 times more S-100, and 200 times more 14-3-2 than any other organ (Moore, 1965; Moore and Perez, 1968).

BLAKE W. MOORE

Brain (1- 5) homogenized in 5 m M t r i s phosphate, pH 7.2 centrifuge

t

Precipitate (discard)

t

Supernatant

I

-

P40, pH 7

ammonium sulfate precipitation

dialyze

Chromatography on DEAEcellulose, pH 7.2, 14-3-2 eluted at 0.15 M NaCl

I 1

P 100, pH 4

dialyze

i

Chromatography on (3-150 Sephadex. 14-3-2eluted at 50,000mol. wt.

Chromatography on DEAEcellulose, pH 7.2 S-100 eluted at 0.4 M NaCl

Chromatography on G-150 Sephadex. S-100 eluted at 24,000 mol. wt. Chromatography on DEAESephadex pH 5.0 separates 14-3-2 and 14-3-3 Chromatography on DEAESephadex, pH 7.2

t I

Desalt and lyophilize

1

14-3-2

t

Desalt and lyophilize

s-100

FIG. 1. Flow chart for purification of S-100 and 14-3-2 from beef brain.

SPECIFIC PROTEINS OF THE NERVOUS SYSTEM

219

IV. Species Distribution of S-100 and 14-3-2

The presence of S-100 and 14-3-2 in brains of a variety of species was tested by looking for cross-reactions with antisera to the beef brain proteins. The S-100 protein was found in a closely cross-reactive form by agar diffusion and C'-fixation in all vertebrate species examined, including mammals, birds, fish, and reptiles. The amount of cross-reactivity indicated a high degree of evolutionary stability for the protein. The 143-2 protein showed close cross-reactions among all mammalian species, weaker reactions in birds, but no detectable cross-reactivity in fish and reptiles. The amount of the proteins showed some variation from species to species. For example, whole rat brain contains about 120 pg S-100 per gni wet brain, while mouse brain has a value of about 20. In general, the levels of 14-3-2 were 2 to 10 times higher than those for S-100. For example, the value for 14-3-2 in rat brain was about 300 p g l g m (compared to 120 for S-loo), while the 14-3-2 level in chicken brain was about 700 pg/gni (compared to 7 for S-100). V. Distribution and Localization within the Nervous System

Both S-100 and 14-3-2 are found in central and peripheral nerve. In general, in human brain, S-100 was high in white matter and low in gray, and the opposite was true for 14-3-2 (Table I ) . Also, in general, S-100 was particularly high in cerebellum in all species examined. HydBn and McEwen (1966) first attempted to localize S-100 to celltype within the nervous system by the immunofluorescence technique and by wet-dissection of neurons and glia, followed by inimunological (agar diffusion) assay, and concluded that S-100 was primarily glial, although he claimed a small amount was present in neuronal nuclei. More-certain localization was obtained using degeneration studies and assays on single cells or cell-types. The cloned rat glioma (astrocytoma) tissue cell culture (C-6) WBS shown to produce S-100 during the stationary phase of the growth cycle (Beiida et nl., 1968). This glionia culture produces little 14-3-2, but neurobIastoma cell cultures produce large amounts of 14-3-2 and no detectable S-100 (M. Goldstein and B. W. Moore, 1972, unpublished data). Thrce types of degeneration studies have been done to demonstrate localization of S-100 and 14-3-2. First, during Wallerian degeneration of rabbit tibia1 nerve (Perez and Moore, 1968), S-100 fell to low levels during axonal degeneration of the distal end of the nerve. On the other hand, during degeneration of rabbit optic nerve (Perez et al., 1970), S-100 rose slightly and 14-3-2 fell, indicating a glial localization for S-100 and axonal for 14-3-2. It is not known why S-100 falls during degenera-

220

BLAKE W. MOORE

S-100

AND

TABLE I 14-3-2 CONTENTS OF VARIOUSPARTS OF HUMAN BRAIN s-100 pg/gm wet weighta

Mean S.E.M.

Nb

Mean S.E.M.

45+ 3 177 f 16 53f 4 190 f 22 61f 4 208 f 1s 49f 4 134 14 154 f 12 131 i 13 59+ 6 105 f s 61k 7 138 5 S 126 f 10 133 f 10 54f 7 68f 9 143 f s

16 16 16 16 16 16 16 16 16 15 16 16 16 16 16 16 16 16 12

191 21 181 f 10 20s f 20 116 f 11 239 f 16 131 & 14 170 13 101 f 10 101 8 196 i 12 271 27 169 11 237 f 19 158 i 13 251 22 213 f 16 186 & 20 201 k 10 205 f 21

Frontal gray Frontal white Parietal gray Parietal white Occipital gray Occipital white Temporal gray Temporal white Corpus callosum Globm pallidus P a t amen Hippo campus Head of caudate nucleus Hypothalamus Thalamus, central nucleus Thalamus, lateral nucleus Cingulate gyrus, rostra1 Amygdaloid body Corpus quadrigemina inferior (L

14-3-2 pg/gm wet weighta

+

*

*

+

Nb 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 11

In terms of beef brain equivalents. Number of brains.

tion of peripheral nerve, but this effect suggests some control of S-100 in Schwann cells (its presumed location in peripheral nerve) by the intact axon. Finally S-100 and 14-3-2 levels were followed during retrograde degeneration of dorsal thalamus (Cicero et al., 1970) in response to degeneration of cortex produced by termination of its blood supply. In this case, where neuronal cell bodies as well as fiber degeneration was followed, S-100 rose slightly and 14-3-2 fell, in agreement with the optic nerve studies. Using the C’-fixation microassay, sensitive to a few picograms of antigen, S-100 and 14-3-2 were measured in single neurons dissected from frozen-dried sections using Lowly’s methods (V. J. Perez and B. W. Moore, unpublished). Neither anteiior holm nor dorsal root ganglion cells contained detectable 5-100,but surrounding neuropil contained large amounts. Both the cell bodies and neuropil contained 14-3-2. These results agree with the other localization studies and provide the most direct evidence of localization to cell type.

SPECIFIC PROTEINS OF THE NERVOUS SYSTEM

221

VI. Developmental Studies

The S-100 level of rat whole brain (Moore, 1969) is low from birth until about 10 days of age, when it rapidly rises, reaching about half the adult level at 30 days and adult levels at about 90 to 120 days of age. Zuckerman et al. (1970) have measured S-100 in areas of human fetal brain and found a caudal-rostra1 order of development, that is, it appeared in cord, pons, medulla, cerebullum, and midbrain at 10-15 weeks of gestational age, but its appearance was delayed until 30 weeks in the frontal cortex. Moore and Perez (1968) found, in 22 of 26 areas of human brain, that S-100 level was positively and linearly correlated with age from 2 to 80 years. A detailed study (Cicero et al., 1966) was made of development of S-100 and 14-3-2 in the chick optic tectum, a developmental system which has been studied intensively with regard to time of cell divisions, growth of fibers, migrations of cells, and layering. Although all cell division ceases by 14 days of incubation, both S-100 and 14-3-2 levels remain low until several days past this point, until at 17 or 18 days they both begin to rise and then increase rapidly before, during, and immediately after hatching (21 days), reaching adult levels 2 to 3 weeks after hatching. The levels of both proteins rise in parallel fashion, although the absolute level of 14-3-2 is much higher than that of S-100. It is significant that some differentiated functions of neurons and glia, namely 14-3-2 and S-100 synthesis, seem to occur relatively late in the development of the nervous system. The same phenomenon is true of developing chick spinal cord (T. J. Cicero, 1972, unpublished data). The levels of $100 and 14-3-2 start to rise some time after cell division has ceased. In fact, the increase seems to parallel the development of electrical activity in the cord and the beginning of limb movements. VII. Turnover Studies of S-100

There is some disagreement in the literature regarding the turnover rate of S-100 in brain. McEwen and HydCn (1966) measured the halflife of S-100 in rat brain by systeinic injection of labeled leucine or lysine and counted the S-100 band after acrylamide gel electrophoresis. Their data indicated a very rapid turnover, with a half-life on the order of hours. Cicero and Moore (1970) injected labeled leucine, via chronic implanted cannulae into awake rats, and purified S-100 for counting by a two-step procedure involving DEAE-cellulose chromatography followed by acrylamide gel electrophoresis. The purity of the isolated S-100 was assessed by quantitative complement fixation assay. This data indicated a half-life for S-100 of about 12-15 days. The discrepancy be-

222

BLAKE

W.

MOORE

tween these two experiments can perhaps be explained by possible contamination of isolated s-100 by more rapidly turning-over proteins in the Hydbn experiment. Herschmann ( 1971) has estimated the synthetic rate of S-100 in C-6 glioma cell cultures and concludes that his data agree closely with the 12-15 day figures. VIII. Chemistry of S-100

The molecular weight of S-100 is approximately 21,000 to 24,000 Daltons (Moore, 1965; Dannies and Levine, 1971a). Levine has presented evidence that it is constructed of three subunits of about 7000 Daltons each (Dannies and Levine, 1969, 1971b). These cannot be identical, since there is a single tryptophan residue per 21,000 Daltons in S-100 (Table 11). Dannies and Levine ( 1971a) have succeeded in separating one subunit from the other two by chromatography in 8 M urea on DEAE-cellulose. This subunit contains the single tryptophan. They have some evidence that the other two subunits might be identical. The amino acid analysis is shown in Table 11. The most characteristic feature is the high content of glutamic and aspartic acid residues, a fact which is reflected in the high mobility of S-100 on gel electrophoresis at alkaline pH. There is a single tryptophan, three tyrosine, and a number of phenylalanine residues. There are three or four cysteine residues and no disulfide bonds in the native molecule. It is highly soluble, even to some extent in saturated ammonium sulfate at neutral pH, although it precipitates at pH 4. The ORD spectrum of native S-100 indicates a helix content of approximately 40% (Kessler et al., 1968). Under some conditions S-100 seems to display multiple forms when electrophoresed on acrylamide gel (Gombos et al., 1966; H y d h and McEwen, 1966; Vincendon et al., 1967). Calissano et al. (1969) showed that the appearance of multiple forms was dependent on Caz+; Mg2+ TABLE I1 AMINOACID ANALYSISOF BEEF5-100Glu Asp Lys His Arg Ser Thr cys Try a

36 21 17 8 3 10 8 3 1

3 1 16 17 6 Val 13 Ala 12 Gly 9 Met 4 Tyr Pro Phe Leu Ileu

Values represent numbers of residues per 24,000 Daltons to the nearest whole

number.

223

SPECIFIC PROTEINS OF THE NERVOUS SYSTEM

TABLE I11 EFFECTOF CA*+ON ANS BINDINGTO S-100 -~

___

~

~~

~

___

~~

Relative fluorescence of ANS (0.1 mM).

No Ca2+ 10 mM Ca2+

NO S-100

100 pg/ml S-100

2.8 3.0

3.5 7.5

The buffer was 20 mM tris C1, pH 8.2. For fluorescence measurements, the primary wavelength was 380 nm and the secondary 480 nm.

had no effect. They also demonstrated that S-100 undergoes a conformational change, during Ca2+ binding which results in exposure of the tryptophan, one of the three tyrosine, and two of the three cysteine residues, all of which are buried in the absence of Ca2+.The dissociation constant for Ca2+as measured either by increase of fluorescence of tryptophan during binding, or by equilibrium dialysis with 45Ca,was about 1 mM in the presence of 60 mM K+, and there were 8-10 binding sites per molecule. No conformational change was seen with Mg2+, and a slight one with Sr2+.Monovalent cations antagonized the Ca2+effect, the order of effectiveness being K+ > Na' > Li'. Dannies and Levine ( 1971b) have shown that aggregation, involving disulfide bond formation, tends to occur in S-100. This aggregation can be reversed by incubating with 0.01 M 2-mercaptoethanol. Calissano et al. (1969) have shown that S-100 together with Ca2+stimulates, to a great degree, the transport of monovalent cations across artificial liposome membranes, as measured by radioactive R b exit. Such experiments may suggest a role of S-100 in ion transport. In the absence of Ca2+, S-100 does not bind the fluorescent hydrophobic probe, 8-anilinonaphthalene sulfonic acid ( ANS ) but ANS is bound in the presence of Ca2+ (Table 111). This experiment suggests that there are few, if any, hydrophobic regions exposed in the absence of Ca2+,and that the conformational change induced by Ca2+binding leads to exposure of hydrophobic residues which are responsible for the ANS binding. This suggestion agrees with the known exposure of aromatic residues during Ca2+binding as measured by tryptophan fluorescence, difference spectra, and spectrophotometric titration of tyrosine. IX. Function of S-100

At the present time, there are no clear clues as to the exact function of $100. H y d h and Lange (1970a,b), on the basis of learning experi-

224

BLAKE W. MOORE

ments in rats, suggested that S-100 might be connected with the process of learning. However it is too early, and the results of such experiments are not conclusive enough, nor well-enough controlled, to permit unequivocal conclusions, although such experiments are interesting and suggest further work. The fact that S-100 is found in a relatively invariant form in the brains of such a wide range of species, and the fact that it is present only in the nervous system and that it is present there in fairly large amounts, suggest that S-100 is related to some general, important, specific nervous system function, The correlation of S-100 development with deveIopment of function in the nervous system tends to support this view. The work on Caz+effects and conformational change, and on effects on monovalent cation transport in artificial membranes, suggests that its function may be connected with ion movements in membranes. REFERENCES Benda, P., Lightbody, J., Sato, G., Levine, L., and Sweet, W. (1968). Science 161, 370. Calissano, P., and Bangham, A. D. ( 1971). Biochem. Biophys. Res. Commun. 43, 504. Calissano, P., Moore, B. W., and Friesen, A. (1969). Biochemistry 8, 4318. Cicero, T. J., and Moore, B. W. (1970). Science 169, 1333. Cicero, T. J., Cowan, W. M., and Moore, B. W. (1966). Brain Res. 24, 1. Cicero, T. J., Cowan, W. M., Moore, B. W., and Suntzeff, V. (1970). Brain Res. 18, 25. Dannies, P. S., and Levine, L. (1969). Biochem. Biophys. Res. Commim. 37, 587. Dannies, P. S., and Levine, L. (1971a). J. Biol. Chem. 246, 6276. Dannies, P. S., and Levine, L. (1971b). J. Biol. Chem. 246, 6284. Combos, G., Vincendon, G., Tardy, J., and Mandel, P. (1966). C. R. SOC. Biol. F . D268, 1533. Herschmann, H. R. ( 1971). J. Biol. Chem. 246, 7569. HydBn, H., and McEwen, B. (1966). Proc. Nut. Acad. Sci. U . S . 55, 354. HydBn, H., and Lange, P. W. (1970a). Proc. Nut. Acad. Sci. US.65, 898. HydBn, H., and Lange, P. W. (1970b). Proc. Nut. Acad. Sci. U.S. 67, 1959. Kessler, D., Levine, L., and Fasman, G. (1968). Biochemistry 7, 758. Levine, L., and Moore, B. W. (1965). Neurosci. Res. Program Bull. 3, 18. McEwen, B., and HydBn, H. (1966). 1. Neurochem. 13, 823. Moore, B. W. (1965). Biochem. Biophys. Res. Cornmiin. 19, 739. Moore, B. W. (1969). “Handbook of Neurocheniistry ” (A. Lajtha, ed.), Vol. 1, pp. 93-99. Plenum Press, New York. Moore, B. W., and McGregor, D. (1965). 1. Biol. Chem. 240, 1647. Moore, B. W., and Perez, V. J. (1966). J. Immunol. 96, 1000. Moore, B. W., and Perez, V. J. (1968). I n “Physiological and Biochemical Aspects of Nervous Integration” (F. D. Carlson, ed.), pp. 343-360. Prentice-Hall, Englewood Cliffs, New Jersey. Moore, B. W., Perez, V. J., and Gehring, M. (1968). 1. Nercrochem. 15, 265. Perez, V. J., and Moore, B. W. (1968). J. Neurochem. 15, 971.

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Perez, V. J., Olney, J. W., Cicero, T. J., Moore, B. W., and Bahn, B. A. ( 1 9 7 0 ) . 1. Neurochem. 17, 511. Vincendon, G., Waksman, A., Uyemiira, K., Tardy, J., and Combos, G. ( 1 9 6 7 ) . Arch. Biochem. Biophys. 120, 233. Zuckerman, J. E., Herschmann, H. R., and Levine, L. ( 1 9 7 0 ) . I. Neurochem. 17,

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THE GENESIS OF THE EEG By Rafael Elul of Anatomy and Brain Research lnrtitu , School a Medicine, University of California at Lor Angeler, Lor Angeles, California

Departmer

I. Introduction . . . . . . . . . . . . 11. Scope of this Review . . . . , . . , . . 111. Unitary Sources of the EEG . . . . . . . . . A. Early Models of Wave Activity in the Brain . . . . . B. Experimental Evidence from Extracellular Microelectrode . . . . . . . . . . . Recordings C. Experiments with Multiple Microelectrodes . . . . D. Intracellular Wave Activity in Cortical Nerve Cells . . . E. Intra- and Extracellular Potentials: Biophysical Considerations . . . . . . . . F. Extracellular Wave Activity Originating in Nerve Cells . . G. Relative Importance of Neuronal and Neuroglial . . . . . Contributions to Extracellular Currents . IV. Site of Production of Wave Activity in Cortical Nerve Cells . . A. “Synaptic Functional Units” as the Elementary Generators of Cortical Wave Activity . . . . . . . . B. Synaptic and Nonsynaptic Potential Fluctuations in Dendrites . V. Biophysical Factors in Summation of Unitary Neuronal Waves . A. Basic Assumptions-Linearity of the Extracellular Medium . B. Nonlinearities due to the Topology of the Extracellular Space . C. Nonlinearities due to Capacitive Effects . . . . . D. Nonlinearities Introduced by the Recording Electrodes . . E. Differences in Spatial Propagation of Spikes and Waves . . VI. Theoretical Analysis of Systems of Unitary Generators . . . A. Two Possible Models: Synchronized and Nonsynchronized . . . . . . . . . Generator Systems . B. Phase Relationship of the Summed Record in Generator . . . . . . . Populations of the Two Types C. Amplitude of the Summed Record in Generator Populations of the Two Types . . . . . . . . VII. Experimental Analysis of Population Behavior of Cortical Wave Generators . . . . . . . . . . . A. Preliminary Comments on Application of Phase and Amplitude . . . . . . . Criteria to Experimental Data B. Phase Relationship between Intracellular Activity and . . . . . . . . . . Surface EEG . C. Amplitude Relations: Experiments with Tetrodotoxin . . . D. A Tentative Model of Generation of the EEG . . . . 227

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241 243 245 245 246 248 249 250 252

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RAFAEL ELUL

VIII. Subcortical Control of the EEG and Possible Functional . . . . . . . . . . . Implications . . . . A. Subcortical Pacemaker for the EEG . B. Possible Functional Role of the EEG in Perception . . IX. Conclusion and Coilsequences for Evaluation of Gross EEG Activity . . . . . . . . . . . . A. Summary of the Results in the Present Review . . . B. Implications for Computer Analysis of EEG Data . . References . . . . . . . . . . .

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264 264 266 267 267 268 270

I. Introduction

A newcomer to the field of electroencephalography may find it somewhat puzzling that the four decades following the initial report on brain waves in man (Berger, 1929) have brought about only limited progress in physiological interpretation of this phenomenon. Certainly when compared with the enormous volume of literature amassed during the same period on clinical, physiological, and psychological applications of the EEG, the dearth of basic information appears even more striking. Admittedly, the physiology of the EEG, its sources and regulatory mechanisms, present a formidable objective. Nonetheless, the last 10 years have yielded some highly significant data on the basic mechanisms underlying the EEG. Examination of these data in the present review suggests that the mechanisms of generation of the EEG may now be clear at least in outline. Moreover, the remaining gaps in our understanding of brain waves involve a number of well-delimited areas, each of which is, by itself, amenable to attack with current neurophysiological techniques, and it is not unreasonable to hope that these too would be resolved in the foreseeable future. After going through this review, the reader may perhaps come to share the author’s evaluation that even the ultimate utilization of brain waves to draw precise inferences on various aspects of brain function, including thought processes, now appears a potential reality. II. Scope of This Review

Past research on the EEG has embraced numerous questions, such as the description of overall appearance of wave activity-and more recently of frequency spectra-in various behavioral situations, pathological modifications in the EEG, and the effects of subcortical drives. However, as significant as these questions are, they bear only indirectly on the physiological origin of wave activity in the brain. In the current review no attempt has been made to provide comprehensive coverage of the existing physiological literature on the EEG. Rather, attention is focused here on the basic processes responsible for this activity. It will

THE GENESIS OF THE EEG

229

emerge from the data presented that the EEG primarily reflects nerve cell activity, and to the extent that neuronal activity in the brain determines behavior (and in turn is determined by it) the EEG may constitute a source of objective information on behavioral states. Clearly, understanding of the mechanisms underlying the EEG is essential for correlation of this activity with brain physiology and behavior, and in the following pages an attempt is made to present and evaluate the available experimental information relevant to the question of generation of spontaneous electrical wave activity in the brain. On the other hand, it must be recognized that, because of the demanding techniques involved, there is at the present time no reliable first-hand information on electrical activity on the microscopical level in the brain of man. For this reason, the present review omits discussion of several EEG phenomena, both physiological and pathological, which cannot be investigated properly in lower mammals, although in relation to human behavior and subjective experience such phenomena naturally present an exceedingly intriguing topic. One prominent example is alpha activity, which in recent years has become the focus of much interest in relation to autoregulation of the EEG in man, as well as the subject of a rather whimsical suggestion that these potentials originate in the external ocular muscles ( Lippold, 1970). The identification of this activity with spindle sleep in the cat [Andersen and Anderson, 1968 (see their Chapter 1)1, although perhaps permissible insofar that both represent morphologically similar rhythmic activity, has little to recommend it from any physiological and behavioral viewpoint. [Incidentally, the probability distribution of amplitude of alpha activity is essentially identical with that of waking EEG in the same subject, whereas both spindles in the cat and the EEG from stage I1 sleep in man have very different probability distributions for amplitude than is the case for activity in the waking state (R. Elul, unpublished; R. G . Bickford, personal communication).I One cannot help but note, however, that alpha-like activity is present in anthropomorphoid apes in the waking state and, to a lesser degree, in other primates. Direct experimental investigation of alpha activity would therefore seem to be feasible, and it is to be hoped that such research would be carried out. Leaving aside for the present these complex aspects of the EEG and restricting discussion to the more fundamental issue of the origin of wave activity in the brain, there appear to be two questions which are basic to understanding the EEG: ( i ) What is the nature of the elementary generators of wave activity; is the EEG produced by cells, or is wave activity a property of extracellular space?

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RAFAEL ELUL

(ii) What relationship exists between the EEG recorded from the scalp with large electrodes and activity on the microscopical level? Does the gross activity essentially constitute a replica of the output of elementary generators, or is there a more complex relationship between events on the microscopic and the gross level? Experimental evidence pertaining to these two questions is presented in the current review. Based on this evidence, an attempt is made to construct a model of the EEG which relates the electrical activity recorded from the scalp to the contributions of single nerve cells in the cerebral cortex and in subcortical centers. Ill. Unitary Sources of the EEG

A. EARLYMODELSOF WAVEACTIVITYIN

THE

BRAIN

Characteristically there are no visible spikes in the EEG. (The “spikes” referred to in this review are to be taken as synonymous with “action potentials,” i.e., rapid, all-or-none depolarizations about 0 . W msec in duration, produced by individual nerve cells or neuronal processes. Although references to “spikes” are common in clinical electroencephalography, these are composite events, rather longer in duration-20 to 100 msec-than true unitary action potentials.) Since spikes constitute the most prominent form of nerve activity, the question whether the EEG reflects summated spike activity arose very early following Berger’s recordings of gross brain activity. At that time it appeared natural to consider a scheme in which wave activity, such as the EEG, may arise through summation of spikes. A model of this type has been put forward by Adrian and Matthews (1934). In essence, their model straightforwardly and elegantly proposed that the gross EEG may represent an envelope of spike activity in the underlying tissue. If occurring in exact synchrony, spikes originating in neighboring cells may summate to produce a gross “spike.” In analogous manner, a rapid sequence of spikes might sum to produce a macropotential of longer duration, Through combination of these two mechanisms, wave-like potentials longer in duration than individual unitary spikes might be produced (Fig. I A ) . An essential consequence of this model has been that large EEG waves should signify intense spike activity, and conversely lulls in the gross activity should correspond to a decrease in firing by individual cells. This corollary found an apparent confirmation in the observations of Adrian and Moruzzi (1939), which revealed good correlation between EEG activity recorded from the pyramidal cortex and the frequency of spike discharge in descending spinal pathways.

THE GENESIS OF THE EEG

231

A CELLULAR ACTIVITY

CELLULAR

FIG. 1. Some early models of the origin of the electroencephalogram. A. Wave activity may be produced through summation of spikes from a large number of nerve cells firing in sequence ( adapted from Adrian and Matthews, 1934). B. Single neurons may be capable of rhythmic wave activity (following Gerard, 1936).

Results obtained nearly at the same time by some other investigators, however, did not agree with this model. It is evident that a smooth, wavelike envelope may be formed only through participation of a rather large number of neurons, all engaged in spike firing. For this reason the observation made by Gerard (1936), of the persistence of wave activity even in small fragments of brain tissue, such as the frog’s olfactory lobe, seemed rather difficult to reconcile with the spike model. Although spikes still were thought to be the cardinal activity of nerve cells, in the late 1930s awareness had begun of the coexistence of wave processes with the more rapid action potentials. In analogy with “caffeine waves,” Gerard (1936) proposed a model of wave activity emanating from individual nerve cells (Fig. 1B). Studies of evoked potentials throughout the next decade have clarified that, in addition to spike discharge, nerve cells are capable of producing wavelike potentials. With the introduction of laminar analysis techniques, it also became clear that these slower potentials often originate in the more superficial layers of the cortex, and wave potentials eventually have come to be considered synonymous with “dendritic potentials” (e.g., Chang, 1951, 1952; Clare and Bishop, 1952, 1955, 1956). While the evidence for wave generation in dendrites has never been conclusive (see Section IV for additional discussion), this attractive possibility has been exceedingly difficult to disprove, so that the concept of dendritic

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origin of wave activity has become entrenched to such a degree that suggestions of spike conduction in dendrites ( Spencer and Kandel, 1961; Purpura et al., 1966; Llinhs et al., 1968) often have been viewed as exceptions to some general rule, and indeed occasionally provoked lively arguments (Llinhs et al., 1968, 1969; Calvin, 1969; Calvin and Hellerstein, 1969; Hellerstein, 1969; Zucker, 1969). The issue of electrogenesis in dendrites is most complex, but there does not appear to be any solid foundation for exclusion of all-or-none events from dendrites (cf. R. Elul and W. R. Adey, 1972, unpublished data).

B. EXPERIMENTAL EVIDENCE FROM EXTRACELLULAR MICROELECTRODE RECORDINGS With the advent of microelectrode recording techniques, extracellular exploration of the cerebral cortex has become feasible. Experiments involving extracellular recording in the cerebral cortex (Li and Jasper, 1953; Li et al., 1956), demonstrated unitary spikes riding on the crest of waves (Fig. 2 ) . During anoxia, spikes could be selectively depressed prior to disappearance of the waves, again suggesting that the two phenomena may not originate in the same cellular mechanism. For the first time, these observations brought up what probably still constitutes the most significant and intriguing feature of the EEG: the nonfocal character of this activity, which seems to permeate throughout the cortex. In exploration with microelectrodes, sites of spike activity can be localized in the cortex with high degree of precision (see Mountcastle, 1957), insofar that the amplitude of the recorded spikes increases as the microelectrode is moved closer to the active cell or axon (indeed, in many instances further movement may result in penetration of the membrane). There are no analogous foci of wave activity that can be charted with microelectrodes and, almost without exception, movement of the microelectrode through the tissue is not associated with any significant changes in amplitude of the recorded EEG. As will be seen below (Section V ) , this feature of the EEG still presents a considerable interpretative hurdle, although one possible explanation, in terms of the

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FIG. 2. Wave and spike activities recorded with extracellular microelectrodes in the cerebral cortex. Cat, light pentobarbital anesthesia. Top and bottom tracings from two steel microelectrodes, insulated except at the tip, 600 p depth, 40 p separation between tips of the electrodes (from EM, 1962).

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geomctry of the extracellular space, appears quite reasonable. At the time of the first explorations of the cerebral cortex with extracellular microelectrodes, though, the results obtained seemed to suggest that the EEG may indeed require the summation of contributions of large numbers of generators which, individually, do not produce wave activity-precisely the mechanism put forward by Adrian and Matthews ( 1934). About the same time, data which were clearly conflicting with this view also became available. Evidence from evoked potential studies, which revealed wavelike potentials arising from synchronized stimulation of nerve cells (rather than the gross spikes which might have been expected from the Adrian-Matthews model), may be cited in this context (Barron and Matthews, 1938; Chang, 1950; Clare and Bishop, 1952). Probably most significant in this direction has been the evidence derived from the demonstration in intracellular recordings of synaptic potentials, first in muscle fibers (Fatt and Katz, 1951), and subsequently in spinal motoneurons ( Brock et ul., 1952). Although constituting only indirect evidence with refercncc to cortical nerve cells, these observ.‘3 t‘ions provided indubitable proof on thc cellular level of R nonspiking, relatively slow, graded process. The synaptic potentials classically reported in spinal neurons are short in duration in comparison to EEG waves (5-10 msec as against 20-200 msec), but more recent studies have revealed long i.p.s.p.’s of several hundred nisec duration (Llinhs and Terzuolo, 1964). It has thus become apparent that nerve cells may sustain wave activity similar to the EEG and, consequently, the suggestion has been made that the EEG may ensue from summation of synaptic potentials ( Purpurn, 1959). However, the capability of nerve cells to produce synaptic potentials does not necessarily indicate that the EEG in fact originatcs in nerve cells. (Indeed, as will be seen below, most of the unitary wave activity in the cerebral cortex does not make any detectable contribution to the gross EEG.) Moreover, since synaptic potentials are prcsent in many neuronal centers, the hypothesis of synaptic origin of the EEG was undermined by the fact that gross wave activity can be detected only in the cerebral cortex and not, say, in the spinal cord, a structure which boasts equally large e.p.s.p.’s and i.p.s.p.’s.

C . EXPERIMENTS WITH MULTIPLEMICROELECTRODES During the 1950s there were rcyorted two sets of conflicting data, from extracellular studies in the cerebral cortex, and, in opposition to this, from intracellular investigations in muscle and in spinal niotoneurons. The technique of siinultaneous recording with multiple extra-

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FIG.3. Limited propagation of wave and spike activity in the cerebral cortex. Siniultaneous monopolar and bipolar records from two microelectrodes. A. Microelectrodes 1 and 2 at 500 p depth, 30 p interelectrode distance. Surface = gross electrode recording from surface of the cortex, I/g = monopolar froin niicroelectrode 1 us. the reference point; 1/2 = bipolar record; g/2 = nonpolar from electrode 2 us. the reference point. Note that polarity was reversed to facilitate comparison with the siinultaneous bipolar recording. B. Connections as in A. Double paper speed. C. Monopolar and bipolar recordings at 300 p interelectrode distance. Note that the fast sequence is more evident in the monopolar than in bipolar reading. D. Bipolar recordings at different interelectrode separations, depth 600-1000 p ( froin EM, 1962 ),

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cellular microelectrodes ( Verzeano, 1956; Verzeaiio and Negishi, 1960) seemed to offer considerable promise for resolving this contradiction. Recording differentially between two microelectrodes, and utilizing amplifiers with high rejection capability for common-mode signals ( i.e., signals arising remotely and thereforc impinging with equal intensity 011 both microelectrodes ) , it is possible efiectively to isolate the electrical activity of small volumes of brain tissue (Elul, 1962). The validity of this cdncept may be seen in Fig. 3, which illustrates an experiment with multiple microelectrodes. Spike activity appears at one of the electrodes, but has not been registered by a second electrode only 3 0 p away, indicating that the electrodes have been recording essentially focal activity. For this reason, as well as on account of the rejection by the amplifiers of conimon-mode signals, difierential recordings from two electrodes should represent, primarily, activity arising in the tissue between these two electrodes. If the EEG is produced through summation of a large nunibcr of spike potentials, as proposed by Adrian and Matthews ( 1934), clearly differential recordings should become increasingly spike-like as the volume of tissue ( and conscquently the number of nerve cells) producing the activity is made smaller. Yet, it became evident in this study that wave activity recorded between two electrodes only 3 0 p apart was as EEG-like in character as that recorded between two electrodes several centinietcrs apart. In fact, decreasing the inter-electrode separation had no appreciable effect on the wave activity recorded differentially between them (Fig. 3D). These results can be interpreted only as a contradiction of the Adrian-Matthews model. Moreover, since in experiments of this type the activity recorded differentially essentially represents the potential differences produced in the tissue between the two microelectrodes, wave activity may be recorded only when a single, unitaiy generator is smalk 7 than the interelectrode separation ( i.e., 30 ,U ) . Thus, these experiments, later confirmed by other investigators (Calvet et al., 1964), provided an cstiniate of the size of unitary generators of thc EEG, and suggested that these generators must be of cellular dimensions. It was not possible on the basis of such experiments, however, to arrive at a positive identification of the generators. For instance, the possibility still could not be rejected that the EEG may represent activity unique to neuroglial cells, as proposed by Galambos (1961).

D. INTRACELLULAR WAVEACTIVITYI N CORTICAL NERVECELLS More definitive information regarding the unitary generators of the EEG became available only subsequent to the extension of the technique

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of intracellular recording to cortical neurons. Sizeable synaptic potentials (10-15 mV) may be recorded in spinal motoneurons, but in the deeply anesthetized, as well as in the decerebrate spinal preparation, background activity in the absence of artificial stimuli is only 1-2 mV (Brock et a t , 1952). I n the cerebral cortex, intracellular recordings reveal “background activity of considerably higher amplitude-5 to 20 niV (Fig. 4). This wave activity qualifies for designation as “background’ only to the extent that, more often than not, it does not lead to discharge of a spike; but as may be seen in Fig. 4, cortical neurons exhibit wave activity continuously, so that waves are at least as characteristic of neurons in the cerebral cortex as spikes. In their overall appearance, neuronal waves closely resemble the EEG recorded adjacent at the surface with a gross electrode (upper tracings in Fig. 4 A-C), and modification of the character of the gross EEG,such as in the transition from wakefulness to sleep, is associated with a corresponding change in the cellular waves (Fig. 4A and 4C). Inspection of intracellular recordings suggest that there may be a close relationship between intracellular waves and the EEG. However, the nature of this relationship is not clear: Are the waves recorded by intracellular microelectrodes merely a reflection of ongoing wave activity in the extracellular medium? Alternatively it may be possible that the waves observed with extracellular microelectrodes reflect intracellular activity much the way that spikes recorded extracellularly originate in cellular activity. Finally, a third possibility may be that the intracellular and extracellular events are largely unrelated. Evaluation of the biophysical factors involved A 1100 pN

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FIG. 4. Neuronal wave activity recorded with intracellular niicroelectrode. Animal awake ( A ) , sleeping ( B ) and intensely aroused ( C ) , with EEG patterns Characteristic of each of these states. Note corresponding changes in form of the neuronal waves. A-C are from same cell in posterior suprnsylvian cortex, 750 p depth, micropipette filled with KCl solution. EEG taken froin anterior suprasylvian cortex on contralateral hemisphere ( from Elul, 1968).

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in the production of iiitracellular potentials is iiccessary for choosing between thesc three possibilities.

E. INTRA-AND EXTRACELLULAR POTENTIALS: BIOPHYSICAL CONSIDERATIONS It is important to stress that iiitracellular recordings merely reflect the potential between a given point in the interior of the cell, and a reference point several centimeters away. Changes in the potential recorded intracellularly may arise in at least three cliflerent ways, depending on the site of potential change: ( i ) The potential of the extracellular space adjacent to the cell may vary with respect to the reference point, and, because the potential of the interior of the cell bears a fixed relationship to the potential at the external surface of the membrane (cf. Hodgkin and Chandler, 1965), the internal potential also may vary relative to the reference point. ( i i ) The potential of the cell interior may vary with respect to the exterior, so that the voltage recorded between the intracellular and reference electrodes would reflect changes of potential across the cell niembrane. Such transmembrane potcntial changes may take place when the resting potential decreases or increases secondary to the action of substances interfering with ion transport either directly, or indirectly through their action 011 iTietabolisni-ouabaiii, for cxample; net charge may be lost or gained in these conditions. Processes of this type, however, generally are rather slow, and ion loss to a large extcnt is compensated by changes in osmolarity (cf. Hodgkin and Horowicz, 1959). ( iii ) An explanation commonly invoked to account for iiitracellular potential changes, based on thc localizcd conductance changes due to the release of synaptic transmitter substance by presynaptic terminals. Conductance changes of this type result in a large potential variation close to the synapse; further away inside the cell the potential change is progressively smaller, and intraccllular sites even more remote may show a significant potential shift in the opposite sense. Unlike (ii) above, this mechanism cntails no significant loss from the cell of ionic charges, insofar that the transniembrane flow of ions at the synapse is compensated by migration of ions in the opposite direction across the passive membrane elsewhere on the cell surface. In other words, the net flux of charge through the cell membrane is zero. A potential change is recorded only because, at a given instant in time, certain regions of the cell havc different charge from the rest of the cytoplasm. If the focal electrode is located at one of these regions, a potential change will be rcgistercd. Theoretically, it should also be possible to find, in the same cell, onc’ or more points which do not show

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any potential change, and a partial experimental corroboration of this assumption has been presented (Elul and Adey, 1966). The waves recorded by intracellular microelectrodes may thus arise either passively or in an active fashion. Passively, wave activity may appear intracellularly as a consequence of large extracellular electric fields which induce current flow across the cell. Alternatively, waves may arise actively, either due to localized conductance changes in synaptic spots on the cell surface, or perhaps to a lesser extent because of a generalized membrane conductance change. In attempting to identify the mechanism underlying the recorded waves, it is important to recognize that in the case of a passive process (i.e., the waves being due to variations in the extracellular potential), an electrode placed in vicinity of the cell, but outside its membrane, should register waves at least equal in magnitude to those recorded intracellularly. Figure 4 reveals neuronal waves which in several instances, particularly in tracing B, exceed 15 mV. If the hypothesis of extracellular (i.e., passive) origin of neuronal waves is correct, it should be possible to record comparable potentials with extracellular microelectrodes. However, in exploration of the cerebral cortex with niicroelectrodes, wave potentials of this magnitude are never encountered extracellularly; there are no reports in the neurophysiological literature of extracellular waves in excess of 1-2 mV [extracellular spikes have been described (Granit and Phillips, 1956) up to 30 mV peak amplitude, but all the evidence in that experiment seems to indicate that the electrode has been very close to, or in direct contact with the cell membrane (see also McIlwain and Creutzfeldt, 1967 and discussion of spike propagation in this review) 1. In nornial circumstances the waves recorded by extracellular electrodes are less than 1 mV and, as may be seen in Fig. 5, any activity recorded prior to penetration of the membrane is markedly lower in amplitude than the activity picked up by the same electrode once it has attained intracellular position. Notwithstanding their somewhat elementary nature, these considerations are quite forceful in excluding the possibility that intracellular waves are induced by extracellular fields. The low impedance presented by the extracellular medium (Ranck, 1963a,b; Van Harreveld et al., 1963; Adey et al., 1965; Nicholson, 1965) ensures that any such fields would be quite extensive; for this reason the patent inability to detect them with extracellular microelectrodes cannot simply be discounted as due to inadequate sampling of the extracellular space, but rather must be construed as concrete evidence against the presence of extracellular fields large enough to account for the iieuronal waves recorded intracellular1y.

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FIG.5. Difference in amplitude of netironal waves in the interior of the cell and immediately outside. Recording obtained during penetration of a cortical nerve cell. Wave activity too small to be seen in this gain with electrode outside cell, but appears immediately following impalement ( arrow in A ) . A-B represent continuous record. Note coincidence in spindling in the gross EEG and in the intracellular record. Anterior sigmoid cortex, 800 (I depth, K-citrate electrode. EEG derived from ipsilateral, anterior suprasylvian cortex (adapted from Elul, 1968).

F. EXTRACELLULAR WAVEACTIVITY ORIGINATING IK NERVECELLS It appears from the preceding discussion that the wave activity recorded with intracellular electrodes most likely originates in the very neurons explored. As already noted, such waves, regardless of the specific mechanism of their production, represent electric potential differences between the local micropipette and a gross reference electrode. The path connecting these two electrodes has a finite resistance, and the associated voltage drop is proportional to both the resistance of the membrane, and the resistance of the extracellular space separating the cell from the gross electrode. Membrane resistance is known to be quite high; for a typical cortical nerve cell it may amount to 1-10 MQ. (If the potential involves only a localized region of the cell, even higher impedances have to be contended with.) The resistance of the extracellular path to the reference point is rather more difficult to evaluate experimentally, because it depends not merely on the specific resistivity of the extracellular fluid, but also on tissue anatomy and particularly on the configuration and size of the space in proximity to the cell (see below). Nonetheless it must be realized that even if the extracellular path had only 1%of the resistance presented by the membrane, the voltage drop there would be sufficient to produce waves of 100-200 pV, i.e., the same as the EEG (cf. Eld, 1968). Estimates made in invertebrate ganglia suggest that an intra- to extracellular resistivity ratio of this order (1:100) may be reasonable [Kuffler and Potter, 1964, pp. 316-317, give glial mcmbrane resistance, and the “junc-

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tion resistance” of the extracellular fluid as 10” and 3 x loJ Q, respectively. In the same preparation neuronal membrane resistance was twice as high (see pp. 298-299 and p. 305), giving an impedance ratio of 6: 100.1

G. RELATIVEIMPORTANCE OF NEURONAL AND NEUROGLIAL CONTRIBUTIONS TO EXTRACELLULAR CURRENTS The above evidence, even when considered jointly with the data from extracellular studies cited in Section 111, C, does not necessarily identify nerve cells as the sole or even as the major source of EEG activity recorded with gross electrodes. One needs also to consider the possible role of glial cells as sources of extracellular current, especially since these cells are known to be sensitive to changes in extracellular potassium concentration secondary to repetitive firing of nerve cells (Kuffler and Potter, 1964; Orkand et al., 1966; Kumer et al., 1966). Conceivably, glial cells might in this way “convert” ongoing spike activity into wave potentials. However, glial potentials have an extremely slow time course, requiring several seconds to reach their peak. For this reason, it appears likely that the major glial contribution is to “steady cortical potentials,” rather than to the EEG from which potentials under 1 Hz generally are excluded. Also, the extracellular potassium concentration which determines the membrane potential of astrocytes ( Kuffler, 1967), undergoes significanc changes only in the course of synchronized activation of neilie cclls (Orkand, 1969). In spontaneous activity it is most unlikely that changes in extracellular potassium concentration would be sufficient to produce any glial potentials; in fact, intracellular recordings from cells which do not exhibit spike activity, and have low membrane resistance and therefore may be presumed to be glial, reveal no wave activity (Elul, 1966, 1968). On the other hand, additional evidence which has become availabIe very recently indicates that the EEG arises from nerve cell activity. This evidence derives from observations on the effects of drugs which block spike activity, such as tetrodotoxin. Although tetrodotoxin and siniilar substances only act 011 nerve cells, they block EEG activity when applied topically to the cortical surface ( E M , 1971, and 1972, unpublished data), These experiments will be described in greater detail below, in relation to the question of neuronal interrelations in production of the EEG. At this point it is possible to conclude from the disappearance of surface EEG activity following topical application of tetrodotoxin, that activity of nerve cells is essential for EEG activity. These results also show that in the absence of neuronal activity, any residual glial contributions are not sufficient to generate any detectable wave potentials.

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In summary, several points emerge from the experiments reviewed in this section: nerve cells in the cerebral cortex produce wave activity; this activity reflects active changes in membrane conductance in these neurons; and artificial blockade of nerve cell activity is associated with disappearance of surface EEG activity. It seems reasonable to conclude from this evidence that nerve cells constitute at least the principal, and perhaps the sole source of wave activity. Although some residual doubts still may linger about possible contributions of glial cells and of processes taking place in the extracellular space, their contributions are not likely to be critical uis-a-vis the contribution of nerve cells to surface EEG activity. IV. Site of Production of Wave Activity in Cortical Nerve Cells

A. “SYNAPTIC FUNCTIONAL UNITS”AS OF CORTICAL WAVEACTIVITY

THE

ELEMENTARY GENERATORS

Undoubtedly the most significant finding described in Section I11 is the presence of sustained wave activity in cortical nervc cells. Three possible explanations for this phenomenon have been considered ( Section 111, E ) ; based on certain experimental evidence reviewed there, it was possible with a high degree of ccrtainty to reject an extracellular origin for the waves. Both remaining alternatives involve intracellular production of the waves. One possibility is that the waves originate at a number of discrete foci on the cell surface as a consequence of synaptic activity; the other is that a uniform wavelike potential change occurs over the entire neuron surface, reflecting net loss or gain of charge from the cell. The question, then, is whether each nerve cell represents a single elementary generator of wave activity, or a number of relatively independent generators. From theoretical arguments it is not possible to reject either of the above alternatives. Potential changes have been demonstrated experimentally, especially in invertebrate preparations, which are caused by net loss of charge following administration of metabolic depressants or cooling (e.g., Hodgkin and Keynes 1955; Baker, 1969); apparently such changes may also b e rhythmical. Synaptic currents, needless to say, also generate large intracellular potentials, although the focal loss of charge is largely conipensated by ion movement through the surroundilig passive membrane. Unfortunately there is a significant hiatus in OUT understanding of this aspect of generation of the EEG. However, the available evidence favors a scheme in which the unitary generator of the EEG may be a single synapse (or more likely a group of synapses),

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rather than the entire neuron. This evidence derives from two sets of observations in cortical neurons: ( i ) Intracellular recordings in the cerebral cortex show quite clearly that different regions in the same nerve cell are not equipotential during spontaneous wave activity (Adey and Elul, 1965; Elul and Adey, 1966, and 1972, unpubhhed data). If the triggering level of successive spikes is measured, variations of several mV are commonly noted ( Fig. 6 ) . On the other hand it has been shown in experiments with intracellular injection of current (Lux and Pollen, 1966) that the threshold for triggering of spikes in cortical neurons is fairly constant. For this reason, the apparent variability in the threshold of spike triggering by spontaneous waves, most likely should be interpreted in terms of the intracellular resistance between the tip of the micropipette and different sites of origin on the membrane of successive waves. Since the resistance is proportional to the distance separating micropipette and synapse, equal flow of current from two synapses at different locations may be registered unequally by the electrode. Thus, these results suggest that there are multiple sites of origin of wave activity on the surface of each nerve cell; it is also quite clear from them that the cell surface is not equipotential, as should have been the case if the waves were due to gain or loss of net charge from the cell. (ii) Laminar analysis of the cerebral cortex, by means of extracellular microelectrodes, suggests that different components of spontaneous wave activity originate at different depths in the tissue (Calvet

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FIG. 7. Correlntion between evoked pottmtial recorded from the cortical surface and intracellular wave activity. The neuronal activity follows with reinarkable fidelity the surface record obtained from contralateral hemisphere. Stimulation of dorsal hippocampus contralateral to the site of intracellular recording (sigmoid cortex). Unanesthetized cat.

et al., 1964). Similar results have been obtained with regard to various components of evoked potentials (e.g., Spencer and Brookhart, 1961). Nonetheless, it is possible to record intracellularly a complex potential in response to sensoiy or subcortical Stimulation (Creutzfeldt et al., 1966b, see especially their Fig. 9; Elul and Adey, 1965; Elul, 1969a; R. A. Cyrulnik, P. A. Anninos, and R. E M , 1972, unpublished data). An example is given in Fig. 7, which shows striking correlation between the potential recorded intracellularly and that recorded from the suiface. It would appear from Fig. 7 that all coniponents of the surface evoked potential may be generated in the same neuron. In extracellular measurements, each component of the evoked potential is encountered at a different depth in the cortex; it is, therefore, likely that these components originate at different sites on the cell surface. This conclusion, again, supports the view that individual synapses act as unitary generators of wave activity, and that the waves seen in intracellular recording already represent the outcome of summation of individual synaptic contributions. It should be remembered, of course, that presynaptic fibers tend to branch and make multiple synaptic contacts with each postsynaptic neuron. Considering that these synapses all will be activated nearly simultaneously whenever the presynaptic fiber is triggered, the unitary generator is likely to be a group of synapses sharing the same presynaptic input rather than a single synapse. It may therefore be helpful to introduce the concept of ‘‘synaptic functional unit” in analogy with the “motor unit” in skeletal muscle. For purposes of the analysis of unitary contributions to the gross EEG, such “synaptic functional units” may be viewed as the elementary unitary generators of wave activity.

B. SYNAPTICAND NONSYNAPTIC POTENTIAL FLUCTUATIONS IN DENDRITES The evidence for diversity of focal sources of wave activity in each nerve cell is quite consistent. However, there are no clues as to which

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part of the neuron plays the principal role in producing wave potentials, The suggestion by Bishop, Chang, and other early investigators that dendrites are the primary source of the EEG has already been mentioned. This view apparently is still widely accepted, although evidence pro or con is meagre. To a large extent the lack of evidence is attributable to the fact that the majority of studies of spontaneous wave activity have been carried out in the cerebral cortex, where there is no certainty about the orientation of cells, unless identified by antidromic stimulation as pyramidal, or unless marked histochemically. Even if a neuron is positively identified by one of these methods, still it is difficult to ascertain whether the apical or basal dendrites are the main source of wave activity, because the complex topology of the extracellular space makes the interpretation of extracellular measurements in the cortex rather difficult. The analysis presented by Pollen (1969) may serve to illustrate this comment. The conclusion was reached by Pollen that potentials evoked by subcortical stimulation ( and by inference-also spontaneous EEG) originate primarily in large, vertically oriented neurons located at depth of the cortex-presumably the large pyramidal cells of layer V. However, a key assumption in this analysis has been that the spatial propagation of spikes and waves in the extracellular medium is comparable. This question is discussed in some detail in Section V, and it will be seen there that theoretical as well as experimental considerations cast serious doubt on the validity of this assumption. A second difficulty is that Pollen has assumed that the contribution of small neurons, such as interneurons, is negligible relative to the contribution of the large pyramids. While this is undoubtedly true on a cell-to-cell basis, it should be kept in mind that a unit volume of cortical tissue is likely to contain many more small neurons and dendritic processes than pyramids. Counts of synapses in unit volume of tissue may well eventually provide information which is more meaningful than the number in the same volume of large pyramids, or of any other subclass of neuron. Rather than additional measurements in the cerebral cortex, a more powerful approach may be through studies of structures, such as the hippocampus or olfactory lobe, where the location of the microelectrode may be equated with high degree of certitude with recording from apical dendrites, the cell body, and the basal dendrites, There have been a number of studies involving laminar analysis in the hippocampus (e.g., Andersen, 1960; Green et al., 1960; Fujita and Nakamura, 1961; Gloor et al., 1963; Andersen et al., 1964, 1966; Andersen and L@mo, 1966; Dichter and Spencer, 1969) but none of these contains significant information on the origin of spontaneous activity in this structure (but see also Rall, 1967; Rall and Shepherd, 1968).

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An additional question, which so far has remained unresolved, is whether wave activity is exclusively attributable to synapses. Data from experiments with tetrodotoxin ( Elul, 1971, and 1972, unpublished data), discussed in greater detail in Section VII, suggest that synaptlc activity may be necessary for presence of measurable EEG. It does not necessarily follow, however, that all ncuronal wave activity must be synaptic in origin. In fact, one possibility considered in Section VII, is that the main role of synaptic input is to synchronize the activities of individual generators. By their nature, excitable membranes are unstable, and under certain conditions there is a tendency for sustained oscillations. Oscillatory instability is implicit in the Hodgkin-Huxley equations ( Hodgkin and Huxley, 1952), and has also been observed experimentally in axons as well as in Aplysia neurons as a consequence of depolarization as well as in other preparations following changes in CaZ+concentration ( Arvanitaki, 1939; Shanes, 1949; Huxley, 1959). The action of synaptic currents is to modify the resting potential; by depolarizing the cell, oscillations might be induced in preparations with such latent instability. Based on estimates of the resting potential in the finer dendritic arborizations of central neurons, the suggestion has been made that such fine branches, on account of their high axial resistance and large surface area, may be at a substantially lower resting potential than the cell body of the same neurons. The lower resting potential is likely to make these elcnients predisposed to oscillatory behavior, and synapses located on these fine branches might act either to induce, or to quench ongoing cscillations ( E M , 1969c, and 1972, unpublished data; Elul and Adey, 1972, unpublished data). In this model, synapses may not be the priine source of wave activity, but they still would retain a key regulatory role, insofar as synaptic action may be needed either to initiate or terminate dendritic potential waves. V. Biophysical Factors in Summation of Unitary Neuronal Waves

A. BASIC ASSUMPTIONS-LINEARITY OF

THE

EXTRACELLULAR MEDIUM

Of the two basic questions formulated at the opening of this review, the material presented up to this point bears on the first topic, unitary generators of the EEG. A second fundamental problem is the relationship between these unitary generators and the gross EEG recorded from the scalp. In Section I11 it has been seen that cortical nerve cells produce rhythmic current flow across the cell membrane. The currents generated by individual neurons may combine ( or, alternatively, cancel) in the extracellular medium surrounding the active neurons. Consideration of the biophysical factors governing summation of the contribu-

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tions of individual generators therefore is an essential prerequisite for the understanding of unitary-gross relationships. This question will now be discussed. An additional aspect of the overall problem emerges from the data presented in Section IV, to the effect that neuronal wave activity results from, or is dependent upon, synaptic input. The possibility of synchronization of unitary generators is therefore important for understanding of the relationship of unitary wave activity and the surface EEG; this question will be dealt with in Section VI. In general it is quite obvious that generators which are close to the placement of a surface electrode should make a larger contribution to the recorded potentials than generators located further away. Conservative electric fields, such as electrostatic ones (in context of the EEG we are primarily concerned with electrostatic fields since the frequencies involved are far too low for electromagnetic effects to develop), superimpose and suni in linear fashion. Thus, for example, if the number of generators active within a given volume of brain tissue is doubled, the activity recorded by a remote gross electrode also will be increased to twice its initial value. As will be seen later, this property of linearity of summation is important in interpretation of variations in EEG amplitude. In context of the summation of activities of single generators to forni the gross EEG, linearity allows extrapolation from experimental observations on a relatively small number of elements, to the behavior of the entire generator population. Specifically, one may assume that: ( i ) extracellular currents emitted by individual neurons generate in the extracellular medium voltage drops corresponding to these currents; (ii) for a given amount of transmembrane current, the voltage drop created between the gross electrode and a reference point is primarily influenced by the proximity of the cell to the gross electrode; and (iii) all other things being equal, a larger number of active generators will result in a larger gross potential. However, as will be seen in the following paragraphs, the situation regarding spread of potentials in the cerebral cortex is rather more complex than might have been expected on the basis of the above three assumptions.

B. NONLINEARITIES DUE TO THE TOPOLOGY OF THE EXTRACELLULAR SPACE One important complicating factor in this elementary frame of reference is introduced by the topology of the extracellular space. TO understand this, it must be appreciated that, while single cells produce electric (or rather ionic) currents flowing in the extracellular medium, the recorders in use today register without exception, potential differences. The potential differences recorded between the focal electrode and a remote reference point reflect the voltage drop produced by flow of the

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unitary currents between these two points. It is evident that only a very small percentage of the current flows between these two electrodes, as the highest current density would be in close proximity to the cell. Yet, this small fraction of current is all the signal that is available at a distance from the active sources. Classically, the approach to problems of calculation of potentials induced by remote sources, such as nerve cells in volume conductor, has been through calculation of the solid angle subtended by the source as “seen” froni the electrode (e.g., Woodbuiy, 1965). However this approach is only valid for homogeneous extracellular media. There is ample evidence from nieasurements of tissue resistance (Nicholson, 1965), as well as from measurenients of spread of activity parallel and perpendicular to the surface by means of microelectrode arrays (Elul, 1962; Calvet et al., 1964) that spread of activity in the cortex is not unifoim in all directions (Fig. 8 ) . Indeed, one could hardly expect this to be the case, for it is well known that the extracellular space of the brain is composed of extremely narrow clefts (100-500 A )

FIG.8. Activity in depth of the cortex; records A-C from the experiment shown in Fig. 7. A. 1 5 0 0 p depth, light pentobarbital anesthesia; spindling evident at both inonopolar derivations but absent from the bipolar one, though the interelectrode distance is as much as 170 p . B. Electrodes in same position as in A; electrode 3 is 250 p deeper than 1 and 2; bipolar reading exhibits spindling to the same extent as its monopolar counterparts. C. Same RS B, faster paper speed; note similarity to surface reading. D. Different experinient. Depth, 1800 &; fast spindling originating at electrode 3 , which is only 50 p deeper than electrode 1. These results demonstrate unisotropic character of spread of wave activity in the cerebral cortex and in the underlying white matter ( froni Elul, 1962 ).

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which separate adjacent membraneous processes. Wave activity ( but not action potentials) is restricted to flow through these tortuous canals, so that the proximity or remoteness of the active source may have little bearing on the preferred path of flow of current. Rather, a more significant factor may be the route which offers the largest number of branching pathways communicating between the source and sink on the cell membrane, thus forming the path of least resistance. Considering that axonal fibers in the brain in general, and particularly in the cerebral cortex, are more likely to pass in certain directions than in others, and dendrites proliferate in rather constant orientation relative to the surface, it is to be expected that spread of extracellular currents would not be uniform. The experimental measurements of Nicholson ( 1965), and histological observations (Marsh et al., 1971; Fleischhauer et al., 1972) in particular lend support to this view. Thus, calculations involving extrapolation from homogeneous medium situation ( e.g., Pollen, 1969) must be treated with certain reserve.

C. NONLINEARITIES DUE

TO

CAPACITIVE EFFECTS

The nonhomogeneous nature of the extracellular medium in the brain becomes even clearer from consideration of the ddferences in propagation of spikes and wave activity in the tissue. One of the most striking features of the EEG, already noted above, is the absence of any focal sites of high-voltage wave activity in exploration of the cortex with gross electrodes; rather, the tissue appears to be completely permeated with wave activity. Even more remarkable is the fact that most extensive explorations with extracellular microelectrodes only reveal wave activity of 100-200 pV, even when these electrodes are in sufficient proximity to a cell to record at the same time relatively large spikes ( L i and Jasper, 1953; see also Figs. 2, 3, pp. 232, 234). A model of the EEG based on spread of activity in homogeneous medium (e.g., Pollen, 1969), would have led to expectation of a gradual increase in amplitude of wave activity as the cell is approached; in reality such an increase is never observed, and as already noted the passage from extra- to intracellular position involves a rather .abrupt change in amplitude (Fig. 5 ) . This order of magnitude increase, by itself, might have been taken as an indication that prior to penetration, the electrode had made contact with an inactive patch of the cellular membrane (cf. Freygang, 1958; Freygang and Frank, 19S9)-which may well be the case. Nonetheless, repeated explorations in the extracellular medium fail to reveal large waves, and there is no detectable gradient in amplitude of the waves prior to impalement of the cell. Spikes, in contrast to waves, do show a significant increase in

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amplitude as the microelectrode is moved through the tissue and especially prior to penetration of an active neuron. Indeed, this increase in amplitude from 100-200 p V to 10-20 mV, is often used in physiological recording as a criterion of proximity to a nerve cell. As will be seen below, with regard to spread of spikes the extracellular space is, for all practical purposes, homogeneous; in fact, the spatial decrement of spikes upon increase in distance from their source, is characteristic of spread in homogeneous medium. On the other hand, with regard to the spread of wave activity, the extracellular space quite clearly does not behave as expected of an homogeneous medium. Since physiological recordings are voltage recordings, and the voltage V = RI, the relative constancy of amplitude of wave activity must imply constancy of the product RI (that is, the product of the resistance of thc extracellular medium and the local current density). No system of focal current sources in homogeneous medium is capable of constancy of RI everywhere in space; but the evidence for focality of the current sources in the present case is very substantial (see Section I V ) . It would therefore appear likely that the medium is not homogeneous. The fixed magnitude of extracellular EEG fields may simply reflect the topology of the tissue, in the sense that the number of branching canals of extracellular medium in the vicinity of truly focal sources must be quite small, but as the distance from the sourcc increases, additional branching canals become available, with the result that the effective resistance of the medium between the source and sink may remain fairly constant, or even significantly decrease, at distance from the generator. It certainly is not compatible with homogeneity of the extracellular space.

D. NONLINEARITIES INTRODUCED BY THE RECORDINGELECTRODES

A third complicating factor in evaluating the generator population contributing to EEG activity at any given point stems from the fact that disc electrodes as typically employed in EEG recordings are not uniformly sensitive to dipole generators at different spatial locations (Fig. 9). Equipotential lines for the location of identical dipole generators oriented at 45" to the plane of the electrode are plotted in Fig. 9, which is drawn from Peronnet et at. (1971). It is seen that the electrode is completely "blind" to dipoles at certain locations, and that dipoles in some locations at substantial distance make an equal contribution to the potential at the electrode as dipoles considerably closer but in less favorable locations. It should be appreciated that this extremely nonlinear state of events prevails with dipoles oriented uniformly at 45" to the electrode; in a more realistic situation, with dipoles in

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FIG.9. Nonlinearity in spatial sensitivity of the disc electrodes used in electroencephalography. Equipotential lines have been drawn for dipoles of identical intensity oriented at 45’ to the plane of the electrode ( a t bottom) and placed in various locations in the volunie conductor which is in contact with the electrode. Dipoles located anywhere on the 0 line are not recorded at all by the electrode; those on the “-5” line indnce in the electrode a potential opposite in polarity to that induced by the same dipole in identical orientation, but placed on the “+5” line (from Peronnet et al., 1971).

random orientations, variability is even greater, and a statistical approach must be used to estimate the contribution from a neuronal population ( Elul, 1966). The preceding discussion suggested that the amplitudc of EEG potentials, even when recorded focally with microelectrodes, is not very helpful in providing an estimate of the distance of the active generators from the electrode; it appears that with gross electrodes it may be even more difficult to evaluate the generator population which contributes to the recorded activity,

E. DIFFERENCES IN SPATIALPROPAGATION OF Simms AND WAVES The possibility of contribution by spikes to gross potentials is often raised. Spikes are not observable in the gross record; it is an experimental observation that EEG may be recorded even in the absence of spikes (Li and Jasper, 1953). Also, the normal EEG does not contain visible

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spikes [although small spikes can be visualized through use of wideband amplifiers, especially with small electrodes ( see Schlag and Balvin, 1963)l. Spikes recorded undcr these conditions are smaller in amplitude than the concomitant EEG waves, whereas, in iiitracellular records, spikes are at least three times largcr than the waves. It is therefore nccessary to explain how it is possible that spikes, which after all involve very substantial transmembrane current, make such an insignificant contribution to the gross activity. A frcquent answer to this question is that the extracellular mediiun has the properties of a low-pass filter (Humphrey, 1968; Pollen, 1969). Unfortunately, this viewpoint is somewhat misleading. There is 110 doubt that cellular membranes present much lowcr impedance to rapid transients than to slow potential changes. Thc reason for this is that a sharp transient is capable of crossing the membrane by capacitive coupling whereas slow potentials involve actual ion movement through pores in the membrane. The high capacitance of cellular membranes ( 1-20 pF/cm’ ) makes them essentially “transparent” to rapid transients. Thus, spikes originating from a ncuronal source are virtually free to propagate through brain tissue across cellular barriers, whereas the slower wave activity is limited to the narrow clefts of the extracellular space. Consequently the propagation of spikes takes place effectively in homogeneous mcdiuni, a 1 ~ the 1 rcsistance presented to thc flow of the extracellular currents produced by spikes is much lower than the resistance of the same tissue to the flow of slow currents. As already noted, electrophysiological recording entails measurement of the voltage drop iiiduced by extracellular currents flowing against the resistance of the medium; for this reason, the voltage drop recorded at a given distaiicc from a point source for a spike is much smaller than that recorded from a wave originating at the same point, even when the instantaneous extracellular current is the same in both instances. The difference in the effective impedance presented by the medium to spikes and to wave activity may explain the marked difference in their spatial decrement discussed in the preceding paragraphs. In an homogeneous medium, activity emanating from a focal source decays inversely with increasing distance ( i n case of dipoles, the spatial decay is an inverse function of the third power of thc distance). Thus one may expect to observe an increase in spike amplitude as the electrode approaches the cell-as indeed is the case. I n contrast, wave activity propagates only through the minute extracellrilar clefts, so that the voltage (the product of current density and effective resistance) may well stay approximately constant at various distances from the active region. An important corollary is that the ratio between the amplitude

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of spikes recorded with iiiicroelectrodes immediately adjacent to the cell membrane, and the wave activity recorded by the same microelectrode, may provide an estimate of the ratio between the transmembrane currents responsible for both processes. This ratio is from 1 : l O to 1:100, and would suggest that the currents responsible for wave activity niight be significantly smaller than those produced by spike discharge. VI. Theoretical Analysis of Systems of Unitary Generators

A. Two POSSIBLE MODELS:SYNCHRONIZED AND NONSYNCHRONIZED GENERATOR SYSTEMS The part played by “synaptic functional units” as the unitary generators of neuronal waves (or at least their regulating action on these unitaiy generators), has been discussed in some length in Section IV. The synaptic involvemcnt in wave production raises the possibility that extracellular currents produced by individual generators may combine in some consistent way to form the gross EEG. For evoked potentials, formal analysis may be feasible, because the extraneous stimulus is likely to reach a large number of generators at nearly the same time (even though this stimulus may well produce outward currents in some synapses and inward currents in others ). Spontaneous activity presents an enormously more complex situation, as there are no a priori grounds to anticipate that any two generators may receive a similar input at any given moment in time. Ideally, a i m p of the inputs to each neilie cell in the cortex should be available, and if this map were consulted, it might be possible to deduce the likely role of various nerve cells in the production of each EEG deflection. Needless to say, not only arc such maps presently unavailable, but even if they were to be had, no computer in existence today would be capable of storing this information. For this reason, we are forced to use at this time a less powerful, but perhaps more general, approach: exploration of the statistical aspects of the relationship between individual generators of wave activity. In view of the sparsity of relevant information on structure of the cortex, it is best to begin by considering two extreme situations: The generator population contributing to the activity recorded at the surface may either be fully synclzroniaed or, alternatively, individual generators may be totally independent of one another. It is easy to see that these two models imply rather different experimental observations on the relationship between activity of any given cell and the gross EEG recorded adjacently: With a synchroi3ized generator population, the gross EEG should be synchronized with the activity of any arbitrarily

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selected generator. On the other hand, if the relationship among the elementary generators is random, no single generator can bc synchronized with the EEG. (Usage of the term “synchronization” here does not necessarily imply that activity of one element is a replica of the other; phase delay is also permissible, provided that it is constant-of course a continuously variable phase h g between two processes amounts to a random relationship-or at the very least to a relationship that is noillinear in time. ) At this point, the reader may be justified in inquiring whether these two models are consistent at all with the information available from intracellular and other observations. It is not immediately obvious that the extracellular currents produced by a population of synchronized generators may appear similar to the gross EEG. Likewise, is it possible for EEG-like activity to arise by summation of contributions from randomly-related generators? Indeed, it appears rather unlikely that mechanisms as disparate as the two proposed above might lead to the same end product. Somewhat unexpcctedly, the evaluation of synchronized and independent generator populations undertaken in the following paragraphs indicates that the output produced in both cases would be closely similar. Fortunately, this analysis also reveal$ certain criteria for distinguishing between thcse two possibilities in experimental data.

B. PHASERELATIONSHIPOF THE SUMMEDRECORDIN GENERATOR POPULATIONS OF THE Two TYPES Assuming first that the gross EEG originates in a synchronized population of nerve cells, clearly tlic wave activity of any given generator should be synchronized with tlic surface record. This statement remains valid even if the individual generators are not uniformly oriented in space, for one of the elementary properties of dipoles is that a dipolc of any arbitrary orientation may be decomposed into two orthogonal c o n ponent dipoles, e.g., one perpendicular to the surface, and a second dipolc parallel to the surface (cf. E M , 1966, 1969a; Pollen, 1969). In this maiincr, a population of generators at random orientations may be rcprcsellted by a set of uniformly oriented elements (Elul, 1966). If thc polarities of the generators may be opposite in certain cases ( e g , synapses on apical dendrites in some cells, and on basal dendrites in other cells), the resultant activity would be smaller in amplitude, owing to mutual cancellation of the contributions of some generators. Nonetheless, even in this situation, any randomly sampled unitary generator would be synchronized with the activity recorded at the surface. With a nonsynchronized generator population, it would appear that

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the summed activity would tend towards zero, as the individual contributions might oppose one another. This however, is not the case, In reality, the summed activity of a group of generators emitting similar, but not synchronized activity, has the same general appearance and frequency content as the activity of any given generator, although this summed activity is not synchronized with any single generator. To understand this apparently paradoxical result, one may consider the sum of two sine waves of identical frequency and amplitude, but emitted by two independent oscillators. If the oscillators happen to be at 180" phase lag, their activities would cancel. At any phase lag other than B O O , the waves would not cancel, and their summation would result in a sine wave of precisely the same frequency, but differing in phase and amplitude from either of the parent waveforms. By combining the resultant sine wave with the output of a third, similar oscillator, etc., this process may be extended to encompass any desired number of generators, the ensuing activity always retaining the fundamental frequency characterizing each of the individual elements. It would be most unlikely for the summed activity to match in phase any given unitary generator except by chance. The situation would be essentially similar with broadband generators, each emitting a wide spectrum of frequencies (as do neuronal wave generators). The summed activity in this situation would be characterized by a similar, broad-frequency spectrum; however, there should not be a constant phase relationship with any single unitary generator. This result is clearly different from that obtained in a synchronized generator population, and the distinction provides the first criterion for distinguishing between synchronized generator systems.

C. AMPLITUDEOF THE SUMMEDRECORDIN GENERATOR POPULATIONS OF THE Two TYPES A second criterion is provided from measurements of the amplitude of the output in the two situations. In a synchronized population, amplitude of the summed activity obviously increases in direct proportion to the number of participating generators. In contrast, as the size of a population of nonsynchronized generators is increased, the output increases only proportionalIy to the square root of the number of unitary generators. For example, when a population of 10,000 generators changes from a synchronized to desynchronized state, its output should decrease 100-fold (i.e., from 10,000 arbitrary units to ( 10,000) = 100 such units). Thus, the output of a generator population may be expected to be substantially larger when this population is synchronized than when it is desynchronized. ''2

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VII. Experimental Analysis of Population Behavior of Cortical W a v e Generators

A. PRELIMINARY COMMENTS ON APPLICATION OF PHASEAND AMPLITUDECRITERIA TO EXPERIMENTAL RATA Because the relationship between activity of a single cell and the gross EEG can be examined readily in intracellular recording, substantial experimental data pertinent to the relationship between unitary generators is available. It must be emphasized that, at the present time, observations of this type constitute our principal source of information on the mechanisms of production of gross wave activity, and for this reason they merit a detailed examin, t’ion. Of the two criteria proposed in Section VI for evaluation of the relationship of the EEG to unitary generators, that is, as regards phase relationships and amplitude, only the first is immediately applicable to data from intracellular recordings. Although the output of synchronized and nonsynchronized generator populations is likely to be very different, the amplitude criterion is difficult to apply in the absence of information on the magnitude of the contribution to the gross potential by an average single generator. As noted in Section IV, there is at present no way to identify, in the gross record, the contribution of a single generator, and information on the approximate size of the generator population contributing to the activity picked up by a gross electrode also is lacking. Without knowledge of these two parameters, it is not possible to predict the expected amplitude of the gross activity in synchronized and nonsynchronized situations, so that one cannot decide from the amplitude observed in experimental data whether the neuronal population is, or is not, likely to be synchronizcd. Nonetheless, it appears reasonable to assunie that a typical population may contain well in excess of 10,000 elements (see below), so that amplitude variations of the order 1:100, or even larger, may be expected. While the absolute level of expected amplitude still remains unknown, the possibility of changes of this magnitude provides a poweiful test of models formulated from measurements of phase relations, as will lie seen later. The initial test, however, is whether synchrony of the unitary generator with surface activity can be detected. This question is amenable to examination in intracellular recordings, but i t must be recalled that an intracellular recording is likely to represent the activity of more than one generator. Indeed, if each synapse, or “synaptic functional unit,” is activated independently of other such elements, each cell would represent the combined activity of many thousands of elementary generators [the synaptic count on central neurons is in the order of lo4 to 10’

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(Kositzyn, 1964)’). In the event that the generator population is synchronized, this need not present any difficulty, for the individual generators should then be synchronized anioiig themselves, so that in this situation any intracellular record, regardless where in the cell it is taken, would replicate typical generator activity. With a nonsynchronized system of generators, the intracellular record also would represent suinmed activity, but because of the random relationship of elementary generators, the intracellular recorded activity would be a composite of individual synaptic potentials; in this case, however, intracellular waves would only bear random relationships to the activity of any given generator, and thus should not be synchronized with the gross EEG. In summary, the detection in the experimental data of synchronization of intracellular recordings with the EEG can provide strong support for viewing the EEG as the product of a synchronized population of unitary generators. On the other hand, failure to detect such correlation between intracellular records and the EEG would suggest just as emphatically that the EEG represents the sum of iionsynchronized generators. B. PHASERELATIONSHIP BETWEEN INTRACELLULAR ACTIVITY AND SURFACEEEG

Which of the two alternatives is supported by experimental findings? Two sets of observations which seem to point in opposite directions have been reported. Working primarily with anesthetized preparations, Creutzfeldt and co-workers ( 1966a,b) have presented evidence for positive correlation between intracellular recordings and EEG activity. Work on unanesthetized, waking cats, on the other hand, has failed to reveal any consistent synchrony or correlation ( E M , 1966, 1968). One possibility, that the differencc bctweeii these results may reflect different international relationships in wakefulness and in sleep, can be ruled out on the basis of the theoretical considerations discussed in Section VII, A. As noted there, a change from nonsynchrony to a synchronized state entails a very marked increase in amplitude. However, in practice, the niaximum difference observed between EEG amplitude in wakefulness and sleep does not exceed 3- to 10-fold. Thus, the disagreement between the two experiments may be explained in terms of a shift from nonsynchronized generator population in wakefulness to a synchronized one in sleep, only if the total generator population were 10100. Without going into detailed consideration of cortical anatomy it is evident that a surface electrode of some 1-5 mm2 (as used in EEG studies) must pick up activity from many times this number of unitary generators, even if the entire neuron, rather than a single synapse, is considered as the unitaiy generator. For this reason, interpretation of

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the two sets of data in terms of synchronization and desynchronization, respectively, is untenable. Apart from the difference in behavioral state of the preparation, the most significant difference between the two experiments has been that positive correlation of neuronal waves with the EEG was described from observations of short duration (1-2 sec; see Creutzfeldt et al., 1966b), whereas the other study involved statistical examination of long epochs ( u p to 10 min; see Elul, 1966, 1968), and it was statistical tests, rather than direct observations, which failed to reveal any consistent relationship between intracellular activity and the EEG. It was noted in this latter study that short periods may be found which exhibit excellent correlation. Figure 10, drawn froin Elul (1966), shows one such case, where in the period delimited by interrupted lines intracellular activity is nearly perfectly synchronized with the EEG. However, when inspection is extended beyond these boundaries, the relationship rapidly deteriorates and subsequently disappears. In fact, if the intracellular wave activity of a single cell is observed over an extended period of time, there are found oiily very isolated instances of correlation. Figure 11 represents the results of a quantitative analysis, utilizing computations of spectral ‘coherence’ ( Goodman, 1957; Walter, 1963; Walter and Adey, 1963). With this approach, spectral decoinpositioii into bands of 0.5 Hz is perfoimied using either digital filtering or Fourier transform techniques. Subsequently, the noiinalized cross-correlntion of corresponding bands in the two activities over the same 10-sec epoch is calculated. The level of cross-correlation attained is plotted as shaded areas with 0.5 Hz resolution, in the contour map in Fig. 11. Although there are found many instances of apparent correlation, it must be realized that such apparent correlations may also arise by chance. For instance, the black-shaded areas in Fig. 11 represent epochs in which the likelihood of correlation arising b y chance is only 5%, but 5% chance correlation implies that 1 out of 20 trials will produce a “false positive.” Since the complex wave-

EEG

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FIG.10. Relationship between the EEG and intracellular activity. Recording conditions as in Fig. 4. Activity between the dashed lines is quite synchronous, but the phase relationship reverses on the right and then can no longer be traced (froin Elnl, 1966).

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FIG. 11. Coherence between EEG and neuronal wave activity of a single cortical neuron computed over a 500-sec continuon\ epoch. Each 10-sec epoch involves 30 separate measurements, corresponding to each 0.5 Hz, and coherence at the 95% confidence level should therefore occur by chance, 1.5 times in each 10-sec epoch; so that even when the co~nnionfrequency hand\ extend over 1 Hz (for example at 40, 60, or 130 sec) or even over 2 Hz ( a t 290 sec) a chance occiirience cannot be excluded with confidence. When, however, high coherence level is found in the saine frequency band over two adjacent 10-sec epochs, a chance occtii ience liecomes far less likely, and the probability of coherence at the 100-120-sec period and at 11.5-12.5 Hz occiiriing by chance is 0.16% (from Elnl, 1968).

foiiii in each 10-sec epoch is deconiposcd into 30 frequency bands, we may expect 30 x 0.5 = 1.5 false positives per 10-sec epoch. Whcn the nuinber of positive correlations at the various significance levels ( solid line in Fig. 12) is compared with the number of apparent correlations which might have been obtained from two entirely unrelated processes (interrupted line in Fig. 12), excellent agreement is found between the two curves. In other words, over long periods of time, the correlations observed in Fig. 10 do not exceed the level expected for two processes which have no causal relationship whatsoever. The uiiavoidable coilclusion is that apparent correlations may arise even between completely independent EEG’s. This conclusion is illustrated graphically in Fig. 13: an EEG recorded from one animal is displayed next to intracellular activity from a second cat, and several instalices of “correlation” are clearly evident. One important point is ignored in statistical analysis of the type iIlustrated in Figs. 11-12: Cross-correlation computations (either crosscorrelation or coherence), perfoinied over a given period, only yield an average score of the level of coincidence in the two processes. These procedures do not distinguish, for instance, betwcen a siiigle brief epoch of intense correlation, and a number of separate epochs of weak cor-

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6007

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EXPERIMENTAL EXPECTED

FIG. 12. Correlation between EEG and unitary iierironal \wives ( solid line) compared to correlation between two independent processes (interrupted line). Further explanation in text.

relation which also niay fall within the period under analysis. Because of this, it is quite possible that similar scores niay be obtained from two sets of data as different as those in Fig. 10 and Fig. 13. Visual inspection, however, reveals the correlation in Fig. 10 to be highly consistent, in contradistinction to the data in Fig. 13, where only occasional “correlation” restricted to one potential peak is found. Although in a record of, say, 10 sec, the total number of these spuriously correlated peaks in Fig. 13 may even exceed the number of correlated peaks in Fig. 10. there is no question that the record of Fig. 10 is less likely to arise by chance alone. Indeed, if analysis is restricted to shorter epochs, e.g., 0.25 sec, the correlation found for the data of Fig. 10 would be manifestly higher than for the data of Fig. 13. Unfortunately, however, data of the type presented in Fig. 10 are rather infrequent in intracellular recordings from cortical neurons. It is therefore difficult to decide whether there is any meaningful relationship between neuronal wave activity and the surface EEG. This problem is illustrated in Fig. 14, redrawn from the paper by Creutzfeldt et al. ( 1966b); the technique of graphical superimposition was utilized by these authors to demonstrate correlation. The top tracing reveals the outcome when successive epochs of intracellular activity were superimposed each time a spindle wave appeared in the gross EEG (Fig. 14A). Agreement between the unitary and gross activities with this procedure was not very inipressive; correlation could be improved 0x11~

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FIG. 13. Apparent correlation between E E C from one cat (top tracings, A-C) and intracellular activity from a second cat (middle tracings). EEG of the second animal is included for coniparison (lower tracings). Voltage calibration 100 pV for EEG and 80 mV for intracellnlar activity. A-C are three difl'erent examples from the same nerve cell. Activity of nerve cell and EEG (middle and lower tracings) were registered with a chart recorder. EEG activity from the other cat (top tracing) was played back off magnetic tape and recorded subsequently. Note that the top tracings often shows better correlation with the intracellular activity than the EEG activity from the same nnimal. This is x consequence of the narrow frequency band of the EEG.

if the EEG waves were selected by the investigators according to their shape (Fig. 14B). It is therefore clear that any statistical test which treats all successive blocks of incoming data indiscriminately would be extremely unlikely to indicate positive correlation between neuronal waves and surface recordings. Yet, fsoni a statistical viewpoint, there is no reason to accord preferential treatment to any particular portion of the data and, indeed, it can be demonstrated that an unbiased treatment is least likely to give rise to statistical errors. The evidence reviewed here may leave the reader in a quandry: Visual inspection suggests that there may be excellent correlation of wave activity in individual nerve cells with the sinface EEG, but that such correlation may occur only rather infrequently and for extremely short periods (under I sec). On the other hand, quantitative analysis of

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A

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FIG. 14. Difficulty of correlating neuronal waves with EEG activity in spindle sleep. Intracellular recording from cat cerebral cortex, pentol)arl~italanesthesia. Short epoch drawn from continuous record and siiperimposed u.;ing EEG spindles (upper tracings in A-B ) as the criterion for temporal aligninent. A. :Ill sriccessive spindles estracted from record. B. Certain spinclles only selected visually nccording t o shape. Note that correlation of nenronal wave activity the surface recording is somewhat loose in A, but quite striking in A ( r e d r a w n follo\ving Figs. 8 ant1 10 of Crer~tzfeldt et al., 196613). \\Titi>

the same experimental data s~eiiisto indicate that there is no correlation there which could not potentially be explained by chance alone.

C. AMPLITUDE RELATIONS : EXPERIMENTS WITH TETRODOTOXIN Experimental exaniination of relationships between unitary generators and the EEG up to this point has only involved phase relations. As previously noted (Section VI, C ) , the range of changes in amplitude which can be observed experimentall y provides a second and independent criterion, but application of this test is hampercd by tlie lack of a franie of reference for evaluation of the amplitucle of gross activity in tcrms of the individual contri1)iitions. Reccntly, however, one c.spcrimcntal situation h a s beconic available whcrc the amplitude criterion inay be usefully applied. This situation i s iii experiments involving administration of tetrodotoxin ( T T X ) into the brain ventricle (Elul, 1971, and 1972, unpub-

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FIG. 15. Eflect of iiitraventricular tetrodotoaiii on EEG. A. Taken just before injection of the drug. B. 10 inin later. C. 20 inin following the injection. Bipolar recording from surface electrodes making contact with the dura ( parietal-parietal ), calibration: 1 sec, 50 pV.

lished data). When administered in this way, TTX induces intermittent flattening of surface EEG (Fig. 15). The episodes of flattening are relatively shoit (1-20 sec), and thus provide an opportunity to record from the same neuron intracellularly during EEG “on” as wcll as “off conditions. If the EEG represents the output of a synchronized generator population, then a sudden reduction in overall amplitude, as illustrated in Fig. 15, may reflect a temporary desyiiclironizatioii of the individual generators. With a nonsynchronized population, on the other hand, reduction in amplitude may only result from a corresponding reduction in unitary output in all generators, or alteiiiativcly from a temporary cessation of all wave activity in a substantial fraction of generators. In the case of TTX, where activity is reduced to approximately oiily 10%of its initial valuc, this would imply cither a 90%reduction in output of each generator, or complete isoelectricity of 90% of the generators. Since changes in generator output must be reflected on the neuronal level, comparison of intracellular wave activity during flattening episodes with the activity just preceding and following them, may provide inipoitant information on the relationship bctwccn single generators and the gross EEG. Data from intracellular recordings during TTX-induced flattening of the EEG is presented in Fig. 16. The top tracing in each record represents the gross EEG, and the lower one rcpresents simultaneously recorded intracellular activity. It will be seen that intracellular activity is not significantly diminished in amplitude during flattening of the gross EEG. Nonetheless, there are some subtle changes in intracellular activity during EEG pauses. The most significant change is a reduction in spike firing rate, occasionally even leading to a complete arrest of

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FIG. 16. Effect of tetrodotoxiii on neuronal \\Live ‘tctivity and (in EEG. Uiianesthetized cat, 950 p depth in signioid cortex; EEG taken from surface about 1.5 nini away. A-B successive episodes of EEG “flattening” (delimited by arrows). Note inconsistent behavior of nerve cell during three episodes.

firing during EEG flattening. It is important to note that the changes in firing rate are not consistent from one flattening episode to the next, and in none of the episodes are they closely synchronized with the onset of the reduction in EEG amplitude. Referring to Fig. 16, the neuron seen there stops firing during one episode of EEG flattening, but persists in spike activity during another episode.

D. A TENTATIVE MODELOF GENERATION OF

THE

EEG

The findings from thc expc>rinients with tetrodotoxin effectively exclude interpretation of the EEG as the output from a nonsynchronized neuron population, considering that the reduction in gross EEG amplitude was not accompanied with a corresponding attenuation of unitary activity. On the other hand, a clecreasc in firing rate was observed. Such decrease in firing would be likely to limit the capability of the observed cell to conimunicate with other nerve cells, and thus result in a desynchronizing effect. However, the firing rate is not consistently affected each time the gross EEG is flattened. Rather, Fig. 16 suggests that the neuron under obscrvation participated in the events underlying EEG flattening on certain occasions only, but was not involved the rest of the time. While this evidence is not conclusive, the tetrodotoxin experiments leave little, if any, doubt that the EEG cannot be the product of a nonsynchronized population. At thc same time, it is necessaiy to take account also of the statistical analysis (Figs. 11-12) which excludes quite emphatically the possibility of consistent synchrony of the neuronal generators with the EEG. These two conflicting results can be reconciled by adoption of a model bascd on sporadic and short-term synchrony of unitary generators. It may be necessaiy to recall that even if only a small fraction of the total population is synchronized at any given moment, the summed output of this fraction may well ovenveigh the combined wave activity of the rest of the neuronal population. For example, consider a generator population of 10,000. Evcn if only 10% of this population are syuchro-

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nized, this segment of the population will produce 10-fold the activity of the remaining 90%of the generators which are not synchronized (10% of 10,000 is 1,000, whereas the activity of a nonsynchronized population of 10,000 would be only (9000)’/2 z 100). For typical generator populations it is thus possible that a relatively small portion of the total neuronal population which is synchronized will produce significant activity. Although the rest of the population is likely to be coiitiiiuously active, the amplitude of this nonsynchronized activity might be so small that it could well remain below the resolution level of EEG recorders. That a comparatively small part of the generator population may be capable of producing activity of the same amplitude as the EEG is indicated by the studies of evoked potentials (R. A. Cyrulnik, P. A. Anninos, and R. Elul, 1972, unpublished data; see Fig. 7, p. 243), where it is known that the gross evoked potential is produced by some 10-20% of the neuronal population, and where there is no doubt that this fraction of the population is synchronized as a consequence of the artificial stimulation. One additional point merits comment in relation to this model. The studies of spontaneous activity, of evoked activity, and of the effects of tetrodotoxin on the EEG cited above, clearly exclude the possibility of consistent synchronization of a fixed group of generators with the EEG. There is thus no specific class of specialized “EEG generators” in the cortex. Rather, the available evidence suggests that all neuronal generators (or at least a large percentage of the population) alternate in this role, intermittently becoming synchronized with the EEG. In this way the critical generator population may be active all the time, but synchronization would shift from one cell group to another every fraction of n second. The experiments with tetrodotoxin also provide some preliminary information on the mechanisms responsible for this shift in synchrony, and suggest some possible behavioral implications, which are discussed in the following section. VIII. Subcortical Control of the EEG a n d Possible Functional Implications

A. SUBCORTICAL PACEMAKER FOR

THE

EEG

The experiments involving administration of tetrodotoxin ( TTX) into the lateral brain ventricle provide important information not only on the relationship between unitary generators and the gross EEG, but also on the mechanism of control of these generators. In Section VII, the conclusion was reached that the EEG recorded from the cortical surface represents intermittent synchronization of relatively snialI groups of neurons. The neuronal mechanisms underlying such intermittent synchronization and desynchronization, and the selection for synchroniza-

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tion of alternate groups of neurons, remain to be considered. Although a comprehensive answer to this question still is not within our reach, the data presently available suggests certain intriguing possibilities. It should be realized in these experiments that tetrodotoxin is applied by microinjection into the ventricle, i.e., some 5 1 0 m m from the cortical surface. Previous studies on the diffusion of tetrodotoxin in brain tissue have revealed it to be exceedingly slow ( Hafenximi et al., 1969). Since flattening of the EEG first appears within 10-20 niin., one would suspect that the flattening may not express a direct effect on cortical neurons. The presence of spikes in cortical ~ieurons,and indeed even during episodes of EEG flattening ( Fig. 16) supports this interpretation, since tctrodotoxin, when present in effective concentration in the tissue, invariably blocks spikes by interfering with the inward sodium current during the action potential (cf. Narahashi et al., 1964). Thus, if TTX were to induce in these experiments EEG flattening by direct action on the cerebral cortex, no spikes should have been present in the cortex. There is, of course, the possibility that, in contrast to all other nerve cells studied in vertebrate and invertebrate preparations, in which without exception tetrodotoxin blocks the action potential ( Kao, 1966; cf. also Blankenship, 1968, for manimalian CNS ) cortical neurons may be ininiune to this effect. However, direct application of TTX on the cortical surface within 1 0 3 0 niin, blocks all spike firing in the underlying tissue (Elul, 1971, and 1972, unpublished data). Hence, the presence of spikes in the cerebral cortex during episodes of EEG flattening (Fig. 16), must be viewed as strong evidence against a cortical action of this drug in the experiments considered here. It is likely, therefore, that the effect of TTX on the EEG in the present experiments is attributable to a subcortical action of the drug. In other words, a subcortical mechanism may exist which is vital for EEG activity and, when blocked by TTX, would bring about disappearance of the EEG. Although the data presently available does not make possible localization of this subcortical mechanism, the slow rate of diffusion of TTX in brain tissue, and the rapid onset of EEG flattening, tend to implicate one or more of the structures imniediately adjacent to the brain ventricles. More specific identification of the subcortical controlling centers responsible for the EEG has been proposed in the monograph by Andersen and Andersson (1968). Rased on the early results by Andersen and Sears (1!364), as well as morc recent information on autorhythmicity in the thalamus ( Andersson and Manson, 19711, these authors conclude that the thalamus is the pacemaker for cortical rhythms (Andersen and Andersson, 1968). Detailed discussion of subcortical mechanisms is beyond the scope of the present review, and the primary objective in

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this section is to consider consequences at the level of the cerebral cortex of the possibility of a subcortical pacemaker. The idea of a subcortical pacemaker for the EEG of course is not novel, and the reader is referred to the monograph cited above for a detailed review of the pertinent literature. It may be relevant to point out, however, that the conclusions of Andersen and Andersson (1968) regarding the role played by the thalamus were arrived at preponderantly from observations on evoked potentials, and on a single type of spontaneous activity-sleep spindleswhich they equate with alpha rhythms in man (erronously, in the opinion of the present author-cf. Elul, 1972c; also Section 11). It is exceedingly difficult from this evidence to exclude the possibility of important contributions from non-thalamic subcortical centers to other typcs of cortical rhythms.

B. POSSIBLEFUNCTIONAL ROLE OF

THE

EEG

IN

PERCEPTION

The results discussed in Section VII led to the conclusion that the EEG is produced through intermittent synchronization of selected groups of cortical nerve cells. In the preceding paragraphs, evidence is presented suggesting that this intermittent synchronization is under subcortical control, possibly in the thalamus. It may, therefore, be relevant to inquire whether intermittent cortical synchronization by subcortical centers may have any functional significance, or might simply be an epiphenomenon of processes otherwise unrelated to cortical function. There does not appear to be an!. concrete evidence bearing on this question, but the fact that different neuronal groups must be synchronized in successive instants in time suggests that subcortical centers may thus effectively perform “scanning” of the cerebral cortex. The concept of scanning was originally introduced by Pitts and McCulloch (1947) with relation to perception. Certainly, the brief synchronizations observed in intracellular recordings, which generally lasted only several hundred nisec, approximate the limits for perception determined in psychophysiological measurements, which otherwise appear grossly below the speed of reaction of which neuronal systems are capable. The striking disparity between cell counts in the cortex and in subcortical centers would make it almost mandatory for any form of communication between the two systems to involve divergence in the subcortical afferents to the cortex, and convergence in the efferents from the cortex. Under these conditions, the processing of cortical output by subcortical centers must confront the difficulty of identifying which specific cortical elements are active at any given moment in time. One possible solution to this problem is by means of “test impulses” from the subcortex to selected groups of cortical neurons, with subsequent sub-

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cortical evaluation of tlie rcsultant volleys returning froin the cortex. The sequential character of the synchronizing drive to different groups of cortical neurons may suggest that perhaps the outputs from a11 these cortical elements might coiiverge on a single subcortical system. This system would thus satisfy at least soiiie of the requisites of a “perception center” or "consciousness center,” insofar that it would be in a position to evaluate a large variety of messages processed at tlie cortex, but would be capable of doing so only in sequential fashion, much as we “turn” our attention at will, first to auditory cucs, next to visual oiies, then to olfactory stimuli and so on. IX. Conclusion a n d Consequences for Evaluation of Gross EEG Activity

A. SUMMARY OF

THE

RESULTSIN

THE

PRESENT REVIEW

This review has been conceriied with the mechanisms of generation of the EEG. There is now very substantial evidence that the EEG is derived from wave activity of nerve cells in the cerebral cortex. Although it appears that the elementary generators of the EEG arc relatively small patches of the cell membrane, probably iiicludiiig several synapses and some passive membrane through which the synaptic current returns into the cell, there is still no definitive evidence as to the approximate size of such “unitary generators,” and their relative distribution on the cell body and dendrites, although it was suggested that they represent “synaptic functional units.” Iiiforniatioii is also lacking on the extent of spread of the activity of individual generators in the extracellular medium, and finally it niust be recognized that the disk electrodes conlinonly used in EEG recording introduce serious distortions in recording of generators in different locations in thc tissue. Because of thesc limitations, it appears productive at the present time to treat the question of summation of generator activities only in a statistical manner, essentially disregarding individual deviations from the “average unitary generator.” Analysis of the relationships between the gross EEG and the wave activity of individual nerve cells indicates that the gross activity is produced through summation of the synchronized activity of a comparatively small fraction of the cerebral neuronal population. Although the rest of the population also is active, their contributions are not synchronized and therefore sunimate much less effectively, probably not reaching the limit of resolution of EEG recorders (i.e., 1-2 pV). There is only sketchy information on the mechanisms of synchronization, but it is clear that they involve subcortical drives and entail sequential activation of different groups of cortical neurons. The possiblc significance of

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such subcortical “scanning” of the cortex in relation to perception has been briefly discussed A b ove. This view at present is rather speculative, as there is yet no direct evidence identifying the specific subcortical centers sending synchronizing drives to the cerebral cortex in different behavioral situations, although the thalamus is involved in production of sleep spindles. Also, in addition to the experimental data available at present, which show only an inconstant relationship between given neurons and the EEG, it would be desirable to obtain more direct evidence that at the instant that a particular group of cells ceases to be synchronized with the EEG, it is replaced with another group of synchronized neurons, etc. It is possible that evidence of this type eventually may be obtained from studies with extracellular electrodes of the microscopic organization of electrical activity in the cortical extracellular inediuni; but until direct evidence is obtained, it would be difficult to reach any conclusive view on the detailed interactions between the cortical generators which produce the EEG. It must be recognized that, whereas intracellular recordings provide reasonably dependable infomiation on the behavior of a single cell, no methods of coniparable precision are currently available for evaluation of intercellular relationships. Analysis with multiple extracellular microelectrodes can yield some valuable information on this question (Verzeano and Negishi, 1960; Elul, 1962; Calvet et al., 1964), but the complex topology of the extracellular space significantly curtails the usefulness of this approach because of the difficulty of knowing, even approximately, the volume of tissue and nuniber of generators scanned by each microelectrode. 8

B. IMPLICATIONS FOR COMPUTER ANALYSISOF EEG DATA Accepting for the present only the actual experimental findings described in this review: the inconsistency of relationship between individual neurons and the EEG, and the data from experiments with ?TX which showed that flattening of the EEG is due to desynchronization; it is clear that the EEG must in some way be produced through intermittent synchronization of cortical neurons, and that different neurons become synchronized with the EEG in successive instants in time. These results lead to a practical conclusion of great importance: Rather than a continuous process, the EEG represents a series of consecutive short segments, each of which is produced from synchronized activity of a different group of cells. In view of this disjointed nature of gross activity it would appear misleading to treat the EEG as a stationary statistical process. Nonstationarity of the EEG, which is also directly detectable in ceitain types of statistical analysis (Elul, 1969b, and 1972, un-

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269

published data; Kawabata, 1972) iiiay perhaps not be totally unexpected. The view of gross activity as a sequence of “evoked potentials” in response to natural stiiiiuli has been prevalent for iiiaiiy years among experiiiicntal psychologists. However, since we are dealing essentially with a sequence of discrete processes, methods presupposing a continuous nature for the analyzed process would appear to have rather limited application in EEG analysis. In particular, frequency power spectra, calculated on the assumption of EEG stationarity using long epochs of EEG activity (5-10 sec) must be exceedingly difficult to interpret in physiological terms insofar as, although the frequcncy spcwtruin of gross activity does rcflect the frequency spectra of the unitary generators ( E M , 1968), the spectrum computed in these circunistaiiccs represents averaging and “smoothing” of the outputs from an unknown number of distinct groups of neurons, active consecutively, and perhaps also to some extent overlapping in their activities. The results from the physiological studies described here would suggest that techniques involving piecemeal treatment of the EEG might be more appropriate. One such method which has recently been developed is the technique of nonlinear power spectra, which has been applied to describe the dynainic changes in the EEG during opening and closing of the eye's ( N . Kawabata, 1972, unpublished data). EEG analysis can specifically address the question of interdependence of successive discrete values of the EEG through the use of amplitude histograms (Elul, 1966); it is perhaps significant to note that this approach reveals that the relationship of EEG values at successive points in time undergoes a niarked change during the performance of mental tasks ( Elul, 1969b), and also is different between normal and meiitally retarded children, perhaps inclicating differences in scanning ability in these two groups (R. E M , 1972, J. Hanley, and J. Q. Simmons, u i i published data). Practically all microelectrode analysis perforiiicd until the present has been limited to single cortical loci. It is, however, to be expccted that, just as different groups of cells at the same locus are consecutively synchronized, a similar process of selection takes place over the entire corticaI surface. If the hypothesis proposed here is tentatively accepted, that the EEG reflects subcortical scanning of cortical information, and as such may provide us with information about attention and consciousness, i t foIlows that rather detailed information on mental processes may be had from the EEG, provided that a large number of cortical loci are sampled simultaneously. At the present time we still do not know how to interpret such information, even in the preliminary attempts made SO far (Petsche and Marko, 1955; Rkmond et al., 1969; Lehmann, 1971), and the number of sites sampled certainly is at least one or two orders of magnitude below that ultimatcly required, but it may not be alto-

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gether unrealistic to hope that an array of several hundred cortical electrodes may provide not only a detailed map of cortical potential distribution in space and time, but also a meaningful physiological correlate of perception and consciousness. ACKNOWLEDGMENTS

I thank Mrs. C. H. Skinner for help in preparation of this manuscript. The unpublished research reported here was supported by grants NS-8012 and 8498 from the National Institutes of Health, and by grant GB-30498 from the National Science Foundation. REFERENCES Adey, W. R., and Elul, R. (1965). The Physiologist 8, 159. Adey, W. R., Kado, R. T., and Walter, D. 0. (1965). Exp. Neurol. 11, 190. Adrian, E. D., and Matthews, B. H. C. (1934). Brain 57, 355. Adrian, E. D., and Moruzzi, G. (1939). J. Physiol. ( L o n d o n ) 97, 153. Andersen, P. (1960). Acta Physiol. Scand. 48, 178. Andersen, P., and Andersson, S. A. (1968). “Physiological Basis of the Alpha Rhythm.” Appleton, New York. Andersen, P., and Lorno, T. (1966). Exp. Brain Res. 2, 247. Andersen, P., and Sears, T. A. (1964). J. Physiol. ( L o n d o n ) 173, 459. Andersen, P., Eccles, J. C., and Loyning, Y. ( 1964). J . Neurophysiol. 27, 592. Andersen, P., Blackstad, T. W., and Lorno, T. (1966). E x p . Brain Res. 1, 236. iindersson, S. A., and Manson, J. R. ( 1971 ). Electroencephalogr. Clin. Neurophysiol. 31, 21. Arvanitaki, A. (1939). Arch. Internut. Physiol. 49, 209. Baker, P. F. (1969). J. Physiol. ( L o n d o n ) 200, 459. Barron, D. H., and Matthews, B. H. C . (1935). J. Physiol. 92, 276. Berger, H. (1929). Arch. Psychiat. 87, 527. Blankenship, J . (1968). J. Neurophysiot. 31, 186. Brock, L. G., Coombs, J. S., and Eccles, J. C. (1952). J. Physiol. ( L o n d o n ) 117, 431. Calvet, J., Calvet, M. C., and Scherrer, J. (1964). Electroencephalogr. Clin. Neurophysiol. 17, 109. Calvin, W. H. (1969). Exp. Nezirol. 24, 248. Calvin, W. H., and Hellerstein, D. (1969). Science 163, 96. Chang, H. T. (1950). J. Neurophysiol. 13, 236. Chang, H. T. (1951). J. Neurophysiol. 14, 95. Chang, H. T. (1952). J. Neurophysiol. 15, 5. Clare, M. H., and Bishop, C. H. (1952). Electroencephalogr. Clin. Neurophysiol. 4, 31. Clare, M. H., and Bishop, G . H. ( 1955). Electroencephabgr. Clin. Neurophysiol. 7. 85. Clare, M. €I., and Bishop, G. H. ( 1956). Electroencephalogr. Clin. Neurophysiol. 8, 583. Creutzfeldt, 0 . D., Watanabe, S., and Lux, H. D. ( 1966a). ElectroenceplialoRr. Clin. Neurophysiol. 20, 1. Creutzfeldt, 0. D., Watanabe, S., and Lux, H. D. (1966b). Electroencephalogr. Clin. Neurophysiol. 20, 19.

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McIlwain, J. T., and Creutzfeldt, 0. D. (1967). J. Neurophysiol. 30, 1. Mountcastle, V. B. (1957). J. Neurophysiol. 20, 408. Narahashi, T., Moore, J. W., and Scott, W. R. (1964). I. Gen. Physiol. 47, 965. Nicholson, P. W. (1965). Erp. Nenrol. 13, 386. Orkand, R. K. (1969). In “Basic Mechanism of the Epilepsies” ( H . H. Jasper, A. A. Ward, and A. Pope, eds.), p. 737. Little, Brown, Boston, Massachusetts. Orkand, R. K., Nichols, J. G., and Kuffler, S. W. (1966). J. Neurophysiol. 29, 788. Peronnet, F., Anninos, P. A,, and EM, R. (1971). Proc. 1st Europ. Biophys. Congr. 5, 231. Petsche, €I., and Marko, H. (1955). W i e n . Z. Neruenheilk. Deren Grenzgeb. 12, 87. Pitts, W., and McCulloch, W. S. (1947). Bull. Math. Biophys. 9, 127. Pollen, D. A. (1969). In “Basic Mechanisms of the Epilepsies” ( H . H. Jasper, A. A. Ward, and A. Pope, eds.), p. 411. Little, Brown, Boston, Massachusetts. Purpura, D. P. (1959). Int. Reu. Newobiol. 1, 47. Purpura, D. P., McMurtry, J. G., Leonard, C. F., and Malliani, A. (1966). 1. Neurophysiol. 29, 954. Rall, W. ( 1967). I. Netrrophysiol. 30, 1138. Rall, W., and Shepherd, G . M. (1968). J. Nerirophysiol. 31, 884. Ranck, J. B., Jr. (1963a). Ex?). Neurol. 7, 144. Ranck, J. B., Jr. (196313). Ex)). Neurol. 7, 153. RCmond, A., Lesevre, N., Joseph, J. P., and Rieger, H. (1969). Electroencephalogr. Clin. Neurophysiol. 26, 245. Schlag, J., and Balvin, R . (1963). E x p . N e r d . 8, 203. Shanes, A. M. (1949). J. Gen. Physiol. 33, 57. Spencer, W. A,, and Brookhart, J. M. (1961). 1. Neurophysiol. 24, 50. Spencer, W. A., and Kandel, E. R. (1961). 1. Neurophysiol. 24, 272. Van Harreveld, A., Murphy, T., and Nobel, K. W. (1963). Amer. J. Physiol. 205, 203. Verzeano, M. (1956). Science 124, 366. Verzeano, M., and Negishi, K. (1960). J. Gen. PhysioE. 43, 177. Walter, D. 0. (1963). E x p . Neurol. 8, 155. Walter, D. O., and Adey, W. R. (1963). E r p . Nezrrol. 7, 481. Woodbury, J. W. (1965). In “Nenrophysiology” (T. C. Ruch, H. D. Patton, J. W. Woodbury, and A. L. Towe, eds.), Chaps. 1 and 2 . Saunders, Philadelphia. Zucker, R. S. (1969). Science 165, 409.

MATHEMATlCAL IDE NTIFICAT10N OF BRAI N STATES APPLIED TO CLASSIFICATION OF DRUGS'

.

By E R . John. P. Walker.

D . Cawood. M. Rush.

and J . Gehrmann

Department of Psychiatry. New York Medical College. New York. N e w Yark

I . Introduction . . . . . . . . I1 Methods . . . . . . . . . A . Subjects. Surgical. and Recording Procedures B . Training Procedures . . . . . . C . Drug Injection Schedule . . . . . D . Testing Procedure . . . . . . E . Selection of Recording Sites . . . . F. Data Analysis Methods . . . . . G . Row Analysis . . . . . . . H Column Analysis . . . . . . . I11. Results . . . . . . . . . A . Behavioral Effects of Drugs . . . . B. General Discussion of Behavioral Results . . C. EEG Findings . . . . . . . D . Frequency Analysis . . . . . . E . Average Evoked Responses . . . . F. Column Factor Analyses-Raw Data . . G . Regression Equations . . . . . . H . Combined Column Analysis . . . . IV. Discussion . . . . . . . . . References . . . . . . . .

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I. Introduction

Among the major problems in the evaluation of electroencephalographic (EEG) and evoked potential (e.p.) studies is the extraction of representative. reliable. and physiologically relevant samples of analog wave forms and the representation of essential features of such data in a precise and comprehensible form . These problems are especially difficult in behavioral experiments involving the acquisition of electrophysiological data from multiple chronically iniplanted electrodes over long time periods. during which enormous amounts of data may be generated . 'This work has been supported by PHS Grant No . MH 08579 and grants froin Mead Johnson and Ciba-Geigy Pharmaceuticals . 273

274

E. R. JOHN, P.

WALKER, D. CAWOOD, M. RUSH,

J. GEHRMANN

Some years ago (John et al., 1963, 1964), we showed that a precise quantitative description of the signal space of the brain was parsimoniously provided by the set of regression equations obtained from a principal component factor analysis of recordings from different brain regions. It was possible to reconstruct the evoked activity recorded from any brain region in terms of a small number of mathematical descriptors common to all brain regions. Subsequently, Donchin (1966) applied these multivariate techniques to the analysis of sets of average visual evoked responses sequentially recorded from the same site, further demonstrating the utility of this method for concise quantitative description of a large body of electrophysiological data. Halas and Beardsley (1969) applied principal component factor analysis to the study of habituation, confirming our report that strong coupling of electrical activity exists between different brain regions, as shown by the finding of high loadings on common factors in different brain regions. Elmgren and Lowenbard (1969) subjected data on the covariance between 23 EEG frequency lines to principal factor analysis in order to extract 7 statistically independent components of the power spectrum. The high degree of stability of this solution was shown by excellent right-left hemisphere reproducibility. Naitoh et al. ( 1971) have similarly used principal component factor analysis to classify differences between the power spectrum in different stages of sleep, providing further confirmation of the utility of this method. Suter (1969, 1970) has used principal component factor analysis to decompose auditory average evoked responses into a set of simple wave forms, which appear to correspond to physiologically defined evoked response components. Bennett et al. ( 1971) have applied principal component factor analysis to decompose the visual evoked response in man into a set of four uncorrelated components. In addition to the fact that factor analysis can provide a precise quantitative description of a body of electrophysiological data, the method possesses a number of unique advantages. First, because factor analysis examines the mathematical implications of the set of covariances between the different elements of the data, it constructs a description of those data in terms of the relationships (factors) between those elements. Since the structure of a coupled system imposes constraints on the independence of its elements, this description might be expected to reveal aspects of the organization of the system. Second, use of the principal component method ( Harmon, 1960), which constructs factors so that each successive factor accounts for the largest possible portion of the residual variance of the original data set, provides a minimum estimate of the true dimensionality of the space in which the observed set of variables was generated (Scheme 1 ) . The number of dimensions thus obtained can be interpreted as the smallest number of uncorrelated

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underlying processes which could have accounted for a specified percentage of the observed data. Third, any set of orthogonal axes can be rotated to an infinity of different positions within the space which it spans. The Varimax procedure (Kaiser, 1958) is a technique by which a selected set of axes is rotated into an orientation such that each axis is aligned as closely as possible to one cluster of signal vectors (Scheme 2). Since only highly intercorrelated vectors will cluster about the same axis, the effect of this rotation is to divide the original data into groupings, which can often be interpreted in terms of characteristics of the actual data generating mechanisms (Bennett et al., 1971; Donchin, 1966; Suter, 1970). Thus, a Varimax rotation of the principal components obtained from factor analysis of a body of electrophysiological data might be expected to provide a precise quantitative description of that data within a framework which emphasized and clarified the physiological processes which varied within that data. In our early work on “physiological factorization,” we were striving toward this goal (Ruchkin et al., 1964; John et al., 1964). See Schemes 1 and 2, pp. 275 and 276. In that work we noted that drug administration caused striking changes in factor loadings. The purpose of the present study was to examine the utility of factor analysis for thc reduction and quantification of large amounts of evoked potential data and to ascertain the adequacy with which the Varimax

SI= %J, +b,,J, Sz=% Ji+ b.2 Jz

SCHEME 1 Where

ail =

~ 2 1 b, 1 2

= -bzz.

In this description, both vectors S, and Sz receive equal contributions from J,, the principal (Jacobi) factor which contributes the largest amount of energy to

this two-vector example. The loadings of upon S, and Sz respectively are h,and b, equal in magnitude but opposite in sign. Thus, S, and S, receive equal contributions from both 3% and J?. The factor analysis spans the space in a way which does not relate any factor selectively to any signal vector.

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rotation of principal components would provide a reliable framework for the quantitative description or classification of drug action upon the electrical activity of the brain. In order to provide a frame of reference to estimate the utility of this method, a number of different kinds of data were compared. These included measures of behavioral performance on approach and avoidance tasks, “typical” samples of EEG recordings, spectral analyses of EEG segments, average evoked responses, principal component factor analysis, and Varimax factor analysis. The results indicated that Varimax factor analysis provided a more precise, reliable, and unique description of the effects of various drugs on the electrical activity of the brain than the other measures studied. This description was markedly different from drug to drug but basically consistent for different doses of the same drug. It. Methods

RECORDINGPROCEDURES Subjects in the experiments to be reported in this paper were three adult female cats obtained from a commercial supplier. After inoculation with feline enteritis vaccine, a 3-week quarantine period was imposed. Using pentobarbital anesthesia, each animal was then subjected

A.

SUBJECTS,

SURGICAL, AND

S,. o;, V, + b;zVz

S,= a;,V,+ b&V2

SCHEME 2 Where

a’21> arll and b‘12 > b’22. The Varimax rotation of J1 and JI is shown. The two Varimax factors, V1 and V?, are located as closely as possible to the vectors S1 and S?. The loadings of V, upon S, and S2 are a’,, and a’21.Thus, V1 contributes markedly more to Sr than to S,, since alz, a’,l. Conversely, the loadings of Vz upon S1 and S? are b,? and b‘z. V? contributes more to S1 since b12 b,?. The factor analysis now spans the space in a way which locates vector S1 closest to factor V2, while vector S, is closest to factor V,.

>

>

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277

to stereotaxic implantation of 34 Teflon-insulated stainless steel electrodes ( 0.007 in. diameter) into selected cortical and subcortical regions. These electrodes were cemented to the skull with acrylic resin, and coilnected by crimping to a Winchester subminiature plug which was then imbedded in acrylic resin (John, 1962). All three cats were implanted in 1968. Electrophysiological rccording was subsequently accomplished by connecting the plug on each cat’s head to appropriate amplifiers, using a cable constructed of Microdot Mininoise wire. A 12-channel Grass Pi’ polygraph was used to amplify the data. All data were routinely recorded on magnetic tape, using 14-channel Mnemotron tape recorders. A voice track commentary and a stimulus trigger channel on the magnetic tape plus a detailed written protocol of every experiment permitted subsequent recovery of data for computer analysis.

B. TRAINING PROCEDURES Cats I and I1 were trained using the observation method, in which initial acquisition of learned behaviors was accomplished by watching a trained animal perform (John et ul., 1968). All procedures were carried out in a metal cubicle, 2 x 2 x 2 ft, with a transparent front work panel, inside a shielded, soundproof room. The animals first learned to press either the left- or right-hand lever on a work panel within 15 sec of the onset of a 5 Hz flickering light ( V l ) , in order to obtain a dipper of food ( approach-CR ) , A low hurdle, perpendicular to the work panel, divided the apparatus into two equal compartnients. After full acquisition of the V, approach response, the animals learned to jump across the hurdle into the unoccupied compartment within 15 sec of the onset of a 2 Hz flicker ( V 2 ) , in order to avoid electric shock to their feet delivered by a floor grid (avoidance-CAR). The flicker stimuli were delivered from a silent flash tube mounted in the apparatus top, causing a rhythmic Auctuation in the luminance of the full visual field, when superimposed upon the constant illumination level inside the apparatus. Note that the animals thus remained fully adapted to light throughout experimental sessions. After sequential establishment of the approach and avoidance behaviors, which took about 8 weeks, cats I and I1 learned to discriminate between V, and V? stiniuli administered in a random sequence, inchding about 80 stimuli in daily training sessions. About 3 months of further training was required to establish rapid and accurate discrimination responses to these differential stimuli. Cat I11 received no training, serving as a control to indicate possible interactions between the effects of conditioning and the drug effects to be studied.

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D. CAWOOD, M.

RUSH,J.

GEHRMANN

C. DRUGINJECTION SCHEDULE For one year after completion of training, cats I and I1 were part of a group of seven trained and six naive cats. These animals were the population of a factor analytic study of the electrophysiological action of drugs, involving a total of 330 experiments with 35 different drugs. During that year, cats I and I1 received injections of 12 to 15 different drugs at intervals of several weeks. Cat I11 received only seven injections during that period. The results of those studies will be published in subsequent articles, but are too lengthy to include herein. In early 1970, cats I, 11, and I11 were withdrawn from the larger study, and designated as the population for the dose-response study being reported here. This study began 3 months after the last drug injection administered in the previous study, to minimize chances of any aftereffects. The study involved the following drugs and dosages: Chlorpromazine-5.0, 2.5, and 1.0 mg/kg; MJ W22-1. (This experimental nonphenothiazine tranquilizer was developed by Mead Johnson & Com2-Pyrimidinyl)-l-pipany. The chemical name of MJ 9022-1 is 8-(4-[4-( perazinyll-butyl ) -8-azaspiro[4.5]decane-7,9-dione Hydrochloride ( Wu et al., 1972).)-5.O, 2.5, and 1.0 mglkg; sodium phenobarbital-20, 10, and 5 mg/kg; methamphetamine-1.0 and 0.5 mg/kg. In the following discussions, these drugs will be abbreviated as CPZ, MJ, PHENO, and METH, respectively. Together with two injections of saline placebo (SALINE), these drugs and doses were administered according to a double blind Latin square design, decoded only after final data processing. Injections were intramuscular, and were standardized for volume and concentration.

D. TESTING PROCEDURE Each animal was subjected to one drug experiment per week, in accordance with the Latin square design. This schedule was interrupted only on those few occasions when illness on the part of the animal or the experimenter interfered. Each experiment was preceded by a 48-hour period of food deprivation, to achieve a uniform and high level of motivation for food. On each experimental day, the animal was placed in the apparatus in the morning, connected to the amplifying and recording equipment, and a random sequence of v, and v, was administered. The latency and appropriateness of approach responses ( C R ) and avoidance responses (CAR) were recorded, together with the electrophysiological data and stimulus trigger markers. This first body of data was denoted as the “2-hour pre-drug” sample. A second body of data was obtained an hour later and is referred to as the “l-hour pre-

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drug” sample. The injection appropriate for that day was then administered and data were gathered in hourly recording sessions for the next 3 to 6 hours. Such data are identified as “n-hour post-drug” samples. The prescribed stimulus presentation sequence was followed, whether or not any behavioral responses were elicited from the cat. About 40 trials constituted the usual session. Trials were about one minute apart. Reinforcement rules were as follows: V,CR-one dipper of food (meatmilk slurry, volume 1 ml); V,NR (no response)-no consequence, V, off after 15 sec; V,CAR (error)-no consequence, V, off after 15 sec; V, CAR-V, off, no electric shock; V,CR-no food, electric shock if no CAR within 15 sec; V2NR-electric shock if no CAR within 15 sec. After drug doses which severely interfered with CAR performance, punishment for V,NR deviated from this schedule. Shock was administered as sparingly as possible, only to test whether shock-escape behavior remained intact. It was felt that such intermittent aversive reinforcement did not cause a significant amount of extinction, because of the known resistance of the CAR to extinction and the fact that CAR performance invariably returned to pre-drug levels as drug effects wore off with time.

E. SELECXIONOF RECORDING SITES For the original study, 34 electrodes had been implanted in each cat to give access to the largest possible variety of recording sites. It was essential to obtain hourly records on a routine basis, with sufficient data for each stimulus and behavioral contingency to constitute an adequate statistical sample. For this purpose, it was necessary to reduce the anatomical structures to be studied to a total of 12 derivations in each cat, since this was the maximum number of channels which could be recorded simultaneously. The 12 derivations selected in each animal were intended to provide good samples of sensory specific, nonsensory specific, and limbic regions and were as follows: Cat I-visual cortex, monopolar (VIS,) and bipolar (VIS,); lateral geniculate body, monopolar ( LG, ) and bipolar ( LG, ); mesencephalic reticular formation, monopolar (MRF,) and bipolar (MRF,); septum, monopolar (SEPT,,); dentate, monopolar (DENT,); subthalamic nucleus, monopolar ( SUBTH,) ; dorsal hippocampus, bipolar ( D HIPP, ) ; nucleus centralis lateralis, bipolar ( CLR); and substantia nigra, bipolar ( SN, ). Cat II-left visual cortex, monopolar ( L VIS), lateral geniculate, monopolar and bipolar ( LG) ; prepyriform cortex, monopolar ( PREPYR); mesencephalic reticular formation, monopolar ( MRF ) ; nucleus reticularis, monopolar and bipolar ( N RET) ; medial forebrain bundle, monopolar ( MFB ) ; ventral dentate, monopolar ( V DENT), right visual cortex, monopolar ( R VIS);

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E. R . JOHN, P. WALKER, 1). CAWOOD, M. RUSH, J . GEHRMANN

and cingulate, monopolar and bipolar ( CING ) . Cat 111-visual cortex, monopolar and bipolar ( R VIS); lateral geniculate, monopolar and bipolar ( L G ) ; prepyrifomi cortex (PREPYR); mesencephalic reticular formation, monopolar and bipolar ( MRF) ; subthalamic nucleus, monopolar ( SUBTH ) ; medial forebrain bundle, monopolar ( MFB) ; dorsal hippocampus, bipolar ( D HIPP) ; ventral dentate, bipolar ( V DENT) ; and nucleus ventralis anterior, bipolar ( VA) .

F. DATAANALYSISMETHODS 1. Behavioral Results

For the trained cats, a separate graph was plotted for each experimental day, showing the percentage of correct responses to V, and V2 in every hourly session.

2. E E G Records Every hourly session began and ended with a one-minute recording of the spontaneous EEG data. Each of these samples of spontaneous activity was studied and examples were selected which contained features judged to be “typical” or representative of activity at that stage of the experiment.

3. Evoked Potential Records The electrographic data recorded during each behavioral trial were studied and examples were selected which were judged to typify the features of the responses elicited by presentation of V, and V, in eveiy hourly session.

4. Power Spectrum Using the spontaneous EEG data recorded during the one-hour predrug session as the “pre-drug sample” and the spontaneous EEG data recorded at the end of the post-drug session in which the largest behavioral effect was measured as the “post-drug sample,” the distribution of power among different bands of the frequency spectrum was measured. This measurement was carried out using a Neurodata Bandpass EEG Filter Model 100 and a Neurodata Model 2200 Symmetry Analyzer. This system provides information about power spectral density as a meter reading ill =

Jr (power in EEG band of interest) JOT

(power in total EEG)

A 10-sec time constant was used in the measurement, and the average reading during the 1-miu sample of spontaneous EEG activity was

BRAIN STATES APPLIED TO DRUG CLASSIFICATION

28 I

recorded. The bandpass filters were constructed to provide 18 dB of attenuation per octave, and the EEG bands were defined as follows: delta ( S ) = 0.5-3.5 Hz; theta ( 0 ) = 3.5-7.0 Hz; alpha ( a ) = 7.0-13.0 Hz; beta ( p ) = 13-25 Hz; ganima ( 7 ) = 2 5 4 0 Hz. While probably a few percent less precise than digital methods for measurement of the absolute amount of power in a specific band, the precision of this analog system was considered ample for the measurement of the relative amount of power in a given band when pre-drug and post-drug data were compared. This system was used to nicasurc~the power spectrum, in prcfercncc to the conventional fast-Fourier transform or period-analysis methods, because it provided a reproducible estimate of the average frequency composition of the EEG across a sufficiently long epoch to compensate for the short-term fluctuations in this measure. This seemed like an acceptable alternative to the practice of selecting a one-second sample for Fourier analysis, which may well not yield an accurate estimate of fluctuations in the EEG over longer intervals of time, or to averaging the results of several such power spectra. However, some samples of data were also subjected to Fourier analysis.

5 . Average Evoked Responses For each hourly session, an average response was computed from each derivation separately for V, and V, trials of all behavioral outcomes displayed during the session. Thus, in one session, examples of V,CR, V,NR, VICAR, V,CAR, V,NR, and V,CR might be obtained. Insofar as was permitted by the amount of data which was available, average responses were composed of 200 evoked potentials. An analysis epoch of 180 msec was used for all averages submitted to factor analysis. Since there were usually six recording sessions per experiment ( two pre-drug and four post-drug), a typical experiment produced 6 sessions x 2 stimuli x 12 derivations, yielding a matrix of 144 average evoked responses, comprising about 28,000 evoked potentials which occurred in about 250 behavioral trials. For each average cvoked response which was computed, a corresponding point-by-point variance curve was computed, representing the variability of the data at each latency point. Variance data were used to estimate whether averages were composed of homogeneous or heterogeneous types of data. Interestingly, although it was clearly demonstrated that the data from certain structures was markedly heterogeneous (John, 1972) , performance stability in these overtrained aniinals was sufficient so that a consistent and reproducible whole trial average response wave form was composed from mixtures of these different singlc evoked potential wave shapes.

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E. R. JOHN, P. WALKER, D.

CAWOOD, M. RUSH,J.

GEHRMANN

6. Factor Analysis On the basis of pilot studies which revealed substantial homogeneity to the post-drug factor structure as a function of time, four average evoked responses from each derivation were selected from each drug experiment, to be utilized for factor analysis: 1-hour pre-drug examples of the average responses evoked by both V, and V2, and post-drug examples of the responses to V, and V, taken from that session in which the behavioral effects of the drug were most pronounced (usually the %hour post-drug data). These data can be arranged in an n x m matrix, with 12 derivations X 2 stimuli ( n = 24), and with 13 pre-drug examples plus 13 postdrug examples ( m = 26), thus: Derivation 1

vi

Pre-drug 1 Post-drug 1

v2

-

-

-

-

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

Derivation 18 v1

. . . . . . . . . . . . . . . . . . . . . . . . . . . . -

v1

-

G. Row ANALYSIS These data represent the total drug experiment sample selected for factor analysis. Note that a row in this n x m matrix contains a set of average response wave shapes recorded under a specified drug or control condition from 12 different brain regions. One-half of the wave shapes in each row were recorded simultaneously, during presentation of a number of V, trials. The other half of the wave shapes in the row were recorded simultaneously intercurrently with the first half, during presentation of a series of V, trials. Thus, a row of this matrix provides an example of the set of 24 average response wave shapes simultaneously or intercurrently produced in this set of interrelated anatomical regions, during a period in which the system occupied a specified control or drug state, and was perturbed by one of two test stimuli. It should be clear that factor analysis of a row (henceforth called row analysis) will yield a dimensionality which reflects the functional neuroanatomical organization which couples the different anatomical regions from which recordings were obtained. If all regions function independently, the dimensionality must equal the number of regions. If coupling between regions exists, the dimensionality will reflect the number and factor loadings will reflect the strength of functional sub-

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systems relating the regions. As transactions and influences between regions alter, when the meaning of a stimulus changes or as a drug is administered, the dimensionality of the signal space and the Ioadings of different regions should change to reflect the new functional organization. These implications and interpretations of row analysis have been elaborated in previous work concerned with row analysis (John et al., 1964; John, 1967). The row analysis of data from the present experiments will not be discussed in this paper, but will be forthcoming later.

H. COLUMNANALYSIS Note that a column in the n x m matrix contains a set of wave shapes recorded from a specified anatoinical locus in response to a particular test stimulus presented under 13 similar control and 13 dissimilar drugdose conditions. We assume that average response wave shapes recorded sequentially from a single region in a system at different times will display comparable features if the state of the system remains reproducible during the several sampling periods. We assume that the state of the system will be reproducible, as a first approximation, if a trained animal responds correctly and with characteristic response latency to a wellovertrained conditioned stimulus. Stability of pre-drug data in cat I and cat I1 will be used to confirm these assumptions. The utility of conditioned response methods for achieving stabilization can be estimated by comparison with like data from cat 111. It should be clear that factor analysis of a column (henceforth called column analysis) will yield a dimensionality which indicates the variety of states caused in the system by the different experimental conditions examined, as reflected in the activity of the anatomical region or derivation corresponding to each column. If none of the conditions investigated changes the state of the system in a way which influences that region, the dimensionality of the signal space representing the column will be equal to unity: the region will display one immutable state. If every condition changes the state of the brain in a uniquely different way, then the dimensionality of the column space must equal the number of conditions. In inteiinediate instances, in which different conditions cause varying amounts of a limited variety of states, the dimensionality of the signal space and the factor loadings for each condition will provide quantitative descriptions which specify each case. Furthermore, there is no reason to assume that all regions of the system reflect changes in state in the same way. Therefore, different dimensionalities might be displayed by different parts of the same system. Careful examination of the column analysis of data recorded from a given brain region under a variety of conditions can provide a quan-

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E. R. JOHN, P.

WALKER, 1). CAWOOD, M. Hum,

J. GEHRMANN

titative classification of conditions. Since similar conditions will be classified similarly, this offers a basis for a quantitative nomenclature of specifiable states in particular brain regions. Combination of a series of column analyses from different regions can provide a quantitative description of the anatomical covariations in states created in the brain under a certain condition, yielding a quantitative description of the effects of that condition on the whole brain. It must be understood that combinations of column analyses from different brain regions, simultaneously studied under the same set of conditions, can only be interpreted in a dimensional sense. The corresponding states of two different regions may merely represent couariation, but may also represent similarity of response processes. Column analyses must be supplemented by row analyses to resolve this ambiguity. Row and column analyses are two different ways of approaching an understanding of the dynamic interrelationships between regions in the brain. The primary purpose of this paper is to examine the technique of column analysis, particularly in order to evaluate its possible utility for the mathematical description and classification of drug action on the brain. All computing procedures were implemented on our PDP-12 computer, using programs developed here.? Ill. Results

A. BEHAVIORAL EFFECTS OF DRUGS Under ordinary conditions, each sessioii included about 40 trials, divided approximately equally between the approach and avoidance stimuli, which were presented in random order. A trial usually occupied 5-10 sec, and the spacing between trials averaged 1 min. The actual number and sequence of trials per session was not rigidly fixed. Changes in the responsivity of the animals due to drug effects not only altered characteristic response latencies, but often made it desirable to deviate from the predetermined stimulus sequence. On occasion, for example, it was of interest to present a series of like stimuli in order to explore a drug-induced behavioral bias. Behavioral results were normalized by calculation of the percentage correct responses to each stimulus in each hourly session. The behavioral graphs summarizing the full set of experiments are presented in Fig. 1 ( t o p ) for cat I and in Fig. 1 (bottom) for cat 11. In both instances, the columns of graphs from left to right show the effects of SALINE, CPZ, MJ, PHENO, and METH, while each rot0

' We

wish to acknowledge our indebtedness to the programming skill of Drs.

D. S. Ruchkin and Paul Easton, and Noel Fleming and Robert Nagel.

CAT

I SALINE

MJ W22-I

CPZ

PHENOBARB

METHAMPHETAMINE

60 40 20

0 PRE WIN

, ' 131

2nd 3rd

cpasioeus-

m

40

PR€MUG

100

-

I -

~+

V, APPROACH SIIMULUS V, AVOIDANCE STIMULUS

M)

ImDfkp

.

1. 2nd 3 d 4th cFmlORU6-

_...

;

’.,j

60

40

40

20

20

PRCDR~S 1.1 2nd 3rd 41h cfQs1WIIN-

PREMU~I* 2nd ~d4th c s a s r MUC

1

FIG. 1A. Each graph shows the behavioral performance of cat I throughout a day's experiment. Although drugs were administered in a Latin square design, these graphs have been grouped according to drug and dosage. Each column of graphs presents the results obtained with a particular drug, with the highest dose at the top of the column. Dotted line s1iou.s the percentage of correct responses to the approach signal while the solid line shows the performance level to the avoidance signal. Injections occurred after the second pre-drug session of each experiment.

YETH4YPHETWIWE

’:v,, &Dlus

Id 2ndM HR

60

40 20

0 MORW

Id 2 d 3 r d l l h

-m-

FIG.IB. As for previous Fig. lA, but for cat 11.

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E. R. JOHN, P. WALKER, D.

CAWOOD, M.

RUSH, J. GEHRMANN

of graphs presents either a replication, in the case of SALINE, or a different dose, in the drug columns. Dose effects are arranged from highest on the top row to lowest on the bottom. Approach performance level is shown by the dotted line, avoidance performance by the solid line. Detailed examination of these graphs reveals several features which merit discussion. The SALINE placebo experiments, in the first columns, show that these animals could sustain a relatively high level of welldifferentiated approach-avoidance performance throughout a lengthy series of closely spaced sessions. In the second columns, the top graph shows that 5 mg/kg of CPZ abolished any behavioral response to either stimulus for the remainder of the day. One hour after 2.5 mg/kg of CPZ, both cats displayed some residual CAR performance although CR performance was abolished. In cat 11, this anomalous response to a strong tranquilizer dose became further accentuated as CAR performance was restored to relatively high levels for the rest of the day while CRs remained absent. Paradoxically, in this cat 1 mglkg of CPZ had the effect conventionally expected, interfering with CAR performance more markedly than with CR and causing a greater effect than the 2.5 mg/kg dose. Even clearer differential effects of the 1 mg/kg dose of CPZ were seen in cat I, with only slight impairment of the CR while the CAR was substantially abolished. The third columns of graphs show the effects of the experimental drug, referred to as MJ. Compared to CPZ, MJ has a shorter duration of action. As before, the data reveal major similarities but contain definite internal inconsistencies. In the case of the highest dose, both cats show an initially complete blockade of both CR and CAR behaviors, with CAR performance returning before CR as the drug wore off. [Cat I1 received an upper dose of 10 mg/kg of MJ because she was more resistant to this drug than was cat I, as judged by the minimal impairment after a dose of 1 mg/kg (not shown).] At the intermediate dose, both cats show greater impairment of CAR than CR. At the low dose, cat I shows parallel early impairment and swift recovery of both behaviors, with a second later deterioration of the CAR. Paradoxically, to the lowest dose of MJ, cat I1 not only showed more marked and prolonged drug effects, but CAR performance returns to high levels while CR remains absent. The fourth columns of graphs show the effects of PHENO. In cat I, the highest dose promptly abolished CAR performance, while causing significantly less interference with the CR. The intermediate dose caused marked deterioration of the CAR with a brief period of complete blockade. CR performance levels followed the CAR but were consistently

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somewhat higher. At the lowest dose, this drug caused definite impairment of CAR in cat I while CR performance remained perfect throughout the day. Cat I1 showed a different profile of effects. A t the highest dose, both CAR and CR showed a brief period of apparent enhancement followed by prolonged blockade of both types of performance. At the intermediate dose, moderate impairment of CAR and severe impairment of CR oceurred. At the lowest dose, a slight but consistent superiority of CAR over CR was observed. Cats I and I1 thus showed qualitatively opposite differential effects of PHENO on the two test behaviors, although the data for each cat across the three doses displayed internal consistency. The effects of the two doses of METH on the two animals, shown in the last column, were quite consistent. In every case, the drug injection was followed by complete blockade of any CR performance, decreasing latency of CAR performance, appearance of spontaneous CAR responses and performance of CAR’S t o all CR stimulus presentations (not graphed).

B. GENERAL DISCUSSION OF BEHAVIORAL RESULTS In spite of broad general agreement between the two bodies of data from cats I and 11, detailed examination of the behavioral results has revealed numerous minor contradictions. Not only do the data contain internal inconsistencies from dose to dose of the same drug in the same animal, but the qualitative effects in one animal were sometimes diametrically opposite from those in the other animal. It is possible that the conclusions which might be drawn from the areas of concordance in the data might suffice for certain purposes. Unquestionably, increasing the size of the population might permit one to label the response configuration displayed by one of these animals as “typical,” although it can never eliminate the “atypical” data from the sample. However, no matter which body of data we choose to regard as typical, we encounter difficulty when we attempt to characterize the behavioral effects of these drugs or to distinguish one drug from the other. In neither cat does either tranquilizer uZauys interfere with CAR more than with CR. The barbiturate has no behavioral effect not seen in the phenothiazine, nor do striking differences emerge between the phenothiazine and the nonphenothiazine. Only the stimulant shows a consistent and characteristic profile of effects which clearly distinguishes it from the other compounds. These findings suggest, therefore, that such measures as differential interference with discriminated approach-avoidance behaviors may not only provide erratic and inconsistent descriptions of the behavioral

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hl. HUSH, J. GEHRMANN

effects of a particular drug, but arc probably of inadequate resolution to permit unequivocal identification of a drug or useful classification of compounds on the basis of other than gross similarities. These shortcomings in no way invalidate the use of behavioral techniques for routine screening and bioassay purposes, or to specify a particular state in the organism, but are pointed out as limitations of the method for the spccific purposes here of concern. C. EEG FINDINGS Using magnetic tape in addition to conventional ink writers, EEG recordings were obtained throughout the two pre- and four post-drug test sessions of every experiment. The voluminous data thus accumulated for 78 sessions per cat were subjected to careful visual examination. Particular attention was paid to the EEG data obtained at the time of maximum behavioral effect (TMBE ) . For a given drug, the qualitative effects of the various doses seemed generally similar, primarily varying with respect to amount and duration. At TMBE, different doses of the same drug usually produced essentially similar effects, but in a number of cases the lower doses produced no grossly visible change in the electrical activity which was observed. Typical data from cat I are illustrated in Figs. 2A (control), 2B (CPZ), and 2C (MJ). Data from cat I1 are illustrated in Figs. 3A (control), 3B (PHENO), and 3C (METH). All data in Figs. 2 and 3 were taken at TMBE. SALINE data closely resembled the control data in all animals. The remaining raw EEG data will not be illustrated here. Inspection of such data revealed qualitative features such as relative amounts of synchrony or desynchronization, theta rhythms, and spindling. Evoked responses were more or less apparent as a function of various factors such as the anatomical region involved, the amount of background or spontaneous activity, and the frequency of the stimulus. Comparison of data from control and drug sessions often revealed clear-cut differences. For example, the EEG of cat I after CPZ has noticeably less high frequency activity than the control records. Marked spindling can be seen in nucleus centralis lateralis (C L ), becoming extremely pronounced during flicker stimulation. However, neither of these features is unique. Cat I showed marked slowing of EEG under PHENO and pronounced CL spindles under MJ. Further, such changes are not always consistent. For example, cat I1 shows extremely large and widespread spindles under PHENO, which caused little or no spindling in cat I. Examination of the evoked responses discernible in these recordings shows clear evoked responses in certain structures, while other structures

CAT 1

I

FIG. 2A. EEG records obtained from cat I during control trials resulting in correct response to the avoidance and approach stimuli. Stimulns artifact is shown on the bottom trace, and calibration marks are at the right. For explanation of abbreviations see pp. 279-280.

290

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E. R. JOHN, P. WALKER, D, CAWOOD, At. HUSH, J. GEHRMANN

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2% E. R . JOHN, P. WALKER, D. CAWOOL), h l . HUSH, J. GEIIRMANN

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METH 1 mg/kg

CAT 11 Vl APPROACH STIMULUS -CAR

M

V2 AVOIDANCE STIMULUS-CAR

I

50 U V

3

U

n 9

8

8

"a Y

!+

-1 SEC

FIG.3C. As Fig. 3A, but after 1 mg/kg of methamphetamine.

BRAIN STATES APPLIED TO DRUG CLASSIFICATION

295

show little or no response to the conditioned stimuli. In general, evoked responses elicited by thc fast CS wcrc~more apparcnt than those caused by the slow CS. Differences in the wave shape of the responses evoked by the two differentiated stimuli were often quite marked. Administration of drugs caused changes in the amount and form of evoked responses in many regions. Evaluation of both EEG and evoked response features by qualitative methods was extremely difficult. Thc, sheer volumc of data constituted a major source of difficulty. No reliable method exists to compress this volume into a concise form or to define the features of a representative example. Short samples could be selected within the usual recording session to illustrate almost any desired phenomenon. Thus, data analysis required inspection of a sufficient body of data to acquire a “gestalt” and selection of an example which typified that universe of data in the judgment of the experimenter. Even after selection of the data, classification by mere visual inspection became increasingly difficult as the number of examples to be compared increased. Therefore, a crude, semiquantitative description was devised. Using only data from the highest dose of each drug, the session in which the maximum behavioral effect occurred was identified, and representative or “typical” records were selected. The amount of synchrony in the ongoing EEG was scored, with values ranging from 0 for desynchronized to 1 for hypersynchrony. Occurrence of marked theta rhythms and spindles were noted. The amount of evoked response to the two different CS’s was likewise scored. Values ranged from 0 if no evoked response was apparent, to 1 if intermittent evoked response could be discerned, to 2 if evoked responses were marked and continuous throughout the period of stiniul at’ion. For each anatomical region, composite scores were obtained by averaging the data from that structure across animals and across stimuli. Those data are summarized in TabIe I. The average amount of synchrony is denoted by %S, and the average amount of evoked response by %E.An overview of the effects of various drugs upon the spontaneouq EEG and the amount of evoked response are provided by Table I. The anatomical regions from which data were obtained have been divided into: Group I-visual cortex and lateral geniculate body; Group 11-mesencephalic reticular formation and nonsensoiy-specific thalamic nuclei; Group IIIlimbic structures and others. 1. Control:

Group 1. Desynchronized EEG and inarked evoked responses noted, ranging from intermittent in monopolar derivations to steady in bipolar

TABLE I Control Structure

Derivation

n %Sa

SALINE CPZ -

%Eb %S

MJ

PHENO

METH

%E

%S

%E

%S

YoE

67

1 1 1 1 1

67 67 50 92 69

3333333333

75 75 92 92 84

67676767 67

58 50 75 100 71

33 58 67 92 69

1 0

25 42 25 25 00 50 28

0

13 33 75 25 25 50 37

50338 1

25 00 00 00 00 00 04

50 67 00 50 100 00 46

70S

%E

%S %E

I Visual cortex Lateral geniculate

M B M B

3 3 3 3

0 0 0 0 0

67 83 58 92 75

M B M

2 3 1 1 1 1

0 0 00O0 0

00 58 25 50 00 00 22

0 0 O N 00 0 0

00 58 25 50 00 00 22

le

13 00 00 00 00 25 00 13 06 50 50 50

ie

13 00 00

Average

83 58 92 75

4

0

"E at

I1 Mesencephalic reticular formation Nucleus reticularis Nucleus ventralis anterior Nucleus centralis lateralis Average

B B B

I 1 0 167

oe 1 1 0 O N

33

1-

1 064

U

111 Dentate

M

M

2 1 2 2 1 1 1 2

M B

2 1

B Hippocampus Prepyriform Cirigulate Septum Medial forebrain bundle Average Subthalamus Substantia nigra Average a

%S

=

b

7,E

=

B M

M B M

18

le O-

0 00 O38

oe 0 0

1e

ie O N

0 O0 O38

oe 0 0

00 00 25 00 13 06 50 50 50

1 1 1 75111 1 97 1 1 1

13

25 13 13 25 25 25 38 22 25 25 25

50 0

5oe O1 1 0-

5oe 44

oe 00

13 00 13 00 00 00 25 25 10 25 25 25

-

00-

1 ,500

501 10 5057

ie0 50

13 00 00

5oe

00 00 00 25 13 06 25 25 25

00 0 0 508 25 508 0 25

0

ie

zS/n; S = EEG synchrony; 0 = desynchronized; 1 = hypersynchrony; e = theta rhythm; = spindle activity. zS/n; E = evoked response to VI and Vz; 0 = no visible response; 1 = intermittent; 2 = steady.

00 00 00 13 50

00 00 13 10 25 50 37

i:

BRAIN STATES APPLIED TO DRUG CLASSIFICATION

297

derivations. The consistently greater amount of cvoked response visible in bipolar derivations is probably to be attributed to a better signalto-noise ratio, since such derivations primarily reflect local activity.

Group IZ. Desynchronized EEG noted, with 2 0 4 0 Hz spindle activity seen in nucleus reticularis. Intermittent evoked responses were consistently visible in bipolar derivations from the mesencephalic reticular formation. Evoked responses were sporadically seen in monopolar records from nuclcus rcticularis and were somewhat more marked in bipolar recordings. Group ZlZ. Hypersynchrony with marked theta rhythms was observed in the spontaneous EEG activity from the dentate and hippocampus electrodes, and from the subthalamus. Other structures in Group I11 showed desynchronized rhythms, with 2 0 4 0 Hz spindle activity quite marked in records from the prepyriforin or cingulate cortex and the medial forebrain bundle. Occasional evoked responses were observed in bipolar derivations from the cingulate and substantia nigra, and in monopolar records from dentate, medial forebrain bundle, and the subthalamus.

2. SALINE: Saline-injected animals showed no changes from the controls.

3. CPZ: Group I . After CPZ, these structures displayed marked slowing of EEG activity, but relatively little change in the amount of evoked response apparent to visual inspection. Group Zl. Synchronous EEG activity appeared in most derivations from this group. Spindle activity disappeared from nucleus reticularis, but was noted in centralis lateralis. Moderate evoked responses appeared in monopolar recordings from the MRF, with some decrease in bipolar records, suggesting somewhat more uniform effects of the stimulus upon this structure. Centralis Iateralis showed a marked increase in evoked response. Group ZZZ. All structures in Group 111 showed markedly hypersynchronous EEG after CPZ. Theta rhythms disappeared, but spindle activity remained in the prepyriform and cingulate cortex. Intermittent but definite evoked responses became apparent in all structures. The general effect of CPZ was therefore to cause widespread hypersynchrony and disappearance of theta activity. Responsiveness to the conditioned stimuli was increased, especially in Group 111.

298 4.

E. R. JOHN, P. WALKER, D. CAWOOD, M. RUSH, J. GEHRMANN

MI:

Group 1. After MJ, all structures in Group I showed a slight increase in synchronous activity with widespread spindling. Evoked responses became more apparent in monopolar derivations, suggesting a more global response to the CS. Group ZI. Spontaneous EEG activity in this set of structures was altered but little by MJ. Derivations from nucleus reticularis displayed slow waves. Bipolar derivations from the MRF showed the appearance of theta rhythms, while spindle activity appeared in centralis lateralis. Evoked responses became visible in all structures, most markedly in nucleus reticularis. Group I l l . EEG activity was slowed in many members of this group of structures, especially in the cingulate. Theta activity and spindles showed a somewhat different distribution from the controls. Slight amounts of evoked response activity became visible in the septum and hippocampus, but the overall level of evoked response was little changed. The general effect of MJ was a moderate increase in hypersynchrony and spindling. The amount of evoked response was increased, especially in Group 11.

5. PHENO: Group 1. After PHENO, all structures in Group I showed markedly slower activity, with occasional large spindle bursts. Evoked responses at the cortical level diminished somewhat but became more pronounced and regular in the lateral geniculate, especially in bipolar derivations.

Group Zl. Marked hypersynchrony appeared in almost all Group I1 structures. The only exception was centralis lateralis which displayed large spindle bursts on a background of fast activity. Spindles and theta rhythms were also observed in the MRF, while spindles appeared in nucleus reticularis. Evoked responses almost completely disappeared, except for brief “on” responses in monopolar recordings from the MRF. The total absence of evoked responses in bipolar records from MRF, which normally showed marked and consistent responses, was particularly noteworthy. Group I l l . A moderate iwrease in hypersynchrony was observed in Group I11 structures, especially in cingulate, medial forebrain bundle, and the subthalamus. Marked spindle bursts and theta rhythms were also seen. Evoked response activity was little altered.

299

BRAIN STATES APPLIED TO DRUG CLASSIFICATION

The general effects of PHENO, thus, were a widespread increase in hypersynchrony and marked spindle bursts. Evoked responses almost disappeared from Group 11, but were little diminished in amount elsewhere. 6. METH: Group I . After METH, all structures in Group I showed low-voltage fast EEG activity. The amount of evoked response activity was little changed. Group ZZ. EEG activity in structures of this group altered little after METH. Marked evoked responses appeared in monopolar records from MRF and in bipolar records from VA, suggesting more massive response in this system. Group ZZZ. Structures in this group showed somewhat more fast activity, less spindling and less theta rhythm after METH. Little change was seen in the amount of evoked response. In general, the effect of METH was to diminish slow activity and to increase the amount of evoked responses in Group I1 structures.

7. Summuy These findings are summarized in the following tabulation: CPZ

MJ

PHENO

-

-

Group

S

Y

E

S

I I1 I11

+

o

+

O

$

0

+

O

E

+ +

+ +

METH

S

E

+ o

0

0

+ o

-

E

+ -

o + 0

Similarities and differences can be seen in these profiles of the action of the different drugs examined in these experiments. CPZ, MJ and PHENO all cause a widespread increase in hypersynchrony, while METH causes an increase in fast activity restricted to the limbic system. MJ and METH caused an increase in the amount or extent of evoked response in nonsensory specific structures, while PHENO abolished almost all response in these regions. CPZ had little effect on the amount of evoked response in Group I1 structures, but caused an increase in the extent of limbic system responses. Thus, qualitative evaluation of the effects of the highest doses of these various drugs suggests that they display somewhat different profiles. However, characterization of gross changes in the features of spon-

300

E. R. JOHN, P. WALKER, D.

CAWOOD, M. RUSH,

J. GEHRMANN

taneous EEG activity failed to show distinctive and consistent patterns which were unique to each drug. Although METH did not cause the EEG slowing which the other three drugs shared, the increased fast activity after METH was not so unequivocal as to suffice for identification. An increase in evoked responses in Group I1 resembling the effect of MJ was also caused by METH. The spindling and reduction of Group 11-evoked responses caused by PHENO was distinctive but was not consistently observed in all animals nor at all doses in the same animal. Further, no measurements were available on the reproducibility of subjective scoring of the data, on the validity of the selected example, or on the interanimal and interdose replicability. Systematic substantiation of these findings would have been prohibitively time-consuming, in view of the low precision of the results which might finally have been obtained. Yet, little question exists that the electrophysiological activity after these various drugs appears somehow different. Clear differences can be discerned, for example, in evoked potential wave shapes. Recourse to more quantitative methods was necessary to ascertain whether it was possible to extract and specify particular features of spontaneous or evoked activity which would reliably and uniquely describe different drugs in a variety of doses.

D. FREQUENCY ANALYSIS Because of widespread reliance on the frequency spectrum or the power spectral density as a descriptor of EEG activity, it was of interest to study our data from this viewpoint. Rather than computing the fastFourier transform of short EEG segments, which poses the problem of selection of representative samples of record and requires extremely time-consuming computation, spontaneous EEG activity during longer periods was analyzed using sharply tuned band-pass filters and a special purpose computer ( Neurodata SA 2200). This instrument calculates the “signal ratio” of the strength of the EEG signal in the bandwidth of interest to the strength of the total signal and presents the result (percent of total signal in the specified band) as a ineter reading. Using a long time constant to smooth the reading (10 sec), the average value of the signal ratio was determined during 1 min of EEG activity. At the end of each hourly session in a day’s experiment, a few minutes of spontaneous EEG activity was recorded. The frequency analysis results reported here were obtained using 1 min of EEG arbitrarily selected from the end of the second hour of control data and 1 min of activity from the end of the second hour after drug administration.

BRAIN STATES APPLIED TO DRUG CLASSIFICATION

301

These data are illustrated for cats I and I1 in Fig. 4A-D. In each case, the solid curve represents the pre- and the dotted curve the postdrug data. Ordinate values are in arbitrary units, with increasing content in the band corresponding to smaller numbers along the vertical axis. Note that because the band-pass filters only cut off 18 DB per CAT II CPZ

CAT I

I

CPZ

LGM 4.0

't,

I

LGS

X WE 0 WST

FIG. 4A. Each set of 12 graphs shows the effects of a particular drug on the frequency composition of the EEG. Each graph shows the distribution of the energy in the EEG recorded from the indicated anatomical placement before ( x ) and after (0) the drug. The vertical axis is proportional to the ratio of the portion of the energy within a particular frequency pass band to the total energy in the signal. The horizontal axis indicates the different bands: 6 = 0.5 to 3.5 Hz; e * 3.5 to 7.0 Hz; CY = 7.0 to 13.0 Hz; p = 13.0 to 25.0 Hz; y = 25 to 40 Hz. Left: Effects of 5 mg/kg of CPZ on cat I. Right: Effects of 5 nig/kg of CPZ on cat 11.

302

E.

R. JOHN,

P.

WALKER, D.

CAWOOD, M. RUSH, J. GEHRMANN

octave, and because the total EEG bandwidth is greater than the sum of the individual pass-bands, the sum of the values is not a constant.

1. CPZ. Figure 4A shows the effect of a 5 mglkg dose of CPZ on the frequency composition of the EEG. In almost every structure, cat I shows markedly diminished high-frequency activity ( y and p ) and increased activity at slower frequencies after this drug. Cat I1 shows less y in five structures, more y in four structures, and unchanged y in three structures. Almost every structure shows an increased amount of slow activity after CPZ. 2. M I ,

Figure 4B shows that in cat I, 5 mg/kg of MJ caused an increase in ,8 and y activity and a marked decrease in 0 and/or 8 in a number of structures. In contrast, cat I1 shows a decrease in p and y and increased slow activity in many structures. 3. P H E N O .

Figure 4C shows that in cat I, 20 mg/kg of PHENO caused an increased amount both of very slow frequency ( 8 ) and of fast frequencies ( p and y ) in some structures, but a decrease of fast frequencies in other structures. In cat 11, PHENO caused an increase in 8 and a decrease in y in many structures. 4. M E T H . Figure 4D shows that in cat I, 1 mglkg of METH caused an increase in y activity in seven structures, but a decrease in three structures. Most structures also showed an increase in very slow activity. In cat I1 similar results were observed. Two facts seem evident from these findings. First, the effects of a specified dose of a given drug upon the frequency spectrum of various brain structures in one cat may be quite different from the effects of the same dose upon similar structures of a different cat. Second, careful examination of the repeated sets of control measurements ( solid curves ) from particular brain structures reveals wide variation in the control spectra from session to session. The scope of this lack of replicability of the frequency spectrum can be better appreciated from Fig. 5, which summarizes all control and post-drug measurements for cats I and 11. For each frequency band, the vertical solid line shows the range of six control measurements on 1-min recordings of spontaneous EEG taken at least a week apart. The

BRAIN STATES APPLIED TO DRUG CLASSIFICATION

303

CAT I MJ 9022-1

CAT IT MJ 9022-1

FIG. 4B. Left: Effects of 5 mg/kg of MJ 9022-1 on cat I. Right: Effects of 5 mg/kg of MJ 9022-1 on cat 11.

vertical dotted line shows the range of six post-drug measurements, including the highest doses of each of the four drugs studied in these experiments and two runs after saline placebos. Examination of Fig. 5 shows that, for most structures, the variation in frequency spectrum observed within a series of measurements repeated under control conditions was approximately as great as the variation between a series of measurements each taken after a dlfferent drug. The diEerences observed between effects of the same drug and dose on different cats may be due to the fact that the drug effect depends to some extent upon the pre-drug state.

304

E. R. JOHN, P. WALKER, D.

CAT I

CAWOOD, M.

RUSH, J. GEHRMANN

CAT II PHENOBARBITAL

PHENOBARBITAL

1.5 20

LO 4.0

20

3.0 4.0 20

3.0 4.0 2.0

3.0 4.0 2 .o

3.0 4.0 2.0

3.0 4.0

4.0

6 X PRE 0 POST

eap X

6

e a p

>

X PRE 0 POST

FIG. 4C. Left: Effects of 20 nig/kg of PHENO on cat I. Right: Effects of 20 nig/kg of PHENO on cat 11.

The poor consistency of these data, derived from relatively long segments of EEG, highlight the need for extreme care when computing frequency spectra or power spectral densities of short data segments by conventional techniques. Because of the implications of these findings, we computed the 120 1-sec EEG segments taken from the preand post-CPZ samples of spontaneous EEG of cat I, using a fast-Fourier transform technique." Mean values and standard deviations of each frequency band were also computed. The extreme variability obtained ' W e wish to thank Noel Fleming, of the University of California at San Diego, for making this computer program available to us.

BRAIN STATES APPLIED TO DRUG CLASSIFICATION

I

I METHAMPHETAMINE I

305

CAT II METHAMPHETAMINE

FIG.4D.Left: Effects of 1 mg/kg of METH on cat I. Right: Effects of 1 mg/!ig of METH on cat 11.

from these computatioiis confirmed the results discussed above. Adequate sampling must be utilized to demonstrate that the rcported results are truly representative of the state of the nervous system throughout the period of interest, not merely for the interval of the sample. In view of this variability, there seemed little basis for an attempt to characterize these drugs on the basis of any possible distinctive features of the EEG frequency spectra observed after their administration.

E. AVERAGE EVOKEDRESPONSES Using sample sizes of 200 evoked potentials, average evoked responses to v, and v, were computed from every hourly session. Each computation included data from 5-10 behavioral trials. Figure 6 shows

306

E. R. JOHN, P.

WALKER,

D.

CAWOOD,

M. RUSH, J. GEHRMANN

FIG. 5. Summary of frequency analysis results for cat I (Natalia) and cat I1 (Audrey). Each graph presents a comparison of the range of six control (solid vertical lines) and six post-drug (dotted vertical lines) measurements of the frequency spectrum for a given electrode placement. Axes as for Fig. 4.

typical average evoked responses which were obtained from cats I, I1 and I11 during a saline placebo experiment. These data illustrate the extremely high reproducibility of average evoked-response wave shape throughout a long series of recording sessions. Note that the untrained animal, cat 111, shows somewhat greater variability than cats I and 11, which were trained. On this stable wave shape baseline, drug effects were rather conspicuous. Such effects, for each drug in this experiment, are illustrated in Fig. 7A-D. Figure 7A shows the effects of 5 nig/kg of MJ on cat I. Drug effects are obvious to visual inspection in most structures, particularly visual cortex (MONO and BIPOLAR leads), lateral geniculate (MONO and

BRAIN STATES APPLIED TO DnuG CLASSIFICATION

307

BIPOLAR leads ) , centralis lateralis, septum, and mesencephalic reticular formation. These effects are quite clear in the first hour post-drug, when the animal displayed no behavioral response ( N R ) to the conditioned stimulus. Effects can still be noticed in the second and third hours after the drug, while the stimulus continues to fail to elicit the behavioral response, but are less obvious. Figure 7B shows the effects of 5 mg/kg of CPZ on cat 11. The changes in average response wave shape are extremely clear-cut, and are apparent for a longer period than the effects of MJ previously illustrated. Figure 7C shows the effects of 20 mg/kg of PHENO on cat 11. The changes in average evoked response are again obvious. Notice the gross similarity between the effects of CPZ and PHENO on evoked response wave shapes, especially in VIS bipolar, nucleus reticularis, and the medial forebrain bundle. Figure 7D shows the effects of 1 mg/kg of methamphetamine on cat 111. Clear-cut changes in evoked response wave shape can be seen in most structures. Notice the extrcme reproducibility of data in this cat after METH, in contrast to the relative variability seen in the cat 111 data shown in Fig. 6. This suggests that some of the variability seen in cat I11 is due to fluctuations in the level of arousal, while the stability of response in cats I and 11 is to be attributed to the stabilization of arousal levels as a result of behavioral conditioning and thc task relevance of the stimuli eliciting evoked responses.

F. COLUMNFACTOR ANALYSES-RAW DATA In terms of average evoked responses, each drug experiment could be reduced to a set of wave shapes representing the hourly change in response to V, and V L ,as illustrated i n Figs. 6 and 7 . For each drug experiment, the post-drug average evoked response from the test session in which the greatest behavioral deficit was observed was selected as the prototypic wave shape to show the drug effect. Since there were 3 CPZ, 3 MJ, 3 PHENO, 2 METH, and 2 SALINE runs, 13 sets of wave shapes served as prototypes of the drug effects on responses to each stimulus on each cat. Similarly, for each stimulus, 13 sets of corresponding pre-drug wave shapes constituted prototypes of the control responses. As discussed on p. 282, these average evoked responses could be arranged into n niatrix of 111 rows ( m = 26; 13 pre-di-ug plus 13 post-drug wave shapes) and 11 columns (12 derivations from which recordings werc obtained). For each cat there were two 26 X 12 matrices, one for V, and one for V 1 responses. Across a row, the matrix contains the simultaneous responses of 12 structures to a given stimulus under a specified drug condition. Down a column, the matrix

308

E. R. JOHN, P. WALKER, D. CAWOOD, M. RUSH, J. GEHRMANN

CAT I

V, APPROACH STIMULUS b U15

WESALINE CR

SALINE CONTROL

L LG

ylr'

PRESALINE CR

I" HR CR

(EVENTRIALS) 1"

nu CT1

(WOW L S I 2"

M? CR

3 ’

HR CR

-&-'-N--

<

4"HRCR

R U15 PRES4LIHE CR

WEaALlNF CP 1" HR CR IEVEV TRIALS1

I" HR CP

lorn TRIALS1 2"HRCR

3" HR CR 4"

m CR

-vwy

w

v4-

FIG. 6. Average evoked responses elicited by V, and VI during each hour of the saline placebo experiment. Each column of data shows the sequence of responses recorded from a different anatomical region. Data on the same row were recorded simultaneously. Each computation consisted of 200 evoked potentials taken from 5-10 behavioral trials. Note the extreme stability of wave shapes. Analysis epoch 180 msec in this and all subsequent e.p. figures, unless noted otherwise. Data from cat I during correct performance of approach responses to stiniulus V,. Data from cat I1 during correct performance of avoidance responses to stimulus V,. Data from cat I11 (untrained) during V, presentation (258 msec epoch).

309

BRAIN STATES APL'LIED TO DRUG CLASSIFICATION CAT I1

V2 AVOIDANCE STIMULUS

SALINE CONTROL

R U15

L2Nv\

pREsAut€IEuLR

KESMWEW 1"HR CM

2"' HRCAR 3"HRCAR 4"HRCM

&

CAT I11 b U15

L LG

PREPYR

PREDRUG

PREDRUG

ISIMR

2nd HR

SALINE CONTROL

"I

5-

9

R UI5

&,-----7

L LG

SUE TH

MFR

?Ic--

F

/\fJ.-

b

kJ---

3rd HR

L MRF

L HRF

%+-v-2

+ D HlPP

FIG. 6 (Contitttrcd),

U

DENT

v--UJ

310

E. R. JOHN, P. WALKER, D. CAWOOD, M. RUSH, J . GEHRMANN

-

Vl APPROACH STIMUWS

M.J. 9022-1 5m p m

L DENT

-

__c

FIG.7A. As Fig. 6, but data from sessions in which drugs were injected. Effects of 5 mg/kg of MJ 9022-1 on the responses of cat I to the V1 approach stimulus.

contains a set of wave shapes all derived from the same brain structure in response to the same stimulus, under a wide variety of states caused by different drugs and also under control conditions over a long span of time, This paper will confine its discussion to results of subjecting columns from these matrices to factor analysis. Row analyses will be discussed in a subsequent paper. Because of the amount of computation involved, the storage capacity of our PDP-12 was taxed, and the columnar data were slightly compressed by deleting the two pre-SALINE control wave shapes. This did not substantially alter the variety of species in the signal space because of the high stability and hence redundancy of the control data, but permitted each column to be reduced to 24 wave shapes (11 predrug plus 13 post-drug). For each cat, the total amount of experimental data was thus reduced to 2 stimuli X 12 structures X 21 wave shapes, a total of 576

311

BRAIN STATES APPLIED TO DRUG CLASSIFICATION

---

CPZ

V,- APPROACH STIMULUS

CAT= L UI5 PREDRUGCR

&

PRElDRKiCR

&h,

L LG

R PRE PY

L

MRF

5mgIkg

L N RET

p"L.J

1"HRN.Q

w

P H R ~

V/LV

R MFE

9 A

. v

3"HRNR-

R

U DENT

R Y15

L LG

L N RET

R ClNG

R

ClNG

FIG.7B. Effects of 5 mg/kg of CPZ on the responses of cat I1 to the V, approach stimulus.

average evoked responses. Then 24 separate column analyses were carried out to provide a quantitative evaluation of this body of data and the results combined into an overall analysis for each cat. Before proceeding with the discussion of the results of column factor analysis, it is interesting to examine some representative columnar arrays of data to gain a qualitative appreciation of the stability of the control data and the nature of the wave shape changes caused by different doses of the various drugs. Figure 8 shows the column data from the bipolar lateral geniculate derivation for V1 (the approach stimulus) and V, (the avoidance stimulus) for cat I. Note the extreme reproducibility of the control wave shapes, shown in the first column of each figure. The differences between the wave shape of the responses to V, and V2, although small, are sufficiently reliable and pronounced to permit discrimination between these two responses both by visual inspection or computer assessment. Careful examination of the right-hand column of post-drug wave shapes shows certain overall similarities between the effects of different doses

312

E. R. JOHN, P. WALKER,

CAT II

I).

CAWOOD, M. RUSH, J. GEHRMANN

V,- APPROACH STIMULUS R Vl5

PREDRUG CR

&

HR NR

2 nd HRNR

3 rd HRNR 4 fh HRNR

PREDRUGCR

L N RET

-*-

---

- - - - - - d

v ".v" v

R U DENT

PREDRUGCR

R tING

- T v c / -

PREDMJG CR I S

R PREPY

-------

- - - - - - I / - - - - - -

L LG

L N RET

R

KING

1 St HR NR

2 M H R NR

I/cL

3 rd HRNR

A

fh HA NR

A

4

FIG. 7C. Effects of 20 mg/kg of PHENO on the responses of cat I1 to the VI approach stimulus.

of the same drug. This can be seen somewhat more clearly in Fig. 8b than 8a. Figure 9 gives other examples of columnar data from this cat, to illustrate the range of variability of wave shapes which is encountered in various brain regions using these procedures. Note the variations in signal size in the control data from the visual cortex (Fig. 9a). Such amplitude differences have negligible effects in the factor analytic computations, which normalize all waves to equal total energy. Figure 10 shows the column data from the bipolar lateral geniculate derivation of cat 11. The control data in the left column show comparable stability to the data from cat I. The post-drug data in the right column show Iess qualitative variation to visual inspection than the data from cat I, and further illustrate the level of analytic difficulty with which this method must cope. The bewildering amount of detaiI and subtle variation in wave shapes encompassed by this body of data are further illustrated by Fig. 11, showing column data from bipolar derivations of the cingulate

-

313

BRAIN STATES APPLIED TO DRUG CLASSIFICATION

CAT ILK

PREDRUG PREDRUG

-

L LG

&

7

PREPYR

L MRF

5UE TH

I mg/kg

NFK

cyc

-fR Ulf

PREDRUG

--

b U15

4"HR

METHAMPHETAMINE "I

L LG

-

L ffRF

D

HIPP

V DENT

PRE,DRUG 7

l* HR 2*HR

3"HR

4"HR

-LA---

0

FIG. 7D. Effects of 1 mg/kg of METH on the responses of cat I11 to the VI stimulus ( 256 msec epoch ) .

cortex and nucleus reticularis. Control data are not always highly replicable nor are drug effects always perfectly evident. So that the reader may have some impression of the differences between data from trained and untrained animals, an example of the column data from the bipolar lateral geniculate records of cat 111 is presented in Fig. 12. In general, it has been our experience that drug effects are most differentially evidenced in the lateral geniculate body, under the conditions of our experiments. Note that in spite of the relatively high variability of the control data in the left-hand column, the wave shapes in the right-hand column tend to group themselves visually into clusters which correspond to the different doses of each drug. Similar clusters can be discerned in data of some of the preceding figures.

G. REGRESSION EQUATIONS In the previous section, the reader has examined cross sections of the matrix of data into which the results of these experiments have been compressed. These examples illustrate several features of the data : some

314

E. R. JOHN, P. WALKER, D. CAWOOD, M. RUSH, J. GEHRMANN

CAT I

CAT I

LLG,

LLG,

VI -APPROACH STIMULUS PREORUG

V, -AVOIDANCE

POST DRUG

DOSE

JJG A

CPZ

MJ 9022-1

kJL

5

DOSE

wkg

CPZ

4

5

?

25 I

MJ 9022-1

5 25

I

PHENOBARB PHENOBARB

POST DRUG

PREDRUG DRUG

DRUG

STIMULUS

20

20 10

5

METH

075

L

~

METH

075

050 SAl INt

b

SAL INk

6

a

FIG. 8. Total array of data from 13 experiments, constituting the input for column factor analysis of the lateral geniculate body (bipolar derivation) of cat I. Each row of data comes from a different experiment. The wave shape on the left illustrates the average response prior to drug administration (usually from the second pre-drug session), while the wave shape on the right shows the average response obtained after drug administration ( usually from the second post-drug session). All averages based upon 200 evoked potentials taken from 5-10 stimulus presentations (trials). a. Data obtained using the V1 approach stimulus. b. Data obtained using the V, avoidance stimulus.

315

BRAIN STATES APPLIED TO DRUG CLASSIFICATION

CAT I

CAT I

R VIS,

LCL, VI -APPROACH STI MUCUS

Vi -APPROACH STIMULUS PREDRUG DRUG

PREDRUG

POSTDRUG DRUG

DOSE

CPZ

POSTDRUG

DOSE pgh

CPZ

5 - 4 4 -

1-

MJ 9022-1

5

25

V

v

PHENOBARB

PHENOBARB

-yi-

YETH

p

SAl IN4

-" I

a

+

20

10

-4.4-

5METH

075

050 SALINE

vim' b

FIG.9. As Fig. 6, but data from other structures in cat I, a. Data obtained from the visual Cortex (monopolar) in response to the V, approach stimulus. b. Data obtained from nucleus centralis lateralis (bipolar) in response to the V, approach stimulus.

but not all structures variously display relatively high stability of control wave shapes, relatively clear changes in wave shapes after drug administration, similarities in the effects of different doses of the same drug, and/or differences between effects of different drugs. If one bears in mind the fact that, for each cat, the experiment yielded 24 sets

316

E. R . JOHN, P.

WALKER,

CAWOOD,

D.

CAT II

hl. HUSH, J . G E H H M A N N

LLGB

V2-AVOlDANCE STIMULUS PREDRUG

DRUG

POSTORUG

DOSE

CPZ

MJ 9022-1

PHENOBARB

ME TH

SALINE

20

075

+

qn I

FIG.10. As Fig. 8, hut data obtained froiii the lateral geniculate body (bipolar) of cat I1 in response to the V, avoidance stimulus.

of columns like those illustrated, it becomes apparent that the full data set contained a bewildering amount of detail and variation. Nonetheless, in some of the examples of columnar data which liave been presented, the reader may have noted a vague tendency for the data to be grouped into clusters which share aspects of visual pattern, which are quite definite evcn if hard to specify. The first step in quantifying this similarity of patterii is to computc thc correlation coefficient betwecn each wave shape and every other wave shape.

317

BRAIN STATES APPLIED TO DRUG CLASSIFICATION

CAT lI

CAT II

k LINGB

LNRET,

V, -AVOIDANCE STIMULUS

VI -APPROACH STIMULUS

POST DRUG

PRE DRUG

DRUG CPZ

A

DRUG

w

CPZ 25 I

MJ 9022-1

MJ 9022-1

5

I

PHENOBARB

fl

d A

PHENOBARB

20

METH METH

075

SALINE

a

b

FIG. 11. As Fig. 10, but data from other structures of cat 11. a. Data froiii cingdate cortex (bipolar) in response t o the V J avoidance stimulus. b. Data from nucleus reticularis (bipolar) in response to the VI approach stimulus.

In order to computc thc, correlation coefficient, each ~ 7 a v cshapc must be digitized into a series of numbers, V, ( t ) ,such that each SUCcessive number V, represents the value of the average evoked response voltage at that time. As i ranges from 0 to T in small steps (usually 1 msec), the series V, ( t ) provides an accurate digital rcprcscntxtion of thc wave.

318

E. R. JOHN, P. WALKER, D. CAWOOD, M. RUSH, J. GEHRMANN

CAT

III

LLG, "I PREDRUG

DRUG

POSTORUG

DOSE

CPZ

MJ 9022-1

'V _nlr PHENOBARB

20

METH

SALINE

+-

FIG.12. As Fig. 8, but data obtained from the lateral geniculate body (bipolar) of cat III in response to the V, stimulus.

As pointed out in a previous publication (John et al., 1964), the series of numbers V ; ( t )dcfiiies a signal vector in a T-dimensional space. If the correlation coefficient, M i j , is now computed between two such series of numbers representing two average response wave shapes, the value of M , , is analogous to the cosine of the angle between the two signal vectors in the T-space. Thc correlation matrix, which contains

BRAIN STATES APPLIED TO DRUG CLASSIFICATION

319

the set of values of M , , obtained by correlating each wave shape in the column against every other wave shape in the column, defines a set of spatial constraints simultaneously imposed upon the corresponding group of signal vectors representing the columnar wave shapes in T-space. Factor analysis uses this set of constraints to make explicit the relationships between these vectors and the space in which they exist. Functionally, two goals are accomplished by principal component factor analysis: first, the niininium number of dimensions ( factors), K , necessary to contain all of the signal vectors of the column is determined. (This will subsequently be referred to as the “dimensionality of the signal space.”) The lower the dimensionality, the smaller the number of independent processes whose interaction can represent the original set of data.) Second, the original set of N signal vectors is described in a set of regression equations. The regression equation which describes a signal vector V, consists of the linear sum of K independent axes or “factors,” F , , each multiplied by the appropriate weighting coefficient a,j. Thus: v, = a,lFI atzFz -k . . . -k a x F K Since all signal vectors can now be described as linear combinations of the same basic set of components, differences between different wave shapes are quantitatively represented by comparing the appropriate sets of coefficients, ah,. I n principal component factor analysis, the axes provided by the technique are mutually orthogonal. As a result, the sum of the squares of the weighting coefficients

+

K

2

(fll?

1-1

must equal 100%of the length of the signal vector (energy of the wave shape). Thus, a precise metric is provided for the estimation of the relative contributions of different factors to a given signal. The principal-component ( Jacobi ) method provides such advantages as the one just mentioned, but has severe shortcomings. Successive factors are oriented so as to maximize the rate of reduction of residual variance in the signal space. This yields a set of axes for the space so oriented as to straddle the largest number of signal vectors with each basis vector or factor. Consequently, differences between signal vector orient at’ions tend to become obscured in the resulting regression equations. The SOcalled Varimax procedure deals with this shortcoming by rotating the set of axes provided by principal-component analysis into an orientation in which each signal vector lies as close as possible to the smallest number of axes. This procedure tends to accentuate functional groupings

m 3

RI-GRESSION EQU.\TIONS

FOR v I S U . \ L CORTEX

TABLE I1 (MONOPOLAR) RTCSPONSES

8 TO v1 I N C.\T I-COLUMS

$

. 4 N \LYSIS

..

Jacvbi coefficients squared

1)rugs (mg/kg)

CPZ 5 2.5 1

21

Varirnax coefficients sqriared

U

Factor

1

2

3

4

5

6

2P

1

2

3

4

5

.04

. T9* -

. 00

.04

.o1

.ol

.93 .77

.03

.04 . 00

.ol

.oo

.oo

9s 95 94

.01

.81 -

.ll .ol

.OO

. 08

.oo .oo

.40 -

.10

.17

.o1 .I9 .3l

-

.OO

,32 .26 .37

.oo .oo .oo

.12 .01 .o0

92 93 93

.09 .I0 .14

.OO .30 .06

.S3 .58 .77 -

.06

.OO

.oo

.oo

9.3 93

.OO

.OO

85

.07 . 00 02

.oo

.oo .02 .OO

-

.02 .17 .05

.37 .30 .07

.07 .3.; .33

.OO .OO .OO

'3s

.OO .OO

95 96 96

.s9 -

,05

.oo .oo

%J 99 97 96

MJ 5 2.5 1

PHENO 20 10 5

.47 .41 .49 .78 .73 .93

-

. 01

.23 .07 . 00

.21 .00

-

.oo .02

.oo .10 .OO

.O1

.52 .16 .52

-

-

I

-

97 100

0

2! d -g g 3

"Z

+ 2

?j

5 3

iLIETH 1

.01

.oo

. 00

. 00

.03

.08

.oo

.40 -

--

.96 -

.05

.02 .l5

.oo

.21 -

97 97

.oo

.57 -

.13 .ll

.02

0.5

SALINE #I

.92 -

.04

. 00

.00

.01

.oo

97

.82 -

.OO

.06

.I0

.00

SALINE #2

.89 -

.0:2

100

. 00

.oo

.01

93

.76 -

.oo

.10

.I3

. 00

.93 -

.01 .Ol .04 .13 .09 ."5 .I0 .02 . 00 .12 , 00

.02 .05 . 00 . 00 .01 .04 . 00 .07 .03 .01 0.5

97 94 96 97 94 93 95 96 97 97 93

.70 -

.OO .03

.04 .06 .08 .05 .01 .ll .06 .03 .04 .08 .04

.24 -

Controls Pre-CPZ 5 Pie-CPZ 2.5 Pre-CPZ 1 Pre-MJ 5 Pre-MJ 2.5 Pre-MJ I Pre-PHENO 20 Pre-PHENO 10 Pre-PHENO 5 Pre-METH 1 Pre-METH 0.5 0

J

=

.87

reconstruction accuracy,

. 00

* Underlining shows heaviest loadings.

.81 -

. 00

.oo

.01

.00 . 00 . 00

.01 .OO .01 .04 .00

.oo

. 00

.OO

.OO .01 .OO .00 .OO .OO

. 00 . 00

.oo

.oo

.03

. 00

.01

.01 .02

. 00

. 00 . 00

.00

.42 .78 -

.8S -

.90 .80 .85 .42 .63 .89 .50

-

.oo .oo .00 .07

.oo .O6 .03 .OO .Ol

.00

. 00

.47 .12

. 00 . 00

.04

. 00

06 . 00 .06 .46 .28 .01 43

. 00 . 00 . 00

-

. 00

-

. 00

. 00

. 00 P

2

322

E. R. JOHN, P. WALKER,

D. CAWOOD, M.

RUSH,J .

GEHRMANN

within the data. Both Jacobi and Varimax procedures have been utilized in this study to permit comparison of the two methods. An example of the regression equations provided by both methods is shown in Table 11. These data are the results of column analysis of the monopolar visual cortex derivation in cat I, in response to the approach CS, V,. In order to understand the tabular material which will constitute the remainder of this paper, it is necessary to discuss this example in detail. Summing the contribution of successive factors to the energy of the set of signal vectors (normalized sum of eigenvectors), it was found that six factors accounted for 97%of the total energy in the signal space. The regression equations which reconstructed all 24 of the data wave shapes in terms of these six Jacobi factors were computed, and the squared coefficients of those equations are shown on the left side of Table 11. The accuracy of reconstruction ( J ) of each original wave shape is given in column 7 of the table as a percentage. Thus, for example, the energy of the wave shape recorded from the visual cortex of cat I, in response to the approach stimulus V1,after a dose of 5 mg/kg of CPZ could be described as:

+

+

+

98% CPZ (5 mg/kg) = 4% FI 79% Fz 4% F4 11% F 5 All contributions from any factor to any wave shape in excess of 20% have been underlined in Table 11. It can be seen that factor 2 accounts for most of the energy in CPZ 5 mg/kg and CPZ 2.5 mg/kg, while most of the energy in CPZ 1.0 mg/ kg comes from factor 1.Although factor 1 accounts for most of the energy in all three MJ doses, the next largest contribution comes from factor 3. Note that CPZ wave shapes derive little energy from factor 3, while MJ wave shapes derive little energy from factor 2, although in the case of MJ 2.5 mg/kg, 23% comes from that source, All PHENO wave shapes derive most of their energy from factor 1, but some energy seems to come from other sources. The higher METH dose derives 81%of its energy from factor 4, a factor which contributes essentially zero to all the CPZ, MJ, and PHENO wave shapes. The lower METH dose derives most of its energy from factor 1, with some contribution from factor 3. The two SALINE placebos derive almost all of their energy from factor 1. All of the pre-drug controls derive most of their energy from factor 1. Only in one case, preMJ 1.0 mg/kg, does any other factor contribute as much as 25%. These data, then, can be interpreted as meaning that the normal or control response of this structure to the approach stimulus is closely approximated by factor 1, which accounts for about 85% of the energy in the usual control wave shape. Higher doses of CPZ ( 5 and 2.5 mg/kg cause the appearance of a new wave shape, well described by factor 2.

BRAIN STATES APPLIED TO DRUG CLASSIFICATION

323

The lowest CPZ dose (1.0 mg/kg) causes no deviation from the control wave shape in this structure. All three MJ doses cause changes in the response wave shape such that only between 4 6 5 0 % of the energy can be accounted for by factor 1. A new component appears in the wave shape, and is described by factor 3. PHENO apparently has relatively little effect at this anatomical level, with most of the energy in the wave shapes observed after all three PHENO doses accounted for by factor 1. The 10 mg/kg dose of PHENO, like the 2.5 mg/kg dose of MJ, has a mild “CPZ-like” effect, seen by the loading on factor 2. The high METH dose apparently causes a new wave shape to appear in this structure, well-described by factor 4. This wave shape component is not elicited by any other drug. The lower METH dose perturbs the visual cortex less from its normal state. This description begins to group different doses of the same drug together, describing them in terms which utilize the same factor to account for significant amounts of their energy. At the same time, different drugs tend to be discriminated, with the major contributions of energy coming from different factors. Yet, these distinctions are not as sharp as one might like them to be. Many drug-induced wave shapes derive most or major portions of their energy from factor 1, when the relative orientation of signal vectors and spatial axes are determined in accordance with the criteria of principal component analysis. The set of axes was then rotated in accordance with the Varimax criteria, and a new set of regression equations computed to describe the signal vectors in terms of this new orientation. The squared coefficients of these equations are presented on the right side of Table 11. As stated earlier, the apparent effect of this rotation is to cause clusters of like wave shapes to group more closely while unlike wave shapes are separated more clearly. The price paid for this enhanced resolution is some diminution in the accuracy with which the original wave shape can be reconstructed. The last column of the table, %J, refers to the percentage of the Jacobi reconstruction accuracy which was achieved by the Varimax reconstruction. Thus, for example, the Varimax regression equation for the 2.5 mglkg dose of CPZ should be read: 97% [95% CPZ (2.5 mg/kg)l

=

1% F I

+ 77% Fz + 1% F3 + 19% F ,

In most cases, the loss of precision in the regression equations is unimportant. Examining the right half of Table 11, it can be seen that factor 2 contributes most heavily to the CPZ 5 mg/kg and 2.5 mg/kg wave shape. The CPZ 1.0 mg/kg wave shape derives the largest amount of its energy from factor 1 and receives a substantial contribution from

324

E. R. JOHN, P. WALKER, D. CAWOOD,

M.

RUSH, J. GEHRMANN

factor 4. All three MJ wave shapes now receive their largest contribution from factor 3. The MJ 2.5 mg/kg wave shape contains 30%of its energy in a form which resembles the predominant response to CPZ, described by factor 2. The PHENO 20 mg/kg dose causes a wave shape which receives its largest loading from factor 1, but contains a substantial amount of energy in a component resembling the MJ effect, described by the 37% weighting of factor 3. The PHENO 10 mg/kg and 5 mglkg doses caused a new wave shape to appear, described by factor 4. The PHENO 10 mg/kg and 5 mg/kg wave shapes are unlike in that the 10 mg/kg wave shape derives 30%of its energy from factor 3, while the 5 mg/kg wave shape derives most of its energy from factor 1. Note the suggestion of a U-shaped curve in the dose-response factor analysis of PHENO action on this structure. The higher METH dose causes a wave shape unlike that caused by any other condition, closely approximated by factor 5, while the lower METH dose causes a wave shape heavily loaded on factor 4. The SALINE wave shapes, and most of the pre-drug controls, derive most of their energy from factor 1. Note that five control cases, however, derive substantial amounts of energy from factor 4. This suggests that the effects of the low CPZ and PHENO doses on this structure can be approximated by normal physiological fluctuations of state, such as drowsiness. Comparing the Varimax and the Jacobi descriptions of the same body of data, the Varimax description seems to group different doses of the same drug more consistently and to discriminate between different drugs somewhat more sharply. The variability of the control wave shape description has somewhat increased, however, with an average of approximately 708 coming from factor 1. These results are in accordance with our expectations from the Varimax method. Although both Varimax and Jacobi descriptions will be provided for the remainder of these data, in general only the Varimax results will be discussed.

H. COMBINED COLUMN ANALYSIS Before proceeding further, a convention must be defined for the identification of Varimax factors. The factor which contributes the heaviest loading to control wave shapes will be called factor 1; the factor other than factor 1 which contributes the heaviest loading to CPZ wave shapes will be called factor 2; the factor other than factors 1 and 2 which contributes the heaviest loading to MJ wave shapes will be called factor 3; the factor other than factors 1 3 which contributes the heaviest loading to PHENO wave shapes will be called factor 4; the factor other than factors 1 4 which contributes the heaviest loading to METH wave shapes will be called factor 5; other factors will be pooled

BRAIN STATES APPLIED TO DRUG CLASSIFICATION

‘‘>

325

as factor 6.” This convention is necessary because the Varimax rotation does not provide the same unambiguous criterion of relative contribution to communality which determines the order of the Jacobi factors. Adoption of this convention permits us to assign like numbers to corresponding Varimax factors obtained in different column analyses. Identification of factors according to this functional definition permits combination of the 24 column analyses into a single table which fully describes all the data obtained from each animal in the whole series of experiments. Such summary tables will constitute the remainder of the data to be presented in this paper. Such combination can bc justified as follows: Factors A1 and B1 from column analysis 1 (structure 1 ) may or may not be identical with factors A2 and B2 from column analysis 2 (structure 2). Suppose drug A loads on factor A1 in analysis 1 and A2 in analysis 2, while drug B loads heaviest on B1 in analysis 1 and on B2 in analysis 2. If the data from both structures were combined into column analysis 3, drug A would load heaviest on a single factor A3 and drug B would load heaviest on a different single factor, B3, provided that A1 and A2 had been identical and B1 and B2 had been identical. If A1 were different from A2, then in the combined column analysis 3 the signal vectors representing the effects of drug A on the two structures would lie in the hyperplane A3A3’. Similarly, if B1 were different from B2, the signal vectors representing the effects of drug B would lie in the hyperplane B3B3’. Column analysis of the combined data set, equivalent to a two-structure row analysis across all conditions, would be necessary to resolve whether A1 = A2 and B1 = B2. However, since the hyperplanes A3A3’ and B3B3‘ are orthogonal, no vector lying in one has components in the other. Thus, vectors which are independent in separate single column analyses will be independent in a combined analysis subsuming the whole body of data. Therefore, the results obtained by combining different column analyses according to the definitions above are dimensionally and conceptually correct. Table I11 shows the result of combining certain aspects of all 24 Varimax analyses for cat I. This table shows only the most salient features of the data, since it presents the percentage of the loadings of each factor, on the set of 24 wave shapes elicited in all structures under each drug condition, which represented the largest contribution to that set. Secondary contributions are ignored in this summary. Each of the 24 experimental conditions (13 drug, 11 control) constitutes a separate column in this table, while the separate factors are shown in successive rows. Thus, in 90% of the cases, the heaviest contribution to the set of CPZ 2.5 mgfkg wave shapes came from factor 2, in 5%of the cases

VAIUMAX SUMMARY:

TABLE I11 HIGHESTLOADINGS AMONG Ma4JOR FACTORS (CIT I)

5

2.5

MJ 1

5

2.5

0

"g

Controls

Drug (mg/kg)

CPZ

LI

P E R C E N T A G E DISTRInUTION OF

PHENO

METH

Pre-drug measures

SALINE 1

2

5

7

S

20

10

5

.75

.5

0 0 0 25 10 25 55 70 50 5 0 5 0 0 0 15 20 20

40 10 10 30 0 10

15 15 15 55 0 0

40 -

10 0 0 0 90 0

_40 _ 95- 85- -SO - 75- 90- 85 - -95 - S8_ 85- 95_

NRNRNR NltNRNR

N R N R NR N l t N I t CAR

3

4

6

1

9

10 11 12 13

Factor 1 2 3

4 5 >6

0 0 70 90 - 10 5 5 0 0 0 15 5

45"

45 5 0 0 5

Behavior V1 N R N R CR Vz N R N R CAR a

- - -

Heaviest loading is underlined in each case.

15 15 35 0 5

-

20 10 0 0 30

CAR CAR CAR CAR

0 1 0 5 0 0 0 0 0 0 0 1 5 2 0 0 0 0 5 5 0 5 [4 [4

0 0 0 0 5 1

0 0 5 0 0

0 0 5 0 5

CR CAR

0 5 0 8 0 0 0 0 0 0 0 0 0 5 1 0 5

95 75 75 0 1 0 1 0 0 0 5 0 0 0 0 0 0 5 1 5 1 0 b

*

BRAIN STATES APPLIED TO DRUG CLASSIFICATION

327

from factor 3, and in 5%of the cases from factor 6, while no wave shape was recorded from any brain region after that dose of CPZ which received its largest contribution of energy from factors 1, 4, or 5. The behavioral responses to the approach stimulus V, and the avoidance stimulus V, are shown in the bottom rows of the table. Examining this table, it can be seen that the heaviest contribution to the sets of wave shapes under control or SALINE placebo conditions came from factor 1. The CPZ 5 mg/kg and 2.5 mg/kg sets received the largest contribution from factor 2. The CPZ 1.0 mg/kg set shares features with both the control set and the set elicited by larger CPZ doses, deriving approximately equal contributions from factors 1 and 2. All three MJ doses produce sets of wave shapes substantially different from the control responses, but with some similarity to CPZ. Thus, the major contribution to the MJ set comes from factor 3, but a strong secondary contribution from factor 2 is apparent. The high and low PHENO doses both produce wave shapes which, over the full set of anatomical structures, bear marked resemblance to the control responses. A suggestion of a bimodal or U-shaped dose-response curve is again apparent. All three PHENO doses cause a new component to appear in some structures, described by factor 4, which makes no marked contribution to wave shapes observed under any other conditions, although some control wave shapes receive a slight contribution from this factor. The high dose of METH produces a set of wave shapes markedly different from all the others, receiving its heaviest loadings from factor 5. The lower dose of METH has a somewhat different effect, causing a set with certain similarities to both control and CPZ sets, but with distinctive features described by factor 6. Thus, we see that the overall description of the effects of different doses of the same drug has strikingly common features, while different drugs are described in markedly different terms. Factor 1 closely approximates the control wave shapes, factor 2 describes a “CPZ-like” effect, factor 3 an “MJ-like” effect, factor 4 a “PHENO-like” effect, and factor 5 a “METH-like” effect. Note that these descriptions often hold true even at lower doses when behavioral performance is still intact, as seen in CPZ 1.0 mg/kg and PHENO 5 mg/kg. These results can be depicted geometrically. If we ignore all dimensions of the signal space other than factors 2, 3, and 4, the three CPZ, MJ, and PHENO doses can be represented in a 2-34 space. The corresponding vectors are shown in Fig. 13. Note that the different doses of the three drugs cluster in different regions of the hyperspace. From Table 111, it became apparent that the factorial description of different doses of the same drug had essentially similar features. There-

328

E. R. JOHN, P. WALKER, D. CAWOOD, M. RUSH, J . GEHRMANN

4 100%

lOrng/kg

IOOX

3

100~1.

2 FIG. 13. Combined results of 24 column analyses represented as a vector for each different drug and dose, using only the factor 2-3-4 space. The component of the vector along each factor axis is proportional to the amount of energy contributed by that factor to the whole set of wave shapes initially recorded from different placements.

fore, the data from different doses of the same drug were combined. Table IV breaks the combined results down into subsets on the basis of anatomical classification of the monitored regions. In the top half of Table IV, results are stated in terms like those of Table 111, i.e., percentage of all coefficients in the regression equations describing a condition for which the highest loadings were on the indicated factor. The bottom half of the table presents the same data in less quantized form, indicating the actual average loading across structures for the contribution of each factor to the wave shapes observed under different drug conditions. Both halves of the table are separated into four sections: All structures; Group I-sensory-specific regions: visual cortex and lateral geniculate; Group 11-nonsensory-specific regions: mesencephalic reticular formation and nucleus centralis lateralis; Group 111-limbic structures. The section of Table IV which describes the factor analysis of all structures grouped together necessarily closely resembles Table 111. Essentially, each different drug corresponds to a different factor. The section dealing with Group I structures shows that CPZ and M J radically alter sensory-specific responses. PHENO and METH leave sensoryspecific structures substantially responsive in modes resembling normal states, although the deviations from normal response are quite marked. Group I1 structures respond somewhat differently to these drugs. CPZ,

TABLE I V VARlMAX SQIJAR13:D COEFFICIICNTS (C.\T

All structrires

Group I*

Factor 71

1

2

3

5

>

6

Percentage highest loadings 5 1 j 0 9 Control 260 CPZ 60 1 7 6 1 7 0 0 1 3 MJ 60 2 2 9 % 0 0 13

80

PHENO IIETH

60 40

.Iverage Ioadiiip Control 2GO CPZ 60 A1J EO P H E N O 60

METH

40

%

10

8

29

19

14

3

2

62

07 02

11 06 14

-

00 00 23 4 2 01 00 09 06 3Z 00 09 01 02

g

104

1

90

2

1

3

4

2

.

1

2 4 1 7 2 4 0 24 0 3 8 E 0

Factor 5

>6

71

0

4

78

0 0

0

0

1

2

3

4

Factor 6

>6

77 8 0 1 0 14 l 8 1 1 ~ 1 1 0 0 6 18 0 3 3 5 0 0 0 17 18 39 11 0 0 6 12 0 25 8 0 17

5

71 j6

8 0

13

8

0

0

0

6

38

0

104

73

03

02

03 00

09 01 01 IS 07 j 9 07 00 18 06 24 3y 00 18 32 I 1 00

00

11

08

03 00 00 00

78 59

24 13 69 03 03 00 24 04 34 4j 01 00 24 5_7 06 13 08 00

2

00 00 00

18

16

4_0 00 00 07 37

05

12

00

2

9 17

03

n

Group 111’

116

24 I6

0

03 02

~

Group

Factor

4

I)

08 09 10

~~~~~

-

-

~~

00

IS

02

TL

78

1

2

‘4

5

3

4

1

.

5

>6

10

0

10

1 8 2 2 3 _ 9 6 0 0 3 3 18 6 1 7 5 6 0 0 22

-

18 22

11

11

33

0

22

12

0

17

0

0

j0

33

78 18 18 I8

53 -

08 02 04

00

10

04 13 06

14

3_1

05

22

07 18

12 41 09 06

31

00 00 00

22

12

02

09 00

00

2

02 02

8

16

I5 28

~

Sensory-specific structures. Visual cortex (monopolar. bipolar recordings). lateral geniculate (monopolar, hipolar recordings). * Xonsensory-specific structures. Mesencephalic reticular formation (monopolar, bipolar recordings), cen tralis lateralis (bipolar recorrlinrr). Limliic striictnres. Hippocampus (IJipolar recording). dentate (monopolar recording), subthalamiia (monopolar recording). Major factor underlined.

w

M

W

T.BLE JACOBI SQU.4RED Group I

All strurtures Factor *I

1

2

3

4

5

40

6

2

3

1

2

3

4

28

2 4 4 2 4 6 8 4 0

0

18

4j 22 22

2

05 01 00 00

2.5

2

0

8

E

2492

4

0

4

0

0

18

45 -

16

52

6

6

25

6

0

12

8

00 00 00 09 01 00 28 09 03 01 04 03 04 00 04 11 1.5 09

00 00

78 18

01

18

00

18 12

00

104

87

02

35

-

10 03

00

00

24

28

43

24

10 03

01

04

24

32

13

10 02

02

00

24

15

06

16 44

* See footnotes to Table II

n

1s

5

0

6

7

10495

0

>

0 0

8

14

5

2 4 4 2 5 4 4 0 0

4

10 09

4

0

22

2

1

0

-

29 46 -

n

Factor

0

0 6 0

-

METH

>

-

Group 111

Group I1

Factor

Percentage highest loadings 92 5 1 0 Control 260 3 6 4 8 1 2 4 CPZ 60 3 7 g 1 2 5 MJ 60 62 1815 1 PHENO 60 AIETII 40 30 16 7 17 Average loading Control 260 CPZ 60 hlJ 60 PHESO 60

I)

COEFFICIENTS (CAT

00

G

2

4 11

22

34

25

8

6 3 09

02

Factor 5

>

6

n

1 0 6

O

1 0 0

7

0 6

0

8

2

9

3

7

4

3

5

0

0

>

0

6

0

18

39

28

22

11

0

0

18

25

47

6

6

0

17

0

0

18

50

28

11

0

11

0

8 42

8

12

25

17

8

17

17

17

72 2

0.5

01

00 00

00

21

13 06

2s

04

00

01

78

2 32

08 01 00 16 17 02 01 17 18 03 00

00 03

18 18

01

08

16 07

2

06

18 12

23

1

01

41

13

23

2 14 -

00

01

03 00

07

17

10 02

02

00

10

10

13

13

13

BRAIN STATES API'LLED TO DRUG CLASSIFICATION

331

MJ, and METH responses each deviate extremely from the control pattern, while PHENO responses occupy an intermediate position, showing some normal features intermixed with features characteristic of PHENO. Group I11 structures are severely and characteristically affected by each of these drugs. As a rough generalization, the data suggest that CPZ and R/IJ act upon sensory-specific, nonsensory-specific, and limbic structures with equal intensity, PHENO acts upon limbic structures more than nonsensory-specific or sensory-specific, while METH acts upon limbic and nonsensory-specific more than sensory-specific. Similar data, but in terms of Jacobi coefficients, are presented in Table V. These results are presented primarily to show that the results of principal component analysis are relatively unclear and inconclusive, while the results of Varimax analysis are clearcut and consistent. Examination of Table V reveals no pattern which clearly discriminates between different drugs. This negative outcome of principal component analysis was consistently observed in the three animals studied in these experiments. For this reason, results of principal component or Jacobi analyses will be presented but not further discussed in this paper. For completeness, Table VI shows the dimensionality of V, and V, signal space as spanned by Variinax and Jacobi factors. Note that, on the average, Varimax descriptions in various structures are as parsimonious as Jacobi descriptions. In view of the great improvement in clarity, the diminution in precision of description must therefore be deemed insignificant. Similar results were obtained from all three animals. Table VII shows the salient features of the Varimax data for cat 11, obtained by calculating the percentage of all the loadings of a given factor upon a set of wave shapes which represented the largest contribution to that set. Comparable data for cat I were shown in Table 111. Inspection of Table VII shows that factor 1 made the greatest contribution to all wave shapes obtained under SALINE placebo or control conditions. The wave shapes recorded after all three CPZ doses received the largest contribution from factor 2, even at the lowest dose when behavioral performance was not affected. The wave shapes recorded under the three MJ conditions received heaviest loadings from factor 3. The largest PHENO dose receives its heaviest loading from factor 2, with substantial contributions from factors 1 and 4. The two smaller PHENO doses receive heaviest loadings from factor 1, with other contributions shared between factors 2, 3 and 4. The highest dose of METH produces a wave shape markedly different from all others, receiving its heaviest loadiiigs from factor 5. The lowcr METH dose causes a

332

E. R. JOHN, P.

WALKER,

I). CAWOOD, hl. HUSH, J. GEHRhlANN

TABLE V I I)IMI:NSION.~LITY O F 20 TESTCONDITIONS I N EICH OF 10 L)I.:RIVATIONSD U R I N G PRKSENT.\TION O F APPETITIVI.: (VI) . i N D AVl:ltSIVE (v,) S T I M U L I (C.IT I)

977, of Energy accounted for by n factor Structure

Right visual cortex Monopolar

Stimulus

Vl

V? Bipolar

Vl

V? Left lat.era1geriiculate Monopolar Bipolar Mesencephalic reticular format,ion Monopolar Bipolar Left ceiitralis lateralis Bipolar

v1 V? V1 V?

Dentate Monopolar Subthalamus Monopolar

Jacobi squared

5 5 7 5

6 6

5 5

6 5

6

7 6

5

s 6

V I

7

7

V? V1 V?

11

11

7

7

15

14

VI

12 13

10 10

V.?

9 12

9 12

V1 V,

12 12

10 10

VI VP

9 32

9 11

V? Hippocampus Bipolar

Varimax squared

V1

wave shape with certain similarities to the control wave shape, but with distinctive features described by factor 6. The overall results from cat I1 generally correspond with the findings previously described for cat I. Different doses of the same drug tend to have similar fcatures, as indicated by qualitative correspondence between their factorial descriptions, while different drugs arc described in markedly different terms. Factor 1 closcly approximates the control wave shape, factor 2 describes a “CPZ-like” cffcct, factor 3 describes an “MJ-like” effect, and factor 5 a “METH-like” cffect. Although PHENO receives a slightly heavier loading from factor 4 than do the other

v \RIM \X S U M M \RY:

TABLE VII DISTRIBUTION OF HIGHI4;ST LOADINGS ZMONG M \JOR F.ICTOM

(cIT

PERClCNT IGI;

IT)

mm k

Conditions

Z

Controls (mg/kg)

Drug (mg/kg)

CPZ

MJ

5

2.5

1

5

2.5

1

20

10

Factor 1 2 3

17 54 13

23 42 It)

15 48 15

13

10

7

3

0 4

0

0

6

15

19 28 50 0 0 3

27 13 50 7 0 3

25 43 11

4

17 2s 41 lo 0 3

40" 13 23 13 0 10

>G

METH

PHENO

1s 0 4

5

1

jl! -

0

23 10 10 0 6

0

0.5

32 8 12 8

1 0 100 0

Is 32 -

CAR CAR

CAR CAI1

SALINE 1

V,

NR NIX CII Nlt NR NIt N R N R NI! NI? N l t CAll NR NI? N R N R NR C.411 Heaviest loading is nrrderlined in each case.

3

4

5

6

- 45- 62 _ -74 -64- 52- -59 18

4

9

5

8

9 0 23

4 8 15

9 0 0 9

Behavior \'I

2

Pre-drug mesures

[4 [4

7 11

3 14 11 14 7 7 1 0 0 0 7 19 14

7

R

9

67 67 10 12 4 10 4 17 0 3 0 8 0 0 0 27 13 13 50

cI? CAll

10 11 12 13

58 71 - 47 - 46 -

13 13 4 0 13

4 10 17 4 20 S 8 6 8 0 3 0 13 13 21

b b

334

E.

R.

JOHN,

P.

WALKER,

D.

CAWOOD,

M. RUSH, J. GEHRMANN

drugs, this factor does not make the largest contribution to PHENO wave shapes. In this case, the factor analysis did not succeed in aligning PHENO wave shapes close to a unique factor, or “basis vector,” but described them in terms utilizing contributions from several different vectors. As before, the similarity across different doses of the same drug seemed to justify combination of these data. Table VIII breaks the combined results of different doses of each drug down into subsets on the basis of anatomical classification of the monitored regions. The section of Table VIII which describes the factor analysis of all structures grouped together closely resembles Table VII. Both “Percentage highest loadings” and “Average loading” representations of these data show that the largest contributions to control wave shapes come from factor 1, to CPZ wave shapes from factor 2, to MJ wave shapes from factor 3, and to METH wave shapes from factor 5. PHENO receives significant contributions from factors 2 and 4, in addition to 1. The section dealing with group I structures shows that CPZ, MJ, and METH alter sensory-specific activity somewhat more than does PHENO. Group I1 structures were relatively less affected in general, with CPZ, MJ, and METH causing the most marked effect. Group 111 structures were also markedly affected by CPZ, MJ, and METH, while PHENO had little effect. This profile of relative drug effects differs somewhat from those seen in cat I. As a rough generalization, in this animal the data suggest that CPZ, MJ, and METH act upon sensory-specific and limbic structures with equal intensity, and somewhat less upon nonsensory-specific midline structures. PHENO acts at all levels in this animal, but most markedly on the sensory-specific structures. Examination of Table IX, which shows the same data as Table VIII represented in Jacobi dimensions, indicates that the principal component representation fails to organize the data with the same functional clarity as the Varimax rotation. The differental effects of these drugs are all but completely obscured by the Jacobi description. Confirming the results shown in Table VI for cat I, Table X shows that there was little or no loss of parsimony in the regression equations describing these wave shapes when the descriptions were couched in terms of Varimax rather than Jacobi factors. Table XI shows the Varimax summary for cat 111. In 12 out of 13 cases, the SALINE and control sets of wave shapes received the largest loadings from factor 1. All three CPZ wave shapes received the heaviest contributions from factor 2. Although all three MJ wave shapes received the largest loading from factor 1, in each case the next largest

TABLE VIII VARIMAX SQUARED COEFFICENTS(CAT 11) .‘dl structures

Group I.

Group IIb

Group 111.

Factor

Factor

2 Factor 11

1

2

3

Percentage highest loadings Control 260 5 CPZ

3I.J I’HENO LIETH

4

3

Factor 3

6

>

0

6

20

1

1

1

104

0

60 60

13 17

21.5

8

0

8

j2

6

0

3

24 24

60

42 -

29

7

13

0

4

24

13

2

6

4

2

38 -

40

19

16

6

.\%rerageloading Control 260

23

2

3

4

3

1

5

0

>

6

3

n

1

.%

2

3

2

4

60

4

17

1J

0

0

13

ti

c,3

13

02

02

52

2

02

33 0

jo

04 04 04

01

13

104

67

02

08 01

0 0

0

0

01

0

24

S

25

1

2

3

47

4

cl

5

>

6

12 17 13 29

5

24

46 - 21

4

25

0

4

16

13

6

6

%

19

24

6

8

8 13 13

0 0 0

2X 23 8

E

%e Fl 0

8

tl

04

04

00

13

02

24

00 03

21

14 09

19

09 32

08 00 07 00

09 09

P+ 2

31 14 04 16 08 04 05

15 00

05

2

4_7

17

cl

2

16

00

04

24

06

jS

17 03 00

00

12

19

46

12 06

5

03

00

03

24

15

16

j’ 01 00

00

12

17

13

47

22

08

I1

00

03

24

26

27

18

09 00

01

12

39

26

02

01

03 03

2

14

16

12 00

52

08

8

18

M)

0 0 -00

Yisual cortex (monopolar. hipolar). lateral geniculate (monopolar. bipolar). (I hfesenrephalic reticular formation (monopolar, bipolar), centralis lateralis (hipolar). = Hippocampus (bipolar), dentate (monopolar), suhthalamns (monopolar). d Major factor nnderlined.

09

00 00 00

13 06

16

2

3.5

00 09

43 -

12

-

104

24

13

a

104

00

60

09 05

n

29 0

07

60

13

6

3

L1.J PHENO METH

40

>

0

CPZ

60

5

10 0 1 2 2 1 z 1 7 8 0 12 25 17 58 0 0 12 33 0 17 0 8 2 5 0 0 0 10

52

9 E 2 1

13 24

4

vl

24

0.5

v,

2

m 5

TABLE IX JACOBISQUARED COEFFICIENTS (CAT 1I)a

Factor 1

n

2

I’ercentaae highest loadings Control 260 15 00 5-2 3 8 CPZ 60 6 AIJ -Y 2 3

80

PHESio

60

1IETII

40

35 78

Average loading Control 280

3

4

Factor 5

>

6

?

0

1

104

1

17 0 2

00 00

IlETH

40

22 - 07 09

15 11

07

~~~

0

See note to Tahle VIII.

01

n

1

2

3

10

0

0

0

-

8

0

0

0

104 74 21 2 4 4 4 3 9

0

0

0

0

22 1 2 5 0 4 2

4

5

3

0

4

8

>

6

8

0

8

R

0

2 4 5 4 3 9

8

4

12

75

-

23

0

0

0

0

24

25

0

0

0

6

16

19

13

0

8

38

75 -

12

0

2.5

25

0

16

19

6

13

6

19

2

03 00 00 00

00

52

17 06 01 00

00

104

02

01

00

12

38

24

12

5’

62

16 00 00 00

12

53

20

00

00 00 00

24 24

24

00 00

25 11 00 02 15 05 03 03

24

2 5 34 2

14 02

46 30 08 00 00 58 17 10 02 00

00

00

06

00

8

19 09 04 28 14

03

16

16 06 08 05

I1

16

57 -

6

1 2 L 5

104

60

>

0

03

hIJ PHENO

5

68

0 0

52

4

0

16

00 00 00 00

3

0

13

01

2

0

1Y

00

1

0

17

01

6 n

0

Y

11

>

0

R

26 08 02 19 07 02

5

0

0

39 5

0 4

4

17

0

F z

3

24

0

60 60

CPZ

2

2 4 2 2 1

3

22

3

99

1

Factor

2 0 0 0

0 0 0

j

~

Group 111

Factor

1 4 0

0 3 4

4

Group I1

Group I

All structures

ir

1

2 4 E 3 3

24

47 sfi

30

16

13 09

04

00

~

22 06 05 02 25 07 01 01 15

02

01 01 00 00

n

2

8

“U

337

BRAIN STATES APPLIED TO DRUG CLASSIFICATION

TABLE X OF 10 DERIVATIONS DURING DIMENSIONALITY OF 20 TESTCONDITIONS IN EACH PRESENTATION OF APPETITIVE(V,) AND AVERSIVE (VZ) STIMULI (Cat 11) 97Oj, of Energy accounted for by n factor

Structure Right visual cortex Monopolar

Stimulus

V,

VL Bipolar

Bipolar

Bipo1ar Right cingulate Bipolar Right medial forebrain bundle Monopolar Tiight dentate Monopolar

7 4 5 5

6 4 5 4

V*

8 9

10

V1

8

7

VP

8

8

Vl VL

7

s

7

7

VI

6

9

6 9

10 12

10 12

13 15

13 15

V, V, V, Vl

vz Vl

V? Prepyriform Monopolar

7

5 5

V,

112

Left nucleus reticularis Monopolar

8 8

Varimax squared

8 4 4

VZ Left lateral geniculate Monopolar

Jacobi squared

v 1

V?

8

contribution came from factor 3. The two higher PHENO doses received their largest loading from factor 4, while the lowest dose showed equal contributions from factors 1 and 2. The larger dose of METH received equal contributions from factors 1 and 5, while the lower dose looks much like the controls. Thus, in this third animal it was once again demonstrated that the descriptions of different doses of a particular drug tended to have siniilar features, while different drugs were described in markedly different terms. Control data were consistent and different from drug data. The gross similarity between different doses of the same drug, seen

VARIMAX SUMMARY: PERCENTAGE

OF

TABLE XI HIGHESTCOEFFICIENTS

FOR THE

MAJORFACTORS (CAT 111)

Dmg (mg/kg) MJ

CPZ 5

Factor 1 2 3 4 5 >6 a

25 -54 17 0 0 4

2.5

1

29 13 58 _ - 38 0 2 1 8 1 3 0 0 4 1 7

5

2.5

PHENO 1

71" 71 67 0

29 0 0 0

Heaviest loading is underlined.

Controls (mg/kg)

8 4 1 3 2 5 8 0 0 0 0 4

2 0 1 0 5

METH 1

.5

29 25 42 42 79 17 17 42 13 8 1725 4 4 4 3 8 2 9 4 0 0 - 0 0 4 4 -2 0 0 4 4 0 8

-

SALINE 1

2

58 - 67 8 13 13 8 8 0 0 4 8 8

Pre-drug measures 3

4

5

6

7

8

9

1 0 1 1 1 2 1 3

21

8 4 0 8

17 8 8 29 13 8 1 3 8 4 4 0 8 1 3 1 3 0 0 0 8 0 4 1 3 8 8 8 8 1

-

29 88 8 8 38 42 - 4 8 2 1 4 1 7 0 0 4 8 4 0 0 0 4 4 0 7 1 3 8 4 8

71 67 54 38 -58 -63 -63 -54 -46 -

a

"i

BRAIN STATES APPLIED TO DRUG CLASSIFICATION

339

in Table XI, justified combination of the data from each drug and analysis of the drug effects upon different groups of anatomical structures, as shown in Table X I . These findings show that in this animal, control data from all structures and subgroups showed the largest contribution from factor 1. CPZ data showed the heaviest contribution from factor 2, with effects most marked in sensory-specific structures of Group I, quite noticeable in limbic structures of Group 111, and least in nonsensory-specific structures of Group 11. MJ data reveal no effect upon Group I or Group I11 structures, but a pronounced effect on Group I1 structures which show a heavy loading on factor 3. PHENO data shows most effect at the Group I level, less at the limbic or Group I11 level, and little effect at the Group I1 level. METH effects are equally apparent at all levels. Table XI11 shows the same data as Table XII, but in terms of Jacobi components. As before, it is apparent that the Jacobi representation fails to reveal the orderly patterns in the data which are so clearly evident in the Varimax data. Finally, Table XIV shows once again that the Varimax representation loses little parsimony or accuracy when compared with the Jacobi treatment. IV. Discussion

The purpose of this study was to see whether it was possible to extract representative, reliable, and physiologically relevant samples of electrophysiological activity from behavioral experiments and to represent essential features of such data in a quantitative form. Were this goal achieved, different states of the central nervous system could be represented in precise terms which would permit neural processes of particular interest to be identified unequivocally and compared analytically. Although this goal has general utility for a wide variety of problems, it was decided to focus this study on the particular problem of whether different doses of each of several drugs caused unique brain states corresponding to each drug, which could be recognized and classified. The problem of drug identification was selected not only because of its potential practical utility, but also because whether or not a given drug was administered can be established unequivocally, whereas many other factors influencing brain states cannot be manipulated with similar ease. In order to establish that the drug effects under study were not so strikingly different as to render trivial any success which might be accomplished by our analytic procedures, the consequences of administration of graded doses of four drugs and two placebos were studied on two trained and one untrained cat, from the viewpoint of a variety of con-

c3 0

m

TABLE XI1 VARIMAXSQUARED COEFFICIENTS (CAT111)

3

~

~

n

All structures

Group 10

Group Ilb

Factor

Factor

Factor

1

2

3

4

5

Percentage highest loadings 5 16 9 Control 312 CPZ 72 2 12 6 23 MJ 72 67 4 2 4 2 33 25 17 20 PHENO 72

2”

METH

- -

48

G_1.

9

20

3_s

2 2 2 12 45 09 04

Average loading Cont,rol 312

CPZ hl J I’HEKO METH

72 72 72 46

~

>

6

2

9

1 0 2

7 4

2

n

1

2

3

4

5

>

18

3

0

2

76

0

0 0

8

4c,

17

4

0

0

17 0 0 2-5

16 1 8 3 9 18 4j

0

1 2 6 7

24 17 1 6 2

0

00

104

02 02

24 24 24

07 j4 15 01 01 6 2 07 09 00 00 21 3 L 09 15 01

02

16

63

2

4s

4

11 02 02 05 20 02 00 15 02 01

1

13 6

10 04 01

03

~

15

2

16

1

24 9 75 2488 4

104

0 27

2

6

.z

17 05

02

05 00 00

00

2

0

01 05

2

18

00

16

04

18

02

12

4

5

>

7

5

5

0

11

5

0 11 0

OF

0

15 07 03 07 00 01 32 04 32 02 00 32 21 14 11 03 06 05 03 22

03 00 07 00 00

0 17 22

8

39 17 2 3 2

15

Factor

11 22 % 0

33 33

78

3

Group 111.

7

0

6 n

1

2

53

15

30 27 3072 30 37

40

130

43

4

9

5

>

4

1

19

3

0

13 0

0

3

5

13 09

30 24 3_2

0 %

04

01

13 11

30 30

54 31

20

32 16 07 00

04 10

b c

5

10 04 03 ‘4 03 00 13 01 24

Right visual cortex (monopolar. bipolar); left lateral geniculate (monopolar. bipolar). Left mesencephalic reticular formation (monopolar. bipolar); ventralis anterior (bipolar). Prepyriform (monopolar); medial forebrain bundle (monopolar); siibthalamus (monopolar); ventral dentate (bipolar); left dorsal hippocampus (bipolar). d Major factor underlined. 0

6

13 3 7 1 3 7 13 13 33 -

2 0 2 2 0 130

3

01 03 04

TABLE XI11 JACOBISQUARED COEFFICIENTS (CAT 111). Group I

All structures

Factor n

1

2

Percentage highest loadings Control 312 2 16 CPZ 72 57 31 72 2 1 3 MJ PHENO 72 2 2 0 METH 48 1_3 9 Average loadng Control 312 CI'Z 72 MJ 72 PHENO 72 METH 48

3

4

>

6

5

2

0

1

4

4

4

0

1

2

3

104 3 24 5 2 38

4

2

0 4

0

0

2.1'36

4

0

4

0 3

2 4 2 2 3

2

1 9

6 6

13 04

02 00

01

42

21 07 05 02 08 06 01 00

00 00

53

18 05 01

00

54

11 04 03

05

00 04

-

104 24 4.5 24 24 '57 16 61

>

0 0 0

2

0

5

1

0

1688

.

6

6

n

1

0 0

7s 18

67 28

57

2

3

22

4

7 0

Factor 5

3 6

>

0 0

6

n

1 0

130 30

1

2

3

4

67 47

23 27

7 13

5

1 7

>

0 7

6

2 0

0

0

1 8 5-0 2 8 1 7

6

0

0

3 0 %

7

3

0

0 0

1 8 7 8 1 7

0

0

0

307_020

7

0

0 3

0

0 0

0 1 7

0

2 0 5 5 2 0

0

5 1 0

10

06 03 00

14 05

-

1 2 2

8

21

6 0

04 01 01 00

00

78

2

27 06 01 02 04 01 00 00

00 00 00

18 IS

4_fi 20 06 07 02

01 00

38

16

00

18

54

18 05 00

00

12

53 -

14 0 2

05 00 00 05 04 03 03 21

5

Group I11

Factor

0

7

62

See footnote to Table XII.

n

6

___________ 0

Group I1

Factor 5

m

12

04 00

00

00

00 08

04

130

55

0

03 01

01

30 30

3j 17 08 07 03 7_0 05 04 00 00

01 00

30 20

47 47

03 04

01

13 05 05 04

08

15 06

342

E. R. JOHN, P. WALKER, D. CAWOOD, M. RUSH, J. GEHRMANN

TABLE XIV DIMENSIONALITY OF 20 TESTCONDITIONS IN EACH OF 12 DERIVATIONS DURING PRESENTATION OF APPETITIVE (Vl) AND AVERSIVE (V,) STIMULI(Cat 111)

97y0of Energy accounted for by n factor ~

Structure Right visual cortex Monopolar Bipolar

Jacobi squared

Varimax squared

11 7 6 5

10 7 6 4

6

7

6 6

v1

7

7

VZ

6

5

VI

6

5

VZ

4

4

7 5

7 5

10 9

10 9

12 12

12 13

Stimulus

v 1

vz

v 1

v2

Left lateral geniculate Monopolar

VI v2

Bipolar

v 1 v2

Left mesencephalic reticular formation Monopolar Bipolar Ventralis anterior Bipolar Prepyriform Monopolar Medial forebrain bundle Monopolar Subthalamus Monopolar

v1

V, Ventral dentate Bipolar Left dorsal hippocampus Bipolar

VL

vz V1 v 2

ventional indexes. Neither behavioral performance, spontaneous EEG characteristics, power spectrum analysis, nor computation of average evoked responses revealed clear and consistent similarities between different doses of the same drug nor marked differences between the effects of different drugs. On the contrary, such analyses yielded an

BRAIN STATES APPLLED TO DRUG CLASSIFICATION

343

enormous volume of inconsistent and self-contradictory findings with little apparent analytic value. The set of average response wave shapes derived from a given brain region under each drug-dose condition was then subjected to principal component analysis. The results were represented using a Varimax rotation, as well as in the initial principal component form. The 24 socalled column analyses, corresponding to the effects of two different stimuli upon 12 recording derivations under 24 different conditions, were then combined into an overall analysis. These analyses were found to possess extremely similar features for different doses of the same drug, but markedly different features for the effects of different drugs. The reproducibility of these findings is illustrated by Fig. 14. A major source of difficulty in conceptualizing the physiological significance of factor analytic results arises from the impossibility of representing an N-dimensional space graphically. However, it is possible to visualize certain portions of such data by constructing threedimensional subspaces to examine by graphic means. Figure 14 illustrates the cordiguration of all drug vectors in the “factor 2, 3, 5” subspace. This figure was constructed from the “percentage highest coefficients” data in the upper sections of Tables IV, VIII, and XII. The figure shows the average of the analyses of different doses of each drug, represented as vectors constructed by treating the loading on each factor as a component along the corresponding axis. Four separate subspaces have been depicted for each cat, describing the results of analyzing wave shapes from “All structures,” Group I, Group 11, and Group 111. These vectorial representations show clearly (1) that the different drugs used in this study caused distinctive effects which can be readily discerned in various groups of anatomical structures, and ( 2 ) that the different cats in this study provided basically similar results. The primary purpose of column analysis is to provide quantitative information about the variety of states displayed by the electrical activity in a particular location. In order to ascertain the relative effects of a specific drug on different brain regions, “row analysis” is the most appropriate method, as discussed above. However, some indication of reIative effects can be obtained by examining the results of separate column analyses of simultaneously recorded data obtained from different anatomical regions. In Table XV,the effects of different drugs on various anatomical structures in each cat have been scored qualitatively. Examination of this table shows that CPZ effects could be demonstrated clearly on all anatomical groups, but appeared to be most marked in the visual cortex and the lateral geniculate body. The data from all

344

E. R. JOHN, P. WALKER, D. CAWOOD, M. RUSH, J , GEHRMANN

ALL STRUCTURES

GROUP I

GROUP II

GROUP III

FIG.14. Representation of the results of combined column analyses in the factor 2-3-5 space. Each row of graphs comes from a different cat. In every graph, the results of the different doses of each drug have been averaged and represented by a single vector. The component of the vector along each factor axis is proportional to the amount of energy contributed by that factor to the whole set of wave shapes. The headings of each column of graphs refer to groups of anatomical structures defined as in Tables IV, VIII, and XII. These graphs constitute a vectorial representation of those results.

three cats were in agreement on this point. The two trained cats showed MJ effects at all levels, most markedly at the level of the visual cortex and the lateral geniculate. Cat 111, the untrained animal, shows marked effects in the reticular formation and the nucleus ventralis anterior but little effect elsewhere. The difference between cats I and I1 and cat I11 in the effect of this drug may reflect the fact that cat I11 is untrained while cats I and I1 are highly trained to discriminate visual stimuli in a stressful situation, or may merely be fortuitous. In all three cats,

345

BRAIN STATES APPLIED TO DRUG CLASSIFICATION

TABLE XV DRUGEFFECTS AT DIFFERENT LEVELS Drug

CPZ MJ PHENO

METH

Cat

Group I

I I1 I11 I I1 I11 I I1 I11 I I1 I11

++" ++ ++ ++ ++ oc

C+d

C++ C+ C+

++ 0

Group I1

Group I11

+ + + + ++ +

+ + + + + 0 ++

+ +

++ +

+b

C+ C+

C+

C+ C+

C+

++, major difference observed.

* +, marked difference from control. c

0, no different from control. C, shows some control features.

PHENO effects left the visual cortex and lateral geniculate showing some features of control data, while drug effects could be discerned at all levels. The effects of METH were quite erratic. Cats I and I11 showed certain control features in Group I activity after METH, while cat I1 showed the greatest effect on Group I structures. All cats showed METH effects on Group I1 and 111 structures. These data again show substantial consistency from cat to cat, and illustrate that this method can provide information about the relative effects of different manipulations upon different anatomical regions. Since our primary purpose was to evaluate the ability of the method to provide a useful and precise description of differing brain states, only three cats were used in this study. In view of the inter-animal variability of certain of the observed drug effects, the description of drug actions provided by this work can only be regarded as a first approximation. A larger population would have to be studied to resolve such inconsistencies before the description could be accepted as satisfactory. These various findings show that principal component factor analysis, using the Varimax procedure, provides a parsimonious and precise quantitative description of the effects of various drugs upon the brain. This quantitative description is reproducible from dose to dose of the same drug, and has basically similar features from cat to cat. Presumably, this reproducibility arises because: ( 1 ) different doses of a given drug tend to affect the same neuroanatomical-neurochemical substrate in the

346

E. R. JOHN, P. WALKER, D. CAWOOD, M.

RUSH,J .

GEHRMANN

brain; ( 2 ) different drugs often act upon different substrates; ( 3 ) all animals of a species share in common the same anatomical and neurochemical substrate upon which these drugs act; and (4) the Varimax rotation maximizes the differences between the quantitative description of various effects provided by factor analysis, in a way which enhances functionally relevant similarities and differences. These findings suggest that a useful quantitative description of different brain states, whether caused by drug administration or any definable condition of interest, can be provided by factor analysis. Such precise quantitative descriptions could be useful for a variety of purposes: they might provide a quantitative nomenclature for drugs, a quantitative nomenclature for neuropathology, a statement of functional equivalence between increased or decreased brain activity of any specified sort and the effects of behavioral, surgical, or pharmacological maneuvers. Such precise descriptions of brain states should have significant practical and theoretical consequences. In spite of the apparent conceptual complexity of this method, the technique is relatively simple and rapid to implement. Admittedly, a small general-purpose computer is essential, but such devices are becoming increasingly available. Programs for the PDP-12 computer, to implement this analysis, can be obtained from the authors upon request. It is hoped these methods will be found useful in a number of research areas in the neurosciences. REFERENCES Bennett, J. R., Macdonald, J. S., Drance, S. M., and Venoyama, K. (1971). IEEE Trans. Bio-Med. Eng. 18, 23. Donchin, E. (1966). IEEE Trans. Bio-Med. Eng. 13, 131. Elmgren, J., and Lowenbard, P. (1969). “A Factor Analysis of the Human EEC,” Rep. No. 2, Psychol. Lab., University of Goteborg, Sweden. Halas, E. S., and Beardsley, J. V. (1969). Psychol. Rec. 19, 47. Harmon, H. H. ( 1960). “Modern Factor Analysis.” Univ. of Chicago Press, Chicago. John, E. R. (1962). In “Theories of the Mind” ( J . M. Sher, ed.), p. 80-121. Free Press, New York. John, E. R. (1964). Methods Med. Res. 2, 251. John, E. R. (1967). “Mechanisms of Memory.” Academic Press, New York. John, E. R. (1972). Science 177, 850. John, E. R., Ruchkin, D. S., and Villegas, J. (1963). Science 141, 429. John, E. R., Ruchkin, D. S., and Villegas, J. (1964). Ann. N. Y. Acad. Sci. 112, 362. John, E. R., Cheder, P., Barlett, F., and Victor, I. (1968). Science 159, 1489. Kaiser, H. F. (1958). Psychometrika 23, 187. Naitoh, P., Johnson, L. C., Lubin, A., and Wyborney, G. (1971). EEG 31, 294. Ruchkin, D. S., John, E. R., and Villegas, J. (1964). Ann. N. Y. Acad. Sci. 115, 799.

BRAIN STATES APPLIED TO DRUG CLASSIFICATION

347

Suter, C. ( 1969). “Computer Analysis of Evoked Potential Correlates of the Critical Band,” Tech. rep., pp. 69-98. Computer Sci. Center, University of Maryland, College Park, Maryland. Suter, C. (1970). E r p . Neurol. 29, 317. Wu, Y. H., Rayburn, J. W., Allen, L. E., Ferguson, H. C., and Kissel, J. W. (1972). 1. Med. Chem. 15, 477.

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AUTHOR INDEX Numbers in italics refer to the pages on which the complete references are listed. Arden, C . B., 138, 156’ A Armstrong, 1).M., 8, 22 Abbott, B. C., 131, 163 Arvanitaki, A,, 245, 270 Abe, S., 192, 194, 198, 213 Asano, Y., 121., 140., 162 Abe, Y., 129, 155 Amnuma, H., 7, 10, 12, 14, 16, 19, 20, A l m ~Zar, M., 129, 156, 163 22, 24, 25, 77, 77, 81 Abzug, C., 7, 22 Aschoff, J. C., 7, 25 Aceves, J., 126, 155 Adelman, W. J., Jr., 110, 113, 115, 116, Atwater, I., 119, 164 Auclair, hl. C., 128, 161 117, 121, 158 Adey, W. R., 150, 165, 238, 242, 243, Austin, L., 197, 201, 202, 208, 212 Aiitilio-Gainbetti, L., 202, 210 257, 270, 271, 272 Autilio, L. A., 202, 208 Adolphe, M., 96, 140, 157 Adrian, E. D., 37, 77, 230, 233, 235, 270 Awapara, J., 167, 184 Axelrod, J., 207, 212 Adrian, R. H., 121, 124, 125, 155, 156 Axelsson, J., 178, 184 Aho, I., 140, 147, 164 Azcurra, J. M., 192, 213 Ajala, G. F., 41, 57, 77 Akert, K., 19, 24 B Albe-Fessard, D., 19, 21 Alberici de Canal, M., 202, 213 Baliicky, A., 200, 210 Albers, R. W., 183, 184 Bachelard, H. S., 197, 202, 208 Albuquerque, E. X., 112, 113, 163 Bahn, B. A., 204, 212,219, 225 Alfrey, V. G., 196, 208 Raker, P. F., 111, 156,241, 270 Allen, G. I., 48, 77 Baker, R. C., 65, 73, 74, 75, 76, 78, 80 Allen, L. E., 347 Balaz, R., 197, 208 Altnian, J., 190, 198, 199, 208 Baldissera, F., 64, 78 Alvord, E, C., 204, 21 1 Balvin, R., 251, 272 Amakasu, O., 89, 91, 96, 165 Banay-Schwartz, M., 20G, 208 Amassian, V. E., 1, 9, 22 Bando, T., 169, 180, 187 Ambache, N., 129, 156 Andersen, P., 1, 2, 4, 7, 9, 10, 13, 14, Baner, H., 204, 213 15, 17, 18, 22, 170, 184, 229, 244, Bangham, A. D., 224 Banna, N., 171, 184 265, 266, 270 Anderson, N. C., 112, 114, 119, 162, 163 Bannard, R. A. B., 88, 89, 92, 94, 116, 156, 157 Andersson, S . A., 2, 4, 7, 9, 13, 14, 18, Baranska, W., 203, 208 22, 229, 265, 266, 270 Andrews, T. M., 194, 195, 208 Barnes, C. D., 56, 78 Angaur, P., 41, 46, GO, 61, 74, 77, 78 Barnhill, R., 117, 159 Angelotti, R., 105, 160 Barnola, F. V., 121, 166 Anninos, P. A., 249, 250, 272 Barondes, S. H., 196, 202, 205, 208, 209 Appel, S. H., 202, 208 Rarron, D. H., 233, 270 Aprison, M. H., 168, 171, 175, 180, 183, Barstad, J. A. B., 86, 122, 156 185, 187 Bartels, E., 113, 120, 122, 124, 137, 139, Aratani, M., 86, 92, 161 ISO, 158, I59 349

550

AUTHOR INDEX

Bartlett, F., 277, 346 Bartscli, A. F., 95, 96, 97, 98, 99, 100, 101, 106, 108, 161 Basford, R. E., 198, 208 Bass, A., 202, 210 Batini, C., 44, 51, 78 Baumann, F., 138, 156 Beani, L., 129, 156 Beardsley, J. V., 274, 346 Beattie, D. S., 198, 208 Beeler, G. W., Jr., 127, 156 Belik, Ya. V., 194, 202, 212 Bell, C., 128, 130, 156 Benda, P., 204, 208, 219, 224 Bender, M. B., 68, 69, 70, 78, 81 Bendich, A., 203, 208 Benington, F., 116, 165 Bennett, G. S., 204, 208 Bennett, J. R., 274, 275, 346 Bennett, M. V. L., 75, 79 Bennett, T., 128, 156 Benolken, R. M., 90, 137, 139, 156 Benson, A. A., 88, 163 Berger, H., 228, 270 Bergner, A. D., 122, 134, 156 Berlin, L., 1, 22 Bernstein, M. E., 128, 156 Bessou, P., 8, 19, 23 Bevan, J. A., 130, 165 Bianchi, C., 129, 156 Binstock, L., 110, 119, 156 Bishop, G. H., 231, 233,270 Bisti, S., 170, 184 Blackstad, T. W., 244, 270 Blair-West, J. R., 128, 156 Blankenship, J., 265, 270 Blankenship, J. E., 149, 151, 156 Blanstein, M. P., 111, 112, 119, 156, 162 Bloedel, J., 136, 156 Blomstrand, C., 197, 198, 199, 208, 210 Bloom, F. E., 173, 183, 184, 184, 187 Bocci, V., 192, 208 Bogoch, S., 203, 208 Boivie, J., 19, 22 Bolton, B. L., 122, 134, 156 Boman, K. K. A,, 9, 23 Bondareff, W., 192, 208 Bondy, S. C . , 194, 196, 213 Borenfreund, E., 203, 208

Borison, H. L., 96, 144, 149, 150, 152, 156, 164 Borisy, G. G., 204, 208, 213 Borun, T. W., 197, 212 Botting, J. H., 128, 156 Bourassa, C. M., 4, 5, 9, 14, 20, 22, 25 Bourillet, F., 96, 97, 104, 108, 114, 120, 122, 125, 133, 134, 140, 141, 142, 155, 157 Bowden, J. P., 89, 92, 93, 94, 104, 162, 164, 165 Bowman, R. E., 192, 210 Bownds, M. D., 181, 185 Bowsher, D., 29, 32, 33, 67, 81 Brackett, B. G., 203, 208 Bradbury, J., 137, 139,162 Bradley, K., 8, 22, 170, 184 Brandt, P. W., 125, 132, 133, 157, 164 Brattgard, S. O., 198, 208 Bray, J. J., 201, 202, 208, 212 Breckenridge, B. McL., 207, 209 Brimijoin, S., 207, 212 Brizzee, K. R., 196, 209 Brleger, L., 86, 156 Brock, L. G., 233, 236, 270 Brodal, A., 4, 17, 18, 22, 23, 28, 29, 32, 33, 36, 38, 41, 42, 44, 46, 47, 48, 50, 54, 55, 59, 60, 61, 65, 66, 67, 73, 74, 77, 78, 79, 80, 81 Brookhart, J. M., 243, 272 Brooks, C., 146, 149, 150, 161 Brooks, V. B., 7, 25 Brooks, V. R., 169, 184 Brouchon, M., 20, 24 Brown, D. M., 194, 195, 196, 205, 213 Brown, H. F., 126, 127, 156 Brown, M. C., 9, 22 Brown, M. S., 85, 89, 93, 96, 122, 156, 158, 164 Brownell, H. H., 88, 92, 94, 157 Bryan, J., 204, 209 Brzin, M., 200, 201, 210 Buchwald, H. D., 84, 85, 86, 89, 90, 96, 99, 155,156,162 Bulbring, E., 129, 131, 157 Bull, R. J., 122, 151, 157 Burdett, A. H., 197, 209 Burdman, J. A., 196, 197, 199, 209 Burn, J. H., 207, 209

351

AUTHOR INDEX

Butcher, R. W., 207, 209 Byzov, A. L., 138, 157

C Cain, J. C., 194, 195, 212 Calissano, P., 222, 223, 224 Calvet, J., 235, 243, 247, 268, 270 Calvet, M. C., 235, 243, 247, 268, 270 Calvin, W. H., 232, 270 Camejo, G., 121, 157, 166 Camougis, G., 91, 115, 116, 157 Campagnoni, A. T., 194, 195, 209 Campbell, J. E., 94, 103, 104, 105, 106, 108, 160, 162 Campbell, M. K., 197, 209 Campbell, P. N., 197, 210 Cangiano, A., 35, 55, 65, 71, 78 Carli, G., 17, 22, 53, 56, 78 Carlson, K., 204, 212 Carmeliet, E., 126, 157 Camay, L., 205, 213 Carpenter, D. O., 134, 161 Carpenter, M. B., 29, 33, 38, 39, 59, 67, 80

Carreras, M., 15, 16, 17, 23 Carter, R. H., 114, 135, 157 Carter, S., 20, 22 Casselman, A. A., 88, 92, 94, 116, 156, 157 Cha, Y. N., 130, 147, 160 Chambers, W. W., 15, 16, 17, 23 Chan, S . L., 122, 145, 151, 157 Chandler, P. T., 88, 163 Chandler, W. K., 118, 124, 156, 157, 237, 271 Chang, H. T., 231, 233,270 Changeux, J. P., 205, 209 Chaubal, K. A,, 191, 211 Cheng, C.P. K., 125,157 Cheng, K. K., 125, 130, 142, 143, 145, 146, 155, 157 Chester, P., 277, 346 Cheung, W. Y., 207, 209 Cheymol, J., 96, 97, 104, 107, 108, 114, 120, 122, 123, 125, 133, 140, 141, 142, 155, 157 Chiarandini, D. J., 125, 157 Chiba, S., 126, 128, 145, 159 Chuah, C. C., 207,209

Chung, S. H., 113, 137, 139,156 Cicero, T. J., 204, 209, 212, 219, 220, 221, 224, 225 Clapham, W. F., 121,157 Clare, M. H., 231, 233, 270 Clark, F. J., 150, 163 Clark, R. B., 155, 157 Clark, W. G., 96, 144, 152, 156 Clouet, D., 200, 209 Cobtantin, L. L., 124, 125, 155, 157 Cocks, W. A., 197,208 Cohen, B., 30, 56, 68, 69, 70, 78, 81 Cole, R. D., 197, 212 Colomo, F., 151, 157 Colquhoun, D., 115, 117, 157 Conti, F., 113, 157 Cook, W. A., Jr., 35, 55, 65, 71, 78 Cooke, J. D., 4, 11, 13, 14, 22 Coombs, J. S., 233, 236, 270 Cooper, J. R., 121, I60 Coppin, C. M. L., 8, 19, 22 Coraboeuf, E., 126, 127, 131, 159, 164 Corriol, J., 122, 160 Costin, A., 118, 150, 151, 159, 165, 265, 271 Cotman, C. W., 202, 209 Covian, M. R., 1, 24 Cowan, W. M., 204, 212, 220, 221, 224 Crawford, J. M., 184 Crawford, M. L. J., 148, 150, 157 Creed, K. E., 128, 157 Crema, A., 129, 156 Cremer, J. E., 193, 199, 209 Creutzfeldt, 0. D., 238, 243, 256, 257, 259, 261, 270, 272 Cuervo, L. A., 110, 113, 115, 116, 117, 121, 158 Cullen, C., 232, 271 Curtis, D. R., 167, 168, 169, 170, 171, 173, 177, 178, 179, 180, 184, 185 D DaCosta, F., 104, 166 DAguanno, W., 134, 158 Dahlstrom, A,, 205, 209 Daly, J. W., 113, 125, 156 Dannies, P. S., 222, 223, 224 Darian-Smith, I., 16, 22 Datta, R. K., 197, 203, 209, 211

352

AUTHOR INDEX

Davidian, N. McC., 198, 209 Davidoff, R. A., 168, 171, 175, 180, 183, 185, 187 Davidson, S. Ya., 201, 213 Davis, L., 47, 80 Deanin, G. G., 197, 202, 210 de Groat, W. C., 171, 177, 178, 180, 184, 185 Deguchi, T., 86, 91, 96, 108, 109, 112, 113, 123, 143, 147, 158, 163 del Castillo, J., 172, 177, 185 Dennis, M. J., 136, 158, 172, 177, 178, 185, 186 De Robertis, E., 202, 205, 207, 209, 210, 213 Desmedt, J. E., 169, 185 Dettbarn, W. D., 85, 108, 113, 120, 122, 124, 137, 158, 200, 201, 210 Deysson, G., 96, 140, 157 Diamond, J., 139, 158 DiBella, F., 207, 210 Dichter, M., 244, 271 Diete-Spiff, K., 17, 22, 53, 56, 78 D’Monte, B., 197, 209 Donchin, E., 274, 275, 346 Down, R. J., 97, 105, 162 Drakontides, A. B., 129, 158 Drance, S. M., 274, 275, 346 Dravid, A. R., 196, 209 Dreifuss, J. J., 135, 158, 168, 170, 175, 185 Droz, B., 190, 198, 199, 200, 209 Drysdale, J. W., 195, 210 Dudar, J. D., 150, 158 Dudel, J., 126, 158 D’Udine, B., 203, 210 Duensing, F., 37, 55, 57, 76, 78 Duggan, A. W., 170, 178, 180, 185 Dull, D. L., 155, 159 Durham, L., 86, 89, 90, 156 Dutcher, J. D., 165 Dutton, G. R., 205, 209

E Easton, D. M., 170, 184 Eccles, J. C., 2, 8, 9, 10, 11, 13, 14, 15, 17, 18, 22, 33, 49, 50, 78, 168, 169, 170, 171, 177, 183, 184, 185, 233, 236, 244, 270

Eccles, R. M., 8, 13, 15, 22, 151, 160 Edelman, G. M., 204, 208 Edstrom, A., 200, 201, 209 Edstriim, J. E., 191, 201, 207, 209, 210 Edwards, C., 139, 158 Egyhazi, E., 196, 209 Eichner, D., 201, 209 Ekholm, R., 194, 209 Eldefrawi, A. T., 205, 209 Eldefrawi, M. E., 205, 209 Elliott, H. A., 85, 165 Elliott, K. A. C., 167, 185 Elmgren, J., 274, 346 Elmquist, D., 133, 158 Elul, R., 232, 234, 235, 236, 238, 239, 240, 242, 243, 244, 245, 247, 249, 250, 253, 256, 257, 258, 259, 261, 265, 268, 269, 270, 271, 272 Endoh, M., 126, 158 Endou, H., 121, 140, 162 Engberg, I., 9, 22, 168, 170, 175, 178, 180, 185, 187 Engstrom, H., 28, 78 Erhardt, K. J., 57, 58, 78 Erlij, D., 126, 155 Emst, W., 138, 156 Errera, M., 196, 211 Erulkar, S. D., 151, 157 Evans, M. H., 86, 88, 94, 95, 96, 97, 103, 104, 107, 108, 111, 125, 133, 134, 135, 142, 143, 147, 148, 150, 155, 157, 158, 166 Evarts, E. V., 21, 22

F Fasman, G., 222, 224 Fatt, P., 170, 185, 233, 271 Feinstein, M. B., 125, 130, 131, 132, 144, 145, 146, 158 Feit, H., 204, 205, 209 Feldnian, D. S., 133, 158 Feldman, M., 70, 78 Felix, D., 170, 171, 173, 178, 179, 180, 185 Felpel, L. P., 47, 48, 50, 55, 62, 64, 67, 76, 78, 80, 81 Feltz, A., 133, 158 Ferguson, F. C . , 205, 213

353

AUTHOR INDEX

Ferguson, H. C., 347 Ferin, M., 41, 78 Ficq, A., 196, 211 Field, J., 122, 140, 158, 166 Figge, U., 28, 79 Fingerman, M., 123, 134, 158 Fischer, H. G., 84, 85, 86, 89, 90, 96, 97, 99, 107, 155, 156, 162, 166 Fischer, J., 200, 210 Fischer, S . , 200, 201, 209 Fiszer, S., 205, 210 Flangas, A. L., 192, 210 Fleischhauer, K., 248, 271 Fleisher, J. H., 96, 122, 129, 134, 158 Flock, A., 39, 78 Florey, E., 179, 185 Fluur, E., 30, 68, 69, 78 Fonnum, F., 181, 182, 185, 187 Ford, D. H., 198, 210 Forester, R. H., 123, 134, 158 Frank, G. B., 148, 150, I58 Frank, K., 248, 271 Frankel, S., 167, 187 Frazier, D. T., 91, 113, 115, 163 Fredrickson, J. M., 28, 48, 61, 78, 79 Freeman, A. R., 110, 111, 114, 132, 134, 136, 158, 163 Freeman, J. A., 232, 271 Freeman, S . E., 124, 158 Frey, P. A., 105, 160 Freygang, W. H., Jr., 248, 271 Friesen, A., 222, 223, 224 Fuerst, R., 167, 184 Fuhrman, F. A., 84, 85, 86, 89, 90, 95, 96, 97, 99, 100, 104, 107, 108, 110, 113, 115, 121, 122, 129, 130, 132, 135, 140, 143, 155, 156, 158, 159, 160,162, 165 Fuhrman, G. J., 155, 158, 159 Fujita, Y., 39, 79, 244, 271 Fukuda, J., 42, 61, 75, 79, 169, 185 Fukuyama, F., 86, 92, 161 Fuller, G., 88, 163 Furness, J. B., 129, 158 Furshpan, E. J., 171, 185 Furst, S . , 200, 211 Furukawa, T., 107, 113, 114, 133, 135, 159, 171, 185 Fumsaki, A., 89, 90,159

G Gaballach, S . , 207, 210 Gacek, R. R., 28, 29, 30, 38, 39, 79 Gage, P. W., 85, 136, 156, 159 Galambos, R., 235, 271 Galindo, A., 170, 177, 179, 180, 185 Gambetti, P. L., 202, 208, 210 Ganoza, M. C., 195, 210 Garel, J. P., 196, 210 Gargouil, Y. M., 126, 127, 131, 159, 164 Gariglio, P., 201, 209 Garnier, D., 126, 127, 131, 159, 164 Garoutte, B., 18, 25 Geduldig, D., 111, 132, 133, 159 Gehring, M., 217, 224 Gelfan, S . , 20, 22 Gentile, J. H., 88, 111, 125, 128, 132, 159, 160, 164 Gerard, R. W., 231, 271 Gernandt, B. E., 37, 55, 56, 62, 64, 79 Gershenfeld, A. M., 177, 187 Gershon, M. D., 128, 129, 157, 158, 159 Ghosh, J. J., 196, 210 Giacobini, E., 191, 210 Giaquinto, S . , 20, 22, 48, 79 Gilman, S., 55, 56, 79 Giorgi, P. P., 192, 198, 199, 210 Girardier, L., 132, 133, 164 Girolamo, A. D., 201, 212 Girolamo, M. D., 201, 212 Giuditta, A., 196, 200, 201, 203, 210, 213 Gleisner, L., 28, 81 Gloor, P., 244, 271 Goldman, L., 110, 119, 156 Golenhofen, K., 135, 159 Gombos, G., 222, 224, 225 Gonatas, N. K., 202, 210 Goncalves, J. M., 125, 157 Gonzalez-Cadavid, N. F., 210 Goodman, D. B. P., 207, 210 Goodman, N. R., 257, 271 Goodwin, G. M., 20, 21, 22, 23 Gordon, G., 2, 10, 17, 23 Gordon, M. W., 197, 202, 210 Goto, K., 30, 69, 78, 81 Goto, T., 86, 89, 92, 155, It59, 161 Grabar, P., 203, 211 Graham, L. T., Jr., 171, 183, 185

354

AUTHOR INDEX

Grampp, W., 3, 4, 5, 9, 23, 124, 132, 137, 139, 156, 159, 201, 207, 209, 210 Granit, R., 21, 23, 238, 271 Grant, G., 4, 19, 22, 23 Gray, J. A. B., 139, 158 Green, J. D., 170, 185, 244, 271 Greengard, P., 207, 211, 212 Greenhalgh, R., 88, 92, 94, 157 Grigorian, R. A,, 41, 78 Grillner, S., 20, 23, 52, 53, 54, 64, 79 Grippo, J., 44, 59, 60, 72, 73, 80 Gruber, C. P., 194, 195, 196, 205, 213 Gruena, R., 111, 133, 159 Grundfest, H., 110, 114, 119, 124, 125, 132, 133, 134, 136, 137, 138, 139, 157, 159, 162, 163, 179, 185 Gul'ko, F. B., 113, 161 Guthrow, C. E., Jr., 207, 210 Gutmann, E., 200, 210 Guttman, R., 117, 159

H Haas, H. G., 86, 111, 112, 113, 119, 120, 133, 162, 163 Haas, H. L., 180, 187 Haber, B., 181, 182, 186 Hafemnnn, D. R., 118, 150, 151, 159, 265, 271 Haffner, J. F. W., 129, 159 Hagiwang, S., 123, 126, 127, 130, 131, 159 Halas, E. S., 274, 346 Hall, V. E., 99, 141, 145, 146, 152, 160 Hall, Z. W., 167, 181, 186 Hallett, M., 205, 213 Halstead, B. W., 84, 85, 86, 97, 108, 122, 133, 141, 144, 147, 154, 159 Hamberger, A,, 192, 194, 197, 198, 199, 208, 210 Hansson, H. A., 192, 210 Harada, R., 86, 89, 90, 156 Harding, R., 128, 156 Harmon, H. H., 274,346 Harris, A, J., 136, 158, 172, 177, 178, 185, 186 Harris, J. P., 124, 159 Harrison, C. R., 1, 24 Harrison, C. S . , 96, 122, 129, 134, 158

Hartman, J., 191, 211 Hartstein, M., 198, 210 Harvey, J. A., 117, 118, 151, 162 Harvey, R. J., 8, 22 Hasegawa, S., 192, 194, 213 Hashimoto, K., 126, 140, 147, 158, 162 Hashimoto, T . , 139, 160 Hashimoto, Y., 85, 86, 87, 94, 95, 103, 104, 126, 128, 145, 155, 159, 161, 163 Ilassler, R., 3, 6, 7, 16, 23 Haufe, F., 76, 78 Hauglie-Hanssen, E., 28, 29, 65, 67, 79 Hayama, T., 152, 159 Heistracher, P., 124, 1% Hellerstein, D., 232, 270, 271 Hensel, H., 9, 23 Herbst, E. J., 121, 160 Herriott, R. M., 203, 210 Herschmann, H. R., 221, 222, 224, 225 Hibuquesque, E. X., 113, 125, 132, 137, 139, 156 Hicks, S. T., 195, 210 Hidaka, T., 131, 165 Highstein, S. M., 42, 61, 75, 76, 79, 81, 169, 170, 178, 179, 180, 183, 185, 186 Higman, H., 108, 113, 120, 122, 124, 137, 139, 158, 159 Hille, B., 90, 109, 110, 111, 113, 114, 115, 117, 118, 119, 120, 121, 155 HilLnan, D., 232, 271 Hillman, H., 192, 210 Hirata, Y., 89, 155, 159 Hiyoshi, K., 148, 161 Hobbard, J. I., 151, 160 Hobbiger, F., 129, 160 Hoddart, H., 125, 160 Hodgkin, A. L., 113, 118, 124, 156, 160, 237, 241, 245, 251, 271 Hokfelt, J., 201, 209 Hoffer, B. J., 172, 184, 184, 185 H@ivik,B., 41, 78 Holland, W. C., 120, 125, 126, 128, 166 Holman, M. E., 9, 24, 130, 160 Holmqvist, B., 4, 11, 14, 23 Hongo, T., 17, 23, 52, 53, 54, 64, 79 Hopkins, E. W., 121, 160

355

AUTHOR INDEX

Horowicz, P., 237, 271 Horsburgh, D. G., 99, 141, 145, 146, 152, 160 Hosli, L., 168, 170, 171, 178, 179, 180, 185, 187 Hosoya, Y., 107, 113, 114, 133, 135, 159 Howard, W. L., 89, 92, 165 Hsii, C. S., 196, 213 Huang, T. F., 128, 143, 160, 166 Huffman, R. D., 169, 185 Huglit?, J., 130, 160 Humphrey, D. R., 251, 271 Hunt, C. C., 124, 159 Hnxley, A. F., 113, 160, 245, 271 HydCn, H., 191, 192, 194, 196, 198, 203, 204, 208, 209, 210, 211, 219, 221, 222, 223, 224

I Iida, Y., 125, 164 Ikuma, S., 89, 160, 165 Ildefonse, M., 124, 164 Inamnra, H., 196, 197, 210 Inman, D. R., 139, 158 Inoko, Y., 108, 148, 165 Inove, A., 87, 94, 103, 159, 161 Inove, S., 86, 92, 161 Iosif, G.. 170, 184 Iranyi, M., 56, 79 Ishihara, F., 108, 160 Ito, M., 32, 33, 34, 35, 42, 43, 44, 46, 47, 48, 49, 50, 54, 59, 61, 64, 75, 76, 78, 79, 81, 168, 169, 170, 171, 174, 181, 185, 186 Ito, Y., 130, 160 Itokawa, Y., 135, 160 Iversen, L. L., 167, 183, 184, 185 Iwasaki, S., 123, 135, 160, 162

J Jabbur, S. J., 15, 23, 25, 171, 184 Jack, J. J, B., 8, 19, 22 Jackini, E., 88, 94, 104, 105, 160 Jacob, M., 196, 210 Janig, W., 15, 23 Jaffe, M., 94, 106, 108, 160 Jakoubek, B., 190, 200, 202, 205, 206, 210 James, T. N. I., 126, 145, 165

Janiszewski, I., 155, 160 Jankowska, E., 15, 23 Jansen, J., 4, 23, 181, 187 Jansen, J. K. S., 1, 10, 11, 1.3, 23 Jarlstedt, J.. 194, 210 Jarvik, M. E., 196, 208 Jasper, H. H., 167, 185, 232, 248, 250, 271 Jensen, E. T., 103, 104, 105, 162 Joanny, P., 122, 160 John, E. R., 274, 275, 277, 281, 283, 346 Johnson, D. E., 192,198,199,200,210,213 Johnson, H. H., 85, 165 Johnson, H. M., 85, 105, 160, Johnson, L. C., 274, 346 Johnston, G. A. R., 168, 170, 171, 178, 179, 185 Johnston, I. H., 168, 170, 171, 175, 177, 178, 185 Johnston, P. V., 192, 193, 199, 209, 210, 211 Jones, E. G., 7, 18, 23 Jones, G. M., 38, 79 Joseph, J. R., 269, 272 Jost, J. P., 195, 212 Journey, L. J., 196, 197, 199, 209 Jouvet, M., 65, 79 Judes, C., 196, 210 Jnkes, M. G. M., 2, 15, 23 Jund, R., 196, 210 Junge, D., 111, 132, 159 Junge, K., 18, 22

K Kadenbach, B., 198, 211 Kado, R. T., 238, 270 Kahn, N., 143, 166 Kaiser, H. F., 275, 346 Kalan, E. B., 88, 163 Kalning, I., 135, 158 Kanieda, K., 7, 25 Kamiya, H., 85, 87, 155, 155 Kandel, E. R., 232, 272 Kanzaki, J., 137, 160 Kao, C. Y.,85, 86, 87, 89, 90,92, 95, 96, 97, 99, 100, 101, 104, 107, 108, 110, 111, 113, 114, 115, 116, 120, 121, 122, 124, 127, 128, 129, 130, 132, 133, 134, 135, 138, 139, 140, 141, 144, 145, 146, 147, 148, 152, 154,

356

AUTHOR INDEX

156, 158, 160, 163, 166, 166, 265, 271 Kasahara, M., 36, 39, 79 Kasby, C. B., 130, 160 Kato, M., 56, 81 Kato, T., 196, 197, 203, 210, 211 Katsuki, Y., 55, 79, 137, 139, 160 Katz, B., 133, 134, 135, 136, 137, 151, 160, 161, 172, 177, 185, 186, 233, 271 Kawai, N., 33, 34, 35, 42, 46, 47, 48, 49, 54, 59, 79, 181, 185 Kawaniura, H., 170, 186 Kawaniura, hl., 89, 91, 96, 101 Kawamrira, Y., 169, 186 Kay, E. R. M., 203, 211, 212 Kazachasvili, M., 191, 211 Keatinge, W. R., 130, 131, 161 Kellaway, C. H., 108, 123, 129, 134, 143, 146, 148, 161 Kellerth, J. O., 169, 186 Kelly, J. S., 135, 158, 168, 170, 175, 180, 185, 186 Kelsey, E., 137, 161 Kendall, J. I., 139, 160 Kenney, F. T., 207, 213 Kerkut, G. A., 206, 211 Kei-nell, D., 206, 211, 212 Kessler, D., 222, 224 Keynes, R. D., 110, 111, 118, 120, 156, 161, 241, 271 Kharetchko, X., 196, 209 Khodorov, B. I., 113, 161 Kidokoro, Y., 42, 75, 79, 169, 186 Kies, M. W., 204, 211 Killos, P. J., 96, 122, 129, 134, 158 Kimberlin, R. H., 196, 211 King, R. B., 171, 187 Kiralis, E. S., 165 Kishi, Y., 86, 89, 92, 155, 159, 161 Kissel, J. W., 347 Kitai, S. T., 169, 180, 187 Kiyohara, T., 169, 180, 187 Klatzo, I., 203, 212 Klebe, R., 204, 212 Klee, C. B., 198, 211 Kleinhaus, A. L., 143, 160 Kobayashi, N., 184, 186 Kobayashi, T., 96, 97, 120, 133, 142, 157

Koelle, G. B., 200, 211 Koenig, E., 191, 200, 201, 211 Koenig, H. L., 202, 209 Kiirner, L., 8, 14, 23 Kofoid, C. A., 86, 165 Kohl, H. H., 192, 211 Koizumi, K., 146, 149, 150, 161 Koketsu, K., 135, 161, 170, 185 Kokoi, H., 86, 92, 161 Kolodny, G. M.,203, 211 Komatsuzaki, A., 70, 78 Konishi, T., 137, 161 Konoso, S., 87, 94, 103, 104, 159, 161,

163 Kopin, I. J., 134, 161 Koppenhofer, E., 109, 114, 161 Koprowski, H., 203, 208 Koritz, S. B., 198, 208 Kornhuber, H. H., 61, 79 Kosinski, E., 203, 211 Kositzyn, N. S., 256, 271 Kottegoda, S. R., 129, 161 Krauss, K. R., 134, 161 Kmvitz, E. A., 167, 181, 185, 186 Kreutzberg, G. W., 205, 211 Kriebel, hl. E., 75, 79 Kristensson, K., 203, 211, 212 Kmjevic, K., 142, 161, 168, 170, 171,

175, 179, 180, 185, 186 Kruger, L., 1, 23 Kubota, K., 169, 186 Kuffler, S. W., 136, 158, 167, 172, 177, 178, 181, 185, 186, 239, 240, 271, 272 Kuno, M., 34, 79, 149, 156 Kuo, J. F., 207, 212 Kuriaki, K., 148, 161 Kuriyama, H., 125, 126, 128, 129, 130,

160, 161 Kuriyania, K., 171, 181, 182, 186 Kurokawa, M., 196, 197, 210 Kusano, K., 136, 161 Kuypers, H. G. J. M., 2, 23

1 Ladpli, R., 36, 38, 55, 67, 73, 79 Lagier, G., 128, 161 Lagnado, J. R., 204, 211 Lajtha, A., 190, 194, 196, 197, 200, 202, 203, 206, 208, 209, 211

357

AUTHOH INDEX

Lamal.re, Y., 19, 21 Landau, E. M., 134, 161 Landgren, S., 2, 3, 4, 7, 8, 9, 13, 14, 18, 19, 22, 23, 28, 79 Landva, A. J., 167, 184 Lane, C. E., 155, 161 Lange, P. W., 191, 204, 210, 222, 223, 224 Laporte, Y., 8, 13, 14, 19, 23 Larson, B., 4, 11, 13, 14, 22 Larson, M. D., 169, 186 Larsson, S., 191, 210 Lasek, R. J., 200, 201, 211 La Torre, J. L., 205, 209 Lavender, A. R., 140, 147, 164 Leake, C. D., 101, 129, 163 Leblond, C. P., 190, 198, 199, 209 Lechat, P., 128, 161 Lee, K. L., 207, 213 Lehmann, D., 269, 271 Lehninger, A. L., 197, 210 Leonard, C. F., 232, 272 Lerman, L., 119, 120, 166 Lesevre, N., 269, 272 Levine, D. G., 146, 149, 150, 161 Levine, L., 204, 208, 217, 219, 221, 222, 223, 224, 225 Levitt, M., 15, 16, 17, 23 Lewis, K. H., 94, 101, 102, 103, 104, 105, 106, 108, 160, 162, 165 Li, C. L., 232, 248, 250,271 Li, K. M., 125, 130, 144, 145, 146, 155, 157, 161 Liakopolou, A., 203, 211 Liebeskind, J., 19, 21 Lightbody, J., 204, 208, 219, 224 Lindeman, H. H., 28, 79 Ling, L. L., 142, 143, 157 Lippold, O., 229, 271 Lipsius, M. R., 130, 146, 147, 152, 161 Litvak, S., 200, 201, 209 Liu, C. N., 15, 16, 17, 23 Livengood, D. R., 136, 161 Livingston, A., 16, 23 Livingston, R. B., 55, 56, 79 LlinAs, R., 41, 42, 65, 75, 78, 79, 80, 136, 156, 169, 186, 232, 233, 271 Lloyd, D. P. C., 15, 23 Lodin, Z., 191, 192, 211, 213

Locblich, A. R., 88, 146, 161, 163 Loewenstein, W. R., 132, 137, 138, 139, 161 Logan, R., 196, 211 Lol, D. V . , 135, 159 L@mo,T., 244, 270 Long, P., 96, 104, 141, 157 Lorente de N6, R., 5, 24, 29, 54, 61, 66, 67, 70, 76, 79, 80 L@vtrup-Rein, H., 196, 197, 199, 211 Lowenbard, P., 274, 346 Lowry, 0. €I., 190, 211 LZyning, Y., 170, 184, 244, 270 Luhin, A., 274, 346 Lund, S., 15, 23, 50, 52, 53, 54, 64, 79, 80 Lundberg, A., 1, 8, 11, 13, 14, 15, 22, 23, 24 Lundh, H., 124, 164 Lundquist, P. G., 28, 81 Lunt, G. S., 205, 209 Luttgau, H. C., 125, 161 Lux, H. D., 242, 243, 256, 257, 259, 261, 270, 271 Lynch, J. M., 86, 89, 92, 93, 94, 104, 162, 164, 165 Lyons, C., 204, 211

M McAfee, D. A,, 207, 211 McCarthy, L. E., 96, 144, 149, 150, 152, 156, 164 McCloskey, D. I., 20, 21, 22, 23 McCulloch, W . S., 266, 272 McCullough, 166, 166 Macdonald, J. S., 274, 275, 346 McEwen, B., 191, 204, 210, 211, 219, 221, 222, 224 McFarren, E. F., 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 115, 155, 161, 162 McCregor, D., 203, 212, 216, 224 Mcilwain, H., 117, 118, 122, 151, 162 McIlwain, J. T., 238, 272 McIntyre, A. K., 1, 8, 9, 19, 22, 24 McKenzie, J. S., 128, 156 McLaughlin, J. J. A., 97, 105, 107, 162 McLennan. H.. 168, 170, 173, 178, 180, 185, 186

358

AUTHOR INDEX

McMasters, R. E., 59, 67, 80 McMurtry, J. G., 232, 272 MacPherson, C. F. C., 203, 211 Maekawa, K., 9, 24 Magni, F., 15,22, 170,186 Magnus, R., 51, 57,80 Magnusson, H. W., 87, 164 Mahler, H. R., 194, 195, 197, 209 Mallart, A., 133,158 Mallart, M. A,, 2, 4, 24 Malliani, A., 232, 272 Maloney, T. E., 88, 159 Mancia, M., 170, 185 Mandel, P., 196, 210, 222, 224 Manil, J., 111, 156 Mano, N., 36, 39, 42, 46, 48, 49, 64, 73, 74, 78, 79, 80 Manson, J. R., 265, 270 Marchesi, G. F., 170, 186 Marco, L. A., 32, 33, 34, 35, 36, 38, 54, 59, 60, 61, 62, 71, 81 Mark, R. F., 9, 24 Markham, C . H., 32, 33, 39, 60, 61, 67, 71, 74, 80 Marko, H., 269, 272 Marks, N., 194, 197, 202, 203, 209, 211 Marsh, R. C., 248, 271 Maruno, F., 121, 140, 162 Mascitti, T. A., 50, 52, 80 Mae, K., 194, 196, 197, 211, 213 Matlovsky, L., 248, 271 Matschinsky, F. M., 207, 209 Matsumoto, K., 86, 89, 93, 165 Matsumura, M., 123, 133, 135, 162 Matsumura, S., 140, 147, 162 Matthews, B. H. C., 230, 233, 235, 270 Matthews, P. B. C., 9, 20, 21, 22, 23, 24 Maurer, J. E., 93, 162 Mauro, A., 137, 139, 162 Maxwell, D. S., 244, 271 Mayr, R., 202, 213 Medcof, J. C., 87, 163 Meizel, S., 203, 212 Mellstrom, A,, 30, 69, 78 Melnechuk, T., 186 Menon, T., 207, 213 Mertis, I., 194, 195, 212 Merton, P. A., 20, 21, 24 Meunier, J. C., 205, 209

Meves, H., 118, 157 Meyer, K. F., 87, 88, 95, 101, 106, 131, 165 Miani, N., 201, 203,212 Migata, M., 25, 159 Miledi, R., 133, 134, 135, 136, 137, 139, 151, 160, 161, 162, 205, 212 Millecchia, R., 137, 139, 162 Miller, R., 17, 23 Milsuni, J. H., 38, 79 Minard, F. N., 194, 195, 212 Mirsky, A. E., 196, 208 Mitchell, J. F., 183, 185, 186 Mitchelson, F., 129, 160 Miyahara, J. T., 34, 79 Miyamoto, E., 207, 212 Miyamoto, J., 7, 25 Miyata, Y., 181, 182, 183, 186 Mochizuki, Y., 148, 161 Mokrash, L. C., 198, 212 Mold, J. D., 87, 89, 92, 93, 94, 104, 162, 164,165 Molinoff, P. B,, 205, 207, 212 Monaco, P., 169, 185 Monnier, R. P., 165 Moore, B. W., 203, 204, 209, 212, 216, 217, 219, 220, 221, 222, 223, 224, 225 Moore, J. W., 84, 86, 91, 101, 110, 111, 112, 113, 114, 115, 116, 118, 119, 120, 121, 132, 141, 162, 165, 265, 272 Moore, W. J., 197, 209 Morgan, I. G., 197, 202,208, 212 Mori, Y., 91, 114, 115, 116, 129, 132, 163 Morimoto, T., 169, 186 Morin, R. D., 116, 165 Morita, T., 197, 213 Moruzzi, G., 44, 51, 78, 230, 270 Mosher, H. S., 84, 85, 86, 89, 90, 92, 96, 97, 99, 107, 122, 155, 156, 158, 159, 162, 166 Mountcastle, V. B., 1, 19, 24, 232, 272 Moyer, G., 195, 212 MiiIler, J., 191, 211 Mugnaini, E., 29, 32, 46, 50, 80, 81 Muhs-Clement, K., 3, 6, 7, 16, 23 Mulberry, G., 105, 160

359

AUTHOR INDEX

Munoz-Martinez, J., 148, 164 Munro, H. N., 195, 210 Murphy, T., 238, 272 Murray, R. K., 195, 212 Murtha, E. F., 96, 100, 104, 125, 127, 142, 144, 145, 148, 152, 162 Murthy, M. R. V., 194, 195, 196, 212

N Nachmansohn, D., 108, 113, I=, 124, 137, 158, 162 Nagaki, J., 57, 80 Nagasawa, J., 129, 130, 146, 147, 160, 162 Naitoh, P., 274, 346 Naka, K., 131, 159 Nakajima, S., 110, 114, 119, 123, 124, 126, 127, 131, 132, 159, 162 Nakamura, Y., 110, 114, 119, 124, 132, 162, 244, 271 Nakatsubo, F., 86, 92, 161 Narahashi, T., 86, 91, 101, 102, 108, 109, 110, 111, 112, 113, 114, 115, 116, 119, 120, 123, 133, 141, 162, 163, 265, 272 Nauta, W. J. H., 5, 24 Neal, M. J., 183, 185 Neff, N. H., 172, 185 Negishi, K., 235, 268, 272 Ng,*M. H., 150, 163 Nicholls, J. G., 172, 186 Nichols, J. G., 240, 271 Nicholson, C., 232, 271 Nicholson, P. W., 238, 240, 247, 248, 272 Nicolaysen, K., 14, 23 Nicoll, R. A., 170, 178, 180, 186 Niedergerke, R., 127, 163 Nishi, K., 137, 138, 163 Nishi, S., 135, 161 Nishiyania, A., 105, 107, 111, 114, 123, 124, 127, 129, 130, 134, 135, 140, 143, 144, 160, 163 Nitta, I., 89, 90, 159 Nobel, K. W., 238, 272 Noble, S. J., 126, 127, 156 Noguchi, T., 86, 87, 94, 103, 104, 159, 161, 163 Norton, W. T., 192, 212

Nozue, M., 33, 34, 35, 54, 59, 79 Nyberg-Hansen, R., 42, 50, 52, 54, 58, 59, 80

0 Oates, K., 125, 160 Obara, S., 132, 137, 139, 163 Obata, K., 42, 43, 44, 46, 47, 79, 123, 162, 168, 170, 171, 172, 173, 174, 175, 176, 178, 179, 180, 181, 182, 186 O’Brien, G. S., 147, 163 O’Brien, R. D., 205, 209 Ochi, R., 42, 46, 47, 79, 168, 170, 171, 174, 186 Ochs, S., 150, 163, 206, 212 Oesterlin, R., 89, 93, 164, 166 Ogata, K., 195, 197, 213 Ogura, Y., 85, 91, 96, 99, 101, 114, 115, 116, 122, 124, 128, 129, 132, 134, 136, 140, 141, 148, 152, 157, 159, 163 Ohkubo, Y., 108, 109, 123, 163 Ohlsson, W. G., 192, 213 Okada, Y., 17, 23, 32, 34, 35, 43, 44, 79 Okamoto, K., 151, 163 Okazaki, H., 194, 212 Oliver, A. P., 173, 187 Oliver, I. T., 207, 209 Olmsted, J. B., 204, 212 Olney, J. W., 204, 212, 219, 225 Olsson, Y., 203, 212 O’Neil, J. J., 122, 134, 156 Ono, T., 184, 186 Oornura, Y., 184, 186 Ooyama, H., 184, 186 Orkand, R. K., 127, 163, 240, 271, 272 Orlovskii, G. N., 21, 25 Osa, T., 125, 126, 128, 129, 161 Osawa, S., 196, 208 Oscarsson, O., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 21, 22, 23, 24 Oshinia, T., 14, 15, 17, 22, 36, 39, 79, 80 Otsuka, M., 167, 181, 182, 183, 186 Ottoson, D., 113, 137, 139, 156, 163 Oza\va, H., 135, 145, 146, 163

360

AUTHOR INDEX

Ozawa, S., 36, 39, 79 Ozeki, M., 110, 114, 132, 134, 136, 137, 138, 163

P Paillard, J., 20, 24 Painire, M., 125, 130, 131, 132, 144, 145, 146, 158 Paine, C. H., 10, 23 Palade, G . E., 194, 195, 212, 213 Palay, S. L., 194, 201, 202, 212 Palladin, A. V., 194, 202, 212 Pappano, A. J., 125, 126, 128, 163 Pappas, G. D., 75, 79 Parnas, I., 131, 163 Paton, W. D. M., 129, 163 Patton, S., 88, 163 Peache, S., 196, 205, 213 Peachey, L. D., 125, 155 Penniall, R., 198, 209 Pepe, M., 203, 210 Peper, K., 126, 158 Perez, V. J., 203, 204, 212, 217, 219, 221, 224, 225 Peronnet, F., 249, 250, 272 Peters, A., 194, 201, 202, 212 Peterson, B. W., 32, 33, 35, 39, 43, 44, 45, 55, 56, 57, 60, 62, 65, 67, 71, 80 Peterson, J. A., 201, 212 Peterson, R. P., 206, 211, 212 Petras, J. M., 50, 59, 80 Petsche, H., 248, 269, 271, 272 Pettis, P., 202, 208 Pevzner, L. Z., 193, 203, 212 Phillips, C. G., 7, 16, 19, 23, 24, 238, 271 Phillis, J. W., 167, 171, 178, 185 Pichon, Y., 110, 132, 163 Pilot, H. C., 195, 212 Pimpaneau, A., 19, 21 Pinsky, C., 148, 150, 158 Pitts, W., 266, 272 Podleki, T., 205, 209 Poduslo, S. E., 192, 212 Pollen, D. A., 242, 244, 248, 251, 271 Pollock, L. J., 47, 80 Polyshchuk, N. A., 138, 157 Pompeiano, O., 18, 20, 22, 28, 35, 41,

42, 44, 46, 47, 48, 50, 51, 52, 53, 54, 55, 56, 59, 60, 61, 64, 65, 66, 71, 78, 79, 80 Popoff, C., 207,210 Poppele, R. E., 10, 23, 41, 57, 77, 80 Poston, R. N., 86, 91, 116, 163 Potter, D. D., 167, 181, 186, 239, 240, 271 Potter, L. T., 205, 212 Powell, T. P. S., 7, 18, 19, 23, 24 Prakash, A., 87, 88, 163 Precht, W., 32, 33, 34, 35, 36, 37, 38, 41, 42, 44, 59, 60, 61, 65, 69, 71, 72, 73, 74, 75, 76, 78, 79, 80, 232, 271 Price, R., 206, 212 Prinzmetal, M., 101, 129, 163 Provini, L., 170, 186 Provins, K. A., 20,24 Pudleski, T., 113, 120, 122, 124, 137, 158 Pullman, T. M., 140, 147, 164 Purpura, D. P., 9, 24, 232, 233, 272

Q Quastel, D. M. J., 136, 156 Quastel, J. H., 122, 145, 151, 157, 163

R Radhakrishnian, N., 96, 144, 152, 156 Ragland, W., 195, 212 Rall, T. W., 207, 213 Rall, W., 244, 272 Ram6ny Cajal, S., 2, 24 Ranck, J. B., Jr., 238, 272 Rand, M. J., 129, 160 Randic, M., 170, 179, 186 Ranney, B. K., 86, 92, 135, 159, 164 Rapoport, H., 86, 89, 93, 165, 166 Rappoport, D. A,, 196,213 Rasmussen, H., 207, 210 Rayburn, J. W., 347 Rdzok, E. J., 194, 195, 212 Rech, R. A., 149, 151,164 Redfern, P., 124, 164 Redman, C. M., 195, 212 Rees, K. R., 196, 212 Reid, B. R., 197, 212 Reilly, J., 104, 166 Rembish, R. A., 125, 126, 128, 163 Rhniond, A., 269, 272

36 1

AUTHOR INDEX

Renard, L. P., 170, 180, 186 Rendi, R., 196, 212 Reuben, J. P., 125, 132, 133, 136, 157, 164, 179, 185 Reuter, H., 126, 127, 156, 164 Rexed, B., 4, 24 Rhines, R., 198, 210 Rhodes, A., 198, 210 Rhodorov, B. I., 113, 161 Richards, C . D., 151, 164 Richter, A,, 72, 73, 80 Ricketts, E. F., 84, 165 Rickles, W. H., Jr., 179, 185 Riegel, B., 87, 89, 92, 93, 94, 104, 162,

165 Rieger, H., 269, 272 Riel, F. S . , 89, 92, 94, 104, 164 Ritchie, J. M., 101, 115, 117, 118, 120, 157, 161 Robbins, E., 197, 212 Roberts, E., 167, 181, 182, 186, 187 Roberts, S., 194, 195, 196, 205, 213 Roberts, T. D. M., 28, 54, 57, 58, 80 Roberts, T. S., 19, 24 Roch-Arveiller, M., 96, 104, 125, 141, 157 Rodriquez, G., 117, 118, 151, 162 Rodriquez de Lores Amaiz, G., 202, 213 Rogier, O., 124, 126, 127, 131, 159, 164 Rojas, E., 101, 118, 119, 120, 161, 164 Roots, B. I., 192, 193, 199, 209, 210, 211 Rose, S. P. R., 192, 193, 198, 199, 213 RosCn, I., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 Rosenbaum, J. L., 204, 212 Rosenberg, J., 39, 79 Rosenberg, P., 108, 113, 122, 124, 158 Rowland, G. F., 196, 212 Ruchkin, D. S., 274, 275, 283, 346 Ruddle, F., 204, 212 Rudel, R., 126, 158 Rudjord, T., 13, 23 Rudomin, P., 148, 164 Ruf, K. B., 135, 158 Rumley, M. K., 203, 213 Russell, C . J., 90, 137, 139, 156 Russell, F. E., 100, 155, 164 Ryall, R. W., 177, 184

s Sabah, N. H., 48, 77 Sakai, K., 89, 91, 96, 143, 166 Sakata, H., 7 , 16, 22, 25, 77, 77 Salganicoff, L., 207, 209 Salmoiraghi, G. C . , 173, 187 Samec, J., 196, 210 Sandlin, R., 205, 213 Sano, T., 125, 164 Santini, M., 48, 79 Sapeika, N., 129, 164 Sasaki, K., 74, 80 Sasaoka, T., 107, 113, 114, 121, 133, 135, 140, 159, 162 Sasner, J. J., Jr., 88, 111, 125, 128, 132, 164 Satnke, M., 192, 194, 198, 212, 213 Sato, A., 96, 143, 164 Sato, G., 204, 208, 219, 224 Sato, M., 137, 138, 163 Sato, N., 168, 170, 171, 174, 186 Satow, Y., 135, 160 Sawicki, W., 203, 208 Sawyer, P. J., 88, 111, 125, 128, 132, 164 Schaeffer, K. P., 37, 55, 57, 72, 78, 80 Schafer, M. L., 101, 102, 103, 104, 105, 162, 165 Schantz, E. J., 86, 87, 88, 89, 92, 93, 94, 101, 102, 103, 104, 105, 106, 108, 116, 121, 122, 140, 155, 165, 162 Scheid, P., 28, 79 Schell, P. L., 203, 213 Scherrer, J., 235, 243, 247, 268, 270 Schever, P. J., 89, 155, 165 Schild, R. F., 8, 22 Schimert, J. S., 50, 52, 80 Schindler, W. I., 244, 271 Schlag, J., 251, 272 Schmidt, R. F., 2, 13, 14, 15, 17, 22, 23, 171, 177, 183, 185 Schmiegel, J. L., 86, 92, 159, 164 Schmitt, F. O., 186, 205, 213 Schnieden, H., 129, 165 Scliolz, H., 126, 165 Schor, R. H., 52, 56, 64, 81 Schorderet, M., 207, 211 Schuett, W., 86, 93, 165

362

AUTHOR INDEX

Schwartz, S., 168, 170, 171, 185, 186 Schwarz, D., 48, 61, 78, 79 Schwarz, J . R., 115, 166 Scott, D., 202, 210 Scott, W. R., 110, 163, 265, 272 Seale, B., 167, 184 Sears, T. A., 9, 10, 18, 22, 265, 270 Segundo, J. P., 39, 79 Sekiya, K., 203, 21 1 Sellinger, 0. Z., 192, 198, 199, 200, 210, 211, 213 Seiniginovskf, B., 190, 202, 205, 206, 210 Severin, F. V., 21, 25 Shanes, A. M., 245, 272 Shank, R. P., 171, 183,185 Shapiro, A., 206, 211 Shapiro, B. I., 113, 163 Shapot, V. S., 201, 213 Shapovalov, A. I., 50, 80 Shariff, G. A., 190, 194, 213 Shavel, J., 89, 92, 164 Shaw, T. I., 101, 111, 118, 120, 156, 162 Shelanski, M . L., 204, 209 Shende, M. C., 171,187 Shepherd, G. M., 139, 163, 242, 272 Sherrington, C. S., 169, 187 Shibata, S., 148, 150, 157 Shik, M. L., 21, 25 Shimazu, H., 18, 25, 30, 32, 33, 34, 35, 36, 37, 38, 39, 61, 67, 71, 73, 74, 78, 79, 80 Shinozaki, H., 168, 170, 171, 172, 173, 175, 176, 178, 180, 186 Siegman, M. J., 130, 143, 146, 147, 152, 160, 161 Siekevitz, P., 195, 213 Siggins, G. R., 172, 184, 184, 185 Silfvenius, H., 2, 3, 7, 8, 14, 18, 19, 22, 23, 25, 28, 79 Simada, Z., 170, 187 Simpson, L. L., 155, 165 Singer, I., 113, 119, 120, 165, 166 Sinha, A. K., 199, 213 Sisken, B., 181, 182, 186 Sjolund, B., 4, 11, 13, 14, 22, 25 Sjostrand, J., 192, 198, 200, 201, 208, 209, 210 Skubalanka, E., 155, 160 Sladek, N., 195, 212

Slusarek, L., 204, 209 Sluto, S., 169, 186 Small, R. C., 129, 165 Smythies, J. R., 116, 117, 155, 165 Sokoloff, L., 198, 211 Solling, H. D., 195, 212 Solomon, S., 86, 87, 88, 90, 95, 101, 106, 131, 139, 140, 165 Soinmer, H., 87, 89, 92, 94, 101, 104, 129, 163,164, 165 Spencer, W . A., 232, 243, 244, 271, 272 Sperti, L., 244, 271 Spiegelstein, M. Y., 86, 92, 116, 129, 130, 135, 146, 147, 160, 162, 165 Spoor, R. P., 205, 213 Srinivasan, V., 183, 185, 186 Stabile, D. E., 96, 100, 125, 142, 144, 145, 148, 152, 162 Stampfli, R., 126, 127, 164 Stanfield, P. R., 124, 130, 160 Stanger, D. W., 87, 89, 92, 93, 94, 104, 162, 164, 165 Stein, B. M., 29, 33, 38, 39, 80 Steinbeck, J., 84, 165 Steiner, J., 9, 24 Steinhardt, R. A., 111, 156 Stenhouse, D., 151, 160 Stephen, N. R., 103, 104, 166 Stevenin, J., 196, 210 Stohler, R., 86, 165 Stoney, S. D., Jr., 7, 22, 25, 77, 81 Storm-Mathisen, J., 182, 187 Stover, J. H., 123, 134, 158 Strata, P., 41, 78, 170, 184, 186 Straughan, D. W., 170, 179, 186 Stroniberg, M. W., 248, 271 Stunipf, C., 244, 271 Su, C., 130, 165 Sugano, H., 194, 213 Sugawara, K., 135, 145, 146, 163 Sugimori, M., 184, 186 Sulg, I., 3, 4, 5, 6, 10, 12, 14, 15, 24 Suniino, R., 109, 186 Suinner, A. J., 19, 25 Suntzeff, V., 220, 224 Suter, C., 274, 275, 347 Sutheis, M. B., 130, 160 Sutherland, E. W., 207, 209, 213 Suzuki, J.-I., 30, 56, 68, 69, 78, 81 Suzuki, T., 143, 160

363

AUTHOR INDEX

Snzuki, Y., 196, 213 Sveen, O., 18, 22 Sweet, W., 204, 208, 219, 224 Swett, J. E., 4, 5, 9, 14, 20, 22, 2.5 Sze, P. Y., 171, 186 Szentlgothai, J., 49, 50, 56, 64, 67, 68, 69, 70, 78, 81 Szerb, J. C., 150, 158 Szuinski, A., 169, 186

T Taehikawa, R., 89, 91, 96, 165 Tahara, Y., 89, 165 Taira, N., 140, 147, 162 Takagi, M., 195, 213 Takahashi, D., 108, 148, 165 Takahashi, K., 123, 159 Takahashi, S., 89, 155, 159 Takahashi, Y.,194, 196, 213 Takata, M., 110, 113, 121, 132, 165, 169, 186 Takeda, K., 168, 170, 171, 172, 173, 175, 176, 178, 180, 182, 186 Takenaku, T., 110, 165 Takeuchi, A., 167, 172, 177, 180, 187 Takeuchi, N., 167, 172, 177, 180, 187 Takman, B. H., 91, 115, 116, 157 Tamura, C., 89, 91, 96, 165 Tanaka, R., 192, 194, 213 Tanaka, Y., 181, 182, 186 Tanino, H., 86, 92, 161 Tarby, T. J . , 118, 150, 151, 159, 165, 265, 271 Tardy, J., 222, 224, 225 Tarifeno, E., 201, 209 Tnrlov, E., 66, 67, 69, 71, 73, 74, 81 Tasaki, I., 110, 113, 119, 120, 165, 166, 205, 213 Tasse, J. R. P., 91, 115, 116, 157 Tata, J. R., 194, 195, 208 Tatum, E. L., 99, 122, 140, 141, 145, 146, 152, 160, 166 Tauc, L., 177, 187 Taylor, D. A,, 202, 209 Taylor, E. W., 204, 208, 213 Tebecis, A. K., 178, 180, 187 Tenant, A. D., 87, 163 Ten Bruggenate, G., 168, 170, 175, 178, 180, 187

Teravainen, H., 122, 16.5 Terkeurs, W. J., 151, 164 Terzuolo, C. A., 10, 23, 132, 137, 138, 139, 158, 161, 233, 271 Tetreault, L., 96,,97, 120, 133, 142, 1.57 Tewari, S., 197, 209 Thach, Toan., 104, 142, 157 Thaller, A,, 180, 185 Therrien, E. F., 111, 112, 119, 120, 163 Thesleff, S., 124, 159, 165, 177, 178, 184, 186 Thompson, W. D., 7, 25, 77, 81 T h i n , E. N., 113, 161 Tiplady, B., 198, 199, 213 Toida, N., 125, 126, 128, 129, 161 Tokuniasu, K., 30, 69, 78, 81 Tomiie, Y . , 89, 90, 159 Tomita, T., 129, 131, 157 Tomlinson, J. C . , 126, 145, 165 Towe, A. L., 15, 23,25 Toynma, K., 48, 77 Trautwein, W., 126, 158 Trevor, A., 122, 151, 157 Trevor, A. J . , 193, 199, 209 Tsuchiya, T., 75, 76, 79, 81, 170, 185 Tsuda, K., 89, 91, 96, 101, 165 Tsukahara, N., 169, 180, 187 Tsurugi, K., 197, 213 Tiierk, J. D., 2, 23 Turner, A. D., 128, 156 Turner, R. J., 124, 1.58 Turner, R. S., 108, 165 Twarog, B. M., 131, 165 Twitty, V. C., 85, 165 U IJddenberg, N., 8, 24 Udenfriend, S . , 167, 187 Udo, M., 42, 46, 47, 48, 49, 64, 79, 181, 185 Ulricht, W., 115, 166 Uraguchi, K., 96, 143, 164 Urakawa, N., 108, 109, 123, 163 Uyemura, K., 222, 225 Uzman, L. L., 203, 213

V Vallbo, A. B., 21, 25 Van Den Root, S., 203, 213 Van Gelder, N. M., 171, 187

364

AUTHOR INDEX

Watanabe, S., 243, 256, 257, 259, 261, 270 Watanabe, Y., 163 Waterfield, C. J,, 95, 97, 104, 107, 108, 166 Watkins, J. C., 167, 171, 173, 184, 185 Watts, J. C., 104, 166 Waxman, S. G., 75, 79 Webster, H. de, F., 194, 201, 202, 212 Weight, E., 64, 78 Wed, C . S . , 97, 166 Weinreich, D., 134, 166 Weisenberg, R. C., 204, 213 Weiss, A. H., 59, 67, 80 Weiss, P. A., 202, 213 Wellhoner, H. H., 113, 132, 137, 166 Welt, C., 7, 25 Werman, R., 136, 161, 168, 171, 175, 183, 185, 187 Wersall, J., 28, 81 Weston, A. H., 129, 165 Whedon, W. F., 86, 165 Wiberg, C. S., 103, 104, 166 Wickremasinghe, G., 204, 21 1 W Wicks, W. D., 207, 213 Wiesendanger, M., 7 , 19, 24 Waelsch, H., 200, 209, 211 Wikholm, D. M., 87, 164 Wagley, P. F., 122, 134, 156 Wilczok, T., 203, 208 Wagner, A., 44, 57, 58, 59, 60, 78, 80 Williams, C. A,, 195, 210 Wagner, H. H., 115, 166 Willis, J. H., 96, 100, 125, 142, 144, 145, Wakely, J. F., 85, 97, 107, 166 148, 152, 162 Waksman, A,, 222, 225 Walberg, F., 2, 25, 28, 29, 32, 33, 41, Willis, W. D., 11, 13, 15, 22, 171, 177, 183, 185 42, 44, 46, 48, 50, 54, 55, 59, 60, 61, 65, 67, 78, 80, 81, 181, 185, 187 Wilson, J. A. F., 130, 160 Wilson, L., 204, 209 Walker, R. J., 206, 211 Wilson, V. J., 31, 32, 33, 34, 35, 36, 38, Wall, P. D., 17, 25, 183, 187 43, 44, 48, 49, 50, 51, 52, 53, 54, Wall$e, L., 1, 10, 11, 14, 23 55, 56, 59, 60, 61, 62, 63, 64, 71, Walter, D. O., 238, 257, 270, 272 81, 184 Walters, D. R., 165 Winsbury, C., 11, 24 Wang, H. H., 143, 166 Wintersteiner, 0. P., 165 Wang, J. C. C., 125, 142, 143, 157 Witkop, B., 113, 125, 156 Wang, S. C . , 143, 166 Witman, G. B., 204, 212 Warecka, K., 204, 213 Wittkowski, W., 248, 271 Warnick, J. E., 125, 156 Wolsk, D., 2, 3, 8, 14, 18, 22, 23, 28, 7 9 Waschawsky, H., 190, 209 Washizu, Y., 128, 132, 137, 138, 139, Wong, J. L., 89, 93, 166 Woodbury, J. W., 247, 272 151, 161, 166 Woodward, R. B., 89, 155, 166 Wassermann, O., 120, 125, 128, 166 Watanabe, A,, 119, 120, 129, 151, 165, Work, T. S., 197, 209 Wu, Y. H., 278, 347 166, 205, 213

Van Harreveld, A., 238, 272 Van Wagtendonk, W. J., 122, 140, 166 Vane, J. P., 130, 160 Vassort, G., 124, 126, 127, 131, 164 Vayvada, G., 86, 89, 93, 165 Veale, J. L., 9, 24 Venoyama, K., 214,275,346 Vera, C. L., 244, 271 Vereecke, J., 126, 157 Verney, J., 129, 156 Verzeano, M., 235, 268, 272 Vesco, C., 196, 213 Victor, I., 277, 346 Vilenkin, S. Y., 113, 161 Villegas, J., 274, 275, 283, 346 Villegas, R., 121, 157, 166 Vincendon, C . , 222, 224,225 Vizi, E. S., 129, 163 Vogel, W., 109, 114, 161 Vogt, J., 196, 209 Von Baumgarten, R., 170, 173, 185, 187 von Euler, C . , 21, 25 Voorhoeve, P. E., 170, 184

AUTHOR INDEX

Wyborney, G., 274, 346 Wyler, R. S., 89, 92, 93, 94, 104, 164 Wylie, R. M., 31, 33, 34, 35, 36, 38, 47, 48, 50, 54, 59, 60, 61, 62, 71, 81

Y Yamada, T., 121, 140, 162 Yamagami, S., 196, 213 Yaniagishi, S., 110, 125, 164 Yaniaguchi, H., 131, 165 Yamamota, S., 123, 133, 135, 162 Yamauchi, T., 56, 81 Yamazaki, T., 184, 186 Yanaga, T., 126, 166 Yanagihara, T., 192, 198, 199, 210 Yanagisawa, K., 137, 160 Yanagisawa, N., 18, 25

365

Yano, 122 Yasumoto, T., 87, 159 Yoasa, H., 151, 166 Yokoo, A,, 85, 89, 166 Yokota, T., 2, 13, 15, 16, 17, 22 Yoshida, A., 121, 140, 162 Yoshida, M., 42, 43, 44, 51, 52, 53, 54, 55, 56, 62, 63, 64, 79, 81 Yoshida, T., 203, 211 Young, W., 122, 166

Z Zenkin, G. M., 138, 157 Zimmerman, M., 15, 23 Zomzely, C. E., 194, 195, 196, 205, 213 Zucker, R. S., 232, 272 Zuckerman, J. E., 221,225

SUBJECT INDEX A Afferents, of forelimb muscle, to cerebral cortex, 1-25 y-Aminobutyric acid (GABA) in inhibitory synaptic transmission,

spatial patterns of convergence a t different levels of pathway, 10-12 synaptic properties, 9-10 transmission control, 15-18 Chlorpromazine, effects on EEG, 290,

167-187

297-298, 302

by convu~sants,178-181

ComP1lter, in analysis of EEG data, 268-

drug administration and, 171-173 electrical changes in neuronal nienibranes and, 173-178 Axons, protein metabolism in, 200-201

Convulsants, as antagonists of inhibitory transmission, 178-181 Cortical nerve cells EEG and, 241-245 experimental analysis, 255-264

chemistry, 181-183

270

B

D

Behavior, drug effects on, 284-288 Bicuculline, as antagonist of inhibitory transmission, 180-181 Brain wave activity in, see also EEG models of, 230-232

Deiters’ nucleus, vestibular pathways and,

C Cardiac muscle, tetrodotoxin and saxitoxin effects on, 125-128 Cat forelimh muscle afferents to cortex of,

1-25 hindlinib muscle afferents to cortex of,

18-19 Cerebellum, vestibular nuclei and, 40-42 Cerebral cortex forelimb muscle afferents to, in cat,

1-25 comparative aspects, 18-20 convergence of excitation from other afferents and, 12-14 cortical neurons of, 4-6 cortical projection areas and, &8 course and relay nuclei of pathway, 2-4 diagram of, 3 functional aspects of, 20-21 inhibition of, 14-15 receptor identification, 8-9

32 Dendrites, potential fluctuations in, 243-

245 Drugs behavioral effects of, 284-288 brain states in evaluation of, 273-347 methods, 276-284 results, 284-339

E EEG biophysical factors in, 245-252 computer analysis of data on, 268-270 cortical nerve cells and, 241-245 experimental analysis, 255-264 genesis of, 227-272 tentative model of, 263-264 gross activity of, evaluation, 267-268 models of, 230-232 in perception, 26G-267 spikes and waves in, differences in,

366

250-252 in studies on drug effects, 288-300 subcortical control of, 264-267 pacemaker, 264-266 synaptic functional units and, 241-243 unitary sources of, 230-241 cellular potentials, 237-239 intracellular wave activity, 235-237

367

SUBJECT INDEX

microelectrode studies, 232-235 theoretical analysis, 252-254 Extraocular niotoneurons, projections of vestibular nuclei to, 66-76

F Fastigial nncleus, projections of, 42

G (:aminn neurons, in vestibular pathways,

53 Glycine, as transmitter for inhibitory synapses, 170

H Horizontal canal-abducens reflex arcs, 72-74

I Inhibitory synapses, with glycine o r GABA as transmitters, 170 Inhibitory synaptic transmission by y-aminobutyric acid, 167-187 chemistry of, 181-183 Invertebrate muscle, tetrodotoxin and saxitoxin effects on, 131-133

1 Labyrinth, input of, into vestibular nuclei, 2 8 4 0 Limb motoneurons, in vestibular pathways, 52

M Medial vestibulospinal tract ( MVST) inputs to neurons of origin, 59-62 origin and course of, 58-59 spinal action of, 62-66 Membranes, neuronal, electrical changes in, 173-178 Meniory, brain protein metabolism and, 189 Methamphetamine, effects on EEG, 294, 299-300, 302-305 Mitochondria, of brain cells, protein metabolism in, 197-198 MJ 9022, effects on EEG.. 291., 298.. 302 Monkey, group I projection in, 19-20

Muscle, tetrodotoxin and saxitoxin effects on, 108-122 Mytilotoxin, see Tetrodotoxin

N Nerves, tetrodotoxin and saxitoxin effects on, 108-122 Nervous system proteins, 215-225 developmental studies on, 221 distribution and localization of, 219220 immunological assays of, 217 preparation of, 217, 218 species distribution of, 219 Neurons of cerebral cortex, forelimb muscle afferents and, 4-6 protein metabolism of, 189-213 proteins specific to, 203-205 metabolism related to brain function, 205-207 wave activity originating in, 239-240 Nuclei, of brain cells, protein metabolism in, 19G-197

P Perception, EEG role in, 266-267 Perikaryon, of nerve cell protein metabolisiii in, 190-200 Phenobarbital, effects on EEG, 293, 298300, 302 Picrotoxin, as antagonist of inhibitory transmission, 179-180 Plankton poison, see Saxitoxin 14-3-2 Protein, 215-225 developmental studies on, 221 distribution and localization of, 219220 iinmunological assay of, 217 preparation of, 217, 218 species distribution of, 219 turnover studies on Protein( s ) neuro-neuronal and nenro-nonneuronal transfer of, 203 specific to neurons, 203-205 Protein metabolism in axons, 200-201 in brain mitochondria. 197-198 i n brain nuclei, 196-197

368

SUBJECT INDEX

in neuron, 189-213 catabolism, 202-203 functional activity and, 205-207 mechanisms, 193-198 perikaryon, 190-200 Puffer fish, tetrodotoxin from, 84-85

R RNA, in nerve cell perikaryon, 194-196 S S-100 protein, 215-225 chemistry of, 222 developmental studies on, 221 distribution and localization of, 219220 function of, 223-224 immunological assay of, 217 preparation of, 217, 218 species distribution of, 219 turnover studies on, 221-222 Saxitoxin ( STX), 83-166 bioassay of, 101-105 chemical assay of, 106 chemical and physical properties of, 92-94 differentiation from tetrodotoxin, 1 0 6 108 effects on cardiovascular system, 144-147 CNS, 147-152 muscle, 122-133 nerve, 108-122 neuromuscular junctions and synapses, 133-137 neuroniuscular systems, 141-144 receptor organs, 137-139 in uiuo effects of, 141-152 lethal doses of, 104-105 sources of, 86-89 structure of, 93 Skeletal ninscle, tetrodotoxin and saxitoxin effects on, 122-128 Smooth muscle, tetrodotoxin and saxitoxin effects on, 128-131 Spinal cord, vestibular nuclear projections to, 42-66

Strychnine, as antagonist of inhibitory transmission, 178-181

T Tarichatoxin, see Tetrodotoxin Tetrodotoxin (TTX), 83-166 bioassay of, 95-101 chemical and physical properties of, 89-92 differentiation from saxitoxin, 106-108 effects on cardiomuscular system, 144-147 CNS, 147-152 muscle, 122-133 nerve, 108-122 neuromuclcular junctions and synapses, 133-137 neuroinuscular systenls, 141-144 receptor organs, 137-139 itr r;iljo effects of, 141-152 lethal dose of, 96-97 sources of, 84-86 structure of, 91 in studies of EEG, 261-263, 264-265

V Vesti bul a 1- nuclei cells in, labyrinthine influence on, 3040 electrical stimulation, 30-36 natural stininlation, 37-40 cerebellovestibnlar pathways through, 41-42 cerebelluni and, 40-42 labyrinthine input to, 2 8 4 0 physiological pathways through, 27-81 projections to extraocular motoneurons, 66-76 projections to spinal cord, 42-66 lateral vestibulospinal tract, 42-58 medial vestibulospinal tract, 58-66 vcstibular nerve fibers within, 28-30 vestibulocerebellar pathways through, 4041 Vestibulo-extraocular reflexes, pathways of, 69-70

CONTENTS OF PREVIOUS VOLUMES Volume 1

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

Volume 2

Regeneration of the Optic Nerve in Amphibia R . Ad. Gaze Experimentally Induced Changes in the Free Selection of Ethanol Jorge hlardones

The Mechanism of Action of the Heinicholiniuins F . W . Schueler 36!J

370

CONTENTS OF PREVIOUS VOLUMES

The Role of Phosphatidic Acid and Phosphoinositide in Transmembrane Transport Elicited by Acetylcholine and Other Humoral Agents Lowell E . Hokitt and Mabel R . Hokin Brain Neurohormones and Cortical Epinephrine Pressor Respoilses as Affected by Schizophrenic Serum Edward J . Walaszek The Role of Serotonin in Ncurobiology Erininio Costa Drugs and the Conditioned Avoidance Response Albert Herz Metabolic and Neurophysiological Roles of 7-Aminobutyric Acid Eugene Roberts and Eduardo Eidelberg Objective Psychological Tests and the Assessment of Drug Effects H . J . Eysenck AUTHOR INDEX-SUB JECT INDEX

Volume 3

Submicroscopic Morphology and Function of Glial Cells Eduardo De Robertis and H. M . Gersclzenfeld Microelectrode Studies of the Cerebral Cortex Vahe E . Amassian Epilepsy Arthur A. Ward, IT. Functional Organization of Somatic Areas of the Cerebral Cortex Hiroshi Nakahama Body Fluid Indoles in Mental Illness R. Rodnight Some Aspects of Lipid Metabolism in Nervous Tissue G. R . Webster Convulsive Effect of Hydrazides: Relationship to Pyridoxine Hurry L. Williams and James A. Bain The Physiology of the Insect Nervous System D . M . Vozdes AUTHOR INDEX-SUB JECr INDEX

CONTENTS OF PREVIOUS VOLUMES

371

Volume 4

The Nature of Spreading Deprcssioll in Neural Networks Sidney Ochs Organizational Aspects of Some Subcortical Motor Areas Werner P . Koella Biochemical and Ncurophysiolofiicnl Dcvclopmcnt of the Brain in the Neonatal Period Williamina A. Hinrdch Substance P: A Polypeptide of Possible Physiological Significance, Especially within the Nervous System F . Lembeck and G. Zelter Anticholinergic Psychotomimetic Agents L. G. Abood and 1. H . Biel Benzoquinolizine Derivatives : A New Class of Monarnime Decreasing Drugs with Psychotropic Action A. Pletscher, A. Brossi, and K . F . Gey The Effect of Adrenochrome and Adrenolutin on the Behavior of Animals and the Psychology of Man A. Hoffer AUTHOR INDEX-SUB JECT INDEX

Volume 5

The Behavior of Adult Mammalian Brain Cells in Culture Ruth S . Geiger The Electrical Activity of a Primary Sensory Cortex: Analysis of EEG Waves Walter J. Freeman Mechanisms for the Transfer of Information dong the Visual Pathways Koiti Motokawa Ion Fluxes in the Central Nervous System F. J . Brinky, 3r. Interrelationships bctween the Endocrine System and Neuropsychiatry Richard P. Michael and James L. Gibbons Neurological Factors in the Control of the Appetite Andre' Soulairac

372

CONTENTS OF PREVIOUS VOLUMES

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

Volume 6

Protein Metabolism of the Nervous System Abel Lajtha Patterns of Mu~c~ilar Innervation in the Lower Chordates Quentin Bone The Neural Organization of the Visual Pathways in the Cat Thomas H. Meikle, Jr. and James M . Sprague Properties of Afferent Synapses and Sensory Neurons in the Lateral Geniculate Nucleus P. C. Bishop Regeneration in the Vertebrate Central Nervous System Carmine D. Clemente Neurobiology of Phencyclidine ( Sernyl), a Drug with an Unusual Spectrum of Pharmacological Activity Edward F . Domino Free Behavior and Brain Stimulation Jose' M . R. Delgado AUTHOR INDEX-SUB JECT INDEX

Volume 7

Alteration and Pathology of Cerebral Protein Metabolism Abel Lajtha Micro-Iontophoretic Studies on Cortical Neurons K . Krnjezjic' Responses from the Visual Cortex of Unanesthetized Monkeys John R. Hughes Recent Development of the Blood-Brain Barrier Concept Ricardo Edstrom

CONTENTS OF PREVIOUS VOLUMES

373

Monoamine Oxidase Inhibitors Gordon R . Pscheidt The Phenothiazine Tranquilizers : Biochemical and Biophysical Actions Paul S . Guth and Morris A. Spirtes Comments on the Selection and Use of Symptom Rating Scales for Research in Pharmacotherapy 1. B. Wittenborn Multiple Molecular Forms of Brain Hydrolases Joseph Bernsohn and Kevin D. Barron AUTHOR INDEX-SUB JECT INDEX

Volume 8

A Morphologic Concept of the Limbic Lobe Lowell E. White, lr. The Anatornophysiological Basis of Somatosensory Discrimination Dauid Bowsher, d t h the collaboration of Denise Albe-Fessard Drug Action on the Electrical Activity of the Hippocampus Cli. Stumpf Effects of Drugs on Learning and Memory James L. McGaugh and Lewis F . Petrinovich Biogenic Aniines in Mental Illness Giinter G. Brune The Evolution of the Butyrophenones, Haloperidol and Trifluperidol, from Meperidine-Like 4-Phenylpiperidines Paul A. J. Janssen Amplitude Analysis of the Electroencephalogram ( Review of the Information Obtained with the Integrative Method) Leonide Goldstein and Raymond A. Beck AUTHOR INDEX-SUB JECT INDEX

Volume 9

DeveIopinent of “Organotypic” Bioelectric Activities in Central Nervous Tissues during Maturation in Culture Stanley M . Crain Thc Unspecific Intralaminary Modulating System of thc Thalamus P . Krupp and M . Monnier

374

CONTENTS OF PREVIOUS VOLUMES

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

Volume 10

A Critique of Iontophoretic Studies of Central Nervous System Neurons G. C . SaZmoiraghi and C . N . Stefanis Extra-Blood-Brain-Barrier Brain Structures Werner P . Koella and Jerome Sutin Cholinesterases of the Central Nervous System with Special Reference to the Cerebellum Ann Silver Nonprimary Sensory Projections on the Cat Neocortex P. Buser and K . E . Bignall Drugs and Retrograde Amnesia Albert Weissman Neurobiological Action of Some Pyrimidine Analogs Harold Koenig A Comparative Histochemical Mapping of the Distribution of Acetylcholinesterase and Nicotinamidc Adenine Dinucleotide-Diaphorase Activities in the Human Brain T . Ishii and R . L. Friede Behavioral Studies of Aiiimal Vision and Drug Action Hugh Brown

CONTENTS OF PREVIOUS VOLUMES

375

The Biochemistry of Dyskinesias G. Curzon AUTHOR INDEXSUBJECT INDEX

Volume 1 1

Syllaptic Transmission in thc Central Nervous System and Its Relevance for Drug Action Philip B. Bradley Exopeptidases of the Nervous System Neville Marks Biochemical Responses to Narcotic Drugs in the Nervous System and in Other Tissues

Doris H . Clouet Periodic Psychoses in the Light of Biological Rhythm Research F . A. l m n e r Endocrine and Neurochemical Aspects of Pineal Function Be'la Mess The Biochemical Investigations of Schizophrenia in the USSR D. V. Lozovsky Results and Trends of Conditioning Studies in Schizophrenia J. Saarma Carbohydrate Metabolism in Schizophrenia Per S . Lingjaercle The Study of Autoiminune Processes in a Psychiatric Clinic S . F . Semenov Physiological Foundations of Mental Activity N . P . Bechtereva and V. B . Gretchin AUTHOR IKDEX-SUBJECT

INDEX

Cumulative Topical Index for Volumes 1-10 Volume 12

Drugs and Body Ternpert 1' urc Peter Lomax Pathobiology of Acute Trietliyltiii Intoxicat-1011 R . Torack, 3. Gordon, and I . Prokop

376

CONTENTS OF PREVIOUS VOLUMES

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

Volume 13

Of Pattern and Place in Dendrites Madge E. Scheibel and Arnold €3. Scheibel The Fine Structural Localization of Biogenic Monoamines in Nervous Tissue Floyd E. Bloom Brain Lesions and Ainine Metabolism Robert Y . Moore Morphological and Functional Aspects of Central Monoaniine Neurons Kjell Fuxe, Tomas Hokfelt, and Urban Ungerstedt Uptake and Subcellular Localization of Ncurotransmitters in the Brain Solomon H . Snyder, Michael 1. Kuhar, Alan 1. Green, Joseph T. Coyle, and Edward G. Shuskan Chemical Mechanisms of Traiisinitter-Rcceptor Interaction lolzn T . Garland and lack Dwell The Chemical Nature of the Receptor Site-A chemistry of Synaptic Mechanisms I. R. Smythies

Study in the Stereo-

CONTEXTS OF I’IIEVIOUS VOLUMES

377

Molecular Mechanisms in Information Processing Georges Ungar Thc Effect of Increased Functional Activity on thc Protcin Mctabolism of the Nervous System B . Jakoubek and B . Semiginovsktj Protcin Transport in Neurons Raymond 1. Lasek Neurochemical Correlates of Bchavior hl. H . Aprison and 1. N . Hingtgen Some Guidelines from System Science for Studying Neural Information Processing Donald 0. Walter and Martin F . Gardiner AUTHOR INDEX-SUB JECT INDEX

Volume 14

The Pharmacology of Thalamic and Geniculate Neurons J. W. Phillis The Axon Reaction: A Revicw of the Principal Features of Pcrikaiyal Responses to Axoil Injury A . R. Lieberman CO, Fixation in the Nervous Tissue Sxe-Chuh Cheng Reflections on the Role of Receptor Systems for Taste and Smell John G . Sinclair Central Cholinergic Mechanism and Behavior S . N . Pradhan and S . N . Duttu The Chemical Anatomy of Synaptic Mechanisms: Receptors I . R . Smythies AUTHOH INDEX-SUB J E C r INDEX

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