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UCLA F O R U M IN M E D I C A L S C I E N C E S

VICTOR E .

HALL,

MARTHA BASCOPÉ-ESPADA,

Editor Assistant

Editor

EDITORIAL BOARD

Forrest H. Adams Mary A. B. Brazier Carmine D. demente Louise M. Darling Morton I. Grossman

William P. Longmire H. W. Magoun C. D. O'Malley Sidney Roberts Emil L. Smith Reidar F. Sognnaes

UNIVERSITY

OF C A L I F O R N I A ,

LOS

ANGELES

THE INTERNEURON

UCLA FORUM IN MEDICAL SCIENCES NUMBER 11

THE INTERNEURON

Proceedings of a Conference held September, 1967 Sponsored by the Brain Research Institute, University of California, Los Angeles

EDITOR

MARY A. B. BRAZIER

UNIVERSITY

OF C A L I F O R N I A

BERKELEY AND LOS ANGELES 1969

PRESS

EDITORIAL NOTE

The present volume contains the proceedings of the first in a series of conferences on Neural Interaction, organized by Arnold B. Scheibel, Carmine D. Clemente, Mary A. B. Brazier and John D. French of the Brain Research Institute, UCLA School of Medicine. Acknowledgement for the support of this conference is owed to the following: Abbott Laboratories; Hoffmann-LaRoche; Merck Sharp & Dohme; Sandoz Foundation; Schering Corporation; Smith, Kline & French; E. R. Squibb & Sons, Upjohn Company and Wallace Laboratories.

CITATION

FORM

Brazier, M. A. B. (Ed.), The Intemeuron. UCLA Forum Med. Sci. No. 11. Univ. of California Press, Los Angeles, 1969

University of California Press Berkeley and Los Angeles, California University of California Press, Ltd. London, England © 1969 by The Regents of the University of California Library of Congress Catalog Card Number: 69-16504 Printed in the United States of America

PARTICIPANTS IN THE CONFERENCE ABNOLD B . S C H E I B E L , Chairman Brain Research Institute,UCLA School of Medicine Los Angeles, California

M A R Y A. B . B R A Z I E R , Editor Brain Research Institute, University of California Los Angeles, California

W . Ross A D E Y Brain Research Institute, UCLA School of Medicine Los Angeles, California

P E R ANDERSEN

Neurophysiological Institute, University of Oslo Oslo, Norway

THEODOR W . BLACKSTAD

Normal-Anatomisk Institut, Aarhus Universitet Aarhus, Denmark

THEODORE H . BULLOCK

Department of Neurosciences, University of California San Diego La Jolla, California

CARMINE D . CLEMENTE

Department of Anatomy, UCLA School of Medicine Los Angeles, California

JOHN D . FRENCH

Brain Research Institute, UCLA School of Medicine Los Angeles, California

M.

G.

F.

FUORTES

Laboratory of Neurophysiology National Institute of Neurological Diseases and Blindness Bethesda, Maryland

SUSUMU H A G I W A B A

Scripps Institution of Oceanography University of California San Diego La Jolla, California VICTOR E . H A L L

UCLA Forum in Medical Sciences, UCLA School of Medicine Los Angeles, California G . ADRIAN HORRIDGE

Gatty Marine Laboratory, University of St. Andrews Fife, Scotland MASAO ITO

Department of Physiology University of Tokyo Faculty of Medicine Tokyo, Japan E R I C R . KANDEL

Departments of Physiology and Psychiatry New York University School of Medicine New York, New York D O N A L D KENNEDY

Department of Biological Sciences, Stanford University Stanford, California L . M . H . LARRAMENDI

Department of Anatomy University of Illinois College of Medicine Chicago, Illinois CHAN-NAO LIU

Department of Anatomy and Institute of Neurological Sciences University of Pennsylvania School of Medicine Philadelphia, Pennsylvania RODOLFO R .

LLINÀS

Department of Neurobiology, Institute for Biomedical Research American Medical Association—Education and Research Foundation Chicago, Illinois ANDERS LUNDBERG

Department of Physiology, University of Göteborg Göteborg, Sweden DAVID S. MAXWELL

Brain Research Institute, UCLA School of Medicine Los Angeles, California

DONALD M .

MAYNARD

Department of Zoology, University of Michigan Ann Arbor, Michigan DOMINICK P . PUBPURA

Department of Anatomy, Albert Einstein College of Medicine New York, New York MADGE E .

SCHEIBEL

Los Angeles, California J O H N D . SCHLAG

Brain Research Institute, UCLA School of Medicine Los Angeles, California ROBERT F . SCHMIDT

II. Physiologisches Institut, Universität Heidelberg Heidelberg, Germany J O S É P . SEGUNDO

Brain Research Institute, UCLA School of Medicine Los Angeles, California S T E N SKOGLUND

Department of Anatomy, Karolinska Institutet Stockholm, Sweden W M , A L D E N SPENCER

Department of Physiology, New York University School of Medicine New York, New York COSTAS S T E F A N I S

Department of Neurology, Athens National University Athens, Greece L . TAUC

Laboratoire de Neurophysiologie Cellulaire Centre d'Études de Physiologie Nerveuse Centre National de la Recherche Scientifique Paris, France C. A. G . WIERSMA

Biology Division, California Institute of Technology Pasadena, California WILLIAM D . WILLIS, JR.

Department of Anatomy The University of Texas Southwestern Medical School Dallas, Texas

FOREWORD Within the frame of contemporary neurophysiology, a conference devoted to the interneuron seems a natural choice. For with the advent of the intracellular microelectrode and recognition of the hyperpolarizing potential as an invariable concomitant, if not causative agent, of postsynaptic inhibition, the presynaptic progenitor of this effect has invariably been identified as a local circuit cell—an interneuron—in the immediate neural environment. A number of different cell families have been implicated in the development of this effect. Internuncials in the intermediate spinal gray, so-called Renshaw cells in the ventral motoneuron pool, basket cells in hippocampus and cerebellum, and stellate cells in neocortex are among those cited. All of these elements are believed to share certain qualities which distinguish them from other neurons. The most important of these are the presumably local (short-axoned) nature of their axonal trajectory, their characteristically intercalated position between long-axoned projection neurons, and their functional capacity for reversing the sign of excitation from facilitation to inhibition. Selection of elements such as these to serve as chapter and verse for this symposium immediately meets with difficulties. In the first place, their number forms but a fraction of that grand ensemble which rightfully deserves the name of "Interneuron." The definition of Bullock & Horridge (1) suggests panoramic diversity: "Interneuron. An internuncial neuron; one which is neither sensory nor (purely) efifector-innervating, but connects neurons with neurons..." At best, this definition allows us to discard from consideration first order sensory cells, motor neurons, and possibly a few central elements whose peripheral processes run a centrifugal course to control gain levels at the receptor stage. All others, and this must constitute the great majority of elements of the vertebrate nervous system, fall within our category. For over three quarters of a century the short-axoned (Golgi II) internuncials have seemed to constitute a cell type in search of a function. But, despite the undoubted advantages implicit in their identification as the obligatory, intercalated interneurons mediating inhibition, facts have begun to handle the theory unkindly. For one thing, the Renshaw cell, long considered the paradigm of this type, can no longer be accepted as a short-axoned cell. If indeed there is a special category of spinal neurons responsible for the burst-type firing and IPSPs generated concomitantly in local ipsilateral xi

xii

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INTERNEURON

motoneuron populations, structural data demand that they be long-axoned projection cells, similar in all respects to other proprioneurons. The same reservation applies to interneurons more dorsally located and implicated in the generation of primary afferent depolarization. For another, the conceptual restraints imposed by Dale's thesis no longer seem so binding in the light of recent data pointing to mediation of both excitatory and inhibitory functions by the same synaptic substance. With the development of the receptor-mosaic notion of postsynaptic membrane organization, so far shown only in invertebrate material, it is true, the presence of the intercalated sign-reversing cell seems less urgent. In fact, careful study of spinal cord sections with degeneration and Golgi methods now suggests that some loops of known inhibitory function may well be direct rather than interneuronmediated. With the advantages of exclusivity already under attack, how wide must we enlarge our set so that no worthy candidate is omitted? Following the definition of Bullock and Horridge, all elements beyond the level of the first order sensory cell, and prior to the final common pathway motoneuron must be considered. This includes most spinal neurons, the brain stem axial core, rostral sensory projection systems, the great descending motor paths, pyramidal and extrapyramidal, and all cortical tissue including cerebellum and cerebral hemispheres. Clearly we have invoked a situation demanding consideration, not of a set, but virtually the set of all possible sets. In similar fashion, the category of functions commonly attributed to interneurons will no longer suffice. In the course of phylogenetic development, only the protozoa, mesozoa, and porifera seem without identifiable nervous systems. The coelenterate nerve nets are made up exclusively of sensory and motor components, fashioned in random nets, some of which are "throughconducting," and others "non-through-conducting." But, even in this primitive matrix, it has been found that activity in the "non-through" may connect directly or progressively to the "through" depending on frequency and (or) repetition rate of the stimulus. Perhaps in this plastic relationship between the two nets where output relates to, but is not precisely the same as, input, we can recognize an Anlage of interneuron function. The next phyletic step, to platyhelminthes presents at once the prospect of a complex ganglionated chain and, with it, of cells whose axonal course is run entirely within ganglia and connectives. Suddenly, many of the familiar characteristics of vertebrate reticular neurons are present including bifurcating axons running long distances rostral and caudal along the chain, and dendrite-like structures immersed in afferent neuropile. And, in successive invertebrate phyla built to this scheme, a bold functional plan emerges. Interneurons are more than the bridge between input and output. They are repositories of the output pattern for which the afferent signal is but the trigger. The sensory input serves only aflip-flopfunction and the master plan, already coded into the interneuron and its connectivity pattern through the effectors, is ac-

FOREWORD

xiii

tivated in invariant form once the sensory input switches to "yes." Here is a class of interneuron activity directly on the route, not just to sign change or relay function but to the most sophisticated strategy-forging levels of cortical operation in the brain. With progressive enrichment of the set, both in number and in function, it becomes increasingly clear that a symposium on interneurons should properly encompass almost the entire phyletic series, and the vertebrate nervous system from conus to cortex. The range of communications which follows gives substance to this belief. M. E. and A. B. Scheibel REFERENCE 1.

T. H., and HORRIDGE, G. A., Structure and Function in the Nervous Systems of Invertebrates. Freeman, San Francisco, 1965: p. 1602.

BULLOCK,

This slide was prepared personally by Santiago Ramón y Cajal and was presented to the Brain Research Institute by Professor Clemente Estable of Montevideo, Uruguay. The sections were impregnated with silver according to the techniques of Golgi and, as can be seen by the label in Cajal's own handwriting, are from a "Cat of 15 days, acoustic cortex". The "bue" is short for "bueno", which Cajal used to label his best preparations.

Contemporary photomicrograph made from slide on facing page, showing a cell in the second section in the left column. It shows a short-axoned neuron of the Golgi type at about X 740 magnification. These cells are among those in the cerebral cortex generally considered to be interneurons.

CONTENTS

T H E INTERPRETATION O F BEHAVIOR IN T E R M S OF INTERNEURONS

1

G. ADRIAN HORRIDGE T H E C O N T R O L O F O U T P U T B Y C E N T R A L NEURONS

21

DONALD KENNEDY E X C I T A T O R Y AND INHIBITORY PROCESSES

37

L. TAUE T H E ORGANIZATION OF SUBPOPULATIONS IN T H E A B D O M I N A L GANGLION

OF APLYSIA

71

ERIC R. KANDEL REGULATIVE M E C H A N I S M S FOR THE DISCHARGES OF S P E C I F I C INTERNEURONS

113

C. A. G . WIERSMA R E F L E X MATURATION

131

A

159

Sten Skoglund

STRUCTURAL ANALYSIS O F S P I N A L INTERNEURONS AND R E N S H A W C E L L S

Madge E. and Arnold B. Scheibel

S P I N A L CORD A F F E R E N T S :

FUNCTIONAL ORGANIZATION AND I N H I B I T O R Y

CONTROL

209

ROBERT F. SCHMIDT CONVERGENCE O F E X C I T A T O R Y AND I N H I B I T O R Y ACTION ON INTERNEURONES IN T H E S P I N A L CORD

231

ANDERS LUNDBERG T H E LOCALIZATION OF FUNCTIONAL G R O U P S OF INTERNEURONS

267

WILLIAM D. WILLIS, JR. E L E C T R O N MICROSCOPIC STUDIES OF C E R E B E L L A R INTERNEURONS

289

L. M. H. LARRAMENDI NEURONS OF C E R E B E L L A R N U C L E I

309

MASAO ITO F U N C T I O N A L ASPECTS O F INTERNEURONAL E V O L U T I O N IN T H E CORTEX

CEREBELLAR 329

RODOLFO R. LLINAS T H E N E R V E C E L L AS AN A N A L Y Z E R O F S P I K E T R A I N S

349

JOSÉ P. SEGUNDO AND DONALD H. PERKEL STUDIES ON T H E H I P P O C A M P U S :

METHODS O F ANALYSIS

Theodor W. Blackstad

xvii

391

xviii

THE

PARTICIPATION

OF

INTERNEURON

INHIBITORY AND EXCITATORY

INTERNEURONES

IN

THE

CONTROL OF HIPPOCAMPAL CORTICAL OUTPUT

415

PER ANDERSEN, GARY N. GROSS, TERJE LPMO AND OLA SVEEN INTERNEURONAL MECHANISMS IN THALAMICALLY INDUCED SYNCHRONIZING AND DESYNCHRONIZING ACTIVITIES

467

DOMINICK P. PURPURA INTERNEURONAL MECHANISMS IN THE C O R T E X

497

COSTAS STEFANIS SUMMATION

527

DOMINICK P. PURPURA N A M E INDEX

543

S U B J E C T INDEX

547

THE INTERPRETATION OF BEHAVIOR IN TERMS OF INTERNEURONS G. ADRIAN HORRIDGE St. Andrews University Fife, Scotland

The aim of neurobiology is the explanation of behavior, and microelectrode techniques now provide the possibility of explaining it at a new level in terms of units which we believe correspond to the actual units of the nervous system. I propose to deal with some implications of this new emphasis. In the physical sciences, vast expense is justified by the expectation of securing an explanation in terms of sub-units of the next size down; chemical properties in terms of molecules, molecules in terms of atoms. From experience we know that this type of explanation does not provide complete answers, or even any answers to many of the outstanding questions, but it makes old questions redundant, provides a particular style of mechanistic understanding, leads to new useful experiments, and sometimes reveals methods whereby we can exert control. Other kinds of explanation are less likely to be fruitful in these valuable ways. Further, to be most effective, the mechanistic explanation must be in terms of units which correspond to real structures, although, like chromosomes or atoms, these units may turn out later to be both divisible and mutable. BACKGROUND

As units in the nervous system we have ready to hand the anatomical neurons of Ramón y Cajal and the physiological all-or-none properties which were worked out historically for the heart, the jellyfish bell, single axons, vertebrate fast muscle fibers and then parts of many other excitable cells. It was a great temptation to endow the anatomical neuron with the physiological all-or-none activity, and call the hypothetical product the functional unit of behavior. Circuits of these units could act as models with some of the properties of nervous systems. Examples are found in the study of interneurons in optokinetic responses by Lorente de Nó (1), in papers by Pitts & McCulloch (3), and by a few even to this day. Studies with intracellular microelectrodes on a few advantageous preparations have now brought a profusion of variables so that many physiological 1

2

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INTERNEURON

properties of units are explained by changes at membrane level. The variables are potentials of all shapes and sizes; the mechanisms are changes in permeability to various ions; the effects are inhibitions and excitations, immediate, delayed or rhythmic. So far as events at membrane level control those at the neuron or unit level, all are inhibitory or excitatory. All are summed in time and in space upon the branches or soma of each neuron by virtue of the electrotonic spread as governed by the time and space constant of the piece of neuron considered. To build up behavior from this is like writing mathematics with limited symbols such as plus, minus, zero and a few digits. We know that success depends upon close attention to the spatial pattern of the symbols or units. What is written at one place in such a system has little significance without the rest of the program. In the nervous system the same dependence on the whole pattern holds. If we are to have explanations of critical choices and operations in small parts of behavioral patterns they must ultimately be expressed in terms of local excitations and local inhibitions in the anatomical context of the active nerve fibers, and in order to exclude alternative explanations the pattern must be known in detail in space and time. This adds an immense anatomical problem, but is the only realistic analysis, in contrast to blanket explanations with black boxes, and it must be stressed over and over again that the principal causal factor seems to be the spatial geometry of the actual synapses and fibers. The problem of analysis is easier in two circumstances which are well worth examination—in very simple examples where all alternatives might easily be explored, for example, the ear of a moth with two receptor cells, and in very specialized examples where the larger part of the system serves a single known function, as in the auditory system of bats. But for all situations in nervous systems a great deal of microanatomical description is required before any exclusive explanation of behavior can emerge. THE PROBLEM

The task involves description of two kinds, (a) anatomically observed connectivity patterns and (b) causal relationships as revealed by the activity of single units. Although we may expect that these refer to the same substrate of investigation, experience warns us to distinguish carefully the actual phenomena seen with different techniques from the inferred entities and their supposed relationships. A unit is a physiological entity defined as the apparent output of a supposed anatomical neuron or axon, and is often recognized by the all-or-none properties of an active propagated response (called a spike); however, we are now moving into a period when units are not necessarily recognizable by spikes. For example, first and second order cells on the visual pathway of insects and vertebrates apparently have only graded non-propagated potentials. Numerous small neurons in all groups of animals may turn out not to

BEHAVIOR

IN TERMS

OF

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3

have spikes but to work by electronic transmission over small distances. Therefore they will have to be analyzed as units. Furthermore, a neuron, defined anatomically as a cell, may have several regions each of which can act as a separate unit. To complicate the issue still more, where several neurons interact in a compact cluster and operate without spikes, neither the anatomical neuron nor the physiological unit is appropriate to understanding the nature of the system of interactions. For example, electron microscopy reveals that in the neuropile of the vertebrate retina or cortex, or in any invertebrate neuropile, the proximity and serial occurrence of the synapses provides a situation where simultaneous complex electrotonic interaction between many inputs may prevail. The ommatidium of Limulus and the optic cartridge of arthropods are other examples of tightly interacting groups of units. The responses of the output neurons as units are observable but are not necessarily predictable from synaptic potentials or other membrane phenomena of any one cell in the group. Despite the complexity of the interactions which bear upon the spike-initiating process, however, the information carried by the spike is the justification for the examination of the output at the unit level. 'Connectivity', like 'synapse', is a term based on either functional or anatomical observations, or both, in every individual instance. As favored by some, for a mathematical relationship, or by investigators of vertebrate CNS for inferred physiological or synaptic interaction, connectivity is a functional term. As favored by others it means what is connected to what, in actuality, by anatomical synapses which can be mapped. Apart from serial sections under the electron microscope we have as yet almost no sure tools with which to study anatomical connectivity. The relatively gross anatomy of which tracts run over or under, which layers contain what fibers, the density, size and shape of neuron branches, and so on, is of little consequence so far as we know. If the feature of interest is what is connected to what, the pattern of axon terminal branching or dendritic tree matters little, although particular growth patterns may represent easy ways of establishing the required anatomical connectivity. I wish it were otherwise, but almost the whole of neuroanatomy of all classes of animals needs to be reworked in terms of anatomical connectivity. The anatomy of neurons revealed by the methylene blue or Golgi techniques even today takes us little further than Cajal or Retzius was able to see. At the detailed level of synapses the anatomical complexity is so great that a complete description is out of the question. Only carefully chosen areas of critically relevant neuropile can be described, and one must hope that they are representative of similar areas under the electrode in physiological experiments. At a gross level this is adequate but the correspondence inevitably breaks down at some level. The question is whether the variability between samples of the same critical region from different animals allows an exact analysis of the physiological results. Electrophysiology employed alone leads to the inference of physiological

4

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pathways and these can be equated exactly with individually identified neurons only in a few relatively simple examples which are mostly giant neurons. In the vertebrates the problem of equating any single physiological pathway with any single anatomical connectivity pattern at unit level is that the same neurons (except Mauthner fibers) cannot be found twice. Who has marked a vertebrate neuron at the conclusion of recording its normal activity, found the part it plays in behavior and where it lies in the pathway, then identified its connections anatomically? The point reached in vertebrate studies is the analysis of the numerically most abundant types of physiological unit and a partial equating with the most abundant types of anatomical neurons. This can be most readily achieved where there are central projections of the body senses or muscles. We prefer to build models and draw arrows between boxes in two dimensional diagrams of a typical digested subsystem which ignores the individuality of the units. The boxes are sometimes drawn as nerve cells. A central nervous system is always marked by a choice of action that is dependent upon its own structure in interaction with the whole history of sensory impressions, and by a behavioral output that is highly adapted to the animal's environment. This adaptive choice of alternative activity patterns distinguishes a CNS from a cardiac, sympathetic, or other peripheral ganglion. Central nervous systems of all animals consist of three components, terminals of axons of sensory neurons, dendrites of motoneurons, and thirdly those neurons which lie between sensory and motor—the interneurons. The proportion of the latter rises with increased behavioral complexity. Interneurons are not just interpolated small inhibitory black boxes on physiological pathways. Beyond this point many definitions do not hold for situations such as nerve nets, outlying ganglia or ciliated cells. The study of interneurons has in practical terms become the interpretation of repeated sampling of units by microelectrodes and the description of all experimentally controlled factors which appear to modify their changes in spike frequency. On the sensory side this has led to the important concept of the receptive field. For each type of stimulus available, the receptive field is the plot of contours of equal stimulus intensity which give selected standard strengths of response. For chemical stimuli, each experimentally applied stimulating substance has to be treated as if it lies in a different dimension. The receptive field is the natural way of expressing what a neuron responds to, and the present effort is largely directed to the full description and classification of the receptive fields. For the numerous units which are spontaneously active, or which have non-specific inputs, the problems of classification are very great. We also have to face the possibility that every neuron in every animal is in some way unique. The classification into categories is a human means of ordering the data, but a particular classification scheme may be unrelated to the animal's organization and will therefore subsequently impede progress until superseded.

BEHAVIOR

IN T E R M S

OF

INTERNEURONS

5

The uniqueness of neurons adds to the fascination of the problem. On account of the geometrical arrangement of sensory neurons on the body, no two sensory units have identical receptive fields. In the retina, in the skin, and along the cochlea, receptors are distributed in particular spatial patterns so that no two can be identical. In vertebrates, even in the chemical senses, it is an experimental finding that every receptor and interneuron is in some small way unique. In vertebrates the ascending sensory interneurons form classes with beautiful progressive changes in receptive field as we pass from unit to unit at one level within a single nucleus, but it is a matter of experience that no two interneurons are exactly duplicated. Considered anatomically, every neuron in the central nervous system has its dendritic field in a different place, and must have its own pattern of anatomical connectivity: every effector neuron runs to its own differently situated muscle or gland cells. Although every analysis has classified units into classes, advance has been most rapid in those systems where each unit is provided with a unique label because it occupies its own position, as in a projection from the retina, the cochlea or from the body surface. Apart from its place in the projection, most vertebrate interneurons are not described in an individually recognizable way by electrophysiological studies. The reason for shunning the issue of uniqueness is that in vertebrates there are as yet no techniques for returning to work over and over again on the same neuron, identified even by receptive field together with projection. This lack of techniques becomes crucial in the experimental study of the growth of neurons that are in process of forming new connections. For example, the distinction between fast and slow vertebrate motoneurons made possible the analysis of how each group connects with and modifies the properties of its own type of muscle fiber. Comparable analysis on central neurons can be made only to the degree of detail which the recognition of individual differences permits. The interneurons recognize each other, as proved by their formation of synapses in particular ways. We have not succeeded in distinguishing the basis of their specificity even after they have accentuated their differences as cells by their establishment of unique anatomical and functional connections. If we had the facility to extend a probe into a distant city and record individual conversations (but not purpose, motive or thoughts) of the inhabitants at random, it would be possible, in time, to gain a good deal of insight into the activity of that city, and to classify even the most individualistic inhabitants. As an aid to understanding mechanisms, the classification would be the essential first step only, and many types of classifications would assist little in the identification of causes and effects in the city's affairs. A street plan would be some help; a map showing the paths of movement and possible communication points of every individual might seem desirable for analysis, but too complex to be useful if secured. The problems of learning anything about behavior from interneuron studies are in some ways similar.

6

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INTERNEURON

This analogy immediately suggests that the most interesting units are likely to be numerically very rare but to have widespread effects. They will be the ones most likely to be omitted from preliminary classifications of numerically numerous types whose actions cannot be understood without them. Vast amounts of work upon irrelevant facets can easily be entirely wasted and yet tiny details can turn out to be of great significance. The problem lies in the choice of features which will be found to be relevant. L o w INFORMATION CONTENT OF BEHAVIOR

Apart from feats of human memory, and the specialized actions of primates and a few other mammals such as elephants, an act of behavior in an experimental test of a cat, rat, octopus or crab is relatively simple. The total number of choices described in all psychological literature could be punched on comparatively few yards of tape. Although justly regarded as marvelous, the behavior of most animals is strictly adapted to the environment in which they live, and special-purpose machines to perform these tasks would not require many components. I feel competent to say this because I am a zoologist and I strive to make the following point. The complexity of the nervous system seems out of all proportion to the job it has to do. A small worm with little interesting life operates with about 100,000 neurons; a sea-anemone has many more. The complexity of impulse patterns and possible cross-correlations in neurons, even in lower animals, is astronomical, like the detail of leaves in woodland undergrowth. There is literally no end to the potential analysis of it. Most of the detail and correlations will therefore not be relevant to the required explanation. Yet for medical reasons alone we must strive to understand the basis of action by the nervous system. The important topic for armchair discussion is how to proceed without waste of effort upon details which later are proved irrelevant. SUBCATEGORIES OF BEHAVIOR

For a long time now a number of terms have been employed in explanations of that highest category, behavior. They are words like arousal, pattern abstraction, sign stimulus, innate behavior pattern, selection of alternatives, attentiveness, reflex, efferent copy, reinforcement, learning. Experimental analysis of behavior is full of such terms. All can be defined satisfactorily; many are open to experimental analysis, but all carry the expectation of being underpinned by a lower category of entities. They all need to be explained at neuronal or unit level. The point is that these terms referring to the behavioral level are very obviously abstractions of the human mind; they are not necessarily components of real mechanisms in the animal. We should therefore not be disappointed that when we start to explain this kind of concept at a lower level we find that each of our magnificent comprehensive terms covers a multitude of possible mechanisms. Gunpowder and ice have in common an ability to change under mechanical pressure, but this is

BEHAVIOR

IN T E R M S

OF

INTERNEURONS

7

hardly explainable by a mutability which they hold in common. Detailed analysis of each, in terms of component units, leads to a different world of interactions, where the mutability feature is not lost but is given less consideration. Arousal is a term which conjures up the vertebrate (or perhaps only mammalian) reticular system, and we might ask whether arousal is necessarily nonspecific. In neuronal terms, if there are particular units which, when excited, arouse others, are they not specific? If they are nonspecific with reference to the stimulus, does that mean a wide receptive field or inability or failure to explore the receptive field? What is the total receptive field of that arousal neuron? What are its total connections and when is it active? Well, of course usually we do not know. It is about as far as we can go to record an electrical or neuronal correlate of arousal at all. There is therefore a temptation to hang on to the term for as long as possible before substituting half a dozen types of spatial summation at terminals of arousal axons with known pathways. Pattern abstraction, as a property of higher order sensory interneurons, is still an exciting new discovery. Some interneurons respond to warble notes falling between certain sound frequencies, others to movement in a particular direction irrespective of contrast. These stimuli are features of the environment of interest to the animal. The responses of interneurons are explicable as the consequence of particular patterns of summation and inhibition of earlier order neurons which impinge upon them. With hindsight it is easy to say that pattern selection is inevitable wherever dendrites have excitatory and inhibitory terminals impinging on them from several sources. To stress that every unit selects a pattern is no longer novel; however there is a considerable amount of spade work still to do, even after the foundations are drawn out. Only recently has it been demonstrated that the directional discrimination of a sound is a type of pattern selection that is made possible by the summation and inhibition of excitation from the two ears upon a common interneuron. Different higher order interneurons select sounds with different time delays between the two ears. There is a population of these neurons such that sounds which originate in different directions are signaled along separate pathways. From this arises a line-labeled representation of the outside world—not necessarily a topographical map. Many other fascinating examples of the same kind wait to be worked out, but why should they be explored if they really are of the same kind? The answer is not because they are there, or because Ph.D. students require topics, but because new principles of explanation in terms of component units emerge only from analysis of those very components. This can only be done by hours of recording of responses to thousands of test stimuli, as the receptive fields are mapped out in all dimensions. During the course of this work, for instance, some of the well-known sign-stimuli of behavioral studies may prove to be examples of pattern abstraction by single interneurons. The in-

s

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tellectual challenge, however, comes from those units (which are already known to be common in all animals) where the receptive fields do not correspond to the obvious structure of the environment, e.g., multimodal units. The reflex as a unit of behavior is easily recognizable as an appropriately adaptive movement in response to a normal environmental stimulus. In terms of interneurons it implies pattern selection on the input side and further selection of patterns of interneurons by motoneurons whereby the appropriate set of motoneurons are excited. Traditionally we are led to believe that in vertebrates the running commentary provided by the receptors of joints, skin and muscles, provides a continuous control of the course of the movement. In arthropods however, where the critical experiments are possible, the general rule is that reflex movements are the result of centrally determined patterns of motor impulses that are substantially unchanged by the removal of all proprioceptors, periphery and musculature. The great change of emphasis in the past five years made by workers on arthropods justifies illustration by a few examples, because it is no longer possible to begin with the assumption that coordinated responses (at least in invertebrates ) are reflexes that are dependent upon continuous sensory monitoring of the environment including their own consequences. Ventilation of the trachea, oscillation of the wings in flight, song of insects and swimming movements, are each the product of a central program of the central ganglia. Moreover, where a rhythm is modified by sense organs these do not necessarily have an effect at the instant when they are excited. As shown by Wilson and his colleagues (5), wind blowing against hairs on the head of a flying locust causes a flow of impulses in second order neurons down the neck nerves to the thoracic ganglia. While these fibers are active, at more or less any frequency or pattern, regular bursts of motor impulses oscillate the wings through the direct wing muscles. Even when the wing muscles have been removed these bursts of impulses resemble those in normal flight. In addition, proprioceptors can influence the efferent pattern. Near the hinge of the wing is a large sensory cell which increases the frequency of the wing beat and shifts the position of certain motor impulses to two of the muscles. By this shift in the position of an impulse in each cycle, lift is added at the time when it is most effective. The receptor fires only once at the top of each wing cycle and its impulse has no immediate effect, acting only when summed over many cycles. Immediately acting reflexes also contribute; for example, contact of the insect's feet with the ground stops the flight rhythm at once and orientation reflexes control turning. There is, therefore, a central rhythm with peripheral control of some aspects. As in all animals, the response as a whole is a result of summation of the mechanical effects of different muscles at the gross anatomical level. A more complex but equally clear central rhythm controls horizontal optokinetic eye movement of the crab. The eyes of the crab are on the ends of movable stalks which can follow the movement of a striped drum rotating

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around the animal. One eye moves towards the midline, the other away from it. The eyes follow slowly for 10° to 15°, then flick back to start again. Nine muscles, each with fast and slow motor axons, and phasic and tonic muscle fibers participate in this regularly repeated movement, which is called the optokinetic response. The eye moves steadily across the orbit because the motor impulses to one of the muscles slowly change in frequency. For each motor frequency the eye is pulled over to an equilibrium position which is determined by the balance between the muscle tensions and the elastic recoil. The forward movement and periodie fast return phase are due to centrally determined programs which depend only upon the history of the total apparent movement seen by either eye. A large movement of all contrasts in the visual field causes a large movement of the eye, during which the sequence of slow forward and fast return phases may run through several cycles. However, for a given stimulus relative to the eye the response is the same whether or not the eye is allowed to move. Numerous tests on the immediate control of eyestalk position revealed no evidence of utilization of proprioceptors. The only information which the animal has about the direction in which the eye points at any moment appears to have been converted into the motor impulse pattern to the nine eye muscles at that moment. A vertical or a horizontal movement of contrasts in the visual field, or a tilt of the crab in any plane adds a little "excitation" of the appropriate motoneurons and, if it is free to move, the eye moves in the appropriate direction. If it is not free, the change in the motor impulse pattern takes place nevertheless. The central program is predetermined to bring the eye to the right place for every relevant stimulus situation. Suppose the input is an instantaneous instruction to move 20° to the right, but the eye can move only 12°: it will flick back and then complete the remaining 8°. This is achieved by an entirely central mechanism with no proprioceptive feedback, and the motor output, including the flick-back, is unaltered by clamping the eye. Recently I have been looking at two aspects of this system. If the eye's traverse is caused by a pattern of central origin it is difficult to see how the positions of the ends of the traverse agree with the shape of the socket. The motor impulse frequency of the pertinent muscle is not constant from crab to crab. The new relevant observation is that when the edge of the eye socket is repeatedly stimulated while the optokinetic response is going on, the fast flick-back soon begins its onset at a position which is earlier in the cycle. This has the consequence that the eye does not move so far in its slow phase across the socket. The change in the onset of flick-back lasts for many minutes whereas the shock treatment lasts for a few seconds. The direction of this long-term change in the central program is such that an eye which touched the edge of the socket would eventually be limited to a more centrally situated track. The brain utilizes no proprioceptive information for the immediate control of onset of flick-back. The long-term adaptive change of

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the central pattern, however, could provide the necessary adjustment to a new shape at each moult of the cuticle. It is a type of learning, without association but with reinforcement that is appropriate for trimming the central pattern to suit the animal's anatomy. The adaptive plasticity of the response is therefore no more than one more item which is written into a centrally determined program. The other new feature of interest appeared during a study of the protective eye withdrawal, in which the eye is pulled back into its socket. As the eye extends to its former position it sweeps across contrasting objects in its visual field. The question is whether the crab distinguishes between real movement and the apparent movement which its own eyecup extension creates. This is an old problem which von Hoist tried to solve by supposing that every efferent signal is accompanied by an efferent copy which is a signal that neutralizes the expected effects of the motor output. In the crab a right eye in process of being voluntarily extended after a protective withdrawal certainly continues to see. This is proved by the observation that if the left eye is blinded both eyes continue to respond to small background oscillations of a surrounding striped drum while the right eye is in process of extending. A voluntary extension of the right eye fails to drive the blind left eye by the amount of apparent movement which the extension generates. When the right eyecup is slowly pushed in a forced extension, however, the blind left eye makes a movement in the opposite direction, which is certainly caused by the apparent movement that is generated. There is therefore a difference between the voluntary and the forced cases. But when the relative movement caused by a voluntary extension of the right eye is forcibly prevented, either by holding the eye or blinding it, the left eye makes no movement. Therefore no actual compensatory "efference copy" is passed to the other side. The inescapable conclusion from these and many related experiments was that the crab somehow has learned, during the course of many spontaneous eye retractions, to distinguish and eliminate the central effect of its own eye movement, and makes use neither of efferent copy nor of proprioceptors. Although the fixed programs are largely inherited, some of their detail and, in particular, compensation for their own movements, may well be later determined by flexible phrases in a central program. To a complex animal with many sense organs in a varied environment, the effects of its own actions must frequently be unpredictable. Many lower animals have startle reflexes which are set off by sensitive receptors. Examples are the jump of the cockroach as triggered by an air current on the anal cerci, and the contraction twitch of the earthworm at the slightest touch. But these giant fiber responses are stilled when the animal itself moves. The automatic cancelling mechanism is not understood even in these simple examples, although it will probably turn out to be wholly central. One can predict that the motor output is accompanied by an inhibition of the giant fiber reflex even when, by

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section of motor nerves, the slow movement is not allowed to take place. In more complicated examples of control by a centrally determined program there must be many types of underscribed plasticity. I am in fact suggesting that the simple type of postural learning, with non-specific reinforcement, that I first described in the insect ventral cord, or which I have just mentioned in the control of the position of onset of the fast flick-back of the crab eye, could be a common mechanism of adjustment of otherwise centrally determined sequences. The central program contains phrases which are adjustable under the influence of inputs that are only remotely related in pattern to the output. I believe that once the technique of repeated recording of detailed central programs from groups of identified neurons is widely utilized it will lead to a new understanding of the part played by centrally fixed patterns of efferent impulses as components of reflexes and of conditioned reflexes, even in vertebrates. The central consistency in the face of variation in sensory input also helps us to ignore one great slice of nervous activity, the sensory excitation which the animal itself ignores. Plasticity controlled by nonspecific inputs evolves easily into learning mechanisms controlled by reinforcement neurons. The electrophysiological analysis of arthropod movement teaches that each neuron can be individually named and each is a unique pattern perceiving neuron. The analysis of "reflexes" by recording from named motoneurons reveals a fixed central mechanism. Thinking of this in terms of interneurons suggests that reflexes and central rhythms grade into each other and each have many mechanisms at the neuronal level. Learning, analyzed at the interneuron level, presents a range of new obstacles which arise from the difficulty of recording from the required units exactly when the learning occurs. Learning is a troublesome term from another method of observation and there is no reason to expect a single mechanism at a neuronal level. Learning is difficult to achieve in experimental situations where electrophysiology is possible and vice versa. If physiological changes are discovered at the time that learning occurs there is no obvious way of knowing whether they are primary causes or not; because units are so numerous the observed changes are almost certainly not primary causes. Moreover, if long-term physiological changes in a unit are suspected as significant, the constancy of the synaptic inputs to that unit and the absence of interfering small cells, must be demonstrated. In electrophysiological tests during a learning experiment the stimulus situation must be presented repeatedly while a number of units are tested. Therefore by the time the units of interest have been selected, they have been modified. At the critical spot in the nervous system, presumably upon part of a neuron, a crucial change in learning must occur at some time during the series of presentations; the problem is to anticipate by probing at that spot before applying the right stimulus. In a nervous system where every neuron can be individually identified, this is not a fundamental uncertainty principle after the Heisenberg

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model, because patient elucidation will eventually discover the unit of interest and make possible experimentation upon it alone before it is modified by repeated test stimuli. However, if units are not individually identifiable, the problem appears to be insuperable by a sampling process. Worse than this, there is no means of knowing what kind of primary change to expect. Changes of activity will certainly be found; perhaps the only way of testing whether they are significant is to introduce them artificially. FEATURES OF INTERNEURONS IN INVERTEBRATES

The numerous interneurons which have been investigated in arthropods, particularly by Wiersma (4) in the crayfish, have the following general characteristics that can probably be extended to many invertebrate phyla. Interneurons, identified by function, are constant in position in the ganglion or in location in a bundle. Many, which are inferred to be of lower order in the hierarchy, have receptive fields which are readily defined and constant. This is a strong practical justification for adhering to their classificiation by receptive field. The receptive fields at higher levels are more complex and are explainable by taking sums and differences of lower order units. Some interneurons have inputs of one modality; others of two or more modalities. In the crayfish, about 10 per cent of interneurons can be identified, and counts of fiber numbers in the electron microscope show that 90 per cent are below the present size limit of recording with microelectrodes. Receptive fields with a geometrical regularity such as inhibitory surrounds or with exclusion-type interactions are rare in invertebrates, though common in mammalian sensory systems. The arthropod interneurons seem to function purely by selection of particular patterns of sensory input but, so far, it has rarely been possible to make sense of their responses in terms of the normal environment or of normal behavior. Every stimulus excites numerous interneurons but, strangely enough most stimuli have no other consequence, and, in particular, no overt behavioral effect. We must therefore infer that a behavioral output of interneuron activity is caused only by certain combinations of stimuli when these act for relatively long periods of time upon ganglia which are in a receptive state. This is unscientific until the "receptive state" is separately definable. More difficult to interpret in terms of known interneurons is the general finding that motor activity in arthropods is largely governed by centrally determined sequences that are relatively independent of immediate sensory pattern. We are led to the conclusion that the interneurons are signaling when their part of the sensory combinations are appropriate for a central sequence to be emitted, allowing that some kind of receptive state is also essential centrally. The motoneuron pool seems to churn out programs like a jukebox but to be comparably deaf to feedback. In such a system we find novel types of plasticity of the detail of central sequences, and we observe diurnal rhythm in their execution but, in contrast, pattern selection by interneurons shows neither plasticity nor diurnal rhythm.

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The large cells of molluscs have revealed other features of intemeuron organization, as more fully outlined by Kandel in this symposium. A single cell with two axons can cause excitation at one terminal but inhibition at the other, apparently by the release of the same transmitter at each. This shows that the nature of the response depends on the postsynaptic cell. Different identified single neurons have quite different sensitivities to drugs and to transmitter substances. They sometimes have different ionic contents, different sensitivity to light and their own characteristic rhythm of activity. Identifiable pairs of cells, with one member on each side of the animal, are physiologically symmetrical where tested. In the case of one pair of cells, an obvious asymmetry proved to be only of the gross anatomical position of the cell body while the physiological pathways to these cells and their membrane properties were similar. As for anatomically symmetrical neurons in many groups of animals, it seems reasonable to conclude that the symmetry of connections in the two sides of the central nervous system arises from a similarity in the DNA-governed differentiation of the symmetrical cells. It is significant that in many animals symmetrical neurons often demonstrate their recognition of the metabolic similarity of their partner and form low resistance pathways from one to the other where they meet, whereas they are less likely to form such pathways with other neurons. DIFFERENCES

BETWEEN INVERTEBRATE AND VERTEBRATE

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Invertebrate interneurons are in many cases individually identifiable, constant in number and receptive field, few of each type, accurately duplicated in different individuals of the same species, and with constant anatomical branching pattern. In contrast, vertebrate interneurons are not identifiable individually, and therefore appear to be less easily defined physiologically: they are more numerous, with many overlapping classes with reference to one stimulus. The receptive fields of invertebrate interneurons are constant and independent of stimuli applied elsewhere (with one or two exceptions); on the other hand, many types of vertebrate interneurons have receptive fields which are modified by excitation in parallel channels or by nonspecific stimuli of all kinds. As we consider interneurons, in coelenterates, worms, molluscs and arthropods and in the classes of vertebrates, it is evident that evolution has taken the form of a progressively greater diversity of types, and that this is often achieved by greater detail of line-labeling. Occasionally there is an apparent simplification of pathways, as in the primate fovea, but in reality the wiring diagram is more specified, by an increased individual exactness of neuron connections in the higher form. There is greater diversity of interneuron classes and their receptive fields are more clearly definable, in more dimensions, in the higher groups of animals. Vertebrate sensory pathways often have a neural gain control system near the receptor terminals, and the higher order interneurons have regularly arranged inhibitory surrounds with a topotopical representation of the periphery in a spatial array of interneu-

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rons. Receptive fields in vertebrates are controlled at many levels by inhibitory feedback circuits. These features of the organization are not typical of invertebrates, where pathways of interaction are mostly linear sequences from receptor to effector. In invertebrates even the control of movement by proprioceptors, where it occurs, is sometimes not achieved by immediately acting feedback arcs to particular subsystems. Some of the above distinct features of vertebrates derive from the amacrine cells of vertebrate sensory pathways. All central nervous systems have nonspecific systems which ramify everywhere, but in vertebrates the specific systems have dendritic fields which are ordered in space, as in most of the nuclei of the mammalian central nervous system. In the invertebrates highly ordered dendritic fields are found only in optic lobes. The meaning of this regular anatomical order in vertebrates is not known but it does not necessarily have any relation to synaptic interaction, in which only the anatomical connectivity pattern is relevant. My own theory is that in a group of interneuron pathways which represent some kind of sensory projection, the necessary distinction of pathways is more easily reached in growth if the terminals and dendrites are spatially separated by a growth pattern which produces a regular array. Certainly cells which are adjacent are those most likely to interact at the next higher level. If the endings representing a projection are all confused together, then each output fiber has a more difficult task of connecting functionally with only its own particular input fiber if the projection is to be maintained. On this theory the regularly repeated arborizations in a vertebrate nucleus are mainly devices to assist in establishing correct anatomical connections with a minimum of individual specifications from the parent cell bodies. Perhaps vertebrates are driven to utilize more obviously regular growth patterns because they have many more neurons in each class than do most invertebrates. PATTERN ABSTRACTION

The outstanding general feature of interneurons that has emerged in the last decade is that all are units of pattern recognition. On account of this property of responding best to a particular class of inputs, each neuron is telling us whether the stimulus is relevant for it, and for all interneurons the stimulus is an excited cluster of neurons that are lower in the hierarchy. Because interneurons are so numerous, the overall relevance of a stimulus to the animal can be determined only by direct testing of all boundary conditions in all dimensions of the stimulus and by recording interneurons all down the physiological pathway. A stimulus configuration which has components of interest but is not quite acceptable may not excite at the final interneuron stage and, as far as concerns this particular response, might as well not be there at all in the early stages. Conversely all stimulus configurations which are just adequate, however varied, are treated by a responding interneuron as a single successful class. Small discrepancies in the stimu-

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lus are ignored so long as the unit's threshold is exceeded. An animal with a range of many partially overlapping interneuron fields (called range fractionation) has therefore a much greater capacity to reject at a later stage what was mistakenly responded to by the earlier stage neurons. In consequence there is a high premium on range fractionation by large numbers of pathways in parallel at each level. Most excitation patterns still cause no response at the motor levels. The above paragraph summarizes what neurons say. What they do not say is equally relevant. Intemeurons as individuals do not make decisions as a result of some computation, either on a basis of the probable needs of the animal or on the statistical properties of the stimulus. They are not comparators; they do not pause to consider before a decision. Usually the size of their response is not an accurate measure of the message which they convey by being active. Despite numerous efforts to establish the importance of temporal pattern, the spacing of impulses does not seem to be particularly important in higher order units, although clearly paramount in auditory directional sensitivity and in rate of movement detection mechanisms. Interneurons respond to the "here and now" as it impinges upon them: even if modified by past experience they still respond only when their own threshold is exceeded. A pattern classifying system of any kind which discriminates between all combinations of n inputs requires at least 2n indicating units to signal its result. Therefore the classification must be adapted to the patterns which are likely to be encountered. This is why animals with a complex behavior pattern are highly adapted to their environment. Lack of universal flexibility caused by paucity of pathways is one of the evolutionary factors which makes adaptation essential, and a restricted repertoire certainly reduces the number of necessary outputs. The auditory and visual pathways of a bird, for example, are extremely complex but the abstractions they make which are of interest to the bird appear to be relatively restricted. In fact the number of outputs indicated by behavior is always fewer than the number of inputs indicated by the sensory interneurons. This is a measure of the extent to which the environment contains predictable situations. Most animals, having freedom of movement, have selected the customary niche to which they are adapted and there they stay. PROBLEMS OF CLASSIFYING

INTERNEURONS

Technical difficulties are too great and numerous to be overlooked. Arthropods and perhaps some other invertebrate groups have unipolar central neurons with cell bodies which are electrically far from the synaptic regions. Therefore recordings must be taken from axons that are too small to be individually localizable in neuropile. Only relatively giant fibers have been identified anatomically as well as functionally; all examples of individually identifiable cells show a complete fixity of anatomy and function; the same neu-

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ron always does the same thing when picked up in different individuals of one species. When recording by blind probing, the realistic responses of a central unit are those with low thresholds or properties obviously related to the normal environment of the animal. With care to avoid strong stimuli that excite widely, the function of an interneuron can be explored by numerous patient tests which define the contours of its response in as many dimensions as possible. The maximum amount of information is obtained in the time for which the unit can be held, although stimulation must not be so frequent as to cause habituation. New experimental stimulus situations come to mind with every new test but once lost the unit cannot for certain be found again. Each time a new significant aspect of the stimulus is discovered, all previous descriptions of similar units have to be brought up to date by revised experiments. There is therefore a continually developing interaction between the preparation and the analysis. By definition, almost, since nervous systems are so complex and permutations and combinations of stimuli mount up, the complete responses of single interneurons can never be known. Having found units which respond, it is impossible to find a significance for most of the electrophysiological detail, such as impulse pattern and rates of adaptation. Responses of interneurons are often not related to anything in the animal's repertoire. When a change in the neuron activity is found, even during an overt behavioral response, there are no rules for deciding whether the change is relevant or not, or whether it is a primary response or a side effect. The analyst usually does not know what would be a significant situation for an animal unlike himself. That is why very specialized animals, such as bats, can be more safely studied in the knowledge that many of their interneurons will play a part in their specialized activity. Interneurons, like sensory fibers, function in groups, and the next higher level abstracts patterns of activity from these groups. If this happens at every stage right through to the motoneurons—as seems probable except for a few giant fiber emergency responses—the only sensible analytical method is to record simultaneously from many interneurons to identify these groups and to concentrate on their responses. This means the abandonment of Müllers concept (originally for sensory nerves) that the nature of a message depends only on whichfiberis stimulated (2). A response requires several motoneurons, and each motoneuron is excited by the appropriate group of interneurons just as each interneuron is excited by a pattern of sensory terminals, or interneurons lower in the hierarchy. Let us call the unit of activity the excitation cluster. The question for the immediate future is whether it is possible to achieve an advance in the analysis of behavior in terms of excitation clusters, as it was once thought possible to make the analysis in terms of single units. The desirability of so doing is clear but simultaneous recording with many electrodes is necessary. An even greater amount of detail will emerge, however, in the form of cross-

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correlations than we now have from single interneurons. Complexity is increased when we reach small neurons which have no spike activity but nonetheless control behavior. There will be an ever increasing discrepancy between the enormous information content obtainable from neuron recording and the relatively puny information content of the behavior which it purports to explain. Somehow we have to learn to ignore about the same fraction of the neuron activity as the animal itself ignores. In my experiments on crabs and locusts this is a depressingly large amount. The best indication of whether or not a stimulus is appropriate is therefore the activity of the next higher-order interneuron along the line, or a motoneuron. However, to pick up the appropriate neuron one stage higher in the hierarchy is usually impossible, so that churning through varieties of stimulus situations and picking the ones with low thresholds is the only available method in the absence of an overt response. For this reason, the analysis of a behavioral pattern which is readily evoked is preferable to analysis of unknown systems in pinned-down animals. Even so the more stimulus situations are tested the greater the complexity to be found, whether relevant to the animal or not. The journals will inevitably publish material which is later found to be of no consequence. PHILOSOPHICAL PROBLEMS ARISING FROM THE SAMPLING

METHOD

Experimental recording reveals physiological pathways only, not anatomical connectivity. In trying to equate physiological pathways with anatomy and with behavioral responses we start with partial descriptions of each, so that some inferences are no more than convenient fictions. It is impossible to be conclusive about the mechanisms that govern interneurons high in the hierarchy or whole-animal responses until all the lower order entities are described in all relevant detail. But the procedure is the reverse: the causal factors at an earlier stage further upstream are largely guessed at, and the functions of units are taken for granted if they resemble in any way the components required to excite responses downstream. One of the best examples, the one-way movement receptors in vision, are proposed as the input for the optokinetic response and the stabilization of the eye. The proof that they are so should include the demonstration that no other suitable mechanism exists in the system, and this demonstration is difficult to achieve. Science depends upon a reproducibility which, in the study of interneurons, breaks down for two technical reasons. A unit once tested will perhaps never be found again, so that sampling methods become of major importance. However, is this year's sample the same as last year's? Statistical tests of the equality of average properties require 10-50 observations but testing the similarity of two distributions requires hundreds of observations. Secondly, at this level of detail every animal's experience can be different from every other's, even of the same species. The causes of the individuality of units in the differences between individuals cannot be discovered by a sta-

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tistical survey. These unhappy facts do not remove interneuron studies from the area of science but they do increase the difficulty of making meaningful explanations of detailed differences. Numerous channels in parallel lie in every situation in every nervous system. Therefore spurious correlations are readily found. Almost any set of observations upon the components can be explained by many possible models or inferred relationships. Because the causative or crucial excitation may travel by a route that is longer than expected, the measurement of latency is of little value except in the simplest examples. The possibility of feedback loops within even the smallest ganglion or central nucleus jeopardizes any hope of fixing a causative sequence from unit to unit. Finally, long term effects, such as habituation and changes in hormone levels, prevent the evaluation of all causal influences because full reproducibility is impossible. When the whole picture is not revealed, the missing parts are perhaps erroneously constructed in the image of the small accessible fraction. The characteristic of every neuron—to select an input pattern in space and time—has other effects on the means of analysis. The continual storm of sensory excitation rushing into any normal animal defies description. Apparently the central nervous system fails to react to most of this—it simply excludes it by pattern selection. At the same time the representation of the stimulus becomes more obscure and is simultaneously present in numerous different forms. The reduction in the amount of information as we pass from sensory pattern to interneuron to motoneuron and to behavior means that it is quite impossible to infer a unique solution of the mechanism of each stage from the output at that stage. There is insufficient information left to define the unknown inputs which have been rejected. From this it follows that the outside world cannot be reconstructed (by the observer) when the responses of interneurons are recorded, because information has been thrown away. The responses of individual interneurons are ambiguous as representations of the stimulus. Faith, however, compels one to suppose that the combined activity of all the neurons gives a unique exact picture of the relevant aspects of the environment. That this is in fact so remains to be demonstrated. The apparent certainty of an animal's actions may frequently originate in a rather haphazard selection of one course from a number of hazily perceived alternatives. Pattern selection by higherorder interneurons has exactly this property—that if the interneuron responds it conveys the impression of certainty down its own line so long as it was excited above threshold. An effective stimulus which is an imperfect pattern is as adequate as a perfect one. The line that is slightly bent appears straight, and most of life's decisions of rapid coordination are made at first glance. LIMITATIONS ON LOGICAL SYSTEMS

Ultimate limits to the analysis of complex nervous systems are set by mathematical or logical propositions. Our inability to examine all the con-

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nections and all contours in all dimensions of the receptive fields implies that interneurons have open-ended properties. None can be fully defined. Therefore, the physical properties of the system at any instant are not determinable. Moreover, the memory of every part is of unknown duration and therefore the physical state is determined by events over an unknown length of time. The state is not determined by quantized jumps: therefore small apparently insignificant drifts can become causes of later events but can never be inferred. Overcoming this latter feature is the secret of accuracy of the digital computer. Limitations apply to any complex system of relationships; for example, no complex logical system can be known to be complete; no question as to how a complex logical system would operate in its entirety can be solved; as principles of action of a logical system are discovered there is no means of telling which will later prove to be inconsistent with each other. There are always combinations which have not been analyzed; in a non-digital system with unknown length of memory, apparently sub-threshold influences necessarily have later effects which leave no clue as to their origin. Limitations on experimental time as well as limitations of logic mean that only the bare essentials of the causative scheme can be worked out. Even a few feedback loops or non-linear properties soon make exact analysis prohibitively complex. In practice theoretical limitations do not impede current efforts to establish principles of action and the basic causal relationships in nervous systems, but they are most significant in the philosophical consideration of how far man may understand the working of every detail of his own brain. CONCLUSION

The lessons to be learned from the anticipations of frustration are those common to all science. Advance is achieved by careful choice of the preparation, technique and combination of anatomical, physiological, behavioral approaches, explained at each step by the best models available. Most of the advances will be remodeled in time. The lesson is to learn to discard our rubbish before the world has a chance to discard it after us. Anatomy from 1880 to 1920, experimental psychology from 1920 to 1967, and every other science during its period of rapid advance and greatest attractiveness has left yards of rubbish in the shelves of periodicals. In the same way, records of interneurons will fill the journals for the next decade, and perhaps longer. The judgment of history is the acid test in a subject with little immediate practical result, and I consider a paper worth writing if it becomes condensed to a sentence in a general text-book a decade or two later. A scientific finding which wins a paragraph after a century is a classic indeed. This process of abstraction of the relevant by the effect of time, usage and sifting action of other minds is the equivalent in society of the interneuronal pattern selection in nervous systems. In conclusion, the aim at each level of organization is to describe the type

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of system of interactions. To have explanatory value the interactions must be between entities which appear to be components at the next lower level of organization. At no level is a complete explanation possible in terms of categories at that level or at any other. Behavior is not satisfactorily explained by technical terms at the behavioral level, and (for reasons given) cannot be completely explained in terms of entities such as neurons, units, chemicals or potentials at a lower level. We can do no more than find out what is going on in as much detail as possible and express the results as a temporary conceptual explanation in so far as a system of interactions is revealed. The part of the description that will persist as relevant will be only a small part of the possible detail, just as a description of a tree or a lawn ignores the detail of leaves and blades of grass. The objective is the elucidation of the mechanisms of the brain and the discovery of the changes which constitute thought. Philosophers and the general public (if only as taxpayers who support much of the effort) are concerned to know the extent to which this objective can be approached. To a neuroanatomist the minute details of anatomy are all-important in revealing relationships but most of the descriptive detail is thereafter of no interest. To the neurophysiologist a detail of activity may be of fundamental importance in elucidating interactions but the other details are subsequently of no interest. Relationships and causal factors are progressively discernible by combination of many techniques, but all results are concepts of the human mind. Valuable explanations are constructive acts of comprehension: details of all causes and interactions at the finest level are for technical reasons beyond the reach of analysis in a reasonable experimental time. In understanding itself the brain is limited by the techniques available, by the time taken to deal with a structure of such complexity and by its own ability to understand the degree of complexity or the subtlety of interaction that may be found. REFERENCES 1. LORENTE DE NO, R., Vestibulo-ocular reflex arc. Arch. Neurol. Psychiat., 1933,

30 : 245-291. J., Physiologie des Gesichtssinnes. Leipzig, 1826. PITTS, W., and MCCULLOCH, W. S., HOW we know universals; the perception of auditory and visual forms. Bull. Math. Biophys., 1947, 9: 127-147. 4. WIERSMA, C. A. G., and IKEDA, K., Interneurons commanding swimmeret movements in the crayfish, Procambarus chrki (Girard). Comp. Biochem. Physiol., 1964, 12: 509-525. 5. WILSON, D. M., The origin of the flight-motor command in grasshoppers. In: Neural Theory and Modeling (R. F. Reiss, Ed.). Stanford Univ. Press, Stanford, 1964 :331-345.

2. 3.

MULLER,

THE CONTROL OF OUTPUT BY CENTRAL NEURONS" DONALD KENNEDY Stanford University Stanford, California

The problem of how movement is controlled is one to which the numerically restricted nervous systems of invertebrates may make a definitive contribution. Ideally, one would like to approach this problem in the same way one attacks the sensory side of the nervous system: by working with single units at levels successively more removed from the periphery. Unfortunately, the stimulation of single nerve cells in the vertebrate central nervous system imposes technical limitations much more severe than those that attend single unit recording. Even in a densely populated mass of cortical gray matter, an extracellular microelectrode readily singles out the activity of one or a very few neurons. When one tries to stimulate by this means, however, the selectivity is immediately lost; even in recent attempts to correlate the two approaches in mammalian motor cortex (2), the number of cells activated by the stimulating currents is large and indeterminate. The results of such relatively crude stimulation in mammals, however, have shown that localized regions of central nervous system can, when activated, release reproducible and coordinated movements. In several invertebrate systems this approach has now been brought to the level of single cells. Wiersma & Ikeda (31) first showed that the rhythmic movements of the abdominal appendages—the swimmerets—could be released by the stimulation of nerve bundles isolated by fine dissection from connectives in the central nervous system. The normal beat of the swimmerets is metachronous; it begins in the 5th segment and sweeps anteriorly with a delay of about 150 msec, per segment. The whole cycle repeats about two times per second. When evoked by central stimulation, these phase relationships are entirely independent of the frequency or phase of the input; the central elements appear to act only as releasers for a pattern whose own form depends upon interconnections between subsequent elements. Because of the consistency with which effective filaments could be located in a given region of a central connective, Wiersma & Ikeda (31) * Supported in part by grants from the U. S. Public Health Service (NB 02944) and the U. S. Air Force Office of Scientific Research (AFOSR-334). The author gratefully acknowledges the collaboration of William Evoy, Howard Fields, Herbert Pabst and Joanna Hanawalt in various phases of this work.

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proposed that single interneurons were involved; they termed them "command fibers". Similar elements trigger coordinated movements of several abdominal segments in the crayfish, and for these direct proof is available that a complete movement may be executed at the command of one cell only (22). It proved possible to isolate fine filaments from the equivalent regions of two connectives, separated by three intact segments. Direct stimulation of either filament while recording from the other revealed the presence of a single, through-conducting element that was common to both. Subsequently, one filament was stimulated at constant frequency and gradually increasing voltage while activity was monitored in the other filament. Motor output appropriate to the behavior appeared at precisely the point where the threshold for a single through-conducting unit was exceeded. This finding has now been replicated in at least 55 experiments on different elements producing flexion and extension of a variety of types. In common with the kind of interneurons described by Wiersma & Ikeda (31), the command fibers for extension and flexion in the crayfish abdomen evoke coordinated movements. Because our knowledge of the motoneuron population mediating these actions is quite complete (13-15, 23), we are able to make some beginning at a definition of the role of command fibers. First, they produce fully reciprocal actions in a given segment. Since the peripheral neuromuscular system in crustacea is somewhat more complex than that in mammals, this involves four central actions rather than two. Flexion commands, for example, centrally excite five flexor motoneurons in each half-segment and also drive the peripheral inhibitor to the extensors; at the same time, the activity of five extensor motoneurons and a flexor inhibitor is suppressed. Extension commands produce just the opposite responses. Second, the behavior usually involves several segments. Normally these are contiguous and the sign of the effect is the same in each one. Thus the total number of efferent neurons whose activity is influenced by the command interneuron is considerable: at least 120 for an element effecting abdominal flexion or extension in all segments. Third, command fibers often have overlapping functions; indeed, some interneurons influencing swimmeret position or the swimmeret rhythm also influence abdominal position (4, 10). Fourth, they evoke distinct behavioral responses at discharge frequencies that are physiologically reasonable, for example, 75/sec. or less. In summary, command neurons may be defined as single cells which, at modest discharge frequencies, release coordinated behavior involving a number of motor output channels. The specificity of the behavior is related to the identity and connections of the cell, but not to its pattern of activity; the role of the command element is to release a motor outflow that depends for its organization upon the connections of a network postsynaptic to the command fiber. Such organizing networks may involve interconnections between motoneurons, but more likely also comprise sets of "driver" neurons

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that are responsible for reciprocal motor outflow in single segments. Command fibers are endowed with the same individuality as sensory interneurons in the same animal—that is, elements having a specific, often unique behavioral action, are consistently found in a given anatomical location. In view of the abundance of such elements in the crayfish central nervous system—and the probability that similar ones are employed in such other stereotyped behaviors as stridulation in crickets (17) and flight in locusts (33)—it is surprising that they have not been found in Aplysia. There, efforts to demonstrate behavioral output upon stimulation of some large somata have met with failure (18). In part, this may be due to the selection of the abdominal ganglion rather than the cerebral for most of the analysis. The remarkable results of Willows (32), who has produced complex movements (withdrawal, turning) in Tritonia with microelectrode stimulation of single brain cells, suggests that we may be able to link the extensive microphysiology of identifiable neurons in gastropod molluscs to the behavioral machinery. DIFFERENTIATION OF C O M M A N D

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The number of interneurons that release coordinated behavior in the crayfish abdomen is strictly limited. It might have been supposed, given the tonic nature of motor output to the postural systems with which we have worked, that almost everv central unit we stimulated would influence that output in some way. On the contrary, it has been our experience that most of the bundles isolated centrally—even those containing a number of interneurons that could be activated by sensory stimulation—had no effect whatever. Our own sample includes specific interneurons that have unique motor effects and in addition can themselves be discharged by particular sensory stimuli (24).* Similar entities have been reported by Atwood & Wiersma (4). The identification of individual interneurons is thus as firm for their motor influence as it is for their receptive fields (28, 30), though it does not involve the same set of cells. Nonetheless, if one records the motor output to postural abdominal muscles in a single segment, one can find a series of command elements that seem to produce roughly similar effects. There is not a single command fiber for abdominal extension, but several—perhaps as many as two dozen. What is the basis for this replication of function? One explanation is apparently related to the specificity of the motor pathways involved—a specificity that as yet we do not fully understand. Most command elements do not produce equal excitation of all the apparently synergistic motoneurons that innervate a particular set of muscles. Some, in fact, are highly specific. The largest motoneuron to the postural flexor muscles of an abdominal segment in the crayfish, for example, may be activated alone or with several smaller ones; other command fibers drive the '

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• Also, D. Kennedy and J. T. Hanawalt, unpublished observations.

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smaller motoneurons very effectively but fail to excite the largest, even at high stimulus frequencies (10). In part, this is correlated with different kinetics of movement, but there may be other explanations. Extensor commands (see below) may differentially excite motor fibers that innervate muscle receptor organs, thus producing "reafference", or they may restrict their actions to exclusively skeletomotor neurons. A probably more important basis for the differentiation of command pathways involves the segmental distribution of their effects. Though two elements may produce, at a given rate of stimulation, the same motor output in a given segment, they usually differ in their influence upon other segments. Some fibers evoke predominantly rostral extension, some predominantly caudal extension, others a more distributed movement (20). Such findings encourage the speculation that interneurons on the motor side of the arthropod nervous system differ one from another in the distribution of their output synapses, just as those on the sensory side differ in the distribution of input synapses (28, 30). It would be unfair not to include in this discussion an admission that there are probably levels of command that introduce a partly spurious element of diversity. Some evidence has already accumulated that interneurons are hierarchically arranged; cells have been found which have weak effects upon motor output themselves, but connect to cells that are powerful command elements. Conversely, there is often a diminishing responsiveness to sensory stimulation as one gets closer to the motor side. Thus it is possible that some weak command fibers—which may, nonetheless, give real motor effects if stimulated at high enough frequency—are in fact interneurons one level removed from the ones we normally designate as command elements. C E N T R A L COMMAND IN RELATION TO PROPRIOCEPTIVE FEEDBACK

The abdomen is equipped with proprioceptive systems that respond to imposed or self-generated changes in shape. The best known comprises the dorsal muscle receptor organs (MROs); a fast and a slow muscle strand span each dorsal intersegment on both sides of the abdomen (1), and these are each associated with single phasic and tonic mechanoreceptor neurons (12, 29). Fields & Kennedy (15) showed that the efferent innervation of each receptor muscle is shared with the working muscle fibers having the same characteristics; that is, the slow MRO is innervated by a branch of one or more of the motor axons that also supply the postural extensors. The reflex role of the MROs has recently been worked out by Fields (13) in terms of the activities of individually identified cells. Activity in the slow (but not the fast) MRO drives one of the five excitatory motoneurons to the postural extensor muscles; this particular motoneuron always has the widest innervation field of the five, and it produces the largest junctional potentials found in nearly all of the polyneuronally innervated fibers. Hence it is a uniquely effective motoneuron in terms of tension production. Significantly, it never

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innervates the receptor muscle of the MRO; positive feedback is thereby avoided in the resistance reflex loop. Fields (13) showed directly that the MRO servo acts to stabilize the position of a segment against imposed flexion, and he demonstrated that the "set point" for the servo could be altered by efferent output to the receptor muscle during natural movements. Often, MRO discharge increases during extension in intact preparations, despite the unloading of the receptor by extensor muscles in parallel with it. A less well-understood class of receptors includes those sensitive to stretch of the ventral nerve cord (21) and those with endings upon the soft cuticle of the ventral abdomen near the insertion of the postural flexor muscles (24). Both these inputs suppress output to the flexor muscles; stretching the nerve cord (which happens during natural extension) also produces some extensor motor outflow. The effect of these endings is therefore unlike that of the MROs: they do not yield resistance reflexes. Whether activity in the superficial flexor muscles is itself excitatory to the endings in soft cuticle is not known, and it is therefore difficult to predict the influence of naturally executed movements upon these sensory endings. We know the MRO circuit in enough detail, however, to test directly some proposals about the role of proprioception in movements. As a general question, of course, this has a long history—particularly with respect to the control of locomotion in animals. Its application to arthropod systems has been discussed by Pringle (25) and by Wilson (33). It is fair to generalize that recent events in the "center versus periphery" controversy have all served to emphasize the importance of the center, and to discount the continuous-reflex role that proprioceptors might play. The motor program for flight in the locust, for example, is quite independent of the inflow of information from receptors in the wings; instead, it depends upon the phylogenetically derived information that was used in development, during the assembly of a set of ganglionic neurons. Even when addressed with perfectly random central stimulation, the ensemble produces a sequence of impulses in several motor nerves that is appropriate for flight (33). The functional overlap in command neurons that produce swimmeret movement as well as postural changes in crayfish demonstrates the intricacy with which single cells may be connected with appropriate sets of elements to produce complex behavior. If proprioceptors are not required to provide a temporal reflex organization for behavior, what are they for? It seems simplest to propose, as Wilson has (33), that they function to supply information about variables which are inherently unpredictable and therefore are unsuited to genetic control. For the present case, external load is the most obvious variable of this sort. Load may vary because the size and weight of the animal is not genetically fixed, or because the animal alternately occupies media of differing buoyancy, or because other neural pathways add load by mobilizing antagonists. In each case, the value of the load from moment to moment cannot be pre-

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dieted, with the result that without feedback the system applying force to it will produce movements of varying amplitudes. In order to produce movements that achieve a final position independent of load, a system of central command elements must rely upon proprioception. It might do this in a simple way, by having proprioceptors that signal completion of the movement and inhibit the central command system. Unfortunately, this would be an uneconomical solution: each position command would seem to require a unique set of proprioceptor elements to supply negative feedback. An all-purpose device, however, can be constructed on the opposite principle: that a single set of proprioceptors supply excitation to the system with an amplification which is proportional to the load. The crayfish MRO is a system that functions on this principle. It has been directly demonstrated (14) that some command pathways for abdominal extension quite selectively excite the one or more motoneurons that innervate the MRO. The result of stimulating such a central neuron is shown in Figure 1; reaiferent excitation of the MRO is strong, as would be expected from the fixed position of the preparation. Figure 2 is a diagram of the segmental proprioceptive servo and its relationship to the command pathway. Suppose that a given excitation is supplied to the shared motoneurons via command fibers that activate them selectively, like the one which was stimulated in the record of Figure 1A. Under lightly loaded conditions, the receptor muscle and the working muscle, which are in parallel, will contract at approximately the same rate. Such a contraction, since it should be nearly isotonic for the receptor muscle, will produce only a weak activation of the MRO, and therefore will supply little added tension via the "resistance reflex" loop involving the unshared motoneuron driven by the MRO. If, on the other hand, the load is heavy, the same amount of excitation delivered by the central command will produce a contraction in which substantial tension is developed in the receptor muscle. That tension is, of course, directly related to the load; in the extreme case of an isometrically loaded muscle the tension has the full value of the load itself. This means that, as the load increases, more and more MRO discharge occurs, and more and more excitation is fed to the muscle across the loop. In theory, such a system should function to cancel the effects of reasonable loads, especially if the gain of the proprioceptive servo is adequate. While we have not been able to measure its value, there are reasons for believing that it is sufficiently high. The input/output ratio for the central synapse between the MRO afferent and the motoneuron it activates is between 5:1 and 10:1; the motoneuron innervates over 90 percent of the muscle fibers, and produces large junctional potentials that require only little facilitation. Since the muscle fibers are typically tonic with long sarcomeres, one would expect them to develop tension at very low values of depolarization ( 3 ) . In the superficial flexors, which resemble the tonic extensors closely in all other respects, discharge rates of 5-15 per second are adequate to develop substantial whole-muscle tension. The contractions are

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Figure 2. Diagram of the position control system in a single segment of the crayfish abdomen. See text.

smoothly fused even at the lower frequencies because the excitation-contraction coupling mechanism has an extremely long time-constant (11). These facts suggest that the amount of tension that can be fed in across the loop is substantial. One would naturally like to know whether such a concept is readily transferable to the control systems for movement in mammals. The arrangement of elements is, of course, quite different in that efferent fibers are for the most part strictly fusimotor or strictly skeletomotor, rather than common to both receptor and functional muscles. Despite this difference, at least one mammalian system—that for the control of respiration—shows some close resemblances to the MRO control circuit. Inspiratory intercostal muscles receive an extremely powerful rhythmic fusimotor discharge during their contractions—so strong that its influence overcomes the unloading effects of a motoneuron discharge, causing the spindles from these muscles to increase their discharge frequency during inspiratory contractions, instead of during contraction of their antagonists. The source of the strong bias has been traced to two different sorts of fusimotor neurons, the action of which can be separated by lignocaine block (9). One class of fibers are tonically active; these y efferents respond to proprioceptive input from the chest wall, and their activity remains after high spinalization (8). A second class show rhythmic activity that is closely linked to that of the respiratory motoneurons; though tonic fusimotor neurons may be easily activated by stimu-

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lation in the anterior cerebellum independent of rhythmic y or a, the latter are less sensitive and tend (with some exceptions involving increases in the a/y ratio) to maintain their close relationship. It seems reasonable to suppose, then, that influences from the medullary respiratory center project in common to the a motoneurons and the rhythmically active fusimotors. A parallel output to working muscles and to the servo is thus assured by congruent central connection instead of by having a single efferent element serve both functions, as in the MRO system. Where large numbers of neurons are involved it is difficult to specify precisely the role of each; so, for example, the identity of the descending elements that command respiratory and rhythmic fusimotor activity is unknown, and it is not clear precisely how their excitation is distributed to the two sets of efferent elements. As in the M R O system, however, the proprioceptive loop involves the skeletomotor efferents exclusively. Thus all the components of a load-compensating system are present; and Corda, Eklund & von Euler ( 7 ) have demonstrated that artificial loading by tracheal obstruction does result in the predicted reflex compensation. Where specific cellular triggers for coordinated behavior are available, one can inquire about the role of proprioception in movement by stimulating command elements centrally, and evaluating the movement or the motor output generating it under varying conditions of peripheral feedback. W e have recently begun this sort of analysis in preparations in which the abdomen is free to move posteriorly; the movements of the free segments, as well as flexor and extensor motor discharge in a fixed anterior one, can be recorded in response to stimulation of interneurons isolated from the first abdominal connective. Movements can be photographed, and then reconstructed from frame-by-frame analysis of the projected film (20). Alternatively, various artificial restraints can be imposed upon the free segments to evaluate proprioceptive influences upon the motor discharge to the recorded one. The first such experiments (10, 20) showed that proprioceptive signals were not required to generate the reciprocal organization of abdominal flexion or extension, or to regulate the general time-course and extent of centrally commanded movements. Because the roots supplying the postural flexor muscles of the abdomen are exclusively motor, it was possible to compare the effects of flexion commands before and after total abdominal deafferentation. Under these conditions, with the abdomen minimally loaded, the absence of proprioceptive input had no influence on the time-course of flexion movements or upon the final positions attained. This does not indicate that proprioceptors play no role at all. It was not possible to examine extension in the same way, because the roots supplying these muscles are mixed; this is unfortunate, because the most prominent proprioceptive circuit is associated with extensors. Furthermore, the experiment does not test whether proprioceptive feedback might operate to amplify loaded contractions.

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We have therefore tried to classify command elements according to whether their motor effects are influenced by abdominal position. In some cases, it is clear that there is no such influence. These commands generate a set pattern of motoneuron discharge—and, a fixed amount of tension—regardless of the conditions at the periphery. Other central elements produce, in the same motoneurons, a discharge that subsides as the final position is reached—even when the command fiber is continuously stimulated. As long as stimulation is continued, imposed departures from the "final" position evoke increased motoneuron activity that subsides when that position is attained once again. These results, though preliminary, make it necessary to assume that, as demonstrated earlier for the isolated MRO loop, certain central commands engage the proprioceptive servo while others do not. We assume that the former sort generate load-compensated movements that achieve a pre-programmed final position, whereas the latter provide for transient changes in position superimposed upon a stable "set point". GANGLIONIC ORGANIZATION

The variety of inhibitory and excitatory influences which command fibers impose upon the motoneurons they affect makes it unlikely that the former synapse with all of the latter directly—though interneurons with direct excitatory and inhibitory outputs are known in molluscan systems (19, 26). Several bits of evidence argue against such direct connections. First, "spontaneous" discharge from flexor excitors and the flexor inhibitor in isolated ganglia often shows some reciprocity, indicating intrinsic organization of the output. Secondly, crossed habituation often occurs when two command fibers having the same action are stimulated sequentially, suggesting that a common set of junctions links them to the motoneurons. Thirdly, Evoy 0 has recently succeeded in recording intracellularly from flexor motoneurons in the neuropile of their ganglia of origin, identifying them by their responses to antidromic stimulation of the superficial third root or by correlating impulses in them with motor discharges in that root. Even when effective command fibers are repetitively stimulated, EPSPs in the motoneurons do not show a 1:1 correlation with impulses in the command element. These findings indicate that the network ensuring reciprocity or motor output is postsynaptic to the command fiber. It would be economical if the arrangements were provided by connections among the motoneurons themselves, but there is substantial evidence against this view. Synergistic motoneurons show weak coupling bilaterally, and similar excitatory influences may link excitatory efferents with the inhibitor to antagonist muscles (11). None of these connections is powerful enough to account for the strict reciprocity observed in reflex actions, or in the responses to command fiber stimulation. We have also tested the possibility that the peripheral inhibitor to a particular muscle might suppress activity in excitatory efferents destined for the same muscle, or vice versa. Evoy has found by intracellular recording, * Personal communication.

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however, that IPSPs in inhibited motoneurons do not correspond with impulses in the peripheral inhibitory neuron, and antidromic stimulation of the inhibitor does not by itself yield the appropriate suppression of motoneurons to the same muscle. We are thus forced to propose that each ganglion contains a local set of elements which drive motoneurons in reciprocal groups. This assumption is an awkward one in some respects. For example, the selectivity with which some command elements drive certain motoneurons demands that the driving network sometimes be bypassed. The results, however, leave us quite convinced that the local network must be present. Unfortunately, we have not found it; and its identification and unraveling are obviously necessary before we can claim a full understanding of how the central nervous systems control movement in these animals. In summary, the postural motor system of the crayfish abdomen provides a useful testing ground for ideas about the central nervous control of muscle action. The behavioral unit comprises a limited number of sensory and motor elements, which can be individually characterized quite fully; these units exist in homologous segmental series, each operated by a ganglionic network upon which command elements impinge. The 26 efferent neurons in each ganglion connect with the driver network and through it to command fibers, and also with segmental proprioceptive inputs. The coordinated behavior of a number of adjacent segments is determined by the levels of activity in command elements, and local proprioceptive connections serve to eliminate the error that arises through variations in load. We now seek to identify the influences that distribute excitation to command channels, and to establish the relationships among them. Bullock: There are a few points I would like to make that are largely based on Dr. Horridge's intentional effort to incite. In addition I would point out the obvious beauty of the analysis that Dr. Kennedy has offered us. The first point I will call "The Importance of Natural History", or we might call it "Case Law". Dr. Kennedy has given an elegant example of the analysis of a case with reference to other cases, such as the fly and locust. Not many of us, probably, have seen a patient with tabes dorsalis, or a mammal with damage to the dorsal columns or to a large portion of the somatic sensory input. Those who have, or who have read about it, certainly must be impressed with the enormous deficit in the performance of the system, and this surely has been largely responsible for the prevailing bias that existed before the era of Dr. Donald Wilson, which has recently revealed the importance of central commands and central patterning as an adequate tape of the details of the phasic relations of performance. Certainly in the era of Gray (16) voices like von Hoist's (27) were mostly ignored and the prevailing notion was that the proprioceptive input was, second by second, millisecond by millisecond, of importance in determining the timing of movement. This was in spite of earlier evidence,

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including some from Sherrington, that the spinal cord is capable of a large amount of phasic command, of sequential command, without proprioceptive input, phase by phase. What I am saying is that I think there must be a full spectrum of cases in the animal kingdom. The purely centrally determined case, the neurogenic origin of movement of von Hoist and others, long ignored in the literature, is one that really exists. We have heard beautiful new examples. Now we are less sure of the truly peripherally determined case, but in the human with tabes dorsalis and in many mammalian experiments the evidence points strongly toward a very real role of the proprioceptive input, at least in shaping and fine control. As I tried to point out some years ago in an essay about the range of possibilities of control of movements (5), there are probably many intermediate cases in which there may be different degrees of proprioceptor control. I do not think we will have a proper perspective until we make the same kind of analysis on a wide variety of preparations that Drs. Wiersma, Kennedy, Wilson and others here have done. There is a prevailing notion, for instance, that it is all very well to talk about flight, or maybe swimming, and it has been granted that there is also a certain amount of pure central origin in the peristaltic rhythm in the earthworm, but when one comes to higher animals and the ambulation of tetrapods and maybe even hexapods on land, then it is different. Then you see proprioceptive control on a grander scale. For my part, I do not think we know this. Even the experiments of Gray on deafferented toads are not conclusive, to my mind. They need to be repeated and reexamined. All this is by way of pointing out the obvious importance of Kennedy's contribution and the need for more like it in a wide variety of cases. The kind of example that Dr. Kennedy has presented with the precise, centrally controlled tapes, constant almost neuron by neuron, raises another question which is real in many of our minds. That is, to what degree is the nervous system probabilistic? We frequently hear that a general principle of operation of the nervous system is that it is a probabilistic mechanism. What is the alternative? What should I call it? Perhaps "quasi-deterministic". In the cases analyzed for the role of individual neurons where, in animal after animal, the identity of the same neuron in the same role is known (or believed), it would be good to know whether the same happens in them as in, let us say, auditory nerve fibers or cochlear nucleus neurons of the mammalian auditory system. When a certain click or tone is repeated time and again, the record varies, giving two spikes one time, three at another time, five the next time and one the next time. You do not get the same response in a given neuron though you can get a very reproducible average. Such a finding when extrapolated to the whole system, leads to the notion that we are basically dealing with a statistical consistency, and an individual neuron is not that consistent. Looking at some of Dr. Kennedy's records, one would wonder. They do

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not look too impressively determinant or consistent time after time, but one wonders if, looking at the right parameter, one would not find a high degree of deterministic operation, particularly when there are so few motoneurons to a given muscle in this animal. I think this is one of the really exciting issues of the time, because it is something one can work on. It is heuristic. One can design experiments which should answer the question to what degree along a spectrum a certain situation is working. Again my expectation is that we will find both extremes actually exist, with cases in between. We need "case law" and "natural history" to get a perspective on the range of nervous systems that nature presents. Closely related to this is a comment in Dr. Horridge's paper about the drift he thought must occur in the operation of units, and therefore the unknowable state of the whole system. While this makes sense in a kind of a priori way, I think we are faced also with the reality of what I will call permanent calibration. That is to say, many systems that we know are, apparently, without any feedback, permanently calibrated to a high degree and, if there is any movement at all, must have limits to the drift. For example, absolute pitch is thought of as generally quite rare, but I think a large number of us have a considerable degree of pitch discrimination which does not drift very much. We have a certain level of sugar in the blood and a level of body temperature which does not drift very much over years. What the feedbacks are that may determine this we do not know. To think of even more simple cases, the frequency of discharge of certain electric fish is predictable day after day, in the same individual, within fantastically small limits. Pacemakers are the most elementary example of a large class of nerve cell properties that are as consistent, maintained and predictable, as the identifiable neurons that Dr. Kennedy referred to in arthropods (and which are now cropping up more and more in insects, snails, Aplijsia and elsewhere). What the basis of this precision can be is another really challenging question. It also will involve perspective from a number of cases before we appreciate it properly. The last comment I would like to make I will call "Neural Codes" (6). What are the ways by which the neurons are communicating with each other? Spike discharges have already been mentioned but without reference to the range of parameters that are candidates for codes, that is, the possible ways in which neurons are influencing each other. I refer to both spike and non-spike forms of influence. Dr. Horridge said: "What really matters is connectivity pattern, not dendrite pattern." If you happen to be working in the cerebellum, you might wonder whether he has given consideration to the precision of orientation of dendrites which, because of its very existence, suggests meaning to that geometry, whereas the connectivity would not require such precision of dendrite orientation.

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One wonders whether a structure like the cerebellar cortex can really be working entirely by spike interaction and connectivity, or whether it is largely operating in an analog manner by non-spike events. This to me is a basically exciting and challenging question. What kinds of events are happening and are available for transmitting information among neurons in addition to the spike parameters and what spike parameters are doing it? The common notion that average frequency represents the information content of spike trains is probably a grossly inadequate statement of the possibilities. The recognition, definition and testing of other candidate parameters of spike trains in single channels and in parallel channels is one of great problem areas for the future and particularly concerns the interneurons as the agencies of the precise mechanisms of command and control that Dr. Kennedy has illustrated. Scheibel: Dr. Kennedy, in terms of the programmed effects obtainable from individual filaments in the connectors, you mentioned 75 per second was the usual stimulating frequency. Is this a frequency-specific effect? Kennedy: So far as we know, no, not with respect to the specific system that we are using as a test object. Atwood & Wiersma (4) have shown that, in polyvalent command elements affecting a number of motor systems, one can get activity in different members of this complex of motor elements by using different frequencies. This may simply be related to the facilitation requirements of the motoneurons involved. In our case we have been unable to demonstrate any specific effect of frequency or, in fact, of pattern. Scheibel: May I raise one other point. The concept of a group of command or elite neural elements programming the behavior of followers can be extended, one order at a time. We could then conceive of a super-elite who program the output of the elite, and so forth—leading to the ultimate absurdity of one final pontifical neuron at the apex of the pyramid of command. Do you in fact find in these systems a hierarchy of command functions or is it simply a dichotomy between populations of follower elements and smaller groups of equipotent command elements? Kennedy: We do not know of any "omnineurons", but the question of hierarchy is an important one which I can deal with briefly. I suspect that there are command elements at different levels. We have a few specific cases in which we have been able to record from two cells at a time; in these cases we know first, that A drives B; second, that A is a weak command fiber whereas B is a strong command fiber; third, that A is easier to excite from the periphery than B; and fourth, that A synaptically excites B. So we can work with short hierarchical chains and we think that this system, at least in part, is organized in that way. As to specificity and precision of motor command, I think we cannot work our way back up the watershed toward the divide to find more and more potency; rather, it is the elements that are closest to the motor side and most difficult to excite by natural sensory stimulation that are the decision elements.

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REFERENCES J. S., Muscle receptor organs in the abdomen of Homarus vulgaris and Palinurus vulgaris. Quart. J. Micr. Sei., 1951, 92: 163-200. ASANUMA, H . , and SAKATA, H . , Functional organization of a cortical efferent system examined with focal depth stimulation in cats. J. Neurophysiol., 1967, 30: 35-54. ATWOOD, H . L . , HOYLE, G . , and SMYTH, T . , JR., Mechanical and electrical responses of single innervated crab-muscle fibres. J. Physiol. (London), 1965, 180 : 449-482. ATWOOD, H . L., and WIERSMA, C. A . G., Command interneurons in the crayfish central nervous system. J. Exp. Biol, 1967, 46: 249-261. BULLOCK, T. H., The origins of patterned nervous discharge. Behaviour, 1961, 17: 48-59. , Signals and neuronal coding. In: The Neurosciences: A Study Program (G. C. Quarton, T. Melnechuk and F. O. Schmitt, Eds.). Rockefeller Univ. Press, New York, 1967 : 347-352. CORDA, M., EKLUND, G., and v. E U L E R , C., External intercostal and phrenic a motor responses to changes in respiratory load. Acta Physiol. Scand., 1965, 63 : 391-400. CORDA, M . , VON EULER, C . , and LENNERSTRAND, G . , Reflex and cerebellar influences on a and on rhythmic' and 'tonic' f activity in the intercostal muscle. J. Physiol. (London), 1966, 184: 898-923. CRITCHLOW, V . , and VON EULER, C . , Intercostal muscle spindle activity and its y motor control. J. Physiol. (London), 1963, 168 : 820-847. EVOY, W. H . , and KENNEDY, D., The central nervous organization underlying control of antagonistic muscles in the crayfish. I. Types of command fibers. J. Exp. Zool., 1967,165: 223-238. EVOY, W. H., KENNEDY, D., and WILSON, D. M., Discharge patterns of neurones supplying tonic abdominal flexor muscles in the crayfish. J. Exp. Biol., 1967, 46: 393-411. EYZAGUIRRE, C., and KUFFLER, S. W., Processes of excitation in the dendrites and in the soma of single isolated sensory nerve cells of the lobster and crayfish. /. Gen. Physiol., 1955, 39: 87-119. FIELDS, H. L., Proprioceptive control of posture in the crayfish abdomen. J. Exp. Biol., 1966, 44 : 455-468. FIELDS, H . L . , EVOY, W . H . , and KENNEDY, D . , Reflex role played by efferent control of an invertebrate stretch receptor. J. Neurophysiol., 1967, 30: 859-874. FIELDS, H . L . , and KENNEDY, D . , Functional role of muscle receptor organs in crayfish. Nature (London), 1965, 206: 1235-1237. GRAY, J., The role of peripheral sense organs during locomotion in the vertebrates. Symp. Soc. Exp. Biol., 1950,4: 112-126. HUBER, F., Untersuchungen über die Funktion des Zentralnervensystems und insbesondere des Gehirnes bei der Fortbewegung und der Lauterzeugung der Grillen. Zsehr, vergl. Physiol., 1960, 44: 60-132. HUGHES, G . M . , and TAUC, L . , An electrophysiological study of the anatomical relations of two giant nerve cells in Aplysia depilans. J. Exp. Biol., 1963, 40: 469-486.

1. ALEXANDROWICZ, 2.

3.

4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

15.

16. 17.

18.

36

19. 20.

21.

22. 23. 24.

25. 26.

27. 28. 29. 30.

31.

32. 33.

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INTERNEURON

R., FRAZIER, W . T., and COGGESHALL, R. E., Opposite synaptic actions mediated by different branches of an identifiable interneuron in Aplysia. Science, 1967, 155: 346-349. KENNEDY, D., EVOY, W . H., D A N E , B., and HANAWALT, J. T., The central nervous organization underlying control of antagonistic muscles in the crayfish. II. Coding of position by command fibers. /. Exp. Zool., 1967, 165: 239-248. KENNEDY, D., EVOY, W . H., and FIELDS, H. L., The unit basis of some crustacean reflexes. Sympos. Soc. Exp. Biol., 1966, 20: 75-109. KENNEDY, D., EVOY, W. H., and HANAWALT, J. T., Release of coordinated behavior in crayfish by single central neurons. Science, 1966, 154: 917-919. KENNEDY, D., and TAKEDA, K., Reflex control of abdominal flexor muscles in the crayfish. II. The tonic system. J. Exp. Biol, 1965, 43 : 229-246. PABST, H., and KENNEDY, D., Cutaneous mechanoreceptors influencing motor output in the crayfish abdomen. Zsehr, vergl. Physiol., 1968, 57: 190-208. PRINGLE, J. W. S., Proprioception in arthropods. In: The Cell and the Organism (J. A. Ramsay and V. B. Wigglesworth, Eds.). Cambridge Univ. Press, Cambridge, 1961: 256-282. STRUMWASSER, F., Post-synaptic inhibition and excitation produced by different branches of a single neuron and the common transmitter involved. In: XXII International Congress of Physiological Sciences, Vol. II: Abstracts of Communications (J. W. Duyff et al., Eds.). Excerpta Medica Foundation, Amsterdam, 1962: No. 801. VON HOLST, E., Die relative Koordination als Phänomen und als Methode zentralnervösen Funktionsanalyse. Ergbn. Physiol., 1939, 42: 228-306. WIERSMA, C. A. G., On the functional connections of single units in the central nervous system of the crayfish, Procambarus clarkii Girard. J. Comp. Neurol., 1958,110: 421-471. WIERSMA, C. A. G., FURSHPAN, E., and FLOREY, E., Physiological and pharmacological observations on muscle receptor organs of the crayfish, Cambarus clarkii Girard. J. Exp. Biol., 1953,30:136-150. WIERSMA, C. A. G., and HUGHES, G. M., On the functional anatomy of neuronal units in the abdominal cord of the crayfish, Procambarus clarkii Girard. /. Comp. Neurol., 1961, 116: 209-228. WIERSMA, C. A. G., and IKEDA, K., Interneurons commanding swimmeret movements in the crayfish, Procambarus clarki (Girard). Comp. Biochem. Physiol., 1964, 12 : 509-525. WILLOWS, A. O. D., Behavioral acts elicited by stimulation of single, identifiable brain cells. Science, 1967, 157: 570-574. WILSON, D. M., The origin of the flight-motor command in grasshoppers. In: Neural Theory and Modeling (R. F. Reiss, Ed.). Stanford Univ. Press, Stanford, 1964: 331-345. KANDEL, E .

EXCITATORY AND INHIBITORY PROCESSES L. TAUC Centre d'Études de Physiologie Nerveuse Centre National de la Recherche Scientifique Paris, France

The designation of a neuron as an interneuron indicates that this latter is situated functionally in between other neuronal structures with which it interacts. There is still a clear tendency to consider this interaction to be synaptic in nature, correlated to a specific anatomical structure and acting by modifying the excitability of the postsynaptic neuron, either in an excitatory or in an inhibitory direction. It is admitted that the efficacy of the synaptic action can often be increased by post-tetanic potentiation or decreased in more limited structures by presynaptic inhibition. If we add the possibility of an electrical junction, these phenomena represent practically all the possibilities which have been considered by neurophysiologists and anatomists when proposing schemata of possible connectivities to explain a response of a given neuronal chain. It appears, however, that the modalities of neuronal interaction seem to be even more numerous than those enumerated above and include as well such factors as the site of action. This suggestion comes from experiments made in the central nervous system of the marine mollusc Aplysia, which combines the functional complexity of a brain with a relative simplicity in neuronal organization and where, consequently, it is possible to analyze the processes of transmission in rigorously controllable conditions in single identifiable neurons. These observations have led to the description of some new mechanisms of neuronal interaction which, besides their intrinsic interest, should be taken into account if functional connections in a group of neurons are being considered. The following is a short review of modalities of interaction limited to the central ganglia of Aplysia but which, in fact, covers most of all possible aspects of neuronal interaction known up to this date. These different modalities are summarized in Figure 3. The observed phenomena of transmission in respect to their possible mechanisms and (or) site of action have been divided in three groups, designed as synaptic, biphasic and episynaptic. The synaptic and biphasic interaction is fully developed in between two neurons, the episynaptic effects need a three-neuron 37

THE

38 TYPE

ELECTRICAL MANIFESTATION

5YNA PTIC

EP5P facilitotion

INTERNEURON

DURATION TRANSMITTER UNITARY ACh 30-500ms

unknown unknown ACh

IP5P

I !>

B I P H ASIC

inhibition

ELECTRICAL P5P Facilitation

PERMEABILITY CHANGES

unknown

30 - 5 0 0 m s

membrane constant

0

I C I ' , Na + I Cl', ( K + )

C a ~ M g + DYNAMIC PROPERTIES Ca+>

DURATION

M g * /primary focilit'

Ca + + < M g " |post-tetanic pot| + / * habituation" +

K+,(Na+)

/primary facilit' PTP

0 rectification

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0

| I i I l i i i l i i

¡LD (inhibition of longue duration) type DILDA e x c < 3 0 0 m s mhRighl ' /connect

\ \ \ \

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,Branchiol

' genital nerve

Abdominal gangl.a

a new powerful mechanism of neuronal interaction. As in presynaptic inhibition, the degree of facilitation, the delay of maximal effect and the maximal duration of the facilitation differ for inputs to the same cell. Also these parameters can be different for the same cell situated in different preparations. A diminution of the efficacy of the priming pathway to produce presynaptic facilitations, was also observed when the mechanism was repetitively activated. Mechanism of Episynaptic

Action

There is little or no direct evidence concerning the functioning of episynapses. It is, however, evident from the duration of the phenomena that a chemical mediator is involved, liberated from pre-episynaptic structures. The disparity in time of the synaptic and episynaptic actions can be attributed to a prolonged effect of a specific transmitter, rather than to the repetitive firing of the pre-episynaptic neuron, although both factors could be relevant, at least in the early phase of the process. The dependency of the recovery time of the episynaptic action on the intensity for the priming stimulus suggests that the transmitter is not destroyed or removed in a fixed period of time, as in a classical synapse; possibly the enzymatic action on the episynapse is weak or absent. In these respects, the episynaptic mechanism is reminiscent of the action of the transmitter responsible for inhibition of long duration ( I L D ) .

52

TIIE

1

A 7

INTEHNEURON

RGC

r 4

2

.

3

• f—••———^

"

1min. 5

6

1 (

2mV. 1mVI

B

2,5min. |

5,5min.

1I

2l

'

25 min. 3

Figure 14. Heterosynaptic facilitation of the EPSP in the light giant cell caused by stimulation of a piece of skin during block of the left connective. Responses in the right and left giaut cells (RGC and L G C ) to the stimulation of the left tentacular nerve are shown before ( A l ) and during ( A 2 ) the block. Before removing the block, the priming stimulation was administered. Records A3-A6 show the heterosynaptic facilitation thus produced on the EPSP in the right giant cell, reaching its peak 2.5 min. after the priming stimulation. No effect is seen when block is applied without administering priming stimulation ( B ) . In records A l , A3 and B l , because of a partial sucrose block causing a decreased conduction velocity, the latency of the EPSP in the right giant cell is increased, thus lengthening the normally constant interval between the two EPSPs. (From Tanc & Epstein, 40.)

Furthermore, the decrease in ability of the priming pathway to produce presynaptic effects suggests that there is an exhaustion of the episynaptic transmitter reserve, unless a phenomenon similar to desensitization takes place. This process might also be similar to events on contacts producing ILD, which also show a decrease in intensity and duration upon repetition after recovery ( 3 7 ) . The modifications produced in different presynaptic fibers by the same priming stimulus vary in magnitude and delay. Such variations might result from different sensibilities of different fibers to the episynaptic transmitter and ( o r ) from structural characteristics, such as differences in distance between the pre-episynaptie and post-episvnaptie membranes. Consequently for some afferents a summation of episvnaptic activity is necessary to produce episynaptic effects. Here again the similarity to ILD mechanisms can be evoked.

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The similarities between presynaptic inhibition and facilitation suggest that the same mechanism of episynaptic action is responsible for both presynaptic phenomena. Their opposite effects might be due to different episynaptic transmitters or to specificities of receptors of the post-episynaptic membrane, or to both of these phenomena. It is indeed significant that cells showing presynaptic inhibition and facilitation belong to two different pharmacological groups. The giant cells are of the H type (43), whereas the cells showing presynaptic inhibition of their excitatory inputs have been demonstrated to belong to the CILDA type (36). The excitatory inputs to these two types of cells are also pharmacologically different. On the other hand, the episynaptic phenomena are not specific to excitatory inputs; we have to remember that presynaptic inhibition has also been seen on inhibitory inputs (31, 32). Thus presynaptic inhibition and facilitation are mediated by different fibers synapsing on different cells; and, in a cell, all EPSPs of the same type show either presynaptic facilitation or inhibition but never both. The nature of the modification which the activation of episynapsis induces in post-episynaptic structures is unknown. It could be a polarization change active on the site of the pre-episynaptic spike and thus on the quantity of synaptic transmitter released; but other mechanisms can be proposed that would exert a more direct action on release of the synaptic transmitter through a biochemical or, more simply, an ionic influence. The physiological role of episynaptic phenomena can only be guessed. Both presynaptic inhibition and facilitation represent powerful mechanisms that permit the temporary modifications of synaptic efficacy in a limited number of neurons. Other related modifications are disclosed by the decreasing effectiveness of the conditioning pathway to produce presynaptic modifications of the test PSP. These modifications of the presynaptic mechanisms can extend over several hours and are of much longer duration than the direct presynaptic facilitatory or inhibitory action. In this respect it might be suggested that the presynaptic phenomena are involved in longlasting modifications of the properties of the CNS, although this affirmation is of little value. But it is significant that both presynaptic phenomena occur under normal physiological conditions. Depression of a test EPSP has been observed to result from a spontaneous activation of interneurons in the absence of any conditioning stimulation (36). Presynaptic facilitation in the giant cells has been produced with physiological conditioning stimuli in the partially isolated CNS (6, 7, 40) or even in the whole animal preparation (24). CONCLUDING REMARKS

The classical and non-classical modalities of neuronal interaction, described in the central ganglia of Aplysia, incite some reflections. With the exception of electrical synapses, all other interaction is transmitted by a

54

THE

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chemical mediator. But it is not necessarily the identity of this transmitter which determines the nature of the modification in the receptive neurons; the decision devolves also on the nature of receptors in these neurons and also, more prosaically, on their ionic content. This is clear from observations on D and H neurons and especially on HILDA cells, where the same transmitter, presumably acetylcholine, combines on the same cell with two different postsynaptic receptors, each of them then opening, simultaneously but not for the same duration, channels for different ions. It is possible that a similar mechanism using a different transmitter, is responsible for inhibition of long duration in DILDA cells. But a hypothesis which would postulate the release of two transmitters from the same presynaptic neuron, can also be considered with some favor, not only as a mechanism producing some biphasic effects, but in respect to presynaptic actions. It was indeed observed that spontaneously appearing presynaptic inhibition is preceded, in the postsynaptic cell, by direct synaptic-like effects having kinetics completely different from presynaptic inhibition (36); two tentative explanations can be proposed. Both will consider the presence of two different specific receptors, one type on the postsynaptic neuron and another type on the presynaptic test pathway, but they will either both combine with the same transmitter released from the conditioning neuron or, in the second alternative, with two different transmitters released by the conditioning neuron. The "two transmitter hypothesis" can find some support in the observation that some neuronal endings in gastropods possess more than one kind of vesicle, usually considered as storage structures for transmitter substances (13). It seems probable, that both mechanisms, liberation of more than one transmitter type from the same neuron and presence of different receptors to the same transmitter on the receptive neurons, are used in the gastropod central nervous system. But, so far, only the second hypothesis has received a direct experimental basis. It is generally considered that neuronal interaction is necessarily related to a specific anatomical structure or synapse, having a specific ultrastructure. Some aspects of transmission concerning episynaptic effects and ILD in molluscan CNS, however, point to a possibility of a particular kind of interaction, which does not seem to be conveyed by synapse-like structures as defined by a rigid anatomical consideration. The corresponding observations are: ILD and presynaptic facilitation and inhibition appear in different cells with a considerable difference in delay (attaining sometimes several tens of seconds) and intensity, a shorter delay being associated with a more intense effect. They both show cumulative properties, especially increase in duration related to the number of initial responses, and appear in delimited cell groups and affect, presynaptically, all inputs of a given type. Moreover, the conductance changes produced by the ILD inhibitory phase, as recorded in the soma, are so important in some cells that they must be due to modifications affecting a large membrane surface, apparently larger

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than that covered by synapses, which in fact are very scarce according to anatomical observations. All these properties can easily be explained by admitting an extrasynaptic release of a transmitter substance, possibly in the absence of a specific anatomical configuration (episynapse in case of presynaptic effects). The transmitter could then reach by diffusion both proximal and less proximal receptive fields of many neurons, without being limited in its action to only one postsynaptic neuron. The possible objections, concerning the specificity of action, extrasynaptic distribution of receptors and the very long duration of effects in a system open to diffusion, can easily be discarded. a. Specificity of action: It is evident that only cells having suitable receptors will be affected by this particular activity, whereas other neurons deprived of adequate receptors will not be influenced. In fact, ILD and presynaptic phenomena appear only on structures with very well-defined pharmacological properties. b. Distribution of receptors: The extrapolation of the work on the neuromuscular junction has given to neurophysiologists the feeling that the synaptic receptors have to be localized on the synapse. The studies on cholinergic transmission in Mollusca have clearly demonstrated that in ganglion cells the localization of cholinoceptive receptors is not limited to the subsynaptic membrane but that these receptors are distributed on the cell body completely deprived of synapses, and that here their distribution occurs with the same concentration as at the synapses (12, 43). Consequently, there is no necessity for a rigid anatomical correlate to insure neuronal chemoception. c. Duration of effect: In fact, there is no contradiction between the duration of a transmission process and the absence of synapses, such duration probably results from the stability of the combination transmitter-receptor. In HILDA cells, the second inhibitory phase produced by a single injection of ACh can last for minutes, and this is an open membrane and in the presence of a powerful destructive enzyme, acetylcholinesterase. Thus a substance liberated extrasynaptically might be able, through diffusion in the intracellular space, and before being destroyed by enzymes if present, to come in contact with several neurons and combine with adequate receptors on those neurons which have them. The time of diffusion would determine the delay of action, and the duration of effect would depend on the properties of the receptors involved and from the quantity of transmitter which reached a given receptor field. This dependence of duration on the quantity of transmitter was indeed observed in the case of ILD. The possibility of extrasynaptic liberation of the transmitter raises doubt about the absolute interpretation of neuronal connectivities as studied by ultramicroscopio methods, and the justification for taking synaptic "active zones" as unique indication of transmission processes. The objection that, in vertebrates, no extrasynaptic liberation of transmit-

56

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ter has so far been proposed is not really serious; the experimental conditions in central vertebrate structures are difficult and do not permit the analysis of elementary phenomena with such security and depth as is possible in Aplysia. Moreover, ILD and episynaptic effects, even in Aplysia, are the properties of a limited number of neurons, whereas synaptic interaction is a general property. Thus isolation of mechanisms different from synaptic transmission could very well have escaped observation, especially as these have mostly been conducted with the conviction that synapses are the unique site of neuronal interaction. In this respect, the remarkable early work of anatomists, using light microscopy, is possibly of permanent importance, as it is limited in showing only where the extremities of neurons extend without compromising conclusions about the nature of neuronal interaction. It is thus evident that, in studying connectivities, the anatomist necessarily needs the help of a physiologist, as there is no general anatomical distinction between inhibitory and excitatory synapses and there is until now no defined anatomical correlate of the episynaptic interaction. On the other hand, only thorough anatomical study in correlation with physiological results will one be able to confirm or deny the concept of extrasynaptic neuronal interaction. Maynard* I shall comment on three topics which are relevant to the two preceding papers. Let me first refer to the old problem of the time element in the nervous system. For a long time now, we have been faced with the situation in which the most obvious and reproducible electrical activity of single neurons—the action potential for example—is of relatively brief duration whereas much of the activity in which the central nervous system as a whole indulges has a time course greater by several orders of magnitude. Many of the attempts to explain long-lasting effects in the vertebrates—and to a lesser extent in other forms—have been in terms of multicellular circuits and interactions which can prolong the traditionally brief unitary activity. Such approaches which combine discrete units in various logical patterns have a certain elegance and intellectual appeal; in many instances they are probably even correct. In the past few years, however, most of us have begun to suspect that the neuron is often not such a pure unit as we might hope (8); that it may be relatively sloppy and be able, not only to produce a variety of responses on occasion, but to distribute its responses differentially among its various processes. Thus the possibility that long-lasting responses may result from intrinsic activity cycles or active responses having long time-courses within single neurons must be seriously considered. Examples of wide variation in the time-course of action potentials from different animals or tissues are legion. The case of the squid axon with an action potential of about 1 msec. • Original work reported here was supported in part by U. S. Public Health Service Research Grants NB-03271 and NB-06017.

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and that of the giant cell of another mollusc, Aplysia, with an action potential ranging from 10 msec, up to 50 msec, or more (30), is particularly apt. More to the point, however, is the increasing number of preparations in which evidence for combinations of long and short duration responses in single elements is relatively good. Perhaps the best known is that of the parabolic burster in Aplysia ( 30 ). This giant neuron, in addition to action potentials lasting about 20-50 msec., shows two apparently intrinsic slow rhythms. One has a period of tens of seconds, the other a period of about 24 hours, or about 10°-107 times the duration of the single spike. A less dramatic range of time-courses has been recently documented in the pacemaker neurons of the cardiac ganglion in certain species of stomatopods (Crustacea) (44, 45). These neurons apparently produce active, slow depolarizations which last hundreds of milliseconds and drive bursts of spikes. The occurrence of a phenomenon in a few special cases, however, does not insure its generality. Nor does the possession of the potentiality for a given kind of response by a neuron assure its utilization in the normal course of events. Although technical difficulties are great, and the accumulation of data may be relatively slow, it will be very interesting to learn the extent of intrinsic slow activity among interneurons of complex central nervous systems in all three phyla, Chordata, Mollusca, and Arthropoda. Perhaps many of the slower responses of the entire organism will prove to be controlled by groups of elements with similarly slow intrinsic responses. Certainly, any final understanding of the functional meaning of interneuron networks will require much fuller knowledge about the time courses of intrinsic activity open to the individual neurons of the network. > My remaining comments will be confined to a brief discussion of two sets of findings on crustacean systems. Unlike Dr. Kennedy, who has concentrated on the derrière of the crayfish, we have looked into the neuropile of the brain and the ganglion of the stomach. Much of our work has involved intracellular recording from processes within the neuropile, relatively near sites of synaptic activity, rather than from the more distant cell bodies. This has particular virtue when the neuropile is well-differentiated as in the brain of the spiny lobster. There is now extensive evidence for presynaptic modulation in the central nervous systems of vertebrates and molluscs, and in the peripheral neuromuscular junction in crayfish (1, 11, 39). I wish to extënd these observations to an example of "presynaptic modulation" in the lobster brain. Figure 15 is a diagram of the anterior quarter of the brain of a spiny lobster ( Panulirus argus) as viewed from the midline. The various lobes need not concern us here, but the antennular nerve is important. It carries motor and sensory fibers to and from the antennule. Among the sensory fibers are some of large diameter (10-20 n in fixed material) which probably originate in various antennular mechanoreceptors and pass deep into the brain before terminal ramifications. It is possible to place microelectrodes within these

58

THE

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Figure 15. Diagram of anterior right quarter of lobster brain (see insert) showing course of the deep sensory fiber tract. The microelectrode is in position to record from one of these sensory fibers near the central terminal arborizations. The dendritic ramifications of a motor neuron in the parolfactory lobe ( P o L ) , and the course of the motor axon in the brain and antennular nerve is also diagrammed. AL: Accessory lobe; aln: antennular nerve; an: antennal nerve; OL: olfactory lobe. Arrows indicate direction of orthodromic transmission in motor and sensory fibers.

sensory fibers near their terminations, and to record changes in excitability and membrane potential when neighboring fibers are active. Figure 16 is a paraffin section of the lobster brain cut across the path of the recording microelectrode after a typical experiment. The electrode path passes through the tract of large sensory fibers from the antennule diagrammed in Figure 15. Corrected depth measurements made at the time of the experiment in conjunction with physiological landmarks during penetration indicate that the electrode tip was within a fiber of this tract when the following recordings were made. This locus is a short distance from the first terminal branchpoints. Figure 17 presents records taken from such a preparation. The antennular nerve was divided into bundles in the periphery; these could be stimulated independently. Current could be passed, via a bridge circuit, through the intracellular recording electrode. Adequate stimulation of the appropriate an-

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Figure 16. Frontal section of lobster brain normal to microelectrode path at presumed level of recording. Arrow points to electrode path in the sensory tract (S) at the inner margin of the parolfactory lobe ( P o L ) . AL: Accessory lobe; M: tract of motor axons just before branching to send dendritic arborizations into PoL. Paraffin section, Masson's trichrome stain. Calibration, 100 p..

tennular nerve bundle elicited an antidromic impulse followed by a prolonged phase of depolarization in the penetrated sensory fiber (Figure 1 7 a l ) . On the other hand, depolarizing current passed through the recording electrode elicited a repetitive spike discharge that must originate near the central terminations of the sensory fiber (Figure 17a2). When peripheral and central stimulation are combined (Figure 17a3) the repetitive discharge resulting from the central depolarization is depressed by the arrival of a volley in the sensory nerve, and there is the suggestion of prolonged hyperpolarization underlying the depression. The extent of such depression is a function of the number of elements active during the input volley, and is totally unrelated to the presence or absence of an impulse in the penetrated fiber. Thus the depression of the sensory terminal cannot represent the effects of an afterpotential, but must be the direct or indirect result of activity in neighboring sensory fibers from the antennule. This is shown more clearly, even with a weaker response, if the input volley arrives over an antennular nerve bundle which does not include the axon of the penetrated unit (Figure 17b). In such a case, the late depolarizing phase of Figure 17 is resolved as a depolarizing post-synaptic potential (PSP, Figure 1 7 b l ) whose time course of decay parallels the depressant effect on the repetitive discharge (Figure 17b3).

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.b!

si

Figure 17. Intracellular records from near central arborizations of sensory axon from antennule. al, cl: Response to supramaximal stimulation of inner bundle of antennular nerve containing axon of penetrated element, bl, dl: Response to supramaximal stimulation of outer bundle of antennular nerve. a2, b2: Repetitive response originating in central region of sensory fiber in response to depolarizing current (5 X 10~9 Amp.) applied through recording electrode. a3: Response to combined stimuli; volley in inner bundle depresses centrally initiated repetitive discharge; note initial depolarizing phase (arrow), h-3: Response to combined stimuli; the effects of a volley in the lateral bundle are not obvious, but do include a slight reduction in the interval between the first and second spike of the repetitive discharge (excitation), and subsequent increase in intervals between spikes 2, 3, and 4 (inhibition); vertical marks above peaks of spikes indicate positions of control spikes. c2: Response to stimulation of inner bundle during hyperpolarization (6.3 X 10 ° Amp.) of central ramifications; note increased amplitude of late depolarization (bridge slightly unbalanced to keep response on screen of CRO). d2: As above, but stimulating outer or lateral bundle. Dots (•) below records indicate instant of peripheral stimulus to antennular nerve bundles. Calibrations; 20 mV, 10 msec.

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Depression is not the sole effect of the input volley, however. There is also a brief initial excitation that is normally represented by a slight depolarization (Figure I7a3, arrow). In Figure 17b3 this depolarization accelerates the second spike of the repetitive discharge. In other preparations the sign of the initial excitatory phase may be more obvious and reach amplitudes of nearly 10 mV. Unlike the later portion of the PSP recorded at normal membrane potentials, the early phase does not significantly decrease with nominal depolarizing current. In Figure 17, c and d show the effects of applied hyperpolarizing current on the amplitude of the late depolarization in Figure 17al and the PSP in Figure 17bl. In each case the amplitude increased significantly, suggesting that the excitability changes, obvious in Figure 17a3 and b3, are mediated by changes in membrane conductance near the electrode site, not by external field effects. If all the data are considered, the following interpretation seems to account for most observations: The sensory fiber terminations receive synaptic input from two kinds of presynaptic element, inhibitory and excitatory. Under the conditions of the experiment, excitatory synapses become active slightly before the inhibitory elements, but the latter are dominant, and the final net result of both inputs is depression of the excitability of the sensory terminal. Observed potential changes are reasonable if the equilibrium potential of conductance changes caused by the inhibitory transmitter lies close to or just below the resting potential of the preparation. Since depolarization of the sensory terminal is associated with presynaptic inhibition in vertebrates (1, 11), we may presume that variations in membrane potential and excitability at sensory "terminals", as described here, similarly modulate synaptic transmission from the sensory fiber to its postsynaptic element. The postsensory neuron, or first order interneuron of this system, has, however, not been identified so we cannot say whether the depolarizing or the hyperpolarizing PSP at the sensory terminal depresses—or stimulates—transmitter release and synaptic transmission in this preparation. Nevertheless, as far as I know, this is the first reasonably clear and direct evidence for both inhibitory and excitatory synapses on the central processes of a single sensory fiber. It suggests that modulation of sensory input in the terminal arborizations may have more possible variations than thus far recognized. My third topic, and second set of experimental results, relates to the papers of both Drs. Kennedy and Tauc. First, it represents an example of the kind of executive system which executes the orders of command fibers, such as those discussed by Dr. Kennedy, by producing an integrated motor output pattern. Second, it is an example of the way in which a variety of neuron properties and synaptic processes, such as those discussed by Dr. Tauc, are utilized to produce an output pattern in a multineuron system. Specifically, I shall examine the kinds of interaction found in a group of

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Figure 18. Diagram of stomatogastric ganglion of Scylla serrata. a: Tracing of experimental preparation showing location of cell bodies of 27 of 30-35 ganglion neurons, the two A-neurons giving large spikes in the periphery are shaded ( A ) ; the B-neuron is stippled ( B ) ; command fibers enter the ganglion neuropile ( N ) via the stomatogastric nerve (nsg); motor axons from the ganglion neurons leave via the dorsal ventricular nerve ( n d v ) ; SG: stomatogastric ganglion, b: Diagram of experimental arrangement with isolated ganglion; Re: external recording electrodes placed on lateral nerve, a branch of the dorsal ventricular nerve; Ri: microelectrode inserted into cell bodies of selected ganglion cells in desheathed ganglion; S: external stimulating electrode placed on stomatogastric nerve.

9-10 stomatogastric ganglion neurons which lead to a repeating, rhythmic, triphasic, sequential output pattern. The experiments were performed about a year ago aboard the R/V ALPHA H E L I X * in collaboration with Dr. Liam Burke of the University of Sidney (28, 2 9 ) . The stomatogastric ganglion of the Australian mud crab, Scylla serrata, contains 30-35 monopolar neurons. These seem to be motor elements; the single process of each cell sends collaterals into the ganglion neuropile, and then divides to innervate one or more of the 30-40 symmetrical gastric muscles on either side of the stomach. The activity of the ganglion is controlled by interneurons or command fibers which originate in the brain or ventral ganglia. Direct sensory input to the ganglion has not been described as yet, though it may occur. Figure 18 is a sketch of the ganglion and a diagram of the preparation. The monopolar cell bodies are located in a single layer in a crescent about the central neuropile. Terminating command fibers enter anteriorly via the stomatogastric nerve. Motor fibers from the ganglion cells pass out to stomach muscles in small lateral nerves and primarily via the posterior dorsal * A portion of the work presented in this paper was done aboard the Research Vessel A L P H A H E L I X of the Scripps Institute for Oceanography, University of California, with support from the National Science Foundation, Grant #GB-5916.

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A

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J L Figure 19. Activity patterns of A-neurons of stomatogastric ganglion, a: Extracellular recording from lateral branch of dorsal ventricular nerve, in vivo; similar activity was obtained from isolated ganglia; note repeating cycle of two large spikes from Aneurons discharging in a burst (A), the single B-neuron discharging repetitively ( B ) , and the several small neurons firing between B and A (s). b: Intracellular recording from a single A-neuron, "spontaneous" bursting; arrow points to single IPSP evoked by spike in B-neuron. c: Simultaneous intracellular recording from two A-neurons (see Figure 1 8 ) ; repetitive stimulation at about 6/sec. of stomatogastric nerve evokes EPSP in both elements; arrows indicate IPSP resulting from B-neuron discharges, d: Simultaneous recording from extracellular electrodes on lateral branch and from microelectrode within A-neuron; note depolarizing "PSP" following spike discharge of other, unpenetrated A-neuron (dotted spikes in extracellular recording). Time calibration of 0.5 sec. in record b applies to a, b, and c. Time calibration of record d is also 0.5 sec. Voltage calibration for b, c, and d in record d is 10 mV. No voltage calibration for record a.

ventricular nerve. The ganglion, with 1-2 cm of the stomatogastric and dorsal ventricular nerves attached, was isolated and placed in a recording chamber. Primary output from the ganglion was monitored by extracellular electrodes on the post-ganglionic ventricular nerve. Intracellular records were taken by microelectrodes inserted into selected cell bodies in the ganglion. Stimulating electrodes on the stomatogastric nerve provided input via command fiber volleys as required. Resting potentials of ganglion neurons were modified via a second, current microelectrode inserted into the cell, or via the recording electrode and a bridge circuit. Figure 19a illustrates a typical output from the ganglion recorded from the dorsal ventricular nerve. The characteristic pattern begins with a burst of repetitive spikes originating in two neurons (A-burst). After a silent period of 50-100 msec, a single fiber discharges several times (B-burst). Finally, a group of about five neurons having relatively small action potentials begin to discharge as the B-neuron stops and continue (s-burst) until the

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next A-burst occurs. The pattern forms a cycling sequence of three different groups of neurons; A,B,s, A,B,s, A, . . . It is much like the patterns which commonly underlie rhythmic behavior associated with locomotion and respiration. When the microelectrode is placed within one of the neurons active during the A-burst (Figure 19, b-d), we find that the spike discharge arises from a slow potential cycle in the ganglion. After a slight hyperpolarizing phase which follows strong bursts, the membrane potential gradually depolarizes until it reaches spike threshold. Spikes then occur repetitively, and both spike frequency and membrane depolarization increase exponentially to a maximum and then abruptly decline. Postsynaptic potentials are not obvious, but depolarizing EPSPs can be recognized during the burst (Figure 19d) following spikes in other A-neurons and throughout the entire cycle when command fibers are stimulated repetitively (Figure 19c). Hyperpolarizing IPSPs following B-neuron spikes, occur after the burst in many but not all A-neurons (Figure 19, b, c ) . The slow potential oscillation, however, does not simply reflect external synaptic input distribution. When two Aneurons are penetrated simultaneously (Figure 19c) they exhibit rough synchronization of both slow potential and spike discharge. The A-neurons, of which there are at least four, two with large obvious impulses (Figure 19 a, d ) and two with small impulses, seem to form a coherent functional group. Figure 20 shows that the A-neurons are electrically coupled. Depolarization of one (Figure 20a, upper trace) produces a maintained depolarization in another. Hyperpolarization effects are symmetrical (Figure 20b). The attenuation factor for a maintained potential change between two A-neurons ranges from two to eight. Coupling is sufficiently strong so that spike potentials in one element appear as depolarizing PSPs in the other. Quantitative calculations indicate that all interaction between A-neurons can be explained by electrical connections only; chemical synapses are not required. As suggested from Figure 20, changing the membrane potential of one Aneuron alters the output pattern of the entire ganglion, including B- and sneuron discharges. The A-neurons, therefore, act as the pacemakers for the entire patterned sequence. The exact form of the A-burst seems to be determined both by connectivity pattern among A-neurons and by intrinsic properties of individual A-neurons. One must now ask what kinds of connections and feedback are there with the other cells involved in the pattern sequence, the B-neuron and the s-neurons. Intracellular recordings by the single B-neuron show that it is under continuous bombardment with IPSPs except immediately following the Aburst (Figure 21a). The background IPSPs (see particularly, Figure 21d) are produced by s-neuron discharge. During the A-burst, there is additional increased hyperpolarization of impressive dimensions. The B-neuron recovers during the following period in which IPSPs are absent, and in many in-

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Figure 20. Simultaneous intracellular recording from two A-neurons. a: Depolarizing current (3 X 10 9 Amp.) passed through recording electrode in cell of upper trace (bridge unbalanced) spreads to cell of lower trace; spikes in upper cell elicited by depolarization appear as PSP in lower cell; lower cell discharges once near end of second period of depolarization, h: Hyperpolarizing current (5 X 10_!> Amp.) also spreads, indicating absence of rectification of electrical junction; note afterdischarge following termination of hyperpolarizing current; spikes in upper cell in record b are attenuated. Voltage calibrations: 5 mV; time calibration: 1.0 sec.

stances there is sufficient post-inhibitory rebound to evoke spikes (Figure 21, b, c ) , the B-burst. The silent period following the A-burst is thus a measure of the recovery time of the B-neuron. As indicated above, the Bneuron makes inhibitory connections with some of the A-neurons. Both the IPSPs resulting from s-neuron impulses and the greater hyperpolarization occurring during A-bursts are diminished by hyperpolarization and augmented by depolarization of the B-neuron. Single A-neuron impulses do not produce recognizable IPSP however, and only occasionally can the slight, long-lasting hyperpolarizations illustrated in Figure 21d be detected. It is possible that the effects of A-neuron activity on the B-neuron are mediated by an intermediate element. There is no evidence of electrical coupling between the A-neurons and the B-neuron. No s-neurons were penetrated, so it is impossible to describe the detailed connections between the s-neurons themselves, or the nature of the A-neuron—s-neuron connection. Since s-neurons cease to fire as the A-neuron burst progresses, it is likely that A-neurons inhibit s-neurons in some manner. If activity in s-neurons acts on the A-neurons, it must do so by some mechanism which does not involve PSP detectable in the cell body. I have omitted many details, but the general pattern as diagrammed in Figure 22 seems reasonably clear. The pacemakers of the entire pattern sequence are the A-neurons. These elements are coupled together in a positive feedback network, and possibly because of this coupling, discharge together in brief high-frequency bursts of impulses. A-neuron activity in turn feeds— probably indirectly—into the B-neuron and the s-neurons, inhibiting both.

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Figure 21. Activity patterns of B-neuron of stomatogastric ganglion. A: Simultaneous recording from extracellular electrodes on lateral branch and microelectrode within B-neuron (see Figure 1 8 ) ; background of IPSP corresponds with s-neuron discharge, and large hyperpolarization is roughly synchronous with A-neuron burst; immediately after A-neuron burst, there is no indication of PSP; B-neuron did not spike in this record. B: Recording as above but at faster speed; two B-neuron action potentials follow A-burst, and are evident in external recording; a single stimulus to command fibers precedes the A-burst, note the manner by which IPSP background maintains B-neuron below firing level. C: Simultaneous intracellular recording of A-neuron and B-neuron; in this instance the B-neuron did not make inhibitory connections with the A-neuron; two stimuli to command fibers occur at beginning of record; command fibers elicit an IPSP—probably indirectly—in B-neuron. D: Simultaneous intracellular recording of A-neuron and B-neurons; PSPs on A-neuron record indicate discharges in other, unpenetrated A-neuron; these are associated with a slight, long-lasting hyperpolarization in B-neuron; the background IPSP resulting from s-neuron discharge provides a point of comparison for amplitude and time-course of this slow response. Voltage calibrations: 5 mV; time calibration: 1 sec. for record A, 0.5 sec. for records B and C, 2 sec. for record D. T h e i n h i b i t i o n o f t h e s - n e u r o n s o u t l a s t s t h a t of t h e B - n e u r o n , a l l o w i n g t h e l a t t e r to r e c o v e r t o p r o d u c e p o s t - i n h i b i t o r y r e b o u n d d i s c h a r g e s . U p o n s - n e u r o n r e c o v e r y , h o w e v e r , t h e B - n e u r o n is a g a i n p a r t i a l l y i n h i b i t e d , c u t t i n g off its a f t e r d i s c h a r g e . T h e s - n e u r o n s r e m a i n a c t i v e u n t i l t h e A - n e u r o n s a g a i n b e g i n t o fire, a n d t h e c y c l e r e p e a t s . T h e o n l y f e e d b a c k to t h e A - n e u r o n s f o u n d t h u s f a r is t h e i n h i b i t o r y e f f e c t o f t h e B - n e u r o n . T h i s , h o w e v e r , does n o t s e e m e s s e n t i a l f o r t h e o u t p u t s e q u e n c e , for it is o f t e n m i s s i n g a n d w h e n p r e s e n t , o c c u r s l o n g a f t e r A - n e u r o n d i s c h a r g e h a s s t o p p e d . P e r h a p s it is i m p o r t a n t in o t h e r p a t t e r n s of a c t i v i t y , or p e r h a p s it a c t s to p r e v e n t p r e m a t u r e A-neuron bursts.

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junctions. Diagram of electrical activity recorded in cell bodies shows slow potentials and spikes, and IPSP from B-neuron in one element. The A-neurons make inhibitory connections, possibly through a hypothetical intermediate ( h ) , with the B-neuron. The B-neuron makes direct inhibitory connections with the A-neuron network. Diagram of B-neuron activity shows IPSP from s-neurons, slower inhibition from A-burst, and postinhibitory rebound discharge. T h e A-neurons also make inhibitory connections with s-neurons, but recovery from such inhibition is slower than that of the B-neuron, permitting time for rebound activation. The s-neurons make direct inhibitory connections with the B-neuron. The number of s-neurons is uncertain, but is probably greater than four; s-neurons were not penetrated, but diagram of activity indicates hypothetical time-course of inhibition. The output of all three cell types passes out to the stomach muscles through the dorsal ventricular nerve. A diagram of the spike discharge pattern recorded in that nerve is shown. To the left are diagrammed equivalent two-element circuits. A-neuron to A-neuron forms a positive excitatory feedback; Aneuron to h-element to B-neuron to A-neuron forms a positive inhibitory feedback, or flip-flop circuit; A-neuron to h-element to s-neuron to B-neuron to A-neuron forms a negative feedback if it can be assumed that inhibition of s-neurons is equivalent to excitation of the B-neuron—the observed responses of the ganglion indicate that this assumption is justified.

To summarize: these nine or ten cells of the stomatogastric ganglion can produce, because of the specific pattern and nature of their connections, a temporally complex, sequential pattern when sufficiently stimulated by nonspecific input from command fibers. The details of interaction are of interest because they give biological meaning to the variety of interactions and postsynaptic responses of which neurons are capable, as Dr. Tauc described, and show how specific properties may play an essential role, in conjunction with the circuit design, in producing given output patterns. It is also important to note that although the observed output of the system seems to be a function of the whole system, nevertheless individual elements whether A-, B-, or sneurons may be removed without basically altering the cyclic pattern in the

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remaining elements; it is a strongly conservative system. Finally, those interested in control systems may also observe that within this limited group of elements there is positive feedback ( A-neuron to A-neuron ), negative feedback (A-neuron, s-neuron, B-neuron circuit), and a flip-flop (A-neuron to B-neuron). REFERENCES 1. ANDERSEN, P., ECCLES, J. C., and SCHMIDT, R. F., Presynaptic inhibition in the

2.

cuneate nucleus. Nature (London), 1962, 194 : 741-743. P., GLOWINSKY, J., TAUC, L., and TAXI, J., Incorporation and libera-

ASCHER,

tion of 3 H- Serotonine from molluscan tissues. Acta Pharmacol. (Kobenhavn), 1968, in press. 3. ASCHER, P., KEHOE, J. S., and TAUC, L., Effects d'injections électrophorétiques

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de dopamine sur les neurones d'Aplysie. J. Physiol. (Paris), 1967, 59: 331-332. BIEDEBACH, M., MEUNIER, J. M., and TAUC, L., Bases ioniques du potentiel postsynaptique biphasique chez l'Aplysie. J. Physiol. (Paris), 1968, in press. BRUNER, J., and TAUC, L., La plasticité synaptique impliquée dans le processus d'habituation chez l'Aplysie. J. Physiol. (Paris), 1965, 57: 230-231. , Long-lasting phenomena in the molluscan nervous system. Sympos. Soc. Exp. Biol., 1966, 20: 457-475. , Habituation at the synaptic level in Aplysia. Nature (London), 1966, 210: 37-39. BULLOCK, T. H., Neuron doctrine and electrophysiology. Science, 1959, 129: 997-1002. CHIARANDINI, D. J., STEFANI, E., and GERSCHENFELD, H. M., Inhibition of membrane permeability to chloride by copper in molluscan neurones. Nature (London), 1967, 213: 97-99. , Ionic mechanisms of cholinergic excitation in molluscan neurons. Science, 1967, 156: 1597-1599. ECCLES, J. C., The Physiology of Synapses. Academic Press, New York, 1964. FRANK, K., and TAUC, L., Voltage-clamp studies of molluscan neuron membrane properties. In: The Cellular Functions of Membrane Transport (J. F. Hoffman, Ed.). Prentice-Hall, Englewood Cliffs, 1965: 113-135. GERSCHENFELD, H. M., Observations on the ultrastructure of synapses in some pulmonate molluscs. Zsehr. Zellforsch., 1963, 60: 258-275. GERSCHENFELD, H. M., ASCHER, P., and TAUC, L., Two different excitatory transmitters acting on a single molluscan neurone. Nature (London), 1967, 213 : 358-359. GERSCHENFELD, H. M., and CHIARANDINI, D. J., Ionic mechanism associated with non-cholinergic synaptic inhibition in molluscan neurons. J. Neurophysiol., 1965, 28: 710-723. GERSCHENFELD, H. M., and STEFANI, E., 5-Hydroxytryptamine receptors and synaptic transmission in molluscan neurones. Nature (London), 1965, 205: 1216-1218.

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and STEFANI, E., An electrophysiological study of 5hydroxytryptamine receptors of neurones in the molluscan nervous system. /. Physiol. (London), 1966, 185: 684-700. 18. GERSCHENFELD, H. M., and TAUC, L., Différents aspects de la pharmacologie des synapses dans le système nerveux central des mollusques. J. Physiol. (Paris), 1964, 56: 360-361. 19. GERSON, I., and GERSCHENFELD, H. M., Ultraestructura de contactos electrotónicos (sinapsis eléctricas) en "Cryptomphallus aspersa". Acta Physiol. Lot. Amer., 1966,16, Supp. 1: 57. 20. HUGHES, G. M., and TAUC, L., An electrophysiological study of the anatomical relations of two giant nerve cells in Aplysia depilans. J. Exp. Biol., 1963, 40: 469-486. 21. , A unitary biphasic post-synaptic potential (BPSP) in Aplysia 'brain'. /. Physiol. (London), 1965, 179 : 27-28P. 22. , A direct synaptic connexion between the left and right giant cells in Aplysia. J. Physiol. (London), 1968, 197: 511-527. 23. KANDEL, E . R., and TAUC, L., Mechanism of prolonged heterosynaptic facilitation. Nature (London), 1964, 202: 145-147. 24. , Heterosynaptic facilitation in neurones of the abdominal ganglion of Aplysia depilans. J. Physiol. (London), 1965, 181: 1-27. 25. , Mechanism of heterosynaptic facilitation in the giant cell of the abdominal ganglion of Aplysia depilans. J. Physiol. (London), 1965, 181: 28-47. 26. KEHOE, J. S., Pharmacological characteristics and ionic bases of a two component postsynaptic inhibition. Nature (London), 1967, 215: 1503-1505. 26a. , Suppression sélective par l'ion tétraéthylammonium d'une inhibition cholinergique résistant au curare. C.R. Acad. Sci. Paris, 1969, in press. 27. KERKUT, G. A., and THOMAS, R. C., The effect of anion injection and changes in the external potassium and chloride concentration on the reversal potentials of the IPSP and acetylcholine. Comp. Biochem. Physiol., 1964, 11: 199-213. 28. MAYNARD, D. M., Neural coordination in a simple ganglion. Science, 1967, 158: 531-532. 29. MAYNARD, D. M., and BURKE, W . , Electrotonic junctions and negative feedback in the stomatogastric ganglion of the mud crab, Scylla serrata. Am. Zool, 1966, 6; 526. 30. STRUMWASSER, F., Types of information stored in single neurons. In: Invertebrate Nervous Systems; Their Significance for Mammalian Neurophysiology (C. A. G. Wiersma, Ed.). Univ. of Chicago Press, Chicago, 1967: 291-319. 31. TAUC, L., Analyses unitaires d'activités synaptiques chez l'Aplysie révélant le mise en jeu de neurones intermédiaires dans le ganglion abdominal. J. Physiol. (Paris), 1958, 50: 541-544. 32 . , Processus post-synaptiques d'excitation et d'inhibition dans le soma neuronique de l'Aplysie et de l'Escargot. Arch. Ital. Biol., 1958, 96: 78-110. 33. , Interaction non synaptique entre deux neurones adjacents du ganglion abdominal de l'Aplysie. C. R. Acad. Sci. Paris, 1959, 248: 1857-1859. 17. GEBSCHENFELD, H. M.,

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L., Sur la nature de l'onde de surpolarisation de longue durée observée parfois après l'excitation synaptique de certaines cellules ganglionnaires de mollusques. C. R. Acad. Sei. Taris, 1959, 249: 318-320. -, Inhibition présynaptique dans les neurones centraux de l'Aplysie. C. R. Acad. Sei. Paris, 1964, 259: 885-888. , Presynaptic inhibition in the abdominal ganglion of Aplysia. J. Physiol. (London), 1965,181: 282-307. , Physiology of the nervous system. In: Physiology of Mollusca, Vol. II (K. M. Wilbur and C. M. Yonge, Eds.). Academic Press, New York, 1966: 387-454. , Some aspects of postsynaptic inhibition in Aplysia. In: Structure and Function of Inhibitory Neuronal Mechanisms, Pergamon, Oxford, 1968: in press. , Transmission in invertebrate and vertebrate ganglia. Physiol. Rev., 1967, 47: 521-593. TAUC, L., and E P S T E I N , R., Heterosynaptic facilitation as a distinct mechanism in Aplysia. Nature (London), 1967, 214: 724-725. TAUC, L., E P S T E I N , R., and M A L L A R T , A., Action des ions Mg++ et Ca++ sur les potentials postsynaptiques unitaires csez l'Aplysie J. Physiol. (Paris), 1965, 57: 284. TAUC, L . , and GERSCHENFELD, H. M . , Cholinergic transmission mechanisms for both excitation and inhibition in molluscan central synapses. Nature (London), 1961, 192 : 366-367. , A cholinergic mechanism of inhibitory synaptic transmission in a molluscan nervous system. J. Neurophysiol, 1962, 25: 236-262. W A T A N A B E , A . , OBARA, S., and AKIYAMA, T., Pacemaker potentials for the periodic burst discharge in the heart ganglion of a stomatopod, SquiUa oratoria. J. Gen. Physiol., 1967, 50 : 839-862. W A T A N A B E , A . , O B A B A , S . , AKIYAMA, T . , and YUMOTO, K . , Electrical properties of the pacemaker neurons in the heart ganglion of a stomatopod, Squilla oratoria. J. Gen. Physiol., 1967, 50: 813-838. TAUC,

THE ORGANIZATION OF SUBPOPULATIONS IN THE ABDOMINAL GANGLION OF APLYSIA ERIC R. KANDEL" N e w York University School of Medicine New York, N e w York

The great value of the abdominal ganglion of Aplysia for cellular electrophysiological and pharmacological studies has been elegantly demonstrated by the work of Arvanitaki (1) and of Tauc (29) and their collaborators, as well as by the recent work of Strumwasser (27). The neurons in this ganglion are exceptionally large, ranging from 50 m to 1 mm in diameter. The cell bodies of these neurons are primarily located on the dorsal and ventral surfaces of the ganglion and are highly pigmented which facilitates visualizing them. Consequently, in any given preparation one can see and penetrate many cells with microelectrodes. In view of these obvious advantages it seemed to my colleagues and to me that this ganglion should also prove useful for studying the patterns of interconnections of a numerically simple nervous system. * We hoped that by studying in detail the organization of a relatively simple ganglion we might learn some principles of neural organization which are sufficiently general to be applicable to other neural aggregates, including those of vertebrates which are currently not as accessible to such direct investigations. We therefore set ourselves the following outline: First, to distinguish functionally distinct neural groups, in the isolated ganglion, on the basis of physiological and morphological criteria. Second, to study the principles which govern the collective behavior of these groups. Third, to relate these neural groups to the peripheral endocrine, sensory, and motor structures in order to understand how different types of neuronal organization give rise to different types of effector behavior. As a beginning in this direction we identified all of the prominent cells (30 cells) and cell clusters (eight clusters) in the ganglion on the basis of a 4

This research was in part supported by Grant No. R-214-67 from the Cerebral Palsy Foundation. Additional support was provided by Career Program Award MH 18, 558-01. The author thanks Dr. Alden Spencer for his helpful comments on an earlier draft of this manuscript, Catherine Hilten for technical assistance, and Rosalie Cordasco for typing the manuscripts, t The electrophysiological work to be described was begun in collaboration with Drs. W. T. Frazier and R. Waziri, and has been continued in collaboration with Dr. I. Kupfermann and Mr. H. Wachtel. We have been particularly fortunate in having the continued collaboration of Dr. R. E. Coggeshall, who has carried out parallel morphological studies on this ganglion. 71

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number of electrophysiological and morphological criteria (12). We then used these cells and cell clusters as a reference population for studying the patterns of organization among the different cell groupings (17), and the relation of the cell groupings to effector functions. In Figure 23, a schematic drawing of the abdominal ganglion illustrates the most common position of the identified cells. As is obvious even on casual inspection, the abdominal ganglion of Aplysia is remarkably similar to the hippocampal-hypothalamic (limbic) system of mammals. As is the case for the limbic system, part of this ganglion is concerned with visceral integration whereas other parts serve as neuroendocrine organs. The early findings of Bottazzi & Enriques (2) on the regulation of visceral activity by the abdominal ganglion have recently been extended by Kupfermann and Kesselman using modern techniques. By stimulating the connectives and peripheral nerves to the ganglion, Kupfermann and Kesselman have shown that the ganglion modulates the movement of a number of organs, such as the heart, the genital apparatus, the siphon and the ctenidium (branchae). In preliminary experiments, Kupfermann and I have also found that some of these motor effects can be produced by direct stimulation of individual identified neurons. The earlier findings of Scharrer (26) on neurosecretory cells in the abdominal ganglia were recently extended by Coggeshall (6, 7 , 1 2 ) in an important electron microscopic investigation of this ganglion. Coggeshall (7) found two morphologically distinct types of clusters in the ganglion: the bag cells and the white cells, both of which contained great numbers of elementary neurosecretory granules and had other morphological characteristics of neurosecretory cells. The function of the white cells is still obscure, but Kupfermann has recently shown that the extracts of the bag cells stimulate egg laying in mature animals (20). Thus, as a result of Coggeshall's work, we could isolate three functionally distinct neural groups among the identified cells, two of these are neurosecretory and a third group, consisting of the remaining non-neurosecretory cells, is primarily concerned with visceromotor integration ( 1 2 ) . " We were therefore in a position to examine the patterns of interconnections used by the constituent cells in each of the three groups. We found that the two neurosecretory clusters, although differing in detail, do not seem to require interneurons for their functioning as a group. By contrast, the visceromotor group is innervated by a number of interneurons, which make connections with the individual cells of the group (the follower cells) as well as with each other. In this report I will first review briefly the organization of the two neurosecretory groups which appear to function without interneurons. Then I will consider the visceromotor groups to illustrate the types of operations which are added to a neuronal population by innervation from even a single inter* Also, I. Kupfermann and E. R. Kandel, report in preparation.

Dorsal Surface

BAG CELLS - \ LRQG „

- BAG CELLS j

B

Ventral Surface L R

Connective

Connective

Rostral

RRQG

LRQG

Siphon nerve

Caudal

Branchial nerve

Genitalpericardial nerve

Figure 23. Schematic drawing of the dorsal and ventral surfaces of the abdominal ganglion of Aplysia californica, indicating the most common position of the 30 identified cells. The identified cells are labeled with an L or R (designating left or right hemiganglion) and with a number (in arbitrary sequence). The right and left hemiganglia are divided into quarters: RRQG, right rostral quarter ganglion; RCQG, right caudal quarter ganglion; LRQG, left rostral quarter ganglion; and LCQG, left caudal quarter ganglion. The white cells ( R 1 3 - R 1 4 ) have been indicated in black. The remaining identified cells, in white, are concerned with visceromotor integration. (From Frazier, Kandel, Kupfermann, Waziri & Coggeshall, 12.)

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neuron. I will use this comparison of subpopulations with and without interneurons to consider the function of interneurons in the integrative actions of neural aggregates in Aplysia. GENERAL STRUCTURE OF THE GANGLION

The abdominal ganglion of Aplysia contains about 1500 neurons. In addition 800 cells make up the two clusters of bag cells which lie at the junction of the right and left connective with the ganglion (Figure 23). The ganglion proper is organized into an interior neuropile which is continuous with the connectives and peripheral nerves and an outer cortex of ganglion cells (6). The ganglion is anatomically divided into two hemiganglia and each hemiganglion can be further subdivided into quarters on the basis of functional considerations. The quarters are the left rostral and caudal quarter ganglia (LRQG and LCQG) and the right rostral and caudal quarter ganglia (RRQG and RCQG). We have also divided the ganglion into smaller cell groupings on the basis of a number of morphological and functional criteria (12). The two most distinct cell groupings are the bag cells and the white cells. The Bag Cell Group The bag cells are located in two clusters at the junction of the left and right connectives with the ganglion (Figures 23 and 24B). In the very small (and young) animal (weighing about 2 g) there are probably less than 100 bag cells in each cluster (12). At this stage the cells are small (2-10 m), they are not yet attended by glial cells, and their process extends only a slight distance from the perikaryon. In contrast to the bag cells of larger animals there is at this stage no morphologic evidence that these cells produce granules. As the animals grow, a striking change occurs in the number and morphology of the bag cells. In mature animals the number of bag cells per

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Figure 24. A: Drawing of the edge of a bag cell cluster; the bag cells send their processes towards the longitudinally oriented axons of the connectives; when the processes meet the axons in the connective, they wrap around them; from this point the bag cell processes either turn laterally into the sheath, or they turn rostrally and travel between the axons of the connective and the sheath, and then laterally. The bag cell processes end within the sheath and no morphologic evidence for functional contact between these processes and other axons or muscle cells within the sheath has been obtained. B: Diagram of the ganglion with the area shown in A outlined. C: The nuclear and perinuclear cytoplasm of a bag cell body; the nucleolus in the bag cell nucleus is remarkable in that many of the particles presumed to be ribosomes are aligned in rows; the cytoplasm contains a number of granules which seem to be formed in the Golgi complex (G.C.); the granules travel into the bag cell processes where they undergo a morphologic change, as is illustrated in Figure 26. (From Frazier, et al., 12.) X 17,000

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cluster increases fourfold and reaches 400 or more and each cell grows to about 50 m in size. The cell bodies and initial segments are now attended by glial cells, and the cells now send a thickened process into the connective tissue sheath which surrounds the ganglion and the connectives. Before ending in the sheath these processes form a stout cuff around and superficial to the axons in the connectives (Figure 24A). Also, the cells now produce large (1500-2500 A), round granules which have moderately electron-dense cores (Figure 2 4 B ) . These granules are transported down the process and, as the granules are traced along the process, their cores become smaller and less electron-dense and the membrane of each granule becomes ruffled or crenated and is frequently broken (Figure 25A). The bag cell processes end within the connective tissue sheath without showing morphological evidence of functional contacts with other axon or effector cells (Figure 2 6 B ) . The sheath is highly vascularized (Figure 26) and, together with neurosecretory processes which end within it, serves to form a neurohaemal organ (Figure 26A) ( 6 ) . The granules of the bag cells bear a slight resemblance to the catecholamine granules described in certain vertebrate postganglionic autonomic neurons. However, Goldstein" failed to find significant amounts of dopamine in the isolated bag cell clusters. Although the biochemical nature of the bag cells granule is unknown, Kupfermann, in our laboratory, has recently shown that bag cell homogenates stimulate egg laying in mature animals, whereas homogenates from the remaining parts of the ganglion are ineffective (20). When we examined the bag cells with intracellular microelectrodes we found them to have remarkably similar electrophysiological properties (21, and report in preparation). When undamaged, the cells have a resting potential of about 40 mV. The cells are silent and do not show spontaneous PSPs even when examined at very high gain. The cells can be made to generate an action potential of up to 70 mV in amplitude in response to intracellular current pulses. The cells do not respond to stimulation of the pe• Unpublished observations.

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Figure 25. A: Electromicrograph of a cross section of the connective at a point where the bag cell processes wrap around it; at the periphery of the connective there are only bag cell and glial processes; the axons of the connective are located centrally (not shown); the granules in the bag cell processes here differ from those in the cell body (see Figure 2 4 C ) by having ruffled, crenated and sometimes broken membranes, and less electron-dense cores; X 37,000. B: From their position surrounding the axons of the connectives, the bag cell processes turn laterally into the sheath and end there; in an almost longitudinal section of an axon in the sheath, numerous bag cell processes characterized by granules with crenated membranes and moderately electron-dense cores can be seen. In the sheath, the nerves containing bag cell processes are joined by white cell processes which possess round granules with very electron-dense cores; serial sections establish that both the bag cell and white processes end within the sheath; X 29,500. (From Frazier, et al., 12.)

Figure 26. A: Drawing illustrating the vascularization of the sheath by branches from the aorta (from Coggeshall, 6 ) . B: Schematic drawing illustrating the termination of a process from a neurosecretory cell in the sheath.

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ripheral nerves; however, strong electrical stimuli to the connectives frequently cause a prolonged discharge of the bag cells (lasting from several minutes to half an hour). The remarkable aspect of this activity is that when ipsilateral bag cells are recorded from, two at a time, they invariably fire synchronously (Figure 2 7 ) . Underlying the synchronization is a prepotential which is similar and simultaneous in all of the bag cells on the same side. This prepotential is all-or-none and the prepotential is progressively decreased in amplitude by large hyperpolarizing currents suggesting that it may be a remote axonal spike rather than an elementary PSP. Attempts to block the synchrony in any one cell by interjecting large hyperpolarizing pulses into that cell have been unsuccessful. Indeed, these large hyperpolarizing pulses could not even completely block spike generation in the impaled cell, indicating that the orthodromic spike was generated at a remote site and perhaps in the fine processes which the cell sends into the connective tissue sheath. Depolarizing or hyperpolarizing currents injected into one bag cell also failed to produce a potential change in another bag cell. This indicated that the synchronization was accomplished either by an electrical connection between the remote processes of the bag cells, perhaps at the cuff which the processes form around the connective, or by an interneuron which innervated all 400 bag cells in the cluster. To differentiate between these two hypotheses we first removed the bag cell clusters from the rest of the ganglion and found that synchronous bag cell activity still occurred in the isolated bag cell—connective preparation. If the synchrony resulted from a common interneuron, it would have to be with the bag cell cluster itself. Repeated attempts to find such an interneuron were unsuccessful. Indeed the synchronization could even be demon-

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strated when a single cluster was split into two halves as long as the cut did not extend into the region of the cuff formed by the bag cell process. This type of procedure is most likely to destroy at least half of the branches of a synchronizing interneuron. Only when the cut extended into the area of the cuff did the synchronous activity disappear. Although these experiments are not conclusive, they suggest that synchrony is most likely due to an electrical interaction in the region of the cuff. The extraordinary degree of synchrony is also most consistent with an electrical mechanism. Attempts by Coggeshall* to define the possible areas of electrical interaction morphologically have not yet revealed any areas of contact (12). In summary, this neurosecretory cluster behaves as a group of interdependent and synchronous parallel elements, which act essentially as one large cell. This cluster can function independently of the ganglion as long as its connections with the connectives are intact. To become active this group requires an external trigger mediated by the connectives, perhaps a set of sensory stimuli directly from the periphery or via higher ganglia. Once triggered, the cluster itself can maintain a prolonged activity without requiring further input. The White Cells Group Although they are not topographically segregated from the rest of the abdominal ganglion as are the bag cells, the second neurosecretory clusters are equally easily recognized. When one examines the ganglion under transilluminated light, all cells in the left hemiganglion appear translucent and orange. However, in the right hemiganglion there are, in addition to the orange-colored cells, a group of cells that appear white and opalescent (darkened cells in Figure 23). These white cellsf (R3-R14) form a structurally and functionally distinct neurosecretory group. All but one (R14) is located in the RRQG. We will here consider only the rostral white cell group but the properties of R14 are quite similar (12). The white cells all contain numerous characteristic granules. These have a markedly electron-dense core resembling the "elementary" neurosecretory granules (Figure 28B). Indeed, it is the high concentration of these granules that is responsible for the white appearance of these cells (7, 12). The white cells send processes into the connective tissue sheath that surround the ganglion much as do the bag cells (Figure 28A). However, these processes spread out diffusely into the sheath and do not come in close apposition as do the bag cell processes in the formation of their cuff. The white cells also * Personal communication. t Cell R15 in the RCQG can be white, and in these circumstances its appearance under the dissecting microscope is similar to that of the rostral white cells and R14. However, unlike those cells, R15 is not white in small animals but yellow and translucent, and it then is found to contain no, or only few, granules when examined with the electron microscope. R15 is white only in large animals and only then does it contain many granules. Moreover, R15 does not send process into the sheath and therefore does not have the morphological characteristics typical of the other neurosecretory cells.

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resemble the bag cells in showing considerable morphological change with maturation. However, whereas the bag cells increase in number with age, the number of white cells does not; rather, each white cell emits a much greater number of processes into the sheath. In a 2 g animal a white cell may emit only a few processes but in a 100 g animal each white cell emits several hundred processes into the sheath. As with the bag cell the rostral white cells fulfill the morphologic criteria for neurosecretory cells. Each of the rostral white cells also sends a single stout axon into the neuropile which emerges in the branchial nerve (Figures 28A and 29C). The peripheral destination of these fibers is not known. Within the neuropile the white cell axons do not seem to synapse on other cells and very few synaptic boutons seem to end on the white cells (12). The electrophysiological properties of the rostral white cells are quite uniform (7, 12). The cells usually fire spontaneously at a slow and highly regular rate (Figure 29A). The rhythm results from an endogenous pacemaker process; its high regularity is due to the absence of modulation by interneurons. Moreover the white cells seem to show only a very weak synaptic response to stimulation of the major pathways to the ganglion (Figure 29, A and B). Both thesefindingsare consistent with the paucity of terminal boutons ending on white cells axons in the neuropile. When two white cells were simultaneously impaled, these rhythms were clearly seen to be independent and there were no common prepotentials (Figure 29A). Changing the membrane potential of one cell had considerable effect on the endogenous rhythm of that cell but no effect on the rhythm of other white cells. The granules of the white cell resemble those found in certain invertebrate cells rich in serotonin. But chemical analysis by Goldstein" of the isolated right rostral quarter ganglion, which contains these cells, showed no significant amounts of either serotonin or dopamine. As a result, neither the chemical composition of the white cell granule nor its neuroendocrine function is understood. The relatively weak response to neural stimulation suggests that these cells may respond to hormonal or osmotic stimuli. In summary, this neurosecretory cluster behaves as a group of independent and non-modulated parallel elements, and appears relatively free from neuronal influence. Despite differences in its functional organization this neurosecretory cluster resembles the bag cell cluster in being, functionally, relatively independent of the remainder of the abdominal ganglion. The Integrative and Visceromotor Cell Group The rostral white cell cluster occupies the RRQG and its cells account for all but two of the identified cells in it. The remaining identified cells are distributed among the other three quarter ganglia. With the exception of R14, the "stray" white cell, the properties of these cells are remarkably different " Unpublished observations.

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from those of the white cells. First, these cells are all, to some degree, pigmented. Second, although some of these cells produce granules, none sends processes into the sheath. Third, all of these cells receive obvious input from interneurons. Although these cells innervate different regions and participate in different effector functions, almost all of these cells share an innervation from two interneurons (interneurons I and I I ) . In this part of the report I will consider first the interaction between each of the two interneurons and their follower cells and second, the interactions between the two interneurons (17,18). INTERACTIONS B E T W E E N INTERNEUKON I AND ITS F O L L O W E R

CELLS

The connections I will first describe are those made by interneuron I on follower cells in the LRQG and the RCQG, respectively. All of the six identified cells in the LRQG receive powerful inhibitory connections. Four of these cells are endogenously active bursting cells and, in the isolated ganglion, the IPSPs provide the major modulating drive of the bursting rhythm. By recording two at a time from the cells in the LRQG we could show that the IPSPs were synchronous; this suggested that each cell received an inhibitory branch from a common interneuron (Figure 30A). One of the several modulating synaptic inputs on the bursting cell of the RCQG (R15) was a unitary depolarizing postsynaptic potential. Simultaneous recordings from the cell in the RCQG (R15) and one of the cells in the LRQG (L3) showed that these EPSPs in R15 and the IPSPs in the cells of the left upper quadrant were produced synchronously, suggesting that they are mediated by different branches of the same interneuron (Figure 30B). By searching among the cells of the ganglion, we were able to find and identify the interneuron (L10) that produced these opposite effects on cells in the LRQG and RCQG respectively (Figure 31). We call cell L10 interneuron I, to distinguish it from ten other interneurons (numbered II-XI), nine of which have been inferred only on the basis of indirect data (17). Figure 32 is a simultaneous high sweep record from three cells, showing > ^

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Figure 28. A: Drawing of two white cells illustrating the processes that extend into the sheath and the single axon that passes through the neuropile to enter the branchial nerve; the processes in the sheath, the cell bodies, and the axons all contain large numbers of granules that are characteristic for these cells. B: White cell granules. The perikarya, processes, and axons of the white cells, R3-R14, contain many granules, which are round, have an average diameter of approximately 2000 A and. in comparison with the granules of other cells in this ganglion, have very electron-dense cores; the granules in these white cells, as well as those of other granule-containing cells, take shape in the Golgi complex (G.C.); in the Golgi cisternae, localized regions of electron-dense material accumulate (unlabeled arrows) and are eventually pinched off, forming the final granule. (From Coggeshall, Kandel, Kupfermann & Waziri, 7.) X 40,000

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through large ventral root fibers of animals more than two weeks of age, the paranodal myelin sheath contour appears rather symmetrically c r e n a t e d having a trefoil-like outline ( F i g u r e 5 5 ) , w h i c h in serial sections can b e shown to retain this shape over several microns of fiber length. Besides internodes of a conventionally described length of 3 0 0 - 4 0 0 n ( 3 9 ) , several verv short internodes 10-50 u long were found in the newborn animals; these showed a most bizarre myelin sheath contour and large disintegrating myelin sheath fragments ( F i g u r e 5 6 A ) . In animals more than seven days of age, such very short myelinated internodes are sparse in the large fibers. T h e r e are, however, several verv short unmyelinated internodes containing a b u n d a n t myelin sheath fragments in different stages of disintegration ( F i g u r e 5 6 B ) . F u r t h e r m o r e , in animals less than 14 days old, there are S c h w a n n cell-like units containing myelin debris closely apposed to some nodes of Ranvier ( F i g u r e 5 6 C ) . No such formations have b e e n observed on large fibers in animals more than one m o n t h old. It is suggested that the very short internodes are affected b y myelin disintegration to the point w h e r e they are eliminated from the fiber. Such an elimination of very short internodes obviously implies a decrease in the n u m b e r of nodes of R a n v i e r a n d m i g h t b e a partial explanation for t h e reduction in reflex time postnatallv.

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Figure 55. Twenty-day-old kitten: ventral lumbar root; cross-cut large paranode. The myelin sheath is crenated and has a trefoil-like outline. There are definite accumulations of mitochondria in the Schwann cell cytoplasm. (From Berthold & Skoglund, 4b.) X 15,000

The paranodal accumulation of Schwann cell mitochondria was studied by a combined analysis of NADFL-tetrazolium reductase activity in teased specimens and the ultrastructural identification of mitochondria. The reductase pattern of mature fibers is shown in Figure 57 and consists of intensively stained paranodal stripes which, by delicate longitudinally running bands, are connected with the pool of activity that surrounds the Schwann cell nucleus. In the newborn kitten, the paranodal reductase activity is diffuse, whereas a definitely paranodal accentuation is present at a fiber size around 4 p. These observations are in good agreement with the increase, found by elec\\\\\ y

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Figure 56. A: Newborn kitten: ventral lumbar root; a heavy myelinated short internode (about 12 ¡i long) is shown; note the bizarre myelin sheath loops filled with axoplasm and the disintegrating myelin sheath fragments (large arrows); X 6000. B: Seven-davold kitten: ventral lumbar root; longitudinal section through an unmyelinated very short internode; the Schwann cell contains, besides its nucleus (Sn), a large number of myelin sheath fragments (arrows); X 4500. C: Same specimen as in B; longitudinal section through large node of Ranvier; a Schwann cell (Sn) is apposed to one side of the axon, the contact zone being marked by small arrows; long arrow points at myelin sheath fragment; X 6000. (From Berthold & Skoglund, 4c.)

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Figure 57. Adult cat: ventral lumbar root. Specimen teased and stained for NADH2 tetrazolium reductase. Paranodally, the activity forms distinct stripes that extend up to the node (see two upper fibers). Lower fiber shows the spider-like configuration of the activity surrounding the Schwann cell nucleus. (From Berthold & Skoglund, 4b.) X 620

tron microscopy, of paranodal Schwann cell mitochondria which reaches the adult range around the end of the first month (compare Figure 51A with Figures 53 and 55). In this connection it is worth noting that, in the thinnest myelinated fibers observed in the newborn animal, a constant finding is an accumulation of relatively large intraaxonal mitochondria in the nodeparanode region (Figure 58), whereas no such observation has been made in the more mature stages. The thinnest myelinated fibers in the newborn animal have a node gap which is only about 200 p in width and closed off from the endoneural space by overlapping cytoplasmic extensions of the meeting Schwann cells (Figure 59A). In large ventral root fibers, on the other hand, the node gap is roomy and occupied by microvilli-like Schwann cell processes arranged at random (Figure 59B). There is also an open communication between the node gap and the endoneural space. If the nodes in fibers of different sizes are compared in a newborn animal, various transitional stages between the closed type of gap and the more roomy open gap, occupied by microvillilike processes, are found. At a fiber size of more than 4 p the node gap contains densely packed, radially arranged processes, a picture close to the mature one (compare Figure 59B with Figure 52). In summing up these changes taking place during the process of nodalization, it can be stated that there is a transformation of the paranodal region including a considerable amount of myelin degeneration, leading to the for-

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Figure 58. Newborn kitten: ventral lumbar root. Longitudinal section through the transition between paranode and node of a very thin myelinated fiber. The narrow, closed node gap is shown to the right (large arrows). Close to the node there is in the axon an accumulation of relatively large mitochondria ( M i ) . (From Berthold, 3.) X 34,000

mation of a system of longitudinal myelin sheath crests and interposed deep furrows in which aggregations of mitochondria appear, while they disappear from the node-paranode axoplasm. The mitochondrion aggregations are in communication with the node gap through a system of microvilli-like processes which become radially arranged around the nodal axon segment. The nodalization is well under way in the fiber when it reaches a size of 4 n, which corresponds to the size: (a) when the fibers attain adult electrical properties as indicated by the absolute refractory period ( b ) after which they are able to convey slowly adapting responses from the sense organs and ( c ) when the first signs of post-tetanic potentiation and facilitation between synergists are present. An interesting finding, made by Dr. G. Schwieler in my laboratory, is that the bronchial nerves convey tonic impulses from the stretch receptors of the lung at fiber sizes equal to those in the sural nerve in the newborn stage. This would be in opposition to the correlation made above if it were not that the bronchial nerves, in spite of their small size, show a nearly mature NADfL-tetrazolium reductase pattern, whereas the similarly thick sural nerve fibers appear quite immature in this respect. I therefore propose that the statement of a correlation between developing function and fiber size should be replaced by one between the stage of the nodalization (as defined here) and function. It is strange, though, that the excitability in the monosynaptic pathway decreases postnatally in spite of the fibers becoming more mature and their function getting better, as judged by their ability to convey tonic discharges from their receptor ends and to carry high frequencies in their central ends. Without further evidence, this cannot be attributed to an increase in inhibi-

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Figure 59. A: Newborn kitten: sural nerve; cross-section through medium sized node of Ranvier; outside the axon (Ax) there is a narrow node gap (large arrows) which is closed off from the surrounding interstitial space by the overlapping edges of the meeting Schwann cells ( S I ) ; X 45,000. B: Sevenday-old kitten: ventral lumbar root; cross-cut large node of Ranvier; the node gap ( N g ) is rather roomy and contains a system of radially arranged, densely packed microvilli-like processes; there is an open communication between the node gap and the endoneural space (arrows). (From Berthold, 3.) X 21,000

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tion or to the establishment of new interneuronal inhibitory connections. Furthermore, these changes start at an age before a transaction of the spinal cord at the thoracic level causes any major changes, at least in the activity of distal hindlimb muscles. Thus, the changes occur prior to the time when an action of supraspinal influences could be the reason for these changes. In an attempt to elucidate this problem further we have taken up morphological studies of the spinal cord. Conradi and I® find, in both the light and the electron miscroscope, that the spinal cord in the newborn stage appears very uniform in regard to the size of the nerve cells, and the ventral horn is practically devoid of myelin (Figure 60). This situation seems to prevail during the whole first fortnight postnatally, whereas the spinal cord, except with regard to neuron size, has a more or less adult appearance after one month. One possible explanation for the change in excitability could be that postnatal increase of the motoneuron size might shift the localization of the boutons which have established functioning monosynaptic contacts. Mellstrom and If studied the growth of the spinal cord both macro- and microscopically and measured the first proximal hundred cells in L 7 and C7 by the method of Schade & van Harreveld (28). Our findings are in good agreement with the electron microscopic impressions. As can be seen in Figure 61, there is no increase in the mean size of the neurons in lamina IX during the first fortnight in contrast with the rapid increase in size during the second fortnight; the cell gray coefficient similarly shows a very small decrease in the lumbar region during the first fortnight and a large one during the second fortnight, f Thus, there is little evidence for a change of the bouton localization due to increase in neuron size being responsible for the change in excitability. However, there is one striking change during this period; this is the rapid increase in the number of mitochondria as judged by the histochemical demonstration of succinic dehydrogenase (Figure 62) which parallels the development in the peripheral nerve fibers (36). Of course, part of the explanation for the changing reflex pattern might, as pointed out earlier (33), be due to a postnatal development of the connection of the dorsal root collaterals, at least functionally. Both inhibitory and facilitatory connections might become functionally mature. Electronmicroscopically, the boutons are smaller than in the adult stage, as can be seen in Figure 63, and they seem to contain less mitochondria in the newly born. The number of synaptic vesicles in each bouton also appears to be less than in the adult stage but they are of the same size as in the adult. Thus, while a maturation of the boutons might be part of the explanation for a change, there is evidently a reduction in their synaptic action, at least in the monosynaptic pathway. Another possibility for a change in excitability could, as pointed out by * S. Conradi and S. Skoglund, report in preparation, t A. Mellstrom and S. Skoglund, unpublished observations.

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Figure 60. Survey picture from 1-day-old kitten ( a b o v e ) , 1-month-old kitten ( c e n t e r ) , and adult cat ( b e l o w ) , showing neuropile just outside a motoneuron in the lumbar spinal cord. Note the increase in myelin and mitochondria. X 5700

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and prolonged (from 150 msec, to 140 sec.) constant force stimuli ranging from 50 to 1500 g. The single unit analysis which preceded the measurements of the overall outflow revealed that three types of receptors with large myelinated afferents can be activated by mechanical stimulation of the central pad: the first group was called PC-receptors, because they resembled in all their properties the Pacinian corpuscles investigated elsewhere. The second group consisted of mechanoreceptors which showed a dynamic response of 50 to 500 msec, duration to a constant force stimulus. They were termed "rapidly adapting receptors", (RA-receptors). The last group consisted of receptors which discharged throughout a constant force stimulus; they were named "slowly adapting receptors", (SA-receptors). The PC- and RA-receptors never discharged spontaneously and were insensitive to rapid cooling of the skin, whereas 80 per cent of the SA-receptors showed a spontaneous discharge and, of these, quite a few responded with a transient burst of impulses when the skin was cooled with ethyl chloride. The complete single unit analysis and all methodological details have been published elsewhere (14). The Total Afferent Outfloic after Short Stimuli The PC-receptors can be excited by very low stimuli. Fifty per cent of the units investigated needed less than 1 n indentation at the point of their maximum sensitivity and 90 per cent needed less than 4 |j. Furthermore, moving the stimulating stylus away from this site for considerable distances required only rather small increases in stimulus strength. Thus, with a five times threshold strength, a PC-receptor could usually be activated from more than half of the central pad surface, and a stimulus of 20 |j applied to the middle of the central pad excited all or nearly all PC-receptors which had their sites of maximal sensitivity on or in the neighborhood of the central pad. On the other hand, the thresholds of the RA- and SA-receptors are higher. Fifty per cent had thresholds from 5 to 10 |j, the others up to 30 m and more. In addition, the stimulus strength required for excitation rose steeply if the stylus was moved from the most sensitive site. Due to these

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characteristics, a 10-20 m stimulus applied to the central pad excites mainly PC-units. In the experiment of Figure 98, the medial plantar nerve was dissected into 11 filaments and the lateral plantar nerve into six filaments. One filament after the other was put on a recording electrode and the number of activated units on 2 msec, stimuli of 0.4 to 16 n indentation was directly read off the oscilloscope screen. Thereafter the number of units of all filaments were added. The graph in Figure 98 plots the relation between the stimulus strength (abscissa) and the number of activated units (ordinate). It can be seen that initially the number of activated units increased steeply with stimulus strength, a 5 p indentation activating about 40 units, whereas thereafter the number of activated units increased more slowly, a 16 m stimulus activating about 65 units. Equivalent results have been obtained in other experiments of this type ( 1 4 ) . They correspond well with the upper range of values found when using a different method of recording (3, 14). It can be concluded from these experiments and from the results reported in the beginning of this chapter, that small stimuli ( < 20 n) applied to the units

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central pad will excite nearly exclusively PC-receptors, the number of activated units depending in a fairly predictable manner on the stimulus strength. With a 20 m stimulus the afferent volley will consist of one single inpulse from probably more than 50 but less than 100 PC-units. In addition, a few impulses from RA- and SA-receptors might be included in the volley. It may be pointed out that an exclusive repetitive activation of this PC-receptor population is also possible. With sinusoidal stimulation, the receptors have minimal thresholds for a 1:1 stimulus-response sequence at frequencies of 150-200 Hz. At these frequencies the RA- or SA-receptors are practically inexcitable. Thus the rhythmic afferent inflow into the spinal cord consists only of PC-impulses. The Afferent Outflow during Constant Force Stimuli The PC-receptors do not respond at all to a constant pressure. The RA-receptors fire a short (50-500 msec.) burst of impulses which, apart from differences from receptor to receptor, depends in frequency and duration on the strength of the stimulus. The SA-receptors have a dynamic phase of activity very similar to that of the RA-receptors but thereafter they continue to discharge at a rate which depends on the stimulus strength. Thus, shortly after the onset of a constant force stimulus to the central pad the activity in the afferent nerves (minus the spontaneous activity) consists only of impulses coming from SA-receptors. An analysis of the stimulus-response relations of the SA-receptors during constant force stimuli (stimulus durations up to 140 sec.) has shown that this relation can be described by a power function with exponents averaging around n = 0.5 (14). It can be expected that the overall output under these conditions reflects the behavior of the individual units. In the experiment of Figure 99A the spike activity of the various small filaments was recorded during three constant force stimuli of 150, 625 and 1100 g. The records were taken on tape and analyzed electronically. It is seen that there was a spontaneous activity of about 250 impulses per second. After the stimulus onset, the impulse frequency rose above 4000/sec. and, due to spike superpositions could not be resolved fully for the first 150 msec. In the first 10 sec. after stimulus onset, the discharge declined considerably but from the 10th sec. until the end of the stimulus, the discharge rates remained rather steady. The complete set of measurements was replotted in Figure 99B. On log-log scales, the force of the pressure stimulus (on the abscissa) was plotted against the total afferent outflow (impulses per second on the ordinate). The discharge rates during the 2nd, 5th and 30th second are shown. At all times the points can be connected by straight lines, indicating that a power function can be fitted to the results. In Figure 99B the exponents increased slightly from n = 0.47 after 2 sec. to 0.52 after 30 sec. The average discharge rates after 10 sec. were for a 500 g stimulus about 1000 impulses per second, and for a 1000 g stimulus about 1700 impulses

214 imp/ s

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Figure 99. Relationship between the strength of constant force stimuli applied to the central pad and the total afferent outflow in the medial and lateral plantar nerves. A. Time-course of the total afferent outflow (impulses per second, ordinate, log scale) for three constant force stimuli (see symbols); the stimulus was lowered onto the pad at 0 of the abscissa and lifted off after 42 sec. B. Average impulse rates (ordinate, log scale) at 2, 5 and 30 sec. after stimulus onset, plotted against the strength of the constant force stimuli (abscissa, log scale). (From Janig, Schmidt & Zimmermann, 14.)

per second (14). The 14 individual SA-receptors analyzed by these authors showed discharge rates from 5 to 60 impulses per second (average 13) 10 sec. after the onset of a 500 g stimulus. From these values, estimates of the total number of SA-receptors in the central pad gave, in four experiments, values of 34 to 110 for the extreme limits and 53 to 77 for the average. It has to be kept in mind, however, that it is not known to what degree the analyzed individual units were representative of the total SA-receptor population. The situation is even worse for the RA-receptors because at present no ways exist for estimating their total number reliably. During the single unit studies they were not found more frequently than the SA-receptors, which may indicate that their number is of the same order of magnitude as the number of SA-units; in addition, the thresholds, receptive fields and conduction velocities of both types were identical, a fact which probably prevented a biased sampling. The Afferent Outflow during the Onset of Constant Force Stimuli As already shown in Figure 99A, the afferent outflow reached its highest value during the onset of constant force stimuli. This onset took about 60-200 msec, in our experiements. During this time the outflow came from PC-, RA- and SA-receptors; with our recording techniques the spikes superposed in the larger filaments so that the electronic counting device gave too low a value for the spike rates. The correct values can be obtained only if

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the nerves are not only split in their natural filaments but into much smaller ones. Possibly this cannot be done without damaging a considerable proportion of the fibers. One may give, instead, an estimate of the number of impulses and of the impulse rates reaching the spinal cord during a pressure increase from 0 to 500 g within 200 msec. Assuming that about 100 PC-, 50 RA-, and 50 SA-receptors are excited, and taking into account the behavior of the individual units, a conservative estimate shows that about 6000 impulses reach the spinal cord during these 200 msec. Correspondingly, the impulse rate is 30,000 impulses per second, that is, about 30 times higher than it is 10 sec. later (Figure 99). The Afferent Innervation of the Central Pad Previous work has shown that mechanical stimuli to the central pad evoked activity from 50 to 100 PC- and from 50-100 SA-units and there was some evidence that the RA-units were approximately equivalent in number. The conduction velocity of the fibers ranged from 40 to 80 meters per second, corresponding to diameters of 6-12 |j. We now asked what proportion of the large myelinated afferents running to the sole of the foot, particularly to the central pad and its subcutaneous tissue, is taken by this population of 150-300 fibers. The 17 multifiber filaments in the experiment of Figure 98 had diameters from 80 to 260 m, as judged from micrometer readings at 50 times magnification. The total cross-section of all filaments was 0.358 mm2. If it is assumed that the fiber spectrum in these filaments was similar to that found in other cutaneous nerves, it can be estimated that the filaments contained 4000 to 5000 or more myelinated fibers. Roughly half of them would belong to the 6-12 m fiber group. It appears from this estimate that the mechano-afferents of the central pad constitute about 10 per cent of all large myelinated fibers present in the plantar nerves. In histological sections parallel to the pad surface, the myelinated afferent fibers cut transversally were counted and their diameters were measured.* A fiber spectrum typical for cutaneous nerves was found with about 250 fibers < 6 m, and 250 fibers > 6 |j in diameter. The sections were made close to the surface, so that it can be assumed that all fibers went to end organs in the skin and that few PC-afferents were included, because these receptors are found deeper in the subcutaneous tissue. The number of large myelinated afferents appears to be larger than the number of RA- and SA-receptor units (250 fibers against 100-200 units), although both values are in the same order of magnitude. Since the estimate of the RA-units rests on very weak evidence only, it may well be that all large myelinated afferents belong only to mechano-sensitive units. The alternative is that another receptor type (cold?), again with some 50-100 units, is also innervated by large afferents. * W. Jänig, unpublished observations.

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T H E S E G M E N T A L P A T H W A Y S AND T H E FUNCTIONAL SIGNIFICANCE P R E S Y N A P T I C I N H I B I T I O N O F MECHANORECEPTOR

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The presynaptic terminal is the earliest possible central site at which the afferent inflow can be modified. It has been shown for various cutaneous reflex systems and projection pathways that presynaptic depolarization can exert a powerful inhibition of the afferent inflow from cutaneous afferents (7). The electrical stimulation of cutaneous nerves used in these studies was inadequate for an analysis of the contribution of the various types of afferents both on the giving and the receiving site. The possibility of selectively exciting phasic and tonic mechanoreceptor populations and our technique of identifying single afferent units, both at the periphery and within the spinal cord (20), enabled us to study the inhibitory influence of these two receptor populations on the afferents of mechano-sensitive units from hairy and hairless skin of the leg. The primary afferent depolarization (PAD) was detected by testing the excitability of the afferent terminals. Details of the methods have been published (13,20,21). The PAD after Activation of Phasic Mechanoreceptors A short single stimulus of 20 n indentation to the central pad, that is activation of about 50-100 PC-receptors (Figure 98), will induce a depolarization in all types of mechanoreceptor afferents from the foot. This depolarization has a time-course similar to that found after single electrical stimulation: a few msec, after the arrival of the afferent volley in the spinal cord the depolarization begins, reaches its maximum within 10-20 msec, and declines within a further 100-150 msec, (for illustrations see references 19, 21). Thus the phasic mechanoreceptors depolarized all mechanoreceptor afferents; however, it will now be shown that they do so to very different extents, depending on whether the depolarized fiber belongs to the phasic or the tonic population. (The maximal PAD amplitude reached during timecourse measurements depends mainly on the distance of the tip of the testing microelectrode from the depolarized site and cannot be used as an index for the strength of the PAD.) The white symbols in Figure 100A plot the relation between stimulus strength (ordinate) and the fall in threshold (increase in excitability) of three phasic mechanoreceptor afferents. The stimulus was always applied to the middle of the central pad. All units showed a steep fall in the threshold with increasing stimulus strength, which was nearly maximal for a 10 |j displacement. The onset of the curves was sharp, lying below 2 p indentation. Corresponding results were obtained in 19 other phasic units. Extrapolation of the strength-response curves yielded threshold stimulus strengths between 0.3 and 3.5 m, the median being 0.8 p. The corresponding numbers of activated PC-receptors were from 1 to 20, the median being 3 (13). In two respects the strength-response curves of tonic mechano-afferents were different (Figure 100A, black circles): the onset of the fall in threshold was not sharp but slow and, with stimulus strength above 10 (j, no leveling

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icT"

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Figure 100. Relationship between the strength of single short stimuli and the size of the presynaptic excitability changes of single mechanoreceptor afferents. In A and B, the central threshold (ordinate) was measured 30 msec, after a conditioning skin displacement at the strength indicated by the abscissa; the stimulus was applied to the middle of the large pad. In A the stimuli ranged up to 27 ¡J. (abscissa, linear scale); the black circles plot the results from a touch corpuscle afferent, the white symbols are from phasic units (o, PC-; • , RA-; A, hair follicle-unit). In B, the conditioning stimuli ranged up to 600 ¡i (abscissa, log scale); the black symbols are from touch corpuscle afferents, the white circles from a PC-receptor and the other white symbols from hair follicle units. (From Janig, Schmidt & Zimmermann, 13.)

off of the curves was seen. Extending the stimulus range up to 600 n (Figure 100B) further emphasized this latter difference: the thresholds of the phasic units (white symbols) changed very little with the higher stimulus strengths, whereas those of the tonic units fell considerably. An interpretation of these findings will be given after the results obtained during constant pressure pulses have been reported. The PAD during Activation of Tonic

Mechanoreceptors

As outlined in connection with Figure 99, a constant force stimulus evokes an afferent input from SA-receptors only. The white and black symbols in Figure 101A plot the time-course of the threshold changes in the afferent terminals of phasic and tonic mechanoreceptor units respectively during the application of a 1000 g constant force stimulus to the central pad. It is seen that, in the tonic afferents, the threshold fall was large and persisted throughout the stimulus whereas, in the phasic afferents, the fall in threshold was smaller and decayed to zero in less than 50 sec. A further step in the analysis of this difference is shown in Figure 101B. The white and black cir-

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15 s

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Figure 101. A: Time-course of the presynaptic excitability changes during constant force stimulation; as indicated by the horizontal bar, a pressure of 1000 g was applied for 140 sec. to the large pad; the ordinate gives the resulting changes in central threshold of phasic and tonic mechanoreceptor units (see symbols). B: Relationship between the strength of constant force stimulation (abscissa) and the size of presynaptic excitability changes (ordinate); the circles are from a touch corpuscle, the measurements were taken 15 sec. (white circles) and 60 sec. (black circles) after the onset of the stimulus; the squares are from a RA-receptor, taken at 15 sec. of pressure application.

cles plot for a tonic afferent unit the relation between stimulus strength and threshold 15 sec. (°) and 60 sec. (•) after stimulus onset. As could be expected from Figure 101A, the changes at 60 sec. were smaller than those at 15 sec., but both curves showed a steep fall in threshold in the stimulus range up to 500 g and little additional change with stronger stimuli. Extrapolation of these and other curves showed that the smallest weight required to produce a threshold change might have been about 20 g. In the steady state, such a small weight produces very few discharges from SA-receptors. A 50 g weight, which always produced a fall in threshold in tonic afferents, evokes about 200 impulses per second, 15 sec. after its application. The white squares in Figure 101B show corresponding measurements on a phasic unit. Although this fiber exhibited clear falls in threshold after phasic volleys (for example, to 84 per cent after an 8 n stimulus), no change in threshold could be seen 15 sec. after the onset of constant force stimulation. It should be mentioned that in other phasic afferents small changes in threshold were detected which usually were independent of the stimulus strength ( 1 3 ) .

The Specificity of the Tonic and Phasic PAD Feedback

Pathways

It is evident from Figure 100 that phasic volleys depolarize preferentially

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the afferent terminals of phasic receptors, while Figure 101 has shown that in the course of constant force stimuli, only the terminals of tonic receptors were continuously depolarized. In Figure 100B, short stimuli were used up to a range in which not only the phasic PC-receptors were activated but, from approximately 20 p upwards, an increasing amount of SA-receptors, which after such a short but vigorous stimulus respond with a burst of impulses. It was seen that this inflow produced very small additional excitability changes in the afferents of phasic units whereas, in parallel to the increasing SA-input, the tonic units were depolarized to a larger and larger extent. It must be concluded from the results presented so far, that two systems exist to generate PAD in cutaneous afferents, both in the character of negative feedback. One system is activated by impulses from rapidly adapting, low threshold receptors and preferentially depolarizes the terminals of such afferents; the other system operates correspondingly on the slowly adapting units. There is evidence that one type of hair follicle receptors takes an intermediate position (13). They behave like phasic units when exposed to a phasic input but they are also depolarized, though to a lesser extent, by tonic impulses. The crosslink from the phasic system to the tonic afferents is very weak with the touch corpuscle units of the hairy skin and usually somewhat larger with the SA-units. The tonic system can be activated, not only by pressure pulses applied to the central pad, but also, and equally well, by pressure pulses to the hairy skin. Thus the tonic system is fed by SA-receptors and touch corpuscle units. As input to the phasic system only the PC-receptors were reliably determined, since it was not possible to activate populations of the other rapidly adapting receptors (RA- and hair follicle units) in isolation. Probably this imperfection applies to all natural stimuli and is, therefore, of minor importance. FUNCTIONAL SIGNIFICANCE

It has been postulated, in the introduction of this paper, that the central nervous system is in need of inhibitory mechanisms to adjust its input sensitivity to the stimulus intensity level. The strong negative feedback character of the PAD generators, described here, makes them excellent candidates for this task. The lack of inhibition at the beginning of an afferent inflow provides a high dynamic sensitivity of the afferent transmission systems, whereas the inhibition during a sustained stimulus (that is, pressure for the tonic, and vibration for the phasic PAD generators) expands the stationary input range. From the viewpoint of information economy this inhibitory adaptive influence means a reduction of redundancy in the course of a steady state stimulus. The independence of the phasic and tonic PAD systems also ensures an optimal performance of the cutaneous transmission systems under varying external conditions. For instance, PC-receptors do not change their thresh-

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olds appreciably when a static skin pressure acts upon the covering skin area (14). By virtue of this property, they are excellently suited for the signaling of small fast changes superimposed on a stationary stimulus. Since the PC-afferent terminals are free of any tonic inhibitory influence, an incoming volley in these fibers always finds the input channel at its highest sensitivity irrespective of a pressure being present or not. ( I t may be added that in respect to the other skin modalities, the distinct separation of the phasic and tonic P A D systems points to a much more detailed organization of the presynaptic inhibition of cutaneous afferents than the experiments using electrical stimulation were able to indicate.) Finally, the topographical organization of the P A D should be mentioned. It has been shown (13, 21) that the reflex pathways of both the phasic and the tonic P A D systems are interconnected in such a way that the feedback inhibition is strongest in the immediate neighborhood of the stimulus and decreases with increasing distance from the stimulus point, thus forming a system of lateral or surround inhibition. The possible significance of this organization has been considered previously (19, 21) and it suffices to say that it is probably important for the restoration of spatial contrast in the stimulus pattern lost at the periphery by the considerable overlap of the receptive fields of the primary receptors. Lundberg: I congratulate Dr. Schmidt on his very interesting results, which give further evidence for the specificity of connections to primary afferent terminals. I think his findings should be of special interest to those who still do not believe that primary afferent depolarization is mediated through specific neuronal pathways. I have two questions: Have you tried to stimulate the interosseus nerve, which contains many afferents from Pacinian corpuscles? M y second question concerns the primary afferent depolarization evoked from the flexor reflex afferents. Which kind of cutaneous afferents are responsible? Schmidt: I also would like to know which peripheral receptors give rise to the flexion reflex. Certainly none of the three I have described, at least not at the stimulus strengths we have been using. The stimuli, as I pointed out, are in the physiological range, where one would not expect to evoke a flexor reflex. In answer to your first question: W e did stimulate the interosseus nerve, but this was very early in the study; at that time we did not yet know about the clear-cut separation between tonic and phasic afferents because we were not applying the right types of mechanical stimulation. Therefore, the results were not different from those obtained by electrical stimulation of cutaneous nerves. Fuortes: I did not quite understand the evidence for your statement that primary afferent depolarization requires activation of interneurons. Schmidt: The primary afferent depolarization of phasic afferent fibers starts after peripheral stimuli between 0.3 (j and 3.5 indentation, the median

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being 0.8 n- Such stimuli will activate between 1 and 20 PC-receptors, 3 being the mean value. Thus it appears possible that a single afferent impulse from a Pacinian corpuscle may activate this P A D reflex pathway. This pathway includes at least two interneurons. Therefore, the more general conclusion from these results is that a single impulse from a phasic receptor may be able to activate multineuronal reflex chains. Fuortes: I think that the primary afferent depolarization is the same phenomenon which used to be called "dorsal root potential". It was shown some time ago (10, 11) that activation of a single fiber originating in skin or in muscle receptors can evoke a dorsal root potential in that same fiber. My question is, then: How can you tell whether the depolarization is evoked primarily in the excited fiber itself or requires activation of interneurons? Schmidt: W e can do that very easily, because in all experiments a continuous recording from the afferent fiber under observation was made, and the P A D was only measured when the peripheral stimulus did not evoke activity in that fiber. But I can also show you records where the excitability changes due to afterpotentials are seen to be clearly different from those induced by PAD. Kennedy: I have a question for the group. I am a collector of instances in which single afferent fibers or single central neurons seem to have a powerful set of postsynaptic actions. This seems to be a good example, and I wonder if there are others in mammalian systems. I recall hearing, for example, that the pinna reflex in cats can be evoked by stimulation of a single hair. One has a number of examples of reflexes that can be evoked by single afferents in invertebrates, but they seem to be rarer in mammals. Schmidt: The movement of a single hair does not necessarily evoke only one action potential. It may result in a whole burst of action potentials. Kennedy: In your preparation, can you be getting only a single action potential? Schmidt: W e get only one with this type of stimulus, which is only 2 msec, long; it never evokes repetitive activity. Increasing the stimulus duration to more than 2-3 msec, results in two impulses, one at the beginning and one at the end of the stimulus, but in our stimulus situation there is only one impulse. Kandel: Could you then review for us what is known about the electrophysiology of the two interneurons in the chain of the P A D reflex? Schmidt: Nothing is known. Eccles, Kostyuk and 1 ( 8 ) made a search for interneurons which could be in the chain of the cutaneous primary afferent depolarization. W e found interneurons which had the required properties, the D-cells, but all these experiments were done with electrical stimulation of peripheral nerves. Now we have to find cells which are activated by phasic or tonic mechanical stimulation only. Kandel: Is it conceivable that the fibers could interact directly without requiring interneurons?

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Schmidt: A direct interaction is difficult to imagine. For instance the central latency is usually between 2 to 3 msec, or longer. Kandel: You are postulating an unusual pathway to begin with, so that you might have a process that has a long delay, even without requiring interneurons. I am not sure what your evidence is for the reflex involving two interneurons. Is that a necessary postulate? Schmidt: It is the postulate that best fits all of the observations that have so far been made on the system. Lundberg: There is some evidence that the process itself does not require a time interval as long as the segmental latency. With intraspinal stimulation close to the terminals that are being recorded from, it is possible to evoke a primary afferent depolarization with considerably shorter latency. There is another type of indirect evidence, which to me strongly suggests that the primary afferent depolarization is mediated by specific interneuronal pathways. Descending volleys or activity in other primary afferents can give selective inhibition of transmission to the terminals of one primary afferent system without interfering with transmission to other afferent terminals. Kandel: But why would you have to postulate interneurons for that? The same could occur by direct terminal interaction. Lundberg: It is difficult to imagine that terminal interaction could give such a selective inhibition. For example, la and lb afferents both terminate in the intermediary region and yet you can obtain an inhibition of transmission to la but not to lb afferent terminals. The most likely explanation is a selective inhibition of interneurons transmitting depolarization to la afferent terminals. I am discussing inhibition produced by neuronal activity, not by drugs. Conditioning volleys do not give any measurable effect in la afferent terminals, they merely prevent the depolarization from being produced in these terminals; however, my interpretation is based on the existing knowledge of synaptic mechanisms in the central nervous system. If new mechanisms, for example neuronal inhibition through receptor blockage, are discovered manyfindingsmust be reinterpreted. Willis: Evidence for at least one interneuron in the presynaptic inhibitory pathway comes from the demonstration of facilitation. For example, it is possible to show that the effects of two separate pathways stimulated simultaneously may exceed the sum of the actions of the same pathways stimulated separately. This has been reported for the presynaptic inhibitory pathways to group la terminals (9). It is more difficult to show facilitation in the pathways to cutaneous afferents because of the large effects produced by small volleys. I would think that Dr. Schmidt should be able to use his preparations to re-examine the question. Another finding which may be pertinent comes from some of van Harreveld's experiments. He recently described a monosynaptic dorsal root reflex (23) which appears after the spinal cord has been asphyxiated. Since dorsal

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root reflexes in unasphyxiated animals have a longer latency, this may be the exception that proves the rule. Purpura: I wonder if anyone can answer my question of the relationship of these interneurons to the different types of elements shown previously by Dr. Scheibel. Schmidt: The D-cells were all found in the same region in the base of the dorsal horn at a depth of 1.65 to 2.5 mm from the dorsal surface. Lundberg: From a physiological point of view there is only one type of interneuron that is reasonably well identified; that is the Renshaw interneuron of the recurrent inhibitory pathway to the motoneuron. For all other pathways investigated you can find candidate interneurons that may do the job, but they have not been identified as belonging to a definite pathway. Segundo: I wonder what Dr. Lundberg means by "identified". Lundberg: I mean identified in a physiological sense as belonging to a certain pathway. It is likely that all afferent systems connect with a variety of neuronal pathways in the spinal cord. The afferents from the pad receptors, investigated by Dr. Schmidt, probably feed interneuronal pathways, not only to primary afferent terminals, but also to motoneurons and ascending pathways. How are you going to tell to which of these pathways an interneuron, activated from the afferents, belongs? Kandel: Has anyone ever shown in the case of the Renshaw cells that stimulating them very selectively produces IPSPs in motoneurons? Lundberg: I doubt that such an experiment would have much value, because whatever region in the spinal cord we stimulate, we probably also activate neurons other than those we are aiming at. Maynard: Am I correct, Dr. Schmidt, that you think it very unlikely that the afferents interact but that you do not entirely exclude the possibility that, in addition, there may be direct interaction without interneurons? Your evidence would not exclude a direct interaction at the terminals. Schmidt: It is at least conceivable that there could be a mechanism, of which we know nothing, which could produce PAD instead of a reflex pathway with interneurons. Our evidence does not exclude it. But according to our present knowledge, as Dr. Lundberg has tried to point out, an interneuronal pathway, an ordinary reflex pathway such as we find everywhere in the spinal cord, is the most likely mechanism to be involved here. Kennedy: As I understand it, Dr. Schmidt has a preparation in which it is highly probable that a single afferent fiber is producing impulse activity in only one interneuron or a chain of two. The Pacinian corpuscle is a type of receptor having very strong tendencies to respond to repeated phasic stimuli; it will follow vibratory stimuli at 75 per sec. So this ought to be an excellent system in which to study the temporal as well as the spatial properties. I wonder whether you have any information on what happens to the primary afferent depolarization when you stimulate a very small number of these phasic receptors repetitively? Schmidt: We have not done much in this direction yet. It was our inten-

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tion to look at the simple situations first and then to get involved later in more complex stimulus patterns. Incidentally, the Pacinian corpuscles you mention will follow rates of stimulation as high as 400 per sec. Andersen: If you want to postulate this reflex pathway, you have to account for the time between the arrival of the afferent impulse only and the onset of the intrafiber depolarization; the place where this can most easily be done is, I think, in the cuneate nucleus. The time you have to explain is a lag of between 2.5 and 3.2 msec. So it is very unlikely, in my opinion, that the primaryfibersalone can contribute this delay. Kandel: Frankly, I am really unhappy about using a latency argument by itself to establish how many interneurons there are in a pathway. This makes assumptions about how long it takes for an action potential to conduct into the terminal, and this one really does not know. Andersen: In this case you do know, because your electrode is so close to the terminal. It is 0.2 mm from the terminal and the myelin is lost about 5 n from the terminal. The fibers lose their myelin only 5 n from the terminal. It is extremely hard to understand how the impulse can use one and a half milliseconds over the distance of 200 |j. Kandel: At the neuromuscular junction of vertebrates and of crustacea, extraordinarily sensitive techniques are required in order to show whether or not conduction occurs in the terminals, even though they can be visualized. This is not the situation in the cuneate where you are recording the whole afferent volley coming into the nucleus. You are not really focusing down on individual terminals, to see whether or not they block. Schmidt: In the cuneate nucleus, individual fibers were impaled within the terminal region. The latency measurements have been published (2). Kandel: But how do you know you are at the terminal? Did you mark this spot in order to identify that particular terminal? Brazier: There is an analogous problem at the neuromuscular junction. Katz (15) has published beautiful microphotographs of his electrode positions in the terminals and the recordings he obtained from them. Although the microanatomy in your case is very different, I expect it is the kind of data that you need for your argument. Schmidt: At the neuromuscular junction, the presynaptic action potential seems to be actively conducted over most of the length of the non-myelinated nerve twigs (5). Andersen: It is possible to obtain such information in the cuneate nucleus because the cells are concentrated in a very narrow area. The electrode cannot be farther away than 500 to 200 n from the terminals, and in those fibers that are no more than 200 m away from the terminals there are still 2.5 msec, of latency that have to be explained. Schmidt: I think we should agree that the possibility exists that some mechanism other than interneurons may be involved, but there is no evidence whatsoever of what kind of mechanism this could be. Can we leave it like that?

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Kandel: Yes. I am just making the point that I think 2.5 msec, is not really a very long time; that conduction into the terminals plus synaptic delay could be longer than 2.5 msec. Scheibel: It is in these last couple of hundred micra within the cuneate nucleus that the fiber bifurcates many hundreds of times, making a dense neuropile field whose individual axonal components may be only a tenth or a hundredth of the diameter of the original fiber. If we assume that a spike enters the nucleus at 40 or 50 meters per second, we can also conceive of a rather catastrophic decay in conduction velocity in that last 200 or 300 mp which would completely throw off your calculations. I think one has to know the exact histology of the last few hundred m|j of the presynaptic fibers before one can make assumptions about propagation into the tips—so in that regard Dr. Kandel's point is very well taken. With regard to the problem of invoking complex systems of interneurons in the spinal cord to account for PAD, the Golgi technique shows an interesting structural paradigm not previously reported. Primary afferent collaterals destined for interneuronal and motoneuronal pools, descend in tightly massed fiber groups which we call "microbundles". These are made up of primary afferent fibers of different sources and of different diameters. Although they are myelinated during the early part of their descending course, they lose their myelin sheaths before reaching their destinations. Contact between fibers of various types and sources is extremely close, and smaller caliber fibers often wind in a spiral-like fashion along the course of larger ones. As seen in sagittal section, the terminal arborizations of these elements are also tightly compressed in the rostro-caudal dimension. The possibilities for axon to axon interaction are so great that we have come to wonder whether this type of structural motif might not serve—in part at least—as substrate for PAD. In this case, delay times might be a function of branch point phenomena, field effects exerted by adjacent parallel elements, and so on. Schmidt: One measurement that can be made of the length of time it takes for the impulse to reach the end of the presynaptic terminal is the delay of the postsynaptic potential. If that delay is measured in the cuneate nucleus, it is found to be much briefer than the delay of the primary afferent depolarization. Kandel: Does not that sound like electrical transmission? Schmidt: Oh, no. The delay is not shorter than that found in other synapses. That means that the impulse runs right into the terminals in the same way as it does at the motoneurons. Andersen: I quite agree with Dr. Scheibel that the cuneate fibers divide and branch and end in various synapses; however, they are very thick fibers and are myelinated all the way down to the synapse. During the last stretch, the internodal lengths are shorter, but not to the extent that will slow conduction seriously. Scheibel: Perhaps we are not talking about the same thing, Dr. Andersen.

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The dorsal column fiber ascending from the spinal cord terminates in a very dense bushy arbor that is totally unmyelinated. The diameter of the individual axonal bifurcations drops almost as a direct function of the number of bifurcations, and it is within this neuropile field that we are suggesting the possibility of developing delay. Andersen: I am basing my ideas on Walberg's determinations with electron microscopy (24). When the dorsal columns have been cut, the degenerating boutons are those belonging to cuneate fibers. These identified synapses are often very large indeed. According to Walberg (although he unfortunately has not done this quantitatively), large numbers of these fibers lost their myelin just within 3 to 10 m from the terminal; this is important, because it means that the outward current has to be extremely close to the terminal. So I cannot see where the loss of milliseconds occurs. But I quite agree that this is not a proof of the terminal invasion. Larramendi: In the cuneate nucleus Walberg (24) has observed axoaxonic contacts upon what he has identified as primary sensory terminals reaching this nucleus. Unless we know the numbers of axoaxonic contacts, statements based on impressions may be misleading. Andersen: That is true. However, we do not know how significant this is, because one finds what one is looking for. Admittedly, in the cuneate, Walberg found very few axoaxonic terminals. Yet there is strong depolarization, as judged by measuring the excitability of single fibers. This can be done in the gracilis nucleus, firing back into the sural nerve and using various mechanical conditioning stimuli. There is a discrepancy between the relative paucity of axoaxonal contacts and the very dramatic fiber depolarization. The axoaxonal contacts have been measured, and the number is on the order of one in several hundred contacts. Clemente: I wish to make a comment regarding the apparent differences between Walberg's reports (24) and what Dr. Scheibel discussed. The initial stages of Wallerian degeneration are characteristically identified in nerve terminals by the swollen appearance of the terminals. This is thought to be due to the imbibition of water, and I wondered if the differences in diameter of the terminals, as observed in the electron microscope and in the Golgi preparations, is not caused by the fact that, in one, the terminals were degenerating and, in the other, they were normal. Andersen: We all are aware that electron microscopy has its pitfalls; the field we see is so small that we do not know exactly what we are looking at. But with the light microscope one finds two classes of synaptic terminals, large ones and small ones; it is mostly the large ones that degenerate following spinal cord transection. But I admit that this is only a qualitative statement. Quantitatively, we do not know what to say for sure. Tauc: Concerning presynaptic inhibition, I would like to ask what this group thinks about the type of contact which exists between interneurons and postsynaptic fibers, or in between these fibers. I ask this question be-

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cause, if I remember rightly, Szentagothai ( 2 2 ) said at Stockholm in 1966 that the serial synapses are not found in places where one finds presynaptic inhibitions. If one finds them at all, they are of opposite polarity. Is there any new information about this problem? Willis: I believe that Ralston (18) at Stanford has shown that axoaxonal complexes are common in the dorsal horn, where presynaptic inhibition of cutaneous afferents appears to take place. In experiments in which dorsal root fibers were cut, he showed that the polarity of the complexes is generally in the opposite direction, although complexes of the opposite polarity are also seen. Tauc: Again on this question of presynaptic inhibition, and with reference to Dr. Schmidt's experiments, I would like to ask him how many fibers show this depolarization on stimulation of the receptor? I get an impression that this is a very diffuse effect, that practically any of these fibers show a change. I do not think that by stimulation of one receptor one could activate 50 interneurons. You have to admit that this interneuron would have to have contact everywhere with any of these fibers, which would be a very complex anatomical structure. Schmidt: This is another story which I did not go into at all. Using appropriate experimental conditions it can be demonstrated that the PAD has a topographic organization: the PAD decreases when the conditioning stimulus is moved away from the receptive field of a unit. The topographic arrangement is found both with the phasic and with the tonic PAD system (21). On the average, stimulating about 50 mm from the unit under study results in about half as much excitability change as when stimulating close to the unit. Llinas: Dr. Andersen mentioned intracellular recordings from presynaptic fibers. What were the equilibrium potentials for the depolarization during presynaptic inhibition? Would you say that the equilibrium potential was near zero resting potential? Andersen: I have personally never seen an intrafiber recording where the spike is more than 15 mV; and I do not know what the membrane potential is—probably about 15 or 20 mV. Llinas: I am interested in the equilibrium potentials because recently Katz has suggested that CI" may be involved in the mechanism for presynaptic inhibition in the central nervous system (16). Lundberg: It is indeed regrettable that our information about the synaptic mechanisms involved is so limited. Nevertheless, I believe that the dorsal column nuclei offer an opportunity for a further analysis because it is possible to record very close to the terminals. In the spinal cord it is probably much more difficult to get close to the terminals. REFERENCES 1. Adrian, E. D., and Zotterman, Y., The impulses produced by sensory nerve

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5.

6.

7.

8.

9.

10. 11. 12.

13.

14. 15.

16. 17. 18. 19.

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endings. Part 3. Impulses set up by touch and pressure. J. Physiol. (London), 1926, 61: 465-483. ANDERSEN, P., ECCLES, J. C . , SCHMIDT, R. F., and YOKOTA, T., Depolarization of presynaptic fibers in the cuneate nucleus. J. Neurophysiol., 1964, 27: 92-106. ARMETT, C . J., GRAY, J. A . B., HUNSPERGER, R. W . , and L A L , S . , The transmission of information in primary receptor neurones and second-order neurones of a phasic system. /. Physiol. (London), 1962, 164 : 395-421. ARMETT, C. J., and HUNSPERGER, R. W., Excitation of receptors in the pad of the cat by single and double mechanical pulses. J. Physiol. (London), 1961,158: 15-38. BRAUN, M., and SCHMIDT, R. F., Potential changes recorded from the frog motor nerve terminal during its activation. Pfliiger Arch. ges. Physiol., 1966, 287: 56-80. DUNCAN, D . , and KEYSER, L . L . , Further determinations of the numbers of fibers and cells in the dorsal roots and ganglia of the cat. J. Comp. Neurol., 1938, 68: 479-490. ECCLES, J. C . , The Physiology of Synapses. Academic Press, New York, 1 9 6 4 . ECCLES, J. C., KOSTYUK, R. G., and SCHMIDT, R. F., Central pathways responsible for depolarization of primary afferent fibres. J. Physiol. (London), 1962,161: 237-257. ECCLES, J. C., SCHMIDT, R. F., and W I L L I S , W . D., Presynaptic inhibition of the spinal monosynaptic reflex pathway. J. Physiol. (London), 1962, 161: 282-297. FESSARD, A., and MATTHEWS, B. H. C., Unitary synaptic potentials. J. Physiol. (London), 1939,95: 39-41P. FUORTES, M. G. F . , Potential changes of the spinal cord following different types of afferent excitation. /. Physiol. (London), 1951, 113 : 372-386. HOLMES, F. W., and DAVENPORT, H . A., Cells and fibers in spinal nerves. IV. The number of neurites in dorsal and ventral roots of the cat. J. Comp. Neurol, 1940, 73: 1-5. JANIG, W., SCHMIDT, R. F., and ZIMMEBMANN, M., Single unit responses and the total afferent outflow from the cat's foot pad upon mechanical stimulation. Exp. Brain Res., 1968, 6: 100-115. Two specific feedback pathways to the central afferent terminals of phasic and tonic mechanoreceptors. Exp. Brain Res., 1968, 6: 116-129. KATZ, B., The transmission of impulses from nerve to muscle, and the subcellular unit of synaptic action. Proc. Roy. Soc. London B, 1962, 155: 455-477. •—•——, Nerve, Muscle, and Synapse. McGraw-Hill, New York, 1966. LANGLEY, J. N., Antidromic action. Part I. J. Physiol. (London), 1923, 57: 428-446. RALSTON, H. J., Dorsal root projections to dorsal horn of cat spinal cord. Anat. Rec., 1966, 154: 406. SCHMIDT, R . F . , The functional organization of presynaptic inhibition of mechanoreceptor afferents. In: Structure and Function of Inhibitory Neural Mechanisms (C. von Euler, S. Skoglund and U. Soderberg, Eds.). Pergamon, Oxford, 1968: 227-233.

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20. 21. 22.

23. 24.

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R. F., SENGES, J., and ZIMMERMANN, M., Excitability measurements at the central terminals of single mechano-receptor afferents during slow potential changes. Exp. Brain Res., 1967, 3: 220-233. , Presynaptic depolarization of cutaneous mechanoreceptor afferents after mechanical skin stimulation. Exp. Brain Res., 1967, 3 : 234-247. SZENTAGOTHAI, J., Synaptic structure and the concept of presynaptic inhibition. In: Structure and Function of Inhibitory Neuronal Mechanisms C. von Euler, S. Skoglund and U. Soderberg, Eds.). Pergamon, Oxford, 1968: 15-31. V A N HARREVEUD, A., and N I E C H A J , A., A monosynaptic component of the dorsal root reflex in the cat. Fed. Proc., 1967, 26: 433. WALBERG, F., Axoaxonic contacts in the cuneate nucleus, probable basis for presynaptic depolarization. Exp. Neurol., 1965, 13: 218-231. SCHMIDT,

CONVERGENCE OF EXCITATORY AND INHIBITORY ACTION ON INTERNEURONES IN THE SPINAL CORD* ANDERS LUNDBERG University of Göteborg Göteborg, Sweden

At an early stage the microelectrode technique was employed in the study of interneurones in the spinal cord (10, 23, 31, 41, 42). Criteria were given for identification of interneurones and their properties and synaptic activation described. This survey will deal with the functional organization of interneuronal pathways in the spinal cord. There are two approaches to this problem. A study of the synaptic actions mediated by polysynaptic pathways permits deductions regarding the interneuronal organization in these pathways. Recording from interneurones is a more direct approach, but there is the difficulty of identifying interneurones as belonging to a certain pathway. A combination of these approaches is often used and has met with noticeable success; it has been possible to find interneurones which conform with the known properties of polysynaptic pathways to motoneurones (6). Before discussing some recent results, it may well be worth considering whether the study of the functional organization of the spinal cord does require a more detailed analysis of interneurones. Is it not sufficient to know that there exist interneurones which may transmit synaptic actions in a given reflex path? The answer is "no". Reflex pathways or other neuronal paths do not function independently but interact. This interaction constitutes one of the main elements in the integration in the central nervous system and the interneurone is of crucial importance as a site for interaction. Evidence for interaction at an interneuronal level can be obtained from indirect evidence, but in order to explore the mechanism of the interaction and the importance it may have under different conditions, it is necessary to study interneurones with intracellular techniques. •Work supported by the Swedish Medical Research Council (Project No. B69-14x-94-05A). Abbreviations used in this paper. ABSm: anterior biceps and semimembranosus; BS: brain stem; co: contralateral; Cx: cortex; DLF: dorsolateral funiculus; DOPA: 1-3-4-dihydroxyphenylalanine; DP: deep peroneal; DRP: dorsal root potential; EPSP: excitatory postsynaptic potential; FDL: flexor digitorum and hallucis longus; FRA: flexor reflex afferents; G-S: gastrocnemius and soleus; HS: hamstring; i: ipsilateral; IPSP: inhibitory postsynaptic potential; NR: nucleus ruber; PAD: primary afferent depolarization; PBSt: posterior biceps and semitendinosus; PI: plantaris; Q: quadriceps; SP: superficial peroneal; Sur: sural; Tib: tibial. 231

THE

232

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I . CONVERGENCE F R O M P R I M A R Y

AFFERENTS

INDIRECT EVIDENCE REGARDING CONVERGENCE ONTO INTERNEURONES

In this section three examples will be given of interactions between neuronal pathways from which it can be inferred that spinal cord interneurones are excited from one primary afferent system and inhibited from another. Two of the examples relate to pathways to primary afferent terminals which in many respects are easier to analyze than the pathways to motoneurones. a. Inhibition from the Flexor Reflex Afferents (FRA) of transmission from la afferents to la afferent terminals. The original observation, illustrated in Figure 102 was that the DRP evoked by volleys in la afferents from flexors is profoundly depressed when preceded by a volley in the FRA (46). From previous investigations there was reason to believe that the conditioning and

cond Sur

L 1 0 0 msec

Sur-»PBSt la DRP % of test

Figure 102. Depressive action by volleys in the FRA on the DRP evoked from la afferents of flexors. Spinal cat under chloralose anaesthesia. The upper traces in A-E are DRPs recorded from the most caudal dorsal rootlet in L6, and the lower traces are recorded from the L7 dorsal root entry zone. In A there is stimulation of the sural nerve; B shows the test DRP evoked by a train of maximal la volleys from PBSt; the stimulus strength is indicated in multiples of threshold for the nerve. In C-E there is combined stimulation of the Sur and the PBSt nerves at different time intervals; the depression of the test la DRP is plotted in the graph. The inset diagram shows the connection through which volleys in the FRA depress transmission from la to la afferents. Terminals with two branches are excitatory. Circles indicate presynaptic terminals on presynaptic endings. In the diagram, a single interneurone may represent a chain. (From Lund, Lundberg & Vyklicky, 46.)

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233

testing volleys did not evoke PAD in the same afferent terminals (cf. Eccles, 8) and it was therefore likely that the DRP depression was caused by inhibition of transmission from la afferents to la afferent terminals. This hypothesis has been fully substantiated by further analysis with intracellular recording from primary afferents. A PAD in an la afferent can be depressed by FRA volleys which do not change the membrane potential in the fiber. This depression is as long-lasting as the DRP evoked from the FRA and this finding led to the suggestion that the inhibition was presynaptic, caused by depolarization of interneuronal terminals (see inset diagram in Figure 102). It has not been possible to test this suggestion further, however, preliminary observations indicate that the inhibition has an earlier onset than the DRP evoked from the FRA. Hence it is possible that an additional inhibitory mechanism operates to inhibit transmission from la afferents to la afferent terminals. b. Alternative pathways from the FRA to primary afferent terminals. An intravenous injection of DOPA causes a profound change of transmission to primary afferents and motoneurones in acute spinal cats (2). The early DRP from the FRA, which is believed to be due mainly to a PAD in the FRA (13, 46) is depressed, and a late large DRP appears (Figure 103). An analysis of the origin of the late DRP, with excitability testing by Wall's method (57), revealed a Pad in la afferent terminals but not in lb or cutaneous afferents (3). These findings have recently been confirmed by the use of the intracellular technique by Bergmans, Burke, Fedina and Lundberg.0 Since the same volleys have effect on different afferent systems before and after DOPA, there must be alternative pathways from these afferents. Our interpretation of the finding is given in the diagram of Figure 103. The short latency pathway in A is normally active in the acute spinal cat and exerts an inhibitory action on the pathway from the FRA to la afferent terminals. DOPA is assumed to act by liberation of the transmitter from a descending noradrenergic pathway, which inhibits transmission in pathway A (1). Hence there will be release of pathway B from the inhibitory action normally exerted from pathway A with the result that pathway B is able to transmit. As far as interneuronal processes are concerned the interesting suggestion to be made is that according to the diagram in Figure 103 some interneurones should be both excited and inhibited from the same primary afferent system. c. Inhibitory interaction between pathways transmitting late reflex actions to extensor and flexor motoneurones. After DOPA there is also an interesting change in transmission from the FRA to motoneurones. The short latency flexor reflex, characteristic of the acute spinal cat, is depressed and a late reflex discharge appears (2, 37). There are probably alternative pathways to motoneurones such as have been inferred above from the pathways to primary afferent terminals (see Fig. 16 in reference 37). The late reflex discharge is evoked from both the ipsilateral and the contralateral FRA, the * Unpublished observations.

234

THE

FRA

INTERNEURON

la

are recorded from the L7 dorsal root entry zone. The recordings shown in A-E were made before DOPA was given, the records F-J immediately below show the corresponding eifects after injection of DOPA (100 mg/kg). F and } are also shown at slower speeds in K and L, respectively. Stimulus strengths (multiples of threshold) for the ABSm nerve are shown beside the relevant records; the sural nerve was stimulated at a strength about ten times threshold. Time scale under I applies to A-}, that under L to K also. Voltage scale applies to all DRPs. The diagram gives a schematic representation of the neuronal pathways involved in the depolarization of la afferent terminals evoked by stimulation of the FRA. Pathway A is activated in the acute spinal cat, and inhibits pathway B, so that normally no depolarization of la terminals results from activation of the FRA. The black interneurone is inhibitoiy, but it is not known if it operates postsynaptically or presynaptically by depolarizing the terminals of interneurones. Pathway A can be partially or completely suppressed by injection of DOPA, thus removing the inhibition of pathway B, which now operates to give a long-latency, long-lasting depolarization of la terminals when the FRA are stimulated. Pathway C is responsible for the depolarization of la terminals evoked by activation of la afferents; it may share interneurones with pathway B. A single interneurone in the diagram may represent a chain of interneurones. (From Andén, Jukes, Lundberg & Vyklicky, 3.)

former gives a discharge in flexor motoneurones, the latter in extensor motoneurones. Experiments with interaction of volleys from the two hindlimbs revealed a reciprocal organization in that either flexor or extensor motoneurones were activated. Intracellular recording from motoneurones was employed for the further analysis. Figure 104 shows records from a flexor motoneurone. An early and a late EPSP is evoked from the ipsilateral FRA but there is no corresponding IPSP from the contralateral FRA ( B ) . Volleys in the contralateral FRA are able completely to inhibit transmission of the longlasting EPSP from the ipsilateral FRA. The time-course of this inhibition is given in the graph. It is of special interest that this inhibition occurs at a conditioning-testing

CONVERGENCE

A 11 i PBSt 1 ®

B

W s r 5msec

cond co C G-S 54

ON

INTERNEURONES

235

test i Sur 5 L , i i t

V

N v

V —

E cond • test F

i

v^y,

v KJ 200 msec

% of test 120-1 100-

80 H

60 40 H

i FRA

200 msec 10 mV

co FRA

T 7 T

• early EPSP o late EPSP

20 0 - aqgpi^-o-jap^i—o 1—oo—| 009100 200 300 400 500

600msec

Figure 104. The effect of conditioning volleys in the contralateral FRA on early and late components of the EPSP evoked from ipsilateral FRA in a flexor motoneurone. Upper traces are intracellular from a PBSt motoneurone and lower traces are from the L7 dorsal root entry zone. DOPA (100 mg/kg) had been given. The effect of conditioning volleys in the contralateral FRA is shown in B. The test responses from the ipsilateral FRA, shown in C and D, were evoked by single volleys in the sural nerve. E-l illustrate the effect of combined stimulation at different intervals of the contra- and ipsilateral nerves. The double records in C, D and I were obtained simultaneously at two different sweep speeds, the arrows marking the beginning of the expanded right sweep, and the time calibration corresponding to 200 msec, in all the records in. B-I. Voltage calibration is for intracellular recording. The graph shows the amplitudes of the early and late components (see records in C, D, I) of the EPSPs from iFRA, conditioned by volleys in coFRA, plotted as a function of the interval between conditioning and testing stimulation and expressed as percentages of control test values. Line below the ordinate indicates duration of the conditioning repetitive stimulation. The diagram shows the organization of the neuronal paths giving longlasting reciprocal activation of flexor (Flex.) and extensor (Ext.) motoneurones. In this diagram an interneurone may represent an interneuronal pool. Excitatory interneurones are indicated by white circles, inhibitory interneurones by black circles. Termination of inhibitory interneurones on cell bodies merely indicates inhibition, with no commitment on whether inhibition is postsynaptic on the cell bodies or presynaptic on the terminals of interneurones. (From Jankowska, Jukes, Lund & Lundberg, 37.)

236

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interval when the early EPSP from the ipsilateral FRA is not depressed. This shows that inhibition is not produced by a conductance change in the motoneurones, there being no change in the membrane potential; neither can this inhibition be due to a PAD. It is true that the FRA volleys evoked a crossed PAD in the FRA (14) but transmission in this pathway is depressed after DOPA and the total duration of this action does not exceed 150 msec, in the unanaesthetized cat. Since the inhibition is evoked at conditioningtesting intervals far exceeding this period, it was postulated that contralateral volleys inhibit transmission from the ipsilateral side to flexor mononeurones at an interneuronal level. Corresponding findings were made on transmission from contralateral FRA to extensor motoneurones, which can be inhibited from the ipsilateral FRA. It was therefore postulated that there is a mutual inhibitory interaction between the interneuronal pathways to flexor and extensor motoneurones (37). This hypothesis is summarized in the diagram of Figure 104. RECORDING F R O M INTERNEURONES

Early investigation of the organization of interneuronal pathways employed mainly extracellular techniques. Two different approaches were made. One aimed at finding interneurones that may be part of a given reciprocal pathway such as the recurrent inhibitory pathway (10) or the reciprocal la inhibitory pathway (11). Other investigations aimed more directly at classifying and describing the location of different types of interneurones. For example, Kolmodin (40) studied interneurones activated from muscle receptors, and Kolmodin & Skoglund (43) interneurones activated from exteroceptors. The following description refers only to interneurones influenced from primary afferents. This is an important limitation because the Renshaw cells (10) are excluded; these are the most extensively investigated of the interneurones in the spinal cord and with a high degree of certainty identified as belonging to the recurrent inhibitory pathway to motoneurones (60). a. Excitatory convergence. In the investigation of the reciprocal la inhibitory pathway Eccles and his collaborators (11) found cells in the intermediate nucleus which conformed with the expected properties of this pathway, in that they were activated from la afferents of one muscle but not from lb afferents. Other interneurones in this region are excited from lb afferents but not from la afferents (9). However, records have been published of an interneiirone which receives excitatory action both from la and lb afferents (Fig. 7 in reference 7). A recent systematic investigation (33) with the intracellular technique has revealed convergence of monosynaptic excitatory action from group I muscle afferents and cutaneous afferents onto many interneurones; this convergence was found in no less than 20 of 40 interneurones with monosynaptic EPSPs from group I muscle afferents. Several investigators have ob-

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ON

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237

served that volleys in the F R A may give a late EPSP in interneurones monosynaptically activated from group I muscle afferents (9, 11, 5 1 ) . In the study referred to above ( 3 3 ) this convergence was found in seven of the 40 interneurones with group I EPSPs. Polysynaptic excitation from the FRA is also common in interneurones with monosynaptic excitation from cutaneous afferents (9, 3 3 ) . The systematic exploration of the spinal gray matter by Kolmodin and Skoglund (40, 41, 4 3 ) , employing adequate stimulation also revealed that excitatory convergence from proprioceptors and exteroceptors is common on interneurones. Finally, it should be mentioned that many of the studies of interneurones have been confined to the dorsal horn and intermediary region where there are many interneurones with monosynaptic action from primary afferents.

2msec 1.9 G

SP 2.3

¡"^v'iMii^, H

j

L

FDL 3.3

5iW

Q 1.8

J

3.3

20msec

K

13.3

i^¡^^mxs^

Figure 105. Interneurone receiving a monosynaptic EPSP from la afferents and an IPSP from the FRA. Intracellular recording (upper traces) with a citrate electrode from a cell in L7 located 2.6 mm from the cord dorsum. The lower traces in D, G, H, K, and L were recorded after withdrawal of the microelectrode to a just extracellular position. The middle traces in the latter records and the lower traces in A-F, I, and J are from the L7 dorsal root entry zone. There is separation in la and lb volleys from DP and a nearly maximal EPSP evoked in C at a strength that is maximal for the la but subthreshold for the lb volley. In I there is no effect by a maximal group I volley from Q, but an IPSP is evoked when the stimulus strength is increased in J and K to activate high-threshold muscle afferents. The intracellular traces in A-D were taken at higher gain (upper calibration) and in I-L at the lower gain, but all the extracellular records in D, G, H, and L were taken at the higher gain. The upper time calibration refers to A-D, the lower to E-L. (From Hongo, Jankowska & Lundberg, 33.)

238

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G-S

15.5

INTERNEURON

PBSt

18.7

_

2msec

9 |2nrV

20msec

Figure 106. Interneurone receiving a monosynaptic EPSP from lb afferents and an IPSP from the FRA. Intracellular recording (upper traces) with a citrate electrode from a cell located 2.5 mm from the cord dorsum. The lower traces in C-G were recorded after withdrawal of the microelectrode to a just extracellular position. The lower traces in A and B and the middle traces in C-G are from the L7 dorsal root entry zone. There is graded stimulation of the Q nerve in A-C; a maximal la volley has no effect in A, but a monosynaptic EPSP is evoked in B and C when the stimulus strength is increased to evoke a lb volley. The IPSPs in E and F are evoked from high-threshold muscle afferents. The upper time calibration refers to A-D, the lower to E-G. Voltage calibration refers to the microelectrode recording. (From Hon go, Jankowska & Lundberg, 33.)

However, the effect from primary afferents on interneurones in the ventral horn have also been dealt with by Kolmodin & Skoglund (41) and by Willis & Willis (59). b. Inhibitory convergence. Many investigators have found pronounced inhibition of the discharges in interneurones either by adequate stimulation or from volleys in exposed nerves (23, 35, 41, 43). To exclude effects caused by a PAD it is necessary to record intracellular^; a systematic investigation with recording from many interneurones has recently been made (33). It is a striking finding that the great majority of the interneurones in the dorsal and intermediary region do receive IPSPs from primary afferents. Three different afferent systems evoke these IPSPs: the FRA, the lb afferents and the low threshold cutaneous afferents. Figures 105 and 106 are examples of interneurones with monosynaptic group I EPSPs from la afferents in Figure 105, and lb afferents in Figure 106. Both interneurones receive IPSPs from the FRA. Group I volleys may also evoke IPSPs in interneurones with monosynaptic group I EPSPs, as is exemplified in Figure 107 in an interneurone with monosynaptic group I EPSPs from PBSt, DP and ABSm. There is also convergence of excitatory action from cutaneous afferents and high thresh-

CONVERGENCE

ON

4 msec

COHS 1.8

V

4.5

239

INTERNEURONES

co Sur

X

20 msec

4mVl

F i g u r e 107. Interneurone receiving monosynaptic EPSPs and disynaptic IPSPs from group I muscle afferents and, in addition, EPSP from the ipsilateral and contralateral FRA. Intracellular recording (upper traces) with a citrate electrode from a neurone located at a depth of 2.4 mm from the cord dorsum. Extracellular traces obtained after withdrawal of the microelectrode to a just extracellular position are shown in D, H, L, P and T. Middle traces in the latter records and lower traces in all other records are from the L 7 dorsal root entry zone. In A-P is shown the effect of graded stimulation of the PBSt, DP, G-S and PI nerves, as indicated. There is a monosynaptic EPSP from PBSt ( A - D ) , DP ( E - H ) , ABSm and FDL, and a small effect from G-S and plantaris, which are best seen in records L and K in comparison with the extracellular traces. Disynaptic IPSPs are evoked from DP (F-H), G-S (J-K), PI (N-P) and Q (T). The later EPSP in H and R is evoked by group II muscle afferents and the EPSP from coH is also evoked from high-threshold muscle afferents, as illustrated in U-Y. T h e stimulus strength in U is maximal for group I, but the incoming volley is recorded at lower amplification than that used in V and X. Time calibration for A-D is shown below C, for E-Y below S. U-Y were taken at the slower speed indicated below. (From Hon go, Jankowska & Lundberg, 33.)

240

THE

Q 12.8

INTERNEURON

PBSt 29.9

Sur 49.2

ABSm 10.2

K/ Nembutal 5 mg/kg

^r* Nl"

20msec



5mVI

Figure 108. The effect of pentobarbital on an interneurone receiving mixed excitatory and inhibitory effects from the FRA. Intracellular recording (upper traces) with a citrate electrode from an interneurone located at a depth of 1.5 mm from the cord dorsum. Lower records in E-H are obtained after withdrawal of the microelectrode to a just extracellular position. Corresponding upper and lower records were evoked by the same volleys before and after intravenous pentobarbital injection of 5 mg/kg. The PSPs in A-C and E-G are evoked from high-threshold muscle afferents. Throughout the recording the resting membrane potential was 65 mV and the spike potential 70 mV. (From Hongo, Jankowska & Lundberg, 33.)

old muscle afferents of ipsi- and contralateral nerves. The IPSPs in this interneurone are evoked both from DP, G-S, P I and Q, in all likelihood via l b afferents. The l b disynaptic inhibition is much more common on interneurones which receive monosynaptic excitatory action from group I afferents than on other interneurones, and it is possible that those few interneurones without group I EPSPs received the effect from a nerve that had not been dissected. The occurrence of group I disynaptic IPSPs in an interneurone does not exclude an IPSP from the FRA. Frequently, as a matter of fact, there is convergence of IPSPs from these two afferent systems. IPSPs from the FRAs are common in all types of interneurones. They are found in cells which receive a monosynaptic EPSP from cutaneous afferents and are found also in cells which receive EPSPs from the FRA. This is illustrated in Figure 108, which also shows that the inhibitory effect is remarkably sensititive to pentobarbital. A small close of pentobarbital suffices to produce a marked decrease of the IPSPs. The convergence of EPSPs and IPSPs from the FRA is rather common and is naturally of considerable interest in relation to the diagram given in Figure 103. Special mention should be made of the inhibitory effect evoked from cutaneous afferents. Many interneurones, with monosynaptic EPSPs from low threshold cutaneous afferents, receive large disynaptic IPSPs from low threshold cutaneous afferents. Sometimes, as in Figure 109, these IPSPs may

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241

Tib 4.2

jv^ -v20msec

DmVI

Figure 109. Interneurone receiving a monosynaptic EPSP from cutaneous afferents, a disynaptic IPSP from cutaneous afferents, and an IPSP from the FRA. Intracellular recording (upper traces) with a citrate electrode from an interneurone located at a depth of 1.9 mm from the cord dorsum. The lower traces in C-H were obtained after withdrawal of the microelectrode to a just extracellular position. The middle traces in these records and the lower traces in A and B are from the L7 dorsal root entry zone. Observe that, with graded stimulation of the SP nerve, an IPSP is evoked in A, and an EPSP only when the stimulus strength is raised to 1.5 times threshold in B. In B there also appears a later IPSP, which increases with stronger stimulation in C. The IPSP from the muscle nerves in F-H are evoked from high threshold afferents. Voltage calibration in D is valid for A-D, the calibration in H for E-H; A-D are taken at the faster speed, E-H at the slower. Resting membrane potential: 35 mV. (From Hongo, Jankowska & Lundberg, 33.)

have a lower threshold than the IPSPs. In all likelihood these disynaptic IPSPs are not part of the F R A response but may be mediated by a special pathway activated only from low threshold cutaneous afferents. This is clearly so in those cases in which impulses in high threshold afferents evoke EPSPs, while low threshold cutaneous afferents evoke the disynaptic IPSP (Figure 110). A similar effect is found on neurones of the spino-cervical tract and has there been proven to be evoked by afferents activated by movement of hair. The low threshold at which the IPSPs are evoked indicates that also in interneurones this inhibitory pathway is activated by these afferents ( 3 6 ) . The findings described above leave no doubt that inhibitory interaction between neuronal pathways in the spinal cord is common. It is, as a matter of fact, difficult to find interneurones in which IPSPs are not evoked from primary afferents. It is true that we may tend to overemphasize these IPSPs because many of the interneurones have low membrane potentials, 30-50 mV, and an inhibitory effect of rather doubtful functional significance may, under this condition, show up as rather large hvperpolarizing responses. Nevertheless, profound effects are common enough to permit the postulate

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Figure 110. Interneurone receiving a monosynaptic EPSP from cutaneous afferents, a polysynaptic EPSP from the FRA, and a disynaptic IPSP from cutaneous afferents. Intracellular recording (upper traces) with a citrate electrode from an interneurone at a depth of 1.4 mm from the cord dorsum. The lower traces in B-E are extracellular, obtained after withdrawal of the microelectrode to a just extracellular position. The middle traces in those records and the lower trace in A are from the L7 dorsal root entry zone. There is a monosynaptic EPSP in D from SP, followed by a disynaptic IPSP and a later wave of EPSP. In A, a group I volley from Q has no effect, but an EPSP is evoked from highthreshold muscle afferents of this nerve in B. (From Hongo, Jankowska & Lundberg, 33.)

that inhibitory interaction at an interneuronal level may play an important role. c. Interneurones transmitting late long-lasting effects from the FRA. Such interneurones have been found in the lateral part of Rexed's layer VII (38). In a few cases it has been possible to follow the discharge in these interneurones during the injection of DOPA. It has been shown that the late longlasting discharge is not evoked in them before DOPA. This suggests that the short- and long-lasting effects evoked from the FRA in motoneurones before and after DOPA represent activity in alternative pathways, just as in the case with transmission to primary afferents (cf. above). With respect to latency and duration of their discharges these interneurones have the characteristics that would be required for mediators of the late long-lasting excitation of motoneurones. Furthermore, activation is supplied either from the contralateral or the ipsilateral FRA and conditioning volleys in the other hindlimb give a profound inhibition. The graph in Figure 111 shows that the time-course of the inhibition is long-lasting. Taken together, these findings virtually prove that these interneurones transmit the late long-lasting excitation evoked in motoneurones from the FRA after DOPA. It has been demonstrated that, during the inhibition, there is complete cessation of the discharge produced by electrophoretic application of glutamate (Figure 112). This shows that there is a large excitability change in the interneurones, which probably is caused by an IPSP, although disfacilitation at present cannot be entirely excluded. Attempts so far made to record intracellularly from these interneurones have failed, probably mainly because of the large pulsations that occur in the spinal cord after an injection of DOPA. An analysis with the intracellular technique is clearly required, not only to find the mechanisms for the reciprocal inhibition, but also in order to analyze the synaptic mechanisms

CONVERGENCE A

ON I N T E R N E U R O N E S test co Sur 4 6

B

243

cond i G-S 105+tMt

m»ec Figure 111. Reciprocal inhibition in an interneurone: the effect of conditioning volleys in iFRA on the discharge evoked in an interneurone activated by the coFRA. Upper traces are extracellular microelectrode recordings and lower traces are from L 7 dorsal root entry zone; 150 mg/kg DOPA had been given. The unconditioned test response to a short train of volleys in coSur nerve is shown in A, and the effect of conditioning volleys in iG-S in B. Stimulus strengths are given as multiples of threshold strengths. The graph shows the number of spikes, counted during the first 4 0 0 msec, of the response to coSur, plotted as a function of the interval between conditioning and testing stimulation. Control values (resting stimulation alone) are indicated at the left by the mean value and the range of variation. (J. Bergmans, L. Fedina and A. Lundberg, unpublished observations.)

causing the long latency and very long duration of the discharge. The discharge may last for 30 seconds, and the most likely hypothesis is that it is caused by a closed-chain positive feedback. The functional significance that reciprocal innervation at an interneuronal level may have in alternating activation offlexorsand extensors has been discussed in the original paper (37). d. Location of interneurones. The recording of the extracellular monosynaptic focal potential evoked in the spinal cord was an important early step in determining the location of different types of interneurones. The assumption that these focal potentials were generated in the areas in which the af-

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resting

E

resting 10 nA glutam. eject

B

test co Sur 6 0

C

cond + test

G

CO Sur

60

i G-S

56

v i

5 0 0 msec D

v

1

cond i G-S 5 6 I mV

Figure 112. Inhibition of an intemeurone activated by glutamate (activated from coFRA and inhibited from iFRA). Upper traces are extracellular microelectrode recordings and lower traces are from the L7 dorsal root entry zone; 90 mg/kg DOPA had been given. There is no activity at rest (A). Stimulation of the coSur nerve gives a late discharge ( B ) , which is inhibited by conditioning stimulation of the iG-S nerve ( C ) . Stimulation of the iG-S nerve alone is without effect (D); E shows resting activity during ejection of glutamate (10 nA) from the recording electrode. Stimulation of the iG-S nerve gives a long-lasting inhibition of the glutamate-induced discharge. Voltage calibration refers to the microelectrode recordings. Electrode filled with 2M solution of glutamate. Stimulus strengths are given as multiples of threshold strengths. (J. Bergmans, L. Fedina and A. Lundberg, unpublished observations.)

CONVERGENCE

ON

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245

ferents established synaptic connections has been confirmed by later work with recording from single interneurones. Volleys in la and lb afferents give a focal potential in the intermediate nucleus of Cajal in Rexed's layer V and VI, the effect from lb afferents being somewhat more widely distributed in a transverse section. These findings were confirmed by Coombs, Curtis & Landgren ( 5 ) , who also found that large cutaneous afferents produced a monosynaptic focal potential in a region dorsal to the intermediary nucleus in Rexed's layer IV and V. These workers also mapped the distribution of focal potentials evoked from group II and III muscle afferents ( 5 ) but these potentials are not monosynaptically evoked. There is no information, from recording either of focal potentials or of discharges, about the termination of group II and III muscle afferents. Further work (33, 58) with unit recording has confirmed that there is a largely differential location of interneurones monosynaptically activated by group I muscle afferents and cutaneous afferents, but a recent study (33) does not give any indication that further subdivisions can be made at present. Interneurones with monosynaptic EPSPs were classed according to other synaptic effects, excitatory or inhibitory. There was, for example, no differential location of interneurones with group I EPSPs depending on whether or not they received disynaptic group I IPSPs, or whether volleys in the FRA evoked EPSPs or IPSPs. Great caution should be exercised in ascribing interneurones with a certain type of response as belonging to a given pathway. The Renshaw interneurones, because of this characteristic pattern of activation from alpha afferents, are well identified as belonging to the recurrent inhibitory pathway and the same holds true for the interneurones transmitting reciprocal excitation to motoneurones after an injection of DOPA. However, no other interneurones in the spinal cord can be described as identified by these criteria. A knowledge of the convergence pattern from primary afferents discussed in this section is important in future attempts to identify them. Experiments aiming at finding spatial facilitation between pathways from group I muscle afferents, cutaneous afferents and the FRA are clearly required. Naturally the inhibitory actions evoked from primary afferents are equally important in this respect. For example, it is not known if the reciprocal la inhibitory pathway to motoneurones can be inhibited or facilitated by volleys in the FRA. Likewise it would be interesting to know if transmission in the lb pathway to motoneurones can be influenced from the FRA, and also if this pathway or other group I pathways can be inhibited by group I volleys. Some difficulties with this further analysis are, however, clear: Effects evoked at an interneuronal level may be obscured by the synaptic actions that the conditioning volley produces in motoneurones. Furthermore, conclusions regarding interneuronal effects can only be drawn if the conditioning volley does not give a PAD in the terminals of the afferents used to evoke the test response.

THE

246 II.

INTERNEURON

CONVERGENCE F R O M DESCENDING

PATHWAYS

INDIRECT EVIDENCE REGARDING CONVERGENCE

a. Facilitatory effects. Three pathways with facilitatory effects on transmission in spinal reflex pathways have been analyzed in some detail. Two of them, the corticospinal and the rubrospinal, have a rather widespread effect, whereas the vestibulospinal pathway has a more specialized action, in that activity in this pathway facilitates the reciprocal la inhibitory pathway from extensor to flexor motoneurones, but affects neither the corresponding la inhibitory pathway from flexors nor any other pathway from primary afferents so far analyzed (29). The vestibulospinal tract has monosynaptic connections with extensor motoneurones (28, 47), and at the same time reciprocal disynaptic inhibition is evoked in flexor motoneurones (29). It seems probable that the facilitatory effect from the vestibulospinal tract serves not so much to facilitate the la inhibitory pathway, but that the same interneurones are employed to transmit effects from two different sources. The effect from the corticospinal tract on reflex transmission was the first one analyzed and has already been discussed in several reviews (48-50). The corticospinal impulses have a facilitatory effect on transmission in a variety of reflex pathways to motoneurones and primary afferents. It is interesting to correlate these findings with well known clinical symptoms following lesions of the corticospinal tract. The disappearance of the toe extensor reflex and the higher threshold for the abdominal and cremaster reflexes could be caused by a removal of tonic facilitation on reflex transmission. It is also known that the threshold for the flexor reflex increases following transection of the pyramids (56). Pronounced effects on reflex transmission are also evoked from the rubrospinal tract (32). In this case there is as well a widespread facilitation with marked effects on the la inhibitory pathway (Figure 113) and on the excitatory and inhibitory pathways from lb afferents to motoneurones. Clear-cut facilitatory effects on the pathways from the FRA to motoneurones have been observed in some cases rubrospinal volleys even gave inhibition of transmission from the FRA. It is of interest that at the same time as this inhibitory effect was evoked on transmission from the FRA, there could be facilitation of transmission from low threshold cutaneous afferents, which suggests that there are pathways from cutaneous afferents which are not part of the FRA pathways. Transmission in some pathways to primary afferent terminals is also facilitated from the rubrospinal tract. b. Inhibitory effects. There are many findings showing that reflex transmission can be very effectively inhibited from higher centers, but in the present context we are not concerned with inhibition caused either by a PAD or by effects to motoneurones. In order to demonstrate a depression that may be ascribed to interneurones, it should be evoked under conditions

CONVERGENCE DP

F

ON

INTERNEURONES

247

w v — - •—^ ^ B

NR

C FDLI.03

D

FDL 1.97 E NR»FDLIS7

FDL 1.97 G NR FDLI.97 H 2mV 5msec

% of test

NR->Ia

IPSP

150140130120110— 100-

3.0

T " 4.0

5.0

" T "

6.0msec

Figure 113. Facilitation from the rubrospinal tract of the la inhibitory pathway. Records A-} are from a DP motoneurone; the upper traces are intracellular records and the lower traces are from the L6 dorsal root entry zone. A, B and C show the PSPs evoked from DP, NR and F D L , respectively. The IPSP from F D L appeared with a slightly more than threshold stimulation; D and F give the IPSP evoked with somewhat higher strength of stimulation and used as a test (note different sweep speed); E and G-J show the effect of the conditioning stimulation of the NR (subthreshold for evoking the EPSP in the motoneurone) when different conditioning-testing intervals were used. The graph gives the time-course of, the facilitation with the amplitudes of the IPSPs (ordinate) plotted against the intervals between the conditioning stimulation of the NR and the arrival of the testing group I volley to the L7 dorsal root entry zone (abscissa). The arrow indicates the time of the arrival of the rubrospinal volleys to the L7 segment. The voltage calibration is for the intracellular records. The time calibrations (5 msec.) are for A, B, C-E, and F-J respectively. (T. Hongo, E. Jankowska and A. Lundberg, unpublished observations.)

when the descending activity neither gives rise to effects to primary afferent terminals nor to motoneurones. A study of the tonic depression of spinal reflex transmission in the decerebrate state led us to postulate inhibition of interneuronal transmission (4, 15). It was difficult to analyze further this tonic inhibition and in more recent experiments electrical stimulation of the brain stem was employed

THE

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INTERNEURON

(18, 2 0 ) . In order to avoid the stimuli producing postsynaptic inhibition in motoneurones, the ventral quadrants of the spinal cord were transected. Also to avoid the stimulation reaching the rubrospinal tract, the dorsal part of the lateral funiculus was transected ipsilaterally to the testing side. In this experimental situation, reticular stimuli may give a profound depression of transmission in reflex pathways without giving a P A D or a postsynaptic inhibition in motoneurones (Figure 1 1 4 ) . There is complete parallelism with the decerebrate tonic control with effect on the pathways from l b afferents and from the F R A to motoneurones, but not on the l a reciprocal inhibitory pathway. T h e system producing this descending inhibition has been denoted the dorsal reticulospinal system ( 2 0 ) . Activity in this system also effectively inhibits transmission in pathways from the F R A to primary afferent terminals but has no effect on the corresponding pathways from l a and l b afferents. Indirect evidence suggests that also other pathways with inhibitory action on interneuronal transmission originate from the brain stem ( 4 9 ) . However, no attempt has yet been made to investigate effects from these pathways by recording from interneurones. It should finally be mentioned that there may exist another type of interaction between descending impulses and primary afferents. Vyklicky and I ( 5 2 ) found that a descending primary afferent depolarization evoked by stimulation of the caudal reticular formation could be removed by volleys in the F R A . It is not yet known if this effect is caused by inhibition from the F R A of the interneurones transmitting the descending effect.

DP

G-S

I.I x

l.4x

test

H brainstem + test lOmvl 2 5 0 ms

25 ms

5 mV 10ms

Figure 114. Inhibition from the reticular formation of transmission from the flexor reflex afferents (FRA) to a flexor motoneurone. A and B, taken simultaneously at different sweep speeds, show the EPSP evoked from high-threshold muscle afferents in the gastrocnemius-soleus (G-S) nerve. In the corresponding lower records E and F, the EPSP is depressed by stimulation of the brain stem. There is no depression of the la EPSP and IPSP, shown unconditioned in C and D, and conditioned by brain stem stimulation in G and II. (From Engberg, Lundberg & Ryall, 18.)

CONVERGENCE RECORDING F R O M

ON

INTERNEURONES

249

INTERNEURONES

It has been found that impulses both in the corticospinal and in the rubrospinal tracts evoke EPSPs in interneurones, a finding which could explain the facilitatory effect on reflex transmission. Monosynaptic EPSPs are more common from the rubrospinal (Figure 115) than from the corticospinal tract (32, 5 1 ) . Both pathways can also give IPSPs in interneurones. There is a parallelism between the occurrence of IPSPs from primary afferents and from these two descending pathways (Figure 116). The more profound the inhibition is from the periphery, the larger also is the inhibitory effect evoked by the descending impulses. This suggests that the IPSPs are caused by excitation of the interneurone, transmitting inhibitory effects from pri-

5 msec Figure 115. Interneurone monosynaptically excited from la afferents from a flexor muscle and by volleys from NR. Intracellular recording with citrate electrode (upper traces) from a cell in L7 located 2.5 mm from the cord dorsum. Lower traces in I and K were recorded when the microelectrode was withdrawn to a just extracellular position. Middle traces in I, K, and the lower traces in other records are from L7 dorsal root entry zone. A-D; EPSPs evoked from PBSt nerve by increasing strengths of stimulation: submaximal for la fibers in A, B, and maximal for la and lb fibers in C and D, respectively; E-H: IPSPs evoked from G-S by increasing strength of stimulation, maximal for group I in H; I: EPSP from high-threshold skin afferents; /, K: EPSPs evoked by a short train of stimuli (/) or single shocks (K) in NR (about 0.1 mA). The latency of the EPSP from NR was 3.8-3.9 msec. Voltage calibration refers to microelectrode recording. Voltage calibration and time calibration for A-H are given in H; for I, } in J, and for K in K. (T. Hongo, E. Jankowska and A. Lundberg, unpublished observations.)

THE

250

A

INTERNEURON

B

Sur

cortex

- X \

3mV 10 msec 5 0 msec Joint

D

ABSm

E

SP

i

F

cortex

G

Joint

^ S S S ^ S ^ S ^

H

cortex+Joint WG^xgZZ^

Figure 116. Inhibitory action from the sensorimotor cortex in an interneurone receiving inhibition from the FRA. The intracellular recordings (upper traces) were obtained from a cell in the dorsal horn of L7. Lower traces in A and F-H and middle traces in B-E were recorded from the dorsal root entry zone in L7. The lower traces in B-E are microelectrode recordings obtained after withdrawal to a just extracellular position; this interneurone was monosynaptically activated by large afferents in the sural nerve (record B ) . The dominating effect of cortical stimulation was an IPSP (record B ) . C-E show that IPSPs were evoked also from peripheral nerves; the effects in C and D were evoked from high-threshold afferents. F-H illustrate spatial facilitation between the paths from the cortex and the periphery; liminal stimulation was used in F and G, and when combined in H the action is larger than the sum of the effects in F and G. Calibration between A and B refers to records B-H. The lower amplification in A was not recorded. (A. Lundberg and U. Norrsell, unpublished observations.) m a r y afferents. Support for this suggestion is given b y the finding that there m a y b e spatial facilitation b e t w e e n the inhibitory pathways from primary afferents and the descending tracts ( F i g u r e 1 1 6 ) . T h e inhibitory effect on interneurones f r o m the corticospinal and rubrospinal tracts is, therefore, in all likelihood part of their general facilitatory action on the reflex paths. W h e n a reflex p a t h w a y is mobilized, there is also facilitation of the inhibitory interactive systems through which this reflex path m a y control other reflexes. A systematic comparison has b e e n made of effects from the corticospinal

CONVERGENCE

A

PBSt 24

ON

INTERNEURONES

W^

B

SP 7.6 C

251

ex D

NR

50 msec

SP 14.3

«/V

G

cx

H

u

NR

H+t-

20msec Figure 117. Similar synaptic actions from NR and from the sensorimotor cortex. Intracellular records (upper traces) from two interneurones in L7 (A-D and E-H) located 1.6 and 2.5 mm from the cord dorsum, respectively. Lower traces in C and D were recorded after withdrawal of the electrode to a just extracellular position. Middle traces in C and D and lower traces in other records are from L7 dorsal root entry zone. NR was stimulated with about 0.1 mA and Cx with 1.3 mA. The cell inhibited from NR and Cx was excited from PBSt (probably group II fibers). SP, Tib and Sur (probably disynaptic EPSPs from skin afferents). Volleys in FRA evoked an IPSP (A, B). The cell excited from NR and Cx received a monosynaptic EPSP from group I afferents from DP (E), G-S, PI and from low threshold skin afferents in SP ( F ) and Tib. Volleys in FRA evoked either an EPSP (E, F) or an IPSP (not illustrated). Voltage calibration (referring to the microelectrode recording) and time calibration are for corresponding rows of records. (T. Hongo, E. Jankowska and A. Lundberg, unpublished observations.)

and the rubrospinal tract in our laboratory. In many interneurones very similar effects are evoked from two pathways (Figure 117), but in some cases differences were found. It is at present not known if the same reflex can be facilitated from two different descending systems, one controlled mainly from the cerebral cortex, the other mainly from the cerebellum, or whether the differences sometimes observed indicate that the two pathways are used to facilitate slightly different reflex pathways. Activity in the dorsal reticulospinal system, evoked by stimulation of the lower brain stem, gives very effective inhibition of the discharges in interneurones activated from the F R A ( 2 1 ) . When these interneurones are impaled, IPSPs are not found in the great majority of cells. It can therefore be concluded that the dorsal reticulospinal system does not inhibit reflex transmission through widespread postsynaptic effects in interneuronal chains. However, corresponding to the inhibition of the discharge, there was an effective depression of the EPSPs (and also of the IPSPs) evoked from the FRA (Figure 118). Since the brain stem stimuli did not evoke a PAD, it is postulated that the inhibition is exerted on interneurones transmitting the synaptic effects from the primary afferents to the interneurones from which the recordings were being made. Regarding the mechanism of the inhibition, it is of considerable interest

252

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B

G-S 35

test —ye**

cond 100 msec E 5 0 msec

25 msec BS 5 0 msec

Figure 118. BS depression of EPSP and IPSP in interneurones. A, C and E are intracellular records from an interneurone (KC1 electrode) at 1.9 mm depth, B, D and F from another (citrate electrode) at 2 mm depth, each with two sweep speeds. The EPSP evoked from joint afferents (A) is depressed about 30% after BS stimulation ( C ) ; B and D show very strong depression of the IPSP evoked from high-threshold muscle afferents, whereas there is no change in the early EPSP (which was monosynaptic from group I afferents). BS stimulation evokes no synaptic potential in any of these cells ( E and F). (From Engberg, Lundberg & Ryall, 21.)

that in five of 78 interneurones IPSPs were evoked from the dorsal reticulospinal system. One example is given in Figure 119. This interneurone, like the other four with IPSPs from the brain stem, received monosynaptic effects from primary afferents. Taken together, our findings suggest that the dorsal reticulospinal system inhibits reflex transmission by producing postsynaptic inhibition in first order interneurones. However, it must be emphasized that our experiments with recording from interneurones were not designed to test if the dorsal reticulospinal system may inhibit interneuronal transmission through other inhibitory mechanisms, for example presynaptic inhibition through depolarization of terminals of interneurones. There is indeed no easy way in which to demonstrate whether such presynaptic inhibition exists or not (see, however, Figure 102). It should, however, be pointed out that one difficulty in its operation would be that in cells with short axons, such as must be rather common among interneurones, the terminal depolarization would spread electrotonically to the soma and could facilitate transmission to the cell, hence activation of a larger number of cells could counteract the inhibition of transmission from each cell. Many interneurones with monosynaptic EPSPs did not receive IPSPs from the dorsal reticulospinal system. In this connection it should be noted that there are many pathways which are not controlled by the dorsal reticulospinal system, for example the l a inhibitory pathway to motoneurones, the pathways from l a and l b afferents to primary afferent terminals and a pathway from low threshold cutaneous afferents to cutaneous afferent terminals

CONVERGENCE

ON

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253

50msec Figure 119. Interneurone with synaptic inhibition from BS. Depth 1.8 mm; intracellular records (citrate electrode). A, B: Synaptic actions from high-threshold muscle afferents; inhibition dominates, but some excitation is also evoked from FDL. C-F: Excitatory and inhibitory actions from cutaneous afferents; the excitation from SP is partly monosynaptic. G: IPSP evoked by three stimulus shocks in BS. //; Onset of IPSP evoked by a single shock in BS (the beginning of the first deflection is marked by an arrow); below is the IPSP evoked by a single stimulus of the contralateral D L F in the lower thoracic region. Third traces in A-D and G are extracellular records. Time calibration for A-D under D, for E and F under F. (From Engberg, Lundberg & Ryall, 21.)

(21). Four of the five interneurones with IPSPs from the dorsal reticulospinal system received monosynaptic EPSPs from cutaneous afferents. Since cutaneous afferents are part of the FRA it is tempting to correlate the finding with the strong inhibitory control of transmission from the FRA that is exerted by the dorsal reticulospinal system. However, most of these interneurones may not belong to FRA paths; the interneurone in Figure 119 is actually inhibited from the FRA. These interneurones may be part of more specialized neuronal paths activated exclusively from cutaneous afferents. There is only a limited knowledge of such paths, one example is provided by the reflex path giving plantar flexion from mechanoreceptors in the central plantar cushion; this reflex is tonically inhibited in the decerebrate state, probably via the dorsal reticulospinal system (17). It is probable that in the flexor reflex pathway there are few first order interneurones and that the widespread effect that these afferents have is caused by divergence from a relatively small number of cells. It is not known if convergence from the different types of afferents included in the FRA (16) occur already on first order FRA interneurones. In this connection, it should be mentioned that the shortest pathway from the

254

THE

INTERNEURON

FRA to flexor motoneurones probably is trisynaptic and not disynaptic as was previously assumed (16). Convergence could occur on second order interneurones. Knowledge of the level at which convergence occurs is important for the understanding of the functional role of the descending control of the FRA pathways. If convergence occurs on second-order interneurones there is the possibility for a differential control of transmission from the various afferent systems included among the FRA. It is interesting to compare the effects evoked in interneurones from the dorsal reticulospinal system (which inhibits reflex transmission) with the effects produced from corticospinal and rubrospinal tracts (which facilitate reflex transmission). It is a somewhat surprising finding that IPSPs are more often evoked from the latter tracts than from the former. The explanation derives from the results described above: The corticospinal and rubrospinal tracts evoke IPSPs through excitation of interneurones transmitting inhibition from primary afferents, hence a process that is initially facilitatory results eventually in inhibition. The dorsal reticulospinal system, on the other hand, is primarily inhibitory; no EPSP was found in any of the 78 intracellularly recorded interneurones, and it gives equally effective inhibition of transmission in excitatory and inhibitory paths to interneurones (Figure 118). A study of the descending effects to reflex pathways and interneurones can undoubtedly supplement the information obtained from work on the convergence from primary afferents and aid future attempts to identify interneurones as belonging to a certain pathway. Of particular interest in this respect is the fact that volleys in the corticospinal tract facilitate the reciprocal la inhibitory pathway to motoneurones but not the pathway from la afferents to primary afferent terminals and that the dorsal reticulospinal system inhibits transmission in lb pathways to motoneurones but not transmission from these afferents to primary afferent terminals. Altogether, this report suffices to show that the interneurones which relay effects from primary afferents are subject to an extraordinary convergence of excitation and inhibition from many different sources. The extensive convergence from different sources strongly suggests that the interneurones in spinal reflex pathways act as an integrative center. It is outside the scope of this review to discuss how this integration may be achieved. Some aspects of it have been dealt with in a previous publication (49), and in a forthcoming paper by Hongo, Jankowska and Lundberg dealing with rubrospinal effects on reflex transmission. SUMMARY

This review deals with the convergence of excitatory and inhibitory actions on lumbo-sacral interneurones. Part I is devoted to the convergence from primary afferents with the main emphasis given to the IPSPs evoked from primary afferents. Three types of primary afferent systems evoke IPSPs

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255

in interneurones of the dorsal horn and intermediary region. Ib afferents, low threshold cutaneous afferents and the flexor reflex afferents. These results are presented in relation to examples of inhibitory interactions between spinal reflex pathways. Brief mention is also made of reciprocal innervation at an interneuronal level between interneuronal paths giving excitation to flexor and extensor motoneurones. In part II the convergence of effects from descending pathways and primary afferents is discussed. Volleys in the corticospinal and rubrospinal tracts evoke EPSPs in different types of interneurones and IPSPs are produced in interneurones which receive this effect from primary afferents. The dorsal reticulospinal system with inhibitory effect on reflex transmission probably acts through inhibition of first order interneurones, IPSPs are evoked from this system in some interneurones in which volleys in primary afferents give monosynaptic EPSPs.* Tauc: In your Figure 103, why should the inhibition go to neurone B? Could it not be presynaptic? Lundberg: Admittedly, our evidence is extremely indirect. Pathway B is tonically inhibited in the acute spinal cat also when the spinal cord is completely de-afferented. This tonic inhibition of pathway B could be due to presynaptic inhibition only if, in this state, there was a tonic primary afferent depolarization in the terminals of the FRA. For many reasons this is very unlikely and I believe our findings are much more easily explained by the assumption of an interneuronal inhibition exerted from pathway A onto pathway B. Lundberg: Cortical stimulation after a brain stem section sparing the pyramids gives the same effect. Purpura: Did you study these interneurones following suprasegmental stimulation? This is especially relevant to the problem of the central mechanism controlling alternating movements. Lundberg: We have not stimulated supraspinal structures, but know from experiments with stimulation of the spinal cord that the interneurones activated after DOPA receive descending connections. It is possible that descending impulses can start the interneurones and give alternate movements of extensors and flexors by operating these segmental mechanisms. However, this problem has not yet been investigated and I think more knowledge is required about the segmental processes before we can tackle it. Llinas: Dr. Lundberg, you mentioned that the reticulospinal system produced inhibition of interneurones and motoneurones at spinal cord level by * Added by Dr. Lundberg in proof: Since the symposium was held, an important advance has been made in the field discussed in this review. The reciprocal la inhibitory pathway is inhibited at an interneuronal level by impulses in recurrent collaterals from motor axon collaterals ( 3 3 a ) . Corresponding recurrent IPSPs are found in interneurones monosynaptically activated from la afferents. These interneurones, which may belong to the la inhibitory pathway, are located, not in the intermediary region, but dorsomedially to the motor nucleus ( 3 3 b ) .

256

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means of an inhibitory interneurone. I wonder what the evidence is for that statement. Lundberg: The inhibition of interneuronal transmission exerted by the dorsal reticulospinal system is probably mediated by one interneurone. W e have calculated the conduction velocity of the descending axon by comparing the latency for the IPSP evoked from the reticular formation with that evoked by stimulation of the descending tract in the lower thoracic region. The latency for the IPSP evoked in interneurones by stimulation of the reticular formation exceeds the conduction time by about 2 msec. Llinas: Did you find direct IPSPs following reticular stimulation, or did you find EPSPs exclusively? I am under the impression, following experiments with Dr. Terzuolo (44), that some pathways produce IPSPs exclusively. I seem to remember that Nyberg-Hansen (53) showed that the bulbar reticulospinal pathway ends directly on motoneurones, and so has a direct action. Since it has been shown that there are direct reticulospinal pathways which are, in fact, very extensive, I wondered if they could exert inhibition directly. Lundberg: Impulses in the dorsal reticulospinal system do not have any effect on motoneurones. Grillner & Lund (30) have found a pathway with a monosynaptic excitatory connection with flexor motoneurones. This pathway probably originates from reticular centers in the lower pontine region. Electrical stimulation of Magoun's inhibitory center in the medullary reticular formation evokes an IPSP in motoneurones after a segmental delay of 1.3 msec.; hence we assume that transmission is disynaptic. Kandel: I was really impressed with the very elegant aspects of the model building that you were employing; that is, trying to find interneurones that fit the models that you have. I do not have enough of a feel for the detailed physiology of the cord to know how unique are these solutions: Do these interneurones uniquely fit into the necessary pathways? Is there enough overlap in the properties of the interneurone so there might be some question as to whether it serves uniquely in one rather than in another pathway? Lundberg: Not one of all the interneurones we have recorded from in the dorsal horn and intermediate region can be identified as belonging to a certain pathway. However, I think there is some hope for the future if we take the total convergence pattern into consideration. Let me give one example. Ib afferents have known pathways to motoneurones and to primary afferent terminals. The dorsal reticulospinal system, that I discussed, effectively inhibits interneuronal transmission in the Ib pathway to motoneurones but has no effect on the pathways from Ib afferents to primary afferent terminals. A further study of the effect of supraspinal control systems and spinal interactive processes may give criteria that will allow us to differentiate between interneurones belonging to these two pathways. Kandel: Could you, for example, try post-tetanic potentiation of the path-

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ways to see whether the interneurones have the necessary properties to give these effects? Or could you try different frequencies in order to see whether there is a parametric fit between the properties of the interneurone and the pathway? Lundberg: To some extent this method was used in the early attempts to identify interneurones of the la and lb pathways. In my opinion, it is a difficult approach and I believe that investigation of the convergence pattern is a more crucial test. Fuortes: In order to understand the significance of Dr. Lundberg's contribution, it may be useful to retrace the steps which have led to our present understanding of spinal cord function. The initial studies on this subject were performed using anatomical methods; they not only describe pathways and cellular aggregates, but also they define the synapse as the anatomical substrate for the functional connections between nerve cells. We all remember the controversies between Ramón y Cajal and Golgi on this topic. One should also remember, however, that Golgi (24) was the first to point out that one of the basic principles of organization of the central nervous system is the convergence of many axons upon one cell and the divergence of the branches of one axon to many cells, a principle which is often ascribed to Sherrington who rediscovered it several years later. Following these anatomical studies, physiological investigations were performed which inferred the functions of the spinal cord from the properties of the muscular contractions induced by different types of stimulation. Using this method, Sherrington (54) described the properties of extensor and flexor reflexes and introduced the now familiar concepts of excitation, inhibition, occlusion, facilitation, and spatial and temporal summation. In an attempt to interpret the more complex features of reflex activity, Sherrington also introduced the two well-known terms: central state and integration. The notion of central state is based on the observation that identical stimuli applied within a short interval of time do not necessarily evoke the same motor response. Sherrington interpreted this observation by stating that the first stimulus had altered the "central state" of the spinal cord. Increase of action was ascribed to central excitatory state and decrease to central inhibitory state, but until recently the nature of these changes was not clearly understood. The second term which Sherrington introduced, but could not precisely interpret, is "integration". Although Sherrington's original definitions are vague, I think that we can agree to define integration as the ability of the central nervous system to coordinate incoming signals in a way appropriate to elicit a response useful for the survival of the organism. Observation of behavior is sufficient to convince us that central states and integrative abilities are important properties of the central nervous system, but elaborate experimentation is required in order to interpret the mechanisms underlying these actions.

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One of the important problems which Sherrington was unable to clarify is the role of interneurones in reflex action. Credit should be given to Alexander Forbes (22) for ascribing to interneurones the role of "delay paths" in the flexor reflex. But a greater advance was made when Lloyd (45) established a clear distinction between monosynaptic and polysynaptic reflexes, and thus gave us a tool for direct comparison of these two actions. It soon became clear that monosynaptic reflexes are simpler, more repeatable, and easier to analyze than polysynaptic actions. For this reason attention was concentrated for many years on monosynaptic reflexes, and many of us remember articles and monographs in which signs of interneuronal activity were referred to as "contamination". This is, of course, perfectly justified if the purpose of the study is to analyze the simpler functions of the spinal cord and, in fact, it was as a result of the investigations performed by Eccles on monosynaptic reflexes that the basic mechanisms of synaptic action were clarified. It is evident, however, that if the spinal cord possessed only monosynaptic reflexes it could not perform the complex and purposeful actions required for survival. In other words, it seems clear that Sherrington's central states and integrative actions must be due largely to the properties of interneurones. With these considerations in mind, several investigators started to study the features of interneuronal activity by recording the electrical changes occurring in interneurones following different types of stimulation. Systematic studies of interneuronal actions were started by Lundberg and his coworkers around 1960. As Dr. Lundberg has told us, there is in general a high degree of convergence on interneurones; some cells are excited by muscle afference and inhibited by fibers from the skin, others are inhibited by both skin and muscle, still others are excited by skin and inhibited by muscle, and so on. If we were to classify the afferent impingement as muscular or cutaneous in origin, as monosynaptic or polysynaptic and so on, and if we were to classify the response of a cell as excitatory or inhibitory, we could summarize the observations in the form of a table or of a matrix, for instance, by writing in each space the percentage of cases in which a particular observation was made. This sort of tabulation would show which actions are common and which are rare or absent. For instance, despite extensive experimentation, Dr. Lundberg has never observed monosynaptic inhibition on either motoneurones or interneurones and thus has confirmed a view that impulses of primary afferent fibers are always excitatory for spinal neurones. In his presentation, Dr. Lundberg has discussed not only segmental actions but also the effects exerted in interneurones by supraspinal stimulations. With these additions the number of possible interactions increases and a systematic tabulation of the results soon becomes impractical. What Dr. Lundberg has very skillfully done in his presentation is to give us examples of the interactions which he has observed in interneurones. He

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has then pointed out that several known properties of the spinal cord could be explained if interneurones possessing certain interactions were part of certain postulated circuits. In order to confirm the existence of these circuits it would be useful to apply to the spinal cord the type of experiments on invertebrates which were described in Dr. Kandel's presentation,1* namely simultaneous recording from one interneurone and one connecting motoneurone. However, this may well be too laborious for practical purposes. Dr. Lundberg has pointed out in several of his papers that microelectrodes may select certain populations of cells at the expense of others. It follows that the features of activity of some cells may remain undetected despite extensive sampling. It is possible, therefore, that some features of interneurones have not yet been described. For instance, it is known that numerous short-axon neurones exist in the spinal cord, and one wonders whether these cells might operate without production of spikes. This possibility was mentioned before in a different context, and it would be interesting to know whether any evidence has been found in the spinal cord. Also, we all remember that several years ago reverberating circuits were frequently postulated in order to explain long-lasting actions, but no mention of this mechanism has been made at this symposium so far, and I should like to ask whether the notion is still tenable or the evidence is now against it. Lundberg: I agree with Dr. Fuortes that a new approach is required if we are to be able to identify interneurones as belonging to a certain pathway. However, I doubt that simultaneous recording from interneurones and motoneurones will suffice. I think it will be necessary also to stimulate single interneurones and record the synaptic effect in motoneurones. May I disagree with you on one point? You said that Sherrington did not define integration. In the introductory chapter to The Integrative Action of the Nervous System (54), there is the statement that "the unit reaction in nervous integration is the reflex", and co-ordination is the compounding of reflexes. Kennedy: I submit that you have just made my point brilliantly, because in fact that is the very worst definition of integration that you could possibly use. You led us to believe that the reflex was in fact the unit of integration, and it is not. It is the set of connections made centrally that produces a pattern. Lundberg: The discussion with Dr. Fuortes was about the concepts that Sherrington operated with and at that time it was quite a reasonable concept, particularly if you include inhibitory interaction between reflexes, as indeed Sherrington did. Brazier: May I recommend your reading Granit's recent book (27) on Sherrington, because he clarifies for us a good deal of Sherrington's rather muddy description of what he thought about integration. The title of Sher* Pages 71-101.

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rington's famous book has, of course, been a stumbling block for students ever since 1906, because in Sherrington's experiments the animals usually had the head cut off. How could there be "integration of the nervous system" without a brain? As Granit points out, the title should have been "Integration in the Nervous System". Adey: I think Dr. Fuortes characterized the situation very well in emphasizing the question of state and the fact that synaptology as such is not the most direct tool by which we can approach questions of state, even though state itself is something which is the product of the synaptic interaction; and, going back once again to Sherrington's notions of integration as something that occurs in space and time, I think we have here in the spinal cord a fascinating opportunity to consider one of the very basic aspects of the genesis of ultimate states. For example, is it something in which the intrinsic interactions are those that occur within a single population of neurones which are themselves diverse, and thus capable of this type of alternations of states by local interaction? Or do we necessarily have to postulate that there will be groupings of neurones of a single characteristic which, by their action at a distance or in domains adjacent to themselves, will manifest this characteristic of alternation or change in state? Spencer: A point about the methodology of Sherrington in contrast to modern approaches. Much of Sherrington's work was done in animals with a closed gamma loop, in many cases using chronic spinal animals, often decerebrated and without anesthesia. In such active preparations it is quite easy to demonstrate reflex changes that last a very long time. I think it would be a mistake for modern investigators to rule out concepts such as interneuronal afterdischarge until more modern work is done in these same kinds of preparation, which are more normal from some points of view than those we use now. Horridge: Following Graham Brown's method (25), the origin of a rhythmical process which is elicited by a constantly maintained stimulus was re-examined in the spinal cord of the cat by Sherrington in 1913 (55). He used a pair of knee extensors, and applied faradic tetanization separately to an afferent nerve on each side, with all remaining muscles immobilized by nerve severance. Rhythmical stepping was more pronounced with both muscles deafferented. By a careful reading of this paper, however, it is not possible to exclude the beat frequency of the stimulators as the origin of the rhythm, and several of his comments about the necessarily exact equality of stimulus intensity suggest that the matter should be re-examined. Lundberg: Sherrington worked on the competition between the flexor reflex and the crossed extensor reflex and came to the idea that reflexes were building blocks of alternate movement. Graham Brown (25) demonstrated that alternate movement could be produced through intrinsic activity in the de-afferented spinal cord. His paired half-centers did not require afferent

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impulses to produce alternate movement. Dr. Horridge's story has no relation to the present discussion. Since Dr. Fuortes discussed the problem of reverberation, I would like to draw your attention to the pathway from the F R A to motoneurones, which opens up after an injection of DOPA. A short train of volleys evokes a discharge usually lasting for 1 sec. but sometimes the discharge goes on for 30 sec. or more. These long-lasting discharges may very well be caused by activity in a closed chain but I do not know how to investigate this problem. W e can certainly not exclude that processes within a single neurone may give long-lasting discharges. For example, Grampp (26) has shown that under certain conditions a single antidromic volley may give very long-lasting discharges in the stretch receptor neurone of the lobster. Brazier: A comment about reverberation: I am surprised not to hear from Dr. Maynard, because I thought this was what was implied by the diagram he showed of a circuit of ten cells in a crayfish, with a reverberation that seemed to me to include both inhibition and excitation, and which he told us went on for a long time. Maynard: I have been concerned about reverberating circuits for a long time. It is important to clarify what they are being proposed to explain: whether it is a matter of simply prolonging an impulse discharge or whether it is a matter of the organization of neuronal action into certain patterns. In the example I gave earlier, I do not think that the reverberation, or the sequential activation, is involved in prolonging activity in any of these cells. It is, however, I think, quite important in determining when a cell will discharge, and what kind of interaction between cells may occur and, in electrotonic connections, reverberation may very well be responsible for the production of the synchronous burst. But this is quite different from the proposal of reciprocal excitation as an explanation of a prolonged and a maintained discharge. It was to account for prolonged discharges that reverberating circuits were first proposed. Wiersma: I would like to support the possibility both of excited states and reverberating circuits. There is evidence that certain cells, when they are sufficiently excited, will go by themselves and run loose. If they are not inhibited they will continue to discharge. To me that is an excited state. Now, if you have an antagonistic system you will get a reverberating circuit. I think this may well be a kind of inhibition, and I think the type of thing Kennedy has described is responsible for these rhythmic movements. I believe that the inhibitory pathway has been overlooked when we were talking about reverberating circuits. Kandel: In continuation of what Dr. Kennedy said, and also as to your point, Dr. Wiersma, in Aplysia, interneurones numbers I and I I are spontaneously active and inhibit each other. Wachtel and I (39) have shown that when interneurone I goes into a burst, it will inhibit the interneurone

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II. The termination of inhibition of interneurone No. II leads to rebound excitation of that interneurone, which in turn inhibits interneurone No. I, so there is an oscillating kind of re-excitation of one neurone by the other which is not dependent on excitatory connections but due to mutually inhibitory connections and rebound excitation in each cell. A more common mechanism for afterdischarge exists in some invertebrate neurones which have endogenous rhythms and this does not require a circuit at all, because an afferent volley can produce a change in the pacemaker rhythm which persists for several seconds or even minutes. I personally doubt whether re-entrant circuits are common. First of all there are so many inhibitory elements in a chain like that, that excitation is quite likely to be damped down; secondly, I think that you can explain many specific examples of afterdischarge by simpler mechanisms than reverberating circuits. Kennedy: I would just add that if we want to continue the historical flavor, the correct reference to what is going on in these situations is not "reverberating circuits" but "paired half-centers".

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spinal cord. 2. A pharmacological analysis. Acta Physiol. Scand., 1966, 67: 387-397. ANDEN, N.-E., JUKES, M. G. M., LUNDBERG, A., and VYKLICKY, L., The effect of DOPA on the spinal cord. 1. Influence on transmission from primary afferents. Acta Physiol. Scand., 1966, 67: 373-386. , The effect of DOPA on the spinal cord. 3. Depolarization evoked in the central terminals of ipsilateral la afferents by volleys in the flexor reflex afferents. Acta Physiol. Scand., 1966, 68 : 322-336. CABPENTER, D., ENGBERG, I., FUNKENSTEIN, H., and LUNDBERG, A., Decerebrate control of reflexes to primary afferents. Acta Physiol. Scand., 1963, 59: 424-437. COOMBS, J. S., CURTIS, D. R., and LANDGREN, S., Spinal cord potentials generated by impulses in muscle and cutaneous afferent fibres. J. Neurophysiol., 1956,19: 452-467. ECCLES, J. C., The Physiology of Nerve Cells. Johns Hopkins Press, Baltimore, 1957. , Central connexions of muscle afferent fibres. In: Symposium on Muscle Receptors (D. Barker, Ed.). Hong Kong Univ. Press, Hong Kong, 1962: 81-101. , Presynaptic inhibition in the spinal cord. Progr. Brain Res., 1964, 12: 65-91. ECCLES, J. C., ECCLES, R. M., and LUNDBERG, A., Types of neurone in and around the intermediate nucleus of the lumbosacral cord. J. Physiol. (London), 1960, 154: 89-114.

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cleus and la afferents to flexor motoneurones. Acta Physiol. Scand., 1966, 68, Suppl. 277: 61. 30. GRILLNER, S., and LUND, S., A descending pathway with monosynaptic action on flexor motoneurones. Experientia, 1966, 22: 390. 31. HAAPANEN, L., KOLMODIN, G. M., and SKOGLUND, C. R., Membrane and action potentials of spinal interneurons in the cat. Acta Physiol. Scand., 1958, 43: 315-348. 32. HONGO, T., JANKOWSKA, E., and LUNDBERG, A., Effects evoked from the rubrospinal tract in cats. Experientia, 1965, 21: 525-526. 33. , Convergence of excitatory and inhibitory action on interneurones in the lumbosacral cord. Exp. Brain Res., 1966, 1: 338-358. 33a. HULTBORN, H., JANKOWSKA, E . and LINDSTROM, S., Inhibition in IA inhibitory pathway by impulses in recurrent motor axon collaterals. Life Sci., 1968, 7:337-339. 33b. — -, Recurrent inhibition from motor axon collaterals in interneurones monosynaptically activated from la afferents. Brain Res., 1968,9: 367-369. of interneurones transmitting effects from the flexor reflex afferents. 34. HUNT, C. C., and KUNO, M., Properties of spinal interneurones. J. Physiol. (London), 1959,147: 346-363. 35. , Background discharge and evoked responses of spinal interneurones. /. Physiol. (London), 1959, 147: 364-384. 36. HUNT, C. C., and MCINTYRE, A. K., An analysis of fibre diameter and receptor characteristics of myelinated cutaneous afferent fibres in cat. J. Physiol. (London), I960, 153: 99-112. 37. JANKOWSKA, E., JUKES, M. G. M., LUND, S., and LUNDBERG, A., The effect of DOPA on the spinal cord. 5. Reciprocal organization of pathways transmitting excitatory action to alpha motoneurones of flexors and extensors. Acta Physiol. Scand., 1967, 70: 369-388. 38. , The effect of DOPA on the spinal cord. 6. Half-centre organization of interneurones transmitting effects from the flexor reflex afferents. Acta Physiol. Scand., 1967, 70: 389-402. 39. KANDEL, E. R., and WACHTEL, H., The functional organization of neural aggregates in Aplysia. In: Physiological and Biochemical Aspects of Nervous Integration (F. D. Carlson, Ed.). Prentice-Hall, Englewood Cliffs, 1968: 17-65. 40. KOLMODIN, G. M., Integrative processes in single spinal interneurones with proprioceptive connections. Acta Physiol. Scand., 1957, 40: Supp. 139. 41. KOLMODIN, G. M., and SKOGLUND, C. R., Properties and functional differentiation of interneurons in the ventral horn of the cat's lumbar cord as revealed by intracellular recording. Experientia, 1954,10: 505-506. 42. , Slow membrane potential changes accompanying excitation and inhibition in spinal moto- and interneurons in the cat during natural activation. Acta Physiol. Scand., 1958, 44: 11-54. 43. , Analysis of spinal interneurons activated by tactile and nociceptive stimulation. Acta Physiol. Scand., 1960, 50: 337-355. 44. LLINAS, R., and TERZUOLO, C. A. Mechanisms of supraspinal actions upon spinal cord activities. Reticular inhibitory mechanisms of alpha-extensor motoneurons. J. Neurophysiol., 1964, 27: 579-591.

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D. P. C., Reflex action in relation to pattern and peripheral source of afferent stimulation. J. Neurophysiol., 1943, 6: 111-120. LUND, S., LUNDBERG, A . , and VYKLICKY, L . , Inhibitory action from the flexor reflex afferents on transmission to la afferents. Acta Physiol. Scand., 1965, 64: 345-355. LUND, S., and POMPEIANO, O . , Descending pathways with monosynaptic action on motoneurones. Experientia, 1965, 21: 602-603. LUNDBERG, A., Supraspinal control of transmission in reflex paths to motoneurones and primary afferents. Progr. Brain Res., 1964, 12: 196-221. , Integration in the reflex pathway. In: Muscular Afferents and Motor Control (R. Granit, Ed.). Wiley, New York, 1966 : 275-305. , The supraspinal control of transmission in spinal reflex pathways. EEG Clin. Neurophysiol., 1967, Supp. 25: 35-46. LUNDBERG, A . , NORRSELL, U . , and VOORHOEVE, P., Pyramidal effects on lumbosacral interneurones activated by ¿omatic afferents. Acta Physiol. Scand., 1962, 56: 220-229. LUNDBERG, A . , and VYKLICKY, L . , Inhibition of transmission to primary afferents by electrical stimulation of the brain stem. Arch. Ital. Biol., 1966, 104: 86-97. NYBERG-HANSEN, R., Functional organization of descending supraspinal fibre systems to the spinal cord; anatomical observations and physiological correlations. Ergebn. Anat. Entwicklungsgesch., 1966, 392: 1-48. SHERRINGTON, C. S., The Integrative Action of the Nervous System. Scribner, New York, 1906. , Further observations on the production of reflex stepping by combination of reflex excitation with reflex inhibition. J. Physiol. (London), 1913, 47: 196-214. TOWER, S. S., The dissociation of cortical excitation from cortical inhibition by pyramid section, and the syndrome of the lesion in the cat. Brain, 1935, 58 : 238-255. W A L L , P. D., Excitability changes in afferent fibre terminations and their relation to slow potentials. J. Physiol. (London), 1958, 142: 1-21. , The laminar organization of dorsal horn and effects of descending impulses. J. Physiol. (London), 1967, 188: 403-423. WILLIS, W . D., and WILLIS, J. C., Properties of interneurons in the ventral spinal cord. Arch. Ital. Biol., 1966, 104: 354-386. WILSON, V. J., Regulation and function of Renshaw cell discharge. In: Muscular Afferents and Motor Control (R. Granit, Ed.). Wiley, New York, 1966: 317-329. LLOYD,

THE LOCALIZATION OF FUNCTIONAL GROUPS OF INTERNEURONS W I L L I A M D. WILLIS,

Jr.*

T h e University of T e x a s Southwestern M e d i c a l School at Dallas Dallas, T e x a s

A major challenge in the study of spinal cord organization is the determination of the role played by particular groups of interneurons in segmental reflex actions and in the transfer of information to and from the brain. Several histologic techniques have been used to show the distribution of interneurons within various parts of the gray matter and the patterns of termination of their axons (1, 38, 39, 41, 43-45). Microelectrode recording of extra- and intracellular activity have aided in the analysis of the synaptic inputs to interneurons following either natural or electrical stimulation of primary afferent fibers (7, 17, 19, 21-23, 26-30, 48-50). Neurons giving rise to ascending tracts have been identified by antidromic activation of their axons (7, 10, 12). One of the tasks which are incomplete is the association of functional categories with histologic groupings of interneurons. METHODS

The approach used in our laboratory has been to record the electrical activity of interneurons in response to synaptic excitation or inhibition and, in some cases, to antidromic activation. The identification of units as interneurons was made according to criteria reported by previous workers (16, 22). The records have been intracellular when possible. The presence of postsynaptic potentials served to indicate that the microelectrode was within or very near the cell body. With extracellular recording, the electrode was considered to be near the cell body when the spike potential had the appropriate configuration. After electrical recording was completed, the approximate position of the interneuron was marked and later found histologically. Several marking techniques were employed (18, 46, 47). Often, a section of spinal cord near the mark was stained for Nissl substance. It was generally possible to assign the location of an interneuron to one of Rexed's laminae (38, 39). Care had to be taken to ensure that the electrical records were related * This research has been supported by Grant NB 0 4 7 7 9 of the National Institute of Neurological Diseases and Blindness, U.S. Public Health Service. The author would like to thank R. R. Grace, M. W . Pocock, R. M. Skinner and J. C. Willis for permission to refer to work, some unpublished, done with their collaboration. 267

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to the appropriate mark. This restricted the number of interneurons studied in any one experiment. A part of this work has been published (52, 53). RESULTS

After the exclusion of records which were probably from axons, the total number of interneurons thus far investigated has reached 104. Satisfactory intracellular records were made from 39 of these. Successful marks were made on 39 occasions, giving the approximate positions of 42 interneurons. The sites marked are summarized in the drawing shown in Figure 120. Since it is difficult to ensure an exact placement of the regions marked upon a single cross-section of the spinal cord, the positions of the interneurons whose potentials are illustrated in Figures 121 to 126 are shown again on outline drawings from the histologic sections. Reference will be made below to the various categories of interneurons indicated by the different symbols in Figure 120. The positions of other interneurons could be estimated from the direction of the microelectrode tracks and depth readings from the micromanipulator, but these will not be considered here. The types of interneurons studied were classified according to the scheme proposed by Eccles, Eccles & Lundberg ( 7 ) . This scheme is no longer so clear-cut, since more recent work has suggested the addition of another type of interneuron (type D; see reference 11), and a considerable overlap between the different categories has been shown to exist (21). Nevertheless,

Figure 120. Diagram showing the positions of various functional categories of interneurons within the spinal cord gray matter. Rexed's laminae are indicated. White circles: type C interneurons; black circles: type A cells; triangles: unidentified interneurons; white squares: Renshaw cells; black squares: tract cells.

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the original scheme of classification has been followed here. Interneurons in the dorsal horn and intermediate region which received monosynaptic excitatory connections from cutaneous afferent fibers were designated C cells, and those receiving similar connections from group la afferents of muscle nerves were termed A cells. No type B cells—monosynaptically excited by group lb afferents from Golgi tendon organs (7,13)—have been located histologically as yet in these experiments. This is no doubt due to the small number of interneurons in the intermediate region which have been sampled. In addition, there were interneurons in the dorsal part of the cord which were excited or inhibited polysynaptically but not monosynaptically by any of the nerves available for stimulation. These cells were considered to be unidentifiable. The main groups of interneurons investigated in the ventral horn were the Renshaw cells (8, 37) and the neurons found in the commissural region in Rexed's lamina VIII (15, 41, 53). There were, in addition, an unidentified interneuron in the ventral horn adjacent to the Renshaw cells and several interneurons in Rexed's lamina X. DORSAL H O R N AND INTERMEDIATE REGION

Type C Interneurons The positions of seven type C interneurons have been determined. Three were in lamina IV, two in lamina V, and two in lamina VI, as indicated by the white circles in the diagram of Figure 120. Intracellular records from one of the type C interneurons in lamina IV are shown in Figure 121. Stimulation of the sural nerve (S) produced a monosynaptic excitatory postsynaptic potential (EPSP), as seen in the upper traces of Figure 121, A-C. The stimulus was graded, so that a series of records was made as fibers of progressively smaller diameter were activated. Three such records are shown, the stimulus strengths being indicated as multiples of the strength required to fire the most excitable fibers in the nerve (Figure 121, A-C). A disynaptic inhibitory postsynaptic potential (IPSP) also resulted from activation of sural afferent fibers (Figure 121, AC). Another purely cutaneous nerve, superficial peroneal (SP), caused the generation of a disynaptic EPSP, which was followed by an IPSP, as shown in Figure 121D. The mixed nerve, plantar (PT), evoked a polysynaptic IPSP (Figure 121E). None of the muscle nerves used had any effect (Figure 121, F-I). The other two type C interneurons in lamina IV were studied by extracellular recording. One was fired monosynaptically by the superficial peroneal nerve and polysynaptically by the plantar nerve and two muscle nerves (peronei-deep peroneal and the combined flexor digitorum longus and plantaris nerves). There was inhibition of the firing produced in the superficial peroneal nerve when a conditioning volley was set up in fibers of the contralateral L7 dorsal root. The other type C unit in lamina IV was discharged monosynaptically by stimulation of the sural nerve. The unit was lost before the effects of other nerves could be tested.

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Figure 121. Type C interneuron. Position was in lamina IV, as shown in diagram J. The upper traces of the electrophysiological records in this and succeeding figures were recorded from the microelectrode either within or near the interneuron. In this case, the records were made intracellularly. The second traces show the afferent volley produced by peripheral nerve stimulation and recorded from the dorsal root entry zone. When there is a third trace, as in C-E, it represents the field potential recorded with the microelectrode just outside the cell. The field potential should be subtracted from the intracellular record to give the transmembrane potential change. Field potentials are shown only when they are significant, although they were routinely recorded. The abbreviations for the nerves stimulated are shown above the appropriate records; the corresponding names are given in the text. When graded stimuli are used, the strength is given as a multiple of that required to excite the most excitable fibers of the nerve. For instance, in A the sural nerve was stimulated with a strength 1.08 times threshold. The time scale below B is for A-B, while the one below I is for the other records. There is a calibration pulse at the beginning of the traces in A-C.

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Intracellular records from one of the two type C interneurons found in lamina V are illustrated in Figure 122. Monosynaptic excitation resulted from stimulation of either of two nerves, sural and superficial peroneal ( F i g ure 122, A and B ) . There were, in addition, disvnaptic excitation produced by the semimembranosus-anterior biceps nerve ( F i g u r e 1 2 2 F ) and a variety of polvsvnaptic actions by other nerves ( F i g u r e 122, C, E, G, H ) , including an I P S P by the contralateral L 7 dorsal root ( F i g u r e 1 2 2 D ) . T h e other type C interneuron in lamina V was excited monosynapticallv bv fibers of the superficial peroneal nerve, disvnaptically by the sural and plantar nerves, and polvsynapticallv bv the superficial peroneal and sural nerves. There were no effects when anv of the muscle nerves available were stimulated. Both of the tvpe C interneurons found in lamina V I were excited monosynapticallv by two different cutaneous nerves, sural and superficial peroneal. One was also excited disynapticallv bv each of two muscle nerves (posterior biceps-semitendinosus and semimembranosus-anterior b i c e p s ) and a mixed nerve ( p l a n t a r ) , and inhibited disynapticallv bv a muscle nerve (flexor digitorum longus-plantaris). T h e other was excited polvsvnaptically by the

CONTRA

Figure 122. Tvpe C interneuron. Position in lamina V, as indicated in I. Field potential records shown below A-D. The time and potential scales are for all the electrical records.

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same cutaneous nerves that produced its monosynaptic excitation and, in addition, it was inhibited polvsynapticallv by the superficial peroneal nerve and by several muscle nerves (posterior biceps-semitendinosus, semimembranosus-anterior biceps, and gastrocnemius-soleus). In neither case were the effects of stimulation of a contralateral dorsal root observed.

Figure 123. Type A interneuron. Position in medial lamina V, as shown in ]. Field potential records shown below D and E. The stimulus was graded for the flexor digitorum longus-plantaris nerve, A-C.

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F i g u r e 124. T y p e A intemeuron. Position in the lateral part of lamina VI, as shown in }. A graded series of stimuli was applied to the combined peroneideep peroneal nerve, A-C. T h e time scale below C is for A-C, while the one below I is for the other records. Ti/pe

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The positions of four tvpe A intemeurons were successfully marked. One of these was in lamina V, two in lamina VI and one in the dorsomedial part of lamina VII, as indicated by the black circles in Figure 120. The intracellular records in Figure 123 were taken during the study of the type A interneuron in lamina V. Monosynaptic E P S P s were generated in this cell when either the flexor digitorum longus-plantaris nerve ( F i g u r e

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F i g u r e 125. T y p e A interneuron. Position was probably in lamina VII, near the border with lamina VIII, as shown in I. The mark was made intracellularly. Several records, resulting from a graded series of stimuli applied to the posterior bieeps-semitendinosus nerve, are shown in A-C.

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123, A - C ) or the plantar nerve (Figure 123D) were stimulated. It was assumed that afferents from small muscles of the foot were responsible for the monosynaptic EPSP from the plantar nerve. However, there is a possibility that both monosynaptic EPSPs were from joint or cutaneous afferents, since the flexor digitorum longus nerve often contains a significant admixture of afferents from the interosseous membrane (24). In this event, the interneuron was misidentified and should be considered a type C interneuron. In addition to the monosynaptic connections, the cell of Figure 123 received disynaptic excitation from the superficial peroneal and sural nerves (Figure 123, E, F ) , disynaptic inhibition from the peronei-deep peroneal (Figure 1231) and plantar (Figure 123D) nerves, and polysynaptic effects from several nerves (Figure 123, C - F ) . One of the two type A interneurons in lamina V I was studied by intracellular recording. It was monosynaptically excited by activation of the peronei-deep peroneal nerve (Figure 124, A - C ) . It was also disynaptically excited by the superficial peroneal (Figure 124E) and plantar (Figure 124F) nerves. Several nerves produced a variety of polysynaptic effects (Figure 124, C - H ) , including an IPSP by the contralateral L7 dorsal root (Figure 1241). The other type A interneuron in lamina V I was studied by extracellular recording. This cell was fired monosynaptically when the flexor digitorum longus-plantaris nerve was stimulated. The discharge was caused by the lowest threshold component of the nerve. The cell was also fired, although polysynaptically, by each of several nerves: peronei-deep peroneal, sural, superficial peroneal, and plantar. The type A interneuron in lamina V I I was not only investigated electrophysiologically by intracellular recording but also marked intracellularly (47). Records made of its activity are illustrated in Figure 125. The monosynaptic EPSP generated following stimulation of the posterior biceps-semitendinosus nerve was already maximal when the stimulus strength was 1.25 times the threshold of the most excitable fibers in that nerve (Figure 125A; cf., B and C ) . Disynaptic IPSPs were evoked by the gastrocnemius-soleus (Figure 125D) and flexor digitorum longus-plantaris (Figure 125E) nerves. Several of the muscle nerves also produced polysynaptic IPSPs (Figure 125, B-E). The sural and superficial peroneal nerves (Figure 125, G and H ) produced disynaptic EPSPs followed by complicated sequences of polysynaptic excitatory and inhibitory potentials. Unidentified

Interneurons

Four interneurons whose positions were marked proved to be unidentifiable. These are indicated by the white triangles in Figure 120. One was in lamina I and three in lamina IV. The unidentified interneuron in lamina I was studied by extracellular recording. It was fired disynaptically by afferents of the gastrocnemius-soleus

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nerve and polysynaptically by several muscle and skin nerves (posterior biceps-semitendinosus, sural, superficial peroneal, and plantar). The unidentified interneurons in lamina IV were excited primarily by cutaneous afferents. Two were studied intracellularly. One of these was found to generate a disynaptic EPSP when the sural nerve was stimulated. It also generated polysynaptic EPSPs in response to the activation of the posterior biceps-semimembranosus-anterior biceps, flexor digitorum longus-plantaris, sural and superficial peroneal nerves. The other was excited disynaptically by the sural, superficial peroneal and plantar nerves; the same nerves also produced polysynaptic effects. The third of these interneurons was examined by extracellular recording. It was fired polysynaptically by the sural, superficial peroneal, and the flexor digitorum longus-plantaris nerves. VENTRAL HORN

Renshaw Cells The locations of 19 marks placed adjacent to Renshaw cells have been identified histologically. These are indicated by the white squares in Figure 120. Almost all of the Renshaw cells were studied by extracellular recording; satisfactory intracellular records were made in just one case. Each of the Renshaw cells was identified by its characteristic burst discharge in response to stimulation of the ventral root of the segment of spinal cord in which it lay (8, 37). Most of the Renshaw cells were located in the ventral part of Rexed's lamina VII, near the region from which the motor axons leave the gray matter en route to the ventral root (46). Three Renshaw cells were located more superficially in lamina VII. This is consistent with the report of Eccles, Fatt & Koketsu (8), who found some Renshaw cells in the dorsal part of the ventral horn. One of the records made extracellularly near a Renshaw cell is shown in Figure 126A. The position of the mark was in the ventral part of lamina VII as indicated in the tracing in Figure 126B made from a histologic section. In a series of experiments (51), the possibility was investigated that afferents in the ventral roots might be responsible for excitation of Renshaw cells, rather than invasion of recurrent collaterals of motor axons. It has been known since Sherrington's work (42) that there are a few afferent fibers in the ventral roots. These can be detected when they degenerate following chronic section of the root in which they travel. The presence of afferent fibers in ventral roots has recently been confirmed both in the cat and in the rat (5,34). In a series of five animals, one or more ventral roots were sectioned 7 to 18 days prior to an acute experiment. An attempt was made in each of the terminal experiments to demonstrate the persistence of the recurrent inhibitory mechanism despite the degeneration of any afferents which might have entered the spinal cord over the ventral roots. Stimulation of the central ends of chronically sectioned ventral roots produced recurrent IPSPs in 15

Figure 126. Renshaw cell. Position in the ventral part of lamina VII, as indicated in B. The extracellular record of spike discharge from this cell in A is preceded bv a calibration pulse having a duration of 1 msec, and an amplitude of 1 mV. The spike is negativepositive.

motoneurons out of the 17 from which intracellular records were made. The motoneurons were indentified by antidromic invasion of an action potential when their motor axons were stimulated in a ventral root ( 2 ) . The recurrent IPSPs were readily demonstrated when the stimulus strength was reduced below threshold of the axon belonging to the motoneuron studied ( 8 ) . The motoneurons investigated had axons in either the L7 or SI ventral roots. In three experiments, records were also made from five Renshaw cells, all activated by stimulation of chronically sectioned ventral roots. The position of one of these was marked; it lay in the ventral part of lamina VII just medial to the motor nucleus. It can be concluded that Renshaw cells do not require the presence of afferents in ventral roots for their activation in response to ventral root stimulation. Nor does their action in producing recurrent inhibition appear to be altered by chronic sectioning of ventral roots.

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Interneurons in Laminae VIII and X In a previous study (53), the marked positions of five interneurons were found to be in laminae VIII and X. All three of the cells in lamina VIII could be activated antidromically by stimulation of the ventral part of the spinal cord at the lower thoracic or upper lumbar level. One was discharged antidromically when the stimulus was restricted to the ventral part of the contralateral half of the spinal cord. In recent experiments, two other interneurons were found which could be fired antidromically by stimulation of the ventral white matter of the cord. One of these was located in lamina VIII. Records were made from the other at the same microelectrode position from which the activity of a Renshaw cell was observed. A mark was made which was later shown to be in lamina VII. Thus, either there are some interneurons giving rise to long ascending axons to be found within lamina VII as well as lamina VIII, or the electrical activity, which was recorded extracellularly, was picked up from a distance and the interneuron was actually in lamina VIII. Alternatively, the activity could have been recorded from a dendrite extending laterally into lamina VII. These tract cells are indicated in the drawing of Figure 120 by the black squares. Some of the interneurons of the commissural region have been the recipients of monosynaptic excitatory connections descending in the ventral white matter of the cord. Activation of the ventral cord with stimuli subthreshold for the ascending axons has provided proof of this for several tract cells. Similar connections were also found in the neurons of lamina X (Figure 120, white triangles below central canal). Afferent fibers from peripheral receptors could be shown to have excitatory or inhibitory effects upon the interneurons of laminae VIII and X in several instances. The postsynaptic potentials were almost all polysynaptic and may have been due to the activation of the flexion reflex afferents (14). One interneuron in lamina VIII was excited monosynaptically by afferent fibers. Several of these interneurons were not affected, so far as could be judged, by any of the afferents stimulated. One piece of negative evidence should be mentioned. There was no effect in any of the cells tested of stimulation of the ventral root of the same segment. Thus, the interneurons of the commissural region are functionally distinct from the Renshaw cells in the adjacent lamina VII. Other Interneurons of the Ventral Horn Several interneurons were studied which did not fit into the categories of Renshaw cell or commissural cell. One is indicated in Figure 120 by the white triangle in lamina VII. It was excited monosynaptically when the dorsal root of the same segment was stimulated. Since the unit was firing spontaneously, it was easy to demonstrate inhibition in response to stimulation of the ipsilateral ventral root. The response to dorsal root stimulation was also inhibited by ventral root stimulation. Inhibition of interneuron discharges

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by ventral root volleys has been reported a number of times (17,23, 26, 56). It is possible that such interneurons produce a tonic inhibition of motoneurons and that their recurrent inhibition is responsible for disinhibition of motoneurons, which is the mechanism of recurrent facilitation (55). Other kinds of interneurons were found in the ventral horn, but their positions were not marked. COMMENT

The findings in the series of experiments just summarized are generally in good agreement with those reported by others. The interneurons in the dorsal horn and intermediate region were distributed approximately as might have been predicted from the field potential studies (3, 9 ) and as found in previous single unit studies (7, 21, 4 8 , 5 0 ) . It might seem from the recent work in Lundberg's laboratory (21) and from this study that there is no evidence that different functional categories of dorsal horn and intermediate interneurons can be localized to discrete regions within Rexed's laminae. About the most that can be said is that there is a tendency for interneurons monosynaptically excited by cutaneous afferents to be situated slightly dorsolaterally to those monosynaptically activated by muscle afferents. However, a better correlation between the histologic arrangement and functional groupings was obtained by Wall ( 4 9 , 5 0 ) , using natural stimulation. He showed that the marginal cells of lamina I were fired from large cutaneous receptive fields and also by proprioceptive stimuli. The interneurons in lamina IV responded to cutaneous stimulation but not to movements of joints. Cells in lamina V were similar to those in lamina IV, except that they seemed to be activated after a longer latency. A marked change occurred in lamina VI, where interneurons were again found which were discharged by either cutaneous or proprioceptive stimuli. If the di- and polysynaptic connections were taken into account, the results of the present study might agree to a large extent with the results of Wall. The main discrepancy would be the presence of effects by muscle afferents in laminae IV and V found in this study and in the work of Hongo, Jankowska & Lundberg (21). It is probable that the different stimulating techniques employed account for this discrepancy. The interneurons of the ventral horn include the very well studied Renshaw cells. These neurons were found to lie, in most instances, in the ventral part of lamina VII, near the point of exodus of the motor axons from the gray matter. This finding is in good agreement with a field potential study ( 8 ) and with the suggestions of several histologists (40, 44); it has already been confirmed (46). Anatomical studies show that there are interneurons in this region which have axons that project ipsilaterally and that terminate within a few segments of the cell body (41). This type of interneuron would be ideally suited to produce recurrent inhibition of motoneurons and of inhibitory interneurons, since it is known that both recurrent inhibition

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and facilitation may be distributed over several segments (6, 57). This spread of recurrent effects cannot be attributed to the projections made by recurrent collaterals, since most of these appear to terminate within about one half a segment of their parent motoneurons (41). Nor can the distribution of recurrent effects be attributed to afferent fibers within the ventral roots, since these have now been ruled out as significant contributors to the activation of Renshaw cells or the production of recurrent inhibition. All of the interneurons located in lamina V I I I so far studied have been found to send an axon at least as far rostrally as the thoraco-lumbar junction. Presumably, these axons form a tract which ascends to the brain, possibly the spinoreticular or the spinothalamic tract (33). Recent evidence shows that the axons of interneurons in this area of the cord ramify within both the ipsilateral and the contralateral ventral horns in addition to the rostral projection (41). These cells would thus be in a position to take part in segmental reflex activity, including reflexes that work on the contralateral side to the afferents stimulated (20), as well as those on the ipsilateral side. Since many of these interneurons are excited monosynaptically by fibers descending in the ventral white matter of the cord, they very likely relay information from the brain. They are an obvious candidate for producing the disynaptic excitation which results in motoneurons when the "bulbospinal correlation system" of Lloyd is stimulated (31, 54). The widespread nature of the axon terminals of these cells, and the wide receptivity of some of them for afferent input remind one of the neurons of the reticular formation (32). Their position in the cord can, in fact, be considered homologous to that of the medial reticular formation. SUMMARY

A. The positions of 42 interneurons investigated electrophysiologically have been located histologically. B. There was no obvious correlation between the positions of type C or A interneurons and the laminar organization of Rexed; this may be due to the use of electrical rather than natural stimulation. C. Renshaw cells were found to lie within Rexed's lamina VII; most were in the ventral part of the lamina, between the motor nucleus and the commissural region; several, however, were more dorsally situated. D. Experiments in animals with chronically sectioned ventral roots showed that ventral root afferent fibers are not required for the production of recurrent inhibition. E. The interneurons of lamina V I I I , the commissural region, all had axons which extended as far rostrally as the thoracic cord; it is likely that these belong to one of the tracts ascending to the brain. F. The commissural interneurons may be excited monosynaptically by fibers in the ventral part of the spinal cord; this may result from activation of descending pathways which are known to relay in the ventral horn. Scheibel: It is a pleasure to see how well Dr. Willis' physiology seems to

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correlate with data derived from Golgi material. The physiology, of course, is non-committal as to the actual nature of the Renshaw cells but, as was mentioned yesterday, the evidence seems very strong from histology that these are projecting interneurons of the propriospinal system, and therefore longaxoned rather than Golgi type II cells. He mentioned afferents in the ventral root, and we just recently had the opportunity to see one completely impregnated in a Golgi section. It is the only one we have seen in many thousands of spinal sections. Adey: Dr. Willis, have you any evidence from your studies about the question of the short-axon versus the long-axon type of interneuron? Willis: I do not think our evidence adds anything on this issue. It is known that recurrent effects spread along the cord for several segments, although the greatest intensity is at the level of the ventral root being activated (6). Since most recurrent collaterals of motor axons seem to be limited in their longitudinal distribution to about half a segment (41), I suppose that the effects seen at distances greater than that can best be explained by the distribution of Renshaw cell axons. If this is the case, Renshaw cells would not qualify as Golgi type II cells, which have axons that terminate very close to the cell bodies. In the earlier work with the large marks, one could see the cell patterns on the same sections as the marks because the sections were stained with a panoramic stain. In the more recent studies, we have left the marked sections unstained, but adjacent sections were stained for Nissl substance and the Rexed laminae traced from these using a projector. Schmidt: In this connection, I have a more general question. Is the Rexed system just a convenient way of expressing locations in the spinal cord, or can we already delegate certain physiological functions of the spinal cord to some of the anatomical groupings which Rexed has described? Willis: It is certainly a convenient way to describe the position of cells within the gray matter of the spinal cord. There are certain keys to assist in defining some of the laminae, such as the bundles of longitudinal fibers in the lateral part of lamina V. In our experiments, as I mentioned, we have not been able as yet to find a very clear lamellar pattern of functional organization in the dorsal horn, but this may be a reflection of the type of stimulation we are using. I would expect that there would be some relationship between layering and function in the spinal cord, just as there is in other parts of the central nervous system. Schmidt: Dr. Scheibel, would you comment on how your results correspond to Rexed's findings? Scheibel: We have been surprised to find how well Rexed's lamination, based on size, shape and distribution of cell bodies alone, agrees with general neuropile configurations. Cytological discontinuities revealed by Nissltype stains in the cord also reflect important changes in distribution and pat-

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tern of axonal and (or) dendritic elements. For this reason we feel that the laminae described by Rexed are physiologically relevant. Kennedy: I would like to discuss the rationale for functional groupings in cells. In the invertebrate preparations, somata are usually so uninvolved in the synaptic field of the cell that connectivity does not really put any constraints on soma position; cells do not have to be very strategically located with respect to one another in order to connect. In Dr. Kandel's preparation, where he showed his map of receptivities of cells,4 it looks as though the clustering involves the availability of a certain kind of receptor site. This is one kind of biochemical competence, presumably depending upon the history of cell lineages in various regions of the ganglion. Otsuka, Kravitz & Potter's (35) results in crayfish suggest another kind of biochemical competence, the competence to make transmitters. These authors found a distinct clustering of inhibitory cells that make GABA, and of excitatory cells whose transmitter is unknown. These are motoneurons, and they cluster according to this property even though cells that are nearest neighbors in terms of soma position may make entirely different connections. For example, two inhibitors that lie next to one another may innervate a postural extensor and a phasic flexor, but never operate in any relation to one another, and can receive entirely independent inputs. I think that these positional specificities relate to the shared history that affects products of a given cell lineage; biochemical competence may be developed early in the lineage. I would like to ask the neuroanatomists who work on the spinal cord how many morphogenetic movements follow the completion of cell division in a developing piece of spinal cord. To what extent do cells differentiate in situ, and to what extent do they move around after the lineages have been completed? Adey: Are you suggesting that the grouping of cells in at least some of these nuclear configurations is determined by a biochemical history? And that these cells would have a common set of transmitter substances? Kennedy: One can assume that before division is complete, one stem cell may already have developed a certain biochemical competence, and there is then a regional affiliation between its offspring. It appears that clustering is on that principle in at least some arthropod systems, whereas in the Aplysia ganglia described by Dr. Kandel the clustering appears to be on the basis of the type of membrane receptors. Larramendi: Regarding the biochemical specificity of cell groups differentiated from a common stem cell, I would like to mention that a similar idea is implicit in Sidman's proposal (33a) that cell groups with a common origin form synapses during their migration. Although this is an interesting idea, there is also the possibility of specification by cell contact, as exemplified by the retrograde specification of motoneurons by muscle. There is, in * Page 94.

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this respect, an old law of Ramón y Cajal's, "the law of the precedence in the development of the cells of the anterior horn over those of the posterior horn" in the spinal cord, which suggests that neural circuits may develop backwards. We are investigating this hypothesis in the cerebellum, where, in terms of observations with the light microscope, the sequence of development seems also to be backwards. Therefore, "cousin cell groups" synaptic affinities, as well as "cell contact" specifications, retrograde or orthograde, are factors to be considered and investigated in the formation of neural circuits. Another aspect of interest is the fact that common stem cells, such as the external granular layer cells in the cerebellum, give rise to excitatory as well as inhibitory cell populations. Purpura: We have not yet discussed in any great detail the question of cell geometry in relation to the various laminae and the mode of impulse initiation in these elements. Wall (49) demonstrated in extracellular recordings that, during spontaneous activity, unit responses might be detectable in one lamina whereas, during evoked responses, the site of origin of these impulses must change as indicated by careful histological analysis of microelectrode tracks and careful depth explorations. The title of Wall's paper, "Impulses originating in the region of dendrites" (49), certainly indicates that the localization of unit discharges in one of Rexed's laminae may have little meaning if the element has dendrites in another lamina and impulses can arise at more than one site. In view of these and other findings suggesting impulse initiation in dendrites under appropriate conditions (36), I think your approach, which is based on examination of dendritic fields is very useful. Surely, one has to come to grips with both the problems of cell geometry and variations in impulse initiation in the analysis of the organization of elements at all neuraxial sites. Scheibel: The dendrite system from which Wall was recording is unusual in one respect. These dorsal dendrites of lamina IV cells are thrust into one of the densest axonal neuropile fields in the central nervous system. The field is generated by terminating, low-threshold, cutaneous afferents which may always have some degree of incoming activity propagating into the terminal region. The dendrites trapped in this milieu must be covered by virtually continuous sheets of presynaptic membrane, an assumption supported by the electron microscope studies. If ever there was a locale where one might expect to see continuous generation of dendritic spikes as an expression of postsynaptic integrative activity, this is it. I do not think that your objection mitigates the basic proposition, but I certainly would agree with you that in the long run it is the organization of the domain which is important, rather than the distribution of somata, which are at best trophic centers for the elements. Adey: I think that it is a very good point, and this question of the organization of the domain, on the basis of Dr. Kennedy's remarks about the chem-

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istry of transmitter substances, is something that the fluorescence microscopy studies of Dahlstrom & Fuxe ( 4 ) have suggested: that there is a streaming of transmitter granule substances, if one would allow that interpretation, and that these are functionally organized in streams that go, for instance, through the medulla to the midbrain and on into the forebrain structures, and in some cases in identifiable streams into the myelencephalon. If that is the case, I wondered when I first saw this type of study whether, in fact, the identification of specially overlapping systems of granules would be one way in which the presence of interneurons, interneuron groups of a particular kind, might be identified. Llinas: Dr. Kennedy, there is some evidence in the cerebellum for differences between excitatory and inhibitory cells. Kilham & Margolis (25) at Dartmouth, have shown that if kittens are infected with what he calls F.A.V. (feline ataxia virus), he can obtain a cerebellum which has essentially no granule cells. That is to say, a large majority of the only excitatory neural elements is gone. However, the inhibitory cells (basket and stellate) still remain. The inhibitory interneurons and the granule cells originate from the same layer, the external granule layer, and then migrate down to the internal granule layer. It is possible that these two types of cells differ in some particular way so that only one type is infected. It is also possible that only those cells which divide are damaged, and so it must be the case that at a given stage of development, only excitatory cells divide.

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around the intermediate nucleus of the lumbosacral cord. J. Physiol. (London), I960, 154: 89-114. ECCLES, J. C., FATT, P., and KOKETSU, K., Cholinergic and inhibitory synapses in a pathway from motor-axon collaterals to motoneurones. J. Physiol. (London), 1954, 126: 524-562. ECCLES, J. C., FATT, P., LANDGBEN, S., and WINSBUKY, G. J., Spinal cord potentials generated by volleys in the large muscle afferents. }. Physiol. (London), 1954, 125: 590-606. ECCLES, J. C., HUBBARD, J. I., and OSCARSSON, O., Intracellular recording from cells of the ventral spinocerebellar tract. J. Physiol. (London), 1961, 158: 486-516. ECCLES, J. C., KOSTYUK, P. G., and SCHMIDT, R. F., Central pathways responsible for depolarization of primary afferent fibres. J. Physiol. (London), 1962, 161: 237-257. ECCLES, J. C., OSCARSSON, O., and WILLIS, W. D., Synaptic action of group I and II afferent fibres of muscle on the cells of the dorsal spinocerebellar tract. J. Physiol. (London), 1961, 158: 517-543. ECCLES, R. M., Interneurones activated by higher threshold group I muscle afferents. In: Studies in Physiology (D. R. Curtis and A. K. Mclntyre, Eds.). Springer, New York, 1965 : 59-64. ECCLES, R. M., and LUNDBERG, A., Synaptic actions in motoneurones by afferents which may evoke the flexion reflex. Arch. Ital. Biol., 1959,97: 199-221.

1 5 . ERULKAR, S. D . , SPRAGUE, J . M . , WHITSEL, B . L . , DOGAN, S., a n d JANNETTA,

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P. J., Organization of the vestibular projection to the spinal cord of the cat. /. Neurophysiol., 1966, 29: 626-664. FRANK, K., and FUORTES, M. G. F., Potentials recorded from the spinal cord with microelectrodes. /. Physiol. (London), 1955, 130 : 625-654. , Unitary activity of spinal interneurones of cats. J. Physiol. (London), 1956,131: 424-435. GALIFRET, Y., and SZABO, T., Locating capillary microelectrode tips within nervous tissue. Nature (London), 1960, 188: 1033-1034. HAAPANEN, L., KOLMODIN, G. M., and SKOGLUND, C. R., Membrane and action potentials of spinal interneurons in the cat. Acta Physiol. Scand., 1958, 43: 315-348. HOLMQVIST, B., Crossed spinal reflex actions evoked by volleys in somatic afferents. Acta Physiol. Scand., 1961,52: Supp. 181. HONGO, T., JANKOWSKA, E., and LUNDBERG, A., Convergence of excitatory and inhibitory action on interneurones in the lumbosacral cord. Exp. Brain Res., 1966,1: 338-358. HUNT, C. C., and KUNO, M., Properties of spinal interneurones. J. Physiol. (London), 1959,147: 346-363. , Background discharge and evoked responses of spinal interneurones. J. Physiol. (London), 1959, 147: 364-384. HUNT, C. C., and MCINTYRE, A. K., Characteristics of responses from receptors from the flexor longus digitorum muscle and the adjoining interosseous region of the cat. /. Physiol. (London), 1960, 153 : 74-87. KILHAM, L., and MARGOLIS, G., Viral etiology of spontaneous ataxia of cats. Am. J. Path., 1966, 48: 991-1011.

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4 5 . SZENTAGOTHAI, J . , 46.

GROUPS

ELECTRON MICROSCOPIC STUDIES OF CERERELLAR INTERNEURONS L. M. H. LARRAMENDI*

University of Illinois College of Medicine Chicago, Illinois

Four main types of nerve cells form the cerebellar cortex in most species from fish to man. Of these cell types, the Purkinje cell is the center of convergence and the final common path for the neural activity reaching the cerebellar cortex. The other cell types: granule, Golgi, stellate and basket cells possess short-axoned processes which form short neuronal paths in the cerebellar cortex, for which reason they can be classified as interneurons. However, it is interesting that Cajal classified Golgi and stellate cells as short-axoned cells but regarded the tiny granules as long-axoned distributing cells. For Cajal, Golgi and stellate cells had a role other than that of distributing cells. He was particularly intrigued by the retrograde loop formed by the axons of the Golgi cell. Since Cajal's neural circuitry did not include inhibitory cells, he believed that short-axoned cells were important in maintaining a continuous and persistent activity in neural centers. He considered interneurons as reinforcers of incoming signals. Ramón y Cajal's cerebellar circuitry is shown in Figure 127. To Cajal's basic description, Scheibel & Scheibel (33) added an important detail: These authors demonstrated that climbing fibers do not make contacts exclusively with Purkinje cells, as originally thought by Cajal, but that they also establish synaptic contact with Golgi, basket and stellate cells. As a result of these observations it became clear that the short-axoned cells of the cerebellar cortex receive external information either directly via the climbing fibers or, indirectly, from mossy fibers via the granule cells. Not until lately has Cajal's cerebral circuitry been validated. The brilliant physiological work by Eccles and his group (4-8) has recently established the inhibitory and excitatory pathways of the cerebellar cortex, thus answering Cajal's questions as to why there are short accessory pathways and why there are Golgi cells. To the physiological work of Eccles and his group, Ito and his colleagues have added the unsuspected contribution that Purkinje cells are themselves inhibitory (19). While Eccles and Ito's laboratories were unraveling the dynamics of the * Work supported by U.S. Public Health Service Research Grant NB 05408. 289

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E

Granule c. Climbing f t( Mossy f.

nr

Stellate c.

Granule c. Mossy f

Mossy f.

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Figure 127. Diagram representing Ramón y Cajal's cerebellar circuitry. In I and II are shown the two extrinsic afferent systems reaching the cerebellar cortex, the climbing and the mossy fibers; climbing fibers contact directly the Purkinje cell, whereas mossy fibers reach the Purkinje cell through granule cells. In III and IV, the short-axon cells are shown; basket and stellate cells receive contacts from parallel fibers, and their axons terminate on neighboring Purkinje cells; in TV, the Golgi cells receive contact from the parallel fibers of granule cells and send their axons back to the granule cell dendrites, thus forming a retrograde loop. The Purkinje collaterals which, according to Cajal, recur upon the dendrites of the Purkinje cell, are omitted.

cerebellar pathways, electron microscopic studies by Palay ( 27 ), Gray ( 13 ) Fox (9, 11), Hâmori & Szentâgothai (14-16) and others, also began to confirm Cajal's synaptic circuitry. Szentâgothai, in a chapter of a book coauthored with Eccles and Ito ( 3 ), and Fox and coworkers ( 10 ) have reviewed the electron microscopy of the cerebellum up to 1966, including their own contributions. In this presentation, I shall go beyond their recent accounts and report some important advances made recently in our laboratory. These advances have been made possible by the systematic and comprehensive

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sampling of synapses in the molecular layer of the mouse cerebellum perfused with glutaraldehyde. IDENTIFICATION OF NERVE TERMINALS IN THE MOLECULAR L A Y E R

By correlating the well-known light microscopic morphology, distribution and orientation of parallel fibers, climbing fibers, basket-stellate axons and Purkinje collaterals in the molecular layer with corresponding electron microscopic profiles, we ( 2 1 ) were able to establish the characteristics of these terminals and, therefore, to identify consistently their synapses in the molecular layer.

Figure 128. Low stellate cell (basket) close to a Purkinje dendrite. Note that in direct apposition to the cell surface, there are many axonal profiles, very small amount of glia, and 7 synaptic boutons. All except one of these boutons are parallel fiber synapses.

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Figure 129. a: Parallel fiber (pf) synapsing with a basket cell; note the aggregation of synaptic vesicles close to the presynaptic membrane; compare the size and shape of synaptic vesicles with those in Figures 130a and 131; boutons of parallel fibers usually make a single synaptic adhesion, h: Climbing fiber synapse ( c f ) on a basket cell soma; note that this also forms a synapse with a spine ( D s ) , originating probably in the neighboring large Purkinje dendrite; this type of nerve terminal usually contains many large, round, clear-core vesicles and some dense-core vesicles which appear empty in this instance; they also contain neurotubules, but not neurofilaments.

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Figure 130. a: Basket nerve terminal (b-s) synapsing on basket cell soma; two other nerve terminals are in direct apposition with the basket cell but do not form synaptic contacts; note the aggregation size and shape of the synaptic vesicle and compare them with those in B and in Figure 129; observe the characteristics of the synaptic adhesion, which shows symmetric pre- and postsynaptic densities; basket and stellate terminals usually make more than one adhesion per bouton. b: Basket cell soma spine (sp) receiving a synaptic contact from a parallel fiber which also is forming a synaptic contact directly on the soma; basket and stellate spines make synaptic contacts only with parallel fibers, usually with more than one.

The internal characteristics of these terminals were found to differ in the following respects: (a) the size, shape and pattern of aggregation of synaptic vesicles, ( b ) the appearance and symmetry of the pre- and postsynaptic densities, (c) the axoplasm density, (d) the proportions of neurotubules (200 A) or neurofilaments (75 A), and (e) the presence or absence of agranular reticulum. The electron micrographs in Figures 128 to 131 show the appearance of parallel fibers, climbing fibers, Purkinje collaterals and

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Figure 131. Purkinje collateral (Pc) synapsing on a basket cell soma. Note the large size of the bouton and the multiple synaptic adhesions. Observe the size, shape and aggregation of the synaptic vesicles and the presence of some compound or spiky vesicles. Compare in the same figure the parallel fiber (pf) and the basket synapses (b-s) on the thin dendrite emerging from the basket cell.

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basket-stellate axons in synaptic contact with basket cells as reported by us (22, 23). From our studies of the synapses upon Purkinje, basket and stellate cells three main generalizations regarding nerve terminals in the molecular layer appear possible. First: each type of terminal in the molecular layer shows distinctive morphological features which permit its identification; however, ascending basket and stellate axon terminals, which originate in closely related types of cells have very similar characteristics. Second: inhibitory and excitatory nerve terminals contain clear core synaptic vesicles which differ in size and shape; the smaller and more elongated synaptic vesicles are found in the inhibitory terminals (basket-stellate and Purkinje collaterals), whereas the larger and round vesicles are seen in the excitatory terminals (climbing and parallel fibers) (20). And third: both excitatory and inhibitory terminals make synaptic contacts on spines as well as directly on the smooth surface of soma and dendrites; however, the great majority of spines receive excitatory synapses; for the Purkinje cell almost all excitatory synapses are made on spines. Whether or not Gray's type I and II synapses correspond to excitatory and inhibitory synapses respectively, as suggested by Eccles ( 2 ) , requires a comparative study of the cerebellar synapses after either osmium or aldehyde fixation. INDEX OF SYNAPTIC EFFICIENCY OF STRENGTH OF BOUTONS

In a recent analysis of the synapses upon basket and stellate cells we observed (23) the size of bouton apposition on the recipient cell, the length of the synaptic adhesions ("active sites" or postsynaptic membrane) and found that the number of adhesions per bouton differed for each type of nerve terminal. For instance, it was found that parallel fiber boutons formed a single, but long synaptic adhesion, whereas boutons of other types of terminals usually formed multiple, but shorter synaptic contacts. Measurements of these parameters for synapses made directly on the soma of the basket cells led to the establishment of an "index of synaptic efficiency or strength of boutons". This index was obtained by multiplying the average length of synaptic adhesions by the average number of adhesions formed by boutons for each type of terminal. A comparison of these indices revealed that synaptic contacts made by climbing fibers and Purkinje collaterals were 1.7 times more efficient than those made on parallel fibers. The efficiency of basket-stellate boutons, relative to parallel fibers, was found to be 1.4. The index of efficiency or strength points out that the number of boutons synapsing on a cell does not necessarily define in morphological terms the strength of the input to that cell. PATTERNS OF DISTRIBUTION OF SYNAPSES

Once the identification of synaptic terminals was consistently obtained (21), the next step was to try to establish the patterns of synaptic distribu-

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tion upon the cells in the molecular layer. In this paper I shall limit my discussion to the distribution of basket and stellate synapses upon the Purkinje cell, and to the pattern of synaptic distributions of the synapses received by basket and stellate cells. In other words, I shall describe the output and input of basket and stellate cells in the mouse cerebellum. Basket and Stellate Axons Synapsing on Purkinje Cells According to Ramón y Cajal's classic description, basket descending axons make synaptic contacts mainly with the Purkinje cell body by the so-called basket synapse. Ascending basket axons and stellate axons are believed by Scheibel & Scheibel (33) to contact Purkinje dendrites and other basket and stellate cells as described by Cajal. Electron microscopic studies by Herndon (17, 18), Hámori & Szentágothai (14), Palay (28) and Fox (10) have verified that basket cells, in fact, form synapses with the Purkinje cell body and occasionally with the axon hillock (29). Hámori & Szentágothai report (14) that basket cells in the cat consistently form synapses only upon the lower part of the Purkinje cell, including the axon hillock. Fox (10) has observed that basket axons also synapse with spiny branchlets (on their nonspiny surface) and on spine dendrites. No identification of stellate axons is reported by any of these authors. In our recent investigations (21-23) we have confirmed the observations reported by previous investigators and have substantially extended them. For instance, it was found that nerve terminals similar to those unquestionably identified as basket axons (and which presumably were stellate terminals) made synaptic contacts with Purkinje dendrites of all sizes. Also, it was observed that basket axons made synaptic contacts with all types of Purkinje dendrites in the lower molecular layer and occasionally, as described by Fox and his colleagues (10), with spines. These spines were traced to Purkinje branchlets. The analysis of the synaptic densities (number of profiles of synaptic boutons per 100 ¡j of perimeter) of basket and stellate terminals on Purkinje cells showed some interesting patterns. A proximo-distal decrease in the synaptic densities of these terminals from the Purkinje cell body (11 profiles per 100 |j) to the distal dendrites 1.3 profiles per 100 p) was observed. This finding indicates that inhibitory terminals on the Purkinje cell extend all over the dendritic tree, although the greater density of inhibitory terminals occurs upon the cell body. It is interesting in this respect to mention that the number of climbing fiber synapses on the Purkinje cell increases from the cell body to dendrites 1.5 n in diameter, and that the number of parallel fibers which synapse upon spines increases progressively from mediumsized Purkinje dendrites to branchlets. As a result of these gradients of synaptic distribution, it appears that in the Purkinje cell the excitatory synapses (climbing and parallel fibers) outnumber by far the inhibitory basket and presumably stellate synapses, and that the ratio of excitatory over inhibitory synaptic densities increases tremendously from the soma, where almost all

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synapses are inhibitory, to the distal dendrites, where the opposite is true. This distribution confirms the spirit, if not the letter, of Andersen & Eccles' postulate (1) stating that the best design of a cell for an effective inhibitory effect should place the inhibitory synapses upon the cell body close to the axon hillock. Patterns of Distribution of Synapses upon Basket and Stellate Cells Golgi studies by Ramón y Cajal (32), followed by those of Scheibel & Scheibel (33) have long since suggested that parallel fibers, climbing fibers and recurrent axons from basket and stellate cells form synapses with basket and stellate cells. While recent electron microscopic studies have confirmed that parallel fibers do, in fact, establish synaptic contacts with basket and stellate cells, some disagreement exists as to the site of these synapses. Hámori & Szentágothai (15, 16) insist that they occur only upon spines, whereas Fox (10) has observed them directly on the dendrites. Our investigations in the mouse confirm that parallelfibersmake synaptic contacts with the smooth surface of the soma and dendrites of basket cells and that the spines of these cells also receive parallel fibers (actually, more than one parallelfibersynapse per spine). Hámori & Szentágothai (15,16) have observed other types of synapses on basket and stellate cells. On the basis of indirect evidence these authors have suggested that climbing fibers and Purkinje collaterals or stellate terminals make synaptic contacts with these cells. In our electron microscopic study in the mouse we have clearly established that basket cells receive synapses from parallel, climbing, Purkinje collaterals and basket-stellate axons both upon the soma and dendrites. Uppermost stellate cells, however, receive synaptic contacts only from parallel fibers and stellate axons. A quantitative analysis revealed that most of the synapses received by basket cells were parallel fibers. Purkinje collaterals and basket-stellate axons each contributed approximately ten per cent of the synapses, and climbing fibers less than five per cent (Figure 132). It was also found that the synaptic density of the cell body was lower than that of the distal dendrites. From the analysis of synapses several patterns emerged regarding the distribution of excitatory (parallel and climbing fibers, 6) and inhibitory synapses (Purkinje, basket and stellate axons, 6, 24) upon these interneurons: (a) Parallel fibers are the main source of excitatory terminals for basket cells and the only source for uppermost stellate cells; (b) basket-stellate axons and Purkinje collaterals are the source of inhibitory terminals to basket cells; (c) stellate cells receive only half the amount of inhibitory input of basket cells, due to their lack of Purkinje collaterals; (d) very low basket cells appear to receive only Purkinje collaterals as a source of inhibition (these observations refer only to the cell body); (e) excitatory synapses outnumber inhibitory synapses by a factor greater than ten; and ( / ) the ratio of synaptic densities of excitatory over inhibitory terminals ( E / I ) in-

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Purkinje cell dendrites

IS. D. : 4.51

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c

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Purkinje cell soma 8 dendrites IS D : 18 .01

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Figure 132. Percentage distribution of synapses from parallel fibers (p.f.)i climbing fibers (cl.), basket or stellate cells (b.-s.), and Purkinje collaterals (P.c.) on the soma of stellate cells at different depths in the molecular layer of the mouse cerebellum. Synaptic density (S.D.) refers to the number of synaptic boutons per 100 ¡J, of cell perimeter. Note that both percentage and synaptic density change in stellate cells at different depths. Dark tracing denotes inhibitory synapses. The cells referred to as "very low baskets" are probably correctly so named, but without definite evidence.

creases in both basket and stellate cells in a proximo-distal direction from the cell body the distal dendrites; this increase in the ratio E / I is due mainly to the increase in the synaptic density of excitatory terminals and not to a decrease in the density of inhibitory terminals.

Comparison of Patterns of Synaptic Distribution in Purkinje and Basket and Stellate Cells The following similarities were observed among the two sets of cells (stellate and basket versus Purkinje cells): ( a ) Dendrites receive more synapses per unit of length than do the somata; ( b ) excitatory synapses far outnumber inhibitory synapses; (c) spines receive excitatory synapses almost exclu-

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sively; (d) excitatory synapses increase in number progressively from the cell body to the distal dendrites whereas inhibitory synapses tend to decrease; and ( e ) axon collaterals recur upon cells of the same class. Thus, it seems that apart from specific differences, both sets of cells have common patterns of synaptic organization. SYNAPTIC PATTERNS AND PHYSIOLOGICAL PHENOMENA

MEDIATED BY BASKET AND

STELLATE C E L L S IN THE MOLECULAR L A Y E R

Our data placed in the context of the neural circuitry of the cerebellar cortex, as shown in Figures 133 and 134, clearly support the concept of inhibition and disinhibition mediated by cerebellar interneurons as described by others. More specifically, our results support the excitatory function of climbing and parallel fibers on basket and stellate as well as on Purkinje cells (4, 6, 7, 25, 26); the inhibitory action of Purkinje collaterals, basket and stellate axons upon Purkinje cells (4, 5, 8 ) ; and the disinhibitory phenomena of the Purkinje cell resulting from the inhibition of basket and stellate cells by Purkinje collaterals (24). Finally, our data suggest that the phenomena of inhibition and disinhibition upon Purkinje cells predominate in the lower portion of the cell rather than in the dendrites of the upper molecular layer. Let us conclude by stating that our results clearly demonstrate that electron microscope techniques can be applied successfully to the study of synaptic patterns upon nerve cells. Tauc: Did you make serial sections to establish the synaptic strength of boutons upon the basket cell soma?

r

Figure 133. Qualitative synaptic connections of parallel fibers, Purkinje cells and stellate cells in the mouse molecular layer as established by our electron microscope studies. The shaded nerve terminals are those observed to contain small, elongated clear synaptic vesicles; according to physiological evidence these nerve terminals are inhibitory. Numbers in the diagram indicate synaptic events in time which would follow a hypothetical discharge of a set of granules. Note the synergic action of Purkinje collaterals and basket and stellate terminals either on the Purkinje cell or on basket and stellate cells themselves. These synaptic connections appear to be the anatomical substrate for the phenomena of inhibition and disinhibition of the Purkinje cell found by the physiologists.

Figure 134. Climbing fibers and their synapses in the mouse molecular layer, as established by our electron microscope studies. Parallel fiber synapses are not included in the diagram. Compare with Figure 133.

Larramendi:

No. All of these measurements were made on a random sam-

Kandel: How do you decide which is a parallel fiber, to begin with? Do you do degeneration studies? Larramendi: No. We make our decision by correlating EM profiles with descriptions of parallel fibers as seen in Golgi impregnations. In this manner we can correlate size, shape, orientation and position of the parallel fibers with corresponding profiles. The fact is that each type of terminal has its own morphological characteristics, at least in the cerebellum. Adey: Dr. Larramendi, what is the standard deviation in the measurements you have reported? Larramendi: We do not have the standard deviations; however, I believe that the spread is small. Kennedy: As you were talking, I thought that you were using these very characteristics to define the types of boutons. It seems to me that there is something a little circular about this. You have used the various characteristics to categorize a bouton, and then you are separating characteristics and telling us about the significance of the difference. Larramendi: The profiles of boutons were identified by the size and shape of the bouton, by the characteristics of the synaptic vesicles and their aggregation, by the presence of the neurotubules or neurofilaments, by the density of the axoplasm and by the characteristics of the pre- and postsynaptic densities. The measurements of the length of the synaptic adhesions were made in already-identified profiles. So, I do not see any circularity in our operations to define the synaptic strength of boutons. »

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Adey: Dr. Larramendi, your figures are most exciting, and I think the principal point that I drew from your presentation, or one of them at least, is the question of very high synaptic density in the tertiary dendrites. This, of course, raises all sorts of problems in terms of excitation and inhibition in the frame of a cable theory of dendritic conduction as proposed by Rail (31) and his colleagues, and more recently the work of Granit (12), for example, on remote inhibition which may perhaps involve peripheral dendritic structures. At least the question of the window that the soma has on its own dendritic tree, and particularly the region of the axon hillock, is one that remains very enigmatic in terms of the way in which such remote structures could exercise their effect upon a centrally situated trigger zone. Therefore, I would invite the contributions of some of the physiologists to this very interesting observation of such a high synaptic density in the peripheral path of the dendritic tree. Llinas: An important point here is that, as far as Purkinje cells are concerned, their dendrites seem to be able to generate action potentials. The evidence for this statement is indirect. If one records the field potentials generated by the antidromic invasion of Purkinje cells, it is very often found that the maximum negative deflection corresponds to the level of the lower dendrites and not to the somatic level. This agrees with the idea of active antidromic invasion of dendrites. There is also other indirect evidence. For instance, different firing levels can be found in the same Purkinje cell when superficial and deep parallel fiber systems are activated. This difference strongly suggests the existence of more than one site for spike initiation. On the other hand, one can imagine that even if the distal synaptic depolarization cannot reach the firing level of any of these sites, they can still function as important modulatory systems, which alter in a very discrete manner the excitability of the cell and thus serve as a very fine regulatory system. Purpura: I have had several problems following your arguments, Dr. Larramendi, and in evaluating some of your conclusions. What probably disturbs me most is the extraordinary facility with which you identify elements in electron micrographs. We always seem to have much greater difficulty in our laboratory. Perhaps you could elaborate on additional criteria for the identification of cellular elements, apart from saying that you look at the Golgi picture. I do not know how you safely bridge the gap between looking at Golgi material and then jumping to the identification of specific elements in electron micrographs. I would also appreciate it if you would elaborate a little on the relationships between adhesion sites, characteristics of different terminals and the organization of different synapses. There is also a specific point of information I would request. Your attempts to show that the climbing fiber engages only the secondary and tertiary spine synapses is somewhat disturbing to me. As you know, in the early postnatal period, Purkinje cell dendrites do not exhibit tertiary branchlets, yet the climbing fiber appears to make effective synaptic contacts (30). Do you envision a progres-

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sive displacement of the climbing fiber synapses from the dendritic trunks and large branches to the tertiary branchlets when the latter develop? Larramendi: The first question is too general. You have to be more specific. For instance, you may ask how I translate the Golgi picture of a parallel fiber to the profiles observed with the EM. Purpura: I have no argument with that relationship. Larramendi: Then I would like to discuss the identification of these terminals one by one. Let me take the climbing fibers first. In the mouse cerebellum perfused with glutaraldehyde, climbing fiber profiles have a distinctive E M appearance. They appear as axonal segments rich in filaments (neurotubules) or as axonal enlargements filled with round synaptic vesicles. These enlargements consistently show synaptic contacts with spine profiles. Nerve terminals with these characteristics are seen close to the apical portion of the Purkinje cell body and, specially, close to the primary and secondary dendrites of the Purkinje cell. Actually, spines from these segments of the Purkinje cell form synaptic contacts with the axonal enlargements of these nerve terminals. Profiles identified as climbing fibers are very seldom seen following Purkinje tertiary or branchlet dendrites. The distribution of these terminals upon the Purkinje cell body and its dendritic tree follows exactly the distribution of climbing fibers as seen in Golgi impregnations. Furthermore, profiles of climbing fibers were not observed in the uppermost portion of thé molecular layer, as could be expected from their known distribution according to Golgi impregnations. Therefore, we are entitled from these correlations to conclude that the profiles of nerve terminals described above belong to climbing fibers. With respect to the developmental point raised by Dr. Purpura, we can say that the activation of Purkinje cells by climbing fibers is entirely possible before secondary and tertiary dendrites are formed. The developing climbing fibers form their synapses first on the soma of the Purkinje cells (pericellular nest stage) and later on primary and secondary dendrites. The interesting finding is that the climbing fibers make synaptic contacts with spinelike processes of the immature Purkinje cell soma which disappear almost entirely in the adult animal. Whether the spines of the Purkinje cell which form synapses with the climbing fibers are reabsorbed or become translocated upwards to the developing dendrites it is not yet completely clear. In conclusion, our developmental evidence in the mouse and the evidence in the frog and alligator observed by Hillman and Llinas® support our contention that climbing fibers form synapses mainly on spines of the soma and large dendrites of Purkinje cells Let me now say something about the identification of basket axons in the mature animal. Profiles of basket axons can be recognized with the E M by their appearance and distribution. They appear as large tubular structures, * Personal communication.

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filled with a clear axoplasm rich in thin neurofilaments. Close to their synaptic site they contain synaptic vesicles which are different from those observed in parallel fibers and climbing fibers. Kandel: When you say "the vesicles are different", can you actually trace in serial sections a process from the cell body to the terminal, to be sure you are in fact describing a distinct terminal? Larramendi: Basket cell axons can be recognized by their position around the cell body of the Purkinje cell. Kandel: There are other synapses coming to the cell body besides the basket axons. Larramendi: No. Kandel: You mean every synapse on the cell body is a basket cell? Larramendi: The overwhelming majority are basket synapses. We have been able to find a few that are climbing fibers but those form synapses with spines of the soma of the Purkinje cell. We have not yet observed either parallel fibers or Purkinje collaterals forming synapses on the Purkinje cell body. Basket axons do actually make, here and there, synaptic contacts with the Purkinje cell body. Skoglund: When you say "making contact here" or "making contact there", what do you actually mean? If these synapses are on one and the same bouton, I do not see what you mean by these contacts being interrupted. We have worked with the spinal cord for several years now, and actually we run into all kinds of trouble if we do not use serial sections. You must require an ideal sectioning to see the whole synaptic cleft all the way when you use random sections. Larramendi: In sections which are longitudinal to the basket axon you can see that they form multiple contacts on the Purkinje cell body. From a random sample you can infer it. Skoglund: That is my point; if you use serial sections you will see where the actual synaptic surface is, whereas otherwise you will see these interruptions because you have not got the ideal sections. Larramendi: From a random sample you can get an average description of the synapses observed, whereas from a serial section you can derive only a description of a single sample. Scheibel: While we all agree that reconstruction techniques have much to offer, let us not forget that random sampling methods have been significant in the past and will continue to be so. Skoglund: I will make only a general statement, and that is that I think that if we are actually to get to know what these contact areas mean, we have to use serial sections, because otherwise we do not know what kinds of dendrites they are, or what kinds of contacts they have. So, I claim that serial sections are actually needed before we are able to make any firm statements. Andersen: May I raise one point with regard to oval and round vesicles.

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Although I would very much like the differentiation into two types of synapses to be true, I wonder whether this division holds true in other areas. Do you have any excitatory terminals with oval vesicles in them? In Oslo I have myself seen pictures of basket cells where the vesicles are partly perfectly round, partly oval. I am not quite sure that you can make a general statement on this histological correlation with a functional characterization of the synapses. Larramendi: The only way to answer your point is by describing what we did to reach the conclusion that basket axons contain elongated vesicles. We collected 350 synaptic vesicles from 20 different basket axon synapses and we measured the major and minor diameters of the vesicles. From the average major and minor diameters of the vesicle population we established an "elongation index". Basket axons and Purkinje terminals had an "elongation index" which varied between 1.3 and 1.5. We also established the average size of the vesicle population. Kandel: I am not sure one can correlate the configurations of a basket cell terminal with a general kind of synaptic action. I do not disagree with you. I think it perfectly all right to say, empirically, "In the cerebellum this is what I find", but not to generalize and say, "All inhibitory synapses will be characterized this way". Larramendi: You have evidence that this may not be so. I am establishing only the empirical correlation in the cerebellum. Segundo: If you make a histogram of all of the indices of elongation, do you get a clearly bimodal curve with two distinctly preferred values? A bimodal histogram would suggest strongly that there are two different subpopulations, one with a big index and one with a small index. If the indices cluster around one value, or are uniformly distributed, it may be arbitrary to postulate two populations. Larramendi: I have not tried it, but my guess is that I will get a bimodal distribution. I would like to say that the differences among vesicle populations are observable by simple inspection of the electron micrographs. For instance, Golgi terminals contain the smallest synaptic vesicles of all types of terminals present in the cerebellar cortex as determined by visual inspection, and this impression was clearly demonstrated in our measurements. In fact, measurements only added a numerical value to otherwise visible differences. Kennedy: I am going to press a little further with this. You said that, irrespective of what a histogram would look like, the measurements prove what visual observations suggested, namely that there is a statistically significant difference between the mean indices of elongation. I wonder if you can show us the numbers that indicate this statistical significance—what are the standard deviations of those two means? Larramendi: I do not have them here, but they can be found in a paper that appeared recently in Science (20).

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Maynard: The populations of terminals you describe are defined on the basis of fine structural parameters within the terminals. In most of the discussion it seems to be implicitly assumed that different terminal populations, as defined above, necessarily come from correspondingly different neuron types. Is it at all possible that these differences, observed in the terminal populations correspond to different stages of age or activity within single cell types? Larramendi: I do not think so. However, the changes in size and form that we have observed in vesicles during maturation could be related to the "age" of the vesicles. Thus far, the "half-life of synaptic vesicles" is not known, and it is not possible to ascertain whether or not vesicles change in form or size with age. The differences in vesicle populations among the various types of terminals may reflect the morphological individuality of the cells from which vesicles arise. For instance, it appears that there are at least two types of vesicle populations among cerebellar inhibitory synapses, one represented by the vesicles of Purkinje and basket terminals, and the other by those present in Golgi terminals. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.

P., and ECCLES, J. C., Locating and identifying postsynaptic inhibitory synapses by the correlation of physiological and histological data. Symp. Biol. Hung., 1965, 5: 219-242. ECCLES, J. C., The Physiology of Synapses. Academic Press, New York, 1964. ECCLES, J. C., ITO, M., and SZENTAGOTHAI, J., The Cerebellum as a Neuronal Machine. Springer, New York, 1967. ECCLES, J. C., LLINAS, R., and SASAKI, K., The excitatory synaptic action of climbing fibres on the Purkinje cells of the cerebellum. /. Physiol. (London), 1966, 182: 268-296. , The action of antidromic impulses on the cerebellar Purkinje cells. J. Physiol. (London), 1966, 182 : 316-345. , The inhibitory interneurones within the cerebellar cortex. Exp. Brain Res., 1966, 1: 1-16. , Parallel fibre stimulation and the responses induced thereby in the Purkinje cells of the cerebellum. Exp. Brain Res., 1966, 1: 17-39. , Intracellularly recorded responses of the cerebellar Purkinje cells. Exp. Brain Res., 1966, 1: 161-183. Fox, C. A., The structure of the cerebellar cortex. In: Correlative Anatomy of the Nervous System (E. C. Crosby, T. Humphrey, and E. W. Lauer, Eds.). Macmillan, New York, 1962: 193-198. ANDERSEN,

1 0 . F o x , C . A . , H I L L M A N , D . E . , SIEGESMUND, K . A . , a n d DUTTA, C . R . ,

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primate cerebellar cortex: a Golgi and electron microscopic study. Progr. Brain Res., 1967, 25: 174-225. 11. Fox, C. A., SIEGESMUND, K. A., and DUTTA, C. R . , The Purkinje cell dendritic branchlets and their relation with the parallel fibers: light and electron microscopic observations. In: Morphological and Biochemical Correlates

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of "Neural Activity (M. M. Cohen and R. S. Snider, Eds.), Hoeber Med. Div., Harper & Row, New York, 1964: 112-141. GRANIT, R., KELLERTH, J.-O., and WILLIAMS, T. D., Intracellular aspects of stimulating motoneurones by muscle stretch. J. Physiol. (London), 1964, 174: 435-452. GRAY, E. G., The granule cells, mossy synapses and Purkinje spine synapses of the cerebellum: light and electron miscroscope observations. J. Anat., 1961, 95: 345-356. HÄMORI, J., and SZENTÄGOTHAI, J., The Purkinje cell baskets: ultrastructure of an inhibitory synapse. Acta Biol. Acad. Sei. Hung., 1965, 15: 465-479. , Identification under the electron microscope of climbing fibres and their synaptic contacts. Exp. Brain Res., 1966, 1: 65-81. — , Participation of Golgi neuron processes in the cerebellar glomeruli: an electron microscope study. Exp. Brain Res., 1966, 2: 35-48. HERNDON, R. M., The fine structure of the Purkinje cell. J. Cell Biol., 1963, 18: 167-180. , The fine structure of the rat cerebellum. II. The stellate neurons, granule cells, and glia. J. Cell Biol., 1964,23: 277-293. ITO, M., YOSHIDA, M., and OBATA, K., Monosynaptic inhibition of the intracerebellar nuclei induced from the cerebellar cortex. Experientia, 1964, 20: 575-576. LARRAMENDI, L. M. H., FICKENSCHER, L., and LEMKEY-JOHNSTON, N., Synaptic vesicles of inhibitory and excitatory terminals in the cerebellum. Science, 1967, 156: 967-969. LARRAMENDI, L. M. H., and VICTOR, T., Synapses on the Purkinje cell spines in the mouse; an electronmicroscopic study. Brain Res., 1967, 5: 15-30. LEMKEY-JOHNSTON, N., and LARRAMENDI, L. M. H., Morphological characteristics of mouse cerebellum stellate and basket cells: an electronmicroscopic study. J. Comp. Neurol., in press. , Types and distribution of synapses upon basket and stellate cells of the mouse cerebellum: an electronmicroscopic study. J. Comp. Neurol., in press. LLINÄS, R., Discussion of Oscarsson, I., Functional significance of information channels from the spinal cord to the cerebellum. In: Neurophysiological Basis of Normal and Abnormal Motor Activities (M. D. Yahr and D. P. Purpura, Eds.). Raven Press, Hewlett, N.Y., 1967: 113-116. LLINÄS, R., and BLOEDEL, J. R., The climbing fibre activation of Purkinje cells in the frog cerebellum. Brain Res., 1967, 3 : 299-302. , Frog cerebellum: absence of long-term inhibition upon Purkinje cells. Science, 1967, 155 : 601-603. PALAY, S. L., The electron microscopy of the glomeruli cerebellosi. In: Cytology of Nervous Tissue. Taylor & Francis, London, 1961: 82-84. , Fine structure of cerebellar cortex of the rat. Anat. Ree., 1964, 148: 491. —•—-, The structural basis for neural action. In: Brain Function, Vol. II: RNA and Brain Function; Memory and Learning (M. A. B. Brazier, Ed.).

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UCLA Forum Med. Sei. No. 2, Univ. of California Press, Los Angeles, 1964: 69-108. 3 0 . PURPURA, D . P . , SHOFER, R . J . , HOUSEPIAN, E . M . , a n d NOBACK, C . R . ,

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parative ontogenesis of structure-function relations in cerebral and cerebellar cortex. Progr. Brain Res., 1964, 4: 187-221. 3 1 . R A L L , W . , Theoretical significance of dendritic trees for neuronal input-output relations. In: Neural Theory and Modeling (R. F. Reiss, Ed.). Stanford Univ. Press, Stanford, 1964: 73-97. 3 2 . R A M Ó N Y C A J A L , S . , Histologie du Système Nerveux de l'Homme et des Vertébrés, Vol. II. Maloine, Paris, 1911. 33. SCHEIBEL, M. E., and SCHEIBEL, A. B., Observations on the intracortical relations of the climbing fibers of the cerebellum. A Golgi study. J. Comp. Neurol., 1954, 101: 733-764.

NEURONS OF CEREBELLAR NUCLEI MASAO ITO University of Tokyo Tokyo, Japan

The cerebellar nuclei form an intermediate stage of the cerebellar efferent pathway; the direct output from the cerebellar cortex is mediated solely by axons of Purkinje cells which pass to the cerebellar nuclei; the projection axons from the cerebellar nuclei in turn innervate brain stem neurons (Figure 135A). This two-stage projection occurs in all of the three phylogenic subdivisions of the cerebellum: (a) from the vermis to the nucleus fastigii and then to vestibular nuclei and reticular formation; (b) from the paravermis to the nucleus interpositus and then to the red nucleus and thalamus, particularly the nucleus ventralis lateralis (VL); and (c) from the hemisphere to the nucleus lateralis (or dentatus) and then to VL. However, there is a direct pathway from the cerebellar cortex to certain brain stem nuclei; Purkinje cells located mainly in the vermal cortex send their axons to Deiters' nucleus as well as to other vestibular nuclei (36). This type of long corticofugal projection seems to be common in the cerebellum of lower vertebrates, such as fish (4,26). On the basis of these anatomical observations, it seems to have been generally thought that the cerebellar nuclei are simple relay stations in the flow of information from the cerebellar cortex to brain stem centers (Figure 135A); however, recent physiological investigation has revealed the direct inhibitory action of Purkinje cell axons upon their target neurons in the cerebellar nuclei (21, 22) as well as in Deiters' nucleus (16, 19, 20). It would be necessary to assume that the cerebellar nuclei are reflex centers by themselves which, under the inhibitory control by Purkinje cells, integrate excitatory signals from various extracerebellar structures, as illustrated diagramatically in Figure 135B (17). In further efforts to identify the sources of excitatory input to cerebellar nuclei, collateral innervation by the cerebellar afferent fibers has gained new importance. Lorente de No (27) indicated that the spino-cerebellar fibers passing through the restiform body supply collaterals to Deiters' nucleus. Brodal & Torvik (8) also showed that the secondary vestibular fibers, arising from the vestibular descending nucleus, terminate both in the fastigial nucleus and in the cerebellar cortex of theflocculo-nodularlobes. Recently, 309

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B

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Figure 135. Neuronal connections between the cerebellar cortex and subcortical centers. Arrows indicate direction of impulse propagation. Abbreviations used in this and subsequent figures: B, basket cell; BS, brain stem neuron; cf, climbing fiber; CN, neuron of the cerebellar nuclei; CN', Golgi type II neuron in the cerebellar nuclei; DVN, descending vestibular nucleus; F , fastigial nucleus; G, Golgi cell; gr, granule cell; I.O., inferior olive; IP, nucleus interpositus; LN, lateral nucleus; LRN, lateral reticular nucleus; mf, mossy fiber; P, Purkinje cell; PN, pontine nucleus; P R F , paramedian reticular formation; RN, red nucleus; VL, nucleus ventralis lateralis.

Szentagothai gave evidence that the spinocerebellar system contributes quite a number of collaterals to the fastigial nucleus, less but still significant numbers to the nucleus interpositus and even some to the lateral nucleus (12). The ponto-cerebellar and olivo-cerebellar, as well as probably the reticulo-cerebellar system, supply the cerebellar nuclei with quite abundant numbers of collaterals (12). There seems to be no specific input to the cerebellar nuclei. This rather peculiar input organization in the cerebellar nuclei is illustrated in Figure 135C. It seems to be important that both of the two distinct types of cerebellar afferents, giving rise in the cerebellar cortex—to mossy and climbing fibers, respectively—contribute collaterals to the cerebellar nuclei. It is also indicated histologically (12) that the collaterals of cerebellar afferents make

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contact with dendrites of the neurons in the cerebellar nuclei, while Purkinje cell axons innervate their somata. Functional implications of these arrangements will be discussed later. Some intrinsic organization within the cerebellar nuclei has also been indicated by two lines of evidence; first, in addition to the large, probably projective neurons, numerous small cells exist in the cerebellar nuclei, particularly in the dentate nucleus. These cells have been assumed to be Golgi type II neurons (23). Small cells with peculiar fine structures have also been recognized in the cerebellar nuclei by electron microscopists (10a);* it should be noted however, that small cells are not necessarily Golgi type II neurons. After transection of the nucleofugal axons in the brachium conjunctivum and restiform body, all cell types, large, medium and small, are involved in the retrograde degeneration (25). Secondly, projection neurons in the cerebellar nuclei issue axon collaterals (30). These collaterals appear to terminate locally within the cerebellar nuclei and not to extend toward the cerebellar cortex (31). The postulate that the cerebellar nuclei extend their axons, either collaterals of projective axons or stem axons of a special cell group, toward the cerebellar cortex as climbing fibers (9) has now to be discarded, because both histological and physiological evidence indicates that the climbing fibers originate from the inferior olive (13, 31). The intranuclear collaterals of projection axons may innervate the projective neurons directly, or indirectly via Golgi type II neurons. If so, the cerebellar nuclei are equipped with a recurrent system such as exists in many other parts of the central nervous system. The functional significance of this system, however, would vary greatly according to the nature of the Golgi type II neurons, which can be either inhibitory or excitatory. ELECTRICAL ACTIVITIES IN CEREBELLAR NUCLEI

Events in the cerebellar nuclei have been studied by us with intracellular microelectrode techniques (21, 22). Their major characteristics are essentially the same as those observed in Deiters neurons which, because of their giant size and the subsequent ease of penetration, have been the subject of thorough intracellular investigation (16-20). In our work, cats were anesthetized by pentobarbital sodium. In order to approach the cerebellar nuclei, microelectrodes were advanced either horizontally in the caudocranial direction with the cat's head fixed in a sphinx position (Figure 136), or vertically in a ventrodorsal direction through the medulla, the cat's head being held upside down (see Figure 138C). Antidromic Invasion The axons from the nuclei lateralis and interpositus project rostrally through the brachium conjunctivum and pass the red nucleus (RN), eventually reaching VL. Stimulation at VL and RN, therefore, induces anti" Also, L. M. H. Larramendi: personal communication.

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Figure 136. Diagram showing how to approach the cerebellar nuclei with microelectrodes, and how to stimulate them antidromically.

dromic invasion of the neurons of these nuclei. This procedure evoked a fast negative field potential within nuclei lateralis and interpositus (Figure 137, A, B ) . Correspondingly, individual neurons of these nuclei generated a negative or positive-negative diphasic extracellular spike (Figure 137, C, D ) , or, when impaled with the microelectrode, a full-sized spike potential with ISSD configuration (Figure 137, E, F ) . Immediately after impalement, the resting potential rose to — 50 mV. It tended to decline rapidly, owing to the injurious effect of penetration. The latency of the antidromic invasion from RN was measured for 30 intracellular and 19 extracellular units of the lateral nucleus (22). It ranged from 0.3 to 1.3 msec., with the mode at 0.6 msec. (Figure 137 H ) . Thirty of these 49 units were activated also from VL at longer latencies. The difference of the latency between RN and VL stimulation was 0.2-1.3 msec. (Figure 137G). Since the distance between these two regions was about 6 mm, the conduction velocity of the lateral nucleus neurons should be 5-30 meters per second. This is in agreement with 30 m/sec. calculated for the excitatory pathway from the interpositus nucleus to the red nucleus (33), and 10-20 m/sec. for that from the lateral nucleus to VL (34). Some lateral nucleus neurons were activated also from the medullary reticular formation, probably through their axon collaterals extending to this region ( 6 ) . Inhibition from the Cerebellar Cortex Stimulation of the cerebellar cortex produces prominent inhibitory postsynaptic potentials (IPSPs) in the underlying cerebellar nuclei neurons

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Figure 137. Antidromic activation of lateral nuclei neurons. A, C, E: Field potential, extracellular and intracellular spikes respectively, evoked by stimulation of RN. B, D, F: Stimulation of V L under the same recording conditions as in A, C, E, respectively. G: Frequency distribution of the latency differential of antidromic invasion from V L and RN; the lower scale on the abscissae is for calculation of the conduction velocity. H: Latency of antidromic invasion from RN.

(Figure 138A) (21, 22). When the stimulus was given to an appropriate location on the cerebellar cortex, the latency of the IPSPs was as brief as 0.71.1 msec. Their exact moment of onset was determined at the diverging point of their time courses taken under normal conditions and during intracellular injection of hyperpolarizing currents and chloride ions, which reversed the IPSPs in a depolarizing direction (Figure 138B). The short latency 6f the IPSPs indicates their monosynaptic origin ( 2 1 , 2 2 ) . Localization of the monosynaptic inhibitory area for each cerebellar nucleus conforms to the cerebellar corticofugal projection by Purkinje cells as determined histologically (24). Individual neurons in the lateral nucleus have their respective monosynaptic inhibitory zones somewhere in the hemispheral cortex (Figure 139, A, B, C ) . The nucleus interpositus is inhibited monosynaptically from the intermediate zone and the nuclei fastigii and Deiters' nuclei from the vermis (16, 21, 22). The running course of the inhibitory fibers from the vermis to the nucleus of Deiters was explored in detail and found to be in excellent agreement with the long corticofugal projection by Purkinje cell axons (16). Delayed IPSPs were often recorded within neurons of the cerebellar nuclei, either in isolation (Figure 139A, record 1) or in superposition upon the monosynaptic IPSPs (Figure 139A, records 2, 3, upward arrows). These

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msec Figure 138. Monosynaptic initiation of IPSPs in lateral nucleus neurons after stimulation of the cerebellar cortex. A: Upper traces, intracellular record; lower traces, extracellular control; pulse stimuli were given to the hemispheral cortex with electrode No. 1 of C. B: Same IPSP as in A, but taken at a faster sweep speed; upper traces taken during passage of a hyperpolarizing current (3 X 1 0 9 Amp.) through the impaling KCl-filled microelectrode; lower traces, original potential; the superposed dotted line indicates the time-course of the upper traces; upward arrow marks the diverging point of the upper and lower traces. Ca: Transverse section of the brain stem and the cerebellum through the lateral nucleus (m: microelectrode track); Cb: another section at 1 mm rostral from a; three concentric electrodes (Nos. 1, 2, 3) were attached at the level of b. (From Ito & Yoshida, 20.)

IPSPs are also attributable to Purkinje cell axons which can be excited by cerebellar stimulation, not only directly but also trans-synaptically via the climbing fibers or through the mossy fiber-granule cell transmission, as shown in the diagram of Figure 139D. In particular, impulses along the climbing fibers excite Purkinje cells monosynaptically with extraordinary high efficacy of transmission ( 1 3 ) . Climbing fiber activation was seen commonly in Purkinje cells around the stimulating electrodes implanted in the cortex ( 1 9 ) . Its latency varied over a wide range from 0.8 to 10 msec. The latency of the IPSPs recorded in Deiters neurons showed similarly wide

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>-4mV Figure 139. Monosynaptic and polysynaptic inhibition of lateral nucleus neurons from the cerebellar cortex. A: Potential changes recorded during stimulation at the spots indicated in the inset diagram; downward arrows mark the onset of monosynaptic IPSPs and the upward ones that of delayed IPSPs. B, C: Plot sizes of the monosynaptic IPSPs for two cells of lateral nucleus on the sites of stimulation; note that the line drawing of B and C represents the right hemisphere of the cerebellum shown in A. D: Diagrammatic illustration of the polysynaptic inhibitory pathway from the cerebellar cortex to the cerebellar nuclei. Abbreviations and convention as in Figure 135.

variation. These wide latency variations appear to be caused by excitation of different branches of climbing fibers within the cerebellar cortex, as well as by reflex activation of the inferior olive neurons through their excitatory interconnections ( 1 3 , 1 9 ) . Action of Olivary Impulses When the inferior olive is stimulated directly, Purkinje cells in the contralateral cerebellar cortex are excited very effectively, presumably through the climbing fiber terminals on Purkinje cells (13, 3 1 ) . This was indicated by the appearance of a large spiky potential deflection in the cerebellar cortex (Figure 140A) at a latency of about 3 msec; and by the characteristic climbing fiber responses in individual Purkinje cells (Figure 140B). Corresponding changes in the neurons of cerebellar nuclei, as well as in Deiters neurons, were a sequence of a brief EPSP and a large, prolonged IPSP, as illus-

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Figure 140. Effects of stimulation of the inferior olive. A: Evoked potential in the cerebellar cortex of the contralateral hemisphere, recorded with a concentric electrode; downward arrow points to the moment of the olivary stimulation. B: Intracellular potential recorded from a Purkinje cell. C: Intracellular record from a neuron of lateral nucleus. D: Same as in C, but at higher amplification. E: Extracellular control taken just after D. F: Upper traces, intracellularly recorded EPSP from a red nucleus neuron ( 3 2 ) ; lower traces, extracellular control. Voltage calibration of 2 mV applies to B-E, that of 5 mV to F. G illustrates diagrammatically the neuronal connections involved in the olivary stimulation.

trated in Figure 140, C, D for a lateral nucleus neuron. The onset of the EPSP (upward arrow in D) preceded the climbing fiber activation of Purkinje cells by about 1 msec, and the start of the IPSP (downward arrows in C, D) by about 2 msec. This EPSP is presumed to be induced monosynaptically through the olivo-cerebellar fibers which, as illustrated in Figure 140G, innervate cerebellar nuclei, probably bv their collaterals (19). The effectiveness of the olivary impulses to activate the cerebellar nuclei was demonstrated by the disynaptic excitation from the inferior olive of red nucleus neurons through the interpositus nucleus (Figure 140F) (32). As a rule, one climbing fiber terminal of the olivo-cerebellar projection

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makes an intimate contact with dendrites of a single Purkinje cell ( 1 2 ) . Accordingly, impulses along climbing libers induce one, or sometimes two, large unitary EPSPs in the Purkinje cell (Figure 140G). On the contrary, the EPSPs evoked in the cerebellar nuclei neurons (Figure 140, C, D ) , or Deiters neurons ( 1 9 ) from the inferior olive, appear to be composed of much smaller unitary EPSPs converging from many presynaptic fibers. This is an example where the same stem fibers supply synaptic terminals of different forms (not the excitatory-inhibitory nature). Similar differentiation can be seen between the axon terminals of motoneurons on the muscle fibers, on the one hand, and on the Renshaw cells, on the other ( 1 1 ) . Action of the Mossy-Fiber Cerebellar

Afferents

Pontine nucleus and lateral reticular nucleus are sources of mossy fibers which innervate the cerebellar hemisphere. As seen in Figure 141, A, B, stimulation of these structures induced, in lateral nucleus neurons, small EPSPs, probably monosynaptically through collaterals of ponto-cerebellar and reticulo-cerebellar fibers. The small amplitude of these EPSPs may be due to

Figure 141. Effects of stimulation of the pontine nucleus and lateral reticular nucleus upon lateral nucleus neurons. A: Upper traces, intracellularly recorded potentials during stimulation of PN; dotted line indicates the baseline given by the extracellular control in the lower traces. B: Intracellular and extracellular records taken in another cell during stimulation of the lateral reticular nucleus; upward arrows point to the moment of onset of EPSPs. C: Neuronal connections involved in stimulation of these nuclei.

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reduction of the membrane potential which occurs rapidly after impalement with a microelectrode, but it should also be relevant that the collaterals of mossy fibers impinge onto the dendrites of cerebellar nuclei neurons but not onto their somata (12). T h e EPSPs were followed by IPSPs (Figure 141A), which, in respect of their relatively long latency of about 4-5 msec., might be produced by excitation of Purkinje cells through mossy fiber-granule cell transmission (14). EPSPs were sometimes induced in cerebellar nuclei after stimulation of the cerebellar cortex, as shown by a small depolarization in record 1 of Figure 139A. These EPSPs may occur by a kind of axon reflex through collaterals of cerebellar afferents, as studied in detail in Deiters neurons ( 1 7 ) . Disinhibition

A slow depolarization can be produced in cerebellar nuclei after stimulation of the cerebellar cortex as well as various extracerebellar structures, as illustrated in Figure 142, A-F. T h e depolarization was reversed by intracel-

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Figure 142. Disinhibition in lateral nucleus neurons induced by stimulation of cerebellar afferents. A: Intracellular record during stimulation of pontine nucleus. B: Upper traces same as in A, but shown at a slower sweep speed; lower traces, extracellular control just after withdrawal. C: Same cell as in A and B, but with double shock stimulation of PN at an interval of 1.5 msec. D: Same cell as in A, but during stimulation of lateral reticular nucleus. E: Same stimulation as in D, but in another cell and at slower sweep. F: Upper traces same as in E, but during passage of hyperpolarizing currents (10 s Amp.) through the impaling KCl-filled microelectrode; lower traces, extracellular control. Time scale of 10 msec, applies to A and D, that of 100 msec, to B and C, and that of 50 msec, to E and F, respectively. G: Pathway for the disinhibition.

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lular injection of hyperpolarizing currents and chloride ions in the same direction as IPSPs (Figure 142F), indicating their common ionic mechanism. The slow depolarization should therefore be induced by removal of tonic inhibition, that is, disinhibition, as occurs in spinal motoneurons (37). The mechanism of the disinhibition was studied in detail in Deiters neurons in relationship with the inhibitory action of Purkinje cells (18). Within the nucleus of Deiters, the Purkinje cell axons of the cortico-vestibular projection were discharging at a rate of 20-90/sec. They were suppressed drastically for 50-100 msec, after stimulation of the cerebellar cortex. The depression of Purkinje cells would be effected through the inhibitory neurons excited within the cerebellar cortex as well as in the inferior olive (Figure 142E); the basket and outer stellate cells inhibit Purkinje cells (2), and Golgi cells disfacilitate Purkinje cells by inhibiting granule cells which otherwise are tonically activating Purkinje cells (14). Impulses firing back along mossy fibers may activate inhibitory neurons within the inferior olive, and thereby stop spontaneous discharges along the climbing fibers toward Purkinje cells. Respective contributions of these three processes would vary according to the stimulating conditions. The disinhibition accounts for most of the facilitatory effects of cerebellar stimulation upon the cerebellar nuclei (16). Tonic inhibition by Purkinje cells was indicated in the classic experiments where, when the cerebellar cortex was removed or cooled (5) or even deprived of blood supply (29), the decerebrate rigidity was enhanced, indicating release from inhibition of the brain stem motor centers. Possible Recurrent Effects Any effect which may be induced through collaterals of projection axons in the cerebellar nuclei neurons should be revealed at relatively short latency after their antidromic invasion. However, stimulation of the red nucleus region was unexpectedly ineffective in producing such effect in the neurons of the lateral nucleus. As shown in Figure 143, B, C, the only effect so far detected was late EPSPs which occurred at latencies of 2.5 msec, or so. Since their latency is longer than that of the antidromic invasion by the amount of 2 msec., the pathway for these EPSPs should be at least disynaptic. It may be formed through the Golgi type II neurons which should then be excitatory (CN', Figure 143A). It should be noted, however, that the recurrent pathway is not exclusive for explaining this facilitation of Figure 143, B, C, because the disynaptic pathway can also be formed through the pontine nucleus or lateral reticular nucleus. The pontine nucleus may be excited through collaterals of the nucleofugal axons (6), while the lateral reticular nucleus is impinged by collaterals of rubrospinal tract fibers which may be involved in the stimulation (35). Thus evidence is scarce for the recurrent system in the cerebellar nuclei. Very late onset of IPSPs, as in Fig-

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Figure 143. Effects of stimulation of red nucleus upon lateral nucleus neurons. A: Possible disynaptic pathways from RN to a cell in the lateral nucleus. B-D: Traces of the records taken from three different lateral nuclei neurons; upper trace, intracellular record during stimulation of RN; upward arrows mark deteriorated antidromic spike; downward arrows point to the onset of EPSPs in B and C, and that of IPSP in D; note that in C the stimulus intensity is just at threshold for the axon of the impaled cell; lower traces, extracellular controls.

ure 143D, was also noticed sometimes after stimulation of the red nucleus region. This may be explained by activation of Purkinje cells through the mossy fibers which originate from the pontine nucleus as well as from the lateral reticular nucleus (Figure 143A). Action of Nucleofugal

Impulses

Intracellular recording from various brain stem neurons revealed that the direct effect of the cerebellar nucleofugal impulses is monosynaptic initiation of EPSPs. This implies that the nature of the projection neurons in cerebellar nuclei is excitatory. This was confirmed for all of the following projections: from fastigial nucleus to vestibular nuclei and reticular formation ( 1 2 ) , from interpositus nucleus to red nucleus (32, 33), from nuclei interpositus and lateralis to VL ( 3 4 ) . IPSPs were also evoked in V L neurons from interpositus nucleus, but only disynaptically ( 3 4 ) . They are presumed to be mediated by inhibitory interneurons within VL itself.

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Figure 144. Tonic facilitation of red nucleus from the nucleus interpositus and its modulation by cerebellar stimulation. A: Intracellular recording from an interpositus axon within RN. B: Same as in A, but with stimulation of the cerebellar cortex at the moment indicated by arrow. C: Intracellular recording from a red nucleus neuron under the same stimulating conditions as in B. D: Illustrates positions of recording and stimulating electrodes. (From Toyama, Tsukahara & Udo, 32.)

Spontaneous Discharges from Neurons of Cerebellar Modulation by Purkinje Cells

Nuclei and Their

One of the conspicuous features of the cerebellar nuclei is spontaneous occurrence of nucleofugal impulses. Under relatively light anesthesia with pentobarbital interpositus axons, impaled within the red nucleus, discharged rhythmically at the relatively high rate of 50-100/sec. ( 3 2 ) . T h e discharge was suppressed after stimulation of the cerebellar cortex at the intermediate zone, as would be expected by the inhibitory action of Purkinje cells upon interpositus neurons (Figure 1 4 4 B ) . After the period of suppression, the discharge rate was increased above the pre-stimulation level (Figure 1 4 4 B ) , corresponding to the phase of the disinhibitory depolarization that might

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occur in interpositus neurons, as in Deiters and lateral nuclei neurons (18, 22). Intracellular recording from red nucleus neurons revealed concomitant hyperpolarization and depolarization as a result of removal and addition of tonic facilitation, respectively (Figure 144C). Spontaneous discharges at 1-30/sec. have been detected from fastigial units in decerebrate non-anesthetized cats ( 3 ) . Dorsal Deiters neurons in decerebellated non-anesthetized preparations were also found to discharge at 10-80/sec. (38). FUNCTIONAL PERFORMANCE BY CEREBELLAR NUCLEI

The major aspects in the synaptic organization in the cerebellar nuclei are that the Purkinje cells supply them with powerful inhibition, and that collaterals of the cerebellar afferents provide excitatory inputs to them. The former occurs in the soma region of the nuclei neurons and the latter in dendrites. Furthermore, both types of cerebellar afferents, mossy and climbing, supply collaterals. It would therefore be difficult to find a specific kind of synaptic organization there. But rather there may be a nonspecific, somewhat diffuse innervation to built up a background facilitation in neurons of the cerebellar nuclei. This view is supported by the finding, in Deiters neurons, that the cerebellar afferents impinging onto these neurons are mainly those fibers which may carry from the lower medulla information that is highly integrated and therefore with mixed modality specificity and poor spatial discrimination (17). Even if any degree of integration occurs at the level of the cerebellar nuclei, it would be much simpler than what takes place in the elaborate neuronal arrangement of the cerebellar cortex. It looks as if the cerebellar afferent signals build up an excitatory background in the cerebellar nuclei during the time that they are being integrated in the cerebellar cortex to form a spatiotemporal pattern of Purkinje cell excitation; then, by Purkinje cell discharge, this pattern in turn is impressed as an inhibitory pattern upon the cerebellar nuclei. The above view is, moreover, in keeping with the observation that the cerebellar nuclei neurons are discharging tonically toward brain stem neurons. Any activity in the cerebellar cortex modulates, either by inhibition or by disinhibition, the frequency of nucleofugal impulses and consequently the membrane potential of brain stem neurons. The cause of the tonic discharges from cerebellar nuclei may simply be spontaneous impingement of cerebellar afferent signals, but it may be facilitated by the following two processes: First, it seems that the cerebellar nuclei have no recurrent inhibitory system such as exists in the spinal cord (11) and thalamus ( 1 ) . Instead, there is a possibility that the recurrent system has a facilitatory effect. This would favor the continuous firing from the cerebellar nuclei neurons. Secondly, as pointed out by histologists, there are mutual projections between the cerebellar nuclei and certain brain stem neurons. For example,

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Figure 145. Mutual projections between the cerebellar and brain stem nuclei.

between the fastigial nucleus and the vestibular descending nucleus (Figure 145A) (7); between the fastigial nucleus and the paramedian reticular formation (Figure 145B) (6); between the nucleus interpositus and the red nucleus (Figure 145C) (10); between the lateral nucleus and the pontine nucleus (Figure 145D) (6). These brain stem nuclei are sources of mossy fibers and, therefore, the nature of their synaptic terminals would be excitatory. The nucleofugal projection is shown to be excitatory (see above). The action of the rubrospinal fibers upon the spinal cord neurons (15) and upon the lateral reticular nucleus is also excitatory.* It is quite likely that reverberation occurs between these structures; however, it may be noted that the reverberation to be expected here would not occur in the form of impulse circulation along a neuronal linkage with high efficacy of transmission. The effect of traveling impulses would be summated and averaged at each end of the mutual projection, affecting the level of the tonic activity. This would help the cerebellar nuclei to maintain the background facilitation to prepare for the inhibitory sculpturing control by the cerebellar cortex. Skoglund: With regard to the IPSP you get from stimulating the cortex or from the mossy fibers, are the duration and the amount of IPSP larger when you have stimulated the mossy fibers? Are there any differences? I am thinking of the spinal motor neurons. If they are slightly depolarized they might give a larger IPSP afterwards. I was wondering, whether this is the case here too. Ito: There is a difference, but I think this difference is not simply due to excitation of mossy fibers. Even when you stimulate the cerebellar cortex, you may excite the mossy fibers also. Schmidt: If it is such a simple system, what can it do? We have two types of interneurons, multifiber and climbing fiber, and one output which is totally inhibitory. Could you comment on what you think is the physiological role of that? * K. Toyama: personal communication.

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Ito: I think the complexity of the higher central nervous system arises from the tremendous number of units working in parallel. The cerebellum of cats includes half a million Purkinje cells, and that of the human being 15 millions. Even if their action is simply inhibitory, information involved in the spatiotemporal patterns of their activity should be an enormous contribution. Schmidt: But there is the other evidence that there are only small parts of the cerebellum involved for one given peripheral input, so the number activated from the peripheral input might be rather small. Is that not true? Ito: Yes, but one square millimeter of the cerebellar cortex includes 500 Purkinje cells. I can also point out that the rostral spino-cerebellar tract fibers branch extensively in the anterior lobe and cover a wide area of the culmen. One afferent fiber of this tract would have relevance to a great number of Purkinje cells. Llinas: W e have recently been working on the vestibulo-cerebellar system, and have found what appears to be a direct vestibulo-dentate pathway. Interestingly enough, there is not very much inhibition of the responses by a preceding shock to the same pathway. This seems to indicate that this input behaves as a nonspecific afferent to the dentate nucleus, which does not activate specific corticonuclear pathways. This finding seems to agree with some of your statements. Purpura: The resting discharges of Purkinje cells that you recorded, Dr. Ito, are of fairly high frequency. You have shown us a regular steady discharge of 50-80 per sec. The "resting" discharges of neurons in the roof nuclei are also at a fairly high frequency. How is this high frequency "resting" discharge maintained? It seems as though there are two high frequency discharge systems in continuous activity. Ito: An impulse along single Purkinje cell axons produces in Deiters neurons an IPSP of a very small size (0.2 mV in amplitude). Each Deiters neuron receives 30-50 Purkinje cell axons, which discharge at a rate of 20-90 impulses per sec. As a consequence, there will be a steady hyperpolarization of only a few millivolts. Llinas: Another point that must be taken into consideration is that it is very difficult to estimate the true spontaneous activity of Purkinje cells. Most cells recorded from have had some injury, especially as a microelectrode is advanced through their dendritic tree. Unless one is very careful to approach the cells from a particular angle, or in some way avoid altogether the dendritic pole, I think it is very difficult to specify the normal discharge patterns. Ito: But we can record from Purkinje cell axons within the nucleus of Deiters, and there are indeed spontaneous discharges up to a rate of 90 impulses per sec. Therefore, Purkinje cells appear to be discharging spontaneously at such a relatively high rate as this even under anesthesia. The cerebellar nuclei neurons are also firing at a rate of 50-100 impulses per sec.

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I assume that the excitation in the deep nuclei surpasses the inhibition from Purkinje cells. There may be more background excitatory inputs to overcome the steady inhibition. Purpura: I think I would feel happier if I could see one record of the discharge frequency of the Purkinje cell. Your records show only the first 10-15 msec., which does not provide information on the background activity. Ito: This is because the size of the EPSPs and IPSPs induced by single presynaptic fibers is very small. An impulse of Purkinje cell axons is responsible for an IPSP of only 0.2 mV. It is difficult to recognize the background activities as discrete potential changes. Scheibel: It is a rather remarkable observation that the Purkinje cell, one of the largest neurons, projects such a very short distance and yet produces such a small postsynaptic disturbance. Ito: Yes. That is rather mysterious, but it is true. We have observed it in many cases. REFERENCES P., and ECCLES, J., Inhibitory phasing of neuronal discharge. Nature (London), 1962, 196: 645-647. ANDERSEN, P., ECCLES, J . C . , and VOORHOEVE, P. E . , Postsynaptic inhibition of cerebellar Purkinje cells. J. Neurophysiol., 1964, 27: 1138-1153. ARDUINI, A . , and POMPEIANO, O., Microelectrode analysis of units of the rostral portion of the nucleus fastigii. Arch. Ital. Biol., 1957, 95: 56-70. ARIËNS KAPPERS, C . U . , H U B E R , G . C . , and CROSBY, E . C . , The Comparative Anatomy of the Nervous System of Vertebrates, Including Man. Hafner, New York, 1960. B R E M E R , F., Contribution a l'étude de la physiologie du cervelet; la fonction inhibitrice du paléo-cerebellum. Arch. Int. Physiol., 1922, 19: 189-226. BRODAL, A . , The Reticular Formation of the Brain Stem; Anatomical Aspects and Functional Correlations. Oliver & Boyd, Edinburgh, 1957. BRODAL, A . , POMPEIANO, O . , and W A L B E R G , F . , The Vestibular Nuclei and Their Connections; Anatomy and Functional Correlations. Oliver & Boyd, Edinburgh, 1962. BRODAL, A . , and TORVIK, A . , Über den Ursprung der sekundären vestibulocerebellaren Fasern bei der Katze. Eine experimentell-anatomische Studie. Zsehr, g es. Neurol. Psychtat., 1957, 195: 550-567. CARREA, R . M . E., REISSIG, M . , and M E T T L E R , F. A., The climbing fibers of the simian and feline cerebellum; experimental inquiry into their origin by lesions of the inferior olives and deep cerebellar nuclei. J. Comp. Neurol., 1 9 4 7 , 8 7 : 3 2 1 - 3 6 5 . COURVILLE, J . , and BRODAL, A . , Rubro-cerebellar connections in the cat: an experimental study with silver impregnation methods. J. Comp. Neurol.,

1 . ANDERSEN, 2.

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5. 6. 7.

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R. P., Discussion of Ito, M . , Neuronal circuitry in the cerebellar efferent system. In: Neurophysiological Basis of Normal and Abnormal Moton Activities (M. D. Yahr and D. P. Purpura, Eds.). Raven Press, Hewlett, N.Y., 1967: 133-134.

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THE

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J. C., The Physiology of Synapses. Academic Press, New York, 1964. as a Neuronal Machine. Springer, New York, 1967. 13. ECCLES, J. C., LLINAS, R., and SASAKI, K., The excitatory synaptic action of climbing fibres on the Purkinje cells of the cerebellum. J. Physiol. (London), 1966,182: 268-296. 14. , The mossy fibre-granule cell relay of the cerebellum and its inhibitory control by Golgi cells. Exp. Brain Res., 1966, 1: 82-101. 15. HONGO, T., JANKOWSKA, E., and LUNDBERG, A., Effects evoked from the rubrospinal tract in cats. Experientia, 1965, 21: 525-526. 16. ITO, M., KAWAI, N., and UDO, M., The origin of cerebellar-induced inhibition of Deiters neurones. III. Localization of the inhibitory zone. Exp. Brain Res., 1968,4: 310-320. 17. ITO, M., KAWAI, N., UDO, M., and MANO, S., Axon reflex activation of Deiters neurones through the cerebellar aiferent collaterals. Exp. Brain Res., in press. 18. ITO, M., KAWAI, N., UDO, M., and SATO, N., Cerebellar-evoked disinhibition in dorsal Deiters neurones. Exp. Brain Res., 1968, 6: 247-264. 19. ITO, M., OBATA, K., and OCHI, R., The origin of cerebellar-induced inhibition of Deiters neurones. II. Temporal correlation between the trans-synaptic activation of Purkinje cells and the inhibition of Deiters neurones. Exp. Brain Res., 1966, 2: 350-364. 20. ITO, M., and YOSHIDA, M., The origin of cerebellar-induced inhibition of Deiters neurones. I. Monosynaptic initiation of the inhibitory postsynaptic potentials. Exp. Brain Res., 1966, 2: 330-349. 21. ITO, M., YOSHIDA, M., and OBATA, K., Monosynaptic inhibition of the intracerebellar nuclei induced from the cerebellar cortex. Experientia, 1964, 20: 575-576. 22. ITO, M., YOSHIDA, M., OBATA, K., KAWAI, N., and UDO, M., Inhibitory control of the intracerebellar nuclei by the Purkinje cell axons. Exp. Brain Res., in press. 23. JAKOB, A., Das Kleinhirn. In: Handbuch der mikroskopischen Anatomie des Menschen, Vol. IV/1 (W. v. Mollendorf, Ed.). Springer, Berlin, 1955: 674-916. 24. JANSEN, J., and BRODAL, A., Experimental studies on the intrinsic fibers of the cerebellum. II. The cortico-nuclear projection. J. Comp. Neurol., 1940, 73 : 267-321. 25. JANSEN, J., and JANSEN, J., JR., On the efferent fibers of the cerebellar nuclei in the cat. J. Comp. Neurol., 1955, 102: 607-632. 26. LARSELL, O., The Comparative Anatomy and Histology of the Cerebellum from Myxinoids through Birds (ed. by J. Jansen). Univ. of Minnesota Press, Minneapolis, 1967. 27. LORENTE DE No, R., Vestibulo-ocular reflex arc. Arch. Neurol. Psychiat., 1933, 30: 245-291. 28. OSCARSSON, O., Functional organization of the spino- and cuneocerebellar tracts. Physiol. Rev., 1965, 45 : 495-522. 29. POLLOCK, L. J., and DAVIS, L., The reflex activities of a decerebrate animal. J. Comp. Neurol, 1930,50: 377-411. ECCLES,

12. ECCLES, J. C., ITO, M., and SZENTAGOTHAI, J., The Cerebellum

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33. 34. 35. 36.

37. 38.

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S . , Histologie du Système Nerveux de THomme et des Vertébrés, Vol. II. Maloine, Paris, 1911. SZENTÁGOTHAI, J., and R A J K O V I T S , K., Über den Ursprung der Kletterfasern des Kleinhirns. Zschr. Anat. Entwicklugsgesch., 1959, 121: 130-141. T O Y A M A , K . , TSUKAHAHA, N., and UDO, M., Nature of the cerebellar influences upon the red nucleus neurones. Exp. Brain Res., 1968, 4: 292-309. TSUKAHARA, N., T O Y A M A , K . , and KOSAKA, K . , Electrical activity of red nucleus neurones investigated with intracellular microelectrodes. Exp. Brain Res., 1967,4: 18-33. UNO, M., YOSHIDA, M., and HDROTA, I., The mode of cerebello-thalamic relay transmission investigated with intracellular recording from cells of the ventrolateral nucleus of the thalamus. Exp. Brain Res., in press. W A L B E B G , F., Descending connections to the inferior olive; an experimental study in the cat. J. Comp. Neurol, 1956, 104: 77-174 W A L B E B G , F . , and JANSEN, J . , Cerebellar corticovestibular fibers in the cat. Exp. Neurol., 1961, 3: 32-52. W I L S O N , V. J., and BUBGESS, P. R., Disinhibition in the cat spinal cord. J. Neurophysiol., 1962, 25: 392-404. W I L S O N , V. J., KATO, M., T H O M A S , R. C., and PETEBSON, B. W . , Excitation of lateral vestibular neurons by peripheral afferent fibres. /. Neurophysiol., 1966, 29: 508-529.

3 0 . RAMÓN Y CAJAL,

31.

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FUNCTIONAL ASPECTS OF INTERNEURONAL EVOLUTION IN THE CEREBELLAR CORTEX RODOLFO R. LLINÂS American Medical Association—Education and Research Foundation Chicago, Illinois

Among the various regions of the central nervous system, the cerebellum is outstanding for the constancy of its microscopic structure throughout evolution. Indeed, it was pointed out by Ramón y Cajal (37) that, "In spite of the large variations in size and macroscopical arrangement of this organ, every detail of the organization of the cerebellar cortex in mammals is faithfully reproduced in the lower vertebrates, thus giving it the stability, vigor and generality of a biological law." Given this fact, it is reasonable to postulate the existence of a "basic cerebellar circuitry" which would be a common denominator in all vertebrate cerebellar cortices. This basic cerebellar circuitry would encompass the two afferent systems which enter in contact with the large Purkinje cells, and the single efferent system represented by the axons of these Purkinje cells (Figure 146). Of these two afferent systems, oneconsisting of the climbing fiber terminals—is known to establish a monosynaptic contact directly with the Purkinje cell dendrites (14, 15, 21, 24, 27, 35, 36). This type of junction is unique in the CNS in as much as there is a one-to-one relation between a climbing fiber teledendron and the dendrites of a given Purkinje cell (37). The second afferent system is the disynaptic pathway formed by the incoming mossy fibers, which contact Purkinje cells through an intermediary relay in the granular layer, the granule cells (34). The granule cells establish, by means of their axons, the parallel fibers' synaptic relation with the Purkinje cell dendrites (34). In impressive contrast to the climbing fiber system, the mossy fiber-granule cell-parallel fiber pathway is arranged in such a manner that a given mossy fiber may exert its action on many thousands of Purkinje cells as opposed to the one-to-one climbing fiber-Purkinje cell relationship (14, 21). Such clear morphological differences have led to the elucidation of some aspects of the functional role of these two input systems onto the Purkinje cell as well as in the general functional organization of the cerebellar cortex. The climbing fibers, which in mammals arise mainly from the olivo-cerebellar pathway (38), have been shown to evoke a prolonged all-or-none burst of spikes on the Purkinje cell (4). This burst, gen329

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erated by a prolonged all-or-none postsynaptic potential (5), was attributed to the very extensive nature of the climbing fiber-Purkinje cell synapse, its all-or-none character being the reflection of the all-or-none character of the action potentials in the climbing fiber. This observation has since been confirmed for the frog (29), alligator,* and pigeon, f Thus, the universality of the climbing fiber activation of Purkinje cells seems established. Regarding the mossy afferents, transfolial electrical stimulation of these fibers at the surface of the cerebellar cortex has demonstrated their excitatory nature onto granule cells (8, 11) as well as the excitatory action of these latter cells via the parallel fibers onto the Purkinje cells (7). As for the climbing fiber system, the excitatory action of the mossy fiber-granule cell pathway has been demonstrated in the frog (30, 31), the alligator* and the pigeon, f Another characteristic of the basic cerebellar circuitry seems to be the constant tridimensional arrangement of the parallel fibers in the molecular layer, which run parallel to the surface of the cortex and parallel to themselves in all planes (Figure 146). Their direction is the same as that of the main axis of the cerebellar folia, which in most cases is perpendicular to the cranio-caudal axis of the animal (this is especially so in the lower vertebrates). Furthermore, these fibers establish synaptic contacts of the cruciform type (16, 18, 19, 24) with spines in the Purkinje cell dendritic tree. The Purkinje cell dendrites themselves are also organized in a very geometrical manner, such that the dendritic tree is oriented vertically on the surface of the cerebellum and is flattened in a plane perpendicular to the axis of the parallel fibers (Figure 146). Study of the comparative microscopic anatomy of this cortex shows, on the other hand, that while this basic cerebellar circuitry does not appear to change with evolution, there is, however, an increase in the complexity of the system resulting from the introduction of new neuronal elements—the short-axon interneurons of the cerebellar cortex. It is well known, for instance, that the cerebellum of the frog and of other lower vertebrates (25, 26, 32) lacks the basket cell interneurons which, as first described by Ramón y Cajal, have axons which run transversely across the folia and which (in birds and mammals) end as a "basket-like" fiber plexus in synaptic contact with the soma and axons of the Purkinje cells (15, 20, 23, 24, 33). Furthermore, only a very few interneurons are found in the molecular layer of these lower forms, the great majority of the cells found in that layer being undifferentiated cells.f A similar situation holds for the interneurons of the granule layer where their number is very small, f In mammals, the granule cell interneurons, which were first described by Golgi in 1886 (17), reside for the most part in the superficial half of the * R. Llinás, W. Precht and S. T. Kitai, unpublished observations, t R. Llinás, unpublished observations. J D. Hillman, personal communication.

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Figure 146. Diagram of the "basic cerebellar circuitry", modified from one of Ramón y Cajal's drawings ( 3 7 ) . CF: Climbing fiber; GC: granule cells; MF: mossy fiber; PC: Purkinje cell; PF: parallel fibers. The arrows indicate the direction of nervous conduction in the different pathways as suggested originally by Ramón y Cajal.

granule layer. Their dendrites reach the molecular layer where they make contact with the parallel fibers (15, 19, 3 7 ) . They are also in direct synaptic contact with the mossy fibers by means of their descending dendrites ( 2 2 ) . Their axons terminate in contact with the granule cells (15, 22, 3 7 ) (see Figure 148, A and C ) . As vertebrates progress in the evolutionary scale, there is a clear tendency to an increase in the number and complexity of the interneurons of the cerebellar cortex until finally, in the alligator, there is a primitive but otherwise complete cerebellar circuitry with clear molecular and granule layer interneurons. This animal, the only representative of the ruling reptiles in existence today, has the first truly complete cerebellar cortex ( 3 2 ) . A serieá of electron microscopic studies in this reptile has demonstrated that, although no true basket cells are present, cells organized transversely to the parallel

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fibers and contacting the dendrites of Purkinje cells can be found.* A similar situation holds for the more superficial stellate cells. Their Golgi cells, on the other hand, seem to be well developed and form part of the cerebellar glomerulus. In birds and mammals, the basket, stellate and Golgi interneurons finally have the very remarkable development so well known to neuroanatomists. For purposes of comparison, three electron micrographs of Purkinje cell somata of frog, alligator and cat are shown in Figure 147. Note the lack of "basket" formation on the frog and alligator Purkinje cells as compared with the very rich plexus formed around the lower soma and axon in the case of the cat. It must be kept in mind that the Purkinje cells form a very dense layer in lower vertebrates. For instance, in the frog the Purkinje cell layer is several cells deep, the alligator being the first reptile to have a single row of cells in this layer (32). Field Potentials Generated by Local Stimulation in Frog and Cat

Cerebellar

From the functional point of view, there are also large differences between the electrophysiology of the lower vertebrate cerebellum and that of the mammalian cerebellar cortex. As an example, the field potentials generated by local cerebellar stimulation in the frog differ in large measure from those recorded in the cat under similar circumstances. Since the early studies by Dow (2), it has been known that local stimulation of the surface of the cerebellum evokes a series of field potentials which can be recorded with the aid of a microelectrode in the molecular layer. Local stimulation of the surface of the cerebellum evokes in the frog (Figure 148B), as has been previously demonstrated for the cat cerebellum (Figure 148D) (1, 7) a positive-negative-positive parallel fiber compound action current followed by a large and long-lasting negativity (Figure 148B) which is generated by the excitatory synaptic currents evoked by the parallel fiber impingement onto Purkinje cells (30). Previous experiments in deafferented cat cerebella have shown some of the characteristics of this potential when recorded at different depths and at different lateral displacements from the beam of par* D. Hillman, personal communication.

///// ^w JK J

Figure 147. Electron micrographs of Purkinje cell in frog, alligator and cat to illustrate the difference in the distribution of the basket cell boutons in these three species. A: Frog Purkinje cell; the soma and axon lack the basket cell terminals; X 3700. B: Alligator Purkinje cell; no filamentous terminals in contact with the soma (this type of terminal is, however, found very frequently in the dendrites); X 4000. C: Cat Purkinje cell; typical basket cell terminals (BT) in synaptic contact with the soma of the Purkinje cell; X 2500 (D. Hillman, unpublished data).

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Figure 148. Diagrams of frog and cat cerebella, experimental arrangements, and comparison of the field potentials recorded at the surface of both cerebella following local cerebellar stimulation. A: Frog cerebellum; GC, granule cell; GL, granule layer; LOC, local stimulating electrode; ME, microelectrode; MF, mossy fiber; ML, molecular layer; PA, Purkinje axon; PC, Purkinje cell; PF, parallel fiber; RE, surface recording electrode, with white matter stimulating electrode; arrows mark the stimulus artifact (from Llinas & Bloedel, 30). B: Field potentials recorded from the surface of a frog cerebellum following Loc. stimuli of increasing amplitudes; the numbers at the left of the traces represent the amplitude of the electrical stimulus in an arbitrary scale (R. Llinas and J. Bloedel, unpublished observations). C: Cat cerebellar cortex; BC, basket cell; GoC, Golgi cell; GrC, granule cell; Loc-1 and 2, surface stimulating electrodes; ME, recording microelectrode; MF, mossy fiber; PA, Purkinje axon; PAC, Purkinje axon collateral; PC, Purkinje cell; SC, stellate cells; SRE, surface recording electrode. D: Field potentials recorded at the surface of the cat cerebellar cortex, and evoked as in B (from Eccles, Llinas & Sasaki, 7 ) . Time and voltage calibration and polarity marked at the lower right side of B and D.

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allel fibers activated. The superficial negativity is present only within the "beam" of activated parallel fibers while the underlying positivity can be recorded up to 400 n out of beam (7). A two-dimensional display of the depth and transverse location of the microelectrode tip (Figure 149, D and I ) and arbitrary numerical representation of the amplitude of the potential recorded at each one of the recorded points, allows the plotting of isopotential contour lines and thus of the distribution of the potential field at right angles to the direction of parallel fiber conduction. The interpretation of the isopotential contours in deafferented cerebella (Figure 1491) led to the conclusion that in the cat, the deep positivity recorded at 200 to 600 n lateral to the beam of activated parallel fibers (Figure 149, F-H) was produced by the synaptic currents generated by the inhibitory synaptic impingement of basket terminals onto Purkinje cells. In the frog, on the other hand, the fields (Figure 149, A-C) were interpreted as generated exclusively by the synaptic depolarization of Purkinje dendrites by the parallel fiber stimulation. This synaptic action would establish a current sink at the tip of the Purkinje cells which would draw current from the lower dendrites and soma, generating thus a "passive" current source at that level. This sink-source relation generates the symmetrical negative-positive dipole seen in the frog cerebellum in the absence of the "active" current source produced by the basket cells (30). Functional Differences between the Purkinje Cell Activity Evoked by Local Cerebellar Stimulation in Frog and Cat Single cell activity recorded intra- or extracellularly from Purkinje cells of different species following local electrical stimulation of the cerebellar cortex, also reveals a very characteristic difference between lower and higher vertebrates. If, for instance, the cerebellar cortex of the frog is stimulated with electrical pulses of increasing strength, the number of Purkinje cell action potentials increases as the stimulus is raised to 2.5 times threshold (Figure 150A). Moreover, when two consecutive stimuli follow one another at short intervals, the second stimulus evokes a larger number of action potentials than it would in the absence of the preceding stimulus (control Figure 150B). That is, there is summation of the excitatory action of consecutive stimuli. This situation is completely different in the cat. There, the number of Purkinje cell action potentials evoked by single shocks to the parallel fibers rarely exceeds two, regardless of the stimulus strength used to evoke this activation. When stimuli are paired at short intervals (Figure 150C), the second Purkinje cell response is inhibited for 20 msec. (7). The difference between the electrophysiological behavior of the Purkinje cells in these two species can be attributed to the different interneuronal population in their molecular layer. Thus in the cat, Purkinje cell inhibition following local stimulation has been postulated as produced by the activation of basket

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reasonably well by the corresponding statistics of the waiting times to reach certain membrane potential. A cell in the Aplysia visceral ganglion was quiet, not showing spontaneous spikes or PSPs. Bursts of Geiger-counterdriven EPSPs were produced by stimulating a connective. Prior to each burst the membrane potential was at resting level (Figure 171, upper right). Stimulation was interrupted as soon as the potential crossed the threshold that was at 16 mV less negative than the resting level. Bursts were separated by periods of over 30 seconds. We measured the waiting times Ti, T2, T 3 and Ti from the first EPSP to the instants at which depolarizations of 8, 10, 12 and 14 mV, respectively, were achieved, as well as the waiting time T, to the first spike. The waiting-time histograms (Figure 171, left column), means and standard deviations (right-lower graph) vary smoothly from Ti through T2, T, and Ti to To. This suggests that in this cell, the main

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issue in firing is the achievement of a depolarization of 16 mV from resting level. (Note that the passage through threshold always occurred during the positive-going swing of the EPSP and, therefore, with practically always the same rate of change. Under other circumstances the rate of change at which threshold is reached may become important.) If one ignores spike consequences like afterpotentials and refractoriness, the sample statistics of the waiting times to reach threshold estimate the corresponding interspike interval statistics that one would observe if the cell were submitted uninterruptedly to the same input. Similarly, the statistics of Ti through T4 estimate the interspike interval statistics if thresholds were at 8 through 14 mV, respectively, from the resting level. The excitability of the postsynaptic spike mechanisms is another important issue. The effect of any EPSP is biased by recent postsynaptic firings. In fact, a postsynaptic spike does not occur simply if a "word" is enunciated, but only if it is enunciated when the postsynaptic cell is responsive (26, 27). The postsynaptic threshold depends essentially upon the times elapsed since recent spikes. Wakabayashi (35) showed that the maximum efficiency of a given number of supra-threshold stimuli is achieved if they arrive only when the excitability has recovered from previous firings. This issue reflects, of course, postsynaptic properties, but its importance depends also upon the presynaptic timing. For example, it is weakly or strongly influential if "words" are generated at intervals usually longer or shorter, respectively, than recovery periods. Accommodation and quasi-conditioning should be kept in mind also, even though not influential in these experiments. In pacemaker cells submitted to large PSPs, the natural shapes of the pacemaker drift and of the PSPs determine the form of a delay function that contains the necessary conditions for the effects described above. The delay function (Figure 172) quantifies the influence exerted by a sequence of n PSPs contained in the interval between two spikes, and expresses it as a function of the "phase" that is, of the time elapsed between the initial spike and the arrival of the first PSP in the sequence (16,22, 25). Only regular sequences (that is, sequences in which all inter-PSP intervals were equal to a given value i) have been studied. There is a different delay function for each pair of values of n and i. The delay is measured either by the amount 8 by which the interval containing the PSPs differs from the natural interval N, or by the time D from the last PSP in the sequence to the next spike. Note that N + 8 = 4> + (n — l ) i + D Figure 170 displays S-delay functions for two cells with no more than one PSP per interval. Intervals with IPSPs or EPSPs are longer or shorter respectively, than N, and the corresponding 8 is positive (graph A ) or negative (graph B), respectively. The first part of both graphs shows a close-to-linear dependence of 8 upon Slopes are usually between —2

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quency of which varied between 200 and 500/sec. Only the fimbrial pathway was in these cases capable of producing a population spike, as seen in the differentiated record (Figure 192, middle). Following this population spike, the non-pyramidal spikes were much smaller in amplitude than those evoked by other pathways. Fimbrial stimulation excited the non-pyramidal cells with very short latency. This was only 1 msec, longer than the latency of the antidromic field potential, thus corroborating the suggestion of Andersen, Eccles & L0yning ( 3 ) that the basket cells may be activated by the axon collaterals of pyramidal cells. If the non-pyramidal repetitive discharges should be associated with the postsynaptic recurrent inhibition produced in pyramidal cells, their discharge pattern should conform to the extracellular positive field potential that can be recorded in the pyramidal layer and that is associated with the IPSPs. In Figure 193, increasing stimulus strengths to the commissural pathway increased the number and decreased the latency of the repetitive discharges, matching the growth of the extracellular positive wave. Each assemblage consists, from above, of the microelectrode, the differentiated records and the surface records, and each contains three superposed sweeps. The first two spikes had a relatively constant latency. When the stimulus strength was raised to about 0.4 mAmp., there appeared a remarkable reduction of the size of the non-pyramidal spikes following the second discharge;

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at 0.6 mAmp. this was very conspicuous. The period with low amplitude spikes appears between the two initial non-pyramidal cell discharges and the individual spikes that compose the incipient population spike. The relation between the stimulus strength and the latency and number of repetitive discharges appears in Figure 194. With increasing stimulus intensities, the initial latency decreased, while both the number of spikes and the frequency increased. When the population spike occurred, the subsequent repetitive non-pyramidal discharges were blocked. The duration of this blockade was a function of the size of the population spike. The reduction in latency with an increase in the stimulus strength suggests convergence of afferent fibres on a single non-pyramidal cell. Since commissural stimulation will excite antidromically, as well as orthodromically conducting fibres, the initial spike probably represents the monosynaptic activation of the non-pyramidal cell by antidromic impulses in pyramidal axons. The remarkably constant latencies of the two initial spikes also favour this conclusion.

In contrast to the behaviour of the pyramidal cells, the non-pyramidal cells show very little if any inhibition. When a single commissural shock ( C O M ) was preceded by a weak commissural shock (Figure 195, 0.08 mAmp.), there was an increase in the number of discharges in the test response as well as a reduced latency of the first spike. These alterations became more apparent when the conditioning shock was increased to 0.1 mAmp. When the conditioning strength was increased further (to 0.15 mAmp.), a remarkable change appeared in the test response. The initial spike underwent an additional decrease in its latency; however, the follow-

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ing few spikes of the test discharge were remarkably reduced in amplitude for a period of about 8-10 msec. At a conditioning strength of 0.2 mAmp. this effect was even more pronounced so that only the initial discharge had its ordinary amplitude. At the conditioning strength of 0.5 mAmp, the reduction of the spike amplitude was evident both in the conditioning and the test responses. On withdrawal of the conditioning shock, the response appeared as in the control, proving that the effect was neither due to an increased recording distance nor to injury of the cell. The most likely explanation is a conductance change of the non-pyramidal cell membrane caused by an intense synaptic depolarization by afferent fibres, probably to a large extent pyramidal axon collaterals. When a double shock was applied to any of the afferent pathways used in the present study, a definite facilitation of the test response appeared as a reduction of the initial spike latency (Figures 195, 196). In Figure 196, A is the control test response to a commissural volley; the response to two similar volleys is seen in B, whereas C is an expanded sweep, containing the test response only. The upper trace in C is the non-conditioned test response and the lower is the test response as changed by a preceding conditioning shock;

HIPPOCAMPAL

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the reduction of latency in the latter instance measured 0.8 msec. The reduction of the initial non-pyramidal spike latency caused by a double fimbrial volley can be seen in Figure 196D. The reduction of the initial spike latency may be taken as a sign of convergence by several afferent pathways onto the non-pyramidal cells. The effect was strongest when the double shocks were delivered to the same pathway, but could also be seen with conditioning volleys through either of the other two pathways. The behaviour of the non-pyramidal cells during frequency potentiation appeared of particular interest. When the hippocampal pyramidal cells are recorded from, recurrent IPSPs are found in virtually all cells. Although many pyramidal cells discharge, as indicated by extracellular population spike responses, ordinary EPSPs are either absent or very small. Through an analysis of intracellular and extracellular laminar recordings, Andersen & L0mo ( 5 ) concluded that the excitatory synapses, which are almost exclusively located on the spines of the relatively thin dendrites, produce their effect by a local EPSP that creates a dendritic spike which travels towards the soma with a relatively low safety factor. Under ordinary circumstances the intracellular electrode, being lodged in the soma, is probably so far distant from the site of synaptic action that no, or very small, EPSPs are seen. However, if the rate of stimulation is increased, a striking increase of the EPSPs develops and the EPSPs can now be recorded by the intrasomatic electrode. With continued repetitive stimulation, the hyperpolarization caused by the IPSPs gives way to a marked and long-lasting depolarization due to the potentiated EPSPs ( 6 ). During repetitive stimulation the behaviour of the non-pyramidal cells differed markedly from that of the pyramidal cells. In Figure 197, each trace in the assemblage is, from above, the microelectrode record, the differentiated microelectrode record, and the surface re-record. The stimulus strength was deliberately kept low, exciting a non-pyramidal cell only. There is a marked difference in threshold between the non-pyramidal and pyramidal cells. As seen in Figure 193, the non-pyramidal cells appeared at a stimulus of 0.08 mAmp., whereas the population spike appeared at about 0.4 mAmp. When the stimulus rate was increased to 10 per sec., there the first effect was an enhancement of the non-pyramidal cell discharges, as expressed by a shorter initial latency and a larger number of spikes. After four seconds of 10 per sec. stimulation three very large negative population spikes appeared. However, the initial spike of the non-pyramidal cell appeared some 2 msec, earlier than the first pyramidal population spike. This can be explained by the antidromic volley elicited by the contralateral stimulation exciting the non-pyramidal cell synaptically. If so, the non-pyramidal EPSP has a shorter rise-time than that of the pyramidal EPSP. In this experiment, the antidromic volley was so small that it is not visible. When the tetanic stimulation was maintained for some seconds, the population spike increased in latency as a sign of fatigue, and the number of population spikes was also de-

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creased. After 10 seconds of stimulation, the initial spike was the only one left of the non-pyramidal cell discharges, and its size was reduced. This decline was even more pronounced at 12 sec., and complete blocking occurred at 14 sec. With maintained tetanic stimulation, the population spike occurred at longer latencies until it finally disappeared after 30 sec. However, as the population spike decreased in size and increased in latency, the initial discharge of the non-pyramidal cell reappeared with normal size in the 30 sec. record. After 32 sec., all pyramidal discharges were gone, as indicated by the lack of any population spike, whereas the non-pyramidal cell fired with a high frequency discharge of long duration and normal size in the 30 sec. record. After 32 sec., all pyramidal discharges were first four discharges of non-pyramidal cells are seen to fall into groups. After a rest of 10 sec., control stimulation at 1 per sec. showed the same condition as before the tetanic stimulation. The main conclusion is that the non-pyramidal cells are brought to increase their discharges by repetitive stimulation but that the discharge is, in some way, inhibited by the occurrence of large population spikes or strong afferent stimulation. This can be taken to mean that the discharges of the

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caudate, cortical and mesencephalic neurons during evoked EEG-synchronizing activities have indicated the rather diffuse involvement of elements at several neuraxial sites during nonspecific thalamic stimulation at low frequencies. As noted earlier, the optimum frequency for eliciting these synchronizing activities is in the range of 6-12/sec. The next problem to be addressed concerns the manner in which these synchronizing PSPs are altered during high-frequency nonspecific thalamic or brain stem stimulation which typically leads to the electrographic picture of cortical activation. The question arises, how do the interneuronal networks which generate EPSP-IPSP sequences in thalamic neurons respond to the higher stimulus frequency? As we all know, the generalized activation pattern is associated with behavioral signs of arousal. This phase of the discussion can profitably begin with a consideration of the changes in PSP patterns in rostral thalamic neurons during the transition from evoked E E G synchronization (recruiting responses) to desynchronization produced by high-frequency MTh-stimulation (22). In other words,

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Figure 240. Persisting excitatory effects of repeated high-frequency medial thalamic stimulation on an intralaminar neuron exhibiting rapid EPSP summation during recruitment. A-E, continuous record. A: Terminal phase of a period of 7/sec. medial thalamic stimulation; first stimulus after spontaneous discharge elicits a prolonged IPSP-EPSP complex, subsequent stimuli evoke EPSP-IPSP sequences. B: Short burst of high-frequency stimulation at x; enhancement of high-frequency evoked repetitive discharges on 80 msec, duration wave of depolarization is succeeded by repolarization; then beginning of spontaneous activity; high-frequency (75/sec.) medial thalamic stimulation indicated by arrows. C: Seven per sec. medial thalamic stimulation initiates powerful excitatory synaptic drives that are associated with brief inactivation phases; a second period of high-frequency stimulation occurs in C. D: Depolarization persists for several seconds. E: Gradual repolarization associated with reappearance of partial spikes, then full spikes. F: Twenty seconds after E. (From Purpura & Shofer, 24.)

stimulation is to the same site in the medial thalamus but the stimulus frequency is abruptly changed. A typical EPSP-IPSP pattern in a thalamic neuron during low-frequency MTh stimulation is shown in Figure 239A; note that the first of a series of high-frequency stimuli elicits an IPSP, but subsequent stimuli produce a sustained depolarization with superimposed partial spikes. When the highfrequency stimulus is terminated and the low-frequency stimulation resumes (Figure 239C) only EPSPs are observed. These EPSPs are greatly facilitated by a second phase of high-frequency MTh-stimulation (Figure 239, D and E ) . Only several seconds later does low-frequency stimulation elicit EPSP-IPSP sequences once again (Figure 239F). The conclusion to be drawn from these observations is that the transition from thalamically induced synchronization to desynchronization is asso-, ciated with a blockade of synchronizing IPSPs and marked enhancement of

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excitatory synaptic drives. This "inhibition of inhibition" is demonstrable in a large proportion of thalamic neurons exhibiting typical EPSP-IPSP sequences during recruiting responses. If one examines the activity of intralaminar neurons during this change in MTh-stimulation, even more prominent excitatory events are detectable along with the blockade of IPSPs (Figure 240). Thus, a cell exhibiting a bursting discharge and subsequent IPSP during low-frequency MTh-stimulation may show a profound, sustained EPSP summation during high-frequency stimulation, as well as prolonged post-activation facilitation (Figure 240). The question of what happens to the IPSP during the high-frequency stimulation is of no little importance. The fact that IPSPs obtained with low-frequency MTh-stimulation may not be observed during high-frequency stimulation might indicate lack of high-frequency responses of the synaptic pathway generating the IPSPs. Obviously, the important test would be, first, to initiate the EPSP-IPSP sequence in identifiable thalamic neurons during evoked EEG-synchronization, and then simultaneously stimulate, at high-frequency, components of the brain stem reticular system remote from the thalamic stimulating site. In effect, the experimental design reproduces the classical experiments of Moruzzi & Magoun (14), who showed suppression or blockade of thalamically evoked recruiting response during high-frequency stimulation of the brain stem reticular formation. A number of different aspects of this experiment are summarized in Figure 241. Recruiting responses and typical EPSP-IPSP sequences initiated by low-frequency MTh-stimulation are shown in Figure 241A. Concomitant high-frequency brain stem reticular (BRS) stimulation is shown in Figure 241B. The results are unambiguous in showing suppression of the synchronizing IPSP during BRS stimulation. There is, additionally, a slight increase in discharge frequency in this thalamic element. It will be recalled from Figure 227 that, with proper conditioning intervals, the IPSP evoked in VLrelay cells during low-frequency MTh-stimulation effectively blocks the monosynaptic excitatory action of brachium conjunctivum stimulation on VL elements; these events are shown in Figure 241, C and D. The action of high-frequency BRS stimulation on the VL relay cell is illustrated in Figure 241E, and the blocking effect of BRS stimulation on the inhibition of VL-relay activity associated with low-frequency MTh stimulation is shown in Figure 241F. The results summarized in this figure indicate that high-frequency brain stem reticular stimulation can effectively inhibit the interneurons involved in the production of IPSPs essential for synchronizing thalamic neuronal discharges during low-frequency medial thalamic stimulation. This "inhibition of inhibition" occurs in most but not all thalamic neurons (Figure 241H). This would suggest that the reticular activating effect is not diffusely distributed to all thalamic neurons capable of participating in the evoked synchronization. Studies of the effects of high-frequency medial thalamic stimulation on

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Figure 241. Intrathalamic synaptic events associated with suppressing effect of highfrequency (50/sec.) brain stem reticular formation (BSRF) stimulation on thalamocortical recruiting responses. A: Unidentified thalamic neuron exhibits EPSP-IPSP sequence during low-frequency medial thalamic (MTh) stimulation. B: Concomitant BSRF stimulation attenuates IPSPs in this element. C-F, effects of BSRF stimulation on a VL-relay cell; C: VL discharge evoked by brachium conjunctivum stimulation, at arrow. D: Blockade of relay discharge during MTh stimulation (at dot). E: BSRF stimulation increases discharge frequency of VL cell. F: BSRF stimulation during low-frequency MTh stimulation inhibits the IPSP shown in D, and thereby permits reactivation of specific relay response, at arrow. G: Superimposed recordings of recruiting response and IPSP evoked by MTh-stimulation and the latter stimulus combined with BSRF stimulation; note marked attenuation of IPSP. H: Another neuron in which BSRF stimulation fails to affect the MTh-evoked IPSP; the BSRF stimulation is generally effective in suppressing the recruiting response to MTh-stimulation. Modified from Purpura, Frigyesi, McMurtry & Scarff (19) and from Purpura, McMurtry & Maekawa (22).

caudate neurons have revealed that caudate elements which show E P S P I P S P patterns during evoked EEG-synchronization ( F i g u r e 2 3 3 ) exhibit blockade of the I P S P and an increase in E P S P s during high-frequency MTh-stimulation (Figure 242). Thus, such caudate neurons mimic in all respects the responsiveness of VA and V L elements to both low and high frequency MTh-stimulation. Finally, if one examines the responses of mesencephalic reticular neurons to high-frequency MTh-stimulation, one finds that, during such stimulation, reticular elements may exhibit a slowly increasing depolarization and an increase in cell discharge ( F i g u r e 2 4 3 ) . Such P S P characteristics are entirely different from those encountered in nonspecific or relay nuclei of the thalamus. I t may b e useful to recapitulate some of the points discussed above in the context of the main subject to the symposium. T h e r e can b e little doubt that

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50msec Figure 242. Alterations in EPSP-IPSP sequences in a ventromedial caudate neuron during transition from low- to high-frequency medial thalamic stimulation; same cell as in Figure 233. A-C, from continuous record. A: First medial thalamic stimulus elicits an IPSP, but subsequent stimuli evoke EPSPs which increase in magnitude in B and produce double discharges, some of which show inflections on rising phase of spike (at arrow). Resumption of low-frequency medial thalamic stimulation as in Figure 233 fails to reveal the IPSP. The E P S P is facilitated for several seconds after the high-frequency medial thalamic stimulation. (From Purpura & Malliani, 21.)

the system we have chosen to study as a model for diffuse synchronization consists of the most complex organizations of interneurons imaginable. But despite this complexity, which is being elegantly explored in the Golgi studies of the Scheibels, there is a basic simplicity in the patterns of synaptic events which occur in different parts of the thalamus and its related organizations. We have attempted in the past five years to characterize these PSP patterns in several structures. From these studies it is clear that organizational differences in interneuronal networks receiving input from nonspecific thalamic nuclei greatly modify this input and confer specific characteristics on the responses of ele-

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Figure 243. Slowly developing excitatory effects of low-intensity medial thalamic stimulation on a mesencephalic reticular neuron. Note slow depolarization which begins to subside during continuation of stimulation. (From Maekawa & Purpura, 9.)

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ments at different sites. A remarkable finding is that neurons of the caudate nucleus may exhibit PSP patterns in response to thalamic stimulation that are also observed in cortical PT-cells. On the other hand, some populations of ventrally located caudate neurons have a "thalamic" type of responsiveness. Thalamo-striate connections antedate, phylogenetically, thalamocortical relations. It is not unlikely that with the development of the neocortex, cortical interneurons activated by nonspecific thalamic stimulation reproduced the patterns established in the caudate. An important point to be emphasized in this summary of synchronizing and desynchronizing events relates to the prominent role of thalamic EPSPIPSP responses in triggering the basic synchronization of elements with caudate, cortical and brain stem connections. W e have shown that this nonspecific thalamic stimulation exerts very specific effects on neurons in fundamentally different organizations. These specific effects are in part related to the activities initiated by interneuronal networks interposed in the pathways between nonspecific thalamic nuclei and the particular organization under examination. It is of considerable interest that the synchronization mechanism discussed here is quite frequency specific. The 8-10/sec. rhythm imposed is, of course, set by the duration of the IPSPs. At slightly higher stimulus frequencies the IPSPs alternate and finally at threshold frequencies for initiating EEG desynchronization and blockade of recruitment, IPSPs are no longer detectable. This inhibition of inhibition at thalamic sites constitutes one of the most impressive features of the synaptic events underlying the transition from EEG-synchronization to EEG-activation. Unfortunately, there are not sufficient data to suggest the nature or organization of the inhibitory system capable of blocking IPSPs so widespread in neurons of the thalamus during synchronization. Schlag: Dr. Purpura, I completely agree with you that EEG desynchronization can be produced by high-frequency stimulation of the medial thalamus. However, in order to interpret the mechanism of this effect, it should be noted that the desynchronization does not outlast the duration of the stimulus train when the experiment is made in a cerveau isolé preparation, or after partial transection of the brain stem at the level of the posterior commissure (28). You have shown very well how the activity of thalamic cells is modulated by what you called the nonspecific system. The question I have is: Is there such a system? It is supposed to be characterized by the production of long-latency cortical surface-negative potentials on repetitive stimulation around 10/sec. but, recently, we have found that such responses can be elicited from many places within the thalamus, and not only from its medial aspect (29). Furthermore, these negative potentials develop around foci of positive-negative potentials which are considered to be characteristic of the stimulation of specific nuclei. Thus the distinction between specific and non-

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specific becomes blurred. I wonder if there is not a similar organization almost everywhere in the thalamus instead of a central, unique, nonspecific system modulating the different thalamic relays to the cortex. I have a second question: It seems that you can elicit discharges from relay cells of nucleus ventralis lateralis when you stimulate the medial thalamus. When you stimulate this nucleus electrically, you get a cortical evoked potential which is positive-negative. When you stimulate the medial thalamus, you induce firing in cells of nucleus ventralis lateralis and there is no positive-negative response on the motor cortex. Is it not possible that something is going on within the cortex as a consequence of the medial thalamic stimulation, in such a way that the usual cortical response is prevented there? Purpura: I am quite familiar with the kinds of questions you are raising, as I have followed your own work closely in this area. It is to be expected that a stimulus in the medial thalamus will have very different effects on different thalamic and rostral cortical areas. Surely, the medial thalamic stimulus is not independently capable of producing all the effects observed. These will depend upon stimulation of direct pathways and synaptic activation of local and internuclear organizations at the thalamic level. Additionally, we have shown involvement of caudate and mesencephalic neurons. Although there is activation and long-latency inhibition of VL cells during medial thalamic stimulation, the excitation of VL relay cells is certainly not as powerful as one would expect from direct electrical stimulation in VL.° Thus, whatever direct synaptic activation of VL cells might occur, the overall motor cortex response to MTh-stimulation is likely to obscure this event. Remember that many VL cells undergo rather profound and prolonged summations of IPSPs which can depress EPSPs below the firing level. It is unreasonable to expect that MTh-stimulation could reproduce the primary and augmenting sequence involving VL motor cortex pathways. Another major point to be made is that, if one examines intracellular recordings from PT cells, there is no question of a fundamentally different pattern of synaptic drive elicited by MTh and VL stimulation (26). Unfortunately, there has been a good deal of confusion in the literature concerning the question of "how nonspecific the nonspecific thalamic projection system really is". In part, this confusion is a consequence of the operational procedures employed to define the properties of "nonspecificity". There are certainly focal cortical projection regions which exhibit short-latency positive responses and surround long-latency negativities during stim* Added by Dr. Purpura in proof: It is quite clear that PSP patterns similar to those observed in VL neurons during nonspecific thalamus stimulation may also be elicited in medial nonspecific neurons by stimulation in VL. Hence the intrinsic interneuronal networks are reciprocally related. However, there is one major distinction. Stimulation in VL generally evokes extremely short-latency prolonged IPSPs in many medical thalamic neurons. This indicates that the VLinduced response must be associated with synaptic activation and inhibition of nonspecific thalamic neurons. It is impossible to consider the augmenting or recruiting responses as independent intrathalamic events. Our most recent studies suggest that the inhibitory effects of VL stimulation on medial thalamic neurons are among the most impressive inhibitory interactions we have detected in thalamic neurons.

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ulation of many specific projection nuclei, as your own studies have indicated. And it is more likely that the same may hold true for nuclear groups designated as "nonspecific". But as long as the method of direct electrical stimulation is employed to define these projections, the problem will, in my opinion, be extremely difficult to clarify. I might add that it is my personal view that prime consideration of surface potential waveform and latency may turn out to be as sterile an approach to this problem as making lesions in nuclear groups which are traversed by many fibers of passage. Leaving aside the question of thalamocortical relations, we can examine the intrathalamic internuclear effects of medial thalamic stimulation without reference to projection systems to cortex. The nonspecific-specific internuclear interactions I discussed above do not depend on cortical projections. The same holds true for thalamo-caudate responses ( 2 1 ) . There is no necessity to introduce the question of what the cortical evoked potentials look like in different regions. The fact is that the medial and intralaminar nuclei do project in part to lateral nuclear groups of the thalamus and to the striatum. Such projections activate organizations of interneurons at these sites which produce PSP patterns capable of synchronizing or desynchronizing neuronal discharges. The chassis of the interneuronal organization may be quite different in these different structures, and that is precisely what we have been attempting to show with intracellular recording of relevant PSPs. Schlag: The main point I want to stress is that this nonspecific system is not so nonspecific. Purpura: We all are now aware of that. We are employing a terminology in relation to a concept that has been of considerable heuristic value. Surely, every system we now call "nonspecific" will eventually be shown to be very specific in function, that is, when the system, is properly identified morphologically and the criteria are defined for determining "specificity". We may have to wait for Dr. Lundberg to identify his interneurons before we can identify ours! The only value in calling a pathway or neuron "nonspecific" is to distinguish it from another in which the connections and functions are relatively well known. If you insist on eliminating the term "nonspecific," that is fine—but substitute something that will aid in communication and not further confound an already difficult subject. Andersen: The work that Andersson, L0mo and I are doing seems to be very much along the line Dr. Schlag suggests. We have had no trouble in synchronizing or desynchronizing the VPL cell by stimulation of the medial thalamus. Perhaps the stimulating electrode has to be a little more lateral, though, than just the midline. But it seems to us, when we use multiple electrodes in a large grid, that stimulation in the caudal part of the thalamus at different depths and at different lateral locations causes widespread but not nonspecific action. One can make a system out of it. I am tempted to explain things in the simplest manner, and I think the cells that Dr. Scheibel described seem to be able to do the job. They come

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from the C M and a region very close to the C M and are distributed through a wide area, including the ventrobasal complex. There they might be able to set up a synchronizing mechanism according to the simple scheme of recurrent inhibition with postsynaptic rebound. At least that is a working hypothesis that can be tested. In my opinion, we seem to have the simplest element that can do the job already described. Purpura: I showed examples of the most typical situation observed by us in ventrobasal cells during medial thalamic stimulation. It was also pointed out that evoked spindle waves produce marked modulation of VB cell activity. If one utilizes very strong stimulating currents, in some preparations medial thalamic stimulation produces a more impressive synchronizing action on VB cells. May I ask what anesthesia you were using? Andersen: W e were using pentobarbital. Purpura: Well, of course, with pentobarbital I expect that you can facilitate spindle waves with any low-frequency stimulus. My main point is that, in the encéphale isolé preparations, medial thalamic stimulation very effectively produces EPSP-IPSP sequences in V L cells, whereas these sequences are very difficult to evoke with time-locked regularity in VB cells. When I say we have had trouble with the VB system, this is not to imply that spindle waves could not be elicited which produced typical effects in VB. I think that we are perhaps in dispute concerning the simple model of recurrent inhibition with post-anodal rebound. It is difficult for me to accept this model in all parts of the thalamus where we observe EPSP-IPSP sequences during synchronization of neuronal discharge. You say that it should be possible to test whether the IPSP sets up post-anodal exaltation. How would you test this hypothesis? Let me put it this way: In order to synchronize discharges you require a collateral of the VB cell that will activate an inhibitory neuron which will in turn generate an IPSP. As the IPSP is terminated, a phase of post-anodal exaltation or rebound excitation triggers the next discharge. How would you test for this? Andersen: I would activate the cell antidromically. Purpura: How would you tell that the response did not involve excitatory elements which set up an EPSP? Andersen: You can do it artificially by hyperpolarizing the cell. Purpura: That is exactly what we considered to be a prime test of postanodal exaltation. When one impales VB neurons most of these tend to be somewhat injured by the impalement. W e consider a VB neuron with 45-50 mV spike potentials fairly healthy. Now, if we hyperpolarize such an element by 30 mV immediately after impalement, when the cell exhibits some signs of injury, we observe some post-anodal discharges (Figure 244B). When the cell has an opportunity to settle down and the spontaneous spike potentials are of larger amplitude, similar strong hyperpolarization produces a depolarizing afterpotential but no cell discharge (Figure 244D). With the onset of some depolarization, the anode-break responses again become prominent ( Figure 244F ).

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h Figure 244. Relationship between injury effects and anode break responses. A, C, E: Characteristics of spontaneous spikes observed at different times after impalement. B, D, F: Effects of injected currents of constant strength at corresponding times. Lemniscal stimulation (arrows) is at the same intensity throughout. A and B were recorded a few seconds after impalement. Injury effect is indicated by a low-amplitude and broad spike (A). B: The lemniscal-evoked EPSP is observed in isolation during the membrane hyperpolarization; termination of the current pulse elicits an anode-break spike. C, D: Recordings obtained during maximum stabilization phase; amplitude of spike potential is larger than in A; termination of the hyperpolarizing current pulse produces a delayed depolarizing potential which is not accompanied by spike discharges; the EPSP has a somewhat slower decay phase than in B. E, F: Recordings obtained during onset of cell deterioration; spike amplitude is slightly reduced and anode-break responses are prominent. (From Maekawa & Purpura, 11.)

Examination of the effects of strong hyperpolarization in another VB cell with a satisfactory resting potential is shown in Figure 245. In this element, the EPSP was followed by a small IPSP. Hyperpolarization to, a level in excess of the reversal potential of the IPSP in this VB cell failed to elicit postanodal discharges. On the basis of these findings, we reject the notion that a phase of post-anodal exaltation is of importance in triggering the subsequent discharge in a repetitive sequence. It is difficult to make this work in the VB

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Figure 245. Effects of induced membrane hyperpolarization on a VB relay neuron. A: Evoked spike potential is followed by a small IPSP, as shown in B. Weak inward current injection blocks spike and reveals EPSP and succeeding IPSP. Increase in strength of hyperpolarizing currents does not augment the EPSP, but IPSP is inverted and adds a phase of depolarization to the initial EPSP. Although the hyperpolarizing current increases membrane potential above the reversal potential of the IPSP, no anode-break responses are observed. (From Maekawa & Purpura, 11.)

cells and it is even more difficult to make the model work in the caudate or at other sites where EPSP-IPSP sequences are observed in a rhythmically recurring pattern. It is my contention that such sequences involve a matrix of excitatory and inhibitory interneurons which operate to set up the rhythmical EPSP-IPSP sequences we have observed. I believe that in Figures 244 and 245 we have provided the test you asked for. I do not know what else we can do. Andersen: I do not think anybody has said, or meant, that hyperpolarization itself may be enough to trigger the cells, but it might increase the membrane excitability so that other impulses may trigger them. Let us put it the other way: If you reject the post-inhibitory rebound hypothesis, then you have to put in another mechanism. This has to explain a very important fact, and that is that the whole group, many hundreds of cells, fire simultaneously after the inhibition. This is very easily shown. If you reject the post-anodal exaltation hypothesis, you just move the problem of phasing to the former nervous station. In order to progress we have to postulate hypotheses and then test them. I admit that your figures have been the major objection to this theory by

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far, but there must be ways of testing this, other than negating the possibility. Purpura: I think Dr. Andersen is perfectly correct. We have been negligent in proposing hypotheses to account for anything more than the basic synchronization, which we believe to be due to activity in a complex network of excitatory and inhibitory interneurons. The point is that post-anodal exaltation is probably of little importance in the synchronizing mechanism. Schlag: I wonder if other thalamic cells, those of nucleus ventralis lateralis, for instance, also failed to show anode-break responses? Purpura: Strong hyperpolarization to the level of the reversal potential of the IPSP in cortical neurons does not trigger discharges in these elements if the cell is not initially depolarized. This is clear in our studies (11) and in the work of Pollen & Lux (15). However, anode-break responses are frequently seen in hippocampal neurons, as was initially shown by Kandel & Spencer (8). This may be a particular characteristic of hippocampal neurons, although it remains to be shown whether this is facilitated by slight traumatic depolarization of the cell. Kandel: I must say I like the inhibitory synchronization hypothesis. In fact, Spencer and I developed a very similar kind of hypothesis for the hippocampus. Recently, Frazier and I had a chance to examine this problem further, in the abdominal ganglion (which is a sort of hippocampus) in Aplysia. We used an identified interneuron and recorded simultaneously from two or three bursting cells which received inhibitory branches. The follower cells all have an endogenous rhythm and when the interneuron does not fire, the bursts in any cell will be out of phase with the bursts in the other cells. We then examined the degree to which one can synchronize the follower cells by producing repeated bursts in the interneuron, and found that the synchronization is quite effective. So that, certainly in very simple cell systems where one can control an inhibitory interneuron sending branches to a population of follower cells, one can get very clear inhibitory synchronization in this way. Lundberg: Dr. Purpura, do you find recovery of the IPSP? The reason I ask is that, even when citrate electrodes are employed for recording, a reversal of IPSP may occur in small cells through leakage of extracellular chloride into the cells. Purpura: There was certainly recovery of the IPSP, after the inhibition of inhibition induced by high-frequency medial thalamic or brain stem stimulation. Recovery may be delayed by 30 or 40 sec., which represents the time during which EEG-desynchronization persists after a five-second high-frequency stimulation. Of course, the IPSP then returns. See, for example, Figure 238F. Stefanis: Did you ever see any units firing during this inhibitory phase? Purpura: I showed an example of a cell in the intralaminar thalamus

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which fired in a burst pattern for a period longer than the latency for IPSPs generated in lateral thalamic neurons. Are you asking whether we impaled neurons that fired during the IPSP in other cells? We have seen some cells of this type in extracellular recordings (18). Stefanis: Did you ever try to use thiopental while you were recording, particularly when you were synchronizing the units, to see if any changes would occur following the administration of the anesthetic, as far as synchronization is concerned? Purpura: We have administered thiopental in order to "prime" the synchronizing system particularly in studies of reticular units because we were unhappy about not seeing IPSPs. Small amounts of this barbiturate did not facilitate the appearance of IPSPs; rather small amounts might eliminate IPSPs when they were present in mesencephalic reticular elements. REFERENCES Observations on thefinestructure of the feline caudate nucleus. Anat. Rec., 1967, 157: 203. ANDERSEN, P . , ECCLES, J. C . , and SEARS, T. A . , The ventro-basal complex of the thalamus: types of cells, their responses and their functional organization. /. Physiol. (London), 1964, 174: 370-399. BROOKHART, J. M . , and ZANCHETTI, A . , The relation between electro-cortical waves and responsiveness of the cortico-spinal system. Electroenceph. Clin. Neurophysiol., 1956, 8: 427-444. COHEN, B . , HOUSEPIAN, E . M . , and PURPURA, D . P . , Intrathalamic regulation of activity in a cerebellocortical projection pathway. Exp. Neurol., 1962, 6: 492-506. CREUTZFELDT, O. D., L U X , H. D., and W A T A N A B E , S., Electrophysiology of cortical nerve cells. In: The Thalamus (D. P. Purpura and M. D. Yahr, Eds.). Columbia Univ. Press, New York, 1966: 209-230. D E M P S E Y , E. W., and MORISON, R. S., The production of rhythmically recurrent cortical potentials after localized thalamic stimulation. Am. J. Physiol., 1942, 135: 293-300. FRIGYESI, T . , and PURPURA, D . P . , Functional properties of synaptic pathways influencing transmission in the specific cerebello-thalamocortical projection system. Exp. Neurol., 1964, 10: 305-324. KANDEL, E. R . , and SPENCER, W . A . , Electrophysiology of hippocampal neurons. II. After-potentials and repetitivefiring.J. Neurophysiol., 1961, 24: 243-259. MAEKAWA, K., and PURPURA, D. P . , Excitatory processes and spike potential variations in reticular neurons. Fed. Proc., 1967, 26: 434. , Intracellular study of lemniscal and non-specific synaptic interactions in thalamic ventrobasal neurons. Brain Res., 1967, 4: 308-323. , Properties of spontaneous and evoked synaptic activities of thalamic ventrobasal neurons. J. Neurophysiol., 1967, 30: 360-381. MAGOUN, H. W., The Waking Brain (2nd ed.). Thomas, Springfield, 1 9 6 3 .

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and D E M P S E Y , E . W . , A study of thalamo-cortical relations. Am. J. Physiol., 1942,135: 281-292. MORUZZI, G . , and MAGOUN, H . W . , Brain stem reticular formation and activation of the EEG. Electroenceph. Clin. Neurophysiol., 1949, 1: 455-473. POLLEN, D . A . , and Lux, H . D . , Conductance changes during inhibitory postsynaptic potentials in normal and strychninized cortical neurons. J. Neurophysiol., 1966, 29: 369-381. PURPURA, D . P . , Nature of electrocortical potentials and synaptic organizations in cerebral and cerebellar cortex. Int. Rev. Neurobiol., 1959, 1: 47-163. , Comparative physiology of dendrites. In: The Neurosciences: A Study Program (G. C. Quarton, T. Melnechuk and F. O. Schmitt, Eds.). Rockefeller Univ. Press, New York, 1967: 372-393. PURPURA, D . P . , and C O H E N , B . , Intracellular recording from thalamic neurons during recruiting responses. J. Neurophysiol., 1962, 25: 621-635. PURPURA, D . P . , FRIGYESI, T . L . , M C M U R T R Y , J . G . , and SCARFF, T . , Synaptic mechanisms in thalamic regulation of cerebello-cortical projection activity. In: The Thalamus (D. P. Purpura and M. D. Yahr, Eds.). Columbia Univ. Press, New York, 1966: 153-170. PURPURA, D. P . , and HOUSEPIAN, E. M., Alterations in corticospinal neuron activity associated with thalamocortical recruiting responses. Electroenceph. Clin. Neurophysiol., 1961, 13: 365-381. PURPURA, D . P . , and MALLIANI, A., Intracellular studies of the corpus striatum. I. Synaptic potentials and discharge characteristics of caudate neurons activated by thalamic stimulation. Brain Res., 1967, 6 : 325-340. PURPURA, D. P . , M C M U R T R Y , J. G . , and M A E K A W A , K., Synaptic events in ventrolateral thalamic neurons during suppression of recruiting responses by brain stem reticular stimulation. Brain Res., 1966, 1: 63-76. PURPURA, D . P . , SCARFF, T . , and M C M U R T R Y , J. G . , Intracellular study of internuclear inhibition in ventrolateral thalamic neurons. J. Neurophysiol., 1965, 28: 487-496. PURPURA, D . P . , and SHOFER, R . J., Intracellular recording from thalamic neurons during reticulocortical activation. J. Neurophysiol., 1963, 26 : 494505. , Cortical intracellular potentials during augmenting and recruiting responses. I. Effects of injected hyperpolarizing currents on evoked membrane potential changes. J. Neurophysiol., 1964, 27: 117-132. PURPURA, D . P . , SHOFER, R . J., and MUSGRAVE, F . S . , Cortical intracellular potentials during augmenting and recruiting responses. II. Patterns of synaptic activities in pyramidal and nonpyramidal tract neurons. J. Neurophysiol., 1964, 27: 133-151. PURPURA, D. P . , SHOFER, R. J . , and SCARFF, T., Properties of synaptic activities and spike potentials of neurons in immature neocortex. J. Neurophysiol., 1965, 28: 925-942. SCHLAG, J . D., and C H A I L L E T , F., Thalamic mechanisms involved in cortical desynchronization and recruiting responses. Electroenceph. Clin. Neurophysiol., 1963, 15: 39-62. SCHLAG, J . , and VILLABLANCA, J . , Cortical incremental responses to thalamic stimulation. Brain Res., 1967, 6: 119-142.

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INTERNEURONAL MECHANISMS IN THE CORTEX* COSTAS STEFANIS Athens National University Athens, Greece

The cerebral cortex is expected to be more complicated in functional organization than a ganglion of a lower invertebrate; this is some consolation to me when I compare our findings on cortical interneuronal mechanisms with the wealth of data presented so far in this symposium. In any case, I will confine my presentation to the motor cortex and more specifically to the interneuronal mechanisms involved in the recurrent activity exerted on the pyramidal tract (PT) neurons. But first, before I discuss interneuronal mechanisms, let me outline the general features of the motor-cortical feedback system of which the interneurons are an integral part. It is well known that PT cell activity can be temporarily arrested or radically modified by stimulating various sites such as the cortical surface, specific and non-specific thalamic nuclei, and the pyramidal tract itself (22-24, 28, 29). Phillips (22, 23) used this latter technique and revealed that depression of PT cell activity following antidromic stimulation is associated with long-lasting hyperpolarization of the PT cell membrane which is often preceded by a small, brief depolarizing phase. He proposed that such membrane potential changes in PT neurons might be induced by activity mediated through the PT cell axon collaterals. Such a system would therefore assign to PT cells self-controlled properties similar to those which were shown to exist in the spinal motoneurons. In order to elucidate this hypothesis further, we studied, with intra- and extracellular microelectrodes, PT cell responses to a variety of conditioning and testing stimuli applied to orthodromic and antidromic pathways to the motor cortex. Identification of the PT cells followed criteria established previously (22, 35). As is known, interpreting results obtained by pyramidal stimulation is difficult because pyramidal tract axons run very close to the afferent pathways, making current spread to dromic pathways possible, and the axons branch collaterally to various subcortical structures connected to the motor cortex. Therefore, caution is needed in assigning to antidromic activation findings which very well might be due to orthodromic excitation of * The research on cortical interneurons was supported by Research Grant NB 11201 from the National Institutes of Health, U.S. Public Health Service. 497

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the PT neurons. In our study, we tried to avoid such complications by using various techniques (35) which assure that effects observed, either intra- or extracellularly, were actually due to antidromic activation. Among the techniques used were stimulation at both the medullary pyramid and the pes pedunculi and deafferentiation of the motor cortex by section of afferent pathways or by coagulation of ventrobasal thalamic nuclei and of corpus striatum. The results to be reported, most of which have been confirmed by other investigators (2, 3, 37, 38), were obtained from intracellular recordings of 162 PT neurons and extracellular recordings from over 500. Most of the results have appeared earlier (35, 36). A more complete account, which includes findings derived from experiments employing intracellular stimulation and microelectrophoretic techniques, is given in a recent monograph (34). Figure 246 shows intracellular recordings of the events following antidromic stimulation. If the current pulse applied to the pyramidal tract is strong or repetitive, the cell fires antidromic spikes with a short and constant latency, followed by a variety of slow membrane potential changes, usually polarizing (A, B, D ) . The polarizing potential changes are frequently preceded by a brief, small depolarizing potential ( C ) . Since this can be obtained independently of a preceding spike, for example by applying stimulus strength subthreshold to the test cell axon, it cannot represent an afterpotential. Blockage of antidromic invasion can also be obtained by employing a testing stimulus, as is the case with the neuron shown later in Figure 250. The fact that slow potential changes can occur independently of membrane conductance changes associated with the spike mechanism, also indicates that they are due to the activity of neighboring axons and presumably mediated to the test cell synaptically through axon collaterals.

Figure 246. Various types of antidromically evoked IPSPs. In Al, antidromic shock is ineffective for resetting the spontaneous rhythm; in A2, three shocks evoke two spikes, followed by a hyperpolarizing potential and arrest of firing; in A3, hyperpolarization and arrest of firing are not preceded by antidromic spikes. In B, the effect of two trains of three antidromic impulses is shown. C shows at high and low gain a depolarizing "hump" preceding the slow IPSP in response to two pyramidal shocks. D shows the effect of increasing the antidromic stimulus strength on the spike of the recurrent IPSP. (From Stefanis & Jasper, 35.)

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The intracellular positive and negative slow waves are characterized by properties which indicate their synaptic origin: they are graded responses dependent on the parameters of stimulation, and they increase in amplitude and (or) duration by increasing either the stimulus strength or the number of pulses applied. As a rule, short bursts of two or more shocks of relatively high frequency are required to evoke them. The dependency of the antidromic slow potential changes on the transmembrane polarization level is indicated by the fact that intracellular negative potentials will change their amplitude according to the membrane polarization level. Membrane potential shifts with depolarizing currents resulted in an increase of the hyperpolarizing slow potential. The reverse was seen with hyperpolarizing current steps applied through the intracellular recording microelectrode. While all these properties indicate that these slow potential changes are true postsynaptic potentials—excitatory (EPSP) when depolarizing and inhibitory (IPSP) when hyperpolarizing, the best evidence is that these IPSPs can be inverted into depolarizing potentials by intracellular injection of chloride ions, or by excessive membrane polarization, produced by passing a current through the recording microelectrode (34). In order to define more precisely the time-course and the functional significance of the recurrent effects in the motor cortex, we studied excitability changes of PT neurons following antidromic stimulation. We used for this purpose various conditioning and testing techniques. In all of them, the conditioning pulses were applied to the pyramidal tract at the medullary or peduncular level. The test stimuli which followed the conditioning at different time intervals were applied at various sites such as the pyramidal tract, the ventrobasal nuclei of the thalamus, and within the cell itself through the recording micropipette. The excitability curves, obtained by applying identical conditioning and testing stimuli at the pyramidal tract, are shown in Figures 247 and 248. The excitability curve of PT neurons was obtained by plotting the probability of invasion of the cell by testing antidromic impulse against the interval between the conditioning and testing stimuli, when the conditioning stimuli were 100 per cent effective in eliciting an antidromic spike response. Both the conditioning and testing stimuli could vary in strength or in the number of pulses. As shown in the example illustrated in Figure 247, the probability of antidromic invasion in response to the testing stimuli failed considerably with a conditioning testing interval of 40-65 msec. The insert, showing intracellular recordings from the PT neuron from which the excitability curve was obtained, demonstrates that the decrease in probability of antidromic invasion of the testing impulse coincided with the peak of the IPSP evoked by the conditioning stimulus. By increasing either the strength of the conditioning stimulus or the number of conditioning shocks, the antidromic IPSPs would become larger and the probability of antidromic invasion of the testing impulse would be lessened. Furthermore, the decrease would start sooner and last longer. This is shown in Figure 248.

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Figure 247. Time-course of recurrent inhibition and sample traces of intracellular recordings (insert) of P T neuron to twin peduncular shocks at different testing intervals. Note the blockage of antidromic invasion with interval of about 6 0 msec, at 3. (From Stefanis & Jasper, 3 6 . )

In this and in many other cases, the maximum decrease in probability of invasion was found to occur about 25-60 msec, after the conditioning stimulus. One can also see in this figure that IPSP hyperpolarization resulted in a rebound firing of the cell. This phenomenon, however, was rather rare. It was not seen except in somewhat deteriorated cells such as this one. In all cases where the membrane potential was constantly above 60 mV during long recordings, all attempts to evoke rebound firing by hyperpolarization failed. In some cases excitability of the cell membrane increased slightly in the late phase of the IPSP, but these were all neurons with relatively low membrane potential. Excitability curves similar to the ones already shown have been obtained by employing orthodromic test stimuli applied on the ventrobasal thalamus and on the peripheral nerves. On a few occasions, the method of testing by direct intracellular stimulation was used to study membrane excitability changes evoked by antidromic activation. The next figure illustrates the results obtained from two neurons by this method. At the top of Figure 249, in A, is the tracing of an IPSP evoked by a pair

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Figure 248. Effect of the number of pyramidal shocks upon the excitability curves of a PT neuron, with sample intracellular records from the same neuron. The dashed curve was obtained with a single shock, and the solid curve with three peduncular shocks. Vertical bar at left of B is calibration of 10 mV. (From Stefanis & Jasper, 36.)

of antidromic pulses subthreshold for the test axon. The curve shown below the IPSP was derived from plotting the reciprocals of the threshold current strength relative to the control strength against the conditioning-testing intervals. It is clear from this curve that the amount of current needed to evoke a spike response increases during the IPSP. It is also clear that excitability decrease is closely related to the membrane potential changes represented at the IPSP on top. In Figure 249B, the testing pulse was set at suprathreshold values for spike responses. It consistently evoked the two spikes when applied before the onset of the recurrent IPSP, as shown in the two right-hand records. This pulse became less effective during the recurrent IPSP. It evoked only a solitary spike response with the longest latency coinciding with the peak of the recurrent IPSP. Llinas: Did you often find that the resistance of the cell is increased during the IPSP, as is shown in the middle record of Figure 249? Stefanis: I am afraid that we cannot rely on these records and draw conclusions as to resistance changes during the IPSP. The bridge is not balanced and we cannot make a definite statement. What I want to show in these records is that, with the same testing current strength, we can consistently have two spike responses before the onset of the IPSP, and only one spike appearing after a long latency during the recurrent IPSP. Lately, we have used another method for testing excitability changes in

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- 55mv

Figure 249. Excitability testing by direct stimulation applied intracellularly through the recording microelectrode. See text. (From Stefanis & Jasper, 36.)

PT neurons during antidromic activation. This combines the conditioning testing technique with the microelectrophoresis technique and has enabled us to study the effects of excitatory (acetylcholine, glutamic acid) and depressant (GABA, 5-HT) substances on the excitability curves of PT neurons following antidromic conditioning. It was found that substances which excite PT cells become less effective during recurrent inhibition and more effective during recurrent facilitation. The opposite was observed with depressant substances. Moreover, the excitatory substances failed in most instances to abolish the inhibition completely. By increasing the intensity of

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the electrophoretic current, the inhibition was shortened, the drug being more effective at the beginning and towards the end of the inhibitory curve. Figure 250 illustrates the effect of various drugs on the spike responses of P T cells to pyramidal tetanization. I t was shown in a previous paper ( 3 6 ) that repetitive stimuli on the pes pedunculi of a reciprocal frequency substantially longer than the refractory period and of suprathreshold current strength, would block antidromic invasion of the cell's soma. Such a blockage is shown in Figure 250, column A. Repetitive antidromic shocks of 3 V each evoked one to one antidromic spikes. However, blockage of antidromic invasion occurs in the middle of the burst when the stimulus strength is increased to 5 V, and occurs terminally when the strength is increased to 6 V. This loss of antidromic spikes is not due to a factor limiting the frequency response of the P T cells, but to a strong recurrent inhibition evoked by the repetitive antidromic stimulation. That this is the case is also evidenced by the fact that the microelectrophoretic application of acetylcholine in the immediate vicinity of the test cell results in progressive recovery of axon-AB transmission as shown in Figure 250, column B. W i t h the cessation of ace-

B

A

ACH O N

No drug

c

D

ACh OFF

GABA

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GABA

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^^uuuUUi* ¡MI^JM}/*^

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^ ^ J l ™ » . 100 msec. Figure 250. Effect of acetylcholine and aminobutyric acid applied microelectrophoretically through a five-barrel micropipette on recurrent inhibition produced by pyramidal tetanization. See text.

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tylcholine administration, recurrent inhibition becomes again effective in blocking antidromic invasion (column C ) . The opposite sequence of events can be observed with microelectrophoretic application of GABA which enhances recurrent inhibition and facilitates antidromic blocking (columns D and E ) . It is very interesting to note that both the blockage of antidromic invasion and the recovery from blockage occur in a definite step pattern. With the recovery, the small A spike appears first and it takes some time before the full A-B spike develops. With blockage, on the other hand, there is first a loss of the B spike while the A remains a while longer. This and other findings dealt with in detail elsewhere (34) indicate that the main recurrent inhibitory input occurs very close to the spike-generating region. We think, however, that not all of the inhibitory input is confined in that region. On many occasions reversal of the recurrent IPSPs, obtained by injection of CI" ions and membrane polarization through the intracellular recording microelectrode, was not entire, a small portion at the end being affected. It is therefore quite conceivable that part of the inhibitory input is widely distributed to PT cell areas which are not affected by the current pulse applied within the soma. So far, we have been discussing mainly recurrent inhibition. In our work (35), evidence has been obtained that PT cells are also subject to recurrent facilitation. The main piece of evidence derived from the finding that subthreshold antidromic stimulation evoked, in about one-third of the tested PT neurons, depolarizing potentials. Usually they were combined with IPSPs. Latent depolarizing potentials could be isolated from IPSPs by injecting thiopental intravenously into the animal. The potentials were classified into various types. The most commonly seen is the one shown in Figure 246C, and in the insert of Figure 247. It is very small, and usually precedes a larger IPSP. As we pointed out in a previous paper (35), displacements of the membrane potential to the hyperpolarizing direction did not produce measurable changes in their size. Contrary to recent observations by others (38), we failed to observe any substantial increase in size following injection of depolarizing current. In addition to depolarizing responses of this type, we have observed in nine PT neurons very large and prolonged EPSPs in response to antidromic stimulation. Displacements of the membrane potential within the tested range of -f-10 and —25 mV failed to produce substantial changes. This was the main reason for suggesting in a previous paper (35) that these potentials represent recurrent EPSPs generated in remote, presumably dendritic, membrane areas. Coming back now to the large, prolonged EPSPs, shown in Figure 251,1 would like to emphasize some of their special characteristics which make us think that they were recorded from a dendritic rather than a somatic region of the PT cell. As one can see in this example, the antidromic spikes, responding to twin short trains of three shocks applied to the pes peduncli, are

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followed by a large depolarization sustained at a nearly constant level of several features worth noticing in this record: (a) whereas antidromic firing brings the membrane potential abruptly down to the resting level. There are several features worth noticing in this record: (a) whereas antidromic firing had no effect on the sustained depolarization, orthodromic firing abolished it completely; (b) the firing level varies in the same neuron within a wide range; and (c) the orthodromic spike responses are larger by several mV and shorter in duration than the antidromic spikes. All these features strongly suggest that, during the recording, the microelectrode tip was located far from the firing zone of the antidromic spike, most likely in a dendritic region. Findings like these also suggest that, whereas IPSPs are mainly distributed at or close to the spike triggering zone, recurrent EPSPs extend to dendritic membrane areas. Figure 251. Large and prolonged depolarizations produced by a twin train of three peduncular shocks. Note that the orthodromic spikes abolish the depolarization. Time scale: 10 msec. The distance from resting membrane potential level to the time scale is equal to voltage difference of 60 mV.

All the results so far presented here, and results published elsewhere (33-36), provide evidence that motor-cortical function is effectively regulated by a feedback system operating through the axon collaterals of pyramidal fibers. This system becomes effective in regulating output discharge frequencies and patterns only when there is adequate excitatory drive upon PT cells to evoke synchronous repetitive discharges of pyramidal axons. Recurrent inhibition is primarily exerted upon the less active or even subliminally excited PT neurons on the fringe of the receptive field. Such a distribution of recurrent inhibition within the receptive field enhances "motor contrast", prevents spread of excitation to wide cortical areas and facilitates precise localized activation. On the other hand, recurrent facilitation seems to be weak and seems to be exerted primarily upon the very active PT cells in the center of the field by the less active PT cells located at the fringe of the receptive field. This topographical arrangement of the facilitatory input will supplement recurrent inhibition by enhancing motor contrast. To us, it seems that it is not so much the size of the PT neuron, as other workers have suggested (37), but its position with respect to the receptive field and its discharge frequency which will determine if it will exert an inhibitory or facilitatory action upon the neighboring PT cells. One further step to elucidate the mechanism of recurrent effects upon the motor cortex was to define the intracortical connections of PT cells' axon collaterals. Anatomy in this respect is not particularly helpful. So far, very little is known from anatomical studies (17, 25, 31) of the exact destination

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of the axon collaterals. Consequently, the answer to the question of whether they are linked to PT cells directly or indirectly through one or several interneurons, has to come from electrophysiological work. The relatively long latencies of PSPs compared with the latencies of antidromic spikes, together with the temporal characteristics of the PSPs (the long and variable duration, dependence on the stimulus parameters, the slope of the rising phase, their step-like development, indicating temporally dispersed synaptic events), are evidence in favor of a polysynaptic pathway. Direct evidence for such a pathway would derive from electrophysiological identification of interneurons engaged in recurrent activity. Whether such neurons exist or not remained for several years a matter of speculation, despite all evidence supporting their existence (3, 23, 28, 34-36). All attempts made so far by several investigators (3, 23, 36) to record from such neurons have been unsuccessful. In the course of our study of PT neurons, and while we were transversing the motor cortex with a microelectrode, we occasionally recorded from neurons which were somehow affected by antidromic stimulation but which could not be made to evoke an antidromic spike. This observation recently prompted us to undertake a more systematic study of these neurons and experiments were performed in which they were specifically sought. One problem we faced from the outset in this study was what criteria to apply in order to define physiologically a cortical neuron as an interneuron of the recurrent pathway. We heard from Dr. Wiersma that an interneuron can be defined as every neuron that has its main input from a neuron and its main output to another neuron. According to this definition, PT cells themselves can be considered interneurons. What we were concerned with in our study, however, were neurons which have a great part of their input from, and a great part of their output to, PT cells. That is, we were concerned with neurons, other than PT cells, which are involved as relays in the recurrent pathways of the PT cells. The criteria, therefore, for their identification would be that, although they are not invaded antidromically, they respond to pyramidal stimulation with spike discharges, the latency, the pattern and the duration of which are dependent on stimulus parameters. The experimental arrangement for recording and stimulation is shown diagrammatically in Figure 252. Stimulating electrodes for antidromic activation were placed in the pyramidal tract at the medullary or peduncular level. Other stimulating electrodes were placed at various thalamic nuclei and at the cortical surface area overlying the recording microelectrode. For intracellular recordings, the glass micropipettes were filled with potassium citrate, while the micropipettes used for extracellular recordings were filled with sodium chloride. On many occasions, two single micropipettes with their tips as close to each other as possible were used for recording. In other cases, a five-barrel micropipette for recording and microelectrophoretic administration of drugs (6, 15, 16, 27) was used in conjunction with the single recording microelectrode. Out of several hundred units recorded in the

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Figure 252. Schematic illustration of electrodes used for stimulation and recording. All cortical electrodes placed in pericruciate cortex. Disk-like object represents electrode for surface polarization. PP: Pes pedunculi.

motor cortex in the course of this study, only 59 met the above mentioned criteria and were classified as interneurons. Intracellular recordings were obtained from 11 of these cells while recordings from the remaining were extracellular. A typical series of responses of the presumed interneurons of the recurrent pathway is shown in Figure 253. These are all extracellular recordings; negativity is signaled by an upward deflection. The records are taken from five different neurons; A, B, C and D show responses to a single pyramidal volley. As can be seen, the cells could not be invaded antidromically, even though very strong pulses were used. They responded, however, with multiple spike discharges of variable and relatively long latency. The number of spikes varied, being dependent on the stimulus strength as will be shown more clearly in the following figure. A single stimulus pulse of average strength value for evoking antidromic spike responses in PT cells usually evoked three to four spikes in these neurons. For a given stimulus strength, the pattern of discharge was regularly repeatable, which is illustrated in the superimposed traces in B and C. The pattern of the evoked spike discharges was not common to all the neurons tested. Neither was it constant in a given neuron under various stimulation parameters. In some, as the case is with the Benshaw cells (8-10), the first cycle was briefer than the second, while in others the opposite was true. The frequency of the evoked spike discharges was usually around 200/sec., though it could be as high as 800/sec. In Figure 254, the spike responses are shown, recorded extracellularly from two interneurons to single pyramidal shocks of variable strength. In Al, the stimulus strength is just threshold for evoking a spike response of long latency. In A2, increase of the stimulus strength by only one-tenth of the threshold value (from 11 to 12 V) results in the firing of a second spike. In A3, a slight further increase from 12 to 12.5 V evoked a burst of five spikes. Despite the increase of the number of spikes, the latency in this par-

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W>

I 50msec

Volt, scale: lmV

100 m s e c

Figure 253. Samples of cortical interneurons responding to pyramidal stimulation. Samples B and D are composed of ten superimposed traces, while E shows an interneuron firing negative spikes at a very high rate. The microelectrode tip occasionally picks up a large spike of positive polarity, presumably fired by a PT cell.

ticular case did not change appreciably. In other cases, however, as in the instance of the neuron shown in B, the increase in strength of the pyramidal shock by 2 V steps (B1-B3) not only increased the number of spikes but also shortened the latency and changed the interspike interval. Both neurons in this figure were recorded from a deafferented motor cortex. Repetition of the stimulus, held at a constant value, at regular intervals frequently resulted in increase of the number of spike responses, as can be

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-fAI^W 0 . 5 mV 50 msec Figure 254. Effect of the peduncular stimulus strength upon spike responses of two different motocortical interneurons. See text.

seen in Figure 255. The maximum increase most frequently occurred at repetition rates of 5-15 cps. A sharp decline was observed at over 30 cps. Neurons which were classified into this group were not capable of following high-frequency trains of stimulation, in contrast with PT neurons which followed frequencies of stimulation of over 100 cps. It was mentioned earlier that the experimental arrangement was such that

Spent

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50 P P >

Figure 255. Effect of pyramidal stimulation frequency on the firing rate of a cortical interneuron. Low-frequency stimulation facilitates and high-frequency stimulation inhibits firing.

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stimulating pulses could also be applied to the cortical surface, to ventrobasal thalamic nuclei and to the cell's membrane through the intracellular recording microelectrode. In this study, advantage of this arrangement was taken to investigate the effect of various pathways upon cells which on the basis of their responses to pyramidal activation were classified as interneurons. Figure 256 illustrates the intracellular responses of two neurons to stimulation applied to various sites. The neuron in column A was located 1.2 mm beneath the cortical surface. A single shock to the pes pedunculi evoked a high-frequency burst of six spikes, the latency of the first spike being over 10 msec. (A2). It is worth noticing in this record that the spikes rise on the large wave of depolarization and they progressively decrease in size, as noted in the previous figure. Here, again, the first cycle of the response is substantially longer than the subsequent ones. The same cell responded to a

B

J so mV

s o mV 5o

msec

Figure 256. Samples from two interneurons recorded intracellularly, demonstrating mode of response to various types of synaptic and direct electrical activation. Al: Response to V L stimulation; A2: response to pyramidal (pes pedunculi) stimulation; A3: response to epicortical cathodal stimulation; note differences in latencies as well as in duration of the evoked burst of spikes. Bl: Response to pes pedunculi stimulation; B2: response to V L stimulation; B3: response to intracellular polarizing pulse. See text.

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single shock applied to VL with a long burst of spikes (Al). Compared with the response evoked by pyramidal stimulation, this one has several distinguishing features. The entire response lasts longer and has an earlier onset. It has no characteristic pattern with regard to the length of the cycles within the burst. There is a tendency at the final portion of the burst for the spike size to diminish and for the interspike interval to become progressively shorter. The response to epicortical stimulation (A3) consists also of six spikes rising on a very large wave of depolarization. Disregarding minor differences, the discharge pattern of this response is similar to the one evoked by pyramidal stimulation. Traces shown in B1 are from another intracellularly recorded neuron located at a depth of about 1.8 mm beneath the pial surface. The response to single pyramidal shock consists of two spikes again rising on a wave of depolarization but, in contrast to the neuron shown in A, the first cycle is shorter than the second one. The response to VL stimulation (B2) consisted of three spikes equally spaced in time, with the first one occurring at a longer latency than the first spike of the response to the pyramidal stimulation. The evoked depolarization is also less conspicuous in this case. The third trace illustrates the effect of hyperpolarizing pulse (6 X 10~10 Amp.) applied through the recording microelectrode to the cell's membrane. It displaced the membrane potential by 7 mV and promptly arrested the spontaneous firing. Compared with the PT neurons, these neurons required less current to produce the same membrane potential displacement, indicating a higher membrane resistance for these neurons than for the PT neurons. The total membrane resistance of the latter was found by us (34) to average less than 7 mQ, a value considerably lower than that reported by others (5). We have not yet completed the study of the spontaneous activity of the presumed interneurons. An account, however, can be given of the data so far accumulated. There is no common pattern to all these neurons with regard to their spontaneous activity. Some of them are silent, responding only to a given stimulus. Others fire at low rates and in an irregular fashion. The majority of them, however, tend to fire in bursts of high frequency, each burst occasionally being preceded by a larger, solitary spike, presumably fired by a PT neuron; this is the case in the examples illustrated in Figure 257. In Figure 257A, the recordings are intracellular (positivity upwards); in B and C, extracellular (negativity upwards). At the beginning of the trace Al, a hyperpolarizing step of current is passed through the recording microelectrode and it evokes a very pronounced rebound response. The cell continues firing in bursts. The spikes of each burst rise on successive waves of depolarization and have a tendency to decrease their height and shorten their interspike interval with the increase in size of the underlying depolarization at the end of the burst. On several occasions, the arrest of firing seemed to result from an intense depolarization. This was evidenced by the

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I Figure 257. Spontaneous firing of various presumed cortical interneurons. In Al, intracellular recording for 1 sec.; vertical bar is voltage calibration of 50 mV; a hyperpolarizing pulse is applied at the beginning of the trace and evokes a strong rebound. The terminal portion of the rebound depolarization is magnified in A2. In B, successive sweeps from bottom to top and from left to right; antidromic stimulation (arrow at bottom trace) evokes an antidromic spike from a PT neuron, followed by a barrage of smaller spikes; spikes of the PT cell and of the non-PT neuron occur spontaneously, either in combination or independently of each other. In C, a high-frequency spontaneous burst of an interneuron is shown.

firing, towards the end of the burst, of abortive spikes as is shown in A2, which illustrates an enlarged portion of the continuous record of Al. In Figure 257B (successive sweeps from bottom to top and from left to right), an antidromic shock (indicated by arrow, lower left) evokes a large solitary spike of short constant latency from a PT neuron followed by a high frequency burst of smaller spikes. The same pattern reappears in the following sweep, but this time spontaneously. A low frequency burst of the large spikes occurs later on in isolation. That the small spikes are fired by a different neuron than the large one is evidenced by the fact that they also occur independently of the large spike, as can be seen in the third sweep from the bottom in the righthand column. A very high-frequency (800 cps) burst is displayed by the neuron in C. It is well established that such discharge patterns are not encountered in PT cells' spontaneous activity. Therefore, whenever present, this pattern of spontaneous activity can be used as an additional criterion for distinguishing the assumed interneurons from PT neurons.

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Although the features outlined so far are not typical of Renshaw cells (8, 9), or Renshaw-like cells (1, 12, 18), they provide adequate support for our assumption that these are interneurons interposed in the recurrent pathway of PT cells. Further evidence derived from experiments in which interactions of PT cells and presumed interneurons were studied. In Figure 258, two examples of such an interaction are illustrated. In A and in the left-hand column (control), the recording microelectrode of a five-barrel micropipette picks up spikes from two neurons in response to a single pyramidal shock. The large one is from a PT cell and the small one from a neuron which, although it was not being invaded antidromically, responded with multiple discharges. The PT cell also fires an orthodromic spike of variable latency. In this case, the firing of the small spikes does not prevent a second (orthodromic) discharge of the PT neuron. In fact, it 5 HT CONTROt

55 :

4

180 lec

r 2 mV I 50 m s e c

B

1 mV

25 msec _i_J—L Figure 258. In A, the effect of 5-HT electrophoretically applied on antidromic and orthodromic responses of a PT cell firing in conjunction with another non-PT neuron is shown. B shows the effect of electrophoretically applied homocysteic acid on the antidromic response of a PT neuron. Records c and d are taken five minutes after a and b; d after the administration of homocysteic acid. See text.

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seems to facilitate it. When 5-HT is applied, both the orthodromic firing of the PT cell and the multiple discharges of the non-PT cell are suppressed, while the antidromic spike is not affected. In the example shown in Figure 258B, the interaction seems to be reciprocal. A PT cell, probably somewhat injured by the tip of the five-barrel micropipette, tends to fire spontaneously at rather unusually high rates for a P T cell ( a ) . It responds to single pyramidal shocks (note stimulus artifacts) with an antidromic spike, which is not followed by the commonly observed silent period. Following application of homocysteic acid by passing a current of 20 nAmp. through another barrel of the five-barrel micropipette, the rate of spontaneous discharge slows down and there is a silent period of over 100 msec, following the antidromic spike. This is contrary to what could be expected from an unspecific excitatory substance. What is even more interesting is that the pyramidal shock evokes a burst of small spikes following the antidromic spike. The phenomenon could be repeated several times. Records c and d were taken five minutes after a and b; c before the administration of homocysteic acid, d, following it. It looks as if, in this case, the repetitive firing of the small neuron was responsible for the silent period following the antidromic invasion of the PT neuron. In the example in Figure 259, a single-barrel micropipette was used in combination with a five-barrel micropipette. The tip of the single pipette was located about 1.8 mm beneath the surface. The five-barrel micropipette was inserted at an angle and the position of its tip could not be accurately determined. According to micrometer recordings, it ought to have been lying about 0.3 mm above the tip of the single microelectrode. In the figure sample traces of unit responses recorded by the single microelectrode are shown in A, while in B are shown traces of spike discharges recorded from another unit by the central barrel of the five-barrel micropipette synchronously with the recordings in A. In A l , prior to the application of glutamate from the five-barrel micropipette, a PT cell is shown responding to a train of three pyramidal shocks with three antidromic spikes. In A2, while glutamate was being administered, a single pyramidal shock evoked an orthodromic spike, followed by a pair of smaller spikes. In A3, as in A l , pyramidal shocks failed to evoke the second antidromic spike, while in A4 only one—the first—antidromic spike is elicited, followed by a barrage of smaller spikes. In A5, a single pyramidal shock evoked an antidromic spike followed by a pair of smaller spikes. This record was taken about 28 sec. after the cessation of the administration of glutamate. The corresponding events taking place at the site of the five-barrel micropipette are shown in B. It can be seen that a cell—not identified—is activated into repetitive firing during the electrophoretic administration of glutamate. It is very tempting in this case to assign the failure of the antidromic vol-

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60

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a f t e r 25 sec Glut, o f f

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Figure 259. Traces taken from an experiment in which both a single- and a five-barrel microelectrode were used. Electrophoretically applied glutamate from three of the barrels of the five-barrel micropipette results in partial blocking of the antidromic invasion of the P T cell, recorded from the single microelectrode, while bringing up multiple spike discharges of another neuron following antidromic stimulation ( A ) . An unidentified cell close to the fivebarrel micropipette's tip is also activated by glutamate and is recorded by the central barrel of the five-barrel microelectrode. See text.

515

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i '•JIP^H OJ.mV 1

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Figure 260. A: Two neuronal elements participating in spontaneous spindle bursts of the cortical surface; one shows large spikes and was identified as a PT neuron; the other, with small spikes firing in high-frequency bursts, is probably an interneuron; both were silent in the interspindle periods; record taken with short time-constant; dots indicate antidromic stimulation. B: The same neuronal elements during the spindle, their activity having been recorded with a long time-constant in order to show relationship to the slow-wave activity.

leys to invade the PT cell to the activation of an interneuronal population by the glutamate ions administered from a distance through the five-barrel micropipette. Possible interaction between PT cells and presumed interneurons were looked for during the cortical spindles. In previous work (14, 32), we had shown that cortical spindle bursts are associated with synchronized alternating EPSPs and IPSPs of PT neurons. The PT cells tend to fire on the crest of the depolarizing potentials in phase with the surface negative waves. It would be interesting to see how a presumed interneuron behaves during the cortical spindling. Figure 260 is one example of this behavior. The record in A was taken with a short time-constant in order to distinguish the small spikes while the record in B was taken with a longer time-constant so that the relationship of spike firing to the slow wave activity will be shown. In both traces, two spikes are recorded synchronously by the microelectrode tip. The larger one is fired by a PT neuron and the smaller ones from another neuron which responded to pyramidal stimulation with a barrage of spikes. As can be seen, their behavioral patterns during the spindle burst are quite distinct. The small spikes, in contrast to the large ones, fire in bursts of very high frequency and tend to occur during the positive phase of the oscillatory rhythm. On the basis of these two records, it is tempting to assume that the small spikes are fired by an inhibitory interneuron and are responsible for the polarizing potentials recorded from the PT cells during the cortical spindle burst. This assumption might, however, be premature, particularly since not all interneurons tested so far have displayed such behavioral patterns during

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().5mV 50 msec

Figure 261. Extracellular recordings from one P T neuron and an interneuron while the microelectrode was moving down from a depth of 1200 ¡J. (a) to a depth of 1500 ¡x (d). While on the position where a was taken, pyramidal stimulation evoked an antidromic spike with a high-frequency response. On the following phase of the slow field potential, a burst of small spikes appeared; this became progressively more pronounced as the microelectrode moved downwards. In b, 100 ¡x. further down, the PT cell spike can barely be seen, whereas the spikes in the burst reach their maximum height. In c, the spikes are still high, though a further downward movement of 50 ¡i took place. The spikes gradually decreased and finally disappeared with further downward movement (d).

cortical spindling. On the other hand, the possibility of interneurons inhibiting inhibitory interneurons, in other words, the possibility of disinhibition (29,41) has always to be kept in mind. The results thus far presented, although somewhat fragmentary, permit a number of conclusions to be drawn: (a) We may say, first, with a degree of confidence, that axon collaterals of PT neurons are connected with cells other than PT cells and can identify cells, distinguished from the latter, by the characteristics of their response to pyramidal stimulation. (b) Volleys arising from pathways other than the pyramidal pathways (VL cortical surface) converge with the collateral volleys upon these cells, ( c ) Their response characteristics, although distinctly different from those of the PT cells, are not typical of Renshaw-type cells. (d) The pattern of their interaction with PT-cells revealed in certain instances indicates that they have an inhibitory effect on PT neurons. However, the duration of their burst-firing could hardly account for the frequently encountered long inhibition of the PT cells during antidromic activation. (e) At least some of the interneurons tested seemed to display in their spontaneous activity a certain pattern of firing which may be used as an additional criterion for their characterization. One question which is readily raised in studies such as this is whether or not there is any anatomical correlate to electrophysiological findings. This is a question which is very difficult to answer. It is known that in the neocortex as many as 60 cell types have been described (17). Others (31) have reduced the number to seven, although this was considered to be an oversimplification (4). According to Colonnier's work (4), fusiform stellate cells,

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Figure 262. Depth distribution of interneurons studied, along the vertical axis o£ the pericruciate cortex. T h e horizontal bars indicate the depth within the cortex, and the length of these bars indicates the relative number of interneurons located at these depths. (Schematic drawing from Colonnier, 4.)

due to their ramified vertical axons, are the most suitable for vertical columnar spread of excitation in the cortex, although the basket-like cells of layers II and IV, due to their horizontally extending axons and their extensive arborization, are the most suitable for surround inhibitory action. In conclusion, I would like to give a very brief account of our attempts to correlate location and function of the tested neurons. It was clear from the beginning of our study that only rarely could one encounter neurons of the type described, located so close to PT cells that an extracellular micropi-

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pette tip could monitor simultaneously the activity of both. This can be taken as an indication that interneurons are not situated within the pyramidal layer. In many cases following the antidromic spike from a PT neuron there was a rippling activity which could be interpreted as interneuronal firing picked up from a distance. By moving the microelectode tip along the vertical axis from the point that such records were obtained, we were in several cases able to record alternately solitary antidromic spikes of a PT neuron and a barrage of multiple spike discharges presumably fired by an interneuron. Such a case is illustrated in Figures 261 and 262. Our numerical findings, with regard to the location of the tested interneurons along the vertical axis of the cortex, were charted on Colonnier's drawing showing the various neuronal elements of the cortex ( 4 ) . It is quite clear from this figure that, on the basis of the gross depth measurements employed in this study, one cannot draw a definite conclusion as to the anatomical characterization of the interneurons tested. It is quite likely that they belong to the basket-like type but further work with more delicate measurement techniques for localization (injection of dyes, and so on) are required before we can draw a complete wiring diagram of recurrent inhibition. Schlag: I want to congratulate Dr. Stefanis for courageously broaching this subject. We have heard a great deal about cortical inhibition but very little about cortical inhibitory interneurons. Probably most of the people who have recorded from cortical units have seen a few cells that they thought were inhibitory interneurons. As we know, identifying these interneurons may be a very difficult task. A first hint is to look for units discharging—probably at a high frequency— when others are silent. This is what Marco, Brown & Rouse (18) have done quite recently in an attempt similar to yours, Dr. Stefanis, but concerned with nucleus ventralis lateralis of the thalamus. They have produced remarkable records showing close cells firing in alternate periods. These authors experimented with intranuclear electrical stimulation and obtained two kinds of response: the first type consisted of one or two short-latency spikes, the second was characterized by a long-latency prolonged barrage. It would be good if this second type represented the inhibitory interneurons, because we would have an easy way to identify them. Dr. Villablanca and I happened to conduct about the same kind of experiment though with a quite different objective in mind (30). Our data fitted quite well with those of Marco's group; however, looking closely at the statistical distribution of the responses in our material, we could not find any convincing evidence for the existence of two cell populations. The latency distribution was bimodal but with no sharp separation between the peaks. The duration of the train of spikes and the number of spikes in each train varied rather monotonously from the smallest to the largest values. Furthermore, a few relay cells, identified by their short-latency activation by the

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cerebello-thalamic input, were found to respond late and repetitively to local stimulation. Certainly, it is not impossible that cells of distinct types happen to fire around the same time and maybe in a similar manner and, conversely, cells of the same type may fire in quite different patterns to a given input. I think that thalamic and cortical cells should be regarded, not only as elements in series in a path, but also as being part of a local network. I would like to expand a little more on these experiments because the point seems relevant also to what Dr. Purpura has reported. While electrically stimulating in nucleus ventralis lateralis, we found that cells around the point of stimulation could be affected very differently. Many did not seem disturbed at all, as shown by the absence of changes in their post-stimulus histogram; some of them, though, were very close (less than 1 mm) from the tip of the stimulating electrode. Others discharged first and then were silenced for more than 100 msec. A third group was immediately silenced by the stimulus. We tried to determine the location of these last cells, but they were scattered, and the only information we could gather was that they were more frequently encountered farther away from the point of stimulation. In fact, the difference in the distribution of initially fired and initially silenced units as a function of distance from the point of stimulation was statistically significant. Incidentally, some of the relay cells of nucleus ventralis lateralis showed this immediate arrest of firing (30). It seems that a stimulation even in the relay nucleus itself can have diverse, sometimes opposite, effects on the activity of relay cells. It is not simply a massive excitation. One cannot overlook the fact that units in a population are probably interacting, and these interactions complicate our efforts tremendously. Ito: I would like to introduce an interesting observation made by Uno and his coworkers* on the inhibition of VL neurons. They recorded intracellularly from VL neurons and found IPSPs of two different forms. One is produced by stimulation of cerebellar nuclei with a disynaptic latency and has a relatively short duration; since the disynaptic latency is maintained even when the pathway is stimulated at the rostral pole of red nucleus, the interneurons for this inhibition are probably located within VL itself. The other is induced after stimulation of the sensorimotor cortex, and in this case its latency is long enough to assume three or more synapses on the pathway and its duration is much prolonged, just like the IPSPs which Dr. Purpura has demonstrated. We therefore suspect that VL includes two types of inhibitory interneurons (11). Of course, a possibility remains that one and the same inhibitory interneuron is activated in different ways after stimulation of the cerebellar nuclei and the sensorimotor cortex. I would also like to present an observation made by Toyama & Matsunami (39) on the visual cortex, in relation to Dr. Stefanis' presentation. In the vi* M. Uno, M. Yoshida and I. Hirota: Personal communication.

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sual cortex they have been recording from a number of cells intracellularly and found that stimulation of the ipsilateral geniculate body produces, in the majority of the cells sampled, prominent EPSPs monosynaptically. This is in good agreement with Dr. Scheibel's recent experimental observation that the specific visual afferents from the lateral geniculate body innervate directly pyramidal cells in the visual cortex. This monosynaptic EPSP is always followed by a prominent IPSP with an additional delay of 1 msec. This indicates that there is a disynaptic feed-forward inhibition in the visual cortex. The inhibitory interneurons for this pathway may be found among the stellate cells which are innervated monosynaptically by the specific visual afferents. Purpura: I would like to comment on the cortical interneuron, particularly in relation to its organization in columnar arrays. Studies in which four or five impalements of cortical interneurons have been made during a single penetration of motor cortex have revealed some very unique features of the columnar organization of excitatory and inhibitory elements. Figure 263 shows such an experiment during stimulation of the VL nucleus of the thalamus (24). The most superficial cells (Figure 263, A and B ) were located at about

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Figure 263. PSP patterns evoked during augmenting responses in four interneurons (nonPT cells) impaled during a single penetration of cortex. Approximately 10-20 min. elapsed between recording each series of responses. A: Cell located at a depth of 0.8 mm exhibits high-frequency repetitive discharges during surface-positivity, and late IPSPs during surface-negativity; discharges reappear prior to stimulation. B: Cell at a depth of 0.85 mm; short-latency complex IPSPs observed throughout period of stimulation. C: Cell at 0.95 mm shows IPSPs during augmentation. D: Cell at 1.00 mm exhibits IPSPs during cortical surface response. Note similar IPSP patterns observed in B-D during third response of each series. Calibration: 0.1 sec; 50 mV. (From Purpura, Shofer & Musgrave, 24.)

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0.8 and 0.85 mm. If one examines the third response in each series one is impressed with the extraordinary similarity in time course of the EPSP and IPSP patterns in these cells at different depths during VL stimulation. These results are consistent with the studies of Mountcastle (21) and of Hubel & Wiesel (13) in showing very similar patterns of activity in columnar arrays of cortical neurons. The point of emphasis is that the inhibitory neurons are linked to these cells at different depths, so as to produce basically similar PSP and discharge patterns. Maynard: In looking at interneurons in the lobster brain I have been very impressed with the pattern of excitation followed by a very prolonged inhibition. This seems to be something which appears again and again in the vertebrate interneurons which we have been discussing here. Obviously, it is difficult to say exactly what this means, but such a pattern does imply that there must be some two, three, or four-neuron connectivity pattern which seems to be rather widespread in some of the higher centers of many animals. Finally, I would like to show an example (Figure 264) of a prolonged response of an interneuron in Crustacea, of the kind Dr. Purpura mentioned earlier in a slightly different context (19, 20). This happens to be an interneuron in the brain of the spiny lobster that is excited monosynaptically by a very large number of converging olfactory axons. As you see, a single volley, when it includes a sufficient number of presynaptic elements, initiates a very large, prolonged depolarization. The depolarization is large enough to depress full spike potentials. Throughout the plateau phase of the depolarization only small, repetitive, local potentials occur. These eventually develop into full spikes only as the prolonged potential decays. A second volley (or many) arriving at the interneuron during its response is ineffective, apparently because of temporary exhaustion of transmitter or inhibition of its release of the presynaptic sensory terminals. Responses similar to this have been described in the stretch receptor elements in crayfish (40). I regard it as significant, however, that they have also been found among some interneurons in various central nervous systems. It would appear as though many neuronal properties that now tend to be considered exotic and unusual may prove to be found generally in specific classes of interneurons—possibly serving common functions—in central nervous systems from very different animals. Kennedy: I want to emphasize one feature of crustacean interneurons that may provide a model—albeit perhaps a little far out—for what happens in mammalian systems where very long dendrites are intersected at different levels by input fibers. If these dendrites spike (and from what one hears it looks more and more as though they may do so), one has to consider the problem of what happens when impulses are running around in the dendritic tree. Most segmental interneurons in Crustacea present a similar situation. An interneuron may receive synapses in each of several ganglia. When Preston

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Figure 264. Prolonged discharge in response to single afferent volley in olfactory fibers from antennule of spiny lobster. Intracellular recording from an interneuron in the olfactory-accessory lobe complex (see Figure 15, p. 5 8 ) . Records a, h, c: responses to maximal volleys applied at frequencies of 0.5, 1.0, and 5 per sec. respectively; stimulus artifact is evident on upper trace in each record; the initial input volley elicits a sharply rising, depolarizing PSP; after two spikes, this PSP produces a prolonged, high-frequency, repetitive discharge of small, "local" potentials; these fuse to produce the thick trace at this recording speed; as the PSP gradually returns toward resting level, the "local" potentials grow in amplitude, decrease in frequency, and eventually drive full spikes; the late spike discharge may last for tens of seconds; input volleys arriving during the first one to two seconds of the PSP are ineffective—the variation in duration of the initial PSP is a result of variations in inter-test recovery time, not input frequency—and then gradually recover partially. Records d and e show that the decline in effectiveness of successive input volleys probably represents failure of, transmitter release at the presynaptic terminal, rather than failure of the postsynaptic interneuron to respond; submaximal volleys produce a response far below that of which the neuron is capable, and vet show almost no summation or facilitation. Voltage calibration, 40 mV; time calibrations, 0.5 sec.

and I first began recording intercellularly in neuropile from such units eight or ten years ago, we were confronted with some very bizarre-looking situations. W e would shock a root containing afferent fibers; this would provide a long EPSP, with spikes rising from it with what looked like a constant firing level. Some later spikes would then arise from a firing level that was very much lower, much like what Dr. Purpura described. W e now have an explanation for this: the later spikes were initiated in a different ganglion, by sensory axons that projected there centrally. Even where input is from a single segment, spike initiating loci may shift; and impulses in the interneuron are propagated in both directions. What would the situation be in a long spiking dendrite with input fibers that intersect it at intervals? If it is stimulated in a linear sequence by the inputs, the frequency of discharge at one end of the dendrite is going to be higher—as much as 50 per cent higher—than that at the other. In fact, if you record at two different points in the cell when the initiation locus is shifting the actual discharge pattern may be different.

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There is also a kind of interaction ( L C ) in which the spikes are followed by depolarizing after-potentials. Such afterpotentials can last for as long as 25 msec, after passage of the spike, and a spike initiated in one place can thus accomplish subthreshold modulation of distant synaptic inputs. All kinds of complicated things can thus be done in integrative regions of nerve cells if action potentials are traveling around in them. Now that we know that action potentials may do just that, I think we have to take seriously what might happen as a result. REFERENCES P., ECCLES, J . C . , and VOORHOEVE, P. E . , Postsynaptic inhibition of cerebellar Purkinje cells. J. Neurophysiol., 1964, 27: 1138-1153. ARMSTRONG, D. M., Synaptic excitation and inhibition of Betz cells by antidromic pyramidal volleys. J. Physiol. (London), 1965, 178: 37-38P. BROOKS, V . B . , and ASANUMA, H . , Recurrent cortical effects following stimulation of medullary pyramid. Arch. Hal. Biol., 1965, 103: 247-278. COLONNIER, M . , The structural design of the neocortex. In: Brain and Conscious Experience (J. C. Eccles, Ed.). Springer, New York, 1966: 1-23. CREUTZFELDT, O. D., L U X , H. D., and NACIMIENTO, A. C., Intracellular Reizung corticaler Nervenzellen. Pfliiger Arch, ges. Physiol., 1964, 281: 129151. CURTIS, D. R., Microelectrophoresis. In: Physical Techniques in Biological Research. Vol. V: Electrophysiological Methods, Part A (W. L. Nastuk, Ed.). Academic Press, New York, 1964: 144-190. CURTIS, D. R., and ECCLES, R. M . , The excitation of Renshaw cells by pharmacological agents applied electrophoretically. ]. Physiol. (London), 1958, 141: 435-445. CURTIS, D . R., and RYALL, R . W . , The synaptic excitation of Renshaw cells. Exp. Brain Res., 1966, 2: 81-96. ECCLES, J . C., ECCLES, R. M., IGGO, A., and LUNDBERG, A., Electrophysiological investigations on Renshaw cells. J. Physiol. (London), 1961, 159 : 461478. ECCLES, J . C., F A T T , P., and KOKETSU, K., Cholinergic and inhibitory synapses in a pathway from motor-axon collaterals to motoneurones. J. Physiol. (London), 1954,126: 524-562. ECCLES, J . C., ITO, M., and SZENTAGOTHAI, J., The Cerebellum as a Neuronal Machine. Springer, New York, 1967. ECCLES, J . C., LLINAS, R., and SASAKI, K., The inhibitory interneurones within the cerebellar cortex. Exp. Brain Res., 1966, 1: 1-16. H U B E L , D . H . , and W I E S E L , T . N . , Shape and arrangement of columns in cat's striate cortex. J. Physiol. (London), 1963,165: 559-568. JASPER, H . , and STEFANIS, C . , Intracellular oscillatory rhythms in pyramidal tract neurones in the cat. Electroenceph. Clin. Neurophysiol., 1965, 18: 541-553. KRNJEVIC, K., and PHILLIS, J . W., Iontophoretic studies of neurones in the mammalian cerebral cortex. J. Physiol. (London), 1963, 165 : 274-304.

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M., and STRAUGHAN, D . W., Nature of a cortical inhibitory process. J. Physiol. (London), 1966, 184 : 49-77. LORENTE DE NÓ, R., Cerebral cortex: architecture, intracortical connections, motor projections. In: Physiology of the Nervous System (J. F. Fulton, Ed.). Oxford Univ. Press, New York, 1943: 274-313. MARCO, L. A., B R O W N , T. S., and ROUSE, M . E., Unitary responses in ventrolateral thalamus upon intranuclear stimulation. J. Neurophysiol., 1967, 30: 482-493. MAYNARD, D. M . , Repetitive responses in central lobster neurons. Fed. Proc., 1963, 22: 220. , Integration in crustacean ganglia. Sympos. Soc. Exp. Biol., 1966, 20: 111-149. MOUNTCASTLE, V . B., Modality and topographic properties of single neurons of cat's somatic sensory cortex. /. Neurophysiol., 1957, 20: 408-434. PHILLIPS, C . G . , Actions of antidromic pyramidal volleys on single Betz cells in the cat. Quart. J. Exp. Physiol, 1959, 44: 1-25. , Some properties of pyramidal neurones of the motor cortex. In: The Nature of Sleep (G. E. W. Wolstenholme and M. O'Connor, Eds.). Churchill, London, 1961: 4-29. PURPURA, D. P . , SHOFER, R. J., and MUSGRAVE, F. S . , Cortical intracellular potentials during augmenting and recruiting responses. II. Patterns of synaptic activities in pyramidal and nonpyramidal tract neurons. J. Neurophysiol, 1964, 27: 133-151. R A M Ó N Y C A J A L , S . , Histologie du Système Nerveux de l'Homme et des Vertébrés, Vol. II. Maloine, Paris, 1911. RANDIC, M., S I M I N O F F , R . , and STRAUGHAN, D. W., Acetylcholine depression of cortical neurons. Exp. Neurol, 1964, 9: 236-242. SALMOIRAGHI, G . C . , and STEFANIS, C . N . , Patterns of central neurons responses to suspected transmitters. Arch, ltal Biol., 1965, 103 : 705-724. SAWA, M . , MARUYAMA, N . , K A J I , S . , and HANAI, T . , Actions of stimulation to medullary pyramid on single neurons in cat's motor cortex. Folia Psychiat. Neurol. Jap., 1960,14: 316-346. SCHLAG, J., Reactions and interactions to stimulation of the motor cortex of the cat. J. Neurophysiol, 1966, 29: 44-71. SCHLAG, J., and VILLABLANCA, J., A quantitative study of temporal and spatial response patterns in a thalamic cell population electrically stimulated. Brain Res., 1968, 8: 255-270. SHOLL, D . A . , The Organization of the Cerebral Cortex. Methuen, London, 1956. STEFANIS, C. N., Relations of the spindle waves and the evoked cortical waves to the intracellular potentials in pyramidal motor neurons. Electroenceph. Clin. Neurophysiol., 1963,15: 1054. , Electrophysiological properties of cortical motoneurons during iontophoretic application of chemical substances. Physiologist, 1964, 7: 263. , Further Investigation on the Electrophysiological Properties of PT Cells and the Mechanism of Integration of Motor Function. Sotiropoulos, Athens, 1965 (in Greek).

K R J N E V I C , K . , RANDIC,

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and JASPER, H . , Intracellular microelectrode studies of antidromic responses in cortical pyramidal tract neurons. J. Neurophysiol., 1964, 27: 828-854. 36. , Recurrent collateral inhibition in pyramidal tract neurons. J. Neurophysiol, 1964, 27: 855-877. 3 7 . TAXAHASHI, K . , Slow and fast groups of pyramidal tract cells and their respective membrane properties. J. Neurophysiol., 1965, 28: 908-924. 3 8 . TAXAHASHI, K . , KUBOTA, K . , and UNO, M . , Recurrent facilitation in cat pyramidal tract cells. J. Neurophysiol., 1967, 30: 22-34. 3 9 . TOYAMA, K . , and MATSUNAMI, K., Synaptic action of specific visual impulses upon cat's parastriate cortex. Brain Res., 1968, 10: 473-476. 40. WASHIZU, Y., BONEWELL, G. W., and TERZUOLO, C. A., Effect of strychnine upon the electrical activity of an isolated nerve cell. Science, 1961, 133: 333-334. 41. WILSON, V. J . , and BURGESS, P. R., Disinhibition in the cat spinal cord. J. Physiol (London), 1963,13: 386-398. 3 5 . STEFANIS, C . ,

SUMMATION DOMINICK P. PURPURA Albert Einstein College of Medicine New York, New York

The concluding chapter of a symposium volume is generally reserved for recapitulation of essential new findings, analysis of their interrelations and definition of emergent principles likely to satisfy Horridge's "Criteria of Significance", that is, survival as a sentence in a textbook one or two decades hence. In surveying the remarkable display of data presented in preceding chapters it is apparent that "interneurons" have not only come of age but they must be accorded all the respect previously conferred upon their more impressive neighbors, the spinal motoneuron, the Purkinje cell, or the pyramidal neuron. This three-day conference was not called to affirm the wellestablished fact that neuronal integration is effected through the activity of interneurons. Rather the purpose of this symposium may be sought in the meaning of "Interneuron Integration," and its expression in the operation of different hierarchies of synaptic organizations that constitute the neural substrate of behavior. How to proceed with the task at hand? It has been argued by Horridge that, since interneurons have open-ended properties, none can be fully defined. And if he is correct in the assumption that most of the detailed studies of interneuron discharge patterns and correlations will not be relevant to the required explanation of behavior, why continue along these lines? The biologist, in order to dispel nihilism or uncertainty, answers: Because only by such examinations will new principles of operation be forthcoming that will ensure elucidation of the mechanisms of neuronal integration. Thus, the outcome sought depends more on the adequate design and choice of experimental material than on the necessity for direct confrontation with the problem of behavior. Nevertheless, those experimental designs which permit the most precise explanations of neuronal interactions, at whatever level of organization are most likely to provide the most satisfactory models for investigating complex integrative activities. The question of "relevancy" to behavior mechanisms may be of little consequence at this stage, however one prefers to think to the contrary. The immediate urgency is for adequate definition of principles of operation. And it is this problem we must address in assessing the contributions of the symposium. 527

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It is pertinent to note that the participants were not specifically troubled with seeking a consensus on a functional or anatomical definition of 'the Interneuron'. Some were, however, so compelled as to include a definition in their presentation perhaps more as an expression of faith than commitment to prejudice. To paraphrase the definition quoted by the Scheibels from Bullock and Horridge, "An interneuron is a neuron which connects neurons with neurons" (with apologies to Gertrude Stein). This, the least restrictive definition, was probably also the least offensive to the participants. Further attempts to define interneuronal properties met with limited success and, in fact, it was quite clear that one could list a number of non-properties of interneurons more readily than one could specify features to distinguish them from other elements. To catalog but a few of the non-properties of interneurons here seems appropriate to avoid future exorcization: A. Interneurons are not necessarily short-axoned cells or elements with limited dendritic spread; B. interneurons are not exclusively commutators of synaptic action in a particular pathway; and C. there are no electrophysiological properties which serve to distinguish interneurons from non-interneurons. This is not to say that interneurons cannot be distinguished from spinal motoneurons with appropriate methods of analysis, but such distinctions are less obvious the more difficult it becomes to effect satisfactory identification of neurons. For the sake of argument the Bullock-Horridge-Stein definition may be taken to indicate that probably all types of elements in the vertebrate central nervous system, with the exception of primary sensory neurons, are "interneurons". Motoneurons might be excluded but for the fact that the vast majority are synaptically related via their axon collaterals to other neurons; and in some species motoneurons may be electrotonically coupled one to the other. Admittedly, this heterodoxy is a reductio ad absurdum and of little more than academic interest. After all, everybody knows what interneurons are, why attempt to define them? Perhaps this tacit assumption was sufficiently powerful as a non-verbal communication to inhibit much discussion of the term "interneu99

ron . It is obvious that the participants attached far more importance to operational definitions of interneurons derived from considerations of afferent and efferent relations, cytoarchitectural features and synaptic actions in different organizations. In what follows an attempt is made to indicate features of interneurons specified in the symposium that have provided important clues to the operation and organization of relatively "simple" as well as highly complex synaptic systems. Some of the most elegant analyses of interneuronal activities described to date have come from studies of invertebrate nervous systems. Identification of elements in terms of input-output relations and excitatory-inhibitory actions has permitted accumulation of a wealth of information on the properties of interneuronal interactions. The reports by Wiersma, Kennedy, Tauc,

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Kandel, and Maynard amply attest to this. The ability to study specific, identifiable interneurons in different preparations as noted by Wiersma, represents an impressive feature of invertebrate studies that is rarely met in studies of higher vertebrates. But this does not imply that it is always desirable to extrapolate findings obtained in studies of Crustacea and Aplysia to higher vertebrates. Thus many CNS interneurons of Crustacea do not show alterations of discharge patterns following stimulation of receptors other than their main sensory input source. Contrast this property with the extraordinary convergence from central and peripheral sources upon interneurons in the spinal cord or brain stem of mammals. Other physiological distinctions may be cited from Wiersma's report. It will hardly be questioned that Wiersma and his associates have probably studied a greater variety and number of interneurons in the crayfish than any other group. Considerable significance may thus be attached to his statement that in only one instance has he observed changes in sensory field-size of a central mechanoreceptor fiber. Such is rarely the case in examination of the receptive fields of interneurons in vertebrates. But it is not to be inferred that complex interactions are not observed in invertebrate systems or that analysis of these interactions is always readily apparent. As Wiersma points out, primary sense fibers distribute to several parts of the CNS in Crustacea, that is, up and down the whole cord to the brain and to ganglia adjoining those in which they enter. Thus, the implications of this for a high degree of parallel computation are more obvious than anticipated from anatomical data. However this may be, there emerges from studies of specific interneurons in Crustacea, recognition of a feature of these elements which has yet to be described in the nervous systems of higher vertebrates. This is the capacity for command interneurons to release complex, coordinated behavior patterns. Both Wiersma and Kennedy have explored this problem in some detail in their presentations. The specificity of behavior evoked by a command interneuron has been shown to depend upon synaptic relations of the interneuron with given sets of "driven" neurons responsible for reciprocal motor outflow in single segments. As might be anticipated, not all command interneurons are equipotent in their effects, there being hierarchial arrangements that introduce possibilities for diversity. One might wonder here why Kennedy appears so hesitant to admit this property of some interneurons in the crayfish, unless it be due to the disturbing element of variability which this introduces in the analysis of "simple" systems. That a broad range of problems is accessible to analysis of such systems is well illustrated by Kennedy in consideration of the role of proprioception in movement in arthropods. In contrast to the situation in vertebrates, proprioceptors in arthropods are not required for reflex organization of motor behavior. (Some might question whether proprioceptors are important in this respect in higher vertebrates in view of the capacity of monkeys to utilize

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their limbs in well organized and complex behaviors following complete deafferentation!) In the case of arthropods, the organization of motor activity is programmed in the ensemble of ganglionic neurons associated with command elements, probably indirectly through an interposed local network whose identity and function, according to Kennedy, remain obscure. Interesting parallels have been drawn by Kennedy between the operation of the muscle-receptor organ control circuit in Crustacea and the proprioceptor control of respiration in mammals. This should serve as a useful analysis of systems with widely divergent properties but with similar overt functions. The object lesson here is that the evolutionary process discards little of value in principles of operation, if not of design. Command interneurons have been found in abundance in the crustacean CNS but, as Kennedy hastens to add, they have not as yet been demonstrated in the abdominal ganglia of Aplysia. (The reader may recognize this as an example of "gamesmanship" on the part of a crayfish-enthusiast.) While this may be the case, it should be recalled that probably more detailed information on the anatomical connections and physiological and pharmacological operation of identifiable neurons has been forthcoming from studies of Aplysia abdominal ganglia than from any other structure in the animal kingdom. And as recent work would suggest, the time is at hand for extrapolation of much of the basic data obtained to the analysis of behavior patterns of Aplysia. The impressive advances made in recent years in the studies of Tauc and Kandel and their associates require little comment. The demonstration of the conjoint excitatory and inhibitory action of a single interneuron, as originally proposed by Tauc and confirmed by Kandel, is likely to be recorded as one of the most decisive experiments in neurophysiology. The principle embodied in this demonstration will undoubtedly survive as more than one sentence in a textbook two decades hence! In a word, the monosynaptic connections of the interneuron with the two follower cells it both excites and inhibits have been traced anatomically, the transmitter released at the interneuron terminals has been specified as ACh, and the ionic permeability changes induced by the transmitter on the different cells has been indicated. Even the most casual reader will appreciate the challenge these findings present to any theory which assigns excitatory or inhibitory functions to an interneuron, either on the basis of its transmitter or the structural configuration of the vesicles in which the transmitter is presumably packaged. What emerges from the studies of Tauc, Kandel and others in this area affirms the important role of postsynaptic membrane receptor sites in determining the nature of the ionic permeability change and consequently the overt physiological effect resulting from the transmitter-induced receptor activation. These data also bear directly on the question of the significance of attempts to distinguish inhibitory and excitatory synapses on the basis of characteristics of presynaptic components as revealed in electron microscope studies (vide infra).

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The review of neuronal interactions presented by Tauc gains in importance when it is recognized that the data derived from studies of central ganglia in Aplysia encompass virtually the entire spectrum of possible neuronal interactions described to date. To the long list of "simple" synaptic interactions, Tauc has added some new possibilities which involve consideration of biphasic synaptic actions of a single transmitter and release of two different transmitters from a single ending with different effects on pre- and postsynaptic membrane. Perhaps one of Tauc's most provocative suggestions is his proposal that long duration "episynaptic" effects and certain prolonged inhibitory actions may not involve classical synapse-like structures. The hypothesis concerning extrasynaptic release of transmitter and diffusion of transmitter to more than one postsynaptic element is compelling indeed, as judged by the evidence marshalled for its support. If such mechanisms of extrasynaptic transmitter or transmitter-like release are operating in other systems the implications for studies of "neuronal interactions" as currently defined, will be dramatic indeed. A major factor conditioning the rapid accumulation of basic information on neuronal interactions in Aplysia would appear to be the facility with which new observations have been confirmed and pursued along different tracks in different laboratories. The example noted above, concerning the conjoint excitatory and inhibitory action of an identifiable interneuron, amply illustrates this point. To the growing body of information on Aplysia neurons and the manner in which they participate in integrative activities in inquiry in efforts to obtain a more precise definition of synaptic relations between identifiable elements and to specify properties of different subpopulations in the ganglia. The results of this ambitious program have been considerable in permitting detailed studies of different types of multiaction interneurons and the manner in which they participate in integrative activities in the ganglion. The demonstration of overt postsynaptic effects dependent, in part, on differential receptor desensitization at biphasic synaptic sites clearly indicates the wide range of interactions, some frequency specific, which can be elucidated with proper experimental design. Consideration of the varieties of neuronal interactions would be incomplete without inclusion of the mechanism of electrotonic coupling for synchronizing activity in a population of elements. The observations of Kandel and his associates on the synchronization of activity in Aplysia bag cells, and of Maynard in studies of the A-neurons in the stomatogastric ganglion of Scylla are of interest in this regard. While the accepted method for demonstrating coupling between cells by passing currents between intracellularly located electrodes has provided positive evidence for coupling of stomatogastric A-neurons, this technique has not yielded the expected evidence in Aplysia bag cells. The problem presented by Kandel to the effect that electronic junctions may be remote from the site of intrasomatic current application has been well considered. Fortunately, in the case of the bag cells, section of the connectives in the neuropile, with subsequent loss of synchroni-

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zation, has provided crucial supporting evidence for the distant location of possible electrotonic junctions involving these elements. It is perhaps of interest that the question of electrotonic interactions was not considered in relation to higher vertebrate neuronal systems except in a speculative fashion in connection with the problem raised by findings of primary afferent collaterals in microbundles, as described by the Scheibels. This might reflect the view that since no morphological evidence for electrotonic junctions has been obtained in neurons of the mammalian central nervous system, electrotonic interactions are not likely to be of importance in the operation of interneuronal organizations in higher vertebrates. Two points are noteworthy in this context: first, electrotonic junctions are by no means essential for electrotonic interactions and, second, no tests for electrotonic coupling have been carried out to date on neurons of the mammalian brain. If there is a take-home lesson to be gleaned from the variety of data presented in studies of invertebrates, it is that multiaction interneurons are capable of effecting a remarkable degree of integrative activity within relatively small populations of neurons. In this context one cannot help but be impressed by the relatively few cells required to achieve a highly integrated output in the stomatogastric ganglion studied by Maynard. Presumably a single multiaction interneuron carries out functions assignable to a number of excitatory and inhibitory interneurons in the vertebrate nervous system. This by no means indicates that multiaction neurons may not exist in higher nervous systems since it has not been technically feasible as yet to examine interneuronal interactions with the precision possible in invertebrate systems. Illustrations of the reports by Tauc and Kandel disclose a wide variety of postsynaptic potentials in different Aplysia elements following different modes of activation. The possible ways in which a postsynaptic element reads some of this input was considered by Segundo in a summary of presynaptic and postsynaptic statistics that reveal several aspects of underlying complex transformations. The importance of this synthetic approach that allows definition of an input responsible for a particular extracellular unit activity is self-evident. However, its application to the analysis of most types of unit studies in the brain might be frustrated by the fact that such activity is usually determined by a complex interplay of excitatory and inhibitory synaptic bombardment of different parts of the neuron. Nevertheless, the net synaptic input could initiate a particular temporal sequence of discharges which might impress some degree of specificity upon "nonspecific" cells such as are encountered in reticular systems. Available data processing techniques, in combination with refined electrophysiological studies, may be expected to enhance further the power of statistical approaches which have already provided new information on input-output transformations in Aplysia neurons and other systems, as Segundo has indicated.

SUMMATION

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On the question of computational methods in neurobiology it is pertinent to recall the admonition of Brazier that, unless more attention is directed to the development of appropriate computer programs and techniques for examining neurohistological material, we may be faced with a hopeless task of defining the intimate relations of neurons as revealed in Golgi or electron microscope studies, such as those described by Blackstad and Larramendi. Statistical methods are required for profile recognition, for studying quantitative relations between neurons, the distribution of axonal and dendritic fields in neuropile and relationships of cell geometry to field potentials. Unfortunately, very few attempts in this direction have been made and with little success. Such methods should supplement good histology carried out by competent neurohistologists. The reader has but to examine the report by the Scheibels on the structural analysis of spinal interneurons and related afferent systems to appreciate the magnitude of the task ahead in developing computer programs that could carry out the analyses described by these workers. The Scheibels have provided us with a definition of a short-axoned interneuron that is quite satisfactory. Short-axoned cells are those whose axons never leave gray areas to enter tracts or nerves. If we accept this definition, then the conclusion follows that such elements are not encountered anywhere in the ventral horn. On the positive side, if a system of interneurons is required for the Renshaw phenomena, then this is most likely to involve interneurons with widely distributing axonal trajectories. There is no disagreement here between neurophysiological and histological data. In point of fact, many of the neurophysiological requirements for the Renshaw phenomena are satisfied by interneurons of the type described by the Scheibels. But what of their findings that large motoneurons may not all be endowed with axon-collaterals or that axon-collaterals of large motoneurons may distribute to other large motoneurons? Need Renshaw inhibition always involve an intercalated interneuron? Clearly, the further exploration of these questions could lead to entirely new avenues of investigation in an area by no means saturated with hard data. The Scheibels' extensive analysis of neuropile patterns of spinal interneurons in different laminae, as derived from careful studies of different planes of section, has previously not been attempted on the spinal cord. This analysis will undoubtedly prove to be of much greater importance than the question of what kind of interneurons reside in the ventral horn. Refreshing approaches to the cytoarchitecture of gelatinosa cells and their glial relations are of particular interest in view of the role these elements may play in the generation of primary afferent depolarization (PAD), a subject of central importance in current neurophysiological studies. Many electron microscopists might take exception to the Scheibels' statements concerning the distribution of axo-axonal contacts in the dorsal horn as revealed in Golgi preparations. Still, their observation that axons of gelatinosa cells at one level

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enter into axo-axonal relations with cutaneous terminals one or two segments away provides strong support for the notion of a gating function effected by gelatinosa cells on input mediated by large cutaneous afferents. A similar histological demonstration of a substrate for presynaptic modulatory control by propiospinal systems distributing within the base of each cutaneous sensory neuropile field adds to the obvious complexity of the presynaptic mechanisms operating upon primary inputs. From these neuroanatomical studies it is little wonder that Lundberg has repeatedly insisted upon the necessity for careful evaluation of the contribution of presynaptic inhibitory processes in all studies of interneuronal interactions in the spinal cord. The provocative suggestion has been made by the Scheibels from their studies of afferent collateral microbundles that spatial arrangements might allow for direct axo-axonal interactions which could account for PAD without the necessity for invoking interposed interneuronal networks. Electrical interactions between dorsal inputs have long been known and may have their explanation, in part, in the anatomical arrangements described by the Scheibels. The issue raised strikes at the core of the problem of the neural basis of the widespread distribution of PAD which, as Schmidt indicates, may be initiated by a few impulses in very few afferents. Schmidt also points out that there is a distinct separation of phasic and tonic PAD systems which suggests a detailed organization of presynaptic inhibitory effects of cutaneous afferents. Such features are not likely to be accounted for by electrotonic interactions in microbundles of afferent collaterals. Another issue is emphasized in the data presented by Schmidt. For inasmuch as adequate physiological stimulation is capable of revealing a more highly structured organization of PAD systems than previously envisioned on the basis of electrical stimulation of nerves, it follows that many of the data on presynaptic inhibition obtained with volley stimulation of whole nerves may require re-examination. The same may hold true for some of the results reported by Willis, who has employed electrical rather than natural stimulation of cutaneous and muscle afferents to examine the monosynaptic inputs and distribution of spinal interneurons. Notwithstanding this, the judicious employ of marking techniques by Willis has provided physiological and anatomical support for the localization of several types of interneurons studied by the Scheibels. On the question of the localization of Renshaw cells, it is of interest that the Scheibels have focused their attention on lamina VIII elements, whereas Willis identified cells with Renshaw features in lamina VII. Lest it be inferred from this that there is a discrepancy in the results, it must be pointed out that the two studies have sought different objectives. For the Scheibels have addressed the question of whether or not there are short-axoned cells anywhere in the vicinity of large motoneurons (and there are not!), whereas Willis has sought the location of neurons activated by ventral root stimulation. It follows from these studies that the cells localized by Willis are not short-axoned neurons, since it is unlikely that the

SUMMATION

535

Scheibels could have missed these elements in the course of examining an astronomical number (25,000!) of Golgi preparations. Doubtless, the issue of "what is a Renshaw cell" will continue to generate debate for some time to come. Fortunately, one aspect of the problem of the mechanism underlying Renshaw inhibition has been unequivocally settled by Willis with the demonstration that afferents in the ventral root do not play a role in the production of the Renshaw phenomena. It remains to be demonstrated whether or not nonsynaptic interactions, possibly mediated via electrotonic junctions, may play a role in the Renshaw effects of antidromic stimulation in the mammalian spinal cord. If the Scheibels' contribution to the symposium is a reflection of the renaissance of interest in the morphological organization of spinal interneurons, then surely the work summarized by Lundberg represents its physiological counterpart. The task of unraveling the complex interactions of spinal interneurons has probably troubled Lundberg more than is evident from his report. How else to explain his necessity to raise the question, posed in another context by Horridge, as to whether the study of the functional organization of the spinal cord requires a detailed analysis of interneurons? His answer is an unqualified affirmative. For reflex pathways interact via interneurons to achieve an integrative output. The theme is recurrent. Lundberg has shown that depolarization of interneuronal terminals on la afferents by interneurons in the flexor reflex afferent pathway indicates a greater degree of presynaptic inhibition within interneuron pathways than has been previously suspected. Complexities are seen in mutual inhibitory interactions in the interneuronal organizations distributing to extensor and flexor motoneurons and in the overt effects of DOPA on descending influences to interneurons. The extraordinary monosynaptic and polysynaptic excitatory and inhibitory convergence of afferent activities on interneurons of the dorsal horn should be examined in the framework of the neuropile analyses of this region presented by the Scheibels. Lundberg correctly cautions against attempts to identify interneurons with a certain type of response as belonging to a given pathway. If the hope exists that knowledge of input pattern may facilitate this identification, there is no question that this will be accomplished only by appropriate efforts to control contaminating effects of PAD or the consequences of postsynaptic effects in motoneurons. The fact that descending projections via corticospinal, rubrospinal, reticulospinal and vestibulospinal pathways must exert their major actions through interneuronal networks has guided Lundberg's work for several years. His data are limited to the cat, but it would be surprising indeed if the facilitatory or inhibitory actions of descending pathways on reflex pathways to motoneurons and primary afferents did not also occur in primates and man. The work described on the convergence of primary afferent and descending pathways on interneurons represents but a summary of the most detailed data currently available on the subject of spinal interneuronal ac-

536

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tivities. All signs point to the fact that the voluminous literature on the motoneuron will soon be surpassed by a new and larger literature on the spinal interneuron. If this is not to represent the "yards of rubbish in the shelves of periodicals" that Horridge warns about, then it will be necessary for future investigators to approach the problem of interneuronal interactions with the same degree of caution and sophistication evidenced in the work of Lundberg. Horridge's shelves of periodicals are not the only repositories of a continuous input of "rubbish". Rubbish-generators are continually active and bombard our nervous system from all quarters. Mechanisms for controlling, shaping, enhancing and dissecting relevant inputs are crucial for information detection and processing. Recurrent and afferent or lateral inhibition as well as presynaptic inhibition are generally recognized as important processes for controlling redundancy and irrelevancy. The requirements for a central detection mechanism that must respond to sensory signals differing in intensity by several orders of magnitude are enormous indeed. Schmidt has provided data which bear on this problem in his analysis of the negative feedback character of presynaptic inhibitory generators. He has shown that the latter are adjusted to the level of the input. Schmidt's demonstration of the adaptive function of presynaptic inhibitory control systems, made possible through the employment of discrete physiological inputs suggests that similar properties of presynaptic inhibition will be found at other neuraxial levels when experimental designs incorporate features of physiological rather than electrical stimulation of inputs. Particularly impressive advances in the understanding of the organization of neuronal systems have been made when physiological studies have been combined with detailed anatomical investigations. When these are combined with ontogenetic and phylogenetic studies, the gain is even more noteworthy. Such a combination is illustrated in the report by Skoglund, who has extended earlier work on the development of spinal reflexes in the kitten to the analysis of peripheral and non-neuronal factors underlying the alterations in reflex patterns. Skoglund has presented electron microscopical and histochemical evidence which challenges the long established view that increases in axon diameter and myelination are the only events underlying the increase in conduction velocity of neurons during development. His demonstration of the nodalization process in immature nerve fibers calls attention to a factor that has escaped careful scrutiny. Skoglund has also examined the possible mechanisms underlying the paradoxical decrease in excitability of the monosynaptic pathway, postnatally. Since he maintains that this cannot be adequately explained by changes in neuronal properties, the possibility has been explored that changes in the ionic milieu may be responsible for the observed phenomenon. The hypothesis that in early stages of development, a high extracellular potassium concentration could account for the increased reflex excitability is novel and worthy of further consider-

SUMMATION

537

ation. It underscores the need for more quantitative data on changes in the ionic environment of the immature nervous system. New and emergent principles of operation of interneurons have been difficult to define in studies of the cerebrum, brain stem or cerebellum. For the most part interest has focused on characterizations of interneuronal organizations in different structures and on studies relating to the contribution of excitatory and inhibitory interneurons in complex activities. In this context, the papers presented on the cerebellum best illustrate the vigorous thrust that has been made in specifying the synaptic relations and functions of interneurons and Purkinje cells in this structure. Llinas has reviewed the basic features of cerebellar circuitry from a phylogenetic standpoint and Ito has examined the intrinsic organization of cerebellar nuclei in considerable detail. These, and the contribution of Larramendi, add to the relatively large volume of new information on the cerebellum which has been sparked by Eccles and his associates during the past four years. Llinas' phylogenetic approach has provided important complementary data to that obtained in the mammal, particularly in specifying consequences of the lack of basket cells in lower vertebrates. Morphophysiological differences in cerebellar interneuronal populations in amphibian, reptile and mammal show correlations with electrophysiological observations that can be extended to analyses of presynaptic relations of Purkinje cells. It is evident from these studies that the major evolutionary feature in the development of the mammalian cerebellum consists in the extraordinary increase in inhibitory interneuron elements. Llinas reminds us that increase in inhibitory functions may be a general feature of the evolution of other parts of the brain as well. Certainly, the important role that inhibition plays in many of the integrative activities in thalamus, as summarized by the present writer, or in hippocampus and neocortex, as discussed by Andersen, Spencer and Stefanis, amply supports this view long held by neurobiologists. Perhaps the most startling observation of recent years served as the point of departure for the studies described by Ito. This reviewer well recalls the day he first learned of Ito's findings on the general inhibitory function of Purkinje cells. Initial disbelief faded to mild skepticism and vanished with final acceptance. No one could have predicted this discovery—while today one wonders how he could have thought otherwise in view of the subsequent work of Ito and his associates. To say the least, the discovery of the inhibitory function of the Purkinje cell, the most elegantly appointed neuron in the brain, has dealt a mortal blow to the commutator theory that inhibitory activities are subserved by intercalated short-axoned interneurons. Examination of Ito's contribution reveals a good deal more than this. It is evident that the physiological and anatomical organization of small and large neurons in the cerebellar nuclei has been a most enigmatic problem. The studies presented, indicate that the cerebellar nuclei receive collaterals from both mossy and climbing fiber inputs. Of particular interest is Ito's finding

53S

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that synaptic terminals of climbing fibers on cerebellar nuclei neurons may produce different forms of EPSPs than the terminals of these fibers on Purkinje cells. This establishes that a single neuron may produce different PSPs depending upon the geometry and characteristics of its terminals. Shades of still another type of multiaction neuron! Clearly, however, the major contribution of Ito's presentation consists in the disclosure of integrating features of cerebellar nuclei neurons, which were presumably considered simply "relays" for cerebellar corticofugal activity arising in Purkinje cells. Although the picture of this integrative activity of cerebellar nuclei neurons has been painted in broad brush strokes by Ito, the details will surely be supplied in short order. These elements are tonically inhibited by Purkinje cells, but excited monosynaptically by collaterals of cerebellar afferents. The data-processing between cerebellar cortex and nuclei must be remarkable indeed and the mechanisms underlying this will undoubtedly serve as a Rosetta stone for the analysis of other complex neuronal interactions in the brain. Meanwhile, examination of the hieroglyphs that convey the information in the 'Stone' proceeds, as we have learned from the report by Larramendi, who has attempted to decipher the cryptic contours and profiles with the electron microscope. Larramendi has provided evidence based on Golgi studies and electron microscopy that he considers important as criteria for identification of synapses of different origin. These include characteristics of vesicle morphology and aggregation, characteristics of pre- and postsynaptic densities, axoplasmic density and other features. The data obtained have been translated into physiological expressions of "synaptic efficiency or strength," and the distribution of functional properties of synapses has been mapped in accordance with these criteria. A previous tour de force of this kind has not been attempted in any structure. It has already been noted above, in discussion of the report by Kandel, that attempts to identify the functional nature of synapses from morphology alone can be hazardous. Larramendi is not only prepared to effect such relations but to indicate the physiological potency of the synaptic actions from morphology. Were it not for the fact that a considerable body of physiological data has provided the necessary background for Larramendi's studies, it is a matter of some conjecture whether he would have survived the day of his presentation! Still, it is to his credit that he has moved so energetically and boldly in an area so pregnant with hope. And if his criteria are established as valid in other structures, the task will be all the more simple. But while Larramendi may be correct in the assumption that all spine synapses on cerebellar neurons are excitatory and all axo-somatic synapses inhibitory, obvious deficiencies exist in attempts to extrapolate this hypothesis to other structures and neuronal organizations. It is already apparent that the generalization is invalidated in the case of developing synaptic systems of the cerebral cortex as indicated below. The rapidity of accumulation of information on interneuronal interactions

SUMMATION

539

in spinal cord and cerebellum may be contrasted with the situation in the cerebrum and upper brain stem. Complexity of organization is but one problem; others are accessibility, identification and method of approach to interneurons at higher levels. Despite these problems, it should be recalled that a cerebral structure, the hippocampus, provided, in the studies of Andersen, Eccles and Lpyning, the first system for attempts to identify the morphological basis of inhibition in the mammalian brain. In that study it was inferred that the prolonged IPSP observed in many hippocampal pyramidal cells, following several modes of stimulation, could be accounted for by the operation of axo-somatic synapses effected by basket cells which were excited by collaterals of pyramidal neuron axons as well as by different pathways. This basket cell-inhibitory interneuron hypothesis has been extended by Andersen to consideration of both excitatory and inhibitory interneurons in the control of hippocampal output. Attempts have been made to identify these functionally different interneurons on the basis of their location and discharge characteristics. Those in the immediate vicinity of pyramidal neurons are considered inhibitory basket cells as noted above. In deep regions of the stratum oriens, smaller interneurons (in view of their predominant axo-dendritic relations with pyramidal neurons) are considered to be excitatory in function. The underlying generalization is basically similar to that encountered in Larramendi's report, namely that axo-somatic synapses are inhibitory, whereas most axo-dendritic synapses, particularly spine contacts, are excitatory. This reviewer has summarized recent electron microscopic and electrophysiological studies on the hippocampus of newborn kittens which challenges this view. In the early neonatal period, hippocampal pyramidal neurons are devoid of axo-somatic synapses, whereas axo-dendritic synapses are well-developed. Intracellular recording from hippocampal neurons in newborn kittens reveals IPSPs in response to fimbrial stimulation that have all the characteristics of IPSPs observed in adult animals. Evidently, then, there are alternatives to the hypothesis that axo-somatic synapses are exclusively concerned with the production of IPSPs in hippocampal neurons, at least in the immature brain. What is required is more of the detailed Golgi- and electron microscopic analysis, such as described by Blackstad. As yet it cannot be decided from immature hippocampal material which of the various types of interneurons described by Andersen are responsible for the observed IPSPs in newborn kitten hippocampus or neocortex. Blackstad has demonstrated important techniques that should permit identification and quantification of the nature and number of different elements in relation to the cell bodies and dendrites of hippocampal pyramidal neurons as well as the distribution of afferent terminals in different hippocampal sectors and the fascia dentata. Both Blackstad's and Larramendi's reports are in keeping with the spirit expressed by Maxwell, to the effect that electron microscopy of the brain has indeed

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come of age. But if it is not to die an untimely death, it must cease to be merely a means of extending the resolution of the light microscope and become an operational tool of the experimentalist, whether he be primarily anatomical or physiological by primary affiliation. Electron microscopy will be particularly suitable, as pointed out by Blackstad, for studies of interneuronal interrelations since these are generally not suited to examination by classical degeneration techniques. Andersen's studies of hippocampal neurons have detailed several features of archicortical synaptic pathways, particularly the role of potentiated axodendritic excitatory synapses in initiating spikes in dendrites which may either fail to invade the soma or propagate into the soma in an all-or-none fashion. The contribution of elements of the fascia dentata to this frequency potentiation has also been examined. Of interest for the analysis of interneuronal activities is Andersen's study of the manner in which repetitive synchronous discharges in pyramidal neurons may overwhelm the spike generating capability of collaterally activated inhibitory interneurons and thereby effectively eliminate their inhibitory feedback upon pyramidal neurons. Spencer indicates in his study of focal epileptogenic discharges in the hippocampus that recurrent inhibition operates to limit their spread, since IPSPs are prominent in pyramidal neurons outside the focus but absent or weak in neurons in or close to the discharging focus. Andersen has also described an additional mechanism of feed-forward inhibition whereby afferents may directly excite inhibitory neurons in the hippocampus. However, intracellular recording in hippocampal inhibitory neurons has not as yet been carried out to reveal the monosynaptic nature of the afferent input to these elements. If the problem of specifying the operation of interneurons in the hippocampus, a relatively "simple" type of cerebral cortex, has been far more complicated than might be inferred from its neurohistology, consider the problem presented by the neocortex! Spartan courage is required for the analysis of its interneuronal interrelations, to say the least. An Athenian's technical skill was all the more appreciated. Notwithstanding imaginative attempts to define neocortical circuitry, the fact remains, as Stefanis indicates, that identification of interneuronal elements involved in a particular neocortical pathway has not as yet been feasible. Still, Stefanis has provided a picture of the operation of recurrent facilitatory and inhibitory effects of pyramidal tract stimulation which clearly establishes the role of interneurons activated by recurrent as well as afferent pathways in the modulation of efferent discharges of the neocortex. A relationship has been shown between recurrent facilitatory efforts and the position of a pyramidal neuron in a receptive field. Such a mechanism supplements recurrent inhibition by enhancing contrast, as Stefanis asserts. In respect to the possible location of inhibitory synapses on pyramidal neurons, the data presented would suggest that part of the inhibitory input is widely distributed on dendrites as inferred from effects of CI" injection on early and late phases of IPSPs.

SUMMATION

541

Stefanis has also discussed the difficulty of relating the long-duration IPSPs observed in neocortical neurons to the repetitive discharges of inhibitory neurons. This problem was also raised during the Symposium in connection with the discrepancy between the long duration of IPSPs in hippocampal pyramidal neuron and the relatively brief discharge of presumed inhibitory interneurons. In this context, it may be noted that bursts of spikes from nearby neurons may be recorded during the IPSP of thalamic neurons partially traumatized by the impalement. However, temporal relationships between spike burst and prolonged IPSPs are not generally concordant, despite some claims to the contrary. Thus, the problem discussed by Stefanis remains poorly defined. What seems obvious is that inhibitory neurons may operate in different ways in different structures and organizations. The complexity of interneuronal interactions demonstrable in reciprocal operations of flexors and extensors and in the control of input-output relations in cerebellum, hippocampus and neocortex probably attains its greatest expression in those organizations of the upper brain stem which subserve generalized control of neuronal activity in several related structures. Studies on intrathalamic, cortical, striatal and reticular organizations involved in these activities have been summarized by the present writer. The general synaptic mechanisms which underlie synchronization and desynchronization of neural discharges in thalamus have been described with particular emphasis on the important role of inhibitory interneurons in the subcortical mechanisms of both EEG synchronization and reticulocortical activation. Attention has also been directed to the problem of how different synaptic organizations impress different PSP patterns in neurons of different structures related to nonspecific thalamic projection systems. Little can be added to this, except to point out obvious deficiencies of the story summarized. Perhaps the greatest deficiency is the lack of a suitable morphological substrate for effecting the widespread EPSP-IPSP sequences observed in thalamic neurons during evoked synchronization, as well as for the synaptic effects underlying desynchronization of neuronal discharges. Inhibition of inhibition and increase in excitatory synaptic drives in many cells has been proposed to account for the transition from EPSP-IPSP sequences to sustained EPSPs in thalamic neurons. But the possible involvement of presynaptic inhibitory processes, such as described in the studies of Lundberg, or of a variety of synaptic interactions of long duration, such as illustrated by Tauc and Kandel, could also account for the changes in PSP patterns produced by changes in stimulus frequency. Clearly, there are sufficient interneurons and interneuronal pathways in the thalamus and brain stem to satiate the appetite of any circuit designer, if circuits are what is required. This reviewer rather doubts that any useful purpose is served by such blueprinting in the absence of adequate neurohistological data. For little is known about the intrinsic cytoarchitecture of thalamus, caudate and brain stem and even less is known concerning the na-

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ture and composition of the trajectories from thalamic nonspecific nuclei to other parts of the thalamus or caudate, cortex and brain stem reticular regions. That multisynaptic pathways of complexly organized inhibitory and excitatory interneurons are involved in the overt effects observed during EEG synchronization and desynchronization seems self-evident. But the operation of oligosynaptic, small fiber pathways is by no means excluded. Combinations of both types of systems are most likely involved, but their detailed examination poses a serious challenge to present microphysiological and neuroanatomical techniques. Consider the many problems discussed by Kandel in his attempt to specify the physiological and anatomical corrections of a single identifiable interneuron (Interneuron I) in Aplysia—and imagine what looms ahead in attempts to unravel the skeins of functional and anatomical relations of cortical, thalamic and brain stem interneurons! This symposium volume has revealed what amounts to the first concentrated effort to define some of the ground rules for this intriguing game. Its raison d'être may be sought primarily in attempts to elucidate properties and principles of interneurons, not in efforts to construct circuit diagrams, however urgent this may seem. Some hypotheses pertaining to interneurons have been quietly laid to rest, others flogged unmercifully even thereafter. Emergent concepts are now poised for the challenge of future rigorous examination. The integrative activities of interneurons have been demonstrated in this volume to be the driving force for the complex machinery whose output is expressed in behavior. Microdissection of the machinery is well under way, as the contributions attest. But the nightmare of synthesizing the details for the understanding of the macrocosm of behavior awaits the dreamer. For this, again, is another story, open-ended and with but little plot. It might well commence with the phrase, "In the Beginning was the Interneuron . .

NAME INDEX 100, 160, 161, 167, 168, 185, 221, 233, 236, 258, 268, 276, 289-290, 295, 297, 415, 420, 422, 438, 460-462, 472, 539 Eccles, R. M 133-135, 268 Ekholm, J 133, 135 Eklund, G 29 Enriques, P 72 Epstein, R 50 Estable, C 345 Evoy, W. H 30

Adey, W. R 260, 281-284, 300, 301 Adinolfi, A 477 Albert, A 201 Alksne, J. F 409, 410 Amassian, V. E 127, 387 Andersen, P 224-227, 297, 303, 304, 415-432, 435, 438, 447, 459-463, 472, 490, 491, 493, 494, 537, 539, 540 Andersson, 490 Ariens Kappers, C. U 179 Arvanitaki, A 71 Asada, J 413 Atwood, H. L 23, 34

F Fatt, P 160, 167, 276 Fedina, L 233 Fetz, E. E 185 Fields, H. L 24, 25 Fink, R. P 405 Forbes, A 100, 258 Fox, C. A 290, 296, 297 Frank, K 168 Frazier, W. T 494 Fuortes, M. G. F. . .168, 220, 221, 257-261 Fuxe, K 284

B Barlow, H. B Bennett, M. V. L Bergmans, J Berthold, C.-H 138, Birks, R Blackstad, T. W 391-413, 533, 539, Blankenship, J. E 87, 107, Bodian, D Bottazzi, F Brazier, M. A. B 105, 224, 259-261, 411, Brinley, F. J., Jr Brodal, A 193, Brown, T. S Bullock, T. H 31-34, 160, 350, 369, 384, Burke, L Burke,

126 413 233 153 412 540 108 147 72

G Gedulding, D. S Gerasimov, V. D Gerschenfeld, H. M Goldstein, M Golgi, C Graham Brown, T Grampp, W Granit, R Gray, E. G Gray, J Grillner, S Gross, G. N

533 415 309 519 528 62 233

Cajal, see Ramón y Cajal, S. Chiarandini, D. J 89, 101, 108 Clemente, C. D 153, 203, 226, 411, 463, 476 Coggeshall, R. E 72, 80, 85, 104-107 Cohen, B 468 Colonnier, M 517, 519 Conradi, S 145, 147, 153 Coombs, J. S 185, 245 Corda, M 29 Curtis, D. R 245

Dahlström, A Dichter, M Dow, R. S

101, 102 101 85, 89, 108 77, 81 257, 331 260 261 259, 260, 301 290, 295, 408 31, 32 125, 256 415-432

H Hagbarth, K.-E 133 Hagiwara, S 101-105 Hamori, J 290, 296, 297 Heimer, L 405 Hemdon, R. M 296 Hillman, D. E 302 Hongo, T 125, 254, 279 Horridge, G. A 1-20, 31, 33, 160, 260, 261, 350, 384, 527, 528, 535, 536 Hubel, D. H 522 Hunt, C. C 168, 412 Hursh, J. B 132, 135 Huttenlocher, P. R 155

284 433 333

I Ikeda, K 21, 22 I to, M 289, 290, 309-325, 520, 521, 537, 538

E Eccles, J. C. 543

THE

544

INTERNEURON

J Jankowska, E Junge, D

254, 279 101, 102

K Kandel, E. R 13, 39, 71-101, 103, 104-109, 154, 191, 221-225, 256, 257, 259, 261, 262, 282, 300, 303, 304, 412, 413, 415, 459-461, 463, 494, 529, 530-532, 538, 541, 542 Katz, B 224, 227, 412 Kehoe, J. S 44 Kennedy, D 21-36 57, 61, 107, 126, 127, 221, 223, 259, 261, 262, 282-284, 300, 304, 522-524, 528-530 Kerr, F. W. L 171 Kesselman, M 72 Kilham, L 284 Koketsu, K 160, 167, 276 Kolmodin, G. M 168, 236-238 Kostyuk, P. J 101, 168, 221 Kravitz, E. A 282 Kuno, M 168 Kupfermann, 1 72, 77, 103, 105, 106

L Landgren, S 245 Langley, J. N 210 Larramendi, L. M. H 226, 282 283, 289-305, 346, 409, 411, 533, 537-539 Lenhossek, M 162 Liu, C.-N 155, 156, 193-203, 412 Llinäs, R. R 227, 255, 256, 284, 301, 302, 324, 329-346, 462, 463, 501, 537 Lloyd, D. P. C 258, 280 L0mo, T 415-432, 490 Lorente de No, R 1, 100, 309, 416, 419 L0yning, Y 415, 420, 422, 438, 539 Lund, S 125, 256 Lundberg, A 125, 126, 156, 168, 185, 193, 198, 220, 222, 223, 227, 231-262, 268, 279, 458, 460, 490, 494, 534-536, 541 Lux, H. D 494

M Maekawa, K 471, 478, 481 Magoun, H. W 256, 485 Maisky, V. A 101 Malcolm, J. L 133 Marco, L. A 519 Margolis, G 284 Marie, P 181 Matsunami, K 520 Matsushita, M 203 Maxwell, D. S 408-411, 539 Maynard, D. M 56-68, 105, 107, 223, 261, 305, 387, 522, 529, 531, 532 McCance, R. A 150

McCouch, G. P McCulloch, W. S Mellgren, S. 1 Mellström, A Melzack, R Meves, H Miledi, R Moruzzi, G Mountcastle, V. B Mugnaini, E Müller, J

412 1 408 145 184 101 412 485 522 410 16

N Nafstad, P. H. J Naka, K.-1 Nelson, P. G Nyberg-Hansen, R

394, 405 132-135, 152, 153 412 256 O

Okomoto, Otsuka, M Oyster, C. W

203 282 126

P Palay, S. L 290, 296 Pappas, G. D 413 Perkel, D. L 349-387 Phillips, C. G 497 Pitts, W 1 Pollen, D. A 494 Potter, D. D 282 Prestige, M. C 165 Preston, 522 Prince, D. A 438 Pringle, J. W. S 25 Purpura, D. P 153-155, 223, 255, 283, 301, 302, 324, 325, 346, 407, 408, 413, 447-460, 467-495, 520-523, 527-542

R Rail, W 301 171, 180, 227 Ralston, H. J Ramón y Cajal, S 1, 3, 100, 159-161, 165, 170, 176, 179, 183, 185, 186, 191, 257, 283, 289, 290, 296, 297, 329, 330, 345, 416, 419, 477, 481 Rasmussen, G. L 155 Renshaw, B 160 Retzius, A. A 3 Rexed, B 162, 242, 245, 267, 280-283 Romero, C 135 Rouse, M. E 519

S Sasaki, K Schadé, J. P Scharrer, B

462 145 72

NAME Scheibel, A. B 34, 126, 159-193, 203, 204, 223, 225, 226, 280283, 289, 296, 297, 303, 325, 345, 346, 463, 479, 487, 490, 521, 528, 532-535 Scheibel, M. E 159-193, 289, 296, 297, 481, 487, 528, 532-535 Schlag, J 488-490, 494, 519, 520 Schmidt, R. F 209-227, 281, 323, 324, 534, 536 Schulman, J 381 Schwieler, G 143 Sears, T. A 472 Segundo, J. P . . 1 0 5 , 107, 127, 223, 304, 349-388, 532 Sherrington, C. S 32, 257-260, 276 Shofer, R. J 468 Sholl, D. A 159 Sidman, R 282 Skoglund, C. R 168, 236-238 Skoglund, S 131153, 155, 156, 303, 323, 412, 447, 536 Spencer, W. A 260, 415, 432-442, 447, 461, 494, 537, 540 Sprague, J. M 197 Stefani, E 89, 101 Stefanis, C 442-447, 463, 494, 495, 497-520, 537, 540, 541 Stein, G 528 Stelzner, D 155 Strumwasser, F 71 Sveen, O 415^32 Szentagothai, J 164, 180, 183, 186, 201, 227, 290, 296, 297, 310

Taue, L 3756, 61, 67, 71, 85, 101, 105-108, 226, 227, 255, 299, 528, 530-532, 541 Terzuolo, C. A 256

545

INDEX Thomas, R. C Torvik, A Toyama, K

168 309 520

u Uno, M

520

V Van Harreveld, A Villabianca, J von Euler, C von Holst, E Vyklicky, L

145, 222 519 29 10, 31, 32 248

w Wachtel, H 87, 93, 107, 108, 261 Wakabayashi, T 379 Walberg, F 226, 409, 410 Waldeyer, H 169, 172, 183 Wall, P. D 168, 171, 180, 181, 183-185, 204, 233, 279, 283 Waller, H. J 127, 387 Westrum, L. E 412 White, L. E., Jr 409 Widdowson, E. M 150 Wiersma, C. A. G 12, 21-23, 32, 34, 108, 113-127, 261, 349, 506, 528, 529 Wiesel, T. N 522 Wilder, B. J 438 Willis, J . C 168,238 Willis, W. D. . . 133, 134, 155, 168, 184, 204, 222, 223, 227, 238, 267-281, 534, 535 Willows, A. O. D 23 Wilson, D. M 8, 25, 31, 32 Wilson, V. J 132, 134, 135, 168

SUBJECT INDEX A

198, 209-221, 233, 236, 255, 260, 276, 284, 296, 311-325, 333-341, 344, 346, 412, 416, 442, 449-460, 536, 539 Catecholamines, 39, 77 Cells A, 64-68, 269, 273-275, 280, 531 amacrine, 14 B, 64-68, 269 bag, 72, 75-81, 100, 101, 103-107, 531 basket, 162, 284, 289-291, 293, 295-299, 302-305, 319, 330, 331, 333, 335, 337, 345, 346, 411, 416, 418-420, 422, 429, 431, 460-462, 518, 537, 539 Betz, 474 C, 269-272, 275, 280 CILDA, 41, 43, 53 Clarke's column, 160, 203 commissural, 278 Cooper-Sherrington, 160 D, 39, 86, 87, 92, 107, 221, 223, 268 Deiters neurons, 311, 314, 315, 317-319, 322, 324 D-H, 92, 107 DINHI, 40 DILDA, 39, 40, 43, 54, 86, 108 DINT, 86 follower, 83-97, 99, 100, 108, 494, 530 giant, 4, 41, 45-47, 50, 53, 57, 101, 108, 376 Golgi, 289, 305, 319, 333, 341-345 granule, 283, 284, 289, 314, 318, 319, 329-331, 341, 345, 346, 400, 402, 428432 H, 39, 86, 87, 89, 92, 107 HILDA, 43-45, 54, 55 integrative, 81, 98-101 long-axoned, 167, 192, 281 marginal, of Waldeyer, 169, 172, 183, 184 mesencephalic, 483 neurosecretory, 72, 80, 81, 94, 98, 105 Purkinje, 174, 179, 289, 291, 293, 295299, 301-305, 309, 311, 313-322, 324, 325, 329, 330, 333, 335, 337-341, 344346, 411, 461-463, 527, 537, 538 pyramidal, 153, 179, 407, 415-463, 474, 497-499, 502-507, 509-517, 527, 537, 539-541 Renshaw, 100, 159-204, 223, 236, 245, 269, 276-281, 317, 507, 513, 517, 534, 535 s, 64-68 Schwann, 135-140, 142, 412, 413 short-axoned, 159-163, 167, 192, 203, 259, 281, 289, 330, 345, 395, 528, 533, 534, 537 stellate, 161, 284, 289, 291, 293, 295-299, 319, 333, 337, 341, 345, 346, 411, 517, 521 white, 72, 75, 80, 81, 83, 94, 100, 101, 105, 107

Accomodation, 379 Acetylcholine, 38-40, 43-45, 54, 55, 85-87, 89, 91-93, 97, 104, 105, 108, 412, 442445, 460, 502-504, 530 Acetylcholinesterase, 45, 55, 408 ACh, see Acetylcholine Activation, 474 Adaptation, 15, 16 Afterdischarge, 133, 260, 262 Afterpotential, 221, 358, 379, 498, 524 Alligator, 330, 331, 333, 338, 341, 344, 345 Alveus, 415-417, 419, 421, 444 Amphibia, 537 see also Frog; Toad Analyzer function, 349-388 Anticholinesterase, 105 Antidromic activation, 30, 105, 153, 155, 267, 277, 278, 301, 311, 312, 319, 339341, 346, 415-417, 423, 425, 428, 431, 434, 436, 454, 461, 474, 491, 497-507, 512, 514, 517, 519, 535 Antidromic blocking, 504 Antidromic depression, 41 Antidromic inhibition, 160, 162, 166 Aplysia, 23, 33, 37, 39, 41, 44, 45, 48, 49, 53, 56, 57, 71-109, 261, 282, 350, 351, 354, 355, 365, 376, 378, 447, 494, 529532, 542 Arousal, 6, 7, 483 Arthropoda, 3, 11, 12, 24, 25, 33, 57, 529 Astrocyte, 171 Astroglia, 400, 402, 404, 407, 410, 412 Atropine, 44, 45 Auditory system, 2, 15, 32 Autocorrelogram, 355 Axon hillock, 192, 296, 301, 345 Axoplasm, 293, 300, 303, 394, 538

B Bat, 2 Behavior, 1-20, 22, 23, 25, 31, 64, 71, 257, 433, 483, 516, 527, 530 Bielchowsky stain, 164 Biphasic postsynaptic potential, 41, 45, 47 Biphasic transmission processes, 37, 41-47 Birds, 333 see also Pigeon Bivalent cations, see Divalent cations BPSP, see Biphasic postsynaptic potential Brain stem, 125, 159, 251, 255, 309, 322, 323, 386, 467, 481, 483, 485, 488, 529, 537, 539, 541, 542

c Calcium, 38, 40, 41, 47, 102, 150, 152, 153 Cat, 6, 126, 131-156, 161, 164, 193, 197, 547

548

THE

INTERNEURON

Central command, 24-30 Cerebellum, 29, 131, 132, 162, 174, 179, 189, 251, 283, 284, 289-305, 309-325, 329-346, 410, 432, 461, 462, 520, 537, 539, 541 granular layer, 283, 330, 331 molecular layer, 291, 295, 296, 299, 330, 331, 335, 338, 346, 397 Cerveau isole, 488 Chloride, 39, 44, 45, 89, 93, 101, 104, 108, 227, 313, 319, 413, 416, 461, 462, 494, 499, 504, 506, 540 Chordata, 57 Cobalt, 102 Cochlea, 5 Cockroach, 10 Command channels, 23, 24 Command elements, 22, 26, 30, 31, 34, 61, 62, 64, 67, 100, 365, 529, 530 Computer simulation, 353-355, 361, 365, 366, 368, 375, 385, 386 Connectivity, 2, 3, 17, 33, 37, 55, 56, 64, 159-204, 282, 391, 405-407, 522 Convergence, 231-257, 416, 419, 420, 423, 424, 460, 535 Cortex, 3, 21, 75, 126, 391, 518, 519, 542 cerebellar, 34, 174, 289-305, 309-325, 329-346, 538 cerebral, 153, 161, 179, 189, 251, 346, 409, 410, 412, 463, 467, 468, 470, 471, 473-483, 488-490, 494, 497-524, 537540 hippocampal, 415-463 motor, 21, 471, 474, 478, 497-499, 505, 507, 508 sensorimotor, 520 Cortical evoked potential, 489, 490 Crab, 6, 8-11, 17, 62, 114, 115 see also Limulus; Podophthalmus; Scilla serrata Crayfish, 12, 22, 23, 25, 31, 57, 100, 113, 115, 116, 118-121, 123-127, 261, 282, 353, 447, 522, 529, 530 Cricket, 23 Crosscorrelation, 16, 17, 360, 365, 383 Crustacea, 22, 57, 101, 113-127, 224, 349, 522, 529, 530 see also Crab; Crayfish; Lobster; Stomatopoda Cryptophallus aspersa, 41 Curare, 44, 45, 87, 90, 97 Cutaneous terminal field, 180-183

D Decerebrate preparation, 131, 132, 135, 156, 253, 260, 319, 322 Degeneration studies, 391-413, 433, 434, 540 Wallerian, 226, 405-407 Disfacilitation, 241 Disinhibition, 279, 299, 318, 319, 321, 322,

428, 482, 485, 488, 517 Divalent cations, 40, 101 see also Calcium, Cobalt Magnesium Divergence, 419 DNA, 13 DOPA (1 -3-4-dihydroxyphenylalanine), 233, 236, 242, 245, 255, 261, 535 Dopamine, 39, 43, 77, 81 Dorsal root potential, 221, 232, 233 Dorsal root reflex, 222, 223

E Earthworm, 10 EEG desynchronization, 467-488, 494, 542 EEG synchronization, 467-488, 541, 542 Electric fish, 101, 102 Electrical junction, 37, 64, 79, 80, 106, 107, 225, 534 Electrical synapse, 41, 47, 53, 103 Electrotonic interaction, 3, 103, 225, 413, 528, 531, 532, 535 Electrotonic spread, 2, 41, 107, 252, 462 Elephant, 6 Encephale isole, 491 Endogenous rhythms, 262 Entorhinal area, 394, 405, 415, 416, 421, 428, 429, 455 Epileptogenic discharge, 540 Episynaptic transmission processes, 37, 4754, 56, 531 EPSP, see Excitatory postsynaptic potential Eserine, 105 Evolution, interneuronal, 309-346 Excitation, 2, 9, 11, 13, 18, 28, 30, 37-70, 93, 97-100, 106, 113, 116, 121-126, 231-255, 257, 261, 262, 267, 269, 271, 272, 276, 278, 280, 282, 289, 296-299, 301, 322, 323, 325, 335, 415-463, 473, 494, 497, 521, 528, 530, 532, 537, 539, 540 recurrent, 435, 436, 441 Excitatory field, 113, 114, 116 Excitatory postsynaptic potential, 30, 37, 41, 47-50, 53, 64, 83, 85, 86, 89-93, 103, 104, 150, 186, 234-242, 245, 249, 251256, 269, 273, 275, 276, 315-320, 325, 337, 354-356, 358-361, 365-381, 386, 416, 425, 430, 431, 436, 441, 455, 456, 458, 460, 468-470, 472-481, 483-486, 488, 489, 491-493, 499, 504, 505, 516, 521-523, 538, 541 Excited cluster, 14, 16 Excited state, 116, 118 Extracellular space, 55, 401

F Facilitation, 26, 50, 51, 53, 103, 120, 123, 133-135, 143, 152, 222, 246, 249, 250, 254, 257, 258, 319, 322, 323, 420, 430, 431, 460, 485 presynaptic, 41, 48, 50, 51

SUBJECT Facilitation (cont.) recurrent, 279, 280, 431, 432, 435, 436, 441, 502, 504, 505 Fascia dentata, 394, 397, 401, 402, 405, 429, 430, 445, 539, 540 Feedback, 12, 14, 18, 19, 29, 33, 64, 66, 99, 119, 160, 166, 168, 218, 220, 356, 497, 505, 540 negative, 26, 28, 210, 219, 536 positive, 25, 65, 68, 243 proprioceptive, 9, 24-30 Fibers climbing, 179, 289, 291, 293, 295-297, 299, 301-303, 310, 311, 314-317, 319, 322, 323, 329, 330, 346, 537, 538 fusimotor, 28, 29 giant, 10, 15 Mauthner, 4 mossy, 289, 310, 314, 317-320, 322, 323, 329-331, 337, 341, 344, 407, 431, 451, 537 motor, 62, 108, 121, 122, 124 movement, 118, 119, 124-126 optokinetic, 121, 122 parallel, 291, 293, 295-301, 303, 329-337, 339, 341, 344, 346, 411 skeletomotor, 28, 29 space control, 119-121 sustaining, 116, 118, 119, 122 Field potential, 437, 438 Fimbria, 153, 416, 421, 422, 425, 445, 454456, 458 Fish, 33, 101, 289 see also Electric fish; Gymnarchus niloticus Flexor reflex afferents, 232-234, 236-238, 240-242, 245, 246, 248, 251, 253-255, 261 Flip-flop control, 68, 69 Fly, 31 Fornix, 433-436, 460 FRA, see Flexor reflex afferents Frequency potentiation, 425 Frog, 114, 123, 193, 195, 196, 330, 333-339, 341, 344

G GABA (Gamma aminobutyric acid), 282, 443, 502, 504 Gastropoda, 23, 41 see also Snail Glia, 75, 77, 153, 171, 391, 401, 410-412, 533 Glial-neuronal relations, 171, 411, 451, 533 Glomerulus, cerebellar, 333, 341-344, 346 Glutamic acid, 242, 442, 445, 502, 514, 516 Glycogen, 400, 402 Golgi technique, 3, 159-204, 281, 391, 395407, 409, 416, 417, 487, 533, 538 Golgi/Kopsch technique, 416 Guinea pig, 408 Gymnarchus niloticus, 101

INDEX

549

H Habituation, 16, 18, 41, 119, 120, 124 Hexamethonium, 39, 107 Hexapoda, 32 Hippocampus, 72, 153, 154, 189, 391-407, 415-463, 494, 537, 539-541 molecular layer, 397, 401, 402, 405 Homocysteic acid, 442, 514 Hypothalamus, 72

I ILD, see Inhibition, long-lasting Inhibition, 2, 7, 10, 13, 22, 30, 31, 37-70, 93, 94, 97-100, 104, 108, 109, 113, 116, 118, 119, 121-126, 133-135, 143, 145, 152, 159, 165-168, 186, 216, 219, 220, 222, 231-259, 261, 262, 267, 269, 272, 278, 279, 282, 289, 291, 296-299, 301, 304, 309, 319, 320, 322, 323-325, 337, 341-346, 381, 415-463, 473, 474, 485, 493, 494, 502, 503, 517-522, 528, 530537, 539-541 forward, 428 long-lasting, 41-45, 51, 52, 54-56 postsynaptic, 248 presynaptic, 37, 48-54, 191, 210, 216-220, 226, 227, 233, 252, 255, 534-536, 541 recurrent, 152, 168, 220, 223, 236, 276, 277, 279, 280, 322, 415, 422, 429, 436, 438-441, 447, 459-461, 491, 502-505, 519, 536, 540 Inhibitory control, 209-227 Inhibitory postsynaptic potential, 31, 42, 47, 48, 64, 65, 83, 85, 87, 89-92, 97,103,104, 134, 223, 234, 238, 240-245, 249, 251256, 269, 271, 275-277, 312-316, 318320, 323-325, 337, 338, 341, 355-359, 379, 381, 415, 416, 422, 424, 428, 429, 436-438, 447, 449, 455-463, 468-470, 472-474, 476, 477, 483-486, 488, 489, 491-495, 499-501, 504, 505, 516, 520522, 539-541 Initial segment, 153, 192, 345 Input-output relation (transformation), 385, 386, 528, 532 Insects, 11, 33 see also Cockroach; Cricket; Fly; Hexapoda; Locust Integration, 100, 114, 122, 123, 257-260, 322, 376, 384, 385, 527 period, 366, 376 Interictal spikes, 433-437, 447 Interval, 351, 353-361, 365-372, 377, 383, 387 histogram, 351, 353-355, 358-360, 368, 369, 371, 372, 377, 378, 383 interspike, 349-388, 508 variability, 357, 361 IPSP, see Inhibitory postsynaptic potential Invertebrates, 8, 12-14, 21, 497, 529, 532

THE

550

INTERNEURON

see also Arfhropoda; Crustacea; Earthworm; Gastropoda; Insects; Jellyfish; Mollusca

J Jellyfish, 1

K Klüver stain, 164

L Learning, 6, 11 Lignocaine, 28 Limbic system, 72 Limulus, 3 Lithium, 50 Lobster, 57-61, 114-116, 118-122, 125, 126, 261, 522 see also Panulirus argus Localization, 267-284 Locomotion, 64 Locust, 8,17, 23, 25, 31

M Magnesium, 38, 40, 41, 47, 104 Mammals, 6, 21, 28, 32, 72, 113, 125, 131, 153, 171, 221, 333, 365, 476, 482, 529, 530, 537, 539 see also Bat; Cat; Elephant; Guinea pig; Mouse; Opossum, Primates; Rabbit; Rat Mechanoreceptor, 24,114, 210, 211, 216-220, 253, 269, 529 Memory, 19 Methylene blue technique, 3 Microglia, 410 Mitochondria, 137, 140, 142, 393, 394 Mollusca, 13, 23, 30, 37, 54, 55, 57, 105 see also Aplysia; Gastropoda; Octopus; Squid; Tritonia Monkey, 161, 193, 529 Motoneuron, 5, 12, 16, 18, 22-31, 33, 34, 108, 125, 147, 152, 153, 155, 160, 161, 164-166, 168, 172, 177, 192-195, 203, 223, 225, 231-236, 242, 245-249, 254256, 259, 261, 277, 279, 280, 282, 317, 31ä, 497, 527, 528, 533-536 Mouse, 161, 291, 296, 297, 302, 346 MRO, see Muscle receptor organ Multifiber interneuron, 323 Muscle receptor organ, 24-30, 133, 135, 191, 530 Myelin sheath, 136-139, 143, 224, 225, 404, 412

N Nauta technique, 193-203, 412 Neocortex, 153-155, 189, 346, 438, 449-460, 463, 476, 488, 517, 536, 537, 539-541

Neonate, 131-135, 139-145, 150, 153-156, 171, 179, 346, 449, 454-458, 460, 539 Neural code, 33 Neurofilaments, 293, 300, 303, 346 Neuromuscular junction, 55, 105, 224, 412, 413 Neuropile, 3, 15, 30, 57, 62, 75, 81, 159, 160, 168, 171, 176, 178-180, 182-184, 186, 189-191, 195, 203, 225, 226, 283. 391, 449, 523, 531, 533-535 Neurosecretory granules, 72, 77, 80 Neurotubules, 293, 300, 302 Nissl body, 408, 417 Nissl stain, 193, 281 Nodalization, 138, 143 Node of Ranvier, 136,139 Nucleus accumbens septi, 476 caudate, 475-480, 483, 486, 488, 489, 493, 541, 542 Clarke's, 197, 201-203 cerebellar, 309-325, 520, 537, 538 cuneate, 224-226 dentate, see n. lateralis, cerebellar fastigii, 309, 310, 313, 320, 323 gracilis, 226 inferior olivary, 315-317, 410 intermediate, of Cajal, 176, 189, 245 internal basal, of Cajal, 185 interpositus, 309-313, 316, 320-323 intralaminar, 490 lätsrälis cerebellar, 309-313, 316, 319-324, 397, 429-431, 445 cervical, 197, 198, 201, 203 mammillary, 415 medial, 490 pontine, 317, 319, 320, 323 red, 309, 311, 312, 316, 319-323, 481, 520 reticular, 317, 319, 320, 360, 387 septal, 415, 476 thalamic intralaminar, 467-495 ventralis anterior, 468, 476,477, 486 lateralis, 309, 311, 312, 320, 468, 470, 471, 476, 477, 485, 486, 489, 491, 494, 511, 517, 519-522 ventrobasal, 471, 472, 491, 492, 499, 500, 510 vestibular, 309, 320, 323 Deiters', 309, 313, 319, 322, 323

o Occlusion, 257 Octopus, 6 Oligodendrogliocyte, 404 Ommatidia, 3,118,119, 124 Opossum, 193 Optokinetic response, 1, 9, 17, 126 Organization, functional, 209-227, 231-262, 281, 329-346 Output control, 21-36

SUBJECT

P Pacemaker, 33, 64, 65, 81, 101, 102, 262, 354-360, 379 Pacinian corpuscle, 211, 220, 221, 223, 224 PAD, see Primary afferent depolarization Panidirus argus, 57 Parabolic burster, 57 Pattern abstraction, 6, 7, 12, 14-16, 18, 19, 21, 31, 34, 67 Penicillin, 433, 436, 441 Pentobarbital, 240, 311, 321, 416, 491 Perforant path, 395, 416, 429, 430 Pigeon, 114, 330 Plasticity, 11 Podophthalmus, 114 Poisson, 375, 387, 388 Post-activation facilitation, see Post-tetanic potentiation Postictal depression, 442, 443, 445 Postsynaptic density, 293, 300, 406, 538 Postsynaptic potential, 48, 53, 59, 61, 64, 65, 77, 79, 85, 89, 90, 92, 93, 96, 97, 99, 267, 278, 330, 351, 352, 354, 374, 378-385, 468, 474-481, 483, 486-490, 506, 522, 538 Post-tetanic potentiation, 37, 41, 50, 134, 135, 143, 256, 343, 416, 424, 483 Potassium, 40, 44, 45, 47, 101, 150-154, 416 Prepotentials, 105, 457, 481 Presynaptic density, 293, 300, 538 Presynaptic field patterns, 178-191 Primary afferent depolarization, 190-192, 216-223, 225, 227, 233, 236-238, 245, 246, 248, 251, 533-535 Primary afferents, 178-204, 232-262, 278 Primates, 6, 535 see also Monkey Procambarus, 355, 381 Propionate, 413 Proprioception, 9, 24-32, 126, 152, 191, 279, 529 Proprioceptors, 8, 25, 26, 191, 529, 530 Prostigmine, 103 PSP, see Postsynaptic potential

R Rabbit, 114, 126, 416 Range fractionation, 15 Rat, 6, 150, 155, 161, 193, 276, 408, 412 Reafference, 24, 119 Receptive field, 4, 5, 7, 8, 12-14, 19, 23, 55, 109, 113, 114, 125, 127, 220, 227, 505, 529 540 Receptor, 8, 10, 14, 25, 28, 45, 53-55, 85, 89, 92, 93, 96, 99, 104, 105, 120, 123, 125, 132, 133, 135, 143, 209-211, 213223, 227, 278, 282, 430, 530 Recruiting response, 467-475, 480, 483, 485 Reflex, 6, 8, 10, 11, 25, 26, 29, 30, 100, 125, 152, 186, 220-224, 231, 233, 246-255, 257-260, 267, 280, 318, 344, 535, 536

INDEX

551

maturation, 131-156 Refractory period, 359, 360, 366, 376, 503 Regulative mechanisms, 113-127 Reinforcement, 11 Renshaw phenomena, 159, 160, 162, 164, 167,168,193, 533-535 Reptiles, 333, 537 see also Alligator Respiration control, 28, 64, 126, 530 Reticular formation, 7, 126, 159, 179, 196, 248, 256, 280, 309, 312, 320, 323, 386, 387,485, 495 Reticulocortical activation, 467, 541 Retina, 3, 5, 116, 119, 122, 124 Reverberation, 259-262, 323 Rexed's laminae, 162-204, 242, 245, 267, 283 Rigidity, decerebrate, 131, 132, 135, 319

s Shafer collateral, 459, 461 Scilla serrata, 62, 531 Seizure, 433, 439, 442, 447, 540 Sensory fields, 113, 114 Serotonin, 39, 81, 502, 514 Slow wave activity, 442-445, 499 Snail, 33, 41, 101 see also Cryptophallus aspersa Sodium, 39, 47, 50, 87, 93, 101, 102, 104, 108, 150, 153, 416, 506 Spike train, 349-388, 519 form, 353-355, 358, 361, 363, 364, 375 pattern, 353, 366, 368, 371, 372 span, 353, 366, 368, 371, 372, 376, 377, 386 timing, 352, 353, 366, 368, 369, 374-376, 383, 386, 387 Spinal cord, 32, 48, 49, 125, 131-156, 159204, 209-227, 231-262, 267-284, 303, 322, 323, 412, 478, 497, 529, 533-535, 539 afferents, 178-204, 209-227 Spines, dendritic, 171, 175, 291, 296-298, 301, 302, 400-402, 404-407, 411, 412, 417, 418, 425 Squid, 56 Statistics, 17, 18, 349-388, 391, 520, 532, 533 Stomatopoda, 57 Stretch receptor, 132, 143, 261, 353, 381, 522 Structural analysis, 159-204 Strychnine, 433 Subiculum, 415, 455 Subpopulation organization, 71-109 Substantia gelatinosa, 155, 170, 171, 173, 175, 176, 180, 181, 183-185, 191, 203, 533, 534 Summation, 257 Synaptic boutons, 81, 145, 155, 163, 171, 185, 295, 296, 299, 300, 393, 394, 402, 404-407, 409-412 Synaptic cleft, 303

552

THE

INTERNEURON

Synaptic distribution, 295-299 Synaptic efficiency, 295, 299, 300, 538 Synaptic transmission, 37-41, 56, 61 Synaptic vesicle, 54, 145, 293, 295, 300, 302-305, 400, 409, 530, 538 Synchronization, 79, 80, 103, 104, 106, 107, 468-474, 477, 478, 485-488, 490, 491, 494, 531, 532, 541

Tight junction, 41, 101, 413 Toad, 32 Transmitter, 13, 38-40, 43, 51-55, 61, 85-87, 89, 91, 93, 97, 99, 103, 104, 233, 282, 284, 409, 430-432, 442, 460, 522, 530, 531 mobilization, 430-432 Tritonia, 23 Two-transmitter hypothesis, 54, 531

T

V

Tabes, dorsalis, 31, 32 Tetrapoda, 32 Tetrodotoxin, 102 Thalamus, 180, 309, 322, 467-495, 499, 500, 510, 521, 537, 541, 542 Thiopental, 495

Vertebrates, 3-5, 11, 13, 14, 49, 55-57, 61, 115, 118, 126, 169, 171, 329, 331, 343, 529 see also Amphibia; Birds; Chordata; Fish; Mammals; Reptiles; Tetrapoda Visual system, 2, 14, 15, 114-125, 521