Progress in Brain Research Volume 44 Understanding the Stretch Reflex

PROGRESS IN BRAIN RESEARCH VOLUME 44 UNDERSTANDING T H E STRETCH REFLEX

PROGRESS IN BRAIN RESEARCH

ADVISORY BOARD

W. Bargmann H.T. Chang E. De Robertis

J.C. Eccles J.D. French

H. Hydkn J. Ariens Kappers S.A. Sarkisov J.P. Schadk F.O. Schmitt

J.Z. Young

Kiel Shanghai Buenos Aires Buffalo (N.Y.)

Los Angeles (Calif.) Goteborg Amsterdam Moscow Amsterdam Brookline (Mass.) London

PROGRESS IN BRAIN RESEARCH VOLUME 44

UNDERSTANDING THE STRETCH REFLEX

EDITED BY S. HOMMA Department of Physiology, School of Medicine, Chiba University, Chiba (Japan)

ELSEVIER SCIENTIFIC PUBLISHING COMPANY AMSTERDAM/OXFORD/NEW YORK 1976

ELSEVIER SCIENTIFIC PUBLISHING COMPANY 335 J A N VAN G A L E N S T R A A T P.O. BOX 211, AMSTERDAM, T H E N E T H E R L A N D S

AMERICAN ELSEVIER PUBLISHING COMPANY, INC. 52 VANDERBILT AVENUE NEW YORK, NEW Y O R K 10017

ISBN 0-444-41456-8

WITH 226 ILLUSTRATIONS AND 16 TABLES COPYRIGHT 0 1976 BY ELSEVIER/NORTH-HOLLAND BIOMEDICAL PRESS, AMSTERDAM

ALL RIGHTS RESERVED. NO PART O F THIS PUBLICATION MAY B E REPRODUCED, S T O R E D IN A R E T R I E V A L SYSTEM, O R TRANSMITTED IN ANY F O R M O R BY ANY MEANS, ELECTRONIC, MECHANICAL, PHOTOCOPYING. R E C O R D I N G , O R OTHERWISE, WITHOUT T H E PRIOR WRITTEN PERMISSION O F T H E PUBLISHER, ELSEVIER/NORTH-HOLLAND BIOMEDICAL PRESS, J A N VAN G A L E N S T R A A T 335. AMSTERDAM

PRINTED I N T H E N E T H E R L A N D S

List of Contributors R. ANASTASIJEVId, Institute for Medical Research, Belgrade, Yugoslavia, T. ARAKI, Department of Physiology, Faculty of Medicine, Kyoto University, Kyoto, Japan. E. ARBUTHNOTT, Institute of Physiology, University of Glasgow, Glasgow, Great Britain. R.W. BANKS, Department of Zoology, University of Durham, Durham DH1 4LE, Great Britain. D.W. BARKER, Department of Zoology, University of Durham, Durham DH1 4LE, Great Britain. R. BENECKE, Department of Physiology 11, University of Gottingen, D-3400 Gottingen, G.F.R. I.A. BOYD, Institute of Physiology, University of Glasgow, Glasgow, Great Britain. F. BUCHTHAL, Institute of Neurophysiology , University of Copenhagen, Copenhagen, Denmark. D. BURKE, Department of Clinical Neurophysiology, Academic Hospital, Uppsala, Sweden. E. ELDRED, Departments of Anatomy and Kinesiology, and Brain Research Institute, University of California a t Los Angeles, Los Angeles, Calif. 90024, U.S.A. P.H. ELLAWAY, Department of Physiology, University College London, London WC1 E 6BT, Great Br,it ain. F. EMONET-DENAND, Laboratoire d e Neurophysiologie, ColEge de France, 7 5231 Paris 05, France. K. ENDO, Department of Physiology, Faculty of Medicine, Kyoto University, Kyoto, Japan. E.V. EVARTS, Laboratory of Neurophysiology, National Institute of Mental Health, Bethesda, Md. 20014, U.S.A. K. EZURE, Department of Neurophysiology, Institute of Brain Research, School of Medicine, University of Tokyo, Bunkyo-ku, Tokyo, Japan. W. FREEDMAN, Krusen Center for Research and Engineering, Temple University, Philadelphia, Pa. 19141, U.S.A. K. FUKUSHIMA, Department of Physiology, Hokkaido University School of Medicine, Sapporo, Japan. M.H. GLADDEN, Institute of Physiology, University of Glasgow, Glasgow, Great Britain. G.M. GOODWIN, University Laboratory of Physiology, Parks Road, Oxford, Great Britain. R . GRANIT, The Nobel Institute for Neurophysiology, Karolinska Institutet, S-10401 Stockholm 60, Sweden. V.S. GURFINKEL, Institute of the Problems of Information Transmission, Academy of Sciences of the U.S.S.R., Moscow, U.S.S.R. A. GYDIKOV, Institute of Physiology, Bulgarian Academy of Sciences, Sofia, Bulgaria. K.-E. HAGBARTH, Department of Clinical Neurophysiology, Academic Hospital, Uppsala, Sweden. D. HARRIS, Department of Physiology, Harvard Medical School, Boston, Mass. 0211 5 , U.S.A. C. HELLWEG, Max-Planck-Institute fur biophysikalische Chemie, Gottingen, G.F.R. H.-D. HENATSCH, Department of Physiology 11, University of Gottingen, D-3400 Gottingen, G.F.R. E. HENNEMAN, Department of Physiology, Harvard Medical School, Boston, Mass. 02115, U.S.A. R. HERMAN, Krusen Center for Research and Engineering, Temple University, Philadelphia, Pa. 19141, U.S.A.

vi K. HIRAYAMA, Departments of Physiology and Orthopedic Surgery, School of Medicine, Chiba University, Chiba, Japan. S. HOMMA, Department of Physiology, School of Medicine, Chiba University, Chiba, Japan. J. HORIKAWA, Department of Biophysical Engineering, Faculty of Engineering Science, Osaka University, Toyonaka, Osaka 560, Japan. J.C. HOUK, Department of Physiology, T h e Johns Hopkins University School of Medicine, Baltimore, Md., U.S.A. M. HULLIGER, University Laboratory of Physiology, Parks Road, Oxford, Great Britain. H. HULTBORN, Department of Physiology, University of Goteborg, Goteborg, Sweden. R.S. HUTTON, School of Physical and Health Education, University of Washington, Seattle, Wash., U.S.A. G.F. INBAR, Department of Electrical Engineering, Technion - Israel Institute of Technology, Haifa, Israel. F. ITO, Department o f Physiology, Nagoya University School of Medicine, Nagoya 466, Japan. K. ITO, Department of Physiology, Faculty of Medicine, Kyoto University, Kyoto, Japan. M. ITO, Department of Physiology, Faculty of Medicine, University of Tokyo, Bunkyo-ku, Tokyo, Japan. Y. ITO, Department of Physiology, Nagoya University School of Medicine, Nagoya, 466, Japan. M. KATO, Department of Physiology, Hokkaido University School of Medicine, Sapporo, Japan. Y. KAWAI, Department of Physical Education, Faculty of Liberal Arts, Yamaguchi University, Yamaguchi, Japan. D. KERNELL, Department of Neurophysiology, University of Amsterdam, Eerste Constantijn Huygensstraat 20, Amsterdam, The Netherlands. D. KOSAROV, Institute of Physiology, Bulgarian Academy of Sciences, Sofia, Bulgaria. Y. LAPORTE, Laboratoire de Neurophysiologie, College de France, 7 5 2 3 1 Paris 0 5 , France. M.I. LIPSHITS, Institute of t h e Problems of Information Transmission, Academy of Sc.iences of t h e U.S.S.R., Moscow, U.S.S.R. L. LOFSTEDT, Department of Clinical Neurophysiology, Academic Hospital, Uppsala, Sweden. P.B.C. MATTHEWS, University Laboratory of Physiology, Parks Road, Oxford, Great Britain. J. MEYER-LOHMANN, Department of Physiology 11, University of Gottingen, D-3400 Gottingen, G.F.R. A. MILBURN, Department of Zoology, University of Durham, Durham DH1 4LE, Great Britain. S. MINASSIAN, Krusen Center for Research and Engineering, Temple University, Philadelphia, Pa. 1 9 1 4 1 , U.S.A. M. MIZOTE, Department of Physiology, School of Medicine, Chiba University, Chiba, Japan. S. MORI, Department of Physiology, Asahikawa Medical College, Asahikawa, Hokkaido, Japan. Y. NAKAJIMA, Department of Physiology, School of Medicine, Chiba University, Chiba, Japan. Y. ODA, Department of Biophysical Engineering, Faculty of Engineering Science, Osaka University, Toyonaka, Osaka 560, Japan. 0 . POMPEIANO, Istituto di Fisiologia Umana, Cattedra 11, Universiti di Pisa, Pisa, Italy. K.E. POPOV, Institute of the Problems of Information Transmission, Academy of Sciences of t h e U.S.S.R., Moscow, U.S.S.R. R.M. REINKING, Department of Physiology, College of Medicine, University of Arizona, Tucson, Ariz. 85724, U.S.A. D.J. REIS, Laboratory of Neurobiology , Department of Neurology, Cornell University Medical College, New York, N.Y. 1 0 0 2 1 , U.S.A.

vii S. SASAKI, Department of Neurophysiology, Institute of Brain Research, School of Medicine, University of Tokyo, Bunkyo-ku, Tokyo, Japan. H. SCHMALBRUCH, Laboratory of Clinical Neurophysiology, Rigshospitalet, Copenhagen, Denmark, Y. SHIGENAGA, Department of Anatomy, Osaka University Dental School, Osaka, Japan. J.L. SMITH, Departments of Anatomy and Kinesiology and Brain Research Institute, University of California at Los Angeles, Los Angeles, Calif. 90024, U.S.A. M.J. STACEY, Department of Zoology, University of Durham, Durham DH1 4LE, Great Britain. M. STANOJEVIC, Institute for Medical Research, Belgrade, Yugoslavia. E.K. STAUFFER, Department of Physiology, School of Medicine, University of Minnesota, Duluth, Minn. 55812, U.S.A. D.G. STUART, Department of Physiology, College of Medicine, University of Arizona, Tucson, Ariz. 85124, U.S.A. C. STUDENT, Department of Physiology 11, University of Gottingen, Gottingen, G.F.R. U. STUDENT, Department of Physiology 11, University of Gottingen, Gottingen, G.F.R. K. TAKANO, Department of Physiology 11, University of Gottingen, Gottingen, G.F.R. K. TANAKA, Research Group o n Auditory and Visual Information Processing, Broadcasting Science Res. Lab., NHK, 1-10-11, Kinuta, Setagaya-ku, Tokyo, Japan. R. TANAKA, Department of Physiology, Hirosaki University Faculty of Medicine, Hirosaki, Japan. N. TANKOV, Institute of Physiology, Bulgarian Academy of Sciences, Sofia, Bulgaria. A. TAYLOR, Sherrington School of Physiology, St. Thomas’s Hospital Medical School, London S.E.l, Great Britain. J.R. TROTT, Department of Physiology, University College London, London WClE 6BT, Great Britain. Y . UCHINO, Department of Physiology, Kyorin University School of Medicine, Mitaka, Tokyo, Japan. M. UDO, Department of Biophysical Engineering, Faculty of Engineering Science, Osaka Un$ersity, Toyonaka, Osaka 560, Japan. J. VUCO, Institute for Medical Research, Belgrade, Yugoslavia. G. WALLIN, Department of Clinical Neurophysiology, Academic Hospital, Uppsala, Sweden. P. WAND, Jstituto di Fisiologia Umana, Cattedra 11, Universitg di Pisa, Pisa, Italy. S. WATANABE, Department of Physiology, School of Medicine, Kyohrin University, Mitaka-shi, Tokyo, Japan. D.G.D. WATT, NASA, Ames Research Center, Moffett Field, Calif. 94035, U.S.A. V.J. WILSON, The Rockefeller University, New York, N.Y. 10021, U.S.A. G.F. WOOTEN, Laboratory of Neurobiology, Department of Neurology, Cornell University Medical College, New York, N.Y. 10021, U.S.A. A. YAFE, Department of Electrical Engineering, Technion - Israel Institute of Technology, Haifa, Israel.

This Page Intentionally Left Blank

Preface

The contents of this volume are a collection of lectures and presentations given at an international symposium: Understanding the Stretch Reflex, November 7-11, 1975, held at the Sasakawa Hall, Mita, Tokyo. The first neurophysiological approach in understanding the stretch reflex should be said to have been started by the late Sherrington about 80 years ago. Since then many neuroanatomists, neurophysiologists and neurologists have been working t o understand the secrets of the whole mechanism of the stretch reflex, a very fundamental piece of neuromuscular machinery. The terms “tonic” and “phasic” types of motoneuron were introduced by Granit more than 20 years ago, at a very early stage of his study in this field which suggested that the contribution of the polysynaptic neural circuit should also be considered, besides the omnipresent monosynaptic one. The “theme emblem” printed in the front page of the first announcement is taken from a schematic illustration used in his paper published in 1957 (J.Neurophysiol., Vol. 20) which he proposed in order to allow the interpretation of many polysynaptic mechanisms. This polysynaptic participation in stretch reflex activity has proved t o be more and more important since then, and in the present symposium too. Among Japanese contributions in this field, I want to mention first the work done by the late Dr. Otani and Dr. Araki of Kyoto, who differentiated IS and SD spikes in frog motoneuron, and who showed a difference in the degree of accommodation in the tonic and phasic motoneurons. At that time, and also a little later I myself had been doing work on muscle stretch at different speeds which, by varying Ia impulse frequency, could change an initial site of excitation at the motoneuron, i.e., from the initial segment to the soma according to the different accommodative characteristics of different portions of the motoneuronal membrane. Efferent innervation of the muscle spindle by gamma fusimotor fibers was discussed extensively by morphologists at the Hong Kong Symposium of 1961, organized by Dr. Barker. The present volume also includes several papers in this important field of stretch reflex research, which were given in the first morning session of the present symposium. Drs. Barker, Boyd and Laporte, all of whom were present at the Hong Kong Symposium too, contributed their recent research performed in the intervening 15 years. In the discussions of this ses-

X

sion readers will find a very nice summary by Dr. Matthews concerningagreements and disagreements between Drs. Barker and Boyd about morphological results. Supraspinal control, stimulatory or voluntary, of the stretch reflex is another important approach for the understanding of it. This was also stressed many times in following sessions. All important discussions throughout the present symposium are incorporated in this publication as far as the size of the volume would allow, although they had t o be condensed considerably. For the hard work of rendering recorded discussions into type-written form I am particularly indebted t o Dr. K. Uemura of Chiba University and t o Dr. M.H. Gladden of Glasgow University. This symposium was held in commemoration of the centenary of Chiba University School of Medicine and of the tenth year of my chair of physiology at the school. I also express my thanks to the Japan Society for Promotion of Sciences which primarily made the present symposium possible by a grant and to both the International Brain Research Organization and Japan Society of Electroencephalography and Electromyography. Both organizations were particularly helpful in arranging the cooperation of so many active participants in the present symposium.

S. Homma Chiba (Japan)

Contents List of Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

V

ix

Opening Address: Relations of reflexes and intended movements E.V. Evarts and R. Granit (Bethesda, Md., U.S.A. and Stockholm, Sweden) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1

Organizer’s Lecture: Frequency characteristics of the impulse decoding ratio between the spinal afferents and efferents in the stretch reflex S. Homma (Chiba, Japan) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

15

Session I -- Muscle Spindle and its Fusimotor Innervation. Part I The mechanical properties of dynamic nuclear bag fibres, static nuclear bag fibres and nuclear chain fibres in isolated cat muscle spindles I.A. Boyd (Glasgow, Great Britain) ............................ Structural features relative t o the function of intrafusal muscle fibres in the cat M.H. Gladden (Glasgow, Great Britain) ......................... Ultrastructural observations of a muscle spindle in the region of a contraction site of a dynamic y axon E. Arbuthnott, I.A. Boyd and M.H. Gladden (Glasgow, Great Britain) . . Studies of the histochemistry, ultrastructure, motor innervation, and regeneration of mammalian intrafusal muscle fibres D. Barker, R.W. Banks, D.W. Harker, A. Milburn and M.J. Stacey (Durham, Great Britain) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Studies on muscle spindle primary endings with sinusoidal stretching G.M. Goodwin, M. Hulliger and P.B.C. Matthews (Oxford, Great Britain) The skeleto-fusimotor innervation of cat muscle spindle Y. Laporte and F. Emonet-Dknand (Paris, France) . . . . . . . . . . . . . . . . . General Discussion

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

33

51 61

67

89 99

107

Session I1 - Muscle Spindle and its Fusimotor Innervation. Part I1 Reflex connections from muscle stretch receptors to their own fusimotor neurones P.H. Ellaway and J.R. Trott (London, Great Britain) . . . . . . . . . . . . . . . Intracellular recordings from intact soleus muscles of cats Y. Nakajima (Chiba, Japan) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effects of FM vibration on muscle spindles in the cat M. Mizote (Chiba, Japan) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of abortive spike on encoding mechanism in frog muscle spindle F. Ito and Y. It0 (Nagoya, Japan) .............................

113 123 133

141

xii Session I11 - Muscular Afferents associated with Stretch Reflex Nature of the persisting changes in afferent discharge from muscle following its contraction E. Eldred, R.S. Hutton and J.L. Smith (Los Angeles, Calif .. U.S.A.) . . . 157 Use of afferent triggerec'. averaging t o study the central connections of muscle spindle afferents A. Taylor, D.G.D. Watt, E.K. Stauffer, R.M. Reinking and D.G. Stuart (Tucson, Ariz., U.S.A.) . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171 Selective activation of group I1 muscle afferents and its effects on cat spinal neurones M. Kato and K. Fukushima (Sapporo, Japan) ..................... 185 Session IV - Information Processing of the Stretch Reflex The relative sensitivity of Renshaw cells to static and dynamic changes in muscle length 0. Pompeiano and P. Wand (Pisa, Italy) ......................... Muscle stretch and chemical muscle spindle excitation: effects on Renshaw cells and efficiency of recurrent inhibition J. Meyer-Lohmann, H.-D. Henatsch, R. Benecke and C. Hellweg (Gottingen,G.F.R.) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Transmission in the pathway of reciprocal Ia inhibition to motoneurones and its control during the tonic stretch reflex H. Hultborn (Goteborg, Sweden) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Recruitment, rate modulation and the tonic stretch reflex D. Kernel1 (Amsterdam, The Netherlands) ....................... Patterns of motoneuronal units discharge during naturally evoked afferent input R. Anastasijevid, M. Stanojevid and J. VuEo (Belgrade, Yugoslavia) . . . .

199

223 235 257 267

Session V - Supraspinal Control of the Stretch Reflex. Part I Single unit spindle responses t o muscle vibration in man K.-E. Hagbarth, D. Burke, G. Wallin and L. Lofstedt (Uppsala, Sweden) 281 Reciprocal Ia inhibition and voluntary movements in man R. Tanaka (Hirosaki, Japan) .................................. 291 An assessment of stretch reflex function J.C. Houk (Baltimore, Md., U.S.A.) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303 Session VI - Supraspinal Control of the Stretch Reflex. Part I1 Parameter and signal adaptation in the stretch reflex loop G.F. Inbar and A. Yafe (Haifa, Israel) .......................... Alpha-gamma linkage in man during varied contraction S. Watanabe and K. Hirayama (Tokyo, Japan) ....................

317 339

xiii Session VII - Significance of Slow and Fast Muscles in the Stretch Reflex. Part I Discharge pattern of tonic and phasic motor units in human muscles upon stretch reflex D. Kosarov, A. Gydikov and N. Tankov (Sofia, Bulgaria) . . . . . . . . . . . . 355 Contraction times of reflexly activated motor units and excitability cycle of the H-reflex F. Buchthal and H. Schmalbruch (Copenhagen, Denmark) . . . . . . . . . . . 367 Identification of fast and slow firing types of motoneurons in the same pool E. Henneman and D. Harris (Boston, Mass., U.S.A.) . . . . . . . . . . . . . . . . 377 Session VIII Part I1

- Significance of Slow and Fast Muscles in the Strech Reflex.

Blood flow in red' and white muscle: relationship t o metabolism development and behavior D.J. Reis and G.F. Wooten (New York, N.Y., U.S.A.) . . . . . . . . . . . . . . 385 Controlled variations of input-output parameters affecting the active tension-extension diagram during muscle strength H.-D. Henatsch, C. Student, U. Student and K. Takano (Gottingen, G.F.R.) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 403 Supraspinal control of slow and fast spinal motoneurons of the cat T. Araki, K. Endo, Y. Kawai, K. Ito and Y. Shigenaga (Kyoto, Japan) . . 413 Session IX - Supraspinal Control of the Stretch Reflex. Part I11 Adaptive control of reflexes by the cerebellum M. Ito (Tokyo, Japan) ...................................... 435 Cerebellar control of locomotion investigated in cats: discharges from Deiters' neurones, EMG and limb movements during local cooling of the cerebellar cortex M. Udo, Y. Oda, K. Tanaka and J. Horikawa (Osaka, Japan) . . . . . . . . . 445 A role of upper cervical afferents on vestibular control of neck motor activity K. Ezure, S. Sasaki, Y. Uchino and V.J. Wilson (Tokyo, Japan) . . . . . . . 461 Session X - New Approach to the Understanding of the Stretch Reflex The state of stretch reflex during quiet standing in man V.S. Gurfinkel, M.I. Lipshits, S. Mori and K.E. Popov (Moscow, U.S.S.R. 473 andHokkaido,Japan) ...................................... Functional stretch reflex (FSR) - a cortical reflex? W. Freedman, S. Minassian and R. Herman (Philadelphia, Pa., U.S.A.) . . 487 Local tetanism, a tool for understanding the stretch reflex K. Takano (Gottingen, G.F.R.) ............................... 491 SubjectIndex

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

503

This Page Intentionally Left Blank

Opening Address

Relations of Reflexes and Intended Movements E.V. EVARTS and R. GRANIT

*

Laboratory of Neurophysiology, National Institute of Mental Health, Bethesda, Md. 20014 (U.S.A.) and The Nobel Institute for Neurophysiology, Karolinska Institutet, S-I 04 01 Stockholm 6 0 (Sweden)

INTRODUCTION Current knowledge of reflex control of movement is in large part based on experiments carried out in spinal or decerebrate animals in which it is impossible to study the interaction between reflexes and intended movements. For an understanding of sensorimotor function in man, however, it is essential to consider both the reflex and the intended components of motor activity. The interaction of intentions and reflexes is especially significant in movements occurring in response to kinesthetic inputs impinging directly on the body parts t o be moved. For example, the wrestler’s strategy determines whether he will react to an opponent’s abrupt application of force by resisting or by giving way. In the intact subject, reflexes are modified by intentions, and intended movements vary depending upon reflex responses to forces and displacements. Effects of intention on reflex responses have been demonstrated both by Hammond (1956) and by Hagbarth (1967) in studies of reflex responses of arm muscles in human subjects. Hammond showed that a 50 msec latency biceps response t o stretch was present or absent depending upon the subject’s intention t o resist or t o give way when the stretch occurred. Hagbarth confirmed Hammond’s findings, and in addition, showed that even the shorter (25 msec) latency tendon jerk varied in accord with the subject’s intended response. In the experiments t o be described in this report, we have used a paradigm similar to that of Hagbarth (1967) t o study the relation of reflex and intended muscle activity recorded from biceps muscle in human subjects. The methods employed are described in detail in the following section, but one essential feature of our experimental paradigm will be introduced here. This feature was dissociation of the reflex effects on the biceps brachii muscle, as caused by a load change, from the intended response which in its turn was triggered by the same load change. This dissociation was achieved by giving the subject an instruction implying contraction or relaxation of the biceps regardless of the direction of the perturbation that triggered him into intended

* This study was conducted while Ragnar Granit was a Fogarty Scholar-in-Residence, Fogarty International Center, National Institutes of Health, Bethesda, Md. 20014, U.S.A.

2

action. For example, when execution of an instruction calling for biceps contraction was triggered by a perturbation which inhibited biceps motoneurons, the intended biceps discharge occurred in spite of rather than because o f the reflex effects of the perturbation. This experimentally produced dissociation of reflex from intended muscle activity has provided certain new insights into both of these components of motor activity. METHODS Subjects grasped a handle which could be rotated by pronation or supination of the forearm and maintained this handle in a vertical orientation, thus obeying a basic instruction t o preserve that level of tonic motoneuron activity necessary to position the handle correctly. Fig. 1provides a schematic illustration of the experimental apparatus. The handle grasped by the subject was coupled to the axle of a brushless DC torque motor (Aeroflex TQ-52) which could generate steady-state torques requiring that the subject exert maintained muscular effort (pronation or supination) in order that the handle be vertically positioned. By regulating the current through the torque motor, the experimenter could control the steady-state activity of supinator (i.e., biceps) and pronator muscles. Following maintenance of the correct handle position for a period of 2-5 sec, either one of two “instruction lamps” was illuminated. One lamp instructed the subject to respond t o a subsequent handle perturbation by pronation, the other t o respond t o it by supination. Thus the intended pronation or supination movement did not occur in response to the instruction itself, but was elicited in response to the perturbation of the handle which followed delivery of an instruction. The interval between the instruction and the perturbation triggering it into action varied unpredictably,between 1.8 and 2.5 sec. Upon elapse of the delay following the instruction, the perturbing ramp of torque was applied t o the handle. This ramp was superimposed on the steady-

EXTERNALLY PRODUCED SUPINATION SHORTENS A N D INHIBITS BICEPS

BICEPS IS EXCITED A N D SHORTENS FOR INTENDED SUPINATION

\ EXTERNALLY PRODUCED PRoNATloN STRETCHES AND EXCITES BICEPS

BICEPS IS INHIBITED AND LENGTHENS FOR INTENDED PRONATION

Fig. 1. Subjects grasped handle and maintained *itin a vertical position while awaiting an instruction. Motor torque during waiting period determined steady-state biceps discharge, with a torque opposing supination demanding biceps discharge and a torque assisting supination demanding pronator discharge and reciprocal quiescence of biceps. An abrupt ramp triggered intended movement. For further details see text.

3 state torque which had existed prior t o the perturbation. Ramp slope and amplitude could be varied, but for the present report we shall deal only with ramps which were quite abrupt (being completed in 1 0 msec) and which involved a large torque change (1.1ft/lb). Upon completion of the 10 msec ramp, the new level of torque was maintained during the subject's response and for 2 sec after the subject had completed the pronation or supination response which had been called for by the prior instruction. At the end of this time the torque returned t o its original steady-state level and the subject initiated a new trial by returning the handle from the supinated or pronated position to the vertical position. Strain gauges mounted on the motor axle monitored torque, and a potentiometer coupled to the axle monitored handle orientation. Muscle activity was picked up by surface EMG electrodes on the biceps. Torque, position, and EMG data were recorded on magnetic tape together with signals providing information as to instructions and perturbations. Data were quantitatively analyzed by a PDP-12 computer program worked out by Vaughn, Sheriff and Evarts and available from the DECUS Program Library of the Digital Equipment Corporation, Maynard, Mass. This program generated a raster display of EMG activity which had been rectified and then converted t o pulse frequency by a Teledynne-Philbrick voltage-to-frequency converter. Frequency of pulse output was 0-1000 Hz. In addition t o providing raster displays of EMG responses, this program displayed average response histograms and computed latencies of muscle responses and levels of statistical significance of these responses.

RESULTS Some early reflexes caused by the triggering perturbation are predictable from present information (see, e.g., Granit (1970) and below): (i) in response to muscle shortening by supination, an EMG silence caused by the segmental so-called unloading reflex; (ii) biceps extension by pronation will elicit a monosynaptic stretch reflex (the tendon jerk) which will be enhanced if there is tonic discharge of biceps motoneurons t o resist a steady-state load on the handle; (iii) reversal of the steady-state load will reduce the stretch reflex. These effects are easily verified in the records. Their latencies are of the order of 20-30 msec. The combinations obtained by interaction of the reflexes with the intended responses are summarized in Table I. In looking at this Table, it should be kept in mind that with the elbow bent at go", biceps is a supinator which will be stretched when the arm is pronated by the action of the torque motor and which will actively contract when the subject carries out an instruction to supinate.

( I ) Intended excitation and the unloading reflex For one of the 4 pairings shown in Table I, the perturbation (supination) inhibited biceps discharge while the intended movement (supination) required

4 TABLE I This table shows the 4 possible pairings of the two perturbations and the two instructions. One of the perturbations (pronation) stretched biceps and, as shown in the column headed “Reflex Biceps Response to Perturbation”, the effect of this perturbation was excitatory (+). The other perturbation (supination) resulted in biceps shortening and had an inhibitory (-) effect. The effects of the two sorts of instruction are shown in the column at the right headed “Intended Biceps Response”, where it is seen that intended supination involved active biceps contraction (+) and intended pronation involved active biceps relaxation (-). Note that dissociation of the reflex and intended movements is seen in the second row, where reflex excitation produced by stretch is associated with intended inhibition necessary for active pronation, and in the third row, where reflex inhibition due to shortening is associated with intended excitation necessary for active supination.

Perturbation

Instruction

Reflex biceps response t o perturbation

Pronation Pronation Supination Supination

Supinate Pronate Supinate Pronate

+

+

-

Intended biceps response

+

-

+

-

9.26.74

Fig. 2 . In all 4 sets of traces biceps was tonically active (opposing steady torque) when the perturbation (delivered at dotted line) abruptly supinated the arm, eliciting an “unloading reflex” at a latency of 25 msec. For the two sets of traces above, the biceps discharge necessary for the intended supination overcame the .reflex inhibition at a latency of 70 msec, whereas in the two sets of traces below, biceps remained virtually silent during the intended pronation movement. The heavy trace below each EMG record indicates handle movement, with supination being indicated by a downward deflection and pronation by an upward deflection. Time marks are a t 50-msec intervals.

5 active biceps discharge; Fig. 2 (top) shows biceps EMG discharge recorded for this combination. The initial effect of the inhibitory perturbation was an unloading reflex which silenced tonic biceps discharge already present at the time the perturbation was delivered. This tonic discharge was caused by the steadystate motor torque requiring counteracting biceps activity. The unloading reflex in Fig. 2 is terminated by an intense EMG burst which marks the start of the intended supination. In Fig. 2 this intended supination occurs with a latency of 70 msec.

CELL 0 2 20 T R I A L S BIN 5 MSEC

EXP 0 2

PEN 02

1024 1

45

EXP 02

12

PEN 0 2 CELL 0 2

SEC

I

20 TRIALS

-------

j..

.. .. .. ....... .. .. .. .. .. .. ..... ..-. .. .. ............ .- .. . ........ ... .......................... ... ........ ... ... ..... ... .. . . . . .. .. . .. .. ... .. .. . .. .. ... .. .. . . . . -.. . ,.:] . . . . . . . . . . . . . . . . ... .. ... .. ... .. ... .. ... .. ... .. .. ... .. ... .. ... .. ... . . . . ............ . . . . . . .._: . . .. .. . . ::: . .::-: . .:. .. ..:..:.. .

1. I

!

45

2

Fig. 3. This figure illustrates results for a series of trials in a single subject with the instruction-perturbation pairing shown in the upper part of Fig. 2. F o r this pairing, the perturbation was inhibitory and was followed by a n unloading reflex with 30:msec latency. The intended response required biceps discharge, and for some of the individual trials shown in the raster this intended discharge occurred a t latencies as short as 60 msec. It is to be noted in the raster that there is considerable trial-to-trial variability for this subject. Such variability was particularly characteristic of intended responses which were.carried o u t against conflicting reflex inputs. Time marks on abscissa occur a t intervals of 50 msec, with the entire display (before and after the perturbation) covering 500 msec.

6 Fig. 2 (lower half) also shows an unloading reflex response t o an inhibitory perturbation, but in this case the prior instruction had called for pronation rather than supination. Here both the reflex and the intended movements involved biceps inhibition, and the massive EMG response of Fig. 2 (top) was absent. The results in Fig. 2 show that an intended motor response specified by a prior instruction can over-ride the segmental unloading reflex and generate an intense muscle discharge with a latency of 70 msec. Latencies for intended biceps discharge occurring in spite of the unloading reflex varied from trial to trial within a given subject and from subject t o subject for the 10 subjects in whom these responses were studied. Response variability for a single subject is illustrated in Fig. 3.

(IZ) In tended inhibition and reflex excitation The conflict between reflex and intended drives shown in Figs. 2 (top) and 3 involved pairing an instruction t o supinate with a perturbing trigger which supinated. Table I shows that pairing an instruction t o pronate with a perturbation which pronates also gives rise t o a conflict between reflex and intended biceps activity, but a conflict which is opposite t o that shown in Figs. 2 (top) and 3. For this new pairing (Fig. 4, lower half) the perturbing pronation stretches biceps and excites biceps motoneurons to produce a tendon jerk via afferent reflex pathways. The intended pronation movement, however, involves quiescence of biceps motoneurons (with reciprocal excitation of pronator

200 MSEC

-4Fig. 4 . Biceps EMG for an intended supination (above) and a n intended pronation (below) triggered by a reflexly excitatory perturbation (pronation) which stretched biceps and elicited tendon jerks with 25-msec latencies. For the trace above (intended supination), t h e tendon jerk merged into the biceps discharge associated with t h e intended supination movement, whereas for the trace below (intended pronation), there was virtually n o biceps discharge following t h e tendon jerk. The handle movements are indicated by heavy trace below EMG, with downward deflection indicating pronation and upward deflection indicating supination.

7 motoneurons) and, as shown in Fig. 4 (lower half), there is little activity following the tendon jerk for this pairing. Sometimes, however, reflex excitation persists for 150 msec following biceps stretch; this is shown in Fig. 5. The duration and intensity of this “unintended” stretch reflex depend on level of tonic biceps discharge. In Fig. 4 (lower half) biceps was silent at the time stretch was delivered, whereas in Fig. 5 biceps was tonically active. The occurrence of unintended biceps excitation in spite of intended inhibition in Fig. 5 (left) does not mean that the subject’s intention is without effect. To determine the effect of the intention, one must compare the biceps response t o the same stretch for the two different directions of intended movement. Fig. 5 (right) shows biceps responses when the intention was biceps discharge, with both the reflex and the intended responses favoring biceps discharge. Here, the biceps discharge following stretch was of greater intensity and more consistent from trial to trial. Comparison of the rasters of 5 (left and right) is particularly revealing as to the way in which reflex and intended movements interact. In Fig. 5 (left), where the intended biceps silence is often overcome by reflex excitation, both

EXP 0 2

PEN 0 2 CELL 0 2

1024

BIN

15 TRIALS

EXP 0 2

PEN 02

CELL 0 2

BIN

5 MSEC

5 MSEC

15 TRIALS

768 512

512

256

256

32

EXP 0 2

f5

PEN 02 CELL 0 2

45

15 TRIAL

EXP 0 2 PEN 0 2

SEC

32

5

15

CELL 0 2

15 TRIAL

SEC

45

5

Fig. 5. A perturbing pronation which stretched biceps triggered pronation a t t h e left a n d supination a t the right. Reflex excitation in the trace a t the left persisted for up t o 150 msec in spite of t h e intended pronation which required biceps relaxation. At t h e right, however, where the intended movement involved biceps discharge, the second phase of excitation was more pronounced and for some trials the silent period following t h e tendon jerk was virtually absent. Presence of prolonged reflex excitation a t the left is related t o the high level of tonic discharge present ( t o left of center line) during the steady state prior t o stretch (delivered a t center line). In Fig. 4 there was n o tonic biceps discharge, but instead the steady-state load required tonic pronator discharge.

the magnitude and the latency of the unintended reflex biceps discharge vary from trial to trial. In some instances, there is a relatively long silent period between tendon jerk and the subsequent burst of unwanted biceps activity, and in a few cases the biceps becomes almost totally silent following the brief discharge associated with the tendon jerk. Thus, the intended biceps silence sometimes obliterates the upintended biceps reflex, and when the reflex does occur, it exhibits a variability commonly associated with intended movements. In contrast, the raster of Fig. 5 (right) shows results when both reflex and intended responses involved biceps discharge. Here, intense biceps discharge occurred on every trial and the latency for discharge of the phase of activity following the tendon jerk was less variable than in Fig. 5 (left). In some responses of Fig. 5 (right) the raster shows the brief suppression of activity after the tendon jerk to be virtually negligible. (111)Intended movement and the “silent period” The silent period after a synchronous discharge of motoneurons, discovered by Paul Hoffmann (1922), is now understood to be a complex phenomenon dominated by the following segmental components: (i) afterhyperpolarization caused by the synchronous firing; (ii) cessation of the spindle discharge induced by the muscle shortening; (iii) Golgi tendon organ inhibition, as demonstrated also by intracellular recording (Granit et al., 1966); (iv) recurrent inhibition (see, e.g., reviews by Granit, 1970; Matthews, 1972). While the results illustrated in Fig. 5 confirm the well-known reduction in EMG activity during the period between the tendon jerk and the second phase of discharge (the silent period), it is also apparent that for some of the trials in Fig. 5 this reduction is more pronounced when tne intended movement calls for biceps relaxation (Fig. 5, left) than when it calls for biceps discharge (Fig. 5, right).

( I V ) Effects o f steady-state load Fig. 5 illustrated responses when the subject maintained a steady state of activity opposing the force generated by the torque motor. The level o f steadystate tonic biceps discharge turned o u t to be a critical factor in biceps responses to arm perturbation. Fig. 6 compares biceps responses to stretch for two different steady-state loads, one load (left) requiring a high level of tonic biceps discharge and the other load (right) requiring a reduced level of tonic biceps discharge. It is apparent in Fig. 6 that the biceps stretch reflex, in spite of an intention calling for relaxation, depends on the level of tonic activity present at the time the stretch is applied. Thus, stretch during a high level of tonic biceps discharge (Fig. 6, left) evokes a considerably greater reflex than stretch delivered on a background of reduced biceps discharge (Fig. 6, right). In considering the two different stretch-evoked responses shown in Fig. 6, it should be kept in mind that the required background activity of the biceps will involve tonic co-activation of alpha and gamma motoneurons (Granit, 1970). The biceps alpha motoneurons will thus be receiving descending instructions t o keep firing as well as

9 15 M6 7 D 04 M X M X

BIN

1 5 M 5 OD 04

1 5 TRIALS

1 0 MSEC

192

M X MX

15 T R I A L S

BIN 10 M S E C 192

V8j

II

32

105

15 M6 7 D 0 4 M X M X

---1

SEC---

105

32

15 TRIALS

15 M5 O D 04 M X M X

--1

1 5 TRIALS

SEC---

Fig. 6. Both a t the left and a t the right a n intended movement of pronation was triggered by a perturbation (pronation) which stretched biceps and evoked biceps discharge via reflex pathways in spite of intended relaxation. The reflex excitation was much more pronounced, however, when stretch was delivered during a high level of tonic biceps discharge (at left) than during a reduced level of biceps discharge (at right). At the right, t h e steady-state load was reduced. Thus, t h e magnitude of the stretch reflex can be seen to depend o n the tonic level of motoneuron discharge present a t the time the stretch is delivered.

peripheral support across the gamma loop via the Ia afferents. In Fig. 6 (left) the instruction to maintain handle position against the force of the torque motor has mobilized the segmental apparatus to a level of activity that enables it to counteract (for awhile) the command to relax the biceps. In this connection it should be recalled that there are lingering aftereffects of fusimotor gamma action that may keep contracting spindles facilitated long after cessation of stimulation (Hunt and Kuffler, 1951, since often confirmed). In the light of what has been pointed out above, the striking effects of load on stretch reflexes seem well explained. ( V ) Changes of tendon jerk depending on intended movement Hammond (1956) did not observe effects of intention on the shortest latency response to muscle stretch (the tendon jerk), whereas Hagbarth (1967) did observe such effects; two important differences in their experimental paradigms may explain this discrepancy. First, Hagbarth gave subjects blocks of trials in which each successive intended movement was the same, whereas in Hammond’s experiment the intended movement varied from trial to trial. Fig. 7 shows a series of biceps stretch responses (records 7-9) in which there is a progressive reduction in the tendon jerk as the subject repeats the same intended pronation triggered by biceps stretch. Note that, on the first intended supination (record

10

4 =TFig, 7. This figure illustrates 1 2 successive responses t o biceps stretch. The first 5 trials involved intended supination, and for all of these trials the tendon jerk was followed by intense biceps discharge associated with intended supination. For trial No. 6 (second row, second trace from left) the instruction called for pronation. On this first intended pronation the tendon jerk was unchanged but later muscle discharge was virtually absent. F o r trial No. 9 (third row, leftmost trace) the tendon jerk had disappeared. Then, o n trial No. 1 0 the intended movement was supination (after a series of 4 pronation movements). Here t h e tendon jerk was virtually absent. On the next trial (No. 11)the tendon jerk amplitude had increased, and o n trial No. 1 2 (third row, rightmost trace) t h e original tendon jerk amplitude had been restored. Handle movements are indicated by t h e heavy trace below EMG records, with downward deflections indicating pronation and upward deflections indicating supination.

10) following a series of pronations, the biceps tendon jerk remains absent. Thus, there is a cumulative effect of prior performance on the tendon jerk. A second difference between the experiments of Hammond and Hagbarth was in the nature of the intended movements. Hammond instructed subjects to resist or to give way in response to the perturbation, whereas Hagbarth instructed subjects to flex or extend the elbow. Thus, Hagbarth called for one active movement or an opposite active movement, whereas Hammond called either for an active response or no response. Clearly our paradigm corresponded to the one employed by Hagbarth. Of the two differences in experimental procedure, the grouping of similar trials seems to be the more important: very striking changes in tendon jerk such as those illustrated by Hagbarth (1967) required that a series of one type of movement be compared with a series of the other type of movement. For the experiments illustrated in Figs. 2 through 6, the successive instructions were delivered in a predetermined pseudorandom order which made it equally probable that a given movement would follow a like or unlike prior movement. This process of randomization tended to minimize the effects of the intended movement on the tendon jerk.

11 DISCUSSION The most general conclusion that can be drawn from these observations is that the responses initiated by a perturbing supination easily fit into the predictions based on present knowledge (above, p. 3). There is the initial unloading reflex, silencing the biceps motoneurons, and, when the instruction is t o supinate, the pause is interrupted by the intended response of contraction (Figs. 2 and 3). When the instruction requires pronation, the active relaxation which elicits reciprocal inhibition of the biceps from contracting pronators adds up with the unloading effect t o produce virtually complete silence (Fig. 2). This may also entail a suprasegmental descending component of biceps inhibition. When the perturbing trigger is a pronation (Figs. 4 and 5), however, the ensuing stretch reflex, succeeded by the well-known transient decrease of activity (the silent period), interacts in a more complex way with the instruction. When the latter is a demand for supination (Figs. 4, top, and 5, right), the combination of maintained stretch with a contraction added should be a powerful stimulus for the muscle’s Golgi tendon organs (Granit, 1950; Hagbarth and Naess, 1950; Granit and Strom, 1951). The segmental effect should consequently be a strong inhibition of the biceps motoneurons. Nothing of the kind is seen. The command t o contract is “allowed” t o be executed as if the tendon organ inhibition itself had been inhibited in the Ib interneurons which, according to Lucas and Willis (1974), are located in Rexed’s lamina V of the spinal cord. Under the circumstances (Figs. 4, top, and 5, right) there is less of a silent period following the monosynaptic stretch reflex; in fact, in some cases it is wholly obliterated. A supraspinal inhibition of the tendon organ interneurons has been postulated by Hufschmidt (1966) t o explain the absence of a silent period in voluntary contraction. In this combination of stretch plus contraction (Figs. 4, top, and 5, right) the stretch reflex has added afferent alpha-gamma linked depolarization of the motoneurons t o the voluntary demand for the same effect. While it may be difficult t o assess the relative magnitudes of the shares contributed from the two sources, it can be shown that the contribution from the Ia afferents definitely is present. This emerges from the experiment of Fig. 5 (left). In it the triggering pronation initially produces the monosynaptic stretch reflex followed by the characteristic depression (the silent period). Tne intended response is active relaxation (pronate) which, as stated, is inhibitory on the biceps motoneurons. In spite of this, the reflex effect may persist for 150 msec. It must have been quite powerful t o compensate for the intended suppression of biceps activity. From the point of view of the demanded action (pronate), this delayed reflex has the character of a “constant error” of the kind underlying motor illusions. Considering these results in the light of the current discussion (see below) of transcortical loops in the stretch reflex, it is clear that with sufficient segmental mobilization of the mechanisms in the spinal cord, here achieved by a tonic discharge to maintain handle position, the stretch reflex does exist as a reflex in its own right. Fig. 6 shows how this effect is reduced with reduced general activation of the biceps motoneurons. This is well understood (Granit, 1970).

12 Similarly, it has been pointed out (Matthews, 1972) that the mere presence of an unloading reflex is a sign of the importance of the segmental contribution from the co-activated muscle spindles. For all of the reflexes referred to above, the effects of intention are apparent with latencies of well under 100 msec from the kinesthetic input. Since response latencies less than 100 msec have commonly been taken as ruling out “voluntary movements”, it has seemed that a “presetting” of the spinal cord reflex mechanism by tonic descending of inputs must explain reflex variations depending on the subject’s intention; this was the explanation proposed by Hammond (1956) and by Hagbarth (1967). In support of this explanation, Tanji and Evarts (1976) have shown that monkey motor cortex pyramidal tract neurons (PTNs) exhibit directionally related responses t o instructions for movements to be made in response to subsequent arm perturbations. The paradigm employed by Tanji and Evarts in monkeys was the same as the one described in the present report for human subjects. These directionally related changes of PTN activity may constitute a mechanism for presetting of spinal cord reflexes as a function of the intended movement. While in no way denying a role for presetting of spinal cord reflexes in genesis of intention-dependent responses t o load disturbances, several recent observations show that short ( E

-100 4

u

5msec

Fig. 4. Intracellular muscle action potentials evoked by single stimulation of ventral rootlets. In every record the to p trace shows zero membrane potential and lower trace the intracellularly recorded muscle action potential. Record 1: RMP -60.0 mV, AP 108.0 mV, L 3.8 msec. Record 2: RMP -68.0 mV, AP 111.2 mV, L 7.9 msec. Record 3: RMP -102.0 mV, AP 155 mV, L 10.0 msec. Record 4: RMP -60 mV, AP 79 mV, L 12.0 msec.

TABLE I Spike potential Responded singly Number of observations

249

Junction potential Responded doubly 7

12

Percentage for total number

(%I Resting membrane potential ( m V) (mean + S.D.)

93.0

4.4

2.6

68.1 t 15.5

62.4

?

12.8

first Latency (msec) (mean t S.D.) Amplitude of potential (mV) (mean A S.D.)

7.0 i 79.0

i

50.7

t

7.5

9.0

t

2.5

second

2.2

9.4

t

1.6

12.8

t

2.7

20.9

62.4

t

19.0

52.1

t

30.8

22.8 i 9.8

128 1

3

2

4

lOmsec

Fig. 5. Double shock stimulation with decreasing time interval. RMP -53 mV, AP 7 8 mV, L 5.0 msec. Record 1: time interval is 9.05 msec, record 2: 4.29 msec, record 3: 2.48 msec, and record 4: 1.92 msec. In every record top trace shows zero membrane potential together with the stimulus artifacts and lower trace t h e intracellularly recorded muscle potential.

Since dantrolene does not suppress a tetanic contraction it was difficult to successfully record the muscle potential continuously. In Fig. 5 one record is shown. It was found from these recordings that the least stimulus interval was 1.92 msec. This kind of test was made on 5 muscle fibers which showed spike potentials with latencies of 5.0-9.6 msec. The least stimulus intervals obtained were between 1.92 and 2.25 msec but their numerical value was not related t o their latency. The same test was tried in one muscle fiber which showed a junction potential. It seemed t o show a summation of junction potentials, but due to the muscle contraction its amplitude of depolarization began t o decrease and it was impossible to be certain of this summation. Statistical calculations were made from the results of Table I. Important and significant statistical differences were found between the latency of the junction potentials and those of the spike potentials which responded singly and between the latency of spike potentials occurring singly and the latency of the first spike of those fibers giving double responses. The difference between the latency of the junction potentials and that of the first occurring spike potentials where double responses were elicited was not statistically significant.

DISCUSSION Since the naturally separate fine fasciculi of ventral roots which were stimulated in this study contained both alpha- and gamma-motor nerves, it would be natural t o consider that the intracellularly recorded muscle potentials consist of ones from extrafusal fibers as well as from intrafusal fibers. In a strict sense, it is necessary to make histological identification of the origin of the potentials as coming from extrafusal or from intrafusal fibers. In these experiments, three kinds of muscle potential were recorded. One of them is a junction potential. It is improbable that stimulation of alpha-motor fibers would evoke junction potentials in the extrafusal muscle fibers of cat soleus muscle. According t o the work of Bessou and Pag& (1972), junction potentials are commonly seen in intrafusal fibers. Another characteristic muscle potential was the one which showed two responses after a single stimulation of the ventral rootlets. Even the first of the

129 double responses had a longer latency compared with that shown by a single action potential. The mean latency of these first occurring spikes of a double response is of the same order as that of the junction potential. If it is considered that a naturally separate ventral fasciculus contains many gamma fusimotor fibers and that one spindle receives a supply of several fusimotor fibers (Boyd, 1962; Bessou and Laporte, 1966; Brown et al., 1969; Barker et al., 1970), it seems easy to explain the nature of the double response to single stimuli (Fig. 3) and repetitive occurrence of the junction potentials (Fig. 2, record 3) as being the responses to more than one motor axon. In this experiment, however, we do not know the origin of the junction potentials. They may be caused by a special type of motor ending or by an electrotonic spread of the action potential propagated along the muscle fiber from the opposite side of one spindle capsule to the site of impalement. As the small diameter of the intrafusal muscle fibers seems to make it difficult to record muscle potentials intracellularly, the probabilities found of detecting junction potentials (4.4%) and double evoked responses (2.6%) seem to be reasonable ones. If we consider that the spike potentials which occurred singly with long latency have been recorded both from extrafusal and intrafusal fibers, then the probability of recording intrafusal action potentials may become higher than 2.6% and the mean latency of the extrafusal action potentials shorter than 7.0 msec. The least stimulus interval to evoke a double response was between 1.92 and 2.25 msec, which is almost the same value quoted by Bessou and Pag6s (1.4 msec) (1972). As these values were obtained from the muscle fibers which evoked spike potentials with short latency, which presumably suggests that the fibers are extrafusal, it seems to indicate that the electrical membrane properties of extra- and intrafusal muscle fibers are not very different.

SUMMARY The experiments were carried out on cats of similar sizes and anesthetized with urethane and chloralose. Soleus muscles were placed in a pool of Ringer’s solution with intact nerve and blood supplies. Naturally separate fine ventral fasciculi were stimulated and potentials of soleus muscles were recorded intracellularly by glass capillary microelectrodes filled with 3 M KC1. To minimize any extrafusal contraction, dantrolene sodium, an excitation-contraction coupling blocker, was injected intravenously. Muscle potentials were recorded from 268 muscle fibers, 4.4% of the muscle fibers evoked junction potentials with a mean latency of 9.0 msec while 2.6% of them showed a double response which could be a spike potential followed by another spike potential or a spike potential followed by a junction potential. The mean latency of the first occurring spikes was 9.4 msec. The remaining muscle fibers evoked single spike potentials with latencies of 2.65-15.6 msec. From these results it was assumed that the junction potential and the double response potential were recorded from intrafusal muscle fibers.

130 ACKNOWLEDGEMENTS

I would like t o thank Dr. P.H. Ellaway (Department of Physiology, University College London) for valuable advice and correcting the English usage during the preparation of this manuscript. REFERENCES Barker, D., Stacey, M.J. and Adal, M.N. (1970) Fusimotor innervation in the cat. Phil. Trans. B, 258: 315-346. Barker, D., Emonet-DBnand, F., Laporte, Y., Proske, V. and Stacey, M. (1973) Morphological identification and intrafusal distribution of the endings of static fusimotor axons in the cat. J. Physiol. (Lond.), 230: 405-427. Bessou, P. and Laporte, Y. (1966) Observations on static fusimotor fibers. In Nobel S y m posium I. Muscular Afferents and Motor Control, R. Granit (Ed.), Almqvist and Wiksell, Stockholm, pp. 81-89. Bessou, P. and Pagss, B. (1972) Intracellular potentials from intrafusal muscle fibers evoked by stimulation of static and dynamic fusimotor fibers. J. Physiol. (Lond.), 227: 709727. Boyd, I.A. (1971a) The mammalian muscle spindle - an advanced study (film). J. Physiol. (Lond.), 214: 1-2P. Boyd, I.A. (1971b) Specific fusimotor control of nuclear bag and nuclear chain fibers in cat spindles. J. Physiol. (Lond.), 214: 30-31P. Boyd, I.A., Gladden, M.H., McWilliam, P.N. and Ward, J. (1973) Static and dynamic fusimotor action in isolated cat muscle spindles with intact nerve and blood supply. J. Physiol. (Lond.), 230: 29-30P. Brown, M.C. and Butler, R.G. (1973) Studies on the site of termination of static and dynamic fusimotor fibers within muscle spindles of the tenuissimus muscle of the cat. J. Physiol. (Lond.), 233: 553-573. Brown, M.C. and Butler, R.G. (1975) An investigation into the site of termination of static gamma fibers within muscle spindles of the cat peroneus longus muscle. J. Physiol. (Lond.), 247: 131-143. Brown, M.C., Lawrence, D.G. and Matthews, P.B.C. (1969) Static fusimotor fibers and the position sensitivity of muscle spindle receptors. Brain Res., 14 : 173-187. Ellis, K.O. and Bryant, S.H. (1972) Excitation-contraction uncoupling in skeletal muscle by dantrolene sodium. Naunyn-Schmiedeberg’s Arch. exp. Path. Pharmak., 274: 107109. Ellis, K.O. and Carpenter, J.F. (1972) Studies on the mechanism of action of dantrolene sodium - a skeletal muscle relaxant. Naunyn-Schmiedeberg’s Arch. exp. Path. Pharmak., 275: 83-94. Nakajima, Y. (1975) Effects upon spindle discharges of electrical stimulation of static fusimotor fibers with concomitant application of muscle vibration. Jap. J. Physiol., 25: 4 17-4 3 3. Lowndes, H.E. (1975) Dantrolene effects on neuromuscular function in cat soleus muscle. Europ. J. Pharmacol., 32: 267-272.

DISCUSSION MATTHEWS: Your personal results are very exciting when we think how much effort you have devoted t o dissecting isolated muscle spindies in order t o impale them, and you of course have some very interesting experiments to d o almost at once. Firstly, if you were to stimulate a single gamma fiber instead of alpha and gamma together, when you could be certain that some of your potentials were coming from a spindle. The other experiment I know

131 you have in mind is that you can label t h e fibers that you recorded from and then see whether it is the spindle or whether it is perhaps equally from a n interesting kind of extrafusal fiber which is not shown u p by stain methods.

NAKAJIMA: I once tried to stimulate a single gamma fiber, but t o record a response from t h e soleus muscle is very difficult. So I stimulated a large number of axons rather than a single fusimotor fiber. LAPORTE: I could suggest that you stimulate 10-15 gamma fibers together. I t is quite possible to d o this. With a single one you have no chance a t all of finding a spindle by it.

This Page Intentionally Left Blank

Effects of FM Vibration on Muscle Spindles in the Cat MUNEAKI MIZOTE Department of Physiology, School of Medicine, Chiba University, Chiba (Japan)

INTRODUCTION Constant frequency vibratory stimulation may allow us to distinguish the response of a primary ending from that of a secondary ending in muscle spindles. But the responses of nuclear bag fibers have never been divided functionally by mechanical stimulations from those of nuclear chain fibers in muscle spindles. It has been suggested that nuclear bag fibers are more viscous and nuclear chain fibers purely elastic in muscle spindles of the cat (Boyd, 1971). The mechanical properties obtained by ramp stretches of muscle spindles led t o a model of intrafusal muscle fibers (Matthews, 1964;Houk, 1966;Crowe, 1968). It is possible, therefore, that the different visco-elastic properties of the two kinds of intrafusal muscle fiber may allow us t o distinguish responses of nuclear bag fibers from those of nuclear chain fibers by using various modes of stretch. It is believed that frequency modulated ( F M ) vibration influences particularly the velocity sensitivity of intrafusal muscle fibers. The present paper shows that responses of primary endings can be divided into categories which correspond to the two types of intrafusal muscle fibers. METHODS The experiments were carried out on cats weighing 2.0-3.5 kg, which were anesthetized intraperitoneally and muscles dissected free of surrounding tissue, while keeping intact as much as possible of the blood supply. All other nerves in that hindlimb were cut except the gastrocnemius and soleus nerves. A laminectomy was performed to expose the dorsal and ventral roots between L5 and S1. They were cut at their entry into the spinal cord.'The dorsal root L7 was split into fine filaments for the isolation of single primary afferent fibers, which were identified as spindle endings by their behavior during twitch contractions of the muscle elicited by stimulating its nerve (Matthews, 1933). They were classified as primary or secondary endings on the basis of the conduction velocity of their afferent fibers. All primary endings studied had afferent fibers which conducted at over 80 m/sec. The peripheral ends of the cut L7 ventral root were subdivided into about 20 approximately equal filaments

134 which were classified as exerting a static or dynamic type of fusimotor action by the effect of stimulation of the filament at 100 Hz on the response of a spindle to different kinds of stretch. Filaments which on stimulation caused a marked increase of the dynamic index to a ramp stretch and a silent period on the releasing phase of 3 Hz sinusoidal stretch (the amplitude is less than 500 pm) were classified as having a dynamic fusimotor action, while those which on stimulation caused a decrease of the dynamic index in response t o 3 Hz sinusoidal stretch and a driving effect were classified as static fusimotor (Crowe and Matthews, 1964a, b). A primary ending was always excited strongly by gamma fusimotor fibers, in spite of the concomitant contraction of the extrafusal fibers due to excitation of alpha motor fibers on stimulating a ventral rootlet. Mechanical sinusoidal vibration was applied to the tendon of the muscle through a steel hook. Vibration of 0.5 sec duration was repeated 5 times or more every 2.0 sec (Homma et al., 1972). The initial muscle length was determined by the single shock stimulus of ventral root. Primary endings discharging spontaneously at the shortest muscle length were then neglected. The threshold of a primary ending to vibratory stimulation was obtained as the smallest amplitude of vibration which elicited only a single Ia spike and discharged a Ia spike of more than 80% on repeated stimulation periods. Two types of vibration were used, characterized by their different frequency components. One was a constant frequency of vibration (upper trace in Fig. 1)and in the other the frequency was modulated by continuously increasing frequency from one value to another (lower trace in Fig. 1).In this paper, they are termed CF vibration and FM vibration respectively. Frequencies of vibration were varied from 1 0 to 100 Hz. The amplitude excursion of vibration was detected electronically by a difference transformer in the vibrator and could be compensated for as the frequency was changed by a feedback system, to an accuracy of the order of 5% in the frequency band used. Amplitude values were indicated on an electronic meter. The amplified action potentials of the single Ia fiber and the displacement due to vibration were recorded simultaneously on separate channels of the magnetic tape recorder.

constant frequency vibration I

I

I

I

I

upward FM vibration

0 Ssec

Fig. 1. Vibration of 0.5 sec duration was repeated 5 times every 2.0 sec. Two types of vibration (CF and FM) were used characterized by their different frequency components. In this figure the action potential of the single Ia fiber and the displacement due t o vibration are shown in the upper and lower trace respectively.

135 RESULTS Fig. 2 shows the threshold amplitude at which a constant frequency of vibration elicits a Ia spike on each occasion of steps of 10 Hz from 10 t o 100 Hz. The abscissa shows the frequency of vibration and the ordinate shows the smallest amplitude of vibration which elicits only the one spike. These frequency characteristics could be divided into two types by CF vibration over this frequency band. One shows a basically hyperbolic curve which decreases monotonically as the frequency increases. The other shows a parabolic curve which opens upward and has the lowest threshold in the middle of the band of applied frequency. In this paper, the former type of curve is labeled B and the latter labeled K. In Fig. 2, 5 primary endings are illustrated for both types of curve. The effects of FM vibration on primary endings showing curves of both type B and K were investigated in Fig. 3. An arrow shows the threshold t o FM vibration of primary endings. The direction of an arrow indicates an increasing frequency for FM vibration. In type B all the arrows cross the curve of a constant frequency vibration. On the other hand, arrows of type K never cross the curve. This means that for type K receptors tested below about 50 Hz the same value of threshold is given by the two methods of determination. In contrast, type B

400

2oc

50

ZHZ

50

‘OOHZ

Fig. 2. The abscissa shows t h e frequency of vibration and the ordinate shows the smallest amplitude of vibration which elicits only t h e one spike. These frequency characteristics were classified by CF vibration and could be divided into tw o types (type B and type K ) over this frequency band.

136 pm

200-

100-

i

200-

100-

50

100

50

HZ

100 Hz

Fig. 3. An arrow shows the threshold to FM vibration of primary endings. The direction of a n arrow indicates an increasing frequency for FM vibration. All t h e arrows cross t h e curve of a constant frequency vibration. O n t h e other hand, in t y p e K, arrows never cross the curve.

receptors are shown as having a lower threshold when tested by CF rather than by FM stimulation. The significance of this difference between types B and K seems of potential interest. For frequencies above 50 Hz the FM method can give no measure of the rising phase of the curve determined by CF and simply indicates the lowest threshold occurring in the “trough” at about 50 Hz. After primary endings were classified as either type B or type K by CF vibration and FM vibration respectively, gamma fusimotor fibers were stimulated electrically at 100 Hz in Fig. 4.

rm 200

100

50

’O0Hz

50

“OHz

Fig. 4. After stimulating static fusimotor fibers the response pattern of a primary ending which showed type B properties (solid line) changes into o n e showing type K properties (dotted line). In contrast, as shown on t h e right figure, type K receptors are not changed by stimulation of static gamma fusimotor fibers.

137

100-

50

l0*HZ

Fig. 5. After eliciting dynamic gamma fusirnotor fiber excitation, type K receptors (solid line) change into type B (dotted line). In contrast, as shown on the right figure, type B receptors remain unchanged by dynamic fusimotor fiber stimulation.

After producing static fusimotor excitation the response pattern of a primmy ending which showed type B properties changes into one showing type K properties, and thresholds are always significantly lower. In contrast, type K receptors are not changed by stimulation of static gamma fusimotor fibers, in spite of thresholds having been lowered. In Fig. 5, after eliciting dynamic gamma fusimotor fiber excitation, type K

1

0

0 1 50 'DOH z Fig. 6. This figure shows the response properties of the separate secondary endings to CF vibration and FM vibration. These response patterns are very similar to those of the type K receptors already described for primary endings. '

138 receptors change into type B and thresholds are also lowered. In contrast, type B receptors remain unchanged by dynamic fusimotor activation. Fig. 6 shows the response property of the separate secondary endings to CF vibration and FM vibration. The response patterns are very similar t o those of the type K receptors already described for primary endings.

DISCUSSION The frequency-response curves of primary endings were not always divided simply into two types. The various complex types in which types B and K were mixed were observed about 30%. But in this paper two typical types are described. The threshold of type B is lower on CF vibration than on FM vibration and is lowest in the highest frequency band. Type B receptors respond effectively to the velocity of vibration. In contrast, in type K the threshold t o CF vibration almost coincides with that to FM and is lowest in the middle of the frequency band. Type K receptors respond effectively to the displacement of vibration. These results indicate that the type B receptors show viscous properties and the type K receptors elastic properties respectively. It has been reported that the nuclear bag fibers are viscous and the nuclear chain fibers are purely elastic (Boyd, 1971). Boyd and Ward (1975) reported that repetitive stimulation of a fusimotor axon produced visible contraction in the bundle of nuclear chain fibers or contraction in nuclear bag fibers, but not in both. On the gamma fusimotor fiber stimulation, it is suggested that changes of type are relative t o those of contractile intrafusal muscle fibers in their study, In the present paper, two kinds of frequency-response curve of primary endings were obtained on sinusoidal stretching (type B and K). On the other hand, Goodwin and Matthews (1971) have obtained the frequency-response curve of primary endings which increased monotonically as frequency increased from 0.1 t o 100 Hz. These differences depend upon the experimental arrangement, namely, they used a very small amplitude of stretch and a very large initial muscle length. In contrast, the author used a large amplitude and a very small muscle length. As a result, type B shows the frequency-response of the nuclear bag fibers and type K of the nuclear chain fibers. SUMMARY (1)Longitudinal C F and FM vibrations were applied t o the de-efferented gastrocnemius or soleus muscles of anesthetized cats while recording the discharge of single afferent fibers from the proprioceptors within the muscle. (2) Frequencies of vibration of 10-100 Hz were used. The maximum amplitude of vibration was 500 pm (peak t o peak) at 20 Hz. (3) The frequency-response of the primary endings t o CF and F M vibration was divided into two types, type B and type K. Type B receptors are very sensitive t o CF vibration but not so t o FM vibration, while, in contrast, type K receptors are very sensitive t o FM vibration.

139 ( 4 ) Type B properties changed into those showing type K properties after static gamma fusimotor fibers were stimulated electrically. On the other hand, type K were changed into type B by the stimulation of dynamic gamma fusimotor fibers. (5) Type B receptors show the response properties of nuclear bag fibers and type K receptors those of nuclear chain fibers.

ACKNOWLEDGEMENTS

I would like to thank Dr. P.B.C. Matthews and Dr. P.H. Ellaway for valuable advice during the preparation of this manuscript. REFERENCES Boyd, I.A. ( 1 9 7 1 ) Specific fusimotor control of nuclear bag and nuclear chain fibers in cat muscle spindles. J. Physial. (Lond.), 214: 30-31P. Boyd, I.A. and Ward, J. (1975) Motor control of nuclear bag and nuclear chain intrafusal fibers in isolated living muscle spindles from the cat. J. Physiol. (Lond.), 244: 83-112. Crowe, A . ( 1 9 6 8 ) A mechanical model of the mammalian muscle spindles. J. theoret. Biol., 2 1 : 21-41. Crowe, A and Matthews, P.B.C. (1964a) The effects of stimulation of static and dynamic f u s h o t o r fibers o n the response t o stretching of the primary endings of muscle spindles. J. Physiol. (Lond.), 1 7 4 : 109-131. Crowe, A . and Matthews, P.B.C. (1964b) Further studies of static and dynamic fusimotor fibers. J. Physiol. (Lond.), 1 7 4 : 132-151. Goodwin. G.M. and Matthews, P.B.C. ( 1 9 7 1 ) Effects of fusimotor stimulation o n the sensitivity of muscle spindle endings t o small-amplitude sinusoidal stretching. J. Physiol. (Lond.), 218: 56-58P. Homma, S., Mizote, M., Nakajima, Y. and Watanabe, S. ( 1 9 7 2 ) Muscle afferent discharges during vibratory stimulation of muscles and gamma fusimotor activities. Agressologie, 13: 45-53. Houk, J.C.( 1 9 6 6 ) A model adaptation in amphibian spindle receptors. J. theoret. Biol., 1 2 : 196-215. Matthews, B.H.C. (1933) Nerve endings in mammalian muscle. J. Physiol. (Lond.), 7 8 : 1-53, Matthews, P.B.C. ( 1 9 6 4 ) Muscle spindles and their motor control. Physiol. Rev., 4 4 : 219288.

DISCUSSION MATTHEWS: Here we have had the second paper o n sinusoidal stretching today, and you may perhaps feel that it conflicts somewhat with what I have already said. But I should emphasize that we are doing different things in different ways. Dr. Mizote is classifying his primary ehdings into t w o distinct groups o n sinusoidal stretching. I have my primary endings in one group with sinusoidal stretching. The first difference between our experimental arrangements, which I have had the opportunity of learning by being in Dr. Mizote’s laboratory, is that we are using different sizes of stretch. He is using what I call a large stretch, 400 pm, but this is an equally acceptable stretch, and so we are working in different parts of the range. That, however, is a doubtful difference. The interesting difference is that we have been working o n nearly the full length of the muscle with everything stretched tight. Dr. Mizote is working with t h e muscle very very slack. In Dr. Mizotc’s experiments, the inside

of t h e muscle spindle must look like the pictures tha t Dr. Gladden has shown us before she put the acetylcholine on, and it is quite possible that when the spindle is made so slack, then t h e branches o n one kind of intrafusal fiber are working and those o n another kind of intrafusal fiber are inactive. So it is possible that Dr. Mizote is showing differences in behavior which one seesin the very slack muscle, but which one does not see when the muscle is made tight.

Role of Abortive Spike on Encoding Mechanism in Frog Muscle Spindle F. I T 0 and Y.I T 0 Department of Physiology, Nagoya University School of Medicine, Nagoya 466 (Japan)

INTRODUCTION Abortive spikes have been found in the frog muscle spindle by Katz (1950), who has considered them t o be the same as the prepotential for triggering propagated spikes. A hypothesis has been proposed on the encoding mechanisms in the frog muscle spindle in which sensory impulses may be initiated at a point in the axon terminal where a summation of abortive spike and generator potential exceeds a threshold, and therefore the site of impulse initiation may vary from a portion along the non-myelinated branches t o the bifurcating node of the myelinated branches, or to the more proximal nodes with different degrees of spindle stretch (It0 e t al., 1974). In accordance with this hypothesis, one would expect that reducing the generator potential would enhance the ratio of the population of abortive to that of propagated spikes in a train of spontaneous static afferent discharges, and would also produce abortive spikes of large amplitude which remain insufficient t o attain the threshold. Evidence should be put forth t o substantiate the above hypothesis. The generator potential may easily be reduced by slackening the spindle receptor, but this simultaneously results in a decrease in the rate of abortive spike discharges. Hyperpolarization of the sensory terminal seems t o be the best method of reducing the generator potential, but it is difficult to insert a microelectrode into a non-myelinated terminal of approximately 1pm or less. Maintaining the normal rate of the discharge by maintaining the spindle length, the generator potential may be cancelled for the duration of the after-hyperpolarization following orthodromic and antidromic impulses, which are known t o invade the sensory nerve terminals through an axon reflex and antidromically (It0 et al., 1974). These authors have demonstrated that the time course of the after-hyperpolarization recorded intracellularly from the myelinated sensory nerve terminal was well coincident with that of the positive after-potential following the propagated spike recorded extracellularly by the paraffin gap method. The existence of the after-hyperpolarization a t nerve terminals is a well-documented phenomenon (cf. Eccles and Krnjevid, 1959a, b; Hubbard and Schmidt, 1963).

142 METHODS Twenty-seven experiments were carried out on semi-isolated preparations of single-type muscle spindles in the frog’s sartorius muscle. The single parent axon of a spindle receptor was isolated in its intramuscular course until the capsule of the spindle receptor was cleared, but the spindle capsule and intrafusal muscle bundle remained intact within the dissected extrafusal muscle fibers. Motor innervations to intra- and extrafusal muscle fibers were removed. This semi-isolated preparation was used to determine the static lengths of muscle spindles. The sartorius muscle was maintained for 3.5 min at different lengths, from slack to 130% of the in situ length (100%).The preparation was placed in a Ringer’s pool in a perspex box, and the isolated nerve was passed into another Ringer’s pool through a liquid paraffin pool of 1mm length. The paraffin pool was situated in a slit of 1mm at the center of a partition between the two Ringer’s pools. The distance from the boundary t o the capsule of the isolated spindle was usually kept within 300 pm. A pair of calomel electrodes was inserted into subsidiary Ringer’s pools, each of which was connected t o the two Ringer’s pools by means of two Ringer-Agar bridges. Six experiments were made on isolated single-type muscle spindles for observing abortive spikes in a semi-blocked condition caused by tetrodotoxin. The isolation and the mounting of the preparation in a chamber have been described in detail previously (Ito et al., 1974). Microapplication of tetrodotoxin was performed with microelectrodes filled with 2 M NaCl containing the toxin at a concentration of 1 X g/ml. The resistance of the electrodes was between 1 0 and 20 M a . The tip of the electrode was brought near the surface of the spindle capsule under binocular microscopic observation, and then exactly into contact with a chosen point on the surface of the intrafusal fiber under inverted microscopic observation. The toxin was applied with a current of 1-100 nA ejected from the microelectrode in series with a 500 M a resistance, connected via a constant current stimulus isolation unit to the Ringer-Agar bridge in the spindle pool. The current was measured by means of an operational amplifier. In some preparations, currents were applied to the surface of the intrafusal fiber through microelectrodes filled with 2 M NaCl alone (resistance 10-20 M a ) as a control. Antidromic stimulation was applied t o a portion of the parent axon approximately 25 mm distant from the spindle capsule, through a pair of platinum electrodes. Antidromic impulses were elicited by pulses of 0.05 msec duration derived from a constant current stimulator. Differences in potential between the two pools were fed into a high input impedance amplifier, displayed simultaneously with time and amplitude calibration on a cathode ray oscilloscope, and photographed on running film. The record during the initial 1 5 sec after stretching the muscle was discarded because the rate of discharges changed markedly depending upon the velocity of the stretch during the initial period. The amplitudes of individual abortive spikes and the intervals between successive spikes were measured from the film with the aid of the calibration. Some of the data were coded into a paper tape which was used for constructing amplitude histograms of abortive spikes by means of a computer (FACOM 270-20). The interval histograms were con-

143 structed by hand from the intervals between a propagated spike and the subsequent propagated (P-P) or abortive spikes (P-A), or those between an abortive spike and the subsequent propagated (A-P) or abortive spikes (A-A).

RESULTS

Amplitudes of abortive spikes during the period o f positive after-potentials It has been found that the positive after-potential attained a maximum amplitude of about 100-300 pV, approximately 15 msec after the orthodromic impulse, and then decayed gradually, lasting for approximately 100 msec (It0 and Kuroda, 1972). A similar time course of after-hyperpolarization was also observed following orthodromic impulses recorded intracellularly from the sensory nerve terminal (It0 et al., 1974). In this experiment, the period of 70 msec following each orthodromic spike was taken as the effectively operating period of the hyperpolarization in the sensory terminal membrane in the in situ length, because it is developed over this period. Fig. 1 represents a conspicuous example of the amplitude histograms of abortive spikes which were measured from a continuous record obtained from a spindle receptor. The amplitude of abortive spikes occurring within 70 msec following each propagated spike fell in two separate groups: from 40 t o 240 pV, and from 420 t o 480 pV (Fig. 1, P-A < 70 msec). The amplitude of abortive spikes occurring independently of the after-hyperpolarization was characterized by one group (Fig. 1, P-A > 70 msec), the distribution of which was almost identical t o that of the smaller group of abortive spikes during afterhyperpolarization. The mean amplitude of the abortive spikes during after-

t

P-A < 7Qmsec '

O

E

C

$

0

s

L

L 100

L I

0

100

200

300

Amplitude ( p V )

400

A-A

500

0

100

200

I

300

-200

300

II

400

Amplitude ( p V )

Fig. 1. Amplitude histograms of abortive spikes. Left side: two kinds of amplitude distribution of abortive spikes occurring within (P-A < 70 msec) and after 70 msec (P-A > 70 msec) following each propagated spike, measured from a continuous record for 3 min. Right side: histograms without (A-A) and during repetitive antidromic stimulation at 1 Hz (S-A) measured from a continuous record for 30 sec.

144 hyperpolarization was 157.9 pV, which was approximately twice the amplitude of those (71.0 pV) occurring in the absence of the after-hyperpolarization. Similar results were obtained from all of the preparations tested. Similar phenomena can also be observed during positive after-potential following antidromic spikes. In a preparation maintained at the in situ length, the amplitude of abortive spikes preceded by abortive spikes were chosen and measured as a control. Two groups of the amplitude histograms of the abortive spikes during spontaneous discharge and during repetitive antidromic stimulation at 1 Hz in Fig. 1were obtained from a series of continuous records for 82 sec respectively in the same preparation. In the intact condition, the amplitudes of abortive spikes fell between 20 and 240 pV. During repetitive antidromic stimulation at 1 Hz, the amplitude distribution (S-A < 70 msec in Fig. 1)of abortive spikes occurring within 70 msec following antidromic stimuli was distinctly larger than that (S-A 2 70 msec in Fig. 1)occurring after 70 msec. It is also noticeable from Fig. 1that the population of abortive spikes during repetitive antidromic stimulation is considerably larger than that during spontaneous discharge.

Effect of stretch upon interval histograms When the muscle was maintained at the in situ length (100%)abortive spikes were followed by subsequent abortive or propagated spikes with short latencies, ranging from a few msec to 220 msec (A-A or A-P in Fig. 2, loo%), while propagated spikes were always followed by a silent period of 60-90 msec, subsequently propagated and abortive spikes occurred within 500 msec. This is in parallel with the fact that the propagated spike is always followed by an afterhyperpolarization but the abortive spike is not. The total numbers of propagated and abortive spikes in 3 min were 750 and 397 respectively in this case. The characteristic pattern in the interval histogram was accentuated when the same muscle was extended to 110%, as shown in Fig. 2 (110%). The interval histograms of P-P and P-A ranged from 30 t o 290 msec with peaks at approximately 100 msec, while those of A-P and A-A showed a skewed distribution with peaks at 10-20 msec. The shorter pause following propagated spikes at 110% in comparison with that at 100% implies that the silent period may not be a fixed phenomenon but may consist of an interaction between a generator potential as a facilitatory mechanism and an after-hyperpolarization as an inhibitory mechanism. The total numbers of propagated and abortive spikes were 1301 and 665 respectively in 3 min. The ratio of propagated t o abortive spikes was approximately 2 : 1, i.e., identical t o that at 100% length in this preparation. Interval histograms with a large population and with a narrow distribution were obtained when the above-mentioned receptor was kept at 120%, as shown in Fig. 2 (120%). The narrow distribution showed a rhythmic discharge, and the large population represents a higher frequency of discharges. The fact that the ratio of abortive spikes (684 in 3 min) t o propagated ones (2499) at 120% length is distinctiy smaller than those at 100 or 110%suggests that many of the abortive spikes may be transformed into propagated spikes with greater mechanical stimulation.

145 100 "1.3

I LA-A 66

P A

t

a

L

236

-

P

crcl

0 100

200

AP

P 515

S1E

100

120 "i,

1 (1 0

300

1 1 0 "1.

P -A

L60

100 P - P 841

100

200

Interval

0

100

Im s e c l

Fig. 2. Interval histograms of successive spikes obtained from a preparation during maintained loo%, 110%and 120% length. Individual histograms consist of the sum of the intervals, characterized by t h e kinds of t h e intervening spikes, obtained from a continuous 3 min record. A-A: intervals between successive abortive spikes; A-P: intervals between abortive and the subsequent propagated spikes; P-A: intervals between propagated and the subsequent abortive spikes; P-P: intervals between successive propagated spikes. The numbers near the above symbol represent the total number in each histogram.

Interval histograms modulated b y repetitive antidromic stimulation A t a spindle stretch of 110%,in which the interval histogram represented the characteristic pattern, the sensory nerve was stimulated antidromically. Repetitive antidromic stimuli at 1 Hz did not produce a significant change in the population and shape of the histograms, though stimuli at 5 Hz did; 357 orthodromic spikes and 384 abortive spikes were followed by orthodromic propagated spikes (S-P) with latencies ranging from 50 t o 195 msec (Fig. 3). The fact that the antidromic stimuli are also followed by a silent period of approximately 50 msec (S-P or S-A) indicates that the antidromic spike is also accompanied by an after-hyperpolarization similar to that following an orthodromic propagated spike. The first orthodromic spikes following an antidromic spike were also accompanied by one or several propagated or abortive spikes (A-A, A-P, P-A or P-P in Fig. 3). The total numbers of orthodromic propagated and abortive spikes were 630 and 511 respectively. Repetitive antidromic stimuli of 10 Hz suppressed orthodromic discharges more strongly, being followed by only 90 propagated and 175 abortive spikes. Only 35 abortive spikes (S-A) survived

146 5 Hz

In

1 10 Ole

A-A 215

C .-0

10 Hz A-A

c,

r

0 v-

0

A-P 5 S-A 167

0

20 H z

L

S-A 35

s - P 85

J Z

[u (L,

n

0

Interval

100

[ J

LLU

0

60

(rnsec)

Fig. 3. Interval histograms without (left side) and during repetitive antidromic stimuli a t 5, 10 and 20 Hz. Individual histograms were obtained from a continuous 3 min record in t h e same preparation as that in Fig. 2, during maintenance a t 110% length. All of t h e symbols are the same as in Fig. 2. S-A: intervals between antidromic stimuli and t h e abortive spikes; S-P: intervals between antidromic stimuli and t h e propagated spikes.

during antidromic stimuli at 20 Hz, and no orthodromic spikes could be detected during stimulation at 25 Hz or more. Antidromic stimulation at 20 Hz or more may be supposed to produce a large hyperpolarization, which has less ripple, at the nerve terminal. At all frequencies of antidromic stimuli, abortive spikes were more resistant t o suppression than propagated spikes. This supports the concept that the after-hyperpolarization may increase the triggering threshold for propagated spikes.

Amplitude histograms of abortive spikes modulated by repetitive antidromic stimulation In the in situ muscle length (loo%), 4 groups of abortive spikes were observed in the amplitude histogram (Fig. 4,100%). During antidromic stimulation of 5 Hz, the populations of abortive spikes with smaller amplitudes appeared t o be depressed more strongly than those with larger amplitudes. The spontaneous discharges disappeared during repetitive antidromic stimulation at 10 Hz or more. The amplitude histogram of abortive spikes at the 100% length consisted of 6 groups. These peaks appeared, in principle, t o be multiples of 50 pV (Fig. 4, 110%). Repetitive antidromic stimulation at 5 Hz reduced the populations of low amplitude, between 40 and 200 pV, but increased those of the two groups between 240 and 320 pV. At 10 Hz, the total population of the latter groups became larger than those of the former .groups. At 1 5 Hz and 20 Hz, the two groups in the histogram at 200 and 250 pV remained during stimuli, and no abortive spikes were observed during stimuli over 25 Hz. A similar depression in the amplitude histogram by antidromic stimuli of 5-25 Hz was also observed

147 m

1 1 0 "10

100 "lo

C

.-0 m al

m 0

120 "lo

_t ll

> L

n

100

'O0r

0

c

0

100

200

300

0

100

200

300

-I

f

.

0

100

I

.&

ZOHz

=

25HZ 200

300

A m p l i t u d e ( pV ) Fig. Amplitude histograms of abortive spikes obtained --om a spindle receptor during maintained muscle length at loo%, 110% and 120%, without (uppermost of each group of histograms) and during repetitive antidromic stimulation a t increasing rates (5, 10, 15, 20 and 25 Hz).

in the histograms constructed from abortive spikes at 120% or more (Fig. 4, 120%). Amplitude histograms o f abortive spikes in the semi-blocked condition produced b y tetrodotoxin Fig. 5A represents a control in which the amplitude histograms of propagated and abortive spikes in an intact preparation consisted of a distribution for propagated spikes, the peaks of which varied between 0.5 and 1.8 mV in different preparations, and of a separate distribution with two or more distinct peaks for abortive spikes which usually ranged between 10-30 and 50-100 pV. At 1 5 sec, during microapplication of the toxin with 5 nA current (Fig. 5B), individual peaks of the amplitude histograms were unchanged, although the populations of the higher amplitudes diminished, In the semi-blocked condition, in which propagated spikes disappeared but abortive spikes survived, small populations of the two lower amplitude groups of abortive spikes survived (Fig. 5C). After cessation of tetrodotoxin application, the amplitude histograms of abortive spikes showed an increased skewness toward higher amplitudes, matching the increase in the rate of abortive spikes (Fig. 5D). A group in the abortive spike histogram, which was always higher (100-150 pV) than the ordinary two groups, occurred during the period when propagated spikes returned (Fig. 5E). As the recovery progressed, the population of the third group with the highest amplitude increased (Fig. 5F). After the population of the third group attained a maximum 1-2 min after cessation of tetrodotoxin application, it diminished,

148

' L

E L 0

E--L

50

100

150

550

I

600

650

AmDlitude ( p V ) Fig. 5. Amplitude histograms of abortive and propagated spikes obtained from a preparation during maintained muscle length at loo%, before (A), during (B at 15 sec, C at 30 sec) and after (D immediately after, E at 15 sec, F at 30 sec, G at 2 min) the microapplication of tetrodotoxin with 5 nA. Each histogram consists of the sum of the amplitudes obtained from a continuous 10 sec record. Sample traces of these spikes are shown near the letter designating the individual stages.

while the populations of the two groups of lower amplitude for abortive spikes and that for the propagated spikes increased (Fig. 5 G ) . The change in the population of the third group of abortive spikes suggests, possibly, that those with the highest amplitude may be replaced by propagated spikes in the course of recovery from the semi-blocked condition. The normal amplitude distributions of the propagated and abortive spikes usually returned within 30 min after cessation of the application of the toxin. The duration of the effect is considered to be evidence that the block was not caused by the electrical current used during the iontophoresis of tetrodotoxin, but rather, due to the tetrodotoxin itself. The above experiments could be observed repeatedly, and the change in the, amplitude histograms was reversible and consecutive. The above results imply that the abortive spikes left in the semi-blocked condition may not differ in principle from those observed in an intact preparation. The amplitude histograms of successive abortive spikes under the semiblocked condition were characterized by elimination of the larger amplitude group with a peak at 200 pV (Fig. 6B, A-A), and also by the absence of the difference between the group occurring within 70 msec following antidromic

149

-

"

f m

'"Er, ; O

-

I

- _

I

f

.

f

=5

10 p

2

-

1-.1

0

71! r , . e c

-

S-?\ 70 msec, S-A < 70 msec) as obtained from two additional 8 2 sec records in the same semi-blocked preparation.

stimuli (S-A < 70 msec in Fig. 6) and that occurring after 70 msec (S-A 2 70 msec in Fig. 6). These results suggest that the antidromic impulses may be unable t o invade into the non-myelinated terminals whereas the after-hyperpolarization following the propagated impulses may do so, cancelling the generator potential. Stretch of muscle spindle in the semi-blocked condition In order to assess whether a generator potential elicits abortive spikes alone or triggers both the abortive and propagated spikes, different amounts of generator potentials are elicited by different degrees of spindle stretch in the semiblocked condition. Fig. 7 illustrates an example of such experiments. Microapplication of tetrodotoxin for 20 sec with a current of 5 nA through a microelectrode of 20 M a removed the propagated spikes, but left the abortive spikes occurring at a mean rate of 1.4/sec, as shown in Fig. 7A. When the muscle spindle was stretched from its in situ length (100%) to 110%length with a constant velocity of 4 mm/sec, a burst discharge of abortive spikes and a train of monophasic spikes, of which the amplitude was approximately 6 times larger than that of abortive spikes, occurred for 0.5 sec near the completion of the stretch (Fig. 7 3 ) . A few propagated spikes, each of which had a larger positive than negative deflection, as was the case in the spontaneous propagated spikes just before completion of blocking by tetrodotoxin microapplication, occurred during a stretch t o 120% length. The rate of abortive spikes in the burst was 8/sec for a 10% stretch and 14/sec for a 20% stretch. Both the large monophasic and positively shifted propagated spikes were blocked by the addition of tetrodo: toxin at 2 X lo-' g/ml to the receptor bath (Ito, 1971).

150 A

J

1mV

0.2 sec

p1

Ii""

II I I: :

Fig. 7 . Responses recorded from a preparation during stretch in the semi-blocked condition. A: spontaneous abortive spike discharges during maintained 100% length. B: responses during stretch of the spindle from 100 to 110%length with a constant velocity of 4 mmlsec. C: during stretch from 100 to 120%length.

These results suggest that the rate of production of abortive spikes depends upon the amplitude of the generator potential. It seems likely that the large monophasic spikes may be due t o an all-or-none response on one of the nonmyelinated branches, which may be triggered by a summation of generator potential and abortive spikes. In intact muscle spindles, spontaneous propagated impulses may be able to arise as a synchronous excitation of the non-myelinated branches beyond the non-subdivided branch. Consequently, the large monophasic spike is possibly a part of the impulse. DISCUSSION

Peripheral inhibition caused by after-hyperpolarization Floyd and Morrison (1974) have investigated the process of collision of orthodromic and antidromic impulses in branches of slowly adapting splanchnic mechanoreceptors. Such a collision of action potentials may contribute slightly to the depression of afferent discharges, but may not be the dominant factor, as has been pointed out by Brokensha and Westbury (1974). Measurements of the depression following orthodromic or antidromic impulses in the present experiments have shown that the depression is 1 0 times longer than the conduction time of the impulse along the isolated axon, which was calculated to be approximately 5 msec from the length of 50 mm at 10 m/sec (cf., Ito et al., 1964). The resulting shortening of the depression, effectively enhancing the excitability of the receptor with static extension of the spindle, suggests that the process of depression may be due to an activity of the axon terminal membrane in competition with the generator potential. The after-hyperpolarization is probably capable of generating such a depression. It has been proposed that

151 repetitive firings in mammalian muscle spindle are paced, or reset, by an afterhyperpolarization following each impulse at the sensory terminal (Fohlmeister et al., 1974). There is much evidence indicating that the after-hyperpolarization is of particular importance for the repetitive impulse firing of spinal motoneurons (e.g., Eccles et al., 1958; Baldissera and Gustafsson, 1971; Kernell and Sjoholm, 1973), and that the after-hyperpolarization is due to potassium permeability changes (e.g., Connor and Stevens, 1971a, by c,; Kernell and Sjoholm, 1972). The same ionic process is also thought to be essential in the after-hyperpolarization following an impulse at the sensory nerve terminal (Fohlmeister et al., 1974).

Abortive spikes and generator potentials in the encoding process The hypothesis that propagated sensory impulses may be generated as the sum of a biasing mechanism of the generator potential and triggered by abortive spikes which may also be elicited by the generator potential, is supported by the following results. (1)At the in situ length, orthodromic and antidromic propagated impulses were always followed by a pause of 60-80 msec in duration, which was identical to that of the after-hyperpolarization, while abortive spikes, without afterhyperpolarization, were not followed by any pause. The duration of the pause was shortened by extending the spindle, by which the amplitude of the generator potential might also be increased. These facts imply that the after-hyperpolarization may be able to negate the effects of generator potential. (2) Temporal summation of the after-hyperpolarization induced by repetitive antidromic stimulation resulted in a distinct reduction in the population of propagated spikes, with less decrease in that of abortive spikes. It is likely that abortive spikes may fail to develop into propagated spikes during reduction of the generator potential by the after-hyperpolarization. Temporal summation of the after-hyperpolarization has been observed in a motoneuron (Ito and Oshima, 1962) and computed in a membrane model (Kernell and Sjoholm, 1973). (3) The amplitudes of abortive spikes occurring during the after-hyperpolarizations following orthodromic impulses were larger than those of abortive spikes in the absence of after-hyperpolarization. As the rate of repetitive antidromic stimulation was increased, the population of abortive spikes decreased, and the amplitude distribution changed from a normal distribution t o one that showed an increased skewness toward larger amplitudes, and then the spikes of relatively small amplitude disappeared. It is difficult for EPSPs t o reach the threshold level of depolarization in a motoneuron until the after-hyDerpolarization following an SD spike has subsided (Eccles, 1953). (4) Stretch of the spindle in the semi-blocked condition resulted in a burst discharge of abortive spikes and half-sized propagated action potentials. This suggests that propagated impulses may be initiated at an area along the nonmyelinated branch where summation of currents, due t o the abortive spikes and generator potentials, exceeds a threshold. In the normal physiological condition the impulse may be able to arise at more proximal nodes along the

152

parent axon with generator currents converging from the terminal branches when the spindle is stretched strongly, especially during dynamic stretching. In this encoding mechanism, the abortive spikes may play a role as a trigger for generating propagated impulses in cooperation with the generator potential. Such a role for abortive spikes is consistent with that of the fast prepotential in the cat’s hippocampal neuron (Spencer and Kandel, 1961) or the dendritic trigger spikes in the hyperpolarized cortical neuron (Purpura and Shofer, 1964). Temporal and/or spatial integration between abortive spikes and the generator potential along the non-myelinated branches may also contribute to a kind of peripheral analysis of the sensory information. SUMMARY Amplitude and interval histograms of abortive and/or propagated spikes were constructed from trains of spontaneous discharges recorded from isolated and semi-isolated single-type spindles in frog sartorius muscle at steady states of various degrees of extension. The amplitude distribution of the spikes occurring within 70 msec following orthodromic propagated spikes, in which after-hyperpolarization was conspicuous, included a group distinctly larger in comparison with that occurring later than 70 msec. This implies that the larger abortive spikes may remain insufficient t o reach the threshold for triggering propagated spikes during the after-hyperpolarization. Static extension of the muscle spindle from 100% t o 130% result9d in a shortening of the pause from 70 msec to 20 msec following individual propagated spikes, and also gave rise t o a prominent increase in the probability of appearance of abortive spikes. During the period of after-hyperpolarization accompanying an antidromic spike, the occurrence of orthodromic spikes was suppressed strongly but smaller effects were observed on abortive spikes; even in a high frequency invasion by repetitive antidromic stimulation a few abortive spikes often survived. The amplitude distribution of abortive spikes which survived during repetitive antidromic invasion shifted to become a group of spikes which were large relative t o those obtained without antidromic invasion. Microapplication of tetrodotoxin produced a semi-blocked condition, in which propagated spikes disappeared but abortive spikes survived. Amplitude histograms of the abortive spikes during this condition indicate that antidromic impulses are unable to invade the nerve terminal. Stretching the muscle spindle in this condition gave rise to a burst discharge of abortive spikes and half-sized action potentials. The above results were discussed to assess the hypothesis that the abortive spikes as a trigger may be summated with the generator potential t o initiate impulses on the sensory nerve terminal, where inhibition may also occur by after-hyperpolarization. ACKNOWLEDGEMENTS The authors wish to thank Dr. L.M. Vernon for improving the English. This study was supported by a Research Grant from the Ministry of Education (010607).

153 REFERENCES Baldissera, F. and Gustafsson, B. (1971) Regulation of repetitive firing in motoneurones by the after-hyperpolarization conductance. Brain Res., 30: 431-434. Brokensha, G . and Westbury, D.R. (1974) Adaptation of the discharge of frog muscle spindles following a stretch. J. Physiol. (Lond.), 2 4 2 : 383-403. Connor, J.A. and Stevens, C.F. (1971a) Inward and delayed outward membrane currents in isolated neural somata under voltage clamp. J. Physiol. (Lond.), 213: 1-19. Connor, J.A. and Stevens, C.F. (1971b) Voltage clamp studies of a transient outward membrane current in gastropod neural somata, J. Physiol. (Lond.), 213: 21-30. Connor, J.A. and Stevens, C.F. ( 1 9 7 1 ~Prediction ) of repetitive firing behaviour from voltage clamp data on an isolated neurone soma. J. Physiol. (Lond.), 213: 31-53. Eccles, J.C. (1953) The Neurophysiological Basis of Mind. The Principles of Neurophysiology, Clarendon Press, Oxford. Eccles, J.C. and KrnjeviE, K. (1959a) Potential changes recorded inside primary afferent fibres within the spinal cord. J. Physiol. (Lond.), 1 4 9 : 250-273. Eccles, J.C. and KrnjeviE, K. (1959b) Presynaptic changes associated with post-tetanic potential in the spinal cord. J. Physiol. (Lond.), 149: 274-287. Eccles, J.C., Eccles, R.M. and Lundberg, M. (1958) The action potentials of the alpha motoneurones supplying fast and slow muscles. J. Physiol. (Lond.), 142: 275-291. Floyd, K. and Morrison, J.F.B. (1974) Interactions between afferent impulses within a peripheral receptive field. J. Physiol. (Lond.), 238: 62-63P. Fohlmeister, J.F., Poppele, R.E. and Purple, R.L. (1974) Repetitive firing: dynamic behavior of sensory neurons reconciled with a quantitative model. J. Neurophysiol., 37 : 1213-1227. Hubbard, J.I. and Schmidt, R.F. (1963) An electrophysiological investigation of mammalian motor nerve terminals. J. Physiol. (Lond.), 1 6 6 : 145-167. Ito, F. (1971) Effects of tetrodotoxin on responses of the frog muscle spindle. Jap. J. Physiol., 2 1 : 349-358. Ito, F. and Kuroda, H. (1972) The positive after-potential following the orthodromic and antidromic propagated impulses in the frog muscle spindle. Jap. J. Physiol., 2 2 : 4 4 1-4 5 2. Ito, F., Toyama, K. and Ito, R. (1964) A comparative study on structure and function between the extrafusal receptor and the spindle receptor in the frog. Jap. J. Physiol., 14: 12-33. Ito F., Kanamori, N. and Kuroda, H. (1974) Structural and functional asymmetries of myelinated branches in the frog muscle spindle. J. Physiol. (Lond.), 241 : 389-405. Ito, M. and Oshima, T. ( 1 9 6 2 ) Temporal summation of after-hyperpolarization following a motoneurone spike. Nature (Lond.), 195: 910-911. Katz, B. (1950) Action potentials from a sensory nerve ending. J. Physiol. (Lond.), 1 1 1 : 2 4 8-2 6 0. Kernell, D. and Sjoholm, H. (1972) Motoneurone models based on ‘voltage clamp equations’ for peripheral nerve. Acta physiol. scand., 8 6 : 546-562. Kernell, D. and Sjoholm, H. (1973) Repetitive impulse firing : comparisons between neurone models based on ‘voltage clamp equations’ and spinal motoneurones. Acta physiol. scand., 8 7 : 40-56. Purpura, D.P. and Shofer, R.J. (1964) Cortical intracellular potential during augmenting and recruiting responses. I. Effects of injected hyperpolarizing currents on evoked membrane potential changes. J. Neurophysiol., 27: 117-132. Spencer, W.A. and Kandel, E.R. (1961) Electrophysiology of hippocampal neurones. IV. Fast prepotentials. J. Neurophysiol., 24: 272-285.

DISCUSSION BUCHTHAL: I would like t o ask you if you observed spontaneous contraction of intrafusal fibers at the same time.

ITO: No, I’ve never observed contraction of the intrafusal muscle fibers, because in my preparation the motor axons are not intact. STUART: How often do you think these abortive spikes are occurring under natural conditions? ITO: In almost all preparations tested by me the abortive spikes were present in frog muscle spindles. However, I don’t know whether abortive spikes occur in mammalian muscle spindles. HENNEMAN: In a few experiments I’ve observed that a prolonged tetanic stimulus t o a muscle nerve results in a definite decrease in firing of afferents from the stretch receptors in the muscle. But my impression was that the recovery was much more rapid than it usually is for a posttetanic potentiation. Do you have any thoughts about that? ITO: I’m sorry I can’t answer that.

BOYD: I would just like t o compliment Prof. Ito on the considerable achievement of getting a microelectrode into the first order of the afferent fiber and I’d like to ask about your showing the branching of the terminals distributed in a rather different way to the two intrafusal fibers that you found in each of these sartorius spindles. Is there anything different about these inputs? Is there any mechanical difference for example? ITO: I’m very interested in that point. However, I can’t answer that because I can’t yet separately stretch each kind of intrafusal fiber. I’d like t o do experiments on this.

SESSION 111

MUSCULAR AFFERENTS ASSOCIATED WITH STRETCH REFLEX

Chairman: Y. Laporte (Paris)

This Page Intentionally Left Blank

Nature of the Persisting Canges in Afferent Discharge from Muscle following its Contraction EARL ELDRED, ROBERT S. HUTTON

* and JUDITH L. SMITH

Departments of A n a t o m y and Kinesiology and Brain Research Institute, University o f California at Los Angeles, Los Angeles, Calif. 90024 (U.S.A.)

One day long ago when I was learning about spindles in Professor Granit’s laboratory, I heard “Professor” exclaim that the isolated spindle afferent we were listening t o on the loudspeaker was “hung up”. We had been following the discharge of this afferent through an isometric contraction induced by stimulation in the brain, and after the muscle relaxed, the firing rate had not returned to the prestimulus level (cf., Hnik et al., 1969). A momentary tug on the tendon brought the firing rate down again. I realized then that under some circumstances the passive spindle shows behavior that is not related in a straightforward way to static and changing muscle length. Hunt and Kuffler, in their exploration of the effects on spindle afferents of single y motoneurons, had noticed that spindle excitation was sometimes followed by a persisting elevation of the discharge (Kuffler et al., 1951) and enhanced sensitivity t o subsequent y efferent stimulation (Hunt and Kuffler, 1951). This “postexcitatory facilitation” disappeared following a brief stretch and they suggested that it was due to some physical change in the intrafusal fibers. Granit et al. (1959) also had the impression that the phenomenon had a mechanical basis which did not necessarily involve intrafusal contraction, since the alteration appeared when stimuli of low strength were applied t o the muscle nerve. They saw at times a converse effect, wherein a rise in discharge appeared upon tapping the muscle tendon. Ten years later, Brown et al. (1967) made a detailed analysis of the postexcitatory effect as seen in single afferent fibers following stimulation of dynamic and static fusimotor fibers. Both types produced a greater discharge and stretch sensitivity, and they suggested that following contraction of the intrafusal fibers, persistence of cross bridges between actin and myosin filaments might leave the motor poles somewhat shortened and stretch the sensory ending. This concept of residual cross linkages had been advanced by Hill (1968) t o explain changes in extrafusal muscle rigidity following contraction. A quite different explanation was introduced by Kidd (1964a, b) t o account for the persistent afferent discharge following contraction of tail muscles in the rat, which was not abolished by brief stretch. He suggested that K’ ions released into interstitial spaces during contraction may have partially depolarized

* Present address: School of Physical and Health Education, University of Washington, Seattle, Wash. 98195, U.S.A.

158 the sensory endings and led to the acceleration in discharge (Kidd et al., 1971a, b). Hnik and his coworkers at about this time had been studying an increase in afferent discha.rge that appeared several weeks after de-efferentation or tenotomy (Hnik et al., 1963; Hnik, 1964b; Hutton et al., 1975). These conditions, of course, are associated with atrophy and they reasoned that the effect on afferent discharge was probably due to release to metabolic products. When Hnik with Payne (1965) and later with KuEera and Kidd (1970) monitored the afferent discharge from muscles undergoing contraction and found a persisting afferent increase, they thought this might have a similar explanation. Supporting these arguments was the demonstration that contraction can cause K‘ ions t o appear in the venous drainage in concentrations that were comparable t o those found intravenously when K’ was given close intra-arterially in amounts sufficient to cause spindle acceleration (Hnik et al., 1969). More recently, Hnik et al. (1972, 1973) have made refined observations on K’ release using an electrode inserted into the muscle, which seem t o show that the concentrations of K’ ions found in interstitial spaces might be adequate, if brought in contact with nerve endings, to cause significant depolarization. A further complication arose as to the identity of the afferents responsible for the postcontraction rise in afferent activity. Hnik and his coworkers (Hnik et al., 1963; Hnik 1964a, b; Hnik and Payne, 1966) in their studies of the effects of tenotomy and de-efferentation had come to the conclusion that the delayed increase in discharge in these conditions for the most part did not arise in the encapsulated muscle receptors, but in some unidentified receptor, perhaps an ending concerned with the metabolic regulation of a muscle. With contraction, too, it was thought that spindles were but little affected and the increased volume of afferent activity arose in some other receptor (Hnik et al., 1969, 1970). In the rat, for instance, increasing the strength of the stimulus applied to the muscle nerve to bring in the fusimotor neurons was found t o have no greater effect than stimuli just sufficient to give a maximal contraction (Hnik and Payne, 1965). Also, enhanced levels of discharge were obtained from muscles experimentally rendered free of spindles, or which had their fusimotor innervation blocked by a local anesthetic. We were led t o look at the postcontraction increase in sensory discharge through our own observations on the effects of tenotomy (Yellin and Eldred, 1970; Estavillo et al., 1973). In the cat, we had not seen a late increase in ongoing afferent activity recorded from the dorsal roots and we turned, therefore, t o look also for the increase in sensory inflow following contraction that Hnik and Payne (1965) had described. In most preparations it was an obvious phenomenon. Our conclusions as to the source of the discharge (Hutton et al., 1973; Smith et al., 1974), nevertheless, differ and will now be described. The typical pattern of the postcontraction sensory discharge (PCSD), as it will be called to avoid premature commitment to any hypothesis of its cause, is seen in the polygraph traces of Fig. 1. Represented are the partially integrated activity from the medial gastrocnemius muscle of the otherwise denervated leg as it was recorded from severed rootlets at the L7 and S1 dorsal levels. At the bar labeled “tetanus”, a 10-sec 100-Hz train of stimuli was applied t o the L7 and S1 ventral roots at twice the twitch threshold, the roots being cut close t o the cord and well-isolated so as to minimize current spread. The leg and ankle

159

n

I

STRETCH

TETANUS

I TAP

Fig. 1. Postcontraction sensory discharge (PCSD) from the cat’s medial gastrocnemius muscle of partially integrated afferent activity recorded from the severed L7 and S1 dorsal rootlets.

were fixed by pins and clamps, so that the muscle length was unchanged, except for that due to intrinsic elasticity. After the stimulus artifact, the afferent activity in three of the roots is seen to be elevated above the control level. A maximum in activity was reached in about 20 sec and thereafter there was a gradual falling away to a plateau that persisted until the tendon was lightly tapped. This brief extension of the muscle, after causing a brief dip in discharge to below the control level (best in the second trace), was followed by a return to the prestimulus level. No PCSD appeared in the rootlet which failed to show a response to stretch of the muscle. Three characteristic features of the PCSD, as it was seen in cat, rat and guinea pig muscles, are demonstrated: the usual delay to a maximum, the persistence over minutes, and the susceptibility t o brief extension of the muscle. Although the PCSD was immediately erased by brief extension of the muscle, temporary shortening of the muscle was without effect. In Fig. 2, upper trace, after tetanus and development of the PCSD, the puller tied t o the severed tendon was released for 30 sec (a) and then returned t o the original position (b). During the fall in tension, of course, a marked decline in level of discharge occurred. Upon re-extension, the activity showed a short dynamic overshoot and then returned to essentially the same level it had before the passive shortening. Further extension of the muscle (c) by just 1mm and return to the baseline length (d) was found to have abolished the PCSD. The lower trace in the figure, from another cat, demonstrates the comparable lack of effect from active muscle shortening. Twitches were induced by stimulating the ventral roots, while the muscle remained attached under light tension to the puller. The elevation in discharge continued unabated until a stretch of comparable brief duration abolished it. The duration of the contraction needed t o elicit a PCSD was brief, but a single twitch was not sufficient. As seen in Fig. 3 (right column), 1sec of tetanization gave a good response and although lengthening the period to 5 or 10 sec was somewhat more effective, further increase in spacing or number of stimuli had no greater effect. The maximum in the response seemed to bear closer

160

1 RELEASE

STRETCH

I I min

T

-

T

-r

T

TWITCH

-

r

T

I

& TAP

Fig. 2. Postcontraction sensory discharge and temporary release of the muscle. Further explanation in the text.

STIMULUS DURATION 5sec

STIMULUS

FREQUENCY

7

VMlk 10

I

&,h__

20

w

n

for 2 sec

,

I min

I

Fig. 3. Varied tetanization and resultant change in postcontraction sensory discharge. See text for further explanation.

161 relation to the termination of the stimulus period than its initiation (left column). The identity of the afferent primarily responsible for this effect became clear upon monitoring the discharge of single units isolated from the dorsal roots. Many spindle afferents showed a discharge pattern consistent with the multiunit responses recorded from dorsal roots. Of the 14 primary afferents isolated in the experiment represented in Fig. 4,only two failed to give a postcontraction response. In a population of 78 primary afferents collected from 8 cats, 46 !showed enhancement of activity. Only two of 21 secondaries gave a response, the several tendon organs tested gave no response, and there were no responding units which could not be identified as stretch receptors. Thus, the PCSD, for the most part, seems to arise in Ia afferents. Although the steady discharge was instantly reduced t o the pretetanic level by a momentary stretch of only 1 or 2 mm, sensitivity of the receptors to stretch did not wholly return to the control status. Moreover, the muscle which demonstrated no elevation in steady discharge under a submaximal stimulus might still exhibit enhanced responses to stretch, as seen in Fig. 5 when the stimulus was at 1.1 X threshold. Enhanced levels were seen until the muscle was lengthened several millimeters beyond the length at which it was held during tetanization. The muscle of Fig. 5 demonstrated increased activity t o stretches over a range of 8 mm. A fundamental step toward understanding the cause of the sensory changes would be to determine whether contraction of the gross muscle was needed if intrafusal fibers alone participated, or if perhaps the activation of both contributed. In the preparation of Fig. 5, graded stimuli were applied t o the ventral

UNIT

L7DR h

7

,

I

1

UNIT

rww*uuy

UNITS

5

1.2

, I

I

UNIT

l3 I4

n

n

I

I

II a i z

L

.

JL 5 TRE TCH

I,

TETANUS

l

m

u I j _ i . "

n

I

" '

- .L

m

'

L JL

Fig. 4. Consistency of postcontraction sensory discharge monitored by single afferent with the multiunit response. See text for further explanation.

162 roots to observe effects of differential stimulation of the large, low-threshold Q fibers and the smaller, higher-threshold and y fibers. With the stimulus only 1.1 X threshold for a twitch contraction, an effect on stretch sensitivity can be detected, even though no certain elevation of the steady discharge is seen until the stimulus strength approached 2.0 X threshold. If it be accepted that the very largest axons lead only t o extrafusal fibers, it may be concluded that their contraction was sufficient to leave an effect on the spindles. A higher stimulus strength, however, was decidedly more effective, as may be seen in the responses in dorsal root and unit activity in the upper half of Fig. 6 to stimuli of different threshold multiples. Moreover, it was noted that as the frequency of stimulation was raised from the 30 Hz needed to yield a smooth tetanus t o 100 Hz, the response was further enhanced. Intrafusal fibers are susceptible to this higher rate of stimulation. Evidence for a fusimotor-intrafusal fiber contribution also was obtained by testing for the presence of PCSD in the course of progressive block by gallamine. This is known to block (Y before y motoneuronal junctions. In the preparation of Fig. 7, gallamine had already been given and as seen from the sensitive tension trace, the muscle showed almost no response t o single stimuli applied t o the ventral root. These single stimuli (a+) failed to arouse a PCSD, but a 2-sec period of stimulation (at e) left a slight aftermath of elevated discharge and with 8 sec of stimulation, a fair discharge was obtained. The tension of the gastrocnemius rose less than 0.5 g. The lower trace in Fig. 6, also taken

NO STIMULUS

S,DR LENGTH TENSION

_

w

SLACK

'

TRACE

zmm

4

NO STIMULUS

,

.I m i n

1

Fig. 5. Postcontraction effect on triceps surae responses to stepwise increase in muscle length after tetanic contraction at stimulus strengths 2.0, 1.4 and 1.1 X threshold for a twitch contraction. The first and last traces, controls with no preceding tetanic stimulation. Tension traces are included for the first and second runs, and these are juxtaposed for easier comparison. Millimeter readings give muscle lengthening beyond the point where on the initial run slack in the thread attached to the tendon was taken up.

163

Fig. 6. Stimuli of different tetanic threshold multiples and changes in the postcontraction sensory discharge. See text for further explanation.

1

i

TENSION

I

I

I

I

I

I

L

-rl

Fig. 7. Postcontraction sensory discharge in gallamine-treated preparation. Explanation in the text.

I min Fig. 8. Effect of vibratory stimulation o n postcontraction sensory discharge during progressive neuromuscular blockade. See text for further explanation.

164 after administration of gallamine, demonstrates that efferents with a fairly high electrical threshold contribute. During the onset of paralysis there was progressive loss in the poststimulation response ( a - c ) which was partially reversed when the stimulus was raised from 6 to 8 times twitch threshold. The last illustration is from another animal undergoing progressive neuromuscular blockade, and when the record was taken, stimulation of the ventral root no longer elicited a contraction. Nevertheless, some PCSD was obtained (at a). A second trial 1 min later elicited a rise half as great. The gain was then reduced by half and the baseline lowered in anticipation of a more marked response when a vibrator attached to the tendon was turned on. A vibratory stimulus is particularly effective for revealing postactivation changes in spindle discharge (Hutton et al., 1973), and in this case was able to elicit an enhanced response when the PCSD would have been hardly detectable. At the next trial, during the progressing block, even this effect had disappeared. CONCLUSION Several conclusions were drawn from these observations. (1)A persisting increase in discharge at a maintained muscle length and in response t o stretch appears after a muscle has undergone contraction, under the condition that the efferent background is otherwise quiet. (2) Fusimotor activation alone, i.e., contraction of intrafusal fibers, can produce this effect, though extrafusal contraction also seems to contribute. (3) The enhanced discharge, as it was recorded, arises to a major extent in Ia afferent fibers. (4)The cause of the effect is probably of a mechanical nature. As the last two points do not entirely accord with opinions formed by Hnik, Kidd and their coworkers (Hnik et al., 1969, 1970; Kidd et al., 1971a; Kidd and Vaillant, 1974), they will be discussed. Our belief that the cause of the postcoqtraction effect is mechanical, rather than due to the sensory ending being affected by a contraction-induced change in the metabolic milieu, rests on these arguments. Momentary stretch of the muscle results in an immediate disappearance of the postcontraction effect; it is unlikely that this action would so rapidly dispel an excess accumulation of metabolite that is capable of such long-lasting effect. The duration of stimulus needed for the response is a few tenths of a second; excitatory levels of K' would probably not build up in this time. The elevated discharge and stretch sensitivity persist for many minutes if the muscle is undisturbed; circulation would be expected to sweep away excess K' or other metabolites more quickly. In leg muscles of the dog, for instance, the period of increased interstitial K' lasts less than 1 0 sec (Mohrman and Albrecht, 1973). The maximum in PCSD is more closely related to the end of the tetanic stimulation period than its onset; an accumulation of metabolite would probably plateau at some point in time where removal and production come into balance. The postcontraction effects are intensified when the stimulus rate is increased above the tetanic fusion frequency; in this situation there should be no additional K' release from extrafusal fibers. And most persuasive, the effects are demonstrable even in the absence of an overt contraction during curarization or after reflex elicitation (Hutton et al., 1975).

165 These arguments relate to production and removal of metabolites. An additional argument concerns the identity of the sensory ending involved. Primary afferents seem to be the major source of the postactivation effects. Yet there is no ostensible reason why metabolites released during contraction should not affect secondary spindle and tendon organ terminals as well. Furthermore, Kidd found that it took several minutes after immersion of a small muscle in high K' solution before an increase in spindle discharge developed, a finding that caused him to propose that the spindle capsule acts as a barrier (Kidd et al., 1971b). The capsule may hinder access of K' ions to the spindle afferent terminals, but there is no such barrier protecting the non-encapsulated endings in the muscle, and these afferents may be excited in the immediate postcontraction period, as various autonomic reflex observations suggest. Our observations only indicate that there is seemingly sufficient effect on spindle afferents to account for the rises seen in integrated multiunit discharge recorded from a dorsal root or peripheral nerve. Virtually all group I and I1 axons supply stretch receptors (Boyd and Davey, 1968), so that any other fibers that might give rise to the postcontraction discharge must be group I11 and unmyelinated axons. Although the proportion of small myelinated afferents in a muscle nerve is fairly large, about 25%, their individual contribution to recorded activity is small, for axon potentials recorded extracellularly diminish with fiber diameter. Considering that some background discharge from group Ia axons is always present (Hnik and Payne, 1966; Hnik et al., 1969) and Ia afferents were demonstrated t o participate in the PCSD, it is evident that extremely vigorous activation of the small axons would be needed t o produce increases in discharge of a mean 73% above baseline level, as found by Hnik and Payne (1965), or a peak reaching 20% of maximum physiological stretch response as we sometimes saw. The argument (Hnik et al., 1970) that the posttetanus activity must arise in another undefined ending because it is seen in rat muscles deprived of encapsulated receptors by crushing the nerve in the neonatal period is not convincing, since the large afferents destined for spindles had presumably regenerated along with the other sensory fibers and may still have given rise to the discharge. While some role for released K' in setting the stage for the postcontraction changes is not excluded, the evidence strongly indicates that these are due to some mechanical event affecting the spindles. What could be the nature of this alteration that can lead to increases in discharge of considerable volume, persist for many minutes, and resist several millimeters of muscle stretch? Perhaps more than one factor contributes. Indeed, this would have t o be concluded from the demonstrations that stimulation at either end of the motor axon spectrum had an effect. The positive findings that we and Granit et al. (1959) obtained with low-strength stimuli and that Hnik et al. (1970) found in the presence of procaine block of small diameter efferents indicate that some change in the gross muscle alone can lead t o the changes. One possibility is that the events in relaxation of the muscle after contraction differ from those following passive stretch. In the gastrocnemius particularly this might occur, for slower motor units (and spindles) predominate in central portions of the muscle, SO that this region should relax more slowly after tetanus than the periphery. This could leave in the muscle a slightly different arrangement of connective tissue

and muscle fascicles than when the muscle relaxes after an imposed stretch. Some alteration in geometry or rigidity within the muscle must occur, since after tetanus there is commonly a small residual elevation in tension (Fig. 2). Those experiments in which fusimotor excitation without muscle contraction resulted in an elevated discharge must be explained, of course, by some event related to the contraction of intrafusal fibers. The appearance on histological sections of the close fit of the poles of the capsule about the longer intrafusal fibers suggests one possibility. The arrangement is presumably tight enough to prevent the specialized intracapsular fluid from escaping under lateral pressure from neighboring extrafusal fibers. Perhaps intrafusal fiber movements in either direction are also hindered. If so, there would be more likelihood for frictional arrest if the movement was slow. Thus, the relatively slow return permitted by declining contraction in the motor poles would be more apt to arrest return of the fiber through the capsule collar than when the fiber is adjusting only to the force of axial bundle elastic tissue following relaxation from passive stretch. A converse situation might arise if in robust shortening of the gross muscle a segment of the intrafusal fiber were forced inward through the capsule collar. Then a tap on the muscle tendon, by pulling the fiber farther out through the collar might result in a slight elevation of the discharge, as Granit et al. (1959) described. The intrafusal fibers in this hypothetical mechanism must pass through the capsule collar, and in the feline spindle these are chiefly nuclear bag fibers (Bridgman et al., 1969). It is these fibers which seem to be most altered in the postcontraction period, since primary afferents show an accelerated discharge, whereas the secondary endings, which lie on chain fibers, are less affected, according to our findings and those of Brown et al. (1969). Proske (1974), also, has reported that the initial burst of a primary afferent to stretch is enhanced when a dynamic fusimotor neuron is stimulated, i.e., bag fibers are primarily activated, when static neurons that primarily innervate chain fiber are excited. Bag fibers would be more apt to produce postactivation effects not only because they pass through the collar, but because their relaxation is slower. Finally, as an explanation for the postcontraction effects is the intriguing idea advanced by Brown et al. (1969) that following activation of the intrafusal fibers, there remain residual cross-linkages between actin and myosin filaments that result in greater rigidity and shorter length of the sarcomeres. One of their observations in particular would be difficult t o account for by the collar friction concept. Two fusimotor fibers affecting a given primary afferent were successively stimulated, each at a different intermediate point in the range of excursion of the muscle. Subsequently, upon stretch of the muscle over the entire range, crests in the afferent activity appeared at both the positions at which stimulation had been carried out. This is understandable only if the two fusimotor fibers produced alterations in two separate intrafusal fibers. A difficulty with application of this explanation to the present findings is that according to illustrations presented by Brown et al. (1969), stretching the muscle by 2 mm was enough to abolish the enhanced afferent response, whereas the changes in sensitivity following contraction of the whole muscle may still be detected after as much as 8 mm of extension (Fig. 5). Short-range stiffness in extrafusal tissue (cat soleus, Rack and Westbury, 1972; rat psoas, Ito and

167 Oyama, 1970) is overcome by about a millimeter of stretch. However, as

Brown et al. (1969) point out, in intrafusal fibers where multiple innervation and localized contractions occur, bonds between myofilaments in different sarcomeres need not rupture simultaneously. The giving away of weaker bonding in some sarcomeres would permit regional lengthening before the bonds in other sections of the muscle fiber reached the threshold for rupture. Extensive stretch might be required t o overcome the acquired rigidity over the entire muscle fiber. In this way, the presence of residual bonds after intrafusal fiber activation may account for much, but probably not all, of the enhancement and sensitivity of sensory discharge from the muscle after its contraction.

REFERENCES Boyd, I.A. and Davey, M.R. (1968) Composition of Peripheral hrerues, Livingstone, Edinburgh. Bridgman, C.F., Shumpert, E.E. and Eldred, E. (1969) Insertions of intrafusal fibers in muscle spindles of the cat and other mammals. Anat. Rec., 164: 391-401. Brown, M.C., Engberg, I. and Matthews, P.B.C. (1967) The relative sensitivity t o vibration of muscle receptors of the cat. J. Physiol. (Lond.), 192: 773-800. Brown, M.C., Goodwin, G.M. and Matthews, P.B.C. (1969) After-effects of fusimotor stimulation o n the response of muscle spindle primary afferent endings. J. Physiol. (Lond.) 205: 677-694. Estavillo, J., Yellin, H., Sasaki, Y. and Eldred, E. (1973) Observations on the expected decrease in proprioceptive discharge and purported advent of non-proprioceptive activity from the chronically tenotomized muscle. Brain Res., 63: 75-91. Granit, R., Homma, S. and Matthews, P.B.C. (1959) Prolonged changes in the discharge of mammalian muscle spindles following tendon taps or muscle twitches. Rcta physiol. scand., 46: 185-193. Hill, D.K. (1968) Tension due t o interaction between the sliding filaments in resting striated muscle. The effect of stimulation. J. Physiol. (Lond.), 199: 637-684. Hnik, P. (1964a) The effect of deafferentation upon muscle atrophy due t o tenotomy in rats. Physiol. bohemoslov., 13: 209-215. Hnfk, P. (1964b) Increased sensory outflow from de-efferented muscles. Physiol. bohemoS ~ O V . ,13: 405-410. Hnifk, P. and Payne, R. (1965) Increased sensory outflow following muscle activity. J. Physiol. (Lond.), 181: 36-37P. Hnifk, P. and Payne, R. (1966) The origin of increased sensory outflow from chronically deefferented muscles. Physiot. bohemoslou., 15: 498-507. Hnik, P., Beranek, R., Vyklickjr, L. and Zelen6, J. (1963) Sensory outflow from chronically tenotomized muscles. Physiol. bohemoslov., 12 : 23-29. H n i l , P., Hudlicka, A., KuEera, J. and Payne, R. (1969) Activation of muscle afferents by nonproprioceptive stimuli. Amer. J. Physiol., 217: 1451-1458. Hnik, P., KuEera, J. and Kidd, G.L. (1970) Increased sensory outflow from muscles following tetanic stimulation of alpha motor nerve fibers. Physiol. bohemoslou., 19 : 4954. H n i l , P., VyskoEil, F., K rii, N. and Holas, M. (1972) Work-induced increase of extracellular potassium concentration in muscle measured by ion-specific electrodes. Brain Res., 40: 559-562. Hnfk, P., K rii, N., VyskoEil, F., Smiesko, V., Mejsnar, J., Ujec, E. and Holal, M. (1973) Work-induced potassium changes in muscle venous effluent blood measured by ionspecific electrodes. Pflugers Arch. ges. Physiol., 338 : 177-181. Hunt, C.C. and Kuffler, S.W. (1951) Further study of efferent small-nerve fibres to mammalian muscle spindles. Multiple spindle innervation and activity during contraction. J. Physiol. (Lond.), 113: 283-297.

168 Hutton, R.S., Smith, J.L. and Eldred, E. (1973) Postcontraction sensory discharge from muscle and its source. J. Neurophysiol., 36: 1090-1105. Hutton, R.S., Smith, J.L. and Eldred, E. (1975) Persisting changes in sensory and motor activity of a muscle following its reflex activation. Pfliigers Arch. ges. Physiol., 353: 327-336. Ito, Y. and Oyama, 0. (1970) Change in stiffness of mammalian muscle fibers by stretch. Nagoya J. med. Sci., 33- 131-137. Kidd, G.L. (1964a) A persistent excitation of muscle-spindle receptor endings in the rat following ventral root filament stimulation. J. Physiol. (Lond.), 170 : 39-52. Kidd, G.L. (1964b) A further study of the persistent excitation of primary muscle-spindle endings in the rat. J. Physiol. (Lond.), 176: 5-6P. Kidd, G.L. and Vaillant, C.H. (1974) The interaction of KC and stretching as stimuli for primary muscle-spindle endings in the rat. J. Anat. (Lond.), 119: 196. Kidd, G.L., Kufera, J. and Vaillant, C.H. (1971a) The susceptibility of muscle spindles t o intra-arterial and external applications of solutions of KCl. J. Physiol. (Lond.), 221 : 15-16P. Kidd, G.L., Kufera, J. and Vaillant, C.H. (1971b) The influence of the interstitial concentration of KCon the activity of muscle receptors. Physiol. bohemoslou., 20: 95-108. Kuffler, S.W., Hunt, C.C. and Quilliam, J.P. (1951) Function of medullated small-nerve fibers in mammalian ventral roots: efferent muscle spindle innervation. J. Neurophysiol., 14: 29-54. Mohrman, D.E. and Abbrecht, P.H. (1973) Time course of potassium release from skeletal muscle following brief tetanus. Fed. Proc., 32: 374. Proske, U. (1974) Potentiation by stretch of responses from muscle spindles in the cat. J. Anat. (Lond.), 119: 197. Rack, P.M.H. and Westbury, D.R. (1972) The short range stiffness of active mammalian muscle. J. Physiol. (Lond.), 229: 16-17P. Smith, J.L., Hutton, R.S. and Eldred, E. (1974) Postcontraction changes in sensitivity of muscle afferents to static and dynamic stretch. Brain Res., 78: 193-202. Yellin, H. and Eldred, E. (1970) Spindle activity of the tenotomized gastrocnemius muscle in the cat. Exp. Neurol., 29: 513-533.

ACKNOWLEDGEMENT Figure 5 is from article by Smith, Hutton and Eldred in Brain Res., 78: 193-204, 1974. The other figures are excerpts from illustrations in Hutton, Smith and Eldred, Pfluegers Arch. ges. Physiol., 353: 327-336,1975.

DISCUSSION HOUK: If the effects are strictly mechanical you may be able to obtain them simply by shortening the muscle and pulling it out. The reason why I suggest that is that we have looked a little bit at postcontraction effects, but mainly we’ve looked at the effects of shortening the muscle and then pulling it out slowly and gently. The twitch and tetanic contraction with causal internal shortening could allow some shortening of sarcomeres in the poles of the spindles which could allow a resetting of the static firing. Would the way to test for a purely mechanical effect be simply mechanically shortening? ELDRED: As for the PCSD, i.e., the ongoing discharge, release of the muscle and re-extension to the same length does not abolish it. The level of discharge is about the same as it was before the momentary relaxation.

169 HOUK: If you can abolish it by stretching, would you be able then to reset it by shortening the muscle and then pulling it o u t once more without any contraction? ELDRED: I don’t know. I don’t think shortening alone results in a PCSD. BOYD: Observing the spindle in t h e muscle directly during alpha contraction, the extrafusal contraction unloads t h e intrafusal bundle. If you then recruit the gamma fibers to the bundle you see a straightening o u t and when you turn off the gamma stimulation it doesn’t relax again. So when you look a t the whole bundle, you require t o restretch the muscle before you bring the intrafusal bundle back where it was. That happens in the nuclear bag fibers, but when I observed that I was not looking for t w o kinds of nuclear bag fiber and I supposed it was t h e dynamic o r bag1 fiber. Secondly I did some experiments o n the effects of potassium ion o n isolated muscle spindles and found rather t o my surprise that changing the concentration by quite considerable amounts had negligible effects o n the isolated spindle. I don’t know why. The third point I’d like just t o ask you. You emphasized several times that it was the Ia fiber. Have you got specific recordings showing tha t t h e effect doesn’t show in the group I1 afferent discharge? ELDRED: T wo of 21 secondaries showed the effect. Brown, Goodwin and Matthews, I believe, saw some effect o n the secondaries, but it was not as marked as o n the Ia’s. May I ask you a question? Do you think there is appreciable friction between the capsule and those intrafusal fibers that pass through the capsule collar? Somehow we must explain t h e observation of Granit, Homma and Matthews that tapping the tendon sometimes results in an increase in discharge. BOYD: I couldn’t answer that not having looked specially a t the end of the capsule. I just observed within the fluid space and was struck by the bundle tha t didn’t relax again until it was pulled. HAGBARTH: I’m wondering if this can be regarded as a normal physiological phenomenon which occurs in normal intact animals. In our recording of muscle spindle afferents in man we have never observed anything similar following a voluntary contraction of the muscle. Could it possibly be due t o t h e temperature of the muscle. Your muscle is isolated. Have you observed any difference in t h e temperature of the muscle? ELDRED: You are asking n o t only us but the others who have seen postcontraction effects, Prof. Granit, Homma, Matthews and Dr. Kidd. I don’t think temperature is concerned. BUCHTHAL: With respect to t h e late changes, there are changes i n the human excitability which are very late after the normal mechanical condition is reinstated. I have t h e feeling that it would be of value if we could know a little bit about sarcomere length under these conditions. I’m just wondering if people who work o n bundles have thought a bout using the laser technique. There is a rather inexpensive laser by which you can read the sarcomere length in a bundle b y a disfraction grating. That might be worthwhile thinking of for observing the high sensitivity. MATTHEWS: Two points. First in reply t o Dr. Hagbarth about whether these occur physiologically. Goodwin and Luschei in Seattle have been recording from Ia afferents in the intact chewing monkey from jaw muscles by having electrodes in mesencephalic V and here it is notable that they find a very strong burst when the muscle is being stretched. There’s not only the coactivation with t h e Ia firing during shortening of the muscle, there is also a burst o n stretching, and in this burst there is a very strong initial burst component which is probably of the same effect, which is much more marked during the chewing of t h e monkey spontaneously than if you stretch t h e jaw yourself. So they partly suspect tha t they are seeing some sign of this postexcitatory facilitation in the intact animal. The second point to Dr. Eldred: Surely we now have a new question coming up, whether there are not t w o separate effects o n the muscle. We have this effect which is undoubtedly fusimotor but is it possible that these are also other, longer-term effects which are not fusimotor which may be some

170 ordinary extrafusal muscle fiber effect coming onto the spindle. It seems t o me that all your experiments have been devoted to saying that is there a fusimotor effect?The answer is yes. Having agreed there is fusimotor effect, then you have to d o a new set of experiments to say that is there also an alpha effect when the alpha’s are not beta fibers going also t o the spindle? Clearly if you could dissect out a number of alpha’s in the way that Prof. Laporte does and compare their effects on stimulating firstly at low frequency, just fusion and secondly a t high frequency of fusion for intrafusal fibers, and difference between those is fusimotor, but if the certainty of alpha’s and stimulating just above threshold is not sure, then you must dissect out alpha fibers. There might be fatigue effects going on because it still gives a very interesting question whether the fatigue of a muscle does change the calibration of its sense organs over above the fusimotor effects. ELDRED: Maybe our evidence for pure extrafusal participation is not strong enough. We saw effects upon stimulating with stimuli of 1.1, even 1.05 times threshold. MATTHEWS: But you did not produce anything akin t o fatigue by these weak stimuli. They were of very brief period of stimulation. They did not produce a fusimotor effect but you did not apply them in a long terminal. ELDRED: I don’t think the muscle has to be fatigued to get long-term effects, even from the extrafusal participation. MATTHEWS: If there is some long-term effect in muscle it comes on when you do tenotomy, when you cut roots, when you fatigue and so on. And you get these changes in sensory discharge which are very interesting. Perhaps they are fusimotor mediated but perhaps they are responding to some metabolic effect in muscle. There is quite a wealth of evidence showing that muscle receptors and various other receptors do something. The question for us is whether any of this is spindle mediated or whether it is group I11 mediated and it seems t o me you have left it an open question whether the alpha fibers on their own can or cannot produce any long-term discharge, granted and agreeing that fusimotor fibers produce these very powerful effects. ELDRED: My feeling is that the extrafusals can produce changes. In addition t o the various metabolic effects I would suggest that in complex muscle, say like the gastrocnemius of the cat, in the relaxation of the muscle following contraction you may have a different alignment of stress and the geometry of the fasciculi than when you’ve pulled it out and let the thing go back. This could itself cause some change perhaps. LAPORTE: I should just like to add one comment on the threshold. The beta axons could explain your effect since we know that some of them d o supply chain fibers.

Use of Afferent Triggered Averaging to Study the Central Connections of Muscle Spindle Afferents ANTHONY TAYLOR *, DOUGLAS G.D. WATT **, EDWARD K. STAUFFER ROBERT M. REINKING and DOUGLAS G. STUART

***,

Department of Physiology, College of Medicine, University of Arizona, Tucson, Ariz. 85724 (U.S.A.)

INTRODUCTION Mendell and Henneman (1968, 1971) showed that when intracellularly recorded synaptic noise from motoneurons is averaged by a computer triggered from the firing of a functionally isolated “in-continuity ” primary (Ia) spindle afferent, a waveform emerges which is an estimate of the homonymous monosynaptic EPSP. This spike-triggered averaging (STA) method was next used by Kirkwood and Sears (1974, 1975) to reveal a previously unsuspected similar connection for secondary (group 11) spindle afferents. We have recently confirmed these findings and have extended them somewhat by use of STA at greater sensitivity than before and by use of animal preparations with a high level of spontaneous interneuronal discharge. This account presents some features of spindle connections found in a study on 50 cats in which the projections of 44 Ia, 2 1 tendon organ (Ib), and 9 spindle group I1 afferents from medial gastrocnemius (MG) were studied by the STA method in 940 motoneurons of various types. Preliminary accounts have been published (Reinking et al., 1975; Stauffer et al., 1975a; Watt et al., 1975a) and further details are available in two full reports (Stauffer et al., 197513; Watt et al., 1975b). METHODS Low spinal cats were prepared under gaseous anesthesia (halothane and oxygen) and subsequent recording undertaken with the preparations anesthetized with a mixture of a-chloralose (35-45 mg/kg) and urethane (350-450 mg/kg). These procedures, together with maintenance of blood pressure at 100-120 mm Hg, resulted in preparations with a high level of synaptic noise in intracellular (IC) recordings from motoneurons (see also Rudomin et al., 1975), and

* Present address: Sherrington School of Physiology, St. Thomas’s Hospital Medical School, London S.E. 1,Great Britain. ** Present address: NASA, Ames Research Center, Moffett Field, Calif. 94035, U.S.A. * ** Present address: Department of Physiology, School of Medicine, University of Minnesota, Duluth, Minn. 55812, U.S.A.

172 spontaneous activity in interneurons, but without motoneuron firing. Synaptic noise in IC recordings was averaged with computer sweeps triggered by action potentials from single afferents recorded from subdivided dorsal rootlets or by extracellular (EC) recording from dorsal root (DR) ganglion cells. Afferent continuity at least to cord entry was checked routinely by averaging a high gain record from the DR entry zone, triggering from the afferent spike. High final display gain of IC and EC recordings (down t o 1.5 pV/cm) was sometimes required and we have confidence in the recovery of calibration pulses as small as 1 p V . For small effects, repeatability was checked by recording 2048 sweeps successively in each of the 4 quarters of the computer memory. Only if the same type of response was seen in 3 or 4 of the averages were they then combined and written out as a single measurement. It was also felt necessary to make identical EC control records immediately after IC averaging.

RESULTS Comparison of monosynaptic l a and spindle group 11 EPSPs Some characteristics of monosynaptic EPSPs due t o Ia and spindle group I1 afferents from MG acting on MG and LGS motoneurons are shown in Table I. Note that EPSP rise times are similar for both groups but the mean EPSP amplitude for spindle group I1 projections is less than 50% of that for the Ia projections. Only 11.5% of the spindle group I1 EPSPs were 2 5 0 pV in contrast to 41% for the Ia group. The smallest previously reported Ia EPSP was 1 7 pV (Mendell and Henneman, 1971) and Kirkwood and Sears (1974, 1975) have published records of spindle group I1 EPSPs down to 10 pV. Table I shows MG and LGS EPSPs down to 4 pV and for the indirect synergist SMAB we have seen one at 2.2 pV. Ten percent of the Ia EPSPs of Table I had an amplitude < l o pV, as did 23% of the spindle group I1 population. As with previous observations (Mendell and Henneman, 1971) Ia afferents were shown to project profusely to homonymous MG (87% probability of connection) and heteronymous LGS (61%probability) motoneurons. The spindle group I1 projections appear not to be so extensive. There was a 52%probability of homonymous and 26% probability of heteronymous connection shown for our admittedly small sample. The mean latency (DR entry t o EPSP onset) was 0.17 msec shorter for the Ia EPSPs than for group 11. High sensitivity recording was of value here because a small, fast, positive-negative wave was frequently seen t o precede EPSPs recorded by STA. It seems likely (Jankowska and Roberts, 1972) that this was due t o the arrival of the impulse in the presynaptic axon or endings and so we have called it the presynaptic spike (Pre-SS) and have used it t o divide central latency into conduction time and synaptic delay. Fig. 1 illustrates the measurement of these intervals and the collected results from 15 spindle group I1 and 18 Ia responses. The Pre-SS is of the order of 1-5 p V . It was generally only seen in association with small PSPs for which high final gain was necessary. There was no statistical difference in synaptic delay for the Ia and spindle

TABLE I COMPARISON OF MONOSYNAPTIC MG AND LGS Ia AND SPINDLE GROUP I1 EPSPs ~

Connection ( N )

Ia (100) Group I1 (23)

Rise time (msec)

Latency (msec)

Amplitude (pV)

‘ v L

* S.D.

Range

X

* S.D.

Range

x

i

0.76 0.96

0.18 0.21

0.4-1.1 0.3-1.4

1.0 1.02

0.5 0.43

0.4-2.5 0.4-2.2

65.4 30.08

68 31.4

S.D.

Range 4-2 8 3 4-131

174 group I1 EPSPs shown in Fig. 1. However, the mean 0.19 msec difference in cord conduction time was statistically significant ( P < 0.001) and in good agreement with the overall latency differences (0.17 msec) for the total samples shown in Table I. It also corresponded to the delays measured antidromically by Fu and Schomburg (1974) from points of lowest electrical threshold within the ventral horn to a point near the DR entry of functionally isolated in-continuity spindle afferents. Elsewhere we have reported on the broad distribution of latency for individual EPSPs and have advanced arguments for setting working limits for Ia (Watt et al., 1975b) and spindle group I1 (Stauffer et al., 1975b) monosynaptic connections. Monosynaptic Ia EPSP latency from cord entry seems t o be within 0.4-1.1 msec and within 0.4-1.4 msec for spindle group I1 EPSPs. It bears emphasis that these boundaries are not rigid and may easily extend to 1.5 msec for Ia connections, and to 1.65 msec for spindle group I1 EPSPs.

Group 1A

0.2 0.4 0.6 0.8

.t

O w l

"1

"1

02 0.4 0.6 0.8

0.2 0.4 0.6 0.8

Fig. 1. Measurements of Ia and spindle group I1 cord conduction times and synaptic delay for monosynaptic EPSPs. At left are IC records from 2 MG cells of Ia (upper) and spindle group I1 (lower) EPSPs averaged from 2048 and 1024 sweeps respectively. Both records show a presynaptic spike (Pre-SS) used to separate cord conduction times (CT) from synaptic delay (SD). Immediately below each IC trace are the EC controls (same voltage calibration and sweeps) which also show the Pre-SS. Notethat the Ia EC record shows a marked excitatory field discussed in detail in Watt et al. (1975b) and Stauffer et al. (197513). The lowermost traces are averages of 4096 sweeps (voltage calibration now shown) for dorsal root entry point to show afferent spike used for timing (first arrow). At right are plotted histograms of the CT and SD intervals for 18 Ia and 1 5 spindle group I1 responses.

175

T

rL--\

JI

\--_

4

+ -

Srnsec

4

Fig. 2. Examples of single and double peaked monosynaptic EPSPs. Single peaked Ia ( A ) and spindle group I1 (C) EPSPs are shown with afferent input limited presumably t o the soma (left) and dendrites (right). For theoretical discussion of such profiles see Rall (1967). Double peaked EPSPs are shown in B (Ia) and D (spindle group 11) t o emphasize t h a t monosynaptic afferent input t o a single cell can also be distributed a t different discrete sites along both the soma and dendrites. F o r all these IC recordings arrows indicate DR entry time. Latency (msec) and number of sweeps are: A left, 0.8 msec - 512; A right, 0.6 msec 2048; B left, 0.5 msec - 1024; B right, 0.5 msec - 1 0 2 4 ; C left, 1.0 msec - 4096; C right, 0.8 msec - 4096; D left, 0.8 msec - 4 0 9 6 ; D right, 1 . 0 msec - 1024.

A puzzling difference between our data and t h a t of Mendell and Henneman ( 1 9 7 1 ) concerns t h e profile of monosynaptic EPSPs. In their sample of Ia EPSPs only 2 of 114 responses failed to fall along a smooth exponential as predicted by t h e Rall ( 1 9 6 7 ) model. This led them t o propose t h a t “ t h e endings of a single Ia fiber are not widely dispersed o n the surface of a motoneuron but are clustered together in a group.” Fig. 2A, C show t h a t in the present data we also saw “pure” o r “Rall-type” EPSPs, but for 53% of t h e Ia and 50% of the spindle group I1 EPSPs there were one or more extra “humps” of depolarization (Fig. 2B, D) which we attribute t o the presence of monosynaptic connections a t different sites on the soma or dendrites.

Disynaptic and later responses Use of STA at high sensitivity in preparations with spontaneous interneuronal discharge permitted demonstration of disynaptic and sometimes polysynaptic PSPs. Fig. 3 shows some examples of these effects and our explanation of them. Although t h e mean firing rate of a spontaneously active interneuron will depend o n the total synaptic input its firing pattern is modulated slightly by each excitatory o r inhibitory

176

0

lnterneuron

0

1

PSTH

E

I.C. noise

F

Fig. 3. Examples of disynaptic Ia and spindle group I1 PSPs and a possible explanation for their manifestation. A: PBST cell with Ia EPSP (latency 2.5 msec, 8192 sweeps). The early positive-negative wave is thought to represent a large Pre-SS from a branch of the axon that passes close by the cell but without making synaptic contacts (further discussion in Stauffer et al., 1975b). B: MG cell with spindle group I1 EPSP (latency 2.1 msec, 4096 sweeps). C: TA-EDL cell with Ia IPSP (latency 1.6 msec, 2048 sweeps). D: LGS cell with a rarely encountered spindle group I1 IPSP (latency 1.5 msec, 4096 sweeps). Arrows indicate DR entry time and onset of PSPs. E: shows schematically the spontaneous firing pattern of a relevant interneuron and the poststimulus time histogram (PSTH) that would result from crosscorrelating its discharge with the afferent trigger spike. The PSTH profile reflects the interneuron’s EPSP response to the afferent. F : shows the noise on the motoneuron IC trace which receives a contribution from the interneuron. The STA PSP response (excitatory o r inhibitory) is considered a partly smoothed version of the interneuron’s EPSP profile, shifted by one synaptic delay and some extra conduction time.

presynaptic impulse. Thus, the profile of a poststimulus time histogram (equivalent t o STA) of interneuronal discharge should reflect the EPSP interneuronal response t o an excitatory “in-continuity” afferent impulse (for theory see Moore et al., 1970). We propose that the emerging motoneuronal PSP (excitatory or inhibitory) is a partly smoothed version of the interneuronal EPSP profile, shifted by one synaptic delay and some extra conduction time. STA should also work through more than two synapses provided that the relevant interneuions are firing. Each additional synapse would progressively attenuate and slow the emergent motoneuronal response.

While not excluding faster responses, we have presented detailed arguments for setting working limits for disynaptic Ia connections at 1.2 to 2.6 msec and spindle group I1 connections at 1 . 5 t o 2.8 msec. Table I1 gives some characteristics of disynaptic Ia and spindle group I1 PSPs. The rise time difference between monosynaptic (Table I) and disynaptic responses is seen only on a pop-

TABLE I1 CHARACTERISTICS OF DISYNAF'TIC Ia AND SPINDLE GROUP I1 PSI'S Connection ( N )

Latency (msec)

X

* S.D.

1.2-2.6

1.49

0.83

0.4-2.8

8.9

7.0

1.5-25.4

1.2-2.4

1.38

0.48

0.6-2.3

4.9

2.5

1.5-10.4

0.36

1.5-2.8

1.6

0.34

0.9-3.0

8.5

5.8

3.0-20.0

0.48

1.5-2.7

1.96

0.90

0.8-2.7

4.28

2.6

1.2-

X

k

Ia EPSPs ont o any motoneuron (17 *)

1.71

0.51

Ia IPSPs ont o TA-EDL (31)

1.81

0.31

Group I1 EPSPs ont o any motoneuron (12)

2.03

Group I1 IPSPs ont o any motoneuron ( 6 )

2.15

* Two potentially

Amplitude (pV)

Rise time (msec)

S.D.

Range

Range

X

monosynaptic EPSPs excluded with amplitudes of 89 and 57 pV, and latencies a t 1.4 msec.

?:

S.D.

Range

9.2

178 ulation basis. It is possible for example to have disynaptic rise times less than the 1.0 msec monosynaptic mean. For the reasons outlined above, the most striking difference between monosynaptic and disynaptic PSPs is in their amplitude. Very few disynaptic or later PSPs as estimated by STA had amplitudes >10 pV and the majority were less than 5 pV. Our spindle group I1 sample sizes are as yet too small to attach any significance to the minor differences shown in Table I1 between Ia and spindle group I1 disynaptic rise times. As subsequent sample sizes accumulate and the STA approach is combined with precise measurements of cord conduction time (Fu and Schomburg, 1974) it may even be possible to show that any EPSP of amplitude greater than approximately 20-25 pV is monosynaptic, even if extreme axonal thinning has resulted in a latency from cord entry beyond the normal monosynaptic range. Two unusually large Ia EPSPs of 1.4 msec latency, for example, have been excluded from Table 11.

Use of STA t o settle old controversies and uncertainties on the central connections of limb afferents (Matthews, 1972) is now imminent because the method obviates the well known problems of selective activation (reviewed by McIntyre, 1974). Our initial study was not directed t o this end except in so far as to point out that tendon organ connections are more complex than hitherto supposed (Watt et al., 1975b) and that inclusion of spindle group I1 afferents in the “flexor reflex afferent” population (Holmquist and Lundberg, 1961) seems no longer tenable (Stauffer et al., 1975b). Fig. 4 shows a further interesting feature of our data. For over 30% of the monosynaptic Ia EPSP responses and 41% of the spindle group I1 EPSPs there were “humps” of depolarization on the falling phase that were too late for any explanation other than that the

5msec Fig. 4. Evidence for operation of polysynaptic la and spindle group I1 autogenetic excitatory pathways in the low spinal cat. Upper traces are IC averages and lower traces EC controls at same gain and number of sweeps. Arrows indicate DR entry time for : A, a monosynaptic Ia EPSP for an MG cell (latency 0.4 msec, 512 sweeps); and B, a monosynaptic spindle group I1 EPSP for an LGS cell (latency 1.2 msec, 4096 sweeps). In each case there is a late hump of depolarization thought due t o polysynaptic excitation.

179 monosynaptic response was running into a polysynaptic one. A variety of previous indirect observations (reviewed by Matthews, 1972) have made a compelling case for a polysynaptic Ia autogenetic excitatory pathway t o motoneurons. Its direct demonstration has nonetheless been elusive. Fig. 4 shows such a pathway in operation in the low spinal cat for both Ia and spindle group I1 projections. The effect has also been seen quite recently in motoneuronal responses to electrically activated “la” volleys in preparations that exhibited an intense background of interneuronal discharge (see Figs. 6a and 9a in Rudomin et al., 1975).

DISCUSSION The study of synaptic connections by STA solves the problems of selective activation and it will be of great interest t o see the extent t o which subsequent studies can bring out di- and trisynaptic connections by manipulation of the activity level of relevant interneurons. During averaging we sometimes saw spontaneous changes in the direction of what appeared to be a growing disynaptic response. With more attention directed to the control of interneurons it may well be possible t o demonstrate alternative interneuronal pathways which can be switched by other influences such as supraspinal stimulation or the use of L-DOPA. We recognize that the study of reflexes by STA raises new problems of interpretation, particularly in the overlap of latencies between monosynaptic, disynaptic and trisynaptic pathways. The extent t o which axonal thinning and impulse slowing takes place in the spinal cord is still largely unexplored. Fu and Schomburg (1974) have made an excellent start in this direction, however, and by combining their antidromic intraspinal stimulation techniques with the presently used STA procedures, it should be possible t o set quite quantitative working limits to these overlaps. If STA is t o have full impact on the study of central connections, it will be necessary to achieve more representative sampling than acquired t o date. A simple anatomical feature should help in this regard. The nerve t o MG divides into 4-7 rostra1 to caudal branches on entering the muscle (Ledbetter, personal communication). A progressively overlapping somatotopic cord-to-muscle relation is preserved in the efferent innervation (Swett et al., 1970) but the relation between afferent position in the muscle and the exact level of entrance t o the cord is largely random (Swett and Eldred, 1959). By cutting some of the intramuscular branches it should be possible to “thin out ” the number of MG impulses recorded from natural subdivisions of the dorsal root filaments such that many in continuity afferents can be recorded simultaneously during periods of EC and IC averaging. Full exploitation of this possibility should contribute greatly to our understanding of proprioceptive reflexes. SUMMARY

(1)The synaptic connections of single identified muscle spindle Ia and group

180 I1 afferents have been studied in low spinal cats by the spike-triggered averaging (STA) of synaptic noise in motoneurons of various types. (2) By using STA at higher sensitivity than before it has been possible t o reveal monosynaptic EPSPs below the previously reported lower limits, down indeed to 2.2 pV. (3) Mean monosynaptic EPSP amplitude for spindle group I1 projections was less than 50% of that for Ia projections. Both groups contained EPSPs < l o pV (23%of the spindle group I1 and 10% of the Ia population), but only 11.5% of the spindle group I1 EPSPs were 2 5 0 pV in contrast t o 41%of the Ia EPSPs. In further contrast, the Ia afferents appear t o make more profuse monosynaptic connections with homonymous (87% of population) and heteronymous (61%) motoneurons than did spindle group I1 afferents (5296, 26% respectively). (4) High sensitivity STA recording further permitted measurement of a presynaptic spike that showed that, while synaptic delay was of similar duration for Ia and spindle group I1 projections, the central conduction time was significantly longer (mean 0.19 msec) for the spindle group I1 impulses. (5) Use of STA in preparations with a high level of spontaneous interneuronal discharge permitted demonstration of disynaptic and sometimes even later excitatory and inhibitory effects. Polysynaptic Ia and spindle group I1 autogenetic excitatory pathways t o motoneurons have been demonstrated. (6) Several new and provocative findings have emerged from the present work and the previous STA studies of Mendell and Henneman (1968, 1971) and of Kirkwood and Sears (1974, 1975). Further advances are expected from manipulation of interneuronal discharge, combining STA with antidromic intraspinal stimulation procedures and with multiple dorsal root afferent spike recording during periods of motoneuronal recording and averaging.

ACKNOWLEDGEMENTS This work was supported by USPHS Grants NS 07888 and FR 05745. D.G.D. Watt held a Canadian MRC Fellowship. A. Taylor was supported by the Stella M. King Fund, the Fan Kane Foundation and a Porter Fellowship (American Physiology Society), together with grants from the Wellcome Foundation and the Muscular Dystrophy Association Inc., N.Y., U.S.A. REFERENCES Fu, T.C. and Schomburg, E.D. (1974) Electrophysiological investigation of the properties of secondary muscle spindle afferents in the cat spinal cord. Acta physiol. scand., 91: 314-329. Holmquist, B. and Lundborg, A. (1961) Differential supraspinal control of synaptic actions evoked by volleys in the flexor reflex afferents in motoneurones. Acta physiol. scand., 54, Suppl. 186: 1-51. Jankowska, E. and Roberts, W.J. (1972) Synaptic actions of single interneurons mediating reciprocal Ia inhibition of motoneurons. J. Physiol. (Lond.), 222 : 623-642. Kirkwood, P.A. and Sears, T.A. (1974) Monosynaptic excitation of motoneurons from secondary endings of muscle spindles. Naiure (Lond.), 252: 243-244.

181 Kirkwood, P.A. and Sears, T.A. (1975) Monosynaptic excitation of mQtoneurons from muscle spindle secondavy endings of intercostal and triceps surae muscles in the cat. J. Physiol. (Lond.), 245: 64-66P. Matthews, P.B.C. (1972) Mammalian Muscle Receptors and Their Cenlral Actions, Arnold, London. McIntyre, A.K. (1974) Central actions of impulses in muscle afferent fibers. In Handbook of Sensory Physiology, ZW2, C.C. Hunt (Ed.), Springer, Berlin, pp. 235-288. Mendell, L.M. and Henneman, E. (1968) Terminals of single Ia fibers: distribution within a pool of 300 homonymous motoneurons. Science, 154: 96-98. Mendell, L.M. and Henneman, E. (1971) Terminals of single Ia fibers: location, density and distribution within a pool of 300 homonymous motoneurons. J. Neurophysiol., 34: 171-187. Moore, G.P., Segundo, J.P., Perkel, D.H. and Levitan, H. (1970) Statistical signs of synaptic interaction in neurons. Biophys. J., 10: 876-890. Rall, W. (1967) Distinguishing theoretical synaptic potentials for different soma-dendritic distributions of synaptic input. J. Neurophysiol., 30: 1138-1168. Reinking, R.M., Stauffer, E.K., Stuart, D.G., Taylor, A. and Watt, D.G.D. (1975) The inhibitory effects of muscle spindle primary afferents investigated by afferent triggered averaging methods. J. Physiol. (Lond.), 248: 20-22P. Rudomin, P., Burke, R.E., NGieez, R., Madrid, J. and Dutton, H. (1975) Control by presynaptic correlation: a mechanism affecting information from Ia fibers to motoneurons. J. Neurophysiol., 38: 267-284. Stauffer, E.K., Watt, D.G.D., Stuart, D.G., Taylor, A. and Reinking, R.M. (1975a) Synaptic effects of single group Ib and I1 muscle afferent fibers onto lumbosacral motoneurons. Neurosci. Abstr., 1: 169. Stauffer, E.K., Watt, D.G.D., Taylor, A., Reinking, R.M. and Stuart, D.G. (1975b) Analysis of muscle receptor connections by spike triggered averaging. 2. Spindle secondary afferents. J . Neurophysiol., in press. Swett, J.E. and Eldred, E. (1959) Relation between spinal level and peripheral location of afferents in calf muscles of the cat. Amer. J. Physiol., 196: 819-823. Swett, J.E., Eldred, E. and Buchwald, J.G. (1970) Somatotopic cord-to-muscle relations in efferent innervation of the cat gastrocnemius. Amer. J. Physiol., 219: 762-766. Watt, D.G.D., Stauffer, E.K., Stuart, D.G., Taylor, A. and Reinking, R. (1975a) Ia disynaptic pathways studied by spike triggered averaging of synaptic noise. Neurosci. Abstr., 1: 169. Watt, D.G.D., Stauffer, E.K., Taylor, A., Reinking, R.M. and Stuart, D.G. (1975b) Analysis of muscle receptor connections by spike triggered averaging. I. Spindle primary and tendon organ afferents. J. Neurophysiol., in press. I

DISCUSSION HULTBORN: Dr. Stuart paid some attention to the ratio between the amplitude and the latency. But of course this ratio must depend on the size of the unitary EPSPs of these interneurons. If the size of the unitary EPSPs is very large so that one EPSP can fire the interneuron, then this relationship between latency and height might not hold. STUART: We have proposed that the emerging PSP (excitatory or inhibitory) is a partly smoothed version of the “average” profile of EPSP for the relevant interneurons, shifted by one synaptic delay and some additional conduction time. Obviously, this viewpoint must now be tested by using STA to measure the amplitudes of monosynaptic EPSPs evoked in interneurons by spindle and tendon organ afferents. HULTBORN: You have described in a short communication t o the Physiological Society that you can see very short-latency hyperpolarizations. You have suggested that they may be monosynaptic Ia IPSPs.

182 STUART: As I recall, the exact wording was that “the possibility of monosynaptic Ia inhibition should be reconsidered” (J.Physiol. (Lond.), 248 (1975) 20-22P). HULTBORN: I think your evidence for short-latency hyperpolarizations in motoneurons is quite strong, but I feel that they are very unlikely to be monosynaptic Ia IPSPs. That would demand not only that the Ia afferents make contact with “wrong” motoneurons but also a different postsynaptic receptor causing an IPSP instead of an EPSP for the same transmitter substance. Furthermore, reciprocal Ia inhibition has never been seen before in some of Dr. Stuart’s combinations (from gastrocnemius-soleus to posterior biceps-semitendinosus). Since I d o not believe that these early hyperpolarizations are IPSPs I am obliged to propose some alternative explanation. Do you think that on intracellular recording from a motoneuron you may record field potentials which are not seen at a just extracellular position before or after the intracellular recording? I am wondering if the dendritic tree may not serve as an “elongation” of your recording microelectrode and thus help to record field potentials rather far from the soma. The dendrites of posterior biceps-semitendinosus motoneurons certainly extend into the motor nuclei of gastrocnemius-soleus and may thus pick up the monosynaptic excitatory field potential there (caused by a gastrocnemius or soleus Ia afferent). On normal extracellular recording the field potential has of course a very short time course in comparison to a synaptic potential, but the time course of a remote field potential recorded via the dendritic tree would probably resemble that of a postsynaptic potential. STUART: Perhaps. We have definite evidence of occasional small “early negative waves (ENWs)” that may be IPSPs if one accepts conventional standards of intracellular-extracellular recording and “neighboring cell” comparisons. We, ourselves have thought of 4 possible origins for this ENW. They may be: (a) excitatory fields due t o Ia excitatory action on nearby cells (which might include focal synaptic potentials of interneurons); (b) exceptionally fast disynaptic IPSPs; (c) due to some unusually fast presynaptic inhibitory effect on other excitatory input to the antagonists; or (d) monosynaptic inhibition of antagonist motoneurons by Ia afferents. It must be emphasized, however, that the fields due to single Ia excitation are complex and we have reservations about interpreting our results on their face value. HENNEMAN: Have you any idea of how many motoneurons in a pool receive a group I1 excitatory connection. STUART: About 50%, in contrast to over 85% for our Ia sample. LAPORTE: I’m very surprised that no one has raised the question of why you used chloralose. Could you tell us why you chose it? STUART: We used a mixture of a-chloralose (35-45 mg/kg) and urethane (350-450 mg/kg). The intention was to secure a preparation with a high level of spontaneous interneuron activity but without motoneuron discharge. In one of my records, I showed a disynaptic inhibition from a group I1 afferent onto a lateral gastrocnemius-soleus cell. I’ve seen that in 5 of 75 extensor cells. I’ve seen the pathway using spike triggered averaging. But in our data we seee the other effect as well. And we believe that there are two pathways, two ways to the motoneuron, and the proof is the difficult thing. We see this with Ib and we see this with group 11. There are alternative pathways and I think the tric now is how to prove it and how to manipulate the cord such that this point can be made. LAPORTE: Concerning this very important problem of group 11, exciting the motoneurons, I’ve one question. What is the conduction velocity of those group I1 axons which do give monosynaptic EPSP? STUART: I should have said we aligned a study of 152 motoneurons with 9 afferents, they range from 20 to 60 m/sec. There are not enough data yet I think for this. LAPORTE: Yes, but 20 mlsec is quite safe, because what I’ve in mind is really of the sim-

ple spindles without secondary endings. If my histological colleagues are here, they can tell that much better than I. How primary ending connected t o an afferent fiber, which is thinner than a usual Ia, and I was just wondering if some of the fibers in the range of 70-60 couldn’t be fibers connected to primary endings and coming from a single spindle. But if you d o say that group I1 afferent fibers of less than 30 or 40 m/sec have monosynaptic excitation, it is much more convincing, but if you d o get that evidence with axons conducting between 60-70 m/sec, I want to be absolutely certain they are secondary endings connected.

STUART: Well, in the 98 monitored, there were 3 in the ~ O ’ Sone , in the 50’s and one of 61 and the others were under. LAPORTE: It’s quite interesting to know you got the same effect with slow conducting axons. STUART: I might add I don’t know how much data Kirkwood and Sears generated. MATTHEWS: Kirkwood and Sears also have slow ones. POMPEIANO: I would like t o know whether these disynaptic EPSPs are coming from gastrocnemius or monosynaptic EPSP from gastrocnemius. Is this by group I1 volleys or were they obtained mainly from phasic motoneurons or from tonic motoneurons? STUART: I have no idea, we didn’t make that. POMPEIANO: I think this is an important point.

This Page Intentionally Left Blank

Selective Activation of Group I1 Muscle Afferents and its Effects on Cat Spinal Neurones M.KATO and K. FUKUSHIMA Department of Physiology, H o k k a i d o University School of Medicine, Sapporo (Japan)

INTRODUCTION In order to study the central effects produced by the discharges of group I1 fibres, it is desirable to block the impulse conduction of group I fibres. In the usual experiment, in which graded electrical stimulation is applied to a peripheral nerve, group I fibres are already activated when the strength of the electrical stimulation reaches the group I1 range, hence the results obtained in such experiments might be contaminated by temporal and spatial summation of the preceding group I impulses. The present series of investigations aimed at studying the effects of electrical stimulation of group I1 fibres on spinal neurones after excluding any such possible contamination.

DIFFERENTIAL BLOCKING A number of papers have already been published concerning the differential blocking of large fibres (cf., Kuffler and Vaughan Williams, 1953; Mendell and Wall, 1964; Casey and Blick, 1969; Manfredi, 1970; Brown and Hamann, 1972; Sassen and Zimmermann, 1973; Jack and Roberts, 1974). Among them, the anodal blocking method seemed t o be the most reliable. After the medial and lateral gastrocnemius and soleus nerves were together dissected carefully from the rest of the nerves at the popliteal fossa, they were mounted on silver bipolar stimulating electrodes (interpolar distance about 1 0 mm) which were symmetrically straddled by polarizing electrodes of cotton soaked with physiological saline solution (interpolar distance about 20 mm), as shown in Fig. 1. The distal polarizing electrode was placed about 10 mm proximal from the cut end of the nerves. These polarizing electrodes were connected to a trapezoid wave current generator, the proximal electrode being positive and the distal one negative. It was found that the most satisfactory blocking of group I fibres was obtained when the current was raised to about 60 pA in about 5 sec. When the current was raised too steeply the current itself excited the nerve. This differential blocking was obtained repeatedly, for individual periods of up to 10 min, without any sign of deterioration of the nerve fibres over periods of sev-

186

[

3

4

f

4

* R

L7,

SI

f&L

P S P

2mv

I msec

Current

+n-P A

@L!L& I

___----.-

5 sec

n

Fig. 1. Method of differential blocking of group I fibres'in gastrocnemius nerve. Stimulating (S) and polarizing (P) electrodes were arranged as shown in this figure. Upper traces in each pair of A-D show mass volley recordings from an L7 dorsal rootlet; lower traces show single fibre recordings from an L7 dorsal root filament which contained t w o group I fibres (denoted 1 and 2, conduction velocity, 107 m/sec) and one group I1 fibre (denoted 3, conduction velocity, 38 mlsec). Each record is a superimposed record of 50-70 sweeps. A stimulus of 5 times threshold of t h e fastest group I fibre was applied t o the nerve a t t h e frequency of 1 Hz a t the arrows. A: control record. B: when the polarizing current was raised t o 20 PA. C: when the current was further raised to 50 PA. D: 60 PA of t h e polarizing current was applied. Upward is negative for all the records.

eral hours. The efficacy of the block was studied both by mass volley recording and by single fibre recording. Even when a successful differential blocking appears t o be obtained on the evidence of mass volley recording, there still remains the possibility that the latencies of individual fibres may increase irregularly due to the polarization, and hence the .block of the mass volley may not necessarily reflect the actual block of all the individual fibres (Fukushima et al,, 1975). Moreover, there is the danger of evoking asynchronous firing by the polarizing current itself. Fig. 1 shows a representative example of differential

187 blocking of group I fibres. According t o the calculated conduction velocity, the first large spike-like volley in the upper trace of A contained all of the group I fibres and a small part of the fastest group I1 fibres. A notch in the descending part of the large volley corresponds t o a conduction velocity of 72 m/sec. All the later volleys may be attributed to group I1 fibres. Group I11 fibres were not activated at this stimulus strength. In the lower trace two single group I fibres (denoted 1 and 2, conduction velocity 107 m/sec) and one group I1 fibre (denoted 3, conduction velocity 38 m/sec) could be recorded from a fine dorsal rootlet of L7 . When the polarizing current was applied, the group I volley began to decrease at B together with the disappearance of fibre No. 1, and disappeared at C together with fibre No. 2 when the polarizing current was raised t o 50 PA; when the polarizing current was further increased to 60 PA, all the group I and the fastest group I1 volleys were completely blocked while most of the slower group I1 fibres are still conducting impulses. The next point is whether a significant prolongation of impulse conduction occurs in individual fibres during the polarization. For 32 single group I fibres from L, dorsal root filaments the greatest prolongation of the stimulus-response interval was 0.3 msec. The faster the conduction velocity of an individual fibre the less tended the prolongation of its impulse conduction to be. The prolongation of the latencies of individual group I fibres did not exceed the duration of the group I mass volley. When the central effects produced by the discharges of group I1 fibres were being examined using this differential blocking method, it was often necessary to maintain the DC blocking current continuously for 10 min or more. Ten single group I1 fibres were therefore studied t o check how much their latencies were affected by the polarization at a constant DC level for about 10 min. In all of these 10 fibres the fluctuation of latencies was less than 3%. Therefore it is possible to examine segmental effects of group I1 fibres using this differential blocking method. EFFECTS OF GROUP I1 FIBRES ON SEGMENTAL INTERNEURONES Interneurones responding t o group I1 fibres of the gastrocnemius nerve were located in the intermediate region, including the dorsal horn, as well as in the ventral horn (Fu et al., 1974; Fukushima and Kato, 1975). One hundred interneurones from varied sites were studied. They all responded orthodromically t o afferent nerve stimulation, but none responded antidromically on stimulating the cord at L, (Fukushima and Kato, 1975), though many were then excited synaptically; that is, only segmental interneurones are under consideration and tract neurones were discarded in the present experiments, although many were seen. Among these 100 interneurones 38 responded to group I fibres, though no systematic investigation on these cells was performed. Six interneurones received input only from group I1 fibres and they usually responded t o only one afferent nerve such as the lateral gastrocnemius nerve. Nineteen neurones responded to group I11 fibres, and on the other 37 neurones there are wide convergences of inputs from fibres with different diameters as well as from many different sensory nerve fibres.

188 10 7

2 T

.. ..

I

.. ..

.. ..

. ... ..

. ... ..

20 T

. .

Block 5T

. . ...

. . .. .

Fig. 2. Dot displays of a representative example from the group of interneurones that respond solely to group I1 fibres of gastrocnemius nerve. Abscissae show latencies from stimulus artefacts. Bars indicating I and I1 show the time when the first group I and I1 volleys reached the recording electrode at dorsal root. This neurone responded t o stimulation of the lateral gastrocnemius nerve only. This neurone responded with one spike during block of group I fibre (block 5 T). Broken line indicating I1 shows the time when the first group I1 volleys reached the recording electrode at dorsal rootlet.

Fig. 2 illustrates a representative unit which responded solely to group I1 fibres. This group of cells showed little or no spontaneous discharge. The interneurone in the figure responded solely to group I1 fibres of the lateral gastrocnemius nerve and even the stimulation of the medial gastrocnemius nerve was ineffective. No response was elicited by stimulation below the strength of 1.8 T (times threshold of the largest fibre). At 2 T one spike was elicited and two spikes were evoked from 3 to 20 T. This result suggests that group I11 fibres have no significant effect on this neurone, although there are some changes in the latencies of the second spikes. When group I fibres were blocked, one spike

189 was elicited at the stimulus strength of 5 T (block 5 T). This neurone responded with quite uniform latencies as is shown in the figure; the average latency of the first spike being 1.0 msec (range 0.9-1.1 msec) from the fastest group 11 fibre volley recorded at the dorsal rootlet before blocking the group I fibres. The latency did not change after the blocking of group I fibres by applying the polarizing current. The central delay of the first spike of this neurone was calculated as 0.6-4.8 msec after subtracting the conduction time in the peripheral nerve and intraspinal part of the group I1 fibres. The intraspinal slowing was calculated on the basis of Fu and Schomburg’s (1974) figures (Fukushima and Kato, 1975). Therefore it seems likely that this interneurone received a monosynaptic excitation from group I1 afferent fibres of the lateral gastrocnemius nerve. Nothing can be said from the present experiment concerning the receptor which was responsible for the excitation. This problem was thoroughly discussed by Matthews (1972) and he pointed out that specific experimental evidence t o clarify the receptor for each particular case is needed. No such attempt was made in the present experiments. There is a second discharge at 3 T and 5 T which was not evoked when group I fibres were blocked (block 5 T, Fig. 2). It is possible that a small fraction of the fast group I1 fibres, which might have been blocked by our method, may be responsible for the difference in the response. Alternatively, the late discharge might have been evoked by a polysynaptic pathway from group I fibres and needed a certain amount of spatial and/or temporal summation of excitatory action t o be fired. The neurone was located at the border of laminae IV and V. An example of another group of interneurones is illustrated in Fig. 3. In this group of neurones spontaneous discharges were frequently observed (spontaneous, Fig. 3). This neurone was not activated at the strength of 1.3 T. At 1.5 T one or two spikes were evoked, as can be seen in the figure, and more and more spikes were added as the stimulus strength increased t o 1 9 T. When the nerve was stimulated at 5 T during the blocking of group I fibres (block 5 T), the response was very little less than that obtained before the blocking (5 T). Therefore it can be said that this interneurone received excitatory inputs from group I1 and group I11 fibres and, possibly, from high threshold group I fibres. The latency of the first spike was about 1.6 msec from the fastest group I1 volley at the dorsal rootlet before the blocking and about 2.0 msec during the blocking of group I fibres. The latencies of the first spikes of the interneurones of this group were from 1.4 to 6.1 msec from the fastest group I1 volley at the dorsal root. For the interneurone with a latency of 1.4 msec, the central delay calculated as above was 1.0-1.2 msec. It is difficult t o say whether this value means monosynaptic or disynaptic connections (Eccles and Lundberg, 1959; Fu and Schomburg, 1974). However, there may still remain a possibility that the interneurone was activated monosynaptically from group I1 fibres, since it is not necessary t o assume that the fastest group I1 fibres evoked the response. Location of this neurone was in lamina V. Fig. 4 shows an example of an interneurone for which an intracellular impalement was successfully achieved for a sufficiently long period of time t o permit the investigation of input convergence. Group I1 and I11 fibres of tibialis anterior nerve (Fig. 4A) and flexor digitorum longus nerve (Fig. 4B, C), group I(D), II(E) and III(F, G, H, I) fibres of gastrocnemius nerve, common peroneal

190 5 T

Spontaneous

..

.. .. .

. 10 T

1.5 T

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

...

.. 19 T

2T

.. ..

..

: .

1 31

L I

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

a

.

.. ..

. ..

.... ..

t

Spontaneous

Block

5T

H 6

011

IP

10

20 msec

I

0

20

10

II

mscc

Fig. 3. An example of another type of interneurone. Explanations of illustration same as in Fig. 2. L1 shows that this interneurone responded orthdromically to the stimulation of descending tracts at L1.See text for details.

nerve (not shown) and group I1 and I11 fibres of sural nerve (not shown) exert excitatory inputs onto this interneurone. The locations of the interneurones which responded t o group I1 fibres were marked by depositing Fast green FCF dye through the recording micropipette (Thomas and Wilson, 1965). There are two locations, one in the laminae IV, V and VI and the other one in laminae VII and IX (Fukushima and Kato, 1975). These results coincide well with the results of Fu et al. (1974), who investigated the distribution of focal potentials which were obtained by electrical stimulation of group I1 fibres. Within these two locations we could not find any clear differences in terms of mono- and polysynaptic connections with

191

I‘

A

F

.ll)-.L------..

B FDL 2T

L;

10T

GF== C

I

H

D GS 1.2T

I

v

......

E

2T

10 msec

10 msec Fig. 4. EPSPs recorded in an interneurone on stimulation of different nerve fibres. Intracellular recording (upper traces) was performed with a K-citrate filled micropipette, lower traces show incoming volleys at the dorsal rootlet. Time scale for I is different from other records. See text for details.

group I1 fibres or the patterns of input convergence. Many questions remain, such as: “what are the receptors of the responsible group I1 fibres?’?;“ are they from muscle spindles or from pressure-pain receptors??’; “what are the relations with supraspinal structures??’, and so on. Nevertheless, these results indicate that there are two kinds of interneurones: one which conveys impulses independently and another which consists of common pathways of many types of afferent nerve fibres as far as the diameters of the afferent nerve fibres are concerned.

192 EFFECTS OF SELECTIVE STIMULATION OF GROUP I1 FIBRES ON EXTENSOR a-MOTONEURONES There are two current problems concerning the segmental actions of group I1 afferent fibres on extensor a-motoneurones upon which we hoped t o throw light by using the present differential blocking method. First, there has been a controversy concerning the effects from group I1 fibres upon extensor a-motoneurones. One group claims that the group I1 fibres exert inhibitory effects on them (cf., Cangiano and Lutzemberger, 1972), while the other group states there is an excitatory effect besides the inhibitory influences (cf., Wilson and Kato, 1965; Westbury, 1972; McGrath and Matthews, 1973). The second point is whether there exist monosynaptic connections with a significant strength of action from the group I1 fibres on the extensor a-motoneurones. Using averaging techniques, Kirkwood and Sears (1974, 1975) recently reported the existence of a monosynaptic EPSP in triceps surae motoneurones evoked by group I1 impulses of its own secondary spindle afferent fibres, although by more classical techniques Lundberg et al. (1975) once again failed t o see signs of such action. Fifty-eight a-motoneurones were investigated in spinal as well as in nembutalized cats (Table I). No excitatory actions from group I1 muscle afferents were observed on the medial gastrocnemius a-motoneurones in the spinal cats, while in the Nembutal cats such excitatory actions were obtained in 5 out of the 32 a-motoneurones (15.6%). Flexor a-motoneurones received predominantly excitatory actions from the group I1 fibres as has been repeatedly reported (Lloyd, 1943; Eccles and Lundberg, 1959; Lundberg et al., 1975). Two extensor a-motoneurones which received EPSPs from group I1 fibres are illustrated in Fig. 5, an MG a-motoneurone being shown in A and B and an FDL a-motoneurone being illustrated in C-G. Motoneurones were identified by an antidromic activation from the ventral root and by the presence of monosynaptic EPSP from a particular afferent nerve. When the motoneurone was orthodromically activated by stimulation of the medial gastrocnemius nerve at the strength of 5 T while the blocking current was still rising, early and late depolarizing potentials were obtained (A). In this stage group I fibres are

TABLE I FLEXOR IN THIS TABLE MEANS a-MOTONEURONES WHICH RESPONDED TO COMMON PERONEAL NERVE N o response

Depolarization

Hyperpolarization

Spinal cat MG

0

5

7

12

Nembutal cat MG FDL Flexor

5 1 6

5 1 2

22 2 2

32 4

Total

Total

10 58

193 A

C

L

ZT

F

D 31

1 E 51

H

Block

;: 4-J10 mssc

5T

*

+ MG

*

MG

-

I 11S.6

FDL

Fig. 5. Two examples of a-motoneurones which responded t o group I1 fibre stimulation. A, B: an MG a-motoneurone, C-H: an FDL a-motoneurone. Upper trace : intracellular recording except for H; lower trace: afferent volleys recorded a t dorsal rootlet. Upper trace in H was obtained after withdrawal of t h e microelectrode from the FDL motoneurone. Voltage calibration for intracellular recordings is 5 mV and for afferent volleys 1 mV.

still conducting impulses, as can be seen in the lower trace of A which was recorded from the dorsal root. When the group I fibres were completely blocked only the late depolarization remains (B). This late depolarization can be attributable to the action of group I1 afferent fibres. Latency from the fastest group I1 volley was 2.0 msec and the central delay, calculated as above, was about 1.0 msec which probably indicates a disynaptic connection. On an FDL motoneurone (C-G), probably group I1 fibres of GS, have weak excitatory effects on this neurone (D). And thin group I1 andlor thick group 111fibres have excitatory actions which are followed by weak inhibitory actions (E). Group I11 fibres have excitatory as well as inhibitory actions (F, G). Fig. 6. illustrates a Renshaw cell which was activated by stimulation of GS afferent nerve fibres. On this Renshaw cell stimulation of the nerve at the strength of below 2 T was ineffective; that is, the group I fibre was ineffective. As the strength increased from 2.1 T t o 5.1 T the number of spikes increased, as is seen in A, B and C. Apparently group I11 fibres have little effect on this neurone (D, E). When the group I fibres were blocked there still remained several spikes (F). The difference of the response in C and F may be interpreted as the group I fibre probably inducing discharges of the a-motoneurones but not being strong enough to cause the Renshaw cell discharges. When group I1 fibre impulses were added t o the motoneurones, a sufficient number of the a-motoneurones were fired due to the spatial and temporal summation of the

194 A 2.1 T

&

D 9.7T

E 3.1 T

14T

10 m..c

C 5.1 T

6J T

4

0.5 m v

1

Fig. 6. An extracellular recording from a Renshaw cell. In A there are two sweeps of the Renshaw cell responses in the upper and the middle traces. In all other pairs upper traces show Renshaw cell discharges and lower traces afferent volleys. The gastrocnemius nerve was stimulated at the strength indicated at the beginning of each sweep. Note the different sweep speed in F.

excitatory actions. Since it is known that group I1 and group I11 fibres exert inhibitory effects on Renshaw cells (Wilson et al., 1964), this excitatory action may be attributed to an indirect action through the motoneuronal collateral pathway; that is, group I1 fibres induced firing of the a-motoneurones, as often observed previously (Wilson and Kato, 1965), although it is not known from the present experiment which of the a-motoneurones, extensor or flexor, in-

195 duced firing of the Renshaw cell. This case once again shows the complexity of the excitatory inputs onto Renshaw cells (Kato and Fukushima, 1974). In summary, the present experiments show that, on occasion, EPSPs may be induced in extensor a-motoneurones from group I1 muscle afferent fibres, yet the present authors could not detect a quantitatively significant monosynaptic connection from extensor group I1 muscle afferent fibres to their own a-motoneurones.

ACKNOWLEDGEMENTS The authors would like to express their gratitudes to Dr. V.J. Wilson of The Rockefeller University for his valuable discussion during the course of the experiments. They also would like to thank Dr. P.B.C. Matthews of Oxford University for his many valuable suggestions to the early version of the manuscripts and for improving the English. REFERENCES Brown, A.G. and Hamann, W.C. (1972) DC-polarization and impulse conduction failure in mammalian nerve fibres. J. Physiol. (Lond.), 222: 66-67P. Cangiano, A. and Lutzemberger, L. (1972) The action of selectively activated group I1 muscle afferent fibres o n extensor motoneurones. Bruin Res., 41 : 475-478. Casey, K.L. and Blick, M. (1969) Observation on anodal polarization of cutaneous nerve. Bruin Res., 1 3 : 155-167. Eccles, R.M. and Lundberg, A. (1959) Synaptic actions in motoneurones which may evoke the flexion reflex. Arch. itul. Biol., 97: 199-221. Fu, T.C. and’ Schomburg, E.D. (1974) Electrophysiological investigation of the projection of secondary muscle spindle afferents in the cat. Actu physiol. scund., 9 1 : 314-329. Fu, T.C., Santini, M. and Schomburg, E.D. (1974) Characteristics and distribution of spinal focal synaptic potentials generated by group I1 muscle afferents. A c t u physiol. scund., 91: 298-313. Fukushima, K. and Kato, M. (1975) Spinal interneurones responding to group I1 muscle afferent fibres in the cat. Bruin Res., 90: 307-312. Fukushima, K., Yahara, 0. and Kato, M. (1975) Differential blocking of motor fibres by direct current. Pflugers Arch. ges. Physiol., 358: 235-242. Jack, J.J.B. and Roberts, R.C. (1974) Selective electrical activation of group I1 muscle afferent fibres. J. Physiol. (Lond.), 241: 82-84P. Kato, M. and Fukushima, K. (1974) Effect of differential blocking of motor axons on antidromic activation of Renshaw cells in the cat. Exp. Bruin Res., 20: 135-143. Kirkwood, P.A. and Sears, T.A. (1974) Monosynaptic excitation of motoneurones from secondary endings of muscle spindles. Nature (Lond.), 252 : 243-244. Kirkwood, P.A. and Sears, T.A. (1975) Monosynaptic excitation of motoneurones from muscle spindle secondary endings of intercostal and triceps surae muscle in the cat. J. Physiol. (Lond.), 245: 64-66P. Kuffler, S.W. and Vaughan Williams, E.M.(1953) Small nerve functional potentials. The distribution of small motor nerves t o frog skeletal muscle, and the membrane characteristics of the fibres they innervate. J. Physiol. (Lond.), 121: 289-317. Lloyd, D.P.C. (1943) Neuron patterns controlling transmission of ipsilateral hind limb reflexes in cat. J. Neurophysiol., 6: 293-315. Lundberg, A., Malmgren, K. and Schomburg, E.D. (1975) Characteristics of the excitatory pathway from group I1 muscle afferents t o alpha motoneurones. Bruin Res., 88: 538542.

McGrath, G.J. and Matthews, P.B.C. (1973) Evidence from the use of procaine nerve block that the spindle group I1 fibres contribute excitation t o the tonic stretch reflex of the decerebrate cat. J. Physiol. (Lond.), 235: 371-408. Manfredi, M. ( 1 9 7 0 ) Differential block of conduction of larger fibres in peripheral nerve by direct current. Arch. ital. Biol., 108: 52-71. Matthews, P.B.C. (1972) Mammalian Muscle Receptors and their Central Actions, Edward Arnold, London. Mendell, L.M. and Wall, P.D. (1964) Presynaptic hyperpolarization; a role for fine afferent fibers. J. Physiol. (Lond.), 1 7 2 : 274-294. Sassen, M. and Zimmermann, M. (1973) Differential blocking of myelinated nerve fibers by transient depolarization. Pfliigers Arch. ges. Physiol. 3 4 1 : 179-195. Thomas, R.C. and Wilson, V.J. (1965) Precise localization of Renshaw cells with a new marking technique. Nature (Lond.), 206: 211-213. Westbury, D.T. (1972) A study of stretch and vibration reflexes of the cat by intracellular recording from motoneurones. J. Physiol. (Lond.), 226 : 37--56. Wilson, V.J. and Kato, M. (1965) Excitation of extensor motoneurones by group I1 afferent fibers in ipsilateral muscle nerves. J. Neurophysiol., 28: 545-554. Wilson, V.J., Talbot, W.H. and Kato, M. (1964) Inhibitory convergence upon Renshaw cells. J. Neurophysiol., 27: 1063-1079.

DISCUSSION MATTHEWS: How large, please Dr. Kato, are your EPSPs compared to Dr. Stuart’s EPSPs. Are you seeing larger ones than he sees? KATO: I think so, since Dr. Stuart’s EPSPs are 25 or 100 pV o r something like that. STUART: Dr. Kato is using the synchronous shock. He is getting many lines o n t o the cells and we are using a single afferent. KATO: Our EPSPs are always at mV. STUART: Well, I would like to comment Dr. Kato o n a very difficult piece of work, because I’ve tried that block myself, the same profile and it’s hard work t o get it. I would like t o d o this o n one preparation. All techniques I think could d o all of this in one instance. LAPORTE: Do you not get any trouble with repetitive firing even from anodal side? You know people who have been playing with blocking are always afraid of getting repetitive firing. KATO: From the peripheral nerve? Yes, we got sometimes that kind of troubles. LAPORTE: You don’t cool the anode, for instance, o r you don’t modify, KATO: What we did was just t o wait for 1 h , then repetitive discharge disappears. I don’t know why. LAPORTE: Do you get the blocking effect quite well from the first trial or have you t o repeat this with reverse occurrence? KATO: We work usually for more than 10 h, using that blocking technique. LAPORTE: Do you have the impression that the easier t o get the selective block the higher the threshold becomes? I mean the longer the experiment lasts. From the beginning you get some good blocks? KATO: Yes, but in some cats, we had some troubles in that we recorded repetitive discharge o n the peripheral nerve.

SESSION IV

INFORMATION PROCESSING OF THE STRETCH REFLEX

Chairman: M. Ito (Tokyo)

This Page Intentionally Left Blank

The Relative Sensitivity of Renshaw Cells to Static and Dynamic Changes in Muscle Length 0. POMPEIANO and P. WAND

Istituto d i Fisiologia Umana, Cattedra II, Universitci d i Pisa, Pisa (Italy)

INTRODUCTION The discharge of mammalian motoneurons in the spinal cord is greatly reduced in time and space by the negative feedback mechanism involving the activity of Renshaw cells (Renshaw, 1941, 1946). These neurons are monosynaptically excited by the recurrent collaterals of motoneurons (Renshaw, 1946; Eccles et al., 1954) and exert their inhibitory influence directly on motoneurons, thus playing a relevant role in the control of posture and movement (Eccles, 1969; Granit, 1970,1972). Most of the work made in order t o evaluate the physiological properties of the Renshaw cells was performed under conditions in which the activity of these interneurons was induced by antidromic volleys elicited either by electrical stimulation of ventral roots or by stimulation of peripheral muscle nerves performed in deafferented animals (cf. Willis, 1971). While these are useful methods of identifying the Renshaw cells from other interneurons in the spinal cord, they undoubtedly represent unphysiological conditions t o induce the Renshaw cells t o fire. This conclusion is supported by the fact that the temporal pattern of discharge of motoneurons activated orthodromically is obviously different from that induced by electrical stimulation of the &-efferentfibers. Moreover, while small tonic motoneurons with small axons are reflexly excited at lower threshold than the larger phasic motoneurons with larger axons (Granit et al., 1957a; Kuno, 1959; Henneman et al., 1965a, b; Tan, 1971; Tan et al., 1972), weak electrical stimuli t o the motor axons, on the other hand, will excite the larger ones first. After the demonstration that electrically induced group I volleys, producing monosynaptic excitation of the motoneurons, disynaptically excited Renshaw cells via motor axon collaterals (Curtis and Ryall, 1966; Haase and Vogel, 1971; Ryall and Piercey, 1971; Ross et al., 1972; Ryall et al., 1972; cf., Wilson, 1966), experiments were performed in our laboratory t o find out whether Renshaw cells which belong t o the monosynaptic reflex pathway originating from the gastrocnemius-soleus (GS) muscle could be excited during both static and dynamic stretch of this muscle. Moreover, since static and dynamic changes in muscle length activate small and large motoneurons in different proportion,

200 we decided to evaluate quantitatively the Renshaw cell discharge elicited by static and dynamic stretch of the GS muscle for comparable frequency of discharge of the primary endings of the corresponding muscle spindles. It was soon realized that a quantitative analysis of this type could have been performed more easily during a static than during a dynamic ramp stretch. For this reason we used muscle vibration as a dynamic stimulus. It is known that longitudinal vibration applied to the gastrocnemius and/or the soleus muscle produces dynamic changes in muscle length leading to reflex contraction of the vibrated muscle in the decerebrate cat (Matthews, 1966, 1967; Barnes and Pompeiano, 1970; cf. Hagbarth and Eklund, 1966). The induced discharge of the Ia afferents, which are selectively driven by small-amplitude, high-frequency vibration (Bianconi and Van der Meulen, 1963; Brown et al., 1967; Stuart et al., 1970) is in fact transmitted monosynaptically to both the homonymous and the heteronymous motoneurons (Barnes and Pompeiano, 1970; Magherini et al., 1972), which are thus induced to discharge repetitively (Hagbarth and Eklund, 1966; Matthews, 1966; Homma et al., 1967). An evaluation of the Renshaw cell discharge elicited during static stretch and during vibration of the same muscle for comparable frequencies of discharge in the group Ia afferents can also help us t o understand why static stretch represents apparently a relatively more potent stimulus than vibration in eliciting reflex muscle contraction, although vibration is a much more effective stimulus for the primary endings of muscle spindles. Quite recently a comparison of the relative strength and the mode of interaction within a single preparation of the myographically recorded reflex responses t o static stretch and t o high-frequency vibration of the soleus muscle has been performed in the decerebrate cat (Matthews, 1967, 1969, 1970a). It appeared in particular that the tension developed in the soleus muscle during the stretch reflex was much higher, when expressed in terms of tension/impulse/sec in the primary endings of the muscle spindles, compared to tension/ impulse/sec resulting from vibration. To explain the discrepancy it was postulated that the secondary endings, which are stimulated during static stretch but not during vibration, contributed excitation t o the stretch reflex, rather than the classical believed inhibition (Lloyd, 1946; Laporte and Lloyd, 1952; Hunt, 1954; Eccles and Lundberg, 1959; Lundberg, 1964). Moreover it was found that the reflex elicited by stretch failed t o occlude that produced by vibration in the manner expected if they both depended in their entirety upon the same afferent pathway (cf. also Westbury, 1972). The conclusions of these studies have been criticized on the grounds that the reflex response t o a stretch may have been obscured by the length-tension relationship of the contracting muscle (Grillner, 1970; Grillner and Udo, 1970, 1971) and arguments related to this problem have been further developed to support (Matthews, 1970b, 1973) or disprove (Grillner, 1973) the original hypothesis. Another kind of indirect approach was used by McGrath and Matthews (1973), who compared the reflex contraction induced by muscle vibration before and after paralyzing small fibers in the muscle nerve, including y-efferents and group I1 afferents. The effect of vibration was reduced after procaine, which would be expected if the spindle group I1 afferents were excitatory in

201

the stretch reflex. However, the reduction in the vibration reflex could be attributed to the abolition, on fusimotor paralysis, of the central facilitation of the a-motoneurons normally set up in the decerebrate animal by the enhanced resting discharge of the primary endings. Unfortunately, in spite of the experimental data so far accumulated, the allocation of an autogenetic excitatory influence to the spindle group I1 fibers originating from an extensor muscle rests mostly upon indirect evidence. Experiments of selective blocking of large fibers have failed t o provide the evidence in support of the group I1 excitatory hypothesis (Laporte and Bessou, 1959; Cook and Duncan, 1971; Cangiano and Lutzemberger, 1972; EmonetDBnand et al., 1972). Observations in favor of this hypothesis, however, have recently been published (Kirkwood and Sears, 1974; see also this Symposium). The possibility that electrically induced group I1 afferent volleys may produce excitation in extensor motoneurons has been reported from time t o time in the literature (Eccles and Lundberg, 1959; Lundberg, 1964; Wilson and Kato, 1965; Lund and Pompeiano, 1970; Lundberg et al., 1975). As a matter of fact latency measurements indicate that some extensor motoneurons may receive a disynaptic excitation from group I1 afferents (Lundberg et al., 1975; see also this Symposium) and that spinal interneurons located in the area of termination of group I1 afferents (cf. Fu and Schomburg, 1974; Fu et al., 1974) can be monosynaptically excited by the group I1 fibers (Fukushima and Kato, 1975). Unfortunately the receptor origin of these afferents was not investigated in these studies. While the role exerted by the secondary endings of muscle spindles on the spinal cord is still unclear, we postulated that the differences in the gain of the stretch reflex obtained during maintained stretch and during vibration might depend upon different amounts of Renshaw inhibition, for comparable frequencies of discharge in the group Ia afferents. It will be shown in the present report that Renshaw cells, which belong t o the monosynaptic reflex pathway made by the Ia afferents from the GS muscle on the homonymous motoneurons, may respond to both static stretch and vibration of the homonymous muscle. However, the Renshaw cell discharge induced by vibration of the GS muscle is much higher than that elicited during static stretch of the same muscle, for comparable frequencies of discharge of the primary endings of the muscle spindles. The possible factors responsible for this difference will be considered at length in the discussion. The experiments reported in the following sections were performed by the authors with the collaboration of Sontag (Pompeiano et al., 1974a, b, 1975a, b). METHODS The experiments were performed on precollicular decerebrate cats, operated under ether anesthesia. The left hindlimb was completely denervated with exception of the corresponding GS muscle, which was isolated from the surrounding tissues. After a lumbosacral laminectomy, ventral roots L6-Sl of the left side were cut and the proximal end of the ventral roots L7-Sl was used either for antidromic stimulation or for recording the monosynaptic reflexes induced by

202

electrical stimulation of the GS nerve or by longitudinal vibration applied t o the corresponding muscle (cf., Morelli et al., 1970). The cats were paralyzed with gallamine triethiodide (Flaxedil, 2-4 mg/kg i.v.) and mechanically ventilated. The electrical activity of Renshaw cells was recorded with glass micropipettes filled with 3 M KCl (2-5 M a ) . After conventional amplification, the unit activity was recorded on film. Besides this, the action potentials from the same units were selected by an amplitude discriminator and their sequence analyzed by a computer using sequential pulse density histograms (Time Histogram, Mod. TH60, Correlatron 1024, Laben). The dwell time per bin varied from 0.5 t o 20 msec in different experimental conditions for a total number of 128 bins per sweep. The data stored in the computer were then recorded through an X-Y recorder after digital-analog conversion, while the corresponding digital data were printed out by a typewriter for further evaluation of the results using a desk-computer (Olivetti, Programme P101). A master timing unit was used t o trigger the oscilloscope and the computer at a given rate. Provision was made for the same timer to trigger the waveform generator (Wavetek, mod. 116) which produced the muscle vibration. The activity of the same Renshaw cells was recorded during static stretch as well as during muscle vibration. Different amounts of static stretch of the GS muscle were expressed in mm of extension, zero extension being considered that length which produced a deflection on the myograph of about 20 g. The same muscle was also submitted to longitudinal vibrations of prolonged duration, whose amplitudes and frequencies were modified while studying their effects on each of the individual units tested. RESULTS Responses of Renshaw cells to electrically induced GS volleys

The electrical activity of 123 Renshaw cells has been recorded from L7 and upper S1 spinal cord segments. Each Renshaw cell was submitted to antidromic volleys in the central end of the cut ventral roots L7-Sl and t o single shock stimulation of the ipsilateral GS nerve. Among the units which responded monosynaptically t o antidromic stimulation, were 72 Renshaw cells also excited by orthodromic stimulation in the intact GS nerve. In 50 out of these units disynaptic excitation by the group I volleys in the GS nerve could be detected. Fig. 1 shows the typical development of the response of one of these Renshaw cells to orthodromic volleys elicited by single shock stimulation of the GS nerve with increasing stimulus intensities, expressed in multiples of the threshold (T) for the orthodromic group I volley. In all the experiments the maximum excitation of the Renshaw cell was obtained at about 1.6-2.0 T, i.e., when all the group Ia afferents leading to the segmental monosynaptic reflex had be.en recruited by the stimulus. On the other hand, a further increase in the intensity of stimulus applied to the GS nerve from 2 up to 10-20 T reduced the intensity of the induced Renshaw cell discharge (see the diagram in Fig. 1).The reduction of the unit discharge did

203

1.2 T

I T

r

% 100

8

2 T

50

6 T

-

10 T

10msec

I

Ly

W

m

5

z

I I

10 msec

0

r

I

I

I

I

1

I

I

I

1

2

3

4

5

6

7

8

9

I

I

1 0 T

Fig. 1. Response of a Renshaw cell to orthodromic volleys elicited by single shock stimulation of the GS nerve with increasing stimulus intensities. Precollicular decerebrate cat with ventral roots L6-Sl cut, paralyzed with Flaxedil. GS muscle slack, A: responses of a Renshaw cell t o orthodromic volleys elicited by single shock stimulation of the GS nerve with 0.2 msec pulses of progressively increasing intensities expressed in multiples of the threshold (T) for the ingoing group I volley, as indicated a t the bottom of C. On the whole 50 sweeps taken a t t he repetition rate of 1every 5 sec were accumulated for each of the computer records, using 1 2 8 bins with the dwell time/bin of 0.5 msec. Note the progressive increase in amplitude of the Renshaw cell discharge for stimulus intensities u p to 2 T, and the reduced duration o f t h e response for stimulus intensities higher than 2 T. The scale next to the last computer record in this and other figures represents the average number of spikes per bin. B: single specimens of t h e averaged records are shown for each of the stimulus intensities used in A. In this and all subsequent records negativity is shown upward. C: monosynaptic reflex recorded from the ipsilateral ventral r o o t L7 following single shock stimulation of the GS nerve a t increasing stimulus intensities. Time calibration in C applies also to A and B. The latency of t h e Renshaw cell discharge with respect to the foot of the segmental monosynaptic reflex, evaluated o n higher sweep speed, corresponded to 0.86 rnsec, indicating tha t the reflex discharge of t h e motoneurons monosynaptically excited t h e Renshaw cell. The diagram illustrates t h e development of t h e response of this Renshaw cell as a function of the intensity of stimulation (T) applied t o the ipsilateral GS nerve. The magnitude of the response is expressed in percent of the maximum number of spikes elicited by the orthodromic GS volley within the first 40 msec after the stimulus. Each d o t represents the mean of 50 trials. The inset record represents the response of the Renshaw cell t o single shock stimulation of the ipsilateral ventral root L7 with a 0.2 msec pulse, 3 times the a-threshold.

204

not affect the early component of the response, due to co-stimulation of slowly conducting high threshold muscle afferents. It is of interest that when repetitive stimulation instead of single shock stimulation of the GS nerve was used, the threshold for this depression decreased indicating that both group I1 and I11 afferents from the GS muscle contributed to it. It is postulated that stimulation of these afferent fibers exerts an inhibitory action on extensors and an excitatory action on flexors (see Eccles and Lundberg, 1959), and that the resulting changes in motoneuronal activity may lead to a reduced discharge of the Renshaw cells linked with the corresponding GS motoneurons (see also pag. 206 and pag. 211).

Response of Renshaw cells to muscle vibration Among the 72 Renshaw cells responding t o electrical stimulation of the GS nerve, 60 responded to longitudinal vibration of the ipsilateral GS muscle fixed at 8 mm of initial extension (vibration at 200/sec and at the peak-to-peak amplitude of 180 pm, supramaximal for the primary endings of muscle spindles). In 50 out of these neurons the latency of the unit discharge with respect to the beginning of the early monosynaptic reflex induced by the first stroke of vibration corresponded to 1.16 0.32 msec (mean ? S.D.), which is quite similar to the mean value observed when Renshaw cells are excited by antidromic volleys in motor nerves (Renshaw, 1946; Eccles et al., 1954; Ryall et al., 1972). The same units also responded disynaptically to electrically induced group I volleys in the GS nerve. Fig. 2 shows a Renshaw cell identified by antidromic stimulation of L7 ventral root (A) and disynaptically excited by orthodromic stimulation of the ipsilateral GS nerve (B). The same Renshaw cell responded also t o vibration of the corresponding GS muscle with a sudden burst of high-frequency discharge (C), which appeared with a latency of 0.84 msec with respect to the segmental monosynaptic reflex induced by the first stroke of vibration (D). This finding suggests that the orthodromic group Ia volley produced by the first sinusoidal stretch (similar t o that induced by single shock stimulation of the ipsilateral GS nerve) disynaptically excited the Renshaw cells via the monosynaptic reflex of motoneurons. The amplitude of the response of these Renshaw cells to muscle vibration was always higher than that induced by repetitive electrical stimulation of all the group I afferents in the GS nerve for the same frequencies of stimulation (200/sec, for stimulus intensities lower than or corresponding to 2 T). This finding can be attributed to the fact that no threshold discrimination between the group Ia and the Ib afferents occurs on electrical stimulation of the GS nerve, contrary to muscle vibration which may excite selectively the spindle receptors with little if any excitation of Golgi tendon organs in the deefferented muscle (Bianconi and Van der Meulen, 1963; Brown et al., 1967; Stuart et al., 1970). It is likely that in the former condition the autogenetic excitation of the homonymous motoneurons induced by the group Ia volleys is partially depressed by autogenetic inhibition of the same motoneurons elicited by costimulation of the Ib afferents and possibly also of low-threshold group I1 afferents which may be recruited by stimulus intensities below 2 T.

*

205

A

C

6 il

L '

-

5 msec

Fig. 2. Response of a single Renshaw cell to antidromic ventral root stimulation and orthodromic GS volleys. Decerebrate cat with ventral roots L6-S1 cut, paralyzed with Flaxedil. GS muscle at 8 mm of initial extension. A: response of the Renshaw cell to single shock stimulation of the central end of L7 ventral root (0.2 msec pulse, 1.6 times the a-threshold). €3: effect of stimulation of the ipsilateral GS nerve with a 0.2 msec pulse, 2 times the threshold for the group I afferents. C: response of the same Renshaw cell to longitudinal vibration of the ipsilateral GS muscle at 202/sec, 180 p m peak-to-peak amplitude. The latency of this discharge corresponds to 0.84 msec if compared with that of the segmental monosynaptic reflex induced by the first stroke of the vibrator, as shown in D. The lower records in C and D represent the output of the photoelectric length meter. In this and the following figures a lengthening movement is indicated by a downward deflection in the record of the sine wave.

It is of interest that vibration of the GS muscle at 200/sec, 180 prn peak-topeak amplitude for 100 msec produced a sudden increase in the discharge frequency of Renshaw cells, which gradually decreased during the late part of the vibration (phasic response). If vibration continued for a total period of 1 sec, this phasic response of the Renshaw cell was followed by a prolonged increase in the discharge frequency which was maintained throughout the period of vibration at a steady albeit lower level than that obtained during the first 100 msec (tonic response). Simultaneous recording from the ipsilateral ventral roots L7-S1 indicated that monosynaptic reflex discharges were induced by each stroke of the vibration throughout the duration of the stimulus (cf. Barnes and Pompeiano, 1970); however, the height of mechanically induced monosynaptic reflexes during prolonged vibration corresponded only t.0 one-fourth or onefifth of the large-amplitude monosynaptic reflex induced by the first stroke of the vibrator, suggesting that a balance was reached at motoneuronal level between the autogenetic excitation produced by the mechanically induced group Ia volleys driven by the stimulus and the postsynaptic inhibitory effect due to recurrent excitation of the Renshaw cells. The responses of the Renshaw cells to muscle vibration did not appear when the GS muscle was slack or when the muscle was pulled from 0 up to 2-4 mm of initial extension. These responses increased by pulling the muscle from 4 to 8 mm, while no further increase and actually a slight decrease in the response

appeared for initial extensions of the muscle of 10-12 mm. While the appearance and the development of the Renshaw cell discharge for initial extensions ranging from 4 to 8 mm indicates that the induced discharge depends upon mechanical stimulation of the primary endings of muscle spindles, the slight depression of the Renshaw cell discharge to muscle vibration, when the muscle was pulled at initial extensions greater than 8 mm, could be attributed to autogenetic inhibition of the extensor motoneurons due to steady excitation of secondary endings of muscle spindles and/or Golgi tendon organs which occurs for these static muscle stretches. With the GS muscle fixed at 8 mm of initial extension, the threshold amplitude of vibration at 200-250/sec responsible for a Renshaw cell response ranged between 5 and 20 pm. By increasing the amplitude of vibration the response increased in magnitude up to a maximum value for amplitudes of about 70-80 pm, i.e., when all the primary endings of the muscle spindles had been recruited by the stimulus (Bianconi and Van der Meulen, 1963; Brown et al., 1967; Stuart et al., 1970). The same values were also responsible for the appearance and the maximum development of the monosynaptic reflexes simultaneously recorded from the ipsilateral ventral roots. It appears therefore that the discharge of the Renshaw cells is proportional t o the amount of synchronous motor activity, as measured by the amplitude of the mechanically induced monosynaptic reflexes elicited by the orthodromic Ia volleys. A further increase in the amplitude of vibration from 70 to 80 pm up to 300 pm did not modify the induced Renshaw cell response (Fig. 3). This finding indicates that the recruitment of the group I1 afferents elicited by increasing the amplitude of vibration did not significantly modify the response.

Quantitative analysis of the Renshaw cell discharge during muscle vibration With the GS muscle pulled at 8 mm of initial extension the activity of each of the Renshaw cells occurring during 1 sec period of vibration was recorded for increasing frequencies of sinusoidal stretch at the peak-to-peak amplitude of 180 pm. Usually 10 or 20 sweeps taken at the repetition rate of 1every 10 sec were accumulated for each of the frequencies of vibration tested (from lO/sec to 300/sec) and sequential pulse density histograms of the induced discharge were obtained using 128 bins with the dwell time of 20 msec per bin. In order to evaluate quantitatively the response of Renshaw cells t o muscle vibration, the digital counts per bin obtained from the analyzer were normalized by conversion into mean frequency of firing (imp/sec), as given by the expression: =

counts in n.__bins n bins X dwell time per bin in sec X number of trials *

Two values were always calculated for evaluation of the mean firing frequency of the Renshaw cells to prolonged vibration of a given frequency: (i) the phasic response (3)evaluated during t h e first 100 msec of the 1sec vibration period, and (ii) the tonic response ( f i i )evaluated during the last 500 msec of the 1 sec vibration period. It appears from all our recorded units that the mean discharge rate of the

207

7 r - - - 1

0

50

100

I

I

150

200

I

250 p

Fig. 3. Effect of changing amplitude of vibration o n t h e discharge frequency of Renshaw cells disynaptically excited by group Ia volleys from the GS muscle. Decerebrate cats with ventral roots L6-Sl cut, paralyzed with Flaxedil. A: excitation of a Renshaw cell during vibration of t h e GS muscle a t 250/sec for 80 msec and a t different amplitudes as indicated a t the b o t t o m of B. The GS muscle was fixed a t 7 m m of initial extension. 50 sweeps were accumulated for each of t h e computer records using 128 bins with t h e dwell time of 2 insec per bin. The series partially illustrated in A has been computed once more and t h e percentage changes in t h e discharge frequency obtained throughout the period of vibration for increasing amplitudes have been plotted in the diagram as indicated by t h e dotted curve (circles). B: same experiment as in A. The upper traces represent t h e ventral root discharges recorded from L7 following vibration of t h e GS muscle a t 200/sec and a t different amplitudes, corresponding to those used to elicit the responses of t h e Renshaw cell in A. The lower traces represent the o u t p u t of t h e photoelectric length meter. The development of t h e GS monosynaptic reflex clearly parallels that of the Renshaw cell response. The curves in the diagram indicated by continuous lines (dots and triangles) refer to Renshaw cells recorded from two other experiments in which vibration a t 250/sec, 80 msec in duration, a t different amplitudes were applied to t h e GS muscle fixed a t 8 m m of initial extension.

Renshaw cells calculated during both the phasic and the tonic components of the response was linearly related t o the frequency of vibration, at least up t o the frequencies of 150--200/sec for the phasic response (see Fig. 4)and 100150/sec for the tonic response. This finding can be matched with the observa-

208 av spikes/ bin

, A

c* v

p.......-.-

:

400

o/

,/’

I ’ ,

-E

a, , , I 0

200

0 0

50

100

150

200

250

Impjssec I a

Fig. 4. Effect of changing frequency of vibration on the discharge rate of a Renshaw cell. Same experiment as in Fig. 3, same unit. The deefferented GS muscle was fixed at 8 mm of initial extension. A : response of a Renshaw cell t o 92 msec vibration of 180-214 pm amplitude and at different frequencies as indicated at the bottom of the corresponding records illustrated in B. 50 sweeps taken at the repetition rate of 1 every 2 sec were accumulated for each of the computer records, using 128 bins with the dwell time of 2 msec per bin. The series has been computed once more and the mean discharge rate of the Renshaw cell (Imp/ sec Rc) calculated during the period of vibration has been plotted in the diagram as a function of the frequency of vibration, i.e., of the frequency of discharge of the Ia afferents (Imp/sec Ia), since for the amplitudes used one cycle of vibration generates one spike in the Ia afferents. Each circle represents the average of 50 trials. B: single specimens of the averaged records are shown for each of the vibration frequencies used in A.

tion that the responses of Renshaw cells are directly proportional to antidromic tetanization of the ventral roots from 5-10/sec up to a maximum of about 30-40 shocks/sec (Granit and Renkin, 1961; Haase, 1963; cf., Granit, 1972). Since the vibration used throughout the experimental series was of sufficient amplitude to produce driving of every primary ending of the GS muscle spindles (Bianconi and Van der Meulen, 1963; Brown et al., 1967; Stuart et al., 1970), one could evaluate the slope of the linear portion of the curve and express it in term of average increase in the discharge rate of the Renshaw cell per imp/sec average increase in Ia firing for each of the GS muscle spindles. The gain constants evaluated for 24 Renshaw cells during both the phasic and the tonic components of the response are reported in detail in Table I. It is clear from this Table that the gain constant corresponding to the phasic re-

TABLE I Serial no. of Renshaw cell (Re)

Responsiveness of R c to vibration. Gain constant: imp/secR,/imp/secIa Total Phasic Tonic Phasic/tonic

Responsiveness of Rc to static stretch. Gain constants: Imp/secR,/mm

Imp/secR, / imp/secla

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

0.93 0.68 1.80 1.41 2.38 1.56 2.65 1.78 1.88 2.28 2.14 0.98 0.67 0.90 2.18 1.19 0.83 1.14 1.05 0.56 0.91 1.08 1.52 2.40

2.05 1.55 3.15 2.72 5.03 3.67 7.32 3.78 2.53 3.81 3.88 2.61 1.05 2.60 2.1 0 2.82 2.77 2.78 2.65 1.82 1.81 2.60 0.51 3.90

0.71 0.50 1.45 1.15 1.86 0.51 2.23 1.38 1.52 1.79 1.87 0.65 0.59 0.82 1.73 0.87 0.56 0.72 0.73 0.36 0.87 0.78 0.30 1.94

2.89 3.10 2.17 2.37 2.70 7.20 3.28 2.74 1.66 2.13 2.07 4.02 1.78 3.17 1.21 3.24 4.95 3.86 3.63 5.06 2.08 3.33 1.70 2.01

0.94 2.22 0.20 0.21 1.14 1.49 4.24 0.15 0.29 0.24 3.43 0.70 0.37 0.40 1.10 1.50 1.30 -0.03 1.06 0.55 -0.63 -1.23 -

0.36 0.85 0.08 0.08 0.44 0.57 1.62 0.06 0.11 0.09 1.31 0.27 0.14 0.15 0.42 0.57 0.50 -0.01 0.40 0.21 -0.24 -0.47 -

Average gain constants

1.45

2.90

1.08

3.01

0.89

0.34

Correlation coefficients

0.975

0.978

0.964

-

0.997

0.997

sponses was on average 3.01 times higher than that obtained during the tonic response. In addition t o the mean frequency of discharge of the Renshaw cells elicited during the phasic and the tonic component of the response t o muscle vibration, the mean discharge rate of the Renshaw cells, occurring during the total 1sec period of stimulation, has been evaluated for different frequencies of vibration and the resulting slope calculated for each of the individual Renshaw cells tested (Fig. 5). The average slope of the linear portion of the curves relating the changes in the discharge frequency obtained from all the 24 Renshaw cells for increasing frequencies of vibration, i.e., for increasing frequencies of discharge in the Ia afferents obtained during the 1 sec period of vibration has been plotted in Fig. 7 (curve 2). The same figure also shows the average slope obtained from the

210 800

600

U

400

200

0 I

I

I

I

0

50

100

150

Imp/sec

200

I a

Fig. 5. Changes in the discharge rate of the Renshaw cells recorded with increasing frequencies of discharge of the group Ia afferents following prolonged vibration of the GS muscle. All the experiments were performed in decerebrate cats with the deefferented GS muscle fixed a t 8 mm of initial extension. Each line represents the calculated slope of the linear part of t h e response of an individual Renshaw cell to 1 sec vibration of 1 8 0 p m peak-to-peak amplitude and a t different frequencies from 10-25/sec u p to 2OO/sec. I n particular 20 sweeps taken a t t h e repetition rate of 1 every 1 0 sec were accumulated for each of the frequencies of vibration tested (10/sec, 25/sec, 50/sec, 75/sec, 100/sec, 150/sec, 2OO/sec), using 1 2 8 bins with the dwell time of 20 msec per bin. The series of responses obtained was computed once more and the mean discharge rate of t h e Renshaw cell (Imp/sec Rc) calculated throughout the period of vibration was plotted as a function of the frequencies of vibration used, i.e., of the frequencies of discharge of t h e Ia afferents (Imp/sec Ia). In all t h e responses of 24 Renshaw cells t o muscle vibration have been evpluated for the whole range of frequencies of t h e mechanically induced group Ia volleys. Slopes were calculated with the method of t h e least squares.

same Renshaw cells during both the phasic and the tonic components of the responses (curves 3 and 4, respectively). In this figure the slopes of the responses of the Renshaw cells t o muscle vibration have been evaluated after transformation of the coordinates, so that the corresponding values were running through zero. It appears in particular that the discharge of the 24 Renshaw cells increased on the average by 1.45 imp/sec for each imp/sec in the Ia afferents during the 1 second period of vibration (Fig. 7, curve 2). On the other hand the discharge of the same Renshaw cells increased on average by 2.90 and 1.08 imp/sec per imp/ sec in the Ia afferents during the phasic and the tonic component of the response (Fig. 7, curves 3 and 4,respectively).

Quantitative analysis of the Renshaw cell discharge during static stretch The same Renshaw cells tested for muscle vibration responded also t o static stretch of the deefferented GS muscle. This finding has been confirmed by sev-

211 era1 authors (Benecke et al., 1974; Hellweg et al., 1974). In order t o study the sensitivity of the Renshaw cells to static stretch, the discharge rate of these neurons in response t o increasing extensions was calculated. In particular the evaluation of the Renshaw cell discharge occurring for a given amount of static extension of the GS muscle (from 0 t o 1 2 mm, in 2-4 mm steps) was begun 5 sec after completion of the stretch and lasted for about 2 min. The discharge rate was then analyzed by the computer using sequential pulse density histograms for a total number of 1 2 8 bins with the dwell time of 1 sec/bin. The discharge rate of the Renshaw cells corresponded on the average t o 31.5 ? 23.2 imp/sec (mean f S.D.) in the preparation with the GS muscle slack. There was no change in the discharge rate of the Renshaw cells at 0 extension with respect t o the value obtained with the muscle slack; on the other hand, the discharge frequency of the Renshaw cell increased for increasing levels of muscle extension. The increase in Renshaw cell discharge was on the average linearly related t o the extension, at least for values ranging from 0 to 8 mm. A slight depression of the response, however, occurred for higher levels of static stretch, probably due t o autogenetic inhibition resulting from stimulation of secondary endings of muscle spindles and/or Golgi tendon organs. The slope of the linear part of the curve corresponded to 0.89 imp/sec/mm, which is the average value obtained from 22 Renshaw cells.

0

50

100

Imp/sec I a

Fig. 6. Changes in the discharge rate of the recorded Renshaw cells for increasing frequencies of discharge of the group Ia afferents following static stretch of the GS muscle. Precollicular decerebrate cats with the ventral roots L6-Sl cut. Each line represents the slope of the responses of individual Renshaw cells to static stretch of the deefferented GS muscle pulled from 0 up to 8 rnm of initial extension. The experimental data were expressed in terms of changes of the discharge frequency of the Renshaw cell (ordinate) as a function of the frequency of discharge of th Ia afferents (abscissa) evaluated for different mm of initial ex'i tension. In all 22 Renshaw ,cells were completely analyzed for increasing muscle ektensions; they belong t o the same group of cells also submitted to different frequencies of muscle vibration and illustrated in Fig. 5. Slopes were calculated with the method of the least squares.

212

0

50 Imp/sec I a

100

Fig. 7. Comparison of the excitatory effect of static stretch and vibration of the GS muscle on the same Renshaw cells. Precollicular decerebrate cats with the ventral roots L6-S1 cut. Relationship between number of Renshaw cell discharges/sec (ordinate) and number of imp/ sec in the Ia afferents (abscissa) during: 1, static stretch (n = 22 ; slope = 0.34 implsec in the Renshaw cells per imp/sec in the Ia afferents; for calculation see text); 2, vibration for 1 sec at the peak-to-peak amplitude of 180 p m (n = 24; slope = 1.45 implsec in Renshaw cells per imp/sec in the Ia afferents; correlation coefficient under linear hypothesis 0.975). The lines 3 and 4 refer to the phasic and the tonic component of the Renshaw cell responses to muscle vibration (n = 24; slopes = 2.90 and 1.08 imp/sec in Renshaw cells per implsec in the Ia afferents; correlation coefficient under linear hypothesis 0.978 and 0.964 respectively). Note the low gain of the slope relating firing rate of Renshaw cells to firing rate of the Ia afferents induced during static stretch with respect t o vibration.

Since the average discharge rate of primary spindle receptors recorded from the deefferented GS mliscle corresponded to 2.62 imp/sec/mm (cf., also Granit, 1958; Matthews and Stein, 1969), we may conclude that the increase in firing of Renshaw cells per imp/sec/mm average increase in Ia firing during static stretch corresponds to 0.34 imp/sec of the Renshaw cells per each imp/sec in the Ia afferents. A detailed illustration of the average changes in the discharge rate of each individual Renshaw cell recorded for increasing frequency of discharge of the Ia afferents following static stretch of the GS muscle is shown in Fig. 6 (see also Table I). It is clear that in most instances the increase in Renshaw cell discharge was linearly related to extension, although in 3 out of the 22 Renshaw cells examined under the condition of static stretch no autogenetic excitation but only autogenetic depression was seen. In Fig. 7 (curve 1)the average changes in the

213 discharge rate of all the Renshaw cells in response to static stretch have been indicated after transformation of the coordinates so that the line is running through zero.

DISCUSSION The present experiments performed in precollicular decerebrate animals have shown that Renshaw cells disynaptically excited by the orthodromic group I volleys induced by low-threshold electrical stimulation of the intact GS nerve responded to vibration applied longitudinally t o the deefferented GS muscle. Even in this instance the Renshaw cells fired with a latency which was compatible with the disynaptic origin of the response, indicating that excitation of the Renshaw cells depended upon monosynaptic reflex discharge of the GS motoneurons following stimulation of the Ia afferents. An analysis of the stimulus-response relationship, evaluated by measuring the discharge rate of the Renshaw cells as a function of the amplitude of vibration, clearly indicates that this response depended upon activation of the primary endings of the muscle spindles. When the muscle was settled at 8 mm of initial extension, the discharge of Renshaw cells appeared at a threshold amplitude of vibration of 5-20 pm for frequencies of 200-250/sec and increased t o a maximum value for amplitudes of about 70-80 pm, i.e., when all the primary endings of the spindles from the GS muscle had been driven by the stimulus (Bianconi and Van der Meulen, 1963; Brown et al., 1967; Stuart et al., 1970). In support of this conclusion is the fact that disynaptic excitation of Renshaw cells increased in parallel with the average amplitude of the mechanically induced monosynaptic reflexes, which also reached the greatest values for vibration amplitudes of about 70-80 pm. It appears therefore that the amount of recurrent inhibition is proportional t o the amount of synchronous motor activity as measured by the amplitude of the segmental monosynaptic reflex (cf. also Haase and Vogel, 1971; Ross et al., 1972; Ryall et al., 1972; Benecke et al., 1974). The same Renshaw cells, which responded to vibration of the GS muscle, responded also t o static stretch of the same deefferented muscle. It appears therefore that Renshaw cells disynaptically excited by orthodromic Ia volleys originating from hindlimb extensor muscle show both a static as well as a dynamic sensitivity t o stretch. In order to compare the relative effectiveness of both static stretch and muscle vibration on these spinal interneurons, the discharge of the Renshaw cells induced during the two experimental conditions was expressed as a function of the frequency of discharge of the group Ia afferents. There was a linear relation between the average increase in Renshaw cell discharge and the muscle stretch up to 8 mm, the slope of the line being 0.89 imp/sec/mm, which is the mean value obtained from 22 Renshaw cells. Since the Ia spindle receptors recorded from the deefferented GS muscle increased their discharge on the average by 2.62 imp/sec/mm, it appears that the same Renshaw cells increased on the average their firing rate by 0.34 imp/sec per each average imp/sec in the la afferents.

214 As t o the sensitivity of the Renshaw cells t o muscle vibration it appeared that the mean discharge rate of the Renshaw cells increased with the frequency of vibration at least for the range from 10 up to 150 c/sec. Since the vibration was of sufficient amplitude to produce driving of nearly all primary endings of the muscle spindles, if applied t o the appropriately extended deefferented muscle (Bianconi and Van der Meulen, 1963; Brown et al., 1967; Stuart et al., 1970), the slope of the stimulus-response curve could be expressed as increase in the discharge rate of Renshaw cells per average imp/sec in the Ia afferents. It appears in particular that the discharge rate of the Renshaw cells increased on the average by 1.45 imp/sec per each imp/sec in the Ia afferents. This mean value is 4.3 times higher than that obtained during static stretch. The discrepancies in the gain factors observed by relating the changes in the firing rate of the same Renshaw cell t o the frequencies of discharge of the Ia afferents induced during static stretch and vibration can be attributed t o differences in motoneuron synchronization. Due t o the synchronous discharge of the spindle receptors, the vibration reflexes produce a greater synchronization of motoneuron discharge than the stretch reflexes and this factor may account for a greater net discharge among Renshaw cells during the vibration responses. There are, however, additional possibilities which must be taken into account. It is known that small and large motoneurons are not equally recruited in the decerebrate animal for comparable frequencies of discharge of the Ia afferents during static stretch and during vibration and this may be reflected in different amounts of Renshaw cell activity induced under the two different experimental conditions (see Pompeiano et al., 1975b, for references). In particular, we may postulate that the spatiotemporal pattern of the stretch receptor input produced asynchronously during static stretch is mainly if not exclusively effective on the small, tonic motoneurons (Granit et al., 1957a, b; Kernell, 1966; Henneman et al., 1965a, b; Burke, 1967, 1968a, b; Burke and ten Bruggencate, 1971; cf., Granit, 1970), while that produced synchronously during muscle vibration is effective not only on small, tonic but also on large, phasic motoneurons (Anastasijevie et al., 1968, 1971; Westbury, 1971, 1972). On the basis of the likely assumption that different amounts of small and large motoneurons are excited during stretch and vibration for the same amount of the Ia input, one may postulate that the higher gain of the Renshaw cell activity, induced during vibration as compared t o static stretch for increasing frequencies of discharge of the Ia afferents, is due to: (i) greater recruitment of large motoneurons in the former than in the latter condition (cf. Anastasijevie et al., 1968; Hellweg et al., 1974; Pompeiano et al., 1975c), (ii) the greater capability of the large motoneurons for following the frequency of stimulation (Anastasijevid et al., 1968; Brown et al., 1968; Homma et al., 1967, 1970a, b, 1971, 1972; cf., Eccles et al., 1958; Granit, 1970, 1972), and (iii) the greater ability of large motoneurons t o excite Renshaw cells with respect t o small motoneurons (Ryall et al., 1972; Hellweg et al., 1974; Pompeiano et al., 1975c; cf. Granit et al., 1975a; Eccles et al., 1961) *. ,

* Attempts were made recently to study differences in the relative effectiveness of different size ranges of motor axons to Renshaw cells by differential blocking of larger fibers of the

215 This last hypothesis is supported by the fact that the response of the Renshaw cell t o muscle vibration is made by a phasic and a tonic component, the former being much greater than the latter (2.90 and 1.08 imp/sec of Renshaw cell discharge per average imp/sec in the Ia afferents respectively). Since most of the large motoneurons discharge only during the early part of vibration (Anastasijevii: et al., 1968), they must contribute significantly to the phasic response, whereas small motoneurons are probably involved in the late tonic component of the response, since they always show a sustained reflex activity during vibration. However, in addition to small tonic motoneurons, there are large phasic motoneurons which discharge reflexly throughout the vibration period (AnastasijeviC:et al., 1968; Brown et al., 1968).This finding may explain why the sensitivity of the Renshaw cell during the tonic response to vibration is still 3.2 times higher than the sensitivity of the same Renshaw cell during static stretch, where probably small tonic motoneurons are mainly recruited by the stimulus. The hypothesis that recurrent collaterals of large phasic motoneurons have a stronger excitatory action on Renshaw cells than do axon collaterals of the smaller tonic motoneurons (Ryall et al., 1972; Hellweg et al., 1974; Pompeiano et al., 1975c; cf., Granit et al., 1957a; Eccles et al., 1961) is further supported by the observation that the Renshaw cell responses are not only length dependent but also rate dependent (Hellweg et al., 1974; Pompeiano et al., 1 9 7 5 ~ ) . Velocity of stretch actually represents the most effective method in exciting Renshaw cells. An analysis of the responses of Renshaw cells, disynaptically excited by the orthodromic group Ia volleys originating in the GS nerve, to a family of individual sinusoidal stretches of the deefferented GS muscle of the same amplitude but of different variable duration has clearly shown that large amplitude sinusoidal stretches, supramaximal for producing excitation of all the primary endings of muscle spindles, produced a sinusoidal modulation in Renshaw cell discharge for very low velocities of stretch. The linearity of the response, however, disappeared and was substituted by a sudden burst-like increase in the Renshaw cell activity which appeared at shorter latency as soon as velocity of stretch raised above a given value (Pompeiano et al., 1 9 7 5 ~ )There . is apparently a critical velocity of stretch at which the pattern of response of Renshaw cells changes from the sinusoidally modulated type t o the burst-like type. Since the size of the cell dictates the order at which recruitment of individual motoneurons occurs reflexly in response t o muscle stretch (Henneman et al., 1965a, b; cf. Granit, 1970) it has been postulated that the smooth changes in the discharge rate of the Renshaw cells to sinusoidal stretches of low velocity depend exclusively upon activation of small tonic motoneurons. On the other hand, the sharp burst-like response of the Renshaw cells t o high-velocity sinusoidal stretches may depend upon recruitment of large phasic motoneurons. gastrocnemius nerve (Kato and Fukushima, 1974). Unfortunately since branching normally occurs in the motor axons to the gastrocnemius muscle (Eccles and Sherrington, 1930; Gilliatt, 1966; Ebel and Gilman, 1969; Copack et al., 1975), the larger motor axons at ventral root level may not necessarily contribute to the larger motor axons at various distances from the muscle.

216 The hypothesis that individual Renshaw cells are more powerfully excited by recurrent collaterals of large phasic a-motoneurons, particularly recruited during vibration, than by collaterals of the small tonic motoneurons activated particularly during static stretch is relevant to the observation that small tonic a motoneurons (Granit et al., 1957a; Kuno, 1959; Eccles et al., 1961; Tan, 1971, 1972; Tan et al., 1972; cf. also Holmgren and Merton, 1954), as well as static y-motoneurons (cf., Pompeiano et al., 1975b for references), are in their turn subject t o greater recurrent inhibition than are the large phasic motoneurons *. We may postulate therefore that for the same amount of discharge of the Ia afferents from the GS muscle the amount of Renshaw inhibition which affects small tonic a-motoneurons as well as static y-motoneurons is greater during vibration than during static stretch. These actions may explain why activation of the primary endings of muscle spindles produced by muscle vibration generates less reflex tension than is obtained by static stretch for comparable frequencies of discharge of the Ia afferents, as reported in the Introduction. The final problem now is to find out whether the group I1 afferents, which are stimulated during static stretch but not during small amplitude vibration of the GS muscle, contribute to this difference. In particular, the group I1 afferents might act at spinal cord level by reducing the gain of the Renshaw cell discharge obtained during static stretch with respect to that obtained during vibration for the same frequency of discharge of the Ia afferents. Indeed there is evidence that the activity of Renshaw cells, disynaptically excited by electrically induced group Ia volleys from the ipsilateral GS muscle, was depressed when high threshold group I1 and I11 muscle afferents had been recruited by the stimulus (cf. also Wilson et al., 1964; Curtis and Ryall, 1966; Ryall and Piercey, 1971; Fromm et al., 1975). Unfortunately this depression obtained by electrical stimulation of the GS nerve cannot by itself be attributed to activation of the secondary endings of muscle spindles, since the group I1 spectrum contains afferents originating from receptor organs different from the spindles. Moreover, the observation that the secondary endings of the muscle spindles, which are recruited by large amplitude muscle vibrations (Stuart et al., 1970), did not modify the response of the Renshaw cells to the mechanically induced group Ia volleys can hardly be evaluated, since for the amplitudes of vibration used the group I1 mediated effect was probably not so strong to overcome the powerful excitation of the Renshaw cells elicited by the group Ia volleys via the monosynaptic reflex discharge of the homonymous motoneurons. The hypothesis that the secondary endings of the GS muscle spindles may depress the activity of Renshaw cells is supported, however, by the finding that the response of these cells to static stretch, as well as to muscle vibration, slightly decreased when the homonymous muscle was pulled at initial extensions greater than 8 mm. This effect can be attributed to some influence that the group I1 afferents (in addition to the Ib afferents) exert on Renshaw cells probably acting via the motoneurons.

* The existence of an autogenetic reciprocal inhibition which acts asymmetrically from large to small motoneurons by utilizing the Renshaw cells (cf., Tan, 1975) is desirable to oppose tonic discharge during rapid voluntary movements, which might otherwise be hindered (cf., Denny-Brown, 1928; Granit et al., 1957a; Eccles et al., 1961).

217 It would be of interest to know whether the secondary endings of the GS muscle spindles may reduce the discharge of Renshaw cells disynaptically excited by the orthodromic group Ia volleys either by inhibiting some extensor motoneurons (cf. Cangiano and Lutzemberger, 1972), thus leading to disfacilitation of the corresponding Renshaw cells or by exciting flexor motoneurons possibly coupled with the extensors by a mutual Renshaw to Renshaw inhibition (Renshaw, 1946; Ryall, 1970; Ryall and Piercey, 1971; Ryall et al., 1971).

SUMMARY

(1)Renshaw cells, monosynaptically excited by ventral root stimulation and disynaptically excited by electric stimulation of the group I afferents in the gastrocnemius-soleus (GS) nerve, were submitted to both dynamic stretch (vibration) and static stretch of the deefferented GS muscle in precollicular decerebrate cats. In particular, the response of these units t o prolonged vibration applied longitudinally to the GS muscle was compared with that elicited by static stretch of the homonymous muscle, for comparable frequencies of discharge of the group Ia afferents. (2) The response of Renshaw cells to 1 sec periods of muscle vibration increased with the frequency of vibration and, over the value of lO/sec, appeared to be linearly related to the frequency of the input, at least up t o the frequency of 150/sec. Since vibration was of sufficient amplitude t o produce driving of all the primary endings of muscle spindles, the responses were expressed as mean increases in the discharge rate of Renshaw cells per average imp/sec in the Ia afferents. The discharge of the Renshaw cells increased on average by 1.45 imp/ sec per each imp/sec in the Ia afferents. (3) The same Renshaw cells tested above responded also with increasing frequencies of discharge to increasing levels of static extension of the GS muscle. In particular the discharge frequency of Renshaw cells was on average linearly related to muscle extension at least for values ranging from 0 t o 8 mm. The mean increase in discharge rate as a function of the static extension corresponded on average to 0.89 imp/sec/mm. Since the discharge rate of the primary endings of muscle spindles recorded from the deefferented GS muscle increased by 2.62 imp/sec/mm, it appears that the mean increase in the discharge rate of Renshaw cells as a function of static extension corresponded on average to 0.34 implsec per each implsec in the Ia afferents. (4) A comparison of the responses of the same Renshaw cells t o static stretch and vibration indicates that the orthodromic excitation of Renshaw cells is on average 4.3 times greater during vibration than during static stretch for comparable increases in firing of the Ia afferents. This finding may explain why activation of the primary spindle receptors by vibration generates less reflex tension than is obtained by static stretch for the same amount of the proprioceptive Ia input. (5) Since the spatiotemporal pattern of the stretch receptor input produced asynchronously during static stretch is mainly effective on small tonic a-motoneurons (length sensitive) while that produced synchronously during muscle vibration is effective not only on small tonic but also on large phasic a-motoneu-

218 rons (rate sensitive), it is postulated that axon collaterals of larger phasic amotoneurons are more powerful in exciting Renshaw cells, than are those originating from smaller tonic a-motoneurons. There is also the possibility that the secondary endings of the GS muscle spindles, which are recruited during static stretch but not during small amplitude vibration, reduce the response of the Renshaw cells to the orthodromic group Ia volleys originating from the GS muscle, thus leading to some disinhibition of extensor motoneurons. ACKNOWLEDGEMENTS The investigations summarized in the present report were supported in part by the European Training Program in Brain and Behaviour Research, in part by the Public Health Service Research Grant NS 07685-08 from the National Institute of Neurological Diseases and Stroke, N.I.H., U.S.A., by a Research Grant from the Consiglio Nazionale delle Ricerche, Italy, and by the Deutsche Forschungsgemeinschaft, SFB 33.

REFERENCES Anastasijevie, R., AnojCik, M., Todorovif, B. and VuEo, J. (1968) The differential reflex excitability of alpha motoneurons of decerebrate cats caused by vibration applied to the tendon of the gastrocnemius medialis muscle. Brain Res., 11: 336-346. Anastasijevie, R., CvetkoviE, M. and VuEo, J. (1971) The effect of short-lasting repetitive vibration of the triceps muscle and concomitant fusimotor stimulation on the reflex response of spinal alpha motoneurones in decerebrated cats. Pflugers Arch. ges. Physiol., 325: 220-234. Barnes, C.D. and Pompeiano, 0. (1970) Presynaptic and postsynaptic effects in the monosynaptic reflex pathway to extensor motoneurons following vibration of synergic muscles. Arch, ital. Biol., 108: 259-294. Benecke, R., Hellweg, C. and Meyer-Lohman, J. (1974) Activity and excitability of Renshaw cells in non-decerebrate and decerebrate cats. Exp. Brain Res., 21: 113-124. Bianconi, R. and Van der Meulen, J.P. (1963) The response t o vibration of the end organs of mammalian muscle spindles. J. Neurophysiol., 26: 177-190. Brown, M.C., Engberg, I. and Matthews, P.B.C. (1967) The relative sensitivity to vibration of muscle receptors of the cat. J. Physiol. (Lond.), 192: 773-780. Brown, M.C., Lawrence, D.G. and Matthews, P.B.C. (1968) Reflex inhibition by Ia afferent input of spontaneously discharging motoneurones in the decerebrate cat. J. Physiol. (Lond.), 198: 5-7P. Burke, R.E. (1967) Motor unit types of cat triceps surae muscle. J. Physiol. (Lond), 193: 141-1 60. aurke, R.E. (1968a) Group Ia synaptic input t o fast and slow twitch motor units of cat triceps surae. J. Physiol. (Lond.), 196: 605-630. Burke, R.E. (1968b) Firing patterns of gastrocnemius motor units in the decerebrate cat. J. Physiol. (Lond.), 196: 631-4554. Burke, R.E. and ten Bruggencate, G. (1971) Electrotonic characteristics of alpha motoneurones of varying size. J. Physiol. (Lond.), 212: 1-20. Cangiano, A. and Lutzemberger, L. (1972) The action of selectively activated group I1 muscle afferent fibers on extensor motoneurons. Brain Res., 41 : 475-478. Cook, W.A., Jr. and Duncan, C.C., Jr. (1971) Contribution of group I afferents t o the tonic stretch reflex of the decerebrate cat. Brain Res., 33: 509-513. Copack, P.B., Felman, E., Lieberman, J.S. and Gilman, S. (1975) Differences in proximal

219 and distal conduction velocities of efferent nerve fibers to the medial gastrocnemius muscle. Brain Res., 91: 147-150. Curtis, D.R. and Ryall, R.W. (1966) The synaptic excitation of Renshaw cells. Exp. Brain Res., 2: 81-96. Denny-Brown, D. (1928) On the Essential Mechanism of Mammalian Posture. D. Phil. Thesis, University of Oxford. Ebel, H.C. and Gilman, S. (1969) Estimation of errors in conduction velocity measurements due t o branching of peripheral nerve fibers. Brain Res., 16: 273-276. Eccles, J.C. (1969) The Inhibitory Pathways of the Central Nervous System, Thomas, Springfield, Iil., 135 pp. Eccles, J.C. and Sherrington, C.S. (1930) Numbers and contraction-values of individual motor-units examined in some muscles of the limb. Proc. roy. SOC.B , 106: 326-357. Eccles, J.C., Fatt, P. and Koketsu, K. (1954) Cholinergic and inhibitory synapses in a pathway from motor-axon collaterals to motoneurones. J. Physiol. (Lond.), 126: 524562. Eccles, J.C., Eccles, R.M. and Lundberg, A. (1958) The action potentials of the alpha motoneurones supplying fast and slow muscles. J. Physiol. (Lond.), 142: 275-291. Eccles, J.C., Eccles, R.M., Iggo, A. and Ito, M. (1961) Distribution of recurrent inhibition among motoneurones. J. Physiol. (Lond.), 159: 479-499. Eccles, R.M. and Lundberg, A. (1959) Synaptic actions in motoneurones by afferents which may evoke the flexion reflex. Arch. ital. Biol., 97: 199-221. Emonet-Dknand, F., Jami, L., Joffroy, M. et Laporte, Y. (1972) Absence de rkflexe myotatique aprds blocage de la conduction dans les fibres du groupe I. C.R. Acad. Sci. (Paris), 274: 1542-1545. Fromm, C., Haase, J. and Wolf, E. (1975) Decrease of the recurrent inhibition of extensor motoneurons due to group I1 afferent input. Pfliigers Arch. ges. Physiol., 359, Suppl.: No. 155, R78. Fu, T.G. and Schomburg, E.D. (1974) Electrophysiological investigation of the projection of secondary muscle spindle afferents in the cat spinal cord. Acta physiol. scand., 91 : 314-329. Fu, T.G., Santini, M. and Schomburg, E.D. (1974) Characteristics and distribution of spinal focal synaptic potentials generated by group I1 muscle afferents. Acta physiol. scand., 91: 298-313. Fukushima, K. and Kato, M. (1975) Spinal interneurons responding t o group I1 muscle afferent fibers in the cat. Brain Res., 90: 307-312. Gilliatt, R.W. (1966) Axon branching in motor nerves. In Control and Innervation of Skeletal Muscle, B.L. Andrew (Ed.), Livingstone, Edinburgh, 1966, pp. 53-63. Granit, R. (1958) Neuromuscular interaction in postural tone of the cat’s isometric soleus muscle. J. Physiol. (Lond.), 143: 387-402. Granit, R. (1970) The Basis o f M o t o r ControE, Academic Press, New York, 1970, 353 pp. Granit, R. (1972) Mechanisms Regulating the Discharge of Motoneurons, Liverpool University Press, Liverpool, 1972, 88 pp. Granit, R. and Renkin, B. (1961) Net depolarization and discharge rate of motoneurones, as measured by recurrent inhibition. J. Physiol. (Lond.), 158: 461-475. Granit, R., Pascoe, J.E. and Steg, G. (1957a) Behaviour of tonic (Y and 7 motoneurones during stimulation of recurrent collaterals. J. Physiol. (Lond.), 138: 381-400. Granit, R., Phillips, C.G., Skoglund, S. and Steg, G. (1957b) Differentiation of tonic from phasic alpha ventral horn cells by stretch, pinna and crossed extensor reflexes. J. Neurophysiol., 20: 470-481. Grillner, S. (1970) Is the tonic stretch reflex dependent upon group I1 excitation? Acta physiol. scand., 78: 431-432. Grillner, S. (1973) Muscle stiffness and motor control-forces in the ankle during locomotion and standing. In Motor Control. A.A. Gydikov, N.T.Tankov and D.S. Kosarov (Eds.), Plenum Press, New York, 1973, pp. 195-215. Grillner, S. and Udo, M. (1970) Is the tonic stretch reflex dependent on suppression of autogenetic inhibitory reflexes? Acta physiol. scand., 79: 13-14A. Grillner, S. and Udo, M. (1971) Motor unit activity and stiffness of the contracting muscle fibers in the tonic stretch reflex. Actaphysiol. scand., 81: 422-424.

Haase, J. (196:) Die Transformation des Entladungsmusters der Renshaw-Zellen bei tetanischer antidromer Reizung. Pfliigers Arch. ges. Physiol., 276: 471-480. Haase, J. und Vogel, B. (1971) Die Erregung der Renshaw-Zellen durch reflektorische Entladungen der a-Motoneurone. Pfliigers Arch. ges. Physiol. 325: 14-27. Hagbarth, K.-E. and Eklund, G. (1966) Motor effects of vibratory muscle stimuli in man. In Muscular Afferents and Motor Control, Nobel Symposium I , R. Granit (Ed.), Almqvist and Wiksell, Stockholm, 1966, pp. 177-186. Hellweg, C., Meyer-Lohmann, J., Benecke, R. and Windhorst, U. (1974) Responses of Renshaw cells t o muscle ramp stretch. Exp. Brain Res., 21: 353-360. Henneman, E., Somjen, G. and Carpenter, D.O. (1965a) Functional significance of cell size in spinal motoneurons. J. Neurophysiol., 28: 560-580. Henneman, E., Somjen, G. and Carpenter, D.O. (1965b) Excitability and inhibitibility of motoneurons of different sizes. J. Neurophysiol., 28: 599-620. Holmgren, B. and Merton, P.A. (1954) Local feedback control of motoneurones. J . Physiol. (Lond.), 123: 47P. Homma, S., Ishikawa, K. and Watanabe, S. (1967) Optimal frequency of muscle vibration for motoneuron firing. J. Chiba med. SOC.,43: 190-196. Homma, S., Ishikawa, K. and Stuart, D.G. (1970a) Motoneuron responses t o linearly rising muscle stretch. Amer. J. phys. Med., 49: 290-306. Homma, S., Kobayashi, H. and Watanabe, S. (1970b) Vibratory stimulation of muscles and stretch reflex. Jap. J. Physiol., 20: 309-319. Homma, S., Kanda, K. and Watanabe, S. (1971) Monosynaptic coding of group Ia afferent discharges during vibratory stimulation of muscle. Jap. J. Physiol., 21 : 405-417. Homma, S., Kanda, K. and Watanabe, S. (1972) Preferred spike intervals in the vibration reflex. Jap. J. Physiol., 22: 421-432. Hunt, C.C. (1954) Relation of function t o diameter in afferent fibers of muscle nerves. J. gen. Physiol., 38: 117-131. Kato, M. and Fukushima, K. (1974) Effect of differential blocking of motor axons on antidromic activation of Renshaw cells in the cat. Exp. Brain Res., 20: 135-143. Kernell, D. (1966) Input resistance, electrical excitability, and size of ventral horn cells in cat spinal cord. Science, 152: 1637-1640. Kirkwood, P.A. and Sears, T.A. (1974) Monosynaptic excitation of motoneurones from secondary endings of muscle spindles. Nature (Lond.), 252 : 243-244. Kuno, M. (1959) Excitability following antidromic activation in spinal motoneurones supplying red muscles. J. Physiol. (Lond.), 149: 374-393, Laporte, Y. et Bessou, P. (1959) Modification d’excitabilit6 de motoneurones homonymes provoqu6es par l’activation physiologique de fibres aff6rentes d’origine musculaire du groupe 11. J. Physiol. (Paris), 51: 897-908. Laporte, Y. and Lloyd, D.P.C. (1952) Nature and significance of the reflex connections established by large afferent fibres of muscular origin. Amer. J. Physiol., 1 6 9 : 609621. Lloyd, D.P.C. (1946) Integrative pattern of excitation and inhibition in two-neuron reflex arcs. J. Neurophysiol., 9 : 439-444. Lund, S. and Pompeiano, 0. (1970) Electrically induced monosynaptic and polysynaptic reflexes involving the same motoneuronal pool in the unrestrained cat. Arch. ital. Biol., 108: 130-153. Lundberg. A. (1964) Supraspinal control of transmission in reflex paths to motoneurones and primary afferents. In Progress in Brain Research, Vol. 12, Physiology of Spinal Neurons, J.C. Eccles and J.P. Schad6 (Eds.), Elsevier, Amsterdam, 1964, pp. 196-221. Lundberg, A., Malmgren, K. and Schomberg, E.D. (1975) Characteristics of the excitatory pathway from group I1 muscle afferents t o alpha motoneurones. Brain Res., 88: 538-542. Magherini, P.C., Pompeiano, 0. and Thoden, U . (1972) The relative significance of presynaptic and postsynaptic effects on monosynaptic extensor reflexes during vibration of synergic muscles. Arch. ital. Biol., 110: 70-89. Matthews, P.B.C. (1966) The reflex excitation of the soleus muscle of the decerebrate cat caused by vibration applied t o its tendon. J. Physiol. (Lond.), 184: 450-472.

221 Matthews, P.B.C. (1967) Vibration and the stretch reflex. In Myotatic, Kinesthetic and Vestibular Mechanisms, Ciba Foundation S y m p o s i u m , A.V.S. de Reuck and J. Knight (Eds.), Churchill, London, 1967, pp. 40--50. Matthews, P.B.C. (1969) Evidence that the secondary as well as the primary endings of muscle spindles may be responsible for the tonic stretch reflex of the decerebrate cat. J. Physiol. ( L o n d . ) , 204: 365-393. Matthews, P.B.C. (1970a) The origin and functional significance of the stretch reflex. In Excitatory Synaptic Mechanisms, P. Andersen and J.K.S. Jansen (Eds.), Universitetsforlaget, Oslo, pp. 301-315. Matthews, P.B.C. (1970b) A reply t o criticism of the hypothesis t h a t the group I1 afferents contribute excitation t o the stretch reflex. A c t a physiol. scand., 79: 431-433. Matthews, P.B.C. (1973) A critique of the hypothesis that the spindle secondary endings contribute excitation t o the stretch reflex. In Conlrol o f Posture and L o c o m o t i o n , R.B. Stein, K.G. Pearson, R.S. Smith and J.B. Redford (Eds.), Plenum Press, New York, pp. 227-243. Matthews, P.B.C. and Stein, R.B. (1969) The sensitivity of muscle spindle afferents t o small sinusoidal changes of length, J. Physiol. ( L o n d . ) , 200: 723-743. McGrath, G.J. and Matthews, P.B.C. ( 1 9 7 3 ) Evidence from the use of vibration during procaine nerve block that the spindle group I1 fibres contribute excitation t o the tonic stretch reflex of the decerebrate cat. J. Physiol. ( L o n d . ) , 235: 371-408. Morelli, M., Nicotra, L., Barnes, C.D., Cangiano, A., Cook, W.A., Jr. and Pompeiano, 0. (1970) An apparatus for producing small-amplitude high-frequency sinusoidal stretching of the muscle. Arch. ital. Biol., 108: 222-232. Pompeiano, O., Wand, P. and Sontag, K.-H. (1974a) Excitation of Renshaw cells by orthodromic group Ia volleys following vibration of extensor muscles. Pflugers Arch. ges. Physiol., 347: 137-144. Pompeiano, O., Wand, P. and Sontag, K.-H. (1974b) A quantitative analysis of Renshaw cell discharges caused by stretch and vibration reflexes. Brain Res., 6 6 : 519-524. Pompeiano, O., Wand, P. and Sontag, K.-H. (1975a) Response of Renshaw cells t o sinusoidal stretch of hindlimb extensor muscles. Arch. ital. Biol., 113: 205-237. Pompeiano, O., Wand, P. and Sontag, K.-H. (197513) The relative sensitivity of Renshaw cells t o orthodromic group Ia volleys caused by static stretch and vibration of extensor muscles. Arch. ital. B i d , 113: 238-279. Pompeiano, O., Wand, P. and Sontag, K.-H. ( 1 9 7 5 ~ The ) sensitivity of Renshaw cells t o velocity of sinusoidal stretches of the triceps surae muscle. Arch. ital. Biol., 113: 280294. Renshaw, B. ( 1 9 4 1 ) Influence of discharge of motoneurons upon excitation of neighboring motoneurons. J. Neurophysiol., 4 : 167-183. Renshaw, B. (1946) Central effects of centripetal impulses in axons of spinal ventral roots. J. Neurophysiol., 9 : 191-204. Ross, H.-G., Cleveland, S. and Haase, J. (1972) Quantitative relation of Renshaw cell discharge t o monosynaptic reflex height, Pfliigers Arch. ges. Physiol., 332: 73-79. Ryall, R.W. (1970) Renshaw cell mediated inhibition of Renshaw cells: patterns of excitation and inhibition from impulses in motor axon collaterals. J. Neurophysiol., 3 3 : 2 57-2 7 0. Ryall, R.W. and Piercey, M.F. ( 1 9 7 1 ) Excitation and inhibition of Renshaw cells by impulses in peripheral afferent nerve fibers. J. Neurophysiol., 34: 242-251. Ryall, R.W., Piercey, M.F. and Polosa, C. ( 1 9 7 1 ) Intersegmental and intrasegmental distribution of mutual inhibition of Renshaw cells. J. Neurophysiol., 34: 700-707. Ryall, R.W., Piercey, M.F., Polosa, C. and Goldfarb, J. (1972) Excitation of Renshaw cells in relation t o orthodromic and antidromic excitation of motoneurons. J. Neurophysiol., 35: 137-148. Btuart, D.G., Mosher, C.G., Gerlach, R.L. and Reinking, R.M. (1970) Selective activation of Ia afferents by transient muscle stretch. E x p . Brain Res., 10: 477-487. Tan, U. (1971) Changes in firing rates of extensor motoneurones caused by electrically increased spinal inputs. Pfliigers Arch. ges. Physiol., 3 2 6 : 35-47, Tan, U.(1972) The role of recurrent and presynaptic inhibition in the depression of tonic motoneuronal activity. Pfliigers Arch. ges. Physiol., 337: 229-239.

222 Tan, U . (1975) Post-tetanic changes in the discharge pattern of the extensor alpha m otoneurones. Pflugers Arch. ges. Physiol., 353: 43-57. Tan, U., Yorukan, S. and Ridvanagaoglu, A.Y. (1972) A quantitative analysis of the m otoneuronal depression produced by increasing the stimulus parameters of afferent tetanization. Pfliigers Arch. ges. Physiol., 333: 240-257. Westbury, D.R. (1971) The response o f a-motoneurones of the cat t o sinusoidal movements of the muscles they innervate. Brain Res., 2 5 : 75-86. Westbury, D.R. ( 1 9 7 2 ) A study of stretch and vibration reflexes of the cat by intracellular recording from motoneurones. J. Physiol., (Lond.), 2 2 6 : 37-56. Willis, W.D. (1971) The case for t h e Renshaw cell. Brain Behau. Evol., 4 : 5-52. Wilson, V.J. (1966) Regulation and function of Renshaw cell discharge. In Muscular A f f e r ents and Motor Control, Nobel Symposium I, R. Granit (Ed.), Almqvist and Wiksell, Stockholm, 1966, pp. 317-329. Wilson, V.J. and Kato, M. (1965) Excitation of extensor motoneurons by group I1 afferent fibers in ipsilateral muscle nerves. J. Neurophysiol., 28: 545-554. Wilson, V.J., Talbot, W.H. and Kato, M. ( 1 9 6 4 ) Inhibitory convergence upon Renshaw cells. J. Neurophysiol., 2 7 : 1063-1079.

Muscle Stretch and Chemical Muscle Spindle Excitation : Effects on Renshaw Cells and Efficiency of Recurrent Inhibition * J. MEYER-LOHMANN, H.-D. HENATSCH, R. BENECKE and C. HELLWEG

**

Department of Physiology 11, University of Gottingen, 0-3400Gottingen (G.F.R.)

The principal events occurring in the stretch reflex are relatively well known, as far as the main stations of the reflex pathway are concerned. At a closer look, however, the situation is complicated by many additional factors, as we know from the studies of numerous workers. One of these factors is the recurrent Renshaw mechanism forming an intraspinal feedback loop from the output side of the motoneurones (Renshaw, 1946). It is the aim of the present paper to analyze, by several independent approaches, some aspects of the functional role of the Renshaw mechanism in the stretch reflex. Let us begin with an observation (Meyer-Lohmann and Henatsch, 1966) obtained from a decerebrate cat which exhibited long-lasting tonic stretch reflexes. In Fig. 1,the 3 upper graphs represent the tonic discharge frequencies of a single a-motoneurone (a-MN) during prolonged stretches of the triceps surae muscle. Stretch lengths of 8, 10 and finally 1 2 mm were used. In spite of the stepwise increase of extension, the 3 curves are quite similar, showing a nearly constant discharge frequency at about the same level during all 3 stretches (see also Denny-Brown, 1929; Grillner and Udo, 1971a; Kernell, this symposium). Obviously a strong stabilizing influence is capable of maintaining a mean frequency level and regularizing the individual discharge intervals within narrow limits. Since we suspected that this effect was due, at least partly, to the recurrent Renshaw feedback, we attempted to block this mechanism by means of the drug dihydro-0-erythroidine (DHE) which is known to suppress rather sufficiently, though not completely, the early Renshaw cell (RC) responses (Eccles et al., 1954, 1956). The result is seen in the lower 3 records, obtained with the same extension steps as before: after DHE, there is not only a very marked irregularity of the individual discharges, but also a more distinct stepwise increase of the mean frequency level with higher extension values. This strongly supports the view that an intact Renshaw mechanism is necessary for the relative constancy of the stretch responses, which contributes to stabilization of the a-MN discharges under these experimental conditions. If this is

* Supported in part by grants from the Deutsche Forschungsgemeinschaft (SFB-33: “Nervensystem und biologische Information”, Gottingen). * * Present address: Max-Planck-Institut fur biophysikalische Chemie, Gottingen, G.F.R.

224 Contr o I

B

A 8 m m Stretch 10.5

-

-..-...... . . -*

.

_

~r

c.p~

lOmm Stretch

11.8 ... ...... -..... ....... .... ..-

~. _.CIc _ ~

~

"I.

I . . .

lo

40

b

40

20

b

40

20

After DHE

E lOmm Stretch 7.7

.

.. . _ _ _

-.

I

.1

*

0

i0

1

40

9

0

_ _ _

c

_._.L

~

- -

"

.".."... .. ..

_.L - 1 2-1

I

F 12 mm Stretch ..... . ..... ._". . .-.-1507 . ...-_. I.

9,5

20

2

1

io

b

20

I

40

Time after Onset of Stretch ( s e c )

Fig. 1. Effects of dihydro-P-erythroidine (DHE, 0.5 mg/kg Lv.) upon the responses of a single a-motoneurone t o sudden maintained stretches of different lengths applied t o the triceps surae muscle. Activity of the a-motoneurone (implsec) recorded from a filament oi the ventral root L7. Dashed lines indicate mean discharge rates, their values are given by numbers. Ventral root L7 cut. Precollicular decerebrate cat (reported by Meyer-Lohmann and Henatsch, 1966).

correct, it can be postulated that under increbed muscle stretches a rising RC activity should occur to counteract the augmented excitatory spindle input t o the a-MNs. It has been conclusively proved (Ryall and Piercey, 1971) that there are no monosynaptic actions of the primary spindle afferents on the RCs. Hence, any stretch-induced RC discharge is most likely triggered orthodromically via reflexly activated motoneurones. Since we have seen that the output frequency of the individual a-MN is relatively independent of different stretch lengths, the remaining possibility to get an enhanced RC action with greater stretches would be by a recruitment of more motor units. That this indeed occurs was demonstrated by several workers (e.g., Denny-Brown, 1929; Grillner and Udo, 1971b) and was also seen in own experiments (Meyer-Lohmann and Henatsch, 1966). In order t o study the stretch-dependent activities of individual RCs themselves, we recorded them extracellularly with capillary microelectrodes (Hellweg et al., 1974). In Fig. 2, the original records in the right half are from a single RC belonging t o the triceps surae pool: A shows the irregular spontaneous activity which is typical for a decerebrate preparation. B, C and D are the RC responses to stretches of 5, 1 0 and 1 4 mm, respectively. There is an early maximum in each record, corresponding roughly t o the dynamic peak of the ramp

225 Renshaw Cell [Imp ~ s e c ] I

Spontaneous A c t i v i t y

I

5 rnrn Muscle S t r e t c h

2 scc

0

4

8 Muscle Stretch

12

[mm]

16

Fig. 2. Responses of Renshaw cells to ramp-and-hold stretches of the triceps surae muscle of 5, 10, and 1 4 mm. Registrations on the right side show the activity of a single cell recorded extracellularly by means of conventional microtechniques in a decerebrate cat. The graph on the left side summarizes the data from 14 Renshaw cells with respect t o their averaged tonic responses plotted against length of muscle stretch. Responses were calculated for at least 2 sec starting 1 sec after the end of the dynamic stretch phase. Spontaneous activity prior to stretch was subtracted. Bars indicate standard deviation. Nine Renshaw cells were recorded in decerebrate cats, 5 in cats anaesthetized with urethane-chloralose. (Modified from Hellweg et al., 1974.)

stretch, followed by a tonic discharge component during the stretch plateau. As expected, the overall response is the more pronounced the greater the stretch length. The same phenomenon can be seen from the left-hand curve summarizing the results obtained from 14 RCs in different preparations. It is a plot of the averaged tonic stretch responses of the RCs against muscle stretch lengths (cf. also Pompeiano, this symposium). Another example, again from a decerebrate cat, is more thoroughly documented in Fig. 3 (Hellweg and Meyer-Lohmann, 1973). The records A and B represent the identification tests for the RC, A showing the typical early response to an antidromic shock stimulation of ventral root S 1 , B the orthodromic response t o stimulation of the peripheral gastrocnemius-soleus (GS) nerve. C is the continuous record of the RC activity before and during a rampand-hold stretch (12 mm) of the muscle. In D, the discharge rate was counted every sec and plotted against time. The stretch period is marked by the shaded bar. The RC activity jumps from the prestretch spontaneous level to an initial peak, followed by a transient fall, and then remains at about 30 imp/sec during the stretch. In the right half, the test procedure is repeated 10 min after DHE. E and F, corresponding to A and B, respectively, show a marked reduction of the antidromic and orthodromic RC response. The stretch response, in G, is also considerably diminished, particularly in its tonic component, while an initial dynamic burst is still present. The graph, in H, shows that almost no net activity change occurred during the stretch. In the preceding part we were concerned with the stabilizing function of the Renshaw mechanism which could be sufficiently demonstrated by the behaviour of individual tonic a-MNs and RCs, under natural stretch conditions. The next part of our presentation will deal with the second important property of

226 10 M I N A F T E R

CONTROL

DHE

A VRS, S t i r n

B G S Stirn

E VRS, S t i r n

F GS S t i r

rw~~~$S.J-.

&44.$.Jaw"-

'+LJJ-

A J W ' .

'

5GS G

( I m p /sec)

n I scc

Fig. 3. Responses of a single Renshaw cell to ramp-and-hold stretches of the triceps surae muscle before (left side) and after (right side) administration of DHE (1.0 mgikg i.v.). Decerebrate cat, curarized and artificially ventilated. Ventral roots L7 and S1 cut. Upper records are Renshaw cell responses to supramaximal stimulation of ventral root S1 ( A and E ) and of gastrocnemius-soleus nerves (B and F). C and G : original records of Renshaw cell responses (upper traces) t o muscle stretch (lower traces). Length of stretch 12 mm, stretch rate 60 mmlsec. D and H : plot of Renshaw cell activity (imp/sec) against time; horizontal bars indicate time of muscle stretch (reported by Hellweg and Meyer-Lohmann, 1973).

the Renshaw feedback, namely, its selecting, functionally focussing action on an entire a-MN pool. For this purpose, we investigated as many single a units as possible, interacting together with their RCs in the control of the muscle under study. Reliable stretch response data of numerous a-motor units can be easily collected and compared by means of the conventional recording technique from ventral rootlet filaments. Since Granit et al. (1957) and many subsequent papers by other authors, we know that the response type of individual a-MNs within the same pool, submitted t o identical stretch tests, can differ widely from purely phasic to extremely tonic behaviour. There are many transitions between the two extremes, and one and the same cell can be brought from phasic to tonic behaviour or vice versa by suitable means (Henatsch et al., 1959). Here we want to stress the fact that one frequently finds either one or the other of two basically different decerebrate preparation types, with respect t o their overall stretch reflex behaviour. The point is illustrated in the next figure. On one hand, there are preparations in which nearly all tested extensor a-MNs have one feature in common: their discharge frequency in response to muscle stretch remains almost constant throughout the long stretch duration, thus representing an overall "persistent-tonic" preparation type. In Fig. 4A, a considerable number of 33 a-MNs were collected from 11preparations of this type and their averaged response curve was plotted. It may be added that the active tension curve during stretch of the muscle in such a preparation (not illustrated) looks like a copy of the curve A. On the other hand, there are other preparations in which the majority of

227 A 20-

c 2033d-MN 11 E x p .

I

.

- .

.? - 10Q

87 d - M N

-

17Exp.

.

* E l &

VlC’St

V

z

i

\ * :

YIC*31

. -.

v PERIODS

X OF

.\;-

,

.‘

v

X

VIBRATION

Fig. 6. The differences between the effect of the trains of antidromic stimuli on the reflex response of the pair of tonically (left) and phasically (right) discharging motoneuronal units to continuous and repetitive vibration of the muscle. Muscle extended by 4 mm. Amplitude of vibration 100 Fm. Pauses in the repetitive series of stimuli 2000 msec.

1000 msec were applied in an intermittent manner both during continuous vibration and simultaneously with short-lasting repetitive vibration of the muscle. The strength of the antidromic stimuli was always just above the threshold for &-fibres. The standard procedure included measurements of the frequencies of the discharge (Fn) during control periods of stretch and vibration and its rate (Fi) during antidromic stimulation. Duration and aelivery of pulse trains and periods of muscle vibration were controlled by a Devices Digitimer 3290. There was a 3 min pause between each series. Fig. 6 shows the reflex response pattern of a pair of motoneuronal units during continuous (Vi,) and during repetitive (Vi,) vibration of the muscle without and with (+St) antidromic stimulation. During continuous vibration of the muscle and intermittent antidromic stimulation, the reflex response of the tonically firing unit of the motoneuronal pair was inhibited by a constant amount, regardless of its rate of discharge. The discharge of the phasically firing unit was soon depressed to zero. By repetitive vibration of the muscle, the reflex activity of both monitored units was kept long-lasting and high enough t o allow a comparison of the normal frequency with the inhibited one. It is evident that in spite of the constant strength of the superimposed inhibitory influence, the differences between the normal and inhibited rate of discharge of the phasic unit diminished with the progression of the trains of both stimuli applied together, while that of the tonically firing unit of the pair remained constant. Differences in the position of the regression lines of Fn against Fi of a group of 11 motoneuronal pairs, during the first (I) and the last (X) trials in the sequences of repetitive vibration and antidromic stimulation applied together, and during continuous vibration of the muscle, are presented in Fig. 7. Due t o the influence of time on the maintenance of the discharge of tonically firing

275 d'

Fi 20

., j

.

,

.;

I.V.

10 -?. /

,