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

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

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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.

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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

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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.

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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

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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

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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 (
13 “unwanted reflex” PTN activity can be quickly suppressed if the intended movement involves quiescence of the PTN. Taken together, these findings are consistent with Phillips’ (1969) proposal that a transcortical pathway may play a role in mediating movements occurring in response to kinesthetic inputs applied to the moving part. The PTNs which constitute the corticomotoneuronal limb of the transcortical pathway have the capacity to influence motoneurons within 25 msec of a kinesthetic input in the monkey. This initial PTN influence will be of the same sign as the segmental reflex effects of the input (Evarts and Tanji, 1976), but a second phase of PTN activity, related to intention, can influence motoneurons in opposition to segmental reflex effects at latencies of 45 msec. In considering the possible role of PTN activity, it must be kept in mind that the corticospinal tract is but one of a number of descending systems which may play a role in rapid but supraspinally mediated responses to limb disturbances. The role other descending systems (mbrospinal, vestibulospinal, reticulospinal) play in control of movement has been reviewed by Lundberg (1966) and more recently by Shapovalov (1975), and will not be dealt with here. But it seems certain that these systems, too, are important in mediating rapid responses via supraspinal circuits.

SUMMARY

(1)A subject is instructed to contract (supinate) or relax (pronate) his biceps brachii while he is holding a vertically placed handle in a position requiring a tonic compensating action against the force of a torque motor. (2) The subject acts upon the instruction when a fast ramp movement of the handle has extended or shortened the biceps, again by pronation or supination, while its mechanical and electrical responses are being recorded. (3) The intended act, triggered by the ramp, is in the same or opposite direction to the latter movement. This implies that a segmental reflex effect (the ramp) may support or clash with the command from above, as illustrated in Table I for the different combinations used. (4) Reflex stretch (trigger pronating) combined with intended contraction (supinate) failed to show any evidence of the tendon organ inhibition that stretch combined with contraction is known to optimize. The initial tendon jerk at 25-30 msec latency was succeeded by the intended contraction at 60-70 msec. (5) The same stretch reflex combined with an intended inhibition (pronate) produced a delayed component lingering on for some 150 msec, provided that the grip on the handle had necessitated sufficient tonic activity. Without such activity the stretch reflex was restricted t o the tendon jerk; the rest of it wholly abolished despite maintained stretch (by pronation). (6) While these two findings were unexpected, those with trigger-induced supination (reflex of unloading) appeared fully to agree with what on current knowledge seemed reasonable to expect. (7) Serial repetition of an intended movement had a definite effect on the earliest segmental responses (at 25-30 msec) modifying them up or down in magnitude depending upon the specification of such movements.

14 (8) In the discussion the statements 4-7 are considered from the point of view of the present knowledge about segmental reflexes and transcortical loops.

REFERENCES Evarts, E.V. (1973) Motor cortex reflexes associated with learned movement. Science, 179 : 50 1-5 0 3. Evarts, E.V. and Tanji, J. (1976) Reflex and intended responses in motor cortex pyramidal tract neurons of monkey. J. Neurophysiol., in press. Granit, R. (1950) Reflex self-regulation of the muscle contraction and autogenetic inhibition. J. Neurophysiol., 1 3 : 351-372. Granit, R. (1970 repr. 1973) The Basis of Motor Control, Academic Press, London. Granit, R. and Strom, G. (1951) Autogenetic modulation of excitability of single ventral horn cells. J. Neurophysiol., 1 4 : 113-132. Granit, R., Kellerth, J.-0. and Szumski, A.J. (1966) Intracellular autogenetic effects of muscular contraction on extensor motoneurones. The silent period. J. Physiol. (Lorid.), 182: 484-503. Hagbarth, K.-E. (1967) EMG studies of stretch reflexes in man. In Recent Advances in Clinical Neurophysiology, Flectroenceph. clin. Neurophysiol., Suppl. 25, L. Widen (Ed.), Elsevier, Amsterdam, pp. 74-79. Hagbarth, K.-E. and Naess, K. (1950) The autogenetic inhibition during stretch and contraction of the muscle. Acta physiol. scand., 21: 41-53. Hammond, P.H. (1956) The influence of prior instruction to the subject on an apparently involuntary neuro-muscular response. J. Physiol. (Lond.), 132: 17P-18P. Hoffmann, P. (1922) Untersuchungen iiber die Eigenreflexe (Sehnenreflexe) menschlicher Musheln, Springer, Berlin. Hufschmidt, H.-J. (1966) The demonstration of autogenetic inhibition and its significance in human voluntary movement. In Muscular Afferents and Motor Control. Nobel S y m posium I, R. Granit (Ed.), Almqvist and Wiksell, Stockholm, pp. 269-274. 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. Lucas, M.E. and Willis, W.D. (1974) Identification of muscle afferents which activate interneurons in the intermediate nucleus. J. Neurophysiol., 37 : 282-293. Lundberg, A. (1966) Integration in the reflex pathway. In Muscular Afferents and Motor Control. Nobel Symposium I, R . Granit (Ed.), Almqvist and Wiksell, Stockholm, pp. 2 7 5-305. Marsden, C.D., Merton, P.A. and Morton, H.B. (1973) Latency measurements compatible with a cortical pathway for the stretch reflex in man. J. Physiol. (Lond.), 230: 58P59P. Matthews, P.B.C. (1972) Mammalian Muscle Receptors and Their Central Actions, Wiley, New York. Phillips, C.G. (1969) Motor apparatus of the baboon’s hand. R o c . roy. SOC. B, 173: 141174. Shapovalov, A.I. (1975) Neuronal organization and synaptic mechanisms of supraspinal motor control in vertebrates. Rev. Physiol. Biochem. Pharmacol., 72 : 1-54. T a n k J. and Evarts, E.V. (1976) Anticipatory activity of motor cortex neurons in relation t o the direction of an intended movement. J. Neurophysiol., in press.

Organizer’s Lecture

Frequency Characteristics of the Impulse Decoding Ratio between the Spinal Afferents and Efferents in the Stretch Reflex S. HOMMA

Department of Physiology, School of Medicine, Chiba Uniuersity, Chiba (Japan)

INTRODUCTION It has been repeatedly confirmed that the stretch reflex is elicited through both mono- and polysynaptic reflex arcs (Granit et al., 1957; Kolmodin, 1957; Tsukahara and Ohye, 1964; Lance et al., 1966; Kanda, 1972; Homma and Kanda, 1973). Impingement of Ia afferent impulses of a certain frequency upon spinal motoneurons is decoded into a motor output of a much lower frequency. The relationship between afferent and efferent frequencies has been shown always to be an integral ratio (Homma et al., 1971). This relation between the two parameters has been expressed by a very simple formula, as below:

1 Mf = ;Iaf

(n = integer 1 , 2 , 3, 4,...)

where Mf and Iaf are the firing frequency of an a-motoneuron and the discharge frequency of Ia afferent fibers, respectively. Since the number n in the above formula was found, experimentally, always to be an integer, the reciprocal of n, i.e. l / n , was called the decoding ratio. The aim of the present report is to study the upper and the lower limits of Iaf within which the above formula is valid. In other words, this report is to study the stretch reflex of the spinal cord from the viewpoint of frequency characteristics of formula (1). It also seemed interesting to study how the frequency characteristics of the above formula change when the reflex center is bombarded by synergistic or antagonistic Ia impulses which either facilitate or inhibit the agonist’s activity. At the same time it is also very probable that such a set of facilitatory or inhibitory combinations might also show their own frequency characteristics in their interference with the above formula. For this particular purpose of investigation, the muscle under observation was quickly stretched by a series of triangular pulses while the synergistic or antagonistic nerve was being stimulated by electric regular pulses. Thus, various ways will be demonstrated in which the decoding ratio in the formula is modified by the Ia afferent impulses from synergistic or antagonistic muscles.

16 METHOD Cats were anesthetized by 3 ml i.v. injection of 1%chloralose and 10% urethane mixture. A moderate degree of anesthesia was maintained throughout the experiment by an appropriate additional injection of the above mixture. Gastrocnemius, soleus and tibialis anterior muscles were carefully separated from the adjoining connective tissues so as to facilitate longitudinal extension of the muscles. Each tendon was cut and was interchangeably connected to an electromagnetic puller with a steel hook. All nerves except those innervating the above three muscles were cut in the same hind limb. Routine laminectomy was performed and temperature of the oil pool was kept at 37-38°C throughout the experiment. Glass micropipettes filled with 3 M KC1 solution (resistance 15-20 M a ) were used for the intracellular recording of the a-motoneuron. For identification of the motoneuron, a brief manual stretch was applied t o both gastrocnemius and soleus tendons. More effective spike generation was used to determine the innervation of the motoneuron under observation. For unitary recording from the filaments, ventral rootlets were cut just before their entry into the spinal dura mater. They were split until a functionally single unit was obtained. For identification of the unit, the same method as used in the intracellular recording experiment was employed. Both sinusoidal wave and triangular pulses were used t o drive the vibrator system. For observation of inhibitory effects on the stretch reflex, the peroneal nerve was stimulated by a regular pulse series.

RESULTS

( I ) Intracellular recording from the a-motoneuron ( A ) Mono- and polysynaptic recording in the stretch reflex It has been proposed that both mono- and polysynaptic reflex arcs participate in the generation of the stretch reflex (Kanda, 1972; Homma and Kanda, 1973). Intracellular experiments have revealed that both fast phasic (vibratory) and slow tonic (augmenting) changes of the membrane potential are very important for an understanding of the stretch reflex. (1) Vibratory EPSPs. Fast sinusoidal stretch of the muscle elicits Ia afferents impulses in the sensory arc. They monosynaptically generate EPSP ripples which have the same frequency as that of the sinusoidal stretch (Homma et al., 1970; Westbury, 1972). Therefore the ripples were called vibratory EPSPs (Homma and Kanda, 1973, or see Fig. 4). As seen in Fig. 1, intracellular recordings revealed that motoneuronal spikes were always elicited after a temporal summation of vibratory EPSPs and that they were invariably triggered at the depolarizing peak of one of the vibratory EPSPs. It was very clear that motoneuronal spikes triggered at these peaks were temporally locked to the sinusoidal stretch. These spikes were called locked spikes (Hirayama et al., 1974). Nonsequential interspike interval histograms of the locked spikes revealed that they were elicited by the intervals which were

17

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EPSP ripples of the same frequency. Motoneuronal spikes were always elicited after a temporal summation of vibratory EPSPs (Homma and Kantla, 1 9 7 3 ) .

integral (n) multiples of the vibratory cyclic time. These facts automatically indicate that the relationship between mass discharge frequency of Ia afferents (Iaf) which are equal to vibratory cycle and that of a-motoneurons (&If)can be expressed by the formula (l),where n was the integer 1, 2, 3, 4,... . For the reasons explained above and already in the introduction, the reciprocal of n, i.e., l / n , was called the decoding ratio (Homma and Kanda, 1973). (2) Augmenting I;PSPs. When vibratory stimulation is continued for some period and, hence, Ia afferent impingement upon the membrane of an a-motoneuron is tonically maintained, tonic depolarization is always observed (Fig. 4). Such a component of depolarization was proposed as an augmenting EPSP. This augmenting EPSP was shown in our previous reports t o be produced by the post-tetanic potentiation of polysynaptic reflex arcs (Homma and Kanda, 1973). It was known that the larger the augmenting EPSP the earlier would the firing level be attained in the motoneuronal membrane potential. In fact it has always been the case that a large augmenting EPSP resulted in a decrease of the integer n and, automatically, an increase of its reciprocal, l / n . Thus, the decoding ratio, l / n , was increased by the large augmenting EPSP. Such a condition is judged from many reasons to be due t o forward facilitation by the activity of polysynaptic reflex arcs. It was often observed that a relatively fast rise in the membrane potential of an a-motoneuron due t o a large augmenting EPSP attained its critical firing level without additional depolarization by the superimposed vibratory EPSPs. It is very clear that these spikes triggered by such augmenting EPSPs as described above have no reason t o be locked t o the vibratory phase, and this was shown t o be true by correlation analysis. Therefore, those spikes triggered by the augmenting EPSPs only were called "unlocked" spikes. An example of data that substantiate this is shown in Fig. 2. In this case the quadriceps femoris muscle was vibrated a t the patellar tendon and contraction due to the tonic vibration reflex, TVR (Ilagbarth and Eklund, 1966a, 1966b; Hirayama et al., 1974), was generated (Fig. 2A). The NMU spikes during this contraction are shown just below it (Fig. 2B). This spike activity was redrawn as a trimmed pulse series in Fig. 2D. Through a window circuit of an appropriate time adjustment, 'activity' of D is divided into two series of locked (Fig. 2E) and unlocked (Fig. 2F) spikes. It is obvious from Fig. 2 F that unlocked spikes appeared rather late, in an augmenting manner. Fig. 2E suggests that spikes triggered by the augmenting EPSP increased tonically along the course of vibratory stimulation.

~

18

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5 sec

Fig. 2. TVR of quadriceps femoris muscle in man. The patella tendon was vibrated at 55 Hz as shown in C. A : isometric contraction of the quadriceps femoris muscle. B: NMU spikes during TVR activity. These spikes were redrawn as a trimmed pulse series in D. Through a window circuit of an appropriate time adjustment the activity of D is divided into two series of locked (E) and unlocked (F) spikes. Unlocked spikes appear rather late in an augmenting manner (Hirayama et al., 1974).

Gradual contamination with the unlocked spikes resulted in gradual degradation of the validity of the formula (1).In an extreme condition, any definite value of the decoding ratio, l f n , cannot be found in the computer-processed data since there will be no available output from the spinal reflex center. (B) Inhibitory influence in the stretch reflex In the experiment of intracellular recording from the a-motoneuron during tonic afferent impingement, an intermittent appearance of hyperpolarizing deflection was recognized in addition t o the above mentioned vibratory and augmenting EPSPs. The time course of these hyperpolarizing potentials revealed that they could be divided into two different categories of IPSPs, phasic and tonic ones, just in the same way as in the case of EPSPs. (1) Phasic IPSP, In the intracellular recording of Fig. 1,motoneuronal spikes were triggered at intervals of an approximately definite number, n, of vibratory EPSPs. However, such a condition of firing interval is lost as indicated by the arrows in Fig. 3. At every arrow the motoneuron was restrained from firing by the incoming hyperpolarizing influence. It can be seen, in this particular case, that the motoneuron fired again after a definite additional number, n, or vibratory EPSPs subsequent to each arrow. Therefore, such a mode of motoneurond

19

~,~..~~,~,.~,,",""...." ,_.,.__.._ ... - .,.

,

,,-..-..",.I ,._,I,.,.

,

-- -

. . . . 4 . , 1 " . . _I.., "I . I . . . . .

_,.,^

^.,..." " , - ~

-

__..I"I...._.__._. "_

-100 msec

I loop

Fig. 3 . Cat gastrocnemius motoneuron. Sinusoidal stretch of muscle is 100 Hz, as shown in the lower trace. Motoneuronal spikes are triggered a t intervals of a n approximately definite number of vibratory EPSPs. But such a condition of firing interval is lost, as indicated by the arrows, and therefore t h e mode of motoneuronal firing can b e expressed as Mf = (1/2n) Iaf. At every arrow the motoneuron is restrained from firing by an incoming hyperpolarizing influence (Homma and Kanda, 1973).

firing can be expressed as below.

M

1

- --af - 2n

On the extreme right of the record the motoneuron was inhibited twice, as indicated by the two successive arrows. With this long interval the coefficient of Iaf must become 1/3n (Homma et al., 1975). An extreme example of inhibitory influence during random quick triangular stretch was obtained by the intracellular recording shown in Fig. 4. In the upper trace, two additional spikes of the motoneuron could very probably have been provided if the very abrupt and deep hyperpolarizing deflections shown by the two arrows had not been present. It was suggested that such an intermittent strong inhibitory influence was produced by a recurrent inhibition from an adjacent motoneuron (Homma and Kanda, 1973). It will be appropriate to propose that such a brief hyperpolarization is designated as phasic IPSP (Fig. 4A). Since the above process of neural impulse is a sort of nullification of the activity of the adjacent motoneuron, this phasic IPSP might play, at any rate, some role associated with "motor contrast" (Granit, 1970). (2) Tonic IPSP. It was also observed that the augmenting EPSP (Fig. 4B)due to continued impingement by Ia impulses was often preceded by temporary hyperpolarization. Repetitive electrical stimulation of the peripheral nerve, especially with the range of higher intensities, was found to have a tendency such that spikes due to the stretch reflex failed to be elicited (Homma et al., 1975). This inhibition was attributed to the activation of group I1 fibers, since the above tendency became pronounced as the intensity of stimuli was increased above the threshold of group I1 fibers. Fig. 4B shows an example of such an inhibition of intracellular spikes of an a-motoneuron when the soleus muscle received intermittent random stretch of triangular excursion. The time course of the resting potential of Fig. 4B also shows that the membrane was very gradually hyperpolarized and the motoneuron remained completely silenced during the period of the gradual hyperpolarization. However, this hyperpolarization was readily counteracted by the continued Ia impulses and the motoneuron resumed its repetitive firing in accordance with the augmenting EPSP (Fig. 4B). Since the muscle stretch used in the present experiment was very minute, it is safe to say that only the group Ia fibers were excited and that activation of

20

C Fig. 4. Cat soleus motoneuron. A and B: intracellular recordings during random triangular stretch of the soleus muscle as shown in the lower trace of A and in C. A shows a record of the inhibited part of the B trace a t an expanded time scale. In A, t w o spikes could have very probably been provided if deep hyperpolarizing deflections shown by the two arrows had n o t been present. Such a hyperpolarization is designated as the phasic IPSP. On the other hand, triangular stretch generates EPSP ripples which correspond t o the triangular waves. The ripples will be designated as vibratory EPSPs as shown by the broken arrow. When triangular stretch is continued for some time, tonic depolarization is observed. Such a component is proposed as a n augmenting EPSP. In B, the augmenting EPSP is preceded by temporary hyperpolarization. Such an augmenting hyperpolarization during triangular stretch is called the tonic IPSP (see in text).

group 11, if any, was very scarce. Therefore it should be pointed out that the responsible neural circuit for the hyperpolarization may be in some way different from the one that explains the former inhibition which was attributed t o activation of group I1 fibers. Some polysynaptic circuits, different from the one responsible for the monosynaptic stretch reflex, may be activated by continued random stretch to produce the inhibition, very probably of a feed forward type. It is proposed that the hyperpolarization of the type exemplified above is called tonic IPSP as shown in the record of Fig. 4. (11)Frequency characteristics of motoneuronal transmission

in the stretch reflex In the previous section it was concluded that, for the discussion of spinal input-output relations, 4 types of the postsynaptic potential must be considered: (1) monosynaptic vibratory EPSPs, (2) polysynaptic augmenting

21 EPSPs, ( 3 ) phasic IPSPs generated by recurrent inhibition from an adjacent motoneuron, and (4) tonic IPSPs. All of these 4 types of PSPs can occur either dependently or independently and their final algebraic summation t o the critical firing threshold can fire the motoneuron. Therefore further analysis of Ia-triggered PSPs are necessary to study the validity of the formula (1)during sustained muscular stretch when it is combined with synergistic and antagonistic afferents. The variability of M, was studied in this section in which varied frequency of Iaf and its relation to the motoneuronal firing were examined. Thus the frequency characteristics of the stretch reflex will be expressed by Mf-Iaf relations. ( A ) Analysis of the time course of the vibratory EPSP and of the Mf-Iaf relation For analysis of the time course of the vibratory EPSP in more detail, each excursion of the quick muscle stretch was standardized at a triangular pull of 5 msec in both rising and falling phases. Such a pull with linear time course was convenient because there was no change in the acceleration and deceleration of the muscle stretch even when the interval between each stretch was varied. A typical intracellular record during such a pull is shown in Fig. 5. The socalled “shape indices” of an independent vibratory EPSP were measured by the “time to peak” and its “duration at half amplitude” (Burke, 1967). The time to peak of the vibratory EPSP due t o a quick triangular stretch as used in the present experiment was measured as around 5-15 msec and its duration at half amplitude as around 7-20 msec. Since the duration at half amplitude was longer than the time t o peak, it is clear that the falling phase is slower than the rising phase. Measurements collected from the vibratory EPSPs due to the soleus and gastrocnemius stretch are shown in Fig. 6.

nisec Q)

c

m

- 10 N

\

h

m

time t o peak

6

Fig. 5. Vibratory EPSPs in gastrocnemius (left) and soleus (right) motoneurons during triangular pull with 5 msec in both rising and falling phases. Fig. 6. Shape indices of the vibratory EPSP. Abscissa shows the time t o peak and the ordinate t h e duration a t a half amplitude of vibratory EPSP. The duration a t half amplitude is longer than the time to peak and therefore the falling phase of vibratory EPSP is longer than t h e rising phase. Filled circles represent the vibratory EPSPs of gastrocnemius motoneurons and open circles those of soleus motoneurons.

22

The amplitude of the triangular stretch used was in the range of 0.1 mm and the size of the vibratory EPSP was 1-2 mV. It may be noted from the figure that vibratory EPSPs due t o the soleus stretch have greater values both in time to peak and half amplitude duration than those of the gastrocnemius stretch. It has been confirmed that the EPSP due t o single Ia fiber activation has average values of 0.1 mV for amplitude and around 0.3-3.0 msec for rising phase (Mendell and Henneman, 1971). From this finding each vibratory EPSP shown in the measurements of Fig. 7 can be judged t o result from almost synchronous summation of several tens of the above “unitary” EPSPs. On the other hand, temporal summation of such vibratory EPSPs has been known to be a very important factor in determining the motoneuronal firing interval. Modes of summation of vibratory EPSPs are shown in Fig. 7 where two successive vibratory EPSPs were elicited by two triangular stretches applied at varied intervals. It is evident that even a minute brief stretch, if applied in quick repetition, can elicit a sufficient amount of depolarization t o trigger firing of the motoneuron. Further test of the variability of firing intervals during varied frequencies of triangular stretch was performed by recording unitary spikes at the split ventral root filament. The results are shown in Fig. 8 where spikes from gastrocnemius efferent nerve were recorded during regular triangular stretch of 20 and 50 Hz. Regular triangular stretches of 20 Hz elicited motoneuronal spikes of 10 Hz, the decoding ratio being f. Triangular stretches of 50 Hz also elicited regular ventral root spikes a t a slightly faster repetition, though the frequency of the

gastrocnemius triangular stretch

20Hz

50Hz

J-fL.--

$2 l l m v 7~

-2Omsec

-100 msec

8

Fig. 7. Summation of vibratory EPSPs. Two successive vibratory EPSPs are elicited by t w o triangular stretches applied a t varied intervals. Fig. 8. Spikes f r o m gastrocnemius efferent nerve during regular triangular stretch a t 20 and 50 Hz. Stretches a t 20 Hz elicit spikes o f 10 Hz, t h e decoding ratio being a t 0.5. The decoding ratios during 50 Hz stretch are measured at 0.33 o r 0.25 except t h e first three spikes.

23

gastrocnemius

I

I

100

95

-

90

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75

70

65

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10 Hz

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5 sec Fig. 9. Spikes from gastrocnemius efferent nerve during regular triangular stretch which was continued for 1 . 2 sec with the interval of about 2 sec and the frequency of stretch was varied as indicated in the figure.

first three spikes was excited at a much faster rate. The decoding ratios during 50 Hz stretch were measured at or i. This relation was further tested by changing the frequency of triangular stretch from 10 to 100 Hz as shown in Fig. 9. Regular triangular stretch was continued for 1.2 sec with an interval of about 2 sec, and the frequency of the stretch was varied as indicated in the figure. Measurement of motoneuronal spike number/sec yielded the Mf -Iaf relation as plotted by the filled circles, and decoding ratios, calculated by Mf/Iaf;l/nIaf relation is shown in the upper trace. The horizontal axis of Iaf is viewed as increasing from right to left. No linear portion in the relation could be found. The whole pattern of the curve revealed that the Mf-Iaf relation is expressed by a quadratic curve in which the maximum existed around 80 Hz. Since motoneuronal spikes are elicited by temporal summation of the EPSP, the increase of Mf as the Iaf is increased from 10 to 80 Hz is due to summation of larger EPSP amplitudes. Therefore, coincidence of the curve of the Mf-Iaf relation with the time course of a typical vibratory EPSP is predicted. A typical vibratory EPSP is shown in the lower trace with the same horizontal time scale. The similarity of the Mf-Iaf curve and the vibratory EPSP suggests that temporal summation of the vibratory EPSPs was most effective when Iaf was approximately equal to a summit of the vibratory EPSP. In order t o compare the Mf-Iaf curve with the time course of vibratory EPSPs, measurements of "shape indices" of Mf-Iaf relations were performed in the same way as used for the measurement of EPSPs, and collected data from soleus and gastrocnemius units are shown in Fig. 11. The duration at one-half Mf is longer than the time t o peak. Therefore the falling phase is slower than the rising phase in the Mf-Iaf relation. Filled circles are values of gastrocnemius and open circles those of soleus. The Mf-Iaf relation was also larger in size and time for the soleus units than for the gastrocnemius units, as observed in the EPSP shape indices of Fig. 7. It can be concluded that the whole profile of the Mf-Iaf relation is intrinsically underlined by the time course of the vibratory EPSP. On the other hand, unitary EPSP of slower decay, as seen in soleus moto-

24 0

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20

50

10 Hz lat lOOmsec

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time to peak

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Fig. 10. Measurement of motoneuronal spike number per second yields the Mf-Iaf relation in the middle trace and the l/n-Iaf relation in t h e upper trace. A typical vibratory EPSP is shown in the lower trace with the same horizontal time scale. Fig. 11. Shape indices of the Mf-Iaf relation. The abscissa shows the time t o peak of t h e Mf-Iaf curve and the ordinate the duration a t a half M f . Filled circles are values of gastrocnemius and open circle those of soleus.

neuron, has been attributed to the synaptic terminals around dendrites and was shown to be well encountered in the tonic motoneuron (Burke, 1968). Differences in the time courses of the vibratory EPSPs between soleus and gastrocnemius motoneurons clearly show differences in their intrinsic nature. Therefore it is concluded that the intrinsic nature‘of the motoneuron is reflected in the frequency characteristics of the stretch reflex measured from the Mf-Iaf relation.

( B )Susceptibility of the M f - l a f relation to inhibitory input It also seemed interesting to study how the frequency characteristics of the Mf-Iaf relation are changed. Typical data of this experiment are shown in Fig. 12. A shows control spikes of a gastrocnemius motoneuron elicited by 5 different frequencies of triangular stretch. B shows the attitude of the same spikes when the peroneal nerve was stimulated at 20 Hz, just a t the threshold for contraction of the tibialis anterior muscle. C shows the same experiment when the peroneal nerve was stimulated at 50 Hz. Measurements of the Mf-Iaf relation from this original record and added data are shown in the curves of Fig. 13. The pronounced downward shift of Mf-Iaf curves seen in the figure indicates that discharge of the motoneuron was greatly inhibited and this inhibition was most remarkable with a low frequency rahge of Iaf. Another interesting point was the gradient of the Mf-Iaf relation, which became more and more steep as the frequency of peroneal stimulation was increased. According t o the data shown in Figs. 11and 12, frequency characteristics of

25

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Fig. 1 2 . A shows control spikes of a gastrocnemius motoneuron elicited by 5 different frequencies of triangular stretch. B shows the attitude of the same spikes when the peroneal nerve was stimulated at 20 Hz, and C shows the same experiment at 50 Hz stimulation.

the Mf-Iaf relation could be said to be almost determined by the EPSP time course. If this notion is applied to the curves of Fig. 13, it may even be possible to say that activation of antagonistic afferents changed the time course of EPSP. Algebraic summation of EPSPs and the IPSP caused by antagonistic Ia afferents

Fig. 13. Mf-Iaf relation measured from the original record as shown in Fig. 12. The gradient of the Mf-Iaf relation becomes more and more steep as the frequencies of the peroneal nerve stimulation are increased.

26

J

100 10

.

20 50

I

Iaf

H2

msec

10 100

Fig. 14. Mf-Iaf relation of gastrocnemius motoneuron during stimulation of the medial gastrocnemius nerve. Pronounced upward shifts of Mf-Iaf curves are seen in the figure.

(Eccles, 1957), may explain such an “imaginary change” of the EPSP time course and also the fact that the “change” becomes more pronounced with higher frequencies of antagonistic stimulation. ( C ) Susceptibility of the Mf-Iaf relation to facilitatory input The sensitivity of the Mf-Iaf relation was further tested during the presence of facilitatory input. For this purpose the central cut end of the medial gastrocnemius nerve was stimulated just above the threshold of muscle contraction which was tested before severing of the nerve. Intact innervation of the muscle by the lateral nerve made further testing of the Mf-Iaf relation possible. Results of this experiment are shown in Fig. 14. Mf values were moderately increased by stimulation of the synergistic nerve. This is suggested by a general upward shift of the M,--Iaf curve. Stimulation with higher frequencies was found to be more effective in eliciting facilitation, as observed in the experiment of inhibition. An increased amount of depolarization by the concurrent facilitatory input may have played an important role in making the time course of EPSP slower, and this may be responsible for the greater gradient of the Mf-Iaf curve.

DISCUSSION Repetitive stretch of gastrocnemius and soleus muscles elicited continuous Ia afferent impingement upon the innervating a-motoneuron, which produced (i) vibratory EPSPs, (ii) augmenting EPSPs, (iii) phasic IPSPs, and (iv) tonic IPSPs. There could be found no disagreement for the notion that muscle stretch produced by a triangular wave form elicits vibratory EPSPs monosynaptically (Homma et al., 1971; Westbury, 1972). Tonic repetition of Ia impulses elicits tonic depolarization of the a-motoneuron through the activity of interneurons.

27 This tonic depolarization was proposed as the augmenting EPSP (Homma and Kanda, 1973). The polysynaptic circuits responsible were located within a relevant segment of the spinal cord (Kanda, 1972). The phasic IPSP was also found to have an influence upon the firing of the a-motoneuron, in rivalry with summation of the vibratory EPSP, and thus alternatively or interdependently decide the motoneuron’s firing level (Homma et al., 1975). A typical record of a phasic IPSP is shown in Fig. 4. Such spontaneous hyperpolarization was judged as an IPSP because it always appeared after repeated stretch, and was thus regarded as recurrent inhibition triggered by another homonymous motoneuron very near the one penetrated. On the other hand, the tonic IPSP was observed during a relatively early phase of continued Ia impulses, and it inhibited firing of the motoneuron for some time. It should be pointed out here that this inhibition observed at the motoneuron is due to the tonic IPSP that has been known to be effected through the cerebellospinal inhibitory circuit because Ia impulses are definitely conducted up t o the cerebellum (Lundberg, 1964; Burke et al., 1971; Kuno et al., 1973). It has also been shown that cerebellar stimulation recruits and augments recurrent inhibition within the spinal cord (Granit et al., 1960; Hasse and Van der Meulen, 1961). Therefore, the above finding should be considered in the present study of possible mechanisms for the generation of the tonic IPSP. Continued Ia impingement also elicited an augmenting EPSP. Pharmacological tests with mephenesin suggested that the augmenting EPSP was elicited by Ia afferents through polysynaptic pathways. Therefore, the augmenting EPSP has been attributed t o some potentiation of the relevant polysynaptic pathways activated during sustained vibratory or triangular stretch of the muscle. Another possibility for generation of the augmenting EPSP is that it might be a phenomenon due to disinhibition (Wilson et al., 1960; Hultborn et al., 1971). However, the phenomenon of disinhibition is primarily associated with flexor motoneurons. In the present study the augmenting EPSP was studied with extensor motoneurons and therefore the possibility of disinhibition can be excluded. The form of the “vibratory” EPSP due to single triangular muscle stretch should be discussed. The EPSP due t o electrical activation of Ia nerve fibers was shown to attain its summit in 2 msec and t o decay with a time constant of 4.9 msec (Eccles, 1957). Muscle stretch was known to elicit an EPSP with a much slower time constant (Homma, 1966). The average size of the unitary EPSP due t o activation of a single Ia fiber was 0.27 mV in amplitude (Kuno and Miyahara, 1969). The size of the unitary EPSP elicited by muscle stretch was identified as being in the range of 25-100 pV (Stuart et al., 1971). The size of the EPSP of the present study, in which a brief triangular stretch was employed, was in the range of 1-2 rnV. Therefore, it is possible t o say that such a large size of EPSP as presented above may be the result of almost synchronous summation of the so-called unitary EPSPs. The falling phase of the unitary EPSP was shown t o decay with a time constant of 5.8 msec (Jack et al., 1971). The falling phase of the EPSPs studied by triangular stretch also indicated that the present EPSP was the synchronous summation of many unitary EPSPs, probably of several tens of them.

28

Even the unitary EPSPs elicited by single Ia fiber activation have been found t o be variable from fiber t o fiber (Burke, 1967; Mendell and Henneman, 1971). Such intrinsic variety of unitary EPSPs may also be seen in the variety of EPSPs recorded in the present study (Fig. 7). Variation in the half amplitude duration used in the present study may be discussed from the results of experiments in which the unitary EPSP with a slower decay was attributed to the synaptic terminals around dendrites (Burke, 1968). I t was also pointed out by the experiment that such dendritic termination was well encountered in the tonic motoneuron. In the present study, also, the soleus motoneuron was found t o have “vibratory” EPSPs of larger shape indices than those of the gastrocnemius. The fact that the Mf--Iaf relation acquired from the soleus motoneuron was generally of a gentle curvature can also be explained by the difference in the shape of EPSPs. Therefore, the above difference in the intrinsic nature of the motoneuron was also reflected in the frequency characteristics of the stretch reflex as seen in the measurements of many Mf-Ia, curves acquired from both soleus and gastrocnemius motoneurons.

SUMMARY Genesis of the stretch reflex during repeated muscle stretch was studied from the viewpoint of modes of the time course of EPSPs recorded at cu-motoneurons. The phasic type of EPSP was called a vibratory EPSP, which was generated by monosynaptic activation of Ia afferent impulses. The augmenting EPSP, the tonic type of EPSP, which was recruited very gradually during repeated muscle stretch, suggested that this potential was of a polysynaptic nature. Temporal summation of vibratory EPSPs elicited motoneuronal firing whose phase was always locked to an arbitrary phase of muscle vibration. The following formula of integral relationship is valid between input and output frequencies of the spinal reflex center,

Since n was always an integer, the reciprocal coefficient, l / n , was called the decoding ratio. Gradual recruitment of the augmenting EPSP elicited a decrease of n, i.e., an increase of the reciprocal, l / n . However, it also elicited gradual contamination of regular spikes with the irregular ones that were not locked to the vibratory phase by their polysynaptic nature. The time course of hyperpolarization was also divided into two categories, phasic and tonic IPSPs. The phasic IPSP inhibited motoneuronal firing by suppressing the summation of vibratory EPSPs just before the motoneuronal firing threshold was attained. The firing mode during this inhibition could be represented as below, I

Mf = % Iaf .

29

Continued impingement of Ia afferent impulses elicited intermittent tonic IPSPs, which suppressed motoneuronal firing for longer periods and decreased the decoding ratios more than above. In conclusion, continuous Ia afferent impulses can elicit vibratory EPSPs, augmenting EPSPs, phasic IPSPs and tonic IPSPs, all through different propriospinal neural circuits. Ventral root spikes were recorded during a triangular muscle stretch at 10-100 Hz. Similarity between Iaf and the frequency of triangular muscle stretch was shown by the form analysis of vibratory EPSP recordings. Frequency characteristics of the stretch reflex could be expressed by an Mf--Iaf relation, which showed a distinctive curve along the axis of Iaf. Mf had its maximum a t around 80 Hz of Iaf and showed a decrease in either direction. The Mf--Iaf relation was known t o be expressed as a coincident phenomenon with the time course of a vibratory EPSP. Average shape indices of both vibratory EPSPs and the M,--Iaf relation were 10 msec in their time to peak and 15 msec in their half amplitude duration, and the soleus had larger values of both time t o peak and duration at a half amplitude. The Mf-Ia, relation was shown t o be shifted in opposite directions during concurrent inflows from the antagonist and the synergist. Concurrent antagonistic inflow decreased Mf considerably and thus decreased the decoding ratio. Concurrent synergistic inflow elicited a moderate increase of Mf and thus increased the decoding ratio. The time course of the vibratory EPSP shows an intrinsic nature in the motoneurons. Thus, the intrinsic nature is reflected in the frequency characteristics of the stretch reflex investigated by the Mf-Iaf relation.

REFERENCES Burke, R.E. (1967) Motor unit types of cat triceps surae muscle. J. Plzysiol ( L o n d . ) , 1 9 3 : 141 -1 60. Burke, R.E. (1968) Firing patterns of gastrocnemius motor units in t h e decerebrate cat. J. Physiol. (Lond.), 1 9 6 : 631-654. Burke, R., Lundhcrg, A. and Weight, F. ( 1 9 7 1 ) Spinal border cell origin o f the ventral spin()cerebellar tract. E x p . Bruin Kcs., 1 2 : 283-294. Eccles, J.C. (1957) The Physiology of N e r l v Cells, Johns Hopkins Press, Baltimore, Md. Granit, R ( 1 9 7 0 ) The Busis o f h l o l o r Control, Academic Press, London. Granit, R., Phillips, C.G., Skoglund, S. and Steg, G. ( 1 9 5 7 ) Differentiation of tonic from phasic alpha ventral horn cells by stretch, pinna and crossed extensor reflexes. J. Neurophysiol., 2 0 : 470-481. Granit, R., Haase, J. and Rutledge, L.T. (1960) Recurrent inhibition in relation t o f r e y u e ~ ~ c y of firing and limitation of discharge rate of extensor motoneurones. J. Physlol. ( [ m i d . ) , 1 5 4 : 308-328. Hagbarth, K.-E. and Eklund, G. (1966a) Motor effects of vibratory muscle stimuli in man. In Nobel S y m p o s i u m on Nltsc.ulurAffererlfs and Motor Control, R. Granit (Ed.), Almyvist and Wiksell, Stockholm, pp. 3 77-186. Hagbarth, K.-E. and Eklund, G. ( 1 9 6 6 b ) Tonic vibration reflexes ( T V R ) in spa5ticity. J h i n Hes.. 2 : 201-203. Hasse, J. and Van der Meulen, J.P. ( 1 9 6 1 ) Effects of supraspinal stimulation on Renshaw cells belonging to extensor motoneurons. J. Neurophysiol., 24 : 51 0-520. Hirayama, K., Homma, S., Mizote, M., Nakajima, Y. and Watanabe, S. ( 1 9 7 4 ) Separation of

30 the contribution of voluntary and vibratory activation of motor units in man by crosscorrelograms. Jup. J. Physiol., 24: 293-304. Homma, S. (1966) Firing of the cat motoneurone and summation of the excitatory postsynaptic potential. In Muscular Afferents and M o t o r Control, R. Granit (Ed.), Almqvist and Wiksell, Stockholm, pp. 235-244. Homma, S. and Kanda, K. ( 1 9 7 3 ) Impulse decoding process in stretch reflex. In M o t o r Control, A. Gydikov, N. Tankov and D. Kosarov (Eds.), Plenum, New York, pp. 45-64. Homma, S., Ishikawa, K. an& Stuart, D.G. (1970) Motoneuron responses to linearly rising muscle stretch. A m e r . J. p h y s . M e d . , 49: 290-306. Homma, S., Kanda, K. and Watanabe, S. (1971) Monosynaptic coding of group Ia afferent discharges during vibratory stimulation of muscles. Jap. J. Physiol., 21: 405-417. Homma, S., Mizote, M. and Watanabe, S. ( 1 9 7 5 ) Participation of mono- and polysynaptic transmission during tonic activation of the stretch reflex arcs. Jup. J. Physiol., 2 5 : 135-1 46. Hultborn, H., Jankowska, E., Lindstrom, S. and Roberts, W. (1971) Neuronal pathway of the recurrent facilitation of motoneurones. J. Physiol. ( L o n d . ) , 218: 495-514. Jack, J.J.B., Miller, S., Porter, R. and Redman, S.J. ( 1 9 7 1 ) The time course of minimal excitatory post-synaptic potentials evoked in spinal motoneurones by group Ia afferent fibers. J. Physiol. ( L o n d . ) , 215: 353-380. Kanda, K. ( 1 9 7 2 ) Contribution of polysynaptic pathways to tonic vibration reflex. Jup. J. Physiol., 2 2 : 367-377. Kolmodin, G.M. (1957) Integrative processes in single spinal interneurones with proprioceptive connections. A c t u physiol. scund., 4 0 , Suppl. 1 3 9 : 1-89. Kuno, M. and Miyahara, J.T. (1969) Analysis of synaptic efficacy in spinal motoneurones from “quantum” aspects. J. Physiol. ( L o n d . ) , 201 : 479-493. Kuno, M., Muiioz-Martinez, E.J. and Randie, M. (1973) Sensory inputs to neurones in Clarke’s column from muscle, cutaneous and joint receptors. J. Physiol. ( L o n d . ) , 228: 3 27-342. Lance, J.W., De Gail, P. and Neilson, R.D. (1966) Tonic and phasic spinal cord mechanisms in man. J. Neurol. Neurosurg. Psychiat., 29: 535-544. Lundberg, A. (1964) Supraspinal control of transmission in reflex paths to motoneurones and primary afferents. In Physiology of Spinel Neurons, J.C. Eccles and J.P. Schad6 (Eds.), Elsevier, Amsterdam, pp. 197-219. 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. Stuart, D.G., Willis, W.P. and Reinking, R.M. (1971) Stretch-evoked excitatory postsynaptic potentials in motoneurons. Brain Res., 33: 115-125. Tsukahara, N. and Ohye, C. (1964) Polysynaptic activation of extensor motoneurones from group Ia fibers in the cat spinal cord. Experienfia (Basel), 20: 628-629. Westbury, D.R. ( 1 9 7 2 ) A study of stretch and vibration reflexes of the cat by intracellular recording from motoneurones. J. Physiol. ( L o n d . ) , 226: 37-56. Wilson, V.J., Talbot, W.H. and Diecke, F.P.J. ( 1 9 6 0 ) Distribution of recurrent facilitation and inhibition in the spinal cord. J. Neurophysiol., 2 3 : 144-153.

SESSION I

MUSCLE SPINDLE AND ITS FUSIMOTOR INNERVATION

Part I Chairman: E. Eldred (Los Angeles, Calif.)

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The Mechanical Properties of Dynamic Nuclear Bag Fibres, Static Nuclear Bag Fibres and Nuclear Chain Fibres in Isolated Cat Muscle Spindles I.A. BOYD Institute of Physiology, University o f Glasgow, Glasgow (Great Britain)

INTRODUCTION The behaviour of individual intrafusal fibres during fusimotor activity, in spindles isolated from the tenuissimus muscle of the cat, has been observed directly in two series of experiments conducted by my research group. In the first series, in which a large number of spindles were studied over a period of 5 years, fusimotor axons were activated in the muscle nerve. The principal qualitative results have already been published (Boyd, 1966,1971; Boyd and Ward, 1969, 1975). The quantitative analysis of all the data on film was completed only recently, however (Boyd, 1976a,b). This will be summarised and the earlier results reviewed. In the second series of experiments, which overlapped in time with the analysis of the first series, dynamic and static fusimotor axons were activated individually in ventral root filaments. Preliminary results have been published (Boyd et al., 1973, 1975a,b). These spinal root/isolated spindle experiments will be reviewed and the preliminary results compared with those of the muscle nerve/isolated spindle experiments. MUSCLE NERVE/ISOLATED SPINDLE PREPARATION The tenuissimus muscle and nerve were removed from the cat and a spindle completely isolated. In most cases the innervation of the spindle was intact, but it had no blood supply. The spindle was bathed in Krebs solution containing glucose, glutamine and amino acids. The fusimotor axons to the spindle were recruited by applying graded stimuli to the whole muscle nerve. Thus, all the fusimotor axons to the spindle (0and y) were activated in many cases, but it was not known which were “dynamic” and which were “static”. Active and passive intrafusal tension In 4 experiments the maximal tension developed by the whole intrafusal bundle was measured. It was in every case less than 10 mg and could be as small as 2 mg. Since the extension of the primary sensory spirals so produced could be as much as 20-2576, as great as has ever been observed in spindles with an

34 intact blood supply, it is concluded that the tension developed by all the intrafusal fibres together is less than 10 mg. The tension transducer was not sufficiently sensitive to allow measurement of the tension developed by individual intrafusal fibres, but the contribution of the nuclear chain fibres appeared to be rather less than that of the nuclear bag fibres. The same 4 spindles were extended in a stepwise fashion until the spindle broke. The breaking strain varied from 1.5 to 4.5 g. A stretch of 25-3096, which is the maximum that spindles are subjected t o in situ (Gladden, personal communication), produced a passive tension of about 500 mg. In one particular spindle 20% stretch in the physiological range produced 200 mg increment in passive tension. Yet about 20% stretch of the primary sensory spirals of the same spindle was produced by an active intrafusal tension of 2 mg. It follows that a very large proportion of the tension applied to a spindle is absorbed by the spindle capsule, and only a small proportion is transmitted to the intrafusal bundle itself.

Time course o f intrafusal contraction Frame by frame analysis of the time course of intrafusal contraction in cine film of 21 spindles was carried out (Boyd, 1975). Contraction in nuclear chain fibres was almost complete in 0.3 sec and complete in about 0.5 sec on average, though sometimes the contraction time was as short as 0.25 sec (Fig. 1).Such contraction appears relatively fast in moving film. Further, at low frequencies of stimulation nuclear chain fibres characteristically exhibit small oscillatory contractions, whereas nuclear bag fibres show a small smooth contraction or none at all (Boyd, 1966).

.. -.*)7

0

0.5

1.0

1.5

2.0

2.5

3.0 sec

Fig. 1. Time course of contraction and relaxation of the slow nuclear bag fibre ( o ) , the fast nuclear bag fibre (O), and the bundle of nuclear chain fibres (A) in the same muscle spindle during stimulation of all its fusimotor axons at 100/sec for the period indicated by the solid bar. Contraction was assessed from the percentage stretch of the spirals of the primary sensory ending surrounding individual intrafusal fibres (see also Figs. 2, 7 and 8a). (Reproduced with permission of the Quart. J. exp. Physiol., Boyd, 197613.)

Fig. 2 . Time course of extension of the spirals of a primary sensory ending due to tetanic contraction of the polar regions when the fusimotor axons t o the spindle were stimulated a t 100/sec (see also Fig. 1). N.B., nuclear bag fibre; N.C., nuclear chain fibres; S, turns of sensory spirals; f.s., fluid space; c, capsule. Scale bar = 5 0 pm. a : rest. Fast N.B. spiral above. Slow N.B. spiral below. N.C. spirals n o t clearly visible in the middle. b: 0.42 see after start of stimulation. Fast N.B. spiral maximally extended 9% (cf. inverted arrows in a, b). Slow N.B. spiral extended 2% (cf. upright arrows): N.C. fibres maximally extended 9%. c : 1.3 sec after start of stimulation. Slow N.B. fibre maximally extended 6%.Fast N.B.’and N.C. fibres as in b.

36 It was possible to compare the time course of activity in the two nuclear bag fibres in 14 spindles. Eleven spindles contained one “fast” fibre and one “slow” fibre; in eight they were separately operated, and in the remaining three there was doubt as t o whether this was so or not. One spindle contained two “slow” nuclear bag fibres, and one contained two “fast” nuclear bag fibres, in both cases operated by the same axon. One spindle contained two nuclear bag fibres operated by the same axon, and a third which was inactive, presumed denervated. The “fast” nuclear bag fibres had a mean contraction time of 0.5 sec and their time course of activity was very similar t o that of the nuclear chain fibres. The contraction in “slow” nuclear bag fibres was almost complete in 0.6 sec and complete in 0.8 sec, on average, though it could be even longer (Fig. 1). The primary sensory ending from which the measurements of Fig. 1 were derived is shown in Fig. 2a-c. About 0.4 sec after the commencement of stimulation the upper “fast” nuclear bag fibre spiral was fully extended (cf., inverted arrows in Fig. 2a and b), as were the nuclear chain spirals in the middle of the intrafusal bundle, whereas the lower “slow” nuclear bag fibre spiral had only just started to open (upright arrows). The “slow” nuclear bag fibre spiral was, however, fully extended 1.3 sec after the start of stimulation (Fig. 2c). The difference between the mean contraction time of “fast” and “slow” nuclear bag fibres was highly significant statistically whereas that between “fast” nuclear bag fibres and nuclear chain fibres was not significant. The difference between the relaxation times of “fast” and “slow” nuclear bag fibres was particularly obvious. The values for the two nuclear bag fibres in 10 numbered spindles are given in Fig. 3. Although there is variation in the absolute values from spindle to spindle it is clear that in all but one spindle there

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0.8 1.0 Relaxation time (sec)

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Fig. 3. Relaxation time of pairs of nuclear bag fibres (below) and the nuclear chain fibres (above) in 10 numbered spindles following a maximal tetanic contraction. Black areas, slower nuclear bag fibre of the pair; hatched areas, faster nuclear bag fibre; stippled areas, nuclear chain fibre bundle. Measurements for nuclear chain fibres were unobtainable in three spindles; in two others (broken boxes) the nuclear chain fibres relaxed rapidly at first and then adhered to the slow nuclear bag fibre and moved with it thereafter.

37 was a marked difference between the two nuclear bag fibres. In the one exception both nuclear bag fibres were “fast”. The relaxation time of the nuclear chain fibres, when this could be studied, was similar to that of the “fast” nuclear bag fibres (Fig. 3) except in two cases where the nuclear chain fibres relaxed rapidly at first and then followed the movement of the “slow” nuclear bag fibre and were clearly physically linked t o it by connective tissue. Smith (1966) activated the intrafusal fibres in rat lumbrical muscle spindles directly with repetitive stimuli at 5O/sec. He observed “fast” and “slow” intrafusal fibres with contraction times of about 200 and 500 msec, respectively. He thought that the “slow” fibres were probably nuclear bag fibres and the “fast” fibres nuclear chain fibres. It now seems likely that some of his “fast” fibres may have been fast nuclear bag fibres and the rest were, no doubt, nuclear chain fibres. ‘%reep” in intrafusal fibres When a ramp and hold passive stretch is applied to an isolated spindle some intrafusal fibres exhibit “creep” after the stretch is complete (Smith, 1966; Boyd and Ward, 1969). This appears to be due to “give” in the region of the fibre beyond the end of the fluid space so that it creeps towards the spindle equator, past the other intrafusal fibres, within the fluid space. Thus, the final extension of the primary sensory spiral around the fibre when the creep is complete is less than it was at the completion of the ramp stretch. Even a small creep of this nature is obvious in moving film (Boyd, 1970). Occasionally the

T i m e from end of stretch

Fig. 4 . Intrafusal “creep” following a ramp and hold stretch of one spindle (1.2 mm at 1 . 8 mmlsec). Note marked creep in the slow nuclear bag fibre (a),the small creep in the fast nuclear bag fibre (o), and absence of creep in the nuclear chain fibres (A). Creep assessed from the approximation of the spirals of the primary ending round individual intrafusal fibres during the maintained stretch of the spindle. Creep amplitude, 32% of the applied stretch. Creep time, 2 . 4 sec.

whole intrafusal bundle shows some creep, but it is usually confined to one of the nuclear bag fibres in any spindle. In three spindles it was established that the fibre showing creep was the “slow” nuclear bag fibre by measurement of its time course of contraction and relaxation in response t o a train of stimuli applied to its fusimotor axon. Creep is illustrated in Fig. 4 which shows graphically the time course of the substantial creep in the primary sensory spiral round the slow nuclear bag fibre, and in Fig. 5 which shows the creep in the slow nuclear bag fibre in the secondary sensory region of the same spindle and its absence in the nuclear chain fibres. Fig. 5a and b show the intrafusal bundle a t the start and end of the stretch, which resulted in a 13% extension of the sarcomeres at this point and slight displacement of the bundle t o the left. Fig. 5c shows the situation after creep was complete; the nuclear chain fibres are exactly as in Fig. 5b but the slow nuclear bag fibre has crept about 10 pm t o the left within the inner capsule towards the spindle equator. Note particularly the displacement of the inverted arrow marking the slow nuclear bag fibre, relative to the upright arrow marking a nuclear chain fibre, in Fig. 5b and c. Creep was studied in detail in 9 spindles. The amplitude of the creep back was measured accurately in 4 slow nuclear bag fibres and amounted t o 5876, 32%, 20% and 18% of the extension of the primary sensory spiral produced by the stretch. In 5 other slow nuclear bag fibres the creep amplitude was between 20% and 30%. In 6 of these 9 spindles the fast nuclear bag fibre showed no creep at all. In the two spindles in which the creep in the slow nuclear bag fibre was over 3096, the fast nuclear bag fibre showed a small creep of the order of 4% (Fig. 4). Possibly this was due t o mechanical coupling between the two fibres which is especially likely in the region of the primary sensory ending. In the remaining spindle the fast nuclear bag fibre could not be seen. In 5 of the 9 spindles the nuclear chain fibres showed no creep at all (Fig. 4),in two they could not be seen clearly, and in the other two there was a small creep (
Fig. 5 . “Creep” in intrafusal fibres following passive stretch (see Fig. 4). Secondary sensory region about 400 p m from t h e spindle equator. Corresponding points on “slow” nuclear bag fibre (N.B. above, inverted arrow) and on one of the nuclear chain fibres (N.C. below, upright arrow). f.s., fluid space; c, capsule. a : before start of stretch. b: a t end of 1 . 2 m m stretch a t 1.8 mmlsec. Sarcomeres 13% extended. c : 2.4 sec after end of stretch when creep was complete. Note creep in slow nuclear bag fibre and absence of creep in nuclear chain fibres (cf. arrows in b and c). Scale bar = 50 pmr

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Fig. 6. Time after the end of a ramp and hold passive stretch for all creep in individual intrafusal fibres to cease, for 8 numbered spindles in which both nuclear bag fibres could be observed. Black area, slow nuclear bag fibres; hatched area, fast nuclear bag fibres; stippled area, nuclear chain fibre bundles.

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41 The effect of intrafusal contraction The spindle with behaviour shown in Fig. 7 and innervation shown in Fig. 8a was typical of those in the first series of experiments in which not only were the nuclear bag fibres and nuclear chain fibres selectively operated but the two nuclear bag fibres were themselves under separate central control. The resting length was such that the nuclear chain fibres were kinked. When axons ys and 7 6 (Fig. 8a) were activated there was local contraction at both poles of the nuclear chain fibre bundle, maximal at 150/sec, around a stationary point about 1.2 mm from the equator (Fig. 7). The contraction occurred over a considerable length of the fibres in the region beyond the end of the fluid space, and the whole of the region within the fluid space was stretched. There was marked displacement towards the poles in the S, and S2 regions, the kinks straightened out and the small spirals of the primary sensory ending opened 9%. If the nuclear chain fibres had been straight initially, the extension of the spirals would have been, more typically, about 15--20%. The two nuclear bag fibres behaved differently. One fibre contracted and relaxed slowly (Fig. I). A single y axon operated both poles of this “slow” nuclear bag fibre and produced maximal local contraction at 75/sec at the 3 distinct foci shown in Fig. 7. The resultant displacement of different regions is shown above the arrows and the opening of the primary sensory spiral was relatively small (8%).The other, “fast” nuclear bag fibre was innervated by separate y axons at the two poles. It contracted and relaxed rapidly (Fig. 1) and maximal contraction was produced at 100/sec. There were 3 discrete foci of contraction, one at each pole more than 2 mm from the spindle equator and a third 0.9 mm from the equator. Attempts to expose the distal contraction sites resulted in the denervation of both. The single remaining focal contraction extended the sensory spiral 11%(Fig. 7). The extension was much greater when contraction occurred at all 3 foci but was not measured precisely. Usually the extension of the primary sensory spiral round the “fast” nuclear bag fibre during maximal contraction is much greater than that of the spiral round the “slow” nuclear bag fibre.

Selective control o f intrafusal fibres On the basis of the study of both poles of 30 spindles we (Boyd and Ward, 1975) concluded that in 30% one nuclear bag fibre had some innervation in common with the nuclear chain fibres, whereas the other nuclear bag fibre was selectively controlled (legend of Fig. 5c). In 60% of spindles the nuclear chain fibres were under separate control from the nuclear bag fibres; in some of these (20%)the two nuclear bag fibres were also selectively controlled (legend of Fig. 5b) while in others (40%) they had innervation in common (legend of Fig. 5a). In the light of the more recent analysis, the proportion of spindles with completely selective innervation of nuclear chain fibres still stands at 60% but the proportion of these in which the two nuclear bag fibres were themselves separately controlled needs to be substantially increased for the reasons now given. In the initial analysis attention was focussed on whether or not the nuclear

42 chain fibres and the nuclear bag fibres were separately operated. The spindle was usually oriented so that one nuclear bag fibre and the bundle of nuclear chain fibres were clearly visible, the other nuclear bag fibre lying underneath. We noticed that the speed and degree of contraction of nuclear bag fibres were very variable, but it never occurred to us that the two nuclear bag fibres in the same spindle might always be functionally different even when Ovalle and Smith (1972) showed that the two nuclear bag fibres in cat spindles were always histochemically different. Since the case was being made for selective intrafusal innervation, when it could not be established clearly whether the two nuclear bag fibres in a particular pole were selectively innervated they were recorded as having innervation in common. When the first experiments (see below) which involved the stimulation of single y axons in the ventral roots (Boyd et al., 1973) suggested, however, that selective innervation of the two

a

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Fig. 8. Motor control and intrafusal behaviour of a, a particular spindle from the work of Boyd and Ward (1975), and b, a typical spindle from the work of Boyd e t al. (1975b). a : spindle operated by 6 axons numbered in order of increasing threshold t o stimulus applied to muscle nerve. Action of 7 3 negligible. Contraction times for fast nuclear bag fibre and nuclear chain fibres the same (0.25 sec); for slow nuclear bag fibre much greater (1.17 sec). Both nuclear bag fibres and nuclear chain fibres selectively innervated. b : spindle operated by one dynamic y axon and three static y axons.' D.N.B., dynamic nuclear bag fibre with slow contraction time; S.N.B., static nuclear bag fibre with fast contraction time; N.C., bundle of nuclear chain fibres. Slow nuclear bag fibre of a corresponds to dynamic nuclear bag fibre of 6.

43 nuclear bag fibres might be frequent, I paid particular attention to this point while carrying out frame by frame time course analysis of much of the early film. As described above, “fast” and “slow” nuclear bag fibres were usually, if not always, selectively operated, and the only definite cases of common innervation related to two nuclear bag fibres which were both either “fast” or “slow”. Further, of the 18 spindles (60%) whose nuclear chain fibres were entirely selectively controlled (Boyd and Ward, 1975; legend of Fig. 5a, b), six had two nuclear bag fibres definitely controlled by different axons, in five the point could not be settled with certainty, and in five both nuclear bag fibres were inactive, presumed denervated. Only two spindles had two nuclear bag fibres definitely operated by the same axon. Thus, the proportion of spindles in which both nuclear bag fibres and the nuclear chain fibres were all controlled selectively (as in Fig. 8a) was at least 2096, and was probably about 50%. SPINAL ROOT/ISOLATED SPINDLE PREPARATION In the second series of experiments (Boyd et al., 1973, 1975a, b) group Ia axons from several spindles in either the tenuissimus or abductor digiti quinti medius muscles were isolated in “single fibre” dorsal root filaments. Several, and in some cases all, of the y fusimotor axons supplying these spindles were isolated in ventral root filaments. In a few experiments 0 fusimotor axons were also sought for in the ventral roots. In all cases the axons were identified by stimulating the muscle nerve and recording their individual action potentials in the spinal root filaments. The fusimotor axons were then classified as “dynamic” or “static” according to their action in increasing or reducing the dynamic index of the Ia afferent discharge, respectively (Matthews, 1962). A length of the muscle containing several spindles was then reflected into a bath in such a way as to permit observation of spindles in the muscle, while at the same time preserving the nerve and blood supply intact. A particular muscle spindle was then isolated, if possible one which was known to be operated by at least one of the dynamic axons and one of the static axons already identified. The behaviour of each of the two nuclear bag fibres and of the nuclear chain fibre bundle during repetitive stimulation of the dynamic and static axons was then observed and recorded on moving film. This whole procedure occupied some 24 h and extended a team of three to near the limit of their physical endurance.

Functional distribution of fusimotor axons In the early experiments of this series (Boyd et al., 1973), using the abductor digiti quinti medius muscle, it quickly became apparent that dynamic y axons selectively innervated nuclear bag fibres, usually one pole of one nuclear bag fibre only. Further, static y axons were observed to operate nuclear chain fibres only, or nuclear chain fibres and one nuclear bag fibre together, in about equal proportions. About the same time Bessou and Pag&s(1973), in similar experiments on the tenuissimus muscle (Table I), obtained similar results. They

44 TABLE I FUNCTIONAL DISTRIBUTION O F FUSIMOTOR AXONS T O TENUISSIMUS SPINDLES B o y d (1971) B o y d and Ward ( 1 9 75)

B o y d et al. (1975b, 1976)

3 dynamic 1 0 y dynamic 1 4 static

Bessou and Pages (1973, 1975)

11 y dynamic 26 C15 static

Nuclear bag fibres only

56

Nuclear chain fibres only

75

12

y static

12

y static

Nuclear bag and nuclear chain fibres

17

8

y static

11

ystatic

148

47

Tot a1

49

found, in addition, several static y axons operating nuclear bag fibres only and a number more which appeared to do so, but there was some doubt as to whether or not they operated nuclear chain fibres as well. Static y axons innervating nuclear bag fibres only in some tenuissimus spindles were found histologically by Barker et al. (1973), and their presence in considerable numbers in tenuissimus was also deduced by Boyd and Ward (1975). In later experiments (Boyd et al., 1975a, b, 1976) using the tenuissimus muscle, a number of static y axons operating one nuclear bag fibre only were encountered as well as the types previously described (Table I). Further, several p axons, all of which were dynamic in action, all produced contraction in one nuclear bag fibre only. In the work of our group, and that of Bessou and Pag& (1973), nuclear bag fibres operated by dynamic' y axons were never operated by static y axons and vice versa. As our ability to isolate in the ventral roots all the fusimotor axons t o a particular spindle, and to isolate the spindle without damaging any of them, improved it gradually became apparent that all the tenuissimus spindles we were studying contained one nuclear bag fibre operated by a dynamic axon (occasionally two such axons) and a second nuclear bag fibre operated by static y axons, some of which selectively innervated this fibre and others of which operated the nuclear chain fibres as well. My colleagues were convinced of this sooner than I was, since from my earlier work I believed that in a proportion of spindles both the nuclear bag fibres were operated by the same axon. But after 5 experiments in a row in which one nuclear bag fibre was operated by one dynamic axon, and the other by one or more static axons, I finally concluded that this must be true for almost all tenuissimus spindles. The picture is clear if the results from our 1 6 most recent experiments are considered. In 1 4 spindles one nuclear bag fibre and the nuclear chain fibres were operated by static y axons only. In 8, or possibly 9, of these spindles one non-selective static y axon, never more, was encountered whereas all the other static y axons operated either one nuclear bag fibre or the nuclear chain fibres selectively. In 11 spindles the second nuclear bag fibre was operated by one dynamic axon only (two in one case), and in the other five it was inactive,

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presumably operated by a missing dynamic axon. Likewise, in one spindle an inactive nuclear bag fibre was presumably operated by missing static y axons since the other was operated by a dynamic y axon. The motor control of a typical spindle is shown in Fig. 8b. One dynamic y axon operates a particular nuclear bag fibre. The other nuclear bag fibre is controlled by one selective static y axon and one non-selective static y axon. The nuclear chain fibre bundle is controlled by the non-selective static y axon at one pole, and selectively by another static y axon at the other pole. Time course of intrafusal activity In every spindle the nuclear bag fibre operated by the dynamic axon had an obviously slower time course of contraction than the one operated by static y axons. Although no accurate measurements of the time course of contraction have yet been made, it is already clear that the “slow” and “fast” nuclear bag fibres of the earlier study are the fibres operated by dynamic and static axons, respectively. Thus it follows that in the spindle in Fig. 8a, axon y1 was a dynamic y axon while all the other fusimotor axons were static y axons. COMPARISON OF ISOLATED SPINDLE STUDIES Dynamic and static nuclear bag fibres The clearcut findings of the spinal root/isolated spindle experiments led us to designate the nuclear bag fibre controlled by the dynamic axon(s) the “dynamic nuclear bag fibre”, and the one controlled by static axons the “static nuclear bag fibre” (Boyd et al., 1975b). In doing so we were influenced by the obvious difference in their time course of contraction evident in both series of experiments, showing that they were mechanically different, and by the usually much larger contraction in the static nuclear bag fibres than in the dynamic ones. Further, creep following passive stretch seems to be characteristic of dynamic nuclear bag fibres. The recent observations of Gladden (1976) lend further weight to this classification, namely that the dynamic nuclear bag fibres are more sensitive to directly applied acetylcholine, and differ in their supporting elastic tissue from the static nuclear bag fibres. It follows, also, that the quantitative measurements of the time course of activity in “slow” and “fast” nuclear bag fibres (Boyd, 1975) may be assumed to apply to dynamic and static nuclear bag fibres, respectively. Selectivity of control In the spinal root/isolated spindle study the two nuclear bag fibres were separately controlled in at least 70%,and probably in loo%, of spindles. In the muscle nerve/isolated spindle study the nuclear bag fibres were separately controlled in at least 5096, and probably in 80%, of spindles. It seems likely that all tenuissimus spindles contain one dynamic nuclear bag fibre and one static nuclear bag fibre, except for a small minority which contain two dynamic

46 nuclear bag fibres or two static nuclear bag fibres controlled by the same axon, and which may contain a third nuclear bag fibre of the opposite type. Non-selective axons innervating nuclear chain fibres and one nuclear bag fibre were encountered in about 50% of the spindles in the spinal rootlisolated spindle study and in about 30% of those in the earlier study. There were also a few atypical forms in the latter study bringing the total with non-selective innervation up to 40%. In both studies there was never more than one nonselective axon to any spindle. The total proportion of these axons t o the tenuissimus muscle out of all the fusimotor axons studied is given in Table I and was 11%(Boyd, 1971; Boyd and Ward, 1975), 17% (Boyd et al., 1975b, 1976) and 22% (Bessou and PagGs, 1973). We may conclude that in about half of the spindles in the tenuissimus muscle all three types of intrafusal fibre are under separate central control (Fig. 8a), but that the other half receives one static y axon operating both nuclear chain fibres and the static nuclear bag fibre in addition to selective innervation (Fig. 8b). Our results agree with those of Barker et al. (1973), who showed that static y axons to the tenuissimus muscle could innervate nuclear chain fibres only, nuclear bag fibres only, or nuclear chain fibres and one nuclear bag fibre. Further, they agree with the results of Brown and Butler (1975) who showed that static y axons t o the peroneus longus muscle could cause glycogen depletion in nuclear chain fibres only, or nuclear chain fibres and one, but not both, of the nuclear bag fibres; they did not, however, encounter any static y axons innervating nuclear bag fibres only. On the other hand, our results are at variance with those of Barker et al. (1975) who concluded that static y axons to tenuissimus could innervate both types of nuclear bag fibre as well as nuclear chain fibres, and with the earlier study of Brown and Butler (1973) on the tenuissimus muscle in which they showed that static y axons could cause glycogen depletion in all the nuclear bag fibres in some spindles.

Motor nerve terminals Motor nerve terminals in cat muscle spindles have been shown by electron microscopy to be of 3 types (Barker et al., 1970); p, plates with well developed junctional folds which are the terminations of3!, axons; p2 plates with shallower, irregular junctional folds; and trail endings with no, or only rudimentary, junctional folds, which were subsequently shown t o be the terminations of static y axons (Barker et al., 1973). Initially I had reservations about this classification because in earlier light microscopy (Boyd, 1962) I concluded that almost all nerve terminals on nuclear bag fibres were plates (yl plates), and those on nuclear chain fibres were trail-like (y2) endings. Barker et al. (1970), however, frequently found trail endings on nuclear bag fibres. Secondly, preliminary electron microscopy in my laboratory showed that a single intrafusal motor end-plate could have junctional folds in one region and none in another. Thirdly, the contraction in both dynamic and static nuclear bag fibres is confined t o discrete foci such as would be consistent with innervation by motor end-plates. The uncertainty in

47

my mind is reflected in the fact that in describing the first series of experiments on isolated spindles (Boyd and Ward, 1975) we depicted all endings on nuclear bag fibres as plates, and those on nuclear chain fibres as trails. In the preliminary account of the second series of experiments (Boyd et al., 1975b) we felt it wise not t o indicate the type of nerve ending of dynamic axons and static y axons at all until we had ourselves studied them by electron microscopy. Now that our own electron microscopic observations are coming to hand (Arbuthnott et al., 1976), the doubts in my mind are resolving. First, provided that a p2 plate is studied throughout its length it can be clearly identified by the presence of irregular junctional folds at some point, and it appears to be the termination of a dynamic y axon, as we had anticipated. Secondly, we have found endings, probably on the static nuclear bag fibres only, without junctional folds and yet which are at least as large as a p2 plate and which could thus have been termed plates in the early light microscopy. The term “trail ending” can be somewhat confusing unless it is realised that it is the axons that trail along the intrafusal fibres and not the synaptic contacts and that the latter vary greatly in size, To signify my growing belief that the classification of Barker et al. (1970) based on junctional folding is reliable, I have depicted the terminals of dynamic y axons in Fig. 8 of this paper as plates, and the terminals of static y axons as trails irrespective of their distribution. SUMMARY Observations on isolated spindles activated through the muscle nerve (Boyd, 1966; Boyd and Ward, 1975) are reviewed, extended and compared with preliminary observations on isolated spindles activated by static and dynamic fusimotor axons in the ventral roots (Boyd et al., l973,1975a, b). Maximal intrafusal tension is less than 10 mg, only about 1%of that produced by passive stretch which extends the sensory spirals by the same amount. Most spindles contain one “slow” and one “fast” nucIear bag fibre with significantly different contraction times and which are operated by different fusimotor axons, and several nuclear chain fibres whose contraction time is not significantly different from “fast” nuclear bag fibres, with which they may sometimes have innervation in common. “Slow” nuclear bag fibres exhibit “creep” following passive stretch whereas other intrafusal fibres usually do not. Dynamic and static fusimotor axons always operate different nuclear bag fibres, named “dynamic nuclear bag fibres” and “static nuclear bag fibres”, which are synonymous with “slow” and “fast” nuclear bag fibres, respectively. Static y axons also operate nuclear chain fibres selectively and, in about 50% of spindles, one of them operates both the nuclear chain fibres and the static nuclear bag fibre. ACKNOWLEDGEMENTS I wish to acknowledge gratefully the expert technical assistance provided by Mr. James Ward and Miss Jess Wilson, and the support of the Muscular Dystrophy Group of Great Britain which financed part of this work.

48 REFERENCES Arbuthnott, E.R., Boyd, I.A. and Gladden, M.H. (1976) Ultrastructural observations of a muscle spindle in the region of a contraction site of a dynamic axon. Progr. Brain Res., 44: 61-65. Barker, D., Stacey, M.J. and Adal, M.N. ( 1 9 7 0 ) Fusimotor innervation in t h e cat. Phil. Trans. B, 258: 315-346. Barker, D., Emonet-Dbnand, F., Laporte, Y., Proske, U. and Stacey, M.J. (1973) Morphological identification and intrafusal distribution of t h e endings of static fusimotor axons in the cat. J. Physiol. (Lond.), 230: 405-427. Barker, D., Bessou, P., Jankowska, E., PagBs, B. and Stacey, M.J. ( 1 9 7 5 ) Distribution of static and dynamic y axons to cat intrafusal muscle fibres. J. Anat. (Lond.), 1 1 9 : 199. Bessou, P. e t PagBs, B. (1973) Nature des fibres musculaires Pusales activkes par des axones fusimoteurs uniques statiques ou dynamiques chez le chat. C. R . Acad. Sci. (Paris), 277: 89-91. Bessou, P. and PagBs, B. (1975) Cinematographic analysis of contractile events produced in intrafusal muscle fibres by stimulation of static and dynamic fusimotor axons. J. Physiol. (Lond.), 252: 397-427. Boyd, I.A. (1962) The structure and innervation of t h e nuclear bag muscle fibre system and the nuclear chain muscle fibre system in mammalian muscle spindles. Phil. Trans. B, 245: 81-136. Boyd, LA. (1966) The behaviour of isolated mammalian muscle spindles with intact innervation. J. Physiol. ( L o n d . ) , 1 8 6 : 109-110P. Boyd, I.A. (1970) The Mammalian Muscle Spindle - A n Advanced S t u d y ( 1 6 m m sound film), University of Glasgow, Glasgow. Boyd, I.A. (1971) Specific fusimotor control of nuclear bag and nuclear chain fibres in cat muscle spindles. J. Physiol. (Lond.), 214: 30-31P. Boyd, I.A. (1976a) Time course of activity in intrafusal fibres in isolated cat muscle spindles. J. Physiol. ( L o n d . ) , 254: 23-24P. Boyd, I.A. (1976b) The response of fast and slow nuclear bag fibres and nuclear chain fibres in isolated cat muscle spindles to fusimotor stimulation, and the effect of intrafusal contraction o n the sensory endings. Quari. J. exp. Physiol., 61 : 203-252, Boyd, LA. and Ward, J. (1969) The response of isolated cat muscle spindles to passive stretch. J. Physiol. (Lond.), 200: 104-105P. Bcyd, LA. and Ward, J. (1975) Motor control of nuclear bag and nuclear chain intrafusal fibres in isolated living muscle spindles from t h e cat. J. Physiol. (Lond.), 244: 83-112. 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: 29-3OP. Boyd, LA., Gladden, M.H., McWilliam, P.N. and Ward, J. (1975a) Study of 0 innervation and of and 7 control of isolated muscle spindles in t h e c a t hind limb. J. Anat. ( L o n d . ) , 1 1 9 : 198. Boyd, I.A., Gladden, M.H., McWilliam, P.N. and Ward, J. (1975b) ‘Static’ and ‘dynamic’ nuclear bag fibres in isolated cat muscle spindles. J. Physiol. (Lond.), 250: 11-12P. Boyd, I.A., Gladden, M.H., McWilliam, P.N. and Ward, J. (1976) Control of static and d y namic nuclear bag fibres and nuclear chain fibres by y and 0axons in isolated cat muscle spindles. J. Physiol. (Lond.), in press. Brown, M.C. and Butler, R.G. (1973) Studies o n the site of termination of static and dynamic fusimotor fibres 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 t h e site of termination of static gamma fibres within muscle spindles of the cat peroneus loiigus muscle. J. Physiol. (Lond.), 247: 131-143. Gladden, M.H. (1976) Structural features relative to the function of intrafusal muscle fibres in the cat. Progr. Brain Res., 44: 51-59. Matthews, P.B.C. (1962) The differentiation of t w o types of fusimotor fibre by their effects o n the dynamic response of muscle spindle primary endings. Quart. J. exp. Physiol., 47: 324-333.

49 Uvalle, W.K. a n d Smith, R.S. (1972) Histochemical identification of three types of intrafusal muscle fibres i n the c a t and monkey based o n the myosin ATPase reaction. Canad. J. Physiol. Pharmacol., 5 0 : 195-202. Smith, R.S. (1966) Properties of intrafusal fibres. In Muscular Affererzis and Moior Control, R. Granit (Ed.), Alniquist and Wiksell, Stockholm, pp. 69-80.

DISCUSSION HOUK: How stretched is the spindle with respect t o the physiological range o f the muscle and have y o u looked a t different amounts of stretch within that range? How docs that relate to the kinking of the chain fibres? BOYD: Stretch of t h e tenuissimus muscle within its physiological range represents a b o u t 25-30% stretch of the spindle. We have looked a t t h e spindles a t different lengths within this range. We d o n o t have definitive measurements of t h e muscle length a t which t h e nuclear chain fibres start to kink. T h a t would be difficult to obtain because if you fix them for histological study they may change in any case during t h e fixation. However, we think t h a t the chain fibres are normally straight but that they must kink during an extrafusal contraction. ELDRED: I agree that histological artefacts make this type of study difficult. Wc have looked a t longitudinal studies of spindles in relaxed muscles and have seen kinking of intrafusal fibres, b u t I don’t specify which type. MATTHEWS: One of the crucial points of dissent, which I think is appearing a t this meeting, is whether your dynamic nuclear bag fibre could have any innervation from static fusimotor axons. The dynamic nuclear bag fibre has a very weak contraction even when activated by dynamic gamma axons. Supposing a static gamma axon also activated it and were to produce a contraction as strong as the dynamic gamma axons produce, you would have seen it. That is very convincing from your film. But if i t produced a contraction 5% as strong as t h e dynamic you would n o t have seen it. What percentage strength of contraction d o you think y o u can recognize reliably? BOYD: This depends o n whether t h e static gamma axon is activating o n e or both poles of the spindle. If it acts o n b o t h poles of t h e spindle and fixes t h e primary ending then y o u can see quite clearly that t h e dynamic nuclear bag sarcomeres d o n o t move. This is particularly convincing during relaxation following contraction produced by static gamma axons. However, if t h e spindle is activated a t one pole only the primary ending is displaced sideways and bunches u p the dynamic bag fibres, and under those circumstances it is much harder t o be sure that there is n o contraction in the dynamic bag fibre. I would n o t like t o p u t a percentage value o n it. I’m sure we would have seen anything which approached the strength o f the normal contraction produced by dynamic gamma axons, but I could n o t be certain t h a t there never was a very weak contraction. MATTHEWS: Is it possible that a dynamic gamma can produce a dynamic action on the primary ending without you seeing a n y movement, because a dynamic action might perhaps be produced just by changing the mechanical properties of the intrafusal fibre? BOYD: It’s a difficult question to answer, because if a motor axon produces n o movement how d o you know it’s there? Every dynamic fusimotor axon which we have tested in the ventral roots always produces a visible contraction. BUCHTHAL: This problem could b e solved by superimposing a very slight vibration o n your spindles and seeing whether you get an increase in stiffness when y o u d o not get movement. BOYD: Yes. Dr. Gladden and I intend to concentrate on t h e dynamic bag fibre next year

t o see what t h e spirals do under different conditions. We have n o t gone further than trying to separate o u t the t w o time courses of contraction, and t h e fusimotor innervation.

ELDRED: Could some of your spindles be tandem spindles so that some of the movement of the intrafusal fibres might be related n o t to t h e primary ending you are looking at b u t to another tandem-linked primary ending. BOYD: No. In my earlier work all the spindles were isolated from a very short length of muscle, barely longer than the spindle itself, perhaps 10-12 mm. We can scan o u t t h e spindle and observe the continuity of the intrafusal muscle fibres, and we can nearly always locate the foci of motor contraction.

Structural Features Relative to the Function of Intrafusal Muscle Fibres in the Cat MARGARET H. GLADDEN Institute of Physiology, Glasgow University, Glasgow (Great Britain)

INTRODUCTION Matthews (1964) proposed that the effects of activating static and dynamic y axons could be explained by supposing that static y axons innervated nuclear chain fibres, that dynamic y axons innervated nuclear bag fibres and that the viscoelastic properties of these muscle fibres are different, The expected differences in mechanical behaviour were later found experimentally (Boyd, 1966; Smith, 1966). Although Baxker et al. (1970) showed that some static y axons innervate both nuclear bag and nuclear chain fibres, Matthews’ explanation could still be true (see Matthews, 1972) since the effects of nuclear chain contraction might predominate, producing a static effect. However, his ideas had to be seriously questioned when Bessou and Pag&s(1973) clearly demonstrated that the contraction of nuclear bag fibres by themselves could produce a static effect, and their finding was soon confirmed by Boyd et al. (1975a). Nevertheless, both these groups agree that in muscle spindles under direct visual observation static and dynamic y axons always operate different nuclear bag fibres, so opening the possibility that there could be two kinds of nuclear bag fibre, separately innervated by static and dynamic y axons. Two kinds of nuclear bag fibre have been distinguished both by histochemistry (see Ovalle and Smith, 1972), by electron microscopy (see Banks et al., 1975) and in terms of their time course of contraction (Boyd, 1976a), but there is disagreement about their innervation. Barker et al. (1975) combined an electrophysiological and histochemical study of the same muscle spindles and concluded that static y axons could innervate both types of nuclear bag fibre and nuclear chain fibres. Boyd et al. (1975b) studied the innervation of muscle spindles by direct observation and found that spindles typically have one nuclear bag fibre operated only by dynamic y or axons and another nuclear bag fibre operated only by static y axons either together or separately from the nuclear chain fibres (see also Boyd, 1976). This paper is a preliminary report on the histology of some of the spindles studied by Boyd et al. (1975a,b), and on the effect of acetylcholine on isolated muscle spindles. The results support the view that nuclear bag fibres innervated by dynamic y axons have quite different physiological and structural properties from those innervated by static y axons.

52 HISTOLOGY OF MUSCLE SPINDLES WITH KNOWN INNERVATION Four muscle spindles from the tenuissimus muscle whose motor innervation had been studied electrophysiologically (Boyd et al., 1975b; Boyd, 1976), each had two nuclear bag fibres, one operated by dynamic y axons and the other by static y axons. One of these fibres, usually the “dynamic” bag fibre, was marked intracellularly with Alcian blue dye (4%) injected iontophoretically through a microelectrode under direct visual observation. These muscle spindles were fixed and embedded in paraffin. Serial 5 pm thick sections were cut and stained for elastic fibres (see Cooper and Gladden, 1974). The dye was present in only 1-3 sections of each sectioned muscle spindle (Fig. l a ) . In the polar regions the nuclear bag fibres innervated by static y axons had in every case more and thicker elastic fibres than those innervated by dynamic y axons. This difference was not always obvious close to the end of the capsule, but was obvious about 1 mm from the end of the capsule (Fig. l b and c), i.e., about 2.5-3.0 mm from the equator. In all 4 spindles the difference was present at both poles. The length of the nuclear bag fibres could not always be determined because, unfortunately, the spindles were not sectioned from end t o end. In three poles the nuclear bag fibres innervated by dynamic y axons could be followed to their ends. They were shorter than the nuclear bag fibres innervated by the static y axons. In two poles the nuclear bag fibres innervated by static y axons could be followed to their ends and the thick elastic fibres around them continued on in a group into the muscle. The nuclear bag fibres innervated by the dynamic y axons did not have a striking elastic fibre “tendon”. Neither kind of nuclear bag fibre was consistently thinner than the other. The relative diameters varied considerably along the length of the spindles.

Fig. 1.a: transverse section of muscle spindle showing site of dye marking. The mark in the centre of the middle fibre is the dye and this fibre is a dynamic nuclear bag fibre. Above it and to the left is a nuclear chain fibre and below is a static nuclear bag fibre. b and c: the same fibres 1 mm past the end of the capsule in the two polar regions: the static nuclear bag (lowermost) fibre has prominent elastic fibres around it. The elastic fibres are stained black.

53 THE EFFECT OF ACETYLCHOLINE ON NUCLEAR BAG FIBRES IN ISOLATED MUSCLE SPINDLES

I also observed the behaviour of the nuclear bag fibres during passive alterations in length in 11 muscle spindles which I isolated from the tenuissimus muscle. I exposed only the encapsulated regions t o avoid damaging the polar regions of the nuclear bag fibres. The muscle together with its nerve lay in a bath perfused with oxygenated Krebs solution. When the spindle is shortened, at each end of the fluid space the nuclear chain fibres can usually be seen t o kink before the nuclear bag fibres (Boyd and Ward, 1975). This kinking can occur because the intrafusal fibres are usually not firmly bound together in this region. I found that if the spindle was shortened still further one nuclear bag fibre kinked before the other. In some spindles this difference was very striking (Fig. Z), but in others it was not so obvious, and probably depended on the extent to which the intrafusal fibres were bound together. This observation might seem unremarkable but in fact it is very useful because it enables one to detect separate movements of the two nuclear bag fibres: this is often difficult because one nuclear bag fibre may be passively pulled by another active one. If acetylcholine (Sigma Chemical Company) in concentrations of lo-’ mg/ml was present in the perfusate the least kinked nuclear bag fibre contracted and the other stayed kinked. This was seen in 8 muscle spindles. The acetylcholine

Fig. 2. Isolated muscle spindle which has been shortened so that all the intrafusal fibres are kinked. The two large diameter fibres are nuclear bag fibres and the small diameter fibres are nuclear chain fibres. This region is at the end of the fluid space.

54

Fig. 3 . a: A and B are t wo kinked nuclear bag fibres of a passively shortened isolated muscle spindle shown at the end of the fluid space. b: after application of acetylcholine mg/ ml) A straightened while B remained kinked. c : B began to straighten about 10 sec later: it is shown here less kinked than in b but before it became fully straight.

55 could be washed out and the same effects observed on reapplication of the drug. In two experiments I succeeded in impaling single nuclear bag fibres with microelectrodes. In the least kinked fibre acetylcholine caused a depolarisation coinciding with the contraction. In the other nuclear bag fibre lo-’ ACh produced a depolarisation of a few millivolts but no contraction. The rate of the contraction varied. On one occasion it was so slow that it would not have been noticed had not the initial position of the bag fibre relative to an eye piece graticule been noted. This variability may be due to different rates at which the drug reaches the fibre and not to a difference in the sensitivity of individual fibres. mg/ml was observed The effect of acetylcholine at a concentration of in 3 muscle spindles (Fig. 3). The nuclear bag fibre which contracted at the lower concentrations contracted first, followed after about 10 sec by a contraction of the other nuclear bag fibre. No contraction of the nuclear chain fibres in response to acetylcholine has so far been observed. As the effects of the acetylcholine could be reversed by washing it out. As the acetylcholine wore off the nuclear bag fibres sometimes began to “kink” again, but often they stayed in the same position as if still contracting. However, if the muscle spindle was stretched and then shortened the nuclear bag fibres would “kink” in the same manner as they did before the acetylcholine was applied. In one preparation after single twitch contractions of the extrafusal fibres the muscle spindle was stretched briefly by the relaxation of the extrafusal fibres. The nuclear bag fibre which was more sensitive t o acetylcholine gave way slowly after the stretching and this phenomenon has been called “creep” by Boyd (1975). The other nuclear bag fibre did not show this creep. In another experiment the more sensitive nuclear bag fibre could be operated by stimulating the muscle nerve after the extrafusal fibres had been denervated. Again the fibre relaxed slowly after its own contraction. This is the behaviour we would expect in a nuclear bag fibre innervated by dynamic y axons and corresponds to the “slow” nuclear bag fibre of Boyd (1975). In three muscle spindles, subsequent histology showed that the more kinked nuclear bag fibres which were less sensitive to acetylcholine had thicker and more numerous elastic fibres in their polar regions. These correspond to the nuclear bag fibres innervated by the static y axons. In the three poles that the relative lengths of the nuclear bag fibres could be determined these were also the longer fibres.

HISTOCHEMISTRY Serial frozen sections of two muscle spindles were stained for elastic fibres and for myosin ATPase as used by Ovalle and Smith (1972). The nuclear bag fibres which had the conspicuous elastic fibres in their polar regions contained ATPase which was stable under both acid and alkaline conditions and would correspond t o the bag, of Ovalle and Smith. The other nuclear bag fibre contained the acid stable, alkali labile form of ATPase and would correspond to their bag,. The procedures which have been described in this paper are outlined in the

56

I---

N B fibres recognised by LACh sensitivity (8) I ~.. . I

; I ... . _J

I

marked ( 1 )

I

unmarked ( 1 )

\

marked ( 4 )

i

marked ( 3 )

[ ‘L.M.7 elastic fibres

(2)

r-

-

1

Histochcmistry

L.

-

i

flow diagram 1. The diagram also indicates the two muscle spindles which have been sectioned for electron microscopy one of which is the subject of a separate paper (Arbuthnott e t al., 1976). The number of experiments on muscle spindles studied in each case is shown in brackets. The preliminary results are shown in Table I. The correlation between features 1-3 and the dynamic and static nuclear bag fibres of Boyd e t al. (1975b) is a direct one, but the correlation between the functional types of fibre and features 4-7 has been inferred indirectly from their elastic fibre distribution, and this is indicated by the arrows. TABLE I Distinguishing fca ture

Elastic surrounding poles Length Diameter

Response to acelylcholine “Kinking” Time course of relaxation IIistochemistry

“Dynamic” nuclear bag

I

“Static” nuclear bag

T1

few shorter variable

striking longer variable

more sensitive less easily slow bag1

less sensitive more easily rapid bag2

-I

I

DISCUSSION Although this paper must be regarded as a preliminary report the results described do compliment each other. The nuclear bag fibre innervated by dynamic y axons has the same histological features as the bag fibre which is more sensitive t o acetylcholine. Acetylcholine causes an increase in the dynamic responsiveness of primary sensory afferents, similar t o the effect of

57 dynamic y stimulation (Rack and Westbury, 1966). One would expect from the present results that high doses of acetylcholine would have a static effect. Rack and Westbury (1966) found that high doses of succinylcholine, which has a similar effect t o acetylcholine, gave responses which were “quite similar to the effect of combined stimulation of powerful static and dynamic fusimotor fibres”. However, very high doses of succinylcholine may have a direct action on sensory endings (Kidd and KuEera, 1969). Succinylcholine was not used in these experiments because the effect takes longer to wear off. Smith (1966) found that succinylcholine produced a contracture of “slow” intrafusal fibres, presumably nuclear bag fibres in isolated rat muscle spindles. He also discovered that “fast” intrafusal fibres, presumably nuclear chain fibres, failed t o respond t o direct electrical stimulation after some minutes. This agrees with Boyd’s observations (personal communication) that succinylcholine consistently abolished the response of nuclear chain fibres to indirect stimulation but was observed to produce contracture of nuclear bag fibres on some occasions. These observations were made before it was realised that there are two types of nuclear bag fibre, but together with the present results they indicate that the membranes of the three types of intrafusal fibre are different. The finding that the “dynamic” nuclear bag fibre kinks less easily than the “static” one, both kinking less easily than the nuclear chain fibres, indicates that the myoplasm of the three types of fibre is different also. A functional significance of this might be that the “static” nuclear bag fibres could operate effectively at shorter muscle lengths than the nuclear chain fibres: there must be some reason why the spindle possesses two types of fibre with the same function. The failure of Cooper and Gladden (1974) to distinguish two types of nuclear bag fibre by the elastic fibre distribution in muscle spindles of the cat was mine, and was because I tried t o find a difference close t o the primary sensory ending and did not look closely enough at the poles. The present results suggest that the connective tissue linkage between the two kinds of nuclear bag fibre and the extrafusal muscle fibres is different. One wonders whether the movement of the extrafusal fibres is transmitted t o the static nuclear bag fibre along its elastic fibre tendon, and if so whether the elastic fibres “give” during stretching. If they do “give” during stretching this together with its mechanical properties, which are different from those of the dynamic nuclear bag fibre, may help t o explain why contraction of the static nuclear bag fibre does not increase the dynamic responsiveness of the primary sensory ending.

SUMMARY Nuclear bag fibres which had been shown electrophysiologically to be operated by either static or dynamic y axons were iontophoretically labelled with dye. Subsequent histology showed that the nuclear bag fibres operated by static y axons had thick elastic fibres around their polar regions which continued past the end of the fibres into the muscle. The dynamic nuclear bag fibres had fewer and thinner elastic fibres around their polar regions. This difference was then used to correlate indirectly other physiological and structural properties of

58 these fibres with their innervation. In this way it was inferred from the preliminary results that the dynamic nuclear bag fibre is more sensitive to direct application of acetylcholine than the static nuclear bag fibre and that the ATPase of the static nuclear bag fibre is stable in both alkaline and acid conditions, whereas the dynamic nuclear bag fibre contains the acid-stable, alkalinelabile form of ATPase. ACKNOWLEDGEMENTS

I should like t o thank Professor I.A. Boyd and Dr. P.N. McWilliam for reading the manuscript. Without the technical assistance of Miss J. Wilson this paper could not have been written. The technical assistance of Mr. J. Ward and Miss R. McCafferty is also gratefully acknowIedged. The work was done partly with a grant from the Muscular Dystrophy Group of Great Britain. REFERENCES Arbuthnott, E.R., Boyd, LA. and Gladden, M.H. (1976) Ultrastructural observations of a muscle spindle in the region of a contraction site of a dynamic y axon. Progr. Brain Res., 44: 61-65. Banks, R.W., Barker, D., Harker, D.W. and Stacey, M.J. (1975) Correlation between ultrastructure and histochemistry of mammalian intrafusal muscle fibres. J. Physiol. (Lond.), 252: 16P. Barker, D., Emonet-DBnand, F., Laporte, Y., Proske, U. e t Stacey, M.J. (1970) Identification des terminaisons motrices des fibres fusimotrices statiques chez le chat. C.R. Acad. Sci. (Paris), 271: 1203-1206. Barker, D., Bessou, P., Jankowska, E., Pagis, B. and Stacey, M.J. (1975) Distribution of static and dynamic y axons to cat intrafusal muscle fibres. J. Anat. (Lond.), 119: 199. Bessou, P. et PagBs, B. (1973) Nature des fibres musculaires fusales activbes par les axones fusimoteurs uniques statiques ou dynamiques chez le chat. C.R. Acad. Sci. (Paris), 277: 89-91. Boyd, I.A. (1966) The behaviour of isolated mammalian muscle spindles with intact innervation. J. Physiol. (Lond.), 186: 109-1lOP. Boyd, I.A. (1976a) Time course of activity in intrafusal fibres in isolated cat muscle spindles. J. Physiol. (Lond.), 254: 23-24P. Boyd, I.A. (1976b) The mechanical properties of dynamic nuclear bag fibres, static nuclear bag fibres and nuclear chain fibres in isolated cat muscle spindles. Progr. Brain Res., 44: 33-50. Boyd, LA. and Ward, J. (1975) Motor control of nuclear bag and nuclear chain intrafusal fibres in isolated living muscle spindles from the cat. J. Physiol. (Lond.), 244: 83-112. Boyd, I.A., Gladden, M.H., McWilliam, P.N. and Ward, J. (1975a) Study of fl innervation and of 0 and y control of isolated muscle spindles in the cat hind limb. J. Anat. (Lond.), 119: 198. Boyd, I.A., Gladden, M.H., McWilliam, P.N. and Ward, J. (1975b) ‘Static’ and ‘dynamic’ nuclear bag fibres in isolated cat muscle spindles. J. Physiol. (Lond.), 250: 11-12P. Cooper, S. and Gladden, M.H. (1974) Elastic fibres and reticulin of mammalian muscle spindles and their functional significance. Quart. J. exp. Physiol., 59: 367-385. Kidd, G.L. and KuEera, J. (1969) The excitation by suxamethonium of non-proprioceptive afferents from caudal muscles of the rat. Experientia (Basel), 25: 158-160. Matthews, P.B.C. (1964) Muscle spindles and their motor control. Physiol. Rev., 44: 219-288. Matthews, P.B.C. (1972) Mammalian Muscle Receptors and their Central Actions, Edward Arnold, London. Ovalle, W.K. and Smith, R.S. (1972) Histochemical identification of three types of intrafusal

59 muscle fibres in the cat and monkey based on the myosin ATPase reaction. Canad. J. Physiol. Pharmacol., 50: 195-202. pack, P.M.H. and Westbury, D.R. (1966) The effects of suxamethonium and acetylcholine on the behaviour of cat muscle spindles during dynamic stretching and during fusimotor stimulation. J. Physiol. (Lond.), 186: 698-713. Smith, R.S. (1966) Properties of intrafusal fibres. In Muscular A f f e r e n t s and Motor Control, R. Granit (Ed.), Almquist and Wiksell, Stockholm, pp. 69-80.

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Ultrastructural Observations of a Muscle Spindle in the Region of a Contraction Site of a Dynamic y Axon ELINOH ARBUTHNOTT, I.A. BOYD and MARGARET H. GLADDEN Instilute of Physiology, Uniuersity of Glasgow, Glasgow (Great Britain)

A region of focal contraction of a nuclear bag fibre produced by a dynamic y axon was found during a study of spindles isolated from the abductor digiti quinti medius muscle of the cat (Boyd et al., 1973). The spindle was fixed and stained for electron microscopy by the method of Hayat and Giaquinta (1970). The same region was then identified in the Araldite block using landmarks (axons and blood vessels) as a guide and was serially sectioned transversely for 1 mm. Ultrathin sections were cut alternately with 4 pm thick sections which were re-embedded and ultrathin sections cut as necessary. One large intrafusal fibre (Fig. 1, fibre I), almost certainly a nuclear bag fibre though it has not yet been traced t o the spindle equator, had a motor nerve ending in two parts each of which had definite, though irregular, junctional folds at some point (Fig. Za). This corresponds t o the “p2 plate” described by Barker e t al. (1970). Towards the edges of this motor nerve ending there were no junctional folds, Thus, t o establish that a motor ending is a p2 plate it is necessary t o section it from end t o end. This particular intrafusal fibre received no other motor terminations in the 1 mm length so far studied. A second, large intrafusal fibre (Fig. 1,fibre 2) presumed t o be a nuclear bag fibre, also, had 3 motor terminations in this region which had no, or only very rudimentary, junctional folds at any point. A transverse section of the largest of these terminations is shown in Fig. 2b. These endings correspond to the synaptic contacts of the “trail endings”of Barker e t al. (1970). All the intrafusal fibres in this region, other than the one operated by the dynamic y axon, were operated by a static y axon, and it is known that static y axons end in trails (Barker et al., 1973). Thus, it may be assumed that fibre 2 was operated by the static y axon. It should be noted that one of the trail terminations was almost as large as the p2 plate. Thus, the presence or absence and nature of junctional folds at some point in the ending is the distinguishing feature between pz plate and trail, and not the size of the synaptic contact. Four small intrafusal fibres (Fig. 1,fibres 4, 5, 6, 7 ) ending in this particular region were almost certainly nuclear chain fibres. One of these received 4 small synaptic contacts with negligible junctional folding, presumably trail terminations of the static y axon. Fibre 3 (Fig. 1)was of intermediate size, and may have been either a third nuclear bag fibre or an unusually long nuclear chain fibre. It received 6 synaptic

62 0 pm

I

250 p m I

500 p m I

750 pm I

1000 p m I

Fig. 1. The distribution and ultrastructural type of m otor nerve endings in 1 mm of the pole of a muscle spindle where focal contraction of one nuclear bag fibre was produced by a dynamic y axon, and all t h e other intrafusal fibres were operated by a static y axon. Isolated spindle fixed for electron microscopy and serially sectioned transversely following experimental study of its fusimotor control (Boyd e t al., 1973). Zero of scale approximately 2.5 m m from spindle equator.

contacts, all with negligible junctional folding, presumably trail terminations of the static y axon. Thus, the static y axon which operated all the intrafusal fibres in this region, save one large fibre, had trail terminations on two of the larger fibres (fibres 2, 3) and one of the small ones (fibre 4).The remaining large fibre (fibre 1)must have been the one supplied by the dynamic y axon which operated one large fibre only. It follows that the pz plate was the termination of the dynamic y axon. To be certain of this it would be necessary to mark the “dynamic nuclear bag fibre” with dye after it had been identified electrophysiologically, which was done in subsequent experiments for which ultrastructural data are not yet available. Alternatively, if the spindle was sectioned further towards the pole, the dynamic and static nuclear bag fibres could be distinguished by the distribution of their supporting elastic fibres. This could not be done in the region sectioned so far which was within the spindle capsule. It should be noted that none of the intrafusal fibres in this region of the spindle received both plate and trail innervation which fits with the electro-

Fig. 2. Ultrastructure of motor nerve endings in an isolated spindle whose fusimotor control had been studied previously. a: transverse section of fibre 1at 325 pm (see Fig. l ) , showing a region of the “p2 plate” with irregular junctional folding (arrows). b: transverse section of fibre 2 at 216 pm (see Fig. 1)showing part of one synaptic contact of the “trail ending”. Little or no junctional folding is evident.

64 physiological finding that individual intrafusal fibres are operated either by dynamic y axons or by static y axons, but qot by both (Bessou and Pagss, 1973; Boyd et al., 1973, 1975). It cannot be said with certainty that fibre 1received no trail terminations at any point until it has been sectioned throughout its length. However, the large fibre operated by the dynamic y axon did not contract in any other region when either the dynamic y axon or the static y axon was stimulated which suggests that this was the only region in which it received motor innemation. SUMMARY

A 1 mm region of a muscle spindle, in which a dynamic y axon had been observed to produce focal contraction of one nuclear bag fibre, and in which all other intrafusal fibres were operated by a static y axon, was studied by electron microscopy. Trail endings with negligible junctional folding, presumably the terminations of the static y axon, were found on one large fibre, one intermediate fibre and one small fibre. A p2 plate with definite irregular junctional folding, presumably the termination of the dynamic y axon, was found on the other large fibre, which was free of trail terminations. ACKNOWLEDGEMENTS We wish t o thank Mr. W. Biddlecombe and Mr. D. Mackay for valuable technological assistance and the Muscular Dystrophy Group of Great Britain for financial support. REFERENCES, Barker, D., Stacey, M.J. and Adal, M.N. (1970) Fusimotor innervation in the cat. Phil. Truns. B, 258: 315-346. Barker, D., Emonet-DCnand, F., Laporte, Y., Proske, U. and Stacey, M.J. (1973) Morphological identification and intrafusal distribution of the endings of static fusimotor axons in t h e cat. J. Physiol. (I,ond.), 230: 405-427. Bessou, P. e t Pages, B. (1973) Nature des fibres musculaires fusales activPes par des axones fusimoteurs uniques statiques ou dynamiques chez le chat. C.R. Acud. Sci. (Puris), 277: 89-91. Boyd, I.A., Gladden, M.H., McWilliam, P.N. and Ward, J. (1973) Static a nd dynamic fusimotor action in isolated cat muscle spindles with intact nerve and blood supply. J. Physiol. (Lond.), 230: 29-30P. Boyd, LA., Gladden, M.H., McWilliam, P.N. and Ward, J. (1975) ‘Static’ and ‘dynamic’ nuclear bag fibres in isolated cat muscle spindles. J. Physiol. (Lond.), 2 5 0 : 11-12P. Hayat, M.A. and Giaquinta, R. (1970) Rapid fixation and embedding for electron microscopy. Tissue and Cell, 2: 191-195.

DISCUSSION BARKER: I agree with the correlations between your dynamic bag fibre and our bag,, and your static bag fibre and our bagz. Your observation o n the kinking of the intrafusal fibres

65 links u p with o u r incidental observations t ha t when we throw 60 p m lengths of spindles into glutaraldehydc in electron microscopic processing, so that shortening is not opposed, the bagl fibre never fully contracts and this is the one which you find is less easily kinked, so that ties up. In which region were y o u r observations on the effect of acetylcholine made? GLADDEN: I’m sorry I didn’t make it clear: I look a t the end of the fluid space. BARKER: So you are in what we would call region A? GLADDEN: Yes. I look a t this region particularly because it’s a part where the bag fibres are not well tied together with connective tissue and so they are able t o kink in fact. In other parts they arc so well tied together they couldn’t possibly kink. BARKER: I would like t o make o n e point about the result of Dr. Arbuthnott’s serial reconstruction. When Ada1 and I first saw fusimotor endings in electron microscopy some had wide folds and some had smooth junctions and their position seemed t o indicate that the former was P, and the latter trail. Now we’ve no doubt a t all, unfortunately, tha t folding is n o t diagnostic of ending type. This is particularly well illustrated by one experiment in which we were collaborating with Bessou and Pages. A chain and a bag fibre were impaled and were both activated by the same static y axon. The fibres were labelled iontophoretically with Procion yellow dye. The static y axon had motor terminals on both muscle fibres, one terminal was folded and the other smooth. We feel tha t folding is possibly related to fibre type and distance from the equator, but that you can’t find folding and say it’s Pz. However, the folding of t h e beta-innervated PI-plate is difrerent. GLADDEN: I should have said that parts of the folded ending found by Dr. Arbuthnott did not in fact have folds and o n e needed t o section right through the ending to be sure tha t it was different. BARKER: That is of course another trap. ELDRED: You wrote that many elastic fibres turn off into the capsule as the intrafusal fibres go through the capsule. It is possible that the elastic fibres are concerned with t h e reaction of the capsule rather than t h e reaction of t h e intrafusal fibres? GLADDEN: This distribution of t h e elastic fibres around the intrafusal fibres within the fluid space is quite complex too, so that may not be completely the answer. I find it rather unsatisfactory to think about these elastic fibre patterns because y o u can fit lots of explanations t o them and I can’t think of a way of deciding which explanation is the right one. ELDRED: Does the capsule act as a barrier to the access of acetylcholine? GLADDEN: 1 think that t h at is t h e reason why there is a lag between the contraction of these t w o types of bag fibre; it takes some time for the concentration of acetylcholine within the fluid space to build up. ELDRED: I was thinking that this was relevant t o Dr. Kidd’s experiments o n the capsule acting as a barrier to potassium ions.

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Studies of the Histochemistry, Ultrastructure, Motor Innervation, and Regeneration of Mammalian Intrafusal Muscle Fibres DAVID BARKER, ROBERT W. BANKS, DAVID W. HARKER, ALICE MILBURN and MICHAEL J. STACEY Department of Zoology, Universiiy of Durham, Durham DHI 3LE (Great Britain)

INTRODUCTION One of the main findings recognized during the Durham Symposium on Muscle Spindles (April 1974) was that mammalian limb spindles possess two types of nuclear bag muscle fibre distinguished by differences in length, diameter, distribution of associated elastic fibres, histochemical profile, ultrastructure, and development. There was some doubt as to how t o classify the fibres into two types, and how they should be named, since the histochemical and ultrastructural observations reported by various workers (see review by Barker, 1974) had been made on separate preparations of different spindles, so that to some extent their correlation was a matter for conjecture. It was agreed that a find decision on naming the two types should await the correlation of histochemical and ultrastructural characteristics in one and the same spindle, preferably in a number of different species (Barker and Laporte, 1975). We begin by summarizing the progress we have made towards this end, and then give some account of other work that we have been engaged in. This includes work on cat fusimotor innervation, in collaboration with our colleagues in Paris and Toulouse, and a study of the degeneration and regeneration of rat spindles following the administration of the local anaesthetic bupivacaine.

HISTOCHEMICAL/ULTRASTRUCTURAL CORRELATIONS We found that a direct comparison between the histochemical profile and ultrastructure of an intrafusal muscle fibre could be made by cutting frozen serial transverse sections in batches at about 15 pm alternating with much thicker ones at about 60 pm. The thin sections could be used for the application of various histochemical techniques, while the thick ones were processed for the observation of ultrastructure in both transverse and longitudinal section. By sectioning according t o this sequence, the histochemical and ultrastructural characteristics of each type of intrafusal muscle fibre can be correlated at all levels from equator t o extreme pole as it is traced through the spindle. Our observations using this technique have so far been made on 34 spindles sampled from various hindlimb muscles of the cat, rabbit and rat. The histochemical

68 profiles examined were those of myofibrillar ATPase following alkaline preincubation (Guth and Samaha, 1970), phosphorylase (Eranko and Palkama, 1961), and glycogen (PAS method). The results show that (i) in addition t o chain fibres, all the spindles contain two types of bag fibre, usually one of each type; (ii) histochemical profiles vary along the length of individual intrafusal muscle fibres; (iii) there are regional differences in ultrastructure in bag fibres; and (iv) mistakes have been made in some previous indirect histochemical/ ultrastructural correlations concerning bag fibres such that ultrastructural and morphological properties have been ascribed t o the wrong histochemical type (Banks et al., 1975, 1976a). In this study we found it convenient to distinguish three regions between the equator and the insertion or origin of a spindle pole, namely, region A , that part of the equatorial region lying between the equator and the equatorial end of the periaxial space; region B, that part of the pole extending from the equatorial end of the periaxial space to the end of the capsule; and region C, the extracapsular part of the pole (see Fig. 1).Since the equatorial length of the periaxial space and the length of the capsule vary according to the number of sensory endings present, it follows that regions A and B are shortest in those spindles that receive a primary ending only (these also have the shortest overall length). Thus in the 8 poles of 4 such spindles from cat tenuissimus the mean distance of the equatorial end of the periaxial space (level A/B) from the equator was 238 pm (range 150-290 pm), and the mean distance of the end of the capsule (level B/C) from the equator was 1278 pm (range 1100-1850 pm). Comparable mean distances from the equator for 4 spindle poles in which region A included two secondary endings were 826 pm (range 655-990 pm) for level A/B and 2165 pm (range 1950-2470 pm) for level B/C (data from Barker, 1974, Fig. 3). Analyses of the sensory innervation of spindles in various cat hindlimb muscles (Barker, 1962; Boyd, 1962) indicate that the most common types of spindle pole are those that receive one secondary ending and those that receive none. The length of such poles is usually 4.0-4.5 mm, with region A occupying up to 0.5 mm, region B 1.5 mm, and region C 2.0-2.5 mm. Ovalle and Smith (1972) distinguished two types of bag fibre in cat and monkey spindles on the basis of their ATPase staining reactions and called them “bag,” and “bag,” fibres. We have adopted these terms, abbreviating them, as convenient, t o b l and b,. A difference of alkaline ATPase staining intensities between bag, (low) and bag, (medium or medium/high) obtains in cat, rabbit and rat spindles, being most marked in regions A and B (see Fig. 2). In region C it is less obvious owing t o an increase in staining intensity of the bag, fibres. In cat spindles the bag, fibres are medium and the chain fibres high, but in rabbit and rat spindles the ATPase profiles of the bag, and chain fibres are very similar over most of their lengths. With the phosphorylase reaction a profile pattern in rabbit spindles of low (bag,), medium (bag,), high (chains) is most evident in region By but in region C it is lost as the staining intensity of the bag, fibres rises to the same medium level as shown by the bag, fibres. In cat and rat spindles the two types of bag fibre are not clearly differentiated by this reaction. The glycogen levels of the Ijag, fibres are generally higher than those of the bag, fibres in rabbit and rat spindles, but in the cat the reverse is true except in region C where they are the same. Further information about the

69

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D M line absent wdMpresmt

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Fig. 1. On the left of the figure t h e equatorial region and pole of a typical cat spindle are schematically represented (muscle fibre width scale X 3 that used for length). The axial bundle consists of one bagl ( b l ) fibre, one bag2 (b2) fibre and 4 chain fibres (one long). Their M line condition is indicated from equator (e) to polar t i p in the regions A , B, and C. Representative transverse sections of a cat tenuissimus spindle through t h e three regions are shown on t h e right of the figure. Note dissociation of bag, fibre from the rest of the axial bundle in region A, and presence of long chain (lc) fibre in region C. ex.m.f., extrafusal muscle fibre; c, chain fibre.

70

rabbit rat cat

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spindle region

Fig. 2. Alkaline ATPase staining intensity in bag, ( b l ) and bagz ( b z ) fibres of rabbit, rat and cat spindles (indicated by symbols as shown in key) in regions A, B and C. Values are average numbers of points awarded o n a scale 0 (absent), 7 (low), 2 (medium), 3 (high) in examining transverse sections sampled from a number of levels in each region.

glycogen profiles of cat intrafusal fibres is given below in connexion with the glycogen depletion work (seep. 75); for full details of the histochemical profiles obtained with all three methods see Banks et al. (1976a). It was possible to form a fairly rapid assessment of the variation in histochemical profile along the length of an intrafusal fibre pole from the serial batches of 15 pm transverse sections. Preparing and processing the thick 60 pm sections for ultrastructural observation of course takes much longer, and our present data have been gathered from regions A, B and C sampled at various levels in the three species studied. We have also confined our observations a t this stage mainly t o variation in condition of the M line. This may be present as a single prominent line, a condition we have designated as “M”. Alternatively the M line may be absent, or present as two faint parallel lines. Since a non-M line sarcomere and one with a faint double M line may be adjacent in the same muscle fibre, both conditions are included under the designation “dM”. The chain fibres show the M condition at all levels sampled in all three species. The condition in the bag, fibres of all spindles is dM for most of region A switching t o M as they approach level A/B. The condition in cat bag, fibres is dM in region A switching t o M towards the polar end of region B; in rabbit bag, fibres a similar switch occurs in the middle of region C; and in rat bag,

71 e

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Fig. 3. Variation in M line condition of bag1 (b, ) a nd bag2 (b, ) fibres in regions A , B and C of the poles of typical rat, rabbit and cat spindles (muscle fibre width scale X 3 lhat used for length). e , equator.

fibres the condition is dM throughout (see Fig. 3). We have not yet obtained preparations that reveal the nature of the transition from a dM t o an M condition. The fact that the transition zone in the bag, fibres of cat and rabbit spindles occurs at a similar distance from the equator (1.0-1.5 mm) suggests that this may be a standard length, determined perhaps by developmental factors associated with the primary afferent. A situation thus occurs in which, owing t o the differences in average spindle length between the three species, the switch from the dM t o the M condition is intracapsular in cat spindles (region B), extracapsular in rabbit spindles, and absent in rat spindles. Micrographs illustrating the ultrastructure of cat bag, and bag, fibres at levels sampled from regions A, B and C are shown in Fig. 4a-f. When the thick sections are fixed for electron microscopy during the application of our technique, contraction cannot be opposed and the sarcomeres of the muscle fibres are greatly shortened. It is interesting t o note, however, that the sarcomeres of bag, fibres in all three species always remain longer under these conditions than those of bag, fibres (compare Fig. 4c and d), chain fibres, and extrafusal fibres. Apart from this, in regions where fibre types have the same M line condition, e.g., as in the case of bl and b2 fibres in part of region A, or all three fibre types in cat region C, there is no obvious difference in their ultrastructure. At this stage of the work our impression is that the change of M line condition in a bag fibre is accompanied by a change of other uitrastructural features. In the transition from dM t o M the mitochondria appear t o change from being small and

72

Fig. 4. Electron micrographs of representative longitudinal sections of bag fibres from regions A, B and C of cat tenuissimus spindles. I n Aa,b the condition of the M line is dM in both types of fibre; in Bc,d the condition is dM in the bag, fibre, but M in t h e bag2 fibre; in C,,f it is M in both types. In c and d the electron micrographs illustrate sections obtained using the combined histochemical/ultrastructural technique of Banks et al. (1976a); the sections illustrated in a , b , e and f a r e from muscle fixed in the normal way for electron microsCOPY.

73 scarce to being larger and more numerous; the amount of interfibrillar sarcoplasm increases; and the sarcotubular system becomes better developed. Variation in histochemical profile along the length of an intrafusal fibre in the reactions that we have examined does not appear t o be correlated with the condition of the ultrastructure. Since there is regional variation in the histochemical profiles of intrafusal muscle fibres, and in the ultrastructure of bag fibres, it is scarcely surprising that some investigators (e.g., Arendt and Asmussen, 1974) have recognized more than three histochemical types, and that others (e.g., Barker et al., 197213) have made erroneous indirect histochemical/ultrastructural correlations. In their ultrastructural study of dog spindles Banker and Girvin (1971) did not distinguish between two types of bag fibre, but they were on the right track when they observed that the bag fibres had M lines in the poles and lost them in the equatorial region. Though, generally speaking, it may be said that there is a hierarchy of length and diameter among the three types of fibre in the sequence bag,-bagl-chain, these characteristics are not an entirely reliable guide as to fibre type. In cat spindles bag, fibres are usually longer than bag, fibres, but in rabbit and rat spindles they are usually about the same length. Also in some cat spindles the length of one of the chain fibres may be similar to, or even longer than, the bag, fibre (see p. 75). Bag, fibres are thicker than bag, fibres, and both types of bag fibre are thicker than chain fibres except in rabbit spindle poles where the diameters of bag, fibres and chain fibres are not significantly different (Banks and James, 1975). A feature that is useful in helping t o identify bag fibres in cat spindles is that in their course through the equatorial region the bag, fibre generally lies somewhat apart from the rest of the axial bundle, whereas the bag, fibre is closely associated with the chain fibres (see Figs. 1, 8). This may be true of all mammalian spindles that have four or more chain fibres. During rat development the three types of intrafusal fibre arise sequentially in the order bag,, bag,, chain. Bag, and chain fibres each develop in association with the older bag, fibre, presumably separating from it in cat spindles t o a greater (bag, ) or lesser (chains) extent after the fusion of their constituent myoblasts. The main characteristics of the three types of intrafusal muscle fibre in cat, rabbit and rat spindles may be summarized as follows.

Bag, fibres Diameter: medium, similar t o chains in rabbit, thicker than chains in cat, rat. Length: usually shorter than b2 fibres in cat, usually same length as b, fibres in rabbit, rat. Deuelopment: second fibre formed, usually dissociates from b2 fibres and chains equatorially in cat. Alkaline ATPase profile: low. M line condition: rat, dM; rabbit, dM switching t o M in extracapsular pole (region C); cat, dM switching t o M in capsule sleeve (region B). Bag, fibres Diameter: always the thickest.

74 Length: usually longest in cat, usually same length as bl fibres in rabbit, rat. Deuelopment: first fibre formed, remains closely associated with chain fibres equatorially in cat. Alkaline ATPase profile: medium in cat, medium/high similar t o chains in rabbit, rat. M line condition: dM for short stretch adjacent t o nuclear bag, otherwise M for rest of length. Chain fibres Diameter: thinnest in cat, rat, similar to bl fibres in rabbit. Length: shortest, but some chains in cat may be as long as b l fibres or longer. Deuelopment: last fibres t o be formed, remain closely associated with b2 fibre equatorially in cat. Alkaline ATPase profile: high. M line condition: M line present throughout length.

CAT FUSIMOTOR INNERVATION In collaboration with Laporte and his colleagues we have analysed the distribution of static and dynamic y axons t o cat tenuissimus spindles using Edstrom and Kugelberg’s (1968) glycogen depletion technique (Barker et al., 1974, 1976). Our study differs in a number of respects from a similar one made by Brown and Butler (1973): the muscle portions containing the activated spindles were quick-frozen and then fixed in absolute ethanol during freeze-substitution in order to avoid the “streaming” of glycogen granules; sampling of y static axons was not restricted to those of relatively fast conduction velocity; and the two types of bag fibre were taken into account in the analysis. In each experiment a single y axon supplying tenuissimus spindles was prepared and its function, static or dynamic, determined by measuring the dynamic index after 2-3 mm ramp stretches applied during repetitive stimulation a t 100/sec. Glycogen depletion in the muscle fibres innervated by the axon was obtained by repetitively stimulating it during several periods of blood occlusion. The activated spindles were located t o within 1-3 mm by gently pulling on the connective tissue near the edge of the stretched muscle and observing the change in frequency of the primary ending discharge. After freeze-substitution the muscle portions were embedded in Paramat and serial transverse 10 pm sections cut, stained for glycogen (PAS method), and examined for depletion. Serial reconstructions were made of each experimental spindle in order to ascertain the levels of glycogen and zones of glycogen depletion in each intrafusal muscle fibre. The glycogen levels of cat tenuissimus intrafusal muscle fibres show some regional variation. In bag, fibres the level is medium in regions A and B, though it may drop to medium/low or low over short stretches. In region C the level is medium rising to medium/high, or occasionally high, towards the polar extremity. The level in bag, fibres is mainly medium/high; there may be short medium

75 stretches in regions A and B, and there is usually a rise t o a high level towards the polar extremity. The chain fibres have the most glycogen, the level being generally high throughout, though there may be occasional stretches where the level drops t o medium/high or medium/low. Long chain fibres are particularly liable to show this variation. There is a gradation of length among cat tenuissimus chain fibres such that the origins or insertions of some lie inside the capsule whereas those of others lie outside. In a sample of 369 chain fibres measured in this study 40.6% of the origins/insertions were intracapsular, 59.3% extracapsular (see Fig. 5). The percentages of origins/insertions lying 0.5 mm or more and 1.0 mm or more beyond the end of the capsule were 30.3 and 7.9, respectively. Long chain fibres beginning or ending 1.0 mm or more beyond the end of the capsule were present in 25 (29.4%)of 85 spindle poles; usually only one was present, occasionally two, rarely three. Control muscles were examined in order t o ascertain whether the glycogen profiles were affected by a regime of blood occlusion without nerve stimulation, or by the standard experimental procedure from which both blood occlusion and nerve stimulation had been omitted. The controls were normal; they lacked any blanched zones such as occur with glycogen depletion. The stimulation of 8 single static y axons (conduction velocity range 19.045.0 m/sec) produced zones of glycogen depletion in 27 whole spindles and 5 half spindles (i.e., spindles cut in two when the muscle portion was excised so as to leave only one complete pole). The stimulation of 4 dynamic y axons (conduction velocity range 23-46 m/sec) produced zones of glycogen depletion in 16 whole spindles and one half spindle. The following results emerged.

region 6

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(150 fibres)

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Fig. 5. Histogram of the origin/insertion points of 369 chain fibre poles belonging to cat tenuissimus spindles (35 complete spindles, 1 3 complete half spindles) reconstructed from serial 10 pm transverse paraffin sections stained for glycogen (PAS method). Transition between region B (intracapsular) and region C (extracapsular) at level B/C indicated by arrowhead.

Static yaxons. (i) Analyses of 27 spindles showed that 1 3 (48.2%)had bag and chain depletions, about twice as many as those in which depletion was restricted to bag fibres only (8, or 29.6%)or to chain fibres only (6,or 22.2%). (ii) Almost as many bag fibres were depleted as chains, and among the bag fibres, both types were equally involved. (iii) Of the 8 spindles in which depletion was restricted to bag fibres, three involved bag, fibres only, one involved a bag, fibre only, and 4 involved both types. It must be noted, however, that in 5 of these spindles (including all those with bag, only depletions) there were patches in some of the chain fibres where the glycogen level dropped from high t o medium. Although this is a feature of the normal profile the possibility cannot be excluded that some of these patches represent zones of partial depletion. (iv) The same static axon usually differed in its pattern of distribution t o each of the spindles it supplied. (v) Bag-fibre involvement was restricted to bag, fibres on stimulating axons with conduction velocities slower than 2 5 m/sec. Dynamic y axons. Analyses of 1 6 spindles showed that depletion was almost exclusively restricted t o bag, fibres. In only 3 spindles were other types of fibre involved; in one this was a bag, fibre, in two it was a long chain fibre. Of the 21 spindle poles in which depletion occurred, the fibre types involved were bag, in 20, bag, fibres in 2, and long chain fibres in 2. The results obtained in the experiments with static y axons agree well with those obtained from a study of the distribution of static y axons t o tenuissimus spindles in which all other motor axons had degenerated (Barker et al., 1973). In that study silver staining was used and a detailed histological analysis made of 30 spindles innervated by 6 static y axons (conduction velocity range 35-48 m/sec). In terms of trail endings supplied t o bag (b, type not specified) and chain (c) fibres the static axons innervated 37 spindle poles as follows: bc poles, 48.7% (19 bc, 1 bbc); b only poles, 24.3% (8 b, 1bb); c only poles, 27.0% (10). This compares with the distribution of blanched zones among the bag and chain fibres of the 44 spindle poles activated by static axons in the glycogen depletion experiments, as follows: bc poles, 41% (15 bc, 3 bbc); b only poles, 34% (12 b, 3 bb); c only poles, 25% (11). The lengths of the zones depleted of glycogen by static y axons varied from 0.1 mm to about 1.5 mm in all three fibre types, the mean length being around 0.5 mm. In the majority of instances the depletion of a fibre was restricted t o a single zone in one pole. Histograms of the distances of the centres of the depleted zones from the equator for each fibre type are shown in Fig. 6 a - c in relation to mean distances of levels A/B and B/C (broken vertical lines) from the equator in each sample of spindle poles concerned. In each fibre type most of the zone centres are seen to lie in region B, the mean distance for all 26 bag, fibre zones being 1548 pm, all 24 bag, fibre zones 1395 pm, and all 51 chain zones 931 pm; for all fibres the mean was 1111 pm. The histograms compare well with the location of trail endings as seen in silver preparations. In 21 silverstained tenuissimus spindle poles the mean distance of the centre of trail-ending areas lay 1101 pm from the equator, the nearest and furthest limits of the areas being at mean distances of 753 pm and 1891 pm, respectively (range 4902330 pm) (data from Barker, 1974, Fig. 3; see also Barker et al., 1970). A similar histogram of the distances from the equator of the centres of the 25 zones depleted of glycogen in bag, fibres by dynamic y axons is shown in

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a-d : histograms showing location o f centres o f blanched zones i n glycogen depletion experiments. e : histogram showing location o f centres o f p 2 plates i n s i l v e r preparations.

Fig. 6. Histograms of the distances from the spindle equator o f the centres of the zones depleted of glycogen in each type of intrafusal muscle fibre o n stimulating single static y axons (a-c) and single dynamic y axons ( d ) (data from Barker e t al., 1976). I n a - c the horizontal line above each histogram represents the range of the limits o f trail-ending areas as measured in 21 silver-stained spindle poles from cat tenuissimus; arrowheads above the line indicate the mean distances from the equator of the nearest and furthest limits of the trailending areas (data from Barker, 1974, Fig. 3). Broad arrowhead below the abscissa of each histogram in a-d indicates the mean distance from the equator of the centres of the depleted zones in the tibre type concerned (excluding the hag, and chain fibres in d). Histogram e shows the distances from the equator of the centres of 35 p2 plates as measured in 1 9 silverstained spindle poles from cat tenuissimus (data from Barker, 1 9 7 4 , Fig. 3); broad arrowhead below the abscissa indicates mean distance. Spindle pole regions A, B and C are shown in each histogram with the levels A/B and B/C indicated by broken vertical lines a t their mean distance from t h e equator for each sample of fibre poles (a-d) or spindle poles (e) concerned.

78 Fig. 6d; the 5 zone centres in the depleted bag, and long chain fibres are included, distinguished by different shading. It will be noted that the zone centres do not lie mostly in region B, as in the case of the static bag, depletions (Fig. 6a), but are about equally distributed through regions B and C and extend over a greater polar length. Their mean distance from the equator also lies further along the pole being extracapsular at 1732 pm. This distribution compares well with that of p2 plates as seen in silver preparations. The characteristic location of these is over a polar length of about 1 mm that includes the transition from regions B and C. Some may occur towards the extreme end of the pole, and others may lie closer in towards the equator, though they do not encroach into region A as do trail endings. Some of these points are illustrated in Fig. 6e, a histogram of the distances from the equator of 35 p, plates as measured in 19 silver-stained tenuissimus spindle poles (data from Barker, 1974, Fig. 3). Owing to the hazards of teasing, the majority of the spindle poles were cut at a level 2.5-3.0 mm from the equator so that some p2 plates with an extreme polar location may have been lost. This probably accounts for the mean distance of the p2 plate centres being extracapsular at 1435 pm, i.e., slightly nearer t o the equator than the equivalent mean of the zone centres of dynamically depleted bag, fibres. In a sample of 50 p, plates examined in silver preparations of spindles teased from cat peroneal muscles Barker et al. (1970) found that “90% were located on bag fibres, 10% on chain. In two instances a p2 fibre was seen t o branch so as to supply one plate to a bag fibre and one t o a chain fibre. One p2 plate was seen to terminate on a bag fibre as well as on an adjacent chain fibre; another spread its terminals over two bag fibres” (p. 331). These observations, taken in conjunction with the distribution and location of the zones depleted of glycogen by dynamic y axons, strongly indicate the probability that such axons terminate in p2 plates. Further evidence of this has come from experiments in which a local contraction in a bag fibre produced by stimulating a dynamic y axon is observed, photographed, and precisely located prior t o processing for examination with electron microscopy (Banks et al., 1976b). The ultrastructure of the terminal present in the activated region is then compared with the ultrastructure of p2 plates previously located and photographed in spindles stained with methylene blue (attempts to stain the dynamically activated spindle itself with methylene blue have so far failed). Longitudinal sections through motor endings found at the site of the observed local contraction produced by stimulating a dynamic y axon show terminals whose length and ultrastructure are very similar to those of p2 plates previously stained with methylene blue (see Fig. 7). The postsynaptic membrane in such plates is mostly smooth and not thrown into wide, shallow folds as described by Barker et al. (1970). Observations of the ultrastructure of y fusimotor endings that we have made since then make it clear that the presence or absence of postsynaptic folding is not a reliable criterion for distinguishing between the terminals of trail endings and p2 plates. It may be that postsynaptic folding is related to muscle fibre type and distance from the equator. By observing the regional M line condition, diameter, and the equatorial relationship with chain fibres, type of bag fibre can confidently be identified in

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Fig. 7 . a-c: light and electron micrographs of pz plates in cat tenuissimus spindles. a: methylene blue preparation of a pz plate in region B. b : electron micrograph of a loiigitudinal section through part of the p2 plate shown in a. Vacuolated appearance of the axon terminals (indicated by arrows) and the bag, muscle fibre is due to damage caused by methylene blue staining. c : electron micrograph of an oblique longitudinal section through part of a m o t o r ending located o n a bag fibre (probably bag, type) a t the site of observed local contraction (2.05 m m from t h e equator) produced by stimulating a dynamic y axon. Position, length and ultrastructure of this ending suggest that it is a pz plate. f.n., fibroblast nucleus; s.p.n., sole-plate nucleus. (From work in progress by Barker, Bessou, Pages and Stacey.)

ultrastructural preparations providing all three criteria are satisfied. On this basis we have identified two p, plates stained with methylene blue, and subsequently examined ultrastructurally, as being positively located on a bag, fibre in one case, and doubtfully so in another (two criteria only satisfied). Of two motor endings located at the sites of local contractions produced by stimulating dynamic y axons and identified ultrastructurally as p, plates, one was positively identified as being situated on a bag, fibre, the other doubtfully considered t o be on a bag, fibre. Using these criteria we have checked on bag fibre type in other work in which muscle fibres were marked by the electrophoretic injection of the fluorescent dye Procion yellow and examined ultrastructurally after recording their membrane responses during fusimotor activation (Barker et al., 1972a, 1975a). In 6 experiments in which dynamic y axons were stimulated a bag, fibre was

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marked in 5 instances, and a chain fibre in a sixth. The fibres marked in these experiments are, of course, not necessarily the only ones activated by the fusimotor stimulation. There is thus strong evidence that the dynamic response t o y fusimotor stimulation in cat spindles is mainly produced by y axons terminating in p, plates on bag, fibres. However, the p, innervation is not exclusively restricted t o bag, fibres, and the evidence from glycogen depletion experiments shows that, though a bag, fibre is always involved, a bag, or a chain fibre may also occasionally participate. The situation with respect to dynamic /3 axons is very similar. Histological evidence indicates that the fusimotor collaterals of /3 axons terminate as p, plates on bag fibres (75%) and chain fibres (25%)(Barker et al., 1970). Glycogen depletion experiments in which the sites of termination of 3 dynamic /3 axons were studied in 1 2 cat tenuissimus spindles (Barker et al., 1975b) showed that depletion was restricted t o bag, fibres in 9 spindles, but that in two spindles a bag, fibre was also depleted, and in one spindle depletion also occurred in one bag, fibre and three chain fibres. Boyd et al. (1975) maintain that all cat spindles contain two functionally distinct types of bag fibre; that those controlled by dynamic axons are never operated by static axons, and vice versa; and that chain fibres are always operated by static axons (see also Boyd and Ward, 1975). They therefore propose that the two bag fibre types be designated “dynamic” nuclear bag fibres and “static” nuclear bag fibres. It is difficult t o accept this concept in view of our own findings. We cannot regard bag, fibres as being operated solely by dynamic axons since the evidence from glycogen depletion experiments shows that they are activated by static axons as often as bag, fibres. Moreover bag fibre involvement is restricted to bag, fibres on stimulating static y axons with conduction velocities slower than 25 m/sec. Nor can we regard bag, fibres or chain fibres as being activated solely by static axons, since on occasion either or both these fibre types may be activated by dynamic axons, y or /3. Nevertheless we must take account of an observation made by Bessou and PagGs (1975), which appears t o support the view that one type of bag fibre is selectively operated by dynamic axons. They found that all dynamic y axons activate bag fibres, but not the same bag fibres that are activated by static y axons. Thus when a dynamic y axon and a static y axon supplying the same spindle were stimulated, the dynamic axon was observed t o activate one of the bag fibres where the static axon activated the other, often together with one or more chain fibres. While acknowledging that some spindles may well have this pattern of motor innervation, we feel that the activation of bag, fibres by static y axons cannot be ruled out on the basis of this observation for the following reasons. (i) The results of the static glycogen depletion experiments show that most of the centres of the depleted zones in bag, fibres lie in regions A and B; 28% are located less than 1 mm from the equator, 76% less than 1.5 mm. This intracapsular area is the least favourable for observing weak local contractions of the type that occur in bag fibres. Bessou and PagGs (1975) acknowledge that such contractions may be missed if chain fibres are strongly contracting simultaneously in the same pole. Furthermore in region A, and for most of region B (i.e., over a length of about 1.5 mm from the equator) the ultrastructure of bag, fibres is different (dM condition) from that found over

81 the rest of its polar length (M condition), and, except for part of region A, different from that of bag, fibres. It is not yet known whether these differences are associated with any differences in contractile properties. (ii) The conditions for observing weak local contractions in bag fibres are much better in region C (extracapsular) than in region B (intracapsular). In our collaborative work most of the contractions produced by stimulating dynamic y axons are located in region C, 3.0-4.0 mm from the equator. However, the centres of zones in bag, fibres depleted of glycogen by dynamic y activation (Fig. 6d) are distributed to regions B and C about equally. (iii) Identification of type of intrafusal muscle fibre by Bessou and PagGs (1975) was made on the basis of length and diameter. In our experience this can give rise t o error, e.g., long chain fibres can be mistaken for bag fibres and vice versa. We are thus left, as usual, with a piece of the jigsaw that just won’t fit. This time it is the bag, fibre. Is it possible that this fibre, depending on the way in which it is activated, can give either a dynamic or a static effect? Bag fibres activated by dynamic axons have not so far been observed t o be also activated by static axons, but that may be due to some of the reasons outlined in the preceding paragraph. Though much more information is required, the evidence at present available to us from glycogen depletion experiments (Fig. 6a, d), ultrastructural observation, and silver preparations (Fig. 8) suggest that bag, fibres can be innervated by either trail endings or pz plates. We do not know whether this may be true of one and the same bag, fibre, though we have observed instances of p, and trail innervation being supplied t o a bag fibre whose type cannot be reliably ascertained (see Barker et al., 1970, Fig. 41). In this connexion the glycogen depletion experiments show that the average distances from the equator of the centres of zones depleted by static axons in bag, and bag, fibres are respectively 30.4% and 30.5% of their total polar lengths, whereas the comparable figure for zones depleted by dynamic axons in bag, fibres is 46.9%. There is now a considerable amount of evidence indicating that of the two kinds of y motor endings, trail endings are involved in the static response and p2 plates in the dynamic response. That is not t o deny that either ending may on occasion be involved in the contrary response, for we must certainly allow for the probability that it is the type of muscle fibre activated that is the relevant factor rather than the type of ending. Regional differences n x y also be of crucial importance. All intrafusal contractions are local; even those associated with propagated action potentials that sometimes occur in chain fibres involve only one pole (Bessou and Laporte, 1965; Bessou and PagGs, 1972, 1975). Hence perhaps consideration should go beyond fibre type and concentrate on the regional characteristics of the contracting zone. In some tandem spindles it occasionally happens that a bag muscle fibre in one of the spindle units will continue through to the next encapsulation as a chain fibre (Barker and Cope, 1962; Eldred, as cited by Barker, 1962). From what is known of spindle development (Barker and Milburn, 1972; Landon, 1972; Milburn, 1973) it seems likely that tandem spindles with two linked capsules form as the result of two primary sensory axons making contact with a primary generation myotube (the future bag, fibre) a t two points some distance apart. If one of the primary axons were t o arrive somewhat later than the

82

Fig. 8. a: the equatorial region and part of one pole (region B) of a spindle from cat peroneus brevis. T h e innervation consists of a primary ending (P) in t h e equatorial region and a trail-ending area (tr.e.a.) in the pole. The bag1 fibre ( b l ) is dissociated equatorially from the bag2 ( b z ) and chain (c) fibres and is individuated by annulospiral terminals. Its course through the pole towards the trail-ending area is plainly visible. b : part of the trail-ending area in a, photographed at higher magnification. Arrowhead indicates a trail ramification terminating o n the bag, fibre. Teased, silver preparation (method of Barker and Ip, 1963).

other, the sequential development of successive generations of myotubes that it engendered would lag behind such development initiated by its earlier partner. In that event the bag, fibre in the former spindle unit would be starting to develop at a time when the first chain fibre was already forming in the latter. In the subsequent polar fusion of myoblasts in the intercapsular region fusion could occur between the polar extremities of the bag, fibre and a chain fibre, especially if this were a long chain fibre. It is unlikely that bag, fibres engage in such fusions since they are already well developed in the muscle primordium before the primary axons reach them. However this may be, we encountered a tandem spindle with a bag,/long chain compound fibre in one of the static glycogen depletion experiments. The proximal pole of a large spindle unit (A) consisted of a bag, fibre, a bag, fibre, and five chain fibres, one of them a long chain. This was linked to a smaller unit (B) whose distal pole consisted of a bag, fibre, a bag, fibre, and four chains. The poles of the two units in the intercapsular region were linked by the bag, fibre, which was continuous, and a compound fibre formed by fusion between the long chain fibre of the large unit and the bag, fibre of the small. In the region of fusion of chain with bag, fibre diameter increased and glycogen level dropped from high to medium. The static y axon activated four muscle fibres in this tandem spindle producing zones of glycogen depletion as follows. Unit A . Bag, fibre, one zone in region B (200 pm long), proximal pole; one in region C (810 pm), distal (intercapsular) pole. Unit B. Compound fibre, bag, portion; two intercapsular zones, one in region C (470 pm), one in region B (420 pm); and one in region B (200 pm), distal pole. Chain fibres, distal pole; one zone 330 pm long in one chain fibre, one 230 pm long in another. The total length of muscle fibre of bag, type activated was thus 2.1 mm as against a total chain length of 0.56 mm. Because of its anomalous nature the data from this spindle, activated by a static y axon with a conduction velocity of 28 m/sec, are omitted from Fig. 6. Did the type of response from its two primary endings depend on the type of motor ending involved? (In this case these were presumably all trail endings.) Or did it depend on the type of muscle fibres activated? If we accept that the latter is more probable and agree with the hypothesis that contractions of bag, fibres are exclusively associated with dynamic responses, and those of chain fibres with static responses, we find ourselves in a dilemma. In view of the large amount of bag, activation we would presumably have t o ignore the relatively slight activation of chain fibres and conclude that, had the two primary responses been individually monitored, both would have been dynamic. But in two other spindles activated by this axon only chain fibres were depleted, and we would have to regard the responses from these as static. However, this would lead t o a situation where the same axon was producing different types of response from different spindles, and all previous reports (Crowe and Matthews, 1964; Brown e t al., 1965; Bessou et al., 1966) are to the effect that a fusimotor axon has the same action on the spindles that it supplies. It could be argued that in those spindles where a fusimotor axon innervates different types of fibre (i.e., in terms of the selective hypothesis, dynamic bag fibres and static bag fibres, or dynamic bag fibres and chain fibres), similarity of action is achieved by the effect of contraction in one fibre type overriding or

84 eliminating the effect of that in another. An explanation on these lines has been put forward to account for the action of those y axons which produce a dynamic response when stimulated at low frequency, and a static response when the rate of stimulation is increased (Emonet-D6nand et al., 1972). But it is difficult to see how such overriding or elimination could operate at the same frequency of stimulation. In view of these uncertainties and contradictions it seems t o us that little further progress can be made until the function of a fusimotor axon is correlated with primary response and pattern of motor innervation in each spindle that it supplies. It would be helpful for all concerned if there could be a restatement of the functional properties of static and dynamic fusimotor axons. We need clearly defined criteria for classifying primary responses as static or dynamic, which take full account of their behaviour during different frequencies of motor stimulation and different methods of applying stretch. If there are different kinds of static and dynamic axons, let these differences be specified in precise terms. Without such a restatement it will be difficult to provide satisfying answers t o the sort of questions posed by some of the recent histological findings. For example, are there any differences between the dynamic responses of cat, rabbit, and rat spindles that might be correlated with the differences in ultrastructure of their bag, fibres? Is the nature of the primary response affected in any way if it is produced by the activation of more than one type of intrafusal muscle fibre? For example, is there any difference in the dynamic response from a cat spindle in which fusimotor activation is confined t o a bag, fibre, t o a response from one in which a bag, fibre, or a long chain fibre, is activated in addition?

EXPERIMENTS WITH BUPIVACAINE If the long-lasting local anaesthetic bupivacaine (Marcain) is applied t o the surface of a muscle, the superficial muscle fibres undergo rapid degeneration followed by complete regeneration (Sokoll et al., 1968; Benoit and Belt, 1970; Libelius et al., 1970). The action of the drug is specifically myotoxic and does not affect the motor innervation. If bupivacaine combined with hyaluronidase is injected into a muscle, the whole muscle degenerates and then regenerates (Hall-Craggs, 1974). It was not clear what happened t o the muscle spindles in such circumstances, and it seemed worthwhile t o investigate since, if the intrafusal muscle fibres were similarly affected, it would provide an experimental model for studying their development in the adult. The results of such an investigation have justified this hope and have unexpectedly provided a possible opportunity for using the model to discover the function of the nuclear bags and chains (Milburn, 1976a, b). The muscle used was adult rat peroneus longus. It was injected with 0.5 ml of bupivacaine prepared in sterile 0.9% saline solution containing 15 IU of hyaluronidase, and then processed for electron microscopy at postoperative intervals varying from 4 hr t o 21 days. Degeneration of the intrafusal muscle fibres begins within 4 hr and is advanced after 2 days. The equatorial nuclei become disorganized and pyknotic and finally disappear so that after 2 days the

85

bags and chains in most spindles are absent. The muscle fibres ultimately become reduced to tubes of often thickened basement membrane containing varying amounts of necrotic, filamentous and granular material. The satellite cells, however, survive this phase, and may become the myoblasts that participate in the subsequent regeneration. The spindle nerve supply is affected differently according t o whether it is sensory or motor. The motor innervation is little affected; the terminals simply wyihdraw from the degenerating muscle fibres and become invested by Schwann cells. But the sensory endings degenerate and the axon branches supplying them also show signs of necrosis. The capsule and the periaxial space remain normal. Three days after injection phagocytes have infiltrated the degenerating muscle fibres to remove debris, and myoblasts have appeared at their periphery within the basement membrane. Muscle fibre regeneration proceeds as the myoblasts fuse t o become myotubes. By the end of the third week three types of muscle fibre have been fully restored in the axial bundle with normal differences in size and ultrastructure, but lacking equatorial nucleation. Instead of bags or chains there is simply an occasional central nucleus lying in a thin bed of sarcoplasm. The motor innervation is restored, but the sensory endings that regenerate lack spirals and differ in ultrastructure from normal terminals. Thus the ultimate effect of bupivacaine injection is t o produce “enucleated” spindles. The regenerating primary axon appears t o lack the morphogenetic influence it possessed during development so that nuclear aggregations do not form at the site of reinnervation. The absence of nuclear bags and chains may in turn account for the failure of the regenerated terminals to develop annulospiral configurations. By reducing the strength of the bupivacaine/hyaluronidase injection it may be possible t o produce enucleation with minimal disturbance to the sensory innervation. Such spindles could provide an experimental model for the study of the part played by the nuclear bags and chains in the production of responses from primary and secondary endings. ACKNOWLEDGEMENT We wish t o thank the Medical Research Council for financial support.

REFE~ENCES Arendt, K.-W. und Asmussen, G. (1974) Enzymhistochemische Untersuchungen an Muskelspindel verschiedener Species. Anal. Anz., 136: 217-228. Banker, B.Q. and Girvin, J.P. (1971) The ultrastructural features of the mammalian muscle spindle. J. Neuropath. exp. Neurol., 30: 155-195. Banks, R.W. and James, N.T. (1975) Rabbit intrafusal muscle fibres. J. A n a t . ( L o n d . ) , 119: 193. Banks, R.W., Barker, D., Harker, D.W. and Stacey, M.J. (1975) Correlation between ultrastructure and histochemistry of mammalian intrafusal muscle fibres. J . Physiol. (Lond.), 252: 16-17P. Banks, R.W., Harker, D.W. and Stacey, M.J. (1976a) A study of mammalian intrafusal muscle fibres using a combined histochemical and ultrastructural technique. J . A n a f . ( L o n d . ) , in press.

86 Banks, R.W., Barker, D., Bessou, P., PagGs, B. and Stacey, M.J. (1976b) Serial-section analysis of cat muscle spindles following observation of the effects of stimulating dynamic fusimotor axons. J. Physiol. (Lond.), in press. Barker, D. (1962) The structure and distribution of muscle receptors. In Symposium on Muscle Receptors, D. Barker (Ed.), Hong Kong Univ. Press, Hong Kong, pp. 227-240. Barker, D. (1974) The morphology of muscle receptors. In Handbook of Sensory Physiology, Vol. I I I / 2 , C.C. Hunt (Ed.), Springer, Berlin, pp. 1-190. Barker, D. and Cope, M. (1362) The innervation of individual intrafusal muscle fibres. In Symposium on Muscle Receptors, D. Barker (Ed.), Hong Kong Univ. Press, Hong Kong, pp. 263-269. Barker, D. and Ip, M.C. (1963) A silver method for demonstrating the innervation of mammalian muscle in teased preparations. J. Physiol. (Lond.), 169: 73-74P. Barker, D. and Laporte, Y. (1975) Introduction t o Symposium on Muscle Spindles. J. Anat. (Lond.), 119: 183-185. Barker, D. and Milburn, A. (1972) Increase in number of intrafusal muscle fibres during the development of muscle spindles in the rat. J. Physiol. (Lond.), 222: 159-16OP. Barker, D., Stacey, M.J. and Adal, M.N.(1970) Fusimotor innervation in the cat. Phil. Trans. B, 258: 315-346. Barker, D., Bessou, P., Jankowska, E., PagBs, B. et Stacey, M.J. (1972a) Distribution des axones fusimoteurs statiques et dynamiques aux fibres musculaires intrafusales, chez le Chat. C.R. Acad. Sci. (Paris), 275: 2527-2530. Barker, D., Harker, D.W., Stacey, M.J. and Smith, C.R. (197213) Fusimotor innervation. In Research in Muscle Development and the Muscle Spindle, B.Q. Banker, R.J. Przybylski, J.P. Van Der Meulen and M. Victor (Eds.). Excerpta Medica, Amsterdam, pp. 227250. Barker, D., Emonet-Dbnand, F., Laporte, Y., Proske, U. and Stacey, M.J. (1973) Identification of the endings and function of cat fusimotor fibres. J. Physiot. (Lond.), 230: 40 5-4 2 7. Barker, D., Emonet-DBnand, F., Harker, D.W., Jami, L. et Laporte, Y. (1974) D6termination par la methode de la depletion glycogbnique de la distribution intrafusale des axones fusimoteurs y chez le Chat. C.R. Acad. Sci. (Paris), 279: 1595-1598. Barker, D., Bessou, P., Jankowska, E., Pag&s, B. and Stacey, M.J. (1975a) Distribution of static and dynamic y axons to cat intrafusal muscle fibres. J. Anat. (Lond.), 119: 199. Barker, D., Emonet-DBnand, F., Harker, D.W., Jami, L. and Laporte, Y. (197513) Intrafusal glycogen depletion elicited by 0 axons in cat spindles. J. Anat. (Lond.), 119: 200. Barker, D., Emonet-Dbnand, F., Harker, D.W., Jami, L. and Laporte, Y. (1976) Distribution of fusimotor axons to intrafusal muscle fibres in cat tenuissimus spindles as determined by the glycogen depletion method. J. Physiol. (Lond.), in press. Benoit, P.W. and Belt, W.D. (1970) Destruction and regeneration of skeletal muscle after treatment with a local anaesthetic, bupivacaine (Marcaine @). J. Anat. (Lond.), 107: 547-5 56. Bessou, P. e t Laporte, Y. (1965) Potentials fusoriaux provoqubs par la stimulation de fibres fusimotrices chez le Chat. C.R. Acad. Sci. (Paris), 260: 4827-4830. Bessou, P. and PagPs, B. (1972) Intracellular potentials from intrafusal muscle fibres evoked by stimulation of static and dynamic fusimotor axons in the cat. J. Physiol. (Lond.), 227: 709-727. Bessou, P. and Pagis, B. (1975) Cinematographic analysis of contractile events produced in intrafusal muscle fibres by stimulation of static and dynamic fusimotor axons. J. Physiol. (Lond.), 252: 397-427. Bessou, P., Laporte, Y. et PagBs, B. (1966) Similitude des effets (statiques ou dynamiques) exerc6s par des fibres fusimotrices uniques sur les terminaisons primaires de plusieurs fuseaux chez le Chat. J. Physiol. (Paris),58: 31-39. Boyd, I.A. (1962) The structure and innervation of the nuclear bag muscle fibre system and the nuclear chain muscle fibre system in mammalian muscle spindles. Phil. Trans. B, 245: 81-136. Boyd, I.A. and Ward, J. (1975) Motor control of nuclear bag and nuclear chain intrafusal fibres in isolated living muscle spindles from the cat. J. Physiol. (Lond.), 244: 83-112.

87 Boyd, I.A., Gladden, M.H., McWilliam, P.N. and Ward, J. (1975) ‘Static’ and ‘dynamic’ nuclear bag fibres in isolated cat muscle spindles. J. Physiol. (Lond.), 250: 11-12P. Brown, M.C. and Butler, R.G. (1973) Studies on the site of termination of static and dynamic fusimotor fibres within spindles of the tenuissimus muscle of the cat. J. Physiol. (Lond.), 233: 553-573. Brown, M.C., Crowe, A. and Matthews, P.B.C. (1965) Observations on the fusimotor fibres of the tibialis posterior muscle of the cat. J. Physiol. (Lond.), 177: 140-159. Crowe, A. and Matthews, P.B.C. (1964) Further studies of static and dynamic fusimotor fibres. J. Physiol. (Lond.), 175: 132-151. Edstrom, L. and Kugelberg, E. (1968) Histochemical composition, distribution of fibres and fatiguability of single motor units. J. Neurol. Neurosurg. Psychiat., 31 : 424-433. Emonet-DBnand, F., Joffroy, M. et Laporte, Y. (1972) Fibres fusimotrices dont I’action sur la sensibilite phasique des terminaisons primaires ddpend de leur frequence de stimulation. C.R. Acad. Sci. (Paris), 275: 89-91. Eranko, 0 . and Palkama, A . (1961) Improved localization of phosphorylase by the use of polyvinyl pyrrolidone and high substrate concentration. J, Histochem. Cytochem., 9 : 585. Guth, L. and Samaha, F.J. (1970) Procedure for the histochemical demonstration of actomyosin ATPase. Exp. Neurol., 28: 365-367. Hall-Craggs, E.C.B. (1974) Rapid degeneration and regeneration of a whole skeletal muscle following treatment with bupivacaine (Marcaine). Exp. Neurol., 43: 349-358. Landon, D.N. (1972) The fine structure of the equatorial regions of developing muscle spindles in the rat. J. Neurocytol., 1: 189-210. Libelius, R., Sonesson, B., Stamenovic, B.A. and Thesleff, S. (1970) Denervation-like changes in skeletal muscle after treatment with a local anaesthetic (Marcaine@). J. Anat. (Lond.), 106: 297-309. Milburn, A. (1973) The early development of muscle spindles in the rat. J. Cell Sci., 12: 175-195. Milburn, A. (1976a) The effect of the local anaesthetic bupivacaine on the adult rat muscle spindle. J. Physiol. (Lond.), 256: 54P. Milburn, A. (1976b) Degeneration and regeneration of muscle spindles in rat, following the administration of the local anaesthetic bupivacaine. J. Neurocytol., in press, Ovalle, W.K. and Smith, R.S. (1972) Histochemical identification of three types of intrafusal muscle fibres in the cat and monkey based on the myosin ATPase reaction. Canad. J. Physiol. Pharmacol., 50: 195-202. Sokoll, M.D., Sonesson, B. and Thesleff, S. (1968) Denervation changes produced in an innervated skeletal muscle by long-continued treatment with a local anaesthetic. Europ. J. Pharmacol., 4: 179-187.

DISCUSSION BOYD: Naturally I’m very interested in your glycogen depletion work, which seems to be the zone in which we are least well in accord. You mentioned that static gamma axons could deplete both types of nuclear bag fibre. It is important to know whether one type is depleted in one spindle and the other type in another spindle. How often are both types of bag fibre depleted? BARKER: We have two spindles where both are depleted. BOYD: And only two bag fibres in those spindles, not three? BARKER: That’s right. BOYD: It was not clear to me how you identify bagl and bagz in your glycogen preparations.

BARKER: There are t w o main ways. One is by the profile in non-depleted areas compared with normal controls. Secondly, by bonus points like dissociation in t h e equatorial region, which certainly distinguishes the bag1 very nicely, and also length and diameter, though we would never rely o n length and diameter alone. BOYD: Then I understand you cannot directly correlate your bagl -bag2 classification with electron microscopy? BARKER: That’s precisely what we’ve done. Once you’ve got yoult index, once you’ve correlated diameter with histochemical profiles and with ultrastructure, then you just subtract ultrastructure and you’ve got the other features there. You can confidently identify t h e bag fibres. BOYD: I won’t pursue that further, b u t this is a n area which we feel is debatable. MATTHEWS: How confident are you about t h e glycogen depletion technique being a mcthod of uniform sensitivity in detecting activity of intrafusal muscle fibres? The bag, fibre, by having very little glycogen to begin with, may be an unduly sensitive indicator of a very small amount of bag1 contraction. Are you perhaps sharpening a small amount of motor innervation of bag, into apparently a rather large effect? BARKER: There is regional variation in all the enzyme activities we’ve looked a t , not just with glycogen. In t h e region where bag, has the dM structure you d o get occasional low stretches in the normal glycogen profile, but we are confident from our controls, particularly in fresh-frozen material, that there is a difference between blanching f r o m activation and t h e paleness of these normal low stretches.

Studies on Muscle Spindle Primary Endings with Sinusoid a1 Stretching G.M. GOODWIN, M. HULLIGER and P.B.C. MATTHEWS University Laboratory o f Physiology, Parks Road, Oxford (Great Britain)

INTRODUCTION Sinusoidal stretching offers an attractive way of characterising the behaviour of muscle spindle afferents since for a linear system the unique frequencyresponse curve, with accompanying phase measurements, says all that there is to say about the input-output relations of the system. For most of the physiological range of movement, however, the primary spindle afferent behaves far from linearly; as shown by Matthews and Stein (1969) and by Poppele and Bowman (1970) it is very much more sensitive t o small movements than to large ones. Such non-linearity would seem greatly to increase its versatility since it allows the ending to signal small movements with a high sensitivity while permitting it also to signal the extent and time course of large movements, falling outside the linear range, without becoming saturated. Although the linear range appears small when expressed as a movement (0.1 mm total extent at 1 Hz for a spindle primary in the 5 cm long cat soleus) yet the high sensitivity of the ending leads to its firing being appreciably modulated by such excursions (say 20-40 imp/sec under above conditions) so that with lowfrequency stretching it transmits a message which may be presumed to be of significance to the central nervous system. At frequencies of stretching above 20-30 Hz the nervous signal probably ceases to be of functional significance for central action, but analysis of the form of the frequency-response relation may still help to throw light upon intrafusal mechanisms. It thus seemed of interest to investigate the effect of fusimotor stimulation on the response of spindle primary endings t o a wide range of frequencies of stretching (0.5-500 Hz), in each case appropriately restricted in amplitude so as t o fall within the linear range. A detailed description of the findings with their various controls is in course of publication (Goodwin et al., 1975); the present article concentrates on the physiological essentials. Preliminary notes on such work have already been published by ourselves (Goodwin and Matthews, 1971) and by Chen and Poppele (1973).

90

QUANTITATIVE OBSERVATIONS Fig. 1 shows, as long known, that the sensitivity of the spindle primary ending t o small-amplitude low-frequency sinusoidal stretching (here 50 pm at 2 Hz) is much greater during stimulation of a single dynamic fusimotor fibre than it is during stimulation of a static fusimotor fibre. During the dynamic stimulation the firing was modulated by about t 2 5 imp/sec making the sensitivity of the ending t o this frequency of stretching about 500 imp/sec/mm. During the static stimulation the total modulation was below about 5 implsec making the sensitivity less than 50 imp/sec/mm. Retrospectively, these responses can be recognised to have fallen more or less within the linear range, both because of the comparative modest modulation of firing and because of the absolute magnitude of stretching. (Inter alia, linearity requires that variation in stimulus amplitude produces a proportional variation in the response without change of its phase.) In contrast, the responses seen in the absence of fusimotor stimulation at the beginning of each record fall outside the linear range of response so no statement can be made about the “passive” sensitivity of the ending; it may be noted that the ending then fails to fire throughout the falling phase of the cycle so that, quite apart from the question of linearity, any fitted sine has to swing into regions of negative frequency of firing. Linear analysis of the passive behaviour necessitates further reduction in the amplitude of stretching. During dynamic fusimotor stimulation the afferent response can be seen t o be slightly “phase advanced” both on the imposed sinusoidal length change and on the resulting modulation of tension in the muscle. No estimate is possible about the phase relations during static stimulation, but when larger movements were used the response could again be seen to be somewhat phase advanced on the stimulus. NEED FOR AVERAGING Precise measurements, especially of phase, are not really possible on records such as those in Fig. 1. A degree of averaging is required of the responses t o successive cycles of stretching with the subsequent fitting of a sine curve t o the response by the method of least squares; both are now readily achieved with the use of a small laboratory computer. Moreover, in order to extend the analysis t o high frequencies of stretching it is necessary t o abandon the measurement of the frequency of firing (the response) by measuring the interval between successive spikes as in Fig. 1, and instead t o use the computer simply t o count the number of spikes occurring in each of a number of bins arranged serially throughout the cycle. Such measurement of spike density is an equally valid measure of frequency, but is one which remains applicable when the interspike interval spans several successive cycles of stretching. The stretching then manifests its action by slightly altering the fine timing of the spikes, so that more tend t o occur on the rising phase of the stretch and fewer on the falling phase, while barely affecting the interspike interval. Such measurement of spike density by averaging the response of a single ending t o successive cycles of stretch is analogous to the averaging done by the CNS (or by individual central

91

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Fig. 1. Records of instantaneous frequency to show the characteristic effect of repetitive stimulation of a dynamic fusimotor (above) and of a static fusimotor fibre (below) o n t h e response of a primary spindle ending to low-frequency small-amplitude sinusoidal stretching ( 2 Hz, 50 p m amplitude). ( F r o m Crowe and Matthews, 1 9 6 4 b . )

neurones) of the response of many similar endings t o a single cycle of stretch. Of course, computer averaging permits such analysis t o be performed for frequencies far beyond those that can be expected to be analysable by the CNS or of interest t o it, but the spindle afferent responses can then be thought of as indicating the time course and magnitude of the underlying receptor potential. FREQUENCY-RESPONSE CURVES Fig. 2 illustrates the characteristic effects of static and dynamic fusimotor stimulation on the frequency-response curves of the primary ending. For each frequency of stretching the amplitude of movement was reduced until the ending was responding linearly and the sensitivity of the ending then measured in imp/sec firing per mm of stretching. The very high values of sensitivity with high-frequency stretching are an expression of the fact that amplitudes of movement of a fraction of a pm were then sufficient t o produce an appreciable alteration in the fine timing of the spikes; they in no way imply that frequencies of firing of 105-106 imp/sec were ever observed experimentally. Such high values of sensitivity are entirely in accordance with the observation that stretching at 100-500 Hz can “drive” the primary ending to fire a spike on every cycle when the total movement is only a few micra (Brown et al., 1967). The “passive” curves largely agree with those published before for the cat (Poppele and Bowman, 1970) while showing additionally a flattening at higher

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Fig. 2. The effects of static and of dynamic fusimotor stimulation o n the sensitivity o f the primary ending to a wide range of frequencies of stretching. T h e ‘‘passive’’ frequency rcsponse curves were obtained in the absence of fusimotor stimulation. A: all threc curves obtained o n the same ending. B: results for fusimotor stimulation from t w o separate preparations with similar passive behaviour. The static fibres o f A and B differed greatly in t h e extent to which they phasically modulated the afferent discharge (i.e., in their tendency to elicit “driving”), yet their effects o n the frequency response curve are similar.

frequencies than have hitherto been explored; such flattening causes n o surprises. During dynamic fusimotor stimulation the sensitivity is slightly decreased for lower frequencies (below 30 Hz) and slightly increased for higher frequencies (above 100 Hz). The smallness of the effects is in many ways remarkable since the dynamic fibres studied had typically powerful dynamic effects when tested on the same endings with large ramp stretches, rather than with small sines. But the dynamic fusimotor fibre is not without action in the small linear range, since its stimulation partly counteracted the decrease in sensitivity which otherwise occurred when the muscle was slackened; static stimulation equally tended t o set the spindle sensitivity t o a uniform level irrespective of the length of the muscle. During static fusimotor stimulation the effects on the curve are more pronounced. At low frequencies (below 30 Hz) the sensitivity is reduced approximately 10-fold from its passive value. At high frequencies (over 100 Hz) the sensitivity is increased slightly, as with dynamic stimulation. In consequence of these opposite shifts a t the two ends of the spectrum the sensitivity in between increased much more rapidly than did that of the passive ending. For frequencies of 20-100 Hz the slope of the passive curve approximates to 102/decade and that of the active curve t o 103/decade;in other words, in this range the passive ending is responding predominantly t o the acceleration component of the stretching and the statically activated ending t o the rate of change of acceleration.

93 IRRELEVANCE OF PULSATILE EFFECTS OF STATIC STIMULATION With a muscle a t constant length, static fusimotor stimulation has two prominent effects on the primary ending. First, it increases the mean rate of firing of the ending. Second, it often produces considerable variability in the afferent interspike interval distribution, because the ending tends t o fire preferentially at certain particular times in relation to the fusimotor stimulus. This occurs, it is presumed, because the unfused contraction of the nuclear chain intrafusal muscle fibres produces a pulsatile mechanical stimulus to the afferent terminals. In some cases, it seems possible that some part of the increase in the mean rate of firing of the ending during static stimulation should be attributed t o such intermittent pulsatile stimulation of the terminals, rather than t o their steady deformation as a result of the intrafusal contraction. It might thus be suggested that the static fusimotor effects on the frequency-response curve arise solely from such pulsatile effects (cf., Chen and Poppele, 1973); the weak effects of dynamic stimulation could then be correlated with the absence of phased afferent modulation on dynamic fusimotor stimulation, since they act via the slowly contracting nuclear bag intrafusal muscle fibres. This seems an unlikely mechanism for static action, since not all static fibres produce such phased afferent modulation yet such fibres still have the typical static effect on the frequency-response curve. For example, the static action of Fig. 2B was associated with a strong fusimotor induced afferent modulation whereas that of Fig. 2A was not, as shown by the flatness of the poststimulus histogram relating the moment of afferent firing t o the time of fusimotor stimulation. A fur-

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Fig. 3. The effect o n t h e frequency response curve of adding an adventitious mechanical signal, transgressing t h e linear range, which t o some extent mimics the pulsatile effects of static fusimotor stimulation. A: adventitious signal a pure sine of 1 2 0 Hz and 1.6 p m total extent; this produced complete afferent silence for half t h e period of its own cycle histogram and increased the mean frequency of firing by about 7 imp/sec. B: adventitious signal band-limited noise of about 6 p m peak amplitude; this increased t h e mean firing rate by about 14 imp/sec. Results from t w o separate endings.

94 ther argument that pulsatile affects, when present, are not responsible for the observed static action on the curve is that the deliberate introduction of adventitious pulsatile movements produces quite a different effect on the curve, as shown in Fig. 3. In Fig. 3A the main “wanted” sinusoid at each frequency was combined with an “unwanted” pulsatile stimulus consisting of a pure sine of 120 Hz with a peak to peak movement of 1.6 pm. Fig. 3B the “wanted” sine was contaminated with random mechanical noise in the range 10 Hz-1 kHz with movements up to 6 pm (but with an uncertain spectral distribution). In both cases the adventitious signal fell outside the linear range of response of the ending and slightly increased its mean rate of firing. However, with both such disturbances the lower end of the frequency response curve was unaltered while its upper end was displaced downwards; the latter finding is in contrast to the upwards displacement of the high-frequency end of the curve with static stimulation. Clearly the mimicking of fusimotor action by the introduction of adventitious signals could be more exact, but the findings strengthen the view that pulsatile intrafusal effects play little part in mediating the typical static action on the curve which should rather be attributed to some more steady aspect of the contraction.

PHASE On using large ramp stretches, the primary ending shows itself to be “dynamically sensitive” in that it fires much more rapidly during the application of a stretch than it does at either the initial or final lengths. One widespread but arbitrary measure of such dynamic sensitivity is the dynamic index which is the fall in firing frequency between the terminal stages of the dynamic phase of a ramp stretch and the value 0.5 sec later with the muscle at the final length (Crowe and Matthews, 1964a). Both from qualitative observation and on measurement of the dynamic index it is well established that the “dynamic sensitivity” of a primary ending is enhanced during dynamic fusimotor stimulation, and diminished during static fusimotor stimulation. But it shoald be recognised that such observations on a non-linear system cannot be immediately transmuted into statements on the effect of fusimotor stimulation on a single parameter uniquely characterising “the velocity sensitivity” of the ending, as we ourselves have at times optimistically tended t o suppose (cf., Schafer, 1973; Hasan and Houk, 1975). In a linear sinusoidal analysis, any velocity and/or acceleration sensitivity of an ending shows itself firstly, as phase advance of the afferent response on the stimulus (a pure velocity receptor, for example, shows a phase advance of 90”) and, secondly, as an upward slope in the frequency-response curve with increasing frequency (sensitivity is expressed in terms of mm of stretching, yet a given amplitude of stretching corresponds to progressively higher velocities of stretching as the frequency is increased). The passive primary ending studied in the linear range satisfies both criteria and is thus properly described as being dynamically sensitive for small stretches as it is for large ones, although in so saying it should be realised that “dynamically sensitive” is a qualitative term which need not, indeed almost cannot, have precisely the same physical mean-

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Fig. 4. Scatter diagram illustrating the approximate constancy of the absolute value of the phase advance of the spindle primary to 1Hz stretching, irrespective of the type or strength or fusimotor action. Each point represents the combination of a separate fusimotor fibre with an afferent. The points at 0 on the abscissa give the mean and standard deviation for the endings when passive, @, points obtained during dynamic fusimotor stimulation; 0,during static stimulation. The abscissa indicated the strength of fusimotor action by showing the amount by which fusimotor stimulation increased the mean frequency of afferent firing when the length of the muscle was constant; this measure undervalues the action of the dynamic fibres all of which produced appreciable increases in the response t o large ramp stretches.

ing for the linear and the non-linear ranges. This apparently purely semantic matter gains immediate emphasis, because in the present experiments fusimotor action was found t o be without systematic effect on the velocity sensitivity of the spindle primary studied within its linear range for the physiologically important frequencies of 0.5-20 Hz. First, the shape of the low-frequency end of the curve was unaltered by fusimotor stimulation so that the difference between the activated and the passive ending remained approximately constant for frequencies up to 20-30 Hz. Second, there was no consistent change in the phase of the response with fusimotor action, as illustrated in Fig. 4 for 1 Hz stretching; such lack of effect on phase was confirmed by statistical testing. In other words, for low-frequency stretching fusimotor action produces an overall change in the sensitivity of the ending to stretching, but without affecting the relative contributions t o the excitation of the length, velocity and acceleration components of the stimulus. At higher frequencies of stretching, however, both kinds of fusimotor action tended to increase the steepness of the frequency response curve, especially the static fibres, and to lead to a phase advance in excess of that of the passive ending. Thus for most physiologically relevant frequencies when movement is confined t o the “linear range”, it becomes inadmissible t o speak of static and dynamic fusimotor fibres as controlling the velocity sensitivity of the primary ending. Rather, static action may be seen as reducing the “gain” of the ending some 10-fold, whereas dynamic action helps stabilise it at slightly below the passive value in the face of variations in the length of the muscle. Nor should this conclusion be dismissed on the grounds that the linear range is unduly small to matter. The high sensitivity of the ending means that small movements

produce appreciable modulation of firing, and in man powerful stretch reflex activity may be seen for comparable amplitudes of movement in terms of the muscle spindle (Joyce and Rack, 1974).

WIDER CONSIDERATIONS The contrast with the responses t o large stretches is probably more terminological than real and arises in part from our having adopted an unduly simple phraseology for describing the behaviour of a non-linear system. There is no difficulty in principle t o explaining the findings in terms of intrafusal mechanisms. Much of the non-linearity probably arises from the change in the relevant mechanical properties of the intrafusal fibres that occurs as the magnitude of the stretch is increased. When the stretch is small most of the deformation applied to the muscle spindle as a whole can probably be taken up in the equatorial region and by the “swinging” of the cross-bridges that connect the actin and myosin filaments in each sarcomere. The dynamic characteristics of the ending then probably depend upon transducer properties alone. Large movements necessitate the continual detachment and re-attachment of the cross-bridges and so introduce an entirely fresh mechanical factor, thus allowing different effects on the ending t o result from the contraction of one and the same intrafusal fibre depending upon the amplitude of stretching. The findings, however, as yet await detailed explanation. In broader functional terms, it may be noted that irrespective of the range under consideration dynamic fusimotor action favours a high sensitivity of the primary ending t o the stimulus of dynamic movement so that an appreciable signal is sent t o the CNS the moment a muscle begins t o be displaced from a pre-existing equilibrium position. This should both aid the recognition that a stretch is being applied and allow the elaboration of an appropriate counteracting muscular response; it would be of some interest t o know whether such high sensitivity is maintained during the course of a slow movement. Static action, in contrast, reduces the sensitivity of muscle feedback while also producing an appreciable increase in the frequency of afferent discharge per se, and enabling the ending t o continue firing as the muscle shortens. Fuller understanding of such matters may perhaps be gained by the continued analysis of the pattern of spindle firing in relation to the “meaning” that is put into it by the central analysing mechanisms. The fact that it may be possible for a physiologist to extract some particular facet of a stimulus by retrospective analysis of nervous signals is no guarantee that the CNS is equipped to, or interested in, performing the same computation. In any such continued study, physiological intuition and assessment of biological and higher neuronal response would appear t o require t o be married t o analytical expertise.

REFERENCES Brown, M.C., Engberg, I.E. and Matthews, P.B.C. (1967) The relative sensitivity t o vibration of muscle receptors in the cat. J. Physiol. (Lond.), 192: 773-800.

97 Chen, W.J. and Poppele, R.E. (1973) Static fusimotor effect on the sensitivity of mammalian muscle spindles. Brain Res., 57: 244-247. Crowe, A. and Matthews, P.B.C. (1964a) The effects of stimulating static and dynamic fusimotor fibres on the response t o stretching of the primary endings of muscle spindles. J. Physiol. (Lond.), 174: 109-131. Crowe, A. and Matthews, P.B.C. (1964b) Further studies of static and dynamic fusimotor fibres. J. Physiol. (Lond.), 174: 132-151. Hasan, Z. and Houk, J.C. (1975) Analysis of response properties of deefferented mammalian spindle receptors based on frequency response. J. Neurophysiol., 38 : 663-72. Goodwin, G.M. and Matthews, P.B.C. (1971) Effects of fusimotor stimulation o n the sensitivity of muscle spindle endings t o small-amplitude sinusoidal stretching. J. Physiol. (Lond.), 218: 56-58P. Goodwin, G.M., Hulliger, M. and Matthews, P.B.C. (1975) The effects of fusimotor stimulation during small-amplitude stretching on the frequency-response of the primary ending of the mammalian muscle spindle. J. Physiol. (Lond.), 253: 175-206. Joyce, G.C. and Rack, P.M.H. (1974) The effects of load and force on tremor at the normal human elbow joint. J. Physiol. (Lond.), 240: 375-396. Matthews, P.B.C. and Stein, R.B. (1969) The sensitivity of muscle spindle afferents to sinusoidal stretching. J. Physiol. (Lond.), 200: 723-743. Poppele, R.E. and Bowman, R.J. (1970) Quantitative description of linear behaviour of mammalian muscle spindles. J. Neurophysiol., 33: 59-72. Schafer, S.S. (1973) The characteristic curves of the dynamic response of primary muscle spindle endings in the absence and presence of stimulation of fusimotor fibres. Brain Res., 59: 395-399.

DISCUSSION TAKANO: You said that the noise frequency does not affect your results but you only showed us the interaction of the noise frequency when the vibration frequency was 2 and 200 Hz. What happens when the signal frequency is near the noise frequency, e.g., 60-70 Hz. * MATTHEWS: There are two points in reply. Firstly perhaps I have not made our averaging methods plain. We average either from the sinusoidal stimulus marker or from the noise marker and what we see depends on what we average on. Secondly, we have not studied as many interactions of noise and signal as we would like because this is very time consuming work. What we hope we are achieving is the generality of saying that with small stretches the muscle spindle is behaving linearly in an engineering sense, and it is a property of linearity that two signals can add together arithmetically. So if we can show linearity under one set of conditions we hope that we can transpose that t o other sets. We find a linear relationship between the size of the response and the size of the stimulus providing the modulation of afferent firing is not more than about 30%. At 50% it is often linear but at 100% it is often going non-linear. BUCHTHAL: I find it most interesting that you have gone over to the range of sinusoidal vibration which is so small that you are within the range of linearity that was used in studying the properties of muscle fibres many years ago. In the linear range perhaps you are studying the mechanical properties of the capsule, because the elastic system of the capsule will react t o linear vibration and in the non-linear range you have mostly the elastic properties of the muscle system itself, where viscosity comes into a much greater degree. Even at small stretches of a muscle fibre you get a certain amount of non-linearity.

* This question refers to Fig. 1 in Goodwin et al. (1975) which was shown as a slide, but is not presently reproduced.

98 MATTHEWS: We can only speculate here because we cannot see our muscle spindle. The muscle may become more linear in this range because when the myofilaments slide past each other the head between the actin and myosin does not have to detach itself. Some of our responses may depend on the mechanical properties of the capsule, but the fact that they change with intrafusal contraction suggests that the muscle properties are doing something. Unfortunately a movement of 1 pm is not easily observed. So it is not a question one can immediately ask of Prof. Boyd. HOUK: Two questions, firstly Henatsch and Schafer pointed out that the response of the primary ending during gamma dynamic stimulation just after the period of stretch was augmented about as much as the response during the dynamic phase of stretch. Do you agree with that, and would it be more appropriate to use different terms than dynamic and static gamma axons? Secondly, you emphasized that there was no phase change at low frequencies with gamma stimulation, but at high frequencies an interesting shift occurs which seems t o fit the frequency plots in that it occurred at the place where the sensitivity of the ending is increasing from a very low value to a very high value, so it fits within the realm of minimum phase type of prediction. Can you rationalise that in terms of the operation of the spindle as well? MATTHEWS: I think this is too speculative a subject for now, but I agree that the findings are perfectly compatible with a minimum phase system. I’ve particularly emphasized that the phase advance at high frequencies can go above 90’; therefore, when we are modelling we need more than one filter element for viscous elasticity. I think we should keep the terms static and dynamic because so much literature has been written with these terms; and I still think they are good terms because if you give a dynamic stimulus, whether it is a small sinusoid or a large stretch, the dynamic fusimotor fibre maintains a high dynamic sensitivity and the static fusimotor fibre reduces the dynamic sensitivity. Now we can play many other mathematical games on the discharge of the spindle. You were referring, I imagine, t o Schafer’s work * in which she plotted dynamic index against velocity of stretch on a log-log scale, thus showing a power function relating dynamic index to velocity and the exponent of which changes in perhaps surprising ways as you stimulate static and dynamic gamma axons. One difficulty I have with some of this work is that the data which she used are different from my own. In particular at a velocity of 100 mm/sec her values of dynamic index on stimulating static and dynamic axons appear the same, whereas I have quite different values. We have a lot of talking to do to say what is the best wag in which we can extract information from the discharge of the spindle; but we should not forget to ask what the CNS thinks of these signals, because it may extract quite different information from that which we are able to extract with a computer.

* Schafer, S.S. (1973). Bruin Res., 59: 395-399.

The Skeleto-fusimotor Innervation of Cat Muscle Spindle Y. LAPORTE and F. EMONET-DENAND Laboratoire d e Neurophysiologie, Coll&ge d e France, 75231 Paris 05 (France)

INTRODUCTION The question whether cat spindles receive branches from skeletomotor axons in addition to their specific fusimotor supply has been much debated. Recent physiological and histological observations, reviewed in this paper, show that a significant proportion of spindles are in fact innervated by collaterals from motor axons that supply extrafusal muscle fibres, i.e., by skeleto-fusimotor or p axons. The following points will be considered: (a) Rate of degeneration of p1 plates after section of the muscle nerve. (b) Experimental demonstration of p axons in large hindlimb muscles. (c) Proportion of spindles supplied by dynamic p axons in the peroneus brevis muscle. (d) Histophysiological determination of the types of muscle fibres innervated by axons, using the glycogen depletion technique. (a) RATE OF DEGENERATION OF PI PLATES AFTER SECTION OF THE MUSCLE NERVE

P I plates are spindle motor endings that are located mainly on nuclear bag intrafusal fibres and which resemble the end-plates of extrafusal muscle fibres. Barker et al. (1970) reported that after section of a muscle nerve, p1 plates and extrafusal end-plates degenerate simultaneously and faster than the other spindle motor endings, p2 plates and trail endings. Since the degeneration rate of nerve endings is apparently related to the diameter of their stem fibre, this observation suggests that axons terminating in p1 plates and axons supplying extrafusal end-plates have comparable diameters, or, in other words, that axons supplying p, plates are larger than y axons. From such degeneration experiments it is impossible to ascertain whether the axons that give p r plates are distributed exclusively to spindles ( a fusimotor innervation) or to both extrafusal and intrafusal muscle fibres (skeletofusimotor or p innervation). Haase and his colleagues (Haase and Schlegel, 1966; Haase et al., 1966) concluded from indirect evidence that spindles in some hindlimb muscles receive an a fusimotor innervation. This view is incom-

100 patible with the observations of Ellaway et al. (1972). They studied over 1800 single motor axons with conduction velocities greater than 50 m/sec in muscles whose spindles were alleged t o have 01 fusimotor innervation, but found that the stimulation of nearly all these axons activated extrafusal muscle fibres. The very few axons which did not elicit a muscle action potential had no action on the discharge of spindles and were most likely axons damaged during the dissection of the muscle nerves. Thus, if it is accepted that the fast rate of degeneration of p1 plates indicates that they are supplied by large-diameter axons, it follows that these axons must be skeleto-fusimotor rather than exclusively fusimotor. In many leg muscles the proportion of spindle poles containing one or more p1 plates exceeds 50% (Barker et al., 1970). (b) EXPERIMENTAL DEMONSTRATION OF p AXONS IN LARGE HINDLIMB MUSCLES Physiological evidence for the existence of 0 axons was provided by the observations of Bessou et al. (1963, 1965) who reported that repetitive stimulation of some motor axons to the first deep lumbrical muscle elicited both the contraction of extrafusal muscle fibres and an acceleration of primary ending discharge. This acceleration was ascribed t o the contraction of intrafusal muscle fibres for two reasons. Firstly, it reached a maximal value with a rate of stimulation well above that necessary t o elicit maximal contraction of the extrafusal motor unit. Secondly, it persisted after the selective elimination of extrafusal contraction had been achieved by the combined effects of repetitive stimulation and of small amounts of a curarizing drug. Possible excitation of Ia afferent fibres by ephaptic stimulation and the activation of primary endings by some illdefined mechanical actions of extrafusal origin could thus both be ruled out. Consequently it was concluded that the acceleration was due t o the contraction of intrafusal muscle fibres. Bessou et al. (1963, 1965) did not study the discharges which often occur before and during the rising phase of a twitch, that are especially noticeable when several a axons are stimulated together, because in this situation it is extremely difficult to appreciate the respective role of 3 possible factors: ephaptic stimulation; activation of sensory endings by extrafusal muscle fibres; and contraction of intrafusal muscle fibres (see Matthews, 1972). Very soon after the investigations of Bessou et al., Ada1 and Barker (1965) obtained incontrovertible evidence for the existence of p axons in deep lumbrical muscles. In teased muscles they traced the course of single motor axons t o their terminal branches, and found that some of them terminated on both intrafusal and extrafusal muscle fibres. Relatively little progress was made during the following years. Beta axons were found in another small muscle of the foot, the superficial lumbrical (Ellaway et al., 1971). An isolated observation was made on a tenuissimus muscle in which all motor axons except a single 0. had degenerated; spindle collaterals of this axon were seen to terminate in p1 plates (Barker et al., 1971). In addition, Brown et al. (1965) mentioned finding a few 0 axons in the tibialis posterior muscle, but there was no systematic search for p axons in large muscles. The de-

101 tection of p axons in a given muscle requires testing the action of as many as possible of its motor axons on the largest possible fraction of the spindle population. This becomes increasingly difficult as the size of the muscle augments. Several technical improvements resulted in an increased probability of finding axons in large muscles: the search was restricted to a fraction of the muscle by preserving only a small nerve branch; in each experiment, 6-12 Ia afferent, fibres were prepared and most of the 20-30 fast-conducting motor axons present in that branch could be consecutively tested; the activation of extrafusal muscle fibres was detected by electromyography because the motor units supplied by p axons are often very small. By these means, p axons were found in flexor hallucis longus, peroneus brevis, tibialis anterior and soleus muscles

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Fig. 1. Dynamic skeleto-fusimotor axon in a peroneus brevis muscle. 1, 2, 3 : action potentials of a single motor axon and of its extrafusal muscle fibres; the first recording electrode was placed under the nerve branch t o the peroneus brevis muscle; the second electrode was on the surface of the muscle. Stimulation of a ventral root filament at different strengths: 1, threshold intensity for the axon, showing the all-or-none behaviour of the nerve and muscle potentials; 2 and 3, supramaximal stimulation showing that the filament did not contain any other slower conducting axons. Conduction velocity of the axon: 56 m/sec. 4, 5: upper trace: primary ending discharge recorded with an instantaneous frequencymeter. Lower trace: muscle action potentials. When the axon was stimulated at 75/sec the discharge of the ending accelerates and the muscle action potential followed the rate of stimulation. The stimulation of the axon at 400/sec resulted in a complete block of the extrafusal neuromuscular junction. The acceleration persisted. 6, 7: responses of the primary ending to a rampand-hold stretch of 2 mm followed by a progressive return t o the original muscle length. 6, no stimulation; 7, the stimulation of the axon at 2OO/sec elicited a marked increase in the dynamic response of the ending.

102

(Emonet-Dhand et al., 1975). The conduction velocity of the p axons identified in that investigation ranged from 39 t o 92 m/sec, but for nearly all of them it was slower than 80 m/sec. Almost all of these 0 axons (72 out of 76) had a dynamic action, and this was also the case for p axons in lumbrical muscles (Bessou et al., 1965), and in tenuissimus and abductor digiti quinti muscles (McWilliam, 1975). The dynamic action was sometimes concealed by the contraction of extrafusal muscle fibres; a few axons were identified as static. A typical example of a dynamic 0 axon is illustrated by Fig. 1. (c) PROPORTION OF SPINDLES SUPPLIED BY DYNAMIC THE PERONEUS BREVIS MUSCLE

AXONS I N

The selective block of extrafusal neuromuscular junctions, which is necessary for identifying the /3 axons, can be obtained without using a curarizing drug (Emonet-Dknand and Laporte, 1974) by stimulating single motor axons a t 300-500/sec for a few seconds. Using this method, which facilitates the study of several 0 axons in the same experiment, a quantitative analysis of innervation was made in the peroneus brevis muscle in which a high incidence of p1 innervated spindles has been reported (Barker et al., 1970). In 5 muscles it was found that out of 32 spindles, 23 (72%) were supplied by at least one dynamic 0 axon. Of 1 3 5 axons supplying extrafusal muscle fibres, 24 (18%)were axons (Emonet-Dhand and Laporte, 1976). Spindles innervated by dynamic /3 axons were found t o be also supplied by dynamic y axons, which suggests that 0 and y components are associated rather than competitive in fusimotor dynamic innervation (Bessou et al., 1965; Emonet-Dbnand and Laporte, 1976). (d) HISTOPHYSIOLOGICAL DETERMINATION OF THE TYPES O F MUSCLE FIBRES INNERVATED BY p AXONS, USING THE GLYCOGEN DEPLETION TECHNIQUE The glycogen depletion technique, originally described by Edstrom and Kugelberg (1968), allows the identification of the muscle fibres innervated by single motor axons or by a group of axons. It consists of mapping, on serial transverse sections stained t o demonstrate glycogen, those extrafusal and intrafusal muscle fibres which, following prolonged contraction elicited by the stimulation of their nerve supply, have been depleted of their glycogen content. In tenuissimus and peroneus brevis muscles it was found that repetitive stimulation of single dynamic /3 axons produced depletion in nuclear bag intrafusal muscle fibres (see Fig, 2) and in extrafusal muscle fibres of the slow oxidative type (Barker et al., 1975; and unpublished observations). The intrafusal muscle fibres most frequently depleted by dynamic 0 axons were nuclear bag fibres with low glycogen content, and low phosphorylase and myofibrillar ATPase activity; such a histochemical profile suggests a slow contractile mechanism. In their preliminary report, Barker e t al. (197'5) referred t o these fibres as intermediate, but to avoid the erroneous impression that their properties are intermediate between the other type of nuclear bag fibre and chain fibres, they will

103

Fig. 2. Glycogen depletion produced in a tenuissimus spindle by prolonged repetitive stimulation of a single dynamic 0 axon. The transverse section is of paraffin-embedded material, stained to demonstrate glycogen with t h e PAS method. The bag, fibre ( b l ) is depleted of glycogen, whereas t h e bag2 fibre (bz ), and t h e chain fibres (c) retain respectively medium/ high and high levels of glycogen. Conduction velocity of the 0 axon 5 3 misec. Bar: 20 pm. (From Barker, Emonet-DBnand, Harker, Jami and Laporte, unpublished observation.)

be in future referred t o as bag, fibres, using the nomenclature of Ovalle and Smith (1972). The extrafusal muscle fibres innervated by dynamic /3 axons are of small diameter; their histochemical profile of low glycogen content, low phosphorylase and myofibrillar ATPase activity, and relatively high succinate dehydrogenase activity resembles that of bag, fibres. The glycogen depletion technique can be used in a different way. Instead of stimulating a single axon previously identified as skeleto-fusimotor, many single axons supplying extrafusal muscle fibres can be stimulated together t o determine whether some of them give collaterals t o spindles; in this case, spindle supply is evidenced by the depletion of intrafusal muscle fibres. In the tenuissimus muscle it was observed (Barker et al., unpublished data) that stimulation of motor axons with a conduction velocity higher than 50 m/sec produced depletion in some bag and chain muscle fibres. Very recently, Harker et al. (unpublished observation) applied this technique t o motor axons with conduction velocities faster than 90 m/sec, because with the present physiological methods (see paragraph b) very few /3 axons have been identified in that conduction velocity range. Experiments were carried out on 6 peroneus tertius muscles. Glycogen depletion was found in approximately 25% of the spindles; it was almost entirely restricted t o one or two of the longest chain fibres in any spindle pole. This observation (see Fig. 3), which fits with the presence of p1 plates on some chain fibres (Barker et al., 1970), shows that

104

C*

L

Fig. 3. Glycogen depletion observed in a peroneus tertius spindle after prolonged repetitive stimulation of a group of 23 motor axons with conduction velocities higher than 90 m/sec. The transverse section is of fresh-frozen material stained with the PAS method. One chain fibre (c*) is depleted of glycogen; the other chain fibres (c) remain high. The bag1 fibre ( b l ) is medium, and the bag2 fibre (b2) is medium/high in glycogen content. Bar: 20 pm. (From Harker, Jami and Laporte, unpublished observation.)

among the very fast-conducting motor axons t o that muscle there must be skeleto-fusimotor fibres. The function of these 0 axons and the type of extrafusal muscle fibres they supply have to be determined. In spite of these recent advances it is not yet possible to assess the functional importance of 0 innervation in cat spindles. Many questions remain unanswered. Are p1 plates exclusively supplied by 0 axons? The observations of Barker et al. (1970) on the rate of degeneration of motor endings are highly suggestive, but it would be most interesting to have more direct information on the endings supplied by p and y dynamic axons. What is the proportion of pinnervated spindles in various muscles? Do fast-conducting skeleto-fusimotor axons exist in many niuscles and what is their function? Are they responsible for part of the early discharge? What is the quantitative importance of 0 innervation in comparison to y innervation? It may prove difficult to obtain satisfactory answers to some of these questions but the present evidence, however incomplete, appears to justify the conclusion that the skeleto-fusimotor system in cat must play a significant role in the control of posture and movement.

REFERENCES Adal, M.N. and Barker, D. (1965) Intramuscular branching of fusimotor fibres. J. Physiol. (Lond.), 1 1 7 : 288-299.

105 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, U. and Stacey, M. (1971) Identification of the endings and function of fusimotor fibres in the cat. J. Physiol. (Lond.), 216: 5 1-5 2P. Barker, D., Emonet-DBnand, F., Harker, D., Jami, L. and Laporte, Y. (1975) Intrafusal glycogen depletion elicited by p axons in cat spindles. J. Anat. (Lond.), 119: 2OOP. Bessou, P., Emonet-DBnand, F. and Laporte, Y. (1963) Occurrence of intrafusal muscle fibre innervation by branches of slow motor fibres in the cat. Nature (Lond.), 198: 594-59 5. Bessou, P., Emonet-Denand, F. and Laporte, Y . (1965) Motor fibres innervating extrafusal and intrafusal muscle fibres in the cat. J. Physiol. (Lond.), 180: 649-672. Brown, M.C., Crowe, A. and Matthews, P.B.C. (1965) Observations on the fusimotor fibres of the tibialis posterior muscle of the cat. J. Physiol. (Lond.), 177: 140-159. Edstrom, L. and Kugelberg, E. (1968) Histochemical composition, distribution of fibres and fatiguability of single motor units. J. Neurol. Neurosurg. Psychiat., 31 : 424-433. Ellaway, P., Emonet-DBnand, F. et Joffroy, M. (1971) Mise en Bvidence d’axones squelettofusimoteurs (axones p ) dans le muscle premier lombrical superficiel du Chat. J. Phy siol. (Paris), 63: 617-623. Ellaway, P., Emonet-DBnand, F., Joffroy, M. and Laporte, Y. (1972) Lack of exclusively fusimotor (ll-axons in flexor and extensor leg muscles of the cat. J. Neurophysiol., 35: 149-1 5 3. Emonet-DBnand, F. et Laporte, Y. (1974) Blocage neuromusculaire selectif des jonctions extrafusales des axones squeletto-fusimoteurs produit par leur stimulation repetitive 2 frequence 6levBe. C.R. Acad. Sci. (Paris), 279: 2083-2085. Emonet-Denand, F. and Laporte, Y. (1975) Proportion of muscle spindles supplied by skeleto-fusimotor axons ( p axons) in the peroneus brevis muscle of the cat. J. Neurophysiol. 38: 1390-1394. Emonet-DBnand, F., Jami, L. and Laporte, Y. (1975) Skeleto-fusimotor axons in hind-limb muscle of the cat. J. Physiol. (Lond.), 249: 153-166. Haase, J. und Schlegel, J.H. (1966) Einige funktionelle Merkmale von a-innervierten Extensor- und Flexor-Spindeln der Katze. Pfliigers Arch. ges. Physiol. 287 : 163-175. Haase, J., Meuser, P. und Tan, U. (1966) Die Konvergenz fusimotorischer &-Impulseauf deeffertierte Flexor Spindeln der Katze. Pfliigers Arch. ges. Physiol., 289 : 50-58. Matthews, P.B.C. (1972) Mammalian Muscle Receptors and their Central Actions, Edward Arnold, London. McWilliam, P.N. (1975) The incidence and properties of p axons to muscle spindles in the cat hind limb. Quart. J. exp. Physiol., 60: 25-36. Ovalle, W.K. and Smith, R.S. (1972) Histochemical identification of three types of intrafusal muscle fibres in the cat and monkey based on the myosin ATPase reaction. Canad. J. Physiol. Pharmacol., 50: 195-202.

DISCUSSION GRANIT: When we see this so-to-speak frog-like reminiscence in a certain muscle we wonder if it would be more important in posture than in movement. You have not done this on a typically postural muscle like soleus yet? LAPORTE: Only once, for technical reasons, but it should be recalled that Barker, Adal and Stacey made some observations on the rate of degeneration of motor endings after nerve section which suggest that p1 plates are innervated by p axons; they have also shown that p1 plates are present in a large proportion of soleus spindles. BOYD: Are any of these fast skeletomotor axons static in action? LAPORTE: We d o not know yet. The experiments showing intrafusal glycogen depletion

106 after stimulation of a group of fast motor axons are very recent. These fast skeleto-fusimotor axons almost exclusively supply chain fibres. I hope they are static. BOYD: So d o I. LAPORTE: Yes, but one must be careful. STUART: In studies on tendon organs in cat medial gastrocnemius and tibialis anterior, it has not been uncommon to find an Ib afferent that fires on the rising phase of the muscle twitch. To exclude the possibility that the afferent is of Ia origin we have usually stimulated the whole muscle at 10-25 pulses/sec with stimulus strength adjusted to fire the majority of (Y fibres and a minimum of y fibres (i.e., an SO-90% contraction). In our viewpoint, this procedure should result in sustained Ib firing whereas gamma activated Ia discharge should either be absent or intermittent. Are we also safe in now assuming that the same procedure would not result in a P-activated spindle discharge? LAPORTE: You raise an interesting point. It is often very difficult to distinguish between a spindle and a tendon organ. In our laboratory we consider that a muscle receptor is undoubtedly a spindle only if it is activated by a y axon. Possibly this is going too far but, while experimenting on single P axons, we always had a few axons available t o check that a receptor activated by the stimulation of a p axon was a spindle and not a tendon organ.

HOUK: Might 0 motoneurones receive different afferent projections than the (Y motoneurones? If not, it would be a clear interaction of a positive feedback loop activity of motor axons causing activity of spindles, which could reinforce on the activity of P motoneurones. I wonder if you had thought about this? LAPORTE: I think you should ask the first question to Professor Henneman himself. As you know, his work with Mendell shows that each Ia fibre from a given muscle does supply almost all the motoneurones supplying extrafusal muscle fibres of that muscle. I assume that motoneurones are no exception. It is very likely that the skeleto-fusimotor system in mammals provides, as it does in batracians and reptiles, a positive feedback to the motoneurones of a muscle as long as the muscle does not shorten. GLADDEN: When one stimulates a p axon it is difficult t o fatigue the spindle response. Do you think that these might be a partial block in the branch of the axon supplying the spindle? I wondered about this because in one experiment we had t o stimulate a 0 axon at 200 imp/sec in order to get a maximum contraction of a fibre, and yet we would normally find that with a y we would only need 100 implsec t o do this. LAPORTE: I cannot give you a definite answer, because we have not studied the frequencygram of primary endings activated by 0 axons. After stimulating y static axons, periodic increases in the frequencygram can be detected for rates of stimulation as high as 400/sec, which implies that the terminal branches of these axons can conduct impulses at that frequency. I don’t see why spindle collaterals of P axons should behave differently, but there is no direct evidence on this point.

General Discussion

MATTHEN’S: I have made a personal wiring diagram which may serve as a useful talking point. BARKER

BOYD

It is now generally agreed that the muscle spindle has three kinds of intrafusal fibres, the chain fibres and two kinds of bag fibres. Barker has a bag,, a bag, and chains determined histochemically and histologically and Boyd has a dynamic and a static bag fibre and chains. We have already agreed today that bag, and dynamic bag fibres are the same and that bag, and the static bag fibres are the same. Our previous problem was how the gamma static could innervate both bags and chains. For Boyd this is totally resolved because the gamma static only goes to the static nuclear bag fibre and in Barker’s case gamma static innervates the bag, which is structurally more similar t o a chain fibre than it is t o the bag1 fibre, although it is n o t a chain fibre. Prof. Laporte has given us the additional information that the beta dynamic innervates the bag1 and some beta axons which we hope are static innervate the chains. However, Barker is quite definite that his gamma (S) goes as much to the bag, as t o the bag,, but if this were withdrawn the t w o wiring diagrams would be the same. BARKER: I entirely agree with Laporte but I d o think we should add these connections.

108 I don’t want to make it more complicated, b u t there is evidence found both by ourselves and by Bessou and Pages that the gamma (D) axons activate both bag1 and bag, and we have evidence from glycogen depletion studies o n both beta (D) and gamma ( D ) axons that these fibres play a minor role. Essentially we are now talking about the dirference between in vivo observations of contractile movements and histological observation o f glycogen depletion and silver. I think we should p u t the names of Bessou and PagPs also o n Boyd’s side because they are very firm that a ganlma (D) activates the bag fibre which the gamma (S) does not. However they d o say that weak local contraction of this bag fibre could be extraordinally difficult to detect when the bag2 and chains are also contracting. In collaborative experiments, Bessou and Pages have found spindles innervated by dynamic gamma axons, and we find the terminals and report o n their ultrastructure and the type o f muscle fibre innervated. In about 20 experiments all the sites of observation were 2 m m away from the equator, but in only one experiment was the site less than 2 m m away. We find them particularly significant. In one experiment in which t h e site was a t about 2 mm, the ending proved to be o n a bag2 fibre and when pressed very hard o n whether any other contraction was visible t h e answer was no. We know from glycogen depletion with both gamma (D) and beta (D) axons that t h e bag2 is always activated in addition t o the bag1 . So one’s suspicion that a bag, contraction has been missed is strengthened.

BOYD: I would like t o modify Dr. Matthews’ drawing in t w o ways. Firstly we have confirmed directly that the beta (D) axon innervates t h e dynamic bag fibre. Now I think it is very significant that in all the structural work - Barker and Laporte’s work o n degenerating all the axons except the gamma (S) axon, in the glycogen depletion work reported today and in Brown and Butler’s work o n peroneus longus - when t h e gamma (S) innervates a hag fibre it is almost always only one of them. We believe it t o be the static bag fibre since we have never observed contraction of a dynamic bag fibre when a static gamma axon is stimulated. A possible explanation might be that a gamma (S) axon supplying chain fibres spreads o n t o one of the bag fibres, n o d o u b t t h e nearest one, so that we could put or in t h e diagram.

c

I suppose if t h e spread is t o the static bag fibre we could see t h e contraction, but if t h e spread were t o the dynamic bag fibre i t s contraction might be missed. However, although t h e dynamic bag fibre contraction can be weak, it is quite possible to see a 2% stretch of t h e primary spirals and so a contraction taking place out in t h e polar regions, even though weak, can almost always be seen in the fluid space. If a contraction is so small t h a t it is n o t transmitted t o the primary sensory ending, I cannot help wondering whether it is functionally significant. Finally, I would like t o comment o n the sites of the contractions. The site of contractions of the gamma (S) axons can be a long way o u t in the poles and t h e gamma (S) and gamma ( D ) contraction sites are often overlapping. Further, the contraction produced by the gamma (S) o n the static bag fibre is focal, just like the o n e produced by the gamma ( D ) axon o n t h e dynamic bag fibre. This does not fit with the idea of trails as t h e only terminations of gamma (S1 axons, and perhaps the gamma (S) axons selectively operating t h e static bag fibres are producing something very similar t o the focal contraction produced by gamma ( D ) axons o n the dynamic nuclear bag fibres, though of course t h e e f k c t s are mechanically different. We would agree with Prof. Barker that these static gamma axons selective to t h e bag fibres tend to be t h e high conduction velocity gamma axons. So summarizing, t h e gamma (D) or beta (D) axons operate the dynamic bag fibres, some gamma (S) axons selectively operate the static bag fibre while the others, which operate the chains, may also

109 simply spill over onto the nearest bag fibre. This would be picked up histologically and if the dynamic bag fibre is involved, whatever the effect, it simply cannot be seen. BARKER: If we look at the histograms showing the distance from the equator of the centres of zones depleted of glycogen and the position of ending identified in silver preparations, two points emerge. Firstly, the two types of bag fibre are very similar in this respect, and secondly, in the bag1 fibre, the majority of glycogen depletions are intracapsular as you would expect from the trail innervation. Finally I would like to show you a tandem spindle which we excluded from our glycogen depletion series because of its anomalous nature. Cope and I and also Eldred in 1962 reported tandem spindles in which a chain fibre continued on into the next unit as a bag fibre. We have seen this several times and one turned up in our glycogen depletion series. As the fibre changed from chain t o bag, its glycogen profile changed from light to medium and the diameter increased. The bag fibre was a bag, and a 2 mm length was depleted following gamma (S) stimulation but only 0.5 mm of the chain. In developmental terms, this is entirely conceivab!e. The bag? is present in the muscle primordium, and if there were sequential development of the two primaries, subsequent fusion of the fibres would be relatively possible. HAGBARTH: What do we know about the mechanical interaction between these fibres? Does the contraction of the bag fibres mechanically affect the chain fibres? And what about the beta fibres? They innervate both the extrafusal and the intrafusal fibres so that the extrafusal contraction is going to unload and at the same time the spindle will be activated. LAPORTE: In the abnormal, experimental condition in which a single axon is stimuIated when there is no tonic activity because the ventral root is cut, a single beta axon when stimulated at 20 implsec does produce unloading and does produce a pause. But if you test during that pause the dynamic sensitivity is still increased, and if you stimulate two or three gamma axons together plus a dynamic beta axon, even at very low frequency, you do see an increase in the discharge. So in fact the spindle integrates all the information given by several gamma axons. You shouldn’t really think of the experimental situation in which unloading is very clear.

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SESSION 11

MUSCLE SPINDLE AND ITS FUSIMOTOR INNERVATION

Part I1 Chairman: D.G. Stuart (Tucson, Ariz.)

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Reflex Connections form Muscle Stretch Receptors to their own Fusimotor Neurones P.H. ELLAWAY and JUDY R. TROTT Department of Physiology, University College London, London N C l E 6BT (Great Britain)

INTRODUCTION The aim of our work is to establish the role of muscle stretch receptors in the reflex control of fusimotor neurones supplying the same muscle. Sporadic reports of such autogenetic control have appeared beginning with Hunt’s (1951) description of fusimotor neurones being inhibited by muscle stretch (for a review see Matthews, 1972). The fact that these reports have not provided a clear or complete picture of the reflex control suggests that connections from muscle afferents to fusimotor neurones do not exert such potent actions as those known to exist from skin afferents and supraspinal structures. Nevertheless there are two reasons why an action of muscle stretch receptors on fusimotor neurones may have been overlooked. Firstly, fusimotor neurones have not been investigated as thoroughly as alpha motoneurones with the intracellular recording technique. Fusimotor neurone cell bodies appear t o be particularly difficult to impale successfully (Eccles e t al., 1960). Secondly, most investigations have employed either electrical stimulation of the muscle nerve or stretch of a muscle and have not therefore achieved selective stimulation of afferents from any of the three principal types of muscle stretch receptor, i.e., the primary and secondary endings of muscle spindles and Golgi tendon organs. The difficulty in obtaining intracellular recordings to detect responses of fusimotor neurones can be avoided since these neurones often exhibit a background or resting discharge in the decerebrated cat. Thus, any response to a stimulus will be evident either as a gross change in the frequency of discharge or, at least, a change in duration of an interspike interval in a train of action potentials. In the latter case many trials may have t o be “averaged” to reveal a statistically significant change or response. Using such techniques we have begun t o investigate the reflex connections to fusimotor neurones from two muscle stretch receptors; primary endings of muscle spindles and tendon organs. Vibration of a muscle can be used t o excite selectively the primary endings of muscle spindles (Brown et al., 1967). Trott (1975, 1976) has shown that some homonymous fusimotor neurones are weakly but consistently excited by vibration while the others remain unaffected. This was an unexpected and intriguing result; unexpected in that previously only inhibitory actions from muscle stretch receptors have been described

114 (Hunt, 1951; Brown et al., 1968; Grillner et al., 1969; Proske and Lewis, 1972; Fromm et al., 1974) and intriguing, since such excitation implies that positive feedback may occur between the motor and sensory innervation of muscle spindles. A recent report of work by Fromm and Noth (1975), where vibration was used t o stimulate muscle receptors, stressed that such a stimulus inhibited some fusimotor neurones although other fusimotor neurones were seen to be excited. Comment on the excitation was postponed by these authors. Autogenetic inhibition has not been seen in this laboratory using amplitudes of vibration which, in control studies, were found to excite selectively the primary endings (Trott, 1975, 1976). However, if the muscle nerve is stimulated at Gp I strength, inhibition of homonymous fusimotor neurones is seen (Ellaway and Trott, 1976). Bearing in mind that intense activation of primary endings (by vibration) results in excitation, the possibility arises that afferents from tendon organs might have autogenetic inhibitory connections to fusimotor neurones (cf., Grillner et al., 1969). We decided to look for such an action even though the inhibition that we had seen with electrical stimulation may have been recurrent inhibition (Ellaway, 1968, 1971) following antidromic conduction of impulses in alpha motoneurone axons. We have used contraction of the muscle with the intention of preferentially exciting tendon organ afferents. Contraction was found to inhibit fusimotor neurones but, as the following results will show, some primary spindle afferents were excited in the form of an early discharge, and such a stimulus was thus not selective. Neither previous studies nor our own results shed any light as yet on whether the secondary endings of muscle spindles participate in the reflex control of fusimotor neurones. This report concentrates on results obtained from experiments designed solely to investigate connections between the afferent axons of Gp I diameter, supplying primary endings and tendon organs, and their homonymous fusimotor neurones.

METHODS Cats of either sex were decerebrated, under halothane in oxygen anaesthesia, by cutting across the brain at an intercollicular level. The brain forward of the cut was removed. On completion of the surgery to expose any required blood vessels and nerves the animals were allowed 1-2 hr to recover before nerve recordings were made. The condition of the animal was monitored continuously by recording blood pressure and body temperature. The nerve supply to the triceps surae in one leg was left intact and the rest of that leg and the tail denervated. In the experiments using vibration, but not those using contraction, denewation was limited to the thigh and lower leg. To obtain recordings of fusimotor neurone discharges, the sheath of a single fascicle of the triceps surae nerve was slit and a bundle of fibres cut and reflected centrally. This bundle was teased until a strand containing a single active unit was isolated. During dissection fusimotor neurones were identified by their small spike size relative to those of alpha motoneurones, their higher frequency,

115 regular, background discharges and their lower reflex threshold to cutaneous and pinna stimulation. Conduction latencies following direct stimulation of the axon were then measured whenever possible. Conduction velocities lay in the range 19-39 m/sec. A continuous indication of mean frequency of discharge of a single neurone was obtained by shaping action potentials into identical pulses which were then fed into an integrating device whose output voltage was linearly related to the frequency of input pulses. Alternatively, a Biomac computer (Data Laboratories) was used to note the occurrence of pulses relative in time to the presentation of a stimulus. Repeated sweeps built up a poststimulus time interval histogram. RESULTS

Excitation o f fusimotor neurones b y primary spindle afferents To achieve selective excitation of primary spindle endings within triceps surae, the Achilles tendon was attached to the probe of an electromagnetic vibrator. Recordings were then made from afferent axons in dorsal roots to establish the parameters of vibration necessary t o excite solely primary endings. A t frequencies of 70-500 Hz a range of amplitudes was found (50-100 pm) which powerfully excited all primary endings studied and drove many of them to fire an impulse to each cycle of vibration. Secondary endings and tendon organs remained unaffected. To achieve driving of the majority of primaries the muscle was stretched so that resting tension developed, the final length of the muscle being a few millimetres short of maximum natural extension. Vibration under these conditions was then used t o study the action of primary endings on their homonymous fusimotor neurones. Decerebrated cats were paralysed with gallamine triethiodide (Flaxedil, 8 mg/kg) so that any alpha motoneurone response to vibration would not elicit muscle contraction. Tendon organ discharges in response to vibration-induced contraction were thus avoided. During paralysis cats were artificially respired. Of 27 fusimotor neurones 19 were consistently excited by vibration, the remainder being unaffected. Fig. 1 illustrates the form of the excitation. The excitation was not always so sharp in onset but typically the frequency of discharge continued to rise for several seconds before reaching a plateau during maintained vibration. On discontinuing vibration the frequency of discharge fell more or less abruptly than is illustrated, to its prestimulation level. Occasionally, simultaneous recordings were made from alpha motoneurones. Excitation of fusimotor neurones was often observed at amplitudes of vibration too low to excite alpha motoneurones. Nevertheless, on raising the amplitude so as to excite alpha motoneurones the excitation of fusimotor neurones usually increased. Two forms of control experiment were regularly carried out. (1) Anaesthetisation of the tendon. Since squeezing the Achilles tendon often excited fusimotor neurones, 5%procaine solution was applied t o the tendon. This invariably abolished the response t o squeezing but left the excitation

116

A

-

Vib r a tio n

(200H2, 5Opm)

G a m m a ( 22m/s)

75 imp/s

discharge

- 50

frequency

10 sec

B Before

vibration

. Du r in g v i b r a t i o n ( 2 0 0 H z , 50 p m )

After

v i b r a t ion

. .. .......

..........,......-. i=

671s

..

..

iI

551s

1 sec

Fig. 1. The excitatory effect of vibration applied t o the tendon of triceps surae o n the discharge of a gastrocnemius medialis (G.M.) fusimoto; neurone. A: duration of vibration signalled by the upward deflection of the marker. Below this is plotted the integrated discharge frequency of the neurone. B: selected portions of the plot in ( A ) showing t h e original discharge. To the right of each record is the mean frequency calculated for the whole 2.2 sec of each record.

to vibration unaffected. Thus, mechanical irritation of the tendon was not the source of reflex excitation produced by vibration. (2) Section of the muscle nerve. Vibration applied to the tendon could be felt at the hip and the belly of the animal. However, after complete section of the triceps surae nerves all reflex excitation disappeared and discharges remained unaffected by vibration.

Inhibition of fusimotor neurones by vibration Brown et al. (1968) reported that certain fusimotor neurones of unknown destinations (recorded in ventral roots) were inhibited by vibration of triceps surae. This was confirmed in the present study where, of 30 fusimotor neurones, 7 were inhibited by vibration. A further 5 neurones were seen t o be excited. Since the preparation and the parameters of vibration were basically the

117 same in the peripheral nerve and ventral root studies, it is improbable that the fusimotor neurones inhibited by vibration of triceps suiae are destined for the same muscles. A recent article (Fromm and Noth, 1975) has reported that some fusimotor neurones can be inhibited by vibration of their own muscle. It was suggested that the inhibition was most probably due to the recurrent pathway (Ellaway, 1968, 1971) with Renshaw cells being activated by collaterals of alpha motoneurones. Alpha motoneurones were seen to respond to the applied vibration. No control recordings of the types of afferent axon excited by vibration were reported. We have since specifically examined fusimotor responses to vibration on extending the muscle to, or just past, its maximum natural limit and increasing the amplitude of vibration above that necessary to drive most primary endings. Two fusimotor neurones examined then proved to be inhibited. Previously one of them had been excited by vibration of 30-100 pm amplitude at a shorter muscle length several millimetres short of the natural limit. We believe that the more powerful stimulus needed to produce inhibition in our experiments may not be exciting only primary endings.

Inhibition of fusimotor neurones on eliciting contraction We have examined the effect of muscle contraction predicting that contraction would cause an increased discharge from tendon organs and a pause in muscle spindle receptor discharges. Once again we confined our attention to the triceps surae and isolated fusimotor neurones supplying the gastrocnemius lateralis/soleus (GLISol) muscle nerves. Near isometric contraction of triceps surae was achieved on stimulating part of ventral roots L7 and S1 or by stimulating the muscle directly via inserted pin electrodes. Fig. 2 shows recordings from two neurones which were inhibited by contraction. In the upper example a clear pause in firing occurs during the rising phase of contraction. In the lower example the inhibition is not so clear but is more typical of the inhibitory responses generally recorded. In these cases a poststimulus time interval histogram was constructed (see Methods). Fig. 3 presents data from the same neurone as is featured in the lower part of the previous figure. The inhibition is revealed as a lowered count of spike occurrences during contraction and this usually represented a lengthening of a single (occasionally two) interspike interval. It is also clear from subsequent oscillations in the histogram that the inhibition tends to re-phase the firing pattern of the cell. When a tetanus was elicited the inhibition was prolonged but the initial inhibition, which it was possible to elicit with twitch contractions, remained the major component. Of 1 6 neurones, 11 were inhibited as contraction occurred, 4 were unaffected and one was excited. In all the 6 cases studied, denervating the muscle abolished the inhibition whether the stimulus was applied to ventral root fibres or the muscle directly. Thus, neither contractions of non-denervated muscles, other than the triceps surae, nor movement of other innervated structures caused by contraction of triceps surae could have been responsible for the inhibition.

118 GL or Sol 23rn/sec

L 0

J

Muscle

tension

100

Gamma discharge

GL or Sol 37rn/sec

1 200 sweeps

Countsf2 msec bin

1

0,3kg

2’

0

250 msec

0

100

msec

3

Fig. 2. The inhibitory effect of isometric contractions of triceps surae on two fusimotor neurones supplying GL or Sol muscles. In both cases the contractions were elicited by double shocks (each 0.1 msec duration, 3 msec apart) applied at time 0 t o peripheral parts of the ventral root supply. In both examples the three traces, from the top, depict action potential discharges, tension developed and time. In the upper example there is a clear pause in fusimotor discharge during the rising phase of contraction. The two large spikes are irrelevant and are direct alpha motoneurone responses to the shocks. In the lower example the pause is much less evident. Fig. 3. Inhibition on eliciting contraction of triceps surae of the GL or Sol neurone conducting at 37 m/sec depicted in Fig. 2. The muscle contraction tension and the fusimotor (gamma) discharge have been traced from original records. The histogram below has been constructed with the same time base. For each action potential in a sweep a single count has been added to the bin which has currently been reached. Each bin is “open” for 2 msec. The histogram is the result of analysing 200 sweeps. The lowered count starting approximately 17 msec after the first shock indicates that the neurone is inhibited. Subsequent oscillations represent partial re-phasing of the rhythm of discharge.

A complication arose in interpreting our results. In all but one case it had been noted that contraction occurred slightly too late t o be responsible for the actual onset of inhibition. On examining afferent responses t o contraction it was discovered that “early discharges” were elicited in Gp I axons in advance of any rise in tension. These were early enough t o be responsible for the inhibition but they occurred in both spindle primary and tendon organ afferents. The tendon organs continued t o fire during contraction while the primaries, with the odd exception, paused. These control experiments, recording musde afferent responses, were performed on some of the cats used to investigate fusimotor responses to contraction.

DISCUSSION These experiments were designed to examine specifically the response of fusimotor neurones to activity from spindle primary endings and Golgi tendon organs of the homonymous muscle or its close synergists.

119 From the experiments using muscle vibration, we can safely conclude that afferents from primary endings have excitatory reflex connections to some homonymous fusimotor neurones. It was found that only primary endings among the three muscle stretch receptor types were excited by amplitudes of vibration that excited fusimotor neurones. Excitation of fusimotor neurones was usually provoked by vibration at a slightly lower amplitude than that needed to excite alpha motoneurones. Although this difference in threshold was not systematically investigated, it suggests that fusimotor excitation is not a consequence of alpha motoneurone firing. An action via alpha recurrent collaterals, such as disinhibition by Renshaw cells, thus seems unlikely. The question remains whether the excitation is purely a segmental reflex or, since decerebrated cats with intact spinal cords have been used, is an action transmitted through supraspinal regions. It is now known that the tonic vibration reflex responses of alpha motoneurones can be elicited in a spinal cat (Goodwin et al., 1973; Ellaway and Trott, 1975) although the pathway is severely depressed on removing the influence of descending pathways. Thus the connections to alpha motoneurones, at least, exist in the cord. One indication of the course of the central pathway would be the latency of the reflex excitation. The onset of fusimotor excitation by vibration can be sharp but, due to the inherent variability in fusimotor discharges, it has not been possible to measure the latency. Experiments are in hand to investigate this point by using brief, small amplitude (100 pm) stretches equivalent t o the onset of vibration. The alternative is t o experiment in the spinal animal but this meets the problem that most fusimotor neurones to triceps surae cease firing following spinal section. The excitation of a fusimotor neurone by vibration can be up to a 50% increase in firing rate, but it should be remembered that it is caused by high frequency firing of most of the population of primary endings. This level of activity would be met naturally only for brief periods during sudden muscle extensions (and possibly contractions?). Of course the intriguing aspect of such autogenetic excitation is that it appears to be positive feedback. A reinforcing cycle of excitation may result since fusimotor neurones, in their turn, excite primary endings. However, until we have some idea of the extent of excitation of fusimotor neurones by primaries within the normal range of muscle activity we do not intend to speculate on the consequences of this potentially unstable mechanism. Turning t o the inhibition observed with contraction we have met a problem of interpretation in that early discharges in spindle afferents are evoked with contraction and thus selective excitation of tendon organs has not been achieved. Activity generated in secondary endings does not arrive at the cord in time to cause the inhibition. Furthermore, we have not seen inhibition with vibration, which selectively and powerfully excites primary endings, making it more likely that the inhibition with contraction is a result of activity generated in tendon organ afferents. The receptors responsible for the inhibition of a few neurones when the muscle is highly stretched and vibrated at large amplitudes remain undisclosed. A possibility is that tendon organs have been excited but an action via other afferents cannot be excluded. For example, the effect of such a potentially

120 painful stimulus on muscle receptors with small diameter axons in the Gp I11 and IV ranges is largely unknown (cf. Paintal, 1962). In conclusion it seems that we may have to consider fusimotor neurones as having similar reflex connections from Gp I afferents supplying muscle stretch receptors as their homonymous alpha motoneurones. However, these connections do not exist t o all fusimotor cells and appear to be weaker than to their alpha motoneurone counterparts. In retrospect it does not seem surprising that the action of stretching a muscle has had no clear-cut effect on fusimotor discharge (Hunt and Paintal, 1958). SUMMARY

(1) Selective and intense activation of muscle spindle primary endings by vibration consistently excited 1 9 out of 27 homonymous fusimotor neurones. (2) Near isometric muscle contraction was found t o inhibit 11 out of 16 homonymous fusimotor neurones with one being excited. ( 3 ) The latency of this inhibition is so short that “early discharges” generated in Gp I afferent axons, as contractions are elicited, may be responsible for the onset of inhibition. (4)Since primary endings as well as tendon organs produce “early discharges” to contraction we cannot be certain that the inhibition is due solely to activity generated in tendon organ afferents. ACKNOWLEDGEMENTS We thank the M.R.C. for financial support and Mr. J.E. Pascoe for much helpful advice during the work and in the preparation of the manuscript. We thank Mrs. Maria Winder for her considerable technical help. Fig. 1 of this paper is reproduced through the courtesy of the editors of the Journal of Physiology. We thank the Wellcome Trust for travel funds for one of us (P.H. Ellaway) t o participate in the conference on stretch reflex mechanisms held in Tokyo. REFERENCES 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: 773-800. 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. Eccles, J.C., Eccles, R.M., Iggo, A. and Lundberg, A. (1960) Electrophysiological studies on gamma motoneurones: A d o physiol. scond., 5 0 : 32-40. Ellaway, P.H. (1968) Antidromic inhibition of fusimotor neurones. J. Physiot. (Lond.), 198: 39-40P. Ellaway, P.H. (1971) Recurrent inhibition of fusimotor neurones exhibiting background discharges in the decerebrate and the spinal cat. J. Physiol. (Lond.), 216: 419-439. Ellaway, P.H. and Trott, J.R. (1975) Facilitation of the tonic vibration reflex in the spinal cat by 5-hydroxytryptophan (5-HTP). J. Physiol. (Lond.), 249: 54-56P.

121 Ellaway, P.H. and Trott, J.R. (1976) Inhibition of fusimotor neurones on eliciting contraction of the homonymous muscle. J. Physiol. (Lond.), 256: 52-5313. Fromm, Chr. and Noth, J. (1975) Vibration induced autogenic inhibition of gamma motoneurones. Brain Res., 83: 495-497. Fromm, Chr., Haase, J. and Noth, J. (1974) Length dependent autogenic inhibition of extensor gamma motoneurones in the decerebrate cat. Pfliigers Arch. ges. Physiol., 346: 251-262. Goodwin, G.M., McGrath, G.J. and Matthews, P.B.C. (1973) The tonic vibration reflex seen in the acute spinal cat after treatment with DOPA. Brain Res., 49: 463-466. Grillner, S., Hongo, T. and Lund, S. (1969) Descending monosynaptic and reflex control of gamma motoneurones. Acta physiol. scand., 75: 592-613. Hunt, C.C. (1951) The reflex activity of mammalian small-nerve fibres. J. Physiol. { L o n d . ) , 115: 456-469. Hunt, C.C. and Paintal, A S . (1958) Spinal reflex regulation of fusimotor neurones. J. Physiol. (Lond.), 143: 195-212. Matthews, P.B.C. (1972) Mammalian Muscle Receptors and their Central Actions, Monograph No. 23, Physiological Society, London, pp. 448-451. Paintal, A.S. (1962) Responses and reflex effects of pressure-pain receptors of mammalian muscles. In Symposium o n Muscle Receptors, D. Barker (Ed.), Hong Kong University Press, Hong Kong, pp. 133-142. Proske, U. and Lewis, D.M. (1972) The effects of muscle stretch and vibration on fusimotor activity in the lightly anaesthetised cat. Brain Res., 46: 55-69. Trott, J.R. (1975) Reflex responses of fusimotor neurones during muscle vibration. J. Physiol. {Lond.), 247: 20-22P. Trott, J.R. (1976) The effect of low amplitude Vibration on the discharge of fusimotor neurones in the decerebrate cat. J. Physiol. (Lond.), 255: 635-650.

DISCUSSION HULTBORN: Have you only confirmed on triceps surae or have you seen this excitation in other muscles? ELLAWAY: We have restricted ourselves t o the triceps surae. Certainly the excitation occurs between the two heads of triceps. HULTBORN: Concerning the similarity between the action to the alpha motoneurones and to the gamma motoneurones, that raises the question whether the Ia excitation of gamma motoneurones is monosynaptic or polysynaptic. ELLAWAY: We have so far done only one experiment, in which we gave the muscle a sharp tap, hoping t o see in the post-stimulus time histogram the very first response of this complicated response. As you can see, compared with inhibition, there is virtually no change in the post-stimulus time histogram. HENNEMAN: I believe Hunt was one of the first people to report an effect on gamma motoneurones, and then later Hunt and Paintal expressed some uncertainty about it, and they thought that the difference between preparations, spinal versus decerebrate, explained the two different kinds of effect. ELLAWAY: As you mentioned that was left completely unsolved. LAPORTE: Are we certain that paciniform corpuscles are not excited by vibration? ELLAWAY: Well they certainly are excited by vibration. The numbers suggested from the histology of the muscle nerves are thought t o be small compared with say those in the interosseous nerve.

LAPORTE: This is true for the Pacinian corpuscles but the paciniforms are not so exceptional. I wonder if there are any receptors connected t o group I11 afferent fibres which are sensitive to vibration. ELLAWAY: The only reference I have t o that was by Paintal. He saw that vibration did not excite group I11 fibres, but this was a remark at a symposium and was not backed up by numbers. I plan t o re-investigate this. MATTHEWS: It seems wide open for further experimentation. We rely on Barker’s figures for the numbers of paciniform corpuscles in triceps and in soleus. HOUK: If primary endings excited the gamma motoneurones and if you paralysed the muscle, it seems you could get possibly a response t o stretch. You must have tried this. ELLAWAY: These were experiments done by Judy Trott. In the early days when the vibrator was attached to the tendon she could not stretch the muscle more than 1 or 2 mm. She did sometimes see excitation on stretching the muscle but this was not a consistent result and we didn’t draw conclusions from it. HAGBARTH: Were the gamma discharges time locked to the vibratory stimulus? We know that the alpha discharges usually are, as shown by Homma. ELLAWAY: Not obviously, but I’ve not looked at that closely. Certainly the frequencies of firing were below the vibration frequencies. TAKANO: I have been trying t o relate your results to ours. I suppose we have two systems: when you stretch the muscle the motoneurone could be depressed, but if you stretch the muscle frequently by vibrating or stretching then you can potentiate the gamma system. GYDIKOV: Have you studied the instantaneous frequency after stretching, because if you use a post-stimulus histogram you only record the frequency of impulses which are locked to the beginning of the stretch and you find then oscillation, which in my opinion does not express the gamma motoneurone inhibition really. ELLAWAY: I quite agree. It’s a useless technique for investigating any long-term effects when the gamma motoneurone continues t o fire, but it is useful to look at the initial response t o a stimulus.

Intracellular Recordings from Intact Soleus Muscles of Cats Y. NAKAJlMA Department of Physiology, School of Medicine, Chiba University, Chiba (Japan)

INTRODUCTION Regarding the innervation of muscle spindles by static fusimotor fibers there have been 4 separate lines O C investigation. They are (1)direct observation of contractions elicited in spindles (Boyd, 1971a, b; Boyd et al., 1973), (2) physiological identification of intrafusal fibers yielding spike or junction potentials recorded by microelectrodes (Bessou and PagGs, 1972), (3) glycogen depletion method (Brown and Butler, '1973,1975), and (4) degeneration method (Barker et al., 1973). In my previous work on soleus muscle spindles (Nakajima, 1975) it was assumed, from the viewpoint of the ability of static fusimotor fibers t o drive spindle afferents, that such I tatic fusimotor fibers would terminate on nuclear chain fibers and not drive on nuclear bag fibers. To make the spindle innervation by static fusimotor fibers in relatively large muscles clearer, in this preliminary report, intrafusal muscle potentials have been recorded intracellularly in soleus muscles of the cat. In order to minimize any extrafusal contraction, dantrolene sodium, an excitation-contraction coupling blocker (Ellis and Carpenter, 1972; Lowndes, 1975) was used. It is known that this substance dDes not alter the electrical membrane properties of the muscle fibers (Ellis and Bryant, 1972).

METHODS The experiments were carried out on adult cats of similar sizes and anesthetized with an intraperitoneal injection of 5 ml/kg of a solution of 10% urethane and 1%chloralose. All hindlimb nerves on the experimental side were cut except the lateral gastrocnemius nerve. After the triceps surae muscle and its tendon were separated from the surrounding tissues, the gastrocnemius muscle was removed completely, care being taken to keep the blood supply to the soleus muscle as far as posijible intact. Skin flaps surrounding the dissected muscle were used t o form a pool containing Ringer's solution which was kept at a temperature of 38--39°C. The femur, tibia and fibula were rigidly clamped. The tendon of the soleus muscle was tied t o a steel plate t o record an isometric

124 contraction. A routine laminectomy was performed between L5 and S 1 to expose the ventral roots, all of which were cut at their entry to the spinal cord. The skin flaps surrounding the exposed spinal cord were used t o form a pool containing paraffin oil which was kept at a temperature of 38--39°C. The L, ventral root was split into naturally separate fine fasciculi which were mounted on stimulating electrodes and stimulated electrically by 0.5 msec rectangular pulses at an intensity of 15 X T (T = the threshold of the motor nerves t o the soleus muscle). While monitoring twitch contraction elicited by stimulation of a ventral root fasciculus, dantrolene sodium, partly in solution in distilled water and partly as a suspension, was injected intravenously to a final concentration of 4 5 - 6 0 mg/kg. The cats were artificially ventilated t o avoid hypoventilation. Thirty minutes after injection of dantrolene, intracellular recordings were made at points 1 cm proximal t o the line of maximal traverse of the muscle and the recording site was narrowly restricted to a volume of 5 mm X 3 mm X 3 mm. Glass capillary microelectrodes filled with 3 M KC1 were used and their resistances were 10-15 M a . Muscle potentials and zero reference potentials together with stimulus artifacts were recorded and stored on magnetic tape by a data recorder (Model 351-FH, TEAC, Tokyo). Later these potentials were led into the averager of a biological computer (ATAC 501-20, Nihon Kohden, Japan) and displayed on a strip chart recorder. RESULTS By using dantrolene sodium and stimulating relatively small ventral roots, muscle potentials were recorded from 268 muscle fibers without injuries t o muscle membranes or destruction of microelectrodes by any concomitant contraction. Since the recording site was chosen as described in the Methods and muscle potentials were recorded without any selection from the surface of the muscle membrane down t o a depth of 5 mm, a latency histogram, as shown in Fig. 1,seems to be based on a random sampling. Two kinds of muscle potential were obtained. They were a spike potential with an overshoot potential and a junction potential. Latencies of spike potentials were distributed between 2.65 and 15.6 msec and those of junction potentials between 6.2 and 12.0 msec. It was clear that the junction potentials in this study were not due to injury of the muscle membrane since extracellularly recorded potentials obtained before impalement of the membrane by the microelectrode always showed the mirror image of the potential recorded intracellularly . In Fig. 2 three kinds of junction potentials that were evoked by single stimulation of ventral rootlets are shown. Record 1 is the most frequent type of junction potential, with a latency of 9.8 msec and a long repolarization phase. Record 2 shows a junction potential with a notch in the repolarization phase. The timing of this notch was not always constant. Record 3 shows three repetitive responses after a single stimulus. The first potential was always seen with constant latency but the second and third ones were not constant either in their occurrence or their latency. Since no repetitive spikes or junction poten-

125

Or ’

n J

2

L

3

Latency

1 5

Fig. lets. half ulus

Junction Fnientlal

-60 -

-

2

1. Latency histogram of muscle potentials evoked by single stimulation of ventral rootUpper half of t h e histogram is composed of the spike potential latencies and the lower of the junction potential latencies. Abscissa is the time from the beginning of the stimpulse t o the beginning of the muscle potential. Ordinate is t h e number of observations in each 0.5 msec interval. Total number of observation is 275.

Fig. 2. Three kinds of junction potential evoked by single stimulation of ventral rootlets. In every record t o p trace shows zero membrane potential together with t h e stimulus artifact and lower trace the intracellularly recorded muscle potential. Voltage calibration in every record is shown o n t h e left and time calibration is shown o n the b o t t o m of record 3. Record 1: resting membrane potential (RMP) - 46.8 mV, amplitude of potential (AP) 20 mV, latency ( L ) 9.8 msec. Record 2 : RMP -63.0 mV, AP 31.5 mV, L 6 . 2 msec. Record 3 : RMP -55.0 mV, AP 14.3 mV, Ls of the first, second and third 7.2, 12.4 and 1 7 . 3 msec, respectively.

tials were seen as a consequence of injury of the muscle membrane, as is frequently seen in neurons, and as no spontaneous muscle potentials were seen, this repetitive generation of the junction potential seems to be one of the characteristics of some kind of muscle membrane or motor terminal. Junction potentials, including records l, 2 and 3 of Fig. 2, were obtained from 1 2 muscle fibers. The ratio of muscle fibers which evoked a junction potential, their mean resting membrane potential and their mean amplitude of depolarization are listed in the right column of Table I. The remaining 256 muscle fibers evoked two kinds of spike potential after single stimuli applied t o ventral rootlets. These are either a single spike potential or a double response. Seven muscle fibers out of the 256 showed a double response, which could be a spike potential followed by another spike potential

126

2

5 msec

Fig. 3. Spike potentials showing a double response t o single stimulation of ventral rootlets. In both records t o p trace shows zero membrane potential together with stimulus artifact and lower trace the intracellularly recorded muscle potential. Record 1: tw o spike potentials with overshoot potentials. RMP -82.0 mV, APs of the first and second, 107.0 mV and 102.0 mV, respectively, Ls of the first and second response 12.5 msec and 15.6 msec, respectively. Record 2: one spike potential with overshoot and one junction potential. RMP -56.5 mV, AP of the spike 6 5 mV, AP of t h e junction potential 12.5 mV, L of the spike potential 7.8 msec, L of the junction potential 12.4 msec.

or a spike potential followed by a junction potential. Record 1 in Fig. 3 shows an example of the former and record 2 of the latter case. The ratio between the former and the latter was 4 : 3. Latencies of the first occurring spike ranged between 7.7 and 12.5 msec and those of the second response between 8.9 and 15.8 msec. The mean latency of the first spike and of the second response, their mean resting membrane potentials and the mean amplitudes of the first spike and second response are shown in the middle column of Table I. The remaining 249 muscle fibers evoked one spike potential after a single stimulus to a ventral rootlet. Four examples with various latencies are shown in Fig. 4. Latencies of these spike potentials were distributed between 2.62 and 12.60 msec. Time intervals from the beginning of stimulation t o the beginning of the spike potential consist of the time due t o conduction of the impulse down the nerve fiber, synaptic delay at the neuromuscular junction and the time due to conduction along the muscle fiber. Since the recording site was narrowly restricted t o a volume of 5 mm X 3 mm X 3 mm at the same location in a muscle as described in the Methods, the main factor in the difference of latency seems to be due to the difference in conduction time along the nerve fiber. The mean latency of the spike potential, the mean resting membrane potential and the mean amplitude of action potential are listed in the left column of Table I. The least stimulus interval which can evoke a double response was examined.

127

> 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.

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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

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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

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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

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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.

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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.

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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

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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-

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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.

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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

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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

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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,

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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

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Fig. 6. Absence of effect of repetitive antidromic stimulation upon the amplitude of abortive spikes during t h e semi-blocked condition. An experimental arrangement and a superimposed record are shown o n the left. A: amplitude histograms of abortive spikes obtained from a continuous 8 2 sec record in an intact preparation as control. B: amplitude histograms of successive abortive spikes without (A-A) and during repetitive antidromic stimulation a t 1 Hz (S-A > 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).

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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)

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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

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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

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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

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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.

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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

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0

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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

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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

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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)

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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

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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.

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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.

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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

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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

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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

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tested a-MNs exhibits a different type of stretch responses: most of them have a falling frequency curve. Some units stop firing after a few seconds, others keep firing until the end of the stretch but reach a relatively flat frequency level not earlier than 30-40 sec after the onset of stretch. We may call this a “decremental-tonic” preparation type. We have collected individual response curves of 87 such a-MNs to 1 4 mm stretch from 1 7 preparations of the described type. In Fig. 4C, again the averaged frequency-time curve, as calculated from these data, is plotted, showing clearly the net decrement of the overall response. The active mechanical tension of the stretched muscle, not shown here but recorded in representative members of this preparation type, follows very closely the time course of curve C. We will now ask how the Renshaw mechanism is involved in the performance of these two different types of preparations. In part D of Fig. 4,one of the longer responding a cells of this population was chosen to test the efficiency of antidromic Renshaw inhibition by means of intermittent tetanic stimulation of the central stump of about one-quarter of the ventral root L, . The antidromic inhibition is clearly of the cumulative type, that is, its efficiency grows with

228

longer stretch times, as described a couple of years ago by Granit and Rutledge (1960). How can we interpret this result? Granit and Rutledge (1960) have proposed that the cumulative type of antidromic Renshaw inhibition might be due t o some kind of “surplus excitation” which initially protects the tonically discharging a-MN against the inhibitory RC action but soon decreases during the ongoing stretch. Surplus excitation might indeed be an important factor which contributes t o the just-mentioned phenomenon. In our opinion, however, another factor, probably likewise essential, has been neglected too much as yet, namely, the variable amounts of so-called “natural recurrent inhibition” (NRI). The data collected from the decremental-tonic preparation type have informed us that in this case a large fraction of MNs starts firing immediately at the onset of stretch, but with very different initial frequencies, ranging from lowest t o highest values. The large number of responding a-MNs will initially activate a large number of RCs. Thus the initial net state of NRI must be quite high, and consequently, some of the a units - those with the weakest excitatory drive - will be silenced very soon. So the amount of NRI begins t o decrease, making it easier for some other units to continue their firing. As time proceeds, further MNs will gradually drop out, dependent partly on the adaptive decrease of stretch-induced afferent input, partly on their individual synaptic thresholds which are governed by Henneman’s well-known size principle (Henneman, 1957). What remains active in the late phase of stretch is a relatively restricted and constant fraction of MNs having optimal tonic properties and maintaining a constant low degree of NRI which might help t o stabilize the final discharge frequencies. In other words, we have described here the second important function of the natural Renshaw mechanism: it has favourably selected from the entire population a limited number of a cells discharging tonically at a preferred frequency range, t o the disadvantage of the rest of the pool. It remains t o say that the cumulative behaviour of the antidromic inhibition can be easily interpreted in terms of a decreasing surplus excitation. According t o our results, however, we also have t o consider that an inverse relation exists between the amount of net NRI and the available inhibitory amount which can be added by antidromic stimulation : if the natural RC activity decreases during the ongoing stretch, whereas the stimulus-induced activity remains constant, then the gap between naturally induced and antidromically induced RC activity would be small in the beginning but would grow with time. This would lead to the cumulative inhibition. The other population prototype, presented in part A and B of Fig. 4, will now require little further comment. Here, the responding fraction of the a-MN pool is concentrated from the beginning on tonically firing MNs, probably due to a generally higher but more selective excitatory drive which might be controlled by supraspinal sources. Surplus excitation and/or NRI are high enough to stabilize the initiated frequencies but not t o cause any drop-out of units. Thus the NRI remains stable, helping t o level out the last irregularities in the discharge patterns of the a-MNs. The implications for the antidromic Renshaw inhibition, which is non-cumulative this time as seen in B, are clear enough and need not be discussed in more detail.

229

In the last part of our presentation we will deal with the effects of succinylcholine (SCh) in order to further support the outlined concept. Since this drug is known t o induce strong excitation of primary muscle spindle endings as well as of secondary ones (Verhey and Voorhoeve, 1963), such experiments can furthermore give some hints as t o the recently disputed influence of the latter spindle afferents on the RCs. Henatsch and Schulte (1958) have shown long ago that under the influence of SCh the tonic stretch reflex responses of most Q units are considerably enhanced and prolonged. Even the large, originally phasic MNs are usually brought to more or less tonic stretch responses some time after SCh (see also Henatsch et al., 1959). A typical experiment is shown in Fig. 5. The two upper records are from two ventral rootlet filaments, containing mainly tonically firing o( cells (first record) and a phasic unit (second record). The third record is the concomitant stretch-induced discharge of a RC, belonging to the same pool. Its response pattern is as described earlier. Shortly after SCh, the reflex responses of the MNs are markedly enhanced: new units are recruited, and even the originally phasic unit now discharges during the plateau of stretch. The RC response is also extremely enhanced and prolonged, which indicates an increase of NRI in the pool. Fig. 6 gives more evidence for this (Meyer-Lohmann, 1965). Again, the antidromic stimulation technique is used to inhibit a tonically responding M N in a decerebrate cat whose general excitability was further enhanced by moderate hypocalcaemia due t o preceding removal of the parathyroid glands (Schulte et al., 1962). The antidromic inhibition is of the rapid cumulative type. After SCh, the stretch response is prolonged, but the flat part of the frequency curve remains at the same or at a slightly lower level as before. The antidromic inhibition is now weak and non-cumulative throughout the long Control

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stretch. In the next few minutes, the original inhibition type is gradually restored, concomitant with the decrease of the SCh effect. After 10 min, the control situation has approximately returned. Chemical spindle excitation by SCh also induces "spontaneous" reflex discharges of a-MNs, that is, in the absence of stretch stimulation. These, however, will in most cases only appear in small amplitude, with respect to stretch tonically responding units, whereas originally phasic types are rarely activated. Fig. 7 shows what happens in such cases lacking the supporting effects of stretch to the microrecorded RCs. Upper and lower halves are from two different experiments, the upper one with a decerebrate cat in good general condition. The filled dots are discharges of a single a-MN which in the control period showed a slow spontaneous background discharge. This was increased after SCh to more than 20 imp/sec and maintained, at slightly lower frequencies, for more

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Fig. 7. Effects of chemical excitation of muscle spindles by means of succinylcholine (1mg/ kg, i.v.) upon the “spontaneous” activity of GS a-motoneurones ( 0 )and homonymous Renshaw cells (0). Two different experiments. Upper part: decerebrate cat; lower part: cat anaesthetized with urethane-chloralose (400 mg/kg + 40 mg/kg i.p.). The activity of one 01motoneurone is shown in the upper part, while the averaged activity of 4 a-motoneurones is plotted in the lower part (reported by Henatsch e t al., 1975).

than 4 min. Other MNs have probably fired for similar periods after the injection. In contrast, the recorded RC activity (open circles) jumps from the previous level of about 20 imp/sec to a peak of 40 imp/sec, but falls back within the first minute. Either the excitatory action was actually restricted to this short time, despite the ongoing a firing, or a counteracting inhibitory effect from the spindles might have suppressed the further activation of the RC. The lower graphs are from a cat in moderate chloralose-urethane anaesthesia. Relatively weak spontaneous a activity appears after SCh, but is distinct for at least 2 min. The RC, however, firing around 20 imp/sec, shows no significant activity change at all after SCh. Again, we might suspect that some balance of excitatory and inhibitory influences prevents an increase of RC discharges. There is indeed a good chance for some hidden inhibitory influence on the RCs under the action of SCh, because this drug typically excites primary as well as secondary spindle endings, but no other known proprioceptors. It is known that the primaries have a t least no inhibitory effect on the RCs. For the secondaries, however, conducted in group I1 fibres, there are some recent experimental findings suggesting an inhibitory synaptic action directly on the

232

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RCs, as reported by Fromm et al. (1975).Our last figure (Fig. 8) gives some independent observations which might support this view: the cat was deeply anaesthetized with Nembutal, no a discharges appeared after SCh. Thus it can be assumed that no excitatory input reached the RC via recurrent a collaterals. The early response of the recorded RC, however, tested with single antidromic shocks, now showed a distinct reduction for 4-5 min after SCh, as shown in the graph. At present, we can think of no other SCh-induced source for this inhibitory effect than the secondary spindle afferents! ACKNOWLEDGEMENTS The authors thank Miss P. Terhaar for checking the English text. REFERENCES Denny-Brown, D. (1929) On the nature of postural reflexes. Proc. roy. SOC. B, 104: 252301. Eccles, J.C. Fatt, P. and Koketsu, K. (1954) Cholinergic and inhibitory synapses in a pathway from motor axon collaterals to motoneurones. J. PhysioZ. (Lond.), 126: 524562. Eccles, J.C., Eccles, R.M. and Fatt, P. (1956) Pharmacological investigations on a central synapse operated by acetylcholine. J. Physiol. (Lond.), 131 : 154-169. Fromm, Ch., Haase, J. and Wolf, E. (1975) Decrease of the recurrent inhibition of extensor motoneurones due to group I1 afferent input. Pfliigers Arch. ges. PhysioZ., Suppl. 359 : R78. Granit, R. and Rutledge, L.T. (1960) Surplus excitation in reflex action of motoneurones as measured by recurrent inhibition. J. Physiol. (Lond.), 154: 288-307. Granit, R., Henatsch, H.-D. and Steg, G. (1957) Tonic and phasic ventral horn c e l s differentiated by posttetanic potentiation in cat extensors. Actu physiol. scund., 37: 114126.

233 Grillner, S. and Udo, M. (1971a) Motor unit activity and stiffness of t h e contracting muscle fibres in the tonic stretch reflex. A c t a p h y s i o l . scand., 8 1 : 422-424. GrilIner, S. and Udo, M. (1971b) Recruitment in t h e tonic stretch reflex. A c t a physiol. scand., 8 1 : 571-573. Hellweg, C. and Meyer-Lohmann, J. ( 1 9 7 3 ) Responses of individual Renshaw cells t o ramp stretch of t h e triceps surae muscle. Pfliigers Arch. ges. Physiol., Suppl. 339: R76. Hellweg, C., Meyer-Lohmann, J., Benecke, R. and Windhorst, U. ( 1 9 7 4 ) Responses of Renshaw cells to muscle ramp stretch. E x p . Brain Res., 21 : 353-360. Henatsch, H.-D. und Schulte, F.J. (1958) Wirkungen chemisch erregter Muskelspindeln auf einzelne Extensor-Motoneurone der Katze. Pfliigers Arch. ges. Physiol., 267: 279294. Henatsch, H.-D., Schulte, F.J. und Busch, G. (1959) Wandelbarkeit des tonisch-phasischen Reaktionstyps einzelner Extensor-Motoneurone hei Variation ihrer Antriehe. Pfliigers Arch. ges. Physiol., 270: 161-173. Henatsch, H.-D., Benecke, R. and Hellweg, C. (1975) Behaviour of lumbar Renshaw cells and Ia-inhibitory interneurones during excitation of muscle spindles by means of succinylcholine. Pfliigers Arch. gees. Physiol., Suppl. 359: R79. Henneman, E. (1957) Relation between size of neurons and their susceptibility t o discharge. Science, 1 2 6 : 1345-1347. Kernell, D. (1976) Recruitment, rate modulation and the tonic stretch reflex. This volume, Ch. 21. Meyer-Lohmann, J. (1965) Die antidrom-recurrente Hernrnbarkeit uon Extensor-AlphaMotoneuronen bei asynchronen Anfriebsarten. Inaugural-Dissertation, Universitat Go ttingen. Meyer-Lohmann, J. und Henatsch, H.-D. ( 1 9 6 6 ) Die Bedeutung der naturlichen recurrenten Hemmung (NRH) fur die Frequenz tonisch entladender Extensor-a-Motoneurone. Pfliigers Arch. ges. Physiol., 2 9 1 : R 3 . Pompeiano, 0. and Wand, P. (1976) The relative sensitivity of Renshaw cells to static and dynamic changes in muscle length. This volume, Ch. 18. Renshaw, B. (1946) Central effects of centripetal impulses in axons of spinal ventral roots. J. Neurophysiol., 2 : 191-204. Ryall, R.W. and Piercey, M. (1971) Excitation and inhibition of Renshaw cells by impulses in peripheral afferent nerve fibers. J. Neurophysiol., 3 4 : 242-251. Schulte, F.J., Kaese, H.-J. und Meyer-Lohmann, J. ( 1 9 6 2 ) Die spinale Motorik hei experimenteller Hypocalcamie. Klin. Wschr., 4 0 : 246-250. VerheyJ3.A. and Voorhoeve, P.E. ( 1 9 6 3 ) Activation of group Ia and group I1 muscle spindle afferents by succinylcholine and other cholinergic drugs. A c t a p h y s i o l . pharmacol. neerl., 1 2 : 23-29.

DISCUSSION GRANIT: The problems we now face are so complicated that what is really necessary is t o record from a single Renshaw cell and stimulate a single motoneuron from inside t o see what really happens. But even then this might be misleading, because Renshaw cells are influenced from others. But if one has penetrated a single motoneuron, stimulated from inside, and finds a Renshaw cell to which it corresponds and finds its conduction velocity and other properties, then one will in the long r u n he able to map out what Renshaw cells d o t o different cells. That work has begun, b u t it is a long-lasting work and it will take some time before we know precisely how individual combinations work. POMPEIANO: I just want t o comment. When succinylcholine is used, we may have two complicating factors. One is, as you know, the occurrence of powerful presynaptic inhibition o n t h e Ia pathway after administration of this drug. The second is that since you use a precollicular decerebrated cat, you may activate with group I1 afferents the spino-bulbo-spinal or

spino-cerebello-spinal mechanisms and this may produce long-loop reflexes in group I1 afferents instead of Ia afferents. MEYER-LOHMANN: In this respect, I don’t think that presynaptic inhibition would b e very important but, o n the other hand, there might be long-loop reflexes acting o n the pathway from secondary endings. M. ITO: The functional significance of Renshaw cell inhibition has been raised in the last t w o papers, As Prof. Granit has pointed o u t , and several others have suggested in t h e past, three functional roles have been particularly emphasized. First of all, stabilization. In this case Renshaw cell inhibition seems to act as a n internal feedback loop by which input and o u t p u t relationship of motoneurons will be improved. Secondly, surrounding inhibition may be assumed. As Meyer-Lohmann has suggested this may select one group of neurons b y inhibiting surrounding groups of neurons. This is surrounding or lateral inhibition. Thirdly, t h e competition between phasic and tonic motoneurons has been assumed. This is based already on an old finding by Denny-Brown and o n this point Prof. Pompeiano and others provided new evidence. Well m y question, here, is how these 3 kinds of ideas are in conflict and how these 3 kinds of ideas are integrated. Is there anyone t o comment on this point? HENNEMAN: If one compares the effect of Renshaw inhibition evoked by antidromic stimulation o n t w o motoneurons from the same pool, in our experience, we have never been able to see that the Renshaw inhibition altered the order of recruitment of those t w o cells whether or not they were close together in threshold or they were far apart. So I have never felt really completely happy about the statement in t h e literature t h a t Renshaw cell inhibition is more powerful o n certain parts of the pool than o n others. It may be, but in the situation that we look a t it we don’t see that. One aspect of t h e problem that is often neglected, I think, is t h e fact that if you have two cells in t h e same pool firing, let’s say one is a small tonic and one is larger cell, t h e tonic cell is firing with a greater safety margin than the bigger cell. Then when you begin to apply a n inhihitory input, you always have t o remember that one of these cells has a safety margin which protects it against inhibition and therefore you don’t see really the effect of the Renshaw inhibition. What you are seeing is t h e safety margin effect.

Transmission in the Pathway of Reciprocal Ia Inhibition to Motoneurones and its Control during the Tonic Stretch Reflex H. HULTBORN Department of Physiology, University of Goteborg, Goteborg (Sweden)

I. INTRODUCTION It is now well established that nerve impulses in large muscle spindle (Ia) afferents give rise t o a monosynaptic excitation of motoneurones innervating the homonymous and synergic muscles and a concomitant short-latency inhibition of motoneurones t o antagonists. Though it was originally believed that Ia afferents have direct inhibitory access to motoneurones (Lloyd, 1941; hence the term “direct inhibition”), later studies proved that an interneurone is interpolated in the Ia inhibitory pathway (Eccles et al., 1956; Araki et al., 1960). The interneurone, henceforth called Ia inhibitory interneurone, was first merely viewed as a commutator-like device t o transform the excitatory action of primary afferents t o an inhibitory action (Eccles e t al., 1956), but it was soon realized that it might also act as a simple “integrative centre” (Eccles and Lundberg, 1958). To elucidate that question an extensive knowledge was needed of the convergence from other neuronal systems onto the Ia inhibitory interneurones. In general there are two different approaches for obtaining such information (Fig. 1A; cf., Lundberg, 1969): (1)a direct approach of recording from interneurones, or (2) the more “indirect” approach of studying how synaptic potentials evoked from primary afferents - in this case group Ia afferents - are influenced from other neuronal systems. I will first consider the latter “indirect” method. A graded electrical stimulation of group Ia afferents will discharge an increasing number of Ia inhibitory interneurones (50-100 interneurones act in parallel, see below) and thus cause a smooth increase in amplitude of the Ia IPSPs in motoneurones of an antagonist. When a weak stimulus strength is used only a small proportion of the interneurones is fired with the rest remaining in the subliminal fringe. Only a small IPSP is then evoked (first trace in Fig. 1B and C). A conditioning stimulation is then applied to another neuronal system, preferably with a stimulus strength so weak that the conditioning volley alone evokes no or almost no effect in the recorded motoneurone (second trace in Fig. 1B and C). If the neuronal system activated by the conditioning stimulus has an excitatory effect onto the Ia inhibitory interneurones the combination of the two stimuli will result in an increased test Ia IPSP (due t o convergence of subliminal excitatory synaptic actions which summate t o discharge common

236 A

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Fig. 1. A: diagram showing the disynaptic Ia inhibitory pathway to motoneurones. The recording electrodes illustrate the technique of direct recording from the interposed interneurones and the indirect technique of recording their synaptic effects in motoneurones. B-C: examples showing the technique of spatial facilitation and inhibition to deduce convergence on the interposed interneurone. D: schematic representation of some synaptic connexions to the interneurones in the Ia inhibitory pathway. i, ipsilateral; co, contralateral; cx, sensorimotor cortex; NR, red nucleus; ND, Deiters’ nucleus; FRA, flexor reflex afferents; cut., cutaneous; R,Renshaw cell. Interrupted lines indicate polysynaptic connexions.

interneurones, see third trace in Fig. 1B). On the other hand, a decrease or an abolishment of the test Ia IPSP on combined stimulation (third trace in Fig. 1C) indicates that the conditioning volley has an inhibitory effect on the interneurones, provided that it neither changes the membrane conductance in the moton’eurone nor gives presynaptic inhibition of transmission from the Ia afferents. The more direct approach is t o record directly from interneurones, but conclusions from such results are often difficult. This is because the projections of the recorded interneurones are usually unknown and therefore it is most often impossible to identify them as belonging t o a defined reflex pathway (see Lundberg, 1969). As will be reviewed below, a combination of both approaches in dealing with the reciprocal Ia inhibitory pathway has met with noticeable success. 11. “INDIRECT” EVIDENCE REGARDING CONVERGENCE ONTO Ia INHIBITORY INTERNEURONES

Fig. 1D summarizes schematically some of the excitatory and inhibitory connections on the Ia inhibitory interneurones revealed with the “indirect” method. I will only briefly comment on these various descending and segmental pathways. Descending excitatory control Volleys in several descending pathways have now been found t o facilitate Ia IPSPs in motoneurones. This was first established for the corticospinal tract

237 (Lundberg and Voorhoeve, 1962) and later also for the rubrospinal tract (Hongo et al., 1969). Facilitation of Ia IPSPs from the cortico- and rubrospinal tracts was seen in motoneurones to both flexors and extensors and occurred with a segmental latency indicating that the minimal linkage onto the Ia inhibitory interneurones is disynaptic (Hongo et al., 1969; Illert and Tanaka, 1976). Fig. 2A exemplifies these observations; in this case the transmission in the Ia inhibitory pathway to a knee flexor motoneurone was facilitated by volleys in the corticospinal tract. A weak stimulus t o the nerve of the antagonist knee extensor (quadriceps, Q; 1.05 X T) only evoked a very small IPSP on its own (second trace). In combination with the conditioning stimulus, however, a very large facilitation was observed (third trace). The transmission in the Ia inhibitory pathways to motoneurones supplying knee, hip and some ankle flexors as well as some hip extensors is facilitated also by volleys in the ipsilateral vestibulospinal tract (Grillner et al., 1966, Grillner and Hongo, 1972). In this case the onset of facilitation has such a brief latency that it must be due to monosynaptic excitation of the Ia inhibitory interneurones. Somewhat surprisingly it has also been found that volleys in the contralateral vestibulospinal tract facilitate the Ia inhibition to the same motor nuclei (Hongo et al., 1971; see also Grillner and Hongo, 1972). The contralateral effect on the Ia inhibitory interneurones is, however, disynaptic as indicated in Fig. 1D. Experiments by Jankowska et al. (1973) and by Illert and Tanaka (1976) have revealed that also some propriospinal fibre systems evoke monosynaptic excitation of interneurones mediating Ia inhibition.

Excitatory and inhibitory control from segmental afferents From the point of view of their reflex actions, high-threshold muscle afferents (groups I1 and 111), high-threshold joint afferents and cutaneous afferents are often grouped together as flexor reflex afferents (FRA, cf., e.g., Eccles and Lundberg 1959; Holmqvist and Lundberg, 1961). It has been shown that activation of polysynaptic ipsilateral and contralateral FRA pathways can facilitate transmission in the Ia inhibitory pathways t o both flexor and extensor motoneurones (Fedina and Hultborn, 1972;ten Bruggencate and Lundberg, 1974; Fedina et al., 1975). These results are exemplified in Fig. 2B, which illustrates facilitation of a Ia IPSP in a knee flexor motoneurone by conditioning stimulation of contralateral high-threshold joint afferents. It was, however, noticed that the facilitation from FRA was very dependent on the type of preparation (Fedina and Hultborn, 1972; Fedina et al., 1975); it was seen regularly in spinal cats under chloralose anaesthesia (Fig. 2B), while it was uncommon in the spinal unanaesthetized state and always absent in decerebrate cats with a low pontine lesion. In the latter preparation the test Ia IPSPs were on the contrary often depressed following a conditioning FRA volley indicating that the Ia inhibitory interneurones may also be inhibited from the FRA. As illustrated in Fig. 2C volleys in ipsilateral low-threshold cutaneous afferents do, however, facilitate Ia IPSPs also in the decerebrate preparation with low pontine lesion (Fedina and Hultborn, 1972). It was therefore postulated that the Ia inhibitory interneurones receive excitatory actions both from the FRA (transmission de-

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pressed in decerebrate state) and through a separate pathway from low-threshold cutaneous afferents. Any detailed discussion of FRA effects on Ia inhibitory transmission is quite complex because of the existence of several alternative FRA pathways and the fact that transmission through them may vary under different circumstances (Lundberg, 1973). The FRA effects on Ia inhibitory transmission were discussed extensively by Fedina et al. (1975) and Hultborn et al. (1976b).

Recurrent inhibition from motor axon collaterals Hultborn et al. (1971a) demonstrated that conditioning ventral root stimulation effectively depresses transmission in the Ia inhibitory pathways t o motoneurones. The effect was found on Ia IPSPs in practically all tested motoneurones, supplying different flexor or extensor muscles of the hindlimb. Since the depression occurred without any concomitant change in the membrane conductance of the recorded motoneurones and without changes in excitability of group Ia primary afferents, it was concluded that the observed effect was due to an inhibitory interaction at the interneuronal level in the Ia inhibitory pathway. By varying the interval between the conditioning and the testing volleys it was found that the depression occurred at an interval corresponding t o a

239 disynaptic linkage from motor axons to the Ia inhibitory interneurones. Furthermore, the time course of the depression was similar t o the time course of recurrent inhibition of motoneurones (Renshaw, 1941; Eccles et al., 1954). This and some other observations led t o the conclusion that the recurrent depression of the Ia IPSP was caused by postsynaptic inhibition of the Ia inhibitory interneurones from Renshaw cells. Thus, the pathway is similar t o the recurrent inhibitory pathway t o a-motoneurones (Eccles et al., 1954). The next step in the analysis was t o establish the relative contribution from different efferent nerves to the depression of transmission in Ia inhibitory pathways to different species of motoneurones (Hultborn et al., 1 9 7 1 ~ )It. was invariably found that the strongest depression of Ia IPSPs was evoked from motor fibres to those muscles whose Ia afferents produced the IPSPs. This means that activity in the same Ia afferents, which directly excites the interneurones, also indirectly evokes their recurrent inhibition (cf., Fig. 1D). The very finding of an extensive convergence from many neuronal systems in the Ia inhibitory pathway raises the question whether all interneurones in the pathway receive similar connexions or if there are functional subgroups characterized by different patterns of convergence. So far an attempt was made t o establish if supraspinal or segmental excitation and recurrent inhibition converge on the same Ia inhibitory interneurones. The method of facilitation and depression of test Ia IPSPs as indicators of an excitatory and inhibitory convergence onto interneurones was modified so that convergence from more than two systems could be tested simultaneously (Hultborn and Udo, 1972). If a test IPSP is due t o spatial facilitation between two excitatory convergent systems and this test is depressed from an inhibitory system, it can be concluded that all three systems involved converge onto the same interneurones. With this method it has been possible to show that the same Ia inhibitory interneurone receives convergence of excitation from any of the supraspinal or segmental pathways discussed above and inhibition from recurrent motor axon collaterals as drawn in Fig. 2D (Fedina and Hultborn, 1972, Hultborn 1972; Hultborn and Udo, 1972). Some results are exemplified in the lower traces of Fig. 2A-C; they show that the facilitated Ia IPSPs are almost obliterated by conditioning antidromic volleys in motor axons (compare third and fourth traces). 111. IDENTIFICATION OF THE INTERNEURONE MEDIATING RECIPROCAL Ia INHIBITION A group of interneurones which receives monosynaptic excitation from group Ia afferents and inhibition from recurrent motor axon collaterals has been found in a region just dorsomedial t o the motor nuclei (large filled circles in the drawing of the spinal cord grey matter in Fig. 3A; Hultborn et al., 1 9 7 1 ~ ) . Intracellular records in Fig. 3A illustrate the convergence of monosynaptic Ia EPSP and recurrent inhibition in an interneurone of this type. On the other hand, no recurrent inhibition could be detected in interneurones with monosynaptic Ia excitation in the intermediate nucleus (points in Fig. 3A). These findings casted doubt on the original hypothesis that inhibition from group Ia afferents t o motoneurones is mediated by the la activated interneurones in the

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Fig. 3. A: the location of interneurones with convergence of monosynaptic Ia excitation and disynaptic recurrent inhibition is shown by large filled circles. The points denote the interneurones without recurrent inhibition. The hatched areas indicate the extent of motor nuclei. The right side of the diagram shows Rexed’s laminae. The records exemplify the convergence of Ia excitation from anterior biceps-semimembranosus (ABSm) and recurrent inhibition in one interneurone located in the ventral horn. The upper traces are intracellular potentials (positivity upwards) and the lower traces cord dorsum potentials t o monitor the incoming volley (negativity upwards). (Modified from Hultborn et al., 1971b.) B: the experiment proving projection of Ia inhibitory interneurones t.0 its target motoneurones. The interneurone was functionally identified since it received monosynaptic Ia excitation and recurrent inhibitioc. Simultaneous recording was made extracellularly from the interneurone and intracellularly from its target motoneurone (diagram). Parallel records of spikes in the interneurone (lower traces) and small unitary IPSPs (dashed lines) in the motoneurone (upper traces). Below is shown averaged records triggered by the spikes in the interneurone. (Modified from Jankowska and Roberts, 197213.)

intermediate region (Eccles et al., 1956). A comparison of the properties and the patterns of convergence onto these two groups of Ia coupled interneurones with those required for interneurones in the Ia inhibitory pathway to motoneurones, as inferred from studies on transmission in the pathway, led Hultborn et al. (1971b) to suggest that reciprocal Ia inhibition to motoneurones is mediated only via the Ia activated interneurones in the ventral horn. Subsequent studies by Jankowska and Roberts (1972a, b) have given the final proof; by simultaneous recording from Ia interneurones in the ventral horn (convergence of Ia excitation and recurrent inhibition) and their supposed target motoneurones they were able t o show that action potentials in the interneurones evoked monosynaptic unitary IPSPs in the motoneurones as illustrated in Fig. 3B. The amplitudes of the unitary IPSPs ranged between 8-220 pV and were 10-200 times smaller than the maximal Ia IPSPs in the same motoneurones thus a large number of Ia inhibitory interneurons seem t o converge onto a

24 1

single motoneurone. The distribution of axonal termination of individual Ia inhibitory interneurones was determined by the area from which they can be antidromically activated (Jankowska and Roberts, 1972a). By a combination of data on axonal distribution and on unitary IPSPs it was estimated that individual quadriceps-coupled Ia inhibitory interneurones project to about 20% of all knee flexor posterior biceps and semitendinosus motoneurones. With the series of experiments just described, the disynaptic Ia inhibitory pathway to motoneurones became the first interneuronal pathway in the mammalian CNS which has been fully identified physiologically in all parts. The morphology of these interneurones was later studied with intracellular injection of Procion Yellow (Jankowska and Lindstrom, 1972). Besides valuable information on the precise location of these interneurones, their shape, size and so on, it was clearly illustrated that the axon may project either t o the ipsilateral ventral or lateral funiculi (depending on cell location) and that some axons bifurcate into an ascending and a descending branch within the funiculi. IV. EXCITATORY AND INHIBITORY CONVERGENCE AS JUDGED BY DIRECT RECORDING FROM IDENTIFIED Ia INHIBITORY INTERNEURONES Direct recording from Ia inhibitory interneurones has now considerably enhanced our knowledge of the control of Ia reciprocal inhibition (Hultborn et al., 1976a, b, c). With direct recording it was much easier t o verify that excitatory actions from several segmental and supraspinal systems converge onto individual Ia inhibitory interneurones. Quantitative comparisons of excitation or inhibition from different sources could easily be obtained and so are the latencies and time courses of the converging effects. Most segmental and descending systems evoke polysynaptic effects in the Ia inhibitory interneurones. It has now been shown (Hultborn et al., 1976a, b, c) that several of these systems share interneurones finally affecting the Ia inhibitory interneurones. These questions have been approached by the technique of spatial facilitation - now applied at interneuronal level. In these experiments several new reflex connexions were also revealed.

Mutual inhibition between “opposite” l a inhibitory interneurones Most surprisingly it was found that the Ia inhibitory interneurones themselves seem t o receive disynaptic Ia inhibition (Hultborn et al., 1976a). These connexions are exemplified in Fig. 4 by extra- and intracellular records from a Ia inhibitory interneurone (cf., the recording microelectrode in Fig. 40). The monosynaptic excitation from the knee flexor PBSt nerve (Fig. 4 A, D, F-H) suggests that the interneurone may inhibit motoneurones to the knee extensor quadriceps. Stimulation of Ia afferents from quadriceps evoked a disynaptic inhibition (Fig. 4 J-L; see also D, E and N), which was depressed by antidromic stimulation of the L5 + L6 ventral roots (Fig. 4L, M). Similar inhibition has been found from group Ia afferents from flexors in interneurones excited from extensors. The muscles from which this inhibition was evoked were al-

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ways strictly antagonistic to those supplying their Ia excitation. Similarly to the Ia inhibition in motoneurones, the Ia inhibition in the Ia inhibitory interneurones was always decreased after conditioning stimulation of ventral roots. Furthermore, transmission of Ia inhibition to the Ia inhibitory interneurones was also facilitated from ipsilateral and contralateral primary afferents as well as several supraspinal pathways analogous t o earlier findings for the la inhibition of motoneurones. The pattern and the control of the Ia inhibition of motoneurones and of Ia inhibitory interneurones do in fact show such striking similarities that it is most likely that identical interneurones are responsible. This in turn leads t o the conclusion that “opposite” Ia inhibitory interneurones (i.e., interneurones monosynaptically connected t o antagonistic muscles) are mutually inhibiting each other as drawn in Fig. 5. If so, an increased activity in Ia afferents from one muscle should secure an increased excitability of homony-

243

Fig. 5 . Schematical drawing of parallel effects o n t o 01- and 7-motoneurones innervating a muscle and t h e Ia inhibitory interneurones inhibiting t h e motoneurones of its antagonist. Note also t h e mutual inhibition between “opposite” Ia inhibitory interneurones.

mous motoneurones not only by direct monosynaptic excitation but also by releasing them from a possible reciprocal Ia inhibition. The possible functional significance of this organization during flexion-extension movements was discussed by Hultborn et al. (1976a). Effects from other segmental afferents (Hultborn et al., 1976b) Volleys in ipsilateral and contralateral flexor reflex afferents evoked a mixture of polysynaptic excitation and inhibition in the Ia inhibitory interneurones. However, interneurones excited monosynaptically from flexor nerves received stronger net excitation from the ipsilateral FRA than did extensor coupled interneurones, while the opposite pattern was seen from the contralateral FRA. These patterns are similar t o those found in flexor and extensor motoneurones respectively. The FRA pathways in the acute spinal cat seem thus t o evoke their effects in parallel in a-motoneurones and in Ia inhibitory interneurones which are monosynaptically excited by la afferents from the same muscles. Effects from descending pathways Stimulation of several supraspinal systems (Hultborn et al., 1976c) has

244

shown that Ia coupled interneurones in the ventral horn - as should be expected - are subject to all descending actions postulated from earlier indirect evidence (see Section 11). Intracellular recording has, however, revealed that the effects are not as simple as previously thought, since volleys in the tested tracts usually evoked a mixture of excitation and inhibition (Hultborn et al., 1 9 7 6 ~ ) . Some findings of special importance for the later conclusions will be summarized in the following. Volleys in the vestibulospinal tract may evoke not only monosynaptic EPSPs (cf., Section 11) but also disynaptic EPSPs in extensor coupled Ia inhibitory interneurones. Flexor coupled interneurones instead receive disynaptic inhibition. This shows again the similarity of effects evoked in a-motoneurones; extensor a-motoneurones receive mono- and disynaptic excitation and flexor motoneurones disynaptic inhibition from the vestibulospinal tract (Grillner et al., 1970). Volleys in the rubrospinal tract evoked disynaptic and polysynaptic EPSPs in flexor as well as extensor coupled interneurones in accordance with earlier “indirect” results (see Section 11). Now it was also revealed that the dominating excitation is usually mixed with di- and polysynaptic inhibition. From spatial facilitation of effects evoked from the red nucleus and from other fibre systems, it was concluded that the same interneurones transmitted these disynaptic rubrospinal EPSPs and IPSPs and excitation and inhibition from primary afferents, especially low-threshold cutaneous afferents. These findings in Ia inhibitory interneurones are thus similar to those previously found for motoneurones (Baldissera et al., 1971). Also the pyramidal tract gave rise to a dominating polysynaptic excitation, usually mixed with inhibition, in flexor as well as extensor coupled Ia inhibitory interneurones. Pyramidal volleys were also shown to facilitate transmission in various segmental reflex pathways to the Ia inhibitory interneurones very much in the same way as earlier described for motoneurones (Lundberg and Voorhoeve, 1962). To summarize: direct recording from the Ia inhibitory interneurones has enabled a detailed description of the convergence of the descending and segmental fibre systems onto them and of the interactions between several of these systems at earlier interneuronal stations. This in turn has permitted a comparison of effects onto a-motoneurones (known since before) and onto Ia inhibitory interneurones. There is indeed a striking parallelism of actions onto a-motoneurones and onto l a inhibitory interneurones with the same monosynaptic l a input, henceforth referred to as “corresponding” a-motoneurones and l a inhibitory interneurones. Even the detailed organization of effects onto these two types of neurones appears to be very similar; thus descending pathways facilitate the corresponding effects from primary afferents in parallel t o both neurones. It is also noteworthy that the parallelism in convergence to corresponding motoneurones and Ia inhibitory interneurones is not restricted to their excitatory input but extends to inhibitory inputs as well; for segmental systems this is exemplified by Renshaw inhibition (Hultborn et al., 1 9 7 1 ~Ia )~ inhibition and FRA inhibition (Hultborn et al., 1976a, b) and for supraspinal systems by inhibition from vestibulospinal, rubrospinal and corticospinal pathways (Hultborn et al., 1 9 7 6 ~ ) .

24 5

V. FUNCTIONAL ASPECTS OF PARALLEL EFFECTS ONTO CORRESPONDING a-MOTONEURONES AND Ia INHIBITORY INTERNEURONES Many descending as well as segmental neuronal pathways have been shown t o evoke parallel effects on a- and y-motoneurones supplying a given muscle (Granit, 1955, 1970; Matthews, 1972). This led Granit (1955) t o postulate that movements often depend on a coactivation of a-and y-motoneurones. According t o this hypothesis a neuronal system with “a-y linked” actions will exert synaptic depolarization of a-motoneurones via two routes, the “direct” a-route and the “indirect” y-route. This implies that the discharge of many a-motoneurones will depend on spatial facilitation from the “direct” a- and the “indirect” y-routes. For a number of naturally occurring movements it has been possible t o demonstrate that the afferent discharge from spindles actually increases during muscle shortening; in quiet breathing (Eklund et al., 1964), in stepping movements (Severin et al., 1967) and finally in voluntary movements in man (Vallbo, 1970a, b). For the present discussion it is not so important to which extent the intrafusal activation is effectuated by coactivated y-motoneurones or by @-fibres,i.e., by motor axons innervating both extra- and intrafusal muscle fibres - the significance of the latter has recently been reemphasized (Emonet-Dknand et al., 1975) and a full discussion of this topic can be found in this volume (Laporte, Session I). What is important is the fact that the tendency for unloading of muscle spindles during extrafusal shortening is well compensated by intrafusal contraction, thus allowing the stretch reflex t o operate even during shortening. If the shortening of a contracting muscle is opposed, an increased discharge in the Ia afferents would further augment motoneuronal firing and thereby add force t o the contraction. To produce well coordinated movements it seems t o be of importance that the reciprocal inhibition of antagonists is coupled t o and coincides with the excitation of agonists. Hongo et al. (1969) and Lundberg (1970) suggested that neuronal pathways which evoke a - y linked movements may achieve a coupling between excitation of agonists and inhibition of antagonists by exciting not only a- and y-motoneurones t o agonists, but also Ia inhibitory interneurones impinging on motoneurones of antagonists (conclusions in Section IV and Fig. 5). Notice that corresponding a-motoneurones and Ia inhibitory interneurones would be governed by the similar convergence from “direct” and “indirect” routes, hence the term “a-y linkage in the reciprocal inhibition” (Hongo et al., 1969). In this scheme the servo-assistance given by the y-loop (cf., Matthews, 1972, pp. 546-611) will thus support not only the contraction of agonists but also the relaxation of antagonists. Though the parallel connexions onto the 3 types of neurones (Fig. 5) certainly exist as required for the idea of an “a-y linkage in the reciprocal inhibition” (summarized in Section IV), our type of experiments cannot show whether the central nervous system indeed operates them in accordance with that hypothesis. The functional interpretation must obviously be tested by other experimental approaches. Fortunately some evidence to this effect is now emerging. Feldman and Orlovsky (1975) recently succeeded in recording from Ia inhibitory interneurones during locomotion in mesencephalic cats. They

found that the firing of the Ia inhibitory interneurones coincides with the activation of the muscle which supplies their la afferent input. From experiments on deefferented hindlimbs, i.e., under conditions when the y-loop was broken, they concluded that the normal activation of the interneurones was due to convergent excitation from group Ia afferents and from the central system which generates the stepping movements, thus demonstrating for the first time the operation of an “0-7linkage in the reciprocal inhibition”. Recent experiments by Tanaka (summarized in this volume, Session V) suggest that “a-y linked reciprocal inhibition” also operates in man.

VI. QUANTITATIVE EVALUATION OF THE RECURRENT INHIBITION OF Ia INHIBITORY INTERNEURONES From experiments with single electrical shocks applied to peripheral nerves to ventral roots (Sections 11-IV), we know that activity in group Ia afferents from a given muscle will cause direct monosynaptic excitation of Ia inhibitory interneurones and recurrent inhibition of the same interneurones if homonymous motoneurones were activated (cf., Fig. 1D). It is not known, however, to which extent an increment in Ia excitation will be counteracted by augmented recurrent inhibition. It should be realized that this problem is not limited t o the excitation from group Ia afferents, which is only a special case of a most general question since several segmental and descending pathways evoke parallel effects onto corresponding a-motoneurones and Ia inhibitory interneurones. In an attempt to elucidate this problem Fu, Hultborn, Larsson and Lundberg (Hultborn and Lundberg, 1972; and unpublished) investigated reciprocal inhibition of knee flexor motoneurones, posterior biceps and semitendinosus (PBSt) during the tonic stretch reflex in the knee extensor quadriceps (Q) muscle. Tonic inhibition of transmission in reflex pathways from the FRA and group Ib afferents in the decerebrate cat (Eccles and Lundberg, 1959) turned out to be important for the interpretations and all the results illustrated in Figs. 6 and 7 and discussed in the following refer to experiments with an efficient tonic inhibition of these pathways. In exceptional cats without stretch reflexes there was a linear relationship between the length of the quadriceps muscle, the frequency of group Ia afferents from this muscle and the inhibition of the monosynaptic test reflex from the posterior biceps-semitendinosus nerves, as illustrated in Fig. 6B ( 0 , Ia frequency; X , reciprocal inhibition). A linear relationship between Ia firing rate and reciprocal inhibition is seen also in graph C during the initial extension. With the appearance of a static stretch reflex at 7-8 mm of extension, however, a plateau appears in the inhibitory curve although both tension (0) and EMG (shown in E) in the quadriceps muscle increase with further extension, The plateau in graph C suggests that at different levels of muscle extension the increments in Ia excitation and recurrent inhibition in the la inhibitory interneurones almost exactly balance each other. In many experiments the plateau was maintained only for a certain range of extensions and with further stretch of the muscle the inhibition increased again as in graph D. For an interpretation of the increasing reciprocal inhibition during vigorous

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stretch reflexes it is of relevance that Grillner and Udo (1971a, b) have shown a pronounced non-linearity in motoneuronal activity during “good” stretch reflexes in the soleus muscle. At increasing lengths the recruitment of new motoneurones was successively reduced and each individual motoneurone stabilized its firing frequency soon after recruitment. The linear relation between length and tension at greater lengths was instead largely due t o the stiffness of already active muscle fibres. In this situation one would expect an imbalance between the two opposing processes controlling the Ia inhibitory interneurones with increasing muscle lengths. The linear increase in Ia excitatory action would be met with a successively decreased increment in recurrent inhibition, which

248 would result in an increased reciprocal inhibition with higher degrees of extension. A closer inspection of the graph in D and of the corresponding EMG records in F shows that the plateau in the inhibitory curve between 4 and 7 mm extension indeed corresponds t o an increase in EMG activity while the EMG appears t o be at least partially saturated with extensions beyond 7 mm. A further analysis indicates that the increment in inhibition at higher degrees of extension was due not only to an increased activity in the Ia inhibitory pathway but also to another mechanism. Excitability measurements (ad modum Wall, 1958) revealed that a depolarization was evoked in terminals of the Ia afferents mediating the test reflex corresponding to the increment in inhibition. Fig. 7 indicates that the presynaptic depolarization would produce a presynaptic inhibition of roughly the same order of magnitude as the increment of inhibition. The EMG activity in the experiment of Fig. 7 commenced at an extension of about 4 mm (but was quite small up t o about 7 mm) and then continued t o grow up t o 1 2 mm. In this extreme case almost no tendency for a plateau could be discerned in the inhibitory curve (X). There was, however, a steady increase in the excitability of the PBSt Ia afferents ( O ) , which roughly paralleled the inThe following procedure was then adopted t o “subcrease in active tension (0). tract” the presynaptic part from the total effects in order t o obtain an estimate of the postsynaptic inhibition. Different degrees of excitability increase in the PBSt Ia terminals were elicited with a short train of graded electrical stimuli (within the group I range) applied t o the deep peroneal nerve. The effect of the same stimuli on the monosynaptic test reflex was then checked. Since the effects of this stimulation on the PBSt monosynaptic reflex should be of purely presynaptic nature, it was possible t o obtain a relation between the increase in excitability of PBSt group Ia afferents and presynaptic inhibition of the PBSt monosynaptic reflex (graph B in Fig. 7). Using this relation the inhibition

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249 caused by primary afferent depolarization during the stretch reflex (the shaded area in Fig. 7A) was subtracted from the total inhibition. The resulting curve (v) should represent the postsynaptic inhibition only. From the diagram it is clear that the postsynaptic inhibition stays virtually constant with extensions from 8 t o 12 mm which again suggests a reasonable balance between the two opposing systems controlling the excitability of Ia inhibitory interneurones. The large shaded area in Fig. 7A indicates that the presynaptic inhibition evoked during the stretch reflex in the quadriceps muscle can be very pronounced, though presynaptic inhibition evoked from extensors in general is weaker than that from flexors (Eccles et al., 1963). In this connexion it is of interest to recall the experiments on reciprocal inhibition by Liddell and Sherrington (1925). They used the stretch reflex in the quadriceps muscle t o monitor the degree of reciprocal inhibition evoked by weak pulling of the hamstring muscles. A quite powerful inhibition was produced, but it proved t o be quite resistant t o strychnine. From our present knowledge of the sensitivity of Ia IPSPs to strychnine (Bradley et al., 1953), it must be concluded that most of the inhibition studied by Liddell and Sherrington was of presynaptic origin. Though the presynaptic inhibition has been treated rather as a complicating factor obscuring the control of the Ia inhibitory interneurones (our primary interest in this study), it should be kept in mind that they of course may be of great significance in the normal motor control. The experiments on the control of Ia inhibitory interneurones during the tonic stretch reflex are still preliminary and an extensive discussion on conceivable complications by activity in group Ib and I1 afferents has t o be postponed. Already at this stage it should, however, be noticed that it is unlikely that Ib inhibition of the Ia inhibitory interneurones is important for the plateau in reciprocal inhibition (Fig. 6C and D). The reason is that even minute Ib IPSPs were rarely seen on intracellular recording from Ia inhibitory interneurones while recurrent inhibition was always very prominent (Hultborn e t al., 197613). Also supporting that opinion is the fact that reciprocal inhibition increases when the EMG saturates (cf., Fig. 6D, i.e., when the excess of Ia excitation is not matched by recurrent inhibition) although active tension continues t o increase. To summarize: the observations made so far seem t o indicate that there is a reasonable balance between the two opposing systems (i.e., Ia excitation and recurrent inhibition) controlling the excitability of the Ia inhibitory interneurones when an excess of Ia excitation produced an increased motoneuronal firing in the tonic stretch reflex. Under the highly speculative assumption that the balance between direct excitation and recurrent inhibition during the stretch reflex also holds for other segmental and descending systems, it would suggest that recurrent inhibition of the Ia inhibitory interneurones may serve as a segmental autoregulatory mechanism that prevents “a-y linked reciprocal inhibition” from getting too deep during increased activity in agonist a-motoneurones. The rationale for such an organization may be related t o the role of inhibition t o prevent firing. A command for increased muscular force obviously requires increased excitation of agonistic motoneurones, but not necessarily a corresponding increase of the reciprocal inhibition. Indeed an “unnecessarily” strong inhibition may com-

250 promise the activation of these motoneurones should the need arise. There are various types of motor performances which may require different degrees of reciprocal inhibition. It can thus be expected that reciprocal inhibition is of importance in “simple” flexion-extension movements while it may rather be deleterious during a cocontraction of antagonists in order to stabilize a joint. There is evidently not only a need for a “constant” reciprocal inhibition during different degrees of agonist contraction, but also for some mechanism t o choose the proper level of inhibition. In this connexion it is of interest that Renshaw cells are subject to spinal as well as supraspinal control (see Hultborn et al., 1971c, for ref.) which can either enhance or depress transmission in the recurrent pathway and thus set the sensitivity of the recurrent autoregulatory mechanism. It may be assumed that there is a need for some excitatory summation at Renshaw cells (i.e., that the motoneuronal activity must reach a certain level) before they start t o transmit recurrent inhibition. When this threshold is reached further increments in Ia excitation and recurrent inhibition may balance each other in the Ia inhibitory interneurones as discussed above. During flexion-extension movements, in which use of effective reciprocal inhibition was postulated, transmission in the recurrent pathway should be inhibited. This would mean that a larger excitation from motor axon collaterals (i.e. more motoneuronal activity) on Renshaw cells is needed in order to overcame the inhibition and allow transmission of recurrent inhibition. Until that increased threshold is reached the reciprocal Ia inhibition can grow unchecked. The recent evidence by Feldman and Orlovsky (1975) that the recurrent inhibition of Ia inhibitory interneurones is depressed during stepping is thus well in keeping with that idea. Facilitation of Renshaw cells would on the contrary diminish the need for excitatory summation from motor axon collaterals. Even the slightest activity of agonist a-motoneurones may give rise t o recurrent inhibition of Ia inhibitory interneurones and thereby keep the reciprocal inhibition very small, as may be needed in non-reciprocal movements with cocontraction of antagonists. EPILOGUE

,

It may be asked where the ever more detailed and seemingly diverse results on the convergence onto single interneurones will lead us. I think, however, that the long series of investigations reviewed in this paper indeed shows how it is possible to bring together the details into a few coherent hypotheses. It is now imperative that these ideas are subjected t o functional tests. The importance of a further analysis is emphasized by recent findings showing that the Ia inhibitory pathway in primates, including man, seems t o be organized in much the same way as in cats (Mizuno et al., 1971; Jankowska et al., unpublished). Indeed, the first evidence of an operating “a-y linkage in the reciprocal inhibition” during voluntary movements was obtained in man (Tanaka, this volume). The recurrent inhibition which is evoked in the Ia inhibitory interneurones following activity in their corresponding a-motoneurones (cf., Fig. 6) seems simple enough to offer a chance of functional understanding. It is probably crucial t o recognize that the recurrent inhibition acts in parallel onto corres-

251

ponding a-motoneurones and Ia inhibitory interneurones and that the functions of these projections probably are inseparable. Although the pathways subserving reciprocal Ia inhibition and recurrent inhibition belong to the best analyzed neuronal pathways in the central nervous system, we are clearly far from a detailed understanding of how they contribute to the neuronal motor control.

SUMMARY The wide excitatory and inhibitory convergence on the interneurones mediating the disynaptic Ia reciprocal inhibition first emerged from studies on facilitation and inhibition of test Ia IPSPs recorded in motoneurones. They revealed that several descending (cortico-, rubro- and vestibulospinal tracts) as well as segmental pathways excite the interneurones mediating reciprocal Ia inhibition. It was also found that antidromic stimulation of ventral roots can depress transmission in the Ia inhibitory pathway by an inhibition of the interposed interneurone. Interneurones with monosynaptic Ia excitation and all postulated convergence from descending and segmental pathways were found in the ventral horn just dorsoinedial to the motor nuclei. These interneurones were later shown to produce monosynaptic IPSPs in motoneurones, thus finally proving that they mediate the reciprocal Ia inhibition to motoneurones. Recent studies with direct recording from these interneurones have considerably increased the knowledge of the synaptic convergence onto them. They led to the important conclusion that there is a striking parallelism of actions onto a-motoneurones and onto Ia inhibitory interneurones with the same monosynaptic Ia input. The functional significance of the excitatory convergence from Ia afferents and descending as well as segmental pathways on the interneurones mediating reciprocal Ia inhibition (“a-y linkage in the reciprocal inhibition”) was discussed in relation to the total a y linked servo-control of movements. The recurrent inhibition of the interneurones mediating the “a-y linked reciprocal inhibition” may serve as a segmental autoregulatory mechanism that prevents the reciprocal inhibition from getting too deep during increased a y linked excitation of agonists. It emerges that the interneurones in the reciprocal Ia inhibitory pathway serve as efficient integrative centres.

ACKNOWLEDGEMENTS The author is indebted to T.-C. Fu, R. Larsson and A. Lundberg for permission to reproduce and discuss unpublished data and t o E. Jankowska, S. Lindstrom and A. Lundberg for valuable comments on the manuscript. Part of this work was supported by the Swedish Medical Research Council (Project No. 4500).

252 REFERENCES Araki, T., Eccles, J.C. and Ito, M. (1960) Correlation of the inhibitory postsynaptic potential of motoneurones with the latency and time course of inhibition of monosynaptic reflexes. J. Physiol. (Lond.), 154: 354-377. Baldissera, F., Bruggencate, G. ten and Lundberg, A. (1971) Rubrospinal monosynaptic connexion with last order interneurones of polysynaptic reflex paths. Brain Res., 27: 390-392. Bradley, K., Easton, D.M. and Eccles, J.C. (1953) An investigation of primary or direct inhibition. J. Physiol. (Lond.), 122: 474-488. Bruggencate, G. ten and Lundberg, A. (1974) Facilitatory interaction in transmission t o motoneurones from vestibulospinal fibres and contralateral primary afferents. Exp. Brain Res., 1 9 : 248-270. Eccles, J.C., Fatt, P. and Koketsu, K. (1954) Cholinergic and inhibitory synapses in a pathway from motor-axon collaterals t o motoneurones. J. Physiol. (Lond.), 126: 524562. Eccles, J.C., Fatt, P. and Landgren, S. (1956) The central pathway for the direct inhibitory action of impulses in the largest afferent nerve fibres to muscle. J. NeurophysioZ., 19: 75-98. Eccles, J.C., Schmidt, R.F. and Willis, W.D. (1963) Depolarization of central terminals of group Ib afferent fibres of muscles. J. Neurophysiol., 26: 1-27. Eccles, R.M. and Lundberg, A. (1958) Integrative patterns of Ia synaptic actions on motoneurones of hip and knee muscles. J. Physiol. (Lond.), 144: 271-298. 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. Eklund, G., von Euler, C. and Rutkowski, S. (1964) Spontaneous and reflex activity of intercostal gamma motoneurones. J. Physiol. (Lond.), 1 7 1: 139-163. Emonet-DQnand, F., Jami, L. and Laporte, Y. (1975) Skeleto-fusimotor axons in hindlimb muscles of the cat. J. Physiol. (Lond.), 249: 153-166. Fedina, L. and Hultborn, H. (1972) Facilitation from ipsilateral primary afferents of interneuronal transmission in the Ia inhibitory pathway to motoneurones. A c t a physiol. scand., 86: 59-81. Fedina, L., Hultborn, H. and Illert, M. (1975) Facilitation from contralateral primary afferents of interneuronal transmission in the La inhibitory pathway to motoneurones. Acta physiol. scand., 94: 198-221. Feldman, A.G. and Orlovsky, G.N. (1975) Activity of interneurones mediating reciprocal Ia inhibition during locomotion. Brain Res., 8 4 : 181-194. Granit, R. (1955) Receptors and Sensory Perception, Yale University Press, New Haven, Conn., 369 pp. Granit, R. (1970) The Basis o f M o t o r Control, Academic Press, London, 346 pp. Grillner, S. and Hongo, T. (1972) Vestibulospinal effects on motoneurones and interneurones in the lumbosacral cord. In Basic Aspects of Central Vestibular Mechanisms, Progress in Brain Res., Vol. 37, A. Brodal and 0. Pompeiano (Eds.), Elsevier, Amsterdam, pp. 244-262. Grillner, S. and Udo, M. (1971a) Motor unit activity and stiffness of the contracting muscle fibres in the tonic stretch reflex. Actaphysiol. scand., 81: 422-424. Grillner, S. and Udo, M. (1971b) Recruitment in the tonic stretch reflex. A c t a physiol. scand., 81: 571-573. Grillner, S., Hongo, T. and Lund, S. (1966) Interaction between the inhibitory pathways from the Deiters’ nucleus and Ia afferents t o flexor motoneurones. A c t a physiol. scand., 68, Suppl. 277: 61. Grillner, S., Hongo, T. and Lund, S. (1970) The vestibulospinal tract. Effects on alphamotoneurones in the lumbosacral cord in the cat. Exp. Brain Res., 10: 94-120. Holmqvist, B. and Lundberg, A. (1961) Differential supraspinal control of synaptic actions evoked by volleys in the flexion reflex afferents in alpha motoneurones. A c t a physiol. scand., 54, Suppl. 186: 1-51. Hongo, T., Jankowska, E. and Lundberg, A. (1969) The rubrospinal tract. 11. Facilitation of

253 interneuronal transmission in reflex paths to motoneurones. Exp. Brain Res., 7 : 365391. Hongo, T., Kudo, N. and Tanaka, R. (1971) Effects from the vestibulospinal tract on the contralateral hindlimb motoneurones in the cat. Brain Res., 31 : 220-223. Hultborn, H. (1972) Convergence on interneurones in the reciprocal Ia inhibitory pathway t o motoneurones. Acta physiol. scand., 85, Suppl. 375. Hultborn, H. and Lundberg, A. (1972) Reciprocal inhibition during the stretch reflex. A c t a physiol. scand., 85: 136-138. Hultborn, H. and Udo, M. (1972) Convergence in the reciprocal Ia inhibitory pathway of excitation from descending pathways and inhibition from motor axon collaterals. Acta physiol. scand., 84: 95-108. Hultborn, H., Jankowska, E. and Lindstrom, S. (1971a) Recurrent inhibition from motor axon collaterals of transmission in the Ia inhibitory pathway t o motoneurones. J. Physiol. (Lond.), 215: 591-61 2. Hultborn, H., Jankowska, E. and Lindstrom, S. (1971b) Recurrent inhibition of interneurones monosynaptically activated from group Ia afferents. J. Physiol. (Lond.), 215 : 6 13-6 36. Hultborn, H., Jankowska, E. and Lindstrom, S. ( 1 9 7 1 ~Relative ) contribution from different nerves to recurrent depression of Ia IPSPs in motoneurones. J. Physiol. (Lond.), 215: 6 37-66 4. Hultborn, H., Illert, M. and Santini, M. (1976a) Convergence on interneurones mediating the reciprocal Ia inhibition of motoneurones. I. Disynaptic inhibition of Ia inhibition interneurons. Acta physiol. scand., 96: 193-201. Hultborn, H., Illert, M. and Santini, M. (1976b) Convergence on interneurones mediating the reciprocal Ia inhibition of motoneurones. 11. Effects from segmental flexor reflex pathways. Acta physiol. scand., 96: 351-367. Hultborn, H., Illert, M. and Santini, M. ( 1 9 7 6 ~Convergence ) on interneurones mediating the reciprocal Ia inhibition of motoneurones. 111. Effects from supraspinal pathways. A c t a physiol. scand., 96: 368-391. Illert, M. and Tanaka, R. (1976) Transmission of corticospinal IPSPs to cat forelimb motoneurones via high cervical propriospinal neurones and Ia inhibitory interneurones. Brain Res., 103: 143-146. Jankowska, E. and Lindstrom, S. (1972) Morphology of interneurones mediating Ia reciprocal inhibition of motoneurones in the spinal cord of the cat. J. Physiol. ( L o n d . ) , 226: 805-82 3. Jankowska, E. and Roberts, W.J. (1972a) An electrophysiological demonstration of the axonal projections of single spinal interneurones in the cat. J. Physiol. ( L o n d . ) , 222: 597-622. Jankowska, E. and Roberts, W.J. (1972b) Synaptic actions of single interneurones mediating reciprocal Ia inhibition of motoneurones. J.Physio2. (Lond.), 222: 6 2 3 4 4 2 . Jankowska, E., Lundberg, A. and Stuart, D. (1973) Propriospinal control of last order interneurones of spinal reflex pathways in the cat. Brain Res., 53: 227-231. Liddell, E.G.T. and Sherrington, C.S. (1925) Further observations on myotatic reflexes. Proc. roy. SOC.B , 97: 267-283. Lloyd, D.P.C. (1941) A direct central inhibitory action of dromically conducted impulses. J. Neurophysiol., 4: 184-190. Lundberg, A. (1969) Convergence of excitatory and inhibitory action on interneurones in the spinal cord. In The Interneurone, UCLA Forum Med. Sci., No. 1 1 , M.A.B. Brazier (Ed.), Univ. of Calif. Press, Los Angeles, pp. 231-265. Lundberg, A. (1970) The excitatory control of the Ia inhibitory pathway. In Excitatory Synaptic Mechanisms, P. Andersen and J.K.S. Jansen (Eds.), Universitetsforlaget, Oslo, pp. 333-340. Lundberg, A. (1973) The significance of segmental spinal mechanisms in motor control. Proc. 4 t h int. Biophys. Congr. Moscow. Lundberg, A. and Voorhoeve, P. (1962) Effects from the pyramidal tract on spinal reflex arcs. Actaphysiol. scand., 56: 201-219. Matthews, P.B.C. (1972) Mammalian Muscle Receptors and their Central Actiorts, Edward Arnold, London.

254 Mizuno, Y., Tanaka, R. and Yanagisawa, N. (1971) Reciprocal group I inhibition on triceps surae motoneurons in man. J. Neurophysiol., 34: 1010-1017. Renshaw, B. (1941) Influence of the discharge of motoneurons upon excitation of neighboring motoneurons. J. Neurophysiol., 4 : 167-183. Severin, F.V., Orlovskii, G.N. and Shik, M.L. (1967) Work of the muscle receptors during controlled locomotion. Biophysics, 12: 575-586. (Engl. transl. of Biofiziha, 1967, 1 2 : 502-51 1) Vallbo, A.B. (1970a) Slowly ddapting muscle receptors in man. Acla physiol. scand., 78: 315-33 3. Vallbo, A.B. (1970b) Discharge patterns in human muscle spindle afferents during isometric contractions. Acta physiol. scand., 80: 552-566. Wall, P.D. (1958) Excitability changes in afferent fibre terminations and their relation t o slow potentials. J. Physiol. (Lond.), 142: 1-21.

DISCUSSION STUART: How can you unequivocally separate recurrent inhibition from autogenic tendon organ inhibition? HULTBORN: It is not possible t o do it unequivocally. It seems, however, unlikely to us that Ib inhibition of Ia inhibitory interneurones is important for the plateau in reciprocal inhibition. The reason is two-fold: firstly, it was difficult to see even the shadow of presumed I b IPSPs on inlracellular recording from the Ia inhibitory interneurones while recurrent inhibition was always very prominent; secondly, the reciprocal inhibition increased with further stretch when the EMG saturates, i.e., when there is no increase in recurrent inhibition, though there is an increase in active tension at this stage. Both these points need, however, further examination. We also attempt to use Dr. Houk’s method t o stimulate the peripheral end of a cut ventral rootlet t o cause an increased tension and thus “selectively” increased activity in Ib afferents. HONGO: I just want to ask you if it is necessary t o consider the influence from group I1 afferents from secondary endings. HULTBORN: Transmission in reflex pathways from tendon organ afferents and secondary spindle afferents is tonically inhibited in the decerebrate cat and it was observed t o operate effectively in all preparations from which the present results originate. Of course this does not exclude effects from group I1 afferents - they are indeed considered by many t o participate actively in the stretch reflex. If there are any group I1 effects I can only hope that they are distributed in parallel to corresponding a-motoneurones and Ia inhibitory interneurones since the distorting effects would then be minimized. In cases where the decerebrate control (especially of inhibitory pathways) was waning, we could see a linear increase in reciprocal inhibition during the whole stretch. In this case there was no modulation of the reciprocal inhibition in relation to the EMG of the stretch reflex suggesting that other interneurones lacking recurrent inhibition were mediating this type of reciprocal inhibition. ELLAWAY: Please correct me if I am wrong. I think early in your talk you mentioned that the recurrent inhibition of the Ia inhibitory interneurones was restricted t o its own muscle. Now this would be one definite point of difference with Renshaw cell axons, about which Prof. Granit pointed out that distributions among gamma motoneurones are certainly not restricted. Did I understand you correctly? HULTBORN: Although the maximal recurrent depression of Ia IPSPs is always evoked from the very nerves whose afferents give the Ia ichibition, recurrent effects are evoked also from some other nerves (Hultborn et al., 1 9 7 1 ~ )According . t o Eccles and collaborators (Eccles et al., J. Physiol. (Lond.), 1961, 195: 479-499) the distribution among motoneurones is re-

255 lated mainly t o their proximity and it was postulated that recurrent inhibition has a general suppressor action on motoneurones regardless of function. A more detailed analysis has prompted us (Hultborn et al., 1 9 7 1 ~ t)o upgrade the importance of muscle function in relation t o distribution of recurrent inhibition. Thus, motor nuclei supplying muscles acting as strict antagonists at the same joint lack recurrent interconnections even when they have a similar rostrocaudal distribution in the cord. On the other hand, motor nuclei to muscles which are linked in Ia synergism are mutually connected by recurrent inhibition regardless of their location in the spinal cord. When the contribution from different nerves t o recurrent inhibition was compared, a strong parallelism was revealed for motoneurones and Ia inhibitory interneurones receiving the same Ia excitatory input. STUART: I am interested in your acceptance of the interneurones as being Ia inhibitory interneurones. If you are going to study the descending effects on the interneurones, and if you wish to know if it really is a Ia inhibitory interneurone, you see first that it is an interneurone, then you see that it is driven by low-threshold electrical stimulation orthodromically, and then you show inhibition antidromically by the Renshaw system. Is that your set of criteria for a Ia inhibitory interneurone? My real question is: “DO you think that there are other interneurones that could satisfy those criteria which would be playing other segmental roles?” HULTBORN: The criteria we have used to identify an interneurone as “la inhibitory interneurone” were discussed by Hultborn et al. (1971b) and most recently by Hultborn et al. (1976a). Despite the difficulties in the technique of Jankowska and Roberts they were able to show positively for a large majority of so identified interneurones that they indeed mediate reciprocal Ia inhibition. Furthermore, recurrent conditioning of other postsynaptic potentials in motoneurones has never given any positive evidence of recurrent inhibition of interneurones in reflex pathways t o motoneurones other than those transmitting reciprocal Ia inhibition. Disynaptic Ia inhibition in Ia inhibitory interneurones (see in the preceding article) and some ventral spinocerebellar tract cells (Gustafsson and Lindstrom, Acta physiol. scund., 1973, 89: 457-481) are depressed by preceding ventral root stimuli, but these IPSPs are held as “collateral” effects from the same Ia inhibitory interneurones which project t o motoneurones of the antagonist. There is thus n o positive evidence for the kind of subgroups you are suggesting, but their existence may be impossible to exclude. A real risk during the course of an experiment, however, is to mistake a motoneurone with blocked antidromic spikes for a Ia inhibitory interneurone. This was always excluded by testing the neurone’s ability t o follow frequencies of more than 300 Hz when stimulating the Ia afferents (see Hultborn et al., 1971b).

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Recruitment, Rate Modulation and the Tonic Stretch Reflex DANIEL KERNELL Department o f Neurophysiology, University of Amsterdam, Eerste Constantijn Huygensstraat 20, Amsterdam (The Netherlands)

In the tonic stretch reflex of limb extensors, the gradation of muscle activation is generally thought t o be due t o changes in the number of recruited motoneurones (“recruitment gradation”). Stretch-evoked changes in motoneuronal firing rate (“rate modulation”) are often relatively unconspicuous, and they have usually been considered to be too small t o be of any practical importance. Such opinions have been widely held since the original studies by Denny-Brown (1929), and the relevant experimental work has largely been done on the triceps surae (particularly soleus) of decerebrate cats. In the present contribution, I will comment upon the results of such experiments from three main points of view. Firstly, I would like t o discuss whether stretch-evoked changes of firing rate actually are too small to be of practical importance. Secondly, I will discuss why a recruitment gradation would commonly be expected t o be accompanied by a modulation of motoneuronal firing rate. Thirdly, I will briefly discuss which mechanisms might be responsible for the amount of rate modulation that occurs in parallel with recruitment gradation within a motoneurone pool (i.e., rate modulation occurring in contractions with a non-complete and changeable recruitment), In some decerebrate cats, a rather wide range of muscle extensions may be applied without causing practically any change in total muscle activation (i.e., neither change in firing rates nor in recruitment; occasional cases noticed by Granit (1958) and Matthews (1959); regularly seen at long muscle lengths by Grillner and Udo (1971a,b)). Results obtained within such a range of muscle extensions do not, of course, say anything concerning the relative importance of recruitment and rate modulation for the gradation of muscle activation. In a given muscle, a tension gradation by recruitment control alone would be indicated if the motoneurones of the muscle were shown to fire constantly at their minimum rate throughout a considerable change in recruitment, up t o and including contractions for which all motoneurones of the muscle were activated (cf., Fig. 6). This does not yet appear t o have been proven for any single motoneurone of triceps surae. Denny-Brown (1929) studied contractions with few recruited motor units, and his results seemed t o suggest that muscle extension rarely affects the firing rates of soleus motor units. Results in later literature strongly indicate, however, that a stretch-evoked rate modulation is of common occurrence among motoneurones of triceps surae. For the gastrocnemius of de-

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Fig. 1. Instantaneous firing rate versus time for single motor unit from soleus (values derived from electromyographic recording). Intercollicularly decerebrated cat. Innervation of soleus muscle intact. Muscle first kept at constant length, then shortened (downward arrow, unit silenced), lengthened (upward arrow, unit resumes firing), slowly lengthened again twice (increases of mean firing rate). Interrupted line: baseline for rate display (0 imp/sec).

cerebrate cats, Alvord and Fuortes (1953) reported that muscle extension would cause the firing rates of homonymous motoneurones t o increase by about 50-250% (range of firing rates seen: 6-40 implsec). For soleus, systematic studies have been scarce, but illustrated examples of a stretch-evoked rate modulation may actually be found in most papers concerning the discharge of single soleus motoneurones during a graded stretch reflex (e.g., rates before and after muscle extension in lower part of Fig. 7 of Granit (1958); Fig. l a of Roberts (1958); Figs. 1-2 of Olson et al., (1968); initial rise of rate with extension in Fig. 1A of Grillner and Udo (1971a)). In illustrated cases from soleus, rate modulation is commonly seen t o occur within the range of 5-10 imp/sec (although rates up t o 15-20 imp/sec have been observed; Denny-Brown, 1929). Similar results were obtained in a few confirmatory experiments of my own. One example is illustrated in Fig. 1. The diagram shows instantaneous firing rate versus time for one single soleus motor unit, and the changes in mean firing rate were produced by changes in muscle extension (see legend of Fig. 1). Among the 7 soleus motor units that I studied in 2 decerebrate cats, the mean minimum rate was 5.5 (range 4.8-6.2) imp/sec and the maximum recordable one during muscle stretch was on average 10.0 (range 9.6-10.5) imp/sec (all rates measured during at least 1 sec of steady firing). My own measurements were all made in rather weak reflex contractions with a non-complete recruitment (reflex-evoked tension less than 15-2076 of maximum tetanic tension). In a stronger stretch reflex, covering a wider range of recruitment, the total amount of rate modulation might have turned out t o be considerably larger. More extensive and systematic measurements are highly desirable. However, even a numerically modest amount of rate modulation in the range of 5-10 imp/sec would be expected t o have a significant influence on the strength of a tonic stretch reflex. At muscle lengt,hs like those employed in experiments on tonic stretch reflexes of soleus, a change of stimulus rate from 5/sec t o lO/sec will typically more than double the mean muscle tension (leftmost curve in Fig. 1 7 of Matthews (1959); curves for ankle angles 90"and 45" in Fig. 9a of Rack and

259 Westbury (1969); confirmed at maximum physiological length in experiments of my own). We do not yet know enough t o be able to calculate accurately the tension-contributions due to recruitment and rate modulation respectively in a tonic stretch reflex. It seems reasonable to conclude, however, that a tonic stretch reflex of soleus is certainly not exclusively graded by a change in the number of recruited motor units. In the tonic stretch reflex as well as in most other maintained contractions that are evoked via the central nervous system, recruitment gradation tends to become accompanied by a significant amount of rate modulation (e.g., for other contractions than stretch reflexes, Adrian and Bronk, 1929; Milner-Brown et al., 1973; Kernel1 and Sjoholm, 1975). What are the possible causes for such a behaviour of motoneurone pools? A few years ago, Mendell and Henneman (1971) showed that each single primary spindle afferent (Ia) from gastrocnemius medialis delivered monosynaptic excitation t o practically every homonymous motoneurone. I will refer t o this as a ‘widespread’ manner of pool innervation. One would certainly expect the findings of Mendell and Henneman (1971) to be applicable to spindle afferents and motoneurones of soleus as well. Furthermore, it seems likely that a rather ‘widespread’ innervation pattern is of common occurrence among spinal reflex systems. Such a ‘widespread’ distribution of synaptic effects has important consequences for the function of a motoneurone pool. Consider, for instance, a motoneurone pool which is influenced by only one set of ‘widespread’ excitatory synapses. Over a certain range, an increased synaptic activation would cause an increased amount of recruitment. Any increase of synaptic excitation to the pool would, however, to some extent occur simultaneously in all the motoneurones, including those that were already firing. In a firing motoneurone, an increase of net maintained excitation would cause a rise in mean firing rate. Thus, in case of a ‘widespread’ distribution of postsynaptic effects within a motoneurone pool, an increase in the number of recruited neurones would commonly be expected to be accompanied by some increase of firing rate among a11 the discharging members of the pool (cf., however, Fig. 6). In a contracting soleus muscle, a given length change tends t o produce a greater relative (e.g., percentagewise) change of tension at low stimulus rates than at higher ones (valid over a wide range of muscle lengths and stimulus rates; cf., Matthews, 1959; Rack and Westbury, 1969; Grillner, 1973). Thus, the stiffness of a given soleus contraction would tend t o be greater if it were caused by many units firing slow rates than if it were produced by fewer units firing at higher rates (Grillner, 1973). Hence, it is of importance for questions of motor control to understand which mechanisms may be responsible for the amount of rate modulation that occurs in parallel with recruitment gradation. This problem has received little attention in earlier literature. I have recently been analyzing questions of this kind by aid of simple models of motoneurone pools. The models that I am dealing with were of a ‘software’ kind, i.e., they consisted of a number of simple equations which were solved by aid of a digital computer. The behaviour of a population of neurones is most easily grasped intuitively if the number of neurones is relatively small. Therefore, I have chosen t o illustrate the results of my theoretical studies by model versions containing only 5 neurones each. It should be stressed, however, that none of the general

260 conclusions that I am going to report were dependent upon the small number of cells within the pool (controlled in separate calculations). Actually, some of the approximations and simplifications (e.g., that recurrent inhibition acts as a steady current, Fig. 5-6) would be more realistic for a large pool than for a very small one. The behaviour of the ‘standard’ version of the present pool model is illustrated in Fig. 2. The diagram of Fig. 2A shows the discharges of the individual neurones when the pool was stimulated by different intensities of postsynaptic excitation. Only steady states of activity were considered, and synaptic influences had the shape of maintained currents (cf., Granit et al., 1967). The current intensities along the abscissa of Fig. 2A refer to the total amount of excitatory current received by all the 5 pool members together (‘total pool excitation’). In the present pool models, all synaptic inputs had a ‘widespread’ distribution among the cells (i.e., any change in the intensity of postsynaptic excitation or inhibition would concern all the cells of the pool), and each cell received a constant percentage of the total pool excitation (or inhibition). In the ‘standard’ version of the model (Fig. 2), each one of the 5 cells received 20% of the total pool excitation. A model neurone would start to discharge repetitively once it became depolarized by 10 mV. When the neurone was just barely activated by steady excitation, it kept discharging at its characteristic minimum firing rate (7 imp/sec for all the cells of Figs. 2 - 6 ) . The amount of total pool

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Fig. 2. From ‘standard’ version of pool model with 5 neurones. A: neuronal firing rate versus intensity of total pool excitation. At pool excitations corresponding to neuronal recruitment thresholds, firing rates were calculated for all discharging neurones of the pool. For each model neurone, computed values were connected by straight lines. B: total rate modulation versus number of recruited motoneurones (‘rate-recruitment curve’). Calculated from values of A as described in text. In the ‘standard’ version of the model, each neurone received 20% of the total pool excitation, and there was no recurrent inhibition. The resting input conductance of the 5 neurones was 30, 40, 50, 60 and 7 0 X lo-* mho respectively. Thus, the resting input resistance was 1.4-3.3 M a , which is similar to the range of experimental values for most soleus motoneurones (Burke, 1967). The slope for the frequency-current relation was 1 imp/sec/nA, which is similar to experimental values for very ‘small’ lumbosacral a-motoneurones (Kernell, 1966; mean value for soleus motoneurones not yet known). The minimum rate of steady firing w’as 7 imp/sec, which is similar to the value expected for a motoneurone with the afterhyperpolarization of an average soleus motoneurone (cf., Eccles et al., 1958; Kernell, 1965). Soleus motoneurones recruited in weak stretch reflexes showed a minimum rate which was slower than the predicted average value (cf., Fig. 1).

excitation needed for just barely activating a given neurone will be referred to as the ‘recruitment threshold’ for that cell. Once a cell had become recruited, a further increase in the net activating current received by the neurone would cause its firing rate to increase along a linear frequency-current relation. In order to make the model as simple as possible, all neurones of a given pool had identical membrane properties, The cells of one and the same pool did, however, differ from each other in size and, hence, in resting input resistance. In the ‘standard’ version of the pool model (Fig. 2), all the cells were simultaneously stimulated by the same absolute amount of excitatory postsynaptic current. Hence, small model neurones were more easily recruited than the larger ones (Fig. 2; valid also for Figs. 3-6), which corresponds to the recruitment order seen during a tonic stretch reflex of soleus (Henneman et al., 1965). The computed values of Fig. 2A (filled circles) show the discharge rates obtained within the pool model each time total pool excitation became just barely strong enough t o recruit yet another neurone. In the present context, it is of particular interest to compare the respective amounts of recruitment and rate modulation. When, for instance, the steady pool excitation of Fig. 2A was just barely strong enough t o recruit 4 neurones, these cells discharged at 7, 8, 9 and 10 imp/sec respectively. Compared t o their minimum rates, the discharge frequencies of these neurones had become increased by 0, 1, 2 and 3 imp/sec respectively. Thus, when the fourth motoneurone became recruited, ‘total rate modulation’ of the pool had become as great as 6 imp/sec (=0 + 1 + 2 + 3). In Fig. 2B, such values for total rate modulation have been plotted versus the number of neurones recruited (values for Fig. 2B derived from those of Fig. 2A). Such a ‘rate-recruitment curve’ (Fig. 2B) gives a quantitative measure of the extent t o which recruitment is accompanied by rate modulation within a neuronal pool. For the model versions of Figs. 2-5 the rate-recruitment curve is deflected upwards. Thus, in these cases, rate modulation tended to become relatively more prominent as a greater proportion of the pool became recruited. In pool models of the present kind, the amount of rate modulation that occurred in parallel with recruitment gradation would become decreased by (i) a decrease in the slope for the frequency-current relation of the neurones, and (ii) a more narrow distribution of recruitment thresholds among the cells (Figs. 3-4). The distribution of recruitment thresholds depended on the distribution of postsynaptic effects (Fig. 3) as well as on the distribution of electrical excitability (Fig. 4 ) among the neurones of the pool. Electrical excitability (Le., the ease with which a model neurone was caused to fire by a stimulating transmembrane current) depended on the values for neuronal input resistance, resting membrane potential, and threshold potential for spike initiation. It is not yet known to what an extent the distribution of recruitment thresholds within a stretch-activated motoneurone pool of soleus might differ from that of other motoneurone pools. With respect t o rhythmic properties, there is earlier evidence indicating that lumbosacral motoneurones with a very high input resistance would tend t o show a comparatively low slope for the frequency-current relation (Kernell, 1966). Thus, the ‘smallest’ a-motoneurones of soleus might have an unusually low slope for the frequency-current relation, and this would help to keep down the amount of rate modulation that occurs

262 B

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Fig. 3. Recruitment and rate modulation in pool model. Values computed and plotted as in Fig. 2. The 5 cells received 16, 18, 20, 22 and 24% respectively of the total pool excitation (cells listed according to amount of resting input conductance, from small t o large). Otherwise, the model was identical to that of Fig. 2. Interrupted line in B: rate-recruitment curve of Fig. 2B. Fig. 4. Recruitment and rate modulation in pool model. Values computed and plotted as in mho reFig. 3. The 5 cells had a resting input conductance of 40, 45, 50, 55 and 60 X spectively. Otherwise the model was identical to that of Fig. 2.

in parallel with recruitment gradation within the soleus muscle. In earlier literature there has been much discussion concerning the role of recurrent inhibition for the general stabilization of motoneuronal firing rate (Granit, 1970; see also Meyer-Lohmann et al., at this symposium). I t has not, however, previously been analyzed to what extent recurrent inhibition might influence the amount of rate modulation that occurs in parallel with recruitment gradation. In my simple pool models, recurrent inhibition had the shape of a steady inhibitory current. The total amount of inhibitory current for the whole pool was proportional to the total number of spikes fired per second within the pool in the presence of the recurrent inhibition. The effect of recurrent inhibition was dependent on the extent to which the distribution of inhibition within the pool differed from that of the excitation driving the discharge. The model version of Fig. 5 was equipped with a strong recurrent inhibition. A given change of pool excitation did, of course, produce a smaller change of firing rate in the presence of this inhibition than it did without recurrent inhibition (cf., Figs. 5A and 2A). Thus, in this general sense, recurrent inhibition had a stabilizing influence on firing rate. It did, however, 'stabilize' recruitment gradation as well (cf., Figs. 5A and 2A) and, in this model version, recurrent inhibition produced no change at all in the rate-recruitment curve (cf., Figs. 5B and 2B). Such a result was always obtained if recurrent inhibition was distributed in the same way as the excitation driving the discharge (i.e., if a cell receiving X% of the total pool excitation did also receive X% of the total pool inhibition). Thus, recurrent inhibition does not necessarily affect the amounl of rate modulation that occurs in parallel with recruitment gradation. For the model version of Fig. 5 this was true in spite of the fact that, among non-discharging neurones, a given total amount of recurrent inhibition would have produced a greater inhibitory postsynaptic potential (IPSP) in small cells than in larger ones. The model version of Fig. 6 demonstrates that, i f appropriately distributed, a

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Fig. 5. Recruitment and rate modulation in pool model with recurrent inhibition. F o r each neurone, t h e net amount of activating current was equal t o t h e difference between t h e amount of excitatory postsynaptic current and t h e amount of inhibitory postsynaptic current. Currents aIong abscissa in A: total postsynaptic excitation of pool (i.e., not n e t amount of activating current). Total inhibitory current for the whole pool was equal to 0.55 times t h e total number of spikes discharged per second within the pool (current given in A, number of spikes calculated, of course, while the pool was influenced by the appropriate amount of recurrent inhibition). Each cell received 20% of the total inhibitory current f o r t h e whole pool. Other values, computations and plots as in Fig. 2.

strong recurrent inhibition may indeed have a very marked effect on the raterecruitment relation. In this case, the distribution of inhibition was such that, at any one moment, the ratio between the inhibitory and the excitatory postsynaptic current was greater for small cells than for the larger ones. In the extreme case illustrated. almost no rate modulation occurred in parallel with the recruitment gradation. This near-complete stabilization of firing rate was, however, only present over the range of stimuli for which recruitment was not yet complete. Stronger stimuli than those needed for recruiting the whole pool caused an increase of firing rate in all the cells.

6 irnp./s 11-

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model with recurrent inhibition. Values computed and plotted as in Fig. 5. The 5 cells received 30, 25, 20, 15 and 10%respectively of t h e total inhibitory current for t h e whole pool (cells listed according to amount of resting input conductance, from small to large). Otherwise the model was identical t o t h a t of Fig. 5. Interrupted line in B: rate-recruitment curve of Fig. 2B. A t the highest current intensity shown in A, a n average value is plotted for t h e firing rates of the 4 smaller cells (actual rates: 7 . 1 2 , 7.09,7.06, and 7 . 0 3 implsec respectively).

264 It remains t o be shown by further experiments to what an extent the raterecruitment relation of the soleus motoneurone pool is dependent on recurrent inhibition (or, perhaps, on autogenetic inhibition from tendon organs). It is, however, not at all certain that such inhibitory mechanisms would be needed for explaining the amount of rate modulation that occurs during a tonic stretch reflex. In the ‘standard’ version of the present pool model, the firing rate of single neurones varied by 4 imp/sec or less within the range of graded recruitment (Fig, 2A). Thus, even in the absence of recurrent inhibition, the single neurones of such a pool model did not show more of a ‘recruitment-parallel’ rate modulation than that seen during a tonic stretch reflex of soleus (cf., Figs. 1 and 2). There are still many gaps in our knowledge Concerning the synaptic organization and properties of soleus motoneurones and further experimental work would be needed for assessing t o what an extent the critical parameters of the ‘standard’ model differ from those of a pool of stretch-activated soleus motoneurones (cf., legend of Fig. 2). As stressed once already, the number of neurones in the pool does not matter much in this context. If the model of Fig. 2 contained 100 neurones, recruitment gradation would still be accompanied by a rate modulation of 4 imp/sec or less in single neurones if all other properties of the model were kept constant (e.g., if, at any one moment, each cell was still influenced by the same absolute amount of postsynaptic current as that acting simultaneously on all other cells of the pool, and if input resistances were still distributed within the same range as that of Fig. 2). SUMMARY Since the studies of Denny-Brown (1929) it is widely believed that the tonic stretch reflex of limb extensors is graded almost exclusively by a change in the number of active motor units (‘recruitment gradation’). Stretch-evoked changes in motoneuronal firing rate (‘rate modulation’) have usually been considered t o be small and unimportant. Previous literature does, however, contain many examples of a stretch-evoked rate modulation in soleus and gastrocnemius motoneurones. This rate modulation may seem numerically small (often within a range between about 5 and 10 implsec for soleus motoneurones; confirmed in present study), but its effect on muscle tension would be quite significant. In a soleus muscle kept close to its maximum physiological length, a rise of stimulus rate from 5/sec to lO/sec will more than double the contractile tension (e.g., Matthews, 1959; Rack and Westbury, 1969; confirmed in present study). For the further analysis of such problems concerning the gradation of muscle activation, it is important to know which mechanisms might be responsible for the amount of rate modulation that occurs within the range of variable and incomplete recruitment of a motoneurone pool (‘recruitment-parallel rate modulation’). In the present study, this was investigated by aid of simple models of motoneurone pools. In these models, the amount of recruitment-parallel rate modulation would be minimized if (i) the various motoneurones were all recruited at rather similar amounts of total pool excitation (i.e., narrow distribution of ‘recruitment thresholds’), or (ii) the recruited motoneurones were rather insensitive to variations in the intensity of stimulating current (i.e., low

slope for frequency-current relation). A recurrent inhibition would not necessarily affect the amount of recruitment-parallel rate modulation. It might, however, d o so if distributed in a manner different from that of the synaptic excitation driving the discharge.

REFERENCES Adrian, E.D. and Bronk, D.W. (1929) The discharge of impulses in motor nerve fibres. Part 11. The frequency of discharge in reflex and voluntary contractions. J. Physiol. (Lond.), 69: 119-151. Alvord, E.C., Jr. and Fuortes, M.G.F. (1953) Reflex activity of extensor motor units following muscular afferent excitation. J. Physiol. (Lond.), 122: 302-321. Burke, R.E. (1967) Motor unit types of cat triceps surae muscle. J. Physiol. (Lond.), 193: 1 41-1 60. Denny-Brown, D. (1929) Nature of postural reflexes. Proc. roy. SOC. B, 104: 252-301. 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. 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 Control, Academic Press, London. Granit, R., Kernell, D. and Lamarre, Y. (1967) Algebraical summation in synaptic activation of motoneurones firing within the “primary range” to injected currents. J. Physiol. ( L o n d . ) , 187: 379-399. 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, pp. 195-215. Grillner, S. and Udo, M. (1971a) Motor unit activity and stiffness of the contracting muscle fibres in the tonic stretch reflex. Actaphysiol. scand., 8 1 : 422-424. Grillner, S. and Udo, M. (1971b) Recruitment in the tonic stretch reflex. A c t u physiol. scand., 81: 571-573. Henneman, E., Somjen, G . and Carpenter, D.O. (1965) Functional significance of cell size in spinal motoneurons. J. Neurophysiol., 28: 560-580. Kernell, D. (1965) The limits of firing frequency in cat lumbosacral motoneurones possessing different time course of afterhyperpolarization. A c t u physiol. scand., 65 : 87-100. Kernell, D. (1966) Input resistance, electrical excitability, and size of ventral horn cells in cat spinal cord. Science, 152: 1637-1640. Kernell, D. and Sjoholm, H. (1975) Recruitment and firing rate modulation of motor unit tension in a small muscle of the cat’s foot. Brain Res., 98: 57-72. Matthews, P.B.C. (1959) The dependence of tension upon extension in the stretch reflex of the soleus muscle of the decerebrate cat. J. Physiol. ( L o n d . ) , 147: 521-546. 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: 1 71-187. Milner-Brown, H.S., Stein, R.B. and Yemm, R. (1973) Changes in firing rate of human motor units during linearly changing voluntary contractions. J. Physiol. ( L o n d . ) , 230: 37 1-390. Olson, C.B., Carpenter, D.O. and Henneman, E. (1968) Orderly recruitment of muscle action potentials. Motor unit threshold and EMG amplitude. Arch. Neurol. (Chic.), 19: 591597. Rack, P.M.H. and Westbury, D.R. (1969) The effects of length and stimulus rate on tension in the isometric cat soleus muscle. J. Physiol. ( L o n d . ) , 204: 443-460. Roberts, T.D.M. (1958) The assessment of changes in the sensitivity of a stretch reflex. J. Physiol. ( L o n d . ) , 144: 2-4P.

266

DISCUSSION HENNEMAN: You referred to t h e distribution of Ia terminals which we did a few years ago. I just observed within t h e last few weeks a study of Mendell and his students in which he showed that the distribution of l a terminals for the soleus pool is l o o % , a very clear example of this sort of things. I want t o ask you whether you o r Dr. Granit, who both injected current into many cells and studied their firing rate, have ever done so for soleus motoneurones. KERNELL: Until now, I have done so for rather few identified soleus motoneurones. I intend to increase t h e size of this sample in the near future. HOUK: I just want t o question you about t h e rate with which you are stretching. There is a l o t of evidence indicating that the slope of the relationship of the discharge rate to any excitatory input is very dependent upon the rate with which the excitatory input is applied. I like t o quote particularly Peter Clamann who has d o n e some work with human biceps muscle. He showed that when the human subjects are tracking force-changes, if they track very rapidly there was a very steep slope between discharge rate and force (using force as a measure of excitatory input). If they were tracking very slowly, there was a shallow relationship and may be that would help t o explain why Sten Grillner got a very shallow slope in his particular case and perhaps you got a steep slope. I don’t know how many units you have done. You’ve shown one or two. KERNELL: I was primarily interested in t h e discharge rate a t different levels of maintained extension, and I typically changed muscle length intermittently (and, then, rather slowly) for t h e 7 motor units quoted in my paper. These studies were complemented by others ones (different cats) in which I investigated the firing rates of single soleus motor units while t h e soleus muscle was kept constantly a t its maximum physiological length. In these units t h e rate of regular firing could generally be decreased by several impulses/sec by the application of a n appropriate amount of steady inhibition (produced by maintained repetitive activation of peroneal nerve a t 200/sec). So far as I can see, m y experimental results are simply confirming those illustrated by others in previous literature. In t h e experiments by Grillner and Udo (1971a,b) there was little activity-gradation of any kind (recruitment o r rate modulation) over most of the range of muscle lengths explored. In their cases, most of the recruitment occurred during the initial portions of t h e applied extensions (Grillner and Udo, 1971b) and, in their illustrated example of a single unit discharge (Fig. 1 A of Grillner and Udo, 1971a), t h e first portion of a n ongoing muscle extension was also accompanied b y a rise of instantaneous firing rate from about 5.5 t o 9.1 imp/sec (minimum rate of steady firing not shown). These data seem quite consistent with t h e presence of a n amount of rate modulation similar t o that seen in my own experiments.

Patterns of Motoneuronal Units Discharge during Naturally Evoked Afferent Input RADMILA ANASTASIJEVIC, MILKA STANOJEVIC and J. VUCO Institute for Medical Research, Belgrade (Yugoslavia)

INTRODUCTION Following the works of Granit et al. (1956, 1957a,b), who showed that some extensor motoneurones of decerebrated cats responded t o steady muscle stretch or electrical tetanisation of muscle nerve by long-lasting discharge while others responded by few initial spikes (the former were named tonic and the

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Fig. 1. Diagram summarizing the main findings from previous experiments on vibration of the muscle (B), sinusoidal stretching or vibration together with fusimotor stimulation (C, D, E). Upper tracings: motoneuronal units reflex spikes. Lower tracings : myography, thickening of the traces indicates duration of vibration. Amplitude of vibration 50 pm. Roman numerals in D indicate ordinal number of the trial in the repetitive series of vibration. A: diagram of preparation used in the described experiments (modified from: Anastasijevie et al., 1968, 1969, 1971; Vuro and Anastasijevie, 1973).

268 latter phasic motoneurones), several new approaches were made in this field of research. On the grounds of the experiments by Henneman et al. (1965a,b), it was firmly established that a-motoneurones formed a spectrum without distinct subgroups of the so-called “tonic” and “phasic” motoneurones. Recently, a new classification scheme was proposed (Burke et al., 1971) which is related to the presence of three groups of motor units in the cat gastrocnemius muscle, two fast contracting and one slow. This evidence suggests that there is no basic difference between the two groups of motoneurones but rather that the firing pattern in decerebrated cats represents the results of quantitative differences in Ia synaptic input, which is distributed t o motoneurones of different sizes in a continuous manner. Bearing this in mind either the terms “tonic” and “phasic” or tonically and phasically discharging units were used, as in the previous works (summarized in Fig. 1)or in those t o be described, in order to explain motoneurones behaviour under certain experimental conditions. As will be seen, the use of these terms in a descriptive or operational sense proved t o be of considerable value for understanding the firing behaviour of motoneuronal units under the influence of a variety of naturally evoked afferent inputs. Therefore, the experiments with extracellular recording were designed so that the reflex response firing pattern of the motoneuronal units was first established by stretching the muscle and subsequently by applying vibration t o its tendon. MOTONEURONAL REFLEX RESPONSE TO MUSCLE VIBRATION AND FUSIMOTOR STIMULATION The use of vibration as a selective stimulus for the primary endings of muscle spindle (Brown et al., 1967) has proven t o be*oneof several valuable means of studying motoneuronal units discharge patterns under the conditions of naturally evoked afferent input both in man and cat (Hagbarth and Eklund, 1966; Lance et al., 1966; Matthews, 1966; Homma and Kanda, 1973). A particular advantage of this procedure is that vibration, with an amplitude of 50 pm or less of the gastrocnemius medialis muscle in decerebrated cats, produces continuous activation of a-motoneurones with smaller reflex spikes. The motoneurones with higher reflex spikes gave only a short-lived discharge at the beginning of muscle vibration (Fig. 1B). Another important advantage of the use of vibration in the study of the motoneuronal reflex response is that the sensitivity of the primary endings to muscle vibration could be greatly increased by concomitant fusimotor stimulation (Granit and Henatsch, 1956; Bianconi and Van der Meulen, 1963; Brown et al., 1967). Thus, the reflex response to vibration of the muscle could serve both as a means of studying the firing properties of the selected motoneuronal units pairs and as an estimate of the extent of gamma influence through the loop. Although the stimulation of the whole ventral root did not allow distinction between the types of fusimotor fibres but favoured an asynchronous barrage of afferent impulses, special features in the reflex response type of tonically and phasically discharging units could still be observed under the variety of experimental conditions as present-

ed in Fig. 1. For example, a differential phase advance between the reflex spikes of a pair of motoneuronal units with respect t o the sinusoidal length changes of the muscle, before and during fusimotor stimulation, indicates their possible specific role in the modulation of the induced movement (Fig. 1C). On the other hand, when under high gamma bias, the primary endings increased their sensitivity t o vibration of the muscle, resulting in an afferent discharge high enough to provide for maximal activity of the spinal motoneurones. The high reflex performance seems to be supported, under conditions of shortlasting repetitive vibration, by both the properties of the primary endings of muscle spindle and special features in the reflex response of tonically and phasically discharging motoneuronal units to the repetitive nature of the applied stimuli (Fig. ID). Additional information concerning the role of the gamma system influences in the regulation of respiratory and other movements (Critchlow and von Euler, 1963; Granit et al., 1966; Severin et al., 1967) can be represented by the response patterns of motoneuronal units during vibration of the contracting muscle. If the gamma loop is fully engaged, the same level and pattern of motoneuronal reflex response t o muscle vibration may be obtained in both the presence and the absence of muscle contraction, the opposing influence of impulses arising from the Golgi tendon organs and of unloaded spindles being completely overwhelmed (Fig. 1E).

Maintained depolarization of spinal motoneurones during fusimotor stimulation and muscle vibration Some further data concerning the extents of gamma influence through the loop could be obtained from the changes of the size of the maintained depolarization of the spinal a-motoneurones innervating triceps muscles, during muscle vibration and simultaneous fusimotor stimulation (Fig. 2A). The distal stumps of the cut ventral roots, either L7or S1, were stimulated by trains of square pulses of 500 psec duration at 200 Hz during selective blockade of transmission in skeletomotor end-plates produced by Flaxedil. Successive periods of fusimotor stimulation, muscle vibration and of both stimuli together, were applied at the selected amplitudes of vibration and muscle lengths. Before the reflex response t o vibration was examined tension in the muscle was raised up t o 70 g by stretching the tendon at an angle of 30" from the horizontal plane. The obtained muscle length is referred t o in this work as the initial or zero (0) muscle length. The frequency of vibration was always 200 Hz. Periods of vibration lasted 1000 msec. Intracellular recordings were obtained by using glass micropipettes filled with 2 M potassium citrate and with resistances of 5-10 M a . The amplitude of the maintained depolarization evoked by muscle vibration and/or fusimotor stimulation was determined by measuring directly on the screen of the storage oscilloscope (or on the film) those membrane potential changes produced by the stimuli over and above that produced by muscle lengthening. Membrane potential was also monitored throughout the investigation and the spike size was measured before and after the procedure. All the cells reported here had a membrane potential of 50 mV or more. When subthreshold amplitudes of vibration (less than the smallest one required to produce membrane depolarization) were applied simultaneously with

270 MICROELECTRODE RECORD1N G

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Fig. 2. Maintained depolarization (B and C), with cut spikes in C of tw o triceps motoneurones, with a spike size of 7 5 mV, during simultaneous fusimotor stimulation and muscle vibration. Upper tracings: maintained depolarization of triceps motoneurones. Lower tracings: myograms during: fusimotor stimulation, muscle vibration and both stimuli applied together. Thickening of the tracings indicates duration of vibration. Amplitude of vibration 50 p m in B and 100 p m in C. Muscle at t he initial length a t B and stretched by 3 m m a t C. A: diagram of t h e preparation in the described experiments.

fusimotor stimulation, a larger depolarization was obtained than when fusimotor stimulation was applied alone (Fig. 2B). Smaller amplitudes of vibration sufficed t o evoke a reflex firing of the motoneurone in the presence of fusimotor stimulation than when this was absent'( Fig. 2C). Further experiments were designed to show the relations between the changes in the size of the maintained depolarization of spinal motoneurones during simultaneous fusimotor stimulation and various amplitudes of vibration of the triceps muscle stretched to different lengths. An example of these relations is shown in Fig. 3. In the absence of fusimotor stimulation there is a steady increase in the size of the maintained depolarization, over and above that produced by lengthening the triceps muscle, obtained by the increase in the amplitude of muscle vibration (- - - - - -). Nevertheless, nearly the same amount of maintained depolarization () was obtained when fusimotor stimulation coincided with various amplitudes of vibration applied on a muscle extended by 3, 6 and 9 mm from its initial length. The greater the amplitudes of vibration and muscle extension, the smaller the differences between the amplitudes of the maintained depolarization established both with and without additional fusimotor stimulation. These experiments undoubtedly show that the effect of fusimotor stimulation on the size of the maintained depolarization caused by muscle vibration was greater when small amplitudes of Vibration were applied t o a muscle which was not greatly extended, i.e., when all the primary endings of the muscle spindles were not yet driven by vibration stimulus. In addition to the fact that these experiments confirm the absence of occlusion between the response t o

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stretch and vibration (Westbury, 1972), they may also indicate that, owing t o the presence of the gamma loop activity, the reflex effect of vibration becomes less dependent on the variations of muscle length. On the grounds of these experiments, which describe a possible way to estimate the extent of gamma influence through the loop, one can suppose that the enhancement of the reflex response t o vibration and fusimotor stimulation applied together is likely to be a consequence of their common action, at least at two sites in the stretch reflex path: firstly, at the level of muscle spindle where the sensitivity of primary endings to small sinusoidal movements is greatly increased by simultaneous intrafusal contraction (Crowe and Matthews, 1964; Matthews and Stein, 1969) and secondly, at the level of motoneurones where shift of the membrane potential toward the firing threshold already occurs during fusimotor stimulation (Granit et al., 1966) so that less additional depolarization is needed to attain it during muscle vibration. THE MOTONEURONAL UNITS REFLEX DISCHARGES DURING MUSCLE VIBRATION AND ANTIDROMIC STIMULATION In view of the recent findings of a higher discharge of Renshaw cells induced by vibration rather than by static stretch of the gastrocnemius-soleus muscles for comparable frequencies of the discharges in the primary endings of muscle spindles (Pompeiano e t al., 1974a), it was desirable to study motoneuronal units reflex discharges during stretch alone and when elicited by vibration of the muscle, while additional recurrent inhibition resulting from trains of antidromic shocks was superimposed upon the naturally occurring one. Experiments were performed on decerebrated adult cats. After laminectomy, the ventral roots L7 and S, on the right side of the spinal cord were cut distally so as to provide filaments for recording as well as a central portion of the same root for

272

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Fig. 4. The difference between the effect of antidromic stimulation on the reflex discharge of the same motoneuronal unit elicited by stretch (B) and vibration of the triceps muscle (C). Records refer to the cut out portion and not to the total periods of counting. Diagram of the preparation used is presented in the top of the figure (A).

placement of the stimulating electrodes (Fig. 4A). Single motoneuronal units were selected from the nerve filaments only if they discharged continuously both during stretch alone and vibration of the muscle. The static stretch of the muscle was achieved by moving manually, with the aid of a handwheel adjustment, the platform t o which a vibrator (Pye-Ling 406) with an attached tendon hook was mounted. To estimate the degree by which antidromic stimulation suppresses the tonic discharge of the motoneuronal units during sustained stretch alone and when vibration up t o 200 pm was superimposed on a barely extended muscle, the procedure shown in Fig. 4 was adopted. Sustained discharge of motoneuronal units has been produced by a 3 mm extension of the muscle from its initial length (see earlier), and 5 trains of antidromic shocks at a rate of 60 Hz, lasting 1 sec and interrupted by a 1 sec pause, were applied at the rest of the ventral root after 1 5 sec latency from the beginning of the stretch (Fig. 4B). Fifteen seconds after the end of the period of antidromic stimulation, the muscle was released and its initial length again reestablished'. The same procedure was then repeated, but when 200 pm vibration of the muscle elicited a maintained firing of the unit of the same frequency as that obtained in both control periods (before and after antidromic stimulation) in Fig. 4B. To achieve this, it was necessary t o extend the muscle by 1.5 mm from its initial length. Extension of the muscle t o that magnitude did not produce reflex firing in the absence of additional vibration. The control frequencies before and after the periods of antidromic stimulation, both during static stretch of the muscle alone and during vibration were, within limits of error, the same: 12.28 2 0.67; 11.91 rt 0.54 and 12.27 0.47; 11.91 rt 0.54 imp/sec, respectively. Statistically insignificant differences were found in the rates of discharge during both control periods in two modes of the applied stimuli. Meanwhile, motoneuronal discharge was reduced by antidromic stimulation to 10.43 f 0.79 imp/sec when it was produced solely by extension, and t o 9.00 ? 0.50 imp/sec when it was elicited by vibration of the muscle. A highly significant difference between those two ( P <

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0.01) indicates the greater inhibitory effect of antidromic stimulation on the discharge of this motoneuronal unit when firing was produced by vibration of the muscle. Fig. 5 shows the mean differences between the suppressive effects of antidromic stimulation on motoneuronal reflex discharge of 8 motoneuronal units during extension of the muscle alone ( 0 ) and when vibration elicited firing of these units (0).Each of the presented units exhibited, during both kinds of stimuli, the same rate of discharge in the control periods. Minor extensions employed t o achieve the predetermined rate of discharge of motoneurones during vibration of the muscle were insufficient to produce reflex firing when applied alone. The motoneuronal unit marked with arrows is the same as presented in Fig. 4. The effect of antidromic stimulation during short-lasting repetitive vibration of the muscle As the effect of recurrent inhibition may depend on the firing properties of the motoneuronal units (Granit et al., 1957a,b), the reflex response of both tonically and phasically discharging units of the same motoneuronal pool was investigated during short-lasting repetitive vibration of the muscle and concomitant antidromic stimulation. For the purpose of this analysis, pairs of motoneuronal units were selected consisting of a unit with smaller spike size discharging continuously during vibration and/or sustained stretch of the triceps muscle and with a larger one responding only by a short-lived burst t o both stimuli. Five series of both continuous and short-lasting repetitive vibration were applied. Duration of the continuous vibration varied from 15 t o 35 sec. Periods of vibration in the interrupted series lasted 1000 msec. They were separated by pauses varying from 500 to 2500 msec. Trains of antidromic shocks lasting

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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

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20 Fn Fig. 7 . Plot of Fi as ordinates against Fn as abscissae for a group of 11 pairs of tonically (at) and phasically ( a p ) discharging motoneuronal units. The values of a and b, from t o p t o bott o m during continuous () vibration for a t are: -0.004, 0.861; 0.808, 0.653; 0.138, 0.580; during repetitive vibration ( - - - - - -): 1.140, 0.716; 0.183, 0.869; -0.764, 0.897; and for a p : -2.250, 0.769; -4.465, 1.184. Numbers in brackets indicate duration of pauses. x = continuous; = repetitive vibration. Duration of pauses: middle row 500 and bottom row 2500 msec.

units during continuous vibration, repetitive trains of antidromic stimulation caused a gradual downward shift of the lines. The comparison between the position of tshese lines obtained during the first and the last stimulation trials indicates that the recurrent inhibition increased with the progression of the trains of the antidromic stimulation. On the other hand, the amount of recurrent inhibition remained nearly the same, regardless of both the progression of stimulation trials and the duration of pauses, when short-lasting repetitive vibration and trains of antidromic shocks were applied together. It is evident that, due t o both the scatter and t o the early disappearance of the reflex spikes, regression lines for high-threshold phasically discharging units could not be determined during continuous vibration of the muscle. Meanwhile, it was quite possible to obtain a good correlation between Fn and Fi of these units when the muscle was subjected t o repetitive vibration. The slope of the regression

276 lines for both examined units, obtained when a pause of 2500 msec was introduced in the repetitive series of both stimuli applied together, indicates that this series of stimulations and pauses is more favourable for opposing the effect of recurrent inhibition than the series containing a 500 msec pause. Previous observation on the pre- and postsynaptic effects of vibration could explain to us the conditions under which sustained reflex firing during muscle vibration is adequately supported in order t o resist the effect of superimposed recurrent inhibition. It is known from the works of Thoden et al. (1972) that presynaptic inhibition may decrease the autogenetic excitation caused by vibration but not during steady stretch of the muscle, On the ground of these findings, one could expect that antidromic stimulation, although of the same strength, may induce a stronger inhibitory effect when motoneuronal unit firing was elicited by vibration than by stretch of the muscle. Since excitatory effects via motor axon collaterals during vibration did not occur when the muscle was slack or fixed at 0-2 mm of initial extension (Pompeiano et al., 1974b), the whole population of Renshaw cells may respond t o the incoming antidromic volleys. On the other hand, the size of the discharging pool of Renshaw cells may not be greatly increased by trains of antidromic shocks, perhaps due to occlusion (Ryall et al., 1972), when, as the result of the extension of the muscle above 3 mm, a sufficient number of Renshaw cells already discharge (Hellweg et al., 1974; Pompeiano et al., 1974b). Due t o the adaptation of motoneuronal reflex discharge and the cumulative inhibitory effect of the successive trains of antidromic shocks, recurrent inhibition was far stronger during continuous rather than during repetitive vibration of the muscle. The cumulative effect could be explained by a gradual increase in the susceptibility of Renshaw cells to respond to the incoming antidromic volleys, although of the same strength, since after an initial burst a gradual decline of their discharge rates occurs (Pompeiano et al., 1974b). Meanwhile, it is evident that when motoneurones were discharging in response t o the repetitive short-lasting vibration of the muscle, concomitant antidromic stimulation revealed the presence of surplus excitation as defined by Granit and Rutledge (1960). As t o the way the surplus excitation of motoneurones is built up during repetitive vibration of the muscle, the following events should be taken into consideration. The susceptibility of Renshaw cells t o respond to the antidromic volleys throughout the duration of the series of repetitive orthodromic stimuli must be lowered since, after each initial rapid increase in the discharge, due t o the short-lasting vibration trials, cells are again ready t o discharge in bursts. As a consequence, the antidromic shocks applied together with trains of repetitive vibration could not produce a discharge of Renshaw cells in excess of that already present. Under these conditions, each of the units of the motoneuronal pairs, highly activated by repetitive vibration of the muscle, contribute by their specific firing properties to the development of the excess of excitation, in spite of the presence of additional recurrent inhibition. Most of the results presented in this study were obtained by observing the motoneuronal units reflex discharges during vibration of the muscle. In spite of the fact that classification of the motoneurones selected in these experiments could not be made according to Burke (1967, 1968a,b), it seems t o us that no essential error was introduced because pairs of motoneuronal units with large

277

differences in spike size and response t o stretch or vibration of the muscle were selected for investigation. The experimental technique employed offered an important advantage over the intracellular one because two cells with different reflex response patterns could be monitored simultaneously and data could be collected from small cells in an equal proportion to those obtained from larger ones. Actually, the aim of this study was t o show those conditions under which both gamma loop activity or additional recurrent inhibition modify the reflex response pattern of motoneuronal units during naturally evoked afferent input. Nevertheless, the main disadvantage of the employed technique is that it showed us what actually happens in the extremes of what is really a continuous distribution of motoneurones’ properties.

SUMMARY This study gives examples of the use of vibration in the investigation of the extent of gamma influences through the loop and the conditions under which additional recurrent inhibition, resulting from trains of antidromic stimuli, can modify the pattern of motoneuroDa1 reflex response t o vibration of the muscle.

REFERENCES Anastasijevie, R., AnojCiC, M., Todorovie, B. and VuEo, J. ( 1 9 6 8 ) The differential reflex excitability of alpha motoneurons of decerebrate cats caused by vibration applied to the tendon of the gastrocnemius medialis muscle. Bruin Res., 1 1 : 336-346. AnastasijeviE, R., AnojEie, M., Todorovie, B. and VuEo, J. ( 1 9 6 9 ) Effect of fusimotor stimulation on the reflex response of spinal alpha motoneurones to sinusoidal stretching of the muscle. Exp. Neurol., 25: 559-570. Anastasijevie, R., CvetkoviE, M. and VuEo, J. ( 1 9 7 1 ) 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. Bianconi, R. and Van der Meulen, J.P. (1963) The response to 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: 773-800. Burke, R.E. (1967) Motor unit types of cat triceps surae muscle. J. Physiol. (Lond.), 193: 141-1 6 0 . Burke, 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. (196813) Firing patterns of gastrocnemius motor units in the decerebrate cat. J. Physiol. (Lond.), 196: 6 3 1 - 6 5 4 . Burke, R.E., Levine, D.N., Zajac, F.E., Tsairis, P. and Engel, W.K. (1971) Mammalian motor units : physiological-histochemical correlation in three types in cat gastrocnemius. Science, 1 7 4 : 709-712. Critchlow, V. and Euler, C. von ( 1 9 6 3 ) Intercostal muscle spindle activity and its motor control. J. Physiol. (Lond.), 1 6 8 : 820-847. Crowe, A. and Matthews, P.B.C. (1964) Further studies of static and dynamic fusimotor fibres. J. Physiol. (Lond.), 1 7 4 : 132-151. Granit, R. and Henatsch, H.D. ( 1 9 5 6 ) Gamma control of dynamic properties of muscle spindles. J. Neurophysiol., 1 9 : 356-366.

278 Granit, R. and Rutledge, L.T. (1960) Surplus excitation in reflex action of motoneurones as measured by recurrent inhibition. J. Physiol. (Lond.), 154: 288-307. Granit, R., Henatsch, H.D. and Steg, G. (1956) Tonic and phasic ventral horn cells differentiated by post-tetanic potentiation in cat extensors. Acta physiol. scund., 37 : 114126. Granit, R., Pascoe, J.E. and Steg, G, (1957a) The behaviour of tonic and 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., 2 0 : 470-481. Granit, R., Kellerth, J.O. and Szumski, A.J. (1966) Intracellular recording from extensor motoneurons activated across the gamma loop. J. Neurophysiol., 29: 530-544. 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, pp. 177-186. Hellweg, C., Meyer-Lohmann, J., Benecke, R. and Windhorst, U. (1974) Responses of Renshaw cells t o muscle ramp stretch. Exp. Bruin 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. Homma, S. and Kanda, K. (1973) Impulse decoding process in stretch reflex. In Motor Control, A.A. Gydikov, N.T. Tankov and D.S. Kosarov (Eds.), Plenum Press, New York, pp. 4 5 - 6 4 . Lance, J.W., de Gail, P. and Neilson, R.D. (1966) Tonic and phasic spinal cord mechanisms in man. J. Neurol. Neurosurg. Psychiat., 29: 535-544. Matthews, P.B.C. (1966) The reflex excitation of the soleus muscle of the decerebrate cat caused by vibration t o its tendon. J. Physiol. (Lond.), 184: 450-472. Matthews, P.B.C. and Stein, R.B. (1969) The sensitivity of muscle spindle afferents to small sinusoidal changes. J. Physiol. (Lond.), 200: 723-743. Pompeiano, O., Wand, P. and Sontag, K.H. (1974a) A quantitative analysis of Renshaw cell discharges caused by stretch and vibration reflexes. Bruin Res., 66: 519-524. Pompeiano, O., Wand, P. and Sontag, K.H. (1974b) Excitation of Renshaw cells by orthodromic group Ia volleys following vibration of extensor muscles, Pflugers Arch. ges. Physiol., 347: 137-144. Ryall, R.W., Piercey, M.F., Polosa, C . and Goldfarb, J. (1972) Excitation of Renshaw cells in relation to orthodromic and antidromic excitation of motoneurons. J. Neurophysiol., 35: 137-148. Severin, F.V., Orlovskii, G.N. and Shik, M.L. (1967) Work of the muscle receptors during controlled movement. Biofiziku, 1 2 : 502-511. Thoden, U., Magherini, P.C. and Pompeiano, 0. (1972) Evidence that presynaptic inhibition may decrease the autogenetic excitation caused by vibration of extensor muscles. Arch. ital. Biol., 110: 90-116. Vur*o, J. and Anastasijevi6, R. (1973) Motoneuronal reflex response to vibration of the contracting muscle. In Motor Control, A.A. Gydikov, N.T. Tankov and D.S. Kosarov (Eds.), Plenum Press, New York, pp. 65-74. Westbury, D.R. (1972) A study of stretch and vibration reflexes of the cat by intracellular recording from motoneurones. J. Physiol. (Lond.), 226: 37-56.

SESSION V

SUPRASPINAL CONTROL O F THE STRETCH REFLEX

Part I

Chairman: E. Henneman (Boston, Mass.)

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Single Unit Spindle Responses to Muscle Vibration in Man KARL-ERTK HAGBARTH, DAVID BURKE, GUNNAR WALLIN and LARS LOFSTEDT Department o f Clinical Neurophysiology, Academic Hospital, Uppsala (Sweden)

INTRODUCTION Earlier studies of spindle afferent behaviour in man (Hagbarth and Vallbo, 1968; Vallbo, 1970, 1971,1974a,b; Hagbarth et al., 1975b) have been directed mainly at activity during voluntary movement. They have established that movement is generated in accordance with the principle of a-y co-activation (Granit, 1970). Thus in non-contracting muscles the responses of spindle afferents to stretch are reproducible, are not subject t o change with changes in attentive state, and are unaffected by local anaesthetic blockade of the muscle nerve proximal to the recording site (Hagbarth et al., 1970; Wallin et al., 1973). These findings suggest that, in relaxed muscles, just as the skeletomotor system is silent, so also is the fusimotor system: there is no “resting fusimotor tonus”. However, with the initiation of movement, be it isometric or isotonic, deliberate or unintentional, fast or slow, the skeletomotor and fusimotor systems appear to be activated in parallel. Similarly, reinforcement manoeuvres do not affect the fusimotor system unless parallel changes in skeletomotor activity occur (Hagbarth et al., 1975a). Studies of stretch reflex mechanisms in normal man were restricted to phasic reflexes such as the tendon jerk or the H-reflex, until the description of the tonic vibration reflex (De Gail et al., 1966; Hagbarth and Eklund, 1966). Although some reservations exist about the precise relationship between the tonic vibration reflex (TVR) and the tonic stretch reflex, at least in the cat (Matthews, 1969; Thoden et al., 1972; Pompeiano et al., 1975), a considerable literature has developed on the motor and perceptual phenomena induced by muscle vibration in man (cf., Delwaide, 1973; Hagbarth, 1973; Homma, 1973; Lance et al., 1973). To date, however, the interpretation of findings in man has rested on data obtained in the cat on muscle stretch receptor sensitivity to vibration (Granit and Henatsch, 1956; Bianconi and Van der Meulen, 1963; Brown et al., 1967), there being only limited information available for the human subject (Hagbarth and Vallbo, 1968; Vallbo, 1970). To rectify this deficiency the responses of muscle spindle afferents to muscle vibration have been examined in awake human subjects using the microneurographic technique originally described by Vallbo and Hagbarth (1968). The results, which are in the course of publication, are summarized in this review.

282 GENERAL PROCEDURES The details of experimental procedures and the techniques of recording and analysis have been discussed in the earlier publications, as have the criteria for identification of afferent fibres. The present review is based upon recordings from 41 spindle afferents. In the absence of the criterion of conduction velocity, afferent fibre identification in man is based solely on the mechanoreceptive characteristics of the unit (including electrical twitch test). A clear differentiation of spindle afferents into two groups, Ia and 11, is not always possible on this basis, some afferents having intermediate properties. Thus 24 afferents have been considered t o originate probably from primary endings, and 11probably from secondary endings. The remaining 6 afferents have been labelled “intermediate”. The afferent potentials were recorded mainly from the peroneal nerve innervating the tibialis anterior and peroneal muscles, but a few were recorded from tibia1 nerve fascicles to the gastrocnemii. Vibration was applied t o the appropriate muscle tendon, usually with a modified pneumatic drill t o which an eccentric weight had been attached so that the amplitude of vibration was approximately 1.5 mm. The frequency of vibration was controlled by regulating the flow of compressed air, and could be varied between 20 Hz and 220 Hz. The vibration was monitored with an accelerometer attached to the vibrator. RESPONSES OF PASSIVE SPINDLE ENDINGS In non-contracting muscles, the discharge of all spindle afferents could be modified by vibration, provided that the vibrator was applied t o the correct tendon and that muscle length was appropriate. During vibration spindle discharge became locked t o a relatively constant, phase of the vibration cycle and remained so even during muscle stretch. Sensitive spindle endings could be activated by vibration applied t o the opposite side of the leg, but this was always a far less potent stimulus. Such endings could also respond at short muscle lengths, but then always at subharmonics of the vibration frequency. Increasing passive stretch increased the response in steps through successive subharmonics. In general, secondary endings appeared less responsive than primaries t o the vibratory stimulus. Primary endings could be driven t o respond t o every vibration cycle at higher frequencies than secondary endings, but there was some overlap. Only one primary ending was unable to follow vibration up t o 50 Hz, but 5 of the 11 secondary endings could not do so. Sensitive primary endings could follow the maximal vibration frequency of around 200 Hz, but the highest frequency t o which a secondary ending could be driven t o respond oneto-one was 130 Hz. This was achieved during a stretching movement and was not maintained during static stretch. Many endings had a “limit frequency”, above which they could not follow the vibration wave. The limit frequency .varied with the degree of muscle stretch, and above this frequency the ending changed its discharge pattern so that it responded at the next subharmonic. At its limit frequency, if an ending was following the vibration one-to-one, an increase in vibration frequency

283 58

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100 90

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Fig. 1. Effect of continuously changing vibration frequency o n discharge rate of a group Ia a€ferent unit from tibidis anterior. Instantaneous frequency plot shows that a t medium muscle length t h e ending responded only to every second cycle of vibration a t frequencies of 58-70 Hz. As vibration frequency rose above 70 Hz the ending failed to respond a t the second subharm o n k and changed t o the third and fourth subharmonics thus producing a decrease in overall discharge rate. As vibration frequency was decreased, the ending changed from responding t o t h e third subharmonic back to the second subharmonic a t approximately 90 Hz, thus producing an increase in overall discharge rate. Duration of vibration indicated by the horizontal bar.

caused the ending t o discharge t o every second cycle, and if already discharging t o every second cycle it began discharging t o every third cycle, i.e., the increase in vibration frequency resulted in a decrease in unit firing frequency (Fig. 1).The reverse process was seen on decreasing the vibration frequency. Consequently, it cannot be assumed that under all conditions a change in vibration frequency will produce a parallel spindle response: on the contrary, for any particular ending the response could involve an opposite change in unit frequency. Under certain conditions in non-contracting muscles, some sensitive primary endings and two ”intermediate” endings discharged more than once during a vibration cycle. Such behaviour was irregular, was seen only with vibration of frequency less than 100 Hz, and was produced usually only during a stretching movement. The two discharges occurred during the same phase of the vibration wave, and the intervals between the discharges could be less than 2 msec, depending on the vibration frequency.

PASSIVE MOVEMENTS As indicated above, increasing static stretch increased the spindle’s ability t o respond to vibration. During a stretching movement additional dynamic changes could be seen for many primary endings such that a greater spindle response was seen during the stretching movement than during maintained static stretch. Only one secondary ending exhibited such behaviour. During muscle shortening, the discharge of primary endings decreased suddenly, usually t o zero, and then slowly recovered to the level appropriate for that muscle length. Such behaviour was unusual for secondary endings.

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1501s c

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Fig. 2. Passive alternating movements. A : group Ia afferent unit (tibialis anterior). Vibration a t 105 Hz throughout trace. Alternating stretch and shortening movements (lower trace) produced periods of high frequency discharge largely confined t o stretching phases. Instantaneous frequency plot shows that the ending responded during stretch to every o r to every other vibration cycle. €3: group I1 afferent unit (peroneus). Vibration at 100 Hz throughout trace. During stretching phases, ending passed slowly through successive subharmonics until it discharged t o every second vibration wave. During shortening, ending decreased its response through successive subharmonics. In both A and B, the vibration frequencies are indicated by arrows on the instantaneous frequency scales. Downward deflection of goniometer signal represents muscle stretch.

With primary endings, presumably as a result of these dynamic properties, passive alternating movements resulted in a high frequency spindle response t o vibration during the stretching phase with relative silence during shortening. On the other hand, the more typical behaviour for secondary endings was a slowly increasing response during the stretching phase with gradual decrease during shortening (Fig. 2). As a consequence, it would appear that during the shortening phase of an alternating movement the secondary ending is potentially the more vibration-sensitive receptor. EFFECT OF VIBRATION ON TAP RESPONSES Since it is well known that muscle vibration tends to suppress phasic monosynaptic reflexes such as the tendon jerk and H-reflex (Delwaide, 1973; Hagbarth, 1973; Lance et al., 1973), the effect of vibration on tendon tap responses has been studied. Provided that the spindle ending was being well acti-

285

vated by the vibratory stimulus, the additional tap stimulus usually failed to generate an additional spindle response. Indeed if the tap fell in the shortening phase of the vibration cycle no response was seen from the ending. If it fell during the stretch phase the tap sometimes altered the timing of discharge, but only occasionally added additional impulses to those produced by the vibration wave. However, immediately after percussion, as the tap-induced dynamic stretch subsided, a temporary pause in the vibration-induced spindle response could be seen, a response analogous t o the loss of primary ending response to vibration which occurs on muscle shortening, as discussed earlier. Thus the interaction of vibration and tendon percussion is inhibitory for some spindle endings, at least under the present conditions of study. Undoubtedly such interference phenomena contribute t o the suppression of tendon jerks by muscle vibration.

VOLUNTARY CONTRACTION Based on the principle of a - y co-activation, the effects of voluntary contraction on the spindle response have been examined using the contraction as a means

50/s 25 0

1 Nrn

Fig. 3. The effect of voluntary contraction of the receptor-bearing muscle o n vibration response. Group Ia afferent unit (tibialis anterior) during vibration a t 55 Hz. A : instantaneous frequency plot of ending responding to every second or third vibration wave when muscle was relaxed. During voluntary contract~ionas indicated by torque change (second trace) and EMG (lower trace), ending responded a t times t o every or to every o t h e r vibration cycle. Vibratory stimulus removed a t the arrow. B: Interdischarge interval histograms of same ending showing responses a t latencies corresponding t o 2 and 3 vibration cycles when a t rest (upper histogram), and a t 1 and 2 vibration cycles during voluntary contraction (lower histogram).

286 of activating fusimotor fibres. Isometric voluntary contraction of the receptorbearing muscle resulted jn an increased spindle response t o vibration, provided that the spindle was responding submaximally prior t o the contraction (Fig. 3). It may be concluded, therefore, that in man, as in the cat (Granit and Henatsch, 1956; Crowe and Matthews, 1964; Brown et al., 1967; Homma et al., 1972),activation of the fusimotor system results in an increased spindle sensitivity to vibration, and that this heightened sensitivity is sufficient t o compensate for the spindle unloading effects of the extrafusal contraction.

THE TONIC VIBRATION REFLEX During many recordings a tonic vibration reflex developed - usually as a result of more prolonged vibration in a relatively stretched position. For approximately one-third of the spindle endings, the appearance of a TVR did not alter spindle response to vibration. For the remainder, both primary and secondary endings, the development of a TVR resulted in a decreased response t o vibration: the reflex contraction unloaded the spindle ending (Fig. 4 ) . Such behaviour cannot be attributed t o a withdrawal of “resting fusimotor tone”, if, as mentioned earlier, there is no resting fusimotor tone in relaxed human subjects. Furthermore it suggests that if a-y co-activation occurs during vibration (cf., Fromm and Noth, 1974, 1976; Trott, 1975), the increased y-drive is insufficient to compensate for unloading from extrafusal contraction. It should be pointed out that as far as possible care was taken t o ensure that the reflex contraction involved the receptor-bearing muscle, and was not a simple unloading response due t o contraction of synergistic muscles. The unloading produced by reflex contractiori of the receptor-bearing muscle is reminiscent of the unloading produced by the phasic stretch reflex (Burg et al., 1973; Hagbarth et a1 , 1975b), and it seems reasonable t o conclude

1: 3

1oo/s

1Nm

2s

Fig. 4. The effect of the development of a TVR in the receptor-bearing muscle. Group Ia afferent unit (tibialis anterior).Vibration at 100 Hz produced initially one-to-one driving of the ending but when a TVR developed, as seen in torque (second trace) and EMG (third trace), the response dropped t o subharmonics of the vibration frequency. On request to suppress the TVR voluntarily (indicated by arrow), torque fell, EMG ceased and the ending resumed a one-to-one response. Duration of vibration indicated by horizontal bar.

287 that the phasic and tonic stretch reflexes are exceptions to the rule of a-y coactivation, the sole exceptions thus far documented. These findings are t o be expected if the stretch reflex is t o operate as a load-compensating mechanism (Von Euler, 1966). On voluntary suppression of the TVR (Fig. 4), spindle response t o vibration returned t o the precontraction level. It can therefore be concluded that voluntary control of the TVR is a central phenomenon, and is not due t o any suppression of the spindle sensitivity t o vibration, a conclusion also reached by Marsden et al. (1969) on indirect grounds. SUMMARY With a vibrator of the type commonly used to elicit tonic vibration reflexes in man, the spindle primary endings can be driven up to firing rates greatly exceeding those resulting from sustained passive stretch alone. Muscle stretch increases the spindle response t o vibration, the spindle primaries following higher vibration frequencies than the secondaries. During muscle shortening, on the other hand, the vibration-induced spindle excitation is minimal, the response being better preserved in the secondaries than in the primaries. Primary endings strongly driven by the vibrator are often unable t o respond t o a tendon tap applied during vibration, but the tap may temporarily unload the spindles on its falling phase, causing a pause in the vibration-induced excitation. Fusimotor coactivation occurring during a voluntary contraction sensitizes spindle endings t o the vibratory stimulus. The TVR resulting from the vibratory stimulus is a contraction of the a-type, which unloads spindle endings, and reduces their sensitivity t o vibration.

ACKNOWLEDGEMENTS This project is supported by the Swedish Medical Research Council (Project NO. B7 6-14X-2881-07B). D.B. is supported by a C.J. Martin Travelling Fellowship from the National Health and MedicaI Research Council of Australia.

REFERENCES Bianconi, R. and Van der Meulen, J.P. (1963) The response to 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: 773-800. Burg, D., Szumski, A.J., Struppler, A. and Velho, F. (1973) Afferent and efferent activation of human muscle receptors involved in reflex and voluntary contraction. Exp. Neurol., 41 : 754-768. Crowe, A. and Matthews, P.B.C. (1964) Further studies of static and dynamic fusimotor fibres. J. Physiol. (Lond.), 174: 132-151. De Gail, P., Lance, J.W. and Neilson, P.D. (1966) Differential effects on tonic and phasic reflex mechanisms produced by vibration of muscles in man. J. Neurol. Neurosurg. Psychiat., 2 9 : 1-11.

Delwaide, P.J. (1973) Human monosynaptic reflexes and presynaptic inhibition. In N e w Developments in EMG and Clinical Neurophysiology, Vol. 3, J.E. Desmedt (Ed.), Karger, Basel, pp. 508-522. Fromm, C. and Noth, J. (1974) Vibration-induced autogenetic inhibition of gamma motoneurons. Brain Res., 8 3 : 495-497. Fromm, C. and Noth, J. (1976) Reflex responses of gamma motoneurones to vibration of the muscle they innervate. J. Physiol. (Lond.), 256: 117-136. Granit, R. (1970) The Basis of Motor Control, Academic Press, London. Granit, R. and Henatsch, H.-D. (1956) Gamma control of dynamic properties of muscle spindles. J. Neurophysiol., 19: 356-366. Hagbarth, K.-E. (1973) The effects of muscle vibration in normal man and in patients with motor disorders. In New Developments in EMG and Clinical Neurophysiology, Vol. 3, J.E. Desmedt (Ed.), Karger, Basel, pp. 428-443. Hagbarth, K.-E. and Eklund, G. (1966) Motor effects of vibratory muscle stimuli in man. In Nobel Symposium I, Muscular Afferents and Motor Control, R . Granit (Ed.), Almqvist and Wiksell, Stockholm, pp. 177-186. Hagbarth, K.-E. and Vallbo, A.B. (1968) Discharge characteristics of human muscle afferents during muscle stretch and contraction. Exp. Neurol., 22: 674-694. Hagbarth, K.-E., Hongell, A. and Wallin, B.G. (1970) The effect of gamma fibre block on afferent muscle nerve activity during voluntary contractions. Acta physiol. scand., 79: 2 7 A-2 8A. Hagbarth, K.-E., Wallin, G., Burke, D. and Lofstedt, L. (1975a) Effects of the Jendrassik manoeuvre on muscle spindle activity in man. J. Neurol. Neurosurg. Psychiat., 38: 1143-1 153. Hagbarth, K.-E., Wallin, G. and Lofstedt, L. (1975b) Muscle spindle activity in man during voluntary fast alternating movements. J. Neurol. Neurosurg. Psychiat., 38: 625-635. Homma, S. (1973) A survey of Japanese research on muscle vibration. In New Deuelopments in EMG and Clinical Neurophysiology, VoZ. 3, J.E. Desmedt (Ed.), Karger, Basel, pp. 463-468. Homma, S., Mizote, M., Nakajima, Y. and Watanabe, S. (1972) Muscle afferent discharges during vibratory stimulation of muscles and gamma fusimotor activities. Agressologie, 13D: 45-53. Lance, J.W., Burke, D. and Andrews, C.J. (1973) The reflex effects of muscle vibration. Studies of tendon jerk irradiation, phasic reflex, inhibition and the tonic vibration reflex. In New Developments in EMG and Clinical Neurophysiology, Vol. 3, J.E. Desmedt (Ed.), Karger, Basel, pp. 444-462. Marsden, C.D., Meadows, J.C. and Hodgson, H.J.F. (1969) Observations on the reflex response t o muscle vibration in man and its voluntary control. Brain, 92: 829-846. Matthews, P.B.C. (1969) Evidence that the secondary as well as the primary endings of the muscle spindles may be responsible for the tonic stretch reflex of the decerebrate cat. J. Physiol. (Lond.), 204: 365-393. Pompeiano, O., Wand, P. and Sontag, K.-H. (1975) The relative sensitivity of Renshaw cells to orthodromic group Ia volleys caused by static stretch and vibration of extensor muscles. Arch. itul. Biol., 113: 238-279. Thoden, U., Margherini, P.C. and Pompeiano, 0. (1972) Evidence that presynaptic inhibition may decrease the autogenetic excitation caused by vibration of extensor muscles. Arch. ital. Biof., 110: 90-116, Trott, J.R. (1975) Reflex responses of fusimotor neurones during muscle vibration. J. Physiol. (Lond.), 247: 20-22P. Vallbo, A.B. (1970) Slowly adapting muscle receptors in man. Acta physiol. scand., 78: 315-333. Vallbo, A.B. (1971) Muscle spindle response at the onset of isometric voluntary contractions in man. Time difference between fusimotor and skeletomotor effects. J. Physiol. (Lond.), 318: 405-431. Vallbo, A.B. (1974a) Afferent discharge from human muscle spindles in non-contracting muscles. Steady state impulse frequency as a function of joint angle. Acta physiol. scand., 90: 303-318. Vallbo, A.B. (197413) Human muscle spindle discharge during isometric voluntary contrac-

289 tions. Amplitude relations between spindle frequency and torque. Acta physiol. scand., 9 0 : 319-336. Vallbo, A.B. and Hagbarth, K.-E. ( 1 9 6 8 ) Activity from skin mechanoreceptors recorded percutaneously in awake human subjects. Exp. Neurol., 21: 270-289. Von Euler, C. (1966) Proprioceptive control in respiration. In Nobel Symposium I , Muscular Afferents and Motor Control, R. Granit (Ed.), Almqvist and Wiksell, Stockholm, pp. 197-207. Wallin, B.G., Hongell, A. and Hagbarth, K.-E. (1973) Recordings from muscle afferents in Parkinsonian rigidity. In New Developments in EMG and Clinical Neurophysiology, Vol. 3, J.E. Desmedt (Ed.), Karger, Basel, pp. 263-272.

DISC US SION GRANIT: Do you always find a close time correlation between spindle activation and alpha contractions during the Jendrassik manoeuvre? Struppler and co-workers report spindle activation without concomitant alpha contraction. What d o you think about that? HAGBARTH: A difference between o u r experiments and those of Struppler and his group is that besides t h e EMG electrodes in the receptor-bearing muscle we had t h e foot attached to a sensitive strain-gauge, detecting even minor mechanical events. The spindle accelerations that we saw were correlated either with contraction in the receptor-bearing muscle or with torque changes indicating that stretch stimuli were acting o n that muscle, for instance due to antagonist contractions. I n all o u r recordings, there was a close correlation between t h e spindle events and the torque signals. POMPEIANO: Long time ago, Dr. Granit and others found that a change of position with respect to gravity may produce modulation of gamma fusimotor activity. Did y o u find in your recordings signs of gamma activation by changing the position? HAGBARTH: F o r technical reasons it is difficult t o test the effect of large positional changes. The recording site is easily lost. However, lifting of the head, for instance, or movements of the arms did not affect spindle firing in the leg muscles, providing these muscles remained relaxed and mechanically uninfluenced by the movements. MATTHEWS: I n one of your records you showed a very clear phase-locking of a secondary ending b y vibration both during stretch and release. What I couldn’t see in the record was how much vibration changed the mean frequency of firing of this secondary ending from what it would have been if you would have done stretching and releasing b u t not vibrating. How much did it change the mean frequency of firing besides altering the timing of t h e firing? HAGBARTH: I did n o t show any slide illustrating a secondary ending responding to stretch alone. However, both secondaries and primaries could generally be brought u p t o higher firing rates by combined stretch and vibration than b y stretch alone. MATTHEWS: In the cat you can sometimes have secondary endings which are quite well phase-locked by vibration but having the mean frequency relatively unchanged. This is quite important t o analyze. HAGBARTH: I n the figure I showed (2B), the secondary ending was during the stretch phase driven at 50 imp/sec. We vibrated a t 1 0 0 Hz and it responded at each second vibration wave. We never saw such high firing rates in secondaries due to the slow passive stretch alone. We may have seen firing rates of 20-30 Hz b u t not more than that.

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Reciprocal Ia Inhibition and Voluntary Movements in Man REISAKU TANAKA

*

Department of Physiology, Hirosaki University Faculty of Medicine, Hirosaki (Japan)

INTRODUCTION This report deals with the identification of the reciprocal Ia inhibition in man and modulation of its activity in relation to voluntary movements. In 1925, Liddell and Sherrington showed in the cat that stretch of a muscle inhibits motoneurones innervating its antagonist muscles. Then, using the monosynaptic reflex testing technique, Lloyd (1946; Laporte and Lloyd, 1952) showed that electrical stimulation of large muscle afferents, group Ia fibres, evoked inhibition of antagonist motoneurones with a central latency of almost 0 msec. Later, this latency was interpreted by Araki et al. (1960) as showing disynaptic linkage. Since then, interneurones mediating this inhibition have been extensively investigated in the cat spinal cord by many investigators (for ref. see Hultborn, 1972). In brief, they are under the control of various supraspinal and segmental systems and considered to play a role of integrative centres in reciprocal innervation. However, little is known regarding the occurrence of the reciprocal Ia inhibition in man. Criteria for identification of Ia inhibition should follow those used in cat experiments: being evoked by low-threshold afferents and with disynaptic latency. In human experiments no such radical procedures as direct access t o the spinal cord and muscle nerves, which enable us t o control selective group I stimulation and t o measure central latency of their effect with ease, are allowed. However, the H-reflex, which is homologous t o the monosynaptic reflex (Magladery et al., 1951), gives a good tool for testing the excitability of spinal motoneurones in man. The lower threshold of the H-reflex than that of the direct motor response (M-wave) in the triceps surae muscle by percutaneous stimulation of the tibial nerve at the popliteal fossa indicates that the group Ia afferents also have a lower threshold than a-efferents by this mode of stimulation. The conduction velocity of Ia fibres should not differ significantly between the tibial and the peroneal nerves. Therefore, the central latency can be estimated from adjustment of the minimal effective interval of conditioningtest shocks by conduction time for Ia volleys t o reach the spinal cord from both sources. In short, the procedure established by Lloyd is applicable t o human experiments and, indeed, found to be successful as shown below. The detailed experimental procedures have been described elsewhere (Mizuno et al., 1971; Tanaka, 1974).

* Present address: Department of Neurobiology, Tokyo Metropolitan Institute f o r Neurosciences, Musashidai 2-6, Fuchu, Tokyq 183, Japan.

292 EXISTENCE OF RECIPROCAL Ia INHIBITION IN MAN The reciprocal Ia inhibitory pathway was first demonstrated in patients with bilateral athetosis (Mizuno et al., 1971). As shown in Fig. lB, the conditioning stimulation in the common peroneal nerve at the level of the caput fibulae evoked a strong inhibition of the H-reflex response of the triceps surae muscle within 5 msec of conditioning-test intervals. The strongest inhibition was observed at 1.5 msec. In other subjects in which the effects with shorter intervals were tested, the minimal effective interval was around 1.0 msec. This time course of the effect is analogous t o the curve for Ia inhibition obtained from the cat spinal cord by Lloyd (e.g., Fig. 7 of Lloyd, 1946), except the onset interval. This interval, however, strongly suggests a disynaptic linkage, provided that the nerve component responsible for the effect be group Ia afferents (Tanaka, 1974). The test reflex is also evoked by Ia volleys. In man, the motor pools of pretibial muscles are situated in the 4th and 5th lumbar segments overlapping those of the triceps surae in the 5th lumbar and 1st sacral segments (Sharrard, 1955). The electrode for stimulation of the peroneal nerve was usually located 5-7 cm distally t o that for the tibial nerve. The conduction velocity of the Ia afferents in the tibial nerve in man is supposed to be about 60 m/sec (Magladery and McDougal, 1950; Smorto and Basmajian, 1972). Therefore, the conduction time for the Ia afferents in the peroneal nerve t o reach the motor pools should be approximately 1.0 msec longer than that of the tibial Ia volleys. Correction of the minimal effective interval of 1 msec by

A normal

B

.

.w I

athetosis

0

. 0

.

8 C ankle dorsiflexion (normal)

0 06

t

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2 Fig. 1. Time course of effects of peroneal nerve stimulation on H-reflex response of the triceps surae muscle in a normal (A) and a patient with bilateral athetosis (B). The amplitude of the test reflex is expressed as a per cent of control amplitude on the ordinate, which is indicated with its standard deviation at the right side of graphs A and B. The abscissa shows the time interval between conditioning and test stimuli. Conditioning stimulus intensity was 1.34 times threshold of the direct motor response, M-wave, ( X motor threshold; X MT) for both cases. The time course of ankle joint movement by the peroneal stimulation from the subject in A is illustrated diagrammatically in C. Upward deflection indicates dorsiflexion. Fig. 2. Same patient as in Fig. 1B. The conditioning-test stimulus interval is fixed at 1.5 msec. The depressive effect is observed at 0.66 X MT and reaches a maximum a t 1.0 X MT (Mizuno et al., 1971).

293 this delay shows that the central latency o f the reciprocal inhibitory effect on the triceps surae motoneurones is about 0 msec. This is just comparable with the central latency of Ia inhibition in the cat hindlimb (Lloyd, 1946; Laporte and Lloyd, 1952), indicating its disynaptic origin. The relationship between the strength of conditioning stimulus and the magnitude of this inhibition at the optimal interval of 1.5 msec from the same subject is given in Fig. 2. The inhibition was evoked with considerably weaker stimuli than the threshold of the M-wave and attained maximal effect around the latter. This clearly indicates that group I afferents, most likely Ia fibres, are responsible, and provides a justification for the above estimation of the central latency. The Ib origin of this inhibition is less likely for the following reasons: the group Ib afferents facilitate the monosynaptic reflex response of motoneurones supplying antagonist muscles in high spinal cats (Laporte and Lloyd, 1952), extensor motoneurones rarely receive inhibitory Ib actions from flexor muscles (Eccles et al., 1957), and the commonest synaptic linkage for Ib actions on motoneurones is trisynaptic (Eccles et al., 1957; cf., Laporte and Lloyd, 1952). Indeed, a facilitatory effect from the peroneal nerve t o the triceps surae H-reflex, which is attributable t o Ib actions, was observed in some cases of bilateral athetosis (Mizuno et al., 1971), spinal cord lesions (Yanagisawa, 1973) and spastic hemiplegia from cerebrovascular lesions (Yanagisawa, Tanaka and Ito, t o be published). The reciprocal Ia inhibition of the triceps surae motoneurones was also observed in some cases with spastic paresis by spinal lesions (Yanagisawa, 1973). In contrast, we failed to reveal this inhibition in normal subjects at rest except with one subject out of 13 so far tested (Mizuno et al., 1971; Tanaka, 1974). Fig. l A , from a normal subject, compared with Fig. l B , indicates a lack of the short-latency inhibition of the triceps surae H-reflex. Multiple volleys in the peroneal nerve were also scarcely effective in revealing the inhibition (Tanaka, 1974). The case which showed Ia inhibition in spite of no signs of neurological disorders was unique in showing H-reflex in pretibial muscles by stimulation of the peroneal nerve. A higher excitability of pretibial motoneurones, in this case as exemplified by the presence of H-reflex responses, might be related t o the revelation of Ia inhibitory effect on the triceps surae motoneurones. Since the presence of H-reflex in pretibial muscles had not been reported in normal subjects at rest (e.g., Hoffmann, 1934; Teasdall et al., 1951) but had in patients with lower brain stem and upper cervical cord lesions (Teasdall et al., 1952), it was suggested that the pretibial motoneurones are tonically suppressed in normal subjects at rest (Teasdall et al., 1952). It was, however, later reported that the pretibial H-reflex occurs in a few normal cases (Hohman and Goodgold, 1960). Coexistence of H-reflex in pretibial muscles and reciprocal Ia inhibition of the triceps surae H-reflex should be confirmed by repeating the experiment in these cases. Reciprocal Ia inhibition of pretibial motoneurones by group I volleys in the tibial nerve was also revealed in subjects who showed H-reflex in pretibial muscles, thus allowing the testing of excitability of pretibial motoneurones. They included one normal subject (see above) and 3 patients hemiplegic from capsular lesions (Tanaka, 1974; Yanagisawa e t al., 1975). Fig. 3 is an example from such a patient. The inhibition was observed by a single tibial stimulus

294

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Fig. 3. Reciprocal inhibition of pretibial motoneurones in a patient with capsular hemiplegia. A: the time course of the effect evoked by single tibial shocks (0.75 X MT). Similar illustration to Fig. 1. Negative values in the abscissa show that the tibial shock followed the peroneal shock and positive values vice versa. B-G: specimen records from pretibial muscles. B, control record by test peroneal shock. Conditioning-test intervals are shown on the left side of each record of C-G (Yanagisawa et al., 1975).

with a strength of 0.75 times threshold for M-wave; slightly supraliminal for the triceps surae H-reflex showing its group Ia origin. The brief time course of the effect was similar t o that of the inhibition of the triceps surae except its onset interval. The effect even appeared when the conditioning tibial shock succeeded the test peroneal shock by 1.0 msec. However, with the calculation given above its central latency could be e s t b a t e d t o be around 0 msec again, indicating a disynaptic linkage. Another example from a normal subject has been presented previously (Tanaka, 1974). In summary, a reciprocal l a inhibitory pathway exists symmetrically between ankle extensor and flexor systems in man, as is the case in cat. The failure of peroneal Ia stimulation to inhibit the triceps surae motoneurones in normal subjects at rest may be related t o the lack of pretibial H-reflex in these subjects and indicates that interneurones of this Ia inhibitory pathway are depressed, as suggested in the case of pretibial motoneurones (Teasdall et al., 1952). Its underlying mechanisms will be referred to in the Comments.

RECIPROCAL Ia INHIBITION DURING VOLUNTARY ANKLE MOVEMENTS Our main aim was to answer the question whether or not the Ia inhibitory system contributes t o the antagonist inhibition actually occurring during voluntary movements. Voluntary ankle dorsiflexion, i.e., contraction of the tibialis anterior muscle, causes an inhibition of its antagonistic triceps surae motoneurones as exemplified by depression of the H-reflex (compare Fig. 4A and C, F;

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Fig. 4. Short-latency reciprocal inhibition revealed during voluntary ankle dorsiflexion. AG: records from the triceps surae muscle. Records on the left are the control for those conditioned by triple and double peroneal stimuli (1.0 x MT, 333/sec) on the right. The third shock is set 1.7 msec prior to the test shock. A and B: at rest. C-E: during a weak contraction. F and G: during a slightly stronger contraction. H and I: responses in pretibial muscles by the conditioning stimuli a t rest and during contraction respectively. An arrow in I indicates the H-reflex response (Tanaka, 1974).

Hoffmann, 1934; Paillard, 1955). If the excitability of Ia inhibitory interneurones t o the triceps surae motoneurones increases together with this antagonist inhibition, it strongly suggests that this system takes part in revelation of the inhibition. Fig. 4 shows the excitability increase of the la inhibitory pathway during voluntary ankle dorsiflexion. Only normal subjects were used for this series of experiments. A t rest, triple peroneal volleys preceding the test tibia1 shock evoked hardly any depression of the H-reflex (compare A and B). During a weak voluntary contraction of the ankle flexors, which produced a decrease of the test reflex ( C ) , the same conditioning shocks effectively reduced the new test reflex, a 16% reduction ( C and D). The withdrawal of the last conditioning shock abolished the depressive effect (E), indicating that this shock was directly linked to the effect. The time interval between the last conditioning and test shocks was 1.7 msec, an optimal one for the Ia inhibitory effect. The time course of the effect which was analogous to the one described above was obtained from this and other subjects. The threshold for the effect was in the group I range. During a slightly stronger dorsiflexion the amplitude of the test reflex further reduced (F) and the peroneal stimuli produced a 33% reduction in this case (G). As has previously been shown (Hoffmann, 1934), an H-reflex was regularly evoked in pretibial muscles by the peroneal nerve stimuli during the active ankle dorsiflexion (I) but not at rest (H). In the exceptional case, that showed the Ia inhibition of the triceps surae in a relaxed state, the threshold for the inhibition was lower during movements than at rest, also indicating an increased interneuronal activity during the movement.

296 This excitability increase of Ia inhibitory interneurones is not a result of generalized excitability increases of the segmental apparatus, but a selective increase of activity of the flexor system since the voluntary ankle plantar flexion was ineffective in revealing the H-reflex inhibition. Reciprocal Ia inhibition of pretibial motoneurones observed in a unique case was abolished by strong dorsiflexion, which augmented the test pretibial H-reflex but abolished the triceps surae H-reflex. The effect of voluntary plantar flexion on the Ia inhibition of flexors could not be tested because it easily abolished the test reflex. RECIPROCAL Ia INHIBITION AT THE ONSET OF VOLUNTARY MOVEMENTS The excitability of pretibial motoneurones starts t o increase 50-100 msec prior to the onset of EMG in these muscles at a voluntary ankle dorsiflexion (Simoyama and Tanaka, 1974), as is the case in the triceps surae muscle at plantar flexion (Kots, 1969; Coquery and Coulmance, 1971; Pierrot-Deseilligny et al., 1971; Gottlieb and Agarwal, 1972). On the other hand, the reciprocal inhibition of the triceps surae motoneurones occurs at or after the onset of voluntary EMG at dorsiflexion (Kots, 1969; Gottlieb and Agarwal, 1972), although some authors reported that the triceps surae H-reflex already started t o reduce prior t o the EMG onset (Pierrot-Desilligny et al., 1971). In our experiments, the inhibition usually started more than 100 msec after the onset of EMG in pretibial muscles. This slow occurrence of inhibition was due t o a rather weak dorsiflexion used here in order t o avoid mechanical artefacts. The inhibition could occur earlier at stronger efforts. The onset of this inhibition

Fig. 5. Reciprocal Ia inhibition of the triceps surae H-reflex, revealed prior to the occurrence of pretibial EMG at ankle dorsiflexion. Upper traces of A and B are records from the triceps surae muscle and the lower traces from pretibial muscles. Arrows indicate the EMG onset in pretibial muscles. The horizontal bar in the lower trace of B indicates an H-reflex evoked in pretibial muscles by conditioning stimuli. In C, the amplitude of the triceps surae H-reflexes (ordinates) is plotted against the intervals between the onset of pretibial EMG and the test H-reflexes (abscissa). Positive values indicate that the H-reflex occurs after the EMG onset. Open circles show the amplitude of the test H-reflexes and filled circles show the conditioned H-reflexes. Control records, obtained in a resting state, are plotted on the right-hand side (Simoyama and Tanaka, 1974).

297 does not necessarily indicate the onset of excitability increase of the responsible inhibitory interneurones. Specimen records of Fig. 5 show the way t o reveal the excitability change in the transmission of the Ia inhibitory pathway t o the ankle extensors. The test tibia1 shock (A) and a pair of conditioning peroneal and test shocks (B) were given alternately at every ankle dorsiflexion and the amplitude of the H-reflexes in each group was compared. The interval of the last conditioning and test stimuli was fixed at 1.5 msec, an optimal interval. The intensity of the peroneal stimuli was kept slightly subliminal for M-wave generation in pretibial muscles and thus limited within the group I range. As shown with open circles in Fig. 5C, the excitability of the triceps surae motoneurones was hardly changed before and until 100 msec after the EMG onset. Combined conditioning stimulation, however, revealed a Ia inhibition of the H-reflex which started 70-80 msec prior t o the EMG onset (compare open and closed circles in Fig. 5C). It was also noticed that the conditioning shocks themselves evoked an H-reflex in the agonist pretibial muscles (horizontal dotted line in the lower trace of Fig. 5B), indicating advanced facilitation of agonist motoneurones. Therefore, the descending command appears t o drive a-motoneurones and Ia inhibitory interneurones in parallel.

COMMENTS Reciprocal inhibition, attributable t o group Ia action, of motoneurones innervating ankle extensor and flexor muscles was revealed in human subjects with or without various neurological disorders. Most prominent findings are that, in normal human subjects, interneurones mediating Ia inhibition t o the ankle extensor motoneurones appear t o be depressed in a resting state but are active in parallel with depression of the extensor motoneurones during a steady voluntary contraction of ankle flexors. Subliminal facilitation of these interneurones already appears nearly 100 msec prior t o the onset of voluntary EMG in the flexors in parallel with that of agonist a-motoneurones. A most likely explanation of these results may be as follows (see Fig. 6A). During a voluntary ankle dorsiflexion of the foot, the brain sends an excitatory command t o the ankle flexor a-motoneurones. The same command should also be aimed at y-motoneurones, producing tonic afferent discharges from the muscle spindle primary endings of the ankle flexor muscles as shown in the forearm muscles in man (Vallbo, 1970). Therefore, the activity of agonist amotoneurones is assisted by these group Ia discharges. This is formulated by Granit (1955) as a-y linkage in muscle contraction. H-reflexes in pretibial muscles reflect the resulting excitability increase of pretibial motoneurones. Ia inhibitory interneurones would also be facilitated by these afferent discharges. They are, however, not the single excitatory source. Since facilitation of the la inhibitory pathway occurs prior t o the onset of voluntary EMG in agonist muscles, while group Ia discharges by y-activation start after the EMG onset (Vallbo, 1971), the interneurones must be activated by the descending command directly and via the y-loop like agonist a-motoneurones (note the thick dotted line in Fig. 6A). The antagonist a-motoneurones would be depressed by this interneuronal activity. This is well in keeping with the hypothesis of

298 B TA .EMG

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TA.MN excit

Fig. 6. A: schematic illustration of neuronal connections. a, a-motoneurones; y, y-motoneurones. Neurones illustrated with filled circles indicate Ia inhibitory interneurones. B: schematic illustration for the mechanism underlying delayed occurrence of actual inhibition of t h e triceps surae motoneurones (GS-MN) in respect to t h e onset of t h e voluntary ankle flexor activity (TA-EMG). MN, motoneurones; Ia, Ia afferents; IN, interneurones; excit., excitability. Horizontal arrows directed to right indicate t h e time point when firing of respective neurones occurs. The one directed to left indicates the onset of inhibition o n GS-MN. Further explanation in text.

a - y linkage in reciprocal inhibition posed by Hongo et al. (1969). A mutual inhibitory connection of Ia inhibitory interneurones belonging t o extensor and flexor systems is also illustrated in Fig. 6A, according to the recent findings in the cat (Hultborn et al., 1974). If this connection also exists in man, the parallel Ia inhibition of antagonist a-motoneurones and Ia interneurones would provide the intended movement with security from antagonist contraction and reciprocal inhibition, respectively, which might be caused by Ia discharges from the antagonist muscles through passive stretch due t o agonist contraction. Delayed occurrence of the actual antagonist inhibition of ankle extensor motoneurones, in respect t o the onset of the flexor EMG, can be explained in this context (Fig. 6B). At weak ankle dorsiflexion, the voluntary effort facilitates ankle flexor a -motoneurones and Ia inhibitory interneurones in parallel (left vertical dotted line), but the facilitation of the latter remains subliminal until the arrival of Ia discharges, which only occur after the onset of the agonist EMG (Vallbo, 1971). Combined facilitatory inputs from the brain and the y-loop now make the interneurones fire (arrow in TA-Ia-IN excit.) and inhibit the extensor a-motoneurones, and possibly extensor Ia interneurones. The occurrence of actual antagonist inhibition after the onset of voluntary agonist EMG is not so surprising, since counteracting Ia discharges from the antagonist muscles would reach the spinal cord only after their stretch by the intended movements. The structure for the a - y linkage in muscle contraction and reciprocal inhibition at voluntary movements is really materialized in the central nervous system. The motor cortex is generally believed t o play a major role in the control of voluntary movements. In the primate, which is akin to humans, corticospinal tract fibres from this area have monosynaptic excitatory connections with a and y-motoneurones (Phillips, 1969) and Ia inhibitory interneurones (Jankowska and Tanaka, 1974). It has also been shown that corticospinal tract neurones

299 in the forelimb area of the motor cortex discharge prior to the contraction of the forelimb muscles contralateral t o the recorded cortex (Evarts, 1966). This descending system is likely to be a major one of the systems responsible for the present findings in man. For the lack of reciprocal Ia inhibition t o ankle extensors in normal subjects at rest, 3 possibilities have been presented (Mizuno et al., 1971; Tanaka, 1974): (i) weak connection in the Ia inhibitory pathway from the deep peroneal nerve to the triceps surae motoneurones, as found in the cat (Eccles and Lundberg, 1959; Hongo et al., 1969) and the crab-eating monkey (Jankowska, Padel and Tanaka, unpublished observations), (ii) existence of a tonic descending inhibition of the Ia inhibitory interneurones in this pathway, as suggested in the case of the baboon (Hongo, Lundberg, Phillips and Thompson, personal communication), and (iii) tonic inhibition of them by antagonist Ia interneurones which are active in the resting state of subjects (Fig. 6A). In spastic patients, in which an excitability increase of the ankle extensor system is clear as exemplified by exaggerated tendon reflexes, an alcohol block t o the lateral and medical gastrocnemius nerves improved the muscle power of the ankle flexors significantly (Yanagisawa et al., 1975). Since reciprocal Ia inhibition of the triceps surae was scarcely observed but that of the pretibial muscles was remarkable, the above finding was explained by the disinhibition of flexor motoneurones due to withdrawal of Ia inflows from the gastrocnemius muscles t o Ia inhibitory interneurones innervating flexor motoneurones by y-block. It is very likely that the third mechanism really works in them. In normal subjects at rest, y-block of the tibial nerve by local anaesthetics decreased the amplitude of the test H-reflex of the triceps surae, suggesting the existence of background a-activity to, and resultant tonic Ia discharges from, the triceps surae muscle (Landau et al., 1960). All together, the third possibility must be fundamental for the lack of Ia inhibition t o ankle extensors in these subjects. The second possibility might be related t o the third. Descending facilitatory influences to the ankle extensor system (thin dotted line in Fig. 6A) would result in tonic inhibition of flexor a motoneurones and Ia interneurones. However, the existence of more direct inhibition from suprasegmental structures t o them is not excluded. The first possibility may also be reponsible. ACKNOWLEDGEMENTS The author is greatly indebted t o Dr. Ito, Dr. Mizuno, Dr. Simoyama and Dr. Yanagisawa for very stimulating collaboration, and t o the people who contributed to the present investigation as subjects. REFERENCES Araki, T., Eccles, J.C. and Ito, M. (1960) Correlation of inhibitory postsynaptic potential of motoneurones with the latency and time course of inhibition of monosynaptic reflexes. J. Physiol. (Lond.), 154: 354-377. Coquery, J.-M. et Coulmance, M. (1971) Variations d’amplitude des reflexes monosynaptiques avant un mouvement volontaire. Physiol. Behau., 6: 65-69.

Eccles, J.C., Eccles, R.M. and Lundberg, A. (1957) Synaptic actions on motoneurones caused by impulses in Golgi tendon organ afferents. J. Physiol. ( L o n d . ) , 1 3 8 : 227252. Eccles, R.M. and Lundberg, A. (1959) Synaptic actions in motoneurones by afferents which may evoke the flexion reflex. Arch. itul. Biol., 9 7 : 199-221. Evarts, E.V. ( 1 9 6 6 ) Pyramidal tract activity associated with a conditioned hand movement in the monkey. J. Neurophysiol., 29: 1011-1027. Gottlieb, G.L. and Agarwal, G.C. (1972) The role of the myotatic reflex in the voluntary control of movements. Bruin Res., 40: 139-143. Granit, R. (1955) Receptors and Sensory Perception, Yale Univ. Press, New Haven, Conn. Hoffmann, P. ( 1 9 3 4 ) Die physiologischen Eigenschaften der Eigenreflexe. Ergebn. Physiol., 36: 15-108. Hohman, T.C. and Goodgold, J. (1960) A study of abnormal reflex patterns in spasticity. Amer. J. phys. Med., 4 0 : 52-55. Hongo, T., Jankowska, E. and Lundberg, A. (1969) The rubrospinal tract. 11. Facilitation of interneuronal transmission in reflex paths to motoneurones. Exp. Bruin Res., 7 : 365391. Hultborn, H. (1972) Convergence on interneurones in the reciprocal Ia inhibitory pathway t o motoneurones. Actu physiol. scund., Suppl. 375: 1-42. Hultborn, H., Illert, M. and Santini, M. (1974) Disynaptic Ia inhibition of the interneurones mediating the reciprocal Ia inhibition o f motoneurones. Actu physiol. scund., 9 1 : 14A-16A. Jankowska, E. and Tanaka, R. ( 1 9 7 4 ) Neuronal mechanism of the disynaptic inhibition evoked in primate spinal motoneurones from the corticospinal tract. Brain Res., 75: 163-1 66. Kots, Ya.M. (1969) Supraspinal control of spinal centres for antagonist muscles in man. Biofizika, 1 4 : 167-172. (English translation by Pergamon Press.) Landau, W.M., Weaver, R.A. and Hornbein, T.F. (1960) Fusimotor nerve function in man. Differential nerve block studies in normal subjects and in spasticity and rigidity. Arch. Neurol. (Chic.), 3 : 10-23. Laporte, Y. and Lloyd, D.P.C. (1952) Nature and significance of the reflex connection established by large afferent fibers of muscular origin. Amer. J. Physiol., 169: 609-621. Liddell, E.G.T. and Sherrington, C.S. (1925) Further observations on myotatic reflexes. Proc. roy. Soc. B, 97: 267-283. Lloyd, D.P.C. ( 1 9 4 6 ) Facilitation and inhibition of spinal motoneurons. J. Neurophysiol., 9 : 421-438. Magladery, J.W. and McDougal, D.B., Jr. ( 1 950) Electrophysiological studies of nerve and reflex activity in normal man. I. Identification of certain reflexes in the electromyogram and the conduction velocity of peripheral nerve fibers. Bull. Johns Hopk. Hosp., 8 6 : 265-290. Magladery, J.W., Porter, W.E., Park, A.M. and Teasdall, R.D. (1951) Electrophysiological studies of nerve and reflex activity in normal man. IV. The two-neuron reflex and identification of certain action potentials from spinal roots and cord. Bull. Johns Hopk. HOSP.,88: 499-519. Mizuno, Y., Tanaka, R. and Yanagisawa, N. (1971) Reciprocal group I inhibition on triceps surae motoneurons in man. J. NeurophysioL, 34: 1010-1017. Paillard, J. ( 1 9 5 5 ) Analyse Clectrophysiologique et cornparaison, chez l’Homme, du reflexe de Hoffmann et du reflexe myotatique. Pfliigers Arch. ges. Physiol., 260: 448-479. Phillips, C.G. (1969) Motor apparatus of the baboon’s hand. Proc. roy. Soc. B, 1 7 3 : 141174. Pierrot-Deseilligny, E., Lacert, P. et Cathala, H.P. ( 1 9 7 1 ) Amplitude et variabilite des reflexes monosynaptiques avant un mouvement volontaire. Physiol. Behuu., 7 : 495-508. Sharrard, W.J.W. ( 1 9 5 5 ) The distribution of the permanent paralysis in the lower limb in poliomyelitis. A clinical and pathological study. J. Bone J t Surg., 37B: 540-548. Simoyama, M. and Tanaka, R. ( 1 9 7 4 ) Reciprocal Ia inhibition at the onset of voluntary movements in man. Bruin Res., 8 2 : 334-337. Smorto, M.P. and Basmajian, J.V. (1972) Clinical Electroneurogruphy: an Introduction to Nerve Conduction Tests, Williams and Wikins, Baltimore, Md.

301 Tanaka, R. (1974) Reciprocal Ia inhibition during voluntary movements in man. Exp. Brain Res., 21 : 529-540. Teasdall, R.D., Park, A.M., Porter, W.E. and Magladery, J.W. (1951) Electrophysiological studies of nerve and reflex activity in normal man. VI. Excitation and inhibition of two-neuron reflexes by impulses in other nerves. Bull. Johns Hopk. Hosp., 88: 538548. Teasdall, R.D., Park, A.M., Languth, H.W. and Magladery, J.W. (1952) Electrophysiological studies of reflex activity in patients with lesions of the nervous system. 11. Disclosure of normally suppressed monosynaptic reflex discharge of spinal motoneurons by lesions of lower brainstem and spinal cord. Bull, Johns Hopk. Hosp., 91: 245-256. Vallbo, A.B. (1970) Discharge patterns in human muscle spindle afferents during isometric voluntary contractions. Acta physiol. scand., 80: 552-566. Vallbo, R.B. (1971) Muscle spindle response at the onset of isometric voluntary contractions in man. Time difference between fusimotor and skeletomotor effects. J. Physiol. (Lond.), 218: 505-531. Yanagisawa, N. (1973) Effects of peroneal nerve stimulation on H reflex in calf muscle in man. Jap. J. EEG EMG, 2: 85-87. (In Japanese, abstract in English.) Yanagisawa, N., Tanaka, R. and Ito, Z. (1975) Reciprocal group I inhibition in the spastic hemiplegia of man. Rinsho-Noha (Clin. EEG), 17: 483-492. (In Japanese.)

DISCUSSION HAGBARTH: I think this is a very nice study and I certainly agree with you that I also think that the Ia reciprocal inhibition is a very important factor in control of voluntary normal movements. But I am a little bit surprised perhaps that in a normal healthy subject you don’t get any inhibition at all from the relaxed flexors over to the extensor muscle. In my experience if you have a voluntary contraction going, steady contraction in the calf muscle, and apply conditioning weak shock to the peroneal muscle to elicit a Ia afferent volley, you tend to get early inhibitory effect of the voluntary contraction. I wonder if you, instead of using Lloyd’s techniques of conditioning and testing H-reflexes, also have looked at the effect upon the voluntary sustained activity. My experience tells me that both when you apply an electrical stimulus to the peroneal nerve, you get the short inhibitory effect of the gastrocnemius voluntary activity and certainly when you vibrate, as I said before, on the relaxed anterior tibia1 muscle, you get inhibitory effect on the voluntary activity if the calf muscle, tonic reciprocal effect is. TANAKA: So far I didn’t use vibration for this system. For example, in your experiments, during the sustained contraction of the triceps, if you apply vibration to the biceps, then you say that inhibition comes. In this case, the most important point is that by vibration there might be many receptors activated, and especially during the vibration, as suggested by many authors, the presynaptic inhibitory mechanism should also work. POMPEIANO: As you know, in the medulla there is a bulbospinal inhibitory mechanism which inhibits postsynaptically the a-motoneurones. Now, as a physiologist, I would like to know if there is any selective supraspinal inhibitory control on these Ia inhibitory interneurones. Now, since you are working on humans, you may have a nice way to test this hypothesis. During the REM sleep, we found that there is a supraspinal tonic inhibition of the 01motoneurones as tested by different means, even in recording H-reflexes. Now, it would be extremely interesting if you could compare in humans during the REM sleep the relative amount of depression of the Ia inhibitory activity, and I am matching the two amounts of inhibition you could probably learn something about the possible existence of supraspinal inhibitory control on the interneurones of the Ia inhibitory pathway. This is simply a suggestion. KERNELL: If you ask subjects to co-contract the pretibial muscles and the triceps surae, what happens then to the inhibition which you have been studying?

TANAKA: Certainly, this will be my future work. I like to know how you can co-contract the pretibial and triceps surae muscles, a most difficult task t o do. KERNELL: If you want to stabilize a joint, you co-contract “antagonistic” muscles, I imagine that can be done in the ankle joint as well.

An Assessment of Stretch Reflex Function JAMES C. HOUK Department of Physiology, The Johns Hopkins University School of Medicine, Baltimore, Md. 21205 (U.S.A.)

INTRODUCTION Most investigators believe that the stretch reflex is an important mechanism in the regulation of posture and movement, but the precise nature of its actions and the importance of this reflex in intact animals and man has remained unclear. My thesis in this paper will be that uncertainty has resulted t o a large extent from an inadequate interpretation of the function of the stretch reflex. Usually it has been assumed that the function is t o compensate for changes in load; in other words, the actions of the stretch reflex are supposed to reduce the influence of load on muscle length, either during the maintenance of a posture or during the execution of a movement, as proposed originally by Merton (1953). I will discuss evidence against this view, and I will also develop the case for an alternative view. The alternative hypothesis states that the main function of the stretch reflex is to compensate for variations in the mechanical properties of skeletal muscle (Houk, 1972). THE DEDUCTION THAT STIFFNESS, RATHER THAN MUSCLE LENGTH, SHOULD BE CONSIDERED THE REGULATED PROPERTY OF THE STRETCH REFLEX An initial assessment of stretch reflex function follows from a consideration of the organization of autogenetic reflex pathways, as summarized in Fig. 1. Assume that an increased load stretches the muscle. The force resisting stretch should increase by way of two well-known mechanisms. One component of increase will result from the inherent mechanical properties of the muscle as emphasized by Grillner (1972), and others before him. A second component of increase will result from the excitatory reflex effect of increased spindle receptor discharge. Formerly this action was attributed solely to primary ending activity, but now there is good evidence that an excitatory action of secondary ending discharge may be a major factor (Matthews, 1973). These two mechanisms oppose length change and, therefore, should assist in load compensation. However, the stretch reflex includes a third well-known mechanism which must be considered in the assessment of function. The inhibitory reflex action provoked

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Fig. 1. A schema illustrating the origins of mechanical responses and reflex actions. The former result from the mechanical dependence of muscular force on muscle length (and velocity), whereas the latter result from a balanced interplay between length-related excitation and force-related inhibition. The efferent signal would be expected to change wherever the muscle fails to produce a particular balance between force change and length change (stiffness). (From Nichols and Houk, 1 9 7 6 . )

by increased discharge of Golgi tendon organs will decrease the force resisting stretch, and consequently will oppose load compensation (Houk, 1972). One way of specifying the function of a feedback control system is to determine what property is regulated by its action. Negative feedback of a signal proportional to a variable of the system acts to regulate that variable. Thus a feedback related to muscle length should provide length regulation, whereas a feedback related to force should provide force regulation. Since both length and force information are fed back in the stretch reflex (Fig. l), neither should constitute the regulated property; instead, some relationship between length and tension should be regulated. We have shown with a theoretical derivation that this regulated property should be stiffness (Nichols and Houk, 1976), which is the ratio of force change to length change.

USE OF THE DECEREBRATE PREPARATION TO DEMONSTRATE THAT REFLEX ACTION COMPENSATES FOR VARIATIONS IN MUSCLE MECHANICAL PROPERTIES Direct experimental evidence regarding stretch reflex function can be obtained from measures of reflex action under different experimental conditions, particularly if these actions are then compared with the performance in the absence of reflex action. Since the net stretch reflex consists of a purely mechanical response together with the reflex actions related to length and t o tension, the latter as a whole can be assessed as the difference between the net reflex and its underlying mechanical component. If length change is the input, the mechanical response is the change in force that would occur if there were no change in the numbers of active motor units or in their firing rates. The mechanical response underlying a stretch reflex cannot be measured directly, but it can be estimated quite precisely if all of the complicating factors are con-

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Fig. 2. Measurement of reflex action in the soleus muscle preparation of the decerebrate cat. The figure is a composite made up of responses to stretch (L+)and to release (L-1. Traces labeled R are net reflex responses whereas those labeled M represent best estimates of the purely mechanical changes in force that underlie the stretch reflex. The differences between the respective R and M traces represent reflex actions. The greater reflex action with stretch, as opposed t o release, compensates for yielding of the mechanical response of the muscle which occurs with stretch, but not with release. (Modified from Nichols and Houk, 1976.)

sidered. For example, the mechanical response of a muscle depends in a highly non-linear manner on the parameters of length change, such as velocity, amplitude and direction. It depends also on the frequency of motor unit discharge, the degree of recruitment and the initial length of the muscle (cf., Joyce et al., 1969). Dr. Nichols and I (1976) have developed experimental procedures for dealing with these factors for the soleus muscle preparation in decerebrate cats. The method makes use of a catalog of recorded responses of an electrically stimulated muscle t o account for different initial muscle lengths, stimulation rates and parameters of length change, together with a simple rule which accounts for the effect of different levels of recruitment. These procedures have enabled us to match any given reflex response with its underlying mechanical component. Fig. 2 summarizes our most important findings. The traces labeled R+ and R- illustrate typical reflex responses t o stretch (L+) and release (L-), respectively. The initial length and force in the two cases was the same; -8 mm represents 8 mm short of maximal physiologic extension and 780 g represents an intermediate force, a little more than one-third the maximal force which the whole soleus can develop at this length. In this experiment the steady initial force was achieved with a crossed extension reflex; similar results were obtained in other experiments when the initial force resulted from a tonic stretch reflex. The magnitude of force increase with stretch was similar t o the magnitude of decrease with release, except during the transient period of length change when the stretch response was often larger. The velocity used is within the range which occurs during walking. The traces labeled M+ and Mrepresent best estimates of the underlying components of mechanical response to stretch and release respectively. Since only the initial conditions were

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matched, the excellent agreement between muscle and reflex responses at early times after the onsets of length change, prior to the time of reflex action, constitutes independent evidence for the validity of the matching procedure. The differences between the respective R and M traces represent reflex actions, expressed in units of force. In the case of stretch, reflex action began just as the mechanical force started to yield, and it was much greater than the mechanical response for the remainder of the record. In the case of release, reflex action began after a similar delay, but its magnitude was considerably less than in the case of stretch. This asymmetric pattern was consistently observed in preparations with good rigidity. The finding that reflex action, in the case of muscle shortening, is smaller in magnitude than is the mechanical response is opposite to what one would expect if load compensation were an important function of the stretch reflex. It is also important to compare the purely mechanical responses in the two cases. The magnitude of the mechanical response to stretch (force increase) is considerably less than the magnitude of the response to release (force decrease). This indicates that the stiffness with which a stimulated muscle resists stretch is generally much less than the stiffness with which it resists release. However, the statement is not true just subsequent to time zero, when the amplitude of length change is small. A difference in stiffness arises whenever the length change is more than about 1%of fiber length (about 400 pm in cat soleus). Such magnitudes are sufficiently large to require turnover of bonds between actin and myosin molecules, which suggests that the asymmetry of muscle mechanical properties may be related t o the kinetics of bond reformation. However, what is important for the present discussion is that the differences in responsiveness t o stretch and release represent large variations in muscle mechanical properties, and these variations are well compensated b y reflex action. We have also studied the compensatory actions when different amplitudes of stretch and release are applied. The remarkable finding was that the highly nonlinear dependence of the underlying mechanical response on amplitude and direction was compensated to a considerable extent by reflex action. We believe that this may be important in the central nervous control of the musculature, since motor control becomes a simpler problem when movement commands are processed by stretch reflex mechanisms which improve linearity, as contrasted with direct commands to skeletal muscles having non-linear properties. The improvement in linearity is an expected outcome of the hypothesis that the function of the stretch reflex is to reduce the system dependence on variations in muscle properties. One class of variation concerns departure from linearity such as the one illustrated, which occurs in association with muscular yielding. IDENTIFICATION OF A DECISION-MAKING PROCESS, THE ACTIONS OF WHICH ARE SUPERIMPOSED UPON STRETCH REFLEXES IN MAN Stretch responses of human subjects depend on the instructions that are given to the subject. Hammond (1960) stretched the elbow flexor muscles and

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compared the force of resistance obtained with the instructions “resist” and “let go”. Differences in force in the two cases appeared as early as 70 msec following the onset of stretch *. Because of the brevity of this latency, Hammond concluded that the differences were not associated with voluntary reactions. The recent estimates of a very rapid conduction time from the periphery t o the motor cortex and back (35 msec in the primate; Evarts, 1973) have prompted us to reinvestigate the question as t o whether the instruction-dependent differences are associated with reactions, or with simple feedback processes (Crago et al., 1976). An equivalent mechanical circuit which portrays our experimental arrangement is shown in Fig. 3. Force loads were applied to the human arm by controlling the current supplied t o a torque motor, and the displacements of the arm were determined from the output of a potentiometer attached t o the motor shaft. The inertial component of the load was constant and resulted mostly from the motor inertia. The subject was required t o establish an initial position in opposition t o a steady force load; this position was perturbed by step-wise changes in the force load, delivered at randomftimes. The direction of load change also was made random in order that the subject would not know in advance in which direction the arm would be deflected. His instructions were to compensate for the deflection; hence, the direction of the appropriate response depended on the randomized direction of the perturbation. We also studied the responses obtained when only one direction of the perturbation was used, in which case the subject knew in advance the correct direction of response. Both of these paradigms differ from the one employed by Evarts and Granit (1976); they used a light to inform the subject the appropriate direction of response in advance of the delivery of a perturbation of random direction. Our particular protocol, which used no initial directional instruction, was chosen t o reveal decision-making processes. It is known that the latency of a “voluntary” choice reaction is longer when the number of choices is greater (Hick, 1952). Thus, if the responses of our subjects represent reac-

* Similar observations are reported earlier in this volume by Evarts and Granit (1976), and these authors also discuss the special conditions under which instruction-dependent differences at monosynaptic latency may be obtained (cf. Hagbarth, 1967).

tions, we would expect the latency to be longer when the direction is not known in advance. In contrast, if the responses reflect actions of a simple feedback process, the latency should be invariant under these two conditions. We found that the mean latency is 10-50 msec longer when the subject does not know the direction of the appropriate response in advance, analogous to findings with choice react‘ons (Leonard, 1959). The most striking evidence that a choice, or decision-making process, produces the responses is the nature of the errors which the subject occasionally makes. These errors are ones of choice which we call inappropriate reactions, since they move the arm farther from the initial position rather than restoring it near to the initial position. Usually we did not stress speed in our instructions, but in a series of experiments in which speed was stressed, our fastest subject produced reactions at a mean latency of 70 msec when the direction of the perturbation was known and 80 msec when it was not known. While our results clearly indicate that these responses represent choice reactions, the short latencies suggest that they may not be fully “voluntary”. Perhaps the volitional aspect is no more than the presetting of a process which then selects an appropriate preprogrammed response based on a simple assessment of afferent information. The reactions which I have just described are superimposed upon a more stereotypic response which I will call automatic. The automatic response to load change is a simple deflection of the arm in the direction of load change. It results in part from the mechanics of the limb and its musculature, but electromyographic (EMG) recordings from the surface of the biceps muscle have confirmed that it is modulated by a reflex mechanism. The latency of EMG change associated with automatic responses ranged from 20 to 65 msec in a sample of 1 2 subjects. This latency did not become shorter when the subject knew in advance the direction of the perturbation. Furthermore, automatic responses were essentially independent of the instructions given to the subject, up to the time at which a reaction began. (The onsets of reactions were detected from acceleration traces, as described by Crago e t al., 1976.) We concluded that automatic responses are controlled by a neural process that is distinct from the decision-making process that generates reactions. We presume that this process is analogous to the decerebrate stretch reflex; but, for the moment, I will refer to it as the automatic process. These various results are summarized in Fig. 4;the traces are not actual records, but are reasonably accurate representations that I have superimposed in order to provide a concise summary. Traces F show the time course of load change in the two directions. The displacements labeled A represent automatic responses which are obtained in pure form when the subject is distracted, or when he is told not to compensate. The dashed lines labeled C represent 3 examples of the reactions which appear superimposed upon automatic responses. Trace I exemplifies an inappropriate reaction; it would have been appropriate if the arm had been unloaded. Trace I also illustrates the type of response obtained when the subject is told to relax, or to let go when the perturbation arrives. Thus, short-latency reactions which move the arm in an opposite direction upon the detection of an identical perturbation can be preset by appropriate instructions. This is in agreement with the observations reported by Evarts and Granit (1976).

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Fig. 4. Diagram summarizing the responses of human subjects to load increases (F+)and decreases (F-). When thesubject was told not to compensate, the responses were compliant dcflections in the directions of load change (A+ and A-), termed automatic responses. When the subject was told to compensate, delayed reactions (C) were superimposed on the automatic responses. Trace I exemplifies an error in choice; this reaction would have been appropriate if the arm had been unloaded; but, since the arm was loaded, it was inappropriate.

REGULATION OF COMPLIANCE BY REFLEX ACTION IN MAN Automatic responses were obtained in quite pure form when the subject was told not to compensate, or not to intervene voluntarily to correct the deflection of the arm. The latter instructions are the ones used by Asatryan and Fel’dman (1965) in their study of the “invariant characteristics” of the elbow joint. These authors found that the elbow displacements in response t o different amplitudes of load change described a simple curve on a plot of elbow moment versus angle. Fel’dman (1966a) also demonstrated that these invariant curves were the expected outcome if he assumed that each of the muscles acting about the elbow was controlled by a stretch reflex of constant shape. Changes in initial position were accounted for by modifications in the thresholds of these stretch reflexes. Crago et al. (1976) have investigated the reflex actions that are responsible for the regulation of stiffness. In these experiments we used changes in EMG activity recorded over the surface of the biceps as an indicator of reflex action, This simplification was justified since recordings over the surface of triceps indicated that this elbow extensor remained quiescent during automatic responses in both directions. Fig. 5 illustrates our typical finding that the arm displacements in response t o symmetrical changes in load were approximately symmetrical. This indicated that the stiffness presented by the elbow musculature together with the automatic regulatory reflexes did not depend appreciably on the direction of movement. In contrast, the EMG changes associated with the automatic responses were always asymmetrical. There was a notable increase in response to loading, particularly during the transient phase when the biceps undergoes stretch. The

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Fig. 5. Automatic responses of a human subject to symmetrical increases ( A ) and decreases (B) in load ( n o t shown). Each trace represents the ensemble average of 10 trials. The displacements represent the net reflex responses, which are basically symmetrical; t h e electromyographic (EMG) traces are indicators of t h e associated reflex actions, which are highly asymmetrical. The asymmetry of reflex action is in a direction appropriate to compensate f o r the expected asymmetry of the underlying mechanical responses of t h e elbow flexors. (From Crago e t al., 1976.)

EMG change in response t o partial unloading, when the biceps shortens, was more complex. An initial small decrease often reversed t o become an increase during the period of continued shortening. Later, after the arm had ceased to move, the respective EMG changes were smaller and more symmetrical. In summary, our results indicated that reflex action is asymmetrical, whereas the net reflex resulting from mechanical properties combined with reflex action is more symmetrical. The greater magnitude ,of reflex action when biceps lengthens is appropriate t o compensate for the expected yielding of muscular force under these conditions, in analogy with reflex action in the decerebrate cat which I described earlier (Fig. 2, R+ and M+). A lesser magnitude of EMG decrease when the biceps shortens would also be appropriate, since a large decrease in force is the expected mechanical response of the muscle (M-, Fig. 2). To this extent the automatic process in man behaves like the stretch reflex in decerebrate cats, but we also noted an important difference. EMG responses which we sometimes recorded in the decerebrate studies did not include a phase of increase in response t o muscle shortening. The phase of EMG increase during shortening represents a period in which reflex action facilitates muscle shortening. Several aspects of this finding require discussion. (1) It appears t o be in agreement with observations by other investigators. For example, Alston et al. (1967) described a phase of EMG increase in response t o complete unloading of the limb. They called this phase the “terminal motor volley” since it terminated a period of EMG silence. We did not observe a silent period, as a result of the lower velocities of shortening prevalent in our experiments (cf., Struppler et al., 1969),but the EMG increases in the two cases are probably due t o similar mechanisms.

311 (2) The period of increase constitutes strong evidence against the hypothesis that the function of this reflex is t o compensate for changes in load. The increase facilitates length change, rather than opposing it. (3) The absence of this phase in the decerebrate cat may result from the depression of transmission through Ib pathways that is a characteristic of the decerebrate state (Eccles and Lundberg, 1959; I-Iongo et al., 1969; Houk et al., 1970). A high gain of force feedback from tendon organs could account for the phase of EMG increase in man, since it occurs at a time when the force in series with tendon organs should be small. It is clear from Fig. 1that the efferent signal would increase if the removal of force-related inhibition were more important than the removal of length-related excitation, under conditions of muscle shortening. This interpretation is consonant with Hufschmidt’s (1966) observations, which suggest that autogenetic inhibition is an important aspect of reflex function in man. Further studies will be required t o determine which mechanisms are responsible for asymmetrical reflex actions; however, the functional significance of the result seems clear. Both the decerebrate stretch reflex and the automatic reflex process in man act to compensate for differences in muscle properties which depend on the direction of length change. One would also expect compensation for other variations in muscle properties, such as those related to muscular fatigue, the frequency of motor unit discharge, the level of recruitment and the length of the muscle. The observation by Matthews (1959a,b) that a variety of extrinsic inputs t o the stretch reflex d o not affect the slope of the relationship between force and length is consistent with this suggestion. It is not enough t o conclude that stretch reflex actions compensate for variations in muscle properties; one would like t o specify what property is regulated. The different results that I have reviewed suggest that the regulated property is the stiffness presented t o load change, or its inverse which is compliance. The term compliance may be preferable t o stiffness, since its connotation is that the position of the limb is not rigidly controlled by reflex action. Load changes applied to human arms clearly result in compliant deflections; rigid control is achieved only when the subject responds t o a deflection by producing a reaction of an appropriate direction and amplitude (Fig. 4). In many instances a compliant response t o load change is well matched t o the requirements for postural stability of the body, since it absorbs the impact of a load change rather than transmitting it t o the body and head. Short-latency reactions, on the other hand, add considerable versatility t o the more stereotypic responses of the stretch reflex. Reactions can result in effective load compensation, and rigid control of limb position; however, with alternative postural goals, reactions can provide a response that is highly compliant instead, as illustrated by trace I in Fig. 4. INTERACTION BETWEEN MOVEMENT CONTROL AND REFLEX ACTION Current evidence suggests that control over the stretch reflex is achieved predominantly by alterat,ions of its threshold (Matthews, 1959a,b; Asatryan and

312

load perturbations Regulated Compliance

Fig. 6. A mechanical analog illustrating the expected interaction between movement commands and the reflex actions which regulate compliance. An increase in load would deflect the arm initially (the spring would be stretched). A reaction in the form of a movement command would have the effect of cranking the rack and pinion (alterations in the thresholds of the associated stretch reflexes). This would result in the initiation, execution and termination of the superimposed movement.

Fel’dman, 1965; Fel’dman, 196613; Fel’dman and Orlovsky, 1972). Other mechanisms may control the compliance of the reflex by changing the gain of reflex pathways, but we were unable to obtain evidence for this mode of control in our experiments with normal human subjects. When we asked our subjects t o make their arms rigid, some accomplished the requested rigidity by producing reactions, while others achieved it by co-contraction of elbow antagonists as judged by EMG recordings (triceps activity was observed, along with an upward shift in biceps activity). However, we did not observe any appreciable modification of the portion of the EMG response that could be associated unequivocally with the automatic process. The question of gain change requires further investigation, but our tentative results suggest that this mechanism of control may not be part of the normal repertoire, in agreement with the reports quoted above. The interaction between movement commands, such as commands for reactions, and the automatic actions of the stretch reflex can be summarized in mechanical terms as shown in Fig. 6. (The major assumption in the construction of this analog is that command signals act by modifying stretch reflex thresholds). Movement commands have the effect of cranking the rack and pinion, whereas the stretch reflex maintains compliance constant. (The compliance must also be damped by dynamic components of reflex action.) According to this model, movement commands control neither the lengths nor the forces of the musculature; instead they offset the. compliant relationship between the two. The simplicity of the analog has an important conceptual value, but it glosses over the intricate neural mechanisms that must be responsible for the expression of these mechanical properties.

313 SUMMARY The main function of the stretch reflex appears to be one of maintaining a constant, compliant relationship between muscle length and force. The evidence in support of this hypothesis consists of simple deductions based on the general organization of autogenetic reflex pathways, direct experimental measures of reflex actions in decerebrate preparations and indirect (EMG) measures of reflex action in normal human subjects. Additional components of response may be superimposed upon reflex responses when load changes are applied to the human arm. These components were termed reactions since they were shown t o represent outputs of a decision-making process, as contrasted with a simple feedback process. The importance of reactions is that they add considerable versatility to the stereotypic responses of the stretch reflex. A simple mechanical analog was proposed t o account for the interaction between movements, such as the reactions, and the actions of the stretch reflex.

REFERENCES Alston, W., Angel, R.W., Fink, F.S. and Hoffman, W.W. (1967) Motor activity following the silent period in human muscle. J. Physiol. (Lond.), 190: 189-202. Asatryan, D.G. and Fel’dman, A.G. (1965) Functional tuning of the nervous system with control of movement or maintenance of a steady posture. I. Mechanographic analysis of the work of the joint on execution of a postural task. Biophysics, 10: 925-935. Crago, P.E., Houk, J.C. and Hasan, Z.(1976) Regulatory actions of the human stretch reflex. J. Neurophysiol., in press. Eccles, R.M. and Lundberg, A. (1959) Supraspinal control of interneurones mediating spinal reflexes. J. Physiol. (Lond.), 147: 565-584. Evarts, E.V. (1973) Motor cortex reflexes associated with learned movements. Science, 179: 501-503. Evarts, E. and Granit, R. (1976) Relation of reflexes and intended movements. This volume. Fel’dman, A.G. (1966a) Functional tuning of the nervous system with control of movement o r maintenance of a steady posture. 11. Controllable parameters of the muscles. Biophysics, 11: 565-578. Fel’dman, A.G. (1966b) Functional tuning of the nervous system during control of movement o r maintenance of a steady posture. 111. Mechanographic analysis of the execution by man of the simplest motor tasks. Biophysics, 11: 766-775. Fel’dman, A.G. and Orlovsky, G.N. (1972) The influence of different descending systems on the tonic stretch reflex in the cat. Exp. Neurol., 37: 481-494. Grillner, S. (1972) The role of muscle stiffness in meeting the changing postural and locomotor requirements for force development by the ankle extensors. Acta physiol. scand., 8 6 : 92--108. Hagbarth, K.-E. (1967) EMG studies of stretch reflexes in man. In Recent Advances in Clinical Neurophysiology, Electroenceph. d i n . Neurophysiol., Suppl. 2 5 , L. Widen (Ed.), Elsevier, Amsterdam, pp. 74-79. Hammond, P.H. (1960) An experimental study of servo action in human muscular control. Proc. 3rd int. Conf. Med. Electronics: 190-199. Hick, W.E. (1952) On the rate of gain of information. Quart. J. exp. Psychol., 4: 11-26. Hongo, T.,Jankowska, E. and Lundberg, A. (1969) The rubrospinal tract. 11. Facilitation of interneuronal-transmission in reflex paths to motoneurones. Exp. Brain Res., 7 : 365391. Houk, J.C. (1972) The phylogeny of muscular control configurations. In Biocybernetics, Vol. 4 , H. Drischel and P. Dettmar (Eds.), Fischer, Jena, pp. 125-144.

314 Houk, J.C., Singer, J.J. and Goldman, M.R. (1970) An evaluation of length and force feedback to soleus muscles of decerebrate cats. J. Neurophysiol., 33: 784-811. Hufschmidt, H.J. (1966) The demonstration of autogenic inhibition and its significance in human voluntary movement. In Muscular Afferents and Motor Control, R. Granit (Ed.), Wiley, New York, pp. 269-274. Joyce, G.C., Rack, P.M.H. and Westbury, D.R. (1969) The mechanical properties of cat soleus muscle during contiolled lengthening and shortening movements. J . Physiol. (Lond.), 204: 461-474. Leonard, J.A. (1959) Tactual choice reactions. I. Quart. J. exp. Psychof., 11: 76-83. Matthews, P.B.C. (1959a) The dependence of tension upon extension in the stretch reflex of the soleus muscle of the decerebrate cat. J. Physiol. (Lond.), 147: 521-546. Matthews, P.B.C. (1959b) A study of certain factors influencing the stretch reflex of the decerebrate cat. J. Physiol, (Lond.), 147: 547-564. Matthews, P.B.C. (1973) A critique of the hypothesis that the spindle secondary endings contribute excitation t o the stretch reflex. In Control of Posture and Locomotion, R.B. Stein, K.B. Pearson, R.S. Smith and J.B. Redford (Eds.), Plenum, New York, pp. 227-243. Merton, P.A. (1953) Speculations on the servo-control of movement. In CZBA Foundation Symposium on the Spinal Cord, Little, Brown and Company, Boston, Mass., pp. 247260. Nichols, T.R. and Houk, J.C. (1976) The improvement in linearity and the regulation of stiffness that results from the actions of the stretch reflex. J. Neurophysiol., 39: 119142. Struppler, A., Landau, W.M. und Mehles, H.O. (1969) Analyse des Enthastungsreflexes am Menschen. Pflugers Arch. ges. Physiol., 313: 155-167.

DISCUSSION GYDIKOV: I found that your theory was very interesting, but I think that you have to control also the activity of the antagonistic muscle, because in many situations we have different levels of balance between agonistic and antagonistic actions, and if you want to say that the muscle response was asymmetrical, we have to know if there are antagonistic actions as well. HOUK: That’s a very important point and we did monitor the triceps as well as biceps activity. When the limb was initially loaded with 10% maximal force pulling on the limb, we found that during the automatic response to both increase and decrease of the load, the triceps muscle remained inactive. Now, that is under a normal situation. When the subjects were asked t o be as rigid as possible, some did succeed in co-contracting the triceps muscle but others did not. I think that usual absence of co-contraction is related to the strong activity of the flexor musculature that was present with our initial condition. The important point is that the EMG responses were asymmetric under conditions in which triceps remained inactive. BUCHTHAL: I found it very stimulating t o introduce mechanical properties as an important parameter, and I wonder if you have included in your consideration, in addition to what you have shown in stretch and release, certain important parameters. Firstly the fact that the stiffness is usually at steady-state proportional to the load and not to the length; and secondly, I wonder whether you have considered that stiffness changes precede changes in tension. Now, how is the time relationship? Are there any adjustments before changes in these parameters? HOUK: With response t o the first question, I recognize that the mechanical properties of muscle are very complicated. That is one of the reasons why we did not try to model the muscle in order t o estimate the underlying mechanical response, but rather made a catalogue of the recorded responses. I would agree with you that stiffness, particularly the stiffness in response t o small change in length, is more closely related to initial force than to initial length. With regard to the second question, you mention the phenomenon first shown by A. V. Hill, that if you stretch a muscle that is just activated, the force can build up more rapidly. In fact we found a very short latency of reflex action, which is partly attributable to the quick-stretch phenomenon.

SESSION VI

SUPRASPINAL CONTROL OF THE STRETCH REFLEX

Part I1 Chairman: P.B.C. Matthews (Oxford)

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Parameter and Signal Adaptation in the Stretch Reflex Loop GIDEON F. INBAR and ALBERT YAFE Department o f Electrical Engineering, Technion -Israel Institute of Technology, Haifa (Israel)

INTRODUCTION Movements are always being carried out in a changing environment, namely changes in loading conditions. Systems, physical or physiological, can cope with large environmental dynamics in one of two ways: (a) by a strong servo action, Le., a system with a very high loop gain, or stiffness, which will follow a wide range of inputs despite changes in load dynamics and b) by adaptation, i.e., a system which can either change its parameters or its control signals - or both - in order t o maintain its performance in the face of large changes in load dynamics. It is the second option (b) which we would like t o investigate here, especially the question of adaptation mechanisms, parameter vs. signal adaptation. Physiological (Wilkie, 1956; Mountcastle, 1968) and engineering (Houk, 1963; McRuer et al., 1968; Stark, 1968; Rosenthal et al., 1970; Wieneke, 1972; Gottlieb and Agarwal, 1973; Inbar and Joseph, 1976)works describing and modelling the muscle control system in posture have come up with sufficient evidence t o suggest that the system is a varying or an adaptive one. Most of these works state this fact but concentrate on the invariant characteristics of various elements of the system. In the present work, using the adaptive system in engineering (Landau, 1972) as a background, a speculative model reference adaptive scheme for the muscle control system is proposed (Inbar, 1972, 1975). The model is then simulated, using physiological data (Inbar, 1975), t o investigate the relative effectiveness and feasibility of parameters and signal adaption. The simulations are followed by experimental results supporting the adaptation concepts presented here in addition t o the manifestation of unconscious learning - equivalent to the building of the model reference in the present scheme. The simulations and experimental results are discussed. THE SYSTEM AS A VARIABLE CONTROL MECHANISM The basic elements involved in posture are the muscles attached to bones around joints which act as fulcrum points. Muscles are basically composed of

318 active (contractile componeilt) and passive (elastic component) elements. The active elements contract and produce force unidirectionally in accordance with neural signals arriving through nerves that make up the alpha efferent system. Because of their unidirectionality muscles are generally arranged in couples called the agonist antagonist pair, enabling reversal of motion direction. During complex movements a number of pairs act together. The basic agonist antagonist pair acts in a feedback loop containing a major sensor; the muscle spindle with its complex gamma efferent innervation. There are other sensors involved in the feedback aspects of the reflex loop; however, only the muscle spindles are incorporated in the model (Inbar and Joseph, 1976). The omission of other sensors has no effect on the basic conclusions of this study. The feedback loop around the muscle made up of the local sensors is closed through the spinal cord. This is called the reflex loop and will be further referred t o as the “local” or “reflex” loop. The different nerve cells (neurones) on the spinal cord have the possibility to be interconnected in various configurations according to neural signals descending from the upper centres. The alpha motor neurone is the sole “signal dispatcher” t o the muscle. All neural signals eventually converge on this motor neurone before being sent t o the corresponding motor unit in the muscle. The internuncial cells act as data collecting and management centres at the spinal level. They can act as excitatory (+) and inhibitory (-) signal suppliers to the alpha motor neurone. They can be interconnected so as t o amplify the neural signal temporally and spacially (Milgram and Inbar, 1975). They can reverse signals from inhibitory t o excitatory and vice versa. Furthermore, connections from the higher centres indicate the possibility of their intervention on the internuncial cell activity, this resulting in an “adaptive” m o d e of control (Mountcastle, 1968). The muscle is a non-linear element. Besides being unidirectional (it can only pull, i.e., produce force in one direction) its performance is governed by the isometric force, neural excitation, isometric forcedisplacement and force-velocity relationships. In this study the isometric force is assumed t o be linearly dependent on neural excitation. The other relations, namely, the non-linear forcevelocity and force-displacement relationships, may be approximated as follows

developed Equation (1)describes the isometric contractile force (Pcontractile) as a function of neural excitation (E) and the length (L) of the muscle. Ln is the muscle natural length. The contractile force vanished at lengths L, and 2L,-L,. C, is a gain constant. Equation (2) describes the passive characteristics of the muscle, i.e., the elastic force developed (Pelastic)as the length (L) of the muscle increases above L,, a characteristic length. C, is another gain constant.

319

At a given isometric situation the total force produced by the muscle is then the sum of the elastic and contractile components as given in equation (3). It is to be stressed that equations (1)and (2) are very approximate, sufficiently accurate around L,, and by no means do they describe the entire behaviour of the muscle under isometric conditions. These equations were chosen because of their simplicity since it is known that under physiological conditions the length L stays within -+5% of L,. Equation (4)is a version of the well-known Hill’s equation (Wilkie, 1957; McRuer et al., 1968). It gives the total output force as a function of velocity of contraction and total isometric force. V, is the maximum velocity of contraction and b is another characteristic muscle constant. The muscle spindle has been measured to generally perform as a linear element, in a limited range of muscle length, this linearity being constantly maintained by the y system. An approximate transfer function (TF) (Houk, 1963) is given by equation (5):

qrs + 1 G(s) = C 7s + 1 where C is the gain, r the time constant, and q 2 1the lead factor. This TF, or similar ones (McRuer et al., 1968; Wieneke, 1972), has been used in previous works on linear models for the reflex loop and is generally quite satisfactory in spite of known non-linearities suggested in the literature (Houk, 1963). Furthermore, this is only an equivalent block, since the muscle spindle which received a number of y inputs (static, dynamic) has two outputs, one responding solely to the length and the other to both the length and velocity of the muscle, the y affecting each output differently. In the equivalent case this e f fect is represented b y the changing o f the parameters (c,q,r) of the TF, thus producing a variable feedback. Small signal linear models suggested for the refIex loop generally use a block characterized by a TF similar to eqn. (5) as the general feedback element and describe the muscle either by an experimentally measured TF (Mountcastle, 1968) or a viscoelastic equivalent derived from its characteristics (Wilkie, 1956; Houk, 1963; McRuer et al., 1968) (eqn. 1-4). Some refinement is obtained by the addition of a non-linear friction element (Inbar et al., 1970). In every linear model for the reflex loop, it has been pointed out that the values of the parameters involved change as the conditions of operation are altered (Houk, 1963; McRuer et al., 1968; Stark, 1968; Rosenthal et al., 1970; Wieneke, 1972; Gottlieb and Agarwal, 1973). All the evidence in the literature on the behaviour of the system in question leads to a solid conclusion, namely that the muscle control mechanism (MCS) is a variable control mechanism (Mountcastle, 1968; Gottlieb and Agarwal, 1973; Inbar, 1975). THE MCS: SIGNAL VS. PARAMETER ADAPTATION Having established that the system is a variable one that can be adjusted within limits at will, we have to define a fairly rigid model which will enable further progress. It is convenient to develop a model in which it will be possible

320 to incorporate existing knowledge on adaptive control schemes. Adaptive control systems can be classified according to a number of criteria (Landau, 1972). We are interested in the classification according to the mode of adaptation, i.e., parameter and signal adaptation. In general, a non-linear system can be adequately described by equation (6), where x and u are the state and input vectors respectively.

(6) h(x(t), 4 t h t) In this case there is only an input vector that can be externally affected. No allusion can be made to “parameters” since in (6) these are not defined. Equation (7) now introduces the X(t) =

X(t) = h(x(t), P(t), 4 t h t)

(7) parameter vector p, representing the independent variable parameters in the system. There is no point in introducing a controllable parameter since this can obviously be treated as a subset of u. As will be presented below we are interested in model reference adaptive control and as such we can now supply a preliminary definition of signal adaptation in such a system. Definition: Given a model (eqn. 8) and a system (eqn. 7), the process of transformation u -+ u *, so as to minimize a criterion involving x,y possibly p,p, and u, is called signal adaptation.

Y(t) = h(Y(t), Pc, u1 (t),t), Pc = const.

(8)

In the specific case of the muscle control mechanism, if eqn. 7 describes the muscle shortening as the output response to neural input, the variable parameter vector p may comprise changing load conditions as well as the biological and chemical states of the muscle etc. When h is a linear function, p(t) is fully contained in the system’s eigenvalues. Since the eigenvalues may also contain externally controllable elements, these can be used to compensate for the changes in p(t). In this case the process is one of parameter adaptation. As will be seen later, the parameters of the linearized MCS, which will be derived from equation 1 through 4, will depend on L and E. These two, together c , q , ~from eqn. 5 , will comprise the controllable parts of the systems eigenvalues . As already mentioned in the overall non-linear control system, controllable parameters can be viewed as being part of the input vector. In the case of the muscle control system, the distinction is even more artificial since the changes in the parameters are carried out by neural signals which are no different from the actuating input signals. The length L and excitation E of the muscle can be set by selectively controlling the a motor neurone output to the antagonists. The parameters of the equivalent muscle spindle can be controlled by the output of the y motor neurones. Furthermore, the a and y systems are physiologically linked and are co-activated. In the following sections, all controllable changes in the parameters of the linear model for the MCS will be referred to as parameter adaptation, no matter their origin. All changes in the small signal actuating input will be referred to as signal aduptation. In the general non .linearsystem there will be no distinction be-

321 tween the two and all changes will be related to the input vector. Having established this basis we can further examine the muscle control system. Basically we can separate the system into two feedback loops. One, the reflex or the local loop closed by the muscle spindle around the muscle through the motor neurones in the spinal cord, and the other closed around the reflex loop by all the sensors involved in muscle control and through the higher centres of the CNS. In this case it is possible to specify the following dynamic equations :

(14)

UZ = f21X)

where x = state vector describing the muscles; y = state vector describing the spindles; pm = independent muscle parameter vector; ps = independent spindle parameter vector; ua = output of the a-motoneurone; uy = output of the ymotoneurone; u1 = output of the higher centres to the a system; u2 = output of the higher centres to the y system. Of all the variables appearing in equations 9-14 only x and ua can be measured on live subjects operating under normal conditions, and even these measurements are hardly accurate (the electromyographic measurements do not yield full information about ua). All the remaining variables are interrelated and no information on their activity can be extracted from the measurable quantities. Therefore any hypothesis involving a specific adaptation mechanism, whether in the linear or in the general models, cannot be experimentally verified. Proposed hypotheses may be justified by considering the possibilities and limitations of the system. Since x and ua are the only measurable quantities, the expected effects according to the hypotheses can be experimentally verified, though by no means contributing any knowledge on the mechanism involved in the process. It should also be noted that x and ua are in fact composed of two components describing the agonist and the antagonist muscles.

THE LINEAR ADAPTIVE MODEL In this section we will develop the theory that the muscle control system is an adaptive one, and will consider parameter adaptation versus signal adaptation in the linear model that will be derived. First it is appropriate to supply the various observations and reasons that lead to the conclusion of an adaptive control scheme.

322 Previous experimental works (Houk, 1963; McRuer et al., 1968; Inbar et al., 1970; Wieneke, 1972; Soechting, 1973; Terzuolo and Viviani, 1973; Tamura and Yoshida, 1974; Inbar, 1975; Milgram and Inbar, 1975; Yafe, 1975) have shown that the system response to the same output varies under different conditions. The dynamics of the linear model with which the system response is approximated are found t o alter. Furthermore, the human subjects can learn t o perform a task, so that when the characteristics of the dynamic system are t o be investigated, precautions are taken so that the subject will not be able t o prepare himself towards the input. Loading conditions, timing of the input and shape of the input are some of the factors which investigators have changed randomly so as to prevent “learning” or “conditioning” of the subject. In this research, the purpose being the investigation of adaptation in the MCS, experimental conditions were expressly kept invariant and the subjects were told to perform as best as they could. Learning was observed. All of the above, enhanced by physiological facts already stated, lead to the speculation that not only is the system an adaptive one but also a model reference adaptive system. The process of learning can then be attributed to the building of the reference model. During actual performance then the system can be adapted according to the different factors involved. Basically there is room for two adaptation modes: (i) adaptation of the parameters of the reflex loop (including the spinal cord); (ii) adaptation of the actuating signal. The latter may be achieved by additional signal generation at various levels of the MCS. The block diagram for the speculative model (Inbar, 1975) of the MCS is given in Fig. 1. The “system” block in this figure represents the entire reflex loop shown in Fig. 2a, and its output is the controlled variable. This is a two input description of the reflex loop. The inputs are internal neural excitation (AE) and external disturbance tension (TL). The output is the displacement of the load. The transfer function coefficients ni, mi, di, pi are derived from the

MODEL EXTERNAL INPUT

-T

I

t

* AL

1 SIGNAL GENERATION at

SPINAL CORD

t

11

GENERATITION

Fig. 1. Block diagram showing the speculative model for the muscle control system functioning as a model reference adaptive system. Signals may be generated in the higher centres, and in the spinal cord. The external input is a force disturbance.

323

a AGONIST

ANTAGONIST

b

Fig. 2. a : the reflex loop block diagram including both internal neural and external tension inputs. The t wo upper blocks are derived from the viscoelastic model of the muscles and the lower one is the one assumed for the muscle spindle. b: the linear viscoelastic muscle models of both t he agonist and antagonist applied t o a load. The viscoelastic parameters are derived from t h e muscle non-linear relations.

324

Kei represents all the series elastic elements of muscle i and is assumed t o be constant. Cfi represents the "gain" of the muscle which translates neural excitation t o force. Kpi represents the parallel elastic characteristics of the muscles as B, represents the viscous characteristics. C , , KPi and Bi can be obtained from equations (1)t o (4)using Taylor's series expansions around a quiescent point vo, Lo, E, and, consequently, Po and Fo and taking only the linear terms. From equation (4):

P can further be expanded around Po : p-p

a'

=-

a L ILo.Eo

(L-Lo)+apI aE

(E-Eo) Lo.Eo

Since P is composed of two components: Pcontractileand Pelastic,the following relations can be obtained :

apelastic _ _ _ - 2Cp(L-L,),

aL

L > L,

Outside the given boundaries the partial derivatives are assumed to vanish. Substituting equations (20) t o (22) into (18) and subsequently into (17) we obtain the form:

The individual muscles given by the above equations can be unified in a single block as shown in Fig. 2a. This is possible because it can be assumed that the neural input t o the muscles, fi, is a differential signal, i.e., A f t = --Af2. If this is not so then it follows that

325

A Af, + Af2 f O 2

Af, =

which can then be added to E, and subtracted from Af, and Af2 t o leave a differential signal. Generally it can be said that the neural signal common to both the muscles serves in the setting of the quiescent or workingpoint in the nonlinear system from which the linear system parameters can be derived. It is to be stressed that the quiescent point is not necessarily constant and may well change as a function of time. The reference model in Fig. 1 contains information of some kind covering all the performance requested from the system. An example of the information contained in the model may be the quiescent point time course of the signals, but many other forms of storage, like response patterns, are well possible. It should be stressed that no speculation is made on how the adaptation is actually being carried out by the systems and, as mentioned above, the model is not stored in the form of differential equations but more likely in the form of

patterns of signals. Operation of the model reference adaptive model for the MCS can be speculated as follows. Given certain criteria set by the higher centres in the CNS, the subject starts “learning”. This process involves identification of the load conditions, the physical state of the physiological system, calculation and checking of criteria. It is an iterative process during which the reference model is constantly being built and modified. When the criteria have been met, the task pertaining to the conditions and goals has been accomplished, the model can be stored for further use, When, at a future time, it is necessary to perform the same task, it is sufficient t o bring forward the existing model and adapt the MCS accordingly to cope with the situation. If new knowledge is obtained, the model is modified. According to what has already been said, one or both of two adaptation modes can be utilized during the performance stage, namely parameter adaptation and signal adaptation. In a discussion on the effectiveness of each option under different conditions, it is obvious that the limitations of the physiological system have to be considered first. The basic limitations on the system are: (1)Whereas the uncontrolled parameters are unlimited in number (the load, for instance, may be of a very high order) and in value, the controlled ones are limited in number (L,E,~,T,C) and also in value. (2) Though not mentioned so far, the velocity of information flow in the nervous system is finite, dependent on nerve size and is rather slow. The delay existing in the reflex loop is about 20-30 msec whereas in the global loop there is up to 200 msec delay (Wieneke, 1972). ( 3 ) The information transmission capacity of the spinal cord is higher than the excitation that would cause tetanic tension in the muscle. In fact, under normal conditions no excitation higher than that that would cause tetanus has been measured in the nerves leading from the motoneurones to the muscles. Besides the above arguments other considerations are also involved, such as the energy lost while activating the antagonist muscle pair against each other in order to ensure a quiescent point.

326 Had there been no limitations, the two modes of adaptation would have been equivalent. As it is, each limitation is followed by a preferred mode of adaptation considering the input. In uolitional control, when the subject has sufficient time t o prepare a set of signals dictated by the model and may or may not need feedback in the global loop, signal adaptation seems preferable over parameter adaptation. On the other hand, when the input is external, the higher centres cannot intervene for a t least the delay period in the global loop. In this case, a pre-set, off-line parameter adaptation, compensating for the expected event in its conditions, will be effective at least during the period the higher centres have not come into action. These two are extreme cases and as examples one can suggest the pianist’s action during a “molto vivo” for the first case and a very short duration blow expected by a boxer for the second. Obviously, in general joint operation can be expected. SIMULATION RESULTS In order to check the possible effects of adaptation, both the linear and the non-linear models describing the MCS were simulated on computer. In the linear case, parameter and signal adaptation t o the pertinent inputs assuming inertial mass M were simulated. For the non-linear case the load chosen included a stiff spring in parallel with the mass M, this was in order t o approximate an isometric tension transducer loading the muscles together with all the mass of the limb. Following the reasoning of the previous section, the simulation for parameter adaptation was performed as a presetting process rather than an on-line adaptation scheme. The reference model was assumed to have the structure shown in Fig. 2a with AE = 0. The system was also assumed t o have the same structure only with a different load value, namely a different M. The adaptation problem was stated as follows: Given h, (t) the impulse response of the model with inertial load My calculate L o , E, , c , ~ , T so that h, (t) describing the impulse response of the system with inertial load M’ # M and the calculated parameters, will minimize J given by equation (25): m

I h,(t) - h,(t)j ’dt

J= -00

In this case, since the frequency responses were available, it was simpler to work in the frequency domain. Using Parseval’s theorem: 1 J=277

m

1 G,(o)-G,(w)]’dw

-00

Furthermore, since it is physiologically known that the frequency range seldom exceeds 30 Hz, it is sufficient to require that the integral in equation (26) be evaluated for -200 < c3 < 200 rps.

1j

FORCE-DISPLACEMENT TF BODE

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100

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10'

HZ ( LOG SCALE )

Fig. 3. Bode plot showing the transfer function from external force input to displacement for an "acceptable" pulse response. This transfer function is then assumed to perform as the reference model.

The model parameters were calculated with M=0.2,

Lo=O.l, Eo=5.0, C = 6 0 , q = 5 ,

~=1/300.

The force-displacement transfer function Bode plot is shown in Fig. 3. The model response to a short duration pulse filtered at 30 Hz is shown in Fig. 4a. When M was changed t o 1 with all the other parameters invariant, the pulse response changed considerably as seen in Fig. 4b. After performing the minimization of equation (26), including bounds on the parameters, the following values were calculated :

Lo = 0.10057,

Eo = 0.163,

C = 61.7,

q = 4.27,

T

= 1/400

The pulse response shown in Fig. 4c clearly displays the fact that the effect of parameter presetting was in this case (as well as in cases with a different M ) quite negligible. The main reason for the ineffectiveness lies in the complex interrelationships between the various parameters in equation (15). Removing the bounds on the controllable variables improved very little the response supporting the above argument. For signal adaptation simulation, the technique used was a simple one, though sophisticated optimal control schemes might have been used. The reasons for choosing the simple technique were: (i) There is no evidence as to the criteria to be preferred to fit physiological phenomena, and the results -though interesting - may well be far-fetched. (ii) Centres whose duties are to generate signals do have a rather large and quite "mysterious" range of possibilities, so that a simple speculative technique may provide an insight into their capabilities with minimum complexity. Since signal adaptation is relevant to neural input, the structure shown in Fig. 2a with T, = 0 was used t o describe the reflex loop. The reference model was assumed t o behave according to the transfer function.

T(s) =

1 s2/w; + (2f/wo)s + 1

1 S/WI + 1

328 OBJECT MODEL PULSE RESPONSE

UNADAPTED, M:: 1

b

TIME ( X

‘4

10-5

2

4

6

8 10 12 TIME(XIO-’)

14

16

PULSE RESPONSE. M I 1 . ADAPTED *Or

C

0

2

4

6

0

10

12

14

16

TIME (X10.0

Fig. 4. a: t h e pulse response of the reference model whose TF is displayed in Fig. 3. b : when t h e load is changed b u t the other parameters kept constant in their previous models the above response is obtained. c: the pulse response obtained after adapting the muscle and spindle parameters is very similar to that in b, suggesting tha t parameter adaptation is ineffective.

w, and were chosen so that the first part of T(s) will be critically damped with a natural frequency of about 10 Hz. The real pole was introduced so that the following final result gave a casual system. The mathematical problem in this case was defined as follows: Given that the requested response is that given by the reference model T(s) when the higher centres supply to it a step input, find the signal to be supplied to the system G(s) so as t o have the same response,

L(s) = T(s) . U(S), where L(s) is the response of T(s) to input U(s),

L’(s) = G(s) * U(s), (29) where L’(s)#L(s). It is now necessary t o define a signal conditioner which will transform U(s) t o U’(s) such that L(s) = G(s) U’(s). (30) Obviously, if U’(s) = (T(s)/G(s))U(s), then the problem is solved. It is naturally necessary that both T(s) and G(s) be well known, and that T(s)/G(s) define a feasible system. The first necessary condition is satisfied by the basic assumption on the

iL73j” REFLEX LOOPBODE

329

OBJECT MODEL NEURAL STEP RESPONSE

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- 124

20

$ 1

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X

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TIME ( X 10”)

CONDITIONER OUTPUT TO STEP

OVERKL RESPONSE C

-

8

a 4

0 .4

0

8

16

24

32

40

48

56

64

TIME (X lo2)

Fig. 5. a: bode plot showing the transfer function from internal neural input t o displacement. b: step response of the T F shown in a. c: the original step input passed through a passive signal conditioner thus producing signal adaptation. The adapted waveform shown here acts as the input t o the TF of a. d: the input from the conditioner c yields this output. This shows that within physiological, feasible limits, signal adaptation can affect the output more than parameter adaptation to yield the desired response.

higher centres, that they are unlimited whereas the second one was satisfied by the addition of the simple pole in equation (27) without essentially changing the influence of the remaining part of T(s). Fig. 5a depicts the transfer function of the reflex loop G(s). Fig. 5b shows the response of the system to a unit step. The step input was preprocessed to provide the signal shown in Fig, 5c which when applied to G(s) produced the required overall response shown in Fig. 5d. In performing experiments on human subjects under normal conditions, there is no way t o measure any of the quantities involved in the small signal model. The only quantities measured are the agonist antagonist excitation through their electromyographical (EMG) activity, and the displacement. For this reason the non-linear system was simulated while using only the relations involving the muscle and not the other elements involved in the reflex loop. The purpose of the simulation was to show the effect of background excitation, or ‘set point’. The tensing of the agonist against the antagonist, affected the agonist signal causing a predetermined response and the small signal parameters of both muscles as calculated from equation (24). The load in this case was set t o be a stiff spring in parallel with inertial mass. Fig. 6 shows the agonist signal time course that results in the same response as in Fig. 5d for two different antagonist excitation levels. Fig. 7 shows the de-

330

0 1 0 - 0 2 1 - 010

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Fig. 6. a: the agonist signal E2 calculated for a given requested output xo and a given antagonist background excitation E l (0.096) in the general non-linear system. b: Same as a but with E l larger (0.5). The variation of E2 in the tw o is quite small, suggesting tha t the actuating signal is not too affected by the quiescent point.

pendence of the small signal parameters on the background excitation levels. The values of the parameters change by an order of magnitude when the antagonist excitation level is changed, whereas the agonist signal is almost invariant. There is stiffening of the system with increased excitation. EXPERIMENTAL RESULTS In order to support the hypothesis presented in this work, a simple experiment was performed. Naive subjects with no previous training were told to follow a pulse of a few seconds duration and qppearing at 0.1 Hz approximately, displayed on an oscilloscope, by pulling on an isometric tension transducer, using their biceps. The force necessary t o follow the amplitude of the pulse was set at 5 kg. The electrical activity of the biceps was measured by surface EMG

331

LEGEND KP1 0 KP2 A

a 16

TIME

LEGENO N

a

KP 1 0 KP2 A

r

b

ow

~ 0 5 om

on om

I

025

030 035 OU)

06 050

TIME

Fig. 7. a : the time course of the parallel elasticities of both muscles ( K p l , K p 2 ) for a given background tension E l (0.096) as a function of time yielding the desired x,, shown in Fig. 6. b : same as a but with E l larger (0.5). The variation of K,: with E l is very strong. K,i dependence o n the control signal is dramatized in its variation with E2 which is quite small (Fig. 6). This shows the difficulty in trying t o identify the system using a small signal linear model.

electrodes. The signal content of the EMG was extracted using a non-linear filtering technique (Kreifeldt and Yao, 1974). This technique enables extraction of the signal content in the presence of multiplicative noise. The EMG signal was fully rectified, taken t o power m and then low pass filtered at 1-2 Hz. It has been shown (Kreifeldt and Yao, 1974) that in EMG best signal-to-noise ratio is obtained with m = 1/2. The tension transducer acts via a stiff spring so that trace a in Fig. 8 represents displacements as well as tension in the limb. Care was taken that the elbow be fixed during the experiment and that the tension produced be solely the result of the biceps activity. Fig. 8 shows the EMG (trace b) which produced the tension (a). Traces c and d are the result of non-linear filtering with m = 1 / 2 and 2 respectively. Two phenomena can be observed in both c and d. Firstly, there is an initial overshoot, apparently necessary t o improve the rise time, and secondly, the steadystate level of the filtered EMG is high in the first tries and drops to a constant

332 a. b.

c.

d. 1

2

3

4

5

6

7

8

a.

b.

c.

d.

a.

b.

C.

d. 9

Fig. 8. Experimental results for 9 consecutive trials. The subject was allowed resting periods not shown here. Duration of each trial is about 5 sec. a: the tension o u t p u t (5 kg) measured by a n isometric tension transducer. b: Raw biceps EMG, c : non-linearly filtered EMG with m = 1/2. d : non-linearly filtered EMG with m = 2. I t is obvious from c and d that there is an initial overshoot and that the background tension diminishes with learning as, apparently, t h e triceps relaxes.

lower level after about 3 or 4 tries. These two results repeated themselves in other subjects.

DISCUSSION OF EXPERIMENTAL RESULTS Simulations on both the linear and non-linear systems showed that the agonist signal has an initial overshoot in order t o produce a step of displacement. In the linear model this was attributed to signal adaptation. Experiments on all the subjects showed that the overshoot invariably appears. The amplitude of

333 the overshoot is apparently the result of some optimization process compromising between an overshoot and a slow response in the output of the system. Unfortunately nothing can be said about its origin. It may originate in the higher centres or the spinal cord, it may be the result of the a system activity or that of the y. All that can be said is that the controlled plant - in this case the biceps muscle - receives appropriate excitation enabling it t o perform as necessary.

CORHtLHTION

LURRELHTILh

CHHPHS

D

GRHFHi

0

-w

9

(CHSE N3)

(CASE N I )

CURRELRTION

CRHPHS

0

(CASE N4)

rn W

ALPHA = 98 OMEGA963

(CRSE

NFI)

ALPHA = 6 7 OMEGA-281

Fig. 9. The progressive increase in motor unit synchronization with learning. Plots of processed EMG autocorrelation function with learning from first trial ( N l ) to the sixth (N6).

The second dimension observed in the experimental results is the dropping of the steady-state level necessary t o maintain the same output tension. Though one may be tempted to ascribe this t o tiring of the muscle, another plausible explanation would be the changing of the quiescent point. Lowering the excitation level reduces the stiffness of the system. Apparently a naive subject attempts to perform an unknown task with a certain amount of stiffness which slackens as he learns to cope with the control problem. Our explanation necessitates the coactivation of the triceps muscle together with the biceps in order to increase the stiffness. The triceps excitation level would then be expected to diminish together with the biceps. Our attempts to measure the triceps excitation were unsuccessful due t o the low signal amplitude from that muscle. Since the two muscles are not identical, and the system is non-linear, an EMG signal of the same order of magnitude as in the biceps is not necessarily produced by the triceps. During the performance of the task the biceps is contracting but the triceps is lengthening. Hill's equation is relevant for contracting velocities. The muscle characteristics are different for lengthening velocities, so that even if the triceps EMG were t o be recorded and processed, not necessarily will it be quantitatively similar to the processed biceps EMG. Another explanation for the reduced level of EMG activity with learning might be the increase in motor units synchronization as was recently observed (Inbar and Wildauer, 1975) by processing the same data shown here in Fig. 8. The synchronization phenomena is seen in Fig. 9 where the autocorrelation function of the processed EMG is shown for the first, third, fourth and sixth trials (N1,N3,N4and N6). The synchronization is seen from: (1)increase in negative value of the function, at about 9 msec, from approximately a value of -4.2 in the first trial to more than -0.4 in the sixth trial; (2) increase in the regularit y and amplitude of the oscillations in the autocorrelation function and (3) decrease in alpha - the exponential coefficient of a best least square fit of the autocorrelation function of the processed EMG by the function e-"t.cos.ot.

CONCLUSION The present study was initiated on the basis of many observations which indicated that the MCS is an adapting system. First a rather raw and primitive approach to modelling the system as a linear adaptive servo mechanism was attempted. Then it was deduced that the adaptive system must be working with a reference model. Attempts to supply a mathematical solution were unsuccesful because of the inherent non-linear nature of the physiological system. For this reason, computer simulations were used as a guiding aid. Simulation results showed that parameter adaptation would be extremely limited in its effect on the system response because of the nature of the system. However, since it had experimentally been observed, it could not be rejected. Thus the hypotheses that volitional control usually employs signal adaptation and that parameter

adaptation was an off-linepresetting procedure was formulated. Based on this hypothesis new simulations were performed, the results of which are shown in this paper. A simple preliminary experiment was performed on naive male subjects and their results were in correlation with each other and

335 the hypothesis. The basic results can be summarized as follows. (i) The parameters of the small signal, linear muscle model are non-linearly and strongly interrelated. (ii) The range of variations of the reflex loop parameters is physiologically bound. (iii) Signal levels in the CNS have higher bounds than those of the reflex loop. (iv) In view of i-iii, it is quite obvious that parameter adaptation schemes developed for engineering purposes are inadequate, and that parameter adaptation is inferior t o signal adaptation. (v) Signal adaptation is feasible in cases unaffected by neural delay. Parameter adaptation helps to modify the response t o external disturbance input, as long as the system is well identified. (vi) In both cases the existence of a conceptual reference model seems to be an essential characteristic. (vii) When subjects were allowed t o learn the effect of signal adaptation (initial overshoot) remained whereas the main factor causing parameter adaptation (background “unnecessary” tension) diminished. It may well be that parameter adaptation is in fact a by-product of another factor. It may be that background tension is applied in order t o enable different signal sensitivities in the muscles, and that this background tension is then translated t o new parameters identified by a viscoelastic model even though this was not the initial purpose.

SUMMARY This paper examines the behaviour of the stretch reflex in an adaptive muscle control system scheme. First a linear model is constructed for the lower stretch reflex loop. The parameters of this model are modified, “adapted”, to compensate for changes in the external load dynamics. Poor results of this parameter adaptation scheme, and other physiological considerations, lead to the study of a signal adaptation scheme. Signal adaptation proves to be an adequate mechanism which also does not violate physiological constraints. The simulation results are followed by a description of experimental results obtained with human subjects. These latter results show both signal adaptation and learning t o be prominent features of the muscle control system. The theoretical and practical difficulties in distinguishing between parameter and signal adaptation are outlined and simulation results show the parameter dependency on the control signal for the non-linear case. The role of parameter adaptation in reflex “presetting” of the muscles working point and the role of signal adaptation and “conditioning” in volitional muscle control are discussed. REFERENCES Gottlieb, G.L. and Agarwal, G.C. (1973) PosturaI adaptation - the nature of adaptive mechanisms in the human motor system. In Control o f Posture and Locomotion, R.B. Stein, K.G. Pearson, R.S. Smith and J.B. Redford (Eds.), Plenum Press, New York, pp. 197-210.

Houk, J.C., Jr. (1963) A Mathematical Modef o f the Stretch Reflex in Human Muscle Systems, M.Sc. Thesis, M.I.T., Cambridge, Mass. Inbar, G.F. (1972) Muscle spindles in muscle control. 111. Analysis of adaptive system model. Kybernetic, 1 1 : 130-141. Inbar, G.F. (1975) Modulation of dynamic parameters of muscle reflex by selective activation of its gamma system. Biol. Cybernetics, 19: 169--180. Inbar, G.F. and Joseph, P.J. (1976) Analysis of a model of the triceps surae reflex control system. IEEE Trans. SMC, 6: 25-33. Inbar, G.F. and Wildauer, E. (1975) A Model f o r Processing the Electrical Activity o f Muscles, Pub. 267, Technion, Haifa. Inbar, G.F., Hsia, T.C. and Baskin, R.J. (1970) Parameter identification analysis of muscle dynamics. Math. Biosci., 7 : 61-79. Kreifeldt, J.C. and Yao, S. (1974) A signal to noise investigation of nonlinear electromyographic processors. IEEE Trans. BME, BME-21: 298-308. Landau, I.D. (1972) Model reference adaptive control systems. A survey, what is possible and why. J. Dynam. Measurement Control, June: 119-132. McRuer, D.T., Magdaleno, R.E. and Moore, G.P. (1968) Small perturbation dynamics of the neuromuscular system in tracking tasks. N A S A Rep., CR-1212. Milgram, P. and Inbar, G.F. (1975) Multichannel information transfer in the nervous system. In Signal Analysis and Pattern Recognition in Biomedical Engineering, G.F. Inbar (Ed.), Wiley, New York. Mountcastle, V.B. (1968) Medical Physiology, Vol. 2, Mosby, St. Louis, Mo. Rosenthal, N.P., McKean, T.A., Roberts, W.J. and Terzuolo, C.A. (1970) Frequency analysis of stretch reflex and its main subsystems in triceps surae muscles of the cat. J. Neurophysiol., 33: 713--749. Soechting, J.F. (1973) Modeling of a simple task in man motor output dependence on sensory input. Kybernetik, 14: 25-34, Stark, L. (1968) Neurological Control Systems, Plenum Press, New York. Tamura, H. and Yoshida, T. (1974) Models of control and the limits of control capability for human subjects. IEEE Trans. SMC, Sept.: 482-488. Terzuolo, C.A. and Viviani, P. (1973) Modeling of a simple motor task in man intentional arrest of an ongoing movement. Kybernetik, 14: 35-62. Wieneke, G.H. (1972) Variations in the Output Impedance o f the Human Motor System, Thesis, Univ. of Utrecht. Wilkie, D.R. (1956) The mechanical properties of muscle. Brit. med. Bull., 12: 177-182. Yafe, A. (1975) Analysis o f an Adaptive Model f o r the Muscle Control System, M.Sc. Dissertation, Technion, Haifa.

DISCUSSION STUART: This is perhaps a trivial point. In your first slide when you set up the mechanical model you said you could ascribe values to the various aspects, were you able to ascribe the values t o the series of elasticity that is in the tendon insertion of each antagonistic muscle in contrast t o the series of the contracting machine itself? INBAR: No, we have not attempted t o separate it. HOUK: I could not help to notice that the Golgi tendon organ and force feedback appear to be absent from the model. The second point was that one of our observations in the decerebrate was that the linearity of the system was greatly improved as a result of reflex action. Now that may relate to your discussion of an adaptor system as regards control function, adaptive control function. That requires a lot of the system as I understand it. It seems to be that improvement in the linearity of the stretch reflex would make the modulation required to do that type of adaptation simpler. I wonder if you could comment on that. INBAR: We could make a model with Golgi tendon organ and just make it more compli-

337 cated by adding more parameters, b u t I d o not think we would gain more insight into the system. In answer to your second question it is true t h a t the range of linearity is extended with t h e stretch reflex, as was shown already b y Terzuolo, however, I don't believe that the linearity is a n aim to itself, or that it makes t h e control aspects easier. Nonlinear systems may have many advantages and are many times being introduced into man made systems. HOUK: My only comment was that with t h e stretch reflex the system to which your system sends command is more linear. I think that might explain nicely your notion that command function adaptation requires the model of the system sending signals to it. Now, if that model would be simpler. BUCHTHAL: There is a certain amount of danger because you get fast components to disappear and if you have interfering activity as you have, and if this interfering activity is dissociated, you get more fast activity or less fast activity. You have t h e experience when you d o this that you d o not record t o o fully any more. So I think that part of what you are missing is simply methodological artifact. INBAR: I think this was shown in t h e last slide. You get mild degree of synchronization. RUCHTHAL: It has nothing t o d o with synchronization. You d o not get these impulses through. If you take, for example, a wire electrode, compared with the point electrode, a wire electrode cannot go up to the frequency with which you wish to record. If you then record synchronization, it's because you do n o t record o n high frequency.

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Alpha-Gamma Linkage in Man during Varied Contract ion SHIROH WATANABE and KEIDAI HIRAYAMA

*

Department of Physiology, School of Medicine, Kyohrin University, Mitaka-shi, T o k y o (Japan)

INTRODUCTION The concept of alpha-gamma linkage has been paid attention to since experiments on supraspinal control of the muscle spindle and its significance by pioneering workers (Eldred et al., 1953). The concept was later expanded t o include even motor cortex where voluntary motor integration is considered t o originate. The first evidence of alpha-gamma linkage in the human voluntary control was given in the findings by Hagbarth and Vallbo (1968) of continuing discharge of Ia afferent impulses during voluntary contraction. We have tried t o actually quantitate alpha and gamma discharge in an indirect way by analyzing the pattern of discharges of single motor units as effected by a vibratory stimulus to the muscle. Voluntary motor unit spikes appearing during tonic vibration reflex are separated into two categories of “alpha” spikes, one driven directly by the voluntary control and the others driven indirectly by the gamma loop (gamma spikes) (Hirayama et al., 1974). The proportions in the alpha-gamma co-activation was then taken as the ratio of the above two categories of motor unit spikes during voluntary contraction. In the assessment, the ratio was estimated around 1 : 2.4 when the normal subjects performed a light contraction and average frequency of motor unit spikes was ca. 9 Hz. A ratio of this magnitude suggested that the participation of the indirect gamma control might be very important in voluntary contraction, although it must be recognized that concomitant vibration was essential in the method. The main purpose of the present report is t o investigate how the above two categories of motor unit spikes change and t o which extent the alpha-gamma co-activation ratio is influenced when the voluntary contraction is performed at various degrees of effort. The ratio will be also compared between normal subjects and spastic patients. METHOD Present data were collected from 5 normal male adults and 5 spastic patients,

* Departments of Physiology and Orthopedic Surgery, School of Medicine, Chiba University, Chiba, Japan.

340 who in turns sat in a specially designed experimental chair. Their feet were tied to a steel footplate, with a strip of soft cloth, to make registration of an isometric voluntary contraction. A Hagbarth-type motor vibrator, 4 cm in diameter and 1 4 cm in length (Hagbarth and Eklund, 1966), was attached and pressed onto the patellar tendon by the experimenter’s hand. A fine Teflon insulated copper wire was inserted into the quadriceps femoris muscle through an injection needle of comparable size. A set of two such copper wire electrodes was used t o record motor unit spikes bipolarly. In all of the present experiments, motor unit spikes, monitored vibration and isometric tension were stored in a 4-channel FM data recorder. Data were reproduced and analyzed by a biological minicomputer, ATAC 501-20, Nihon Kohden, Tokyo, to make (i) non-sequential interspike interval histograms, and (ii) cross-correlograms between motor unit spikes and vibratory stimuli. Subjects were asked to perform voluntary contraction of ca. 20-30 sec at various degrees of voluntary force. Vibratory stimulation of 15 sec duration was applied in the middle of voluntary contraction. When a set of voluntary spikes during vibration showed a good cross-correlation, the data were reprocessed through a special gate circuit to divide them into the two categories of spikes, locked and unlocked ones. Gate time of the circuit was set by t and A t adjustments, t being an arbitrary interval from some arbitrary peak of the vibratory excursion, A t being the gate period. Both values were set according to information from the original cross-correlogram. By these conditions of spike filtration, motor unit spikes between t and t + A t have been called ‘locked’ spikes and the others ‘unlocked’ spikes. For separation of alpha motor unit spikes into two categories of genesis, calculation of spikes according to the following three formulae was adopted as before. (1)Voluntary spikes without vibration = alpha spikes + gamma loop elicited alpha spikes. (2) Voluntary spikes with vibration = locked spikes + unlocked spikes. (2i) Locked spikes = gamma loop elicited alpha spikes + vibratory reflex spikes. (2ii) Unlocked spikes = alpha spikes. (3) Gamma loop elicited spikes = (1)- (2ii) From calculation shown above, a good set of data gave a ratio of alphagamma co-activation. For evaluation of data of change of alpha-gamma components of spike numbers due to varied voluntary contraction, calculated spike numbers were processed by a HITAC 10-11 computer and the regression line was found by the programme for the method of the least squares of the computer.

RESULTS

A preliminary experiment for measurement of aipha-gamma co-activation ratio It has been shown that the histograms of non-sequential inter-spike intervals in discharge of a voluntary motor unit have a typical normal distribution

341 (Homma et al., 1973), the median of which depends on the mean interval of the analyzed data, Concomitant vibratory stimulation, however, markedly changes the pattern of the histogram in that a considerable decrease in the median peak and an appearance of few to several added peaks were observed. For further understanding of the events underlying this pattern, the following experiment was a necessary preliminary.

Demonstration of cross-correlation between spikes and vibration Search for voluntary motor unit spikes with good cross-correlation to the concomitant vibratory stimulation was performed and typical data with good cross-correlation are shown in Fig. 1. Start of voluntary contraction is indicated by the recorded motor unit spikes and gradual development of muscle tension too. Concomitant vibratory stimulation is shown by the thick part of the monitored vibration. During vibration additional development of muscle tension can be seen from the figure. Cross-comelogram test of the motor unit spikes during voluntary effort only is shown by the cross-correlogram of D-1 and that of spikes during concomitant vibratory stimulation by the crosscorrelogram of D-2. In D-1, no clear cross-correlation can be found with vibration since the processed data for D-1 were generated simply by the voluntary control. On the other hand, very clear cross-correlation can be seen in the processed data of D-2. It should be noted, however, that, even in the cross-correlogram of D-2, there was a minor proportion of the units that was not correlated to vibra-

B

C

5 sec

D-1

D-2

Fig. 1. A sustained voluntary contraction of human quadriceps femoris muscle and concomitant vibratory stimulation of t h e muscle in t h e middle of voluntary contraction. A : time course of tension, calibration of which is shown t o t h e extreme right. B: m o t o r unit spikes of the quadriceps femoris. C: monitored vibration which is indicated by the thick part. D: cross-correlograms between m o t o r unit spikes and an arbitrary phase of vibration. F o r further explanation of D-1 and D-2, see text.

342 tory stimulation. In other words, it can be said that voluntary unit spikes during concomitant vibration contained motor unit spikes of 2 categories, (i) spikes well locked to some phase of vibratory stimulation, (ii) spikes unlocked to it. Separation of “locked ” and (bunlocked” spikes

For separation of “locked” and “unlocked” spikes, a special gate circuit was essential. The principle of operation of the circuit is schematically illustrated in Fig. 2. A part of the cross-correlogram which showed good correlation with vibration is illustrated in A. A setting of reference level at the computer input (horizontal line of A) automatically differentiated between motor unit spikes of “good” and “poor” correlation. An example of reference time with vibratory phase is shown by the vertical line superimposed on a certain peak phase of the sinusoidal curve of B. Time setting of t and A t is illustrated by the two dotted vertical lines in A and C. Time settings of t and A t were so adjusted as to fit the criterion used above in the cross-correlogram of A. Spikes between t and t + At, or within At, were electronically separated from the original spike series of C and the output is illustrated in D. Series of original spikes subtraced by D, hence ‘unlocked’ spikes, is indicated by E. A typical example of such electronic operation of motor unit spikes is shown in Fig. 3. Record A shows reproduced motor unit spikes processed through an

D E

1

Fig. 2. Schematic illustration to explain function of a special “window”, t, which discriminates between “locked” and “unlocked” spikes. A: part of cross-correlogram in which a good correlation between spikes and vibration was found. Horizontal dotted line indicates employed level for determination of correlation which automatically decided both timing and duration of t. B: vertical bars superposed on the sinusoidal curve indicate each arbitrary phase of start of correlation search of the computer. C: original series of spikes. Each set of two dotted vertical lines indicates duration of the window for the locked spikes. D: locked spikes. E: unlocked spikes.

343

Fig. 3. Reprocessed series of original voluntary spikes (A) and their separation into locked ( B f and unlocked (C) spikes. Duration of vibratory stimulation is indicated by the thick part. Numbered part (... 1 ...) of A, series 2 and 3 (B and C respectively) are very important for the present calculation of the alpha-gamma co-activation ratio. See text for detailed explanation of the calculation.

amplitude limiting circuit. Mean frequency of these voluntary motor unit spikes without vibratory stimulation was 9.2 Hz. This value was increased up to 11 Hz during concomitant vibratory stimulation. Results of electronic separation of voluntary spikes during concomitant vibration into ‘locked’ and ‘unlocked’ spikes are shown in B and C respectively.

Calculation of alpha-gamma co-activation ratio For calculation of the ratio, significance of the difference of the genesis of voluntary motor unit spikes with and without vibratory stimulation should be considered. This consideration is already very briefly described in Methods. However, fundamental ideas associated with those formulae can be described as below. (1)Voluntary motor unit spikes solely during voluntary command may be subdivided into two groups: (i) those activated by the descending alpha path, and (ii) those activated through the gamma loop and the ensuing spindle activity. (2) Voluntary motor unit spikes during concomitant vibration can be considered to include (i) spikes driven by the direct alpha path; (ii) those driven by the indirect gamma loop; and (iii) those activated by monosynaptic or other Ia afferent impulses. It has been established that Ia impulses can be driven by gamma efferents which supposedly belong to the static fusimotor fibres (Crowe and Matthews, 1964; Bessou et al., 1968). It has been suggested that almost all such Ia impulses, once biased by gamma innervation, would readily become locked t o the needed vibratory stimuli and thus their firing phase would be vibration-correlated (Homma et al., 1973; Nakajima, 1975). Therefore it is very natural that both gamma loop elicited and reflexively activated spikes are vibration-correlated. Although voluntary spikes during concomitant vibration should be devided into three categories from genesis point of view as explained above, they were, by technological limitation, divided into two categories only, those well locked and those unlocked to the vibratory phase.

344 Series of voluntary spikes before vibration are indicated by portion (1)of A of Fig. 3 . Series of voluntary spikes during concomitant vibration in A are to be judged by the part which corresponds t o the thick part of monitored vibration D during when vibratory stimulation was applied t o the muscle. Separated locked spikes are shown by B and unlocked spikes by C. It is possible here to suppose that the locked spikes of B are the sum of motor unit spikes activated directly by the vibratory Ia afferent impulses plus motor unit spikes activated by some other Ia impulses whose firing phase became locked t o the vibratory stimuli. (3) The unlocked spike of the record of C, independent of the vibratory period, may belong to the one or several of the following three categories. (i) Motor unit spikes activated by the alpha path. (ii) Spikes activated by the Ia impulses, driven by the gamma efferent impulses during voluntary command but remaining unlocked t o the superimposed vibratory stimuli. (iii) Spikes driven by the polysynaptic pathway which are mobilized by continued vibration. These spikes due to polysynaptic activation are very few in number compared with the number of locked spikes. Therefore it will be not much of an error if in order to prevent confusion, these spikes were to be attributed to the alpha path of our category (i). Through above consideration and after minor simplifications mentioned immediately above, calculation of alpha-gamma co-activation ratio can be shown by the following 5 formulae: (1)Voluntary spikes = CY spikes + y loop elicited spikes. (2) Locked spikes = y loop elicited spikes f vibratory spikes. (3) Unlocked spikes = CY spikes. (4) y loop elicited spikes = (1)- (2). (5) Alpha-gamma co-activation ratio = (4)/( 3) *. The motor unit spikes in formula (4) thus show the number of motor unit spikes elicited by the gamma loop command during the voluntary effort. They were thus known t o be 2.4 times greater than the number of pure alpha spikes in our previous work, This ratio of 1 : 2.4 permits u s t o assume that in a slight voluntary contraction activities through direct alpha path and indirect gamma loop may be expressed by that ratio in the motor integration of alpha-gamma linkage.

Alpha-gamma co-activation ratio during varied voluntary contraction in normal subjects It seemed very interesting to see how the alpha-gamma co-activation ratio varies when subjects performed their voluntary contractions at different degrees of voluntary effort. Measurements were performed from very slight contraction to the maximum limit, just before contamination by other multi-units of various amplitudes started t o appear. Measurements from 30 such experiments are shown in Table I. Classified values from these data are also shown by the plotted dots in Fig. 4A, B and C. Numbers of gamma loop elicited spikes per each cross-correlogram are plotted in A along the axis of mean motor unit spike frequency changed by varied voluntary contraction. These numbers of alpha spikes are plotted on B and the

-

* In mathematical sense, it is gamma-alpha ratio. See also Table I.

345 TABLE I CALCULATIONS O F MEAN VOLUNTARY UNIT SPIKES FROM 30 VARIED VOLITIONAL CONTRACTIONS, THEIR ‘ALPHA’ AND ‘GAMMA’ COMPONENTS AND THEIR RATIOS ~~

Total 1 2 3 4 5 6

I

8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 21 28 29 30

7.4 6.2 5.4 4.4 6.9 1.2 5.4 6.5 5.4 1.3 9.8 9.2 8.6 9.6 9.2 9.0 10.0 9.4 8.6 9.7 9.0 1.5 7.1 7.2 6.7 8.6 12.3 9.2 11.0 13.2

~~

Alpha

Gamma

Ratios ( y h )

1.7 2.1 2.8 1.3 3.0 1.6 2.5 1.9 2.9 3.0 2.9 2.1 2.1 2.3 2.5 2.8 3.4 3.3 2.5 2.1 2.4 1.9 2.3 2.4 1.6 3.1 4.0 3.5 4.5 3.4

5.1 4.1 2.6 3.1 3.9 5.6 2.9 4.6 2.5 4.3 6.9 6.5 5.9 7.3 6.1 6.2 6.6 6.1 6.1 7.0 6.6 5.6 4.8 4.8 5.1 5.5 8.3 5.1 6.5 9.8

3.4 1.95 0.9 2.4 1.3 3.5 1.2 2.4 0.9 1.4 2.4 2.4 2.2 3.2 2.1 2.2 1.9 1.8 2.4 2.6 2.8 2.9 2.1 2.0 3.2 1.8 2.1 1.6 1.4 2.9

*

* See footnote of previous page. ratio of alpha-gamma co-activation in C. Since coherent linear relation could be found in only a few series of experiments of voluntary contraction, all data were independently plotted in Fig. 4A, B and C. Therefore statistical analysis of overall data was performed further by computer analysis. The thick continuous lines in A and B are regression lines given by the plotted data found after the least square method computation of HITAC 10-11 computer. Although the same analysis was performed on the data of the ratios of alpha and gamma spikes, shown by the points in C, the data were shown t o be too much dispersed for the specific programme of the computer in that no discrete line could be given by the method. Nevertheless direct observation suggests that the ratio is scattered within some limited values as shown by the illustration. Therefore, in very general sense, it should be pointed out that the alpha-gamma coactivation ratio is controlled within the values shown in Fig. 3C and Table I, i.e., within 0.9 and 3.4,its minimum and maximum respectively.

346

2

4

6

8

10

12

1 4 irnGec

. .

Y =023X+OI3 I

I

I

I

I

I

2

4

6

8

10

12

K-X

GO-ACTIVATION

I

1 4 lrnvec

RATIO

.. 2

. 2

4

..

. *. * .

6

8

10

Fig. 4.Shift of ‘alpha’ and ‘gamma’ components in the voluntary motor unit spike frequency measured by the present method. Abscissae: mean frequency of motor unit spikes varied by changing voluntary effort. Ordinates: mean frequency of ‘alpha’ and ‘gamma’ components of spikes per each volitional contraction. Points are collected from Table I. For explanation of the thin dotted lines, see text.

Alp ha-gamma co-activat ion ratio during varied vol un tary contract ion in spastic patients The same experiments were performed on patients with spastic quadriceps femoris muscles. Regression lines for the spastic muscles were found by the same method. The lines for alpha and gamma recruitment during varied contraction are shown by the thin dotted lines in Fig. 4B and A respectively. The regression line for alpha in B was slightly steeper than that of normal subject and the line for gamma in A was slightly gentler than the normal ones. This indicated that recruitment of gamma loop elicited motor unit spikes was slightly more increased during weak contraction in spastic patients than in the normal subjects, whereas it was subnormal in spastic patients during strong effort for

347 voluntary contraction. Thus smooth recruitment of alpha motoneuronal activity is also shown to be disturbed (Fig. 4B). Detailed report about the patient study will be published elsewhere (Hirayama et al., 1976).

DISCUSSION In this study the presence of locked and unlocked discharges of motor unit spikes during vibratory stimulation in man, as had earlier been shown in cat, was demonstrated. The ratio of these was moreover found to be rather constant at different degrees of voluntary effort. We interpret this as indicating that the alpha-gamma linkage was comparably equally strong in human voluntary contraction. The arguments why locked spikes reflect gamma activity primarily will be discussed first and then the notion that the unlocked spikes are primarily volitional in origin.

Locked spikes The concept of the ‘decoding ratio’ in the input and output relation of the stretch reflex centre during the tonic vibration reflex (TVR) was proposed by Homma et al. (1971). This relation, found in cats, has now been demonstrated for man also since the interspike interval histogram of the EMG during TVR was composed of integer multiples of the vibratory cyclic period. The existence of the integer ratios between motor unit spike frequency and vibratory cycles in the cat has been explained as due to temporal summation of integral numbers of the EPSP ripples to vibratory excursions leading to the motoneurone eventually attaining its firing threshold (Homma et al., 1970; Homma and Kanda, 1973). Generation of these EPSP ripples indicates that the monosynaptic activation of motoneurone by rather synchronous Ia impulses was, dominant, even though there must be considerable temporal dispersion in the entry of the afferent discharges at the spinal cord due to variation in afferent fibre diameters and in initiation of the discharge to a vibratory excursion. The interspike intervals of a motoneurone are dependent upon (i) the degree of temporal summation in facilitatory influence, (ii) duration of afterhyperpolarization, and (iii) the course of recurrent inhibition from Renshaw cells. The intervals should also depend upon (iv) the height and rate of rise of the EPSP t o a vibratory excursion, (v) the slope of the background ‘slow’ depolarizing shift, (vi) the variability of firing threshold of the motoneuronal membrane, and (vii) the accommodation of which it is capable. Observations with intracellular recordings indicate that the former three factors are the most important ones (Homma and Kanda, 1973). The shortest interspike interval during TVR activity of quadriceps femoris muscle in man was about 50 msec at all vibratory frequencies. This indicates that human motor units have a preferred maximum rate of discharge at 20 Hz although the precise frequency may differ from motoneurone t o motoneurone, perhaps in relation to their size. To describe this the term ‘preferred firing frequency’ of alpha motoneurone was introduced in an earlier paper (Homma et al., 1972a). The gradual increase of firing frequency of a single alpha moto-

348 neurone during TVR is accompanied by a parallel increase of the preferred firing frequency both in man and cat (Homma and Kanda, 1973). Slow, but steady depolarization of the cell membrane of the alpha motoneurone with approach of the membrane potential toward the critical threshold shortened even the unlocked firing intervals, thereby increasing the preferred frequency. It has been pharmacologically shown that the slow increment of depolarization in response t o a vibratory stimulus can be abolished by an i.v. injection of Mephenesin without influencing the size of the vibratory EPSPs (Homma and Kanda, 1973). Although the injection slowed down the frequency of Ia impulses it did not change the size of the monosynaptic EPSP, and the slow depolarization that was abolished by Mephenesin must, therefore, have an origin different from that of the vibratory EPSP. Since Mephenesin is known to cause selective blockade of polysynaptic pathways, the abolition of the slow depolarization in TVR may also be dependent on the effect on polysynaptic circuits.

Reflex unlocked spikes That the TVR is a tonic stretch reflex has been shown in the studies of Ia afferent discharge in the human nerve (Hagbarth and Vallbo, 1968). As described above, a pharmacological experiment also showed that polysynaptic neuronal circuit is involved during TVR of the cat. Indeed, participation of polysynaptic neuronal circuit has long been recognized (Granit et al., 1957; Tsukahara and Ohye, 1964; Kanda, 1972). In the recent study of the TVR, Homma and Kanda (1972) called the steadily incrementing slow depolarization found in motoneurone the ‘augmenting’ EPSP. During continued vibratory stimulation, the gradual increase in the frequency of ‘locked’ spikes is due t o the algebraical summation of the monosynaptic EPSP and polysynaptic EPSP. The gradual shift of the baseline toward depolarization should allow the motoneurone to eventually discharge to even a small additional input from the polysynaptic pathways. The interval determined by such a firing mechanism needs not to be conform t o the integer multiple activation principle. It is suggested that reflex component in discharge of the unlocked spikes was primarily determined by this imprecise time course of the slow augmenting EPSP.

Vo 1itio nal unlocked spikes In the present study, in which voluntary contraction of the quadriceps femoris muscle was performed at various degrees of volitional effort, it was shown that the intervals of the voluntary motor unit spikes conformed to a normal distribution by non-sequential interval histogram analysis (Homma et al., 1972b). The more intense volitional effort simply led to a shorter median in an unvariable normal distribution. It is clear, therefore, that the final output of volitional effort, presumably directed from the motor cortex, is expressed by a spike sequence in which the intervals always conform to a normal chance distribution. On the other hand, it has been shown in the baboon (Clough et al., 1968)

349 that a good convergence monosynaptic projection from the motor cortex can elicit a large size of EPSP at the spinal motoneurone, although a roughly parallel deviation of EPSP size with ‘command’ EPSP was found at the motoneurone by Ia afferent stimulation. It is possible, therefore, that during human voluntary contraction ‘volitional’ EPSPs of large amplitude may be arriving at the motoneurones continuously, the sequence of spikes due t o such activation being quite independent of the arrival of the vibratory Ia afferents. Therefore, the unlocked component of the voluntary motor unit spikes occurring during concomitant vibratory stimulation, demonstrated in the cross-correlogram, can be regarded as the spikes triggered by such EPSPs of relatively large size due t o descending impulses coming through the direct ‘alpha’ path.

General consideration about factors for locked and unlocked spikes Many factors might conceivably influence the generation of locked vs. unlocked firing patterns of the motoneurone during, and in correlation with, vibratory stimulation. Conditions which might increase the numbers of unlocked spikes are: (i) volitional effort; (ii) segmentally initiated polysynaptic neural connections of short delay (2-4 synapses, i.e., 1.2-3 msec) that are activated during TVR; (iii) descending facilitatory influences from the reticular formation and other supraspinal centres; (iv) pathological conditions marked by muscular rigidity. On the other hand, proportionately more locked spikes would be expected from: (i) any increase in the passive dynamic characteristics of la afferent endings; (ii) active increase in ending sensitivity due to fusimotor activation; (iii) more abrupt muscle excursion, i.e., better as locked by triangular as compared t o sinusoidal extension; (iv) more synchronized transmission of discharges, as favoured by shorter length of conduction (e.g., jaw vs. leg muscles), or rate of conduction; and (v) higher average in the conduction rate in both efferent and afferent pathways, i.e., the faster the better locked.

CONCLUSION

(1)A cross-correlogram study has been made of motor unit spikes in quadriceps femoris muscle of man during voluntary contraction with and without vibratory stimulation of the muscle. (2) A method of analysis whereby the proportion of spikes locked t o vibratory excursion and those unlocked was used. (3) It was found that a ratio of locked t o unlocked spikes seemed t o be controlled within some limited range of values even at different degrees of voluntary effort in the normal subjects. (4) Arguments are advanced which would indicate that, t o a large extent, the locked spikes represent the degree of fusirnotor activation and unlocked spikes background or volitional facilitation of the alpha motoneurones. With this assumption, it may be concluded that the alpha-gamma linkage is constantly assumed within all ranges of volitional isometric contraction.

350

ACKNOWLEDGEMENT The authors wish to thank Prof. Earl Eldred for many helpful discussions. REFERENCES Bcssou, P., Laporte, Y. and Pages, B. (1968) Frequencygrams of spindle primary endings elicited by stimulation of static and dynamic fibres. J. Physiol. (Lond.), 1 9 6 : 4 7 - 6 3 . Clough, J.F.M., Kernell, D. and Phillips, C.G. (1968) The distribution of monosynaptic excitation from the pyramidal tract and from primary spindle afferents t o motoneurones of the baboon’s hand and forearm, J. Physiol. ( L o n d . ) , 1 9 8 : 145-166. Crowe, A. and Matthews, P.B.C. (1964) Further studies of static and dynamic fusimotor fibres. J. Physiol. (Lond.), 174: 132-151. Eldred, E., Granit, R. and Merton, P.A. (1953) Supraspinal control of the muscle spindles and its significance. J. Physiol. (Lond.), 1 2 2 : 498-523. Granit, R., Phillips, C.G., Skoglund, S. and Steg, G. (1957) Differentiation of tonic from phasic alpha ventral horn cells by stretch, pinna and crossed extensor reflex. J. Neurophysiol., 2 0 : 470-481. Hagbarth, K.-E. and Eklund, G. (1966) Motor effects of vibratory muscle stimulation in man. In Muscular Afferents und Motor Control, Nobel Symposium I , R. Granit (Ed.), Almqvist and Wiksell, Stockholm, pp. 177-186. Hagbarth, K.-E. and Vallbo, A.B. (1968) Discharge characteristics of human muscular afferents during muscle stretch and contraction. E x p . Neurol., 22: 674-694. Hirayama, K., Homma, S., Mizote, M., Nakajima, Y. and Watanabe, S. (1974) Separation of the contributions of voluntary and vibratory activation of motor units in man by cross-correlograms. Jup. J. Physiol., 24 : 293-304. Hirayama, K., Tamaki, T., Yamane, T., Kobayashi, H., Egashira, T. and Noguchi, T. ( 1 9 7 6 ) Quantitative measurement of alpha and gamma linkage in spastic patients during voluntary contraction with concomitant TVR. In The 2nd int. S y m p . on Tonic Vibration Reflex, Saikon Publishing Company, Tokyo, in prass. Homma, S. and Kanda, K. (1973) Impulse decoding process in stretch reflex. InMotor Control, A.A. Gydikov, N.T. Tankov and D.S. Kosarov (Eds.), Plenum Press, New York, pp. 45-54. Homma, S., Ishikawa, K. and Stuart, D.G. (1970) Motoneuron response to linearly rising muscle stretch. Amer. J. phys. Med., 4 9 : 290-306. Homma, S., Kanda, K. and Watanabe, S. (1971) Monosynaptic coding of group Ia afferent discharges during vibratory stimulation of muscle. Jup. J. Physiol., 21: 405-417. Homma, S., Kanda, K. and Watanabe, S. (1972a) Preferred spike intervals in vibration reflex. Jup. J. Physiol., 22: 421-432. Homma, S., Kanda, K. and Watanabe, S. (1972b) Integral pattern of coding during tonic vibration reflex. In Neurophysiology Studied in Man. G.G. Somjen (Ed.), Excerpta Medica, Amsterdam, pp. 345-349. Homma, S., Mizote, M., Nakajima, Y. and Watanabe, S. (1973) Muscle afferent discharges during vibratory stimulation of muscles and gamma fusimotor activities. Agressologie, 13: 45-53. Kanda, K. (1972) Contribution of polysynaptic pathways t o the tonic vibration reflex. Jup. J. Physiol., 2 2 : 367-377. Nakajima, Y. ( 1 9 7 5 ) Effects upon spindle discharges of electrical stimulation of static fusimotor fibers with concomitant application of muscle vibration. Jup. J. Physiol., 25: 4 17-433. Tsukahara, N. and Ohye, C. (1964) Polysynaptic activation of extensor motoneurons from group Ia fibers in the cat spinal cord. Experientiu (Basel), 20: 628-629.

351 DISCUSSION GRANIT: Switching motor to gamma side one did not know at a time how strongly gamma effect could be. So one must be careful not to go too far. But I still believe that there is such a switch somewhere. We have a means of putting on the gamma separately. I think that this experiment on the isotonic contraction is quite interesting. Slow isotonic comes immediately to full treatment. After switching on this machinery, slowly the alpha machine mounts up tension. I think that this is our most important experiment to indicate the switching over. You just decide now that there is a force t o put on the gamma machinery? HAGBARTH: First I would like to say that David Berg in Uppsala has recently made a similar study in normal subjects. They have during voluntary contraction in the calf muscle added vibration, and, in a similar way you described, they see many motor unit potentials which are time-locked to the vibrator and others which are not time-locked. We have also studied similar phenomena in human masseter muscle. If you have a voluntary contraction going here and add vibration, then practically all the motor unit spikes are locked. It is very difficult to find any motor unit spikes which are not locked in the masseter muscle. We believe that one of the important factors for this difference is the long conduction distance of the afferent in the case of the leg muscle and the short conduction distance in the case of the masseter muscle. What I’m wondering about now is if you, just by looking at the number of time-locked spikes, can get the correct measure of the extent to which the gamma loop is involved in a voluntary movement. I think another factor that is of importance also is the conduction distance and the dispersion of vibration-induced afferent inflow that must occur in a long afferent conduction loop and so the timing is less pronounced. KERNELL: I am also wondering how your interesting observations could lead to a precise quantitative measure of the extent t o which a motoneurone is excited via the gamma loop. A motoneurone spike might conceivably be time-locked to, and triggered off by, a small vibratory EPSP occurring on top of a large amount of steady excitation. In such a case, the timing of the spike might be decided by the small EPSP while most of the total excitation needed for discharging the spike actually came from elsewhere (for instance, independently of the gamma loop). WATANABE: Since we have classified only single unit spikes, our classification is a statistical one. So we think that purely alpha command triggered spikes generate irrespective of this vibratory EPSP, meaning that superimposed vibratory EPSP doesn’t really approach the peak. So this kind of spike does not occur at the peak of EPSP. Statistical consideration is the basis of our present viewpoint we took, i.e., that unlocked spikes are purely alpha command generated and locked spikes by indirect gamma loop which means directly activated by monosynaptic EPSP. MATTHEWS: I think we all agree that there are two routes converging on the motoneurone, the alpha route and the gamma loop coming back. There must be contribution from alpha route and gamma route t o measure. Any question is whether this particular method is 100% reliable or whether it has certain technical snags which we have t o sort out. Very ingenious method, indeed. WATANABE: We are always conscious of the fact that this is an indirect method.

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SESSION VII

SIGNIFICANCE OF SLOW AND FAST MUSCLES IN THE STRETCH REFLEX

Part I Chairman: H. Hultborn (Goteborg)

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Discharge Pattern of Tonic and Phasic Motor Units in Human Muscles upon Stretch Reflex DIMITER KOSAROV, ALEXANDER GYDIKOV and NICOLAS TANKOV Institute of Physiology, Bulgarian Academy of Sciences, Sofia (Bulgaria)

INTRODUCTION The stretch reflex plays an important role in sustaining the muscle tone and body posture, as well as in muscle response t o unexpected external mechanical forces. The rise in the activity of the muscles during stretch is due t o an increase in the discharge frequency of the a-motoneurons and the contractions of the corresponding motor units (MUs) and also t o recruitment of new cu-motoneurons and MUs. Animal experiments revealed t o a great degree the possible mechanisms of a-motoneuron excitation upon muscle stretch. Of basic significance are the primary endings of the muscle spindles which excite the motoneurons monosynaptically (Lloyd, 1943, 1946a,b; Granit, 1950, 1956; Hunt, 1952). A polysynaptic excitation via these endings is added t o this effect too (Granit et al., 1957a,b; Tsukahara and Ohye, 1964). The secondary endings from muscle spindles activated during stretch have an excitatory effect on the cu-motoneurons of the flexors and an inhibitory effect on the cu-motoneurons of the extensors. These effects are polysynaptic (Hunt, 1952; Eccles and Lundberg, 1959; Laporte and Bessou, 1959). Excitation of primary and secondary endings is highly dependent on y motor innervation of the spindles. The shortening of the muscle during stretch reflex unloads the spindles. During extrafusal contraction spindle afferents may stop t o discharge depending on the level of gamma efferentation (Leksell, 1945; Granit, 1956). The extrafusal contraction activates also the Golgi receptors (Granit, 1950; Hunt, 1952) having an inhibitory disynaptic influence on a-motoneurons. The EMG of human muscles during stretch reflex shows a typical rise of the summated activity, followed by a “silent period”. There is no information about the discharge frequency of different a-motoneurons and the corresponding MUs during stretch reflex in human beings, which may help in understanding the real mechanisms of the reflex under normal physiological conditions. A method of selective recording of impulses from separate MUs a t high muscle activity has been worked out in the course of earlier studies (Gydikov and Kosarov, 1972, 1974). Two types of MUs, phasic and tonic, were distinguished in human muscles by means of this method (Gydikov and Kosarov, 1974). It was interesting to study in the stretch reflex the pattern of discharge of these

356 two types of MUs which are probably activated by phasic and tonic cr-motoneurons (Granit et al., 1956). METHOD The studies were carried out on human m.biceps brachii. The subjects were clinically healthy men and women aged between 21 and 44 years, without deviations from the normal EMG. The study undertaken covered a total of 40 motor units, selectively recorded in 7 subjects. The experimental setup is illustrated in Fig. 1. The subject is sitting on a chair. His arm is in supination, bent at the elbow at a joint angle of 90”. It maintains the load PI which is partly balanced by the smaller load P, . The P I and P, quantities are varied in the course of the experiment. At the initial position the load on the arm is P = PI - P, . The quantity of P is selected in two manners, namely: (1)the muscle tension of the biceps being almost equal to, though somewhat below, the threshold of activation of the MU studied; and (2) the muscle tension being above the threshold and creating a certain desired average frequency of discharge of the investigated MU. An additional loading of the arm S is obtained by cutting the link between the arm and P,, S being equal to P, . We varied S upon keeping P constant and we also varied P upon keeping S constant. The additional loading of S suddenly unfolds the arm at the elbow joint and thereby stretches the biceps. With constant S, the stretch was almost constant. The activity of the MUs in the muscle rises due to the stretch reflex. This is expressed both in the recruitment of new MUs, and in raising the frequency of discharge of those MUs which were active before that also. The intervals between the individual tests were not constant, and all signals which could have served as a warning for the additional loading were avoided. Recording was made from a three-channel “Disa” electromyograph provided

Fig. 1. Experimental setup for stretching the muscle by additional loading. PI,load; Pz, counter-load. The arrow shows the place of cutting the tie of the counter-load.

357

10 rnrn

Fig. 2 . Multielectrode with 4 selective electrodes and one non-selective electrode.

with mean voltage unit and switchbox. We used electrodes with small leadingoff surface with the technic for selective recording, location and size dtermination of MUs as described earlier. At the beginning we determined to which one of the two groups belongs the unit investigated, i.e., tonic MU (small, with low threshold, and with plateau in the frequency-tension dependence) and phasic MU (large, with high threshold, and without plateau in the frequency-tension dependence), A detailed description of this procedure was given by Gydikov and Kosarov (1974). Twenty of the 40 MU investigated were tonic and the remaining 20 were phasic. As for the studies of the stretch reflex caused by additional loading, on one

P = 3500

S 13000 0.25

I

B

A Fig. 3. A: stretch reflex which raises the discharge frequency of a tonic MU. B: stretch reflex raising the discharge frequency of a phasic MU. Above: selective leading-off from one MU. Below: nonselective summated EMG. In the middle: displacement output and mean voltage of the EMG.

3 58 channel of the electromyograph we recorded selectively the discharge of the investigated MU, on another channel we recorded the summated EMG led off from the non-selective electrode in the composition of the multielectrode shown on Fig. 2, while on a third, by means of the mean voltage unit, we recorded the joint displacement (Fig. 3). In leading off the displacement we used a potentiometric transducer attached t o the elbow joint, having a sensitivity of 2 mm for 1". Recording was done a t 5 cmlsec speed. Measurements were made of the instantaneous frequency and of its variations a t different P and S. The points expressing the instantaneous frequency for each inter-impulse interval were related t o that moment of time a t which the interval had ended and stored as a function of time. Twenty responses were accumulated for every combination of S and P. We averaged also the corresponding joint displacements, measuring the values of the displacement for every 1 mm. RESULTS There is great difference between the responses of tonic and phasic MUs upon their reflex activation as a result of stretching through additional loading.

Tonic MUs When the initial load is selected in such a manner as t o be just below the threshold of a tonic MU, the latter is switched on into activity upon additional P.1500

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C E! Fig. 4. Instantaneous frequency of an MU, and joint displacement upon stretch reflex caused by additional loading. Storing and averaging from 20 reactions. Tonic MU, with sub-threshold initial load of 1500 g. Various additional loading: A, 3000 g; B, 2000 g; C, 1000 g. A

359 loading. At high values for S the first one, two or three intervals are short, then follows a drop in the frequency of the impulses and a new rise. A typical curve is obtained upon the storage of the values of the instantaneous frequencies for 20 reactions of an MU (Fig. 4)- switching-on starts at a high frequency which gradually decreases, reaching a minimum at about 180-200 msec after the beP =1500

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Fig. 5. Tonic MU upon suprathreshold initial load: A, B, C, initial load 1500 g; D, initial load 3500 g; E, initial load 5500 g. A, D, E, additional loading 3000 g; B, additional loading, 2000 g; C, additional loading, 1000 g. The rest as in Fig. 4.

360 ginning of the stretch and then the steady-state frequency corresponding t o the total load after the additional loading. The maximum frequencies at S = 3000 g reach 25 imp/sec, the minimum ones being about 6-7 imp/sec. Upon a decrease of the S/2000 g and 1000 g/ the MU is switched on later. The initial small intervals disappear. Specifically at S = 1000 g the first interval is, as a rule, longer than the second one. It can be seen from Fig. 4C that, despite the absence of the initial high-frequency phase, the phase of frequency reduction is preserved almost unchanged compared with the cases of S = 2000 g and S = 3000 g. If we select the initial load in such a manner that the MU investigated discharges at a given average frequency, then the additional loading evokes an initial rise in the frequency which does not reach the high frequencies accompanying the recruitment of the unit (Fig. 5). Then follows a decline in the frequency which is again t o be observed with a minimum at the 180th-200th msec after the beginning of the additional loading, and a new rise in the frequency which transcends into the steady-state level of discharge (corresponding to the increased load). The influence of the size of S can be seen upon comparing Fig. 5A with Fig. 5B and C. At the transient process after S = 2000 g the changes in the frequency differ little from those after S = 3000 g. However, the differences increase at S = 1000 g, the initial rise in the frequency being not so marked. Naturally, the

s=3ooo

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Fig. 6. Summation of the instantaneous frequencies from the first responses of 20 tonic MUs at stretch reflex.

361 steady-state frequency after the transient process is different upon a different S since the load after the additional loading is different. The significance of the initial load P is to be seen upon comparing Fig. 5A with Fig. 5D and E. At an initial load of P = 1500 g the initial frequency was about 1 0 imp/sec, at an initial load of P = 3500 g it is about 1 2 imp/sec, and at initial load of P = 5500 g it is about 1 4 imp/sec. A maximum of up t o 20 imp/ sec is reached upon raising the frequency in all three cases. The frequency which is to be observed during the steady-state after stretching rises with the increase of the total load P from 4500 t o 6500 g, but it shows no marked change upon P reaching 9500 g. The accumulation of the responses, obtained many times from one and the same MU (20 responses, Fig. 5) and from all the 20 tonic MUs investigated (20 responses, storing of the first response of each MU investigated, Fig. 6), has almost one and the same course.

Phasic M Us At an initial load (P) which is smaller than the threshold the additional loading switches on the unit investigated (Fig. 7A). The separate responses for one ' ~ 6 5 0 0S.3000

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Fig. 7. Phasic MU at stretch reflex caused by additional loading (3000 g) A: at subthreshold initial load of 3000 g. .B: at suprathreshold initial load of 6500 g. The rest as on Fig. 4.

362 M U differ considerably. No systematic tendency is t o be observed for the first interval to be shorter or longer than the second. One interesting feature upon the accumulation of 20 responses is the great dispersion of the points. This is due both to the greater dispersion of the consecutive intervals upon one realization and to differences in the average frequencies in the separate transient processes. The only feature characteristic of the transient process in comparison with the steady-state is the greater dispersion of the inter-impulse intervals. The character of the response does not depend on the size of S. The essential difference between tonic and phasic MUs can still be seen more clearly when the MU is active before the additional loading (Fig. 7B). The basic difference between the transient process and the steady-state after it is the appreciably greater dispersion of the inter-impulse intervals during the transient process. Furthermore, a certain stratification is also t o be observed. Immediately after loading, the predominant frequencies are either from 10 to 20 imp/sec or from 28 to 50 imp/sec, while frequencies of 20 t o 28 imp/sec are relatively rare. Fig. 3B represents a transient process of a phasic MU. Typical in the transient process is the alternation of brief and long inter-impulse intervals. It is precisely these alternations, which for a separate MU and even for separate responses of one and the same phasic MU occur at different times, are the cause of the result shown on Fig. 7B. The character of the transient processes does not depend essentially on the size of P and of S. Discharge frequency of MUs and joint displacement The arm is unfolded at the elbow joint upon the additional loading. From the initial state at 90" flexion the joint angle increases to its maximum after about 120-130 msec. The increased muscle force, as a result of the stretch reflex, gradually overcomes the increased load and its inertness, and the extension is followed by flexion. This flexion reaches its maximum after about 270 msec from the beginning of the stretching. A slight extension again follows after that. A comparison of the displacement output with the changes in the frequency of discharge of the tonic MUs shows a number of important characteristics. The frequency may decline even before the maximum of the extension has been reached. The minimum of the frequency shows a delay in relation to the maximum of the extension. On the whole, there is a marked dependence between the frequency of impulsation of the tonic MUs and the displacement output, a dependence which is not at hand in the phasic MUs. The maximum of the extension grows with the increase of S, as should be expected, but it decreases with P. There is a tendency for the arm t o remain in extension at a low P. The maximum of the flexion does not reach the initial position of 90". A t a high P the tendency exists for the arm to remain, after the transient process, in a state close to the initial one.

DISCUSSION The results described show a marked difference in the pattern of discharge of tonic and phasic MUs during stretch activation. Tonic units respond with an

363 initial high-frequency phase, inhibitory phase during which the frequency drops below the initial one (in case of MUs which were active before the additional loading) and a phase of secondary rise in the frequency followed by the steadystate discharge. Phasic MUs respond with a phase of high frequency and high dispersion of the inter-impulse intervals followed by the steady-state discharge. Maybe the differences in the input and the properties of tonic and phasic a-motoneurons could account for the results described. Tonic a-motoneurons are small, low-threshold, with long afterhyperpolarization. Phasic a-motoneurons are big, high-threshold, with shorter afterhyperpolarization (Granit et al., 1956; Eccles et al., 1957b; Kernell, 1965). The monosynaptic excitation of primary endings is more strongly expressed on tonic a-motoneurons (Eccles et al., 1957a; Granit et al., 1966). Recurrent inhibition is strongly expressed also on tonic motoneurons (Granit et al., 1957a,b; Granit and Rutledge, 1960). The initial rise of the discharge frequency after additional loading in tonic and phasic MUs is explainable by the activation of the spindles. The low-frequency phase of discharge in tonic MUs corresponds t o the silent period in the summated EMG. A well expressed silent period evidently is possible, if during stretch reflex only low-threshold tonic MUs are drown in activity. Many mechanisms may underlie this inhibitory phase, for example, afterhyperpolarization, recurrent inhibition, inhibition by activation of Golgi tensoreceptors, unloading of the spindles, y-motoneuron inhibition. The results described show that any of these mechanisms cannot be suggested as a sole explanation of the low-frequency phase. Fig. 4C shows that a low-frequency phase is present with low S despite the absence of the initial high-frequency phase. Evidently the afterhyperpolarization and the recurrent inhibition of the motoneuron caused by its own recurrent axonal branch cannot offer an explanation of the low-frequency phase in this case. Figs. 4 and 5 show that minimum frequency during the low-frequency phase does not depend on S, as it should be expected, if the recurrent inhibition or the Golgi tensoreceptors inhibition are the predominant influences causing the phase described. Spindle unloading may contribute t o a high degree, but frequency drop starts before flexion. It is not possible to exclude an inhibition of ymotoneurons. On the basis of the considerations made probably many factors contribute t o the described drop of the discharge frequency of the tonic MUs. The second maximum of the discharge frequency of the tonic MUs is still more difficult t o explain. Long reflex arcs and even voluntary activity are possible for consideration. An important feature of tonic MUs is, that at a given initial loading and a given additional loading the pattern of discharge during repeated stretch and also in regard t o all units of this type shows relatively small deviations (Fig. 6). Each inter-impulse interval depends on the time between the moment of additional loading and the onset of the interval. The excitation and inhibition develop synchronously in all a-motoneurons innervating tonic MUs. On the contrary the phasic MUs show a greater dispersion of the interimpulse intervals, greater variety of the pattern of discharge upon repeated stretch and an independence of the discharge of separate units of this type. The discharge frequency of tonic MUs stands in correlation with the joint displacement. Of course the joint displacement depends both on tonic and

364 phasic MUs discharge, as well as on the recruitment order of different MUs, but the influence of the discharge of the low-threshold tonic units is evidently predominant.

CONCLUSIONS

(1)Considerable differences in the discharge pattern of tonic and phasic MUs have been found upon stretch reflex caused by additional loading. (2) The tonic MUs discharge in a common manner in accordance with a general principle which is the same for each one of them. Each given interval is dependent on the time between the beginning of the stretching and the beginning of the interval. The discharge pattern is expressed in a phase of rising frequency, a phase of falling frequency, and a phase of secondary rise in the frequency followed by a steady discharge. This sequence is valid for MUs which had been active before the stretch and upon the recruitment of a new tonic MU. (3) The discharges of the phasic MUs are independent of one another. The dispersion of the consecutive intervals grows sharply during the initial high-frequency phase after the stretch. This increased frequency takes the form of alternation of brief and long intervals. SUMMARY The study undertaken covers the discharge of motor units in m.biceps brachii upon stretch reflex caused by additional loading. Essential difference has been found in the discharge pattern of the tonic and phasic MU. The tonic MUs respond with an initial rise in the frequency followed by a sharp drop and by a second rise which passes into a steady staie. The length of the individual inter-impulse intervals depends only on the time which has elapsed from the moment of the additional loading to the initial impulse of the interval. Typical of the discharge of phasic MUs at a stretch reflex is the increase in the dispersion of the inter-impulse intervals. The changes in the frequency of the tonic MUs only is correlated with the displacement output.

REFERENCES Eccles, J.C., Eccles, R.M. and Lundberg, A. (1957a). The convergence of monosynaptic excitatory afferents on too many different species of alpha motoneurons. J. Physiol. (Lond.), 137: 22-50. Eccles, J.C., Eccles, R.M. and Lundberg, A. (1957b) Duration of afterhyperpolarization of motoneurons supplying fast and slow muscles. Nature (Lond.), 179: 866-868. Eccles, R.M. and Lundberg, A. (1959) Synaptic actions in motoneurones by afferents which may evoke the flexion reflex. Arch. ital. BioL, 9 7 : 199-221. Granit, R. (1950) Reflex self-regulation of the muicle contraction and autogenetic inhibition. J. Neurophysiol., 13: 351-372. Granit, R. (1955) Receptors and Sensory Perception, Yale Univ. Press, New Haven, Conn.

Granit, R. and Rutledge, L.T. ( 1 9 6 0 ) Surplus excitation in reflex action of motoneurons as measured by recurrent inhibition. J. Physiol. (Lond.), 154: 288-307. Granit, R., Henatsch, H.D. and Steg, G. (1956) Tonic and ventral horn cells differentiated by post-tetanic potentiation in cat extensors. A c t a physiol. scand., 37: 114-126. Granit, R., Pascoe, Y.E. and Steg, G. (1957a) The behavior of tonic a and y motoneurons during stimulation of recurrent collaterals. J. Physiol. (Lond.), 1 3 8 : 381-400. Granit, R., Phillips, C.G., Skoglund, S. and Steg, G. (195713) Differentiation of tonic from phasic alpha ventral horn cells by stretch, pinna and crossed extensor reflexes. J. Neurop hysiol., 2 0 : 470-48 1. Granit, R., Kernell, D. and Lamarre, Y. (1966) Synaptic stimulation superimposed on motoneurones firing in the secondary range to injected current. J. Physiol. (Lond.), 1 8 9 : 4 0 1-4 1 5. Gydikov, A. and Kosarov, D. ( 1 9 7 2 ) Studies on the activity of the alpha motoneurons in man by means of new EMG methods. In Neurophysiology Studied in Man, G. Somjen (Ed.), Excerpta Medica, Amsterdam, pp. 321-329. Gydikov, A. and Kosarov, D. (1974) Same characteristics of different motor units in human m.biceps brachii. Pfliigers Arch. ges. Physiol., 341 : 75-88. Hunt, C.C. (1952) The effect of stretch receptors from muscle on the discharge of motoneurons. J. Physiol. (Lond.), 117: 359-379. Kernell, D. (1965) The limits of firing frequency in cat lumbosacral motoneurones possessing different time course of afterhyperpolarization. A c t a physiol. scand., 65 : 87-100. Laporte, Y. et Bessou, P. (1959) Modification d’excitabilite de motoneurones homonymes provoquees par l’activation physiologique de fibres afferentes d’origine musculaire du groupe 11. J. Physiol. (Paris), 51: 897-908. Leksell, L. (1945) The action potential and excitatory effect of the small ventral root fibres t o skeletal muscle. Acta physiol. scand., 10, Suppl. 31 : 1-84. Lloyd, D.P.C. (1943) Conduction and synaptic transmission of reflex response to stretch in spinal motoneurons in cat. J. Neurophysiol., 6 : 317-326. Lloyd, D.P.C. (1946a) Facilitation and inhibition of spinal motoneurons. J. Neurophysiol., 9 : 421-438. Lloyd, D.P.C. (194613) Integrative pattern of excitation and inhibition in two neuron reflex arc. J. Neurophysiol., 9 : 439-444. Tsukahara, H. and Ohye, C. (1964) Polysynaptic activation of extensor motoneurons from group IA fibres in the cat spinal cord. Experientia (Basel), 20: 628-629.

DISCUSSION LAPORTE: You said that when you are moving the two recording electrodes, you found a position from which you did not record any muscle action potential. If I understood you correctly, you said that was near the endplate region. GYDIKOV: If we take one motor fiber, the endplate is here. At symmetrical points the monopolar recorded potentials are one and the same. By bipolar recording these were cancelled, by strictly symmetrical positions of the two electrodes. If the distance of one electrode from the endplate is greater, the action potential of positive and negative phases is greater. So, if the bipolar electrode positions are not symmetrical from the endplate, those potentials were subtracted. In some motor units the position of the endplate is apart from others. KERNELL: Is it generally possible with your technique t o record simultaneously and selectively from more than one motor unit of the same muscle? Can that be done during strong voluntary contractions? GYDIKOV: This is possible, but it is very difficult, and especially it is more possible in static conditions, isometric conditions, than during small changes of angles because this change of angle displaces electrodes from a selective position.

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Contraction Times of Reflexly Activated Motor Units and Excitability Cycle of the H-reflex FRITZ BUCHTHAL and HENNING SCHMALBRUCH Institute of Neurophysiology, University of Copenhagen, and the Laboratory o f Clinical Neurophysiology, Rigshospitalet, Copenhagen (Denmark)

I would like to report briefly two sets of observations from our laboratory relevant to the stretch reflex in man. The one is based on experiments I have made with Schmalbruch (Buchthal and Schmalbruch, 1970b), and concerns the type of muscle fibre activated by reflex contractions as compared with those activated by stimuli delivered to the efferent nerve. The other is based on observations made by Diamantopoulos and Zander Olsen (1967a,b) and concerns the excitability cycle of the H-reflex. In cat, Granit et al. (1956) and Henneman et al. (1965) have demonstrated that motoneurones activated by the stretch reflex have smaller axons than phasic motoneurones, and Henneman and Olson (1965) have shown that tonic motoneurones are the first ones to be activated; their axons conduct slower and their muscle fibres have slow contraction times. In man, Homma and Kano (1962) have shown that the reflexly activated twitch of the whole calf muscle has a longer contraction time than when activated by stimulation of the efferent nerve. In our experiments mechanical responses were recorded by inserting a needle in the Achilles tendon under local anaesthesia (Buchthal and Schmalbruch, 1970a). The needle was connected to a semiconductor strain gauge fixed in a stand above the tendon. Fig. 1 shows the set up for recording of contraction times in small bundles of the biceps muscle. It is in principle the same as the one used for the soleus muscle. The transducer measured the displacement of the needle as the tendon was stretched by an isometric twitch. Care was taken that deviations from pure isometric conditions were negligible, that displacement of the needle was within the range of linearity, that time-to-peak tension was reproduced undistorted, and that the intramuscular temperature was kept constant. A threshold stimulus applied in the end-plate region activated a small bundle of muscle fibres with reproducible contraction time (Fig. 2). If the stimulating electrode was moved to another site within the end-plate zone, as shown in a, or if the stimulus was slightly increased, as shown in b, other fibres were activated with shorter or longer contraction times. The smallest force recorded in this way was 0.5-1% of the maximal force produced by a twitch, in the soleus muscle corresponding t o the average force exerted by one motor unit. From contraction times of at least 20 different bundles of muscle fibres we

368 Transducer

, I . -

Fig. 1. Above: set-up for recording isometric twitches evoked in different small bundles by stimuli at various points in the end-plate region (dotted) in the brachial biceps muscle. The transducer was fixed in an adjustable stand above the tendon and the needle was inserted into the tendon. The action potentials were recorded between an 80 p m wire inserted through the muscle and a remote electrode or by a concentric electrode. Below: mechanoelectrical transducer. N, needle to be inserted into the tendon; SG, semiconductor strain gauge (magnified below) cemented on the rod (R); S, shaft. (From Buchthal and Schmalbruch, 1970a.)

have then obtained a spectrum of contraction times which is representative for a given muscle. The range of contraction times in different muscles was compared with the cross-sectional area occupied by muscle fibres of different type. Thus, as shown in Fig. 3, the brachial triceps muscle which contains only very few fibres that are rich in mitochondrial enzymes has faster contraction times than the soleus muscle. However, histochemical differences give only a gross picture of variations in the physiological properties of different fibres in a muscle. Thus, the soleus muscle, which histochemically seems nearly homogeneous in man, containing only fibres rich in mitochondrial enzymes, has a wide spectrum of contraction times, but short contraction times were absent. The question was then whether the H-reflex selectively activates muscle fibres with long contraction times.

369

a

1 rnV

50 rnsec

u 50rnsec

u 50rnsec

50 msec

Fig. 2. Twitches of fibre bundles with different contraction times evoked by near-threshold stimuli in the long head of the brachial biceps muscle (single sweep recordings 37OC). a : same stimulus strength applied at two points in the end-plate zone (subject H.J., female, 18 years old). b: stimuli of different strengths applied at the same point in the end-plate zone (subject P.N., male, 19 years old). c and d: twitches evoked by stimuli applied a t two points in the end-plate zone (subject O.C., male, 23 years old). Ordinates a and b: output of the strain gauge in mV, the peak force was 1 4 % of maximum twitch force. c and d : the force in g calculated to the insertion of the tendon at 90" flexion at the elbow (less than 1%of maximum twitch force). (From Buchthal and Schmalbruch, 1970a.)

U 0 I f , l , j , l , l j l l [ , I , 16 20 21 28 32 36 LO 11 18 52 56 60 61 68 72 76 80 8L 88 92 96 100

msec Fig. 3. Distribution of contraction times of small fibre bundles in m.triceps brachii and rn. soleus of normal subjects. The intramuscular temperature was 36-37OC. (From Buchthal et al., 1973.)

370 The presence of an H-reflex was ascertained by recording it simultaneously with the action potential of the soleus muscle. The action potential was used as point of reference for the onset of tension when the reflex twitch was contaminated by a preceding M-response. For this purpose the recording electrode on the belly of the soleus muscle was moved until the spike of the action potential of the M-response coincided with the onset of the twitch. The timeto-peak tension of the reflex twitch is then given by the time interval from the negative peak of the action potential preceding the reflex twitch t o the peak of the force of the compound response (H + M). When the rate of stimulation was low (0.5/sec) every stimulus evoked both a direct and a reflex twitch. When the rate of stimulation was increased only every fifth stimulus evoked a reflex response and the small M-response on which the reflex twitch was superimposed could be isolated. In this way contraction times of near-threshold reflex responses were compared with those of M-responses of the same amplitude, either obtained by stimulation of the efferent nerve or by stimuli applied within the end-plate zone. Fig. 4 shows to the left the reflex twitch superimposed on a small M-response. The M-response shown below was isolated by increasing the rate of stimulation. To the right is shown the shorter contraction time of an M-response evoked by a stimulus in the end-plate zone. The contraction time of reflex twitches of different amplitude is shown by the triangles of Fig. 5. It was about 100 msec, 30% longer than contraction times of M-responses either evoked by stimulation in the end-plate zone, shown by the open circles, or by weak stimuli t o the efferent nerve shown by the full circles. Stronger reflex responses could only be compared with twitches of equal amplitude evoked by stimulation in the end-plate zone because stimuli t o the nerve, strong enough to elicit twitches of the same amplitude as the reflex twitch, activated the gastrocnemius muscle as .well. The distribution of contraction times obtained in 8 normal subjects is shown in Fig. 6. The upper histogram gives the contraction times activated by reflex

1 mV

+

I

50msec

stirn. nerve

5017-1sec

stim. endplate

Fig. 4. Left: H + M-twitch response of the soleus muscle evoked by an H-reflex superimposed on a small M-response. The lower twitch is the M-response isolated by increasing the rate of stimuli. Ap, action potentials of the M- and of the H-responses. Right: Me, response evoked by a stimulus in the end-plate zone; SG, output by the strain gauge in mV. Subject I.M., male, 1 9 years old. The intramuscular temperature was 37OC. (From Buchthal and Schmalbruch, 1970b. )

371

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12

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68

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Fig. 6. Histogram of contraction times (t) of twitches in the human soleus muscle (3637OC). Above: evoked by H-reflexes. Below: evoked in small bundles activated by stimuli to the end-plate zone (white columns) and to the efferent nerve or the end-plate zone in the same subjects in whom the H-reflexes shown above were obtained (striped columns). n denotes the number of fibre bundles examined. (From Buchthal and Schmalbruch, 1970b.)

372 twitches of different amplitude in 2 subjects. They are confined t o the longer contraction times and are even longer than obtained in these muscles by direct activation of the efferent nerve or the end-plate region. The lower histogram shows contraction times of small bundles of 8 muscles, the striped columns representing responses to stimuli t o the efferent nerve or the end-plate zone of the 2 muscles from which H-reflexes were obtained (above); the white columns represent twitches evoked in 6 other subjects by stimuli t o the end-plate zone. In summary, only the slowest fibres within the soleus muscle contribute to the reflex twitch. Model experiments with paired stimuli ascertained that the longer contraction times were neither due t o the small additional stretch of the tendon by the preceding M-response nor to the shortening of the muscle. It is of interest that the motor units recruited initially during voluntary effort of the brachial biceps muscle were not among those with the longest contraction time, but rather had contraction times near the average of the wide spectrum of contraction times found in this muscle (Fig. 7). The second set of observations I would like t o report is on the excitability cycle of the action potential of the H-reflex in man. The H-reflex bypasses the

bic.br.

i

k

i

J

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30+ -

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20msec

Fig. 7 . Above: average of 200 twitches ( 3 - 6 / s e c ) in a single motor unit of the brachial biceps muscle during weak voluntary effort. Below: average of 64 identical fasciculations. Normal subject, intramuscular temperature 36-37OC. Note the different time base of the action potentials and the twitches. (From Buchthal and Schmalbruch, 1970a.)

373 muscle proprioceptors and makes it possible t o measure the exzitability of the motoneurone more directly than by the mechanically evoked stretch reflex, which in addition depends on the excitability of the muscle spindles. When the conditioning and test stimuli were both of a strength near the threshold for the H-reflex, excitability was depressed u p to 80 msec after a weak conditioning stimulus except for a period of early facilitation 5-8 msec after the first stimulus (Fig. 8). The test reflex increased transiently 100-300 msec after the conditioning stimulus, t o be depressed again for a period of 500-800 msec. The second return of excitability, 100-300 msec after the test stimulus, has been considered to be due t o the phasic response of primary afferents a t the onset of relaxation of muscle after the reflex twitch. The subsequent late depression was attributed t o tonic firing of secondary afferents from muscle spindles inhibitory t o motoneurones (Bianconi et al., 1964). The paired stimuli were given a t intervals of several seconds, such that the return to normal excitability could be evaluated. Not until 1 sec after the conditioning stimulus were unconditioned values again obtained, the long-lasting change being consistent with persisting alterations in discharge of primary spindle afferents after a single tendon tap or twitch (Granit et al., 1959). Nevertheless the long-lasting change in excitability is noticeable because the mechanical properties of the muscle have again reached resting conditions already 300 msec after the conditioning stimulus.

o---I

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100 200 Intervals (nisec 1

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Fig. 8 . Recovery of the H-reflex in normal subjects and in patients with spasticity. a : mean values of reflexes just above threshold in 1 3 normals and in 8 patients. b : mean values of maximal reflexes in 19 normal subjects and 10 patients. Abscissa: time intervals between stimuli of the pair, logarithmic scale. Ordinate: amplitude of the test reflexes as percentage of their conditioning reflexes. The vertical bars denote mean errors. (From Diamantopoulos and Zander Olsen, 1967a.)

374 When conditioning and test stimuli were of a strength t o elicit maximal Hreflexes, the early facilitation occurred less regularly, the H-reflex returned 40 msec after the conditioning stimulus and reached maximum at 200 msec after the conditioning stimulus. The second facilitation was more prominent and the subsequent depression less marked than with threshold reflexes. The course of excitability was the same whether or not there was a small M-response associated with the reflex response, indicating that antidromic activation of motoneurones was of short duration, consistent with the finding in cat that excitation was 80% of normal as early as 60 msec after antidromic activation. In patients with spasticity, both the early and the late facilitation were more prominent than in normal subjects (Fig. 8). Spinal shock in man causes an interesting dissociation between the electrically evoked H-reflex and the stretch reflex: 1-4 days after spinal shock, ankle jerks were absent but single stimuli t o the posterior tibia1 nerve in the popliteal fossa elicited normal H-reflexes (Weaver e t al., 1963; Diamantopoulos and Zander Olsen 196713) (Fig. 9). This indicates normal excitability of motoneurones associated with diminished fusimotor activity. On the other hand, both for threshold and for maximal H-reflexes, the recovery after a conditioning stimulus was severely retarded indicating a depressed excitability of interneurones associated with diminished fusimotor activity and normal excitability of the motoneurones. One t o 4 weeks after onset the tendon reflex had returned and excitability was normalized. Two to 3 months later excitability was increased as in spasticity.

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Fig. 9. Recovery of the maximal test H-reflex after a conditioning reflex. Nine patients 1-4 days, 1 0 patients 1-4 weeks and 5 patients 2-3 months after the onset of spinal shock. Abscissa: time interval between conditioning and .test stimuli, logarithmic scale. Ordinate : amplitude of the test H-reflexes as percentage of their conditioning reflexes. The vertical bars denote the mean errors. Interval A: phase of early facilitation in normals. Interval B: phase of the second return of the H-reflex in normals. Interval C: phase of late depression of the test reflex. (From Diamantopoulos and Zander Olsen, 196713.)

375 REFERENCES Bianconi, R., Granit, R. and Reis, D.J. (1964) The effects of extensor muscle spindles and tendon organs on homonymous motoneurones in relation t o gamma-bias and curarization. A c t a physiol. scand., 61: 331-347. Buchthal, F. and Schmalbruch, H. (1970a) Contraction times and fibre types in intact human muscle. A c t a physiol. scand., 79: 435-452, Buchthal, F. and Schmalbruch, H. (1970b) Contraction times of twitches evoked by H-reflexes. A c l a physiol. scand., 80: 378-382. Buchthal, F., Dahl, K. and Rosenfalck, P. (1973) Rise time of the spike potential in fast and slowly contracting muscle of man. A c t a physiol. scand., 87 : 261-269. Diamantopoulos, E. and Zander Olsen, P. (1967a) Excitability of spinal motor neurones in normal subjects and patients with spasticity, Parkinsonian rigidity and cerebellar hypotonia. J. Neurol. Neurosurg. Psychiat., 30: 325-331. Diamantopoulos, E. and Zander Olsen, P. (196713) Excitability of motor neurones in spinal shock in man. J. Neurol. Neurosurg. Psychiat., 30: 427-431. Granit, R., Homma, S. and Matthews, P.B.C. (1959) Prolonged changes in the discharge of mammalian muscle spindles following tendon taps of muscle twitches. A c t a physiol. scand., 46: 185-193. Henneman, E. and Olson, C.H. (1965) Relations between structure and function in the design of skeletal muscle. J. Neurophysiol., 28: 581-598. Henneman, E., Somjen, G. and Carpenter, D.O. (1965) Functional significance of cell size in spinal motoneurones. J. Neurophysiol., 28: 560-580. Homma, S. and Kano, M. (1962) Electrical properties of the tonic reflex arc in the human proprioceptive reflex. In S y m p o s i u m o n Muscle R e c e p f o r s , D. Barker (Ed.), Hong Kong University Press, Hong Kong, pp. 167-174. Weaver, R.A., Landau W.M. and Higgins, J.F. (1963) Fusimotor function. Part 2. Evidence of fusimotor depression in human spinal shock. Arch. Neurol. (Chic.), 9: 127-132.

DISCUSSION ELDRED: Did I note that the lateral and medial gastrocnemius edge had a high percentage of oxidative fibres, almost as many as the soleus? Does that mean that the gastrocnemius in human has a slow contraction comparable to that of soleus? BUCHTHAL: Yes. We have, since this table was produced some years ago, confirmed that, in man the gastrocnemius muscle contains more slow fibres than in cat; in man 80-90% of the muscle fibres are rich in mitochondria1 enzymes. KERNELL: I am just continuing on the same question. Do you get such a similarity between the human gastrocnemius and soleus with respect to ATPase as well? BUCHTHAL: Yes, we do. We always do ATPase stain in addition. You really don’t know what you are doing with ATPase stain, but you get the same situation. ELDRED: If the histochemical picture is similar in gastrocnemius and soleus and the contraction picture is similar, maybe this is a favourable opportunity to throw some light on why, certainly in cats anyway, you have such a great difference in size of the motor axons between gastrocnemius and soleus. What is known about the difference in fibre caliber spectra of those muscles in human beings? And in size of motor units? BUCHTHAL: I hope I have made myself clear. There is a difference in the gastrocnemius between human and cats, because in the cat it has been shown very convincingly that it does contain fast large nerve fibres to this extent. In man I don’t know of any measurement of difference in calibre of nerve fibres between gastrocnemius and soleus.

376 HULTBORN: May I just postulate that the size of H-reflex may be very different in different subjects. How does that interfere? If you have a very large H-reflex, can you also see rather quick contraction times? BUCHTHAL: We found a slight decrease in contraction time with increasing force ( P < 0.05, Fig. 5).

Identification of Fast and Slow Firing Types of Motoneurons in the Same Pool E. HENNEMAN and D.HARRIS Department of Physiology, Harvard Medical School, Boston, Mass. 021 15 (U.S.A.)

INTRODUCTION Many functionally significant properties of motor units depend on the size of their motoneurons (Buller et al., 1960; Henneman and Olson, 1965; Henneman et al., 1965a, b; McPhedran et al., 1965; Somjen et al., 1965; Wuerker et al., 1965; Kernell, 1966; Davis, 1971; Mendell and Henneman, 1971; MilnerBrown et al., 1973; Clamann et al., 1974a, b; Henneman et al., 1974; Freund et al., 1975). This body of work might suggest that a pool of motoneurons is a homogeneous population of cells differing only in size. This paper presents evidence that a pool is not homogeneous, but, rather, consists of more than one species of motoneuron, with properties that are not entirely dependent on size. METHODS The demonstration of size-independent qualities in motoneurons depends upon the use of a new technique for measuring the size of motoneurons with a quantitative precision of 1-2% under ideal experimental conditions. In each experiment we first measured the total monosynaptic output of the plantaris pool in a decerebrated preparation. Any subsequent monosynaptic reflex could then be described as a percentage of this maximum discharge. When a single motoneuron from the same pool was recorded simultaneously with the whole pool’s output, it was found that the individual motoneuron never discharged if the per cent output was below a certain critical level and always discharged when it was above this level (the critical firing level or CFL). This technique was detailed in a series of three papers in 1974 (Clamann et al., 1974a, b; Henneman et al., 1974). Clamann and Henneman in a paper now in press (1976) show that the CFL is linearly related to the conduction velocity and axon diameter of the motoneuron and, therefore, is a good measure of motoneuron size (Ram6n y Cajal, 1909). With the CFL technique we could isolate a large number of units and measure their CFLs. Whenever two units were found that differed by less than 2% in CFL, we placed them on separate pairs of recording electrodes and simultaneously compared their monosynaptic and repetitive thresholds t o stimula-

378 tion of the plantaris nerve as an additional check on their close similarity in size. We then stimulated the plantaris nerve at 5OO/sec with shocks adjusted to elicit maximal firing rates. The same parameters of stimulation almost always elicited maximal firing rates from both units. RESULTS Initially, plantaris units within any portion of the 0--100% range of CFLs were compared if they appeared to be nearly identical in size. Two contrasting results were obtained, more or less at random. In some cases the firing rates (FRs) of the two units did not differ by more than 1-2 pulses/sec (pps), averaged over a full second. In these pairs there was no evidence of a difference in rate that was independent of size. In other cases, however, a striking difference in the rates of the two units was apparent, one of them discharging at more than twice the rate of the other. This difference in rate was very significant in magnitude and clearly independent of size. Observations on a number of pairs indicated that each pair either differed very little in rate or differed by a factor of 2-3 : 1. This impression was enhanced by several experiments in which we were able to isolate 4 pairs of units of nearly equal size. In these instances 1-3 of the units fired at almost the same rates, whereas the other 1-3 units fired at much higher rates. These results, though small in number, suggested that the units under comparison were being sampled randomly from different populations of motoneurons that discharged at widely different rates. Unfortunately, it was not possible t o isolate more than 4 pairs of plantaris units of nearly equal size in a single experiment, so that we could not plot a histogram of their rates and determine directly whether there were two or more distinct populations of motoneurons. Data from different experiments could not be pooled because decerebrate preparations differ widely in FR. In order to isolate a sufficient number of units for statistical analysis in a single experiment, the criteria for similarity in size were relaxed. In a single experiment 10-15 units with CFLs all in the 0-896 range could be isolated in a single experiment. These units differed somewhat in size, but were obviously still quite similar. The procedure which was ultimately adopted was the well known technique of proof by contradiction. (1)It was first assumed that the maximal FRs in a single experiment could best be described statistically by a single, normal distribution. (2) Logical steps were then taken which led t o a clear contradiction of the original assumptions. (3) This contradiction forced the alternative conclusion, that the data came from more than one distribution. In each experiment the data (maximal FRs of individual units) were plotted. Assuming that they were sampled randomly from a single, normal distribution, a calculation was carried out t o plot this actual distribution, hereafter called the “prediction density”. Finally a plot was constructed illustrating the difference between the actual number of units with a particular FR and the number expected from the prediction density. This difference was called the “prediction error”.

379 In all of the 1 2 experiments of this type the plot of prediction error exhibited two large positive peaks about 10 pps apart, indicating that more units having these particular rates were isolated than expected from a normal distribution. In a small sample two peaks such as these could easily occur by chance. The occurrence of the same pattern in each of the 1 2 experiments, with separation of about 1 0 pps, eliminated the possibility that chance was responsible. It should be emphasized that this contradiction does not depend on the assumption of a single, normal distribution. Consistent contradictions arose when a variety of single, modal distributions were assumed, including exponential, chi-squared, several gamma distributions and a uniform distribution. When different distributions were assumed, of course, different patterns of prediction error were found. Accordingly, there seemed t o be little doubt that there must be more than one population of units in the 0-876 range of CFLs in plantaris units. Pooling of all the data indicated that there was a clear bimodal distribution of units whose FRs differed significantly. There was a small range of FRs for each pDpulation due to the differences in size of the motoneurons in the 0-876 range of the CFLs. The two species of cells appeared t o be co-extensive, though not necessarily equally dense in the 0--8% range.

DISCUSSION A study of single motor units in the medial gastrocnemius muscle of the cat by Wuerker et al. (1965) indicated that there were two populations of small units in that muscle, one contracting slowly, the other more rapidly. There is, thus, a close analogy between the earlier studies of motor units whose size, speed of contraction, and axonal diameter were used as the bases for their distinction and the present study in which FRs of the motoneurons alone were distinguished. Finally, a growing number of clinical studies show selective involvement of specific types of muscle fibers. Examples are central core disease, which singles out slow, red fibers (Engel, 1965) and untreated collagen disease, which attacks fast, pale fibers selectively (Engel and McFarlin, 1966). Engel and his colleagues have found many other examples (Fenichel and Engel, 1963; Engel, 1965) of selective destruction of one type of muscle fiber, with sparing of the other types. There is some evidence that certain diseases of muscle are, in fact, secondary to involvement of motoneurons (McComas et al., 1973). Hence, the practical implications of these studies for clinical neurology are obvious. SUMMARY Simultaneous comparisons of the maximal firing rates of single plantaris motoneurons of approximately the same size or of motoneurons, whose critical firing levels fell between 0 and 876, indicate that the pool of plantaris motoneurons is not a homogeneous population of cells, but consists of more than one species of motoneuron with properties that are not entirely dependent on size.

380 REFERENCES Buller, A.J., Eccles, J.C. and Eccles, R.M. (1960) Interactions between motoneurones and muscles in respect of the characteristic speeds of their responses. J. Physiol. (Lond.), 150: 417-439. Clamann, H.P. and Henneman, E. (1976) Electrical measurement of axon diameter and its use in relating motoneuron size to critical firing level. J. Neurophysiol., in press. Clamann, H.P., Gillies, J.D., Skinner, R.D. and Henneman, E. (1974a) Quantitative measures of output of a motoneuron pool during monosynaptic reflexes. J. Neurophysiol., 37 : 1328-1 337. Clamann, H.P., Gillies, J.D. and Henneman, E. (1974b) Effects of inhibitory inputs on critical firing level and rank order of motoneurons. J. Neurophysiol., 37: 1350-1360. Davis, W.J. (1971) Functional significance of motoneuron size and soma position in swimmeret system of the lobster. J. Neurophysiol., 34: 274-288. Engel, W.K. (1965) Histochemistry of neuromuscular disease-significance of muscle fiber types. In Proceedings of the VIII International Congress of Neurology, Vienna, Excerpta Medica, Amsterdam, pp. 67-101. Engel, W.K. and McFarlin, D.E. (1966) Skeletal muscle pathology in myasthenia gravis histochemical findings. Ann. N . Y . Acad. Sci., 135: 68-75. Fenichel, G.M. and Engel, W.K. (1963) Histochemistry of muscle in infantile spinal muscular atrophy. Neurology (Minneap.), 13: 1059-1066. Freund, H.J., Budingen, H.J. and Dietz, V. (1975) Activity of single motor units from human forearm muscles during voluntary isometric contractions. J. Neurophysiol., 38 : 9 3 3-94 6. Henneman, E. and Olson, C.B. (1965) Relations between structure and function in the design of skeletal muscles. J. Neurophysiol., 28: 581-598. 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. Henneman, E., Clamann, H.P., Gillies, J.D. and Skinner, R.D. (1974) Rank order of motoneurons within a pool: law of combination. J. Neurophysiol., 37: 1338-1349. Kernell, D. (1966) Input resistance, electrical excitability, and size of ventral horn cells in cat spinal cord. Science, 152: 1637-1639. , McComas, A.J., Sica, R.E.P. and Upton, A.R.M. (1973) Comparisons of motor unit populations in disease. In Programme Abstracts of the 9 8 t h Annual Meeting of the American Neurological Association and the Eighth Canadian Congress of Neurological Sciences, Montreal: 30. McPhedran, A.M., Wuerker, R.B. and Henneman, E. (1965) Properties of motor units in a homogeneous red muscle (soleus) of the cat. J. Neurophysiol., 28: 71-84. 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. Milner-Brown, H.S., Stein, R.B. and Yemm, R. (1973) The orderly recruitment of human motor units during voluntary isometric contractions. J. Physiol. (Lond.), 230: 359370. Ram6n y Cajal, S. (1909) Histologie du Syste'me Nerveux d e 1'Homme e t des Verte'brb, Vol. 1 , Maloine, Paris. Somjen, G., Carpenter, D.O. and Henneman, E. (1965) Responses of motoneurons of different sizes t o graded stimulation of supraspinal centers of the brain. J. Neurophysiol., 28: 958-965. Wuerker, R.B., McPhedran, A.M. and Henneman, E. (1965) Properties of motor units in a heterogeneous pale muscle (m. gastrocneqius) of the cat. J. Neurophysiol., 28: 8599.

381 DISCUSSION BUCHTHAL: Can I get you t o speculate about the function with respect t o contraction time? Do you assume that these slower firing units are associated with a slower contraction time? HENNEMAN: I am n o t sure whether I can explain that or not, b u t 1 0 years ago when we studied medial gastrocnemius in the cat, we found that there were t w o kinds of small units. There were fast contracting a n d slow contracting types, and they were both clearly at the bottom end of the size range, both very small cells. So I think there is an analogy between what we saw in examining the whole motor unit in the case of the medial gastrocnemius and in looking just a t the motoneuron of plantaris. HULTBORN: Does that correspond to the slide you showed with weak voluntary contraction? HENNEMAN: Yes. KERNELL: How certain is it that all t h e units you studied belonged to plantaris? HENNEMAN: We had that in mind when we designed the experiment. It was one of the reasons we did not use the gastrocnemius. We chose the plantaris, because all of its efferent fibers as a rule come o u t in L 7 ventral root. So we thought that there was a very small chance that we could pick u p a unit of another muscle and get it confused with what we are dealing with. KERNELL: What are the reflex connections between plantaris and soleus? HENNEMAN: We found nothing in the L7 VR of cats t h a t could be called synergists of plantaris. BARKER: Do you have any general comments t o offer o n how gamma fusimotor neurons and perhaps beta fit into your scheme? HENNEMAN: Gamma motoneurons are discharged a t lower thresholds under many experimental conditions, presumably because they are very small and therefore very excitable, I presume that they are in the pool b u t are n o t fired monosynaptically, b u t I don’t know if anyone is really assured of that point. Do y o u have anything t o add? BARKER: No. GYDIKOV: We have recently found in human opponens pollicis, a method t o stimulate single motor units, and we improved this method in order t o be sure that there is only a single unit responding and we found three types of units. T w o of them have short contraction time and one has a long contraction time. We were surprised that when we also estimated the sizes of the motor unit, we found both small motor units which have short contraction times and long contraction times, and I was very pleased t o hear that you also had the same results that correlate to motor firing rate. HENNEMAN: Thank you. There are a number of points in which we seem to be in close agreement. HAGBARTH: I want t o ask you if the frequency of the stimulus possibly had any relation t o the frequency of motor unit. Firing frequency of motor units is anyway subharmonic t o the stimulus.

HENNEMAN: The frequency of the stimuli did have some relation t o the firing frequency of the motor unit, but not as a subharmonic. Decerebrate preparations in which firing rates were fastest, were easiest to work with and most easy t o separate two types of units. In experiments with weakly decerebrate animals, firing rates were only slighthly different, b u t beyond that I don’t have any particular comment. KERNELL: When you studied two units simultaneously, did you then get the maximal frequency of both units with the same stimulation? HENNEMAN: Yes, in general we did. We have tried quite a few tests of this sort and we never, in any single pair, found that b y changing stimulation parameters we could make t h e faster o n e dower or the slower one faster. LAPORTE: In most muscles we know that there are three types of motor units. When we turn to the motoneuron side, we are confronted here with two types of behavior. There is something missing. Can you say what is missing? HENNEMAN: I think what is missing are the units between 8 and 100%. In other words, we are only looking a t the bottom end of the size scale and we are doing that deliberately so that the size differences would play a very small role. Size plays some role but a very small one.

SESSION VIII

SIGNIFICANCE OF SLOW AND FAST MUSCLES IN THE STRETCH REFLEX

Part I1 Chairman: J.C. Houk (Baltimore, Md.)

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Blood Flow in Red and White Muscle: Relationship t o Metabolism Development and Behavior DONALD J. REIS and G.F. WOOTEN Laboratory of Neurobiology, Department of Neurology, Cornell University Medical College, New York, N . Y . 10021 (U.S.A.)

INTRODUCTION It has been long recognized that red and white skeletal muscle * differ in their physiological, metabolic, and histochemical properties (Denny-Brown, 1929; Cooper and Eccles, 1930; Buller et al., 1960a; Ogata, 1960; Domenkos and Latzkovits, 1961; George and Talesara, 1961; Beatty et al., 1963; Dawson and Romanul, 1964; Ogata and Mori, 1964; Romanul, 1964, 1965; Buller and Lewis, 1965; Henneman and Olson, 1965; McPhedran et al., 1965; Wuerker e t al., 1965; Kugelberg, 1973; Burke and Tsairis, 1974; Hilton, 1974). Red muscles, with slower contractile properties and lower fusion frequencies and velocities of shortening, depend upon aerobic oxidative metabolism for the energy required for their tonic activity, participation in posture and in tonic stretch responses. White muscles, in contrast, with rapid contractile properties, possess a relatively low oxidative capacity and depend upon glygolytic metabolism for the energy required for rapid bursts of contraction in phasic movements. That red and white skeletal muscle might differ in blood flow requirements has long been suspected, particularly in view of histological evidence that red muscles possess a richer capillary density than white (Romanul, 1965,1968). However, it was not until 1967 (Reis et al., 1967) that we established that the nutrient blood flow of red muscles in awake and unrestrained animals is considerably greater than that of white, a finding confirmed independently by others in anesthetized animals (Folkow and Halicka, 1968; Hilton and Vrobova, 1968). Over the next several years, in a series of investigations, we sought t o establish the quantitative relationship between blood flow, oxidative capacity, capillary density and twitch characteristics of different muscles (Reis and Wooten, 1970; Wooten and Reis, 1972a,b) t o determine whether or not differences in blood flow in red and white muscle existed at birth or developed postnatally in parallel with the differentiation of histochemical and contractile properties (Wooten

*

Histochemically, few red or white muscles are truly homogenous (i.e., consist of only red or white muscle fibers); rather they contain varying proportions of fibers capable of aerobic or anaerobic metabolism. In most limb muscles of cat and rodent, however, one group of fibers predominates. Thus, the whole muscle may be broadly characterized biochemically or functionally as red or white.

386 and Reis, 1972b), and t o establish if blood flow in red and white muscle was independently regulated by the brain (Reis et al., 1969; Sheridan and Reis, 1972). In this paper we shall review these studies all of which have been published separately elsewhere. METHODS Our studies were performed in cats, rats and rabbits. The methods have been extensively detailed in our publications (see Reis e t al., 1969; Reis and Wooten, 1970; Wooten and Reis, 1972a,b) and will only be summarized. In our investigations we sought t o utilize a technique for measuring nutrient muscle blood flow (i.e., non-shunt) which could determine flow in very small muscles or portions of muscles and was suitable for studying muscle blood flow in chronically prepared, unrestrained and behaving animals. The method which we adapted was the isotope dilution technique developed by Sapirstein (1958) using 86Rbas indicator. The principle of this method is based on the fact that Rb, like K, rapidly distributes itself in the intracellular compartment of muscle and remains at a constant concentration for up t o 2 min. Since, in most organs, most of the injected 86Rbis extracted in one circulation, the radioactivity of any organ or part of an organ is proportional t o the percentage of the cardiac output reaching that tissue. The ratio of organ activity t o injected activity is termed the fractional flow (FF) and is calculated: organ activity (counts/min/g) X 100 FF = total injected activity (counts/min/mg) ' If the cardiac output is known, it is possible to estimate absolute blood flow by the calculation: Absolute flow (ml/min/100 g) = F F X cardiac output X100. The use of 86Rb for the measurement of nutrient flow t o muscle has been validated in anesthetized and unanesthetized animals (Reis e t al., 1967). The values for nutrient flow in red and white muscle obtained by this technique are entirely comparable with those obtained by other methods including "Kr washout (Reis et al., 1967), or by measurement of venous effluent (Folkow and Halicka, 1968; Hilton and Vrobova, 1968). When flow measurements are made in several muscles simultaneously in the same animal, the cardiac output can be considered a constant. Thus, comparison of fractional flow values alone can serve t o measure differences in blood flow distribution to various muscles in the same animal when cardiac outputs are difficult t o obtain. In the usual experiment, in a preliminary operation under anesthesia, a polyvinyl cannula was threaded down the right external jugular vein until the tip was estimated t o lie right above the right atrium. The cannula was fixed in deep tissue, brought up posteriorly through a stab wound in the neck, and fixed by a collar. The cannula was filled with heparinized saline and flushed periodically

387 to avoid clotting. In animals in whom the defense reaction was elicited, electrodes were implanted in the hypothalamus during this operation. In animals in whom changes during a blood flow distribution were studied during sleep, appropriate electrodes for recording EEG, extraocular movements, and neck muscle EMG were implanted. After recovery from surgery, the terminal experiment was performed. For studies in quiet behavior, the animals were petted and, while calm, approximately 1-3 pCi of 86RbC1were injected. One minute later the animal was killed instantaneously by injection of saturated KC1 into the heart through the jugular cannula. Similarly, when blood flow distribution was measured during excited behavior or sleep, the behavior was established, 86Rbinjected and then the animal rapidly killed. Following sacrifice, tissues were dissected, weighed, and counted for radioactivity and appropriate calculations of FF made. Absolute blood flow was estimated from measurements of cardiac output from animals run in parallel or, when appropriate, from values published by others. As an index of the relative oxidative metabolism in muscles, we measured the myoglobin content (Lawrie, 1953). The relative capillary density of muscles was assessed by a biochemical technique in which the relative activity of the enzyme alkaline phosphatase was determined. The rationale of this technique is that, within skeletal muscle, alkaline phosphatase can be demonstrated by histochemical techniques to be contained only within capillaries (Romanul, 1965). Speed of contraction was measured in single twitches elicited by electrical stimulation either as time t o peak and/or the time of peak to half relaxation (Buller e t al., 1960a,b) often by use of the technique of Bridgeman and Eldred (1965) in which contraction speed is obtained by measurement of intramuscular pressure.

RESULTS AND DISCUSSION

Blood flow in red and white skeletal muscles in mature animals In cat and rabbit (Reis and Wooten, 1970; Wooten and Reis, 1972a,b) fractional flow (FF) and hence, absolute blood flow, measured in up to 24 selected muscles of the limb and trunk, falls onto a continuum with, in cat, 7- 10-fold differences between the highest and lowest values. When grouped according to color, the flow to red muscles is always substantially greater than that t o white muscle, usually 3-4-fold, a feature also found in rat (Table I). Red limb muscles always have a greater blood flow than white limb muscles regardless of whether they are in hind- or forelimb, proximal or distal, or in portions of a single muscle. Muscles of an intermediate color, usually localized to the trunk, have blood flow values falling somewhere in between. Blood flow, for comparison, was also measured in extraocular muscles and in myocardium. As noted in Fig. 1, in cat, blood flow to red limb muscles was only approximately 60% that of the extraocular muscles in the cat which in turn was only at 40% that of the myocardium. In other words, myocardial blood flow is almost 15 times as great as that of blood flow t o a white limb muscle.

TABLE I BLOOD FLOW TO SELECTED RED AND WHITE MUSCLES IN CAT, RABBIT AND RAT

Blood flow (rnllrnin/lOO g)

Muscles

Cat

Red limb Triceps (short, medial head) Soleus Crureus Grouped red

36.3 t 34.9 f 29.6 t 27.88 i

White limb Gastrocnemious (lat) Triceps (long, internal head) Tibialis anterior Grouped white

1 0 . 9 r 1.4 * 11.6 i 2.1 * 7.6 t 1 . 0 * 9.06 i 0.57 **

4.1 * 1.4 * 3.5 * 1.8 **

i

S.E.M.

Rabbit

***

Rat

56.12 i 42.9 i 43.9 f 47.3

3.9 2.4 3.9 2.0

67.7 -

19.5 18.1 16.1 11.2

2.4 3.9 2.4 1.5

13.3 i 1.3 -

i i i i

f

5.2

* From Reis and Wooten, 1970.

** From Reis et al., 1969. *** From Wooten and Reis, 1972a,b.

t From Snyder, Doba and Reis (unpublished).

* I

Left ventiicle

Extraocular Diaphragm muscle

Soleus Gastrocnemius

Fig. 1. Comparison of blood flow between myocardium, extraocular muscle, diaphragm, and representative red (soleus) and white (gastrocnemius) skeletal muscle in awake cat. (From Wooten and Reis, 1972a.)

389 The relationship between nutrient blood flow and oxidative metabolism in skeletal muscles In order t o quantitatively determine the relationship between nutrient blood flow and the oxidative metabolic requirements of any muscle, we sought to establish the relationship between the myoglobin content of the muscle and its blood flow (Reis and Wooten, 1970; Wooten and Reis, 1972a,b). Myoglobin served as a convenient biochemical indicator of oxidative metabolism since the myoglobin content in any muscle is directly related to the capacity of the muscle to synthesize energy-rich phosphate bonds aerobically (Lawrie, 1953). (Indeed, it is the myoglobin which gives muscle its redness.) In both cat and rabbit the concentration of myoglobin, in up to 19 muscles, varied over a 3-4-fold range being lowest in white and highest in red muscles. However, when muscles were grouped according to color, significant differences in the myoglobin concentration were found amongst muscles of red ;_id white coloration. As demonstrated in Fig. 2 the relationship between blood flow (as indicated by the FF) and the myoglobin concentration in individual skeletal muscles of limb and trunk was direct and linear. The only exceptions were t o be found in extraocular muscles and myocardium: the myoglobin values in these were less than would be predicted by the high blood flow. When left and right ventricular and extraocular muscles were excluded from consideration, the regression analysis of the relationship yielded a curve with an extremely high correlation coefficient which was highly significant.

y 0 756

+

0 4 3lx

u 0 85, p 001

I

I

040 Fractional blood flow

020

I

060

I

080 I% of cardiac output lOOgm muscle1

lbO 166 226

Fig. 2. Relationship between fractional blood flow and myoglobin concentration in different muscles of quiet, alert cat. In this and subsequent illustrations the slope and intercept of curve calculated by regression analysis by method of least squares and the correlation coefficient ( r ) and its P value are indicated. Abbreviations: EOM, extraocular muscle; LV, left ventricle; RV, right ventricle. (From Reis and Wooten, 1970.)

390 The relationship of blood flow to capillary density and contractile properties As with myoglobin content there was a direct and linear correlation of high significance between the blood flow t o any given muscle, (other than extraocular muscle and myocardium), the capillary density of that muscle and the twitch characteristics (Reis and Wooten, 1970) (Fig. 3 ) . Our study therefore was able to demonstrate quantitatively what had been surmised in the past, namely that there was a direct relationship between frac-

8

4

,

.LV

RV

Eon1

+ 1320.51~ u = 0 . 7 7 pCOO1

y-24.81

Fractional blood flow I%of cardiac output f lOOgm muscle1

345

100 --

z -E

80 -

-

60-

5

time to peak y 18 6 t 1747Jx u 0 85 p < 001

A time of peak to half decay y -1 82 + ( 8 0 1 ) ~

u 0 87 p -= 001

-

' Oii

I 020

I

I

MO 060 080 Fractional blood flow 1% of cardiac output / lOOgm muscle1

Fig. 3. Upper graph: relationship between fractional blood flow and capillary density as indirectly assessed by alkaline phosphatase activity as indicated in Methods. Lower graph: relationship between fractional blood flow and speed of contraction in different muscles. Fractional flow measured in cat in quiet behavior. (From Reis and Wooten, 1970.)

39 1 Fractional blood flow

IMyoglobtn Imgigrn) 0 Alkaline phosphatase p

(sigma unttsigm protein1 Twitch time (m secl

Fig. 4. Relationship between fractional blood flow, myoglobin concentration, capillary density (alkaline phosphatase) and twitch time expressed as % of maximal values, in muscles of different color in cat. Muscles from triceps through crureus are red limb and from supraspinatous to semimembranosus white limb muscles. Intercostal and sacrospinalis are trunk muscles of intermediate color. (Data from Reis and Wooten, 1970.)

tional blood flow, capillarity, myoglobin concentration, and the duration of contraction of skeletal muscles. The findings demonstrated that some of the relationships were so linear and closely correlated that by knowing the value for one variable, it would be possible to predict the others. The interrelationships of all of these variables are illustrated in Fig. 4. Developmental changes in blood flow in red and white muscle

At birth mammalian skeletal muscles are histochemically and mechanically homogenous having the characteristics of red muscle, including oxidative metabolic patterns and slow contraction times. Histochemical and physiological differentiation into red and white muscles occurs within the first week in early development with histochemical and physiological maturity achieved in rat and cat during the fourth t o sixth week of life and somewhat earlier in the rabbit and occurring in a rostral-caudal direction (Buller et al., 1960a,b; Close, 1964; Nystrom, 1966, 1968; Dubowitz, 1967; Karpati and Engel, 1967; Friedman et al., 1968; Hajek et al., 1969; Hudlicka et al., 1973). In view of the difference between red and white flow in the adult we sought t o establish if the blood flow differences between red and white muscles of adults were inherent and therefore established at birth, or developed postnatally. Moreover, if postnatal, whether they developed in advance of, or behind the changes in metabolism and twitch characteristics. We therefore analyzed the development of blood flow to different muscles in developing rabbits (Wooten and Reis, 1972b). The rabbit was selected on the

392

.Triceps. Short. medial head I t S E l o Triceps, long, internal head 1 i S E l

0.6 -

Soleur I f SEI *Gastrocnemius I t S E l

o

10-

0.5

t

08-

E'

5

-

0.4 -

06-

0.3 -

3

-

L 04-

02-

0.2

-

0.1

-

b

+ ----Q

0

0

'

; o "

" M

20

" 00

'

J Adult

@I)

'

,b'

nr

nr

A .~ m ldavsl

ns

ns

p< t2

p< OI

p< 02

p< t2

p< 01

MI

'

Age ldaysl

ns

MI

p
' p< 01

'

A0 p r G5

F

'

-I

Adult

p< 01

Fig. 5. Left panel: time course during development of change of fractional flow in a red (triceps, short, medial head) and white (triceps, long, internal head) heads of forelimb muscle of unanesthetized rabbit. Right panel: same for paired red (soleus) and white (gastrocnemius) hindlimb muscles. Note that no difference in fractional flow between red and white pairs is present at birth but occur postnatally with forelimb preceding hindlimb. Also note that fractional flow decreases to all muscles in development. (From Wooten and Reis, 1972b.)

basis of size and also the fact that the postnatal development occurred earlier than in any other mammalian species. During the first 6 weeks of life there was a progressive diminution of fractional blood flow to all skeletal muscles (Fig. 5). However, when pairs of closely opposed muscles, one destined to become red and the other white, were compared at birth, no difference in fractional flow was observed. Differences appeared many days postnatally. Moreover, the differentiation occurred in a rostrocaudal direction. For example, as illustrated in Fig. 5, no significant differences in the FF are observed between the short medial head (red) and long internal head (white) of the triceps of the forelimb up to 12 days of age. Between 12-21 days, however, a significant difference in FF appears which is maintained throughout the remainder of development until adult values are approximated by the 42nd day of 1if.e. In hindlimb the differences in flow t o red and white hindlimb muscles also appeared after several weeks. In hindlimb muscles, however, the differentiation occurred about a week later than in forelimb, i.e., during the period from 21 to 28 days. Thus, at birth there is no difference in blood flow between red and white skeletal muscles in rabbit, as in chicken and cat (Hajek et al., 1969; Hudlicka, 1969; Hudlicka et al., 1973). The differences in flow between muscles charac-

393 teristic of the adult evolves, like the physiological and histochemical differences, during the first few weeks of life, and proceeds rostrocaudally. The postnatal differentiation of the histochemical and biochemical characteristics of muscle predominantly results from disproportionate changes in the properties of white muscle relative t o the neonatal pattern (Nystrom, 1968). Similarly, most of the changes which result in the differences in blood flow between adult muscle types also appear t o occur in white muscle. Fractional flow of white muscle gradually diminishes relative to the body growth in contrast to the fractional flow of red muscle which increases in proportion to body growth. The mechanisms accounting for the reduction in flow t o white muscle remain obscure. Three possible mechanisms would appear possible: (a) There could be a greater net increase in fiber size of white muscle relative t o red muscle in the presence of a capillary bed of constant size. The disproportionate growth would result in fewer capillaries surrounding white fibers and would result in less blood flow per unit weight in white than red muscle. This mechanism seems unlikely since the average fiber size in red is greater than in white muscle throughout development (Nystrom, 1968), and recent evidence suggesting that the capillary density around individual muscle fibers does not differ between red and white muscle (Plyley and Groom, 1975); (b) The greater capillary density in red muscle (Romanul, 1965; Reis and Wooten, 1970) could result from a decrease in capillary proliferation in white relative t o red muscle during growth and development. Failure of capillary growth would occur concomitantly with the metabolic differentiation of fibers into oxidative and glycolytic types so that the density of the capillary network around every fiber might be matched t o the oxidative metabolism of that fiber. That the capillary network can change with biochemical evolution in muscle has been demonstrated (Romanul and Van der Meulen, 1967); (c) The development of greater vasoconstrictor tone in white muscle might account, at least in part, for the observed flow differences. In the adult cat, a greater range of vasoconstrictor control is found in white muscle (Folkow and Halicka, 1968). The precise mechanism governing the reduction of relative blood flow t o white muscle, however, still remains obscure. Another important concomitant of our study relates to the time course of development of blood flow differences. Since rabbit muscle shows some histochemical differentiation at birth (Dubowitz, 1967) and blood flow differences between red and white muscles d o not appear until the third to fourth week of life, it is likely that the metabolic differentiation precedes that of blood flow in rabbits as in chicks and kittens (Hajek et al., 1969; Hudlicka e t al., 1973). This observation lends further credence t o the view that the metabolic requirements of individual muscle fibers may exercise a measure of control over the degree of capillarity and of the blood flow in their immediate vicinity. The apparent rostrocaudal course of development of blood flow differences between red and white muscles are of interest in view of the fact that spontaneous and reflex skeletal movements also develop rostrocaudally and with essentially the same time course (Skoglund, 1960). One would predict, therefore, that tonically active muscles would begin t o maintain a higher basal blood flow than phasically active muscles. Whether the postnatal differentiation in blood flow is neurally directed by maturation of dilator and constrictor mechanism or

394 is secondary to changes in capillarity induced by metabolic differentiation of muscle remains to be determined. Both mechanisms may well be operative.

Differential regulation of blood flow to red and white muscle in behavior; evidence for a differential supersegmental control That the blood flow regulation t o red and white muscle may be differentially regulated by the central nervous system has been supported by studies in which we analyzed changes in the distribution of muscle blood flow in two strikingly opposite behaviors: the rapid eye movement (REM) phase of sleep and the defense response elicited by hypothalamic stimulation (Reis et al., 1969). In REM sleep we observed that there were notable changes in the distribution of cardiac output in different types of muscle (Fig. 6, Table 11). The result was a dissociation between the changes in fractional flow t o red and white muscles in this behavior. Blood flow t o red limb muscles was reduced by about a third, whereas that of white limb muscles was unaffected. Of the other muscles, only the extraocular muscles showed a comparable diminution of flow. As a consequence of this selective reduction of flow t o red muscle, the difference between the blood flow of red and white limb muscle approached unity and the approximately 3 : 1 difference in flow between these muscular species, characteristic of the awake animal, was reduced t o 1 : 1. In contrast were the changes in distribution of blood flow to muscle during the defense reaction. In this behavior there was a general redistribution of blood flow to white limb muscle, extraocular muscles and myocardium, but not t o the red limb muscle. As a consequence of the disproportionate increase in

O “r

, Red muscle

T

0White muscle Myocardium Extraocular muscle

-

160

.c., L

“

0

”.0 .

E 120 a

nL a .

80

40

n Excited

REM

Fig. 6. Changes in fractional blood flow t o red and white skeletal muscle, myocardium and extraocular muscle in the defense reaction (excited) and REM sleep. (Data from Reis et al., 1969.)

395 TABLE I1 MEAN FRACTIONAL BLOOD FLOW IN RED AND WHITE MUSCLES IN CAT, RABBIT AND RAT N.S., not significant. Adapted from Reis e t al. (1969). Behavioral state

Red muscle 100 g muscle

Quiet, alert REM sleep Excitement

6.80 2.06 8.42

f

f

f

0.44 0.17 0.68

White muscle 02]

[”

of100 cardiac g muscle output

2.21 ? 0.14 1.89 0.14 6.16 ? 0.63

Red/white

x

1021

3.1 * 1.1(N.S.) 1.4 (N.S.)

* Significant differences ( P < 0.05). white muscle blood flow, the ratio of red : white was reduced, as in REM sleep, so that the red : white ratio of 3 : 1 , characteristic of the alert quiet animal, was reduced t o 1.4 : 1, a difference which was no longer significant. Muscle blood flow, therefore, can be selectively redistributed to red or white muscle in behaviors in which muscle blood flow, in general, has been known to be altered. In REM sleep, at a time when fractional flow is increasing to other viscera (Reis et al., 1969), probably as a result of widespread reduction in sympathetic tone (Iwamura et al., 1966), there is a striking decrease in flow in red limb and extraocular muscles t o about 30% of control. The fall in red muscle flow cannot be attributed t o absence of muscular activity since it persists in the anesthetized cat (Reis e t al., 1967). The reduction of flow in limb muscle has been demonstrated to be due to increased sympathetic activity (Baccelli et al., 1974) indicating that the activity of the sympathetic nervous system is dissociated in REM sleep. In contrast, in the defense reaction there is an increase in fractional flow to white skeletal muscles in the limbs as well as to muscles of respiration and t o myocardium. Red muscle, however, fails to increase its fractional flow significantly, since in the defense reaction cardiac output increases 2-fold (Folkow et al., 1968). There will be some “passive” elevation of flow in red muscle in proportion to the altered cardiac output, and total flow will be expected t o double. However, the increased flow to other muscles and myocardium will be disproportionately larger by comparison. White limb muscle should, on the other hand, increase 6-fold. These estimations are consistent with the observation of Folkow et al. (1968) that muscle blood flow in the hindlimbs of cat rises &fold in the defense reaction. The most parsimonious interpretation to explain the different behavior of the vascular beds of red and white muscle in REM sleep and the defense reaction is t o assume that at rest the nutrient blood flow in white muscle lies near the lower extreme of its dynamic range, whereas in red muscle nutrient flow approaches the upper limit of its dynamic range. Hence, in behavior in which the fractional flow t o muscle is increased such as in the defense reaction, only white muscle would be capable of signaling an increase in fractional flow, whereas conversely, when fractional flow to muscle falls as in REM sleep, it

396 would only be reflected in red muscle. The observation by Hilton and Vrobova (1968) of a failure of soleus t o show postexercise hyperemia could also be explained in this manner by assuming a widely dilated capillary bed in soleus. This interpretation, however, is untenable in view of the demonstration (Folkow and Hadlicka, 1968) that both red and white limb muscle in cat have the capacity t o significantly increase or decrease nutrient blood flow well beyond the range of flow which could be predicted by our findings. Thus, we must conclude that the selective changes in blood flow distribution which we have seen in red and white skeletal muscle during sleep or in excitement reflects the fact that the peripheral vascular beds in each type of muscle are governed independently of each other and are under independent suprasegmental control involving at least regions of hypothalamus, lower brain-stem, and, as demonstrated elsewhere, cerebellum (Sheridan and Reis, 1972). Relationship of pattern of blood flow, myoglobin, capillarity and twitch characteristics to behavior There is another and more interesting implication of the differential control of flow in red and white muscle in behavior. While it appears that blood flow is closely matched to metabolism and function in different skeletal muscles, it is evident that this relationship only holds in quiet wakefulness (Fig. 7). The relationship between blood flow and metabolism entirely disappears in REM sleep or excitement as a result of the selective redistribution of blood flow in each type of muscle. These facts suggest some relationship between the type of metabolism and behavior: muscle blood flow can change almost instantaneously (Folkow and Halicka, 1968) with changes in muscle activity or sympathetic

5-

IA

A REM sleep B A Excitement C A Awake, r e s t i n g

_,4...-c 2;

I

4

*III*

-

k .

A/A

g3

/ '

I

1

1 0

0

I'

I

I

I

040

060

080

I

020

Fractional blood f!ow

I%of

100

cardiac output 1100gm muscle)

Fig. 7 . Relationship between fractional blood flbw and niyoglobin concentration in different red and white muscles in cat in: rapid eye movement (REM) sleep (line A, filled circle); excitement (line B, filled triangle); quiet wakefulness (line C, open triangle). (From Reis and Wooten, 1970.)

TABLE I11 CHANGES IN FRACTIONAL BLOOD FLOW OF SOLEUS AND GASTROCNEMIUS IN RAT FOLLOWING DENERVATION AND INACTIVATION N.S. = not significant. Experimental group

Soleus

FF

Naive control 24 h post-denerv. t 4 wks post-denerv. t 4 wks post-pinning tt

k

Gastrocnemius

S.E. (n)

P*

FF

t

S.E. (n)

(% cardiac output/

(% cardiac output/

g muscle)

g muscle)

0.745 f 0.244 f 0.466 i 0.882 t

* Difference from naive control. ** Difference between red and white.

0.076 (10) 0.074 ( 4) 0.051 ( 6) 0.114( 6)

<0.01 <0.02

N.S.

0.282 i 0.444 * 0.326 k 0.543 r

0.024 (10) 0.083 ( 4) 0.020 ( 6) 0.041 ( 6)

t Denervation was produced by transection of posterior tibia1 nerve and branches. tt Pinning of the hindlimb was by the method of Fishbach and Robbins (1969).

P*

<0.05 N.S. <0.001

Red/ white

2.6 0.5 1.4 1.6

P

**

<0.001 N.S. < 0.05 <0.02

398 nerve activity and, in general, its distribution reflects the minute t o minute level of activity in different muscles; on the other hand, the metabolic characteristics, including the myoglobin content, the density of the capillary network, and the twitch time, are relatively static properties of muscle, and as already pointed out, appear t o be adapted t o the relative, average distribution of activity between red and white muscle. Changes in the static properties when they occur after nerve transection or cross-union take days or weeks (Buller e t al., 1960b; Bass, 1962; McPherson and Tokunaga, 1967). In view of the different time constants for change of blood flow and metabolism in muscle, it is likely that the behavior in which blood flow and metabolism are entirely proportional is the one t o which muscle metabolism as well as capillarity and twitch characteristics are adapted. On this assumption, our results suggest that the metabolism of individual skeletal muscles is adapted t o the activity of that muscle in quiet, alert behavior, rather than REM sleep or excitement. Segmental mechanisms At the present time the nature of the segmental control of blood flow in red and white muscles is unclear. While sympathetic mechanisms probably control the vasoconstriction occurring during REM sleep (Baccelli et al., 1974), it is not the entire mechanism. Denervation of the soleus produced in rat (Table 111; Wooten and Reis, unpublished) results in a persistent reduction in flow t o red muscle which cannot be attributed t o inactivity since it is unchanged after pinning, a manipulation which will halt muscle activity (Fishbach and Robbins, 1969). Such lesions will interrupt much of the sympathetic innervation of the soleus. In contrast, denervation of white muscle will transiently increase flow, as will pinning. These findings would be consistent with a view that the elevated flow in red muscle is maintained by some neural process probably other than sympathetic cholinergic vasodilation, since red muscle flow is not increased in the defense reaction (Reis et al., 1969). The elucidation of factors maintaining flow in red muscle is a problem most worthy of pursuit.

SUMMARY In quiet, alert behavior blood flow to skeletal muscles varies widely in cat, rat and rabbit. In general, flow to red muscles is 3-5 times greater than that of white muscles. The blood flow in any muscle is directly related t o the myoglobin content (and hence the dependence of that muscle on oxidative metabolism) t o the capillary density and inversely t o the speed of contraction. Differences of flow in adult red and white muscle develop postnatally with a rostralcaudal progression. Change in flow appears later than the developmental differentiation of biochemical and physiological characteristics, thereby demonstrating that differences in blood flow probably occur in response to metabolic demands. Flow in red and white muscle can be differentially changed during behavior: in the defense reaction, produced by hypothalamic stimulation, only flow in white muscle is increased; in the rapid eye movement (REM) phase of sleep or after cerebellectomy, red muscle flow is reduced. As a consequence of

either excitement of REM sleep, the 3--5-fold differences of red and white muscle blood flow approach unity and the linear relationship between flow, oxidative metabolism, capillary density and twitch characteristics are lost. This fact suggests that the metabolism of any skeletal muscle is adapted t o its activity during quiet, alert behavior rather than sleep or excitement. The segmental mechanisms controlling flow in red and white muscle are only partially defined. Studies on the effects of denervation and/or inactivation suggest that while both red and white muscles are under sympathetic vasoconstrictor regulation, however, the high resting flow of red muscle appears due t o some as yet undefined vasodilator mechanism. ACKNOWLEDGEMENT This research was supported by grants from the NIH and NASA.

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DISCUSSION BUCHTHAL: If you look at the spectrum of contraction time switch I showed before, for example in the soleus muscle which has a spectrum which goes from 60 t o about 120 msec, and you arrest the circulation for about 20-40 min, you find that the spectrum changes in such a way that the long contraction time is simply abolished and appears in all mixed muscles to the same extent especially in the brachial biceps. But the interesting thing is that even in an entirely red muscle there is a differentiation in that the fibers representing these contraction times are much more susceptible to hypoxia. I think it gives an interesting aspect of peripheral regulation when you have a strong contraction. You didn’t speak about your experience if during strong contraction blood flow is inhibited, practically entirely inhibited. Did you find any differences there and in the soleus and more white muscle in the cat, when these inhibited units will not respond, because they are simply not contracting. Finally I would like t o mention that there is a difference in temperature dependence of contraction time of these highly oxidative units and of the more glycogenetic units. HENNEMAN: I think this paper is full of interesting points. I would like to comment that, if you don’t happen to know the paper, there is an interesting study by Rose Eccles and several collaborators in which she showed that as the motor neurons in the kitten mature and enlarge up to about the 20th day, the changes in the muscle seemed to go hand in hand with that. I think your results may suggest what happens t o some extent during training, if capillarity is made more effective in the muscle. I would like to ask you also whether you think you could selectively kill off red fibers in muscle by hypoxia and leave the white. REIS: With regard to the work of Eccles, I am familiar with that and perhaps I didn’t emphasize that also one of the features of the developmental curve is that the metabolic differentiation of muscle is in advance of vascular. One could make a hypothesis that perhaps the difference in blood cells would then somehow by providing substrate induce certain enzyme patterns, but this does not seem t o be the case. So the muscle differentiation occurs and blood flow will match t o that. Capillary changes in training, I rather doubt that. I am so petalling a little bit our capillary data, though via an indirect method, one which permits estimation of large bulk of muscle. Recently there is a paper in the American Jourmal of Physiology claiming that the geometry of capillary to muscle ratio, in any kind of muscle, is the same, each muscle fiber being surrounded by exactly three capillaries in some glid form. So it’s probably the relationship of the number of muscle fibers that would ultimately determine the number of capillaries, but the ratio of capillary to muscle may be constant. The final point was about the capacity to kill off red muscle at the expense of white. One can use

402 metabolic poisoning selectively and dose response. I t sounds very feasible. Somebody should try it, not me. HOUK: Would more blood flow not be required for developmental processes, too? I am just wondering about your notion of the causality. It seems to me that the tissue might cause blood flow because they are more active. REIS: I d o not think that it would be the cause, because white muscles are hard t o explain under those circumstances. I think that any function of any tissue is multiple. We can ascribe many functions t o it and I just raised a possibility that very large blood flow through red muscle might relate t o generation of heat. POMPEIANO: What is the mechanism with which cerebellum maintains blood flow? Is it due t o fastigial facilitation of some vegetative mechansim or, did you try t o make asymmetrical lesions of the cerebellum which produce hypotonia o n one side and hypertonia o n the other side. Could you evaluate the same control in the same experiment by testing the left and right side? REIS: No, we didn’t d o that. ELDRED: Did you see what happens when deafferented? How much control did you use? REIS: No, that is what we are doing at present. It is the purpose t o look into the peripheral mechanism, reflex dependent upon muscle contraction, and what is the transmitter. Something very funny about this red muscle control doesn’t follow the prediction of normal sympathetic tone. That’s all I can say. I think different mechanisms are involved peripherally. ELDRED: One other thing occurred to me in reference to causality mentioned here. What can you, or maybe a histologist, make o u t of the fact that the axial bundle in spindles, at least in some species, seems t o be very much devoid of blood vessel capillaries and here within the capsule of spindles we have two types of fibers, one of which contracts faster than probably any other muscle fiber in the body and the other is probably t h e slowest. I n this case we don’t have any kind of relationship between blood supply and contractility. Would anybody know something about this? REIS: All I can say is that this technique will work. If you can dissect o u t two types of fiber, you can determine the difference in the flow between the two. HENNEMAN: When you look a t the cross-section of the muscle stained appropriately, it really makes you wonder how the large tail fibers can get along, because frequently they have n o capillary at all associated with them and near response some distance away. HOWd o they manage metabolically? REIS: Diffusion. I don’t know really.<