Australian National University, Canberra, Australia

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1 430 J. Phy8iol. (1965), 179, pp With 6 text-figures Printed in Great Britain MUSCLE STRETCH AND THE PRESYNAPTIC INHIBITION OF THE GROUP Ia PATHWAY TO MOTONEURONES BY M. S. DEVANANDAN, ROSAMOND M. ECCLES AND T. YOKOTA* From the Department of Physiology, Australian National University, Canberra, Australia (Received 16 November 1964) It has been shown that the monosynaptic EPSP which was evoked in a motoneurone by a volley in its muscle nerve could be depressed by conditioning volleys in other muscle nerves without any evidence of postsynaptic inhibitory changes in the motoneurone (Frank & Fuortes, 1957; Eccles, Eccles & Magni, 1960, 1961). Eccles et al. (1961) described the time course of this EPSP depression and related the depression to volleys in nerves from muscles with different functions. In general, activity in nerves from flexors evoked greater depression of the monosynaptic EPSP than volleys in nerves from extensors. It seemed that a correlation existed between the EPSP depression and the dorsal root reflex (DRR) and that both these phenomena are due to the same process, viz. depolarization of the primary afferent fibres within the spinal cord. Eccles, Magni & Willis (1962) provided conclusive evidence of the depolarization of central terminals of group I primary afferents from muscle. This depolarization followed the same time course as the EPSP depression and is evoked under the same experimental conditions. This phenomenon was designated 'presynaptic inhibition'. Devanandan, Eccles & Yokota (1965) observed that depolarization of the central terminals of primary afferent fibres can be produced by muscle stretch. It will be shown in this paper that muscle stretch may effectively depress the monosynaptic EPSP and the monosynaptic reflex. This depression of the monosynaptic reflex was strychnine resistant, thereby establishing that it was not due to post-synaptic inhibition (Bradley, Easton & Eccles, 1953). METHODS The experimental techniques have been described in full in the earlier paper (Devanandan et al. 1965) as well as in other publications (Eccles, Kostyuk & Schmidt, 1962; Eccles, Schmidt & Willis, 1963). * Rockefeller Fellow.

2 DEPRESSION OF GROUP Ia PATH WA Y 431 To record monosynaptic reflexes, the ventral roots L 6-S 1 were cut and the central ends of the L 7 and S 1 ventral roots were prepared for recording. For studies on the monosynaptic EPSPs the ventral roots were often left intact so that the motoneurone impaled could be accurately identified by antidromic invasion (Eccles, Eccles & Lundberg, 1957a). Presynaptic inhibition was demonstrated by the depression of the monosynaptic excitatory post-synaptic potential (EPSP), the membrane potential remaining constant, and by the depression of the monosynaptic reflex. When recording reflexes selective depression of post-synaptic inhibition was effected by strychnine in an initial intravenous dose of 0-08 mg/ kg, and this dosage was later increased in order to show that the inhibition was truly strychnine resistant. Generally strychnine could not be given during intracellular recording because strychnine convulsions often dislodged the micro-electrode from the motoneurone. However, six motoneurones were examined after intravenous strychnine (0-1 mg/kg) and one of these motoneurones is illustrated in Fig. 6. RESULTS Depression of the monosynaptic EPSP The presynaptic inhibitory pathways have been shown to depolarize the primary afferent fibres (Eccles et al. 1961; Eccles, Magni & Willis, 1962), thereby causing a reduction of transmitter release from the primary afferent fibres (Takeuchi & Takeuchi, 1962; Eccles, 1964). This leads to a reduction in size of the monosynaptic EPSP of a motoneurone without an alteration in its membrane potential (Frank & Fuortes, 1957; Eccles et al. 1961). In Fig. 1 the monosynaptic EPSP evoked by a volley in the plantar nerve was preceded by a stretch of the DP muscles at the indicated intervals, the recording conditions being given by Fig. 1 of the preceding paper (Devanandan et al. 1965). The EPSP was depressed to about 83 % of the control height in 30. The depression had a total duration of about 130. From the inset it can be seen that stretch of the DP muscle evoked only a small brief EPSP in the motoneurone under observation. In Fig. 2 a volley of group I strength in the GS nerve evoked an EPSP in a gastrocnemius motoneurone that was conditioned by a stretch applied to the PDP muscles. The presynaptic inhibition was greater, the EPSP being depressed to 70 % of the control height, but both the maximum depression at 30 and duration of 140 were similar to those observed in flexor motoneurones (Fig. 1). Stretch of the PDP muscles produced a post-synaptic inhibitory potential (IPSP) of short duration (see inset). Finally, in Fig. 3 the effects of conditioning with applied stretches to either ST or PDP are recorded. The monosynaptic EPSP in the plantaris motoneurone was small but constant. The peaks of the EPSP depressions in response to stretches of ST at 40 and of PDP at 35 were slightly later than those recorded in Figs. 2 and 3. The durations were long, though not observed as a smooth recovery over The earlier

3 432 M. S. DEVANANDAN AND OTHERS depression evoked by a PDP stretch (Fig. 3D) was probably due to the post-synaptic inhibitory action by PDP group I a fibres on to motoneurones of the antagonistic group, i.e. ankle flexors on to ankle extensors. It seemed that conditioning with applied stretch of the PDP muscle was not as effective in producing EPSP depression as a tetanus applied to the cut PDP nerve (Eccles et al. 1961). It was found that, if electrodes were placed around the intact PDP nerve, application of stimuli to these electrodes always evoked much larger EPSP depressions than muscle stretch. A limv ~~~~F7..~~~~~~~] 1..I0 CON 2fK 36/N B \1 80 so. 20 * o so MMiseonds Fig. 1. In the graph (B) are the plotted heights of the monosynaptic EPSPs in a plantar motoneurone evoked by a volley in the plantar nerve, and preceded by activation of the muscle receptors of DP muscles. Samples of the records are given above (A) with numbers indicating the time between conditioning and testing stimuli in. Potential and time scales the same for all records. The 100 % of the graph is the size of the control EPSP. Duration of stretch of DP muscle was 10 as shown. Inset is a record of the effect of the muscle stretch on the membrane potential of the impaled motoneurone. The arrow marks the end of the pull artifact. The EPSP produced by the stimulation of the plantar nerve is seen as the large deflexion at the end of the record, the upstroke having disappeared. The voltage calibration is the same as in A. Pull used about 160 g. Reflex testing In a monosynaptic reflex the number of discharging motoneurones is approximately proportional to the size of the spike potential in the appropriate ventral root. The monosynaptic reflex is set up by stimulation of the muscle nerve in the periphery. The largest afferent fibres (I a) in the muscle nerve synapse directly on to the motoneurones. If the excitation is above a threshold level, a spike is initiated and propagates down the axon. Any alteration in the excitability of the afferent fibres, or their

4 DEPRESSION OF GROUP Ia PATH WA Y 433 terminals, or the motoneurones will be signalled by a change in the reflex size. Consequently a monosynaptic reflex is a valuable guide to excitability changes in the largest afferent fibres from muscle (Ia). In Fig. 4 the testing situation was the facilitated monosynaptic reflex from stimulating posterior biceps nerve (PB). Since the unfacilitated PB reflex was rather unstable, it was facilitated by an earlier conditioning volley in the PB nerve which alone did not evoke a reflex. A stretch was applied to the DP muscles at various intervals preceding the facilitated PB reflex. The depression evoked is shown by the graph (open circles, A CON/k 38\ B C_>j5JK~JX%SmV k Milliseconds Fig. 2. The monosynaptic EPSPs evoked in a gastrocnemius motoneurone by a volley in gastrocnemius nerve were conditioned by stretches applied to the PDP muscle at the times indicated on the graph B. The 100 % level was the size of the control EPSP. Duration of the stretch was lo. Some ofthe EPSPs from which the graph was constructed are shown above with their potential and time scales. Inset is a record of the effect of the muscle stretch on the membrane potentials of the GS motoneurone. As the record shows, a transient hyperpolarization is evoked by the muscle stretch. The voltage calibration is the same as in A. Pul] used about 395 g. Fig. 4). Typical responses from which this graph was derived are given in Fig. 4A. The depression reached a maximum at about 50 and recovered first quickly and from 90 onwards more slowly. It is realized that activity of the afferent fibres either by stretching the stretch receptors or stimulating the nerve (Eccles et al. 1963) may evoke both post-synaptic effects and presynaptic effects in the spinal cord. For example, impulses in lb fibres of a flexor muscle can depress the excitability of the PB Ia afferent fibres (i.e. presynaptic inhibition). Also, impulses in Ib fibres from a muscle flexor can evoke post-synaptic excitation of PB motoneurones (i.e. post-synaptic excitation up to 40 in 28 Physiol. 179

5 434 M. S. DEVANANDAN AND OTHERS ACOJ6' B 10io ok- 0 [1 mv ImsJcLLLLL 80 F 60 K. Nel///A Milliseconds CON D fcon J b-> 2 m es [1 mv 1 0 7t_ 80 F F l0,il I IItI1lI$ I. V'7,YYA Milliseconds Fig. 3. The intracellular monosynaptic EPSPs from a plantaris motoneurone evoked by a volley in plantaris nerve were conditioned at different intervals by applied stretches to the ST muscles (A and B) or the PDP muscles (C and D). In B and D the full time course of the effectiveness of stretches applied to ST muscle (B) and PDP muscle (D) are plotted. Duration of stretch again was 10. In A and C are the examples of some of the EPSPs recorded. Note that A and C have their own potential and time scales. The insets show the effect ofmuscle stretch on the membrane potential of the impaled cell, the voltage calibration in each case being the same as that given above the respective graph. Pull of ST muscle about 177 g. Pull of PDP muscles about 450 g. t

6 DEPRESSION OF GROUP Ia PATH WA Y 435 graph of Fig. 4) though some inhibitory effects may be seen (Eccles, Eccles & Lundberg, 1957 b). One can distinguish between these inhibitions in two ways: the post-synaptic inhibitions are generally shorter than presynaptic inhibitions (Eccles, Schmidt & Willis, 1962) and strychnine blocks postsynaptic inhibitions and usually increases presynaptic inhibitions (Eccles et at. 1963). Since the depression of the facilitated PB reflex (Fig. 4, open circles) showed a second component at 80, strychnine (0-1 mg/kg) was administered intravenously. This increased the inhibition and also greatly lengthened the time course (Eccles et al. 1963). It will be noted that strychnine increased the earlier phase of excitation (cf. the records in C. 5 8CONA 28j\ 53 A 58 A 83 fn 120' A 151 A [0-5mV Osj 'b~~~,00.o-~_j \0-.8J1~~~~~~~tJ 'JJ--,~~~~MSC Z~~~~~ g 60F o0 n0 Milliseconds Fig. 4. The monosynaptic reflex evoked by PBST was recorded in the central end of S1VR (CON in A) and conditioned by a pull (10 in duration) applied to the DP muscles (row A and open circles). Row B and filled circles on graph were recorded after a dose of strychnine, 0-1 mg/kg. Open circles on graph, before strychnine. Same potential and time scales for A and B. Pull about 160 g. Fig. 4A and B at 28 ). This is a typical result of strychnine administration and probably indicates an increased excitability of the motoneuronal membrane due to an increased background of interneuronal activity. Often the sizes of the monosynaptic reflexes were increased after strychnine. If this occurred the control reflex was kept approximately to the same size as that before strychnine administration (i.e. the control reflex of the B series would equal the control reflex of the A series). 28.2

7 436 M. S. DEVANANDAN AND OTHERS Since, in this particular experiment, the control reflexes were approximately equal and no adjustment of reflex size was required, it was probable that the majority of motoneurones were firing even before the drug was given. CON C [2mV 60 \%* *a o lovio. U - 40 \/ 20 \ \ oo ~ ~ ~ Milliseconds ~ X \ /\ Volts Fig. B. The monosynaptic reflex evoked by stimulation of the nerve from the GS muscle was recorded in the central end of SlVR. In A and filled rectangles in C the effect of conditioning with ST muscle stretch (10 duration) was investigated to weak pulls (2-5 V about 46 g) and to stronger pulls about 177 g (B and open rectangles (C)). The effect of increasing strength of the muscle stretch (abscissa) is plotted (in D) against the depression recorded (ordinate). In Fig. 5 after a conditioning stretch of the ST muscle the facilitated reflex to stimulation of the GS nerve was recorded in the S1VR. When a small pull (2 5V) was applied to the ST muscle the inhibition was less than

8 DEPRESSION OF GROUP Ia PATHWAY % (Fig. 5C or the record taken at 29 in Fig. 5A). The time course for the inhibition was 100. If the strength of pull was altered to 10V there was a much larger depression (open squares, Fig. 5C), with almost complete inhibition of the GS reflex at 25 and later at The early depression could definitely be post-synaptic inhibition since no strychnine was given to this animal, but the long, late, second depression seems to have a time course similar to those described already for presynaptic inhibition. Even at 140 the reflex is depressed by more than 40 %. At a fixed interval in Fig. 5D when the strength of pull was altered there was a sharp distinction between 3 and 5V. This was quite repeatable so that one can assume that a voltage of 2 5V stimulates only the most sensitive receptors whilst for all stretches evoked by 5V or more a maximum effect is obtained. These changes on the monosynaptic reflex could be due either to a motoneuronal change or to an alteration in the excitability of the afferent fibres. It seemed likely to be presynaptic in origin since the excitability changes (Devanandan et al. 1965) were not unlike those associated with presynaptic inhibition, particularly regarding the long durations observed, and strychnine was ineffective in reducing the inhibitions observed (Eccles, Schmidt & Willis, 1962, 1963). Finally, in Fig. 6, the relation between EPSP depression and reflex depression is illustrated when all post-synaptic inhibition would have been eliminated by strychnine (Bradley et al. 1953). The monosynaptic EPSP from an impaled GS motoneurone was displayed on one beam of the oscilloscope, and the unfacilitated monosynaptic GS reflex recorded in the S, ventral root was recorded on the other. A 1OV pulse was employed to elicit stretches of the PDP muscles. The EPSP was depressed to about 80 % of the control, whereas the reflex was depressed much more (about 40 % of the control). This illustrates that, though the depression of the individual EPSPs may not appear to be large when conditioned with a muscle pull, the excitability of the whole motoneurone pool may be considerably depressed. DISCUSSION The motoneurone pool is subject to several influences that converge on to it from the periphery, from within the spinal cord and from supra-spinal regions. The net result of these several factors will tend to activate or depress a given motoneurone pool. Since the index of activity has been the monosynaptic reflex or monosynaptic EPSP, only the group I a afferent fibres in the stimulated peripheral nerve will be responsible for this activity (Lloyd, 1943). This reflex or EPSP has been preceded by a muscle stretch at various intervals, and the resulting depression of the reflex or

9 *~~~ * 0~~~~~~~~~~ 438 M. S. DEVANANDAN AND OTHERS EPSP noted. The control monosynaptic reflexes and EPSPs taken at frequent intervals during the experiments were very stable. Therefore, the depression noted, when the conditioning muscle stretch was applied, was CON [2mV a AU. 1J~llv~ [0.1mV , 60 -No - 0 g,'i o,' 40 cs - 0' Q 20 20~~~~~~~ _ 0 O 0 Il II l Milliseconds Fig. 6. The monosynaptic EPSP of a GS motoneurone was recorded on one beam of the oscilloscope, upper row of records. Simultaneously the unfacilitated GS monosynaptic reflex was recorded in S1VR and displayed on the other beam, lower row of records. The effect of stretching the PDP muscles with a pull of about 450 g is plotted on the graph. The filled circles follow the time course of the EPSP depression; the open circles the time course of the reflex depression. Inset is the effect of the muscle stretch on the membrane potential of another GS motoneurone close by, as the motoneurone used for the records illustrated in the graph was lost before the effect of muscle stretch on the membrane potential could be recorded. Note strychnine (0-1 mg/kg) was administered intravenously before this motoneurone was impaled. due to a change in the synaptic events involved rather than to any spontaneous variation in the excitability of the motoneurones. In the present series of experiments, monosynaptic EPSPs in GS, PL or plantar motoneurones have been conditioned by stretches of the flexor

10 DEPRESSION OF CROUP Ia PATH WA Y 439 muscles, ST, DP or PDP. Care was always taken to check whether the muscle pull produced an IPSP in motoneurones in each case (see insets in Figs. 1, 2 and 3). In agreement with Eccles & Lundberg (1959) it can be seen that the effect of muscle stretch on the plantar motoneurone was excitatory, and the stretch of the ST muscle had no effect on the PL motoneurone. On the other hand, the stretch of the PDP muscles did evoke IPSPs in the PL and GS motoneurones. However, the depression of the monosynaptic EPSP is unlikely to have been greatly affected as the EPSP remained depressed long after the IPSP had disappeared. Also it has been shown that direct inhibition will reduce the size of the EPSP only if the EPSP falls within 2 of the start of the IPSP (Coombs, Eccles & Fatt, 1955). When the testing situation was the monosynaptic reflex, post-synaptic inhibition was generally discounted by the use of strychnine (Bradley et at. 1953). Also, the group I afferents from the ST have little or no postsynaptic effects on the motoneurones of the physiological extensors of the ankle (Eccles & Lundberg, 1959), and the post-synaptic actions of the DP muscle afferents are, if anything, excitatory on the PB motoneurones (Eccles & Lundberg, 1959). Therefore, the EPSP depression and reflex depression reported here are probably due to presynaptic inhibition. It has been shown (Fig. 5C and D) that increasing the intensity of the pull to a certain level increases the amount of inhibition. This has been observed constantly. When the responses from the muscle stretch receptors were analysed by recording from filaments in the dorsal root, it was always noticed that the increase in inhibition coincided with the activation of the Ib afferents (Fig. 6; and Devanandan et al. 1965). This is in agreement with previous results from electrical stimulation of muscle afferent fibres (Eccles, Schmidt & Willis, 1962). The differences in the experiments reported above and those in a communication from Granit, Kellerth & Williams (1964) may be in the techniques employed. Granit et al. (1964) stated that no presynaptic inhibition was observed when the behaviour of motoneurones was directly observed during applied stretches to muscle. These authors employed only large stretches with slow rates of rise and maintained for several seconds. In contrast, only rapid brief stretches (10 in duration) have been employed here. In our experiments we have been only concerned with the period subsequent to the muscle stretch so that the long-lasting depressions would be recorded, after 40 or so, uncontaminated by post-synaptic events on the motoneurones themselves. These long-lasting depressions of both the monosynaptic EPSPs and reflexes have been shown to be presynaptic inhibitory effects since they persist after strychnine administration.

11 440 M. S. DEVANANDAN AND OTHERS Since it has been shown that muscle stretch can evoke presynaptic inhibition, it becomes necessary to study it in greater detail taking into consideration the patterns of activation of the muscle stretch receptors and their reflex regulation through the y-motoneurones. The response of the primary spindles to stretch can be divided into a dynamic response and a static response (Jansen & Matthews, 1961; Matthews, 1964). Renkin & Vallbo (1964) have suggested that they register the instantaneous velocity of stretch and the instantaneous length of the muscle. The presynaptic inhibition evoked by the I a afferents must therefore be analysed in regard to these two different responses, both with y-loops intact or interrupted. Jansen & Rudjord (1964) suggested that the Golgi tendon organs are contraction receptors rather than stretch receptors. The presynaptic inhibition evoked by the lb afferents should therefore be examined during muscle contraction as well as muscle stretch. SUMMARY 1. The monosynaptic EPSPs of motoneurones of extensor muscles of the ankle, and of a flexor muscle of the foot were conditioned by stretches of flexor muscles of the knee or the ankle. 2. The EPSPs were depressed by the muscle stretches over a time course of about , the peak being at about The monosynaptic reflexes of extensor muscles of the ankle and flexor muscles of the knee were similarly reduced by conditioning with applied stretches of flexor muscles of the ankle or the knee. 4. This depression of the reflex and the EPSP could not be abolished by strychnine and therefore was not due to post-synaptic inhibition. 5. It is suggested that these depressions are due to presynaptic inhibition. REFERENCES BRADLEY, K., EASTON, D. M. & ECCLES, J. C. (1953). An investigation of primary or direct inhibition. J. Physiol. 122, COOMBS, J. S., ECCLES, J. C. & FATT, P. (1955). The inhibitory suppression of reflex discharges from motoneurones. J. Physiol. 130, DEVANANDAN, M. S., ECCLES, R. M. & YOKOTA, T. (1965). Depolarization of afferent terminals evoked by muscle stretch. J. Physiol. 179, ECCLES, J. C. (1964). The Physiology of Synapses. Berlin, G6ttingen, Heidelberg: Springer. Verlag. EccLEs, J. C., ECCLES, R. M. & LUNDBERG, A. (1957a). The convergence of monosynaptic excitatory afferents on to many different species of alpha motoneurones. J. Physiol. 137, ECCLES, J. C., ECCLES, R. M. & LUNDBERG, A. (1957b). Svnaptic actions on motoneurones caused by impulses in Golgi tendon organ afferents. J. Physiol. 138, ECCLES, J. C., ECCLES, R. M. & MAGNI, F. (1960). Presynaptic inhibition in the spinal cord. J. Physiol. 154, 28P.

12 DEPRESSION OF GROUP la PATHWAY 441 ECCLES, J. C., ECCLES, R. M. & MAGNI, F. (1961). Central inhibitory action attributable to presynaptic depolarization produced by muscle afferent volleys. J. Phy8iol. 159, ECCLES, J. C., KOSTYUK, P. G. & SCHMIDT, R. F. (1962). Central pathways responsible for depolarization of primary afferent fibres. J. Physiol. 161, ECCLES, J. C., MAGNI, F. & WILLIs, W. D. (1962). Depolarization of central terminals of group I afferent fibres from muscle. J. Phy8iol. 160, EccLEs, J. C., SCHMIDT, R. F. & WmILis, W. D. (1962). Presynaptic inhibition of the spinal monosynaptic reflex pathway. J. Physiol. 161, EccLEs, J. C., SCHMIDT, R. F. & WILLIS, W. D. (1963). Pharmacological studies on presynaptic inhibition. J. Physiol. 168, ECCLES, R. M. & LUNDBERG, A. (1959). Synaptic actions in motoneurones by afferents which may evoke the flexion reflex. Arch. ital. Biol. 97, FRANK, K. & FUORTES, M. G. F. (1957). Presynaptic and postsynaptic inhibition of monosynaptic reflexes. Fed. Proc. 16, GRANIT, R., KELLERTH, J.-O. & WILLIAMS, T. D. (1964). 'Adjacent' and 'remote' postsynaptic inhibition in motoneurones stimulated by muscle stretch. J. Physiol. 174, JANSEN, J. K. S. & MATTHEWS, P. B. C. (1961). The dynamic responses to slow stretch of muscle spindles in the decerebrate cat. J. Physiol. 159, 20-22P. JANSEN, J. K. S. & RUDJORD, T. (1964). Some properties of Golgi tendon organs of the soleus in the cat. J. Physiol. 171, 41P. LLOYD, D. P. C. (1943). Neuron patterns controlling transmission of ipsilateral hind limb reflexes in cat. J. Neurophy8iol. 6, MATTHEWS, P. B. C. (1964). Muscle spindles and their motor control. Phy8iol. Rev. 44, RENKIN, B. Z. & VALLBO, A. B. (1964). Simultaneous responses of group I and 11 cat muscle spindle afferents to muscle position and movement. J. Neurophysiol. 27, TAKEUCHI, A. & TAKEUCHI, N. (1962). Electrical changes in pre- and postsynaptic axons of the giant synapse of Loligo. J. gen. Physiol. 45,