Activity of interneurons mediating reciprocal 1a inhibition during locomotion

Brain Research, 84 (1975) 181-194

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© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

ACTIVITY OF INTERNEURONS MEDIATING RECIPROCAL la INHIBITION DURING LOCOMOTION

A. G. F E L D M A N AND G. N. ORLOVSKY

Institute of Problems of Information Transmission, Academy of Sciences, and Interfaculty Laboratory of Mathematical Methods in Biology, M. V. Lomonosow Moscow State University, Moscow (U.S.S.R.) (Accepted September 30th, 1974)

SUMMARY

The activity of interneurons monosynapticallyexcited by 1a afferents and mediating the reciprocal inhibition of motoneurons of the antagonistic muscles was recorded in mesencephalic cats. The activity was studied during locomotion evoked by stimulation of the 'locomotor region' of the mesencephalon. The neurons turned out to be active in a definite phase of the step simultaneously with that muscle which supplied them by la afferent input. In preparations with de-efferent hindlimbs, stimulation of the 'locomotor region' evoked a periodic process in the spinal cord ('fictive' locomotion); in this case bursts of activity in the interneurons also coincided with the activity of motoneurons of corresponding muscles. Under such conditions excitation of 1a afferents by the passive stretch of the muscle or by electric stimulation of the muscle nerve resulted in an increase of the activity of interneurons, which depended on the phase of the cycle. The data obtained show that during locomotion there are at least two sources of inhibition of motoneurons of antagonistic muscles: (i) the activity of la afferents of the active muscle, mediating by corresponding interneurons; and (ii) signals coming through the same interneurons from the central mechanisms generating stepping movements.

! NTRODUCTION

The reciprocal interaction between muscles was first investigated by Liddell and Sherrington who demonstrated that passive extension of the muscle resulted in the inhibition of its antagonist13. This inhibitory influence is determined by activation of the large afferent fibers from the extending muscle14-16, which were identified as la endings of the muscle spindlesS,~0. The la afferents inhibit alpha-motoneurons of

182

the antagonist not directly but by exciting a special group ofinhibitoryinterneurons 1,e, located in the ventral horn of the spinal cordll,lL The system of reciprocal inhibition is considered to be of great importance for the control of movements (cf refs. 13, 17), since an alternating, reciprocal activity of muscles is observed in many types of movements. The efficiency of the reciprocal inhibition in many cases is rather high 13,18. Besides, the system of reciprocal inhibition seems to be very flexible since its elements, i.e. the muscle spindles and the spinal inhibitory interneurons, are under powerful central control. Activity of the la afferents may be regulated by the gamma-efferent innervation of the spindles (cf. ref. 18) while that of the inhibitory interneurons is regulated by a large number of spinal and supraspinal inputs (cf ref. 7). The present series of experiments was undertaken in order to investigate the activity of the system of reciprocal inhibition during locomotion. The experiments were carried out on the decerebrate (mesencephalic) cats, in which locomotion could be evoked by stimulation of the 'locomotor region' of the mesencephalon~9, a0. Activity of the interneurons mediating reciprocal inhibition was recorded during locomotion. To establish the relative contribution from central and peripheral sources to the activity of inhibitory interneurons, the experiments were performed, not only in preparations having intact innervation of the hindlimbs, but also in preparations in which both hindlimbs were immobilized by de-efferentation. In such preparations, stimulation of the 'locomotor region' results in generation of rhythmic bursts of motoneurons with a period of about 1 sec comparable to the duration of the step during normal locomotion 23. This intraspinal rhythmic process may be considered as an expression of the central program of stepping ('fictive' locomotion24). METHODS

Preparation with intact innervation of limbs These experiments were carried out on mesencephalic cats in which stimulation (30 pulses/sec, 1 msec pulse duration, 10-20 V, bipolar electrode) of the 'locomotor region' (LR) of the mesencephalon evoked locomotion 29,30. Transection of the brain stem at approximately the A5 level and laminectomy at the level of the L4-L6 vertebrae were performed under ether anesthesia. The head of the cat was then put in a stereotaxic device, and the lumbar vertebrae and the pelvis were fixed. The legs of the cat were lowered to make contact with the treadmill which could move at a speed of up to 5 km/h. Activity of interneurons excited by la afferents from the knee extensor m. quadriceps (Q) was recorded. These neurons mediating the reciprocal inhibition from Q to its antagonists will be called RI neurons of Q or, if there are no special reservations, simply RI neurons. A schematic representation of the experimental arrangement is given in Fig. 1A. A stimulating electrode with flexible terminals was put on the Q nerve. An afferent wave evoked by this stimulation was recorded by means of the small ball electrode placed near the dorsal roots 5 and 6 entry zone. Approximately one-third of VL6 was

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Fig. 1. A and B: schematic representation of the experiments with real (A) and 'fictive' locomotion (B). Locomotion is evoked by stimulation of the 'locomotor region' of the mesencephalon (1). RI neurons are recorded extracellularly by means of the microelectrode (2), RI neurons of Q being recorded in the L5 and L6 segments while those of PBSt in the L7 segment. The ball electrode (3) is used to record the afferent wave evoked by stimulation of the Q nerve (4); when the PBSt nerve is stimulated this electrode is placed in the L7 segment. The electrode (5) is used for stimulation of motor axons. At real locomotion the EMG of Q (6) and the limb movement (7) are recorded. At 'fictive' locomotion the monosynaptic response in the L5 or L7 ventral roots (8, 9) to stimulation of Q nerve (4) or G nerve (10) correspondingly is recorded. C-E: an example of identification of an RI neuron of Q. C: the response (upper trace) of the neuron to stimulation of Q nerve with a strength of 1.4 threshold for la afferents; lower trace shows an afferent wave at the VL6 entry zone. The stimulating impulse is of 0.1 msec duration. D: responses of the neuron to repetitive (300/sec) stimulation of Q nerve with the same parameters of the stimulating impulse. E: stimulation of the VL5 inhibits activity of the neuron (superposition of several responses). Time calibration: 6 msec (C), 12 msec (D) and 60 msec (E).

cut a n d separated f r o m the rest o f the r o o t a n d the p r o x i m a l end was m o u n t e d o n the s t i m u l a t i n g electrode. The exposed spinal cord was covered with w a r m m i n e r a l oil. T h i n copper wires were inserted into Q, for E M G recording. The m o v e m e n t s o f the l i m b were recorded by a p o t e n t i o m e t e r transducer. R e c o r d i n g o f R I n e u r o n s was performed extracellularly with p l a t i n u m microelectrodes (tip diameter, 10-20/~m; resistance, 100-500 kf~). The electrode was inserted into the L 5 - L 6 segments o f the spinal cord, 1.3-1.5 m m f r o m the midline, with a n angle o f 10-15 ° t o w a r d the lateral side, at a depth o f a b o u t 2.5 m m . Just in this region o f the ventral h o r n R I n e u r o n s o f Q were foundg,11,1L R I n e u r o n s were identified according to the k n o w n criteria 9. (i) They r e s p o n d e d m o n o s y n a p t i c a l l y ( i n t r a s p i n a l delay less t h a n 1 msec) to s t i m u l a t i o n o f l a afferents; (ii) n e u r o n s could follow a high rate o f s t i m u l a t i o n o f l a fibers (300-500/sec); (iii) s t i m u l a t i o n o f m o t o r axons (in VL6) i n h i b i t e d b o t h the resting discharge o f a n e u r o n

184 and the discharge evoked by the stretch of Q. An example of the identification of an RI neuron is given in Fig. 1C-E.

Preparation with hindlimbs de-efferented In this series the same procedures were used. After the capability of locomotion with stimulation of LR had been tested, a wide lumbosacral laminectomy was performed and all ventral roots at both sides, starting from L3, were cut. In these preparations, stimulation of LR evoked a rhythmic process in lumbosacral spinal cord ('fictive' locomotion). If the stimulation was not strong, in half of the cases this process was localized at the lumbosacral level so that the stepping mechanism of forelimbs was not involved. But in other experiments 'fictive' locomotion of hindlimbs and 'real' locomotion of forelimbs were aroused simultaneously 23. Since the hindlimbs were immobilized, in these experiments activity of the motoneurons was studied indirectly by recording the monosynaptic reflex (MSR). To activate la afferent fibers, the Q nerve was rhythmically stimulated (10-20/sec) and the response in the VL6 was recorded. In part of the experiments, motoneurons of another extensor, i.e.m, gastrocnemius (G), were tested monosynaptically to prevent the direct influence of the stimulation on RI neurons. (It is known that during locomotion all extensors are active mainly in phase3,4.) During stimulation of LR the rhythmic modulation of MSR appeared (see Results) which allowed us to establish the phase of the 'step': a maximal MSR corresponded to the extensor ('stance') phase. A schematic representation of the experimental arrangement for these preparations is given in Fig. lB. Unlike the case of Fig. 1A, in order to identify RI neurons, not a part but the whole VL6 (or sometimes VL5) was stimulated. To analyze the contribution of muscle afferents to the activity of RI neurons, the tendon of Q was cut and the muscle was stretched with a force of up to 0.5 kg. The main results have been obtained while recording interneurons in the pathway of reciprocal inhibition from Q to its antagonists. Besides, two interneurons mediating reciprocal inhibition in the 'opposite' direction have been recorded. These interneurons located in L7 were monosynaptically excited by I a afferents of n. biceps posterior and semitendinosus (PBSt). They could be inhibited by stimulation of VI7. These cells will be called RI neurons of PBSt. All results were obtained in 11 successful experiments, 2 of which were performed on decerebellate cats. RESULTS

Preparation with intact innervation of limbs In this series of experiments, 11 RI neurons activated by la afferents of Q were recorded during locomotion. The resting discharge of these cells was 10-30 pulses/sec and was strongly dependent on the limb position. An example of the record is given in Fig. 2. With passive knee flexion (A) the RI neuron increased its firing rate up to 100 pulses/sec. During locomotion evoked by LR stimulation ([3) the discharge frequency alternated according to the rhythm of stepping: the neuron was much more

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active during the stance phase of the step than during the swing phase. The frequency in the stance phase reached 180 pulses/sec. At the stance phase the extensor group of muscles is active3, 4, including Q whose EMG is also represented in Fig. 2B. Thus during locomotion, the neuron mediating reciprocal inhibition from Q was active almost simultaneously with this muscle. However, some non-coincidence between the activity of the RI neuron and that of the Q alpha-motoneurons could be found. Thus, after LR stimulation was turned off, stepping movements decreased gradually, and the last burst of the activity of the RI neuron appeared without any corresponding activity of Q (Fig. 2C). Fig. 2D and E show the activity of another RI neuron of Q during passive knee flexion (D) and during locomotion (E). In both cases the discharge frequencies were smaller than those for the first neuron; however, during locomotion the neuron was also active in the stance phase (in E, extension, i.e. stance phase, corresponds to downward deflection of the beam). Corresponding results were obtained in all RI neurons tested. All of them showed modulation of the discharge with a maximal activity in the stance phase. Most cells (8 out of 11) were completely silent in the swing phase but in 3 units depression in this phase was not complete. The maximal frequencies were observed usually in the middle of the stance phase; these frequencies varied from 50 to 250 pulses/sec, with the average value of 150 pulses/sec. In some cases an increase in the activity of RI neurons after the start of LR stimulation appeared before any limb movements began. The results described above show that during locomotion RI neurons are active at the stance phase of the step, on the whole simultaneously with alpha-motoneurons

186 of 'their' muscle, i.e. the muscle whose la afferents terminate the RI neurons. The la afferents of extensors are known to be modulated during locomotion with a maximal activity at the stance phase, simultaneously with extensor muscles '~7. Such a pattern of the la afferent discharge is determined by the influence of gamma-motoneurons which are active in the stance phase like alpha-motoneurons, and thus enhance the discharge of the spindle afferents in this phase. That is why the modulation of RI neurons during locomotion could be explained by rhythmic alternations of the afferent flow coming to them through la fibers. On the other hand the modulation of RI neurons can be determined not only by peripheral but also by central factors (both spinal and supraspinal). To establish the contribution of these sources, activity of RI neurons was investigated in preparations with motionless hindlimbs.

Preparation with hindlimbs de-efferented (a) Central modulation of RI neurons. In this series of experiments 25 RI neurons were recorded: 23 RI neurons of Q and 2 neurons of PBSt. Resting discharge frequency of these neurons was usually of 5-50 pulses/sec depending on the limb position. The L R stimulation resulted in tonic activation of RI neurons and after that a periodic modulation of the activity appeared. A typical pattern of discharge is shown in Fig. 3A-D (upper trace). A simultaneous recording of the MSR evoked in

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D Fig. 3. Activity of an RI neuron of Q during 'fictive' locomotion of the preparation with the hindlimbs de-efferented. The upper trace in A - D is the activity of the neuron; the lower trace is the MSR in the L7 ventral root to stimulation of the G nerve with a strength of 1.5 threshold (the threshold is determined by appearance of an afferent wave). After the LR stimulation is started (marked by an arrow in A) activity of the neuron and an amplitude of the MSR increase. In the recording of B (which is the continuation of A) the 'fictive' locomotion arises. C: the same neuron with stronger LR stimulation. D: the same neuron during non-stable locomotion. E and F: the MSR recorded with high time resolution, at rest (E) and soon after the LR stimulation was started (F); the lower trace is the recording from the L7 ventral root entry zone.

187 the L7 ventral root by stimulation of the G nerve is also presented (Fig. 3A-D, lower trace; cf. Fig. 3E and F, upper trace). After L R stimulation was started (arrow in Fig. 3A) the M S R gradually increased in parallel with the activity of the R I neuron. Then the rhythmic process arose in the spinal cord resulting in periodic oscillations of the M S R (Fig. 3B) - - 'fictive' locomotion. H a d the hindlimbs not been immobilized they would have performed stepping movements. At the same time periodic alternations of activity of the R I neuron appeared: the neuron discharged in the phase when the M S R was maximal. Stronger L R stimulation resulted in an increase of the frequency of 'stepping' (Fig. 3C); the bursts of the neuron activity also became more frequent, frequency within the bursts increased, but phase relations did not change; the neuron was active when the MSR was large. A close correlation between the activity o f extensor alpha-motoneurons and that of R I neurons is also seen in Fig. 3D where stepping was not regular. Twenty-two out of 23 recorded RI neurons of Q were modulated. Most of them (18 out of 22) discharged by bursts and were silent in between the bursts, like the neuron in Fig. 3 (cf. Figs. 5B, C and 7B). In a few neurons the discharge frequency in the phase of minimal activity did not fall to zero (Fig. 5E). In the middle of the phase of maximal activity frequencies of the bursts were 50-200 (sometimes up to 300) pulses/see, with an average of 120 pulses/see. In all cases when activity of an RI neuron was recorded simultaneously with the M S R evoked in the ventral root by stimulation of the extensor nerve (Q or G), the phase of the maximal activity of the neuron coincided with that of the maximal MSR, as in Fig. 3. Fig. 4A shows the activity of an R I neuron of PBSt during 'fictive' locomotion (upper trace) as well as an M S R in VL6 to stimulation of the Q nerve (lower trace). One can see that the neuron was active at minimal values of MSR, i.e. when the activity of the extensor motoneurons was minimal. The second recorded RI neuron of PBSt had a similar pattern of discharge. This data indicates that R I neurons o f PBSt are active in the flexor ('swing') phase of the 'step' in contrast to R I neurons o f Q which are active in the extensor ('stance') phase. The present data show that the activity of R I neurons has distinct periodic

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188 modulation without any periodic changes in the afferent flow coming from the hindlimbs. In part of the experiments stimulation of LR evoked not only periodic alternations of the MSR but also stepping movements of the forelimbs (see Methods). However, since modulation of the activity of RI neurons was observed not only in these cases, periodic changes of the afferent flow from the forelimbs seem not to be necessary for the modulation. The data presented here clearly show that the modulation of RI neurons has a central origin. To clear the role of spinal and supraspinal mechanisms in modulation of RI neurons, their activity was studied in preparations in which the cerebellum was removed. In these preparations no noticeable modulation of activity in descending pathways was revealed (vestibulospinal, reticulospinal and rubrospinal) correlated to stepping movements during locomotion 19-zl. This finding showed that in decerebellate cats generation of stepping is performed by the spinal mechanism only. An example of the activity of an RI neuron of PBSt in decerebellate cat with the hindlimbs de-efferented is given in Fig. 4B (activity of the same neuron prior to cerebellar ablation is shown in Fig. 4A). With LR stimulation a periodic process arose in the spinal cord that is shown by changes of the MSR evoked in VL6 by stimulation of the Q nerve (lower trace); activity of the RI neuron was also periodically modulated in the same manner as prior to decerebellation. All 3 neurons recorded in decerebellate preparations had periodic modulation (one of PBSt and 2 of Q). Both RI neurons of Q were active in extensor phase. Thus rhythmic supraspinal influences are not the only reason for modulation of the RI neurons. This modulation can arise within the spinal cord in parallel with rhythmic modulation of corresponding motoneurons. As result, RI neurons of Q are activated simultaneously with Q motoneurons in that phase which, in real locomotion, would be a stance phase, while RI neurons of PBSt are activated in the opposite phase. (b) Contribution of muscle afjerents to the excitation of RI neurons. In the experiments described above an afferent inflow to RI neurons was weak: it was determined by the activity of afferents of the relaxed muscle deprived of the gammaefferent innervation. In one experiment this input was completely abolished by cutting the Q nerve behind the stimulating electrode. The modulation of RI neurons was observed in this case as well. So the effect of modulation was not determined by any periodic changes in presynaptic inhibition of la terminals. In real locomotion the RI neurons of Q would receive during the stance phase a powerful afferent input from the muscle spindles activated in this phase by gammamotoneurons 27. To approximate this real case we stretched the Q muscle with a force of about 0.5 kg applied to its tendon. Fig. 5A shows that at rest, before LR stimulation was started, such extension resulted in a considerable activation of the RI neuron (the frequency increased from 15 to 50 pulses/sec). Then the rhythmic process ('fictive' locomotion) was evoked in the spinal cord by the LR stimulation; the RI neuron periodically generated bursts of impulses with a frequency of about 80 pulses/ sec (Fig. 5B). Now the stretch of the Q with the same force led to an increase of the activity of the neuron, the bursts became 1.5-2 times longer, the frequency became 2 0 - 3 0 ~ higher, the amount of pulses within the burst was approximately 2 times

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190 A role of the afferent input from muscles was also studied in one RI neuron o f PBSt. Fig. 6A shows that the passive extension at the knee joint (i.e. the stretch of knee flexors) resulted in a reflex excitation of the neuron. During 'fictive' locomotion when the neuron activity was periodically modulated the knee extension was still effective: in all phases the frequency increased about 2 times. These results show that the afferent flow coming to RI neurons from the extended muscle increases their activity. It seems obvious that the main contribution to this effect belongs to la afferents, since the l a afferents compose the main input to R I neurons 7. However, other types o f receptors are also activated during stretching. To obtain the effects of the selective activation of la fibers, stimulation of the Q nerve was tried with a strength exciting only la afferents. Fig. 6 shows the results of such an experiment with continuous recording of the activity of an RI neuron of Q (lower trace) and with a high speed sweep triggered by the stimuli to the Q nerve (upper traces). At rest, before L R stimulation was started, the neuron responded to each stimulus (A). During 'fictive' locomotion evoked by LR stimulation (B) the neuron generated periodically bursts of pulses separated by phases of silence. Now stimulation of the Q nerve was effective only during the bursts of activity of the neuron or near the burst. It is also seen that the latency of the response changes decreasing gradually from the beginning of the burst toward its middle and then increasing toward the end of burst; that may be explained by periodic changes of the excitability of the neuron. These results show clearly that the selective activation of la afferents

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Fig. 8. Inhibitory effects evoked in RI neurons by stimulation of the ventral root (hindlimbs de-efferented). A-C: RI neuron of Q, stimulation of the VL6 with frequency of 70 pulses/sec (marked by horizontal lines). There are shown effects at rest (A) and 'fictive' locomotion with weak (B) and strengthened (C) LR stimulation. D and E: RI neuron of PBSt, stimulation of the VL7 with frequency of 40 pulses/see (marked by artifacts). There are shown effects at rest (D) and during 'fictive' locomotion (E). results in an increase of the amount of impulses generated by the R I neuron. It seems important that the sensitivity of the neuron to the afferent input varies considerably during the 'step': the neuron reacts easily to the impulses from l a afferents coming in the phase when extensors must be active, and does not react in the opposite phase. The findings lead one to assume that, in real locomotion, discharge of RI neurons is determined by two main factors: (i)central influences; and (ii)impulses coming by la fibers. An exact evaluation of the relative contribution of both sources is difficult, but they seem to be of the same order since activation o f muscle receptors in natural physiological limits usually led to two-fold increase of the activity of R I neurons. We do not compare mean frequencies obtained in experiments with intact and de-efferented limbs (150 and 120 pulses/sec) since in the first case the intensity of locomotion was not high enough (rnicroelectrode recording during energetic movements were difficult to perform). (c) Ej~ciency of the recurrent inhibition of RI neurons. It is known that the recurrent inhibition of alpha-motoneurons is suppressed during locomotion zs, which can be explained by the inhibition o f the Renshaw cells. Renshaw cells are known to mediate the recurrent inhibition of R I neurons too s,9, that is why one could expect a decrease of the recurrent inhibition of R I neurons during locomotion. Fig. 8 A - C shows the effect of stimulation of the VL6 on the RI neuron of Q. At rest, before LR stimulation was started, repetitive stimulation of the ventral root inhibited completely the activity of the RI neuron evoked by Q extension (A). The L R stimulation elicited a periodic process in the spinal cord. When this process was rather weak (B) stimulation of the ventral root was still effective. With stronger L R stimu-

192 lation the generation became more intensive (C) and efficiency of the recurrent inhibition of the RI neuron decreased. This test was used with 4 neurons. The efficiency of the recurrent inhibition during 'fictive' locomotion decreased in two RI neurons of Q and did not change in one RI neuron of Q and one RI neuron of PBSt (Fig. 8D, E).

DISCUSSION

(a) Central program o f activity o f R I neurons. The main result of this study is

a demonstration of strongly expressed modulation of RI neurons during locomotion. This modulation turned out to be determined by two main factors: (i) central influences, i.e. influences from the mechanisms generating stepping movements, and (ii) afferent flow from muscles. Existence of the central program of modulation of RI neurons was demonstrated in the experiments carried out on preparations with the hindlimbs immobilized. This central modulation is shown to be determined, to a large extent, not by supraspinal commands but by intraspinal rhythmic processes, since the modulation was found also in decerebellate preparations which practically have no rhythmic alternations of activity in the descending pathwayslg-~L According to the central program, RI neurons are activated in the same phase of the step as alpha-motoneurons of the corresponding muscle; in the opposite phase, when motoneurons are not active, activity of RI neurons is also decreased or completely inhibited. Thus RI neurons controlled by the spinal mechanisms perform the inhibition of the motoneurons of antagonistic muscles in that phase when their 'own' motoneurons are active. In this way RI neurons, even deprived of the afferent input, participate in generation of the basic pattern of stepping, i.e. alternating, reciprocal activation of flexors and extensors. Gamma-motoneurons are known to discharge in parallel with alpha-motoneurons of the same muscle during locomotion25-zL Periodic modulation of their activity is also determined, at least partly, by the central program of stepping, since it may be seen not only in preparations with intact innervation of limbs 27, but also in those with deafferented limb 24. This data together with the results presented in this paper lead one to conclude that during locomotion there exists a central program of co-activation of alpha-motoneurons, gamma-motoneurons and RI neurons, belonging to an extensor (or to a flexor) group of muscles. The finding that several neuronal pathways, both spinal and supraspinal, evoke parallel effects in alpha-motoneurons, gamma-motoneurons and RI neurons, led Hongo et al. 6 to the conception of 'alpha-gamma-linked reciprocal inhibition'. According to the conception, the three mentioned groups of neurons are expected to be active simultaneously in those types of movements where inhibition of the antagonist muscles is useful. Our results confirm this hypothesis. Presented data also confirm the suggestion that the central control of RI neurons, to some extent, can be mediated by Renshaw cells7. Indeed, the example has been presented which show the decrease of efficiency of the recurrent inhibition

193 of RI neurons through the collaterals of motor axons during 'fictive' locomotion (Fig. 8C), that indicates the inhibition of Renshaw cells.

(b) RI neurons periodically 'open and close' the pathway for the reflex inhibition of antagonistic muscles. At that phase of the cycle when RI neurons are excited by central program they are very sensitive to the signals coming through la fibers (cfi Fig. 7). That is why activity of la afferents at this phase results in strong inhibition of motoneurons of antagonistic muscles. On the contrary, at the phase when activity of RI neurons is suppressed by the central program, impulses in la fibers do not inhibit motoneurons of antagonistic muscles. The efficiency of such a mode of regulation of the reflex inhibition is obvious: in the first case, according to the basic pattern of muscular activity in stepping, the antagonist must be suppressed and the system of reflex inhibition promotes this; in the opposite phase the antagonist must be active, and the pathway for reciprocal inhibition is interrupted. How strong is the reciprocal inhibition during locomotion? Our results show that even during rather weak locomotion the discharge rate of RI neurons in the phase of maximal activity reaches 50-250 pulses/sec. These frequencies are higher than those obtained in the same neurons during passive stretch of corresponding muscle. On the other hand, it is known that passive stretch of a muscle in decerebrate and spinal cat results in considerable inhibition of the antagonist13, is. These data suggest that during locomotion RI neurons efficiently inhibit the motoneurons of antagonistic muscles. ACKNOWLEDGEMENT

We wish to thank Dr. I. B. Kozlovskaja for helpful discussion as well as for assistance during preparation of the manuscript.

REFERENCES 1 ECCLES,J. C., FATT, P., AND LANDGREN,S., The central pathway for the direct inhibitory action of impulses in the largest afferent nerve fibres to muscle, J. Neurophysiol., 19 (1956) 75-98. 2 ECCLES, R. M., AND LUNDBERG, A., The synaptic linkage of 'direct' inhibition, Acta physiol. scand., 43 (1958) 204-215. 3 ENGBERG,J., AND LUNDBERG,A., An electromyographic analysis of muscular activity in the hindlimb of the cat during unrestrained locomotion, Acta physiol, scand., 75 (1969) 614-630. 4 GAMBARIAN,P.P., ORLOVSKY, G . N . , PROTOPOPOVA, T. G., SEVERIN, F. V., AND SHIK, M.L., Muscular activity at different kinds of cat locomotion, and adjusting changes of the organs of movements in the family Felidae, Proc. zool. Inst. U.S.S.R., 48 (1971) 220-239. 5 GRANIT, R., Reflex self-regulation of muscle contraction and autogenetic inhibition, J. Neurophysiol., 13 (1950) 351-372. 6 HONGO, T., JANKOWSKA,E., AND LUNDBERG, A., The rubrospinal tract. II. Facilitation of interneuronal transmission in reflex paths to motoneurones, Exp. Brain Res., 7 (1969) 365-391. 7 HULTBURN, H., Convergence on interneurones in the reciprocal la inhibitory pathway to motoneurones, Acta physiol, scand., Suppl. 375 (1972) 1-42. 8 HULTBURN, H., JANKOWSKA, E., AND LINDSTR~M, S., Recurrent inhibition from motor axon collaterals of transmission in the la inhibitory pathway to motoneurons, J. Physiol. (Lond.), 215 (1971) 591~512. 9 HLrLTBURN,n., JANKOWSKA,E., AND LINDSTROM,S., Recurrent inhibition of interneurons monosynaptically activated from group la afferents, J. Physiol. (Lond.), 215 (1971) 613-636.

194 10 HUNT, C. C., The effect of stretch receptors from muscle on the discharge of motoneurones, .L Physiol. (Lond.), 117 (1952) 359-379. 11 JANKOWSKA,E., AND ROBERXS,W,, Function of single interneurones established by their monosynaptic inhibitory effects on motoneurones, Acta physiol, scand., 82 (1971) 24-25A. 12 JANKOWSKA,E,, AND ROBERTS,W. J., Synaptic actions of single interneurones mediating reciprocal la inhibition of motoneurones, J. Physiol. (Lond.), 222 (1972) 623-642. 13 LIDDELL, E. G. T., AND SHERRINGTON, C. S., Reflexes in response to stretch, Proe. roy. Soc. B, 96 (1924) 212-242. 14 LLOYD, D. P. G., A direct central inhibitory action of dromically conducted impulses, J. Neurophysiol., 4 0941) 184-190. 15 LLOYD,D. P. C., Reflex action in relation to pattern and peripheral source of afferent stimulation, J. Neurophysiol., 6 (1943) 111-120. 16 LLOYD, D. P. C., Neuron patterns controlling transmission of ipsilateral hind limb reflexes in cat, J. Neurophysiol., 6 (1943) 293-315. 17 LUNDBERG,A., Reflex control of stepping. In The Nansen Memorial Lecture, U niversitetsforlaget, Oslo, 1969, pp. 1-42. 18 MATTHEWS, P. B. S., Mammalian Muscle Receptors and their Central Actions, Arnold, London, 1972, pp. 1-630. 19 ORLOVSKY, G. N., Activity of reticulo-spinal neurons during locomotion, Biophysics, 15 (1970) 761-771 (Eng. trans, of Biofizika, 15 (1970) 728-737). 20 ORLOVSKY,G. N., Influence of the cerebellum on the reticulospinal neurons during locomotion, Biophysics, 15 (1970) 928-936 (Eng. trans, of Biofizika, 15 (1970) 894-901). 21 ORLOVSKY, G. N., Activity of vestibulospinal neurons during locomotion, Brain Research, 46 (1972) 85-98. 22 ORLOVSKY, G. N., Activity of rubrospinal neurons during locomotion, Brain Research, 46 (1972l 99-112. 23 ORLOVSKY, G. N., AND FELDMAN, A. G., On the role of afferent activity in generation of stepping movements, J. Neurophysiol., 4 (1972) 401-409. 24 PERRET, C., Analyse des Mdcanismes d'une Aetivitd de Type locomoteur chez le Chat, Th6se Doct. Sci., CNRS A08342, Paris, 1973, pp. 1-259. 25 PERRET, C., ANO BUSER, P., Static and dynamic fusimotor activity during locomotor movements in the cat, Brain Research, 40 (1972) 165-169. 26 PERRET, C., AND BERTHOZ, A., Evidence of static and dynamic fusimotor actions on the spindle response to sinusoidal stretch during locomotor activities in the cat, Exp. Brain Res., (1973). 27 SEVERIN, F. V., ORLOVSKY, G. N., AND SHIK, M. L., Work of the muscle receptors during controlled locomotion, Biophysics, 12 (1967) 575-586 (Eng. trans, of Biofizika 12 (1967) 502-511 ). 28 SEVERIN,F. V., ORLOVSKY,G. N., AND SHIK, M. L., Recurrent inhibitory effects on single motoneurones during an electrically evoked locomotion, Bull. exp. Biol. Med., 66 (1968) 3-9. 29 SHIK, M. L., ORLOVSKY, G. N., AND SEVERIN, F. V., Control of walking by means of electrical stimulation of the mid-brain, Biophysics, II (1966) 756-765 (Eng. trans, of Biofizika, 11 (1966) 659-666). 30 S,~K, M. L., SEVERIN, F. V., AND ORLOVSKY, G. N., Structures of the brain stem responsible lbr evoked locomotion, Sechenov physiol. J. U.S.S.R., 53 (1967) 1125-1132.