Recurrent inhibition between motor nuclei innervating opposing wrist muscles in the human upper limb

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1 5571 Journal of Physiology (1997), 499.1, pp Recurrent inhibition between motor nuclei innervating opposing wrist muscles in the human upper limb C. Aymard*t, B. Decchit, R. Katzt, C. Lafittet, A. Penicaudt, S. Raoult and A. Rossit t Laboratorio di Neurofisiologia, Istituto di Scienze Neurologiche, Universita' di Siena, V Le Bracci, I-531 Siena, Italy, t Clinical Neurophysiology, Reeducation, H6pital de la Salpetriere, 47 boulevard de l'h6pital, F , Paris cedex 13; France and * Universite de Bourgogne, 7 rue Jeanne d'arc, 21 Dijon, France 1. Effects of conditioning motor volleys of increasing amplitude on antwonistic motor nuclei were tested at the wrist and elbow level in man. 2. The modifications of biceps, triceps, flexor carpi radialis (FCR) and extensor carpi radialis (ECR) motoneurone excitability were tested both in post-stimulus time histogram (PSTH) and rectified averaged EMG experiments. 3. Conditioning motor volleys were evoked in biceps, triceps, FCR and ECR muscles by electrical stimuli applied to the corresponding nerves. The intensity of the conditioning stimuli was systematically varied from an intensity just below motor threshold to one giving rise to a motor response equal to half of the maximal direct motor response (Mmax). 4. The effect of the injection of a cholinergist agonist, L-acetylcarnitine (L-Ac), on antagonistic conditioning motor volleys was tested for each motor nucleus. 5. Results obtained at the wrist and elbow level were strikingly different. Antagonistic motor volleys resulted in an early and long-lasting inhibition enhanced by L-Ac injection in wrist motor nuclei, while no modification was observed in elbow motor nuclei. 6. The characteristics of the early and long-lasting inhibition evoked in wrist motor nuclei by antagonistic motor volleys suggest that they are due to the activation of Renshaw cells by the conditioning motor volleys. 7. It is therefore concluded that these experiments provide further evidence that the interconnections between motoneurones, interneurones mediating reciprocal inhibition and Renshaw cells at the wrist level are different from those described elsewhere. Indeed, it has already been shown that the interneurones mediating reciprocal inhibition between FCR and ECR are not inhibited by Renshaw cells, and the results presented here suggest that FCR-coupled Renshaw cells inhibit ECR motoneurones and vice versa. Reciprocal Ia inhibition, recurrent inhibition and their interactions have been extensively described in the lumbar spinal cord of the cat (for references, see Baldissera, Hultborn & Illert, 1981). Their features appear to follow a general rule for motor nuclei innervating limb muscles, not only in the spinal cord of the cat, but even in monkey and man, although results were more scattered in monkey or man. In summary, (i) homonymous recurrent inhibition is present in all motor nuclei, except those innervating the intrinsic muscles acting at digits, (ii) recurrent inhibition is present in synergistic motor nuclei but never in motor nuclei innervating antagonistic muscles acting at the same joint, and (iii) Renshaw cells inhibit not only motoneurones, but also Ia inhibitory interneurones (Hultborn, Jankowska & Lindstrom, 1971). However, muscles acting at the human wrist joint seemed to differ from this schema: indeed, homonymous recurrent inhibition is present in both flexor carpi radialis (FCR) and extensor carpi radialis (ECR) motor nuclei (Katz, Mazzocchio, P6nicaud & Rossi, 1993) and activation of their Ia fibres results in a disynaptic reciprocal inhibition (Baldissera, Campadelli & Cavallari, 1983; Day, Marsden, Obeso & Rothwell, 1984), but Aymard, Chia, Katz, Lafitte & Penicaud (1995) failed to show any depression of this To whom correspondence should be addressed.

2 268 C. Aymard and others J Physiol. 49S9. 1 reciprocal inhibition by stimulation of FCR and ECR Renshaw cells. The question thus arises whether projections of wrist motor nuclei-coupled Renshaw cells also differ when taking into account their projections to motoneurones, i.e. if FCR-coupled Renshaw cells project to ECR motoneurones and vice versa. This study attempts to answer this question. To highlight the results obtained at wrist level, the homologous experimental protocol was first applied at elbow level, since at this level it has been already shown that the projections of Renshaw cells exhibit similar projections to those described in the cat lumbar spinal cord. METHODS General experimental procedure Experiments were carried out partly in the Siena laboratory (seven subjects) and partly in the Paris laboratory (twelve subjects). The pharmacological experiments were all performed in Siena. All subjects were healthy adult volunteers aged 23-5 years who had given their written, informed consent to the experimental procedure, which was approved by the respective appropriate institutional local ethics committees. The subjects were comfortably seated in an armchair. The examined (right) arm was rested on an armrest with a shoulder abduction of about 6 deg; the elbow was semi-flexed and the wrist extended. In all the experiments, except in those with pharmacological stimulation of recurrent inhibition (see below), the surface electromyogram (EMG) was recorded by two non-polarizable disc electrodes ( 9 cm diameter) placed 1-5 cm apart and secured to the skin over the belly of the triceps, biceps, FCR and ECR muscles. Conditioning stimuli Conditioning stimuli were electrical shocks of 1 ms duration applied to the musculo-cutaneous nerve, the branch of the radial nerve innervating the triceps, and the median and radial nerves. The musculo-cutaneous and triceps nerves were stimulated through stimulating electrodes placed a few centimetres below the shoulder, ensuring that even using the maximal intensity delivered by the stimulators, there was no encroachment on other nerves (i.e. no motor response and/or paraesthesia at wrist or digit level) (Katz, P6nicaud & Rossi, 1991). The median and radial nerves were stimulated at elbow level (Cavallari & Katz, 1989). The current delivered by constant current stimulators was measured by a current probe (Tektronix 621) and the stimulus intensity was expressed in multiples of the threshold intensity (T) of the motor wave. The EMG of the muscle innervated by the nerve involved in the conditioning stimulus was recorded and averaged by a Digital Signal Averager (Nicolet 37) to detect the direct motor and/or the reflex response evoked in this muscle by the conditioning stimulation. Study of single motor units The effects of a conditioning stimulus on a voluntarily activated motor unit can be determined by constructing a time histogram of the occurrence of motor unit spikes following repeated presentation of the stimulus. The post-stimulus time histogram (PSTH) extracts from the naturally occurring spike train only those changes in firing probability that are time locked to the stimulus (Stephens, Usherwood & Garnett, 1976). The validity of the method to detect postsynaptic potentials in motoneurones has been established by intracellular recordings (Fetz & Gustafsson, 1983). A detailed description of the particular method used in this section has been already published (Fournier, Meunier, Pierrot-Deseilligny & Shindo, 1986) and will be only summarized here. The EMG from single motor units was recorded while the subject performed a very weak (below 5% of maximal voluntary force) steady contraction. The EMG potentials of single motor units were converted into standard pulses by a discriminator with variable trigger levels, and used to trigger first a computer (Quadra 95) and then the stimulators delivering nerve stimulations. The motor unit potential and the trigger pulse were continuously monitored to detect false triggers due to other active units and to ensure that the motor unit shape and trigger position remained constant within and between sequences. In the method described by Fournier et al. (1986) the nerve stimulation is delivered at a fixed time interval after a previous motor unit discharge, thus allowing the choice of delay at which the probability for a new discharge is high and to reduce the number of trials. In this method the probability of discharges in the PSTH depends not only on the postsynaptic potentials evoked by the stimulation, but also on the motoneurone membrane trajectory during the interspike interval. To take the latter into account, a control histogram of firing probability was constructed in the absence of stimulation (see Fig. 5B). The control and the different conditioning situations were randomly presented within a sequence. The control histogram represented the background firing probability with which the results following stimulation were compared. To clarify the stimulus-induced changes, the control value in each bin was subtracted from that obtained after stimulation. Within different time interval windows, a x2 test was used to determine to what extent the distribution of firing probability after stimulation differed from that obtained in the control situation. Such an analysis was performed after having checked that in the control situation the firing probability within the window of analysis did not differ from the mean probability of discharge of the unit. The onset of the changes in firing probability following the conditioning stimulation was visually identified. It was then required that (1) the number of counts in the bins following the onset was significantly different from the control values and (2) the number of counts in the bins preceding this onset did not differ significantly with and without stimulation. To perform the statistical analysis, the duration of the bin window was increased by summing individual bins so that there were at least five counts (validity conditions for x2 test). Rectified averaged EMG experiments Multi-unit EMG activity was recorded during maintained selective isometric tonic contractions of the relevant muscles. The force developed during these tonic contractions was roughly 2% of the maximum voluntary force. The EMG was filtered (1 Hz to 1 khz), full-wave rectified and averaged against the conditioning stimuli over 6 ms, using a sampling rate of 1-2 khz. The stimulus sequence consisted of random alternation every 4 ms of a control trial (without stimulation), and a trial with conditioning stimulation. The averages obtained with conditioning stimulations were expressed as a percentage of the control average EMG activity. Each series usually consisted of 2 trials and was repeated to ensure reproducibility. To test the significance of the changes in the EMG activity, the rectified EMG was integrated for the duration of the inhibition and a variance analysis was performed to compare the values obtained in control and conditioned situations. Rectified averaged EMG experiments were performed to support the results obtained in PSTH experiments. Indeed, PSTH experiments deal with the modifications of excitability of slower motor units, while

3 J: Physiol Recurrent inhibition between opposing wrist muscles in man 269 rectified averaged EMG experiments deal with the modifications of excitability of a population of faster motor units, including faster ones than those studied in PSTH experiments. Study of recurrent inhibition The experimental protocol used to study the projections of Renshaw cells coupled to a given motor nucleus onto other motor nuclei has been set up in PSTH experiments (Meunier, P6nicaud, Pierrot- Deseilligny & Rossi, 199) and has already been used to study the distribution of recurrent inhibition among motor nuclei innervating various muscles of the human upper limb (Creange, Katz, Meunier, Penicaud & Pierrot-Deseilligny, 1992; Katz et al. 1993). The first step of this study was to establish that the experimental protocol set up in PSTH also satisfied averaged EMG experiments. 2 B e 2 c.o. _ o b-2 o E E C 2 z $o1 -.5 _O 15- u.6o E D F I -1 mv 1 ms It co o wo w. -- = cuoao (Y'B; G I Latency (ins) H J II IW Figure 1. Changes in triceps motor pool excitability induced by an electrical stimulation of the median nerve A-D, changes in firing probability evoked in a voluntarily activated triceps motor unit by electrical median nerve stimulation. PSTHs were obtained in the control situation and after stimulation. Each bar represents the difference between the two histograms (see Fig. 6B, C and D). The zero of the abscissa corresponds to the latency of the heteronymous monosynaptic peak. A, PSTH of the discharge of the unit in response to a median nerve stimulation just below motor threshold (B). C, PSTH of the discharge of the unit in response to an FCR reflex discharge (D); B and D are averaged EMG response in ECR. E-J, changes in rectified averaged triceps EMG. E, G and I, the EMG of the triceps muscle was recorded during steady tonic voluntary contractions equal to 2% of the maximal voluntary contraction during the control situation and after stimulation, and the figure represents the differences between the two situations. The zero of the abscissa corresponds to the latency of the monosynaptic effects. Ordinate: size of EMG expressed as a percentage of its control value; each trace represents the mean of 2 measurements. F, H and J, averaged motor and reflex responses in the FCR. Number of triggers: A, 132; C, 288; E, Gand I, 4.

4 27 C. Aymard and others J Phy8iol The main features of the experimental protocol are summarized in Fig. 1, where the effects of the activation of FCR Renshaw-coupled cells onto triceps motoneurones are illustrated. The intensity of the conditioning stimulus (applied to the median nerve in the example illustrated here) was adjusted to be just below the threshold of motor or reflex response, and thus below the activation of Renshaw cells (Fig. 1B). As already shown by Katz et al. (1993), a slight increase in firing probability of the test triceps motor unit appeared (Fig. 1A, first bin), which was ascribed to heteronymous Ia facilitation. When the intensity of the conditioning stimulus applied to the median nerve was increased and gave rise to a FCR monosynaptic reflex response, and therefore to an activation of Renshaw cells via the reflex volley in the motor axons (Fig. 1D), it resulted in a long-lasting decrease in firing probability of the test triceps motor unit (Fig. 1 C). This long-lasting inhibition was linked to that of a volley in motor axons and its size and duration increased with the size of the motor response. It is therefore probably due to recurrent inhibition (see Katz et al and Discussion). Exactly the same kind of results were obtained when the effects of similar conditioning stimuli were tested on rectified averaged EMG. When the intensity of the median conditioning stimulus gave rise to a reflex response (Fig. 1H), a long-lasting inhibition of the triceps EMG appeared (Fig. 1 G), which increased with the size of the conditioning motor response (Fig. 1I and J), whereas it was not seen (Fig. 1E) when the intensity of the conditioning stimulus was just below motor and reflex threshold (Fig. 1F). Pharmacological stimulation of recurrent inhibition Test EMG activity was recorded by high-impedance needle electrodes (single-fibre needle electrodes) inserted percutaneously into the muscle so as to give selective recordings and to minimize contamination and far-field effects. Voluntary contraction was measured and monitored by surface electrodes (3 cm interelectrode distance) placed near the needle electrodes. Muscle activity was kept constant between 5 and 1% of maximum voluntary contraction. Test EMG activity usually consisted of three or four single action potentials. In fact, because of the conditioning twitch induced in the antagonistic muscle, a stable needle recording from an isolated single fibre could only occasionally be maintained throughout the experimental session. The raw signal was full-wave rectified and low-pass filtered at 3 Hz before averaging. Conditioning stimuli were applied as described above. The size of the conditioning motor response in FCR and ECR (ranging from 9 to 48% of the maximal direct motor response, Mmax; mean+ S.D., %) was chosen so as to evoke a long-lasting inhibition 3-5% of its maximum size. For biceps and triceps nerves, conditioning motor response was kept between 3 and 4% of Mmax (36x + 3 6%). Intravenous administration of L-acetylcarnitine (L-Ac), a cholinergic agonist, was used to potentiate recurrent inhibition. The method has been described in detail by Mazzocchio & Rossi (1989) and only its main features will be summarized here. Two infusion sets were prepared and connected to the subjects via a two-way tap. The first bottle contained normal saline and the second a solution of 2 g L-Ac (saline acetate buffer) dissolved in 1 ml of sterile distilled water. Continuous infusion of normal saline was begun after setting appropriate stimulation and recording parameters. This was followed by L-Ac administration for 8-14 min. Fifteen to twenty minutes before, during and 7-9 min after L-Ac infusion, a series of 2 pre- and post-stimulus periods of activity (5 ms analysis time) applied in a pseudorandom sequence were recorded. During each experimental phase, surface EMG recording, single-fibre activity, as well as conditioning motor responses, were monitored. Responses to conditioning stimulation were defined as the difference between the rectified and averaged conditioned and unconditioned single-fibre activity. The size of the short latency inhibition in FCR and ECR was measured in terms of area. This was computed offline after setting the analysis window between the first and the second intersection of the trace with the baseline (from 2-25 to 35-4 ms; see Fig. 6B and C). The same analysis window was also used to measure the area of the conditioning triceps and biceps EMG activity. RESULTS Rectified averaged EMG experiments The main finding of these experiments is illustrated in Fig. 2 and originates from a comparison of the results obtained from the homologous experimental protocol at elbow and wrist level. Indeed, at elbow level, the excitability of motoneurones was not modified by the activation of antagonistic motor axons, while at wrist level the excitability of motoneurones was deeply depressed by the activation of antagonistic motor axons. In Fig. 2A, the modifications of the biceps EMG recorded during a voluntary steady tonic contraction following electrical stimulations of the triceps nerve are represented. The intensity of these electrical stimulations was: (a) below motor threshold (upper trace); (b) adjusted to give rise to a triceps direct motor response equal to Mmax/4 (middle trace); and (c) a triceps direct motor response, equal to Mmax/2 (lower trace). Whatever the intensity of the antagonistic nerve stimulation, no significant modification of the biceps EMG was revealed. The size of conditioned and control biceps on-going rectified 2 ms of the traces EMG was calculated during the first given in Fig. 2A. The ratio between conditioned and control EMG was % (mean + S.E.M.) just below motor threshold, % when the conditioning triceps motor volley was equal to Mmax/4 and % for a conditioning motor volley equal to Mmax/2. In Fig. 2B, the modifications of the triceps EMG following electrical musculo-cutaneous nerve stimulation of increasing intensities are shown. The results are exactly the same as those represented in Fig. 2A. The ratio between conditioned and control triceps on-going rectified EMG was %, just below motor threshold, % for a conditioning biceps motor volley equal to Mmax/4 and P7 % for a conditioning motor volley equal to Mmax/2. In Fig. 2C, the modifications of the FCR EMG following radial nerve stimulation of increasing intensities are represented. No long-lasting inhibition was seen when the intensity of the radial nerve stimulation was just below motor threshold; the ratio between unconditioned and control FCR on-going rectified EMG was equal to %. However, contrary to what was seen at elbow level, the appearance of an ECR direct motor response resulted in a long-lasting inhibition of the FCR EMG. The greater the ECR motor response, the Ap,eper the inhibition (cf. middle and lower traces in Fig. 2C).

5 J Physiol Recurrent inhibition between opposing wrist muscles in. man.ks c 1 (!-' 8 W 6 4 C) w.5 c - c) a O- u. o A Bicens IM= 4 At% 14U B Triceps 1 -,,, 6., C M= M,1/ ~~~ ,,, 6, C M= M 1/ L FCR M = 14 12, "- 6 8 D ECR 2 I, M = MmI,/ [ uj o 1 F 6 F 2 L.5 I ooi- /. CD -'o 6-3.w 2 - r 1 6,, I I.~~~~~~~ M = M,,/2 12 r Figure 2. Changes in rectified averaged EMG induced by antagonistic nerve stimulation A, biceps EMG voluntary activity is conditioned by an electrical stimulation applied to the triceps nerve. B, triceps EMG voluntary activity is conditioned by an electrical stimulation of the biceps nerve. C, FCR EMG voluntary activity is conditioned by an electrical stimulation applied to the radial nerve. D, ECR EMG voluntary activity is conditioned by an electrical conditioning stimulation applied to the median nerve. Upper traces, the conditioning stimulation is adjusted to be just below motor threshold (M is the motor response). Middle traces, the conditioning stimulation gave rise to a direct motor response equal to Mmax/4 (Mmax is the maximal motor response). Lower traces, the conditioning stimulation gave rise to a direct motor response equal to Mmax/2. Number of triggers: A, 4; B, 8; C, 4; D, 8.

6 272 C. Aymard and others J. Phy8ioL 49t9.1 Table 1. Mean values of inhibition of on-going rectified EMG evoked by antagonistic motor volleys Intensity of the antagonistic motor volleys On-going EMG T Mmax/4 Mmax/2 Biceps Triceps FCR ECR T, motor threshold. In each experiment, the amount of inhibition was determined as the ratio between ongoing rectified EMG obtained following antagonistic and control situations (i.e. without stimulation). The rectified EMG was integrated for 2 ms following the conditioning stimulus for biceps and triceps and during the inhibition for ECR and FCR. The values presented are mean values of all the experiments + 1 standard error of the mean. The ratio between unconditioned and control FCR EMG was equal to % for a conditioning intensity equal to Mmax/4 and 69x % for a conditioning intensity stimulus equal to Mmax/2. In Fig. 2D, the modifications of the ECR EMG following median nerve stimulation are exactly the same as those represented in Fig. 2C; a longlasting inhibition of the ECR EMG appeared and increased with the appearance and increase of the FCR direct motor response (Fig. 2D, middle and lower traces). The ratio between conditioned and control FCR on-going EMG was % for a conditioning stimulus intensity just below motor threshold, % for a conditioning stimulus intensity equal to Mmax/4 and 85x5 + 1x3% for a conditioning stimulus intensity equal to Mmax/2. The effects of triceps nerve electrical stimulation of increasing intensity were tested in eight experiments performed in seven subjects. In all subjects, as illustrated in Fig. 2A, no change in the biceps EMG appeared with the appearance of a triceps direct motor response. In two of them, a short-duration inhibition appeared below motor threshold and remained unchanged when the intensity of the conditioning stimulus was increased. This short duration inhibition has been described previously in PSTH experiments (see Katz et al and below) and is very likely to be due to reciprocal Ia inhibition. In another experiment, a late inhibition (1 ms after the latency of the homonymous monosynaptic effect) appeared below motor threshold and remained unchanged when the intensity of the conditioning stimulus was increased. This late inhibition may have been induced by the activation of cutaneous afferents. The effects of musculo-cutaneous electrical stimulation of increasing intensity on triceps EMG were tested in eight experiments performed in seven subjects. The results were similar to those illustrated in Fig. 2B. In three experiments, a short-duration inhibition appeared below motor threshold and remained unchanged when the intensity of the conditioning stimulus was increased. In the other experiments, a late inhibition (more than 15 ms after the homonymous monosynaptic peak) appeared below motor threshold and remained unchanged when the intensity of the conditioning stimulus was increased. For each experiment, the ratio between the amount of conditioned and control ongoing EMG has been calculated as in the example illustrated in Fig. 2. In Table 1, the mean values of all the experiments are given. No significant inhibition was exhibited whatever the strength of the antagonistic nerve stimulus in biceps and triceps motoneurones. In short, at elbow level, activation of antagonistic motoneurones (and thus to antagonistic Renshaw-coupled cells) never resulted in a long-lasting inhibition of the tested motoneurones. The effects of radial nerve electrical stimulation of increased intensities were tested in twelve experiments performed in eight subjects. In all but two experiments, the appearance of an ECR motor response resulted in a long-lasting inhibition (at least 15 ms when the ECR conditioning motor discharge was equal to Mmax/4) that increased with the size of the ECR motor discharge. In two other experiments, an inhibition was evoked in the FCR EMG when the ECR motor discharge was present, but its duration was shorter (7 and 1 ms). No long-lasting inhibition was seen in any experiment where the conditioning stimulus did not evoke a motor response. The effects of median nerve electrical stimulation of increased intensity on ECR EMG were tested in fourteen experiments performed in eight subjects. In all of them, the appearance of an FCR motor response resulted in a long-lasting inhibition (at least 2 ms) of the ECR EMG, while it did not exist when the intensity of the conditioning stimulus was below motor threshold. The mean values of all the experiments are presented in Table 1. In ECR and FCR, a highly significant inhibition was present when the intensity of the conditioning stimulus was equal to Mmax/4 and the inhibition was larger when the

7 J Physiol Recurrent inhibition between opposing wrist muscles in man 273 Table 2. Mean values of the differences in on-going EMG inhibition T - Mmax/4 T - Mmax/2 Mmax/4 - Mmax/2 FCR 97 (P < -1) 16-3 (P < '5) 6-6 (P < 3) ECR 11 6(P<-2) 21 1 (P<-6) 9-8(P<-1) Mean values (and statistical significance) of the differences of inhibition obtained (i) at motor threshold (T) and with a conditioning motor volley equal to Mmax/4; (ii) at motor threshold and with a conditioning motor volley equal to Mmax/2; (iii) with conditioning motor volleys equal to Mmax/4 and Mmax/2. The statistical significance of the differences was tested with a Wilcoxon test. intensity of the conditioning stimulus was increased. A Wilcoxon test, the results of which are presented in Table 2, was used to determine whether the difference between the mean values obtained: (i) at motor threshold and Mmax/4; (ii) at motor threshold and Mmax/2; Mmax/4 and Mmax/2 were statistically significant. All their differences were statistically significant for ECR and FCR. In four subjects, the amount of inhibition obtained in wrist muscles by antagonistic nerve stimulation with a conditioning stimulation intensity giving rise to maximal motor response was compared with that obtained with a conditioning stimulus intensity equal to 12% of that giving rise to the maximal motor response. The amount of inhibition was similar for both conditioning intensities, thus suggesting that the inhibition was saturated when the motor response became maximal. To summarize the results at wrist level, the activation of antagonistic motor axons (and thus of antagonistic Renshawcoupled cells) resulted in a long-lasting inhibition of the tested motoneurones. PSTH experiments The experimental protocol was identical to that described in the preceding section, except that the effects of the conditioning stimulus on the motoneurone excitability were tested not on the amplitude of voluntarily averaged EMG but on the firing probability of a voluntarily activated motor unit. A typical example of the results obtained in PSTH experiments is represented in Fig. 3, which is set out as in Fig. 2. At elbow level (Fig. 3A and B), antagonistic motor discharges did not modify the probability of discharge of the test motor unit, while at wrist level they resulted in a longlasting decrease in firing probability of the test motor unit (Fig. 3C and D, middle and lower traces). A x2 test was performed to determine to what extent the distribution of firing probability after each kind of conditioning stimulation (just below motor threshold, Mmax/4, Mmax/2) differed from that obtained in the control situation. The window analysis was 2 ms for biceps and triceps and for the duration of the inhibition for ECR and FCR. No statistically significant difference was found for biceps and triceps, while a highly significant difference (P < 1) was found for FCR and ECR when the conditioning antagonistic motor volleys were equal to Mmax/4 and Mmax/2. The effects of triceps nerve electrical stimulation of increasing intensity were tested in eighteen motor units recorded in six subjects, whereas the effects of musculocutaneous nerve stimulation of increasing intensity were tested in fifteen motor units recorded in seven subjects. In all of them, antagonistic motor axon stimulations never resulted in a long-lasting decrease in firing probability of the test motor units, and no statistically significant differences were revealed using a x2 test. In roughly half of the motor units, antagonistic nerve stimulations at an intensity just below motor threshold resulted in a short duration decrease in firing probability, which remained unchanged when the intensity of the conditioning was increased. The effects of radial nerve electrical stimulation of increasing intensity were tested in twenty-seven FCR motor units recorded in nine subjects. In nineteen of them, i.e. at least once in each subject, ECR direct motor responses resulted in a long-lasting decrease in firing probability (between 14 and 27 ms, mean value 18-6 ms when the antagonistic motor response was equal to Mmax/2). These long-lasting decreases were highly significant with antagonistic motor volleys equal to Mmax/4 (P < 1), except in four cases (P < 5). The same results were found with antagonistic motor volleys equal to Mmax/2 (P< 1), except in two cases (P < 1 and P < 5). In six FCR motor units, a short decrease in firing probability (between 3 and 9 ms, mean value 4X8 ms) appeared for an intensity of radial nerve stimulation just below motor threshold and remained unchanged when the intensity of the conditioning stimulus was increased. Such a low threshold inhibition was also present in five motor units in which antagonistic motor volleys resulted in a long-lasting decrease in firing probability. In the two remaining FCR motor units, no effect was observed whatever the intensity of the conditioning stimulus. The effects of median nerve electrical stimulation of increasing intensity were tested in fourteen ECR motor units recorded in five subjects. In nine ECR motor units, i.e. at least once in each subject, the appearance of an FCR

8 274 C. Aymard and others J Physiol A Biceps M = B Triceps CD 6-6 M = MmaxI4-6 - ju) co cj DC6-. *J p]...,.. M = Mmax/ FCR e>'. -6-I - u U al) -6 - C 4 M = M = MmaxI4 D 2 4 ECR 6 - O -.-@< -6 J,- 6 ZA c -6 -I.c2 2 4 M=Mm=/ J I Figure 3. Changes in firing probability of isolated voluntarily activated motor units following antagonistic nerve stimulation A, changes in firing probability of a biceps voluntarily activated motor unit following an electrical stimulation applied to the triceps nerve. B, changes in firing probability of a triceps voluntarily activated motor unit following an electrical stimulation applied to the musculo-cutaneous nerve. C, changes in firing probability of a FCR voluntarily activated motor unit following an electrical stimulation applied to the radial nerve. D, changes in firing probability of a ECR voluntarily activated motor unit following an electrical stimulation applied to the median nerve. Same presentation as in Fig. 2; the zero of the abscissa corresponds to the latency of the homonymous monosynaptic peak. Ordinate as in Fig. 2A and C. Number of triggers: A, upper trace, 2; middle trace, 518; lower trace, 44; B, upper trace, 392; middle trace, 284; lower trace, 438; C, upper trace, 444; middle trace, 4; lower trace, 682; D, upper trace, 44; middle trace, 1564; lower trace, 114.

9 J Physiol Recurrent inhibition between opposing wrist muscles in man 275 direct motor response resulted in a long-lasting decrease in firing probability (between 16 and 24 ms, mean value 19-7 ms when the amplitude of the FCR motor response was equal to Mmax/2). These long-lasting decreases in firing probability were highly significant with antagonistic motor volleys equal to Mmax/4 (P< 1), except in two cases (P < 1), as well as with antagonistic motor volleys equal to Mmax/2 (P < 1), except in three cases (P < 1 and P < 5). In four ECR motor units, a short decrease in firing probability was seen when the intensity of the radial nerve stimulation was just below motor threshold (between 3 and 6 ms, mean value 541 ms) and remained unchanged when the intensity of the conditioning stimulus was increased. Such a low threshold inhibition was also found in two ECR motor units in which antagonistic motor volleys resulted in a long-lasting depression in firing probability. No modification in firing probability was seen in the last ECR motor unit. To summarize, the results obtained in PSTH experiments are fully in accordance with those obtained in EMG experiments. At elbow level, antagonistic motor responses never resulted in a long-lasting decrease in firing probability of flexor and extensor motor units while, at wrist level, antagonistic motor discharges resulted in a long-lasting decrease in firing probability of the majority of the ECR and FCR motor units. In the following section, results of complementary experiments performed to specify the characteristics of the long-lasting inhibition evoked in wrist motor nuclei by antagonistic motor volleys are presented. C.)_ X 1 mv w A 5 2- o - - W -2I Figure 4. Effect of a steady mechanical vibration, applied to the FCR distal tendon, on long-lasting inhibition evoked in the ECR by an FCR reflex discharge A, B and C, before 2 min vibration; D, E, F and G, after 2 min vibration. A, FCR conditioning H reflex evoked by median nerve stimulation. B, long lasting inhibition of a single ECR fibre induced by the FCR conditioning reflex discharge. C, monosynaptic response of the same single ECR fibre evoked by subthreshold stimulation ( 8T) of the radial nerve. Note the coincidental latencies of the ECR inhibitory and excitatory responses. After 2 min of vibration, the same conditioning stimulation to median nerve failed to evoke any FCR H reflex (D) as well as ECR short-latency inhibition (E). Eleven minutes after the end of vibration, the conditioning H reflex started to recover (F) and an ECR short-latency inhibition again appeared (C). Each trace is the mean of 2 responses. B, C, E and G are rectified and are average responses from which the same number of unconditioned, rectified and averaged sweeps have been subtracted. H reflex rate; pseudorandom sequence; minimum time interval was 1 s.?,%, UV1 e> *5~ 1 mv I w -2 ] W5t Q 21.5 C) 1 mv DI E F w a?, UV > 2- G CD - -2 J

10 C. Aymard and others 276 J Physiol ) o 5 C._ A B. E ' = - a Latency (ins) CO en a1) M en c.2 C 2 E - 4 ō D nln n h1nhn ---.t " LU " ~~ ~ '' -u -ji G -95T I-- n a) en CD c.l 8 o - E', z -5- E [l..l H 1lT 1-n ) Co a.2') C) CD o3 -E E: z I- -- r---t I A_-1T Figure 5. Comparison between the latencies of the increase in firing probability in response to homonymous nerve stimulation (A) and of the decrease in firing probability in response to antagonistic nerve stimulations (D, E and F) just below motor threshold (G), giving rise to antagonistic motor discharges of increasing amplitude (H and I) A-F, time histograms of the discharge of a given voluntarily activated FCR motor unit. B, histogram obtained in control conditions (i.e. without stimulation). C, histogram obtained in response to radial nerve stimulation just below motor threshold. A, D, E and F, difference between histograms obtained in control conditions and after stimulation: in each 1 ms bin, the control value was subtracted from that obtained after stimulation (as in Figs 1 and 3). A, changes in firing probability following an electrical stimulation of the median nerve: the vertical arrow indicates the bin corresponding to the onset of the increase in firing probability. This bin was visually identified and statistical analyses were performed to determine to what extent the distribution of firing probability after stimulation differed from that in the control situation. No significant difference between the control situation and that after stimulation was seen in 2, 21, 22 and 23 ms bins (o < x2 < 2413), whereas the increase in 24 and 25 ms bins were each highly significant (X2 = 14-4 and x2 = 13X3, P < 1). D, Eand F, the zero of the abscissa corresponds to the latency of the homonymous monosynaptic peak. Similar calculations were made for PSTH represented in E and F No significant difference between the control situation and that after stimulation was seen in the and 1 ms bins - or in the two preceding bins, not represented (X5 x < 2419) - whereas the decrease in 2, 3 and 4 ms bins, was each significant (3-13 < x2 < 6-67, P < 5 or P < -1). For the PSTH represented in D, no significant difference was seen in each of the 1 first ms bins - and in the two preceding ones, not represented ( S x2 < 1P38). Number of triggers: A, 18; B and C, 25; D, 54; E, 1272; F, 752. Abscissa: A, latency after stimulation; D, E and F, latency after the onset of the homonymous monosynaptic peak. Ordinate: number of counts expressed as a percentage of the number of triggers; G, Hand I, averaged reflex and motor responses obtained in ECR.

11 J. Phy8iol Recurrent inhibition between opposing wrist muscles in man 277 Complementary experiments Electrophysiological experiments The long-lasting inhibition is also evoked with a conditioning reflex discharge. It is well known that during a voluntary contraction of a given muscle, the monosynaptic reflex evoked in its antagonist is strongly depressed or abolished. Furthermore, even at rest, monosynaptic reflexes are not easily evoked in the ECR and finally, for technical reasons (size of the mirror image of the reflex in the EMG antagonistic muscle), PSTH experiments are not suited to study the modifications of the excitability of motoneurones induced by an antagonistic reflex discharge. Nevertheless, experiments were performed with an EMG protocol in which it was possible to evoke an antagonistic reflex of sizeable amplitude during the voluntary contraction of the test muscle. This was achieved in only five experiments (four with a conditioning FCR H reflex and one with a conditioning ECR H reflex). In all experiments, the appearance of an antagonistic monosynaptic reflex resulted in a long-lasting inhibition of the test EMG that increased in size and duration with the size of the conditioning monosynaptic test reflex. Another argument favouring the hypothesis that the long-lasting inhibition induced by antagonistic nerve stimulation is linked to the antagonistic motor axon discharge, originates from the experiment illustrated in Fig. 4. Coppin, Jack & MacLennan (197) showed in the cat that a long-lasting mechanical vibration applied to a muscle tendon selectively increases the electrical threshold of I a afferents. In man, Heckman, Condon, Hutton & Enoka (1984) showed that after such prolonged tendon vibration, the monosynaptic soleus H reflex was deeply depressed. This artifice was used to abolish the conditioning H reflex without any modification of the conditioning stimulus, and to see what happened to the long-lasting inhibition evoked in the antagonistic motor nucleus. In one subject, a clear-cut FCR H reflex was recorded during the voluntary contraction of the ECR muscle (Fig. 4A). Its appearance resulted in a long-lasting inhibition in the ECR muscle fibre needle recording (Fig. 4B). After 2 min of steady vibration of the FCR tendon (1 Hz, 1-5 mm peak, see Rossi, Mazzocchio & Parlanti, 1991, for further details), the same conditioning median nerve stimulation failed to evoke the FCR H reflex (Fig. 4D) and the ECR inhibition disappeared (Fig. 4E). Only. a longer latency inhibition, presumably of cutaneous origin (see above) was observed (Fig. 4E). Eleven minutes after the end of the vibration, the FCR H reflex partially recovered (Fig. 4F) and the ECR long-lasting inhibition also began to develop (Fig. 4G). Determination of the latency and duration of the longlasting inhibition. The latency of the inhibition induced by antagonistic motor volleys in the motor nuclei innervating wrist muscles was determined in PSTH experiments that allow better resolution time interval measurements. A representative example of the procedure is shown in Fig. 5. The test motor unit was a FCR motor unit. Firstly, the latency of the homonymous monosynaptic peak was determined by studying the effects of an electrical conditioning stimulus applied to the median nerve on the firing probability of an FCR motor unit (Fig. 5A). With an intensity just below motor threshold, an early and brief increase in firing probability was revealed, which is probably due to the monosynaptic Ia effect (Mao, Ashby, Wang & McCrea, 1984; Malmgren & Pierrot-Deseilligny, 1988). The onset of the homonymous peak was abrupt and its duration was short. Nevertheless, statistical analyses (see Methods) were performed in the time interval window surrounding the peak: in the four bins preceding its onset (arrow in Fig. 5A), it was verified using the x2 test that the distribution of firing probability after stimulation did not differ from that in the control situation, while the probability significantly increased during the peak and its first bin was significant (P < 1). Secondly, the effects of a conditioning stimulation applied to the radial nerve were tested in the same FCR motor unit, the site of the stimulation of the radial nerve being slightly more proximal than that of the median one. When the radial conditioning stimulus gave rise to a ECR motor response (Fig. 5H and I), a long decrease in firing probability of the test FCR motor unit appeared, which began 2 ms after the onset of the monosynaptic peak (Fig. 5E and F). The onset of these antagonistic decreases was visually identified and it was verified that: (i) there was no difference in the distribution of firing probability between control and after stimulation in each of the four bins preceding the onset of the decrease of firing probability; and (ii) a significant decrease (P < 5 or P < 1) was seen in each of the two first bins of the decrease in firing probability (or at least in a time interval window including these two first bins). In all the experiments, the decrease in firing probability of FCR motor units induced by ECR antagonistic motor volleys began between and 3 ms (mean value, 1-3 ms) after the homonymous monosynaptic peak. Exactly the same protocol was performed with test ECR motor units. In all the experiments, the onset of the decrease in firing probability induced by the conditioning FCR motor volley was between and 3 ms (mean value, 1-6 ms) after the ECR monosynaptic peak. Taking into the account the differences in afferent conduction peripheral times (sites of the homonymous and antagonistic stimulation, conduction velocity of the fibres involved by the conditioning stimuli) and the fact that bins of 1 ms were used, these results strongly suggest that the long-lasting inhibition is mediated through a few synaptic pathways. The duration of the decrease in firing probability was measured as the interval between the first and the last bin of an uninterrupted sequence of decreased firing probability and the statistical significance of the inhibition was measured for the whole duration (P < 1 in the great majority of the cases). Furthermore, when the decrease in firing probability was not immediately followed by an increase, the last bin of the decrease was determined using a similar statistical procedure to that used to determine the first bin of the decrease of firing probability.

12 278 C. Aymard and others J Physiol For a similar number of triggers, a procedure relying on an average of traces (rectified averaged EMG method) is less reliable than a PSTH technique to determine the exact onset of a facilitation or an inhibition. Furthermore, in some cases, the conditioning motor volleys gave rise to a 'mirror image' in the test EMG that could interfere with the onset of the inhibition. However, it has been verified in EMG experiments that the onset of the inhibition visually identified (for example, 2 ms after the latency of the monosynaptic effect in Fig. 3C, lower trace) corresponded to statistically significant differences between control and conditioned A O in c C: a) N Time from onset of L-Ac injection (min) B C 27 2 mvl 1 ms Figure 6. Effects of the L-acetylcarnitine (L-Ac) on elbow and wrist motor nuclei A, filled symbols: grand mean of L-Ac-induced enhancement of the long-lasting inhibition of FCR (A) and ECR (L). The size (area) of the inhibition, expressed as a percentage of its control value before injection, is plotted against the time from onset of L-Ac injection. Each symbol represents the grand mean + S.E.M. of 2-3 conditioned responses recorded from each subject (n = 6). The asterisks above the S.E.M. bars mark significant points (Student's t test) on the ECR; asterisks below S.E.M. bars denote significant points of the FCR curve. Open symbols: effect of the same injection of L-Ac on triceps () and biceps (A) muscles. Each point is the grand mean of 2-3 conditioned responses recorded from each subject (n = 4). Inset, amplitude (x) and duration (y) of potentiation of the FCR short-latency inhibition, over the 1 min interval, corresponding to the maximum effect of L-Ac in each subject. Values are expressed as a percentage of their control values (abscissa) before L-Ac injection: x = %; y = %. B, example of FCR short-latency inhibition, before (upper trace), 4 min after (middle trace) and 6 min after (lower trace) L-Ac injection. Conditioning stimulation of the radial nerve evoked a ECR response 24% of its maximum motor wave (left-hand traces). C, ECR short-latency inhibition at the same interval. Conditioning motor response of FCR, by stimulation of the median nerve, was 32% of its maximum motor wave (left-hand traces). Each trace is the rectified and averaged response of 2 sweeps, from which the same number of unconditioned, rectified and averaged, sweeps have been subtracted.

13 J Physiol Recurrent inhibition between opposing wrist muscles in man 279 EMG in each of the two adjacent 1 ms time interval windows (2-3 ms and 3-4 ms window in Fig. 3C, lower trace), or at least in a time interval window including the two first adjacent bins. Variance analysis (f test) and Scheff6's test were used for statistical analysis. Threshold of the long lasting inhibition. It has been suggested that the long-lasting inhibition is linked to the appearance of an antagonistic motor discharge. However, experiments were performed to determine whether or not the threshold of the long-lasting inhibition was the same as that of the conditioning motor response. A representative result of the experiments is shown in Fig. 5. When the intensity of the electrical conditioning stimulus was adjusted to be just below motor threshold (f95t) and did not evoke any reflex response (Fig. 5G), no modification in the firing probability of the test FCR motor unit was seen (Fig. 5D). When the intensity of the radial conditioning stimulus was slightly increased (up to 1 1 T), it gave rise to an ECR motor response (Fig. 5H) and resulted in a longlasting decrease in firing probability of the test FCR motor unit (Fig. 5E). A further increase in the intensity of the radial conditioning stimulus (up to 1 f5t) resulted in a larger ECR motor response (Fig. 5I) and in a larger decrease in the firing probability of the test FCR motor unit (Fig. 5F). Exactly the same results, i.e. that the threshold of the inhibition is linked to that of the antagonistic motor volley, were found with an ECR test motor unit and a conditioning stimulation applied to the median nerve. In this situation, when the threshold of the FCR H reflex was below that of the FCR motor response during the activation of the ECR motor units, the threshold for the long-lasting zlecrease in firing probability was that of the FCR H r4flex. Some experiments were also performed with the EMG protocol and the results were similar: the threshold for the inhibition was linked to that of the antagonistic motor discharge (evoked by a reflex or a direct motor discharge). Pharmacological experiments L-Ac is a cholinergic agonist, a substance therefore potentiating recurrent inhibition. Its action in man has already been described (Mazzocchio & Rossi, 1989; Katz et al. 1993). The following experiments were thus performed to see if the long-lasting inhibition between FCR and ECR was increased by the injection of a substance known to potentiate recurrent inhibition. As shown in Fig. 6A (filled symbols), long-lasting inhibition of both these muscles underwent strong potentiation, starting between 3 and 4 min after the onset of L-Ac injection, reaching its maximum 4-5 min after it and exhausting 6-8 min later. These results were observed in twelve out of thirteen experiments performed in six subjects. From the inspection of the traces in Fig. 6B and C, it is apparent that both amplitude and duration of the inhibition increased during L-Ac-induced potentiation. In two control experiments on FCR, it was verified that administration of L-Ac did not modify the EMG response after conditioning stimulation of the radial nerve at an intensity below (-95T) the ECR motor response (not illustrated). The long-lasting inhibition following antagonistic motor volleys was thus strongly enhanced by L-Ac injection. On the contrary, at elbow level, in eight experiments in four subjects, no significant change of biceps and triceps EMG activity conditioned by motor response (3-4% of Mmax) was found after L-Ac injection (open symbols in Fig. 6A). In one case, which exhibited a short-lasting inhibition of the biceps, reflecting I a reciprocal inhibition, a slight decrease of this inhibition was observed 45 and 55 min after L-Ac injection. Such a depression of Ia reciprocal inhibition by a substance that potentiates recurrent inhibition has already been described and discussed in a previous paper (Rossi, Decchi, Zalaffi & Mazzocchio, 1995). This may be due to the potentiation by L-Ac of triceps Renshaw-coupled cells, which in turn inhibit Ia interneurones and thus depress Ia reciprocal inhibition (cf. Fig. 7B). DISCUSSION The main finding of this series of experiments is that antagonistic motor volleys of increasing amplitude evoke long-lasting inhibition of increasing size in motoneurones innervating wrist muscles, while they do not modify the excitability of motoneurones innervating elbow muscles. As already stressed by Meunier et al. (199), Creange et al. (1992) and Katz et al. (1993), the long-lasting inhibition evoked by motor volleys in motoneurones is probably Renshaw in origin. Indeed, the inhibition revealed both by PSTH and EMG experiments in wrist muscles appeared and increased with the conditioning motor discharge (whether reflex or motor response), had a short latency and a long duration. Motor and reflex discharges were obtained for different intensities of the conditioning stimulus and the fact that the appearance of the inhibition was linked to that of the motor discharge (whether reflex or motor response), suggests that it is not dependent on the conditioning stimulus intensity per se. Furthermore, in the experiment represented in Fig. 4, the intensity of the radial nerve conditioning stimulus was kept constant and the changes in amplitude of the ECR conditioned reflex discharge were obtained by an artifice; when the reflex was present, the inhibition was present, when the reflex vanished, the inhibition disappeared. As already stressed by others (Meunier et al. 199; Meunier, Pierrot-Deseilligny & Simonetta-Moreau, 1994), none of these arguments is able to demonstrate recurrent inhibition in isolation, but their congruence makes it probable. Moreover, there are cogent arguments against a significant contribution of other mechanisms or pathways to the inhibition described here: (i) an effect whose threshold is above motor threshold is probably not due to activation of group I fibres; (ii) Marque, Pierrot-Deseilligny & Simonetta- Moreau (1996) suggested that in man, as in the cat, the conduction velocity of I a fibres is roughly double that of

14 28 C. Aymard and others J Physiol group II fibres, so that group II effects immediately following the monosynaptic peak can be ruled out; (iii) similarly, the twitch-induced Ib inhibition would reach the motoneurone about 1 ms after the monosynaptic I a facilitation (time between the onset of the twitch and the activation of Golgi tendon organs (Binder, Kroin, Moore & Stuart, 1977)). Moreover, despite the fact that voluntary contraction strongly depressed antagonistic reflexes, it has been possible in some cases, both in PSTH and EMG experiments, to evoke a conditioning H reflex. In such cases, the twitchinduced afferent discharge following the reflex response cannot manifest itself before several tens of milliseconds (i.e. the time taken from and to the spinal cord (see Meunier et al. 199)); (iv) cutaneous effects onto propriospinal neurones appeared slightly above perception threshold (i.e. below motor threshold, had a longer latency and a specific pattern: radial cutaneous afferents inhibit propriospinal motoneurones projecting to ECR motoneurones and not to FCR motoneurones (Pierrot-Deseilligny, 1996)). Thus the other reflex pathways activated by a conditioning stimulus above motor threshold (i.e. group I fibres, group II fibres, twitch-induced afferent volleys, cutaneous afferents) would not seem to be responsible for the long-lasting inhibition induced at wrist level by antagonistic motor volleys. The pharmacological experiments with L-Ac, a cholinergic substance able to potentiate recurrent inhibition, also support the hypothesis that the long-lasting inhibition observed between ECR and FCR is due to recurrent inhibition. In other terms, L-Ac potentiates the activation of ECR Renshaw cells which, via their heteronymous projections, inhibit FCR motoneurones and vice versa (Fig. 7A). Two main arguments support this conclusion. Firstly, the time course of this potentiation is virtually identical to the L-Ac-induced potentiation of homonymous A B K ) Cure 7. Schematic diagram summarizing the connections ~~~~~~~~Figoween Renshaw cells, motoneurones and interneurones ldiating reciprocal inhibition connections at human wrist level. B, connections in the cat lumbar nal cord (for references, see Baldissera et al. 1981). Large open nbols, motoneurones; large filled circles, Renshaw cells; hatched le, interneurones mediating reciprocal inhibition.

15 J Physiol Recurrent inhibition between opposing wrist muscles in man 281 recurrent inhibition observed in many different lower and upper limb motor nuclei (Mazzocchio & Rossi, 1989; Rossi & Mazzocchio, 1991; Katz et al. 1993; Rossi, Zalaffi & Decchi, 1994; Rossi et al. 1995), including FCR and ECR motor nuclei (Katz et al. 1993). Secondly, in a previous paper (Rossi et al. 1995), the effects of L-Ac injection were tested in parallel on reciprocal inhibition between biceps and triceps and on reciprocal inhibition between FCR and ECR. While L-Ac depressed I a reciprocal inhibition from triceps to biceps, it had no effect on reciprocal inhibition from ECR to FCR. In the present experiments, in which L-Ac was tested on the motoneurones after activation of Renshaw cells coupled to the 'antagonistic' motor nucleus, exactly the opposite occurred: L-Ac injection modified the long-lasting inhibition between FCR and ECR and had no effect at elbow level. These results can be easily explained (Fig. 7A), assuming that FCR and ECR are reciprocally linked by recurrent projections that do not contact interneurones mediating the reciprocal inhibition between them (Aymard et al. 1995); at elbow level, however, triceps and biceps are not linked by recurrent projections, whereas the interneurones mediating reciprocal inhibition at their level are depressed by Renshaw cells (Katz et al and Fig. 7B). Finally, further indirect support comes from the observation that both amplitude and duration of inhibition increased in parallel (see Fig. 6). In fact, this could reflect the tendency of Renshaw cells to enhance the frequency and duration of their discharge in response to increasing depolarizing stimuli (e.g. Hultborn & Pierrot-Deseilligny, 1979). In conclusion, both electrophysiological and pharmacological studies favour the hypothesis that the long-lasting inhibition induced in wrist motoneurones by antagonistic motor volleys is due to recurrent inhibition (see Fig. 7A) while - as was regularly found in the cat lumbar spinal cord (see Fig. 7B) - no recurrent inhibition was seen between antagonistic elbow muscles. The interconnections between reciprocal inhibition and recurrent inhibition at wrist level is schematized in Fig. 7A, taking into account the results obtained by Aymard et al. (1995) and those presented in this paper. ECR Renshawcoupled cells project onto ECR motoneurones, onto FCR motoneurones and not onto interneurones mediating reciprocal inhibition from ECR to FCR (and vice versa for the FCR Renshaw-coupled cells). An indirect argument reported below suggests that, in the cat wrist also, FCR and ECR are not interconnected like other antagonistic muscles. The depression of reciprocal I a inhibition by Renshaw cells (Fig. 7B) was first described in 1962 by Wilson & Burgess as 'recurrent facilitation'. In 1989, Illert & Wietelmann observed recurrent facilitation between motor nuclei innervating elbow muscles but not between wrist muscles. This observation suggests that Renshaw cells do not project to interneurones mediating reciprocal inhibition between wrist muscles in the cat either. Functional significance In a previous paper (Aymard et al. 1995), evidence was presented that reciprocal inhibition between FCR and ECR does not correspond to the classical organization by which strict antagonists (as for example triceps and biceps) are linked. This is in accordance with the fact that FCR and ECR, acting at a joint with several degrees of freedom, may behave as antagonists or agonists according to the tasks. In particular, when ECR and FCR act as synergists, i.e. during wrist abduction, their reciprocal Renshaw projections (Fig. 7A), could provide a similar distribution of recurrent inhibition on both motor nuclei. Taking into account the hypothesis that the Renshaw system primarily serves as a variable gain regulator at the motoneuronal level (Hultborn, Lindstrom & Wigstrom, 1979), the similar distribution of recurrent inhibition to FCR and ECR may contribute to set their output gain to the same level. This may be important when a strictly balanced contraction of these muscles is required, as for a pure wrist abduction-adduction trajectory. On the other hand, when they act as antagonists, e.g. during a wrist extension, the prevailing recurrent activity from ECR to FCR may contribute, in parallel with group I inhibition, to prevent activation of FCR. However, even when they behave as antagonists, there are important differences with respect to other antagonistic muscle pairs. Indeed, to produce a given change in a joint angle the central nervous system can use the 'reciprocal inhibition strategy' or 'co-contraction strategy', providing low levels and high levels of joint stiffness, respectively (co-contraction strategy allows greater adaptability to unanticipated changes in external forces or loads). Since Renshaw cells inhibit interneurones mediating reciprocal inhibition, it follows that when recurrent activity is high, the co-contraction strategy is favoured. Accepting that Renshaw cells act as variable output gain regulators, then it also follows that joint stiffness and output gain are not fully independent variables when Renshaw cells project onto both homonymous motoneurones and I a interneurones (see Fig. 7B). This is not the case at the wrist joint (Fig. 7A), where the absence of Renshaw cells projecting to interneurones mediating reciprocal inhibition makes an independent control of these two parameters possible. This can contribute to the enhancement of the flexibility of movements at this level. AYMARD, C., CHIA, L., KATZ, R., LAFITTE, C. & PENICAUD, A. (1995). Reciprocal inhibition between wrist flexors and extensors in man: a new set of interneurones? Journal of Physiology 487, BALDISSERA, F., CAMPADELLI, P. & CAVALLARI, P. (1983). Inhibition of H-reflex in wrist flexors by group I afferents in the radial nerve. Electromyography and Clinical Neurophysiology 23, BALDISSERA, F., HULTBORN, H. & ILLERT, M. (1981). Integration in spinal neuronal systems. In Handbook of Physiology, section 1, The Nervous System, vol. 2, Motor Control, ed. BROOK, V. B., pp The American Physiological Society, Bethesda, MD, USA.

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Journal of Neurophysiology 25, Acknowledgements The authors wish to express their gratitude to Dr Leonor Mazieres for reading and commenting upon the manuscript. Our thanks are also due to A. F. Rigaudie and M. Dodo for their unfailing assistance during the course of the experiments, and to David MacGregor for scrutinizing the English. This work was supported by grants from MESR (Ministere de l'enseignement Superieur et de la Recherche), AP-HP (Assistance Publique-Hopitaux de Paris), INSERM (Institut National de la Sante et de la Recherche Medicale; CRI ) and IRME (Institut pour la Recherche sur la Moelle Epiniere). A. Rossi and B. Decchi were supported by a grant from Regione Toscana (Ricerca Sanitaria Finalizzata), Italy. Received 13 March 1996; accepted 28 October 1996.