Increase in reciprocal I a inhibition during antagonist contraction in the human leg: a study of motor units and the H reflex

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1 433 Journal of Physiology (1995), 489.1, pp Increase in reciprocal I a inhibition during antagonist contraction in the human leg: a study of motor units and the H reflex Masaomi Shindo, Sohei Yanagawa, Hiroshi Morita and Nobuo Yanagisawa Department of Medicine (Neurology), Shinshu University School of Medicine, Asahi 3-1-1, Matsumoto 39, Japan 1. The change in reciprocal I a inhibition of soleus motoneurones produced by stimulation of the common peroneal nerve was investigated by the use of twenty-three soleus motor units as well as the soleus H reflex in six normal subjects during tonic pretibial contraction. 2. In the motor unit experiments, motoneuronal excitability was measured as the 'critical firing stimulus' (FS), which is the difference between the test stimulus intensity needed to reach the threshold for the lowest threshold I a fibres and the intensity which evokes firing of a motor unit with the probability of 5%. The conditioning effect, assessed from the change in the FS, was expressed as a percentage of the unconditioned FS. 3. At a conditioning intensity of 95 times the motor threshold value, there was I a inhibition in sixteen of the twenty-three motor units (69-6%) at rest. Of these sixteen motor units, twelve showed increases in inhibition at intervals below 2- ms during pretibial contraction. In four of the remaining seven units, inhibition first appeared during contraction. There was no significant decrease in inhibition at any time during contraction. 4. Based on the conventional H reflex, reciprocal I a inhibition increased during very weak (below 2 % of the maximum) voluntary dorsiflexion and continued to increase at a slightly stronger (3-8 % of the maximum) contraction, then decreased continuously when contraction was strengthened further. Maximal inhibition occurred at a relatively strong contraction when a weak conditioning stimulus was used, and vice versa. 5. We conclude that the activity of reciprocal I a inhibitory interneurones increases during tonic antagonist contraction. The previous controversy about this inhibition is the result of occlusion at the I a interneuronal level. The motoneuronal excitability of a muscle is clearly inhibited when its antagonistic muscles contract (Kots, 1969) and reciprocal I a inhibition has been assumed to be an important mechanism for antagonistic inhibition. Since the first descriptions by Mizuno, Tanaka & Yanagisawa (1971) and by Kots & Zhukov (1971) of reciprocal I a inhibition in humans, extensive studies have been done on the modulation of the excitability of this pathway by voluntary contraction of the agonists or antagonists in the arm (Day, Rothwell & Marsden, 1983; Day, Marsden, Obeso & Rothwell, 1984; avallari, Fournier, Katz, Pierrot-Deseilligny & Shindo, 1984) and in the leg (Tanaka, 1974; Simoyama & Tanaka, 1974; Iles, 1983, 1986; Shindo, Harayama, Kondo, Yanagisawa & Tanaka, 1984; rone, Hultborn & Jespersen, 1985; rone, Hultborn, Jespersen & Nielsen, 1987; rone & Nielsen, 1989a; Nielsen, Kagamihara, rone & Hultborn, 1992). In the arm, the efficacy of transmission has been shown to increase due to voluntary contraction of antagonistic muscles through activation, at least in part, of the supraspinal descending pathway. This parallel control of a-motoneurones and the corresponding I a interneurones is known as the 'a-y linkage in reciprocal inhibition' (Hongo, Jankowska & Lundberg, 1969). Results reported for tonic antagonist contraction of the leg, however, are controversial. Tanaka (1974) was the first to report facilitation of the reciprocal I a inhibitory pathway to the soleus motoneurones during tonic voluntary contraction of the antagonist pretibial muscles. In light of this, Shindo et al. (1984) showed that the efficacy of reciprocal Ia inhibition is facilitated during antagonist contraction and depends on the strength of the contraction. Later studies, however, reported no increase in reciprocal Ia inhibition during the same type of contraction (rone et al. 1985, 1987; Iles, 1986; rone & Nielsen, 1989a; Nielsen et al. 1992). In particular, rone et al. (1987) reported that during tonic ankle dorsiflexion, inhibition always increased at conditioning-test stimulus intervals of more than 2-5 ms but only rarely at shorter intervals, even at various

2 276 M. Shindo and others J. Phy8iol strengths of contraction. They concluded that reciprocal I a inhibition did not increase during tonic antagonist contraction and that the subsequent increase was caused by some other type of inhibition. More recently, the same research group applied ischaemia to the leg during tonic pretibial contraction to block peripheral feedback of the I a afferent discharge from the contracted muscle and demonstrated that I a inhibition did not increase during the contraction before ischaemia but did increase during the contraction after it (Nielsen et at. 1992). On the basis of these results they attributed the lack of increase during contraction in the intact condition to post-activation depression (rone & Nielsen, 1989 b). In considering the different conclusions in reports, the effects of 'pool problems' have yet to be assessed. These include the dependence of the conditioning effect on the test reflex size (rone, Hultborn, Mazieres, Morin, Nielsen & Pierrot-Deseilligny, 199), the population of sampled motoneurones as a test reflex (T. Hashimoto, M. Shindo, S. Yanagawa & N. Yanagisawa, unpublished observation), and the non-linear relationship of input/output in the motoneuronal pool (Kernell & Hultborn, 199; Nielsen, Hultborn & Gossard, 199). To overcome these problems, we studied the modulation of reciprocal I a inhibition on the single motor units involved in the compound H reflex during tonic contraction of the antagonists. Moreover, we investigated I a inhibition using the conventional H reflex to determine what mechanisms account for the discrepancies in the previous results. Part of this work was published in abstract form (Shindo, Yanagawa, Morita & Y,anagisawa, 1993). METHODS As the principle and the methodological issues for motor unit experiments have been described in detail elsewhere (Shindo, Yanagawa, Mlorita & Hashimoto, 1994), they are given here briefly. Subjects Eight normal subjects, including three of the authors, participated in the experiments. All the subjects were men aged years. All had been informned in advance of the purpose of the study and the procedure to be used and gave their consent to be studied. The experiments were approved by the Ethics ommittee of Shinshu University School of Medicine (Matsumoto). All the procedures used followed the guidelines given in the Declaration of Helsinki. The subject was seated comfortably in a reclining armchair in front of which an oscilloscope had been placed to monitor the electromyogram (EMG) of the pretibial muscles. The knee and ankle joints were kept at angles of 15 and 1 deg, respectively. The foot was fixed wa-ith a band to an immobile foot-plate. Stimulation and recording Test stimuli of constant voltage and with a duration of 1 ms were applied to the tibial nerve in the popliteal fossa every 1P5 s (for motor unit studies) and every 3-4 s (for the conventional H reflex studies) to evoke H r-eflexes in the soleus muscle. Preceding each test stimulus, a conditioning stimulus of 5 ms duration was delivered bipolarly to the common peroneal nerve through a pair of surface electrodes at the level of the caput fibulae. The intensity of the conditioning stimulus was expressed as a multiple of the threshold for the direct M-response (T) in the tibialis anterior muscle. are was taken to place the conditioning electrode such that the threshold for the M-response of the tibialis anterior muscle Nras lower than that for the peroneal one. The distance between the test and conditioning stimulation sites was 7-1 cm. The soleus H reflex was recorded with a pair of surface electrodes placed 3 cm apart longitudinally on the dorsal aspect of the leg, one being on the Achilles tendon. H reflex size was measured as the area after full-wave rectification and integration of the EMG and expressed as a percentage of the maximal M-response (Mmax). For the motor unit recordings, a tungsten microelectrode (FH, Brunswick, Maine, USA) was inserted into the soleus muscle between the two surface electrodes used for the H reflex and the reference surface electrode on the opposite aspect of the leg. In later experiments a bipolar needle electrode (Medelec E/ND1, Old Woking, UK) was used for the motor unit recordings. The pretibial EMG was also recorded with the surface electrodes used to monitor the efficacy of the conditioning stimuli and provide the subject feedback control of the strength of voluntary contraction of the pretibial muscles. The strength of contraction was determined from the integrated EMG. Assessment of the excitability of a single motoneurone A single motor unit was identified within the compound H reflex by the constancy of latency and its wave form, and its all-or-none occurrence. The range of test stimulus intensities from the firing threshold for the I a fibres to the intensity that evokes a motor unit firing with the probability of 5% (FP5%) was the index used to evaluate the excitability of the motoneurone. The intensity at FP5% was determined with a microcomputer (NE P-981 VM2, Tokyo, Japan) such that the stimulus intensity was augmented or weakened by one step, depending on the effect of the previous stimulus. When the motor unit fired, the stimulus was decreased automatically, but when there was no firing it was augmented. To make the firing probability converge to 5%, 5-1 stimuli were needed in each trial. To avoid technical bias, it vas sometimes necessary to exclude several sequential responses before reaching the intensity levels with and without firing. The threshold for I a fibres in the tibial nerve was determined to be the threshold for homonymous I a facilitation by using a post-stimulus time histogram (PSTH) technique during weak tonic voluntary contraction of the soleus muscle (Fournier, AIeunier, Pierrot- Deseilligny & Shindo, 1986). This value was subtracted from the intensity at FP5%. The resulting intensity, termed the 'critical firing stimulus' (FS), was measured in volts. Assessment of the conditioning effect in a single motoneurone onditioning effects were assessed as the change in the FS produced by the conditioning stimuli. When the effect was inhibition with IPSPs, the motoneurone required a stronger test stimulus to fire (a stronger FS) than it did without conditioning stimuli. The conditioning effect was expressed as the percentage of the control FS without conditioning. Inhibition, expressed as vralues of more than 1 % the value of the control FS, is shown downswards in the figures in order to provide an image similar to that of the IPSP. The extent of inhibition was obtained by subtracting the 1% (control FS) value from the conditioned FS value.

3 J; Physiol Ia inhibition during antagonist contraction 277 The threshold of the motor unit was determined from the critical firing level (FL) which gives the rank order of excitability of the motor unit within the motoneuronal pool (Henneman, lamann, Gillies & Skinner, 1974). Here it is represented by the size of the compound H reflex as a percentage of the MnImax for a firing probability of 5 %. The motor unit potentials were first converted to single pulses through a triggering device then fed into the computer. PSTHs were constructed for the reflex-evoked motor unit in order to obtain their latencies from the test stimuli. Procedures The experiments on motor unit recording usually consisted of sessions for the time courses both at rest and during antagonist contraction as well as for the PSTHs used to determine the threshold for I a fibres. In the time course session, conditioning-test stimulus intervals were varied systematically and randomly in the same experimental session, the conditioning intensity being fixed. In some experiments, to determine the afferents responsible for the effect, intensity curves were obtained when conditioning intensities were varied systematically and the interstimulus interval was fixed. Voluntary contraction of the pretibial muscles was done tonically and isometrically. The strength of a contraction, monitored with integrated EMG, was 1-2% of the maximum for each subject in order not to displace the needle electrode. During the contraction, the subject was provided with visual feedback of the integrated EAIG on an oscilloscope in order to maintain a constant strength of contraction. o-contraction of the triceps surae muscle was checked carefully and, when found, data for that session were discarded. After alternate sessions of rest and contraction, the threshold for the Ia fibres in the tibial nerve was determined. During weak tonic contraction of the triceps surae muscle, PSTHs were constructed on a single motor unit with electrical stimuli to the tibial nerve. As almost all the I a fibres seem to connect to the homonymous motoneurones as shown in the cat (Scheibel & Scheibel, 1969; Mendell & Henneman, 1971), the minimal stimulus intensity that caused a significant increase in the firing of the motor unit at about the latency of monosynaptic I a facilitation was considered to be the threshold for the I a fibres. The individual sessions did not exceed 15 min. Because the motor units examined were confined to those with low firing thresholds, and contraction was limited to very weak because of technical difficulties, the conventional H reflex was also used to examine the modulation of reciprocal Ia inhibition as a group effect and thus to make comparison possible with previously reported results. The extents of the conditioning effects were examined in the resting state and during tonic isometric voluntary dorsiflexion of various strengths ranging from 5 to 2% of the maximum. In some subjects, the effects caused by conditioning stimuli of 2-3 intensity grades at a fixed conditioning-test stimulus interval (within 7 ms after their latencies) were explored in a single experimental session either of rest or dorsiflexion of a particular strength. The size of the test H reflexes in each situation (rest and contraction) was kept constant, usually 2-4 % of Mmax, by adjusting the intensity of the test stimuli. Since strengths of contraction were measured mostly by torque in the previous studies, an additional experiment was done in three subjects to explore the relationship between the amount of EMG activity and the torque during voluntary contraction to make comparison possible. The torque values measured were: 5-7, 1 5-2, 3-3 6, 6-6 9, 1-1 5, , and N m corresponding to strength of contraction values of 1, 2, 4, 6, 8, 1, 15 and 2% (measured with EMG as a percentage of the maximum). Analysis The data were analysed on line, and at the end of each session the time courses of the effect and the PSTHs were shown as tentative results on a computer display. The test intensities, presence or absence of unit firing, latency of the unit from the application of stimuli and the sizes of the H reflexes were stored on a floppy diskette for re-evaluation of the results and further analyses. Student's t test and ANOVA were used to analyse the results. RESULTS Inhibition of single motoneurones at rest Thirty-one motor units from six subjects were investigated. The time course and intensity curve of the conditioning A (I) ILco. ~LL ) - o - a1) 95 r 1 V 15 [ 11 F 115 F B L on onditioning-test stimulus interval (ms) L on onditioning stimulus intensity (xt) 1-1 Figure 1. Time course (A) and intensity curve (B) of inhibition for a single motor unit at rest A, FSs are plotted against conditioning-test stimulus intervals (absciss. The conditioning intensity to the common peroneal nerve was -95T. Inhibition appeared at 58 ms, increased up to 1-5 ms, then decreased. The distance between the conditioning and test stimulation sites was 8 cm. B, FSs plotted against the conditioning intensity at the fixed interstimulus interval of 1P5 ms. Inhibition appeared at the intensity of -75T, increased up to 1l5T, then remained at a plateau. onditioned FSs are expressed as percentages of the unconditioned FS. A and B shown, data for the same motor unit with a FL 16-6% of the "max. The circles with bars show the means + S.D. on, control.

4 278 M. Shindo and others J Physiol r cn LL 3 F cd.- 25 F 2 - c._. 15 F. x w 1o 5. * ritical firing level (% of Mmax) 2 Figure 2. Extent of reciprocal I a inhibition of soleus motoneurones produced by stimulation to the common peroneal nerve at rest The extent of inhibition, expressed as a percentage of the control FSs with the control value (1%) subtracted, is plotted against the FLs of all 23 motor units tested. The conditioning intensity was -95T for all the units. Interstimulus intervals of ms were set so that maximal inhibition was obtained. *, significant inhibition;, non-significant inhibition. Sixteen of the twenty-three motor units received reciprocal Ia inhibition in the resting state. There was no correlation between the existence or extent of inhibition and the FLs of the motor units. Note the logarithmic scale on the abscissa. effects produced by stimulation of the common peroneal nerve in one motor unit are shown in Fig. 1. For the time course, the conditioning intensity was fixed at O95T. Inhibition started at the conditioning-test stimulus interval of O8 ms (the distance between the conditioning and test stimulation sites was 8 cm) and increased to 1 5 ms, after which it decreased (Fig. 1A). The intensity curve for a fixed interstimulus interval of 1-5 ms shows that the inhibition threshold was O75T and that inhibition increased to 1-5T, after which it remained at a plateau (Fig. 1B). 4 r 45 F S E cn :._ 5 55 F on X4 onditioning-test stimulus interval (ms) 5 Figure 3. Effect of tonic antagonist contraction on I a inhibition in a single motor unit A typical time course for reciprocal I a inhibition at rest () and during tonic voluntary contraction of pretibial muscles at 2% of the maximum strength (). The conditioning intensity was '9T. In the resting state, there was slight inhibition starting at 1' ms. Inhibition was enhanced significantly (P < 5, ANOVA) during voluntary contraction. Note that inhibition increased throughout the time course, including the initial part. The control FS increased from 45'9 V at rest to 53 1 V during contraction. The circles with bars show the means + S.D.

5 J Physiol Ia inhibition during antagonist contraction 279 Time courses were obtained for twenty-three motor units, and intensity curves for eight units. The latency of inhibition ranged from O8 to 1-5 ms, and the duration of inhibition was 5-6 ms. The thresholds ranged from 65 to 83T with a mean of 74T. In the sixteen motor units whose time courses showed significant inhibition, maximal inhibition at the conditioning intensity of 95T ranged from 13-1 to % of the control FS (mean 19-7 %) at interstimulus intervals of 1P2-3- ms in the resting state. The distributions of the percentage inhibition at the conditioning intensity of 95Tfor all twenty-three motor units together with their FLs are shown in Fig. 2. The FLs of the units investigated ranged from 3 to 2 9% of Mmax, the mean value being 5 9%. In the resting state, sixteen of the twenty-three motor units (69-6 %) received significant inhibitory effects from I a afferents in the common peroneal nerve, but there was no significant correlation between the FLs of the motor units and the existence or extent of inhibition. The other seven motor units showed no significant inhibitory modification at rest. No motor unit received facilitatory effects from the common peroneal nerve. hange in I a inhibition on single motoneurones during tonic ankle dorsiflexion Typical time courses of reciprocal I a inhibition on one motor unit at rest and during weak tonic voluntary contraction of the pretibial muscles at 2 % of the maximum contractile strength are shown in Fig. 3. The intensity of the conditioning stimulus was 9T. In the resting state, slight inhibition occurred at a latency of -6 ms, maximal inhibition being 19-1 % of the control FS at 1P5 ms. During pretibial contraction, the control FS of this unit increased from 45 9 to 53 1 V (the Ia fibre threshold subtracted), and inhibition increased significantly (P < 5, ANOVA). It should be noted that an increase in inhibition occurred throughout the time course, including the very early part, restoration almost reaching the control level at 5 ms. The effect of tonic voluntary antagonist contraction on all twenty-three motor units is shown in Fig. 4. hange in reciprocal I a inhibition was assessed at intervals during the 7 ms after its onset at conditioning intensities of 8-95Twhich produced relatively little inhibition. The continuous lines show significant enhancement of inhibition, and the dotted lines show non-significant enhancement. Sixteen of the twenty-three motor units showed significant increases in inhibition. It should be noted that inhibition did not decrease significantly in any of the motor units during antagonist contraction. Of the twenty-three motor units tested, I a inhibition of soleus motoneurones from common peroneal nerve stimulation was found in sixteen units at rest, inhibition increasing during tonic antagonist contraction in twelve of these units. In the remaining four units, I a inhibition was not changed by contraction. In contrast, of the seven motor units which did not show significant inhibition at rest, inhibition first appeared in four units at interstimulus intervals of 1 2 and 2 ms, but the remaining three units 2 ll 4- c 15F x w 5 - I ritical firing level (% of Mmax) 6 Figure 4. hange in I a inhibition during tonic antagonist contraction in the 23 motor units Similar to the presentation in Fig. 2. Open symbols, data at rest; filled symbols, during contraction. ircles, significant inhibition; triangles, non-significant inhibition. The interstimulus intervals were fixed within 7 ms after the onset of inhibition. onditioning intensities were set at 8-95Tso that slight inhibition could be recorded for each motor unit. Data at rest and during contraction for the same motor unit are connected by lines. ontinuous lines indicate significant changes between rest and contraction, dotted lines non-significant change. No significant correlation was found between the increase in inhibition and the FLs of the motor units.

6 28 M. Shindo and others J Physiol received no conditioning effect from the common peroneal nerve at rest or during ankle dorsiflexion. In a motor unit that showed relatively large inhibition at rest (maximum % of the control FS at 2 ms), there was no significant increase in inhibition during tonic antagonist contraction at the conditioning intensity of -9T; inhibition at rest was % of the control FS, but % during ankle dorsiflexion. At the conditioning intensity of -8T, however, inhibition increased significantly during pretibial contraction ( % at rest and % during contraction). hange in I a inhibition of the motoneurone pool during tonic dorsiflexion Because inhibition increased at much weaker contractions than in the experiments published previously and because the motor units examined were limited to those with low thresholds (the FLs, at most 2% of Mmax), change in the extent of inhibition was also examined using the conventional H reflex. Figure 5A shows the time course of inhibition for one subject. In the resting state, the H reflex started to be depressed at 1P5 ms, maximal inhibition being reached at 2-5 ms. During voluntary tonic pretibial contraction at 2 % of the maximum contractile strength, the extent of inhibition increased throughout the time course, beginning at 1 ms. This increase in inhibition was statistically significant (P < -1, ANOVA). Figure 5B gives the results for the eight subjects examined. In three subjects the experiment was repeated several times to check the reproducibility of the results, and each time the results were almost identical when the conditioning intensities were low enough and the voluntary contractions weak enough. The strengths of the contractions were below 2 % of the maximum, and it was verified that no cocontraction of the antagonistic muscles occurred. The sizes of the control reflexes were equilibrated, between 2 and 4% of Mmax, for the rest and contraction sessions. In each subject, the conditioning-test stimulus interval was set within 7 ms from the onset of inhibition, and the conditioning intensity was set so as to obtain only small inhibition at rest (-7--95T). In seven of the eight subjects inhibition increased significantly during very weak contraction (1-2% of the maximum). In the other subject the extent of inhibition also tended to increase during voluntary contraction but did not reach statistical significance. When the conditioning intensity was relatively strong, such as P T, and/or when the contraction was relatively strong (5-2% of the maximum), inhibition sometimes did not increase during antagonist contraction: in one subject the extent of inhibition at 1was T as much as 43 2 % of the control H reflex at rest. During weak tonic contraction of the antagonists (2% of the maximum) the extent of inhibition decreased significantly to 9-1 %, whereas with the weaker conditioning stimuli of -85T it increased significantly, from 1 2 % of the control H reflex in the resting state to 21-5% during contraction of the same strength (not illustrated). A 15 x 1- I g 95. _ 9* X I 75. Nl on onditioning-test interval (ms) B -, W. 15 c c o Rest DF Figure 5. hange in reciprocal I a inhibition of the soleus motoneuronal pool by voluntary tonic pretibial contraction A, time courses of reciprocal I a inhibition of the soleus motoneurones in the resting state () and during contraction at 2% of the maximum () for one subject. Ordinate, the size of the conditioned reflex. At rest inhibition started between ms, peaking at 2-5 ms. During contraction inhibition increased throughout the time course of inhibition, starting at 1- ms. Each symbol with a bar represents a mean and S.E.M. of 3 measurements. B, change in the extent of inhibition with weak conditioning intensity which produced only slight inhibition at rest (rest) and during weak dorsiflexion (DF) in all 8 subjects. The conditioning-test stimulus intervals were set within 7 ms after the latency of inhibition in each subject. The extent of inhibition increased significantly (continuous lines) or non-significantly (dotted line) during voluntary antagonist contraction.

7 J Physiol Ia inhibition during antagonist contraction 281 Effect of contraction strength on I a inhibition of the motoneuronal pool To analyse the factors responsible for the differing results in the present study and previous reports, the effect of contraction strength on the change in the extent of inhibition of the soleus motoneuronal pool was examined systematically in subjects who showed strong inhibition at rest. Figure 6 shows the results. onditioning stimulation to the common peroneal nerve at an intensity of 1 OT produced inhibition which started between 1P and 1P5 ms and peaked at 2- ms in the resting state. hange in the extents of inhibition produced by tonic voluntary contraction of the pretibial muscles was examined during contractions of various strengths ranging from 1 to 2% of the maximum, with three grades of conditioning intensity:.9, 1. and 11 T. During contraction of a particular strength, effects produced by conditioning stimuli of the three intensities were evaluated in a single experimental session in order to equalize the effect of contraction on the inhibition caused by conditioning stimuli of these three intensities. The sizes of the unconditioned control H reflexes were kept at about 22% of Mmax during contractions of all strengths as well as at rest. With the conditioning intensity of -9T, inhibition started to increase at the contraction strength of 2 %, gradually increasing to peak inhibition at 8% contraction. At stronger contractions, the extent of inhibition decreased progressively. In contrast, at 1 and 11 T, inhibition was maximal at a contraction of 2 %, gradually decreasing with the contraction strength. It should also be noted that the extents of inhibition obtained with the three conditioning intensities at contractions above 6 % of the maximum were almost the same and that at 2% contraction they were all less than the values for the resting state. Grading of the contraction strength experiments was done for five subjects. In all of them inhibition increased significantly at very weak contractions of -5-2% of the maximum, and was strongest at a slightly stronger contraction, thereafter decreasing at stronger contractions. The strengths of contraction needed to obtain maximal inhibition were always greater for weak conditioning intensities. In three subjects, the extent of inhibition during contractions 2 % of the maximum was smaller than that in the resting state. The difference between the extents of inhibition produced by relatively weak and strong conditioning stimuli became smaller as the strength of contraction was increased a1) I - Q c._ n x UJ Strength of dorsiflexion (% of maximal contraction) Figure 6. Effect of contraction strength on I a inhibition with three different conditioning intensities The extents of inhibition produced by tonic pretibial contraction of strengths from 1 to 2% of the maximum for three grades of conditioning intensity, 9 (), 1 (A) and 1 1 T (), are plotted against the strength of contraction. onditioning stimuli of the three intensities were applied randomly during a single experimental session of rest or of contraction of a particular strength. The control reflex sizes were about 22% of Mmax in each situation. With the conditioning intensity of 9T, inhibition started to increase at the contraction strength of 2 %, gradually increasing to peak inhibition at 8% contraction. The extent of inhibition decreased progressively at stronger contractions. At the intensity of 1x and 1 1 T, inhibition was maximal at 2 % contraction, gradually decreasing with contraction strength. It is noteworthy that the extents of inhibition produced by the three conditioning intensities at contractions above 6% of the maximum were almost the same and that at 2% of maximum contraction they were all less than the values in the resting state. Each symbol represents the mean and S.E.M. of 2-6 measurements.

8 282 M. Shindo and others J Physiol DISUSSION As the sensitivity of the FS method for evaluating conditioning effects is the same as that of the H reflex method (Shindo et al. 1994), the short latency and low threshold of inhibition on single soleus motor units found in our study are a strong indication that the inhibition observed is reciprocal I a inhibition. Interpretation of data using motor units is simpler than that of data in previous reports on reciprocal I a inhibition determined by the H reflex technique because it is not necessary to evaluate 'pool problems' as was the case in previous studies (Shindo et al. 1984; rone et al. 1985, 1987; Iles 1986; rone & Nielsen, 1989a; Nielsen et al. 1992). Distribution of I a inhibition in single soleus motoneurones from the common peroneal nerve Although the motor units tested were limited to earlyrecruiting ones because of methodological difficulties in isolating single motor units with high thresholds, the incidence of inhibition of the soleus motoneurones by the I a afferents from the common peroneal nerve could be delineated by the proposed FS method. In the resting state, nearly two-thirds of the twenty-three motor units investigated received significant inhibitory effects from I a afferents in the common peroneal nerve. In four motor units of the remaining one-third, inhibition first appeared during voluntary dorsiflexion. Therefore 87 % of the motor units in the soleus muscle receive reciprocal I a connections from the common peroneal nerve. This is a new finding for human subjects. Increase in reciprocal I a inhibition during tonic antagonist contraction To estimate the change in 'pure' reciprocal I a inhibition, we focused on determining the onset of inhibition. Our results for the motoneuronal pool based on the conventional H reflex method and for single motoneurones based on the new FS method differ from the conclusions made in several recent papers (rone et al. 1985, 1987; Iles, 1986; rone & Nielsen, 1989 and show that the reciprocal I a inhibitory pathway from the common peroneal nerve to the soleus motoneurones is facilitated during ankle dorsiflexion. As for the data obtained by the FS method, it must be noted that the increase in inhibition is underestimated about twofold. Firstly, because inhibition was expressed as a percentage of the control FS (which was larger during contraction than at rest), the equal percentage indicates that inhibition actually increased during contraction. Therefore, even in the four motor units that showed Ia inhibition at rest and no increase during ankle dorsiflexion, there may have been an increase because if the inhibition that occurred during contraction was expressed as a percentage of the control FS at rest, all four motor units would show significant increases. Secondly, post-activation depression (rone & Nielsen, 1989b) most probably depresses the effect of conditioning stimuli to the common peroneal nerve during pretibial contraction. onsequently, we conclude that activity of the I a inhibitory interneurones which are driven by I a afferents from the pretibial flexors increases during tonic voluntary contraction of the pretibial muscles. The validity of comparing the conditioning effects among the different situations by use of the FS method certainly depends on the linearity of the relationship between the test stimulus intensity and the size of the test EPSP within a particular motoneurone. Naturally the relationship reflects the distribution of Ia fibres in the peripheral nerve stimulated. According to Eccles, Eccles & Lundberg (1958), the relationship is linear, but in fact on close examination it could be sigmoid as is usually the case for many physiological phenomena. A non-linear relationship might be expected when the stimulus intensity is either very weak or very strong. The intensity we used was such that the firing probability of motoneurones with low thresholds (FLs were well below the size of the maximal H reflex in each subject) was 5%, indicative that the stimulus intensity (thus the size of the test EPSP) is neither too weak (small) nor too strong (large). We therefore expect that in the FS method the relationship between the stimulus intensity and the EPSP size would be in a linear part. The change in extent of the conditioning effect during contraction, measured using the FS method, depends also on other factors than those described above. The fact that the unconditioned FSs of the motor units were stronger during voluntary contraction of the antagonists implies that the resting membrane potential of motoneurones were hyperpolarized. As the IPSP size decreases when the resting membrane potential of a motoneurone is hyperpolarized (Eccles, 1957), the same conditioning inhibitory input to the motoneurone will produce a smaller IPSP. Hyperpolarization of the motoneurones will thus lead to underestimation of the increase in inhibition. However, because an increase in presynaptic inhibition on soleus I a terminals during tonic antagonist contraction (Meunier & Morin, 1989) will cause increase in the unconditioned FS, the increased FS found during contraction cannot be ascribed solely to hyperpolarization of the motoneurone. Furthermore, the membrane conductance of the motoneurone may be increased (Araki, Eccles & Ito, 196) following various inhibitory inputs during contraction, and this factor might increase the conditioning IPSPs with resulting overestimation of the conditioning effects. Precise evaluation of these factors remains to be clarified. The progressive decrease in I a inhibition, seen with the conventional H reflex method, after peaking during the strengthening of contraction (Fig. 6) is very probably due to occlusion at the level of I a inhibitory interneurones. This is also a sign of increased activity of the I a inhibitory interneurones (see below). It may, however, be partly explained by the activity of Renshaw cells. During tonic pretibial contraction, discharge of the motoneurones that innervate the pretibial muscles fires Renshaw cells, which in turn suppress the activity of the inhibitory I a interneurones (Hultborn, Jankowska & Lindstrom, 1971 a,b). According to Hultborn & Pierrot-Deseilligny (1979) Renshaw cell activity is activated or depressed during voluntary contraction depending on the strength of the contraction: activated most during weak contraction (1% of the maximum), moderately during 4% contraction, and depressed at 8% contraction. Inhibition

9 J Physiol Ia inhibition during antagonist contraction 283 of I a interneurones through Renshaw cells during relatively weak contraction employed in our study (1-2% of the maximum) may thus explain the progressive decrease in I a inhibition during contraction, but change in Renshaw cell activity during this contraction range is not known. Factors accounting for different conclusions in the literature What factors account for the different conclusions seen in the results of reported studies? As suggested previously (rone et al. 1985, 199), the first factor is the 'pool problem'. In the studies by Tanaka (1974) and Shindo et al. (1984) the reduced test H reflex during antagonist contraction was used as it was; this contrasts with Danish researchers (rone et al. 1985, 1987; rone & Nielsen, 1989a; Nielsen et al. 1992) who equilibrated the test reflex size for the state of rest and contraction by controlling the intensity of the test stimuli. Because the extent of the conditioning effects is expressed as a smaller percentage of the Mmax when the test reflex decreases in size (e.g. below 2% of Mmax (rone et al. 199)), the 1984 results of Shindo et at. must be underestimations. If the motoneuronal populations with equilibrated test reflexes (rone et al. 1985, 1987; rone & Nielsen, 1989a; Nielsen et al. 1992) were not identical for the testing done at rest and during contraction, this would explain the difference; but, even though the motoneuronal populations may not be exactly the same for both these tests, the equilibration manoeuvre would not make a significant change in the motoneuronal population (T. Hashimoto, M. Shindo, S. Yanagawa & N. Yanagisawa, unpublished observation). The non-linear input/output relationship or recruitment gain of the motoneuronal pool (Kernell & Hultborn, 199; Nielsen et al. 199) also might explain the difference - the sensitivity of the H reflex may differ from that in the resting state. No change in recruitment gain during tonic voluntary contraction, however, has yet been reported. Furthermore, our present study using the conventional H reflex with equilibration of the test reflex size showed the same results as those for single motor units. The 'pool problem' is therefore very unlikely to be responsible for the different conclusions in the literature. The second possibility is the post-activation depression reported by rone & Nielsen (1989 b). These authors studied modulation of reciprocal I a inhibition by tonic antagonist contraction before and after ischaemia to the leg. Tonic antagonist contraction prior to ischaemia did not increase inhibition, but when given afterwards it did. The authors attributed the lack of increase in inhibition by contraction before ischaemia to post-activation depression (Nielsen et at. 1992). Similarly, in our study the extent of inhibition did not increase significantly, or it decreased at the relatively strong contractions used (Fig. 6). The present results on the grading of voluntary contraction cannot, however, be explained readily by post-activation depression. It must have affected inhibition to the same extent as long as the strength of contraction remained the same. We studied the inhibitions produced by conditioning stimuli of three different intensities in the same contraction, but the effects of contraction on the modulation of inhibition differed, depending on the conditioning intensities. Post-activation therefore cannot explain the lack of increase in inhibition during contraction, although its contribution cannot be denied. The third possibility is that an occlusion mechanism acts at the I a interneuronal level. If the excitability of the I a interneurones is very high and produces strong inhibition at rest, the descending facilitatory command during voluntary contraction would result in interneuronal firing. When many interneurones have been fired by descending commands and by peripheral I a discharge through a-y co-activation during contraction, only a few interneurones would be available to respond to peripheral conditioning stimuli. Hence the fact that there was a marked difference in the extent of inhibition in the resting state for both groups is very important. The percentage inhibition was very small (at most 1% of the control reflex) in early reports, whereas it has been shown to be as much as 4 % of the control reflex (mean value 14-9 %) in a later report (rone et al. 1987). In our study, inhibition was enhanced during very weak antagonist contraction (1-2 % of the maximum), evidence of strong facilitatory effects on I a interneurones from both the descending tract and the peripheral I a afferents. Furthermore, the effect of contraction strength on the change in inhibition (Fig. 6) strongly supports the occlusion hypothesis, i.e. stronger conditioning stimuli can fire the I a interneurones more easily upon background excitation by voluntary descending facilitatory inputs and peripheral I a impulses, resulting in earlier saturation of the interneurones. Once some interneurones are fired by the descending inputs alone, only the remaining interneurones can be fired by peripheral stimulation. The number of these interneurones is expected to decrease progressively when contraction is strengthened, as is seen in the right half of Fig. 6. It should be noted that weak contraction, as employed in the present study, has not been explored in previous reports with the exception of several subjects in rone et al. (1987). It is thus very likely that such differences in experimental conditions led to different conclusions being drawn in the literature. Furthermore, the mode of change in reciprocal I a inhibition in the ischaemia experiment (Nielsen et al. 1992) is also explained by the occlusion hypothesis: ischaemia decreases peripheral I a discharge during contraction. The fourth possibility is that there is co-contraction of the soleus muscle during contraction of the pretibial muscles. Very weak tonic contraction of the isolated muscle was seldom easy for the subjects. rone et al. (1987) also found very weak contraction with no increase in inhibition during antagonist contraction, but they did not report whether

10 284 M. Shindo and others J Physiol there was co-contraction. When the examined muscle cocontracts, I a inhibition must decrease (Nielsen & Kagamihara, 1992). In our experiment, when co-contraction occurred inhibition did not increase, but it sometimes decreased during 'antagonist' contraction (not shown). We suggest that lack of increase in I a inhibition during tonic antagonist contraction in the previous studies is mainly due to occlusion at the I a interneuronal level and, to some extent, to co-contraction of the triceps surae muscle during tonic pretibial contraction. Recently Iles & Pisini (1992), using transcranial electrical stimulation, studied interaction of the two inhibitory inputs, corticospinal tract and peripheral I a fibres, onto the soleus motoneurones. They demonstrated that inhibition was facilitated when cortical and spinal inhibitory actions were weak, but reversed to occlusion when both actions were strong. The present results will further support their conclusion during actual voluntary contraction. In this connection it would be worthwhile to point out the apparent discrepancy with our previous report (Shindo et al. 1984) which showed progressive increase in inhibition during tonic voluntary dorsiflexion even at contraction strengths of 1-3 % of the maximum. The extent of inhibition at rest was generally small in the previous report (Shindo et al. 1984); this contrasts with the fact that only those subjects having rather strong inhibition in the resting state were included in the present experiment for the grading of contraction strength; none of the subjects in the 1984 paper were included in the present study. Moreover, in the 1984 paper the contraction strength was expressed as a percentage of the maximum torque around the ankle. The relationship between the torque and EMG percentages for the strength of contraction was then examined in the present study in one of the 1984 subjects; the 1, 2 and 3% contraction by torque was equivalent to respectively 5, 8 and 12 % of the maximum when the EMG was used. lose examination of Fig. 4B in the 1984 paper, showing data on different dates for the same subject, reveals that inhibition increased during contraction of 15 or 25% of the maximum torque when no inhibition was observed at rest on one day; however, although the inhibition increased during 1% contraction, it decreased during 25 % contraction when inhibition was relatively strong on the other day. These findings are in line with those of the present experiments, and implicate the importance of the excitability level of I a inhibitory interneurones in each subject. Functional significance Parallel facilitation of the motoneurones of the prime mover and the corresponding I a interneurones (Hongo et al. 1969) was confirmed during tonic contraction of antagonists as it was at the onset of contraction. The fact that this took place even during very weak contraction indicates that a reciprocal I a inhibitory pathway does function in ordinary movements and that it suppresses actively antagonist motoneurones. In the case of isolated contraction, the enhanced activity of this reflex pathway would suppress the antagonist stretch reflex caused by contraction of the prime mover thereby contributing to the smoothness of movement. ARAKI, T., ELES, J.. & ITO, M. (1 96). orrelation of the inhibitory post-synaptic potential of motoneurones with the latency and time course of inhibition of monosynaptic reflexes. Journal of Physiology 154, AVALLARI, P., FOURNIER, E., KATZ, R., PIERROT-DESEILLIGNY, E. & SHINDO, M. 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