Muscle fatigue changes cutaneous suppression of propriospinal drive to human upper limb muscles

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1 J Physiol (2007) pp Muscle fatigue changes cutaneous suppression of propriospinal drive to human upper limb muscles P. G. Martin, S. C. Gandevia and J. L. Taylor Prince of Wales Medical Research Institute and the University of New South Wales, Sydney 2031, Australia Some voluntary drive reaches human upper limb muscles via cervical propriospinal premotoneurones. Stimulation of the superficial radial nerve can inhibit these premotoneurones selectively and the resultant suppression of voluntary drive to motoneurones changes on-going electromyographic (EMG) activity. We investigated whether muscle fatigue changes this cutaneous-induced suppression of propriospinal drive to motoneurones of upper limb muscles. EMG was recorded from the extensors and flexors of the wrist and elbow. In the first study (n = 10 subjects), single stimuli (2 perception threshold; 2PT) to the superficial radial nerve were delivered during contraction of the wrist extensors, before and after sustained fatiguing contractions of wrist extensors. In the second study (n = 10), similar stimuli were applied during elbow extension, before and during fatigue of elbow extensors. In the final study (n = 10), trains of three stimuli (2PT) were delivered during contractions of wrist extensors, before and while they were fatigued. With fatigue of either the wrist or elbow extensors, EMG suppression to single cutaneous stimuli increased significantly (by 75%) for the fatigued muscle (P < 0.05). Conversely, in the other muscles, which were coactivated but not principally involved in the task, inhibition decreased or facilitation increased. Trains of stimuli produced greater suppression of on-going wrist extensor EMG than single stimuli and this difference persisted with fatigue. A control study of the H reflex in extensor carpi radialis showed that the mechanism responsible for the altered EMG suppression in fatigue was not at a motoneurone level. The findings suggest that the proportion of descending drive mediated via the disynaptic propriospinal pathway or the excitability of inhibitory interneurones projecting to propriospinal neurones increases substantially to fatigued muscles, but decreases to other active muscles. This pattern of changes may maintain coordination during multimuscle movements when one group of muscles is fatigued. (Resubmitted 10 December 2006; accepted after revision 11 January 2007; first published online 11 January 2007) Corresponding author J. Taylor: Prince of Wales Medical Research Institute, University of New South Wales, Barker Street, Randwick, Sydney, NSW 2031, Australia. jl.taylor@unsw.edu.au In humans, the corticospinal tract represents the primary pathway between the motor areas of the cortex and the spinal cord (for review see Lemon et al. 2004). This pathway makes monosynaptic connections to most upper limb motoneurones (e.g. Palmer & Ashby, 1992; de Noordhout et al. 1999). In addition to these direct cortico-motoneuronal connections, there is likely to be a parallel disynaptic path from the cortex to motoneurones. This pathway is believed to transmit a component of the descending drive for voluntary movement through cervical premotoneurones located rostral to motoneurones (for reviews see Pierrot-Deseilligny, 1996, 2002; Fig. 6). This set of interneurones may be analogous to the system of C3 C4 propriospinal neurones which have been precisely characterized in the cat (for review see Alstermark & Lundberg, 1992) and more recently the macaque monkey (Alstermark et al. 1999; Sasaki et al. 2004; Isa et al. 2006), though that analogy has been disputed (Maier et al. 1998; Nakajima et al. 2000; Olivier et al. 2001). In the cat, these propriospinal neurones have divergent projections onto motoneurones of muscles operating at different joints (Alstermark et al. 1990; Tantisira et al. 1996) and a system of divergent projections has been proposed for humans (Mazevet & Pierrot-Deseilligny, 1994; Pierrot-Deseilligny, 1996). The interneurones that relay the descending drive also receive excitation and inhibition from peripheral afferents (Gracies et al. 1991; Burke et al. 1992a). During weak voluntary contractions, removal of some of the drive relayed via cervical propriospinal neurones can reduce muscle activity by 30% in healthy subjects but substantially more in some patient groups (Pol et al. 1998; Mazevet et al. 2003; Stinear & Byblow, 2004). Therefore, it has DOI: /jphysiol

2 212 P. G. Martin and others J Physiol been proposed that this pathway plays an important role in the control of human movement (Burke et al. 1994; Pierrot-Deseilligny & Burke, 2005). Muscle fatigue is an exercise-induced reduction in maximal voluntary muscle force and it is accompanied by changes at cortical and spinal levels (for review see Gandevia, 2001). While processes at the muscle cause much of the reduction in force for limb muscles, changes in the ability of the central nervous system to drive the muscle maximally are also important. A reduced efficacy of corticospinal drive to motoneurones has been demonstrated during sustained (Gandevia et al. 1996) and intermittent maximal (Taylor et al. 2000; see also Todd et al. 2003) and submaximal contractions (Loscher et al. 1996; Sogaard et al. 2006). However, it is unknown whether drive conveyed to motoneurones through the cervical propriospinal pathway changes with fatigue. Stimuli delivered to cutaneous afferents in the superficial radial nerve briefly suppress both voluntary on-going electromyographic (EMG) activity and responses to cortical stimulation in various upper limb muscles (e.g. Burke et al. 1994; Pol et al. 1998; Marchand-Pauvert et al. 1999; Mazevet et al. 2003). If this suppression were due to inhibition exerted directly on motoneurones, responses to stimulation of homonymous Ia afferents should be similarly reduced. However, these cutaneous volleys have minimal effects on the H reflex of extensor carpi radialis (e.g. Burke et al. 1994; Mazevet et al. 2003) or the tendon reflex evoked in biceps or triceps brachii (Pierrot-Deseilligny, 1996). Instead, the suppression is exerted on premotoneurones which transmit part of the voluntary drive to the motoneurone pool, in parallel with the monosynaptic cortico-motoneuronal pathway. Hence, selective inhibition of propriospinal neurones by specific cutaneous inputs can interrupt some of the propriospinal component of descending drive without inhibiting the target motoneurones (Nielsen & Pierrot-Deseilligny, 1991; Burke et al. 1994). The relationship between the cutaneous-induced suppression of descending excitation at the propriospinal level and the resulting disfacilitation is complex, and the percentage of the descending drive that is relayed through this indirect system cannot be equated with the percentage of EMG suppression. However, a greater propriospinal contribution to corticospinal drive allows a greater disfacilitation of the motoneurones in response to the cutaneous inputs (Burke et al. 1994; Mazevet et al. 1996; Nicolas et al. 2001). In the current studies, we used stimulation of the superficial radial nerve to investigate whether muscle fatigue changes the cutaneous-induced suppression of propriospinal drive to motoneurones of various upper limb muscles. To explore the mechanism for changes in cutaneous-induced inhibition with fatigue, the effects of single stimuli or brief trains of stimuli were investigated in separate experiments. Suppression from a train of stimuli increases temporal summation at inhibitory interneurones projecting to propriospinal neurones. Comparisons between the magnitude of EMG suppression in response to single stimuli or trains have been used previously to infer the mechanisms responsible for changes in the propriospinal pathway that occur at the offset compared to the onset of voluntary movements (Pierrot-Deseilligny, 1996) and in patients with hemiplegia (Mazevet et al. 2003) and Parkinson s disease (Pol et al. 1998). Because altered EMG suppression in fatigue could be due to inhibition exerted directly on motoneurones, a control study assessed changes in the extensor carpi radialis Hreflex. Methods Ten healthy subjects (3 females; 31 ± 9 years, mean age ± s.d.) participated in three main studies performed on separate days. The technique used in these experiments was developed by Burke et al. (1994). To explore changes in the disynaptic propriospinal pathway with muscle fatigue, cervical propriospinal neurones were inhibited by a cutaneous volley during tonic contractions of the wrist or elbow extensors. Changes in the depth of the cutaneous-induced suppression of the EMG reflect changes in the proportion of descending drive relayed via the propriospinal pathway or changes in the excitability of interneurones involved in mediating feedback inhibition to propriospinal neurones (for reviews see Pierrot-Deseilligny, 1996, 2002; Pierrot-Deseilligny & Burke, 2005). The procedures were approved by the local ethics committee and the study was conducted according to the Declaration of Helsinki. All subjects gave their informed consent to participate. Force and EMG recordings For wrist extension, the subjects sat with the right shoulder and elbow flexed at 45 deg and the forearm resting on a table with the hand open. The forearm was supported in the neutral position and the hand was clamped in an isometric myograph, which measured the torque of wrist extension. For elbow extension, the right arm was held by the side with the elbow at 90 deg and the forearm semipronated. The forearm was held in an isometric myograph which measured elbow extension torque. Subjects were instructed to perform wrist or elbow extensions but were not given specific instructions related to the muscle groups required to perform the task. During maximal voluntary contractions (MVCs), subjects received visual feedback of torque and were encouraged to perform maximally throughout the contraction. EMG was recorded from extensor carpi radialis (ECR), flexor carpi radialis (FCR), biceps brachii and triceps brachii by

3 J Physiol Propriospinal drive with fatigue 213 surface electrodes secured to the skin over the belly of each muscle (Ag AgCl, 10 mm diameter). EMG activity was filtered ( Hz) and sampled at 2 khz for off-line analysis using customized software (CED 1401 with Signal software; Cambridge Electronic Design, Cambridge, UK). Stimulation Electrical stimuli of 1 ms duration (Digitimer DS7 constant-current stimulator, Digitimer Ltd, Welwyn Garden City, UK) were delivered to the superficial radial nerve through bipolar surface electrodes (Ag AgCl, 10 mm diameter) placed on the skin of the radial edge of the forearm, 3 cm proximal to the wrist. The stimulus intensity was 2 perception threshold (2PT). Perception threshold was determined by increasing stimulus intensity until the stimulus produced radiating paraesthesiae in the dorsal side of the hand. At 2PT paraesthesiae were often felt in the dorsum of the hand and the first three fingers. This intensity ( ma) was used for both single volleys and trains (three stimuli at 300 Hz). Protocol Study 1. Subjects performed three to four brief MVCs of the wrist extensors. The on-going rectified integrated EMG activity (time constant of 100 ms) from ECR was displayed on an oscilloscope and a target corresponding to the amount of EMG produced during a 30% MVC of the wrist extensors was displayed. Subjects performed a series of test contractions to this target each held for 15 s (Fig. 1). Single electrical stimuli were delivered to the superficial radial nerve during these contractions. The stimulus sequence consisted of control trials (without stimulation) and trials with superficial radial stimulation in random order (once every second). The contractions were separated by 60 s rest to avoid fatigue and any effects of habituation to the stimulus (Burke et al. 1994). The data recorded were averaged to produce a single run containing 150 conditioned responses and a run containing EMG with no conditioning. Subjects then performed pairs of contractions comprising a sustained MVC of the wrist extensors held until torque dropped to 60% of the initial maximum followed by a 15 s test contraction during which they matched their integrated EMG to the same target as prior to fatigue (i.e. EMG corresponding to 30% of initial MVC; Fig. 1). Again, single stimuli were delivered during these test contractions and trials without stimulation were also recorded. Because the sustained MVCs varied in length, the pairs of contractions were separated by 60 s intervals, which ensured that for both non-fatigued and fatigued conditions, contractions with conditioning stimuli were always at least 60 s apart. Again, the data were averaged to produce a single run containing 150 conditioned responses and a run of unconditioned EMG. Study 2. The procedures for study 2 were identical to those for study 1, except that subjects performed contractions of the elbow extensors instead of the wrist extensors and integrated EMG from triceps was displayed rather than from ECR. Conditioning stimuli were still delivered to the superficial radial nerve. Study 3. The procedures for study 3 were identical to those for study 1, except that trains (three stimuli at 300 Hz) rather than single stimuli were delivered to the superficial radial nerve and only 105 conditioned trials were collected in both non-fatigued and fatigued sets. Figure 1. Experimental protocol Subjects (n = 10) performed a series of 20 (studies 1 and 2) or 14 (study 3) test contractions at 30% of maximal torque (MVC) each lasting 15 s. During these test contractions, single electrical stimuli (studies 1 and 2) or 3 stimuli at 300 Hz (study 3) were delivered randomly to the superficial radial nerve (indicated by arrows). To avoid habituation to the stimulus the 30% MVCs were separated by 60 s rest intervals. Subjects then performed pairs of contractions comprising a sustained MVC held until torque dropped to 60% of the initial maximum followed by a 15 s contraction in which they matched integrated EMG to the EMG produced during 30% MVCs prior to fatigue. Again, stimuli were delivered randomly during these contractions. Because the sustained MVCs varied in length, at least 60 s rest was given between the end of a 30% MVC and the start of the next sustained MVC.

4 214 P. G. Martin and others J Physiol H reflex control study. Nine of the subjects also performed a control study to assess whether cutaneous afferent volleys caused direct inhibition of motoneurones. The ECR H reflex was elicited by bipolar stimulation of the radial nerve in the spiral groove during voluntary contraction of ECR. The size of the reflex was measured as the peak-to-peak amplitude. Prior to each experiment, the input output relationship between stimulus intensity and the amplitude of the H reflex was defined and a stimulus intensity corresponding to the midpoint of this recruitment curve was chosen for the subsequent experiment. This procedure ensured that the stimulus intensity chosen was on the steepest part of the recruitment curve and maximized the chance of detection of small changes in the H reflex. The same stimulus intensity was used throughout the experiment. At the completion of the experiment, this stimulus intensity was increased and an observable increase in the size of the H reflex confirmed that this intensity remained on the sensitive part of the recruitment curve for the fatigued condition. The maximal compound muscle action potential (maximal M-wave; M max ) evoked by supramaximal stimulation of the radial nerve was also measured before and during fatigue to check for changes at the muscle which would affect the size of thehreflex. The procedures for this control study were identical to study 1, except that the stimulus sequence consisted of control trials (H reflex alone) and trials with superficial radial nerve stimulation (H reflex and cutaneous stimulation) in random order delivered every 2 s. The interval between the cutaneous stimulus and H reflex stimulus was varied from 3 ms to 15 ms to define the time course of any cutaneous effects on the H reflex. At shortest interstimulus intervals (ISIs) the cutaneous and H reflex volleys should arrive together at the same segmental level as the ECR motoneurone pool, based on differences in peripheral conduction time for the cutaneous afferents to the stimulus site for the H reflex ( 4 ms; Burke et al. 1994). The longest ISI corresponded with the end of the window of analysis of the on-going EMG in study 1. Approximately 15 responses were evoked at each ISI during both non-fatigued and fatigued trials. Additional control study. Nine subjects also performed another control study in which responses to transcranial magnetic stimulation (TMS) were evoked. Differences in the latencies of these potentials were used to calculate the appropriate windows of analyses for cutaneous inhibition (see below). A circular coil (13.5 cm outside diameter) positioned over the vertex elicited motor evoked potentials (MEPs) recorded from ECR, FCR, biceps and triceps muscles (Magstim 200, Magstim Co., Dyfed, UK). The stimulus intensity was adjusted to evoke potentials of 1 mv peak-to-peak amplitude during weak contractions of the target muscle. Subjects performed two weak ( 20% MVC) contractions of each muscle separately, during which five stimuli were delivered at 0.2 Hz. The latency of the onset of the MEP was calculated for each muscle for potentials evoked during contractions of the target muscle. Data analysis Voluntary on-going EMG activity from ECR, FCR, biceps and triceps was full-wave rectified and averaged for each subject to produce two runs, one containing conditioned responses and the other unconditioned EMG. To assess the level of suppression of on-going EMG following stimulation an appropriate window of analysis was calculated. For ECR this window corresponded with that previously used for single stimuli and trains of stimuli delivered to the superficial radial nerve at wrist level (Mazevet et al. 2003). Hence, the window started 26 ms after the single stimulus and 32 ms after the first stimulus in the train (i.e. 26 ms after the last stimulus). To calculate the expected onset of the cutaneous-induced EMG suppression in the other muscles, the differences in the latency of MEPs were measured. Like the MEPs, differences in the onset of the cutaneous-induced effect in each muscle should only reflect the different distances of conduction from the spinal cord to the recording electrodes. The differences in onset latencies of MEPs were 0.5 ± 0.9 ms, 3.6 ± 0.7 ms and 3.5 ± 0.7 ms earlier for FCR, biceps and triceps, respectively, when compared to ECR. Therefore, the onset of the window for analysis for FCR EMG was the same as for ECR, whereas it was set 4 ms earlier for biceps and triceps. The duration of the window of analysis was limited to 9 ms in order to avoid late effects due to inhibition exerted at the cortical level by the test input (Maertens de Noordhout et al. 1992; Burke et al. 1994). The effect evoked by the conditioning volley on the voluntary EMG was assessed as the area from the baseline within the window of analysis in the averaged run (Fig. 2). Baseline EMG was defined as the mean EMG obtained over a 40 ms period from the averaged run of the unconditioned trials. Conditioned EMG was expressed as a percentage of control EMG. For each unconditioned sweep, torque and rectified EMG from each muscle were measured for a 250 ms window and the means for non-fatigued and fatigued sets were found. For the target muscle in each study, additional analyses compared the mean rectified EMG produced in unconditioned sweeps during each test contraction in each condition (i.e. non-fatigued or fatigued). Statistics Group data are presented as means ± standard deviations (s.d.) in the text and the table and means ± standard

5 J Physiol Propriospinal drive with fatigue 215 Table 1. Changes in torque and EMG during test contractions before and after sustained fatiguing contractions Torque ECR EMG FCR EMG Triceps EMG Biceps EMG (N m) (μv) (μv) (μv) (μv) Study 1 Non-fatigued wrist extensors 4.0 ± ± ± 6 34 ± ± 30 Fatigued wrist extensors 3.2 ± ± ± 7 47 ± ± 20 Study 2 Non-fatigued elbow extensors 13.2 ± ± 9 11 ± ± ± 11 Fatigued elbow extensors 11.8 ± ± ± ± ± 13 Study 3 Non-fatigued wrist extensors 3.5 ± ± ± 4 19 ± ± 38 Fatigued wrist extensors 2.9 ± ± ± 6 34 ± ± 30 Mean (± S.D.) torque and rectified EMG produced during contractions in which the EMG from the target muscle (bold) was matched to that produced during a 30% maximal voluntary contraction prior to fatigue. Values show the mean torque and EMG produced across all sets before and after sustained isometric wrist (study 1 and 3) or elbow extensions (study 2). Significant difference from non-fatigued values: P < P < P < error of the mean (s.e.m.) are shown in the figures (with n in the legends). Student s t test for paired data was performed to compare the difference between the amount of cutaneous-induced EMG suppression, torque or background rectified EMG, prior to and with fatigue in each study. Separate repeated measures ANOVAs (with Student Newman Keuls post hoc tests) were used to compare the amount of cutaneous-induced suppression or background rectified EMG produced in the target muscle across multiple test contractions before and during fatigue. A two-way repeated measures ANOVA was used to compare the amplitude of the control H reflex, prior to or during fatigue, with H reflexes evoked at various ISIs following cutaneous stimulation. Statistical significance was set at P < Results Cutaneous-induced suppression of propriospinal drive to motoneurones by stimuli delivered to the superficial radial nerve at twice perceptual threshold was investigated before and during fatigue of elbow or wrist extensor muscles. Force and EMG Subjects successfully matched EMG throughout the studies. The amount of EMG produced in the target muscle during test contractions was not different between non-fatigued and fatigued sets for any of the three studies (P > 0.05; Table 1). Further analyses revealed no differences in the level of EMG produced in the target muscles across the non-fatigued contractions (P = 0.995, study 1; P = 0.211, study 2; P = 0.098, study 3) or across the test contractions with fatigue (P = 0.259, study 1; P = study 2; P = 0.328, study 3). The amount of torque produced by a matched level of EMG decreased following sustained maximal contractions of the target muscle in all three studies (P < 0.05; Table 1). This suggests that the sustained maximal contractions produced significant fatigue in motor units involved in producing the EMG during test contractions. The amount of EMG in muscles in which EMG was not deliberately matched tended to increase. This increase was significant for FCR in both studies involving wrist extension (P < 0.05; Table 1). In addition, biceps EMG increased in studies involving wrist extension or elbow extension, although this increase was more variable in study 3 (Table 1). Study 1. In the first study, single cutaneous stimuli were delivered to the superficial radial nerve during tonic contractions of the wrist extensors, before and after sustained fatiguing contractions. Figure 2A shows the typical amount of cutaneous-induced suppression, expressed with respect to baseline EMG, for one subject. For the group of subjects, the stimuli reduced the on-going EMG by 8.4 ± 4.5% (Fig. 2D, filled circles). The amount of cutaneous-induced suppression of ECR EMG changed following sustained fatiguing contractions of the wrist extensors. For the typical subject, as shown in Fig. 2A, there was a more marked decline in EMG following cutaneous stimuli during fatigued sets (dashed line) than non-fatigued sets (continuous line). For the group of subjects, EMG suppression increased by 75% to 14.3 ± 3.6% (P < 0.001; Fig. 2D, open circles). The increase in the level of EMG suppression with fatigue was evident in all 10 subjects (Fig. 3A). EMG suppression was also assessed across repeated test contractions and this comparison revealed no progressive changes in suppression evoked in ECR either before (P = 0.586) or with fatigue (P = 0.573). Whereas EMG suppression in the target muscle increased with fatigue, for muscles that were coactive during wrist extension the cutaneous-induced suppression decreased or, in cases where stimuli produced

6 216 P. G. Martin and others J Physiol facilitation, this facilitation increased (Fig. 4A). On average, the amount of suppression in triceps halved from 7.6 ± 3.8% to 4.0 ± 6% (P < 0.05) with fatigue of the wrist extensors and a small facilitation evoked in FCR prior to fatigue (0.5 ± 8%) increased substantially (4.9 ± 9%; P < 0.05). Small changes for the level of suppression in biceps were also observed, but were not significant. Study 2. To test whether the changes observed in the target and cocontracted muscles with fatigue were also evident for other types of contractions in the human upper limb, single stimuli to the superficial radial nerve were applied during tonic contractions of the elbow extensors. Figure 2B shows, for a single subject, that a small suppression of on-going triceps EMG produced by single stimuli (continuous line) was increased with fatigue (dashed line). For the group of 10 subjects, the level of suppression evoked in triceps, the muscle predominantly involved in the task, increased by 75% from 7.2 ± 3.6% to 12.5 ± 5.9% (P < 0.05; Fig. 2E, openversus filled circles). This increase was evident in 8 out of 10 subjects (Fig. 3B). There was no progressive change in the amount of suppression produced during repeated test contractions before (P = 0.291) or during (P = 0.496) fatigue. Opposite changes were again evident in the muscles cocontracted during elbow extension. The suppression evoked in ECR decreased from 7.5 ± 5.9 to 2.9 ± 5.7% (P < 0.05) and the small suppression in FCR (1.9 ± 2.6%) was reversed to a small facilitation (2.3 ± 6.5%; P < 0.05; Fig. 4B). Changes in biceps were not significant. Figure 2. Suppression of EMG activity by cutaneous inputs Modulation of the on-going EMG by cutaneous stimuli to the superficial radial nerve. The difference between the conditioned EMG and the mean control EMG over 40 ms (expressed as a percentage of the control EMG) is plotted against the latency after stimulation. A C, results obtained before (continuous line) and after (dashed line) sustained fatiguing contractions in the same subject. D F, group data for 10 subjects before ( ) and after ( ) sustained fatiguing contractions. Mean values (± S.E.M.) are plotted. A and D, on-going EMG from ECR was conditioned by single stimuli at twice perceptual threshold (2PT) during wrist extension. B and E, on-going EMG from triceps was conditioned by single stimuli (2PT) during elbow extension. C and F, on-going EMG from ECR was conditioned by a train of three stimuli (300 Hz, 2PT) during wrist extension.

7 J Physiol Propriospinal drive with fatigue 217 Study 3. The effects of a temporal summation elicited by a train of three stimuli were investigated during contractions of the wrist extensors before and after sustained fatiguing contractions of these muscles. The train of stimuli (Fig. 2C and F) produced a much larger suppression of the on-going EMG compared to single stimuli (24.0 ± 18.8% versus 8.4 ± 4.5%; P < 0.01; Fig. 2A and D). This suppression increased further by 40% to 33.3 ± 15.6% with fatigue of the wrist extensors (Fig. 2C, broken line, Fig. 2F, open circles) and was evident in 9 out of 10 subjects (Fig. 3C). Decreased EMG suppression or increased facilitation was again observed in the other muscles (Fig. 4C) and was significant for both FCR (P < 0.01) and triceps (P < 0.05). H reflex control study. If the superficial radial induced suppression of the on-going EMG, either before or with fatigue, resulted from inhibition exerted directly on motoneurones then the H reflex should also be depressed. Figure 5 shows changes in the H reflex induced by superficial radial nerve stimulation at 2PT during tonic contractions of ECR. The arrow indicates the expected simultaneous arrival of the cutaneous and H reflex volleys at the level of the ECR motoneurone pool (see Methods), and the broken line indicates the expected onset of EMG suppression. The cutaneous volley did not alter the amplitude of H reflexes at any ISI, either before (P = 0.881) or after (P = 0.628) sustained fatiguing contractions (Fig. 5). The amplitude of the control H reflex (without cutaneous stimulation) was not different before or with fatigue (1.2 ± 0.5 mv versus 1.1 ± 0.4 mv; P = 0.155). Similarly, when the control H reflexes were expressed relative to M max they did not differ before or with fatigue (13.2 ± 3.7% M max versus 12.3 ± 3.9% M max ; P = 0.251, paired t test). Discussion The main novel finding is that the pattern of suppression of the on-going EMG activity recorded from various upper limb muscles in response to stimuli applied to the superficial radial nerve changes dramatically with fatigue. The two important observations made during fatigue were that (i) the suppression of on-going EMG evoked in the fatigued target muscle increased by as much as 75% and (ii) for muscles not directly fatigued but cocontracted during tonic contractions, the suppression of on-going EMG decreased and any facilitation (if present) increased. These results suggest that either the proportion of cortical command to fatigued and cocontracted muscles passing through the cervical propriospinal pathway or the excitability of interneurones mediating the inhibition to propriospinal neurones is altered with fatigue. During fatigue, there will be various changes at a segmental level, which may change the excitability of motoneurones (for review see Gandevia, 2001). For the Figure 3. Cutaneous modulation of the on-going EMG before and after sustained fatiguing contractions Cutaneous modulation of the on-going EMG before and after sustained fatiguing contractions for each subject and the group of subjects. The vertical scales in each graph show the conditioned on-going EMG (expressed as a percentage of the control EMG) before and after sustained fatiguing contractions. The mean for 10 subjects is shown by the dark line and filled circles. The on-going EMG was conditioned by single stimuli (2PT) (A and B) ora train of three stimuli (300 Hz, 2PT) (C) to the superficial radial nerve. A and C, subjects performed wrist extensions and on-going EMG was recorded from ECR. B, subjects performed elbow extensions and on-going EMG was recorded from triceps brachii.

8 218 P. G. Martin and others J Physiol A B C principal muscle involved in the task, muscle activity was kept constant before and during fatigue. However, due to changes at the motoneurones a different level of descending drive may be required to produce this same muscle activity. Descending drive to the other muscles of the arm may also have changed, as EMG was not deliberately matched and sometimes increased significantly. Importantly, a change in contraction strength does not alter the proportion of descending drive mediated via the propriospinal pathway (Mazevet et al. 2003). This suggests that a change in overall descending drive does not, by itself, alter the proportion of drive mediated via the indirect pathway. Hence, for some muscles during the fatigued contractions, there may be an increase in the absolute magnitude of drive conveyed to motoneurones by both monosynaptic and disynaptic pathways, but our conclusions, based on changes in the proportion of EMG suppressed by inhibition of the propriospinal pathway, should not be affected by these alterations. Furthermore, fatigue-related changes in excitability of the motoneurones will influence the efficacy of direct descending drive, but also that of drive through the propriospinal pathway. Thus, the observed differences in the relative contributions of these inputs to muscle activity are likely to reflect changes within the propriospinal pathway as a direct consequence of fatigue. Stimuli to the superficial radial nerve at the wrist suppress the EMG recorded from ECR during tonic wrist extension (e.g. Burke et al. 1994; Mazevet et al. 2003). Here, Figure 4. Modulation of the on-going EMG in target and cocontracted muscles Modulation of on-going EMG of ECR, FCR, triceps brachii and biceps brachii by cutaneous inputs before (black bar) and after (grey bar) sustained fatiguing contractions. Subjects (n = 10) performed wrist extensions (A) or elbow extensions (B) and the EMG was conditioned by single stimuli (2PT) to the superficial radial nerve. C, subjects (n = 10) performed wrist extensions and the EMG was conditioned by a train of three stimuli (300 Hz, 2PT) to the superficial radial nerve. The bars show the difference between conditioned responses and control EMG (expressed as a percentage of the control EMG) during a 9 ms window at the expected time of suppression for each muscle (see Methods). The dashed box shows the major muscle involved in each task. Significantly different from suppression prior to fatigue, P < Figure 5. Suppression of the H reflex by cutaneous inputs Modulation of the H reflex of ECR by single cutaneous stimuli to the superficial radial nerve (2PT) before ( ) and after ( ) sustained fatiguing contractions of the wrist extensors. H reflexes were elicited during wrist extension. The difference between conditioned and control H reflexes (expressed as a percentage of control reflexes) is plotted against the interstimulus interval (ms). Vertical dashed line corresponds to the beginning of the window of analysis used for the on-going EMG in study 1. Vertical arrow indicates the expected simultaneous arrival of the cutaneous and the H reflex volleys at the same segmental level as the ECR motoneurone pool. Mean values (± S.E.M.) are plotted.

9 J Physiol Propriospinal drive with fatigue 219 we used this technique to assess whether muscle fatigue changes cutaneous-induced suppression of propriospinal drive to motoneurones of various upper limb muscles. Previous studies have used weak contractions against gravity (Burke et al. 1994; Mazevet et al. 2003), whereas we used stronger isometric contractions (30% MVC). Despite these differences, the magnitude of cutaneous-induced suppression of ECR EMG prior to fatiguing exercise was similar to that described elsewhere for single stimuli (Marchand-Pauvert et al. 1999) and trains (Mazevet et al. 2003). After sustained fatiguing contractions of the wrist extensors, the EMG suppression in ECR evoked by a single stimulus increased by 75%. Thus, the cutaneous input was able to interrupt a greater proportion of cortical descending drive to the target muscle. To confirm that this change was not unique to the ECR muscle when fatigued, we investigated on-going triceps EMG before and after sustained fatiguing contractions of the elbow extensors. Single stimuli to the superficial radial nerve at the wrist briefly suppressed triceps EMG and this again increased 75% with fatigue. Together, these results suggest that changes specifically related to fatigue of these muscles, and which are not unique to wrist extensors, increase the proportion of descending drive disrupted by cutaneous inputs to propriospinal neurones. It is believed that the suppression of on-going ECR EMG by stimuli delivered to the superficial radial nerve can be attributed to disfacilitation of motoneurones produced by disruption of descending drive relayed via cervical propriospinal neurones rather than to direct inhibition of motoneurones because the same single stimulus (Burke et al. 1994) or brief train of stimuli (Mazevet et al. 2003) suppresses the on-going EMG but has little effect on the H reflex recorded during tonic contractions. However, during fatigue the possibility that an independent, new set of inhibitory circuits provides cutaneous inhibition directly to motoneurones was investigated in a control study. Our results confirm that under control conditions (i.e. without fatigue), the cutaneous volley produces minimal effects on the H reflex. Furthermore, with fatigue, the single cutaneous volley did not significantly affect the size of the H reflex at the ISIs investigated. These results suggest that the increased EMG suppression with fatigue was due to factors at a premotoneuronal level. Cervical propriospinal neurones receive monosynaptic excitation from corticospinal pathways (Pauvert et al. 1998; Nicolas et al. 2001) and peripheral afferents (Malmgren & Pierrot-Deseilligny, 1988a,b). In addition, feedback inhibitory interneurones, which receive monosynaptic excitation from peripheral and corticospinal inputs, project to these propriospinal neurones (Malmgren & Pierrot-Deseilligny, 1988b; Nicolas et al. 2001; Fig. 6). Increased suppression of on-going EMG with fatigue could be explained by either an increase in the magnitude of the component of descending command relayed through propriospinal neurones (Fig. 6, a) or an increase in the excitability of interneurones mediating feedback inhibition (Pierrot-Deseilligny, 2002; Fig. 6, b). Although, in human subjects, there is no direct way to differentiate between these possibilities, some conclusions have been drawn by comparison of the effects of single cutaneous stimuli to the effects of brief trains of stimuli. For example, at the onset of a voluntary movement the weak inhibition evoked by a single volley is significantly increased by the use of a train (temporal facilitation at the inhibitory interneurones). Conversely, at the offset of the movement, the inhibition evoked by a single volley was strong but not significantly increased when using a train. The latter result is interpreted as reflecting an increased descending excitatory drive on inhibitory interneurones; if the spatial facilitation between this increased descending input and the first peripheral volley recruits most inhibitory interneurones, temporal summation due to the train can no longer manifest itself (Pierrot-Deseilligny, 1996). In an attempt to clarify the mechanism for changes with fatigue in EMG suppression by cutaneous inputs, we performed similar comparisons. Trains produced larger suppression of on-going ECR EMG than single stimuli, but the suppression elicited by both single pulses and trains increased with fatigue. Such a finding makes it difficult to identify unequivocally the mechanism involved in the change with fatigue. One possibility is that the interneurones mediating feedback inhibition to propriospinal neurones become more excitable (Fig. 6, b) and therefore more effective at suppressing drive through the propriospinal pathway. Such an effect could be mediated by an increase in descending (Fig. 6, c) or peripheral excitation (Fig. 6, d) to these interneurones. As fatigue is associated with changes in the firing of various muscle afferents, a possible candidate is a change in the balance of excitation provided by muscle afferents (Fig. 6, d). A second possibility is that with fatigue the proportion of drive relayed via the propriospinal pathway increases (Fig. 6, a), which would lead to greater suppression elicited by a single volley or a brief train even if the level of excitation of the inhibitory interneurones remained unchanged. A final possibility is that presynaptic inhibition of Ia afferents projecting to propriospinal neurones (Burke et al. 1992b) decreased during fatigue, increasing the excitability of propriospinal neurones (Fig. 6, e). Although this possibility cannot be excluded, it is unlikely, given that any decreased presynaptic inhibition would be offset by waning Ia afferent firing during fatigue (Bongiovanni & Hagbarth, 1990; Macefield et al. 1991). Changes in feedback inhibition or changes in propriospinal drive are both plausible explanations for our results. Future studies of the level of excitation necessary to excite maximally the interneurones providing feedback inhibition to propriospinal neurones may provide a way to differentiate the mechanisms. If a train of

10 220 P. G. Martin and others J Physiol three pulses provides maximal or near maximal excitation to most inhibitory interneurones mediating inhibition to propriospinal neurones, then a further increase in EMG suppression with fatigue could only be due to an increase in the component of drive relayed via this pathway. Alternatively, an investigation of the disynaptic excitation of motoneurones evoked by isolated peripheral stimulation and that produced by combining peripheral stimulation and descending inputs (Nicolas et al. 2001) may provide further evidence. The suppression of the target muscle by the cutaneous inputs increased during fatigue, but the pattern reversed in muscles which were not deliberately fatigued but which cocontracted during the task. Hence, for muscles in which cutaneous volleys produced inhibition (usually triceps or ECR), the inhibition decreased, whereas for muscles in which they produced facilitation (usually FCR) this facilitation increased. This pattern was consistent for fatigue of wrist or elbow extensor muscles and for single stimuli or trains of stimuli. These changes are consistent with either a decrease in descending drive to propriospinal neurones or a decrease in the excitability of interneurones mediating the cutaneous inhibition. While the absolute level of EMG in the cocontracted muscles was not always identical before and during fatigue, two arguments suggest that this was probably not the primary f a b c e d Figure 6. Connections of the propriospinal system and possible changes with fatigue of the wrist extensors Excitatory synapses are represented by Y-shaped bars and inhibitory synapses by small filled circles. Propriospinal neurones (PN) project to motoneurones (MN) and receive monosynaptic excitation from corticospinal (thick continuous line) and peripheral afferents (thin continuous line). Feedback inhibitory interneurones (large filled circles) projecting to propriospinal neurones receive monosynaptic excitation from peripheral and corticospinal inputs, whereas feedforward inhibitory interneurones (large striped circles) projecting to propriospinal neurones receive monosynaptic excitation from the same descending tracts as propriospinal neurones. With fatigue of the wrist extensors, there is an increase in the cutaneous-induced suppression of propriospinal drive to motoneurones of wrist extensors but a decrease for elbow extensors. As presented in Discussion (in order a f) several factors may contribute to changes in the cutaneous-induced suppression of propriospinal drive with fatigue. a, the magnitude of descending corticospinal drive to propriospinal neurones; b, the excitability of inhibitory interneurones projecting to propriospinal neurones; c, the magnitude of descending corticospinal excitation of the feedback inhibitory interneurones; d, the balance of inputs from muscle and cutaneous afferents projecting to feedback inhibitory interneurones (and to propriospinal neurones, dashed line); e, the magnitude of presynaptic inhibition of Ia afferent volleys to propriospinal neurones; f, the magnitude of drive to feedforward inhibitory interneurones.

11 J Physiol Propriospinal drive with fatigue 221 factor determining changes in these muscles. First, the degree of cutaneous-induced suppression of ECR EMG during wrist extension is unaffected by the background level of EMG, and this indicates that the proportion of voluntary drive provided by the propriospinal system does not change (Mazevet et al. 2003). Second, the pattern of changes was seen for muscles that showed no significant increase in EMG (e.g. triceps during tonic wrist extension). Therefore, we propose that the pattern of changes reflects a co-ordinated response to fatigue that is mediated via the propriospinal system. During sustained voluntary contractions, muscle relaxation slows and lower motor unit firing rates are required to maintain force fusion (e.g. Edwards et al. 1972; Bigland-Ritchie et al. 1983, e.g. Bigland-Ritchie & Woods, 1984). Furthermore, at least in maximal efforts, firing rates become unduly low in relation to the speed of muscle contraction (Fuglevand & Keen, 2003) and central fatigue increases (for review see Gandevia, 2001). During fatigue, greater unsteadiness and tremor develops (e.g. Buchthal & Madsen, 1950; Lippold, 1970) and the stabilizing effect of the stretch reflex declines (Matthews, 1997). In addition, there is progressive recruitment of other muscles during the task. Despite these changes, some indices of the accuracy of performance show little or no change (e.g. Skinner et al. 1986; Sharpe & Miles, 1993; Carpenter et al. 1998; Jaric et al. 1999). Synergies involving multiple muscles may change during peripheral fatigue to maintain the quality of performance (e.g. Bonnard et al. 1994; Forestier & Nougier, 1998; Huffenus et al. 2006). For example, the accuracy of throwing can be maintained despite fatigue of either distal or proximal upper limb muscles (Huffenus et al. 2006). The propriospinal system with its wide range of inputs and diffuse distribution (Gracies et al. 1991) presents a conceptually attractive means by which the CNS can help distribute the appropriate level of corticospinal drive to muscles involved in a task. Our results suggest that during fatiguing exercise, corticospinal drive through this pathway is focused to certain muscles either by increasing the level of descending excitation to propriospinal neurones (Fig. 6, a) or alternatively, by releasing the inhibition provided to them by feedback interneurones (Fig. 6, b)or feedforward inhibitory interneurones (Alstermark et al. 1984; Lundberg, 1999; Nicolas et al. 2001; Fig. 6, f ). If our task activated propriospinal neurones which project to motoneurones of both fatigued and non-fatigued muscles, similar effects should have been seen in these muscles. This was not observed. Thus, different sets of propriospinal neurones (Burke et al. 1992a) may distribute the drive to fatigued and non-fatigued muscles. This system would allow peripheral inputs to modulate the command to different muscles simultaneously during fatigue. In addition, if a greater proportion of drive relays through propriospinal neurones or the excitability of feedback interneurones increases, the gain of this pathway increases, such that any sudden change in the size of the peripheral input, will result in rapid adjustments, particularly in the fatigued muscle. The altered gain may compensate for changes in other reflex pathways. In conclusion, we present novel findings on changes in cutaneous-induced suppression of disynaptic propriospinal drive to motoneurones during human muscle fatigue. Our results reveal co-ordinated changes to both fatigued and non-fatigued muscles of the upper limb. 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