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[Revised submission of 2015-270569]

Unexpected factors affecting the excitability of human motoneurones in voluntary and stimulated contractions

Serajul I. Khan, Janet L. Taylor and Simon C. Gandevia Neuroscience Research Australia and University of New South Wales, Randwick, NSW, Australia

Running head: Recurrent discharge of human motoneurones Key words: muscle fatigue, motoneurone, F-waves Number of words in the manuscript, excluding references and figure legends: 4843 Table of contents category: Neuroscience: behavioural/systems/cognitive

Correspondence: Prof. Simon C. Gandevia Neuroscience Research Australia Barker Street

This is an Accepted Article that has been peer-reviewed and approved for publication in the The Journal of Physiology, but has yet to undergo copy-editing and proof correction. Please cite this article as an 'Accepted Article'; doi: 10.1113/JP272164.

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Randwick, Australia, 2031 E-mail: [email protected] Tel.: +61 2 9399 1617 Fax: +61 2 9399 1027

Key points summary  The output of human motoneurone pools decreases with fatiguing exercise, but the mechanisms are uncertain. We explored depression of recurrent motoneurone discharges (F-waves) after sustained maximal voluntary contractions.  Maximal voluntary contraction (MVC) depressed the size and frequency of F-waves in a hand muscle but a submaximal contraction (at 50% MVC) did not.  Surprisingly, activation of the motoneurones antidromically by stimulation of the ulnar nerve (at 20 or 40 Hz) did not depress F-wave area or persistence.  Furthermore, a sustained (3-min) MVC of a hand muscle depressed F-waves in its antagonist but not in a remote hand muscle.  Our findings suggest that depression of F-waves after voluntary contractions is not simply due to repetitive activation of the motoneurones but requires descending voluntary drive. Furthermore, this effect may depress nearby, but not distant, spinal motoneurone pools. [Word count 134]

Abstract There are major spinal changes induced by repetitive activity and fatigue which could contribute to ‘central’ fatigue but the mechanisms are poorly understood in humans. Here we confirmed that the recurrent motoneuronal discharge (F wave) is reduced during relaxation immediately after a sustained maximal voluntary contraction (MVC) of an intrinsic hand muscle (abductor digiti minimi, ADM) and explored the relation between motoneurone firing

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and the depression of F-waves in three ways. First, the depression (both in F-wave area and persistence) was present after a 10-s MVC (initial decrease 36.4±19.1%; mean±SD) but not after a submaximal voluntary contraction at 50% maximum. Second, to evoke motoneurone discharge without volitional effort, 10-s tetanic contractions were produced by supramaximal ulnar nerve stimulation at the elbow at physiological frequencies of 25 Hz and 40 Hz. Surprisingly, neither produced depression of F-waves in ADM to test supramaximal stimulation of the ulnar nerve at the wrist. Finally, a sustained MVC (3 min) of the antagonist to ADM (4th palmar interosseous) depressed F-waves in the anatomically close ADM (20% ±18.2) but not in the more remote first dorsal interosseous on the radial side of the hand (FDI). We argue that depression of F-waves after voluntary contractions is not due to repetitive activation of the motoneurones but requires descending voluntary drive. Furthermore, this effect may depress nearby, but not distant, spinal motoneurone pools and it reveals potentially novel mechanisms controlling the output of human motoneurones.

Abbreviations ADM, abductor digit minimi; CMEP, cervicomedullary motor evoked potential; EMG, electromyographic activity; FDI, first dorsal interosseous; 4PI, fourth palmar interosseous, Mmax, maximal compound muscle action potential; MVC, maximal voluntary contraction; 5-HT, 5-hydroxytryptamine (serotonin)

Introduction The output of human motoneurone pools decreases during and after sustained voluntary isometric fatiguing contractions and the major mechanisms for this reduction include both supraspinal and spinal factors (Gandevia, 2001). Some fatigue-related changes at the spinal motoneurones have been demonstrated with subcortical stimulation of the corticospinal tract at the level of cervicomedullary junction (Gandevia, 2001; Butler et al. 2003). Cervicomedullary motor evoked potentials (CMEP) are reduced in size during the latter half


of a sustained 2 min MVC (Butler et al. 2003; Martin et al. 2006). More dramatic changes in CMEPs occur when a transcranial magnetic stimulus to the motor cortex is used to interrupt descending voluntary drive and cause transient disfacilitation of the motoneurone pool. During this transient disfacilitation, CMEPs are easily evoked prior to fatigue but are abolished after ~ 20 s of maximal voluntary effort (McNeil et al. 2009; McNeil et al. 2011a; McNeil et al. 2011b). These changes in the CMEP during an MVC suggest altered motoneurone excitability which may reflect changes in the intrinsic properties of the motoneurones. However, changes in the size of the CMEP do not necessarily correspond to changes in the excitability of motoneurones because the CMEP can also be influenced by premotoneuronal changes presumably at the corticomotoneuronal synapse (Gandevia et al. 1999; Petersen et al. 2003). In an attempt to assess changes produced in motoneurones more directly, we have previously measured the recurrent discharge of motoneurones (Renshaw, 1941; Eccles, 1955; McLeod & Wray, 1966; Trontelj, 1973), following repetitive activity produced by maximal voluntary contractions (MVC). These discharges are known as F-waves when measured in the EMG in humans (McLeod & Wray; see also Espiritu et al. 2003). Animal studies indicate that when motoneurones are stimulated antidromically the recurrent discharge is generated in the nonmyelinated proximal segment of the axon initial segment (Coombs et al. 1957) or first node of Ranvier (Gogan et al. 1984). Our study showed that F-waves were strongly depressed after MVCs in both an upper and lower limb muscle and, furthermore, the depression was activity-dependent being greater after longer efforts (Khan et al. 2012). Reductions in Fwaves after MVCs were also reported by Rossi and colleagues (Rossi et al. 2012). We proposed that activity-dependent hyperpolarisation at the soma or the axon initial segment depresses the recurrent discharge of human motoneurones. This proposal fits with many studies showing that repetitive activity hyperpolarises axons (e.g. Gasser, 1935; Bostock & Grafe, 1985; Kiernan et al. 2004; Milder et al. 2014) likely via altered activity of the Na+K+ pump. The aim of the current studies was to test the link between repetitive activity of motoneurones and depression of F-waves in three ways. First, we determined whether the strength of a


voluntary contraction of an intrinsic hand muscle affects the depression of F-waves. For these muscles a voluntary contraction beyond about 50% maximum recruits most motoneurones so that a stronger contraction requires an increase in the discharge frequency of motoneurones (e.g. Kukulka & Clamann, 1981; De Luca et al. 1982). This would be expected to produce greater axonal hyperpolarisation and hence F-wave depression. Second, we predicted that antidromic activation of motoneurones by nerve stimulation would produce similar depression in F-waves to that observed after voluntary contractions, and that the depression would increase with the frequency of the tetanic stimulus. Finally, because we found that antidromic activation of motoneurones (unlike voluntary activity) did not depress F-waves, we tested whether strong voluntary contraction of an anatomically ‘nearby’ muscle would depress F-waves in the target muscle. A preliminary version of the results has been presented (Gandevia et al. 2014). Methods A total of 53 healthy subjects (23 females) participated in the study. Fifteen subjects (31±7 years; mean ± SD; 9 females) participated in the first study which examined the effect of contraction strength (50% and 100% MVC) on F-wave depression in abductor digiti minimi (ADM). Seven of these subjects plus a further five (33±6 years; 7 females) participated in the second study which examined whether the post-exercise depression of F-waves required activation of the motoneurones through voluntary drive. Twenty seven subjects (10 from study 1 plus a further 17 [29±7 years; 10 females]) initially participated in the third study which examined F-waves in ADM after a 3-min maximal voluntary contraction (MVC) of the fourth palmar interosseous (4PI) which adducts the little finger and is the functional antagonist to ADM. As indicated below, based on performance on the training and experiment days only 19 subjects were included in analysis. Written informed consent to the experimental procedures was obtained from each subject. The study was approved by the local human research ethics committee and was conducted according to the Declaration of Helsinki (2008). Experimental setup


In studies 1 and 3, subjects sat in an adjustable chair with the right forearm and fingers rested on a table and the elbow flexed in a comfortable position. The forearm was pronated and secured by straps. The thumb rested in a midflexed position and the second to fourth fingers were secured by a strap. The little finger was placed firmly in a custom-designed finger splint with an axis of rotation about the metacarpophalangeal joint in a horizontal plane. The splint was connected to a force transducer to measure isometric finger abduction and adduction force. In study 2, subjects were seated with their right forearm rested in a neutral position on a padded L shaped support which was affixed to a table and supported the right forearm and hand from the elbow to the fingertips. The forearm was secured tightly to the support with a strap at the elbow and the wrist to prevent any movement from stimulation. The thumb rested in a neutral position and a vacuum pillow secured the other fingers. A padded clamp on the wrist prevented ulnar deviation during tetanic stimulation. Surface EMG was recorded from ADM, or both ADM and first dorsal interosseous (FDI) using Ag/AgCl disk electrodes (6 mm in diameter) filled with conducting gel. Recordings from FDI were required in Study 3 as we needed to record from a ‘remote’ ulnar-innervated muscle on the thenar side of the hand. The cathode was placed over the motor-point of each muscle and the anode 2 cm distal to the motor-point. A large ground electrode was placed on the anterior aspect of the wrist. The EMG signals were recorded in two ways. First, EMG signals were amplified (×1000), band-pass filtered (200–1000 Hz; 2-pole Bessel filter; CED 1902 amplifiers) and sampled at 5 kHz (Fig. 1, lower panel; see also Fig. 1 in Khan et al. 2012). The high-pass filter was set at 200 Hz to remove the low frequency tail of the M wave to obtain a more stable baseline, so that it was easier to identify the onset of individual Fwaves and to measure their amplitude and area (filtered F-waves; Fig. 1B). Measurements made in this way are reported in the text and figures. Second, so that the M wave could also be assessed in the conventional way, the EMG signals were amplified (×300), band-pass filtered (10–1000 Hz; 2-pole Butterworth filter; CED 1902 amplifiers) and sampled at 2 kHz (Fig. 1, top panel). The experimental interventions in the three studies (described below) did not significantly change the duration of the maximal M wave (data not shown). For study 3,


we quantified co-contraction of ADM and FDI during the sustained MVC of 4PI using the EMG amplitudes derived from this less filtered signal. Force data were sampled at 1000 Hz. All signals were stored on a computer (CED 1401 Plus, Cambridge Electronic Design Ltd. Cambridge, UK) and Spike 2 software (version 6.13; Cambridge Electronic Design). Stimulation In all studies, the ulnar nerve at the wrist was stimulated to evoke maximal compound muscle action potentials (Mmax) and F-waves at rest in ADM. The best location for the cathode was identified by stimulation with a hand-held electrode on the ulnar side of the forearm. Single electrical stimuli (100 µs duration) were delivered by a constant current stimulator (Digitimer DS7AH, Welwyn Garden City, Herfordshire, UK) through a custom-designed bar electrode (interelectrode distance 4 cm) with the cathode over the ulnar nerve and just proximal to wrist. With the subject at rest, the stimulus intensity was gradually increased until the compound muscle action potential failed to increase in peak-to-peak amplitude despite an increase in current. To maintain supramaximal stimulation despite any effect of activityinduced hyperpolarisation of the motor axons during a sustained contraction, the intensity of stimulation to evoke F-waves was set at 150% of the intensity that evoked Mmax (20 45 mA). In study 2, the ulnar nerve at the elbow was also stimulated using a constant-current stimulator (Digitimer DS7AH) to activate ADM. Electrical stimuli (500 µs duration) were delivered through Ag-AgCl electrodes with the cathode and anode proximal and distal to the medial epicondyle of the humerus, respectively. The best position was identified using a hand-held electrode to minimise stimulus current require to evoke the maximal compound muscle action potential (Mmax). The size of Mmax was determined and the intensity of stimulation was set at 150% of the intensity required to produce a maximal M-wave (1530 mA). Stimulation at the elbow was used (see below) because current levels for maximal stimulation were much lower at this site than at the wrist and hence prolonged tetanic stimulation was more comfortable.


Protocol Study 1: F-waves in ADM after 10-s submaximal and maximal contractions This assessed the effect of the strength of a voluntary contraction of ADM on F-wave depression. Initially, subjects performed 3 brief MVCs of 2-3 s each separated by at least 1 min rest. The peak force of the 3 MVCs was measured and a target level of the rectified integrated EMG required to reach 50% MVC was set on a visual feedback display. Two control sets of F-waves, separated by 1 min, were collected prior to the voluntary contraction. For each set, subjects received 30 supramaximal stimuli (150% Mmax) to the ulnar nerve at the wrist delivered at 0.5 Hz with the muscle relaxed. After another min of rest, subjects performed a 10-s submaximal voluntary contraction to the target level (50% MVC). They relaxed immediately after the contraction, and starting after ~ 2 s of relaxation, 6 postcontraction sets of F-waves, each separated by 30-s rest, were collected. After a rest of 30 min, the protocol was repeated with a 100% MVC. Study 2: F-waves in ADM after a 10-s supramaximal tetanic stimulation of ulnar nerve This assessed whether the depression of F-waves required activation of the motoneurones through voluntary drive. F-waves were examined in relaxation before and after supramaximal tetanic stimulation of ulnar nerve at the elbow for 10 s at 25 Hz and 40 Hz. In brief, F-waves were collected with sets of 30 supramaximal ulnar nerve stimuli (150% Mmax) at the wrist delivered at 0.5 Hz (i.e. as in Study 1). Initially, two control sets were collected, with a 1 min rest between sets. After 25-Hz tetanus subjects relaxed immediately, and six more sets of F-waves were collected. The protocol was repeated with a 40-Hz tetanus after a rest of 30 min. Study 3: F-waves in ADM and FDI after contraction of the fourth palmar interosseous Because the tetanic stimulation did not depress F-wave area or persistence despite repetitive antidromic activation of all motoneurones supplying ADM (see Results 2), we further explored the effects of voluntary activity on F-waves. We measured F-waves in ADM and in


a remote ulnar-innervated hand muscle, the first dorsal interosseous (FDI) after a sustained 3-min MVC of the fourth palmar interosseous (ADM’s antagonist). There was a training and an experimental session, separated by at least 2 days. In the training session, 27 subjects practiced maximal sustained adductions of the little finger so that co-contraction of the functional antagonist (ADM) was low. This was judged from the surface EMG (root mean square with 100 ms time constant) with a level ≤ 10 % that in a maximal contraction of ADM considered acceptable. Twenty-three subjects could perform the task and continued to the experimental session, in which F-waves were collected from ADM and FDI before and after a sustained 3-min MVC of the fourth palmar interosseous. As in study 1, sets of 30 supramaximal ulnar nerve stimuli (150% Mmax) at the wrist were delivered at 0.5 Hz. Two control sets were collected before and 10 sets after the sustained MVC. At the end of the protocol, subjects performed brief isometric MVCs of ADM and FDI. Data from 19 subjects were used for ADM: 2 subjects whose ADM EMG exceeded 10% that in a maximal contraction of ADM were excluded and 2 subjects withdrew as the supramaximal stimuli were too uncomfortable. Data from 15 subjects were used for FDI as 4 subjects had low persistence of F-waves in this muscle (less than 50%). Data analysis and statistics During off-line analysis, Signal software (version 3.05; Cambridge Electronic Design) was used to determine all measures (area, amplitude and persistence). To measure the area and the amplitude of F-waves, responses to 30 supramaximal stimuli were overdrawn on the screen at high gain and the cursors were set at the beginning and the end of the responses that showed a clear deflection from the baseline. Maximal compound muscle action potentials (Mmax) were superimposed, and cursors were set at the start and the end of the responses to determine the area and peak-to-peak amplitude. For both F- waves and Mmax, measurements were made from individual potentials. To identify any EMG activity at rest, root mean square EMG was calculated over 50 ms immediately before each stimulus. The area and peak-to-peak amplitude of F-waves measured from the heavily filtered EMG showed similar results, and area is reported throughout the text. Because of activitydependent changes in muscle fibre action potentials during voluntary efforts, the area of each


F-wave was normalised to the area of the corresponding Mmax (also heavily filtered) and then expressed as a percentage of mean control values (mean of control set 1 and 2) obtained prior to the MVC (Taylor et al. 1999; Khan et al. 2012). For each subject, the mean area of the 30 F-waves in each set were used for statistical analysis. To illustrate in more detail the time course of changes, figures show the F-wave area averaged for each 10 consecutive stimuli (Espiritu et al. 2003). In addition, as F-waves are not seen after every supramaximal stimulus, we measured the frequency with which F-waves occurred in each set of 30 consecutive stimuli for ADM. This value (usually termed ‘persistence’) was expressed as a percentage. Assessment based on 30 trials can be considered sufficient (review Panayiotopoulos & Chroni, 1996). We identified an F-wave as present if a response with an appropriate latency (minimum of 22 ms for ADM) had an amplitude ≥ 20 µV. Standard Fwave characteristics at baseline from subjects used in studies 1, 2 and 3 are given in Table 1. Statistical comparisons were made using repeated measures ANOVA (SYSTAT version 13; San Jose, CA). For studies 1 and 2, F-wave area was analysed with two-way repeated measures ANOVA with time as one factor and contraction strength as the other. Because data for F-wave persistence were not normally distributed, we rank-transformed the data and performed a two-way parametric repeated measures ANOVA. Whenever the ANOVA showed a significant main effect or a significant interaction, post-hoc Student -NewmanKeuls tests were used to identify differences between the control and subsequent time points or between contractions. For study 3, F-wave area and persistence were analysed using oneway repeated measures ANOVA to assess the main effect for time. These were followed by post-hoc Student-Newman-Keuls test to identify differences between the control and subsequent time points. Data are represented as mean ± SD in the text and as mean ±SEM in the figures. Statistical significance was set at P