Respiratory Physiology & Neurobiology 216 (2015) 15–22
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TMS-evoked silent periods in scalene and parasternal intercostal muscles during voluntary breathing Billy L. Luu a,b , Julian P. Saboisky a,b , Janet L. Taylor a,b , Simon C. Gandevia a,b , Jane E. Butler a,b,∗ a b
Neuroscience Research Australia, Barker St, Randwick 2031, NSW, Australia The University of New South Wales, Sydney 2052, NSW, Australia
a r t i c l e
i n f o
Article history: Received 22 January 2015 Received in revised form 13 May 2015 Accepted 18 May 2015 Available online 27 May 2015 Keywords: Magnetic stimulation Cortical Respiratory Hypercapnia Inhibition Drive
a b s t r a c t Transcranial magnetic stimulation (TMS) during voluntary muscle contraction causes a period of reduced electromyographic (EMG) activity (EMG). This is attributed to cortical inhibition and is known as the ‘silent period’. Silent periods were compared in inspiratory muscles following TMS during voluntary inspiratory efforts during normocapnia, hypercapnia, and hypocapnia. TMS was delivered during isometric and dynamic contractions of scalenes and parasternal intercostals at 25% maximum inspiratory pressure. Changing end-tidal CO2 did not affect the duration of the silent period nor suppression of EMG activity during the silent period. In scalenes, silent periods were shorter for dynamic compared to isometric contractions (p < 0.05); but contraction type did not alter the degree of suppression of EMG during the silent period. In parasternal intercostal, no significant differences in silent period parameters occurred for the different contraction types. The lack of effect of end-tidal CO2 suggests that descending drive from the medullary respiratory centres does not independently activate the inspiratory muscles during voluntary inspiratory efforts. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Breathing is usually controlled involuntarily. Rhythmic activity originating from a network of pontomedullary neurons activates the respiratory muscles via bulbospinal pathways (for reviews, see Feldman and Del Negro, 2006; Richter and Smith, 2014). The activity of these respiratory neurons is modulated by arterial CO2 to control breathing and maintain normocapnia. However, like the skeletal muscles of the limbs, the respiratory muscles also receive cortical drive during behavioural tasks that require controlled breathing such as swimming, fine motor tasks, and pursed-lip breathing. It is generally observed that deliberate attention to breathing normally shifts the behaviour from the automatic regulation of respiration to voluntary control by the motor cortex (Macefield and Gandevia, 1991; Petersen et al., 2011; Raux et al., 2007). The voluntary command from the motor cortex may act via corticospinal pathways that bypass the medullary respiratory centres (Gandevia and Plassman, 1988; Gandevia and Rothwell, 1987; Murphy et al., 1990; Straus et al., 2004) and/or via inhibitory
∗ Corresponding author at: Neuroscience Research Australia, Barker St, Randwick 2031, NSW, Australia. Tel.: +61 2 93991608. E-mail address: [email protected] (J.E. Butler). http://dx.doi.org/10.1016/j.resp.2015.05.010 1569-9048/© 2015 Elsevier B.V. All rights reserved.
corticobulbar projections that modulate the activity of respiratory neurons (Bassal and Bianchi, 1982; Orem, 1989; Orem and Netick, 1986). It is not known how the cortical drive to respiratory motoneurones is integrated with the ongoing automatic control of breathing during a voluntary breath. It is possible that medullary output to the motoneurones summates with the descending drive from the motor cortex, or that medullary output is suppressed and replaced by the descending cortical drive. The interaction between the cortical and medullary facilitatory drives to the respiratory muscles has been investigated previously. Corfield et al. (1998) and Lefaucheur and Lofaso (2002) explored a potential cortico-medullary interaction by examining separately the two components of the muscle response to transcranial magnetic stimulation (TMS): the motor evoked potential and the subsequent silent period. Corfield et al. (1998) compared diaphragmatic motor evoked potentials, an indicator of overall corticospinal excitability, during normocapnia and hypocapnia, where reduced CO2 has been shown to attenuate the diaphragmatic muscle activity evoked by a transient hypoxic stimulus (Roberts et al., 1995). Corfield et al. found that hypocapnia did not attenuate the motor evoked potential and therefore suggested that voluntary activation of the diaphragm bypasses the medulla. However, a limitation of their study was that in order to expect a difference in the motor evoked potential they had assumed that TMS activates an
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excitatory projection to bulbar respiratory premotoneurones, a circuitry which has yet to be demonstrated, and that this pathway would be attenuated by hypocapnia in the same way as the response to a transient hypoxic stimulus. Lefaucheur and Lofaso (2002), on the other hand, focused on the silent period, which is the electrical silence in ongoing electromyographic (EMG) activity following TMS. The suppression of EMG activity during the silent period is due to an early spinal component (Fuhr et al., 1991; Inghilleri et al., 1993; Uncini et al., 1993; Ziemann et al., 1993) followed by a later supraspinal inhibition due to the activation of intracortical inhibitory neurons with consequent inhibition of corticospinal output (Kujirai et al., 1993). Temporary inhibition of corticospinal output with TMS during inspiration would highlight the reliance placed on this pathway during voluntary breathing and also reveal the contribution of non-cortical inputs to respiratory motoneurones through the remaining ongoing EMG activity. Lefaucheur and Lofaso showed that silent period durations during matched involuntary and voluntary activation of the diaphragm were similar. However, a shortcoming of this study is that the diaphragmatic silent periods were ∼60 ms in duration from the onset of the stimulus. This is significantly shorter than the 100 ms duration threshold that is considered to reflect intracortical inhibition of corticospinal output (Fuhr et al., 1991; Inghilleri et al., 1993). The present study reconsiders the use of the silent period in understanding the interaction between the motor cortex and medullary respiratory neurons during voluntary breathing. Here, we determine whether distinct cortical silent periods are present in the obligatory inspiratory muscles of scalenes (De Troyer and Estenne, 1984; Gandevia et al., 1996; Raper et al., 1966) and parasternal intercostals (De Troyer and Sampson, 1982; Gandevia et al., 2006). Surprisingly, clear silent periods could only be achieved during substantial voluntary muscle activity. Hence, stimuli were delivered with inspiratory EMG activity of 25% maximum during voluntary hyperventilation or during single, large voluntary inspiratory efforts that were interspersed during eupnoea and CO2 -driven breathing. Subjects were studied at different levels of end-tidal CO2 (PET CO2 ) to alter the chemical feedback acting on the medullary respiratory neurons. We posited that if the cortical and medullary drives during voluntary inspiratory efforts were independent and summated at a motoneurone level, then the increase in medullary drive during hypercapnia compared to hypocapnia would reduce the level of corticospinal drive needed to produce the designated EMG activity. Thus, inhibition of this reduced level of corticospinal drive by TMS would be less effective at suppressing EMG activity since the CO2 -driven increase in medullary drive would still activate the motoneurones when corticospinal output was suppressed. From this rationale, if the silent periods in the scalenes and parasternal intercostal EMGs were shorter during hypercapnia than during hypocapnia and/or there were less reduction in EMG activity following TMS, we would conclude that the medullary drive independently activates inspiratory muscles during voluntary inspiratory efforts.
2. Methods Ten healthy adults (seven males) aged between 24 and 47 years (mean 29.2; s.d. 7.1) participated in the experiments. All procedures were approved by the University of New South Wales Human Research Ethics Committee and conducted in accordance with the Declaration of Helsinki (2008). Subjects provided written informed consent before participating.
2.1. Experimental setup Respiratory measurements were performed with the subject seated comfortably on a physiotherapy chair inclined at 54◦ from horizontal with the head resting against a pillow (Fig. 1A). Wearing a nose clip, the subject breathed through a mouth piece connected in series with a pneumotachometer (Series 3813, Hans Rudolph Inc, Kansas City, USA) and a 3-way directional stopcock valve (Series 2100, Hans Rudolph Inc). Inspiratory flow measured from the pneumotachometer and a differential pressure transducer (DP45-16, Validyne Engineering Corp., Northridge, USA) was integrated on-line to provide a signal of change in lung volume. PET CO2 , measured with a capnometer (Normocap, Datex Instrumentarium Corp., Helsinki, Finland), was used to estimate arterial CO2 , and along with inspiratory mouth pressure (DP15-34, Validyne Engineering Corp.), was measured close to the mouth piece. Surface electromyograms of the right scalenes and parasternal intercostal (PSIC) muscles were made using bipolar Ag/AgCl electrodes (Cleartrace, ConMed Corp, Utica, USA). For scalenes, the active electrode was placed in the posterior triangle of the neck at the level of the cricoid cartilage with the reference electrode on the clavicle in line with the long axis of the scalene muscle group (Murray et al., 2008). The active electrode for PSIC muscle activity was placed on the second intercostal space with the reference electrode adjacent on the sternum. Electrocardiograms (ECG) were monitored using a bipolar configuration with surface electrodes on the left clavicle and the right ninth intercostal space near the midaxillary line. An electrode placed over the right shoulder served as a common ground for ECG and EMG recordings. TMS was delivered to the motor cortex with a round stimulating coil (mean diameter 90 mm) centred over the vertex. The coil was oriented so that the current flowed through the coil in a counter-clockwise direction. To minimise changes in the coil’s position between pulses, an experimenter held the coil horizontally and a marker pen was used to trace the inner diameter of the coil and several references points onto the scalp. A single, high-intensity pulse was generated by simultaneously discharging two Magstim 200 stimulators connected through a Bistim module (Magstim Co. Ltd., Whitland, UK) programmed with an inter-stimulus interval of 0 ms. Data were acquired (Power 1401, Cambridge Electronic Design, Cambridge, UK) and recorded to computer with Spike2 software (Cambridge Electronic Design). Respiratory data were sampled at 1000 Hz. ECG and scalenes EMG signals were amplified ×1000 and PSIC EMGs were amplified ×3000, band-pass filtered at 16–1000 Hz, and sampled at 2000 Hz. 2.2. Protocol To evoke long and clear silent periods in muscle activity with TMS, subjects were required to make voluntary inspiratory efforts at 25% of their maximum inspiratory pressure (MIP). This level of activation was greater than normal tidal breathing (∼5% MIP), but pilot data indicated that silent periods were difficult to obtain consistently even at 10% MIP (Fig. 1C). Each subject’s MIP was determined beforehand as the best out of 3 maximal inspiratory efforts performed against a closed valve at function residual capacity (FRC). The output intensity of the Bistim was adjusted for each subject (between 70% and 100%) to achieve silent periods longer than 100 ms (from the time of the stimulus) in scalenes EMG during quasi-isometric inspiratory efforts at 25% MIP. Of the ten participants, eight satisfied this criterion and completed the remainder of the study. Once set, TMS intensity was then kept constant throughout the respiratory experiments. TMS was delivered during voluntary inspiratory efforts in three experimental conditions: normocapnia, hypercapnia accompanied
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Fig. 1. Experimental setups. (A) Subjects were seated comfortably. The head and arms were restrained to minimise movement artifacts in the electromyograms of the scalene and parasternal intercostal muscles. The pneumotachometer recorded respiratory air flow as subjects breathed though the mouth piece. A stopcock valve directed air flow between conditions to facilitate re-breathing of dead-space air through an open-ended tube. Inset: Transcranial magnetic stimuli (arrows) were delivered during isometric inspiratory efforts performed at functional residual capacity following expiration (e) and during dynamic inspiratory efforts as subjects increased inspiration (i) above tidal volume (Vt). (B) The hand and forearm were restrained for contractions of the first dorsal interossous (FDI). The index finger rested on a platform that could be made rigid for isometric contractions of the FDI or mobile for dynamic abductions of the finger against the resistance of an elastic string. (C) Raw data from a single subject during isometric contractions of the scalenes and FDI. Traces from the first 5 magnetic pulses (arrow) are superimposed for each condition. Scalenes contractions were performed at 10% and 25% of maximum inspiratory pressure (MIP) with a stimulus intensity of 90% maximum stimulator output. FDI contractions were performed at 10% peak muscle activity during a maximal voluntary contraction (MVC) with a stimulus intensity of 60% maximum. The black horizontal bar represents the duration of the silent period evoked by transcranial magnetic stimulation and the grey horizontal bar represents the period of attenuated muscle activity.
by reflex hyperventilation, and hypocapnia induced by voluntary hyperpnoea. Hypercapnia was achieved by having subjects rebreathe via an added deadspace of ∼1.4 l or ∼2.2 l (diameter 35 mm, Fig. 1A) until PET CO2 and ventilation stabilised. This re-breathing approximately doubled ventilation as PET CO2 increased from a mean of 38.8 mmHg (s.d. 3.0) during normal breathing to a mean of 51.7 mmHg (s.d. 5.3). Hypocapnia was achieved by having subjects voluntarily increase ventilation; using real-time visual feedback of lung volume to track an analogue signal that would produce the same tidal volume, respiratory rate, and inspiratory time as recorded during hypercapnia-driven reflex hyperventilation. This voluntary hyperpnoea was performed with air flow diverted away from the tube attachment by the stopcock (Fig. 1A). Once stabilised, matched voluntary hyperpnoea reduced PET CO2 to a mean of 24.3 mmHg (s.d. 3.8). These three conditions were conducted in random order with at least a 5-min rest period to allow for the respiratory variables to return to baseline. For those subjects who were randomly assigned the hypocapnia condition first, a brief stable period of re-breathing dead-space air was performed initially without TMS to determine the parameters for the tracking signal.
A period of rest allowed PET CO2 to normalise before commencing the voluntary hyperpnoea protocol. A custom Spike2 (Cambridge Electronic Design) script triggered the TMS pulse based on two criteria, (i) an EMG threshold of 25% of the maximum root-mean-squared (RMS) amplitude (time constant: 50 ms) of scalenes activity recorded during a MIP, and (ii) the detection of the R wave in the ECG within a window between 150 and 300 ms prior to the trigger to avoid contamination of the silent period by an ECG artifact. For each condition, TMS was delivered during a block of 10 isometric and then 10 dynamic contractions of the inspiratory muscles (Fig. 1A, inset) with the order randomised to compare tonic activation of these muscles, as is common for recruitment of limb muscles, to the phasic activation observed during eupnoeic breathing. Subjects were prompted, at an average frequency of one in five breaths, to perform these contractions. Since the quasi-isometric inspiratory efforts (i.e. with minimal change in lung volume) were performed against a closed valve at FRC, visual feedback of scalenes muscle activity was provided on an oscilloscope so that subjects could maintain a steady voluntary contraction at the target EMG threshold. During dynamic
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inspiration, the level of activation required to trigger the TMS pulse could not be easily reached during normal or hypercapnic breathing so subjects were prompted to make a single, larger voluntary breath until scalene muscle activity reached the target EMG threshold. In all conditions, subjects were instructed to react “as fast as possible” to the TMS pulse by producing a maximal inspiratory effort to reveal the end of the silent period. 2.3. Silent periods in a non-respiratory muscle On a different day, a control experiment was performed in which TMS was delivered during contractions of the right first dorsal interosseous (FDI), an intrinsic muscle of the hand. The effect of an isometric and dynamic contraction on the TMS-evoked silent period in the FDI was compared to that in the scalene and parasternal intercostal muscles during normocapnia. A separate setup (Fig. 1B) was used with the subject seated with the pronated forearm resting on a custom-designed table. The hand was positioned with the index finger at a neutral angle (0◦ abduction, 0◦ flexion) while the wrist, forearm, and middle to little fingers were held in place with Velcro straps. The index finger rested on top of a movable aluminium platform which rotated with minimal friction about an axis that passed vertically through the first metacarpophalangeal joint. The abduction angle of the index finger was recorded with a potentiometer fixed to the rotational axis of the platform. A fixed post prevented adduction of the thumb and provided leverage for abduction of the index finger. Surface EMG of FDI muscle activity was recorded with electrodes placed over the muscle belly and distal tendon. Isometric contractions involved isometric activation of the FDI to generate an abduction force while the aluminium platform was made rigid. Using the same Bistim setup, TMS (40–70% of maximal intensity) was delivered while the subject maintained a brief isometric contraction at 10% of the peak RMS amplitude of FDI EMG, as determined from the best of three maximal voluntary abductions of the index finger. Visual feedback of FDI EMG was provided on an oscilloscope with a cursor indicating the target contraction intensity. Dynamic contractions involved abduction of the index finger with a mean angular displacement of 15.0◦ (s.d. 1.4◦ ) at a frequency of 0.2 Hz and 0.4 Hz to simulate the movement frequency of the inspiratory muscles during breathing. Feedback of abduction angle was provided on an oscilloscope so that subjects could maintain the desired frequencies by tracking a superimposed sine wave. An elastic string attached to the moving platform provided a small resistance against abduction to mimic the elastic recoil of the lung and chest wall, increasing peak EMG activity in the FDI to between 19% and 35% of that produced maximally by each subject. TMS was again delivered, at an average rate of one in five oscillations, during abduction of the index finger at an EMG threshold of 10% of maximum rather than the 25% target in the scalenes. The isometric and dynamic tasks were performed in random order, with a set of 10 TMS pulses recorded in each condition. Subjects were instructed to react as fast as possible to the TMS pulses by maximally abducting the index finger. 2.4. Data analysis Raw EMG traces were processed off-line in Spike2 software (Cambridge Electronic Design) to help identify the onset and duration of EMG suppression during the silent period. A 50 Hz high-pass digital FIR filter was applied to all EMG signals. Filtered data were rectified, and then averaged within each condition to produce mean responses for each subject. The duration of the silent period was calculated from the onset of the magnetic stimulus to the termination of EMG suppression as it recovered above the pre-stimulus mean (averaged over a 100 ms window).
TMS did not evoke periods of absolute electrical silence in the scalene and parasternal intercostal muscles for all subjects. However, prolonged periods of attenuated muscle activity relative to the pre-stimulus mean were clearly visible in these subjects. To maintain conventional terminology, TMS-evoked periods of attenuated muscle activity are referred to as ‘silent periods’ here. For each condition, the mean amplitude of the residual low-level EMG during the silent period from the end of the motor evoked potential to the recovery of rectified EMG above the pre-stimulus mean was normalised to its pre-stimulus mean. Thus, activity in the ‘silent period’ is referred to as ‘low level EMG’ activity. Group mean data are presented as means with 95% confidence intervals in square brackets. Statistical analyses were performed using Statistica 6.0 software (StatSoft. Inc., Tulsa, USA). For the respiratory muscles, two-way repeated measure ANOVAs were performed to determine whether there were significant differences in the duration or amplitude of the silent period due to the main effects of PET CO2 and the type of muscle contraction (isometric vs. dynamic). The data for silent period duration and low-level EMG amplitude during the silent period did not violate Mauchly’s sphericity test. Each factor used in the two-way repeated measures ANOVA passed the Shapiro–Wilk normality test. Cohen’s d effect size calculations were performed on silent period duration and low-level EMG amplitude during the silent period at hypercapnia and hypocapnia for both isometric and dynamic scalene contractions. For the FDI, one-way repeated measure ANOVAs were performed to compare the effect of muscle contraction type on the duration and amplitude of the silent period, with Tukey’s HSD post-hoc test used to compare between groups. The level of statistical significance was set at p < 0.05.
3. Results 3.1. Responses in respiratory muscles During eupnoea, the scalenes reached a peak of 5.3% [3.6, 7.0] of the maximal EMG recorded during maximum inspiratory pressure (MIP). This increased to 8.7% [5.5, 11.9] during hypercapnia-driven reflex hyperventilation and to 15.3% [9.2, 21.4] during voluntary hyperpnoea to induce hypocapnia. A similar trend occurred for parasternal intercostal (PSIC), with peak EMG activity during quiet breathing increasing from 5.6% [3.0, 8.2] of maximum to 8.4% [5.0, 11.8] during hypercapnia and 11.4% [8.8, 14.0] during hypocapnia. Mean ventilation across subjects was 11.8 l min−1 [10.3, 13.4] during eupnoea, which increased to 26.3 l min−1 [21.7, 30.9] during hypercapnia and 30.0 l min−1 [24.3, 35.6] to induce hypocapnia. TMS evoked prolonged decreases in the EMGs of the scalene and parasternal intercostal muscles during voluntary inspiratory efforts at 25% MIP. Data for a single subject are shown in Fig. 2 for isometric and dynamic contractions of the scalenes during the three experimental conditions. Across subjects, TMS produced consistently long silent periods in the scalenes during isometric normocapnic inspiratory efforts (Fig. 3), as required by the inclusion criterion of a silent period lasting 100 ms. The mean duration of the scalenes silent period was 175.7 ms [151.9, 199.6]. For isometric inspiratory efforts, TMS during hypercapnia and hypocapnia produced little difference (Cohen’s d = 0.18) in silent period duration, 165.1 ms [141.8, 188.4] compared to 159.0 ms [133.8, 184.1], respectively. For corresponding dynamic contractions, TMS evoked relatively shorter silent periods in scalenes EMG of 124.7 ms [92.3, 157.2] during normocapnia, 127.1 ms [92.6, 161.6] during hypercapnia, and 136.2 ms [101.6, 170.9] during hypocapnia. Similar to isometric contractions, there was little difference in silent period duration between hypercapnia and hypocapnia (Cohen’s d = −0.27). There was a significant difference in the duration of the silent period for
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Fig. 2. Silent periods in the scalene muscle for a single subject. Raw data are shown for the three experimental conditions. Transcranial magnetic stimulation (arrows) produced a motor evoked potential followed by a period of suppressed muscle activity (grey horizontal bars). The electromyographic responses evoked from the first 5 stimuli are superimposed for each condition with the averaged rectified response of all 10 stimuli below. Dashed horizontal lines represent the pre-stimulus mean averaged over 100 ms. The mean duration of the silent period (black horizontal bars) was shorter for dynamic contractions of the scalenes (short vertical lines) than during the corresponding isometric contractions (long vertical lines). Solid horizontal lines represent 0 V.
Fig. 3. Silent period duration and low-level EMG amplitude for respiratory muscles. Transcranial magnetic stimulation was applied during isometric (filled symbols) and dynamic (unfilled symbols) inspiratory efforts at 25% maximum inspiratory pressure. Group mean data with 95% confidence intervals are shown for each condition with individual subject data adjacent. The duration of the evoked silent periods are presented in the top panel with normalised amplitudes of the low-level EMG during the silent periods presented across the bottom panel. For scalenes, the duration of the silent period was significantly shorter during dynamic than isometric contractions (F1,7 = 10.717, p = 0.014). End-tidal CO2 had no significant effect on the duration or amplitude of the evoked silent period for both muscles. Horizontal line represents the threshold for the subject inclusion criterion during isometric contractions of the scalenes during normocapnia.
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isometric and dynamic contractions (F1,7 = 10.717, p = 0.014), but not for different PET CO2 . Silent periods evoked in PSIC EMGs were shorter compared to the scalenes (Fig. 3). The difference in duration of the silent period between isometric and dynamic contractions was small for this muscle and not statistically significant. Similarly, there was no significant effect of PET CO2 on silent-period duration. Varying PET CO2 had a limited effect on the mean amplitude of the low-level EMG during the silent period (Fig. 3). For isometric inspiratory efforts (25% maximum), the mean amplitudes for scalenes low-level EMG were 40.7% [32.8, 48.6] during normocapnia, 41.7% [31.7, 51.7] during hypercapnia, and 45.3% [36.7, 54.0] during hypocapnia, and these were not significantly different (F2,14 = 0.216, p = 0.808). Cohen’s effect size (d = −0.27) also indicates that the difference in mean amplitude between hypercapnia and hypocapnia was small. For dynamic inspiratory efforts (up to 25% of maximum), the mean amplitudes of low-level EMG were comparable to the isometric contractions, albeit with greater variability between subjects, and the difference in mean amplitude between hypercapnia and hypocapnia was also small (Cohen’s d = 0.07). The PSIC behaved similarly with no significant differences between the mean amplitudes of the low-level EMG at different PET CO2 or between isometric and dynamic contractions.
3.2. Responses in a non-respiratory muscle Long silent periods could be evoked in the first dorsal interosseous (FDI) with TMS at lower stimulus intensities (40–70% maximal output) than in the respiratory muscles (70–100% maximal output). Fig. 4 shows there were significant differences in the duration of the silent period based on the type of contraction (F2,14 = 8.861, p = 0.003). Thus, unlike the respiratory muscles, in FDI the silent periods were significantly longer during dynamic than isometric contractions. The duration of the silent period for isometric FDI contractions was 185.6 ms [178.1, 193.2], and this increased to 198.6 ms [191.8, 205.4] with dynamic contractions at 0.2 Hz and to 203.2 ms [193.1, 213.2] at 0.4 Hz. No significant differences (F2,14 = 1.836, p = 0.20) were observed in the mean amplitude of the low-level EMG during the silent period when dynamic contractions at 0.2 Hz (11.3% [10.7, 11.9]) and 0.4 Hz (11.5% [10.7, 12.3]) were compared with the isometric contraction (12.2% [11.5, 12.9]).
Fig. 4. Silent period duration and low-level EMG amplitude for first dorsal interosseous. Group means with 95% confidence intervals and individual subject data of the silent periods evoked by transcranial magnetic stimulation are shown for isometric (filled symbols) and dynamic (unfilled symbols) abductions of the index finger at 10% of maximum. The type of contraction affected the duration of the silent period but not the amplitude. Post-hoc comparisons with Tukey’s HSD show that the silent period was longer for dynamic contractions at 0.2 Hz (p = 0.024) and 0.4 Hz (p = 0.003) than during isometric contractions, as indicated by * .
4. Discussion This study has determined that TMS can suppress EMG in human scalene and parasternal intercostal muscles for longer than 100 ms, which suggests a cortical component to the suppression. While difficult to obtain during eupnoea in most subjects, long and distinct periods of EMG suppression were present at higher inspiratory efforts following high-intensity TMS, with the duration of the suppression being shorter for dynamic compared to isometric contractions. Although low-level EMG during the ‘silent period’ is clearly present, increasing PET CO2 by 27 mmHg from hypocapnia to hypercapnia did not shorten the silent period, despite the increased reflex chemical drive to medullary respiratory centres, nor did PET CO2 affect the amplitude of the low-level EMG during the silent period. Together, these results show that when TMS suppresses voluntary inspiratory drive, a hypercapnic stimulus to respiratory premotoneurones does not independently activate the scalene and parasternal intercostal muscles. 4.1. Why is the silent period not silent in respiratory muscles? To obtain silent periods of greater than 100 ms following TMS over the motor cortex, subjects were required to activate the inspiratory muscles voluntarily above tidal breathing. It is not clear why there would be a contraction threshold for the production of a silent period in scalene and parasternal intercostal muscles. The duration of the silent period in limb muscles is dependent on the intensity of the magnetic stimulus rather than the level of prior muscle activity (Haug et al., 1992; Inghilleri et al., 1993; Säisänen et al., 2008; Taylor et al., 1997), although greater activity after the silent period tends to shorten it. This was controlled for by having subjects react as fast as possible to the TMS pulse by producing a maximal inspiratory effort. One possible explanation is that the low-level EMG during the silent period in the scalenes, approximately 10% of MIP in this study, is not cortically driven, making it difficult to identify any loss in activity due to intracortical inhibition for contractions at or near that intensity. Werhahn et al. (1995) had noted a similar residual level of EMG during the silent period in intramuscular recordings of mentalis and genioglossus, but not sternocleidomastoid, when these muscles were activated voluntarily at 20% of maximum. This feature of respiratory muscles would explain the absence of long silent periods in the previous study by Lefaucheur and Lofaso (2002, ∼60 ms) who used lower contraction strength and stimulus intensities. If the low-level EMG during the silent period is not cortically driven, it also seems unlikely that this residual activity reflects ongoing drive from the respiratory centres in the medulla. The mean amplitude of low-level EMG did not increase with CO2 as would be expected from a reflex increase in chemical drive to the medullary respiratory neurons. The other possibility is that the low-level EMG is sustained by an intraspinal network which is not affected by intracortical inhibition. DiMarco and Kowalski (2011, 2013) have identified a network of propriospinal neurons that, when electrically stimulated, activate inspiratory motoneurones in a pattern that is similar to spontaneous breathing. Thus, while it is difficult to produce cortical silent periods in inspiratory muscles, this is not because of ongoing descending drive from the medulla. Several other factors may prevent the complete suppression of respiratory muscle activity during the silent period. The large between-subject variability in silent period duration in this study is comparable to that observed in the FDI at moderate stimulus intensities (Orth and Rothwell, 2004), which suggests that, even with the Bistim (Magstim Co. Ltd.) at or close to maximal output, the stimulus was close to the threshold for activating the intracortical inhibitory neurons associated with respiratory motor areas.
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Moreover, despite using a stimulus above motor threshold, the residual low-level EMG (∼10% MIP) during the silent period may indicate that this stimulus did not recruit a significant proportion of intracortical GABAB inhibitory neurons (McDonnell et al., 2006) to completely suppress descending respiratory drive from the motor cortex. Whether this is due to stimulus parameters or fewer GABAB inhibitory neurons in the motor cortex is not clear. Petersen et al. (2011) found that it is possible to suppress scalene muscle activity below 10% of MIP during a voluntary inspiratory effort by preferentially targeting GABAA receptors with TMS delivered at below resting motor threshold (Ilic´ et al., 2002). In comparison to inspiratory muscles, using a magnetic stimulus above motor threshold in the current study reduced the mean amplitude of low-level EMG in FDI to ∼1% of maximum during the silent period for contractions at 10% of maximum. Further investigations using paired-pulse TMS protocols to assess cortical excitability are needed to understand the lack of complete suppression of scalene and parasternal intercostal EMGs during the silent period. However, the conclusions in this study remain valid since changes in cortical excitability would not affect the reflex medullary drive to the inspiratory motoneurones with changes in arterial CO2 .
4.2. Cortical silent periods in respiratory muscles While changes in PET CO2 did not affect the duration of the silent period, dynamic voluntary inspiratory efforts shortened periods of EMG suppression in the scalenes more than during isometric efforts. Although the amplitude of low-level EMG was not affected, this difference due to contraction type is consistent with the reflex facilitation of motoneurones by muscle spindles that is believed to contribute to the low-level EMG during the silent period. This has been described for lengthening contractions of the elbow flexors (Butler et al., 2012) and observed in the distal muscles of the upper and lower limbs (Holmgren et al., 1990; Sammut et al., 1995; Wilson et al., 1995). During dynamic inspiration, the scalenes contract and shorten to elevate the rib cage. Since TMS was delivered at lung volumes above FRC in these contractions, the higher intrathoracic pressure relative to atmospheric pressure at the onset of EMG suppression in the silent period would cause air to be expelled passively from the lungs, lowering the rib cage and stretching the scalenes. Any spindle induced reflex facilitation would only appear towards the end of the EMG suppression, i.e. after the spinal component of the silent period (Fuhr et al., 1991; Inghilleri et al., 1993; Uncini et al., 1993; Ziemann et al., 1993), resulting in a shortened period of EMG suppression. Presumably, this spindle-induced reflex facilitation during the silent period also occurs for isometric inspiratory efforts, albeit to a lesser extent as lung volume was held constant at FRC where intrathoracic and atmospheric pressures are equal when the respiratory muscles are relaxed. A contraction-type effect was also observed for EMG suppression produced in scalenes by low-intensity TMS at sub-motor threshold where the duration of suppression was shorter for dynamic compared to isometric inspiratory efforts (Petersen et al., 2011). For FDI, silent periods were of longer duration in the dynamic rather than the isometric tasks. This effect is opposite to that predicted by a spindle-induced reflex facilitation as the muscle lengthens during the silent period. Whether this differential effect between the FDI and scalenes is a real phenomenon is difficult to determine from the present study. However, it seems more plausible that the lack of shortening of the silent period during dynamic contractions of the FDI is due to the low stiffness of the elastic string which may not have recoiled fast enough to produce a spindle-induced reflex facilitation rather than being a musclespecific effect.
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4.3. Voluntary control of breathing Our main finding was that hypercapnia did not affect the duration of the silent period or amplitude of low-level EMG in the silent period during voluntary inspiratory efforts. We postulated that if the cortical and medullary drives to the muscles were independent then a CO2 -driven increase in medullary drive would still activate the motoneurones when cortical drive was suppressed with TMS. Thus, we expected the silent period during hypercapnia to be of shorter duration and show more low-level EMG than during hypocapnia. This lack of a CO2 -driven effect on the silent period indicates that, despite doubling ventilation, the reflex increase in chemical drive to the medulla when PET CO2 was elevated by 13 mmHg did not reach the motoneurones during voluntary breaths. The lack of a differential effect of CO2 on the silent period can be interpreted in two ways. Either the medullary drive was inhibited by the voluntary activation of the scalene and parasternal intercostal muscles or TMS activated an inhibitory pathway to the respiratory neurons in the same way as a breath hold (Orem, 1989; Orem and Netick, 1986). Voluntary control of a motor system that is usually regulated automatically, such as breathing, may have inhibited or gated task-dependent bulbospinal pathways to the motoneurones. This behaviour has been observed in the balance system where postural muscles also receive both cortical and subcortical inputs. The reflex muscle response to electrical vestibular stimulation was absent when the plantarflexors were activated voluntarily to perform a motor task that was equivalent to standing (Fitzpatrick et al., 1994). The alternative explanation of a TMSinduced inhibition of medullary drive would require corticobulbar projections to the medulla that operate with a time constant similar to or longer than the suppression of cortical drive such that no difference in silent period was detected. These two processes are not mutually exclusive, but both are consistent with cortical drive that bypasses the medulla, as was suggested by Corfield et al. (1998). Even if only a portion of the cortical drive went via the medulla, so that this portion of descending drive was not inhibited or gated, we would still expect hypercapnia to decrease the duration of the silent period or increase the low-level EMG amplitude in it. A limitation of this study was the inability to evoke a visible silent period in the scalene and parasternal intercostal muscles during spontaneous breaths in hypercapnia. A direct comparison of the silent period between spontaneous breaths and matched voluntary activation of the respiratory muscles during hypocapnia would have provided a clearer indication of the interaction between the descending cortical and medullary drives. Nevertheless, our results indicate that, despite the increased chemo-reflex input to the medulla present in hypercapnia, the change in reflex drive does not reach the inspiratory motoneurones during the silent period to independently activate the inspiratory muscles when taking a voluntary breath. Acknowledgement This work was funded by the National Health and Medical Research Council (of Australia). References Bassal, M., Bianchi, A.L., 1982. Inspiratory onset or termination induced by electrical stimulation of the brain. Respir. Physiol. 50, 23–40. Butler, J.E., Petersen, N.C., Herbert, R.D., Gandevia, S.C., Taylor, J.L., 2012. Origin of the low-level EMG during the silent period following transcranial magnetic stimulation. Clin. Neurophysiol. 123, 1409–1414. Corfield, D.R., Murphy, K., Guz, A., 1998. Does the motor cortical control of the diaphragm ‘bypass’ the brain stem respiratory centres in man? Respir. Physiol. 114, 109–117.
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De Troyer, A., Estenne, M., 1984. Coordination between rib cage muscles and diaphragm during quiet breathing in humans. J. Appl. Physiol. 57, 899–906. De Troyer, A., Sampson, M.G., 1982. Activation of the parasternal intercostals during breathing efforts in human subjects. J. Appl. Physiol. 52, 524–529. DiMarco, A.F., Kowalski, K.E., 2011. Distribution of electrical activation to the external intercostal muscles during high frequency spinal cord stimulation in dogs. J. Physiol. 589, 1383–1395. DiMarco, A.F., Kowalski, K.E., 2013. Spinal pathways mediating phrenic activation during high frequency spinal cord stimulation. Respir. Physiol. Neurobiol. 186, 1–6. Feldman, J.L., Del Negro, C.A., 2006. Looking for inspiration: new perspectives on respiratory rhythm. Nat. Rev. Neurosci. 7, 232–242. Fitzpatrick, R., Burke, D., Gandevia, S.C., 1994. Task-dependent reflex responses and movement illusions evoked by galvanic vestibular stimulation in standing humans. J. Physiol. 478, 363–372. Fuhr, P., Agostino, R., Hallett, M., 1991. Spinal motor neuron excitability during the silent period after cortical stimulation. Electroencephalogr. Clin. Neurophysiol. 81, 257–262. Gandevia, S.C., Hudson, A.L., Gorman, R.B., Butler, J.E., De Troyer, A., 2006. Spatial distribution of inspiratory drive to the parasternal intercostal muscles in humans. J. Physiol. 573, 263–275. Gandevia, S.C., Leeper, J.B., McKenzie, D.K., De Troyer, A., 1996. Discharge frequencies of parasternal intercostal and scalene motor units during breathing in normal and COPD subjects. Am. J. Respir. Crit. Care Med. 153, 622–628. Gandevia, S.C., Plassman, B.L., 1988. Responses in human intercostal and truncal muscles to motor cortical and spinal stimulation. Respir. Physiol. 73, 325–337. Gandevia, S.C., Rothwell, J.C., 1987. Activation of the human diaphragm from the motor cortex. J. Physiol. 384, 109–118. Haug, B.A., Schonle, P.W., Knobloch, C., Kohne, M., 1992. Silent period measurement revives as a valuable diagnostic tool with transcranial magnetic stimulation. Electroencephalogr. Clin. Neurophysiol. 85, 158–160. Holmgren, H., Larsson, L.E., Pedersen, S., 1990. Late muscular responses to transcranial cortical stimulation in man. Electroencephalogr. Clin. Neurophysiol. 75, 161–172. ´ T.V., Meintzschel, F., Cleff, U., Ruge, D., Kessler, K.R., Ziemann, U., 2002. ShortIlic, interval paired-pulse inhibition and facilitation of human motor cortex: the dimension of stimulus intensity. J. Physiol. 545, 153–167. Inghilleri, M., Berardelli, A., Cruccu, G., Manfredi, M., 1993. Silent period evoked by transcranial stimulation of the human cortex and cervicomedullary junction. J. Physiol. 466, 521–534. Kujirai, T., Caramia, M.D., Rothwell, J.C., Day, B.L., Thompson, P.D., Ferbert, A., Wroe, S., Asselman, P., Marsden, C.D., 1993. Corticocortical inhibition in human motor cortex. J. Physiol. 471, 501–519. Lefaucheur, J.P., Lofaso, F., 2002. Diaphragmatic silent period to transcranial magnetic cortical stimulation for assessing cortical motor control of the diaphragm. Exp. Brain Res. 146, 404–409. Macefield, G., Gandevia, S.C., 1991. The cortical drive to human respiratory muscles in the awake state assessed by premotor cerebral potentials. J. Physiol. 439, 545–558. McDonnell, M.N., Orekhov, Y., Ziemann, U., 2006. The role of GABAB receptors in intracortical inhibition in the human motor cortex. Exp. Brain Res. 173, 86–93.
Murphy, K., Mier, A., Adams, L., Guz, A., 1990. Putative cerebral cortical involvement in the ventilatory response to inhaled CO2 in conscious man. J. Physiol. 420, 1–18. Murray, N.P., McKenzie, D.K., Gorman, R.B., Gandevia, S.C., Butler, J.E., 2008. Reproducibility of the short-latency reflex inhibition to loading of human inspiratory muscles. Respir. Physiol. Neurobiol. 162, 216–222. Orem, J., 1989. Behavioral inspiratory inhibition: inactivated and activated respiratory cells. J. Neurophysiol. 62, 1069–1078. Orem, J., Netick, A., 1986. Behavioral control of breathing in the cat. Brain Res. 366, 238–253. Orth, M., Rothwell, J.C., 2004. The cortical silent period: intrinsic variability and relation to the waveform of the transcranial magnetic stimulation pulse. Clin. Neurophysiol. 115, 1076–1082. Petersen, N.C., Taylor, J.L., Murray, N.P., Gandevia, S.C., Butler, J.E., 2011. Differential effects of low-intensity motor cortical stimulation on the inspiratory activity in scalene muscles during voluntary and involuntary breathing. Respir. Physiol. Neurobiol. 175, 265–271. Raper, A.J., Thompson Jr., W.T., Shapiro, W., Patterson Jr., J.L., 1966. Scalene and sternomastoid muscle function. J. Appl. Physiol. 21, 497–502. Raux, M., Straus, C., Redolfi, S., Morelot-Panzini, C., Couturier, A., Hug, F., Similowski, T., 2007. Electroencephalographic evidence for pre-motor cortex activation during inspiratory loading in humans. J. Physiol. 578, 569–578. Richter, D.W., Smith, J.C., 2014. Respiratory rhythm generation in vivo. Physiology 29, 58–71. Roberts, C.A., Corfield, D.R., Murphy, K., Calder, N.A., Hanson, M.A., Adams, L., Guz, A., 1995. Modulation by central PCO2 of the response to carotid body stimulation in man. Respir. Physiol. 102, 149–161. Säisänen, L., Pirinen, E., Teitti, S., Kononen, M., Julkunen, P., Maatta, S., Karhu, J., 2008. Factors influencing cortical silent period: optimized stimulus location, intensity and muscle contraction. J. Neurosci. Methods 169, 231–238. Sammut, R., Thickbroom, G.W., Wilson, S.A., Mastaglia, F.L., 1995. The origin of the soleus late response evoked by magnetic stimulation of human motor cortex. Electroencephalogr. Clin. Neurophysiol. 97, 164–168. Straus, C., Locher, C., Zelter, M., Derenne, J.P., Similowski, T., 2004. Facilitation of the diaphragm response to transcranial magnetic stimulation by increases in human respiratory drive. J. Appl. Physiol. (1985) 97, 902–912. Taylor, J.L., Allen, G.M., Butler, J.E., Gandevia, S.C., 1997. Effect of contraction strength on responses in biceps brachii and adductor pollicis to transcranial magnetic stimulation. Exp. Brain Res. 117, 472–478. Uncini, A., Treviso, M., Di Muzio, A., Simone, P., Pullman, S., 1993. Physiological basis of voluntary activity inhibition induced by transcranial cortical stimulation. Electroencephalogr. Clin. Neurophysiol. 89, 211–220. Werhahn, K.J., Classen, J., Benecke, R., 1995. The silent period induced by transcranial magnetic stimulation in muscles supplied by cranial nerves: normal data and changes in patients. J. Neurol. Neurosurg. Psychiatry 59, 586–596. Wilson, S.A., Thickbroom, G.W., Mastaglia, F.L., 1995. An investigation of the late excitatory potential in the hand following magnetic stimulation of the motor cortex. Electroencephalogr. Clin. Neurophysiol. 97, 55–62. Ziemann, U., Netz, J., Szelenyi, A., Homberg, V., 1993. Spinal and supraspinal mechanisms contribute to the silent period in the contracting soleus muscle after transcranial magnetic stimulation of human motor cortex. Neurosci. Lett. 156, 167–171.
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Respiratory Physiology & Neurobiology journal homepage: www.elsevier.com/locate/resphysiol
TMS-evoked silent periods in scalene and parasternal intercostal muscles during voluntary breathing Billy L. Luu a,b , Julian P. Saboisky a,b , Janet L. Taylor a,b , Simon C. Gandevia a,b , Jane E. Butler a,b,∗ a b
Neuroscience Research Australia, Barker St, Randwick 2031, NSW, Australia The University of New South Wales, Sydney 2052, NSW, Australia
a r t i c l e
i n f o
Article history: Received 22 January 2015 Received in revised form 13 May 2015 Accepted 18 May 2015 Available online 27 May 2015 Keywords: Magnetic stimulation Cortical Respiratory Hypercapnia Inhibition Drive
a b s t r a c t Transcranial magnetic stimulation (TMS) during voluntary muscle contraction causes a period of reduced electromyographic (EMG) activity (EMG). This is attributed to cortical inhibition and is known as the ‘silent period’. Silent periods were compared in inspiratory muscles following TMS during voluntary inspiratory efforts during normocapnia, hypercapnia, and hypocapnia. TMS was delivered during isometric and dynamic contractions of scalenes and parasternal intercostals at 25% maximum inspiratory pressure. Changing end-tidal CO2 did not affect the duration of the silent period nor suppression of EMG activity during the silent period. In scalenes, silent periods were shorter for dynamic compared to isometric contractions (p < 0.05); but contraction type did not alter the degree of suppression of EMG during the silent period. In parasternal intercostal, no significant differences in silent period parameters occurred for the different contraction types. The lack of effect of end-tidal CO2 suggests that descending drive from the medullary respiratory centres does not independently activate the inspiratory muscles during voluntary inspiratory efforts. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Breathing is usually controlled involuntarily. Rhythmic activity originating from a network of pontomedullary neurons activates the respiratory muscles via bulbospinal pathways (for reviews, see Feldman and Del Negro, 2006; Richter and Smith, 2014). The activity of these respiratory neurons is modulated by arterial CO2 to control breathing and maintain normocapnia. However, like the skeletal muscles of the limbs, the respiratory muscles also receive cortical drive during behavioural tasks that require controlled breathing such as swimming, fine motor tasks, and pursed-lip breathing. It is generally observed that deliberate attention to breathing normally shifts the behaviour from the automatic regulation of respiration to voluntary control by the motor cortex (Macefield and Gandevia, 1991; Petersen et al., 2011; Raux et al., 2007). The voluntary command from the motor cortex may act via corticospinal pathways that bypass the medullary respiratory centres (Gandevia and Plassman, 1988; Gandevia and Rothwell, 1987; Murphy et al., 1990; Straus et al., 2004) and/or via inhibitory
∗ Corresponding author at: Neuroscience Research Australia, Barker St, Randwick 2031, NSW, Australia. Tel.: +61 2 93991608. E-mail address: [email protected] (J.E. Butler). http://dx.doi.org/10.1016/j.resp.2015.05.010 1569-9048/© 2015 Elsevier B.V. All rights reserved.
corticobulbar projections that modulate the activity of respiratory neurons (Bassal and Bianchi, 1982; Orem, 1989; Orem and Netick, 1986). It is not known how the cortical drive to respiratory motoneurones is integrated with the ongoing automatic control of breathing during a voluntary breath. It is possible that medullary output to the motoneurones summates with the descending drive from the motor cortex, or that medullary output is suppressed and replaced by the descending cortical drive. The interaction between the cortical and medullary facilitatory drives to the respiratory muscles has been investigated previously. Corfield et al. (1998) and Lefaucheur and Lofaso (2002) explored a potential cortico-medullary interaction by examining separately the two components of the muscle response to transcranial magnetic stimulation (TMS): the motor evoked potential and the subsequent silent period. Corfield et al. (1998) compared diaphragmatic motor evoked potentials, an indicator of overall corticospinal excitability, during normocapnia and hypocapnia, where reduced CO2 has been shown to attenuate the diaphragmatic muscle activity evoked by a transient hypoxic stimulus (Roberts et al., 1995). Corfield et al. found that hypocapnia did not attenuate the motor evoked potential and therefore suggested that voluntary activation of the diaphragm bypasses the medulla. However, a limitation of their study was that in order to expect a difference in the motor evoked potential they had assumed that TMS activates an
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excitatory projection to bulbar respiratory premotoneurones, a circuitry which has yet to be demonstrated, and that this pathway would be attenuated by hypocapnia in the same way as the response to a transient hypoxic stimulus. Lefaucheur and Lofaso (2002), on the other hand, focused on the silent period, which is the electrical silence in ongoing electromyographic (EMG) activity following TMS. The suppression of EMG activity during the silent period is due to an early spinal component (Fuhr et al., 1991; Inghilleri et al., 1993; Uncini et al., 1993; Ziemann et al., 1993) followed by a later supraspinal inhibition due to the activation of intracortical inhibitory neurons with consequent inhibition of corticospinal output (Kujirai et al., 1993). Temporary inhibition of corticospinal output with TMS during inspiration would highlight the reliance placed on this pathway during voluntary breathing and also reveal the contribution of non-cortical inputs to respiratory motoneurones through the remaining ongoing EMG activity. Lefaucheur and Lofaso showed that silent period durations during matched involuntary and voluntary activation of the diaphragm were similar. However, a shortcoming of this study is that the diaphragmatic silent periods were ∼60 ms in duration from the onset of the stimulus. This is significantly shorter than the 100 ms duration threshold that is considered to reflect intracortical inhibition of corticospinal output (Fuhr et al., 1991; Inghilleri et al., 1993). The present study reconsiders the use of the silent period in understanding the interaction between the motor cortex and medullary respiratory neurons during voluntary breathing. Here, we determine whether distinct cortical silent periods are present in the obligatory inspiratory muscles of scalenes (De Troyer and Estenne, 1984; Gandevia et al., 1996; Raper et al., 1966) and parasternal intercostals (De Troyer and Sampson, 1982; Gandevia et al., 2006). Surprisingly, clear silent periods could only be achieved during substantial voluntary muscle activity. Hence, stimuli were delivered with inspiratory EMG activity of 25% maximum during voluntary hyperventilation or during single, large voluntary inspiratory efforts that were interspersed during eupnoea and CO2 -driven breathing. Subjects were studied at different levels of end-tidal CO2 (PET CO2 ) to alter the chemical feedback acting on the medullary respiratory neurons. We posited that if the cortical and medullary drives during voluntary inspiratory efforts were independent and summated at a motoneurone level, then the increase in medullary drive during hypercapnia compared to hypocapnia would reduce the level of corticospinal drive needed to produce the designated EMG activity. Thus, inhibition of this reduced level of corticospinal drive by TMS would be less effective at suppressing EMG activity since the CO2 -driven increase in medullary drive would still activate the motoneurones when corticospinal output was suppressed. From this rationale, if the silent periods in the scalenes and parasternal intercostal EMGs were shorter during hypercapnia than during hypocapnia and/or there were less reduction in EMG activity following TMS, we would conclude that the medullary drive independently activates inspiratory muscles during voluntary inspiratory efforts.
2. Methods Ten healthy adults (seven males) aged between 24 and 47 years (mean 29.2; s.d. 7.1) participated in the experiments. All procedures were approved by the University of New South Wales Human Research Ethics Committee and conducted in accordance with the Declaration of Helsinki (2008). Subjects provided written informed consent before participating.
2.1. Experimental setup Respiratory measurements were performed with the subject seated comfortably on a physiotherapy chair inclined at 54◦ from horizontal with the head resting against a pillow (Fig. 1A). Wearing a nose clip, the subject breathed through a mouth piece connected in series with a pneumotachometer (Series 3813, Hans Rudolph Inc, Kansas City, USA) and a 3-way directional stopcock valve (Series 2100, Hans Rudolph Inc). Inspiratory flow measured from the pneumotachometer and a differential pressure transducer (DP45-16, Validyne Engineering Corp., Northridge, USA) was integrated on-line to provide a signal of change in lung volume. PET CO2 , measured with a capnometer (Normocap, Datex Instrumentarium Corp., Helsinki, Finland), was used to estimate arterial CO2 , and along with inspiratory mouth pressure (DP15-34, Validyne Engineering Corp.), was measured close to the mouth piece. Surface electromyograms of the right scalenes and parasternal intercostal (PSIC) muscles were made using bipolar Ag/AgCl electrodes (Cleartrace, ConMed Corp, Utica, USA). For scalenes, the active electrode was placed in the posterior triangle of the neck at the level of the cricoid cartilage with the reference electrode on the clavicle in line with the long axis of the scalene muscle group (Murray et al., 2008). The active electrode for PSIC muscle activity was placed on the second intercostal space with the reference electrode adjacent on the sternum. Electrocardiograms (ECG) were monitored using a bipolar configuration with surface electrodes on the left clavicle and the right ninth intercostal space near the midaxillary line. An electrode placed over the right shoulder served as a common ground for ECG and EMG recordings. TMS was delivered to the motor cortex with a round stimulating coil (mean diameter 90 mm) centred over the vertex. The coil was oriented so that the current flowed through the coil in a counter-clockwise direction. To minimise changes in the coil’s position between pulses, an experimenter held the coil horizontally and a marker pen was used to trace the inner diameter of the coil and several references points onto the scalp. A single, high-intensity pulse was generated by simultaneously discharging two Magstim 200 stimulators connected through a Bistim module (Magstim Co. Ltd., Whitland, UK) programmed with an inter-stimulus interval of 0 ms. Data were acquired (Power 1401, Cambridge Electronic Design, Cambridge, UK) and recorded to computer with Spike2 software (Cambridge Electronic Design). Respiratory data were sampled at 1000 Hz. ECG and scalenes EMG signals were amplified ×1000 and PSIC EMGs were amplified ×3000, band-pass filtered at 16–1000 Hz, and sampled at 2000 Hz. 2.2. Protocol To evoke long and clear silent periods in muscle activity with TMS, subjects were required to make voluntary inspiratory efforts at 25% of their maximum inspiratory pressure (MIP). This level of activation was greater than normal tidal breathing (∼5% MIP), but pilot data indicated that silent periods were difficult to obtain consistently even at 10% MIP (Fig. 1C). Each subject’s MIP was determined beforehand as the best out of 3 maximal inspiratory efforts performed against a closed valve at function residual capacity (FRC). The output intensity of the Bistim was adjusted for each subject (between 70% and 100%) to achieve silent periods longer than 100 ms (from the time of the stimulus) in scalenes EMG during quasi-isometric inspiratory efforts at 25% MIP. Of the ten participants, eight satisfied this criterion and completed the remainder of the study. Once set, TMS intensity was then kept constant throughout the respiratory experiments. TMS was delivered during voluntary inspiratory efforts in three experimental conditions: normocapnia, hypercapnia accompanied
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Fig. 1. Experimental setups. (A) Subjects were seated comfortably. The head and arms were restrained to minimise movement artifacts in the electromyograms of the scalene and parasternal intercostal muscles. The pneumotachometer recorded respiratory air flow as subjects breathed though the mouth piece. A stopcock valve directed air flow between conditions to facilitate re-breathing of dead-space air through an open-ended tube. Inset: Transcranial magnetic stimuli (arrows) were delivered during isometric inspiratory efforts performed at functional residual capacity following expiration (e) and during dynamic inspiratory efforts as subjects increased inspiration (i) above tidal volume (Vt). (B) The hand and forearm were restrained for contractions of the first dorsal interossous (FDI). The index finger rested on a platform that could be made rigid for isometric contractions of the FDI or mobile for dynamic abductions of the finger against the resistance of an elastic string. (C) Raw data from a single subject during isometric contractions of the scalenes and FDI. Traces from the first 5 magnetic pulses (arrow) are superimposed for each condition. Scalenes contractions were performed at 10% and 25% of maximum inspiratory pressure (MIP) with a stimulus intensity of 90% maximum stimulator output. FDI contractions were performed at 10% peak muscle activity during a maximal voluntary contraction (MVC) with a stimulus intensity of 60% maximum. The black horizontal bar represents the duration of the silent period evoked by transcranial magnetic stimulation and the grey horizontal bar represents the period of attenuated muscle activity.
by reflex hyperventilation, and hypocapnia induced by voluntary hyperpnoea. Hypercapnia was achieved by having subjects rebreathe via an added deadspace of ∼1.4 l or ∼2.2 l (diameter 35 mm, Fig. 1A) until PET CO2 and ventilation stabilised. This re-breathing approximately doubled ventilation as PET CO2 increased from a mean of 38.8 mmHg (s.d. 3.0) during normal breathing to a mean of 51.7 mmHg (s.d. 5.3). Hypocapnia was achieved by having subjects voluntarily increase ventilation; using real-time visual feedback of lung volume to track an analogue signal that would produce the same tidal volume, respiratory rate, and inspiratory time as recorded during hypercapnia-driven reflex hyperventilation. This voluntary hyperpnoea was performed with air flow diverted away from the tube attachment by the stopcock (Fig. 1A). Once stabilised, matched voluntary hyperpnoea reduced PET CO2 to a mean of 24.3 mmHg (s.d. 3.8). These three conditions were conducted in random order with at least a 5-min rest period to allow for the respiratory variables to return to baseline. For those subjects who were randomly assigned the hypocapnia condition first, a brief stable period of re-breathing dead-space air was performed initially without TMS to determine the parameters for the tracking signal.
A period of rest allowed PET CO2 to normalise before commencing the voluntary hyperpnoea protocol. A custom Spike2 (Cambridge Electronic Design) script triggered the TMS pulse based on two criteria, (i) an EMG threshold of 25% of the maximum root-mean-squared (RMS) amplitude (time constant: 50 ms) of scalenes activity recorded during a MIP, and (ii) the detection of the R wave in the ECG within a window between 150 and 300 ms prior to the trigger to avoid contamination of the silent period by an ECG artifact. For each condition, TMS was delivered during a block of 10 isometric and then 10 dynamic contractions of the inspiratory muscles (Fig. 1A, inset) with the order randomised to compare tonic activation of these muscles, as is common for recruitment of limb muscles, to the phasic activation observed during eupnoeic breathing. Subjects were prompted, at an average frequency of one in five breaths, to perform these contractions. Since the quasi-isometric inspiratory efforts (i.e. with minimal change in lung volume) were performed against a closed valve at FRC, visual feedback of scalenes muscle activity was provided on an oscilloscope so that subjects could maintain a steady voluntary contraction at the target EMG threshold. During dynamic
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inspiration, the level of activation required to trigger the TMS pulse could not be easily reached during normal or hypercapnic breathing so subjects were prompted to make a single, larger voluntary breath until scalene muscle activity reached the target EMG threshold. In all conditions, subjects were instructed to react “as fast as possible” to the TMS pulse by producing a maximal inspiratory effort to reveal the end of the silent period. 2.3. Silent periods in a non-respiratory muscle On a different day, a control experiment was performed in which TMS was delivered during contractions of the right first dorsal interosseous (FDI), an intrinsic muscle of the hand. The effect of an isometric and dynamic contraction on the TMS-evoked silent period in the FDI was compared to that in the scalene and parasternal intercostal muscles during normocapnia. A separate setup (Fig. 1B) was used with the subject seated with the pronated forearm resting on a custom-designed table. The hand was positioned with the index finger at a neutral angle (0◦ abduction, 0◦ flexion) while the wrist, forearm, and middle to little fingers were held in place with Velcro straps. The index finger rested on top of a movable aluminium platform which rotated with minimal friction about an axis that passed vertically through the first metacarpophalangeal joint. The abduction angle of the index finger was recorded with a potentiometer fixed to the rotational axis of the platform. A fixed post prevented adduction of the thumb and provided leverage for abduction of the index finger. Surface EMG of FDI muscle activity was recorded with electrodes placed over the muscle belly and distal tendon. Isometric contractions involved isometric activation of the FDI to generate an abduction force while the aluminium platform was made rigid. Using the same Bistim setup, TMS (40–70% of maximal intensity) was delivered while the subject maintained a brief isometric contraction at 10% of the peak RMS amplitude of FDI EMG, as determined from the best of three maximal voluntary abductions of the index finger. Visual feedback of FDI EMG was provided on an oscilloscope with a cursor indicating the target contraction intensity. Dynamic contractions involved abduction of the index finger with a mean angular displacement of 15.0◦ (s.d. 1.4◦ ) at a frequency of 0.2 Hz and 0.4 Hz to simulate the movement frequency of the inspiratory muscles during breathing. Feedback of abduction angle was provided on an oscilloscope so that subjects could maintain the desired frequencies by tracking a superimposed sine wave. An elastic string attached to the moving platform provided a small resistance against abduction to mimic the elastic recoil of the lung and chest wall, increasing peak EMG activity in the FDI to between 19% and 35% of that produced maximally by each subject. TMS was again delivered, at an average rate of one in five oscillations, during abduction of the index finger at an EMG threshold of 10% of maximum rather than the 25% target in the scalenes. The isometric and dynamic tasks were performed in random order, with a set of 10 TMS pulses recorded in each condition. Subjects were instructed to react as fast as possible to the TMS pulses by maximally abducting the index finger. 2.4. Data analysis Raw EMG traces were processed off-line in Spike2 software (Cambridge Electronic Design) to help identify the onset and duration of EMG suppression during the silent period. A 50 Hz high-pass digital FIR filter was applied to all EMG signals. Filtered data were rectified, and then averaged within each condition to produce mean responses for each subject. The duration of the silent period was calculated from the onset of the magnetic stimulus to the termination of EMG suppression as it recovered above the pre-stimulus mean (averaged over a 100 ms window).
TMS did not evoke periods of absolute electrical silence in the scalene and parasternal intercostal muscles for all subjects. However, prolonged periods of attenuated muscle activity relative to the pre-stimulus mean were clearly visible in these subjects. To maintain conventional terminology, TMS-evoked periods of attenuated muscle activity are referred to as ‘silent periods’ here. For each condition, the mean amplitude of the residual low-level EMG during the silent period from the end of the motor evoked potential to the recovery of rectified EMG above the pre-stimulus mean was normalised to its pre-stimulus mean. Thus, activity in the ‘silent period’ is referred to as ‘low level EMG’ activity. Group mean data are presented as means with 95% confidence intervals in square brackets. Statistical analyses were performed using Statistica 6.0 software (StatSoft. Inc., Tulsa, USA). For the respiratory muscles, two-way repeated measure ANOVAs were performed to determine whether there were significant differences in the duration or amplitude of the silent period due to the main effects of PET CO2 and the type of muscle contraction (isometric vs. dynamic). The data for silent period duration and low-level EMG amplitude during the silent period did not violate Mauchly’s sphericity test. Each factor used in the two-way repeated measures ANOVA passed the Shapiro–Wilk normality test. Cohen’s d effect size calculations were performed on silent period duration and low-level EMG amplitude during the silent period at hypercapnia and hypocapnia for both isometric and dynamic scalene contractions. For the FDI, one-way repeated measure ANOVAs were performed to compare the effect of muscle contraction type on the duration and amplitude of the silent period, with Tukey’s HSD post-hoc test used to compare between groups. The level of statistical significance was set at p < 0.05.
3. Results 3.1. Responses in respiratory muscles During eupnoea, the scalenes reached a peak of 5.3% [3.6, 7.0] of the maximal EMG recorded during maximum inspiratory pressure (MIP). This increased to 8.7% [5.5, 11.9] during hypercapnia-driven reflex hyperventilation and to 15.3% [9.2, 21.4] during voluntary hyperpnoea to induce hypocapnia. A similar trend occurred for parasternal intercostal (PSIC), with peak EMG activity during quiet breathing increasing from 5.6% [3.0, 8.2] of maximum to 8.4% [5.0, 11.8] during hypercapnia and 11.4% [8.8, 14.0] during hypocapnia. Mean ventilation across subjects was 11.8 l min−1 [10.3, 13.4] during eupnoea, which increased to 26.3 l min−1 [21.7, 30.9] during hypercapnia and 30.0 l min−1 [24.3, 35.6] to induce hypocapnia. TMS evoked prolonged decreases in the EMGs of the scalene and parasternal intercostal muscles during voluntary inspiratory efforts at 25% MIP. Data for a single subject are shown in Fig. 2 for isometric and dynamic contractions of the scalenes during the three experimental conditions. Across subjects, TMS produced consistently long silent periods in the scalenes during isometric normocapnic inspiratory efforts (Fig. 3), as required by the inclusion criterion of a silent period lasting 100 ms. The mean duration of the scalenes silent period was 175.7 ms [151.9, 199.6]. For isometric inspiratory efforts, TMS during hypercapnia and hypocapnia produced little difference (Cohen’s d = 0.18) in silent period duration, 165.1 ms [141.8, 188.4] compared to 159.0 ms [133.8, 184.1], respectively. For corresponding dynamic contractions, TMS evoked relatively shorter silent periods in scalenes EMG of 124.7 ms [92.3, 157.2] during normocapnia, 127.1 ms [92.6, 161.6] during hypercapnia, and 136.2 ms [101.6, 170.9] during hypocapnia. Similar to isometric contractions, there was little difference in silent period duration between hypercapnia and hypocapnia (Cohen’s d = −0.27). There was a significant difference in the duration of the silent period for
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Fig. 2. Silent periods in the scalene muscle for a single subject. Raw data are shown for the three experimental conditions. Transcranial magnetic stimulation (arrows) produced a motor evoked potential followed by a period of suppressed muscle activity (grey horizontal bars). The electromyographic responses evoked from the first 5 stimuli are superimposed for each condition with the averaged rectified response of all 10 stimuli below. Dashed horizontal lines represent the pre-stimulus mean averaged over 100 ms. The mean duration of the silent period (black horizontal bars) was shorter for dynamic contractions of the scalenes (short vertical lines) than during the corresponding isometric contractions (long vertical lines). Solid horizontal lines represent 0 V.
Fig. 3. Silent period duration and low-level EMG amplitude for respiratory muscles. Transcranial magnetic stimulation was applied during isometric (filled symbols) and dynamic (unfilled symbols) inspiratory efforts at 25% maximum inspiratory pressure. Group mean data with 95% confidence intervals are shown for each condition with individual subject data adjacent. The duration of the evoked silent periods are presented in the top panel with normalised amplitudes of the low-level EMG during the silent periods presented across the bottom panel. For scalenes, the duration of the silent period was significantly shorter during dynamic than isometric contractions (F1,7 = 10.717, p = 0.014). End-tidal CO2 had no significant effect on the duration or amplitude of the evoked silent period for both muscles. Horizontal line represents the threshold for the subject inclusion criterion during isometric contractions of the scalenes during normocapnia.
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isometric and dynamic contractions (F1,7 = 10.717, p = 0.014), but not for different PET CO2 . Silent periods evoked in PSIC EMGs were shorter compared to the scalenes (Fig. 3). The difference in duration of the silent period between isometric and dynamic contractions was small for this muscle and not statistically significant. Similarly, there was no significant effect of PET CO2 on silent-period duration. Varying PET CO2 had a limited effect on the mean amplitude of the low-level EMG during the silent period (Fig. 3). For isometric inspiratory efforts (25% maximum), the mean amplitudes for scalenes low-level EMG were 40.7% [32.8, 48.6] during normocapnia, 41.7% [31.7, 51.7] during hypercapnia, and 45.3% [36.7, 54.0] during hypocapnia, and these were not significantly different (F2,14 = 0.216, p = 0.808). Cohen’s effect size (d = −0.27) also indicates that the difference in mean amplitude between hypercapnia and hypocapnia was small. For dynamic inspiratory efforts (up to 25% of maximum), the mean amplitudes of low-level EMG were comparable to the isometric contractions, albeit with greater variability between subjects, and the difference in mean amplitude between hypercapnia and hypocapnia was also small (Cohen’s d = 0.07). The PSIC behaved similarly with no significant differences between the mean amplitudes of the low-level EMG at different PET CO2 or between isometric and dynamic contractions.
3.2. Responses in a non-respiratory muscle Long silent periods could be evoked in the first dorsal interosseous (FDI) with TMS at lower stimulus intensities (40–70% maximal output) than in the respiratory muscles (70–100% maximal output). Fig. 4 shows there were significant differences in the duration of the silent period based on the type of contraction (F2,14 = 8.861, p = 0.003). Thus, unlike the respiratory muscles, in FDI the silent periods were significantly longer during dynamic than isometric contractions. The duration of the silent period for isometric FDI contractions was 185.6 ms [178.1, 193.2], and this increased to 198.6 ms [191.8, 205.4] with dynamic contractions at 0.2 Hz and to 203.2 ms [193.1, 213.2] at 0.4 Hz. No significant differences (F2,14 = 1.836, p = 0.20) were observed in the mean amplitude of the low-level EMG during the silent period when dynamic contractions at 0.2 Hz (11.3% [10.7, 11.9]) and 0.4 Hz (11.5% [10.7, 12.3]) were compared with the isometric contraction (12.2% [11.5, 12.9]).
Fig. 4. Silent period duration and low-level EMG amplitude for first dorsal interosseous. Group means with 95% confidence intervals and individual subject data of the silent periods evoked by transcranial magnetic stimulation are shown for isometric (filled symbols) and dynamic (unfilled symbols) abductions of the index finger at 10% of maximum. The type of contraction affected the duration of the silent period but not the amplitude. Post-hoc comparisons with Tukey’s HSD show that the silent period was longer for dynamic contractions at 0.2 Hz (p = 0.024) and 0.4 Hz (p = 0.003) than during isometric contractions, as indicated by * .
4. Discussion This study has determined that TMS can suppress EMG in human scalene and parasternal intercostal muscles for longer than 100 ms, which suggests a cortical component to the suppression. While difficult to obtain during eupnoea in most subjects, long and distinct periods of EMG suppression were present at higher inspiratory efforts following high-intensity TMS, with the duration of the suppression being shorter for dynamic compared to isometric contractions. Although low-level EMG during the ‘silent period’ is clearly present, increasing PET CO2 by 27 mmHg from hypocapnia to hypercapnia did not shorten the silent period, despite the increased reflex chemical drive to medullary respiratory centres, nor did PET CO2 affect the amplitude of the low-level EMG during the silent period. Together, these results show that when TMS suppresses voluntary inspiratory drive, a hypercapnic stimulus to respiratory premotoneurones does not independently activate the scalene and parasternal intercostal muscles. 4.1. Why is the silent period not silent in respiratory muscles? To obtain silent periods of greater than 100 ms following TMS over the motor cortex, subjects were required to activate the inspiratory muscles voluntarily above tidal breathing. It is not clear why there would be a contraction threshold for the production of a silent period in scalene and parasternal intercostal muscles. The duration of the silent period in limb muscles is dependent on the intensity of the magnetic stimulus rather than the level of prior muscle activity (Haug et al., 1992; Inghilleri et al., 1993; Säisänen et al., 2008; Taylor et al., 1997), although greater activity after the silent period tends to shorten it. This was controlled for by having subjects react as fast as possible to the TMS pulse by producing a maximal inspiratory effort. One possible explanation is that the low-level EMG during the silent period in the scalenes, approximately 10% of MIP in this study, is not cortically driven, making it difficult to identify any loss in activity due to intracortical inhibition for contractions at or near that intensity. Werhahn et al. (1995) had noted a similar residual level of EMG during the silent period in intramuscular recordings of mentalis and genioglossus, but not sternocleidomastoid, when these muscles were activated voluntarily at 20% of maximum. This feature of respiratory muscles would explain the absence of long silent periods in the previous study by Lefaucheur and Lofaso (2002, ∼60 ms) who used lower contraction strength and stimulus intensities. If the low-level EMG during the silent period is not cortically driven, it also seems unlikely that this residual activity reflects ongoing drive from the respiratory centres in the medulla. The mean amplitude of low-level EMG did not increase with CO2 as would be expected from a reflex increase in chemical drive to the medullary respiratory neurons. The other possibility is that the low-level EMG is sustained by an intraspinal network which is not affected by intracortical inhibition. DiMarco and Kowalski (2011, 2013) have identified a network of propriospinal neurons that, when electrically stimulated, activate inspiratory motoneurones in a pattern that is similar to spontaneous breathing. Thus, while it is difficult to produce cortical silent periods in inspiratory muscles, this is not because of ongoing descending drive from the medulla. Several other factors may prevent the complete suppression of respiratory muscle activity during the silent period. The large between-subject variability in silent period duration in this study is comparable to that observed in the FDI at moderate stimulus intensities (Orth and Rothwell, 2004), which suggests that, even with the Bistim (Magstim Co. Ltd.) at or close to maximal output, the stimulus was close to the threshold for activating the intracortical inhibitory neurons associated with respiratory motor areas.
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Moreover, despite using a stimulus above motor threshold, the residual low-level EMG (∼10% MIP) during the silent period may indicate that this stimulus did not recruit a significant proportion of intracortical GABAB inhibitory neurons (McDonnell et al., 2006) to completely suppress descending respiratory drive from the motor cortex. Whether this is due to stimulus parameters or fewer GABAB inhibitory neurons in the motor cortex is not clear. Petersen et al. (2011) found that it is possible to suppress scalene muscle activity below 10% of MIP during a voluntary inspiratory effort by preferentially targeting GABAA receptors with TMS delivered at below resting motor threshold (Ilic´ et al., 2002). In comparison to inspiratory muscles, using a magnetic stimulus above motor threshold in the current study reduced the mean amplitude of low-level EMG in FDI to ∼1% of maximum during the silent period for contractions at 10% of maximum. Further investigations using paired-pulse TMS protocols to assess cortical excitability are needed to understand the lack of complete suppression of scalene and parasternal intercostal EMGs during the silent period. However, the conclusions in this study remain valid since changes in cortical excitability would not affect the reflex medullary drive to the inspiratory motoneurones with changes in arterial CO2 .
4.2. Cortical silent periods in respiratory muscles While changes in PET CO2 did not affect the duration of the silent period, dynamic voluntary inspiratory efforts shortened periods of EMG suppression in the scalenes more than during isometric efforts. Although the amplitude of low-level EMG was not affected, this difference due to contraction type is consistent with the reflex facilitation of motoneurones by muscle spindles that is believed to contribute to the low-level EMG during the silent period. This has been described for lengthening contractions of the elbow flexors (Butler et al., 2012) and observed in the distal muscles of the upper and lower limbs (Holmgren et al., 1990; Sammut et al., 1995; Wilson et al., 1995). During dynamic inspiration, the scalenes contract and shorten to elevate the rib cage. Since TMS was delivered at lung volumes above FRC in these contractions, the higher intrathoracic pressure relative to atmospheric pressure at the onset of EMG suppression in the silent period would cause air to be expelled passively from the lungs, lowering the rib cage and stretching the scalenes. Any spindle induced reflex facilitation would only appear towards the end of the EMG suppression, i.e. after the spinal component of the silent period (Fuhr et al., 1991; Inghilleri et al., 1993; Uncini et al., 1993; Ziemann et al., 1993), resulting in a shortened period of EMG suppression. Presumably, this spindle-induced reflex facilitation during the silent period also occurs for isometric inspiratory efforts, albeit to a lesser extent as lung volume was held constant at FRC where intrathoracic and atmospheric pressures are equal when the respiratory muscles are relaxed. A contraction-type effect was also observed for EMG suppression produced in scalenes by low-intensity TMS at sub-motor threshold where the duration of suppression was shorter for dynamic compared to isometric inspiratory efforts (Petersen et al., 2011). For FDI, silent periods were of longer duration in the dynamic rather than the isometric tasks. This effect is opposite to that predicted by a spindle-induced reflex facilitation as the muscle lengthens during the silent period. Whether this differential effect between the FDI and scalenes is a real phenomenon is difficult to determine from the present study. However, it seems more plausible that the lack of shortening of the silent period during dynamic contractions of the FDI is due to the low stiffness of the elastic string which may not have recoiled fast enough to produce a spindle-induced reflex facilitation rather than being a musclespecific effect.
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4.3. Voluntary control of breathing Our main finding was that hypercapnia did not affect the duration of the silent period or amplitude of low-level EMG in the silent period during voluntary inspiratory efforts. We postulated that if the cortical and medullary drives to the muscles were independent then a CO2 -driven increase in medullary drive would still activate the motoneurones when cortical drive was suppressed with TMS. Thus, we expected the silent period during hypercapnia to be of shorter duration and show more low-level EMG than during hypocapnia. This lack of a CO2 -driven effect on the silent period indicates that, despite doubling ventilation, the reflex increase in chemical drive to the medulla when PET CO2 was elevated by 13 mmHg did not reach the motoneurones during voluntary breaths. The lack of a differential effect of CO2 on the silent period can be interpreted in two ways. Either the medullary drive was inhibited by the voluntary activation of the scalene and parasternal intercostal muscles or TMS activated an inhibitory pathway to the respiratory neurons in the same way as a breath hold (Orem, 1989; Orem and Netick, 1986). Voluntary control of a motor system that is usually regulated automatically, such as breathing, may have inhibited or gated task-dependent bulbospinal pathways to the motoneurones. This behaviour has been observed in the balance system where postural muscles also receive both cortical and subcortical inputs. The reflex muscle response to electrical vestibular stimulation was absent when the plantarflexors were activated voluntarily to perform a motor task that was equivalent to standing (Fitzpatrick et al., 1994). The alternative explanation of a TMSinduced inhibition of medullary drive would require corticobulbar projections to the medulla that operate with a time constant similar to or longer than the suppression of cortical drive such that no difference in silent period was detected. These two processes are not mutually exclusive, but both are consistent with cortical drive that bypasses the medulla, as was suggested by Corfield et al. (1998). Even if only a portion of the cortical drive went via the medulla, so that this portion of descending drive was not inhibited or gated, we would still expect hypercapnia to decrease the duration of the silent period or increase the low-level EMG amplitude in it. A limitation of this study was the inability to evoke a visible silent period in the scalene and parasternal intercostal muscles during spontaneous breaths in hypercapnia. A direct comparison of the silent period between spontaneous breaths and matched voluntary activation of the respiratory muscles during hypocapnia would have provided a clearer indication of the interaction between the descending cortical and medullary drives. Nevertheless, our results indicate that, despite the increased chemo-reflex input to the medulla present in hypercapnia, the change in reflex drive does not reach the inspiratory motoneurones during the silent period to independently activate the inspiratory muscles when taking a voluntary breath. Acknowledgement This work was funded by the National Health and Medical Research Council (of Australia). References Bassal, M., Bianchi, A.L., 1982. Inspiratory onset or termination induced by electrical stimulation of the brain. Respir. Physiol. 50, 23–40. Butler, J.E., Petersen, N.C., Herbert, R.D., Gandevia, S.C., Taylor, J.L., 2012. Origin of the low-level EMG during the silent period following transcranial magnetic stimulation. Clin. Neurophysiol. 123, 1409–1414. Corfield, D.R., Murphy, K., Guz, A., 1998. Does the motor cortical control of the diaphragm ‘bypass’ the brain stem respiratory centres in man? Respir. Physiol. 114, 109–117.
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