Transcranial magnetic stimulation reduces masseter motoneuron pool excitability throughout the cortical silent period

Clinical Neurophysiology 119 (2008) 1119–1129 www.elsevier.com/locate/clinph

Transcranial magnetic stimulation reduces masseter motoneuron pool excitability throughout the cortical silent period Paul F. Sowman, Stanley C. Flavel, Christie L. McShane, Timothy S. Miles, Michael A. Nordstrom * Discipline of Physiology, School of Molecular & Biomedical Sciences, Research Centre for Human Movement Control, The University of Adelaide, Adelaide SA 5005, Australia Accepted 21 December 2007 Available online 4 March 2008

Abstract Objective: To evaluate the time-course of changes in masseter motoneuron pool excitability following transcranial magnetic stimulation of motor cortex, and relate this to the duration of the masseter cortical silent period (CSP). Methods: Surface EMG was recorded bilaterally from masseter and digastric muscles in 13 subjects. Focal TMS was applied at 1.3 active motor threshold (AMT) to motor cortex of one hemisphere to elicit a muscle evoked potential (MEP) and silent period bilaterally in masseter as subjects maintained an isometric bite at 10% maximum. With jaw muscles relaxed, a servo-controlled stretcher evoked a stretch reflex in masseter which was conditioned by TMS (1.3 AMT) at 14 different conditioning–testing intervals. There were 20 trials at each interval, in random order. TMS evoked no MEP in resting masseter, but often produced a small MEP in digastric. Results: Mean (±SE) masseter CSP was 67 ± 3 ms. The masseter stretch reflex was facilitated when stretch preceded TMS by 8 and 10 ms, which we attribute to spatial summation of corticobulbar and Ia-afferent excitatory inputs to masseter. Masseter stretch reflex amplitude was reduced when TMS was given up to 75 ms before stretch, and for up to 2 ms afterwards. Conclusions: We conclude that descending corticobulbar activity evoked by TMS acts bilaterally on brainstem interneurons that either inhibit masseter motoneurons or increase pre-synaptic inhibition of Ia-afferent terminals for up to 75 ms after TMS. The reduction of masseter motoneuron pool excitability following TMS has a similar time-course to the CSP. Significance: In contrast to the situation for spinal and facial (CN VII) muscles, the masseter CSP appears to have no component that can be attributed exclusively to cortical mechanisms. Abnormalities in the masseter cortical silent period observed in neurological conditions may be due to pathophysiological changes at cortical and/or sub-cortical levels. Ó 2008 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved. Keywords: Silent period; GABA; Mastication; Trigeminal; Corticobulbar; Stretch reflex

1. Introduction The cortical silent period (CSP) is a period of reduced electromyographic (EMG) activity induced in a voluntarily active muscle by transcranial magnetic stimulation (TMS) of the motor cortex. The suppression of ongoing activity can last up to 300 ms in limb muscles (Inghilleri et al., *

Corresponding author. Tel.: +61 8 8303 4567; fax: +61 8 8303 3356. E-mail address: [email protected] (M.A. Nordstrom).

1993) and is due to a combination of factors operating at the level of the spinal cord (and brainstem for cranial muscles) that affect motoneuron excitability, as well as cortical inhibitory processes affecting the corticofugal descending drive. The contribution of changes in motoneuron pool excitability to the CSP is assessed by testing reflex responses at intervals during the cortical silent period after TMS. Responses differ between muscles. In upper limb muscles, H-reflexes are markedly suppressed early in the silent period following TMS and return to control levels before the end of the CSP (Fuhr et al., 1991; Cantello

1388-2457/$34.00 Ó 2008 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.clinph.2007.12.019

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et al., 1992; Uncini et al., 1993). In resting soleus, Hreflexes are unchanged or facilitated throughout the CSP (Roick et al., 1993; Ziemann et al., 1993). Cortical inhibitory mechanisms contribute to the CSP and predominate in the later component when segmental reflex excitability is normal in spinal muscles (Inghilleri et al., 1993; Roick et al., 1993; von Giesen et al., 1994; Chen et al., 1999). In facial muscles (CN VII), the R1 component of blink-like reflexes induced by cutaneous trigeminal stimulation is not reduced during the CSP, which suggests a largely cortical origin for the CSP in these muscles (Leis et al., 1993; Cruccu et al., 1997). GABAB receptors are thought to be involved in the cortical inhibition of the CSP (Werhahn et al., 1999). The cortical silent period is abnormal in a number of neurological conditions (reviewed by Abbruzzese and Trompetto, 2002; Cantello, 2002; Curra` et al., 2002), which is believed to reflect pathophysiological changes in cortical inhibition based on the evidence cited above. TMS evokes a CSP in the trigeminally innervated masseter muscle (Cruccu et al., 1989, 1997; Desiato et al., 2002; Pearce et al., 2003; Jaberzadeh et al., 2008). The corticobulbar projection to the trigeminal motor pools is bilateral, with stronger excitatory effects evoked contralaterally following focal TMS (reviewed by Nordstrom, 2007), but with symmetrical CSPs in masseter muscles on both sides (Jaberzadeh et al., 2008). The masseter CSP is shorter than those in hand and some other cranial muscles at comparable intensities of focal TMS (Jaberzadeh et al., 2008). This may reflect a relatively weaker influence of cortical inhibitory systems on corticobulbar neurons of the trigeminal motor system than corticospinal neurons. This interpretation is not clear at present, however, because the relative importance of cortical and sub-cortical factors in the trigeminal CSP has not been directly tested. The muscle-specific differences in the effects of TMS on segmental reflexes (vide supra), and differences in reflex and descending control of trigeminal motoneurons compared with spinal and other cranial systems (Luschei and Goldberg, 1981), mean that the results from other systems cannot be simply extrapolated to the trigeminal system. The aim of the present study was to assess masseter motoneuron pool excitability during the CSP following TMS. Because H-reflexes are technically difficult to elicit in the mandibular nerve, we used a servo-controlled muscle stretcher to evoke a stretch reflex in resting masseter muscles at intervals before and after focal TMS. The timecourse of stretch reflex inhibition and recovery after TMS provides an indication of the component of the masseter CSP that can be attributed to cortical inhibitory mechanisms (Fuhr et al., 1991; Cantello et al., 1992; Uncini et al., 1993). This information is important for interpreting the site of pathophysiologic changes in the trigeminal motor system associated with changes in masseter cortical silent periods, such as its shortening in amyotrophic lateral sclerosis (Desiato et al., 2002). The masseter CSP also has potential application in assessment of pathophysiological

changes in other disorders involving the masticatory muscles, such as cranial dystonias (Curra` et al., 2000), bruxism (Lobbezoo and Naeije, 2001; Lavigne et al., 2003) and temporomandibular dysfunction (Raudino, 1994). 2. Methods A total of 38 volunteers (22 females and 16 males) aged 25.6 ± 1.6 (SD) years was recruited to the study with their informed, written consent. The study was approved by the Human Research Ethics Committee at The University of Adelaide, and procedures were in accordance with the Declaration of Helsinki. All subjects completed a modified version of the TMS Adult Safety Screen Questionnaire (Keel et al., 2001) to ensure that there was no contraindication for TMS. After preliminary testing, 25 subjects were excluded due to either (a) small or absent stretch reflex in one or both resting masseter muscles; or (b) our inability to elicit muscle evoked potentials (MEPs) in active masseter muscles on both sides with focal TMS without direct ipsilateral activation of the trigeminal nerve in the cranial fossa (root MEP; Cruccu et al., 1989). Most subjects were excluded due to the first criterion, which reflects a limitation of the servo-controlled muscle stretcher (vide infra). Data from 13 subjects are reported herein (9 females and 4 males; age 23.0 ± 2.3 years). 2.1. Apparatus and recording Subjects sat upright in a chair with their upper and lower incisor teeth resting comfortably on metal bite plates connected to a servo-controlled electromagnetic device that imposed a controlled displacement of the lower jaw (Miles et al., 1993). We have used this system previously to investigate stretch reflexes in the active masseter muscles (Poliakov and Miles, 1994; Miles et al., 1995). The metal bite plates were coated with a semi-rigid dental impression material (3M ExpressTM, Michigan) individually moulded to each subject’s teeth. This ensured that only the incisor teeth were in contact with the bars during the experiment, and that the position of the upper and lower jaws on the bars remained constant throughout. Force and acceleration were measured from sensors mounted on the lower bite plate, digitized at a sampling rate of 5 kHz and stored on a computer using a CED 1401 laboratory interface (Cambridge Electronic Design, Cambridge, UK). In two subjects a piezoelectric accelerometer was attached to the mental protuberance of the mandible for direct measurement of jaw movements. Adhesive EMG electrodes (DuotrodeÒ, Myo-Tronics Inc, Western Australia, 2 cm inter-electrode distance) were affixed to the skin over right and left masseter muscles in a superior–inferior configuration along the longitudinal axis of the muscle fibres, 0.5 cm posterior to the anterior border of the muscle. Electrodes were also affixed to the skin over both anterior digastric muscles. The inter-electrode impedance was less than 5 kX in all experiments. EMG signals

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were amplified (5000) with a custom-built artefact-suppressing amplifier based on a design reported by Millard et al. (1992). The stimulus artefact was suppressed by reducing the gain of the EMG amplifier to unity from 1 ms before the TMS pulse until up to 2 ms after the TMS. EMG signals were filtered (20–1000 Hz), and digitized at a sampling rate of 5 kHz/ch. Rectified EMG from left masseter was passed through a leaky integrator with a time constant of 100 ms and displayed to the subject on an oscilloscope as feedback of muscle activation level. The masseter EMG level during maximal voluntary contraction (MVC) was determined before the start of the experiment by having the subjects perform three maximal bites of 5-s duration in full occlusion. The highest EMG value in the three trials was taken as the MVC, and this was used to scale masseter EMG target levels for subject feedback. TMS was performed with a Magstim 200 stimulator (Magstim Company Limited, UK) and a focal figure-8 stimulating coil (outer coil diameter 90 mm). Magnetic stimuli were given at the optimal site for eliciting motor evoked potentials (MEPs) in masseter during weak contraction of the masseter muscles. The coil was oriented at an angle of 45° relative to the parasagittal plane, with the handle pointing posteriorly. Care was taken with coil positioning to ensure that TMS did not activate the ipsilateral trigeminal nerve root, as evidenced by the appearance of a short latency (2 ms) response in the ipsilateral masseter EMG at rest (rMEP: Cruccu et al., 1989). TMS was applied to the left motor cortex (M1) in 9 subjects and right M1 in 4 subjects to assess masseter cortical silent periods and the effect of TMS on the masseter stretch reflex. The right hemisphere was used for subjects who displayed an ipsilateral rMEP or small masseter MEP with left hemisphere stimulation. 2.2. Protocol Each experiment consisted of three components. First, a series of jaw displacements of varying amplitude and velocity were applied to establish the stretch parameters needed to elicit a consistent, non-saturated stretch reflex in the resting masseter muscles. This consisted of a series of five ramp stretches (1.0, 1.5, 2.0, 2.5 and 3.0 mm amplitude) with a 20-ms rise-time, in a random sequence. The minimum inter-stimulus interval was 1.5 s. Each stretch amplitude was repeated 20 times, giving 100 trials in total. Subjects sat with their incisor teeth lightly touching the bite bars, and were instructed to keep their masseter muscles relaxed with the aid of visual feedback of EMG. The inter-incisal separation (12 mm) imposed by the thickness of the dental impression and the bite bars resulted in sufficient passive tension for the teeth to remain in contact with the device without the need for active contraction of the masticatory muscles. Masseter EMG was averaged for a 1-s epoch around the time of the stimulus ( 300 ms to +700 ms) and the peak-to-peak amplitude of the stretch reflex measured for the five different stretches. The ramp

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stretch that elicited the largest-amplitude stretch reflex in each subject was used for the remainder of that experiment. In cases where the stretch reflex amplitude plateaued at the higher stretch amplitudes, the lower stretch amplitude in the plateau phase was used. In more than half the subjects tested we were unable to elicit a reliable stretch reflex in the resting masseter muscles on both sides and the experiment was terminated. This was due to a limitation of the rate of muscle stretch that could be delivered by our stretcher, which was designed to test stretch reflexes in active masseter muscles (Poliakov and Miles, 1994; Miles et al., 1995) in which reflexes are facilitated by increased motoneuron pool excitability and elevated fusimotor drive to muscle spindles, rather than the passive muscles in this experiment. TMS active motor threshold (AMT) was assessed for the contralateral masseter during a constant background contraction of 10% MVC, and was defined as the lowest TMS intensity that produced a masseter MEP P 50 lV in amplitude for 3 out of 5 consecutive stimuli. To assess the masseter cortical silent period, focal TMS was applied (10 stimuli at 1.3 AMT, 0.2 s 1) while subjects activated the masseter muscles at 10% MVC (Jaberzadeh et al., 2008). A conditioning–testing protocol was used to examine whether TMS modulated the masseter stretch reflex, and to determine the time-course of this effect in relation to the cortical silent period. TMS (1.3 AMT) was used to condition the stretch reflex (test response) at 14 different conditioning–testing (C–T) intervals. Muscle stretch was delivered after TMS (C–T intervals +2, +10, +25, +50, +75, +100, +150 ms), coincident with TMS (C–T interval 0 ms), and prior to TMS (C–T intervals 50, 10, 8, 6, 4, 2 ms). Two additional conditions were included in the protocol: a TMS pulse (1.3 AMT) in isolation (for stimulus artefact subtraction in the EMG) and a muscle stretch in isolation (for normalisation of reflex size). These 16 conditions were randomly presented in blocks of 50 trials each separated by a minimum of 5 s. Blocks of 50 trials, separated by a 2-min rest period, were repeated until each condition had been tested 20 times (320 trials in total). During the trials, subjects were instructed to keep masseter muscles relaxed with the aid of visual feedback of EMG. 2.3. Data analysis Analyses were conducted off-line from the digitized records. Estimation of the masseter CSP duration was performed by computer using a modified CUSUM method described by Brinkworth and Tu¨rker (2003), and implemented using code written in LabViewTM (National Instruments Corporation, TX, USA). EMG signals were zero-phase filtered (5–500 Hz), full-wave rectified, averaged with respect to TMS (±250 ms epoch), and smoothed with a zero-phase 11-bin moving average filter (ca. 200-Hz low-pass). The CUSUM procedure was performed on the EMG records to identify periods in which the EMG was

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significantly above or below the mean level in the 250-ms epoch preceding the stimulus. The program defined a significant event if the vertical distance between two consecutive CUSUM turning points (EMG crossing the prestimulus average) was more than 100% of the maximum variation in the pre-stimulus epoch (Brinkworth and Tu¨rker, 2003). CSP duration was measured from the TMS pulse until the end of the last significant period of EMG suppression identified in the CUSUM by the program (see also Jaberzadeh et al., 2008). For C–T trials, the masseter and digastric EMG were averaged for 300 ms before and 700 ms after the onset of muscle stretch (or TMS pulse in the no stretch condition). The averaged TMS stimulus artefact was assessed from the TMS-only condition, and subtracted from the averaged EMG records for all other conditions. The peak-to-peak amplitude of the masseter stretch reflex was measured with cursors on the averaged EMG records for each C–T interval. These values were normalised to the stretch reflex amplitude obtained in the stretch-alone trials, and the normalised stretch reflex size was used for statistical analyses. Two-factor repeated measures ANOVA was used to determine the effect of C–T INTERVAL (14 levels) and SIDE (ipsilateral or contralateral to the hemisphere stimulated by TMS) on the size of the normalised stretch reflex. Analyses were conducted with SPSS version 13.0 for Windows (SPSS Inc., Chicago, IL) with the level of statistical significance set at 5%. In two subjects, a masseter stretch reflex was not consistently seen in the masseter muscle on one side. These subjects were excluded from the initial analysis, but included for subsequent analysis once it was clear that SIDE did not affect the results. Mauchly’s W test was computed to check for violations of the sphericity assumption. When Mauchly’s W test was significant, the Greenhouse–Geisser correction was applied. Results of tests adjusted in this way are reported with degrees of freedom rounded to the nearest integer. To identify C–T intervals at which the normalised stretch reflex was either suppressed or facilitated relative to baseline, an a priori linear contrast matrix was incorporated into the ANOVA whereby 13 C–T intervals were compared to a reference C–T interval ( 50 ms, in which the stretch reflex was elicited before TMS was given). To establish that the stretch reflex with a C–T interval of 50 ms was not different to that obtained in stretch-alone trials, the mean amplitudes were compared using a paired t-test. Results are presented as means ± SEM unless otherwise stated.

Table 1 Summary of masseter stretch reflex, MEP and cortical silent period data Side

Stretch reflex amplitude (lV)

Stretch reflex onset latency (ms)

MEP onset latency (ms)

Cortical silent period duration (ms)

Ipsilateral

72 ± 28 n = 11 76 ± 32 n = 13

12.1 ± 0. 7 n = 11 12.9 ± 0.8 n = 13

7.2 ± 0.2 n = 13 7.7 ± 0.3 n = 13

64 ± 4 n = 13 70 ± 6 n = 13

Contralateral

Values are means ± SEM.

3. Results The masseter stretch reflex data, MEP onset latency and CSP durations are summarised for the 13 subjects in Table 1. Two subjects did not have a detectable stretch reflex in the ipsilateral masseter during the C–T trials. Stretch reflex and MEP onset latencies did not differ between sides (paired t-tests, P > 0.05). The mean difference in onset latency of the stretch reflex and MEP was 5 ± 1 ms for the ipsilateral muscle and 5 ± 1 ms for the contralateral muscle, with no significant difference between sides (paired t-test, P = 0.99, n = 11). All subjects had a significant reduction of EMG following TMS during voluntary muscle activation (see Fig. 1A). Masseter CSP duration ranged from 43 to 80 ms in the ipsilateral muscle, and 39 to 116 ms in the contralateral muscle. Mean CSP duration did not differ between sides (Table 1) and averaged 67 ± 3 ms for both muscles. 3.1. Conditioning of the masseter stretch reflex by TMS Fig. 1 shows representative examples from one subject. Fig. 1A shows the CSPs in both weakly contracting masseter muscles. Fig. 1B and C shows the masseter and digastric EMGs, respectively, during the C–T trials (i.e., in resting muscles). The lowermost traces (Fig. 1D) show the average jaw displacement during the ramp stretch, with traces in Fig. 1B–D aligned to the signal triggering the stretcher (time 0). In this subject the masseter CSP duration was 71 ms in the ipsilateral muscle and 116 ms in the contralateral muscle with TMS of 1.3 AMT (Fig. 1A). The stretchevoked responses in the left and right masseter are shown in Fig. 1B, with the uppermost row showing the stretchalone trial, and the remaining rows showing responses with TMS conditioning of the stretch at each of the C–T inter-

" Fig. 1. Representative results from a single subject showing the masseter CSP and stretch and TMS evoked responses in resting masseter and digastric muscles. Data from muscles ipsilateral to focal TMS are shown on the left, contralateral muscles are shown on the right. (A) Average rectified EMG recorded from masseter muscles in response to 20 TMS at 1.3 AMT during a 10% MVC. The vertical dotted line shows the termination of the CSP, and numbers indicate CSP duration in ms. (B) Masseter EMG averaged (n = 20) with respect to stretch delivered at time 0. Time of the stretch with respect to conditioning TMS (C–T interval, ms) is shown on the left of each trace. Positive C–T intervals indicate stretch followed TMS, negative intervals indicate stretch preceded TMS. (C) Averaged EMG from digastric muscles, arranged as for (B). (D) Average displacement of the lower bite bar imposed by the stretcher. Downward deflection indicates jaw-opening. Amplitude calibration bars represent: (A) 25 lV; (B) 250 lV; (C) 250 lV; and (D) 1.5 mm.

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vals indicated. Positive C–T intervals indicate stretch followed TMS, negative intervals indicate stretch preceded

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TMS. The stretch reflex with a C–T interval of 50 ms was similar in size to that seen in the stretch-alone trials.

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At C–T intervals 8 and 10 ms the stretch reflex was larger than control in the contralateral masseter, and this was also the case for the ipsilateral masseter at a C–T interval of 8 ms. The stretch reflex was clearly smaller in both masseter muscles for C–T intervals between 4 ms and +50 ms. The masseter stretch reflex was either facilitated or at control amplitude for C–T intervals +75 ms to +150 ms. As expected, no stretch reflexes were evoked in digastric (Fig. 1C). TMS elicited a small MEP in the resting digastric muscles on both sides, that varied in onset with respect to time 0, corresponding to the timing of the TMS for each C– T interval. Note that TMS did not evoke a MEP in the resting masseter muscles (Fig. 1B); hence the analysis of the stretch reflexes was not complicated by the relative refractoriness of the masseter motoneurons. The data from the 11 subjects (7 females and 4 males; age 23.0 ± 2.3 years) with bilateral stretch reflexes were included in the initial repeated measures ANOVA. The main effect of C–T interval was significant (F3,26 = 6.7, p < 0.001), but there was no significant effect of SIDE (F1,10 = 2.3, P = 0.16) or significant interaction between C–T interval and side (F4,36 = 1.7, P = 0.18). Therefore, the results from ipsilateral and contralateral muscles were equivalent, and the data from the two subjects who had a stretch reflex in contralateral masseter but not ipsilateral masseter were included in a subsequent one-way repeated measures ANOVA with C–T interval as the only factor.

The data from the 13 subjects are summarised in Fig. 2. The main effect of C–T interval was significant in the ANOVA (F3,79 = 10.6, P < 0.001), indicating that the effect of TMS on the masseter stretch reflex was a function of the relative timing of the TMS and the stretch. There was no significant difference in absolute stretch reflex amplitude in the stretch-alone trials and those with C–T interval of 50 ms (paired t-test, p = 0.47, n = 24), indicating that this C–T interval is a valid reference point for comparison of the effect of TMS on the normalised stretch reflex. The a priori defined contrast matrix showed the normalised stretch reflex was significantly different from the reference C–T interval of 50 ms at a number of C–T intervals (Fig. 2). Fig. 2 shows that the normalised masseter stretch reflex was significantly suppressed at all C–T intervals from 2 ms to +75 ms. The largest reduction was 42% at a C– T interval of +25 ms. The C–T intervals producing significant suppression of the masseter stretch reflex fall approximately within the period of the masseter CSP duration induced by TMS in these subjects (Fig. 2). In contrast, C–T intervals of 8 and 10 ms resulted in significant facilitation of the masseter stretch reflex. The presence of a small MEP in the resting digastric muscles in most subjects (e.g., Fig. 1) raised the possibility that a jaw-opening movement followed TMS, and that this altered the stretch imposed on the masseter muscles by the servo-controlled stretcher, for example by removing the lower teeth from contact with the lower bite bar of

Fig. 2. Effect of conditioning TMS at 1.3 T on the amplitude of the masseter stretch reflex. Normalised peak-to-peak amplitude of the masseter stretch reflex is shown for the 14 conditioning–testing (C–T) intervals used in the experiment. Data are means ± SE from 22 muscles in 12 subjects. Positive C–T intervals indicate stretch followed TMS, negative intervals indicate stretch preceded TMS. The shaded area indicates the mean duration of the masseter CSP in these subjects with TMS at 1.3 T. The reference category for testing of a priori defined linear contrasts was the C–T interval of 50 ms, indicated by the solid black bar. Significant differences from this reference value are indicated by asterisks: *p < 0.05; **p < 0.01; ***p < 0.001. The masseter stretch reflex is suppressed following TMS for a period of time that approximates the mean duration of the masseter cortical silent period.

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the stretcher. A sufficiently large jaw-opening movement may have stretched masseter spindles and evoked a stretch reflex in masseter. To investigate these possibilities, we tested two subjects with an accelerometer glued to the skin over the mental protuberance of the mandible to provide a direct measure of mandibular movements. Examples from one subject are shown in Fig. 3. The masseter stretch reflex (Fig. 3A) was facilitated at C–T interval 10 ms, and suppressed at C–T intervals +25 and +50 ms, compared to

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control (C–T interval 50 ms). The digastric (Fig. 3B) shows a small MEP elicited by TMS in three of four corresponding trials. The mandibular acceleration (Fig. 3C) was very similar in each trial, and corresponded well with the displacement of the lower bite bar imposed by the stretcher (Fig. 3D). There was no jaw-opening movement or stretch reflex in masseter time-locked to TMS. Similar results were seen in the other subject tested. We conclude that the presence of a digastric MEP did not produce a jaw-opening movement sufficient to evoke a masseter stretch reflex or interfere with the mandibular displacement imposed by the stretcher. 4. Discussion Focal TMS at 1.3 AMT produced a CSP that was similar in the weakly contracting masseter muscles on both sides, and lasted 67 ms on average. TMS at that intensity did not elicit a MEP in the masseter muscles at rest, but modulated the size of the masseter stretch reflex in a manner that depended on the timing of TMS with respect to muscle stretch. This evidence suggests that the TMS pulse evoked descending activity in the corticobulbar axons that transiently facilitated the motoneuron pool at short latency (C–T intervals 10 to 8 ms), and subsequently suppressed motoneuron pool excitability for up to 75 ms. As this exceeds the average duration of the masseter CSP in these subjects (67 ± 3 ms), we are unable to ascribe any part of the masseter cortical silent period exclusively to cortical mechanisms. 4.1. TMS at 1.3 AMT evoked descending activity in corticobulbar neurons at rest

Fig. 3. Effects of TMS and muscle stretch on jaw kinematics. Data are from a single subject in whom TMS evoked a MEP in resting digastric, and no MEP in resting masseter. (A) Masseter EMG averaged (n = 20) with respect to stretches triggered at time 0 (dashed vertical line). Time of stretch with respect to conditioning TMS delivered to the contralateral hemisphere (C–T interval, ms) is shown on the left of each trace. Positive C–T intervals indicate stretch followed TMS, negative intervals indicate stretch preceded TMS. The masseter stretch reflex was facilitated at C–T interval 10 ms, and suppressed with C–T intervals +25 and +50 ms, compared with control (C–T interval 50 ms). (B) Averaged EMG from the digastric contralateral to TMS, arranged as for (A). The was no stretch reflex in digastric, but 3 of 4 traces show a MEP elicited by TMS. (C) Mandibular acceleration. Downward deflection indicates jaw-opening. (D) Average displacement of the lower bite bar imposed by the stretcher for the four C–T intervals (traces superimposed). Mandibular acceleration was similar for all trials, despite the small MEP in digastric. Calibration bars represent: (A) 100 lV; (B) 100 lV; (C) 10 ms 2; and (D) 2.5 mm.

The TMS intensity used for the CSP measurements and to condition the stretch reflex was 1.3 AMT. At rest, TMS at 1.3 AMT evokes large, multiple descending volleys in the corticospinal pathway and elicits a MEP in hand muscles (Di Lazzaro et al., 1998). We often observed a small MEP in the resting digastric muscles (Figs. 1 and 3), which is consistent with previous findings (Gooden et al., 1999) and is evidence that the corticobulbar projection was activated by TMS at rest (i.e., at least the component directed to digastric). TMS at 1.3 AMT did not produce a MEP in masseter at rest, consistent with previous reports that it is rarely possible to elicit a MEP in resting masseter (Cruccu et al., 1989; Butler et al., 2001). TMS evokes a single descending volley in corticobulbar fibres projecting to masseter (Pearce et al., 2003), rather than the multiple D- and I-wave volleys evoked in corticospinal neurons by TMS which are needed for a resting MEP (Nakamura et al., 1997; Di Lazzaro et al., 1998). This, combined with relatively weak excitatory projections (compared to hand muscles), explains the absence of the resting MEP in masseter with TMS at 1.3 AMT. The facilitation of stretch reflex amplitude at certain C–T intervals (vide

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infra) is indirect evidence that the corticobulbar projection to masseter was activated by TMS at this intensity. 4.2. Facilitation of the masseter stretch reflex amplitude by TMS In the active masseter, focal TMS evoked a MEP with a mean onset latency of 7.4 ms (Table 1), consistent with previous reports of a fast, monosynaptic corticobulbar projection to masseter motoneurons on each side (Pearce et al., 2003). The mean stretch reflex latency in the resting masseter was 12.5 ms (Table 1), which compares with previously reported stretch reflex latencies of 10–12 ms in active masseter muscles with the same stretcher (Poliakov and Miles, 1994). Inspection of the mandibular acceleration records (Fig. 3) revealed there was 6 ms delay between the signal triggering the stretcher and movement of the mandible. When this delay is subtracted, the masseter stretch reflex latency is similar to the H-reflex latency in active masseter (5.5–6.4 ms) reported by Scutter et al. (1997). In the present study, the mean difference in onset latency of TMS- and stretch-evoked responses in masseter was 5 ms. The Ia-afferent volleys arising from muscle stretch and a descending volley in corticobulbar fibres evoked by TMS would therefore arrive coincidently at the alpha motoneurons when TMS was given 5 ms after the stretch. This corresponds to a C–T interval of 5 ms. With C–T intervals of 8 and 10 ms, the stretch reflex was significantly facilitated (Fig. 2). We consider that this is due to activation of the descending corticobulbar projection to masseter by TMS, which at C–T intervals 6, 8 and 10 ms would result in the corticobulbar EPSP occurring in the motoneurons slightly after the Ia-afferent EPSP, and summating with it to recruit more motoneurons to the excitatory response. The sustained ramp stretch would produce repetitive firing of Ia-afferents and a compound EPSP with a sustained depolarisation on the rising phase. This may explain why the reflex is more facilitated at C–T intervals of 10 and 8 ms than 6 ms, as increased time between the two inputs allows greater depolarisation and a larger subliminal fringe of motoneurons facilitated by the Ia-afferent input and brought to threshold by the corticobulbar EPSP. 4.3. Reduction of the masseter stretch reflex amplitude by TMS The masseter stretch reflex was reduced in amplitude with C–T intervals 2 ms to +75 ms (Fig. 2). These intervals correspond to trials in which the descending corticobulbar activity evoked by TMS arrived at the brainstem level before the Ia-afferent volley evoked by the stretch, and thereby suppressed the stretch reflex. As the corticobulbar volley evoked by TMS did not elicit an action potential in resting masseter motoneurons, several mechanisms that may potentially contribute to reduced motoneuron pool excitability following TMS can be excluded. These include refractoriness of the motoneuron due to

the after-hyperpolarization following an action potential, and recurrent inhibition of motoneurons via axon collateral branches onto Renshaw cells. In fact, both animal and human evidence suggest the trigeminal motoneurons lack recurrent inhibition (Kidokoro et al., 1968; Iwata et al., 1995; Kamogawa et al., 1998; Tu¨rker et al., 2007). Several other proposed mechanisms for reduced motoneuron pool excitability following TMS involve reflexes from sensory afferents activated by the muscle twitch. These include a reduction in tonic Ia excitatory drive due to a pause in homonymous spindle Ia-afferent discharge as the muscle shortens, an increase in Ia-inhibitory interneuron activity due to stretch of spindles in the antagonists, and inhibition from Golgi tendon organ Ib afferents. These factors did not play a role in the present study as TMS did not evoke a contraction in the resting masseter muscles. The lack of muscle spindles and stretch reflexes in the antagonist digastric, and absence of reciprocal Ia-afferent inhibition in the trigeminal motor system (Luschei and Goldberg, 1981) further eliminates the possibility of spindle-mediated reflexes from the antagonists. Adult jaw muscles have few if any Golgi tendon organs, and lack Ib-mediated inhibition (Luschei and Goldberg, 1981). TMS commonly evoked a MEP in resting digastric muscles (Figs. 1 and 3), which posed two potential problems. If this elicited jaw-opening it may have stretched the masseters (evoking a masseteric stretch reflex), or displaced the lower teeth from the bite bars and reduced the effectiveness of the muscle stretcher. Analysis of mandibular acceleration and EMG records (Fig. 3) showed that the small digastric MEP did not produce jaw-opening sufficient to evoke a masseter stretch reflex in the inactive muscle, or interfere with the jaw movements imposed by the stretcher. By exclusion, the reduction of the stretch reflex amplitude at C–T intervals 2 to +75 ms is therefore most likely to be due to effects mediated at the brainstem level by the corticobulbar fibres that result in increased pre-synaptic inhibition of Ia-afferent terminals onto motoneurons, and/or post-synaptic inhibition of these motoneurons. At rest, Ia-afferent terminals in the spinal cord are subject to tonic pre-synaptic inhibition via axo-axonal synapses from segmental interneurons. During selective voluntary activation pre-synaptic inhibition is reduced for Ia terminals of agonist motoneurons, and increased at Ia terminals onto motoneurons of muscles not involved in the movement (Hultborn et al., 1987). This effect is under the influence of descending commands as it precedes movement onset (Nielsen and Kagamihara, 1993). Activation of the corticospinal pathway with TMS reduces pre-synaptic inhibition of Ia-afferent inputs to soleus (Iles, 1996; Meunier and Pierrot-Deseilligny, 1998) and increases pre-synaptic inhibition of Ia-afferent terminals to wrist flexor motoneurons (Meunier and Pierrot-Deseilligny, 1998). There are no reports of corticobulbar control of pre-synaptic inhibition of Ia-afferent terminals in the trigeminal motor system. The organization of stretch reflexes is different in the trigeminal motor system compared with the limbs, with no stretch reflexes

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in the antagonist (digastric), and no Ia-mediated reciprocal inhibition (Luschei and Goldberg, 1981). Although there is no direct supporting evidence, it is possible that the TMS-evoked corticobulbar volley stimulated brainstem interneurons to induce a prolonged increase in pre-synaptic inhibition of Ia-afferent terminals onto masseter motoneurons, thereby suppressing the masseter stretch reflex throughout the C–T intervals 2 to +75 ms. It is worth noting here that the subjects included in this study were selected in part by a low threshold for eliciting a stretch reflex in resting masseter. If this reflects higher fusimotor tone at rest, masseter motoneuron pool excitability may be more susceptible to increased pre-synaptic inhibition of Ia-afferent terminals in these subjects than in the excluded subjects. If TMS suppresses masseter motoneuron pool excitability by modulating pre-synaptic inhibition it may be less effective in other subjects. Corticospinal fibres produce di- or oligosynaptic inhibition of spinal motoneurons via projections onto segmental interneurons (Jankowska et al., 1976). Corticobulbar fibres induce a similar inhibition in trigeminal motoneurons. The most common response to low-intensity microstimulation of pre-central face area of cortex in rhesus monkeys is jawopening, with bilateral inhibition of jaw-closer muscles (Clark and Luschei, 1974). This begins at short latencies (7 ms) with time for only a few intervening synapses (Hoffman and Luschei, 1980). In the rat, cortical stimulation induces inhibitory post-synaptic potentials lasting about 10 ms in masseter motoneurons, with an estimated time for one synaptic relay (Ohta, 1984). Therefore, the suppression of the masseter stretch reflex by the descending corticobulbar volley could be due to interneuron-mediated, post-synaptic inhibition of masseter motoneurons. The identity of the inhibitory interneuron responsible for this in the trigeminal motor system is not known. For limb muscles, the Ia-inhibitory interneuron mediates di-synaptic inhibition of motoneurons following corticospinal activation (Jankowska et al., 1976), however this class of interneuron is not found in the trigeminal system (Luschei and Goldberg, 1981). 4.4. Contribution of cortical and sub-cortical mechanisms to the masseter CSP While we are unable to identify the precise mechanism for the inhibition of the masseter stretch reflex by TMS, we have demonstrated for the first time a reduction of human trigeminal motoneuron pool reflex excitability following TMS, and the time-course of this effect. The timecourse of stretch reflex suppression approximately matched the duration of the masseter CSP induced by TMS during voluntary activation (Table 1 and Fig. 2). Unlike the situation in other cranial and spinal muscles (see Section 1), we are therefore unable to identify a component of the masseter CSP that may be attributed exclusively to cortical mechanisms. Our data do not preclude a contribution to the masseter CSP from cortical inhibition. The trigeminal system exhib-

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its short-interval intracortical inhibition to paired-pulse TMS with similar features to limb muscles (Jaberzadeh et al., 2007), and this is thought to be mediated by GABAA systems in motor cortex (Chen, 2004). The CSP is thought to reflect GABAB inhibition in motor cortex (Werhahn et al., 1999), although the pharmacological evidence is not definitive (Boroojerdi, 2002). In the present study the maximum reduction of the masseter stretch reflex following TMS was 42% at +25 ms. This is much weaker than the effect of TMS on H-reflexes in upper limb muscles, which are reduced by 70–90% for up to 50 ms after the MEP (Fuhr et al., 1991; Uncini et al., 1993). The masseter CSP with focal TMS over M1 can last around 100 ms (Jaberzadeh et al., 2008), while deep brain stimulation of the corticobulbar tract in the internal capsule of Parkinson’s disease patients produced a silent period in masseter of around 45 ms duration (Costa et al., 2007) with a similar size muscle evoked potential in the two studies. TMS that is sub-threshold for activation of descending corticobulbar fibres can elicit a masseter CSP (Jaberzadeh et al., 2008). These observations suggest that cortical mechanisms contribute to the masseter CSP. The recovery of stretch reflex amplitude at C–T intervals +100 and +150 ms indicates that motoneuron pool excitability has returned to control levels at these time-points after TMS, as is the case in other muscles. The relatively short masseter CSP compared to hand muscles and some other cranial muscles (Jaberzadeh et al., 2008) therefore probably reflects reduced strength of cortical inhibition in the trigeminal motor system. The CSP has been used to assess pathophysiological changes in patients with a number of neurological conditions (reviewed by Abbruzzese and Trompetto, 2002; Cantello, 2002; Curra` et al., 2002), including some affecting cranial muscles (Curra` et al., 2000; Desiato et al., 2002). Because the late component of the CSP can be attributed to cortical mechanisms in limb and facial (CN VII) muscles, abnormal CSPs are interpreted as indicating pathophysiological changes at the level of the cortex. Our study shows that changes in the masseter cortical silent period, as for example in amyotrophic lateral sclerosis (Desiato et al., 2002), may be due to pathophysiological changes at cortical and/or sub-cortical levels. Acknowledgment This work was funded by a project Grant (349451) from the NHMRC of Australia. References Abbruzzese G, Trompetto C. Clinical and research methods for evaluating cortical excitability. J Clin Neurophysiol 2002;19(4):307–21. Boroojerdi B. Pharmacologic influences on TMS effects. J Clin Neurophysiol 2002;19(4):255–71. Brinkworth RS, Tu¨rker KS. A method for quantifying reflex responses from intra-muscular and surface electromyogram. J Neurosci Methods 2003;122:179–93.

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