Transcranial magnetic stimulation induced early silent period and rebound activity re-examined.

Despite being widely studied, the underlying mechanisms of transcranial magnetic brain stimulation (TMS) induced motor evoked potential (MEP), early cortical silent period (CSP) and rebound activity are not fully understood. Our aim is to better characterize these phenomena by combining various analysis tools on firing motor units. Responses of 29 tibialis anterior (TA) and 8 abductor pollicis brevis (APB) motor units to TMS pulses were studied using discharge rate and probability-based tools to illustrate the profile of the synaptic potentials as they develop on motoneurons in 24 healthy volunteers. According to probability-based methods, TMS pulse produces a short-latency MEP which is immediately followed by CSP that terminates at rebound activity. Discharge rate analysis, however, revealed not three, but just two events with distinct time courses; a long-lasting excitatory period (71.2 ± 9.0 ms for TA and 42.1 ± 11.2 ms for APB) and a long-latency inhibitory period with duration of 57.9 ± 9.5 ms for TA and 67.3 ± 13.8 ms for APB. We propose that part of the CSP may relate to the falling phase of net excitatory postsynaptic potential induced by TMS. Rebound activity, on the other hand, may represent tendon organ inhibition induced by MEP activated soleus contraction and/or long-latency intracortical inhibition. Due to generation of field potentials when high intensity TMS is used, this study is limited to investigate the events evoked by low intensity TMS only and does not provide information about later parts of much longer CSPs induced by high intensity TMS. Adding discharge rate analysis contributes to obtain a more accurate picture about the characteristics of TMS-induced events. These results have implications for interpreting motor responses following TMS for diagnosis and overseeing recovery from various neurological conditions.

Introduction

Transcranial magnetic stimulation (TMS) of motor cortex brings about a short-latency excitatory response in skeletal muscles known as motor-evoked potential (MEP) [1,2]. When elicited during voluntary contraction, MEP is followed by a period of reduced electrical activity or silence, known as cortical silent period (CSP). The duration of this silent period is dependent on the stimulus intensity and can last as long as 300 ms [3-5]. Both intracortical and spinal mechanisms have been suggested to contribute to this silent period. The initial portion has been suggested to be due to a contribution from spinal mechanisms involving changes in motoneuron excitability, afferent input and recurrent inhibition [6,7]. The remainder of the silent period has been suggested to be due to inhibition of motor cortical output mediated by GABAB receptors (for review see Ziemann [8]). Based on these claims, the duration of the CSP has been claimed to reflect strength of inhibition within cortex [7,9,10] and MEP to indicate net excitability of corticospinal pathway [11]. TMS induced MEP and CSP, therefore, are proposed as indicators for evaluating functions of central nervous system in plasticity [12,13] and various neurological diseases in clinical applications including, amyotrophic lateral sclerosis (ALS), Parkinson’s disease and stroke [14-17].

Precisely because the size of MEP and duration of CSP are being used in the literature as indicators reflecting excitability of cortical circuitries, it is imperative that we know for sure whether or not they reflect real phenomena. For MEPs and CSPs values to be useful, it is imperative that the methods used to record and analyze them must be valid. The classical methods, averaged surface EMG (SEMG) and peristimulus time histogram (PSTH), used to study these phenomena have been assumed to be valid [18]. Using these analysis methods, it is commonly assumed that peaks and troughs in an averaged response to a stimulus indicate synaptic excitation and inhibition, respectively (reviewed in Türker and Powers [19]).

However, it has long been recognized that peaks and troughs in response to an afferent stimulus can reflect not only direct synaptic effects, but also secondary effects arising from the discharge statistics of the pre- and postsynaptic cells [20]. Direct synaptic effects on spike probability lead to subsequent changes in probability from phase advanced or delayed spikes (count-related errors), followed by secondary and tertiary peaks and troughs due to synchronization of the spikes in relation to the stimulus (i.e., the synchronization-related errors) [19]. It has been shown in regularly discharging motoneurons in brain slices that the probability-based methods (averaged rectified SEMG and PSTH) contain significant errors for indicating underlying synaptic potentials [21]. The errors in estimation using probability-based analyses, however, have been shown to be minimized if discharge rate analysis, peristimulus frequencygram (PSF), is also used on the same data [19]. This is since the PSF is made up of superimposition of individual discharge rate points and these points do not add onto one other. Therefore, PSF does not generate count and synchronization-related errors as shown to be embedded in the classical probability-based methods [19,21].

In our previous work, we used threshold TMS intensity to study MEP and CSP in a hand [22] and a leg muscle [23] and analyzed the data using both probability and discharge-rate based analyses methods. Using stimulus intensities around MEP threshold, both of our previous studies showed increased rate of single motor unit (SMU) discharge following MEP lasting for tens of milliseconds [22,23]. Similarly, TMS pulses induced long-lasting facilitation in alert non-human primates [24] and cat primary visual cortex [25] using discharge rate-based analysis methods.

The current study was, therefore, planned to further characterize the properties of not only the CSP but also the MEP and rebound activity using suprathreshold TMS at varying intensities. We will be questioning whether CSP may include a long lasting excitatory period due to increased rate of SMU discharge as reported [22,23]. Therefore, we aimed to closely examine the motor unit discharge characteristics to better understand the synaptic potentials evoked by TMS in tibialis anterior (TA) and abductor pollicis brevis (APB) motor units. We hypothesize that the CSP may not reflect an inhibitory postsynaptic potential (IPSP) only but include a period of net excitatory postsynaptic potential (EPSP), induced by single-pulse magnetic stimulation of the motor cortex.

Materials and methods

The Human Ethics Committee of Koç University approved the experimental procedure (2017.124.IRB2.038). Experiments were performed in the neurophysiology laboratory of Koç University on subjects who signed informed consent forms. Total of 24 subjects (14 male and 10 female) participated in this study. Subjects were in the 18–30 age groups, had no known neuromuscular disease, brain injury or nervous system related disorder, had normal body habitus and were not regularly using prescribed medication. The experiments were performed on TA muscle of right leg (in 20 subjects) or APB muscle of the right hand (in 4 subjects).

Recording configurations and subject preparation

Software Spike2 7.20 (Cambridge Electronic Design, England) was used to perform data acquisition and offline analyses. CED 1902 Quad System MKIII amplifier and CED 3601 Power 1401 MKII DAC were used for recording. To record the activity of the muscle, SEMG and intramuscular EMG were used. Two of the standard SEMG electrodes (Ag/AgCl) were placed on either muscle after rubbing with sandpaper, cleaning with alcohol and applying electrode gel to reduce the impedance. The electrodes were placed 4 cm apart for optimal signal recognition for TA muscle [26] but they were 2 cm apart for APB muscle due to its smaller size. In addition, tip-active Teflon insulated silver fine-wire electrodes (75 μm in core diameter; Medwire, USA) were used as intramuscular EMG electrodes. Those sterile bipolar wire electrodes were placed in the muscles via 25 G surgical needles in between two SEMG electrodes. After insertion, the needle was removed immediately by leaving a pair of fish-hooked fine wires inside the muscle. Sterile lip clip was used as a ground electrode in all trials for both SEMG and intramuscular EMG [27]. Both SEMG and intramuscular EMG recorded at a sampling frequency of 20,000 Hz. The recordings were filtered with a cut-off frequency of 20–10,000 Hz for SEMG and 200–10,000 Hz for intramuscular EMG.

Isometric force during dorsiflexion of the right foot was measured using a linear strain gauge (Model LC1205-K020, A & D Co. Ltd., Tokyo, Japan: linear to 196 N). The foot was restrained, and subjects dorsiflexed the foot against a plate that was positioned parallel to the base of the foot. Force signals were amplified (x 1,000), filtered (DC-100 Hz), and sampled at 2,000 Hz using the same data acquisition system.

Determination of the optimum stimulation configurations

Each subject sat comfortably on a chair. The experiment began with three brief (5 seconds) maximal voluntary isometric contractions. Maximal efforts were separated by approximately 60 seconds of rest to avoid fatigue. Then, subjects performed isometric contraction that resulted in the firing of one or two SMUs which were easily recognizable (). A transcranial magnetic stimulator (Magstim 2002, Magstim Co., Whitland, UK) with double cone coil electrode (110mm Double Cone Coil) was used to stimulate the corresponding region of the left hemisphere for right TA or figure of eight coil (D70mm Alpha Coil) for right APB muscle. MEPs were measured as the peak-to-peak amplitude of non-rectified recordings for each trial to determine optimum stimulation region. To find that point, we located the motor cortex according to the center of the head which was determined using the midpoint of the nasion-inion line. For APB, the stimulation site on the head was found using suprathreshold stimuli around the C3 region according to the international 10–20 system [28]. The figure-of-eight-shaped coil was oriented 45-degrees relative to the nasion-inion line compared to midpoint. On the other hand, we placed the double cone coil around the midpoint of the nasion-inion line, but slightly angled in line with the C3 region, to stimulate the deeper side of the left motor cortex which is responsible for innervation of the right TA muscle.

A sample TA recording from a subject.

(A) Top trace is a typical intramuscular EMG recording where each line represents SMU potential. Figure below is the blown-up version of the intramuscular EMG where asterisk (*) shows SMU potentials. MEP and the CSP are indicated with horizontal arrows. (B) The figure above is the simultaneously recorded SEMG. Bottom figure is the closer view of the SEMG where MEP and the CSP are displayed. Higher intensity stimuli evoked complex field potentials at MEP period in intramuscular EMG recordings that made discrimination of SMU spikes very difficult.

The coil was positioned over the corresponding motor area of the left hemisphere as described. Single pulse TMS was delivered during weak isometric contraction for both muscles, separately. Initially, stimuli were delivered randomly between 4 and 6 seconds and at an intensity of approximately 150% of active motor threshold. The active motor threshold was defined as the minimum stimulus intensity at which 5 out of 10 consecutive stimuli evoke MEP of at least 100 μV in amplitude during weak muscle contraction in SEMG (). Then, the optimum location was marked and used throughout the experimental protocol.

Experimental design

We performed several recording sessions per subject using suprathreshold TMS intensities. After deciding the optimal point, subjects performed weak muscle contraction throughout stimulation period in all sessions. The desired voluntary contraction intensity was determined when only one or two SMUs were active and at least one of them could be identified by its amplitude (see ). This information was provided to subjects as visual feedback to help them fire these low threshold SMUs regularly.

Stimulation

Stimulus intensity was determined according to the real-time triggering of SMU channel during on-line recording. TMS-triggered averaging window was monitored for the SMU channel where the number of occurrences of SMU potentials could be visualized during MEP period. For that purpose, simultaneous superimposition of the traces in the SMU channel was updated after each stimulus delivered and was determined. Various stimulus intensities that induced between 10 and 50 SMU potentials (at the time period where MEP was expected to occur) out of 100 TMS were used. This method was used to determine optimal stimulation intensity in order to track the full profile of the synaptic potentials. These intensities corresponded to an average strength of 33.56 ± 1.37% and 38.13 ± 1.17% (mean ± SEM) in terms of the maximum stimulator output for TA and APB, respectively.

Force recording and sham stimulation

Also, 4 subjects (1 female and 3 male) were recruited for twitch force recordings and sham TMS protocol for TA muscle using similar procedure. Sham TMS was applied using a homemade placebo coil that is similar in operation to the normal coil. The placebo coil provides minimal scalp sensation and an auditory click similar to that made by the normal coil, but without stimulating the cortex. The purpose for repeating the protocol with sham TMS was to determine whether other factors, such as the auditory click of the stimulator, contribute to the alteration of voluntary EMG observed after suprathreshold TMS. In addition to sham TMS, normal TMS were applied to these 4 subjects while twitch was recorded in order to determine the time course of synaptic potentials to compare with the muscle force generation time. We detected a visible twitch in 4 experiments (average twitch amplitude was; 0.93± 0.21% MVC).

Stimulus and unit numbers

Low intensity, suprathreshold stimuli of 541 ± 19 for TA and 320 ± 32 for APB were delivered to the motor cortex at each session. A total of 29 TA SMUs and 8 APB SMUs were recorded from 16 and 4 subjects respectively, using intramuscular EMG electrodes. For TA, only one SMU from each of 9 subjects were extracted. However, some other participants (7 out of 16) could clearly evoke different SMUs in each of the recording sessions in TA muscle. For the APB experiments, we analyzed two SMUs from each of the subjects (8 units from 4 different subjects in total).

Data processing

To build both PSF and PSTH, electrical activity from the intramuscular fine wire electrodes was displayed and a shape of an individual motor unit action potential defined as a template in the Spike2 program. During the experiment and during off-line analysis, any spike whose shape matched this pre-established template, generated acceptance pulses in the program. The acceptance pulses from the discriminated units were used to construct PSTHs and PSFs around the time of stimulation using scripts of PSTH and PSF in Spike2. Basically, acceptance pulses around stimuli were placed into time bins to obtain PSTH. PSF was obtained by superimposing the instantaneous discharge rate values around the stimulus. In short, while PSTH is a histogram indicating the timing of occurrence of spikes against the stimulus, PSF is made up of superimposition of the instantaneous discharge rates of a selected unit around the time of the stimulus [29].

PSTH and SEMG records make significant errors in estimating synaptic potentials as stimulus-induced synchronization of spikes generate secondary and tertiary peaks and trough related to autocorrelation function of the synchronous spikes rather than genuine synaptic events. Each discharge rate that make up of PSF record however represents an independent data point which indicates the membrane excitability at that time. Therefore, PSF does not include synchronous events and hence avoids the drawbacks of PSTH and SEMG [19].

Peak-to-peak amplitude and latency of MEPs were determined. For sustained contraction, SEMG analysis involved extraction of a period (-200 ms to +200 ms) from around each stimulus and averaging the signals. Intramuscular EMG analysis involved identification of SMU potentials using Spike2 algorithms. Analysis of each SMU involved extraction of a defined period from around each stimulus (-600 ms to +600 ms) followed by construction of a PSTH and PSF. Both PSF and PSTH were built with 0.1 ms binwidths.

Cumulative sums (CUSUMs) were then constructed from PSTH and PSF data. SMU firing probability (PSTH) and SMU discharge rate (PSF) responses were compared with SEMG responses. In addition, the effect of discharge rate on the duration of the CSP was investigated using PSF. CUSUMs were also calculated from averaged SEMG traces to illustrate subtle reflex responses [30]. While CUSUM for PSTH illustrates subtle but consistent changes in bin counts, CUSUM for PSF pinpoints subtle but consistent changes in discharge rate. Basically, CUSUM and CUSUM related analyses were performed in the following steps. First, the prestimulus average bin value in microvolts (for SEMG), in number of events (for PSTH), and in discharge rates (for PSF) was determined. Then the prestimulus average bin value was subtracted from each of the bin values in the entire analysis period (usually -200 to +200 ms period). Then the residual values left in each bin were integrated to obtain CUSUM. Therefore, CUSUM calculations simply sum up the differences of each bin value from the prestimulus average bin value and hence clearly indicate any subtle but continuous changes in the poststimulus period that are not normally visible. Maximum deflection in the prestimulus CUSUM is used to build an error box which is then used to determine the significant poststimulus deflections [31,32]. Any post stimulus deflection that is larger than the largest prestimulus deflection and appears before the reaction time to this stimulus is considered as a genuine / significant response to stimulus [31].

Latency of a significant event in the CUSUM records is defined as the time of the turning point. Duration of a significant event is the horizontal distance between its first significant turning and the next significant turning. Strength of an event correspond to the vertical distance between the two CUSUM turning points and is expressed as a percentage of the maximum possible event [32]. If significant deflections are up-going, they are classified as ‘excitation’ and if they are down-going, as ‘inhibition’. This is a conservative but accurate method for pinpointing genuine poststimulus deflections that underlie stimulus-induced changes in the motoneuron activity [21].

Statistical analyses

We, firstly, searched the optimum TMS intensity which evokes several motor unit firings at the MEP period. For this purpose, we randomly adjusted stimulus intensity that generated SMUs at MEP period between 0 (sham) and around 50 SMUs per 100 stimuli, which was suggested to represent the injection of 0 to 5 mV EPSPs into regularly discharging motoneurons in previous studies [33,34]. Then, we calculated discharge rates of SMUs that are above prestimulus firing rate in the CSP region in all 29 SMUs of TA using nonparametric Wilcoxon matched-pairs signed rank test, after testing the normality using Shapiro-Wilk test. The duration of the CSP obtained with various analysis tools were compared using nonparametric Friedman test where multiple comparisons were corrected using Dunn’s test. We have also checked if the background discharge rates in PSF influence the duration of CSP using regression analysis. Statistical significance was set at p<0.05. All statistical analyses were performed using GraphPad Prism 7.

Results

Optimum stimulus intensity to trace synaptic potentials

To decide which intensity that could correctly trace and identify the event clearly, four different TMS intensities were used that generated 50, 30, 20 and 10 SMU occurrences at the MEP period out of 100 TMS pulses as well as control stimulation using sham coil in TA (. The highest intensity (50 SMU occurrences out of 100 stimuli at the MEP period) stimulus evoked a large MEP but was too strong to allow us to trace the post-MEP events as seen in However, the second highest stimulus intensity (generating 30 SMU occurrences for every 100 TMS) clearly traced the post-MEP events (. The intensity that induced 20 SMU occurrences out of 100 TMS also showed clear MEP and higher discharge rates during the post-MEP event period but relatively lower in number compared to the 30/100 stimulus intensity (). Other intensity (10/100) neither clearly showed the MEP nor the post-MEP events, so this was not the ideal intensity to investigate the post-MEP event characteristics (). Hence, due to better representation of MEP and following events in 20/100 and 30/100 intensities were used for investigating the MEP and post-MEP events. On the other hand, sham TMS application did not induce any response ().

Representation of discharge rates (PSF) in different TMS intensities in TA muscle.

The horizontal arrows indicate duration of the TMS induced CSP. Latency was calculated using PSTH-CUSUM and the duration using PSF-CUSUM. The intensities which evoked (A) 50, (B) 30, (C) 20, (D) 10-unit potentials at the MEP period per 100 stimuli, and (E) sham TMS without any MEP. Note that in 30/100 intensity, the motor unit responses during MEP clearly indicate I1 and I2 responses. In all cases, average the discharge rates of SMUs and scalebars that indicate calibrations in each trace are shown.

The cortical silent period and rebound activity

PSF, PSTH, and SEMG with their CUSUMs were also used to investigate late post-MEP events ( PSF clearly showed that the units were firing at higher discharge rates at the CSP region than at the prestimulus region especially when stimulus intensities that delivered 20–30 SMU occurrences out of 100 stimuli. However, SEMG and PSTH analyses indicated that the electrical activity was lower during CSP hence suggesting an inhibitory period due to their definition for inhibition, i.e., lower number of spike events during a period compared with the prestimulus spike counts ().

Representation of MEP and early (1: CSP) and late (2: Rebound activity period) post-MEP events with a stimulus intensity that generated 30-motor unit occurrences at the MEP period per 100 stimuli in TA. Black vertical dashed lines indicate the post-MEP event onsets and endpoints while red dashed line indicates MEP latency. Black horizontal arrows present CSP (1) and Rebound activity period (2). Early post-MEP period is the classical CSP and the late period is the classical ‘Rebound Activity’ that terminates the CSP. (A) PSF illustrates the frequency pattern of the unit together with its CUSUM (top trace). Horizontal dashed lines indicate 2 x SD according to the pre-stimulus firing rates and yellow line is average background discharge rate which was 9.8 Hz. (B) PSTH represents the firing probability of the unit, represented with its CUSUM (orange trace above). Dashed lines in CUSUM indicate the error box limits (see Methods). (C) Averaged-rectified SEMG-CUSUM response shows the MEP latency and reduced activity (black trace above).

Again, in the classical probability-based analysis there was a rebound excitatory period (secondary peak) immediately after the CSP (). Unlike the SEMG and PSTH, PSF results showed that the secondary peak was actually indicating an inhibitory event ( as the discharge rates of spikes during this period were lower than the average prestimulus discharge rate (for definition of inhibition see and Kudina [35]). We observed this secondary inhibitory event clearly in 19 out of 29 SMUs in TA and 6 out of 8 SMUs in APB with duration of 57.9 ± 9.5 ms for TA and 67.3 ± 13.8 ms for APB.

Pre-stimulus vs early post-stimulus discharge rate of motor units

We compared the number of units fired 2xSD above the background discharge rate at prestimulus and early poststimulus (CSP, see ) region in TA muscle. Prestimulus time was selected as the time between -250 to 0 ms, while poststimulus time is the duration of the CSP which was calculated using PSF-CUSUM. The number of high frequency firings (i.e. above 2xSD) obtained at both regions was then normalized timewise to the 100 ms of duration. Shapiro-Wilk test revealed non-normal distribution for both prestimulus region (W = 0.8969, p = 0.0083) and CSP (W = 0.9013, p = 0.0106). A significantly higher firings during CSP was found in TA muscle (p = 0.0042, Wilcoxon matched pairs signed rank test) compared to prestimulus period ().

Average number of units firing above 2xSD for different TMS intensities at prestimulus region and early poststimulus region (CSP) in TA muscle.

The number of high-frequency firings during prestimulus time of 250 ms (top left figure, units in gray rectangular shape) was compared with the average number of high-frequency firings at the CSP region (top right figure, units in blue rectangular shape) after normalization to 100 ms (see text for details). The average number of high-frequency events at prestimulus and CSP regions for 29 units are shown in gray and blue column at the bottom, respectively **p<0.01. Error bars are SEM and N = 29.

Different analysis tools and duration of the early cortical silent period

We calculated the duration of the CSP using CUSUMs in all 29 units from 20 subjects for TA muscle (p = 0.0117, Friedman). The longest CSP durations was obtained in PSF (71.2 ± 9.0 ms) and PSTH (49.2 ± 3.9 ms) with insignificant difference (p>0.99, Dunn). On the other hand, the duration in SEMG (46.4 ± 7.2 ms) was significantly shorter compared to the duration that was calculated using PSF (p = 0.0175, Dunn) but not shorter than the duration in PSTH (p = 0.0543, Dunn).

Similarly, in 8 of APB SMUs from 4 subjects, no significant difference was observed when duration of CSP calculated in PSF (42.1 ± 11.2 ms), PSTH (40.0 ± 8.0 ms) and SEMG (35.5 ± 8.2 ms) (p = 0.7943, Friedman and p>0.99 for all multiple comparisons, Dunn).

Background firing rate and duration of the early cortical silent period

The relationship between the CSP and discharge rate of the units was investigated (). Linear regression revealed a significant inverse relationship between the CSP and background discharge rate in PSF for both muscles (TA: p = 0.0005 and APB: p = 0.0221).

The correlation between the background discharge rate of the SMUs and duration of the CSP measured using PSF-CUSUM.

Left figure shows the relationship for 29 units measured in TA muscle, while the figure on the right represents 8 APB units.

Discussion

Several unique findings have been proposed in this study. Firstly, we found that background discharge rate of motor units significantly altered CSP duration. Secondly, even though the number of spike occurrences was lower than average pre-stimulus spike number during CSP, discharge rate of motor units was higher than the pre-stimulus discharge rate. Therefore, early CSP may not be an inhibitory period but may instead represent a net excitation induced by the TMS pulse and possibly contributed by both spinal and cortical mechanisms. Both TA and APB had similar CSP characteristics. Lastly, our findings suggest that the rebound activity at the end of the CSP indicates existence of a net IPSP, again possibly contributed by both spinal and cortical mechanisms. This study illustrates the importance of combining the techniques of SEMG, PSTH, and PSF to accurately characterize net synaptic events evoked by TMS.

Background discharge rate of motor units significantly alters the duration of cortical silent period

It has been previously suggested that the duration of CSP is strongly influenced by stimulus intensity [36,37]. Therefore, standardization of some parameters is needed to determine reliable comparison between health and disease as MEP and CSP can vary with the exact stimulation point, level of stimulus intensity and prestimulus contraction level [37,38]. In the current study, while SEMG and PSTH analyses did not indicate any change in CSP duration with the level of prestimulus contraction, PSF displayed a significant decrease in CSP duration with an increase in the background discharge rate of motor units. This correlation may explain reported reduction in CSP duration in spastic patients since these patients display increased motor unit discharge rates especially on the less-affected side [39].

The duration of CSP we found in this study was shorter than CSP duration measured in classical studies for both upper and lower limb muscles [5,40-42]. The reason for this difference could be due to the lower stimulus intensity that was used in the current study. Both this study and a study by van Kuijk et al. [40] clearly showed that the duration of CSP is shorter when lower stimulus intensities are used. Therefore, to reduce the possibility of large at MEP and post-MEP event periods, which interferes with correct recognition of SMU spikes, we were limited to use lower stimulus intensities around 30–40% of maximum stimulator output. This has led to shorter CSP durations in the current study.

Early cortical silent period may represent a net excitatory postsynaptic potential

In the literature, it has been suggested that both intra-cortical and spinal mechanisms contribute to the TMS induced CSP where spike occurrence is low. Reduced spike occurrences after a large MEP may not however indicate an inhibitory event as the MEP can cause phase advancement of spikes to an earlier time period hence generating a gap in discharge probability immediately after MEP. Furthermore, when a motor unit fires at a given time it cannot fire for tens of milliseconds due to its afterhyperpolarization property [43,44].

While the classical analysis methods, SEMG and PSTH, identified the CSP with reduced spike activity; frequency analysis (PSF), on the other hand, suggested that CSP may represent a mixed event in which compound net excitation during CSP may be a combination of the falling phase of a large EPSP (rising phase of which induces the MEP) and some corticospinal inhibitory circuits that are activated by the TMS pulse. Similar long lasting excitatory periods have been described in animal studies where activity of single cortical neurons were recorded in response to TMS [24,25]. The rationale for suggesting CSP to represent a net excitatory event comes from the fact that the discharge rates of small number of spikes that fire during CSP were significantly higher than the average prestimulus discharge rate (). This indicates that a low number of spikes following MEP may be due to being in the shadow of a large net EPSP that usually crosses the firing threshold during its rising phase. Threshold crossings during the falling phase of such a large net EPSP can only occur due to synaptic noise and only in rare occasions (). This situation was observed in both TA and APB muscles. This finding suggests that the CSP may not be an inhibitory event only, instead, it may represent a combination of excitations and inhibitions resulting in a net EPSP.

Hypothetical motoneuron discharge to illustrate the effect of TMS induced net EPSP on ongoing action potentials.

Rising phase of the EPSP crosses firing threshold at most cases as it is larger than the synaptic noise and also is rapidly-rising. Threshold crossing by rapidly-rising phase of EPSP effectively brings action potentials that were to occur later to an earlier time (phase advance of spikes). This creates a period of low firing probability (cortical silent period; CSP) immediately after the rising phase of EPSP as spikes that were to fire in that period moved to occur earlier, generating MEP in the SEMG. Threshold crossing during falling phase of an EPSP is only possible when fast-rising phase fails to cross the threshold and the falling phase of the EPSP crosses the threshold with the help of an up-going synaptic noise. This is an extremely rare event and hence most of the threshold crossings will be achieved during the rising phase of an EPSP especially when EPSP is large.

Previously proposed mechanisms for the CSP, especially for its late part, have focused on cortical inhibition [45-47]. Inghilleri et al. [45] illustrated that the longest duration of CSP they observed was around 300 ms, and the first 50 ms was mediated by spinal circuitries upon brainstem stimulation. Similarly, paired TMS with epidural recordings of indirect waves (I-waves) showed no changes in I-waves in the first 50 ms of the CSP, whereas, these waves were reduced in the later parts of the CSP (after 100 ms) [46]. Moreover, there was no observable effect of Vigabatrin, a selective GABAergic drug, neither on the peripheral motor excitability nor on the early part of CSP [47]. This indicates that the late cortical component of the CSP is likely an inhibitory phenomenon mediated by GABA.

In detail, it has been argued that the cortical inhibition is presynaptic to the cortico-spinal neurons, rather than due to a decreased excitability of these cortico-spinal neurons [7,48,49]. Other neuropharmacological modulation in healthy subjects have suggested that the CSP reflects GABAB-mediated intracortical inhibition [50-53]. Siebner et al. [50] observed a marked prolongation of the CSP during continuous intrathecal administration of high doses of the GABAB receptor agonist baclofen in a patient with generalized dystonia. Werhahn et al. [51] showed in healthy subjects that the ingestion of a single dose of tiagabine (which inhibits the uptake of GABA from the synaptic cleft) prolonged the TMS-induced silent period duration. Both groups argued that this prolongation must reflect increased intracortical inhibitory changes. This prolonged inhibition could reflect the changes in the later-onset event, suggested as the rebound activity (net IPSP) in this study.

Our suggestion about the net EPSP during CSP is made up of a contribution from the falling phase of MEP-induced EPSP and TMS-induced IPSPs in cortical and spinal networks. One of the contributing IPSPs may come from the Renshaw circuitries in the spinal cord [54] which may explain prolongation in CSP after GABA-specific drug administration that may cause enhanced/longer recurrent inhibition.

Rebound activity may indicate an inhibitory postsynaptic potential

Rebound activity observed in this study marks the end of CSP in the classical studies [18,36,48,55]. As can be seen in , reduction in the discharge rate during this period as indicated in PSF has been claimed to be a rebound excitation in PSTH and SEMG analyses. However, during this period of rebound activity, the discharge rate of motor units decreases below the level of prestimulus discharge rate (Phase 2 in Figs . PSF method clearly shows that the rebound activity following CSP is made up of synchronous occurrence of phase delayed spikes. The reason for the phase delay in spike timing is due to a net IPSP.

The proposed mechanisms behind rebound activity (net IPSP).

About 30 ms after TMS pulse; a large and long-lasting net EPSP (MEP induced EPSP–recurrent inhibition and other inhibitions in the circuitry) is recorded from the muscle (CSP, 1). Following this net EPSP, a second PSP (net IPSP) develops on motoneurons (rebound activity), 2) which could be due to autogenic inhibition + intracortical inhibition + other unknown inhibitory and excitatory mechanisms. Left: Force trace and PSF as well as its CUSUM of a unit recorded from TA. Right: proposed mechanism responsible for the net EPSP and net IPSP. Black parabolic curve represents imaginary profile of TMS-induced net EPSP while red curve shows the imaginary profile of net IPSP. Blue and red arrows show durations of net EPSP and IPSP, respectively. Black horizontal dashed line in the PSF is the average background firing rate.

Several hypotheses can be put forward about the mechanism of this late inhibition (. The effective inhibition may come from the activation of tendon organs, Renshaw circuitry, other spinal inhibitory circuits and/or cortical inhibition mediated by GABAB. It is possible that muscle contraction that is initiated by TMS pulse can activate the tendon organs within the muscle. It is known that the tendon organ activation induces IPSP on homonymous muscles [56] and its latency (incorporating: electromechanical delay + afferent conduction time + synapses) is similar to the latency of the rebound activity described in this study even though the muscle twitch was rather weak due to smaller stimulus intensity. Our findings suggest that the net IPSP should be arriving at the muscle 75–100 ms after TMS pulse, therefore, GABAB-mediated intracortical inhibition might also contribute to this late-onset inhibition as previously reported by epidural recordings [46] as well as other approaches show the late part of the IPSP is cortical origin [6,45,57].

The time delay for recurrent inhibition to show its effect should be several milliseconds following MEP and recurrent IPSP can last for about 40–50 ms on firing motoneurons [54]. At around the time onset of IPSP reported in this study, the effect of recurrent inhibition would wither away. Therefore, recurrent mechanism is unlikely to be the cause of the late-onset IPSP observed in the current study though it can be involved in the net EPSP during CSP, due to its timing.

In summary, this study proposed that the early part of the CSP may not be an inhibitory event; instead, it may represent a long-lasting net EPSP. Moreover, following CSP, motor units fired at lower discharge rates and therefore this period of rebound activity may represent existence of a net IPSP. This study illustrated the complexity of the mechanisms underlying MEP, CSP and rebound activity which are confounded by the activation of several networks both in the cortical and spinal levels. What we record from the motoneurons as the final end product is a combined net effect of all these TMS-evoked spinal and supraspinal circuitries.

Limitations of the study

Due to the problem with the field potential, we could not use high stimulus intensities. High stimulus intensities induced a response not only on the regularly discharging SMUs but also on other previously non-active motor units. Therefore, high-level TMS pulses generated a field potential () at the MEP latency which made recognition of SMU potentials impossible. This limitation also restricted us from directly comparing the MEP and CSP sizes and durations with the literature as they have used much stronger stimulus intensities than this study. This means our analysis was constrained to the first 50 ms of the CSP, whereas CSP duration is typically in the order of several hundreds of milliseconds. Although GABAB-mediated cortical inhibition is distributed within most of the corticospinal neurons, the inhibition evoked in the current study using low intensity TMS might have been relatively weak to express itself in the TMS-induced net response. However, we are satisfied to suggest that TMS stimulation within the limits of the current framework induces a long-lasting net EPSP which is followed by a net IPSP. We propose that stronger stimuli would increase the size of the EPSP and hence make it impossible to record action potentials in the falling phase of the net EPSP (see for instance a stronger intensity induced CSP in ).

6 Aug 2019

PONE-D-19-18148

TRANSCRANIAL MAGNETIC STIMULATION INDUCED SILENT PERIOD AND REBOUND ACTIVITY RE-EXAMINED

PLOS ONE

Dear Prof. Turker,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The main issue is that CSP duration by SEMG was only 46.4 ms (mean), whereas in many previous studies the CSP duration were over 100 ms and in some cases over 200 ms. Previous studies already demonstrated potential “spinal” inhibition in the first 50 ms of the SP and your study adds further information on this early phase of inhibition. However, it does not provide information to the later stages of CSP (~100 ms). There is some discussion in the paper of the low intensity used but this largely relates to technical issues of not able to test higher intensities with the methods used. It appears unlikely that the mechanisms proposed in the present study can account for the much longer CSP durations reported in other studies in the literature. This need to be clearly acknowledged in the abstract and in the discussion.

The abstract indicates that “the CSP may denote a continuation of the excitatory period initiated by TMS-induced MEP”. While this is technically correct, it is confusing as it suggest that the excitability during CSP is increased but there is decreased number of motor units firing possibly due to the refractory period. Part of the CSP may be related to the falling phase of net EPSP induced by TMS should be mentioned in the abstract.

Please discuss the proposed mechanisms for increased firing rate for the small number of units that fires during the CSP. The statement that “the silent period may not represent a genuine inhibitory period” is an overstatement because there is other evidence for cortical inhibition for example from epidural recording of D and I waves as noted by one of the reviewers.

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PLOS ONE

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Reviewers' comments:

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Reviewer #1: Partly

Reviewer #2: Partly

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Reviewer #1: Yes

Reviewer #2: I Don't Know

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Reviewer #2: No

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5. Review Comments to the Author

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Reviewer #1: This study used three different techniques to analyze the same set of data with recordings after a transcranial magnetic brain stimulation. The three techniques were surface electromyography, peristimulus time histogram and peristimulus frequencygram. The recording was a motor evoked potential followed by a cortical silent period in the tibialis anterior or abductor pollicis brevis muscle. The main result was that the well-known suppression of background discharge during silent period cannot be observed with the frequencygram. Instead, the firing rate analysis revealed a long-lasting excitatory period and a long-latency inhibitory period starting at the middle of silent period seen in the surface electromyographic recording. The conclusion was that the silent period may

denote a continuation of the excitatory period initiated by the motor evoked potential. Rebound activity may represent tendon organ inhibition induced by muscle contraction due to the magnetic stimulation or long-latency intracortical inhibition. This is an interesting study with a conclusion different from many (almost all) other studies in the field. I have a few minor comments, mainly for the interpretation of the results.

The authors’ previous studies investigated how the electromyographic recordings in a muscle might estimate the synaptic potential and how surface recording and peristimulus time histogram may lead to the error in the estimation. I think these background knowledges should be briefly reviewed in the introduction. The point should also be discussed with the present results.

Similarly, it was missed in the method part how the technique with peristimulus frequencygram is performed and how it is technically different from the peristimulus time histogram.

One technical issue is that this study used very low stimulus intensity. Therefore, only motoneurons with low firing threshold were recorded. The widely accepted GABAB mediated inhibition during silent period may act on the majority of the corticospinal neurons but not on this group of neurons with low threshold. I wonder if this should be further considered.

The mechanism with the function of tendon organ inhibitory interneuron was extensively discussed. However, I feel the discussion was speculative as the muscle contraction with the very low stimulus intensity should be subtle. By the way, the model illustrated in the last figure was somewhat different from the discussion and could be removed entirely.

Minor points:

Current orientation of the stimulation for the tibialis anterior muscle should be mentioned.

I doubt the 4 cm distance between two electrodes for recording in the abductor pollicis brevis muscle.

Stimulus intensity related to the maximal device output should be reported for both muscles.

Force output related to the maximal output should also be reported.

Reviewer #2: The authors hypothesise that “the CSP may not reflect an inhibitory postsynaptic potential (IPSP) only but include a period of excitatory postsynaptic potential (EPSP)”. In the discussion they suggest that “that the silent period may not represent a genuine inhibitory period, instead it may be due to a falling phase of a compound net EPSP generated by the TMS”. The rebound phase would be related to “twitch-induced autogenic inhibition by tendon organ inhibitory interneurons”.

While the CSP is likely composed of cortical and spinal phenomena, I think the interpretation here doesn’t sufficiently acknowledge the wealth of previous evidence of a cortical IPSP. If one is to reconceptualise the CSP, how do the authors account for the finding that epidural volleys indicate that cortical output is reduced from 50-200ms (Chen et al., 1999)? This is critical, because it is a direct demonstration of reduced cortical output during this time. Peripheral observations on the other hand can only comment on the net effects of cortical, spinal (GTO inhibition etc.) and local phenomena in muscle units. Similarly, TMS-EEG studies have demonstrated inhibitory correlates of the CSP. This evidence needs to be included and interpretation adjusted accordingly. Certainly, as the authors note, pharmacological studies elicit systematic changes in GABA - but what of the study by Pierantozzi et al., 2004 which concluded that the observed effects were not driven by peripheral effects of the drug? Such findings suggest that the CSP is likely related to long lasting IPSP at the cortex. These findings represent just some of the wealth of evidence for cortical inhibitory contributions to the CSP. The authors could perhaps refocus their paper to better acknowledge this evidence and then the additional information that is provided by their measures. The authors work has value in providing further evidence that the CSP may be a messy measure that is confounded by spinal contributions.

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Reviewer #1: No

Reviewer #2: No

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3 Sep 2019

Response to Reviewers

TRANSCRANIAL MAGNETIC STIMULATION INDUCED SILENT PERIOD AND REBOUND ACTIVITY RE-EXAMINED

Reviewer #1

1. The authors’ previous studies investigated how the electromyographic recordings in a muscle might estimate the synaptic potential and how surface recording and peristimulus time histogram may lead to the error in the estimation. I think these background knowledges should be briefly reviewed in the introduction. The point should also be discussed with the present results.

Please see the second and third paragraphs of the Introduction. Also, please refer to the second paragraph of Discussion in the “Cortical silent period may represent a net excitatory postsynaptic potential” section where we discussed the current findings in the light of the previous studies.

2. Similarly, it was missed in the method part how the technique with peristimulus frequencygram is performed and how it is technically different from the peristimulus time histogram.

Please see the Methods “Data processing” section, first and second paragraphs are extended with the information requested.

3. One technical issue is that this study used very low stimulus intensity. Therefore, only motoneurons with low firing threshold were recorded. The widely accepted GABAB mediated inhibition during silent period may act on the majority of the corticospinal neurons but not on this group of neurons with low threshold. I wonder if this should be further considered.

At high stimulus intensities, the stimulus generated large field potentials at latencies where MEP and CSP responses were normally observed. These field potentials are made up of synchronously activated several motor unit potentials hence making it impossible to distinguish selected unit potentials from the field potentials. Therefore, using relatively smaller TMS intensities is a technical limitation in this study. This statement has been added to the Limitations section in the Discussion along with the statement about corticospinal neurons.

4. The mechanism with the function of tendon organ inhibitory interneuron was extensively discussed. However, I feel the discussion was speculative as the muscle contraction with the very low stimulus intensity should be subtle. By the way, the model illustrated in the last figure was somewhat different from the discussion and could be removed entirely.

The discussion about the tendon organ is shortened and the last figure is updated in line with the discussion.

5. Minor points:

Current orientation of the stimulation for the tibialis anterior muscle should be mentioned.

A brief statement has been included. Please see the Materials and Methods Section, Page 8, the first paragraph.

I doubt the 4 cm distance between two electrodes for recording in the abductor pollicis brevis muscle.

It was about 2 cm for APB muscle and 4 cm for TA, section is updated (Page 6, the second paragraph).

Stimulus intensity related to the maximal device output should be reported for both muscles.

Please see the Materials and Methods Section, Page 10, subheading: stimulation.

Force output related to the maximal output should also be reported.

Updated. Please see the Materials and Methods Section, Page 11, the first paragraph.

Reviewer #2

1. While the CSP is likely composed of cortical and spinal phenomena, I think the interpretation here doesn’t sufficiently acknowledge the wealth of previous evidence of a cortical IPSP. If one is to reconceptualise the CSP, how do the authors account for the finding that epidural volleys indicate that cortical output is reduced from 50-200ms (Chen et al., 1999)? This is critical, because it is a direct demonstration of reduced cortical output during this time. Peripheral observations on the other hand can only comment on the net effects of cortical, spinal (GTO inhibition etc.) and local phenomena in muscle units. Similarly, TMS-EEG studies have demonstrated inhibitory correlates of the CSP. This evidence needs to be included and interpretation adjusted accordingly.

The discussion is now extended with the information about the previous cortical IPSPs together with epidural recording reference of Chen et al. 1999 and some others. The net EPSP during the CSP and late-onset IPSP (referred to as rebound activity) is now updated with the information stating that both the CSP and rebound activities are mixture of excitations and inhibitions originating from both spinal and supraspinal circuits. Several paragraphs in the Discussion.

2. Certainly, as the authors note, pharmacological studies elicit systematic changes in GABA - but what of the study by Pierantozzi et al., 2004 which concluded that the observed effects were not driven by peripheral effects of the drug? Such findings suggest that the CSP is likely related to long lasting IPSP at the cortex. These findings represent just some of the wealth of evidence for cortical inhibitory contributions to the CSP. The authors could perhaps refocus their paper to better acknowledge this evidence and then the additional information that is provided by their measures. The authors work has value in providing further evidence that the CSP may be a messy measure that is confounded by spinal contributions.

We updated the entire Discussion between Pages 22 and 29 in line with the pharmacological studies, indicating the complex structure of CSP which is a net EPSP followed by a net IPSP, the latter possibly due to the mechanisms of intracortical GABAB mediated inhibition contributed by tendon-organ induced autogenic inhibition and other mechanisms.

Academic Editor

1. The main issue is that CSP duration by SEMG was only 46.4 ms (mean), whereas in many previous studies the CSP duration were over 100 ms and in some cases over 200 ms. Previous studies already demonstrated potential “spinal” inhibition in the first 50 ms of the SP and your study adds further information on this early phase of inhibition. However, it does not provide information to the later stages of CSP (~100 ms). There is some discussion in the paper of the low intensity used but this largely relates to technical issues of not able to test higher intensities with the methods used. It appears unlikely that the mechanisms proposed in the present study can account for the much longer CSP durations reported in other studies in the literature. This need to be clearly acknowledged in the abstract and in the discussion.

We propose that the CSP is a net EPSP made up of the falling phase of the MEP-induced EPSP and IPSPs originating from cortical / spinal circuitries. Our study however does not provide information about these effects individually. Similarly, the so-called rebound activity seems to be a net IPSP possibly made up of GABAB mediated cortical inhibition (as shown by previous studies such as by epidural recording) and/or autogenic inhibition. However, due to field potential generation by high intensity stimulation, we technically could not study the long CSPs as most of the literature talks about. We have now stated these restrictions in the Limitations section of discussion as well as in the abstract.

2. The abstract indicates that “the CSP may denote a continuation of the excitatory period initiated by TMS-induced MEP”. While this is technically correct, it is confusing as it suggest that the excitability during CSP is increased but there is decreased number of motor units firing possibly due to the refractory period. Part of the CSP may be related to the falling phase of net EPSP induced by TMS should be mentioned in the abstract.

An action potential can occur during any part of the TMS induced EPSP, this can be during the rising phase or the falling phase. Once it occurs, however, we do not expect another action potential until the end of that cell’s after-hyperpolarization duration (normal discharge after one inter-spike interval). Therefore, we can suggest that the reduced number of action potentials are not due to refractory period but is due to the phase advancement of spikes. Spikes that were to occur during the ‘silent period’ phase advanced as a result of the extra excitation on the cell’s depolarization trajectory, hence generating a gap after the rising phase of the EPSP (as represented in Figure 6).

We have updated the statement mentioned by the editor in the Abstract: “We propose that part of the CSP may relate to the falling phase of net excitatory postsynaptic potential induced by TMS”

3. Please discuss the proposed mechanisms for increased firing rate for the small number of units that fires during the CSP. The statement that “the silent period may not represent a genuine inhibitory period” is an overstatement because there is other evidence for cortical inhibition for example from epidural recording of D and I waves as noted by one of the reviewers.

Low number of high discharge rate spikes can only if there is extra excitation on its trajectory. From the well-known current-frequency relationship of neuronal discharge, the time period where spike discharge is higher than normal must represent a net excitation affecting the motoneuron. The reason why the number is low has been illustrated in Figure 6 and its legend: “Rising phase of the EPSP crosses firing threshold at most cases as it is larger than the synaptic noise and also is rapidly-rising. Threshold crossing by rapidly-rising phase of EPSP effectively brings action potentials that were to occur later to an earlier time (phase advance of spikes). This creates a period of low firing probability (cortical silent perriod; CSP) immediately after the rising phase of EPSP as spikes that were to fire in that period moved to occur earlier, generating MEP in the SEMG. Threshold crossing during falling phase of an EPSP is only possible when fast-rising phase fails to cross the threshold and the falling phase of the EPSP crosses the threshold with the help of an up-going synaptic noise. This is an extremely rare event and hence most of the threshold crossings will be achieved during the rising phase of an EPSP especially when EPSP is large.”

This study illustrated the complexity of the mechanisms underlying MEP, CSP and rebound activity which are confounded by the activation of several networks both in the cortical and spinal levels. What we record from the motor units as the final end product is a combined net effect of all the TMS-evoked spinal and supraspinal circuitries. This statement is now included just before the Limitations.

29 Oct 2019

PONE-D-19-18148R1

TRANSCRANIAL MAGNETIC STIMULATION INDUCED SILENT PERIOD AND REBOUND ACTIVITY RE-EXAMINED

PLOS ONE

Dear Prof. Turker,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please review comments and suggestions from Reviewer 2. The statement that the study “does not provide information about later parts of much longer CSPs induced by high intensity TMS” is now included in the Abstract. I suggest that you include a similar statement in the limitation section of the Discussion

We would appreciate receiving your revised manuscript by Dec 13 2019 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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Robert Chen

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PLOS ONE

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Comments to the Author

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Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: (No Response)

Reviewer #2: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: (No Response)

Reviewer #2: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: (No Response)

Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: The authors have incorporated many of the reviewer suggestions. I felt that the importance of some of the points raised by reviewers wasn’t fully reflected in the updated manuscript. In particular, the following inclusion just glosses over this point “Previously proposed mechanisms for the cortical silent period have focused on theories regarding cortical inhibition [7, 10, 45, 46].” Actually, I think that in the spirit of transparent/clear science, it is absolutely critical to more clearly and explicitly acknowledge that cortical inhibitory mechanisms contribute to the late CSP, in particular, it should be specifically stated that there is clear evidence that epidural volleys are reduced in amplitude during the CSP (Chen et al, 1999) and that Pierantozzi et al., 2004 concluded that the drug effects they observed were not driven by peripheral effects – this indicates that the late cortical component of the CSP is likely an inhibitory phenomenon mediated by GABA. CSP duration is typically in the order of 100-200ms (Chin et al., Brain Res, 2012) and the time-course of spinal contributions has been partially illustrated using brainstem stimulation (Inglhilleri; 1993; JPhysiol). This leaves plenty of scope for the authors to still demonstrate the mechanisms of the spinal contribution and does not detract from this.

I have a few further other suggestions (in astereisks), which in my view clarify an important distinction relative to the bulk of the literature on this topic:

-Title: “Transcranial magnetic stimulation induced *early* silent period and rebound activity re-examined”

-Abstract – “Our aim is to better characterize the *early* CSP phenomena by combining various analysis tools on firing motor units.”

“Discharge rate analysis, however, revealed not three, but just two events with distinct time courses; a long-lasting excitatory period (71.2 ± 9.0 ms for TA and 42.1 ± 11.2 ms for APB) and a long-latency inhibitory period.” - *insert total duration at end of sentence (i.e. around 46ms). The early phase of the CSP is conventionally considered as the first 50ms, whereas the remainder up to several hundred ms is considered mostly cortical. I think the article is still not making this distinction sufficiently clear.

-In the discussion, the following header could be retitled as follows for clarity: “*Early* cortical silent period may represent a net excitatory postsynaptic potential”

“This limitation also restricted us from directly comparing the MEP and CSP sizes and durations with the literature as they have used much stronger stimulus intensities than this study. *This means our analysis was constrained to the first 50ms of the CSP, whereas CSP duration is typically in the order of 100-200ms.*”

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Reviewer #1: Yes: Zhen Ni

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

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3 Nov 2019

Response to Reviewers

TRANSCRANIAL MAGNETIC STIMULATION INDUCED EARLY SILENT PERIOD AND REBOUND ACTIVITY RE-EXAMINED

Reviewer #2

The following inclusion just glosses over this point “Previously proposed mechanisms for the cortical silent period have focused on theories regarding cortical inhibition [7, 10, 45, 46].” Actually, I think that in the spirit of transparent/clear science, it is absolutely critical to more clearly and explicitly acknowledge that cortical inhibitory mechanisms contribute to the late CSP, in particular, it should be specifically stated that there is clear evidence that epidural volleys are reduced in amplitude during the CSP (Chen et al, 1999) and that Pierantozzi et al., 2004 concluded that the drug effects they observed were not driven by peripheral effects – this indicates that the late cortical component of the CSP is likely an inhibitory phenomenon mediated by GABA. CSP duration is typically in the order of 100-200ms (Chin et al., Brain Res, 2012) and the time-course of spinal contributions has been partially illustrated using brainstem stimulation (Inglhilleri; 1993; JPhysiol). This leaves plenty of scope for the authors to still demonstrate the mechanisms of the spinal contribution and does not detract from this.

A paragraph to make the contribution of the cortical inhibitory mechanisms to the late CSP clearer was included in line with the suggestions of the Reviewer in Page 26.

-Title: “Transcranial magnetic stimulation induced *early* silent period and rebound activity re-examined”.

-Abstract – “Our aim is to better characterize the *early* CSP phenomena by combining various analysis tools on firing motor units.”

“Discharge rate analysis, however, revealed not three, but just two events with distinct time courses; a long-lasting excitatory period (71.2 ± 9.0 ms for TA and 42.1 ± 11.2 ms for APB) and a long-latency inhibitory period.” - *insert total duration at end of sentence (i.e. around 46ms). The early phase of the CSP is conventionally considered as the first 50ms, whereas the remainder up to several hundred ms is considered mostly cortical. I think the article is still not making this distinction sufficiently clear.

-In the discussion, the following header could be retitled as follows for clarity: “*Early* cortical silent period may represent a net excitatory postsynaptic potential”

“This limitation also restricted us from directly comparing the MEP and CSP sizes and durations with the literature as they have used much stronger stimulus intensities than this study. *This means our analysis was constrained to the first 50ms of the CSP, whereas CSP duration is typically in the order of 100-200ms.*”

All included.

Submitted filename: Response to Reviewers v2.docx

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7 Nov 2019

Response to Reviewers TRANSCRANIAL MAGNETIC STIMULATION INDUCED EARLY SILENT PERIOD AND REBOUND ACTIVITY RE-EXAMINED

PONE-D-19-18148R2

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19 Nov 2019

PONE-D-19-18148R2

Transcranial magnetic stimulation induced early silent period and rebound activity re-examined

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