The Effects of Individualized Theta Burst Stimulation on the Excitability of the Human Motor System

Brain Stimulation 7 (2014) 260e268

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The Effects of Individualized Theta Burst Stimulation on the Excitability of the Human Motor System Philip W. Brownjohn a, b, John N.J. Reynolds b, c, Natalie Matheson b, c, Jonathan Fox a, b, Jonathan B.H. Shemmell a, b, * a

The School of Physical Education, Sport and Exercise Sciences, University of Otago, New Zealand The Brain Health Research Centre, University of Otago, New Zealand c Department of Anatomy, University of Otago, New Zealand b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 14 October 2013 Received in revised form 10 December 2013 Accepted 10 December 2013 Available online 16 January 2014

Background: Theta burst stimulation (TBS) is a pattern of repetitive transcranial magnetic stimulation that has been demonstrated to facilitate or suppress human corticospinal excitability when applied intermittently (iTBS) or continuously (cTBS), respectively. While the fundamental pattern of TBS, consisting of bursts of 50 Hz stimulation repeated at a 5 Hz theta frequency, induces synaptic plasticity in animals and in vitro preparations, the relationship between TBS and underlying cortical firing patterns in the human cortex has not been elucidated. Objective: To compare the effects of 5 Hz iTBS and cTBS with individualized TBS paradigms on corticospinal excitability and intracortical inhibitory circuits. Methods: Participants received standard and individualized iTBS (iTBS 5; iTBS I) and cTBS (cTBS 5; cTBS I), and sham TBS, in a randomised design. For individualized paradigms, the 5 Hz theta component of the TBS pattern was replaced by the dominant cortical frequency (4e16 Hz; upper frequency restricted by technical limitations) for each individual. Results: We report that iTBS 5 and iTBS I both significantly facilitated motor evoked potential (MEP) amplitude to a similar extent. Unexpectedly, cTBS 5 and cTBS I failed to suppress MEP amplitude. None of the active TBS protocols had any significant effects on intracortical circuits when compared with sham TBS. Conclusion: In summary, iTBS facilitated MEP amplitude, an effect that was not improved by individualizing the theta component of the TBS pattern, while cTBS, a reportedly inhibitory paradigm, produced no change, or facilitation of MEP amplitude in our hands. Ó 2014 Elsevier Inc. All rights reserved.

Keywords: Transcranial magnetic stimulation Cortical plasticity Primary motor cortex Electroencephalography

Introduction When applied as single and paired pulses, transcranial magnetic stimulation (TMS) can be used to assess the health and excitability

Author contributions: PB planned and conducted experiments, analyzed and interpreted data and wrote the manuscript. JR conceived of the study, interpreted data and approved of the final manuscript. NM interpreted data and approved of the final manuscript. JF conducted experiments and approved of the final manuscript. JS conceived of the study, planned and conducted experiments, interpreted data and wrote the manuscript. This project was funded by the Neurological Foundation of New Zealand. Financial disclosures: All authors report no biomedical financial interests or potential conflicts of interest. * Corresponding author. The School of Physical Education, Sport and Exercise Sciences, University of Otago, P.O. Box 56, Dunedin 9054, New Zealand. Tel.: þ64 3 479 8388. E-mail address: [email protected] (J.B.H. Shemmell). 1935-861X/$ e see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.brs.2013.12.007

of the human motor cortex and corticospinal tract. When applied repetitively however, TMS (repetitive TMS; rTMS) induces neural plasticity that is able to outlast periods of stimulation. One pattern of rTMS that has received significant attention is theta burst stimulation (TBS), consisting of high frequency gamma bursts of low intensity stimulation, repeated at a theta frequency. It has been demonstrated that when applied to the human motor cortex using TMS, TBS in an intermittent (iTBS) or continuous (cTBS) pattern is able to facilitate or depress, respectively, corticospinal excitability for up to 1 h following application [1e8]. The application of TBS as an adjunct therapy in stroke patients has been explored with some success, with reports of transient improvement in corticospinal excitability in paretic limbs [9,10]. Effects on function are, however, highly variable, task specific, and in one recent study, absent altogether [11]. While some of this variability may be due to the heterogenous nature of stroke patients, it has also been shown that the neurophysiological effects of TBS between even healthy

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participants can be highly variable, and may not be as pronounced or generalizable as first described [12]. It is believed that the opposing effects of iTBS and cTBS are induced at the cortical level, and reflect long term potentiation (LTP) and long term depression (LTD) -like processes [2e4,13]. The traditional TBS pattern of 50 Hz triplicates is repeated at 5 Hz, either continuously (cTBS), or in 2 s bursts intermittently at 0.1 Hz (iTBS) [5]. This pattern was originally based on the theta firing rate of hippocampal neurons in animals [14], which is particularly prominent during exploration of new environments, and is thought to reflect the encoding of new information [15]. While these TBS patterns have subsequently been shown to induce LTP in hippocampal and cortical preparations from animals when applied electrically [16,17], the relationship of this pattern of stimulation with underlying human cortical rhythms has yet to be elucidated. It is known that cortical rhythms in the theta and alpha ranges recorded from humans are subject to great task-specific and interindividual variation [18], yet a standardized 5 Hz theta pattern continues to prevail in human TBS studies, without consideration for underlying cortical oscillations. By optimizing the frequency that TBS is applied for each individual, it may be possible to improve the effect profile and reduce the variability of TBS protocols [19]. Utilizing a within-participants design, we aimed to compare neurophysiological measures following standard 5 Hz TBS protocols with intermittent and continuous TBS protocols individualized for each participant to the dominant underlying cortical frequency recorded from the motor cortex of each participant. We hypothesized that individualizing the theta component of TBS to the underlying cortical rhythms would improve the magnitude, duration or both, of the effects of intermittent and continuous TBS protocols. Furthermore, we hypothesized that the efficacy of standard 5 Hz TBS protocols would be related to the peak underlying cortical rhythm of each participant. Initially, we compared the efficacy of standard 5 Hz TBS with TBS individualized to the dominant peak of cortical activity in each participant, on corticospinal excitability. Second, we investigated the interaction of the efficacy of standard 5 Hz TBS protocols with underlying basal cortical rhythms. Third, we assessed the effects of each intervention on intracortical inhibitory circuits, proposed to mediate the neurophysiological effects of TBS. Methods and materials Participants Participants were 10 neurologically healthy volunteers (9 males; 1 female) aged between 22 and 37 years (mean 26.9 years (SD 4.7)), who gave written informed consent. Eight participants were right handed, and two left handed, as determined using the Edinburgh Handedness Inventory [20]. All procedures were performed in accordance with the Declaration of Helsinki, and were approved by the University of Otago Human Ethics Committee. Stimulation and recording Participants were seated in a comfortable chair during each experimental session. Electromyographs (EMGs) were recorded from the non-dominant first dorsal interosseous (FDI) muscle using disposable bipolar AgAg/Cl electrodes. EMG was filtered and amplified (high pass filter 0.2 Hz, low pass filter 10 kHz, gain 500e2000) using NeuroLog preamplifiers (Digitimer Ltd, Welwyn Garden City, UK) and sampled at 4 kHz using a PowerLab 8/30 data acquisition system coupled with LabChart 7 software (ADInstruments, Dunedin, NZ).

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Transcranial magnetic stimulation was applied over the representation of the first dorsal interosseous within the motor cortex contralateral to the target muscle. Single and paired pulse TMS was performed using linked Magstim 200 stimulators with a 70 mm remote coil, while repetitive TMS was performed using a Magstim Rapid2 with an air-cooled 70 mm coil (Magstim Company, Whitland, UK). Coils were held tangentially to the scalp, with the handle facing posteriorly and laterally, in order to evoke electrical currents in a posterioreanterior direction within the motor cortex. The FDI representation ‘hotspot’ was defined as the position at which magnetic stimulation produced the largest motor evoked potentials (MEPs) in the contralateral FDI muscle. This ‘hotspot’ was located and marked on the scalp independently with both magnetic coils, using a custom laser-guided positioning system to ensure accurate relocation of the coils to the same positions throughout the experiment. Stimulation intensity of the theta burst interventions was set to 80% of active motor threshold (AMT) during each session [5]. AMT was defined as the minimum stimulus intensity evoking a discernible MEP (>100 mV amplitude) in at least half of trials during mild contraction (approximately 10e20% maximum voluntary contraction) of the FDI muscle, and was calculated independently for each stimulator. Electroencephalograms (EEGs) were recorded with a 3 electrode array: the active recording electrode was fixed to the FDI motor hotspot, the reference electrode to the ipsilateral mastoid, and the ground electrode to the contralateral forehead. Electrodes were filled with conducting gel, and impedance lowered to <10 kU. EEG signals were filtered and amplified (high pass filter 0.2 Hz, low pass filter 10 kHz, gain 1000) as for EMG above, save for the addition of a 50 Hz notch filter, and sampled at 10 kHz in LabChart 7 (ADInstruments, Dunedin, NZ). Three-minute EEG recordings were taken at rest, with the participant viewing a static, circular visual target in order to focus attention, while wearing ear plugs to reduce disruptive auditory input. Recordings were analyzed for artifacts within the 4e16 Hz range, before fast Fourier transformation using a Cosine-Bell windowing function and 64 k window size to determine frequency components. Spectral analysis was performed on the final window of each EEG recording, based on the assumption that stable resting cortical rhythms would have developed by this stage in the recording. Peak cortical frequency was defined as the frequency at which the greatest amplitude of EEG power density occurred, between 4 and 16 Hz (lower limit set as the onset of normal theta activity; upper limit set as the maximum frequency that can practically be delivered in a theta burst protocol). EEG electrodes were then removed from the scalp prior to TMS. Experimental parameters Corticospinal excitability Corticospinal excitability was assessed by measuring the peakto-peak amplitude of MEPs from the non-dominant FDI at rest, using single pulse TMS. A short recruitment curve was performed in order to identify the stimulus intensity that produced MEPs of approximately 50% maximal amplitude, henceforth referred to as SI50. This SI50 intensity, determined within each experimental session, sat on the linear portion of the stimuluseresponse curve [21], and thus provided the greatest dynamic range for detecting positive or negative changes in corticospinal excitability. This method produced average MEP amplitudes of 1.25 mV (SD 0.75), with a range of 0.20e3.59 mV, at baseline across all sessions. Thirty MEPs, recorded in 3 sets of 10, were measured at baseline, and 20 MEPs, recorded in 2 sets of 10, were recorded at 0e12 min, 15e27 min, 30e42 min and 60e72 min following intervention. MEPs were elicited at random intervals between 4 and 8 s (whole seconds only) in order to minimize anticipation effects. All traces were visually inspected for

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Figure 1. Timeline of experimental procedures for each session. Sets of outcome measures (consisting of 10 single stimuli for MEPs and CSPs, and 10 conditioned and 10 unconditioned stimuli for SICIs) were always recorded in the same order: MEPs, CSPs, SICIs. This order was repeated three times at baseline, and twice at each post-intervention time point.

background motor activity by a blinded experimenter. MEPs were discarded if there was EMG activity present in the 100 ms prior to stimulus, or if they were recorded during times of spontaneous motor unit activity.

had caused the algorithm to provide obvious under- or over estimations.

Short interval cortical inhibition (SICI) SICI was assessed using a paired pulse protocol as previously described [22], with an interstimulus interval of 3 ms. A SICI curve was performed at the beginning of each session in order to identify the conditioning pulse intensity required to inhibit the SI50-induced MEP by 50%, in order to avoid floor and ceiling effects of inhibition [23]. This corresponded to a mean conditioning pulse intensity of 84% AMT (SD 13). While the conditioning pulse intensity remained constant throughout each experimental session, test pulse intensity was altered as necessary to produce unconditioned MEPs of approximately the same amplitude as SI50 at baseline. SICI was measured in 3 blocks of 10 paired stimuli at baseline, and 2 blocks of 10 paired stimuli at every time point post intervention, intermixed with 10 unconditioned test stimuli. Averaged conditioned MEP amplitude was normalized to averaged unconditioned MEP amplitude at each time point. MEPs were discarded if there was EMG activity in the 100 ms prior to stimulus, or if they were recorded during times of spontaneous motor unit activity. The discovery of substantial motor unit activity during SICI measurements in one participant led us to exclude this participant from analysis of this outcome measure. In trials where the conditioned MEP was inhibited to the point of being undetectable above the level of signal noise, we estimated MEP amplitude as being 50% of the amplitude of the signal noise. This method of estimation was based on the fact that the amplitude of an undetected response could vary in amplitude from zero to the level of signal noise, and the assumption that the distribution of MEP amplitudes in this range across a number of trials would be normally distributed.

Five experimental theta burst protocols were examined: standard 5 Hz iTBS; individualized iTBS; standard 5 Hz cTBS; individualized cTBS; sham cTBS. Every participant was treated with each of the 5 conditions in randomised ordered sessions, each separated by at least 3 days (50 sessions in total were administered). A timeline of the experimental protocol within each session is presented in Fig. 1. Interventions were delivered while participants observed the static visual target described in Stimulation and recording section. Participants were blinded to the treatment being applied in each session. TBS protocols were administered at 80% AMT and consisted of 600 pulses in total, as described by Huang et al. [5]. Standard 5 Hz iTBS consisted of bursts of 3 pulses at 50 Hz repeated at 5 Hz for 2 s trains every 10 s. For individualized iTBS, the 50 Hz triple pulse was repeated at the peak underlying cortical frequency of that participant, determined at the beginning of the session. Standard 5 Hz cTBS consisted of bursts of 3 pulses at 50 Hz repeated continuously at 5 Hz. For individualized cTBS, the 50 Hz triple pulse was repeated at the peak underlying cortical frequency. Sham cTBS was performed at 5 Hz over the same region using a 70 mm placebo coil (Magstim Company, Whitland, UK).

Cortical silent period Cortical silent periods were induced by delivering single TMS pulses over the FDI representation hotspot, contralateral to the non-dominant FDI at SI50, during tonic EMG activity. Participants were instructed to maintain a constant mild contraction of the FDI muscle (approximately 10e20% maximum voluntary contraction) while receiving visual feedback of EMG activity. Thirty cortical silent periods, recorded in 3 sets of 10, were measured at baseline, and 20 cortical silent periods, recorded in 2 sets of 10, were recorded at 0 min, 15 min, 30 min and 60 min following intervention. Silent periods were elicited at random intervals between 4 and 8 s in order to minimize anticipation effects. The duration of each cortical silent period was measured from the onset of the MEP to the return of tonic motor activity [24]. These durations were calculated by a custom algorithm in MATLABÒ 7.12.0 (MathWorksÒ, MA, USA) which determined MEP onset and recovery of activity as the point at which activity exceeded 2 standard deviations of background measured either 100 ms prior to stimulus, or during the silent period, respectively. These durations were checked by visual inspection, and corrected if anomalous data points

Theta burst stimulation

Role of muscle activity on TBS effects Due to the previously described interaction of TBS protocols with subsequent muscle activity [6], an additional experimental session was performed on 5 of the participants who participated in the main study, in which the effect of standard 5 Hz cTBS on corticospinal excitability was assessed in the absence of cortical silent period measurement, which required periods of voluntary muscle activity before and after intervention. Experimental methods in this session were identical to those described for the standard 5 Hz cTBS session, except for the omission of EEG, SICI and cortical silent period measurements. Resting MEPs were recorded in three sets of 10 at baseline and at each post intervention time point, with each set separated by a 5 min break in order to account for the approximate time taken to record CSP and SICI measures in the main study. AMT determination, which also required voluntary muscle activation prior to intervention, was still performed in these sessions, and a wash out period of at least 15 min was introduced to minimize interactions between muscle activity required for AMT determination, and TBS intervention. Changes in corticospinal excitability were compared with sham treatment sessions conducted in the same participants. Data analysis Raw data were extracted and processed by an experimenter blinded to the intervention order, using custom scripts in MATLABÒ

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Figure 2. Time course of the effects of standard 5 Hz (iTBS 5) and individualized (iTBS I) intermittent theta burst stimulation on MEP amplitude (A), and relationship between dominant underlying cortical rhythm and 30 min grand average of change in MEP amplitude after standard 5 Hz iTBS (iTBS 5) (B). (A) Both iTBS 5 and iTBS I paradigms significantly increased MEP amplitude. Planned comparisons revealed a significant increase in MEP amplitude 15e27 min after iTBS 5 (*P < 0.05 vs. sham) and iTBS I (yyyP < 0.001 vs. sham). Data expressed as mean  SEM. Inset: Typical response profile of one participant to iTBS I; dashed line represents average MEP amplitude at baseline, and solid line represents average MEP amplitude 15e27 min following iTBS I. (B) No significant correlation exists between the dominant peak of cortical activity at baseline, and the effect of iTBS 5 on MEP amplitude (Kendall’s tau correlation coefficient; r2 ¼ 0.073, P ¼ 0.281). n ¼ 10 participants.

7.12.0 (MathWorksÒ, MA, USA). Data was analyzed using SPSS StatisticsÒ, version 19 (IBM Corporation, NY, USA) and GraphPad PrismÒ, version 6 (GraphPad Software, Inc., CA, USA). Data from iTBS and cTBS experiments were assessed separately for each outcome measure. Corticospinal excitability and cortical silent period data were normalized to baseline within each experimental session before analysis. Data were initially probed using a ShapiroeWilk normality test, and log transformed if necessary to stabilize deviations from normality. Conscious that the time course of any TBS effect is highly variable between individuals and would not be expected to last the full 60 min post-intervention period probed in this study [12], we performed planned Bonferronicorrected comparisons between treatment groups at each postintervention time point instead of an ANOVA which is concerned with main effects. Correlation analyses were performed between the dominant underlying cortical EEG peaks and the grand average MEP ratio generated 0e42 min after standard TBS protocols [12], using Pearson’s correlation coefficient in the case of cTBS, and Kendall’s tau in the case of iTBS, data from which deviated significantly from normality and could not be rectified. The distribution of cortical silent period data also could not be normalized using standard transformation methods, and was therefore analyzed with Bonferroni-corrected Wilcoxon signed-rank comparisons between treatments at each post-intervention time point. Results iTBS Corticospinal excitability Standard 5 Hz and individualized iTBS treatments significantly facilitated MEP amplitude to a similar magnitude (Fig. 2A). Planned comparisons demonstrated that iTBS 5 Hz and individualized iTBS treatment had a significant facilitatory effect on MEP amplitude compared with sham 15e27 min post treatment (P < 0.05 and P < 0.001, respectively). There were no significant differences in MEP amplitude between individualized and 5 Hz iTBS treatment at any post-intervention time point. There was no significant interaction between the underlying dominant cortical frequency (range 4.3e8.4 Hz) and the combined effect of standard 5 Hz iTBS up to 42 min post intervention (Fig. 2B), confirmed by a non-significant Kendall’s tau correlation coefficient (r2 ¼ 0.073, P ¼ 0.281). Furthermore, there was no significant relationship between the efficacy of standard 5 Hz iTBS up to 42 min post intervention and either the test stimulus intensity (SI50) used to probe resting MEPs

(Kendall’s tau correlation coefficient; r2 ¼ 0.002, P ¼ 0.856), or average baseline MEP amplitude (Kendall’s tau correlation coefficient; r2 ¼ 0.012, P ¼ 0.655). Inhibitory pathways Standard 5 Hz and individualized iTBS protocols had no overall effect on SICI (Fig. 3A). Planned comparisons revealed that SICI was not significantly different between treatments at any postintervention time point (P > 0.05 for all comparisons). Matching of unconditioned test stimulus MEP amplitudes was effective, with a mean of 1.45 mV at baseline and 1.30, 1.24, 1.33 and 1.20 mV at each post-intervention time point. Moreover, standard and individualized iTBS treatments had no significant effect on cortical silent period duration (Fig. 3B). Wilcoxon signed-rank comparisons between treatments at each post-intervention time point showed no statistically significant differences (P > 0.05 for all comparisons). cTBS Corticospinal excitability Standard 5 Hz cTBS and individualized cTBS had no significant effect on MEP amplitude (Fig. 4A). Planned comparisons revealed no significant differences between treatments at any postintervention time point (P > 0.05 for all comparisons). There was no significant interaction between the underlying dominant cortical frequency (range 4.1e9.9 Hz) and the combined effect of standard 5 Hz cTBS up to 42 min post intervention (Fig. 4B), confirmed by a non-significant Pearson’s correlation coefficient (r2 ¼ 0.029, P ¼ 0.641). Moreover, there was no significant relationship between the efficacy of standard 5 Hz cTBS up to 42 min post intervention, and either the test stimulus intensity (SI50) used to probe resting MEPs (Pearson’s correlation coefficient; r2 ¼ 0.018, P ¼ 0.734), or average baseline MEP amplitude (Pearson’s correlation coefficient; r2 ¼ 0.032, P ¼ 0.622). Inhibitory pathways Neither cTBS intervention had any significant effect on SICI (Fig. 5A). Analysis with planned comparisons revealed no treatment differences at any time point (P > 0.05 for all comparisons). Matching of unconditioned test stimulus MEP amplitudes was effective, with a mean of 1.37 mV at baseline and 1.27, 1.23, 1.41 and 1.31 mV at each post-intervention time point. Furthermore, neither cTBS treatment had any effect on cortical silent period (Fig. 5B). Wilcoxon signed-rank comparisons of normalized cortical silent period duration between treatments at each post-intervention time

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Figure 3. Standard 5 Hz (iTBS 5) and individualized (iTBS I) intermittent theta burst stimulation had no significant effects on SICI (A) or CSP duration (B). Data expressed as mean  SEM. n ¼ 9 participants for (A); 10 participants for (B).

point revealed no significant differences (P > 0.05 for all comparisons). Effect of muscle activation on cTBS changes in corticospinal excitability Conscious that interactions between voluntary muscle activity, such as that performed to obtain cortical silent period measurements, and cTBS protocols have previously been shown to affect corticospinal excitability changes induced by theta burst protocols [6,7], we conducted an additional standard (5 Hz) cTBS session assessing corticospinal excitability alone in 5 participants. In the absence of voluntary muscle activity immediately prior to or following intervention, standard cTBS had a facilitatory effect on MEP amplitude (Fig. 6). Planned comparisons revealed that standard cTBS significantly facilitated MEP amplitude 60e72 min post intervention when compared with sham treatment (P < 0.05). Discussion We report that intermittent TBS significantly facilitates MEP amplitude when applied in a 5 Hz or individualized burst pattern. There were no significant differences in time course, or magnitude of facilitation between standard and individualized iTBS protocols, and moreover, underlying cortical frequency had no effect on the efficacy of 5 Hz iTBS. Unexpectedly, continuous TBS did not depress

MEP amplitude, and was even facilitatory in the absence of potentially confounding voluntary motor activity immediately prior to or following intervention. None of the tested TBS protocols had any significant effect on intracortical inhibitory circuits. iTBS The facilitatory effects of iTBS on MEP amplitude reported here are somewhat consistent in magnitude and duration with previous reports, which describe approximately 50e100% MEP amplitude facilitation for up to 30 min following administration [1,2,4e7]. While we describe a slightly less robust facilitation than others, it should be noted that there is a high inter-individual variability of effects induced by iTBS protocols [2,12], and several studies have reported weak or null effects of iTBS on corticospinal excitability [12,24,25]. So while sub-motor-threshold iTBS could be categorized as being a generally facilitatory cortical stimulation protocol, its effects are highly individual and vary according to variables that are not fully understood. The mechanisms behind iTBS-induced increases in corticospinal excitability are not clear, but are thought to be driven by LTP or LTDlike phenomena, given a reliance on N-methyl D-aspartate (NMDA) receptor activation [4], an essential component of LTP/LTD induction. Given that iTBS produces strong effects on corticospinal excitability despite application at intensities well below resting and

Figure 4. The effects of standard 5 Hz (cTBS 5) and individualized (cTBS I) continuous theta burst stimulation on MEP amplitude (A) and relationship between dominant underlying cortical rhythm and 30 min grand average of change in MEP amplitude after standard 5 Hz cTBS (cTBS 5) (B). (A) cTBS treatments had no significant effects on MEP amplitude. Data expressed as mean  SEM. (B) There was no significant correlation between the dominant peak of cortical activity at baseline, and the effect of cTBS 5 on MEP amplitude (Pearson’s correlation coefficient; r2 ¼ 0.029, P ¼ 0.641). n ¼ 10 participants.

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Figure 5. Standard 5 Hz (cTBS 5) and individualized (cTBS I) continuous theta burst stimulation had no significant effects on SICI (A) or CSP duration (B). Data expressed as mean  SEM. n ¼ 9 participants for (A); 10 participants for (B).

active motor thresholds, it has long been suggested that the effects are mediated through changes in local intracortical circuits, similar to those activated during paired pulse inhibition or facilitation [22]. A unique study by Di Lazzaro et al. [2] took advantage of the rare opportunity to record corticospinal volleys from patients implanted with epidural electrodes. They assessed iTBS-induced changes in cortically generated direct (D) waves resulting from direct activation of corticospinal axons, and early and late indirect (I) waves, resulting from transsynaptic activation of the same cells, which all contribute to MEP generation [26]. They report that iTBS facilitated late I waves, while leaving D waves and early I waves unaffected, suggesting that iTBS, performed at low intensity, preferentially modulates the excitability of intracortical circuits [2]. We explored the possibility that iTBS was selectively modulating intracortical inhibitory circuits through the assessment of SICI and cortical silent period duration, thought to reflect two independent circuits mediated through GABAA [27] and GABAB [28] receptors, respectively. While initial studies reported increases in SICI following iTBS [5,29], we were unable to replicate this finding, with both standard 5 Hz and individualized iTBS failing to induce any change in SICI compared with sham TBS. However, our results are consistent with the majority of studies that have also failed to

detect significant changes in SICI following iTBS in healthy participants [1,8,24,30e32]. It should be noted that previous reports that have detailed iTBSmediated SICI modulation report very short effects, of 5e10 min duration [5,29]. Therefore we cannot discount the possibility that subtle effects of iTBS on SICI were missed due to the time required to record multiple outcome measures (up to 12 min). Furthermore, it should be noted that while TBS-induced effects on SICI have been observed using the same 3 ms ISI as in this study, use of this particular interval runs a greater risk of contamination from short interval cortical facilitation (SICF) than a 2 ms ISI [33]. While significant contamination by SICF at this ISI is not usually observed when the conditioning stimulus intensity is below 90% AMT (conditioning stimulus intensities in this study were 84% of AMT on average), we cannot rule out the possibility that some contamination by SICF reduced our power to detect TBS-induced changes in SICI, and a 2 ms ISI may have been a more suitable choice for avoiding this confound, given that SICF contamination is minimal at this interval [33]. Our findings that both forms of iTBS had no significant effects on contralateral cortical silent period duration are consistent with the results of the only other study to have examined this and found no effect of iTBS on silent period duration [1]. The phenomenon of long interval cortical inhibition (LICI), which is thought to reflect an additional distinct GABAB associated intracortical inhibitory circuitry [34], was not tested in the current study, and to our knowledge, has not been assessed after iTBS in the stimulated motor cortex. Taken together, the results of this and previous studies provide no evidence to suggest that iTBS selectively modulates intracortical inhibitory circuits. cTBS

Figure 6. The effect of standard 5 Hz cTBS (cTBS 5) on MEP amplitude in the absence of muscle activity. cTBS 5 significantly facilitated MEP amplitude in the absence of voluntary muscle activity. There was a significant increase in MEP amplitude 60e72 min post cTBS 5 compared with sham treatment (*P < 0.05). Data expressed as mean  SEM. n ¼ 5 participants.

In contrast to iTBS, the effects of cTBS on MEP amplitude reported here differ significantly from previous reports using this protocol. While we report no effect of either standard or individualized cTBS on MEP amplitude, almost all previous studies have reported inhibitory effects for up to 1 h following administration [1,3e7]. There are several possible explanations that may account for this discrepancy. Firstly, one of our outcome measures for assessment of intracortical inhibitory circuits was cortical silent period duration. Recording of this measure required the participant to maintain a mild contraction of the FDI muscle for approximately 2 min per block, and was measured three times prior to intervention, and twice at every post intervention time point. It has previously been shown that voluntary muscle contraction interacts with cTBS [6,7]. To investigate this potential confound, we performed an

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additional cTBS experimental session in five of the participants, in which the FDI muscle was kept relaxed for the duration of the session (after AMT determination and 15 min wash out), and only MEP amplitude recorded. We report that in the absence of voluntary contraction, cTBS, performed as originally reported by Huang et al. [5], was facilitatory when compared with sham TBS, a result similar to that obtained by Gentner et al. [35] for a shortened version of cTBS; cTBS300 [35]. While the majority of previous studies have not employed contraction-based outcome measures, a comprehensive analysis of rTMS paradigms by Di Lazzaro et al. [1] analyzed cortical silent period duration over a similar timescale to this study, yet still reported cTBS-induced inhibition of MEP amplitude as expected. Combined with our activity-absent control trials, this makes it unlikely that an interaction of cTBS with voluntary motor activity contributed to the unexpected null effects of cTBS in our main experiment. Secondly, it has been reported that the intensity at which TBS is applied is a key determinant in the outcome of both iTBS and cTBS protocols. We were careful to apply our protocols at 80% AMT as originally described [5], and to properly determine AMT based on well-established methods [36]. Even if our effective applied intensities had been as low as 70% AMT, where negligible effects on MEP amplitude are reported, cTBS is reportedly still able to modulate inhibitory pathways [32] and we saw no such effects. Likewise, cTBS at 90% AMT is reportedly still inhibitory in stroke patients [9]. We believe that the most parsimonious explanation for these inconsistent results, therefore, lies not in methodological inconsistencies, but in the high inter-participant variability of TBS protocols on neurophysiological outcome measures. Recent studies have indicated that the effects of both intermittent and continuous TBS protocols on corticospinal excitability are considerably more variable in magnitude and duration than first reported [5], and are often non-existent [2,12,25]. A number of theories have been proposed to explain the inter-individual variation in response to TBS, including genetic polymorphisms. For example, it has been shown that a single nucleotide polymorphism of the brain-derived neurotrophic factor (BDNF) gene is a potentially important correlate in the induction of neuroplasticity induced by motor learning [37] and rTMS [38], although there is conflicting evidence specifically linking BDNF polymorphisms to variations in TBS-induced neuroplasticity [39e41]. A comprehensive recent study presents evidence for an intriguing hypothesis that as much as half of the variability in response to TBS stems from inter-individual differences in the recruitment of interneuron networks, with some participants responding ‘as expected’ to TBS protocols, some responding opposite to expected, and others responding as expected to one form, and opposite to the other, all dependent on the relative recruitability of circuits generating late I waves [12]. It is possible that our participant pool contained mostly participants that, based on the relative recruitability of their interneuron networks, would have been predicted to have facilitatory responses to both iTBS and cTBS stimulation. This is a possibility which we did not test for, but should be considered in future studies. As with iTBS, the mechanisms behind cTBS-induced suppression of corticospinal excitability as reported by others are yet to be elucidated, but are again thought to be underpinned by LTP or LTDlike processes in intracortical circuits [3,4]. We probed the effects of standard 5 Hz and individualized cTBS on intracortical inhibitory circuits, and again report no significant effect of cTBS on SICI or cortical silent period. While a number of reports have detailed a reduction in SICI immediately following cTBS [5,29,32,42,43], an equal number of studies have not replicated this finding [1,8,24,31]. The effects of cTBS on GABAB mediated circuits have not been investigated in as much depth, however Di Lazzaro et al. [1] report

no significant effect on cortical silent period, and Goldsworthy et al. [42] report no change in LICI, consistent with the null effects on cortical silent period we report in this study. As with iTBS, there is limited and conflicting evidence to suggest that cTBS selectively modulates intracortical inhibitory circuits in any way. Theta rhythm Theta burst protocols are based on the theta firing pattern of hippocampal neurons in animals during learning or ‘LTP-inducing’ situations [15], and have subsequently been used successfully to induce LTP in animal brain slice preparations [16,17]. Theta firing patterns have also been described in Layer 5 pyramidal neurons from the neocortex [44], and cortical rhythms of 4e7 Hz are detectable in human EEG recordings, particularly during encoding and retrieval of information [45,46]. It therefore follows that a theta pattern of stimulation may be optimal for inducing plasticity in the human motor cortex. The exact frequency of theta, however, changes not only between species, but also between individuals as a function of age, cognitive performance, and wakefulness (see review by Ref. [18]). Therefore, the use of a standard 5 Hz theta burst protocol may not be optimal for inducing plasticity in each individual at any particular point in time. We hypothesized firstly that a relationship may exist between underlying cortical frequencies and an individual’s response to prevailing 5 Hz TBS protocols, and secondly that individualizing the inter-burst frequency of TBS to the dominant underlying cortical frequency would enhance the magnitude and duration of plasticity, and potentially decrease inter-participant variability of this intervention. We instead found no relationship between the dominant underlying cortical rhythm recorded with EEG and individual responses to the 5 Hz TBS protocols described by Huang et al. (2005), and, furthermore, no advantage in individualizing the ‘theta’ component of iTBS or cTBS to this underlying dominant frequency. A similar recent study examined the relationship between baseline spectral power in each frequency band and a participant’s response to cTBS, and found that response to cTBS was not predicted by or correlated with baseline spectral power in delta, theta, alpha or beta frequency bands [47]. Based on the proposed mechanisms of TBS-induced modulation of corticospinal excitability, these results perhaps seem surprising, but a number of factors may have contributed to the null finding in the current study. Using a simple, practical approach, we measured EEG with a three electrode array under the same conditions under which we would apply TBS; that is relaxed, eyes open, with a static visual target to focus attention. Under these conditions, we recorded a mixture of EEG rhythms, which generated large variations in peak frequency both between and within participants (Supplementary Fig. 1), and likely reflected different states of arousal or cognitive processing within each participant session. An almost identical recording approach was used by McAllister et al. [47], where baseline resting EEG was recorded in a relaxed, eyes open, fixated state, with an active electrode over the motor cortex at C3, and a reference over Fz. The authors of that study rightly identified that a simple EEG electrode array such as this represents the ongoing activity of a large population of neurons underlying the electrodes, and does not specifically reflect neural circuits projecting to the target muscle [47]. While EEG is temporally sensitive, it has a low spatial resolution, and it may be of benefit in future studies of this type to elicit rhythms more specifically from the motor cortex, perhaps by motor imagery or training of the target muscle(s). Also contributing to the large variation in EEG spectral peaks was the fact that we chose not to limit the selection of the dominant frequency to theta range, and instead measured the dominant underlying frequency peak that occurred between 4 Hz

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(corresponding to early theta) and 16 Hz (the maximum inter-burst frequency possible with a triple stimulus 50 Hz burst). This rationale was based on the debatable importance of a strict ‘theta’ pattern on neuroplasticity modulation in the cortex, as exemplified by the recent observation that alpha (10 Hz) and beta (20 Hz) frequencies applied over the auditory cortex were sometimes more effective in suppressing tinnitus than theta (5 Hz) patterns [48]. If we had constrained our choice of underlying peak to a specific frequency band (i.e. theta), or elicited frequencies within a desired frequency band, then we may not only have reduced intra and interparticipant peak variability, but may perhaps have measured cortical rhythms more relevant to plasticity induction through the corticospinal motor pathway. In any case, while we found no evidence to suggest that the efficacy of current TBS protocols is related to peak underlying cortical frequencies, or that individualizing existing protocols to these frequencies is beneficial, it is possible that future optimization of measurement and recording techniques may tease out more subtle relationships between ongoing cortical activity and TBS-induced plasticity. Acknowledgments We would like to thank Mr Brian Niven, from the Department of Mathematics and Statistics for his statistical advice, Mr Glenn Braid, from the School of Physical Education, Sport and Exercise Sciences for his construction of the laser-guided coil positioning system, and the participants who volunteered for this study. Our sincere thanks also to the reviewers of this manuscript, whose feedback has improved our interpretation of the data. Appendix. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.brs.2013.12.007. References [1] Di Lazzaro V, Dileone M, Pilato F, Capone F, Musumeci G, Ranieri F, et al. Modulation of motor cortex neuronal networks by rTMS: comparison of local and remote effects of six different protocols of stimulation. J Neurophysiol 2011 May;105(5):2150e6. [2] Di Lazzaro V, Pilato F, Dileone M, Profice P, Oliviero A, Mazzone P, et al. The physiological basis of the effects of intermittent theta burst stimulation of the human motor cortex. J Physiol 2008 Aug 15;586(16):3871e9. [3] Di Lazzaro V, Pilato F, Saturno E, Oliviero A, Dileone M, Mazzone P, et al. Thetaburst repetitive transcranial magnetic stimulation suppresses specific excitatory circuits in the human motor cortex. J Physiol 2005 Jun 15;565(Pt 3):945e50. [4] Huang YZ, Chen RS, Rothwell JC, Wen HY. The after-effect of human theta burst stimulation is NMDA receptor dependent. Clin Neurophysiol 2007 May;118(5):1028e32. [5] Huang YZ, Edwards MJ, Rounis E, Bhatia KP, Rothwell JC. Theta burst stimulation of the human motor cortex. Neuron 2005 Jan 20;45(2):201e6. [6] Huang YZ, Rothwell JC, Edwards MJ, Chen RS. Effect of physiological activity on an NMDA-dependent form of cortical plasticity in human. Cereb Cortex 2008 Mar;18(3):563e70. [7] Iezzi E, Conte A, Suppa A, Agostino R, Dinapoli L, Scontrini A, et al. Phasic voluntary movements reverse the aftereffects of subsequent theta-burst stimulation in humans. J Neurophysiol 2008 Oct;100(4):2070e6. [8] Murakami T, Muller-Dahlhaus F, Lu MK, Ziemann U. Homeostatic metaplasticity of corticospinal excitatory and intracortical inhibitory neural circuits in human motor cortex. J Physiol 2012 Nov 15;590(Pt 22):5765e81. [9] Ackerley SJ, Stinear CM, Barber PA, Byblow WD. Combining theta burst stimulation with training after subcortical stroke. Stroke 2010 Jul;41(7):1568e72. [10] Talelli P, Greenwood RJ, Rothwell JC. Exploring Theta Burst Stimulation as an intervention to improve motor recovery in chronic stroke. Clin Neurophysiol 2007 Feb;118(2):333e42. [11] Talelli P, Wallace A, Dileone M, Hoad D, Cheeran B, Oliver R, et al. Theta burst stimulation in the rehabilitation of the upper limb: a semirandomized, placebo-controlled trial in chronic stroke patients. Neurorehabil Neural Repair 2012 Oct;26(8):976e87.

267

[12] Hamada M, Murase N, Hasan A, Balaratnam M, Rothwell JC. The role of interneuron networks in driving human motor cortical plasticity. Cereb Cortex 2013 Jul;23(7):1593e605. [13] Stagg CJ, Wylezinska M, Matthews PM, Johansen-Berg H, Jezzard P, Rothwell JC, et al. Neurochemical effects of theta burst stimulation as assessed by magnetic resonance spectroscopy. J Neurophysiol 2009 Jun;101(6):2872e7. [14] Kandel ER, Spencer WA. Electrophysiology of hippocampal neurons. II. Afterpotentials and repetitive firing. J Neurophysiol 1961 May;24:243e59. [15] Hill AJ. First occurrence of hippocampal spatial firing in a new environment. Exp Neurol 1978 Nov;62(2):282e97. [16] Hess G, Aizenman CD, Donoghue JP. Conditions for the induction of long-term potentiation in layer II/III horizontal connections of the rat motor cortex. J Neurophysiol 1996 May;75(5):1765e78. [17] Larson J, Wong D, Lynch G. Patterned stimulation at the theta frequency is optimal for the induction of hippocampal long-term potentiation. Brain Res 1986 Mar 19;368(2):347e50. [18] Klimesch W. EEG alpha and theta oscillations reflect cognitive and memory performance: a review and analysis. Brain Res Brain Res Rev 1999 Apr;29(2e3):169e95. [19] Cash RF, Ziemann U, Murray K, Thickbroom GW. Late cortical disinhibition in human motor cortex: a triple-pulse transcranial magnetic stimulation study. J Neurophysiol 2010 Jan;103(1):511e8. [20] Oldfield RC. The assessment and analysis of handedness: the Edinburgh inventory. Neuropsychologia 1971 Mar;9(1):97e113. [21] Devanne H, Lavoie BA, Capaday C. Input-output properties and gain changes in the human corticospinal pathway. Exp Brain Res 1997 Apr;114(2):329e38. [22] Kujirai T, Caramia MD, Rothwell JC, Day BL, Thompson PD, Ferbert A, et al. Corticocortical inhibition in human motor cortex. J Physiol 1993 Nov;471:501e19. [23] Fisher RJ, Nakamura Y, Bestmann S, Rothwell JC, Bostock H. Two phases of intracortical inhibition revealed by transcranial magnetic threshold tracking. Exp Brain Res 2002 Mar;143(2):240e8. [24] Hasan A, Hamada M, Nitsche MA, Ruge D, Galea JM, Wobrock T, et al. Directcurrent-dependent shift of theta-burst-induced plasticity in the human motor cortex. Exp Brain Res 2012 Mar;217(1):15e23. [25] Todd G, Flavel SC, Ridding MC. Priming theta-burst repetitive transcranial magnetic stimulation with low- and high-frequency stimulation. Exp Brain Res 2009 May;195(2):307e15. [26] Patton HD, Amassian VE. Single and multiple-unit analysis of cortical stage of pyramidal tract activation. J Neurophysiol 1954 Jul;17(4):345e63. [27] Ziemann U, Lonnecker S, Steinhoff BJ, Paulus W. The effect of lorazepam on the motor cortical excitability in man. Exp Brain Res 1996 Apr;109(1):127e35. [28] Siebner HR, Dressnandt J, Auer C, Conrad B. Continuous intrathecal baclofen infusions induced a marked increase of the transcranially evoked silent period in a patient with generalized dystonia. Muscle Nerve 1998 Sep;21(9):1209e12. [29] Murakami T, Sakuma K, Nomura T, Nakashima K, Hashimoto I. High-frequency oscillations change in parallel with short-interval intracortical inhibition after theta burst magnetic stimulation. Clin Neurophysiol 2008 Feb;119(2):301e8. [30] Zamir O, Gunraj C, Ni Z, Mazzella F, Chen R. Effects of theta burst stimulation on motor cortex excitability in Parkinson’s disease. Clin Neurophysiol 2012 Apr;123(4):815e21. [31] Doeltgen SH, Ridding MC. Modulation of cortical motor networks following primed theta burst transcranial magnetic stimulation. Exp Brain Res 2011 Dec;215(3e4):199e206. [32] McAllister SM, Rothwell JC, Ridding MC. Selective modulation of intracortical inhibition by low-intensity Theta Burst Stimulation. Clin Neurophysiol 2009 Apr;120(4):820e6. [33] Peurala SH, Muller-Dahlhaus JF, Arai N, Ziemann U. Interference of shortinterval intracortical inhibition (SICI) and short-interval intracortical facilitation (SICF). Clin Neurophysiol 2008 Oct;119(10):2291e7. [34] McDonnell MN, Orekhov Y, Ziemann U. The role of GABA(B) receptors in intracortical inhibition in the human motor cortex. Exp Brain Res 2006 Aug;173(1):86e93. [35] Gentner R, Wankerl K, Reinsberger C, Zeller D, Classen J. Depression of human corticospinal excitability induced by magnetic theta-burst stimulation: evidence of rapid polarity-reversing metaplasticity. Cereb Cortex 2008 Sep;18(9):2046e53. [36] Rossini PM, Barker AT, Berardelli A, Caramia MD, Caruso G, Cracco RQ, et al. Non-invasive electrical and magnetic stimulation of the brain, spinal cord and roots: basic principles and procedures for routine clinical application. Report of an IFCN committee. Electroencephalogr Clin Neurophysiol 1994 Aug;91(2):79e92. [37] Kleim JA, Chan S, Pringle E, Schallert K, Procaccio V, Jimenez R, et al. BDNF val66met polymorphism is associated with modified experience-dependent plasticity in human motor cortex. Nat Neurosci 2006 Jun;9(6):735e7. [38] Jayasekeran V, Pendleton N, Holland G, Payton A, Jefferson S, Michou E, et al. Val66Met in brain-derived neurotrophic factor affects stimulus-induced plasticity in the human pharyngeal motor cortex. Gastroenterology 2011 Sep;141(3):827e836.e1e3. [39] Cheeran B, Talelli P, Mori F, Koch G, Suppa A, Edwards M, et al. A common polymorphism in the brain-derived neurotrophic factor gene (BDNF) modulates human cortical plasticity and the response to rTMS. J Physiol 2008 Dec 1;586(Pt 23):5717e25.

268

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[40] Lee M, Kim SE, Kim WS, Lee J, Yoo HK, Park KD, et al. Interaction of motor training and intermittent theta burst stimulation in modulating motor cortical plasticity: influence of BDNF Val66Met polymorphism. PLoS One 2013;8(2):e57690. [41] Mastroeni C, Bergmann TO, Rizzo V, Ritter C, Klein C, Pohlmann I, et al. Brain-derived neurotrophic factor e a major player in stimulation-induced homeostatic metaplasticity of human motor cortex? PLoS One 2013;8(2):e57957. [42] Goldsworthy MR, Pitcher JB, Ridding MC. Neuroplastic modulation of inhibitory motor cortical networks by spaced theta burst stimulation protocols. Brain Stimul 2013 May;6(3):340e5. [43] Suppa A, Ortu E, Zafar N, Deriu F, Paulus W, Berardelli A, et al. Theta burst stimulation induces after-effects on contralateral primary motor cortex excitability in humans. J Physiol 2008 Sep 15;586(Pt 18):4489e500.

[44] Silva LR, Amitai Y, Connors BW. Intrinsic oscillations of neocortex generated by layer 5 pyramidal neurons. Science 1991 Jan 25;251(4992):432e5. [45] Kahana MJ, Sekuler R, Caplan JB, Kirschen M, Madsen JR. Human theta oscillations exhibit task dependence during virtual maze navigation. Nature 1999 Jun 24;399(6738):781e4. [46] Klimesch W, Doppelmayr M, Russegger H, Pachinger T. Theta band power in the human scalp EEG and the encoding of new information. Neuroreport 1996 May 17;7(7):1235e40. [47] McAllister SM, Rothwell JC, Ridding MC. Cortical oscillatory activity and the induction of plasticity in the human motor cortex. Eur J Neurosci 2011 May;33(10):1916e24. [48] De Ridder D, van der Loo E, Van der Kelen K, Menovsky T, van de Heyning P, Moller A. Theta, alpha and beta burst transcranial magnetic stimulation: brain modulation in tinnitus. Int J Med Sci 2007;4(5):237e41.