Potential participants were recruited from a local university. Twenty young healthy participants (Group 1: age = 27.40 ± 2.07, two women and eight men; Group 2: age = 27.10 ± 2.08, two women and eight men) were recruited. All of them were postgraduate and met all of the following criteria: 1) 18 to 30 years old; 2) right-handed, according to the Edinburgh handedness inventory ; and 3) normal or corrected-to-normal vision. Participants were excluded if they met any of the following criteria: 1) any contraindication to TBS, such as a history of seizures, metal implants, and pregnancy. All participants were screened by a standard safety checklist before enrollment ; 2) previous history of any neurological or psychiatric diseases; 3) presence of upper limb injuries in the past three months; and 4) presence of congenital deformities of the bilateral upper extremities. This study was approved by the Human Subjects Ethics Committee, The Hong Kong Polytechnic University (reference number: HSEARS20190326003). All participants voluntarily consented to participate in this study and their written informed consent was obtained before participation commenced.
Participants were randomly allocated to one of the following two groups by drawing lots: Group 1: cTBS followed by iTBS; and Group 2: sham cTBS followed by iTBS. All participants had to attend four consecutive TBS sessions and two EEG assessments before and immediately after 4 daily sessions of stimulation.
Motor threshold assessment
The stimulation site for iTBS was the right motor cortex. The optimal position was defined as the coil position eliciting the largest MEP, with the coil rotated 45° from the sagittal plane. The stimulation position was maintained by a neuro-navigation system (Localite, Bonn, Germany). Resting motor threshold (RMT) is defined as the minimum intensity over the hot spot that elicits an MEP of no less than 50 μv in three out of six trials over the contralateral first dorsal interosseous (FDI). MEPs were visualized and measured through the MEP monitor (MagVenture, Denmark), with an inter-pulse interval of at least five seconds.
Daily serial sessions of iTBS were delivered by MagPro X100 stimulator (MagVenture, Denmark) with a standard butterfly-shape coil (C-B60), over the right M1 for four consecutive days. Repeated applied stimulation sessions were used in order to obtain observable modulatory effects and to imitate the design usually applied in clinical intervention  .We followed previous studies, using four daily sessions of iTBS for healthy adults [30-32]. The standard 600-pulse TBS protocol was followed . The stimulation intensity of iTBS was set at a safety limit of 70% of the individual’s RMT . We did not set the intensity based on the active motor threshold (AMT), because a previous study has shown that pre-stimulation muscle contraction during the measure of AMT could influence the after-effects of TBS . Sham stimulation was delivered using the same coil that delivers only 20% of the individual RMT. All participants were told that TBS was a subthreshold stimulation that could not induce significant arm movements or somatosensory perception. The interval between priming and stimulation sessions was 10-minute. We choose the 10-minute interval based on a previous in vitro study about reversal of synaptic plasticity in response to TBS , and followed a previous human neurophysiological study about priming iTBS . Participants were asked to complete a questionnaire regarding the side effects of iTBS they experienced upon the completion of each stimulation session.
EEG was captured with a 64-channel cap, using a Digital DC EEG Amplifier and Curry 7 (Compumedics Neuroscan, USA). Electrode impedance was kept below 10 kOhm and the signal was sampled at 1000 Hz. Participants were seated upright in an electromagnetic shielded room and required to minimize any body movements during the recording. Movement-related and MVF-induced sensorimotor ERD were evaluated. Left index finger tapping and incongruent (i.e., mirrored) visual observation of the right index finger tapping were used to elicit the ERD over bilateral sensorimotor areas, with a possibly right dominant lateralization [32, 35]. For movement-related ERD, participants were instructed to tap on a computer keyboard three times with their left index finger, in response to 60 auditory cues (i.e., 300-ms beep sounds) delivered at random intervals (from seven seconds to 10 seconds) and to relax the finger after the completion of the movement. Participants were asked to focus on a centrally located fixation cross in a computer monitor placed in front of them.
For MVF-induced ERD, participants were asked to tap on a computer keyboard three times with their right index finger, in response to 60 auditory cues delivered at random intervals (from seven seconds to 10 seconds), and to relax the finger after completing the movement (Reference to finger tapping tasks). Movements were performed under two conditions. 1) A mirror view of the movement: Participants performed right-index tapping while simultaneously looking at the MVF of their moving finger. The MVF was created using a physical mirror (406 × 432 mm) placed over their midsagittal plane, between both arms. A black curtain was used to block the view of their moving hand. 2) A direct view of the movement: Participants performed right-index tapping while looking at the direct visual feedback (DVF) of their moving finger. Their left hand was hidden by a non-transparent board (see Figure 1 for the EEG set-up). The order of conditions was randomized by drawing lots. A total of 60 movements were collected for each condition, with 180 movements in total.
EEG time-frequency analysis
Clean trials were analyzed in a time-frequency domain. The event-related spectral perturbation (ERSP) method was used to compute ERD power . The power was baseline corrected. We selected a baseline period from -1500 ms to -1000 ms for correction, to avoid the contamination of neural oscillations caused by auditory cues delivered prior to the execution of movement. Subsequently, the power was averaged across all included trials and converted to log power (see the following formula).
where n is the number of trials, and Fk (f, t) is the spectral estimation of the kth trial at frequency f and time t. ERD was further computed using the following formula :
where F represents the frequency band of interest. We defined four frequency bands of interest, including mu-1 (8-10 Hz), mu-2 (10-12 Hz), beta-1 (12-16 Hz), and beta-2 (16-30 Hz) based on our previous studies [26, 32]. T represents the time interval of interest and a window from 0 to 1000 ms was selected to reflect the movement stage. N is the number of time-frequency bins in a selected two-dimension rectangular matrix. Following previous literature [22, 24, 32], we extracted averaged ERD powers at two electrodes C3 and C4 to represent the left and right sensorimotor activation, respectively. In accordance with previous studies, an asymmetric index (i.e., no hemispheric effect) was calculated with the following formula and used in further statistical analyses . A more positive value indicates more activation of the right sensorimotor area.
A statistical analysis was performed using SPSS version 23.0. GraphPad Prism version 7 and custom Matlab scripts were used for the figure visualization. Analysis of variance (ANOVA) was performed separately for each frequency band and the asymmetric index was used as the dependent variable. The level of significance was p < 0.05. Violation of sphericity was corrected by Green-Geisser. Potential between-group difference of the dependence variable at baseline was tested by independent t tests. Two-way repeated measures analysis of variance (ANOVA) with time (pre vs. post) as a within-subject factor and group (Group 1 vs. Group 2) as a between-subject factor was used to analyze the movement-related ERD. Three-way repeated measures ANOVA with time (baseline vs. post-stimulation) and condition (mirror view vs. direct view) as within-subject factors and group (Group 1 vs. Group 2) as a between-subject factor was used to analyze the MVF-induced ERD. In case of any significant effect found, paired t tests were used for the post hoc comparisons. If any of the dependent variable showed significant between-group difference at baseline, analysis of covariance (ANCOVA) with the baseline value as the covariance would be used instead. Missing data were imputed using a last observation carried forward (LOCF) method; that is, if a subject dropped out, the missing value was replaced by the last assessment results.