DTI Acquisition: -Single Shot Spin Echo EPI Pulse Sequence (TR = 3800 Ms, TE = 105 Ms)

e-Methods

DTI acquisition: -single shot spin echo EPI pulse sequence (TR = 3800 ms, TE = 105 ms) with 12 uniformly distributed Stejskal–Tanner (Stejskal and Tanner, 1965) motion-probing gradient orientations (b = 1000 s/mm2). The FOV was 256 mm, with voxel dimensions of 2.0* 2.0 *5.0 mm.

DTI analysis was performed using standard probabilistic tractography with FMRIB’s Diffusion Toolbox (FDT) from the FSL package. First, data was preprocessed for motion artifacts, and distortions due to eddy currents. Non-brain tissue was removed using the brain extraction tool (BET). DTI data was calculated for each voxel after fitting the diffusion tensor model to each voxel. Fractional anisotropy (FA) and tract volume valueswere obtained using probtracx (FSL) to delineate transcallosal fiber and corticospinal tract (CST) of the hand and lower extremity representative areas. For transcallosal fiber tracts, seed and target ROIs were obtained for the left and right whole motor cortex regions. To obtain corpus callosum ROI as a waypoint mask, the corpus callosal map was projected from the motor cortex using the seed classification method (e-1). For CST, we drew ROIs at three different levels: the first level was at the motor cortex, separately the M1 hand knob area and the lower extremity area; the second level was at the middle of the pons, and the last level was at the lower pons. The distortion corrected FA map was aligned to a template brain using nonlinear registration and then registered to the MNI152 atlas template using a linear registration approach.

TMS experiment: For investigating inter-hemispheric inhibition (IHI), two Magstim 200 Mono Pulse stimulators connected to a Bistim module and to two figure-of-eight coils (Peak magnetic field 2.0 T; Magstim Co Ltd, UK) was used and for investigating the propagation of TMS-generated brain activity to the homologous contralateral area, TMS compatible EEG recording (Nexstim Ltd, Helsinki, Finland) was used.

IHI experiment: Sites of stimulation were determined through motor mapping of both the hemispheres and corresponded to the hot-spot for first dorsal interosseous (FDI) muscles. The resting motor threshold (RMT) was determined for each side. Then for each hand, two conditions were performed: (i) a batch single (unconditioned) pulses over the contralateral hot-spot (baseline) and (ii) a batch of pulses over the contralateral hot-spot, preceded, 6, 10 or 14 ms before, by a conditioning pulse over the ipsilateral, homologous area. All the pulses were given at an intensity of 120% of RMT. To avoid uncontrolled intervening factors, in both conditions the two coils were positioned on the patient’s scalp with the same orientation. Motor-evoked potentials (MEPs) were measured by recording EMG simultaneously. Raw EMG signals were amplified and band-pass-filtered between 20 and 2000Hz, digitized and sampled at a rate of 5000 Hz. EMG analysis was performed using an EMG-dedicated software (Scope, ADinstruments, Australia). Amplitudes of MEPs in response to conditioned pulses were then compared to the amplitudes of MEPs in response to unconditioned pulses (t-test).

TMS-EEG experiment: A 60-channels TMS-compatible EEG system (Nexstim Ltd, Helsinki, Finland) was used.The signals were sampled at 1450 Hz and referenced to a forehead electrode. The impedance of each electrode was kept below 5 kΩ. Contamination of TMS-evoked potentials by auditory responses to the clicks produced by the TMS coil’s discharge was reduced by earplugs. EEG data were processed offline using the EEGlab toolbox (e-2) running in a MATLAB environment (Mathworks). The EEG signals were high-pass filtered (1 Hz); continuous data was epoched from 200 ms before the TMS pulse to 600 ms after (baseline correction based on pre-TMS). Independent component analysis (ICA) was performed to remove electrical artifacts and eye movements/blink artifacts from physiological response to TMS pulse (9 components out of 60). Finally, data were average-referenced

(e-1)Behrens TE, Johansen-Berg H, Woolrich MW, et al. Non-invasive mapping of connections between human thalamus and cortex using diffusion imaging. Nat Neurosci 2003;6:750-757.

(e-2) Delorme A, Makeig S. EEGLAB: an open source toolbox for analysis of single-trial EEG dynamics including independent component analysis. J Neurosci Methods 2004;134:9-21.