Mechanisms of Non-Invasive Neuromodulation Interventions: Influence on Human Neurochemistry and Functional Connectivity
Phase: NA
Conditions: Non-invasive Neuromodulation in Healthy Subjects
Sponsor: University of Minnesota

Official Title: Mechanisms of Non-Invasive Neuromodulation Interventions: Influence on Human 
Neurochemistry and Functional Connectivity 
NCT Number: NCT02677740 
Date: June 1st, 2019 

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Protocol procedures 
Experimental Design, safety and confidentiality 
Adult, healthy, right-handed males were recruited for the study, following the criteria listed in the relevant 
section of the protocol registered on clinical-trial.gov. The protocol was approved by the Institutional 
Review Board: Human Subjects Committee of the University of Minnesota in accordance with the 
recommendations of The Code of Federal Regulations and the Declaration of Helsinki. A written 
informed consent was provided by each subject right before the beginning of the study.  
The study consisted of one study visit during which resting state fMRI and MRS data were collected 
before and after the rTMS intervention. The subject first underwent the pre-rTMS MRI session, then was 
transferred to another room for the rTMS intervention, and finally was returned to the MR scanner room 
for the post-rTMS MRI session. For both pre- and post-rTMS MRI sessions, data were acquired in the 
following order: anatomical MRI, MRS from left then from right motor cortex and finally rsfMRI.  
Each participant was evaluated for any adverse event throughout the study. A medical director was 
present throughout the rTMS session and the second imaging session. The medical director reviewed all 
participant outcomes including responses to report of symptoms questionnaires after the MRI and rTMS 
sessions, along with the vital signs (blood pressure before rTMS, blood oxygenation during the MRI 
scan), and approved continuation of the study after each session. A designated medical monitor was 
assigned to review any adverse event that occurred. 
The records of this study are kept private and only available to the PI. All experimental data related to the 
study are de-identified prior to being stored on PC or emails. Hard copies of protected health information 
(PHI) are locked in a file cabinet in the PI’s office. 
rTMS intervention 
TMS was delivered using a 70-mm figure-eight TMS coil connected to a Magstim 200 machine. The coil 
was positioned over the hand region of the left motor cortex (approximately 5 cm lateral to each 
individual vertex of the head), contralateral to the dominant hand in all studied participants. TMS-induced 
motor evoked potentials (MEPs) were recorded by placing surface EMG electrodes over the first dorsal 
interosseous muscle of the participant’s dominant hand, contralateral to the side of TMS. The position of 
the coil was moved systematically to find the location that evoked the largest and most reliable MEP 
(“hotspot”). The resting motor threshold (RMT) was then measured as the minimum TMS intensity 
required to produce a 50 µV MEP while the participant was at rest. Immediately after completion of the 

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RMT assessment, the rTMS intervention began. Participants received either 1-Hz or 5-Hz rTMS, applied 
over the motor cortex hotspot contralateral to the dominant arm for 20 minutes. Corticospinal excitability 
was finally re-tested within no more than 10 minutes after the rTMS session.  
MRI/MRS acquisitions 
The imaging sessions were performed using a 7-T/90-cm magnet (Agilent/Magnex Scientific, UK) 
interfaced to a Siemens Syngo console. A single channel transmit/32-channel receive head (NOVA 
Medical, Wilmington, MA). Head motion was minimized using a molded foam head cushion and padding 
placed snugly around the participant’s head. Participants were asked to close the eyes but remain awake 
during both the resting state fMRI and MRS acquisitions. 
High-resolution 3D-MPRAGE images were first obtained to visualize the anatomical structure of the 
motor cortices. Then automatic B0 field mapping and adjustment of 1st- and 2nd-order shims were 
achieved by FASTMAP (Gruetter and Tkac, 2000). Spectroscopy voxels of interest (24 x 22 x 17 mm3) 
were selected in left and right motor cortices based on anatomical landmarks. 1H-MRS data were acquired 
using the semi-LASER localization (Bednarik et al., 2015; Oz and Tkac, 2011). Unsuppressed water 
signal was also acquired for eddy current correction and as an internal reference for metabolite 
quantification. 
Resting state fMRI was performed with a 2D single-shot gradient echo EPI sequence to collect blood 
oxygenation level dependent (BOLD) data. A total of 280 volumes were acquired, corresponding to an 
acquisition time of approximately 6 min.  
MRI/MRS data processing 
Single-scan MRS data were corrected for small frequency and phase variations, summed and finally 
corrected for the residual eddy current effects using unsuppressed water signal (Klose, 1990). Brain 
metabolites were quantified by LCModel (Pfeuffer et al., 1999; Provencher, 1993; Provencher, 2001; 
Tkac et al., 2009). Only metabolite concentrations quantified with Cramèr-Rao lower bounds (CRLB) 
below 50% were included in further analysis. rTMS-induced changes in GABA concentrations, separately 
in left and right motor cortices, were the primary MRS outcomes.  
Resting-state data were preprocessed with the HCP “minimal-preprocessing pipeline” (Glasser et al., 
2013), denoised with the FMRIB’s independent component analysis (ICA)-based X-noiseifier (FIX) 
(Salimi-Khorshidi et al., 2014), and smoothed with an isotropic Gaussian kernel at 4 FWHM. Resting-
state activity was then quantified by examining the fractional amplitude of low-frequency fluctuations 

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(fALFF) (Zou et al., 2008), which is thought to reflect the intensity of spontaneous brain activity. fALFF 
was calculated via AFNI function 3dRSFC (Cox, 1996) as the ratio of the power spectrum within the 
frequency range of interest (0.008 < f <0.09 Hz) to that of the entire spectrum. fALFF was obtained for 
each voxel in the brain and then averaged within the left and right motor cortices, separately. These two 
regions of interest (ROIs) were identified in the upper limb region of the precentral gyrus, as defined in 
the Brainnetome atlas (Fan et al., 2016).  
Statistical analyses 
Pre- vs post-rTMS comparisons of metabolite concentrations and functional connectivity metrics were 
carried out with 2-sided paired t-test separately for left and right motor cortices. Given the nature of the 
study as a pilot investigation of safety and feasibility, multiple comparison corrections were not applied.  
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