Repetitive transcranial magnetic stimulation (rTMS) is a non-invasive neuromodulation technique that has the ability to alter cortical excitability and plasticity¹. Depending on the stimulation protocol used, the neuromodulatory effects of rTMS may outlast the period of stimulation through the induction of plastic changes within the cortical network². This makes rTMS a favorable therapeutic approach to modulate brain activity in several neurological disorders associated with alterations in cortical excitability and functional connectivity. Although promising treatment results have been reported, knowledge about the precise mechanism of action of rTMS, including its effect on structural and functional brain parameters, is incomplete. Thus, to further explore these mechanisms, the aim of our study was to assess the effects of low-frequency (inhibitory) rTMS on hemodynamics and functional connectivity in stimulated cortical tissue.

Animal procedures were conducted according to the guidelines of the European Communities Council Directive and approved by our institution’s Ethical Committee on Animal Experiments. Four naive male Sprague Dawley rats (380-400 g) were subjected to 20 minutes of low frequency (1 Hz, 1200 pulses) rTMS for 4 consecutive days, while under isoflurane (1%) anesthesia. Animals were stimulated with a Neuro-MS/D stimulator (Neurosoft Ltd., Ivanovo, Russia) using a 25 mm figure-of-eight coil (inner diameter, 18 mm; outer diameter, 25 mm) that was positioned lateral to the midline, with the center of the coil positioned over the right sensorimotor cortex.

Prior to and following the application of rTMS, we acquired structural, functional and perfusion MRI data at 9.4 T (Varian Instruments), whilst animals were anesthetized with isoflurane (1.5%). Resting-state (rs-fMRI) images were executed with a 3D gradient echo, echo planar imaging sequence (800 images, TR/TE=26.1/15 ms, flip angle=13°, resolution=600 µm isotropic). For perfusion MRI we applied dynamic susceptibility contrast-enhanced MRI with a 2D gradient echo, echo planar imaging sequence (800 images, TR/TE=330/11 ms, flip angle=40°, resolution=764x764x1 µm) combined with an intravenous bolus injection of Gadobutrol (Gadovist®, Bayer Schering Pharma AG, Berlin, Germany) (0.35 mmol/kg bodyweight).

Inter- and intrahemispheric functional connectivity [Fisher-transformed correlation (z’) of low-frequency BOLD fluctuations (0.01<f<0.1 Hz) derived from rs-fMRI] were calculated between the primary (M1) and secondary (M2) motor cortices, the forelimb region of the somatosensory cortex (S1FL), the secondary somatosensory cortex (S2), thalamus (Th) and caudate putamen (CPu)³. Relative cerebral blood flow (CBF) in the stimulated sensorimotor cortex was calculated from perfusion MRI data using tracer arrival time-sensitive deconvolution⁴. Contralateral sensorimotor cortex was used as a control. rTMS-induced changes in perfusion and functional connectivity were evaluated using a one-way repeated measures ANOVA (SPSS, IBM SPSS Statistics version 23).

Four days of 1 Hz (inhibitory) rTMS resulted in reduced interhemispheric functional connectivity between homotopic regions of interest (M1, M2, S1FL, S2, Th and CPu) (Fig. 1). The loss of interhemispheric connectivity after rTMS is clearly displayed in the functional connectivity maps of the right M1 (Fig. 2) and S1FL (Fig. 3). There was a trend towards reduced intrahemispheric functional connectivity between certain stimulated regions of interest (e.g. between M1 and M2), but this was not uniformly observed (Fig. 4A-C). Relative CBF in the stimulated sensorimotor cortex slightly dropped from 110±7% (pre-stimulation) to 106±16% (post-stimulation), however this was not statistically significant.

Our preliminary data in rats show that repetitive, low-frequency TMS reduces functional connectivity in the stimulated hemisphere, which may be due to its inhibitory effect on cortical excitability. Information about the degree to which brain activity can be modulated by rTMS may prove helpful in the development of treatment options⁵ for several neurological disorders that are associated with alterations in cortical excitability and functional connectivity. However, currently little is known about the influence of rTMS on neural networks in health and disease.