Influence of repetitive transcranial magnetic stimulation on functional connectivity andhemodynamics in the rat brain
Julia Boonzaier, Geralda A.F. van Tilborg, Mark J. Bouts, Petar I. Petrov, Caroline L. van Heijningen,
Gerard van Vliet, Annette van der Toorn, Sebastiaan F. Neggers, Rick M. Dijkhuizen

Synopsis
Repetitive transcranial magnetic stimulation (rTMS) is a non-invasive neuromodulation techniquewith the ability to change cortical excitability, however its precise mechanism of action is notcompletely understood. Therefore, by acquiring resting-state fMRI and perfusion MRI data weassessed the influence of unilateral low-frequency (inhibitory) rTMS on functional connectivity andhemodynamics in stimulated cortical tissue in rats. After four consecutive days of rTMS we measured
reduced interhemispheric functional connectivity between homotopic sensorimotor regions, while
cerebral blood flow remained largely unaffected. This reduction in interhemispheric functional
connectivity may be due to the inhibitory effect of low-frequency rTMS on cortical excitability.

Introduction
Repetitive transcranial magnetic stimulation (rTMS) is a non-invasive neuromodulation techniquethat 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 theinduction 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 beenreported, knowledge about the precise mechanism of action of rTMS, including its effect on
structural and functional brain parameters, is incomplete. Thus, to further explore thesemechanisms, the aim of our study was to assess the effects of low-frequency (inhibitory) rTMS on
hemodynamics and functional connectivity in stimulated cortical tissue.

Methods
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. Fournaive 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 werestimulated with a Neuro-MS/D stimulator (Neurosoft Ltd., Ivanovo, Russia) using a 25 mm figure-ofeight 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%). Restingstate (rs-fMRI) images were executed with a 3D gradient echo, echo planar imaging sequence (800images, TR/TE=26.1/15 ms, flip angle=13 , resolution=600 µm isotropic). For perfusion MRI weapplied 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 withan 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 lowfrequency BOLD fluctuations (0.01<f<0.1 Hz) derived from rs-fMRI] were calculated between theprimary (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 fromperfusion MRI data using tracer arrival time-insensitive deconvolution4. Contralateral sensorimotor
cortex was used as a control. rTMS-induced changes in perfusion and functional connectivity wereevaluated using a one-way repeated measures ANOVA (SPSS, IBM SPSS Statistics version 23).

Results
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 ofinterhemispheric connectivity after rTMS is clearly displayed in the functional connectivity maps ofthe right M1 (Fig. 2) and S1FL (Fig. 3). There was a trend towards reduced intrahemisphericfunctional connectivity between certain stimulated regions of interest (e.g. between M1 and M2),
but this was not uniformly observed (Fig. 4A-C).
Relative cerebral blood flow (CBF) in the stimulated sensorimotor cortex slightly dropped from110±7% (pre-stimulation) to 106±16% (post-stimulation), however this was not statisticallysignificant.

Conclusion
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 inthe development of treatment options5, however currently little is known about its influence onneural networks in health and disease.

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3. van Meer MPA, van der Marel K, Wang K, et al. Recovery of sensorimotor function after
experimental stroke correlates with restoration of resting-state interhemispheric functional
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Figures

Figure 1: Interhemispheric functional connectivity (z’) between homotopic regions of interest
pre- and post-rTMS. The bars represent mean functional connectivity values +/- standarddeviation.

Figure 2: Average functional connectivity maps, using the right, stimulated (*) primary motor
cortex (M1) as a seed region, pre- and post-rTMS. R, right; L, left.

Figure 3: Average functional connectivity maps, using the right, stimulated (*) forelimb region ofthe somatosensory cortex (S1FL) as a seed region, pre- and post-rTMS. R, right; L, left.

Figure 4: Intrahemispheric functional connectivity (z’) between regions of interest pre- and postrTMS. The bars represent mean functional connectivity values +/- standard deviation.