Temporal interference stimulation over the motor cortex enhances cortical excitability in rats

Abstract

Temporal Interference Stimulation (TIS) represents a novel non-invasive brain stimulation technique that deeply targets specific brain regions using the differential beat frequency of two high-frequency stimulation pairs. This study investigated the neuromodulatory effects of TIS at different beat frequencies on cortical excitability in the rat motor cortex. Rats were randomly assigned into four groups, receiving TIS at alpha (10 Hz), beta (20 Hz), gamma (70 Hz), or sham frequencies targeting the motor cortex for 20 min under anesthesia. Cortical excitability and inhibition were evaluated by measuring motor-evoked potentials (MEPs), input-output (I/O) curves, and long-interval intracortical inhibition (LICI) before and after TIS. Additionally, immunohistochemistry was performed for neural biomarkers c-Fos and glutamic acid decarboxylase (GAD-65) to confirm targeted neural activation following TIS. We also examined glial fibrillary acidic protein (GFAP)-positive cells in the stimulated region to assess astrocyte responses associated with TIS. Alpha and gamma TIS significantly increased MEP amplitudes compared to sham stimulation. The analysis of I/O curves revealed a significant enhancement in the area under the curve (AUC) post-stimulation in the alpha and gamma TIS groups. Notably, only gamma TIS significantly reduced intracortical inhibition, indicated by an increased LICI ratio post-stimulation. Immunohistochemical analysis demonstrated a significant 35% increase in c-Fos-positive cells in the stimulated motor cortex regions after TIS compared to sham, whereas no significant changes in GAD-65-positive cells or GFAP expression were observed. These findings indicate that a single session of alpha or gamma TIS effectively modulates cortical excitability, highlighting its potential for targeted neuromodulation applications.

Keywords: Cortical excitability; Intracortical Inhibition; Motor-evoked potential; Neuromodulation; Temporal interference stimulation.

Fig. 1

(A) Schematic representation of the experimental design. Thirty-two rats were divided into four groups of eight animals each. Each group received a respective stimulation protocol for 20 min under anesthesia. Electrophysiological recordings, including motor-evoked potential (MEP), input-output curve (I/O Curve), and long-interval intracortical inhibition (LICI), were measured pre-and post-stimulation. (B) The top view of the rat skull shows the positioning of epidural screw electrodes for stimulation and electrophysiological recordings. (C) A detailed diagram illustrates the electrode placements with specific coordinates on the skull over the targeted motor cortical region.

Fig. 2

(A) Schematic representation of TIS protocols and (B) TIS stimulator.

Fig. 3

Schematic representation of recording electrophysiological responses.

Fig. 4

(A) Time-course changes in raw MEP signals across four groups. MEP amplitudes increased after alpha TIS, beta TIS, and gamma TIS but did not after sham TIS compared to baseline. (B) The average normalized MEP amplitudes for the contralateral limb across the four intervention protocols (alpha, beta, gamma, and sham TIS). (C) The total responses of cortical excitability were calculated for each intervention group within 30 min following stimulation on the contralateral limb. Asterisks (*) indicate statistically significant differences when comparing alpha TIS, beta TIS, and gamma TIS with the sham group at the same time point. The data are presented as means, with error bars representing the standard error of the mean (SEM); * p ≤ 0.05.

Fig. 5

(A) Representations of MEP responses at five thresholds (90%, 100%, 110%, 120%, and 130% RMT) pre- and post- stimulation in all groups. (B) Pre- and post-TIS changes in the I/O curve. Average MEP amplitudes across the five thresholds (90%, 100%, 110%, 120%, and 130% RMT) in the four conditions (sham, alpha, beta, and gamma TIS). (C) AUC was calculated for each TIS condition before and after stimulation. The data are presented as means, with error bars representing the standard error of the mean (SEM); *p ≤ 0.05, **p ≤ 0.01.

Fig. 6

(A) Representations in long-interval intracortical inhibition (LICI) showing unconditioned and conditioned MEPs response conditioning stimulus (CS) and testing stimulus (TS) pre- and post-stimulation in the sham, alpha TIS, beta TIS, and gamma TIS groups. (B) Changes of long interval intracortical inhibition (LICI). The ratio of conditioned stimulus to test stimulus alone when the inter-stimulus interval was set to 200 ms. The data are presented as means and the standard error of the mean (SEM); *p ≤ 0.05.

Fig. 7

Changes in cortical expression of (A) c-Fos and (B) GAD-65 following TIS. Asterisks (*) indicate statistically significant differences, either between the stimulated- and non-stimulated regions of the brain within a group, or when compared to the sham group on the same side of the brain. Data are presented as means and the standard error of the mean (SEM); *p ≤ 0.05.

Fig. 8

Changes in GFAP expression following TIS. Data are presented as means and the standard error of the mean (SEM).