INVESTIGATING THE MECHANISM OF TEMPORAL INTERFERENCE STIMULATION USING FUNCTIONAL CALCIUM IMAGING IN HUMAN IPSC-DERIVED NEURONS

Temporal interference stimulation (TIS), has emerged as a promising non-invasive brain stimulation technique for targeting deep brain regions while avoiding activation of overlaying structures [1]. However, the cellular mechanisms remain poorly understood, limiting clinical translation. Here, we investigated the functional response of human iPSC-derived excitatory neurons to TIS using calcium imaging.

Neurons were cultured in PDMS-based microfluidic structures and transfected with Cal-520 for activity monitoring. TIS was applied using two stimulation channels with frequencies f_c=1-2 kHz and f_c+Δf, where Δf=0–5 Hz.

In the suprathreshold regime, excitatory neurons responded to both TIS and high-frequency stimulation (HFS; Δf=0). However, TIS uniquely synchronized neuronal activity at the beat frequency, suggesting temporal modulation. Increasing either the carrier or beat frequency elevated the stimulation threshold, and HFS elicited stronger responses compared to TIS—supporting the hypothesis that suprathreshold TIS is effectively pulsed HFS.

We further investigated current steering, a distinctive feature of TIS, wherein the location of the electric field maximum amplitude modulation shifts toward the channel with lower current amplitude. Although maximal calcium responses remained close to the electrode, beat-frequency-specific power was maximized at intermediate locations where the two field amplitudes are comparable, confirming a dissociation between response strength and modulation-specific entrainment.

These findings support an interpretation of suprathreshold TIS as a temporally structured variant of HFS in in vitro excitatory iPSC-derived cells. Given that in vivo studies report TIS effects at much lower field amplitudes, future work should test whether TIS can modulate ongoing activity through subthreshold mechanisms, when continuous HFS cannot.