Differential glutamatergic and GABAergic responses drive divergent prefrontal cortex neural outcomes to low and high frequency stimulation

Background Repetitive brain stimulation is hypothesized to bidirectionally modulate excitability, with low-frequency trains decreasing and high-frequency (>5 Hz) trains increasing activity. Most insights on the neuroplastic effects of repetitive stimulation protocols stem from non-invasive human studies (TMS/EEG) or data from rodent slice physiology. Here, we developed a rodent experimental preparation enabling simultaneous imaging of cellular activity during stimulation in vivo to understand the mechanisms by which brain stimulation modulates excitability of prefrontal cortex. Methods Repetitive trains of intracortical stimulation were applied to the medial prefrontal cortex using current parameters mapped to human rTMS electric-field estimates. Calcium imaging of glutamatergic (CamKII) and GABAergic (mDLX) neurons was performed before, during, and after stimulation in awake rodents (n=9 females). Protocols included low-frequency (1 Hz, 1000 pulses) and high-frequency (10 Hz, 3000 pulses), with sham stimulation as a control. Results Glutamatergic neurons were differentially modulated by stimulation frequency, with 10 Hz increasing and 1 Hz decreasing activity. Post-stimulation, 1 Hz suppressed both glutamatergic and GABAergic activity, whereas 10 Hz selectively suppressed GABAergic neurons. Conclusions These findings provide direct evidence that clinical brain stimulation protocols induce long-term modulation of cortical excitability, with low-frequency stimulation broadly suppressing activity and high-frequency stimulation preferentially inhibiting GABAergic neurons after stimulation.