Single-cell and population-level neuromodulation dynamics in dual-electrode intracortical microstimulation

Intracortical microstimulation (ICMS) is widely used to activate cortical networks and evoke sensory percepts. Despite its successful clinical outcomes, how individual neurons respond to adjacent multi-electrode ICMS remains unclear. In this study, we combined two-photon calcium imaging with dual-electrode in mouse visual cortex, allowing us to directly observe how neurons behave converging electrical stimulation inputs. This specific setting revealed that at low stimulation levels (5–7 µA), neural activity changed only modestly during dual-electrode stimulation input compared to single-electrode stimulation. As stimulation increased to intermediate levels (10–15 µA), neurons exhibited diverse nonlinear interactions—yet the overall population response shifted toward enhancement. At stronger stimulation (15–18 µA), the proportion of modulated neurons increased in both enhancement and attenuation directions, however, the net effect at the population level was neutral. Neurons that responded strongly to single-electrode ICMS tended to be attenuated during dual-electrode stimulation, whereas weaker responders were often enhanced. The most pronounced modulatory effects occur at intermediate distances between the two electrodes. Together, our results indicate nonlinear neuromodulatory effects from dual-electrode, reflecting complex circuit-level interactions. This data provides better understanding of single-cell modulation demonstrating how cortical networks integrate multiple ICMS inputs, informing necessary strategies to optimize neuroprosthetic performance.