Optimal interphase delay in biphasic current pulses facilitates neural circuit activation induced by microstimulation in the mouse visual cortex

Cathodic-first biphasic current pulses are commonly employed in intracortical microstimulation. Due to the intricate arrangement of axons, somata, and dendrites in the cerebral cortex, membrane polarizations induced by the cathodic and subsequent anodic phases of biphasic pulses may interact in a location-, magnitude-, and timing-dependent manner. Introducing an interphase delay between the two phases has been proposed to mitigate counteracting interactions between these membrane polarizations. Previous clinical studies on visual prostheses have demonstrated that such a delay lowers the stimulus threshold required for percept induction. However, despite this functional outcome, direct physiological observations of cathodic-anodic interactions in cortical circuit activation remain limited. Here, we employed voltage-sensitive dye imaging in mouse brain slices to visualize membrane excitation elicited by biphasic current pulses with varying interphase delays. The results demonstrated that an optimal interphase delay nonlinearly facilitated cortical circuit activation in response to both single and repetitive pulses. At 10 μA/phase and 200 μs/phase, the biphasic pulse elicited larger excitation with a 500-600-μs interphase delay than with shorter or longer delays or the cathodic monophasic pulse. At 20 μA/phase, the cathodic monophasic pulse and biphasic pulses with interphase delays > 800 μs elicited larger excitation than all other conditions. Pharmacological experiments suggested that trans-synaptic excitation contributes to this facilitative effect. These findings provide evidence that an optimally timed cathodic-anodic interaction enhances cortical circuit activation, beyond simply negating opposing phase effects. Such timing optimization of stimulus pulses may improve neural recruitment efficiency while minimizing charge delivery, offering insights for intracortical prosthetic design.