MULTIELECTRODE INTRACORTICAL MICROSTIMULATION ENABLED BY FLEXIBLE MULTISHANK PROBES DURING TWO-PHOTON CALCIUM IMAGING

Modern neuromodulation approaches increasingly rely on multielectrode microstimulation, which is widely used in neural implants to restore impaired sensory functions. However, the spatiotemporal effects of advanced stimulation strategies—such as bipolar stimulation and current steering—on cortical networks remain poorly understood.
To address this gap, we developed polymer-based, flexible multishank iridium oxide electrode arrays integrated with a custom high-density neurostimulator. The platform was validated using two-photon calcium imaging in layer 2/3 of the visual cortex of both anesthetized and awake, head-fixed transgenic GCaMP6 mice. The polyimide shanks featured sharp tips to minimize tissue damage, brain dimpling, and mechanical mismatch with neural tissue.
Monopolar stimulation reliably evoked robust neuronal activation that depended on stimulation current and pulse duration, recruiting increasing numbers of neurons at greater distances from the stimulation site. In contrast, bipolar stimulation between electrodes on separate shanks produced low overlap between activated neuronal populations, even when electrode pairs were spatially close.
Current steering, achieved by varying the current ratio between electrode pairs, enabled controlled shifts in the centroid of activated neuronal populations, with greater neuronal recruitment near the electrode delivering higher current.
In awake, head-fixed animals, increasing monopolar stimulation current similarly recruited more neurons and increased mean calcium response amplitudes. While overall response trends were consistent across sessions, substantial variability in absolute response magnitudes was observed.
Together, this platform provides a versatile framework for probing local neuronal responses to complex electrical stimulation paradigms and supports the development of neural prostheses with improved spatial precision.