Calcium activation of frequency dependent temporally phasic, localized, and dense population of cortical neurons by continuous electrical stimulation

Electrical stimulation of the brain has become a mainstay of fundamental neuroscience research and an increasingly prevalent clinical therapy. Despite decades of use in basic neuroscience research over acute time scales, and the growing prevalence of neuromodulation therapies, gaps in knowledge regarding activation or inactivation of neural elements over time in the vicinity of the electrode limit the ability to adequately interpret evoked downstream responses or fine-tune stimulation parameters to focus on the desired response. In this work, in vivo two-photon microscopy was used to image Thy1-GCaMP activity in Layer 2/3 neurons of S1 cortex during 30 s of continuous electrical stimulation at varying frequencies. We show that during continuous stimulation, stimulation frequency influences a distinct spatial and temporal pattern of somatic activation. Our results elucidate conflicting results from prior studies reporting either dense spherical activation of somas biased towards somas near the electrode, or sparse activation of somas at a distance via axons near the electrode. These findings indicate that the neural element specific temporal response local to the stimulating electrode as a function of changes in charge density applied, frequency and temporal patterning need to be considered to properly interpret downstream circuit responses for basic science understanding, or determining mechanisms of action for clinical therapeutic applications.