Extraocular Electrical Stimulation Activates Retinal Ganglion Cells In Vivo

Objective

Here, we directly demonstrate that extraocular electrical stimulation can reliably activate retinal ganglion cells (RGCs) in vivo and systematically identify optimal stimulation waveforms that maximize RGC activation at tolerable amplitudes. Approach: Using transpupillary two-photon calcium imaging in Thy1-GCaMP6f rats, we directly visualized RGC activation during extraocular electrical stimulation with single-cell resolution. We tested symmetric (SCB 1:1) and asymmetric (ACB 1:4) charge-balanced waveforms across frequencies ranging from 20 to 5,000 Hz and amplitudes from 1 to 300 μA, correlating cellular calcium responses with behavioral outcomes during awake stimulation. Main results: ACB 1:4 stimulation at lower frequencies (20-50 Hz) robustly and reliably activates RGC somas in vivo, producing larger calcium responses at lower amplitudes than SCB 1:1. At comparable amplitudes, ACB stimulation generated 1.8-fold greater calcium responses. In contrast, SCB stimulation required higher amplitudes that exceeded animal tolerance before reliable RGC activation could be achieved.