Year: 2026
Type: preprint
Abstract: 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. Significance: These findings provide direct evidence that extraocular electrodes can reliably activate RGCs in vivo. Notably, stimulation parameters previously associated with full-length optic nerve regeneration were found to be minimally effective at activating RGCs in vivo, suggesting increased gains could be had with newer approaches. The results further demonstrate that waveform asymmetry improves the efficiency of optic nerve stimulation by engaging RGC somas at lower, more tolerable, amplitudes. By combining two-photon imaging with behavioral tolerance testing, this work defines a practical therapeutic window for extraocular stimulation of the eye and establishes asymmetric charge-balanced waveforms as a more clinically translatable strategy for visual pathway neuromodulation.
Language: English
Subfield: Cellular and Molecular Neuroscience
Field: Neuroscience
Domain: Life Sciences
Open Access status: green
Awards: G111028698, G2893077671, GCR: Reprogramming Biological Neural Networks with Field-Based Engineered Systems, G4238164613, G853170728, G8565433924, G9000159425, Center Core Grant for Vision Research, A Translational 3D Map of Hippocampal Cell Types To Drive Investigations of Alzheimer's Disease, Restoration of Optic Nerve Function Driven by In Vivo Multimodal Electrical Stimulation, NEI UCI Center Core Grant for Vision Research
OpenAlex ID: W7213425838