In Vivo Evaluation of Naïve and Diseased RGC Activities at Single-Cell Level

Abstract

Purpose : The characteristic feature of optic nerve injury and glaucoma is the progressive degeneration of RGCs and their axons. However, the neuronal activities of RGCs and their changes in disease models have not been studied longitudinally in vivo. Here we demonstrate the feasibility of directly visualizing light-evoked RGCs activities by cSLO-mediated high throughput RGC Ca²⁺ imaging. The powerful population and single-cell analytic strategies we developed revealed, for the first time, distinct dynamic RGC activity changes in optic nerve crush(ONC) and glaucoma models.

Methods : Due to the superb optical accessibility of the retina, fluorescence-labeled RGCs can be imaged noninvasively in vivo by cSLO. We specifically delivered Ca²⁺ indicators, jGCaMP7s, to the mouse RGCs by AAV2 and mSncg promoter. Thus, we employed cSLO customized with UV stimulation and jGCaMP7s, to optically record light-evoked activities of about 1200 RGCs/retina simultaneously in naïve living mice, as well as longitudinal recording RGC activities at multiple time points in ONC and glaucoma mice.

Results : In naïve retinas, 47 RGC clusters were recognized by unsupervised algorithms, and then hierarchically re-grouped into 9 groups with distinct ON, OFF, and ON-OFF responses to UV stimulation. Consistent with distinct transcriptomic changes in trauma and glaucoma, population analysis of longitudinal RGC Ca²⁺ imaging reveals initial increase, but later rapid loss of ON-RGC activities in ONC, versus mild decreased but well-preserved ON- and OFF-RGC activities in glaucoma. More strikingly, single-cell Ca²⁺ imaging tracing uncovers unprecedented conversions of RGC activities, majority of ON-to-OFF conversion in trauma and OFF-to-ON conversion in glaucoma. Thus, not static as assumed, individual RGC’s light-evoked activity is rather vibrant with unique transformation patterns in different diseases.

Conclusions : Our results demonstrate the potential of in vivo RGC Ca²⁺ imaging as a reliable, sensitive, direct and noninvasive RGC functional measurement at single-cell level, but with high throughput capability. This proof-of-concept study lays out the foundation for in vivo RGC function classification and evaluation with more visual stimuli under normal, disease, and therapeutic conditions, at both population and single-cell levels, which is invaluable in understanding RGC pathophysiology and identifying functional biomarkers for diverse optic neuropathies

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.