PROJECT SUMMARY Glaucoma, the most common worldwide cause of irreversible blindness, is characterized by progressive dysfunction and death of retinal ganglion cells (RGCs). We recently employed confocal scanning laser ophthalmoscopy (cSLO) to successfully obtain in vivo Ca2+ imaging with mouse RGCs expressing jGCaMP7s, a genetically encoded calcium indicator. Thousands of ON, OFF, and ON-OFF RGCs with characteristic responses to light stimulation are readily detected in living animals through this non-invasive in vivo imaging platform. Here we seek to develop a more advanced, first-of-its-kind two-photon (2P)-SLO platform with patterned stimulation and multiple detection channels. Through a multidisciplinary collaboration with expertise in in vivo optical imaging, RGC pathophysiology, and retinal neural circuitry and visual processing, we will use complementary imaging techniques and state-of-the-art analysis protocols to understand naïve RGC physiology in real time. We recently extended our original mouse silicone oil-induced pupillary blocking and ocular hypertension (SOHU) model to recapitulate phenotypes of two forms of glaucoma: a chronic model with moderate IOP elevation and mild RGC neurodegeneration; and an acute model with greatly elevated IOP and severe neurodegeneration. Importantly, SO removal reduces IOP to normal almost immediately, allowing better exploration of the effects of IOP lowering treatment and combined treatment with neuroprotection strategies. Thus, we will determine the longitudinal functional and metabolic changes of glaucomatous RGCs, under clinically relevant models, both before and after IOP normalization and/or neuroprotective treatments. These data will deepen our understanding of the pathophysiology of glaucoma, towards finding much-sought biomarkers to better predict progression, and create more relevant endpoints for developing treatment to restore RGC physiology in vivo.