A novel tool to study bipolar cell responses in glaucoma

Abstract

AbstractPurpose: Retinal ganglion cell death has been well studied in glaucoma, however how the calcium (Ca2+) dynamics in living inner retinal neurons alter to ocular hypertensive stress is poorly understood. Here we characterized a novel adeno‐associated virus (AAV) mediated Ca2+ sensor to investigate light‐evoked Ca2+ responses in bipolar cells with two‐photon laser scanning microscopy (TPLSM).Methods: To visualize ON bipolar cell specific Ca2+ expression, we bioinformatically designed a serotype 2 AAV vector carrying GCaMP6f, a Ca2+indicator, under human MiniPromoter: Ple265 (PCP2, 986 bp, AAV.Ple265.GCaMP). The AAV.Ple265.GCaMP6 was administered intravitreal to adult mice (2 μl/eye). Two and 4 weeks later (n = 3 mice/group), eyes were collected, and retinal cross sections (16 μm) were prepared for immunohistochemistry using the following primary antibodies: GFP to visualize GCaMP6f, and bipolar cell‐specific markers PCP2 or PKCa. Furthermore, we monitored real time light‐evoked Ca2+ responses in bipolar cell terminals at 4 weeks after the injection and amplitude (ΔF/F0) was calculated.Results: At 2 weeks after the viral injection, GCaMP6f was clearly visualized in bipolar cells, but its expression was limited to the cell body and dendritic trees. Four weeks post‐injection, GCaMP6f expression was abundantly detected in many bipolar cell bodies, dendrites, axons, and axonal terminals. TPLSM allowed visualization of bipolar cell‐specific GCaMP6f expression in axonal terminals in vivo, and light‐evoked sustained ON‐responses were recorded in terminal microdomains (average intensity = 1.25 ΔF/F0).Conclusions: Our study reveals that the AAV.Ple265.GCaMP is a powerful tool to monitor ON bipolar cell specific light‐evoked Ca2+ responses in bipolar cells, and can be potentially used for studies aimed at understanding the response of these neurons to glaucomatous damage.