CHARACTERIZATION OF AAV2-GCAMP3 FOR FUNCTIONAL IMAGING IN RETINAL GANGLION CELLS

Abstract

The loss of retinal ganglion cells (RGCs) is the hallmark of optic neuropathies, such as glaucoma. Monitoring RGC function and subsequent dysfunction over time is an invaluable tool in experimental optic neuropathies and potentially in clinical disease. This research characterizes a non-invasive technique for delivering a functional fluorescent marker to RGCs to optically record function. We tested the hypothesis that the genetically encoded calcium (Ca2+) indictor, GCaMP, could be exogenously delivered to the retina with intravitreal injection and produce functional GCaMP3 proteins capable of responding to external stimuli. C57Bl/6 mice were injected with an AAV2-CAG-GCaMP3 viral vector and monitored weekly with in vivo confocal scanning laser ophthalmoscopy over five weeks to quantify the transduction of the virus into retinal cells. Following five weeks, Ca2+ imaging was performed in retinas of a subset of mice injected with AAV2-CAG-GCaMP3 and well as in Thy1-GCaMP3 transgenic mice. Transient increases of intracellular Ca2+ evoked by 50 μM kainic acid (KA) treatments were recorded. In another subset of AAV2-GCaMP3 injected mice, retinas were processed for immunohistochemistry with antibodies against RBPMS (a marker for RGCs), ChAT (a marker for cholinergic amacrine cells), and GFP (to enhance the GCaMP signal). In all injected mice, labelled cells were visible with in vivo imaging one-week following injection. The density of transduced cells increased significantly over five weeks, and the labelling persisted to six-months post-injection. Ex vivo Ca2+ imaging demonstrated that the transduced GCaMP3 was functional in response to treatments of 50 μM KA. The transduced GCaMP3 produced similar mean KA-induced-transients when compared to Thy1-GCaMP3 transgenic mice. Immunohistochemical analysis determined that a mean (SD) of 79 (6)% of GFP-positive cells were RBPMS-positive, 9 (4)% were ChAT-positive, and 12 (7)% did not co-localize with either RBPMS or ChAT. This research demonstrates that exogenous functional fluorescent markers such as GCaMP can be delivered to the retina, be visualized with in vivo imaging and be used to record functional responses from RGCs. It paves the way for examining single cell functional responses, both in vivo and non-invasively, for evaluating experimental optic neuropathies and gauging the effects of potential neuroprotective avenues. Finally, it is proof-of-principle for translation to clinical medicine.