Retinal Characterization of the Thy1-GCaMP3 Transgenic Mouse Line

Abstract

The Thy1-GCaMP3 transgenic mouse expresses the genetically encoded Ca2+ indicator GCaMP3 in excitatory projection neurons in the central nervous system. This line has the potential to be a valuable research tool for retina research, however very little is known about its performance in the retina. Therefore, the objective of this thesis was to characterize the anatomical and functional properties of GCaMP3 expressed in retinal ganglion cells and to assess whether these properties change in a model of ganglion cell damage, optic nerve transection. Retinas from Thy1-GCaMP3 mice were examined with conventional Ca2+ imaging. In a subset of animals, ONT was performed in one eye one, three, five or seven days prior to sacrifice and calcium imaging. Retinas were mounted and transient increases of [Ca2+]i evoked by superfusion of kainic acid (KA; 10 μM, 50 μM, 100 μM). After Ca2+ imaging, retinas were fixed and processed for immunohistochemistry with antibodies against RBPMS (an RGC specific marker) and ChAT (a selective cholinergic amacrine cell marker). In Thy1-GCaMP3 mice, GCaMP3 was widely and uniformly expressed in the ganglion cell layer, and responded to elevated [Ca2+]i evoked by KA with robust increases in fluorescence intensity. Following ONT, baseline fluorescence reported by GCaMP3 was not altered, and at three, five and seven days post-ONT there was a significant decrease in transient amplitude evoked by all concentrations of KA tested. This suggests that RGCs display a decline in cellular responsiveness prior to cell death. In addition, fewer cells were able to respond to modest concentrations of applied extracellular KA indicating that functional experiments to accompany anatomical data in injury models is critical to developing the most accurate timelines of injury progression. Immunohistochemical analysis revealed that GCaMP3 was expressed in many, but not all RBPMS+ RGCs, and rarely in ChAT+ amacrine cells in both control and ONT retinas suggesting expression is in RGCs and not cholinergic amacrine cells. Furthermore, the proportion of GCaMP+ cells that were also labeled with RBPMS or ChAT did not change following ONT. Therefore, the current work provides evidence that GCaMP3 persistently reports intracellular calcium transients evoked by KA application in RGCs both in the absence and presence of axonal injury, and that GCaMP3 expression is likely restricted to RGCs, and does not change following ONT. The use of the Thy1-GCaMP3 transgenic mouse as a tool for future retina research is promising as it will allow functional assessment of RGCs over time.