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Stephanie N Blandford, Spring R Farrell, Michele L Hooper, Balwantray C Chauhan, William H Baldridge; Retinal characterization of the Thy1-GCaMP3 mouse after optic nerve transection. Invest. Ophthalmol. Vis. Sci. 2016;57(12):607.
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© ARVO (1962-2015); The Authors (2016-present)
The genetically encoded calcium indicator GCaMP3 was a developed as a tool for monitoring intracellular calcium concentration ([Ca2+]i) dynamics. The Thy1-GCaMP3 transgenic mouse has been used extensively in central and peripheral nervous system research, but very little in the retina. We therefore wanted to assess the properties of GCaMP3 in retinal ganglion cells (RGCs) and, in particular, to determine if GCaMP3 expression and response to elevated [Ca2+]i, are maintained in a model of RGC damage, optic nerve transection (ONT).
Retinas from Thy1-GCaMP3 (B6; CBA-Tg(Thy1-GCaMP3)6Gfng/J; Jackson Laboratories) mice were examined with conventional calcium imaging. In a subset of animals, ONT was performed on the left eye 5, 6 or 7 days prior to sacrifice and calcium imaging. Retinas were flat mounted and transient increases of [Ca2+]i evoked by superfusion with kainic acid (KA; 50 μM). After calcium imaging, retinas were fixed and processed for immunohistochemistry with antibodies against RBPMS (RNA-binding protein with multiple splicing, an RGC specific marker) and ChAT (choline acetyltransferase, a selective amacrine cell marker).
Baseline GCaMP3 fluorescence was lower in retinas 5, 6 and 7 days after ONT compared to control retinas. There was no difference in the density of GCaMP3-expressing cells 5, 6 and 7 days post-ONT in retinas examined by calcium imaging. The mean (SE) GCaMP3 expressing cells in ONT retinas compared to controls (1181 (66) vs. 2000 (145) cells/mm2, ONT vs. control, respectively; p<0.05, t-test). Furthermore, a lower percentage of GCaMP-expressing neurons showed increased fluorescence in response to 50 μM KA after ONT (42.5% vs. 98.5%, ONT vs. control, respectively). Also, the peak increase of GCaMP3 fluorescence in response to 50 µM KA was less in ONT compared to control retinas. Immunohistochemical analysis of control retinas demonstrated that GCaMP3 in the Thy1-GCaMP3 mouse is expressed in many, but not all, RBPMS-immunoreactive neurons (RGCs) but not in ChAT-immunoreactive amacrine cells.
In Thy1-GCaMP3 transgenic mouse retinas, GCaMP3 is expressed in RGCs which show increases in [Ca2+]i in response to KA. After ONT, the number of GCaMP3-expressing cells was decreased, baseline GCaMP3 fluorescence was reduced, and KA-evoked responses were diminished.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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