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Stephanie N. Blandford, Michele L. Hooper, Takeshi Yabana, Balwantray C. Chauhan, William H. Baldridge, Spring R. M. Farrell; Retinal Characterization of the Thy1-GCaMP3 Transgenic Mouse Line After Optic Nerve Transection. Invest. Ophthalmol. Vis. Sci. 2019;60(1):183-191. doi: 10.1167/iovs.18-25861.
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© ARVO (1962-2015); The Authors (2016-present)
GCaMP3 is a genetically encoded calcium indicator for monitoring intracellular calcium dynamics. We characterized the expression pattern and functional properties of GCaMP3 in the Thy1-GCaMP3 transgenic mouse retina.
To determine the specificity of GCaMP3 expression, Thy1-GCaMP3 (B6; CBA-Tg(Thy1-GCaMP3)6Gfng/J) retinas were processed for immunohistochemistry with anti-green fluorescent protein (anti-GFP, to enhance GCaMP3 fluorescence), anti-RBPMS (retinal ganglion cell [RGC]–specific marker), and antibodies against amacrine cell markers (ChAT, GABA, GAD67, syntaxin). Calcium imaging was used to characterize functional responses of GCaMP3-expressing (GCaMP+) cells by recording calcium transients evoked by superfusion of kainic acid (KA; 10, 50, or 100 μM). In a subset of animals, optic nerve transection (ONT) was performed 3, 5, or 7 days prior to calcium imaging.
GFP immunoreactivity colocalized with RBPMS but not amacrine cell markers in both ONT and non-ONT (control) groups. Calcium transients evoked by KA were reduced after ONT (50 μM KA; ΔF/F0 [SD]; control: 1.00 [0.67], day 3: 0.50 [0.35], day 5: 0.31 [0.28], day 7: 0.35 [0.36]; P < 0.05 versus control). There was also a decrease in the number of GCaMP3+ cells after ONT (cells/mm2 [SD]; control: 2198 , day 3: 2224 , day 5: 1383 , day 7: 913 ; P < 0.05). Furthermore, the proportion of GCaMP3+ cells that responded to KA decreased after ONT (50 μM KA, 97%, 54%, 47%, and 58%; control, 3, 5, and 7 days, respectively).
Following ONT, functional RGC responses are lost prior to the loss of RGC somata, suggesting that anatomical markers of RGCs may underestimate the extent of RGC dysfunction.
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