Purchase this article with an account.
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: https://doi.org/10.1167/iovs.18-25861.
Download citation file:
© 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.
This PDF is available to Subscribers Only