Department of Medical Neuroscience, Dalhousie University, Halifax, Nova Scotia, Canada.
Retina and Optic Nerve Research Laboratory, Dalhousie University, Halifax, Nova Scotia, Canada.
Invest Ophthalmol Vis Sci. 2019 Jan 2;60(1):183-191. doi: 10.1167/iovs.18-25861.
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 [453], day 3: 2224 [643], day 5: 1383 [375], day 7: 913 [178]; 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.
GCaMP3 是一种用于监测细胞内钙动力学的基因编码钙指示剂。我们对 Thy1-GCaMP3 转基因小鼠视网膜中 GCaMP3 的表达模式和功能特性进行了表征。
为了确定 GCaMP3 表达的特异性，用抗绿色荧光蛋白（anti-GFP，增强 GCaMP3 荧光）、抗 RBPMS（视网膜神经节细胞 [RGC]-特异性标志物）和抗中间神经元标志物（ChAT、GABA、GAD67、突触素）对 Thy1-GCaMP3（B6；CBA-Tg（Thy1-GCaMP3）6Gfng/J）视网膜进行免疫组织化学处理。通过灌流 kainic acid（KA；10、50 或 100 μM）记录钙瞬变，钙成像用于表征 GCaMP3 表达（GCaMP+）细胞的功能反应。在一组动物中，视神经横断术（ONT）在钙成像前 3、5 或 7 天进行。
GFP 免疫反应性与 RBPMS 共定位，但与中间神经元标志物在 ONT 和非 ONT（对照）组均不共定位。KA 诱导的钙瞬变在 ONT 后减少（50 μM KA；ΔF/F0 [SD]；对照：1.00 [0.67]，第 3 天：0.50 [0.35]，第 5 天：0.31 [0.28]，第 7 天：0.35 [0.36]；P < 0.05 与对照相比）。ONT 后 GCaMP3+细胞数量也减少（细胞/mm2 [SD]；对照：2198 [453]，第 3 天：2224 [643]，第 5 天：1383 [375]，第 7 天：913 [178]；P < 0.05）。此外，ONT 后对 KA 有反应的 GCaMP3+细胞比例下降（50 μM KA，97%、54%、47%和 58%；对照，第 3、5 和 7 天）。
ONT 后，功能性 RGC 反应在 RGC 体丢失之前丧失，表明 RGC 的解剖学标志物可能低估了 RGC 功能障碍的程度。
