GCaMP3 expressing cells in the ganglion cell layer of Thy1-GCaMP3 transgenic mice before and after optic nerve injury

Abstract

The genetically encoded green fluorescent protein-based calcium sensor, GCaMP, has been used to detect calcium transients and report neuronal activity. We evaluated the specificity of GCaMP3 expression to retinal ganglion cells (RGCs) of the transgenic Thy1-GCaMP3 mouse line in healthy control animals and in those after optic nerve transection (ONT). Retinas from control mice (n = 4) were isolated and stained for RNA-binding protein with multiple splicing (RBPMS) and choline acetyltransferase (ChAT), specific markers for RGCs and cholinergic amacrine cells, respectively. GCaMP3 expression was enhanced with green fluorescent protein (GFP) immunoreactivity. In one subset of animals, ONT was performed 3, 7, or 14 days before sacrifice (n = 4, 4, 4, respectively). Cells positive for GCaMP3, RBPMS, ChAT, as well as the population of co-labeled cells, were quantified. In another subset of animals (n = 4), in vivo confocal scanning laser ophthalmoscope imaging was performed in the same mice at baseline and at 3, 7 and 14 days after ONT. The mean (SD) densities of GCaMP3, RBPMS, and ChAT expressing cells in control retinas were 2663 (110), 3401 (175), and 1041 (47) cells/mm², respectively. Of the GCaMP3+ cells, 92 (1)% were co-labeled with RBPMS, while 72 (1)% of RBPMS-labeled cells expressed GCaMP3. ChAT expressing cells were not co-labeled with GCaMP3. The number of GCaMP3+ and RBPMS+ cells decreased dramatically after ONT; 78%, 39%, and 18% of GCaMP3+ and 80%, 40%, and 15% of RBPMS+ cells, relative to control retinas, survived at 3, 7, and 14 days after ONT. However, the number of ChAT+ cells did not change. There was a progressive decrease in GCaMP3 fluorescence after ONT in in vivo images. The majority of RGCs in the ganglion cell layer of Thy1-GCaMP3 mice express GCaMP3. There was an expected progressive and specific loss of GCaMP3 expression after ONT. Thy1-GCaMP3 transgenic mice have potential for longitudinal assessment of RGCs in injury models.

Keywords: Confocal scanning laser ophthalmoscope; Fluorescent protein; Optic nerve transection; Retina; Retinal ganglion cell; Transgenic mice.