Abstract
Purpose :
Intrinsically photosensitive retinal ganglion cells (ipRGCs) are photoreceptors important for the function of circadian rhythms, pupillary constriction, and other non-image forming visual behaviors. Recent molecular and electrophysiological analyses suggest the existence of uncharacterized subtypes, indicating an incomplete understanding of ipRGC subtypes. We combined an intersectional genetic strategy and fluorescence in situ hybridization (FISH) to characterize ipRGC subtypes with a focus on dorsal GlyT2-ipRGCs (Berry et al., 2023). These experiments further inform the contributions of ipRGC subtypes to mouse visual processing.
Methods :
Mouse lines with Cre or Flpo under the control of the glycine transporter 2 (GlyT2), vesicular glutamate transporter2 (vGluT2), vesicular GABA/glycine amino acid transporter (vGAT/VIAAT), or opsin 4 (OPN4) promoter were bred with FLTG or Ai80d intersectional reporter mice to generate triple transgenic mice targeting distinct cell populations. A modified FISH protocol from a commercial kit was used to label flat-mount retinas.
Results :
GlyT2-ipRGCs were labeled in GlyT2-C:vGluT2-F:FLTG mouse retinas. Of GFP+ cells, 99% expressed melanopsin (n=144, 2 animals), indicating the intersectional approach restricted labeling to RGCs. Few GFP+ cells expressed melanopsin in GlyT2-C:vGAT-F:FLTG (0.4% of GCL cells, n=248, 3 animals) and OPN4-C:vGAT-F:Ai80d (0.8% of all GFP+ cells, n=647, 2 animals) retinas, suggesting ipRGCs do not express vGAT. RNA was visualized in GlyT2-C:vGluT2-F:Ai80d and wild-type (WT) retinas. GlyT2 mRNA was present in GFP+ cells in transgenic retinas, but OPN4 mRNA+ somas in WT did not contain GlyT2 mRNA (0%, n=42 cells, 2 animals), suggesting ectopic expression of GlyT2 mRNA in GlyT2-Cre mice. To determine if the GlyT2-ipRGC population contained the neuromedin b (NMB) expressing “M1dup” subtype (Tran et al., 2019), NMB and OPN4 mRNA were visualized in GlyT2-C:vGluT2-F:Ai80d mice. NMB was only detected in OPN4 mRNA+ somas (n=10 cells, N=1 animals), and NMB+ somas were enriched in the ventral retina. NMB was not detected in most GFP+ cells (9%, n=11 cells, 1 animal).
Conclusions :
These studies indicate that a combined intersectional and FISH approach can further refine retinal cell populations, providing provide rich information on the spatial localization of RGC types.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.