A Viral Method for Optogenetic Control of Intrinsically Photosensitive Retinal Ganglion Cells

Abstract

Purpose: We sought a method for selective optogenetic control of intrinsically photosensitive retinal ganglion cells (ipRGCs). Cre-dependent viruses are an attractive option, given the availability of mice with Cre knocked into the melanopsin locus (Opn4^Cre/+; Ecker et al 2010). However, this cre is ‘leaky’ in some contexts, so specificity is not guaranteed. Also, some cell types resist infection by certain viral serotypes, so neither is efficacy assured. We assessed the efficacy and specificity of a cre-dependent optogenetic virus by comparing morphology and projections of infected cells to those of the 6 known ipRGC types.

Methods: We made intravitreal injections of AAV2-EF1a-DIO-hChR2(H134R)-mCherry (UNC Vector Core) in adult Opn4^Cre/+ mice carrying one Z/EG allele (Jax003920), a transgene that expresses GFP cre-dependently. After 4-6 weeks, we assessed infection from the mCherry which, along with GFP, we immuno-enhanced. Melanopsin was visualized by immunolabeling with tyramide signal amplification (TSA). The distributions of these markers were studied in confocal image stacks.

Results: The virus infected all types of ipRGCs, recognizable from their stratification, morphology, melanopsin staining and projections, though M1 cells were underrepresented. Labeled dendrites stratified only in the ON and/or OFF melanopsin bands. We never encountered cells morphologically distinct from ipRGCs. Though most virally labeled cells were co-labeled with GFP by the cre-dependent Z/EG reporter, about 40% were not; these were nonetheless ipRGCs as well, mainly M5 and M6 cells. Thus, Z/EG underreports Opn4^Cre. Axons of infected RGCs terminated exclusively in structures known to receive ipRGC input, including the suprachiasmatic nucleus (SCN), intergeniculate leaflet (IGL), ventral lateral geniculate (vLGN), and olivary and posterior pretectal nuclei (OPN; PPN). Appropriate laminar specificity was seen for terminals in the superior colliculus (SC, especially stratum opticum) and dorsal lateral geniculate nucleus (dLGN; limited to the expected ventromedial sector).

Conclusions: Convergent evidence shows that the viral vector tested here selectively and effectively targets channelrhodopsin to ipRGCs. We now have a method for targeted optogenetic stimulation of ipRGC afferents in the brain. Variants of this viral system should permit selective ipRGC silencing, killing, or other genetic manipulations.