Intrinsically Photosensitive Retinal Ganglion Cells Detect Light With a Vitamin A–Based Photopigment That is Most Likely Melanopsin

Abstract

Abstract: : Purpose: In mammals, intrinsically photosensitive retinal ganglion cells (ipRGCs) mediate non–image forming visual functions such as pupillary light reflex (PLR) and circadian photoentrainment. This photosensitivity requires melanopsin, an opsin–like protein expressed by the ipRGCs. It remains debated, however, whether melanopsin functions as a photopigment or a photoisomerase, the latter possibility being enhanced by a recent report that melanopsin is expressed also in the mouse retinal pigment epithelium (RPE). To investigate whether a vitamin A–based chromophore is required for photoreception by ipRGCs, and to ask whether melanopsin is the signaling photopigment in these cells, we have studied a knock–out mouse line that lacks RPE65(rpe65^–/–), a key protein for the regeneration of 11–cis–retinal in the RPE. Methods: We compared the rpe65^–/– mouse line with the melanopsin knock–out line (opn4^–/–) with respect to the intrinsic photosensitivity of single ipRGC before and after administration of exogenous 9–cis–retinal, an analog of 11–cis–retinal. In order to isolate the intrinsic light signals from ipRGCs for PLR measurement, we also bred rpe65^–/– mice into a gnat1^–/–cnga3^–/– background, which has non–functional rods and cones as a result of targeted deletions of the genes for the rod transducin α subunit (gnat1) and the cone cyclic GMP–gated channel A3 subunit (cnga3). Results: Rpe65^–/– ipRGCs were ∼20–40–fold less photosensitive at both single–cell and behavioral (PLR) levels, but sensitivity was rescued by exogenous 9–cis–retinal, an 11–cis–retinal analog, In contrast, 9–cis–retinal could not restore intrinsic photosensitivity to melanopsin–ablated ipRGCs, thus inconsistent with melanopsin functioning merely as an photoisomerase in photopigment regeneration. Finally, we did not detect any melanopsin in the RPE or any changes in rod and cone sensitivities resulting from melanopsin ablation in ERG and single cell recordings. Conclusions: These results indicate the requirement of vitamin A–based chromophore for ipRGC photosensitivity and melanopsin is most likely the photopigment in the ipRGCs.

Keywords: photoreceptors • ganglion cells