Novel Phototransduction Pathway in Intrinsically Photosensitive Retinal Ganglion Cells (ipRGCs)

Abstract

Purpose : M1-ipRGCs utilize a rhabdomeric-photoreceptor-like phototransduction pathway mediated by melanopsin, G_q-subfamily members, PLCβ4, and TRPC6,7 channels. Phototransduction in the other subtypes of ipRGCs has not been explored. Here we report in mouse that the intrinsic photocurrent in M4-ipRGCs, as well as a substantial portion of it in M2-ipRGCs, involves HCN channels instead of TRPC channels or PLCβ4.

Methods : The photocurrents of M2- and M4-ipRGCs were recorded by whole-cell, patch-clamp recording from flat-mount mouse retina in the presence of synaptic blockers. Photo-uncaging experiments used intracellular caged cyclic nucleotide delivered from whole-cell pipette. To disrupt HCN channel function, mouse retina was infected by intravitreal injection of AAV2 virus carrying a mutant HCN channel subunit. Animal circadian photoentrainment was monitored from wheel-running activity. PLR (pupillary light reflex) was recorded as described in Xue et al., Nature 479: 67-73, 2011.

Results : We found that M2- and M4-ipRGCs had a persistent intrinsic photocurrent in PLCβ4^-/- or TRPC6,7^-/- genotype, even in TRPC1,3,4,5,6,7^-/- genotype. This photocurrent was insensitive to Ruthenium Red, a wide-spectrum TRPC-channel blocker, but completely blocked by ZD7288, a HCN-channel blocker. The voltage dependence of the Trpc6,7^-/- photocurrent was consistent with HCN-channel properties, and its amplitude was positively correlated with the hyperpolarization-induced I_h current. Immunostaining and/or genetic labeling also revealed HCN4-channel, but not CNG channel, expression in retinal melanopsin-positive cells. A virally-expressed mutant HCN channel subunit significantly reduced the light responses of M2- and M4-ipRGCs. We found that phototransduction in M1-ipRGCs, but not M2- or M4-ipRGCs, depends on Gα_q, Gα₁₁ and Gα₁₄. Photo-uncaged cyclic nucleotide in Opn4^-/- M2- or M4-ipRGCs induced an inward current similar to the melanopsin-mediated response. Finally, Trpc6,7^-/-;rd/rd mice (which have lost rod/cone signals and TRPC6,7-dependent signals in ipRGCs) still exhibited PLR and circadian photoentrainment, although both functions were partially impaired.

Conclusions : In M2- and M4- ipRGCs, there is a novel phototransduction pathway mediated by HCN channels and cyclic nucleotide. This pathway makes a significant contribution to in vivo PLR and circadian photoentrainment.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.