PROJECT SUMMARY It is now clear that the mammalian retina has at least one other photoreceptor class besides rods and cones, consisting of a subpopulation of retinal ganglion cells (RGCs) that express the pigment melanopsin (OPN4) and are intrinsically photosensitive (ipRGCs). These cells project predominantly to non-image-vision centers in the brain, serving functions such as circadian photoentrainment and pupillary light reflex. At the same time, ipRGCs project moderately to the brain's image-vision centers, presumably serving subtle image- forming visual functions such as possibly providing information about absolute light intensity in the visual scene. IpRGCs are known so far to comprise 6 subtypes, M1-M6, which differ in the level of melanopsin content, photosensitivity, somatic and dendritic-field size, exact locations of their dendritic arborizations in the retina's inner plexiform layer, and the detailed locations of their axonal projections in the respective brain targets. To fully understand the ipRGC system, it is important to know their light-response properties and the underlying mechanisms. In recent years, we have made great progress in understanding M1-ipRGCs, including the molecular identities of several key components in phototransduction such as Gαq-subfamily members, the PLCβ4 enzyme and the TRPC6,7 channels. These components closely resemble those found in fly-eye phototransduction. Most recently, we have made the exciting discovery of yet another phototransduction pathway, found in M2- and M4-ipRGCs. This pathway involves a light-induced elevation of cyclic nucleotide, which opens HCN channels (channels gated by membrane hyperpolarization and cyclic nucleotide) to produce membrane depolarization and action potentials. This application constitutes a continuation of these successful investigations. Aim 1 is to seek some details of the HCN-phototransduction pathway still outstanding, including establishing (i) whether cGMP or cAMP is the second messenger, (ii) the identity of the effector enzyme that controls the second messenger, and (iii) the identity of the G protein upstream of the HCN-pathway. Aim 2 is to characterize the phototransduction mechanism(s) still unknown in M3-, M5- and M6-ipRGCs. Our speculation is that the same two pathways are involved, but this requires verification. Aim 3 is to quantify the relative importance of the two signaling pathways in different cell subtypes. We shall study sensitivity, intensity-response relation, single- photon response, and response kinetics of each pathway, hopefully eventually to derive correlations with the respective subtype's macroscopic functions.