Physiology and Development of Inner Retinal Photoreception
Sexton, Timothy Jack
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Melanopsin is the photopigment in intrinsically photosensitive retina ganglion cells (ipRGCs). Melanopsin endows ipRGCs with a photo-response that can act independently or be summed directly with input from rods and cones, with ipRGCs acting as the conduit for all non-visual photic information. ipRGCs project primarily to the suprachiasmatic nuclei and the olivary pretectal nuclei, subserving circadian entrainment to lighting cycle and the pupillary light response respectively. Here I examined at 3 aspects of in situ melanopsin function in whole retina: 1) the melanopsin photocycle, 2) ipRGC recovery from light and 3) mechanisms of melanopsin adaptation. First, I found that melanopsin is resistant to bleaching by visible light. Conversely, ultraviolet light (UV) can substantially bleach melanopsin. Using UV bleaching and retinoid supplementation techniques, <italic>cis</italic>-retinal was determined to be the <italic>in vivo</italic> chromophore preference of apo- melanopsin. Neither all-<italic>trans-</italic>retinal nor the Müller glial photocycle product, <italic>cis</italic>-retinol, can restore ipRGC function after UV bleaching. Melanopsin is resistant to chemical bleaching with hydroxylamine both in light and in darkness, a characteristic of bistable pigments. In sum, these experiments are consistent with the hypothesis that melanopsin is a bistable pigment requiring 11-<italic>cis</italic>-retinal to bind the apo-form to constitute a functional photopigment. Secondly, I found the ability of ipRGCs to recover from short and long light exposures decreases over the course of development from postnatal (P) day 8 to P30. A concurrent decrease in photosensitivity is observed over the same period as measured by on-latency, maximal firing rate, off-latency and total spike count during light exposure in response to a 1-min duration, 480 nm light stimulus. The age dependent decrease in recovery cannot be reversed with exogenous 9-<italic>cis</italic>-retinal supplementation but can partially be overcome with a 10-fold increase in light intensity, i.e., ipRGCs did not bleach but adapted to light. This observation suggests that adaptation mechanisms but not photocycle mechanisms change in ipRGCs over time. The most dramatic difference is found in the ability of P8 Type 1 ipRGCs to recover nearly completely from a 1-hr 480 nm light exposure within 20-min of incubation in the dark. At all ages studied, all other ipRGC subtypes recover to more modest levels of 60% or less of the pre-exposure levels over the course of a 1-hour dark incubation period. Finally, I found that the G-protein related kinase 2 (GRK2) plays a small role in melanopsin inactivation. I thus investigated whether the age dependent changes in adaptation were regulated by GRK2 by knocking out GRK2 specifically in melanopsin expressing cells. Previous <italic>in vitro</italic>work indicated GRK2 was responsible for melanopsin inactivation. However, in the mutant only small increases in the response decay constant and in the off-latencies were found, indicating that GRK2 is either not the primary, or not the only kinase responsible for melanopsin phosphorylation.