Determining the role of phosphorylation of mouse melanopsin in non-image forming vision

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UMBC Theses and Dissertations

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2017-01-01

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dissertations
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Biological Sciences

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Biological Sciences

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Abstract

Purpose: Melanopsin is a visual pigment, expressed in intrinsically photosensitive retinal ganglion cells (ipRGCs) of the mammalian retina, that plays a major role in non-image forming visual behaviors like the pupillary light reflex, circadian photoentrainment, and sleep. It is hypothesized that melanopsin-mediated phototransduction is terminated by the phosphorylation of melanopsin'sC-terminus by a G-protein coupled receptor kinase, followed by ?-arrestin activation and binding. Little is known about the contribution of melanopsin phosphorylation to ipRGC physiology and its influence on non-image forming behaviors. We investigated the role of melanopsin C-terminus phosphorylation on non-image forming behaviors by generating a phosphorylation deficient melanopsin mutant that lacks all putative C-terminal phosphorylation sites (C-phosphonull). Method: C-phosphonull mice were generated by expressing the C-terminal phosphorylation deficient melanopsin mutant in Opn4Cre/Cre mice by intravitreal delivery of an AAV2 construct for targeted expression of melanopsin in ipRGCs. Results: We found that C-terminal phosphorylation of melanopsin has a direct influence on the dilation kinetics of the pupillary light reflex, due to the observation that the C-phosphonull mice exhibit prolonged pupil constriction to a high intensity blue light stimulus, and have a half-time of dilation of 9.43min as compared to only 6s in wildtype animals. This phenotype is reflected in the physiology of ipRGCs as determined by both single cell and multielectrode recordings, where ipRGCs sustain action potentials for at least 15min to a 5s blue light stimulus. We also found that exogenous expression of melanopsin in melanopsin knockout animals enhances their ability to shift their activities to a delayed light dark cycle (a jet lag paradigm) and rescues a deficit in negative masking (reduction in activity exhibited by nocturnal animals in response to aberrant light during subjective night). Conclusion: Our results suggest that C-terminal phosphorylation of melanopsin influences the kinetics of some non-image forming behaviors and physiology, but does not completely explain deactivation of melanopsin and that there may be alternate mechanisms that play a role in its absence.