Vertebrate Ancient Opsin (VA): Not Just a Teleost Opsin

Abstract

Purpose: : It has been known since the 1930s that birds possess deep brain photoreceptors. Further studies confirmed that these photoreceptors are located within the hypothalamus and regulate both circadian and reproductive responses to photoperiod. Their cellular and molecular identity has remained elusive, although an opsin/vitamin A based pigment has been implicated. Vertebrate ancient (VA) opsin first isolated from Atlantic salmon and subsequently from many species of teleost fish is expressed in the retina, pineal and the sub-ependymal cells of the hypothalamus. They form functional photopigments with a _max of 460-500nm. The distribution of VA within the teleost brain and the absence of a strong molecular candidate for the avian deep brain photoreceptor, prompted a search for a VA gene in birds.

Methods: : Bioinformatic approaches, confirmed using molecular biology techniques including 5’ and 3’ RACE, were used to identify VA opsin from adult chicken. A previously described in vitro expression system was used to determine whether chicken VA could form a functional photopigment in Neuro-2A cells. Immunohistochemistry and Western blotting were then used to determine tissue localisation.

Results: : A 972bp fragment obtained from adult chicken brain cDNA, shows 70% identity to zebrafish VA, and encodes a predicted protein of 323 aa containing all the features expected in an opsin. Whole-cell patch clamp recordings from transfected Neuro-2A cells demonstrated that chicken VA can indeed elicit a retinal-dependent light response in vitro. Analysis of adult chicken tissue by Western blotting, using an antibody (VA-85), identified a single protein which resolved at ~50kDa in hypothalamus. Immunohistochemistry with VA-85 demonstrated that chicken VA is expressed within a population of hypothalamic neurones with extensive projections to the median eminence.

Conclusions: : Here we report the isolation and functional expression of an avian orthologue of VA opsin. Further, we show that in chicken this photopigment is expressed within a population of hypothalamic neurones. These results will allow a transition from studies which reveal the existence of deep brain photoreceptors to experiments that define how this photoreceptor system functions.

Keywords: opsins • circadian rhythms