Novel Inner Retinal Photoreceptors in Non-mammalian Vertebrates

RÍOS MAXIMILIANO; MOERA LUIS P.; DIAZ, NICOLÁS; DANIELA M VERRA; CONTIN MARIA ANA; GUIDO MARIO EDUARDO

Tucumán

Congreso; Tercera Reunión de Fotobiólogos Moleculares Argentinos - 2016; 2016

Grupo Argentino de Fotobiología, GRAFOB

The vertebrate retina contains three different types of photoreceptors: the visual photoreceptors cones and rods, and the intrinsically photosensitive retinal ganglion cells (ipRGCs) expressing the photopigment melanopsin (Opn4) that converged through evolution to regulate visual and non-image forming tasks (Diaz et al 2015). In the chicken, there are two Opn4 genes: Opn4m and Opn4x, the mammalian and Xenopus orthologs respectively. We have previously shown that the chicken retina expressed both proteins with Opn4m confined to the GC layer and Opn4x expressed in the GC layer at first and in horizontal cells (HCs) at later developmental stages (Verra et al. 2011). Embryonic RGC primary cultures expressing both Opn4s respond to light through a photocascade involving phospholipase C activation and calcium mobilization (Contin et al 2006, 2010). Here, we further characterize primary cultures of both Opn4x (+) populations of inner retinal cells (RGCs and HCs) and investigate their intrinsic photosensitivity as well as the visual cycle. For this, we obtained highly enriched Opn4x (+) cells by a chemical gradient (Morera et al 2012) and immunopanning, and assessed positive light responses by calcium fluorescence imaging as compared with dark controls. The expression of different circadian markers such as the clock genes Bmal1, Clock, Per2 and Cry1, and the key melatonin synthesizing enzyme, arylalkylamine N-acetyltransferase (AA-NAT), appears very early in development in both cell populations as well as components of the non-visual phototransduction cascade, the mRNAs for the G protein q (Gq) and Opn4x . When we performed calcium imaging, positive light responsiveness at different light intensities and exposure times were observed in both primary cultures fed exogenous all-trans retinal (atRal) as compared with controls kept in the dark. Moreover, RGC cultures were able to isomerize atRal into 11cRal in the presence of light and to further metabolize it to all-trans retinal and all-trans retinyl palmitate. These results support the idea of a light dependent mechanism of chromophore regeneration in addition to bistability described in invertebrate opsins, non-visual Opn4 photoreceptors and endogenous clocks converge all together in these inner retinal cells at early developmental stages, and moreover, ipRGCs and HCs acting as non-classical photoreceptors may cooperate to detect light that regulates diverse non-visual functions. Referencias 1-Contin M. A., Verra D. M. and Guido M.E., The FASEB J 20: 2648-2650, 20062-Contín MA, Verra DM., Salvador G, Ilincheta M, Giusto NM. and Guido ME. IOVS, 51(11):5491-8, 20103-Verra DM, Contín MA, D. Hicks, Guido ME. IOVS 52(8):5111-204-Morera L.P., Diaz N.M., Guido M.E., Exp Eye Res, 101:44-8.20125-Díaz N. M.; Morera L. P. and Guido M.E., Photochem and Photobiol, 92(1):29-44, 2015