Light responses of the non-visual opsin Opn3 in avian Müller cells

MARCHESE, N.A.; RIOS, M.N.; GUIDO, M.E.

Porto

Congreso; XIV European meeting on glial cells in health and disease; 2019

Network Glia

Müller cells are the main glial cell type present in vertebrates? retina. They are located all along the inner retina playing a crucial role in its metabolism by debris phagocytosis, K+ uptake, and trophic factors and neurotransmitters release. Opsin-based pigments in vertebrates? retina act as photoreceptors in different cell types regulating image- and non-image forming tasks. These lasts responses have been related to the non-visual photopigments melanopsin (Opn4), neuropsin (Opn5) and encefalopsin (Opn3). Particularly, the photosensitive horizontal and retinal ganglion cells expressing melanopsin x are implicated in the photic entrainment of daily activity rhythms, pupillary reflex and pineal melatonin suppression observed in blindness conditions. The present work aims to further characterize the intrinsic photoreceptor capacity of the inner retina in vertebrates; therefore, the expression, localization and the light-response of the non-canonical opsin Opn3 was addressed in primary cultures of chicken Müller cells by PCR, WB, immunocytochemistry, and fluorescence calcium imaging microscopy. Opn3 and Opn5 were found to be expressed in enriched Müller cell primary cultures, kept for two weeks from embryonic day 8. When exposed to blue light for 1h Opn3 expression levels were significantly increased and its subcellular localization was modified from the nucleus to the cytoplasm. When observed 1h after light exposure (in dark conditions), Opn3 showed further increased levels; whereas its nuclear localization was restored. These responses are dependent on protein synthesis as cycloheximide treatment prevented the light-induced increase and cellular re-distribution of the opsin. Interestingly, in another set of experiments, a Müller cell subpopulation in culture (26%) responded to a brief blue light pulse (20sec) by increasing intracellular Ca2+ levels up to 20% from basal levels; a response that was sustained for at least 100 sec and was also accompanied by changes in cell volume. Furthermore, HEK-293 cells, that do not express Opn3, were not able to respond to blue light in the experimental conditions tested but they did so in the presence of ionomycin. Overall, our results show that the novel opsin Opn3 is expressed in glial cells of the chicken retina, with protein levels strongly regulated by light and with a direct photic responses to blue light stimulation; thus, suggesting new roles for Müller cells in retinal physiology, possibly in visual and nonvisual processes regulated by light such as the setting of the biological clock.