SENSING LIGHT BY HORIZONTAL CELLS IN THE CHICKEN RETINA: A NEW PLAYER IN THE PHOTORECEPTIVE SYSTEM

MORERA L P; DIÁZ N M; GUIDO M E.

córdoba

Congreso; 16 International Congress of Photobiology; 2014

Retinal ganglion cells (RGCs) expressing the photopigment melanopsin (Opn4) display intrinsic photosensitivity. In the chicken retina, two Opn4 genes, Opn4x and Opn4m have been described of which Opn4x was phound to be confined to the forming GC layer and optic nerve at early embryonic days (E), but by E15 its expression was mostly in Prox1 (+) horizontal cells (HCs) [1]. The aim of this work was to obtain HC primary cultures and to characterize them by biochemical and morphological assays. Disaggregated chicken retinas at E15 were subject to a discontinuous 1 to 4% bovine serum albumin (BSA) gradient [2]. Phases were examined with specific antibodies against Opn4x and HC markers. Then, immunopanning against Opn4x with the 2,5 % phase was performed to obtain cultures of HCs that express this photopigment. Primary cultures were also examined by flow cytometry and RT-PCR. Finally HC cultures were exposed to light or kept in the dark to assess intrinsic photosensitivity by Calcium Imaging. The results show that only the fraction corresponding to the 2.5% BSA contained most cells displaying PROX-1 immunoreactivity. Also Opn4x-immunoreactivity was observed in this phase. Cultures obtained by immunopanning were highly enriched in Opn4x (+) HCs and exhibited positive light responsiveness as compared with dark controls. In conclusion, by means of this methodological procedure we obtained primary cultures of HCs expressing Opn4x and we also provide the first insight about their intrinsic light sensitivity.