HIGHLY ENRICHED PRIMARY CULTURES OF CHICKEN RETINAL HORIZONTAL CELLS EXPRESS THE PHOTOPIGMENT MELANOPSIN X (Opn4x)

MORERA L P; DÍAZ NM; GUIDO M E.

Córdoba

Congreso; Sociedad Argentina de Neurociencias; 2012

SAN

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, Opn4m was restricted to the GC layer whereas Opn4x was limited 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) (Verra et al., 2011). The aim of this work was to purify HCs from the chicken retina to obtain primary cultures enriched in these cells. Disaggregated chicken embryonic retinas at E15 were subjected to a discontinuous 1 to 4% bovine serum albumin (BSA) gradient (Morera et al., 2012). Cells from the different phases were cultured and characterized by immunochemistry and morphology. The results show that only the fraction corresponding to 2.5% BSA contained most cells displaying PROX-1 and Islet-1 (+) immunoreactivities with a typical HC morphology resembling typical HCs. 30% of cells from the whole disaggregated retina and 80% from the 2.5% BSA gradient phase were Prox-1 (+). Strikingly, Opn4x-immunoreactivity was observed in cultures from both the 2.5 and 3 % BSA gradient phases. Cells from the 3% phase express the neuronal filament of 200 KDa (NF200) and display longer processes resembling RGCs. In conclusion, by means of this method we selectively separated specific retinal cell types and obtained primary cultures highly enriched in HCs expressing the non-visual opsin Opn4x.