NOVEL ISOLATION OF CHICKEN RETINAL HORIZONTAL CELLS

LUIS P. MORERA; NICOLÁS M. DÍAZ; MARIO E. GUIDO

Córdoba

Encuentro; Cuarto encuentro de Jóvenes Investigadores en Neurociencias de Córdoba: Un encuentro Interdisciplinario. Universidad Nacional de Córdoba.; 2012

óvenes Investigadores en Neurociencias

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 highly 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). After centrifugation, cells collected from the different phases were cultured for 4 days and characterized by immunochemistry and morphology. Phases were examined with specific antibodies against Opn4x, HC markers (PROX-1, Islet-1, calretinin) and markers for other retinal cell populations. Prox-1 (+) cells from both the whole retina and the BSA gradient were quantified by flow cytometry. 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 H1- and H3-type HCs (axon-bearing “brush-shaped” and axon-less “candelabrum-shaped” respectively). 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 typical RGCs. 30% of cells from the whole disaggregated retina and 80% from the 2.5% BSA gradient phase were Prox-1 (+). 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.