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

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

Berlin

Congreso; XX Biennial meeting of the Internartional Society for Eye Research.; 2012

ISER

PRIMARY CULTURES OF CHICKEN RETINAL HORIZONTAL CELLS EXPRESS THE PHOTOPIGMENT MELANOPSIN X Mario E. Guido, Luis P. Morera and Nicolás M. Díaz. CIQUIBIC-Department of Biological Chemistry, Faculty of Chemical Sciences, National University of Cordoba-CONICET, 5000 Córdoba, Argentina Email: mguido@fcq.unc.edu.ar, TEL 54-351-4334171 ext 3429/3430/FAX ext 3406 Purpose: Retinal ganglion cells (RGCs) expressing the photopigment melanopsin (Opn4) display intrinsic photosensitivity. In the chicken retina, two Opn4 genes, the Xenopus (Opn4x) and the mammalian (Opn4m) orthologs were described. Opn4m was shown to be restricted exclusively to the GC layer whereas Opn4x was limited to the forming GC layer and optic nerve at embryo day 8 (E8), but by E15 its expression was mostly in Prox1 (+) horizontal cells (HCs) (Verra et al., 2011). The aim in this work was to purify HCs from the chicken retina to obtain primary cultures for further characterization. Methods: Disaggregated chicken embryonic retinas at E15 were subjected to a discontinuous bovine serum albumin (BSA) gradient of 1 to 4% concentrations (Morera et al., 2012). After centrifugation, cells collected from the different phases were cultured for 4 days and characterized by immunochemistry and cell 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 expression was also determined by flow cytometry in two populations, cells from whole dissagregated retina and retinal cells from the 2.5% BSA gradient. Results: The results show that only the fraction corresponding to 2.5% BSA contained most cells displaying PROX-1 and Islet-1 positive immunoreactivities with a typical HC morphology. Based on an accurate morphological analysis, a number of cells in this fraction resembled H1- and H3-type HCs (axon-bearing “brush-shaped” and axon-less “candelabrum-shaped” HCs respectively). Strikingly, Opn4x-immunoreactivity was observed in cultures from both the 2.5 and 3 % BSA gradient phases. It is noteworthy that the 3% phase contains cells that express the neuronal filament of 200 KDa (NF200) and display longer processes that morphologically resemble RGCs. In flow cytometry we found a 30% of cells labeled with Prox-1 in whole dissagregated retina (data accordingly with literature) and a 80% of expression of Prox-1 in cells obtained of the 2.5% of BSA gradient. Conclusions: In conclusion, by means of this method we selectively separated specific retinal cell types and obtained primary cultures highly enriched in HCs that express the non-visual opsin Opn4x. Supported by ANPCyT-FONCyT PICT Bicentenario 2010 Nr. 647, PICT, CONICET, SeCyT-UNC, and MinCyT of Córdoba.