CIQUIBIC   05472
CENTRO DE INVESTIGACIONES EN QUIMICA BIOLOGICA DE CORDOBA
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
HIGHLY ENRICHED PRIMARY CULTURES OF CHICKEN RETINAL HORIZONTAL CELLS EXPRESS THE PHOTOPIGMENT MELANOPSIN X (Opn4x)
Autor/es:
LUIS P. MORERA; NICOLÁS M. DÍAZ; MARIO E. GUIDO
Lugar:
Cancún
Reunión:
Congreso; FALAN; 2012
Institución organizadora:
FALAN
Resumen:
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. (Supported by ANPCyT-FONCyT
PICT 2010 Nr. 647, CONICET, SeCyT-UNC, and MinCyT of Córdoba).