Reversal photoisomerization of all-trans retinal in primary cultures of chicken retinal ganglion cells

DIAZ NM; MORERA LP; TEMPESTI T; BAUMGARTNER MT; GUIDO ME

Seattle, Washington

Congreso; Annual meeting Association for Research in Vision and Ophthalmology (ARVO); 2013

Association for Research in Vision and Ophthalmology (ARVO)

Purpose: In previous studies, we demonstrated the presence of intrinsically photosensitive retinal ganglion cells (ipRGCs) expressing the photopigment melanopsin (OPN4) in the wild type (WT) chicken retina as well as light responses mediated by the inner retina of GUCY1* (blind) birds (Contin et al., 2006, 2010; Valdez et al., 2009; Verra et al 2011). Furthermore, we found that the inner retina of chicken has the capability of synthesizing 11-cis retinal in a light dependent manner (ARVO 2012). In the present study, we investigated the ability of primary retinal ganglion cell (RGC) cultures to take up all-trans retinal from the medium and to isomerize it upon light stimulation. Methods: RGC cultures were obtained by immunopanning with a melanopsin X antibody (Contin et al., 2006) to specially enhance the amount of ipRGCs from chicken retinas at embryonic day 8. The cultures were characterized by RT-PCR and immnocytochemistry for different cellular markers (NeuN, NF-200, Tuj1, PROX1, GABA, Glutamine synthase, OPN4X). At day 3, all-trans retinal was added to the cultures. After that, cells were irradiated with white light (1200 lux) or maintained in the dark. Retinoids were extracted and analyzed by HPLC. Results: Primary cultures obtained were highly enriched in RGCs expressing different neuronal markers (NeuN and NF-200) with a typical RGC morphology displaying long processes after 4 days. Other retinal cell types were not significantly detected in the cultures. After cells were fed all-trans retinal overnight and exposed to a 1 h light pulse, we found detectable levels of 11-cis retinal in the cultures by HPLC. Conclusion: As we had previously reported in the chicken inner retina, the RGC cultures exhibit the capacity to isomerize 11-cis retinal from all-trans retinal under light stimulation strongly suggesting the presence of a photoisomerase activity in these cells that would be responsible for the light conversion of retinal in the RGCs. Supported by Agencia Nacional de Promoción Científica y Tecnológica (FONCyT) (PICT 2004 No 967 and PICT Bicentenario 2010 No 647), CONICET, SeCyT-UNC, MinCyT Cordoba