Sensing light by horizontall cells in the chicken retina: a new player in the photoreceptive system

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

Encuentro; ARVO 2015 Annual Meeting; 2015

arvo

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 found in the GC layer at embryo day 8 (E8), but mostly in Prox1 (+) horizontal cells (HCs) by E15. The aim of this work was to obtain HC primary cultures and to characterize them by biochemical assays.Methods:Disaggregated chicken retinas at E15 were subject to a discontinuous 1-4% bovine serum albumin (BSA) gradient. Phases were examined with specific Opn4x and HC marker antibodies. Immunopanning against Opn4x with the 2.5% phase was performed to obtain HC cultures expressing this opsin. HC cultures previously characterized by flow cytometry and RT-PCR were exposed to light to assess intrinsic photosensitivity by calcium imaging with Fluo-4 AM.Results:The 2.5% BSA phase contained most cells displaying PROX-1 and Islet-1 positive immunoreactivities with a typical HC morphology. Moreover, a number of cells in this fraction resembled typical axon-bearing ?brush-shaped? H1- and axon-less ?candelabrum-shaped? H3-type HCs. Strikingly, Opn4x-immunoreactivity was observed in cultures from both the 2.5 and 3% BSA phases. By flow cytometry we found a 30% of cells labeled with Prox-1 in whole dissagregated retinas and 80% of Prox-1 (+) cells from the 2.5% BSA phase. Then, by immunopanning we obtained highly enriched Opn4x (+) HC cultures. When we examined components of the non-visual phototransduction cascade, the mRNAs for the G protein q (Gq) and Opn4x were observed. Finally when we performed calcium imaging in the HC cultures, positive light responsiveness at different light intensities and exposure times were observed as compared with controls kept in the dark.Conclusions:Results show that primary cultures of HCs obtained at E15 strongly express Opn4x and respond to light by significantly increasing Ca2+ levels. Overall, these observations provide the first evidence about the intrinsic photosensitivity of HCs suggesting the existence of a novel type of photosensitive cells in the avian retina, novel connections between outer and inner retina and a higher degree of complexity in mechanisms of light detection, processing and transmission to the brain.