Abstract
Purpose: :
In previous studies, we demonstrated the presence of intrinsically photosensitive retinal ganglion cells (ipRGCs) expressing the photopigment melanopsin 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). Therefore, bistable photopigments presumably responsible for photosensitivity may use a novel visual cycle for chromophore regeneration that relies upon distinct isomerases through light-dependent or -independent processes (Wang et al., 2010). In the present study, we investigated the expression of the photoisomerase RGR and zebrafish RPE65c (cone cycle isomerase, Takahashi et al., 2011) as well as the activity of visual cycle enzymes and levels of retinal chromophores (11-cis RAL and all-trans RAL) in the inner retina of WT birds.
Methods: :
Dark-adapted young WT chickens were exposed to a 30 min light pulse (2000 lux) or kept in the dark and sacrificed under dim red light. Eyes were dissected and lyophilized, and the inner retina or different cell layers (ganglion cell layer: GCL, inner nuclear layer: INL) obtained by the Scotch tape method (Guido et al., 2001). Samples were processed for RNA extraction, RT-PCR, immunochemical (RPE65c antibody from Dr. Ma at UOHSC) or enzyme activity assays. Retinoids extracted were analyzed by HPLC.
Results: :
Levels of 11-cis RAL were higher in the GCL and INL samples from light-adapted birds as compared with levels of all-trans RAL which were higher in the dark samples. Also, we observed the presence of RGR and RPE65c-like protein in different inner retinal cell layers: RGR in the GCL while RPE65c-like protein in the INL and IPL. Isomerase activity was found in the INL only while acyl CoA:retinol acyltransferase (ARAT) was observed in both INL and GCL.
Conclusions: :
These results demonstrate that in the chicken, different inner retinal cell layers differentially express RGR and RPE65c-like proteins and selectively display enzyme activities for isomerase and ARAT. The different light-dark effects on retinoids and the differential distribution of photoisomerases present in the retina strongly suggest that light-induced isomerization of retinal in the chicken inner retina differs from those described in the classical and cone-visual cycles. These results suggest that a novel visual cycle exists in the chicken inner retina to support retinoid isomerization in ipRGCs or other photosensitive cells.
Keywords: photoreceptors • ganglion cells • retinoids/retinoid binding proteins