PHOTOSENSITIVE INNER RETINAL CELLS IN AN AVIAN MODEL OF BLINDNESS ARE INVOLVED IN THE NON-VISUAL CONTROL OF PHYSIOLOGY AND BEHAVIOR
Mario E. Guido1, Diego Valdez1, Daniela Verra1, Maria A Contin1, Nicolas Diaz1, Paula Nieto1, Victoria Acosta1, Gabriela Salvador2, Monica Ilincheta2 and Norma Giusto2.
1CIQUIBIC-Department of Biological Chemistry, Faculty of Chemical Sciences, National University of Cordoba-CONICET, 5000 Córdoba, Argentina
2 INIBIBB -CONICET, Universidad Nacional del Sur, Bahía Blanca, Argentina
Email: mguido@fcq.unc.edu.ar, TEL 54-351-4334171/FAX 54-351-4334074
Background and aims: In the vertebrate retina, there exist two classes of photoreceptors, cones and rods involved in day and night vision respectively. Recent evidences in mammals (Berson et al., 2002) and from our group in the chicken have shown that the retina contains a subset of intrinsically photosensitive retinal ganglion cells (RGCs) (Contin et al., 2006). Here, we investigated light responses and the presence of photopigments in GUCY1* birds, a blindness model of Leber Congenital Amaurosis, and wild type controls, as well as in primary cultures of RGCs.
Methods: Chicken embryonic retinas from days 4 to 18 (E4 to E18) and post hatching (P5-10) were processed for RNA extraction and RT-PCR, protein detection by WB and immunochemistry, or biochemical assays. Primary cultures of chicken RGCs were obtained at E8 by anti-Thy-1 immunopanning and processed. In the GUCY1* chickens, we assessed the pupillary light responses in dark-adapted animals (consensual reflex) to monochromatic bright light of different wavelengths, and the daily rhythms of feeding after synchronization to diverse LD cycles with cool white and blue fluorescent light of high intensities.
Results: GUCY1* birds lacking functional cones and rods, still perceive light through the inner retina. This photosensitivity is responsible for the synchronization of daily feeding rhythms and regulation of pupil reflexes by using a vitamin A-based opsin with a λmax ~484 nm. Light responses persist with the complete pineal gland occlusion but are lost with enucleation. RGCs express two genes for the photopigment melanopsin (Opn4m and Opn4x) and the G protein q likely involved in the non-visual photocascade. The GUCY1* retina shows a high and widespread expression of Opn4x- protein. Primary cultures of RGCs from chicken embryos at E8 express Opn4m and Opn4x, and have intrinsically photosensitive cells acting through a phototransduction cascade which involves the activation of the phosphoinositide cycle with a significant and very rapid increase in levels of IP3, different phospholipids (PA, PIP and PIP2), and Ca2+ mobilization.
Conclusions: Intrinsically photosensitive cells are present in the inner retina of blind birds, express the photopigments Opn4x and Opn4m. Remarkably, these cells appear very early in the development before any sign of vision may occur, and form part of a non-visual circuit involved in diverse non-image forming functions.
Acknowledgments: Supported by ANPCyT-FONCyT, PICT 04 No 967 and PICT05 Nº 31971, CONICET, SeCyT-UNC, and MinCyT of Córdoba. MG is a fellow of the John Simon Guggenheim Memorial Foundation, 2009.
