Circadian phototransduction and melatonin synthesis in chicken retinal ganglion cells. Maria A. Contin, Daniela Verra and Mario E. Guido. Department of Biological Chemistry-CIQUIBIC, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba-CONICET, Córdoba, ARGENTINA. mguido@mail.fcq.unc.edu.ar
The retina plays a major role in the vertebrate circadian system; it is responsible for photoreception of the environmental lighting conditions that synchronize central oscillators and it is rhythmic itself, containing circadian clocks that generate daily rhythms in molecular and physiological parameters. At present, it remains uncertain whether the different retinal cell types contain functional circadian clocks and non-visual photoreceptors and whether they both may converge into the same cell population. In the chicken retina, it has been shown that photoreceptors (PRCs), retinal ganglion cells (RGCs) and inner nuclear layer cells (INL) display daily rhythms in gene expression and metabolic activities. We have investigated whether RGCs may contain autonomous circadian oscillators and non-visual photoreceptors, and if so, the nature of the photo-cascade involved. We found that primary cultures of immunopurified chicken embryonic RGCs at day 8 express ancestral rhabdomeric photoreceptor markers: the transcription factors Pax6 and Brn3, the photopigment melanopsin and the G-protein q but not markers for ciliary PRCs (a-transducin and Crx). RGC cultures synchronized by medium exchange synthesized 3H-melatonin from 3H-tryptophan with levels peaking 8 h later after synchronization and displayed self-sustained daily rhythms in mRNA expression of the key melatonin-synthesising enzyme, serotonin N-acetyltransferase for at least three cycles with a period near 24 h (Garbarino et al., 2004). To investigate the photoreceptive capability of RGCs, we assessed the direct effect of light on 3H-melatonin synthesis in RGC cultures synchronized to 12:12 h light dark cycles. After synchronization, RGCs maintained in constant darkness exhibited a daily variation in 3H-melatonin levels peaking at subjective day, which was significantly inhibited by light. This light effect was further increased by the chromophore all-trans retinal, and suppressed by specific inhibitors of the invertebrate photo-cascade involving phosphoinositide hydrolysis and Ca2+ mobilization. The results demonstrate that chicken RGCs contain autonomous circadian oscillators and are intrinsically photosensitive operating via an invertebrate-like phototransduction cascade that uses a vitamin A-based chromophore. Remarkably, these oscillatory and photoreceptive capabilities can be essential for the embryo to temporally regulate vital developmental programs and physiology in response to the environmental illumination conditions before any sign of formal vision may occur.
