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M.A. Contin, D.M. Verra, M.E. Guido; Regulation of Melatonin Synthesis by Light in Cultured Retinal Ganglion Cells. Phototransduction Cascade . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5524.
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Melatonin is synthesized in the pineal gland and retina of vertebrates. In the retina, melatonin seems to play a major role in the circadian regulation of retinal physiology. The melatonin synthesis is rhythmic and driven by an endogenous circadian clock, which is also regulated by the environmental photic input. Many reports suggest that melatonin synthesis takes place in photoreceptors (PRCs) only; however, we have recently shown that immunopurified retinal ganglion cells from chicken embryos (eRGCs) also synthesize 3H–melatonin from 3H–tryptophan under a circadian bases with higher levels during the day indicating that they are autonomous circadian oscillators (Garbarino et al., 2004). Recently it was found that a subset of mammalian RGCs that project to brain areas involved in a non–visual circuitry, are intrinsically photoreceptive (Berson et al., 2002). Our goals were to investigate if eRGC in cultures may respond to light and if so, what type of phototransduction cascade may take place.
RGCs obtained from E8 by Thy–1 immunopurification were cultured in B27 supplemented DMEM (GIBCO) and synchronized for 4 days to a 12:12 LD cycle; at day five, cells were released to DD or kept under the LD cycle and fed 3H–trypotophan to assess the biosynthesis of 3H–melatonin at different times.
<We found that eRGC cultures synchronized to a LD cycle for 4 days and released to DD, biosynthesized 3H–melatonin in a rhythmic manner with higher levels during the subjective day, whereas light exposure significantly inhibited this synthesis. To investigate the potential phototransduction pathway occurring in the eRGC cultures, we used different effectors to pharmacologically modulate the light effect on melatonin synthesis, such as PLC inhibitors (625 µg/ml neomicyn or 5 µM U73122); Ca2+ chelator (10 µM BAPTA–AM), etc. The data suggest that an invertebrate–like cascade may be acting in this photic pathway. In addition, 11–cis–retinal showed an increase in the inhibitory light effect on melatonin synthesis. To further characterize the RGCs as rhabdomeric photoreceptors, we examine the presence of the ancient transcription factors Pax 6 and Brn 3, which are expressed in the eRGCs together with the G–protein q (Gq), involved in the invertebrate phototransduction cascade.
<The results indicate that chicken eRGCs are intrinsically photosensitive cells similar to rhabdomeric photoreceptors .
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