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G.G. Tosini, K. Sakamoto, C. Liu; Aa–nat mRNA regulation and melatonin synthesis in dystrophic rat retinas. . Invest. Ophthalmol. Vis. Sci. 2004;45(13):4643.
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Purpose: Previous investigations have demonstrated that the mammalian retina synthesizes melatonin. In mammals retinal melatonin synthesis is under control of a circadian pacemaker located within the retina. In non–mammalian vertebrates it has been demonstrated that melatonin synthesis and its circadian control are localized in the photoreceptors. In the present study we investigated the effect of photoreceptors degeneration on melatonin synthesis and circadian control in the rat retina. Methods: Royal College of Surgeons (RCS) rat with normal (rdy+) and dystrophic retinas (rdy) 60 days old were used in this study. At this age degeneration of the retina in rdy+ has advanced to the point where photoreceptors are both histologically and functionally undetectable. Rats (rdy) and (rdy+) were sacrificed at 6 different time points in light: dark (LD) cycles or in constant darkness (DD), retinas were collected and Aa–nat mRNA levels were measured using real–time quantitative PCR. Localization of Aa–nat transcripts within the retina of rdy and rdy+ were carried out by in situ hybridization. Rdy and rdy+ retinas were cultured at constant temperature (33 oC) in DD for 4 days using a flow–through apparatus. Perifusated samples were collected at 3 hours intervals and melatonin levels were measured by radioimmunoassay. Results: Aa–nat mRNA levels showed a clear rhythm in both rdy and rdy+ in LD and in DD. Data obtained using in situ hybridization technique indicated that in rdy+ retinas Aa–nat transcripts were rhythmic in some cells of the inner retina. Cultured rdy and rdy+ retinas showed clear circadian rhythmicity in melatonin release. Conclusions: Our results demonstrate that in RCS rat Aa–nat mRNA levels are rhythmic in rdy+ animals where photoreceptors are both histologically and functionally undetectable. Such rhythmicity is likely to be generated by cells located in the inner nuclear layer or in the ganglion cell layer. The fact that cultured rdy+ retinas show a clear circadian rhythm in melatonin release in vitro demonstrates that circadian rhythmicity can be generated by these dystrophic retinas.
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