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Kenkichi Baba, Susana Contreras-Alcantara, Gianluca Tosini; Dopamine Entrains The Circadian Rhythm of PER2::LUC Bioluminescence in The Mouse Retinal Pigment Epithelium. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2019.
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The retinal pigment epithelium (RPE) plays an important role in the maintenance of the health and function of photoreceptors. Previous studies have shown that the RPE is also involved in the regulation of disc shedding, a process that is vital for photoreceptor health. This process has been shown to be under circadian control. We have recently reported that Per2 mRNA levels in the RPE-choroid show a clear circadian rhythm in vivo, and the culturing of RPE-choroid tissue obtained from PERIOD 2::LUCIFERASE (PER2::LUC) mice showed robust circadian rhythm in bioluminescence. Additional studies have indicated that the PER2::LUC rhythm was not phase-shifted by light, suggesting that other signals are used by the retina to entrain this circadian rhythm. In this study we investigated whether dopamine (DA) and/or melatonin (MLT) are capable to phase-shift this circadian rhythm.
Eyes were obtained from PER2::LUC mice, the eye-cups containing RPE were dissected, flattened and cultured at 37 oC with 199 medium containing D-Luciferin K salt. The bioluminescence emitted by the tissue was recorded by a luminometer (Lumicycle). After 3-4 days of culture, DA (100uM), MLT (100nM) and DA receptor agonists (SKF38393: 50uM, Quinpirole: 50uM) were added to the culture at different circadian times, and the recording was continued another 5 days.
Administration of DA was able to phase-shift PER2::LUC bioluminescence rhythm in the RPE-choroid during the early subjective day CT0-CT6, the action of DA appeared to be mediated by D2-like receptors, since Quinpirole (D2-like agonist) induced a significant phase-shift during the early subjective day, whereas SKF38393 (D1-like agonist) was not able to produce a significant phase-shift of the PER2::LUC bioluminescence rhythm. Consistently with these results, we observed that Period1 mRNA level was up-regulated significantly up-regulated 1 hour after the addition of DA to the culturing medium. MLT did not produce any significant phase-shift on the circadian rhythm in PER2::LUC bioluminescence.
Our data indicate that DA via D2-like receptors can phase-shift the circadian rhythm in PER2::LUC bioluminescence rhythms in the mouse RPE. Our study also indicates that this new preparation may be useful for elucidating the cellular and molecular mechanisms responsible for the regulation of the physiological rhythmic event in the RPE.
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