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G.-X. Ruan, S. Yamazaki, D. G. McMahon; Regulation of Circadian Clock in the Mammalian Retina. Invest. Ophthalmol. Vis. Sci. 2007;48(13):5973.
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mPer2Luc C57BL/6J mice were backcrossed with C3H rd mice once to produce mPer2Luc knockin mice that were heterozygous for the rd gene and were genetically capable of producing melatonin. Retinas from these mice were isolated, cultured and assayed for circadian rhythms of the transgenic PER2::Luc reporter by luminescence. Drugs were applied to retinal explant cultures at the trough between the 2nd and 3rd peaks of PER2 expression rhythms in vitro, and then were left in the cultures.
Robust circadian rhythms of PER2::LUC expression sustained for up to 15 cycles in vitro without medium change with our modified retinal explant culture procedure. Circadian PER2 expression rhythms persisted during continuous activation or blockade of receptors for melatonin (10nM-10µM), dopamine (10µM-100µM), glycine (1mM-5mM), and glutamate (100µM-500µM). However, continuous application of γ-aminobutyric acid (GABA) inhibited PER2 expression in a dose-dependent manner. Prolonged application of 3 mM GABA reversibly disrupted the PER2 expression rhythms and stopped the retinal circadian clock based on restarted phase after washout. The inhibitory function of GABA was blunted by blockade of chloride channel-coupled GABAA and GABAC receptors.
Our results indicate that intra-retinal communication via melatonin, dopamine, glycine, or glutamate is not necessary for rhythms generation by retinal circadian clocks and suggest that GABA may play an important role in regulating the circadian clock in the mammalian retina.
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