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G.-X. Ruan, S. E. Yanni, J. S. Penn, D. G. McMahon; A Circadian Clock in Müller Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3233.
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© ARVO (1962-2015); The Authors (2016-present)
The mammalian retinal circadian clock controls multiple local circadian rhythms at the physiological, cellular, and molecular levels. Despite its widespread influence, the cellular location of the clock in the mammalian retina is not well understood. The purpose of the present study was to investigate whether Müller cells in the mouse retina contain self-sustained circadian clocks.
mPer2Luc knockin mice, on C57BL/6J background harboring a transgenic PERIOD2::LUCIFERASE (PER2::LUC) fusion protein as a real-time reporter of circadian gene dynamics, were crossed with C3H rd1 mice to produce reporter mice that were heterozygous for the rd1 gene and were genetically capable of producing melatonin. Primary Müller cell cultures were prepared from 6 to 8 neonatal mice at P4 to P7, cultured in low-glucose DMEM medium containing 10% fetal bovine serum (FBS), and maintained at 37°C in a 5% CO2 incubator. At passage 2 or 3, Müller cells were transferred to medium 199 and loaded into a LumiCycle multichannel luminometer for time-lapse bioluminescence assay. Drugs were applied to Müller cell cultures at the beginning of the rising phase of the third cycle recorded in the LumiCycle, and then left in the cultures. The expression of 6 core clock genes in passage 3 Müller cells was examined by TaqMan real-time RT-PCR.
Robust circadian rhythms of Müller cell PER2::LUC expression were observed for up to 10 cycles without medium changes in primary Müller cell culture (> 99% purity). Serum shock was not necessary to induce PER2::LUC rhythms in Müller cells. Distinct from whole-mount retinal culture, the phase and amplitude of PER2::LUC rhythms in Müller cells were not significantly altered by the dopamine D1 receptor agonist SKF-38393 (50 uM; n = 4) or GABA (1 mM or 3 mM; n = 4 for both) treated at the beginning of the rising phase of the third cycle. Real-time RT-PCR revealed that 6 core clock genes, Per1, Per2, Cryptochrome (Cry) 1 and 2, Clock, and Bmal1, but not nitric-oxide synthase (NOS, a negative control gene) were expressed in primary Müller cell culture.
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