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R. Dorenbos, H. Hirasawa, M. Contini, S. Gustincich, E. Raviola; Retinal Dopaminergic Amacrines Are Circadian Clock Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5495.
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The retina contains an autonomous circadian clock that regulates the synthesis and release of dopamine. Retinal dopaminergic amacrine (DA) cells appear to express some of the clock–related proteins and therefore may have a role in the circadian control of retinal function. In this study we identify by single–cell RT–PCR the transcripts of the most common circadian clock genes in DA–cells and confirm the presence of their corresponding proteins by immunocytochemistry.
Retinas from transgenic C57Bl/6J mice that express human placental alkaline phosphatase (PLAP) under the control of the promoter for tyrosine hydroxylase (TH) were dissociated by enzymatic digestion and mechanical trituration. DA cells were stained with a fluorescent monoclonal antibody to PLAP and harvested after patch clamping. Only DA cells were selected that spontaneously generated action potentials. Reverse transcription was followed by two successive rounds of amplification using intron–spanning primers for the circadian clock genes Bmal1, Clock, Cry1, Cry2, Per1, and Per2. In the second round, primers internal to the first pair were used to increase specificity. Identity of the cells was confirmed by carrying out simultaneously RT–PCR for TH. RNA–dependency of the amplification was established by omitting reverse transcriptase during first–strand synthesis. RT–PCR products were digested with the appropriate restriction enzymes to confirm specificity. As a negative control, single–cell RT–PCR showed that rod photoreceptors were consistently negative for the six transcripts, whereas they were positive for rhodopsin message. Expression of the clock genes listed above was investigated by double–label immunocytochemistry of formaldehyde–fixed retinas using anti–TH antibodies to identify DA cells. Specificity of the antibodies to the clock proteins was confirmed by absorption of epitope–containing peptides and, in the case of Bmal1, by staining the retina of a Bmal1–/– mouse.
DA cells contained transcripts for all six circadian clock genes. The percentages of cells positive for each gene were as follows: Bmal1 17.6%, n=17; Clock 55.6%, n=9; Cry1 20.0%, n=15; Cry2 62.5%, n=8; Per1 88.9%, n=18; Per2 36.4%, n=11. Immunocytochemistry showed that all the corresponding proteins were present in DA cells.
The most important clock genes are expressed in DA cells. This suggests that these neurons possess an autonomous circadian clock that modulates dopamine synthesis and release in anticipation of the changes in daily lighting conditions.
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