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Jingjing Zang, Jennifer Keim, Stephan C F Neuhauss; The Role of Circadian Rhythms in Regulating the Cone Phototransduction Cascade Shutoff in Zebrafish Retina. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5005. doi: https://doi.org/.
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A variety of visual behaviours in different species are regulated by the circadian clock. Zebrafish, as a model organism for studying vertebrate circadian timing system, is used to investigate the molecular mechanism underlying how the circadian rhythms influence photoresponse shutoff which requires the phosphorylation of visual pigment by opsin kinase and subsequent binding of Arrestin, and hydrolysis of PDE-TαGTP complex by GTPase.
qRT-PCR has been used to quantitatively evaluate mRNA expression level at 8 time points during the day. In situ hybridization has been used to compare the difference in mRNA expression level at different time points. Western Blot was preformed to show the expression changes at the protein level. Electroretinography was recorded for the functional analysis.
qRT-PCR resultes show that in both 5 days post fertilization larvae and adult eyes, the expression of several key genes which are involved in regulating phototransduction deactivation fluctuates in a 24-hour rhythm. These key genes encode Recoverin, cone opsin kinase, cone specific Arrestin and the GTPase-accelerating protein. Changes in the mRNA expression level throughout the day are maintained when larvae are kept in continuous darkness for 5 days starting the first evening after fertilisation, indicating that this oscillation is endogenous and circadian rhythm dependent. The qRT-PCR results are confirmed by in situ hybridization staining for the larvae or adult eyes fixed at different time points. Preliminary western blot results show that protein levels also change at 24-hour cycle. Electroretinography recording from 5 days post fertilization larvae demonstrates the photoresponse recovery is delayed in the evening and accelerated in the morning. This phenomenon has been observed by spectrum electroretinography which stimulates only the UV cones and white light electroretinography which is dominated by double cone response, suggesting a common feature for cone photoresponse recovery.
The expression level of several important regulators for cone photoresponse recovery shows robust circadian rhythms, which may be correlated with the photoresponse kinetic change throughout the day.
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