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Stephan C F Neuhauss, Jennifer Keim, Jingjing Zang; The Role of Circadian Rhythms in Regulating the Cone Phototransduction Cascade Shutoff in Zebrafish Retina. Invest. Ophthalmol. Vis. Sci. 2016;57(12):No Pagination Specified. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
A variety of visual behaviors in different species are regulated by the circadian clock. Here we performed electrophysiological and behavioral studies to investigate the molecular mechanism underlying how circadian rhythms influence photoresponse shutoff in the cone vision This shutoff requires the phosphorylation of visual pigment by opsin kinase and subsequent binding of Arrestin, followed by hydrolysis of PDE-Tα-GTP-complex by GTPase.
In situ hybridization and qRT-PCR has been used to (semi-)quantitatively evaluate mRNA expression levelsl throughout the day on 5dpf larvae and adult fish. Changes in protein level were assessed by western blotting. Electroretinograms (ERG) and optokinetic response (OKR) served as functional and behavioral assessment.
Expression levels of over 10 key phototransduction regulatory genes (recoverins, arrestins, opsin kinases GRK and GTPase-accelerating proteins) fluctuate in a circadian rhythm which is maintained in complete darkness. In extreme cases, e.g. GRK7a, mRNA decreases 98.4±0.01% (mean ± SED) during the day. Western blot results show that protein levels also change at 24-hour cycle with a delayed peak concentration to the mRNA expression peak, e.g. the reduction for GRK7a is 82.6±6% (mean ± SED). ERGs from larvae demonstrate that the photoresponse recovery is delayed in the evening and accelerated in the morning. When the interval between two stimuli is 1 second, the response recovery in the evening is only 62.6% (p<0.01) compared to the morning. This phenomenon is maintained in continuous darkness (p<0.01), while constant light exposure disrupts the normal oscillating expression pattern. GRK7a mRNA declines only 13.8±7% (mean ± SED) over 12 hours. Consistently there is no significant difference (p>0.05) in photoresponse kinetics between morning and evening under constant light conditions. An inverted light cycle can successfully reverse the mRNA fluctuations and a delayed photoresponse recovery and OKR recorded right before the light is off can still be observed (p<0.05).
The expression level of several important regulators for cone photoresponse recovery shows robust circadian rhythms, which may be correlated with the observed photoresponse kinetic and visual behavioral change throughout the day.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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