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John O'Brien, W Wade Kothmann, Yanran Wang; Rapid light-driven regulation of Connexin35 phosphorylation in zebrafish retina. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2249.
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© ARVO (1962-2015); The Authors (2016-present)
Cx35 and its mammalian counterpart Cx36 form electrical synapses in many types of retinal neuron. Some undergo large changes in coupling through day/night and circadian cycles, modifying retinal circuitry to function in different light regimes. The dominant mechanism to regulate coupling mediated by Cx35 appears to be changes in Cx35 phosphorylation. In this study we test the hypothesis that short-term light adaptation drives changes in Cx35 phosphorylation.
Adult zebrafish of wild type strain AB were maintained on a 12:12-hr light:dark cycle. We utilized an isolated, superfused eyecup preparation to examine response to 10 minute exposure to photopic light. At the end of experiments, eyecups were fixed, embedded in OCT, and cryostat sectioned for immunohistochemistry. Sections were labeled with a monoclonal anti-Cx35 antibody and antibodies against either Cx35-phosphoSer110 or Cx35-phosphoSer276, and imaged with a Zeiss LSM 510 confocal microscope. Image analysis was performed in SimplePCI.
The inner plexiform layer of zebrafish retina contains abundant Cx35 gap junctions in all sublaminae, including small populations of very large gap junctions in two layers. These resemble the giant gap junctions in bass retina that undergo changes in phosphorylation between daytime and nighttime (Kothmann et al., 2007), and were selectively studied using a minimum area threshold of 0.75 µm2. In nighttime retina, 10 minute exposure to photopic light significantly reduced Cx35 phosphorylation of the large plaques at both Ser110 and Ser276 sites (n=5 animals per condition). The change in phosphorylation was larger at the Ser276 site than at the Ser110 site (37% vs. 18%). When all gap junctions in the IPL were considered, 10 minute light exposure significantly reduced phosphorylation at Ser276 but not at Ser110. Application of 10 mM SCH23390, a dopamine D1 receptor antagonist, resulted in a significant reduction in phosphorylation at Ser110 of the overall population of Cx35 gap junctions, but did not specifically affect the large plaques.
Brief adapting exposures to photopic light trigger changes in phosphorylation of regulatory sites on Cx35 in some populations of gap junctions. While Ser110 and Ser276 work together to regulate Cx35 coupling, regulation of their phosphorylation state is somewhat separate, suggesting that the sites may preferentially respond to different signaling pathways.
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