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W. Kothmann, B. Trexler, W. Li, S. C. Massey, J. O'Brien; Presynaptic Activity Drives Increased Phosphorylation of Connexin 36 in AII Amacrine Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1208.
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Connexin 36 (Cx36) gap junctions couple AII amacrine cells (AII ACs) and are a necessary component of the primary rod pathway. Coupling between AII ACs is regulated by background light levels, and we have previously demonstrated a direct relationship between phosphorylation of Cx36 at Ser293 (Cx36-P) and coupling in the AII network. We also showed that dopamine-stimulated uncoupling of AII ACs is mediated by dephosphorylation of Cx36. We now test the hypothesis that presynaptic activity drives increased Cx36 phosphorylation, and thus coupling, in AII ACs.
Rabbit retina-sclera pieces were incubated with pharmacologic agents for 20 min in bubbled Ames medium at 35°C. Some experiments were done under IR illumination only; prior to these, the retina was dark adapted inside a light-tight chamber for 120 min in bubbled Ames medium at RT. Retina pieces were fixed and labeled with antibodies against Cx36, phosphoSer293-Cx36 (P-293), and calretinin. Cx36-P on AII ACs was quantified as the average ratio of P-293 to Cx36 antibody intensity. Other experiments used antibodies to phosphoThr286-CamKII, NMDAR1, and kinesin II.
Blockade of AMPA and NMDA receptors (NBQX & CPP, both 10 uM) significantly reduced Cx36-P in light adapted retina. CPP alone was sufficient to cause this reduction. CamKII inhibition (KN-93, 10 uM) also reduced Cx36-P to a similar extent. These effects persisted when dopamine D1 receptors were blocked (SCH23390, 50 uM). Immunolabeling showed that both NMDARs and activated CamKII colocalize with Cx36 in AII ACs. These NMDARs were extrasynaptic, as they were not closely apposed to kinesin II-labeled synaptic ribbons. Dark adapting the retina greatly reduced Cx36-P. Activation of ON bipolar cells (CPPG, 200 uM) in dark-adapted retina increased Cx36-P back to light-adapted levels. Exposure of dark-adapted retina to photopic light for 15 minutes also increased Cx36-P; this was blocked by application of L-AP4 (50 uM).
We conclude from these data that NMDAR activation modulates Cx36-P in AII amacrine cells. One potential mechanism for this is calcium influx through NMDARs near Cx36 gap junctions, followed by activation of CamKII, and we show evidence that both proteins are appropriately localized for such a pathway. Modulation of dopaminergic amacrine cell activity cannot account for our results. We provide evidence that both ON bipolar cell activity and acute light increments drive increased Cx36-P in AII ACs. This may occur through activation of the NMDAR pathway described above.
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