Tetenborg Stephan, Yadav Shubhash Chandra, Brüggen Bianca, Zoidl Georg R, Hormuzdi Sheriar G, Monyer Hannah, van Woerden Geeske M, Janssen-Bienhold Ulrike, Dedek Karin
Animal Navigation/Neurosensorics, Institute for Biology and Environmental Sciences, University of Oldenburg, Oldenburg, Germany.
Department of Biology & Center for Vision Research, York University, Toronto, ON, Canada.
Front Mol Neurosci. 2019 Aug 28;12:206. doi: 10.3389/fnmol.2019.00206. eCollection 2019.
Neuronal gap junctions formed by connexin36 (Cx36) and chemical synapses share striking similarities in terms of plasticity. Ca/calmodulin-dependent protein kinase II (CaMKII), an enzyme known to induce memory formation at chemical synapses, has recently been described to potentiate electrical coupling in the retina and several other brain areas phosphorylation of Cx36. The contribution of individual CaMKII isoforms to this process, however, remains unknown. We recently identified CaMKII-β at electrical synapses in the mouse retina. Now, we set out to identify cell types containing Cx36 gap junctions that also express CaMKII-β. To ensure precise description, we first tested the specificity of two commercially available antibodies on CaMKII-β-deficient retinas. We found that a polyclonal antibody was highly specific for CaMKII-β. However, a monoclonal antibody (CB-β-1) recognized CaMKII-β but also cross-reacted with the C-terminal tail of Cx36, making localization analyses with this antibody inaccurate. Using the polyclonal antibody, we identified strong CaMKII-β expression in bipolar cell terminals that were secretagogin- and HCN1-positive and thus represent terminals of type 5 bipolar cells. In these terminals, a small fraction of CaMKII-β also colocalized with Cx36. A similar pattern was observed in putative type 6 bipolar cells although there, CaMKII expression seemed less pronounced. Next, we tested whether CaMKII-β influenced the Cx36 expression in bipolar cell terminals by quantifying the number and size of Cx36-immunoreactive puncta in CaMKII-β-deficient retinas. However, we found no significant differences between the genotypes, indicating that CaMKII-β is not necessary for the formation and maintenance of Cx36-containing gap junctions in the retina. In addition, in wild-type retinas, we observed frequent association of Cx36 and CaMKII-β with synaptic ribbons, i.e., chemical synapses, in bipolar cell terminals. This arrangement resembled the composition of mixed synapses found for example in Mauthner cells, in which electrical coupling is regulated by glutamatergic activity. Taken together, our data imply that CaMKII-β may fulfill several functions in bipolar cell terminals, regulating both Cx36-containing gap junctions and ribbon synapses and potentially also mediating cross-talk between these two types of bipolar cell outputs.
由连接蛋白36（Cx36）形成的神经元缝隙连接和化学突触在可塑性方面具有显著的相似性。钙/钙调蛋白依赖性蛋白激酶II（CaMKII）是一种已知能在化学突触处诱导记忆形成的酶，最近有研究表明它能增强视网膜和其他几个脑区中Cx36的磷酸化，从而增强电耦合。然而，单个CaMKII亚型在这一过程中的作用仍不清楚。我们最近在小鼠视网膜的电突触中鉴定出了CaMKII-β。现在，我们着手鉴定含有Cx36缝隙连接且也表达CaMKII-β的细胞类型。为确保精确描述，我们首先在缺乏CaMKII-β的视网膜上测试了两种市售抗体的特异性。我们发现一种多克隆抗体对CaMKII-β具有高度特异性。然而，一种单克隆抗体（CB-β-1）能识别CaMKII-β，但也与Cx36的C末端尾巴发生交叉反应，使得用这种抗体进行定位分析不准确。使用多克隆抗体，我们在双极细胞终末中鉴定出了强烈的CaMKII-β表达，这些终末分泌素和HCN1呈阳性，因此代表5型双极细胞的终末。在这些终末中，一小部分CaMKII-β也与Cx36共定位。在假定的6型双极细胞中也观察到了类似的模式，不过在那里，CaMKII的表达似乎不太明显。接下来，我们通过量化缺乏CaMKII-β的视网膜中Cx36免疫反应性斑点的数量和大小，来测试CaMKII-β是否影响双极细胞终末中的Cx36表达。然而，我们发现不同基因型之间没有显著差异，这表明CaMKII-β对于视网膜中含Cx36的缝隙连接的形成和维持不是必需的。此外，在野生型视网膜中，我们观察到双极细胞终末中Cx36和CaMKII-β经常与突触带，即化学突触相关联。这种排列类似于例如在Mauthner细胞中发现的混合突触的组成，在Mauthner细胞中，电耦合受谷氨酸能活动调节。综上所述，我们的数据表明CaMKII-β可能在双极细胞终末中发挥多种功能，调节含Cx36的缝隙连接和带状突触，并且可能还介导这两种双极细胞输出之间的相互作用。
