Shields Colleen R, Klooster Jan, Claassen Yvonne, Ul-Hussain Mahboob, Zoidl Georg, Dermietzel Rolf, Kamermans Maarten
Retinal Signal Processing, Netherlands Institute for Neuroscience, Netherlands Royal Academy of Sciences, 1105BA Amsterdam, The Netherlands.
J Comp Neurol. 2007 Apr 10;501(5):765-79. doi: 10.1002/cne.21282.
Connexins in retinal horizontal cells (HC) function in the processing of visual information. For example, gap junction-forming connexins may contribute to the spatial integration of visual stimuli. Additionally, connexin hemichannels have been hypothesized to participate in the feedback pathway from HCs to cones. To verify the identities of the zebrafish HC connexins, we performed promoter expression and immunohistochemical studies of connexin 52.6 (Cx52.6) and Cx55.5. Zebrafish embryos were microinjected with Cx52.6 or Cx55.5 promoter sequences and a green fluorescent protein reporter construct. Light and electron microscopic (EM) analysis showed green fluorescent protein expression exclusively in retinal HCs. Immunohistochemistry confirmed that HCs express Cx52.6 and Cx55.5 proteins. Light microscopy revealed Cx52.6 and Cx55.5 in the retinal inner nuclear and outer plexiform layers. Double labeling for Cx55.5 or Cx52.6 and cell-specific markers (tyrosine hydroxylase, protein kinase C-alpha, or GluR2) demonstrated that these connexins do not localize to interplexiform or ON bipolar cells, but most likely are present in HCs. Preembedding immuno-EM confirmed the HC-specific expression of Cx52.6 and Cx55.5 and illustrated the presence of these two connexins in gap junctions between HCs. The EM data also revealed robust labeling for Cx55.5 in hemichannels on HC dendrites in photoreceptor synaptic terminals. Voltage-clamp experiments in cultured cells demonstrated that Cx55.5-containing hemichannels can open at physiological membrane potentials. These results offer the first in vivo demonstration of the HC-specific activities of the Cx52.6 and Cx55.5 promoters. Furthermore, these data provide the first proof at the protein level for retinal HC-specific connexins in the zebrafish.
视网膜水平细胞（HC）中的连接蛋白在视觉信息处理中发挥作用。例如，形成间隙连接的连接蛋白可能有助于视觉刺激的空间整合。此外，有人推测连接蛋白半通道参与从水平细胞到视锥细胞的反馈通路。为了验证斑马鱼水平细胞连接蛋白的身份，我们对连接蛋白52.6（Cx52.6）和Cx55.5进行了启动子表达和免疫组织化学研究。将斑马鱼胚胎显微注射Cx52.6或Cx55.5启动子序列以及绿色荧光蛋白报告构建体。光镜和电镜（EM）分析显示绿色荧光蛋白仅在视网膜水平细胞中表达。免疫组织化学证实水平细胞表达Cx52.6和Cx55.5蛋白。光镜显示视网膜内核层和外网状层中有Cx52.6和Cx55.5。对Cx55.5或Cx52.6与细胞特异性标记物（酪氨酸羟化酶、蛋白激酶C-α或GluR2）进行双重标记表明，这些连接蛋白并不定位于网间或ON双极细胞，而很可能存在于水平细胞中。包埋前免疫电镜证实了Cx52.6和Cx55.5在水平细胞中的特异性表达，并说明了这两种连接蛋白存在于水平细胞之间的间隙连接中。电镜数据还显示在光感受器突触终末的水平细胞树突上的半通道中有强烈的Cx55.5标记。培养细胞中的电压钳实验表明，含有Cx55.5的半通道可在生理膜电位下开放。这些结果首次在体内证明了Cx52.6和Cx55.5启动子在水平细胞中的特异性活性。此外，这些数据首次在蛋白质水平上证明了斑马鱼视网膜水平细胞特异性连接蛋白的存在。
