Connexins and electrical synapses in the retina

描述(申请人提供)：在视网膜中，缝隙连接由许多类型的细胞建立，并可能以各种方式参与视网膜回路。缝隙连接由连接蛋白组成，由高度相关的基因家族的20个成员编码。其中只有一种，连接蛋白36(Cx36)，已经在视网膜神经元中得到了确凿的证明。利用带有组织化学报告基因的Cx36基因敲除(KO)小鼠，我们发现Cx36存在于大多数或全部无长突细胞、许多光感受器、开和关锥双极细胞的亚群和少量的神经节细胞中。然而，水平细胞和许多神经节细胞建立缝隙连接，但不表达Cx36。利用RT-PCR，我们发现Cx45和Cx57在小鼠视网膜中显著表达。我们建议表征这些连接蛋白的细胞分布，并确定其他缝隙连接通道形成蛋白是否可能存在于视网膜神经元中。 许多证据表明，杆状光感受器信号利用多种途径到达神经节细胞。在我们的Cx36 KO中，神经节细胞对暗视刺激的反应被完全消除，这表明Cx36在所有参与ROD ON信号传递的通路中都是必需的。我们发现，双极细胞上AII和视锥细胞之间的缝隙连接偶联在KO中被取消，这被认为是杆状感光信号的主要途径。由于提出的“替代”途径涉及视杆和视锥之间的缝隙连接，而且我们发现Cx36在光感受器中表达，我们的发现与提出的“替代”途径是一致的。然而，最近对基因改变为缺乏视锥的小鼠视网膜的研究表明，视杆-视锥耦合对视杆感光信号没有贡献。此外，测量小鼠视网膜中光感受器之间的连接耦合在技术上是不可行的。因此，为了解决当前“替代”途径模型之间的差异，我们将1)开发一种方法来直接评估WT和Cx36 KO小鼠的视杆-视锥耦合；2)产生条件性基因敲除，从视锥细胞或AII无长突细胞中特异性地消除Cx36。然后，可以评估视网膜中神经节细胞对光的反应，其中主要或替代路径被消融。

DESCRIPTION (provided by applicant): In the retina, gap junctions are established by many cell types and may participate in retinal circuitry in a variety of ways. Gap junctions are composed of connexins, encoded by a >20-member family of highly related genes. Only one of these, connexin36 (Cx36), has been conclusively demonstrated in retinal neurons. Using a Cx36 knockout (KO) mouse incorporating a histochemical reporter, we found Cx36 to be present in most or all All amacrine cells, many photoreceptors, subsets of ON and OFF cone bipolar cells and a low number of ganglion cells. However, horizontal cells and many ganglion cells establish gap junctions yet do not express Cx36. Using RT-PCR, we have found Cx45 and Cx57 to be prominently expressed in mouse retina. We propose to characterize the cellular distribution of these connexins and determine if other gap junction channel-forming proteins may be present in retinal neurons. Many lines of evidence indicate that rod photoreceptor signals utilize multiple pathways to reach ganglion cells. In our Cx36 KO, ON ganglion cell responses to scotopic stimuli are completely eliminated, indicating that Cx36 is required in all pathways contributing to rod ON signaling. We showed that gap junctional coupling between AII and cone ON bipolar cells, the presumed 'primary' pathway for rod photoreceptor signaling, was abolished in the KO. Since a proposed 'alternative' pathway involved gap junction between rods and cones, and since we found Cx36 expression in photoreceptors, our findings were consistent with the proposed 'alternative' pathway. However, recent studies of retinas from mice genetically altered to lack cones suggest that rod-cone coupling does not contribute to rod photoreceptor signaling. In addition, it has not been technically feasible to measure junctional coupling between photoreceptors in mouse retinas. Therefore, to resolve the discrepancy between current models of the 'alternative' pathway, we will 1) develop a method to directly assess rod-cone coupling in WT and Cx36 KO mice and 2) produce conditional knockouts that eliminate Cx36 specifically from either cones or from AII amacrine cells. Then, ganglion cell responses to light can be assessed in retinas where either primary or alternative pathways are ablated.