Connexins and electrical synapses in the retina

视网膜中的连接蛋白和电突触

• 批准号: 6820588
• 负责人: DAVID L PAUL
• 金额: $ 25.43万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6820588
• 关键词: amacrine cells; cone cell; gap junctions; gene expression; genetically modified animals; horizontal cell; immunocytochemistry; in situ hybridization; laboratory mouse; neural transmission; polymerase chain reaction; retina; retinal bipolar neuron; retinal ganglion; rod cell; synapses

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.

描述（由申请人提供）：在视网膜中，缝隙连接由许多细胞类型建立，并且可以以多种方式参与视网膜回路。间隙连接由连接蛋白组成，由一个>20个成员的高度相关基因家族编码。其中只有一个，连接蛋白36（Cx 36），已被最终证明在视网膜神经元。使用Cx 36基因敲除（KO）小鼠纳入组织化学报告，我们发现Cx 36是目前在大多数或所有的无长突细胞，许多光感受器，开和关锥双极细胞的子集和少量的神经节细胞。然而，水平细胞和许多神经节细胞建立间隙连接，但不表达Cx 36。用RT-PCR方法检测了Cx45和Cx 57在小鼠视网膜中的表达。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，视杆细胞信号通过多种途径到达神经节细胞。在我们的Cx 36 KO中，ON神经节细胞对暗视刺激的反应被完全消除，表明Cx 36在所有有助于视杆ON信号传导的途径中都是必需的。我们发现，AII和锥ON双极细胞之间的间隙连接耦合，杆光感受器信号转导的假定的“主要”途径，在KO中被废除。由于提出的“替代”途径涉及视杆细胞和视锥细胞之间的间隙连接，并且由于我们发现Cx 36在光感受器中表达，因此我们的研究结果与提出的“替代”途径一致。然而，最近的研究表明，从老鼠的视网膜基因改变，缺乏锥细胞的杆-锥耦合不有助于杆光感受器信号。此外，测量小鼠视网膜中光感受器之间的连接偶联在技术上还不可行。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Cx 36 KO小鼠中的视杆-视锥偶联，2）产生条件性敲除，从视锥细胞或AII无长突细胞中特异性消除Cx 36。然后，可以在视网膜中评估神经节细胞对光的反应，其中主要或替代途径被消融。