Plasticity of Electrical Synapses

描述（由申请人提供）：本提案的目标是研究听觉系统中间隙连接介导的电突触的作用和特性。听觉传入终止为大的混合（电和化学）突触终端的金鱼Mauthner细胞非常适合这些研究，因为不像哺乳动物的电突触，实验的可访问性使得它可以量化在体内的变化，在不同的生理条件下发生的交界处电导，并将它们与解剖，超微结构和分子分析。引人注目的是，这些模型电突触的电导是在神经元活动的精细调节控制之下。电传递由连接蛋白35（Cx 35）介导，连接蛋白35是存在于听觉系统中的哺乳动物连接蛋白36（Cx 36）的鱼类直系同源物，这表明哺乳动物听觉电突触可以类似地调节。该建议涉及了解的分子机制的双向控制交界处的电导在这些终端集中在调节运输的间隙连接通道的作用。目的一是通过超微结构和药理学方法研究在体天然电突触中缝隙连接通道的运输。我们的初步结果表明，存在一个积极的营业额的间隙连接通道，这构成了第一个证据，这种现象在一个天然的突触。目的2是确定管制贩运活动依赖的电传输增强的贡献。它将测试增强的表达是否需要胞吐机制，以及它是否需要与调节激酶CaM-KII和支架蛋白ZO-1的直接相互作用。相反，目标3将调查管制贩运活动依赖性抑制电传输的可能贡献。使用类似的方法，我们将调查是否需要的机制的内吞作用的活性依赖性抑郁症的连接电导，以及直接蛋白质-蛋白质相互作用的潜在作用。此外，CaM-KII和ZO-1通过Cx 35和Cx 36的保守区域的直接相互作用表明，其功能可能是电传输的基本和广泛的性质的基础，也与哺乳动物电突触有关。因此，拟议的研究解决了新的概念，电突触的强度是通过动态调节间隙连接通道的运输。由于电突触已被证明能促进协调的神经元活动，因此这种调节的功能障碍可能具有深刻的病理学意义，导致听觉障碍、癫痫和认知（精神）和发育障碍 公共卫生相关性：该提案探讨了间隙连接介导的电突触调节所涉及的分子机制。此外，该提案还探讨了间隙连接通道作为其潜在机制的受管制贩运的作用。这一过程已被证明参与化学突触的调节，但没有证据表明它参与电突触。因此，该提案调查了化学和电突触共享共同调节机制的可能性。由于电突触已被证明可以促进协调的神经元活动，其调节功能障碍可能具有深刻的病理学意义，有助于癫痫和认知（精神）和发育障碍。

DESCRIPTION (provided by applicant): The goal of this proposal is to investigate the role and properties of gap junction-mediated electrical synapses in the auditory system. Auditory afferents terminating as large mixed (electrical and chemical) synaptic terminals on the goldfish Mauthner cell are ideally suited for these studies since unlike mammalian electrical synapses, the experimental accessibility makes it possible to quantify in vivo changes in junctional conductance that occur under different physiological conditions and to correlate them with anatomical, ultrastructural and molecular analysis. Strikingly, the conductance of these model electrical synapses is under the fine regulatory control of neuronal activity. Electrical transmission is mediated by connexin 35 (Cx35), the fish ortholog of the mammalian connexin 36 (Cx36) which is present in the auditory system, suggesting that mammalian auditory electrical synapses could be similarly regulated. This proposal deals on understanding the molecular mechanisms underlying the bi-directional control of junctional conductance at these terminals by focusing in the role of regulated trafficking of gap junction channels. Aim 1 is to investigate the existence of trafficking of gap junction channels at native electrical synapses, in vivo, by combining ultrastructural and pharmacological approaches. Our preliminary results suggest the existence of an active turnover of gap junction channels, which constitute the first evidence of this phenomenon in a native synapse. Aim 2 is to determine the contribution of regulated trafficking to activity-dependent potentiation of electrical transmission. It will test if mechanisms of exocytosis are required for the expression of the potentiation and if it requires of direct interactions with the regulatory kinase CaM-KII and the scaffold protein ZO-1. Conversely, Aim 3 will investigate the possible contribution of regulated trafficking to activity-dependent depression of electrical transmission. Using similar approaches, we will investigate if mechanisms of endocytosis are required for activity-dependent depression of junctional conductance, as well the potential roles of direct protein-protein interactions. Furthermore, the direct interactions of CaM-KII and ZO-1 through conserved regions of both Cx35 and Cx36 suggests that its function might underlie a fundamental and widespread property of electrical transmission, also relevant to mammalian electrical synapses. Thus, the proposed research addresses the novel concept that the strength of electrical synapses is achieved by dynamically regulating the trafficking of gap junction channels. Because electrical synapses have been shown to promote coordinated neuronal activity, dysfunction of this regulation could have profound pathological implications, contributing to auditory impairment, epilepsy and cognitive (psychiatric) and developmental disorders PUBLIC HEALTH RELEVANCE: The proposal explores the molecular mechanisms involved in the regulation of gap junction-mediated electrical synapses. Further, the proposal explores the role of regulated trafficking of gap junction channels as their underlying mechanism. This process has been shown involved in the regulation of chemical synapses, but no evidence suggests its participation in electrical synapses. Thus, the proposal investigates the possibility that both chemical and electrical synapses share common regulatory mechanisms. Because electrical synapses have been shown to promote coordinated neuronal activity, dysfunction of their regulation could have profound pathological implications, contributing to epilepsy and to cognitive (psychiatric) and developmental disorders.