GAP JUNCTIONS AND ASTROCYTES

本研究的目的是分析间隙的生理作用 由星形胶质细胞形成的连接。最重要的假设是， 连接在关键的星形胶质细胞功能中很重要， 耦合的中断将对这些功能产生不利影响。我们将 应用间隙连接通信的标准调制方法， 所有这些都至少部分地在本计划中得到了发展， 包括通过同源重组和反义核酸的基因敲除 战略布局一个重要的进一步方法将是恢复耦合 通过用野生型Cx43瞬时转染或病毒转导， 与突变型Cx43或与另一种功能上不同于Cx43的连接蛋白 Cx43具体目标1。表征以下表达的连接蛋白： 星形胶质细胞星形胶质细胞中的主要连接蛋白是Cx43; Cx40也有表达，尽管程度较低。 Cx43基因敲除小鼠的星形胶质细胞表现出罕见的弱电 和染料耦合;连接通道特性不同于 关于CX43具体目标2。检验缝隙连接的假设 星形胶质细胞之间的耦合介导Ca 2+波的传播， 促进神经营养物质的分泌和 神经传递素缝隙连接通讯的阻断剂， 连接蛋白的核苷酸或蛋白质序列将允许高度特异性 结耦合块。Cx43基因敲除小鼠的星形胶质细胞 还提供了具有降低的偶联的制剂。具体目标3。测试 在培养中，假设神经元和其他细胞分泌的因子 细胞类型调节星形胶质细胞间隙的水平、分布和性质 交叉点星形胶质细胞之间缝隙连接的发生率变化 在体内创伤后， 体外神经递质我们将尝试在体外繁殖 星形胶质细胞连接蛋白的变化发生在反应性神经胶质增生中。具体 目标4。表征星形胶质细胞在反应性 神经胶质增生我们将确定耦合是否有变化 与Cx43分布和免疫反应性的变化有关。 我们将确定胶质增生的其他变化，如- GFAP的调节受到修饰偶联的影响。具体目标5. 验证星形胶质细胞之间的缝隙连接减少的假设 对癫痫刺激的敏感性。我们将使用大脑切片 培养和测定修饰的 结耦合这项广泛的研究将大大提高我们的 了解星形胶质细胞的作用，并提供 神经保护免受创伤的治疗策略， 预防神经变性和改善疾病 流程.

The goal of this project is to analyze the physiological roles of gap junctions formed by astrocytes. The overriding hypothesis is that gap junctions are important in critical astrocytic functions and that disruption of coupling will impact adversely on these functions. We will apply standard methods of modulation of gap junctional communication, all at least partially developed in this Program, and new methods including gene knockout by homologous recombination and antisense strategies. An important further approach will be to restore coupling by transient transfection or viral transduction with wild type Cx43, with mutant Cx43 or with another connexin that differs functionally from Cx43. Specific Aim 1. Characterize the connexin(s) expressed by astrocytes. The principal connexin in astrocytes is Cx43; we have strong evidence that Cx40 is also expressed, although to a lesser degree. Astrocytes from a Cx43 knockout mouse exhibit infrequent weak electrical and dye coupling; the junctional channel properties are distinct from those of Cx43. Specific Aim 2. Test the hypothesis that gap junctional coupling between astrocytes mediates propagation of Ca2+ waves and facilitates secretion of neurotrophic substances and uptake of neurotransmitters. Blockers of gap junctional communication that depend on connexin nucleotide or protein sequences will allow highly specific block of junctional coupling. Astrocytes from the Cx43 knockout mouse also provide a preparation with reduced coupling. Specific Aim 3. Test in culture the hypothesis that factors secreted by neurons and other cell types modulate levels, distribution and properties of astrocyte gap junctions. The incidence of gap junctions between astrocytes changes following trauma in vivo and coupling can be modulated by neurotransmitters in vitro. We will attempt to reproduce in vitro the changes in astrocyte connexins that occur in reactive gliosis. Specific Aim 4. Characterize the changes in coupling of astrocytes in reactive gliosis. We will determine whether there are alterations in coupling associated with the changes in Cx43 distribution and immunoreactivity. We will determine whether other changes in gliosis, such as up- regulation of GFAP, are affected by modifying coupling. Specific Aim 5. Test the hypothesis that gap junctions between astrocytes decrease susceptibility to epileptogenic stimuli. We will use brain slices in culture and determine effects on seizure thresholds of modifying junctional coupling. This broad based study should greatly increase our understanding of the roles of astrocytes and provide possibilities of therapeutic strategies for neural protection against trauma, for prevention of neurodegeneration and for amelioration of disease processes.