REGULATION OF NEURITE OUTGROWTH AND CONNECTIVITY

该提案的总体目标是定义细胞内的 调节神经元生长和连接的机制。 集中于神经纤维伸长尖端的生长锥的运动 来揭示控制它们功能的机制 这些结构包含 神经突中的运动机制和决策机制 延伸、路径发现和突触发生。这个假设 测试的是，生长锥运动的改变是由 内部Ca的变化。 这些实验利用了大量已识别的 蜗牛Helisoma的神经元培养。 这些神经元发育得很大 生长锥的结构和运动可以定量研究。 此外，识别出的神经元提供了一个机会来揭示共同的， 以及独特的再生特性， 神经元 第一个具体目标解决了钙如何调节生长锥 运动在过去四年中取得的进展， 对生长锥有广泛影响的刺激。的影响 这些刺激引起的生长锥运动的分级改变， 内部钙水平将通过成像生长锥加载 fura-2。 第二个具体目标集中在轴突中Ca的作用， 使用新开发的制剂进行再生， 研究与轴突的神经突延伸相关的动态事件 树桩 这种制备在培养中复制了再生， 通常发生在轴突受损后的体内。 Ca的重要性 允许调节因子诱导轴突残端的生长， 接受检查。 具体目标3将研究如何在增长的变化， 视锥细胞骨架与生长锥运动的变化有关。 生长锥的结构和运动性取决于 计算机增强DIC和荧光显微镜 将用于揭示细胞骨架的动态变化如何调节 生长锥的结构和功能。假设， 内部钙影响细胞骨架，从而改变生长锥 结构和运动将使用多种刺激进行测试， 被认为是生长调节剂。

The overall objective of this proposal is to define the intracellular mechanisms that regulate neuronal growth and connectivity.The experiments focus on movements of growth cones at the elongating tips of nerve fibers to reveal mechanisms that govern their function. These structures contain the motile machinery and decision making apparatus involved in neurite elongation, path finding, and synaptogenesis. The hypothesis that will be tested is that alterations in growth cone movements are mediated by changes in internal Ca. The experiments exploit the large identified neurons of the snail Helisoma in culture. These neurons develop large growth cones whose structure and movements can be studied quantitatively. Moreover, identified neurons provide an opportunity to reveal common, as well as unique, regenerative properties based upon study of individual neurons. The first Specific Aim addresses how Ca regulates growth cone movement. Progress over the last four years has established multiple stimuli that have a broad range of effects on growth cones. The effects of graded alterations in growth cone movements caused by these stimuli on internal Ca levels will be tested by imaging growth cones loaded with fura-2. The second Specific Aim focuses on the role of Ca in axonal regeneration using a newly developed preparation that makes it possible to study dynamic events associated with extension of neurites from an axon stump. This preparation duplicates in culture the regeneration that normally occurs in vivo after an axon is damaged. The importance of Ca in allowing conditioning factors to induce outgrowth from the axon stump will be examined. Specific Aim 3 will investigate how changes in the growth cone cytoskeleton are related to changes in growth cone movement.The structure and motility of growth cones depends on the organization of cytoskeletal components.Computer enhanced DIC and fluorescence microscopy will be used to reveal how dynamic changes in the cytoskeleton regulate growth cone structure and function. The hypothesis that changes in internal Ca influence the cytoskeleton and thereby alter growth cone structure and movement will be tested using multiple stimuli that have been identified as growth regulators.