HORMONAL INFLUENCES ON INSECT MOTOR NEURONS DEVELOPING IN CELL CULTURE

类固醇激素对神经回路和行为有强大的影响 通过它们在胚胎后期发育过程中对中枢神经系统的作用。 确定这些激素的细胞靶点和分子 它们采取行动的机制是努力实现以下目标的重要组成部分 了解胚胎后中枢神经系统是如何改变以适应正常的 神经功能和行为成熟，然而这些努力通常是 受阻于细胞的异质性和复杂的相互作用 描述一下中枢神经系统的特征。在提议的实验中，我们将利用 一个特性良好的昆虫模型系统，在那里识别出神经元 经历可预测的结构和功能修改阶段 对明确的类固醇激素信号的反应。胸腿运动神经元 六纹夜蛾经历了戏剧性的树突和轴突退化 并在变态期间重新生长以适应突触的变化 输入焦油会得到肌肉。我们已经证明，使用原代细胞培养， 类固醇激素，20-羟基蜕皮激素(20-HE)，直接作用于 运动神经元调节轴突生长。的主要目标是 拟议的实验是为了表征细胞的后果 类固醇激素的体外作用及其与细胞机制的关系 参与腿的结构和功能重塑 体内的运动神经元。在第一个具体目标中，视频分析将是 用于确定20-HE是否促进培养腿的生长 通过刺激分支形成或促进保留运动神经元 现有分支机构。在体外放置的运动神经元恰好在 还将分析轴突退化，而不是增长 比较一下。染料注入的激光扫描共聚焦显微镜图像 运动神经元将被用来在体内检查类似的问题。第二 具体目的是将类固醇激素对轴突生长的影响与 在组织和稳定性方面进行了修改的回归 细胞骨架。免疫荧光，结合共聚焦显微镜，将 用于获取微管和肌动蛋白的高分辨率图像 20-HE诱导培养的小腿运动神经元的细丝 轴突生长或退化。互补性实验将采用 电子显微镜显示已识别的细胞骨架结构 处于体内发育关键阶段的运动神经元。最终的目标是 验证20-HE引起电压门控性钙、钾离子变化的假说 通道和随之而来的钙离子内流变化是 20-HE调节血管重构的细胞内途径 腿部运动神经元。我们将确定这些水平的变化是否 已经描述的Ca~(2+)和K~+电流由20- 他和是否钙离子诱导的钙释放和K=电流，有 已经描述过，是由20-HE介导的，以及钙离子是否诱导 内层钙离子的释放对调节细胞功能具有重要意义 结构重塑。这些研究揭示的基本机制将 有助于我们理解中枢神经系统在正常成熟过程中的可塑性， 以及在受伤或疾病之后。

Steroid hormones exert powerful influences on neural circuits and behavior through their actions on the CNS during postembryonic development. Identifying the cellular targets of these hormones and the molecular mechanisms through which they act are essential components of efforts to understand how the CNS is modified postembryonically to accommodate normal neural function and behavioral maturation, yet these efforts are often hindered by the cellular heterogeneity and complex interactions that characterize the CNS. In the proposed experiments we will take advantage of a well-characterized insect model system, where identified neurons undergo predictable phases of structural and functional modifications in response to defined steroid hormonal cues. The thoracic leg motoneurons of the moth, Manduca sexta, undergo dramatic dendritic and axonal regression and regrowth during metamorphosis to accommodate changes in their synaptic inputs an tar get muscles. We have shown, using primary cell culture, that the steroid hormone, 20-hydroxyecdysone (20-HE ), acts directly on the motoneurons to regulate neurite outgrowth. The principal goal of the proposed experiments is to characterize the cellular consequences of steroid hormone action in vitro and relate them to the cellular mechanisms that are involved in the structural and functional remodeling of the leg motoneurons in vivo. In the first specific aim, video analysis will be employed to determine whether 20-HE enhances the growth of cultured leg motoneurons by stimulating branch formation, or promoting the retention of existing branches. Motoneurons placed in vitro just prior to the phase of neurite regression, rather than growth, will also be analyzed in comparison. Laser-scanning confocal microscopy images of dye-injected motoneurons will be used to examine similar questions in vivo. The second specific aim is to correlate steroid hormone effects on neurite growth and regression with modifications in the organization and stability of the cytoskeleton. Immunofluorescence, coupled with confocal microscopy, will be used to obtain high-resolution images of microtubules and actin filaments in cultured leg motoneurons that are undergoing 20-HE induced neurite growth or regression. Complementary experiments will employ electron microscopy to reveal cytoskeletal structures within identified motoneurons at crucial stages of development in vivo. The final aim is to test the hypothesis that 20-HE induced changes in voltage-gated Ca2+ and K+ channels, and consequent changes i Ca2+ influx, are essential elements of the intracellular pathway through which 20-HE regulates the remodeling of the leg motoneurons. We will determine whether changes in the levels of Ca2+ and k+ currents, that have already been described, are mediated by 20- HE and whether Ca2+-induced release of Ca2+ and K= currents, that have already been described, are mediated by 20-HE and whether Ca2+ -induced release of Ca2+ from internal stories is important for regulating structural remodeling. The basic mechanisms revealed by these studies will contribute to our understanding of CNS plasticity during normal maturation, and following injury or disease.