HORMONAL INFLUENCES ON INSECT MOTOR NEURONS DEVELOPING IN CELL CULTURE

类固醇激素对神经回路和行为产生强大的影响 通过它们在胚后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靶点， 他们采取行动的机制是努力的重要组成部分， 了解中枢神经系统是如何修改胚后，以适应正常的 神经功能和行为成熟，但这些努力往往是 受到细胞异质性和复杂相互作用的阻碍， 描述CNS。 在拟议的实验中，我们将利用 一个特征良好的昆虫模型系统，其中识别的神经元 经历可预测的结构和功能修饰阶段， 对类固醇激素的反应。 胸腿运动神经元 天蛾经历了戏剧性树突和轴突退化 并在变态过程中重新生长，以适应它们突触的变化。 输入一个焦油得到肌肉。 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 运动神经元来调节神经突的生长。 的主要目标 提出的实验是表征细胞的后果， 甾体激素在体外的作用及其与细胞机制的关系 参与了腿部的结构和功能重塑 体内运动神经元 在第一个具体目标中，视频分析将 用于确定20-HE是否促进培养腿的生长 运动神经元通过刺激分支的形成，或促进 现有的分支机构。 运动神经元放置在体外之前的阶段， 神经突退化，而不是生长，也将在 对比 激光扫描共聚焦显微镜图像的染料注入 运动神经元将被用来检查类似的问题在体内。 第二 具体目标是将类固醇激素对神经突生长的影响与 回归与修改的组织和稳定性， 细胞骨架 免疫荧光结合共聚焦显微镜， 用于获得微管和肌动蛋白的高分辨率图像 在培养的腿部运动神经元中的细丝正在经历20-HE诱导 神经突生长或退化。 补充实验将采用 用电子显微镜观察细胞骨架结构， 运动神经元在体内发育的关键阶段。 最终目标是 检验20-HE诱导电压门控性Ca 2+和K+变化的假设 通道，以及随之而来的变化，钙离子内流，是必不可少的元素， 20-HE通过细胞内途径调节 腿部运动神经元 我们将确定， 已经描述的Ca 2+和K+电流由20- HE和Ca 2+是否诱导Ca 2+和K=电流的释放， 已经描述过的，是由20-HE介导的，无论是Ca 2+诱导的 Ca 2+从内部故事中的释放对于调节 结构重塑 这些研究揭示的基本机制将 有助于我们理解正常成熟过程中的CNS可塑性， 以及受伤或患病后。

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.