HORMONE INFLUENCES ON POSTEMBRYONIC INSECT MOTOR NEURONS

项目3：激素对昆虫胚后发育过程中运动神经元的影响.类固醇激素通过对中枢神经系统的作用对神经回路和行为产生强大的影响。确定这些激素的细胞靶点和它们作用的分子机制对于理解CNS如何在胚胎后被修饰以适应正常的神经功能是必不可少的，这些努力通常受到CNS特征的细胞异质性和复杂相互作用的阻碍。在拟议的实验中，我们将利用两个良好的特点昆虫模型系统，提供互补的进步。在蛾的变态过程中，Manduca，它们的突触输入和目标肌肉发生变化。我们已经使用原代细胞培养显示，类固醇激素20-羟基蜕皮激素（20 E）直接作用于适当阶段的运动神经元，以调节神经突生长和分支，以及电压门控Ca 2+电流的水平。拟议实验的主要目标是表征类固醇激素作用的细胞内机制以及激素与细胞间相互作用在调节这种重塑中的相对影响。而Manduca提供了大尺寸的优点，便于操作和电生理学以及良好表征的内分泌滴度。果蝇也经历了类似的运动神经元重塑，为探索信号通路提供了强有力的分子遗传学方法。在第一个具体的目标，一个新的器官培养协议和细胞成像系统将被用来描述识别飞行运动神经元在Manduca和果蝇在其自然环境中，在体内的树突状建模，并确定特定的突触输入和20 E的作用。在第二个具体目标，细胞内20 E反应途径将通过描述蜕皮激素受体的时间表达模式和一个关键的主要响应关键BRC在确定的运动神经元，因为它们正在重塑检查。将在原代细胞培养中检查反应途径中关键要素的类固醇激素调节。与项目4的研究人员合作，通过检查适当的果蝇突变体来确定对特定初级应答基因的需求。第三个具体的目标如下，从我们的观察，20 E影响钙电流和持续时间的内部Ca 2+的增加，并在Ca 2+电流水平的变化，在体内，Ca 2+水平，Ca 2+从内部存储的释放，和Ca 2+依赖性酶，CAM激酶II，蜕皮激素诱导的树突重塑使用电压钳，Ca 2+成像和遗传方法。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，以及损伤或疾病。

PROJECT 3: Hormonal influences on insect motoneurons during post- embryonic development. Steroid hormones exert powerful influences on neural circuits and behavior through their actions on the CNS. Identifying the cellular targets of these hormones and the molecular mechanisms through which they act are essential for understanding how the CNS is modified post-embryonically to accommodate normal neural function, these efforts are often hindered by the cellular heterogeneity and complex interactions that characterize the CNS. In the proposed experiments we will exploit two well characterized insect model systems that offer complementary advances. During metamorphosis in the moth, Manduca, changes in their synaptic inputs and target muscles. We have shown, using primary cell culture, that the steroid hormone, 20-hydroxyecdysone (20E) acts directly on the motoneurons of the appropriate stage to regulate neurite outgrowth and branching, as well as the levels of voltage- gated Ca2+ currents. The principal goals of the proposed experiments are to characterize the intracellular mechanisms of steroid hormone action and the relative influences of hormonal vs. inter-cellular interactions in regulating this remodeling. Whereas Manduca offers the advantages of large size for manipulation and electrophysiology and well-characterized endocrine titers. Drosophila, which undergoes similar motoneuron remodeling, offers powerful molecular-genetic approaches to exploring signaling pathways. In the first specific aim a new organ culture protocol and cell imaging system will be used to describe the dendritic modeling of identified flight motoneurons in Manduca and Drosophila in their natural context, in vivo, and to determine the roles of specific synaptic inputs and 20E. In the second specific aim, the intracellular 20E response pathway will be examined by describing the temporal expression patterns of ecdysteroid receptors and a key primary response key BRC in identified motoneurons as they are being remodeled. Steroid hormone regulation of key elements in the response pathway will be examined in primary cell culture. The requirement for specific primary response genes will be determined by examining appropriate Drosophila mutants, in collaboration with investigators of project 4. The third specific aim follows from our observation that 20 E affects both Ca2+ currents and the duration of internal Ca2+ increases, and that changes in Ca2+ current levels, in vivo, Ca2+ levels, Ca2+ release from internal stores, and the Ca2+-dependent enzyme, CAM-kinase II, in ecdysteroid-induced dendritic remodeling using voltage clamp, Ca2+ imaging and genetic approaches. The basic mechanisms revealed by these studies will contribute to our understanding of CNS plasticity during normal maturation, and following injury or disease.