HORMONE INFLUENCES ON POSTEMBRYONIC INSECT MOTOR NEURONS

激素对胚胎后昆虫运动神经元的影响

基本信息

批准号：
7090677
负责人：
RICHARD B LEVINE
金额：
$ 17.85万
依托单位：
UNIVERSITY OF ARIZONA
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-07-01 至 2007-06-30
项目状态：
已结题

项目摘要

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.

项目 3：胚胎后发育过程中激素对昆虫运动神经元的影响。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。识别这些激素的细胞靶点及其作用的分子机制对于理解中枢神经系统如何在胚胎后进行修饰以适应正常的神经功能至关重要，但这些努力往往受到中枢神经系统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，我们将利用两个特征良好的昆虫模型系统，它们可以提供互补的进步。在天蛾的变态过程中，其突触输入和目标肌肉发生变化。我们通过原代细胞培养证明，类固醇激素 20-羟基蜕皮酮 (20E) 直接作用于适当阶段的运动神经元，调节神经突的生长和分支，以及电压门控 Ca2+ 电流的水平。所提出的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。而曼杜卡则具有大尺寸的优势，便于操作和电生理学以及良好表征的内分泌滴度。果蝇经历了类似的运动神经元重塑，为探索信号通路提供了强大的分子遗传学方法。在第一个具体目标中，将使用新的器官培养方案和细胞成像系统来描述曼杜卡和果蝇中已识别的飞行运动神经元在体内自然环境中的树突建模，并确定特定突触输入和 20E 的作用。在第二个具体目标中，将通过描述蜕皮类固醇受体的时间表达模式和已识别的运动神经元在重塑时的关键初级反应关键 BRC 来检查细胞内 20E 反应途径。将在原代细胞培养物中检查类固醇激素对反应途径中关键元件的调节。对特定初级反应基因的要求将通过与项目 4 的研究人员合作检查适当的果蝇突变体来确定。第三个具体目标来自我们的观察，即 20 E 影响 Ca2+ 电流和内部 Ca2+ 增加的持续时间，并且这会改变Ca2+ 电流水平、体内 Ca2+ 水平、内部储存的 Ca2+ 释放以及 Ca2+ 依赖性酶 CAM 激酶 II 在使用电压的蜕皮类固醇诱导的树突重塑中钳夹、Ca2+ 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中枢神经系统在正常成熟过程中以及受伤或疾病后的可塑性。