HORMONE INFLUENCES ON POSTEMBRYONIC INSECT MOTOR NEURONS

激素对胚胎后昆虫运动神经元的影响

• 批准号: 6589571
• 负责人: RICHARD B LEVINE
• 金额: $ 23.19万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-15 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6589571
• 关键词: Drosophilidae; Manduca; arthropod genetics; calcium channel; calcium flux; confocal scanning microscopy; dendrites; developmental genetics; developmental neurobiology; ecdysone; electron microscopy; gene expression; hormone regulation /control mechanism; immunocytochemistry; metamorphosis; motor neurons; neuroanatomy; protein kinase; protein structure function; steroid hormone receptor; synapses; tissue /cell culture; transcription factor; video microscopy; voltage /patch clamp; voltage gated channel

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。在提出的实验中，我们将利用两个具有良好特征的昆虫模型系统，提供互补的进展。在蛾的变态过程中，它们的突触输入和目标肌肉的变化。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（20E）直接作用于适当阶段的运动神经元，以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的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具有操作和电生理的大尺寸优势，并且具有良好的内分泌滴度特征。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有力的分子遗传学方法。在第一个特定目标中，一个新的器官培养方案和细胞成像系统将用于描述曼杜卡原虫和果蝇在自然环境中识别的飞行运动神经元的树突模型，并确定特定突触输入和20E的作用。在第二个特定目标中，细胞内20E反应途径将通过描述外皮甾体受体的时间表达模式和确定的运动神经元中关键的主要反应关键BRC来进行检查，因为它们正在被重塑。在原代细胞培养中，将对反应途径中关键要素的类固醇激素调节进行研究。与项目4的研究人员合作，将通过检查适当的果蝇突变体来确定对特定初级反应基因的需求。第三个具体目标来自我们的观察，即20e影响Ca2+电流和内部Ca2+增加的持续时间，Ca2+电流水平的变化，在体内，Ca2+水平，Ca2+从内部储存释放，以及Ca2+依赖性酶，cam -激酶II，在使用电压钳，Ca2+成像和遗传方法的激素诱导的树突重塑中。这些研究揭示的基本机制将有助于我们理解正常成熟期间以及损伤或疾病后中枢神经系统的可塑性。