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: 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
• 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
• 来源: https://reporter.nih.gov/project-details/7090677
• 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：胚胎后发育过程中对昆虫运动神经元的影响。类固醇激素通过对CNS的作用对神经回路和行为产生强大的影响。鉴定这些激素的细胞靶标和其作用的分子机制对于理解后孔子进行修饰以适应正常神经功能至关重要，这些努力通常受到细胞异质性和复杂的相互作用的阻碍。在拟议的实验中，我们将利用两个具有互补进展的良好特征昆虫模型系统。在飞蛾的变形过程中，甘达（Manduca）的突触输入变化并靶向肌肉。我们已经使用原发性细胞培养表明，类固醇激素20-羟基肌酮（20E）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，以及电压盖的CA2+电流的水平。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时的相对影响。而曼达杜（Manduca）为操纵和电生理学和表征良好的内分泌滴度提供了大尺寸的优势。经过类似运动神经元重塑的果蝇为探索信号通路提供了强大的分子遗传学方法。在第一个特定的目标中，将使用新的器官培养方案和细胞成像系统来描述在自然环境中，体内确定的甘达和果蝇中鉴定出的飞行运动神经元的树突形成建模，并确定特定的突触输入和20E的作用。在第二个特定目的中，将通过描述ecdysteroid受体的时间表达模式和鉴定运动神经元中的关键主要反应键BRC来检查细胞内20E响应途径。在原代细胞培养中将检查反应途径中关键元素的类固醇激素调节。 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使用电压夹，Ca2+成像和遗传方法，ecdysteroid诱导的树突状重塑。这些研究揭示的基本机制将有助于我们在正常成熟期间以及受伤或疾病后对CNS可塑性的理解。