Postembryonic development of drosophila motoneurons

描述(申请人提供)：正常的大脑功能需要精确的树突形状和电学特征。神经元必须在最初的发育过程中获得这些特性，并在一生中保持它们，除非需要功能可塑性。该项目的长期目标是了解神经元活动在调节电压门控离子通道的树突形状和分布中的作用。我们已经开发了在遗传易驯化的模式生物果蝇中跟踪已识别运动神经元在变态期间的树突发育的技术，并证明了活动水平对树突发育有显著影响。无论是在体内还是在原代细胞培养中，神经元的活动和钙依赖的转导通路都可以被精确地操纵。具体目标是：1.确定已识别的飞行运动神经元的功能属性在变态过程中如何改变。将采用全电池电流和电压钳位技术。2.确定电压门控性钾通道和钙通道在不同发育阶段的表达、它们在运动神经元中的分布以及类固醇激素对它们的调节。将通过RT-PCR测量通道的表达，并通过强制表达标记有四个半胱氨酸基序的通道来确定通道的分布。3.刻画树突状细胞发育的活性依赖性调控。识别的运动神经元的活动模式将通过驱动修饰的离子通道的表达以及通过体内和体外的生长和功能测量来确定的效果而增加或减少。4.确定钙依赖信号通路的作用。活性依赖性钙内流的来源和性质将使用遗传编码的钙指示剂、神经元离子通道的高度保守性以及调节发育过程的分子途径来确定，这表明从这个模型系统获得的基本信息将为人类健康提供见解。与公众健康相关：正常的大脑功能需要神经元形成精确的树突结构和明确的电特性阵列。中风和疾病，如癫痫，可能会导致这些属性的不当维护。拟议的研究试图确定确保适当发展和保持这些特征的机制的特点。

DESCRIPTION (provided by applicant): Normal brain function requires precise dendritic shape and electical characteristics. Neurons must acquire these properties during initial development and maintain them through life, except under circumstances where functional plasticity is required. The long-term objectives of this project are to understand the role of neuronal activity in regulating dendritic shape and distribution of voltage-gated ion channels. We have developed techniques for following the dendritic development of identified motoneurons during metamorphosis in the genetically-tractable model organism, Drosophila melanogaster, and demonstrated that activity levels influence dendritic development significantly. Neuronal activity and calcium-dependent transduction pathways can be manipulated precisely, both in vivo and in primary cell culture. Specific aims are: 1. To determine how the functional attributes of identified flight motoneurons are modified during metamorposis. Whole-cell current and voltage-clamp techniques will be employed. 2.To determine which forms of voltage-gated potassium and calcium channels are expressed at different stages, their distributions within the motoneuron and their regulation by steroid hormone. Channel expression will be measured by RT- PCR, and distribution determined by forcing the expression of channels tagged with a tetrad-cystiene motif. 3. Characterize the acitivity-dependent regulation of dendritic development. The activity patterns of identified motoneurons will be increased or decreased by driving the expression of modified ion channels and the effect determined with both in vivo and in vitro measurements of growth and function. 4. Determine the role of calcium-dependent signalling pathways. The source and nature of activity-dependent calcium influx will be determined using genetically-encoded calcium indicators, the highly conserved nature of neuronal ion channels and the molecular pathways that regulate developmental processes suggests that basic information derived from this model system will provide insights into human health. Relevance to public health: Normal brain function requires that neurons develop a precise dendritic structure and a defined array of electrical characteristics. Stroke and diseases, such as epilepsy, can result in improper maintenance of these properties. The proposed research seeks to characterize mechanisms that ensure proper development and maintenance of these characteristics.