Electrical Signaling in a Circadian Pacemaker Circuit

描述（由申请人提供）：虽然对昼夜节律分子钟的核心组成部分和操作及其在模式生物果蝇中的神经解剖学位置了解很多，但对这种昼夜节律分子钟如何与起搏神经元中的电信号相互作用知之甚少。昼夜节律分子钟如何控制起搏神经元的电活动和突触的输出，从而最终控制动物的行为，这是一个悬而未决的问题。将通过膜片钳和成年全脑神经元成像的直接生理分析以及果蝇昼夜节律起搏神经元中修饰离子通道的转基因表达来研究起搏神经元电信号与昼夜分子钟和动物行为之间的生理相互作用。已经设计了转基因策略以（1）电沉默起搏神经元和（2）电超兴奋起搏神经元。每一种对起搏神经元电活动的操纵都会引起昼夜节律行为的显著变化。 具体目标是：（1）通过电沉默、神经递质标记物和免疫细胞化学绘制起搏神经回路的功能子集;（2）确定电超兴奋诱导的节律分裂和NaChBac表达对电沉默起搏神经回路的功能拯救的机制;（3）绘制野生型和修饰的表达通道的果蝇起搏器的电生理特性。 这些研究将阐明在一个充分研究的模式生物中决定昼夜节律行为的生理机制。最终，这项工作可能为神经回路的一般研究提供强大的新工具，并为研究和治疗异常细胞电兴奋性的人类疾病提供新的分子遗传学策略。

DESCRIPTION (provided by applicant): While much is known about the core components and the operation of the circadian molecular clock and its neuroanatomical location in the model organism Drosophila, very little is known about how this circadian molecular clock interacts with electrical signaling in the pacemaker neurons. It is an open questions as to how circadian molecular clock controls pacemaker neuronal electrical activity and synaptic output that ultimately controls animal behavior. The physiological interactions between pacemaker neuron electrical signaling and the circadian molecular clock and animal behavior will be studied by direct physiological analysis using patch clamp and imaging of adult whole brain neurons as well as transgenic expression of modified ion channels in the circadian pacemaker neurons of Drosophila. Transgenic strategies have been devised to (1) electrically silence pacemaker neurons and (2) electrically hyper-excite pacemaker neurons. Each of these manipulations of pacemaker neuronal electrical activity causes striking changes in circadian behavior. The Specific Aims are to: (1) Map the functional subsets of the pacemaker neural circuit by electrical silencing, neurotransmitter markers, and immunocytochemistry; (2) Determine the mechanism of electrically hyper-excitation induced rhythm splitting and NaChBac expression's functional rescue of the electrically silenced pacemaker neural circuit; (3) Map the electrophysiological properties of wild-type and modified channel-expressing Drosophila pacemakers. These studies will elucidate the physiological mechanisms that determine circadian behavior in a well studied model organism. Ultimately, this work may provide powerful new tools for the general study of neural circuits and novel molecular-genetic strategies for studying and treating human diseases of aberrant cellular electrical excitability.