Electrical Signaling in a Circadian Pacemaker Circuit

描述(申请人提供)：虽然对模式生物果蝇中昼夜节律分子钟的核心组件和工作原理及其神经解剖学位置知道得很多，但对这种昼夜节律分子钟如何与起搏器神经元中的电信号相互作用知之甚少。关于昼夜分子钟如何控制起搏器神经元的电活动和突触输出，最终控制动物的行为，这是一个悬而未决的问题。起搏器神经元电信号与昼夜节律分子钟之间的生理相互作用将通过膜片钳直接生理学分析和成体全脑神经元的成像以及转基因离子通道在果蝇昼夜节律起搏器神经元中的表达来研究。转基因策略已被设计用于(1)电沉默起搏神经元和(2)电超兴奋起搏神经元。这些对起搏器神经元电活动的每一种操作都会导致昼夜行为的显著变化。 其具体目的是：(1)通过电沉默、神经递质标记物和免疫细胞化学定位起搏器神经回路的功能亚群；(2)确定电高兴奋诱导节律分裂的机制和NaChBac表达对电沉默起搏神经回路的功能挽救；(3)定位野生型和改良通道表达的果蝇起搏器的电生理特性。 这些研究将阐明在一个经过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，这项工作可能会为神经回路的一般研究提供强大的新工具，并为研究和治疗人类异常细胞电兴奋性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.