Electrical Signaling in a Circadian Pacemaker Circuit

描述(申请人提供)：健康的睡眠/清醒周期取决于控制唤醒和睡眠的神经回路的适当整合。对这些回路之间相互作用的洞察将促进我们治疗睡眠障碍的能力。最近发表的工作表明，果蝇起搏器回路中的一部分弱昼夜节律神经元对感觉输入具有生理反应，并有助于行为唤醒(Sheeba等人，2008；Parisky等人，2008；Sang等人，2008)。提出了一种分子、生理和行为方法的综合组合，以阐明昼夜节律和觉醒神经回路之间的接口。为了达到这一目标，描述了在全脑标本和分离的神经元培养中电生理记录果蝇起搏神经元的新方法。最近的另一项关键技术进步是成像每个启动子在单细胞分辨率下对整个表达PER的中央大脑昼夜节律回路进行数天的循环。这是通过将高度敏感的微光成像系统与我们最近共同开发的强大的长期果蝇全脑培养系统相结合而实现的(Ayaz等人，2008年)。据我们所知，这是第一个允许对感官激活的整个神经回路进行长期多天成像的准备工作。具体目标是：特定目标1.确定隐色素依赖光诱导起搏神经元放电频率快速变化的特异性、偶联性和潜在的信号转导。明确目标2.确定大的外侧腹侧神经元是否有助于行为唤醒。具体目的3.通过全脑Per-Luc成像，以高空间和时间分辨率确定整个昼夜节律回路对光激活和外源性诱导的大LNV觉醒神经元放电的Per循环模式。这些研究将揭示发生在觉醒和昼夜节律之间的关键操作。这项拟议的工作可能会以快速和经济有效的方式为哺乳动物昼夜生物学提供新的见解，这里描述的分子遗传学和生理学工具将在未来被其他实验室用于研究神经回路和电兴奋性障碍。 与公共健康相关：睡眠中断可能是由于唤醒神经元的异常活动延迟睡眠开始并降低睡眠维持率，因此了解唤醒、昼夜节律和睡眠回路之间的相互作用至关重要。我们最近的工作发现了一组光驱动的神经元，它们连接果蝇的昼夜节律和唤醒电路，并与哺乳动物睡眠调节唤醒神经元有许多共同的生理特征。我们建议确定这些觉醒神经元的功能特性以及它们对昼夜节律回路的调节。

DESCRIPTION (provided by applicant): Healthy sleep/wake cycles depend on the proper integration of neural circuits that control arousal and sleep. Insight on the interactions between these circuits will promote our ability to treat sleep disorders. Recent published work shows that a subset of weakly circadian neurons in the Drosophila pacemaker circuit is physiologically responsive to sensory input and contributes to behavioral arousal (Sheeba et al., 2008; Parisky et al., 2008; Shang et al., 2008). An integrative combination of molecular, physiological, and behavioral approaches to illuminate an interface between circadian and arousal neural circuits is proposed. Towards this goal, novel methods to electrophysiologically record Drosophila pacemaker neurons in whole brain preparations and in dissociated neuronal cultures are described. Another key recent technical advance is to image per promoter cycling for the entire PER-expressing central brain circadian circuit at single cell resolution for multiple days. This was achieved by combining a highly sensitive low-light imaging system with a robust long-term Drosophila whole brain culture system that we recently co-developed (Ayaz et al., 2008). To the best of our knowledge, this is the first example of a preparation that permits long-term multi-day imaging of a sensory-enabled entire neural circuit. The Specific Aims are: Specific Aim 1. Determine the specificity, coupling, and underlying signal transduction of CRYPTOCHROME-dependent light-induced rapid changes in pacemaker neuron firing rate. Specific Aim 2. Determine whether large lateral ventral neurons contribute to behavioral arousal. Specific Aim 3. Determine the pattern of PER cycling for entire circadian circuit in response to light activation and exogenously induced firing of the large LNv arousal neurons at high spatial and temporal resolution by whole brain per-luc imaging. These studies will reveal critical operations that occur between arousal and circadian circuits. The proposed work will likely provide new insights for mammalian circadian biology in a rapid and cost effective manner and the molecular genetic and physiological tools described herein will be used in the future by other laboratories for studying neural circuits and disorders of electrical excitability. PUBLIC HEALTH RELEVANCE: Sleep disruption can occur due to aberrant activity of arousal neurons which delay sleep onset and lower sleep maintenance, thus understanding the interactions between arousal, circadian and sleep circuits is of critical importance. Our recent work identifies a group of light-driven neurons that interface circadian and arousal circuits in Drosophila and share many physiological characteristics with mammalian sleep regulating arousal neurons. We propose to determine the functional properties of these arousal neurons and their modulation of the circadian circuit.