Electrical Signaling in a Circadian Pacemaker Circuit

描述（由申请人提供）：健康的睡眠/觉醒周期取决于控制觉醒和睡眠的神经回路的适当整合。深入了解这些回路之间的相互作用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弱昼夜节律神经元的子集在生理上响应于感觉输入并有助于行为唤醒（Sheeba等人，2008; Parisky等人，2008; Shang等人，2008年）。分子，生理和行为的方法来照亮昼夜节律和唤醒神经回路之间的接口的综合组合提出。为了实现这一目标，新的方法来电生理记录果蝇起搏神经元在全脑制剂和分离的神经元培养。最近的另一个关键技术进步是以单细胞分辨率对整个表达PER的中枢脑昼夜节律回路的每个启动子循环进行多天成像。这是通过将高度灵敏的低光成像系统与我们最近共同开发的稳健的长期果蝇全脑培养系统相结合来实现的（Ayaz等人，2008年）。据我们所知，这是第一个允许对整个感官神经回路进行长期多日成像的制备实例。具体目标是：具体目标1。确定光敏色素依赖性光诱导的起搏神经元放电率快速变化的特异性、偶联和潜在信号转导。具体目标2。确定大的侧腹神经元是否有助于行为唤醒。具体目标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响应于光激活和大LNv唤醒神经元的外源诱导放电的PER循环模式。这些研究将揭示唤醒和昼夜节律回路之间发生的关键操作。拟议的工作可能会提供新的见解哺乳动物昼夜节律生物学在一个快速和成本效益的方式和分子遗传学和生理学的工具，本文所述的将在未来由其他实验室用于研究神经回路和电兴奋性障碍。 公共卫生相关性：睡眠中断可能是由于唤醒神经元的异常活动而发生的，这些异常活动延迟睡眠开始并降低睡眠维持，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。我们最近的工作确定了一组光驱动的神经元接口的昼夜节律和唤醒电路在果蝇和哺乳动物睡眠调节唤醒神经元共享许多生理特征。我们建议确定这些唤醒神经元的功能特性和它们对昼夜节律回路的调制。

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.