Electrical Signaling in a Circadian Pacemaker Circuit

描述（由申请人提供）：健康的睡眠/觉醒周期依赖于控制觉醒和睡眠的神经回路的适当整合。了解这些神经回路之间的相互作用将提高我们治疗睡眠障碍的能力。最近发表的研究表明，果蝇起搏器回路中的弱昼夜节律神经元子集对感觉输入有生理反应，并有助于行为唤醒（Sheeba et al., 2008; Parisky et al., 2008; Shang et al., 2008）。提出了一种分子、生理和行为方法的综合结合，以阐明昼夜节律和觉醒神经回路之间的接口。为了实现这一目标，描述了在全脑制备和游离神经元培养中电生理记录果蝇起搏器神经元的新方法。最近的另一项关键技术进步是在单细胞分辨率下对整个表达per的中枢大脑昼夜节律回路的每个启动子周期进行多天的成像。这是通过将高灵敏度的低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实现的（Ayaz等人，2008）。据我们所知，这是第一个能够对整个神经回路进行长时间多天成像的制备。具体目标是：确定特异性，耦合和潜在的信号转导依赖于cryptochrome光诱导的起搏器神经元放电速率的快速变化。具体目标2。确定大外侧腹侧神经元是否有助于行为唤醒。具体目标3。利用全脑逐路成像技术，在高时空分辨率下确定大LNv觉醒神经元在光激活和外源诱发放电下整个昼夜节律回路的PER循环模式。这些研究将揭示在觉醒和昼夜循环之间发生的关键操作。这项工作可能会以一种快速和经济有效的方式为哺乳动物昼夜节律生物学提供新的见解，本文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。

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.