Circuitry and Modulation of the Circardian Clock Network of D. melanogaster

描述(由申请人提供)： 单个已识别基因的变化可以对行为产生可测量的和生物相关的影响，这一发现改变了我们对行为控制方式的理解。这些基因的作用如何统领神经系统的复杂回路是神经科学中一个长期存在的问题，我希望开发一个研究计划来解决这个问题。一个由大约150个神经元组成的网络组织了黑腹蛇的日常运动行为模式。虽然人们对支持细胞内计时的时钟基因知道得很多，但关于时钟神经元如何与中枢神经系统的其他部分传递一天中的时间信息却知之甚少。神经肽色素分散因子(PDF)由16个时钟神经元表达，是正常昼夜运动行为所必需的，但其靶点尚不清楚。最近，PDFr，PDF的G蛋白偶联受体(G-Protein-Couted-Receptor，GPCR)被发现。PDFr通过营地发出信号。我已经开发了cAMP成像方法来测量已识别的、活的苍蝇神经元中的GPCR信号。这些方法与苍蝇研究人员可用的强大遗传工具相结合，现在可以直接绘制活着的大脑中的多肽感受性图谱，并将能够对中枢神经系统中与行为相关的GPCR信号进行分子/遗传表征。为了了解神经时钟网络的组织，PDF的作用机制，以及神经系统中GPCR信号的基本问题，我提出了以下目标：i)开发实时成像方法来映射PDF和其他多肽、胺和贯穿整个大脑的递质的感受性；ii)我将识别和表征神经时钟网络的神经化学调节器；iii)我将研究在昼夜节律时间尺度上所确定的时钟神经元内的cAMP和钙信号动力学。Iv)我将对已识别的时钟神经元内的GPCR/cAMP信号转导机制进行遗传分析。初步结果证明了所提出研究各方面的可行性。公共卫生相关性：鉴于GPCR信号对中枢神经系统功能的核心重要性，这项工作将解决神经科学的基本方面。这里概述的工作将很好地延伸到我提议的研究的独立阶段。苍蝇时钟网络功能电路的表征以及时钟神经元内GPCR/cAMP信号的遗传解剖，将一直是我职业生涯的重点，并将基于我在指导研究期间开发的方法和候选递质/基因。

DESCRIPTION (provided by applicant): The discovery that changes in single identified genes can have measurable and biologically relevant effects on behavior has transformed our understanding of how behavior is controlled. How the actions of such genes marshal the complicated circuitry of the nervous system is an abiding problem in Neuroscience, and I hope to develop a research program to address it. A network of approximately 150 neurons organizes the daily pattern of locomotor behavior in D. melanogaster. Though much is known about the clock genes that support intracellular timekeeping, little is known about how clock neurons communicate time-of-day information to the rest of the CNS. The neuropeptide pigment dispersing factor (PDF) is expressed by 16 clock neurons and is required for normal circadian locomotor behavior, but its targets are unknown. Recently, PDFr, PDF's G-protein-coupled-receptor (GPCR) was identified. PDFr signals through cAMP. I have developed cAMP imaging methods to measure GPCR signaling within identified, living fly neurons. These methods, in conjunction with the powerful genetic tools available to the fly researcher, now permit direct mapping of peptide receptivity within the living brain and will allow for a molecular/genetic characterization of behaviorally relevant GPCR signaling within the CNS. To understand the organization of the neuronal clock network, the mechanisms of PDF action, and fundamental questions of GPCR signaling in the nervous system, I propose the following aims: I) I will develop live imaging methods for the mapping of receptivity to PDF and other peptides, amines, and transmitters throughout the brain of D. melanogaster; II) I will identify and characterize neurochemical modulators of the neuronal clock network; III) I will investigate cAMP and Ca2+ signaling dynamics within identified clock neurons over circadian timescales. IV) I will employ a genetic analysis of GPCR/cAMP signal transduction mechanisms within identified clock neurons. Preliminary results establish the feasibility of all aspects of the proposed research. PUBLIC HEALTH RELEVANCE: Given the central importance of GPCR signaling for CNS function, this work will address fundamental aspects of Neuroscience. The work outlined here will extend well into the independent phase of my proposed research. The characterization of the functional circuitry of the flies clock network and the genetic dissection of GPCR/cAMP signaling within clock neurons, will long be the focus of my career and will be based on the methods and candidate transmitters/genes developed during my mentored research.