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.