Project Summary/Abstract Hypothalamic suprachiasmatic nucleus (SCN) neurons express a cell-autonomous molecular clock that generates circadian rhythms and regulates physiological rhythms throughout the body. The molecular clock produces a circadian pattern of neuronal activity that feeds back onto the molecular circadian clock and strengthens its activity. Intercellular communication between SCN neurons and astrocytes further strengthens and synchronizes these neuronal rhythms. This integrated SCN network activity is critical for generating precise circadian timing signals, stabilizing the circadian clock, and determining an animal's behavioral circadian phenotype. Although small in size, the SCN expresses a diverse population of neurons with unique functional properties, spatial locations, and efferent projections that regulate different physiological and behavioral rhythms. SCN neurons expressing vasoactive intestinal peptide (VIP+) or arginine vasopressin (AVP+) are the most extensively studied. These neurons have distinct SCN locations and unique roles in photic entrainment, circadian timing maintenance, and different downstream circadian rhythms. The unique functional properties of the dorsal and ventral SCN regions reflects differences in the number and the coupling mechanisms and strength of oscillating neurons. Most SCN neurons utilize GABA as a neurotransmitter, and GABAergic neurotransmission in the SCN is rhythmic at synaptic and extrasynaptic GABAA receptors and shows significant regional variation. Astrocytes regulate GABA neurotransmission by releasing transmitters that modify GABA release and expressing GABA transporters that control the extrasynaptic GABA concentration. Multiple small-molecule transmitters and neuromodulators regulate GABA neurotransmission, but the cellular mechanisms of this regulation are poorly understood. GABA refines the action potential firing pattern, a critical component in refining the SCN circadian clock output. A complete understanding of how the SCN network generates circadian timing signals requires more detailed knowledge of the signaling pathways that mediate communication between SCN neurons and astrocytes and a deeper understanding of how these signaling pathways differ in different parts of the SCN. Our research's long-term goal is to identify the signaling pathways by which neurons and astrocytes communicate to generate and entrain circadian rhythms. Our short-term goal is to determine the mechanisms mediating GABA neurotransmission and regulating the coupling strength between individual SCN neuronal oscillators and SCN regions. The Specific Aims of the application are: 1) Investigate the different roles of synaptic and tonic GABA receptor-mediated neurotransmission in regulating the activity of SCN. 2) Investigate the mechanisms regulating GABA transporter activity in astrocytes and whether GABA released from astrocytes contributes to the tonic GABA current. 3) Examine the role of glutamate r...