PROJECT SUMMARY Although slow wave activity (SWA) in the EEG has been linked to homeostatic sleep regulation, the neurobiological substrate of sleep homeostasis is little understood. In three different species, we have identified a rare population of cortical GABAergic interneurons that expresses c-FOS only during sleep. These sleep-active cells express the enzyme neuronal nitric oxide synthase (nNOS) and NK1R, the receptor for Substance P. Cortical nNOS/NK1R neurons seem to monitor the homeostatic sleep drive that accumulates during wakefulness because the percentage of nNOS/NK1R cells that express cFOS during sleep is proportional to prior wake duration. Cortical nNOS/NK1R cells are exceptional among cortical GABAergic neurons in that they have widespread intracortical projections. We have proposed that cortical nNOS/NK1R neurons are critical for orchestrating EEG SWA during sleep through release of an amino acid transmitter (GABA), a peptidergic transmitter (Neuropeptide Y), and/or a gaseous transmitter (NO). Thus, cortical nNOS/NK1R neurons may be part of the long- sought neuroanatomical circuit underlying the sleep-dependent ?Process S? and may provide insight into the neural circuitry underlying homeostatic sleep regulation. Determination of the inputs to cortical nNOS/NK1R neurons will be central to understanding their regulation and function. In the present application, we will test the hypotheses that Substance P excites cortical nNOS neurons through NK1R and that cortical nNOS/NK1R neurons are both sufficient and necessary for propagation of EEG SWA. To address these hypotheses, we will use a combination of in vitro physiology, cellular pharmacology, functional neuroanatomy, microendoscopic Ca2+ imaging, optogenetic stimulation and inhibition in vitro and in vivo, and cell-specific ablation using both wildtype and multiple transgenic mouse strains. Together, these experiments will provide further insight into the afferent regulation of cortical nNOS/NK1R neurons, as well as the necessity and sufficiency of these cells for EEG SWA. The results will not only enhance our understanding of sleep/wake regulation, but may also have implications for understanding sleep disorders and neurological and psychiatric diseases involving the cerebral cortex.