Project Summary/Abstract Sleep is an active process requiring the participation of delimited nodes of sleep-promoting cell populations. Work over the last twenty years has demonstrated that galanin expressing neurons in the ventrolateral preoptic (VLPOGal) nucleus are necessary for normal sleep. While a portion of VLPO galanin neurons is concentrated in a cluster, the so-called VLPO core (cVLPO), others are scattered dorsally and medially to that core, a region that came to be known as the extended VLPO (eVLPO). Lesion and optogenetic studies have demonstrated that the galanin expressing neurons in the cVLPO and those in the eVLPO are two functionally distinct cell groups with the cVLPOGal neurons required for non-REM sleep (NREM) while the eVLPOGal neurons required for REM sleep. Anatomical tracing studies have shown that the eVLPOGal neurons innervate brainstem nuclei implicated in REM sleep regulation and through these descending projections, the eVLPOGal neurons are thought to promote REM sleep by the disinhibition of the REM-generating neurons in the pontine reticular formation. There remain however several fundamental gaps in our understanding of the cellular and synaptic basis by which eVLPOGal neurons drive REM sleep. Studying the eVLPOGal population has been proven to be quite challenging as the cVLPO and the eVLPO are small regions located in close proximity and until now, there were no markers for selective targeting. We have recently discovered a potential marker for the eVLPOGal neurons that will allow us to manipulate the eVLPOGal neurons independently from their neighboring cVLPOGal neurons. Specifically, we have found that: 1) the eVLPOGal neurons selectively express the neuropeptide neurotensin and, 2) that chemogenetic inhibition of the neurotensin eVLPO neurons reduces REM sleep. We have also found that eVLPO neurotensin neurons send robust projections to regions of the midbrain and pons that are known to suppress REM sleep including the ventrolateral periaqueductal gray (vlPAG) and the locus coeruleus (LC). On the basis of these results, we propose that the eVLPO neurotensin neurons promote REM sleep by disinhibiting REM-generating neurons in the pontine reticular formation via inhibition of GABA neurons of the vlPAG and monoaminergic neurons of the LC. The current proposal thus seeks to identify, first in vitro and then in vivo, the brainstem efferent outputs through which the eVLPO neurotensin neurons drive REM sleep. To do so, we will first employ, in Specific Aims 1, in vitro circuit mapping to identify the postsynaptic neurons that are targeted by the eVLPO neurotensin neurons. In Specific Aim 2, we will use in vivo optogenetics to test whether the circuits first identified in the in vitro studies are sufficient to produce REM sleep. Given the large knowledge gap this proposal seeks to fill, we expect results from this work to provide 1) an important interpretative framework for future studies on the eVLPO neurotensin pa...