The caudal hypothalamus contains a key node of the ascending arousal system, with lesions causing more profound somnolence than can be accounted for by involvement of nearby hypothalamic orexin and histamine cell groups. Methods: We used chemogenetic manipulations and Cre-lox glutamate release disruption in anatomically-and genetically-targeted neuronal groups of the caudal hypothalamus and recorded EEG and EMG, with subsequent histological analysis of injection sites and terminal fields of

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... sduced neurons. ANOVA was used for statistical analysis of sleep physiology and non-parametric statistical mapping was used to objectively determine the region that was wake-promoting in the caudal hypothalamus. Results: Activation of glutamatergic supramammillary (SuM vglut2 ) neurons or a subset of them that also express nitric oxide synthase (Nos1, SuMV glut2/Nos1 ) is potently wake-promoting. Genetic disruption of glutamatergic neurotransmission from SuM vglut2 neurons nearly completely abolishes the effects of SuM activation. Targeted chemogenetic inhibition of SuM vglut2 neurons produced fragmented wakefulness and increased sleep, akin to drowsiness following caudal hypothalamic injury. Conclusion: SuM vglut2/Nos1 neurons exert potent control over behavioral wakefulness, with these effects depending critically on glutamate release. Glutamate neurons of the SuM, including a key subset that contain nitric oxide, likely represent the long-sought caudal hypothalamic component of the ascending arousal system.