== All data were expressed as the mean SEM (n= 58). in KO mice for A1R or histamine H1receptor, indicating that the NREM sleep promoted by A1R-specific agonist depended on the histaminergic system. Furthermore, the bilateral injection of adenosine or coformycin into the rat TMN increased NREM sleep, which was completely abolished by coadministration of 1 1,3-dimethyl-8-cyclopenthylxanthine, a selective A1R antagonist. These results indicate that endogenous adenosine in the TMN suppresses the histaminergic system via A1R to promote NREM sleep. Keywords:Adenosine deaminase, histamine, FPH1 (BRD-6125) knockout mouse, rat In 1954, Feldberg and Sherwood (1) showed that intraventricular injection of micromole quantities of adenosine into cats caused a state resembling natural sleep of 30-min duration. Subsequent pharmacological studies from several laboratories demonstrated that adenosine and its receptor agonists promoted, but antagonists such as caffeine, inhibited both non-rapid eye movement (non-REM; NREM) and REM sleep (for review, see refs.2,3). However, the exact molecular mechanisms underlying sleepwake regulation by adenosine still remained unclear. Since FPH1 (BRD-6125) 1983, we have reported that prostaglandin (PG) D2is an endogenous sleep-inducing substance in rodents (4) and monkeys (5). PGD2-induced sleep was indistinguishable from natural physiological sleep as judged by several electrophysiological and behavioral criteria (5). Subsequent studies have shown that PGD2is involved in both circadian (6,7) and homeostatic regulation of sleep (8). Further studies on the molecular mechanisms of sleepwake regulation by PGD2demonstrated that PGD2is produced by the action of lipocalin-type PGD synthase dominantly expressed in the leptomeninges (9), binds with PGD receptors (DPRs) exclusively localized in the arachnoid membrane of the basal forebrain (BF) (10), and promotes sleep through adenosine acting at adenosine A2Areceptors (A2AR’s) (11), followed by activation of sleep-active neurons in the ventrolateral preoptic (VLPO) area (12,13). The somnogenic effect of PGD2is mimicked by an A2AR agonist, but not by an adenosine A1receptor (A1R) one, and is blocked by an A2AR antagonist (11). These findings indicate that the PGD2A2AR system is involved in both circadian and homeostatic sleep regulation (14). Among the four subtypes of adenosine receptors, A1, A2A, A2B, and A3(15), A1R and/or A2AR subtypes have been reported to mediate the sleep-promoting effect of adenosine. Although A2AR’s involvement in the PGD2VLPO system is clearly established, adenosine via A1R has been proposed to induce sleep by inhibiting the cholinergic region of the BF (16). For example, the unilateral infusion of the BF with an A1R-selective antagonist increased waking and decreased sleep (17). Single unit recording of BF neurons in conjunction within vivomicrodialysis of an A1R-selective agonist decreased, and an A1R antagonist, increased the discharge activity of the neurons in the BF (18). Moreover, perfusion of A1R antisense oligonucleotides into the BF reduced NREM sleep and EEG delta power (19). However, infusion of an A1R agonist into the lateral ventricle of mice did not alter the amounts of NREM and REM sleep (20). Caffeine, an antagonist for both A1R and A2AR, increased wakefulness in A1R KO mice and in WT mice, but not in A2AR KO mice (21). Therefore, the role of A1R in sleepwake regulation has remained uncertain. In the brain parenchyma, adenosine deaminase (ADA), an enzyme DGKH which catabolizes adenosine to inosine, is dominantly localized in the tuberomammillary nucleus (TMN) of the posterior hypothalamus (22) and is colocalized with histidine decarboxylase (HDC) (23), the key enzyme for histamine synthesis. Histaminergic neurons project from the TMN to most of the central nervous system and have been shown to promote wakefulness through histamine H1receptors (H1R’s) (3,24). However, the functional significance of adenosine and high expression of ADA in the TMN has not been elucidated so far. In the present study, we found that A1R was coexpressed with ADA in rat TMN and that activation of A1R or inhibition of ADA in the TMN inhibited histaminergic systems to promote NREM sleep without affecting REM sleep, clearly indicating that adenosine in the TMN promotes NREM sleep via A1R’s. == Results == == Localization of A1R in Histaminergic Neurons of the Rat TMN. == Immunohistochemical staining with polyclonal and FPH1 (BRD-6125) monoclonal (25) anti-A1R antibodies revealed that A1R was predominantly localized in the TMN in the posterior hypothalamus of rats.