Adenosine in the tuberomammillary nucleus inhibits the histaminergic system via A1 receptors and promotes non-rapid eye movement sleep Yo Oishia,b, Zhi-Li Huanga,c,1, Bertil B. Fredholmd, Yoshihiro Uradea,b, and Osamu Hayaishia,1 aDepartment of Molecular Behavioral Biology, Osaka Bioscience Institute, Suita, Osaka 565-0874, Japan; bDepartment of Aging Science, Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan; cState Key Laboratory of Medical Neurobiology and Department of Pharmacology, Shanghai Medical College of Fudan University, Shanghai 200032, China; and dDepartment of Physiology and Pharmacology, Karolinska Institute, S-171 77 Stockholm, Sweden Contributed by Osamu Hayaishi, October 30, 2008 (sent for review October 6, 2008) Adenosine has been proposed to promote sleep through A1 re- promotes sleep through adenosine acting at adenosine A2A ceptors (A1R’s) and/or A2A receptors in the brain. We previously receptors (A2AR’s) (11), followed by activation of sleep-active reported that A2A receptors mediate the sleep-promoting effect of neurons in the ventrolateral preoptic (VLPO) area (12, 13). The prostaglandin D2, an endogenous sleep-inducing substance, and somnogenic effect of PGD2 is mimicked by an A2AR agonist, but that activation of these receptors induces sleep and blockade of not by an adenosine A1 receptor (A1R) one, and is blocked by them by caffeine results in wakefulness. On the other hand, A1R an A2AR antagonist (11). These findings indicate that the has been suggested to increase sleep by inhibition of the cholin- PGD2–A2AR system is involved in both circadian and homeo- ergic region of the basal forebrain. However, the role and target static sleep regulation (14). Among the four subtypes of adenosine receptors, A1,A2A, sites of A1R in sleep–wake regulation remained controversial. In this study, immunohistochemistry revealed that A R was ex- A2B, and A3 (15), A1R and/or A2AR subtypes have been reported 1 to mediate the sleep-promoting effect of adenosine. Although pressed in histaminergic neurons of the rat tuberomammillary A R’s involvement in the PGD –VLPO system is clearly es- nucleus (TMN). In vivo microdialysis showed that the histamine 2A 2 tablished, adenosine via A R has been proposed to induce sleep release in the frontal cortex was decreased by microinjection into 1 6 by inhibiting the cholinergic region of the BF (16). For example, the TMN of N -cyclopentyladenosine (CPA), an A1R agonist, aden- the unilateral infusion of the BF with an A1R-selective antag- osine or coformycin, an inhibitor of adenosine deaminase, which onist increased waking and decreased sleep (17). Single unit catabolizes adenosine to inosine. Bilateral injection of CPA into the recording of BF neurons in conjunction with in vivo microdialysis rat TMN significantly increased the amount and the delta power of an A1R-selective agonist decreased, and an A1R antagonist, density of non-rapid eye movement (non-REM; NREM) sleep but increased the discharge activity of the neurons in the BF (18). did not affect REM sleep. CPA-promoted sleep was observed in WT Moreover, perfusion of A1R antisense oligonucleotides into the mice but not in KO mice for A1R or histamine H1 receptor, indicating BF reduced NREM sleep and EEG delta power (19). However, that the NREM sleep promoted by A1R-specific agonist depended infusion of an A1R agonist into the lateral ventricle of mice did on the histaminergic system. Furthermore, the bilateral injection of not alter the amounts of NREM and REM sleep (20). Caffeine, adenosine or coformycin into the rat TMN increased NREM sleep, an antagonist for both A1R and A2AR, increased wakefulness in which was completely abolished by coadministration of 1,3- A1R KO mice and in WT mice, but not in A2AR KO mice (21). dimethyl-8-cyclopenthylxanthine, a selective A1R antagonist. Therefore, the role of A1R in sleep–wake regulation has re- These results indicate that endogenous adenosine in the TMN mained uncertain. In the brain parenchyma, adenosine deaminase (ADA), an suppresses the histaminergic system via A1R to promote NREM sleep. enzyme which catabolizes adenosine to inosine, is dominantly localized in the tuberomammillary nucleus (TMN) of the pos- terior hypothalamus (22) and is colocalized with histidine de- Adenosine deaminase ͉ histamine ͉ knockout mouse ͉ rat carboxylase (HDC) (23), the key enzyme for histamine synthesis. Histaminergic neurons project from the TMN to most of the n 1954, Feldberg and Sherwood (1) showed that intraventric- central nervous system and have been shown to promote wake- Iular injection of micromole quantities of adenosine into cats fulness through histamine H1 receptors (H1R’s) (3, 24). How- caused a state resembling natural sleep of 30-min duration. ever, the functional significance of adenosine and high expres- Subsequent pharmacological studies from several laboratories sion of ADA in the TMN has not been elucidated so far. demonstrated that adenosine and its receptor agonists pro- In the present study, we found that A1R was coexpressed with moted, but antagonists such as caffeine, inhibited both non-rapid ADA in rat TMN and that activation of A1R or inhibition of eye movement (non-REM; NREM) and REM sleep (for review, ADA in the TMN inhibited histaminergic systems to promote see refs. 2, 3). However, the exact molecular mechanisms NREM sleep without affecting REM sleep, clearly indicating underlying sleep–wake regulation by adenosine still remained that adenosine in the TMN promotes NREM sleep via A1R’s. unclear. Since 1983, we have reported that prostaglandin (PG) Results D2 is an endogenous sleep-inducing substance in rodents (4) and Localization of A1R in Histaminergic Neurons of the Rat TMN. Immu- monkeys (5). PGD2-induced sleep was indistinguishable from natural physiological sleep as judged by several electrophysio- nohistochemical staining with polyclonal and monoclonal (25) logical and behavioral criteria (5). Subsequent studies have shown that PGD2 is involved in both circadian (6, 7) and Author contributions: Y.O., Z.-L.H., Y.U., and O.H. designed research; Y.O. and Z.-L.H. homeostatic regulation of sleep (8). Further studies on the performed research; B.B.F. contributed new reagents/analytic tools; Y.O., Z.-L.H., Y.U., and molecular mechanisms of sleep–wake regulation by PGD2 dem- O.H. analyzed data; and Y.O., Z.-L.H., Y.U., and O.H. wrote the paper. onstrated that PGD2 is produced by the action of lipocalin-type The authors declare no conflict of interest. PGD synthase dominantly expressed in the leptomeninges (9), 1To whom correspondence may be addressed. E-mail: [email protected] or binds with PGD receptors (DPRs) exclusively localized in the [email protected]. arachnoid membrane of the basal forebrain (BF) (10), and © 2008 by The National Academy of Sciences of the USA 19992–19997 ͉ PNAS ͉ December 16, 2008 ͉ vol. 105 ͉ no. 50 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0810926105 Downloaded by guest on September 30, 2021 demonstrated that A1R was colocalized with HDC or ADA in the TMN. These results indicate that A1R was expressed in HDC-positive neurons and in ADA-positive ones in the TMN. Inhibitory Action of A1R in Histaminergic Systems of the Rat TMN. To monitor the regulation of histaminergic systems by A1R or ADA, we determined the histamine release from the frontal cortex (FrCx) after a bolus injection of a selective A1R agonist, N6-cyclopentyladenosine (CPA), its natural agonist, adenosine, or an ADA inhibitor, coformycin, into the TMN in urethane- anesthetized rats (Fig. 1J). The CPA administration into the TMN (0.17–1.5 nmol/side) induced a remarkable dose- dependent decrease in the histamine release from the FrCx for 2 h after the injection (Fig. 1K). The adenosine administration (2.7 or 4.5 nmol/side) also induced a significant decrease in the histamine release for1haftertheinjection in a dose-dependent manner (Fig. 1L), although the inhibition of the histamine release was weaker and shorter than in the case of CPA. The injection of coformycin (1.3 or 4 nmol/side) also decreased the histamine release for1hfrom1haftertheinjection (Fig. 1M). These results indicate that the stimulation of A1R’s in the TMN with CPA or adenosine and the inhibition of ADA with cofor- mycin suppressed the activity of histaminergic systems in the TMN to decrease the histamine release in the FrCx. NREM Sleep Promotion by Activation of A1R in the Rat TMN with CPA. We then examined the sleep–wake profile after a bilateral injection of CPA into the TMN of freely moving rats at 22:00, when such animals spend most of their time in wakefulness. Typical examples of EEG, electromyogram (EMG), and hypno- grams from rats given vehicle or CPA at a dose of 1.5 nmol/side are shown in Fig. 2A. The CPA (1.5 nmol/side) injection remarkably increased NREM sleep for3hfrom22:30 to 01:30, as compared with the case of the vehicle injection. As shown in Fig. 2B, CPA at 1.5 nmol/side significantly increased the hourly NREM sleep time by 4.6-, 2.1-, 2.5-, and 2.1-fold during the first, second, third, and fourth hour, respec- tively, after the injection as compared with the vehicle injection. Enhancement of NREM sleep by the CPA injection concomi- Fig. 1. Immunohistochemistry of A1R, A2AR, and HDC in the rat TMN (A–H) and in vivo microdialysis to measure histamine release in the rat FrCx (I–M). (A) tantly decreased wakefulness, but did not affect REM sleep. No Low- and high- (inset to A) magnification views of rat TMN immunostained additional disruption of sleep architecture was observed during with polyclonal A1R antibody. (B) Low- and high- (inset to B) magnification the subsequent period. Similar time course profiles were ob- views of rat TMN after incubation with anti-A2AR antibody.