Orexin neurons are exclusively localized in the lateral hypothalamic area and project their fibers to the entire central nervous system, including the histaminergic tuberomammillary nucleus (TMN). Dysfunction of the orexin system results in the sleep disorder narcolepsy, but the role of orexin in physiological sleep–wake regulation and the mechanisms involved remain to be elucidated. Here we provide several lines of evidence that orexin A induces wakefulness by means of the TMN and histamine H ₁ receptor (H1R). Perfusion of orexin A (5 and 25 pmol/min) for 1 hr into the TMN of rats through a microdialysis probe promptly increased wakefulness for 2 hr after starting the perfusion by 2.5- and 4-fold, respectively, concomitant with a reduction in rapid eye movement (REM) and non-REM sleep. Microdialysis studies showed that application of orexin A to the TMN increased histamine release from both the medial preoptic area and the frontal cortex by ≈2-fold over the baseline for 80 to 160 min in a dose-dependent manner. Furthermore, infusion of orexin A (1.5 pmol/min) for 6 hr into the lateral ventricle of mice produced a significant increase in wakefulness during the 8 hr after starting infusion to the same level as the wakefulness observed during the active period in wild-type mice, but not at all in H1R gene knockout mice. These findings strongly indicate that the arousal effect of orexin A depends on the activation of histaminergic neurotransmission mediated by H1R.

Histaminergic neurons play an important role in the regulation of sleep–wake behavior through histamine H₁receptors (H₁R). Blockade of the histamine H₃receptor (H₃R) is proposed to induce wakefulness by regulating the release of various wake-related transmitters, not only histamine. In the present study, we characterized sleep–wake cycles of H₁R knockout (KO) mice and their arousal responses to an H₃R antagonist. Under baseline conditions, H₁R KO mice showed sleep–wake cycles essentially identical to those of WT mice but with fewer incidents of brief awakening (<16-sec epoch), prolonged durations of non-rapid eye movement (NREM) sleep episodes, a decreased number of state transitions between NREM sleep and wakefulness, and a shorter latency for initiating NREM sleep after an i.p. injection of saline. The H₁R antagonist pyrilamine mimicked these effects in WT mice. When an H₃R antagonist, ciproxifan, was administered i.p., wakefulness increased in WT mice in a dose-dependent manner but did not increase at all in H₁R KO mice.In vivomicrodialysis revealed that the i.p. application of ciproxifan increased histamine release from the frontal cortex in both genotypes of mice. These results indicate that H₁R is involved in the regulation of behavioral state transitions from NREM sleep to wakefulness and that the arousal effect of the H₃R antagonist completely depends on the activation of histaminergic systems through H₁R.

Infusion of prostaglandin (PG) D ₂ into the lateral ventricle of the brain induced an increase in the amount of non-rapid eye movement sleep in wild-type (WT) mice but not in mice deficient in the PGD receptor (DP). Immunofluorescence staining of WT mouse brain revealed that DP immunoreactivity was dominantly localized in the leptomeninges (LM) of the basal forebrain but that PGD synthase immunoreactivity was widely distributed in the LM of the entire brain. Electron microscopic observation indicated that DP-immunoreactive particles were predominantly located on the plasma membranes of arachnoid trabecular cells of the LM. The region with the highest DP immunoreactivity was clearly defined as bilateral wings in the LM of the basal forebrain located lateral to the optic chiasm in the proximity of the ventrolateral preoptic area, one of the putative sleep centers, and the tuberomammillary nucleus, one of the putative wake centers. The LM of this region contained DP mRNA 70-fold higher than that in the cortex as judged from the results of quantitative reverse transcription–PCR. PGD ₂ infusion into the subarachnoid space of this region increased the extracellular adenosine level more than 2-fold in WT mice but not in the DP-deficient mice. These results indicate that DPs in the arachnoid trabecular cells of the basal forebrain mediate an increase in the extracellular adenosine level and sleep induction by PGD ₂ .

Prostaglandin (PG) D ₂ has been proposed to be essential for the initiation and maintenance of the physiological sleep of rats because intracerebroventricular administration of selenium tetrachloride (SeCl ₄ ), a selective inhibitor of PGD synthase (PGDS), was shown to reduce promptly and effectively the amounts of sleep during the period of infusion. However, gene knockout (KO) mice of PGDS and prostaglandin D receptor (DP ₁ R) showed essentially the same circadian profiles and daily amounts of sleep as wild-type (WT) mice, raising questions about the involvement of PGD ₂ in regulating physiological sleep. Here we examined the effect of SeCl ₄ on the sleep of WT and KO mice for PGDS and DP ₁ R and that of a DP ₁ R antagonist, ONO-4127Na, on the sleep of rats. The i.p. injection of SeCl ₄ into WT mice decreased the PGD ₂ content in the brain without affecting the amounts of PGE ₂ and PGF _2α . It inhibited sleep dose-dependently and immediately after the administration during the light period when mice normally sleep, increasing the wake time; and the treatment with this compound resulted in a distinct sleep rebound during the following dark period. The SeCl ₄ -induced insomnia was observed in hematopoietic PGDS KO mice but not at all in lipocalin-type PGDS KO, hematopoietic and lipocalin-type PGDS double KO or DP ₁ R KO mice. Furthermore, the DP ₁ R antagonist ONO-4127Na reduced sleep of rats by 30% during infusion into the subarachnoid space under the rostral basal forebrain at 200 pmol/min. These results clearly show that the lipocalin-type PGDS/PGD ₂ /DP ₁ R system plays pivotal roles in the regulation of physiological sleep.

Adenosine has been proposed to promote sleep through A₁receptors (A₁R's) and/or A_2Areceptors in the brain. We previously reported that A_2Areceptors mediate the sleep-promoting effect of prostaglandin D₂, an endogenous sleep-inducing substance, and that activation of these receptors induces sleep and blockade of them by caffeine results in wakefulness. On the other hand, A₁R has been suggested to increase sleep by inhibition of the cholinergic region of the basal forebrain. However, the role and target sites of A₁R in sleep–wake regulation remained controversial. In this study, immunohistochemistry revealed that A₁R was expressed in histaminergic neurons of the rat tuberomammillary nucleus (TMN).In vivomicrodialysis showed that the histamine release in the frontal cortex was decreased by microinjection into the TMN of N⁶-cyclopentyladenosine (CPA), an A₁R agonist, adenosine or coformycin, an inhibitor of adenosine deaminase, which catabolizes adenosine to inosine. Bilateral injection of CPA into the rat TMN significantly increased the amount and the delta power density of non-rapid eye movement (non-REM; NREM) sleep but did not affect REM sleep. CPA-promoted sleep was observed in WT mice but not 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 1,3-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.

To examine the function of prostaglandin (PG) D synthase (PGDS) gene, as well as endogenously produced PGD ₂ in sleep regulation in vivo , we generated transgenic (TG) mice that overexpress human PGDS gene to study their sleep behavior. Although no difference was observed in the sleep/wake patterns between wild-type and TG mice, a striking time-dependent increase in non-rapid eye movement (NREM), but not in rapid eye movement (REM), sleep was observed in two independent lines of TG mice after stimulation by tail clipping. Concomitantly, the spontaneous locomotor activity of TG animals was drastically decreased in response to the tail clip. Induction of NREM sleep in TG mice was positively correlated with the PGD ₂ production in the brain. Sleep, locomotion, and PGD ₂ content were essentially unchanged in wild-type mice after tail clipping. The results with TG mice demonstrate the involvement of the PGDS gene in the regulation of NREM sleep.