Dopamine (DA) and its D2 receptor (R) are involved in cognition, reward processing, and drug addiction. However, their roles in sleep-wake regulation remain unclear. Herein we investigated the role of D2R in sleep wake regulation by using D2R knock-out (KO) mice and pharmacological manipulation. Compared with WT mice, D2R KO mice exhibited a significant decrease in wakefulness, with a concomitant increase in non-rapid eye movement (non-REM, NREM) andREMsleep and a drastic decrease in the low-frequency (0.75-2 Hz) electroencephalogram delta power of NREM sleep, especially during the first 4 h after lights off. The KO mice had decreased mean episode duration and increased episode numbers of wake andNREMsleep, many stage transitions between wakefulness andNREMsleep during the dark period, suggesting the instability of the wake stage in these D2R KO mice. When the KO mice were subjected to a cage change or an intraperitoneal saline injection, the latency to sleep in the KO mice decreased to half of the level for WT mice. The D2R antagonist raclopride mimicked these effects inWTmice. When GBR12909, a dopamine transport inhibitor, was administered intraperitoneally, it induced wakefulness inWTmice in a dose-dependent manner, but its arousal effect was attenuated to one-third in theD2RKO mice. However, these 2 genotypes showed an identical response in terms of sleep rebound after 2, 4, and 6 h of sleep deprivation. These results indicate that D2R plays an essential role in the maintenance of akefulness, but not in homeostatic regulation of NREM sleep.

L-stepholidine, an active ingredient of the Chinese herb Stephonia, is the first compound known to havemixed dopamine D1 receptor agonist/D2 antagonist properties and to be a potential treatment medication forschizophrenia. In schizophrenic patients insomnia is a common symptom and could be partly related to thepresumed over-activity of the dopaminergic system. To elucidate whether stepholidine modulates sleepbehaviors, we observed its effects on sleep–wake profiles in mice. The results showed that stepholidineadministered i.p. at doses of 20, 40 or 80 mg/kg significantly shortened the sleep latency to non-rapid eyemovement (non-REM, NREM) sleep, increased the amount of NREM sleep, and prolonged the duration ofNREM sleep episodes, with a concomitant reduction in the amount of wakefulness. Stepholidine at doses of40 and 80 mg/kg increased the number of state transitions from wakefulness to NREM sleep andsubsequently from NREM sleep to wakefulness. However, stepholidine had no effect on either the amount ofREM sleep or electroencephalogram power density of either NREM or REM sleep. Immunohistochemistrystudy showed that stepholidine dose-dependently increased c-Fos expression in neurons of the ventrolateralpreoptic area, a sleep center in the anterior hypothalamus, as compared with the vehicle control. Theseresults indicate that stepholidine initiates and maintains NREM sleep with activation of the sleep center inmice, suggesting its potential application for the treatment of insomnia.

Adenosine has been proposed to promote sleep through A1 receptors(A1R’s) and/or A2A receptors in the brain. We previouslyreported that A2A receptors mediate the sleep-promoting effect ofprostaglandin D2, an endogenous sleep-inducing substance, andthat activation of these receptors induces sleep and blockade ofthem by caffeine results in wakefulness. On the other hand, A1Rhas been suggested to increase sleep by inhibition of the cholinergicregion of the basal forebrain. However, the role and targetsites of A1R in sleep–wake regulation remained controversial. Inthis study, immunohistochemistry revealed that A1R was expressedin histaminergic neurons of the rat tuberomammillarynucleus (TMN). In vivo microdialysis showed that the histaminerelease in the frontal cortex was decreased by microinjection intothe TMN of N6-cyclopentyladenosine (CPA), an A1R agonist, adenosineor coformycin, an inhibitor of adenosine deaminase, whichcatabolizes adenosine to inosine. Bilateral injection of CPA into therat TMN significantly increased the amount and the delta powerdensity of non-rapid eye movement (non-REM; NREM) sleep butdid not affect REM sleep. CPA-promoted sleep was observed in WTmice but not in KO mice for A1R or histamine H1 receptor, indicatingthat the NREM sleep promoted by A1R-specific agonist dependedon the histaminergic system. Furthermore, the bilateral injection ofadenosine 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 TMNsuppresses the histaminergic system via A1R to promote NREMsleep.

Modafinil is a wake-promoting compound with low abuse potential used in the treatment of narcolepsy. Although the compound isreported to affect multiple neurotransmitter systems such as catecholamines, serotonin, glutamate, GABA, orexin, and histamine,however, the molecular mechanism by which modafinil increases wakefulness is debated. Herein we used dopamine (DA) D2 receptor(D2R)-deficient mice combined with D1R- and D2R-specific antagonists to clarify the role of DA receptors in the arousal effects ofmodafinil. In wild-type mice, intraperitoneal modafinil induced wakefulness in a dose-dependent manner. Pretreatment with eitherD1Rantagonist SCH23390 [R-(_)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine] at 30 _g/kg or D2R antagonistraclopride at 2 mg/kg blocked the arousal effects of low-dose modafinil at 22.5 and 45 mg/kg. When modafinil was given at 90 and180 mg/kg, pretreatment of D1R antagonist did not affect the wakefulness at all, whereas D2R antagonist significantly attenuated thewakefulness to the half level compared with vehicle control. Similarly,D2R knock-out (KO) mice exhibited attenuated modafinil-inducedwakefulness. However, pretreatment of D2R KO mice with D1R antagonist completely abolished arousal effects of modafinil. Thesefindings strongly indicate that dopaminergic D1R and D2R are essential for the wakefulness induced by modafinil.

To avoid the stress encountered during oral drug administration, we implanted chronically a catheterinto the stomach, and recorded electroencephalogram (EEG) and electromyogram, in freely moving ratsto evaluate their sleep–wake pattern. Rats with catheters in their stomach did not exhibit any changesin sleep–wake profiles in terms of sleep amount, number of episodes and EEG power spectra. Whenadministered through the catheter, caffeine (6 mg/kg) statistically increased wakefulness, as comparedwith the vehicle control. However, when given orally by hand restraint and gavage, it caused no increasein wakefulness, owing to the masking effect of this method, which caused increased wakefulness whensaline was used by handling animals. These results indicate that oral administration through a chronicstomach catheter is a useful way for evaluating wake-promoting components.