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The median preoptic nucleus (MnPN) and the ventrolateral preoptic area (VLPO) are two hypothalamic regions that have been implicated in sleep regulation, and both nuclei contain sleep-active GABAergic neurons. Adenosine is an endogenous sleep regulatory substance, which promotes sleep via A1 and A2A receptors (A2AR). Infusion of A2AR agonist into the lateral ventricle or into the subarachnoid space underlying the rostral basal forebrain (SS-rBF), has been previously shown to increase sleep. We examined the effects of an A2AR agonist, CGS-21680, administered into the lateral ventricle and the SS-rBF on sleep and c-Fos protein immunoreactivity (Fos-IR) in GABAergic neurons in the MnPN and VLPO. Intracerebroventricular administration of CGS-21680 during the second half of lights-on phase increased sleep and increased the number of MnPN and VLPO GABAergic neurons expressing Fos-IR. Similar effects were found with CGS-21680 microinjection into the SS-rBF. The induction of Fos-IR in preoptic GABAergic neurons was not secondary to drug-induced sleep, since CGS-21680 delivered to the SS-rBF significantly increased Fos-IR in MnPN and VLPO neurons in animals that were not permitted to sleep. Intracerebroventricular infusion of ZM-241385, an A2AR antagonist, during the last 2 h of a 3-h period of sleep deprivation caused suppression of subsequent recovery sleep and reduced Fos-IR in MnPN and VLPO GABAergic neurons. Our findings support a hypothesis that A2AR-mediated activation of MnPN and VLPO GABAergic neurons contributes to adenosinergic regulation of sleep.

The hypocretin (HCRT) system of the perifornical-lateral hypothalamic area (PF-LHA) has been implicated in the facilitation of behavioral arousal. HCRT neurons receive serotonergic afferents from the dorsal raphe nucleus. Although in-vitro pharmacological studies suggest that serotonin (5-HT) inhibits HCRT neurons, the in-vivo effects of 5-HT on HCRT neurons in the PF-LHA and associated behavioral changes have not been described. We examined the effects of 5-HT delivered locally into the PF-LHA using reverse microdialysis on its neuronal activity and the consequent sleep-wake changes in rats. First, we quantified Fos expression (Fos-IR) in HCRT and other PF-LHA neurons following unilateral 5-HT perfusion in awake rats. Second, we determined the transient effects of 5-HT perfusion on the extracellular activity of the PF-LHA neurons recorded via microwires placed adjacent to the microdialysis probe. Third, we examined the effects of 5-HT perfusion into the PF-LHA on the sleep-wake profiles of the rats during the lights-off period. Unilateral perfusion of 5-HT into the PF-LHA in awake rats dose-dependently decreased the number of HCRT neurons exhibiting Fos-IR. 5-HT also inhibited the discharge activity of four of five responsive wake-related, putative HCRT neurons. However, unilateral perfusion of 5-HT into the PF-LHA did not produce significant behavioral changes during the 2-h recording period. These results confirm the in-vitro findings that 5-HT exerts inhibitory influences on HCRT neurons but further suggest that the inactivation of a limited number of HCRT neurons by unilateral 5-HT microdialysis may not be sufficient to induce behavioral changes.

The perifornical-lateral hypothalamic area (PF-LHA) has been implicated in the regulation of arousal. The PF-LHA contains wake-active neurons that are quiescent during nonREM sleep and in the case of neurons expressing the peptide hypocretin (HCRT), quiescent during both nonREM and REM sleep. Adenosine is an endogenous sleep factor and recent evidence suggests that adenosine via A 1 receptors may act on PF-LHA neurons to promote sleep. We examined the effects of bilateral activation as well as blockade of A 1 receptors in the PF-LHA on sleep-wakefulness in freely behaving rats.The sleep-wake profiles of male Wistar rats were recorded during reverse microdialysis perfusion of artificial cerebrospinal fluid (aCSF) and two doses of adenosine A 1 receptor antagonist, 1,3-dipropyl-8-phenylxanthine (CPDX; 5μM and 50μM) or A 1 receptor agonist, N 6 -cyclopentyladenosine (CPA; 5μM and 50μM) into the PF-LHA for 2h followed by 4h of aCSF perfusion. CPDX perfused into the PF-LHA during lights-on phase produced arousal (F=7.035, p <0.001) and concomitantly decreased both nonREM (F=7.295, p<0.001) and REM sleep (F=3.456, p<0.004). In contrast, CPA perfused into the PF-LHA during lights-off phase significantly suppressed arousal (F = 7.891; p <0.001) and increased nonREM (F = 8.18; p <0.001) and REM sleep (F = 30.036; p = <0.001). These results suggest that PF-LHA is one of the sites where adenosine, acting via A 1 receptors, inhibits PF-LHA neurons to promote sleep.

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The perifornical-lateral hypothalamic area (PF-LHA) plays a central role in the regulation of behavioral arousal. The PF-LHA contains several neuronal types including wake-active hypocretin (HCRT) neurons that have been implicated in the promotion and/or maintenance of behavioral arousal. Adenosine is an endogenous sleep factor and recent evidence suggests that activation and blockade of adenosine A1 receptors within the PF-LHA promote and suppress sleep, respectively. Although, an in vitro study indicates that adenosine inhibits HCRT neurons via A1 receptor, the in vivo effects of A1 receptor mediated adenosinergic transmission on PF-LHA neurons including HCRT neurons are not known. First, we determined the effects of N6-cyclopentyladenosine (CPA), an adenosine A1 receptor agonist, on the sleep-wake discharge activity of the PF-LHA neurons recorded via microwires placed adjacent to the microdialysis probe used for its delivery. Second, we determined the effects of CPA and that of an A1 receptor antagonist, 1,3-dipropyl-8-phenylxanthine (CPDX) into the PF-LHA on cFos-protein immunoreactivity (Fos-IR) in HCRT and non-HCRT neurons around the microdialysis probe used for their delivery. The effect of CPA was studied in rats that were kept awake during lights-off phase, whereas the effect of CPDX was examined in undisturbed rats during lights-on phase. CPA significantly suppressed the sleep-wake discharge activity of PF-LHA neurons. Doses of CPA (50μM) and CPDX (50μM) that suppressed and induced arousal, respectively, in our earlier study (Alam et al., 2009), significantly suppressed and increased Fos-IR in HCRT and non-HCRT neurons. These findings suggest that wake-promoting PF-LHA system is subject to increased endogenous adenosinergic inhibition and that adenosine acting via A1 receptors, in part, inhibits HCRT neurons to promote sleep.