Neuropeptide S Activates Histaminergic Neurons In The Tuberomammillary Nuecleus Through Its Receptor To Promote Wakefulness

Neuropeptide S (NPS)is a newly identified neuromodulator located in the brainstem. NPS selectively binds with high affinity to its receptor (NPSR), involving in the regulation of multiple central functions. The NPS-NPSR system is proposed as a newly identified arousal system. However, the effects of NPS on the architectures of sleep-waking states and the mechanisms involved remain unclear.First, the sleep-waking states and related EEG power spectrum were defined by the electroencephalogram(EEG)and electromyogram(EMG)signal recordings during light-phrase (8:00-20:00h) in freely moving rats following its intracerebroventricular(i.c.v.)injection,and ex-vivo Fos immunohistochemistry was made to identify the potential neuronal targets for NPS in the brain involved in arousal.Second, guided by c-Fos expression, sleep-waking studies following TMN microinjections of NPS,quantitative real-time PCR (RT-PCR),neuropharmacology and immunohistochemistry assays were applied to comprehensively demonstrate the profiles of NPS-NPSR system in regulating sleep-wake cycles, and the pathways and key targets of sleep-wake regulations from molecular to the overall levels, together aiming to reveal the mechanisms of NPS-NPSR system in regulating sleep-wake stages.Results:Central administration of NPS(0.1-1nmol) dose dependently induced an enhancement of wakefulness for54.7±3.2and64.9±2.1min respectively, characterized by the increase of constant high frequency and fast wave EEG activities (14.5-60Hz). In relation to saline (41.4±2.5min).the amount of wakefulness was increased by32.1%(P<0.01) and56.8%(P<0.001),whereas slow wave sleep(SWS) and paradoxical sleep(PS) characterized as FEG delta (0.5-4Hz) and theta (4.5-8.5Hz) activities were suppressed during the first2h section post injection. As compared with saline-treated rats,central administration of NPS increased c-Fos expression in histaminergic neurons by76.0%in the ventral tuberomammillary nucleus (TMN) and57.8%in the dorsal TMN, and in orexinergic neurons by28.2%in the perifornical nucleus (PeF),24.3%in the dorsal-medial hypothalamic nucleus (DMH), and13.7%in the lateral hypothalamic area (LH) of the posterior hypothalamus.RT-PCR assay revealed that NPSR mRNA was highly expressed in the TMN. Dual-immunofluorescences showed that91.5%HAergic neurons expressed NPSR in the TMN.Bilateral injection of NPS (0.1-1nmol) into the TMN dose dependently induced a remarkable wakefulness for72.1±2.8and96.5±1.2min respectively, accompanied by an increase in EEG high frequency activities(14.5-60Hz).As compared with saline (41.4±2.5min), the amount of wakefulness was increased by78.5%(P<0.001)and138.9%(P<0.001)respectively,and SWS and PS characterized by EEG delta (0.5-4Hz) and theta (4.5-8.5Hz) activities were concomitantly suppressed during the first2h following injection. The amount of wakefulness induced by NPS0.1nmol microinjected into TMN was1.1fold greater than by NPS1nmol i.c.v. administration.[D-Val5]NPS (20-40nmol)dose-dependently antagonized the arousal effect of NPS (0.1nmol)when co-injected into the TMN.Pyriiamine (H1R antagonist)5mg/kg or ranitidine (H2R antagonist)10mg/kg were administered i.p.respectively and significantly blocked the arousal-promoting effect of intra-TMN injection of0.1nmol NPS.Conclusion:NPSR mRNA is highly expressed in the TMN,and most of the HAergic neurons express NPSR.In comparison with i.c.v injection of NPS,a comparable wake-promoting effect is obtained with a10fold smaller dose by intra-TMN administration, and antagonists of NPSR. H1R and H2R receptors block the arousal effect of NPS, suggesting that the NPS-producing neurons in the brainstem project to the TMN histaminergic neurons in the posterior hypothalamus to promote wakefulness via activation of its cognate receptor. The TMN histaminergic neurons play a key role in the NPS-NPSR system in the regulation of sleep-wake stages.