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Objective The effect of neuropeptide S (NPS) on characteristics and mechanisms in the regulation of sleep-waking cycle are unclear, though the initial findings that NPS promotes arousal associated to locomotor activity and a genuine anxiolytic-like action have recently been documented.We hypothesized that NPS activated posterior hypothalamic arousal systems involved histaminergic (HAergic) neurons in tuberomammilary nucleus (TMN) and orexinergic (OXergic) neurons in perifornical (PeF) and lateral hypothalamic (LH) area, where NPSR mRNA has been reported to be highly expressed, to regulate sleep-wakefulness cycle.The present studies were designed to provide more information regarding the NPS-induced sleep-waking characteristics and potential targets in posterior hypothalamus by analyzing each state across sleep-waking cycle, especially focusing on active waking (aW) and quiet waking (qW) states since the rather unique behavioral profile challenges the common idea that anxiolytics are also sedative or that stimulants are also anxiogenic, during the 24 h period after a range dose ofNPS intracerebroventricular (ICV) injection and by observing earlier genes c-fos expression.Methods Four stainless-steel screws as electroencephalogram (EEG) electrodes were screwed into the animals skull and three silver wire as electromyogram (EMG) electrodes were inserted into the dorsal cervical neck muscles to monitor the sleep-wake cycle, and the EEG and EMG signals were amplified and digitalized with a resolution of 256 and 128 Hz respectively using a CED 1401 MK Ⅱ.After sleep-waking recording, c-fos immunereactive (Fos-IR), histidine decarboxylase (HDC)-IR, orexin-A (OX-A)-IR, and dual labeling of Fos-IR+HDC-IR and Fos-IR+OX-A-IR neurons were respectively labeled to identify the active sites in the brain for the NPS effects on arousal.Results The sleep-wake profile after an ICV injection of NPS at 10:00 h was examined, when rats with saline injection spend most of their time in sleep.NPS given at 0.1 and 1.0 nmol induced a dose-dependent increase in aW time for 36.18 ± 1.69 min (P < 0.01) and 49.33 ± 1.69 min (P < 0.01, compared with 18.3 ± 0.87 min for saline treatment), a decrease in light slow-wave sleep (SWS1) time for 6.81 ± 1.14 min (P < 0.01) and 2.94 ± 0.58 min (P < 0.01, compared with 13.06 ± 1.02 min for saline treatment), deep slow-wave sleep (SWS2) time for 10.56 ± 1.09 min (P < 0.01) and 2.72 ± 0.78 min (P < 0.01, compared with 19.22 ± 1.21 min for saline treatment) during the first hour, and a rebound of SWS2 time (32.38 ± 1.86 and 33.56 ± 1.92 min, P < 0.01, compared with 20.39 ± 1.49 min for saline treatment) at 2 h.In addition, NPS 1.0 nmol consistently suppressed paradoxical sleep (PS) for 3 h and increased a W time (P < 0.01, 22.83 ± 2.47 min, compared with 6.56 ± 1.59 min for saline treatment) again at 3 h.In contrast, there was no significant difference in qW between range dose of NPS and saline treatment.Cortical power spectra showed that NPS administration caused a markedly increase in neocortical fast rhythms including β and γ waves (14.5-60 Hz), a suppression in α wave (9-14 Hz, spindles), θ wave (4.5-8.5 Hz) and δ wave (0.5-4 Hz) at 1 h compared with saline, and cortical slow activity increase at 2 h.Furthermore, neocortical fast rhythms increased again at 3 h accompanied by suppression in δ wave following NPS 1 nmol treatment.NPS administration increased the number of Fos-IR neurons and Fos-IR+HDC-IR dual labeling in HDC-IR neurons in the ventral and dorsal TMN (vTMN, dTMN) by 6.6-and 5.9-fold (P < 0.01) and by 76% and 58% (P < 0.01); and the number of Fos-IR and Fos-IR+OX-A-IR dual labeling in OX-A-IR neurons in the PeF and LH by 5.2-and 3.6-fold (P < 0.01), and by 28% and 14% (P < 0.01), respectively, compared with saline treatment.Conclusion We firstly report that NPS mainly induces a dose-dependent increase in aW time and a concomitant decrease in SWS1 and SWS2, but no influence in qW.High dose of NPS induces a respective rebound in SWS2 time at 2 h and aW time at 3 h, and consistently suppresses PS/REM sleep for 3 h.The increase of number of Fos-IR neurons in HA-containing neurons in the TMN and OX-containing neurons in the PeF and LH after NPS central administration suggests that NPS activates histaminergic and orexinergic neurons in the posterior hypothalamic waking center through its receptor to promote aW.