神经肽S及受体系统抗焦虑样行为与睡眠的作用和机制

发布时间：2018-01-04 04:22

本文关键词：神经肽S及受体系统抗焦虑样行为与睡眠的作用和机制　出处：《兰州大学》2017年博士论文　论文类型：学位论文

【摘要】：睡眠障碍是多数焦虑症患者的主诉,失眠为常见的临床表现。正常个体睡眠剥夺可导致焦虑,且多数焦虑患者常处于失眠(睡眠剥夺)状态,表明焦虑与睡眠障碍共存并相互影响。神经肽S(neuropeptide S,NPS)与其受体(NPS receptor,NPSR)是新近发现内源性的促觉醒和抗焦虑神经调质系统,NPSR mRNA在焦虑行为与睡眠的关键调节中枢杏仁核的高表达,提示NPS-NPSR系统在焦虑样行为与睡眠以及抗焦虑样行为与睡眠中起重要的调节作用,但其作用途径和机制仍不明。本研究目的主要揭示NPS-NPSR系统抗焦虑样行为与睡眠的作用和机制。第一部分研究采用改良的多平台水环境法(modified multiple platform method,MMPM)对成年雄性SD大鼠进行异相睡眠/快动眼睡眠剥夺(paradoxical sleep/rapid eye movement sleep deprivation,PSD/REMSD)24小时(8:00-8:00 h),运用开场实验(open field test,OFT)和明暗箱实验(light-dark box,LDB)的行为学检测,脑电(electroencephalogram,EEG)和肌电(electromyogram,EMG)记录(80:00-8:00 h),EEG能谱分析和睡眠觉醒时相解析,分别评价PSD引起的焦虑样行为与睡眠模式,以及侧脑室注射NPS抗PSD引起焦虑样行为与睡眠的作用特征。RT-qPCR检测PSD引起杏仁核内NPSR mRNA表达变化。即刻早基因c-Fos免疫组化标记侧脑室注射NPS引起活动神经元在杏仁核的数量变化,NPSR与c-Fos免疫荧光双标记NPS引起的活动神经元是否载有NPSR。依据第一部分研究结果所提示NPS引起活动神经元表达NPSR的主要核团部位,第二部分研究采用选择性NPSR拮抗剂,[D-Val5]NPS,大鼠基底外侧杏仁核(basolateral amygdala,BLA)核团注射,运用高架十字迷宫(elevated plus maze,EPM)、OFT和LDB行为学检测,EEG、EMG记录(20:00-20:00 h),EEG能谱分析和睡眠觉醒时相解析,分别评价抑制BLA的NPSR神经元活动是否引起焦虑样行为与睡眠改变,以及NPS联合NPSR拮抗剂BLA注射是否消除NPSR神经元被抑制引起的焦虑样行为与睡眠改变。结果:PSD引起大鼠在OFT中心场时间和直立次数减少,理毛时间增加,LDB明箱停留时间和明暗箱之间穿梭次数降低为特征的焦虑样行为。PSD大鼠焦虑样睡眠的特征为有较长的慢波睡眠(slow wave sleep,SWS)潜伏期,24小时睡眠觉醒周期的日间PS和夜间SWS、PS显著增加,这种PS增加伴有EEG的theta活动(4.5-8.5 Hz)增强,主要是由于PS片段发生次数增加。中枢注射NPS(1 nmol)抑制了PSD导致的焦虑样行为,NPS仍引起PSD大鼠第1小时约40分钟觉醒,随后睡眠恢复期的日间PS和EEG theta活动显著减少,但夜间PS和SWS增加。PSD引起杏仁核NPSR mRNA表达水平显著上升。侧脑室注射NPS引起BLA,中央杏仁核(central amygdala,CeA)和内侧杏仁核(medial amygdala,MeA)的Fos标记阳性神经元数量显著增加。免疫荧光双标记显示BLA77.9%,CeA 65.1%和MeA 64.8%的Fos标记神经元共表达NPSR。双侧BLA注射[D-Val5]NPS(10 nmol/side)引起大鼠焦虑样行为,与对照组(saline)比较,表现为在EPM开臂停留时间百分比和进入开臂次数百分比降低,在OFT中心场时间和直立次数减少,理毛时间增加,在LDB明箱停留时间和明暗箱之间穿梭次数降低。24小时睡眠觉醒周期表现为SWS潜伏期缩短,给药后第1小时SWS增加,W降低,PS无显著变化,但PS片段发生次数显著增加而片段持续时间降低;夜间W总量降低,SWS总量增加。BLA联合注射NPS(0.25 nmol/side)和[D-Val5]NPS(10 nmol/side),[D-Val5]NPS引起的焦虑样行为完全消失,睡眠觉醒时相表现为SWS和W与对照组比较无统计学差异。结论:PSD导致的焦虑样行为和睡眠与引起的杏仁核NPSR mRNA表达上调密切相关,NPS经激活杏仁核负载NPSR的神经元抗PSD引起的焦虑样行为与睡眠。抑制BLA的NPSR神经元引起焦虑样行为与睡眠改变,表明BLA是NPS-NPSR系统调节焦虑行为与睡眠的关键中枢部位。
[Abstract]:Is the most complained of sleep disorders in patients with anxiety disorder, insomnia is a common clinical manifestation of normal individuals. Sleep deprivation can lead to anxiety, and most often in patients with anxiety insomnia (sleep deprivation), anxiety and sleep disorders that coexist and influence each other. The neuropeptide S (neuropeptide S NPS) and its receptor (NPS, receptor, NPSR) is a newly discovered endogenous neuromodulator and anti anxiety arousal system, high expression of NPSR mRNA in the key of anxiety behavior and sleep regulation of the central amygdala, suggesting that NPS-NPSR system play an important role in anxiety like behavior and sleep and anti anxiety like behavior and sleep, but the mechanism is still unknown. And the mechanism of action and the main purpose of this study is to reveal the mechanism of the NPS-NPSR system of anti anxiety like behavior and sleep. The first part of the multi platform water environment improved method (modified multiple platform method, M MPM) of paradoxical sleep / REM sleep deprivation on adult male SD rats (paradoxical sleep/rapid eye movement sleep deprivation, PSD/REMSD) 24 hours (8:00-8:00 h), using the open field test (open field test, OFT) and Ming (light-dark box, LDB experimental box) detection behavior, EEG (electroencephalogram EEG (electromyogram, EMG) and EMG recording (80:00-8:00) H), EEG energy spectrum analysis and phase analysis of sleep arousal, anxiety like behavior and sleep patterns were evaluated by PSD, expression of NPSR mRNA in amygdala caused changes and characteristics of.RT-qPCR and sleep anxiety like behavior induced by intracerebroventricular injection of anti NPS PSD detection PSD immediate early gene c-Fos. Immunohistochemical markers of intracerebroventricular injection of NPS caused by changes in the number of neurons in the amygdala activity, NPSR and c-Fos immunofluorescence double labeling neurons induced by NPS with NPSR. according to whether According to the first part of the study results suggest that NPS caused the main nucleus location of NPSR expression neurons, the second part of the selective NPSR antagonist, [D-Val5]NPS, in the rat basolateral amygdala (basolateral, amygdala, BLA) microinjection, using the elevated plus maze (elevated plus, maze, EPM, OFT and LDB) detection, behavior EEG, EMG (20:00-20:00 h), EEG records the energy spectrum analysis and phase analysis of sleep wake, were evaluated for inhibition of BLA NPSR neuronal activity and sleep can cause anxiety like behavior change, and combination of NPS and NPSR antagonist BLA injection is the elimination of NPSR neurons were inhibited with anxiety like behavior caused by sleep changes. Results: rats the decrease in the OFT center field time and vertical frequency caused by PSD, grooming time increased, the residence time and between LDB light light dark box shuttle to reduce the number of anxiety like anxiety like behavior in.PSD rats Sleep characterized by slow wave sleep longer (slow wave sleep, SWS) incubation period, 24 hours of sleep and wakefulness during the day and night PS SWS, PS increased significantly, the PS increased with EEG theta (4.5-8.5 Hz) increased, mainly due to the increase in the number of PS fragments occurred. Central injection of NPS (1 nmol) inhibited the anxiety like behavior induced by PSD, NPS still induced PSD rats first hours for about 40 minutes after awakening, sleep recovery period of daytime PS and EEG theta activity were significantly decreased, but SWS increased by.PSD night PS and amygdala NPSR mRNA expression level was significantly increased. Intracerebroventricular injection of NPS caused by BLA, the central nucleus of the amygdala (central amygdala, CeA) and the medial amygdala (medial amygdala, MeA) the number of Fos positive neurons increased significantly. Double immunofluorescence showed that BLA77.9%, Fos 65.1% and MeA 64.8% CeA labeled neurons were expressed in bilateral NPSR. BLA [D-Val5] injection NPS (10 nmol/side) induced anxiety like behavior, and the control group (saline), to open arms stay time in EPM and the percentage of open arm entries were decreased, the decrease in the OFT center field time and rearing, grooming time increase in LDB light and clear black box shuttle between the time stay times reduce.24 hours of sleep wake cycle shortened SWS latency, first hours after administration of SWS increased, W decreased, PS showed no significant change, but PS fragment frequency increased significantly while the segment duration decreased; at night the total W decreased, SWS increased the total combined injection of.BLA NPS (0.25 nmol/side) and [D-Val5]NPS (10 nmol/side). Anxiety like behavior induced by [D-Val5]NPS completely disappeared, the sleep wake phase performance for SWS and W compared with the control group had no significant difference. Conclusion: the up regulation of PSD induced anxiety like behavior and sleep and caused NPSR mRNA expression in the amygdala Closely related, NPS activates the amygdala NPSR neurons to resist PSD induced anxiety like behavior and sleep. Inhibition of BLA NPSR neurons causes anxiety like behavior and sleep changes, indicating that BLA is the key central part of NPS-NPSR system to regulate anxiety behavior and sleep.

【学位授予单位】：兰州大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R740

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