【作者】 赵正卿；

【导师】 赵忠新；

【作者基本信息】 第二军医大学 ， 神经病学， 2010， 博士

【摘要】 睡眠是人类不可缺少的生理过程。睡眠不仅可以维持个体生存及正常的生理功能,还具有促进生长发育、易化学习、形成记忆的功能。睡眠和觉醒是一个包括复杂的神经环路和多种神经化学成分的行为反应。神经递质在此过程中发挥了极其重要的作用。神经肽S (neuropeptide S, NPS)是在2004年最新研究发现的一种与睡眠和觉醒相关的神经肽,其相应的G蛋白偶联受体即为神经肽S受体(neuropeptide S receptor, NPSR)。最早的研究发现,神经肽S广泛分布在脊椎动物(人、小鼠、大鼠、鸡、猩猩、牛)中,在成年大鼠的脑组织、甲状腺、唾液腺和乳腺中都较高表达。神经肽S与其特异性受体结合发挥其生理作用,能够调节睡眠觉醒、减少惊恐发作、影响摄食行为、调节炎性反应及变态反应等。而其中尤以它与睡眠／觉醒方面的作用最为突出和明确。由于神经肽S发现时间较晚,目前对于神经肽S在中枢神经系统中的分布及功能研究很少,仅有少数研究报道。为进一步深入对神经肽S功能的认识,本研究首先系统研究了神经肽S在大鼠中枢神经系统中的表达和分布特点,为深入研究其功能打下基础。在此实验中,我们对大鼠脑组织和脊髓做连续切片,首次全面研究神经肽S在大鼠整个神经系统中的表达分布情况。其次,鉴于神经肽S已被发现的最主要的作用——调节睡眠／觉醒的作用,首次研究了不同时程REM期睡眠剥夺后大鼠下丘脑内神经肽S的表达变化,以期进一步研究神经肽S与睡眠觉醒的关系。最后,首次研究了中枢给予神经肽S是否可以改善REM睡眠剥夺导致的大鼠认知功能损害,并探索其可能的分子机制。本研究使用免疫组织化学、原位杂交技术研究神经肽S蛋白和mRNA在大鼠中枢神经系统中的表达和分布特点。实验结果显示,神经肽S广泛分布于大鼠中枢神经系统中,且免疫组织化学结果和原位杂交结果具有高度一致性。在嗅球、初级嗅皮质区域,神经肽S均呈阳性染色,阳性染色主要集中在细胞胞体,证实神经肽S可能参与中枢嗅觉系统的兴奋传递过程。神经肽S强阳性染色集中在大脑运动皮质区、躯体感觉皮质区、梨状皮质和嗅皮质,尤其是在运动皮质区、躯体感觉皮质区第Ⅴ层存在致密染色,提示神经肽S可能参与神经局部环路的构建。在海马中,在CA1-CA3区锥体细胞的胞体和轴突均呈现不同程度的神经肽S阳性染色,提示神经肽可能参与海马的兴奋性神经递质传递,也可能与认知功能有关。在丘脑的很多神经核团中,尤其是前腹侧核、前内侧核、前背侧核和腹外侧核中发现神经肽S广泛表达；在下丘脑的室旁核和背内侧核也有神经肽S阳性神经元表达。神经肽S在这些部位的分布提示它可能参与机体多种生理活动,包括调节体温、水盐代谢、活动能力、饮食、睡眠／觉醒和激素分泌等。在脑干中,神经肽S受体强阳性染色分布在蓝斑、外侧臂旁核、三叉神经中脑核、三叉神经运动核。在小脑皮质中,浦肯野细胞中神经肽S阳性染色最强,几乎所有的浦肯野细胞均为阳性染色。致密染色集中在细胞胞体和轴突上。在脊髓颈部、胸部、腰部、骶部灰质区均有神经肽S阳性细胞分布,在背角染色较强。其次,使用免疫组织化学、原位杂交、RT-PCR等技术观察不同时程REM睡眠剥夺对大鼠下丘脑神经肽S蛋白及mRNA表达量的影响。将成年雄性SD大鼠随机分为正常对照组(CC)、环境对照组(TC)、REM睡眠剥夺组(SD),每组又分为1天、3天、5天组。采用改良多平台水环境睡眠剥夺法(MMPM)建立大鼠的REM睡眠剥夺模型,完成相应时间的REM睡眠剥夺后,测定大鼠下丘脑神经肽S表达量的变化。免疫组织化学及原位杂交结果显示：在REM期睡眠剥夺后,下丘脑的背内侧核和室旁核的NPS蛋白和mRNA表达发生改变。在这两个核团,CC组和TC组的NPS表达较少,散在分布于核团中。而REM期睡眠剥夺1天后,大鼠背内侧核和室旁核NPS蛋白和mRNA阳性细胞数仍较少。到REM期睡眠剥夺的第3天,可以观察到大鼠背内侧核和室旁核NPS蛋白和mRNA阳性细胞数量均增多,在REM期睡眠剥夺的第5天,仍呈现这样的趋势。RT-PCR结果显示,CC组和TC组下丘脑内NPSmRNA的表达量很少,在REM期睡眠剥夺1天后,大鼠下丘脑的NPSmRNA的表达也较少。而在REM期睡眠剥夺3天和5天组,大鼠下丘脑中NPSmRNA的表达量较CC组,TC组和SD1天组明显升高。最后,我们研究了中枢给予神经肽S对REM睡眠剥夺导致的大鼠认知功能损害是否有改善作用,并探索其可能的分子机制。SD大鼠随机分为正常对照组(CC)、环境对照组(TC)、REM睡眠剥夺组(SD),其中每组又分为神经肽S侧脑室给药组(NPS)和人工脑脊液对照组(aCSF)。总共分为六组：CC-aCSF组,TC-aCSF组,SD-aCSF组,CC-NPS组,TC-NPS组和SD-NPS组。睡眠剥夺时间为72小时。各组大鼠侧脑室置管,恢复后采用改良多平台水环境睡眠剥夺法(MMPM)建立大鼠的REM睡眠剥夺模型,同时侧脑室给予神经肽S或人工脑脊液。选用Morris水迷宫的定位航行实验对所有大鼠进行空间认知学习能力的测定,72小时的REM睡眠剥夺结束后,利用Morris水迷宫的空间探索实验测定大鼠的空间记忆能力。迷宫测试完毕,运用免疫组织化学和蛋白质免疫印迹(Western-blot)等方法测定大鼠海马磷酸化的CREB(p-CREB)表达的变化。实验结果表明：72小时REM期睡眠剥夺后,大鼠的空间学习和记忆能力与正常对照和环境对照组的大鼠相比明显减退；正常对照组与环境对照组大鼠的空间记忆能力无明显差异(p>0.05)；CC—NPS组大鼠和TC—NPS组大鼠的空间记忆能力比CC—aCSF组和TC—aCSF组有所提高,而空间学习能力无差别；SD—NPS组大鼠的空间记忆能力较SD—aCSF组明显增强,空间学习能力也有所提高；72小时REM期睡眠剥夺后,大鼠海马的p-CREB表达下降,SD—NPS组大鼠海马的p-CREB表达量较SD-aCSF组增高。本课题联合使用免疫组织化学和原位杂交技术,首次系统研究了神经肽S蛋白和mRNA在大鼠中枢神经系统中的表达和分布特点,发现该神经肽广泛分布于中枢神经系统的神经元中,在皮质、杏仁核、海马、丘脑、下丘脑、脑干、脊髓、嗅球都有分散表达。提示神经肽S可能参与多种神经生理活动。首次研究了REM期睡眠剥夺后,大鼠下丘脑内NPS蛋白和mRNA的表达变化情况,发现在持续REM期睡眠剥夺状态下,下丘脑内的NPS表达增加,证实了NPS与觉醒的相互关系,在受到外因刺激无法正常睡眠时,机体会自身调节,分泌足量的NPS来维持觉醒。我们还发现,REM睡眠剥夺对大鼠空间学习记忆能力有损害作用,神经肽S可以缓解REM期睡眠剥夺造成的认知功能损伤,REM期睡眠剥夺后,海马的p-CREB表达下调,NPS可以部分逆转睡眠剥夺引起的p-CREB表达下调。提示神经肽S可能通过调节p-CREB的表达来改善REM睡眠剥夺造成的认知功能损伤。更多还原

【Abstract】 Sleep plays important roles during physiological and pathophysiological processes. It is well known that sleep plays a key role in learning and memory.Neuropeptide, a member of a class of protein-like molecules made in the brain, are tightly related with sleep and wakefulness. Neuropeptide S (NPS) and its receptor (NPSR) form a newly discovered neuropeptide system. The 20 residue peptide NPS, is the endogenous ligand of the NPSR, a former orphan G-protein-coupled receptor. NPS can be found in all vertebrate species (human, rat, rat, chick, chimpanzee and cattle), mainly distributing in brain, glandula thyreoidea, salivary gland and mammary gland. NPS/NPSR system modulates many physiological functions, such as wakefulness and sleep, motoring, anxiety, pain, food intake and oxidative damage. The most important one is modulating wakefulness and sleep. However, as a newly discovered neuropeptide, controversy exists about the expression and functions of neuropeptide S in the central nervous system (CNS).As the complete expression pattern and the distribution of NPS and NPS mRNA in the in the rat central nervous system has not been described in detail so far, we planed to investigate the expression of NPS in CNS to lay foundation for future functional researches. We describe in this paper a comprehensive anatomical mapping of NPS in rat CNS using immunohistochemistry and in situ hybridization together. We investigated the effects of different durations of rapid eye movement (REM) sleep deprivation on the expression of NPS and NPSmRNA in rat hypothalamus. We also discussed the connection between cognition disorder and hippocampal p-CREB changes caused by REM sleep deprivation and studied the role of NPS in modulating hippocampus-dependent spatial learning and memory by the MWM, determined whether NPS could affect spatial learning and memory impairments induced by REMSD and examined the change of p-CREB expression in the rat hippocampus in the NPS treated group after 72h REMSD.The present study investigates the NPS expression pattern in the rat CNS using immunohistochemistry and in situ hybridization histochemistry. We found that NPS were widely distributed in rat CNS, and the distribution patterns and levels of NPS immunolabeling of native protein had significant correlation with its mRNA counterpart. The main olfactory bulb showed NPS immunostaining. The immunostaining was observed mainly in the dendrites and cell bodies. Heavy NPS immunostaining was observed in the motor, somatosensory, piriform and entorhinal cortex. Dense immuno labeling in layers V, suggests a role for NPS in interconnection of local cortical areas. In the hippocampus, the cell body and distal dendrites of CA1-4 pyramidal cells had different levels of NPS immunostaining, suggesting that NPS may contribute to hippocampal excitatory neurotransmission or modulation in learning and memory. NPS was expressed extensively in most subnuclei of the thalamus, particularly heavy immunostaining was observed in the anteroventral, anteromedial, anterodorsal and ventral-lateral thalamic nuclei. In different regions of the hypothalamus, NPS immunostaining neurons and fibers were widely. NPS immunostaining was widely distributed in the rat brain stem, heavy NPS immunostaining was detected in the locus caeruleus, parabrachial nucle, mesencephalic trigeminal nucleus, motor trigeminal nucleus. The study showed heavy immunostaining for NPS in the Purkinje cell.NPS immunostaining was observed at the cervical, thoracic, lumbar and sacral levels. The most prominent staining was seen in the dorsal horn.Adult male Sprague-Dawley rats were randomly divided into cage control group (CC), tank contral group (TC), REM sleep deprivation group (SD).Each group were divided into three duration groups:1d,3d and 5d. The rats were deprived of REM sleep for different durations by the modified multiple platform method (MMPM). Immunohistochemistry, in situ hybridization histochemistry and RT-PCR were used to test the expression of neuropeptide S in rat hypothalamus. Resluts show that:1. There was no significant difference between CC and TC in the expression of neuropeptide S in rats hypothalamus; 2. The expression of neuropeptide S in rats hypothalamus of SD1 were not significantly different compared to that of CC and TC3; 3. The expression of neuropeptide S in rats hypothalamus of SD3 and SD5 were significantly higher than that of SD1,CC and TC.At last, we used the Morris water maze (MWM) to determine the effects of NPS on spatial learning and memory following intracerebroventricular (i.c.v.) injection in rats after 72 h REMSD. We also investigated the changes of phosphorylation of cAMP-response element binding protein (CREB) in the rat hippocampus in the NPS treated group after 72h REMSD. Adult male Sprague-Dawley rats were randomly divided into rats were randomly divided into six groups (CC-aCSF group, TC-aCSF group, SD-aCSF group, CC-NPS group, TC-NPS group and SD-NPS group). Rats were deprived of REM sleep by the modified multiple platform method (MMPM) for 72h while NPS or aCSF was injected every day. The Morris water maze task was performed to test the spatial learning and memory. After certain duration of REM sleep deprivation, hippocampal CREB phosphorylation level and quantity were analyzed by immunohistochemistry and Western-blot. Results show that 1.72h REMSD impaired spatial learning in rats and NPS mitigated the deficit.2.72h REMSD impaired hippocampus-dependent spatial memory, and NPS mitigated spatial memory impairment induced by REMSD in rats.3. By immunostaining, There were p-CREB-ir cells in CC-aCSF group, TC-aCSF group, CC-NPS group and TC-NPS group in CA1 and DG, fewer positive cells were observed in SD-aCSF group and positive cells increased in the SD-NPS group.4. After 72h REM sleep deprivation, the expression of p-CREB in the hippocampus significantly decreased, while the expression of p-CREB increased in the SD-NPS group compared to the SD-aCSF group.Firstly, this is the first extensive study undertaken to investigate the combined distribution of NPS protein and mRNA in the rat CNS using immunohistochemistry and in situ hybridization histochemistry together. The distribution of NPS shown here considerably extends previous studies which reported NPS exists in limited populations of neurons and may show the directions for explore the new function of NPS.Secondly, we first found that after 72h REM sleep deprivation, the expression of NPS and NPS mRNA increased in rat hypothalamus. This convinced the relationship between sleep and NPS. NPS may be participates in the body self-regulation after REM sleep deprivation.Lastly, in summary, our findings demonstrate that neuropetide S plays an important role in REM sleep deprivation-induced memory impairment, and imply that NPS/NPSR system may be a potential target for treatment of spatial memory impairment caused by REM sleep deprivation. The protective effect of NPS on spatial memory impairment in REMSD rats may be mediated by increasing the p-CREB expression in the hippocampus.更多还原