【作者】 段新；

【导师】 马光瑜；

【作者基本信息】 汕头大学 ， 司法精神病学与精神药理学， 2003， 硕士

【摘要】 背景与目的：中枢胆碱能系统被证明是影响学习和记忆的重要神经系统，已有资料表明，中枢乙酰胆碱(Ach)是学习记忆的重要神经递质，主要证据是M—胆碱受体阻断剂东莨菪碱能使大脑乙酰胆碱含量下降，并导致记忆障碍，而胆碱酶抑制剂(AchE)能增强记忆，到目前为止，治疗老年性痴呆(AD)最有效的方法是胆碱酯酶制剂。它保护AD病人大脑中正在减少的Ach数量，改善AD病人记忆。 有关阿片类药物成瘾及其脱毒治疗的相关研究发现阿片系统也参与和调制学习记忆的过程。许多实验研究证明吗啡降低额叶皮层、隔区，海马等部位细胞外Ach，其是，至少是部分抑制了中枢胆碱能神经元活力而损害学习记忆。非选择性阿片受体拮抗剂纳络酮能够逆转这种作用。纳络酮有利于记忆的保持、巩固，微脑透析实验发现，纳络酮能显著增加海马Ach的释放，并呈剂量依赖性。可能通常情况下阿片系统对胆碱能神经元有紧张性抑制作用，纳络酮解除了阿片肽对乙酰胆碱释放的抑制。阿片系统和胆碱能系统对学习记忆影响的机制尚不清楚。 乙酰胆碱主要在神经末梢合成，其合成的关键酶是胆碱乙酰转移酶(ChAT)。ChAT在神经元胞体合成，籍轴突运输运送到神经末梢的胞浆中。是否阿片系统对胆碱能神经元胞体ChAT的合成或轴突ChAT的运输或神经末梢ChAT活性存在影响，对神经元突触可塑性有何影响，目前尚未见文献报道。 基于以上理论及资料，纳洛酮改善东莨菪碱所致学习记忆障碍与其促进乙酰胆碱在突触的释放有关，本课题拟对其机制进行一些探讨：纳洛酮是否对ChAT的合成或者运输有影响，对学习记忆相关的神经元突触可塑性有何影响。从而为纳洛酮改善老年性痴呆的临床应用提供理论依据，为进一步阐明在学习记忆过程中阿片系统对胆碱能系统的调节机制进行有益的探索。同时比较纳洛酮和加兰他敏对东莨菪碱所致大鼠学习记忆障碍的改善效果。汕头大学医学院硕士学位论文材料和方法: 实验一Morris水迷宫工作记忆试验 2月龄SD大鼠56只按Morris水迷宫工作记忆试验方法训练，通过训练，每只大鼠表现稳定，随机分成4组(n=14):正常对照组、东蓖若碱组、纳洛酮治疗组(东食若碱+纳洛酮)和加兰他敏治疗组 (东食若碱+加兰他敏)。东蓑若碱按0.4mg/kg、纳洛酮按3mg/kg，加兰他敏按2 mg/kg，正常对照组予等量生理盐水，均在实验前30分钟腹腔给药，然后开始水迷宫延缓性匹配作业，记录每只大鼠前后两次逃避潜伏期。四组大鼠各取半数(n=7)，1 .5小时后重复Morris水迷宫工作记忆实验，以观察纳洛酮治疗效果持续时间。 采用免疫组化方法和计算机图像分析技术定量检测大鼠海马CAI、以3区和前额叶皮层区胆碱乙酞转移酶(ChAT)，透射电镜观察海马以1区锥体细胞超微结构。 实验二Morris水迷宫参照记忆试验 1月龄sD大鼠14只，随机分为正常对照组、东蓖若碱模型组 (下7)，东蓖若碱模型组大鼠每天皮下注射东蓑若碱Zmg/kg，一天两次，正常对照组予等量生理盐水，连续21天。然后按Morris水迷宫参照记忆试验方法，观察定位航行、空间探索和可见平台实验。 采用Nissl染色法进行海马以l、以3区锥体细胞计数，常规苏木素一伊红(hematoxylin一eosin，HE)染色进行海马CAI、CA3区组织病理学观察，透射电镜观察海马以1区锥体细胞超微结构。结果:实验一结果: 四组大鼠空间工作记忆结果，前后逃避潜伏期比较:正常组呈极显著性差异(p<0.001)，东莫若碱组无差异(乃0.05)，说明工作记忆受损害;纳洛酮治疗组呈显著性差异(p<0.05)，说明东蓖若碱所致工作记忆损害得到明显恢复;加兰他敏治疗组仍无差异(乃0.05)，说明受到损害的工作记忆并无改善。四组Triall逃避潜伏期比较:其他三组与正常组比较(Dunnett T test)，均无显著性差异，组间最大差异(LSD检测)比较，东莫蓉碱组、加兰他敏治疗组显著小于正常组(p<0 .05)，东蓖若碱组、纳洛酮治疗组和加兰他敏治疗组间无差异。汕头大学医学院硕士学位论文 胆碱乙酞转移酶(C hAT)的灰度、阳性单位数、阳性单位/面积在四组大鼠海马以1、以3区锥体细胞和前额叶皮层无显著性差异(P>0.05);其他三组与正常组t匕较(Dunnett T test)，均无显著性差异;组间最大差异(LSD检测)比较亦无显著性差异。说明各组以1、以3区锥体细胞、前额叶皮层神经元ChAT量、ChAT表达阳性数目及密度无差别。 四组大鼠海马CAI区超微结构的变化:四组大鼠锥体细胞胞体细胞核及细胞器正常，但CAI区神经元突触超微结构有明显变化，正常组突触间隙清晰，囊泡大小均一，并向突触前膜聚集，突触后致密物质均匀，致密。东蓖若碱组突触前后膜模糊，融合，间隙不清;囊泡明显大小不一，突触前膜处聚集明显，密度增加，远隔处囊泡大而稀疏，突触后致密物质不均、模糊，边界朦胧。纳络酮组，突触间隙清楚，囊泡大小均一，稀疏，向突触前膜聚集，突触后致密物质均匀，边界整齐清楚。加兰他敏组，突触间隙清楚，但前后膜模糊似有融合;囊泡大小欠均一，分布弥散，无明显突触前膜聚集现象，突触后致密更多还原

【Abstract】 Background and objective: septo-hippocampal cholinergic pathway plays a crucial role in spatial memory. Lesions and pharmachological blockades of the pathway result in the deterioration in learning and memory. Scopolamine, a non-specific muscarinic receptor antagonist, can impair the spatial memory. Studies have showed that age-related impairments and scopolamine-induced impairments may operate through similar mechanisms.Experiments have discovered that extracellular Ach releasing in septal and hippocampal regions is closely associated with cognition and behavioral state of animal.Morphine can decrease acetylcholine(Ach) turnover in parietal cortex, occipital cortex, septal region and hippocampus, etc. Morphine infusion into medial septum produces memory deficit. The effect may be due, in part, to opioid inhibition of cholinergic neurons projecting to hippocampus. Naloxone, a mu-opioid receptor antagonist, can release this inhibition and reverse morphin-induced memory damage. Brain microdialysis in vivo found that naloxone could increase Ach level in hippocampus and facilitate memory retention and recall. It is obvious that opioidergic system is also involved in the regulation of septo-hippocampal cholinergic activity.Either naloxone or galanthamine, a AchE inhibitor, can ameliorate scopolamine-induced memory deficits. Data strong support that interaction between opioid and cholinergic systems influences learning and memory function. However, the mechanism of the interaction is still unclear.The question was raised whether endogenous opioid system exerts influence on choline acetyltransferase (ChAT) activity because ChAT is the only enzyme for Ach synthesis.Scopolamine, which due to its cholinergic bloking effects, is widely accepted as providing a model of cognitive impairment seen in the elderly and has been extensively used as a tool to test the efficacy of new drugs which may have cognition enhancing potential.In present study, acute and chronic effects of scopolamine on memory were assessed using Morris water maze tasks in rats. Meanwhile, pharmaceutical effects of naloxone was compared with galanthamine on scopolamine-induced impairment of spatial memory. The assumption that naloxone can increase extracellular Ach due to enhance ChAT expression was tested. Therefore, the mechanism of interaction between opioid and cholinergic system was explored.Materials and methods: the effects of a single dose of scopolamine alone and in combination with naloxone, galanthanmine were tested in working memory version of the Morris water maze in experiment 1. All naive rats were accepted the reference memory version of navigation task: on each trial, the rat was placed into the water between quadrants, with the drop location changing for each trial but the platform location remaining constant. The rat had to swim until it found and climbed onto the escape platform. Rats were guided to the platform if they failed to locate it within 60 s. The rat was allowed to stay on the platform for 15 s. Rats were trained two sessions (three trials per session) a day for 2 days. Then the training on the working memory version of navigation was followed. A delayed-matching-to sample variant of Morris water maze was adapted. Rats were tested 2 sessions per day for 4 days, with each session consisting of two trials separated by a 10s interval, during which rats can utilize working memory to locate the platform at the second trials. On day 7, all rats were divided at random into four groups (n=14) : normal group, scopolamine group, naloxone treatment group and scopolamine treatment group. The different chemical drugs were delivered intodifferent groups (i.p.) respectively prior to the sessions(the normal group rats received the same volume saline injection). Then the escape latencies were record.Level of ChAT expression in rats hippocampal subfield CA1,CA3 and in forebrain cortex were measured quantitatively by using the immunohistochemistry method and computerized image technique. Pathomorphological change更多还原