【作者】 江刚；

【导师】 舒斯云；

【作者基本信息】 第一军医大学 ， 细胞生物学， 2004， 博士

【摘要】 脑纹状体边缘区(marginal division，MrD)是舒斯云等1987年在研究大鼠脑纹状体时发现的位于新纹状体尾内侧、环绕苍白球头外侧的一个由梭形细胞构成的新亚区，随后在猫、猴和人脑内也发现存在类似的结构，其富含多种神经肽类阳性纤维、终末和胞体，与脑内包括海马、杏仁核、Meynert氏基核和前额叶皮质等多个学习记忆相关脑区存在着结构和／或功能上的联系，是一个在细胞形态、神经递质和受体等生物大分子的分布，以及纤维联系方面均有别于纹状体其他部位的独特的亚区。而且，己证实大鼠的MrD参与了电Y迷宫厌暗逃避条件反射联合型学习，在相同的行为模式训练后可观察到大鼠MrD细胞有c-fos、c-jun即早基因的表达。最近，又进一步发现MrD涉及人的听觉数字工作记忆，临床上也观察到因MrD受损而出现认知功能障碍的患者。但是，MrD参与学习记忆功能时的信号转导机制仍未完全明确，而且，在相同的行为模式中，MrD与其他学习记忆相关脑区在学习记忆功能方面又有何异同?由于学习记忆是脑的一项高级的神经功能，目前，海马记忆系统、前额叶皮层、间脑、杏仁核、小脑和Meynert基底核等不同的脑部结构相继被证实参与了中枢学习记忆过程的调控，它们之间主要以组成神经环路或网络的方式在学习记忆中发挥重要作用。因此，对上述两个问题的解决，无疑将会有助于在细胞和分子水平上了解MrD完成学习记忆功能的机制，以及有助于探讨MrD与其他学习相关脑区在中枢学习记忆功能中的关系问题。 环磷酸腺苷反应单元结合蛋白(c-AMP responsive element binding protein，CREB)是一种重要的核蛋白，这种核转录因子具有调节包括学习记忆在内的广泛的生物学功能，是细胞内多种信号通路的一种关键成分。目前，来自海兔、果蝇、小鼠以及大鼠的大量研究已表明，细胞内构成长期记忆的变化需要依赖CREB的转录，CREB是长期记忆形成过程所必需的一种关键的调节分子。Ser- 133磷酸化的CREB(phosph娜lated CREB，pCREB)是eREB活化的主要方式，受pC既B调节的下游靶基因至少有包括即刻早期基因c一、、c犷un在内的100多个，这些靶基因具有控制神经传递、细胞结构、信号转导、转录以及代谢等多种功能。谷氨酸是中枢神经系统中最为丰富的内源性兴奋性氨基酸，同时也是脑内主要的兴奋性神经递质，己证实其涉及神经突触的可塑性、生长发育及兴奋毒性等多个方面的作用，特别是其离子型的N一甲基一D-门冬氨酸伽一methyl一D一asParate，NMDA)受体，在学习记忆功能的调控上具有十分重要的地位。 如上所述，由于我们己证实大鼠的MrD涉及电Y迷宫厌暗逃避性学习记忆行为模式，而这种行为模式的训练可诱发大鼠MrD细胞中c一fos、c-jun即早基因的表达，且大鼠MrD细胞含有丰富的NMDA受体。因此，本课题的第一部分拟在以下三个方面进行研究:l.在电Y迷宫厌暗逃避性学习记忆行为模式中，大鼠脑纹状体MrD内是否有pcREB的表达;2.这种表达与MrD细胞的NMDA受体是否有关系;3.如果有关，那么通过应用NMDA受体拮抗剂阻断这种表达后，是否会影响动物的长期记忆?以探索在这种行为模式中，MrD学习记忆信号转导机制的细胞膜受体和核转录因子问题。然后，在课题的第二部分，同样在电Y迷宫厌暗逃避性学习记忆行为模式中，分别从行为学和训练后pcREB表达的动态的角度，分析比较MrD与海马的异同，以便为了解MrD与海马在中枢学习记忆功能中的关系问题，提供有价值的资料。方法和结果第一部分大鼠脑纹状体边缘区逃避性学习记忆信号转导机制的探讨1.逃避性学习记忆训练后pcREB在大鼠脑纹状体边缘区的表达 将成年SD大鼠首先在电Y迷宫中进行连续3天的逃避性学习记忆强化训练，然后应用免疫组织化学方法检测pCREB在MrD的表达情况。结果显示训练后MrD内即有明显的pcREB表达，阳性表达的细胞呈密集的带状分布。在表达的数量上训练组与对照组和假训练组有显著性差异(P<0.01)。此外，其他学习相关脑区如海马、前额叶皮质及扣带回皮质等处也见有较多的pcREB阳性细胞。2.逃避性学习记忆训练后腹腔内注射MK一801对大鼠脑纹状体边缘区pCREB表达的影响 成年SD大鼠在电Y迷宫中进行强化训练后，立即分别接受腹膜腔内注射MK一801(0. 3 mg瓜g)或等量生理盐水，30 min后处死，然后采用免疫组织化学的方法比较两组纹状体MrD内pcREB表达的差异。结果MK一801组大鼠与对照组相比，其纹状体特别是MrD内pCREB的阳性表达明显减少(P<0.01)。3.逃避性学习记忆训练后脑纹状体边缘区内微量注射MK一801对大鼠逃避性记忆功能巩固性的影响 成年SD大鼠在电Y迷宫中进行强化训练后，立即在10%水合氯醛腹腔注射全麻下，向双侧边缘区内分别注射MK一801(2 pg/侧)或等量生理盐水，5d后再复查比较电Y迷宫试验成绩。结果MK一801组动物的正确次数明显少于对照组(P<0.05)。第二部分大鼠纹状体边缘区与海马逃避性学习记忆功能的比较研究1.大鼠纹状体边缘区与海马逃避性学习记忆功能的行为学比较研究 在电Y迷宫训练成年SD大鼠后即刻或24h后，用海人藻酸化学损毁双侧MrD或切断双侧弯窿海马伞(fomi对更多还原

【Abstract】 The marginal division of the striatum (MrD) is a newly identified structure at the caudal most edge of the neostriatum of the rat brain. It was firstly discovered by Shu et al in 1987. The MrD was also successively observed in the brains of cat, monkey and human. Many neuropeptides densely packed in the fibers, terminals and neuronal somata of the MrD. It has structural and/or functional connections with many learning and memory related brain areas including hippocampus, amygdala, basal nucleus of Meynert (NBM) and prefrontal cortex. Thus, MrD is distinguished from the rest of the stiratum by spindle-shaped neurons, special connections and intensely expressed many neurotransmitters and related receptors. Functionally, MrD was proved to be involved in learning and memory function of rat brain by a double blinded Y-maze avoidance task. The expression of immediate-early genes c-fos and c-jun was observed in the MrD of rat striatum immediately after the Y-maze avoidance training. The MrD was proved involving in the auditory digital working memory in human. Lesions in human MrD might cause disorder of cognitive function. However, the mechanism of the signal transduction in the MrD during learning and memory is not clear. It is unknown that whether there are differences between the MrD and other memory-related brain areas especially hippocampus in the learning and memory functions of the brain. Learning and memory function are the most important and fundamental mental processes of the brain. At present, the hippocampus and other brain areas e.g. the medial temporal lobe memory system, prefrontal cortex, diencephalon, amygdala, cerebellum and NBM have been found to be involved in learning and memory in succession. These memory -related centers were not only thought to play different roles-9-in learning and memory function of the brain, but there were close functional and structural connections among them. Thus, the solution of the above two problems could contribute to know the mechanism of learning and memory function on cellual and molecular level in the MrD, and the relationship between the MrD and other memory-related centers in the learning and memory function of the brain.The cAMP responsive element binding protein (CREB) is a important nuclear protein. This transcription factor is a key component of intracellular signaling events that regulate a wide range of biological functons including leraning and memory. Evidences from Aplysia, Drosophila, mice, and rats showed that CREB-dependent transcription is required for the cellular events underlying long-term but not short-term memory. CREB may be a key modulater of processes required for long-term memory formation. The phosphorylation of Ser 133 is a critical step in CREB activaton. The putative CREB target genes were observed over 100, including genes that control neurotransmission, cell structure, signal transduction, transcription, and metabolism et al. The glutamate is most abounded among the excitatory amino acid in the central nervous system. It is the major excitatory neurotransmitter in the brain. And it has be proved that the N-methyl-D-aspartate (NMDA) subtype of glugamate receptors in the mammalina brain plays a central role in synaptic plasticity underlying refinement of neuronal connections during development, or processes like long-term potentiation (LTP), learning and memory.As mentioned above, we have proved that the MrD was involved in learning and memory function of rat brain by a double blinded Y-maze avoidance task. The expression of immediate-early genes c-fos and c-jun was observed in the MrD of rat striatum immediately after the Y-maze avoidance training. It was showed that mRNA of NMDA receptor subnits of NR1, NR2A and NR2B proteins were expressed in the MrD of rat striatum. In thepart one of this study, to investigate role of the membrane receptor and nuclear transcriptional factor in the mechanism of signal transduction in MrD within the process of long-term memory consolidation by Y-maze avoidance task, the study was designed i更多还原