【作者】 何绘敏；

【导师】 王滨；

【作者基本信息】 大连医科大学 ， 人体解剖学与组织胚胎学， 2010， 博士

【摘要】 目的：学习记忆是人类和动物生存不可缺少的重要功能。学习(learning)是获得新信息和新知识的神经过程,而记忆(memory)则是对所获取信息的编码、巩固、保存和读出的神经过程。大脑具有多重记忆系统,如记忆可分为陈述性记忆(declarative memory)和非陈述性记忆(non-declarative memory)等,不同类型的记忆在不同的脑区中贮存,海马、丘脑、杏仁核、前额叶皮层及基底神经节等都与学习记忆有关。近年来研究发现丘脑前核在空间学习记忆中发挥重要作用。1973年Bliss和lomo首次在兔海马观察到突触传递的长时程增强(Long term potentiation LTP)现象,长时程增强很可能是学习与记忆的神经生理基础,而长时程增强现象的发生及维持过程与突触前膜释放谷氨酸的增加有关。本实验室前期通过胶体金免疫电镜等技术发现丘脑前核与边缘皮质学习记忆环路间存在的兴奋性神经递质是谷氨酸,但丘脑前核内是否存在NMDA受体研究较少。越来越多的研究证实,MAPK/ERK信号通路与脑内长时程增强的形成以及学习记忆功能有重要的联系。如经水迷宫训练后的大鼠,海马CA1/CA1区ERK被激活,使用PD098059抑制MAPK/ERK级联反应则p-ERK蛋白含量降低,并且长期空间记忆的形成受损。丘脑前核在学习记忆过程中,ERK通路是否参与胞内信号的转导目前仍不清楚。即早基因(Immediate early genes IEGs)被认为是核内的第三信使,如大鼠海马内注射c-Fos的反义寡核苷酸后发现大鼠的长时记忆巩固过程障碍。丘脑前核在空间学习记忆即早基因是否参与仍不清楚。突触可塑性包括两个方面,长时程增强(Long-term potentiation LTP)和长时程抑制(Long-term depression LTD), LTP和LTD对一个完整的学习记忆神经网络均不可缺少,LTD在学习记忆中发挥着不断纠正错误的作用。γ-氨基丁酸(γ-Amino butyric acid, GABA)是中枢神经系统内主要的抑制性神经递质,GABA与LTD有关。目前丘脑前核内GABA及其受体的表达仍不明确。本实验目的是在前期发现丘脑前核内存在谷氨酸神经递质基础上,探讨丘脑前核内NMDA受体表达,Morris水迷宫训练后ERK1／2的变化以及c-Fos和c-Jun的表达。并探讨抑制性神经递质GABAA受体在丘脑前核的表达。方法1.通过原位杂交观察大鼠丘脑前核内NR1,NR2A及NR2BmRNA的表达及分布特点。2.成年雄性SD大鼠31只,分为3组：PD组：侧脑室内注射5ul PD098059溶液(PD098059 3ug／只)；PD对照组：侧脑室内注射5ulDMSO溶剂;正常组：未进行侧脑室注射。在Morris水迷宫中,定位航行训练后,行侧脑室内注射,24小时后空间探索实验。免疫组化检测p-ERK的变化。3.成年雄性SD大鼠分为3组,正常组：未经Morris水迷宫训练；训练组：通过Morris水迷宫训练；假训练组：撤掉平台,每天游泳次数和游泳时间与训练组相同。训练后一半免疫组化检测c-Fos和c-Jun的表达。另一部分行Westwern-Blot检测c-Fos和c-Jun蛋白的表达。4.原位杂交观察丘脑前核内GABAAR-alpha 1和GABAAR-beta2受体mRNA的表达。结果1.NR2A,NR2B及NR1mRNA在丘脑前核都有表达,阳性神经元分布较密集,细胞形态较一致,整体分布尚均匀。NR2AmRNA及NR2BmRNA在丘脑前背侧核和腹侧核的分布前者稍显密集,胞体略大。同时在海马CA1,CA3及DG和扣带后回也观察到NR2A,NR2B及NR1 mRNA的表达。空白对照切片为阴性。2.在空间探索实验中PD组寻找平台次数与对照组及正常对照组相比明显减少(P<0.01)。PD组在靶象限停留时间与对照组及正常组相比明显减少(P<0.01)。正常组在靶象限停留时间和穿越平台次数与对照组相比没有显著差异,p>0.05。p-ERK阳性表达神经元呈棕黄色,为圆形或椭圆形,PD对照组与PD组与正常对照组比较,在丘脑前核p-ERK组表达明显增强,光密度分析有显著性差异(P<0.01),PD组与正常对照组比较,在丘脑前核p-ERK无显著差别。在PD对照组,丘脑前背侧核(AD)的p-ERK免疫反应阳性产物与丘脑前腹侧核(AV)相比,表达显著增强,光密度分析有显著性差异(P<0.05)。空白对照切片为阴性,未见p-ERK表达阳性神经元。3.c-Fos免疫组化阳性表达位于细胞核内,呈褐色的圆形或椭圆形。训练组c-Fos阳性细胞在AD和AV均有表达,但AD与AV相比表达显著增强,经图像平均光密度有显著差异(P<0.05)。训练组丘脑前核c-Fos阳性细胞表达与正常对照组和假训练组比较明显增强,经图像平均光密度分析明显增强(P<0.05)。c-Jun免疫组化阳性表达位于细胞核内,呈褐色的圆形或椭圆形。训练组c-Jun阳性细胞在AD和AV均有表达,AD与AV相比染色略强,细胞稍显密集,形态略大。正常对照组和假训练组AD和AV的c-Jun阳性细胞都有弱的表达;但训练组与正常对照组和假训练组比较染色明显增强,经图像分析平均光密度明显增强(P<0.05)。4.原位杂交：GABAAR-alphal和GABAAR-beta2 mRNA原位杂交阳性反应产物为棕黄色,主要分布在神经元胞浆中。在AD和AV,阳性神经元分布较密集,细胞形态较一致,在AD和AV的分布前者稍显密集。同时大脑其他学习记忆脑区如海马CA1,CA3及DG和扣带后回也观察到GABAAR-alpha 1和GABAAR-beta2mRNA的表达。结论1.大鼠丘脑前核内存在NR1,NR2A.NR2BmRNA的表达。提示丘脑前核在空间学习记忆信号转导中细胞膜受体可能以NR1-NR2A, NR1-NR2B或NR1-NR2A-NR2B形式存在。2.大鼠通过Morris水迷宫训练及应用PD098059后,大鼠空间参考记忆受损,p-ERK表达减弱,提示丘脑前核空间参考记忆细胞内信号转导可能与MAPK途经有关。在PD对照组,Morris水迷宫训练后p-ERK在丘脑前背侧核表达较丘脑前腹侧核表达显著增强(P<0.001)。3.Morris水迷宫训练后大鼠丘脑前核内c-Fos和c-Jun蛋白表达显著增加,提示核内第三信使可能以Fos-Jun异源二聚体的形式,作用于靶基因AP1结合位点,启动靶基因进一步转录。水迷宫训练后丘脑前背侧核c-Fos表达与丘脑前腹侧核显著增强,但丘脑前背侧核与前腹侧核在学习记忆中的具体作用有待进一步探讨。4.丘脑前核内存在GABAAR-alpha 1和GABAAR-beta2表达,提示在学习记忆中丘脑前核可能受到抑制性氨基酸的调控更多还原

【Abstract】 Background and purpose:Learning and memory are indispensable function of survival to human and animals. Study is to obtain new information and new knowledge of neural processes. Memory is the access to information while encoding,consolidation, storage and reading out the neural process. The brain has multiple memory systems, such as declarative memory and non-declarative memory, etc. Different types of memories are stored in different brain regions, Hippocampus, thalamus, amygdala, prefrontal cortex and basal ganglia are all related to learning and memory. Recent studies have found that anterior thalamic nucleus(ATN) played an important role in spatial learning and memory.It was not until 1973 that long-term potentiation (LTP)of synaptic efficacy was originally described in the hippocampus of rabbits by Bliss and lomo. LTP is likely to be neurophysiological basis of learning and memory, the maintenance of LTP is related to an increased release of glutamate of presynaptic membrane.In our laboratory by immunogold electron microscopy techniques showed that the excitatory neurotransmitter is glutamate between learning and memory neural circuits of the ATN and limbic cortex. However, it is not clear if the ATN expresses NMDA receptors.A growing body of evidence has recently been accumulating that MAPK/ERK1/2 pathway activity is involved in LTP and the formation of memory. The present study found that ERK is activated in hippocampal CA1 after water maze training in rats, PD098059 inhibited p-ERK1/2 to affect the ERK1/2 pathway activity and impaired long-term spatial memory. It is not clear if ERK pathway is involved in the process of learning and memory of ATN.Immediate early genes (IEGs) are considered the nucleus of third messenger. After injecting c-fos antisense oligonucleotide into the hippocampus of rats, long-term memory consolidation in rats was impaired after Morris water maze training. During spatial learning and memory, it is not clear whether IEGs express in ATN.Synaptic plasticity consists of LTPand long-term depression(LTD). LTP and LTD are essential to a complete neural network of learning and memory, y-aminobutyric acid (GABA) is major inhibitory neurotransmitter, GABA is related to LTD. The expression of GABA and its receptor in the ATN are not clear.The aim of this study is to explore the expression of NMDA receptors and GABAA receptor expression in the ATN; And to explore that MAPK/ERK signal transduction and IEGs are related to spatial lerning and memory in the ATN.Methods:1.To observe the expression and distribution of NR1, NR2A and NR2BmRNA in the ATN of rats by in situ hybridization.2. The rats were divided into 3 groups:①PD group:Rats were injected intraventricularly with PD098059(3μg/5ul) after Place navigation training.②PD control group:intraventricular injection of 5ul DMSO solvent;③Normal group:no intraventricular injection. After 24 hours to do space exploration training. p-ERK was detected by means of immunohistochemistry.3. Adult male SD rats were divided into 3 groups:①Control group:without the Morris water maze training;②Training group:Morris water maze training;③Sham training group:removed platform, swim times and time with the same as training group. After training, half of the rats to detect the expression of c-Fos and c-Jun by means of immunohistochemistry. Another half to detect the expression of c-Fos and c-Jun by means of Westwen-Blot.4. GABAAR-alphal and GABAAR-beta2 receptor mRNA expression were determined through situ hybridization in the ATN. Results:1. The mRNA of NMDA receptor subunits NR1, NR2A and NR2B distributed in the intensively and homogeneously in the ATN. The mRNA of NMDA receptor subunits NR1, NR2A and NR2B were also visible in the hippocampus and retrosplenial granular cortex. Control sections were negative for the expression of mRNA of NMDA receptor subunits NR1, NR2A and NR2B.2. In the probe trial performance, the platform crossings in PD group were significantly reduced compared to the PD control group and normal control group (P<0.01). The percentage of time of PD group spent in the correct quadrant decreased significantly compared to the PD control group and the normal group (P<0.01).The platform crossings and the percentage of time of spent in the correct quadrant in PD control group showed no significantly different compared to normal control group (P> 0.05). p-ERK positive neurons were brown, round or oval. p-ERK expression in ATN of PD control group was significantly increased compared to PD group and the normal control group (P<0.01). p-ERK showed no significant difference between PD group and the normal control group. In the PD control group, p-ERK in the anterior dorsal nucleus (AD)was stronger than in anterior ventral nucleus(AV) (P<0.05).No expression of p-ERK-positive neurons was found in negative control sections.3. After Morris water maze training,The expression of c-Fos was distributed in the AD and AV. But AD was significantly enhanced compared to AV(P<0.05). The expression of c-Fos of the training group in the ATN was dramatically stronger compared to the normal control group and the sham training group (P<0.05). The expression of c-Jun was visible in the AD and AV. The expression of c-Jun protein in the AD was stained slightly stronger than in the AV. In the normal control group and sham training group. The expression of c-Jun protein was weak. The expression of c-Jun in the training group significantly increased compared to the control group and the sham training group(P<0.05).4. In situ hybridization:GABAAR-alpha1 and GABAAR-beta2 mRNA in situ hybridization-positive brown reaction products, mainly in the cytoplasm of neurons.In AD and AV, positive neurons were more intensive, more consistant morphology, the distribution in the AD and AV former slightly intensive. while such as the hippocampus and retrosplenial granular cortex the expressions of GABAAR-alphal and GABAAR-beta2 mRNA were also observed in the other parts of brain related to learning and memory.such as hippocampus and retrosplenial granular cortex.Conclusions1. The results suggest that the neurons of ATN are capable of producing NMDA receptors subunits of NR1,NR2A and NR2B.During signal transduction of learning and memory in the ATN, the cell membrane receptors are likely to be NR1-NR2A,NR1-NR2B,or NR1-NR2A-NR2B patterns.2. Post-training application ERK cascade inhibitors (PD098059) in rats, the spatial reference memory was impaired, and the expression of p-ERK was decreased. Suggesting a likely crucial role of MAPK/ERK signalling passway in spatial reference memory process of ATN. After Morris water maze training in the PD control group, the expression of p-ERK in the AD was significantly increased compared to AV.3. After Morris water maze training, the expression c-Fos and c-Jun in the ATN were increased dramatically, which indicates Fos form heterodimeric complexes with Jun to constitute the AP-1, which further regulates gene expression. After Morris water maze training, the expression of c-Fos in the AD was significantly increased compared to the AV, but the functions of AD and AV respectively in learning and memory are still further studied.4. GABAAR-alphal and GABAAR-beta2 mRNA were expressed in the ATN, suggesting that learning and memory process in the ATN was likely regulated by GABA.更多还原