大鼠丘脑前核参与学习记忆的分子生物学研究

发布时间：2018-03-26 20:04

本文选题：丘脑前核　切入点：NMDA受体　出处：《大连医科大学》2010年博士论文

【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(P0.01)。PD组在靶象限停留时间与对照组及正常组相比明显减少(P0.01)。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,p0.05。 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(P0.01),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(P0.05)。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(P0.05)。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(P0.05)。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(P0.05)。 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(P0.001)。 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
[Abstract]:Objective : To study memory is an important function of human and animal survival . Learning is a neural process to acquire new information and new knowledge , and memory is a neural process for coding , consolidating , preserving and reading information . The brain has multiple memory systems , such as memory , memory and non - declarative memory . The brain has multiple memory systems , such as memory , hippocampus , thalamus , apricot kernel , frontal cortex and basal ganglia . In recent years , it has been found that the anterior nucleus of thalamus plays an important role in spatial learning and memory .

In 1973 , Bliss and lomo observed synaptic transmission in the hippocampus of rabbits for the first time . The long - term enhancement was probably the neurophysiological basis of learning and memory , while the occurrence and maintenance of long - term enhancement was related to the increase of the release of glutamic acid in the presynaptic membrane .

More and more studies have shown that MAPK / ERK signaling pathway is associated with the formation of intracellular long - term programming and learning and memory function . For example , in rats after water maze training , ERK activation in the CA1 / CA1 region of hippocampus , the inhibition of MAPK / ERK cascade reaction by PD098059 , and the formation of long - term spatial memory are impaired . In the process of learning and memory , it is still unclear whether ERK pathway is involved in intracellular signal transduction .

Early gene ( IEGs ) is considered to be the third messenger in the nucleus , such as the injection of c - fos antisense oligonucleotides into the hippocampus of rats , and the long - term memory consolidation process in rats is impaired . It is still unclear whether the early genes involved in the spatial learning and memory of the thalamus .

The synaptic plasticity includes two aspects : Long - term long - term LTP and Long - term depression LTD , LTP and LTD are indispensable to a complete learning and memory neural network . LTD exerts a constant error in learning and memory . GABA is the main inhibitory neurotransmitter in the central nervous system . GABA is related to LTD . The expression of GABA and its receptors in the anterior nucleus of the thalamus is still unclear .

The purpose of this study was to investigate the expression of NMDA receptor in the anterior nucleus of thalamus and the expression of c - fos and c - Jun after Morris water maze training , and to explore the expression of the inhibitory neurotransmitter GABAA receptor in the anterior nucleus of thalamus .

method

1 . The expression and distribution of NR1 , NR2A and NR2B mRNA in the anterior nucleus of the thalamus were observed by in situ hybridization .

2 . 31 adult male SD rats were divided into three groups : PD group : PD group : 5ul PD098059 solution ( PD098059 3ug / ) ;
PD control group : 5ulDMSO solvent was injected intracerebroventricular injection ; normal group : no lateral ventricle injection was performed . In Morris water maze , after the navigation training , the lateral ventricle was injected , and the space exploration experiment was conducted after 24 hours . The change of p - ERK was detected by immunohistochemistry .

3 . Adult male SD rats were divided into three groups : normal group : no Morris water maze training ;
Training group : Through Morris water maze training ;
The expression of c - fos and c - Jun was detected in half of the training group , and the expression of c - fos and c - Jun protein was detected by Westwern - Blot .

4 . In situ hybridization , the expression of GABAergic receptor mRNA in the anterior nucleus of the thalamus was observed .

Results

1 . NR2A , NR2B and NR1mRNA were expressed in the anterior nucleus of thalamus . The distribution of NR2AmRNA and NR2B mRNA was more uniform and the overall distribution was uniform . NR2AmRNA and NR2B mRNA were slightly more densely distributed in the ventral nucleus and ventral nucleus of the thalamus .

2 . In the space exploration experiment , the number of the PD group was significantly decreased compared with the control group and the normal control group ( P0.01 ) . Compared with the control group and the normal control group , the residence time of PD group was significantly decreased compared with the control group ( P0.01 ) .

In PD control group , the expression of p - ERK immunoreaction was significantly higher in PD control group than in normal control group ( P0.01 ) .

3 . The expression of c - fos positive cells in the training group was significantly higher than that of the normal control group and the sham training group , but the expression of c - Jun positive cells in the training group was significantly increased compared with the normal control group and the sham training group .

4 . In situ hybridization : the positive reaction products of GABAza - alphaal and GABAergic - beta2 mRNA were brown - yellow , mainly distributed in the cytoplasm of neurons . In AD and AV , the distribution of positive neurons was more dense , the morphology of cells was more consistent , the former was slightly more intense in the distribution of AD and AV .

Conclusion

1 . The expression of NR1 , NR2A and NR2B mRNA in the anterior nucleus of the thalamus suggests that the receptor of the cell membrane may exist in the form of NR1 - NR2A , NR1 - NR2B or NR1 - NR2A - NR2B .

2 . After Morris water maze training and PD098059 , the spatial reference memory of the rats was impaired , and the expression of p - ERK decreased , suggesting that the signal transduction in the anterior hypothalamic nucleus space reference memory cells might be related to MAPK pathway . In PD control group , the expression of p - ERK in the anterior dorsal thalamus of the thalamus was significantly enhanced ( P0.001 ) .

3 . After Morris water maze training , the expression of c - fos and c - Jun protein in the anterior nucleus of the thalamus increased significantly , suggesting that the third messenger in the nucleus could act on the binding site of the target gene AP1 and activate the target gene for further transcription .

4 . In the anterior nucleus of the thalamus , the expression of GABAergic - alpha - 1 and GABAergic - beta2 was found , suggesting that the anterior nucleus of the thalamus might be regulated by inhibitory amino acids in learning and memory .

【学位授予单位】：大连医科大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R341

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