本文選題：丘腦前核　切入點：NMDA受體　出處：《大連醫(yī)科大學》2010年博士論文

【摘要】： 目的：學習記憶是人類和動物生存不可缺少的重要功能。學習(learning)是獲得新信息和新知識的神經過程,而記憶(memory)則是對所獲取信息的編碼、鞏固、保存和讀出的神經過程。大腦具有多重記憶系統(tǒng),如記憶可分為陳述性記憶(declarative memory)和非陳述性記憶(non-declarative memory)等,不同類型的記憶在不同的腦區(qū)中貯存,海馬、丘腦、杏仁核、前額葉皮層及基底神經節(jié)等都與學習記憶有關。近年來研究發(fā)現(xiàn)丘腦前核在空間學習記憶中發(fā)揮重要作用。 1973年Bliss和lomo首次在兔海馬觀察到突觸傳遞的長時程增強(Long term potentiation LTP)現(xiàn)象,長時程增強很可能是學習與記憶的神經生理基礎,而長時程增強現(xiàn)象的發(fā)生及維持過程與突觸前膜釋放谷氨酸的增加有關。本實驗室前期通過膠體金免疫電鏡等技術發(fā)現(xiàn)丘腦前核與邊緣皮質學習記憶環(huán)路間存在的興奮性神經遞質是谷氨酸,但丘腦前核內是否存在NMDA受體研究較少。 越來越多的研究證實,MAPK/ERK信號通路與腦內長時程增強的形成以及學習記憶功能有重要的聯(lián)系。如經水迷宮訓練后的大鼠,海馬CA1/CA1區(qū)ERK被激活,使用PD098059抑制MAPK/ERK級聯(lián)反應則p-ERK蛋白含量降低,并且長期空間記憶的形成受損。丘腦前核在學習記憶過程中,ERK通路是否參與胞內信號的轉導目前仍不清楚。 即早基因(Immediate early genes IEGs)被認為是核內的第三信使,如大鼠海馬內注射c-Fos的反義寡核苷酸后發(fā)現(xiàn)大鼠的長時記憶鞏固過程障礙。丘腦前核在空間學習記憶即早基因是否參與仍不清楚。 突觸可塑性包括兩個方面,長時程增強(Long-term potentiation LTP)和長時程抑制(Long-term depression LTD), LTP和LTD對一個完整的學習記憶神經網(wǎng)絡均不可缺少,LTD在學習記憶中發(fā)揮著不斷糾正錯誤的作用。γ-氨基丁酸(γ-Amino butyric acid, GABA)是中樞神經系統(tǒng)內主要的抑制性神經遞質,GABA與LTD有關。目前丘腦前核內GABA及其受體的表達仍不明確。 本實驗目的是在前期發(fā)現(xiàn)丘腦前核內存在谷氨酸神經遞質基礎上,探討丘腦前核內NMDA受體表達,Morris水迷宮訓練后ERK1／2的變化以及c-Fos和c-Jun的表達。并探討抑制性神經遞質GABAA受體在丘腦前核的表達。 方法 1.通過原位雜交觀察大鼠丘腦前核內NR1,NR2A及NR2BmRNA的表達及分布特點。 2.成年雄性SD大鼠31只,分為3組：PD組：側腦室內注射5ul PD098059溶液(PD098059 3ug／只)；PD對照組：側腦室內注射5ulDMSO溶劑;正常組：未進行側腦室注射。在Morris水迷宮中,定位航行訓練后,行側腦室內注射,24小時后空間探索實驗。免疫組化檢測p-ERK的變化。 3.成年雄性SD大鼠分為3組,正常組：未經Morris水迷宮訓練；訓練組：通過Morris水迷宮訓練；假訓練組：撤掉平臺,每天游泳次數(shù)和游泳時間與訓練組相同。訓練后一半免疫組化檢測c-Fos和c-Jun的表達。另一部分行Westwern-Blot檢測c-Fos和c-Jun蛋白的表達。 4.原位雜交觀察丘腦前核內GABAAR-alpha 1和GABAAR-beta2受體mRNA的表達。 結果 1.NR2A,NR2B及NR1mRNA在丘腦前核都有表達,陽性神經元分布較密集,細胞形態(tài)較一致,整體分布尚均勻。NR2AmRNA及NR2BmRNA在丘腦前背側核和腹側核的分布前者稍顯密集,胞體略大。同時在海馬CA1,CA3及DG和扣帶后回也觀察到NR2A,NR2B及NR1 mRNA的表達。空白對照切片為陰性。 2.在空間探索實驗中PD組尋找平臺次數(shù)與對照組及正常對照組相比明顯減少(P0.01)。PD組在靶象限停留時間與對照組及正常組相比明顯減少(P0.01)。正常組在靶象限停留時間和穿越平臺次數(shù)與對照組相比沒有顯著差異,p0.05。 p-ERK陽性表達神經元呈棕黃色,為圓形或橢圓形,PD對照組與PD組與正常對照組比較,在丘腦前核p-ERK組表達明顯增強,光密度分析有顯著性差異(P0.01),PD組與正常對照組比較,在丘腦前核p-ERK無顯著差別。在PD對照組,丘腦前背側核(AD)的p-ERK免疫反應陽性產物與丘腦前腹側核(AV)相比,表達顯著增強,光密度分析有顯著性差異(P0.05)。空白對照切片為陰性,未見p-ERK表達陽性神經元。 3.c-Fos免疫組化陽性表達位于細胞核內,呈褐色的圓形或橢圓形。訓練組c-Fos陽性細胞在AD和AV均有表達,但AD與AV相比表達顯著增強,經圖像平均光密度有顯著差異(P0.05)。訓練組丘腦前核c-Fos陽性細胞表達與正常對照組和假訓練組比較明顯增強,經圖像平均光密度分析明顯增強(P0.05)。c-Jun免疫組化陽性表達位于細胞核內,呈褐色的圓形或橢圓形。訓練組c-Jun陽性細胞在AD和AV均有表達,AD與AV相比染色略強,細胞稍顯密集,形態(tài)略大。正常對照組和假訓練組AD和AV的c-Jun陽性細胞都有弱的表達;但訓練組與正常對照組和假訓練組比較染色明顯增強,經圖像分析平均光密度明顯增強(P0.05)。 4.原位雜交：GABAAR-alphal和GABAAR-beta2 mRNA原位雜交陽性反應產物為棕黃色,主要分布在神經元胞漿中。在AD和AV,陽性神經元分布較密集,細胞形態(tài)較一致,在AD和AV的分布前者稍顯密集。同時大腦其他學習記憶腦區(qū)如海馬CA1,CA3及DG和扣帶后回也觀察到GABAAR-alpha 1和GABAAR-beta2mRNA的表達。 結論 1.大鼠丘腦前核內存在NR1,NR2A.NR2BmRNA的表達。提示丘腦前核在空間學習記憶信號轉導中細胞膜受體可能以NR1-NR2A, NR1-NR2B或NR1-NR2A-NR2B形式存在。 2.大鼠通過Morris水迷宮訓練及應用PD098059后,大鼠空間參考記憶受損,p-ERK表達減弱,提示丘腦前核空間參考記憶細胞內信號轉導可能與MAPK途經有關。在PD對照組,Morris水迷宮訓練后p-ERK在丘腦前背側核表達較丘腦前腹側核表達顯著增強(P0.001)。 3.Morris水迷宮訓練后大鼠丘腦前核內c-Fos和c-Jun蛋白表達顯著增加,提示核內第三信使可能以Fos-Jun異源二聚體的形式,作用于靶基因AP1結合位點,啟動靶基因進一步轉錄。水迷宮訓練后丘腦前背側核c-Fos表達與丘腦前腹側核顯著增強,但丘腦前背側核與前腹側核在學習記憶中的具體作用有待進一步探討。 4.丘腦前核內存在GABAAR-alpha 1和GABAAR-beta2表達,提示在學習記憶中丘腦前核可能受到抑制性氨基酸的調控
[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 .

【學位授予單位】：大連醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2010
【分類號】：R341

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相關期刊論文 前2條

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本文編號：1669371