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

【摘要】： 目的：學(xué)習(xí)記憶是人類和動物生存不可缺少的重要功能。學(xué)習(xí)(learning)是獲得新信息和新知識的神經(jīng)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(jīng)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(jīng)節(jié)等都與學(xué)習(xí)記憶有關(guān)。近年來研究發(fā)現(xiàn)丘腦前核在空間學(xué)習(xí)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(qiáng)(Long term potentiation LTP)現(xiàn)象,長時程增強(qiáng)很可能是學(xué)習(xí)與記憶的神經(jīng)生理基礎(chǔ),而長時程增強(qiáng)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(guān)。本實(shí)驗(yàn)室前期通過膠體金免疫電鏡等技術(shù)發(fā)現(xiàn)丘腦前核與邊緣皮質(zhì)學(xué)習(xí)記憶環(huán)路間存在的興奮性神經(jīng)遞質(zhì)是谷氨酸,但丘腦前核內(nèi)是否存在NMDA受體研究較少。 越來越多的研究證實(shí),MAPK/ERK信號通路與腦內(nèi)長時程增強(qiáng)的形成以及學(xué)習(xí)記憶功能有重要的聯(lián)系。如經(jīng)水迷宮訓(xùn)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(yīng)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(xué)習(xí)記憶過程中,ERK通路是否參與胞內(nèi)信號的轉(zhuǎn)導(dǎo)目前仍不清楚。 即早基因(Immediate early genes IEGs)被認(rèn)為是核內(nèi)的第三信使,如大鼠海馬內(nèi)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(xué)習(xí)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(qiáng)(Long-term potentiation LTP)和長時程抑制(Long-term depression LTD), LTP和LTD對一個完整的學(xué)習(xí)記憶神經(jīng)網(wǎng)絡(luò)均不可缺少,LTD在學(xué)習(xí)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(jīng)系統(tǒng)內(nèi)主要的抑制性神經(jīng)遞質(zhì),GABA與LTD有關(guān)。目前丘腦前核內(nèi)GABA及其受體的表達(dá)仍不明確。 本實(shí)驗(yàn)?zāi)康氖窃谇捌诎l(fā)現(xiàn)丘腦前核內(nèi)存在谷氨酸神經(jīng)遞質(zhì)基礎(chǔ)上,探討丘腦前核內(nèi)NMDA受體表達(dá),Morris水迷宮訓(xùn)練后ERK1／2的變化以及c-Fos和c-Jun的表達(dá)。并探討抑制性神經(jīng)遞質(zhì)GABAA受體在丘腦前核的表達(dá)。 方法 1.通過原位雜交觀察大鼠丘腦前核內(nèi)NR1,NR2A及NR2BmRNA的表達(dá)及分布特點(diǎn)。 2.成年雄性SD大鼠31只,分為3組：PD組：側(cè)腦室內(nèi)注射5ul PD098059溶液(PD098059 3ug／只)；PD對照組：側(cè)腦室內(nèi)注射5ulDMSO溶劑;正常組：未進(jìn)行側(cè)腦室注射。在Morris水迷宮中,定位航行訓(xùn)練后,行側(cè)腦室內(nèi)注射,24小時后空間探索實(shí)驗(yàn)。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(jīng)Morris水迷宮訓(xùn)練；訓(xùn)練組：通過Morris水迷宮訓(xùn)練；假訓(xùn)練組：撤掉平臺,每天游泳次數(shù)和游泳時間與訓(xùn)練組相同。訓(xùn)練后一半免疫組化檢測c-Fos和c-Jun的表達(dá)。另一部分行Westwern-Blot檢測c-Fos和c-Jun蛋白的表達(dá)。 4.原位雜交觀察丘腦前核內(nèi)GABAAR-alpha 1和GABAAR-beta2受體mRNA的表達(dá)。 結(jié)果 1.NR2A,NR2B及NR1mRNA在丘腦前核都有表達(dá),陽性神經(jīng)元分布較密集,細(xì)胞形態(tài)較一致,整體分布尚均勻。NR2AmRNA及NR2BmRNA在丘腦前背側(cè)核和腹側(cè)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(dá)。空白對照切片為陰性。 2.在空間探索實(shí)驗(yàn)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(dá)神經(jīng)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(dá)明顯增強(qiáng),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(cè)核(AD)的p-ERK免疫反應(yīng)陽性產(chǎn)物與丘腦前腹側(cè)核(AV)相比,表達(dá)顯著增強(qiáng),光密度分析有顯著性差異(P0.05)�？瞻讓φ涨衅瑸殛幮�,未見p-ERK表達(dá)陽性神經(jīng)元。 3.c-Fos免疫組化陽性表達(dá)位于細(xì)胞核內(nèi),呈褐色的圓形或橢圓形。訓(xùn)練組c-Fos陽性細(xì)胞在AD和AV均有表達(dá),但AD與AV相比表達(dá)顯著增強(qiáng),經(jīng)圖像平均光密度有顯著差異(P0.05)。訓(xùn)練組丘腦前核c-Fos陽性細(xì)胞表達(dá)與正常對照組和假訓(xùn)練組比較明顯增強(qiáng),經(jīng)圖像平均光密度分析明顯增強(qiáng)(P0.05)。c-Jun免疫組化陽性表達(dá)位于細(xì)胞核內(nèi),呈褐色的圓形或橢圓形。訓(xùn)練組c-Jun陽性細(xì)胞在AD和AV均有表達(dá),AD與AV相比染色略強(qiáng),細(xì)胞稍顯密集,形態(tài)略大。正常對照組和假訓(xùn)練組AD和AV的c-Jun陽性細(xì)胞都有弱的表達(dá);但訓(xùn)練組與正常對照組和假訓(xùn)練組比較染色明顯增強(qiáng),經(jīng)圖像分析平均光密度明顯增強(qiáng)(P0.05)。 4.原位雜交：GABAAR-alphal和GABAAR-beta2 mRNA原位雜交陽性反應(yīng)產(chǎn)物為棕黃色,主要分布在神經(jīng)元胞漿中。在AD和AV,陽性神經(jīng)元分布較密集,細(xì)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(xué)習(xí)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(dá)。 結(jié)論 1.大鼠丘腦前核內(nèi)存在NR1,NR2A.NR2BmRNA的表達(dá)。提示丘腦前核在空間學(xué)習(xí)記憶信號轉(zhuǎn)導(dǎo)中細(xì)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(xùn)練及應(yīng)用PD098059后,大鼠空間參考記憶受損,p-ERK表達(dá)減弱,提示丘腦前核空間參考記憶細(xì)胞內(nèi)信號轉(zhuǎn)導(dǎo)可能與MAPK途經(jīng)有關(guān)。在PD對照組,Morris水迷宮訓(xùn)練后p-ERK在丘腦前背側(cè)核表達(dá)較丘腦前腹側(cè)核表達(dá)顯著增強(qiáng)(P0.001)。 3.Morris水迷宮訓(xùn)練后大鼠丘腦前核內(nèi)c-Fos和c-Jun蛋白表達(dá)顯著增加,提示核內(nèi)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(jié)合位點(diǎn),啟動靶基因進(jìn)一步轉(zhuǎn)錄。水迷宮訓(xùn)練后丘腦前背側(cè)核c-Fos表達(dá)與丘腦前腹側(cè)核顯著增強(qiáng),但丘腦前背側(cè)核與前腹側(cè)核在學(xué)習(xí)記憶中的具體作用有待進(jìn)一步探討。 4.丘腦前核內(nèi)存在GABAAR-alpha 1和GABAAR-beta2表達(dá),提示在學(xué)習(xí)記憶中丘腦前核可能受到抑制性氨基酸的調(diào)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(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R341

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