本文選題：sigma-1受體(σ1R) + 基底外側(cè)杏仁核(BLA)�。� 參考：《南京醫(yī)科大學(xué)》2017年碩士論文

【摘要】：背景sigma-1受體(σiR)主要在海馬、下丘腦及杏仁核的神經(jīng)細(xì)胞中表達(dá)。σ1R活化能增強(qiáng)海馬CA1區(qū)(CornuAmmonis 1 field)錐體細(xì)胞N-甲基-D-天門(mén)冬氨酸受體(NMDA受體)介導(dǎo)的鈣離子(Ca2+)內(nèi)流,增加鈣調(diào)蛋白(calmodulin)的活性,提高細(xì)胞外調(diào)節(jié)蛋白激酶(Extracellularregulated protein kinases,ERK1/2)的磷酸化水平和環(huán)磷腺苷效應(yīng)元件結(jié)合蛋白(cAMPresponsive element binding protein,CREB)的轉(zhuǎn)錄水平,易化突觸可塑性長(zhǎng)時(shí)程增強(qiáng)(Long-term potentiation,LTP)的誘導(dǎo)。同時(shí),σ1R活化對(duì)GABAA受體有負(fù)向調(diào)控作用。此外,σ1R活化能增強(qiáng)電壓門(mén)控鈣離子通道開(kāi)放來(lái)增加鈣離子內(nèi)流。σ1R基因敲除小鼠有抑郁樣的表型,但是迄今有關(guān)其分子機(jī)制仍然沒(méi)有闡明�；淄鈧�(cè)杏仁核(Basolateral amygdala,BLA)神經(jīng)元被認(rèn)為是調(diào)節(jié)情感行為的重要腦區(qū)之一,小鼠BLA的錐體神經(jīng)元表達(dá)σ1R。BLA主要包括兩種類(lèi)型的神經(jīng)元:谷氨酸能神經(jīng)元和GABA能中間神經(jīng)元。大量的研究證明,來(lái)自于皮層外囊(External capsule,EC)的神經(jīng)纖維末梢與BLA谷氨酸能神經(jīng)元構(gòu)建了興奮性神經(jīng)通路,GABAA受體介導(dǎo)了 BLA的抑制性局部回路。谷氨酸能神經(jīng)元的突觸可塑性LTP和長(zhǎng)時(shí)程抑制(Long-term depression,LTD)都參與了恐懼記憶的形成和抑郁-焦慮行為的發(fā)生,如LTP是恐懼記憶形成的分子基礎(chǔ),而LTD的誘導(dǎo)能消除形成的恐懼記憶。LTP誘導(dǎo)依賴于NMDA受體介導(dǎo)的Ca2+的內(nèi)流,GABAA受體介導(dǎo)的抑制性局部回路與LTD的誘導(dǎo)密切相關(guān)。此外,NMDA受體的鈣離子內(nèi)流通過(guò)活化突觸后致密蛋白(Postsynaptic density-95,PSD-95),促進(jìn)PSD-95與神經(jīng)元型一氧化氮合酶(Neuronal NO synthase,nNOS)的結(jié)合從而增加一氧化氮(Nitric oxide,NO)的產(chǎn)生和逆行性釋放,這可以增加突觸前膜的谷氨酸釋放�；谏鲜龇治�,本研究提出"σ1R缺失有可能通過(guò)影響B(tài)LA的突觸可塑性誘發(fā)抑郁樣行為"的科研設(shè)想。研究目的1.明確σ1R缺失對(duì)BLA神經(jīng)回路和突觸可塑性的影響及其分子機(jī)制;2.闡明σ1R缺失影響B(tài)LA的突觸可塑性在小鼠抑郁樣行為發(fā)生中的作用。實(shí)驗(yàn)方法1.σ1R基因敲除小鼠(σ1R-KO小鼠)由美國(guó)California的Zollila教授提供。提取小鼠尾巴的DNA,用PCR方法進(jìn)行小鼠的基因型鑒定。2.行為學(xué)檢測(cè):用曠場(chǎng)實(shí)驗(yàn)(Open field test,OFT)評(píng)價(jià)小鼠的自主運(yùn)動(dòng)和探知行為。用懸尾試驗(yàn)(Tail suspension test,TST)和強(qiáng)迫游泳試驗(yàn)(Forced swimming test,FST)檢測(cè)小鼠的抑郁樣行為。3.形態(tài)學(xué)檢測(cè):制備BLA石蠟?zāi)X片,用甲苯胺藍(lán)染色觀察BLA區(qū)神經(jīng)元的形態(tài);進(jìn)行BLA區(qū)的σ1R免疫組織化學(xué)染色觀察σ1R的表達(dá)。4.用場(chǎng)電位記錄外囊和BLA谷氨酸神經(jīng)元的突觸(EC-BLA突觸)傳遞效應(yīng),雙脈沖易化(Paired-pulse facilitation,PPF,評(píng)價(jià)突觸前谷氨酸釋放)和雙脈沖抑制(Paired-pulse inhibition,PPI,評(píng)價(jià)抑制性神經(jīng)回路功能),用高頻刺激(High frequency stimulation,HFS,100 Hz,1min×5 times)誘導(dǎo) LTP,用低頻刺激(Low frequency stimulation,LFS,1Hz,15 min)誘導(dǎo) LTD。5.用ELISA試劑盒檢測(cè)BLA腦區(qū)的NO水平。6.用免疫共沉淀(Coimmunoprecipitation,Co-IP)和蛋白印跡(Western blotting,WB)的方法檢測(cè)BLA區(qū)nNOS和PSD-95的結(jié)合能力;檢測(cè)nNOS、PSD-95、calmodulin、NMDA受體亞基NR2A和NR2B的磷酸化水平。7.用實(shí)時(shí)定量聚合酶鏈反應(yīng)(RT-PCR)檢測(cè)AMPA受體、NMDA受體、GABAA受體的mRNA水平。實(shí)驗(yàn)結(jié)果1.σ1R免疫組織染色結(jié)果顯示,野生型(WT)小鼠BLA區(qū)的大型神經(jīng)元(谷氨酸神經(jīng)元)表達(dá)σ1R。BLA腦區(qū)甲苯胺藍(lán)細(xì)胞染色結(jié)果顯示,與同齡WT小鼠相比,σ1R-KO小鼠神經(jīng)元的數(shù)量和形態(tài)都沒(méi)有明顯改變。2.與WT小鼠相比,σ1R-KO小鼠BLA的EC-BLA突觸傳遞的興奮性突觸后電位(fEPSP)斜率明顯降低,雙脈沖易化(PPF)值明顯增加,提示σ1R缺失→減少谷氨酸釋放→突觸傳遞功能降低。3.與 WT 小鼠相比,σ1R-KO 小鼠 BLA 的 AMPA 受體(GluR1、GluR2)、NMDA受體(NR1、NR2B、NR2A)和GABAA受體 GABAAR-A4、GABAAR-δ)mNRA水平?jīng)]有明顯改變,然而NR2B亞基的磷酸化水平顯著降低,NR2A亞基的磷酸化水平?jīng)]有改變,提示σ1R缺失能降低NMDA受體功能。4.與 WT 小鼠相比,盡管σ1R-KO 小鼠 BLA 的 nNOS、PSD-95 和 calmodulin的蛋白水平?jīng)]有明顯改變,然而nNOS和PSD-95的結(jié)合水平顯著降低,NO水平也降低。NMDA受體激動(dòng)劑NMDA的BLA區(qū)微注射可以恢復(fù)nNOS和PSD-95的結(jié)合和NO產(chǎn)生,提示σ1R缺失→NMDA受體下調(diào)→降低nNOS與PSD-95結(jié)合→減少NO生成。5.在σ1R-KO小鼠腦片,NMDA或者NO供體DETA/NO處理能恢復(fù)fEPSP斜率和PPF值;nNOS抑制劑7-NI可以阻斷NMDA的作用,提示σ1R缺失→減少NO生成→抑制谷氨酸釋放。6.HFS能誘導(dǎo)WT小鼠產(chǎn)生NMDA受體依賴性LTP,但是在σ1R-KO小鼠HFS不能誘導(dǎo)LTP。在σ1R-KO小鼠腦片,NMDA處理能恢復(fù)LTP的誘導(dǎo),7-NI會(huì)拮抗NMDA的作用,提示σ1R缺失→NMDA受體下調(diào)→阻止LTP誘導(dǎo)。7.與WT小鼠相比,σ1R-KO小鼠BLA的PPI值明顯增加。GABAA受體激動(dòng)劑muscimol和NMDA處理能恢復(fù)σ1R-KO小鼠的PPI值。在WT小鼠腦片,GABAAR阻斷劑bicuculine處理能增加PPI值,提示σ1R缺失→減弱GABAAR介導(dǎo)的抑制性神經(jīng)回路。8.LFS能誘導(dǎo)WT小鼠產(chǎn)生LTD,但是在σσ1R-KO小鼠LFS不能誘導(dǎo)LTD。bicuculine 能阻斷 WT 小鼠的 LTD 誘導(dǎo)。muscimol,NMDA 和 DETA/NO 均可以恢復(fù)σ1R-KO小鼠LTD,提示σ1R缺失→減弱GABAA受體介導(dǎo)的抑制性回路→損害LTD的誘導(dǎo)。9.與WT小鼠相比,σ1R-KO小鼠在強(qiáng)迫游泳和懸尾試驗(yàn)中的不動(dòng)時(shí)間明顯延長(zhǎng)。在σ1R-KO小鼠BLA區(qū)注射N(xiāo)MDA、DETA和muscimol可以逆轉(zhuǎn)強(qiáng)迫游泳和懸尾試驗(yàn)中的時(shí)間延長(zhǎng),7-NI能拮抗NMDA的作用,提示σ1R缺失→阻礙BLA的LTD誘導(dǎo)→引起抑郁樣行為。結(jié)論1.BLA谷氨酸神經(jīng)元的σ1R缺失減少了 NMDA受體的鈣離子內(nèi)流從而降低nNOS與PSD-95結(jié)合導(dǎo)致NO生成減少;2.σ1R缺失減少NO的釋放從而減少了突觸前谷氨酸的釋放導(dǎo)致突觸傳遞功能減弱和LTP誘導(dǎo)障礙;3.σ1R缺失減少NO的釋放從而減弱GABAA受體介導(dǎo)的抑制性回路導(dǎo)致LTD誘導(dǎo)障礙并發(fā)生抑郁樣行為。
[Abstract]:Background sigma-1 receptor (sigma iR) is mainly expressed in the neurons of the hippocampus, hypothalamus and amygdala. The activation of sigma 1R enhances the flow of calcium ion (Ca2+) mediated by N- methyl -D- aspartate receptor (NMDA receptor) in the pyramidal pyramidal cells of the hippocampal CA1 region (CornuAmmonis 1 field), increases the activity of the calmodulin (calmodulin) and improves the extracellular modulating protein excitation. The phosphorylation level of the enzyme (Extracellularregulated protein kinases, ERK1/2) and the transcriptional level of the binding protein of the adenosine effect element binding protein (cAMPresponsive element binding protein, CREB) can induce the induction of the synaptic plasticity long time potentiation (Long-term potentiation, LTP). In addition, sigma 1R activation enhances the opening of voltage gated calcium channels to increase the flow of calcium ions. The sigma 1R knockout mice have a depressive phenotype, but their molecular mechanisms are still not elucidated. The basal lateral amygdala (Basolateral amygdala, BLA) neurons are considered to be one of the most important brain areas for regulating emotional behavior, and BLA in mice. The expression of sigma 1R.BLA in pyramidal neurons mainly includes two types of neurons: glutamatergic neurons and GABA intermediate neurons. A large number of studies have shown that the nerve fibers from the External capsule (EC) and BLA glutamic acid neurons construct an excitatory neural pathway, and the GABAA receptor mediates the inhibitory part of BLA. The synaptic plasticity LTP and Long-term depression (LTD) of glutamate neurons all participate in the formation of fear memory and the occurrence of depression and anxiety, such as LTP is the molecular basis of the formation of fear memory, and LTD induction can eliminate the formation of fear memory.LTP induced Ca2+'s internal flow dependent on NMDA receptor, G. G The inhibitory local loop mediated by ABAA receptor is closely related to the induction of LTD. In addition, the calcium influx of the NMDA receptor activates the binding of Postsynaptic density-95 (PSD-95) to the binding of PSD-95 and neuronal nitric oxide synthase (Neuronal NO synthase, nNOS) to increase the production of nitric oxide (Nitric). Birth and retrograde release, which can increase the release of glutamate in the presynaptic membrane. Based on the above analysis, this study suggests that "Sigma 1R deletion may induce depression like behavior induced by BLA's synaptic plasticity". Purpose 1. study the effect of sigma 1R deletion on BLA neural circuit and the plasticity of sudden touch and its molecular mechanism; and 2. clarify Sigma 1R The role of BLA's synaptic plasticity in the occurrence of depressive behavior in mice. Experimental methods 1. Sigma 1R knockout mice (sigma 1R-KO mice) were provided by Professor Zollila of California in the United States. The DNA of mouse tail was extracted and PCR method was used to detect the genotype of mice in.2. behavior test: a open field experiment (Open field test, OFT) was used to evaluate the behavior of mice. The autonomous movement and the behavior of the mice were detected. The Tail suspension test (TST) and the forced swimming test (Forced swimming test, FST) were used to detect the depressive behavior of mice. The morphology of the BLA paraffin brain slices was prepared, the morphology of the BLA neurons was observed with toluidine blue staining, and sigma immunohistochemical staining of the BLA region was used to observe the sigma. 1R expression.4. uses field potential to record synapse (EC-BLA synapse) transfer effect of outer capsule and BLA glutamic neuron, double pulse susceptibility (Paired-pulse facilitation, PPF, evaluation of pre synaptic glutamic acid release) and double pulse inhibition (Paired-pulse inhibition, PPI, evaluation of inhibitory neural circuit function), high frequency stimulation (High frequency) HFS, 100 Hz, 1min x 5 times) induced LTP with low-frequency stimulation (Low frequency stimulation, LFS, 1Hz, 15 min) to induce LTD.5. to detect the level of the brain region. D-95, calmodulin, NMDA receptor subunit NR2A and NR2B phosphorylation level.7. use real-time quantitative polymerase chain reaction (RT-PCR) to detect AMPA receptor, NMDA receptor, GABAA receptor mRNA level. Experimental results 1. Sigma 1R immuno tissue staining results showed that large neurons (glutamate neurons) in wild type mice expressed sigma brain region toluidine The results of blue cell staining showed that compared with the WT mice of the same age, there was no significant change in the number and morphology of the neurons in the sigma 1R-KO mice. Compared with the WT mice,.2. was significantly lower in the EC-BLA synaptic potential (fEPSP) slope of the EC-BLA synaptic transmission in the BLA of the sigma 1R-KO mice, and the value of the double pulse susceptibility (PPF) was obviously increased, indicating that the sigma 1R deletion and glutamic acid release were reduced. The level of AMPA receptor (GluR1, GluR2), NMDA receptor (NR1, NR2B, NR2A) and GABAA receptor (BLA) in the BLA of sigma 1R-KO mice did not change significantly, but the level of phosphorylation of BLA was significantly reduced, and the level of phosphorylation of 1R-KO was not changed. The decrease of NMDA receptor function.4. was compared with that of WT mice. Although the protein levels of PSD-95 and calmodulin in the BLA of the 1R-KO mice were not significantly changed, the binding level of nNOS and PSD-95 decreased significantly. The loss of NMDA receptor decreased to the binding of nNOS to PSD-95 and reduced NO to produce.5. in the sigma 1R-KO mouse brain slices, NMDA or NO donor DETA/NO treatment could restore fEPSP slope and PPF value. Sex LTP, but in sigma 1R-KO mice HFS can not induce LTP. in the sigma 1R-KO mouse brain slices, NMDA treatment can restore the induction of LTP, 7-NI will antagonize the effect of NMDA, suggesting that sigma 1R deletion and NMDA receptor downregulation. The PPI value of mice. In WT mouse brain slices, GABAAR blocker bicuculine treatment can increase the PPI value, suggesting that 1R deletion and weakened GABAAR mediated inhibitory neural circuit.8.LFS can induce LTD in WT mice, but it can not be induced in sigma and sigma 1R-KO mice. The LTD of sigma 1R-KO mice was restored, indicating that sigma 1R deletion, weakened GABAA receptor mediated inhibitory circuit and induced.9. that damage LTD, compared with WT mice, the time of immobility of sigma 1R-KO mice in forced swimming and tail suspension test was obviously prolonged. NMDA in the BLA region of the sigma 1R-KO mice, DETA and the time could reverse the time of forced swimming and tail suspension test. 7-NI can antagonize the effect of NMDA, suggesting that the deletion of sigma 1R, which hinders the LTD induction of BLA, induces depressive behavior. Conclusion the loss of the sigma 1R in the 1.BLA glutamic neurons reduces the calcium influx of NMDA receptors and reduces the decrease of nNOS and PSD-95 binding to NO generation, and 2. Sigma 1R deletion reduces the release of presynaptic glutamic acid. The release of 3. Sigma 1R reduces the release of NO and reduces the inhibition of the GABAA receptor mediated inhibitory circuit to LTD induced disturbance and depressive behavior.

【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R749
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本文編號(hào)：1832838