sigma-1受体基因缺失诱发抑郁样行为的机制研究

发布时间：2018-05-02 06:49

  本文选题：sigma-1受体(σ1R) + 基底外侧杏仁核(BLA)　； 参考：《南京医科大学》2017年硕士论文

【摘要】：背景sigma-1受体(σiR)主要在海马、下丘脑及杏仁核的神经细胞中表达。σ1R活化能增强海马CA1区(CornuAmmonis 1 field)锥体细胞N-甲基-D-天门冬氨酸受体(NMDA受体)介导的钙离子(Ca2+)内流,增加钙调蛋白(calmodulin)的活性,提高细胞外调节蛋白激酶(Extracellularregulated protein kinases,ERK1/2)的磷酸化水平和环磷腺苷效应元件结合蛋白(cAMPresponsive element binding protein,CREB)的转录水平,易化突触可塑性长时程增强(Long-term potentiation,LTP)的诱导。同时,σ1R活化对GABAA受体有负向调控作用。此外,σ1R活化能增强电压门控钙离子通道开放来增加钙离子内流。σ1R基因敲除小鼠有抑郁样的表型,但是迄今有关其分子机制仍然没有阐明。基底外侧杏仁核(Basolateral amygdala,BLA)神经元被认为是调节情感行为的重要脑区之一,小鼠BLA的锥体神经元表达σ1R。BLA主要包括两种类型的神经元:谷氨酸能神经元和GABA能中间神经元。大量的研究证明,来自于皮层外囊(External capsule,EC)的神经纤维末梢与BLA谷氨酸能神经元构建了兴奋性神经通路,GABAA受体介导了 BLA的抑制性局部回路。谷氨酸能神经元的突触可塑性LTP和长时程抑制(Long-term depression,LTD)都参与了恐惧记忆的形成和抑郁-焦虑行为的发生,如LTP是恐惧记忆形成的分子基础,而LTD的诱导能消除形成的恐惧记忆。LTP诱导依赖于NMDA受体介导的Ca2+的内流,GABAA受体介导的抑制性局部回路与LTD的诱导密切相关。此外,NMDA受体的钙离子内流通过活化突触后致密蛋白(Postsynaptic density-95,PSD-95),促进PSD-95与神经元型一氧化氮合酶(Neuronal NO synthase,nNOS)的结合从而增加一氧化氮(Nitric oxide,NO)的产生和逆行性释放,这可以增加突触前膜的谷氨酸释放。基于上述分析,本研究提出"σ1R缺失有可能通过影响BLA的突触可塑性诱发抑郁样行为"的科研设想。研究目的1.明确σ1R缺失对BLA神经回路和突触可塑性的影响及其分子机制;2.阐明σ1R缺失影响BLA的突触可塑性在小鼠抑郁样行为发生中的作用。实验方法1.σ1R基因敲除小鼠(σ1R-KO小鼠)由美国California的Zollila教授提供。提取小鼠尾巴的DNA,用PCR方法进行小鼠的基因型鉴定。2.行为学检测:用旷场实验(Open field test,OFT)评价小鼠的自主运动和探知行为。用悬尾试验(Tail suspension test,TST)和强迫游泳试验(Forced swimming test,FST)检测小鼠的抑郁样行为。3.形态学检测:制备BLA石蜡脑片,用甲苯胺蓝染色观察BLA区神经元的形态;进行BLA区的σ1R免疫组织化学染色观察σ1R的表达。4.用场电位记录外囊和BLA谷氨酸神经元的突触(EC-BLA突触)传递效应,双脉冲易化(Paired-pulse facilitation,PPF,评价突触前谷氨酸释放)和双脉冲抑制(Paired-pulse inhibition,PPI,评价抑制性神经回路功能),用高频刺激(High frequency stimulation,HFS,100 Hz,1min×5 times)诱导 LTP,用低频刺激(Low frequency stimulation,LFS,1Hz,15 min)诱导 LTD。5.用ELISA试剂盒检测BLA脑区的NO水平。6.用免疫共沉淀(Coimmunoprecipitation,Co-IP)和蛋白印迹(Western blotting,WB)的方法检测BLA区nNOS和PSD-95的结合能力;检测nNOS、PSD-95、calmodulin、NMDA受体亚基NR2A和NR2B的磷酸化水平。7.用实时定量聚合酶链反应(RT-PCR)检测AMPA受体、NMDA受体、GABAA受体的mRNA水平。实验结果1.σ1R免疫组织染色结果显示,野生型(WT)小鼠BLA区的大型神经元(谷氨酸神经元)表达σ1R。BLA脑区甲苯胺蓝细胞染色结果显示,与同龄WT小鼠相比,σ1R-KO小鼠神经元的数量和形态都没有明显改变。2.与WT小鼠相比,σ1R-KO小鼠BLA的EC-BLA突触传递的兴奋性突触后电位(fEPSP)斜率明显降低,双脉冲易化(PPF)值明显增加,提示σ1R缺失→减少谷氨酸释放→突触传递功能降低。3.与 WT 小鼠相比,σ1R-KO 小鼠 BLA 的 AMPA 受体(GluR1、GluR2)、NMDA受体(NR1、NR2B、NR2A)和GABAA受体 GABAAR-A4、GABAAR-δ)mNRA水平没有明显改变,然而NR2B亚基的磷酸化水平显著降低,NR2A亚基的磷酸化水平没有改变,提示σ1R缺失能降低NMDA受体功能。4.与 WT 小鼠相比,尽管σ1R-KO 小鼠 BLA 的 nNOS、PSD-95 和 calmodulin的蛋白水平没有明显改变,然而nNOS和PSD-95的结合水平显著降低,NO水平也降低。NMDA受体激动剂NMDA的BLA区微注射可以恢复nNOS和PSD-95的结合和NO产生,提示σ1R缺失→NMDA受体下调→降低nNOS与PSD-95结合→减少NO生成。5.在σ1R-KO小鼠脑片,NMDA或者NO供体DETA/NO处理能恢复fEPSP斜率和PPF值;nNOS抑制剂7-NI可以阻断NMDA的作用,提示σ1R缺失→减少NO生成→抑制谷氨酸释放。6.HFS能诱导WT小鼠产生NMDA受体依赖性LTP,但是在σ1R-KO小鼠HFS不能诱导LTP。在σ1R-KO小鼠脑片,NMDA处理能恢复LTP的诱导,7-NI会拮抗NMDA的作用,提示σ1R缺失→NMDA受体下调→阻止LTP诱导。7.与WT小鼠相比,σ1R-KO小鼠BLA的PPI值明显增加。GABAA受体激动剂muscimol和NMDA处理能恢复σ1R-KO小鼠的PPI值。在WT小鼠脑片,GABAAR阻断剂bicuculine处理能增加PPI值,提示σ1R缺失→减弱GABAAR介导的抑制性神经回路。8.LFS能诱导WT小鼠产生LTD,但是在σσ1R-KO小鼠LFS不能诱导LTD。bicuculine 能阻断 WT 小鼠的 LTD 诱导。muscimol,NMDA 和 DETA/NO 均可以恢复σ1R-KO小鼠LTD,提示σ1R缺失→减弱GABAA受体介导的抑制性回路→损害LTD的诱导。9.与WT小鼠相比,σ1R-KO小鼠在强迫游泳和悬尾试验中的不动时间明显延长。在σ1R-KO小鼠BLA区注射NMDA、DETA和muscimol可以逆转强迫游泳和悬尾试验中的时间延长,7-NI能拮抗NMDA的作用,提示σ1R缺失→阻碍BLA的LTD诱导→引起抑郁样行为。结论1.BLA谷氨酸神经元的σ1R缺失减少了 NMDA受体的钙离子内流从而降低nNOS与PSD-95结合导致NO生成减少;2.σ1R缺失减少NO的释放从而减少了突触前谷氨酸的释放导致突触传递功能减弱和LTP诱导障碍;3.σ1R缺失减少NO的释放从而减弱GABAA受体介导的抑制性回路导致LTD诱导障碍并发生抑郁样行为。
[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.

【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R749
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本文编号：1832838