【作者】 何婷；

【导师】 陈焕新；

【作者基本信息】 西南大学 ， 基础心理学， 2020， 硕士

【摘要】 目的:抑郁症(major depression disorder,MMD)作为一种以情感持续性低落、思维迟缓、运动性抑制为核心症状的精神疾病,已经是全世界致残和健康不佳的主要原因之一,并且因其治疗效果的不佳严重加剧了对医疗保健系统的社会经济负担,所以当前抑郁症的药物研究和治疗是亟待解决的。众多抑郁症患者被发现前扣带回皮质(anterior cingulate cortex,ACC)的体积明显减少,突触数量减少,突触传递减弱,与其相关神经网路连接减弱,表明前扣带回皮层及其神经网路的连接可能是抑郁症的重要靶点。人类和动物研究表明东莨菪碱(scopolamine)是一种非特异性毒蕈碱胆碱能受体(muscarinic cholinergic receptor,m ACh R)拮抗剂,具有一定的快速抗抑郁作用。在前扣带回皮层中普遍存在毒蕈碱胆碱能受体,其在抗抑郁治疗中的具体作用机制仍然是不明的。内侧前额叶皮质和前扣带皮层的γ-氨基丁酸能中间神经元在重度抑郁症中起着重要作用,但是γ-氨基丁酸能中间神经元的突触活动如何受到m ACh R活动的影响仍有待进一步研究。在本研究中,我们通过利用毒蕈碱(muscarine)和scopolamine来激活或拮抗M型胆碱受体,从而研究探讨m ACh R的活动对前扣带皮层中生长抑制激素(somatostatin,SST)和小清阳性蛋白(Parvalbumin,PV)中间神经元突触传递的影响。实验方法:我们通过基因杂交技术获得转基因SST-cre/Ai9-RFP小鼠和PVcre/Ai9-RFP小鼠,并通过脑片细胞染色和聚合酶链式反应技术(polymerase chain reaction,PCR)确认小鼠种系;借助免疫荧光染色验证记录的SST和PV中间神经细胞及其ACC脑区位置,凭借脑片膜片钳电生理技术毒蕈碱胆碱能受体的激活或拮抗对PV和SST中间神经元的兴奋性突触传递和抑制性突触传递的影响,并且通过对成年期和青春期C57BL/6J小鼠与转基因SST-cre小鼠腹腔注射东莨菪碱(3mg/kg),进行强迫游泳实验,测试小鼠木僵水平,重复东莨菪碱在快速抗抑郁中的行为实验结果。结果:我们发现,在膜片钳电生理实验中,m ACh R的激动剂muscarine对自发性兴奋性突触后电位(s EPSCs)的频率有显著增加的影响(p<0.01),但对微小性兴奋性突触后电位(m EPSCs)的频率和幅值没有影响(p>0.05)。这种作用会被m ACh R的拮抗剂scopolamine所阻断。给药灌流scopolamine对s EPSCs和m EPSCs的频率和振幅影响不显著(p>0.05)。然而,在两种类型的中间神经元中,muscarine会减少诱发性兴奋性突触后电位(e EPSCs)幅值(p<0.01),这伴随着双脉比率的增加变化(p<0.05)。给药灌流muscarine可显著降低SST中间神经元微小性抑制性突触后电位(m IPSCs)和自发性抑制性突触后电位(s IPSCs)的频率(p<0.05),但对PV中间神经元无影响(p>0.05)。Scopolamine能显著增加SST中间神经元m IPSCs和s IPSCs的频率(p<0.05),而对PV中间神经元无明显影响。此外,muscarine降低了PV中间神经元的诱发性兴奋性突触后电位(e IPSCs)幅值(p<0.01),而在SST中间神经元中未见。然而,在SST间神经元中,e IPSCs的配对脉冲比增加(p<0.05),而在PV中间神经元中则未见。在强迫游泳实验中,腹腔注射东莨菪碱可显著降低青春期和成年期小鼠的木僵行为(p<0.01)。此外,在免疫荧光实验和电生理实验中,我们明确了SST-cre/Ai9-RFP小鼠和PVcre/Ai9-RFP小鼠ACC脑区的表达红色荧光蛋白(RFP)SST和PV中间神经元的形态和电生理特性。结论:目前实验结果表明,m ACh R的活动会促进ACC脑区的SST和PV中间神经元的兴奋性突触功能,减弱SST中间神经元的抑制性突触活动,但不显著地影响PV中间神经元,在快速抗抑郁的机制中不同类型的中间神经元可能起不一样的作用。然而Scopolamine通过中间神经元的M型胆碱受体所起到快速抗抑郁作用仍然需要我们更加完善去探究的,从而为当前抑郁症的药物研发和治疗提供一定的思考和依据。更多还原

【Abstract】 Objective:Major depression disorder(MDD)as an emotional persistent low thinking slow motility inhibition as the core symptoms of mental illness,has already been one of the main causes of the world disabled and poor health,and the social and economic burden on the health care system is severely increased because of its poor treatment effect,so the current drug research and treatment of depression has been urgently needed.In many patients with depression,the significant decreased volume of the anterior cingulate cortex,the decreased number of synapses,the decreases in synaptic transmission,and the weakened connections between the anterior cingulate cortex and its neural networks,suggesting that the connections between the anterior cingulate cortex and its neural networks may be important targets for depression.Human and animal researches have indicated that scopolamine is a nonspecific muscarinic cholinergic receptor(m ACh R)antagonist,which has a rapid antidepressant effect.Muscarinic cholinergic receptors are commonly found in the anterior cingulate cortex,and the mechanism of their action in antidepressant therapy is still unknown.GABAergic interneruons in the medial prefrontal cortex and anterior cingulate cortex play an important role in major depression,but how the synaptic activity of GABAergic interneruons is affected by m ACh R activity remains to be further investigated.In this study,we activated or antagonized m-type choline receptors by muscarine and scopolamine,so as to investigate the effect of m ACh R activity on synaptic transmission of both somatostatin(SST)and parvalbumin(PV)containing interneurons in the ACC.Methods: We obtained transgenic SST-cre mice and PV-cre mice by gene hybridization,and identified the mouse germline by brain block cell staining and polymerase chain reaction(PCR).With immunofluorescence staining test target record between SST and PV cells and their position in the ACC,with brain slice of patch clamp electrophysiological technique a muscarinic cholinergic receptor activation or antagonism between the PV and SST neuronal excitatory and inhibitory synaptic transmission,and through adulthood and adolescence C57BL/6J mice with genetically modified(gm)SST-cre mice celiac injection of scopolamine(3 mg/kg),by forced swimming test,tests immobilized behaviors in mice and repeats scopolamine in the rapid antidepressant action experimental results.Results: We found that muscarine,in the patch clamp electrophysiological test,an agonist of m ACh R significantly affected frequency,but not amplitude,of s EPSCs(p < 0.01),but not m EPSCs(p > 0.05),was significantly increased by bath application of muscarine in both SST and PV interneurons.The effect was blocked by scopolamine,an antagonist of m ACh R.Bath application of scopolamine did not have an evident effect on frequency and amplitude of s EPSCs and m EPSCs(p > 0.05).However,muscarine reduced evoked EPSCs in both types of interneurons,which was accompanied by increased changes in the paired-pulse ratio.Bath application of muscarine significantly reduced the frequency of m IPSCs and s IPSCs in SST interneurons(p < 0.01),but not in PV interneurons.Scopolamine significantly increased the frequency of m IPSCs and s IPSCs in SST interneurons(p < 0.05),but not in PV interneurons.Moreover,muscarine reduced evoked IPSCs in PV interneurons(p < 0.01),but not seen in SST interneurons.However,it was an increase in the paired-pulse ratio of evoked IPSCs in SST interneurons(p < 0.05),but not seen in PV interneurons.In forced swimming experiments,intraperitoneal injection of scopolamine significantly reduced immobilized behavior in adolescent and adult mice(p < 0.01).In addition,in immunofluorescence and electrophysiological experiments,we identified the morphological and electrophysiological characteristics of SST-cre/Ai9-RFP mice and PV-cre/Ai9-RFP mice expressing red fluorescent protein(RFP)in ACC brain regions of SST and PV interneurons.Conclusion: Current experimental results indicate that the activity of m ACh R facilitates the function of the excitatory synapses in both SST and PV interneurons in the ACC region,but attenuates the activity of the inhibitory synapses only in SST interneurons without significant effect on that in PV interneurons.Different types of interneurons may play different roles in the mechanism of rapid depression.However,Scopolamine’s rapid antidepressant effect through the m-type choline receptor of interneurons still needs to be further explored,so as to provide certain thinking and basis for drug development and treatment of depression.更多还原