杏仁核不同神经元集群在大鼠PTSD样行为中的作用

发布时间：2018-03-19 02:02

本文选题：创伤后应激障碍　切入点：单次长时程应激　出处：《天津医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:本课题采用单次长时程应激(single prolonged stress,SPS)作为创伤后应激障碍模型,综合行为学、化学遗传学方法、免疫荧光等技术手段观察杏仁核不同的神经元集群在大鼠PTSD样症状中的作用及相关神经生物学机制,旨在为开发新的PTSD临床治疗手段和方案提供理论依据。方法:首先,采用SPS模型进行造模,将SD大鼠随机分成未造模对照组,SPS模型造模后1d组,造模后14d组,利用旷场实验、高架十字迷宫实验检测大鼠PTSD的焦虑样行为改变,利用免疫荧光技术检测杏仁核的c-fos蛋白表达变化,并用c-fos来表征脑区的激活,探究杏仁核的激活在大鼠PTSD样行为发生中的作用。其次,采用化学遗传学方法,即DREADDs技术(designer receptors exclusively activated by designer drugs)来抑制杏仁核脑区,检测其是否影响PTSD的发生。通过脑区注射腺相关病毒,将改造的人M4毒蕈碱受体hM4Di表达在大鼠杏仁核CeA脑区,并通过腹腔注射hM4Di的配体氯氮平-N-氧化物(clozapine-N-oxides,CNO)来实现杏仁核的抑制。采用SPS模型进行造模,利用旷场实验、高架十字迷宫检测PTSD的焦虑样行为学改变,验证杏仁核参与PTSD样症状的发生。最后,利用免疫荧光技术检测BLA和CeA的c-fos表达变化,并用来表征其脑区激活;同时应用谷氨酸能神经元的分子标签CaMKII和GABA能神经元的分子标签GAD67和c-fos共标来表征不同的神经元集群的激活情况,探究相关机制。结果:1.同未经SPS造模的对照组大鼠相比较,SPS造模后14d的大鼠在旷场中央区和高架十字的开臂停留时间均显著的缩短(P0.05),SPS造模后1d组没有明显差异,表明SPS可诱导大鼠产生延迟性的焦虑样行为。c-fos免疫荧光结果显示在CeA脑区,同未经SPS造模的对照组大鼠相比,造模14d后c-fos的表达含量增高(P0.05),但是与造模后1d的大鼠相比,没有明显差异。而BLA脑区神经元集群的激活模式未发现任何显著性改变。2.利用DREADD技术抑制杏仁核CeA脑区,同其他未干预组相比,SPS造模后14d大鼠在旷场中央区和高架十字的开臂停留时间均显著的增加(P0.05),表现出抗焦虑。3.大鼠在SPS模型后,在免疫荧光技术检测试验中,GAD67+c-fos阳性/c-fos阳性的比值显示在CeA脑区,同未造模动物相比,造模14天后GAD67+c-fos阳性/c-fos阳性的比值明显增高[F(2,16)=13.06,P0.001,],有统计学意义,但是造模后第1天没有表现明显差异,而BLA脑区GABA能神经元集群的激活模式未发现任何显著性改变。CaMKⅡ+c-fos阳性/c-fos阳性的比值显示在CeA脑区和BLA脑区,谷氨酸能神经元集群的激活模式都未发现任何显著性改变。结论:1.大鼠PTSD样行为的发生同时伴随有中央杏仁核的激活,这可能参与了PTSD发生。2.大鼠中央杏仁核CeA脑区的抑制,出现抗焦虑作用,中央杏仁核参与PTSD的发生。3.中央杏仁核的GABA能神经元的激活比例的失调很可能参与了PTSD样行为的发生。其因果联系需要进一步的化学遗传学进一步证明。
[Abstract]:Objective: this topic by a single minister Cheng Yingji (single prolonged stress, SPS) as a model of posttraumatic stress disorder, comprehensive behavioral methods, chemical genetics, immunofluorescence technique was used to observe the different clusters of amygdala neurons in rat PTSD like symptoms in use and bioneurological mechanism, aiming to provide a theoretical basis for the development of the new PTSD clinical treatment methods and programs. Methods: firstly, SPS model is used to model, SD rats were randomly divided into non model control group, SPS model group 1D, model group 14d, using the open field test, the change of anxiety like behavior in the elevated plus maze test in rats PTSD to detect the expression of amygdala, c-fos protein by immunofluorescence technique, and using c-fos to characterize the activation of brain regions, the activation of the amygdala in rats on PTSD like behavior. Secondly, using chemical genetics Methods, namely DREADDs Technology (designer receptors exclusively activated by designer drugs) to suppress the amygdala, detect whether or not influence the occurrence of PTSD. Through the brain injection of adeno-associated virus, the transformation of the human M4 muscarinic receptor hM4Di expression in the amygdala of CeA rat brain, and the ligand clozapine were intraperitoneally injected with hM4Di (-N- oxide clozapine-N-oxides, CNO) to achieve the amygdala inhibition by SPS model. Modeling, using the open field test, the change of anxiety like behavior in the elevated plus maze test PTSD, test PTSD like symptoms in the amygdala. Finally, the BLA expression of CeA and c-fos by immunofluorescence technique, and used to characterize the brain activation at the same time; application of glutamatergic neurons and molecular labels CaMKII and GABA neurons GAD67 and c-fos labeled molecular tags to neuron clusters of different characterization of the bowel Live, to explore the related mechanism. Results: 1. without rats compared with the control of SPS, SPS after the model of 14d rats were significantly reduced in the open arm time, open field and elevated central cross (P0.05), there was no significant difference in SPS model group 1D, showed that SPS rats can induce delayed anxiety like behavior of.C-fos immunofluorescence results showed CeA in brain regions, without SPS model control group rats compared with model 14d expression increased the content of c-fos (P0.05), but with the mice in 1D rats compared to no significant difference. BLA clusters of neurons in brain activation patterns did not find any significant change in.2. using DREADD technology to inhibit amygdala CeA brain regions, compared with other non intervention group, after SPS 14d rats in the open arm time central open field and elevated plus were significantly increased (P0.05),.3. exhibited anti anxiety rats in SPS In the model, immunofluorescence assay test, the ratio of GAD67+c-fos positive /c-fos positive display in the CeA brain regions, compared with an animal model, modeling the ratio of GAD67+c-fos positive /c-fos positive after 14 days were significantly increased [F (2,16) =13.06, P0.001, have statistical significance, but not first days after modeling the obvious difference, while the GABA BLA brain neurons cluster activation patterns did not find any significant change in the ratio of.CaMK II +c-fos positive /c-fos positive CeA and BLA displayed in the brain areas of the brain, glutamate can activate the pattern of neuronal clusters are found no significant changes. Conclusion: 1. rat PTSD like behavior at the same time accompanied by the activation of the central nucleus of the amygdala, which may be involved in the suppression of PTSD central amygdala CeA cortex of.2. rats, have anti anxiety effects, the central nucleus of the amygdala is involved in the development of PTSD.3. GABA central amygdala neurons The maladjustment of the activation ratio of the element is likely to be involved in the occurrence of PTSD like behavior. The causal link requires further evidence of chemical genetics.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R749.5

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