乙酰胆碱对小鼠嗅球神经环路的调控作用

发布时间：2018-04-20 05:09

本文选题：嗅球 + 乙酰胆碱　；参考：《桂林医学院》2016年硕士论文

【摘要】：目的:乙酰胆碱是中枢神经系统内一种重要的神经递质。基底前脑胆碱能神经元通过释放乙酰胆碱来调节注意、学习和记忆等功能。主嗅球接受来自基底前脑斜角带核水平支大量的胆碱能和GABA能离心纤维的投射,这一投射系统已经被证明在嗅觉信息的处理和嗅觉记忆中发挥至关重要的作用。动物行为学实验表明乙酰胆碱影响动物的嗅觉分辨和嗅觉记忆能力。但是,基底前脑斜角带核水平支胆碱能神经元对嗅球中神经元以及神经环路的调控作用仍然未知。近来研究发现,阿尔茨海默病等神经退行性疾病的患者早期多伴有胆碱能神经元的损伤和嗅觉的减退缺失。因此,了解乙酰胆碱在嗅觉信息处理中所起的作用对探索神经退行性疾病的发病机制有一定的临床意义。本文的主要目的是探索乙酰胆碱对嗅球内部神经环路的作用。方法:通过脑立体定位注射,将腺相关病毒注射到小鼠的基底前脑,待病毒稳定表达后,通过灌流取材和冰冻切片观察胆碱能纤维在嗅球中的分布。采用离体脑片的膜片钳记录,制备厚度为300?m的嗅球脑片用于记录。使用含有生物素的记录电极记录位于嗅小球层的球周细胞和僧帽细胞层的僧帽细胞,再利用给药电极在细胞附近局部喷射乙酰胆碱等药物。记录完成之后,将脑片用4%PFA固定,用Cy3的红色二抗进行免疫染色,用激光共聚焦显微镜扫描图像,确定细胞形态和类型。所有的电生理数据用Clampex10.2软件(Axon)进行分析处理。结果:胆碱能神经纤维在嗅球各层都有投射,主要集中在嗅小球层。局部给予乙酰胆碱使表达VGAT的球周细胞自发放电和诱发放电频率显著增加,并产生内向电流,这一反应可被mAChR的拮抗剂阿托品阻断,突触传递的阻断剂不能消除该反应。尼古丁则对球周细胞无此作用。乙酰胆碱还可通过激活mAChR使球周细胞接受的兴奋性输入的频率增加。嗅球的投射神经元僧帽细胞放电被乙酰胆碱抑制,这种抑制可被GABAA受体的阻断剂和mAChR的拮抗剂阻断。嗅球中另一类多巴胺能的球周细胞,其放电可被乙酰胆碱直接抑制。结论:乙酰胆碱通过激活mAChR从而增加GABA能球周细胞的兴奋性,这一兴奋性的增加不依赖于任何突触传递。乙酰胆碱通过兴奋GABA能球周细胞间接抑制僧帽细胞,从而调节嗅球的信息输出。多巴胺能的球周细胞则受到乙酰胆碱的抑制。乙酰胆碱对嗅球神经环路的调节作用,可能影响到动物的嗅觉分辨和嗅觉记忆能力。
[Abstract]:Objective: acetylcholine is an important neurotransmitter in the central nervous system. Basal forebrain cholinergic neurons regulate attention, learning and memory by releasing acetylcholine. The main olfactory bulb receives a large number of cholinergic and GABA energy centrifugal fibers from the horizontal branch of the basal diagonal band nucleus. This projection system has been shown to play a crucial role in the processing of olfactory information and olfactory memory. Animal behavioral experiments show that acetylcholine affects olfactory resolution and olfactory memory in animals. However, the regulation of horizontal cholinergic neurons on the olfactory bulb neurons and the neural loop is still unknown. Recent studies have found that patients with neurodegenerative diseases such as Alzheimer's disease are associated with cholinergic neuron damage and loss of olfaction. Therefore, understanding the role of acetylcholine in olfactory information processing has a certain clinical significance in exploring the pathogenesis of neurodegenerative diseases. The main purpose of this paper is to explore the effect of acetylcholine on the neural loop in olfactory bulb. Methods: adeno-associated virus was injected into the basal forebrain of mice by stereotactic injection. After stable expression of the virus, the distribution of cholinergic fibers in olfactory bulb was observed by perfusion and frozen sections. Using patch-clamp recording of isolated brain slices, the olfactory bulb slices with thickness of 300 m were prepared for recording. A recording electrode containing biotin was used to record the peribulbar cells located in the olfactory pellet layer and the mitral cells in the mitral cell layer. The acetylcholine and other drugs were sprayed near the cells by the administration electrode. After the recording was completed, the brain slices were fixed with 4%PFA and stained with the red second antibody of Cy3. The images were scanned by laser confocal microscope to determine the morphology and type of cells. All electrophysiological data were analyzed with Clampex10.2 software. Results: cholinergic nerve fibers were projected in all layers of olfactory bulb, mainly in the layer of olfactory bulb. Local administration of acetylcholine significantly increased the frequency of spontaneous and evoked discharges in peripheral cells expressing VGAT and produced inward currents, which could be blocked by atropine, an antagonist of mAChR, which could not be eliminated by synaptic transmitters. Nicotine had no such effect on peribulbar cells. Acetylcholine can also increase the frequency of excitatory inputs accepted by peribulbar cells by activating mAChR. The firing of mitral cells of olfactory bulb projecting neurons was inhibited by acetylcholine, which could be blocked by antagonists of GABAA receptor and mAChR. In the olfactory bulb, another type of dopaminergic peribulbar cells whose discharges are directly inhibited by acetylcholine. Conclusion: acetylcholine increases the excitability of GABA peracyclic cells by activating mAChR, which is independent of any synaptic transmission. Acetylcholine regulates the information output of olfactory bulb by stimulating GABA and inhibiting mitral cells indirectly. Dopaminergic peribulbar cells were inhibited by acetylcholine. The effect of acetylcholine on olfactory bulb loop may affect olfactory resolution and olfactory memory.
【学位授予单位】：桂林医学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R741

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