【作者】 张丽君；

【导师】 田波；

【作者基本信息】 华中科技大学 ， 神经生物学， 2022， 博士

【摘要】 背景:条件性恐惧（Fear conditioning system,FCS）可以帮助学者们用认知科学的方法来研究大脑是如何处理恐惧这种情绪状态,所以FCS既是研究恐惧学习的行为学范式,也是研究焦虑和抑郁等的行为学范式。情绪应激可以强烈地作用于认知过程而干扰恐惧学习,并促进恐惧相关精神疾病的发生和发展。因此,研究情绪应激与FCS的关系以已成为生命科学研究的热点之一。在本研究中,我们重新设计了三箱-替代性社交失败（Three chamber-vicarious social defeat stress,3C-VSDS）模型,以研究长期情绪应激作用下的小鼠在FCS中特异的神经环路和神经机制。FCS的环路机制研究已经将多巴胺（Dopamine,DA）系统确定为大脑中参与FCS的重要组成部分,但DA的来源尚不完全清楚。未定带（Zona incerta,ZI）近年来成为人们关注和研究的焦点。ZI内侧边界有一群DA能神经元,在FCS学习中被广泛的关注。ZI还含有丰富的γ氨基丁酸（Gamma-aminobutyric acid,GABA）能神经元,是迄今为止还未被充分认识的重要抑制性核团。但是ZI^DA能神经元和ZI^GABA能神经元在FCS中是否起作用至今还未被研究。目的:在C57BL/6J小鼠和3C-VSDS小鼠中,研究ZI及其神经环路在FCS学习过程中的作用,发现调节FCS的新环路,为阐明情绪应激及应激相关障碍的神经机制提供新的思路。方法:（1）C57BL/6J小鼠进行FCS检测,包括适应、习得、提取、消退、再提取五个阶段。基于Cre/Lox P原理,使GCa MP6m病毒分别在小鼠左侧ZI^DA能神经元和右侧ZI^GABA能神经元中表达,体内光纤钙成像的方法同时记录左侧ZI^DA能神经元和右侧ZI^GABA能神经元在FCS过程中的钙信号变化,并记录小鼠在FCS中的行为学表现,观察ZI^DA能神经元和ZI^GABA能神经元与FCS的相关性。（2）恐惧习得CS阶段分为CS早期（1-14s）和CS后期（15-28s）,在CS后期（15-28s）抑制ZI^DA能神经元,观察对FCS的影响;利用顺行示踪和逆行示踪的方法,找到ZI^DA能神经元的下游核团基底内侧杏仁核（Basomedial amygdala,BMA）,同样在恐惧习得阶段CS后期（15-28s）抑制ZI-BMA的多巴胺能轴突末梢,观察对FCS的影响,验证ZI^DA能神经元以及ZI-BMA多巴胺能神经环路在FCS中的作用。（3）体内光纤钙成像的方法观察3C-VSDS模型小鼠ZI^DA能神经元在FCS中的作用。在恐惧习得阶段CS后期（15-28s）激活3C-VSDS小鼠ZI-BMA的多巴胺能轴突末梢,观察对FCS的影响。结果:（1）C57BL/6J小鼠FCS过程中,恐惧习得阶段CS后期（15-28s）,ZI^DA能神经元的活性逐渐增加,而ZI^GABA能神经元的活性没有明显的变化,提示ZI^DA能神经元可能与FCS的恐惧习得有关,尤其是CS后期（15-28s）恐惧预期阶段。（2）在CS后期（15-28s）抑制双侧ZI^DA能神经元能够阻碍小鼠FCS过程。顺行示踪和逆行示踪的方法确定BMA是ZI^DA能神经元的下游靶点,通过光遗传学的病毒策略抑制双侧ZI-BMA的多巴胺能轴突末梢也可以阻碍FCS过程。（3）3C-VSDS小鼠表现出明显的FCS学习障碍,经过CS和US的配对,3C-VSDS小鼠的僵直率明显的低于对照组。同时3C-VSDS小鼠ZI^DA能神经元的活性在CS后期（15-28s）也明显的低于对照组,提示3C-VSDS模型小鼠的FCS学习障碍可能与ZI^DA能神经元CS后期（15-28s）的低活性有关。在CS后期（15-28s）激活双侧ZI-BMA的多巴胺能轴突末梢能够改善3C-VSDS小鼠的FCS学习障碍。结论:C57BL/6J小鼠和3C-VSDS模型小鼠的FCS恐惧学习都需要ZI^DA能神经元以及ZI-BMA多巴胺能神经环路的参与,该环路是调节FCS恐惧学习尤其是恐惧预期的潜在靶点。ZI^DA能神经元的低活性是3C-VSDS模型小鼠的FCS学习障碍的原因之一。激活ZI-BMA多巴胺能神经环路可以挽救3C-VSDS模型引起的FCS学习障碍。更多还原

【Abstract】 Background:Fear conditioning system（FCS）has helped scholars to use cognitive science methods to study how the brain processes fear as an emotional state,therefore,FCS is not only a model for studying fear learning,but also a model for studying emotional stress-related disorders such as anxiety and depression.Emotional stress can strongly affect the cognitive process,interfere with the aversive learning,so that promote the occurrence and development of fear-related disorders.Therefore,the relationship between emotional stress and FCS has become one of the hotspots in life science.In this study,we redesigned three-chamber vicarious social defeat stress（3C-VSDS）model to investigate the circuit mechanism of FCS under chronically emotional stress.Decades of research on the neuronal circuits involved in FCS have identified dopamine（DA）neuron systems as the main brain structures associated with FCS,the source of DA is not clear.Zona incerta（ZI）has become the focal point of research in recent years.At the medial border of the ZI lies a group of DA neurons known as the A13 cell population.ZI also contains abundant gamma-aminobutyric acid（GABA）neurons,which is an important inhibitory nucleus that have not been fully recognized so far.However,whether DA neurons and GABA neurons of ZI are involved in FCS is unknown.Objective:To study the role of ZI and its neural circuits in the process of FCS in C57BL/6J mice and 3C-VSDS mice,discover a new circuit for regulating FCS,so as to providing a new idea to elucidate the neural mechanism of emotional stress and stress-related disorders.Methods:（1）All wild-type mice were subjected to FCS procedure,which included five stages of Habituation,Conditioning,Retrieval,Extinction and Recall.Based on the Cre/Lox P system,GCa MP6m virus were expressed in the left DA neurons while the same virus was expressed in the right GABA neurons of the ZI region.In vivo calcium fiber photometry was used to record the activity of ZI^DA neurons and ZI^GABA neurons respectively.At the same time,the behavioral performance of experimental mice in FCS paradigm was also recorded,and the correlation of ZI^DA neurons and ZI^GABA neurons and fear learning were evaluated.（2）We divided CS during conditioning procedure into early stage of CS（1-14s）and late stage of CS（15-28s）.We inhibit ZI^DA neurons at the late stage of CS（15-28s）to observe the effect on FCS.The downstream basomedial amygdala（BMA）of ZI^DA neurons were found by anterograde and retrograde tracing method.Similarly,we inhibit the dopaminergic（DAergic）axon terminals of ZI-BMA at the late stage of CS to observe the effect on FCS,so as to verify the role of ZI^DA neurons and ZI-BMA DAergic circuit in FCS.（3）In vivo calcium fiber photometry was used to measure the activity of ZI^DA neurons during FCS in 3C-VSDS mice,behavioral performance in FCS was also recorded.We activate the DAergic axon terminals of ZI-BMA at the late stage of CS to observe the effect on FCS.Results:（1）During the Conditioning phase in wild-type mice,the activity of ZI^DA neurons increasingly in late stage of CS,while the activity of ZI^GABA neurons did not change.This suggests that ZI^DA neurons may be related to Conditioning phase in FCS,especially in the late CS stage.（2）Inhibition of ZI^DA neurons bilaterally in the late stage of CS blocked process of FCS in mice.BMA was identified as the downstream target of ZI^DA neurons by anterograde and retrograde tracing method,and inhibition of the DAergic axon terminals of ZI-BMA could also block FCS procedure.（3）The 3C-VSDS model mice showed obvious FCS learning deficit,the freezing level in the 3C-VSDS model mice was significantly lower than that in the control group after CS and US pairing.At the same time,the activity of ZI^DA neurons in the late CS stage of 3C-VSDS mice was also significantly lower than that in the control group.These results suggest that the FCS learning deficit in 3C-VSDS mice may be related to the low activity of ZI^DAneurons during late stage of CS.Activation of the dopaminergic（DAergic）axon terminals of ZI-BMA could ameliorates FCS learning deficits in 3C-VSDS mice.Conclusion:FCS learning in both wild-type and 3C-VSDS mice requires ZI^DA neurons and the DAergic projection of ZI-BMA circuit,which is a potential target for regulating FCS learning.The low activity of DAergic projection of ZI-BMA circuit is at least one of the reasons for the 3C-VSDS-induced FCS learning deficits.Activation of DAergic projection of ZI-BMA circuit can rescue the FCS learning deficit caused by 3C-VSDS model.更多还原