【作者】 陈鹏；

【导师】 周江宁；

【作者基本信息】 中国科学技术大学 ， 神经生物学， 2017， 博士

【摘要】 应激反应是一种高度保守的生理机制,它促使机体能够在不断变化的环境中维持生存。机体调节生理应激反应的最后共同通路是下丘脑和自主神经系统。内侧前额叶负责协调自主神经系统和下丘脑-垂体-肾上腺(HPA)轴来调节情绪反应和执行功能以应对外界环境的刺激。下丘脑促肾上腺皮质激素释放因子(CRF)或激素(CRH)是HPA轴主要的驱动力,在协调神经内分泌应激反应中具有重要的作用。对CRF在大脑分布的解剖学研究显示,除下丘脑外,CRF在中枢神经系统的边缘区域和皮层区域都有广泛的表达。CRF不仅仅是一类神经内分泌应激调节因子,还作为神经调控因子调节大脑的神经活性和应激反应中的情绪和认知功能。目前关于CRF神经元在前额叶(特别是内侧前额叶)对应激反应的调控尚不清楚。本研究采用形态学染色、全细胞膜片钳记录和行为学测试等方法,探究内侧前额叶CRF神经元形成的神经环路在应激反应中的作用及其可能机制。本研究以CRF转基因小鼠为研究对象,发现CRF神经元的胞体主要分布在内侧前额叶的Ⅱ层和Ⅲ层(Layer Ⅱ/Ⅲ),并具有多种形态。根据其电生理特性,CRF神经元被分为四种类型。急性束缚应激提高Ⅱ-b型regular-spiking CRF神经元的神经兴奋性,却不改变Ⅱ-a型regular-spiking CRF神经元的兴奋性。免疫荧光显示急性束缚应激增加内侧前额叶CRF神经元c-Fos蛋白表达。同时急性束缚应激提高内侧前额叶锥体神经元的诱发动作电位速率和静息膜电位,降低阈电流强度。阻断CRF1型受体(CRFR1)不改变正常生理状态下内侧前额叶锥体神经元的兴奋性,但降低急性束缚应激小鼠内侧前额叶锥体神经元的兴奋性。灌流给药50 nM CRF可以模拟急性应激的效应,即提高锥体神经元的神经兴奋性,这种效应可以被CRFR1拮抗剂所阻断。孵育CRF促进磷酸化ERK的表达。上述研究结果表明,急性束缚应激激活内侧前额叶CRF神经元释放CRF作用于锥体神经元细胞膜上CRFR1,进而通过激活胞浆ERK信号通路提高锥体神经元的兴奋性。进一步采用病毒注射方法特异性操控内侧前额叶CRF神经元的活性来探究CRF神经元的行为学功能。发现特异双侧毁损内侧前额叶CRF神经元降低小鼠的社会交往记忆能力,而不改变社会交往能力本身。同时特异性双侧毁损内侧前额叶的CRF神经元增强焦虑样和抑郁样行为,提高小鼠对于低阈值急性应激的敏感性。双侧急性抑制内侧前额叶的CRF神经元具有和特异毁损CRF神经元同样的效果。双侧急性兴奋内侧前额叶的CRF神经元可以降低焦虑样和抑郁样行为,同时双侧慢性兴奋内侧前额叶的CRF神经元可以逆转重复社会击败应激所引起的社会交往能力的下降和焦虑水平的上升。这些结果表明内侧前额叶的CRF神经元在调节应激敏感性和抑郁相关行为方面具有重要的作用。更多还原

【Abstract】 Stress response is a highly conserved physiological process that promotes survival despite uncontrollable and often unpredictable changes in the environment.Hypothalamus and autonomic nervous systems were the final common pathways of physiological stress response.The medial prefrontal cortex plays a key role in coordinating activity between the autonomic and hypothalamic-pituitary-adrenocortical(HPA)axis to regulate both emotional/reactive and executive functions in response to environmental stimuli.CRF(also referred to as corticotropin-releasing hormone-CRIH)in hypothalamus is the major physiological activator of the HPA axis,and coordinates the neuroendocrine response to stress.The anatomical distribution of CRF in the brain suggests that CRF is expressed throughout the central nervous system(CNS)including most limbic and cortical structures besides hypothalamus.CRF not only acts as a key neuroendocrine stress mediator,but is also able to regulate neuronal activity and emotional and cognitive components of the stress response in a neuromodulatory fashion.However,it is not yet known the regulation mechanism of CRF neurons in medial prefrontal cortex in stress response.The present study applied morphological methods,whole-cell patch-clamp recordings and behavioral tests to explore the function and possible mechanism of CRF neural circuit in medial prefrontal cortex in response to stress.In the present study,we observed that the cell bodies of CRF neurons were mainly distributed in layers Ⅱ and Ⅲ(Layer Ⅱ/Ⅲ)of medial prefrontal cortex with a variety of morphological features by using CRF transgenic mice.Based on electrophysiological characteristics of current-evoked firing,CRF neurons could be divided into four categories.Acute restraint stress elevated the neural excitability of one type of regular-spiking CRF neurons in medial prefrontal cortex.By using CRF transgenic mice and immunofluorescent staining,we found that the percentage of the immediate early gene c-Fos positive/CRF positive neurons of CRF neurons in restraint stressed mice was increased significantly.Under acute restraint stress,the rate of evoked action potential and rest membrane potential of pyramidal neurons in medial prefrontal cortex were increased,while the threshold current was decreased.CRFR1,but not CRFR2,antagonists NBI27914 depressed the neural excitability of pyramidal neurons in medial prefrontal cortex of stressed mice.While in normal physiological condition,NBI27914 had no effect on the neural excitability of pyramidal neurons in medial prefrontal cortex.Perfusion of 50 nM CRF could simulate the effect of acute restraint stress by enhancing the neural excitability of pyramidal neurons in medial prefrontal cortex.NBI27914 blocked the effect of perfusion of 50 nM CRF.Phosphorylation level of ERK kinases of neurons in medial prefrontal cortex was increased after CRF treatment.These results suggest that acute restraint stress elevates the level of CRF peptide which was released from CRF neurons in medial prefrontal cortex.The elevated CRF increases the neural excitability of pyramidal neurons through CRFR1 and the downstream ERK signaling pathway.In the following research,to investigate the behavioural function of CRF neurons in medial prefrontal cortex,we used virus delivery method to manipulate CRF neurons.We found that ablation of CRF neurons bilaterally in medial prefrontal cortex impaired social memory while had no effect on social affiliation and sociability.Ablation of CRF neurons bilaterally in medial prefrontal cortex enhanced anxiety-like and depression-like behaviours in elevated plus maze test and forced swimming test and increased stress vulnerability during subthreshold social defeat stress.Acute inhibition of CRF neurons bilaterally in medial prefrontal cortex caused the same effects on anxiety-like and depression-like behaviours with ablation of CRF neurons bilaterally.Acute activation of CRF neurons bilaterally in medial prefrontal cortex decreased anxiety-like and depression-like behaviours in open field test,elevated plus maze test and forced swimming test.Meanwhile,activation of CRF neurons bilaterally in medial prefrontal cortex during repeated social defeat stress could reverse social avoidance and decreased anxiety induced by social defeat stress.Altogether,these results indicate that CRF neurons in medial prefrontal cortex play a key role in regulating stress vulnerability and depression related behaviours.更多还原