【作者】 张娟；

【导师】 汪凯；

【作者基本信息】 安徽医科大学 ， 神经病学， 2024， 博士

【摘要】 背景:抑郁症(Major depressive disorder,MDD)是一种常见的精神疾病,可以影响人的情感、思维和日常行为,甚至可能导致自杀。电休克治疗(Electroconvulsive therapy,ECT)是目前治疗重型抑郁症的有效方法之一。大量临床研究表明,ECT可改善抑郁患者抑郁症状,并改变治疗后奖赏相关环路的内在神经活动。奖赏环路的异常可能是介导抑郁发作重要的神经机制之一。其中伏隔核(Nucleus Accumbens,NAc)是控制奖励反应、动机和情绪的关键脑区,内侧前额叶(The medial prefrontal cortex,mPFC)通过“上-下”的调控作用参与调节情绪、动机和行为等。奖赏环路正常化可能在ECT改善抑郁中起关键作用。目前关于ECT抗抑郁治疗具体的神经机制,多集中于微观层面,鲜有文献在动物实验中从介观层面探索ECT抗抑郁作用的具体机制。目的:本研究基于前额叶-伏隔核神经环路在抑郁发生中的重要性。假设:前额叶皮层-伏隔核神经环路可能参与了ECT抗抑郁过程。本研究目的:采用临床实验结合动物实验,来探索mPFC-NAc神经环路是否参与ECT抗抑郁过程中的作用及机制。从介观层面解析ECT抗抑郁的机制可能为临床优化治疗提供新思路。方法:(1)研究一:我们招募了45例重症抑郁患者,所有人在ECT治疗前、后都接受了一系列的神经心理学评估和功能磁共振扫描。我们采集患者基线伏隔核与背侧前额叶的功能连接,利用支持向量回归分析(Support vector machine regression,SVR)来预测ECT的疗效。(2)研究二:利用不可逃避足部电击建立习得性无助(Learned Helplessness,LH)大鼠模型,以及可逃避足部电击来建立主动穿梭回避(Two-Way Shuttle Avoidance,TWSA)大鼠模型,探究不同应激条件下大鼠的行为表现并结合相关行为学筛选出习得性无助大鼠。(3)研究三:对习得性无助大鼠模型,进行动物电休克治疗(Electroshock therapy,ECS),结合糖水偏好实验和强迫游泳等一系列行为学验证ECS是否改善大鼠抑郁样行为。(4)研究四:采用免疫荧光技术检测ECS治疗后抑郁组及对照组大鼠mPFC、NAc内即早基因蛋白c-Fos表达与分布;通过脑内立体定位注射Muscimol分别局部抑制mPFC、NAc脑区神经活动,观察两脑区在ECS改善大鼠抑郁样行为中的作用;最后使用化学遗传实验,在mPFC脑区注射h M4D(Gi)病毒,ECS治疗前30分钟在NAc脑区给予CNO,抑制mPFC-NAc神经环路,验证mPFC-NAc神经环路是否参与ECS改善习得性无助大鼠抑郁样行为。(5)研究五:利用蛋白质免疫印迹(Western blotting,WB)技术来观察ECS治疗后模型组及对照组大鼠mPFC、NAc、海马和杏仁核脑区α-氨基-3-羟基-5-甲基-4-异噁唑吁基丙酸受体(Alpha-amino-3-hydroxy-5-methyl-4-isoxazolysopypropionic acid receptor,AMPAR)亚基(GluA1、GluA2、总的S831磷酸化GluA1、总的S845磷酸化GluA1)、N-甲基-D-天冬氨酸受体(Nmethyl-d-aspartate receptor,NMDAR)亚基(NR1、NR2A、NR2B)、PSD95及synaptophysin蛋白表达水平;采用全细胞膜片钳实验记录mPFC及NAc脑区细胞兴奋性。结果:(1)研究一:抑郁患者经ECT治疗后HRSD评分较治疗前显著降低。以基线伏隔核与背侧前额叶的功能连接作为特征,发现右侧伏隔核与右背侧前额叶的功能连接可较好预测ECT后HRSD的改善率。(2)研究二:本研究发现不可逃避的足部电击可以诱导大鼠产生习得性无助行为,表现为高逃避失败率。与此相反,在主动穿梭回避的训练条件下,大鼠表现出较高主动回避率,说明可以通过行为训练改善或预防习得性无助的产生。(3)研究三:本研究发现ECS不影响习得性无助大鼠习得性无助行为,但ECS可有效改善习得性无助大鼠在糖水偏好实验中的糖水偏好率、强迫游泳实验中的不动时间、高架十字迷宫中的开臂次数和进入开臂潜伏期,说明ECS可有效改善习得性无助大鼠抑郁样行为。(4)研究四:本研究发现习得性无助大鼠mPFC和NAc脑区c-Fos表达显著增高,ECS治疗后mPFC和NAc脑区c-Fos表达显著降低;局部抑制NAc脑区可阻断ECS改善习得性无助大鼠糖水偏好指数,局部抑制mPFC脑区可阻断ECS改善习得性无助大鼠糖水偏好指数和强迫游泳实验的不动时间;下调mPFC-NAc神经元活动能够有效阻断ECS改善习得性无助大鼠糖水偏好指数和强迫游泳实验的不动时间。(5)研究五:本研究发现ECS治疗后模型组和对照组大鼠mPFC、NAc、海马和杏仁核脑区AMPAR亚基(GluA1、GluA2)、NMDAR亚基(NR1、NR2A、NR2B)蛋白表达降低,但不影响PSD95及synaptophysin蛋白表达;ECS治疗可增加习得性无助大鼠mPFC神经元兴奋性。结论:临床研究证明了基线右侧伏隔核与右背侧前额叶的功能连接可用来预测ECT干预抑郁的疗效;动物研究表明ECS可改善习得性无助大鼠抑郁样行为,mPFCNAc神经环路参与了ECS改善习得性无助大鼠抑郁样行为中的作用;并伴随mPFC、NAc、海马以及杏仁核脑区AMPAR亚基(GluA1、GluA2)、NMDAR亚基(NR1、NR2A、NR2B)蛋白表达降低及mPFC神经元兴奋性增加。综上,本研究通过临床实验及动物实验揭示了mPFC-NAc神经环路以及突触相关蛋白在电休克治疗抑郁症中的重要性。更多还原

【Abstract】 Background:Major depressive disorder(MDD)is a common mental illness that affects an individual’s emotions,thinking,and daily behavior,and can even lead to suicide.Electroconvulsive therapy(ECT)is currently one of the effective treatments for severe depression.Numerous clinical studies have shown that ECT can improve depressive symptoms in patients and alter the intrinsic neural activity of reward-related circuits after treatment.Abnormalities in the reward circuit may be one of the key neural mechanisms mediating depressive episodes.The nucleus accumbens(NAc)is a critical brain area that controls reward response,motivation,and emotion,while the medial prefrontal cortex(mPFC)participates in regulating emotions,motivation,and behavior through top-down control.Normalization of the reward circuit may play a crucial role in the improvement of depression through ECT.Currently,research on the specific neural mechanisms of ECT’s antidepressant treatment is mostly focused at the microscopic level,with few studies exploring the specific mechanisms of ECT’s antidepressant effects at the mesoscopic level in animal experiments.Objectives:This study is based on the significance of the prefrontal cortex-septal nuclei neural circuit in the onset of depression.Hypothesis: The prefrontal cortex-nucleus accumbens neural circuit may be involved in the antidepressant process of ECT.Purpose of the study: To explore whether the mPFC-NAc neural circuit participates in the antidepressant process of ECT,this research combines clinical trials with animal experiments to investigate the roles and mechanisms involved.Analyzing the mechanisms of ECT’s antidepressant effects from a mesoscopic perspective may provide new insights for optimizing clinical treatments.Methods:Study One: We recruited 45 patients with severe depression,all of whom underwent a series of neuropsychological assessments and functional magnetic resonance imaging(fMRI)scans before and after treatment with ECT.We collected baseline functional connectivity data between the nucleus accumbens and the dorsal prefrontal cortex of the patients.Using support vector machine regression(SVR),we predicted the therapeutic efficacy of ECT.Study Two: Utilizing inescapable foot shocks,we established a rat model of learned helplessness(LH)and a model of active two-way shuttle avoidance(TWSA)using escapable foot shocks.We investigated the behavioral responses of rats under different stress conditions and combined this with behavioral screenings to identify rats exhibiting learned helplessness.Study Three: In a learned helplessness rat model,electroshock therapy(ECS)was applied,combined with a sucrose preference test and forced swim test among other behavioral assessments,to verify whether ECS improves depressive-like behaviors in rats.Study Four: Using immunofluorescence technology,we assessed the expression and distribution of the immediate early gene protein c-Fos in the mPFC and NAc of rats in the depression group and control group post-ECS treatment.Intracerebral stereotaxic injections of Muscimol were used to locally inhibit neuronal activity in the mPFC and NAc,observing the role of these brain areas in ECS-mediated improvement of depressive-like behaviors in rats.Lastly,a chemogenetic experiment was conducted by injecting the h M4D(Gi)virus into the mPFC and administering CNO in the NAc 30 minutes before ECS treatment to inhibit the mPFC-NAc neural circuit,confirming its involvement in the amelioration of depressive-like behaviors in learned helplessness rats.Study Five: Western blotting(WB)was employed to observe changes in protein expression levels in the mPFC,NAc,hippocampus,and amygdala of model and control rats post-ECS treatment.This included the subunits of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor(AMPAR)(GluA1,GluA2,total S831 phosphorylated GluA1,and total S845 phosphorylated GluA1)and subunits of the Nmethyl-D-aspartate receptor(NMDAR)(NR1,NR2 A,NR2B),as well as PSD95 and synaptophysin.Whole-cell patch clamp experiments were conducted to record the excitability of cells in the mPFC and NAc brain regions Results:Study One: After treatment with ECT,patients with depression exhibited significantly reduced Hamilton Depression Rating Scale(HRSD)scores compared to before treatment.Utilizing the functional connectivity between the nucleus accumbens and the dorsal prefrontal cortex as a feature,it was found that the connectivity between the right nucleus accumbens and the right dorsal prefrontal cortex could effectively predict the improvement rate of ECT.Study Two: This study observed that inescapable foot shocks could induce learned helplessness behavior in rats,characterized by a high rate of escape failure.In contrast,under conditions of active shuttle avoidance training,rats exhibited a higher active avoidance rate,suggesting that behavioral training could improve or prevent the development of learned helplessness.Study Three: This study found that ECS does not affect the learned helplessness behavior in rats,but ECS effectively improves the sucrose preference rate in the sucrose preference test,immobility time in the forced swim test,the number of entries into the open arms,and the latency to enter the open arms in the elevated plus maze.These findings suggest that ECS can effectively alleviate depression-like behaviors in rats with learned helplessness.Study Four: We observed a significant increase in c-Fos expression in the mPFC and NAc of LH rats,which significantly decreased after ECS treatment.Local inhibition of the NAc blocked the improvement in sucrose preference index by ECS,and local inhibition of the mPFC blocked both the improvement in sucrose preference index and the reduction in immobility time in the forced swim test by ECS.Downregulation of neuron activity in the mPFC-NAc pathway effectively blocked the improvements in sucrose preference index and immobility time in the forced swim test achieved by ECS.Study Five: Post-ECS treatment,we found reduced expression of AMPA receptor subunits(GluA1,GluA2)and NMDA receptor subunits(NR1,NR2 A,NR2B)in the mPFC,NAc,hippocampus,and amygdala of both model and control rats,without affecting PSD95 and synaptophysin expression.ECS treatment increases the excitability of neurons in the mPFC of rats with LH.Conclusions:Clinical studies have demonstrated that the functional connectivity between the right septal nucleus and the right dorsolateral prefrontal cortex at baseline can be used to predict the therapeutic efficacy of ECT in depression.Animal studies indicate that ECS can improve depression-like behaviors in rats with learned helplessness,with the mPFC-NAc neural circuit playing a role in this improvement.This is accompanied by reduced protein expression of AMPAR subunits(GluA1,GluA2)and NMDAR subunits(NR1,NR2 A,NR2B)in the mPFC,NAc,hippocampus,and amygdala,as well as increased excitability of neurons in the mPFC.In summary,through clinical and animal experiments,this study has revealed the importance of the mPFC-NAc neural circuit and synaptic proteins in the electroconvulsive treatment of depression.更多还原