【作者】 陈晨；

【导师】 朱东亚；

【作者基本信息】 南京医科大学 ， 药理学， 2015， 博士

【摘要】 恐惧是一种由现实或想象中的威胁诱发的情绪行为,是人类情感的正常组成部分。适度的恐惧反应能警示动物面对类似于先前经历的有害环境,及时评估潜在的危险并做出保护反应,而病理性恐惧记忆的产生将会导致创伤后应激综合征（posttraumatic stress disorder,PTSD）、惊恐障碍（panic disorder）、恐惧症（phobia）等恐惧相关疾病。流行病学研究显示,普通人群PTSD的终生患病率约为5%-10%,曾遭受创伤事件（如战争）的特定人群PTSD的患病率高达10%-40%。近年来,随着突发事件的增加,PTSD等恐惧相关疾病的发生率明显增加,给个人、家庭及社会带来沉重的负担。然而,恐惧相关疾病的治疗仍面临巨大挑战。一方面,恐惧记忆获得极快,可以长期稳定存在,并且容易泛化;另一方面,目前临床采用的基于恐惧消退原理的暴露疗法仍存在众多缺陷,如起效慢,治疗效果有限,且疗效不能维持等。因此,研究病理性恐惧记忆消退机制对PTSD等恐惧相关疾病的治疗具有重要意义。基于巴甫洛夫条件反射原理建立的经典的条件性恐惧（Cued-fear conditioning）以及在此基础上发展出的背景关联恐惧（Contextual fear conditioning）是最常见研究恐惧相关疾病的动物模型,广泛应用于恐惧学习和记忆的神经、分子机制研究。海马是大脑边缘系统结构之一,在情感和认知功能中具有重要作用。既往研究表明,海马与PTSD发生、发展及治疗密切相关,其中包含海马CA3、DG的三突触通路对于背景关联型恐惧记忆至关重要。然而,海马在恐惧记忆消退中的作用及神经、分子学机制所知甚少。神经元型一氧化氮合酶（neuronal nitric oxide synthase,nNOS）是中枢神经系统中主要的NO合酶,在脑边缘系统中大量表达,参与调控多种情感和认知行为。药理学及遗传学研究发现,nNOS及NO信号通路在恐惧记忆的形成和消退中扮演重要角色。nNOS N-端的PDZ（post-synaptic density protein discs-large,ZO-1）结构域可以与突触后密度蛋白95（post-synaptic density protein 95,PSD-95）结合形成复合蛋白nNOS-PSD-95。PSD-95是谷氨酸能突触后密集区的核心脚手架蛋白,连接突触后膜受体蛋白与胞内蛋白,介导谷氨酸能突触可塑性,对突触功能、树突棘发生、生长等起调控作用,与阿尔茨海默症、精神分裂症、亨廷顿病以及PTSD的发生密切相关。研究表明,PSD-95是恐惧记忆形成、稳定及消退过程的一个关键调节分子。另外,N-甲基-D-天冬氨酸（N-methyl-D-aspartic acid,NMDA）受体介导的突触可塑性是恐惧记忆消退的核心机制,我们实验室研究发现,NMDA受体激活诱导nNOS-PSD-95耦联,那么nNOS-PSD-95相互作用可能在调节恐惧记忆消退中起重要作用。为证明此推测,本课题设计两部分研究内容:1)研究nNOS-PSD-95耦联在恐惧记忆消退中的作用;2)探讨NOS-PSD-95耦联调控恐惧消退的分子机制。第一部分我们采用背景关联型恐惧记忆模型进行恐惧消退训练,结果发现背景关联型恐惧消退后背侧海马nNOS-PSD-95耦联水平显著下降。恐惧记忆检测后立即腹腔注射nNOS-PSD-95耦联阻断剂ZL006,在消退训练前预先给药7d,其后在每次消退训练后立即给药,显著加快条件性恐惧和背景关联型恐惧消退。为了明确阻断nNOS-PSD-95耦联作用于恐惧消退的具体阶段,我们分别在消退训练前连续7d预先给予ZL006,或每次消退训练后立即给予ZL006,进行行为学检测。结果显示,仅在消退训练前给予ZL006显著促进背景关联型恐惧消退,而在消退训练期间给予ZL006并不影响恐惧消退。说明阻断nNOS-PSD-95耦联抑制消退记忆的获得。另外,在恐惧训练前,腹腔注射ZL006,并不影响两种恐惧记忆获得。为了进一步研究海马调控恐惧消退的关键亚区,我们通过微量给药系统,分别在背侧海马CA1、CA3、DG区阻断nNOS-PSD-95耦联,观察对恐惧消退的作用。行为学检测发现,采用背景关联型恐惧记忆模型,在海马CA3区给予ZL006、解耦联多肽Tat-nNOS_1-133,以及构建重组病毒LV-nNOS_1-133-GFP阻断nNOS-PSD-95耦联,均显著加快背景关联型恐惧消退。在nNOS-/-小鼠CA3区给予ZL006,对背景关联型恐惧消退的促进作用消失。而在CA1或DG区给予Tat-nNOS1-133或ZL006并不影响背景关联型恐惧记忆消退。另外,在CA3区给予LV-nNOS1-133-GFP不影响背景关联型远期恐惧记忆的消退。采用条件性恐惧记忆模型,在小鼠CA3区给予ZL006同样显著加快条件性恐惧消退。结果说明,海马CA3区nNOS-PSD-95耦联调控恐惧记忆的消退,阻断CA3的nNOS-PSD-95耦联显著促进恐惧记忆消退。第二部分我们采用背景关联型恐惧记忆模型,初步探讨了阻断nNOS-PSD-95耦联促进恐惧消退的分子机制。蛋白免疫印迹分析（Western blot,WB）结果显示,背景关联型恐惧消退训练显著上调背侧海马BDNF表达水平。消退训练前,在背侧海马CA3区给予LV-nNOS_1-133-GFP或连续四天给予ZL006,行为学检测发现在消退训练第一（E1）或（E2）,阻断nNOS-PSD-95耦联显著加快恐惧消退。于E1（ZL006）或E2(LV-nNOS_1-133-GFP)后1h取CA3组织进行检测。WB结果显示,阻断nNOS-PSD-95耦联显著增加BDNF表达。同样实验方案,在nNOS-/-小鼠CA3区给予ZL006对BDNF表达无影响。为了证明BDNF确实是其机制,采用微量给药系统,在消退训练过程中于CA3区给予BDNF清除剂TrkB-FC,行为学检测发现,TrkB-FC完全逆转LV-nNOS_1-133-GFP对恐惧消退的促进作用。另外,给予特异性TrkB受体拮抗剂ANA-12,阻断BDNF下游信号通路,与TrkB-FC作用一致,行为学结果显示,ANA-12逆转了LV-nNOS_1-133-GFP对恐惧消退的促进作用。以上结果说明,BDNF上调介导nNOS-PSD-95解耦联对背景关联型恐惧记忆消退的促进作用。HDAC2通过表观遗传学机制调控BDNF表达,而我们实验室前期研究发现nNOS-PSD-95解耦联能调控HDAC2表达和活性。因此,我们还进一步研究了HDAC2是否参与BDNF介导的nNOS-PSD-95耦联对恐惧记忆消退的调控。蛋白免疫印迹分析（Western blot,WB）及免疫荧光（Immunofluorescence,IF）结果显示,背景关联型恐惧消退训练显著下调HDAC2表达。采用与BDNF相同的研究方式,发现给予ZL006阻断nNOS-PSD-95耦联显著抑制HDAC2表达。同样实验方案,在nNOS-/-小鼠CA3区给予ZL006,WB及免疫荧光检测发现,对nNOS-/-小鼠CA3区HDAC2表达无影响。为了明确HDAC2在恐惧消退中的重要作用,我们采用重组病毒AD-HDAC2-flag过表达CA3区HDAC2,与对照组给予无活性片段（AD-HDAC2（7989-1）-flag）相比,不仅逆转ZL006对恐惧消退的促进作用,且消退水平显著低于溶剂对照组。以上结果表明,海马CA3区HDAC2下调介导nNOS-PSD-95解耦联对背景关联型恐惧记忆消退的促进作用。综上所述,海马CA3区nNOS-PSD-95蛋白相互作用负调控恐惧记忆消退;HDAC2下调和BDNF上调共同参与nNOS-PSD-95解耦联对恐惧记忆消退的促进作用。更多还原

【Abstract】 As a normal part of human emotions,fear is a mental state induced by an actual or imaginary threat.Learning to fear allows animals to assess the risk of situations in time and organize appropriate defensive behaviors to threats.However,pathological fear memories may lead to fear-related disorders,including posttraumatic stress disorder（PTSD）,panic disorder and phobia.According to epidemiological data,lifetime prevalence rates of PTSD have been estimated at 5-10%in the community,and around 10-40%in specific trauma populations（such as veterans）.In recent years,there has been a sensible rise in the rates of PTSD along with the increased traumatic events.The cost of such disorder on the individual and society can be tremendous.Yet treatment of fear-related disorders is a considerable challenge insofar.Fear memories are rapidly acquired,temporally enduring,and can generalize across contexts.Moreover,exposure therapy based on fear extinction is often slow to develop,short lived,and context dependent.Given the high rates and therapeutic status of fear-related disorders,there is an urgent need for a deeper understanding of the pathological fear mechanisms.In the laboratory,Pavlovian fear conditioning and contextual fear conditiong are powerful animal models of fear-related disorders for understanding the underlying mechanisms of pathological fear memories.As one of components of the limbic system,the hippocampus plays an important role in mood and cognitive disorders.Previous work has shown that hippocampus is involved in the development and treatment of PTSD.The trisynaptic excitatory circuits including CA3 in hippocampus is necessary for contextual fear conditioning.Yet the precise hippocampus circuits and mechanisms involved in fear extinction remain poorly understood.Neuronal nitric oxide synthase（nNOS）,a main NO synthase in the central nervous system,is enriched in the limbic system.Pharmacological and genetic studies reveal that nNOS and NO signaling may contribute to fear acquisition and extinction.Post-synaptic density protein-95（PSD95）,a multivalent excitatory synaptic scaffolding protein and core component of the post-synaptic density,can link to nNOS via direct PDZ-PDZ domain,forming nNOS-PSD-95 complex.PSD-95 has an integral functional role within postsynaptic machinery mediating glutamatergic plasticity.Recent studies indicate that PSD-95 is a key contributor to the regulation of synaptic functions critical for fear memory formation,stability and extinction.NMDA receptor-mediated synaptic plasticity may be the core mechanism of PTSD.In addition,We find that the nNOS-PSD-95 interaction is related to NMDAR activation.Therefore,it is possible that nNOS-PSD-95 interaction may be implicated in regulation of fear extinction.Accordingly,we investigated（1）whether hippocampal nNOS-PSD-95 coupling regulates fear extinction;（2）the molecular mechanism underlying regulation of fear extinction by nNOS-PSD-95 coupling.In part one,we began by testing the contribution of nNOS-PSD-95 coupling to fear memory extinction processes.To this end,we subjected adult mice to contextual fear extinction trials.These mice was sacrificed 1h after the last extinction trial and assessed the nNOS-PSD-95 complex levels in dorsal hippocampus by Coimmunoprecipitation.We found contextual fear extinction significantly decreased nNOS-PSD-95 complex levels.To investigate whether nNOS-PSD-95 coupling regulates fear extinction,immediately after memory test,we treated mice with a small molecule ZL006（20mg/kg,i.p.）for 7 days before extinction trials and immediately after each extinction trial to block nNOS-PSD-95 binding.The data showed that systemic administration of ZL006 facilitated both contextual fear extinction and cued fear extinction.To examine the real phase of extinction regulated by nNOS-PSD-95uncoupling,conditioned mice were respectively treated with ZL006 for 7 days before extinction,or immediately after each extinction trial.We found that treatment of ZL006 before extinction facilitated contextual fear extinction,while within extinction sections had no effects.Otherwise,treatment of ZL006（20mg/kg,i.p.）for 7 days before fear conditioning had no influence on contextual-fear and cued-fear acquisition.To explore the key subregion of hippocampus involved in fear extinction,we disrupted the nNOS-PSD-95 interaction respectively in dorsal hippocampal CA1,CA3 and DG by implanting microcannula into these regions.Three agents including a small molecule named ZL006,a small peptide named Tat-nNOS1-133 and a lentiviral vector called LV-nNOS_1-133–GFP were applied in researches to block the interaction of nNOS with PSD95.Behavior results revealed that mice treated with ZL006 in CA3showed a significant reduction of freezing time in contextual fear extinction.Similar results were acquired in Tat-nNOS_1-133-treated and LV-nNOS_1-133–GFP-treated mice.But the effect of ZL006 in CA3 disappeared in nNOS gene knock-out（nNOS-/-）mice.Interestingly,no reduction of freezing behavior was observed in mice injected with Tat-nNOS_1-133-133 in CA1 or ZL006 in DG.Notably,treatment with LV-nNOS_1-133–GFP did not affect extinction of remote contextual fear memory.Otherwise,injection of ZL006 in CA3 also facilitated cued-fear extinction.In conclusion,these observations suggest that nNOS-PSD-95 coupling in hippocampal CA3 negatively regulates fear extinction via affecting acquisition of extinction.In part 2,we explored the possible mechanism by which nNOS-PSD-95uncoupling regulates contextual-fear extinction.Firstly,we divided conditioned mice equally into two groups after contextual fear conditioning,no-extinction and extinction group.Western blot was carried out to detect the protein expression in dorsal hippocampus.Compare to no-extinction group,contextual fear extinction trials led to high levels of BDNF.Next,we subjected mice to contextual fear conditioning procedure,and infused LV-nNOS1-133–GFP into hippocampal CA3 by microcannula before extinction trials.Infusion of LV-nNOS_1-133–GFP diminished freezing behavior at the first extinction trial（E1）.Then the mice were killed 1h after E1 and the expression of BDNF in CA3was measured by WB.As expect,treatment with LV-nNOS_1-133–GFP induced BDNF overexpression.Similar results were obtained when mice were injected with ZL006.As previous,conditioned mice were injected with ZL006 for four days.Freezing behavior was remarkably declined at E2.Then we killed mice 1h after E2 to assess BDNF level.Consistent with the effect of LV-nNOS_1-133–GFP,mice displayed enhanced BDNF level in CA3.Meanwhile,the expression of BDNF in nNOS-/-mice treated with ZL006 is not affected.These data indicate that BDNF may account for regulation of contextual-fear extinction by nNOS-PSD-95 uncoupling.To exclude the possibility that upregulation of BDNF contributes to the modulation of extinction by nNOS-PSD-95 uncoupling,we explored TrkB-FC,a BDNF scavenger,into CA3 within extinction trials.The enhancement of intra-CA3LV-nNOS_1-133–GFP in fear extinction was completely reversed by TrkB-FC.Moreover,the effect of ANA-12,a specific TrkB receptor antagonist,was similar to that of TrkB-FC.Thus,BDNF does be a key regulator implicating in the role of nNOS-PSD-95 coupling in contextual-fear extinction.BDNF expression may also be regulated by HDAC2 under epigenetic mechanisms.Our previous studies demonstrated that nNOS-PSD-95 disassociation led to changes in HDAC2 expression and activity.To gain further insight into the underlying mechanisms of fear extinction,we investigated the HDAC2 role in the regulation of extinction by nNOS-PSD-95 uncoupling.As previous procedure,conditioned mice were equally divided into two groups,no-extinction and extinction group.WB and immunofluorescence assay were performed to detect the HDAC2expression.Compared to no-extinction,contextual-fear extinction training downregulated HDAC2 in dorsal hippocampus.Next,we examined the effect of nNOS-PSD-95 disassociation on HDAC2expression.We subjected mice to contextual fear conditioning and microinjected ZL006 into CA3 for four days before extinction training.Behavior results showed a significant decline at E2 in ZL006-treated mice.Then we prepared hippocampal lysates at 1h after E2.The HDAC2 level in ZL006-treated mice was only 30%of controls.In addition,WB and immunofluorescence assay revealed that ZL006 was ineffective in nNOS-/-mice.To further confirm the role of HDAC2 in nNOS-PSD-95 uncoupling regulation,we used adenoviral vectors expressing HDAC2,named AD-HDAC2-flag,and inactive HDAC2 extract,named AD-HDAC2（7989-1）-flag.Conditioned mice were microinfused with AD-HDAC2-flag or AD-HDAC2（7989-1）-flag,and systemic administration with ZL006 for seven 7days before extinction.We observed the enhancement of ZL006 in fear extinction was completely reversed by AD-HDAC2-flga.Moreover,compared to vehicle controls,AD-HDAC2-flag even significantly inhibited fear extinction.Taken together,HDAC2 downregulation and BDNF upregulation in hippocampal CA3 contribute to the promotion of fear extinction by nNOS-PSD-95 disassociation.Overall,our data indicate that nNOS-PSD-95 coupling in hippocampal CA3negatively regulates fear extinction.HDAC2 downregulation and BDNF upregulation account for the promotion of fear extinction by nNOS-PSD-95 disassociation。更多还原