【作者】 冯婷；

【导师】 丛斌；

【作者基本信息】 河北医科大学 ， 病理学与病理生理学， 2014， 博士

【摘要】 创伤后应激障碍（Posttraumatic stress disorder，PTSD）是慢性的精神障碍性疾病，病因是由于遭受应激和创伤经历造成的，如家庭暴力、自然灾害或战争等相关的创伤。PTSD是以过度恐惧为特征的，并伴随敏感性增高，从而损害生活质量，并且引起多种疾病、社会和职业问题。目前，人们常把PTSD定义为焦虑障碍性疾病，但大量研究证明PTSD更适合归为学习和记忆障碍类的疾病，因为它包括恐惧记忆的过度巩固和消退的受损。恐惧反应的改变可能导致侵入性记忆和倒叙，增强了回避提示线索和自主觉醒症状。许多研究证明杏仁核是影响PTSD的关键核团。尤其是杏仁核基底外侧核（BLA）对于情感记忆的形成非常重要，在条件恐惧的获得和表达中起重要作用。所以，目前BLA在PTSD的研究中更受关注。在过去的研究中，胆囊收缩素（Cholecystokinin, CCK）被认为是与学习记忆、焦虑和恐惧等脑高级活动有密切关系的神经肽类物质。CCK及其受体在大脑中广泛分布，如，皮质、海马和杏仁核等。过去的一些研究证实了在BLA内的神经元存在高浓度的CCK和CCK mRNA。杏仁核内的CCK阳性神经元对调节锥体神经元的兴奋性和突触的整合具有很强的作用。以往的研究证明激活BLA内的CCK受体可以损伤恐惧记忆的消退。动物模型和人类影像学研究证据表明PTSD的一个潜在机制就是突触可塑性的异常。而先前的研究也已表明CCK可以增强海马的长时程增强并且增强实验动物的空间记忆能力。所以我们有理由认为CCK系统与PTSD的形成机制密切相关。然而，BLA内的CCK系统对PTSD的形成机制的影响尚无深入研究。过去的数据表明PTSD需要从病因研究入手，因为恐惧记忆的起始与巴普洛夫条件恐惧有相似的过程。因此我们建立了环境相关的条件恐惧模型作为PTSD的动物模型。在此模型的基础上我们进一步探讨了BLA内CCK系统在PTSD发生发展过程中的作用及机制，同时在细胞水平上明确CCK受体在引起PTSD相关症状后的相关信号转导机制。以期为临床治疗PTSD选择有效的靶点提供生物学依据。第一部分创伤后应激障碍大鼠杏仁核内CCK系统及相关神经递质的变化目的：以巴甫洛夫条件模型为基础建立与PTSD恐惧症状相关的动物模型；在此模型的基础上观察给予厌恶刺激后内源性CCK系统的变化，包括细胞外和细胞内CCK水平，以及CCK受体；同时也观察了给予厌恶刺激后其他相关的神经递质的变化，包括儿茶酚胺类神经递质及其代谢产物，HPA轴的相关递质，以及与学习记忆相关的神经递质。方法：①建立PTSD的动物模型，通过大鼠对电流强度和电流时间的反应，确立合适的足底电击程序。最后确定为给予大鼠1.2mA的电流，每120秒给予一个60秒的电击，最后180秒自由活动，总共30min。所有的动物被随机分为5组：1天组、7天组、14天组、28天组和对照组；②应用微透析实验收集大鼠模型前及模型后1天和28天在BLA内的细胞外液；③应用高效液相色谱、液质联用和放射免疫技术，检测透析液内各神经递质（包括肾上腺素、去甲肾上腺素、多巴胺、5-HT及其代谢产物，皮质酮和CRH，以及谷氨酸、天冬氨酸和乙酰胆碱）的含量；④应用放射免疫、免疫荧光和免疫组织化学的方法分别观察透析液中CCK的含量，BLA内CCK中间神经元的数量，以及CCK受体的阳性表达。结果：①通过大鼠对电流强度和电流时间的反应，我们确定在给予1.2mA电流强度，10次60秒电击的这种刺激方式下，大鼠不能消退环境相关的恐惧记忆。②在给与大鼠共30min的足底电击程序（1.2mA的电流，每120秒给予一个60秒的电击，最后180秒自由活动）后，大鼠不动时间百分比（freezing%）可以从1天维持到28天并呈持续增长趋势。③BLA内的去甲肾上腺素、多巴胺在电击后1天的水平有所增高，而肾上腺素无明显变化。④BLA内细胞外液的CRH分别在电击后1天和28天均明显高于基础水平。⑤多巴胺的代谢产物DOPAC和HVA，以及5-HT及其代谢产物5-HIAA在大鼠电击前后的BLA内并没有明显变化。⑥谷氨酸、天冬氨酸和乙酰胆碱在电击后在BLA均有明显升高，并且一直伴随着环境相关的恐惧记忆从1天一直持续到28天。⑦电击后1天和28天的BLA细胞外液的CCK水平明显增加；双标的免疫荧光显示了在BLA内含有CCK的神经元在给予足底电击后CCK免疫活性明显比电击前增高；BLA神经元内CCK2受体免疫活性在电击后1天和28天均明显增加，而CCK1受体仅在28天有少量变化。小结：本部分实验成功建立了一个与创伤经验后的联想恐惧有关的PTSD条件性恐惧大鼠模型。证明了应激相关神经递质参与了PTSD形成的起始。同时在条件恐惧的进展过程伴随着CCK及其受体的高水平表达，与此同时与学习记忆相关的神经递质参与了整个PTSD的发展过程。提示内源性CCK系统可能是通过影响恐惧记忆参与了PTSD的形成和发展。第二部分CCK系统在条件性恐惧过程中的作用及其受体机制目的：本部分实验进一步观察内源性CCK是否通过其受体影响了恐惧记忆的获得和表达，以及在该模型下是否改变了BLA内的突触结构可塑性，以明确内源性CCK系统在恐惧记忆的获得和巩固中的作用及机制。方法：①应用在体RNAi技术，分别敲减CCK的1和2受体，观察条件性恐惧大鼠模型的恐惧记忆的获得和巩固。②进一步通过对RNAi的大鼠进行自发活动测试、高架十字迷宫测试及Morris水迷宫测试来判断其在自发活动、焦虑和空间学习记忆上有无变化。③应用高尔基染色的方法观察内源性CCK系统对条件性恐惧大鼠树突棘密度的影响。④应用电镜观察内源性CCK系统对条件性恐惧大鼠杏仁核BLA内突触超微结构的影响。⑤应用液质联用观察内源性CCK系统对条件性恐惧大鼠杏仁核BLA内谷氨酸、天冬氨酸和乙酰胆碱的影响。结果：①CCK1R和CCK2R的siRNA在体干扰效率分别为65%和72%。最佳敲减时间为转染后3天，后续的所有实验均在转染siRNA后3天进行。CCK1R和CCK2R在体干扰后CCK1R和CCK2R的蛋白表达水平明显低于正常对照组，证明CCK受体敲减模型成功。②手术和转染试剂对正常大鼠运动能力和恐惧反应均无影响。③对正常大鼠和BLA内CCK受体基因敲减的大鼠进行条件恐惧实验，发现电击1天或28天后，BLA内CCK2受体敲减后的大鼠在足底电击后1天和28天时不动时间百分比明显降低，说明对BLA内CCK2受体基因敲减能导致大鼠恐惧记忆的巩固受损。同时发现电击后1小时的各组大鼠不动时间百分比无明显变化，以及每组足底电击时间内的每一次电击后的各组大鼠不动时间百分均无明显变化。说明BLA内CCK受体基因的敲减不影响恐惧记忆的获得。④BLA内CCK2受体基因的敲减对动物自发活动无影响；BLA内CCK2受体基因的敲减不能使动物产生焦虑行为；BLA内CCK2受体基因的敲减不能损害动物空间记忆。⑤双侧BLA内注射CCK2受体的siRNA后的大鼠BLA内的神经元与正常大鼠的神经元均排列整齐致密，细胞形态完整，边界清晰，尼氏小体丰富，胞核大而圆，核仁清晰，基本没有观察到神经损伤。⑥遭遇足底电击后大鼠BLA内的兴奋性神经递质尤其是Glu、Asp和Ach均有明显增加，但是仅有Glu和Asp能够在CCK2受体基因的敲减后有所下降。⑦在条件恐惧后28天，大鼠BLA内的树突棘密度和树突长度明显增加，但在BLA内CCK2受体基因敲减后进行条件性恐惧，BLA内的树突棘密度和树突长度与单纯电击组相比明显减少。⑧条件性恐惧对突触形态的影响：条件性恐惧后28天U型突触数量、突触后致密物的厚度及突触前膜囊泡的数量均能显著增加，但BLA内CCK2受体基因敲减后突触形态则无明显变化。小结：本部分实验在第一部分实验建立的条件性恐惧大鼠模型的基础上，观察了CCK系统对大鼠条件性恐惧的影响。研究结果证实，BLA内的CCK2受体是影响条件性恐惧记忆巩固的关键环节，而且是通过改变BLA内突触的结构可塑性而发挥作用的。第三部分CCK2受体参与条件性恐惧过程中恐惧记忆巩固的分子机制目的：本部分研究在细胞水平通过激活原代杏仁核神经元上的CCK2受体，观察其下游信号转导机制，以明确CCK2受体参与条件性恐惧记忆巩固的机制。方法：①培养大鼠原代杏仁核神经元，免疫细胞化学法对神经元进行鉴定，及对原代杏仁核神经元有无CCK及CCK2受体进行鉴定。②shRNA-CCK2R腺病毒载体的构建，及转染到原代杏仁核神经元，应用实时荧光定量PCR检测转染效率。③应用CCK受体的高效激动剂CCK-8激活原代杏仁核神经元的CCK2受体。④应用DiI染色法观察CCK2受体对原代杏仁核神经元树突棘的影响。⑤应用非放射性标记法PKA检测试剂盒，检测CCK2受体激活后对原代杏仁核神经元PKA活性的影响。⑥应用in cell western的方法检测CCK2受体对原代杏仁核神经元内CREB磷酸化的影响。⑦应用钙成像检测CCK2受体对原代杏仁核神经元细胞内Ca2+的影响。⑧应用western blot法检测CCK2受体对原代杏仁核神经元内CamkⅡ磷酸化和突触素的影响。⑨应用western blot和免疫荧光检测CCK2受体对原代杏仁核神经元内PSD95的影响。结果：①用神经元特异性标记物MAP-2多克隆抗体标记神经元，结果显示神经元细胞纯度＞90%；激光共聚焦显微镜下显示，原代杏仁核神经元含有丰富的CCK2受体和CCK阳性表达。②pAD-CCK2R-shRNA构建成功，并且能够应用于原代神经元细胞，最佳MOI值为100时CCK2受体的干扰效率为71.4%。③CCK2受体激活后，原代杏仁核神经元树突的二级分支上的树突棘密度明显增高。④CCK2受体激活后通过使PKA活性升高进一步增加原代杏仁核神经元细胞内CREB磷酸化。⑤CCK2受体激活后能刺激原代杏仁核神经元细胞的内钙释放，而引起细胞内钙增多，从而引起CamkⅡ磷酸化水平增加。⑥CCK2受体激活能增加原代杏仁核神经元细胞内突触素和PSD95的表达从而影响杏仁核神经元突触结构可塑性的变化。小结：本部分实验在体外通过培养大鼠的原代杏仁核神经元，探讨了CCK2受体引起杏仁核神经元突触重塑及结构可塑性的重要机制。CCK2受体可以通过激活cAMP–PKA-CREB及Ga2+–CaMKⅡ-CREB信号转导通路介导PSD95和的突触素翻译增多从而使杏仁核树突棘结构改变。同时也可能由于CCK2受体介导的CaMKⅡThr-262位点的自身磷酸化引起杏仁核神经元突触后致密物质的变化，引起杏仁核神经元突触结构可塑性的改变。结论：本实验系统研究了CCK受体对创伤后应激障碍大鼠恐惧记忆的影响，并对其触发的BLA神经元突触结构可塑性的机制加以深入探讨，得出以下结论：成功建立了一个与创伤经验后的联想恐惧有关的PTSD条件性恐惧大鼠模型，在此模型基础上证明了内源性的CCK系统可能是通过影响恐惧记忆参与了PTSD的形成和发展。并进一步证明杏仁核BLA内的CCK通过CCK2受体影响BLA内神经元的突触重塑及结构可塑性而促进了条件恐惧记忆的巩固，通过对其机制进行深入的探讨，为PTSD的病理生理学机制提供依据，为其临床治疗提供潜在的药物靶标。更多还原

【Abstract】 Posttraumatic stress disorder (PTSD) is a chronic, disabling disorderthat results from exposure to life-threatening trauma. The excessive andcontextually inappropriate triggering of fear memories and associatedbehaviours characteristic of PTSD impairs quality of life. It is thought that thefear response may result in the intrusive memories and flashbacks, enhancedavoidance of reminder cues and autonomic hyperarousal. The initial formationof fear memories similar to those activated by Pavlovian fear conditioning, sowe established an animal model of contextual fear in which the fear response(freezing) was unusually strong and stable. The basolateral amygdala (BLA) iscritical for the formation of emotional memories, and plays an important rolein the acquisition and expression of conditioned fear。A lines of human andnon-human studies suggests that amygdala damage abolishes the developmentof PTSD and the hyperactivity in amygdala plays a causal role in thepathophysiology of PTSD.Cholecystokinin (CCK) is an important neuropeptide that performnumerous regulatory functions in the nervous systems, with particularly highconcentrations distributed throughout the limbic system, such as medialprefrontal cortex, hippocampus and amygdala complex. High concentrationsof CCK or CCK mRNA have been observed in amygdalar neurones.Cholecystokinin-positive interneurons can strongly modulate pyramidal cellexcitability as well as synaptic integration in the BLA.Evidence from animal models and human neuroimaging studies suggeststhat one of the underlying mechanisms of PTSD is aberrant synaptic plasticity.In the nervous system, CCK signaling, activated by two major G-proteincoupled CCK receptors, has been implicated in learning and memory,anxiogenesis, and nociception, and may in addition participate in pathways mediating anxiety and fear. Our earlier work demonstrated that chronicCCK-8treatment has a significant effect on spatial memory and augmentshippocampal long-term potentiation (LTP). Therefore, we predicted thatamygdalar CCK was involved in fear memory in PTSD model as well.However, the neural, synaptoplastic, and molecule mechanisms through whichCCK signalling impairs fear extinction and exacerbates fear responses areunknown.With this in mind, a key challenge in dentifying the etiology of PTSD isto first understand the gentic and celluer systems which regulate fear memoryitselt. One approach to this problem is to fevelop animal models that mightallow us to understand thes fundamental systems. One promising animalmodel focuses on learning of fearful memories. Classic fear conditioning,also known as Pavlovian fear conditioning, is used in our study to discuss therelationship of PTSD and CCK system. In order for the clinical treatment ofPTSDselect a valid target provided biologic evidence.Part1Stress-induced enhancement of fear conditioning activates theamaygdala cholecystokinin system and others neurotransmittersin a rat model of posstraumatic stress dissorderObjective: In this study, we examined if PTSD-like behaviours (strong,persistent contextual fear) are associated with enhanced CCK signalling in theBLA. Indeed, establishment of PTSD-like conditioned fear was associatedwith prolonged upregulation of CCK and CCK receptor expression.Methods:①We developed an animal model of PTSD using multiplefootshocks at1.1mA. All treated rats were randomly assigned to fourexperimental groups, starting1day,7days,14days or28days after theaversive encounter and no shock rats of a control group.②Microdialysiswas used to collect the dialysates with three times:basal transmitter effluxbefore footshock and1day and28day after footshock.③The quantificationof the neurontransmitters in the dialysates was performed by HPLC,HPLC-MS/MS and radioimmunoassays (RIA)；④Quantification ofCCK-like immunoreactivity in the dialysates were analyzed by RIA; Immunofluorescence and immunohistochemical were used to detect CCK andCCK receptors.Results:①Rats show persistent fear when subjected to the strongconditioning protocol, which was exposured to an1.1mA electric footshocks(60s duration) every160s for total of10footshocks.②Rats exposed to ourstrong US protocol maintained their contextual fear memory for a long time.③Significant increases in BLA norepinephrine and dopamine levels wereobserved post-conditioning.④Significantly elevated CRH in the BLA onboth day1and28⑤DOPAC, HVA，5-HT and5-HIAA were notsignificantly increased by the conditioning protocol.⑥The level of Glu, Aspand Ach were increased.⑦There was a significant increase in theextracellular levels of CCK after footshocks1day and28day. Dual labelingCCK immunofluorescence in BLA neurons revealed that an increased CCKimmunoreactivity indicated enhanced synthesis after conditioning compared toa no-shock group. Dual labeling immunhistochemical staining showed thatCCK1and2receptors were densely up expressed in the BLA.Conclusion: In summary, we established an animal model to adressquestions related to the mechanism of PTSD induction. Our results suggestthat stress-induced elevation of NE in the BLA triggers a sustained increase inCCK signalling that may in turn maintain the condition fear response,resulting in PTSD. Our current results provide a framework to further explorethe role of stress hormones and neuropeptides on PTSD induction and theexpression and maintenance of symptoms.Part2Amygdalar CCK2receptor was involved in fear memoryconsolidation in fear conditioningObjective: This part is order to investigate weather endogenous CCKreceptor affected by the acquisition and expression of fear memory. And wealso declosed the the mechanism of acquisition and consolidation withsynaptic plasticity.Methods:①We applied the RNAi to knock down CCK1R and CCK2R,in order to investigate acquisition and consolidation of fear memory in fear conditioning.②Application of locomotor activity, elevated plus maze andMorris water maze is detected locomotor activity, anxiety and learing andmemory in RNAi rats.③The level of Glu, Asp and Ach were examined inour model process.④Synaptic plasticity in structure was examined byGolgi’s stain and electron microscopy.Results:①The rats of CCK2R knock down was not increasefreezing%, compare to nomal rats.②The rats of CCK2R knock down wasnot impaired in locomotor activity, anxiety and learing and memory.③There was no damge in BLA neurons in CCK2R knock down rats.④Synaptic plasticity in structure was increased in BLA neurons.⑤The levelof Glu, Asp were not increased in CCK2R knock down rats after fearconditioning.Conclusion: Our study provides the first behavioral evidence for thepromoting effects of CCK2R on fear memory consolidation, induced byimpact on structure of sunaptic plastic.Part3The mechanisms of CCK2R involved in fear memory consolidationObjective: This part is order to discussion the mechanisms by activationCCK2R in primary amygdala neuron to observe singnal pathway.Methords:①The primary cultured amygdala neurons were culturedfor21d in neurobasal medium with2%B27supplement as describedpreviously. Then, the neurons were identified by immunocytochemistry orimmunofluorescence with antibody against microtubule associated protein-2(MAP2), which is marker for neurons.②The primary cultured amygdalaneurons were transfection pAD-shRNA-CCK2R.③Application of DiI staindetected spine dentsity.④Ca2+signaling in primary neurons was examined.⑤The PKA actity was examined in primary neurons.⑥2The total orphosphorylated protein levels of CamkⅡ, CREB were examined by westernblotting.⑦Syn and PSD95were examinedResults:①Activated CCK2R significantly increassed density ofdendritic spines in the primary amygdala neuron; Knock down CCK2r couldobviously reversed effect in the primary amygdala neuron.②Activated CCK2R can activate PKA activity to affect synaptic plasticity.③CCK2Rleads to increase intracellular calcium through promoting the relases of Ca2+from calciumstore.④CCK2R stimulated phosphorylation, i.e., activation ofCamkⅡ, CREB, and the phosphorylation of these proteins by knock downCCK2R were effectively inhibited.⑤Syn and PSD95were upregulated byactivated CCK2R.Conclusion: The Ga2+–CaMKⅡ-CREB and cAMP–PKA-CREBsignaling pathway activated by CCK2R is involved in amygdara neuronsynaptic plasticity.In summary, our studies provide important views in deeply understandingthe role of CCK2R in fear memory consolidation induced the progress ofPTSD. It will offer a preliminary rationale for studies assessing maintainsPTSD-like symptoms by activated CCK2R enhancing and stabilizing thesynaptoplastic changes in the amygdala mediating contextual fear.更多还原