【作者】 关晓伟；

【导师】 李胜男；

【作者基本信息】 南京医科大学 ， 药理学， 2009， 博士

【摘要】 研究背景可卡因是一种具有兴奋中枢神经系统作用的局部麻醉药。因为它可以引起精神兴奋、产生快感,所以成为吸毒者滥用的毒品之一。近年来,随着实验手段的飞跃发展,科研工作者从神经心理学、神经生理学、发育生物学、生物化学和分子生物学等各个方面对可卡因的神经毒性作用机制进行了广泛深入的研究。可卡因滥用可导致中枢的镐赏和动机相关的神经环路发生可塑性变化,提示可卡因成瘾是一种生理功能和精神紊乱疾病。目前,有关可卡因神经毒性作用的脑区的研究主要集中在多巴胺能神经通路。其中,中脑-边缘叶通路（由中脑腹后被盖区投射到腹侧纹状体和边缘系统）,中脑-大脑皮质通路及黑质-纹状体通路为研究的主要靶点。这三条通路中,纹状体成为重要的交通关联要道。目前公认的可卡因毒性机理多巴胺学说认为:可卡因及其代谢产物主要通过阻断多巴胺转运体的活动,引起突触间隙多巴胺聚集增多,进而改变突触前后多巴胺受体活动发挥神经毒性作用的。但有关滥用可卡因如何引起神经环路发生适应性变化的分子调控机制的研究还十分贫乏。近年,胞外信号调节蛋白激酶（Extracellular signal-regulated protein kinase,ERK）,尤其是ERK1/2在可卡因成瘾中的作用引起了广泛关注。ERK1/2是一种在体内广泛分布的信号激酶,对机体的发育,生长,细胞的迁移和成熟具有重要的调控作用,参与多种神经精神性疾病的病理变化。有报道,特异性抑制ERK1/2激活可以阻断可卡因诱发的条件偏爱行为,也可以减弱可卡因引起的皮质树突形态学改变,提示ERK1/2是可卡因引发神经环路发生适应性改变的重要的调控因子。目前对ERK1/2在可卡因毒性作用机制的研究主要应用慢性可卡因给药动物模型。值得注意的是,可卡因引起的ERK1/2的激活呈一过性变化,所以应用急性可卡因给予模型探讨ERK1/2的时程变化和机制研究十分必要。有报道,急性给予可卡因可引起腹侧纹状体ERK1/2的激活,认为这种ERK1/2活性的变化是引发慢性可卡因毒性作用的启动调控因子。但腹侧纹状体又分为壳和核两部分,这两部分接受的纤维投射和功能均不同,所以有必要观察在可卡因给予模型中的壳和核两部分ERK1/2表达和功能的变化。另外,近年来背侧纹状体在可卡因成瘾机制的研究中也渐渐受到重视。但在可卡因成瘾过程中,有关背侧纹状体中ERK1/2的表达变化,变化机制及功能意义还缺乏系统的研究。同其它滥用药物相似,可卡因滥用的主要特征表现为长期依赖和反复复发。有的患者在长期戒断后,由于环境或心理的诱因导致再次滥用,这提示可卡因成瘾机制中包含学习记忆强化过程。长时程增强（Long term potentiation, LTP）与长时程抑制（Long term depression, LTD）被认为是形成学习记忆过程的分子机制之一。目前多用LTP和LTD模型进行可卡因引发学习记忆行为改变的机制研究。有报道,滥用可卡因可引起海马的神经环路LTP可塑性变化,近而引起陈述性记忆的紊乱。还有研究表明可卡因可引起皮质纹状体depotentiation过程减弱,进而损伤习惯性学习记忆行为。可卡因是通过何种机制影响海马和皮质纹状体通路的LTP或LTD变化?有哪些调控因子参与这一过程?罕有报道回答这些问题。值得注意的是,很多患者的复吸诱因是应激情绪。而且,药物滥用后的戒断反应也是一种应激状态。所以,应激与药物成瘾之间可能共享一些相同的机制。促肾上腺皮质激素释放因子（corticotrophin-releasing factor, CRF）相关肽是参与哺乳动物应激反应的主要神经肽。据报道,CRF肽类家族主要通过与相应的七个跨膜片断的G-蛋白耦联CRF受体相结合（CRF₁和CRF₂）,调节机体内分泌,应激反应等生理功能。几十年来,科研工作者已大量报道HPA轴的CRF相关肽及其受体的变化对可卡因发挥毒性作用的重要性。但越来越多的证据表明,丘脑外的CRF相关肽及其受体在中枢分布广泛,并且在生理和病理状态发挥着强大的调控作用。那么在可卡因成瘾过程中,丘脑外的CRF相关肽及其受体是否参与可卡因诱发的学习记忆行为改变呢?目前有关这个问题的研究还是个空白。研究目的1.观察急性可卡因处理对背侧纹状体中p-ERK1/2表达的影响,时程和定位;探讨背侧纹状体中ERK1/2的活动对可卡因诱发的行为功能变化的影响;探讨可卡因引发背侧纹状体中p-ERK1/2改变的上游机制;探讨可卡因引发背侧纹状体中p-ERK1/2改变的下游因子。2.探讨在可卡因给予过程中,腹侧纹状体ERK1/2对可卡因引发的转录因子的变化的影响;探讨可卡因诱发的p-ERK1/2在腹侧纹状体Core与Shell不同通路的作用。3.观察慢性可卡因处理及可卡因戒断对海马CA1区LTP的影响;探讨CRF相关肽及其受体在这一过程中的作用。4.观察急性可卡因处理,慢性可卡因处理及可卡因戒断对皮质纹状体通路LTP的影响;探讨CRF相关肽及其受体在这一过程中的作用。研究方法1.急性（10分）可卡因给予,慢性（连续14d或连续7d）可卡因给予,可卡因短期戒断（3d）和长期戒断（14d）的动物模型。2.免疫组织化学和免疫荧光双标:观察p-ERK1/2在纹状体的分布和定位。3. Western Blots:观察p-ERK1/2和t-ERK1/2, c-Fos, p-CREB, p-Elk-1的表达。4. RT-PCR:观察背侧纹状体（伏隔核）c-fos mRNA的含量。5.全细胞膜片钳记录:观察海马CA1锥体神经元和背侧纹状体中小棘神经元的静息膜电位和输入阻抗。6.脑片的场电位记录:观察海马Scharffer通路和皮质纹状体通路LTP的情况。实验结果1.急性给予可卡因引起背侧纹状体p-ERK1/2表达增多,而对t-ERK1/2的表达没有影响。2.急性给予可卡因诱发的p-ERK1/2具有时间依赖性,给药后5分种出现,10分种达到高峰,20分种恢复至正常水平。3.急性给予可卡因5分种后,背侧纹状体中的p-ERK1/2主要表达在中小棘神经元的胞浆中;10分种后,主要出现在胞核中。4.特异性阻断ERK1/2激活可部分削弱急性可卡因诱发的运动增多行为。5.急性给予可卡因诱发的p-ERK1/2主要表达在背侧纹状体中多巴胺I型受体标记的中小棘神经元,极少量表达在多巴胺II型受体标记的中小棘神经元。6.特异性阻断多巴胺I型受体可以完全抑制急性可卡因诱发增多的背侧纹状体p-ERK1/2;特异性阻断多巴胺II型受体可以部分抑制急性可卡因诱发增多的背侧纹状体p-ERK1/2。7.特异性阻断ERK1/2激活可以部分降低急性可卡因诱发的背侧纹状体c-Fos表达增多;部分降低急性可卡因诱发的CREB激活;完全抑制急性可卡因诱发的背侧纹状体Elk-1激活。8.急性给予可卡因引起腹侧纹状体（伏隔核）的c-Fos蛋白和mRNA表达增强;特异性阻断ERK1/2活性可以完全降低急性可卡因诱发伏隔核的c-fos mRNA增多,部分降低壳区的c-Fos蛋白增多,完全降低核区增多的c-Fos蛋白。9.慢性可卡因给予和短期可卡因戒断对海马CA1区的锥体神经元的基本电生理特性（静息膜电位、输入阻抗）没有影响。10.急性与慢性给予可卡因对海马Scharffer通路的LTP没有影响;短期给予可卡因戒断可增强海马CA1区LTP的幅度。11.体外给予CRF₁特异性拮抗剂于海马脑片,可以阻断短期可卡因戒断组及其对照组的LTP幅度; CFR1特异性拮抗剂的存在情况下,短期可卡因戒断组来源的海马脑片的LTP幅度与对照组相比,仍然具有显著意义。12.体外给予CRF₂特异性拮抗剂于海马脑片,可以部分阻断短期可卡因戒断组海马CA1区的LTP幅度;对对照组来源的海马脑片的LTP幅度没用影响。13.急性、慢性可卡因给予和长期可卡因戒断对背侧纹状体中小棘神经元的基本电生理特性（静息膜电位、输入阻抗）没有影响。14.急性、慢性可卡因给予和长期可卡因戒断对皮质纹状体通路的LTP振幅没有影响。15.体外给予皮质纹状体脑片20-80 nM的CRF孵育,可以显著性增强可卡因戒断组及其对照组来源的皮质纹状体通路的LTP振幅;可卡因戒断处理后,增加的幅度显著高于对照组。16.特异性阻断CRF₁可以削弱CRF在可卡因戒断组和对照组皮质纹状体脑片诱发增加的LTP振幅。17.特异性阻断CRF₂可以削弱CRF在可卡因戒断组皮质纹状体脑片诱发的增加的LTP振幅;对照组皮质纹状体脑片的CRF诱发的增加的LTP振幅没有影响。18.给予皮质纹状体脑片80 nM的urocortin 2 （UCN2）孵育,可以显著增加可卡因戒断组来源皮质纹状体脑片的LTP振幅;对照组来源的皮质纹状体脑片的LTP振幅没有影响。19.特异性阻断CRF₂可以抑制UCN2在可卡因戒断组皮质纹状体脑片诱发的增加的LTP;对照组皮质纹状体脑片的LTP未受影响。实验结论1.可卡因激活的ERK1/2是可卡因滥用引起背侧纹状体发生神经适应性变化的重要调控因子,同时对可卡因诱发的行为学异常具有重要的调控作用。多巴胺I型受体是可卡因在背侧纹状体激活ERK1/2的主要上游因子;而可卡因激活的ERK1/2可通过增加转录因子（如c-Fos,CREB和Elk-1）的表达或活性发挥其调控作用。2. ERK1/2是可卡因处理引起腹侧纹状体（伏隔核）活动增强的重要调控因子。相对于伏隔核的壳区,ERK1/2的这种调控作用在伏隔核的核区更为明显。3.可卡因戒断可引发海马CA1区LTP幅度增强。CRF₁和CRF₂均参与可卡因戒断所诱发的海马CA1区LTP增强。其中,CRF₁既参与正常生理条件下又调控可卡因戒断所触发的病理条件下海马CA1区LTP的形成。CRF₂对可卡因戒断过程中海马CA1区LTP的调控作用呈特异性特征,提示可卡因戒断可以通过改变CRF₂的功能活动来引发海马的可塑性改变,最终导致学习记忆的行为改变。4.急性、慢性可卡因给予和可卡因戒断均不能改变皮质纹状体通路的LTP的强度。外源性CRF或UCN2可以显著性增强可卡因戒断来源皮质纹状体脑片的LTP振幅,这一过程是CRF₂特异性介导的。更多还原

【Abstract】 BackgroundCocaine is one of commonly abused drugs due to its easy-getting and strongly addictive effects. Last several decades, scientists did a lot of work to investigate the mechanisms of cocaine addiction. Exposure to cocaine causes plasticity in neural circuits related to reward and motivation, supporting the idea that addiction is a physiological and psychological disorder.Till now, most studies focus on the role of dopaminergic system in cocaine addiction. Mesolimbic pathway （originating from Ventral Tegmental Area to limbic area）, mesocortical pathway and substantia nigra-striatum pathway have been regarded as key neural targets for mediating neuroadaptation induced by cocaine abuse. Notably, striatum is the essential bridge among the three pathways. By binding to the dopamine transporter （DAT）, cocaine blocks the reuptake of released dopamine （DA）, causing a long-lasting rise of synaptic DA concentrations. Accordingly, DA receptors can be activated for a long time during and after cocaine administration. The molecular mechanism of the neuroadaptations in neural circuits caused by cocaine abuse remains largely unknown. Recently, the roles of extracellular signal-regulated protein kinase （ERK）, expecially ERK1/2, in cocaine addiction attract much attention. ERK1/2 is one of important kinases that is widely distributed in the brain, and plays an important role in cell migration and matures, and is involved in mediating the physiological changes in biological disorder. Exposure to cocaine has been reported to induce ERK1/2 activation in the dopaminergic system. Importantly, blockade of ERK1/2 pathway has been reported to attenuate abnormal behaviors associated with cocaine treatment, including hyperlocomotor activity and conditioned place preference （CPP）, suggesting that ERK1/2 pathways may play an important role in incubation of cocaine addiction. Notably, most previous studies focused on the role of ERK1/2 in long-term changes induced by chronic drug exposure in the brain. ERK1/2 was significantly activated only at the early time point after acute cocaine treatment, and it returned to normal level after repeated cocaine administration, indicating that early ERK1/2 activation may be critical for the subsequent long-term changes after cocaine exposure. However, how ERK activation leads to long-term changes remains unclear. Thus, it is essential to identify the mechanisms of ERK1/2 activation after acute cocaine treatment. It has been reported that acute cocaine treatment upregulates the level of p-ERK1/2 in ventral straitum （nucleus accumbence, NAc）. However, NAc is divided into shell and core. The core and shell regions might mediate distinct processes associated with cocaine-induced behavioral abnormalities, thus it is important to investigate the function of cocaine-induced p-ERK1/2 in the core and shell of NAc, respectively. In addition, the dorsal striatum attracts more and more attention about its roles in cocaine addiction. However, the studies about the function and mechanisms of ERK1/2 in dorsal striatum need systematic investigation.Similar to other abused drugs, main characteristics of cocaine addiction are compulsive drug use despite adverse consequences and high rates of relapse during periods of abstinence. Environment changes or emotional problem easily lead to cocaine relapse after long term withdrawal, indicating that a pathological process of learning and memory is involved in formation of cocaine addiction. Long term potentiation （LTP） in hippocampus has been an important and useful cellular model of synaptic plasticity and is considered as one of the cellular substrates of learning and memory. Previous studies showed that LTP in hippocampus is significantly enhanced after cocaine self-administration, and this enhancement even persists after extinction of cocaine administration. Cocaine withdrawal is reported to impair the habit learning and memory process by attenuating the depotentiation in corticostriatal synapses. However, the mechanisms by which cocaine abuse leads to the electrophysiological changes in hippocampus and corticostriatal synapses remain unclear. Stress is a well-known risk factor for drug abuse and relapse. On the other hand, stress can be a result of cocaine abuse, especially during cocaine withdrawal. More recent efforts have begun to identify the relationships between neural activity during stress and drug relapse outcomes. Corticotrophin-releasing factor （CRF）-related peptides are primary regulator of the stress response. Till now several CRF-related peptides have been identified: CRF, urocortin 1 （UCN1）, UCN2 and UCN3. These CRF-related peptides actions are mediated through two different G-protein-coupled receptors, CRF₁ and CRF₂. Last decades, abundant evidences showed that the CRF related peptides and their receptors in HPA play an important role in cocaine addiction. However, recent studies find that CRF related peptides and their receptors are widely distributed outside the thalamus, and this extra-thalumal CRF system carry out important regulating roles under both physiological and pathological conditions. Till now, no data have been reported about the roles of CRF related peptides and their receptors in cocaine-induced changes of learning and memory.Aim1. The first aim of this study is to investigate the expression, time course and location of p-ERK1/2 in dorsal striatum after acute cocaine treatment and identify the effects of ERK1/2 on cocaine-induced hyperlocomotor behavior. Furthermore, it is to investigate the upstream and downstream of cocaine-induced p-ERK1/2.2. The second aim of this study is to determine the respective effect of ERK1/2 in the shell and core of NAc on cocaine-induced changes in c-Fos expression.3. The third aim of this study is to investigate the changes of the LTP in hippocampal CA1 slices from chronic cocaine treatment rats and cocaine withdrawal rats. Furthermore, it is to investigate the roles of CRF related peptides and their receptors in the process.4. The last aim of this study is to investigate the changes of LTP in corticostriatal slices from acute cocaine treatment rats, chronic treatment rats and cocaine withdrawal rats. Furthermore, it is to identify the roles of CRF related peptides and their receptors in the process.Methods1. Setting up different cocaine treatment models, including acute cocaine treatment （one single injection）, chronic cocaine treatment （consective 7d or 14d）, short term cocaine withdrawal treatment （3d withdrawal after chronic cocaine treatment） and long term cocaine withdrawal treatment （14d withdrawal after chronic cocaine treatment） models.2. Immunostaining: to observe the distribution and location of p-ERK1/2 in striatum.3. Western blots: to observe the expression of p-ERK1/2, t-ERK1/2, c-Fos, p-CREB, p-Elk-1.4. RT-PCR: to observe the level of c-fos mRNA in ventral striatum （nucleus accubence, NAc）5. Whole-cell patch clamp: to observe the resting membrane potaentials （RMP） and input resistance of pyramid neuron in hippocampus and medium spiny neurons （MSNs） in striatum. 6. Field EPSP recordings: to observe the LTP of hippocampal CA1 slices and corticostriatal slices.Results1. Acute cocaine injection results in up-regulation of p-ERK1/2 in dorsal striatum, but has no effects on t-ERK1/2.2. The level of acute cocaine-induced p-ERK1/2 is time-dependent; the level of p-ERK1/2 is increased at 5 min, and reaches to a peak at 10 min, then returns to basal level at 20 min after acute cocaine injection.3. The p-ERK1/2 positive staining is located in the cytosotic part of medium spiny neurons （MSNs） at 5 min after acute cocaine injection, and is mainly detected in the nucleus of MSNs at 10 min after cocaine treatment.4. Specific blockade of ERK1/2 activation partially attenuates acute cocaine-induced hyperlocomotor behavior.5. Acute cocaine-induced p-ERK1/2 is mainly located in dopamine D1 receptor （D1R）-positive MSNs, and little is collocated with D2R in dorsal striatum.6. Specific blockade of D1R significantly inhibits acute cocaine-induced p-ERK1/2 in dorsal striatum, and the similar results occur in the presence of specific antagonist for D2R.7. Specific blockade of ERK1/2 activation significantly attenuates acute cocaine-enhanced c-Fos, p-CREB, and p-Elk-1 in dorsal striatum.8. Acute cocaine injection increases the level of c-Fos protein and c-fos mRNA in nucleas accubence （NAc）; specific blockade of ERK1/2 activation significantly attenuates acute cocaine-induced c-fos mRNA in NAc, and partially reduces cocaine-induced c-Fos protein in the shell, but completely reduces cocaine-induced c-Fos protein in the core of NAc. 9. There is no effect of chronic cocaine treatment and cocaine withdrawal on the basic properties of hippocampal CA1 pyramid neurons （RMP and input resistance）.10. The magnitude of LTP in hippocampal CA1 is significantly enhanced by cocaine withdrawal, but not affected by chronic cocaine treatment.11. Specific blockade of CRF₁ in vitro significantly attenuates the magnitude of LTP in hippocampal slices from controls and cocaine withdrawals; In the presence of CRF₁ antagonist, the magnitude of LTP in hippocampal slices from cocaine withdrawal groups is still greater than that from controls.12. Specific blockade of CRF₂ in vitro significantly attenuates the magnitude of LTP in hippocampal slices from cocaine withdrawals.13. The basic properties of MSNs in dorsal striatum （RMP and input resistance） are not affected by acute or chronic cocaine treatment or cocaine withdrawal.14. The magnitude of LTP in corticostriatal slices is not affected by acute or chronic cocaine treatment or cocaine withdrawal.15. Application of CRF （20-80 nM） in vitro significantly increases the magnitude of LTP in corticostriatal slices from controls and cocaine withdrawal.16. Specific blockade of CRF₁ in vitro significantly attenuates CRF-enhanced magnitude of LTP in corticostriatal slices from controls and cocaine withdrawal groups.17. Specific blockade CRF₂ in vitro significantly attenuates CRF-enhanced magnitude of LTP in corticostriatal slices from cocaine withdrawal groups.18. Application of UCN2 in vitro significantly increases the magnitude of LTP in corticostriatal slices from cocaine withdrawal groups.19. Specific blockade of CRF₂ significantly attenuates UCN2-enhanced magnitude of LTP in corticostriatal slices from cocaine withdrawal groups. Conclusions1. Acute cocaine-induced p-ERK1/2 in dorsal striatum is a major mediating factor for cocaine abuse-caused neuroadaptations, also contributes to acute cocaine-induced abnormal behavior. In dorsal striatum, D1R is one main regulator to mediate p-ERK1/2 activation induced by acute cocaine treatment. In addition, the increased p-ERK1/2 carrys out its subsequent mediating roles via changing the expression of transcripts, including c-Fos, CREB and Elk-1.2. ERK1/2 plays an important mediating role in acute cocaine-caused hyperactivity in ventral striatum （NAc）. ERK1/2 carrys out its mediating function mainly in the core part of NAc.3. Cocaine withdrawal enhances the magnitude of LTP in hippocampal slice. CRF₁ and CRF₂ both are involved in the process. CRF₁ contributes to the formation of LTP in hippocampal slices both under physiological and pathological conditions. Cocaine withdrawal partially induces the changes of hippocampal synaptic plasticity via changing the CRF₂ activity, which leads to the change of learning and memory.4. CRF₂ is the main receptor type involved in the CRF and UCN2-induced enhancement of the magnitude of LTP in corticostriatal slices in cocaine withdrawal.更多还原