【作者】 刘路英；

【导师】 陈忠；

【作者基本信息】 浙江大学 ， 药理学， 2007， 博士

【摘要】 组胺是体内广泛存在的自体活性物质和炎症介质，在20世纪80年代两个研究组同时用免疫组织化学的方法证实了组胺能神经元主要分布在下丘脑，组胺在中枢神经系统中的作用才逐渐为人们所承认和重视。目前认为脑内组胺能神经元广泛分布于下丘脑的结节乳头核(nucleus tuberomammillaris，TM)，而且TM是脑内组胺能神经元分布的唯一区域，但其轴突可以投射到脑内的绝大部分区域，包括丘脑、杏仁核、海马结构、大脑皮层等。组胺在脑内由其前体组胺酸(L-histidine)脱羧生成，组胺酸脱羧酶(histidine decarboxylase，HDC)是目前发现的唯一组胺合成酶。在中枢神经系统，组胺有三种受体(H1、H2、H3)，均已得到分离鉴定，它们属于G蛋白偶联受体超家族，具有七个跨膜区域。H1受体与磷脂酶C(phospholipase C)、蛋白激酶C(protein kinase C)藕联，H2受体与cAMP、蛋白激酶A(protein kinase A)耦联。H3受体与Gi／Go藕联，是突触前自身受体，对组胺的释放有抑制作用，也影响组胺的合成。同时，它也是异源性受体，在黑质纹状体、杏仁核、大脑皮层等区域调节其它递质的释放。总体上，H1和H2受体对神经元起兴奋性作用，或增强其兴奋性传入。相反，H3受体激活对TM神经元产生负反馈抑制，并能抑制神经递质的释放。组胺作为神经系统的递质或调质，在许多中枢活动中起着重要的调节作用，如神经内分泌调节、饮水摄食调节、体温调节、觉醒—睡眠、运动、攻击行为以及学习记忆等。学习记忆是脑的高级功能，在许多发病率较高的中枢神经系统疾病中，病人的学习记忆功能均受到不同程度的损害。因此，组胺与学习记忆的关系成为越来越多的研究者致力的热点问题。但随着研究的深入，人们发现组胺系统对学习记忆的影响十分复杂，对组胺以及组胺系统的相关药物在行为学方面的研究均出现不一致的结果。其中较为重要的原因是，组胺系统的药理学工具特异性相对较低，并没有选择性的、能够显著持久的抑制脑内组胺合成，及同时拮抗所有组胺受体的工具药。因此，越来越多的研究者采用HDC基因敲除(HDC-KO)小鼠来探讨组胺长期缺乏后对学习记忆的影响。研究表明，HDC-KO小鼠对物体的辨别记忆能力较其野生型(wild type，WT)小鼠下降，而在水迷宫和被动回避反应的学习记忆能力增强，并认为其与多巴胺的负性增强有关。但是这种假设需要在其它学习记忆模型中进一步证明，同时这些研究均停留在行为学水平，没有探讨其增强效应的深入机制。由于背景和空间学习记忆与海马结构的突触可塑性密切相关，在组胺的长期缺乏的情况下是否影响HDC-KO小鼠的海马的突触可塑性，而且在学习记忆的行为学任务中，这种突触可塑性是否发生改变，如何改变，至今仍不清楚。因此为了进一步研究组胺长期缺乏对学习记忆的影响及其深入机制，本研究采用海马依赖的背景恐惧记忆模型，观察HDC-KO小鼠的学习记忆改变；同时用细胞外电生理记录HDC-KO小鼠突触可塑性的改变，探讨行为学改变的生理机制。第一部分HDC基因敲除小鼠背景恐惧记忆的改变近年来，恐惧条件反射模型(Pavlovian fear conditioning)逐渐成为研究学习记忆神经生物学机制的主要行为学模型，分为背景恐惧条件反射(cntextual fearconditioning)和线索恐惧条件反射(cued fear conditioning)。在这个模型中，小鼠需要在训练中将仪器背景刺激及声音刺激(conditioned stimulus)和足底电刺激(unconditioned stimulus)联系起来，产生对背景或声音的恐惧记忆，当小鼠再次暴露到相同背景或者在不同背景中的声音刺激时，小鼠会表现出呆滞反应(freezing)，其持续时间的长短是衡量恐惧记忆能力的标志。背景恐惧记忆而不是线索恐惧记忆依赖于海马。为了进一步研究组胺长期缺乏在海马依赖的学习记忆的重要作用，我们采用背景恐惧条件反射模型，观察HDC-KO小鼠的学习记忆是否发生改变。RT-PCR和高效液相结果显示HDC-KO小鼠不含有HDC基因，而且体内组胺含量极低。免疫印记发现HDC-KO小鼠海马细胞膜H2受体上调。行为学结果显示，给予电刺激后，HDC-KO小鼠的呆滞反应时间百分率从2h开始较WT小鼠升高，至训练后1d有显著性升高，并且一直持续到训练后14d。这些结果提示HDC-KO小鼠对背景恐惧记忆较WT小鼠显著增强，其时程的改变提示组胺长期缺乏可能参与海马的获得和巩固。我们进一步在训练前、训练后以及测试前脑室内注射组胺来确定背景恐惧记忆的增强，同时探讨组胺的缺乏将影响获得、巩固和再现中的哪个过程。结果发现，在训练前、训练后注射组胺明显降低HDC-KO小鼠呆滞反应的时间百分率，在测试前注射没有影响，证明组胺长期缺乏可能影响了海马的获得和巩固过程，对再现过程没有影响。对线索恐惧记忆的结果表明，HDC-KO小鼠在未受电击的环境中受到声音刺激时，其呆滞反应时间也延长，提示组胺长期缺乏导致的学习记忆增强对海马的依赖可能不是特异性的。第二部分HDC基因敲除小鼠的突触可塑性改变海马在学习记忆过程中突触可塑性的形成重要作用，海马的长时程增强(Long-term potentiation，LTP)是研究的最为广泛的活性依赖的突触可塑性的模型，被认为是学习记忆的神经基础。海马齿状回、CA3、CA1均有习得性长时程增强的突触效应，其中以Schaffer侧支与CA1形成的通路与空间背景学习记忆关系最为密切。在第一部分中，我们已经发现组胺的长期缺乏增强HDC-KO小鼠的背景学习记忆，并影响获得和巩固过程。在本部分实验中，主要研究行为学改变的突触可塑性机制。细胞外电生理结果显示，HDC-KO小鼠的基本输入输出曲线(input-output curve)较WT小鼠没有明显改变，即CA1区的基本突触传递并没有因为敲除HDC基因而改变；在TBS刺激后HDC-KO小鼠的群峰电位增强的幅度明显高于WT小鼠，即HDC-KO小鼠的LTP较WT小鼠显著增强，并可以持续2h直到记录结束；10μM组胺灌流小鼠海马脑片，给予TBS刺激诱导LTP后，其幅度明显下降，说明HDC-KO小鼠LTP的增强是由组胺的长期缺乏特异性引起的；HDC-KO小鼠的双脉冲易化在LTP诱导前后有明显差异，特别是诱导后较WT小鼠显著下降，提示在LTP诱导后，HDC-KO小鼠的突触前谷氨酸释放增加，参与了LTP的增强效应；而在训练后1d时，两种基因型小鼠的LTP较正常时均明显下降，但WT小鼠的LTP幅度反而明显高于HDC-KO小鼠的LTP，提示HDC-KO小鼠的背景恐惧记忆明显增强时，突触传递已经维持在较高的水平，LTP出现饱和现象，进一步证明其行为学的改变，以及LTP与联合性学习记忆的相关性。总结1．内源性组胺长期缺乏导致背景恐惧记忆增强，这种增强作用从训练后2h开始，一直持续到14d。组胺脑室内注射提示，组胺长期缺乏对海马的获得和巩固过程有明显影响。HDC-KO小鼠对声音的恐惧记忆亦增强，提示组胺长期缺乏对背景恐惧记忆的影响并不是特异性的，其不仅对海马的功能有影响，也可能影响杏仁核的功能。2．两种基因型小鼠海马脑片的电生理记录显示，虽然HDC-KO小鼠的输入输出曲线左移，但与WT对照小鼠比较没有显著性差异，提示HDC-KO小鼠的基本电生理特性没有发生明显改变；内源性组胺长期缺乏导致HDC-KO小鼠海马脑片CA1区LTP增强，而LTD没有发生明显变化；LTP诱导前和诱导后的PPF下降，以及生化结果发现行为学训练后1d和4d海马内谷氨酸含量增加，提示突触前谷氨酸释放增加可能参与背景恐惧记忆和LTP的增强效应；3．HDC-KO小鼠训练后1d海马脑片电生理记录显示，LTP幅度较WT小鼠明显降低，提示训练后1d背景恐惧增强时其突触效能已经明显增强，LTP与联合性学习记忆的密切相关。4．本研究进一步阐明HDC-KO小鼠对海马依赖的背景恐惧记忆影响，并首次揭示了本实验和以往实验中学习记忆改变的机制，即内源性组胺长期缺乏可能导致海马CA1区突触传递的增强，对比以往局限于外源性组胺的研究，我们的结果从相反的角度深化了组胺与突触可塑性关系的认识，为今后组胺在学习记忆方面的基础和临床研究提供了新的思路。更多还原

【Abstract】 Histamine is one of the most widely distributed inflammatory substances in the body. Compared with other aminergic systems, the histaminergic system in the CNS gained general acceptance only in 1984, after the immunohistochemical demonstration that the tuberomamillary （TM） nucleus was the sole seat of histaminergic neurons and the origin of the widely distributed histaminergic projections. It is now thought that their efferent fibers project to almost the entire brain, including the hypothalamus, septum, thalamus, cortex, amygdala, and hippocampus. Histamine is synthesised in brain from L-histidine by the enzyme histidine decarboxylase （HDC）, and up to now HDC is the only enzyme catalyzed the process. Four histamine receptors have been cloned （H1-H4）, and the H1, H2 and H3 histamine receptors are all expressed in distinctive patterns in the brain. The H1 receptor is a 486-491 amino acid protein encoded by an intronless gene, and is coupled to the Gq/11 protein and phospholipase C. The H2 receptor is coupled to Gs and protein kinase A and the protein consists of 358-359 amino acids. The H3 autoreceptor is coupled to Gi/Go, displays a significant constitutive activity, and controls histamine release and synthesis. The H3 receptor regulates the release of several transmitters in brain areas, such as the substantia nigra, amygdala and cerebral cortex.Histamine controls a variety of neurobiological functions and behavioral responses including sleep-wake cycle, water consumption, food, motor activity, and nociception. Histamine is also involved in learning and memory, and many researchers have contributed a lot in this field. However, these studies yielded to contradictory results. For instance, histamine improved and impaired active avoidance conditioning. The HDC-blocker α-iluoromethylhistidine （α-FMH） also has those effects in a radial-maze task. Furthermore, H₁ receptor antagonism has different influence on spatial memory performance and some emotional tasks. There were also discrepancies found with H₂ receptor antagonists and agonists and H3 receptor. The mechanisms underlying these differences seem to be very complex, which may be in part due to the methods used and the approaches selected in the experiments. Most histaminergic agents, and lesions of the TM and hippocampus, not only influence the histaminergic system but also affect non-histaminergic systems in the brain. So, recently histidine decarboxylase knockout （HDC-KO） mice have been developed to study the role of the histaminergic system in learning and other behaviors more specifically and to explain these discrepancies. Previous research showed that HDC-KO mice had improved water maze performance during both hidden and cued platform tasks, but exhibit deficient object discrimination based on temporal relationships. It is proposed that disruption of brain histamine synthesis may actually have bidirectional effects on learning and memory related to the reinforcement contingencies inherent in the task. However, this hypothesis needs further confirmation. Furthermore, these studies have not explained the mechanisms of the enhancement of learning and memory in the behavioral tasks. Contextual and spatial learning and memory is closely related to the synaptic plasiticity, and it is still unclear whether long-term histamine deficiency influences the synaptic plasiticity during the behavioral performance in HDC-KO mice.In the present study, we investigated the change of learning and memory in hippocampal-dependent contextual fear conditioning, and the synaptic plasiticity in hippocampal CA1 region using electrophysiological recording to explain the mechanisms of behavioral performance in HDC-KO mice.Part I Contextual fear conditioning in HDC-KO micePavlovian fear conditioning has emerged as a leading behavioral paradigm for studying the neurobiological basis of learning and memory. It can be divided into contextual fear conditioning and cued fear conditioning according to the conditioned sitmulus. Contextual but not cued fear memory is dependent on the hippocampus. In this paradigm, mice were given the opportunity to associate both a tone （cue） and the apparatus （context） with footshock in a single training trial. Memory was assessed by scoring the percent time mice spent immobile （freezing, a fear reaction） upon reexposure either to the context or the cue in a distinct context. In this part of investigation, we used contextual fear conditioning to examine learning and memory ability in HDC-KO mice. The data showed that mice lacking histamine exhibited improved contextual fear memory, and this improvement was maintained for a long period from 2h to 14d. The intracerebroventricular injection of histamine before and immediately after training reversed the improvement, while the injection before testing had on effects. HDC-KO mice also showed enhancement of cued fear compared with the wildtype （WT） mice. The results indicated that contextual fear memory increased in HDC-KO mice from 2h to 14d after training, and long-term histamine deficiency may upregulate hippocampal acquisition and consolidation of contextual fear. While these influences may be not specific to the hippocampus, amygdala may also be involved in the process. Part II synaptic plasiticity in the hippocampal CA1 region inHDC-KO miceHippocampus plays an important role in synaptic plasiticity. Physiologial activity-dependent long-term changes in synaptic transmission, as long-term potentiation （LTP） are thought to be the substrate of learning and memory. Contextual and spatial learning and memory are closely related to the LTP in the synapse from the Schaffer collateral pathway to CA1 cells. We found that long-term histamine deficiency increased the acquisition and consolidation of contextual fear in HDC-KO mice. In this part, we investigated the synaptic plasiticity in hippocampal CA1 region to explain the behavioral performance in HDC-KO mice. The data showed that there was no difference in input-out curve and LTD between the two genotypes. While under normal conditions, LTP was stronger in HDC-KO mice than that in WT mice 120 min after induction, and 10 μM histamine perfusion reversed this enhancement. Paired-pulse facilitation （PPF） was significantly decreased after the LTP induction in HDC-KO mice compared with that in WT mice. 1 day after training, LTP in HDC-KO mice was decreased compared with WT mice, which implied involvement of activity-dependent LTP in associative learning and memory. And HPLC analysis showed that hippocampal glutamate content increased in HDC-KO mice 1d and 4d after training. The results indicated that LTP increase in HDC-KO mice, and presynaptic gluatamate release may be also involved during the process. The changes in synaptic plasiticity in the hippocampus may contribute to the improvement in learning and memory. Summary1. Contextual fear memory increased in HDC-KO mice from 2h to 14d after training, and long-term histamine deficiency may upregulate hippocampal acquisition and consolidation of contextual fear. While these influences may be not specific to the hippocampus, amygdala may also be involved in the process.2. The electrophysiological recording showed that there was no difference in input-out curve between the two genotypes, which indicated the basal synaptic transmission do not change in HDC-KO mice. And long-term histamine deficiency may increase LTP in HDC-KO mice, while LTD did not change. Hippocampal glutamate content increase 1d and 4d after training, and PPF decrease after LTP induction indicated that presynaptic glutamate release may be involved in the enhancement of contexutal fear and LTP.3. LTP in HDC-KO mice was significantly decreased compared with that in WT mice 1d after training. These results indicated that the synaptic efficiency increases 1d after contextual fear training, and this phenomenon may be closely related to activity-dependent LTP in associative learning and memory.4. In the present study, we provide additional evidence that histamine is involved in acquisition and consolidation of contextual fear memory in a hippocampal -dependent manner. Fourthermore, we demonstrated that long-term histamine deficiency may contribute to improved contextual fear memory and spatial memory in HDC-KO mice. Compared with the effects of exogenous histamine on learning and memory, we elucidated the relationship between endogenous histamine deficiency and synaptic plasiticity, which provide a new aspect in this field.更多还原