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题名	KMBZ-009改善心理应激所致认知障碍和抑郁样行为的研究银杏叶提取物及复方制剂改善老年鼠空间学习记忆的突触可塑性机理
作者	王永富
学位类别	博士
答辩日期	2007-02
授予单位	中国科学院研究生院
授予地点	北京
导师	蔡景霞 ; 徐林
关键词	应激 突触可塑性 长时程增强 长时程抑制 学习记忆 兴奋性突触后电流 抑制性突触后电流 多巴胺 去甲肾上腺素 抑郁样行为 氧化应激
其他题名	Mechanism study on the anti-stress-like and anti- depressant-like effects of Phenchlobenpyrrone Synaptic plasticity mechanism of EGb 761- or Chinese- Tranditional- Medicin-Fufang on age-induced cognitive deficit in rats
学位专业	动物学
中文摘要	1、KMBZ-009改善高台应激所致认知障碍和应激相关的抑郁样行为及其相关机理研究。 虽然适当的应激会提高动物的学习记忆功能，但过量的应激特别是无法逃避的应激，往往导致依赖海马或前额叶的学习记忆功能受损，这与应激改变脑内应激激素（皮质酮，皮质醇等）和神经递质的释放，影响突触传递和可塑性（包括长时程增强和长时程抑制，LTP和LTD）有关。一些疾病的发生、发展和恶化，比如抑郁症（Depression）、创伤后应激障碍（PTSD），往往也和应激相关联，其神经化学基础被证实与内分泌系统和单胺类（如五羟色，去甲肾上腺素，多巴胺）神经递质系统的功能密切相关。遗憾的是，到目前为止还没有发现能治疗应激的药物。本实验室过去的研究证实：KMBZ-009(申报新药时的名称为芬克罗酮，英文名Phenchlobenpyrrone)——一种新的取代吡咯烷酮类化合物，通过调节细胞内钙，改变脑内神经递质的释放，从而影响脑高级功能。KMBZ-009对神经递质释放影响是否能减轻应激导致的认知障碍及应激相关疾病的发生还没有进行研究。本研究采用Morris水迷宫、行为操作箱、绝望游泳、膜片钳和活体电生理技术研究了KMBZ-009对高台应激所致认知障碍和应激相关的抑郁样行为的影响及其相关机理。 研究结果发现，高台应激或皮质酮注射造成大鼠空间记忆提取障碍，这与其导致的海马CA1区突触可塑性改变有关，而KMBZ-009能成剂量依赖性地逆转应激对空间记忆提取的损伤作用，这与它阻断应激或皮质酮异化的LTD和恢复应激或皮质酮损伤的LTP密切相关。KMBZ-009能部分地降低因应激而升高的血清皮质酮含量，此外，KMBZ-009对大鼠海马CA1区锥体神经元的兴奋和抑制电流的影响可能也参与了其对应激的调节作用。KMBZ-009能显著增加海马CA1区锥体神经元上AMPA受体介导的兴奋性突触后电流（EPSC）的幅度，但不影响其动力学特性。NMDA受体介导的EPSC不受KMBZ-009的影响；GABA受体介导的抑制突触后电流（IPSC）的幅度几乎不受KMBZ-009的影响，而其受体动力学特性明显被KMBZ-009改变，表现为IPSC恢复的时间显著延长。KMBZ-009对CA1区兴奋抑制电流的调节作用，使大鼠海马细胞具有更强的维持细胞稳态的能力，从而避免应激导致神经元功能的损害。KMBZ-009对抗应激对认知得损伤作用提示其可能会减少动物的抑郁样行为，本实验结果发现，KMBZ-009确实能明显减少小鼠在强迫游泳（FST）中的不动时间，增加大鼠在72秒低频差式强化（DRL-72s）模型中的强化率，并降低其反应率。其机制是KMBZ-009增加正常动物中枢神经系统胞外NE水平，激活alpha和beta肾上腺素受体，从而使得实验动物的抑郁样行为明显减少。 2、KMBZ-009减轻氧化应激对细胞活力、线粒体电位及海马LTP的损伤作用。 前人的研究表明，氧自由基过多是导致老年痴呆患者和老年人神经细胞凋亡与认知障碍的因素之一。KMBZ-009和阿尼西坦是吡咯烷酮类化合物，研究显示均具有促智作用。有报道指出阿尼西坦能减少神经胶质细胞在缺血缺氧时氧自由基的生成，从而避免细胞受到氧应激损伤。本研究采用神经元原代培养和离体电生理学方法，观察了KMBZ-009和阿尼西坦对氧应激神经元的保护作用。结果发现，KMBZ-009和阿尼西坦均能保护氧应激神经元的线粒体的功能，对抗氧自由基对神经元细胞活力的损伤，从而有效逆转了氧化应激对海马脑片CA1区LTP的损伤作用。KMBZ-009的作用效果比阿尼西坦的效果强10倍。 3、银杏叶提取物及复方制剂改善老年大鼠空间学习记忆的突触可塑性机理。 有研究表明，银杏叶和三七叶提取物能调节神经系统的功能。本研究采用Morris水迷宫和活体电生理技术研究了银杏三七复方制剂及银杏叶提取物（以标准银杏叶提取物——金纳多作为阳性对照药）改善老年大鼠空间学习记忆障碍的突触可塑性机理研究。结果发现：老年大鼠空间学习记忆能力较差，高频诱导不能在其海马CA1区引发LTP，当长期服用金纳多或复方制剂一个月后，老年动物的空间学习记忆功能得到明显改善，这可能与药物增强海马LTP有密切关系。复方制剂的作用效果与金纳多的效果相当。 4、悬尾应激损伤避暗作业学习行为的多巴胺D1受体机制。 近年来的研究表明，DA系统对应激非常敏感，应激改变PFC内DA的含量，从而导致依赖于PFC的工作记忆受损。但目前尚不知道应激对DA系统的影响是否涉及依赖杏仁核和海马的情绪学习记忆功能。因此，我们采用被动回避作业和行为药理学的方法，初步探讨了此问题。结果发现：和对照组动物相比，随着悬尾应激持续时间的增加（5min、10min、20min），动物在避暗作业作业重测试中的步入潜伏期明显缩短，当动物被悬尾应激后回到鼠笼中休息20min，其步入潜伏期无明显变化；腹腔注射DA D1受体拮抗剂SCH23390呈剂量依赖性地缩短动物的步入潜伏期，但SCH23390腹腔注射和悬尾应激共同处理实验动物时，此种D1受体拮抗剂能有效逆转应激对步入潜伏期的影响；进一步的研究发现，应激或D1受体拮抗剂对痛觉感受的影响不是其改变动物步入潜伏期的主要因素。本研究结果表明悬尾应激导致脑内多巴胺释放过度增加，杏仁核（可能还有海马及相关神经回路）内的D1受体被过度激活，从而导致小鼠在操作被动回避任务时的记忆获得障碍。
英文摘要	Part 1. Mechanism study on the anti-stress-like and anti-depressant-like effects of KMBZ-009. Although the acute response to stress (e.g., heightened cognition) is an adaptive mechanism, excessive stress, in particular uncontrollable stress induces many psychological and physiological problems. Specially, hippocampus- or prefrontal cortex-dependent learning and memory are dramatically impaired by stress. And its mechanism was associated with stress-induced release changes of stress hormone (corticorsterone etc.) and neurotransmitters and deficit of synaptic plasticity (such as LTP and LTD). Many studies have proved that several psychiatric disorders such as depression were related to stress, and the functions of HPA axis and monoamine neurotransmitters system were its underline mechanisms. Previous works of our Lab have proved that KMBZ-009 (a novel compound of pyrrolidone derivatives. It was named phenchlobenpyrrone) changed neurotransmitters release in brain by modulating intracellular Ca2+ concentration, and as a consequence, the higher functions of the brain such as learning and memory were changed by KMBZ-009. However, they are unclear so far whether KMBZ-009 ameliorates stress-induced memory deficit and whether it has anti-depressant-like effect. In the present study, using Morris water maze tasks, operant box paradigm, forced-swim test, patch-clamp in vitro recording and in vivo electrophysiological techniques, we study the effect of KMBZ-009 on acute stress-induced spatial memory deficit and depressive behaviors, as well as its underlining mechanisms. The data showed that acute inescapable stress or a dose of exogenous corticosterone (CORT, 10 mg kg-1) significantly decreased the search times in the target quadrant during retest period. Protective doses of KMBZ-009 (20, 40 mg kg-1), administrated 30 min before acute inescapable stress or CORT injection, abolished memory retrieval deficits seen in untreated rats. Memory is believed to dependent on synaptic plasticity. Here, it was showed that long-term depression (LTD) in the CA1 region of the hippocampus in vivo was facilitated by acute inescapable stress, sub-acute footshock stress and CORT. Pretreatment with KMBZ-009 (10, 20, 40 mg kg-1) inhibited that facilitation. In addition, acute inescapable stress also blocked induction of hippocampal long-term potentiation (LTP), pretreatment with 20 mg kg-1 KMBZ-009 30 min before acute inescapable stress normalized hippocampal LTP induction. Immunoassay results revealed an expected stress-induced CORT increase, and stress-increased CORT level was significantly diminished by KMBZ-009 (10, 20 and 40 mg kg-1) administration. Moreover, KMBZ-009 modulates the functions of AMPARs and GABAARs. The results showed that KMBZ-009 had no effect on NMDARs-mediated EPSCs, but it increased the amplitude of AMPARs-mediated EPSCs without changing of its kinetics at 400μmol L-1 concentration. KMBZ-009 did not change the amplitude of GABAARs-mediated IPSCs, however, the decay time of GABAARs-mediated IPSCs were prolonged by KMBZ-009 at the dose of 100μmol L-1. For GABAARs-mediated miniature IPSCs (mIPSCs), the decay time was also significantly prolonged by KMBZ-009 (100μmol L-1), but it had no effects on the rise time, amplitude and frequency of mIPSCs. The effect of KMBZ-009 on both excitatary and inhibitary currents contributes maintaining normal functions of pyramidal neurons in hippocampus when underdoing stress. Exposure to stress has been linked to the pathophysiology of depression and anxiety disorders. The ameliorating effect of KMBZ-009 on stress suggests that it may be useful in clinical treatment for stress and stress-related psychiatric disorders such as PTSD, depression/anxiety. Data showed that Treatments of animals with KMBZ-009 (5, 10, 20mg kg -1 p.o.) dose-dependent reduced the time of immobility and increased the latency to immobility of mice in FST, without any significant effect on locomotor activity of mice. The dose of 20 mg kg -1 of KMBZ-009 significantly increased the reinforcement rate and decreased the response rate of rats in DRL-72s. DL-propranolol (2 mg kg -1 i.p.; a β-adrenoceptor antagonist) and phentolamine (1 mg kg -1 i.p.; an α-adrenoceptor antagonist) significantly attenuated the KMBZ-009- induced antidepressant-like effect in FST. These data suggests that KMBZ-009 may possess an antidepressant-like effect, mediated by increase of brain norepinephrine. Part 2. KMBZ-009 attenuates oxidative stress-induced deficiency of neuron viability, mitochondria potential and hippocampal long-term potentiation of mice in vitro It is known that the free radicals are involved in neurodegeneration and the cognitive dysfunction, as seen in Alzheimer's disease (AD) and aging. Aniracetam, a compound of pyrrolidone derivatives, was reported to antagonize the ischemia-induced astrocytes degeneration at least in part by reducing the ROS production. Primary cell cultures and electrophysiological technique were used to examine the protective effects of aniracetam or KMBZ-009 against H2O2-induced toxicity to neuron viability, mitochondria potential and hippocampal long-term potentiation (LTP). Data showed that H2O2 exposure impaired the viability of neurons, reduced mitochondria potential and decreased LTP in the CA1 region of hippocampus. These deficient effects were significantly rescued by pre-treatment with aniracetam or KMBZ-009. Part 3. The in vivo synaptic plasticity mechanism of Chinese-Tranditional- Medicin-Fufang- or EGb 761-induced enhancement of spatial learning and memory in aged rats It has not been uniform to date that the Ginkgo biloba extracts enhance cognitive function in aged animals, and the mechanisms of action remain difficult to elucidate. In this study, the Morris water maze task and electrophysiological methods were used to study the effects of repeated daily administration of EGb 761, a standardized extract from G. biloba leaves, or Chinese-Tranditional-Medicin-Fufang (CTMF) on hippocampal-dependent spatial learning and memory and synaptic plasticity of aged rats. It was found that The aged subjects perform the Morris water maze task worse than adult rats, as a cellular mechanism, the hippocampal long-term potentiation (LTP) of aged animals is impaired. In addition, the spatial learning and memory of aged rats that had been fed on an EGb 761- or an CTMF-supplemented diet (60 mg kg-1) for 30 days were significantly better than those of control aged rats. The magnitude of LTP recorded in vivo from the hippocampus CA1 area of aged rats was significantly enhanced by EGb 761 or by CTMF(60 mg kg-1). Part 4. Memory acquisition in one trial step-through test was impaired by tail-hanging stress via dopamine D1 receptors over stimulation in mice Although it is well known that dopamine system is sensitive to stress, and leads to changes in the working memory function that is dependent on prefrontal cortex, it is unclear whether stress via dopamine system impairs emotion memory that is mainly dependent on amygdala and hippocampus. In the current study, the one trial step-through test is adopted to examine the effects of tail-hanging stress on acquisition of emotion memory and the mechanisms of dopamine in mice. The results show that the tail-hanging stress, and a dopamine D1 receptor antagonist SCH23390 significantly impair the memory acquisition, respectively. However, SCH23390 effectively reversed the memory deficit caused by the tail-hanging stress. The present data suggest that the acquisition of emotional memory related to amygdala, hippocampus or associated neural circles was impaired by stress due to the over stimulation of D1 receptors.
语种	中文
公开日期	2010-10-14
内容类型	学位论文
源URL	[http://159.226.149.42:8088/handle/152453/6104]
专题	昆明动物研究所_学习记忆的分子神经机制
推荐引用方式 GB/T 7714	王永富. KMBZ-009改善心理应激所致认知障碍和抑郁样行为的研究银杏叶提取物及复方制剂改善老年鼠空间学习记忆的突触可塑性机理[D]. 北京. 中国科学院研究生院. 2007.

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