【作者】 廖凯；

【导师】 王建枝；

【作者基本信息】 华中科技大学 ， 病理学与病理生理学， 2011， 博士

【摘要】 [背景]嗅觉系统是哺乳动物重要且基本的感觉系统。解剖学上,嗅神经的神经纤维除了投射到与嗅觉相关的脑区外,还大量投射到海马、杏仁核这些与学习记忆相关的脑区。所以,嗅觉系统除了维系正常的嗅觉功能外,也在学习和记忆过程中发挥着重要的作用。环境刺激法作为一种物理疗法,广泛运用于防治认知功能障碍和提高学习记忆能力等领域。已证实通过体育锻炼、新奇环境等途径的外界刺激,达到增强神经元可塑性和促进神经发生,从而改善学习和记忆。但通过嗅觉途径的环境刺激能否改善学习记忆还未见报道。[目的]研究不同持续时间和不同性质的嗅觉刺激对学习和记忆能力的影响及可能的机制。[方法]在3个相对封闭的房间中建立3种不同性质的嗅觉刺激环境：1)正常环境,房间中无特别气味；2)大鼠喜好的嗅觉刺激环境,房间中充满某种大鼠喜好的气味；3)大鼠厌恶的嗅觉刺激环境,房间中充满某种大鼠厌恶的气味。8周龄、体重约200克的大鼠随机分为4个组(每组15-20只)：1)对照组：所处环境无特殊气味刺激；2)丰富气味刺激组：所处环境随机选择为大鼠喜好或厌恶的嗅觉刺激环境中的一种,每24-48小时更换其所处的嗅觉刺激环境；3)喜好气味刺激组：所处环境为大鼠喜好的嗅觉刺激；4)厌恶气味刺激组：所处环境为大鼠厌恶的嗅觉刺激。刺激方式为将不同性质的气味置于各自对应的房间中,24小时不间断持续刺激。每24-48小时更换为同性质的其他气味,刺激持续时间分别为40天(短期)和80天(长期)。刺激结束后,用水迷宫、场景和声音恐惧条件反射测试检测嗅觉刺激对学习和记忆能力的影响,用高尔基染色观察树突棘的数目和形态的变化,用免疫印迹和免疫组织化学技术检测记忆相关蛋白和某些相关酶类和信号分子的表达和活性水平。[结果]1)短期丰富嗅觉刺激提高海马依赖的空间学习能力,但对空间记忆能力无影响；有情感记忆能力增强；突触前蛋白如synapsinⅠ、synaptophysin的表达水平升高、cAMP依赖蛋白激酶(PKA)活性增强和synapsin I的磷酸化程度增高、瘦长型和总树突棘数目增加。长期丰富嗅觉刺激能提高海马依赖的空间学习和记忆能力,同时伴随有情感记忆的增强；突触前蛋白如synapsinⅠ、synaptophysin,突触后蛋白PSD95、PSD93的表达水平水平升高,钙离子-钙调素依赖性蛋白激酶Ⅱ(CaMKII)的激活以及synapsinⅠ和cAMP反应原件结合蛋白(CREB)的磷酸化水平增高；瘦长型、蘑菇型和总树突棘数目均增加。2)短期喜好气味刺激能提高大鼠空间学习和记忆能力,同时伴随有情感记忆的增强；突触前蛋白synapsinⅠ、synaptophysin,突触后蛋白PSD95、PSD93的表达水平和PKA、CaMKⅡ、CREB活性的增加；蛋白磷酸酯酶2A(PP2A)和糖原合成激酶3(GSK-3)的活性无改变。短期厌恶气味刺激能提高大鼠的空间学习能力,但削弱空间记忆和情感记忆能力；同时伴有突触前蛋白如synapsinⅠ、synaptophysin表达水平和PKA活性的增加,但突触后蛋白PSD95、PSD93的表达水平和CREB、CaMKII活性却降低；PP2A活性增加,GSK-3活性无改变。3)长期喜好气味刺激能提高大鼠空间学习和记忆能力,同时伴随有情感记忆的增强；突触前蛋白synapsinⅠ、synaptophysin,突触后蛋白PSD95、PSD93的表达水平和CaMKII、CREB活性的增加；PKA,PP2A,GSK-3活性无改变。长期厌恶气味刺激能提高大鼠学习和记忆能力,但情感记忆减退；突触前蛋白synapsinⅠ、synaptophysin,突触后蛋白PSD95、PSD93的表达水平和CaMKⅡ、CREB活性增加；GSK-3活性降低,PP2A活性无改变。[结论]1)丰富嗅觉刺激能促进空间学习记忆和情感记忆能力,且短期和长期的丰富嗅觉刺激可能分别通过激活PKA和CaMKII及CREB信号途径来提高学习或记忆能力。2)喜好气味的嗅觉刺激能促进情感记忆和空间学习和记忆能力；在短期刺激时通过同时激活PKA和CaMKII及CREB信号途径来提高空间学习和记忆能力；在长期刺激时只激活CaMKII及CREB信号途径来提高空间学习和记忆能力。3)厌恶气味的嗅觉短期刺激损伤情感记忆能力；同时,可能通过激活PKA提高空间学习能力,而通过激活PP2A使CaMKII失活,削弱空间记忆能力。厌恶气味的长期嗅觉刺激也损伤情感记忆,但提高空间记忆能力,后者可能与激活CaMKII及CREB信号途径,同时使GSK-3失活有关。更多还原

【Abstract】 [Background]Olfactory plays an important role in the physiological function in mammalians. Olfactory neurons project axons to various brain regions including amygdala and hippocampus, both of them are essential for the learning and memory. Therefore, olfactory system not only correlative with olfactory function, but also with learning and memory function. Environmental enrichment is a commonly used experimental paradigm known to improve brain cognitive function through the enhancement of neuronal plasticity, learning, memory, and neurogenesis in the adult brain. But how odor enrichment could affect the learning and memory is still not clear.[Objective]It was to study the effects of enriched odor exposure on learning and memory and underlying mechanisms.[Methods]We carried out three independent environments:1) Control, normal environment without extra odor exposure.2) Pleasant odor exposure, an odor exposure environment fill of a single odor that rats like 3) Disgust odor exposure environment, an odor exposure environment fill of a single odor that rat dislike. Adult male Sprague Dawley rats aged 8 weeks were randomly assigned to four experimental groups:1) Control group, housed in the control environment; 2) Enriched odor exposure group, randomly housed in the pleasant odor exposure or disgust odor exposure environment and odor exposure environment replace ever 24-48h; 3) Pleasant odor exposure group, housed in the pleasant odor exposure environment 4) Disgust odor exposure group, housed in the disgust odor exposure environment. The enriched odor exposure group consisted of rats housed in an odor exposure environment for 40 d as a short-term exposure or 80 d as a long-term exposure and exposed daily for 24 hr to different odors which replace ever 24 h. After enrichment, Morris Water maze and Fear conditioning task was used to examine the effects of enriched odor exposure on hippocampus dependent learning/memory and emotional memory, respectively. Golgi staining was used to examine the the shape and morphology of spine. Western blotting and Immunohistochemistry was applied to detect the expressions and activities of presynaptic proteins, postsynaptic proteins, synaptic kinases and memory-associated signals.[Results]1) Short-term enriched odor exposure improves hippocampus dependent spatial learning and emotional memory but not memory, with increased the number of thin dendritic spines and total spines. Short-term enriched odor exposure also increased the levels of presynaptic protein synapsin I, synaptophysin and caused synapsin I phosphorylation by activation of cAMP-dependent protein kinase (PKA). Long-term enriched odor exposure improves hippocampus dependent spatial learning/memory and emotional memory, with increased the number of thin spines, mushroom dendritic spines and total spines. Long-term enriched odor exposure also increased the protein levels of presynaptic protein synapsin I, synaptophysin, postsynaptic proteins PSD93, PSD95 and caused cAMP response element binding (CREB) and synapsin I phosphorylation by activation of calmodulin-dependent protein kinaseⅡ(CaMKII).2) Pleasant odor short-term exposure improves hippocampus dependent spatial learning/memory and emotional memory, with increased the protein levels of presynaptic protein synapsin I, synaptophysin, postsynaptic proteins PSD93. PSD95 and with activation of PKA, CaMKII and CREB. Disgust odor short-term exposure improves hippocampus dependent spatial learning but impairs emotional and spatial memory, with increased the protein levels of presynaptic protein synapsin I, synaptophysin, decreased the protein levels of postsynaptic proteins PSD93, PSD95. It is also increased the activation of PKA and prorein phosphatase type 2A (PP2A), decreased the activation of CaMKII.3) Pleasant odor long-term exposure improves hippocampus dependent spatial learning/memory and emotional memory, with increased the protein levels of presynaptic protein synapsinⅠ, synaptophysin, postsynaptic proteins PSD93, PSD95 and with activation of CaMKII and CREB. Disgust odor short-term exposure improves hippocampus dependent spatial learning/memory but impairs emotional memory, with increased the protein levels of presynaptic protein synapsin I, synaptophysin, postsynaptic proteins PSD93, PSD95 and with activation of CaMKII and CREB. It is also decreased the activation of GSK-3[Conclusions]Enriched odor exposure could improve spatial learning/memory and emotional memory. Short-term and long-term enriched odor exposure time differentiated improves spatial learning and/or memory by activating PKA and CaMKII/CREB synaptic signals, respectively. Pleasant odor exposure could improve emotional memory, and improve spatial learning and memory by activating PKA and CaMKII/CREB synaptic signals in short-term enrichment. In long-term enrichment, it improves learning and memory only by activating CaMKII/CREB synaptic signals. Disgust odor exposure impair emotional and spatial memory by activating PP2A and inactivating CaMKII in short-term enrichment, but in long-term enrichment, it could improve spatial learning by activating PKA, and improve spatial memory by activating CaMKII and inactivating GSK-3.更多还原