【作者】 蔡睿；

【导师】 孙心德； 周晓明；

【作者基本信息】 华东师范大学 ， 生理学， 2011， 博士

【摘要】 出生后脑的结构和功能经历一个不断发育、成熟过程,环境、经验在脑的发育和可塑性中扮演重要角色。生后早期经验-依赖发育可塑性是感觉神经系统研究的重要领域。特别是丰富环境诱导脑可塑性引起人们的关注,它不仅具有重要理论意义,且被认为有广泛、潜在的应用前景。本论文以生后早期丰富环境暴露动物为模型,采用声源-方位分辨行为,在体和离体脑片细胞外记录以及分子生物学等技术,在行为、突触传递、细胞和分子的不同水平上,研究了生后早期丰富环境暴露对大鼠听觉功能发育可塑性的影响,及其可能的细胞分子机制。论文主要包括以下三个部分：一、早期丰富环境暴露增强大鼠听空间方位分辨行为和听皮层神经元听空间敏感性实验在12只生后早期丰富环境暴露动物(EE动物)和11只正常环境饲养的对照动物(CON动物)上进行。自大鼠出生后第7天(P7),将其置于丰富环境(enriched environment, EE)中饲养,直至成年(P56),之后,分别进行行为学和电生理学实验。对照动物为正常环境饲养的同龄大鼠。声源-方位分辨(sound-azimuth discrimination)行为测试结果显示,经过丰富环境暴露的动物,听空间敏感性(auditory spatial sensitivity)较对照动物明显提高。在完成声源-方位分辨作业中表现为,正确率(percent correct)提高,反应时(reaction time)缩短,水平方位角度分辨偏差(azimuth deviation)减小。电生理学实验结果显示,初级听皮层神经元频率调谐曲线(frequency tuning curve)和水平方位选择性曲线(azimuth selective curve)明显锐化,神经元的听空间感受野(auditory spatial receptive field)明显缩小。我们推测,生后发育关键期内,丰富环境暴露促进了神经回路的发育、成熟,显著地提高了听皮层神经元的听空间敏感性。这一效应的产生很可能是通过调节相关兴奋性和抑制性神经回路的发育平衡实现的。二、早期丰富环境暴露增强大鼠听空间敏感性的细胞分子机制实验采用分子生物学蛋白免疫印迹(western blotting)技术和离体脑片记录方法,研究了早期丰富环境暴露诱导大鼠听空间敏感性增强的细胞分子机制。免疫印迹检测结果表明,生后早期丰富环境暴露,可明显上调听皮层NMDA受体NR1、NR2A、NR2B亚单位和AMPA受体GluR2亚单位蛋白质的表达；抑制性GABAA受体α1、β3亚单位表达；NR2A/NR2B以及GABAΛα1/α3表达比值显著提高。中脑下丘的检测结果显示,NR1、NR2A、NR2B、GluR2、GABAΛα1和GABAAβ3的表达也都明显上调,呈现与听皮层相一致的表达变化趋势。离体脑片LTP记录结果显示,早期丰富环境暴露,明显提高了刺激白质(white matter, WM)所诱导的听皮层Ⅱ/Ⅲ层(layersⅡ/Ⅲ) LTP幅度,在相同浓度的NMDA受体拮抗剂APV (D-2-amino-5-phosphono-valeric acid)和AMPA受体拮抗剂DNQX (6, 7-dinitroquinoxaline-2,3-dione)作用下,EE动物LTP被抑制的幅度明显小于CON组和PE组动物。以上结果表明,生后早期丰富环境暴露可显著地上调听皮层兴奋性和抑制性受体亚单位的表达,提高突触传递的效率。提示丰富环境可能是通过影响听觉中枢兴奋性和抑制性信息传递系统发育的平衡,实现对听觉功能可塑性调控的。三、早期丰富环境暴露诱导听觉功能可塑性的保持幼年动物在生后第7天(P7)开始饲养于丰富环境中,成年后(P56)再返回到正常环境中饲养,至115天龄(P115)左右,分别进行行为学和电生理学和分子生物学实验。结果显示：1.在声源-方位分辨作业中,与同龄对照动物相比,EE动物完成作业的正确率(percent correct)高,反应时(reaction time)和方位分辨偏差(azimuth deviation)小；2.听皮层神经元频率调谐曲线(frequency tuning curve)和水平方位选择性曲线(azimuth selective curve)明显锐化,水平方位调谐深度(azimuth tuning depth)增加,听空间感受野(auditory spatial receptive field)缩小；3.听皮层抑制性GABAA受体α1、β3亚单位,GABAΛα1/GABAΛα3表达比值显著增加；兴奋性NMDA受体NR1、NR2A、NR2B和AMPA受体GluR2亚单位蛋白的表达量均有不同程度的上调。结果提示,听觉中枢兴奋性NMDA受体、AMPA受体和GABAA受体亚单位表达调控参与了早期丰富环境诱导的大鼠听空间敏感性增强效应的保持。脑功能发育可塑性及其细胞分子机制研究是神经科学重要的研究领域,它不仅对最终揭示脑发育和可塑性的奥秘有重要理论意义,而且,可能为有效矫治、修复脑感觉功能障碍、研发改善、治疗感觉功能障碍疾病的新策略和手段提供神经生物学实验依据,具有现实的指导意义和积极的应用前景。更多还原

【Abstract】 Enriched environment (EE) has an important role in the development and plasticity of the brain, while the early experience-dependent plasticity during the critical period is a major research field for sensory system. These two highly fruitful fields, namely, sensory-cortical plasticity and EE, are combined together in a novel attempt to investigate the effects of the EE-induced plasticity. The present study used rats as animal model to find out the underlying mechanism of how early EE exposure influenced the functional plasticity of central auditory system, through behavior, electro-physiological and molecular biological methods.This dissertation includes three parts as follow:1. Enriched environment-induced auditory spatial sensitivity of the ratRats were raised in enriched environment from postnatal day 7 to day 56, the EE condition was renew in several days to keep novelty. We used 12 EE rat and 11 CON rats for the study. By testing behavioral tasks via auditory cues, we have shown that EE improved the number of correct scores, but decreased the reaction time and azimuth deviation in behavioral performance of sound-azimuth discrimination. By in vivo extracellular recording, we have shown that EE enhanced the directional sensitivity of neurons in the primary auditory cortex. For example, EE rats had a smaller spatial receptive field, sharper frequency tuning curve and directional selective curve of auditory neurons compared with normal rats. Our findings indicate that early exposure to EE increases directional sensitivity. These results provide an insight into developmental plasticity in the auditory system.2. Molecular mechanism of improved spatial sensitivity of the rat by early EE exposureBy western blotting and brain slice recording, we investigate the expression level changes of several cortical developmental related receptors and amplitude changes of the long-term potentiation between the EE, CON and PE rats. We found that early EE exposure significantly increased the expression level of NMDA receptor subunits, NR1, NR2A and NR2B; AMPA receptor subunit GluR2; GABAA receptor subunits, α1 andβ3 of the auditory cortex. In addition, the ratios between NR2A and NR2B, GABAAα1 and GABAAα3 were also increased. The expression changes tendency in the inferior colliculus was almost the same with the auditory cortex, that is, significant increases were found in NR1, NR2A, NR2B, GluR2, GABAAα1 and GABAAβ3. The results of LTP recording indicated that, the EE rats have higher fEPSP amplitude than CON and PE rats under normal induced condition. When incubated with APV (an antagonist of NMDA receptor) or DNQX (an antagonist of AMPA receptor), the increase amplitude of LTP were both partly inhibited in the two groups. However, compared with the CON and PE rats, the amplitudes were still higher in the EE rat in those cases. All these results above indicated that early EE exposure may influence the functional plasticity of the auditory system by regulating the excitatory and inhibitory circuits simultaneously.3. Maintenance of enriched environment-induced auditory functional plasticity of the ratIn this part, we used EE recovery rats (which return to normal condition for two months) as model, investigated the maintenance of early EE exposure induced changes of auditory system by behavior, electrophysiological and molecular biological methods. We found that, compared with the age-matched control, the EE rats still had better performance. As to A1 neurons, the spatial sensitivity was enhanced, expressed by a sharper frequency tuning curve, smaller spatial receptive field, and a more selective directional curve of the EE rats. In addition, we also detected significant increases in GABAAreceptor al,β3 subunits; NMDA receptor NR2A, NR2B subunits; AMPA receptor GluR2 subunit protein expression; and in the ratios of GABAAα1/GABAAα3 and NR2A/NR2B. In particular, the variation rate of all inhibitory receptor expressions was significantly higher than that of the excitatory receptor expressions in early EE exposed rats. These observations indicate the persistent higher expression levels of the GABAergic and glutamatergic receptors expression induced by early EE exposure, especially enhancement of GABAergic inhibition in the auditory cortex, might be responsible for the maintenance of improved effects in auditory spatial sensitivity.更多还原