【作者】 崔一蕾；

【导师】 孙心德；

【作者基本信息】 华东师范大学 ， 生理学， 2003， 博士

【摘要】 本文以动物听空间方位分辨行为、听皮层神经元听空间方向敏感性、NMDA受体亚单位mRNA组分／比值的表达变化为指标，以早期听觉经验动物为模型，采用Elvax缓释技术、多彩色荧光原位杂交、神经电生理学和行为学等技术，从受体、分子、细胞和整体水平上，研究NMDA受体在经验一依赖性听觉功能发育可塑性中的作用，为进一步揭示脑功能发育可塑性的机制提供实验依据。 论文第一部分，研究早期听觉经验对动物脑听觉功能发育的影响。主要内容包括： 一、听觉经验对生后不同周龄动物听空间分辨行为的效应 实验在24只SD大鼠上进行。分别在声音-食物朝向反射建立前、后和消退三个阶段，详细考察了各实验组动物听空间辨别行为。结果显示，在生后发育关键期，给予动物前方听空间右侧45°、频率3kHz短串纯音听觉经验训练，可使动物听空间分辨行为呈现经验-依赖性变化。表现为对3kHz声音、45°朝向偏好明显，45°方位朝向反射建立时程短，平均反应时缩短、朝向方位偏差减小、反应正确率提高、朝向反射消退时程延长等。结果提示，生后早期听觉经验可明显诱导大鼠听觉行为经验-依赖性的变化。 二、听觉经验对不同周龄动物听皮层神经元反应特性的效应 实验在32只SD大鼠上进行。结果表明，早期听觉经验可使动物听皮层神经元呈现经验-依赖性变化。与正常对照组相比，最佳频率近3kHz的神经元数目明显增多，听反应中心在听空间45°处的分布集中、频率调谐曲线锐度增大、阈值降低、平均Q_ndB值增高。显示了与行为学测试相一致的变化趋势。提示发育关键期的听觉经验，可使听皮层A1区神经元听空间方向敏感性提高。 三、听觉经验对不同周龄动物听皮层NMDA受体亚单位mRNA时空表达的效应 实验采用多彩色原位杂交技术，考察了3、5、8W听觉经验组动物两侧听皮层NMDARs单标、双标、多标阳性神经元密度及组份／比值变化。与正常对照组相比，NMDARs mRNA表达呈现非等比增加趋势。尤其是3W听觉经验组NMDARs mRNA组份及比值变化更加明显。提示早期听觉经验使动物NMDARs mRNA表达上调，其变化趋势与电生理及整体行为学研究结果相一致。提示听觉经验与NMDARs mRNA时空表达密切相关。NMDA受体在大鼠经验一依赖性脑听觉功能发育可塑性中的作用中文摘要 论文第二部分，采用Elvax加载药物缓释技术，在动物生后发育“关键期”，慢性阻断一侧听皮层NMDAF匕n1RNA表达，分析NMDA受体在听觉功能发育可塑性中的功能意义。主要研究内容包括:一、NMDA受体拮抗剂对经验一依赖性听空间分辨行为的效应 实验分别在生后不同年龄埋药组和对照组动物上，考察声音一食物条件反射建立前、后和消退三个阶段听空间分辨行为表征。结果表明，生后发育关键期，在动物左侧听皮层植入NMDA受体拮抗剂APV，可显著地影响动物对听空间右侧45“方位、3kHz短串纯音依赖性的听觉经验的建立。表现为声音一食物朝向条件反射建立时程延长，平均反应时延长、反应角度偏差增大、反应正确率降低，朝向反射消退时程缩短等。埋水组与听觉经验组动物听空间分辨行为几乎无异，说明手术未对动物行为造成可见影响。结果提示，APV对经验一依赖性听觉行为可塑性具明显影响。二、NMDA受体拮抗剂对经验一依赖性听皮层神经元反应特性的影响 实验采用电生理学方法，研究了生后不同年龄埋药动物和对照组动物听皮层神经元的听空间敏感性。结果表明，生后发育关键期埋药动物，与对照组相比，听皮层神经元反应特性不再呈现听觉经验一依赖性变化。表现为最佳频率与早期经验相一致的3kHz神经元数量不再增加，听反应中心分布弥散，听感受野显著扩大、频率调谐曲线锐度减小、平均QndB值降低、平均放电脉冲数减少等。提示生后发育关键期，一侧听皮层植入NMDA受体拮抗剂APV，可引起听皮层神经元听空间方向敏感性显著降低，其中对3周龄动物的影响尤其显著，显示了与行为学测试相一致的结果。三、NMDA受体拮抗剂对听皮层NMDARsn1RNA表达及其组份/比值的影响 实验考察了埋药组动物和对照组动物NMDARs表达及组份/比值变化。结果表明，埋水组和听觉经验组动物NMDARs组份表达及比值变化相似，均呈非等比上调趋势，并表明手术未对动物NMDARslnRNA表达产生可见影响。统计了埋药组动物两侧听皮层NMDARs单标、双标、多标阳性神经元密度及组份比值变化，结果表明，均呈非等比下调趋势，其中NRZB变化尤为显著。3W埋药组较5、SW埋药组NMDARsm丑NA组份及比值变化更加明显。结果提示，动物发育早期在听皮层缓释NMDA受体拮抗剂妙V，显著地下调NMDARs mRNA表达，改变NMDARS组份/比值。NMDA受体在大鼠经验一依赖性脑听觉功能发育可塑性中的作用甲义洞安四、NMDA受体拮抗剂对听皮层神经元细胞形态的影响 实验采用快速HE冰冻切片Sheehcm染色法，比较了正常对照组、埋水组及埋药组动物的细胞形态，未见埋植APV对听皮层神经元形态学的明显影响。 综上所述，生后早期听觉经验可明显增强大鼠听空间分辨行为，并伴随听皮层神经元听空间方向更多还原

【Abstract】 Using Elvax chronic releasing method, multicolor fluorescence in situ hybridization (McFISH), electrophysiological and behavioral methods, we studied the role of NMDA receptor in experience-dependent auditory developmental plasticity on early auditory experience experimental animal model at levels of receptor, molecular, cell and system, with animals’ auditory spatial azimuth discriminating ability, auditory spatial orientation sensitivity of auditory neurons and the change in components/ratio of NMDA receptor subunits mRNAs expression as indexes, giving experimental evidence to determine mechanisms of brain functional developmental plasticity.The first part of this paper focused on the effects of early auditory experience on animal’s auditory functional development, with the main contents as followed:1. Effects of auditory experience on auditory spatial discrimination behavior in rats of different postnatal-week ageThe auditory spatial discrimination ability of different animal groups were investigated in detail during the pre-foundation stage, post-foundation stage and fadeaway stage of sound-alimental conditional reflex in 24 SD rats. The results indicated the experience-dependent changes in animals’ auditory spatial discrimination behavior caused by training with short-duration pure-tune of 3kHz at the right 45?azimuth in front of animals during postnatal developmental critical period, which were demonstrated by the significant preference to 3kHz sound of right 45? the shortened course needed in foundation of 45?orientation conditional reflex, the shorter mean reaction time, smaller orientation error, improved response success rate and the prolonged course needed in the orientation reflex fading-away. The results suggests that early postnatal experience cause the experience-dependent changes in auditory behavior of rats.2. Effects of auditory experience on response properties of neurons in auditory cortex in animals of different postnatal-week ageData obtained from 32 SD rats revealed that early auditory experience induced experience-dependent changes in response properties of auditory cortex neurons. Compared with normal group, the amount of 3 kHz-BF neurons became larger, response centers centralized to 45?in auditory space, with the sharper frequency tuning curves, lower threshold and larger QndB value. This trend displayed above was in accordance with the behavioral tests, suggesting that auditory experience in developmental critical periodwould improve the auditory spatial orientation sensitivity of neurons in auditory cortex AI area.3. Effects of auditory experience on space-time expression of NMDA receptor subunits mRNAs in auditory cortex in animals of different postnatal-week ageBy multicolor fluorescence in situ hybridization method, our research determined the changes in density and components/ratio of NMDARs single-labeled, double-label -ed and multi-labeled positive neurons in bilateral auditory cortex of auditory experienced animals of postnatal week (PNW) 3, 5 and 8, and found an unevenly increasing trend compared with normal group, with the change in PNW 3 especially larger. It means early auditory experience changed the expression of NMDARs mRNA, accordant with the results of electrophysiological and behavioral experiments, suggesting NMDA receptor play an important role in experience-dependent brain functional developmental plasticity.The second part of the paper studied the functional significance of NMDA receptor in auditory developmental plasticity after chronically blocking NMDARs expression in unilateral auditory cortex during animals’ postnatal developmental critical period through Elvax-loaded drug chronic releasing technique. Followed are the main contents:1. Effects of NMDA receptor antagonist on experience-dependent auditory spatial discrimination behaviorThe auditory spatial discrimination behavior of drug-implanted group and control group at different PNW were investigated during the pre-foundation stage, post-foundation stage and fadeawa更多还原