【作者】 夏交；

【导师】 孔维佳；

【作者基本信息】 华中科技大学 ， 耳鼻咽喉科学， 2008， 博士

【摘要】 第一部分红外可视脑片膜片钳术中大鼠脑干脑片的改良制备方法目的:改良脑片制备方法,探讨影响脑片活性的实验因素;方法:在国内外报道常用方法的基础上进行改良,制备含有前庭内侧核的大鼠脑干脑片,从肉眼观、镜下观和放电特性三个方面判断脑片活性,观察鼠龄、生理溶液、温度等因素对脑片活性的影响;结果:在不同年龄组动物建立了稳定可靠的脑片制备方法,通过红外微分干涉相差技术可直接观察脑片表层下50-100μm前庭内侧核神经元的立体影像,并可进行长时间稳定的电生理记录;鼠龄越小神经元活性越易恢复,在成年鼠制备脑片时采取低温人工脑脊液心脏灌注、使用高蔗糖溶液、较高温度预孵育等措施可明显提高脑片活性;孵育和记录温度可直接影响神经元的兴奋性,在一定范围内提高温度可提高神经元兴奋性,但会缩短脑片存活时间;结论:改良后的脑片制备方法简单可行,可为电生理研究提供大量具有良好活性的前庭内侧核神经元;根据试验目的选择合适年龄的试验动物,脑片制备方法的选择和温度、外液实验等环境的控制应根据所选用动物的年龄而进行调整。第二部分大鼠前庭内侧核神经元的基本膜特性和自发放电活动目的:观察大鼠前庭内侧核神经元的基本膜特性及其在静息膜电位下的放电活动,探讨前庭系统生理功能的可能机制。方法:运用红外微分干涉相差技术,可视状态下对前庭内侧核神经元进行全细胞记录,观察电流钳模式下神经元在正常人工脑脊液和低钙高镁脑脊液中的放电活动,按平均动作电位形状对神经元进行分型,比较不同类型神经元基本膜特性和放电活动的差异。结果:在正常人工脑脊液中大鼠前庭内侧核神经元存在规律的放电活动,在低钙高镁人工脑脊液中神经元表现出较不规律的放电活动,放电活性也明显降低;神经元可被分为具有单相后超极化及A样整流特性的A型(n=17,33%),有双相后超极化的B型(n=32,63%),以及同时拥有或不拥有以上特征的其他型(n=2,4%);A、B型神经元的部分主动膜特性存在明显差异。结论:大鼠前庭内侧核神经元的放电为基于其内在膜特性的自发活动,其放电模式受到胞外钙浓度的影响;大部分神经元的放电表现为典型的A型或B型,但仍有少量非典型放电存在。第三部分大鼠前庭内侧核神经元对模拟传入信号的放电反应目的:观察大鼠前庭内侧核神经元对模拟传入刺激信号的放电反应动力学特征,探讨中枢前庭系统生理功能的可能机制。方法:运用红外微分干涉相差技术,可视状态下对前庭内侧核神经元进行全细胞记录,在电流钳模式下记录神经元的放电活动,向神经元胞内注入短暂超级化脉冲模拟突触后抑制性电流,注入线性去极化电流模拟头部作直线加速运动时外周前庭的传入信号,注入正弦刺激电流以模拟头部作匀速旋转运动时外周前庭的传入信号,观察不同类型神经元对刺激电流的放电反应。结果:前庭内侧核神经元在超级化脉冲后放电的后超级化幅度加深,并表现出抑制后反弹现象;神经元放电频率随去极化电流的增加而增加,在同等强度去极化电流刺激下B型神经元比A型神经元表现出较少的衰减现象和较强的超射现象;A、B型神经元的放电反应都可与输入正弦刺激形成共振并引起相位的移动。结论:头部做直线加速运动和匀速旋转运动时,前庭内侧核A、B型神经元对不同的传入刺激都可产生主动反应,放电反应表现出多样性,A、B型神经元放电反应动力学的差异是它们执行不同生理功能的基础。更多还原

【Abstract】 Part 1: Modified Preparing Methods of Brainstem Slices for Visual Patch Clamp TechniquesObjective: To approach a modified method of preparing brainstem slices, and to discuss the factors affecting slices health. Methods: Brainstem slices with medial vestibular nuclei were made on the basis of reported methods, and the quality of slices was judged by naked eye observation, light microscope observation and firing activities recordings. The effects of rats age, physiological fluids and temperature upon slices health were observed. Results: A simple and reliable preparation procedure of brain slices was established. The medial vestibular nucleus neurons were visualized by infrared differential interference contrast technique and readily-patched at a depth of 50-100μm underneath the surface of slices. Brain slices were easy to obtain from juvenile animals, while performing intracardiac perfusion with cold artificial cerebrospinal fluid, using high-sucrose solution and pre-incubating slices at a warmer temperature were useful when prepared slices from adult rats. Within definite temperature rage, the excitability of neurons improved with the increasing of temperature during incubation and recording, while the survival time shortened. Conclusions: Modified preparing methods could supply sufficient healthy medial vestibular nucleus neurons for electrophysiological recordings. The preparation procedures and the controlling of physiological surroundings should be adjusted according to rats age. Part 2: Spontaneous Firing Activities and Basic Membrane Properties of Rat Medial Vestibular Nucleus Neurons in Brain SlicesObjective: To study the basic membrane properties of rat medial vestibular nucleus(MVN) neurons and their firing activities at resting membrane potential, and to discuss how the intrinsic membrane properties contribute to physiological functions in central vestibular system. Methods: By using infrared differential interference contrast technique, whole-cell recordings were made from rat MVN neurons. Firing activities were recorded by current clamp mode in artificial cerebrospinal fluid (ACSF) and low Ca2+-high Mg2+ ACSF. On the basis of their averaged action potential shapes, the MVN neurons were classified. The differences of intrinsic membrane properties and firing mode were observed between two types. Results: Regular discharge activities were recorded in MVN neurons in ACSF. In low Ca2+-high Mg2+ ACSF, Neurons showed more irregular and depressive firing activities. MVN neurons were classified as type A (n=17, 33%) characterized by a single deep after-hyperpolarization (AHP) and A-like rectification, or type B (n=32, 63%) characterized by double AHP, and another two neurons (4%) with all or none of the characters. The passive membrane properties were not significantly different between type A and type B neurons, while part of active membrane properties was significantly different. Conclusions: The discharge activities of MVN neurons were initiated by their intrinsic membrane properties, and calcium mechanism contributed to firing mode. Most MVN neurons were classified as type A and B, while several showed unrepresentative firing properties. The differences of membrane properties between neurons determined their different physiological functions. Part 3: Firing Responses of Rat Medial Vestibular Nucleus Neurons to Simulated Input SignalsObjective: To observe the firing response dynamics of rat medial vestibular nucleu(sMVN) neurons to simulated input signals, and to discuss how the firing dynamics contribute to physiological functions in central vestibular system. Methods: By using infrared differential interference contrast technique, whole-cell recordings were made from rat MVN neurons under direct observation. Firing activities were recorded in current clamp mode. Hyperpolarizing currents were injected into MVN neurons to simulated inhibitory postsynaptic currents (IPSC). Depolarizing currents and sinusoidal currents were injected into neurons to simulate the input signals from peripheric vestibular organs when head in linear accelerated motion and uniform rotation motion. Firing dynamics of MVN neurons to stimulus currents were recorded. Results: After hyperpolarizing currents injected into neurons, the after-hyperpolarization amplitude of action potentials increased and post-inhibitory rebound was recorded. Neurons firing rate increased with the depolarized current. Compared with type A neurons, type B neurons showed the weaker adaptation and the stronger overshot phenomenon to same depolarizing currents. Firing rate of neurons resonantly oscillated with sinusoidal currents, and phase lead was observed. Conclusions: When head made linear accelerated motion and uniform rotation motion, the MVN neurons responded actively to the input signals from peripheric vestibular organs. Differences of firing dynamics between type A and B neurons determined their different physiological functions.更多还原