【作者】 郑小波；

【导师】 田心；

【作者基本信息】 天津医科大学 ， 生物医学工程， 2010， 博士

【摘要】 [目的]神经细胞膜离子通道的活动特性与神经元的功能直接相关,本论文研究发育早期4个周龄组(1w,2w,3w,4w)大鼠海马CA1区锥体神经元全细胞跨膜总电流、全细胞钠通道电流、全细胞钾通道电流、瞬时外向钾电流和全细胞钙通道电流的变化规律,包括激活、失活、失活后恢复等方面的特征变化,探索大鼠海马锥体细胞膜Na+, K+,Ca2+电压门控型离子通道发育的关键期,为研究学习记忆的发育提供支持。[方法]实验动物是出生后1w,2w,3w,4w,4个周龄组的SD大鼠,每组8只，分别制备海马脑片。应用膜片钳技术Axon 700B膜片钳系统记录脑片海马CA1区锥体神经元全细胞跨膜总电流、全细胞钠通道电流、全细胞外向总钾电流、瞬时外向钾电流和全细胞钙通道电流及其激活、失活等特性变化。应用Clampfit 10.2和Origin 7.5软件获取全细胞钠通道电流、瞬时外向钾电流和全细胞钙通道电流的I-V曲线,激活、失活、及失活后恢复动力学曲线,应用曲线拟合方法得到与之对应的半数激活电压、半数失活电压、斜率因子、失活后恢复时间常数等相关参数,进行统计学检验各组间是否存在差异。[结果]1.用发育早期4个周龄组大鼠制备的脑片活性良好,显微镜下海马区结构清晰,CA1区锥体神经元饱满、轴突明显、立体感强。2.Na+通道：(1)随着周龄的增大钠通道电流的I-V曲线向左移动,钠通道最大电流密度增大,1w,2w,3w,4w组对应的最大电流分布密度(pA／pF)分别为：-139.89±9.47,-147.98±9.39,-229.20±15.42,-271.54±22.78。以生后2w至3w电流密度增大最为显著；(2)钠通道电流的激活曲线向复极化的方向移动,4个组对应的半数激活电压(mV)分别为：-44.65±0.39,-47.42±0.56,-54.79±0.61,-58.44±0.57；(3)失活后恢复时间缩短,4个组对应的恢复时间常数(ms)分别为：9.30±0.642,11.15±0.84,7.95±0.55,7.18±0.56；(4)失活动力学没有显著变化,4个组对应的半数失活电压(mV)分别为：-52.29±0.73,-51.98±0.94,-51.44±0.72,-49.65±0.73。3.K+通道：(1)4个组全细胞瞬时外向钾最大电流密度随周龄增大而增大,1w,2w,3w,4w组对应的值(pA／pF)分别为133.11±14.36,182.10±8.55,212.80±15.98,235.86±14.60；(2)4个组半数激活电压(mV)分别为：6.25±2.38,10.88±1.71,5.31±2.46,2.690±2.75；(3)4个组半数失活电压(mV)分别为：-58.41±0.59,-56.35±0.90,-58.06±2.13,-60.37±0.90；(4)4个组恢复时间常数(ms)分别为33.14±3.19,33.78±4.22,35.13±3.92,36.19±3.62。4.Ca2+通道：(1)4个组全细胞钙通道最大电流密度随周龄的增大而增大,1w,2w,3w,4w组对应的最大电流密度(pA／pF)分别为：-8.02±0.56,-10.02±0.88,-27.59±1.03,-32.59±1.04,以2w至3w电流密度增大最为显著；(2)全细胞钙电流的激活曲线失活曲线均向左移动,4个组对应的半数激活电压(mV)分别为-6.94±0.11,-4.72±0.41,-11.72±1.05,-12.36±1.32,(3)4个组对应的半数失活电压(mV)分别为-5.35±2.06,5.43±1.93,-17.80±3.37,-24.88±2.43。[结论]1.发育早期(1w,2w,3w,4w)大鼠海马CA1区锥体神经元的Na+,K+,Ca2+等电压门控离子通道分布,通道的激活动力学,失活动力学随周龄增大而变化,反映了离子通道的发育状态。2.出生后2-3w是大鼠海马CA1区锥体神经元钠通道的快速发育期,此期间锥体神经元钠离子通道的分布显著增大,钠通道的激活曲线左移,半数激活电压显著降低,以上结果表明钠通道更容易被激活,失活后恢复曲线左移,恢复时间常数变小,钠通道的恢复更为迅速.3.大鼠脑发育早期,从1w到4wCAl锥体神经元细胞膜上钾离子通道的分布密度随出生周龄增大而增大,但激活和失活以及失活后恢复曲线特征未见有明显特征变化。4.大鼠出生后2-3w是海马CA1区锥体神经元钙离子通道处于快速发育期,期间钙离子通道最大电流密度显著增大,钙离子通道的激活、失活曲线均左移,半数激活、失活电压显著降低表明在此期间钙离子通道更易被激活,也更易失活。更多还原

【Abstract】 【Objective】The characteristics of the ion channels on cell membrane are direct related to the change of the neurons’functions. This dissertation explores the characters of whole cell transmembrane total currents, whole cell sodium current, whole cell potassium currents, transient outward potassium currents and whole cell calcium currents of hippocampal CA1 pyramidal neurons of S-D rats at various ages in the early stages of development. The aim of this study is to explore the critical period of development of ion channel in hippocampus of rats and provide support for studying the development of learning and memory.【Methods】The subject of this dissertation is hippocampus CA1 region pyramidal neurons of rats at different ages, respectively 1,2,3,4 weeks after birth.Characters of whole-cell transmembrane total current, sodium channel current, whole cell potassium channel currents, transient outward potassium channel currents and whole cell calcium channel currents of hippocampal CA1 pyramidal neurons were recorded using patch clamp technique.Curves of I-V, activation, deactivation and recovery after deactivation of whole-cell sodium channel current, transient outward potassium channel current and calcium channel current were obtained via Clampfitl0.2 and Origin7.5.The related parameters, such as half activation voltage, half deactivation voltage, slope factor, recovery time constant, were fitted according to appropriate equations. Statistical tests were performed to study whether there were differences among groups.【Results】1. In the early stages of development, bioactivity of hippocampal slices at different ages were excellent. Structure of hippocampus, Body and axon of the neurons can be observed clearly. The boundary of the neurons has good refractive index and strong sense of three-dimensional under microscope.2. In the early stages of development, I-V curve of sodium channel shifts to the left with increasing age. Maximum current density of sodium channel increases, the corresponding value of maximum current density (pA/pF) is:-139.89±9.47,-147.98±9.39,-229.20±15.42,-271.54±22.78. We can find current density on CA1 neurons of 2-3 weeks after birth increases more significantly than others; The activation curve of sodium channel shift to the repolarization, the corresponding value of half activation voltages (mV) is:-44.65±0.39,-47.42±0.56,-54.79±0.61,-58.44±0.57; Recovery time constant of sodium channel reduces with increasing age, the corresponding value of recovery time constant (ms) is:9.30±0.64,11.15±0.84, 7.95±0.55,7.18±0.56; We found there are no significant changes in deactivation curve, the corresponding value of half deactivation voltages (mV) in different group is:-52.29±0.73,-51.98±0.94,-51.44±0.72,-49.65±0.73.3. Maximum current density of whole-cell transient outward potassium current increases with increasing age, the corresponding value of maximum current density (pA/pF) is:133.11±14.36,182.10±8.55,212.80±15.98,235.86±14.60; The corresponding value of half activation voltages (mV) is::6.25±2.38,10.88±1.71, 5.31±2.46,2.69±2.75; The corresponding value of half deactivation voltages (mV) is:-58.41±0.59,-56.35±0.90,-58.06±2.13,-60.37±0.90; The corresponding value of recovery time constant (ms) is:33.14±3.19,33.78±4.22,35.13±3.92,36.19±3.62.4. Maximum current density of whole-cell calcium current increases with increasing age, the corresponding value of maximum current density (pA/pF) is:-8.02±0.56,-10.02±0.88,-27.59±1.03,-32.59±1.04, We can find current density on CA1 neurons of 2-3 weeks after birth increases more significantly than others; Both the activation and deactivation curves of whole-cell calcium channel shift to the left, The corresponding value of half activation voltages (mV) is:-6.94±0.11,-4.72±0.41,-11.72±1.05,-12.36±1.32, The corresponding value of half deactivation voltages (mV) is:-5.35±2.06,5.43±1.93,-17.80±3.37,-24.88±2.43.【Conclusions】1. In the early stages of development, the distribution of ion channel and its characters of activation, deactivation, recovery in hippocampal CA1 region pyramidal neurons change with age. That is, ion channel is in developing mode.2. The period of 2-3week after birth is fast developing period of sodium channel in hippocampal CA1 region pyramidal neurons. During this time, the distribution of sodium channel increases significantly; Activation curve of sodium channel shifts to the left as well as half activation voltage remarkably decreases. It indicates that sodium channel is prone to be activated; Recovery curve and recovery time constant show recovery of sodium channel is quicker with age.3.The density of potassium channel in hippocampal CA1 region pyramidal neurons increases significantly with increasing age,. But, no remarkable changes were found in characters of activation, deactivation and recovery.4.During the period of 2-3weeks after birth, calcium channel in hippocampal CA1 region pyramidal neurons develops rapidly. During this time, maximum current density of calcium channel increases significantly. Both the activation and deactivation curves of calcium channel shift to the left, the value of half activation and deactivation voltage obviously decrease. Which indicates that calcium channel is apt to be activated and deactivated.更多还原