以膜片钳技术急性分离新生大鼠尾核神经元

背景：许多离子通道研究需要单个分散的神经元。人工原代培养的神经细胞受环境的影响，自身特性改变较大，而急性分离的神经元却能相对保持完好的生理特性。 目的：建立急性分离尾核神经元的方法，用膜片钳技术研究尾核神经元离子通道及其信号转到机制，利于深入了解尾核的功能。 设计、时间及地点：以细胞为对象的观察实验，于2008-02/12在三峡大学医学院实验中心完成。 材料：新生7~10 d Wistar乳鼠12只，雌雄不限。 方法：取7~10 d的大鼠，采用酶和机械分离法制备分散的单个尾核神经元，利用全细胞膜片钳技术记录电压依赖性钙电流。 主要观察指标：用全细胞膜片钳急性分离大鼠尾核神经元的形态学视察和电生理特性。 结果：用链蛋白酶(Protease)消化及机械分离法，急性分离的新生大鼠尾核神经元，表面光洁,胞膜完整，有较长的突起，形态和生理特性良好。利用急性分离的新生大鼠尾核神经元观察L-钙离子通道电流，所记录的电学参数在正常生理范围内，较好地保存电压依赖性钙离子通道活性。 结论：分离出的神经元形态正常，有较长的轴突；保存了主要的离子通道活性，成功建立了一种适用于膜片钳技术的大鼠尾核神经元急性分离方法。

Acute isolation of caudate nucleus neurons with patch-clamp technique in neonate rats

BACKGROUND: Single scattered neurons are needed in most studies on ion channels. However, artificial primary cultured neurons, for the influence from the environment, have a lot of changes in their characteristics. Comparatively, acutely isolated neurons can keep intact physiological characteristics. OBJECTIVE: To establish an ideal method for acute isolation of caudate nucleus neurons and to study their ion channels and signal transduction mechanism with patch clamp technique for the benefit of deeply understanding functions of caudate nucleus. DESIGN, TIME AND SETTING: An observational experiment on cells was performed at the Central Laboratory, Medicine Science College of China Three Gorges University from February to December in 2008. MATERIALS: Twelve 7- to 10-day-old neonatal Wistar rats, of either gender, were selected for this study. METHODS: Scattered single caudate nucleus neuron was obtained from 7- to 10-day-old neonatal Wistar rats with a combination of enzymatic and mechanical separation method. Voltage-dependent calcium currents were determined with whole-cell patch clamp technique MAIN OUTCOME MEASURES: Observation of morphology and electrophysiological characteristics of acutely isolated caudate nucleus neurons with whole-cell patch clamp. RESULTS: The caudate nucleus neuron acutely isolated with protease digestion and mechanical isolation method was found to have regular configuration, intact cell membrane, long neuraxons, excellent morphology and physiological nature. Their L-calcium ion channel current observation showed electrical parameters within normal physiological range, and activity of voltage-dependent calcium ion channel was well preserved. CONCLUSION: As neurons isolated in this way have normal morphology and long neuraxons, and activities of primary iron channels are well preserved, we conclude that an acute isolation method of caudate nucleus neurons from neonate rats with patch clamp technique is successfully established.