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背景:海马是涉及学习、记忆的重要脑区,已有研究者描述了急性分离海马神经元的方法。但这些方法需要多种酶联合使用、分离过程复杂。目的:建立一种适用于膜片钳研究,简单、快速地分离海马神经元的方法。设计:动物实验观察。单位:西安交通大学医学院生理与病理生理学系。材料:实验于2004-03/10在西安交通大学医学院医学实验中心完成。实验动物选用出生10～15d的Sprague-Dawley大鼠,性别不限。方法:急性分离海马神经元;应用全细胞记录模式记录延迟整流钾电流以及电压门控钙电流。主要观察指标:观察急性分离海马神经元的形态;记录海马神经元延迟整流钾电流、电压门控钙电流。结果:①倒置显微镜观察急性分离的神经元具有光滑、透亮的表面,胞体呈锥体性,有一个较长的顶树突和几个基树突。②分离过程未破坏其电生理特性,钳制电压-90mV,给予时程200ms,阶跃10mV,由-70～+20mV去极化脉冲刺激激活钙电流得到电压门控钙电流。钳制电压-80mV,给予-50mV时程50ms去极化预刺激失活瞬时外向钾电流,再给予时程200ms,阶跃10mV,由-60mV至+50mV去极化脉冲刺激激活钾电流,得到延迟整流钾电流。结论:本方法分离的神经元,适合应用膜片钳技术进行离子通道研究。
BACKGROUND: Hippocampus is an important brain area involved in learning and memory. Researchers have described a method for acutely isolating hippocampal neurons. However, these methods require a combination of enzymes, the separation process is complicated. OBJECTIVE: To establish a simple and rapid method for patch-clamp study of hippocampal neurons. Design: Animal experiment observation. Unit: Department of Physiology and Pathophysiology, Xi'an Jiaotong University School of Medicine. MATERIALS: Experiments were performed at Medical Experimental Center, Xi'an Jiaotong University, March 03, 2004. Sprague-Dawley rats of 10 ~ 15 days of age were selected as experimental animals. Their sex was not limited. Methods: The hippocampal neurons were isolated acutely; delayed rectifier potassium current and voltage-gated calcium current were recorded using whole-cell recording mode. MAIN OUTCOME MEASURES: The morphology of acutely isolated hippocampal neurons was observed. The delayed rectifier potassium currents and voltage-gated calcium currents were recorded in hippocampal neurons. Results: ①Inverted microscopy showed that the acutely isolated neurons had a smooth and translucent surface with a pyramidal cell body with a long apical dendrites and several basal dendrites. The separation process did not destroy the electrophysiological characteristics, clamping voltage -90mV, given the time course of 200ms, step 10mV, -70 ~ +20 mV depolarization pulse-activated calcium current to obtain voltage-gated calcium current. Clamped voltage -80mV, given -50mV 50ms depolarization pre-stimulation inactivation of transient outward potassium current, and then given the time 200ms, step 10mV, from -60mV to +50 mV depolarization pulse-activated potassium current activation, delayed Rectifier potassium current. Conclusion: The neurons isolated by this method are suitable for ion channel studies using patch clamp technique.