全细胞记录急性分离海马锥体神经元电压门控通道电流(英文)  

Whole-cell recording for voltage-gate channel current in acutely isolated hippocampal pyramidal neurons

作　　者：蔡青[1] 朱忠良[1] 樊小力[1] 白转丽[1] 贾宁[1] 李霞[1] 

机构地区：[1]西安交通大学医学院生理与病理生理学系,陕西省西安市710061

出　　处：《中国临床康复》2006年第10期186-188,共3页Chinese Journal of Clinical Rehabilitation

基　　金：国家自然科学基金(30270445)~~

摘　　要：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。BACKGROUNDt Hippoeampus is an important region involved in learning and memory. Some methods for freshly isolated hippocampal pyramidal neurons have been described. These methods need multiple enzymes or the procedure is complex. OBJECTIVE： To acquire large quantities of hippocampal neurons from rats suitable for patch clamp study. DESIGN： An animal experimental observation. SETTING： Department of Physiology and Pathophysiology, School of Medicine, Xi ＇an Jiao Tong University. MATERIALS： The experiments were carried out in the Medical Expertmental Center, School of Medicine, Xi＇an Jiao Tong University between March and October 2004. The animals were the 10-15-day-old Sprague- Dawley. rats, both male and female. METHODS： Hippocampal pyramidal neurons were freshly isolated. Delayed rectifier potassium current and voltage-gated Ca^2＋ currents were recorded by whole-cell patch clamp configuration. MAIN OUTCOME MEASURES： Acute isolated neurons were observed with a Leica microscope. Recordings of delayed rectifier potassium currents and voltage-gated Ca^2＋. currents were obtained. RESULTS： （1） Under the inverted microscope, the acute isolated neurons had a smooth and glassy surface, a three-dimensional contour, a pyramidal shape with a longer apical dendrite and several basal dendrites. （2） After the whole cell recording configuration was formed, voltage-gated barium currents through calcium channels were elicited by 200 ms depolarizing potential from -70 mV to ＋20 mV, with an increment of 10 mV. a holding potential was -90 mV. Delayed rectifier potassium current （Ikd） was elicited by a protocol consisting of a multiple depolarizing pulses （-60 mV to ＋50 mV）, preceded by a single prepulse step to -50 mV, with inactivated transient outward potassium currents from a holding potential of -80 mV. CONCLUSION： This method is simple and ideal for the dissociation of neurons from rat hippocampus, and offers a powerful tool for functional analysis of ion channels by patc

关 键 词：膜片钳术 海马 钙通道 

分 类 号：R741[医药卫生—神经病学与精神病学]