新生大鼠海马神经元原代培养及膜片钳全细胞记录

The primary culture for hippocampal neurons of neonatal rats and patch clamp whole-cell recording

目的探讨一种适用于膜片钳全细胞记录的海马神经元培养方法。方法选择鼠龄在1d内的Wistar大鼠,迅速断头,取双侧海马,神经基础培养基添加B-27及L-谷氨酰胺进行神经元原代培养,培养至第10天时,使用免疫荧光技术配合神经元特异性核蛋白NeuN进行细胞鉴定;神经元的电生理特征由膜片钳全细胞记录。结果该方法所培养神经元状态良好,经鉴定发现纯度可高达100%,通过膜片钳全细胞法可记录到自发性动作电位及自发性兴奋性突触后电流。结论本方法操作简便、高效,所培养的海马神经元状态良好,适用于膜片钳全细胞记录。

Objective To discuss a culture method of hippoeampal neurons,which is suitable for patch clamp whole-cell record- ing. Methods Neonatal Wistar rats（〈1 d） were decollated and bilateral hippocampus were separated rapidly. Neural basal media supplemented withB-27 and L-glutamine were used for hippocampal neurons primary culture. On day 10 ,neurons were identified by immunofluorescence of neuronal nuclei（NeuN）. Electrophysiological properties of neurons were recorded by patch clamp whole-cell recording. Results The cultured neurons were in good condition. By identifying, the purity of cultured neurons was almost high to 100%. Action potential and spontaneous excitatory postsynaptic current were recorded by whole-cell patch clamp. Conclusion This is a simple and efficient method, and the cultured neurons are in good condition. Moreover, neurons are suitable for patch clamp whole-cell recording.