体外癫痫神经元细胞膜显微结构的研究

目的 利用原子力显微镜观察体外癫痫神经元细胞膜显微结构.方法 将培养14 d的神经元放入"无镁"细胞外液处理3 h后,再重新放回含镁的正常细胞外液培养,利用膜片钳记录神经元的自发性电活动.将"无镁"细胞外液处理3h的神经元定为实验组,将未经"无镁"外液处理的神经元定为对照组.分别在80 μm×80 μm,2μm×2 μm和500 nm×500 nm扫描范围,对两组神经元细胞膜表面进行扫描,同时测量两组神经元表面相关结构的直径和深度.结果 膜片钳提示"无镁"细胞外液处理3 h和恢复正常外液14 d时,神经元存在自发的癫痫样放电.在扫描范围为80 μm×80 μm时,两组神经元细胞膜表面光滑;在2 μm×2 μm时,两组神经元细胞膜表面出现一些小凹;在500 nm×500 nm时,实验组神经元表面小凹的直径和深度分别为(114.86±9.33)nm和(5.71±0.69)nm,对照组为(116.4±9.13)nm和(5.69±0.71)nm,两组相比无显著性差异(P＞0.05).结论 原子力显微镜是进行细胞膜显微结构观察的良好工具;"无镁"外液处理神经元3 h,神经元细胞膜显微结构未发生改变.

Microstructural observation of epileptic neurons in vitro by atomic force microscopy

OBJECTIVE: To observe the microstructure of the cell membrane of epileptic neurons using atomic force microscopy (AFM). METHODS: Model of epileptic neurons was established by subjecting the neurons culture for 14 days in vitro to magnesium-free media treatment for 3 h. Patch clamp technique was applied to record the electrophysiological activity of the epileptic neurons. AFM was performed to observe and measure the microstructure of the cell membrane of the epileptic neuron. RESULTS: After a 3-hour treatment with magnesium-free media, the epileptic neurons displayed sustained epileptiform discharge, which continued after the neurons were returned to normal medium culture on day 14. Under AFM scanning size of 80 microm x 80 microm and 2 microm x 2 microm, no obvious difference in the morphology of the cell membrane was noted between epileptic and normal neurons; under the scanning size of 500 nm x 500 nm, small pits occurred in the cell membrane in both groups, but no significant difference was found in the dimension of the pits between the two groups (the diameter and depth of the pits was 114.86-/+9.33 nm and 5.71-/+0.69 nm in epileptic neurons, and 116.4-/+9.13 nm and 5.69-/+0.71 nm in the control neurons, respectively, P>0.05). CONCLUSION: AFM provides a new method for observing neuronal membrane microstructure at nanometer resolutions. No significant alterations occur in the membrane of the neurons after a 3-hour magnesium-free media treatment.