光刺激体外培养的NG2细胞对海马神经元内Ca^(2+)活性和电活动的影响

Influence of Ca^(2+) and electrical activity in neurons of hippocampus by stimulating NG2 cells with blue light in vitro

目的观察选择性光刺激NG2细胞对海马神经元内Ca2+活性和电活动的影响,并初步探讨可能的作用机制。方法体外纯化培养新生SD大鼠NG2细胞,通过脂质体转染的方法,在NG2细胞中特异性表达光敏感通道(channelrhodopsin-2,ChR2)蛋白DNA,并与体外培养7 d的海马神经元共培养48 h,采用蓝光(470 nm、5 s、5 m W/cm2)选择性刺激表达ChR2蛋白的NG2细胞,利用Ca2+成像和电生理的方法记录NG2细胞周围海马神经元中的Ca2+浓度变化和电流改变情况,同时比较加入/未加入γ-氨基丁酸A型受体抑制剂情况下神经元的电流变化特点。结果当蓝光刺激表达ChR2蛋白的NG2细胞后,首先NG2细胞内的Ca2+信号增强,随后其周围海马神经元内的Ca2+升高约20%;Ca2+缓慢下降后再次出现一个Ca2+峰,幅度较第1次小;NG2细胞周围海马神经元的电流频率增加,振幅增大。而当预先在细胞外液中加入50μmol/Lγ-氨基丁酸A受体抑制剂后,海马神经元的电流频率和振幅较未加抑制剂组明显减小(P<0.05)。结论选择性光刺激表达ChR2蛋白的NG2细胞可使其兴奋,且可能通过释放γ-氨基丁酸来调节神经元活动。

Objective It has been investigated that the change of Ca 2+and electrical activity in neurons of hippocampus influenced by NG2 cells which expressed channelrhodopsin-2 (ChR2) and were stimulated by blue light, and then discussed the probable mechanism.Methods We cul-tured NG2 cells from the cortex of neonatal rats, and transfected ChR2 DNA to NG2 cells through li-posomes, then co-cultured NG2 cells expressed ChR2 protein with hippocampal neurons for 48 hours.By way of calcium imaging and electrophysiological recording, we could observe the change of calcium concentration and current in hippocampal neurons around the NG2 cells expressed ChR2 which were stimulated by blue light (470 nm, 5 s,5 mW/cm 2 ).Meanwhile, we compared the change of current in hippocampal neuron pre-adding inhibitor in extracellular fluid with no inhibitor. Results In calcium imaging experiment, we found that there was an elevation of Ca 2+concentration in NG2 cells expressed ChR2 as soon as the blue-light stimulus, and then Ca 2+concentration in neu-rons around NG2 cells increased by 20%, following a second Ca 2+ peak which its amplitude was smaller than the first.In the electrophysiological experiment, we found that it was bigger of the fre-quency and amplitude of current in neurons around NG2 cells expressed ChR2 which were stimulated by blue light than those with no blue light.However, if we added γ-amino-butyric acid (GABA) A receptor inhibitor (bicuculin, 50 μmol/L) in extracellular fluid in advance, the frequency and am-plitude of current in neurons around NG2 cells expressed ChR2 which were stimulated by blue light (5 s, 5 mW/cm 2 )were significantly smaller than those with no inhibitor (P<0.05).Conclusion Selective stimulating NG2 cells expressed ChR2 protein with blue light is able to excite NG2 cells and may regulate neural activity by releasing GABA.