摘要
目的研究铅(Pb^(2+))对大鼠皮质神经元γ-氨基丁酸(GABA)A型受体介导电流(I_(GABA))及GABA能突触传递的抑制作用及其机制。方法①分离出生0 d的SD大鼠大脑皮质神经元进行原代培养,培养7~14 d用膜片钳系统记录神经元GABA激活的I_(GABA),检测不同浓度Pb^(2+)(1,5,10,50和100μmol·L^(-1))(Y管给药,作用时间20 s)对GABA(100μmol·L^(-1))激活I_(GABA)的影响,并检测Pb^(2+)50μmol·L^(-1)(Y管给药,作用时间20 s)对不同浓度GABA(1,10,50,100,500和1000μmol·L^(-1))激活I_(GABA)的影响。②取15~19 d日龄雄性SD大鼠大脑制作厚度为350μm的脑片样本,记录自发抑制性突触后电流(sIPSC)、微小抑制性突触后电流(mIPSC)和注入电流诱导的动作电位(AP),检测Pb^(2+)10μmol·L^(-1)(灌流速度2 mL·min^(-1))处理前和处理5 min后sIPSC和mIPSC振幅和频率及AP频率。结果①在10,50和100μmol·L^(-1)浓度时,随浓度升高,Pb^(2+)抑制原代培养神经元I_(GABA)的作用增强,IC_(50)值为(68±20)μmol·L^(-1)。②Pb^(2+)50μmol·L^(-1)抑制GABA最大激活电流(P<0.01),升高GABA的EC50值,由无Pb^(2+)组的(20±6)μmol·L^(-1)增加到(87±39)μmol·L^(-1),表明Pb^(2+)可能以非竞争性机制抑制I_(GABA)。③脑片实验中,与处理前比较,Pb^(2+)10μmol·L^(-1)处理5 min后可逆地抑制神经元sIPSC的频率(P<0.01)而未影响其振幅,而mIPSC的频率和振幅均未受到影响。此外,Pb^(2+)10μmol·L^(-1)抑制AP的频率(P<0.01),降低神经元的整体兴奋性。结论Pb^(2+)对原代培养神经元I_(GABA)有明显的抑制作用;Pb^(2+)可能通过抑制皮质神经元的AP抑制sIPSC的频率;提示Pb^(2+)对原代培养神经元I_(GABA)的抑制以及对脑片神经元sIPSC频率的抑制可能存在不同的机制,反映了Pb^(2+)中毒机制的复杂性。
OBJECTIVE To investigate the inhibitory effect and mechanism of lead(Pb^(2+))onγ-amino⁃butyric acid(GABA)A receptor-mediated currents(I_(GABA))and GABAergic synaptic transmission in rat cortical neurons.METHODS①The cortical neurons from 0 d Sprague Dawley(SD)rats were cultured for experiments.The cultured cells(7-14 d)were recorded using the patch-clamp technique to analyze the effects of Pb^(2+)at different concentrations(1,5,10,50 and 100μmol·L^(-1))on I_(GABA)induced by GABA 100μmol·L^(-1).②The effects of Pb^(2+)50μmol·L^(-1)on I_(GABA)induced by GABA at different concentrations(1,10,50,100,500 and 100μmol·L^(-1))were detected.③Brain slices(350μm)were prepared from SD rats(15-19 d).The spontaneous inhibitory post-synaptic currents(sIPSCs),miniature inhibitory postsynaptic currents(mIPSCs)and current injection-induced action potential(AP)were recorded to detect the effects of Pb^(2+)10μmol·L^(-1)on the amplitude and frequency of sIPSCs and mIPSCs,and the frequency of AP.RESULTS①Pb^(2+)inhibited I_(GABA)in a concentration-dependent manner,and IC_(50)was(68±20)μmol·L^(-1).②Pb^(2+)also suppressed the maximum current induced by GABA(P<0.01),with a significant increase of the GABA′s EC50 from(20±6)μmol·L^(-1)to(87±39)μmol·L^(-1),indicating that Pb^(2+)might inhibit I_(GABA)in a non-competitive mechanism.③Pb^(2+)10μmol·L^(-1)inhibited the frequency(P<0.01)rather than the ampli⁃tude of sIPSCs reversibly,but had no effect on eigher the frequency or amplitude of mIPSCs.In addition,Pb^(2+)decreased the frequency of evoked AP by current injection(P<0.01)and reduced the overall excitability of rat cortical neurons.CONCLUSION Pb^(2+)can significantly inhibit I_(GABA)in primary cultured neurons.In the brain slice experiment,Pb^(2+)may affect sIPSCs frequency by inhibiting the AP of cortical neurons,suggesting that there are different intrinsic mechanisms through which Pb^(2+)inhibits both I_(GABA)in primary cultured neurons and the frequency of sIPSCs in brain slice neurons,which points to the complexity of the mechanism of Pb^(2+)poisoning.
作者
高文良
张红
袁谊
郭蕊
刘兴阳
邓显华
孙灏
GAO Wenliang;ZHANG Hong;YUAN Yi;GUO Rui;LIU Xingyang;DENG Xianhua;SUN Hao(No.2 Maternal and Child Healthcare Hospital of Jinan City,Jinan 271100,China;School of Life Sciences,Anhui Normal University,Wuhu 241000,China;School of Medicine,Xiamen University,Xiamen 361005,China)
出处
《中国药理学与毒理学杂志》
CAS
北大核心
2024年第1期31-38,共8页
Chinese Journal of Pharmacology and Toxicology
基金
国家自然科学基金(81471160)。
关键词
铅
γ-氨基丁酸A型受体
神经元
自发抑制性突触后电流
微小抑制性突触后电流
lead
γ-aminobutyric acid A receptor
cortical neurons
spontaneous inhibitory postsynaptic currents
miniature inhibitory post-synaptic currents
