铅对大鼠皮质神经元γ-氨基丁酸A型受体介导电流及GABA能突触传递的抑制作用OACSTPCD
Inhibitory effect of lead on GABA A receptor-mediated currents and GABAergic synaptic transmission in rat cortical neurons
目的 研究铅(Pb2+)对大鼠皮质神经元γ-氨基丁酸(GABA)A型受体介导电流(IGABA)及GABA能突触传递的抑制作用及其机制.方法 ①分离出生0d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IGABA,检测不同浓度Pb2+(1,5,10,50和100 μmol·L-1)(Y管给药,作用时间20s)对GABA(100 μmol·L-1)激活IGABA的影响,并检测Pb2+ 50 μmol·L-1(Y管给药,作用时间20s)对不同浓度GABA(1,10,50,100,500和1000 μmol·L-1)激活IGABA的影响.②取15～19 d日龄雄性SD大鼠大脑制作厚度为350 μm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Pb2+ 10 μmol·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100 μmol·L-1浓度时,随浓度升高,Pb2+抑制原代培养神经元IGABA的作用增强,IC50值为(68±20)μmol·L-1.②Pb2+ 50 μmol·L-1抑制GABA最大激活电流(P<0.01),升高GABA的EC50值,由无Pb2+组的(20±6)μmol·L-1增加到(87±39)μmol·L-1,表明Pb2+可能以非竞争性机制抑制IGABA.③脑片实验中,与处理前比较,Pb2+10 μmol·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,Pb2+ 10 μmol·L-1抑制AP的频率(P<0.01),降低神经元的整体兴奋性.结论 Pb2+对原代培养神经元IGABA有明显的抑制作用;Pb2+可能通过抑制皮质神经元的AP抑制sIPSC的频率;提示Pb2+对原代培养神经元IGABA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Pb2+中毒机制的复杂性.
OBJECTIVE To investigate the inhibitory effect and mechanism of lead(Pb2+)on γ-amino-butyric acid(GABA)A receptor-mediated currents(IGABA)and GABAergic synaptic transmission in rat cortical neurons.METHODS ①The 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 Pb2+ at different concentrations(1,5,10,50 and 100 μmol·L-1)on IGABA induced by GABA 100 μmol·L-1.② The effects of Pb2+ 50 μmol·L-1 on IGABA induced by GABA at different concentrations(1,10,50,100,500 and 100 μmol·L-1)were detected.③Brain slices(350 μm)were prepared from SD rats(15-19 d).The spontaneous inhibitory post-synaptic currents(sIPSCs),miniature inhibitory post-synaptic currents(mIPSCs)and current injection-induced action potential(AP)were recorded to detect the effects of Pb2+ 10 μmol·L-1 on the amplitude and frequency of sIPSCs and mIPSCs,and the frequency of AP.RESULTS ①Pb2+ inhibited IGABA in a concentration-dependent manner,and IC50 was(68±20)μmol·L-1.②Pb2+ 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 Pb2+ might inhibit IGABA in a non-competitive mechanism.③Pb2+ 10 μmol·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 addi-tion,Pb2+ decreased the frequency of evoked AP by current injection(P<0.01)and reduced the overall excitability of rat cortical neurons.CONCLUSION Pb2+ can significantly inhibit IGABA in primary cultured neurons.In the brain slice experiment,Pb2+ may affect sIPSCs frequency by inhibiting the AP of cortical neurons,suggesting that there are different intrinsic mechanisms through which Pb2+ inhibits both IGABA in primary cultured neurons and the frequency of sIPSCs in brain slice neurons,which points to the complexity of the mechanism of Pb2+ poisoning.
高文良;张红;袁谊;郭蕊;刘兴阳;邓显华;孙灏
济南市第二妇幼保健院,山东 济南 271100安徽师范大学生命科学学院,安徽 芜湖 241000厦门大学医学院,福建 厦门 361005
药学
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
leadγ-aminobutyric acid A receptorcortical neuronsspontaneous inhibitory post-synaptic currentsminiature inhibitory post-synaptic currents
《中国药理学与毒理学杂志》 2024 (001)
31-38 / 8
国家自然科学基金(81471160) National Natural Science Foundation of China(81471160)
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