谷氨酸毒性研究:单个神经元O2消耗量、细胞内Ca2+浓度和线粒体膜电位的同时记录-技术前沿-资讯-生物在线

谷氨酸毒性研究:单个神经元O2消耗量、细胞内Ca2+浓度和线粒体膜电位的同时记录

作者:旭月(北京)科技有限公司 2009-06-16T00:00 (访问量:6320)

非损伤微测技术用于神经毒性机制的研究
谷氨酸毒性研究:单个神经元O2消耗量、细胞内Ca2+浓度和线粒体膜电位的同时记录
点击查看大图

神经元是可兴奋性细胞,在突触活动及产生动作电位后,神经元要产生大量的ATP驱动离子泵以提高胞内的Na+和Ca2+水平。谷氨酸(Glu)是重要的神经递质,负责快速突触传递及突触传递强度的长期变化,并参与认知和记忆等过程;但如果过度激活谷氨酸受体,谷氨酸会导致离子平衡破坏、细胞死亡等毒性反应。

本文为明确谷氨酸神经毒性的机制,将非损伤微测技术与激光共聚焦技术结合,以大鼠幼崽大脑皮层的神经元为被测样品,用非损伤微测技术(a self-referencing O2 electrode)检测单个神经元O2消耗量(即O2内流),而用激光共聚焦技术检测其胞内Ca2+浓度和线粒体膜电位。


点击查看大图
单个神经元细胞在Glu作用下O2消耗量、胞内Ca2+浓度
和线粒体膜电位的变化过程

研究发现,在谷氨酸作用下,细胞内Ca2+浓度上升,随后O2消耗量增加,这期间线粒体膜电位也发生相应改变。该结论直接证实了下述谷氨酸毒性机理模型:谷氨酸受体被激活后能引起Ca2+内流,导致细胞内ATP损耗。

将非损伤微测技术与激光共聚焦等技术相结合,检测生物样品内部和外部离子分子或其他信息的变化情况,已经成为揭示生命过程机理机制的重要手段。

关键词:非损伤微测技术(a self-referencing O2 electrode), 氧气消耗量(oxygen consumption), 谷氨酸(glutamate);
参考文献:Gleichmann M, et al. J Neurochem, 2009,109: 644-655

Abstract:
In order to determine the sequence of cellular processes in glutamate toxicity, we simultaneously recorded O2 consumption,cytosolic Ca2+ concentration ([Ca2+]i), and mitochondrial membrane potential (mDw) in single cortical neurons. Oxygen consumption was measured using an amperometric selfreferencing platinum electrode adjacent to neurons in which [Ca2+]i and mDw were monitored with Fluo-4 and TMRE+,respectively, using a spinning disk laser confocal microscope.Excitotoxic doses of glutamate caused an elevation of [Ca2+]i followed seconds afterwards by an increase in O2 consumption which reached a maximum level within 1–5 min. A modest increase in mDw occurred during this time period, and then, shortly before maximalO2 consumption was reached, themDw, as indicated by TMRE+ fluorescence, dissipated. Maximal O2 consumption lasted up to 5 min and then declined together with mDw and ATP levels, while [Ca2+]i further increased. mDw and [Ca2+]i returned to baseline levels when neurons were treated
with an NMDA receptor antagonist shortly after the [Ca2+]i increased. Our unprecedented spatial and time resolution revealed that this sequence of events is identical in all neurons, albeit with considerable variability in magnitude and kinetics of changes in O2 consumption, [Ca2+]i, and mDw. The data obtained using this new method are consistent with a model where Ca2+ influx causes ATP depletion, despite maximal mitochondrial respiration, minutes after glutamate receptor activation.
Keywords: excitotoxicity, glutamate, oxygen consumption.

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