單個(gè)神經(jīng)元O2消耗量、細(xì)胞內(nèi)Ca2+濃度和線粒體膜電位的同時(shí)記錄

非損傷微測(cè)技術(shù)用于神經(jīng)毒性機(jī)制的研究 谷氨酸毒性研究：?jiǎn)蝹�(gè)神經(jīng)元O2消耗量、細(xì)胞內(nèi)Ca2+濃度和線粒體膜電位的同時(shí)記錄 神經(jīng)元是可興奮性細(xì)胞，在突觸活動(dòng)及產(chǎn)生動(dòng)作電位后，神經(jīng)元要產(chǎn)生大量的ATP驅(qū)動(dòng)離子泵以提高胞內(nèi)的Na+和Ca2+水平。谷氨酸（Glu）是重要的神經(jīng)遞質(zhì)，負(fù)責(zé)快速突觸傳遞及突觸傳遞強(qiáng)度的長(zhǎng)期變化，并參與認(rèn)知和記憶等過程；但如果過度激活谷氨酸受體，谷氨酸會(huì)導(dǎo)致離子平衡破壞、細(xì)胞死亡等毒性反應(yīng)。 本文為明確谷氨酸神經(jīng)毒性的機(jī)制，將非損傷微測(cè)技術(shù)與激光共聚焦技術(shù)結(jié)合，以大鼠幼崽大腦皮層的神經(jīng)元為被測(cè)樣品，用非損傷微測(cè)技術(shù)（a self-referencing O2 electrode）檢測(cè)單個(gè)神經(jīng)元O2消耗量（即O2內(nèi)流），而用激光共聚焦技術(shù)檢測(cè)其胞內(nèi)Ca2+濃度和線粒體膜電位。 單個(gè)神經(jīng)元細(xì)胞在Glu作用下O2消耗量、胞內(nèi)Ca2+濃度 和線粒體膜電位的變化過程 研究發(fā)現(xiàn)，在谷氨酸作用下，細(xì)胞內(nèi)Ca2+濃度上升，隨后O2消耗量增加，這期間線粒體膜電位也發(fā)生相應(yīng)改變。該結(jié)論直接證實(shí)了下述谷氨酸毒性機(jī)理模型：谷氨酸受體被激活后能引起Ca2+內(nèi)流，導(dǎo)致細(xì)胞內(nèi)ATP損耗。 將非損傷微測(cè)技術(shù)與激光共聚焦等技術(shù)相結(jié)合，檢測(cè)生物樣品內(nèi)部和外部離子分子或其他信息的變化情況，已經(jīng)成為揭示生命過程機(jī)理機(jī)制的重要手段。 關(guān)鍵詞：非損傷微測(cè)技術(shù)(a self-referencing O2 electrode), 氧氣消耗量(oxygen consumption), 谷氨酸(glutamate); 參考文獻(xiàn)：Gleichmann M, et al. J Neurochem, 2009,109: 644-655 全文下載：

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.