單個神經元O2消耗量、細胞內Ca2+濃度和線粒體膜電位的同時記錄

非損傷微測技術用于神經毒性機制的研究 谷氨酸毒性研究：單個神經元O2消耗量、細胞內Ca2+濃度和線粒體膜電位的同時記錄 神經元是可興奮性細胞，在突觸活動及產生動作電位后，神經元要產生大量的ATP驅動離子泵以提高胞內的Na+和Ca2+水平。谷氨酸（Glu）是重要的神經遞質，負責快速突觸傳遞及突觸傳遞強度的長期變化，并參與認知和記憶等過程；但如果過度激活谷氨酸受體，谷氨酸會導致離子平衡破壞、細胞死亡等毒性反應。 本文為明確谷氨酸神經毒性的機制，將非損傷微測技術與激光共聚焦技術結合，以大鼠幼崽大腦皮層的神經元為被測樣品，用非損傷微測技術（a self-referencing O2 electrode）檢測單個神經元O2消耗量（即O2內流），而用激光共聚焦技術檢測其胞內Ca2+濃度和線粒體膜電位。 單個神經元細胞在Glu作用下O2消耗量、胞內Ca2+濃度 和線粒體膜電位的變化過程 研究發現，在谷氨酸作用下，細胞內Ca2+濃度上升，隨后O2消耗量增加，這期間線粒體膜電位也發生相應改變。該結論直接證實了下述谷氨酸毒性機理模型：谷氨酸受體被激活后能引起Ca2+內流，導致細胞內ATP損耗。 將非損傷微測技術與激光共聚焦等技術相結合，檢測生物樣品內部和外部離子分子或其他信息的變化情況，已經成為揭示生命過程機理機制的重要手段。 關鍵詞：非損傷微測技術(a self-referencing O2 electrode), 氧氣消耗量(oxygen consumption), 谷氨酸(glutamate); 參考文獻：Gleichmann M, et al. J Neurochem, 2009,109: 644-655 全文下載：http://www.xuyue.net/xylt/viewthread.php?tid=535&extra=page%3D1

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.