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J Neurochem. 2009 Apr;109(2):644-55. doi: 10.1111/j.1471-4159.2009.05997.x. Epub 2009 Feb 16.

Simultaneous single neuron recording of O2 consumption, [Ca2+]i and mitochondrial membrane potential in glutamate toxicity.

Author information

1
Laboratory of Neurosciences, National Institute on Aging Intramural Research Program, Biomedical Research Center, Baltimore, Maryland, USA.

Abstract

In order to determine the sequence of cellular processes in glutamate toxicity, we simultaneously recorded O(2) consumption, cytosolic Ca(2+) concentration ([Ca(2+)](i)), and mitochondrial membrane potential (mDeltapsi) in single cortical neurons. Oxygen consumption was measured using an amperometric self-referencing platinum electrode adjacent to neurons in which [Ca(2+)](i) and mDeltapsi were monitored with Fluo-4 and TMRE(+), respectively, using a spinning disk laser confocal microscope. Excitotoxic doses of glutamate caused an elevation of [Ca(2+)](i) followed seconds afterwards by an increase in O(2) consumption which reached a maximum level within 1-5 min. A modest increase in mDeltapsi occurred during this time period, and then, shortly before maximal O(2) consumption was reached, the mDeltapsi, as indicated by TMRE(+) fluorescence, dissipated. Maximal O(2) consumption lasted up to 5 min and then declined together with mDeltapsi and ATP levels, while [Ca(2+)](i) further increased. mDeltapsi and [Ca(2+)](i) returned to baseline levels when neurons were treated with an NMDA receptor antagonist shortly after the [Ca(2+)](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 O(2) consumption, [Ca(2+)](i), and mDeltapsi. The data obtained using this new method are consistent with a model where Ca(2+) influx causes ATP depletion, despite maximal mitochondrial respiration, minutes after glutamate receptor activation.

PMID:
19226367
PMCID:
PMC2805059
DOI:
10.1111/j.1471-4159.2009.05997.x
[Indexed for MEDLINE]
Free PMC Article

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