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PLoS One. 2012;7(4):e34465. doi: 10.1371/journal.pone.0034465. Epub 2012 Apr 4.

Investigation of mitochondrial dysfunction by sequential microplate-based respiration measurements from intact and permeabilized neurons.

Author information

  • 1Department of Anesthesiology and Center for Shock, Trauma and Anesthesiology Research, University of Maryland School of Medicine, Baltimore, Maryland, United States of America.

Abstract

Mitochondrial dysfunction is a component of many neurodegenerative conditions. Measurement of oxygen consumption from intact neurons enables evaluation of mitochondrial bioenergetics under conditions that are more physiologically realistic compared to isolated mitochondria. However, mechanistic analysis of mitochondrial function in cells is complicated by changing energy demands and lack of substrate control. Here we describe a technique for sequentially measuring respiration from intact and saponin-permeabilized cortical neurons on single microplates. This technique allows control of substrates to individual electron transport chain complexes following permeabilization, as well as side-by-side comparisons to intact cells. To illustrate the utility of the technique, we demonstrate that inhibition of respiration by the drug KB-R7943 in intact neurons is relieved by delivery of the complex II substrate succinate, but not by complex I substrates, via acute saponin permeabilization. In contrast, methyl succinate, a putative cell permeable complex II substrate, failed to rescue respiration in intact neurons and was a poor complex II substrate in permeabilized cells. Sequential measurements of intact and permeabilized cell respiration should be particularly useful for evaluating indirect mitochondrial toxicity due to drugs or cellular signaling events which cannot be readily studied using isolated mitochondria.

PMID:
22496810
[PubMed - indexed for MEDLINE]
PMCID:
PMC3319583
Free PMC Article

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