Analysis of regional brain mitochondrial bioenergetics and susceptibility to mitochondrial inhibition utilizing a microplate based system

Andrew Sauerbeck, Jignesh Pandya, Indrapal Singh, Kevin Bittman, Ryan Readnower, Guoying Bing, Patrick Sullivan

Research output: Contribution to journalArticlepeer-review

59 Scopus citations

Abstract

The analysis of mitochondrial bioenergetic function typically has required 50-100 μg of protein per sample and at least 15. min per run when utilizing a Clark-type oxygen electrode. In the present work we describe a method utilizing the Seahorse Biosciences XF24 Flux Analyzer for measuring mitochondrial oxygen consumption simultaneously from multiple samples and utilizing only 5 μg of protein per sample. Utilizing this method we have investigated whether regionally based differences exist in mitochondria isolated from the cortex, striatum, hippocampus, and cerebellum. Analysis of basal mitochondrial bioenergetics revealed that minimal differences exist between the cortex, striatum, and hippocampus. However, the cerebellum exhibited significantly slower basal rates of Complex I and Complex II dependent oxygen consumption (p< 0.05). Mitochondrial inhibitors affected enzyme activity proportionally across all samples tested and only small differences existed in the effect of inhibitors on oxygen consumption. Investigation of the effect of rotenone administration on Complex I dependent oxygen consumption revealed that exposure to 10. pM rotenone led to a clear time dependent decrease in oxygen consumption beginning 12. min after administration (p< 0.05). These studies show that the utilization of this microplate based method for analysis of mitochondrial bioenergetics is effective at quantifying oxygen consumption simultaneously from multiple samples. Additionally, these studies indicate that minimal regional differences exist in mitochondria isolated from the cortex, striatum, or hippocampus. Furthermore, utilization of the mitochondrial inhibitors suggests that previous work indicating regionally specific deficits following systemic mitochondrial toxin exposure may not be the result of differences in the individual mitochondria from the affected regions.

Original languageEnglish
Pages (from-to)36-43
Number of pages8
JournalJournal of Neuroscience Methods
Volume198
Issue number1
DOIs
StatePublished - May 15 2011

Keywords

  • Bioenergetics
  • Brain
  • Malonate
  • Mitochondria
  • Regional differences
  • Rotenone
  • Seahorse
  • XF24

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