title: Analysis of regional brain mitochondrial bioenergetics and susceptibility to mitochondrial inhibition utilizing a microplate based system.
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.
authors.affiliation: Department of Anatomy and Neurobiology, University of Kentucky, Lexington, KY 40536, USA.
authors.forename: Andrew
authors.initials: A
authors.lastname: Sauerbeck
authors.forename: Jignesh
authors.initials: J
authors.lastname: Pandya
authors.forename: Indrapal
authors.initials: I
authors.lastname: Singh
authors.forename: Kevin
authors.initials: K
authors.lastname: Bittman
authors.forename: Ryan
authors.initials: R
authors.lastname: Readnower
authors.forename: Guoying
authors.initials: G
authors.lastname: Bing
authors.forename: Patrick
authors.initials: P
authors.lastname: Sullivan
mesh_terms: D000818:Animals; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods; D001921:Brain / Q000033:anatomy & histology / Q000648:ultrastructure*; D042967:Electron Transport Complex I / Q000378:metabolism; D042963:Electron Transport Complex II / Q000378:metabolism; D004734:Energy Metabolism / Q000187:drug effects / Q000502:physiology*; D005453:Fluorescence; D008297:Male; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology*; D010100:Oxygen / Q000378:metabolism*; D051381:Rats; D011916:Rats, Inbred F344; D012402:Rotenone / Q000031:analogs & derivatives / Q000494:pharmacology; D013997:Time Factors
chemical_list: D012402:Rotenone; C028404:rotenolone; D042963:Electron Transport Complex II; D042967:Electron Transport Complex I; D010100:Oxygen