Nitrogen disruption of synaptoneurosomes: an alternative method to isolate brain mitochondria.
Mitochondria are known to be localized in synaptic and non-synaptic compartments in the brain. Synaptoneurosomes, which contain high numbers of mitochondria, may act as a major contaminant of currently used isolation techniques. Currently, there is no method employed to successfully disrupt synaptoneurosomes and isolate both synaptic and non-synaptic mitochondria without structural or functional damage. A novel method is reported here for disruption of synaptoneurosomes and isolation of total brain mitochondria from synaptic and non-synaptic sources using a nitrogen decompression technique. Nitrogen gas was dissolved into crude mitochondrial preparations and maintained under constant, moderate pressure. After a short incubation, the pressure was released causing the nitrogen to come out of solution as growing bubbles, which ruptures cellular and synaptoneurosomal membranes. Mitochondria isolated using this rapid technique were bioenergetically competent and exhibited functional characteristics comparable to mitochondria isolated using traditional techniques. This nitrogen decompression technique will allow for further characterization of synaptic pools of mitochondria, which are almost exclusively neuronal in origin.
Brown
Maile R
MR
Ph.D. Program in Gerontology, Lexington, KY 40536, USA.
Sullivan
Patrick G
PG
Dorenbos
Kristina A
KA
Modafferi
Edward A
EA
Geddes
James W
JW
Steward
Oswald
O
D000818:Animals; D015153:Blotting, Western / Q000379:methods; D001921:Brain / Q000166:cytology*; D003584:Cytological Techniques / Q000379:methods*; D004072:Digitonin / Q000494:pharmacology; D003576:Electron Transport Complex IV / Q000378:metabolism; D008297:Male; D008854:Microscopy, Electron / Q000379:methods; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009584:Nitrogen / Q000494:pharmacology*; D010101:Oxygen Consumption; D018272:Porins; D051381:Rats; D013347:Subcellular Fractions; D013574:Synaptosomes / Q000187:drug effects*; D050994:Voltage-Dependent Anion Channels
D009419:Nerve Tissue Proteins; D018272:Porins; D050994:Voltage-Dependent Anion Channels; C033652:postsynaptic density proteins; D003576:Electron Transport Complex IV; D004072:Digitonin; D009584:Nitrogen