A rapid Percoll gradient procedure for isolation of synaptosomes directly from an S1 fraction: viability of subcellular fractions.
The metabolic and functional viability of synaptosomes was examined in 5 subcellular fractions obtained after centrifugation of an S1 fraction from rat cerebral cortex on a discontinuous Percoll gradient (Brain Research, this volume, 1987). Fraction 4 was the most enriched for viable synaptosomes since, although it accounted for only 11.8% of the total protein recovered from the gradient, this fraction contained 23.7% of the basal synapsin I phosphorylation activity, the greatest degree of depolarisation-stimulated increase in synapsin I phosphorylation, 36.1% of the total [3H]noradrenaline uptake capacity and 46.9% of the total [3H]noradrenaline release capacity. Noradrenaline release from fraction 4 was consistent with a neuronal mechanism as it was increased with increasing K+ concentrations and was dependent on calcium. Fractions 1 and 2 contained few viable synaptosomes as judged by their capacity for noradrenaline uptake and release, yet these fractions accounted for some 62.6% of the endogenous content of noradrenaline. In part their lack of viability was due to a low content of intrasynaptosomal mitochondria, while their high content of endogenous noradrenaline was due to the presence of synaptic vesicles released from damaged nerve terminals. The synaptosomes in fraction 3 were metabolically and functionally viable, but their capacity for uptake and release of noradrenaline was lower than for fraction 4. The synaptosomes in fraction 5 showed only a small depolarisation-stimulated release of noradrenaline, suggesting a lack of viability. Part of the capacity for uptake of [3H]noradrenaline into fraction 5 was attributed to the presence of extrasynaptosomal mitochondria.(ABSTRACT TRUNCATED AT 250 WORDS)
Harrison
S M
SM
Neuroscience Group, Faculty of Medicine, University of Newcastle, New South Wales, Australia.
Jarvie
P E
PE
Dunkley
P R
PR
D000818:Animals; D002458:Cell Fractionation / Q000379:methods; D002499:Centrifugation, Density Gradient / Q000379:methods; D002540:Cerebral Cortex / Q000378:metabolism / Q000648:ultrastructure*; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009638:Norepinephrine / Q000378:metabolism; D010766:Phosphorylation; D011205:Povidone; D051381:Rats; D011919:Rats, Inbred Strains; D012822:Silicon Dioxide; D013574:Synaptosomes / Q000378:metabolism / Q000648:ultrastructure*
D009419:Nerve Tissue Proteins; C016039:Percoll; D012822:Silicon Dioxide; D011205:Povidone; D009638:Norepinephrine