A rapid Percoll gradient procedure for isolation of synaptosomes directly from an S 1 fraction: viability of subcellular fractions

Abstract

The metabolic and functional viability of synaptosomes was examined in 5 subcellular fractions obtained after centrifugation of an S 1 fraction from rat cerebral cortex on a discontinous 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 46.99% 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 62.2% 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 for uptake of [ 3H]noradrenaline into fraction 5 was attributed to the presence of extrasynaptosomal mitochondria. The endogenous content of dopamine across the gradient in general paralled that of endogenous noradrenaline except for fractions 4 and 5, suggesting that nerve terminals enriched in different neurotransmitter types may have been separated. These studies provide evidence for a unique feature of the S 1-Percoll procedure, namely its ability to separate viable from non-viable synaptosomes.