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

Abstract

A method for preparation of synaptosomes from rat cerebral cortex, on a discontiinuous Percoll gradient, was previously developed for use with a P 2 pellet ( Brain Research, 372 (1986) 115–129). Here the Percoll method has been adapted for use with an S 1-supernatant which eliminates a potentially damaging resuspension step and saves over 30 min, representing a third of the total preparation time. The homogeneity of the synaptosomes in each of the 5 subcellular fractions obtained with the S 1-Percoll method was determined biochemically by analysis of the distribution of total protein, myelin basic protein, synapsin I and pyruvate dehydrogenase across the gradient. Electron microscopy was also used to determine the homogeneity of the synaptosomes, as well as to determine their morphological characteristics. Fraction 4 was the most enriched in synaptosomes and contained the lowest level of contamination by myelin, extrasynaptosomal mitochondria and plasma membranes. The yield of synaptosomes in fraction 4 with the S 1-Percoll method was 1.4-fold greater than with the P 2-Percoll method. While all other fractions contained some synaptosomes the major additional content in fractions 1–3 and 5 was, respectively, unidentified small membranes, myelin, synaptic plasma membranes and extrasynaptosomal mitochondria. Fraction 1 was enriched for very small synaptosomes (0.34 μm mean diameter) only 8% of which contained mitochondria, while fractions 2–4 progressively included larger synaptosomes containing more mitochondria. Fraction 5 synaptosomes were approximately the same size as those in fraction 4 (0.63 μm mean diameter), but 83% contained mitochondria, significantly more than in fraction 4. The synaptosomes in fraction 5 were found to be relatively resistant to hypotonic lysis, explaining a previously observed lack of phosphorylation of synapsin I in this fraction. The differences in homogeneity and morphological characteristics of the synaptosomes in fractions 1–5 suggest that the basis for their fractionation on Percoll gradients is different from that achieved with the more traditional procedures for isolating synaptosomes and that unique synaptosomal fractions are obtained with the S 1-Percoll procedure.