Rapid isolation of synaptoneurosomes and postsynaptic densities from adult mouse hippocampus

Abstract

Previous postsynaptic density (PSD) isolation methodologies have utilized either whole brain or discrete brain regions of relatively large mammals such as dogs and rats. The present report details a simple and highly effective procedure for the rapid isolation of PSDs from small amounts of adult mouse hippocampus that has several advantages. First, by substituting synaptoneurosomes for synaptosomes as starting material, we have decreased the steps, time, and amount of tissue required to isolate PSDs. Second, by modifying critical steps in the synaptic isolation protocols we were able to isolate PSDs from less than 200 mg of mouse hippocampi in 3 h. Electron micrographs of isolated synaptoneurosomes showed presynaptic vesicles and densely stained membranes representing PSDs. Morphological examination of these PSDs by electron microscopy revealed a preparation that seems to be quite pure, with little or no membrane contamination. A comparison by Western blot analysis of synaptoneurosome and PSD fractions suggests that this technique yields a purified sample. Moreover, two different protocols using swing and fixed bucket rotors were used for this small-scale PSD isolation and both resulted in a very pure partition, supporting the idea that this procedure is reliable and consistent.

Fig. 1

Schematic depicting protocols for the isolation of (A) synaptoneurosomes and (B) postsynaptic densities (PSDs) from mouse hippocampus.

Fig. 2

Electron micrographs of representative sample of synaptoneurosomes isolated from mouse hippocampus. (A) A typical large field view of synaptoneurosomes (2500×). Arrow indicates synaptic junctions. Mitochondria (m) also are present. (B) A close up view of a complete synaptoneurosome. (C) Higher magnification of the synaptic site where the presynaptic terminal displays vesicles and a postsynaptic membrane contains PSDs (25,000×). Synaptosome (S), presynaptic vesicles (pv), neurosome (N), and densely stained membranes that characterize PSDs (PSD) are shown.

Fig. 3

Electron micrographs of representative samples of PSDs isolated from mouse hippocampus. (A) A typical large field view of isolated PSDs showing densely stained structures that are enriched in the sample (2500×). Arrow indicates membrane contamination. (B) A close up view of a PSD preparation (25,000×).

Fig. 4

Expression of presynaptic and postsynaptic markers in homogenate, synaptoneurosome, and PSD preparations. (A) Representative Western blot analysis of homogenate (H), synaptoneurosome (S), and PSDs (P) isolated using fixed rotor and swing bucket protocols. Equal amounts of protein (7.5 μg/lane for PSD-95, tubulin, αCaMKII, and GFAP, 10 μg/lane for the detection of synaptotagmin) were loaded in each sample (n = 8). (B) Group data (mean ± S.E.M., n = 8) of synaptotagmin and PSD 95 immunoreactivity in Western blots of mouse hippocampus. Immunoreactivity was normalized to the homogenate. ‘*’ denotes statistical significance (p < 0.05 by Student’s t-test).