Rapid isolation of synaptoneurosomes and postsynaptic densities from adult mouse hippocampus.
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.
Villasana
Laura Elena
LE
Department of Molecular Physiology & Biophysics, Baylor College of Medicine, Houston, TX 77030, USA.
Klann
Eric
E
Tejada-Simon
Maria Victoria
MV
D000818:Animals; D015153:Blotting, Western / Q000379:methods; D002458:Cell Fractionation / Q000379:methods*; D002498:Centrifugation / Q000379:methods; D006624:Hippocampus / Q000502:physiology / Q000648:ultrastructure*; D066298:In Vitro Techniques; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D046529:Microscopy, Electron, Transmission / Q000379:methods; D009419:Nerve Tissue Proteins / Q000378:metabolism; D013569:Synapses / Q000648:ultrastructure*; D013570:Synaptic Membranes / Q000378:metabolism / Q000648:ultrastructure*; D013574:Synaptosomes / Q000378:metabolism / Q000648:ultrastructure*
D009419:Nerve Tissue Proteins