SUBCELLULAR FRACTIONATION ON PERCOLL GRADIENT OF MOSSY FIBER SYNAPTOSOMES - MORPHOLOGICAL AND BIOCHEMICAL-CHARACTERIZATION IN CONTROL AND DEGRANULATED RAT HIPPOCAMPUS

A method for preparation of hippocampal mossy fiber synaptosomes direc tly from the postnuclear pellet is presented. This method represents a n adaption of that previously described for the isolation of synaptoso mes by centrifugation through Percoll gradients directly from the supe rnatant fraction. We have characterized by electron microscopy two fra ctions, PII and PIII, enriched in mossy fiber synaptosomes; fraction P III had 75% mossy fiber synaptosomes with well-preserved morphology (l arge size 3 mum, complex morphology, high synaptic vesicle density, mu ltisynapses), whereas fraction PII contained 12%. These fractions were enriched in lactate dehydrogenase activity indicating that the integr ity of synaptosomes was preserved. Compared with the other synaptosoma l fractions, these fractions showed greater levels of dynorphin A (1-8 ) immunoreactivity and endogenous zinc, which are particularly concent rated in hippocampal mossy fiber terminals. Furthermore, we prepared s ynaptosomes from adult hippocampus after neonatal irradiation, which d estroys the majority of granule cells and associated mossy fibers. The levels of dynorphin and zinc decreased by 88 and 70% in fraction PII and by 95 and 90%, respectively, in PIII. These results suggest that t he rapid Percoll procedure is convenient for the purification of mossy fiber synaptosomes.