Immunocytochemical localization of S-100 protein binding sites in synaptosomal fractions and subfractions

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Abstract
Immunocytochemical evidence is presented of the ultrastructural localization of binding sites for S-100 protein in synaptosomal fractions and subfractions. Synaptosomes or isolated synaptosomal subfractions were first incubated with S-100, then centrifuged to remove unbound S-100, and finally fixed before treatment with anti-S-100 antiserum, using the unlabeled antibody peroxidase-antiperoxidase (PAP) method. When intact synaptosomes were used, the immunoreaction product was localized to the postsynaptic density including the postsynaptic membrane. In some reactive synaptosomes, the presynaptic membrane was labeled as well, in the region of synaptic contact. No reaction deposit was found in the synaptic cleft or on intrasynaptosomal structures. Divalent cations (Ca2+ and Mg2+) were essential for S-100 to interact with synaptosomes. Of the synaptosomal subfractions tested, i.e., synaptic vesicles and intraterminal mitochondria, only synaptic vesicles showed immunoreactivity when treated with S-100 and anti-S-100 antiserum as described above. The S-100 immunoreactivity in synaptic structures documented in this report parallels the distribution of the high-affinity binding sites for radiolabeled S-100 in synaptosomal fractions and subfractions.