Detection of zinc in isolated nerve terminals using a modified Timm's sulfide-silver method

An ultrastructural method for detecting the presence of zinc in isolated nerve terminals from the mammalian brain is described. This method is based on the well-known Timm's sulfide-silver technique that has been used by many investigators to detect and localize zinc-containing pathways in sections of intact brain tissue. We report here a modification of this technique that we have used to assess the homogeneity, at the electron microscopic level, of a zinc-enriched synaptosomal fraction from the rat hippocampus. This technique allows biochemical assays to be performed on samples of the same tissue if desired, and also provides the large amounts of tissue needed for synaptosomal isolation. Results indicated that all of the mossy fiber synaptosomes, identified on the basis of their large size and characteristic morphology, stained for zinc using this method, as did about one-third of the smaller synaptic profiles present in the same fraction. The method described here should be useful for determining zinc retention and localization in isolated synaptosomes from other regions of the mammalian central nervous system.