A sulphide-osmium procedure for detection of metal-containing synaptic boutons in the lizard cerebral cortex

Two procedures of the Timm sulphide-silver method for the ultrastructural location of heavy metals and a sulphide-osmium procedure proposed here were applied to studying the Timm-reacting axonal boutons of the lizard cerebral cortex. It was shown that metal-containing boutons which would be labelled when using the Timm method, could be labelled by the sulphide-osmium procedure as well, i.e. perfusion and immersion in a glutaraldehyde fixative saturated with H2S vapour and then osmium postfixation. When the sulphide-osmium procedure was used, Timm-reacting boutons displayed an electron-dense loading in some of their synaptic vesicles. The histochemical significance of this labelling is suggested; metal contents (possibly zinc) in the synaptic vesicles are first transformed into metal sulphides that afterwards interact with OSO4 and render an electron-dense labelling. Moreover, chain-like electron-dense aggregates, which could not be detected in the Timm method, have been shown in endoplasmic reticulum cisternae using the sulphide-osmium procedure. The importance of both the Timm method and the sulphide-osmium procedure in ultrastructural studies in the lizard cerebral cortex could be paramount, as they define specifically one of the projection systems in the reptilian cortex. The sulphide-osmium procedure could be more useful than the Timm method since it is a simple procedure that avoids physical development, allows a better ultrastructural quality and a more precise location of the labelling.