The spatial relationship between the perineuronal proteoglycan network and the synaptic boutons as visualized by double staining with cationic colloidal iron method and anti-calbindin-D-28K immunohistochemistry in rat cerebellar nuclei

The present study demonstrated the precise spatial relationship between meshes in the perineuronal proteoglycan network and the terminal boutons of synaptically associated axons. Sections from the rat cerebellum were stained with cationic colloidal iron (pH 1.0-1.5), and successively immunostained with anti-calbindin-D-28K monoclonal antibody. Cationic iron stained sulfated proteoglycans around the nerve cell of the medial cerebellar nucleus, whereas the anti-calbindin antibody labeled the Purkinje cells including their axons terminating on large neurons in the cerebellar nucleus. It was found that each synaptic bouton fits into a mesh of the perineuronal network. The individual meshes appeared to be divided by partitions faintly stained with the colloidal iron. Electron microscopy of cationic colloidal iron-stained ultrathin sections revealed that the synaptic boutons were separated from each other by the proteoglycan matrix and that each of them was further divided into two or more contact areas of presynaptic membrane by the same matrix. This suggests that individual synapses are protected against the effects of adjacent synaptic transmission, and that each of them may be subdivided by this manner of partitioning, like pads of a cat's paw.