Conventional and high resolution scanning electron microscopy and cryofracture techniques as tools for tracing cerebellar short intracortical circuits.

The present paper shows the potential contribution of conventional and high resolution scanning electron microscopy (SEM) to trace short intracortical circuits in cryofractured fish, primate and human cerebelli. Conventional SEM slicing technique allowed us to identify afferent mossy and climbing fibers and their synaptic relationship in the granular layer. SEM freeze-fracture method exposed the mossy glomerular synapses and the axo-dendritic connections of climbing fibers. At the Purkinje cell layer, the cryofracture process removed the satellite Bergmann glial cell layer, displaying a partial view of the supra- and infra-ganglionic plexuses of Purkinje cells and the ascending pathways of climbing fibers. High resolution SEM (HRSEM) showed the specimen specific secondary electron (SE-I) image of axosomatic synapses on Golgi cell surface. At the molecular layer, the outer surface of parallel fiber synaptic varicosities were distinguished, establishing the cruciform en passant synaptic contact with the Purkinje cell dendritic spines. HRSEM showed the fractured parallel fiber synaptic varicosities containing spheroidal synaptic vesicles embedded in a high dense extravesicular material. Conventional SEM and gold-palladium coating are useful to trace intracortical circuits. With HRSEM and chromium coating, it is possible to study the outer and inner surfaces of synaptic connections.