Contribution of conventional and high resolution scanning electron microscopy and cryofracture technique to the study of cerebellar synaptic junctions.

Abstract

The cerebelli of teleost fishes, primates and humans were processed for conventional and high resolution scanning electron microscopy (SEM) to study the outer and inner surfaces of axo-dendritic, glomerular and axosomatic synapses. The cryofracture technique, either by slow or fast freezing, exposed the hidden neuronal surfaces of synaptic connections, selectively removing the glial ensheathment. Axo-dendritic junctions of climbing fibers and Golgi axonal ramifications were studied in gold-palladium and chromium coated samples. Chromium coating showed different mass density and topographic contrast between axonal and dendritic profiles. Conventional SEM of cryofractured glomerular synapses exhibited the outer surface view of en passant mossy fibers glomeruli, in which granule cell dendrites appear surrounding the afferent mossy fibers. The cryo-fracture method also exposed the axosomatic contacts of basket axonal collaterals upon the Purkinje cell somatic surface and the climbing fiber bulbous endings upon tertiary Purkinje dendrites. Field emission high resolution SEM of parallel fiber-Purkinje cell dendritic spines showed the inner organization of pre-synaptic endings and the three-dimensional structure of the synaptic membrane complex. The spheroidal synaptic vesicles appeared embedded in a homogeneous axoplasmic substance. Round subunits, 15-20 nm in diameter, were observed as intrinsic components of the post-synaptic membrane and associated with the post-synaptic density. High resolution SEM offers SE-I images comparable in resolution to thin section electron microscopy and freeze-etching replicas at intermediate magnifications.