Confocal, scanning and transmission electron microscopic study of cerebellar mossy fiber glomeruli

A correlative microscopic study of vertebrate cerebellar mossy fiber glomeruli has been carried out to obtain a three-dimensional view of the multisynaptic contacts formed by afferent mossy fibers with the granule and Golgi cell dendrites and by the monosynaptic relationship of Golgi cell axonal ramifications with granule cell dendrites. Samples of mice, hamsters, teleost fishes and human species were studied by means of one of the following procedures: confocal laser scanning microscopy (CLSM), ethanol-cryofracturing technique and conventional scanning electron microscopy (CSEM) and transmission electron microscopy (TEM) by ultrathin sections and freeze-etching replicas. CLSM, by means of montages of z-series of the cerebellar granular layer, provided a new approach to explore mossy fiber trajectory and branching bifurcation pattern and the quantitative relationship between mossy fibers and granule cell dendrites. CSEM and freeze-fracture method for SEM offered a more detailed in-depth, higher resolution image of outer and inner surface organization of mossy fiber glomeruli. TEM, either by ultrathin sections or freeze-etching replicas, was used as complementary technique for proper orientation, comparative purposes and rational identification of pre- and postsynaptic structures. Freeze-etching replicas showed in addition the real extent of glial cell cytoplasm encapsulating the synaptic glomeruli. The integrated microscopy approach offers a new and more comprehensive view of three-dimensional morphology, organization and quantitative aspects of mossy fiber glomeruli.