Scanning Microscopy


Mouse and teleost fish cerebelli were processed by the freeze-fracture methods for scanning and transmission electron microscopy in order to study the three-dimensional morphology and intramembrane features of climbing fiber-Purkinje spine synapses. In addition, Alcian Blue and ruthenium chloride stainings were applied to mouse cerebellar tissue to investigate the polyanion composition of these excitatory synapses under the transmission electron microscope. In the granular layer, tendril and glomerular collaterals of climbing fibers were observed. In the molecular layer climbing fibers exhibited a characteristic crossing-over or arborescence pattern type of bifurcation, Scheibel's collaterals and multiple thorn synapses with Purkinje spiny dendrites. At the synaptic active zones of climbing fiber-Purkinje spine synapses the freeze-etching replicas showed focal aggregates of intramembrane particles at the E and P faces of the pre-and past-synaptic membranes. Membrane protuberances and pits were also observed at the pre-synaptic membrane. Ultracytochemical study of the climbing fiber synaptic varicosities revealed an Alcian Blue and ruthenium chloride positive material which appeared at the axoplasm surrounding the synaptic vesicles, at the pre-and post-synaptic densities and in the synaptic cleft. The axoplasmic material was sensitive to testicular hyaluronidase, therefore it would correspond to glycosaminoglycans (hyaluronic acid and/or chondroitin sulphates), which have been earlier reported in other cerebellar excitatory systems as those of mossy fiber-granule cell and parallel fiber-Purkinje dendritic spine.

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