Freeze-fracture scanning electron microscopy and comparative freeze-etching study of parallel fiber-Purkinje spine synapses of vertebrate cerebellar cortex.

The three dimensional surface morphology, topographical arrangement and intramembrane features of parallel fiber-Purkinje spine synapses of teleost fishes and Swiss albino mice cerebellar cortex have been complementarily studied with the scanning electron microscope (SEM) using the freeze-fracture method and with transmission electron microscope (TEM) by means of freeze-etching replica and thin-sectioning techniques. The SEM fractographs revealed for the first time the outer surface morphology of non-synaptic segments and synaptic varicosities of parallel fibers, which showed clusters of spheroidal synaptic vesicles joined by an amorphous substance. Freeze-etching preparations showed an 'en face' view of P face parallel fiber varicosity (presynaptic P face) characterized by a depressed synaptic active zone with exocytotic vesicle sites, round IMPs (7.5-15 nm), and rod particles (38.5-53 nm in length). Conversely the presynaptic E face exhibited a smooth surface, randomly distributed pits and very low density distribution of round IMPs. The P face of Purkinje spine membrane (postsynaptic P face) showed high density distribution of round or ovoid IMPs, 8-18.5 nm in diameter, and rod IMPs up to 79.2 nm in length. IMPs were also observed beneath the postsynaptic membrane (subsynaptic IMPs) corresponding to the localization of postsynaptic density. The E face of Purkinje spine membrane (postsynaptic E face) showed aggregation of round IMPs, 5.6-17.4 nm in diameter, at the synaptic active zone. Some of these IMPs, particularly the large ones, may morphologically represent the glutamate-receptor ion channels complex. The subsynaptic IMPs may correspond to protein kinases. Granular material and fine filaments were observed in the postsynaptic density. A better insight into the topographical relationship between pre- and postsynaptic structures was obtained of flat and invaginated spine synapses and beside a more realistic view of their glial ensheathment. The SEM fractographs and freeze-etching images were compared with corresponding TEM thin sections for orientation and proper identification of pre- and postsynaptic structures.