Structural components in the synaptic cleft captured by freeze-substitution and deep etching of directly frozen cerebellar cortex

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Abstract
Structural components in the synaptic cleft were examined in cerebellar excitatory synapses by conventional electron microscopy and by rapid freezing followed by freeze-substitution or deep etching. Two transverse components and one parallel element were identified in the clefts of rapidly frozen and freeze-substituted synapses: (i) bridging fibrils, 4–6 nm in diameter, that span the cleft; (ii) columnar pegs, 4–6 nm wide and 8–15 nm high, projecting from the postsynaptic surface; and (iii) intervening fine fibrils running parallel to the apposed synaptic membranes. These were more clearly visible in deep-etched synapses, although the postsynaptic pegs were difficult to distinguish from intramembrane particles in the cross-fractured postsynaptic membranes. Deep etching also revealed other fibrils on the cytoplasmic surface of the postsynaptic membrane. These appear to contact the membrane surface or the intramembrane particles. Freeze-substituted materials also displayed the fibrillar components in the postsynaptic dense fuzz, but failed to display the presynaptic dense projections typically observed in thin sections or deep-etched replicas of the conventionally fixed materials. The bridging fibrils are likely to play a mechanical role in holding the synapse together, while the short pegs may be integral parts of the receptor molecules.