Large synaptosome fractions were isolated from the cerebellar and cerebral cortices of rats and were incubated with Clostridium botulinum type A neurotoxin in vitro. The binding of the neurotoxin to the synapses was observed by electron microscopy, using the double-sandwich immunocytochemical method. Botulinum neurotoxin was preferentially bound to the presynaptic membrane in the large synaptosome fraction. The binding regions for the neurotoxin were localized on both the extrajunctional and junctional areas of the presynaptic membranes and appeared as patches of various sizes. However, they did not exist on the postsynaptic membranes. Botulinum neurotoxin is proposed to be a useful analytical tool for understanding the characteristics of the presynaptic membranes in the central nervous system.

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