Further Studies on Electron Microscopic Localization of Acetylcholine-Like Cations in Frog Neuromuscular Junctions.

Abstract

In the present work we have localized at the electron microscope level acetylcholine (ACh) in frog neuromuscular junctions, simultaneously in storage (synaptic vesicles) and releasing space (synaptic cleft) by a cytochemical method, previously described (19), which involved the precipitation of ACh-like cations with silicotungstic acid. The fixation took place in different physiological states (resting, during electrical stimulation and recovery). At rest, ACh was localized in the synaptic vesicles as point-like precipitates and in the synaptic cleft beneath some but not all the “active zones” as laminar precipitates. We think that the precipitates seen in the synaptic vesicles correspond to stored ACh, while those in the synaptic cleft to ACh released through the “active zones”. After a prolonged tetanic electrical stimulation, laminar precipitates covered the whole axoplasm of the nerve terminals, and it appeared that they extend beyond the nerve terminal membrane in contact with Schwann cells. The laminar precipitates found in the nerve terminals might correspond to a newly synthetized axoplasmic ACh (or leaked into the axoplasm from the synaptic vesicles) while the precipitates which filled whole synaptic cleft are presumed to be ACh released by electrical stimulation of the motor nerve. During the recovery period, the distributions of both types of precipitates became similar to those observed at rest. This cytochemical fixation method of ACh-like cations appears to be a useful “tool” for the quick capture of the neurotransmitter ACh in the synaptic vesicles, axoplasm and synaptic cleft, and therefore helpful for identification of cholinergic nerve terminals.