Cd2+- and K+-evoked ACh release induce different synaptophysin and synaptobrevin immunolabelling at the frog neuromuscular junction

Abstract

Synaptophysin and synaptobrevin, two integral proteins of synaptic vesicles, have been used as immunocytochemical markers of the synaptic vesicle membrane during Cd²⁺- or K⁺-induced ACh release at the frog neuromuscular junction. ACh release was stimulated in cutaneous pectoris nerve-muscle preparations by: (1) 1 mM Cd²⁺ in Ca²⁺-free medium for a period of 3 h, (2) 25 or 40mM K⁺ in normal Ringer's solution. Synaptophysin and synaptobrevin were immunolabelled in single fibres teased from fixed muscles using rabbit antisera raised against synaptophysin and synaptobrevin revealed with fluoresceinconjugated IgG. The postsynaptic ACh receptors were simultaneously labelled with rhodaminated α-bungarotoxin. Unstimulated and K⁺-stimulated preparations showed synaptophysin and synaptobrevin immunolabelling only after membrane permeabilization with 0.1% Triton X-100. In preparations stimulated with Cd²⁺ in Ca²⁺-free medium, the immunofluorescence was also observed in non Triton X-100 treated muscle fibres. Confocal laser scanning microscopy analysis revealed that in unstimulated and K⁺-stimulated preparations, synaptophysin and synaptobrevin immunofluorescence appears as bands regularly spaced along the permeabilized nerve terminals and that their distribution corresponds to clusters of synaptic vesicles. After Cd²⁺ stimulation in Ca²⁺-free medium, labelling for both proteins is irregularly distributed, being more intense at the lateral margins of swollen nerve terminals, suggesting a translocation of synaptic vesicle proteins to the axolemma. At the electron microscopic level, Cd²⁺ stimulation in Ca²⁺-free medium produces nerve terminal swelling and synaptic vesicle depletion. The results show that when ACh release is stimulated under an impairment of synaptic vesicle recycling, which leads to synaptic vesicle depletion, synaptophysin and synaptobrevin translocation occurs. These findings are in favour of a permanent incorporation of synaptic vesicle membrane into the axolemma. In contrast, after K⁺ stimulation, the immunofluorescence and the normal synaptic vesicle population observed, suggest that a double process of synaptic vesicle exo-endocytosis rapidly occurs, without incorporation of synaptic vesicle components into the axolemma.