The effects of prolonged repetitive stimulation in hemicholinium on the frog neuromuscular junction.

1. Cutaneous pectoris nerve-muscle preparations from the frog were stimulated for prolonged periods in solutions with curare alone, curare and hemicholinium no. 3 (HC-3), or curare and glucose plus choline. End-plate potentials (e.p.p.s) and miniature end-plate potentials (m.e.p.p.s) were recorded intracellularly. Black widow spider venom (BWSV) was applied to determine the degree of depletion of the transmitter stores. 2. The ultrastructure of the neuromuscular junctions was studied in the electron microscope. Some of the preparations were fixed immediately at the end of the period of stimulation and others were fixed about an hour after BWSV had been applied. In some experiments horseradish peroxidase (HRP) was present during the period of stimulation and the fixed tissue was treated to reveal the distribution of the tracer. 3. The amplitude of the e.p.p. fell rapidly to almost zero during 2 hr of stimulation at 2/sec in 100 muM HC-3 and little recovery occurred during a subsequent hour of rest. About 2-7 times 10-5 quanta were secreted. The e.p.p.s usually persisted throughout the period of stimulation in the other solutions and 2-2-6 times as much transmitter was secreted. 4. When BWSV was applied immediately at the end of the period of stimulation in HC-3, almost no m.e.p.p.s were discharged and only small m.e.p.p.s were discharged when the venom was applied after an hour of rest. 5. When BWSV was applied to unstimulated terminals that had been bathed in HC-3, or to terminals that had been stimulated and rested for an hour in glucose plus choline, m.e.p.p.s of nearly normal amplitude were discharged. 6. Terminals stimulated for 2 hr at 2/sec in 100 muM HC-3 contained a normal complement of synaptic vesicles and a large proportion of vesicles were labelled with HRP when the tracer was present during the period of stimulation. 7. BWSV induced the almost complete depletion of vesicles from terminals that had been stimulated in HC-3. 8. Depletion of vesicles also occurred when terminals were stimulated for 20 min at 10/sec after they had been previously stimulated for 2 hr at 2/sec in HC-3. These terminals showed extensive infolding of the axolemma and they contained swollen mitochondria. 9. These results indicate that stimulation in HC-3 depletes terminals of their store of transmitter but not of their population of vesicles and that vesicles empty of transmitter can fuse with and reform from the axolemma of the nerve terminal.