Facilitation by 3,4-diaminopyridine of regenerative acetylcholine release from mouse motor nerve

1. Effects of 3,4-diaminopyridine (DAP) on endplate potentials (e.p.ps) were studied in mouse phrenic nerve-hemidiaphragms. 2. In cut muscle preparations, low concentrations of DAP (2-20 microns) increased the amplitude of e.p.ps and shifted the curve relating Ca2+ concentration to e.p.p. amplitude leftward. 3. High concentration of DAP (40-4000 microns) prolonged the duration of e.p.ps dose-dependently up to one hundred fold (ca. 200 ms), yielding, in addition to the normal phasic e.p.p., a prolonged plateau depolarization component which was often preceded by an upstroke depolarization. During the plateau depolarization, nerve stimulations did not evoke any e.p.p. 4. The plateau component of prolonged e.p.ps was suppressed by tubocurarine, verapamil, nifedipine, Mn2+ and Cd2+ (but not by atropine) at low concentrations that had negligible effect on the amplitude of miniature e.p.ps or the phasic component of e.p.ps. Abolition of the plateau component by these agents restored the capability of the nerve terminal to evoke e.p.ps on nerve stimulation. 5. Low concentrations of neostigmine (0.01-0.02 microns) markedly lengthened DAP-prolonged e.p.ps. However, the regenerative endplate depolarization evoked in the presence of high concentrations of neostigmine (0.3-0.5 microns) was not prolonged by DAP. 6. Tetraethylammonium (1 mM) did not provoke prolonged e.p.ps but acted cooperatively with DAP to prolong the duration of plateau depolarization. At a high concentration (3 mM), tetraethylammonium depressed the amplitude of miniature e.p.ps and abolished DAP-prolonged e.p.ps. 7. In uncut muscle preparations, DAP apparently did not modify the time course and amplitude of miniature e.p.ps. Upon direct stimulation by current injection at endplate, DAP increased the muscle action potentials by only about 30%, but induced no prolonged depolarization. 8. These results suggest that the prolonged e.p.ps induced in the presence of DAP are due to a regenerative release of acetylcholine from motor nerve and the induction probably involves a presynaptic Ca2+ channel different from that for normal e.p.ps. It may be inferred that the regenerative acetylcholine release is recruited by Ca2 + channels modulated by nicotinic receptors and K+ channels.