Neuromuscular transmission in a mammalian preparation in the absence of blocking drugs and the effect of D-tubocurarine.

1. Neuromuscular transmission in the presence and absence of D-tubocurarine was examined in cut muscle preparations of the rat diaphragm using intracellular recording techniques. The method of localizing the end-plates is described and evidence is presented indicating that end-plate potential (e.p.p.) recordings were made close to the end-plate region and that reliable estimates of the quantum content of e.p.p.s could be made.2. During repetitive nerve stimulation at rates between 2 and 10 Hz, e.p.p. amplitudes declined in response to the first 6-8 stimuli to reach a plateau level which was maintained for 1500 stimuli. At higher rates of stimulation some 500-700 stimuli were needed before a plateau level of amplitude was reached. It was demonstrated that these amplitude changes reflected similar changes in the quantal release of ACh.3. Tubocurarine in a concentration of 4 x 10(-8) g/ml. had no effect on quantal release but did reduce quantal size. In the presence of tubocurarine, 8 x 10(-8) g/ml., small reductions in the quantum content of the first e.p.p. in response to a train of stimuli and the maintained quantal release were observed. In the presence of tubocurarine 4 x 10(-7) g/ml., the quantum content of the first e.p.p. and the maintained release were further reduced, to the level previously described for curarized preparations. At this concentration a significant effect of tubocurarine upon the presynaptic measurements could be detected (t test). Studies on single junctions revealed that the post- and presynaptic effects of tubocurarine developed with different time courses.4. Neuromuscular preparations in vivo have been reported to be much more resistant to maintatined stimulation than curarized neuromuscular preparations in vitro. It is suggested that this disparity is explained by the presynaptic action of tubocurarine.5. From the quantal release rates of the present investigation and reports of ACh release/stimulus/end-plate in previous investigations it can be calculated that a quantum of ACh contains between 12,000 and 21,000 molecules.