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1. The frequency (0-5-150 Hz) and calcium dependence (0-5-2-0 mM) of the effects of the nicotinic antagonist tubocurarine (0f2 /uM) on acetylcholine (ACh) liberation from motor nerve terminals has been examined using binomial analysis of quantal transmitter release. 5. Binomial analysis revealed that all the changes in EPC quantal content associated with both nicotinic antagonists were due to changes in the size of the pool of quanta in the nerve terminal available for immediate release with no effect on the probability of release of an individual quantum. Stertz, Peper, Chau & Wilson, 1973;Glavinovic, 1979c; Magleby, Pallotta & Zhang, 1990). This is manifest in twitch tension Terrar, 1981;Gibb & Marshall, 1984 L. Tian, C. Prior, J. Dempster and I. C. Marshall colleagues (for reviews see Bowman, Gibb, Harvey & Marshall, 1986;Bowman, Marshall, Gibb & Harborne, 1988;Bowman, Prior & Marshall, 1990) have contended that tetanic fade and EPP or EPC amplitude run-down are consequences of tubocurarine inhibiting a nicotinic ACh autoreceptor located on the motor nerve terminal. It is proposed that these putative receptors form part of a positive-feedback system, activation of which by ACh enhances the mobilization and release of ACh during highfrequency motor nerve stimulation. This conclusion is based largely on the ability to detect electrophysiologically a decrease in the quantal release of ACh from motor nerve terminals towards the latter part of high-frequency bursts of motor nerve stimulation . On the other hand, it has been reported that tubocurarine can increase, rather than decrease, ACh release from rat (Wilson, 1982;Wilson & Thomsen, 1991), cat (Blaber, 1970;, frog (Matzner, Parnas & Parnas, 1988) and mouse (Ferry & Kelly, 1988) (Wilson, 1982;Wilson & Thomsen, 1991 have attributed the tubocurarine-induced increase of quantal content, and subsequent increased run-down of EPPs, to a frequency-dependent action of tubocurarine on a negativefeedback motor nerve terminal nicotinic ACh autoreceptor. Blaber (1973) also reported that tubocurarine increased the fractional release of ACh during high-frequency nerve stimulation, although in this study the initial quantal content was not measured. Blaber (1973) proposed that the phenolic moieties of tubocurarine were responsible for its ability to increase fractional release, as trimethyltubocurarine did not increase release. He suggested that these phenolic groups, absent in trimethyltubocurarine, were responsible for the enhancement of the fractional release of ACh, either by increasing the stimulus-evoked influx of calcium ions into the nerve terminal or by increasing the activity of intracellular calcium ions.In an attempt to shed more light on the prejunctional effects of tubocurarine at the neuromuscular junction, we have studied the frequency-and calcium-dependent effects of tubocurarine on the quantal release of ACh from motor nerve terminals. Quantal ACh release at a range of motor nerve stimulation frequencies (0-5-150 Hz) has been determined from the amplitud...

We investigated whether recycled cholinergic synaptic vesicles, which were not refilled with ACh, would join other synaptic vesicles in the readily releasable store near active zones, dock, and continue to undergo exocytosis during prolonged stimulation. Snake nerve-muscle preparations were treated with 5 microM vesamicol to inhibit the vesicular ACh transporter and then were exposed to an elevated potassium solution, 35 mM potassium propionate (35 KP), to release all preformed quanta of ACh. At vesamicol-treated endplates, miniature endplate current (MEPC) frequency increased initially from 0.4 to >300 s-1 in 35 KP but then declined to <1 s-1 by 90 min. The decrease in frequency was not accompanied by a decrease in MEPC average amplitude. Nerve terminals accumulated the activity-dependent dye FM1-43 when exposed to the dye for the final 6 min of a 120-min exposure to 35 KP. Thus synaptic membrane endocytosis continued at a high rate, although MEPCs occurred infrequently. After a 120-min exposure in 35 KP, nerve terminals accumulated FM1-43 and then destained, confirming that exocytosis also still occurred at a high rate. These results demonstrate that recycled cholinergic synaptic vesicles that were not refilled with ACh continued to dock and undergo exocytosis after membrane retrieval. Thus transport of ACh into recycled cholinergic vesicles is not a requirement for repeated cycles of exocytosis and retrieval of synaptic vesicle membrane during prolonged stimulation of motor nerve terminals.

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