There is electrophysiological and biochemical evidence to suggest that nicotinic ACh receptor antagonists modulate the release of ACh from motor terminals (see e.g. Prior et al., 1995). However, biochemical assays of ACh release can be distorted by release from non-neuronal sources whilst electrophysiological techniques require an inference of prejunctional changes from postjunctional observations. Therefore, the aim of this project was to use fluorescent microscopy to study the prejunctional actions of nicotinic ACh receptor antagonists in a model of ACh release that is devoid of these problems.

FM1-43 is a fluorescent dye that is used to monitor secretory activity in motor terminals (Betz & Bewick, 1993). In our studies, the dye was loaded into synaptic vesicles within motor terminals of the isolated mouse (C57/BL6, male, 20-25g) hemidiaphragm by exposure to 4µM FM1-43 during 1 min of 50Hz nerve stimulation or 6 min of 30mM KCl. Loaded motor terminals were exposed to 30mM KCl to trigger exocytosis (destaining). Destaining was evoked in the absence or presence of vecuronium (1µM),
-bungarotoxin (125nM) or hexammethonium (200µM). All experiments were performed at 17 – 22 °C in normal Krebs-Hense solution. Images of motor terminals were collected every 2 min during destaining to determine the effects of the test agents on the time course of exocytotic activity.

In controls, following exposure to KCl, there was a short delay (lasting over 4 minutes) before there was any detectable loss of FM1-43 from the motor terminals. Vecuronium abolished this delay in destaining and also significantly attenuated the rate of destaining (Table 1). Maximum loss of staining intensity was seen within 20 minutes and this was not affected by any of the compounds studied (Table 1). All the effects of vecuronium were also seen with -bungarotoxin, but not with hexamethonium.

Table 1: Effect of nicotinic AChR antagonists on KCl-induced loss of FM1-43 staining.

The vecuronium- and -bungarotoxin-induced decrease in the rate of release of the dye is consistent with an action of the agents to inhibit the mobilisation of ACh for release. Thus, we have shown that FM1-43 can be used to monitor the action of nicotinic ACh receptor antagonists on mouse motor terminals. However, the physiological process that underlies the delayed destaining with KCl and its attenuation by vecuronium and -bungarotoxin is unexplained and requires further study.

Betz, W. J. & Bewick, G. S. (1993). J. Physiol., 460: 287-309.
Prior, C. et al. (1995). Gen. Pharmacol., 26: 659-666.