Fluorescent dyes, such as FM1-43, that label synaptic vesicles in an activity-dependent fashion (Betz & Bewick, 1993) represent a novel optical approach for studying pharmacological effects on transmitter release. After loading, the dye can be liberated by excitation of the terminal and this destaining reflects vesicle exocytosis. The aim of this study was to evaluate the use of FM1-43 to optically monitor nerve- and KCl-evoked release of ACh from motor endings in the C57/BL6 mouse hemidiaphragm.

Motor terminals in isolated mouse (male, 20-25g) hemidiaphragm muscles (17 – 22 °C in normal Krebs-Henseleit solution) were stained with 4µM FM1-43 by exposure to 30mM KCl for 6 min or by electrical field or direct nerve stimulation (20 - 50 Hz for 1 min). Following loading, and one hour of wash, motor terminals were exposed to 30mM KCl or 20Hz nerve stimulation to elicit exocytosis and consequently dye destaining. Images of motor terminals were captured every 1 or 2 min and terminal fluorescence in each image was expressed as a percent of the value from the image collected immediately prior to destaining.

Maximum loss of fluorescent intensity was seen within 20 min of the start of destaining. For terminals stained using nerve stimulation or destained using KCl, the maximum dye loss was consistently around 25 %. In contrast, for motor endings loaded using KCl, there was little dye release with nerve stimulation (Table 1, one-way ANOVA). However, in these muscles, the subsequent application of KCl produced a decrease in intensity to 72.6 ± 1.3 % control (n=6) which is the same as that seen with KCl alone.

Table 1:Effect of staining and destaining protocol on FM1-43 staining intensity.

For KCl-evoked destaining, but not nerve-evoked destaining, there was a slight delay in the start of the decrease in fluorescent intensity after the start of the destaining protocol. Thus, at 4 min after the start of destaining, intensity was reduced with nerve stimulation but not with KCl (Table 1, one sample Student’s t test).

We suggest that KCl causes some FM1-43 to be loaded into a vesicular compartment that can not be subsequently released by high frequency nerve stimulation. This points to a fundamental difference in vesicular recycling that is promoted by elevated K⁺ and by nerve stimulation. The delay in destaining with KCl but not nerve stimulation, also suggests differences in ACh mobilization with the two destaining procedures.

Betz, W. J. & Bewick, G. S. (1993). J. Physiol., 460, 287-309.