Fig. 8.

Presynaptic phalloidin injection inhibits exocytosis and compensatory endocytosis. A: an axon was first impaled with an electrode containing KCl (Ai; no phalloidin signal) and stimulated (2,000 stimuli) in FM1-43 to label presynaptic vesicle clusters (Aii). B: FM1-43 fluorescence at puncta in Aii. Axon stimulated intracellularly (16,000 stimuli, 20 Hz) and intensity of puncta declined. The presynaptic electrode was removed and replaced with one containing phalloidin, which was pressure injected (black arrow). Intracellular phalloidin application was confirmed by imaging its Alexa Fluor 568 label (Ci; image at red arrow in B), which marked the same puncta previously labeled with FM1-43 (Aii). The tissue was again superfused with FM1-43 and the axon stimulated (2,000 stimuli). This revealed the same puncta (labeled with FM1-43) but with significantly reduced fluorescence than in Aii. A further 16,000 stimuli failed to destain these puncta. Ci: phalloidin labels structures associated with vesicle clusters in the same axon as A. Cii: FM1-43 puncta labeled in same axon as A but after destaining and restaining following phalloidin injection. D: mean normalized data from 3 preparations treated as for B. E: summary of staining and destaining data from 3 preparations; control destaining (green) and control restaining (grey) of the same puncta following the destaining protocol. After phalloidin injection, restaining was minimal (1st red bar). Subsequent destaining (2nd red bar) was also minimal.