Clathrin-mediated endocytosis near active zones in snake motor boutons

We have used the activity-dependent probe FM1-43 with electron microscopy ( EM) to examine endocytosis at the vertebrate nerve-muscle synapse. Preparat ions were fixed after very brief neural stimulation at reduced temperature, and internalized FM1-43 was photoconverted into an electron-dense reaction product. To locate the reaction product, we reconstructed computer renderi ngs of individual terminal boutons from serial EM sections. Most of the rea ction product was seen in 40-60 nm vesicles. All of the labeled vesicles we re clathrin-coated, and 92% of them were located within 300 nm of the plasm a membrane, suggesting that they had undergone little processing after retr ieval from their endocytic sites. The vesicles (and by inference the sites) were not dispersed randomly near the plane of the membrane but instead wer e clustered significantly near active zones. Additional reaction product wa s found within putative macropinosomes; these appeared to form from deep me mbrane invaginations near active zones. Thus two mechanisms of endocytosis were evident after brief stimulation. Endocytosis near active zones is cons istent with the existence of local exo/endocytic cycling pools. This mechan ism also might serve to maintain alignment of active zones with postsynapti c folds during periods of activity when vesicular and plasma membranes are interchanged.