A. Endocytosis was dissociated from exocytosis by removing extracellular Ca²⁺ at the end of a 20-minute period of high frequency (20 Hz) action potential stimulation. The preparation was kept in zero Ca²⁺ solution for 90 minutes. Note the expansions of the presynaptic membrane marked by asterisks and the lack of coated pits. B. The plasma membrane from a synapse treated as in A and then incubated in normal Ca²⁺-containing solution for 40 seconds. Arrows mark clathrin coated pits with varying morphology, representing different stages of coated vesicle formation.
C-I. Different reagents were microinjected into reticulospinal axons followed by action potential stimulation at 5 Hz for 30 min (except in I) to induce synaptic vesicle recycling. C, D. Injection of the SH3-domain of human amphiphysin in C (GSTamphSH3) and of a 15-mer proline-rich peptide from rat dynamin 1 in D (PP-15) induced a massive accumulation of uncollared constricted coated pits. E. Shallow coated pits were accumulated on the plasma membrane in synapses injected with anti-endophilin antibodies (anti-endo). Coated intermediates of later stages were seen on deep invaginations of the plasma membrane (asterisk) and occasionally also coated vesicles (arrowhead). F. In synapses injected with a peptide from the proline-rich domain of synaptojanin (PP-19), a large number of free coated vesicles were observed along with uncollared, constricted coated pits. G. Free coated vesicles were also seen after injection of anti-synaptojanin antibodies (LSJ-1). H. Accumulation of coated pits with longer ‘necks’ surrounded by electron-dense collars (curved arrows) in synapses injected with the SH3-domain of endophilin (GSTendoSH3). I. Injection of anti-synapsin antibodies disrupted the distal pool of the synaptic vesicle cluster.

Asterisks indicate synaptic vesicle membrane internalized into the plasma membrane. GST and non-immune antibodies were also injected and had little or no effects as compared to uninjected synapses.