The kinetics of exocytosis and endocytosis in the synaptic terminal of goldfish retinal bipolar cells

1. The kinetics of exocytosis and endocytosis were studied in the giant syn aptic terminal of depolarizing bipolar cells from the goldfish retina. Two techniques were applied: capacitance measurements of changes in membrane su rface area, and fluorescence measurements of exocytosis using the membrane dye FM1-43. 2. Three phases of exocytosis occurred during maintained depolarization to 0 mV. The first component was complete within about 10 ms and involved a po ol of 1200-1800 vesicles (with a total membrane area equivalent to about 1. 6% of the surface of the terminal). The second component of exocytosis invo lved the release of about 4400 vesicles over 1 s. The third component of ex ocytosis was stimulated continuously at a rate of about 1000 vesicles s(-1) 3. After short depolarizations (< 200 ms), neither the FM1-43 signal nor th e capacitance signal continued to rise, indicating that exocytosis stopped rapidly after closure of Ca2+ channels. The fall in capacitance could there fore be used to monitor endocytosis independently of exocytosis. The capaci tance measured after brief stimuli began to fall immediately recovering to the pre-stimulus baseline with a rate constant of 0.8 s(-1) 4. The amount of exocytosis measured using the capacitance and FM1-43 techn iques was similar during the first 200 ms of depolarization, suggesting tha t the most rapidly released vesicles could be detected by either method. 5. After a few seconds of continuous stimulation, the net increase in membr ane surface area reached a plateau at about 5%, even though continuous exoc ytosis occurred at a rate of 0.9 % s(-1). Under these conditions of balance d exocytosis and endocytosis, the rate constant of endocytosis was about 0. 2 s(-1). The average rate of endocytosis during maintained depolarization w as therefore considerably slower than the rate observed after a brief stimu lus. 6. After longer depolarizations (> 500 ms), both the capacitance and FM1-43 signals continued to rise for periods of seconds after closure of Ca2+ cha nnels. The continuation of exocytosis was correlated with a persistent incr ease in [Ca2+](i) in the synaptic terminal, as indicated by the activation of a Ca2+-dependent conductance and measurements of [Ca2+](i) using the flu orescent indicator furaptra. 7. The delayed fall in membrane capacitance after longer depolarizations oc curred along a double exponential time course indicating the existence of t wo endocytic processes: fast endocytosis, with a rate constant of 0.8 s(-1) and slow endocytosis, with a rate constant of 0.1 s(-1). 8. Increasing the duration of depolarization caused an increase in the frac tion of membrane recovered by slow endocytosis. After a 100 ms stimulus, al l the membrane was recycled by fast endocytosis, but after a 5 s depolariza tion, about 50 % of the membrane was recycled by slow endocytosis. 9. These results demonstrate the existence of fast and slow endocytic mecha nisms at a synapse and support the idea that prolonged stimulation leads to an increase in the amount of membrane retrieved by the slower route. The r ise in cytoplasmic Ca2+ that occurred during longer depolarizations was cor related with stimulation of continuous exocytosis and inhibition of fast en docytosis. The results also confirm that transient and continuous component s of exocytosis coexist in the synaptic terminal of depolarizing bipolar ce lls.