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  • Title: The kinetics of exocytosis and endocytosis in the synaptic terminal of goldfish retinal bipolar cells.
    Author: Neves G, Lagnado L.
    Journal: J Physiol; 1999 Feb 15; 515 ( Pt 1)(Pt 1):181-202. PubMed ID: 9925888.
    Abstract:
    1. The kinetics of exocytosis and endocytosis were studied in the giant synaptic terminal of depolarizing bipolar cells from the goldfish retina. Two techniques were applied: capacitance measurements of changes in membrane surface 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 pool 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 involved the release of about 4400 vesicles over 1 s. The third component of exocytosis was stimulated continuously at a rate of about 1000 vesicles s-1. 3. After short depolarizations (< 200 ms), neither the FM1-43 signal nor the capacitance signal continued to rise, indicating that exocytosis stopped rapidly after closure of Ca2+ channels. The fall in capacitance could therefore be used to monitor endocytosis independently of exocytosis. The capacitance 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 techniques was similar during the first 200 ms of depolarization, suggesting that the most rapidly released vesicles could be detected by either method. 5. After a few seconds of continuous stimulation, the net increase in membrane surface area reached a plateau at about 5 %, even though continuous exocytosis occurred at a rate of 0.9 % s-1. Under these conditions of balanced exocytosis and endocytosis, the rate constant of endocytosis was about 0.2 s-1. The average rate of endocytosis during maintained depolarization was therefore considerably slower than the rate observed after a brief stimulus. 6. After longer depolarizations (> 500 ms), both the capacitance and FM1-43 signals continued to rise for periods of seconds after closure of Ca2+ channels. The continuation of exocytosis was correlated with a persistent increase in [Ca2+]i in the synaptic terminal, as indicated by the activation of a Ca2+-dependent conductance and measurements of [Ca2+]i using the fluorescent indicator furaptra. 7. The delayed fall in membrane capacitance after longer depolarizations occurred along a double exponential time course indicating the existence of two 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 fraction of membrane recovered by slow endocytosis. After a 100 ms stimulus, all the membrane was recycled by fast endocytosis, but after a 5 s depolarization, about 50 % of the membrane was recycled by slow endocytosis. 9. These results demonstrate the existence of fast and slow endocytic mechanisms 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 rise in cytoplasmic Ca2+ that occurred during longer depolarizations was correlated with stimulation of continuous exocytosis and inhibition of fast endocytosis. The results also confirm that transient and continuous components of exocytosis coexist in the synaptic terminal of depolarizing bipolar cells.
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