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

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¢. 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 Ca¥ 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¢. 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¢. Under these conditions of balanced exocytosis and endocytosis, the rate constant of endocytosis was about 0·2 s¢. 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 Ca¥ channels. The continuation of exocytosis was correlated with a persistent increase in [Ca¥]é in the synaptic terminal, as indicated by the activation of a Ca¥-dependent conductance and measurements of [Ca¥]é 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¢, and slow endocytosis, with a rate constant of 0·1 s¢. 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 cyt...

Synaptic transmission at retinal ribbon synapses

“…First, the fusion of this physiologically defined vesicle pool is not prevented by the addition of millimolar exogenous calcium buffer EGTA to the presynaptic cytosol, consistent with a vesicle location very near to the sites of calcium entry (Mennerick and Matthews, 1996;Sakaba et al, 1997). Secondly, there is an excellent correlation between the total number of vesicles docked at the base of a bipolar cell's synaptic ribbons and in physical contact with the plasma membrane (≈1000-1400, for a small and large terminal respectively) and the number of vesicles that comprise the rapidly releasing pool of vesicles as determined by capacitance measurements (≈1100) ; see also Neves and Lagnado, 1999). It is also noteworthy that the rate of fusion of vesicles in this pool is so rapid, that it is hindered by the activation kinetics of the presynaptic calcium channels (Mennerick and Matthews, 1996).…”

“…In addition to the releasable pool, a smaller, faster pool of synaptic vesicles has been identified in bipolar cells (Mennerick and Matthews, 1996;Sakaba et al, 1997;Neves and Lagnado, 1999). This pool, referred to as the ultrafast or rapidly releasable pool, was discovered by examining the exocytotic response to a relatively brief (<50 ms) activation of voltage-gated calcium channels (Mennerick and Matthews, 1996).…”

Rosiglitazone balances insulin-induced exo- and endocytosis in single 3T3-L1 adipocytes

“…During exocytosis the luminal vesicle membrane become exposed to external medium and gets stained with FM1-43, thus contributing to the increase in the FM1-43 fluorescence intensity and to the increase in the plasma membrane area. Simultaneously, fluorescently stained membranes get internalized via endocytosis, hence the entire cell fluorescence intensity increase persists and the steady increase in fluorescence signal provides a cumulative record of exocytosis (Neves and Lagnado, 1999).…”

“…2A, right), consistent with previous experiments on primary adipocytes (Chowdhury et al, 2002(Chowdhury et al, , 2005. Some of the dye is internalised by endocytosis, therefore we measured the fluorescence intensity of entire cell to obtain a report of cumulative exocytosis (Neves and Lagnado, 1999). In controls, the addition of Veh as a bolus resulted in a small change in the slope of the FM1-43 fluorescence intensity signal (+Veh).…”

Visual signals in the retina: from photons to synapses

“…To improve the temporal resolution of the FM1_43 technique, Guilherme Neves and I used a photomultiplier tube instead of a camera to record the fluorescence from the terminal (Neves & Lagnado, 1999a). The upper record in Fig.…”