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J.H. Singer, J.S. Diamond; Multi-Vesicular Release From Mammalian Rod Bipolar Cell Terminals . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1008.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Ribbon synapses are thought to be specialized to release large numbers of vesicles over an extended period. We sought to determine whether a single synapse releases more than one vesicle at once and what influence multivesicular release (MVR) has on synaptic transmission in the mammalian rod circuit. Methods: Voltage-clamp recordings were made from synaptically connected rod bipolar cells (RBCs) and AII amacrine cells in thin slices of light-adapted rat retina at 20 C. Strychnine, picrotoxin, tetrodotoxin, and L-AP4 were included in all solutions to isolate excitatory inputs from the presynaptic RBCs. Results: Tail Ca2+ currents elicited in RBCs (Vm=-70 mV) evoked EPSCs (tEPSCs) with a quantal content of ~35 in AIIs (Vm=-90 mV). Deconvolution of miniature EPSCs (mEPSCs) from tEPSCs revealed that exocytosis occurred in a 1 ms window with a peak release rate of ~20 quanta/ms. We propose that tEPSCs reflect synchronous release from a readily releasable vesicle pool; given the RBC-AII connectivity, it is probable that multiple vesicles are released at the same synapse. To assay MVR directly, RBCs were stepped for 100 ms to a potential (usually -55 mV) that activated Ca2+ channels weakly, and multiple, small EPSCs were recorded in the AII. These events were larger in amplitude than mEPSCs, but their time course was the same, indicating that they represented the synchronous release of more than one vesicle. When RBCs were stepped to -10 mV for 100 ms, EPSCs displayed a large, transient peak followed by a small sustained component comprising single synaptic events. In the presence of cyclothiazide (50 uM) to prevent AMPA receptor desensitization, kyneurenic acid (KYN, 2 mM), a low-affinity glutamate receptor antagonist, sped the decay of the transient component and blocked the sustained component preferentially. When paired tail Ca2+ currents were elicited (25 ms interval), tEPSCs showed pronounced paired-pulse depression (PPD), and KYN (250 uM) blocked the second response more than the first. Both of these results are consistent with a reduced cleft glutamate concentration under conditions of lowered release probability (PR). Conclusions: RBCs appear capable of well-synchronized MVR, and our results indicate that the contents of vesicles released at the same synapse pool and increase the cleft glutamate concentration. We cannot determine, however, whether MVR arises from multiple, synchronous exocytotic events or from compound vesicle fusion within the terminal and a single release event. As the extent of MVR depends upon PR, cleft glutamate concentration varies with the synapse's activity history. Thus, MVR may broaden the operating range of RBC synapses.
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