Optical postsynaptic measurement of vesicle release rates for hippocampal synapses undergoing asynchronous release during train stimulation.

Developing hippocampal neurons in microisland culture were found to undergo rapid depression of excitatory synaptic activity caused by consumption of their readily releasable pool (RRP) of vesicles in response to 20 Hz trains of stimulation. Associated with depression was a switch to an asynchronous release mode that maintained transmission at a high steady-state rate equivalent to approximately 2.1 RRPs per second. We have applied postsynaptic Ca2+ imaging to directly monitor these asynchronous release events to estimate both the steady rate of transmitter release and the number of quanta within the RRP at individual hippocampal synapses. Based on the frequency of asynchronous release measured at individual synapses postsynaptically using Ca2+ imaging (5-17 sec after train stimulation) and with knowledge of the time course by which asynchronous release rates decay, we estimate that individual hippocampal synapses exhibit (in response to train stimulation) peak release rates of up to 21 quanta per second from an RRP that contains, on average, 10 quanta. Use-dependent block of evoked synaptic activity by MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d]cyclohepten-5,10-imine maleate] confirmed that synapses undergoing asynchronous release are not significantly different from the general population with regard to their composition of NMDA receptor and/or release probability. Given that high-frequency trains deplete the synapse of readily releasable quanta (and that these release rates can only be maintained for a few seconds), these high rates of asynchronous release likely reflect refilling of vesicles from a reserve pool and not necessarily the continuous action of a relatively slow clathrin- and endosome-dependent process.