One of the most studied central synapses which have provided fundamental insights into cellular mechanisms of neural connectivity is the “giant” excitatory connection between hippocampal mossy fibers (MFs) and CA3 pyramidal cells. Its large presynaptic bouton features multiple release sites and is densely packed with thousands of synaptic vesicles, to sustain a highly facilitating “detonator” transmission. However, whether glutamate release sites at this synapse act independently, in a stochastic manner, or rather synchronously, remains poorly understood. This knowledge is critical for a better understanding of mechanisms underpinning presynaptic plasticity and postsynaptic signal integration rules. Here, we use the optical glutamate sensor SF-iGluSnFR and the intracellular Ca2+ indicator Cal-590 to monitor spike-evoked glutamate release and presynaptic calcium entry in MF boutons. Multiplexed imaging reveals that distinct sites in individual MF giant boutons release glutamate in a probabilistic fashion, also showing use-dependent short-term facilitation. The present approach provides novel insights into the basic mechanisms of neurotransmitter release at excitatory synapses
