Asynchronous glutamate release is enhanced in low release efficacy synapses and dispersed across the active zone.

The balance between fast synchronous and delayed asynchronous release of neurotransmitters has a major role in defining computational properties of neuronal synapses and regulation of neuronal network activity. However, how it is tuned at the single synapse level remains poorly understood. Here, using the fluorescent glutamate sensor SF-iGluSnFR, we image quantal vesicular release in tens to hundreds of individual synaptic outputs from single pyramidal cells with 4 millisecond temporal and 75 nm spatial resolution. We find that the ratio between synchronous and asynchronous synaptic vesicle exocytosis varies extensively among synapses supplied by the same axon, and that the synchronicity of release is reduced at low release probability synapses. We further demonstrate that asynchronous exocytosis sites are more widely distributed within the release area than synchronous sites. Together, our results reveal a universal relationship between the two major functional properties of synapses - the timing and the overall efficacy of neurotransmitter release.