Title: Asynchronous glutamate exocytosis is enhanced in low release probability synapses and is widely dispersed across the active zone. Authors (original) (raw)

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 (presynaptic boutons) from single pyramidal cells in culture with 4 millisecond temporal resolution, and localise vesicular release sites with ~ 75 nm spatial resolution. We find that the ratio between synchronous and asynchronous synaptic vesicle exocytosis varies extensively among presynaptic boutons supplied by the same axon, and that asynchronous release fraction is elevated in parallel with short-term facilitation at synapses with low release probability. We further demonstrate that asynchronous exocytosis sites are more widely distributed within the presynaptic release area than synchronous sites. These findings are consistent with a model in which functional presynaptic properties are regulated via a synapsespecific adjustment of the coupling distance between presynaptic Ca 2+ channels and releaseready synaptic vesicles. Together our results reveal a universal relationship between the two major functional properties of synapses-the timing and the overall probability of neurotransmitter release. Main text Synaptic transmission provides the basis for neuronal communication. When an actionpotential propagates through the axonal arbour, it activates voltage-gated Ca 2+ channels (VGCCs) located in the vicinity of release-ready synaptic vesicles docked at the presynaptic active zone 1. Ca 2+ ions enter the presynaptic terminal and activate the vesicular Ca 2+ sensor Synaptotagmin 1 (Syt1, or its isoforms Syt2 and Syt9), thus triggering exocytosis of synaptic vesicles filled with neurotransmitter molecules. Neurotransmitter diffuses across the synaptic cleft, binds postsynaptic receptors and evokes further electrical or chemical signalling in the postsynaptic target cell. This whole process occurs on a time scale of a few milliseconds. Recent data demonstrate that such speed and precision are in large part achieved via the formation of nanocomplexes that include presynaptic VGCCs, vesicles belonging to a readily releasable pool (RRP) and postsynaptic neurotransmitter receptors 2-4. In addition to fast, synchronous release, which keeps pace with action potentials, many synapses also exhibit delayed asynchronous release that persists for tens to hundreds of milliseconds 1, 5. Asynchronous release is potentiated during repetitive presynaptic firing and is triggered via activation of multiple sensors with both low (e.g. Syt1) and high (e.g. Syt7) Ca 2+ affinity 6. Accumulating evidence demonstrates that the balance between synchronous and asynchronous release plays an important role in coordinating activity within neuronal networks, for example, by increasing the probability of postsynaptic cell firing and/or modulating action potential precision 7-10. It is well established that asynchronous release levels vary among different types of neurons 1, 10, 11. Interestingly, recent data show that the ratio between asynchronous and synchronous release can also be differentially regulated among presynaptic boutons supplied by the same axon and depends on the identity of the postsynaptic cell, which contributes to target cell-specific communication in the brain 7, 8. The mechanisms that control the relative contributions of synchronous and asynchronous release at the level of single synapses are however poorly understood. Variability in asynchronous release among different neuronal types has been attributed to differences in