Spatiotemporal Regulation of Synaptic Vesicle Fusion Sites in Central Synapses

Abstract

The number and availability of vesicle release sites at the synaptic active zone (AZ) are critical factors governing neurotransmitter release; yet, these fundamental synaptic parameters have remained undetermined. Moreover, how neural activity regulates the spatiotemporal properties of the release sites within individual central synapses is unknown. Here, we combined a nanoscale imaging approach with advanced image analysis to detect individual vesicle fusion events with ∼27 nm localization precision at single hippocampal synapses under physiological conditions. Our results revealed the presence of multiple distinct release sites within individual hippocampal synapses. Release sites were distributed throughout the AZ and underwent repeated reuse. Furthermore, the spatiotemporal properties of the release sites were activity dependent with a reduction in reuse frequency and a shift in location toward the AZ periphery during high-frequency stimulation. These findings have revealed fundamental spatiotemporal properties of individual release sites in small central synapses and their activity-dependent modulation.

Subsynaptic spatial organization as a regulator of synaptic strength and plasticity

“…Consistent with this, a novel imaging approach called pHuse ( pH luorin u ncovering s ites of e xocytosis), using vesicle-resident sensors to map sites of single-vesicle fusion events within individual synapses, demonstrated that action potentials evoked vesicle fusion within a smaller area of the presynaptic bouton than spontaneous release in hippocampal terminals [2**]. Critically, trains of stimuli prompt release from repeatedly used sites [3*]. Thus, release probability (P r ) in a single AZ is spatially heterogeneous.…”

Transcellular Nanoalignment of Synaptic Function

“…Analysis of putative fusion events at synapses that are fixed with high pressure freezing within ∼10 ms of optogenetic stimulation and analyzed by electron microscopy have revealed a tendency for release towards the center of the active zone (Watanabe et al, 2013). Superresolution localization of individual exocytotic events either aggregated from separate events in multiple synapses (Park et al, 2012) or many events in single synapses (Maschi and Klyachko, 2017; Tang et al, 2016) have shown that individual fusion events are not confined to a single spot, but may be spread out over a subarea of the active zone, perhaps with differences between action potential-mediated and spontaneous fusion events.…”

“…Because these estimates are indirect, they may bias the analysis to sites with a relatively high release probability, for example those closer to Ca 2+ channels. Two recent studies have used microscopy to determine the localization of individual vesicle fusion events at hippocampal synapses, with the resolution limited by the size of a synaptic vesicle (Maschi and Klyachko, 2017; Tang et al, 2016). One study subsequently used clustering analysis to determine how many distinct sites were present in a single active zone (Maschi and Klyachko, 2017).…”

“…Two recent studies have used microscopy to determine the localization of individual vesicle fusion events at hippocampal synapses, with the resolution limited by the size of a synaptic vesicle (Maschi and Klyachko, 2017; Tang et al, 2016). One study subsequently used clustering analysis to determine how many distinct sites were present in a single active zone (Maschi and Klyachko, 2017). It concluded that, if the diameter of a site is limited to 70 nm, an active zone has approximately ten exocytotic sites.…”

“…Second, it remains unclear to what extent the numbers of presynaptic release sites are in accordance with trans-synaptic units in the cleft and receptor clusters in the postsynaptic density. As described above, presynaptically, estimates of release site numbers range from 1 to ∼15 depending on the method employed (Imig et al, 2014; Maschi and Klyachko, 2017; Miki et al, 2016; Oertner et al, 2002; Tang et al, 2016). Postsynaptically, the view has emerged that an average hippocampal excitatory synapse contains ∼2–3 AMPAR clusters.…”