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Before release, one or several synaptic vesicles (SVs) are thought to bind to each docking site, forming the readily releasable pool (RRP). Determining the RRP size per docking site has important implications for short-term synaptic plasticity. Here, using mouse cerebellar slices, we take advantage of recently developed methods to count the number of released SVs at single glutamatergic synapses in response to trains of action potentials (APs). In each recording, the number of docking sites was determined by fitting with a binomial model the number of released SVs in response to individual APs. After normalization with respect to the number of docking sites, the summed number of released SVs following a train of APs was used to estimate of the RRP size per docking site. To improve this estimate, various steps were taken to maximize the release probability of docked SVs, the occupancy of docking sites, as well as the extent of synaptic depression. Under these conditions, the RRP size reached a maximum value close to two SVs per docking site. The results indicate that each docking site contains two distinct SV-binding sites that can simultaneously accommodate up to one SV each. They further suggest that under special experimental conditions, as both sites are close to full occupancy, a maximal RRP size of two SVs per docking site can be reached. More generally, the results validate a sequential two-step docking model previously proposed at this preparation.<\/jats:p>","DOI":"10.7554\/elife.91087","type":"journal-article","created":{"date-parts":[[2023,9,19]],"date-time":"2023-09-19T07:28:36Z","timestamp":1695108516000},"update-policy":"https:\/\/doi.org\/10.7554\/elife.91087","source":"Crossref","is-referenced-by-count":16,"title":["A maximum of two readily releasable vesicles per docking site at a cerebellar single active zone synapse"],"prefix":"10.7554","volume":"12","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-2687-8650","authenticated-orcid":true,"given":"Melissa","family":"Silva","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01a4enz31","id-type":"ROR","asserted-by":"publisher"}],"name":"Universit\u00e9 Paris Cit\u00e9, SPPIN-Saints P\u00e8res Paris Institute for the Neurosciences, CNRS","place":["Paris, 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