Highlights d Electron tomography enables the dissection of vesicle pools at synaptic active zones d Docked and primed vesicle availability contributes to initial release probability d The ratio of docked and tethered vesicles may co-determine short-term plasticity d Hippocampal mossy fibers contain three morphological types of docked vesicles

Highlights d High-pressure freezing of cultured brain tissue without cryoprotectants for EM d Flash-and-freeze functional EM of identified synapses in complex neural circuits d Direct correlation of activity-induced functional states and ultrastructure d Dissection of the spatiotemporal organization of endocytosis in identified synapses

Despite similarities in the composition of the molecular release machinery, synapses can exhibit strikingly different functional transmitter release properties and short-and long-term plasticity characteristics. To address the question whether ultrastructural differences could contribute to this functional synaptic heterogeneity, we employed a combination of hippocampal organotypic slice cultures, high-pressure freezing, freeze substitution, and 3Delectron tomography to resolve the spatial organization of vesicle pools at individual active zones (AZ) in two functionally distinct synapses, namely Schaffer collateral (SC) and mossy fiber (MF) synapses. We found that mature MF and SC synapses harbor equal numbers of docked vesicles at their AZs, MF synapses at rest exhibit a second pool of possibly 'tethered' vesicles in the AZ vicinity, and MF synapses contain at least three morphological types of docked vesicles, indicating that differences in the ultrastructural organization of MF and SC synapses may contribute to their respective functional properties and corresponding plasticity characteristics. similarities with functional definitions of heterogeneous SV pools, e.g. 'reluctantly/slowly' vs.