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During the previous funding period we have established a workflow to efficiently image synapses by cryo-electron tomography (cryo-ET). We will capitalize on these developments to investigate the native organization of the synaptic cytoskeleton. We will systematically map the organization of synaptic actin and microtubules and their associations with all major synaptic components. Altogether, we aim to provide a detailed cartography of the synaptic cytoskeleton and its roles in synaptic function.