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The physical basis for these communications is the synapse, a multi-component structure requiring coordinated interactions between diverse cell types. However, many aspects of three-dimensional (3D) synaptic organization remain poorly understood. Here, we developed an approach, seamless\n                  <jats:italic>in situ<\/jats:italic>\n                  trimming and milling (SISTM), to reliably fabricate sufficiently thin lamellae for mapping of the 3D nanoarchitecture of synapses in mouse, monkey and human brain tissue under near-native conditions via cryo-electron tomography (cryo-ET). We validated SISTM in a mouse model of Huntington\u2019s disease, demonstrating distinct 3D alterations to synaptic vesicles and mitochondria. By successfully applying SISTM to macaque brain, we described the 3D architecture of a tripartite synapse within the cortex. Subtomogram averaging (STA) enabled spatial mapping of astrocyte-neuron contacts within the tripartite synapse, revealing neurexin-neuroligin complexes as potential constituents that tether the two cell types. Finally, we showed that the defining features of synaptic nanoarchitecture were conserved across species and evident in human brain tissue obtained postmortem. Combining SISTM with cryo-ET and STA is a starting point for a new understanding of brain organization, disease-induced structural alterations and the development of rational, structure-guided therapeutics.\n                <\/jats:p>","DOI":"10.1101\/2025.03.09.642162","type":"posted-content","created":{"date-parts":[[2025,3,11]],"date-time":"2025-03-11T08:25:40Z","timestamp":1741681540000},"source":"Crossref","is-referenced-by-count":3,"title":["Uncovering synaptic and cellular nanoarchitecture of brain tissue via seamless\n                  <i>in situ<\/i>\n                  trimming and milling for cryo-electron tomography"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8244-7230","authenticated-orcid":false,"given":"Jiying","family":"Ning","sequence":"first","affiliation":[{"name":"Department of Psychiatry, University of Pittsburgh, Pittsburgh, PA 15213, 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