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Here, we present SynaptoTagMe, a suite of genetic tools in\n                    <jats:italic>Caenorhabditis elegans<\/jats:italic>\n                    to fluorescently label and conditionally ablate the vesicular transporters for glutamate, GABA, acetylcholine, and monoamines. Using a structure-guided approach informed by protein topology and evolutionary conservation, we engineered endogenously tagged versions for each transporter that maintain their physiological function while allowing for cell-specific, bright, and stable visualization. We also developed conditional knockout strains that enable targeted disruption of neurotransmitter synthesis or packaging in single neurons. We applied this toolkit to map co-expression of vesicular transporters across the\n                    <jats:italic>C. elegans<\/jats:italic>\n                    nervous system, revealing that over 10% of neurons exhibit co-transmission. Using the ADF sensory neuron as a case study, we demonstrate that serotonin and acetylcholine are trafficked in partially distinct vesicle pools. Our approach provides a powerful platform for mapping, monitoring, and manipulating neurotransmitter identity and use in vivo. The molecular strategies described here are likely applicable across species, offering a generalizable approach to dissect synaptic communication in vivo.\n                  <\/jats:p>","DOI":"10.7554\/elife.108675","type":"journal-article","created":{"date-parts":[[2025,11,6]],"date-time":"2025-11-06T14:30:39Z","timestamp":1762439439000},"update-policy":"https:\/\/doi.org\/10.7554\/elife.108675","source":"Crossref","is-referenced-by-count":0,"title":["SynaptoTagMe, a toolkit for in vivo mapping and modulating neurotransmission at single-cell resolution"],"prefix":"10.7554","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4847-0031","authenticated-orcid":true,"given":"Andrea","family":"Cuentas-Condori","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/03v76x132","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Neuroscience and Department of Cell Biology, Yale University School of Medicine","place":["New Haven, United 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