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Using neonatal mice P8\u2010P12, we developed a slicing technique\u2014cutting spinal cords at 150\u00b0 from the coronal plane (30\u00b0 from the horizontal plane in the agarose block), for maximizing EUS\u2010MNs captured per slice. Using transgenic mice co\u2010expressing channelrhodopsin\u20102 in inhibitory interneurons (VGAT\u2010ChR2) and GFP in cholinergic neurons (ChAT\u2010GFP), we investigated inhibitory synaptic transmission onto EUS\u2010MNs. Optogenetic activation evoked robust inhibitory postsynaptic potentials (IPSPs), classified as sustained or transient based on temporal profiles. Pharmacology revealed that sustained IPSPs contained both glycinergic and GABAergic components, while GABAA receptors predominantly mediated transient IPSPs. Strychnine (1\u2009\u03bcM) selectively blocked glycinergic transmission, while bicuculline (10\u2009\u03bcM) eliminated GABAergic components. Insensitivity to glutamatergic antagonists (CNQX and AP5) confirmed purely inhibitory responses. Our findings demonstrate segregation of inhibitory inputs onto EUS\u2010MNs, with glycinergic and GABAergic transmission contributing to sustained and transient inhibition, respectively, establishing the methodological foundation for investigating inhibitory circuit dynamics in pathological conditions such as spinal cord injury with deficient inhibitory control.<\/jats:p>","DOI":"10.14814\/phy2.70703","type":"journal-article","created":{"date-parts":[[2025,12,20]],"date-time":"2025-12-20T07:41:50Z","timestamp":1766216510000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Characterization of optogenetically activated inhibitory inputs onto cholinergic motor neurons in the spinal dorsolateral nucleus"],"prefix":"10.14814","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-6587-7231","authenticated-orcid":false,"given":"Tadanobu C.","family":"Kamijo","sequence":"first","affiliation":[{"name":"Department 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