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We hypothesized that the neuroprotective effect of FN stimulation involves activation of cholinergic pathways, which increases reactive oxygen species (ROS) production by opening mitochondrial K\n              <jats:sup>+<\/jats:sup>\n              <jats:sub>ATP<\/jats:sub>\n              channels, thus leading to an increase in UCP4 (uncoupling protein 4) expression and subsequent neuroprotection.\n            <\/jats:p>\n          <\/jats:sec>\n          <jats:sec xml:lang=\"en\">\n            <jats:title>Methods<\/jats:title>\n            <jats:p xml:lang=\"en\">FN stimulation was performed for 1\u00a0hour in rats. The UCP4 protein and mRNA levels were measured by western blot, dot blot, and in situ hybridization. Carbachol was applied following UCP4\u2010promoter tdTomato reporter vector transfection of the rat primary cortical cell culture (in vitro) and rat brain (in vivo). We observed cellular UCP4 expression using fluorescence microscopy. UCP4 expression in the cell culture in response to diazoxide application was determined by a reverse transcription\u2010polymerase chain reaction and western blotting.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec xml:lang=\"en\">\n            <jats:title>Results<\/jats:title>\n            <jats:p xml:lang=\"en\">\n              Whereas FN stimulation increased UCP4 protein and mRNA levels, carbachol administration induced UCP4 expression in vitro and in vivo. The attenuation of this effect by atropine suggests that FN\u2010induced UCP4 expression involves the cholinergic pathway. The opening of mitochondrial K\n              <jats:sup>+<\/jats:sup>\n              <jats:sub>ATP<\/jats:sub>\n              channels with diazoxide increased the production of ROS and led to increased UCP4 expression. In contrast, quenching ROS with superoxide dismutase reversed the effect of diazoxide on UCP4 expression. Therefore, the opening of mitochondrial K\n              <jats:sup>+<\/jats:sup>\n              <jats:sub>ATP<\/jats:sub>\n              channels increased ROS production, which subsequently enhanced UCP4 expression and attenuated ROS generation.\n            <\/jats:p>\n          <\/jats:sec>\n          <jats:sec xml:lang=\"en\">\n            <jats:title>Conclusion<\/jats:title>\n            <jats:p xml:lang=\"en\">\n              Neuroprotective effect of FN stimulation involves activation of the cholinergic pathways, which increases ROS production by opening mitochondrial K\n              <jats:sup>+<\/jats:sup>\n              <jats:sub>ATP<\/jats:sub>\n              channels, leading to increased expression of neuroprotective UCP4.\n            <\/jats:p>\n          <\/jats:sec>","DOI":"10.1161\/svin.122.000362","type":"journal-article","created":{"date-parts":[[2022,10,27]],"date-time":"2022-10-27T10:40:23Z","timestamp":1666867223000},"update-policy":"https:\/\/doi.org\/10.1161\/crossmarkpolicy","source":"Crossref","is-referenced-by-count":1,"title":["The Cholinergic Pathway and MitoK\n            <sub>ATP<\/sub>\n            Induce UCP4 Expression Involved in Neuroprotection of FN Stimulation in Rats"],"prefix":"10.1161","volume":"2","author":[{"given":"Yasuko","family":"Fukushi","sequence":"first","affiliation":[{"name":"Department of Innovative Medical Photonics Preeminent Medical Photonics Education &amp; Research Center Hamamatsu University School of Medicine Hamamatsu Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Eugene V.","family":"Golanov","sequence":"additional","affiliation":[{"name":"Department of Neurosurgery Houston Methodist Hospital Houston TX"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shinichiro","family":"Koizumi","sequence":"additional","affiliation":[{"name":"Department of Neurosurgery Hamamatsu University School of Medicine Hamamatsu Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Min","family":"Thura","sequence":"additional","affiliation":[{"name":"Department of Innovative Medical Photonics Preeminent Medical Photonics Education &amp; Research Center Hamamatsu University School of Medicine Hamamatsu Japan"},{"name":"Institute of Molecular and Cell Biology Agency for Science Technology and Research Singapore Singapore"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hayato","family":"Ihara","sequence":"additional","affiliation":[{"name":"Department of Physiology Hamamatsu University School of Medicine Hamamatsu Japan"},{"name":"Radioisotope Laboratory Center Wakayama Medical University Wakayama Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8634-0150","authenticated-orcid":false,"given":"Seiji","family":"Yamamoto","sequence":"additional","affiliation":[{"name":"Department of Innovative Medical Photonics Preeminent Medical Photonics Education &amp; Research Center Hamamatsu University School of Medicine Hamamatsu Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"276","reference":[{"key":"e_1_3_2_2_2","doi-asserted-by":"publisher","DOI":"10.1038\/jcbfm.1991.139"},{"key":"e_1_3_2_3_2","doi-asserted-by":"crossref","first-page":"1020","DOI":"10.1038\/jcbfm.1993.128","article-title":"Reductions in focal ischemic infarctions elicited from cerebellar fastigial nucleus do not result from elevations in cerebral blood flow","volume":"13","author":"Yamamoto S","year":"1993","unstructured":"Yamamoto S, Golanov EV, Reis DJ. 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