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A major obstacle to advancing such studies is the absence of a methodology for precise, cell-type and brain-region-specific quantification of critical epitranscriptomic regulators under these complex brain conditions. To overcome this challenge, we developed a super-resolved, three-dimensional spatial transcriptomics method to quantify key epitranscriptomic switches in intact brains. Using this method, we quantified the expression of\n                  <jats:italic>Mettl3<\/jats:italic>\n                  , an N6-methyladenosine (m6A) methyltransferase enzyme recently shown to be upregulated in the amygdala after adolescent intermittent ethanol (AIE) exposure in rats. We observed a significant increase in cytoplasmic\n                  <jats:italic>Mettl3<\/jats:italic>\n                  mRNA in neurons, but not in astrocytes or microglia, within the adult central amygdala and the CA1 and dentate gyrus of hippocampus following AIE. In contrast, no significant changes were observed across neurons, astrocytes, or microglia within the basolateral amygdala or the hippocampal CA3.\n                <\/jats:p>\n                <jats:p>\n                  Additionally, we found both the cytoplasmic density and subcellular localization of\n                  <jats:italic>Mettl3<\/jats:italic>\n                  mRNA were dependent on the specific cell types and brain subregions examined. These results suggest that AIE increases\n                  <jats:italic>Mettl3<\/jats:italic>\n                  expression in a highly cell-type-specific and spatially heterogeneous manner, underscoring the necessity of high-resolution spatial transcriptomics methods for studying transcriptomic and epitranscriptomic regulations under neurological conditions.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>Significance Statement<\/jats:title>\n                  <jats:p>\n                    Epitranscriptomics plays a crucial role in neuronal functions by influencing the splicing, stability, and translation of genes. However, the exact role of epitranscriptomic mechanisms, such as m6A RNA methylation, in brain disorders remains unclear, particularly in a cell-type and circuitry-specific manner. Here we developed a super-resolved, three-dimensional spatial transcriptomics method and applied it to a model of alcohol exposure. We found differential cell-type- and brain-region-specific modulation of\n                    <jats:italic>Mettl3<\/jats:italic>\n                    , a key m6A enzymatic switch, across major brain regions following adolescent intermittent ethanol exposure in adulthood. Our findings, coupled with our pipeline, are expected to address existing methodological limitations and knowledge gaps, thereby accelerating brain transcriptomic and epitranscriptomic studies under various psychiatric and neurological conditions.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2026.01.22.698893","type":"posted-content","created":{"date-parts":[[2026,1,23]],"date-time":"2026-01-23T15:00:18Z","timestamp":1769180418000},"source":"Crossref","is-referenced-by-count":0,"title":["Super-resolved, three-dimensional spatial transcriptomics reveals cell-type and brain-region-specific modulation of key epitranscriptomic switches following adolescent alcohol exposure"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1154-5773","authenticated-orcid":false,"given":"Jamuna","family":"Tandukar","sequence":"first","affiliation":[{"name":"Department of Biological Sciences, University of Illinois Chicago, Chicago, IL 60607, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2815-2458","authenticated-orcid":false,"given":"Anam","family":"Islam","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences, University of Illinois Chicago, Chicago, IL 60607, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2291-2716","authenticated-orcid":false,"given":"Emir","family":"Malovic","sequence":"additional","affiliation":[{"name":"Center for Alcohol Research in Epigenetics (CARE), Department of Psychiatry, University of Illinois Chicago, Chicago, IL 60622, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-3956-8591","authenticated-orcid":false,"given":"Bisma","family":"Afzal","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences, University of Illinois Chicago, Chicago, IL 60607, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Huaibo","family":"Zhang","sequence":"additional","affiliation":[{"name":"Center for Alcohol Research in Epigenetics (CARE), Department of Psychiatry, University of Illinois Chicago, Chicago, IL 60622, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7139-8464","authenticated-orcid":false,"given":"Subhash C.","family":"Pandey","sequence":"additional","affiliation":[{"name":"Center for Alcohol Research in Epigenetics (CARE), Department of Psychiatry, University of Illinois Chicago, Chicago, IL 60622, USA"},{"name":"Jesse Brown Veterans Affairs Medical Center, Chicago, IL 60612, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7137-2512","authenticated-orcid":false,"given":"Ruixuan","family":"Gao","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences, University of Illinois Chicago, Chicago, IL 60607, USA"},{"name":"Department of Chemistry, University of Illinois Chicago, Chicago, IL 60607, USA"},{"name":"Laboratory of Integrative Neuroscience, University of Illinois Chicago, Chicago, IL 60607, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026020503403993000_2026.01.22.698893v1.1","unstructured":"Alon S et al. 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