Super-resolved, three-dimensional spatial transcriptomics reveals cell-type and brain-region-specific modulation of key epitranscriptomic switches following adolescent alcohol exposure

Epitranscriptomic mechanisms dynamically regulate neuronal function through gene expression, but their precise roles in neuropsychiatric and neurological disorders remain to be fully elucidated. 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 Mettl3, 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 Mettl3 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. Additionally, we found both the cytoplasmic density and subcellular localization of Mettl3 mRNA were dependent on the specific cell types and brain subregions examined. These results suggest that AIE increases Mettl3 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. Significance StatementEpitranscriptomics 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 Mettl3, 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.