METHADONE WITHDRAWAL INDUCES REGION-SPECIFIC TRANSCRIPTIONAL AND EPIGENETIC CHANGES IN THE RAT BRAIN

Methadone, a synthetic mu-opioid receptor agonist, is widely used to treat opioid use disorder. However, like other opioids, prolonged methadone exposure can induce enduring adaptations in the central nervous system, including epigenetic remodeling and altered transcription factor activity. Nevertheless, the long-lasting molecular changes associated with chronic methadone exposure and subsequent abstinence remain poorly defined. We therefore characterized the spatiotemporal dynamics of transcriptional and epigenetic alterations following methadone exposure and withdrawal. Adult rats received methadone or saline for 10 days, followed by withdrawal periods of 1 day, 1 week, 4 weeks, or 12 weeks. Immunohistochemistry was used to assess transcription factor-related markers, including cAMP response element-binding protein (CREB), phosphorylated CREB (p-CREB), methyl-CpG-binding protein 2 (MeCP2), and DeltaFosB. In parallel, DNA and proteins were co-isolated from the same brain regions using a TRIzol reagent-based workflow. Extracted DNA was used to quantify 5-methylcytosine (5mC), while protein extracts were subjected to downstream analyses of epigenetic regulators involved in DNA methylation and hydroxymethylation. Our results indicate that CREB immunoreactivity is region-specific and time-dependent during abstinence, with the most pronounced alterations observed in the prefrontal cortex, particularly at early and late withdrawal time points. In contrast, changes in the hippocampus emerged at later stages of abstinence. Pilot protein profiling further suggests regionally divergent modulation of epigenetic regulators associated with DNA methylation and hydroxymethylation. Together, these findings indicate that prolonged methadone exposure followed by withdrawal induces lasting, region- and time-specific molecular adaptations that persist well beyond drug discontinuation.