Clonidine Ameliorates Anxiety-Like Behavior via GRIN2B Demethylation and NR2B Activation in a Two-Vessel Occlusion Rat Model

Abstract Post-stroke anxiety (PSA) is a prevalent neuropsychiatric complication that impairs neurological recovery and quality of life. Clonidine, an α2-adrenergic receptor (α2-AR) agonist, has demonstrated anxiolytic effects; however, the underlying molecular mechanisms remain elusive. In this study, we established a PSA paradigm using a two-vessel occlusion (2-VO) rat model to investigate whether clonidine exerts its anxiolytic effects through epigenetic modulation of the NMDA receptor subunit NR2B. Behavioral assessments, including the open field test and elevated plus maze, confirmed that 2-VO induced significant anxiety-like behaviors, which were attenuated by clonidine pretreatment (40 µg/kg/d, intraperitoneal). These protective effects were abolished by co-administration of yohimbine, a selective α2-AR antagonist, confirming a receptor-specific mechanism. Mechanistically, 2-VO rats exhibited reduced NR2B expression at both protein and mRNA levels in the prefrontal cortex (PFC) and hippocampus, accompanied by decreased membrane-bound NR2B and phosphorylated NR2B (p-NR2B). Clonidine reversed these deficits, enhancing functional membrane insertion of NR2B in neurons. Immunofluorescence revealed that clonidine restored NR2B immunoreactivity in PFC neurons and hippocampal subfields (CA1, CA3, and dentate gyrus). Furthermore, clonidine differentially modulated KIF17-mediated dendritic trafficking of NR2B in the hippocampus and suppressed aberrant CaMKII/CREB signaling in a region-specific manner. Notably, 2-VO induced global DNA hypermethylation and upregulation of DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B), which were reversed by clonidine. Methylation-specific PCR demonstrated that clonidine specifically reversed GRIN2B promoter hypermethylation in both the PFC and hippocampus, effects that were blocked by yohimbine. These findings establish a novel epigenetic-synaptic axis whereby α2-AR activation suppresses DNMT-mediated GRIN2B promoter hypermethylation, thereby restoring NR2B transcription, membrane trafficking, and phosphorylation to alleviate anxiety-like behaviors. Our results position the α2-AR–DNMT–GRIN2B axis as a promising therapeutic target for precision treatment of PSA and related neuropsychiatric sequelae of cerebral ischemia.