Esketamine alleviates the pain-depression dyad: insights from exploratory transcriptomic profiling and Wnt4-associated molecular alterations within the anterior cingulate cortex

Objective The molecular mechanisms driving esketamine (ESK) efficacy in the pain-depression dyad (PDD) remain incompletely understood. This study investigated the effects of a sub-anesthetic ESK regimen on cortical expression profiles and exploratory signaling markers within the anterior cingulate cortex (ACC). Methods Male Sprague-Dawley rats were randomized into Sham, PDD, and PDD + ESK groups. The PDD model, established via chronic constriction injury (CCI), was validated using a behavioral test battery tracking mechanical allodynia and depression-like phenotypes. On day 21 post-surgery, PDD + ESK rats received a single esketamine injection (15 mg/kg, i.p.). Multi-regional c-Fos expression mapping across the PFC, HPC, and ACC identified the primary responsive structure, followed by dual immunofluorescence for cellular phenotyping. Finally, ACC excitatory neuronal calcium dynamics, exploratory transcriptomic profiles, and Wnt4-associated molecular alterations (including GSK3β and mTOR phosphorylation alongside synaptic markers) were evaluated using fiber photometry, RNA sequencing, and Western blotting. Results ESK alleviated pain hypersensitivity, behavioral despair, and anhedonia in PDD rats. Regional c-Fos profiling identified the ACC as the primary reactive locus, with activation localized predominantly to CaMKIIα-positive neurons. Fiber photometry confirmed that ESK attenuated functional hyperactivation in these ACC excitatory neurons. Exploratory transcriptomic profiling (unadjusted P < 0.05) revealed candidate altered transcripts, identifying Wnt4 as an exploratory molecular marker for further investigation. Subsequent orthogonal protein validations demonstrated that ESK modulated Wnt4 expression alongside concomitant alterations in GSK3β and mTOR phosphorylation, parallel with the attenuation of pathologically elevated synaptic protein markers and a restorative trend in synaptic ultrastructure. Conclusion A single sub-anesthetic ESK dose effectively alleviates PDD behavioral phenotypes in rats. This therapeutic efficacy is associated with Wnt4-related molecular adjustments coupled with putative downstream GSK3β-mTOR signaling alterations within the ACC CaMKIIα-positive neurons, providing exploratory transcriptomic and molecular insights into cortical adaptations in pain-related affective disorders.