Opioid Use Disorder (OUD) continues to be a pressing public health crisis, marked by high relapse rates and limited treatment options. Ketamine, a non-competitive N-Methyl-D-Aspartate receptor (NMDAR) antagonist, has emerged as a promising therapeutic for numerous psychiatric illnesses due to its robust antidepressant properties and glutamate-mediated synaptic plasticity. However, ketamine's therapeutic mechanisms remain elusive, with emerging evidence implicating the endogenous opioid system. Furthermore, few studies have investigated ketamine's potential as an intervention for OUD. Accordingly, this study assessed ketamine's ability to rewire endogenous opioid and glutamatergic circuitry and reduce heroin-seeking activity in male Long-Evans rats. Using a multi-cohort design, we investigated ketamine's effects on opioid-related transcriptional, protein, and signaling properties in key brain regions associated with reward and addiction, and subsequently on heroin-seeking using a self-administration paradigm. To investigate mechanisms of plasticity, we also quantified transcriptional changes to NMDAR subunits. Molecular analyses demonstrated increased expression of endogenous opioid system-related genes and endogenous opioid peptides at the protein level, elevated mu-opioid receptor protein levels, and enhanced G protein activity, suggesting increased receptor function. Notably, transcriptional changes were particularly prominent in the shell subregion of the nucleus accumbens (NAc). We also revealed that ketamine altered NMDAR subunit expression in the medial prefrontal cortex and NAc, indicating circuit-level synaptic remodeling. Behavioral analyses revealed that ketamine significantly reduced heroin-primed, but not cue-induced, seeking activity. Together, these findings suggest ketamine reduces acute relapse vulnerability to opioids through coordinated modulation of opioid and glutamatergic systems, highlighting its potential as a novel intervention for OUD.
