PROJECT SUMMARY Recently, opioid use and dependence has increased to epidemic levels. A prominent contributor to this trend is oxycodone, (Oxy), a semisynthetic opioid analgesic and most widely-prescribed opioid painkiller. A major hurdle in addiction treatment, including for opioid addicts, is a long-lasting vulnerability to relapse. Evidence indicates that the drugs of abuse produce long-lasting maladaptive plasticity in nucleus accumbens (NAc) synaptic function that underlie compulsive drug seeking. Such plasticity accounts for the fact that cue-induced craving progressively intensifies (‘incubates’) over weeks/months of forced abstinence or withdrawal in both humans and animals, for many drug classes. During withdrawal from extended-access Oxy self-administration, rats exhibit a progressive intensification (incubation) of cue-induced Oxy craving that is accompanied by adaptations in in AMPA receptor subunit composition in the NAc, similar to other drugs. A key challenge is how to reverse this plasticity in a translationally-relevant manner. Growing evidence suggests that subanesthetic doses of the glutamate-NMDA receptor antagonist ketamine (Ket) may be a potential long-lasting treatment option for substance use disorders. Our preliminary data show systemic subanesthetic Ket, given daily during withdrawal from Oxy self-administration in rats, reduces incubation in a seeking test one day after the last Ket injection. There are 3 objectives of this proposal: (1) characterize the time course of Ket’s ability to reduce Oxy incubation, (2) determine an effective dosing regimen and timepoint for therapeutic intervention, and (3) test potential mechanisms for Ket’s effect. The central hypothesis is that altered activity and/or altered protein translation in the reward circuitry is responsible for Oxy incubation and that Ket may be exerting its effects by normalizing pathological changes. Aim 1 will determine the duration of incubation reduction by repeated Ket and test a role for projection-specific changes in glutamate afferents to the NAc. cFos (an indirect marker of neural activity) immunohistochemistry will be combined with retro-AAV viral tracing to examine input-specific activity in key regions of the reward circuitry that project to the NAc: orbitofrontal cortex, basolateral amygdala, and ventral subiculum of the hippocampus. Activity will be assessed at 4 points after discontinuing Oxy self-administration: withdrawal day 1 (WD1), before adaptations have occurred, WD15, when craving has “incubated”, or at WD30 or WD45, to assess the persistence of incubation. The effects of daily subanesthetic Ket will also be examined to determine the duration of its effects. Aim 2 will characterize protein synthesis during Oxy incubation. Puromycin labeling will be used to measure protein translation of the key AMPA receptor subunits GluA1 and GluA2 in NAc tissue on WD1 and WD15. Daily Ket’s impact on AMPAR translation will also be examined. Aim 3 will exam...