Acetazolamide inhibition of carbonic anhydrase 4 reverses opioid-induced synaptic rearrangements in nucleus accumbens and reduces drug-seeking behavior.

Persistent vulnerability to drug-seeking is driven by enduring synaptic adaptations, yet current μ-opioid receptor-targeting pharmacotherapies provide limited efficacy against these neuroadaptations. Thus, there is a critical need for mechanistically distinct, non-opioid interventions. We recently found that carbonic anhydrase 4 (CA4) disruption reduces cocaine-induced synaptic adaptations and drug-seeking. Building on this foundation, we sought to determine whether deleting CA4 or pharmacological inhibition with acetazolamide (AZD), a clinically employed carbonic anhydrase inhibitor-could mitigate opioid withdrawal-associated plasticity and thus might reduce relapse vulnerability. We studied synaptic and behavioral adaptations to withdrawal from oxycodone in mice and found that prolonged withdrawal from oxycodone increased the AMPAR/NMDAR ratio and promoted synaptic incorporation of Ca2+-permeable AMPARs in nucleus accumbens core (NAcC) medium spiny neurons (MSNs). We found synaptic changes after protracted withdrawal from multiple opioids, which were most pronounced in D1-expressing MSNs, and were prevented by CA4 disruption. Moreover, AZD reversed withdrawal-induced synaptic alterations both in vitro and in vivo, in a CA4- and acid-sensing ion channel 1A (ASIC1A)-dependent manner. Unlike withdrawal from cocaine, withdrawal from oxycodone did not alter dendritic spine density in NAcC MSNs, suggesting a distinct mode of plasticity. Finally, following oxycodone self-administration, both CA4 deletion and a single systemic AZD dose reduced drug-seeking after prolonged abstinence. Together, these findings identify CA4 as a regulator of opioid-induced synaptic adaptations and suggest AZD as a promising, readily translatable pharmacological intervention. By targeting a pathway independent of classical opioid receptor signaling, CA4 inhibition represents a mechanistically distinct strategy that may reduce relapse vulnerability in OUD.