Role of piriform cortex and its afferent projections in relapse to fentanyl seeking after food choice-induced voluntary abstinence.

We previously demonstrated a role of piriform cortex (Pir) in relapse to fentanyl seeking after food choice-induced voluntary abstinence. Here, we used this model to further study the role of Pir and its afferent projections in fentanyl relapse. We trained male and female rats to self-administer palatable food pellets for 6 days (6 h/day) and fentanyl (2.5 µg/kg/infusion, i.v.) for 12 days (6 h/day). We assessed relapse to fentanyl seeking after 12 voluntary abstinence sessions, achieved through a discrete choice procedure between fentanyl and palatable food (20 trials/session). We determined projection-specific activation of Pir afferents during fentanyl relapse with Fos plus the retrograde tracer cholera toxin B (injected into Pir). Fentanyl relapse was associated with increased Fos expression in anterior insular cortex (AI) and prelimbic cortex (PL) neurons projecting to Pir. We used an anatomical disconnection procedure to determine the causal role of these two projections (AI→Pir and PL→Pir) in fentanyl relapse. Contralateral but not ipsilateral disconnection of AI→Pir projections decreased fentanyl relapse but not reacquisition of fentanyl self-administration. In contrast, contralateral but not ipsilateral disconnection of PL→Pir projections modestly decreased reacquisition but not relapse. Fluorescence-activated cell sorting and qPCR data showed molecular changes within Pir Fos-expressing neurons associated with fentanyl relapse. Finally, we found minimal or no sex differences in fentanyl self-administration, fentanyl vs. food choice, and fentanyl relapse. Our results indicate that AI→Pir and PL→Pir projections play dissociable roles in non-reinforced relapse to fentanyl seeking versus reacquisition of fentanyl self-administration after food choice-induced voluntary abstinence. SIGNIFICANCE STATEMENT: We previously showed a role of piriform cortex (Pir) in fentanyl relapse after food choice-induced voluntary abstinence in rats, a procedure mimicking human abstinence or a significant reduction in drug self-administration due to the availability of alternative non-drug rewards. Here, we aimed to further characterize the role of Pir in fentanyl relapse by investigating the role of Pir afferent projections and analyzing molecular changes in relapse-activated Pir neurons. We identified dissociable roles of two Pir afferent projections (AI→Pir and PL→Pir) in relapse to fentanyl seeking vs. reacquisition of fentanyl self-administration after voluntary abstinence. We also show molecular changes within Pir Fos-expressing neurons associated with fentanyl relapse.