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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 d (6 h/day) and fentanyl (2.5 µg/kg/infusion, i.v.) for 12 d (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 next 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 quantitative PCR 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 versus food choice, and fentanyl relapse. Our results indicate that AI→Pir and PL→Pir projections play dissociable roles in nonreinforced relapse to fentanyl seeking versus reacquisition of fentanyl self-administration after food choice-induced voluntary abstinence.SIGNIFICANCE STATEMENTWe previously showed a role of 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 because of the availability of alternative nondrug 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 versus reacquisition of fentanyl self-administration after voluntary abstinence. We also characterized molecular changes within Pir Fos-expressing neurons associated with fentanyl relapse.

The brain µ-opioid receptor (MOR) is critical for the analgesic, rewarding, and addictive effects of opioid drugs. However, in rat models of opioid-related behaviors, the circuit mechanisms of MOR-expressing cells are less known because of a lack of genetic tools to selectively manipulate them. We introduce a CRISPR-basedOprm1-Creknock-in transgenic rat that provides cell type-specific genetic access to MOR-expressing cells. After performing anatomic and behavioral validation experiments, we used theOprm1-Creknock-in rats to study the involvement of NAc MOR-expressing cells in heroin self-administration in male and female rats. Using RNAscope, autoradiography, and FISH chain reaction (HCR-FISH), we found no differences inOprm1expression in NAc, dorsal striatum, and dorsal hippocampus, or MOR receptor density (except dorsal striatum) or function betweenOprm1-Creknock-in rats and wildtype littermates. HCR-FISH assay showed thatiCreis highly coexpressed withOprm1(95%-98%). There were no genotype differences in pain responses, morphine analgesia and tolerance, heroin self-administration, and relapse-related behaviors. We used the Cre-dependent vector AAV1-EF1a-Flex-taCasp3-TEVP to lesion NAc MOR-expressing cells. We found that the lesions decreased acquisition of heroin self-administration in maleOprm1-Crerats and had a stronger inhibitory effect on the effort to self-administer heroin in femaleOprm1-Crerats. The validation of anOprm1-Creknock-in rat enables new strategies for understanding the role of MOR-expressing cells in rat models of opioid addiction, pain-related behaviors, and other opioid-mediated functions. Our initial mechanistic study indicates that lesioning NAc MOR-expressing cells had different effects on heroin self-administration in male and female rats.SIGNIFICANCE STATEMENTThe brain µ-opioid receptor (MOR) is critical for the analgesic, rewarding, and addictive effects of opioid drugs. However, in rat models of opioid-related behaviors, the circuit mechanisms of MOR-expressing cells are less known because of a lack of genetic tools to selectively manipulate them. We introduce a CRISPR-basedOprm1-Creknock-in transgenic rat that provides cell type-specific genetic access to brain MOR-expressing cells. After performing anatomical and behavioral validation experiments, we used theOprm1-Creknock-in rats to show that lesioning NAc MOR-expressing cells had different effects on heroin self-administration in males and females. The newOprm1-Crerats can be used to study the role of brain MOR-expressing cells in animal models of opioid addiction, pain-related behaviors, and other opioid-mediated functions.

The brain µ-opioid receptor (MOR) is critical for the analgesic, rewarding, and addictive effects of opioid drugs. However, in rat models of opioid-related behaviors, the circuit mechanisms of MOR-expressing cells are less known because of a lack of genetic tools to selectively manipulate them. We introduce a CRISPR-basedOprm1-Cre knock-in transgenic rat that provides cell-type specific genetic access to MOR-expressing cells. After performing anatomical and behavioral validation experiments, we used theOprm1-Cre knock-in rats to study the role of nucleus accumbens (NAc) MOR-expressing cells in heroin self-administration in male and female rats.Using RNAscope, autoradiography, and fluorescencein situhybridization chain reaction (HCR-FISH), we found no differences inOprm1expression in NAc, dorsal striatum (DS), and dorsal hippocampus, or MOR receptor density (except DS) or function betweenOprm1-Cre knock-in rats and wildtype littermates. HCR-FISH assay showed thatiCreis highly co-expressed withOprm1(95-98%). There were no genotype differences in pain responses, morphine analgesia and tolerance, heroin self-administration, and relapse-related behaviors. We used the Cre-dependent vector AAV1-EF1a-Flex-taCasp3-TEVP to lesion NAc MOR-expressing cells and report sex-specific effects: the lesions decreased acquisition of heroin self-administration in maleOprm1-Cre rats and had a stronger inhibitory effect on the effort to self-administer heroin in femaleOprm1-Cre rats.The validation of anOprm1-Cre knock-in rat enables new strategies for understanding the role of MOR-expressing cells in rat models of opioid addiction, pain-related behaviors, and other opioid-mediated functions. Our initial mechanistic study with these rats suggests a sex-specific role of NAc MOR-expressing cells in heroin self-administration.Significance statementThe brain µ-opioid receptor (MOR) is critical for the analgesic, rewarding, and addictive effects of opioid drugs. However, in rat models of opioid-related behaviors, the circuit mechanisms of MOR-expressing cells are less known because of a lack of genetic tools to selectively manipulate them. We introduce a CRISPR-basedOprm1-Cre knock-in transgenic rat that provides cell-type specific genetic access to brain MOR-expressing cells. After performing anatomical and behavioral validation experiments, we used theOprm1-Cre knock-in rats to show a potential sex-specific role of nucleus accumbens MOR-expressing cells in heroin self-administration. The newOprm1-Cre rats can be used to study both the general and sex-specific role of brain MOR-expressing cells in animal models of opioid addiction, pain-related behaviors, and other opioid-mediated functions.