Fentanyl

After a week of recovery following surgery, we trained rats to self-administer fentanyl for 10 days as described above. For the first cohort of animals (n = 12) we trained rats to self-administer fentanyl (Sigma Aldrich) at 2.5 μg/kg/infusion. For the second cohort (n = 16), fentanyl was not available from our original source. fentanyl sourced from Cayman Chemical visibly occluded rats from responding at 2.5 μg/kg/infusion. Thus, for cohort 2 we reduced the concentration of fentanyl (Cayman Chemical) to 2.0 μg/kg/infusion for days 3 and 4 of self-administration and to 1.5 μg/kg/infusion for the rest of the self-administration phase. Since the terminal levels of responding for both cohorts were not significantly different (see section “Supplementary Results”), we pooled the data together. During training, Active Lever presses resulted in fentanyl infusion paired with a compound 5s tone-light cue located above the Active Lever.
Incubation extinction tests: After 10 days of training, we injected vehicle intracranially into the BNST of all rats 15 minutes prior to Day 1 of forced abstinence extinction test. After 30 days of forced abstinence (Day 30), we retested the same rats and injected either intra-BNST vehicle in n = 13 rats or intra-BNST R121919 in n = 15 rats in a mixed within-between subject design.

The cDNA for the β₂ adrenergic, GLP-1, MOR, NOP, and V₂ vasopressin receptors, and the cDNA for GRK2, was obtained from the cDNA Resource Center (Bloomsburg University, Bloomsburg, PA). In some experiments receptors modified to include the C-terminal tail of the V₂ vasopressin receptor were used. This involved substituting the last C-terminal residues with the last 29 C-terminal residues of the V₂ receptor (Oakley et al., 2000 (link)) (last 72 amino acids of the β₂ adrenoceptor, and the last 29 amino acids of the MOR, NOP and GLP-1 receptors). DAMGO, met-enkephalin, endomorphin-2, morphine, hydromorphone, oxymorphone, fentanyl, buprenorphine, vasopressin, N/OFQ and exendin-4 were all obtained from Cayman Chemical (Ann Arbor, MI). Isoproterenol was from Millipore Sigma.

We injected 5 μg/kg i.p fentanyl (Cayman Chemical) or vehicle in rats (n=4) 5 min before PLA sessions. We selected this dose based on pilot experiments. In two counterbalanced PLA sessions, we gave the rats either i.p injection of fentanyl or saline.