2.5. The icv injections

All groups underwent stereotaxic surgery after the extinction phase. After anesthetization with pentobarbital (50 mg/kg, ip, Sigma, St. Louis, MO), the rats were fixed in a stereotaxic frame (Model 51600; Stoelting Co., Wood Dale, IL). As shown in Figure ​Figure2,2, a stainless steel injection cannula (28G) was inserted into the right lateral ventricle (coordinates, 1.5 mm lateral to the midline, − 0.8 mm posterior to the bregma, and −4.6 mm ventral to the skull surface).24 Then, a single icv dose of adenovirus or normal saline was administered (volume, 10 μL; rate, 0.5 μL/minute) using a microinfusion pump (Bioanalytical Systems, Inc., West Lafayette, IN) and a 10‐μL microsyringe (Hamilton, Bonaduz, Switzerland). The cannula was left in place for 5 minutes after the injection was finished and pulled out intermittently. A new cannula filled with saline implants was secured and affixed with TitanBond and dental cement thereafter. The rats were given postoperative care for 7 days. β‐FNA (CAS 72786‐10‐8; Santa Cruz, CA), which is a κ opioid receptor agonist and an irreversible μ opioid receptor antagonist (10 mg/kg diluted in 5 μL of sterile saline) was icv injected via cannula 24 hours before the behavioral test under isoflurane anesthesia in the β‐FNA + Ad‐Null and β‐FNA + Ad‐NEP groups.25, 26 Additionally, nor‐BNI (CAS 105618‐26‐6; Abcam, Cambridge, UK), which is a selective κ opioid receptor antagonist (5 mg/kg diluted in 5 μL of sterile saline) was icv injected via cannula 24 hours before the behavioral test under isoflurane anesthesia in the nor‐BNI + Ad‐Null and nor‐BNI + Ad‐NEP groups.27, 28 At the end of the experiments, the rats were administered an icv injection of 5% Evans Blue (5 μL) and then killed by cervical dislocation for confirmation of the cannula location. The behavioral result from that particular animal was excluded if the cannula was incorrectly positioned.

The peak of β‐EP expression in targeted neurons and the CSF β‐EP concentration after Ad‐NEP icv injection in morphine‐primed relapse rats. (A) β‐EP‐ir neurons in the lateral ventricle wall, subventricular zone and adjacent choroid plexus in the Sham (A1), NS (A2), Ad‐Null (A3), and Ad‐NEP (A4) groups on day 7 after icv injection. (B) Statistical analysis of the number of β‐EP‐ir neurons on day 7 after icv injection in all groups. (C) Statistical analysis of the β‐EP concentration in the CSF on day 7 after icv injection in all groups. ***Compared with the Sham group; P < 0.001. Ad‐NEP, adenovirus vector; β‐EP, β‐endorphin; CSF, cerebrospinal fluid; icv, intracerebroventricular