2.2. Guide cannula implantation and intra-BNST drug injections in awake mice

Guide cannula implantation was performed by the methods we have previously reported [16],[27],[32],[33]. Since we implanted two guide cannular in this study, the method was modified for performing to the bilateral drug injections. For bilateral BNST drug injection in awake mice, we implanted the two guide cannulas into the BNST at least 5 days before the behavioral test. Mice were anesthetized with a mixture of medetomidine hydrochloride and butorphanol tartrate (0.3 and 5.0 mg/kg, respectively; Wako Pure Chemical Industries Ltd., Tokyo, Japan) and midazolam (4.0 mg/kg; Sandoz Ltd., Yamagata, Japan) to ensure the loss of sensation, including loss of pain sensation and immobilization during procedures. After the anesthesia, mice were placed in a stereotactic frame, and four holes were made in the skull using a dentist drill. Two holes were used for placement of steel guide cannulas (AG-8; length = 8 mm, i.d. = 0.4 mm; o.d. = 0.5 mm; Eicom, Kyoto, Japan), and the other two holes were made to anchor the stabilizing screws. For bilateral BNST injection of artificial cerebrospinal fluid (aCSF) or drug injection (Figure 1A) of freely-moving mice (without the anesthesia), two guide cannulas were implanted at the site, followed by dummy cannulas with cap nuts. However, because each cap nut had an o.d. of 5 mm, the cap nuts interfered with each other when inserted vertically. Therefore, these guide cannulas were inserted at an angle of 60° to the anterior and posterior sides, respectively (Figure 1A). These stereotaxic coordinates (mm), according to the Paxinos mouse brain atlas [34] were as follows: for intra-BNST injection from the anterior side for the right hemisphere, anteroposterior (AP): +1.6 mm, lateral (L): −0.5 mm from bregma, depth (DV): −4.1 mm; from the posterior side for the left hemisphere, AP: −3.05 mm, L: −0.5 mm from bregma, DV: −4.4 mm. Coordinates refer to the bregma and the dura surface (Figure 1A) [34],[35]. Each cannula was held in position by dental cement (GC Unifast II, GC Dental Products Corp., Tokyo, Japan) attached to the stabilizing screw. The dummy cannulas (AD-8; Eicom) were inserted into the guide cannulas and fixed with cap nuts (AC-1; Eicom) until the behavioral experiments. To reduce the pain by surgery, we applied the 5% of EMLA cream which contains the two different active local anesthetics, lidocaine and prilocaine, twice every day until behavioral experiments after the surgery. After waking from anesthesia, mice were caged until the behavioral assessment, and experiments were performed. Guide cannula-implanted mice were handled individually, once daily, for 2 days before surgery, and then twice daily during the next 4 days after anesthesia recovery until the behavioral tests were performed.

The drug was dissolved in aCSF. It has been demonstrated that the administration of a 0.1 µL volume over 1 min gave a 0.12 mm³ (≈ 0.493 mm × 0.493 mm × 0.493 mm) volume [36]. Microinjection was carried out by infusing 0.125 µL of drug solution (0.1 µL/1 min) using an Eicom cannula swivel unit (SSU-20) attached to an injector and a 5-µL Hamilton syringe. A dialysis probe was used for drug infusion, and the tip of the dialysis membrane part (AI-802, o.d. = 0.35 mm; Eicom) was cut and adjusted to 8 mm (the same length as the guide cannula). A polytetrafluoroethylene coiling tube (CT-20; Eicom) was used to infuse aCSF or drugs, and the mouse was allowed to move freely during drug infusion in the home cage. After drug injection, the injection probe was kept in place for at least 5 min, to minimize the spread and leaking of the drug along the injection track [37]. The drug was infused 30 min before the behavioral tests. The probe was removed 5 min after drug injection, and each mouse was kept in the home cage and monitored until the behavioral tests.