Mice and viral expression in CeA regions
Adult male C57BL/6 mice (6-8 week-old, 20-25 g, Vital River Inc., Beijing, China) were used in this study. Mice were housed 6 per cage in the animal facility and free to food and water. Surgeries including virus and nanoheaters injection were conducted using a standard stereotaxic apparatus under aseptic conditions. The mice were anesthetized through an intraperitoneal injection of pentobarbital sodium (40 mg/kg). Before in vivo magnetothermal neurostimulation, AAV5 virus encoding TRPV1His-mCherry was packaged and injected into CeA region for getting TRPV1His-expressing mice. The stereotaxic coordinate for the CeA was as follows: – 1.25 AP, – 2.75 ML, – 4.30 DV. A small hole was drilled in the skull at the coordinate of CeA and a 32-gauge needle was lowered into the hole. 300 nL of AAV5 virus were injected into the CeA region at 0.1 μL/min using a microsyrine pump. After lifted by 0.1 mm, the syringe was remained for 10 minutes within the brain before slow withdrawal. At least 4 weeks postviral expression, the anti-His-FVIOs in PBS (2 mg/mL) was injected into the same CeA region. The skin tissue was closed with sutures. TRPV1-expressing and FVIOs-loaded mice were prehoused in a closed box with a diameter of 12 cm under a reverse 12-hour light/dark cycle for 3 days as a habituation stage. Animal husbandry and all experimental manipulation of the animals were performed with the approval of the Animal Care and Use Committee of the Chinese Academy of Military Medical Science and Northwest University.