Stereotaxic frame

At least 2 weeks before recording, mice were chronically implanted with custom designed headpieces for head fixation during awake recordings using standard methodology. Briefly, mice were anesthetized with 2.5% and maintained at 1.5% isoflurane in oxygen, and secured a stereotaxic frame (Narishige). Local anesthesia (0.25 ml of 0.625 mg bupivacaine) and antiseptic (Betadine) were applied. Periosteum was exposed and additional local anesthetic (0.25 ml of 2% Lidocaine gel) was applied. Bregma and lambda as well as the site of the future craniotomy (+1 mm to −5 mm AP, +0.25 mm to +6 mm ML left of bregma) were marked. The exposed skull was scored and the headpiece was attached using dental cement (Metabond) and 3 skull screws. Cyanoacrylate adhesive (Loctite 495) was applied over any remaining exposed skull. Mice were given 0.5 mg cefazolin and 0.125 mg meloxicam, and 7 ml of normal saline SQ after surgery. Animals were left to recover for a week before starting the habituation protocol. Mice were habituated to head fixation with body restraint with visual stimuli gradually over the course of 4 days. By the end of day 4, mice tolerated awake head fixation and visual stimuli for 45 min uninterrupted without any apparent distress.

To eliminate hypothalamic progenitor cell proliferation, cytosine arabinoside (AraC, Sigma, St Louis, MO, USA) was chronically infused into the intracerebral ventricle by using an osmotic pump (MODEL2006, Alzet, CA, USA). Rats were anesthetized, the skull was exposed and a small hole (bregma; 0.8 mm posterior and 1.4 mm lateral) was drilled according to the atlas of Paxinos and Watson using a stereotaxic frame (Narishige, Japan). The osmotic pump contained 235 μL of AraC dissolved in saline (1 μg/μL) and was infused at an infusion rate of 0.15 μL/h over 42 days (AraC rats). This dose of AraC effectively blocked cell proliferation in the hypothalamus without causing overt adverse effects on health [11 (link), 14 (link)]. Saline-infused rats were used as a vehicle group (Veh rats). Non-operated rats (Naive rats) and sham-operated rats (Sham rats) were also prepared. All rats were allowed to recover from surgery for 10 days. After the recovery period, AraC and Veh rats were subjected to a constant T_a of 32.0 ± 0.2°C for 40 days (AraC+HA and Veh+HA), while respective CN rats (AraC+CN and Veh+CN) were kept at a T_a of 24.0 ± 0.1°C. After 40-day of heat exposure, rats were subjected to a heat tolerance test and the behavioral thermoregulation of rats was investigated. Subsequent to this, brains were removed and used for immunohistochemical analysis.

The methods in the present study were approved by the Institutional Animal Care and Use Committee of Fukuyama University (H26-A-5, 2019-A-5), and the animals were treated in accordance with the guidelines of the United States National Institutes of Health regarding the care and use of animals for experimental procedures.
In total, 292 male ddY mice (Nihon SLC, Shizuoka, Japan) were used. Mice were housed at 22 ± 1°C under a 12-h light and dark cycle with free access to food and water. The mice were anesthetized by intraperitoneal injection of a mixture of medetomidine (0.3 mg/kg, Kyoritsu Seiyaku, Tokyo, Japan), midazolam (4 mg/kg, Alfresa, Osaka, Japan), and butorphanol tartrate (5 mg/kg, Meiji Seika Pharma, Tokyo, Japan), and then placed in a stereotaxic frame (Narishige, Tokyo, Japan). Collagenase type VII (0.03 U/3 μL saline) or the same amount of saline was injected via a Hamilton syringe into the unilateral striatum (0.2 mm anterior, 2.2 mm lateral from the bregma suture, and 3.5 mm depth from the skull) for 3 min [11 (link)]. The syringe was slowly removed 1 min after the injection, and then the scalp incision was sutured. An IDO inhibitor, 1-MT (1 mg), was dissolved in saline using 0.1 N NaOH solution and injected subcutaneously 1 h after collagenase injection and then daily. Control mice were injected with saline instead of 1-MT.

Following an intraperitoneal injection of sodium pentobarbital (40 mg/kg body weight), the anaesthetized rats were placed in a stereotaxic frame (NARISHIGE, Japan). Using a glass micropipette (internal tip diameter: 15–25 μm) that was attached to a 1 μl Hamilton microsyringe, 0.6–0.8 μl of 10% TMR (D-3308, 3000 MW; Molecular Probes, Eugene, OR, USA) dissolved in 0.1 M of citrate-NaOH (pH 3.0) was injected into the right thalamus, and 0.2 μl of 4% FG (80,014, Biotium, Hayward, CA, USA) dissolved in normal saline was injected into the left PBN. After each injection, the glass micropipette was maintained in place for 15 min. All 30 rats injected with TMR and FG were allowed to survive for 7 days. Furthermore, the rats were equally divided into two groups. While lightly anaesthetized with ethyl ether, 0.1 ml of normal saline was injected into the upper lip ipsilateral to the FG injection site of the 15 rats in the first group, whereas the rats in the second group were subcutaneously injected with 0.1 ml of 4% formalin dissolved in normal saline into the upper lip ipsilateral to the FG injection site. The animals subsequently survived for 2 h prior to euthanasia. The results of the tract tracing were obtained from 6 rats in which the tracer was injected properly into the two target areas; the remaining 24 rats were discarded because of inappropriate injection sites.