Adult male mice, aged 7–12 weeks, including histidine decarboxylase-IRES-Cre (HDC-IRES-Cre, #021198, Jackson Laboratory) [36] and C57Bl/6 J mice (Japan SLC, Hamamatsu, Japan) were housed individually. They were kept under a 12-h light–dark cycle, with lights turning on at 07:00 AM, and had unrestricted access to food and water. Behavior tests were conducted during the light phase of this cycle. Animal experiments were performed with the approval of the Institutional Animal Care and Use Committee of Hokkaido University (approval number: 16–0043) and Nagoya City University (approval number: 22–018). The study adhered to the Hokkaido University and Nagoya City University guidelines for the care and use of laboratory animals and complied with several national guidelines: the Guidelines for Proper Conduct of Animal Experiments (Science Council of Japan), the Fundamental Guidelines for Proper Conduct of Animal Experiments and Related Activities in Academic Research Institutions (Ministry of Education, Culture, Sports, Science and Technology, Notice No. 71 of 2006) and, the Standards for Breeding and Housing of and Pain Alleviation for Experimental Animals (Ministry of the Environment, Notice No. 88 of 2006).

CNO (Enzo Life Sciences) was prepared in a solution of 0.5% DMSO in saline and administered to the mice via intraperitoneal injection at a dose of 0.01 ml/g body weight. The control treatment consisted of an identical volume of 0.5% DMSO in saline. The chosen CNO dose (1 mg/kg) was based on prior studies [37]. Ranitidine hydrochloride (Tokyo Chemical Industry, Tokyo, Japan) was dissolved in saline and directly administered into the PRh. The control group received a comparable volume of saline. The dose of ranitidine was selected based on our previous study [4].

The viral vectors pAAV-hSyn-DIO-hM3D(Gq)-mCherry and pAAV-hSyn-DIO-mCherry, kindly provided by Bryan Roth (Addgene viral prep # 44361-AAV8, 50459-AAV8) [38] were used in the study. Mice were anesthetized with isoflurane (0.8–1.5%) and secured in a stereotaxic frame (SR-6 M-HT, Narishige, Tokyo, Japan). To minimize pain, lidocaine (2%; Aspen Japan, Tokyo, Japan) was applied topically to the scalp. We injected AAV8-hSyn-DIO-hM3D(Gq)-mCherry (2.2 × 1013 virus molecules/mL, 0.5 µL) or AAV8-hSyn-DIO-mCherry (4.1 × 1013 virus molecules/mL, 0.5 µL) into the bilateral TMN (A/P: -2.5 mm, M/L: ± 0.92 mm, D/V: -5.34 mm) at a rate of 0.1 µL/min. After injection, the infusion cannulas (33 gauge) were left in place for at least 10 min to ensure effective distribution of the solution. Behavioral tests were carried out after a waiting period of at least 3 weeks, allowing time for the expression of the transgenes in the mice.

To infuse the H2R antagonist into the PRh, guide cannulas were implanted bilaterally 1 mm above the PRh (A/P: -3.05 mm, M/L: ± 4.55 mm, D/V: -2.8 mm) and secured with a self-curing adhesive resin cement (Super-Bond, SUN MEDICAL, Moriyama, Japan). Dummy cannulas (33-gauge) were then inserted into each guide cannula to prevent clogging. Mice were given at least 7 days for postoperative recovery.

The test was conducted similarly to that in our previous study [4] with minor modifications. Mice first underwent habituation sessions for three consecutive days, during which they explored an open field (32 cm × 32 cm × 35 cm) for 15 min each day. During the training session, they were placed in the field with two identical objects and allowed to explore for 15 min. In the test session, they explored for 5 min in the presence of one familiar object and one novel object. These objects were similar in texture and size but distinct in shape. The roles of familiar and novel objects were counterbalanced among the mice. A discrimination score was calculated for each mouse as the ratio (T2-T1)/(T1 + T2) [T1 = time spent exploring the familiar object, T2 = time spent exploring the novel object]. The test area and objects were cleaned with 70% ethanol solution between trials. All sessions were recorded by a camera, and the video was analyzed by using either Noldus Ethovision XT 10 software (Fig. 1) or DeepLabCut [39] (Fig. 3). Exploration was defined as the mouse’s nose being within 4 cm of an object’s center, excluding sitting on the object.

The test was conducted similarly to that in our previous study [40] with minor modifications. Mice were positioned at the center of the elevated plus maze, which consisted of a central area (8 cm by 8 cm) and four extending arms. Two of these arms were open, each measuring 8 cm wide and 25 cm long, while the other two were enclosed, having the same dimensions but with 25 cm-high walls on the sides and end. At the start of each test, the mice were placed in the central section, facing one of the enclosed arms. The animals’ movements were tracked over 5 min using a camera fixed above the maze’s center. The duration the animal spent in the open and closed arms, and the number of entries into the arms were calculated using Noldus Ethovision XT 10 software. An entry into any arm was considered valid when the animal placed all four paws into that arm.

The Barnes maze test was conducted using a circular platform, which was brightly lit at 360–390 lx and 90 cm in diameter, and elevated 76 cm above the floor. This platform featured 20 holes, each 4.5 cm in diameter, positioned 5 cm from its edge. On the first day, the mice were familiarized with an escape box measuring 15 × 9 × 6 cm for 3 min. From the second to the fifth day, they underwent training sessions. During these sessions, the platform had an escape box placed beneath one of the holes. The location of the escape box was fixed across 4 days of training and different among the mice. The mice, initially placed at the platform’s center and covered by a holding chamber, were given 10 s before the chamber was removed. They then had 180 s to freely explore and find the escape box. The session concluded when a mouse fully entered the escape box. These sessions were conducted thrice daily, at 20-min intervals. On either the sixth or twelfth day, a probe test was carried out, during which the escape box was removed from the platform. The procedure mirrored the training sessions but lasted only 90 s. The mice’s behavior was captured by a camera, and the amount of time spent within 5 cm from the center of each hole was analyzed using Noldus Ethovision XT 10 software.

For the microinfusions, 0.5 µL of the solution was administered to each side using 28-gauge infusion cannulas. These cannulas extended 1 mm below the guide cannulas and were operated with a pump for 2 min. To ensure effective diffusion of the solutions, the infusion cannulas remained in place for at least 2 min following the infusion.

After the behavioral experiments, the mice were deeply anesthetized using either pentobarbital or an anesthetic mixture of medetomidine (0.75 mg/kg), midazolam (4.0 mg/kg), and butorphanol (5.0 mg/kg). They were then transcardially perfused with phosphate-buffered saline (PBS), followed by 4% paraformaldehyde (PFA) solution. The brains were post-fixed in 4% PFA overnight at 4 °C, then cryoprotected in 15% and 30% sucrose solutions dissolved in PBS at 4 °C for 48–72 h. Coronal slices, each 40 µm thick, were prepared using a cryostat (CM3050S, Leica). Slices were mounted on slides and stained for nuclei with 4′,6-diamino-2-phenylidole (DAPI) (0.3 µg/ml, 4′,6-diamidino-2-phenylindole, Cat. #340–07971, Dojindo Laboratories, Kumamoto, Japan). To confirm the exact locations of mCherry expression and cannula implantation, images of these slices were captured using a fluorescence microscope (BZ-X700, Keyence) or confocal microscope system (A1RS + , Nikon) at 10 × magnification.

Immunohistochemistry was carried out following tissue processing. Tissue sections were incubated in PBST (0.1% Triton X-100 in PBS) for 15 min at room temperature (RT). For HDC immunostaining, they were then blocked using PBS-BX (3% BSA, 10.25% TritonX-100 in PBS) for 1 h at RT. The sections were incubated with a rabbit polyclonal antibody against HDC (dilution 1:800, POG, Cat. #16045) overnight at 4 °C. Following antibody incubation, the sections were washed three times with PBS-BX for 15 min each, then incubated with AlexaFluor 488 Goat anti-rabbit IgG (dilution 1:1000, Invitrogen, Cat. #A32731) for 2 h at RT. This was followed by 5-min staining with DAPI in PBS. For c-Fos immunostaining, the sections were then blocked using PBST containing 10% normal goat serum (Abcam, Cat. #ab7481) for 1 h at RT. They were treated with a rabbit polyclonal antibody against c-Fos (1:1000, Millipore, Cat. #ABE457) overnight at 4 °C. Following antibody incubation, the sections were washed three times with PBST for 15 min each, then incubated with AlexaFluor 488 Goat anti-rabbit IgG for 2 h at RT. This was followed by 5-min staining with DAPI in PBS. After two additional 5-min washes in PBS, the sections were mounted on glass slides with a mounting medium (20 mM Tris, 0.5% N-propyl gallate, 90% glycerol, pH 8.0). Imaging was performed using a laser-scanning confocal microscope (A1RS + , NIKON, Tokyo, Japan). The proportion of HDC or c-Fos positive cells within mCherry positive cells in the TMN was calculated.

Values are reported as mean ± SEM (standard error of the mean). Statistical analysis was performed using two-way analysis of variance (ANOVA), repeated-measures ANOVA, Tukey’s test, Sidak’s test, and two-sided unpaired t-test, where appropriate.