50 ml plastic tube

Measurement of core body temperature and heart rate was performed using a radio-telemetry transmitter (TA11ETA-F10; DataSciences International, St Paul, MN, USA) and Dataquest A.R.T. 4.30 (DataSciences International) according to a method^{5 (link)}. Briefly, a radio-telemetry transmitter was implanted following the surgical procedure described by the manufacturer. After the surgery, the mice were allowed to recover for ~10 days before testing. On the test day, each mouse was placed in a separate test cage (17.5 × 10.5 × 15 cm) without a lid placed in a chemical fume hood, habituated for 10 min, and subjected to presentation of filter paper containing test compound (Fig. 1b, e and Supplementary Fig. 1).
For the restrained condition, mice were implanted with radio-telemetry probes approximately 10 days prior to the experiment, as described above. On the test day, each mouse was placed in a separate test cage (17.5 × 10.5 × 15 cm) without a lid, allowed to habituate for 10 min, and physiological parameters were analyzed for 10 min. Immediately afterward, mice were restrained in ventilated 50-ml plastic tubes (Becton Dickinson and Company, Franklin Lakes, NJ, USA), and physiological parameters were analyzed for 30 min (Fig. 1d, f).
Physiological parameters were automatically transmitted from the device every 10 s using Dataquest A.R.T. software (DataSciences International).

Cutaneous blood flow was analyzed using a laser Doppler blood flow monitor (ALF21D; Advance Company Ltd, Tokyo, Japan). Five days prior to the test day, C57BL/6 mice were anesthetized with pentobarbital (50 mg/kg, IP) and the fur on the back was removed with a chemical hair remover. On the next day, mice were restrained in ventilated 50-mL plastic tubes (Becton, Dickinson and Company) and allowed to habituate for 40 min. In addition to ventilation holes, a small window (1.5 × 2.5 cm²) was opened in the tube to allow access to the skin on the back. This habituation procedure was repeated once per day for 4 days. On the test day, mice were restrained in the tube, and a laser Doppler probe was affixed to the back using adhesive tape. After stable blood flow signals were obtained during the habituation period (~30 min), mice were subjected to three consecutive odor presentations: (1) no-odor for 10 min, (2) eugenol for 10 min, and (3) test odorant (271 µmol 2MT [unpaired] or 271 µmol Anis that had been previously paired with FS for 20 min) (n ≥ 8 each). Each odorant was presented to the mouse’s nose using a filter paper. Blood flow signals were automatically extracted every 10 s using the included software. Mean blood flow values during no-odor presentation (10 min) and test odorant presentation (20 min) were calculated. Control (no-odor) values were set to 100%.

The experimental schedule is presented in Table 1. ACMS was performed with a daily combination treatment of ACTH and CMS, as described in the Introduction. Individual stressors in CMS are exposed to animals at various timings and methods, depending on the laboratory [25 (link)]. To avoid excessive impairments induced by the combination of ACTH, we used a CMS protocol, which did cause moderate weight loss in the preliminary experiments. To elevate low corticosterone levels in the light phase, ACTH (Cortrosyn, 0.45 mg/kg, Daiichi Sankyo, Tokyo, Japan) was subcutaneously administered once daily at 9:30–11:30 based on previous reports [21 (link)]. The CMS protocol consisted of one or two daily exposures to random stressors between 9:30–14:00 (short stressor) and overnight (overnight stressor), including the following: (1) 2 h of restraint in a 50 mL plastic tube (Falcon) with openings for ventilation on both sides, (2) 15 min of forced swimming (28 °C), (3) wet bedding, (4) night lighting, (5) cage tilting from horizontal to 45°, (6) water deprivation, and (7) food deprivation. All stressors were repeated throughout the 31-day experiment.

As previously described, rats were exposed to one of the following stressors daily for 28 days in an unpredictable manner, but with some modifications [56 (link),57 (link)]. Exposure to the same stressor was not carried out for more than two consecutive days.
CUMS exposure: Tilted cage—home cages were tilted at a 45° angle for 5 h; Shaking—4 groups of rats were placed in a plastic box container and placed in an orbital shaker for 30 min at 150 rpm; Restraint—rats were placed in a 50 mL plastic tube (Falcon) with openings on both sides for breathing for 3 h; Social defeat—rats were placed in a transparent and perforated plastic container to avoid further physical contact for 8 h; Inverted light cycle—regular room light was switched off during the day time and put on during night time for 2 days.
The initial and final body weight of the rats were recorded during the experimental period.

Mice of the four test groups (excluding the controls) were housed in individual cages and exposed to the CUMS protocol for 8 weeks [30 (link)]. The protocol included the daily exposure to a single random stressor. Stress procedures included (1) 2 h of physical restraint in a 50 ml plastic tube (Falcon) with openings in both sides for breathing, (2) 10 min exposure to hot air steam from a hairdryer on a low setting, (3) 3–4 h with damp bedding, (4) 6 h overnight illumination, (5) 4 h tilting the mouse's cage at a 45° from the horizontal, (6) 10 min warm water forced swimming, (7) 5 min cold water (4 °C) swimming, after which the mice were toweled dry, and (8) 24 h light/dark alterations. All stressors were randomly scheduled over a one-week period and repeated throughout the eight-week experiment (Table 1) to ensure the unpredictability of the stressors.Table 1

The CUMS protocol-induced depression mouse model

Day	Stressor	Duration
Saturday	Physical restraint	2 h
Sunday	Hot air steam	10 min
Monday	Damp bedding	3–4 h
Tuesday	Overnight illumination	6 h
Wednesday	Warm water swimming	10 min
	or cold water (4 °C) swimming	5 min
Thursday	Tlting cage	3–4 h
Friday	Light/dark alterations	24 h

Mice were placed in an adequately ventilated 50 mL plastic tube (FALCON) for 20 min. They could rotate from a prone to supine position and back again but not turn head to tail.