Animals
Soluble ST2 overexpression mice (ST2 Tg) were generated on a C3H/HeJ background [13]. At least twenty generations of ST2 Tg mice were crossed onto the BALB/c genetic background (Japan SLC Inc., Shizuoka, Japan) and maintained as heterozygote. ST2 transgene-negative and ST2 transgene-positive littermates were designated as WT and ST2 Tg mice, respectively. WT and ST2 Tg mice were housed in separate cages after 4 weeks of age. Mice were housed three to five per cage in a specific pathogen-free animal facility under controlled temperatures (23 ± 3 °C) and controlled light schedule (lights on from 7 A.M. to 7 P.M.), with food and water available ad libitum. Male mice at 8–11 weeks of age were used for the experiments.
All experimental procedures were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals, under the animal protocols approved by the Institutional Animal Care and Use Committee of Jichi Medical University.
Reverse transcription (RT)-PCR analysis
DNase I-treated total RNAs were isolated from indicated tissues, and subjected to RT-PCR analysis as described previously [10]. The PCR primers were as follows: ST2 (forward for exogenous and endogenous 5′-TGGCATGATAAGGCACACCATAAGGCT-3′, reverse for exogenous 5′-GCAGCCTGCACCTGAGGAGTGAA-3′, reverse for endogenous 5′-GTTAGTGTCTCTCTCCCTCCCATGC-3′), and β-actin (forward 5′-ATCTACGAGGGCTATGCTCT-3′, reverse 5′-TACTCCTGCTTGCTGATCCA-3′). The PCR products were separated on 2% agarose gels.
Enzyme-linked immunosorbent assay (ELISA)
The concentration of soluble ST2 in serum was measured by a sandwich ELISA as described previously [23].
Behavioral tests
Before beginning testings, each mouse was observed in a clean cage for a general health check and neurological screening tests according to methods of our previous reports [24, 25]. The neurological screening tests were designed to detect any gross abnormalities in physical function. The ear-twitch reflex occurred when the pinna was touched with a cotton swab from behind, resulting in immediate movement of the ear. The eye-blink reflex occurred when a cotton swab was approaching the eye, resulting in blinking. The postural reflex was evaluated by placing the mouse in an empty cage and shaking the cage, eliciting the extension of all four legs to keep an upright, balanced position. The righting reflex was tested by turning the mouse over onto its back, eliciting an immediate turnover response to restore the upright posture on all four feet. The whisker-touch reflex was tested by lightly touching whiskers of a freely moving mouse. Normal mice will stop moving their whiskers and turn the head to the side on which the whiskers were touched.
In Experiment I, comprehensive behavioral phenotyping was performed to reveal the role of soluble ST2 on sensorimotor functions, anxiety-like behaviors, a depression-like behavior, social behaviors, and learning and memory functions. In a visual placing test, a Preyer reflex test, a von Frey filament test, an olfactory habituation dishabituation test, a rotarod test, a wire hang test, a light-dark exploration test, a general activity monitoring, a novel place/object recognition test, social interaction test, and a social recognition test, the same mice were used (WT: n = 9, ST2 Tg: n = 9). In a hot-plate test and a forced swimming test, the same mice were used (WT: n = 9, ST2 Tg: n = 11). In a tube test, paired encounters of WT and ST2 Tg mice (WT: n = 10, ST2 Tg: n = 10) were staged based on body weight. In a novel place/object recognition test, social interaction test, and a social recognition test, the same mice were used (WT: n = 5, ST2 Tg: n = 5). In Experiment II, to confirm the role of soluble ST2 on depression-like behaviors and to check the reproducibility of result of the forced swimming test, a depression test battery (two bottle choice test, forced swimming test, and tail suspension test) was applied to the same mice (WT: n = 12, ST2 Tg: n = 12). The behavioral assessments were performed by experimenters blinded to the genotype. Information about the test order and time of day were provided in Supplementary Table 1.
Experiment I: comprehensive behavioral phenotyping
Visual placing test
The visual placing test was used to assess the visual function [25]. Each mouse was suspended by the tail approximately 30 cm above a flat table surface and then gradually lowered to the Table. A normal mouse extends its forepaws for a “soft landing” as the table surface approaches. A blind mouse does not see the approaching surface so that the mouse does not extend its forelimbs until its whiskers or nose touch the table. Extension of the forepaws was recorded as a yes or no response by the investigator.
Preyer reflex test
The Preyer reflex test was used to assess the auditory function [25]. The Preyer reflex is a flinch response to the sound of a loud hand clap. The reflex was recorded as a yes or no response by the investigator.
Von Frey filament test
The von Frey filament test was used to assess tactile function [25]. Each mouse was placed on an elevated platform with wide gauge wire mesh surface. A von Frey hair (2.9 N) was inserted through the mesh holes from underneath to poke the undersurface of a hind paw. A normal response was defined as the mouse quickly flicking its paw away from the von Frey hair. If the mouse showed the normal responses in two out of three consecutive trials, its tactile ability was recorded as normal.
Olfactory habituation/dishabituation test
The olfactory habituation/dishabituation test was used to assess olfaction [25]. The odorant stimuli were tap water, vanilla extract (Kyoritsu-foods, Tokyo, Japan) diluted 1:100 in tap water, and rum extract (Kyoritsu-foods) diluted 1:100 in tap water. The odorant stimuli and the dilution factors were determined based on pilot experiments in our laboratory. Experimenters presented stimuli by dipping a cotton swab in the stimulus solution and then placing it through the wire lid of the cage. The cotton swab was positioned 4.4 cm from the bottom of the cage. Each stimulus was presented for 3 min and then replaced with a fresh swab scented with the same odorant for a total of three presentation of each stimulus and nine total olfactory stimulus presentations. The order of presentation was water (3x), vanilla (3x), and rum (3x). An experimenter recorded the cumulative time that the mouse spent in sniffing the cotton swab with a stopwatch. Sniffing was defined as (a) tilting the head upward with the nose oriented toward and within 2 cm of the swab, (b) rearing with the nose oriented toward and within 2 cm of the swab, and (c) physically contacting the muzzle to the swab if the mouth was closed. Occasional open-mouth contacts were considered to be chewing and not included in the cumulative sniff time.
Hot-plate test
Thermal pain sensitivity was assessed by a hot-plate test [26]. The test was performed according to the method described by our previous report [25]. The mouse was placed on a metal surface (19 cm in diameter, Muromachi Kikai, Tokyo, Japan) maintained at 54 ± 0.1 °C. The hot plate was surrounded by a transparent plastic barrier 20-cm in diameter and 25-cm in height. The latency to jumping off the plate or licking a hind paw was recorded. Sixty seconds was used as a cut-off time to protect the paw against injury.
Rotarod test
Motor coordination and balance were assessed using an accelerating rotarod (Muromachi Kikai Co., LTD) as described previously [25]. Mice were placed on a cylinder that slowly accelerated from 4 to 40 rpm the latency to fall recorded for a maximum of 300 s. When a mouse is placed on a rotating cylinder, the mouse must continuously walk forward to keep from falling off the rotarod. Each mouse performed three trials.
Wire hang test
Motor function was assessed using the wire hang test, which requires balance and grip strength [25]. A standard wire cage lid was used for this test. Masking tape placed around the perimeter of the lid prevented the mouse from walking off the edge. The test was performed by placing the mouse on the top of a wire cage lid. The investigator shook the lid lightly three times to cause the mouse to grip the wires and then turned the lid upside down. The upside-down lid was held approximately 40 cm above the cage litter, high enough to prevent the mouse from easily climbing down but not high enough to cause harm in the event of a fall. Each mouse performed two trials. The investigator used a stopwatch to time the latency in falling off the wire lid with a maximum of 60 s. The average of two trials was used as an indicator.
General activity monitoring
The general activity monitoring was performed using a cubical box (30 × 36 × 17 cm) made of white Plexiglas. Black lines were drawn on the floor with a marker. The lines divided the floor into nine 10 × 12 cm squares. The mouse was placed in the central square in the middle of the cubical box, and the number of line crossing was measured for 10 min under dim lighting (4 lx). A line-crossing was counted only when the animal crossed the line with all four paws.
Light-dark exploration test
The light-dark exploration test was performed as described previously [25]. The apparatus consisted of a polypropylene cage (44 × 21 × 21 cm) separated into two compartments by a partition with an aperture (12 × 5 cm) at floor level. The larger compartment (29 cm long) was open-topped, transparent, and brightly illuminated by white light from a 40 W desk lamp (1000 lx). The smaller compartment (15 cm long) was close-topped and painted black. Mice were placed individually in the center of the light compartment, facing away from the partition, and allowed to freely explore the apparatus for 10 min. The number of light-dark transitions between the two compartments and the total time spent in the dark compartment were recorded.
Forced swimming test
To evaluate depression-like behavior of mice, behavioral immobility termed “learned helplessness” was assessed by the Porsolt forced swimming test [27, 28]. The test was performed according to the method described in our previous report [25]. Each mouse was placed in individual glass cylinders (24.5 cm tall, 19 cm in diameter) filled with water (23–25 °C water) to a depth of 15 cm. The mice could not support themselves by placing their paws on the floor of the cylinder. Behavioral scoring employed a standard 6-min test duration. The duration of immobility during the last 4 min of the test period was quantified. A mouse was judged to be immobile when the mouse stopped swimming and remained floating with minimal movements necessary to keep its head above water.
Tube test
Social dominance was assessed by a tube test. The tube test assay was adapted from Lindzey et al. [29]. The test was performed according to the method described in our previous report [25]. The test employed a transparent Plexiglas tube 30 cm in length with a 3-cm inside diameter, which is sufficient to permit one adult mouse to pass through and to keep from reversing direction. For training, each mouse was released at alternating ends of the tube to run through the tube, sometimes with the help of a plastic stick pushing at its back. Each mouse was given eight training trials. Then, the mice were given the test trial. In the test trial, two mice were released simultaneously at opposite ends, and care was taken to ensure that they met in the middle of the tube. The mouse that first retreated from the tube within 2 min was designated the “loser”. In rare cases when no mouse retreated within 2 min, the tests were repeated. If the game was not resolved after two matches, a draw was made. Between each trial, the tube was cleaned with 70% ethanol. In WT mice, five WT mice (11 weeks of age) were cagemates and the other five WT mice (10 weeks of age) were cagemates. In ST2 Tg mice, five ST2 Tg mice (11 weeks of age) were cagemates and the other five ST2 Tg mice (10 weeks of age) were cagemates. Between the WT and ST2 Tg mice (WT n = 10, ST2 Tg n = 10), paired encounters were staged based on body weight and age not using a round-robin design. Those with similar weights competed.
Novel place/object recognition test
To evaluate learning ability of mice, recognition memory about place and object was assessed by a novel place/object recognition test [30]. The test was performed according to the method described in our previous report [25]. Briefly, the test was performed using a cubical open field (30 × 36 × 17 cm). The test consisted of three sessions with intertrial intervals of 2 min. During the habituation session, four different plastic objects were presented in open field. Exploration of four different plastic objects in open field was measured for 15 min under dim lighting (4 lx). For the place recognition session, the four objects initially placed in a square arrangement were reconfigured into a polygon-shaped pattern by moving two objects (displaced objects, DOs). The remaining two objects were left at the same location (non-displaced objects, NDOs). The time spent exploring the DOs and NDOs was recorded for 5 min. In the object recognition session, one of the NDOs was replaced with a novel object (NO), and the two DOs were removed. The time examining the NO or familiar NDO was recorded for 5 min. Exploratory behavior was defined as (a) tilting the head upward with the nose oriented toward and within 2 cm of the object and (b) physically contacting the muzzle or forelimb to the object. The percentage of novelty preference was calculated as follows: (novel place or object exploration duration) / (novel place or object exploration duration + familiar place or object exploration duration) × 100.
Social interaction test and social recognition test
To examine social interest to a same-sex BALB/c mouse (8–11 weeks of age), each mouse was allowed to explore an unfamiliar male mouse in a cubical box (30 × 36 × 17 cm) for 10 min. Two identical, wire cup-like containers with removable lids large enough to hold a single mouse were used. These were placed vertically inside the apparatus, one in each side chamber, and contained a naïve mouse or not. Each container was comprised of metal wires to allow for air exchange between the interior and exterior of the cylinder but small enough to prevent direct physical interactions between the inside animal and outside. Time spent exploring the mouse was recorded. Exploratory behavior was defined as (a) tilting the head upward with the nose oriented toward and within 2 cm of the mouse and (b) physically contacting the muzzle or forelimb to the mouse.
Recognition memory of other individuals was assessed by a social recognition test [25]. One hour later following the social interaction test, the mouse was returned to the cubical box and exposed to the mouse encountered at the social interaction test and a novel mouse for 10 min. Two identical, wire cup-like containers were also used. These contained the same, or novel mouse. Time spent exploring each mouse was recorded. The percentage of novelty preference was calculated as follows: (novel individual exploration duration) / (novel individual exploration duration + familiar individual exploration duration) × 100.
Experiment II: depression test battery
Depression-like behavior of mice were assessed by the following three tests: (1) Two bottle choice test was performed to examine hedonic reaction to sucrose [24]. The test was performed according to the method described in our previous report [24]. Briefly, the mouse was placed in a square plastic cage and habituated for approximately 24 h. During this habituation period, the mouse was allowed to drink from two bottles containing water. After the habituation period, one of the two bottles was replaced with a bottle containing 5% sucrose and consumption monitored for 24 h. The preference for sucrose was calculated using the following formula: Preference (%) = sucrose consumption (g) / total consumption (g) × 100. Water and food consumption were also measured during this habituation period. After 24 h from the onset of the habituation procedure, the mouse was briefly removed from its cage and weighed, and the amount of food remaining including any on the bottom of the cage was recorded using an electronic analytical scale (EK-610i, A&D Company, Tokyo, Japan). Food consumption was calculated as the difference between the amount of food at the start and at the end of habituation period. The two bottles containing water were also weighed using the same electronic analytical scale at the onset and end of the habituation period to calculate the water consumption. The water and food consumption were calculated using the following formula: Water or food consumption (g/g) = water or food consumption (g) / body weight (g). (2) Antidepressant activity was assessed by the Porsolt forced swimming test [27, 28]. (3) Antidepressant activity was also assessed by the tail suspension test [31, 32]. Briefly, mice were securely fastened to a flat metallic surface by the tip of the tail using medical adhesive tape and suspended 30 cm above the ground. The time of immobility was quantified.
Statistical analysis
The differences in the mean between two groups were tested using Welch’s t-test after transformation to ranks. Two-way ANOVA with repeated measures was used to assess tests containing two variables. A chi-square analysis was used for tube test. P-values less than 0.05 were regarded as statistically significant.
