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Online resource 2: Behavioral test battery

Elevated plus maze: Elevated plus maze test (EPM) was used to measure unconditioned anxiety-like behavior in mice. The EPM apparatus consisted of a four armed (arm size 30 x 5 cm) maze connected with a central platform (5 x 5 cm). Two opposite arms were fitted with 15 cm high transparent side walls (closed arms); the remaining two arms were open. The maze was raised to 38.5 cm above the floor. Mouse was placed in the central platform facing one of the closed arms. Number of entries to the arms, latency to first entry to open arm, travelled distance as well as time spent in different parts of the maze were measured for 5 min using the video-tracking system (Noldus EthoVision 3.0, Noldus Informative Technology, The Netherlands). An entry was defined as the mouse having entered the arm with all four paws.

Open field test: Open field test was used to analyze spontaneous exploratory locomotor activity in a novel environment. In addition, anxiety signals were monitored by measuring entries to and time spent in the central zone. The mouse was placed on well-lit open field arena (35 x 20 x 20 cm), and spontaneous activity (distance travelled, cm), percent of distance travelled in zones, time spend in both zones, number of entries to centre, latency to first entry to centre, time spend in vertical activity were measured for 30 min in Activity chambers (Version 5, MedAssiociates, St. Albans, GA, USA).

Acoustic startle response: Acoustic startle response test was conducted to test the ability to hear and respond to an aversive stimulus using Acoustic Startle Reflex System (Med Associates, St. Albans, GA, USA). The mouse was placed in a round acrylic holder situated on the piezoelectric platform. Acoustic stimulus and background noise (65 dB) were delivered through a loudspeaker. After a habituation period of 5 min, the animal was exposed to 36 stimuli (white noise, 20 ms) randomly varying from 68 dB to 110 dB in 8-15 s intervals. The flinch amplitudes were measured and averaged for each stimulus intensity level.

Prepulse inhibition of acoustic startle response: Prepulse inhibition of acoustic startle response (PPI) test was used to measure sensorimotor gating in Acoustic Startle Reflex System (Med Associates, St. Albans, GA, USA). Animals fixed in the round acrylic holders were placed on piezoelectric platform inside the isolated chamber. Background white nose was 65 dB. Testing was performed in three blocks after a 5 min acclimatization period. The first block consisted of 5 white noise acoustic startle stimuli (SS, 105 dB, 40 ms) presented alone with an intertrial interval of 8-15 s. The second block included 50 trials of 5 different types. One of them was a startle stimulus (SS) alone as in the Block 1. In four other trial types the startle stimuli were preceded by an acoustic prepulse (PPS, 20 ms, white noise burst) of 68, 72, 76 or 80 dB. The delay interval between PP and SS was 10 ms. The third block was exactly as the first one. The startle response was averaged over 10 trials from Block 2 for each trial type. The prepulse inhibition for each prepulse stimulus intensity level was calculated by using the following formula: PPI = 100 -[(PPS + SS startle response / SS alone startle response)] x 100.

Tube test: Tube test was chosen to measure social dominance in mice. Two mice of same sex were placed in opposite ends of a 30 x 3.5 cm transparent plastic tube and released simultaneously. The match ended when one mouse completely retreated from the tube. The mouse remaining in tube is designated the winner, and the retreated mouse is the loser. Each animal was tested against all animals from the opposed group. The percent of retreated matches as well as aggressive postures were scored for each animal. Matches lasting more than 2 min or in which animals crossed over each other were not scored.

Assessment of barbering behavior: Barbering behavior was assessed by examining the hair and whisker loss in group-housed males. Barbering was scored as 0 = intact hair and whiskers, 1 = loss of whiskers.

Resident-intruder test: Resident-intruder test was used to measure social activity and aggression in male mice. An intruder mouse was put in the cage where the test mouse had been acclimatizing for 30 min. Attack behavior, time spent in social activity (sniffing, heterogrooming) and non-social activity (digging, grooming, and rearing) was recorded for 5 min.

Social novelty test: The social novelty test apparatus consisted of three rectangular compartments (18 x 35 x 18 cm) divided by Plexiglas walls with small openings allowing the animal to move between compartments. The test mouse was first allowed to habituate to the apparatus for 10 min. After the habituation period, an unfamiliar mouse (stranger 1) that had no prior contact with the test animal was placed in one of the side compartments. The location of stranger 1 in either of the side compartments varied systematically between the trials. The stranger 1 mouse was enclosed in a small grid cage (7.5 cm diameter, 10 cm high) that allowed snout contact between the bars but not biting or other fighting behavior. The test mouse was then allowed to explore the whole apparatus for next 10 min. The time spent in and entries into each compartment as well as time spent sniffing near the unfamiliar mouse were recorded. After the first test, a second unfamiliar mouse (stranger 2) was placed in a cage in the chamber that was previously empty. In the second test, the test mouse had a choice between an already-investigated unfamiliar mouse (stranger 1) and non-investigated unfamiliar mouse (stranger 2). Again, entries into compartments as well as time spent in each compartment and time spent sniffing were recorded. All data were obtained using video-tracking system (Noldus EthoVision 3.0, Noldus Informative Technology, The Netherlands). With EthoVision 3.0 system it is possible to score behaviors manually during video-tracking. We used manual scoring for calculation of sniffing time during sociability and novelty preference test. The C57BL/6J mice serving as unfamiliar animals had been earlier habituated to the test conditions. Strangers for sociability and the social novelty tests were taken from separate cages.

Stress-induced hyperthermia: Stress-induced hyperthermia was assessed by measuring the rectal temperature of the mice twice (Bioseb rodent thermometer with rectal probe for mice, Bioseb, France). The first measurement was on one hand baseline measurement and on the other hand the hyperthermia-inducing stressor. The second measurement was carried out 10 min later to reveal the stress-induced hyperthermia reaction.

Assessment of nest-building behavior: To assess nest-building behavior of the single-housed males, each mouse was provided with Nestlet pressed cotton squares 1 hour before the dark period. Nest construction was scored the morning after using a 5-point scale (Deacon 2006) as follows:

1. Nestlet not noticeably touched (>90% intact).

2. Nestlet partially torn up (50–90% remaining intact).

3. Mostly shredded but often no identifiable nest site: <50% of the Nestlet remains intact but <90% is within a quarter of the cage floor area, i.e. the cotton is not gathered into a nest but spread around the cage. Note: the material may sometimes be in a broadly defined nest area but the critical definition is that 50–90% has been shredded.

4. An identifiable, but flat nest: >90% of the Nestlet is torn up, the material is gathered into a nest within a quarter of the cage floor area, but the nest is flat, with walls higher than mouse body height (curled up on its side) on less than 50% of its circumference.

5. A (near) perfect nest: >90% of the Nestlet is torn up, the nest is a crater, with walls higher than mouse body height on more than 50% of its circumference.

Reference:

Deacon RM (2006) Assessing nest building in mice. Nat Protoc 1:1117-1119