Three-Chamber and Three-Trial Social Interaction Test

Social interaction test was conducted as previously described (Nadler et al., 2004; Moy et al., 2007; Li et al., 2015). The box (62 cm L x 40 cm W x 22 cm H) was divided by two equally spaced white Plexiglas panels with retractable rectangular doorway (5 cm W x 22 cm H) allowing free access into each chamber (20 cm L x 40cm W x 22 cm H), for the convenience of applying multi-electrode recording and optogenetics in three-chamber social interaction experiments. Each of the two side chambers contained an empty wire cylindrical Plexiglas cage (15 cm H, 10 cm bottom diameter, and 15 bars spaced 1 cm apart) at the corner of same side to enclose strangers. At the end of each test, the apparatus was cleaned with 75% ethanol and dried with paper towels. Behavioral testing was carried out in a sound-proof, dimly lit (less than 20 lux) animal behavioral test room.

The standard three-chamber social interaction test includes three trials, each for 10-min. The subject mouse was first placed in the middle chamber and allowed to explore all chambers for 10-min habituation. After trial 1, the doorway was closed and the subject mouse was kept in the middle chamber. An age-match unfamiliar conspecific male mouse (stranger 1, S1) was enclosed in one of the wire cages. Then, the doors were raised again and the subject mouse was free to explore the chambers for a 10-min session. After trial 2, the subject mouse was kept in the middle chamber again and a second stranger mouse (stranger 2, S2) was placed in another wire cage. The subject mouse then was left to explore the chambers for another 10 min session in trial 3. All the social processes were videotaped from above and stored by Ethovision XT8 video-tracking system (Noldus Information Technology, Wageningen, Netherlands). The sniff zone was defined as proximity (2 cm) with the cylindrical cage. The time of directing the nose to the stranger within sniff zone and/or touching the cage with the nose was hand scored by a skilled technician who was blind to the genotypes. The positions of stranger 1 were alternated between left and right sides across each of the subject mice. Each stranger was placed in the cages no more than three times each day.

For the delay-dependent social memory test, there was an inter-trial interval (ITI) of 1 min, 5 min, 30 min, or 6 h between trial 2 and trial 3. In within-subject test, each subject mouse was tested once a week but twice at most. The ITI between trial 2 and trial 3 in the rest of three-chamber social interaction tests was 5 min unless mentioned elsewhere. During ITI 1 min, 5 min, and 30 min, subject mouse was kept in the middle chamber. During ITI 6 h, subject mouse was returned to home cage, the selected familiar littermate, the subject mice from the same cage and the strangers were kept separately with food and water access ad libitum. For position-dependent social memory test, at the end of trial 2, stranger 2 (S2) was placed into the cage which enclosed stranger 1 in trial 2; stranger 1 (S1) was switch to the cage which was empty in trial 2. For novelty-dependent social memory test, at the end of trial 1, a littermate male mouse (familiar, F) was enclosed in one of the empty cages; after trial 2, a stranger mouse (S) was placed in the wire cage that had been empty in trial 2. For position-independent social memory test, at the end of trial 2, a stranger mouse (S) was placed into the cage which enclosed familiar mouse (F) in trial 2; the familiar mouse (F) was switched to the cage which was empty in trial 2. For behavioral characteristics of stranger 2 in social interaction, a mouse-sized wooden object (Ob), or a familiar mouse (F) was treated as stranger 2 respectively. All the stranger 1, stranger 2 and familiars were termed as stimuli mice.

Three weeks after the injection of pAAV-CaMKIIα-NpHR3.0-EYFP and/or pAAV-CaMKIIα-EYFP, two optical fibers (NA = 0.37, Φ = 200 μm; Fiblaser, Shanghai, China) linked to a communicator was connected to the pre-planted ceramic ferrules. The light pulse given by a yellow light laser (MBL-II, Brain-King Limited Co., Beijing, China) was controlled by a stimulator (GRASS S88X, Astro-Med, Inc, West Warwick, United States).