Behavioral tests

Social interaction test (SIT). SIT was conducted as previously described with minor modifications ^{17, 44}. Of note, the data in Fig. 1C and ​andEE in the present study, in which the experiments were conducted on two consecutive days, were different to previous data, in which the experiments were conducted on a single day ¹⁷. The subject mouse was introduced into a chamber apparatus (60 cm × 40 cm × 35 cm), and was allowed to habituate to the chamber for 30 min for three consecutive days before testing. During habituation, the subject mouse for SIT with calcium imaging was connected to a plastic “dummy” microscope for training. In the S-trial, the subject mouse encountered a novel mouse in a wire cage and an empty wire cage in a chamber for 10 min. In the SN-trial, the subject mouse encountered a familiar mouse, which was co-housed with the subject animal on the day before the SN-trial, and a novel mouse in the wire cage. The parameters analyzed were time spent sniffing each cage (total interaction time), number of visits to each cage, and interaction time per visit to the cage. Measurements were taken using the Ethovision XT 15 software (Noldus). Age- and sex-matched unstressed mice were used as familiar and novel mice. Sociability and social novelty indexes were calculated as follows: (time interacting with mouse or novel mouse cage – time interacting with empty or familiar mouse cage) / (time interacting with mouse or novel mouse cage + time interacting with empty or familiar mouse cage), meaning that indexes of 0 indicated no preference, while positive indexes indicated increased sociability or social novelty behavior, and negative indexes indicated social avoidance or deficits in social novelty behavior.

For SIT without calcium imaging, S- and SN-trials were conducted on postpartum days seven and eight (Fig. 1A). For SIT with calcium imaging, S-trials were conducted on postpartum days seven and eight, and SN-trials were conducted on postpartum days nine and ten. The trials on one of these two days were conducted with optogenetic stimulation and counterbalanced as described in Supplementary Figures S3C and S12A.

Novel objective recognition test (NOR). NOR in Supplementary Figures 12 A–D was conducted on postpartum days seven, 11/12, or 12/13 as previously described with minor modifications ^{46, 47}. The trials on one of the two days were conducted with the light stimulation and counterbalanced. Each trial consisted of three phases: habituation, training, and test phases (10 min each). During the habituation phase, the subject mouse was introduced into a chamber apparatus (60 cm × 40 cm × 35 cm) and was allowed to habituate for 10 min. During the training phase, two identical objects were introduced into the chamber apparatus. In the test phase, one of two objects was replaced by a novel object (Supplementary Fig. 12 E). For NOR in Supplementary Figures 12H, only one object was introduced into the chamber apparatus during the training phase. This modification was made to be analogous to the method for SIT. The parameters analyzed were time spent sniffing the object (total interaction time), number of visits to each object, and interaction time per visit to the object. The time spent sniffing each object was analyzed using the Ethovision XT 15 software. The discrimination index was calculated as follows: (time sniffing novel object − time sniffing familiar object / (time sniffing novel object + time sniffing familiar object), meaning that indexes of 0 indicated no recognition of a novel object, while positive indexes indicated increased novel object recognition and negative indexes indicated deficits in novel object recognition.

Real-time place preference test (RTPP). RTPP was conducted on postpartum day 13 or 14, as previously described with minor modifications ⁴⁵. The subject mouse was introduced into a chamber apparatus (60 cm × 40 cm × 35 cm) and was allowed to habituate for 10 min. Each test consisted of two consecutive 15-min sessions. In the first session, mice were allowed to freely explore the two compartments for 15 min, during which entering into one half of the chamber triggered the light stimulation. Exiting the stimulated chamber immediately terminated the light stimulation. During the second session, the opposite side was paired with optogenetic stimulation. The results of these two sessions were combined, and time spent in the unstimulated and stimulated sides was evaluated using the Ethovision XT 15 software.