All behavioral procedures were carried between 8:00 a.m. and 17:00 p.m. under dim light conditions. Male animals were moved to the testing room in their home cages 30 min before the test begins. The same animals have been used for all adulthood behavioral tests, starting at 6 weeks old. All experiments and analysis were done in blind genotyping by only one experimenter.

To induce vocalization, pups were isolated individually from their mother at P2 to P12. They were placed into an isolation box (23 cm × 28 cm × 18 cm) located inside a sound-attenuating isolation cubicle (54 cm × 57 cm × 41 cm; Coulbourn Instruments, Allentown, PA, USA). An ultrasound microphone sensitive to frequencies of 10 to 250 kHz (Avisoft UltraSoundGate Condenser microphone capsule CM16/CMPA, Avisoft Bioacoustics, Berlin, Germany) was placed in the roof of the box. Vocalizations were recorded for 3 min using the Avisoft Recorder software (version 4.2) with a sampling rate of 250 kHz in 16-bit format. Recordings were transferred to SASLab Pro (version 5.2, Avisoft Bioacoustics), and a fast Fourier transformation (FFT) was conducted (512 FFT-length, 100% frame, Hamming window and 75% time window overlap, high-pass cutoff frequencies 20 kHz) before analyzing the number of calls emitted by mice.

For the open-field test, the mice were individually placed in the square field (38.5 cm × 38.5 cm; Noldus, The Netherlands). Recording began 10 s after the mouse was placed inside the apparatus for a 10-min trial. Behaviors were recorded by a video camera fixed above the apparatus and analyzed using the EthoVision 11.5 software (Noldus, The Netherlands). After each trial, the open field was cleaned with a solution containing 70% ethanol. Anxiety- and locomotor-like behaviors were analyzed using three parameters: the time spent in the center (12.8 cm × 12.8 cm), the number of entries in the center, and the distance travelled.

For the social interaction tests, we performed the three-chamber test as previously described (46). The test was conducted in an apparatus (59 cm × 39.5 cm; Noldus, The Netherlands) divided in a central empty compartment (19.5 cm × 39.5 cm) and two side compartments (19.5 cm × 39.5 cm) containing a plastic cup–like cage for the strangers. The strangers were of the same sex and age as the tested mice and were habituated during 15 min (one habituation per day) to the plastic cup–like cage 4 days before the beginning of the test. The mouse was recorded during four consecutive trials of 5 min. In trial 1 (habituation), the tested mouse was placed in the empty compartment with the access to the other compartments closed. In trial 2 (sociability testing), a first stranger was placed in a plastic cup–like cage in one of the two-side compartments, and the tested mouse could explore freely all the compartments. In trial 3 (posttest), the tested mouse could explore freely its environment and be habituated for a longer time to stranger 1 (familiar stranger). Last, in trial 4 (social novelty testing), the second stranger (novel stranger) was placed in the second plastic cup–like cage in the opposite side compartment. For each tested mouse, the side compartments where the strangers were placed were alternated to avoid any side preferences. All trials were recorded by a video camera placed above the apparatus by using the EthoVision 11.5 software (Noldus, The Netherlands), and the time spent in each chamber was manually analyzed. Trials where the mice have returned less than once to the chamber were removed.

The splash test was performed as previously described (66). Mice were sprayed in the dorsal coat with a 10% sucrose solution. The viscosity of this solution dirties the mice and initiates the grooming behavior. After being sprayed, mice were individually placed in a plexiglass cylinder (15 cm × 45 cm; Form X.L., France), and their behaviors were recorded for 5 min. Self-care and motivational behaviors were manually analyzed using three parameters: the duration of grooming, the latency to start the first grooming, and the frequency of grooming events. A grooming event was defined as at least one episode of any category of grooming (paw licking, head wash, body groom, leg licking, and tail/genital licking).

To test seizure susceptibility, we used convulsant agent flurothyl (2,2,2-trifluoroethyl ether, Sigma) that is widely used to study epilepsy in different animal models (67, 68). The advantage of the flurothyl, as compared to other proconvulsive agents, is that it could be used to induce epilepsy-like activity in juvenile P0 to P30 rats and mice (67, 69). Because the latencies of the effects of flurothyl depend on large number of parameters including atmospheric pressure, humidity, temperature, movements of the air in experimental chamber, animal weight, and age (69), all experiments were performed using pairs of littermate males (P15 and P30) that were placed in transparent, ventilated but hermetically sealed, cages, into which the epileptic agent flurothyl was delivered. The progressive injection of the flurothyl into the cage produced a stereotypical behavioral manifestation of limbic seizure episodes that varied depending on the animal age and genotype. At P15, these episodes commenced in all studied animals with forelimb and/or tail extension and rigid posture (stage 1) followed by period of tranquility with one to two irregular low-amplitude body jiggles (stage 2), one to three brief (1 to 2 s) myoclonic jerks (stage 3), and severe tonic-clonic seizures (stage 4) lasting 5 to 20 s that ended with falling and immobility of the animal (stage 5) (Fig. 4A). At P30, seizure episodes started from animal immobility and ended by severe tonic-clonic seizures (stage 4). The intermediate stages (rigid posture, tranquility period, jiggles, and jerks) were absent or not detectable at this age. Ten seconds after beginning of tonic-clonic seizures in more resistant animal from each pair, the injection of flurothyl was discontinued and cage was inhalated with fresh air. Two to 5 min after stopping of the exposure to flurothyl, most of the mice returned to their four-limb horizontal position but remained immobile during 5 to 10 min. Thereafter, all mice started moving and exploring the cage, although their moving activity was not scored.

Flurothyl was progressively injected into the cage using a nano-pump (Harvard Apparatus) and homogeneously distributed using a mini-ventilator incorporated into the chamber. Behavioral responses were recorded using a video camera. The latency of tonic-clonic seizures was determined post hoc as the time of the first body convulsion in continuous series of tonic-clonic seizures.

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