Longitudinal analysis of ultrasonic vocalizations in mice from infancy to adolescence: Insights into the vocal repertoire of three wild-type strains in two different social contexts

Abstract

Ultrasonic vocalizations (USV) are emitted by mice under certain developmental, social and behavioral conditions. The analysis of USV can be used as a reliable measure of the general affective state, for testing the efficacy of pharmacological compounds and for investigating communication in mutant mice with predicted social or communication deficits. Social and communication studies in mice have focused mainly on the investigation of USV emitted by neonatal pups after separation from the dam and during social interaction between adult males and females. Longitudinal USV analysis among the different developmental states remained uninvestigated. In our study, we first recorded USV from three inbred mouse strains C57BL/6N, DBA/2 and FVB/N during the neonatal stages after separation from the littermates and then during a reunion with one littermate. Our results revealed significant strain-specific differences in the numbers and categories of USV calls. In addition, the USV profiles seemed to be sensitive to small developmental progress during infancy. By following these mice to the adolescent stage and measuring USV in the three-chamber social test, we found that USV profiles still showed significant differences between these strains in the different trials of the test. To study the effects of social context on USV characteristics, we measured USV emitted by another cohort of adolescent mice during the direct social interaction test. To this end, this study provides a strategy for evaluating novel mouse mutants in behavioral questions relevant to disorders with deficits in communication and sociability and emphasizes the important contribution of genetics and experimental contexts on the behavioral outcome.

Fig 1. USV analysis of C57BL/6N, DBA/2 and FVB/N pups during infancy after separation and reunion with a littermate.

(A) A schematic drawing of the experimental protocol. The recording of USV during infancy was done at P4, P8 and P12. The recording of USV for adolescent mice was done at P24 after 24 h of isolation of mouse from the littermates, VP: vaginal plug, PST: pup separation test. (B) A schematic drawing of the experimental setup for USV recording during infancy. USV were recorded for pups during separation phase (trial 1) for 5 min followed by a reunion with a littermate (trial 2). (C-F) USV analysis including numbers (C), durations (D), frequencies (E) and amplitudes (F) of calls at P4, P8 and P12 during separation phase (trial 1) and reunion with a littermate (trial 2); n = 7 for C57BL/6N (2 males and 5 females), n = 8 for DBA/2 (4 males and 4 females) and n = 11 for FVB/N (6 males and 5 females), all data as mean ± SEM (two-way ANOVA, *p< 0.05, **p< 0.01).

Fig 2. Analysis of USV categories of C57BL/6N, DBA/2 and FVB/N pups.

(A) Representative sonograms of different calls categories with time in milliseconds (ms) represented on the X-axis and frequency in kHz on the Y-axis. (B_1-10) Percentages of different call categories emitted by each strain at P4, P8 and P12 during separation (trial 1) and reunion with a littermate (trial 2); n = 7 for C57BL/6N (2 males and 5 females), n = 8 for DBA/2 (4 males and 4 females) and n = 11 for FVB/N (6 males and 5 females), all data as mean ± SEM (two-way ANOVA, *p< 0.05, **p< 0.01, ***p< 0.001).

Fig 3. USV analysis of adolescent mice from C57BL/6N, DBA/2 and FVB/N strains during the three-chamber social test.

(A) A schematic drawing of the experimental setup for USV recording in adolescent mice. USV were recorded for 10 min in the apparatus as a habituation phase (trial 1), followed by a social contact phase for 10 min with a same-sex littermate on the right compartment (trial 2) followed by another trial for 10 min with an adult unfamiliar mouse from another strain (trial 3). The intertrial interval between trials was 15 min. (B-E) USV analysis including number (B), duration (C), frequency (D) and amplitude (E) of calls during the three trials. (F) Percentages of different call categories emitted by each strain during each trial; n = 7 for C57BL/6N (2 males and 5 females), n = 8 for DBA/2 (4 males and 4 females) and n = 11 for FVB/N (6 males and 5 females), all data as mean ± SEM (two-way ANOVA, *p< 0.05, **p< 0.01).

Fig 4. USV analysis of adolescent mice from C57BL/6N, DBA/2 and FVB/N strains during the direct social interaction test.

(A) A schematic drawing of the experimental setup for USV recording in adolescent mice. USV were recorded for 2 min in a new arena as a habituation phase (trial 1), followed by a social contact phase for 5 min with a same-sex littermate (trial 2). (B-E) USV analysis including number (B), duration (C), frequency (D) and amplitude (E) of calls at P24 during the habituation and social contact phases. (F) Percentages of different calls categories emitted by each strain during social contact phase; n = 25 for C57BL/6N (14 males and 11 females), n = 14 for DBA/2 (7 males and 7 females) and n = 18 for FVB/N (7 males and 11 females), all data as mean ± SEM (two-way ANOVA, *p< 0.05, **p< 0.01, ***p< 0.001).