2022 Hood et al. BBV Playback

Published: 19 April 2022| Version 1 | DOI: 10.17632/562yyxr2bt.1
Kayleigh Hood,


Although male vocalizations during opposite- sex interaction have been heavily studied as sexually selected signals, knowledge of female vocal signals produced in this context is limited. During intersexual interactions between mice, males produce a majority of ultrasonic vocalizations (USVs), while females produce a majority of human-audible squeaks, also called broadband vocalizations (BBVs). BBVs may be produced in conjunction with defensive aggression, making it difficult to assess whether males respond to BBVs themselves. To assess the direct effect of BBVs on male behavior, we used a split-cage paradigm in which high rates of male USVs were elicited by female presence on the other side of a barrier, but which precluded extensive male-female contact and the spontaneous production of BBVs. In this paradigm, playback of female BBVs decreased USV production, which recovered after the playback period. Trials in which female vocalizations were prevented by the use of female bedding alone or of anesthetized females as stimuli also showed a decrease in response to BBV playback. No non-vocal behaviors declined during playback, although digging behavior increased. Bursts of white noise (WN) evoked different behavioral responses than BBVs, in that WN caused larger decreases than BBVs themselves. USVs suppression had two distinct temporal components. When grouped in 5-second bins, USVs interleaved with bursts of stimulus BBVs. USV suppression also adapted to BBV playback on the order of minutes. Adaptation occurred more rapidly in males that were housed individually as opposed to socially for a week prior to testing, suggesting that the adaptation trajectory is sensitive to social experience. These findings suggest the possibility that vocal interaction between male and female mice, with males suppressing USVs in response to BBVs, may influence the timecourse of sexual behavior.


Steps to reproduce

Statistical analyses were performed in SAS (Version 9.3) and Statistica software (Tibco Data Science, Pao Alto, CA). In order to investigate changes in vocal and non-vocal behaviors in response to playback and other contextual factors such as stimulus type and dominance status, we used general linear mixed models (Proc GLMM) in SAS. Due to the high level of individual variation in USV production, models were run with Male Identity as a repeated measure to identify consistent patterns of changes in behavior across experiments among males with different baselines. Vocal behavior counts and non-vocal behavior durations were summed into 5-minute time periods that correspond to the baseline, playback, and recovery sections of the experiments. Each behavior was modeled as the dependent variable with time period (baseline, playback, recovery), male dominance status, Exemplar BBV type (A, B, or C), and the interaction between time period and Exemplar BBV type included as independent factors. A total of 6 models were run, 2 to investigate changes in vocal behaviors (i.e., USV production, 50 kHz USV production) and 4 to investigate changes in non-vocal behaviors across all BBV Playback subjects. Significant main effects of independent factors were further explored with post-hoc t-tests. Benjamini-Hochberg corrections with a Q value of 0.05 were used to adjust for multiple comparisons [25]. A separate general linear model was used to assess the effect of BBV versus non-BBV stimuli on USV production, using time period as a within-subjects factor and stimulus type (BBV, silence, or WN) as a between-subjects factor. Repeated measures ANOVAs were used to assess changes in USV rates across subsequent one-minute rounds of the playback. To assess the distribution of USVs across time periods in the presence of awake versus anesthetized females, USVs in each time bin were normalized to the total number of USVs in a given trial, and differences assessed with a factorial ANOVA with time bin and condition (anesthetized versus awake female) as cofactors. Fisher’s Least Significant Difference (LSD) test were used for posthoc comparisons of groups in the general linear models. A Pearson’s correlation was used to establish the relationship between initial USV rate and change in calling rate across trials with BBV playback, and with BBV and WN playback. Residuals from the regression were used to assess the difference between BBV and WN playback, using a one-way ANOVA. Levene’s test for homogeneity of variance was used to assess whether males with high versus low initial USV rates had greater variability in response to BBV playback. An Analysis of Covariance (ANCOVA) was used to compare the regression of male versus female investigation time across the baseline, playback, and recovery time periods. Variation in values is expressed as the standard error of the mean (s.e.m.).


Indiana University Bloomington


Animal Communication, Animal Sexual Behavior, Mouse