Acoustic features of C57Bl/6 mouse courtship songs

A. Spectrogram of a segment of ultrasonic male vocalizations used for stimulation

B. Distribution of individual ultrasonic syllable durations across a large set of male mouse vocalizations (n = 2373 syllables, light grey histogram; red line is smoothed distribution), including the stimulus set used in subsequent playback experiments (n = 957 syllables, dark grey), emitted in response to the presentation of urine samples from females in estrus.

C. Distribution of syllable peak frequency (point of maximum amplitude across the call element in kHz) across all recorded syllables (n = 2373, light grey), and the subset of syllables used for playback (n = 957, dark grey).

D. Distribution of inter-syllable interval durations. The analysis was restricted to syllables with a subsequent syllable starting within 2 seconds (all recorded syllables: n = 2322, light grey; playback stimulus set: n = 942, dark grey).

Female mice preferentially approach playbacks of male songs over silence

A. Video frame showing the testing box with a soundproof partition (middle) positioned between two ultrasonic loudspeakers. White outlines show the two “speaker zones” used as regions of interest for quantifying the animal’s position.

B. Experimental timeline

C. Tracking of the animal’s position during an example behavioural session, in which a silent baseline period (leftmost panel) is followed by the playback of intact songs from one side contrasted with silence from the other side (coloured panels). Colour saturation indicates time since start of the experimental period of interest. Dark grey outlines indicate the speaker zones.

D. Time the animal spent in the speaker zones during the silent baseline period in C.

E. Index quantifying the animal’s relative place preference to the left vs right speaker zones during the silent baseline. This index was computed as the difference between the time spent in either speaker zones, normalized by the sum of the time in both speaker zones (see Methods). A preference index value close to zero indicates no preference to either side.

F. Time spent in the speaker zones corresponding to song playback and silence during each song presentation trial. Error bars: standard error of the mean.

G. Preference index in response to the playback of intact songs compared to silence over the course of the example session. The preference index is the difference between the time in the song playback and the silent speaker zones, divided by the sum of the time in both speaker zones. A positive index value reflects the animal’s preferential approach to the sources of song playback over silence.

H. Population summary of female approach response to playback of intact male songs (positive values) over silence (negative). Each circle is the preference index displayed by individual animals in one behavioural session (median of 4 sound presentation trials). The red circle corresponds to the example session in C & G. Open circles: sound playback at 58 dB SPL, filled circles: 68 dB SPL. Bar plot shows mean preference index across sessions and 95% confidence interval (C.I.). One-sample two-tailed t-test, **: p < 0.01.

I. Temporal profile of approach behaviour over the four sound presentation trials in the example session in C, calculated as the cumulative sum of time in the intact song playback (positively weighted) vs silent (negatively weighted) speaker zone.

J. Trial-averaged profile of approach behaviour to song playback in the example session, calculated as in I.

K. Population-averaged approach behaviour time course in response to intact mouse songs vs silence, calculated as in J. The dark grey trace indicates the mean of trial-based temporal profiles across all sessions (n = 29). For each session, the median of 4 sound presentation trials (e.g., black trace in J) was normalized to its maximal amplitude. The horizontal black bar indicates time bins during the course of a sound playback trial in which the cumulative approach behaviour significantly deviates from zero (one-sample two-tailed t-test).

L. Normalized temporal profiles of approach behaviour to mouse songs vs silence over the course of 4 sound presentation trials (x-axis, coloured bars) for each of the behavioural sessions (y-axis, n = 29), calculated as in I. Sessions (lines) are ordered by the amplitude of their last element.

F, G, I, J: Black traces indicate the session average (median) across the four sound presentation trials (coloured traces)

Female approach behaviour is not affected by changes to global song structure or the removal of syllable spectro-temporal dynamics

A. Female approach behaviour during simultaneous playback of intact male songs (top) and corresponding randomized syllable sequences (bottom,), displayed as in Fig. 2H. Each circle is the preference index displayed by individual animals in one behavioural session (median of 4 sound presentation trials). Open circles indicate sound playback at 58 dB SPL, filled circles at 68 dB SPL. Bar plot shows mean preference index across sessions, and error bar show 95% confidence interval (C.I.). One-sample two-tailed t-test, ns: non-significant, p > 0.05.

B. Population timecourse of approach behaviour during the playback of intact (positively weighted) vs randomized songs (negatively weighted), displayed as in Fig. 2K. The dark grey trace indicates the population mean of trial-based temporal profiles across all sessions (n = 21 sessions). For each session, the median of 4 sound presentation trials (e.g., black trace in J,) was normalized to the absolute value of its maximal amplitude. At no time bins during the course of a sound playback trial did the cumulative approach behaviour significantly deviate from zero (one-sample two-tailed t-test, all p>0.05).

C Playback of intact songs (top) contrasted with temporally reversed songs (bottom)

D. Typical timecourse of approach behaviour during the playback of intact (positively weighted) vs reversed songs (negatively weighted).

E. Playback of intact songs (top) and sequences of phase-scrambled syllables (bottom).

F. Typical timecourse of approach behaviour during the playback of intact (positively weighted) vs phase-scrambled syllable sequences (negatively weighted).

G. Playback of intact songs (top) contrasted with sequences of pure tones (bottom).

H. Typical timecourse of approach behaviour during the playback of intact (positively weighted) vs pure tone sequences (negatively weighted).

Female approach behaviour is sensitive to disruption of courtship temporal regularity.

A. Distribution of inter-syllable interval (ISI) durations across the set of temporally irregular songs, calculated as in Fig. 1D.

B. Sequential relationships of ISI durations in the intact (shaded grey dots) and temporally irregular (shaded blue dots).

C. Distribution of ISI durations across the super-regular song set.

D. Sequential relationship of ISI durations in the intact (shaded grey dots) and super-regular (shaded red dots).

A-D: n = 957 syllables

E. Female approach behaviour during simultaneous playback of intact male songs (top) and temporally irregular songs (bottom), displayed as in Fig. 2H. Each circle indicates the preference index displayed by individual animals in one behavioural session (median of 4 sound presentation trials). Open circles: sound playback at 58 dB SPL, filled circles: 68 dB SPL. One-sample two-tailed t-test. **: p < 0.01, ns: non significant, p > 0.05. Bar plots show means, and error bars show 95% C.I.

F. Population-averaged time course of approach behaviour in response to intact (positively weighted) vs temporally irregular (negatively weighted) mouse songs, displayed as in Fig. 2K. The dark grey trace indicates the mean of normalized, trial-based temporal profiles across all sessions (n = 24). The black bar indicates time bins in which the cumulative approach behaviour significantly deviates from zero (one-sample two-tailed t-test).

G. Temporal profiles of approach behaviour to intact vs temporally irregular songs over the course of 4 sound presentation trials (x-axis, coloured bars) for each of the behavioural sessions (y-axis. n = 24), displayed as in Fig. 2L.

H. Female approach behaviour during simultaneous playback of intact songs (top) and temporally super-regular songs (bottom), displayed as in panel E.

I. Population time course of approach behaviour to intact (positively weighted) vs temporally super-regular (negatively weighted) mouse songs, displayed as in panel F.

J. Temporal profiles of approach behaviour to intact vs temporally super-regular songs over the course of 4 sound presentation trials, displayed as in panel G.