Why do mice squeak? Toward a better understanding of defensive vocalization

Abstract

Although mice mostly communicate in the ultrasonic range, they also emit audible calls. We demonstrate that mice selectively bred for high anxiety-related behavior (HAB) have a high disposition for emitting sonic calls when caught by the tail. The vocalization was unrelated to pain but sensitive to anxiolytics. As revealed by manganese-enhanced MRI, HAB mice displayed an increased tonic activity of the periaqueductal gray (PAG). Selective inhibition of the dorsolateral PAG not only reduced anxiety-like behavior but also completely abolished sonic vocalization. Calls were emitted at a fundamental frequency of 3.8 kHz, which falls into the hearing range of numerous predators. Indeed, playback of sonic vocalization attracted rats if associated with a stimulus mouse. If played back to HAB mice, sonic calls were repellent in the absence of a conspecific but attractive in their presence. Our data demonstrate that sonic vocalization attracts both predators and conspecifics depending on the context.

Keywords: behavioral neuroscience; biological sciences; neuroscience; systems neuroscience.

Graphical abstract

Figure 1

Mice bred for high anxiety-related behavior have a higher disposition for sonic vocalization (A) Sonic calls were triggered during a 5-min tail suspension and recorded using an ultrasound microphone. (B) Percentage of male mice of various mouse lines emitting sonic calls during tail suspension. HAB, high anxiety-related behavior; NAB, normal anxiety-related behavior; B6N: C57Bl/6. Numbers in parenthesis indicate group sizes. (C) Body weight of HAB (n = 20) and NAB (n = 26) mice. (D) Mobility behavior during the tail suspension. (E) Latency to hind paw flicks or licking during a hot plate test (n = 20 HAB, n = 12 NAB). Data are presented as a percentage of total (B), individual data with mean ± SEM (C and D), and median (E). ^$$$$p < 0.0001 (Chi square test), ∗∗∗∗p < 0.0001 (unpaired t test).

Figure 2

Mice bred for high anxiety-related behavior produce less female-induced ultrasonic vocalization (A) Percentage of male high anxiety-related behavior (HAB; n = 11) and normal anxiety-related behavior (NAB; n = 9) mice emitting ultrasonic calls in the presence of a same-strain female. (B) Number of ultrasonic calls emitted during 10 min of interaction (not corrected for outliers). (C) Total duration of anogenital and facial sniffing by male mouse. Data are presented as a percentage of total (A), individual data with median (B), and mean ± SEM (C). ^$p < 0.05 (Chi square test), ∗∗p < 0.01 (Mann-Whitney test), ∗∗∗∗p < 0.0001 (unpaired t test).

Figure 3

Sonic vocalization is sensitive to anxiolytics Percentage of high anxiety-related behavior (HAB) mice emitting sonic calls (A, D, and G), number of calls emitted per mouse (B, E, and H), and mobility behavior (C, F, and I) during tail suspension (2.5 min) 60 min after systemic administration of diazepam (A–C), FAAH inhibitor URB597 (D–F), or CB1 receptor antagonist/inverse agonist SR141716A (G–I). Data are presented as percentage of total (A, D, G) and individual data with median (B and C, E and F, H and I). ∗p < 0.05 (Mann Whitney test). Animal numbers per treatment group are indicated in parentheses in the figures.

Figure 4

The dorsolateral periaqueductal gray controls sonic vocalization (A–C) Manganese-enhanced magnetic resonance imaging (MEMRI) comparing high anxiety-related behavior (HAB) vs. normal anxiety-related behavior (NAB) mice. (A) HAB (n = 31) and NAB (n = 26) mice received daily systemic manganese injections (8 days, 30 mg/kg per day) and underwent MRI scanning 24 h after the last injection. (B–C) Increased tonic neuronal activity in the caudal periaqueductal gray (PAG) of HAB mice. Statistical threshold is set at p < 0.0001, cluster extent >50. d/ilSC, deep/intermediate layers of superior colliculus; IPN, interpeduncular nucleus; SCRN, superior central raphe nucleus. Numbers (#) indicate image numbers of corresponding reference images in the Allen Brain Atlas. See also Figure S1. (D–I) Local inhibition of the dlPAG using muscimol (MUSC). (D) MUSC was bilaterally injected via guide cannulas into the dlPAG 45 min before the TST and EMP (n = 14). (E) Representative image of the injection site. Scale bar: 1 mm. See also Figure S2. (F) Percentage of HAB mice emitting sonic calls, (G) the number of calls emitted per mouse, and (H) mobility behavior during the tail suspension (5 min). (I) Open arm time on the Elevated Plus Maze (EPM) during the first 5 min of the test. Data are presented as percentage of total (F) or individual data with median (G–I). ^$$$$p < 0.0001 (Chi square test), ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001 (Mann-Whitney test).

Figure 5

Mice bred for high anxiety-related behavior emit sonic calls when caught by the tail (A) Representative spectrogram displaying various high anxiety-related behavior (HAB) calls. (B) Relative frequency of calls emitted by HAB mice (n = 20) over the course of a 5-min tail suspension. (C) Mean fundamental frequency of HAB calls. (D) Mean duration of HAB calls per animal. (E) Number of calls emitted per mouse during tail suspension. Data are presented as relative occurrence (B), individual data with mean ± SEM (C and D), or median (E).

Figure 6

Sonic mouse calls are appetitive to rats and mice in the presence of a social stimulus (A) High anxiety-related behavior (HAB) squeaks (SQ) or control time- and amplitude-matched white noise (WN) served as stimulus sounds (depicted here schematically). See also Figure S5. (B) Setup for rat (n = 13) playback experiment with two playback compartments and a connecting corridor. (C) Playback experiment with HAB (n = 9) and normal anxiety-related behavior (NAB; n = 8) mice were performed in a Y-maze with two playback compartments and a start arm where no sound was presented. (D) During the baseline stage (BL), animals could freely explore the maze without sound presentation or stimulus mouse. (E) Squeaks or white noise were played back once the animal had entered the respective zone during the playback stage (PB) without additional stimulus mouse. (F) During the last stage (Playback + Stimulus, PB + S), the sounds were presented with an additional male stimulus mouse being present in each playback zone. (G–R) Percentage of time spent in each zone is presented for rats (G–I), HAB (K–M), and NAB (O–Q). The squeak zone score for each stage of rats (J), HAB (N), and NAB (R). See also Figure S6. Data are presented as individual data with mean ± SEM (G–I, K–M, O–Q) or individual data with mean (J, N, and R). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 (paired t test for I, L, M, and Q; RM 1-way ANOVA followed by Tukey’s post hoc test for J, N, R), ^#p < 0.05 (one sample t test for N and R).