Determining Ultrasonic Vocalization Preferences in Mice using a Two-choice Playback Test

Summary

Ultrasonic vocalizations (USVs) in mice differ depending on age, sex, condition, and genetic background. Using two ultrasound emitters broadcasting simultaneously in different locations, this two-choice test can evaluate murine recognition and preference responses to different characteristics of USVs.

Abstract

Mice emit ultrasonic vocalizations (USVs) during a variety of conditions, such as pup isolation and adult social interactions. These USVs differ with age, sex, condition, and genetic background of the emitting animal. Although many studies have characterized these differences, whether receiver mice can discriminate among objectively different USVs and show preferences for particular sound traits remains to be elucidated. To determine whether mice can discriminate between different characteristics of USVs, a playback experiment was developed recently, in which preference responses of mice to two different USVs could be evaluated in the form of a place preference.

First, USVs from mice were recorded. Then, the recorded USVs were edited, trimmed accordingly, and exported as stereophonic sound files. Next, the USV amplitudes generated by the two ultrasound emitters used in the experiment were adjusted to the same sound pressure level. Nanocrystalline silicon thermo-acoustic emitters were used to play the USVs back. Finally, to investigate the preference of subject mice to selected USVs, pairs of two differing USV signals were played back simultaneously in a two-choice test box. By repeatedly entering a defined zone near an ultrasound emitter and searching the wire mesh in front of the emitter, the mouse reveals its preference for one sound over another. This model allows comparing the attractiveness of the various features of mouse USVs, in various contexts.

Introduction

Many animals use vocalizations for intraspecific communication. In the mouse Mus musculus, one important type of communication signal is ultrasonic vocalizations (USVs), which have frequencies higher than 20 kHz. USVs emitted by mice are considered a component of social recognition in male-female^1-4, female-female^{1, 5}, and male-male^{1, 6} interactions. USVs are also emitted by pups when they are isolated from their mother, which increases her pup-retrieving behavior, and therefore pup survival ⁷. Although many reports have analyzed and categorized mouse USVs^8,9, the behavioral responses and neural mechani....

Protocol

All procedures were approved by the Ethics Committee of Azabu University. All experiments were carried out in a soundproof chamber.

1. Animal Preparation

1. Males for Recording
   1. Obtain sexually mature male mice experienced in mating.
2. Female Subjects
   1. Obtain virgin female mice that have been housed with 2 to 5 littermates per cage (usually 8 - 12 weeks old).
   2. Obtain vaginal smears daily to determine the phase of the estrous cycle before the test, according to McLean²¹. Care should be taken to minimize the vaginal stimulation to avoid pseudopregnancy.
   3. Stain....

Results

The USVs recorded from one BALB-male (161 syllables per 20 sec), as well as background noise were used as playback sounds in the representative experiment shown in Figure 3. In this experiment, 7 female B6 mice were used at 9 weeks of age. To determine the best duration of testing of female response to playback sounds, the behavioral parameters were analyzed separately for the first and last five min of the total 10 min test time.

First, there was no significant difference in .......

Discussion

Here, the results of a representative test showed that female mice can discriminate between artificial male USVs and background noise (Figure 3). The conclusion to be drawn from these results is that the discrimination signal is reflected in the duration of stay in the room and sound zone, and in the duration of searching the mesh in the first 5 min of testing, but not in the second 5 min (Figure 3C, E and F). These data indicate that mice become habituated to the playback sounds, possib.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Soundproof chamber	Muromachi Kikai
Small cage	CLEA Japan	CL-0113-1
Middle cage	CLEA Japan	CL-0103-1
Ultrasound condenser microphones	Avisoft Bioacoustics	CM16/CMPA
A/D converter	Avisoft Bioacoustics	UltraSoundGate116H
Audio software	Avisoft Bioacoustics	RECORDER USGH
Adobe Audition 3.0 / Audio editing software	Adobe Systems	Adobe Audition 3.0
Nc-Si emitter	Original		not commercially available but it is planned to be so in near future
D/A converter	National Instruments	NI USB-6251 BNC
Attenuator	Original
Amplifier	Yamatake
PC	Windows 7 professional		Intel® core i7-2600K CPU @ 3.4GHz, 8GB RAM, 64-bit operating system
Event recorder Excel-macro / Event-scoring software	original		Programmed by Naoto Akagawa & Takeru Yamamoto
CCD Camera
Rubber plates (made of elastomer resin)	Tokyo bouon	TI-75BK B4	Cut them to the proper size http://www.piano-bouon.jp/shopping/?pid=1329272401-447630&ca=6&p=3
Giemsa's azur eosin methylene blue solution	Merck Millipore	1.09204.0500