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 mechanisms of the receiving animal have been less documented^{10, 11}. The latter is necessary to clarify the biological significance of the various characteristics of USVs. To reveal these mechanisms, the playback experiment is an efficient method. Recent playback studies have revealed that female mice are attracted to USVs¹², and that they prefer USVs from males that are different from their parents^{13, 14}.

This article explains the playback test used to evaluate USV preference in mice. A two-choice test box was developed in which two different USVs can be played back simultaneously in two compartments of a test enclosure, as shown in Figure 1. This type of test box prevents sound contamination by dividing the test area in three sub-chambers, using lead walls. The ultrasound emitters are located outside each room. In the wall between the rooms and ultrasound emitters are holes covered with wire mesh. Mice can move freely in the three rooms, and show a “searching the mesh” behavior, as if to respond to USVs played back by the ultrasound emitters. In this test, mice stay for periods of different duration close to one sound emitter or the other. These parameters can be logged to obtain a sensitive measure of sound preference.

To play the USVs back, nanocrystalline silicon thermo-acoustic emitters (i.e., “nc-Si emitter”) were used as in previous studies^15-17. These devices are composed of a thin-film heater electrode, a nano-porous silicon layer, and a single-crystalline silicon wafer. The digital sound file is converted to an analog signal and then passed through the heater electrode. The device converts the resulting voltage-dependent thermal signals into significant sound pressure with low distortion. This device is unique in that, unlike common sound generators that depend on mechanical vibrations, it can reproduce sound without the need of a diaphragm. The emitter exhibits a flat sound pressure level at frequencies from 20 to 160 kHz (Figure 2), and can reproduce digitally recorded murine USVs very accurately in terms of duration, frequency, and sound pressure level^{15, 18, 19}.

In a representative experiment shown in Figure 3, C57BL/6 (B6) females were allowed to choose between BALB/c (BALB) male USVs and background noise. In addition, Figure 4 shows the choice of B6 and BALB females between simultaneous USV playbacks from a BALB and a B6 male, as reported in a previous study¹⁴. The characteristics of male USVs differ between B6 and BALB strains²⁰. As shown by these results, the attractiveness of USVs can be assessed with the present protocol, in which sounds are recorded from a live individual, acoustically analyzed, and played back to other individuals.

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 the smears with Giemsa, and determine phase of the estrous cycle based on the presence or absence of leukocytes, cornified epithelial cells, and nucleated epithelial cells, according to Nelson²².

2. Devices (Figure 1)

Figure 1. Schematic of the Two-choice Test Box and Devices. Mice can access rooms A and B through the small gates located between them and the neutral zone. The two-choice box and two ultrasound emitters are placed on the floor of a soundproof chamber. The microphone is suspended inside the soundproof chamber. Abbreviation: PC, personal computer. Please click here to view a larger version of this figure.

1. For Recording
   1. Connect an ultrasonic condenser microphone to a PC using a USB A/D converter.
   2. Set the gain level of the A/D converter to “7”.
   3. Record acoustic data in 16-bit format files with a sampling rate of 400 kHz using audio capture software.
2. For Playback
   1. Connect a D/A converter to the PC using a USB interface.
   2. Connect two amplifiers to the D/A converter through an attenuator. (The latter device serves to prevent overload.)
   3. Connect an ultrasound emitter to each amplifier.

3. USV Recording

1. Place a male mouse in a small- (17 x 10 x 11 cm) or medium-sized (21 x 14 x 13 cm) cage with a 6 cm-diameter hole in the sidewall and the hole covered with a 0.5 cm wire mesh. When using a medium-sized cage, construct a partition across the center of the cage to reduce the area.
2. Do not use bedding in the cage to avoid rustling noises that could contaminate the recordings.
3. Place a microphone beside the mesh.
4. Begin ultrasonic vocalization monitoring with the recording setup shown in step 2.1.
5. Place a sexually mature female mouse in either metestrus or diestrus in the cage with the male mouse.
6. Record USVs for 3 – 5 min.

4. Test Box

1. Build the test box using acrylic board (Figure 1).
   Note: The test box (35 x 20, and x 20 cm high) is divided in three compartments: a neutral zone (15 x 20 cm), and rooms A and B (20 x 10 cm, each). Rooms A and B each have a 4 cm-diameter hole that is covered with a 0.5cm wire mesh. The holes and mesh are located at each end of rooms A and B. Make gates (5 x 5 cm each) between the neutral zone and rooms A and B.
2. To prevent sound leakage insulate each ultrasound emitter, except for the side facing the mesh, using rubber plates, sealing them to the box around the perimeter of each mesh.
3. Place lead seals on both sides of the wall between rooms A and B for sound insulation.

5. Preparing for Sound Playback

1. Creation of Playback Sounds
   1. Capture a 20 sec segment of USV from the recorded file using digital audio editing software.
   2. Select and capture background noise (0.35 sec) from the 20 sec USV segment file. Create a 20 sec background noise file by repeating the noise segment.
   3. To play back the two sound files simultaneously, export them as a single stereo sound file, with one file for the “left ear” and the other file for the “right ear.”
   4. Filter the sound files using as high-pass filter with a cutoff of 40 kHz.
   5. Reduce noise using the noise-reduction tool in post-processing software. Select and capture a segment of the file that contains no USVs as the noise-reduction profile, and run noise reduction with level 40.
2. Calibration of Playback Sounds
   1. Ensure that the space between ultrasound emitters and microphone is 10 cm.
   2. To calibrate sound pressure levels from the ultrasound emitters, perform ultrasonic monitoring using the configuration described in Step 2.1. Measure the sound pressure level by the microphone in decibels (dB).
   3. Using the attenuator and amplifiers, adjust the volume of the USVs generated by the ultrasound emitters to the same sound pressure level as the male USVs recorded in Step 3.
   4. When using the two ultrasound emitters and a sound file composed of USVs and background noise, confirm that the two ultrasound emitters exhibit the same sound pressure level using a calibration sound (e.g., a 75-kHz pure tone) before performing the behavioral test.
   5. When reproducing a sound file composed of two streams of USVs, adjust the two generated sound pressure levels to the same level, before testing.

6. Two-choice Test

1. Place the ultrasound emitters behind the meshes of the test box.
2. Close the gates to rooms A and B with an acrylic board, and habituate the female subject to the neutral zone for 30 min. Cover the box with acrylic board to avoid that the mouse can escape.
3. Start video recording using a CCD camera mounted above the box. The camera (not shown in Figure 1) covers the area of all three compartments.
4. Remove the gate and cover-board and allow the female to explore freely the test box, including the sound zones.
5. Once the female has investigated both meshes, and has returned to the neutral zone, start playing a 20 sec sound file repeatedly for 5 or 10 min.
6. Conduct behavioral monitoring for 5 or 10 min.
7. To reduce undesired olfactory cues deposited from the previous subject, clean the test cages between the tests with 70% ethanol.
8. Switch the place of ultrasound emitters A and B randomly to avoid inherent side bias effect towards rooms A or B.

7. Statistical Analyses

1. Using behavioral event-scoring software, analyze the following parameters: total number of entries in each room; total duration of stay in each middle zone; total duration of stay in each sound zone; and total duration of search of each mesh in front of ultrasound emitter. Figure 1 illustrates what is meant by “middle zone” and “sound zone.”
   Note: In cases where the mouse stays on the dividing line between the two zones (Figure 1, dashed line) decide the location by the direction of the head. When the head faces the mesh side, it is scored as a stay in the sound zone.
2. For each behavioral parameter, compare responses for rooms A and B using a Wilcoxon signed-rank test, or paired t-tests with a significance level of 0.05.

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