This work was supported by a Grant-in-Aid for JSPS Fellows to A.A.; by Grants-in-Aid for Scientific Research on Innovative Areas for JSPS Fellows (No. 4501 and No. 25132712) to T.K.; and by a research project grant awarded by Azabu University. Figure 2 is reprinted from Kihara, T., Harada, T., &#38; Koshida, N. Wafer-compatible fabrication and characteristics of nanocrystalline silicon thermally induced ultrasound emitters. In: Sensors and Actuators A: Physical, volume 125, Elsevier, p. 426, (2006), with permission from Elsevier.

All procedures were approved by the Ethics Committee of Azabu University. All experiments were carried out in a soundproof chamber.
1. Animal Preparation
<ol>
	<li>Males for Recording
	<ol>
		<li>Obtain sexually mature male&#160;mice experienced in mating.</li>
	</ol>
	</li>
	<li>Female Subjects
	<ol>
		<li>Obtain virgin female mice that have been housed with 2 to 5 littermates per cage (usually 8 - 12 weeks old).</li>
		<li>Obtain vaginal smears daily to determine the phase of the estrous c...

<ol>
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N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unusual+repertoire+of+vocalizations+in+adult+BTBR+T+tf/J+mice+during+three+types+of+social+encounters.">Unusual repertoire of vocalizations in adult BTBR T+tf/J mice during three types of social encounters.</a> Genes Brain Behav. 10 (1), 44-56 (2010).</li><li>Hammerschmidt, K., Radyushkin, K., Ehrenreich, H., Fischer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+and+usage+of+female+and+male+mouse+ultrasonic+vocalizations+reveal+only+minor+differences.">The structure and usage of female and male mouse ultrasonic vocalizations reveal only minor differences.</a> PLoS One. 7 (7), e41133 (2012).</li><li>Merten, S., Hoier, S., Pfeifle, C., Tautz, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+ultrasonic+vocalisation+in+social+communication+and+divergence+of+natural+populations+of+the+house+mouse+(Mus+musculus+domesticus).">A role for ultrasonic vocalisation in social communication and divergence of natural populations of the house mouse (Mus musculus domesticus).</a> PLoS One. 9 (5), e97244 (2014).</li><li>Amato, F. 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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...

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-female1-4, female-female1, 5, and male-male1, 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 USVs8,9, the behavioral responses and neural mechanisms of the receiving animal have been less documented10, 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 parents13, 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 &#8220;searching the mesh&#8221; 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., &#8220;nc-Si emitter&#8221;) were used as in previous studies15-17. 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 level15, 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.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Soundproof chamber</td>
			<td>Muromachi Kikai</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Small cage</td>
			<td>CLEA Japan</td>
			<td>CL-0113-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Middle cage</td>
			<td>CLEA Japan</td>
			<td>CL-0103-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound condenser microphones</td>
			<td>Avisoft Bioacoustics</td>
			<td>CM16/CMPA</td>
			<td></td>
		</tr>
		<tr>
			<td>A/D converter</td>
			<td>Avisoft Bioacoustics</td>
			<td>UltraSoundGate116H</td>
			<td></td>
		</tr>
		<tr>
			<td>Audio software</td>
			<td>Avisoft Bioacoustics</td>
			<td>RECORDER USGH</td>
			<td></td>
		</tr>
		<tr>
			<td>Adobe Audition 3.0 / Audio editing software</td>
			<td>Adobe Systems</td>
			<td>Adobe Audition 3.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Nc-Si emitter</td>
			<td>Original</td>
			<td></td>
			<td>not commercially available but it is planned to be so in near future</td>
		</tr>
		<tr>
			<td>D/A converter</td>
			<td>National Instruments</td>
			<td>NI USB-6251 BNC</td>
			<td></td>
		</tr>
		<tr>
			<td>Attenuator</td>
			<td>Original</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Amplifier</td>
			<td>Yamatake</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PC</td>
			<td>Windows 7 professional</td>
			<td></td>
			<td>Intel&#174; core i7-2600K CPU @ 3.4GHz, 8GB RAM, 64-bit operating system</td>
		</tr>
		<tr>
			<td>Event recorder Excel-macro / Event-scoring software</td>
			<td>original</td>
			<td></td>
			<td>Programmed by Naoto Akagawa &#38; Takeru Yamamoto</td>
		</tr>
		<tr>
			<td>CCD Camera</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rubber plates (made of elastomer resin)</td>
			<td>Tokyo bouon</td>
			<td>TI-75BK B4</td>
			<td>Cut them to the proper size http://www.piano-bouon.jp/shopping/?pid=1329272401-447630&#38;ca=6&#38;p=3</td>
		</tr>
		<tr>
			<td>Giemsa&#39;s azur eosin methylene blue solution</td>
			<td>Merck Millipore</td>
			<td>1.09204.0500</td>
			<td></td>
		</tr>
	</tbody>
</table>

determining ultrasonic vocalization preferences in mice using a two-choice playback test

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.

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 ...

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

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.

Mice emit ultrasonic vocalizations (USVs) during a variety of conditions, such as pup isolation and adult social interactions. These USVs differ with age, ...

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11.0K visualizações.  Azabu University. 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.