这项工作是由支持的格兰特提供的援助为日本学术振兴会研究员为AA;通过赠款的援助科学研究的创新领域的JSPS院士（第4501号和第25132712）传统知识;并荣获由麻布大学， 图2的一个研究项目补助，由木原，T.，原田，T。，越田，北晶圆兼容的制造和纳米硅热致超声波发射器的特点转载。在： 传感器和执行器答：物理 ，成交量125，爱思唯尔，页。 426，（2006），与从爱思唯尔许可。

<html>所有程序都经麻布大学的伦理委员会。所有实验进行了在一个隔音室中。 1.动物的制备<ol><li>男性录制<ol><li>获得经验交配性成熟雄性小鼠。 </li></ol></li><li>女性受试者<ol><li>获取处女雌性小鼠已安置与每笼2-5同窝（一般为8 - 12周龄）。 </li><li>获取阴道涂片每天以确定试验前的发情周期的阶段，根据麦克莱恩21。应注意，以尽量减少阴道的刺激，以避免假孕。 </li><li>染色用吉姆萨涂片，并确定基于存在或不存在的白细胞，角化上皮细胞，和有核上皮细胞的发情周期的阶段，根据纳尔逊22。 </li></ol></li></ol> 2.设备（图1） _content“&gt; <img alt="图1" src="/files/ftp_upload/53074/53074fig1.jpg" /> 图1.原理图的两个选择题测试盒及器件。小鼠可通过位于它们和中立区之间的小门进入房间A和B。二选择框和两个超声发射器被放置在一个隔音室的地板上。话筒隔音室内暂停。缩写：PC，个人电脑<a href="https://www.jove.com/files/ftp_upload/53074/53074fig1large.jpg" target="_blank">，请点击此处查看该图的放大版本。</a> <ol><li>对于录音<ol><li>超声波电容式麦克风连接到使用USB的A / D转换器的PC。 </li><li>设置A / D转换器，以“7”的增益电平。 </li><li>在16位格式的文件，采用音频采集软件400 kHz的采样率记录声学数据。 </li></ol></li><li>对于播放<ol><li>连接数/使用A转换器到PC一个USB接口。 </li><li>连接两个放大器到D / A转换器通过一个衰减器。 （后者装置用于防止过载。） </li><li>超声波发射器连接到每个放大器。 </li></ol></li></ol> 3. USV录音<ol><li>放置一个雄性小鼠在一个小型（17×10×11厘米）或中型（21×14×13厘米）笼与在侧壁一个厘米直径的6孔和覆盖有一个0.5厘米的丝网的孔中。当使用中型笼，构造一个分割整个笼的中心，以减少该区域。 </li><li>不要使用的床上用品在笼子里，以避免沙沙的声音，可能污染记录。 </li><li>放置网格旁麦克风。 </li><li>首先在步骤2.1所示的记录设置超声波发声监控。 </li><li>将一个性成熟的雌性小鼠在任一或发情后期间情期与雄性小鼠笼子里。 </li><li>记录USVs为3 - 5分钟。 </li></ol> 4.Ť美国东部时间盒<ol><li>生成使用亚克力板（图1）的检验框。 注：测试盒（35×20，和×20厘米高）被划分在三个室：一个中性区（15×20厘米），和房间A和B（20×10厘米，各自）。房间A和B各自具有被覆盖有0.5厘米丝网一个4厘米直径孔。孔和网格位于房间A和B的中立区，客房A和B之间两端做门（5×5厘米的） </li><li>为了防止漏音绝缘每个超声发射器，除了面对网格的一侧，用橡胶板，将它们密封到周围各网格的周边的框。 </li><li>将铅封的房间A和B隔声间墙体的两侧。 </li></ol> 5.准备声音播放<ol><li>创建播放声音的<ol><li>从使用的数字音频编辑软件录制的文件捕获USV的20秒片段。 </li><li>选择并没有捕捉背景伊势（0.35秒）从20秒USV段文件。通过重复噪声段创建一个20秒的背景噪音文件。 </li><li>以同时播放两个声音文件，将其导出作为单个立体声文件，其中一个文件，为“左耳朵”和其他文件，为“右耳”。 </li><li>用高通滤波器40千赫的截止过滤声音文件。 </li><li>使用后处理软件的降噪工具，降低噪音。选择和拍摄包含不USVs作为降噪信息文件的片段，并运行降噪用40级。 </li></ol></li><li>播放声音校准<ol><li>确保超声发射器和麦克风之间的空间为10公分。 </li><li>校准从超声波发射器的声压水平，使用步骤2.1中描述的配置中执行超声波的监测。测量由以分贝麦克风（分贝）的声压水平。 </li><li> USI纳克衰减器和放大器，调整由超声发射器产生的相同的声压水平记录在步骤3中的阳USVs的USVs的体积。 </li><li>当使用两个超声波发射器和USVs和背景噪声组成的声音文件，确认两个超声波发射器具有使用进行行为学测试前校准的声音（例如，一个75-kHz的纯音）相同的声压级。 </li><li>当再现的USVs由两个流组成一个声音文件，调整两个生成的声压水平，以相同的水平，在测试之前。 </li></ol></li></ol> 6.两个选择题测试<ol><li>将测试盒的网眼背后的超声发射器。 </li><li>关闭大门，房间A和B具有亚克力板，并且，观察者女性受到中立区30分钟。盖上压克力板箱，以避免鼠标能逃脱。 </li><li>使用CC开始录像立体相机安装在上面的复选框。摄像机（ 图 1中未示出）覆盖所有三个隔室的面积。 </li><li>取下门和覆盖板，并允许女性探索自由的测试中，包括声音的区域。 </li><li>一旦女性已调查两个网格，并返回到中立区域，开始反复播放一个20秒的声音文件为5或10分钟。 </li><li>进行行为监测为5或10分钟。 </li><li>以减少从以前的主题不希望的沉积嗅觉暗示，清洁用70％乙醇的测试之间的测试笼中。 </li><li>开关超声波发射器A的地方，B随机地避免对房间A或B侧的固有偏见的影响</li></ol> 7.统计分析<ol><li>使用行为事件得分软件，分析下列参数：在每个房间的条目总数;留在每一个中间地带的总持续时间;留在每个声区的总的持续时间;和搜索在超声波发射器前面每个网格的总时间。 图1说明了什么是“中间地带”的意思是“健全的区域。” 注意：如遇有鼠标停留在这两个区域之间的分界线（图1，虚线）由头部的方向决定的位置。当头部面向网格的一面，它的得分为留在声区。 </li><li>对于每一个行为参数，比较的房间A和响应b使用一个Wilcoxon符号秩检验或配对 t -tests与0.05的显着性水平。 </li></ol>

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The authors declare that they have no competing interests.

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...

许多动物使用的发声种内通信。在小鼠小家鼠，通信信号中的一个重要类型是超声波发声（USVs），其具有的频率高于20kHz。被老鼠发出的USVs被认为是社会认可的男女1-4，女女1，5，和男男性1,6交互的组成部分。 USVs由幼崽也发出时，他们都从他们的母亲，这增加了她的小狗，检索行为中分离，因此小狗存活7。虽然许多报告分析和归类鼠标USVs 8，图9中，行为反应，并接收动物的神经机制已少记载10,11，后者是必要明确USVs的各种特性的生物学意义。揭示这些机制，播放实验是一种有效的方法。最近播放的研究表明，雌小鼠被吸引到USVs 12，并且它们倾向于从男性对不同于他们的父母13,14 USVs。 本文介绍了用于评估USV偏好小鼠播放测试。两选择测试盒被开发，其中两个不同USVs可以同时在测试外壳的两个室播放，如图1中所示。这种类型的测试盒防止声音污染除以测试区在三个子室，使用铅墙。超声波发射器位于每间客房外。在房间和超声波发射器之间的墙上都布满了铁丝网孔。老鼠可以在三个房间自由移动，并显示“搜索网”的行为，仿佛由超声波发射器播放USVs回应。在该试验中，将小鼠住不同的持续时间接近一个声音发射器或其他的周期。这些参数可以被记录，以获得一个敏感measu<html>再完善的偏爱。 要播放USVs背面，纳米晶硅热声发射器（即，“纳米硅发射极”）用作在以前的研究15-17。这些装置是由一个薄膜加热器电极，纳米多孔硅层，和一个单晶硅晶片。数字声音文件被转换成模拟信号，然后通过加热器电极。该装置所产生的电压依赖性的热信号转换成具有低失真显著声压。这个装置是独特的，不同于依赖于机械振动共同发声器，它可以再现声音无隔膜的需要。发射器表现出在频率从20到160千赫（图2）的平坦的声压水平，并且可以在时间，频率和声压级15，18，19而言重现数字记录的鼠USVs非常精确。 ve_content“&gt; 在图3所示的代表性实验，C57BL / 6（B6）的女性被允许的BALB / c（BALB）之间进行选择雄性USVs和背景噪声。此外，图4显示之间B6和BALB雌性的选择从BALB和B6的男性同时USV回放，据报道在先前的研究14。雄性USVs的特性B6之间不同和BALB菌株20。因此，如这些结果，USVs的吸引力可与本协议进行评估，在这种声音是从个人生活记录，声学分析，并播放给其他人。

<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.

所述USVs从一个BALB-雄（每20秒161音节），以及背景噪声记录被用来作为重放图 3中所示的代表性实验的声音。在该实验中，将7只雌性B6小鼠用于在9周龄。以确定女性响应的测试，以播放声音的最佳持续时间，行为参数被用于总10分钟的测试时间，第一和最后五个分分别进行分析。 首先，有在播放过程中（图3A）之前研究的时间留在房间A或B的总时间不?...

Watch this Scientific Journal Video about Determining Ultrasonic Vocalization Preferences in Mice using a Two-choice Playback Test at JoVE.com

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.

采用双选择播放测试确定超声波发声首小鼠

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

determining-ultrasonic-vocalization-preferences-mice-using-two-choice

Research

JoVE Journal

Behavior

11.0K 次观看.  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.