Name: determining ultrasonic vocalization preferences in mice using a two-choice playback test
Uploaded: 2015-09-03T15:00:00.000+00:00
Duration: 8 min 16 s
Description: Watch this Scientific Journal Video about Determining Ultrasonic Vocalization Preferences in Mice using a Two-choice Playback Test at JoVE.com

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.remotexs.ntu.edu.sg/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, possibly because of lack of social contact occurring near the ultrasound emitter. Therefore, the optimal testing time is 5 min. 
These findings might be accounting for the fact that USVs are just &#8220;more novel sounding than noise.&#8221; However, a previous playback study showed that female mice were attracted to the sounds of male USVs, but not to novelty artificial sounds12. It is assumed that merely novel sounds are not affective to female mice.
As expected, USV preference was dependent on the phase of the estrous cycle, since social investigation towards a male is enhanced by moderate levels of estrogen23. A previous study has revealed that females in pro-estrus or estrus do not show the preference for USVs of males of a different strain observed here14. Therefore, only females in diestrus were used in the present study; in general, when using females for this test, the phase of the estrous cycle should be known.
The limitation of this test is that USVs generated by the ultrasound emitter are one-way playback sounds. This test is not appropriate to evaluate the role of USVs in complex social interactions.In addition, inbred strains of mice such as B6, can exhibit a severe hearing loss after 12 weeks of age24. Therefore, it is recommended that all mice used in this test are less than 12 week-old.
In case of failure to observe differences in response related to the playback sounds, the addition of other social cues could help make the subject more sensitive to the sounds. For instance, it is recommended that the test subject be exposed to mouse odor during the habituation time in the neutral zone. In a previous study, the preference of females for some USVs over others was potentiated when they were exposed to male-soiled bedding, or to the male pheromone ESP125 before behavioral testing. In the absence of male-odor stimuli, females did not show a preference for USVs of males of a different strain14.
This model allows comparing the various features of murine USVs. The characteristics of mouse USVs change with maturation from pup to adult26, and differ across strains1, 20, 27. In addition, the female estrous cycle influences male courtship USVs28. Moreover, the rate of male production of USVs, and the incidence of a specific syllable type show a temporal increase related to male mounting behavior28, 29. Research using models of communication disorders have demonstrated unusual repertoires of USV profiles2, 29-33. However, the question of whether the receiver mice can discriminate and show a specific response to these different USVs remains to be elucidated. The present protocol increases the usefulness of mouse USVs for understanding the context-dependence of social signal production and auditory reaction, and for uncovering the characteristics of attractive vocal communications in mice.

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><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&amp;reg; 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 &amp;amp; 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&amp;amp;ca=6&amp;amp;p=3</td></tr><tr><td>Giemsa&#039;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的总时间不显著的差异，这表明，有没有副作用bias.The结果还显示，有背景噪音侧没有区别和室内的条目数，分析第一5分钟，最后5分钟，总测试时间（图3B）的阳USVs侧。 图3C示出 durat累积秒留在每个房间的播放过程中离子（即，留在中间区的总的持续时间，而在声区）。受试者花费显著更长的时间在BALB阳USV侧比在背景噪声侧时分析所述第一5分钟（p值= 0.043），或总的测试时间（p值= 0.043）。有在当最后5分钟，分析这些参数不显著差异。有在中间区（图3D）的背景噪声侧和公USVs侧之间没有显著差异。然而，B6雌性花显著更长的时间，在声区和搜索在BALB阳USV侧的网，相对于背景噪声侧，当分析前5分钟（声区，p值= 0.018， 图3E;网，P = 0.018， 图3F）或总测试时间（音区，P = 0.028， 图3E;网格，P = 0.043， 图3F）。结果清楚地表明，一个B6女接近更再现男性USVs比背景噪声，特别是在第一个5分钟。 这两个选择测试是有用的比较USVs的特性。在先前的研究中，这个试验是用来确定USV偏好B6和BALB雌性小鼠用于从这些菌株11的男性USVs。当从B6（133音节每20秒）和一个只BALB雄（每20秒108音节），雌性小鼠每这些菌株的使用USVs的重放组合显示更长搜索时间为其他应变比男性的USVs自己的一（ 图4）。 <img alt="图2" src="/files/ftp_upload/53074/53074fig2.jpg" /> 图2.测声压级（SPL）产生由纳米硅发射极作为频率的函数的大约80的几乎恒定的声压级- 90 dB的是40和160千赫之间观察到。 AC输入POW呃是在这种情况下，1.3 W。发射器被放置在从一个高频电容式麦克风的距离为20mm，用它的中心对齐。这个数字已经被修改木原19。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53074/53074fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53074/53074fig3.jpg" /> 图3. B6女性应对背景噪声和BALB男USVs。 （一）留在房间A或B的过程中播放前调查时间总持续时间。（B）在播放过程中房间的条目数。（C）留在房间的总时间。（D）逗留的中间地带总持续时间（E）留在声区的总持续时间。（F）的搜索网格的总持续时间。每个测量个人给出点图;在每个小区的水平条表示均值; N = 7; *表示背景噪音和男性USVs的分析参数之间显著差异（P &lt;0.05）; Wilcoxon符号秩检验。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53074/53074fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53074/53074fig4.jpg" /> 图4女应对男性的USVs从不同的应变与一脉相承的。B6（N = 6）和BALB（N = 10），女性暴露在测试前男性弄脏被褥间情期表现出了持续时间较长的搜索时间其他应变USVs。显示数值为平均值±标准差; *表示显著差异（P &lt;0.05）; Wilcoxon符号秩检验。这个数字已经被修改麻场<s达&gt; 14。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53074/53074fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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

Nanyang Technological University

Research

JoVE Journal

Behavior

11.1K 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.

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

Assessment of Ultrasonic Vocalizations During Drug Self-administration in Rats

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and relapse. Various types of behavioral activity are commonly utilized as measures of drug motivation in animals. However, a crucial component of drug abuse relapse in abstinent cocaine users is "drug craving", which is difficult to model in animals, as it often occurs in the absence of overt behaviors. Yet, it is possible that a class of ultrasonic vocalizations (USVs) in rats may be a useful marker for affective responses to drug administration, drug anticipation and even drug craving. Rats vocalize in ultrasonic frequencies that serve as a communicatory function and express subjective emotional states. Several studies have shown that different call frequency ranges are associated with negative and positive emotional states. For instance, high frequency calls ("50-kHz") are associated with positive affect, whereas low frequency calls ("22-kHz") represent a negative emotional state. This article describes a procedure to assess rat USVs associated with daily cocaine self-administration. For this procedure, we utilized standard single-lever operant chambers housed within sound-attenuating boxes for cocaine self-administration sessions and utilized ultrasonic microphones, multi-channel recording hardware and specialized software programs to detect and analyze USVs. USVs measurements reflect emotionality of rats before, during and after drug availability and can be correlated with commonly assessed drug self-administration behavioral data such lever responses, inter-response intervals and locomotor activity. Since USVs can be assessed during intervals prior to drug availability (e.g., anticipatory USVs) and during drug extinction trials, changes in affect associated with drug anticipation and drug abstinence can also be determined. In addition, determining USV changes over the course of short- and long-term drug exposure can provide a more detailed interpretation of drug exposure effects on affective functioning.

Drug self-administration and ultrasonic vocalizations (USV) are used as behavioral assessments in animal research, but rarely in combination. The purpose of this article is to describe the advantages of recording USVs during drug self-administration procedures to assess affective responses to drug experience.

在大鼠的药物自我管理的评估超声发声

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and ...

科研

神经科学

Recording Mouse Ultrasonic Vocalizations to Evaluate Social Communication

Mice emit ultrasonic vocalizations in different contexts throughout development and in adulthood. These vocal signals are now currently used as proxies for modeling the genetic bases of vocal communication deficits. Characterizing the vocal behavior of mouse models carrying mutations in genes associated with neuropsychiatric disorders such as autism spectrum disorders will help to understand the mechanisms leading to social communication deficits. We provide here protocols to reliably elicit ultrasonic vocalizations in pups and in adult mice. This standardization will help reduce inter-study variability due to the experimental settings. Pup isolation calls are recorded throughout development from individual pups isolated from dam and littermates. In adulthood, vocalizations are recorded during same-sex interactions (without a sexual component) by exposing socially motivated males or females to an unknown same-sex conspecific. We also provide a protocol to record vocalizations from adult males exposed to an estrus female. In this context, there is a sexual component in the interaction. These protocols are established to elicit a large amount of ultrasonic vocalizations in laboratory mice. However, we point out the important inter-individual variability in the vocal behavior of mice, which should be taken into account by recording a minimal number of individuals (at least 12 in each condition). These recordings of ultrasonic vocalizations are used to evaluate the call rate, the vocal repertoire and the acoustic structure of the calls. Data are combined with the analysis of synchronous video recordings to provide a more complete view on social communication in mice. These protocols are used to characterize the vocal communication deficits in mice lacking ProSAP1/Shank2, a gene associated with autism spectrum disorders. More ultrasonic vocalizations recordings can also be found on the mouseTube database, developed to favor the exchange of such data.

Mouse ultrasonic vocalizations are used as proxies to model the genetic bases of vocal communication deficits in mouse models for neuropsychiatric disorders. The present protocol describes three experimental contexts that reliably elicit ultrasonic vocalizations from pups (throughout development) and adult mice (same-sex interactions, male-estrus female interactions).

录制鼠标超声波发声评价社会交往

Mice emit ultrasonic vocalizations in different contexts throughout development and in adulthood. These vocal signals are now currently used as proxies ...

行为学

Targeted Training of Ultrasonic Vocalizations in Aged and Parkinsonian Rats

Voice deficits are a common complication of both Parkinson disease (PD) and aging; they can significantly diminish quality of life by impacting communication abilities. 1, 2 Targeted training (speech/voice therapy) can improve specific voice deficits,3, 4 although the underlying mechanisms of behavioral interventions are not well understood. Systematic investigation of voice deficits and therapy should consider many factors that are difficult to control in humans, such as age, home environment, age post-onset of disease, severity of disease, and medications. The method presented here uses an animal model of vocalization that allows for systematic study of how underlying sensorimotor mechanisms change with targeted voice training. The ultrasonic recording and analysis procedures outlined in this protocol are applicable to any investigation of rodent ultrasonic vocalizations.
The ultrasonic vocalizations of rodents are emerging as a valuable model to investigate the neural substrates of behavior.5-8 Both rodent and human vocalizations carry semiotic value and are produced by modifying an egressive airflow with a laryngeal constriction.9, 10 Thus, rodent vocalizations may be a useful model to study voice deficits in a sensorimotor context. Further, rat models allow us to study the neurobiological underpinnings of recovery from deficits with targeted training.
To model PD we use Long-Evans rats (Charles River Laboratories International, Inc.) and induce parkinsonism by a unilateral infusion of 7 &mu;g of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle which causes moderate to severe degeneration of presynaptic striatal neurons (for details see Ciucci, 2010).11, 12 For our aging model we use the Fischer 344/Brown Norway F1 (National Institute on Aging).
Our primary method for eliciting vocalizations is to expose sexually-experienced male rats to sexually receptive female rats. When the male becomes interested in the female, the female is removed and the male continues to vocalize. By rewarding complex vocalizations with food or water, both the number of complex vocalizations and the rate of vocalizations can be increased (Figure 1).
An ultrasonic microphone mounted above the male's home cage records the vocalizations. Recording begins after the female rat is removed to isolate the male calls. Vocalizations can be viewed in real time for training or recorded and analyzed offline. By recording and acoustically analyzing vocalizations before and after vocal training, the effects of disease and restoration of normal function with training can be assessed. This model also allows us to relate the observed behavioral (vocal) improvements to changes in the brain and neuromuscular system.

Voice disorders are debilitating in aging and Parkinson disease. The ultrasonic vocalizations of rats, also affected by these conditions, can be used to study these voice disorders, their neural substrates, and the nature of functional recovery with behavioral intervention.

老年帕金森病大鼠的超声波发声的有针对性的培训

Voice deficits are a common complication of both Parkinson disease (PD) and aging; they can significantly diminish quality of life by impacting ...

Eliciting and Analyzing Male Mouse Ultrasonic Vocalization (USV) Songs

Mice produce ultrasonic vocalizations (USVs) in a variety of social contexts throughout development and adulthood. These USVs are used for mother-pup retrieval1, juvenile interactions2, opposite and same sex interactions3,4,5, and territorial interactions6. For decades, the USVs have been used by investigators as proxies to study neuropsychiatric and developmental or behavioral disorders7,8,9, and more recently to understand mechanisms and evolution of vocal communication among vertebrates10. Within the sexual interactions, adult male mice produce USV songs, which have some features similar to courtship songs of songbirds11. The use of such multisyllabic repertoires can increase potential flexibility and information they carry, as they can be varied in how elements are organized and recombined, namely syntax. In this protocol a reliable method to elicit USV songs from male mice in various social contexts, such as exposure to fresh female urine, anesthetized animals, and estrus females is described. This includes conditions to induce a large amount of syllables from the mice. We reduce recording of ambient noises with inexpensive sound chambers, and present a quantification method to automatically detect, classify and analyze the USVs. The latter includes evaluation of call-rate, vocal repertoire, acoustic parameters, and syntax. Various approaches and insight on using playbacks to study an animal&#39;s preference for specific song types are described. These methods were used to describe acoustic and syntax changes across different contexts in male mice, and song preferences in female mice.

Eliciting and Analyzing Male Mouse Ultrasonic Vocalization USV Songs

Mice produce a complex multisyllabic repertoire of ultrasonic vocalizations (USVs). These USVs are widely used as readouts for neuropsychiatric disorders. This protocol describes some of the practices we learned and developed to consistently induce, collect, and analyze the acoustic features and syntax of mouse songs.

引导和分析男性超声波声（USV）歌曲

Mice produce ultrasonic vocalizations (USVs) in a variety of social contexts throughout development and adulthood. These USVs are used for mother-pup ...

Introducing Clicker Training as a Cognitive Enrichment for Laboratory Mice

Establishing new refinement strategies in laboratory animal science is a central goal in fulfilling the requirements of Directive 2010/63/EU. Previous research determined a profound impact of gentle handling protocols on the well-being of laboratory mice. By introducing clicker training to the keeping of mice, not only do we promote the amicable treatment of mice, but we also enable them to experience cognitive enrichment. Clicker training is a form of positive reinforcement training using a conditioned secondary reinforcer, the "click" sound of a clicker, which serves as a time bridge between the strengthened behavior and an upcoming reward. The effective implementation of the clicker training protocol with a cohort of 12 BALB/c inbred mice of each sex proved to be uncomplicated. The mice learned rather quickly when challenged with tasks of the clicker training protocol, and almost all trained mice overcame the challenges they were given (100% of female mice and 83% of male mice). This study has identified that clicker training for mice strongly correlates with reduced fear in the mice during human-mice interactions, as shown by reduced anxiety-related behaviors (e.g., defecation, vocalization, and urination) and fewer depression-like behaviors (e.g., floating). By developing a reliable protocol that can be easily integrated into the daily routine of the keeping of laboratory mice, the lifetime experience of welfare in the mice can be improved substantially.

The development of new refinement strategies for laboratory mice is a challenging task that contributes towards fulfilling the 3R principle. This protocol introduces clicker training as a cognitive enrichment program for laboratory mice.

引进响片训练为实验室老鼠认知富集

Establishing new refinement strategies in laboratory animal science is a central goal in fulfilling the requirements of Directive 2010/63/EU. Previous ...

Habituation and Prepulse Inhibition of Acoustic Startle in Rodents

The acoustic startle response is a protective response, elicited by a sudden and intense acoustic stimulus. Facial and skeletal muscles are activated within a few milliseconds, leading to a whole body flinch in rodents1. Although startle responses are reflexive responses that can be reliably elicited, they are not stereotypic. They can be modulated by emotions such as fear (fear potentiated startle) and joy (joy attenuated startle), by non-associative learning processes such as habituation and sensitization, and by other sensory stimuli through sensory gating processes (prepulse inhibition), turning startle responses into an excellent tool for assessing emotions, learning, and sensory gating, for review see 2, 3. The primary pathway mediating startle responses is very short and well described, qualifying startle also as an excellent model for studying the underlying mechanisms for behavioural plasticity on a cellular/molecular level3.
We here describe a method for assessing short-term habituation, long-term habituation and prepulse inhibition of acoustic startle responses in rodents. Habituation describes the decrease of the startle response magnitude upon repeated presentation of the same stimulus. Habituation within a testing session is called short-term habituation (STH) and is reversible upon a period of several minutes without stimulation. Habituation between testing sessions is called long-term habituation (LTH)4. Habituation is stimulus specific5. Prepulse inhibition is the attenuation of a startle response by a preceding non-startling sensory stimulus6. The interval between prepulse and startle stimulus can vary from 6 to up to 2000 ms. The prepulse can be any modality, however, acoustic prepulses are the most commonly used.
Habituation is a form of non-associative learning. It can also be viewed as a form of sensory filtering, since it reduces the organisms' response to a non-threatening stimulus. Prepulse inhibition (PPI) was originally developed in human neuropsychiatric research as an operational measure for sensory gating7. PPI deficits may represent the interface of "psychosis and cognition" as they seem to predict cognitive impairment8-10. Both habituation and PPI are disrupted in patients suffering from schizophrenia11, and PPI disruptions have shown to be, at least in some cases, amenable to treatment with mostly atypical antipsychotics12, 13. However, other mental and neurodegenerative diseases are also accompanied by disruption in habituation and/or PPI, such as autism spectrum disorders (slower habituation), obsessive compulsive disorder, Tourette's syndrome, Huntington's disease, Parkinson's disease, and Alzheimer's Disease (PPI)11, 14, 15 Dopamine induced PPI deficits are a commonly used animal model for the screening of antipsychotic drugs16, but PPI deficits can also be induced by many other psychomimetic drugs, environmental modifications and surgical procedures.

Habituation and prepulse inhibition of startle are operational measures of sensory gating. Sensory gating is disrupted in schizophrenia, and some other mental disorders and neurodegenerative diseases. We here describe a standard protocol to assess short-term and long-term habituation as well as prepulse inhibition of acoustic startle responses in rats and mice.

在鼠害声不惊人死不休的习惯和前脉冲抑制

The acoustic startle response is a protective response, elicited by a sudden and intense acoustic stimulus. Facial and skeletal muscles are activated ...

A Behavioral Assay for Investigating the Role of Spatial Memory During Instinctive Defense in Mice

Evolution has selected a repertoire of defensive behaviors that are essential for survival across all animal species. These behaviors are often stereotyped actions elicited in response to innately aversive sensory stimuli, but their success requires enough flexibility for adapting to different spatial environments, which can change rapidly. Here, we describe a behavioral assay to evaluate the influence of learned spatial knowledge on defensive behaviors in mice. We have adapted the widely used Barnes maze spatial memory assay to investigate how mice navigate to a shelter during escape responses to innately aversive sensory stimuli in a novel environment, and how they adapt to acute changes in the environment. This new assay is an ethological paradigm that does not require training and exploits the natural exploration patterns and navigation strategies in mice. We propose that the set of protocols described here are a powerful means of studying goal-directed behaviors and stimulus-triggered navigation, which should be of interest to both the fields of instinctive behaviors and spatial memory.

This protocol describes a behavioral assay, based on the Barnes maze test, to study how instinctive defensive actions are modified by the knowledge of spatial environment.

一种研究空间记忆在小鼠本能防御中作用的行为分析法

Evolution has selected a repertoire of defensive behaviors that are essential for survival across all animal species. These behaviors are often ...

A Low Cost Setup for Behavioral Audiometry in Rodents

In auditory animal research it is crucial to have precise information about basic hearing parameters of the animal subjects that are involved in the experiments. Such parameters may be physiological response characteristics of the auditory pathway, e.g. via brainstem audiometry (BERA). But these methods allow only indirect and uncertain extrapolations about the auditory percept that corresponds to these physiological parameters. To assess the perceptual level of hearing, behavioral methods have to be used. A potential problem with the use of behavioral methods for the description of perception in animal models is the fact that most of these methods involve some kind of learning paradigm before the subjects can be behaviorally tested, e.g. animals may have to learn to press a lever in response to a sound. As these learning paradigms change perception itself 1,2 they consequently will influence any result about perception obtained with these methods and therefore have to be interpreted with caution. Exceptions are paradigms that make use of reflex responses, because here no learning paradigms have to be carried out prior to perceptual testing. One such reflex response is the acoustic startle response (ASR) that can highly reproducibly be elicited with unexpected loud sounds in na&iuml;ve animals. This ASR in turn can be influenced by preceding sounds depending on the perceptibility of this preceding stimulus: Sounds well above hearing threshold will completely inhibit the amplitude of the ASR; sounds close to threshold will only slightly inhibit the ASR. This phenomenon is called pre-pulse inhibition (PPI) 3,4, and the amount of PPI on the ASR gradually depends on the perceptibility of the pre-pulse. PPI of the ASR is therefore well suited to determine behavioral audiograms in na&iuml;ve, non-trained animals, to determine hearing impairments or even to detect possible subjective tinnitus percepts in these animals. In this paper we demonstrate the use of this method in a rodent model (cf. also ref. 5), the Mongolian gerbil (Meriones unguiculatus), which is a well know model species for startle response research within the normal human hearing range (e.g. 6).

A fast and inexpensive method for the behavioral determination of hearing parameters like hearing thresholds, hearing impairments or phantom perceptions (subjective tinnitus) is described. It uses pre-pulse inhibition of the acoustic startle response and can be easily implemented in a personal computer using a programmable AD/DA-converter and a piezo sensor.

在啮齿动物的行为测听的低成本设置

In auditory animal research it is crucial to have precise information about basic hearing parameters of the animal subjects that are involved in the ...

研究具有改良恐惧条件反射的小鼠的主动防御行为

Modified Fear Conditioning for Inducing Flight Behaviors in Mice

The appropriate manifestation of defensive behavior in a threatening situation is critical for survival. The prevailing theory suggests that an active defensive behavior, such as jumping or rapid darting, is expressed under high threat imminence or actual threat, whereas passive defensive behavior, such as freezing, is expressed when the threat is predicted, but the threat imminence is relatively low. In classical fear conditioning, subjects typically exhibit freezing as a conditioned defensive response, with little expression of active defensive behavior in most cases. Here, we introduce a modified fear conditioning procedure for mice to observe the transition from freezing to flight and vice versa, involving five repetitive pairings of conditioned stimuli (CS; continuous tone, 8 kHz, 95 dB SPL (sound pressure levels)) and unconditioned stimuli (US; foot shock, 0.9 mA, 1.0 s) over two days. This modified fear conditioning procedure requires a relatively large number of conditioning sessions and conditioning days but does not necessitate a high-intensity foot shock for modest expression of flight behavior. Using the same context for conditioning and salient CS presentations is essential to elicit flight behaviors. This modified fear conditioning procedure is a reliable method for observing active defensive behaviors in mice, providing an opportunity to elucidate the fine mechanisms and characteristics of such behaviors in a fearful context.

作者聚焦：探索恐惧学习和灭绝中防御行为的神经相关性

To study flight behaviors in a fearful context, we introduce a modified fear conditioning protocol. This protocol ensures that mice consistently exhibit flight behaviors during cue presentation in the fear conditioning.

改良的恐惧条件反射诱导小鼠的飞行行为

The appropriate manifestation of defensive behavior in a threatening situation is critical for survival. The prevailing theory suggests that an active ...

采用双选择播放测试确定超声波发声首小鼠

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引进响片训练为实验室老鼠认知富集

在鼠害声不惊人死不休的习惯和前脉冲抑制

一种研究空间记忆在小鼠本能防御中作用的行为分析法

在啮齿动物的行为测听的低成本设置

改良的恐惧条件反射诱导小鼠的飞行行为

改良的恐惧条件反射诱导小鼠的飞行行为