霍华德·休斯医学研究所资助EDJ的这项工作得到了支持。我们感谢Pr。 Sylvie Granon（NeuroPSI - 大学巴黎南XI - 法国）为我们提供扬声器硬件。我们还感谢Jarvis实验室的成员对这项工作的支持，讨论，更正和评论，特别是Joshua Jones Macopson的帮助和数据和测试。我们感谢Gustavo Arriaga博士对Mouse Song Analyzer软件的帮助，将其升级到V1.3，以及此协议的其他方面。该软件的v1.0由Holy和Guo开发，v1.0和v1.3由Arriaga开发。

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<li>Hammerschmidt, K., Radyushkin, K., Ehrenreich, H., Fischer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Female+mice+respond+to+male+ultrasonic+%27songs%27+with+approach+behaviour%5BTitle%5D)+AND+%22Biology+Letters%22%5BJournal%5D)">Female mice respond to male ultrasonic 'songs' with approach behaviour.</a> Biology Letters. 5, 589-592 (2009).</li>
<li>Ey, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Autism+ProSAP1%2FShank2+mouse+model+displays+quantitative+and+structural+abnormalities+in+ultrasonic+vocalisations%5BTitle%5D)+AND+%22Behav.+Brain+Res%22%5BJournal%5D)">The Autism ProSAP1/Shank2 mouse model displays quantitative and structural abnormalities in ultrasonic vocalisations.</a> Behav. Brain Res. 256, 677-689 (2013).</li>
<li>Chabout, J., Sarkar, A., Dunson, D. B., Jarvis, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Male+mice+song+syntax+depends+on+social+contexts+and+influences+female+preferences%5BTitle%5D)+AND+%22Front.+Behav.+Neurosci.+9%22%5BJournal%5D)">Male mice song syntax depends on social contexts and influences female preferences.</a> Front. Behav. Neurosci. 9. 76, (2015).</li>
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<li>Whitney, G., Coble, J. R., Stockton, M. D., Tilson, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ULTRASONIC+EMISSIONS%3A+DO+THEY+FACILITATE+COURTSHIP+OF+MICE%5BTitle%5D)+AND+%22J.+Comp.+Physiolog.+Psych%22%5BJournal%5D)">ULTRASONIC EMISSIONS: DO THEY FACILITATE COURTSHIP OF MICE.</a> J. Comp. Physiolog. Psych. 84, 445-452 (1973).</li>
<li>Asaba, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Developmental+social+environment+imprints+female+preference+for+male+song+in+mice%5BTitle%5D)+AND+%22PloS+ONE%22%5BJournal%5D)">Developmental social environment imprints female preference for male song in mice.</a> PloS ONE. 9, 87186(2014).</li>
<li>Neunuebel, J. P., Taylor, A. L., Arthur, B. J., Egnor, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Female+mice+ultrasonically+interact+with+males+during+courtship+displays%5BTitle%5D)+AND+%22eLife%22%5BJournal%5D)">Female mice ultrasonically interact with males during courtship displays.</a> eLife. 4, (2015).</li>
<li>Burkett, Z. D., Day, N. F., Penagarikano, O., Geschwind, D. H., White, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((VoICE%3A+A+semi-automated+pipeline+for+standardizing+vocal+analysis+across+models%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">VoICE: A semi-automated pipeline for standardizing vocal analysis across models.</a> Scientific Reports. 5, 10237(2015).</li>
<li>Grimsley, J. M., Gadziola, M. A., Wenstrup, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Automated+classification+of+mouse+pup+isolation+syllables%3A+from+cluster+analysis+to+an+Excel-based+%22mouse+pup+syllable+classification+calculator%22%5BTitle%5D)+AND+%22Front.+Behav.+Neurosci%22%5BJournal%5D)">Automated classification of mouse pup isolation syllables: from cluster analysis to an Excel-based "mouse pup syllable classification calculator".</a> Front. Behav. Neurosci. 6, (2012).</li>
<li>Grimsley, J. M. S., Monaghan, J. J. M., Wenstrup, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+social+vocalizations+in+mice%5BTitle%5D)+AND+%22PloS+ONE%22%5BJournal%5D)">Development of social vocalizations in mice.</a> PloS ONE. 6, (2011).</li>
<li>Portfors, C. V., Perkel, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+ultrasonic+vocalizations+in+mouse+communication%5BTitle%5D)+AND+%22Curr.+Opin.+Neurobiol%22%5BJournal%5D)">The role of ultrasonic vocalizations in mouse communication.</a> Curr. Opin. Neurobiol. 28, 115-120 (2014).</li>
<li>Merten, S., Hoier, S., Pfeifle, C., Tautz, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+role+for+ultrasonic+vocalisation+in+social+communication+and+divergence+of+natural+populations+of+the+house+mouse+%28Mus+musculus+domesticus%29%5BTitle%5D)+AND+%22PloS+ONE%22%5BJournal%5D)">A role for ultrasonic vocalisation in social communication and divergence of natural populations of the house mouse (Mus musculus domesticus).</a> PloS ONE. 9, 97244(2014).</li>
<li>Neilans, E. G., Holfoth, D. P., Radziwon, K. E., Portfors, C. V., Dent, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Discrimination+of+ultrasonic+vocalizations+by+CBA%2FCaJ+mice+%28Mus+musculus%29+is+related+to+spectrotemporal+dissimilarity+of+vocalizations%5BTitle%5D)+AND+%22PloS+ONE%22%5BJournal%5D)">Discrimination of ultrasonic vocalizations by CBA/CaJ mice (Mus musculus) is related to spectrotemporal dissimilarity of vocalizations.</a> PloS ONE. 9, 85405(2014).</li>
<li>Holfoth, D. P., Neilans, E. G., Dent, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Discrimination+of+partial+from+whole+ultrasonic+vocalizations+using+a+go%2Fno-go+task+in+mice%5BTitle%5D)+AND+%22J.+Acoust.+Soc.+Am%22%5BJournal%5D)">Discrimination of partial from whole ultrasonic vocalizations using a go/no-go task in mice.</a> J. Acoust. Soc. Am. 136, 3401(2014).</li>
<li>Asaba, A., Kato, M., Koshida, N., Kikusui, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Determining+Ultrasonic+Vocalization+Preferences+in+Mice+using+a+Two-choice+Playback%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Determining Ultrasonic Vocalization Preferences in Mice using a Two-choice Playback.</a> J Vis Exp. , (2015).</li>
</ol>

作者没有什么可以披露的。

该方案提供收集，量化和研究实验室中各种大多数女性相关刺激的雄性小鼠求爱发声的方法。如之前在Chabout， et al。在代表性的结果中，使用这种方法使我们能够发现与接收女性相关的上下文相关发声和语法。这些刺激的标准化将提供可靠数量的USV的收集，并允许详细分析男性的求爱歌曲和曲目。 当一个活着的女性与男性在一起时，协议不允许我们清楚地识别发声器的声音。然而，以前的研究表明，大多数在这种情况下发出的发声是男性26 29 。大多数使用特定（男性或女性）作为男性的刺激，相信在这些情况下，女性发声的数量可以忽略不计4，5，22，30。然而，最近的一篇论文使用三角测量来定位群体居住条件中发射器的发声31 ，并表明在一个二进制中，女性贡献了约10％的USV。在本议定书中，使用麻醉的女性允许用户在没有她的发声的情况下研究女性在场的男性发声。与最近的研究31的预期相反，我们发现FE和AF条件之间发音的音节数量没有差异23 。活的女性可能没有显着地贡献录音，或男性在活着的女性与麻醉的女性的存在下发出较少的声音啤酒。然而，我们认为，未来的实验应考虑使用这种三角测量方法来评估女性贡献的潜在影响。 还有其他可用的软件可以做一些我们所概述的步骤，尽管我们不相信我们使用三个程序组合的问题的方式足够:软件A，鼠标乐谱分析器软件脚本C使用软件B，使用自定义电子表格软件D + E计算的语法分析软件，以及使用R的语法解码顺序。例如，最近的一篇文章提出了一种名为VoICE的软件，该软件允许用户从声像图自动提取声变量，或者直接在手动单元上提取声变量由用户32选择。但是，自动或半自动序列分析并不像我们的方法那么详细。一些商业软件可以自动分析声学特征，但不提供自动化音节的分类;用户之后必须对不同的音节进行排序。 Grimsley，Gadziola， et al。 33开发了一种基于表的虚拟鼠标声乐器程序，其基于共享声学特征聚集音节，但不提供音节的自动检测。他们的节目34是独特的，因为它使用马尔可夫模型从录制的歌曲创建新的序列，因此具有比简单编辑更高级的特征。 大多数以前关于小鼠的沟通研究集中在发射体的侧面35,36 。很少有研究探讨了接收方30,37,38 。播放和辨别协议提供了一个简单的测试来研究接收方，如最近由Asaba，Kato 等人描述的人。 39 。在这项研究中，作者使用了一个双声道测试箱，用隔音泡沫代替这里描述的Y型迷宫盒。两种选择设置都有优缺点。首先，Y迷宫不会将声音从一个手臂隔离到另一个手臂，而是双选框。然而，通过使用Y迷宫设计，动物可以快速评估同时播放的两首歌曲，并向首选方向移动。然而，回放实验通常帮助实验者确定为特定动物产生的声音的意义和功能。总之，掌握本协议的技术和分析之后，读者应该能够解决影响小鼠USV的背景，遗传和神经生物学的许多问题。 使用B6D2F1 / J小鼠，女性相关刺激几乎总是触发我们在实验室测试的男性的USV。对于大学来说至关重要ct足够的音节（&gt; 5分钟&gt; 100）可以获得强大的统计学分析。对于故障排除，如果没有记录USV（或不足够），请检查配置以确保记录声音。在进行刺激后，通过观察计算机屏幕上的实时超声波图像，对录像过程中发生的情况进行现场检查。否则，尝试将男性重新暴露于性成熟/接受性的女性一夜之间，然后将他们独自存放数天或长达一周，然后再录制。根据轶事观察，我们发现有一些男性在一天（几乎全部5分钟）唱了很多，第二天没有多少，然后再一天。我们不知道为什么会出现主题变异的原因，但我们认为这可能是男性的动机或季节性，女性尿液的发情状态。如果没有记录USV，请尝试在几天内记录动物以接收这些可变效应。 UNL在鸣鸟中，我们没有注意到基于时间的歌唱量的明显差异。我们发现，在7周龄之前，男性不会唱多少（5分钟内&lt;100个音节）。 这里提出的检测方法可以在几分钟内提取数千个音节和所有的声学参数。但是作为任何自动检测方法，它对背景噪声非常敏感。使用具有嘈杂录音的鼠标乐谱分析仪检测软件（例如从记录有床上用品的动物）可能需要调整检测"阈值"以允许更多的灵活性。然而，这也将增加假阳性音节的数量，并且自动检测可能会失败。在这种情况下，可以使用手动编码。 如前所述，根据应变，发声的数量，余数和延迟量变化很大，因此可能需要改变参数（记录长度，刺激，自动音节检测等 ），以确保用于统计分析的最佳记录。

相对于人类，小鼠产生低频和高频发声，后来被称为高于我们的听力范围的超声发声（USV）。 USVs是在各种情况下生产的，包括从母婴检索，青少年互动，到相反或同性成人的交互4,12。这些USV由多种多音节曲目组成，可以手动分类9或自动分类10,11 。近年来，这些USV在交流中的作用日益增加。这些包括使用USV作为神经精神，发育或行为障碍7,8以及内部动机/情绪状态的小鼠模型的阅读。 USV被认为在e上传达可靠的信息mitter的状态对于接收器14,15是有用的。 2005年，Holy和Guo 11提出了成年男性小鼠USVs被组织为连续的多音节调用元素或类似于鸣鸟的音节的想法。在许多物种中，多音节曲目允许发射器以不同的方式组合和排序音节，以增加歌曲携带的潜在信息。这种语法的变化被认为具有与性行为和伴侣偏好有关的病理学意义16,17 。随后的研究表明，雄性小鼠能够改变在女性5,18之间，之前和之后产生的音节类型的相对组成。也就是说，成年男性小鼠使用他们的USV进行求偶行为，以吸引或与女性保持紧密接触，或促进交配19,20,21 。他们也是在男性与男性的互动中排除，可能在交互过程中传达社会信息4 。为了捕捉这些变化，科学家通常测量光谱特征（声学参数，如振幅，频率等 ），USV音节数或呼叫数，以及第一个USV的延迟。然而，很少有人仔细看这些USV的序列动态22 。最近我们的小组开发了一种测量USV音节序列23动态变化的新方法。我们表明，一首关于（即语法）的歌曲中的音节顺序不是随机的，它根据社会语境而变化，听力动物将这些变化发现为病理学相关性。 我们注意到在许多研究动物传播的调查人员，并没有将术语"语法"与人类言语中使用的语法相同的确切含义。对于动物交流研究，我们只是用一些规则来表示有序，非随机的声音序列。此外，对于人来说，已知特定的序列具有特定的含义。我们不知道这是否是老鼠的情况。 在本文和相关视频中，我们的目标是提供可靠的协议来记录雄性小鼠在各种环境下的求爱USV，并执行播放。使用三个顺序使用的软件:1）自动录音; 2）音节检测和编码;和3）对音节特征和语法的深入分析被证明（ 图1 ）。这使我们能够了解更多有关雄性小鼠USV结构和功能的信息。我们认为，这样的方法可以缓解数据分析，并可能开辟新的视野，以表征缪尔正常和异常的声音通信分别通信和神经精神障碍模型。

<table><tbody><tr><td>Sound proof beach cooler</td><td>See Gus paper has more info on specific kind</td><td></td><td>Inside dimensions (L 27 x W 23 x H 47 cm):</td></tr><tr><td>Condenser ultrasound microphone CM16/CMPA</td><td>Avisoft Bioacoustics, Berlin, Germany</td><td>#40011</td><td>Includes extension cable&amp;nbsp;</td></tr><tr><td>Ultrasound Gate 1216H sound card</td><td>Avisoft Bioacoustics, Berlin, Germany</td><td>#34175</td><td>12 channel sound card</td></tr><tr><td>Ultrasound Gate Player 216H</td><td>Avisoft Bioacoustics, Berlin, Germany</td><td>#70117</td><td>2 channels playback player</td></tr><tr><td>Ultrasonic Electrostatic Speaker ESS polaroid</td><td>Avisoft Bioacoustics, Berlin, Germany</td><td>#60103</td><td>2 playback speakers</td></tr><tr><td>Test cage&amp;nbsp;</td><td>Ace</td><td>#PC75J</td><td>30 x 8 x 13 cm height; plexiglas</td></tr><tr><td>plexiglas separation</td><td>home made</td><td>-</td><td>4 x 13 cm plexiglas with 1 cm holes</td></tr><tr><td>Video camera</td><td>Logitech</td><td>C920</td><td>logitech HD Pro webcam C920</td></tr><tr><td>Heat pad&amp;nbsp;</td><td>Sunbeam</td><td>722-810-000</td><td></td></tr><tr><td>Y-maze&amp;nbsp;</td><td>Home made</td><td>-</td><td>Inside dimensions (L 30 x W 11 x H 29 cm):</td></tr><tr><td>Tweezers</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;&lt;u&gt;Software&lt;/u&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Avisoft Recorder (Software A)</td><td>Avisoft Bioacoustics, Berlin, Germany</td><td>#10101, #10111, #10102, #10112</td><td>http://www.avisoft.com</td></tr><tr><td>MATLAB R2013a (Software B)</td><td>MathWorks</td><td>-</td><td>MATLAB R2013a (8.1.0.604)</td></tr><tr><td>Mouse Song Analyzer v1.3 (Software C)</td><td>Custom designed by Holy, Guo, Arriaga, &amp;amp; Jarvis; Runs with software B</td><td></td><td>http://jarvislab.net/wp-content/uploads/2014/12/Mouse_Song_Analyzer_&lt;br /&gt;v1.3-2015-03-23.zip</td></tr><tr><td>Microsoft Office Excel 2013 (Software D)</td><td>Microsoft</td><td>-</td><td>Microsoft Office Excel</td></tr><tr><td>Song Analysis Guide v1.1 (Software E)</td><td>Custom designed by Chabout &amp;amp; Jarvis. Excel calculator sheets, runs with software D</td><td></td><td>http://jarvislab.net/wp-content/uploads/2014/12/Song-analysis_Guided.xlsx</td></tr><tr><td>Syntax decorder v1.1 (Software F)</td><td>Custom designed by Sakar, Chabout, Dunson, Jarvis - in R studio</td><td></td><td>https://www.rstudio.com/products/rstudio/download/</td></tr><tr><td>Graphiz (Software G)</td><td>AT&amp;amp;T Research and others&amp;nbsp;</td><td></td><td>http://www.graphviz.org</td></tr><tr><td>Avisoft SASLab (Software H)</td><td>Avisoft Bioacoustics, Berlin, Germany</td><td>#10101, #10111, #10102, #10112</td><td>http://www.avisoft.com</td></tr><tr><td>&lt;strong&gt;&lt;u&gt;Reagents&lt;/u&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Xylazine (20 mg/mL)</td><td>Anased</td><td>-</td><td></td></tr><tr><td>Ketamine HCl (100 mg/mL)</td><td>Henry Schein</td><td>#045822</td><td></td></tr><tr><td>distilled water</td><td></td><td></td><td></td></tr><tr><td>Eye ointment&amp;nbsp;</td><td>Puralube Vet Ointment&amp;nbsp;</td><td>NDC 17033-211-38</td><td></td></tr><tr><td>Cotton tips</td><td></td><td></td><td></td></tr><tr><td>Petri dish</td><td></td><td></td><td></td></tr></tbody></table>

eliciting and analyzing male mouse ultrasonic vocalization (usv) songs

在整个发展和成年期间，小鼠在各种社会环境中产生超声发声（USV）。这些USV用于母婴检索1 ，少年交互2 ，相反和相同的性交互3，4，5和领土交互6 。几十年来，USVs被调查人员用作研究神经精神和发育障碍或行为障碍7,8,9 ，最近才了解脊椎动物之间的声音通信的机制和演变10 。在性交互作用中，成年雄性小鼠产生USV歌曲，其中有一些类似于歌鸟11的求爱歌曲的功能。使用这种多音节乐器可以增加他们携带的潜在的灵活性和信息，因为它们可以在元素如何组织和重组中变化，即语法。在该协议中，描述了在各种社会环境中引出来自雄性小鼠的USV歌曲的可靠方法，例如暴露于新鲜的女性尿液，麻醉的动物和发情女性。这包括从小鼠中诱导大量音节的条件。我们通过廉价的音箱减少环境噪音的记录，并提出了一种自动检测，分类和分析USV的量化方法。后者包括呼叫率，声乐曲目，声学参数和语法的评估。描述了使用播放来研究动物对特定歌曲类型的偏好的各种方法和洞察。这些方法用于描述男性小鼠不同背景下的声学和语法变化，以及女性小鼠的歌曲偏好。

在本协议中，对男性B6D2F1 / J小鼠的声乐行为和语法的变化进行了表征。一般来说，使用这个方案，平均每男性每5分钟一次记录675±98.5个音节，分别为女性UR，615.6±72 FE，AF为450±134，AM为75.6±38.9，男性UR为±0.1（男性12例）。女性UR为130音节/分钟，FE为120音节/分钟，AF为100个音节/分钟（ 图5A ）。相对于过夜收集的尿10,23，男性产生大量的音节以响应新鲜收集的尿液。在麻醉的男性或新鲜的男性尿液的存在下，男性也唱得相当少。男性也改变了他们的语境23 。例如，B6D2F1 / J男性显着增加<html>在女性尿液条件下产生多个跳跃"m"类音节（ 图5B ）。他们还改变了单个音节的语音特征。例如，B6D2F1 / J男性在女性尿液环境中以更高的幅度和带宽唱歌曲，并且与其他女性相比，清醒女性背景中的更高的光谱纯度（ 图6 ） 23 。 该协议还提供了一种用于测量序列的动态特征以及语法变化的方法。使用Ey 等人的适应方法22 ，我们使用ISI定义序列之间的差距（ 图7A ） 23 ，然后使用差距来区分和分析音节序列的时间模式。我们显示更长的序列长度在清醒的女性环境中产生（ 图7B ）这种技术允许我们计算复杂序列（由至少2次"m"音节类型组成）与简单序列（由一个或不是"m"型组成，并且因此主要由"我们发现，与B6D2F1 / J男性相比，女性尿液中的上下文触发了比其他人更高的比例（ 图7C ） 23 ，表明它们在女性尿液条件下产生更复杂的音节，而且这些音节也分布在超过更多的序列。 我们还可以计算从一个音节类型到另一个音节类型的条件转换概率（包括从"静音"状态转换到"静音"状态）的24个转换类型总和）。我们发现，在不同的语境中，老鼠对给定起始音节的过渡类型的选择是不同的，而且有更多的语法div女性尿液状况较差（ 图8 ） 23 。这些观察结果与以前的报告一致，表明男性可以根据不同的刺激和经验，改变其发声的声学特征或曲调组成4,5,24。 最后，本协议提供了通过播放测试女性偏好的指导。我们发现B6D2F1 / J女性喜欢更复杂的歌曲（包含2个或更多的"m"音节）相对于简单的歌曲23 。大多数女性选择更多地留在玩迷迭代的Y迷宫的一边（ 图4B ）。 <img alt="图1" src="/files/ftp_upload/54137/54137fig1.jpg" /> 图1:索菲流程图tware使用和分析。给每个程序和相关代码一个字母名称，以帮助解释他们的身份和在正文中的使用。 In（）是我们在协议中使用的具体程序。 <a href="http://ecsource.jove.com/files/ftp_upload/54137/54137fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" src="/files/ftp_upload/54137/54137fig2.jpg" /> 图2:设置用于录制男性小鼠歌曲。 （A）声音衰减记录盒的图片，并设置记录USV发声。 （B）使用软件A（ 表1 ）制作的记录的示例声像图，包括由"Mouse Song Analyzer v1.3"计算的详细光谱特征:持续时间，音节间间隔（ISI），峰值频率最小值（Pf min）峰值频率最大值（Pf max），峰值频率起始（Pf起始），峰值频率<html> （Pf结束）和带宽。 （C）另一个男性唱歌的声音图，在同一个房间里的实验室工作台上的一个活的女性，在衰减箱内部和盒子外面。我们的轶事观察表明，同一动物盒子中的录音显示体积较大（强度更高）和谐波较少，但没有声音回声的声音没有声音泡沫。 <a href="http://ecsource.jove.com/files/ftp_upload/54137/54137fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图3" src="/files/ftp_upload/54137/54137fig3.jpg" /> 图3:"Mouse Song Analyzer v1.3"的屏幕截图whis_gui显示可用于分析的不同选项的窗口。所显示的参数是用于在图中记录雄性USV并且提供的数据分析（除了最小记录持续时间为3ms）。<html> .com / files / ftp_upload / 54137 / 54137fig3large.jpg"target ="_ blank"&gt;请点击此处查看此图的较大版本。 <img alt="图4" src="/files/ftp_upload/54137/54137fig4.jpg" /> 图4:复杂和简单歌曲播放之间的女性选择。 （A）使用的Y迷宫装置的图片和尺寸测量。 （B）女性在每只手臂中花费的时间，来自同一男性的复杂（女性尿液）或更简单（醒目的女性诱发）歌曲。数据以n = 10 B6D2F1J雌性小鼠为平均值±SE，其个体值也显示; 10位女性中有9位表示偏爱更复杂的音节/顺序歌曲。 * p &lt;0.05配对学生t检验。从Chabout 等人修改的图23许可。空白"&gt;请点击此处查看此图的较大版本。 <img alt="图5" src="/files/ftp_upload/54137/54137fig5.jpg" /> 图5:跨条件发出和汇编的音节数 。 （A）不同背景下男性的音节生产率。 （B）在存在女性尿液（UR），麻醉女性（AF），清醒女性（FE）和麻醉男性（AM））情境的情况下，男性的汇辑组合物。数据以平均值±SEM表示。 * p &lt;0.03; ** p &lt;0.005;在本杰明和霍赫伯格校正后（n = 12男），*** p &lt;0.0001用于事后配对学生t检验。来自Chabout， et al。 23许可。 <a href="http://ecsource.jove.com/files/ftp_upload/54137/54137fig5large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> together.within页= "1"&gt; <img alt="图6" src="/files/ftp_upload/54137/54137fig6.jpg" /> 图6:不同背景下的光谱特征示例。 （A）幅度。校正后的特设配对学生t检验的p &lt;0.025。 （B）频率范围或带宽。 *:p &lt;0.041; **:p &lt;0.005; ***:矫正后p &lt;0.0001。 （C）音节的光谱纯度。 * p:&lt;0.025; **:p &lt;0.005; ***:矫正后p &lt;0.0001。缩写:女性尿液（UR），麻醉女性（AF），清醒女性（FE）和麻醉男性（AM）。从Chabout， et al。 23许可。 <a href="http://ecsource.jove.com/files/ftp_upload/54137/54137fig6large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> /&gt; 图7:序列测量。 （A）使用ISI分离序列。短ISI（SI）和中等ISI（MI）用于分离序列内的音节，并且超过250毫秒（LI）的长ISI分离两个序列。 （B）序列的长度，以每个序列的音节数测量，由不同上下文中的男性产生。 *:p &lt;0.025; ** p &lt;0.005; ***校正后，p &lt;0.0001。 （C）复杂歌曲与男性在不同背景下制作的简单歌曲的比例。 * p &lt;0.041; ** p &lt;0.005; ***校正后，p &lt;0.0001。数据以平均值±SEM（n = 12个男性）表示。来自Chabout， et al。 23许可。 <a href="http://ecsource.jove.com/files/ftp_upload/54137/54137fig7large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> 图8:基于每个上下文的条件概率的序列的音节语法图。箭头厚度与从n = 12男性平均的每个上下文中的转换类型的条件概率发生成比例:P（给出起始音节的转换的发生）。为了清楚起见，未示出低于概率0.05的罕见转变。来自Chabout， et al。 23许可。 <a href="http://ecsource.jove.com/files/ftp_upload/54137/54137fig8large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about Eliciting and Analyzing Male Mouse Ultrasonic Vocalization USV Songs at JoVE.com

Eliciting and Analyzing Male Mouse Ultrasonic Vocalization USV Songs

小鼠产生超声发声（USV）的复杂多音节曲目。这些USV被广泛用​​作神经精神障碍的读数。该协议描述了我们学习和开发的一些实践，以始终如一地引导，收集和分析鼠标歌曲的声学特征和语法。

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 ...

eliciting-and-analyzing-male-mouse-ultrasonic-vocalization-usv-songs

Universidad de Cartagena UDEC

Research

JoVE Journal

Behavior

Eliciting and Analyzing Male Mouse Ultrasonic Vocalization (USV) Songs

15.7K 次观看.  Duke University. 小鼠产生超声发声（USV）的复杂多音节曲目。这些USV被广泛用​​作神经精神障碍的读数。该协议描述了我们学习和开发的一些实践，以始终如一地引导，收集和分析鼠标歌曲的声学特征和语法。 

视频: Eliciting and Analyzing Male Mouse Ultrasonic Vocalization USV Songs

Mouse Short- and Long-term Locomotor Activity Analyzed by Video Tracking Software

Locomotor activity (LMA) is a simple and easily performed measurement of behavior in mice and other rodents. Improvements in video tracking software (VTS) have allowed it to be coupled to LMA testing, dramatically improving specificity and sensitivity when compared to the line crossings method with manual scoring. In addition, VTS enables high-throughput experimentation. While similar to automated video tracking used for the open field test (OFT), LMA testing is unique in that it allows mice to remain in their home cage and does not utilize the anxiogenic stimulus of bright lighting during the active phase of the light-dark cycle. Traditionally, LMA has been used for short periods of time (mins), while longer movement studies (hrs-days) have often used implanted transmitters and biotelemetry. With the option of real-time tracking, long-, like short-term LMA testing, can now be conducted using videography. Long-term LMA testing requires a specialized, but easily constructed, cage so that food and water (which is usually positioned on the cage top) does not obstruct videography. Importantly, videography and VTS allows for the quantification of parameters, such as path of mouse movement, that are difficult or unfeasible to measure with line crossing and/or biotelemetry. In sum, LMA testing coupled to VTS affords a more complete description of mouse movement and the ability to examine locomotion over an extended period of time.

Locomotor activity (LMA) is a simple and easily performed measurement of behavior in mice. Coupling of video tracking software (VTS) and LMA allows for the improvement of specificity and sensitivity, especially when compared with the manual, line crossing method of LMA analysis. Additionally VTS allows long-term tracking of mouse LMA.

鼠标短期和长期活动性分析视频跟踪软件

Locomotor  activity (LMA) is a simple and easily performed measurement of behavior in mice  and other rodents. Improvements in video tracking software ...

科研

行为学

Contextual and Cued Fear Conditioning Test Using a Video Analyzing System in Mice

The contextual and cued fear conditioning test is one of the behavioral tests that assesses the ability of mice to learn and remember an association between environmental cues and aversive experiences. In this test, mice are placed into a conditioning chamber and are given parings of a conditioned stimulus (an auditory cue) and an aversive unconditioned stimulus (an electric footshock). After a delay time, the mice are exposed to the same conditioning chamber and a differently shaped chamber with presentation of the auditory cue. Freezing behavior during the test is measured as an index of fear memory. To analyze the behavior automatically, we have developed a video analyzing system using the ImageFZ application software program, which is available as a free download at http://www.mouse-phenotype.org/. Here, to show the details of our protocol, we demonstrate our procedure for the contextual and cued fear conditioning test in C57BL/6J mice using the ImageFZ system. In addition, we validated our protocol and the video analyzing system performance by comparing freezing time measured by the ImageFZ system or a photobeam-based computer measurement system with that scored by a human observer. As shown in our representative results, the data obtained by ImageFZ were similar to those analyzed by a human observer, indicating that the behavioral analysis using the ImageFZ system is highly reliable. The present movie article provides detailed information regarding the test procedures and will promote understanding of the experimental situation.

This article presents a protocol for a contextual and cued fear conditioning test using a video analyzing system to assess fear learning and memory in mice.

背景和线索恐惧制约试验小鼠中使用视频分析系统

The contextual and cued fear conditioning test is one of the behavioral tests that assesses the ability of mice to learn and remember an association ...

Irrelevant Stimuli and Action Control: Analyzing the Influence of Ignored Stimuli via the Distractor-Response Binding Paradigm

Selection tasks in which simple stimuli (e.g. letters) are presented and a target stimulus has to be selected against one or more distractor stimuli are frequently used in the research on human action control. One important question in these settings is how distractor stimuli, competing with the target stimulus for a response, influence actions. The distractor-response binding paradigm can be used to investigate this influence. It is particular useful to separately analyze response retrieval and distractor inhibition effects. Computer-based experiments are used to collect the data (reaction times and error rates). In a number of sequentially presented pairs of stimulus arrays (prime-probe design), participants respond to targets while ignoring distractor stimuli. Importantly, the factors response relation in the arrays of each pair (repetition vs. change) and distractor relation (repetition vs. change) are varied orthogonally. The repetition of the same distractor then has a different effect depending on response relation (repetition vs. change) between arrays. This result pattern can be explained by response retrieval due to distractor repetition. In addition, distractor inhibition effects are indicated by a general advantage due to distractor repetition. The described paradigm has proven useful to determine relevant parameters for response retrieval effects on human action.

The distractor-response binding paradigm is described. It can be used to shed light on the influence irrelevant stimuli, competing with targets for a response, can have on human action. Both response retrieval effects and distractor inhibition effects can be analyzed within the paradigm.

不相干的刺激和动作控制：通过分心 - 响应绑定范式忽略刺激的影响分析

Selection tasks in which simple stimuli (e.g. letters) are presented and a target stimulus has to be selected against one or more distractor stimuli are ...

Morris Water Maze Test: Optimization for Mouse Strain and Testing Environment

The Morris water maze (MWM) is a commonly used task to assess hippocampal-dependent spatial learning and memory in transgenic mouse models of disease, including neurocognitive disorders such as Alzheimer&#8217;s disease. However, the background strain of the mouse model used can have a substantial effect on the observed behavioral phenotype, with some strains exhibiting superior learning ability relative to others. To ensure differences between transgene negative and transgene positive mice can be detected, identification of a training procedure sensitive to the background strain is essential. Failure to tailor the MWM protocol to the background strain of the mouse model may lead to under- or over- training, thereby masking group differences in probe trials. Here, a MWM protocol tailored for use with the F1 FVB/N x 129S6 background is described. This is a frequently used background strain to study the age-dependent effects of mutant P301L tau (rTg(TauP301L)4510 mice) on the memory deficits associated with Alzheimer&#8217;s disease. Also described is a strategy to re-optimize, as dictated by the particular testing environment utilized.

This manuscript describes a Morris water maze (MWM) protocol tailored for use with a commonly used mouse model of Alzheimer's disease. The MWM is widely used in transgenic mouse models. Implementation of a procedure sensitive to the background strain of the mouse model is essential for detecting group differences.

Morris水迷宫实验:优化小鼠品系和测试环境

 The Morris water maze (MWM) is a commonly used task to assess hippocampal-dependent spatial learning and memory in transgenic mouse models of disease, ...

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.

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, ...

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 ...

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 ...

神经科学

An Automated Rapid Iterative Negative Geotaxis Assay for Analyzing Adult Climbing Behavior in a Drosophila Model of Neurodegeneration

Neurodegenerative diseases are frequently associated with a progressive loss of movement ability, reduced life span, and age-dependent neurodegeneration. To understand the mechanism of these cellular events, and their causal relationships with each other, Drosophila melanogaster, with its sophisticated genetic tools and diverse behavioral features, are used as disease models for assessing neurodegenerative phenotypes. Here we describe a high-throughput method to analyze Drosophila adult negative geotaxis behavior, as an indication for possible motor defects associated with neurodegeneration. An automated machine is designed and developed to drive fly synchronization using an initial electric impulse, later allowing the recording of negative geotaxis behavior over a course of secs to mins. Images from the digitally recorded video are then processed with the self-designed RflyDetection software for statistical data manipulation. Different from the manually controlled negative geotaxis assay based on single fly, this precise, fast, and high-throughput protocol allows data acquisition from more than hundreds of flies simultaneously, providing an efficient approach to advance our understanding in the underlying mechanism of locomotor deficits associated with neurodegeneration.

An Automated Rapid Iterative Negative Geotaxis Assay for Analyzing Adult Climbing Behavior in a Drosophila Model of Neurodegeneration

This step-by-step protocol analyzes Drosophila negative geotaxis behavior using an automated multi-cylinder system that hosts hundreds of flies and synchronizes their action by an electric motor. Upon synchronization, fly negative geotaxis behavior is assayed, digitally recorded, and analyzed using the self-designed RflyDetection software.

一种自动快速迭代阴性 Geotaxis 分析方法在神经的果蝇模型中的成人攀爬行为

Neurodegenerative diseases are frequently associated with a progressive loss of movement ability, reduced life span, and age-dependent neurodegeneration. ...

Tickling, a Technique for Inducing Positive Affect When Handling Rats

Handling small animals such as rats can lead to several adverse effects. These include the fear of humans, resistance to handling, increased injury risk for both the animals and the hands of their handlers, decreased animal welfare, and less valid research data. To minimize negative effects on experimental results and human-animal relationships, research animals are often habituated to being handled. However, the methods of habituation are highly variable and often of limited effectiveness. More potently, it is possible for humans to mimic aspects of the animals&#39; playful rough-and-tumble behavior during handling. When applied to laboratory rats in a systematic manner, this playful handling, referred to as tickling, consistently gives rise to positive behavioral responses. This article provides a detailed description of a standardized rat tickling technique. This method can contribute to future investigations into positive affective states in animals, make it easier to handle rats for common husbandry activities such as cage changing or medical/research procedures such as injection, and be implemented as a source of social enrichment. It is concluded that this method can be used to efficiently and practicably reduce rats&#39; fearfulness of humans and improve their welfare, as well as reliably model positive affective states.

This article demonstrates the standardized application of playful handling, a tickling technique designed to mimic rat rough-and-tumble play. This technique is effective at reducing fearful reactions to humans and generating positive affect when rats are handled for common husbandry activities and medical and research procedures such as injection.

痒, 一种诱发大鼠正向影响的技术

Handling small animals such as rats can lead to several adverse effects. These include the fear of humans, resistance to handling, increased injury risk ...

Assessment of Sexual Behavior of Male Mice

Sexual behavior is highly species-specific. Although rodents have slightly different sexual behaviors, mice and rats have a similar sexual behavioral pattern. The purpose of this article is to describe the hormone-induced estrus ovariectomized female model and the experimental procedure for the assessment of sexual behavior of male mice. The most important sexual behavioral elements are demonstrated in the video and illustrations. The critical steps, advantages, and limitations of the sexual behavior test are explained as well. Finally, the behavior parameters are presented, and mounting, intromission, and ejaculation processes in mating are distinguished. Behavioral parameters are assessed in terms of the occurred duration and counts during the test period.

This article describes how to perform sexual behavior tests in male mice.

雄性小鼠性行为评估

Sexual behavior is highly species-specific. Although rodents have slightly different sexual behaviors, mice and rats have a similar sexual behavioral ...