这项工作得到了 KAKENHI Grants JP22K15795（T.F.）、JP22K09734（N.K.）、JP21K07489（R.Y.）、金泽医科大学（C2022-3、D2021-4，R.Y.）和内藤基金会（T.F.）的部分支持。

该协议是根据日本生理学会的指导原则进行的，并获得了金泽医科大学动物护理委员会（2021-32）的批准。所有程序均按照ARRIVE指南进行。该研究使用了成年雄性 C57BL/6J 小鼠（3-6 个月大），并且先前证实这些小鼠表现出本手稿中描述的飞行行为15.
1.动物准备
<ol><li>群居小鼠（每个笼子3-4只;保持在23-27°C;在12小时的光/暗循环下;提供自由获取食物和水）直到实验开始。</li>
	<li>在进行这种改良的恐惧调节之前，将每只小鼠单独饲养在有机玻璃笼子（14 cm × 21 cm × 12 cm）中至少 3 天。</li>
</ol>2. 设置工具/设备
<ol><li>恐惧调节箱（图1A）<ol><li>使用封闭在消音盒（67 厘米× 53 厘米× 55 厘米）中的恐惧调节室（25 厘米× 25 厘米× 25 厘米）（见 材料表）。</li>
		<li>需要两个上下文（A 和 B）。对于上下文 A，通过将白色塑料纸板与黑色胶带（3 厘米宽，一块板上有 4 个）连接在墙上创建黑白条纹。地板使用白色光滑的塑料板。</li>
		<li>每次疗程前用庚醇（1%）擦拭墙壁和地板。 注意： 不使用主动通风系统。在疗程结束时用酒精清洁可减少庚醇气味。</li>
		<li>对于上下文 B，通过移除上下文 A 中使用的板，使墙壁的外观完全黑色。地板是网格地板。 注意： 除了用于清洁的轻微酒精气味外，没有特定的气味。</li>
		<li>使用头顶白色发光二极管（LED，240勒克斯）照亮实验箱（见 材料表）。</li>
	</ol></li>
	<li>令人 震惊<ol><li>将扰频减震器（见材料表）连接到由不锈钢棒组成的网格地板。这用于提供脚部震动。足部电击强度设定为0.9 mA，遵循常见的恐惧调节方法10,11,12,13。</li>
	</ol></li>
	<li>音频发生器<ol><li>在天花板上放置一个扬声器（参见 材料表）。所有声学刺激都被放大。</li>
		<li>使用 1/4 英寸麦克风对每个刺激的整体振幅进行数字修改和校准，以产生声压级 （SPL，re： 20 μ Pa）。通过此扬声器呈现连续的音调爆发。 注意：校准声音扬声器对于检查声音刺激在这种改良的恐惧条件反射期间对防御行为的良好影响至关重要。</li>
	</ol></li>
	<li>传感器<ol><li>将测试室的地板放在传感器上（参见 材料表）以检测振动。来自换能器的信号被传输到采样频率为 8 kHz 的声卡，以记录行为振动。</li>
	</ol></li>
	<li>摄像机<ol><li>将CMOS相机（见 材料表）放置在天花板上，以跟踪拍摄对象的运动并将声音记录在调节箱中。</li>
	</ol></li>
	<li>触发系统<ol><li>使用声音软件（见 材料表）在预定的时间触发音调或脚震。 注意：任何市售的刺激器都适用于此。</li>
	</ol></li>
</ol>3.行为实验
<ol><li>计划为期四天的恐惧调节程序：习惯化（1 天，5 次试验）、条件反射（2 天，每次 5 次试验）和测试/灭绝课程（1 天、5 或 15 次试验）。试验间隔在60-75秒之间变化（图1B）。 注意：优选地，在一组中包括十个或更多受试者，以获得可靠的行为倾向。根据研究的目的，需要两组或三组。<ol><li>在调节会话期间，在条件刺激 （CS） 终止后立即呈现无条件刺激 （US） （1 s， 0.9 mA）（连续音调突发，8 kHz，20 s，95 dB SPL），如 图 1B 所示。在调理日内提供五对 CS-US。</li>
		<li>在第 5 次脚电击结束后，将对象留在上下文中 1 分钟，然后将其放回主笼。每次行为测试后，用 70% 酒精擦拭腔室进行清洁。 注意：CS 和 US 强度可以根据研究目的进行修改。以前的报告表明，较强的 CS 强度比较软的 CS 强度触发更积极的防御行为15。调理天数也可以延长。</li>
	</ol></li>
	<li>按照下面提到的时间表在 CS 演示期间诱导飞行行为。 注意：本实验中使用了CS（95 dB SPL）和US（0.9 mA）。<ol><li>在第 1 天，让受试者在上下文 A 中接受 5 项单独 CS 试验。</li>
		<li>在第 2 天和第 3 天，在上下文 B 中对受试者进行 5 项 CS-US 关联试验。</li>
		<li>在第 4 天，让受试者在上下文 B 中接受 5 次单独 CS 试验以进行召回会议。在测试记忆消退的情况下，让受试者单独进行 15 次 CS 试验。 注意：要测试内存稳定性，将消光会话延长 2-3 天会有所帮助。此外，在一周后而不是在第 4 天测试内存可以进一步确认内存稳定性。</li>
	</ol></li>
</ol>4. 防御行为分析
注意：分析了 CS 演示期间的运动、冻结百分比和跳跃次数。详情如下所述。如果可能的话，以双盲方式进行分析会更好。
<ol><li>使用视频中记录的音调开始同步视频中事件的时间和刺激的时间（CS 和 US）。</li>
	<li>利用自定义代码根据帧中主体轮廓的质心差异来计算小鼠的平均运动和总运动。 注意：由于运动速度取决于短片的采样率，因此此测量使用任意单位。</li>
	<li>要测量冻结百分比，请及时使用换能器信号。<ol><li>使用 20-500 Hz 带通滤波器对传感器信号进行预处理。</li>
		<li>及时计算每个 50 ms bin的传感器信号的均方根幅度。</li>
		<li>设置信号幅度的阈值以检测不动时间。不动的持续时间是低于阈值超过 1 秒的信号周期。</li>
		<li>通过观看视频手动测量冻结的持续时间。</li>
		<li>通过比较手动测量的冻结百分比和从换能器信号计算的百分比来调整冻结信号幅度的阈值。</li>
	</ol></li>
	<li>计算从视频文件中手动跳转的次数。 注意：计算飞镖的数量也有助于评估飞行响应。</li>
</ol>5. 统计分析
<ol><li>将统计显著性设置为 p < 0.05。</li>
	<li>对于多组和多个因素之间的比较，请进行多因素方差分析，然后进行事后检验。如果测试了调节计划的特定日期，请执行多个比较测试或排列测试。</li>
</ol>

研究具有改良恐惧条件反射的小鼠的主动防御行为

<ol>
	<li>Fanselow, M. S., Lester, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Evolution and learning. , 185-212 (1988).</li><li>Fanselow, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+organization+of+the+defensive+behavior+system+responsible+for+fear.">Neural organization of the defensive behavior system responsible for fear.</a> Psychonomic Bulletin &amp; Review. 1 (4), 429-438 (1994).</li><li>Mobbs, D., Headley, D. B., Ding, W., Dayan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Space,+time,+and+fear:+Survival+computations+along+defensive+circuits.">Space, time, and fear: Survival computations along defensive circuits.</a> Trends in Cognitive Sciences. 24 (3), 228-241 (2020).</li><li>Black, D. W., Grant, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dsm-5+guidebook:+The+essential+companion+to+the+diagnostic+and+statistical+manual+of+mental+disorders.">Dsm-5 guidebook: The essential companion to the diagnostic and statistical manual of mental disorders.</a> American Psychiatric Pub. , (2014).</li><li>Bienvenu, T. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+advent+of+fear+conditioning+as+an+animal+model+of+post-traumatic+stress+disorder:+Learning+from+the+past+to+shape+the+future+of+ptsd+research.">The advent of fear conditioning as an animal model of post-traumatic stress disorder: Learning from the past to shape the future of ptsd research.</a> Neuron. 109 (15), 2380-2397 (2021).</li><li>Johnson, L. R., Mcguire, J., Lazarus, R., Palmer, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pavlovian+fear+memory+circuits+and+phenotype+models+of+ptsd.">Pavlovian fear memory circuits and phenotype models of ptsd.</a> Neuropharmacology. 62 (2), 638-646 (2012).</li><li>Yehuda, R., Ledoux, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+variation+following+trauma:+A+translational+neuroscience+approach+to+understanding+ptsd.">Response variation following trauma: A translational neuroscience approach to understanding ptsd.</a> Neuron. 56 (1), 19-32 (2007).</li><li>Ledoux, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emotion+circuits+in+the+brain.">Emotion circuits in the brain.</a> Annual Review of Neuroscience. 23 (1), 155-184 (2000).</li><li>Gruene, T. M., Flick, K., Stefano, A., Shea, S. D., Shansky, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sexually+divergent+expression+of+active+and+passive+conditioned+fear+responses+in+rats.">Sexually divergent expression of active and passive conditioned fear responses in rats.</a> Elife. 4, e11352 (2015).</li><li>Fadok, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+competitive+inhibitory+circuit+for+selection+of+active+and+passive+fear+responses.">A competitive inhibitory circuit for selection of active and passive fear responses.</a> Nature. 542 (7639), 96-100 (2017).</li><li>Hersman, S., Allen, D., Hashimoto, M., Brito, S. I., Anthony, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulus+salience+determines+defensive+behaviors+elicited+by+aversively+conditioned+serial+compound+auditory+stimuli.">Stimulus salience determines defensive behaviors elicited by aversively conditioned serial compound auditory stimuli.</a> Elife. 9, e53803 (2020).</li><li>Trott, J. M., Hoffman, A. N., Zhuravka, I., Fanselow, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+and+unconditional+components+of+aversively+motivated+freezing,+flight+and+darting+in+mice.">Conditional and unconditional components of aversively motivated freezing, flight and darting in mice.</a> Elife. 11, e75663 (2022).</li><li>Borkar, C. D., Fadok, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+pavlovian+fear+conditioning+paradigm+to+study+freezing+and+flight+behavior.">A novel pavlovian fear conditioning paradigm to study freezing and flight behavior.</a> Journal of Visualized Experiments. 167, e61536 (2021).</li><li>Totty, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+and+brain+mechanisms+mediating+conditioned+flight+behavior+in+rats.">Behavioral and brain mechanisms mediating conditioned flight behavior in rats.</a> Scientific Reports. 11 (1), 8215 (2021).</li><li>Furuyama, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+factors+contribute+to+flight+behaviors+during+fear+conditioning.">Multiple factors contribute to flight behaviors during fear conditioning.</a> Scientific Reports. 13 (1), 10402 (2023).</li><li>Cain, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Avoidance+problems+reconsidered.">Avoidance problems reconsidered.</a> Current Opinion in Behavioral Sciences. 26, 9-17 (2019).</li><li>Ledoux, J. E., Moscarello, J., Sears, R., Campese, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+birth,+death+and+resurrection+of+avoidance:+A+reconceptualization+of+a+troubled+paradigm.">The birth, death and resurrection of avoidance: A reconceptualization of a troubled paradigm.</a> Molecular Psychiatry. 22 (1), 24-36 (2017).</li><li>Bouchekioua, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotonin+5-ht2c+receptor+knockout+in+mice+attenuates+fear+responses+in+contextual+or+cued+but+not+compound+context-cue+fear+conditioning.">Serotonin 5-ht2c receptor knockout in mice attenuates fear responses in contextual or cued but not compound context-cue fear conditioning.</a> Translational Psychiatry. 12 (1), 58 (2022).</li><li>Bouchekioua, Y., Nishitani, N., Ohmura, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditioned+lick+suppression:+Assessing+contextual,+cued,+and+context-cue+compound+fear+responses+independently+of+locomotor+activity+in+mice.">Conditioned lick suppression: Assessing contextual, cued, and context-cue compound fear responses independently of locomotor activity in mice.</a> Bio-Protocol. 12 (23), e4568 (2022).</li></ol>

作者声明没有竞争利益。

本文介绍的改进的恐惧条件协议是一种在恐惧环境中研究飞行行为的稳定方法。通过采用这种协议，我们发现小鼠在恐惧环境中的飞行行为是由显着刺激触发的，并且取决于上下文。飞行行为的特征没有得到很好的研究，因为没有合适的协议来观察飞行行为。该协议将是研究恐惧环境中积极防御行为的合适方法之一。
最近，除了本协议之外，还引入了一些协议。在小鼠和大?...

<a href="https://app.jove.com/t/6598">This corrects</a> the article <a href="https://dx.doi.org/10.3791/66272">10.3791/66266</a>

An erratum was issued for: <a href="https://www.jove.com/t/66266">Modified Fear Conditioning for Inducing Flight Behaviors in Mice</a>. The Abstract section was updated.

Erratum: Modified Fear Conditioning for Inducing Flight Behaviors in Mice

在威胁情况下适当选择防御行为对于所有动物的生存至关重要。防御行为根据威胁接近程度逐渐从一种转变为另一种，例如冻结和飞行行为之间的过渡 1,2,3。在各种精神障碍中经常观察到这些行为的失调4.创伤后应激障碍 （PTSD） 是一种以夸张的防御行为为特征的疾病，例如对非威胁性刺激的恐慌反应4.
啮齿动物的经典恐惧条件通常用作 PTSD5、6、7 的模型，但啮齿动物在此模型中不表达逃跑（恐慌样）行为8。因此，经典的恐惧条件反射模型，通常被称为“啮齿动物创伤后应激障碍模型”，对人类创伤后应激障碍缺乏面部有效性，特别是在捕捉逃跑或恐慌样症状方面，这些症状尚未得到充分研究。
最近，几种改进的恐惧调节方案已成功证明啮齿动物受试者在这些过程中表现出飞行行为。例如，条件刺激 （CS） 和无条件刺激 （US） 在一天内重复关联 7 次，使雌性大鼠表现出类似于飞行行为的飞奔行为9。在使用连续复合刺激（SCS;由音调和噪音组成）的为期两天的恐惧条件反射中，小鼠在SCS演示的噪音部分开始表现出飞行行为10,11,12。SCS方法的详细说明在协议报告13中提供。用SCS进行为期三天的恐惧调节也对大鼠诱导飞行行为起作用14。但是，这些新协议有一些局限性。例如，使用串行提示表示不允许排除接近估计对防御行为的影响。在大鼠中CS-US关联七次的情况下，大多数飞行反应是在雌性而不是雄性中观察到的。
鉴于这些考虑，我们为小鼠引入了一种改进的恐惧条件反射方案，以研究恐惧环境中的飞行行为。雄性小鼠在我们改良的恐惧条件反射期间始终表现出飞行行为。在此协议中，突出音调用作 CS 而不是 SCS。此外，每天至少需要五对 CS-US，持续至少两天，以及条件环境的恐惧增强。该协议为研究飞行行为提供了另一种选择，根据研究目的补充了以前的协议。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Audio speaker</td>
			<td>Fostex</td>
			<td>FT17H</td>
			<td></td>
		</tr>
		<tr>
			<td>Amplifier</td>
			<td>Sony</td>
			<td>TA-F500</td>
			<td></td>
		</tr>
		<tr>
			<td>CMOS camera</td>
			<td>Sanwa Supply Inc.</td>
			<td>CMS-V43BK</td>
			<td></td>
		</tr>
		<tr>
			<td>Fear conditioning chamber</td>
			<td>Panlab S.L.U.</td>
			<td>LE116</td>
			<td></td>
		</tr>
		<tr>
			<td>Food pellets</td>
			<td>Nosan</td>
			<td>Labo MR standard</td>
			<td></td>
		</tr>
		<tr>
			<td>LED</td>
			<td>Yamazen</td>
			<td>LT-B05N</td>
			<td></td>
		</tr>
		<tr>
			<td>Microphone</td>
			<td>ACO</td>
			<td>type 4156N</td>
			<td></td>
		</tr>
		<tr>
			<td>Scramble shocker</td>
			<td>Panlab S.L.U.</td>
			<td>LE 100-26</td>
			<td></td>
		</tr>
		<tr>
			<td>Sound card</td>
			<td>Behringer</td>
			<td>UMC202</td>
			<td></td>
		</tr>
		<tr>
			<td>Sound software</td>
			<td>Syntrillium Software</td>
			<td>Cool Edit 2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Transducer</td>
			<td>Panlab S.L.U.</td>
			<td>LE 111</td>
			<td></td>
		</tr>
	</tbody>
</table>

modified fear conditioning for inducing flight behaviors in mice

在威胁情况下，防御行为的适当表现对于生存至关重要。流行的理论认为，主动防御行为，如跳跃或快速飞奔，是在高威胁迫在眉睫或实际威胁下表达的，而被动防御行为，如冻结，是在预测威胁时表达的，但威胁迫在眉睫相对较低。在经典的恐惧条件反射中，受试者通常表现出冻结作为条件反射的防御反应，在大多数情况下几乎没有主动防御行为的表达。在这里，我们为小鼠引入了一种改进的恐惧条件反射程序，以观察从冻结到飞行的转变，反之亦然，涉及五对重复的条件刺激（CS;连续音调，8 kHz，95 dB SPL（声压级））和无条件刺激（US;脚电击，0.9 mA，1.0 s）在两天内。这种改良的恐惧调节程序需要相对大量的调节课程和调节日，但不需要高强度的足部电击来适度表达飞行行为。使用相同的上下文进行条件反射和突出的 CS 演示对于引发飞行行为至关重要。这种改良的恐惧条件反射程序是观察小鼠积极防御行为的可靠方法，为阐明恐惧环境中此类行为的精细机制和特征提供了机会。

按照 图1C所示的时间表，在雄性小鼠（C57BL / 6J;3-6个月大）中用改良的恐惧条件获得的结果。该实验旨在研究条件环境如何影响飞行行为的表达。分配了两组：第 1 组 （n = 10） 和第 2 组 （n = 10）。本实验中使用了 CS （95 dB SPL） 和 US （0.9 mA）。
在第 1 天，所有小鼠在上下文 A 中单独暴露于 5 项条件刺激 （CS） 试验。在此之后，所有小鼠在第 2 天和第 3...

Watch this Scientific Journal Video about Modified Fear Conditioning for Inducing Flight Behaviors in Mice at JoVE.com

作者聚焦：探索恐惧学习和灭绝中防御行为的神经相关性

为了研究恐惧环境中的飞行行为，我们引入了一种改进的恐惧条件反射协议。该协议确保小鼠在恐惧条件反射的线索呈现期间始终表现出飞行行为。

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 ...

modified-fear-conditioning-for-inducing-flight-behaviors-in-mice

Research

JoVE Journal

Behavior

Modified Fear Conditioning for Inducing Flight Behaviors in Mice

729 次观看.  Kanazawa Medical University. 1 我们正在研究恐惧学习和消退中各种防御行为 3 的神经相关性 2。4 本方案可以帮助开发 5 焦虑症 6 和创伤后应激障碍的新治疗方法。7 我们的方案使用两个声压级 8 的条件刺激来重置 9 冻结、飞镖、 和飞行。10 因此，我们的技术比经典的恐惧条件反射更能评估各种防御行为 11。12 我们的实验室专注于恐惧学习和灭绝的神经相关性 13。14 ?...

视频: 作者聚焦：探索恐惧学习和灭绝中防御行为的神经相关性

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 ...

科研

行为学

Trace Fear Conditioning in Mice

In this experiment we present a technique to measure learning and memory. In the trace fear conditioning protocol presented here there are five pairings between a neutral stimulus and an unconditioned stimulus. There is a 20 sec trace period that separates each conditioning trial. On the following day freezing is measured during presentation of the conditioned stimulus (CS) and trace period. On the third day there is an 8 min test to measure contextual memory. The representative results are from mice that were presented with the aversive unconditioned stimulus (shock) compared to mice that received the tone presentations without the unconditioned stimulus. Trace fear conditioning has been successfully used to detect subtle learning and memory deficits and enhancements in mice that are not found with other fear conditioning methods. This type of fear conditioning is believed to be dependent upon connections between the medial prefrontal cortex and the hippocampus. One current controversy is whether this method is believed to be amygdala-independent. Therefore, other fear conditioning testing is needed to examine amygdala-dependent learning and memory effects, such as through the delay fear conditioning.

In the following experiment we describe a protocol for trace fear conditioning in mice. This type of associative memory includes a trace period that separates the neutral stimulus and the unconditioned stimulus.

跟踪恐惧条件小鼠

In this experiment we present a technique to measure learning and memory. In the trace fear conditioning protocol presented here there are five pairings ...

The Modified Hole Board - Measuring Behavior, Cognition and Social Interaction in Mice and Rats

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure multiple dimensions of unconditioned behavior in small laboratory mammals (e.g., mice, rats, tree shrews and small primates). This paradigm is a valuable alternative for the use of a behavioral test battery, since a broad behavioral spectrum of an animal&#8217;s behavioral profile can be investigated in one single test.
The apparatus consists of a box, representing the &#8216;protected&#8217; area, separated from a group compartment. A board, on which small cylinders are staggered in three lines, is placed in the center of the box, representing the &#8216;unprotected&#8217; area of the set-up. The cognitive abilities of the animals can be measured by baiting some cylinders on the board and measuring the working and reference memory. Other unconditioned behavior, such as activity-related-, anxiety-related- and social behavior, can be observed using this paradigm. Behavioral flexibility and the ability to habituate to a novel environment can additionally be observed by subjecting the animals to multiple trials in the mHB, revealing insight into the animals&#8217; adaptive capacities.
Due to testing order effects in a behavioral test battery, na&#239;ve animals should be used for each individual experiment. By testing multiple behavioral dimensions in a single paradigm and thereby circumventing this issue, the number of experimental animals used is reduced. Furthermore, by avoiding social isolation during testing and without the need to food deprive the animals, the mHB represents a behavioral test system, inducing if any, very low amount of stress.

This protocol describes the modified hole board, which is a behavioral test set-up that comprises the characteristics of an open field and a traditional hole board. This set-up enables the differential analysis of unconditioned behavior of small laboratory mammals as well as the analysis of cognitive abilities.

修改后的孔板 - 测量行为，认知和社会互动小鼠和大鼠

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure ...

Quantifying Social Motivation in Mice Using Operant Conditioning

In this protocol, social motivation is measured in mice through a pair of operant conditioning paradigms. To conduct the experiments, two-chambered shuttle boxes were equipped with two operant levers (left and right) and a food receptacle in one chamber, which was then divided from the second chamber by an automated guillotine door covered by a wire grid. Different stimulus mice, rotated across testing days, served as a social stimulus behind the wire grid, and were only visible following the opening of the guillotine door. Test mice were trained to lever press in order to open the door and gain access to the stimulus partner for 15 sec. The number of lever presses required to obtain the social reward progressively increased on a fixed schedule of 3. Testing sessions ended after test mice stopped lever pressing for 5 consecutive minutes. The last reinforced ratio or breakpoint can be used as a quantitative measure of social motivation. For the second paradigm, test mice were trained to discriminate between left and right lever presses in order to obtain either a food reward or the social reward. Mice were rewarded for every 3 presses of each respective lever. The number of food and social rewards can be compared as a measurement of the value placed upon each reward. The ratio of each reward type can also be compared between mouse strains and the change in this ratio can be monitored within testing sessions to measure satiation with a given reward type. Both of these operant conditioning paradigms are highly useful for the quantification of social motivation in mouse models of autism and other disorders of social behavior.

This article describes a new protocol for the quantitative measurement of social motivation in mice using operant conditioning for a social reward. The protocol is useful for the measurement of social motivation in mouse models of autism and other disorders of social behavior.

量化的社会动机小鼠使用操作条件反射

In this protocol, social motivation is measured in mice through a pair of operant conditioning paradigms. To conduct the experiments, two-chambered ...

Whisker-signaled Eyeblink Classical Conditioning in Head-fixed Mice

Eyeblink conditioning is a common paradigm for investigating the neural mechanisms underlying learning and memory. To better utilize the extensive repertoire of scientific techniques available to study learning and memory at the cellular level, it is ideal to have a stable cranial platform. Because mice do not readily tolerate restraint, they are usually trained while moving about freely in a chamber. Conditioned stimulus (CS) and unconditioned stimulus (US) information are delivered and eyeblink responses recorded via a tether connected to the mouse's head. In the head-fixed apparatus presented here, mice are allowed to run as they desire while their heads are secured to facilitate experimentation. Reliable conditioning of the eyeblink response is obtained with this training apparatus, which allows for the delivery of whisker stimulation as the CS, a periorbital electrical shock as the US, and analysis of electromyographic (EMG) activity from the eyelid to detect blink responses.

The preparation presented here for whisker-signaled eyeblink conditioning in head-fixed mice precisely stimulates specific whiskers while allowing mice to ambulate on a cylindrical treadmill. A whisker stimulation conditioned stimulus (CS) paired with a periorbital shock unconditioned stimulus (US) results in reliable associative learning on this apparatus.

在头固定小鼠胡须信号眨眼经典条件反射

Eyeblink conditioning is a common paradigm for investigating the neural mechanisms underlying learning and memory. To better utilize the extensive ...

Novel Object Exploration as a Potential Assay for Higher Order Repetitive Behaviors in Mice

Restricted, repetitive behaviors (RRBs) are a core feature of autism spectrum disorder (ASD) and disrupt the lives of affected individuals. RRBs are commonly split into lower-order and higher-order components, with lower order RRBs consisting of motor stereotypies and higher order RRBs consisting of perseverative and sequencing behaviors. Higher order RRBs are challenging to model in mice. Current assays for RRBs in mice focus primarily on the lower order components, making basic biomedical research into potential treatments or interventions for higher-order RRBs difficult. Here we describe a new assay, novel object exploration. This assay uses a basic open-field arena with four novel objects placed around the perimeter. The test mouse is allowed to freely explore the arena and the order in which the mouse investigates the novel objects is recorded. From these data, patterned sequences of exploration can be identified, as can the most preferred object for each mouse. The representative data shared here and past results using the novel object exploration assay illustrate that inbred mouse strains do demonstrate different behavior in this assay and that strains with elevated lower order RRBs also show elevated patterned behavior. As such, the novel object exploration assay appears to possess good face validity for higher order RRBs in humans and may be a valuable assay for future studies investigating novel therapeutics for ASD.

Higher order restricted, repetitive behaviors (RRBs) disrupt the lives of affected individuals. These behaviors are challenging to model in rodents, making basic biomedical research into potential treatments or interventions for RRBs difficult. Here we describe novel object exploration as a potential assay for higher order RRBs in mice.

新对象的探索在小鼠电位测定高阶重复的行为

Restricted, repetitive behaviors (RRBs) are a core feature of autism spectrum disorder (ASD) and disrupt the lives of affected individuals.  RRBs are ...

The Unpredictable Chronic Mild Stress Protocol for Inducing Anhedonia in Mice

Depression is a highly prevalent and debilitating condition, only partially addressed by current pharmacotherapies. The lack of response to treatment by many patients prompts the need to develop new therapeutic alternatives and to better understand the etiology of the disorder. Pre-clinical models with translational merits are rudimentary for this task. Here we present a protocol for the unpredictable chronic mild stress (UCMS) method in mice. In this protocol, adolescent mice are chronically exposed to interchanging unpredictable mild stressors. Resembling the pathogenesis of depression in humans, stress exposure during the sensitive period of mice adolescence instigates a depressive-like phenotype evident in adulthood. UCMS can be used for screenings of antidepressants on the variety of depressive-like behaviors and neuromolecular indices. Among the more prominent tests to assess depressive-like behavior in rodents is the sucrose preference test (SPT), which reflects anhedonia (core symptom of depression). The SPT will also be presented in this protocol. The ability of UCMS to induce anhedonia, instigate long-term behavioral deficits and enable reversal of these deficits via chronic (but not acute) treatment with antidepressants strengthens the protocol&#39;s validity compared to other animal protocols for inducing depressive-like behaviors.

Here we present the unpredictable chronic mild stress protocol in mice. This protocol induces a long-term depressive-like phenotype and enables to assess the efficacy of putative antidepressants in reversing the behavioral and neuromolecular depressive-like deficits.

诱导小鼠快感缺失的不可预测慢性轻度应力协议

Depression is a highly prevalent and debilitating condition, only partially addressed by current pharmacotherapies. The lack of response to treatment by ...

Visual Classical Conditioning in Wood Ants

Several species of insects have become model systems for studying learning and memory formation. Although many studies focus on freely moving animals, studies implementing classical conditioning paradigms with harnessed insects have been important for investigating the exact cues that individuals learn and the neural mechanisms underlying learning and memory formation. Here we present a protocol for evoking visual associative learning in wood ants through classical conditioning. In this paradigm, ants are harnessed and presented with a visual cue (a blue cardboard), the conditional stimulus (CS), paired with an appetitive sugar reward, the unconditional stimulus (US). Ants perform a Maxilla-Labium Extension Reflex (MaLER), the unconditional response (UR), which can be used as a readout for learning. Training consists of 10 trials, separated by a 5-minute intertrial interval (ITI). Ants are also tested for memory retention 10 minutes or 1 hour after training. This protocol has the potential to allow researchers to analyze, in a precise and controlled manner, the details of visual memory formation and the neural basis of learning and memory formation in wood ants.

We present a protocol for the classical conditioning of harnessed ants that permits researchers to study visual learning and memory formation at a level of analysis not possible with freely moving individuals.

木蚁视觉经典调理

Several species of insects have become model systems for studying learning and memory formation. Although many studies focus on freely moving animals, ...

A Chronic Immobilization Stress Protocol for Inducing Depression-Like Behavior in Mice

Depression is not yet fully understood, but various causative factors have been reported. Recently, the prevalence of depression has increased. However, therapeutic treatments for depression or research on depression is scarce. Thus, in the present paper, we propose a mouse model of depression induced by movement restriction. Chronic mild stress (CMS) is a well-known technique to induce depressive-like behavior. However, it necessitates a complex procedure consisting of a combination of various mild stresses. In contrast, chronic immobilization stress (CIS) is a readily accessible chronic stress model, modified from a restraint model that induces depressive behavior by restricting movement using a restrainer for a certain period. To evaluate the depressive-like behaviors, the sucrose preference test (SPT), the tail suspension test (TST), and the ELISA assay to measure stress marker corticosterone levels are combined in the present experiment. The described protocols illustrate the induction of CIS and evaluation of the changes in behavior and physiological factors for the validation of depression.

This article provides a simplified and standardized protocol for induction of depressive-like behavior in chronically immobilized mice by using a restrainer. In addition, behavior and physiological techniques to verify induction of depression are explained.

诱导小鼠抑郁行为的慢性固定性压力协议

Depression is not yet fully understood, but various causative factors have been reported. Recently, the prevalence of depression has increased. However, ...

Fear Incubation Using an Extended Fear-Conditioning Protocol for Rats

Emotional memory has been primarily studied with fear-conditioning paradigms. Fear conditioning is a form of learning through which individuals learn the relationships between aversive events and otherwise neutral stimuli. The most-widely utilized procedures for studying emotional memories entail fear conditioning in rats. In these tasks, the unconditioned stimulus (US) is a footshock presented once or several times across single or several sessions, and the conditioned response (CR) is freezing. In a version of these procedures, called cued fear conditioning, a tone (conditioned stimulus, CS) is paired with footshocks (US) during the training phase. During the first test, animals are exposed to the same context in which training took place, and freezing responses are tested in the absence of footshocks and tones (i.e., a context test). During the second test, freezing is measured when the context is changed (e.g., by manipulating the smell and walls of the experimental chamber) and the tone is presented in the absence of footshocks (i.e., a cue test). Most cued fear conditioning procedures entail few tone-shock pairings (e.g., 1-3 trials in a single session). There is a growing interest in less common versions involving an extensive number of pairings (i.e., overtraining) related to the long-lasting effect called fear incubation (i.e., fear responses increase over time without further exposure to aversive events or conditioned stimuli). Extended fear-conditioning tasks have been key to the understanding of fear incubation&#8217;s behavioral and neurobiological aspects, including its relationship with other psychological phenomena (e.g., post-traumatic stress disorder). Here, we describe an extended fear-conditioning protocol that produces overtraining and fear incubation in rats. This protocol entails a single training session with 25 tone-shock pairings (i.e., overtraining) and a comparison of conditioned freezing responses during context and cue tests 48 h (short-term) and 6 weeks (long-term) after training.

We describe an extended fear-conditioning protocol that produces overtraining and fear incubation in rats. This protocol entails a single training session with 25 tone-shock pairings (i.e., overtraining) and a comparison of conditioned freezing responses during context and cue tests 48 h (short-term) and 6 weeks (long-term) after training.

对大鼠使用扩展的恐惧调节协议进行恐惧孵化

Emotional memory has been primarily studied with fear-conditioning paradigms. Fear conditioning is a form of learning through which individuals learn the ...