这项工作得到了国家卫生研究院的资助 [R01 AR048179 和 R01 HL126204 到钢筋混凝土 W, T32 AR059033 和 F32 AR069469to 福斯特 G] 和肌肉萎缩症协会美国 [274143 和416364至 R.C.W.]。

这里描述的方法得到了加州大学洛杉矶分校动物保育和使用委员会的批准。野生类型 (C57BL/6J) 和mdx小鼠 (C57Bl/10ScSn 背景) 是从商业来源购买的。mdx: utrophin-Tg 小鼠是来自 Ervasti 的礼物, 得到了凯戴维斯的许可, 并在mdx背景上进行了维护。6月龄雄性小鼠进行了步行试验。老鼠在 Terasaki 生命科学 Vivarium 被维护了由动物看护和用途委员会建立的指南在加利福尼亚大学洛杉矶 (批准 #2000-029-43) 和批准为这些研究被授予由加州大学洛杉矶分校动物福利保证 (批准 #A3196-01)。
1. 分庭的筹备工作
<ol>
	<li>在一天的9的一致时间内, 在安静、温度控制的房间中执行数据收集。对被测试的小鼠进行基因型和治疗的盲测试。</li>
	<li>对任何开放的野外室系统进行适应性分析。 注意: 在这项研究中, 我们使用一个廉价的, 易于运输的记录室建造从一个大的垃圾箱。野外竞技场是一个托盘设置在笼或类似的高架平台。</li>
	<li>把相机放在房间上方的铁丝网上, 在整个竞技场上记录下来。</li>
	<li>每次试验前用消毒剂清洗记录室。</li>
</ol>
2. 演习前的协议和数据收集
<ol>
	<li>(可选) 在活动录制之前, 使用肌肉激活协议挑战每个鼠标。 注意: 这是可选的, 但在涉及mdx小鼠的实验中推荐使用。<ol>
		<li>允许鼠标握住数字力计的拉杆, 然后轻轻地向后拉动, 直到松开拉杆。每次试用重复此步骤五次。</li>
		<li>记录每项试验的峰值张力 (N)。 注意: 除了作为运动试验的挑战, 在这一握力测定过程中, 动物施加的力可以作为一个额外的功能结果测量2,10。</li>
		<li>对每只老鼠进行五次总试验, 每次试验1分钟休息。</li>
	</ol>
	</li>
	<li>紧跟握力测定或其他运动协议后, 将鼠标置于活动室。</li>
	<li>启动在会议厅竞技场的鼠标移动的视频记录。允许鼠标自由探索6分钟。</li>
	<li>停止录制在6分钟, 并将鼠标返回到其主笼。</li>
</ol>
3. 视频分析
<ol>
	<li>(可选) 通过降低帧速率为分析准备视频。为了减少跟踪分析的帧速率, 请在视频编辑软件 (例如、iMovie 或类似程序) 中使用以下协议, 将视频帧速率降低2倍。 注意: 根据视频的长度和帧速率, 在分析前降低视频帧速率可能是很有用的。在本研究中, 录像记录在30帧/秒 (6 分钟录音, 约10800帧总数)。<ol>
		<li>将视频加载到软件中。</li>
		<li>在 "速度" 菜单下, 选择 "速度: 快速", 并设置速度为2x。</li>
		<li>以. mp4 文件格式导出被破坏的视频以进行跟踪分析。</li>
	</ol>
	</li>
	<li>在软件程序中打开视频进行分析。使用 "线条" 工具设置视频录制的校准。沿着房间的一侧画一条线。右键单击该行, 然后选择 "校准测量..."以厘米为单位输入实际大小的腔室侧。</li>
	<li>要开始半自动跟踪鼠标位置, 请单击 "移动" 光标。 从视频的初始帧开始, 右键单击要跟踪的点;跟踪点用蓝色圆圈标记。 注意: 在本研究中, 每只动物的位置都通过跟踪尾巴的底部来跟踪。</li>
	<li>通过单击键盘上的右箭头来前进框架;跟踪点应根据尾部底座的位置自动移动。<ol>
		<li>如果跟踪位置与给定帧中的兴趣点不一致, 请手动将蓝色圆圈与尾部的底座对齐。 注意: 根据视频的质量和鼠标的速度, 跟踪可能需要不同级别的用户输入, 以保持与感兴趣点的对准。鼠标的路径应该在视频过程中前进。</li>
	</ol>
	</li>
	<li>完整视频的跟踪完成后, 保存视频和跟踪覆盖。通过选择 "导出到电子表格" 导出跟踪的位置数据。</li>
</ol>
4. 数据分析
<ol>
	<li>在电子表格软件程序中打开位置数据。移动分析软件在每个框架中报告鼠标的 X、Y 坐标位置。</li>
	<li>要计算帧移动的距离, 请使用以下公式 (其中第一帧的位置为 x1, y1 , 第二帧为 x2, y2): 
	<img alt="Equation" src="/files/ftp_upload/56772/56772eq1.jpg"> 注: 随着时间的推移, 累积的距离可以通过增加每个帧之间的距离来计算。</li>
</ol>

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M., 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=Sarcolemma-localized+nNOS+is+required+to+maintain+activity+after+mild+exercise.">Sarcolemma-localized nNOS is required to maintain activity after mild exercise.</a> Nature. 456 (7221), 511-515 (2008).</li><li>Belanto, J. J., 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=Microtubule+binding+distinguishes+dystrophin+from+utrophin.">Microtubule binding distinguishes dystrophin from utrophin.</a> Proc Natl Acad Sci U S A. 111 (15), 5723-5728 (2014).</li><li>McDonald, C. 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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+hitchhiker's+guide+to+behavioral+analysis+in+laboratory+rodents.">A hitchhiker's guide to behavioral analysis in laboratory rodents.</a> Genes Brain Behav. 5, 5-24 (2006).</li><li>Gibbs, E. M., 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=High+levels+of+sarcospan+are+well+tolerated+and+act+as+a+sarcolemmal+stabilizer+to+address+skeletal+muscle+and+pulmonary+dysfunction+in+DMD.">High levels of sarcospan are well tolerated and act as a sarcolemmal stabilizer to address skeletal muscle and pulmonary dysfunction in DMD.</a> Hum Mol Genet. 25 (24), 5395-5406 (2016).</li><li>Gillis, J. 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作者没有什么可透露的。

野外试验提供了一种有效而非侵入性的方法来检测肌肉疾病的小鼠模型中的运动功能, 而这种检测的变异可以作为临床前研究的一项重要的终点措施。这类化验的一个主要限制是活动监测系统的高成本和有限的可及性。在这项研究中, 我们展示了一个低成本的开放现场活动分析系统, 可以产生类似的结果, 现有的商业软件和设备。该系统可以在 vivaria 的地方使用, 从而避免了将动物转移到核心设施的需要。这一分析的多功能性也使得在各种研究和教育设置中使用视频跟踪, 包括以动物生理学和行为为重点的本科生实验课程。
在这项研究中, 我们发现, 野生类型和mdx小鼠之间的自愿运动的差异, 可以很容易地发现后, 通过运动的恶化。这一强大的差异已经报告了其他团体的研究类似的活动结果, 包括 hour-long 开放现场化验和隔夜饲养化验。虽然我们观察到未野生类型和mdx小鼠之间的自愿移动没有区别, 但这与其他组13的结果是一致的。一些研究人员报告了未野生类型和mdx鼠标之间在自愿移动距离上的差异,7, 而其他报告没有差异13。我们的发现支持在测试之前使用肌肉激活, 以便可靠地检测mdx活动中的统计学意义差异。
在这项研究中, 我们提出了一个新的开放领域环境的小鼠。使用这一新的环境协议, 我们观察到小组内部很少有变化。但是, 当鼠标放置在不熟悉的环境中时, 它们的活动往往会提高14。如果同样的动物在多个疗程中被测试, 动物应该习惯于记录室并且在记录数据之前多次接受完整的测试协议。
从野外试验中评估数据的一个重要的考虑因素是它对动物的行为和情绪状况非常敏感, 尤其是焦虑样的行为。mdx可能对新环境具有不同的行为响应, 而不是野生类型的控制动物。然而, 我们另外测试了肌肉特异的抢救, mdx: utr-Tg 老鼠, 表达 utrophin 在肌肉特定促进者之下。这些小鼠的表现和野生类型的动物一样, 表明在mdx小鼠的任何行为差异都不会推动自愿移动的改变。
野外试验广泛应用于行为范式以及运动功能的研究中, 有多种参数可以从活动监测系统中记录。在商业活动分析软件中常用的一些测量方法包括自动检测特定行为、转向频率、垂直移动以及特定感兴趣区域的偏好。本研究采用的6分钟步行距离法, 其输出量主要限于水平移动距离和速度。
此外, 由于在本协议中半自动跟踪所需的用户输入级别, 这种检测可能不太适合在较长时间内进行活动监视或同时测量多个动物。在这项研究中, 我们发现, 训练有素的用户可以处理和提取数据的视频约15-30 分钟每记录。虽然这只代表了 small-scale 实验的适度时间负担, 但对于在较长时间内观察大型样本集的研究而言, 这一水平的分析将很快变得望而却步。虽然更多的全自动商业跟踪系统通常成本超过2万美元, 这些解决方案可能是更成本-和时间为实验室的移动经常测试。然而, 对于只测试少量动物的实验室和教育环境, 这种方法的简单设置和重现性为评估运动行为提供了一个有价值的工具。
可利用的数据分析类型是在确定临床前研究的适当终点化验中的重要考虑因素。当我们能够通过6分钟移动距离 (野生类型、 mdx和mdx: utr-tg) 来一致地检测出三种基因型之间的差异时, 对 longer-term 活动的更复杂的分析可能是可取的, 当看微妙自愿行动的变化。但是, 这种低成本设置的易用性和简单性使它成为mdx鼠标模型中前临床研究的有用和高度相关的功能测试。

可靠和可重复的测量肌肉功能是关键的评估潜在的治疗 DMD 的疗效。DMD 是由肌营养不良蛋白基因突变引起的遗传性疾病, 导致渐进性肌肉无力, 移动丧失, 最终心肺衰竭。最广泛使用的 DMD 的动物模型是肌营养不良蛋白-空mdx鼠标。在mdx鼠标以及其他营养和病的类似动物模型中, 一组功能测试已成为评估疾病进展的常规方法。通常使用的在体内测试包括测量前肢握力, 导线悬挂时间, rotarod 最大, 在跑步机跑步的时间和运动活动跟踪的疲劳。在这一领域, 为了使这些测试标准化, 有了很大的努力, 目的是减少前临床研究的变异性, 提高在小鼠体内测试的治疗的转化潜能1,2。
临床前测试的一个重要类别是测量自愿运动, 这一参数在小鼠肌肉萎缩症模型中经常被改变。这通常是通过基于野外活动监测的分析来测试的, 并且可以在几分钟或数小时的过程中评估水平 (行走) 或垂直 (饲养) 运动,2,3,4。一些研究表明, 在mdx小鼠中, 特别是在运动之后, 自愿改变的运动, 这些测量结果对药物治疗和疾病进展很敏感。执行这些化验的一个主要限制是需要专门的、高成本的设备。在这里, 一个低成本的方法, 跟踪鼠标移动使用现成的资源。
6分钟步行距离是一种常用的标准作为临床评估工具的个人杜氏肌营养不良症5,6。这项措施的修改已被用来评估杜氏动物模型的结果, 包括mdx小鼠7和金毛猎犬肌肉萎缩症 (GRMD) 狗8。在这项研究中, 我们记录自愿开放野外运动在6分钟后, 紧接着一个温和的运动挑战。然后使用自由开源软件计算移动距离, 以测量随时间推移的水平移动。
这种方法的主要优点是可以在不同的环境中测试动物, 而无需专门的设备或昂贵的商业软件进行分析。这一分析的一个重要方面是, 它可以在一个基本的实验室设置, 而无需移动或转移到一个专门的核心设施 vivarium 小鼠。这里描述的视频跟踪协议非常适合于对相对较短的青年进行移动评估, 并且能够检测出野生类型和mdx小鼠之间的活动差异, 并揭示了救援中的功能改进模型的 DMD。

<table>
	<tbody>
		<tr>
			<td>Video camera</td>
			<td>Apple Inc.</td>
			<td>ME553LL/A</td>
			<td>For recording ambulation video, iPhone 5S Plus (or equivalent)</td>
		</tr>
		<tr>
			<td>Kinovea software (version 0.8.15)</td>
			<td>Kinovea Association</td>
			<td></td>
			<td>Open source video analysis software. Free download, PC compatible (Version 0.8.15, www.kinovea.org)</td>
		</tr>
		<tr>
			<td>iMovie (version 10.0.6)</td>
			<td>Apple Inc.</td>
			<td></td>
			<td>Any similar software can be used to reduce video frame rate (optional)</td>
		</tr>
		<tr>
			<td>Roughneck 32 Gallon Black Round Trash Bin (Open field chamber)</td>
			<td>Rubbermaid</td>
			<td># 1778013</td>
			<td>Any open field chamber system can be adapted for recording. This study uses a recording chamber constructed out of a tray on a platform, at the bottom of a large trash bin.</td>
		</tr>
		<tr>
			<td>Avant White Plastic Tray 15&#34;W x 10&#34;D x 1.45&#34;H (Open field chamber)</td>
			<td>US Acrylic, LLC</td>
			<td></td>
			<td>Any open field chamber system can be adapted for recording. This study uses a recording chamber constructed out of a tray on a platform, at the bottom of a large trash bin.</td>
		</tr>
		<tr>
			<td>C57BL/6J</td>
			<td>Jackson Laboratory</td>
			<td>#000664</td>
			<td>Male 6 month mice</td>
		</tr>
		<tr>
			<td>C57BL/10ScSn-Dmd/J (mdx)</td>
			<td>Jackson Laboratory</td>
			<td>#001801</td>
			<td>Male 6 month mice</td>
		</tr>
		<tr>
			<td>mdx: utrophin-Tg (fiona)</td>
			<td>Gift from from James Ervasti, with permission from Kay Davies</td>
			<td></td>
			<td>Male 6 month mice</td>
		</tr>
	</tbody>
</table>

a simple and low-cost assay for measuring ambulation in mouse models of muscular dystrophy

在肌营养不良症的治疗中, 测量功能性结局是临床前测试的一个重要方面。在小鼠模型中自愿移动的评估是一种非侵入性的和可重复的活性测定方法, 与患者步行6分钟的测量和相关的移动评分直接类似。许多测试鼠标移动速度和距离的常用方法都是基于野外试验, 在这个测试中, 动物在竞技场中的自由运动是随着时间的推移而测量的。这种方法的一个主要缺点是, 用于高分辨率运动跟踪的商用软件和设备成本高昂, 可能需要将鼠标转移到专门的设备进行测试。在这里, 我们描述了一个低成本, 视频的测量鼠标移动的系统, 利用自由和开源软件。使用这个协议, 我们证明, 自愿移动在肌营养不良蛋白-无效的假mdx小鼠模型的杜氏肌肉萎缩症 (DMD) 是减少相对于野生类型的鼠标活动。在表达 utrophin 转基因的mdx小鼠中, 没有观察到这些活动的缺陷, 并且总的移动距离与野生型小鼠是无法区分的。这种方法是有效的测量变化的自愿步行与不良病理学, 并提供了一个多才多艺的平台, 可以很容易地适应不同的研究设置。

开放野外试验的变化已被证明是一种有效的方法来测试疾病进展的模型肌肉萎缩症。在这里, 我们演示了一种通用的视频跟踪方法, 用于分析 two-dimensional 鼠标移动使用现成的记录设备和开源软件 (图 1A-B)。与类似分析报告的结果一致, 我们显示mdx小鼠在测试之前, 在一个6分钟的野外任务中, 比野生型小鼠更少的自愿运动挑战 (图1C,图 2)。随着握力挑战的轻度消耗, mdx鼠标在移动协议的最初几分钟内通常仍然保持不动, 并且在移动时的动作量会适度增加4到 5 (图 3A)。在测试野生类型和mdx动物时, 如果没有运动挑战, 我们检测到的总距离没有差异 (图 2)。
这是与其他组的报告相一致的, 研究类似的运动分析, 未mdx显示与野生型小鼠没有区别或小的减少。我们还对表达高水平 utrophin 的转基因小鼠进行了评估, 以测试我们的检测灵敏度 (mdx: utr-tg, 菲奥娜线)11,12。mdx: utr-tg 是一种具有少量不良特征的mdx表型的健壮的救援模型。在练习之后, 表达 utrophin 的mdx小鼠与野生类型是无法区分的, 在总的距离 (图 3B) 或所测量的任何累计时间点 (图 3A) 上没有显著差异。
视频跟踪依赖于帧的可变性, 通过手动观察评分或自动软件检测来衡量。自由和开放源码运动分析软件允许对动物运动进行半自动检测, 在这种情况下, 在生成活动轨迹时, 可以手动纠正未被察觉或高估的运动。为了最大限度地减少数据处理在这个分析, 我们缩减原始的录影从被记录的 30 fps 到 15 fps。为了确定降低采样率的效果, 我们测量了1分钟全帧速率视频的总距离, 然后比较了从缩减版本的数据集 (图 4A) 导出的距离测量。我们发现, 将帧速率降低一半 (抽取 2) 将测量的距离降低了 5.0%, 并将帧速率降低为 7.5 fps 保留了近90% 的原始距离计算 (89.2% fps 视频的距离)。在这一点之后, 测量距离的精确度急剧下降, 虽然即使是高度毁灭的数据集仍然接近动物的路径 (图 4B)。这些观察强调了在评估动物活动时考虑帧速率和信号处理的重要性, 并证明了高空间分辨率可以保留在缩减的视频数据中。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56772/56772fig1.jpg"> 
图 1: 具有代表性的野外移动距离6分钟的痕迹.(A) 在本研究中使用的活动记录室的设置。握力仪是用于运动的挑战之前的化验, 然后自由移动记录在开放的野外平台。(B) 在运动跟踪软件中使用跟踪分析的6分钟视频录制的复合图像。(C) 覆盖两个代表性的痕迹 (重量, 灰色, n = 1;mdx, 红色, n = 1)。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56772/56772fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56772/56772fig2.jpg"> 
图 2: 运动对mdx移动的影响。6 分钟的移动距离被记录在6月的年龄有或没有事先锻炼 (non-exercised mdx n = 5, 重量n = 4; 行使了mdx n = 5, 重量n = 5)。在未时, 小波变换和mdx之间没有显著差异, 但在检测前的小鼠中有显著差异 (无运动: 体重 1077.0±106.4 厘米, mdx 971.0 ±36.16 厘米; 运动: 重量770.2 ±30.75 厘米,mdx 127.8 ±36.16 厘米)。数据代表的意思是± SEM. 统计数据使用 two-way 方差分析, 其次是 Tukey 的多个比较的测试 (*p和 #60; 0.05, ** p 和 #60; 0.01, *** p 和 #60; 0.001 和 ***p和 #60; 0.0001)。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56772/56772fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56772/56772fig3.jpg"> 
图 3: utrophin 的转基因表达改善了mdx鼠标的移动.6分钟的移动距离被记录在6月的年龄使用开放的野外活动室 (重量n = 5, mdx n = 5, mdx: utr-Tg n = 4)。(A) 累计距离 (分钟)。在小波和mdx之间没有发现明显的差异: utr-tg 在任何一点。统计数据使用 two-way 方差分析, 其次是 Tukey 的多个比较的测试 (*p和 #60; 0.05, **p和 #60; 0.01, ***p和 #60; 0.001 和 ***p和 #60; 0.0001)。(B) 总的开放字段距离超过6分钟 (重量770.2 ± 30.75 cm, mdx 127.8 ± 36.16 cm, mdx: utr-Tg 701.3 ±33.54 厘米)。数据代表的意思是± SEM. 统计数据使用 one-way 方差分析, 其次是 Tukey 的多个比较的测试 (*p和 #60; 0.05, ** p 和 #60; 0.01, *** p 和 #60; 0.001 和 ***p和 #60; 0.0001)。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56772/56772fig3large.jpg" target="_blank">请点击这里查看更大版本的这个数字。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56772/56772fig4.jpg"> 
图 4: 采样率对移动跟踪的影响.在一个野生类型的鼠标, 1 分钟的移动距离记录和跟踪使用运动跟踪软件在全帧速率 (30.0 fps, 1819 总帧)。确定了鼠标在每个帧中的 two-dimensional 位置, 然后这些数据被摧毁以在较低帧速率的视频中模拟跟踪 (30、22.5、20、15、10、7.5、6、3、1.5 和 0.75 fps)。(A) 从每个缩减数据集中的坐标计算出的总距离。虚线显示了从全帧速率视频计算出的距离的5% 以内的值。这项研究的数据是从视频中被摧毁的 15 fps (红色箭头) 获得的。(B) 覆盖的跟踪比较全帧速率跟踪与 1.5 fps (上面) 和 15 fps (下)。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56772/56772fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about A Simple and Low-cost Assay for Measuring Ambulation in Mouse Models of Muscular Dystrophy at JoVE.com

A Simple and Low-cost Assay for Measuring Ambulation in Mouse Models of Muscular Dystrophy

一种简便、低成本的肌营养不良小鼠步行模型的测定方法

该协议描述了一个灵活的, 低成本的测量鼠标移动在野外活动的测试系统。我们显示, 基于这个系统的6分钟的步行试验检测到在mdx小鼠中自愿运动的减少, 并准确地区分了这些动物的肌肉特异性抢救的改进。

Measuring functional outcomes in the treatment of muscular dystrophy is an essential aspect of preclinical testing. The assessment of voluntary ambulation ...

a-simple-low-cost-assay-for-measuring-ambulation-mouse-models

Nanyang Technological University

Research

JoVE Journal

Behavior

9.8K 次观看.  University of California. 该协议描述了一个灵活的, 低成本的测量鼠标移动在野外活动的测试系统。我们显示, 基于这个系统的6分钟的步行试验检测到在mdx小鼠中自愿运动的减少, 并准确地区分了这些动物的肌肉特异性抢救的改进。

视频: A Simple and Low-cost Assay for Measuring Ambulation in Mouse Models of Muscular Dystrophy

Nest Building as an Indicator of Health and Welfare in Laboratory Mice

The minimization and alleviation of suffering has moral and scientific implications. In order to mitigate this negative experience one must be able to identify when an animal is actually in distress. Pain, illness, or distress cannot be managed if unrecognized. Evaluation of pain or illness typically involves the measurement of physiologic and behavioral indicators which are either invasive or not suitable for large scale assessment. The observation of nesting behavior shows promise as the basis of a species appropriate cage-side assessment tool for recognizing distress in mice. Here we demonstrate the utility of nest building behavior in laboratory mice as an ethologically relevant indicator of welfare. The methods presented can be successfully used to identify thermal stressors, aggressive cages, sickness, and pain. Observation of nest building behavior in mouse colonies provides a refinement to health and well-being assessment on a day to day basis.

We demonstrate the utility of nest building behavior in laboratory mice as an indicator of welfare. Nest scoring is a sensitive technique that is altered by temperature, illness, and aggression. The time to integrate into nest test (TINT) is a simple cage-side assessment that can detect postoperative pain.

The minimization and alleviation of  suffering has moral and scientific implications. In order to mitigate this  negative experience one must be able to ...

科研

行为学

Barnes Maze Testing Strategies with Small and Large Rodent Models

Spatial learning and memory of laboratory rodents is often assessed via navigational ability in mazes, most popular of which are the water and dry-land (Barnes) mazes. Improved performance over sessions or trials is thought to reflect learning and memory of the escape cage/platform location. Considered less stressful than water mazes, the Barnes maze is a relatively simple design of a circular platform top with several holes equally spaced around the perimeter edge. All but one of the holes are false-bottomed or blind-ending, while one leads to an escape cage. Mildly aversive stimuli (e.g.&#160;bright overhead lights) provide motivation to locate the escape cage. Latency to locate the escape cage can be measured during the session; however, additional endpoints typically require video recording. From those video recordings, use of automated tracking software can generate a variety of endpoints that are similar to those produced in water mazes (e.g.&#160;distance traveled, velocity/speed, time spent in the correct quadrant, time spent moving/resting, and confirmation of latency). Type of search strategy (i.e.&#160;random, serial, or direct) can be categorized as well. Barnes maze construction and testing methodologies can differ for small rodents, such as mice, and large rodents, such as rats. For example, while extra-maze cues are effective for rats, smaller wild rodents may require intra-maze cues with a visual barrier around the maze. Appropriate stimuli must be identified which motivate the rodent to locate the escape cage. Both Barnes and water mazes can be time consuming as 4-7 test trials are typically required to detect improved learning and memory performance (e.g.&#160;shorter latencies or path lengths to locate the escape platform or cage) and/or differences between experimental groups. Even so, the Barnes maze is a widely employed behavioral assessment measuring spatial navigational abilities and their potential disruption by genetic, neurobehavioral manipulations, or drug/ toxicant exposure.

The dry-land Barnes maze is widely used to measure spatial navigation ability in response to mildly aversive stimuli. Over consecutive days, performance (e.g.&#160;latency to locate escape cage) of control subjects improves, indicative of normal learning and memory. Differences between rats and mice necessitate apparatus and methodology changes that are detailed here.

巴恩斯迷宫测试策略与小型和大型啮齿类动物模型

Spatial learning and memory of laboratory rodents is often assessed via navigational ability in mazes, most popular of which are the water and dry-land ...

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 ...

The Treadmill Fatigue Test: A Simple, High-throughput Assay of Fatigue-like Behavior for the Mouse

Fatigue is a prominent symptom in many diseases and disorders and reduces quality of life for many people. The lack of clear pathogenesis and failure of current interventions to adequately treat fatigue in all patients leaves a need for new treatment options. Despite the therapeutic need and importance of preclinical research in helping identify promising novel treatments, few preclinical assays of fatigue are available. Moreover, the most common preclinical assay used to assess fatigue-like behavior, voluntary wheel running, is not suitable for use with some strains of mice, may not be sensitive to drugs that reduce fatigue, and has relatively low throughput. The current protocol describes a novel, non-voluntary preclinical assay of fatigue-like behavior, the treadmill fatigue test, and provides evidence of its efficacy in detecting fatigue-like behavior in mice treated with a chemotherapy drug known to cause fatigue in humans and fatigue-like behavior in animals. This assay may be a beneficial alternative to wheel running, as fatigue-like behavior and potential interventions can be assessed in a greater number of mice over a shorter time frame, thus permitting faster discovery of new therapeutic options.

Fatigue is a common, undertreated and frequently poorly-understood symptom in many diseases and disorders. New preclinical assays of fatigue may help to improve current understanding and future treatment of fatigue. To that end, the current protocol provides a novel means of measuring fatigue-like behavior in the mouse.

跑步机疲劳试验:疲劳类似的行为，为鼠标操作简单，高通量检测

Fatigue is a prominent symptom in many diseases and disorders and reduces quality of life for many people. The lack of clear pathogenesis and failure of ...

Battery of Behavioral Tests Assessing General Locomotion, Muscular Strength, and Coordination in Mice

Behavioral testing is used in pre-clinical trials to assess the phenotypic effects and outcomes that a particular disease or treatment has on the animal&#39;s wellbeing and health. There are numerous behavioral tests that may be applied. We selected a test for general locomotion, the open field test (OFT); a test for muscular strength, the mesh test (MT); and a test for coordination, the rotarod test (RR). Testing can be accomplished on a weekly or monthly basis. As a test for general locomotion, the OFT works by objectively monitoring movement parameters while the mouse is in an open field apparatus. The field is generally a 2&#39; x 2&#39; box, and the movements are recorded through laser sensing or through video capture. The mouse is placed in the center of the open field and allowed to move freely for the test. The MT uses the latency for a mouse to fall off an inverted screen as a measure of muscular strength. A mouse is placed on a screen, which is inverted over a clear box, and is timed for their latency to fall. Three trials are performed, with the best of the three trials scored for that day. A score of 60 s is the maximum time a mouse is left inverted. Mice are given a 5-min rest period between mesh test trials. Lastly, an accelerated protocol on the RR assesses motor coordination and endurance. During a trial, a mouse walks on a rotating rod as it increases in speed from 4 rpm to 40 rpm over 5 min. The trial ends when the mouse touches the magnetized pressure sensor upon falling. Each mouse undergoes three trials, and the best trial is scored for that day. This combined behavioral data allows for the global assessment of mobility, coordination, strength, and movement of the test animals. At least two out of the three behavioral testing measures must show improvement for an animal to qualify as having overall improved motor function.

Behavioral testing is used in pre-clinical trials to assess the phenotypic effects of a disease or treatment on an animal's wellbeing. In order to globally assess motor functioning, we selected tests for general locomotion, muscular strength, and coordination: the open field test, the mesh test, and the rotarod test, respectively.

用于评估一般运动、肌肉力量和小鼠协调性的行为测试电池

Behavioral testing is used in pre-clinical trials to assess the phenotypic effects and outcomes that a particular disease or treatment has on the ...

Automated, Long-term Behavioral Assay for Cognitive Functions in Multiple Genetic Models of Alzheimer's Disease, Using IntelliCage

Multiple factors&#8212;such as aging and genes&#8212;are frequently associated with cognitive decline. Genetically modified mouse models of cognitive decline, such as Alzheimer&#39;s disease (AD), have become a promising tool to elucidate the underlying mechanisms and promote the therapeutic advances. An important step is the validation and characterization of expected behavioral abnormality in the models, in the case of AD, cognitive decline. The long-term behavioral investigations of laboratory animals to study the effect of aging demand substantial efforts from researchers. The IntelliCage system is a high-throughput and cost-effective test battery for mice that eliminates the need for daily human handling. Here, we describe how the system is utilized in the long-term phenotyping of a genetic Alzheimer&#39;s disease model, specifically focusing on the cognitive functions. The experiment employs repeated battery of tests that assess spatial learning and executive functions. This cost-effective age-dependent phenotyping allows us to identify the transient and/or permanent effects of genes on various cognitive aspects.

This paper describes a protocol for cognitive assessments for genetic models of the Alzheimer&#39;s disease using the IntelliCage system, which is a high throughput automated behavioral monitoring system with operant conditioning.

基于 IntelliCage 的阿尔茨海默病多基因模型认知功能的自动、长期行为检测

Multiple factors&#8212;such as aging and genes&#8212;are frequently associated with cognitive decline. Genetically modified mouse models of cognitive ...

Measuring Active and Passive Tameness Separately in Mice

Domesticated animals such as dogs and laboratory mice show a high level of tameness, which is important for humans to handle them easily. Tameness has two behavioral components: a reluctance to avoid humans (passive tameness) and a motivation to approach humans (active tameness). To quantify these components in mice, we previously developed behavioral tests for active tameness, passive tameness, and the willingness to stay on a human hand, each designed to be completed within 3 min. The data obtained were used for selective breeding, with a large number of mice analyzed per generation. The active tameness test measures the movement of the mouse toward a human hand and the contact it engages in. The passive tameness test measures the duration of time that a mouse tolerates human touch. In the stay-on-hand test, a mouse is placed on a human hand and touched slowly using the thumb of that hand; the duration of time that the animal remains on the hand is measured. Here, we describe the test set-up and apparatus, explain the procedures, and discuss the appropriate data analysis. Finally, we explain how to interpret the results.

Tameness in animals includes a reduction of avoidance responses to humans (passive tameness) and an increase in active approaches to humans (active tameness). Here, we describe detailed protocols for three behavioral tests (active tameness, passive tameness, and stay-on-hand tests) to separately measure the active and passive tameness of mice.

小鼠主动和被动驯服的测定

Domesticated animals such as dogs and laboratory mice show a high level of tameness, which is important for humans to handle them easily. Tameness has two ...

Continuous Noninvasive Measuring of Crayfish Cardiac and Behavioral Activities

A crayfish is a pivotal aquatic organism that serves both as a practical biological model for behavioral and physiological studies of invertebrates and as a useful biological indicator of water quality. Even though crayfish cannot directly specify the substances that cause water quality deterioration, they can immediately (within a few seconds) warn humans of water quality deterioration via acute changes in their cardiac and behavioral activities.
In this study, we present a noninvasive method that is simple enough to be implemented under various conditions due to a combination of simplicity and reliability in one model.
This approach, in which the biological organisms are implemented into environmental evaluation processes, provides a reliable and timely alarm for warning of and preventing acute water deterioration in an ambient environment. Therefore, this noninvasive system based on crayfish physiological and ethological parameter recordings was investigated for the detection of changes in an aquatic environment. This system is now applied at a local brewery for controlling&#160;quality of the water used for beverage production, but it can be used at any water treatment and supply facility for continuous, real-time water quality evaluation and for regular laboratory investigations of crayfish cardiac physiology and behavior.

This article presents a noninvasive biomonitoring system for the continuous recording and analyses of crayfish cardiac and locomotor activities. This system consists of a near-infrared optical sensor, a video-tracking module, and software for evaluating crayfish heartbeats that reflects its physiological condition and characterizes crayfish behavior during heartbeat fluctuations.

小龙虾心脏的连续无创测量与行为活动

A crayfish is a pivotal aquatic organism that serves both as a practical biological model for behavioral and physiological studies of invertebrates and as ...

Low-cost Protocol of Footprint Analysis and Hanging Box Test for Mice Applied the Chronic Restraint Stress

Gait disturbance is frequently observed in patients with movement disorders. In mouse models used for movement disorders, gait analysis is important behavioral test to determine whether the mice mimic the symptoms of patients. Motor deficits are often induced by stress when no spontaneous motor phenotype is observed in the mouse models. Therefore, gait analysis followed by stress loading would be a sensitive method for evaluating the motor phenotype in mouse models. However, researchers face the requirement of an expensive apparatus to obtain quantitative results automatically from gait analysis. For stress, stress loading by simple methods without expensive apparatuses required for electric shock and forced running is desirable. Therefore, we introduce a simple and low-cost protocol consisting of footprint analysis with paper and ink, hanging box test to evaluate motor function, and stress loading defined by restraint with a conical tube. The motor deficits of mice were successfully detected by this protocol.

The low-cost protocol consisting of footprint analysis and hanging box test after restraint stress is useful for evaluating the movement disorders of mouse model.

小鼠应用慢性约束应激的低成本足迹分析和吊箱试验协议

Gait disturbance is frequently observed in patients with movement disorders. In mouse models used for movement disorders, gait analysis is important ...

Paw-Print Analysis of Contrast-Enhanced Recordings (PrAnCER): A Low-Cost, Open-Access Automated Gait Analysis System for Assessing Motor Deficits

Gait analysis is used to quantify changes in motor function in many rodent models of disease. Despite the importance of assessing gait and motor function in many areas of research, the available commercial options have several limitations such as high cost and lack of accessible, open code. To address these issues, we developed PrAnCER, Paw-Print Analysis of Contrast-Enhanced Recordings, for automated quantification of gait. The contrast-enhanced recordings are produced by using a translucent floor that obscures objects not in contact with the surface, effectively isolating the rat&#8217;s paw prints as it walks. Using these videos, our simple software program reliably measures a variety of spatiotemporal gait parameters. To demonstrate that PrAnCER can accurately detect changes in motor function, we employed a haloperidol model of Parkinson&#8217;s disease (PD). We tested rats at two doses of haloperidol: high dose (0.30 mg/kg) and low dose (0.15 mg/kg). Haloperidol significantly increased stance duration and hind paw contact area in the low dose condition, as might be expected in a PD model. In the high dose condition, we found a similar increase in contact area but also an unexpected increase in stride length. With further research, we found that this increased stride length is consistent with the bracing-escape phenomenon commonly observed at higher doses of haloperidol. Thus, PrAnCER was able to detect both expected and unexpected changes in rodent gait patterns. Additionally, we confirmed that PrAnCER is consistent and accurate when compared with manual scoring of gait parameters.

Paw-Print Analysis of Contrast-Enhanced Recordings PrAnCER: A Low-Cost, Open-Access Automated Gait Analysis System for Assessing Motor Deficits

We describe a novel gait analysis system, Paw-Print Analysis of Contrast-Enhanced Recordings (PrAnCER), an open-access automated system for the quantification of gait characteristics in rats that utilizes a novel semitransparent floor to automatically quantify gait. This system was validated using the haloperidol model of Parkinson&#8217;s Disease.

对比度增强记录 (PrAnCER) 的爪打印分析:用于评估电机缺陷的低成本、开放访问自动步态分析系统

Gait analysis is used to quantify changes in motor function in many rodent models of disease. Despite the importance of assessing gait and motor function ...