R01AA016837（JCB）和P20AA017828（AGD和JCB）:这些研究是由美国国立卫生研究院酒精中毒和酒精滥用国家研究院​​的资助。

步骤1.在分析之前执行的日<ol><li>挑L4阶段蠕虫新鲜线虫生长培养基（NGM）板接种OP50 E.的草坪大肠杆菌 ，并对其进行培养，在20 CO / N。每个实验条件要求10蠕虫;挑过量蠕虫允许O / N损失的蠕虫。 <ol><li>只有实验动物，是第一天的成年人;许多突变体生长以较慢的速度比野生型。调整拾取对于具有发育迟缓，使所有测试的动物是第一天的成人菌株的定时。 </li></ol></li></ol>执行对检测的第2天步骤<ol><li>对于实验准备: <ol><li>在标准的非种子60×15毫米NGM板进行测定。干燥所有的板的情况下使用，在37℃，2小时后，用盖子关闭。每个实验试验，采用四干NGM板;这些将是0 20mM和400mM的乙醇测定板及其相应的驯化板。 注意:使用NGM板是很重要的位置;在该板的成分的差异，特别是它们的渗透压，可以强烈地影响对行为乙醇的剂量反应，这是由于，至少部分，以变化的乙醇由动物20中累积的量。 NGM琼脂160毫渗量。 </li><li>干燥后，称量各未播种NGM板的在测定中使用，并记下重量。确定的媒体中基于空盘的重量在板的体积。以近似重量的琼脂至体积的转化，假设介质的重量为相同的等体积水。 注:我们最一致的结果，已发现与已脱水足够的原始10毫升卷有8.3-8.9毫升之间的干燥后体积NGM媒体。一种替代的2小时的干燥时间是干燥直至板达到这个体积范围来解释在孵化的差异。 </li><li>熔体4铜环（1内径。6厘米）插入每个板的表面上以充当畜栏对于不同基因型或worms.Grasp每个环用钳子和热的治疗组中的火焰（来自本生灯强烈的火焰效果很好）约3秒。立即将戒指放在盘子里，而仍持有环钳，以防止"跳跃"。 <ol><li>确保该环靠在平贴在琼脂表面通过按下轻轻用钳子以环上的几个点。当放置戒指，要小心留有余地，另外三个圈。 </li><li>融化三个附加铜环进入板的表面上，同时注意把它们作为并拢尽可能使所有4将在视摄像机的视场。 注意:在制作与琼脂良好的密封是至关重要的，以保持在所述环中的蠕虫的测定期间。环即跳过围绕上盘不可能进行正确的密封与琼脂和可能疤痕琼脂，这使得蠕虫洞穴和干扰可视化的蠕虫。 </li><li>对于每个测定板制备一个伴随"驯化"板，它应被干燥和未接种的和将得不到乙醇。放在这些板块4铜环。 </li></ol></li><li>标签板的底部旁边的每个环与蜗杆应变环中所用的，并注意在视环本身的领域不写。匹配试验板的标签，其伴随的驯化板。 </li><li>计算的100％乙醇的量添加到每个测定板，以使最终浓度为0毫米或400毫米的乙醇（重量比体积）。对于每个实验（N = 1），将有1 0毫和一个400毫乙醇板;驯化板没有收到乙醇。添加乙醇，吹打它围绕板的表面上。用实验室膜以密封所述板，并允许它以平衡地置于工作台顶部2小时。 </li><li>当1.5小时过去后，开始检测，步2.2的驯化一步。 </li></ol></li><li>进行运动检测:测定板<ol><li>小心转移10蠕虫每个实验组，以在驯化板铜环的中心。删除任何食物，通过与蠕虫挑轻轻刮琼脂在这一点上可见。变化都带上跨实验性试验的板，其中所述实验组，使得相同的菌株不能把在平板上在各试验相同的顺序的顺序。 注意:我们的目标是将动物用食物数量最小转移到板，因为如果在驯化板食物被转移到测定板中的蠕虫会聚集周围的食物和运动的药物的作用将被遮蔽。 </li><li>要小心，不要打破挑的琼脂表面，因为这将允许蠕虫打洞，所以会影响检测。孵育蠕虫在室温30分钟。 </li><li>确保有各板的灌顶之间保持适当的间隔。一个有经验的实验者可以移动40动物，10 /环4环的&lt;1.5分，但任何间隔可达2分钟是可以接受的。标准测定具有电影记录在10-12分钟，并暴露于两者0 20mM和400mM的乙醇30-32分钟。 </li><li>启动驯化板为0毫曝光和驯化板为400毫米曝光约2分钟和30秒的间隔，以允许用于保存前的第二电影必须开始的第一个电影文件。 </li><li>在30分钟驯化期后，从驯化板蠕虫转移到测定板中。转移的相同顺序，它们被添加到驯化板，跟踪各板的完井之间的定时的蠕虫到测定板（0毫米或400毫米乙醇）。与实验室薄膜密封板，以减少乙醇损失汽化。 </li><li> ü瑟用细边扁平虫挑收集蠕虫的扁平挑顶部铲动作。在不使用的细菌，其通常在转印蠕虫使用，因为它有助于胶蠕虫拾取的执行蠕虫从非种子驯化板转移至测定板。 注:在该动物在这个步传送速度是非常重要的，因为在板的第一蠕虫将暴露于乙醇比加入到板的最后蠕虫更长，并且较早的时间点可能表现出一些时间依赖性影响。这是用于旋转该菌株被横跨实验重复放置在平板上第一的主要原因。 </li></ol></li><li>执行步态分析:拍摄<ol><li>用显微镜/摄像机组合，其允许所有四个环中的视场（约42x42毫米2平方），的同时成像诸如0.5倍的显微镜物镜，0.8倍的放大倍率和一个照相机与2048×2048 7.481;万像素CCD。 </li><li>使用，即使在整个视场，这在强度阈步骤有助于在物体识别照度（见下文）。 A 3"X3"背光效果很好。 </li><li>像一盘定位媒体朝上的蠕虫（盖下），产生对比正在使用与传输光源的传统显微镜相比，背光时丢失。 </li><li>准备的图像分析软件来捕获的移动蠕虫时间推移电影。设置软件来捕获12位灰度图像每1秒，如2分钟（120帧）的电影。以减小输出文件的大小，而同时仍保留足够的图像分辨率，使用2x2融合模式拍摄1024×1024像素的图像。 </li><li>录制的电影。开始记录第一板（0毫乙醇）的最后一个蠕虫后10分钟被放置在该板的一个120帧的电影。保存的电影文件。 </li><li>记录所述第二板（400毫乙醇）。重复此过程两个板开始最后的蠕虫30分钟后，被放置在第一板（0毫乙醇）来捕获用于每个曝光的30-32分钟的时间点。 注:允许有足够的时间开始下盘记录进行分析之前，每个捕获会话后保存图像文件。 </li><li>为适应未来发展的存档电影，以允许打开和其他公共领域的开源图像处理软件程序，如ImageJ的28分析了这些电影，每部电影的拷贝转换成8位256级灰度TIFF文件。 注:这里所描述的图像分析软件使用一种专有的文件格式。 </li></ol></li><li>分析使用ImagePro软件电影。 <ol><li>一旦捕获，分析使用的Image-Pro Plus软件或等值的电影。 注意:各种其他对象跟踪软件是可用的已被成功地用于跟踪多个C.线虫一次29和这种软件可以合理替代的是，这里所描述的对象识别步骤基于类似的原理。描述的方法涉及到的Image-Pro Plus软件版本6.0-6.3和7.0，新版本的的Image-Pro Plus软件可能有细微的差别。 </li><li>在2分钟（120架）段分析电影。首先，将过滤器应用于图像变平的背景和增强蠕虫的对象的对比度。选择过滤器，如下所示:菜单&gt;过程&gt;过滤器&gt;增强&gt;拼合:参数:背景=黑暗;特征宽度= 20 Pix的。 </li><li>重新保存电影的过滤步骤后，但始终保持在其未经过滤的形式原始影片。 <ol><li>在每个环带的感兴趣区域（ROI）的圆形区域被放置和尺寸与铜环重叠分别分析动物的运动。识别和跟踪的菜单&gt;办法蠕虫&gt;跟踪物体...命令;这带来了跟踪ðATA表窗口。跟踪选项按钮允许特定的曲目被排除和限制任何实验文物。 </li></ol></li><li>根据自动跟踪选项卡，使用下列参数:轨道参数:速度极限（搜索半径）= 400微米/帧，加速度限制=自动，最小总轨道长度= 400微米，主要运动型=混乱;在轨道参数的对象:允许部分曲目= YES，最小长度= 21帧，跟踪预测深度= 1架。 </li><li>要启动跟踪过程中，单击查找所有曲目自动功能按钮，弹出计算/尺寸选项对话框和跟踪对话框。选择手动选项，在计算/尺寸对话框，它提供了一个使用的蠕虫像素的灰度强度，突出分析蠕虫对象，忽视了更轻的背景像素的重要门槛一步的强度范围选择。 <ol><li>调整强度阈值SLI德尔斯（一到设定的上限，一到设定下限）创建一个具有包容性的范围，突出全暗的对象。中0-1,500的规模为0-4,095一系列是一个很好的起点，更精细的调整。 </li><li>适用的尺寸过滤器，以排除对象是比单个蠕虫对象更大和更小，如铜环和在平板上的任何碎屑。设置两个尺寸参数做到这一点，包括大小个别蠕虫在各种姿势与计量&gt;的计算/尺寸选项对话框中选择测量菜单项。 （面积范围:28,000-120,000微米2和周边范围:600-2,500微米）。 </li><li>如果一个突变株是显著小于或大于其它动物更大的板加以分析，扩大的过滤器设置的范围为对象的识别，以容纳不同尺寸的菌株的第一追踪动物之前，使设置不需要改变中期分析。 </li></ol> &lt;/ li&gt; <li>单击继续在跟踪对话框中完成了跟踪过程。直观地比较输出音轨与影片中的每一个蠕虫的进展，以确保每一个蠕虫表示，除非有基于自动过滤器设置一个合理的理由来排除它。手动删除由满足大小的过滤标准确定非蠕虫物体的存在被生产的轨道。 </li><li>使用软件来计算每个帧之间的每个蠕虫的速度（行驶距离为每帧之间1秒的对象的质心），并显示在所有磁迹的平均流速为每个轨道和平均速度在蠕虫的人口铜圈。考虑这最终平均为 n = 1的实验性试验条件。将数据导出到的统计分析和数据归档的电子表格程序。 </li><li>一旦数据已被记录为在板的第一环中，将ROI的到下一个环和重复跟踪处理，而无需改变任何参数。 </li><li>计算运动通过相对速度: </li><li>相对速度（％）=处理（400毫米）的速度/未处理（0毫米）的速度×100。 注:不同的遗传操作往往改变动物的基础运动速度。为了确定乙醇对动物的运动速度的影响，并能够比较不同基因型或条件下，这些效果，计算出相对速度。 </li></ol></li></ol>

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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=Ethanol+metabolism+and+osmolarity+modify+behavioral+responses+to+ethanol+in+C.+elegans.">Ethanol metabolism and osmolarity modify behavioral responses to ethanol in C. elegans.</a> Alcohol. Clin. Exp. Res. 36, 1840-1850 (2012).</li><li>Morgan, P. G., Sedensky, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+affecting+sensitivity+to+ethanol+in+the+nematode,+Caenorhabditis+elegans.">Mutations affecting sensitivity to ethanol in the nematode, Caenorhabditis elegans.</a> Alcohol. Clin. Exp. Res. 19, 1423-1429 (1995).</li><li>Mitchell, P. H., 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+concentration-dependent+effects+of+ethanol+on+Caenorhabditis+elegans+behaviour.">The concentration-dependent effects of ethanol on Caenorhabditis elegans behaviour.</a> Pharmacogenomics J. 7, 411-417 (2007).</li><li>Vidal-Gadea, A., 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=Caenorhabditis+elegans+selects+distinct+crawling+and+swimming+gaits+via+dopamine+and+serotonin.">Caenorhabditis elegans selects distinct crawling and swimming gaits via dopamine and serotonin.</a> Proc. Natl. Acad. Sci. U S A. 108, 17504-17509 (2011).</li><li>Bettinger, J. C., Leung, K., Bolling, M. H., Goldsmith, A. D., Davies, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+environment+modulates+the+development+of+acute+tolerance+to+ethanol+in+Caenorhabditis+elegans.">Lipid environment modulates the development of acute tolerance to ethanol in Caenorhabditis elegans.</a> PLoS ONE. 7, e35129 (2012).</li><li>Davies, A. G., 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=Different+genes+influence+toluene-+and+ethanol-induced+locomotor+impairment+in+C.+elegans.">Different genes influence toluene- and ethanol-induced locomotor impairment in C. elegans.</a> Drug Alcohol Depend. 122, 47-54 (2012).</li><li>Newlin, D., Thomson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alcohol+challenge+with+sons+of+alcoholics:+a+critical+review+and+analysis.">Alcohol challenge with sons of alcoholics: a critical review and analysis.</a> Psychol. Bull. 108, 383-402 (1990).</li><li>Ponomarev, I., Crabbe, J. C. <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+method+to+assess+initial+sensitivity+and+acute+functional+tolerance+to+hypnotic+effects+of+ethanol.">A novel method to assess initial sensitivity and acute functional tolerance to hypnotic effects of ethanol.</a> J. Pharmacol. Exp. Ther. 302, 257-263 (2002).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nat Methods. 9, 671-675 (2012).</li><li>Husson, S. J., Costa, W. S., Schmitt, C., Gottschalk, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keeping+track+of+worm+trackers.">Keeping track of worm trackers.</a> WormBook. , (2012).</li></ol>

The authors have nothing to disclose.

简单的神经生物学和遗传学工具C.提供线虫使蠕虫病毒在其中研究乙醇对行为影响的分子基础的极好的模型。在这里，我们描述了已被用于识别急性行为反应的几种分子和环境介质为乙醇8,10,20,24,25的测定。这种方法允许分化和同时检查两个不同乙醇响应行为表型，初始灵敏度和急性官能耐受的发展，它们一起模拟响应在哺乳动物的水平的复合型。相同的测定，可以很容易地?...

Alcohol use disorders (AUD) are widespread and produce serious health, social, and economic problems. In humans, the susceptibility to developing an AUD is heavily influenced by both genetics and the environment1,2. A strong physiological predictor of abuse liability is the initial level of response (LR) to alcohol (ethanol) that is exhibited by na&#239;ve drinkers3-5. This LR phenotype is influenced by genetics and non-genetic components6. Determining the molecular mechanisms that influence the LR to ethanol is an important goal of the study of ethanol response behaviors.
The nematode, Caenorhabditis elegans, has been increasingly used as a model for studying the effects of ethanol on behavior7-9. There is strong molecular conservation in the machinery of nervous system function between worms and mammals, and several genes that have been shown to influence the LR to ethanol in worms have been shown to influence LR to ethanol in mammals10-16, and have been implicated in abuse liability in humans17-19. 
Ethanol intoxicates worms, which is reflected in a decrease in their locomotion speed. Several different laboratories have developed behavioral assays that differ in several ways, for example, the locomotion behavior that they study (crawling versus swimming11,12,14,20,21) or in the composition of the solutions in which the assays are performed (nematode growth medium versus Dent&#8217;s saline20,22). Interestingly, these diverse assays have yielded somewhat different dose response profiles for the effects of ethanol. These results have pointed to important differences in the underlying behaviors of crawling and swimming9,23, as well as a role for the environmental variable osmolarity in ethanol responses20, and have highlighted the importance of describing experimental detail of the various assays.
An assay to measure the acute effects of ethanol on crawling behavior is presented here. This assay has been used extensively to study the genetic and environmental influences on the LR to ethanol8,10,20,24,25. The mammalian LR phenotype is a composite of at least two components, initial sensitivity to ethanol and acute functional tolerance to ethanol26,27. In worms, the LR phenotype has been shown to be separable into these two components through the use of this behavioral assay. The influences of genetic and environmental manipulations on both phenotypes can be examined using this single assay. Importantly, these two phenotypes are genetically separable.

<table><tbody><tr><td>C. elegans strains</td><td>Caenorhabditis Genetics Center</td><td></td><td></td></tr><tr><td>60 x 15 mm Petri plates, triple vented</td><td>Greiner Bio-One</td><td>628161</td><td>Other plate brands will suffice.</td></tr><tr><td>NGM agar</td><td>Various</td><td></td><td>NaCl (3 g/L), agar (17 g/L), peptone (2.5 g/L), 1 ml cholesterol (5 mg/ml in ethanol), 1 ml (1 M) MgSO&lt;sub&gt;4&lt;/sub&gt;, 1 ml (1 M) CaCl&lt;sub&gt;2&lt;/sub&gt;, 25 ml (1 M) KPO&lt;sub&gt;4&lt;/sub&gt;, pH=6, 975 ml H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Forceps</td><td>Various</td><td></td><td>&lt;I&gt;e.g.&lt;/I&gt;, Fisher Scientific #10300</td></tr><tr><td>37 &amp;deg;C Incubator</td><td>Various</td><td></td><td>For drying agar</td></tr><tr><td>Digital balance</td><td>Various</td><td></td><td>For determining plate weights and agar volume</td></tr><tr><td>Copper rings</td><td>Plumbmaster</td><td>STK#35583 (48 cap thread gasket)</td><td>1.6 cm inner diameter, 1.8 cm outer diameter copper rings</td></tr><tr><td>100% ethanol</td><td>Various</td><td></td><td></td></tr><tr><td>Parafilm M</td><td>Bemis</td><td>PM996</td><td></td></tr><tr><td>CCD camera</td><td>QImaging</td><td>RET-4000R-F-M-12</td><td>This camera has a large field of view.</td></tr><tr><td>Stereomicroscope with C-mount and 0.5X objective</td><td>Leica</td><td>MZ6</td><td>Discontinued model, M60 is current equivalent.</td></tr><tr><td>Light source</td><td>Schott</td><td>A08923</td><td>3&amp;rdquo;x3&amp;rdquo;&amp;nbsp; backlight for even illumination across the field of view</td></tr><tr><td>Imaging and tracking software</td><td>Media Cybernetics</td><td>ImagePro-Plus v6.0-6.3</td><td>Newer versions of the software have tracking functions.</td></tr></tbody></table>

an assay for measuring the effects of ethanol on the locomotion speed of caenorhabditis elegans

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the development of more effective treatments is the relative lack of understanding of the molecular underpinnings of the effects of ethanol on behavior. The nematode, Caenorhabditis elegans (C.&#160;elegans), provides a useful model in which to generate and test hypotheses about the molecular effects of ethanol. Here, we describe an assay that has been developed and used to examine the roles of particular genes and environmental factors in behavioral responses to ethanol, in which locomotion is the behavioral output. Ethanol dose-dependently causes an acute depression of crawling on an agar surface. The effects are dynamic; animals exposed to a high concentration demonstrate an initial strong depression of crawling, referred to here as initial sensitivity, and then partially recover locomotion speed despite the continued presence of the drug. This ethanol-induced behavioral plasticity is referred to here as the development of acute functional tolerance. This assay has been used to demonstrate that these two phenotypes are distinct and genetically separable. The straightforward locomotion assay described here is suitable for examining the effects of both genetic and environmental manipulations on these acute behavioral responses to ethanol in C.&#160;elegans.

从几个不同的基因型和它们的配对控制代表数据（ 图1）呈现8,24;数据特别选择在检测动物亮点的差异。的效果，在暴露10分钟的程度被认为是一个菌株，其上示出在图1B-G的左侧轴的初始灵敏性。突变菌株与相对速度比在10分钟的控制较大被认为是乙醇抗性（ 图1F，G），而突变菌株具有相对速度小于被认为是过敏乙醇（ 图1D的控制，...

Watch this Scientific Journal Video about An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans at JoVE.com

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

C. elegans is a useful model for studying the effects of ethanol on behavior. We present a behavioral assay that quantifies the effects of ethanol on the locomotion speed of crawling worms; both initial sensitivity and the development of acute functional tolerance to ethanol can be measured with this assay.

测量乙醇的影响上的步态速度的测定<em&gt;秀丽隐杆线虫</em

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

8.6K 次观看.  Virginia Commonwealth University. C. elegans is a useful model for studying the effects of ethanol on behavior. We present a behavioral assay that quantifies the effects of ethanol on the locomotion speed of crawling worms; both initial sensitivity and the development of acute functional tolerance to ethanol can be measured with this assay. 

视频: An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

A Simple Way to Measure Ethanol Sensitivity in Flies

Low doses of ethanol cause flies to become hyperactive, while high doses are sedating. The sensitivity to ethanol-induced sedation of a given fly strain is correlated with that strain s ethanol preference, and therefore sedation is a highly relevant measure to study the genetics of alcohol responses and drinking. We demonstrate a simple way to expose flies to ethanol and measure its intoxicating effects. The assay we describe can determine acute sensitivity, as well as ethanol tolerance induced by repeat exposure. It does not require a technically involved setup, and can therefore be applied in any laboratory with basic fly culture tools.

A simple assay to measure the sedating effects of ethanol on Drosophila flies, based on the loss of righting reflex, is described.

一个简单的方法来衡量在果蝇的乙醇灵敏度

Low doses of ethanol cause flies to become hyperactive, while high doses are sedating. The sensitivity to ethanol-induced sedation of a given fly strain ...

科研

神经科学

Measuring Caenorhabditis elegans Sensitivity to the Acetylcholine Receptor Agonist Levamisole

At the neuromuscular junction (NMJ), the binding of the excitatory neurotransmitter acetylcholine (ACh) to postsynaptic receptors leads to muscle contraction. As in vertebrate skeletal muscle, cholinergic signaling in the body wall muscles of the model organism Caenorhabditis elegans is required for locomotion. Exposure to levamisole, a pharmacological agonist of one class of ACh receptors on the body wall muscles, causes time-dependent paralysis of wild-type animals. Altered sensitivity to levamisole suggests defects in signaling at the NMJ or muscle function. Here, a protocol for a liquid levamisole assay performed on C. elegans grown in 24-well plates is presented. Vigorous swimming of the animals in liquid allows for the assessment and quantitation of levamisole-induced paralysis in hundreds of worms over a one-hour time period without requiring physical manipulation. This procedure can be used with both wild-type and mutants that have altered sensitivity to levamisole to demonstrate the functional consequences of altered signaling at the NMJ.

Measuring Caenorhabditis elegans Sensitivity to the Acetylcholine Receptor Agonist Levamisole

The present protocol describes an assay to determine response to levamisole, a pharmacological agonist of one class of Caenorhabditis elegans acetylcholine receptors. In this liquid levamisole swim assay, researchers visually observe and quantitate the time-dependent paralysis of animals cultivated in 24-well plates.

测量 秀丽隐杆线虫 对乙酰胆碱受体激动剂左旋咪唑的敏感性

At the neuromuscular junction (NMJ), the binding of the excitatory neurotransmitter acetylcholine (ACh) to postsynaptic receptors leads to muscle ...

遗传学

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Caenorhabditis elegans is a simple genetic organism amenable to large-scale forward and reverse genetic screens and chemical genetic screens. The C. elegans genome includes potential antipsychotic drug (APD) targets conserved in humans, including genes encoding proteins required for neurotransmitter synthesis and for synaptic structure and function. APD exposure produces developmental delay and/or lethality in nematodes in a concentration-dependent manner. These phenotypes are caused, in part, by APD-induced inhibition of pharyngeal pumping1,2. Thus, the developmental phenotype has a neuromuscular basis, making it useful for pharmacogenetic studies of neuroleptics. Here we demonstrate detailed procedures for testing APD effects on nematode development and pharyngeal pumping. For the developmental assay, synchronized embryos are placed on nematode growth medium (NGM) plates containing APDs, and the stages of developing animals are then scored daily. For the pharyngeal pumping rate assay, staged young adult animals are tested on NGM plates containing APDs. The number of pharyngeal pumps per unit time is recorded, and the pumping rate is calculated. These assays can be used for studying many other types of small molecules or even large molecules.

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Approaches for testing the effects of antipsychotic drugs (APDs) in Caenorhabditis elegans are demonstrated. Assays are described for testing drug effects on development and viability and on pharyngeal pumping rate. These methods are also applicable for pharmacogenetic experiments with drug classes other than APDs.

方法研究抗精神病药物的作用机制在<em&gt;秀丽隐杆线虫</em

Caenorhabditis elegans is a simple genetic organism amenable to  large-scale forward and reverse genetic screens and chemical genetic screens. The C.  ...

High-Throughput Method for Measuring Alcohol Sedation Time of Individual Drosophila melanogaster

Drosophila melanogaster provides an excellent model to study the genetic underpinnings of alcohol sensitivity. In contrast to studies in human populations, the Drosophila model allows strict control over genetic background, and virtually unlimited numbers of individuals of the same genotype can be reared rapidly under well-controlled environmental conditions without regulatory restrictions and at relatively low cost. Flies exposed to ethanol undergo physiological and behavioral changes that resemble human alcohol intoxication, including loss of postural control, sedation, and development of tolerance. Here, we describe a simple, low-cost, high-throughput assay for assessing alcohol sedation sensitivity in large numbers of single flies. The assay is based on video recording of single flies introduced without anesthesia in 24-well cell culture plates in a set-up that enables synchronous initiation of alcohol exposure. The system enables a single person to collect individual ethanol sedation data on as many as 2,000 flies within an 8 h work period. The assay can, in principle, be extended to assess the effects of exposure to any volatile substance and applied to measure effects of acute toxicity of volatiles on other insects, including other fly species.

High-Throughput Method for Measuring Alcohol Sedation Time of Individual Drosophila melanogaster

Current methods to measure alcohol sensitivity in Drosophila are designed to test groups of flies. We present a simple, low-cost, high-throughput assay for assessing alcohol sedation sensitivity in large numbers of single flies. The method does not require specialized tools and can be performed in any laboratory using common materials.

测量个体果蝇黑色素气器酒精镇分时间的高通量方法

Drosophila melanogaster provides an excellent model to study the genetic underpinnings of alcohol sensitivity. In contrast to studies in human ...

行为学

Comparative Analysis of Experimental Methods to Quantify Animal Activity in Caenorhabditis elegans Models of Mitochondrial Disease

Caenorhabditis elegans is widely recognized for its central utility as a translational animal model to efficiently interrogate mechanisms and therapies of diverse human diseases. Worms are particularly well-suited for high-throughput genetic and drug screens to gain deeper insight into therapeutic targets and therapies by exploiting their fast development cycle, large brood size, short lifespan, microscopic transparency, low maintenance costs, robust suite of genomic tools, mutant repositories, and experimental methodologies to interrogate both in vivo and ex vivo physiology. Worm locomotor activity represents a particularly relevant phenotype that is frequently impaired in mitochondrial disease, which is highly heterogeneous in causes and manifestations but collectively shares an impaired capacity to produce cellular energy. While a suite of different methodologies may be used to interrogate worm behavior, these vary greatly in experimental costs, complexity, and utility for genomic or drug high-throughput screens. Here, the relative throughput, advantages, and limitations of 16 different activity analysis methodologies were compared that quantify nematode locomotion, thrashing, pharyngeal pumping, and/or chemotaxis in single worms or worm populations of C. elegans at different stages, ages, and experimental durations. Detailed protocols were demonstrated for two semi-automated methods to quantify nematode locomotor activity that represent novel applications of available software tools, namely, ZebraLab (a medium-throughput approach) and WormScan (a high-throughput approach). Data from applying these methods demonstrated similar degrees of reduced animal activity occurred at the L4 larval stage, and progressed in day 1 adults, in mitochondrial complex I disease (gas-1(fc21)) mutant worms relative to wild-type (N2 Bristol) C. elegans. This data validates the utility for these novel applications of using the ZebraLab or WormScan software tools to quantify worm locomotor activity efficiently and objectively, with variable capacity to support high-throughput drug screening on worm behavior in preclinical animal models of mitochondrial disease.

Comparative Analysis of Experimental Methods to Quantify Animal Activity in Caenorhabditis elegans Models of Mitochondrial Disease

This study presents protocols for two semi-automated locomotor activity analysis approaches in C. elegans complex I disease gas-1(fc21) worms, namely, ZebraLab (a medium-throughput assay) and WormScan (a high-throughput assay) and provide comparative analysis among a wide array of research methods to quantify nematode behavior and integrated neuromuscular function.

线粒体疾病脑 膜炎基因 模型中量化动物活动的实验方法比较分析

Caenorhabditis elegans is widely recognized for its central utility as a translational animal model to efficiently interrogate mechanisms and therapies of ...

An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

Alcohol use disorder (AUD) is a serious health challenge. Despite a large hereditary component to AUD, few genes have been unambiguously implicated in their etiology. The fruit fly, Drosophila melanogaster, is a powerful model for exploring molecular-genetic mechanisms underlying alcohol-related behaviors and therefore holds great promise for identifying and understanding the function of genes that influence AUD. The use of the Drosophila model for these types of studies depends on the availability of assays that reliably measure behavioral responses to ethanol. This report describes an assay suitable for assessing ethanol sensitivity and rapid tolerance in flies. Ethanol sensitivity measured in this assay is influenced by the volume and concentration of ethanol used, a variety of previously reported genetic manipulations, and also the length of time the flies are housed without food immediately prior to testing. In contrast, ethanol sensitivity measured in this assay is not affected by the vigor of fly handling, sex of the flies, and supplementation of growth medium with antibiotics or live yeast. Three different methods for quantitating ethanol sensitivity are described, all leading to essentially indistinguishable ethanol sensitivity results. The scalable nature of this assay, combined with its overall simplicity to set-up and relatively low expense, make it suitable for small and large scale genetic analysis of ethanol sensitivity and rapid tolerance in Drosophila.

An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

Straightforward assays for measuring ethanol sensitivity and rapid tolerance in Drosophila facilitate the use of this model organism for investigating these important ethanol-related behaviors. Here, a relatively simple, scalable assay for measuring ethanol sensitivity and rapid tolerance in flies is described.

一种廉价的，可扩展的行为分析测量乙醇镇静灵敏度和快速耐受<em&gt;果蝇</em

Alcohol use disorder (AUD) is a serious health challenge. Despite a large hereditary component to AUD, few genes have been unambiguously implicated in ...

C. elegans Chemotaxis Assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior.
 The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress 1-10. The method described here aims to address these issues by modifying the assay developed by Bargmann et al.1. A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

C. elegans Chemotaxis Assay

A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest.

线虫趋含量的

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

生物学

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity1 and vibration2. In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

测量细菌的影响<em&gt; C。线虫</em&gt;行为使用一个鸡蛋保留含量

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...

Touch Habituation Assay: A Method to Study Non-Associative Learning in C. elegans

Source: Mersha, M. D., et al. <a href="https://jove.unicartagenaproxy.elogim.com/video/56771/long-term-behavioral-reproductive-consequences-embryonic-exposure-to">Long-term Behavioral and Reproductive Consequences of Embryonic Exposure to Low-dose Toxicants.</a> J. Vis. Exp. (2018).
This video introduces the touch habituation assay, a C. elegans behavioral method of assessing mechanosensation and quantifying time to habituation.

触摸习惯化试验:一种研究秀丽隐杆线虫非联想学习的方法

Source: Mersha, M. D., et al. Long-term Behavioral and Reproductive Consequences of Embryonic Exposure to Low-dose Toxicants. J. Vis. Exp. (2018).
This ...

JoVE Encyclopedia of Experiments

秀丽隐杆线虫（线虫）

Electrotaxis Assay: A Method to Observe Locomotion in C. elegans

Source: Tong, J,&#160;et al. <a href="https://jove.unicartagenaproxy.elogim.com/video/50226/microfluidic-based-electrotaxis-for-on-demand-quantitative-analysis">Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of&#160;Caenorhabditis elegans&#39; Locomotion.</a>&#160;J. Vis. Exp.&#160;(2013).
Researchers can trigger on-demand movement in C. elegans by applying an electric field that worms sense and respond to.&#160; This video introduces electrotactic behavior and shows a sample protocol that is done in a microfluidic device.

趋电性测定:一种观察秀丽隐杆线虫运动的方法

Source: Tong, J,&#160;et al. Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of&#160;Caenorhabditis elegans&#39; Locomotion.&#160;J. ...

测量乙醇的影响上的步态速度的测定

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一个简单的方法来衡量在果蝇的乙醇灵敏度

测量秀丽隐杆线虫对乙酰胆碱受体激动剂左旋咪唑的敏感性

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一种廉价的，可扩展的行为分析测量乙醇镇静灵敏度和快速耐受

线虫趋含量的

测量细菌的影响

触摸习惯化试验:一种研究秀丽隐杆线虫非联想学习的方法

趋电性测定:一种观察秀丽隐杆线虫运动的方法

测量乙醇的影响上的步态速度的测定

摘要

探索更多视频

此视频中的章节

一个简单的方法来衡量在果蝇的乙醇灵敏度

测量 秀丽隐杆线虫 对乙酰胆碱受体激动剂左旋咪唑的敏感性

方法研究抗精神病药物的作用机制在

测量个体果蝇黑色素气器酒精镇分时间的高通量方法

线粒体疾病脑 膜炎基因 模型中量化动物活动的实验方法比较分析

一种廉价的，可扩展的行为分析测量乙醇镇静灵敏度和快速耐受

线虫趋含量的

测量细菌的影响

触摸习惯化试验:一种研究秀丽隐杆线虫非联想学习的方法

趋电性测定:一种观察秀丽隐杆线虫运动的方法

测量秀丽隐杆线虫对乙酰胆碱受体激动剂左旋咪唑的敏感性

线粒体疾病脑膜炎基因模型中量化动物活动的实验方法比较分析